Abstract Book for the 27 ^th Congress of the European Hematology Association

Abstract

Presidential Symposium S100: QUIZARTINIB PROLONGED SURVIVAL VS PLACEBO PLUS INTENSIVE INDUCTION AND CONSOLIDATION THERAPY FOLLOWED BY SINGLE-AGENT CONTINUATION IN PATIENTS AGED 18-75 YEARS WITH NEWLY DIAGNOSED FLT3-ITD+ AML H. Erba1,*, P. Montesinos2, R. Vrhovac3, E. Patkowska4, H.-J. Kim5, P. Zak6, P.-N. Wang7, T. Mitov8, J. Hanyok9, L. Liu9, A. Benzohra9, A. Lesegretain9, J. Cortes10, A. Perl11, M. Sekeres12, H. Dombret13, S. Amadori14, J. Wang15, M. Levis16, R. Schlenk17 1Duke Cancer Institute, Durham, NC, United States of America; 2La Fe University and Polytechnic Hospital, Valencia, Spain; 3University Hospital Centre Zagreb, Zagreb, Croatia; 4Institute of Hematology and Blood Transfusion, Warsaw, Poland; 5The Catholic University of Korea, Seoul St. Mary’s Hospital, Seoul, South Korea; 6University Hospital Hradec Kralove, Hradec Kralove, Czechia; 7Chang Gung Medical Foundation, Linkou, Taiwan; 8Daiichi Sankyo UK Ltd, Uxbridge, United Kingdom; 9Daiichi Sankyo, Inc, Basking Ridge, NJ; 10Augusta University Medical Center, Augusta, GA; 11University of Pennsylvania, Philadelphia, PA; 12University of Miami Health System, Miami, FL, United States of America; 13Saint Louis Hospital, University of Paris, Paris, France; 14Tor Vergata Polyclinic Hospital Rome, Rome, Italy; 15Institute of Hematology and Blood Diseases Hospital, Tianjin, China; 16Johns Hopkins University, Baltimore, MD, United States of America; 17Heidelberg University Hospital and German Cancer Research Center, Heidelberg, Germany Background: Quizartinib (Quiz) is an oral, highly potent, and selective type II FLT3 inhibitor with single-agent activity in relapsed/refractory FLT3–internal tandem duplication positive (FLT3-ITD+) acute myeloid leukemia (AML). This is the first report of the global, randomized, double-blind, placebo (PBO)-controlled phase 3 QuANTUM-First trial (NCT02668653). Aims: QuANTUM-First aimed to determine if the addition of Quiz to standard induction (IND) and post remission (including allogeneic hematopoietic cell transplant [allo-HCT]) in first complete remission [CR1]) consolidation followed by single-agent continuation therapy for up to 3 years improved survival compared with chemotherapy alone in patients (pts) with newly diagnosed FLT3-ITD+ AML. Methods: Pts aged 18-75 y with newly diagnosed AML were centrally screened for FLT3-ITD prior to initiation of IND with cytarabine 100 mg/m2/day (200 mg/m2/day if institutional standard) for 7 days and anthracycline (daunorubicin 60 mg/m2/day or idarubicin 12 mg/m2/day) for 3 days. Pts at 193 sites in 26 countries who were FLT3-ITD+ provided informed consent and were randomized to Quiz (40 mg/day days 8-21) or PBO and were stratified by region (North America, Europe, and Asia/Other regions), age (<60 y, ≥60 y), and white blood cell count (<40×109/L, ≥40×109/L) at diagnosis. A second IND was allowed if residual AML was noted at the post-IND marrow exam. Pts who achieved CR or CR with incomplete hematologic recovery (CRi) received up to 4 cycles of high-dose cytarabine plus Quiz (40 mg/day) or PBO and/or allo-HCT followed by up to 3 y of continuation therapy with Quiz (30-60 mg/day) or PBO. The primary endpoint was overall survival (OS). Results: Between September 2016 and August 2019, 3468 pts were screened, and 539 pts with FLT3-ITD+ AML were randomized to Quiz (n=268) or PBO (n=271). The median age was 56 y (range, 20-75 y). Baseline pt and disease characteristics, including FLT3-ITD variant allele frequency, were balanced between the 2 arms. At data cutoff (August 2021), the median follow-up was 39.2 months and 58 pts remained on continuation therapy. OS was significantly longer in the Quiz arm than the PBO arm (hazard ratio [HR], 0.776; 95% CI, 0.615-0.979; 2-sided P=.0324). Median OS was 31.9 mo with Quiz vs 15.1 mo with PBO (Figure). CR/CRi rates were 71.6% and 64.9%, respectively. Allo-HCT in CR1 was performed in 157 pts (Quiz, 31%; PBO, 27%). When censored for allo-HCT, OS trended longer with Quiz vs PBO (HR, 0.752; 95% CI, 0.562-1.008; 2-sided P=0.055). Relapse-free survival was longer with Quiz than PBO (HR, 0.733; 95% CI, 0.554-0.969). Although rates of grade ≥3 adverse events (AEs) were similar across arms, grade ≥3 neutropenia was more frequent in the Quiz arm (18.1% vs 8.6%). Discontinuations due to AEs occurred in 20.4% of Quiz and 8.6% of PBO pts. A total of 56 treatment-emergent AEs were associated with a fatal outcome (Quiz, 11.3%; PBO, 9.7%), mostly due to infections. Grade 3/4 electrocardiogram QT prolonged occurred in 3.0% of Quiz vs 1.1% of PBO pts. Image: Summary/Conclusion: These pivotal findings show that the addition of Quiz to standard chemotherapy and up to 3 years of continuation therapy yielded statistically significant and clinically meaningful improvements to OS in adults with newly diagnosed FLT3-ITD+ AML up to age 75 y. The manageable safety profile further supports use of Quiz in combination with standard therapy, including allo-HCT, in FLT3-ITD+ AML. S101: GENETIC AND EPIGENETIC FACTORS DRIVING PRIMARY MEDIASTINAL B-CELL LYMPHOMA PATHOGENESIS AND OUTCOME D. Noerenberg1,*, F. Briest1, C. Hennch1, K. Yoshida2,3, J. Nimo1, R. Hablesreiter1, Y. Takeuchi2, D. Sasca4, H. Ueno2, L. Mansouri5, Y. Inoue2, L. Wiegand1, A. M. Staiger6,7, B. Casadei8,9, M. Ziepert10, F. Asmar11, P. Korkolopoulou12, M. Kirchner13, P. Mertins13, J. Weiner14, E. Toth15, T. Weber16, A. Warth17, T. Schneider18, R.-M. Amini19, W. Klapper20, M. Hummel21,22, V. Poeschel23, G. Kanellis24, A. Rosenwald25, G. Held23,26, E. Campo27,28,29, K. Stamatopoulos5,30, I. Anagnostopoulos21,25, L. Bullinger1,22, N. Goldschmidt31, P. L. Zinzani9,32, C. Bödor33, R. Rosenquist5,34, T. P. Vassilakopoulos35, G. Ott6, S. Ogawa2,36,37, F. Damm1,22 1Department of Hematology, Oncology and Cancer Immunology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany; 2Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 3Wellcome Trust Sanger Institute, Hinxton, United Kingdom; 4Department of Hematology, Oncology, and Pulmonary Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany; 5Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 6Department of Clinical Pathology, Robert-Bosch-Krankenhaus; 7Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology Stuttgart, and University of Tuebingen, Stuttgart, Germany; 8Istituto di Ematologia “Seràgnoli”, IRCCS Azienda Ospedaliero-Universitaria di Bologna; 9Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy; 10Institute of Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany; 11Department of Hematology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 12First Department of Pathology, National and Kapodistrian University of Athens, Laikon General Hospital, Athens, Greece; 13Core Unit Proteomics, Berlin Institute of Health, Charité - Universitätsmedizin Berlin and Max-Delbrück-Center for Molecular Medicine; 14Core Unit Bioinformatics, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany; 15National Institute of Oncology, Budapest, Hungary; 16Department of Internal Medicine IV, Haematology and Oncology, University Hospital Halle (Saale), Martin-Luther-University Halle-Wittenberg, Halle; 17Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; 18National Institute of Oncology, Budapest, Hungary; 19Department of Immunology, Genetics and Pathology, Uppsala University and University Hospital, Uppsala, Sweden; 20Department of Pathology, Hematopathology Section and Lymph Node Registry, Universitätsklinikum Schleswig-Holstein, Kiel; 21Department of Pathology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin; 22German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg; 23Department of Internal Medicine 1 (Oncology, Hematology, Clinical Immunology, and Rheumatology), Saarland University Medical School, Homburg/Saar, Germany; 24Department of Hematopathology, Evangelismos General Hospital, Athens, Greece; 25Institute of Pathology, University of Würzburg and Comprehensive Cancer Center (CCC) Mainfranken, Würzburg; 26Department Internal Medicine I, Westpfalzklinikum Kaiserslautern, Kaiserslautern, Germany; 27Centro de Investigacion Biomedica en Red en Oncologia (CIBERONC), Madrid; 28Hospital Clinic of Barcelona, University of Barcelona; 29Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 30Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece; 31Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 32Istituto di Ematologia “Seràgnoli”, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 33HCEMM-SE Momentum Molecular Oncohematology Research Group, 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary; 34Clinical Genetics, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden; 35Department of Hematology and Bone Marrow Transplantation, National and Kapodistrian University of Athens, Laikon General Hospital, Athens, Greece; 36Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan; 37Department of Medicine, Centre for Haematology and Regenerative Medicine, Karolinska Institutet, Stockholm, Sweden Background: Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive lymphoma affecting predominantly young female patients. Previous studies in this rare entity have focused on single genes or were limited in cohort size. Aims: To unravel the underlying genetic pathogenesis and its impact on outcome, we embarked on a comprehensive large-scale genetic investigation. Methods: Specimens of 486 previously untreated PMBCL patients were analyzed by paired tumor/normal whole-genome (WGS, n=14), whole-exome (WES, n=78) and targeted sequencing (TS, n=486). To understand the consequences of highly recurrent mutations in the chromatin-modifying gene ZNF217, we conducted functional and multi-omics analyses in CRISPR/Cas9 engineered cell lines. Results: WGS/WES revealed a complex genomic landscape in PMBCL with a median of 85 structural variants, a mutational burden of 5 mutations/Mb, 12 mutated coding candidate driver genes (CDG) and 4 focal somatic copy-number aberrations per sample (Fig.1a). Besides known targets, significant breakpoints were identified in genes previously not implicated in B-lymphomagenesis such as TOX and TP73 (36% and 21%). In addition, non-coding mutations clustered within the PAX5 enhancer region. With the identification of 50 recurrently mutated CDGs, we significantly expand the repertoire of known PMBCL drivers. The 10 most frequently mutated CDGs were SOCS1 (86%), B2M (67%), ITPKB (64%), ACTB (58%), STAT6 (58%), IGLL5 (56%), TNFAIP3 (53%), NFKBIE (49%), GNA13 (47%), and ZNF217 (36%), respectively. The operative mutational processes were attributed to aging, AID/APOBEC activity, defective MMR, and an unexpected infidelity of DNA-Polymerase ε. Next, we performed TS in 486 samples using a PMBCL-specific 106-gene panel. Recurrent lesions in 25 epigenetic modifiers were found in >90%, with ZNF217 being among the most frequently mutated genes (Fig.1b). After knockdown of ZNF217 in Karpas1106P and L428 cells, we demonstrated altered proliferation, migration, and apoptosis. Using mass spectrometry, we showed that ZNF217 is acting in a LSD1, CoREST and HDAC containing histone modifier complex. Accordingly, knockout of ZNF217 led to global changes in chromatin accessibility with an enrichment of differentially accessible motifs for crucial lymphoma-associated transcription factors, especially of the NF-κB, BATF/AP1, and IRF family, but also of CTCF, a major regulator of global 3D chromatin architecture. Resulting gene expression was characterized by changes in interferon-responsive genes and inflammation-associated transcription (Fig 1c). Clinical data were available for 329 cases, including 84 cases from clinical trials. Multivariate analysis using an IPI-corrected Cox regression model was performed. The estimated 5-year PFS and OS were 77% and 86%. Among the genetic lesions with the strongest association for poor outcome, we identified patients with mutatedCD58 having a significantly shorter survival (PFS: HR 2.96; p<.001; OS: HR 2.55; p=.006). In contrast, mutated DUSP2 indicated longer survival (PFS: HR 0.28; p=.002; OS: HR 0.15; p=.011) (Fig1d). Notably, DUSP2 mutated patients (25%) showed a similar outcome for CR rate, PFS and OS when comparing CHOP-like and intensified treatment regimens, suggesting no further benefit from treatment intensification in this very-low risk patient population. Image: Summary/Conclusion: Here, we present the genetic landscape of PMBCL highlighting a previously underappreciated role of chromatin modifying genes, identify novel treatment targets and provide a solid basis for guiding precision medicine approaches. S102: COMPREHENSIVE GENOME CHARACTERIZATION REVEALS NEW SUBTYPES AND MECHANISMS OF ONCOGENE DEREGULATION IN CHILDHOOD T-ALL P. Pölönen1,*, A. Elsayed1,2, L. Montefiori1, S. Kimura1, J. Myers3, D. Hedges3, J. Xu4, Y. Hui3, Z. Cheng3, Y. Fan3, Y. Chang1, R. Shraim5, M. Devidas6, S. Winter7, K. Dunsmore8, J. J. Yang9, T. L. Vincent10, K. Tan10,11,12,13,14, C. Chen10, H. Newman15, M. Loh16, E. Raetz17, S. P. Hunger18, E. Rampersaud3, T.-C. Chang3, G. Wu3, S. Pounds2, C. G. Mullighan1,19, D. T. Teachey5,12,20 1Pathology; 2Biostatistics; 3Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis; 4Perelman School of Medicine at the University of Pennsylvania; 5Department of Pediatrics and the Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia; 6Global Pediatric Medicine, St. Jude Children’s Research Hospital, Memphis; 7Minnesota Research Institute and Cancer and Blood Disorders Program, Children’s Minnesota Research Institute, Minneapolis; 8University of Virginia Children’s Hospital, Charlottesville; 9Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis; 10Division of Oncology and Center for Childhood Cancer Research; 11Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia; 12Perelman School of Medicine; 13Institute for regenerative medicine; 14Penn Epigenetics Institute, University of Pennsylvania; 15Division of Oncology and Center for Childhood Cancer, Children’s Hospital of Philadelphia, Philadelphia; 16Department of Pediatrics, Benioff Children’s Hospital and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco; 17Department of Pediatrics and Perlmutter Cancer Center, NYU Langone Health, New York; 18Department of Pediatrics and the Center for Childhood Cancer Research, Children’s Hospital of Philadelphia and The Perelman School of Medicine at The University of Pennsylvania, Philadelphia; 19Hematological Malignancies Program, St. Jude Children’s Research Hospital, Memphis; 20Divisions of Hematology and Oncology, Children’s Hospital of Philadelphia, Philadelphia, United States of America Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy including leukemias of early T cell precursor acute lymphoblastic leukemia (ETP-ALL) and transformed thymocytes. Prior genomic studies of T-ALL had limited cohort size, excluded refractory disease and focused on alterations in coding parts of the genome. Aims: Investigate the genomic basis of T-ALL by identifying both coding and non-coding alterations and defining T-ALL subtypes. Methods: We performed whole-genome sequencing (WGS), whole-exome sequencing (WES), RNA-sequencing of 1,313 cases enrolled on the Children’s Oncology Group AALL0434 trial. Results: Uniform Manifold Approximation and Projection (UMAP) and gene expression clustering analyses of RNA-seq data identified 16 subtypes, of which 4 have not been reported previously. Furthermore, we could divide existing subtypes into smaller subgroups with common subtype-defining alterations, such as structural variation (SV) and copy number variation (CNV). TLX1 activation was linked with TLX1-TCR SVs and deletions in the TLX1 chromosomal domain, whereas TLX3 deregulation was associated with TLX3-BCL11B enhancer hijacking, but also through TLX3-TCR, TLX3-CDK6 rearrangements. We discovered two separate NKX2-1 deregulated groups, one characterized by TCR rearrangements and RPL10 mutations and the other by NKX2-1 CNVs, chromosome 14 chromothripsis, or MYB-TCR rearrangements. We also observed a distinct group of 9 cases aged 1-2 years, with recurrent STAG2-LMO2 rearrangements and a patient with inactivating STAG2 mutation and CELF1 enhancer hijacking by LMO2. Moreover, we found a group of 22 cases that were highly enriched for ETP-ALL with the following hallmark lesions: BCL11B enhancer amplification, BCL11B locus SVs involving enhancer hijacking of ARID1B, CCDC26, and novel CD34+ enhancer hijacking of lincRNA locus in chromosome 6. Gene expression-based clustering was unable to stratify patients based on TAL1, TAL2, LMO1, LMO2, LYL1 expression, and their respective activation mechanisms. However, WGS enabled further characterization of these patients, by identifying several types of activation mechanisms and co-occurring alterations for each oncogene. We identified canonical events, such as TAL1-STIL fusions, TCR rearrangements, and activation by TAL1/LMO1/LMO2 regulatory region indels, but also novel events, such as CD34 specific enhancer duplications downstream of TAL1 and BCL11B enhancer hijacking by LMO2. We also observed two smaller clusters with TAL1/LMO2 activation, where one with 39 cases was associated with TAL1/LMO2 activation and RPL10 mutations and the other with 8 cases had refractory disease (Day 29 MRD >5%). HOXA gene expression-associated subtypes were defined by fusions involving KMT2A or MLLT10 and PICALM/DDX3X or HOXA9-TCR SVs. Furthermore, our analysis revealed segregation of patients by HOXA13 or ZFP36L2 rearrangements and NUP98/NUP214 fusions. Interestingly, HOXA locus breakpoints involving HOXA13 and HOXA9 were in different chromatin compartments and were associated with mutually exclusive activation of either HOXA13 or other HOXA genes. Interestingly, HOXA13-deregulation was associated with ETP-ALL and frequent BCL11B enhancer hijacking, whereas HOXA9 breakpoints typically involved TCRg and were non-ETP. Image: Summary/Conclusion: Large-scale analysis of all children enrolled on the AALL0434 study has identified new subtype-defining lesions in T-ALL, including candidate novel enhancer hijacking events and enhancer duplications that are likely to result in oncogene deregulation in T-ALL. S103: EFFICACY AND SAFETY OF ARI0002H, AN ACADEMIC BCMA-DIRECTED CAR-T CELL THERAPY WITH FRACTIONATED INITIAL THERAPY AND BOOSTER DOSE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA C. Fernandez De Larrea1,*, V. González-Calle2, A. Oliver-Caldés1, V. Cabañas3, P. Rodríguez-Otero4, M. Español-Rego1, J. L. Reguera5, L. López-Corral2, B. Martin-Antonio1, B. Paiva4, S. Inogés4, L. Rosiñol1, A. López-Díaz de Cerio4, N. Tovar1, M. López-Parra2, L. G. Rodríguez-Lobato1, A. Sánchez-Salinas3, S. Varea1, V. Ortiz-Maldonado1, J. A. Pérez Simón5, F. Prósper4, M. Juan1, J. M. Moraleda3, M. V. Mateos2, M. Pascal1, A. Urbano-Ispizua1 1Hospital Clínic de Barcelona. IDIBAPS. University of Barcelona, Barcelona; 2Hospital Universitario de Salamanca, Instituto de Investigacion Biomedica de Salamanca (IBSAL), Centro de Investigación del Cancer (IBMCC-USAL, CSIC), Salamanca; 3Hospital Clínico Universitario Virgen de la Arrixaca. IMIB-Arrixaca. University of Murcia, Murcia; 4Clínica Universidad de Navarra, Centro de Investigacion Medica Aplicada (CIMA), IDISNA, CIBERONC Pamplona, Pamplona; 5Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS/CSIC/CIBERONC), University of Sevilla, Sevilla, Spain Background: ARI0002h is a lentiviral autologous CAR T-cell product with a 4-1BB co-stimulatory domain and a humanized single chain variable fragment targeting BCMA. In pre-clinical studies, this academic CAR-T has demonstrated potent in vitro and in vivo activity. Aims: We report the safety and efficacy results of the CARTBCMA-HCB-01 multicenter clinical trial for patients with relapsed/refractory multiple myeloma (RRMM) (NCT04309981) who received ARI0002h in 5 Spanish centers. Methods: Patients (pts) aged 18-75 years old with RRMM were eligible for this study if they had measurable disease, received ≥2 prior regimens, including a proteasome inhibitor, an immunomodulatory drug and an anti-CD38 antibody, and were refractory to the last line of treatment. Bridging therapy was allowed after apheresis. Cyclophosphamide (300 mg/m2) and fludarabine (30 mg/m2) were used as lymphodepletion regimen. The targeted dose was 3x106/kg CAR+ cells and was administered in a fractionated manner (10%/30%/60%), with at least 24h between infusions. A second dose of 3x106 CAR+ cells/kg was planned at least 4 months after the first dose in pts who achieved any grade of response any response and had not or serious complications after the first administration. Primary objectives were overall response rate (ORR; at least partial response -PR-) within 3 months of the first infusion and rate of cytokine release syndrome (CRS) and/or neurological toxicity in the first 30 days. Response was assessed as per IMWG criteria and bone marrow minimal residual disease (MRD) was analyzed by next-generation flow (NGF). Results: As of February 9th 2022, 35 pts (median age 61 years) with RRMM were included in the trial. Four pts could not receive ARI-0002h due to MM progression and one died of infection. Therefore, 30 pts received ARI0002h cells (modified intention-to-treat population), of which 47% received bridging therapy. Median CAR-T cell production time was 11 days (range 9-14) with a 100% manufacture success. Median follow-up after ARI0002h administration for surviving pts was 16 months. The ORR of 30 evaluable pts was 100%, with a stringent complete remission (sCR) plus very good partial response (VGPR) rate of 90%. Median time to first response was one month. Of 26 MRD-evaluable pts at day +100, 92% were MRD-negative in bone marrow by NGF. 53% of patients were alive and without progression at 16 months. Median overall survival (OS) was not reached and the 16-month OS rate was 80% (Figure 1). AEs reported in >70% of pts were CRS (87%; grade [gr] 3/4 0%; gr 1 73%), neutropenia (97%; gr 3/4 100%), anemia (85%; gr 3/4 43%), and thrombocytopenia (79%; gr 3/4 70%). Median duration of CRS was 4 days (range 1-12). No CAR-T cell-related neurotoxicity cases were reported. Tocilizumab and corticosteroids were administered in 76% (mainly for persistent grade 1 CRS) and 12% of pts, respectively. ARI0002h cells demonstrated peak expansion on day 14 (range 7 days-6 months). 24 out of 28 eligible pts (86%) received the second dose (range 1.2-3x106 CAR+ cells/kg). Median time after first infusion was 4 months and 38% received a second lymphodepletion regimen. No relevant toxicities after second infusions were reported. 7 pts (29%) improved their response after reinfusion. Image: Summary/Conclusion: ARI0002h is the first European academic CART for RRMM that has demonstrated deep and durable responses and a favorable safety profile, including the absence of neurotoxicity and the feasibility of a second booster dose. S104: RBPS DYSREGULATION CAUSE HYPER-NUCLEOLI AND RIBOSOME GAIN-OF-FUNCTION DRIVING BONE MARROW FAILURE P. Aguilar-Garrido1,*, M. Velasco1, M. Hernández Sánchez2, M. Á. Navarro Aguadero1, P. Malaney3, M. JL Aitken3, X. Zhang3, K. H Young3, R. Duan3, P. Hu3, S. Kornblau3, A. Fernández1, A. Ortiz1, Á. Otero-Sobrino1, P. J. de Andrés2, D. Megías1, M. Pérez1, J. Gómez1, G. Mata1, J. Martínez López1, S. Post3, M. Gallardo1 1CNIO; 2UCM, Madrid, Spain; 3MD Anderson, Houston, United States of America Background: Nucleoli and ribosome cross-talk regulates cell translation capacity. Its dysregulation and impairment drive ribosome and nucleolus stress (NS), related to the biological mechanism of cancer. Ribosomopathies are a group of diseases characterized by ribosome defects leading to complex syndromes that include bone marrow failure. hnRNP K is an RNA binding protein (RBP) in charge of processing nascent RNAs (nucleoli) into mature mRNAs (ribosome). Our research found a novel ribosome gain-of-function ribosomopathy phenotype by hyper-nucleoli generation due RBP hnRNP K dysregulation. Aims: We aim to elucidate how hnRNP K dysregulation impact on haematopoietic stem cell biology. Methods: Hnrnpk overexpression was established in MEFs using CRISPR/SAM (Konermann S. et al, Nature). Global protein synthesis was tested using a Click-iT OPP and proteasome function was evaluated by Proteasome 20S activity NS hallmarks were analyzed by confocal microscopy evaluating Ncl, NS sensor marker. To evoke NS in our cells, we used Actinomycin D insult. Cell cycle FACS analysis (DAPI) and senescence assays (β-galactosidase staining) were performed. Molecular mechanism underlying was elucidated by qRT-PCR and WB (p21, p16, c-Myc, and mTor). To study the impact of hnRNP K overexpression in vivo, we developed an inducible tamoxifen mouse model activated 30-60 days after birth (Hnrnpk Tg-Ubc-creERT2). Survival was evaluated by Kaplan-Meier, and phenotype described by symptoms, signs, CBC and bone marrow IHC panel (CD34, Gr1, B220, MPO). Results: Hnrnpk overexpressing cells led to an increment in protein and gene expression of Ncl, mTor and c-Myc (Figure 1A-B). Moreover, we found an increase in global protein synthesis (Figure 2C-D). Nevertheless, the elevation of hnRNP K inversely correlated with the proteasome function, which dropped significantly (Figure 1E). NS induction promoted higher hnRNP K expression. Additionally, hnRNP K overexpressing cells showed NS hallmarks associated with an increase of the number of nucleoli, and total area of the nucleoli and nucleus (Figure 1F-G). Cell cycle analysis confirmed an increment of arrested G2/M phase cells (Figure 1H-I), linked to an increment in p21 and p16 levels all leading towards a senescent cell phenotype (Figure 1J-L). HnrnpkTg-Ubc-creERT2 mice had widespread Hnrnpk overexpression (Figure 1M) and a reduction in lifespan (Figure 1N), mainly due to dysplastic and bone marrow failure phenotype, with dramatic reduction of CD34 and b-cells, leukopenia, anaemia and thrombocytopenia. (Figure 1O-Q). Image: Summary/Conclusion: The overexpression of hnRNP K drives to an increase in nucleoli activity, leading to ribosome biogenesis and higher global translation by the regulation of molecules involved in both systems: Ncl, c-Myc or mTor. Our work found that hnRNP K overexpression in vivo drives a bone marrow failure phenotype, promoting the exhaustion of haematopoietic stem cells by ribosome dysregulation that triggered cell senescence. Of note, this is the first time reported that a nucleoli/ribosome-gain-of-function induce bone marrow failure ribosomopathies phenotype. This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295), ISCIII Miguel Servet (CP19/00140), Cancer Research UK [C355/A26819], FC AECC and AIRC under the Accelerator Award Program and National Cancer Institutes of Health Award (R01CA207204, SMP) Leukemia and Lymphoma Society (6577-19, SMP). Oral Sessions S105: IN VIVO PDX CRISPR/CAS9 SCREENS REVEAL MUTUAL THERAPEUTIC TARGETS TO OVERCOME HETEROGENEOUS ACQUIRED CHEMO-RESISTANCE A.-K. Wirth1,*, L. Wange2, S. Vosberg3, A. K. Jayavelu4, W Enard2, T Herold5, I Jeremias1 1Apoptosis in Hematopoietic stem cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), Munich; 2Anthropology and Human Genomics, Faculty of Biology, Ludwig Maximilian University (LMU), Martinsried, Germany; 3Clinical Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; 4Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried; 5Department of Medicine III, and Laboratory for Leukemia Diagnostics, Ludwig Maximilian University (LMU), Munich, Germany Background: Acquired resistance to conventional polychemotherapy regimens leads to relapse and poor prognosis, and remains a major unmet clinical need. Aims: To identify therapeutic options to overcome acquired chemo-resistance. Methods: We studied acute lymphoblastic leukemia (ALL) as model disease and combined long-term in vivo treatment in orthotopic patient-derived xenograft (PDX) models with multi-omics profiling and functional genomic CRISPR/Cas9 screens. Results: We adapted conventional chemotherapeutic protocols to allow treatment of mice for up to 18 consecutive weeks, using a combination of the widely used drugs cyclophosphamide and vincristine. Three luciferase-transgenic PDX models were monitored by repetitive in vivo imaging. Polychemotherapy strongly reduced PDX ALL cells within the first weeks, proving initial sensitivity of the sample. Under continuous treatment, tumor load persisted in mice at the level of minimal residual disease for several weeks, until tumors resumed growth despite treatment, indicating acquired resistance. In an exemplary PDX model, eight resistant derivatives were generated in replicate mice and characterized individually. Genomic profiling revealed profound genomic heterogeneity between distinct derivatives; individual resistant derivatives acquired different copy number alterations in regions associated with resistance and distinct point mutations, e.g. in TP53. In contrast to genomic heterogeneity, transcriptome and proteome profiling identified a group of genes differentially expressed between sensitive and resistant cells, but similar across all derivatives. To gain insights into underlying mechanisms and to identify therapeutic targets to overcome acquired resistance, a customized CRISPR/Cas9 in vivo dropout screen was performed in individual resistant PDX derivatives, to test the relevance of around 200 candidate genes under treatment. Among others, sgRNAs targeting BCL2, BRIP1 or COPS2 dropped out in the in vivo screen specifically during treatment; single knockout experiments confirmed that knockout of either BCL2, BRIP1 or COPS2 re-sensitized PDX ALL cells towards chemotherapy. Of direct translational relevance, treatment of mice with the BCL2 inhibitor ABT-199 sensitized resistant PDX cells towards polychemotherapy in vivo. Interestingly, BCL2 inhibition restored treatment response in resistant derivatives independently from the highly diverse underlying genetic alterations, e.g., in clones with and without mutation in TP53. Summary/Conclusion: Taken together, we established a highly clinically relevant PDX in vivo model of acquired resistance to conventional chemotherapy. Using this model, we demonstrate that heterogeneous genomic alterations evolved in parallel in replicate mice, which could be overcome by a single therapeutic approach to re-sensitize tumors towards conventional chemotherapy. S106: UBTF-ATXN7L3 GENE FUSION DUE TO 17Q21.31 DELETION DEFINES NOVEL HIGH-RISK ALL SUBTYPE AMENABLE TO MRD-BASED TREATMENT INTENSIFICATION L. Bastian1,*, A. Hartmann1, T. Beder1, S. Hänzelmann1, J. Kässens1, M. Bultmann1, M. P. Höppner2, S. Franzenburg2, M. Wittig2, A. Franke2, I. Nagel3, M. Spielmann3, N. Reimer4, H. Busch4, S. Schwartz5, B. Steffen6, A. Viardot7, K. Döhner7, M. Kondakci8, G. Wulf9, K. Wendelin10, A. Renzelmann11, A. Kiani12, H. Trautmann1, M. Neumann1, N. Gökbuget6, M. Brüggemann1, C. Baldus1 1Department of Medicine II, Hematology and Oncology, University Medical Center Schleswig-Holstein, Kiel; 2Institute for Clinical Molecular Biology, Kiel University, Kiel; 3Institute of Human Genetics, University Medical Center Schleswig-Holstein, Kiel and Lübeck, Kiel and Lübeck; 4Medical Systems Biology Group and Institute for Cardiogenetics, University of Lübeck, Lübeck; 5Department of Hematology, Oncology and Tumor Immunology (Campus Benjamin Franklin), Charité - Universitätsmedizin Berlin, Berlin; 6Department of Medicine II, Hematology/Oncology, Goethe University Hospital, Frankfurt / Main; 7Department of Internal Medicine III, University Hospital Ulm, Ulm; 8Department of Hematology, Oncology and Clinical Immunology, University Hospital Düsseldorf, Düsseldorf; 9Department of Hematology and Oncology, University Hospital Göttingen, Göttingen; 10Medical Department V, Hospital Nürnberg, Paracelsus Medizinische Privatuniversität, Nürnberg; 11Medical Department Oncology and Hematology, University Medical Center Oldenburg, Oldenburg; 12Department of Medicine IV, Hematology/Oncology, Klinikum Bayreuth, Bayreuth, Germany Background: Response to induction chemotherapy assessed by quantification of minimal residual disease (MRD) is the strongest independent prognosticator in B precursor acute lymphoblastic leukemia (BCP-ALL). Molecular underpinnings of MRD poor response are insufficiently understood. Aims: We aimed to identify novel high-risk subtypes in adult BCP-ALL as cell-intrinsic determinants of MRD poor response. Methods: Adult BCP-ALL patients (n=565) were treated according to pediatric inspired protocols of the German Acute Lymphoblastic Leukemia Study group (GMALL) and profiled for integrative analyses by RNA-Seq (n=565), SNP-arrays (n=115), whole exome sequencing (WES; n=84) and whole genome sequencing (WGS; n=3). Results: Concordance between transcriptomic and genomic profiles was used to allocate samples to one of 15 established molecular driver subgroups (Figure A). Unsupervised clustering of gene expression from the remaining samples revealed a distinct cluster (n=12/565, 2.1%) with an in-frame gene fusion between upstream binding transcription factor (UBTF) and ataxin-7-like protein 3 (ATXN7L3) as exclusive event in these patients. Both fusion partners are in direct neighborship at 17q21.31. WGS revealed a 10.08 kb genomic deletion which truncated UBTF at exon 17/21 and comprised most of the intergenic region between both genes (Figure B). UBTF-ATXN7L3 rearranged cases frequently harbored 1q gains (n=5/7). Further genomic profiling showed a remarkable paucity of additional cooperating events compared to other molecular subtypes, supporting a prominent driver function of the newly identified fusion. UBTF and ATXN7L3 are global epigenetic regulators involved in transcriptional control. Both genes were highly expressed across the entire cohort. The gene fusion was associated with a marked increase of Caudal Homeobox 2 (CDX2) expression. Analysis of functional modules related CDX2 to upregulated HOXA9 and MEIS1, described essential co-regulators of KMT2A-driven leukemogenesis. NTRK3 expression was also strongly upregulated, suggesting a possible rationale for specific inhibitors. UBTF-AXTN7L3 rearranged patients were older, more frequently female and presented with normal leukocyte counts, low bone marrow infiltration and pro-B immunophenotypes or common ALL with reduced CD10 expression. Response to induction chemotherapy in evaluable patients (n=11) was poor with only 3 patients achieving MRD negativity after consolidation I compared to n=271/402 (67%; p=0.019) in the remaining cohort. Four patients suffered either cytologic (n=2) or molecular (n=2) relapse. Immunotherapeutic treatment intensification using blinatumomab (n=5) or inotuzumab ozogamizin (n=1) and / or allogenic stem cell transplantation (n=7) in MRD poor responders or relapsed cases resulted in an overall survival probability of 80% (+/- 12%) vs. 73% (+/- 2%; p=0.07) in the remaining cohort. Heterogeneous MRD responses were observed for other molecular subtypes (poor: ZNF384 - 48.2% MRD neg., p=0.056; Ph-like - 54.0% MRD neg., p=0.003; KMT2A - 55.8% MRD neg., p=0.127 / good: High Hyperdiploid - 90.9% MRD neg., p=0.01; TCF3-PBX1 - 94.1% MRD neg., p=0.016) indicating how molecular drivers affect chemo-sensitivity in adult BCP-ALL. Image: Summary/Conclusion: Molecular driver alterations determine sensitivity to induction chemotherapy in adult BCP-ALL. UBTF-ATXN7L3 ALL represents a novel subtype with poor induction chemotherapy response which could be successfully salvaged by MRD-based treatment intensification using immunotherapeutic strategies. S107: SOD2 PROMOTES ACUTE LEUKEMIA ADAPTATION TO AMINO ACID STARVATION THROUGH THE N-DEGRON PATHWAY N. K. Ibrahim1,*, S. Schreek1, B. Cinar1, L. Loxha1, B. Fehlhaber1, J.-P. Bourquin2, B. Bornhauser2, C. Eckert3, G. Cario4, M. Forster5, M. Stanulla1, A. Gutierrez6, L. Hinze1 1Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany; 2Department of Pediatric Hematology and Oncology, University Children’s Hospital Zurich, Zurich, Switzerland; 3Department of Pediatric Hematology and Oncology, Charité Universitätsmedizin, Berlin; 4Department of Pediatrics I, Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein; 5Institute of Clinical Molecular Biology, Kiel, Germany; 6Division of Pediatric Hematology and Oncology, Boston Children’s Hospital, Boston, United States of America Background: The ability of cells to tolerate amino acid starvation is fundamental for survival under cellular stress conditions. Some cancer cells are vulnerable to asparagine depletion, which is exploited therapeutically using asparaginase. However, the mechanisms of adaptation to amino acid starvation in leukemia cells remain incompletely understood. Aims: We recently performed a genome-wide CRISPR/Cas9 loss-of-function screen in the resistant T-ALL cell line CCRF-CEM to identify molecular pathways that promote asparaginase resistance. We found that Wnt-dependent stabilization of proteins (Wnt/STOP) induces a profound therapeutic vulnerability to asparaginase in acute leukemias and colorectal cancers (Hinze et. al., 2019; Hinze et al., 2020). Another unrelated gene on the top of the screen included SOD2, a mitochondrial superoxide dismutase. Intriguingly, to date, SOD2 activity has not been linked to a cellular amino acid starvation response, whose biologic basis we thus sought to further investigate. Methods: To evaluate the significance of SOD2 in mediating an asparaginase response, we employed genetic epistasis experiments as well as phenotypic assays including short hairpin RNA (shRNA) mediated knockdowns, quantitative PCRs, Western blots, amino acid starvation, and viability assays. Results: Knockdown of SOD2 (shSOD2) resulted in a profound sensitivity to asparaginase in several T-ALL and B-ALL cell lines (p<0.0001), and an increase in apoptosis, as assessed by caspase 3/7 activity (p<0.001). The sensitization was rescued by either overexpressing SOD2 cDNA (p<0.0001), or by adding the functional SOD2 mimetic MnTBAP (p<0.01). Of note, shSOD2 mediated sensitization was selective to asparaginase, as it could not be observed for other commonly used chemotherapeutic agents including vincristine, doxorubicin, dexamethasone, and 6-mercaptopurine (p=ns). Due to the selectivity to asparagine depletion, we then investigated whether SOD2 inhibition mediates a broader amino acid starvation response. Indeed, culturing SOD2-inhibited T-ALL cells in the absence of essential amino acids (EAA) or non-EAA, induced a significant decrease in cell viability (p<0.05). Sensitization appeared to be specific to the SOD2 isoform, and distinct from known SOD2-associated pathways including reactive oxygen species, cell cycle changes, alterations of mTOR signaling, or glutamine anaplerosis. To better understand the molecular underpinnings of SOD2 in regulating an amino acid starvation response, we leveraged the Bioplex Interactome database (Huttlin et al., 2020), and identified UBR2, an E3 ubiquitin ligase in the N-degron pathway, as a unique binding partner of SOD2. Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation (Yang et al., 2010). Indeed, SOD2 and UBR2 were co-immunoprecipitated, suggesting the formation of a complex that can drive proteasome-dependent protein catabolism. In line, inhibition of SOD2 significantly decreased ubiquitin levels, suggesting that SOD2 positively regulates catabolic protein degradation through the N-degron pathway to promote cancer cell fitness in amino acid starved conditions. Summary/Conclusion: The interaction of SOD2 and the N-degron pathway represents a previously unknown molecular adaptation of cancer cells in response to amino acid starvation. These results serve as a strong proponent for an in-depth characterization of the N-degron pathway in mediating leukemia cell fitness upon amino acid starvation and thus provide a basis for therapeutic intervention in refractory leukemias. S108: PEDIATRIC T- ALL RELAPSE: CONSTITUTIONAL CANCER PREDISPOSITION AND HYPERMUTATATOR PHENOTYPES P. Richter-Pechanska1 2,*, J. Kunz1 2, T. Rausch2 3, B. Erarslan-Uysal1 2, B. Bornhauser4, V. Frismantas4, Y. Assenov5, M. Zimmermann6, M. Happich1, C. von Knebel-Doeberitz1, N. von Neuhoff7, R. Koehler8, M. Stanulla6, M. Schrappe9, G. Cario9, G. Escherich10, R. Kischner-Schwabe11, C. Eckert11, S. Avigad12, S. Pfister5 13, M. Muckenthaler1 2, J.-P. Bourquin4, J. Korbel2 3, A. Kulozik1 2 5 1University Hospital Heidelberg; 2MMPU; 3EMBL, Heidelberg, Germany; 4University Children’s Hospital, Zürich, Switzerland; 5DKFZ, Heidelberg; 6Hannover Medical School, Hannover; 7University Hospital, University of Duisburg-Essen, Essen; 8University Heidelberg, Heidelberg; 9University Hospital Schleswig-Holstein, Kiel; 10University Medical Center Hamburg-Eppendorf, Hamburg; 11Charite, Berlin, Germany; 12Schneider Children’s Medical Center of Israel, Petah Tikva, Israel; 13KiTZ, Heidelberg, Germany Background: Relapse is the main cause of death from pediatric acute precursor T-cell leukemia (T-ALL), but the underlying mechanisms of disease evolution from initial disease to relapse remain incompletely understood and show remarkable interpatient heterogeneity. Aims: As cross-sectional studies failed to identify unifying determinants of relapse, we adopted a longitudinal strategy and performed multi-omic analyses in 13 matched pairs of initial diagnosis and relapse samples and their matched PDXs. We extended this set by WES and methylome analyses in an additional cohort of 25 matched DNA samples from patient cells collected at initial diagnosis, remission, and relapse. Methods: Thirty-eight patients were recruited from the ALL‐BFM 2000/2009, CoALL97/03/09, and ALL‐REZ BFM 2002 trials or from Schneider Children’s Medical Center of Israel at time points of initial diagnosis, remission, and relapse. Material from 13 matched pairs of PDXs (RNA, cells) was used for multi-omic analyses, including DNA-Seq (WES), RNA-Seq, ATAC-Seq and methylation analysis with EPIC arrays. Results: Based on the profile of SNVs and InDels we distinguished 18 (47%) type-1 (derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). We observed stronger remodeling on the way to type 2 than to type 1 relapses reflected by more evident changes in methylation, chromatin accessibility and gene expression. At the time of relapse, 3/20 type 2 patients exhibited a hypermutator phenotype, probably caused by gains of mutations in TP53, BLM and BUB1B combined with PMS2. Moreover, type 2 T-ALLs were predominantly TAL1-driven (4/8) in contrary to type 1 (0/5). T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. The fraction of subclonal mutations of those T-ALLs that later developed into a type-2 relapse was significantly higher already at the time of initial diagnosis than in those T-ALLs that later developed into a type-1 relapse (p=0.0387; Fisher’s exact), a difference that became even more pronounced at the time of relapse (p<0.0001). On the other hand, relapse type 1 T-ALLs exhibited overexpression of IL7R, its ligand HGF, and repressors of cytokine signaling (SOCS1, SOCS2, SOCS3) which regulates the IL7R pathway via negative feedback loop. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. Moreover, an analysis of remission samples revealed a significant enrichment of mutations in constitutional cancer predisposition genes (CPG) in type 2 patients, thus indicating fundamental differences between these two groups of patients. In both types of relapse, we observed known and novel drivers of drug resistance including MDR1 and MVP and NT5C2. Image: Summary/Conclusion: In sum, our comprehensive analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia. Leukemias of patients with type-1 relapses were often characterized by upregulation of the IL7R pathway, whereas type-2 relapses were characterized by (i) an enrichment of TAL-1 fusion, (ii) and of constitutional mutations in CPG, (iii) divergent genetic and epigenetic remodeling, and (iv) an enrichment of somatic hypermutator phenotypes. S109: ONCOGENIC DEUBIQUITINATION CONTROLS TYROSINE KINASE SIGNALING AND THERAPY RESPONSE IN ACUTE LYMPHOBLASTIC LEUKEMIA P. Ntziachristos1,*, Q. Jin2, B. Gutierrez3 1Ghent University, Ghent, Belgium; 2MD Anderson, Houston; 3Northwestern University, Chicago, United States of America Background: Dysregulation of kinase signaling pathways via mutations favors tumor cell survival and resistance to therapy and it is common in cancer. Our data unveil how dysregulated deubiquitination controls signaling pathways, leading to cancer cell survival and drug non-response, and suggest novel therapeutic combinations towards targeting T-cell acute lymphoblastic leukemia (T-ALL). Here, we reveal a novel mechanism of post-translational regulation of kinase signaling and nuclear receptor activity via deubiquitination in acute leukemia. Aims: This study aims at 1) characterizing the function of an oncogenic complex composed by two deubiquitinating enzymes in in vitro and in vivo leukemia systems and 2) testing the association of deubiquitinase activity with resistance to therapy in acute lymphoblastic leukemia. Methods: We use genetic mouse and human:mouse xenograft models of T-cell leukemia, biochemical studies (quantitative global proteomics, phosphoproteomics and ubiquitination analysis) and high-throughput molecular biology (chromatin conformation capture (HiC), chromatin accessibility (ATAC-Seq) and gene expression (RNA-Seq)) analyses. Results: We observed that the ubiquitin specific protease 11 (USP11) is highly expressed in lymphoblastic leukemia and associates with poor prognosis in this disease. USP11 ablation inhibits leukemia growth in vitro and in vivo, sparing normal hematopoiesis and thymus development, suggesting that USP11 could be a therapeutic target in leukemia. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK). Deubiquitination of LCK controls its activity, thereby altering T cell receptor signaling. Impairment of LCK activity leads to increased expression of the glucocorticoid receptor transcript, culminating into transcriptional activation of pro-apoptotic target genes, and sensitizes cells to glucocorticoids in T cell leukemia patient samples. The transcriptional activation of pro-apoptotic target genes, such as BCL2L11, is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Pharmacological inhibition of USP7 or genetic knockout of USP7 in combination treatment of glucocorticoid displayed improved anti-T-ALL efficacy in vivo. Image: Summary/Conclusion: Our data unveil how dysregulated deubiquitination controls signaling pathways, leading to cancer cell survival and drug non-response, and suggest novel therapeutic combinations towards targeting T-cell leukemia. S110: A NOVEL AND SUCCESSFUL CD7 GENE KNOCKOUT CAR-T CELL THERAPY FOR RELAPSED OR REFRACTORY T-CELL HEMATOLOGIC MALIGNANCIES J. Yang1, J. Li1, X. Zhang1, L. Qiu1, P. Lu1,* 1Hebei Yanda Lu Daopei Hospital, Langfang, China Background: T cell malignancies represent a group of hematologic cancers with high relapse and mortality rates. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T due to unintended CAR-T fratricide. Here, we develop a fratricide-resistant anti-CD7 CAR-T modified by CD7 ablation through CRISPR/CAS9 gene editing (KO7CAR). Aims: In a phase I clinical trial, we explored the efficacy and safety of KO7CAR T-cells for relapsed or refractory (R/R) T-cell malignancies (NCT04916860 & NCT04938115). Methods: Peripheral blood mononuclear cells were collected from patients (n=13) or the transplant donor (n=2) by leukapheresis. CD7-ablated CAR T cells (KO7CAR) were derived by electroporation of bulk T cells with CD7-targeting Cas9-gRNA RNP 24 hours before 7CAR transduction. This KO7CAR is a second-generation CAR-T with the co-stimulatory domain of 4-1BB and CD3ζ targeting CD7. Intravenous fludarabine (30mg/m2/d) and cyclophosphamide (300mg/m2/d) were given to all patients on day -5 to day -3 prior to KO7CAR-T cells infusion. Results: From Oct. 2020 to Oct. 2021, 15 patients with T-cell acute lymphoblastic leukemia (n=10), T-cell lymphoblastic lymphoma (n=3), and mixed phenotype acute leukemia (n=2) were enrolled and received KO7 CAR-T cells (Table 1). The median age was 28 (8-46) years old. Four patients had prior hematopoietic stem cell transplantation (HSCT). At enrollment, 10 patients had bone marrow (BM) blasts >5% by morphology, and 10 patients had the extramedullary disease (EMD, diffuse involvement, n=8, and bulky mediastinal masses, n=2). Both patient- and donor-derived KO7CAR-T cells were successfully generated with a transduction efficiency of 59.0% (22.5%-97.4%). A single dose of KO7CAR-T cells was infused to patients at low dose (1.5~5x105 cells/kg, n=8), medium dose (1x106 cells/kg, n=6) or high dose (2x106 cells/kg, n=1). On day 28, 15/15 (100%) patients achieved minimal residual disease (MRD) negative complete remission (CR). Among the 10 patients with EMD, 7 achieved EMD CR on day 30, 2 achieved partial response (PR), and 1 who relapsed post 2nd transplant had no response on day 35 then withdrew. Up to data cutoff Feb.10, 2022, the median follow-up time was of 309 days (35~407 days). About 2 months post KO7CAR, 12 patients bridged into allogeneic HSCT, and all remained progression-free after a median time of 253 (30~388) days after HSCT except for 1 who relapsed on day 147 then died from intracerebral hemorrhage on day 249. The other 2 patients without subsequent HSCT (all had a prior transplant) died from infection on day 78 and GVHD on day 103, respectively, post KO7CAR. Mild cytokine release syndrome (CRS, ≤grade II) occurred in 10/15 (66.7%) patients, and 5/15(33.3%) patients had grade III CRS. One patient had grade I neurotoxicity, and 2 had grade III/IV neurotoxicity. All was controlled after the administration of corticosteroids and/or tocilizumab. Following infusion, the median peak of circulating KO7CAR-T cells was 1.77×105 (0.279~14.3×105) copies/μg genomic DNA which occurred around day 20 (day10~ day25) and 63.47% (23.1%~94.18%) occurring on day15 (day 9~day25) by q-PCR and flow cytometry respectively. Image: Summary/Conclusion: This study demonstrated KO7CAR-T therapy had a high efficacy for CD7+ T-cell malignancies even for those who relapsed post-transplant. Safety was manageable, however, more data on additional patients and longer observation time are needed to evaluate the efficacy of KO7 CAR-T products further. S111: REPEATED INFUSIONS OF ESCALATING DOSES OF EXPANDED AND ACTIVATED AUTOLOGOUS NATURAL KILLER CELLS IN MINIMAL RESIDUAL DISEASE-POSITIVE PH+ ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS. A GIMEMA PHASE 1 TRIAL G. F. Torelli1,*, S. Chiaretti1, N. Peragine1, W. Barberi1, L. Santodonato2, G. D’Agostino2, E. Abruzzese3, M. I. Del Principe4, A. Mancino5, M. Matarazzo1, M. S. Bafti1, M Mancini1, M. Messina5, L. Castiello2, A. Guarini1, R. Foà1 1Hematology, Department of Translational and Precision Medicine, Sapienza University; 2FaBioCell Cell Factory, Istituto Superiore di Sanità; 3Hematology, Sant’Eugenio Hospital, ASL Roma2, Tor Vergata University; 4Hematology, Department of Biomedicine and Prevention, Tor Vergata University; 5Fondazione GIMEMA Onlus, Rome, Italy Background: Due to age and co-morbidities, many Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients are ineligible to undergo high-dose chemotherapy or allogeneic transplant as consolidation treatment. Our group reported the promising results of the chemo-free scheme D-ALBA based on dasatinib/blinatumomab in induction/consolidation, underlying the potential role of immunotherapy in this setting (Foà et al, NEJM 2020;383:1616-23). Considerable interest has been raised by natural killer (NK) cells. We developed a GMP protocol for NK cell ex vivo expansion in the presence of IL-2 and IL-15, and report the results of a phase 1 protocol of adoptive immunotherapy with activated and expanded autologous NK cells for Ph+ ALL patients in complete hematologic remission (CHR) but with persistent/recurrent minimal residual disease (MRD) ≥60 years or ineligible for other post-CHR treatment modalities. Aims: The primary endpoint was to determine the maximum tolerated dose of NK cells and the recommended dose for subsequent studies. Secondary endpoints were the assessment of safety and tolerability of the treatment, the immunologic modifications induced by the procedure and the clinical response to treatment. Methods: The planned 6 patients were enrolled: 5 in 1st CHR and 1 in 2nd CHR. Patients underwent repeated infusions (maximum 5) of escalating doses of NK cells, ranging from 1x106 to 5x107/kg of body weight (BW). No conditioning therapies were administered before the infusion; patients were allowed to continue tyrosine kinase inhibitors. Patients underwent a comprehensive MRD monitoring by Q-RT-PCR with a one-year follow-up. Immunophenotypic analysis on the NK cell product was performed before and after the expansion. Intracellular cytokine production and PBMC cytotoxic activity against K562 cells, allogeneic and autologous blasts were evaluated after expansion and at time 0 and 7 days from each NK cell infusion. Results: NK cells presented a 12.3-fold ex vivo expansion. Expanded cells showed an increased expression of activating receptors and measurable cytotoxicity against primary allogeneic and autologous blasts. One patient received a maximum NK cell dose of 5x106 cells/kg, 2 patients 1x107 cells/kg and 3 5x107 cells/kg/BW. No patient experienced infusion-related toxicities. Two adverse events were recorded (grade 1 and 2), both judged not treatment-related, that resolved after TKI suspension. The higher cell dose infusion resulted in a significantly increased expression of natural cytotoxicity receptors, a greater cytokine production by NK, T and NKT cells, and in an increased capacity of PBMC to lyse K562 cells. These modifications appear persistent over time. At a 1-year follow-up from the last infusion, 5/6 patients are alive in CHR (Table 1). The MRD levels reduced over time and 4/6 patients reached a complete molecular response (CMR) or a positive-not-quantifiable (PNQ) status during the study period. At a median follow-up of 30.8 months from the last infusion, the 5 patients who received the NK treatment in 1st CHR are still in CMR or PNQ, though 1 patient required additional treatment. The patient in 2nd CHR at the time of the infusions showed a rise in MRD and died of disease progression. Image: Summary/Conclusion: This phase 1 study demonstrates that autologous NK cells can be efficiently expanded ex vivo from MRD-positive Ph+ ALL patients in CHR. The infusion of these expanded cells is safe and induces a marked in vivo host immune response, suggesting that this approach represents a tolerable and feasible model worthy of being investigated in larger clinical studies. S112: TISAGENLECLEUCEL IN PEDIATRIC AND YOUNG ADULT PATIENTS (PTS) WITH RELAPSED/REFRACTORY (R/R) B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL): FINAL ANALYSES FROM THE ELIANA STUDY S. Rives1,*, S. L. Maude2, H. Hiramatsu3, A. Baruchel4, P. Bader5, H. Bittencourt6, J. Buechner7, T. Laetsch2, B. De Moerloose8, M. Qayed9, H. E. Stefanski10, K. L. Davis11, P. L. Martin12, E. Nemecek13, C. Peters14, G. Yanik15, A. Balduzzi16, N. Boissel17, S. L. Khaw18, J. Krueger19, J. Levine20, S. Davies21, G. D. Myers22, A. Yeo23, D. O’Donovan24, R. Ramos23, M. Pulsipher25, S. Grupp2 1Department of Pediatric Hematology – Oncology, Hospital Sant Joan de Déu Barcelona, and Institut de Recerca Sant Joan de Déu, Barcelona, Spain; 2Division of Oncology, Center for Childhood Cancer Research and Cancer Immunotherapy Program, Children’s Hospital of Philadelphia and Department of Pediatrics, Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 3Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 4University Hospital Robert Debré (APHP) and Université de Paris, Paris, France; 5Division of Stem Cell Transplantation and Immunology, Hospital for Children and Adolescents, University Hospital Frankfurt, Frankfurt, Germany; 6Department of Pediatrics, Faculty of Medicine, University of Montreal, and the Hematology Oncology Division and Charles-Bruneau Cancer Center, Centre Hospitalier Universitaire Sainte-Justine Research Centre, Montreal, QC, Canada; 7Department of Pediatric Hematology and Oncology, Oslo University Hospital, Oslo, Norway; 8Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium; 9Aflac Cancer and Blood Disorders Center, Emory University, Atlanta, GA; 10National Bone Marrow Donor Program, Be the Match, Division of Pediatric Blood and Marrow Transplant, University of Minnesota, Minneapolis, MN; 11Division of Hematology, Oncology and Stem Cell Transplant, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA; 12Pediatric Transplant and Cellular Therapy, Duke University Medical Center, Durham, NC; 13Oregon Health and Science University, Portland, OR, United States of America; 14Stem Cell Transplantation Unit, St. Anna Children’s Hospital, Vienna, Austria; 15Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, MI, United States of America; 16Clinica Pediatrica Università degli Studi di Milano Bicocca, Fondazione MBBM, Ospedale San Gerardo, Monza, Italy; 17Saint-Louis Hospital (APHP) and Université de Paris, Paris, France; 18Children’s Cancer Centre, Royal Children’s Hospital and Murdoch Children’s Research Institute, Parkville, VIC, Australia; 19Division of Haematology/Oncology/Bone Marrow Transplantation, Department of Paediatrics, The Hospital for Sick Children, Toronto, ON, Canada; 20Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY; 21Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 22Children’s Mercy Hospital and Clinics, Kansas City, MO; 23Novartis Pharmaceuticals Corporation, East Hanover, NJ, United States of America; 24Novartis Pharmaceuticals Corporation, Dublin, Ireland; 25Division of Pediatric Hematology and Oncology, Intermountain Primary Children’s Hospital, Huntsman Cancer Institute at the University of Utah, Salt Lake City, UT, United States of America Background: Pediatric and young adult pts with R/R B-ALL experience a treatment journey characterized by diminishing likelihood of cure and increasing morbidity. Tisagenlecleucel is an autologous CD19-directed chimeric antigen receptor (CAR) T-cell therapy approved for use in pediatric and young adults with B-ALL and adults with B-cell lymphomas. Tisagenlecleucel provided high rates of remission (>80%) in children and young adults with R/R B-ALL in ELIANA, with 62% of responders remaining relapse-free at 24 mo (Grupp et al, Blood, 2018). Aims: Here, we report the final efficacy and safety analyses in pts followed up to 5.9 years post-tisagenlecleucel infusion. Methods: ELIANA (NCT02435849) was a pivotal, Phase II, open-label, multicenter, global study of tisagenlecleucel in pediatric and young adult pts with R/R B-ALL. Pts received a single infusion of tisagenlecleucel at 0.2-5.0×106 CAR+ viable T cells/kg body weight for pts ≤50 kg and 0.1-2.5×108 CAR+ viable T cells for pts >50 kg. Endpoints included overall remission rate (ORR) within 3 mo, relapse-free survival (RFS), duration of remission (DOR), overall survival (OS), persistence of B-cell aplasia, and short- and long-term safety events. Results: Results: As of September 24, 2021, 97 pts were enrolled and 79 pts (81%) received tisagenlecleucel. Median time from infusion to data cutoff was 5.5 y; 64 pts had ≥5 y of follow-up. At study entry, the median age was 11 y (range, 3-24). Pts were heavily pretreated with a median of 3 prior lines of therapy (range, 1-8) and 61% had a history of prior stem cell transplant (SCT). ORR (complete remission [CR] or CR with incomplete hematologic recovery within 3 mo after infusion) was 82% (95% CI, 72-90). Among pts in remission (CR/CRi), the 5y RFS rate was 49% (95% CI, 34-62), and the median RFS was not reached (Figure, 46.8 mo when censoring for SCT; n=15). The median time to B-cell recovery was 38.6 mo (95% CI, 23-not reached) and the probability of B-cell aplasia at 6 mo and 12 mo was 83% (95% CI, 71-91) and 71% (95% CI, 57-82), respectively. Pts with B-cell recovery (<6 mo, n=10; 6-12 mo, n=4; >12 mo, n=7) experienced a 2y cumulative incidence of relapse of 25.2% (with SCT treated as a competing risk). Among all pts, the 5y EFS and OS rates were 42% (95%CI, 29-54) and 55% (95% CI, 43-66), respectively. There were no significant differences in any efficacy endpoint between pediatric (<18 y; n=65) and young adult (≥18 y; n=14) pts. No new or unexpected AEs were reported during long-term follow-up. Among pts in remission, the most commonly reported grade ≥3 AEs occurring >1 y post-infusion were infection (20%) and cytopenias (6%). Ten (14%) pts in remission experienced long-term cytopenias persisting for >1 y; however, none of these pts experienced cytopenias persisting for >5 y (median 2 y; range, 1.1-5y). Eighty-two percent of pts received IVIG any time post-infusion. Image: Summary/Conclusion: This >5 y follow-up study demonstrates continued durable efficacy of tisagenlecleucel without late adverse effects in heavily pretreated pediatric and young adult pts with R/R B-ALL. Tisagenlecleucel continues to be a potentially curative treatment option for pediatric and young adult patients with R/R B-ALL. S113: NATIONAL PEGASPARGASE-MODIFIED RISK-ORIENTED PROGRAM FOR PHILADELPHIA-NEGATIVE ADULT ACUTE LYMPHOBLASTIC LEUKEMIA/LYMPHOBLASTIC LYMPHOMA (PH− ALL/LL). GIMEMA LAL 1913 FINAL RESULTS. R. Bassan1,*, S. Chiaretti2, I. Della Starza3, O. Spinelli4, A. Santoro5, L. Elia3, M. S. De Propris3, A. M. Scattolin1, F. Paoloni6, M. Messina7, E. Audisio8, L. Marbello9, E. Borlenghi10, P. Zappasodi11, C. Vetro12, G. Martinelli13, D. Mattei14, N. Fracchiolla15, M. Bocchia16, P. De Fabritiis17, M. Bonifacio18, A. Candoni19, V. Cassibba20, P. Di Bartolomeo21, G. Latte22, S. Trappolini23, A. Guarini24, A. Vitale3, P. Fazi6, M. Vignetti6, A. Rambaldi4, R. Foà3 1Hematology, Ospedale dell’Angelo, Venice; 2Translational and Precision Medicine, Sapienza Univesrity of Rome; 3Translational and Precision Medicine, Sapienza University of Rome, Rome; 4UOC Ematologia, ASST-Papa Giovanni XXIII, Bergamo; 5Divisione di Ematologia con UTMO, Ospedali Riuniti Villa Sofia-Cervello, Palermo; 6GIMEMA Data Center; 7GIIMEMA Data Center, Fondazione GIMEMA – Franco Mandelli Onlus, Rome; 8Ematologia, Città della Salute, Torino; 9Hematology, Niguarda Ca’ Granda Hospital, Milan; 10Hematology, Spedali Civili, Brescia, Italy, Spedali Civili, Brescia; 11Hematology, Foundation IRCCS Policlinico San Matteo, Pavia; 12General Surgery and Medical-Surgical Specialties, University of Catania, Catania; 13Institute of Hematology, L. and A. Seràgnoli, Bologna; 14Hematology, Ospedale S. Croce, Cuneo, Italy, Cuneo; 15UOC Oncoematologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milan; 16Hematology Unit, Azienda Ospedaliera Universitaria Senese, Siena; 17Hematology Division, S. Eugenio Hospital, Rome; 18Ospedale Policlinico “G.B. Rossi”, University of Verona, Verona; 19Clinica Ematologica, Azienda Sanitaria Universitaria Integrata di Udine, Udine; 20Divisione di Ematologia, Ospedale Civile, Bolzano; 21Oncology Hematology, Ospedale Civile, Pescara; 22Hematology, S. Franceso Hospital, Nuoro; 23Clinica di Ematologia, Azienda Ospedaliero - Universitaria Ospedali Riuniti Umberto I, Ancona; 24Molecular Medicine, Sapienza University of Rome, Rome, Italy Background: Pediatric-inspired chemotherapy is standard of care for younger adults with Ph− ALL/LL. An essential component of these regimens is pegaspargase, here incorporated into a national treatment program for patients 18-65 years. Aims: To assess in the GIMEMA Phase 2 LAL 1913 study the feasibility and efficacy of a pegaspargase-containing induction and consolidation regimen sustaining a risk-oriented strategy for adult Ph− ALL/LL (ClinicalTrials.gov ID NCT02067143). Methods: Our prior, reference 8-block chemotherapy protocol (Blood Cancer J 2020;10:119) was modified to include pegaspargase 2000 IU/m2 at courses 1 (d10), 2 (d8), 5 (d3, with HD-MTX) and 6 (d8), with dose reductions in patients >55 years (pegaspargase 1000 IU/m2). Serum drug activity was not assessed in this study. Responders were risk-stratified for allogeneic stem cell transplantation (SCT) or maintenance according to a mixed risk model based on WBC count, immunophenotype, genetics and post-remission molecular minimal residual disease (MRD): patients with high-risk (HR) features or MRD ≥ 10-4 at weeks 10-16 or positive at week 22 were eligible to SCT; standard-risk (SR) patients were eligible to maintenance. Results: Two hundred and three patients entered the study (median age 39.8 years; 139 B- and 64 T-phenotype). The complete remission (CR) rate was 91% (100% in T-ALL/LL), with a 3-year cumulative relapse incidence and non-relapse mortality of 24.2% and 12.6%, respectively; 60 patients underwent a SCT. Overall (OS), event-free (EFS) and disease-free (DFS) survival were 66.7% (95% CI, 60.1-74.1%), 57.7% (95% CI, 51.0-65.3%) and 63.3% (95% CI, 56.3-71.1%) at 3 years. HR class (n=95) and LL diagnosis (n=20) did not affect prognosis. T-cell phenotype (CR 100%, P=0.001; EFS 67.1%, P=0.038), age 18-40 years (EFS 72.6%, P<0.0001) and MRD <10-4 after courses 1 (55%: DFS 77.9%, P=0.023) and 3 (79%: DFS 75.2%, P=0.048) were prognostically favorable. One hundred and eighty-seven patients had pegaspargase at course 1 (92.1%, 11 delayed, 3 reduced), 154 at course 2 (84.6%; 11 delayed, 12 reduced), 110 at course 5 (83.9%; 2 delayed, 11 reduced) and 73 at course 6 (68.8%; 3 delayed, 7 reduced). Dose reductions and delays were related to high-risk profile (liver dysfunction/steatosis, obesity etc.) or treatment toxicity. Toxicity of grade 2 or more was mainly observed at course 1 (hepatic 12.8%, coagulation/thrombosis 3.2% [enoxaparin prophylaxis recommended with platelets >30-50], pancreatic 1.6%), contributing to an induction death in 3 patients (1.4%), but was rare afterwards. Image: Summary/Conclusion: This pegaspargase-based ALL regimen was safely applicable to the majority of study patients, resulting in 3-year OS, EFS and DFS rates >50% in a patient population aged 18-65. The results were more favorable in patients up to the age of 55, especially in those aged 18-40 years, and in those who achieved maximum MRD response regardless of age (Figure). Subsequently, a pegaspargase dosing algorithm based on patient age, body mass index, hepatosteatosis and selected toxicities at first or prior drug exposure was developed to minimize toxicity, and was used in a successor GIMEMA trial of sequential chemotherapy-blinatumomab for CD19+ adult B-ALL (EHA Congress 2021, abstract S114). S114: PONATINIB AND BLINATUMOMAB FOR PATIENTS WITH PHILADELPHIA CHROMOSOME-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA: UPDATED RESULTS FROM A PHASE II STUDY N. Short1,*, H. Kantarjian1, M. Konopleva1, N. Jain1, F. Ravandi1, X. Huang2, W. Macaron1, W. Wierda1, G. Borthakur1, T. Kadia1, K. Sasaki1, G. Issa1, G. Montalban-Bravo1, Y. Alvarado1, G. Garcia-Manero1, C. Dinardo1, J. Thankachan1, R. Delumpa1, E. Mayor1, W. Deen1, A. Milton1, J. Rivera1, L. Waller1, C. Loiselle1, R. Garris1, E. Jabbour1 1Department of Leukemia; 2Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Ponatinib and blinatumomab are both highly effective therapies for Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). The combination of these two agents may offer an effective chemotherapy-free strategy in these patients (pts). Aims: We evaluated the efficacy and safety of ponatinib and blinatumomab in pts with newly diagnosed (ND), relapsed/refractory (R/R) Ph+ ALL or CML in lymphoid blast phase (CML-LBP). For pts with ND Ph+ ALL, the primary endpoint was the complete molecular response (CMR) rate. For pts with R/R Ph+ ALL, the primary endpoint was the CR/CRi rate. Secondary endpoints included safety, event-free survival (EFS) and overall survival (OS). Methods: In this phase II study, adults with ND Ph+ ALL, R/R Ph+ ALL, or CML-LBP were eligible. Pts were required to have a performance status of ≤2, total bilirubin ≤2x the upper limit of normal (ULN), and ALT and AST ≤3x the ULN. Pts with uncontrolled cardiovascular disease or clinically significant central nervous system (CNS) comorbidities (except for CNS leukemia) were excluded. Pts received up to 5 cycles of blinatumomab as a continuous infusion at standard doses. Ponatinib 30mg daily was given during cycle 1 and was decreased to 15mg daily once CMR was achieved. After 5 cycles of blinatumomab, ponatinib was continued for at least 5 years. Twelve doses of prophylactic IT chemotherapy with alternating cytarabine and methotrexate were administered. Results: Between 2/2018 to 1/2022, 55 pts were treated (35 with ND Ph+ ALL, 14 with R/R Ph+ ALL and 6 with CML-LBP). Baseline characteristics are shown in Table 1. Among the 35 pts with ND Ph+ ALL, 12 were in CR at enrollment (including 2 pts in CMR). 22 of the 23 evaluable pts (96%) achieved CR/CRi. One pt died on day 18 from intracranial hemorrhage in the setting of chemotherapy administered prior to enrollment. After one cycle, 21/33 pts (64%) achieved CMR, and 28/33 pts (85%) achieved CMR at any time. 11 of 15 tested pts (73%) also became MRD-negative by an NGS assay with sensitivity of 1x10-6. CR/CRi was achieved in 12/13 (92%) evaluable pts with R/R Ph+ ALL. CMR was achieved in 10 pts (71%) after cycle 1 and in 11 pts (79%) overall. 5 of 6 pts with CML-LBP achieved CR/CRi, and 1 pt achieved PR as best response. 2 pts (40%) achieved CMR. In the ND Ph+ ALL cohort, 1 of 34 pts who received at least 1 full cycle died in CR; the other 33 are in ongoing hematologic remission. Only one pt underwent stem cell transplant (SCT) in first remission for persistently detectable BCR/ABL1 transcripts. Among 13 responding pts in the R/R Ph+ ALL cohort, 6 proceeded to SCT, 4 did not undergo SCT and subsequently relapsed, 1 died in CR, and 2 are in ongoing remission without SCT. In the CML-LBP cohort, 3 of the 5 responding pts subsequently relapsed. The median follow-up is 11 months (range, 1-46+). For ND Ph+ ALL, the 2-year EFS and OS are both 93% (Figure 2). There were no relapses or leukemia-related deaths in this cohort. In the R/R Ph+ ALL cohort, the 2-year EFS rate was 42% and the 2-year OS rate was 61%. In the CML-LBP cohort, the 2-year EFS was 33% and the 2-year OS was 60%. The treatment was well-tolerated, and most toxicities were grade 1-2 and consistent with the known toxicities of the two agents. Two pts discontinued ponatinib due to toxicity (1 due to stroke and 1 due to DVT). One pt discontinued blinatumomab due to persistent grade 2 tremor. Image: Summary/Conclusion: The chemotherapy-free regimen of simultaneous ponatinib and blinatumomab is safe and effective in pts with Ph+ ALL. For pts with ND Ph+ ALL, SCT does not appear to be needed in first remission. S115: MUTANT NPM1 BINDS CHROMATIN AND COOPERATES WITH MLL1 TO REGULATE ONCOGENIC TRANSCRIPTION H. Uckelmann1,*, S. Armstrong1, E. Haarer1, E. Wong1, C. Hatton1, F. Perner1, C. Marinaccio1, C.-W. Chen2 1Pediatric Oncology, Dana-Farber Cancer Institute, Boston; 2Department of Systems Biology, Beckman Research Institute, City of Hope, United States of America Background: The dysregulation of stem cell and self-renewal associated genes is a common phenomenon during leukemia development. In acute myeloid leukemia (AML) around 50 % of cases express high levels of HOXA cluster genes and MEIS1. Most of these AML cases harbor an NPM1 mutation (NPM1c), which encodes for an oncogene that is mislocalized from the nucleolus to the cytoplasm. Hence, most studies of NPM1c have focused on a potential cytoplasmic role. However, it remains unclear how NPM1c expression in hematopoietic cells leads to its characteristic gene expression pattern. Furthermore, NPM1c AMLs are highly sensitive to the disruption of the MLL1 histone methyltransferase complex. Small molecule inhibitors that block the interaction between MLL1 and its adaptor protein Menin have been shown to impair binding of MLL1 to a subset of its target genes and to inhibit leukemia cell proliferation and self renewal. Several MLL1-Menin inhibitors are currently in Phase I/II clinical trials and show promising activity in patients with NPM1c AML. The effectiveness of these molecules in NPM1c AML prompts the question whether NPM1c and the wildtype MLL complex cooperate directly on chromatin to drive leukemic self-renewal. Aims: In this study we investigated the potential role of NPM1c in regulating oncogenic transcription on chromatin and the interplay between NPM1c and the histone methyltransferase complex KMT2A (MLL1). Methods: We used an endogenously degrader tagged NPM1c leukemia cell line that allows rapid small molecule induced degradation to show that NPM1c occupies specific chromatin targets in AML. To characterize the effects of NPM1c degradation on the chromatin landscape and transcriptional output at genomic loci that are bound by NPM1c we used ChIPseq, PROseq and nascent RNAseq methods. Results: Our results show that endogenous NPM1c directly binds to chromatin at specific target genes, such as HOXA9 and MEIS1, which are highly expressed in NPM1c patient samples. The loss of NPM1c from its targets leads to specific alterations in active chromatin marks and RNA Polymerase II (Pol II) chromatin occupancy which are accompanied by rapid changes in gene expression as well as Pol II transcriptional activity. The recruitment of NPM1c to chromatin is dependent on the nuclear exporter CRM1 as well as one of the acidic domains of NPM1c. We further show that NPM1c is lost from specific loci after treatment with small molecules that disrupt the MLL1-Menin complex interaction thus functionally linking targeted epigenetic therapy and NPM1c function. Summary/Conclusion: Overall, we demonstrate that NPM1c directly regulates a network of leukemia self-renewal associated genes through direct chromatin interaction. We further found that NPM1c acts in collaboration with the MLL1 complex and define the mechanism by which MLL1-Menin small molecule inhibitors produce clinical responses in patients with NPM1-mutated AML. S116: CELLULAR AND MOLECULAR MECHANISMS OF EVI1-EXPRESSING MLL-REARRANGED ACUTE MYELOID LEUKEMIA Hugues-Etienne Châtel-Soulet1, Sabine Juge1, Ana Luisa Pereira2, Frederik Otzen Bagger3, Alexandar Tzankov4, Mineo Kurokawa5, Athimed El Taher6, Jonathan Seguin1, César Nombela Arrieta2, Juerg Schwaller1 1Biomedicine, University Children’s Hospital, Basel, Switzerland; 2Medical Oncology & Hematology, University Hospital, Zürich, Switzerland; 3Center for Genomic Medicine, University Hospital, Kopenhagen, Denmark; 4Institute for Pathology, University Hospital, Basel, Switzerland; 5Hematology & Oncology,The University of Tokyo, Tokyo, Japan; 6Biomedicine, University of Basel, Basel, Switzerland Background: Expression of a doxycycline (DOX)-inducible acute myeloid leukaemia (AML)-associated iMLL-AF9 fusion transgene in long-term haematopoietic stem cells (LT-HSC) can lead to an invasive and chemo-resistant disease expressing the transcription factor EVI1. High EVI1 expression has been suggested as marker of poor outcome in AML patients even without rearrangements of the EVI1 locus at 3q26. Aims: We addressed the association between EVI1 expression, the cellular origin and poor disease outcome in AML driven by the iMLL-AF9 fusion gene. Methods: The role of EVI1 expression was studied in iMLL-AF9 transgenic mice carrying an Evi1-IRES GFP reporter in vitro using flow cytometry, colony formation and RT-qPCR assays, ex vivo with high-resolution bone marrow (BM) imaging, and in vivo, by transplantation of enriched naïve Evi1+ iMLL-AF9 hematopoietic stem and progenitor cells (HSPC) into irradiated recipients on DOX. Haematopoiesis of symptomatic mice was analysed by flow cytometry and histology. For mechanistic studies, single cell and bulk RNA sequencing was performed on enriched HSPC or BM samples from diseased mice. Results: Analysis of BM cells from Evi1-IRES-GFP reporter mice revealed that not only the mostly quiescent LT-HSC but also fractions of the more proliferating multipotent progenitors (MPP1-3) express abundant Evi1 (“Evi1high”). Induction of the iMLL-AF9 fusion did not result in significant changes in numbers of Evi1+ cells nor levels of Evi1 mRNA expression in the LT-HSC and MPP1 compartments. However, in colony assays, Evi1high iMLL-AF9 cells retained a more immature phenotype and produced more colonies with an invasive morphology than Evi1low cells (n=11, p<0.05). While Evi1 expression did not influence disease induction upon transplantation of LT-HSC, recipients of Evi1+ MPP1 cells developed AML earlier than Evi1- MPP1 (n=11, 79 vs. 269d, p<0.05). Disease induced by Evi1+ cells presented with more extensive leukemic organ infiltration than Evi1- AML. Evi1 expression also correlated with in vitro Ara-C resistance. We also examined whether some exogenous factors may increase AML susceptibility by expanding the Evi1+ HSPC. Although a single injection of recombinant mouse thrombopoietin (TPO) only increased the number of LT-HSC and not of MPP1, the Evi1high cell fraction was enlarged in both compartments (LT-HSC: 23 vs. 50%; MPP1: 22 vs. 47%; n=29, p<0.0001) supported by high-resolution imaging. Interestingly, increased TPO induced HSPC cycling was confined to the Evi1high cell population (n=3, p<0.05). Transplantation of TPO-treated iMLL-AF9 LT-HSC or MPP1 resulted in a significantly faster induction of Evi1+ AML than controls (n=19, MPP1: 35 vs. 79d, p<0.001; LT-HSC: 41 vs. 90d, p<0.001). To better understand mechanisms of aggravated AML after TPO mediated expansion of Evi1+ HSCP we performed multiplexed single cell RNA sequencing of highly enriched HSPC cells in iMLL-AF9 and control mice. While we observed no changes of cellular cluster organisation 2 days after TPO injection, we found some differentially expressed genes in TPO-stimulated cycling iMLL-AF9 HSPC, including potential stemness regulators. Summary/Conclusion: Our results suggest that expansion of Evi1-expressing HSPC by exogenous factors can result in a more aggressive MLL-AF9-driven AML. Ongoing data exploration and validation may characterize aberrantly expressed genes in TPO-stimulated Evi1+ iMLL-AF9-expressing HSPC as potential therapeutic targets to impair stemness of AML cells. S117: EVI1 DRIVES LEUKEMOGENESIS THROUGH ABERRANT ERG ACTIVATION J. Schmoellerl1,*, I. Barbosa1, M. Minnich1, F. Andersch1, L. Smeenk2, M. Havermans2, T. Eder3, T. Neumann1, J. Jude1, M. Fellner1, A. Ebert1, M. Steininger1, R. Delwel2, F. Grebien3, J. Zuber1 1Research Institute of Molecular Pathology (IMP), Vienna, Austria; 2Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, Netherlands; 3Institute for Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria Background: Chromosomal rearrangements leading to overexpression of EVI1 (MECOM) on chromosome 3q26 define a distinct subtype of acute myeloid leukemia (AML) that is associated with chemotherapy resistance and a 2-year survival of <10%. While genetic events driving aberrant expression of EVI1 are increasingly understood, the molecular functions of EVI1 that drive leukemogenesis are unclear, which has so far precluded the development of targeted therapeutics. Aims: We aimed to elucidate transcriptional programs that are maintained by aberrant EVI1 expression and to systematically identify vulnerabilities of EVI1-driven AML. Methods: We developed a panel of mouse models that recapitulate phenotypic and transcriptional hallmarks of patients suffering from EVI1-driven AML, allow tetracycline-controllable EVI1 expression and the functional interrogation of genetic targets using CRISPR/Cas9. We mapped transcriptional programs upon acute EVI1 repression in vivo and in vitro, profiled global EVI1 chromatin occupancy in human AML cell lines and primary patient-derived AML cells and performed comparative genome-wide CRISPR/Cas9-based loss-of-function screens in murine and human EVI1-driven AML. Results: Integration of these datasets revealed a conserved core of genes that is transcriptionally regulated by EVI1 in murine and human AML, among which we identified the ETS transcription factor ERG as the only dependency that is highly selective for EVI1-driven AML. Suppression of ERG specifically triggered cellular differentiation and apoptosis of EVI1-driven leukemia cells while other AML cell lines were unaffected. Strikingly, ectopic expression of ERG was sufficient to functionally rescue loss of EVI1 in EVI1-driven AML cells, suggesting that the major oncogenic function of EVI1 in AML is the aberrant activation of ERG. Summary/Conclusion: Interfering with the EVI1/ERG regulatory axis may provide entry points for the development of rational targeted therapies that are urgently needed for this group of AML patients. S118: IDENTIFICATION OF DIRECT TRANSCRIPTIONAL TARGET GENES OF NUP98-KDM5A REVEALS REGULATORY NETWORKS IN ACUTE MYELOID LEUKEMIA S. Troester1,*, J. Schmoellerl2, T. Eder1, G. Manhart1, G. Winter3, J. Zuber2, F. Grebien1 1Institute for Medical Biochemistry, University for Veterinary Medicine Vienna; 2Research Institute of Molecular Pathology (IMP); 3Research Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), Vienna, Austria Background: Oncogenic fusion proteins involving the Nucleoporin 98 (NUP98) gene are recurrently found in acute myeloid leukemia (AML) with a particular prevalence in pediatric patients. A chromosomal rearrangement resulting in the fusion of NUP98 to the gene encoding the lysine-specific demethylase 5A (KDM5A) is the most frequent NUP98-fusion in infant leukemia and is associated with particularly poor prognosis. The urgent need for the development of tailored treatments requires a better understanding of the effects of NUP98-KDM5A on the deregulation of gene expression programs. Although it has been shown that oncogenic NUP98-fusion proteins act as transcriptional regulators, it is unclear if and how NUP98-KDM5A directly regulates gene expression to drive leukemia. Aims: In this study, we aimed to identify immediate critical effectors of the NUP98-KDM5A fusion protein and to characterize the transcriptional programs through which they regulate the development and maintenance of NUP98-KDM5A-driven AML. Methods: We conducted a genome-scale CRISPR/Cas9 loss-of-function screen in a NUP98-KDM5A-driven murine AML cell line to unravel functional genetic dependencies that could be exploited to target leukemia cells. In parallel, we developed a new model for degradation tag (dTAG)-mediated ligand-induced degradation of the NUP98-KDM5A protein to gain a detailed understanding of direct transcriptional effects of NUP98-fusion-dependent gene regulation. We used this model to measure immediate changes in transcription upon acute NUP98-KDM5A degradation by nascent RNA-seq (SLAM-seq). An inducible shRNA system was used to assess the requirements of direct NUP98-KDM5A target genes for leukemia cell growth and to measure global gene expression changes by RNA-seq. Results: Analysis of the CRISPR/Cas9 screen identified 4105 genes that are required for the proliferation and survival of NUP98-KDM5A-driven AML cells. Complete loss of the dTAG-NUP98-KDM5A fusion protein was achieved within one hour after ligand addition, resulting in cell cycle arrest, terminal differentiation and apoptosis of leukemia cells. Global analysis of nascent mRNA expression by SLAM-seq revealed 45 immediate NUP98-KDM5A target genes, as their transcription was significantly downregulated upon fusion protein degradation. Among these genes, 12 were classified as essential factors for NUP98-KDM5A cell growth from the CRISPR/Cas9 screen. This list included known target genes of NUP98-fusion proteins, such as members of the Hoxa gene cluster, but also other transcription factors, enzymes and RNA binding proteins. shRNA-mediated knockdown of the 12 candidate genes confirmed their essentiality for NUP98-KDM5A leukemia cell proliferation. Through RNA-seq studies, we found that the knockdown of a small subset among the 12 candidate genes was able to recapitulate global patterns of gene deregulation that are induced by NUP98-KDM5A knockdown. Summary/Conclusion: Using a combination of CRISPR/Cas9 screening and ligand-induced degradation, we identified direct transcriptional target genes of NUP98-KDM5A that are functionally essential in AML. Multi-layered investigations of the interplay between the members of this small network of genes using a variety of models including primary patient samples will allow us to further dissect their role in the regulation of aberrant gene expression in NUP98-KDM5A-expressing leukemia cells and might identify novel therapeutic targets. S119: CEBPA AND TET2 MUTATIONS COOPERATE TO INDUCE AGGRESSIVE AML VIA GATA-2 DOWNREGULATION E. Heyes1,*, A. S. Wilhelmson2 3 4, A. Wenzel2 3 4, M. B. Schuster2 3 4, M. Ali3 4, T. D’Altri2 3 4, T. Eder1, G. Manhart1, E. Rzepa1, L. Schmidt1, M. Meggendorfer5, T. Haferlach5, G. Volpe6, C. Nerlov7, J. Frampton6, K. Jae Won3 4, F. Grebien1, B. Porse2 3 4 1Institute of Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria; 2The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences; 3Biotech Research and Innovation Center (BRIC); 4Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 5MLL Munich Leukemia Laboratory, München, Germany; 6Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham; 7MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom Background: The transcription factor CCAAT-enhancer-binding protein alpha (C/EBPα) is a master regulator of granulopoiesis and is mutated in 10-15 % of Acute Myeloid Leukemia (AML) patients. N-terminal frameshifts represent the predominant type of lesions and ablate the expression of the full-length protein p42, leading to overexpression of the shorter isoform p30. Mutations in the C-terminal basic-region leucine zipper (bZip) can disrupt the DNA-binding ability of C/EBPα. AML patients harbor mono- or biallelic CEBPA mutations (CEBPAmo or CEBPAbi). The most commonly co-occurring mutations are loss-of-function mutations in the methylcytosine dioxygenase TET2, resulting in adverse overall survival. We hypothesized that combinatorial effects of CEBPA mutations together with TET2 loss specifically rewire transcriptional and epigenetic circuitries in AML cells, thereby strongly influencing disease outcome. Aims: We aimed to elucidate the molecular mechanisms behind cooperative effects of CEBPA and TET2 mutations through state-of-the-art transcriptomic and epigenomic analyses of relevant in vitro and in vivo models as well as data from AML patients. Methods: We used the CRISPR/Cas9 technology to introduce Tet2 mutations in murine cell lines expressing only p30 (Cebpa p30/p30 ) or mimicking biallelic CEBPA mutations (Cebpa p30/C-mut. ) to study the functional cooperation of these mutations in vitro. A Cebpa -/p30 Tet2 -/- mouse model was used to study effects of Tet2 loss in CEBPA mutated AML. We performed RNA-, C/EBPα-ChIP-, ATAC-, Bisulfite-, CUT&RUN and CRISPR/Cas9-mediated enhancer screens to generate a comprehensive dataset for in-depth comparative analysis and correlation with relevant patient data from the beatAML collection. Results: Integration of transcriptomic and epigenomic data from in vitro and in vivo models of CEBPA-TET2 co-mutated AML, in combination with gene expression analyses in AML patients, identified the transcription factor GATA2 as a conserved target of the CEBPA-TET2 axis. p30 and TET2 were strongly bound to the -77 kb enhancer of the Gata2 gene, and CRISPR/Cas9-induced enhancer deletions diminished Gata2 expression. Furthermore, TET2 loss reduced chromatin accessibility and increased DNA methylation of the Gata2 promoter, resulting in decreased Gata2 mRNA levels. RNAi-mediated silencing revealed a dose-dependent effect of Gata2 expression on leukemia cell fitness in vivo. Reduction of Gata2 levels by 25-50 % provided a strong competitive advantage to Cebpa p30/p30 cells while near-complete downregulation of Gata2 expression (>75 %) dramatically reduced cellular fitness. Finally, treatment with the demethylating agent 5-azacytidine restored Gata2 expression in an AML model with Cebpa and Tet2 mutations and caused a significant survival benefit. Summary/Conclusion: The datasets generated from these models enable deeper insights into the epigenetic and transcriptomic changes that depend on CEBPA and TET2 mutations in a physiologically relevant mutational context. Our results reveal that mutational disruption of CEBPA and TET2 results in down-regulated GATA2 expression, causing aggressive AML. We propose a mechanism in which C/EBPα p30 mediates recruitment of TET2 to regulatory regions in the GATA2 gene to maintain its expression. Conversely, loss of TET2 leads to reduced GATA2 levels, which is restored by 5-azacytidine treatment. Thus, interference with the C/EBPα-TET2 axis may provide entry points for the development of rational targeted therapies in AML patients with these mutations. S120: ACUTE MYELOID LEUKEMIA REPRESENTS A FERROPTOSIS-SENSITIVE CANCER ENTITY RAISING THE POSSIBILITY FOR NOVEL TARGETING STRATEGIES A. Narr1 2 3,*, H. Alborzinia1 2, E. Donato1 2, F. Vogel4, T. Boch1, A.-M. Leppä1 2, A. Waclawiczek1 2, S. Renders1 2, A. Schulze4, A. Trumpp1 2 1Division of Stem Cells & Cancer, German Cancer Research Center (DKFZ); 2Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH); 3University Heidelberg; 4Division of Tumor Metabolismus und Microenvironment, German Cancer Research Center (DKFZ), Heidelberg, Germany Background: Despite advances in the treatment of Acute Myeloid Leukemia (AML), the 5-year patient survival rate remains poor with 15-20%. The majority of patients develop recurrence partly due to resistance mechanisms to classical cell death programs. Exploiting new forms of cell death therefore may allow novel therapeutic options to limit survival or occurrence of resistant clones. Ferroptosis has been identified as non-apoptotic and iron-dependent form of cell death, characterized by an excessive ROS-induced peroxidation of cell membranes. Recently it has been implicated with a higher sensitivity in cancer stem cells and drug-resistant cancer cells, potentially making ferroptosis-inducing (FIN) therapies a promising option. Aims: We aim to elucidate whether AML represents a ferroptosis-sensitive cancer entity and is susceptive for potential FIN-therapies. Methods: IC50 values of ferroptosis inducers were determined and used in combination with the TCGA-LAML and a pan-cancer CRISPR screen dataset (CERES) to describe ferroptosis sensitivity of AML. A drug screen with known ROS-inducers was performed in 9 AML cell lines to identify ferroptosis induction. To mechanistically characterize ferroptotic cell death by the identified drug on the genetic and metabolic level, we performed SLAM-RNA-, RNA-Sequencing, Mass Spectrometry and Metabolite Tracing (Cystine/Ser/Gln) in HL-60 cells in vitro and in vivo. Genetic (CRISPR-Cas9-KO) and chemical inhibition were used to characterize the role of identified hits and pathways, aiming to design new synergistic combination therapies. Results: We find that AML belongs to the most dependent cancer entities for various ferroptosis-associated genes and represents a ferroptosis-sensitive cancer entity. Furthermore, a 12-gene-ferroptosis-signature allows us to predict the Overall Survival of AML patients and shall help to identify a potential cohort for FIN-therapies. By screening known ROS-inducing drugs for ferroptosis induction, we identify one drug capable of inducing lipid peroxidation and subsequently ferroptosis in multiple AML and other tumor cell lines. Notably, in detail characterization of the drug’s mechanism on the genetic and metabolic level reveal the activity of the iron-metabolism gene HMOX-1 as essential for ferroptosis induction. Mechanistically, we further find that this drug directly inhibits GPX-4, one of the major ferroptosis-suppressive regulators, and facilitates its degradation. Furthermore, we demonstrate that drug treatment results in an upregulation of different metabolic pathways involved in glutathione (GSH) synthesis; namely (1) SLC7A11-mediated Cystine-uptake, (2) transsulfuration pathway as well as (3) glutaminolysis. Genetical and chemical inhibition of these pathways together with drug treatment in in vitro as well as in vivo studies shows strong synergistic effects marking them as new targetable vulnerabilities in AML. Using these insights of the drug’s mechanism of action, we design a synergistic combination therapy and demonstrate its tumor-eradicating capacity in AML cell lines (in vitro & in vivo) and in primary de novo as well as relapse AML patient samples (ex vivo). Summary/Conclusion: These findings classify AML as ferroptosis sensitive and reveal the capacity of this drug to induce ferroptosis. Detailed characterization of the drug’s mechanism on the genetic and metabolic level allows us to design an effective combination therapy exploiting newly identified vulnerabilities in AML. These results provide a rational basis for the additional pre-clinical and subsequent clinical evaluation of FIN-therapies in AML patients. S121: CD123-CD33 COMPOUND CAR-T CELLS WITH NOVEL ANTIGEN BINDING DOMAINS PROVIDE A NEW HOPE FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA Z. Wang1,*, Y. LU1, S. Qiu1, M. Wang1, D. Xiong1, J. Wang1 1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Tianjin Key Laboratory of Cell Therapy for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology and Blood Diseases Hospital, TianJin, China Background: CAR-T therapy needs to be optimized to treat myeloid malignancies. Antigen-escape mediated relapse after CAR-T treatment is the vital mechanism responsible for the nondurable response. Targeting CD33 and CD123 simultaneously could cover almost all patients with AML. The off-target cytotoxicity of CD123 CAR-T and CD33 CAR-T, especially on HSPCs, has always been concerned. Several researchers have explored this issue and the conclusions are inconsistent in different studies. Aims: To improve the outcome of relapse and/or refractory acute myeloid leukemia and explore the influence of CD123-CD33 CAR-T on hematopoiesis. Methods: We develpoed a panel of anti-CD123 monoclonal antibodies (mAbs) using hybridoma technology, named 6E11, 8D7, 12H7 and 13C3 and incorporated each of the four scFvs into 4-1BB/CD3ζ signaling domain to generate 4 second generation CARs. T cells were infected with lentiviral supernatants to generate CAR-T cells. Comparative analysis were made from the expression property of CAR protein, in vitro and in vivo anti-leukemia efficacy to screen out the best candidate CD123 CAR. Next, we generated a CD123-CD33 dual targeting CAR in a compound way. We established an escape-simulation model to explore whether the compound CAR-T could reduce the risk of relapse associated with antigen loss. Flowcytometry analysis, CFU test and single cell RNA sequecing methods were jointly used to explore the influence of our CD123-CD33 CAR-T on hematopoiesis. Results: CD123 CAR constructs with four different novel scFvs differed in CAR protein expression ratio. A changing trend of CAR proteins from dropping to rising after encountering target cells was observed in 3 CAR-T cell group except in 8D7 CAR-T. 13C3 CAR was the most suitable construct from the perspective of basic and dynamic expression. 123CAR-T cells with different scFvs mediated variable anti-leukemia effect in vitro and in vitro. 123CAR and 33CAR proteins expressed efficiently on the compound CAR-T cell surface without codon optimization. The compound CAR-T showed superiority in the treatment of antigen-escape mouse model. FCM analysis showed no significant difference of residual CD34+ cells proportion between NCT cell group and compound CAR-T group. scRNA-seq analysis showed that the most undifferentiated HSC population barely changed among each group. GSEA revealed that the HSC population in CAR-T groups had no difference in the enrichment of quiescence, cell cycle, G2M checkpoint related genes compared with that of NCT group. In addition, no downregulation/enrichment of TGF-beta signaling pathway/ PI3K-AKT-mTOR signaling pathway was observed in compound CAR-T group compared with NCT group.The CFU assay demonstrated that there were still quite numbers of hematopoietic colonies formed in compound CAR-T treatment groups. Image: Summary/Conclusion: Taken together, in this study we developed a panel of second generation CD123 CARs with novel scFvs on the basis of 4 novel anti-CD123 mAbs. We proved that scFv sequence has a significantly impact on CAR expression both originally and dynamically and further affects CAR-T function, addressing the importance of scFv development in CAR-T therapy refining. The compound CAR-T in our study minimize the risk of antigen-loss mediated escape. Notably, wildtype DNA sequence of cCAR without codon optimization brings no potential risk such as immunogenic complications. scRNA-seq and CFU assay convincingly demonstrated that the CD123-CD33 compound CAR-T had limited toxicity on hematopoiesis. Thus, the CD123-CD33 compound CAR-T with novel scFvs provides new hope for R/R AML patients. S122: ROCK INHIBITORS TARGET SRSF2 LEUKEMIA BY DISRUPTING CELL MITOSIS AND NUCLEAR MORPHOLOGY M. Su1 2,*, T. Fleisher3, I. Grosheva4, M. Horev4, M. Olszewska5, B. Haim6, A. Plotnikov6, S. Carvalho6, Y. Moskovitz3, M Minden7, N. Chapal-Ilani3, E. Papapetrou8, N. Dezorella9, T. Cheng10, N. Kaushansky3, B Geiger4, L. Shlush11 12 13 1Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China; 2Molecular and Cellular Biology, Weizmann Institute of Science, Rehovot; 3Molecular and Cellular Biology, Weizmann Institute of Science; 4Immunology, Weizmann Institute of Science, Rehovot, Israel; 5Icahn School of Medicine at Mount Sinai, New York, United States of America; 6Wohl Institute for Drug Discovery, Weizmann Institute of Science, Rehovot, Israel; 7University Health Network (UHN), Toronto, Canada; 8Icahn School of Medicine at Mount Sinai, New York, United States of America; 9Electron Microscopy Unit, Weizmann Institute of Science, Rehovot, Israel; 10Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China, Tianjin, China; 11Molecular and Cellular Biology, Weizmann Institute of Science, Rehovot; 12Division of Hematology, Rambam Healthcare Campus, Haifa; 13Molecular hematology clinic, Maccabi Healthcare, Tel Aviv, Israel Background: Spliceosome machinery mutations are common early mutations in myeloid malignancies, however effective targeted therapies against them are still lacking in clinical settings. Aims: Exploring safe and efficient methods to target hematopoietic cells carrying SRSF2 mutations might be a powerful tool not just for leukemia prevention and treatment, but also to understand the mechanisms behind SRSF2 mutations. Methods: We generated five SRSF2 Mut (P95H) isogenic cell lines with CRISPR/CAS9 system and performed high throughput drug screen with 3988 compounds in a single dose. After we narrowed down our targets, dose response assay was performed on four different ROCK inhibitors (ROCKi). The leading ROCKi compound was validated in vivo with SRSF2 Mut AML xenograft models. Next, we aimed at understanding why ROCKi target SRSF2 Mut cells and used proteomics, gene expression and imaging of the cytoskeletal system to study the effect of ROCKi on Mut and WT cells. Results: In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified ROCK inhibitors (ROCKi) as selective inhibitors of SRSF2 Mut in MOLM14 and AML2 cells. To study the efficacy of RKI-1447 on human samples, we conducted six AML patient-derived xenograft (PDX) experiments, with SRSF2 Mut samples. In four out of the six primary AML samples, RKI-1447 significantly reduced engraftment compared to the untreated group and inhibited the engraftment of both leukemic blasts and pre leukemic-HSPCs (preL-HSPCS). RKI-1447 was not toxic to mice nor human cells. ROCKi induced mitotic catastrophe (G2M arrest and multipolar spindles) through their apparent effects on microtubules and nuclear organization, and leading to cell death. Confocal imaging and transmission electron microscopy (TEM) data revealed that SRSF2 mutations induce deep nuclear indentation and segmentation, which is reminiscent of the nuclear shape of Pelger–Huët anomaly (PHA). The nuclear volume and area were significantly higher in SRSF2 Mut compared with WT MOLM14 cells regardless of ROCKi. To investigate why SRSF2 Mut cause the structural changes, we looked into the cytoplasmic intrusions that segment the nuclei, and the structures that connect the lobes of the nuclei by TEM. This examination revealed enrichment of microtubule bundles inside the nuclear indentations. More importantly, these microtubules were located at the tip of the cytoplasmic intrusions suggesting that they take an active role in the nuclear segmentation process. After exposure to RKI-1447, the cytoskeletal and nuclear morphological abnormalities of SRSF2 Mut are augmented to a level which are incompatible with cell survival. Image: Summary/Conclusion: We believe it is the first report describing in high resolution the changes in the nucleus after SRSF2 mutations are introduced. The mechanisms we identified are most probably relevant to other SMMs and to the dysplastic phenotype observed in MDS and AML. Accordingly, our findings have wide implications as ROCKi might be even more useful than describe here. Altogether, we provide data from many directions that ROCKi should be tested in clinical trials. S123: DECODING TRANSCRIPTOMIC AND EPIGENETIC CONSEQUENCES OF STRUCTURAL VARIANTS IN CK-AML AT SINGLE-CELL RESOLUTION A.-M. Leppä1,*, K. Grimes2, H. Jeong2, T. Boch1, D Karpova1, A. Jauch3, F. Grünschläger1, A. Dolnik4, L. Bullinger4, A. Krämer5, A. D. Sanders2, J. O. Korbel2, A. Trumpp1 1Division of Stem Cells and Cancer, German Cancer Research Center; 2Genome Biology Unit, European Molecular Biology Laboratory; 3Institute of Human Genetics, University of Heidelberg, Heidelberg; 4Hämatologie, Onkologie und Tumorimmunologie, Charité Universitätsmedizin Berlin, Berlin; 5Molecular Hematology/Oncology, German Cancer Research Center, Heidelberg, Germany Background: Acute myeloid leukemia (AML) with complex karyotype (CK) is a heterogenous AML subgroup with dismal response to standard treatment. CK-AML has remained poorly characterized at the molecular level, with mainstream techniques struggling to unravel the complexity of the structural variants (SVs) and link them to phenotypic characteristics. To improve our understanding of molecular dynamics in CK-AML, we established an integrated single cell multi-omics approach that combines the analysis of SVs and nucleosome occupancy (NO) profiling (scNOVA) with concurrent immunophenotypic and transcriptomic profiling (CITE-seq). Aims: To characterize the genetic and non-genetic intra-patient heterogeneity at single cell resolution in CK-AML. Methods: For scNOVA analysis, blasts from four CK-AML patients were cultured in the presence of BrdU for the duration of one cell division, followed by single-cell template strand-sequencing (Strand-seq). For CITE-seq analysis, cells were stained with 38 antibody-derived tags for combined analysis of single cell transcriptome and immunophenotype. For functional studies, xenografts of the same CK-AML patients were generated by injection of AML cells into the femoral BM cavity of sublethally irradiated NOD.Prkdc scid .Il2rg null (NSG) mice. Results: To characterize cellular hierarchies together with epigenetic, transcriptomic, and surface protein features of CK-AML, we applied an integrated single-cell analytical framework, termed scNOVA-CITE, on over 15,000 leukemic cells from four diagnostic CK-AML patient samples. We detected complex clonal evolution patterns driven by simple and complex SVs in all patients. Three out of four patients harbored SVs leading to loss of one TP53 wild-type allele, often accompanied by loss-of-function mutations in the remaining allele. One patient showed parallel evolution of TP53-deleted and TP53 wild-type subclones in the same sample, with the latter clone harboring a lower abundance of SVs compared to the TP53-deleted cells. Moreover, based on NO we inferred lowest activity of TP53 pathway in the cells with TP53 aberrations, emphasizing the role of TP53 in intra-patient cytogenetic evolution as well as inter-patient heterogeneity. Thereby, we unraveled novel single-cell SV landscapes in CK-AML and explored their epigenetic impact. To assess in depth the functional outcomes of the genetic complexity, we also characterized the transcriptome and cell surface proteome using scNOVA-CITE. Two patients showed high genetic intra-patient heterogeneity and harbored multiple subclones at diagnosis with unique transcriptomic features. When transplanted into NSG mice, we detected predominantly monoclonal engraftment and strongly reduced genomic instability in the respective patient-derived xenografts. The engrafting clones could be traced back to a small subclone in the original patient samples. Molecular analysis of the subclones in the diagnostic samples by scNOVA-CITE revealed converging characteristics leading to stem-like phenotypes. Taken together, these data indicate that cytogenetic evolution resulting in stemness phenotype in CK-AML is advantageous for leukemic stem cell expansion in mice, unfolding novel ways to study regulation of stemness and to identify potential therapeutic targets. Summary/Conclusion: By developing scNOVA-CITE, we established an integrative single-cell multi-omics framework allowing to characterize genetic, epigenetic, transcriptomic and cell surface proteomic properties of CK-AML patient cells. We provide unique insights into the genetic complexity of CK-AML, and evidence of the resulting functional outcomes. S124: BONE MORPHOGENETIC PROTEIN 2 TRIGGERS OFF AN IMMUNOSUPPRESSIVE EFFECT OF ΓΔ T CELLS IN ACUTE MYELOID LEUKEMIA S. Liang1 2,*, T. Dong1, K. Yue1, H. Gao1, N. Wu1, R. Liu1, J. Liu1, X.-J. Huang1 2 1Peking University People’s Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key laboratory of Hematopoietic Stem Cell Transplantation; 2Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking university, Beijing, China Background: Description of immune landscapes that indulge or restrain survival and proliferation of malignant cells is critical to the improvement of therapeutic strategies. Acute myeloid leukemia (AML) remains a severe life-threatening malignancy and often confronts treatment dilemma in clinic. Although γδ T cells exhibit independent and potent cytotoxicity against leukemic cells in vitro and in the mouse models, efficacy of γδ T cell-based immunotherapy on AML patients has seemed unsatisfying so far. How the anti-AML capacity of γδ T cells is suppressed in vivo is unknown. Aims: In this study, we aim to dissect the abnormal changes and functions of intrinsic γδ T cells in the context of AML and dig out the correlated factors in the AML tumor environment. Methods: 1. Immunophenotyping analyses of γδ T cells in bone marrows from newly diagnosed AML patients (n = 62) were detected using flow cytometry, compared with healthy donors (n=51). The levels of TGF-β family members in the supernatants of bone marrows were detected by ELISA kits. 2. Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were stimulated with AML blasts and morphogenetic protein 2 (BMP2) to validate clinical phenotypes. Functional experiments were performed to depict the role of a BMP2-induced subset of γδ T cells in the anti-AML activity of effector γδ T cells. 3. To confirm the findings described above, we established human CD34+ cells-implanted mice models followed by injection with luciferase and GFP co-expressing AML cells. Results: The proportions of total γδ T cells and Vδ1+ fraction were not significantly different between AML patients and donors. In contrast, the percentage of Vδ2+ T cells was markedly decreased in AML patients. Meanwhile, a key activating receptor of γδ T cells, NKG2D, was dramatically decreased and regulatory receptors CD25 and PD-1 were increased on Vδ2+ T cells in AML patients. The expansion capacity of Vδ2+ T cells was profoundly impaired in AML patients. The functional impairments of γδ T cells were accompanied with an abnormally increased levels of BMP2, rather than TGF-β, BMP4 and ActivinA, in AML patients. Furthermore, we demonstrated that BMP2 directly induced an aberrant γδ T cells subset expressing Vδ2+CD25+CD127low (abbreviated as BMP2-Vδ2) in PBMCs from healthy donors. Consistently, BMP2-Vδ2 cells were significantly increased in the bone marrows of AML patients compared with donors. The frequencies of BMP2-Vδ2 cells correlated to soluble BMP2 levels and the disease status. Distinct from effector Vδ2 cells, BMP2-Vδ2 not only lost the ability of killing AML cells, but also was able to reduce the anti-AML activity of effector Vδ2 cells after co-cultures. In AML-loaded humanized mice, treatment with BMP2 induced the occurrence of BMP2-Vδ2 cells and reduced the survival rate of mice. Injection of BMP2-Vδ2 cells significantly attenuated the activity of effector Vδ2 cells on the elimination of AML cells in mice. Together, these results demonstrated the immunoregulatory effect of BMP2 on the phenotype and function of γδ T cells in AML microenvironment. Summary/Conclusion: We first reported that dysregulated BMP2 in AML triggered an immunosuppressive subset of γδ T cells, leading to the impairment of anti-AML function of effector γδ T cells. Our findings provide a novel insight into the mechanisms of immunosuppression in the context of leukemia and suggest potential targets for the treatment of AML and other hematopoietic malignancies. S125: 10-DAY DECITABINE VS. CONVENTIONAL CHEMOTHERAPY (“3 + 7”) FOLLOWED BY ALLOGRAFTING (HSCT) IN AML PATIENTS ≥60 YEARS: A RANDOMIZED PHASE III STUDY OF THE EORTC LEUKEMIA GROUP, GIMEMA, CELG, AND GMDS-SG M. Lübbert1,*, P. Wijermans2, M. Kicinski3, S. Chantepie4, W. van der Velden5, R. Noppeney6, L. Griskevicius7, A. Neubauer8, M. Crysandt9, R. Vrhovac10, M. Luppi11, S. Fuhrmann12, E. Audisio13, A. Candoni14, O. Legrand15, R. Foà16, G. Gaidano17, D. van Lammeren-Venema2, E. F. Posthuma18, M. Hoogendoorn19, A. Giraut3, M. Stevens-Kroef20, J. H. Jansen21, E. Ammatuna22, J.-P. Vilque4, R. Wäsch1, H. Becker1, N. Blijlevens5, U. Dührsen6, F. Baron23, S. Suciu3, S. Amadori24, A. Venditti24, G. Huls22 1Department of Hematology, Oncology and Stem Cell Transplantation, Faculty of Medicine, University Medical Center Freiburg, University of Freiburg, Freiburg, Germany; 2Department of Hematology, Haga Teaching Hospital, The Hague, Netherlands; 3EORTC Headquarters, Brussels, Belgium; 4Institut d’Hématologie de Basse Normandie, Centre Hospitalo-Universitaire de Caen, Caen, France; 5Department of Hematology, Radboud University Medical Centre, Nijmegen, Netherlands; 6Klinik für Hämatologie, Universitätsklinikum Essen, Essen, Germany; 7Department of Hematology, Oncology and Transfusion Medicine Center, Vilnius University Hospital Santaros Klinikos, Vilnius University, Vilnius, Lithuania; 8Department of Internal Medicine, Hematology, Oncology and Immunology, Philipps University Marburg and University Hospital Gießen and Marburg, Campus Marburg, Marburg; 9Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Medical Faculty, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), Aachen, Germany; 10Department of Haematology, University Hospital Centre Zagreb, Zagreb, Croatia; 11Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell’Adulto, University of Modena and Reggio Emilia, Azienda Ospedaliera Universitaria, Modena, Italy; 12Department of Hematology and Oncology, HELIOS Hospital Berlin-Buch, Berlin, Germany; 13SC Ematologia Città della Salute e della Scienza Torino, Torino; 14Clinica Ematologica Azienda Sanitaria Universitaria Integrata di Udine, Udine, Italy; 15Service d’Hématologie Clinique et de Thérapie cellulaire, Hôpital Saint Antoine, APHP, Paris, France; 16Ematologia, Dipartimento di Medicina Traslazionale e di Precisione, “Sapienza” Università di Roma, Rome; 17Division of Hematology, Department of Translational Medicine, Università del Piemonte Orientale and Azienda Ospedaliero-Universitaria Maggiore della Carità, Novara, Italy; 18Department of Internal Medicine, Reinier de Graaf Hospital, Delft; 19Department of Hematology, Medical Center Leeuwarden, Leeuwarden; 20Radboud University Medical Center, Nijmegen, Netherlands; 21Dept laboratory Medicine, Lab Hematology, Radboud University Medical Center, Nijmegen; 22University Medical Center Groningen, Groningen, Netherlands; 23University of Liège, Liège, Belgium; 24Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy Background: Older, fit AML patients (pts) treated with induction chemotherapy (IC) have poor long-term survival unless HSCT is performed. DNA-hypomethylating agents have become the backbone of AML therapy in pts unfit for IC. Promising outcomes have been reported for the 10-day decitabine (DEC) schedule, suggesting it may be a better treatment prior to HSCT as compared to IC. Aims: To compare efficacy and safety of 10-day DEC followed by allografting to IC followed by allografting in older fit AML pts. Methods: This was an international open-label randomized phase III trial (NCT02172872). Key inclusion criteria were newly diagnosed AML, age ≥60 years, eligible for IC, WHO performance status 0-2. DEC was administered 10 days consecutively in cycle 1 (20 mg/m2), 10 or 5 days in subsequent cycles (depending on bone marrow blast clearance at day 28). IC treatment was daunorubicin 60 mg/m2 x 3 days, cytarabine 200 mg/m2 x 7 days, followed by 1-3 additional chemotherapy cycles. Pts who had an HLA-matched donor and at least stable disease were encouraged to undergo HSCT after ≥1 treatment cycle. Pts from the DEC arm not receiving HSCT could continue DEC treatment. The primary endpoint was overall survival (OS). Pts were randomized 1:1, stratified by de novo AML vs. secondary AML, age (60-64 vs 65-70 vs ≥70 yrs), and institution. The statistical design aimed to detect a hazard ratio (HR) for OS of 0.75 (HR<1 indicates longer survival for DEC), requiring 441 deaths (one-sided alpha 0.025, 85% power). Due to the slow accumulation of deaths, the final analysis was performed with a clinical cut-off (CCO) date June 30, 2021, following the Data Monitoring Committee recommendation. Results: Between 12/2014 and 8/2019, 606 pts were randomized, 303 in each arm. Median follow-up was 4.0 yrs. Median age was 68 yrs (range 60-81), 34% of pts were ≥70 yrs old and 57% were male, 21% and 32% had good and adverse ELN 2017 risk profile, respectively. A median of 3 DEC cycles (Q1-3: 2-5) and 2 IC cycles (Q1-3: 1-2) were administered. The CR/CRi rate was 48% with DEC and 61% with IC. HSCT as part of the protocol was performed in 122 pts (40%, 30 of them not in CR/CRi) from the DEC and 118 (39%, 11 of them not in CR/CRi) from the IC arm, and in 52% in both arms at any time. By the CCO, 423 deaths occurred. The OS was not significantly different between DEC and IC groups (HR=1.04, 95% confidence interval [CI]: 0.86-1.26; 2-sided p=0.68). The median OS was 15 months (95% CI: 13-18) in the DEC and 18 months (95% CI: 14-22) in the IC group. The OS rates (%) after 1, 2, 3 and 4 years for the DEC and IC groups were 58 vs 59, 38 vs 40, 30 vs 33, and 26 vs 30, respectively. In age subgroups, the estimated HR for OS for DEC vs IC was 1.34 (99% CI: 0.79-2.28) for pts aged 60-64, 1.14 (99% CI: 0.77-1.69) for pts aged 65-69, and 0.84 (99% CI: 0.55-1.26) for pts aged ≥70 yrs (p-value for trend: 0.058). Notable differences in the incidence of grade 3-5 adverse events (%) reported (before HSCT) were: febrile neutropenia (37% for DEC vs 57% for IC), decrease in platelets (24% for DEC vs 32 % for IC), oral mucositis (2% for DEC vs 10% for IC), diarrhea (1% for DEC vs 8% for IC), decrease in neutrophils (19% for DEC vs 13% for IC). The 30-day mortality rate was 3.6% for DEC and 6.4% for IC. The incidence of grade 5 treatment-related adverse events after HSCT was comparable in both treatment arms (25% for DEC and 22% for IC). Summary/Conclusion: Treatment with DEC resulted in a similar OS and HSCT rate but a better safety profile compared to IC in older AML pts ≥60 yrs, eligible for IC. S126: GEMTUZUMAB-BASED INDUCTION CHEMOTHERAPY COMBINED WITH MIDOSTAURIN FOR FLT3 MUTATED AML. RESULTS FROM THE NCRI AML19 “MIDOTARG” PILOT TRIAL N. Russell1,*, C. Wilhelm-Benartzi2, J. Othman3, R. Dillon3, N. Potter3, J. Jovanovic3, A. Gilkes4, L. M. Batten2, J. Canham2, E. L. Hinson2, P. Kottaridis5, J. Cavenagh6, C. Arnold7, M. Dennis8, S. Knapper4 1Haematology, Guy’s and St Thomas’ NHS Foundation Trust, London; 2Centre for Trials Research, Cardiff University, Cardiff; 3Department of Medical and Molecular Genetics, Kings College London, London; 4School of Medicine, Cardiff University, Cardiff; 5Haematology, University College London Hospitals NHS Foundation Trust; 6Department of Haematology, St Bartholomew’s Hospital, London; 7Haematology, Belfast City Hospital, Belfast; 8Haematology, The Christie NHS Foundation Trust, Manchester, United Kingdom Background: Following the RATIFY study Midostaurin in combination with “7 + 3” like chemotherapy has become the standard of care for patients with newly diagnosed FLT3 mutated AML. The ALFA 0701 trial also suggested a benefit for Gemtuzumab Ozogamicin (GO) in FLT3 mutated AML however the combination of GO-based induction with Midostaurin has not been formally assessed. Aims: To assess the safety of midostaurin in combination with Gemtuzumab and intensive induction therapy and to assess impact of the combination on MRD kinetics Methods: The NCRI AML19 trial randomised patients to receive DA 3 + 10 (Daunorubicin 60mg/m2 on days 1,3,5 plus AraC 100mg/m2 bd on days 1-10) plus a single dose of GO 3mg/m2 on day 1 (DAGO1) or 2 doses (3mg/m2, maximum 5mg) on days 1 and 4 (DAGO2) plus 50mg bd of midostaurin (m) for 14 days following completion of chemotherapy. Eligibility included age 18-60 years with a FLT3-ITD or TKD mutation. Enhanced pharmacovigilance (PV) was performed for four weeks following the first induction course. Midostaurin was also given following second induction (DA 3 + 8 without GO) and 2 courses of HDAC consolidation and as maintenance for 12 cycles in non-transplanted patients. From November 2020 to November 2021, 77 patients were enrolled into the Midotarg pilot receiving DAGO1m (n=39) or DAGO2m (n=38). 59 had a FLT3 ITD and 22 a FLT3-TKD (and 4 had both). 59 patients have completed course 1 and are evaluable. RT-qPCR MRD monitoring for patients with an NPM1 mutation (n=48) was performed following each cycle of chemotherapy. A descriptive comparison is presented here of toxicity and MRD kinetics with FLT3 mutated patients receiving DAGO1 or DAGO2 without Midostaurin in the same trial. Results: Treatment compliance in course 1 was 88% for DAGO1m and 100% for DAGO2m. One patient did not receive any Midostaurin because of colitis during induction. Dose interruption for QTc prolongation occurred in 1 patient. A total of 17 SAEs (CTC grade 3 or greater) were reported (GO1, n=11, GO2 n= 6). Day 60 mortality was 0%. No cases of VOD were reported. Blood count recovery was not delayed. Time to neutrophil recovery to 1 x 109/L was 31 and 32 days with DAGO1m and DAGO2m compared to 31 and 32 days in DAGO1 and DAGO2 .. Time to platelet recovery to 100 x 109/L was 28 and 29 days with DAGO1m and DAGO2m respectively compared to 30 days in DAGO1 and DAGO2. Complete remission (CR plus CRi) was achieved in 51/59 (88%) evaluable patients who have completed induction with DAGOm. In 44 evaluable patients in remission with NPM1 mutation, 34 (74%) were MRD negative in the peripheral blood after course 2. This compares with 63% in 54 evaluable patients with DAGO only. Bone marrow NPM1 transcript levels after courses 1 to 4 were compared with FLT3 mutated patients receiving DAGO1 and DAGO2 without Midostaurin (Figure 1) with a higher proportion of patients becoming MRD negative from course 2 onwards and 81% being MRD negative after course 4. Image: Summary/Conclusion: The addition of midostaurin to DAGO using a single or fractionated dose of GO was well tolerated with no evidence of increased toxicity, high response rate and with encouraging clearance of NPM1 mutant transcripts. A randomised study of DAm versus DAGOm in FLT3 mutated AML is planned S127: QUIZARTINIB WITH DECITABINE AND VENETOCLAX (TRIPLET) IS ACTIVE IN PATIENTS WITH FLT3-ITD MUTATED ACUTE MYELOID LEUKEMIA - A PHASE I/II STUDY M. Yilmaz1,*, M. Muftuoglu1, H. Kantarjian1, C. DiNardo1, T. Kadia1, G. Borthakur1, N. Pemmaraju1, N. Short1, Y. Alvarado1, A. Maiti1, L. Masarova1, G. Montalban Bravo1, S. Loghavi2, K. Patel2, S. Kornblau1, E. Jabbour1, G. Garcia-Manero1, M. Andreeff1, N. Daver1 1Leukemia; 2Hematopathology, MD Anderson Cancer Center, Houston, United States of America Background: Quizartinib (QUIZ), a potent 2nd generation FLT3 inhibitor (FLT3i) demonstrated synergy with venetoclax (VEN) in AML cell lines and PDX models (Mali Haematologica 2020). Aims: To evaluate the safety and efficacy of Decitabine (DAC) + VEN + QUIZ triplet in patients (pts) with newly diagnosed (ineligible for intensive induction chemotherapy) or relapsed/refractory (R/R; up to 5 prior chemotherapies) FLT3 ITD mutated AML. Methods: All pts received 10 days of DAC (20 mg/m2) in Cycle 1. Pts underwent day 14 bone marrow (BM) biopsy, and VEN (400 mg/day starting from day1) was put on hold in pts with BM blasts ≤ 5% or aplasia. Those with day14 BM blast >5% continued VEN for 21 days during cycle 1. In subsequent cycles, DAC was reduced to 5 days. QUIZ (30 or 40 mg/day) was administered daily continuously. Results: Overall 28 pts were enrolled and evaluable at the time of this report. Of 23 pts with R/R AML (median 3 prior Rx, 78% with ≥1 prior FLT3i including prior gilteritinib in 70%, and 39% had a prior alloSCT), 78% achieved CRc (3 CR, 15 CRi) with 6/16 and 5/18 responders achieving FLT3-PCR and multicolor flow cytometry negativity, respectively. The CRc rates were 75% and 72% in pts who received prior gilteritinib and prior HMA+VEN, respectively. 60-day mortality rate was 5%. Of 5 pts with newly diagnosed AML (median age 69), all achieved CRc (2 CR, 3 CRi) with 4/5 and 2/4 responders achieving FLT3-PCR and MFC negativity, respectively. 60-day mortality in frontline was 0. RAS/MAPK mutations appear to drive both primary and secondary resistance. Pts with underlying RAS/MAPK mutations had the lowest response rates, at 40% compared to 94% in those without. None of the six pts who achieved a durable remission (> 6months) had RAS/MAPK mutations at baseline (Figure A). 4 of 16 (25%) of pts who relapsed (< 6 months of remission) or were refractory to this triplet regimen had baseline RAS/MAPK mutations. We had pre- and post-treatment NGS data from 8 pts who had a response but then relapsed (Figure B). Emergent RAS/MAPK mutations were noted in 37% of relapses (3/8), while emergent FLT3 F691L gatekeeper mutations was noted in 25% of relapses (2/8). Interestingly, there were no emergent FLT3 TKD mutations. No pts developed a dose limiting toxicity (DLT) with 30 mg/day quizartinib, however with the 40mg/day quizartinib 2 pts developed hematologic DLT (grade 4 neutropenia with a <5% cellular bone marrow lasting ≥42 days). Hence, quizartinib 30 mg/day dose was determined to be the recommended phase 2 dose for the triplet. Most common Grade 3/4 non-hematologic toxicities included lung infections (42%) and neutropenic fever (30%). No QTcF prolongations >480 msec were noted. With a median follow-up (f/u) of 13 months, the median OS was 7.6 months in R/R cohort. Median OS in prior Gilt exposed pts was 6.3 months and ≥1 prior FLT3i exposed pts was 6.3 months. 8/18 R/R pts (including 5/8 prior Gilt exposed pts) underwent ASCT with a median OS of 19 vs 8 months in pts who underwent ASCT versus not (p=0.26). Of the 5 frontline responding pts median OS was 14.5, 2 were alive in CR, 1 died in CR1 post-ASCT, 2 died due to progressive disease at the last f/u. Image: Summary/Conclusion: DAC + VEN + QUIZ is active in R/R FLT3-ITD mutated AML pts, with CRc rates of 78% and the median OS of 7.6 months. The high response rate was maintained in prior Gilteritinib exposed pts. Interestingly, RAS/MAPK mutations but not emergent TKD mutations were associated with primary and secondary resistance to the triplet. Accrual continues and updated clinical, NGS and mass cytometry (CyTOF) data will be presented. S128: A RANDOMISED COMPARISON OF CPX-351 AND FLAG-IDA IN HIGH RISK ACUTE MYELOID LEUKAEMIA. RESULTS FROM THE NCRI AML19 TRIAL N. Russell1,*, C. Wilhelm-Benartzi2, S. Knapper3, L. Batten2, J Canham2, E. Hinson2, U. Malthe Overgaard4, J. Othman5, R. Dillon5, P. Mehta6, P. Kottaridis7, J. Cavenagh8, C. Hemmaway9, C. Arnold10, M. Dennis11 1Guy’s and St Thomas’ NHS Foundation Trust, Department of Haematology, London; 2Centre for Trials Research; 3School of Medicine, Cardiff University, Cardiff, United Kingdom; 4Copenhagen University Hospital, Copenhagen, Denmark; 5Department of Medical and Molecular Genetics, Kings College London, London; 6University Hospitals of Bristol and Weston NHS Trust, Bristol; 7University College London Hospitals NHS Foundation Trust; 8Department of Haematology, St Bartholomew’s Hospital, London, United Kingdom; 9Aukland Hospital, Aukland, New Zealand; 10Belfast City Hospital, Belfast; 11The Christie NHS Foundation Trust. On behalf of the NCRI AML Working Group, Manchester, United Kingdom Background: Liposomal daunorubicin and cytarabine (CPX-351) has shown a survival advantage for older patients (>60years) with secondary AML compared to 3 + 7 chemotherapy however in younger patients there is a lack of randomised evidence for benefit. We have previously reported improved survival with FLAG-Ida treatment as treatment intensification for younger patients identified with high risk (HR) AML following induction therapy and for patients with secondary AML (Burnett AK et al. Leukemia. 2018 Dec;32(12):2693-2697) and considered this regimen an appropriate comparator for trials in younger patients. Aims: We compared CPX-351 with FLAG-Ida in a randomised fashion in patients who were either HR at trial entry based on cytogenetics or identified as HR following induction or at relapse. Methods: The AML19 trial (ISRCTN78449203) randomised CPX-351 vs FLAG-Ida in 635 patients mainly <60 years with HR AML or MDS (>10% blasts) (median age 53.6 yrs). Three groups of HR patients were randomised 2:1 in favour of CPX with the aim of proceeding to allogeneic transplant. Group 1 (n=195) had known adverse risk cytogenetics (Grimwade et al, Blood 2010,116, 354) and were randomised at diagnosis between 4 courses of CPX-351 and 2 courses of FLAG-Ida followed by MACE/MidAC consolidation. Group 2 (n=263) were HR by validated risk score, had FLT3-ITD without an NPM1 mutation, had refractory disease and were randomised after induction course 1. Group 3 (n=177) were randomised after course 2 if they had persisting MRD at the time of relapse. Here we present results for Group 1. Group 1 was not powered to claim statistical significance; therefore, these results are intended to be exploratory and hypothesis generating. Results: Group 1 included 49.2% with de novo AML 20.3% patients with secondary AML and 30.5% with HR MDS. The Overall response rate(CR/CRi) was 64.8% for CPX-351 and 74.4% for FLAG-Ida (univariate OR:0.57, 95%CI 0.30-1.10, p=0.09). Overall survival (OS) at 3 years was 32% and 24%, median OS was 13.3 months vs 10.2 months (univariate HR:0.83, 95%CI 0.58-1.18, p=0.3) for CPX -351 and FLAG-Ida respectively (Figure 1a). Event free survival (EFS) was not significantly different (HR:0.91 95%CI: 0.50-1.64, p=0.76). Relapse free survival (RFS) at 3 years was 43% and 28%, median RFS was 22.1 months vs 14 months (univariate HR:0.66, 95%CI 0.41-1.06, p=0.09) for CPX -351 and FLAG-Ida respectively (Figure 1b). RFS was significant when adjusting for NPM1 mutation status or FLT3 mutation status using multivariable cox regression model with RFS being better with CPX-351 compared to FLAG-Ida (HR:0.58, 95% CI0.36-0.95, p=0.03). Median duration of remission favoured CPX and was 319.5 days vs 167 days (p=0.046) for CPX vs FLAG-Ida respectively. Haematological toxicity was greater in course 1 with CPX-351 with platelet recovery to 100x109/L at 34.5 days versus 29 days (p<0.001) and neutrophil recovery to 1.0x109/L at 32 days vs 30 days.(p=0.12). Day 30 and day 60 mortality were not significantly different between arms with 4.8% vs 7.3% (p=0.46) and 12.4% vs 11.0% (p=0.77) for CPX-351 and FLAG-Ida respectively.Compliance was better with CPX-351 with 90.7% vs 83.0% receiving the scheduled course 1 dose. More patients receiving CPX-351 were transplanted (50.5% vs 41.5%) with the median number of courses given prior to transplant 2 in both arms. Image: Summary/Conclusion: In patients with adverse cytogenetics CPX-351 did not improve response, OS or EFS compared to FLAG-Ida but was associated with better duration of remission and RFS. Further follow-up is needed to determine the clinical significance of those differences. S129: TAKEAIM LEUKEMIA- A PHASE 1/2A STUDY OF THE IRAK4 INHIBITOR EMAVUSERTIB (CA-4948) AS MONOTHERAPY OR IN COMBINATION WITH AZACITIDINE OR VENETOCLAX IN RELAPSED/REFRACTORY AML OR MDS G. Garcia-Manero1,*, E. S. Winer2, D. J. DeAngelo2, S. Tarantolo3, D. A. Sallman4, J. Dugan5, S. Groepper6, A. Giagounidis6, K. Götze7, K. H. Metzeler8, C.-C. Li9, L. Zhou9, E. Martinez9, M. Lane9, R. von Roemeling9, M. Bohme9, A. S. Kubasch10, A. Verma11, U. Platzbecker10 1Luekemia, MD Anderson Cancer Center, Houston; 2Dana-Faber Cancer Institute, Boston; 3Nebraska Cancer Specialist, Omaha; 4Moffitt Cancer Center, Tampa; 5Novant Health Cancer Institute, Forsyth Medical Center, Winston-Salem, United States of America; 6Marien Hospital/ Univ. of Dusseldorf Germany, Dusseldorf; 7Faculty of Medicine Technical University of Munic, Munich; 8Hematology, Cellular Therapy and Hemostaseology, University Hospital Leipzig, Leipzig, Germany; 9Curis, Lexington, United States of America; 10University Hospital Leipzig, Leipzig, Germany; 11Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, United States of America Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) and FLT3. IRAK4 is critical in triggering inflammation, oncogenesis, and survival of cancer cells. Genetic mutations in the splicing factors SF3B1 and U2AF1 drive overexpression of a highly active long isoform of IRAK4 and have been associated with disease progression and poor prognosis of high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML). Aims: Assessment of safety, clinical activity, and Recommended Phase 2 Dose (RP2D) of emavusertib as monotherapy or in combination with azacitidine (AZA) or venetoclax (VEN). Methods: This is an open-label, phase 1/2a dose escalation and cohort expansion trial (NCT04278768). In phase 1 Dose Escalation, patients with R/R AML or HR-MDS are treated with emavusertib monotherapy. Phase 1b includes 2 arms of combination therapy: emavusertib + AZA and emavusertib + VEN. The primary objectives of this study are to assess the safety, clinical activity, and identify the RP2D of emavusertib as monotherapy or in combination with AZA or VEN in R/R AML or HR-MDS. The Phase 2a Dose Expansion includes patients for emavusertib monotherapy: R/R AML with FLT3 mutation, or AML and HR-MDS R/R to HMA with U2AF1 or SF3B1 mutations. Results: As of December 16th, 2021, 49 patients have been treated in the phase 1 portion, of whom 43 started by September 30th, allowing 2 on-study disease assessments. The median number of prior therapies was 2 (range 1-5). Four monotherapy dose levels of emavusertib were tested (200 to 500 mg orally BID). No dose-limiting toxicities were observed at 200 mg and 300 mg BID. No Grade 4 or 5 treatment-related AEs (TRAEs) were reported, and all the TRAEs were manageable. Reversible, manageable Grade 3 rhabdomyolysis occurred in 1/26 (4%) patients at 300 mg BID, 2/17 (12%) at 400 mg BID, and 1/3 (33%) at 500 mg BID. RP2D was determined as 300 mg BID. Of 43 patients starting before Sept 30th, 2021, 14 had SF3B1, U2AF1 or FLT3 mutations and demonstrated more promising efficacy. In the 5 evaluable AML patients with spliceosome mutations, 40% reached CR/CRh (1 CR, 1 CRh), both with study duration >6 months. In the 7 spliceosome-mutated HR-MDS patients, 57% reached marrow CR, including 1 with RBC transfusion independence and 1 proceeding to HSCT. One of the three FLT3-mutated AML reached CR, and 2 became FLT3-negative. Among the 29 patients without SF3B1/U2AF1/FLT3 mutations, 1 reached CR and 2 PR. Phase 1b and Phase 2a are ongoing. RNA-seq on selected samples showed decrease in relative expression of IRAK4-long isoforms with response to emavusertib. Summary/Conclusion: Emavusertib is well tolerated and effective in heavily pretreated AML and HR-MDS patients, especially in those with U2AF1/SF3B1/FLT3 mutations. No dose-limiting myelosuppression was reported, suggesting emavusertib may be a candidate for combination therapy. Accrual of Phases 1b and 2a is ongoing. S130: NPM1 MUTATED AML: IMPACT OF CO-MUTATIONAL PATTERNS - RESULTS OF THE EUROPEAN HARMONY ALLIANCE A. Hernández-Sánchez1 2,*, Á. Villaverde-Ramiro2, J. Martínez Elicegui2, T. González2 3, A. Benner4, E. Sträng5, G. Castellani6, C. A. Heckman7 8, J. Versluis9, M. Abáigar2 3, M. Sobas10, R. Azibeiro1 2, L. Tur11, P. J. Valk9, K. H. Metzeler12, R. Ayala13, D. Dall’Olio6, J. Tettero14, J. Martínez-López13, H. Dombret15, M. Pratcorona16, F. Damm5, K. I. Mills17, J. Mayer18, C. Thiede19, M. T. Voso20, G. F. Sanz21 22, F. Calado23, K. Döhner24, V. I. Gaidzik25, M. Heuser26, T. Haferlach27, A. T. Turki28 29, D. Reinhardt28, R. Villoria Medina11, M. van Speybroeck30, R. Schulze-Rath31, M. Barbus32, J. E. Butler33, J. M. Hernández Rivas1 2 3, B. J. Huntly34, G. J. Ossenkoppele14 35 36, H. Döhner24, L. Bullinger5 1Hospital Universitario de Salamanca; 2Instituto de Investigación Biomédica de Salamanca (IBSAL); 3Centro de Investigación del Cáncer (CIC), Salamanca, Spain; 4German Cancer Research Center (DKFZ), Heidelberg; 5Charité Universitätsmedizin Berlin, Berlin, Germany; 6University of Bologna, Bologna, Italy; 7University of Helsinki; 8Institute for Molecular Medicine Finland, Helsinki, Finland; 9Erasmus University Medical Center Cancer Institute, Rotterdam, Netherlands; 10Wroclaw Medical University, Wroclaw, Poland; 11GMV Innovating Solutions, Valencia, Spain; 12University of Munich, Munich, Germany; 13Hospital Universitario; 12de Octubre, Madrid, Spain; 14Amsterdam University Medical Center, Amsterdam, Netherlands; 15EA3518 Leukemia Translational Laboratory, Paris, France; 16Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 17Queens University Belfast, Belfast, United Kingdom; 18University Hospital Brnoand Masaryk University, Brno, Czechia; 19University of Technics Dresden Medical Dept., Dresden, Germany; 20University of Rome “Cattolica S. Cuore”, Rome, Italy; 21Hospital Universitario y Politécnico La Fe; 22Instituto de Salud Carlos III (CIBERONC), Valencia, Spain; 23Novartis, Oncology Region Europe, Basel, Switzerland; 24University Hospital of Ulm; 25Ulm University Hospital, Ulm; 26Hannover Medical School, Hannover; 27MLL Munich Leukemia Laboratory, Munich; 28Essen University Hospital; 29West-German Cancer Center, Essen, Germany; 30Janssen Pharmaceutica N.V., Beerse, Belgium; 31Bayer Pharma AG, Berlin; 32AbbVie Germany GmbH & Co. KG, Wiesbaden; 33Bayer AG, Berlin, Germany; 34Wellcome - MRC Cambridge Stem Cell Institute, Cambridge, United Kingdom; 35VU University Medical Center; 36Cancer Center Amsterdam, Amsterdam, Netherlands Background: Acute myeloid leukemia (AML) is a heterogeneous disease in terms of clinical features, outcomes and genetics. While mutations of NPM1 are usually considered as a favorable prognostic marker, the vast majority of the patients carry several co-mutations that might influence the prognosis. Therefore, a better understanding of the NPM1 mut AML mutational landscape is warranted. The large cohort of AML patients collected within the European HARMONY Alliance provides an excellent basis for this purpose. Aims: To identify clinically significant co-mutational patterns in NPM1 mut AML in order to establish a revised risk stratification model. Methods: From the HARMONY Alliance AML database, a total of 1001 NPM1 mut intensively treated patients were selected. Clinically significant co-mutations were evaluated using graphical patterns created with the Gephi tool and confirmed by detailed survival analysis using Kaplan-Meier and Cox regression models. Finally, a novel multi-state risk stratification model for NPM1 mut AML was established. Results: The study population of 1001 NPM1 mut AML patients included 57% females and median age was 53 years. Regarding ELN2017 classification, 68% of patients were classified into the favorable, 29% intermediate and 3% adverse risk groups. The most frequent co-mutations were DNMT3A (54%), followed by FLT3-ITD (38%). In total, 24% of patients presented with a high allelic mutant-to-wildtype ratio ≥0.5 (FLT3-ITDhigh) while 14% had low allelic ratio <0.5 (FLT3-ITDlow). Other frequent co-mutations were NRAS (21%), TET2 (20%) and PTPN11 (15%). The triple mutation pattern of NPM1 mut + FLT3-ITDhigh + DNMT3A mut identified a subgroup with adverse prognosis (2-year OS of 25%), similar to NPM1 mut + TP53 mut. The combination of FLT3-ITDlow + DNMT3A mut or FLT3-ITDhigh + DNMT3A wt was associated with intermediate prognosis (2-year OS of 45% and 53% respectively). Notably, mutations of NRAS, KRAS, PTPN11 or RAD21 were identified to be associated with better OS. However, in the context of NPM1 mut + DNMT3A mut these mutations did not affect the prognosis when a FLT3-ITD was present. This information is summarized in a 3-category risk classification model (Figure 1). The revised NPM1 mut favorable group presented with a 2-year OS of 73%, while for intermediate and adverse groups the OS was 54% and 27% respectively (p<0.001). Regarding relapse free survival (RFS), the median was not reached in the favorable group, while it was 23 months for intermediate and 6 months for adverse group (p<0.001). It should be noted that 171 patients in the NPM1 mut intermediate group would be considered as favorable according to the ELN2017 criteria, as well as 162 patients in the NPM1 mut adverse group were previously classified as intermediate risk. Therefore, our model was able to reclassify 33% of NPM1 mut AML patients in comparison to ELN2017 criteria. Multivariate analysis of OS in NPM1 mut AML identified the following independent prognostic factors: NPM1 mut model (taking favorable group as reference, HR 1.6 for intermediate and HR 2.7 for adverse group, p<0.001); secondary or therapy-related AML (HR 1.8, p<0.001), WBC at diagnosis >100x103/μL (HR 1.5, p<0.001) and age >60 years (HR 1.4, p<0.001). Image: Summary/Conclusion: Analysis of large NPM1 mut AML cohorts allows the discovery of co-mutation patterns associated with prognostic outcome. In accordance, we propose a new genetic stratification model for NPM1 mut AML that identifies 3 groups with different OS and RFS. This model improves ELN2017 criteria as it is able to correctly reclassify 33% of NPM1 mut AML patients. S131: CLINICAL IMPLICATIONS OF SECONDARY-AML TYPE MUTATIONS IN PATIENTS WITH DE NOVO ACUTE MYELOID LEUKEMIA K. Sun1,*, C.-H. Tsai1, M.-Y. Lo1, M.-H. Tseng1, Y.-Y. Kuo2, M.-C. Liu3, C.-C. Lin1, C.-L. Cheng1, S.-J. Wu1, C.-Y. Chen1, B.-S. Ko1 4, M. Yao1, W.-C. Chou1, H.-A. Hou1, H.-F. Tien1 1 1Hematology; 2Oncology; 3Pathology, National Taiwan University Hospital; 4Hematology, National Taiwan University Cancer center, Taipei, Taiwan Background: More and more gene mutations have been identified in patients with de novo acute myeloid leukemia (AML). Among them, a set of gene mutations, including SRSF2, ZRSR2, SF3B1, U2AF1, ASXL1, EZH2, STAG2, and BCOR mutation, are categorized as secondary AML (sAML)-type mutations, for their distinct distribution in secondary AML, compared to primary AML (Lindsley et al, Blood 2015), but the reports regarding the prognostic impact have been scanty, especially in younger patients. Aims: In this study, we aimed to explore the clinical significance and prognostic implication of sAML-type mutations in non-M3 AML patients. Methods: We consecutively enrolled 921 de novo non-M3 AML patients; 368 were 60 years or older (older patients) and 553 were younger. Patients with an antecedent history of hematologic diseases, or therapy-related AML were excluded. sAML-type mutations were identified by targeted next-generation sequencing of 54 myeloid malignancies related gene mutations. Results: A total of 243 (26.4%) patients harbored sAML-type mutations (ST group), 40.2% in older patients and 17.2% in younger ones. Patients in the ST group were significantly older, had a lower WBC count, peripheral blast count and lactate dehydrogenase level at diagnosis. sAML-type mutations were negatively correlated with inv(16), monosomy 17, and complex karyotype, but positively associated with 2017 European LeukemiaNet (ELN)-defined unfavorable-risk genetic category. Among the patients receiving standard chemotherapy (n=686, 74.5%), the ST group had a significantly lower CR rate (62.9% vs. 81.5%, P< 0.01), especially among younger patients (70.8% vs. 86.7%, P<0.01), but only a trend in older patients (49.0% vs. 59.6, P=0.17). With a median follow-up of 4.7 years, patients in the ST group had a shorter overall survival (OS, median, 2.1 years vs. not reached, P< 0.01) and disease-free survival (DFS, median, 0.4 years vs. 0.9 years, P< 0.01) than the non-ST group. Subgroup analyses showed that sAML-type mutations conferred a significantly poorer DFS (median, 0 years vs. 0.5, P=0.03) and a trend of shorter OS (0.8 years vs. 1.1 years, P=0.08) in older patients, and a significantly worse OS (not reached vs. not reached, P=0.03) and a trend of shorter DFS (0.7 years vs. 1.0 years, P=0.16) in younger patients. The numbers of sAML-type mutations had prognostic impacts on both OS (median, 5.8 years vs. 1.5 vs. 1.3 for patients with 0, 1, and ≥2 mutations, respectively, P<0.01) and DFS (median, 0.9 years vs. 0.6 vs. 0 for those with 0, 1, and ≥2 mutations, respectively, P< 0.01). Intriguingly, these findings were valid among both the younger and older patients. Furthermore, the ELN-defined intermediate-risk patients with sAML-type mutations had similar poor OS and DFS to the ELN unfavorable-risk patients in total cohort (Figure 1), as well as in the older and younger patients. Among the patients with sAML-type mutations, allogeneic hematopoietic stem cell transplantation did improve their outcome (median OS 3.0 vs. 0.8 years, P< 0.01). Image: Summary/Conclusion: AML patients with sAML-type mutations had distinct clinical features and poorer outcomes. Incorporating sAML-type mutations can further refine the 2017 ELN risk stratification. It is suggested that AML patients with sAML-type mutations receive more intensive treatment, such as allo-HSCT, and/or novel therapies. S132: TOLERABILITY AND EFFICACY OF THE FIRST-IN-CLASS ANTI-CD47 ANTIBODY MAGROLIMAB COMBINED WITH AZACITIDINE IN FRONTLINE PATIENTS WITH TP53-MUTATED ACUTE MYELOID LEUKEMIA: PHASE 1B RESULTS N. G. Daver1,*, P. Vyas2, S. Kambhampati3, M. M. Al Malki4, R. Larson5, A. Asch6, G. Mannis7, W. Chai-Ho8, T. Tanaka9, T. Bradley10, D. Jeyakumar11, E. Wang12, G. Xing13, M. Chao13, G. Ramsingh13, C. Renard13, I. Lal13, J. Zeidner14, D. Sallman15 1The University of Texas MD Anderson Cancer Center, Houston, United States of America; 2University of Oxford, Oxford, United Kingdom; 3Healthcare Midwest, Kansas City; 4City of Hope National Medical Center, Duarte; 5University of Chicago, Chicago; 6University of Oklahoma, Oklahoma City; 7Stanford University, Stanford; 8University of California Los Angeles, Los Angeles; 9University of California San Diego, San Diego; 10University of Miami, Miami; 11University of California Irvin, Irvine; 12Roswell Park Comprehensive Cancer Center, Buffalo; 13Gilead Sciences, Inc., Foster City; 14Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill; 15Moffitt Cancer Center, Tampa, United States of America Background: Patients (pts) with TP53-mutated acute myeloid leukemia (AML) have a poor prognosis, with limited responses to currently available therapies and low survival outcomes, representing a significant unmet medical need. Magrolimab is a monoclonal antibody blocking CD47, a “don’t eat me” signal overexpressed on cells in cancers such as AML. This blockade induces phagocytosis of tumor cells and is synergistic with azacitidine (AZA) via upregulation of “eat me” signals. Aims: To report tolerability and efficacy data from a phase 1b trial of magrolimab + AZA in frontline pts with TP53-mutated AML unsuitable for intensive chemotherapy (NCT03248479). Methods: Frontline pts with AML not suitable for intensive chemotherapy received magrolimab IV starting with a priming dose (1 mg/kg) followed by ramp-up to 30 mg/kg QW or Q2W as the maintenance dose. AZA 75 mg/m2 was given IV or SC on days 1-7 of each 28-day cycle. Primary endpoints were safety/tolerability and complete remission (CR) rate by European LeukemiaNet (ELN) 2017 criteria. Results: 72 pts with TP53-mutated AML were treated (Table). Common all-grade treatment-emergent adverse events (TEAEs) were constipation (52.8%), diarrhea (47.2%), febrile neutropenia (45.8%), nausea (43.1%), fatigue (37.5%), decreased appetite (37.5%), thrombocytopenia (31.9%), peripheral edema (30.6%), and cough (30.6%). Most common grade ≥3 TEAEs were febrile neutropenia (37.5%), anemia (29.2%; grade 3, 26.4%; grade 4, 2.8%), thrombocytopenia (29.2%), pneumonia (26.4%), and neutropenia (20.8%). Grade 3 hemolysis was reported in 1 pt (1.4%); no grade 4 hemolysis was reported. Objective response rate by intent to treat was 48.6% (CR, 33.3%; CR with incomplete hematologic recovery [CRi]/CR with partial hematologic recovery [CRh], 8.3%; morphologic leukemia-free state [MLFS], 1.4%; partial remission, 5.6%). Stable disease and progressive disease (PD) were reported in 16.7% and 5.6% of pts, respectively; 30- and 60-day mortality rates were 8.3% and 18.1%, respectively. Response assessments were unavailable in an additional 4.2% of pts who discontinued due to AEs and 6.9% due to other reasons, prior to the cycle 3 day 1 assessment. Median time to CR/CRi was 2.2 months (range, 1.7-7.2 months) and to CR was 3.0 months (range, 1.8-9.6 months); 14 of 31 (45.2%) evaluable pts with CR/CRi/CRh/MLFS achieved negative MRD by flow cytometry (investigator reported). Of 24 pts with CR, 8 had a longitudinal TP53 variant allele frequency (VAF) assessment and 5 of 8 (63%) had VAF decreased to ≤5%. Treatment was stopped due to stem cell transplant (9 [12.5%]), PD (26 [36.1%]), death (8 [11.1%]), AE (13 [18.1%]), and other (14 [19.4%]). Median durations of CR and CR/CRi were 7.7 months (95% CI, 4.7-10.9 months) and 8.7 months (95% CI, 5.3-10.9 months), respectively. Median overall survival (OS) in 72 pts was 10.8 months (95% CI, 6.8-12.8 months) (figure), with median follow-up of 8.3 months. Table. Baseline characteristics N=72 Age (range), years 73 (31-89) ECOG, n (%) 0-1 61 (84.7) 2 11 (15.3) ELN cytogenetic risk, n (%) Favorable 1 (1.4) Intermediate 2 (2.8) Adverse 57 (79.2) Unknown 12 (16.7) AML with MDS-related changes, n (%) 34 (47.2) Therapy-related AML, n (%) 15 (20.8) ECOG, Eastern Cooperative Oncology Group; MDS, myelodysplastic syndrome. Image: Summary/Conclusion: In high-risk frontline pts with TP53-mutated AML unsuitable for intensive chemotherapy, magrolimab + AZA showed durable responses and encouraging OS in a single-arm study. A Phase 3 trial of this combination vs standard of care in TP53-mutated AML (ENHANCE-2; NCT04778397) is ongoing. S133: OFF-THE-SHELF CD33 CAR-NK CELL THERAPY FOR RELAPSE/REFRACTORY AML: FIRST-IN-HUMAN, PHASE I TRIAL R. Huang1,*, Q. Wen1, X. Wang1, H. Yan1, Y. Ma1, M. Wang1, X. Han1, L. Gao1, L. Gao1, C. Zhang1, X. Zhang1 1Medical Center of Hematology, Xinqiao Hospital of Army Medical University, Chongqing, China Background: The primary results of CAR-T therapy for patients with R/R AML has shown limited efficacy and severe side-effect. One of the main challenges is that current targets for myeloid malignancies are either widely expressed on healthy hemopoietic stem cells such as CD33 or specific for a group of tumor cells presented as Lewis Y antigen, which could cause lasting bone marrow depression induced by “on target off tumor” side-effect or target negative relapse. Therefore, to receive a balance, we designed a CD33 CAR to recognize AML cells and using NK cells to replace T cells as the carrier to eliminate tumor cells. The CD33 CAR NK cells have combined the wide-expression advantage of CD33 target and the safety of NK cells. Aims: To evaluate the safety and primary efficacy of CD33 CAR NK cells Methods: 5 qualified subjects with R/R AML aged between 18 and 65 years-old were enrolled and received round(s) of infusion of anti-CD33 CAR NK cells (6×108, 1.2×109 or 1.8×109 cells per round after the precondition with Fludarabine (30mg/m2) and Cytoxan 300-500mg/m2 for 3 days to 5 days, determined by tumor burden at baseline. We investigated the response rate at D28 and treatment related side-effect after the CAR NK cell infusion and the long-term efficacy. Results: As of data cut (February 26, 2021), 5 pts have finished CAR NK cells infusion. The median age was 43 (18-65) years-old and the median tumor burden before infusion is 31% (21%-77.5%). 4 of 5 patients have received MRD negetive CR at day 28 assessment. In dose group one, three patients have received 3 rounds of CAR NK cells (6×108, 1.2×109 and 1.8×109 cells) with the interval of 7 days after last round, and only patient 1 developed grade 1 CRS represented as fever after the infusion of 1.8×109 CAR NK cells and alleviated within 24h after symptomatic treatment. Patient 1 and patient 2 received MRD negetive CR at day 14 and 21, but patient 2 relapsed at day 43 after first round infusion. In dosage group 2, patient 4 and patient 5 both received one dose of 1.8 ×109 cells CD33 CAR NK cells after precondition, patient 5 developed grade 2 CRS presenting as lasting fever for 6 days after infusion and alleviated after 5mg Dexamethasone I.V., both patient 4 and patient 5 recieved MRD negetive CR at day 28 after infusion. At the time of this abstract being uploaded, three patients remain MRD negetive CR. Image: Summary/Conclusion: Our primary data of the phase I trial have proved the primary efficacy and safety of CD33 CAR NK cells for patients with R/R AML. The efficacy needs expanded samples and longer follow up. S134: INTRA-PATIENT FUNCTIONAL HETEROGENEITY OF AML DETERMINES FIRST-LINE TREATMENT RESPONSE Y. Severin1,*, Y. Festl1, M. Roiss2, T. Benoit3, T. Heinemann1, R. Wegmann1, A.-K. Kienzler3, M. Bissig3, M. Scharl3, M. Manz3, A. M. Müller3, B. Snijder1 1Institute of Molecular Systems Biology, ETH Zurich; 2Medical clinic of Oncology and Haematology, University Hospital Zurich; 3Medical clinic of Oncology and Haematology, University Hospital Zurich, Zürich, Switzerland Background: Acute myeloid leukemia (AML) is a heterogeneous disease with limited treatment options and poor long-term survival. AML is characterized by a fast clonal expansion of premature myeloid cells with highly variable combinations of chromosomal aberrations and somatic mutations. Patient-to-patient variability is further complicated by the fact that AML partially recapitulates myeloid maturation with a rare leukemic stem cell population at the top of the hierarchy. Although eradication of bulk tumor cells is necessary, matched targeted therapies against the AML stem cell compartment are likely essential for a long lasting cure of patients. Despite this large tumor heterogeneity in de-novo AML patients, most patients receive the same standard-of-care treatment resulting in large differences of treatment success and overall survival. Functional ex vivo drug-response profiling offers a possible route to tailor individual treatments for AML patients. However, the intra-patient functional heterogeneity of divergent AML cell subpopulations, and their contribution to clinical outcome, have not yet been systematically studied. Aims: This study has two aims: 1) To analyze the intra-patient functional heterogeneity of AML blasts, and their contribution to the response to first-line treatment. 2) To improve the clinical response predictions from functional ex vivo drug response profiling by explicit analysis of functional tumor heterogeneity. Methods: In this prospective, non-interventional study, 180 AML patient biopsies were collected with written informed consent (from 44 patients with newly diagnosed AML undergoing intensive induction chemotherapy). Patient matched bone marrow and blood samples were obtained for each donor at three different time points across the course of the treatment. For each sample, we perform ex vivo drug-response profiling analyzed by multiplexed immunofluorescence, automated microscopy, and deep learning-based morphological profiling to determine drug sensitivities on a single-cell level. The drug library includes 100 distinct drugs and drug combinations, including standard of care induction chemotherapy, and single-cell drug sensitivity data is analyzed as a function of cellular clone and blast maturation state. The drug response data is integrated with patient-matched serum cytokine profiling, RNA-sequencing, and clinical data, allowing to identify the cellular subpopulations whose functional response is predictive of treatment response. Results: Our workflow excelled at predicting patient responses to first line treatment, with an overall predictive power of 85% accuracy and a diagnostic odds ratio of 47. Molecular and morphology-based phenotyping of cancer subpopulations strengthened clinical associations and revealed blast maturity as well as immune activation to be strong predictors of treatment success and the risk of chemotherapy-related infectious complications. Furthermore, the screening of 100 unique drugs and combinations enabled individualized ex vivo treatment predictions, suggesting more potent treatment options than those currently deployed. Image: Summary/Conclusion: Maturation-associated intra-tumor drug response heterogeneity is a strong predictor of clinical response to first-line intensive induction chemotherapy in AML. Our multiplexed ex vivo drug screening framework is highly flexible, allowing us to discover, molecularly describe, and target clinically relevant AML subpopulations on a personal basis, leading to improved personalized treatment recommendations. S135: CRISPR/CAS9 EDITING REVEALS DEPENDENCE OF HUMAN RICHTER SYNDROME AND MURINE CLL CELLS ON SIGNALS FROM B CELL RECEPTOR, CXCR4 RECEPTOR AND MACROPHAGES BUT NOT FROM TOLL-LIKE RECEPTORS IN VIVO C. Martines1,*, S. Chakraborty1, V. Guastafierro1, M. Vujovikj2, T. Vaisitti3, S. Deaglio3, L. Laurenti4, A. Dimovski2, D. Efremov1 1Molecular Hematology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; 2Research Center for Genetic Engineering and Biotechnology, Macedonian Academy of Sciences and Arts, Skopje, North Macedonia, Republic of; 3Functional Genomics Unit, Department of Medical Sciences, University of Turin, Torino; 4Department of Hematology, Catholic University Hospital “A. Gemelli”, Rome, Italy Background: Numerous signals from the microenvironment have been identified that can increase the survival or induce the proliferation of chronic lymphocytic leukemia (CLL) cells in vitro. These signals typically represent various secreted or cell surface ligands that are expressed by different cell types present in the lymph node tumor microenvironment, such as T cells, macrophages and stromal cells, or molecules that would be expected to be released by apoptotic cells, such as apoptosis associated autoantigens or CpG-unmethylated mitochondrial DNA. However, the extent to which these different signals contribute to the growth and survival of the leukemic cells in vivo has still not been fully established. Aims: To determine the relevance of signals from Toll-like receptors (TLRs), the B cell receptor (BCR) and the chemokine receptor CXCR4 for the growth of murine Eμ-TCL1 CLL cells and human Richter Syndrome (RS) patient-derived xenograft (PDX) cells in vivo. Methods: To understand the impact of pharmacological inhibition of the TLR pathway, immunocompetent C57BL/6 or immunodeficient NSG mice were transplanted with murine Eμ-TCL1 CLL or human RS-PDX cells, respectively, and were treated with an inhibitor of the kinase IRAK4 or vehicle control. To determine the effects of genetic disruption of the TLR, BCR and CXCR4 pathways, the IRAK4, IgM heavy chain (IGHM) and CXCR4 genes were disrupted by CRISPR/Cas9 editing in the murine Eμ-TCL1 CLL cells or the human RS-PDX lines RS9737, RS1316 and IP867/17 prior to transplantation. The effects of genetic disruption of these pathways on the growth of the malignant cells in vivo were investigated by analyzing the proportion of mutant and wild type alleles in different anatomical compartments of the transplanted mice. Results: Treatment with the IRAK4 inhibitor significantly prolonged the survival of C57BL/6 mice transplanted with murine Eμ-TCL1 CLL cells and significantly delayed the growth of the human RS-PDX lines RS9737 and RS1316 xenografted in immunodeficient NSG mice. However, genetic disruption of IRAK4 in the human RS-PDX lines RS9737, RS1316 and IP867/17 or of the TLR adaptor MyD88 in the murine Eμ-TCL1 CLL cells did not result in negative selection of these cells in vivo, suggesting that these tumors do not receive or do not rely on TLR signals for their growth and that the therapeutic activity of the IRAK4 inhibitor is not caused by disruption of TLR signaling in the malignant cells themselves. In contrast, genetic disruption of the IGHM or CXCR4 gene was associated with significantly reduced growth of these cells compared to their wild type counterparts and resulted in almost complete disappearance of the mutated cells at later timepoints following transplantation. Analysis of the effects of the IRAK4 inhibitor on other cell types from the tumor microenvironment revealed a significant reduction in the number of macrophages, which in co-culture experiments were shown to strongly protect human Richter syndrome and murine Eμ-TCL1 leukemia cells from spontaneous apoptosis. Summary/Conclusion: These data provide evidence that signals from the BCR, CXCR4 and macrophages support the growth and survival of CLL and RS cells in vivo and argue against a role for TLR signals in the pathogenesis of CLL. In addition, they suggest that targeting the TLR pathway may potentially provide a therapeutic benefit in CLL, but that this benefit would be derived from inhibition of TLR signaling in monocytes and macrophages rather than inhibition of TLR signaling in the malignant cells themselves. S136: CONSTITUTIVE VLA-4 ACTIVATION IN CHRONIC LYMPHOCYTIC LEUKEMIA. THE OTHER SIDE OF BCR AUTONOMOUS SIGNALING. E. Tissino1,*, R. Bomben1, P. C. Maity2, F. Pozzo1, G. Forestieri3, A. Nicolò2, A. Härzschel4, T. Bittolo1, F. M. Rossi1, M. Datta2, F. Zaja5, G. Capasso1, G. D’Arena6, A. Chiarenza7, F. Di Raimondo7, H. Jumaa2, D. Rossi3, G. Del Poeta8, T. N. Hartmann4, A. Zucchetto1, V. Gattei1 1Clinical and Experimental Onco-Hematology, Centro di Riferimento Oncologico, Aviano, Italy; 2Institut für Immunologie, Universitätsklinikum Ulm, Ulm, Germany; 3Division of Hematology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 4Department of Internal Medicine, Faculty of Medicine, University Medical Center Freiburg, University of Freiburg, Freiburg, Germany; 5Dipartimento di Scienze Mediche e Chirurgiche e della Salute, University of Trieste, Trieste; 6IRCCS Centro Di Riferimento Oncologico Della Basilicata, IRCCS Centro Di Riferimento Oncologico Della Basilicata, Rionero In Vulture; 7Divisione di Ematologia, Ospedale Ferrarotto, A.O.U. Policlinico-OVE, Università di Catania, Catania; 8Division Hematology, S.Eugenio Hospital and University of Tor Vergata, Rome, Italy Background: In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator and key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its ligands. In CLL, beside the canonical antigen (Ag)-dependent mechanism, BCR signaling has been recently demonstrated to occur via an autonomous Ag-independent manner. Aims: To investigate the role of autonomous BCR signaling on constitutive VLA-4 activation state in CLL. Methods: VLA-4 activation/affinity was determined by flow cytometry (FC) using the conformation-sensitive anti-CD29 mAb HUTS21 and/or by “real-time” (FC) measuring the binding of the VLA-4 ligand LDV-FITC as reported (Tissino et al, J Exp Med, 2018) in: i) 1,984 consecutive CLL all with IGHV gene mutations/BCR features available; ii) sequential samples (0, 14, 30, 60 90 days) from CLL patients (n=26) treated in vivo with ibrutinib (IB) in real-world and from a clinical trial (NCT02827617). HUTS21 staining was also performed in the presence of: plasma depletion/replacement, soluble (s) VCAM-1, fibronectin (FN) and blocking anti-CD49d (HP1/2) mAbs. ELISA assays were used to quantify sVCAM-1 in plasma samples (n=122). BCR signaling were investigated by Ca++ influx assay in a 4-OH tamoxifen (4-OHT)-inducible murine TKO cell model (Dühren-von Minden M et al, Nature, 2012). Results: Out of 1,984 CLL, 1,070 (54%) expressed CD49d (cutoff 30%) and, among them, 250/1,070 (23%) were HUTS21+ (cutoff 20%), indicating an activated VLA-4 conformation. HUTS21 staining was: i) impaired by depletion of plasma from whole blood samples, and reconstituted by specific plasma components (sVCAM-1, FN); ii) impaired by pre-incubation with anti-CD49d HP1/2 blocking mAbs before addition of plasma, sVCAM-1 and FN. sVCAM-1 was higher in CD49d+ vs CD49d- CLL (p<0.0001); among CD49d+ cases, sVCAM-1 was lower in HUTS21+ (i.e. VLA-4 activated) cases (p=0.0096), suggesting ligand sequestration by activated surface VLA-4. CLL with mutated IGHV expressed higher levels of activated VLA-4 compared to unmutated IGHV CLL (p=0.001). Higher levels of activated VLA-4 were found in CLL using the IGHV3 and IGHV4 families, compared to cases using the IGHV1 family (p=0.043 and p=0.004). Finally, analysis of BCR stereotypy highlighted higher VLA-4 activation levels in CLL from subset#2 compared to CLL from subset#1 (p=0.02). To validate these data, murine TKO cells, expressing high VLA-4 levels, were transfected with different BCRs derived from 4 CLL with high level of constitutively activated VLA-4 (TKO-high) and 4 CLL with low level of constitutively activated VLA-4 (TKO-low). Compared to TKO-low cells, TKO-high cells showed a higher autonomous Ca++ influx (p=0.03), and consistently higher VLA-4 affinity (p=0.01). IB treatment impaired both BCR autonomous signaling and VLA-4 affinity. Notably, anti-IgM stimulation induced high Ca++ influx and high VLA-4 affinity state in both TKO-high and TKO-low, irrespective of IB treatment. According to TKO data, a decreased constitutive VLA-4 activation was observed in CLL cells collected at pre-treatment and at day 14, 30, 60 and 90 from patients on IB, confirming an IB-dependent impairment of VLA-4 activation via BCR signal. Summary/Conclusion: The presence of a constitutively activated form of VLA-4 is observed in a fraction of CD49d+ CLL, due to a continuous VLA-4 inside-out stimulation derived from autonomous BCR signaling. S137: SMALL EXTRACELLULAR VESICLES IN THE LEUKEMIA MICROENVIRONMENT SUSTAIN CLL PROGRESSION BY HAMPERING T CELL-MEDIATED ANTI-TUMOR IMMUNITY E. Gargiulo1,*, E. Viry1, P. E. Morande1 2, A. Largeot1, S. Gonder1 3, F. Xian1 4, N. Ioannou5, M. Benzarti1 3, F. Kleine Borgmann1 3 6, M. Mittelbronn1 3 7, G. Dittmar1 3, P. V. Nazarov1, J. Meiser1, B. Stamatopoulos8, A. G. Ramsay5, E. Moussay1, J. Paggetti1 1Luxembourg Institute of Health, Luxembourg, Luxembourg; 2Instituto de Medicina Experimental, Buenos Aires, Argentina; 3University of Luxembourg, Luxembourg, Luxembourg; 4University of Vienna, Vienna, Austria; 5King’s College London, London, United Kingdom; 6Centre Hospitalier de Luxembourg, Luxembourg; 7Laboratoire national de santé, Dudelange, Luxembourg; 8Université Libre de Bruxelles, Brussels, Belgium Background: Small extracellular vesicles (sEV) are nano-sized particles released by every cell and found in all biofluids. Given their composition and abundance, sEV are commonly involved in cell-to-cell communication through the transfer of genetic material and proteins. Furthermore, sEV possess direct functions carried out by sEV-ligands capable to affect the biological functions of targeted cells. In cancer, tumor-derived sEV are involved in the re-education of microenvironment (ME) cells promoting tumor proliferation, immune escape and metastasis. We previously demonstrated that leukemia-derived sEV are involved in the re-education of surrounding cells and increased immune escape. Indeed, chronic lymphocytic leukemia (CLL)-derived sEV induce stromal cell conversion into cancer-associated fibroblasts (Paggetti et al., Blood, 2015), and modulate PD-L1 expression in monocytes (Haderk et al. Science Immunology, 2017). Aims: The goal of the present work was to characterize leukemia ME-derived sEV (LME-sEV) and to evaluate their role in the disease development and progression in vivo. Methods: To obtain a biological representation of sEV in CLL microenvironment, we isolated LME-sEV directly from spleens of leukemic mice, obtaining a complex mix of sEV released by both CLL and ME cells alike. Small EV characterization was performed using a wide range of techniques, including qPCR, mass spectrometry and single sEV flow cytometry (FC). The effect on target cells was evaluated both ex vivo and in vivo using high-throughput techniques, FC, qPCR and cytotoxic assay. Small EV impact on CLL development in vivo was evaluated by generating a novel preclinical mouse model in which sEV release is genetically impaired due to Rab27a/b knock-out. Finally, we analyzed the expression of sEV-related genes in a cohort of 144 CLL patients using qPCR followed by regression analysis. Results: LME-sEV showed a distinct proteome (A) and RNA contents compared to healthy counterparts (HCME-sEV), including miRNA enriched in the plasma of CLL patients. Furthermore, FC-based immune checkpoint (ICP) screening showed the presence of multiple ICP ligands anchored on CLL-derived sEV (CD20+ subset of LME-sEV) (B), while high expression of the corresponding ICP receptors was found on T cells from matching LME. We also found that LME-sEV are internalized by different T cell subsets, thus we performed in vivo and ex vivo functional studies to assess sEV impact on T cells. High-throughput analysis of cells isolated from spleens of control mice treated with LME-sEV revealed considerable physiological changes mainly in CD8+ T cells. Indeed, CD8+ T cells showed alterations in their transcriptome, proteome and metabolome leading to cell exhaustion, decreased functions and survival. In line with this, absence of sEV dramatically delayed CLL progression in vivo. This effect was due to CLL inability to escape immune surveillance in absence of sEV and this was rescued by LME-sEV treatment (C). Finally, we identified a consistent sEV gene signatures in CLL patients correlating with treatment-free survival, overall survival, and with unfavorable clinical parameters routinely used in CLL diagnosis and prognosis (D). Image: Summary/Conclusion: By using different preclinical murine models and strategies, our results demonstrated for the first time that sEV in CLL ME play a key pro-tumoral role in leukemia development by negatively affecting the anti-tumor immune response. Furthermore, high expression of sEV-related genes correlated with poor survival and clinical parameters in CLL patients, suggesting sEV profiling as prognostic tool in CLL. S138: MICROENVIRONMENT-REGULATED TRANSCRIPTIONS FACTORS AHR AND HIF-1Α EXPRESSION IN TREGS PROMOTE CLL PROGRESSION BY IMPAIRING CD8+ T-CELL MEDIATED ANTI-TUMOR IMMUNITY G. Pagano1,*, M. Wierz1, I. Fernandez Botana1, S. Gonder1, E. Gargiulo1, E. Moussay1, J. Paggetti1 1Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg Background: Chronic Lymphocytic Leukemia (CLL) progression is highly dependent on complex interactions between tumor cells and the tumor microenvironment (TME). Indeed, CLL cells can modify stromal cells and immune cells to promote the survival of the leukemic clone and to escape from the immune system surveillance. Within the TME, regulatory T cells (Tregs) represent a subtype of CD4+ T cells with immunosuppressive abilities, causing the evasion of cancer cells from the immune system. We previously characterized extensively the immune microenvironment of pre-clinical CLL mouse models using mass cytometry, and we described a significant increase in the Tregs subsets with an enhanced immunosuppressive and activated phenotype compared to non-leukemic animals (Wierz et al., Blood, 2018). Interestingly, TIGIT+ Tregs are more immunosuppressive than their TIGIT- Treg counterparts and express higher levels of several transcription factors, including Ahr and Hif1α (Joller et al., Immunity, 2014), both involved in the cellular response to microenvironment-mediated stimuli. Aims: The aim of the present study is to investigate the role of AHR and HIF-1α in the suppressive ability of Tregs during CLL development. Methods: We generated conditional knock out mice (cKO) lacking Ahr or Hif1a genes exclusively in Tregs (Foxp3 YFP-Cre Ahr fx/fx and Foxp3 YFP-Cre Hif1a fx/fx mice). We then performed adoptive transfer (AT) of CLL cells obtained from diseased Eµ-TCL1 mice into cKO and control mice. In order to decipher the mechanism by which AHR and HIF-1α pathways in Tregs affect CLL progression, we analyzed the splenic TME of recipient mice and evaluated immune checkpoint expression and cytokine production. Finally, we evaluated the suppression ability of the regulatory T cells with ex vivo suppression assays. Results: Generated cKO mice showed no sign of abnormalities or autoimmune phenotypes, and immune cells phenotyping revealed no major differences. However, we showed that CLL growth in cKO mice was drastically delayed compared to the control mice (A). Interestingly, this decrease was mitigated when CD8+ T cells were depleted. The analysis of the splenic TME in recipient cKO mice revealed an increase in IL-17 and TNF-α production, two major T-cell cytokines, in CD4+ T cells as compared to their WT counterparts. We also measured the expression of immune checkpoints and activation markers in the different T cell subpopulations and observed that Tregs lacking AHR or HIF-1α show decreased levels of the immune checkpoints CTLA-4 and TIGIT, two key proteins for the suppressive functions of Tregs. Ex vivo suppression assays demonstrated an increased proliferation of CD8+ T cells in the presence cKO Tregs (B-C). This result confirmed the decreased suppressive ability of Tregs in absence of the two transcription factors, explaining the observed delay in CLL progression in cKO mice. Image: Summary/Conclusion: Altogether, these results indicate that the TME-regulated transcription factors AHR and HIF-1α in Tregs are crucial for CLL development by promoting escape to anti-tumor immune response, and therefore represent potential therapeutic targets during CLL progression. S139: BCOR DELETION SUSTAINS NOTCH1 SIGNALLING ACTIVATION TO ACCELERATE CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) PROGRESSION TOWARD RICHTER TRANSFORMATION IN MICE C. Rompietti1,*, D. Sorcini1, F. De Falco1, E. Dorillo1, F. M. Adamo1, E. C. Silva Barcelos1, A. Stella1, A. Scialdone1, A. Esposito1, R. Arcaleni1, B. Bigerna2, G. Martino3, L. Moretti2, M. G. Mameli2, C. Geraci1, L. Sandoletti1, A. Cipiciani1, E. Rosati4, B. Falini1, P. Sportoletti1 1Medicine and Surgery, University of Perugia (Center for Hemato-Oncology Research); 2Hospital of Perugia, Perugia; 3Pathology Unit, Azienda Ospedaliera Santa Maria di Terni, University of Perugia, Terni; 4Medicine and Surgery, University of Perugia, Perugia, Italy Background: BCL6 co-repressor (BCOR) is a transcription factor involved in various biological processes including lymphoid development. BCOR disruptive mutations were found in up to 2% of CLL and frequently associated with aberrations of NOTCH1, one of the most common genetic alterations with poor prognosis in CLL and Richter transformation (RT). Recent evidence also indicates that BCOR is involved in NOTCH signalling suppression during embryogenesis. These data indicate the need for further investigation on the role of BCOR mutation and its interplay with NOTCH1 in CLL pathogenesis. Aims: We aim to elucidate the impact of Bcor deficiency in CLL and RT, focusing on the role of active NOTCH1 signalling. Methods: We used a conditional knockout mouse of Bcor (Sportoletti et al, Leukemia 2021) crossed with CD19-Cre mice, to specifically delete Bcor in B-cells, and with the Eμ-TCL1 mouse model of CLL. Mice were characterized for disease phenotype (using flow-cytometry and histological analyses), overall survival and drug response. NOTCH1 activity was assessed by western blot (WB) for the NOTCH1-intracellular domain (NOTCH1-IC) and HES1 expression. Results: B-cell restricted loss of Bcor in Eµ-TCL1 mice significantly expanded leukemic CD5+CD19+ cells in the peripheral blood (PB; p<0.05), spleen (p<0.01), bone marrow (BM; p<0.001), compared to Eµ-TCL1 control mice. No leukemic cells were found in BcorCD19Cre+ mice. Cellular changes resulted in a poorer survival of double mutant mice compared to single-mutant and wild-type (WT) controls (median survival of 334 days vs unreached in BcorCD19Cre+;Eμ-TCL1 vs the other groups, respectively). Adoptive transfer of spleen cells (CD5+CD19+ leukemic burden >50%) resulted in a more rapidly lethal disease in recipient of BcorCD19Cre+;Eμ-TCL1 compared to Eμ-TCL1 cells (median survival of 37.5 vs 74 days, respectively, p<0.05; Figure1A). At necropsy, double mutant mice presented massive splenomegaly, whose histopathological examination revealed a diffuse infiltration by high-mitotic blastoid cells, significantly increased in size, defining a high-grade lymphoid malignancy distinct from the leukemia of Eμ-TCL1 counterparts (Fig.1B). In order to gain mechanistic insight relating to the phenotypic observations, we measured NOTCH1 activity. BcorCD19Cre+;Eμ-TCL1 splenic CD5+CD19+ cells showed significantly increased levels of the active NOTCH1-IC and the up-regulation of its direct target HES1, compared to leukemic cells from Eμ-TCL1 mice and CD19+ cells from non-leukemic BcorCD19Cre+ and WT mice used as control (Fig.1C). In vivo treatment with the NOTCH1 inhibitor Bepridil resulted in a significant growth delay of CD5+CD19+ cells in the PB of mice transplanted with BcorCD19Cre+;Eμ-TCL1 leukemic cells compared to vehicle. At sacrifice, Bepridil caused a significant reduction of spleen dimensions and CD5+CD19+ cellularity, associated with reduced levels of active NOTCH1-IC, c-MYC and HES1 compared to vehicle (28%, 54% and 38% reduction, respectively). NOTCH1 inhibition significantly improved the survival of diseased mice (median survival of 39 vs 33 days in Bepridil vs vehicle, respectively; N=4, p<0.05). Image: Summary/Conclusion: We showed for the first time the tumour suppressor activity of Bcor in a CLL mouse model, ultimately leading to transformation towards a high-grade lymphoma, mimicking human RT. Mechanistically, we implied NOTCH1 signalling activation in Bcor loss mediated tumorigenesis with potential for targeted treatment for high-risk CLL and RT patients. S140: SINGLE-CELL MULTIOMICS ANALYSES REVEAL COMPLEX INTRA-PATIENT HETEROGENEITY IN RELAPSED CLL FOLLOWING VENETOCLAX THERAPY R. Thijssen1 2,*, L. Tian1 2, C. Flensburg1 2, M. A. Anderson1 2 3 4, A. Jarratt1 2, H. Peng1 2, I. Majewski1 2, C. Tam3 4, J. Seymour3 4, P. Blombery3 4, M. Ritchie1 2, D. Huang1 2, A. Roberts1 2 3 4 1The Walter and Eliza Hall Institute of Medical Research; 2Medical Biology, University of Melbourne; 3Clinical Haematology, Royal Melbourne Hospital and Peter MacCallum Cancer Centre; 4Medicine, University of Melbourne, Melbourne, Australia Background: Venetoclax (VEN) is the first approved-in-class BH3-mimetic therapy that inhibits pro-survival BCL2 to induce apoptosis. It is now a standard of care for patients with chronic lymphocytic leukaemia (CLL) and acute myeloid leukaemia. Despite high remission rates, secondary resistance remains problematic with disease progressing on continuous therapy in most patients. While previous studies by ourselves and others demonstrated a BCL2 mutation (Blombery et al., 2019 Cancer Disc) or MCL1 amplification (Guièze et al., 2019 Cancer Cell) confer resistance in patient samples, it is clear that these only account for a fraction of tumour cells at disease progression and are not present in all leukaemias. Given that these changes only provide partial explanations, other mechanisms must operate to subvert the action of VEN. Aims: To uncover why VEN fails in patients by assessing heterogeneity, distinguishing cells with known changes (e.g., BCL2 mutation, MCL1 amplification) and to elucidate what is happening in the cells not harbouring these alterations. Methods: We applied a single-cell (sc) sequencing method that simultaneously measures gene expression and full-length transcripts to determine genotype on samples from patients with progressive CLL with long term follow-up on trials of continuous VEN therapy (Figure 1). Results: By applying scRNA-seq to 161,499 tumour cells, we discovered a high degree of inter- and intra-patient heterogeneity. The majority of CLL cells displayed an altered transcriptional profile at relapse compared to before VEN treatment. 8/13 VEN-relapse samples demonstrated a complex sub-clonal architecture (Figure 1). By applying scLong-read sequencing, we detected BCL2 mutations in 4/13 samples at relapse. We also identified subclones with loss of NOXA or BAX, but overall, alterations in genes for the pro-apoptotic proteins were uncommon. In contrast, altered expression of the pro-survival genes was common and high MCL1 expression was seen in most of the CLL cells in 11/13 relapses. This could only be fully explained by MCL1 amplification in 1 patient and partially in 2 (Figure 1). At VEN resistance, we identified near universal activation of NF-ΚB in circulating tumour cells and this was highly correlated with MCL1 expression. We next demonstrated that the MCL1 locus was transcriptionally targeted by NF-ΚB accounting for the MCL1 increase. NF-ΚB activation and associated high MCL1 was also observed in cells harbouring the BCL2 mutations. Strikingly, NF-ΚB activation (and consequent increased MCL1) dissipated after VEN cessation and commencement of a BTK inhibitor as the leukaemic cells substantially recovered their in vitro sensitivity to VEN. Consistent with the hypothesis that VEN was driving these changes, NF-ΚB activation (and high MCL1) was not detected in a patient (CLL26) with disease relapse 3 years after ceasing VEN in CR. The disease in this patient responded promptly to VEN reinduction. Image: Summary/Conclusion: Taken together, our findings provide a much clearer understanding of resistance to VEN and provide impetus for improved VEN-based clinical management of CLL patients. Given the multiple ways a CLL population in a patient can become resistant to ongoing VEN, it seems unlikely that simply adding other drugs at relapse will be durably effective in most patients. The data pinpoint NF-ΚB activation as a biomarker for in vivo VEN-resistance and provide a specific biological rationale for ceasing VEN in deep response, as is currently being used in time-limited and explored in response-directed regimens. S141: ELICITING ANTI-TUMOR T CELL ACTIVITY IN CHRONIC LYMPHOCYTIC LEUKEMIA WITH BISPECIFIC ANTIBODY-BASED COMBINATION THERAPY D. Papazoglou1,*, L. Ysebaert2, N. Ioannou1, B. Apollonio1, P. Patten3, S. Herter4, M. Bacac4, A. Deutsch5, C. Klein4, A. Vardi6, A. Quillet-Mary2, A. Ramsay1 1Hemato-oncology, King’s College London, London, United Kingdom; 2Centre de Recherches en Cancérologie de Toulouse, Toulouse, France; 3King’s Health Partners, King’s College Hospital, London, United Kingdom; 4Roche Innovation Center Zurich, Roche Pharma Research and Early Development, Zurich, Switzerland; 5Medical University of Graz, Graz, Austria; 6Hematology Department and HCT Unit, G. Papanikolaou Hospital, Thessaloniki, Greece Background: Identifying effective combination immunotherapy could offer hope to chronic lymphocytic leukemia (CLL) patients who relapse on previous lines of therapy. Although prior studies have described an exhausted and pro-tumor T cell state, there may be potential to stimulate anti-CLL cytolytic T cell activity with novel therapy. Aims: Investigate the ability of the CD20-targeted T-cell-engaging bispecific antibody glofitamab (CD20-TCB) to overcome T cell exhaustion and tumor microenvironment (TME)-mediated immunosuppression in CLL. Methods: We pre-clinically investigate CD20-TCB efficacy in CLL utilizing immune and TME model assays that include in vitro functional T cell assays, patient-derived xenografts and lymph node organotypic models. Results: Our previous studies of the CLL lymph node (LN) TMEs suggested that low numbers of infiltrated CD8+ T cells likely contribute to the generation of a “cold” TME that could represent a barrier to effective therapy. Here, we show that CD20-TCB treatment induced the activation, proliferation and migration of previously exhausted patient CD4+ and CD8+T cells and triggered the release of immunostimulatory cytokines related to immune cell recruitment and cytotoxic function. Additionally, we detected enhanced cytolytic (perforin+) immune synapse activity in CD20-TCB-treated cultures which correlated with high levels of T cell-mediated CLL cell death. Importantly, bispecific antibody treatment also induced high levels of anti-CLL T cell activity in both ibrutinib responding and refractory patient samples. Given the emerging evidence that endothelial cells (ECs) that reside within the TME can possess immunomodulatory activity, we next evaluated CD20-TCB-mediated T cell responses in the presence of this stromal cell compartment. We established triple culture assays that allowed patient T and CLL cells to be cultured with CLL-activated ECs (CLL-ECs). These assays revealed that TCB-activated T cells in the presence of CLL-ECs showed a reduced ability to kill target CLL cells compared to healthy ECs. Flow cytometric and multicolor confocal microscopy analysis revealed that CLL-ECs in vitro and in situ expressed increased levels of ICAM-1 and PD-1 ligands that we demonstrated contribute to T cell suppression. In order to overcome this TME-mediated inhibition and enhance anti-CLL CD20-TCB activity, we next investigated combination immunotherapy. Combination of CD20-TCB with a CD19-4-1BBL fusion protein (tumor-targeted co-stimulatory drug) resulted in enhanced CLL cell killing in both T:CLL and triple T:CLL-EC culture assays. Patient-derived xenograft models further confirmed the ability of bispecific antibody combination therapy to induce higher levels of anti-CLL T cell activity compared to monotherapy. Additionally, the pairing of CD20-TCB with CD19-4-1BBL promoted the proliferation and activation of patient T cells within splenic TMEs. Finally, we established 3D organotypic cultures of excess diagnostic LN biopsies that preserve the intact TME cellular composition and spatial organization. LNs of treatment naïve, as well as ibrutinib and venetoclax relapsed CLL patient samples, were treated ex vivo with CD20-TCB combination for subsequent 3D image analysis. Importantly, combination immunotherapy triggered improved anti-tumor T cell killing function and promoted an inflammatory cytokine-rich “hot” TME. Summary/Conclusion: Collectively, this preclinical study supports the concept of triggering cytolytic T cell activity in CLL with bispecific antibody combination immunotherapy that could help overcome T cell exhaustion and the immunosuppressive TME. S142: DECIPHERING THE COMPLEXITY OF T CELLS IN BLOOD AND LYMPH NODES OF PATIENTS WITH CLL BY INTEGRATIVE SINGLE-CELL RNA-SEQ AND MASS CYTOMETRY ANALYSES L. Llaó Cid1, J. Wong1, M. Wierz2, Y. Paul1, S. Gonder2, I. Fernandez Botana2, T. Roider3, S. Dietrich3, D. Colomer4, P. Lichter1, M. Zapatka1, E. Moussay2, J. Paggetti2, M. Seiffert1,* 1Molecular Genetics, B060, German Cancer Research Center, Heidelberg, Germany; 2Department of Oncology, Luxembourg Institute of Health, Luxembourg, Luxembourg; 3Department of Medicine V, University Clinic Heidelberg, Heidelberg, Germany; 4Hematopathology Unit, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Background: Abnormal distribution and impaired function of T cells are key features of chronic lymphocytic leukemia (CLL), a malignancy of mature B lymphocytes that develops in secondary lymphoid tissue and blood. These defects have been linked to failure of immune control and resistance to immunotherapy including immune checkpoint or CAR T-cell therapy. A better characterization of T cells and their loss of function in CLL will help to improve such treatment approaches. Aims: The goal of this study was an in-depth characterization of the T-cell compartment in blood and tissue samples of patients and mouse models with CLL to gain insights into the spectrum of phenotypes and transcriptional programs of T cells and the underlying mechanisms of their development. Methods: We performed mass cytometry (CyTOF) with a panel of 35 antibodies to characterize T cells in blood (n=8), bone marrow (n=3), and lymph nodes (n=21) of CLL patients, as well as reactive lymph nodes of non-tumor patients (n=13). We further generated single-cell transcriptome and T-cell receptor sequencing data of T cells from lymph nodes of CLL patients (n=5) and spleen samples of the Eµ-TCL1 mouse model (n=3), and performed integrative analyses of all data sets. Results: CyTOF analysis allowed for the identification and quantification of 15 clusters of CD4+ and 14 clusters of CD8+ T cells in blood, lymph node and bone marrow samples. T cells in blood and bone marrow were clearly distinct from lymph node derived cells, and several cell subsets showed a positively correlated abundance. A comparative analysis revealed an accumulation of several regulatory T-cell subsets, as well as T cells harboring an exhausted and dysfunctional phenotype in CLL lymph nodes. Single-cell transcriptome and T-cell receptor sequencing demonstrated the presence of clonally expanded and gradually exhausted T-cell clusters in human and murine CLL, and provided insights into the transcriptional programs and regulatory networks of these cell subtypes. Using CellPhoneDB (Efremova et al., Nature Protocols, 2020) and CellChat (Jin et al., Nature Communications, 2021) repositories, we identified novel ligand-receptor-interactions between CLL and T-cell clusters with impact on anti-tumor immune control which are currently tested in preclinical models for intervention therapy. Summary/Conclusion: Altogether, our study provides a detailed characterization of the T-cell compartment in CLL that helps us to understand T-cell exhaustion and suggests novel targets to improve immunotherapy for patients with CLL and likely also other malignancies. MZ, EM, JP and MS share senior authorship. S143: TRANSCRIPTOMIC CHARACTERIZATION OF MRD RESPONSE AND NON-RESPONSE IN PATIENTS TREATED WITH FIXED-DURATION VENETOCLAX-OBINUTUZUMAB O. Al-Sawaf1 2 3,*, H. Y. Jin4, C. Zhang1, Y. Choi4, S. Balasubramanian4, S. Robrecht1, A. Kotak5, N. Chang5, A.-M. Fink1, E. Tausch6, C. Schneider6, M. Ritgen7, K.-A. Kreuzer1, B. Chyla8, J. Paulson4, B. Eichhorst1, S. Stilgenbauer6, Y. Jiang4, M. Hallek1, K. Fischer1 1Department I of Internal Medicine and Center of Integrated Oncology Aachen Cologne Bonn Duesseldorf, University Hospital of Cologne, Cologne, Germany; 2Cancer Institute, University College London; 3Francis Crick Institute, London, United Kingdom; 4Genentech Inc., South San Francisco, CA, United States of America; 5Roche Products Ltd, Welwyn Garden City, United Kingdom; 6Department III of Internal Medicine, Ulm University, Ulm; 7Department II of Internal Medicine, University of Schleswig Holstein, Kiel, Germany; 8AbbVie Inc., North Chicago, IL, United States of America Background: Minimal residual disease (MRD) is a key surrogate for the depth of remission of CLL. The highest rates of undetectable MRD have been observed with regimens using the BH3 mimetic venetoclax. In CLL14, most patients (pts) receiving venetoclax-obinutuzumab (Ven-Obi) had undetectable MRD (uMRD <10-4) at the end of treatment (EoT). However, a subgroup of pts remained MRD positive, regardless of TP53 aberrations or unmutated IGHV status. The biological drivers of response or non-response have so far not been elucidated. Aims: This study explores whether differential transcriptomic profiles can discriminate between pts with vs without deep MRD remissions, particularly after Ven exposure. This might eventually allow for biology-informed treatment development for pts in this high-risk disease setting. Methods: NGS-based MRD measurements (clonoSEQ) from peripheral blood (PB) at follow-up month 3 were grouped into uMRD <10-6 (MRD6), 10-6 ≤ and <10-4 (MRD4/5), and detectable MRD ≥10-4 (MRD+). Pre-treatment CD19-enriched PB samples, and samples collected at relapse from a subset of pts, were subjected to bulk RNAseq on the Illumina NovaSeq platform. Transcriptomic clustering, linear modeling, differential expression (DE), and gene set enrichment analyses were run using the R packages Seurat, DESeq2, fGSEA, and GSVA. Top DE genes were selected based on P-values <0.05 and log2-fold-change >0.5, comparing MRD+ and MRD6 populations. Results: Within the intention-to-treat (ITT) population after a median observation time of 65.4 months, pts with MRD6 had a significantly longer PFS than pts who had MRD4/MRD5 (A). Pre-treatment RNAseq data were available for 405 of the 432 pts in the ITT population (202 Clb-Obi, 203 Ven-Obi), and at relapse for 41 pts (14 Clb-Obi and 27 Ven-Obi). The pre-treatment transcriptomic profile correlated with the IGHV status, trisomy 12, and del13q (B). No clustering according to MRD status was observed, suggesting that individual pathways/genes rather than global differences are associated with MRD response. Individual gene-level analysis of pre-treatment cohorts showed that MRD+ status was associated with resistance markers such as multi-drug response (MDR) gene or ABCB1, but MRD6 was associated with pro-apoptotic BCL2L11 (BIM) (C). Hallmark apoptotic pathways (P53 and Apoptosis), as well as canonical oncogenic pathways (MYC, mTORC1, TNFɑ/NFκB), were enriched in MRD6 pts in both Ven-Obi and Clb-Obi-treated pts (D). In contrast, inflammatory pathway gene sets (Inflammatory response, IFNγ response, IL2/STAT5) were enriched in MRD+ pts in the Ven-Obi arm, suggesting their role in Ven resistance. Gene sets enriched at progression compared to baseline were mostly related to cell proliferation and oncogenic signaling (E). Moreover, in a GSVA analysis, inflammatory pathway gene sets were also markedly upregulated at progression compared to baseline, providing an orthogonal validation that inflammatory signaling might be associated with resistance to venetoclax therapy (Fig F). Common leading edge genes included IL2RB, LCP2, BST2, IRF7, CASP3, CD86, IRF8, and NCOA3. Image: Summary/Conclusion: This analysis of a large, prospective CLL RNAseq dataset confirms that global transcriptomic profiles cluster according to IGHV, trisomy 12 and del13q. Response and non-response to therapy, as assessed by NGS-based MRD status, was associated with a distinct transcriptomic profile that was characterized by upregulated oncogenic pathways and inflammatory signaling, respectively. These data suggest possible biological vulnerabilities that could be leveraged to overcome resistance to venetoclax. S144: IMMUNE RESTORATION AND SYNERGISTIC ACTIVITY WITH FIRST-LINE (1L) IBRUTINIB (IBR) PLUS VENETOCLAX (VEN): TRANSLATIONAL ANALYSES OF CAPTIVATE PATIENTS WITH CLL I. Solman1,*, R. Singh Mali2, L. Scarfo3, M. Choi4, C. Moreno5 6, A. Grigg7, J. P. Dean1, E. Szafer-Glusman1 1Pharmacyclics LLC, an AbbVie Company, South San Francisco, CA; 2AbbVie, North Chicago, IL, United States of America; 3Ospedale San Raffaele, Milan, Italy; 4University of California San Diego, San Diego, CA, United States of America; 5Hospital de la Santa Creu I Sant Pau, Autonomous University of Barcelona; 6Josep Carreras Leukaemia Research Institute, Barcelona, Spain; 7Austin Hospital, Heidelberg, VIC, Australia Background: Ibr and Ven work synergistically through their distinct and complementary modes of action: BTK inhibition by Ibr decreases levels of anti-apoptotic MCL-1 and BCL-XL proteins, but not BCL-2, and therefore increases sensitization to BCL-2 inhibition by Ven in vivo. Single agent Ibr has been shown to normalize CLL-associated immune cell alterations in number and function but the impact of the Ibr plus Ven combination (I+V) on immune cells has not been evaluated. Aims: To 1) confirm BCL-2 sensitization by single-agent Ibr and 2) monitor changes in the cellular immune profile of patients (pts) treated with I+V in CAPTIVATE (NCT02910583), a multicenter phase 2 study evaluating 1L I+V treatment (tx) in CLL and SLL. Methods: Ibr (420mg po daily) effects on anti-apoptotic proteins were evaluated by flow cytometry with samples from 4 previously untreated pts with CLL treated for 1 cycle (28 days). Immune restoration was evaluated in 79 previously untreated pts with CLL enrolled in the CAPTIVATE Minimal Residual Disease (MRD) cohort. Pts received 3 cycles of Ibr, followed by 12 cycles of I+V (Ibr 420 mg/d orally; Ven ramp-up to 400 mg/d orally). Pts who met criteria for confirmed undetectable MRD (Confirmed uMRD) were then randomized 1:1 to placebo fixed-duration (FD) tx or Ibr at cycle 16; pts who did not meet Confirmed uMRD criteria (uMRD Not Confirmed) were randomized 1:1 to Ibr or I+V. Cryopreserved peripheral blood mononuclear cells at baseline (pre-dose cycle 1) and day 1 of cycles 4, 7, 16, 20, 23, and 29 in each of the 4 arms were analyzed by high-dimensional flow cytometry (34-color panel). Immune cell subset counts of B cells, T cells, monocytes, dendritic cells (DCs), myeloid-derived suppressor cells, natural killer cells, and innate lymphoid cells were calculated and compared to those of 20 age-matched healthy donors. Median changes from baseline are reported. Results: In pts treated with single-agent Ibr for 1 cycle, MCL-1, BCL-XL and BCL-2 expression decreased by 74%, 95% and 10%, respectively, in lymph node emigrant (CD5+ CXCR4dim) CLL cells, confirming increased BCL-2-dependence and corresponding inhibitor sensitization. In CAPTIVATE pts treated with I+V, a rapid and significant decrease in circulating CLL cells was observed within the first 3 cycles of Ven tx initiation (Fig 1A, cycle 7). From cycle 16 and onwards, Confirmed uMRD pts had CLL cell counts similar to healthy donors (≤0.8 CLL cell/mL), while in uMRD Not Confirmed pts counts were 1.5 to 22.9 CLL cell/mL. Subsequently, normal B-cell count restoration occurred in Confirmed uMRD pts randomized to placebo (FD tx) with a recovery to levels similar to healthy donors (+332% at cycle 29; Fig 1B). Across all arms, abnormal counts of T cell subsets, classical monocytes and conventional DCs characteristic of CLL were normalized to healthy donor levels (‒49%, +101% and +91% respectively) within the first 6 months of tx and were maintained thereafter regardless of randomized tx. Plasmacytoid DCs counts recovered by cycle 20 (+598%) in all 4 arms. Image: Summary/Conclusion: Sensitization to BCL-2 inhibitor effects by Ibr supports the synergistic activity of I+V. The FD regimen (Confirmed uMRD, placebo arm) of I+V effectively eradicated CLL cells to healthy donor levels and enabled sustained regeneration of normal B cell counts in contrast to ongoing therapy in the remaining tx arms. Combination I+V also allowed for normalization of other critical immune cells, including T cell subsets, classical monocytes, and conventional and plasmacytoid DCs. These data demonstrate promising evidence of immune restoration with FD I+V tx. S145: THE COMBINATION OF IBRUTINIB PLUS VENETOCLAX RESULTS IN A HIGH RATE OF MRD NEGATIVITY IN PREVIOUSLY UNTREATED CLL: THE RESULTS OF THE PLANNED INTERIM ANALYSIS OF THE PHASE III NCRI FLAIR TRIAL P. Hillmen1,*, A. Pitchford2, A. Bloor3, A. Pettitt4, P. Patten5, F. Forconi6, A. Schuh7, C. Fox8, N. Elmusharaf9, S. Gatto10, B. Kennedy11, J. Gribben12, N. Pemberton13, O. Sheehy14, G. Preston15, D. Howard16, A. Hockaday2, D. Cairns2, S. Jackson2, N. Greatorex2, N. Webster17, S. Dalal17, J. Shingles17, K. Cwynarski18, S. Paneesha19, D. Allsup20, A. Rawstron17, T. Munir21 1St James’s University Hospital; 2Leeds Cancer Research UK Clinical Trials Unit, Leeds Institute of Clinical Trials Research, Leeds; 3University of Manchester, Manchester; 4Clatterbridge Cancer Centre NHS Foundation Trust, Liverpool; 5King’s College Hospital, London; 6University of Southampton, Southampton; 7Oxford University Hospital NHT Trust, Oxford; 8Nottingham University Hospitals NHS Trust, Nottingham; 9Wales Teaching Hospital; 10University Hospital of Wales, Cardiff; 11Leicester Royal Infirmary, Leicester; 12St Bartholomew’s Hospital, London; 13Worcestershire Acute Hospitals NHS Trust, Worcester; 14Belfast City Hospital, Belfast; 15Aberdeen Royal Infirmary, Aberdeen; 16Roche, Reading; 17HMDS, Leeds Cancer Centre, Leeds; 18University College London, London; 19Birmingham Heartlands Hospital, Birmingham; 20Castle Hill Hospital, Hull; 21UK NCRI CLL sub-group, Leeds, United Kingdom Background: Ibrutinib (I) and venetoclax (V) improve outcome in CLL. I rarely eradicates measurable residual disease (MRD), whereas V (alone or with anti-CD20) can eradicate MRD permitting time limited therapy. Small studies suggest synergy between I and V, as I+V results in MRD negativity in many patients (pts). Aims: The primary aim was to compare the MRD eradication rate between I and I+V. Key secondary aims were IWCLL overall (ORR), complete response (CR) and safety. Methods: FLAIR (ISRCTN01844152), a phase III, randomised, controlled trial for previously untreated CLL requiring therapy by IWCLL criteria. Pts >75 yrs or with >20% 17p del were excluded. FLAIR was adapted in July 2017 to add two arms, I monotherapy and I+V. I was given at 420mg/day. For I+V, V was added after two months of I with dose escalation to 400mg/day over 5 weeks. The duration of therapy (DOT) was defined by MRD with treatment for up to 6 years. MRD was assessed centrally by flow cytometry, MRD negativity was defined as <1 CLL cell in 10,000 leucocytes (IWCLL criteria), was assessed in peripheral blood (PB) and bone marrow (BM) at 9 months post-randomisation, in PB at 12 months and then 6 monthly. When PB was MRD negative, this was repeated after 3 months and then in both PB & BM 3 months later. If PB & BM were negative the time to MRD negativity was calculated (treatment start to first MRD negative PB) and DOT was twice this. The earliest therapy could stop was 2 years post-randomisation. A formal interim analysis was performed when 50% pts in I and I+V arms had reached 2 yrs post-randomisation and a p-value of <0.005 was statistically significant. Results: 523 pts were randomised to I or I+V. We report the interim analysis in the first 274 pts (I [n=138] and I+V [n=136]) reaching 2 yrs post-randomisation from 83 UK Centres from 13/07/17 to 15/03/19. 72.3% male, median age 63 yrs (34.3% >65yo) and 40.9% Binet C. IGHV were available for 256 (93.4%) pts - 48.2% IGHV unmutated (≥98% homology to germline), 45.3% IGHV mutated and 9.1% Subset 2. Hierarchical FISH testing revealed 16.1% 11q del, 19% trisomy 12, 21.9% normal and 36.9% 13q del with 6.2% failed. The arms were well-balanced for all variables. For I+V arm, MRD negativity was achieved within 24 months in BM in 89/136 (65.4%) and PB in 97/136 (71.3%) compared to no pts for I (p<0.0001). MRD negativity for I+V in BM within 24 months was 51/64 (79.7%) for IGHV unmutated and 31/55 (56.4%) for IGHV mutated. At 9 months post-randomisation 49/136 (36%) I+V pts were MRD negative in BM and 56/136 (41.2%) negative in PB compared to 0/138 with I (p<0.0001). ORR at 9 months in 120/136 (88.2%) I+V pts and 119/138 (86.2%) I pts (p=0.6157). At 9 months CR in 81/136 (59.6%) for I+V and 11/138 (8%) for I (p<0.0001). For I+V CR at any time was 93.4%. At 24 months, 54/136 (39.7%) stopped I+V due to meeting MRD stopping criteria. SAEs were reported in 41.5% I+V and 38.2% I pts. Infectious SAEs 14.8% vs 19.9% and cardiac SAEs 11.9% vs 8.1% of pts for I+V & I respectively. Laboratory TLS was reported in 6/136 (4.4%) of I+V pts and none with I. There were no cases of clinical TLS. Most frequent any grade AEs within 12 months of randomisation differing between I+V & I were diarrhoea (52.6% I+V pts, 29.4% I), anaemia (28.9% vs 16.9%), leucopenia (36.3% vs 8.8%), thrombocytopenia (23.7% vs 14%). Leucopenia was the only grade ≥3 SAE reported in >10% of pts (27.4% I+V and 5.1% I). Image: Summary/Conclusion: Ibrutinib plus venetoclax is an effective and well tolerated combination resulting in a high rate of MRD negativity in blood (71.9%) and marrow (65.4%) in the first 2 years of treatment. S146: VENETOCLAX IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA WITH 17P DELETION: 6-YEAR FOLLOW-UP AND GENOMIC ANALYSES IN A PIVOTAL PHASE 2 TRIAL S. Stilgenbauer1,*, E. Tausch1, A. W. Roberts2, M. S. Davids3, B. Eichhorst4, M. Hallek4, P. Hillmen5, C. Schneider1, S. Böttcher6, R. Popovic7, M. T. Ghanim7, M. Moran7, W. J. Sinai7, X. Wang7, N. Mukherjee7, B. Chyla7, W. G. Wierda8, J. F. Seymour2 1Division of CLL, Internal Medicine III, Ulm University, Ulm, Germany; 2Peter MacCallum Cancer Centre, Royal Melbourne Hospital, and University of Melbourne, Melbourne, Australia; 3Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, United States of America; 4Department of Internal Medicine, Center of Integrated Oncology Köln Bonn, University Hospital of Cologne, Cologne, Germany; 5Leeds Teaching Hospitals, NHS Trust, Leeds, United Kingdom; 6Division of Internal Medicine, Medical Clinic III-Hematology, Oncology and Palliative Medicine, Rostock University Medical Center, Rostock, Germany; 7AbbVie Inc, North Chicago, IL; 8The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America Background: Patients (pts) with chronic lymphocytic leukemia (CLL) with del(17p) and/or mutated TP53 have adverse prognosis and are a population of interest for improving outcomes. In a pivotal Phase 2 study (NCT01889186) evaluating venetoclax (Ven; a selective oral BCL2 inhibitor) monotherapy in del(17p) CLL (N=158), overall response rate (ORR) was 77% (median time on study, 26.6 mo; Stilgenbauer. J Clin Oncol. 2018;17:1973). Aims: To report final analyses after 5 y from last pt enrolled, including post-hoc subgroup analyses of prognostic markers. Methods: Adults with R/R or previously untreated del(17p) CLL received Ven 400 mg (via ramp-up) orally daily until progressive disease (PD) or intolerance. Main cohort comprised pts with R/R CLL; safety expansion cohort included pts with R/R or untreated CLL. Primary endpoints were ORR (main) and safety (safety expansion). Peripheral blood (PB) and bone marrow (BM) were analyzed pre-Ven for somatic mutations via targeted next-generation sequencing (NGS). Minimal residual disease (MRD) was assessed by flow cytometry, ASO-PCR, and/or clonoSEQ NGS; undetectable MRD (uMRD) is reported at <10−4. Results: In all, 158 pts received Ven (main, n=107; safety expansion, n=51). Data cutoff was 15 December 2020. Pts had median 2 (range, 0−10) prior lines of therapy (LOT); 5 had untreated CLL (first-line [1L]). Common mutations (n/N) were TP53 (113/138 [82%]; 35/113 [31%] had >1), SF3B1 (28/137 [20%]) NOTCH1 (22/137 [16%]), ATM (11/120 [9%]), and BIRC3 (6/120 [5%]); 93/115 (81%) had unmutated IGHV and 118/158 (75%) had ≥20% del(17p). Median time on Ven was 27.4 (range, 0–79.3) mo. At study close, 77 pts were alive; 26 remained on Ven. No new safety signals were observed. With 70 mo median follow-up (f/u), investigator-assessed ORR was 77% (95% CI, 70−84); 21% achieved complete remission (CR)/CR with incomplete blood count recovery (CRi). Median duration of response was 39.3 mo (95% CI, 31.1−50.5); 28% had ongoing response at 60 mo. Of 61 evaluable pts with a PB MRD assessment, 25% had uMRD at ~2 y (24−30 mo). Median progression-free survival (mPFS) was 28.2 mo (95% CI, 23.4−37.6; Figure). Median overall survival (mOS) was 62.5 mo (95% CI, 51.7−NR; 5-y PFS and OS rates, 24% and 52%). Of 5 1L pts, 4 were alive and progression-free. In pts with CR/CRi (n=37), mPFS was 62.2 mo (95% CI, 53.1−NR); in pts with partial remission (PR)/nodular PR (n=85), mPFS was 27.6 mo (95% CI, 22.8−38.0). Overall, 98/158 (62%) pts had PD, including 24/158 (15%) with Richter transformation. Of pts with PD (n=98) or whose disease was refractory to Ven (n=11), 73 received another LOT, most commonly ibrutinib (n=41); mOS from ibrutinib initiation was 28.0 mo. In pts with both del(17p) and TP53 mutation (n=111) vs those with either (n=19), ORR was 75% vs 79%; mPFS was 27.4 vs 22.8 mo (P=.8). In pts with mutated (n=22) vs unmutated IGHV (n=93), mPFS was 40.4 vs 26.9 mo (P=.11). No significant difference in ORR, PFS, or OS was observed in pts with vs without ≥20% del(17p), >1 TP53 mutation, NOTCH1, ATM, or BIRC3 mutations; mPFS was shorter in pts with mutated SF3B1 (n=28) vs without (n=109; 16.4 vs 30.2 mo [P=.0071]). Multivariate analysis is ongoing. Image: Summary/Conclusion: At end of study (median f/u, 70 mo), 48% of pts were alive, 24% were progression-free, and 16% remained on Ven, confirming the long-term activity of Ven in this high-risk population with del(17p) CLL and median 2 prior LOT. Except SF3B1 mutation, other adverse features (eg, >1 TP53 mutation, NOTCH1 mutations, unmutated IGHV) did not influence outcomes with Ven treatment in this cohort. S147: PIRTOBRUTINIB, A HIGHLY SELECTIVE, NON-COVALENT (REVERSIBLE) BTK INHIBITOR IN PREVIOUSLY TREATED CLL/SLL: UPDATED RESULTS FROM THE PHASE 1/2 BRUIN STUDY A. R. Mato1,*, J. M. Pagel2, C. C. Coombs3, N. N. Shah4, N. Lamanna5, T. Munir6, E. Lech-Maranda7, T. A. Eyre8, J. A. Woyach9, W. G. Wierda10, C. Y. Cheah11, J. B. Cohen12, L. E. Roeker1, M. R. Patel13, B. Fakhri14, M. A. Barve15, C. Tam16, D. Lewis17, J. N. Gerson18, A. J. Alencar19, C. Ujjani20, I. Flinn21, S. Sundaram22, S. Ma23, D. Jagadeesh24, J. Rhodes25, J. Taylor19, O. Abdel-Wahab1, P. Ghia26, S. J. Schuster18, D. Wang2, B. Nair2, E. Zhu2, D. E. Tsai2, M. S. Davids27, J. R. Brown27, W. Jurczak28 1Memorial Sloan Kettering Cancer Center, New York; 2Loxo Oncology at Lilly, Stamford; 3University of North Carolina at Chapel Hill, Chapel Hill; 4Medical College of Wisconsin, Milwaukee; 5Herbert Irving Comprehensive Cancer Center, Columbia University, New York, United States of America; 6Haematology, St. James’s University Hospital, Leeds, United Kingdom; 7Institute of Hematology and Transfusion Medicine, Warsaw, Poland; 8Oxford University Hospitals NHS Foundation Trust, Churchill Cancer Center, Oxford, United Kingdom; 9The Ohio State University Comprehensive Cancer Center, Columbus; 10MD Anderson Cancer Center, Houston, United States of America; 11Linear Clinical Research and Sir Charles Gairdner Hospital, Perth, Australia; 12Winship Cancer Institute, Emory University, Atlanta; 13Florida Cancer Specialists/Sarah Cannon Research Institute, Sarasota; 14University of California San Francisco, San Francisco; 15Mary Crowley Cancer Research, Dallas, United States of America; 16Peter MacCallum Cancer Center, Royal Melbourne Hospital, and University of Melbourne, Melbourne, Australia; 17Plymouth Hospitals NHS Trust - Derriford Hospital, Plymouth, United Kingdom; 18Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia; 19University of Miami Miller School of Medicine, Miami; 20Fred Hutchinson Cancer Research Center, Seattle; 21Sarah Cannon Research Institute, Nashville; 22Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York; 23Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago; 24Cleveland Clinic, Cleveland; 25Northwell Health Cancer Institute, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell Health, New Hyde Park, United States of America; 26Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy; 27Dana-Farber Cancer Institute and Harvard Medical School, Boston, United States of America; 28Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland Background: Covalent BTK inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but many patients (pts) will require additional treatment. Pirtobrutinib is a highly selective, non-covalent (reversible) BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency. Aims: BRUIN is a phase 1/2 multicenter study (NCT03740529) of oral pirtobrutinib monotherapy in pts with advanced B-cell malignancies who have received ≥2 prior therapies. Methods: Pirtobrutinib was dose escalated in a standard 3 + 3 design in 28-day cycles. The primary objective for phase 1 was to determine the recommended phase 2 dose (RP2D) and the primary objective of phase 2 was overall response rate (ORR); secondary objectives included duration of response, progression-free survival, overall survival, safety and tolerability and pharmacokinetics. Response was assessed every 8 weeks from cycle 3, and every 12 weeks from cycle 13 and was measured according to the iwCLL 2018 criteria, including PR with lymphocytosis (PR-L). Safety was assessed in all pts. Results: As of 27 September 2020, 323 pts with B-cell malignancies (170 CLL/SLL, 61 MCL, 26 WM, 26 DLBCL, 13 MZL, 12 FL, 9 RT, and 6 other) were treated on 7 dose levels (25-300mg QD). Among the 170 pts with CLL/SLL, the median age was 69 (36-88) years. Median number of prior lines of therapies was 3 (1-11). Majority of the CLL/SLL pts had received a prior BTKi (86%), an anti-CD20 antibody (90%), or a chemotherapy (82%). High risk molecular features such as 17p deletion, TP53 mutation, and unmutated IGHV were present in 25% (20/81), 30% (27/91), and 88% (71/81) of pts, respectively. No dose-limiting toxicities (DLTs) were reported and maximum tolerated dose (MTD) was not reached (n=323). 200mg QD was selected as the RP2D. Fatigue (20%), diarrhea (17%) and contusion (13%) were the most frequent treatment-emergent adverse events (TEAEs) regardless of attribution or grade seen in ≥10% of pts (n=323). The most common adverse event of grade ≥3 was neutropenia (10%). Treatment-related hemorrhage and hypertension occurred in 5 (2%) and 4 (1%) pts, respectively. 1% pts discontinued due to TEAEs. 139 CLL/SLL pts were efficacy-evaluable with a median follow up time of 6 months (0.16-17.8+). The ORR was 63% (95% CI 55-71) among the 139 efficacy evaluable pts with 69 PRs (50%), 19 PR-Ls (14%), 45 SDs (32%), and 1 PD (1%), and 5 (4%) discontinued prior to first response assessment. Among the 121 BTKi pretreated pts, the ORR was 62% (95% CI 53-71). Responses deepened over time with an ORR of 86% among the pts with at least 10 months follow-up. ORR was similar in pts who discontinued prior BTKi due to progression (67%), or adverse events or other reasons (52%). Of the 88 responding pts, all except 5 remained on therapy (4 progressed and 1 achieved a PR and electively discontinued treatment to undergo transplant). The longest-followed responding patient had been on treatment for 17.8+ months. Summary/Conclusion: Pirtobrutinib demonstrated promising efficacy in heavily pretreated CLL/SLL pts following multiple prior lines of therapy and in pts with BTK C481 mutations. Pirtobrutinib was well tolerated and exhibited a wide therapeutic index. Updated data, including approximately 100 new pts with CLL and an additional 10 months since the prior data cut will be presented. S148: VENETOCLAX-OBINUTUZUMAB FOR PREVIOUSLY UNTREATED CHRONIC LYMPHOCYTIC LEUKEMIA: 5-YEAR RESULTS OF THE RANDOMIZED CLL14 STUDY O. Al-Sawaf1 2 3,*, C. Zhang1, S. Robrecht1, A. Kotak4, N. Chang4, A.-M. Fink1, E. Tausch5, C. Schneider5, M. Ritgen6, K.-A. Kreuzer1, B. Chyla7, B. Eichhorst1, Y. Jiang8, S. Stilgenbauer5, M. Hallek1, K. Fischer1 1Department I of Internal Medicine and Center of Integrated Oncology Aachen Cologne Bonn Duesseldorf, University Hospital of Cologne, Cologne, Germany; 2Cancer Institute, University College London; 3Francis Crick Institute, London; 4Roche Products Ltd, Welwyn Garden City, United Kingdom; 5Department III of Internal Medicine, Ulm University, Ulm; 6Department II of Internal Medicine, University of Schleswig Holstein, Kiel, Germany; 7AbbVie Inc., North Chicago, IL; 8Genentech Inc., South San Francisco, CA, United States of America Background: One-year fixed-duration venetoclax-obinutuzumab (Ven-Obi) has demonstrated significant improvement of progression-free survival (PFS) as compared to chlorambucil-obinutuzumab (Clb-Obi) in patients with previously untreated chronic lymphocytic leukemia (CLL) and coexisting conditions in the CLL14 trial. As high rates of undetectable minimal residual disease (uMRD) suggested deep remissions, long-term efficacy data including patients with high-risk disease is of particular interest. Aims: The aim of this report is to provide updated efficacy and safety data from the ongoing follow-up of the CLL14 study with all patients being off study treatment for ≥ 4 years. Methods: Patients with previously untreated CLL and coexisting conditions were randomized 1:1 to 12 cycles of venetoclax with 6 cycles of obinutuzumab or 12 cycles of chlorambucil with 6 cycles of obinutuzumab. The primary endpoint was investigator-assessed PFS. Secondary endpoints included safety, rates of MRD response (measured every 3-6 months up to 9 years after last patient enrolment), time to next treatment (TTNT) and overall survival. Follow-up is ongoing, all patients are off study treatment. Results: Of the 432 enrolled patients, 216 were randomly assigned to receive Ven-Obi and 216 to receive Clb-Obi. With a current median follow-up of 65.4 months (interquartile range 52.6-69.4), PFS remained significantly superior for Ven-Obi compared to Clb-Obi (median not reached [nr] vs 36.4 months; hazard ratio [HR] 0.35 [95% CI 0.26-0.46], p<0.0001). At 5 years after randomization, the estimated PFS rate was 62.6% after Ven-Obi and 27.0% after Clb-Obi. Overall, 52 cases of progressive disease (PD) with 28 required second-line treatments occurred in the Ven-Obi arm and 132 with 86 second-line treatments in the Clb-Obi arm. TTNT was significantly longer after Ven-Obi (5-year TTNT 72.1% vs 42.8%; HR 0.42, 95% CI 0.31-0.57, p<0.0001). In both arms, the majority of next-line therapies were BTK inhibitors (54.3% in the Ven-Obi arm, 47.1% in the Clb-Obi arm). The PFS and TTNT difference was maintained across all risk groups, including patients with TP53 mutation/deletion (5-year PFS 40.6% vs 15.6%; 5-year TTNT 48.0% vs 20.8%) and unmutated IGHV status (5-year PFS 55.8% vs 12.5%; 5-year TTNT 66.2% vs 25.1%). A multivariable analysis indicated 17p deletion and high disease burden as independent prognostic factors for PFS in patients treated with Ven-Obi. Four years after treatment completion, 39 (18.1% of the intention-to-treat population) patients in the Ven-Obi arm still had uMRD (<10-4 by NGS in peripheral blood), 27 (12.5%) had low (L)-MRD (≥ 10-4 and < 10-2) and 41 (19.0%) high (H)-MRD (≥ 10-2), compared to 4 (1.9%) uMRD, 13 (6.0%) L-MRD and 24 (11.1%) H-MRD in the Clb-Obi arm. Overall, 40 deaths were reported in the Ven-Obi arm (8 PD related) and 57 in the Clb-Obi arm (23 PD related); at 5 years after randomization the estimated OS rate was 81.9% in the Ven-Obi arm and 77.0% in the Clb-Obi arm (HR 0.72 [0.48-1.09], p=0.12). Second primary malignancies were reported in 44 (20.8%) patients in the Ven-Obi arm and 32 (15.0%) in the Clb-Obi arm. No new safety signals were observed. Image: Summary/Conclusion: These data confirm that over 60% of patients who had received 1-year fixed-duration Ven-Obi have remained in remission four years after end of therapy. The majority of patients treated with Ven-Obi still have not required a second line of CLL therapy. Hence, the 1-year Ven-Obi regimen continues to be an effective fixed-duration option for patients with CLL and coexisting conditions, also in the context of high-risk disease. S149: LONG TERM OUTCOMES OF IFCG REGIMEN FOR FIRSTLINE TREATMENT OF PATIENTS WITH CLL WITH MUTATED IGHV AND WITHOUT DEL(17P)/TP53 MUTATION N. Jain1,*, P. Thompson1, J. Burger1, A. Ferrajoli1, K. Takahashi1, Z. Estrov1, G. Borthakur1, P. Bose1, T. Kadia1, N. Pemmaraju1, K. Sasaki1, M. Konopleva1, E. Jabbour1, N. Garg2, X. Wang3, R. Kanagal-Shamanna4, K. Patel4, W. Wang4, S. Wang4, J. Jorgensen4, W. Lopez1, A. Ayala1, W. Plunkett5, V. Gandhi5, H. Kantarjian1, S. O’Brien6, M. Keating1, W. Wierda1 1Leukemia; 2Radiology; 3Biostatistics; 4Hematopathology; 5Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston; 6Chao Family Comprehensive Cancer Center, University of California Irvine Medical Center, Orange, United States of America Background: Chemoimmunotherapy with FCR has been an effective treatment for patients (pts) with CLL. Pts with mutated IGHV (IGHV-M) have favorable long-term outcomes after receiving FCR. We designed an investigator-initiated, phase 2 trial with ibrutinib, fludarabine, cyclophosphamide, and obinutuzumab (iFCG) for previously untreated pts with IGHV-M CLL (NCT02629809). We report here long-term outcomes with a median follow-up of 56.8 months. Aims: Investigate the role of combined chemoimmunotherapy and targeted therapy in CLL. Methods: Eligibility included age ≥18, IGHV-M, no del(17p)/TP53 mutation. Pts received 3 courses of iFCG. Pts achieving CR/CRi with undetectable MRD (U-MRD) in bone marrow (4-color flow-cytometry, sensitivity 10-4) after 3 courses of iFCG received ibrutinib with obinutuzumab (iG) for 3 cycles, followed by ibrutinib monotherapy for 6 months. All other pts received iG for 9 cycles (C4-12). Pts with marrow U-MRD at end of Cycle 12 stop all therapy. Response assessment was per 2008 iwCLL criteria with BM and CT scans every 3 months during the first year. After completion of all therapy, pts were followed by exam, blood counts and peripheral blood MRD every 6 months. NGS MRD (sensitivity 10-6) was performed in bone marrow in pts with available samples. Results: 45 pts initiated treatment. Median age was 60 [range, 25-71]. 69% had del(13q). After three cycles of iFCG, 39/45 (87%) pts achieved marrow U-MRD. Responses improved with continued therapy with 40/45 (89%) and 41/45 (91%) achieving marrow U-MRD after Cycles 6 and 12, respectively. Overall, 44/45 (98%) pts achieved marrow U-MRD as best response at any time during the study. The 5-year PFS and OS are 97.7% (95% CI 94–100%) and 97.8% (95% CI 94–100%), respectively. No pt had CLL progression or Richter transformation. One pt developed therapy-related MDS; this pt is being monitored for 38+ months without any therapy for MDS with normal blood counts. The sole event noted on both the PFS and OS curve is a pt death from heart failure. 41/45 pts completed 12 cycles of treatment (4 pts came off study prior to C12). All 41 pts achieved marrow U-MRD4 by flow-cytometry and per protocol, all 41 pts discontinued ibrutinib. After a median follow-up of 44.2 mos post-discontinuing ibrutinib, 6 pts had an MRD recurrence (defined as 2 consecutive values of ≥0.01% in peripheral blood by flow cytometry) at a median of 27.2 mos (range, 20.7-49.0 mos) after stopping all therapy. All 6 pts are being monitored with no clinical progression or active therapy; notably, all 6 pts were MRD+ at 10-6 by marrow NGS after the completion of 3 cycles of iFCG and 4/5 (1 missed sample) were MRD+ at 10-6 by marrow NGS after the completion of Cycle 12. Of the 16 pts who were marrow NGS MRD+ at 10-6 after 3 cycles of iFCG, 6/16 had an MRD recurrence in blood in follow-up vs. 0/20 who were NGS MRD negative/indeterminate (p = 0.004). Of the 12 pts who were marrow NGS MRD+ at 10-6 after Cycle 12, 4/12 had an MRD recurrence in blood in follow-up vs. 1/26 who were NGS MRD negative/indeterminate (p = 0.02). Image: Summary/Conclusion: The iFCG regimen, using only 3 cycles of chemotherapy (as opposed to 6 cycles of chemoimmunotherapy) achieves a very high rate of U-MRD in previously-untreated pts with CLL with IGHV-M CLL. No pt had disease progression with a median follow-up of close to 5 years. The 5-year PFS is 97.7%; this is favorable compared to 5-year PFS of ≈65% with FCR (CLL10), ≈70% with ibrutinib (A041202 trial), and 81% with ibrutinib (RESONATE-2) for IGHV-M CLL. Not unexpectedly, MRD recurrence during follow-up correlated with MRD positivity by NGS during therapy. S150: BIOLOGY AND FUNCTION OF CIRCULAR PCMDT1 IN CHRONIC MYELOID LEUKEMIA IN BLAST CRISIS (CML-BC) A. Prathap Urs1,*, D. Papaioannou2, R. Buisson3, S. Khanal1, M. Karunasiri1, S. Kauppinen4, A. Dorrance1 5, R. Garzon1 5 1Comprehensive Cancer Center, The Ohio State University, Columbus; 2NYU Langone Health, Laura and Isaac Perlmutter Cancer Center, New York; 3Department of Biological Chemistry, University of California, California, United States of America; 4Department of Clinical Medicine, Aalborg University, Copenhagen, Denmark; 5Department of Internal Medicine, The Ohio State University, Columbus, United States of America Background: The prognostic and biologic significance of circular RNAs (circRNAs) in patients with acute myeloid leukemia (AML) has been reported. Circular PCMTD1 (cPCMTD1) was among the circRNAs that were prognostic in this disease. Aims: To study the functional role of cPCMTD1 in leukemias. Methods: We used RNase H-recruiting, locked nucleic acid-modified oligonucleotides (gapmers) for knock-down (KD) experiments. Cell cycle analyses were performed with bromodeoxyuridine and 7-actinomycin D staining. Chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU) were used for DNA fiber assays. Results: To study whether cPCMTD1 is important for leukemia, we KD-cPCMTD1 using gapmers that specifically degraded the circular transcript without affecting the levels of the linear PCMTD1. Among the 8 AML cell lines that were tested, we found that depletion of the cPCMTD1 led to a decrease in the proliferating fraction and potent G2/M blockade of only K-562 and LAMA-84 CML-BC cells. In contrast, linear PCMTD1-KD did not have any effect on cell cycle. Mass cytometry (CyTOF) experiments validated the cell cycle blockade after cPCMTD1-KD and identified an increase of H2AX phosphorylation. We validated this finding with western blots and could also show that cPCMDT1-KD led to an increase in the phosphorylation of the ATM, ATR, CHK1, DNA-PK and RPA32 proteins. Further, DNA fiber assays detected a global decrease in the length of CldU- and IdU-labeled fibers as well as of the IdU/CldU ratio in the cPCMTD1-KD cells compared to controls. These results were confirmed by COMET assays. Taken together these data indicate that cPCMTD1-KD causes impaired DNA replication in CML-BC. Then, we examined whether we found in CML-BC cell lines was also true for patients. Thus, we measured cPCMDT1 expression in a panel of CML patients in chronic phase (n=15), accelerated phase (n=4) and blast crises (n=7) and we found a significant increase of cPCDMT1 in CML-BC with respect to chronic and accelerated phases. Next, we performed cPCMDT1-KD in CML-BC patient samples and found increase of H2AX phosphorylation. Like the cell line data, cPCMDT1-KD in 3 samples from AML patients had no functional effect. Finally, after confirming that cPCMDT1 is most abundant in the cytoplasm, in silico analysis and polysome profiling experiments indicated that cPCMDT1 encodes for a small protein. We generated a custom antibody that detected the predicted protein of 30kD, which was depleted after cPCMDT1-KD. Immunoprecipitation experiments followed by mass spectrometry analysis using our custom antibody showed a strong and specific enrichment of the BTR complex (BLM, TOP3A and RMI1), which is implicated in DNA replication and repair. cPCMDT1-KD had no effect on the total amount of each of the three proteins but reduced their interaction and the amount of the formed BTR complex. To validate the therapeutic potential of this strategy we developed a CML-BC PDX model by transplanting a CML-BC patient sample into NSGS mice. This PDX is very aggressive, and mice die from disease by 20 weeks. We are currently treating these mice in vivo with lipid nanoparticle anti-cPCMDT1 gapmers. Results will be updated in the meeting. Summary/Conclusion: cPCMDT1 is a circRNA with protein-coding potential that is over-expressed in CML-BC. cPCDMT1 is critical for the proliferation of CML-BC through the regulation of DNA replication and may represent a novel target for therapy. S151: HIGH-THROUGHPUT EVALUATION OF THE POTENTIAL OF CANCER DRUGS TO ENHANCE NATURAL KILLER CELL IMMUNOTHERAPY IN CHRONIC MYELOID LEUKEMIA. P. Nygren1 2,*, J. Bouhlal1 2, E. Laajala1 2, A. Ianevski3, J. Klievink1 2, H. Lähteenmäki1 2, K. Saeed1 2, D. Lee4, T. Aittokallio3 5 6, O. Dufva1 2, S. Mustjoki2 7 1Hematology Research Unit Helsinki, University of Helsinki and Helsinki University Hospital; 2Translational Immunology Research Program; 3Institute for Molecular Medicine Finland, FIMM, HiLIFE, University of Helsinki, Helsinki, Finland; 4Division of Hematology, Oncology, and BMT, Nationwide Children’s Hospital, Columbus, United States of America; 5Institute for Cancer Research, Department of Cancer Genetics, Oslo University Hospital; 6Centre for Biostatistics and Epidemiology (OCBE), Faculty of Medicine, University of Oslo, Oslo, Norway; 7Hematology Research Unit Helsinki, University of Helsinki and Helsinki University Hospital, Helsinki, Helsinki, Finland Background: Natural killer (NK) cell immunotherapies are promising novel cancer treatments and complete remission has been achieved in patients with relapsed/refractory myeloid leukemias. The significance of NK cells in chronic myeloid leukemia (CML) patients has been highlighted in several studies. CML patients with a higher percentage of mature NK cells have increased relapse-free survival after treatment discontinuation. NK KIR receptor profiles have also been associated with the achievement of remission in response to tyrosine kinase inhibitor (TKI) therapy. Moreover, complex interactions between TKIs and NK cell function have been discovered in CML. For example, dasatinib and imatinib have been suggested to enhance NK cell cytotoxicity through regulation of activating and inhibitory receptors. NK cell responses are usually short-lived and attempts have been made to enhance their function against malignant cells using cytokines, bi- and tri-specific antibodies, and small molecule inhibitors. However, a large-scale drug screening evaluating the combinatorial effects of NK cells and cancer drugs has not been previously conducted. Aims: To evaluate the potential of small molecule cancer drugs to synergize with NK cell immunotherapy in CML and to discover which drugs can enhance or inhibit cytotoxicity in CML cells. Methods: A high-throughput drug sensitivity and resistance testing (DSRT) screen was used to evaluate the effect of 528 investigational and approved cancer drugs on the cytotoxicity of NK cells. CML cells (K562) expressing luciferase and primary expanded NK cells were co-cultured for 24 hours on 384 well drug plates. Target cell viability was measured using a luciferase-based readout, and results were run through a custom pipeline to calculate differential drug sensitivity scores (dDSS) between co-cultured cells and target cells alone, for each drug. Most promising drugs with highest and lowest dDSS were selected for further analysis with single-cell RNA sequencing to determine their mechanism of action. Results: Out of 528 screened drugs, 161 had an inhibitory effect on cytotoxicity (dDSS < -2.5), 325 had no effect, and 42 had an enhancing effect (dDSS > 2.5). The SMAC mimetics birinapant and LCL-161 were the most potent enhancers of NK cell cytotoxicity and had no effect on target cells alone. Individual drugs with other mechanisms, such as PKC activators, were also effective enhancers of NK cytotoxicity. On the contrary, cytotoxicity-inhibiting drugs included previously known immunosuppressive drugs dexamethasone and prednisolone, as well as novel candidates, such as sotrastaurin, a PKC inhibitor. Single-cell RNA sequencing of drug-treated co-cultures revealed the transcriptomic phenotypes of NK cell and target cells under SMAC mimetic treatment. NF-kB target genes such as BIRC3, NFKB2, MHC II genes, and the T/NK cell-recruiting chemokines CXCL9, CXCL10, and CXCL11 were activated in target cells exposed to both birinapant and NK cells, compared to target cells exposed to NK cells alone (p value adj < 0.05). These data suggest that SMAC mimetics in combination with NK cells may induce an immune-inflamed phenotype in addition to sensitizing CML cells to NK cell cytotoxicity. Image: Summary/Conclusion: We discovered novel drug classes, which had both activating and inhibitory effects on the NK cell cytotoxicity against CML cells in vitro. Defining NK cell - drug - CML cell interactions in a high-throughput setting provides a framework for future combination immunotherapies to further improve treatment-free remission in CML. S152: SETD2/H3K36ME3 DEFICIENCY SUSTAINS GENOMIC INSTABILITY AND ENHANCES CLONOGENIC POTENTIAL OF CHRONIC MYELOID LEUKEMIA (CML) PROGENITORS M. Mancini1,*, S. De Santis2, C. Monaldi2, S. Bruno2, F. Castagnetti2, G. Gugliotta1, A. Iurlo3, M. Cerrano4, S. Galimberti5, S. Balducci5, F. Stagno6, G. Rosti7, M. Cavo8, S. Soverini2 1IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; 2Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna; 3UO Onco-ematologia, Fondazione IRCCS Ca’ Granda - Ospedale Policlinico, Milano; 4A.O.U. Citta della Salute e della Scienza di Torino, Torino; 5Clinical and Experimental Medicine, Hematology, University of Pisa, Pisa; 6Ematologia Universitaria e Trapianto Midollo Osseo, Ospedale Gaspare Rodolico, Catania; 7Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” - IRST Srl Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Meldola (FC); 8IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy Background: Genomic instability is a hallmark of chronic myeloid leukemia (CML) cells since the chronic phase (CP) of the disease and results in BCR-ABL1 mutations and/or additional genetic and genomic aberrations that may drive resistance to tyrosine kinase inhibitors (TKIs) and progression to blast crisis (BC). Genomic instability is also a feature of CML stem cells and may underlie their persistence. We have recently reported that virtually all CML patients (pts) in BC display loss of function of SETD2, an enzyme that trimethylates histone H3 lysine 36 (H3K36me3). SETD2 is a tumor suppressor implicated in several neoplastic conditions. H3K36me3 is required for homologous recombination (HR) repair of double strand breaks and for DNA mismatch repair (MMR). Aims: We investigated SETD2/H3K36me3 status in CD34+ progenitors of CP CML pts and whether SETD2/H3K36me3 deficiency may play a role in genomic instability in CML models. Methods: CD34+ progenitor cells were isolated from 20 newly diagnosed CP CML pts and screened for SETD2 and H3K36me3 by Western blotting (WB). SETD2-proficient (LAMA84) and -deficient (KCL22) CML cell lines were also studied. SETD2 knock-down and SETD2 forced expression in CD34+ progenitors and cell lines were performed by RNAi and nucleofection, respectively. DNA damage and DNA repair activation were assessed by WB and immunofluorescence (IF). Clonogenic capacity was evaluated by clonogenic assays. Results: Loss of SETD2 protein expression and function (the latter assessed using loss of H3K36me3 as surrogate marker) were detected by WB in the CD34+ cell fraction of 20/20 newly diagnosed CP CML pts, as compared to the corresponding total mononuclear cell fraction or to the CD34+ fraction obtained from a pool of healthy donors. To investigate whether SETD2/H3K36me3 loss impinges on the activation and proficiency of HR, we used UV rays to induce DNA damage in SETD2 siRNA-depleted LAMA 84 (SETD2-proficient) cells. Compared to control cells, cells silenced for SETD2 displayed an increase in the expression of the DNA damage maker γH2AX associated with a loss of RAD51 (HR) and MSH6 (MMR) repair foci. To confirm the role of SETD2 as a tumor suppressor implicated in maintaining genomic stability in CML, we transfected KCL22 (SETD2-deficient) cells with an ectopic SETD2 plasmid. SETD2 forced expression induced more than 50% reduction in cell doubling time and a significant reduction in clonogenic potential. Moreover, SETD2 overexpression was able to restore DNA damage response, as demonstrated by WB and immunofluorescence detection of H2AX phosphorylation, RAD51 (HR) foci, THEX1 (DNA replication) and MSH6 (MMR) observed after UV exposure. In line with the effects of SETD2 deficiency/overexpression observed in cell line models, CML progenitor cells displaying SETD2/H3K36me3 deficiency showed hyper-phosphorylation of H2AX and the overexpression of RAD51 (HR) and MSH6 (MMR) did not result in DNA repair foci. Moreover, forced overexpression of SETD2 restored proliferation control in CD34+ cells from 5 CP CML pts, since after nucleofection clonogenic assays showed a >50% reduction in clonogenic potential. Summary/Conclusion: SETD2/H3K36me3 deficiency is a novel, BCR-ABL1-independent mechanism of genetic instability in CML. SETD2 behaves like a true tumor suppressor in CD34+ progenitor cells of CP CML pts. Supported by AIRC IG 2019 grant (23001) and Italian Ministry of Health, “Bando Ricerca Finalizzata 2016”, project GR-2016-02364880. S153: DECRYPTING THE ROLE OF HSP90Α AND Β ISOFORMS TO OVERCOME RESISTANCE IN BCR-ABL1 LEUKEMIA M. Vogt1 2,*, N. Dienstbier1, J. Schliehe-Diecks1, K. Scharov1, J.-W. Tu1, P. Gebing1, J. Hogenkamp1, D. Picard1, T. Lenz3, K. Stühler3 4, R. Wagener1 5, A. Pandyra1 5, J. Hauer6, U. Fischer1 5, A. Borkhardt1 5, S. Bhatia1 5 1Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich Heine University Düsseldorf, University Hospital; 2DSO - Düsseldorf school of oncology; 3Molecular Proteomics Laboratory, Biological-Medical Research Center, Heinrich Heine University Düsseldorf; 4Institute of Molecular Medicine, Proteome Research, Medical Faculty and University Hospital, Heinrich Heine University Düsseldorf; 5German Cancer Consortium (DKTK), partner site Essen/Düsseldorf, Düsseldorf; 6Department of Pediatrics, Technical University of Munich, School of Medicine, Munich, Germany Background: The BCR-ABL1 fusion protein, which results from the chromosomal translocation t(9;22)(q34;q11) is detected in over 95% of CML, 25% of adult BCP-ALL and 3-5% of childhood BCP-ALL patients. The development of tyrosine kinase inhibitors (TKi) has enabled targeted treatment, however development of resistance is common and the BCR-ABL1 leukemia subtype is still associated with poor prognosis. Among the chaperone proteins involved in maintaining the homeostasis of cancerous cells, heat shock protein 90 (HSP90) have been widely studied, due to its crucial involvement in stabilizing a variety of oncogenic proteins, including BCR-ABL1 and STAT3/5. Therefore, inhibition of HSP90 can be an effective alternative approach to target TKi resistant and BCR-ABL1T315I mutant expressing cells. In the past, over 17 HSP90 inhibitors (HSP90i) have been evaluated in clinical trials, however associated resistances (e.g. HSR) and dose limited toxicity have thus far precluded their clinical approval. Aims: In mammalian cells, there are two predominant cytosolic isoforms of HSP90, a stress-inducible HSP90α and a constitutively expressed HSP90β isoform. In the past, the distinct roles of the isoforms were studied by applying pan- and isoform-targeted HSP90i. We aimed to decrypt the role of cytosolic HSP90 isoforms using genetically edited models and to identify novel therapeutic vulnerabilities to target therapy refractory BCR-ABL1+ leukemia. Methods: To study the kinetic implications of the resistance mechanism associated with pharmacological targeting of HSP90, we generated a transient knockdown (Kd) and a stable CRISPR-Cas9 based knockout (KO) model of HSP90α/β in BCR-ABL1+ cells. The edited cells were analyzed in ex vivo functional assays (e.g. IF staining and WB) and their transplantation efficiency was determined in NSG mice. Global transcriptomic and proteomic profiling was performed in conjunction with high throughput drug screening (HTDS) to identify therapeutic vulnerabilities upon loss of HSP90α/β. Results: HSP90α/β-KO or -Kd did not affect the expression of other HSP90 paralogues (TRAP1 and GRP94). Only HSP90β-KO cells displayed hyperactive pro-survival HSR. Other reported HSP90 isoform-dependent clients, such as Survivin, CDK4 and CDK6 were also not affected upon KO or Kd of HSP90α/β. As HSP90 is involved in stabilizing, proper folding and subcellular localization of BCR-ABL1 protein, immunofluorescence imaging demonstrated two fold higher abundance of BCR-ABL1 foci (cytoplasmic/nucleocytoplasmic region) in HSP90α KO cells, with hyperactive BCR-ABL1 and downstream signaling (pBCR-ABL and pSTAT5a). When KO cells were transplanted into NSG mice (n=5 mice per arm) the engraftment of the HSP90α-KO cells was significantly reduced with a prolonged overall survival of the animals (19 days, p=0.0083) as compared to HSP90β-KO and control group. To find possible target (s) in order to circumvent resistance associated with the use HSP90i, we performed HTDS with HSP90α/β KO cells, which revealed distinct sensitivities toward certain classes of inhibitors. One of these classes was CDK7i, which was differentially active in HSP90α KO cells. These results can be explained by our global transcriptomic results, which revealed hyperactive androgen receptor signaling in HSP90α KO cells (GSEA), which is known to be a prognostic marker for CDK7 inhibition. Summary/Conclusion: In vivo targeting of HSP90α is a promising approach to target BCR-ABL1+ leukemia cells. Moreover, the combination with CDK7i may help to overcome the resistance associated with clinical use of pan HSP90i. S154: IMATINIB-RESISTANT CLONES ISOLATED FROM A MODEL OF BLAST CRISIS OF CHRONIC MYELOID LEUKAEMIA DIFFER IN MUTATIONS IN BCR::ABL1 AND OTHER CANCER RELATED GENES AND IN THEIR SENSITIVITY TO BH3-MIMETICS A. Laznicka1 2,*, N. Curik1 3, V. Polivkova1, J. Koblihova1, P. Burda1 3, A. Dolnikova3, E. Pokorna3, C. Salek1, H. Klamova1, D. Srbova1, P. Klener3 4, K. Machova Polakova1 3 1Institute of Hematology and Blood Transfusion; 2Second Faculty of Medicine, Charles University; 3Institute of Pathological Physiology, First Faculty of Medicine, Charles University; 41st Dep. of Medicine – Dep. of Hematology, First Faculty of Medicine, Charles University, and General University Hospital, Prague, Czechia Background: The treatment of blast crisis of chronic myeloid leukaemia (BC-CML) remains difficult and the outcome of patients is poor. A dysregulated apoptosis leads to survival of malignant cells. BH3-mimetics inhibit antiapoptotic proteins of BCL-2 family. These drugs have been recently introduced in the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia. This work assumed that these drugs may have a treatment potential for myeloid and/or lymphoid BC-CML. Aims: The aim is to test the sensitivity of imatinib-resistant (IR) clones of KCL-22, model of BC-CML, and primary blast cells to BH3-mimetics and describe a mechanism of sensitivity/resistance using protein analysis of apoptotic pathways. Methods: Isolated IR-clones (n=10) of KCL-22 were characterized by NGS and cultivated with 4µM imatinib (IM) and dilution series of BH3-mimetics (venetoclax – anti-BCL-2, S63845 – anti-MCL-1, A-1155463 - anti-BCL-XL) for 72 hours. IC50 was determined based on proliferation. Protein expression of BCL-2 family (n=10) was conducted in 4 IR-clones cultivated with IM and with/without BH3-mimetics. For in vivo experiments NOD-SCID-gamma mice (n=16) were subcutaneously injected with 106 cells of BCR::ABL1-T315I clone and divided into control group and treated groups. Primary cells of patients with BC-CML (n=4) were exposed to IM and BH3-mimetics for 7 days and LC50 was estimated. Results: IR-clones of KCL-22 differ in their sensitivity to BH3-mimetics (Table 1). The majority of clones (8/10) including 4 clones with T315I were sensitive to MCL-1 inhibitor. A reduced sensitivity was observed in the T315I clone carrying mutated GATA2 and other clone with mutation Y253H and mutated BCOR. Moreover, two T315I clones were sensitive to venetoclax, but other T315I clones with mutations in other cancer related genes showed insensitivity (n=2) or decreased sensitivity (n=1). Interestingly, two T315I clones with mutations in other cancer related genes insensitive to venetoclax were sensitive to BCL-XL inhibitor and in general, it seems that clones insensitive to venetoclax were sensitive to BCL-XL inhibitor and vice versa. Protein analysis of parental KCL-22IR cells and 4 clones showed a high expression of BCL-2 and BCL-XL and undetectable MCL-1 expression. Except for the T315I clone (B8, Table 1), other analysed clones expressed MCL-1 after exposure to BH3-mimetics. The sensitivity of T315I clone to anti-MCL-1 can be explained by detection of cBAX (an active form of apoptotic effector) after exposure to the inhibitor. The parental cell line showed dissociation of antiapoptotic complex MCL-1/BIM after exposure to MCL-1 inhibitor. The released BIM accelerates the apoptosis as an apoptotic activator. In vivo analysis revealed a decreased tumour growth of xenografted T315I clone (B8) in IM, venetoclax or combined treatment compared to control. An additive effect of dual therapy against monotherapy was not observed. This can be explained by IM inhibition of non-mutated BCR::ABL1 in KCL-22IR carrying 2 Ph chromosomes. LC50 analysis of primary blasts (4 patients) showed anti-MCL-1 to be the most potent inhibitor. Mutations in other cancer related genes and/or changes in karyotype increased LC50 for venetoclax and BCL-XL inhibitor or venetoclax, respectively. Image: Summary/Conclusion: The preliminary data of this study showed that the MCL-1 inhibitor S63845 seems to be the most potent BH3-mimetic to induce apoptosis in BC-CML. The combined therapy of TKI and BH3-mimetics should reflect mutation status of BCR::ABL1 and other cancer related genes. S155: EFFICACY AND SAFETY RESULTS FROM ASCEMBL, A PHASE 3 STUDY OF ASCIMINIB VS BOSUTINIB IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE AFTER ≥2 PRIOR TYROSINE KINASE INHIBITORS: WK 96 UPDATE D. Rea1,*, A. Hochhaus2, M. J. Mauro3, Y. Minami4, E. Lomaia5, S. Voloshin6, A. Turkina7, D.-W. Kim8, J. F. Apperley9, J. E. Cortes10, A. Abdo11, L. M. Fogliatto12, D. D. H Kim13, P. le Coutre14, S. Saussele15, M. Annunziata16, T. P. Hughes17, N. Chaudhri18, L. Chee19, V. García-Gutiérrez20, K. Sasaki21, S. Kapoor22, A. Allepuz23, S. Quenet23, V. Bédoucha23, C. Boquimpani24 1Hôpital Saint-Louis, Paris, France; 2Universitätsklinikum Jena, Jena, Germany; 3Memorial Sloan Kettering Cancer Center, New York, United States of America; 4National Cancer Center Hospital East, Kashiwa, Japan; 5Almazov National Medical Research Centre; 6Russian Research Institute of Hematology and Transfusiology, St. Petersburg; 7National Medical Research Center for Hematology, Moscow, Russia; 8Uijeongbu Eulji Medical Center, Geumo-dong, Uijeongbu-si, South Korea; 9Centre for Haematology Imperial College London, London, United Kingdom; 10Georgia Cancer Center, Augusta, United States of America; 11Instituto do Câncer do Estado de São Paulo (ICESPSP), São Paulo; 12Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, Brazil; 13Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Canada; 14Charité–Universitätsmedizin Berlin, Berlin; 15III. Medizinische Klinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany; 16Division of Hematology, AORN Cardarelli, Naples, Italy; 17South Australian Health and Medical Research Institute and University of Adelaide, Adelaide, Australia; 18King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia; 19Peter MacCallum Cancer Center and The Royal Melbourne Hospital, Victoria, Australia; 20Servicio de Hematología, Hospital Universitario Ramón y Cajal (IRYCIS), Madrid, Spain; 21The University of Texas MD Anderson Cancer Center, Houston; 22Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 23Novartis Pharma AG, Basel, Switzerland; 24HEMORIO, State Institute of Hematology Arthur de Siquiera Cavalcanti, Rio de Janeiro, Brazil Oncoclínica Centro de Tratamento Oncológico, Rio de Janeiro, Brazil Background: Asciminib is the 1st BCR::ABL1 inhibitor to Specifically Target the ABL Myristoyl Pocket (STAMP). In the ASCEMBL primary analysis, asciminib had superior efficacy and better safety/tolerability vs bosutinib (BOS) in patients (pts) with chronic myeloid leukemia in chronic phase (CML-CP) after ≥2 prior tyrosine kinase inhibitors (TKIs). Major molecular response (MMR) rate at wk 24 was 25.5% on asciminib vs 13.2% on BOS; the difference in MMR rates after adjusting for baseline major cytogenetic response (MCyR) was 12.2% (95% CI, 2.19%-22.30%; 2-sided P=.029). Fewer grade ≥3 adverse events (AEs) and AEs leading to treatment discontinuation occurred on asciminib vs BOS. After a median follow-up of 2.3 years (16.5 months’ additional follow-up since the primary analysis), we report updated efficacy and safety results (cutoff: October 6, 2021). Aims: The key secondary objective was to compare MMR rate at wk 96 on asciminib vs BOS. Methods: Eligible pts provided informed consent, were adults with CML-CP after ≥2 prior TKIs, with intolerance or lack of efficacy per 2013 European LeukemiaNet recommendations. They were randomized 2:1 to asciminib 40 mg twice daily or BOS 500 mg once daily, stratified by baseline MCyR status (Ph+ metaphases ≤35%). Results: 233 pts were randomized to asciminib (n=157) or BOS (n=76). At cutoff, treatment was ongoing in 84 (53.5%) and 15 (19.7%) pts, respectively; the most common reason for discontinuation was lack of efficacy in 38 (24.2%) and 27 (35.5%) pts, respectively. MMR rate at wk 96 (per ITT) was 37.6% on asciminib and 15.8% on BOS, meeting the key secondary objective. The difference after adjusting for baseline MCyR was 21.7% (95% CI, 10.5%-33.0%; 2-sided P=.001). Preplanned subgroup analyses showed that MMR rate at wk 96 was consistently higher with asciminib than BOS in all demographic and prognostic subgroups, including all prior lines of TKI therapy, and regardless of the reason for discontinuation of the last TKI (Figure). At wk 96, more pts on asciminib than BOS had BCR::ABL1 IS ≤1% (45.1% vs 19.4%) (Table). Responses were durable, with a probability (95% CI) of maintaining MMR and BCR::ABL1 IS ≤1% for ≥72 wk of 96.7% (87.4%-99.2%) and 94.6% (86.2%-97.9%), respectively, on asciminib and 92.9% (59.1%-99.0%) and 95.0% (69.5%-99.3%), respectively, on BOS. Median time to treatment failure was 24 months on asciminib and 6 months on BOS. Median duration (range) of exposure was 103.1 (0.1-201.1) wk on asciminib and 30.5 (1.0-188.3) wk on BOS. Despite asciminib’s longer duration of exposure, its safety/tolerability continued to be better than that of BOS (Table). Fewer pts on asciminib than BOS had AEs leading to treatment discontinuation (7.7% vs 26.3%). No new on-treatment deaths were reported since the primary analysis. Most frequent (>10%) grade ≥3 AEs on asciminib vs BOS were thrombocytopenia (22.4%, 9.2%), neutropenia (18.6%, 14.5%), diarrhea (0%, 10.5%), and increased alanine aminotransferase (0.6%, 14.5%). Image: Summary/Conclusion: After >2 years of follow-up, asciminib continued to show clinically and statistically significant, superior efficacy and better safety/tolerability vs BOS. Responses were durable, and MMR was more than double on asciminib than BOS. The difference in MMR rates between the 2 arms increased from 12.2% at wk 24 to 21.7% at wk 96. A higher proportion of pts had BCR::ABL1 IS ≤1%, a milestone response in later lines that is associated with improved long-term survival. These results further support the use of asciminib as a new CML therapy, with the potential to transform standard of care. S156: INTERNATIONAL, PROSPECTIVE STUDY COMPARING NILOTINIB VERSUS IMATINIB WITH EARLY SWITCH TO NILOTINIB TO OBTAIN SUSTAINED TREATMENT-FREE REMISSION IN PATIENTS WITH WITH CHRONIC MYELOID LEUKEMIA F. Pane1,*, F. Castagnetti2, L. Luciano1, A. Russo Rossi3, E. Abruzzese4, R. Bassan5, G. Binotto6, G. Caocci7, G. Cimino8, P. Fazi9, A. Gozzini10, M. Lunghi11, R. Marasca12, B. Martino13, M. Bonifacio14, F. Cavazzini15, F. Paoloni16, G. Saglio17, S. Sica18, A. Tafuri19, D. Vallisa20, M. Vignetti9, P. Westerweel21, G. Rosti22, M. Breccia23 1Dpt. of Clinical Medicine and Surgery, University of Naples, Naples; 2Department of Experimental, Diagnostic, and Specialty Medicine, University of Bologna, Bologna; 3Hematology and Transplantation Unit, University of Bari, Bari; 4Division of Hematology, Ospedale S. Eugenio, Rome; 5Hematology Unit, Ospedale Dell’Angelo, Venezia-Mestre; 6Hematology Unit, University of Padova, Padova; 7Department of Medical Sciences and Public Health, Hematology, Businco Hospital, University of Cagliari, Cagliari; 8UOC Ematologia e trapianto, S.Maria Goretti Hospital, Latina; 9GIMEMA Data Center, Fondazione GIMEMA Franco Mandelli Onlus, rome; 10Department of Cellular Therapy and Transfusional Medicine, AUO Careggi, Florence; 11Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont, Novara; 12Section of Hematology, Department of Medical Sciences, University of Modena and Reggio Emilia, Modena; 13Division of Hematology, Azienda Ospedaliera ‘Bianchi Melacrino Morelli’, Reggio Calabria; 14Section of Hematology, Department of Medicine, University of Verona, Verona; 15Hematology Section, Department of Medical Sciences, University of Ferrara, University Hospital Arcispedale S. Anna, Ferrara; 16Biostatistic Central Office, Fondazione GIMEMA Franco Mandelli Onlus, Rome; 17Hematology Division, University of Turin, Turin; 18Ematologia, FONDAZIONE POLICLINICO UNIVERSITARIO AGOSTINO GEMELLI IRCCS, Roma; 19Department of Clinical and Molecular Medicine, Hematology, Sant’Andrea University HospitalSapienza University of Rome, Rome; 20Unit of Hematology, Department of Oncology and Hematology, Guglielmo da Saliceto Hospital, Piacenza, Italy; 21Internal Medicine, Albert Schweitzer Hospital, Dordrecht, Netherlands; 22Hematology Unit, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola; 23Translational medicine OUC Hematology, La Sapienza university, Rome, Italy Background: Treatment free remission (TFR) is one of the most important goals of the CML treatment but, so far, the best treatment to reach this aim is still undefined. It is widely accepted that a sustained deep molecular remission (DMR) is the pre-requisite to discontinue TKI, and it is expected that in patients who start treatment with imatinib (IMA) and fail early molecular remission (EMR), a switch to a second generation TKI may improve the probability of achieving a DMR Aims: We launched in November 2016 an international, prospective, interventional, randomized, two arms, study to evaluate both the depth of the molecular response and the rate of TFR in newly diagnosed CP-CML patients treated with a second generation TKI (Nilotinib, NIL) or with IMA followed by switching to NIL in absence of optimal response (defined according the ELN 2013 criteria (Clinical Trial number 02602314). Methods: The patients are randomized 1:1 between NIL and IM according to Sokal risk score (high versus intermediate/low risk) and country. All the patients who obtain a residual disease reduction greater than 4.0 logs (MR4.0) within the first three years of treatment and maintain this level of response up to the end of the fourth years of therapy qualify for the discontinuation phase of the study. The study has two primary end-points: a) the rates of molecular response (MR4.5) at 24 months, and b) the rate of patients who remain in sustained treatment free remission (≥MR3.0) without molecular relapse 12 months after entering the TFR phase. The molecular relapse is defined as loss of MMR or confirmed loss of MR3.0 (figure). Results: From November 2016 to January 2021, 457 patients with newly diagnosed CP-CML patients were enrolled into the study and 448 of these (228 and 220 randomized to the NIL and IMA arms, respectively) were evaluable (mean age 54.2 yrs - range 19.4 – 85.8). At baseline, 183 (40.8%), 191 (42.6%) and 72 (16.1%) patients were classified as low, intermediate, or high-risk Sokal, respectively, while 278 (62.3%), 128 (28.7%) and 40 (9.0%) had a low, intermediate, or high-risk ELTS risk score. The median follow-up of the whole cohort of patients is 30.4 mo. Fifthy-six (25.4%) of the 220 patients of the IMA arm did not fulfill the ELN criteria for optimal response within the first 12 mo. of treatment and, according to the protocol, switched to NIL therapy. At the last analysis of the protocol database (February 2022), 59 patients had had stopped the protocol treatment since their decision, death (24), toxicity (23), progression (9), uncontrolled second neoplasia (2) or protocol violation (1), 69 patients had not reach 24 mo. of follow-up and other 15 had missing data. Of the remaining 304 patients, 35 showed non optimal response to therapy. At the 24 mo. of follow-up, 76 of the 322 patients with an available molecular response (23.6%), reached a MR4.5 response that showed a significantly higher frequency within the patients randomized to the NIL arm (48 vs 28; p=0.015) (first primary co-endpoint of the study). Image: Summary/Conclusion: This is the first and, so far, the unique prospective study comparing not only the rate of DMR but, more important, also the rate of TFR according to treatment: a second generation TKI frontline vs IMA frontline followed by the same second generation in case of non-optimal response. The analysis of the first co-primary endpoint indicates that, despite the early switch in the IMA randomized patients, NIL therapy is more effective to induce DMR. Subsequent analysis will clarify whether the higher rates of DMR in the NIL arm may translate into a higher rate of TFR. S157: BCR::ABL1 DIGITAL PCR IDENTIFIES CHRONIC PHASE CML PATIENTS SUITABLE FOR AN EARLY TKI DISCONTINUATION ATTEMPT: A PATIENT-LEVEL META-ANALYSIS C. Kockerols1,*, S. Dulucq2, S. Bernardi3, M. Farina3, I. Civettini4, G. Colafigli5, S. Mori6, P. Valk7, F.-X. Mahon8, C. Gambacorti-Passerini4 9, F.-E. Nicolini10, M. Breccia5, D. Russo3, P. E. Westerweel1 1Internal Medicine, Albert Schweitzer Hospital, Dordrecht, Netherlands; 2Laboratory of Hematology, Hôpital Haut Lévêque, University hospital of Bordeaux, Pessac, France; 3Clinical and Experimental Sciences, Hematology; 2and Bone Marrow Transplant Center, University of Brescia, ASST Spedali Civili of Brescia, Brescia; 4University of Milano Bicocca, Monza; 5Translational and Precision Medicine, Az. Policlinico Umberto I-Sapienza University, Rome; 6University of Milano-Bicocca, Monza, Italy; 7Molecular Biology, Erasmus University Medical Center, Rotterdam, Netherlands; 8Hematology, Institut Bergonié, Bordeaux, France; 9Hematology, S. Gerardo Hospital, Monza, Italy; 10Hematology, Centre Léon Bérard and CRCL, Lyon, France Background: Digital PCR (D-PCR) is an emerging technique that delivers a highly accurate BCR::ABL1 quantification, even in CML patients with low residual disease. This is crucial in the context of treatment-free remission (TFR) for the selection of patients who may successfully discontinue TKI therapy. However, it is unclear how its prognostic value relates to time variables such as treatment duration prior to the TFR attempt. Current guidelines suggest aiming for a TKI treatment duration >6 years to increase TFR success rate. Aims: In current analysis, we aimed to assess the prognostic value of BCR::ABL D-PCR in relation to prior TKI treatment duration. Methods: We performed an Individual Patient Data Meta-Analysis (IPD-MA) combining data from different study cohorts in which BCR::ABL1 was assessed by D-PCR prior to TKI discontinuation. Eligibility criteria included age ≥18 years and CML diagnosed in chronic phase. Data of the participating studies were pooled and stratified based on D-PCR and/or treatment duration. BCR::ABL1 D-PCR was dichotomized based on each study-defined prediction cut-off. Strata were assessed for molecular relapse (MolR) with Kaplan-Meier estimates and with cox regression analysis including a frailty term for correction for between-study heterogeneity and including confounding variables: age at diagnosis, gender, Sokal score, TKI generation, treatment duration, DMR duration (model 1) and BCR::ABL1 transcript type (model 2). MolR was defined as a BCR::ABL1 >0,1%IS or a 1-log BCR::ABL1 increase in two consecutive analyses. Patients were censored at last follow-up. Results: For this meta-analysis, data were combined from five cohorts: four published cohorts (STIM2 cohort [Nicolini et al.], n=175; ISAV cohort [Diral et al.], n=107; Bernardi et al., n=111; Colafigli et al., n=50) and 1 unpublished cohort (Dutch cohort; n=40). The pooled dataset comprised 483 patients (Table). A total of 205 patients (42%) experienced MolR with a median time to relapse of 3 months. Median follow-up duration for TFR patients was 27 months. MolR patients had a significantly shorter treatment duration prior to TKI discontinuation (6,7 vs 7,9 years, p=0.006) and more often presented a BCR::ABL1 D-PCR above the study-defined prediction cut-off (34% vs 19%, p<0.001). Interestingly, the median treatment durations were almost identical in patients with a BCR::ABL1 below or above the cut-off (7.0 vs 7.5 years, p = 0.470). The probability of MolR at 24 months was 38% versus 58% for patients with a D-PCR BCR::ABL1 below versus above the prediction cut-off (p <0.001). In the cox regression analysis, the HR of D-PCR BCR::ABL1 below the cut-off for MolR was 0.48 (95% CI 0.35-0.66, p<0.001). Treatment duration and BCR::ABL1 transcript type also remained independent predictors, but TKI generation and DMR duration did not. When stratifying into 4 groups based on the D-PCR result and treatment duration, patients with a TKI treatment for ≥6 years and low D-PCR result had the lowest MolR rate (33% at 24 months, figure). Patients treated <6 years and with a low D-PCR result had a rate of 48% at 24 months, while patients treated <6 years and a D-PCR result above the cut-off had the highest MolR rate of 72% at 24 months. Image: Summary/Conclusion: These combined patient-level data of multiple CML cohorts further support the independent prognostic value of BCR-ABL1 D-PCR for TFR success rate. Importantly, patients with a TKI treatment duration <6 years and a low D-PCR result were found to have a clinically acceptable MolR rate (48%). S158: FINAL RESULT OF TKI DISCONTINUATION TRIAL WITH DASATINIB FOR SECOND ATTEMPT OF TREATMENT FREE REMISSION AFTER FAILING FIRST ATTEMPT WITH IMATINIB: TREATMENT-FREE REMISSION ACCOMPLISHED BY DASATINIB D. Kim1,*, E. Atenafu2, D. Forrest3, I. Bence-Bruckler4, L. Savoie5, M.-M. Keating6, L. Busque7, R. Delage8, A. Xenocostas9, E. Liew10, P. Laneuville11, K. Paulson12, T. Stockley13, J. Lipton1, B. Leber14 1Princess Margaret Cancer Centre, Toronto, Canada; 2Statistics, Princess Margaret Cancer Centre, Toronto; 3Vancouver General Hospital, Vancouver; 4Ottawa Hospital Research Institute, Ottawa; 5University of Calgary, Calgary; 6Queen Elizabeth II Health Sciences Centre, Halifax; 7Hôpital Maisonneuve-Rosemont, Montreal; 8Centre Universitaire d’Hématologie et d’Oncologie de Québec, Quebec; 9London Health Sciences Centre, London; 10University of Alberta, Edmonton; 11McGill University Health Centre, Montreal; 12CancerCare Manitoba, Winnipeg; 13Division of Clinical Laboratory Genetics, University Health Network, Toronto; 14Juravinstki Cancer Centre, Hamilton, Canada Background: The Canadian tyrosine kinase inhibitor (TKI) discontinuation (DISC) trial evaluated if Dasatinib (DA) therapy can lead to a successful second treatment-free remission (TFR2) after failing a Imatinib (IM) DISC for first TFR (TFR1) attempt. We previously reported that 1) The 12-month molecular relapse free survival (mRFS) rate for TFR1 was 58.0%; 2) doubling time (DT) at 2 months after IM DISC correlates with TFR1 failure. Aims: Here, we report the final result of TFR2 rate after DA DISC. The null hypothesis was a TFR2 rate of 17.5% while the alternative hypothesis was a TFR2 rate of 35.0%, and the study was designed to reject our null hypothesis if > 28% of pts remain in TFR2 after DA DISC. Methods: This prospective clinical trial (BMS CA180-543, NCT#02268370) had 3 phases: 1) IM DISC phase, 2) DA rechallenge phase, 3) DA DISC phase. Key inclusion criteria included: 1) CML in chronic phase at original diagnosis, 2) total duration of IM therapy of minimum 3 years, 3) total duration of MR4.5 or deeper response over 2 years. Molecular relapse was defined as an increase in BCR-ABL qPCR > MR4 on 2 consecutive occasions, or a single increase in BCR-ABL qPCR > MR3. DA treatment was started at 100mg once daily after molecular relapse was confirmed, and continued for at least 12 months after achieving ≥ MR4 until DISC for TFR2. Results: The study was launched on March 2015 and completed all participants’ planned visits as of Feb 2022 with a median follow-up duration of 27.5 months (range 2-51 months). 1) In the IM DISC phase, 58 of 131 pts (44.3%) experienced molecular relapse with a median onset of 3.53 months, thus the remaining 73 pts achieved TFR1 (55.7%): The mRFS rate at 12 months was 56.8% (95% CI, 47.8-64.8 %). Distribution of monthly DT in 6 months is presented in Fig A. 2) In the DA rechallenge phase, out of the 58 pts who failed TFR1, 51 pts received DA. At 3 months, 98.0%, 87.9%, and 75.4% of pts achieved MMR, MR4 and MR4.5. The halving time (HT) after DA re-therapy is summarized in Fig B. Notably, HT at 2 and 3 months was 10.6 and 10.7 days, consistent with rapid reduction of BCR-ABL qPCR in first 3 months. 3) In the DA DISC phase, 35 pts who attained MR4 or deeper response for ≥ 12 months discontinued DA for a TFR2 attempt. Out of 35 pts, only 4 pts (11.4%) has maintained the molecular response at last follow-up, while the remaining 31 pts lost the molecular response with a median 3.65 months. The actuarial mRFS rate at 6 and 12 months was 22.9% (10.8-37.6%) and 10.0% (2.7-23.1%). Monthly DT within 6 months after DA DISC is presented in Fig C. Based on the final result of 11.4% TFR2 rate, we conclude that 12 months’ DA re-therapy could not improve TFR2 rate significantly. 4) For DT after IM DISC, average DT at 2/3 months was much shorter in those lost molecular response (10.6 / 10.7 days) than those who did not (23.5 / 30.1 days). For DT after DA DISC, while average DT was 7.6 and 14.3 days at 2/3 months in overall population, it was much shorter in those lost molecular response within 6 months (3.2 and 13.3 days at 2/3 months). Image: Summary/Conclusion: The final result indicates that re-challenge with DA after TFR1 failure with IM DISC is effective in restoring deep molecular response as most cases rapidly regained at least MR4. However, 12 months’ DA re-therapy does not significantly improve TFR2 rate. Further studies should consider increasing MR4 duration before TFR2 attempt, adding other therapeutics and refining risk factor for TFR2 failure. S159: QUÉBEC CML RESEARCH GROUP ANALYSIS OF TREATMENT PATTERNS IN CHRONIC MYELOGENOUS LEUKEMIA: SWITCHING IS DRIVEN BY INTOLERANCE AND SIMILAR ACROSS TYROSINE KINASE INHIBITORS AND LINES OF TREATMENT L. Busque1 2,*, M. Harnois2, N. Szuber1 2, R. Delage2 3, L. Mollica1 2, H. Olney2 4, P. Laneuville2 5, S. Sirhan2 6, G. Cournoyer2 7, I. Chamakhi2 8, M. Lalancette2 3, D. Talbot2 9, V. Éthier2 10, P. Desjardins2 11, S. Assouline2 6 1Hematology, Hôpital Maisonneuve-Rosemont, Université de Montréal; 2Groupe Québécois de Recherche en LMC-NMP, GQR-LMC-NMP, Montreal; 3Hematology, Centre Hospitalier Universitaire de Québec (CHUQ), Université Laval, Québec; 4Hematology, Centre Hospitalier Universitaire de Montréal (CHUM), Université de Montréal; 5Hematology, McGill University Health Center (MUHC), McGill University; 6Hematology, Jewish General Hospital, McGill University, Montreal; 7Hemato-oncology, Hôpital regional de St-Jérôme, St-Jérôme; 8Hemato-oncology, Hôpital du Sacré-Cœur de Montréal, Université de Montréal, Montreal; 9Hemato-oncology, Hôpital de la Cité-de-la-Santé, Laval; 10Hemato-oncology, Centre Hospitalier Universitaire de Sherbrooke (CHUS), Université de Sherbrooke, Sherbrooke; 11Hemato-oncology, Hôpital Charles-Lemoyne, Université de Sherbrooke, Greenfield Park, Canada Background: In 2001, imatinib was the first tyrosine kinase inhibitor (TKI) approved for the treatment of chronic myelogenous leukemia (CML) which translated into a revolution in the management of this disease. The arsenal of TKIs was progressively reinforced with the addition of other TKIs such as dasatinib, nilotinib, bosutinib, ponatinib and recently asciminib. Two decades of clinical trials have provided critical insight into differential efficacies and specific toxicity profiles of these TKIs supporting the development of guidelines such as the European Leukemia Net (ELN) and the national comprehensive cancer network (NCCN). Real-world evidence studies provide unique and complementary information on treatment patterns, efficacy, side effects and may help identify unmet medical needs of CML patients Aims: This study was aimed at studying frequency of TKI switching, reason for switch, duration of treatment without switching as a function of line of treatment and specific TKI using the real-world data of the Québec registry created in 2009 Methods: Patients with Philadelphia positive (Ph+) CML were recruited with informed consent in the Québec registry. 795 patients were included in this analysis. Data from the registry were extracted July 31ist 2021. Switching was defined as a change of a specific TKI to another. Reasons to initiate switching were categorized as resistance (primary and secondary) or intolerance (hematological or non-hematological). Standard statistical methods were used to evaluated bivariate and continuous variables. Kaplan-Meier curve were used to evaluate overall survival and survival without switching (JMP® Pro 14.1 software, SAS Institute, Cary, NC, USA). Results: At the time of data collection, the median time of follow-up was 7.5 years. Proportion of switching per line of treatment: 1st: 357/795 (44.9%); 2nd: 157/357 (43,9%); 3rd: 59/157 (37,6%); 4th:23/59 (39%). The reason for switching (ratio intolerance/resistance) for each line were: 1st: 1.33; 2nd: 4.3; 3rd: 2.4; 4th: 2.7. 20 patients switched serially across all lines for intolerance and only 3 for serial resistance. Survival without switching was evaluated using a Kaplan-Meier curve by line of treatment and by specific TKI (Figure 1). The survival without switching was similar for patients on imatinib, dasatinib or nilotinib in first line as in second line. In third line, there was no difference between imatinib, nilotinib, dasatinib or bosutinib although numbers of patients were small. We then compared the survival from diagnosis of patients that remained in first or second line versus the patients that reached third or more lines of treatment. Although the mean age at diagnosis and SD were identical between the two groups (54,8 years and SD 15,2) there was a statistically significant difference in survival in favor of patients needing only 1 or 2 lines of treatment (P= 0.0254). Image: Summary/Conclusion: We demonstrate: (i) that switching of TKI is frequent and mainly driven by intolerance in all lines of treatment; (ii) serial intolerance is 6.6 time more frequent than serial resistance suggesting a class effect for intolerance in some patients; (iii) all TKIs have a similar «retention level» in all lines of treatment; (iv) that patients necessitating 3 or more lines of treatment have a survival disadvantage. Our results suggest that one of the most important unmet medical need in CML management is availability of better tolerated drugs S160: MOLECULAR DETERMINANTS OF DISEASE PROGRESSION AFTER HYPOMETHYLATING AGENT THERAPY IN RAS PATHWAY MUTANT CHRONIC MYELOMONOCYTIC LEUKEMIA AT THE SINGLE-CELL LEVEL G. Montalban-Bravo1,*, F. Ma2, I. Ganan-Gomez1, R. Kanagal-Shamana3, V. Adema1, N. Thongon1, H. Yang1, K. A. Soltysiak1, C. Bueso-Ramos3, H. Kantarjian1, G. Garcia-Manero1, S. Colla1 1Leukemia, The University of Texas MD Anderson Cancer Center, Houston; 2Molecular Biology Institute, University of California, Los Angeles; 3Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Most patients (pts) with chronic myelomonocytic leukemia (CMML) have incomplete or transient responses to hypomethylating agent (HMA) therapy. CMML cases driven by mutations in RAS pathway signaling genes or ASXL1 have a higher risk of failure and progression to acute myeloid leukemia (AML). Development of effective alternative therapies has been delayed, owing to an incomplete understanding of how different hematopoietic populations contributes to disease maintenance and progression. Aims: We aimed to dissect the cellular and molecular mechanisms underpinning CMML maintenance and progression in RAS mutant CMML. Methods: We performed single-cell RNA sequencing (scRNA-seq) analysis of lineage-negative (Lin-) CD34+ hematopoietic stem and progenitor cells (HSPCs) and BM mononuclear cells (MNCs) isolated from RAS pathway mutant CMML pts (n=5 and 6, respectively) and age-matched healthy donors (HD; n=2 and 3, respectively). CMML samples were obtained at the time of diagnosis and HMA failure. Additionally, we performed scATAC-seq analysis of Lin-CD34+ HPSCs isolated at the times of diagnosis and progression (n=1). Results: Our analysis revealed that CMML HSPCs had a predominantly granulomonocytic differentiation route with increased frequencies of myeloid-monocytic progenitors, at the expense of hematopoietic stem cells (HSCs) (Fig 1a), and upregulated expression of genes involved in the oxidative phosphorylation, type I interferon (IFN) and IFNg pathways. Consistent with these results, scRNA-seq analysis of MNCs revealed expanded populations of myelomonocytic progenitors and monocytes and upregulated expression of genes involved in IFNg response and NF-kB activation (Fig 1b), along with upregulation of the NF-kB transcriptional effector BCL2A1 (Fig 1c). Assessment of ligand-receptor interactions using the CellPhoneDB repository identified that CMML monocytes established a high number of cell-cell interactions (n=638) with dendritic cells, NK cells, and HSPCs via chemokines, cytokines, and inhibitory molecules known to induce NF-kB signaling and NK-cell exhaustion. Disease progression was associated with expansion of lympho-myeloid progenitors (LMPPs) (Fig 1d) characterized by the highest levels of IFNg response, NF-kB survival signaling, and cell cycle regulators. scATAC-seq of Lin-CD34+ confirmed higher activity of transcriptional factors associated with monocytic differentiation and NF-kB signaling (Fig 1e-f). Accordingly, scRNA-seq analysis of MNCs showed increased frequencies of HSPCs and myelomonocytic precursors, a reduction of T cells (Fig 1f), and emergence of a monocyte population characterized by the highest expression of NF-kB signaling and its effectors MCL1 and BCL2A1. BCL2A1 protein expression at progression was confirmed by immunohistochemistry (Fig 1g). CellPhoneDB analysis identified a high number of cell-cell interactions (n=2978) involving cytokines, chemokines, and surface proteins known to elicit NF-kB activation and immune evasion between expanded monocytes, LMPPs, myelomonocytic precursors, and immune cells during progression. Image: Summary/Conclusion: Our data suggests that CMML is maintained through metabolically active HSPCs, which leads to monocytes’ reprograming and survival through NF-kB signaling activation. We showed that disease progression arises from the expansion of NF-kB dependent immature myeloid progenitors, which leads to therapy resistance and immune evasion. This study has implications for the development of therapies targeting downstream effectors of NF-kB–mediated survival pathway to overcome treatment failure. In vitro validation is ongoing. S161: ZRSR2 AND TET2 MUTATIONS PROMOTE MDS BY DYSREGULATING GENE EXPRESSION AND ABERRANT ALTERNATIVE SPLICING IN MICE C. Garcia-Ruiz1,*, C. Martínez-Valiente1, A. Liquori1, A. Gutiérrez-Adán2, J. Cervera3, A. Sanjuan-Pla1 1Hematology Research Group, IIS La Fe, Valencia; 2Animal Reproduction Department, INIA, Madrid; 3Genetics Unit, Hospital Universitario y Politécnico La Fe, Valencia, Spain Background: Mutations in splicing factors and epigenetic regulators are the most frequent genetic alterations in patients with myelodysplastic syndromes (MDS). The minor spliceosome factor ZRSR2 and the epigenetic regulator TET2 appear significantly associated in MDS patients. However, the functional impact of such mutations in the hematopoietic system and MDS have been scarcely studied. To address this question, we established a murine model (Zrsr2 m/m Tet2 −/− ) carrying mutations in both genes, which exhibited signs compatible with MDS disease in mice. However, the molecular disease mechanism has not yet been elucidated. Aims: To interrogate the impact of ZRSR2 and TET2 mutations in gene expression and alternative splicing in the context of hematopoiesis and MDS. Methods: Whole transcriptome sequencing was performed to investigate changes in gene expression and alternative splicing patterns. Lin-Sca-1+c-kit+ (LSK) cells (50,000 per sample) were sorted from pools of 3 mice, and mRNA was sequenced in an Illumina NovaSeq 6000 platform. Differential gene expression was determined using DESeq2 with adjusted P-value ≤ 0.05, log2FoldChange ≥ 0. Functional enrichment from differentially spliced genes was identified with KEGG, and the enrichment cut-off p-value was set as adjusted P-value ≤ 0.05. Relevant alternative splicing events were validated by RT-PCR. Results: RNA-seq analysis of Zrsr2 m/m Tet2 −/− cells revealed 2952 differentially expressed genes (DEG), compared to 571 in Zrsr2m/m and 1203 in Tet2−/−. From 2952, 1327 were up-regulated and 1625 were downregulated. Interestingly, relevant genes for hematopoietic lineage specification were significantly dysregulated. In particular, genes related to lymphoid lineage (Flt3, Notch1, and Tlr7) were downregulated, while those related to myeloid lineage (Ccl9, Mpo, Ms4a6b, and Prtn3) and megakaryocytic-erythroid lineage (Pdfgrb, Itgb3, Optn, and Tgfbr3) were upregulated. This suggests an early myeloid and megakaryocyte-erythroid priming of LSK towards the production of cells from these lineages in Zrsr2 m/m Tet2 −/− mice. Enrichment analysis of DEG using Go enrichment analysis identified ribosome function, inflammation, and migration/motility processes as the most significantly altered. Further, KEGG pathway enrichment pointed ribosome, the MAPK family, and pro-inflammatory pathways as significantly enriched in Zrsr2 m/m Tet2 −/− cells. Finally, KEGG pathway enrichment of alternative splicing targets identified the MAPK family and the Fanconi anemia pathway as the most altered targets in Zrsr2 m/m Tet2 −/− cells. Importantly, a total of 9 genes related to the MAPK pathway were identified as mis-spliced, from which Dusp1, Tgfbr2, and Fgf11 were validated in this study. Summary/Conclusion: In this study, we broaden our previous report and show that concurrent mutations in Zrsr2 and Tet2 dysregulate normal gene expression and cause aberrant mRNA splicing. Gene expression analysis identified ribosome function, inflammation, and migration/motility as the most altered processes in Zrsr2 m/m Tet2 −/− cells. Alternative splicing analysis identified the MAPK and the Fanconi Anemia pathway as key targets of aberrant splicing. All in all, gene expression dysregulation and aberrant mRNA splicing disturb important biological pathways and drive the molecular pathomechanism in Zrsr2 m/m Tet2 −/− mice. S162: SOMATIC GENETIC LANDSCAPE IN GATA2 DEFICIENCY PATIENTS L. Largeaud1 2,*, M. Collin3, N. Monselet4, F. Vergez5, L. Larcher6, P. Hirsch7, N. Duployez8, J. Bustamante9, C. Bellanné-Chantelot10, J. Donadieu11, F. Sicre de Fontbrune12, M. Nolla13, C. Fieschi14, F. Delhommeau7, E. Delabesse15, M. Pasquet13 1Clinical Haematology laboratory, Toulouse Hospital; 2UMR1037, Cancer Research Center of Toulouse, Toulouse, France; 3Haematology Department, Institute of Cellular Medicine Newcastle University, Newcastle, United Kingdom; 4Bureau des essais cliniques, Claudius Rigaud Institut; 5Haematology Department, Toulouse Hospital, Toulouse; 6Clinical Haematology laboratory, Saint Louis Hospital APHP; 7Clinical Haematology laboratory, Saint-Antoine Hospital, APHP, Paris; 8Clinical Haematology laboratory, CHU Lille, Lille; 9Unité centre d’études des déficits immunitaires, Necker Hospital APHP; 10Centre de génétique moléculaire et chromosomique, Hôpital Pitié-Salpêtrière, APHP; 11Haematology Department, Trousseau Hospital, APHP; 12Service d’hématologie greffe, Saint-Louis Hospital, APHP, Paris; 13Service d’hématologie et immunologie pédiatrique, Toulouse Hospital, Toulouse; 14Immunologie clinique, Saint-Louis Hospital, APHP, Paris; 15Clinical Hematology laboratory, Toulouse Hospital, Toulouse, France Background: Heterozygous germline GATA2 mutations strongly predispose to myeloid malignancies, immunodeficiency, and/or lymphedema. The progression towards haematological diseases seems to be correlated with the acquisition of molecular and cytogenetic abnormalities. Aims: Our study therefore focuses on the biological characterization of the different progression stages of germline GATA2 deficiency patients and their correlation with clinic features. Methods: We describe here a cohort of 78 patients (62 from the French-Belgian cohort and 16 British patients). Molecular analysis by next generation sequencing (targeting 90 genes frequently mutated in myeloid malignancies with a sensitivity = 1%) was performed in 76 patients. Cytogenetic analyses were determined for 76 patients. Results: Median age of our cohort was 21.8 years [6months–62years]. 44 had missense mutations, 32 had null mutations, one intronic and one synonymous mutation. Our results showed a trend toward a younger age at diagnosis in patients harboring null mutations compared to patients with missense mutations (13 vs 17 years, p=0,086) and more chronic infections during follow-up (74% vs 25%, p<0,001). 55 karyotypic abnormalities were identified including monosomy 7 (29%), trisomy 8 (16%) and der (1;7) (9%). Moreover, 141 somatic mutations were identified targeting STAG2 (38%), ASXL1 (13%), SETBP1 (8%), EZH2 (4%) and RUNX1 (3%) genes. To better stratify patients, we defined 3 spectra based on morphological bone marrow analysis: 12 (15%) patients had spectrum 0 (normal bone marrow), 47 (60%) had spectrum 1 (hypoplastic marrow and/or low-grade myelodysplasia (MDS)) and 19 (25%) were classified as spectrum 2 (MDS with excess blasts, AML and CMML). We found a genotype-phenotype correlation: spectrum 1 is more associated with null mutations and spectra 0 and 2 with missense mutations (p=0.023). Patients in spectrum 0 exhibited no acquired molecular and karyotypic abnormalities. Spectrum 2 was enriched with mutations of SETBP1, RAS pathway genes, and RUNX1 as well as more other cytogenetic abnormalities excluding -7, tri8 and der(1;7) (p<0,001). The proportion of monosomy 7 was higher in spectrum 2 than in spectrum 1 without reaching the significance level (47% vs 28%). Conversely, STAG2 mutations were mainly found in spectrum 1. 53 STAG2 mutations were identified in 25 patients (1 to 8 mutations per patient), mostly at low mutated cell fraction (median: 6%). Follow-up of 3 patients with STAG2 clones in spectrum 1 showed no progression toward spectrum 2. Moreover, clonal hierarchy of 3 patients with STAG2 mutations in spectrum 2 showed that STAG2 mutations were subclonal events. Our results suggested that STAG2 mutation did not act as driver and could be related to clonal hematopoiesis. Interestingly, Stag2 KO mice showed an enrichment of GATA2 binding motif (Ochi et al. Cancer Discov 2020) which could lead to an increase of GATA2 activity. We speculate that in GATA2 deficiency syndrome, STAG2 mutations could induce a mechanism of indirect Somatic Genetic Rescue (SGR) by compensating the loss of GATA2 activity induced by the mutated allele. Image: Summary/Conclusion: This study reported that STAG2 mutations were recurrent in GATA2 deficiency clonal hematopoiesis. Some genetic abnormalities are associated with the leukemic transformation stage such as SETBP1, RAS pathway genes and other cytogenetic abnormalities. An improved ability to identify patients with high risk of developing leukemic transformation has the potential to improve clinical outcomes and help clinicians determine the optimal timing of bone marrow transplantation. S163: EFFICACY OF JAK 1/2 INHIBITION IN MURINE IMMUNE BONE MARROW FAILURE E. Groarke1,*, X. Feng1, N. Aggarwal1, A. L. Manley1, Z. Wu1, S. Gao1, B. Patel1, J. Chen1, N. Young1 1Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, United States of America Background: Immune aplastic anemia (AA) is a severe blood disorder characterized by cytotoxic T-lymphocyte mediated stem cell destruction. Therapies including hematopoietic stem cell transplant and immunosuppression are effective but entail costs and risks, and are not effective or possible in all patients. The Janus Kinase (JAK) 1/2 inhibitor ruxolitinib (RUX) suppresses cytotoxic T cell activation and inhibits production of interferon gamma and tumor necrosis factor alpha in models of graft-versus-host disease. Aims: Assess RUX in murine immune AA for potential therapeutic benefit. Methods: In a murine major histocompatibility complex mismatched C67BL/6(B6) to CByB6F1 lymph node (LN) cell infusion AA model, and a C.B10 minor histocompatibility antigen mismatched B6 to C.B10 LN cell infusion AA model, RUX was administered as a food additive (Rux-chow), which achieves therapeutic levels in 48-72 hours after administration. Control BMF mice received chow without RUX. Animals were fed with Rux-chow two days before LN cell infusion as a prophylaxis (BMF+RUXD-2), or two days after LN cell infusion as therapy (BMF+RUXD+2). Recipient mice were either bled and euthanized at day 14 following LN infusion to collect tissues or were kept for 56 days to record animal survival. Blood counts were measured biweekly. Samples for flow cytometry, histology, and gene expression assays were collected. Results: In both AA murine models, RUX attenuated bone marrow hypoplasia, ameliorated peripheral blood pancytopenia, and prevented mortality when used either prophylactically or therapeutically. Treated mice had significantly higher blood counts (neutrophils, red blood cells, hemoglobin, platelets) as well as bone marrow (BM) and RBM (residual BM, excluding T-cells), and cellularity relative to control BMF mice. RUX suppressed infiltration, proliferation and activation of effector T cells in the bone marrow and mitigated Fas-mediated apoptotic destruction of target hematopoietic cells. All mice who received RUX were alive at 56 days while control BMF mice all died (Figure 1). With the exception of two mice with low RBC at day 56, discontinuing Rux-chow at day 28 or day 42 did not affect animal blood counts measurements, nor survival. Gene expression in mice who received RUX revealed downregulated T cell function and JAK/STAT pathway-related genes (Stat1, Stat3, Stat4, Fas, Ly6a, Infg, Gzmb, Gzma, Gzmk, Infgr1, Il2rb, Il2rg, and Lag3). On network analysis of differentially expressed genes in downregulated pathways, Stat1 and Ifn-g genes were at the center of the network, connecting immune responses and cell cycle pathways. When toxicity was assessed, RUX exerted modest suppression of lymphoid and erythroid hematopoiesis in normal and irradiated CByB6F1 mice, but the drug showed impressive clinical efficacy despite this. Image: Summary/Conclusion: RUX showed striking therapeutic efficacy, improving blood counts and prolonging survival in two different BMF murine models. In patients, clonal T cell expansion and activation, IFN-g and TNF-α upregulation, and Fas mediated cell death, lead to severe BM destruction and the development of AA. Our study demonstrated that JAK 1/2 inhibition with RUX produced its suppressive effects by inhibiting T cell activation, reducing inflammatory cytokine secretion, and limiting FasL/Fas-mediated BM destruction. Given these results, JAK 1/2 inhibition with RUX is an exciting and novel potential therapy for BMF patients based on a well characterized mechanism of action. S164: CLONAL DYNAMICS, IMMUNE PHENOTYPES, AND TARGETS OF BONE MARROW-INFILTRATING T CELLS IN ACQUIRED APLASTIC ANEMIA A. Ben Hamza1,*, C. Welters1, M. Brüggemann2, K. Dietze1, L. Bullinger1 3, T. H. Brümmendorf4 5, J. Strobel6, H. Hackstein6, K. Dornmair7, F. Beier4 5, L. Hansmann1 3 1Department of Hematology, Oncology and Cancer Immunology, Charité - Universitätsmedizin Berlin, Berlin; 2Department of Medicine II, Hematology and Oncology, University Hospital Schleswig Holstein, Kiel; 3German Cancer Consortium (DKTK), partner site Berlin, and German Cancer Research Center (DKFZ), Heidelberg; 4Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University; 5Center for Integrated Oncology, Aachen Bonn Cologne Düsseldorf (CIO ABCD), Aachen; 6Department of Transfusion Medicine and Haemostaseology, University Hospital of Erlangen, Friedrich Alexander University Erlangen-Nürnberg (FAU), Erlangen; 7Institute of Clinical Neuroimmunology, Biomedical Center and University Hospital, Ludwig-Maximilian-University Munich, Munich, Germany Background: Acquired aplastic anemia is a hematological disease characterized by hypocellular bone marrow with insufficient hematopoiesis and pancytopenia. High response rates to immunosuppressive therapy with anti-thymocyte-globulin and cyclosporin A suggest a key role for T cells in disease pathogenesis. However, bone marrow-infiltrating clonal T cell expansion, associated immune phenotypes, and T cell targets remain poorly understood. Aims: To defined clonal T cell expansion, associated immune phenotypes, and potential T cell targets in bone marrow specimens of aplastic anemia patients before and after immunosuppressive therapy. Methods: Within bone marrow of 15 patients with acquired aplastic anemia, we determined T cell clone-associated immune phenotypes by multi-parameter flow cytometry single cell index sorting for subsequent T cell receptor (TCR) αβ and cytokine/transcription factor sequencing. T cell clones of 10 patients were monitored before and after immunosuppressive therapy (median time span: 9 months) by TCRβ repertoire sequencing. Twenty-seven TCRs of expanded clones were re-expressed in reporter cell lines to determine recognition of autologous hematopoietic precursor cells. Results: We detected oligoclonal T cell expansion in all patients and 92.9% of all expanded clones were CD8+, while only 9.6% were CD4+. Frequencies of dominant T cell clones varied between patients (1.1-20.6% of CD8+ T cells). Expanded CD8+ clones were almost exclusively CCR7- PD1- TIM3- CD39- and frequently expressed cytokines and transcription factors associated with cytotoxic effector differentiation (IFNG, GZMB, PRF1, TBX21). More than 50% of the CD45RA+ clones (TEMRA) were CD57+, while CD45RA- clones (TEM) were mainly CD28+ and smaller in size. While TEM clones showed similar frequencies in bone marrow and peripheral blood, TEMRA displayed higher localization-dependent differences in size. Interestingly, almost all (91.1%) expanded clones with frequencies >1% of all T cells persisted in the bone marrow after immunosuppressive therapy independent of clinical response. Similarly, highly expanded CD4+ clones, albeit low in numbers, were stable in frequencies before and after therapy. To determine targets of CD8+ T cell clones with strong expansion, we re-expressed twenty-seven TCRs of eight patients and found five to be specific for immunodominant epitopes of cytomegalovirus. Strikingly, two TCRs recognized hematopoietic progenitor cells expanded from CD34-enriched autologous bone marrow. Summary/Conclusion: We tracked T cell clonotypes and associated immune phenotypes under immunosuppressive therapy at the single cell level. Expanded CD8+ clones showed cytotoxic effector differentiation and persisted in the bone marrow after immunosuppressive therapy even in situations of therapeutic response, challenging the hypothesis that clinical response follows the decline of highly expanded autoreactive T cell clones. We identified two expanded T cell clones that recognized hematopoietic progenitor populations providing experimental proof of concept that expanded autoreactive T cell clones can be critically involved in aplastic anemia pathogenesis. FB and LH contributed equally. S165: INTEGRATED GENETIC DIAGNOSTICS OF PATIENTS WITH EARLY ONSET OF DE NOVO MYELODYSPLASTIC SYNDROMES. E. Attardi1,*, L. Tiberi2, D. Formicola3, R. Artuso3, V. Santini1 1MDS Unit, Hematology, AOU Careggi - Department of Experimental and Clinical Medicine; 2Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence; 3Medical Genetics Unit, Meyer Children’s University Hospital, Florence, Italy Background: Cytogenetic and molecular alterations determine myelodysplastic syndrome (MDS) prognosis and must be evaluated during disease history. Relevance of germline (GM) predisposition in MDS was stressed in WHO classification, but its actual incidence is probably underestimated. Recently, the widespread availability of large-scale genomic sequencing techniques has facilitated the investigation of GM variants, and the identification of novel GM variants predisposing to MDS is expected. In de novo MDS patients (pts) with an early onset of the disease (age ≤55 yrs) when no syndromic signs drive the genetic assessment, whole exome sequencing (WES) analysis of bone marrow (BM) and saliva can constitute an optimal diagnostic procedure, improving the performance of targeted next-generation sequencing (t-NGS) approach. Aims: We adopted integrated diagnostics to define the cytogenetic profile, and WES to investigate possible GM predisposition of a cohort of 30 de novo MDS pts with an unusual young age at disease onset (≤55 yrs). Standard karyotype (SK) was paralleled by low coverage whole genome sequencing (lc-WGS) on plasma cell-free DNA (cf-DNA), as non-invasive screening test for detection of structural chromosome abnormalities. Methods: Patient BM-DNA / peripheral blood DNA (PB-DNA) was analysed by WES and compared to DNA extracted from saliva (S-DNA) samples as control for GM variants. To identify high-confidence MDS variants, we attributed an internal deleteriousness score to define variants, from “of uncertain significance” to “pathogenic” (ACMG guidelines). Variants were suspected as GM where variant allele frequency was > 0.3 and had been confirmed for genes detected in both S-DNA and BM/PB-DNA. To confirm the somatic nature of some variants, deep t-NGS of 27 myeloid custom panel gene was applied in 26/30 MDS cases on BM-DNA. SK and lc-WGS (0.2x) on plasma cf-DNA were assessed in 27/30 pts. Results: The small cohort of “young” MDS pts here analysed (Table 1). The majority of pts, 17/30 (57%) presented at least one high-confidence variant evaluated as GM for MDS. A total of 26 GM variants were recognized. Patients could be grouped on the basis of the type of variant as follows: 11/30 pts (37%) presented variants in genes involved in DNA repair defects and cancer predisposition (ATM, ATR, FANCA, FANCM, PARN, BRCA1, BRCA2, CHEK2); 4/30 (13%) had variants involved in genetic predisposition to myeloid neoplasm - GATA2, ANKRD26 and RBBP6 - the latter classically associated to MPN familial cases; one presented compound heterozygous variants in SBDS; 4 presented variants related to hereditary red blood cell defects. In our cohort, we identified cases with a complex mode of inheritance (4/17) as well as high confidence GM variants in 4 pts in 2 new interesting cancer predisposition genes: RBBP6 and PARN. Lc-WGS of plasma cf-DNA confirmed all the cytogenetic alterations found by SK (8/27) and identified a 21q22.12 deletion involving RUNX1 locus in a patient, not revealed by SK. Image: Summary/Conclusion: By means of an integrated diagnostics, defined as the convergence of diagnostic techniques with advanced information technology (WES, t-NGS, lc-WGS), as a proof of principle, it was possible to fully characterize the genetic asset of a particular subgroup of 30 MDS cases. We also demonstrated that lc-WGS of plasma cf-DNA has an excellent sensitivity allowing to perform periodic cytogenetic evaluations in MDS pts without invasive maneuvers. We showed that, by the use of WES, GM alterations were demonstrated in a small cohort of “young” non-syndromic MDS pts in 57% of cases, a proportion unexpectedly high. S166: MAGROLIMAB IN COMBINATION WITH AZACITIDINE FOR PATIENTS WITH UNTREATED HIGHER-RISK MYELODYSPLASTIC SYNDROMES (HR MDS): 5F9005 PHASE 1B STUDY RESULTS D. A. Sallman1,*, M. M. Al Malki2, A. S. Asch3, E. S. Wang4, J. G. Jurcic5, T. J. Bradley6, I. W. Flinn7, D. A. Pollyea8, S. N. Kambhampati9, T. N. Tanaka10, J. F. Zeidner11, G. Garcia-Manero12, D. Jeyakumar13, L. Gu14, A. Tan14, M. Chao14, C. O’Hear14, I. Lal14, P. Vyas15, N. Daver12 1Moffitt Cancer Center, Tampa; 2City of Hope National Medical Center, Duarte; 3Stephenson Cancer Center, Oklahoma University Health, Oklahoma City; 4Roswell Park Comprehensive Cancer Center, Buffalo; 5Columbia University Medical Center, New York; 6Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami; 7Tennessee Oncology, Nashville; 8University of Colorado School of Medicine, Denver; 9Sarah Cannon Research Institute, Kansas City; 10University of California San Diego Moores Cancer Center, San Diego; 11Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill; 12The University of Texas MD Anderson Cancer Center, Houston; 13University of California Irvine, Orange; 14Gilead Sciences, Inc., Foster City, United States of America; 15Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom Background: Magrolimab is a monoclonal antibody that blocks CD47, a “don’t eat me” signal overexpressed on cancer cells. CD47 blockade by magrolimab induces macrophage-mediated phagocytosis of tumor cells and is synergistic with azacitidine (AZA) in the upregulation of “eat me” signals. A high unmet need exists to build on current standard-of-care AZA frontline therapy to increase efficacy while maintaining a tolerable safety profile in patients (pts) with HR MDS. Aims: To report the final safety/tolerability and efficacy data from a phase 1b trial of magrolimab + AZA in pts with untreated HR MDS (NCT03248479). Methods: Pts with previously untreated intermediate, high, or very high–risk MDS per the Revised International Prognostic Scoring System (IPSS-R) received magrolimab IV as a priming dose (1 mg/kg) followed by ramp-up to a 30-mg/kg QW or Q2W maintenance dose. AZA 75 mg/m2 was administered IV or SC on days 1-7 of each 28-day cycle. Primary endpoints were safety/tolerability and complete remission (CR) rate. Results: A total of 95 pts (median age, 69 y [range, 28-91 y]) were treated. IPSS-R risk was intermediate, high, or very high in 27%, 52%, and 21% of pts. MDS was therapy related in 22% of pts; 26% had a TP53 mutation, and 62% had poor-risk cytogenetics. Median number of cycles was 6 (range, 1-27). The most common treatment-emergent adverse events (TEAEs) included constipation (68%), thrombocytopenia (55%), anemia (52%), neutropenia (47%), nausea (46%), and diarrhea (44%). The most common grade 3/4 TEAEs included anemia (47%), neutropenia (46%), thrombocytopenia (46%), and white blood cell count decreased (30%). Six pts discontinued treatment due to AEs. The 60-day mortality rate was 2%. Median hemoglobin change from baseline (BL) at first postdose sample was −0.7 g/dL (range, −3.1 to 2.4 g/dL). CR and objective response (OR) rates were 33% and 75%, with 31% of OR-evaluable pts with abnormal cytogenetics at BL having cytogenetic CR. Median time to first OR, duration of CR (DCR), duration of OR, and progression-free survival (PFS) were 1.9, 11.1, 9.8, and 11.6 mo. Overall survival (OS) rates at 12 and 24 mo were 75% and 52%; median OS was not reached (NR) with 17.1 mo of follow-up (figure). In pts evaluated with sequential whole-exome sequencing with a variant allele frequency (VAF) cutoff of 5%, 3 of 3 pts with TP53 mutation who achieved CR had TP53 VAF <5% by cycle 5 day 1. Favorable outcomes were observed both in pts with TP53 mutation (CR rate, 40%; median OS, 16.3 mo) and wild-type TP53 (CR rate, 31%; median OS, NR) (table). Table. Outcomes in patients with previously untreated HR MDS treated with magrolimab + AZA Outcome All patients N=95 a Wild-type TP53 n=61 TP53 mutation n=25 OR rate, % b 75 79 68 CR rate (95% CI), % 33 (23-43) 31 (20-44) 40 (21-61) Marrow CR rate, % 32 38 20 Stable disease with HI rate, % 11 10 8 Median DCR (95% CI), mo 11.1 (7.6-13.4) 12.9 (8.0-NR) 7.6 (3.1-13.4) Marrow CR rate with HI/any HI, % 17/59 20/61 12/56 Converted to red blood cell transfusion independence, % 14 10 24 Median PFS (95% CI), mo 11.6 (9.0-14.0) 11.8 (8.8-16.6) 11.0 (6.3-12.8) Median OS (95% CI), mo NR (16.3-NR) NR (21.3-NR) 16.3 (10.8-NR) a a 9 patients had missing TP53 status. b Defined as CR+PR + marrow CR+SD with HI. HI, hematologic improvement. Image: Summary/Conclusion: Magrolimab + AZA was well tolerated with promising efficacy in pts with untreated HR MDS, including those with TP53-mutated and –wild-type disease. A phase 3 trial of magrolimab/placebo + AZA (ENHANCE: NCT04313881) is ongoing. S167: PREDICTION OF RELAPSE AFTER ALLOGENEIC STEM CELL TRANSPLANTATION USING INDIVIDUALIZED MEASURABLE RESIDUAL DISEASE MARKERS; THE PROSPECTIVE NORDIC STUDY NMDSG14B M. Tobiasson1,*, T. Pandzic2, J. Illman3, L. Nilsson4, S. Weström2, K. Sollander2, E. Ejerblad5, A. Olsnes Kittang6, G. Olesen7, O. Werlenius8, A. Björklund9, J. Wiggh1, C. lindholm1, F. Lorentz10, B. Rasmussen11, J. Cammenga12, D. Weber13, D. Grönnås14, M. Dimitriou15, S. Kytölä16, G. Walldin15, P. Ljungman9, K. Groenbeck17, S. Mielke9, S. E. Jacobsen15, F. Ebeling3, L. Cavelier2, L. Smidstrup Friis17, I. Dybedal18, E. Hellström-Lindberg1 1Hematology, Karolinska University Hospital, Stockholm; 2Department of Immunology, Genetics and Pathology, Science for life, Uppsala, Sweden; 3Hematology, Helsinki University Hospital, Helsinki, Finland; 4Hematology, Lund University Hospital, Lund; 5Hematology, Akademiska sjukhuset, Uppsala, Sweden; 6Hematology, Haukeland University Hospital, Bergen, Norway; 7Hematology, Arhus University Hospital, Århus, Denmark; 8Hematology, Sahlgrenska University Hospital, Gothenburg; 9Centre for allogeneic stem cell transplantation, Karolinska University Hospital, Stockholm; 10Hematology, Norrlands University Hospital, Umeå; 11Hematology, Örebro University Hospital, Örebro; 12Hematology, Universitetssjukhuset, Linköping, Sweden; 13Hematology, Odense University Hospital, Odense, Denmark; 14Institution for environmental medicine; 15Centre for hematology and regenerative medicine, Institution for medicine, Huddinge, Karolinska Institute, Stockholm, Sweden; 16HUS Diagnostic Center, HUSLAB, Helsinki University Hospital, Helsinki, Finland; 17Hematology, Rigshospitalet, Copenhagen, Denmark; 18Hematology, Oslo University Hospital, Oslo, Norway Background: One third of patients with myelodysplastic syndrome (MDS) relapse after allogeneic stem cell transplantation (HCT). Early detection of impending relapse would enable pre-emptive treatment and potentially reduce relapse risk but is limited by the lack of sensitive markers for measurable residual disease (MRD). We developed a pipeline where patient-specific mutations, as determined by a myeloid next generation sequencing (NGS) panel are tracked using digital droplet PCR (ddPCR). Aims: To evaluate if personalized MRD detection by ddPCR can predict clinical relapse earlier than conventional methods. Methods: The prospective study (NCT02872662) enrolled patients with MDS, MDS/MPN or MDS-AML with < 30% marrow blasts undergoing HCT. Patients were included before HCT, and serial bone marrow (BM) samples were collected every third month post-HCT for 2 years. Peripheral blood (PB) samples were collected monthly. MRD results were not available for the treating physician. Results: We screened 286 pts between 2016 and 2020, whereof 20 were excluded mainly due to lack of genetic aberration or no HCT performed. 266 pts were included from 12 HCT centers. Median age was 64 (18-78) years and 59% were male. Myeloid panel NGS screening identified a median of 2 (0-9) mutations. The most common mutations were TET2 (n=85), ASXL1 (n=73) and SRSF2 (n=59). Median time of follow up was 886 (4-1934) days. Sixty pts relapsed after a median of 189 (53-1281) days and 46 died due to non-relapse mortality after a median of 121 (4-1036) days. Remaining pts (n=160) were in continuous complete remission (CCR) after a median follow-up of 1053 (479-1934) days. Estimated 1 and 2y overall survival was 79%, and 71%, respectively, while estimated 1 and 2y relapse-free survival (RFS) was 75% and 66%, respectively. MRD data was missing in 46 pts; no post-HCT samples available (n=15), no mutation detected (n=14) and difficulties to design ddPCR primers (n=11). 221 pts were available for MRD analysis with a median number of 4 (0-13) and 5 (0-23) samples from BM and PB, respectively. Of 53 clinical relapses with MRD results available, 42 were preceded by pos MRD (>0.1%) with a median of 70 (range 20-425) days between first pos MRD and clinical relapse. For the 11 remaining pts, 8 were inadequately sampled with a median time of 189 (82-397) days between last sampling and clinical relapse. One patient had an extramedullary relapse only. Of 31 pts who died without relapse, 19 were consistently MRD neg, while 5 were borderline positive (MRD > 0.1% and <0.5%) during the first 100 days but negative thereafter. Four MRD+ patients died without clinical relapse. Three pts were initially MRD+ but turned negative, all of which had chronic GVHD (cGVHD). Of 136 CCR patients, 94 were consistently MRD neg; 26 were borderline pos (MRD > 0.1% and <0.5%) during the first 100 days followed by neg samples; 16 were MRD positive (either > 0.5% during the first 100d or > 0.1% after 100d) of which 10 had a transition from pos to neg samples (all had cGVHD); one patient was treated for a molecular relapse detected by clinical routine method (FISH) and five patients were MRD positive at time of last follow-up. MRD used as a time-dependent co-variate was negatively associated with RFS (HR 7.1, p<0.01). Estimated cumulative incidence of relapse and non-relapse mortality 2y after pos MRD was 60% and 7% respectively (see figure). Image: Summary/Conclusion: We report the development of a highly functional personalized MRD pipeline based on patient-specific mutations showing a high sensitivity to predict relapse and relapse-free survival. S168: ERYTHROPOIETIN STIMULATION AGENTS SIGNIFICANTLY IMPROVES OUTCOME IN LOWER RISK MDS. H. Garelius1,*, A. Smith2, T. Bagguley2, A. Taylor2, P. Fenaux3, D. Bowen4, A. Symeonidis5, M. Mittelmann6, R. Stauder7, J. Čermák8, G. Sanz9, S. Langemeijer10, L. Malcovati11, U. Germing12, R. Itzykson13, A. Guerci-Bresler14, D. Culligan15, I. Kotsianidis16, K. Koinig Mag17, C. van Marrewijk18, S. Crouch2, T. de Witte19, E. Hellström-Lindberg20 1Section of Hematology, Specialist Medicine, Sahlgrenska University hospital, Göteborg, Sweden; 2Department of Health Sciences, University of York, York, United Kingdom; 3Service d’Hematologie, Hôpital Saint-Loius, Assistance Publique des Hopitaux de Paris (AP-HP) and Universite Paris; 7, Paris, France; 4St.James’s Institute of Oncology, Leeds Teaching Hospitals, Leeds, United Kingdom; 5Department of Medicine, Division of hematology, University of Patras Medical Scholl, Patras, Greece; 6Department of Medicine, Tel Aviv Sourasky (Ichilov) Medical Center and Sackler Medical Faculty, Tel Aviv University, Tel Aviv, Israel; 7Department of Internal Medicine V (Haematology and Oncology), Innsbruck Medical University, Innsbruck, Austria; 8Dep. of Clinical Hematology, Institute of Hematology & Blood Transfusion, Prague, Czechia; 9Department of Haematology, Hospital Universitario y Politécnico La Fe, Valencia, Spain; 10Department of Hematolog, Radboud university medical center, Nijmegen, Netherlands; 11Department of Hematology Oncology, Fondazione IRCCS Policlinico San Matteo,University of Pavia, Pavia, Italy; 12Department of Haematology, Oncology and Clinical Immunology, Universitätsklinik Düsseldorf, Düsseldorf, Germany; 13Service d’Hématologie, Hôpital Saint-Louis, Assistance Publique des Hôpitaux de Paris (AP-HP) and Université Paris; 7, Pari, Paris; 14Service d’Hématologie, Centre Hospitalier Universitaire Brabois Vandoeuvre, Nancy, France; 15Department of Haematology, Aberdeen Royal Infirmary, Aberdeen, United Kingdom; 16Dep. of Hematology, Democritus University of Thrace Medical School, University Hospital of Alexandroupolis, Alexandroupolis, Greece; 17Department of Internal Medicine V (Hematology and Oncology), Medical University Innsbruc, Innsbruck, Austria; 18Department of Haematology, Radboud university medical center; 19Department of Tumor Immunology - Nijmegen Center for Molecular Life Sciences, Radboud university medical cente, Nijmegen, Netherlands; 20Department of Medicine, Div. Hematology, Karolinska Institutet, Stockholm, Sweden Background: The EUMDS Registry started in 2008 as a prospective, non-interventional longitudinal study, enrolling newly diagnosed patients with IPSS low or intermediate-1 MDS from 16 European countries and Israel. Aims: The aim of the present analysis was to see how treatment with or without Erythropoietin Stimulating Agents (ESAs) and/or red blood cell transfusions (RBCT) impact overall survival (OS) and quality of life (QoL). Methods: Patient management was recorded electronically every 6 months (“visit”) in a central database, including treatment, transfusions, blood values, and health related quality of life (HRQoL) using the EQ-5D 3-Level index and Visual Analog Scale (VAS). Patients were eligible to be included in the analyses if their hemoglobin was recorded as less than <10 g/dl at a visit. To overcome potential confounding by non-random allocation of ESA treatment, propensity score matching was performed to ensure that treated and untreated patients had similar characteristics. Only patients with comparable propensity scores were included in the analyses to estimate the effects of ESA treatment on outcomes using standard time to event analyses; OS was estimated from the first visit a Hb value of <10g/dl was recorded. OS was examined for patients treated with ESA stratified by their transfusion status prior to commencing ESA treatment (no RBCT, <4 units, ≥4 units). Patients were separated into 4 groups at each clinical visit, depending on the treatment received in the interval leading up to that visit; no ESA nor RBCT, ESA only, ESA and RBCT and RBCT only. HRQoL at each visit according to the treatment status was summarized for patients who had completed a questionnaire at visit 1 and 2; mean values were examined by treatment group. Results: Of 2562 patients registered by November 2021, 2448 were diagnosed before July 2019 and included in the analysis; these patients were divided into two groups: ESA untreated (n=1265) and ESA treated (n=1183). Patients whose Hb remained above 10g/dl were excluded leaving 529 untreated patients and 749 ESA treated; after propensity score matching was applied two comparable groups were produced: ESA untreated (n= 426) and ESA treated (n= 742). Median OS from reaching the eligibility criteria in the ESA treated vs untreated groups were 44.9 and 34.8 months respectively (Fig 1a), giving a clear survival advantage to the ESA-treated group. (p<0.003). In the ESA-treated group, OS was poorer in those who had been transfused prior to commencing ESA (Fig 1b, p<0.001). Fig 1c shows the number of patients at each visit who had been treated with transfusions or ESA; 647/1278 had received neither at visit 1, the figure shows the “flow” of patients by treatment for the first 6 visits. HRQoL was examined for the 695 patients who had completed a questionnaire at both visit 1 and 2 up to visit 6; differences were seen by treatment (Fig 1d). Patients who had received no treatment reported, on average, the highest mean HRQoL, in contrast, patients who had RBCT had the lowest (p<0.001). Image: Summary/Conclusion: This unique large prospective registry study clearly shows a significant survival advantage for lower-risk MDS patients exposed to ESA treatment at onset of anemia (Hb <10g/dL) but before onset of transfusion therapy, strongly supporting recommendations to start ESA treatment early. The effect on patients with an early transfusion need warrants further studies. Moreover, ESA exposure is associated with maintained QoL, while RBCT development with or without ESA exposure is associated with significantly deterioration in QoL. S169: CLINICAL AND MOLECULAR MARKERS FOR PREDICTING RESPONSE TO ROMIPLOSTIM TREATMENT IN LOWER-RISK MYELODYSPLASTIC SYNDROMES A. S. Kubasch1 2 3,*, A. Giagounidis2 3 4, G. Metzgeroth5, A. Jonasova6, R. Herbst7, J. M. T. Diaz8, B. De Renzis9, K. S. Götze2 3 10, M.-L. Huetter-Kroenke11, M.-P. Gourin12, B. Slama13, S. Dimicoli-Salazar14, P. Cony-Makhoul15, K. Laribi16, S. Park17, K. Jersemann18, D. Schipp19, K. H. Metzeler20, O. Tiebel21, K. Sockel2 22, S. Gloaguen2 3, A. Mies22, F. Chermat23, C. Thiede22, R. Sapena23, R. F. Schlenk24 25, P. Fenaux3 23 26, U. Platzbecker2 3 20, L. Ades3 23 26 1Department of Hematology, Cellular Therapy and Hemostaseology, University Hospital Leipzig; 2German MDS Study Group (D-MDS); 3The European Myelodysplastic Syndromes Cooperative Group (EMSCO), Leipzig; 4Department of Oncology, Hematology and Palliative Care, Marien Hospital, Düsseldorf; 5Department of Hematology and Oncology, University Medical Centre, Mannheim, Germany; 61st Medical Department - Hematology, General Hospital, Prague, Czechia; 7Medizinische Klinik III, Klinikum Chemnitz, Chemnitz, Germany; 8Department of Hematology and Oncology, CHU de Poitiers, Poitiers; 9Service d’Hématologie Clinique Adulte, Clermont Ferrand, France; 10Department of Medicine III, Technical University of Munich, Munich; 11Department of Hematology, Oncology, and Tumor Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany; 12CHU Limoges, Limoges; 13Service d’Hématologie, Centre Hospitalier d’Avignon, Avignon; 14University Hospital Bordeaux, Pessac; 15Centre Hospitalier Annecy-Genevois, Pringy; 16Centre Hospitalier Du Mans, Le Mans; 17Department of Hematology, CHU Grenoble, Grenoble, France; 18GWT-TUD GmbH, Dresden; 19DS-Statistics, Rosenthal-Bielatal; 20Department of Hematology, Cellular Therapy and Hemostaseology, Leipzig University Hospital, Leipzig; 21Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty, Technical University Dresden; 22Department of Internal Medicine I, University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany; 23Groupe Francophone des Myélodysplasies, Paris, France; 24Department of Internal Medicine V, University Hospital of Heidelberg; 25NCT-clinical trials office, German Cancer Research Center, Heidelberg, Germany; 26Hématologie Clinique, Hôpital Saint-Louis, Paris, France Background: In about half of patients with lower-risk (LR) myelodysplastic syndromes (MDS), thrombocytopenia is present at the time of diagnosis, being associated with shortened survival and a higher risk of progression to acute myeloid leukemia (AML). Romiplostim (ROM), a thrombopoietin receptor agonist (TPO-RA), has shown safety and clinical efficacy in prospective trials in LR-MDS. Post-hoc analyses have demonstrated hematologic improvement of platelets (HI-P) after ROM treatment contingent on endogenous thrombopoietin (TPO) levels and platelet transfusion events (PTE) (Sekeres et al. BJH 2014). Aims: The prospective EUROPE multicenter phase 2 trial (NCT02335268) investigated the predictive value of biomarkers like endogenous TPO levels, PTE and molecular markers on the clinical efficacy of single-agent ROM treatment within the `European Myelodysplastic Syndromes Cooperative Group` (EMSCO) network. Patients with IPSS low or intermediate 1 risk were eligible if baseline bone marrow blast count was <5% (central morphology) and platelet count was ≤30 Gpt/L or ≤50 Gpt/L in case of a bleeding history. Methods: According to a previously published model of response to TPO-RA (Sekeres et al. BJH 2014), patients were assigned into two different cohorts at the time of screening based on previous PTE and centrally assessed TPO serum levels (cohort A: TPO<500 ng/l and PTE<6 units/past year; cohort B: TPO>500 ng/l, and/or PTE36 units/past year). The primary efficacy endpoint was the rate of HI-P according to IWG 2006 criteria lasting for 38 weeks. ROM was initiated at a dose of 750 μg weekly by subcutaneous injection, the dose was adjusted based on the patient`s platelet counts. Results: From 2015 to 2019, a total of 77 patients were included at 29 different trial sites in Germany, France and the Czech Republic. Regarding the primary endpoint, 32 out of 77 (42%) responded (HI-P) with a numerically higher response rate in cohort A (47%, n=24) vs. cohort B (31%, n=8) (p=0.2953). At 16 weeks of ROM treatment, three (4%) and seven (9%) patients had additional neutrophil (HI-N) and erythroid (HI-E) responses, respectively. None of the patients achieved trilineage responses (HI-P, HI-E and HI-N). Median duration of response was significantly longer for patients in cohort A (351 days) compared to cohort B (315 days) (p=0.006, log-rank-test). Mutated SRSF2 was significantly more frequent in responders (41%) compared to non-responders (16%) (p=0.018, Fisher’s exact test). In patients with an SRSF2 mutation, the probability to achieve HI-P was 65% compared to 33% inpatients with SRSF2 wildtype (Figure 1A). Comparing responders vs. non-responders, we found no significant changes of variant allelic burden of variants detected pre- and post-ROM (Figure 1B). Finally, we developed a response prediction model to ROM therapy with the aim to improve personalized patient stratification in the future. The percentage of correctly predicted HI-P was highest for the model, which included the variables platelet count, SRSF2 mutation status and the hemoglobin level using the threshold of 11.4 g/dl and resulted in an overall accuracy of 70 % for a correct ROM response prediction (Figure 1A). Image: Summary/Conclusion: In conclusion, this prospective study confirms the efficacy and overall safety of ROM in this subgroup of LR-MDS patients with thrombocytopenia. To avoid overfitting of variables and to confirm our results, the here presented response prediction model needs to be validated in an external independent cohort. * U.P. and L.A. contributed equally to this study as senior authors S170: DYNAMIC INTERPLAY BETWEEN TUMOR AND MICRO-ENVIRONMENT DURING MYELOMA DISEASE PROGRESSION M. C. Köse1,*, I. Bergiers2, M. Malfait3, B. Heidrich4, D. De Maeyer2, N. Fourneau2, B. Verbist2, J. Van Houdt2, G. Vanhoof2, R. Verona4, M. Delforge5, J. Depaus6, N. Meuleman7, J. Van Droogenbroeck8, P. Vlummens9, C. J. Heuck4, N. Bahlis10, J. Caers1, T. Casneuf2 1Laboratory of Hematology, University of Liege, Liege; 2Janssen Research & Development, Beerse; 3Department of Applied Mathematics, Computer Science and Statistics, University of Ghent, Ghent, Belgium; 4Janssen Research & Development, Spring House, United States of America; 5University Hospital Leuven, Leuven; 6Department of Hematology, CHU UCL Namur, Yvoir; 7Service d’Hématologie, Université Libre, Brussels; 8Departmentof Haematology, AZ Sint-Jan Brugge-Oostende AV, Brugge; 9Department of Clinical Hematology, Ghent University Hospital, Ghent, Belgium; 10Department of Hematology and Oncology, University of Calgary, Calgary, Canada Background: Multiple Myeloma (MM) is an incurable plasma cell (PC) malignancy that evolves from two premalignant stages: Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). The disease progression has been characterized to be driven by intrinsic genomic events in the myeloma cells and by gradual dysregulation of the immune system. Aims: We investigated how the interplay between tumor cells with their microenvironment and the underlying complex and dynamic immune biology evolve during this process. Methods: Single cell multi-omics profiling, including RNA, B-cell receptor (BCR) and antibody barcode-tagged 10x sequencing, was conducted on human bone marrow (BM) aspirates collected at 6 Belgian centers from 4 cohorts: 31 healthy elderly and 28 MGUS, 32 SMM and 32 newly diagnosed MM. Mononuclear cell isolation, freezing and transport to central facilities was optimizedand data were integrated and filtered using Scanpy and Scirpy. The main immune cell types were identified from the RNA and antibody data using SingleR. Further functional subtyping was done using Leiden clustering. Differential pathway expression analysis was performed with Muscat and FGSEA. Results: From the tumor cell transcriptomes, our analyses confirmed the previously documented myeloma molecular hallmarks, such as MYC and IFN-a signaling, cell proliferation, energy metabolism and oxidative phosphorylation. Evidence was found for transcriptomic similarities and within-and between-patient malignant PC transcriptomic heterogeneity, as well as the existence of multiple transcriptomic clones in several patients. We observed a positive correlation between the antigen processing mechanism in the PCs with IFN response, suggesting that this mechanism associates with initiation of the immune recognition and activation against the tumor.The gradually increasing differential gene expression was also observed in the immune microenvironment: dysregulation of signaling pathways initiates early in MGUS and spreads throughout the various cell types surrounding the tumor cells. Cell population shifts were also found. In the CD1C+ DCs, that play a role in cancer immune control, a functional shift was observed that correlated with disease progression towards a more mature and antigen presenting phenotype with higher levels of CD83, HBEGF, MCL1 and CXCL16 as well as increased TNF-a pathway. Similarly, a shift was observed in the macrophage population, toward M1 state showing high IFN response along with expression of MS4A4A, STAT1, TNFSF13B and TRAIL in more severe disease. Interestingly, in the CD8+ T cells, we detected a pre-dysfunctional subpopulation with high expression of GZMK, activation markers CD69, CCL4, CXCR4 and genes associated with T cell pre-dysfunctionality NR4A2, RGS1, TOX and TIGIT, that was found to be associated with progression (Figure). In the CD4+ cytotoxic T cells, a proportion change was observed with more severe disease. Image: Summary/Conclusion: With the atlas of healthy, precursor and active MM patient BM samples, we generated a comprehensive and granular view of the various cell types involved in disease progression and provide evidence for early and gradually increasing immune dysregulation and activation of oncogenic driver pathways. Our data evidence the co-divergence and reciprocal stimulation of transcriptomes of tumor and microenvironment and support the postulation of microenvironment as a central modulator of cancer cell growth, survival and metastasis. S171: MOLECULAR CLUSTERS OF IGM MONOCLONAL GAMMOPATHIES PRESENT DISTINCT BIOLOGIC, IMMUNE AND METABOLIC FEATURES P. Mondello1,*, J. Paludo1, J. Novak1, K. Wenzl1, S. Jalali1, J. Krull1, E. Braggio2, S. Dasari3, M Manske1, J. Abeykoon1, S. Vivekananda1, P. Kapoor1, A. Paulus4, C. Reeder2, S. Ailawadhi4, A. Chanan-Khan4, R. Kyle1, M. Gertz1, Z.-Z. Yang1, A. Novak1, S. Ansell1 1Medicine, Mayo Clinic, Rochester; 2Medicine, Mayo Clinic, Phoenix; 3Bioinformatics, Mayo Clinic, Rochester; 4Medicine, Mayo Clinic, Jacksonville, United States of America Background: IgM MGUS and Waldenstrom Macroglobulinemia (WM) represent a wide range of conditions whose management varies from observation to immunochemotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. Aims: To shed light on the biology that may account for the clinical differences, we performed the first comprehensive multi-omics analysis of IgM monoclonal gammopathies. Methods: We used bone marrow (BM) CD19+CD138+ sorted cells and matched BM plasma from 32 pts (7 IgM MGUS, 25 WM) and 5 healthy controls to perform whole exome sequencing, RNA-seq, proteomic and metabolomic analysis. 7 matched WM and 4 IgM MGUS pts were also evaluated using mass cytometry (CyTOF). Results: Applying principal component analysis to gene expression profiling, most of WM pts clustered together, while a small subset of them grouped separately with MGUS pts, suggesting a biologic dichotomy within WM. The controls formed a group distinct from most WM and MGUS pts. Fig1A We then applied a non-negative matrix factorization consensus clustering to the gene expression data and identified three robust clusters. Cluster 1 (C1) included only pts with WM, cluster 2 (C2) included pts with both WM and MGUS, and cluster 3 (C3) included all normal controls as well as a small number of WM and MGUS pts. Fig1B-C When mutations commonly identified in WM were analyzed, there was no difference among the three groups (excluding controls) in mutation burden of MYD88 L265P and CXCR4. Interestingly, aberrant expression of TNFAIP3 was a distinct feature of C1 as deletion of 6q (which encodes for TNFAIP3) and TNFAIP3 mutations were each significantly enriched in C1 (47%) compared to C2 (0%) and C3 (20%; p=0.04). Individual clusters associated with specific transcriptional signatures and clinical features. While C1 displayed enrichment of RNA processing, downregulation of inflammatory pathways and aggressive clinical behavior, C2 showed increased inflammatory signaling and senescence with indolent clinical behavior. C3 had intermediate features with combined proliferative and antigen response signatures. Fig1D In accordance with transcriptomics, the proteomic hallmark of C1 was upregulation of proteins involved in proliferation (eg AKT, MAPK) and downregulation of inflammatory proteins (eg IL4, IL10) while the opposite was observed in C2. Once more, C3 confirmed intermediate features with combined upregulation of proliferation and inflammatory proteins. The metabolism was rewired towards mitochondrial anabolism in C1 and C3, while towards glycolysis in C2. Accordingly, C1 and C3 showed undetectable concentrations of 3-hydroxybutyric acid as opposed to C2 which had increased levels of lactic acid, as end products of fatty acid oxidation and glycolysis respectively. Next, we explored whether C1 and C2 displayed a distinct immune profile. tSNE analysis showed that CD4+ T cells were more abundant in C2 compared to C1 while the opposite was observed for CD8+ T cells. Among CD4+ T cells, activated follicular helper (TFH; p=0.02) and regulatory (Treg; p=0.008) cells were predominantly expressed in C2. In contrast, C1 showed a higher expression of senescent T effector memory (TEM) cells. Fig1E In support of this, SPADE clustering analysis identified three clusters including TFH, Treg and TEM cells. Image: Summary/Conclusion: We have identified three molecular clusters in IgM monoclonal gammopathies with distinct clinical, proteomic, metabolomic and immune features, suggesting a potential biologic classification that may have therapeutic implications. S172: FUNCTIONAL SUBSETS OF PLASMA CELLS ASSOCIATED WITH AMYLOID PRODUCTION AND X. Wang1 2 3,*, H. Han1 3, J. Sun3 4, Q. Wang2, X.-M. Gao1 3, K.-N. Shen1 3, L. Zhang1 3, Y. Zhao2, X.-X. Cao1 3, M. Qian5, Y. Chen2, J. Li1 3 1Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; 2The State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College; 3State Key Laboratory of Complex Severe and Rare Diseases; 4Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; 5School of Mathematical Sciences, Peking University, Beijing, China Background: Light chain amyloidosis (AL amyloidosis, AL) is a life-threatening plasma cell dyscrasia characterized by misfolded monoclonal immunoglobulin light chain production by pathogenic bone marrow plasma cells (BMPCs), leading to irreversible damage in multiple organs. Until now, the pathogenic BMPCs in AL haven’t been elucidated, let alone the functional roles of BMPC subsets. Aims: We aimed to identify intra- and inter-individual heterogeneities in BMPCs, especially those related to AL development, light chain production, organ tropism, and chemotherapy response. Methods: Here, we conducted single-cell RNA sequencing and image flow cytometry analysis of BMPCs from patients with AL (n=3), compared with that from monoclonal gammopathy of undetermined significance (MGUS) (n=2) and healthy controls (n=21). All the subjects provided informed consent. Results: We identified 7 functional subsets of BMPCs in AL, MGUS or healthy controls (Fig. 1a). These subsets had over-lapping but distinct functions, including DNA repair, cell proliferation, drug response, osteoclast differentiation, and immunoglobulin production (Fig. 1b). Subsets enriched in AL showed up-regulation of amyloidosis-associated genes (Fig. 1c), such as the amyloid-beta binding protein-encoding Apolipoprotein E (APOE), and showed plasmablastic morphology (Fig. 1d). Subsets defined by aberrant light chain production up-regulated the pathways related to neutrophil degranulation, transportation to and modifications in the Golgi apparatus, and asparagine N-linked protein glycosylation. High expression of Cyclin D1 (CCND1), CD79A, and V-Set Pre-B Cell Surrogate Light Chain 3 (VPREB3) were observed in the predominant subset of AL predicted sensitive to venetoclax, while Cyclin D2 (CCND2), S100 Calcium Binding Protein A6 (S100A6), Cystatin C (CST3) were up-regulated in that of AL predicted resistant (Fig. 1e). In an independent cohort of bulk RNA sequencing (n=29), clinical subgroups of patients with AL were defined by the proportion of functional subsets, including 2 major subgroups that were consistent with the aforementioned AL with differential sensitivity to venetoclax (Fig. 1f). Co-expression of CCND1, CD79A, and VPREB3, and a larger predominant subset, were in the venetoclax-sensitive subgroup. The up-regulation of CCND2 and amyloidosis-associated genes, including S100A6 and CST3, were in the venetoclax-resistant subgroup (Fig. 1g). Image: Summary/Conclusion: These functional subsets of BMPCs provide mechanistic insights into AL pathogenesis, light chain production, and chemotherapy response. These results suggest potential avenues for the exploration of clinical subgroups among AL patients. S173: T-CELL ACTIVATION AND MYELOMA CELL KILLING CONFIRM THE MODE OF ACTION OF RG6234, A NOVEL GPRC5D T-CELL ENGAGING BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA I. Dekhtiarenko1,*, I. Lelios2, J. Attig2, N. Sleiman2, D. Lazzaro2, I. Clausen2, N. Gräfe3, H.-J. Helms2, E. Schindler2, S. Belli2, T. Fauti1, J. Eckmann3, P. Umana1, W. Jacob3, M. Schneider2, C. Hasselbalch Riley4, M. Hutchings4, S.-S. Yoon5, Y Koh5, S. Manier6, T. Facon6, S. J. Harrison7, J. Er7, F. Volzone8, A. Pinto8, C. Montes9, E. M. Ocio9, A. Alfonso-Pierola10, P. Rodríguez Otero10, F. Offner11, A. Guidetti12, P. Corradini12, C. Titouan13, C. Hulin13, C. Touzeau14, P. Moreau14, R. Popat15, S. Leong15, R. Mazza16, C. Carlo-Stella16, A.-M. E. Bröske3 1Roche Pharma Research and Early Development, Roche Innovation Center Zurich, Zurich; 2Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland; 3Roche Pharma Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany; 4Rigshospitalet, Copenhagen, Denmark; 5Seoul National University College of Medicine, Seoul, South Korea; 6Lille University Hospital, Lille, France; 7Peter MacCallum Cancer Center and Royal Melbourne Hospital, and Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia; 8Istituto Nazionale dei Tumori, Fondazione Pascale, IRCCS, Napoli, Italy; 9Hospital Universitario Marques de Valdecilla (IDIVAL), Universidad de Cantabria, Santander; 10Clinica Universidad de Navarra, Navarra, Spain; 11Universitair Ziekenhuis Gent, Gent, Belgium; 12Istituto Nazionale dei Tumori, Milano, Italy; 13CHU de Bordeaux, Bordeaux; 14CHU de Nantes, Nantes, France; 15University College London Hospitals NHS Foundation Trust, London, United Kingdom; 16Department of Biomedical Sciences, Humanitas University and Department of Oncology and Hematology, IRCCS Humanitas Research Hospital, Milano, Italy Background: RG6234 is a novel T-cell engaging bispecific antibody targeting G protein-coupled receptor 5D (GPRC5D) with a unique 2:1 format. GPRC5D is highly expressed on multiple myeloma (MM) cells and concurrent binding of RG6234 to GPRC5D and CD3 on T cells results in immunological synapse formation and potent T-cell directed tumor cell killing. An ongoing Phase 1 dose-escalation study (NCT04557150) is investigating the safety, clinical activity, pharmacodynamics (PD), and pharmacokinetics of RG6234 monotherapy in patients (pts) with relapsed/refractory MM (RRMM). Clinical activity was observed during dose escalation, and the safety profile was manageable (Riley et al. EHA 2022). Aims: Here, we present preliminary clinical biomarker data highlighting PD effects after intravenous (IV) administration that confirm the mechanism of action and high potency of RG6234. Methods: Exploratory biomarker analyses included data from pts treated with RG6234 doses ranging from 0.006mg to 4.8mg in dose escalation. RG6234 was administered as an IV infusion under a step-up dosing regimen, reaching the target dose not later than 2 weeks after the priming dose. Peripheral biomarkers were evaluated using whole blood flow cytometry (n=28), plasma cytokine Protein Simple ELLA (n=33), and plasma sBCMA Protein Simple ELLA (n=26). MM cells were assessed at baseline and on-treatment by bone marrow (BM) aspirate flow cytometry and by BM biopsy CD138/CD8 immunohistochemistry. Informed consent was obtained from participating pts. The clinical cut-off date for the current analysis was January 31, 2022. Results: PD changes were observed in peripheral blood at all tested doses. Cytokines (IFNg, TNFa, CXCL10, IL6, IL10, IL2, IL8) and sCD25 peaked at 4 to 24 hours (h) following the first administration, while cytokine peak magnitudes decreased at subsequent administrations. Cytokine release was followed by transient reduction of circulating T cells at 4 h after infusion, with partial recovery of peripheral T-cell counts by Day 8 after first administration. Elevation of sCD25 and IFNg in plasma (~3.2 and 33 median fold change from baseline, respectively) together with increase in T-cell proliferation (~4 median fold increase of Ki67+CD8+ T-cells) within 72 h post first infusion indicated T-cell activation. Analysis of a limited number of paired baseline and on-treatment BM biopsies (n=13) revealed that the density of CD8+ tumor-infiltrating T cells increased upon treatment in responders, indicating T-cell recruitment towards the tumor. RG6234 induced rapid depletion of MM cells, as demonstrated by a decrease of sBCMA in the plasma of responding pts already 8 days after the first administration (median 33.5% reduction from baseline in responding pts; n=15). Moreover, at the end of Cycle 1, the majority of pts (14/15) had <1% of MM cells in BM based on flow cytometry readout. GPRC5D expression was detected at baseline in all pts with evaluable bone marrow aspirate and >20 detectable MM cells (n=16). Updated exploratory biomarker data will be presented. Summary/Conclusion: Cytokine release, T-cell activation, BM infiltration, and MM cell depletion are early PD changes seen after treatment with RG6234 and precede clinical responses. These PD changes indicate that RG6234 leads to T-cell engagement in the BM of pts with RRMM and clearly demonstrate rapid and effective T-cell mediated anti-MM activity. S174: HIGH LEVEL OF CIRCULATING TUMOUR DNA AT DIAGNOSIS CORRELATES WITH DISEASE SPREADING AND DEFINES MULTIPLE MYELOMA PATIENTS WITH POOR PROGNOSIS M. Martello1,*, A. Poletti1, D. Bezzi2, E. Borsi1, B. Taurisano1, V. Solli1, S. Armuzzi1, I. Vigliotta1, G. Mazzocchetti1, I. Pistis3, L. Pantani3, S. Rocchi1, K. Mancuso1, P. Tacchetti3, I. Rizzello1, M. Cavo1, E. Zamagni1, C. Nanni2, C. Terragna3 1IRCCS - Azienda Ospedaliero Universitaria di Bologna - Department of Experimental, Diagnostic and Specialty Medicine - University of Bologna; 2IRCCS - Azienda Ospedaliero Universitaria di Bologna - Department of Nuclear Medicine; 3IRCCS - Azienda Ospedaliero Universitaria di Bologna, Bologna, Italy Background: Multiple Myeloma (MM) is a plasma cell (PC) disorder characterized by the presence of skeletal involvement at the time of diagnosis, as detected by MRI and/or FDG PET/CT, in most of the patients. The patchy nature of the disease is probably related to the ability of fitter clones to spread into peripheral blood reaching distant sites, where favourable microenvironment conditions might promote clones’ seeding. Recently, cell-free DNA (cfDNA) has been proven to resume the heterogeneity of spatially distributed clones. However, it has to be determined to which extent cfDNA correlates with disease distribution and its possible implications with patients’ outcome. Moreover, the potential of cfDNA to track the evolutionary dynamics and the heterogeneity of MM, possibly anticipating the emergence of therapy resistant residual cells, remains to be confirmed. Aims: Aim of this study is to quantitatively and qualitatively evaluate cfDNA at diagnosis and during follow-up in correlation with imaging data to possibly define the integration of this approach with molecular bone marrow (BM) and whole-body residual disease assessment. Methods: A total of 88 newly diagnosed MM patients were screened at baseline with 18F-FDG PET/CT, and molecularly assessed by Ultra Low Pass-Whole Genome Sequencing (ULP-WGS). In a subgroup of 22 patients, cfDNA was monitored monthly, whereas PET/CT was reassessed after induction therapy to evaluate metabolic tumor response. For each pts, ULP-WGS was used to characterize both the neoplastic PC clone(s) in the BM (gDNA) and the cfDNA from peripheral blood. Data were analysed by ichorCNA and Clonality R packages. Results: At diagnosis, the cfDNA tumor fraction (TF) was significantly lower as compared to gDNA TF [median (M) TF: 4.4 vs. 59.7%, respectively]. Nevertheless, high cfDNA TF levels (> 4.4% cfDNA TF values; range: 4.4-84.3%) correlated with high gDNA TF levels (>65.7% gDNA TF values; range: 65.7-96.7%). This observation was further confirmed by a significant correlation between cfDNA TF and the percentage of BM CD138/CD38 positive plasma cells (r=0.47; p<.0001). Interestingly, patients with high cfDNA TF at baseline were more likely to present with extramedullary disease (EMD) and a higher number of focal lesions, and also featured a more active tumour metabolism, as compared to pts with low TF (EMD 4/44=9% vs. 1/44=2.3%, p=ns; M n. PET lesions: 1.7 vs. 2.5, p= 0.003; SUVmax: 5.2 vs. 9.6, p = 0.01). Despite an overall concordance between cfDNA and BM genomic profiles (80/88=90.9%), those patients with high cfDNA TF showed more frequently evidence of spatial heterogeneity, as highlighted by a substantial divergence of copy number alterations profiles between cfDNA and gDNA (7/8=87.5% of pts with divergent profiles). Finally, high cfDNA TF at diagnosis predicted for poorer prognosis as compared with low cfDNA TF (PFS at 20 months: 67% vs. 86%, p=0.05; OS at 20 months: 90% vs. 100%, p=0.04). Interestingly, after bortezomib-based induction therapy, imaging data and cfDNA TF levels were concordant; notably, in 4/10 (40%) patients with median SUVmax 5.8 and >2 PET lesions, cfDNA TF was still detectable (>1% TF), thus corroborating a possible role for cfDNA in monitoring response to therapy. Summary/Conclusion: In conclusion, patients with high cfDNA TF displayed imaging data that overall suggested a higher propensity to a metastatic spread of the disease, which finally correlated with poorer prognosis. Future studies will be addressed to exploit the use of cfDNA in disease monitoring. Acknowledgements: AIRC IG2019, AIL BOLOGNA ODV S175: ATLAS: A PHASE 3 RANDOMIZED TRIAL OF CARFILZOMIB, LENALIDOMIDE, AND DEXAMETHASONE VERSUS LENALIDOMIDE ALONE AFTER STEM-CELL TRANSPLANT FOR MULTIPLE MYELOMA D. Dytfeld1,*, T. Wróbel2, K. Jamroziak3, T. Kubicki1, P. Robak4, A. Walter-Croneck5, J. Czyż6, A. Tyczyńska7, A. Druzd-Sitek8, K. Giannopoulos9, A. Nowicki1, T. Szczepaniak1, A. Łojko-Dankowska1, M. Matuszak1, L. Gil1, B. Puła10, J. Rybka2, M. Majcherek2, L. Usnarska-Zubkiewicz10, L. Szukalski11, A. Końska10, J. M. Zaucha7, J. Walewski8, D. Mikulski4, O. Czabak5, T. Robak4, U. Grzybowska1, O. B. Lahoud12, J. A. Zonder13, K. Griffith14, A. Stefka15, A. Major15, B. A. Derman15, A. J. Jakubowiak15 1Poznan University of Medical Sciences, Poznan; 2Wroclaw Medical University, Wroclaw; 3Medical University of Warsaw, Warsaw; 4Medical University of Lodz, Lodz; 5University of Medical Sciences in Lublin, Lublin; 6Collegium Medicum, Bydgoszcz; 7Medical University of Gdansk, Gdansk; 8Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw; 9Medical University of Lublin, Lublin; 10Institute of Hematology and Blood Transfusion, Warsaw; 11Medical University of Bydgoszcz, Bydgoszcz, Poland; 12Memorial Sloan Kettering Cancer Center, New York; 13Karmanos Cancer Institute, Detroit; 14University of Michigan, Ann Arbor; 15University of Chicago, Chicago, United States of America Background: Treatment following autologous stem cell transplantation (ASCT) for multiple myeloma (MM) remains an area of active investigation. We have shown that extended post-ASCT treatment with carfilzomib, lenalidomide, and dexamethasone (KRd) after KRd induction improved the depth and duration of response (Jasielec et al, Blood 2020), suggesting a benefit of post-ASCT KRd therapy. Aims: We report the results of ATLAS, a multicenter, international, open-label randomized phase 3 study to determine the efficacy and safety of post-ASCT KRD vs standard lenalidomide (R) maintenance (NCT02659293). Methods: Newly-diagnosed MM patients (pts) who received any induction therapy for up to 12 months (mo) followed by single ASCT and achieved at least stable disease within 100 days afterward were randomized to receive either KRd or R, stratified by post-transplant response (≥VGPR vs <VGPR) and cytogenetic risk [standard risk (SR) vs high [HR: presence of t(4;14), t(14;16), or del(17p)]. Pts randomized to KRd received carfilzomib 36 mg/m2 on days (D) 1,2,8,9,15,16 for 4 cycles (C) then D1,2,15,16 starting C5; R 25 mg D1-21, and dexamethasone 20 mg D1,8,15,22 in 28-day cycles. KRd pts with SR who reached IMWG-defined MRD-negativity after C6 de-escalated therapy to R alone after C8 (KRd->R); the rest continued KRd through C36 followed by R alone until progression. Pts randomized to R received lenalidomide 10 mg C1-3 and then 15 mg daily. The primary endpoint was progression free survival (PFS) rate: based on historical PFS rates, a sample size of 180 Pts was calculated to provide 85% power with 2-sided alpha 0.05. Secondary endpoints included minimum residual disease (MRD) negativity response rates and treatment-related adverse events. Results: 180 pts were enrolled (R n=87; KRd n=93) through 10/21/20; data cutoff was 12/31/21. Pt characteristics in the KRd and R arms were balanced for median age (58 vs 59 yrs), ≥VGPR (88% vs 92%), and HR (23% vs 21%). After 6 cycles, 47% pts in the KRd arm and 29% in the R arm achieved MRD-negativity (p=0.017). 34 KRd pts eligible for de-escalation converted to R alone after C8 and were analyzed on the KRd arm per intention-to-treat. At median follow-up of 33.8 mo, 23 pts (25%) on the KRd arm and 38 pts (44%) on the R arm progressed; estimated median PFS was 59.0 mo for KRd vs 41.4 mo for R (Hazard Ratio 0.56, logrank p=0.026). At cutoff, 90% of KRd and 87% of R pts were alive; no deaths were treatment-related. All-grade toxicities were generally comparable between arms. The most common grade 3+ AEs and those of special interest were neutropenia (KRd 47%; R 59%), thrombocytopenia (KRd 13%; R 7%), infections (KRd 15%; R 6%), cardiovascular toxicities (KRd 4%, R 5%), and secondary malignancies (KRd 2, R 2). Image: Summary/Conclusion: This is the first randomized phase 3 trial demonstrating superior PFS with extended post-transplant KRd therapy compared to R maintenance. Therefore, MRD/risk-adapted post-ASCT extended KRd treatment may represent a new standard of care. S176: DARATUMUMAB CARFILZOMIB LENALIDOMIDE AND DEXAMETHASONE AS INDUCTION THERAPY IN HIGH-RISK TRANSPLANT ELIGIBLE NEWLY DIAGNOSED MYELOMA PATIENTS: RESULTS OF THE PHASE 2 STUDY IFM 2018-04 C. Touzeau1,*, A. Perrrot2, C. Hulin3, S. Manier4, M. Macro5, M.-L. Chretien6, L. Karlin7, O. Decaux8, C. Jacquet9, M. Tiab10, X. Leleu11, L. Planchce12, H. Avet-Loiseau2, P. Moreau13 1CHU Nantes - Hotel Dieu, CHU Nantes - Hotel Dieu, Nantes; 2Hematology, IUCT Oncopole, Toulouse; 3Hematology, CHU Bordeaux, Bordeaux; 4Hematology, CHU Lille, Lille; 5Hematology, CHU Caen, Caen; 6Hematology, CHU Dijon, DIjon; 7Hematology, CHU Lyon, Lyon; 8Hematology, CHu Rennes, Rennes; 9Hematology, CHU Nancy, Nancy; 10Hematology, CHD, La Roche sur Yon; 11Hematology, CHU Poitiers, Poitiers; 12Biostatistic; 13Hematology, CHU Nantes, Nantes, France Background: High-risk (HR) cytogenetic is associated with poor outcome in transplant eligible (TE) newly diagnosed myeloma multiple myeloma (NDMM). The triplet combination carfilzomib lenalidomide and dexamethasone (KRD) plus transplantation demonstrated high efficacy with favorable safety profile in TE-NDMM patients (FORTE). The addition of daratumumab (Dara) to frontline therapy also improved response rate and progression free-survival in TE-NDMM patients (CASSIOPEIA, GRIFFIN). Double transplant also improved outcome of HR TE NDMM patients (EMN02, STAMINA). Aims: The phase 2 trial 2018-04 from the Intergroupe Francophone du Myelome (IFM) is evaluating an intensive strategy with Dara-KRD induction and consolidation plus double transplant in HR TE NDMM (NCT03606577). Methods: HR MM was defined by the presence of del17p, t(4;14) and/or t(14;16). Stategy includes Dara-KRD induction (6 cycles), autologous stem cell transplantation (ASCT), Dara-KRD consolidation (4 cycles), second ASCT, Dara-lenalidomide maintenance. The primary endpoint was the feasibility of this intensive strategy. Here, we report efficacy and safety analysis of Dara-KRD induction. Results: Fifty patients with previously untreated NDMM were included from july 2019 to march 2021 in 11 IFM centers Median age was 57 (range 38 -65). ISS stage 3 was present in 12 (24%) patients. Based on inclusion criteria, all patients had HR cytogenetic, including 17p deletion (n=20, 40%), t(4;14) (n=26, 52%) or t(14;16) (n=10,20%). Forty-six patients completed Dara-KRD induction. Two patients discontinued treatment due to severe adverse event (COVID-19 infection, n=1; drug-induced hepatitis, n=1) and 2 patients discontinued treatment due to disease progression. Grade 3-4 treatment related adverse event (>5% of patients) were neutropenia (38%), anemia (14%), thrombocytopenia (8%), infection (6%), renal insufficiency (6%) and deep-vein thrombosis (6%). Two patients (6%) experienced stem-cell collection failure. Overall response rate was 96%, including 92 % ≥ very good partial response. Among 37/46 evaluable patients post induction, Minimal Residual Disease negativity rate (NGS, 10-5) was 62%. Summary/Conclusion: Dara-KRD as induction prior ASCT is safe and allows deep responses in TE NDMM patients with high-risk cytogenetic profile. IFM 2018-04 study is ongoing and longer follow-up is needed to evaluate safety and efficacy of the overall strategy with Dara-KRD induction and consolidation plus double transplant in this subset of HR patients. S177: EVALUATING SERUM FREE LIGHT CHAIN RATIO AS A BIOMARKER FOR MULTIPLE MYELOMA T. Akhlaghi1,*, K. Maclachlan2, N. Korde2, S. Mailankody2, A. Lesokhin2, H. Hassoun2, S. X. Lu2, D. Patel2, U. Shah2, C. Tan2, A. Derkach3, O. Lahoud4, H. J. Landau4, G. L. Shah4, M. Scordo4, D. J. Chung4, S. A. Giralt4, S. Z. Usmani2, O. Landgren5, M. Hultcrantz2 1Department of Internal Medicine, Icahn School of Medicine, Mount Sinai Morningside and West; 2Myeloma Service, Department of Medicine; 3Department of Epidemiology and Biostatistics; 4Adult Bone Marrow Transplant Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York; 5Myeloma Service, Sylvester Comprehensive Cancer Center, University of Miami, Miami, United States of America Background: In 2014, the definition of multiple myeloma (MM) was updated to include serum free light chain (FLC) ratio ≥100 as a myeloma defining biomarker, based on retrospective data indicating a 2-year progression rate of 80% and a median time to progression (TTP) of 12 months associated with this marker. As a result, patients previously diagnosed as smoldering MM (SMM), were considered to have MM requiring treatment if they had a FLC ratio ≥100. However, two recent studies have reported lower 2-year progression rates, 30-44%, and a longer median TTP of 40 months in patients with FLC ratio ≥100, raising concern about the FLC criteria. Aims: To assess the risk of progression in patients with SMM and a FLC ratio ≥100. Methods: We performed a retrospective analysis of patients diagnosed with SMM at Memorial Sloan Kettering Cancer Center between January 2000 and December 2017. Diagnosis of SMM and progression to active MM requiring therapy was defined according to the International Myeloma Working Group (IMWG) criteria at the time of diagnosis. Kaplan-Meier method was used to assess TTP and generate survival curves, with log-rank test for comparison between groups. Results: A total of 438 patients were included in the study, with a median follow-up time of 52 months. While all patients with FLC ≥100 (n=66) had elevated involved FLC levels, 35 (53%) had an involved FLC concentration >100 mg/L. Per current diagnostic criteria, we only included patients with an involved FLC concentration >100 mg/L in the FLC ratio ≥100 group and found a median TTP of 31 months (95% confidence interval [CI] 16-59 months) and a 2-year progression rate of 49% (CI 28-63%). In a sensitivity analysis including all 66 cases with FLC ratio ≥100 (independent of involved FLC concentration), we found the median TTP to be 41 months (CI 30-72 months), compared to 101 months for those with a FLC ratio <100 (CI 78-127 months; p<0.0001). Furthermore, the risk of progression within 2 years was 35% (CI 22-46%) compared to 18% (CI 14-23%; p<0.0001) for those with a FLC ratio <100. Of note, 22 patients with a FLC ratio ≥100 were monitored expectantly for >4 years, among whom 12 patients had involved FLC levels >100 mg/L. Ten patients (7 with involved FLC level >100 mg/L) were followed over a period ranging from 4 to 8.5 years before eventually progressing, and 12 patients (5 with an involved FLC level >100 mg/L) were followed between 4 and 8 years and did not progress during the study period. Image: Summary/Conclusion: While FLC ratio ≥100 is associated with a high risk of progression in patients with SMM, it does not infer an imminent risk of progression, defined by the IMWG as median TTP of 12 months and 2-year progression rate of at least 80%. On the contrary, select patients with FLC ratio ≥100 can be followed for many years without progressing and some may never progress despite long-term follow-up. These findings suggest that in patients where FLC ratio ≥100 is the only myeloma-defining event, other high-risk features as well as the evolution of FLCs over time should be considered in the decision to start a patient on treatment. S178: SAFETY AND EFFICACY OF BELANTAMAB MAFODOTIN IN COMBINATION WITH RD IN NEWLY DIAGNOSED, TRANSPLANT INELIGIBLE MULTIPLE MYELOMA PATIENTS: A PHASE 1/2 STUDY BY THE HELLENIC SOCIETY OF HEMATOLOGY E. Terpos1,*, M. Gavriatopoulou1, I. Ntanasis-Stathopoulos1, P. Malandrakis1, D. Fotiou1, N. Kanellias1, S. Gkolfinopoulos2, K. Manousou2, E. Kastritis1, M.-A. Dimopoulos1 1Department of Clinical Therapeutics, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 2Health Data Specialists, Dublin, Ireland Background: Belantamab mafodotin (belamaf), a multi-modal antibody-drug conjugate targeting BCMA, has shown efficacy and tolerability in pretreated patients (pts) with relapsed/refractory multiple myeloma. In pts refractory to immunomodulatory drugs, proteasome inhibitors, and antiCD38, belamaf plus pomalidomide and dexamethasone induced a very good partial response (VGPR) or better of 69.2% and a median progression-free survival of 16.2 months (Trudel S, ASH 2021). Preclinical evidence suggests that belamaf plus lenalidomide enhances antimyeloma activity, with no overlapping toxicities. Aims: To evaluate the safety and efficacy of belamaf plus lenalidomide and dexamethasone (Rd) in transplant-ineligible (TI) pts with newly diagnosed multiple myeloma (NDMM). Methods: The ongoing, prospective, open-label, 2-part, phase 1/2 BelaRd study (NCT04808037) aims to enroll 66 pts with TI NDMM from a Greek center. Eligible are adult pts with Eastern Cooperative Oncology Group status 0–2 and adequate organ function. Part 1 (dose selection) evaluates the safety/tolerability of 3 belamaf doses (2.5, 1.9, and 1.4 mg/kg on Day 1 of every other 28-day cycle) plus Rd (each dose regimen administered to a cohort of 6 pts) over ≥4 weeks of follow up; subsequently, an additional 6 pts are enrolled in each dose cohort to establish the recommended phase 2 dose (RP2D). Part 2 (dose expansion) evaluates the safety and clinical activity of belamaf RP2D plus Rd in 30 additional pts. This descriptive analysis presents the safety data for all Part 1 pts and the efficacy data for all Part 1 pts with ≥2 post-baseline efficacy assessments by the cut-off date (14/01/2022). Results: Of 36 pts included, 35 (97.2%) continued study treatment by the cut-off date, and 1 (2.8%) had died due to a belamaf unrelated adverse event (AE). The pt median age was 72.5 years (range 64.0–86.0). Of pts with available data (30 [83.3%]), pts at revised International Staging System stages I, II, and III were 6 [20.0%], 21 [70.0%], and 3 [10.0%], respectively, and 3 (10.0%) had high-risk cytogenetics (i.e., del17p13, t(4;14), t[14;16]). Median duration of therapy was 4.2 months (range 0.5–11.9) and median number of cycles reached was 5.0 (range 1.0–11.0). Twenty-two (61.1%) pts experienced at least one grade (Gr) 3–4 AE. One (2.8%) pt experienced a Gr 5 AE (pneumonia), unrelated to belamaf. Most common (≥ 10.0% of pts) Gr 3–4 AE were fatigue (13 [36.1%] pts), visual acuity reduced (6 [16.7%] pts), and rash (5 [13.9%] pts). Gr 3–4 thrombocytopenias and infections were not reported, as were any Gr infusion-related reactions (Table). Pts with Gr 1–2 ocular symptoms, visual acuity reduced, and keratopathy, were 27 (75.0%), 21 (58.3%), and 18 (50.0%), respectively. Pts with Gr 3–4 ocular symptoms, visual acuity reduced, and keratopathy were 0 (0.0%), 5 (13.9%) and 0 (0.0%), respectively. Of all pts, 28 (77.8%) were evaluable for efficacy (Table). At a median follow up of 4.2 months (range 0.5–11.9), the overall response rate was 96.4% (27/28 pts; complete response [CR]: 14.3% [4/28 pts]; VGPR: 35.7% [10/28 pts]; partial response [PR]: 46.4% [13/28 pts]); no disease progression was reported. Of pts achieving VGPR, 6/10 (60.0%) had negative status serum/urine immunofixation electrophoresis. Median time to PR or better was 1.0 months (range 0.9–2.0). Image: Summary/Conclusion: In pts with TI NDMM, belamaf every 2 months plus Rd showed an improved safety profile, especially at lower doses. Rapid and deep hematological responses were recorded. S179: RANDOMIZED COMPARISON BETWEEN KRD AND KTD INDUCTION, FOLLOWED BY K MAINTENANCE OR OBSERVATION IN TRANSPLANT NON-ELIGIBLE PATIENTS WITH NDMM (AGMT-MM02 TRIAL) H. Ludwig1,*, T. Melchardt2, S. Sormann3, N. Zojer4, J. Andel5, B. Hartmann6, C. Tinchon7, E. Gunsilius8, K. Podar9, A. Egle2, W. Willenbacher8 10, E. Wöll11, M. Schreder4, R. Ruckser12, B Bozic12, M.-T. Krauth13, A. Petzer14, C Schmitt15, S. Machherndl-Spandl16, H. Agis17, M. Fillitz18, W. Pönisch19, S. Knop20, B. Paiva21, R. Greil2 1Wilhelminen Cancer Research Institute, Vienna; 2Department of Internal Medicine III with Haematology, Medical Oncology, Haemostaseology, Infectiology and Rheumatology, Oncologic Center, Salzburg Cancer Research Institute – Laboratory for Immunological and Molecular Cancer Research (SCRI-LIMCR), Paracelsius Medical University, Cancer Cluster Salzburg, Salzburg; 3Department of Internal Medicine, University Clinic Graz, Graz; 4Department of Medicine I, Clinic Ottakring, Vienna; 5Department of Internal Medicine IIr, Pyhrn-Eisenwurzen Klinikum Steyr, Steyr; 6Department of Internal Medicine II, LKH Rankweil, Rankweil; 7Department of Internal Medicine, LKH Hochsteiermark, Leoben; 8Department of Internal Medicine V, Medical University Innsbruck, Innsbruck; 9Department of Internal Medicine II, University Hospital Krems, Karl Landsteiner University of Health Sciences, Krems; 10Oncotyrol, Center for personalized Cancer Medicine, Innsbruck; 11Department of Internal Medicine, KH Zams, Zams; 12Department of Medicine II, Clinic Donaustadt; 13Department of Internal Medicine I, Division Hematology & Hemostaseology, Medical University Vienna, Vienna; 14Department of Internal Medicine I, Ordensklinikum Linz Barmherzige Schwestern; 15Clinic for Internal Medicine; 3, Kepler University Clinic Linz; 16Ordensklinikum Linz Elisabethinen, Linz; 17Department of Internal Medicine I, Division of Oncology, Medical University Vienna; 18Department of Internal Medicine, Hanusch Krankenhaus, Vienna, Austria; 19Medical Clinic and Policlinic I, University Clinic Leipzig, Leipzig; 20Medical Clinic and Policlinic II, University Clinic Würzburg, Würzburg, Germany; 21Clinica Universidad de Navarra, Pamplona, Spain Background: Carfilzomib (K)-based combinations have been established as effective frontline and relapse regimens in pts with multiple myeloma (MM). Aims: In this randomized phase II trial we evaluated the impact of either Revlimid (R) or Thalidomide (T) as combination partner for K and dexamethasone (KRd or KTd) on outcome in pts with newly diagnosed MM (NDMM) not eligible for autologous transplantation (TNE). Further, we evaluated the role of one year K maintenance therapy compared to observation. Methods: One hundred twenty two pts have been enrolled (ITT population). Median age was 75 yrs, ISS stage I/II/III: 29 (23.8%)/48 (39.3%)/45 (36.9%), ECOG stage 0/1: 64 (52.5%) / 58 (47.5%). t(4;14) ± del17p was noted in 15 (16.3%) of 92 pts with results available. Pts were randomized to 9 cycles of KRd or KTd, and 107 pts received at least one full cycle. Carfilzomib (K) was started with 20mg/m2 at d 1 of cycle 1, and was continued with 27mg/m2 for the first 2 cycles (d 1 + 2, 8 + 9, 15 + 16 schedule); followed by K administration at 57mg/m2 once weekly for a 28 d cycle. Thalidomide 100mg/d (50mg in pts >75 yrs of age), d 1-28, or Revlimid 25mg/d (15mg in pts 375 yrs of age) d 1-21. Dexamethasone 40mg (20mg in pts 375 yrs of age) once/week. After induction, pts with 3SD were randomized to K maintenance (d 1 and 15) for 12 cycles or observation. MRD was assessed by NGF with a sensitivity of 10-6 in pts with ≥VGPR. Survival estimates were calculated according to Kaplan-Meier and survival curves were compared with the log-rank test. PFS and OS results presented are given for the ITT population. This trial is registered on clinicaltrials.gov (NCT02891811). Results: Median follow-up was 25.3 mos, 15 pts discontinued therapy within the first cycle due to patient (3) or investigator (1) decision, AE/toxicity (8), death or progressive disease (2) or other reason (1). Overall response rate was 91.3% in the entire group with available data (n=115). Results for sCR, CR, VGPR, PR, and ORR for KRd and KTd were similar between both groups (7.3%/10.0%, 27.3%/33.3%, 38.2%/35.0%, 14.5/16.7%, 87.3%/95.0%, respectively). Minor response was noted in 4 (3.5%), stable disease in 5 (4.3%) and progressive disease in 1 (0.8%) pts. PFS (median 26.9 and 23.5 mos, p=0.832) and OS (not reached vs 52.2 mos, p=0.398) were similar between the KRd and KTd group, respectively. The OS rate at 36 mos was 82% in both groups. MRD testing was performed in 57 pts at time of CR/VGPR. Of those, 43.9%, (20.5% of the ITT group) pts were found to be MRDneg. PFS was significantly longer in MRDneg vs. MRDpos pts (p=0.003). Seventy six pts were randomized to K maintenance therapy or observation. Median PFS was numerically higher in the pts with K maintenance treatment (median 33.0 vs 24.0, p=0.714), but the difference was not statistically significant. Data on OS are not mature yet (only 9 events). Grade 3/4 hematologic AEs were anemia (4.1%), leukopenia (0.8%), thrombocytopenia (7.4%), while non-hematologic grade 3/4 AEs were infection (20.5%), GI-disorders (7.4%), hypertension (7.4%), renal and cardiac impairment/failure (6.6% and 8.2% respectively). Image: Summary/Conclusion: Our data show similar high efficacy of KRd and KTd in elderly NTE NDMM pts, including no difference in ORR (KRd and KTd, 87.3% and 95%, respectively), PFS and OS. Overall survival rate at 3 yrs was 82%. Median PFS was significantly longer in MRDneg pts. PFS was numerically, but not statistically longer in pts on K maintenance vs observation. Treatment was associated with an acceptable tolerance profile. S180: RG6234, A NOVEL GPRC5D T-CELL ENGAGING BISPECIFIC ANTIBODY, INDUCES RAPID RESPONSES IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA: PRELIMINARY RESULTS FROM A FIRST-IN-HUMAN TRIAL C. Hasselbalch Riley1,*, M. Hutchings1, S.-S. Yoon2, Y. Koh2, S. Manier3, T. Facon3, S. J. Harrison4, J. Er4, F. Volzone5, A. Pinto5, C. Montes6, E. M. Ocio6, A. Alfonso-Pierola7, P. Rodriguez Otero7, F. Offner8, A. Guidetti9, P. Corradini9, C. Titouan10, C. Hulin10, C. Touzeau11, P. Moreau11, R. Popat12, S. Leong12, R. Mazza13, A.-M. E. Bröske14, I. Dekhtiarenko15, H.-J. Helms16, S. Belli16, T. Vardar16, T. Fauti15, J. Eckmann14, T. Moore14, M. Schneider16, W. Jacob14, M. Weisser14, C. Carlo-Stella13 1Rigshospitalet, Copenhagen, Denmark; 2Seoul National University College of Medicine, Seoul, South Korea; 3Lille University Hospital, Lille, France; 4Peter MacCallum Cancer Center and Royal Melbourne Hospital, and Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia; 5Istituto Nazionale dei Tumori, Fondazione Pascale, IRCCS, Napoli, Italy; 6Hospital Universitario Marques de Valdecilla (IDIVAL), Universidad de Cantabria, Santander; 7Clinica Universidad de Navarra, Navarra, Spain; 8Universitair Ziekenhuis Gent, Gent, Belgium; 9Istituto Nazionale dei Tumori, Milano, Italy; 10CHU de Bordeaux, Bordeaux; 11CHU de Nantes, Nantes, France; 12University College London Hospitals NHS Foundation Trust, London, United Kingdom; 13Department of Biomedical Sciences, Humanitas University and Department of Oncology and Hematology, IRCCS Humanitas Research Hospital, Milano, Italy; 14Roche Pharma Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany; 15Roche Pharma Research and Early Development, Roche Innovation Center Zurich, Zurich; 16Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland Background: Patients (pts) with relapsed/refractory multiple myeloma (RRMM) are in need of effective treatment options. RG6234 is a T-cell engaging bispecific antibody targeting GPRC5D with a novel 2:1 format. NCT04557150 is an ongoing first-in-human Phase 1 trial. We present initial results from the dose-escalation part using intravenous (IV) administration. Aims: To characterize the safety of RG6234 and to identify a recommended Phase 2 dose (RP2D), and to characterize the pharmacokinetics, pharmacodynamics, and preliminary clinical activity. Methods: Eligible pts with RRMM received RG6234 in escalating weekly step-up doses followed by a q2w regimen at a peak ‘target dose’ for up to 1 year. A mCRM model was used for overdose control. Adverse events (AEs) were graded per CTCAE v5.0 and cytokine release syndrome (CRS) per ASTCT criteria (Lee et al. 2019). Response was assessed by the investigator according to IMWG criteria. All pts provided informed consent. Results: As of January 31, 2022, 41 pts had received RG6234 IV (0.006mg to 10mg). Median age was 63 years (range: 27 to 78) and 61% had R-ISS stage II or III at study entry. High-risk cytogenetics (t(4;14), t(14;16), del(17p), 1q21 gain) were present in 17/29 (58%) evaluable cases. Median number of prior therapies was 5 (range: 2 to 15). Pts were triple-class refractory in 67% of cases and penta-class refractory in 36%. Six pts (14.6%) had prior BCMA-directed therapy. CRS occurred in 85.4% of pts (G1 56.1%, G2 24.4%, G3 2.4%). CRS was generally confined to the first cycle, with a median time to onset of 4.2 hours (range: 0 to 64.2), and 14 pts (34%) requiring tocilizumab. Three pts (7.3%) experienced CNS toxicity related to RG6234 (G1 and G3 headache; G1 confusion). G3/4 neutropenia, thrombocytopenia, and anemia occurred in 9.8%, 19.5%, and 12.2% of pts, respectively. Infections were reported in 46.3% of pts (≥G3 14.6%). One patient died of an E. coli sepsis not considered related to RG6234. AEs probably related to target expression included skin-related AEs in 66% of pts (G3 7.3%), dysgeusia/ageusia (36.6%, all G1/2), dry mouth (36.6%, all G1/2), dysphagia (17.1%, all G1/2), and nail changes (12.2%, all G1/2). No discontinuations due to RG6234-related AEs occurred. The maximum tolerated dose (MTD) was not reached. Within the tested target-dose range, RG6234 serum exposure generally showed a dose-proportional increase, with a preliminary IV terminal T½ of ～5 days (mean value, n=22). At the predicted active-dose range (>0.162mg), RG6234 induced profound and rapid reductions in serum free light chains (FLC) and sBCMA. After the first treatment cycle, 93% of evaluable pts (14/15) had <1% of multiple myeloma cells in bone marrow based on flow cytometry. Overall response rate in 34 efficacy evaluable pts across all doses was 68%, and 50% achieved a VGPR or better response, including 2/6 BCMA pre-treated pts. Responses occurred rapidly, with a median time to first response of 1.3 months (95% CI: 1.1, 1.6). Median follow-up was 3.5 months (range: 0.03 to 11.7). Treatment was ongoing for 58.5% of pts at the time of the data cut. Duration of response was not mature. Response was ongoing in 18/23 (78.3%) responding pts, with a longest duration of response of 10 months. Summary/Conclusion: RG6234, a novel T-cell engaging bispecific antibody targeting GPRC5D, demonstrates compelling clinical activity with manageable safety in heavily pretreated pts with RRMM. Dose escalation via IV and subcutaneous routes continues, and data will be updated. S181: MODAKAFUSP ALFA (TAK-573): UPDATED CLINICAL, PHARMACOKINETIC (PK), AND IMMUNOGENICITY RESULTS FROM A PHASE 1/2 STUDY IN PATIENTS (PTS) WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM) J. L. Kaufman1,*, S. Atrash2, S. A. Holstein3, O. Nadeem4, D Benson5, K. Suryanarayan6, Y. Liu7, C. Li8, L. Yang8, X. Parot8, D. T. Vogl9 1Winship Cancer Institute of Emory University, Atlanta; 2Haematology and blood disorders, Levine Cancer Institute, Charlotte; 3University of Nebraska Medical Center, Omaha; 4Dana-Farber Cancer Institute Boston, Boston; 5Hematology, Ohio State University Comprehensive Cancer Center, Columbus; 6Oncology Clinical Research; 7Statistical and Quantitative Sciences; 8Takeda Development Center Americas, Inc. (TDCA), Lexington; 9Hematologic Malignancies and Bone Marrow Transplant Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, United States of America Background: Modakafusp alfa (TAK-573) is a first-in-class immunocytokine designed to deliver attenuated interferon alpha-2b to CD38+ cells. In this phase 1/2 study (NCT03215030), the maximum tolerated dose of modakafusp alfa was defined as 3 mg/kg every 4 weeks (Q4W); preliminary data from 29 pts treated at 1.5 mg/kg Q4W (5 in dose escalation, 24 in dose expansion) showed single-agent, anti-myeloma activity with an overall response rate (ORR) of 38% after a median follow-up of 4.2 months (mos) (Vogl ASH 2021, #898). Aims: We present a clinical update on the efficacy and safety of modakafusp alfa 1.5 mg/kg Q4W after a median follow-up of 4.5 mos. We also present PK, immunogenicity, and dose-exposure-response data from the overall study. Methods: Eligible pts had received ≥3 prior lines of treatment. Pts received modakafusp alfa as a 1–4-hour IV infusion at 10 dose levels from 0.001–6 mg/kg at weekly (cycles 1–2 only; 0.001–0.75 mg/kg), every-2-week (0.2–0.3 mg/kg), every-3-week (Q3W; 0.4–0.75 mg/kg), or Q4W (0.75–6 mg/kg) dosing intervals; an expansion cohort was opened at 1.5 mg/kg Q4W after anti-myeloma activity was observed in 3/5 pts in the escalation cohort at that dose. Serum samples prepared from blood were used for PK and anti-modakafusp alfa antibody (ADA) assessments. Modakafusp alfa concentrations were determined via a validated enzyme-linked immunosorbent assay; ADA were detected using a validated bridging electrochemiluminescence assay. Results: As of Oct 2021, 30 pts had received modakafusp alfa 1.5 mg/kg Q4W. The median number of prior lines was 7 (range 3–16); 90% of pts were refractory to an anti-CD38 monoclonal antibody (mAb), while 37% were exposed to an anti-B-cell maturation agent (BCMA). Grade (G) 3–4 treatment-emergent adverse events (TEAEs) were reported in 25 pts (83%); the most frequent G3–4 TEAEs were neutropenia in 19 (63%), thrombocytopenia in 13 (43%), leukopenia in 12 (40%), anemia in 9 (30%), and decreased lymphocyte count in 9 (30%) pts. As reported previously, 1 pt in the 1.5 mg/kg Q4W cohort had a G3 bleeding event, while 3 pts had G3 infections. Among all pts, the ORR was 40% (complete response, 7%; very good partial response, 20%; partial response, 13%); among anti-CD38 mAb-refractory and anti BMCA-exposed pts, the ORR was 37% and 27%, respectively. Median progression-free survival was 6 mos and median duration of response had not been reached (Kaplan-Meier estimate, 91% at 6 mos). Based on pooled available data for the Q3W and Q4W cohorts (all doses) within the escalation and expansion phases, there was a strong trend for a dose-exposure-response relationship for ORR, with the apparent inflection point at 1.5 mg/kg Q4W dosing. No apparent relationship between dose-exposure-response and G3/4 thrombocytopenia or neutropenia was observed across all doses, whereas a correlation between dose-exposure-response and incidence of infusion-related reactions across the 0.4–6 mg/kg Q3W and Q4W dosing cohorts was found. There was an apparent non-linear (more than dose proportional) increase in exposure in the dose range of 0.1–3 mg/kg with a geometric mean terminal half-life of 13 hours in serum for modakafusp alfa 1.5 mg/kg. Based on the available data, 14% and 60% of pts were ADA positive at baseline and post-treatment, respectively. Summary/Conclusion: Modakafusp alfa has a novel mechanism of action and has shown encouraging activity with a manageable safety profile in pts with RRMM. Characterization of the potential clinical impact of immunogenicity is ongoing. The optimal dose for single-agent modakafusp alfa will be further investigated in a phase 2 study. S182: TALQUETAMAB, A G PROTEIN-COUPLED RECEPTOR FAMILY C GROUP 5 MEMBER D X CD3 BISPECIFIC ANTIBODY, IN RELAPSED/REFRACTORY MULTIPLE MYELOMA: UPDATED EFFICACY AND SAFETY RESULTS FROM MONUMENTAL-1 M. C. Minnema1,*, A. Krishnan2, J. G. Berdeja3, A. Oriol4, N. W. van de Donk5, P. Rodríguez-Otero6, D. Morillo7, M.-V. Mateos8, L. J. Costa9, J. Caers10, D. Vishwamitra11, J. Ma11, S. Yang11, B. W. Hilder11, J. Tolbert11, J. D. Goldberg12, A. Chari13 1University Medical Center Utrecht, Utrecht, Netherlands; 2City of Hope Comprehensive Cancer Center, Duarte, CA; 3Sarah Cannon Research Institute and Tennessee Oncology, Nashville, TN, United States of America; 4Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain; 5Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 6Clínica Universidad de Navarra, Navarra; 7Hospital Universitario Fundación Jiménez Díaz, Madrid; 8University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca, Spain; 9University of Alabama at Birmingham, Birmingham, AL, United States of America; 10University of Liège, Liège, Belgium; 11Janssen Research & Development, Spring House, PA; 12Janssen Research & Development, Raritan, NJ; 13Mount Sinai School of Medicine, New York, NY, United States of America Background: G protein-coupled receptor family C group 5 member D (GPRC5D) is a promising therapeutic target for multiple myeloma (MM) immunotherapy. GPRC5D has limited expression in healthy human tissue but is highly expressed on malignant plasma cells. Talquetamab (JNJ-64407564) is a first-in-class, GPRC5D x CD3 bispecific IgG4 antibody that induces T cell–activated lysis of GPRC5D+ MM. MonumenTAL-1 is a phase 1 trial evaluating the safety and preliminary efficacy of talquetamab in patients with relapsed/refractory MM (RRMM) (NCT03399799). Aims: We report updated results from MonumenTAL-1 with additional patients and longer follow-up. Methods: Patients with RRMM or who were intolerant to standard therapies were eligible; prior treatment with B-cell maturation antigen-directed therapies was allowed. The primary objectives were to identify the recommended phase 2 doses (RP2Ds) (part 1) and assess the safety and tolerability of talquetamab at the RP2Ds (part 2). Based on the collective safety, preliminary efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) data, 2 RP2Ds of talquetamab were identified: 405 μg/kg SC QW (n=30) and 800 μg/kg SC Q2W (n=44). To mitigate severe cytokine release syndrome (CRS), step-up dosing was used. Administration of required premedications were limited to step-up doses and the first full dose of talquetamab. Adverse events (AEs) were graded by CTCAE v4.03, and CRS events were graded per Lee et al 2014 criteria. Responses were assessed by the investigators as per International Myeloma Working Group criteria. Results: As of Jan 17, 2022, patients in the 405 μg/kg /800 μg/kg groups, respectively, received a median of 6/5 prior lines of therapy, 100%/98% were triple-class (TC) exposed, and 77%/75% were TC refractory, and median follow-up (range) was 11.7 (1.0–21.2)/4.2 (0.7–13.7) months. AEs were mostly grade 1 or 2, and cytopenias and CRS were the most commonly reported AEs. Cytopenias, including neutropenia (67%/36%; grade 3/4: 53%/23%), were mostly limited to step-up and cycle 1–2 doses, reversible, and generally resolved within 1 week. CRS (77%/80%; grade 3: 3%/0%) were mostly reported during step-up dosing. Infections were reported in 47%/34% (grade 3/4: 7%/9%) of patients. 83%/75% of patients reported skin-related and nail disorder AEs (most commonly skin exfoliation: 37%/39% [all grade 1 and 2]). Dysgeusia (63%/57%) was generally mild and managed with dose adjustments. Response-evaluable patients had overall response rates of 70% (21/30 patients)/64% (28/44 patients). Very good partial response or better rates were 57%/52%, and median time to first confirmed response (range) was 0.9 (0.2–3.8)/1.2 (0.3–6.8) months. Median duration of response will be reported. No deaths from drug-related AEs were reported. Both RP2Ds showed comparable PK and PD profiles. Summary/Conclusion: Both RP2Ds of talquetamab appear tolerable with comparable safety and PK profiles. Talquetamab demonstrated highly promising efficacy as a novel, first-in-class therapy for heavily pretreated patients with RRMM. S183: NOVEL COMBINATION IMMUNOTHERAPY FOR THE TREATMENT OF RELAPSED/REFRACTORY MULTIPLE MYELOMA: UPDATED PHASE 1B RESULTS FOR TALQUETAMAB (A GPRC5D X CD3 BISPECIFIC ANTIBODY) IN COMBINATION WITH DARATUMUMAB N. W. van de Donk1,*, N. Bahlis2, M.-V. Mateos3, K. Weisel4, B. Dholaria5, A. L. Garfall6, H. Goldschmidt7, T. G. Martin8, D. Morillo9, D. E. Reece10, D. Hurd11, P. Rodríguez-Otero12, M. Bhutani13, A. D’Souza14, A. Oriol15, E. Askari16, J. F. San-Miguel17, K. M. Kortüm18, D. Vishwamitra19, S. Xin Wang Lin19, T. J. Prior19, L. Vandenberk20, M.-A. D. Smit21, J. D. Goldberg22, R. Wäsch23, A. Chari24 1Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 2Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, AB, Canada; 3University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca, Spain; 4University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 5Vanderbilt University Medical Center, Nashville, TN; 6Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 7University Hospital Heidelberg, Internal Medicine and National Center for Tumor Diseases, Heidelberg, Germany; 8University of California, San Francisco, San Francisco, CA, United States of America; 9Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain; 10Princess Margaret Cancer Centre, Toronto, ON, Canada; 11Section on Hematology and Oncology, Department of Internal Medicine, Comprehensive Cancer Center of Wake Forest University, Winston-Salem, NC, United States of America; 12Clínica Universidad de Navarra, Navarra, Spain; 13Levine Cancer Institute/Atrium Health, Charlotte, NC; 14Medical College of Wisconsin, Milwaukee, WI, United States of America; 15Institut Català d’Oncologia and Institut Josep Carreras and Hospital Germans Trias i Pujol, Badalona, Barcelona; 16Department of Hematology, Fundacion Jimenez Diaz University Hospital, Madrid; 17Clínica Universidad de Navarra, CCUN, CIMA, CIBERONC, IDISNA, Pamplona, Spain; 18Medizinische Klinik II, Universitätsklinikum Würzburg, Würzburg, Germany; 19Janssen Research & Development, Spring House, PA, United States of America; 20Janssen Research & Development, Beerse, Belgium; 21Janssen Research & Development, Los Angeles, CA; 22Janssen Research & Development, Raritan, NJ, United States of America; 23University of Freiburg, Freiburg, Germany; 24Mount Sinai School of Medicine, New York, NY, United States of America Background: Talquetamab (tal; JNJ-64407564) is a first-in-class, bispecific IgG4 antibody that binds both to G protein-coupled receptor family C group 5 member D (GPRC5D), a receptor highly expressed on malignant plasma cells but with limited expression in healthy tissue, and CD3 to mediate T-cell–activated lysis of GPRC5D+ multiple myeloma (MM) cells. Daratumumab (dara) is an anti-CD38 mAb with direct on-tumor and immunomodulatory actions. Initial clinical results from the phase 1b multicohort TRIMM-2 study identified the recommended phase 2 doses (RP2Ds) of tal as 400 μg/kg weekly or 800 μg/kg Q2W and support the combination of tal + dara for the treatment of RRMM, with manageable safety, no overlapping toxicities, and promising efficacy. Aims: Here we report updated results for both RP2Ds of tal + dara in TRIMM-2 with additional patients (pts) and longer follow-up. Methods: Eligible MM pts (aged ≥18 years) had received ≥3 prior lines of therapy (LOT; including a PI and IMiD) or were double refractory to a PI and an IMiD, and could not have received anti-CD38 therapy within 90 days. Pts received dara SC 1800 mg per approved schedule and tal (400 μg/kg weekly or 800 μg/kg Q2W) with step-up dosing. The primary objectives were to identify the RP2D(s) of tal for combination therapy and evaluate safety of the combination. AEs were graded per CTCAE v5.0; cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) were graded per ASTCT guidelines. Responses were assessed by IMWG criteria. Results: At data cutoff (Jan 13, 2022, N=46), median follow-up was 4.0 months (range 0.4-16.3), median age was 65 years (range 47-81), and 48% were female. Pts received a median of 5 prior LOT (range 2-16); 83% were triple-class exposed, 61% penta-drug exposed, 37% anti-BCMA non–CAR-T exposed, and 4% anti-BCMA CAR-T exposed. 96% of pts had ≥1 AE (gr 3/4: 67%). The most frequently reported AEs (≥30% across tal + dara cohorts) were CRS (65%; all gr 1/2; median time to onset: 2 days; median duration: 2 days), dysgeusia (57%), thrombocytopenia (35%; gr 3/4: 20%), anemia (39%; gr 3/4: 20%), and dry mouth (44%). Infections occurred in 50% of pts (gr 3/4: 13%). Skin disorders were reported in 72% of pts (gr 3/4: 11%): skin exfoliation in 26% (all gr 1/2) and nail disorders in 11% (all gr 1/2). Two ICANS events were reported in the 800 Q2W group (both gr 1 and resolved within 1 day). 3 pts discontinued due to AEs. Response rates were consistent across both RP2Ds supporting their equivalence (Table). Median time to first response across dosing cohorts was 0.95 months (range 0.9-9.7); median duration of response was not reached. Upregulation of CD38+/CD8+ T cells and proinflammatory cytokines was observed with tal + dara, supporting potential synergy of the combination in pts with prior anti-CD38 exposure. Updated results will be presented. Image: Summary/Conclusion: Longer follow-up with additional patients shows comparable efficacy and safety across both RP2Ds, with no new safety signals, strengthening the benefit-risk profile of tal + dara as a novel immunotherapy-based approach for heavily pretreated pts with RRMM. S184: EVALUATING TECLISTAMAB IN PATIENTS WITH RELAPSED/ REFRACTORY MULTIPLE MYELOMA FOLLOWING EXPOSURE TO OTHER B-CELL MATURATION ANTIGEN (BCMA)-TARGETED AGENTS C. Touzeau1,*, A. Krishnan2, P. Moreau1, A. Perrot3, S. Z. Usmani4, S. Manier5, M. Cavo6, C. Martinez-Chamorro7, A. Nooka8, T. Martin9, L. Karlin10, X. Leleu11, N. Bahlis12, B. Besemer13, L. Pei14, R. Verona15, S. Girgis15, C. Uhlar15, R. Kobos14, A. Garfall16 1University Hospital Hôtel-Dieu, Nantes, France; 2City of Hope Comprehensive Cancer Center, Duarte, United States of America; 3Centre Hospitalier, Universitaire de Toulouse, Service d’Hematologie, Toulouse, France; 4Levine Cancer Institute/Atrium Health, Charlotte, United States of America; 5University of Lille, Lille, France; 6IRCSS Aziena Ospedaliero-Universitaria di Bologna, Bologna University School of Medicine, Bologna, Italy; 7University Hospital Quirónsalud, Pozuelo de Alarcón, Madrid, Spain; 8Winship Cancer Institute, Emory University, Atlanta; 9University of California, San Francisco, San Francisco, United States of America; 10Service d’Hématologie Clinique, Centre Hospitalier Lyon Sud, Pierre-Bénite; 11Centre Hospitalier Universitaire de Poitiers, Poitiers, France; 12Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada; 13University of Tuebingen, Tuebingen, Germany; 14Janssen Research & Development, Raritan; 15Janssen Research & Development, Spring House; 16Abramson Cancer Center, Perelman School of Medicine, Philadelphia, United States of America Background: Teclistamab (JNJ-64007957) is a T cell redirecting bispecific antibody that targets both B-cell maturation antigen (BCMA) and CD3 receptors to induce T cell mediated cytotoxicity of BCMA-expressing myeloma cells. MajesTEC-1 is an open-label, multicohort, phase 1/2 study evaluating teclistamab in patients (pts) with relapsed/ refractory multiple myeloma (RRMM) previously treated with ≥3 prior lines of therapy (LOT). An overall response rate (ORR) of 62.0% in pts with no prior anti-BCMA treatment (tx) was previously reported in a pooled analysis from phase 1 and phase 2 cohort A at a median follow-up of 7.8 mo. Aims: We report efficacy and safety results of teclistamab from cohort C, which enrolled patients who had prior exposure to anti-BCMA treatment. Methods: Eligible pts (age ≥18 y) had multiple myeloma (MM) per IMWG criteria and were previously treated with ≥3 prior LOT, including a PI, IMiD, anti-CD38 antibody, and anti-BCMA treatment (chimeric antigen receptor T cell therapy [CAR-T] or Ab drug conjugate [ADC]). Pts were enrolled using a Simon’s stage design to receive weekly subcutaneous teclistamab 1.5 mg/kg (step-up doses of 0.06 and 0.3 mg/kg). ORR per IMWG 2016 criteria was the primary endpoint. All AEs were graded per CTCAE v4.03; immune effector cell–associated neurotoxicity syndrome (ICANS) and cytokine release syndrome (CRS) were graded per ASTCT guidelines. Results: In cohort C, 38 pts (median age 63.5 y [range 32–82]; 63% male) received teclistamab (median prior LOT 6 [range 3–14]) at the data cutoff date of Sep 7, 2021. Of the 38 patients, 25 (66%) were refractory to an anti-BCMA treatment, and 32 (84%) were refractory to last LOT. Among 25 efficacy-evaluable patients, 16 (64%) received prior ADC, 11 (44%) received prior CAR-T, and 2 pts received both. The ORR was 40% (95% CI, 21–61) at a median follow-up of 6.9 mo (range 0.7–8.7). Complete response or better were observed in 5 pts (20%). In ADC-exposed and CAR-T-exposed pts, the ORR was 38% (95% CI, 15–65) and 45% (95% CI, 17–77), respectively. Responses were rapid in most, with deepening of responses over time in 7 of 25 pts. While the median duration of response was not reached, median time to first response was 1.2 mo (range, 0.2–4.9) and to best response was 2.1 mo (range, 1.1–5.7). No new safety concerns were observed, and the safety profile was comparable with that of BMCA tx-naive pts. Infections were reported in 16 pts (42%; grade 3/4, 26%). Most common AEs (n=38) were CRS (63%; all grade 1/2; median time to CRS onset: 3 d [range, 2–6], duration of CRS: 2 d [range, 1–4]), thrombocytopenia (42%; grade 3/4, 29%), neutropenia (55%; grade 3/4, 50%), lymphopenia (40%; grade 3/4, 37%), and anemia (39%; grade 3/4 29%), Grade 3 ICANS was reported in 1 pt which resolved with supportive care and the pt remained on tx. Anti-teclistamab Abs were not detected in any pts. Baseline BCMA expression levels were comparable with those reported in BCMA tx-naive pts. Updated efficacy and safety results will be presented for 40 pts. Summary/Conclusion: These preliminary results observed with serial targeting of BCMA with teclistamab following ADC or CAR-T tx suggest a promising ORR with early responses that deepen over time. Additionally, a well-tolerated safety profile was observed in patients treated with anti-BCMA tx. S185: CARTITUDE-2 COHORT B: UPDATED CLINICAL DATA AND BIOLOGICAL CORRELATIVE ANALYSES OF CILTACABTAGENE AUTOLEUCEL IN PATIENTS WITH MULTIPLE MYELOMA AND EARLY RELAPSE AFTER INITIAL THERAPY M. E. Agha1,*, N. W. van de Donk2, A. D. Cohen3, Y. C. Cohen4, S. Anguille5, T. Kerre6, W. Roeloffzen7, J. M. Schecter8, K. C. De Braganca8, H. Varsos8, P. Mistry9, T. Roccia9, E. Zudaire10, C. Corsale8, M. Akram11, D. Geng11, T Nesheiwat11, L. Pacaud11, P. Sonneveld12, S. Zweegman2 1UPMC Hillman Cancer Center, Pittsburgh, PA, United States of America; 2Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 3Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, United States of America; 4Tel-Aviv Sourasky (Ichilov) Medical Center and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 5Vaccine and Infectious Disease Institute, University of Antwerp, Edegem, Belgium, Center for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, Edegem, Belgium; 6University Hospital Ghent, Ghent, Belgium; 7University Medical Center Groningen, Groningen, Netherlands; 8Janssen R&D, Raritan, NJ, United States of America; 9Janssen R&D, High Wycombe, United Kingdom; 10Janssen R&D, Spring House, PA, United States of America; 11Legend Biotech USA, Piscataway, NJ, United States of America; 12Erasmus MC University and Medical Center, Rotterdam, Netherlands Background: CARTITUDE-2 (NCT04133636) is a phase 2, multicohort study evaluating the efficacy and safety of the chimeric antigen receptor (CAR) T-cell therapy ciltacabtagene autoleucel (cilta-cel) in patients (pts) with multiple myeloma (MM). Cohort B enrolled pts who had early relapse after initial therapy. These pts have functionally high-risk disease, as early relapse following autologous stem cell transplantation (ASCT) is associated with poor prognosis, representing an unmet medical need. Aims: To present updated results from CARTITUDE-2 cohort B. Methods: All pts provided informed consent. Pts had MM, received 1 prior line of therapy (proteasome inhibitor and immunomodulatory drug required), had disease progression per International Myeloma Working Group criteria (either ≤12 months after ASCT or ≤12 months after initiation of anti-myeloma therapy for pts not treated with ASCT), and were naive to CAR-T or other anti-B-cell maturation antigen therapies. After lymphodepletion, a single cilta-cel infusion was administered at a target dose of 0.75×106 CAR+ viable T cells/kg. Efficacy and safety were evaluated. The primary endpoint was minimal residual disease (MRD) negativity at 10-5. Patient management strategies were used to minimize risk of movement and neurocognitive adverse events (MNTs). Pharmacokinetic (PK) analyses, including Cmax and Tmax of CAR+ T-cell transgene levels in blood are being performed. Additional assessments include levels of cytokine release syndrome (CRS)-related cytokines (eg, IL-6) over time, peak levels of cytokines by response and CRS, association of cytokine levels with immune effector cell-associated neurotoxicity syndrome (ICANS), and CAR+ T cell CD4/CD8 ratio by response, CRS, and ICANS. Results: 19 pts (median age: 58.0 years [range: 44-67]; 74% male) received cilta-cel as of January 2022; 79% received prior ASCT. Median follow-up was 13.4 months (range: 5.2-21.7). Overall response rate was 100.0%, with 90% of pts achieving ≥complete response, and 95% achieving ≥very good partial response. Median time to first response was 0.95 months (range: 0.9-9.7) and median time to best response was 5.1 months (range: 0.9-11.8). Among 15 MRD-evaluable pts, 14 (93%) achieved MRD 10-5 negativity. Median duration of response was not reached. The 12-month progression-free survival rate was 90%; 12-month event-free rate was 88.9%. CRS occurred in 16 (84.2%) pts (1 grade 4); median time to onset was 8 days (range: 5-11) and all events resolved. ICANS (grade 1) and MNT (grade 3, previously reported) occurred in 1 pt each. 1 pt died post cilta-cel (progressive disease, day 158). Preliminary PK data showed peak expansion of CAR-T cells on day 13.1 (range: 8.96-209.9); median persistence was 76.9 days (range: 40.99-221.8). Summary/Conclusion: This functionally high-risk pt population with early relapse after initial therapy experienced deep and durable responses with manageable safety following a single cilta-cel infusion. Cilta-cel led to responses in pts with ineffective or insufficient response to ASCT. Follow-up is ongoing and responses continue to deepen. We will present updated and detailed PK, cytokine, and CAR-T subset analyses as well as clinical correlation to provide insight into biological correlates of efficacy and safety in these pts. S186: UPDATED RESULTS OF A MULTICENTER FIRST-IN-HUMAN STUDY OF BCMA/CD19 DUAL-TARGETING FAST CAR-T GC012F FOR PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM) J. Du1,*, H. Jiang1, B. Dong2, L. Gao3, L. Liu4, J. Ge5, A. He6, L. Li1, J. Lu1, X. Chen2, M. Sersch7, H. Zhang7, L. Shen7, J. Liu7, W. Fu1 1Changzheng Hospital, Shanghai; 2Xijing Hospital, Xian; 3Chongqing Xinqiao hospital, Chongqing; 4Tangdu Hospital, Xian; 5The first affiliated hospital of Anhui medical University, Anhui; 6The second affiliated hospital of Xian Jiaotong University, Xian; 7Gracellbiotechnologies Ltd, Shanghai, China Background: GC012F is a B cell maturation antigen (BCMA) and CD19 dual-targeting CAR-T developed on the novel FasT CAR-T platform enabling 22-36h manufacturing designed to improve depth of response and overall efficacy. Data was presented at ASCO and EHA 2021 for initial 19 pts. Here we present updated data for study (NCT04236011; NCT04182581) with longer follow up and 9 additional pts treated (total n=28) in 3 different dose levels. Aims: The study aims to assess safety and preliminary efficacy for FAST-CAR GC012F in RRMM patients. Methods: From October 2019 to November 2021, 28 heavily pretreated RRMM pts (age 27-76) with a median of 5 prior lines (range 2-9) were treated on this single-arm, open label, multicenter Investigator Initiated Trial receiving a single infusion of GC012F. 89.3% (25/28) were high risk (HR- mSMART), 8 pts had EM disease, 3 had never achieved a CR including after transplant, 1 pts presented with plasma cell leukemia, 24/28 pts were refractory to last therapy, 3 pts primary refractory. 9/28 pts had received prior anti-CD38, 27/28 pts prior IMiDs. 26/28 pts were refractory to PI, 26/28 pts to IMiDs. After lymphodepletion over 2-3 days (30 mg/m2/d, 300mg/ m2/d Flu/Cy) GC012F was administered as single infusion at 3 dose levels: 1x105/kg (DL1) n=2, 2x105/kg (DL2) n=10 and 3x105/kg (DL3) n=16. Results: As of Jan 26th 2022 cut-off, 28 pts - median follow-up (f/u) 6.3 mths (1.8-29.9) - had been evaluated for response. Overall response rate (ORR) in DL1 was 100% (2/2)- DL 2 -80% (8/10) DL 3 -93.8% (15/16) with 27 pts MRD negative by flow cytometry (sensitivity 10-4-10-6). 100% of MRD assessable pts (27/27) achieved MRD negativity. One patient out of 28 could not get assessed. At d28, 21/24 assessable patients were MRD negative (81.5%), 4/28 pts could not get d28 MRD assessment f/u due to COVID-19 restrictions however were assessed at a later timepoint. To date best response is MRD- sCR in 21/28 patients (75.0%) across all dose levels. Some pts after short f/u show responses that are still deepening. Cytokine Release Syndrome (CRS) was mostly low grade: gr 0 n=3 (10.7%), gr 1-2 n=23 (82.1%), gr 3 n=2 (7.1%) – no gr 4/5 CRS and no ICANs were observed (Graded by ASBMT criteria). Median duration of CRS was 3 d (1-8 d). PK results showed no difference amongst dose levels DL1 to DL3. Overall, CAR-T median Tmax was 10 d (range 8-14 d), median peak copy number (Cmax) was 97009 (16,011-374,346) copies /ug DNA with long duration of persistence of up to d793 (data cut-off). CAR-T geometric mean AUC0-28 for DL1, DL2 and DL3 were 468863, 631540 and 581620 copies/ug DNA×day, respectively. Pts continue to be monitored for safety and efficacy including DOR. Summary/Conclusion: BCMA-CD19 dual FasT CAR-T GC012F continues to provide deep and durable responses with a favorable safety profile in additional RRMM pts across all dose levels demonstrating a very high MRD negativity rate including in pts refractory to anti-CD38, PI and IMIDs. Based on these promising results GC012F is being studied in earlier lines of therapy as well as additional indications. S187: UPDATED PHASE 1/2 DATA OF SAFETY AND EFFICACY OF CT103A, FULLY HUMAN BCMA-DIRECTED CAR T CELLS, IN RELAPSED/REFRACTORY MULTIPLE MYELOMA C. Li1,*, D. Wang1, Y. Song2, J. Li3, H Huang4, B. Chen5, J. Liu6, K. Hu7, H. Ren8, X. Zhang9, Z. Li10, D. Zou11, Q. Yin2, L. Chen12, Y. Hu4, Y. Xu5, Q. Cheng6, Y. Guo7, Y. Dong13, L. Gao9, S. Chen14, A. Xu15, S. Cai15, M. Wu15, J. Guo15, Z. Yao15, W. Wang15, J. Wang1, L. Chen1, J. Zhou1, L. Qiu11 1Department of Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan; 2Department of Hematology, Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Zhengzhou; 3Department of Hematology, Pukou CLL Center, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing; 4Department of Hematology, Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou; 5Department of Hematology, Nanjing University Medical School, the Affiliated Nanjing Drum Tower Hospital, Nanjing; 6Department of Hematology, The Third Xiangya Hospital of Central South University, Changsha; 7Department of Hematology, Beijing Boren Hospital; 8Department of Hematology, Peking University First Hospital, Beijing; 9Department of Hematology, Medical Center of Hematology, Xinqiao Hospital, State Key Laboratory of Trauma, Burn and Combined Injury, Army Medical University, Chongqing; 10Department of Hematology, Affiliated Hospital of Xuzhou Medical University, Xuzhou; 11Department of Hematology, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Blood Diseases Hospital & Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin; 12Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Nanjing; 13Department of Hematology, Peking University First Hospital, Beijing; 14Department of Hematology; 15Nanjing IASO Biotherapeutics Ltd, Nanjing, China Background: CT103A, a fully human BCMA-directed CAR-T therapy, showed excellent safety and promising efficacy in the ongoing phase 1/2 FUMANBA-1 study (ChiCTR1800018137, NCT05066646) in patients with relapsed/refractory multiple myeloma (Wang, ASH, 2021). Aims: We report updated data from the FUMANBA-1 study with more subjects and a longer duration of follow-up. Methods: Enrolled patients had received three or more prior lines of anti-myeloma therapy, including a proteasome inhibitor and an immunomodulatory agent, and were required to be refractory to the last line of therapy. CT103A was administered at RP2D of 1.0×106 CAR+ T cells/kg. Lymphodepletion with cyclophosphamide (500 mg/m2) and fludarabine (30 mg/m2) was used for 3 consecutive days. Study objectives included safety and efficacy profile. Pharmacokinetics, pharmacodynamics, and immunogenicity were also explored. MRD in bone marrow aspirate was detected by EuroflowTM standardized flow cytometry with the sensitivity of 10-5. Adverse events (AEs) were graded using CTCAEv5.0, except that CRS and ICANS were graded by ASTCT criteria. Results: As of Jan 21, 2022, 79 patients received CT103A with the median follow-up of 9.6 months (range 1.2, 40.0). 28 (35.4%) patients had high-risk cytogenetic abnormality defined as del(17p), t(4;14), or t(14;16), and 11 (13.9%) had extramedullary plasmacytomas. Patients received a median of 5 (range 3, 23) prior lines of therapy. Notably, 13 (16.5%) had received prior non-human BCMA-targeted CAR-T cell therapy. ORR was 94.9% (75/79), with 55 (69.6%) patients achieving CR/sCR, and 71 (89.9%) achieving ≥VGPR. Median TTR was 16 days (range 11, 123), and the median time to CR/sCR was 95 days (range 14, 557). For 13 patients who had prior CAR-T therapy, ORR was 98.5%, with 6 (46.2%) achieving CR/sCR. For 11 patients with extramedullary disease at baseline, ORR was 100%, with 9 (81.8%) achieving CR/sCR. All patients with CR/sCR were MRD-negative. The median duration of MRD negativity was not reached. The probability of MRD negativity at month 12 was 88.7% (95% CI 65.9%, 96.6%). CRS occurred in 75 (94.9%) patients, of which only 2 (2.5%) had grade 3 CRS. The median time to onset of CRS was six days (range 1, 12) with a median duration of 5 days (range 2, 30). ICANS occurred in only 2 (2.5%) patients, of which one was grade 1, and the other was grade 2. All CRS were resolved, with the use of tocilizumab in 19 (25.3%) patients and steroids in 47 (62.7%). The most common ≥ grade 3 treatment-related AEs were hematological toxicities, including neutropenia (78.5%), leukopenia (74.7%), lymphopenia (55.7%), thrombocytopenia (54.4%), and anemia (46.8%). CT103A reached the peak at a median of 12 days (range 0, 26) after infusion and were still detectable in 40 of 61 evaluable patients (65.6%) at month 6 and 13 of 21 (61.9%) at month 12. Soluble BCMA decreased to the lower limit of quantification (LLOQ) at a median of 2 months and can maintain bellow LLOQ over 24 months. 11 (13.9%) patients were positive for anti-drug antibodies, of whom 1/76 (1.3%) developed anti-drug antibodies at month 3, 5/43 (11.6%) at month 6 and 5/16 (31.3%) at month 12. Among these, neutralizing antibodies were only identified in one patient (at month 12). Summary/Conclusion: Updated data from the Phase 1/2 FUMANBA-1 study continue to show encouraging efficacy of CT103A with a favorable safety profile in relapsed/refractory multiple myeloma. CT103A led to a deepening and durable response with robust expansion and prolonged persistence. Patients with prior BCMA CAR-T therapy could still benefit from CT103A. S188: TECLISTAMAB IN COMBINATION WITH DARATUMUMAB, A NOVEL, IMMUNOTHERAPY-BASED APPROACH FOR THE TREATMENT OF RELAPSED/REFRACTORY MULTIPLE MYELOMA: UPDATED PHASE 1B RESULTS P. Rodriguez Otero1,*, A. D’Souza2, D. Reece3, N. W. van de Donk4, A. Chari5, A. Krishnan6, T. Martin7, M. V. Mateos8, D. Morillo9, D. Hurd10, L. Rosinol11, A. S. Balari12, R. Wäsch13, D. Vishwamitra14, S. X. Wang Lin14, T. Prior14, L. Vandenberk15, M.-A. D. Smit16, A. Oriol17, B. Dholaria18 1University of Navarra, Navarra, Spain; 2Medical College of Wisconsin, Milwaukee, WI, United States of America; 3Princess Margaret Cancer Centre, Toronto, ON, Canada; 4Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 5Mount Sinai School of Medicine, New York, NY; 6City of Hope Comprehensive Cancer Center, Duarte, CA; 7UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA, United States of America; 8University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca; 9Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain; 10Comprehensive Cancer Center of Wake Forest Baptist Health, Winston-Salem, NC, United States of America; 11Hospital Clínic, Institut d’investigacions Biomèdiques August Pi i Sunyer, Barcelona; 12Institut Català d’Oncologia – Hospitalet, IDIBELL, University of Barcelona, Barcelona, Spain; 13Freiburg University Medical Center, Freiburg, Germany; 14Janssen Research and Development, Spring House, PA, United States of America; 15Janssen Research & Development, Antwerp, Belgium; 16Janssen Research & Development, Los Angeles, CA, United States of America; 17Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain; 18Vanderbilt University Medical Center, Nashville, TN, United States of America Background: Teclistamab (JNJ-64007957) is a B-cell maturation antigen (BCMA) × CD3 T-cell redirecting bispecific antibody currently under investigation in patients with relapsed/refractory multiple myeloma (RRMM). Daratumumab is a CD38-targeting monoclonal antibody with direct on-tumor and immunomodulatory mechanisms of action. The preliminary results from the phase 1b multicohort TRIMM-2 study showed tolerable safety with no overlapping toxicities, and encouraging efficacy, supporting the combination of teclistamab with daratumumab for the treatment of RRMM. Aims: We report updated results from the TRIMM-2 study with additional patients and longer follow-up. Methods: Eligible patients were ≥18 years of age with a MM diagnosis and previously treated with ≥3 prior lines of therapy (including a proteosome inhibitor [PI] and immunomodulatory drug [IMiD]) or were double-refractory to a PI and IMiD. Patients who had received anti-CD38 therapy ≤90 days prior were excluded. Written informed consent was obtained from all eligible patients. Patients received subcutaneous (SC) daratumumab 1800 mg per approved schedule and teclistamab SC 1.5–3 mg/kg once weekly or every 2 weeks. Primary objectives of the study were to identify the recommended phase 2 dose for the teclistamab and daratumumab combination and to assess safety of the combination. Responses were assessed by IMWG criteria. Adverse events (AEs) were graded per CTCAE v5.0, except for cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS), which were graded per ASTCT guidelines. Results: At the Jan 13, 2022 data cutoff, the median follow-up was 7.2 months (range 0.1–16.6). Among the safety population (N=46), 52% were females, and the median age was 67 years (range 50–79). Patients received a median of 6 prior lines of therapy (range 2–17); 74% of patients were triple-class exposed; 63% were penta-drug exposed, and 15% were anti-BCMA exposed. Overall, 91% of patients had ≥1 AE of any grade; 78% had grade 3/4 AEs. The most common AE was CRS (61%; all grade 1/2); median time to onset was 2 days and median duration was 2 days. Other AEs included neutropenia (54%; grade 3/4 50%), anemia (46%; grade 3/4 28%), thrombocytopenia (33%; grade 3/4 28%), and diarrhea (33%; grade 3/4 2%). Infections occurred in 29 patients (63%; grade 3/4 28%). One patient had grade 1 ICANS that was fully resolved. Among 37 response-evaluable patients, the overall response rate was 78% (29/37); 27 patients (73%) had very good partial response (VGPR) or better (Table). While the median duration of response was not reached, median time to first response across dosing cohorts was 1.0 month (range 0.9–2.8). Upregulation of CD38+/CD8+ T cells and proinflammatory cytokines was observed after teclistamab dosing in combination with daratumumab, supporting potential synergy of the combination in patients with prior anti-CD38 exposure. Updated results will be presented. Image: Summary/Conclusion: Teclistamab in combination with daratumumab is a novel immunotherapy approach that may yield improved clinical efficacy in heavily pretreated patients with RRMM. S189: EARLY, DEEP, AND DURABLE RESPONSES, AND LOW RATES OF CRS WITH REGN5458, A BCMAXCD3 BISPECIFIC ANTIBODY, IN A PHASE 1/2 FIRST-IN-HUMAN STUDY IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA J. A. Zonder1, J. Richter2,*, N. Bumma3, J. Brayer4, J. E. Hoffman5, W. I. Bensinger6, K. L. Wu7, L. Xu8, D. Chokshi8, A. Boyapati8, D. Cronier8, Y. Houvras8, K. Rodriguez Lorenc8, G. S. Kroog8, M. V. Dhodapkar9, S. Lentzsch10, D. Cooper11, S. Jagannath2 1Karmanos Cancer Institute, Detroit; 2Icahn School of Medicine at Mount Sinai, New York; 3The Ohio State University Comprehensive Cancer Center, Columbus; 4H. Lee Moffitt Cancer Center, Tampa; 5University of Miami Health System, Miami; 6Swedish Center for Blood Disorders and Stem Cell Transplants, Seattle, United States of America; 7Ziekenhuis Netwerk Antwerpen Stuivenberg, Antwerp, Belgium; 8Regeneron Pharmaceuticals, Inc, Tarrytown; 9Emory University School of Medicine, Atlanta; 10Columbia University Medical Center, New York; 11Rutgers Cancer Institute of New Jersey, New Brunswick, United States of America Background: Despite recent advances in treatment options, multiple myeloma (MM) remains incurable. REGN5458 is a BCMAxCD3 bispecific antibody currently under investigation in relapsed/refractory MM (RRMM) in an ongoing Phase 1/2 trial (NCT03761108). Preliminary data suggest that REGN5458 has a manageable safety profile with early, deep and durable responses in heavily pretreated patients (pts). Aims: Here we describe updated safety, overall response and response durability in pts treated with REGN5458 in the Phase 1 portion of this ongoing trial. Methods: The Phase 1 primary objectives are to assess safety, tolerability and occurrence of dose-limiting toxicities of REGN5458 and to determine a recommended Phase 2 dose regimen (RP2DR). Key secondary objectives include: assessment of objective response rate as determined by the investigator, duration of response (DOR) and minimal residual disease status; pharmacokinetic evaluation; and characterization of immunogenicity. Pts with progressive RRMM, who are double- or triple-refractory, or intolerant to prior lines of systemic therapy including a proteasome inhibitor, immunomodulatory agent, and anti-CD38 antibody are treated with REGN5458 monotherapy following a modified 3 + 3 dose-escalation design (4 + 3). Treatment consists of 16 weekly infusions of REGN5458 followed by q2w dosing until disease progression. Response assessments are measured using modified International Myeloma Working Group criteria. Results: At data cut-off (September 30, 2021), 73 pts were treated with REGN5458 in the dose escalation cohort with full doses ranging from 3–800 mg. Median age at enrollment was 64 years (range, 41–81) and 20.5% pts were ≥75 years. As per Revised International Staging System, stage was 1, 2 or 3 in 15.0%, 57.5% and 23.3% of pts respectively. Pts had a median of 5 prior lines of systemic therapy (range, 2–17) with 38.4% of pts being penta-refractory (Table). Median duration of follow-up was 3.0 months (range, 0.7–22.1). Treatment-emergent adverse events (TEAE) were reported in 73 pts (100%), Grade (Gr) 3 in 31 pts (42.5%), Gr 4 in 24 pts (32.9%). The most common Gr 3/4 TEAEs were hematologic (39.0%). The most frequent TEAEs were fatigue in 33 pts (45.2%), Gr 1/2 in 31 pts (42.5%), Gr 3 in 2 pts (2.7%); cytokine release syndrome (CRS) in 28 pts (38.4%), Gr 1 in 25 pts (34.2%), Gr 2 in 3 pts (4.1%). No pt had Gr ≥3 CRS or discontinued treatment due to CRS. There were no Gr ≥3 neurotoxicity events. Nausea was reported in 24 pts (32.9%), Gr 1 in 17 pts (23.3%), Gr 2 in 7 pts (9.6%). Responses were observed at all dose levels. Across all dose levels, 86.5% (n=32/37) of all responders achieved at least a very good partial response and 43.2% (n=16) of responders had a complete response (CR) or stringent CR. Amongst pts treated at the 200–800 mg dose levels, the response rate was 75.0% (n=18/24). The Kaplan–Meier estimated probability of responders being in response for 8 months or more was 90.2% (95% confidence interval: 72.6–96.7), and median DOR was not reached. Image: Summary/Conclusion: REGN5458 shows a manageable safety and tolerability profile, with Gr 2 CRS in only 4.1% of pts and no Gr ≥3 CRS or neurotoxicity events. No new safety signals were observed during the additional follow-up period. Early, deep and durable responses were seen in triple- to penta-refractory pts with RRMM, with a 75.0% response rate at the combined 200–800 mg dose levels. The Phase 2 portion of the study is currently recruiting. S190: ASXL1 MUTATIONS ACCELERATE BONE MARROW FIBROSIS VIA EGR1-TNFA AXIS MEDIATED INFLAMMATION AND FIBROCYTE GENERATION IN MYELOPROLIFERATIVE NEOPLASMS Z. Shi1 2,*, J. Liu1, Y. Zhao3, L. Yang1, Y. Cai1, P. Zhang4, Z. Xu1, T. Qin1, S. Qu1, L. Pan1, J. Wu1, X. Yan1, Z. Li3, W. Zhang1, Y. Yan1, H. Huang1, G. Huang5, B. Li6, X. Wu7 8, Z. Xiao1 1MDS/MPN center, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Tianjin; 2Department of Hematology, the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing; 3Department of Cell Biology, Tianjin Medical University; 4pathology center, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Tianjin, China; 5Divisions of Pathology and Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States of America; 6MDS/MPN center, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College; 7Department of Cell Biology, Tianjin Medical University; 8State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin, China Background: Apart from the central role of activated JAK/STAT signaling pathway, ASXL1 mutations are the most recurrent additional mutations in myeloproliferative neoplasms (MPNs) and occurred much more common in myelofibrosis (MF) than essential thrombocythemia (ET) and polycythemia vera (PV) patients. However, the mechanism of the association with ASXL1 mutations and bone marrow (BM) fibrosis remains unknown. Aims: To investigate the role of ASXL1 mutations in MF acceleration and explore the underlying mechanism. Methods: We studied the clinical data of 302 consecutive MF patients in our hospital. For mouse model study, Vav1-Cre mice, Asxl1 flox/flox and Jak2 V617F/+ knockin alleles were used to achieve hematopoietic cell-specific Jak2 V617F/+ /Asxl1 flox/flox (Asxl1-/-Jak2 VF ), Jak2 V617F/+ (Jak2 VF ), and Asxl1 flox/flox (Asxl1-/-) mice. Integrated analysis of RNAseq, transposase-accessible chromatin using sequencing (ATAC-Seq) and chromatin immunoprecipitation (ChIP)-seq were performed to explore the underlying mechanism. Results: 98 of 302 (32.5%) patients harbored ASXL1 mutations. ASXL1 mutations are correlated with higher monocyte counts, increasing CD34+ cells in peripheral blood (PB), larger spleen sizes, and higher MF grades. Consistent with clinical findings, Asxl1-/-Jak2 VF mice showed lower platelet counts, higher monocyte counts and c-kit+ cells in PB compared with Jak2 VF mice. Hematopoietic stem and progenitor cells (HSPCs) compartment analysis revealed increased granulocyte/macrophage progenitors (GMPs), and megakaryocyte /erythroid progenitors (MEPs) in spleens of Asxl1-/-Jak2 VF , not in BM compared with Jak2 V617F mice (Figure 1A), in line with increased spleen weights of Asxl1-/-Jak2 VF mice (Figure 1B). Histology analysis revealed reticulin infiltration in BM of Asxl1-/-Jak2 VF mice, not in other age-matched genotypes. Both ASXL1 MT MF patients and Asxl1-/-Jak2 VF mice showed increased monocytes/macrophages in BM (Figure 1C-D), accounting for more severe inflammation in ASXL1 MT MF patients and mouse models. In vitro differentiation assay revealed monocyte/ macrophage differentiation bias of Asxl1-/-Jak2 VF HSPCs (Figure 1E). In fibrosis driving cells analysis, ASXL1 MT MF patients showed comparable mesenchymal stromal cells (MSCs) derived myofibroblasts while increased monocyte derived fibrocytes in BM compared with ASXL1 WT MF patients (Figure 1F). In vitro fibrocyte differentiation assay (Figure 1G) and flow cytometry analysis confirmed increased fibrocytes in BM of Asxl1-/-Jak2 VF mice compared with other genotypes. Mechanismly, Asxl1 deletion in Jak2 VF HSPCs leads to enhancer activation of polycomb group (PcG) target genes, such as Egr1 (Figure 1H). The upregulation of Egr1, which was validated in mouse models and patients (Figure 1I-J), in turn accounts for increased HSPCs commitment to monocyte/macrophage lineage (Figure 1K). Moreover, Egr1 induces the activation of Tnfa (Figure 1L) and thereby further drives the differentiation of monocytes to fibrocytes (Figure 1M). Accordingly, combined TNFR antagonist (R-7050) with ruxolitinib significantly reduces fibrocytes production in vitro in Asxl1-/-Jak2 VF mice and ASXL1 MT MF patients (Figure 1N-O). Image: Summary/Conclusion: Our study illustrated the crucial role of ASXL1 mutation in MPN phenotypes and BM fibrosis. ASXL1 mutations activate the EGR1-TNFA axis in MPNs, leading to monocyte/macrophage mediated inflammation and fibrocyte induced BM fibrosis. Ruxolitinib along with TNFR antagonist may ameliorate fibrocytes, providing an attractive theoretical approach for antifibrosis treatment. S191: CALRETICULIN-MUTATED HEMATOPOIETIC CELLS ARE VULNERABLE TO THE COMBINED INHIBITION OF THE PROTEASOME AND THE IRE1A-XBP1 AXIS OF THE UNFOLDED PROTEIN RESPONSE J. S. Jutzi1,*, A. E. Marneth1, M. J. Jimenez-Santos2, A. Guerra-Moreno1, S. A. Myers3 4, S. A. Carr3, P. van Galen1, F. Al-Shahrour5, A. S. Nam6, A. Mullally1 1Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Boston, United States of America; 2Bioinformatics Unit, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; 3The Broad Institute of MIT and Harvard, Cambridge; 4Center for Autoimmunity and Inflammation, La Jolla Institute for Immunology, La Jolla; 5Bioinformatics Unit, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid; 6Weill Cornell Medicine, New York, United States of America Background: Mutations in the endoplasmic reticulum (ER) chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). These mutant CALR proteins have impaired chaperone function. Concordant with this, transcriptional upregulation of the unfolded protein response (UPR) has been reported in patients with CALR-mutated MPN. However, it is not understood how CALR-mutated cells counter-balance ER stress resulting from impaired chaperone function. Despite the frequency of CALR mutations in MPN, there are currently no treatment strategies to preferentially target CALR-mutant cells over healthy cells. Aims: To determine the mechanisms by which mutant CALR cells alleviate ER stress and to exploit these mechanisms selectively by pharmacological intervention in vivo. Methods: Long-term HSCs (LT-HSCs) from Calr Δ52 knockin mice were isolated for RNA-Seq. Calr Δ52 bone marrow (BM) was differentiated ex vivo, subsequently enriched for megakaryocytes, and subjected to quantitative proteomic analysis. Pathway analyses of CALR-mutant megakaryocyte progenitors (MkPs) were performed on Genotyping of Transcriptome (GoT) data from patients with Essential Thrombocytosis (ET). Calr Δ52 VAVCre mice and chimeric transplanted Calr Δ52 mice were treated with a proteasome inhibitor, an IRE1a inhibitor, or both. Results: To identify dysregulated pathways in Calr Δ52/Δ52 LT-HSCs, we performed RNA-seq and found that Xbp1s downstream of IRE1a and the proteasome pathway was significantly upregulated in Calr Δ52/Δ52 animals. We confirmed elevated XBP1s protein level in heterozygous Calr Δ52 knockin mice by flow cytometry. Since elevated and abnormal megakaryopoiesis are hallmarks of CALR-mutant MPN, we performed quantitative proteomics on megakaryocyte-enriched cells from Calr Δ52 mice. We confirmed upregulation of both proteasome and ER chaperone proteins in Calr-mutant cells. To verify these findings in human MPN, we interrogated a published GoT data set and found that in addition to XBP1s, the proteasome pathway was also differentially upregulated in CALR-mutated patient MkPs compared to the wildtype MkPs (p<2e-16). To investigate the functional consequences of IRE1a or proteasome inhibition, we treated CALR-mutant hematopoietic cell lines with KIRA6 (IRE1a inhibitor) and/or bortezomib (proteasome inhibitor) and found CALR-mutant cells to be differentially sensitive as compared to isogenic controls. We subsequently found that treatment with bortezomib leads to the accumulation of misfolded and ubiquitinylated proteins in mutant Calr BM, resulting in pro-apoptotic priming. In vivo treatment of Calr Δ52/+ mice with bortezomib resulted in a significant reduction in platelets. Finally, we combined IRE1a and proteasome inhibition in a chimeric BM transplantation in vivo model using CD45.2 Calr Δ52/+ MxCre GFP and CD45.1 wild-type competitor cells. Following combination therapy, we found a significant reduction in Calr-mutant donor chimerism, specifically in LT-HSCs and platelets, accompanied by reductions in LT-HSC frequency and platelet count. Summary/Conclusion: In summary, we have found that disrupted proteasis in CALR-mutated MPN cells results in a dependency on the IRE1-XPB1 axis of the UPR and proteasome activity. Using pre-clinical MPN models, we found that combined inhibition of both pathways in vivo normalizes important features of MPN and preferentially targets Calr Δ52 over wild-type cells. These findings highlight combined inhibition of IRE1 and the proteasome as a promising therapeutic strategy in CALR-mutant MPN. S192: SINGLE-CELL RNA PROFILING OF MYELOFIBROSIS PATIENTS REVEALS PELABRESIB-INDUCED DECREASE OF MEGAKARYOCYTIC PROGENITORS AND NORMALIZATION OF CD4+ T CELLS IN PERIPHERAL BLOOD O. Zavidij1,*, N. J. Haradhvala2, R. Meyer1, J. Cui1, S. Verstovsek3, S. Oh4, A. Mead5, P. Taverna1 1Constellation Pharmaceuticals a MorphoSys Company; 2Harvard Graduate Program in Biophysics, Harvard University, Boston, MA; 3Leukemia Department, University of Texas MD Anderson Cancer Center, Houston, TX; 4Hematology Division, Washington University, St Louis, MO; 5NIHR Biomedical Research Centre, University of Oxford, Oxford, United States of America Background: Abnormal differentiation of the megakaryocytic (MK) lineage in conjunction with overproduction of proinflammatory cytokines (Ck), resulting in bone marrow fibrosis, anemia, extramedullary hematopoiesis and often hepatosplenomegaly, are the main biological and clinical characteristics of myelofibrosis (MF). Bromodomain and extraterminal domain (BET) proteins play a key role in the regulation of neoplastic myeloproliferation and proinflammatory Ck production. Pelabresib (CPI-0610) is a potent, selective BET inhibitor under investigation in MF patients (pts) as monotherapy and in combination with the Janus kinase inhibitor (JAKi) ruxolitinib (RUX) in the ongoing MANIFEST Phase 2 study (NCT02158858). Arm 1: pelabresib as monotherapy in RUX-intolerant, ineligible or refractory MF pts; Arm 2: pelabresib in combination with RUX as an ‘add-on’ in MF pts with suboptimal response to RUX; Arm 3: pelabresib combination therapy with RUX in JAKi-naïve MF pts. Pelabresib as monotherapy and in combination with RUX improved spleen volume reduction, symptoms and hemoglobin levels (Talpaz M, ASH 2020). The effects of pelabresib on hematopoietic stem/progenitor cells (HSPCs) and immune cell populations in MF pts are presented here. Aims: We aimed to characterize the cellular composition and transcriptional alterations occurring in peripheral blood (PB) obtained from MF pts enrolled in the MANIFEST trial. Methods: We performed single-cell RNA sequencing on 234,904 CD34+ HSPCs and 135,970 CD34- mature PB cells (Figure). Cells were obtained from a random pool of 20 pts (Arm 1 n=5, Arm 2 n=8, Arm 3 n=7), which included a baseline sample (BL) and samples collected during treatment (range: C3 through C12) for each pt. Mobilized PB cells from healthy donors (HD) were used as a control (n=11). Results: Analysis of CD34+ HSPCs at BL demonstrated a statistically significant increase of MK, neutrophilic and erythroid progenitors in MF pts compared with HDs and decreased numbers of myeloid and B cell lineage progenitors. Pelabresib as monotherapy and in combination with RUX led to a significant reduction of MK-, neutrophilic and erythroid progenitors as compared with BL in all pts analyzed. Analysis of CD34- cells from MF pts identified a significantly lower proportion of CD4+ T cells and increased numbers of erythroid cells at BL compared with HD. Individual pts also exhibited reduction in natural killer cells and CD16+ monocytes as well as elevated MK lineage cells. Pelabresib as monotherapy and in combination with RUX increased the proportion of CD4+ T cells, and more importantly, reduced MK lineage cells compared with BL in both treatment-naïve and RUX relapsed/refractory (r/r) pts. In MF pts at BL, a larger spleen volume was observed in pts with lower numbers of CD4+ T cells and increased numbers of MK- and myeloid CSF3R+ cells. Updated results of the cell composition analysis and transcriptional reprogramming during pelabresib treatment, as well as correlation with effects on cytokine plasma levels and clinical efficacy data, will be presented. Image: Summary/Conclusion: The single-cell profiling of a subset of MF pts enrolled in the MANIFEST study suggests that pelabresib alone and in combination with RUX induces an improvement of the myeloid-lymphoid imbalance in both JAKi-naïve and r/r MF pts. The observed effects of pelabresib on HSPCs from MF pts confirm the ex vivo activity on erythroid and MK differentiation of stem cells (Keller P, EHA 2021; Verstovsek S, ASH 2021) and implies a potential disease-modifying effect warranting further investigation. S193: GENOMIC AND FUNCTIONAL IMPACT OF TP53 INACTIVATION IN JAK2V617F MYELOPROLIFERATIVE NEOPLASMS: A TRANSGENIC MOUSE MODEL APPROACH. P. GOU1, D. LIU1,*, E. Lauret2, N. Maslah3, V. Montcuquet4, V. meignin5, J.-J. Kiladjian6, B. Cassinat3, S. Giraudier3 1U1131, 25Université de Paris, Institut Cochin, INSERM U1016, CNRS UMR 8104, INSERM; 3U1131, INSERM Univrsité de Paris and APHP; 4Animal Facility Unit, Université de Paris; 5Histo-pathological department, Hôpital Saint Louis; 6Centre Investigations Cliniques, Hôpital Saint-Louis, Paris, France, INSERM Univrsité de Paris and APHP, Paris, France Background: MPN are characterized by increased proliferation of myeloid cells and a risk of transformation to AML. MPN are due to the acquisition of mutations (JAK2, CALR, MPL) leading to JAK2 pathway activation but cooperating mutations involved in MPN evolution have been described. Mutations of TP53 in JAK2V617F patients are linked to AML transformation, illustrated by development of leukemias after retroviral overexpression of JAK2V617F in TP53 inactivated (KO) cells transplanted in irradiated animals, thus a possible cooperation between TP53 inactivation and JAK2 signaling in transformation process has been postulated. However, JAK2V617F signaling has also been reported as able to reduce p53 expression per sesuggesting a redundant role of p53 inactivation in JAK2V617F cells. Aims: Determine the role of TP53 in JAK2V617F induced genetic and phenotypic modifications. Methods: To better understand the role of TP53 in MPN phenotype and evolution in steady state hematopoiesis, we developed a transgenic model of vav-cre induced JAK2V617F expression in TP53 knock-out (KO) animals and analyzed phenotype, survival, genetic modifications using stem cell compartments RNA-Seq and response to therapy of this triple transgenic mice. Results: JAK2V617F vav-inductible-Tg/TP53 KO mice were generated to recombine JAK2V617F at an endogenous level in TP53 inactivated hematopoietic cells. During follow-up, JAK2V617F/TP53KO mice developed the same phenotype than JAK2V617F transgenic mice without leukemia. In competitive repopulations with grafts with different ratio of JAK2V617F, JAK2V617F/TP53KO and wild type cells, we observed that JAK2V617F/TP53KO cells outcompete JAK2V617F cells. In order to better define TP53-dependent and independent pathways linked to these phenotypes, LT-HSC, ST-HSC, MPP, CMP, MEP and GMP from normal, JAK2V617F and JAK2V617F/TP53 KO mice were cell sorted and RNA-Seq analysis was performed. Principal component analysis demonstrated that JAK2V617F/TP53KO cells were closer to normal cells than JAK2V617F cells whatever the (stem) cell compartment, illustrating that JAK2V617F-induced modifications are largely p53 dependent. Further analysis demonstrated that approximatively half of JAK2V617F deregulated genes in the different compartments are TP53 dependent including the IFN pathways. In order to validate this finding, mice repopulated with a mix of WT and JAK2V617F (either p53 KO or wild-type) cells were treated for 8 weeks with recombinant murine pegylated IFN-a. JAK2V617F reconstituted animals entered in complete hematological remission while JAK2VF/TP53KO reconstituted animals did not, illustrating that loss of TP53 in this context induced resistance to IFN-a. On the other hand, since MPN develop in the same way in JAK2V617F-only and in JAK2V617F/TP53KO mouse, this suggests that JAK2V617F-specific pathways also found differentially expressed in JAK2V617F/TP53KO are linked to the MPN phenotype. KEGG and GO analysis demonstrated that these genes were mainly implicated in cytokine response, cell proliferation, differentiation, and leukemia evolution illustrating that the development of MPN and its possible risk of transformation in this mouse model is largely TP53 independent. Summary/Conclusion: Taken together, our results show that a large part of genetic modifications induced by JAK2V617F mutation are p53 dependent but MPN phenotype is not, TP53 loss is insufficient to induce quick leukemic transformation in steady-state hematopoiesis in Jak2 V617F MPN despite it increases LT-HSC cell proliferation and finally that TP53 loss could be involved in IFN resistance in MPN. S194: INTEGRATIVE CLINICAL PROTEOTYPING AND DRUG RESPONSE PROFILING IDENTIFIES TARGETABLE BIOLOGY UNDERLYING MYELOPROLIFERATIVE NEOPLASMS M. H. Wildschut1 2 3,*, M. van Oostrum2, J. Settelmeier2 4, J. Mena1, C. Dördelmann5, Y. Festl1, A. Ring3, R. C. Skoda6, M. Lopes5, B. Wollscheid2 4, B. Snijder1, A. Theocharides3 1Institute of Molecular Systems Biology, Department of Biology; 2Institute of Translational Medicine, Department of Health Science and Technology, ETH Zurich; 3Department of Medical Oncology and Hematology, Division of Hematology, University Hospital Zurich, Zurich; 4Swiss Institute of Bioinformatics, Lausanne; 5Insititute of Molecular Cancer Research, University of Zurich, Zurich; 6Department of Biomedicine, Experimental Hematology, University Hospital Basel and University of Basel, Basel, Switzerland Background: Myeloproliferative neoplasms (MPN) are a family of hematopoietic stem cell diseases characterized by frequent driver mutations in Calreticulin (CALR) and JAK2. While patients with essential thrombocythemia (ET) have a more favorable outcome, the prognosis of patients with advanced myelofibrosis (MF) is particularly poor with limited therapeutic options. Aims: Here, we set out to elucidate biology underlying MPN and translate this into vulnerabilities that can be exploited by targeted therapies. By integration of proteomic analyses and drug response profiling we aimed to correlate MPN protein alterations directly to targeted drug responses, and subsequently validate pathway alterations using dedicated follow-up experiments. Methods: We gathered extended MPN patient and matched healthy donor (HD) cohorts (n = 119) and a follow-up MF-specific cohort (n = 44) to investigate both general and subgroup-specific alterations. We performed proteomics on isolated granulocytes, hematopoietic stem and progenitor cells (HSPCs), and T-cells, and drug screening on peripheral blood mononuclear cells (PBMCs). For follow-up experiments, we established a CALR mutant-specific antibody for use in high-content immunofluorescence imaging. Results: We identify a specific signature of disease- and mutation-specific protein alterations present in MPN patients by machine learning. Among the identified alterations, we find strong upregulation of proteins belonging to the MCM complex, a helicase involved in DNA replication, to be specifically present in a subgroup of MF patients that are clinically characterized by a worse prognosis. In these patients, a proteomic signature related to DNA replication and cell cycling is found to be elevated across blood cell types. We proof that cells from these patients are a) characterized by high proliferation rates, and b) respond well to drugs targeting this process (e.g. topoisomerase inhibitors). Furthermore, we find upregulation of proteins involved in endoplasmic reticulum (ER) stress to be significantly correlated to the CALR mutation burden across HSPCs, granulocytes, and T-cells. Using an advanced imaging approach we show that on a single cell level, the amount of ER stress corresponds to the level of CALR mutant protein expression. This results in a higher sensitivity of CALR-mutated cells to drugs targeting ER stress or the corresponding unfolded protein response (UPR). Summary/Conclusion: Using large-scale MPN patient cohorts, we are able to elucidate biology underlying MPN disease. We here show specific proteomic signatures of proliferation and ER stress to be present in subsets of MPN patients and translate this to specific biological alterations and correlated therapeutic vulnerabilities. Our findings can be instrumental to both improved stratification of response and discovery of novel targeted therapeutics for MPN patients. S195: MOMENTUM: PHASE 3 RANDOMIZED STUDY OF MOMELOTINIB (MMB) VERSUS DANAZOL (DAN) IN SYMPTOMATIC AND ANEMIC MYELOFIBROSIS (MF) PATIENTS PREVIOUSLY TREATED WITH A JAK INHIBITOR S. Verstovsek1,*, A. Vannucchi2, A. Gerds3, H. K. Al-Ali4, D. Lavie5, A. Kuykendall6, S. Grosicki7, A. Iurlo8, Y. T. Goh9, M. Lazaroiu10, M. Egyed11, M. L. Fox12, D. McLornan13, A. Perkins14, S.-S. Yoon15, V. Gupta16, J.-J. Kiladjian17, R. Donahue18, J. Kawashima18, R. Mesa19 1The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America; 2Center Research and Innovation of Myeloproliferative Neoplasms, AOU Careggi, University of Florence, Florence, Italy; 3Cleveland Clinic Department of Hematology and Medical Oncology, Avon, OH, United States of America; 4University Hospital of Halle, Halle, Germany; 5Hadassah Hebrew University Medical Center, Jerusalem, Israel; 6Moffitt Cancer Center, Tampa, FL, United States of America; 7Medical University of Silesia, Katowice, Poland; 8Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 9Singapore General Hospital, Singapore, Singapore; 10Policlinica de Diagnostic Rapid Brasov, Brasov, Romania; 11Somogy County Mór Kaposi General Hospital, Kaposvár, Hungary; 12Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain; 13Guy’s and Saint Thomas’ NHS Foundation Trust, London, United Kingdom; 14Monash University, Melbourne, Australia; 15Seoul National University Hospital, Seoul, South Korea; 16Princess Margaret Cancer Centre, Toronto, ON, Canada; 17Saint-Louis Hospital (AP-HP), Paris, France; 18Sierra Oncology, Inc., San Mateo, CA; 19UT Health San Antonio Cancer Center, San Antonio, TX, United States of America Background: MMB, a novel oral ACVR1/ALK2 and JAK1/2 inhibitor, showed clinical activity on MF symptoms, red blood cell (RBC) transfusion requirements (anemia), and spleen volume in the SIMPLIFY trials. Aims: This pivotal phase 3 study of MF patients (pts) previously treated with a JAK inhibitor (JAKi) tested MMB vs DAN on key symptom, anemia, and spleen volume endpoints at 24 weeks (wks). Methods: Eligibility: Primary or post-ET/PV MF; DIPSS high risk, Int-2, or Int-1; MF Symptom Assessment Form Total Symptom Score (MFSAF TSS) ≥10; hemoglobin (Hgb) <10 g/dL; prior JAKi for ≥90 days, or ≥28 days if RBC transfusions ≥4 units in 8 wks or Gr 3/4 thrombocytopenia, anemia, or hematoma; palpable spleen ≥5 cm. Stratification: TSS (≥22 vs <22), palpable spleen (≥12 cm vs <12 cm), and RBC units transfused (0, 1-4, and 5+). JAKi taper and washout was ≥21 days. Randomization: 2:1 to MMB 200 mg QD plus DAN placebo or DAN 600 mg QD plus MMB placebo for 24 wks, after which pts could receive open-label MMB. Assessments: Pt reported symptoms using a daily eDiary and spleen volume by MRI or CT. The primary endpoint was TSS response (≥50% reduction from baseline [BL]) rate at wk 24. Secondary endpoints, assessed sequentially at wk 24, were RBC transfusion independence (TI) rate, splenic response rate (SRR; ≥25% reduction in volume from BL), change from BL in TSS, SRR (≥35% reduction from BL) and rate of zero transfusions since BL. Informed consent was obtained from all participants. Results: 94 of 130 (72%) MMB pts and 38 of 65 (58%) DAN pts completed the 24-wk randomized treatment (RT) phase. Median BL TSS were 28 (MMB) and 26 (DAN), Hgb were 8.1 (MMB) and 7.9 (DAN) g/dL, and platelets were 97 (MMB) and 94 (DAN) x109/L. BL TI was 13% (MMB) and 15% (DAN). BL mean spleen volume was 2367 (MMB) and 2288 (DAN) cm3. Prior JAKi was ruxolitinib in 195 pts (100%) and fedratinib in 9 pts (5%); mean duration of prior JAKi was 134 weeks. All primary and key secondary endpoints were met (Table). Most common Gr ≥3 treatment-emergent adverse events (TEAEs) in the RT phase of the study were thrombocytopenia (MMB, 22%; DAN, 12%) and anemia (MMB, 8%; DAN, 11%). Gr ≥3 infections occurred in 15% of MMB and 17% of DAN pts. Peripheral neuropathy (PN) occurred in 5 (4%) of MMB (all Gr ≤2) and 1 (2%) of DAN (Gr ≤2) pts in the RT phase, and none discontinued study drug due to PN. Overall, TEAEs led to study drug discontinuation in 18% of MMB and 23% of DAN pts, and serious TEAEs were reported in 35% of MMB and 40% of DAN pts, in RT phase. A trend toward improved OS up to wk 24 was seen with MMB vs DAN (HR=0.506, p=0.0719). Image: Summary/Conclusion: In symptomatic and anemic MF pts, MMB was superior to DAN for symptom responses, transfusion requirements, and spleen responses with comparable safety and favorable survival. MMB may address a critical unmet need, particularly in MF pts with anemia. NCT04173494. S196: ROPEGINTERFERON ALFA-2B ACHIEVES PATIENT-SPECIFIC TREATMENT GOALS IN POLYCYTHEMIA VERA: FINAL RESULTS FROM THE PROUD-PV/CONTINUATION-PV STUDIES H. Gisslinger1,*, C. Klade2, P. Georgiev3, D. Krochmalczyk4, L. Gercheva-Kyuchukova, M. Egyed6, P. Dulicek7, A. Illes8, H. Pylypenko9, L. Sivcheva10, J. Mayer11, V. Yablokova12, V. Empson2, K. Krejcy2, H. Hasselbalch13, R. Kralovics14, J.-J. Kiladjian15 1Department of Internal Medicine I, Division of Hematology and Blood Coagulation, Medical University Vienna; 2AOP Orphan Pharmaceuticals GmbH, Vienna, Austria; 3Medical University of Plovdiv, Plovdiv, Bulgaria; 4Teaching Unit of the Hematology Department, University Hospital in Krakow, Krakow, Poland; 5Clinical Hematology Clinic, Multiprofile Hospital for Active Treatment “Sveta Marina”, Varna, Bulgaria; 6Department of Internal Medicine II, Kaposi Mor County Teaching Hospital, Kaposvar, Hungary; 7Department of Clinical Hematology, University Hospital Hradec Kralove, Hradec Kralove, Czechia; 8Department of Hematology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; 9Department of Hematology, Regional Treatment and Diagnostics Hematology Centre, Cherkasy Regional Oncology Centre, Cherkasy, Ukraine; 10First Department of Internal Medicine, Multiprofile Hospital for Active Treatment - HristoBotev, Vratsa, Bulgaria; 11Clinic of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czechia; 12Department of Hematology, Yaroslavl Regional Clinical Hospital, Yaroslavl, Russia; 13Department of Hematology, Zealand University Hospital, University of Copenhagen, Roskilde, Denmark; 14Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria; 15Centre d’Investigations Cliniques, INSERM, CIC1427, Université de Paris, AP-HP, Hôpital Saint-Louis, Paris, France Background: Treatment of polycythemia vera (PV) aims to prevent thromboembolic complications, reduce the risk of progression to acute leukemia or myelofibrosis - of particular concern to patients - and ameliorate the symptom burden; specifically, to improve quality of life, therapy should address the most clinically important symptoms while reducing phlebotomies to avoid iron-deficiency symptoms. Long-term efficacy and safety of ropeginterferon alfa-2b have been demonstrated in PROUD-PV/CONTINUATION-PV; the final analysis applied a patient-focused approach. Aims: To analyze the patient-relevant benefit of ropeginterferon alfa-2b versus hydroxyurea (HU)/best available treatment (BAT) over 6 years. Methods: Patients diagnosed with PV according to WHO 2008 criteria who were cytoreduction-naïve or hydroxyurea pre-treated and gave written informed consent were randomized 1:1 to ropeginterferon alpha-2b or control treatment (HU) for one year in PROUD-PV. In CONTINUATION-PV, control arm patients could switch from HU to BAT. Patient-reported PV symptom burden was assessed based on adverse events documented in the patient diary and recorded at each visit; items defined in the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (MPN-SAF TSS; fatigue, concentration problems, early satiety, inactivity, night sweats, itching, bone pain, abdominal discomfort, weight loss, and fevers) and medical synonyms were evaluated post-hoc. Efficacy assessments included Kaplan-Meier analysis of event-free survival, phlebotomy need and JAK2V617F allele burden. Analyses were conducted on the CONTINUATION-PV full analysis set over 6 years of treatment. Results: The full analysis set comprised 95 patients in the ropeginterferon alfa-2b arm and 74 in the control arm. Patient-reported symptoms defined in the MPN-SAF TSS were present in a small minority (9.5%) of patients per arm at baseline (up to Week 4 of treatment) in this early-stage PV population. Occurrence of the defined symptoms remained low over long-term treatment, reported in 15.7% of patients in the ropeginterferon alfa-2b arm and 20.7% in the control arm during the 6th year of treatment. No phlebotomies were required to maintain hematocrit <45% in the 6th year of treatment in 81.4% of patients receiving ropeginterferon alfa-2b compared with 60.0% of patients in the control arm (p=0.005). Depletion of the JAK2V617F alle burden, which may lower the risk of progression to myelofibrosis, was observed in ropeginterferon alfa-2b treated patients; JAK2V617F allele burden <1% at 6 years was achieved in 19/92 (20.7%) patients in the ropeginterferon alfa-2b arm with baseline allele burden >10%. One patient met this threshold in the control arm (1/70 [1.4%]; p=0.0001). Event-free survival (risk events: disease progression, death and thromboembolic events) over ≥6 years of treatment was significantly higher among ropeginterferon alfa-2b treated patients than the control group (risk events reported in 5/95 vs. 12/74 patients, respectively; p=0.04 [Log-Rank]; Fig 1). Image: Summary/Conclusion: Long-term ropeginterferon alfa-2b therapy fulfils treatment goals important to patients with PV: a good quality of life as indicated by a low symptom burden and phlebotomy requirement, the potential to influence myelofibrosis risk, and better event-free survival versus BAT. S197: NAVITOCLAX PLUS RUXOLITINIB IN JAK INHIBITOR-NAÏVE PATIENTS WITH MYELOFIBROSIS: PRELIMINARY SAFETY AND EFFICACY IN A MULTICENTER, OPEN-LABEL PHASE 2 STUDY F. Passamonti1,*, J. Foran2, A. Tandra3, V. De Stefano4 5, M. Laura Fox6, A. Mattour7, M. F. McMullin8, A. C. Perkins9, G. Rodriguez-Macías10, H. Sibai11, Q. Qin12, J. Potluri12, J. How13 1Department of Medicine and Surgery, University of Insubria, Varese, Italy; 2Mayo Clinic, Jacksonville, FL; 3Indiana Blood and Marrow Transplant, Indianapolis, United States of America; 4Section of Hematology, Department of Radiological and Hematological Sciences, Catholic University; 5Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy; 6Department of Hematology, Hospital Universitari Vall d’Hebron, Experimental Hematology, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Hospital Campus, Barcelona, Spain; 7Henry Ford Hospital, Detroit, MI, United States of America; 8Centre for Medical Education, Queenʼs University Belfast, Belfast, United Kingdom; 9Australian Centre for Blood Diseases, Monash University and The Alfred Hospital, Melbourne, Australia; 10Department of Hematology, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 11Medical Oncology and Hematology, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada; 12AbbVie Inc, North Chicago, IL, United States of America; 13Division of Hematology, McGill University Health Center, Montreal, Canada Background: Ruxolitinib, a Janus kinase (JAK) 1/2 inhibitor, is the current standard of care for patients with myelofibrosis (MF) that improves disease symptoms in approximately 40% of patients with limited impact on disease biology. Many patients lose response over time, highlighting an unmet need for novel therapies. Navitoclax is a first-in-class, oral, small molecule that binds with high affinity to BCL-XL and BCL-2, key pro-survival proteins in the apoptotic pathway, and has a synergistic effect when used in combination with JAK inhibitors to enhance malignant cell death in MF. This ongoing, open-label, multicenter, phase II trial (NCT03222609) is evaluating the efficacy and safety of navitoclax with or without ruxolitinib in patients with MF and previously published data suggest clinically meaningful activity of the combination in patients with prior ruxolitinib failure (Pemmaraju et al., ASH 2020). Here, we report results from patients who were JAK inhibitor-naïve and treated with navitoclax and ruxolitinib combination therapy. Aims: N/A Methods: Enrolled patients had primary or secondary MF with splenomegaly (DIPSS ≥ Intermediate-1) and did not receive prior therapy with JAK-2 or bromodomain and extraterminal motif (BET) inhibitors. Patients initiated navitoclax at 100 mg once per day (QD) or 200 mg QD if baseline platelet count was ≤150 × 109/L or >150 × 109/L, respectively. Ruxolitinib was orally administered twice daily at a starting dose based on baseline platelet count as per the local ruxolitinib label. The primary endpoint was spleen volume reduction of ≥35% (SVR35) from baseline at week 24. SVR35 was assessed at weeks 12, 24, and once every 24 weeks thereafter. Key secondary endpoints were ≥50% reduction in total symptom score (TSS50), bone marrow fibrosis reduction, and anemia response (per International Working Group criteria). Adverse events (AEs) were monitored throughout the study. Results: As of Oct 04, 2021, 32 patients received navitoclax plus ruxolitinib. The median duration of follow-up was 6.1 (range, 1.9 to 18.6) months. Twenty-eight (88%) patients received a navitoclax dose of 200 mg, and 4 (13%) received 100 mg OD. The median age was 69 years (range, 44 to 83), median spleen volume was 1889.08 cm3 (range, 645.6 to 7339.6), and 38% had at least 1 prior line of therapy. The median navitoclax and ruxolitinib exposures were 24.1 (range, 5.1 to 80.9) and 24.1 (5.1 to 80.9) weeks, respectively. Thirty-one (97%) patients experienced ≥1 AE, 25 (78%) had Grade ≥3 AEs, and 6 (19%) had serious AEs. The most frequent Grade ≥3 AEs were anemia (34%), thrombocytopenia (31%), and neutropenia (19%). Three (9%) and 2 (6%) patients experienced an AE that led to navitoclax and ruxolitinib discontinuation, respectively, and 2 (6%; 1 progressive disease, 1 cardiac disorder unrelated to navitoclax) AEs led to death ≤30 days after the last dose of navitoclax. SVR35 was achieved by 52% of evaluable patients at week 24 (SVR35 in DIPSS Intermediate-2, 50%; High-risk, 33%) and by 76% at any time on the study (Table). The median time to first SVR35 was 12.1 (range, 11 to 47) weeks. Image: Summary/Conclusion: The combination of navitoclax and ruxolitinib was well tolerated and demonstrated early and robust reductions in spleen volume, anemia, and BM fibrosis in patients without prior JAK-2 inhibitor exposure. SVR35, anemia, and bone marrow fibrosis improved over time. S198: BET INHIBITOR PELABRESIB (CPI-0610) COMBINED WITH RUXOLITINIB IN PATIENTS WITH MYELOFIBROSIS — JAK INHIBITOR-NAÏVE OR WITH SUBOPTIMAL RESPONSE TO RUXOLITINIB — PRELIMINARY DATA FROM THE MANIFEST STUDY J. Mascarenhas1,*, M. Kremyanskaya1, A. Patriarca2, C. Harrison3, P. Bose4, R. K. Rampal5, F Palandri6, T. Devos7, F. Passamonti8, G. Hobbs9, M. Talpaz10, A. Vannucchi11, J.-J. Kiladjian12, S. Verstovsek13, R. Hoffman1, M. E. Salama13, D. Chen14, P. Taverna15, A. Chang15, G. Colak15, S. Klein15, V. Gupta16 1Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America; 2Hematology Unit, Department of Translational Medicine, University of Eastern Piedmont and AOU Maggiore della Carità, Novara, Italy; 3Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom; 4MD Anderson Cancer Center, Houston, TX; 5Memorial Sloan-Kettering Cancer Center, New York, NY, United States of America; 6IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy; 7University Hospitals Leuven and Laboratory of Molecular Immunology (Rega Institute), KU Leuven, Leuven, Belgium; 8University of Insubria, Varese, Italy; 9Division of Hematology/Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 10University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, United States of America; 11Azienda Ospedaliero-Universitaria Careggi, University of Florence, Florence, Italy; 12Hôpital Saint-Louis, Université de Paris, Paris, France; 13Leukemia Department, University of Texas MD Anderson Cancer Center, Houston, TX; 14Mayo Clinic, Rochester, MN; 15Constellation Pharmaceuticals a MorphoSys Company, Boston, MA, United States of America; 16Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada Background: Myelofibrosis (MF) is characterized by bone marrow (BM) fibrosis, anemia, splenomegaly and constitutional symptoms. Progressive BM fibrosis results from aberrant megakaryopoiesis and proinflammatory cytokine expression, two processes that are tightly regulated by bromodomain and extraterminal domain (BET) protein-mediated expression of genes (e.g. NF-κB, MYC and BCL-2) and often lead to myeloproliferation and cytopenias. While many MF patients (pts) receive the Janus kinase inhibitor (JAKi) ruxolitinib (RUX) as the current standard of care, the depth and durability of responses, along with the percentage of pts that achieve ≥35% spleen volume reduction from baseline (BL; SVR35) and ≥50% total symptom score reduction from BL (TSS50) are limited; thus, a significant unmet medical need exists. Pelabresib (CPI-0610) is an oral, small-molecule, investigational BET inhibitor that downregulates NF-κB gene expression and other relevant genes involved in MF disease pathways. Aims: Data (Sep 2021 data cut) on the safety and efficacy of pelabresib in combination with RUX in pts with MF from Arms 2 and 3 of the ongoing, open-label Phase 2 MANIFEST study (NCT02158858). In Arm 2, MF pts with suboptimal response to RUX are treated with pelabresib as ‘add-on’ to RUX (Arm 2A: transfusion dependent [TD]; Arm 2B: non-TD). In Arm 3, JAKi-naïve MF pts are treated with pelabresib in combination with RUX. Methods: The primary endpoints are SVR35 at Week (Wk) 24 for Arm 3 and Arm 2B (non-TD cohort) and TD to transfusion independence (TI) in Arm 2A (TD cohort). The key secondary endpoint is TSS50 per Myelofibrosis Symptom Assessment Form v4.0 at Wk 24; in Arm 2A (TD cohort), SVR35 is an additional key secondary endpoint. BM biopsies to assess BM fibrosis and safety data are also evaluated. Results: At Wk 24 in Arm 3 (N=84), 68% (57/84) pts achieved SVR35 (median change: –50%; Figure), and 56% (46/82) pts achieved TSS50 (median change: –59%). Twenty-four percent of pts had a mean hemoglobin increase ≥1.5 g/dL from BL over 12 weeks without transfusions. At Wk 24 in Arm 2 (N=86), 20% (16/81) pts achieved SVR35 (median change: –18%; Figure), and 37% (30/81) pts achieved TSS50 (median change: –47%). In Arm 2A, the TD to TI rate was 16% (6/38). BM fibrosis improvement by ≥1 grade was achieved in 31% (16/52) and 25% (9/36) pts in Arm 3 and 2, respectively. Further central review and exploratory analyses of BM pathology from a more mature data set, including clinical correlations, will be presented. The most common hematologic treatment-emergent adverse event (TEAE) of any grade was thrombocytopenia, reported in 52% (≥Grade 3: 12%) and 52% (≥Grade 3: 33%) pts in Arm 3 and 2, respectively. Anemia was reported in 42% (≥Grade 3: 35%) and 27% (≥Grade 3: 19%) pts in Arm 3 and 2, respectively. Low-grade gastrointestinal TEAEs and respiratory infections in Arm 3 and 2 were observed but rarely a reason for treatment discontinuation. Image: Summary/Conclusion: Based on these interim Phase 2 data, pelabresib in combination with RUX, in both RUX treatment-naïve and -experienced pts with MF, resulted in splenic and symptom responses and BM fibrosis improvement, and appeared to be well tolerated. Based on data from MANIFEST Arm 3, the randomized, double-blind, active-control Phase 3 MANIFEST-2 study was initiated to further evaluate the safety and efficacy of pelabresib in combination with ruxolitinib in JAKi treatment-naïve pts with MF (NCT04603495). S199: A NATIONAL RETROSPECTIVE COHORT STUDY OF MPN-SVT: RESULTS FROM THE UK MYELOPROLIFERATIVE NEOPLASMS ASSOCIATED SPLANCHNIC VEIN THROMBOSIS (MASCOT) REGISTRY R. Hargreaves1,*, M. Subhan2, S. Alimam1 2, S. Shapiro3 4, A. Robinson5, M. Carter5, A. Godfrey5, M. Ul-Haq6, M. Jain6, G. Greenfield7, A. McGregor8, H. K. Hussein9, D. Tripathi10, H. Network11, A. Doyle12, K. White12, N. Curto-Garcia12, D. Patch13, M. Sekhar1 2 1Haematology, Royal Free Hospital NHS Foundation Trust; 2Haematology, University College London Hospitals NHS Foundation Trust, London; 3Haematology, Oxford University Hospitals NHS Foundation Trust, NIHR Biomedical Research Centre; 4Radcliffe Department of Medicine, University of Oxford, Oxford; 5Haematology, Cambridge University Hospitals NHS Foundation Trust, Cambridge; 6Haematology, Leeds Teaching Hospitals NHS Trust, Leeds; 7Patrick G Johnston Centre for Cancer Research, Queen’s University Belfast, Belfast; 8Haematology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne; 9Haematology; 10Hepatology, University Hospitals Birmingham NHS Foundation Trust, Birmingham; 11Haematology Specialty Trainee Audit and Research, HaemSTAR Network, Nationwide; 12Haematology, Guy’s and St Thomas’ NHS Foundation Trust; 13Hepatology, Royal Free Hospital NHS Foundation Trust, London, United Kingdom Background: Patients with myeloproliferative neoplasms (MPN) are at increased risk of splanchnic vein thrombosis (SVT), defined as abdominal thrombosis in portal, splenic, mesenteric or hepatic veins. MPN-SVT carries a significant mortality and morbidity burden and poses clinical management challenges. Detailed studies of this patient population are lacking. Aims: To establish a national web-based clinical registry for MPN-SVT (MASCOT Registry) and use this to investigate demographics, clinical features, co-morbidities, outcome & UK treatment practices for MPN-SVT. Methods: The MASCOT Registry is a UK-wide retrospective cohort of patients with MPN-SVT from 9 large haematology/hepatology centres. Participating centres entered pseudo-anonymised data for patients with MPN-SVT. New and historical cases were added; demographic, radiologic, clinical and outcome data were collected. Results: 232 patients with MPN-SVT were registered on the online database between May 2019 and January 2022. 1 centre registered 92 cases (40%) and 2 centres each registered 39 cases (17%) with the remaining 62 cases (27%) from 6 centres. 57% of patients were female and 43% male and the age of SVT onset was 49 years or less in 70% of patients. Median follow up was 7.3 years (range 35 days-31.8 years). SVT was diagnosed in 2009 or later in 72%. MPN was diagnosed first in 30% of patients, SVT first in 42% of patients and simultaneous diagnosis in 28%. Figure 1 shows the subtypes of MPN associated with SVT. Multiple splanchnic vein thromboses were observed in 42% of patients. Of those with single vein thrombosis, the portal vein was affected in 26% and the hepatic vein in 23%. Initial anticoagulation treatment was in line with local policy. TIPSS (transjugular intrahepatic portosystemic shunt) was performed in 21% and thrombolysis in 7%. Warfarin only was the preferred long-term anticoagulant (53%). Cytoreduction at registration comprised 44% of patients on hydroxycarbamide, 18% on Pegylated interferon, 16% on ruxolitinib and 8% on no therapy. 37 patients required major abdominal surgery and 17 received liver transplants. 71 patients (33%) suffered from other non-SVT thromboses (34% pre-SVT, 9% simultaneously and 57% post-SVT), comprising 84 thrombotic events (60 venous, 24 arterial). Grade 3+ haemorrhage was observed in 60 patients (28%), of which the majority (48%) occurred within the first year of SVT diagnosis. 111 patients (51%) were re-imaged at least once within 12 months post-SVT, demonstrating clot extension in 12%, recanalization in 25% and stable appearances in 54%. Overall survival was 88% with 14 deaths, of which 6 were disease-related. Median age at death was 72 years and death occurred at a median of 12 years post SVT diagnosis. Image: Summary/Conclusion: This is one of the largest cohort studies of patients with MPN-SVT to date with over 70% diagnosed in the last 13 years. Similar to previous published studies, our evidence corroborates the female predominance and relatively young age at diagnosis. Our data highlight the significant morbidity and mortality burden incurred by patients with MPN-SVT, including a substantially lower rate of re-canalisation compared to a recent meta-analysis of all patients with SVT and high rates of thrombosis recurrence. We have highlighted variations in clinical practice including choice of anticoagulant, preferred cytoreductive agent and use of thrombolysis and TIPSS. Future studies to further understand this patient population should be focused on access to optimal care, the aetiology of thrombosis, optimum anticoagulation management and strategies to minimise bleeding. S200: IMPROVED OVERALL SURVIVAL WITH FIRST-LINE BRENTUXIMAB VEDOTIN PLUS CHEMOTHERAPY IN PATIENTS WITH STAGE III/IV CLASSICAL HODGKIN LYMPHOMA: 6-YEAR ANALYSIS OF ECHELON-1 M. Hutchings1,*, S. M. Ansell2, D. J. Straus3, J. M. Connors4, W. S. Kim5, A. Gallamini6, R. Ramchandren7, J. W. Friedberg8, R. Advani9, A. M. Evens10, P. Smolewski11, K. J. Savage4, N. L. Bartlett12, H.-S. Eom13, J. S. Abramson14, C. Dong15, F. Campana15, K. Fenton16, M. Puhlmann16, J. Radford17 1Department of Haematology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark; 2Division of Hematology, Mayo Clinic, Rochester, MN; 3Department of Medicine, Lymphoma Service, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 4BC Cancer Centre for Lymphoid Cancer and Department of Medical Oncology, Vancouver, Canada; 5Division of Hematology-Oncology, Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 6Research and Innovation, Antoine-Lacassagne Cancer Centre, Nice, France; 7The University of Tennessee Graduate School of Medicine, Knoxville, TN; 8James P. Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY; 9Department of Medicine, Division of Oncology, Stanford University, Stanford, CA; 10Division of Blood Disorders, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United States of America; 11Department of Experimental Hematology, Medical University of Lodz, Lodz, Poland; 12Washington University School of Medicine Siteman Cancer Center, St Louis, MO, United States of America; 13Center for Hematologic Malignancy, National Cancer Center, Goyang, South Korea; 14Massachusetts General Hospital, Boston, MA; 15Takeda Development Center Americas, Inc. (TDCA), Lexington MA; 16Seagen Inc., Bothell, WA, United States of America; 17The University of Manchester and the Christie NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom Background: In classical Hodgkin lymphoma (cHL), improved overall survival (OS) with first-line treatment using new combinations has seldom been observed compared with existing approaches. Five-year data from the randomized phase 3 ECHELON-1 study (NCT01712490) supported long-term progression-free survival (PFS) with brentuximab vedotin, doxorubicin, vinblastine, and dacarbazine (A+AVD) versus doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD), in patients (pts) with previously untreated stage III/IV cHL, regardless of interim positron emission tomography (PET) status. The long-term safety profile of A+AVD was manageable, and numerically fewer secondary malignancies were reported versus ABVD, as well as a greater number of pregnancies (Straus et al, Lancet Haematol 2021). Aims: To report the pre-specified OS analysis from ECHELON-1, as well as relevant long-term safety data, after approximately 6 years of follow-up (cut-off June 1, 2021). Methods: Pts were randomized 1:1 to receive up to 6 cycles of A+AVD (n=664) or ABVD (n=670) on day 1 and 15, every 28 days. PFS per investigator was reported for long-term follow-up. The key secondary end point was OS, which was an event-driven, pre-specified, alpha-controlled analysis in the intention-to-treat population, with a prespecified interim analysis after 103 deaths. Analysis of OS in prespecified subgroups was exploratory and was not adjusted for multiplicity. Informed consent was obtained for all pts. Results: In total, 39 OS events in the A+AVD arm and 64 in the ABVD arm had occurred at a median follow-up of 73 months, significantly favoring A+AVD (hazard ratio [HR] 0.590; 95% CI 0.396–0.879; p=0.009). There was a consistent OS benefit for A+AVD vs ABVD across prespecified subgroups, including: stage III (HR: 0.863; 95% CI 0.452–1.648) and stage IV disease (HR: 0.478; 95% CI 0.286–0.799) at diagnosis, pts who were PET-negative at cycle 2 (PET2)-negative (HR: 0.583; 95% CI 0.338–0.856) and PET2-positive (HR: 0.163; 95% CI 0.037–0.717), pts aged <60 years (HR: 0.509; 95% CI 0.291–0.890), pts aged ≥60 years (HR: 0.829; 95% CI 0.469–1.466), and across all geographies, including Europe (HR: 0.783; 95% CI 0.467–1.315) and North America (HR: 0.327; 95% CI 0.153–0.699). The 6-year PFS estimate was 82.3% (79.1–85.0) vs 74.5% (70.8–77.7) with A+AVD vs ABVD, respectively (HR: 0.678; 95% CI: 0.532–0.863). The long-term safety profile of A+AVD was comparable to that of ABVD. In both A+AVD and ABVD groups, treatment-emergent peripheral neuropathy continued to resolve or improve, with 86% (379/443) and 87% (249/286) of cases in the A+AVD and ABVD arms either completely resolving (72% vs 79%) or improving (14% vs 8%) by last follow-up. Overall, 23 secondary malignancies in the A+AVD arm and 32 in the ABVD arm were reported. Pregnancies and live births were reported by a greater number of female pts in the A+AVD group vs the ABVD group (49 vs 28 and 42 vs 19, respectively) and there were no stillbirths reported during the study. There were no new safety signals. Summary/Conclusion: There was a statistically significant 41% reduction in the risk of death with A+AVD vs ABVD, with a consistent OS benefit across prespecified subgroups. The long-term safety profile was manageable, consistent with prior reports. These data support A+AVD as a preferred option for previously untreated stage III and IV cHL. S201: CAMIDANLUMAB TESIRINE: UPDATED EFFICACY AND SAFETY IN AN OPEN-LABEL, MULTICENTER, PHASE 2 STUDY OF PATIENTS WITH RELAPSED OR REFRACTORY CLASSICAL HODGKIN LYMPHOMA (R/R CHL) C. Carlo-Stella1,*, S. Ansell2, P. L Zinzani3, J. Radford4, K. Maddocks5, A. Pinto6, G. P. Collins7, V. Bachanova8, N. Bartlett9, I. Bence-Bruckler10, M. Hamadani11, J. Kline12, J. Mayer13, K. J. Savage14, R. Advani15, P. Caimi16, R.-O. Casasnovas17, T. Feldman18, B. Hess19, M. Bastos-Oreiro20, S. Iyengar21, S. Eisen22, Y. Negievich22, L. Wang23, J. Wuerthner22, A. F. Herrera24 1Department of Oncology and Hematology, Humanitas Clinical and Research Center - IRCCS and Humanitas University, Rozzano, Milano, Italy; 2Division of Hematology, Mayo Clinic, Division of Hematology, Minnesota, United States of America; 3Hematology, Institute of Hematology Seràgnoli University of Bologna, Bologna, Italy; 4NIHR Clinical Research Facility, the Christie NHS Foundation Trust and University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom; 5Division of Hematology, Ohio State University Medical Center, Columbus, United States of America; 6Fondazione G. Pascale, IRCCS, Istituto Nazionale Tumori, Naples, Italy; 7Oxford Cancer and Haematology Centre, NIHR Oxford Biomedical Research Centre, Churchill Hospital, Oxford, United Kingdom; 8Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis; 9Division of Oncology, Washington University School of Medicine in St. Louis, St. Louis, United States of America; 10Ottawa-Hospital General Campus, The Ottawa Hospital-General Campus, Ottawa, Canada; 11Medical College of Wisconsin, BMT & Cellular Therapy Program, Department of Medicine, Milwaukee; 12Department of Medicine, The University of Chicago, Chicago, United States of America; 13Internal Medicine, University Hospital and Masaryk University, Brno, Czechia; 14Department of Medical Oncology, BC Cancer and University of British Columbia, Vancouver, Canada; 15Division of Oncology, Department of Medicine, Stanford University, Stanford; 16Cancer Center, Cleveland Clinic/Case Comprehensive Cancer Center, Cleveland, United States of America; 17Department of Hematology, University Hospital F. Mitterrand and Inserm UMR 1231, Dijon, France; 18John Theurer Cancer Center, Hackensack Meridian Health, Hackensack; 19Division of Hematology and Medical Oncology, Department of Medicine, Medical University of South Carolina, Charleston, United States of America; 20Haematology Department, Gregorio Marañón Health Research Institute, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 21Royal Marsden Hospital, London, United Kingdom; 22ADC Therapeutics SA, Epalinges, Switzerland; 23ADC Therapeutics America, Inc, Murray Hill; 24Department of Hematology & Hematology & Hematopoietic Cell Transplantation, City of Hope Comprehensive Cancer Center, Duarte, United States of America Background: Camidanlumab tesirine (Cami), an antibody-drug conjugate comprising a human IgG1 anti-CD25 monoclonal antibody conjugated to a pyrrolobenzodiazepine (PBD) dimer, displayed antitumor activity and manageable toxicity in a phase 1 trial in lymphoma, including R/R cHL (Hamadani et al. 2021). Aims: Present updated efficacy and safety data from a phase 2 study of Cami monotherapy in patients (pts) with R/R cHL (NCT04052997). Methods: Pts with R/R cHL and ≥3 prior systemic therapies, including brentuximab vedotin (BV) and anti-PD-1 (≥2 therapies if hematopoietic cell transplantation [HCT] ineligible), were enrolled. Interim data was reported (Herrera et al. 2020, Zinzani et al. 2021). The primary endpoint is overall response rate (ORR by 2014 Lugano criteria; central review). Secondary endpoints include duration of response (DOR), progression-free survival (PFS), and frequency and severity of adverse events (AEs). Pts received Cami 45 µg/kg (30-min infusion) on Day 1 of each 3-week cycle (2 cycles), then 30 µg/kg (subsequent cycles) for up to 1 year. Results: As of Nov 1, 2021, enrollment was complete (N=117). Median age (range) was 37 years (19-87), 62% of pts were male, and 95% had an ECOG score of 0-1. Pts had received a median (range) of 6 (3-19) prior therapies and received a median (range) of 5.0 (1-15) Cami cycles. Fourteen pts (12.0%) withdrew to undergo HCT (of which 12 [10.3%] received HCT and were censored). In the all-treated population (N=117), ORR was 70.1% (82/117; 95% CI: 60.9-78.2), and 33.3% (39/117) had complete response (CR) (Fig 1A). At median (range) follow-up of 10.7 (1.2-25.2+) months (mos), the median (95% CI) DOR was 13.7 mos (7.4-14.7) for all responders, 14.5 (7.4-not reached, NR) mos and 7.9 (3.8-NR) mos for pts with CR or PR, respectively (Fig 1B). Median (95% CI) PFS was 9.1 (5.1-15.0) mos. All-grade treatment-emergent AEs (TEAEs) in ≥25% of 117 pts were fatigue (45, 38.5%), maculopapular rash (MR, 38, 32.5%), pyrexia (35, 29.9%), nausea (32, 27.4%), and rash (31, 26.5%). Grade ≥3 TEAEs in ≥5% of pts were thrombocytopenia (11, 9.4%), anemia (10, 8.5%), hypophosphatemia (9, 7.7%), neutropenia (9, 7.7%), MR (8, 6.8%), and lymphopenia (6, 5.1%). TEAEs leading to Cami dose delay/reduction or discontinuation occurred in 66 (56.4%) and 32 (27.4%) pts, respectively. TEAEs considered PBD-associated were skin/nail reactions (any grade, 87, 74.4%; grade ≥3, 24, 20.5%), hepatobiliary test abnormalities (any grade, 34, 29.1%; grade ≥3, 8, 6.8%), and edema/effusion (any grade, 20, 17.1%; grade ≥3, 0). TEAEs considered immune-related (IR) occurred in 38 (32.5%) pts. Grade ≥3 IR AEs (TEAEs and non-TEAEs; 10, 8.5%) included diabetic ketoacidosis/type 1 diabetes (2, 1.7%); drug-induced liver injury (2, 1.7%); tubulointerstitial nephritis (2, 1.7%); and aplastic anemia, autoimmune hemolytic anemia or hepatitis, and lichenoid keratosis (1 each, 0.9%). Guillain–Barré syndrome (GBS)/polyradiculopathy was seen in 8 (6.8%) pts (Grade 2, n=2; grade 3, n=3; grade 4, n=3). In 4 pts, onset was after 2 Cami cycles (range: 2-7) and median (range) duration was 115 days (43-523). At data cutoff, 4 cases had recovered, and 4 had not recovered. Image: Summary/Conclusion: With median follow-up of 10.7 mos, Cami demonstrated an ORR of 70.1% (CR: 33.3%) in heavily pretreated R/R cHL after BV and PD-1 blockade failure, with an encouraging median DOR of 13.7 mos and median PFS of 9.1 mos. Safety is consistent with prior findings, including similar incidence rates of GBS/polyradiculopathy. S202: CHILDBEARING AMONG CLASSICAL HODGKIN LYMPHOMA SURVIVORS TREATED WITH BEACOPP AND ABVD IN SWEDEN, DENMARK, AND NORWAY J. P. Entrop1,*, C. E. Weibull1, K. E. Smedby1, L. H. Jakobsen2 3, A. K. Øvlisen2, D. Molin4, I. Glimelius4, A. Marklund5, K. B. Smeland6, T. C. El-Galaly2, S. Eloranta1 1Clinical Epidemiology Division, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden; 2Department of Hematology, Clinical Cancer Research Center, Aalborg University Hospital; 3Department of Mathematical Sciences, Aalborg University, Aalborg, Denmark; 4Department of Immunology, Genetics and Pathology, Unit of Experimental and Clinical Oncology, Uppsala University, Uppsala; 5Department of Reproductive Medicine, Division of Gynecology and Reproduction, Karolinska University Hospital, Stockholm, Sweden; 6Department of Oncology, Oslo University Hospital, Oslo, Norway Background: Recent studies have shown decreased childbearing rates in some classical Hodgkin lymphoma (cHL) survivors compared to the general population. This decrease is hypothesized to be caused by administered intensive chemotherapy. Understanding if currently used treatment protocols lead to reduced post-treatment childbirth potential is important as differential impact on childbearing may influence treatment decisions for younger patients. Firm conclusions from previous population-based studies have been hampered by low power for specific treatment contrasts. Aims: To increase understanding of differences in childbearing patterns among male and female HL survivors treated with ABVD and BEACOPP in a study combining population-based register data from Sweden, Denmark, and Norway. Methods: In this cohort study 2,937 individuals aged 18-40 years with a recorded diagnosis of cHL in the Swedish lymphoma register (SLR), the Danish lymphoma registry (LYFO), or the clinical database at Oslo University Hospital (OUH) between 1995 and 2019 were included. Information on first-line chemotherapy treatment was available in the registers. Treatment regimens were classified as ABVD, 6-8 BEACOPP, or other. The SLR, LYFO, and OUH databases were linked to national medical birth registers to obtain information on childbirths. Follow-up started 9 months after cHL diagnosis and was accrued until date of first childbirth, death, or administrative censoring (December 2017, 2018, or 2019 for Norway, Sweden, and Denmark, respectively) or after 10 years. Females were additionally censored at date of relapse or stem cell transplantation due to the limited accessibility of assisted reproductive techniques for female patients experiencing one of those events. Stratified Cox models allowing for effect modification between sex and treatment and adjusted for stage, age at diagnosis, year of diagnosis, parity, and performance status were used to estimate hazard ratios (HRs). Flexible parametric survival models were used to estimate marginal childbirth rates and standardized cumulative incidence of childbirth across treatment groups. A comprehensive study analysis plan been published and pre-registered on the Open Science Framework (https://osf.io/eumy5/). Results: In summary, 75% of HL patients were treated with ABVD and 10.8% with 6-8 BEACOPP. An additional 14.2% received other treatments or had missing treatment information and were excluded from further analyses. The rate of first childbirth per 1,000 person-years for males and females was 45.5 and 50.0 respectively in the ABVD group, and 23.8 and 43.5 in the 6-8 BEACOPP group. The adjusted HR of first childbirths for males and females, comparing patients treated with 6-8 BEACOPP to ABVD were 0.54 (95% CI 0.37-0.79) and 0.92 (95% CI 0.61-1.39) respectively. Plots of the cause-specific cumulative incidence functions (CIF) of childbirth show a constantly lower proportion of male patients with childbirths over time since HL diagnosis comparing patients treated with 6-8 BEACOPP and ABVD. However, this difference was not present among female patients. Adjusted marginal estimates of the cause-specific CIF of childbirth showed the same pattern. Image: Summary/Conclusion: BEACOPP treatment is associated with decreased childbearing rates in male but not female HL patients. Infertility counselling for this group is advisable. S203: ABSCOPAL EFFECT OF RADIOTHERAPY AND NIVOLUMAB IN RELAPSED OR REFRACTORY HODGKIN LYMPHOMA: PRE-PLANNED INTERIM ANALYSIS OF THE INTERNATIONAL GHSG PHASE II AERN TRIAL P. Bröckelmann1 2 3 4,*, I. Bühnen3, J. Zijlstra5, A. Fossa6, J. Meissner7, S. Mathas8, J. Rosenbrock9, C. Kobe10, M. Fuchs3, A. Plütschow3, S. Marnitz9, P. Borchmann11, A. Engert11, C. Baues9 1Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), University Hospital of Cologne; 2Max Planck Institute for the Biology of Ageing; 3German Hodgkin Study Group (GHSG), University of Cologne; 4Mildred Scheel School of Oncology Aachen Bonn Cologne Düsseldorf (MSSO ABCD), Cologne, Germany; 5Amsterdam UMC, Department of Hematology, Cancer Center Amsterdam, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 6Department of Oncology, Oslo University Hospital, Oslo, Norway; 7Faculty of Medicine and University Hospital Heidelberg, Department V of Internal Medicine, University of Heidelberg, Heidelberg; 8Department of Hematology, Oncology and Tumor Immunology, Charité – Universitätsmedizin Berlin, and Experimental and Clinical Research Center (ECRC), Berlin; 9University Hospital Cologne, Department of Radiation Oncology and Cyberknife Center; 10Department of Nuclear Medicine; 11Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), University of Cologne, Faculty of Medicine, Cologne, Germany Background: Failure of anti-PD1 checkpoint blockade in relapsed or refractory Hodgkin lymphoma (r/r HL) constitutes an unmet medical need. It has been postulated that anti-PD1 and localized radiotherapy (RT) may work synergistically to induce a systemic anti-lymphoma effect resulting in distant and systemic (“abscopal”) response outside the RT field. Aims: We aimed to formally investigate safety and efficacy of nivolumab and RT in r/r HL failing anti-PD1 treatment in a proof-of-concept study. Methods: AERN is an ongoing international investigator-sponsored two stage phase II trial enrolling adult patients with histologically confirmed r/r HL (NCT03480334). All patients must have failed anti-PD1 treatment by either showing progressive disease (PD) or stable disease (SD) >6 months as best response at the time of enrollment. Presence of two distinct FDG-avid lesions with one eligible for 20Gy localized RT and the other >5cm apart and outside the prescribed 95%isodose for measurement of an abscopal response must be confirmed by the GHSG reference panel. After enrollment patients receive 240mg nivolumab 2-weekly and 20Gy RT in 2Gy fractions is started on day 6 after the first infusion on trial. Treatment is continued until PD or unacceptable toxicity or for a maximum of 1.5 years. The primary endpoint is abscopal response rate (ARR) at first interim restaging after six infusions, secondary endpoints include objective response rate (ORR), progression-free survival (PFS), overall survival and safety. Target enrollment are nine and 20 patients in a Simon’s two-stage desig. A pre-planned interim analysis was conducted after completion of stage I and is reported herein. Results: Of 11 patients enrolled into stage I of the trial, two terminated trial participation early (screening failure, withdrawal of informed consent) and nine patients at a median age of 34 (range 25-82) years were evaluable for stage I. All patients presented with stage III/IV and a median of 4 (3-15) prior lines of treatment. While all patients had received prior anti-PD1 antibodies (100%), the majority also received brentuximab vedotin (89%) and high-dose chemotherapy plus autologous stem-cell transplantation (ASCT, 56%). Trial treatment was well tolerated. The most frequently reported °1/2 toxicities were those affecting the skin (55%), one case of °3 gastrointestinal and no °4/5 toxicities. Patients received a median of 19 (7-36) nivolumab infusions with a median treatment duration of 9 (3-18) months. At first interim restaging, five patients had an abscopal response (ARR 56%, 95%CI 16.9-74.9%) by central response assessment and 56% achieved a PR (ORR 56%). Most patients experienced a reduction in disease burden at first interim restaging with a mean decrease of SPD -36.3% (0 to -81.2%). Four out of five patients discontinuing treatment experienced PD at the last restaging. With a median observation time of 15.5 months, the 1-year PFS was 42.3% and all patients were alive at data cut off. Summary/Conclusion: Treatment with nivolumab and 20Gy RT at a single lesion was well tolerated and led to an abscopal response in the majority of patients (56%) previously failing anti-PD1 treatment for r/r HL. An ORR of 56% and 1-year PFS of 42.3% further highlight the potential of this innovative treatment strategy. After meeting the primary efficacy endpoint in this pre-planned interim analysis, AERN trial enrollment is currently continuing internationally. S204: PEMBROLIZUMAB IN CHILDREN AND YOUNG ADULTS WITH NEWLY DIAGNOSED CLASSICAL HODGKIN LYMPHOMA WITH SLOW EARLY RESPONSE TO FRONTLINE CHEMOTHERAPY: THE PHASE 2, OPEN-LABEL, KEYNOTE-667 STUDY L. Vinti1,*, S. Daw2, C. Sabado Alvarez3, F. Fagioli4, A. Beishuizen5, G. Michel6, M. L. Moleti7, M. Cepelova8, A. Thorwarth9, C. Rigaud10, D. Plaza Lopez de Sabando11, J. Landman Parker12, Y. Zhu13, P. Pillai13, A. Nahar13, C. Mauz-Koerholz14 15 1IRCCS Ospedale Pediatrico Bambino, Gesu, Italy; 2University College London Hospitals NHS Foundation Trust, London, United Kingdom; 3Hospital Universitari Vall d Hebron, Barcelona, Spain; 4Ospedale Infantile Regina Margherita, Turin, Italy; 5Princess Máxima Centrum, Utrecht, Netherlands; 6CHU de Marseille Hopital de la Timone Enfants, Marseille, France; 7Universita degli Studi di Roma La Sapienza, Rome, Italy; 8Fakultni nemocnice v Motole, Prague, Czechia; 9Charite-Universitaetsmedizin Berlin Campus Virchow-Klinikum, Berlin, Germany; 10Gustave Roussy Cancer Campus, Villejuif, France; 11Hospital Universitario La Paz, Madrid, Spain; 12Hopital d’Enfants Armand Trousseau, Paris, France; 13Merck Sharp & Dohme, Corp., a subsidiary of Merck & Co., Inc., Kenilworth, United States of America; 14Justus-Liebig University of Giessen, Giessen; 15Medical Faculty, Martin-Luther-University of Halle-Wittenberg, Halle, Germany Background: Patients with classical Hodgkin lymphoma (cHL) with slow early response (SER) to initial chemotherapy (chemo) are at higher risk of relapse, and the burden of late organ toxicities may be higher after dose intensification and radiotherapy (RT). The phase 2, open-label KEYNOTE-667 (NCT03407144) study is evaluating the efficacy and safety of pembrolizumab (pembro) plus chemo in patients with cHL and SER to frontline chemo. Aims: To present results of an interim analysis of efficacy and safety in patients with high risk cHL (Group 2) who were SER. Methods: In Group 2, eligible patients aged 3-17 (children) or 18-25 years (young adults) with newly-diagnosed stage IIEB, IIIEA, IIIEB, IIIB, IVA, or IVB cHL were enrolled to receive induction with vincristine, etoposide, prednisone/prednisolone, doxorubicin (OEPA) for 2 cycles. Response assessment after induction (early) and consolidation (late) therapy was done by PET/MRI/CT. After induction treatment, patients with rapid early response (RER) received non-study therapy and patients with SER received consolidation with 4 cycles cyclophosphamide, vincristine, prednisone/prednisolone, dacarbazine (COPDAC-28) plus pembro 2 mg/kg up to 200 mg IV Q3W (3-17 years of age) or 200 mg IV Q3W (18-25 years of age). After consolidation therapy, patients with PET-positivity (Deauville score 4-5) received modified involved-site RT to late PET-positive residua; RT was omitted in patients with PET-negativity. All patients with SER received maintenance pembro Q3W, for a total of 17 doses. Safety analyses included all patients with SER treated with pembro, while efficacy analyses was based on all patients who had completed late response assessment. All patients provided informed consent. The primary endpoint was ORR by BICR per Cheson 2007 IWG criteria in patients with SER. Secondary endpoints included rate of PET-negativity after consolidation, details of RT, and safety. Data cut-off was Nov 22, 2021. Results: At data cut-off, median (range) follow-up was 9.6 mo (2.5-21.2). A total of 30 patients with high-risk cHL with SER were included. Median (range) age was 15y (6-19), 13 (43%) had bulky disease, 19 (63%) had Ann Arbor stage IV disease. A total of 6 (20%) patients had completed, and 24 (80%) were ongoing on treatment, with median time on treatment of 3.3 mo (range, 0-11.8). Of 30 patients, 25 (83%) had a late response assessment, of which 17 (68%) were PET-negative by BICR; 18 (72%) PET-negative by investigator. All cause AEs occurred in 23 (77%) patients, with 14 (47%) having a treatment-related AE. Grade ≥3 AEs occurred in 6 (20%) patients, with 3 (10%) having an SAE. Grade ≥3 treatment-related AEs occurred in 2 (7%) patients. One (3%) pt had a grade 2 immune-mediated AE of hypothyroidism. Summary/Conclusion: In pediatric patients with high-risk cHL and SER to standard OEPA induction, pembro in combination with COPDAC 28 consolidation therapy was well tolerated and resulted in 68% of patients having a PET-negative response at end of chemo, and being spared RT. This early data suggests that addition of pembro potentially may have the ability to augment responses in this high-risk subgroup of patients. S205: ZANUBRUTINIB + OBINUTUZUMAB (ZO) VS OBINUTUZUMAB (O) MONOTHERAPY IN PATIENTS (PTS) WITH RELAPSED OR REFRACTORY (R/R) FOLLICULAR LYMPHOMA (FL): PRIMARY ANALYSIS OF THE PHASE 2 RANDOMIZED ROSEWOOD TRIAL P. L. Zinzani1,*, J. Mayer2, R. Auer3, F. Bijou4, A. C. de Oliveira5, C. R. Flowers6, M. Merli7, K. Bouabdallah8, P. S. Ganly9, Y. Song10, H. Zhang11, R. Johnson12, A. M García-Sancho13, M. Provencio14, M. Trněný15, S. Yuen16, H. Tilly17, E. Kingsley18, G. Tuyman19, S. E. Assouline20, E. Ivanova21, P. Kim22, J. Huang22, R. Delarue21, J. Trotman23 24 1Institute of Hematology “Seràgnoli”, University of Bologna, Bologna, Italy; 2Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital, Brno, Czechia; 3St. Bartholomew’s Hospital, Barts Health NHS Trust, London, United Kingdom; 4Institut Bergonié, Bordeaux, France; 5Institut Catala d’Oncologia (ICO) Hospital Duran I Reynals, Hospital, Barcelona, Spain; 6Department of Lymphoma/Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America; 7Hematology, University Hospital “Ospedale di Circolo e Fondazione Macchi” - ASST Sette Laghi, University of Insubria, Varese, Italy; 8Hôpital Haut-Lévêque, CHU Bordeaux, Pessac, France; 9Department of Haematology, Christchurch Hospital, Christchurch, New Zealand; 10Beijing Cancer Hospital, Beijing; 11Tianjin Medical University Cancer Institute & Hospital, Tianjin, China; 12St. James’s University Hospital Trust, Leeds, United Kingdom; 13Hospital Universitario de Salamanca, Salamanca; 14Hospital Universitario Puerta de Hierro — Majadahonda, Madrid, Spain; 15Vseobecna fakultní nemocnice v Praze, Prague, Czechia; 16Calvary Mater Newcastle, Waratah, New South Wales, Australia; 17Centre Henri-Becquerel, Rouen, France; 18Comprehensive Cancer Centers of Nevada, Las Vegas, NV, United States of America; 19Department of Chemotherapy of Hemoblastosis, Blokhin Russian Cancer Research Center, Moscow, Russia; 20Jewish General Hospital, Montreal, Canada; 21BeiGene Switzerland GmbH, Basel, Switzerland; 22BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA, United States of America; 23Concord Repatriation General Hospital; 24Department of Haemotology, University of Sydney, Concord, New South Wales, Australia Background: FL is the most common type of indolent non-Hodgkin lymphoma. Approved treatment options are limited for pts with R/R FL. In a phase 1b trial (Blood Adv. 2020;4(19):4802-4811), ZO was found to be tolerable and associated with early signal of efficacy. Aims: To present a primary analysis of ROSEWOOD (BGB-3111-212; NCT03332017), a phase 2, randomized study designed to assess efficacy and safety of ZO vs O in pts with R/R FL. Methods: Pts with R/R FL who received ≥2 lines of therapy, including an anti-CD20 antibody and an alkylating agent, were randomized 2:1 to receive either ZO or O. O was given in both arms on Days 1, 8, and 15 of Cycle 1, Day 1 of Cycles 2-6, and then every 8 weeks up to 20 doses maximum. Z (160 mg twice daily) was given until progressive disease (PD) or unacceptable toxicity; pts with confirmed PD or no response within 12 months in the O arm were allowed to crossover to ZO. Primary endpoint was overall response rate (ORR) by independent central review. Secondary endpoints included complete response rate (CRR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Exploratory endpoint included ORR by investigator after crossover. Primary analysis cutoff was October 8, 2021. All pts gave informed consent. Results: A total of 217 pts were randomized to ZO (n=145) or O (n=72). Median study follow-up was 12.5 mo; median age was 64 yrs. Incidence of high FL International Prognostic Index score was 53% (ZO) and 51% (O). Pts received a median of 3 prior lines of therapy, with 28% (ZO) and 25% (O) of pts receiving >3 lines. Proportion of pts refractory to rituximab, refractory to the most recent line of therapy, or with PD within 24 mo of initiation of first-line therapy was 54%, 32%, and 35% with ZO and 50%, 40%, and 42% with O, respectively. The study met its primary endpoint: ORR was 68.3% with ZO vs 45.8% with O (p=0.0017). CRR was 37.2% (ZO) vs 19.4% (O); 18-mo DOR rate was 70.9% (ZO) vs 54.6% (O); and median PFS was 27.4 mo (ZO) vs 11.2 mo (O; hazard ratio [HR], 0.51 [95% CI, 0.32-0.81], p=0.0040). Median time to new anti-lymphoma therapy or crossover was not evaluable (ZO; NE) vs 12.1 mo (O; HR, 0.37 [95% CI, 0.23-0.60], p<0.0001). ORR for 29 pts who crossed over to ZO was 24.1%. Median OS was NE; 18-mo OS probability was 85.4% (ZO) vs 72.6% (O). Most common any-grade adverse events (AEs) with incidence >10% in the ZO arm were thrombocytopenia (34.3%), neutropenia (27.3%), diarrhea (16.1%), fatigue (14.0%), constipation (13.3%), cough (11.9%), pyrexia (11.2%), and dyspnea (10.5%). Grade ≥3 AEs with incidence >5% with ZO were neutropenia (22.4%) and thrombocytopenia (14.0%); incidence of atrial fibrillation was 0.7% and major hemorrhage was 1.4%. Incidence of treatment-emergent AEs leading to death was 5.6% (ZO) and 9.9% (O). Summary/Conclusion: ZO demonstrated superior efficacy to O in treatment of pts with R/R FL. ZO had a favorable benefit-risk profile and represents a potential combination therapy for pts with R/R FL. S206: OBINUTUZUMAB PLUS CHEMOTHERAPY DEMONSTRATES LONG-TERM BENEFIT OVER RITUXIMAB PLUS CHEMOTHERAPY IN PATIENTS WITH PREVIOUSLY UNTREATED FOLLICULAR LYMPHOMA: FINAL ANALYSIS OF THE GALLIUM STUDY W. Townsend1,*, W. Hiddemann2, C. Buske3, G. Cartron4, D. Cunningham5, M. J. Dyer6, J. Gribben7, E. Phillips8, M. Dreyling2, J. F. Seymour9, A. Grigg10, T.-Y. Lin11, X.-N. Hong12, D. Kingbiel13, T. G. Nielsen13, A. Knapp13, M. Herold14, R. Marcus15 1Cancer Research UK and UCL Cancer Trials Centre, University College Hospitals London, London, United Kingdom; 2Ludwig-Maximilians-University Hospital Munich, Munich; 3Universtitätsklinikum Ulm, Ulm, Germany; 4CHU Montpellier, Montpellier, France; 5Royal Marsden Hospital, Sutton; 6Ernest and Helen Scott Haematological Research Institute, University of Leicester, Leicester; 7Barts Cancer Institute, Queen Mary University of London, London; 8University of Manchester, The Christie Hospital and National Institutes of Health Research Manchester Biomedical Research Centre, Manchester, United Kingdom; 9Peter MacCallum Cancer Centre and the Royal Melbourne Hospital, Melbourne; 10Austin Hospital, Austin, Australia; 11Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, and Collaborative Innovation Center for Cancer Medicine, Guangzhou; 12Fudan University Shanghai Cancer Center, Shanghai, China; 13F. Hoffmann-La Roche Ltd, Basel, Switzerland; 14HELIOS-Klinikum Erfurt, Erfurt, Germany; 15Kings College Hospital, London, United Kingdom Background: Immunochemotherapy with rituximab plus chemotherapy (R-chemo) has significantly improved outcomes for patients (pts) with previously untreated follicular lymphoma (FL). However, most pts still experience disease relapse, progression, or death. Obinutuzumab (G) is a type II anti-CD20 monoclonal antibody with enhanced direct cell killing, antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis versus R. The safety and efficacy of G-chemo versus R-chemo was assessed in pts with previously untreated, advanced stage FL in the randomized, Phase III GALLIUM (NCT01332968) study. In the primary analysis, G-chemo demonstrated a significant improvement in progression-free survival (PFS) versus R-chemo, with a manageable safety profile (Marcus, et al. 2017); this efficacy benefit was maintained after 5 years of observation (Townsend, et al. 2020). Aims: To report the final analysis of the GALLIUM study after a median observation time of 8 years. Methods: Pts aged ≥18 years with previously untreated histologic Grade 1–3a FL requiring treatment were enrolled. Pts were randomized 1:1 to receive G 1000mg intravenously (IV; Days [D]1, 8 and 15 of Cycle 1 and D1 of subsequent cycles) or R 375mg/m2 IV (D1 of each cycle) plus chemo for 6 or 8 cycles depending on the chemo backbone selected at each institution (cyclophosphamide, doxorubicin, vincristine, and prednisolone [CHOP]; cyclophosphamide, vincristine, and prednisolone [CVP]; or bendamustine). Pts attaining a complete or partial response received maintenance with the same antibody every 2 months for 2 years or until disease progression (PD). The primary endpoint was investigator-assessed PFS; secondary endpoints included time-to-next anti-lymphoma treatment (TTNLT), overall survival (OS) and incidence of adverse events (AEs). All pts provided written informed consent. Results: 1202 pts with FL were enrolled (G-chemo, n=601; R-chemo, n=601). As of July 30, 2021, median observation time was 8 years. Seven-year PFS was improved with G-chemo (63.4%) versus R-chemo (55.7%; hazard ratio [HR], 0.77; 95% confidence interval [CI]: 0.64–0.93; p=0.006; Figure). TTNLT was also improved with G-chemo versus R-chemo (HR, 0.71; 95% CI: 0.58–0.87; p=0.001); the proportion of pts who had not started their next treatment at 7 years was 74.1% and 65.4%, respectively. Disease transformation was observed in 4.2% of pts with G-chemo and 5.0% of pts with R-chemo. Seven-year OS was similar in both arms, 88.5% with G-chemo versus 87.2% with R-chemo (HR, 0.86; 95% CI: 0.63–1.18; p=0.36). Seventy-five pts in the G-chemo arm and 86 pts in the R-chemo arm had died, most commonly due to PD (4.2% and 6.0%, respectively). The incidence of serious AEs (SAEs) was 48.9% with G-chemo (28.2% and 24.4% during induction and maintenance, respectively) and 43.4% with R-chemo (24.6% and 21.7%, respectively). Rates of fatal AEs were similar with G-chemo (4.4%) and R-chemo (4.5%); pneumonia was the most common fatal AE (0.8% [n=5] and 0.2% [n=1], respectively) and SAE (5.9% [n=35] and 6.2% [n=37], respectively). Second malignancies occurred in 13.1% of pts in the G-chemo arm and 9.9% of pts in the R-chemo arm. After adjusting for observation time, rates were similar between arms. These safety findings are consistent with previous analyses. Image: Summary/Conclusion: After a median observation time of 8 years, a meaningful improvement in PFS was maintained with G-chemo versus R-chemo in pts with previously untreated FL, confirming the role of G-chemo as a standard of care for the first-line treatment of pts with FL. S207: EFFICACY AND SAFETY OF A THIRD GENERATION CD20 CART (MB-106) FOR TREATMENT OF RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA (FL) M. Shadman1,*, C. Yeung1, M. Redman2, S. Y. Lee2, D. H. Lee2, S. Ra2, D. Qian2, C. Ujjani1, B. Dezube3, C. Poh1, E. H. Warren1, A. Chapuis1, D. Green1, A. Cowan1, R. Cassaday1, H.-P. Kiem1, V. Chow1, J. Gauthier1, C. Turtle1, R. Lynch1, S. Smith1, A. Gopal1, D. Maloney1, B. Till1 1Fred Hutchinson Cancer Research Center / University of Washington; 2Fred Hutch, Seattle; 3Mustang Bio, Worcester, United States of America Background: Follicular lymphoma (FL) is incurable and novel treatments with high efficacy and low toxicity are needed. Chimeric antigen receptor T-cells (CAR-T) targeting CD19 are effective, and axicabtagene ciloleucel is currently approved by FDA for treatment of patients (pts) with relapsed FL, but toxicities like cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) may limit its use. MB-106 is a fully human 3rd-generation CD20-targeted CAR-T product with both 4-1BB and CD28 costimulatory domains. Aims: We present the results of the FL cohort from our ongoing phase I/II clinical trial investigating MB-106 for B-cell lymphoma/CLL. Methods: Pts with R/R B-cell malignancies including FL were eligible after confirmation of CD20 expression. Prior treatment with CD19 CAR-T is permitted. Lymphodepletion (LD) consists of cyclophosphamide (Cy) ± fludarabine (Flu). CAR-T cells are administered at one of 4 dose levels (DL): DL1: 3.3x105, DL2: 1x106, DL3: 3.3x106, DL4: 1x107 CAR T cells/kg. A continual reassessment method dose escalation design was used to find the maximally tolerated dose. CAR-T was infused in the outpatient setting except for the first pt of each dose cohort (overnight observation). Initial treatment response is assessed on day 28 and best response is reported here. CRS and ICANS are graded per ASTCT. Results: Between Dec 2019 and Dec 2021, 16 pts with FL were treated and had day 28 assessment. Median age was 61.5 years (range: 45 – 81). High-risk features included pts with progression of disease within 24 months of first-line chemotherapy (POD24) (n=11; 69%), history of histologic transformation (n=3; 19%), prior treatment with a CD19 targeted CAR-T (n=1; 6%). 6 pts (37.5%) had a PI3 kinase inhibitor. Median time between leukapheresis and LD was 15 days (range: 9-21) and 2 pts received bridging therapy with lenalidomide (1) and high-dose corticosteroids (1). All DLs were reached (DL0=1, DL1=1, DL2=4, DL3=7, DL4=2), with no dose-limiting toxicities. All CRS events were grade 1 (n=4; 25%) or grade 2 (n=1; 6%), with no grade ≥ 3 CRS events. There was no occurrence of ICANS of any grade. No pts had tumor lysis syndrome or Gr 3-4 infections. In the first 28 days, thrombocytopenia (Gr 3-4: 12.5%) and neutropenia (Gr 3-4: 94%) were common but there were no bleeding complications, and the rate of febrile neutropenia was 25%. Overall response (ORR) rate was 94% (15/16) and a complete response (CR) rate was 75% (12/16). Pts who received DL3 or DL4, had an ORR 100% and CR rate of 89%. The single patient who had previously been treated with a CD19 targeted CAR-T achieved a CR. With median follow-up of 10 months, one patient died, from complications of myelodysplastic syndrome (MDS) while still in remission; the MDS was attributed to the prior treatments (7 lines of chemo-based therapy and radioimmunotherapy). Most of the remissions are ongoing and 4 pts have relapsed during the follow-up median 7 months after treatment (range 3-10.5). CAR T cells were detectable by flow cytometry at last available timepoint (up to 2 years) for 15 of 16 patients. One patient had undetectable CAR T cells by day 201. Summary/Conclusion: Treatment with MB-106, a third generation CD20 targeting CAR-T, resulted in high ORR and CR rates and CAR-T persistence in FL pts and was associated with favorable safety profile with no occurrence of Gr 3 or Gr 4 CRS and no ICANS event of any grade. A multicenter trial for treatment of B-cell malignancies including FL will start enrollment in 2022. S208: EFFICACY AND SAFETY OF ZANDELISIB ADMINISTERED BY INTERMITTENT DOSING (ID) IN PATIENTS WITH RELAPSED OR REFRACTORY (R/R) FOLLICULAR LYMPHOMA: PRIMARY ANALYSIS OF THE GLOBAL PHASE 2 STUDY TIDAL A. Zelenetz1,*, W. Jurczak2, V. Ribrag3, K. Linton4, G. Collins5, J. Lopéz-Jiménez6, N. Reddy7, A. Mengarelli8, T. Phillips9, G. Musuraca10, O. Sheehy11, J. Li12, W. Xu12, M. Azoulay13, R. Ghalie12, P. L. Zinzani14 1Memorial Sloan Kettering Cancer Institute, New York City, United States of America; 2Maria Sklodowska Curie National Research Institute of Oncology, Krakow, Poland; 3Institut Gustave Roussy, Villejuif, France; 4Manchester Cancer Research Centre, Manchester; 5GenesisCare, Oxford, United Kingdom; 6Hospital Universitario Ramon y Cajal, Madrid, Spain; 7Vanderbilt University, Nashville, United States of America; 8Regina Elena National Cancer Institute, Roma, Italy; 9University of Michigan Health System, Ann Arbor, United States of America; 10Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori I.R.S.T., Meldola, Italy; 11Belfast Health and Social Care Trust, Belfast, United Kingdom; 12MEI Pharma, Inc., San Diego; 13Kyowa Kirin Co., Princeton, United States of America; 14University of Bologna, Bologna, Italy Background: Zandelisib, a PI3Kδ inhibitor with high target-binding affinity, is administered by intermittent dosing (ID) on days 1 to 7 of 28-day cycles to potentially enable regulatory T-cell repopulation and lower the risk of immune-related adverse events (irAEs) associated with continuous PI3Kδ inhibition. In a phase 1b study in 37 patients (pts) with R/R FL, zandelisib administered daily for two 28-day cycles for tumor debulking then on ID achieved an overall response rate (ORR) of 87% (78% as single agent and 95% with rituximab), a median duration of response (DOR) not reached at median follow-up of 16.9 months, and a low rate (<10%) of grade 3 irAEs (Pagel et al. ICML 2021; #113). Aims: We conducted the TIDAL study (NCT03768505) to further evaluate in a global trial the efficacy and safety of zandelisib in larger group of pts with R/R FL and marginal zone lymphoma (MZL). The MZL cohort is still enrolling and not reported here. Methods: Eligible pts ≥18 years with FL Grade I-IIIA, ECOG performance status 0-1, progressive disease after ≥2 prior therapies including an anti-CD20 antibody and chemotherapy, and no prior PI3Kδ inhibitor, provided consent and then received zandelisib 60 mg daily for two 28-day cycles then on ID. Another study arm evaluating zandelisib 60 mg daily continuously was closed to enrollment early and is not reported here. The planned sample size in FL was 120 pts on ID, with the primary efficacy population (PEP) pre-defined as the first 91 pts treated and the safety population consisting of all FL pts treated. The primary efficacy endpoint was ORR as assessed by independent review using the Lugano criteria and analyzed 6 months after completing enrollment in the PEP. Results: 121 FL pts were enrolled. In the PEP (N=91 pts), the median number of prior therapies was 3 (range 2-8), 21 pts (23%) have received prior stem cell transplant, 42 pts (46%) were refractory to last therapy, 31 pts (34%) had tumors ≥5 cm, and 51 pts (56%) were POD24. The ORR was 70.3% (N=64) (95% CI 59.8-79.5%), with 32 pts (35.2%) achieving a complete response (CR). Responses occurred early, with 85.5% (N=56) achieved in the first 2 cycles of therapy and 75% of CRs (N=24) achieved the first 4 cycles. The data are still immature to estimate accurately the DOR. With a median follow-up of 9.4 months (range 0.8-24) in the safety population of 121 pts, 12 pts (9.9%) discontinued therapy due to any drug-related AE. Grade 3 AEs of special interest (AESI) were diarrhea in 6 pts (5%), colitis in 2 (1.7%), cutaneous rash in 4 (3.3%), stomatitis in 3 (2.5%), and 1 (0.8%) each for AST and ALT elevation, and non-infectious pneumonitis. Grade 3 AESIs primarily (15 of 18, 83%) occurred in cycles 1-3, during daily dosing, with only 3 cases reported on ID in Cycles ≥4. Summary/Conclusion: Zandelisib on ID achieved high ORR (70.3%) and CR rate (35.2%) in heavily pretreated R/R FL pts, and was associated with a low rate (<10%) of grade 3 AESI and discontinuations due to AEs, results comparable to the Phase 1b study. Longer follow-up is needed to estimate median DOR. This profile supports evaluation of zandelisib as a single agent and in combination regimens in various B-cell malignancies, both in R/R disease and in earlier lines of therapy. Zandelisib plus rituximab vs chemoimmunotherapy is being evaluated in the phase 3 study COASTAL in R/R FL and MZL (NCT04745832). S209: PRIMARY RESULTS FROM THE PHASE 3 SHINE STUDY OF IBRUTINIB IN COMBINATION WITH BENDAMUSTINE-RITUXIMAB (BR) AND R MAINTENANCE AS A FIRST-LINE TREATMENT FOR OLDER PATIENTS WITH MANTLE-CELL LYMPHOMA M. L. Wang1,*, W. Jurczak2, M. Jerkeman3, J. Trotman4, P. L. Zinani5, D. Belada6, C. Boccomini7, I. W. Flinn8, P. Giri9, A. Goy10, P. A. Hamlin11, O. Hermine12, J.-Á. Hernández-Rivas13, X. Hong14, S. J. Kim15, D. Lewis16, Y. Mishima17, M. Özcan18, G. F. Perini19, C. Pocock20, Y. Song21, S. E. Spurgeon22, J. M. Storring23, J. Walewski24, J. Zhu21, R. Qin25, T. Henninger25, S. Deshpande25, A. Howes25, S. Le Gouill26, M. Dreyling27 1The University of Texas MD Anderson Cancer Center, Houston, United States of America; 2Maria Sklodowska-Curie National Research Institute of Oncology, Karkow, Poland; 3Skane University Hospital and Lund University, Lund, Sweden; 4Concord Repatriation General Hospital, University of Sydney, Sydney, Australia; 5IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale Università di Bologna, Bologna, Italy; 64th Department of Internal Medicine - Haematology, Charles University, Hospital and Faculty of Medicine, Hradec Králové, Czechia; 7SC Ematologia, AOU Città della Salute e della Scienza di Torino - Presidio Molinette, Torino, Italy; 8Sarah Cannon Research Institute and Tennessee Oncology, Nashville, United States of America; 9Royal Adelaide Hospital, Adelaide, Australia; 10John Theurer Cancer Center, Hackensack; 11Memorial Sloan Kettering Cancer Center, New York, United States of America; 12Department of Hematology, Hôpital Necker, Assistance Publique - Hôpitaux de Paris and Institut Imagine, Université de Paris, INSERM UMR1183, Paris, France; 13Department of Hematology, Hospital Universitario Infanta Leonor, Universidad Complutense, Madrid, Spain; 14Fudan University Shanghai Cancer Center, Shanghai, China; 15Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 16University Hospitals Plymouth NHS Trust, Plymouth, United Kingdom; 17Department of Hematology Oncology, Cancer Institute Hospital of Japanese Foundation for Cancer Research, Toyo, Japan; 18Ankara University School of Medicine, Ankara, Turkey; 19Hospital Israelita Albert Einstein, Sao Paulo, Brazil; 20Kent and Canterbury Hospital, Canterbury, United Kingdom; 21Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Lymphoma, Peking University Cancer Hospital & Institute (Beijing Cancer Hospital), Beijing, China; 22Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, United States of America; 23The Research Institute of the McGill University Health Centre, McGill University, Montreal, Canada; 24Maria Sklodowska-Curie National Research Institute of Oncology, Warszawa, Poland; 25Janssen Research & Development, Raritan, United States of America; 26Institut Curie comprehensive cancer center, Paris, France; Hospitalier Universitaire de Nantes, Centre de Recherche en Cancérologie et Immunologie Nantes Angers, INSERM, Université de Nantes, Nantes, France; 27Department of Medicine III, Klinikum der Universität München, LMU, Munich, Germany Background: Elderly patients (pts) with mantle-cell lymphoma (MCL) are unsuitable for intensive chemotherapy or transplantation due to excessive toxicities. Single-agent ibrutinib (Ibr), a first-in-class, oral Bruton’s tyrosine kinase inhibitor (BTKi), has transformed the care of pts with relapsed or refractory MCL with durable activity. Aims: We conducted a phase III trial (SHINE; NCT01776840) to evaluate combining Ibr with a standard chemoimmunotherapy (BR) and R maintenance in older pts with untreated MCL. Methods: Pts aged ≥ 65 years, enrolled between May 2013 and November 2014 from 183 sites across all geographical regions, were stratified by simplified MIPI score (low vs intermediate vs high risk) and were randomized 1:1 to Ibr (560 mg orally daily) or placebo (Pbo), plus 6 cycles of B (90 mg/m2) and R (375 mg/m2). Pts who achieved an objective response received R maintenance, administered every 8 weeks for up to 12 additional doses in both arms. Ibr and Pbo were administered until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS) assessed by the investigators. Results: A total of 523 pts were randomized to Ibr + BR (n = 261) or Pbo + BR (n = 262). Median age was 71 years (range, 65–87), 65.6% of pts had low/intermediate simplified MIPI, and 8.6% had blastoid/pleiomorphic histology. At the primary analysis, median follow up was 84.7 months. The primary endpoint was met as PFS was significantly improved in the Ibr arm vs the Pbo arm (hazard ratio, 0.75; one-sided P = 0.011) (Figure). Median PFS was 80.6 months with Ibr in combination with BR and R maintenance, a 50% improvement over Pbo in combination with BR and R maintenance (median PFS of 52.9 months). The complete response rate was 65.5% in the Ibr arm and 57.6% in the Pbo arm (P = 0.0567). There was no difference in overall survival between treatment arms (P = 0.648). Time to next treatment was longer in the Ibr arm compared with the Pbo arm (P < 0.001). Fifty-two (19.9%) and 106 (40.5%) pts received subsequent anti-lymphoma therapy in the Ibr and Pbo arms, respectively; 41/106 (38.7%) received a second-line BTKi in the Pbo arm. Rates of grade 3 or 4 treatment-emergent adverse events were 81.5% and 77.3% in the Ibr and Pbo arms, respectively. Of adverse events of clinical interest for BTKis, atrial fibrillation was reported in 13.9% and 6.5% of pts in the Ibr and Pbo arms, respectively. Rates of major hemorrhage, hypertension, arthralgia, and secondary primary malignancies were similar in both arms. Quality of life was also similar in both arms. Image: Summary/Conclusion: This phase III study in untreated MCL demonstrated that Ibr combined with BR and R maintenance significantly improved PFS compared with standard chemoimmunotherapy, with a median PFS of 6.7 years. The safety profile was consistent with the known profiles of the individual drugs. S210: CAR T-CELLS ASSOCIATED ACUTE TOXICITY IN B-CELL NON-HODGKIN LYMPHOMA: REAL-WORLD STUDY FROM THE DESCAR-T REGISTRY P. SESQUES1,*, R. DI BLASI2, S. LE GOUILL3, G. CARTRON4, G. MANSON5, D. BEAUVAIS6, F. LE BRAS7, F. X. GROS8, S. CHOQUET9, P. BORIES10, M. T. RUBIO11, R. O. CASASNOVAS12, L. BOUNAIX13, M. MOHTY14, M. JORIS15, J. ABRAHAM16, C. CASTILLA LLORENTE17, M. LOSCHI18, S. CARRAS19, A. CHAUCHET20, L. DRIEU LA ROCHELLE21, J. ZERBIT22, O. HERMINE23, S. GUIDEZ24, T. GASTINNE25, J. J. TUDESQ26, P. FOGARTY27, F. BROUSSAIS28, F. MORSCHHAUSER29, R. HOUOT30, C. THIEBLEMONT31, E. BACHY1 1CHU LYON SUD, LYON; 2Hematology, Assistance Publique Hôpitaux de Paris; 3Hematology, INSTITUT CURIE, PARIS; 4HEMATOLOGY, Département d’Hématologie clinique, CHU de Montpellier, MONTPELLIER; 5CHU de Rennes, RENNES; 6Hématologie clinique, CHU de Lille, LILLE; 7Lymphoid Malignancies, CRETEIL; 8Hématologie Clinique et Thérapie cellulaire, MERIGNAC; 9APHP la Pitié Salpêtriere, PARIS; 10Hematology Laboratory, Onco-occitanie Network, TOULOUSE; 11Service Hématologie, NANCY; 12Department of Hematology, DIJON; 13Department of Hematology, Clermont-Ferrand University Hospital, CLEMRONT FERRAND; 14Hôpital St Antoine, PARIS; 15Hematology department, CHU Amiens, AMIENS; 16CHU DE LIMOGES - HOPITAL DUPUYTREN, LIMOGES; 17GUSTAVE ROUSSY CANCER CAMPUS GRAND PARIS, VILLEJUIF; 18CHU NICE, NICE; 19CHU DE GRENOBLE, GRENOBLE; 20CHU JEAN MINJOZ, BESANCON; 21CHU BRETONNEAU, TOURS; 22APHP HOPITAL COCHIN; 23APHP HOPITAL NECKER, PARIS; 24CHU DE POITIERS - HOPITAL DE LA MILETRIE, POITIERS; 25Nantes University Hospital Clinical Hematology, NANTES; 26Hematology Department, Montpellier University Hospital, Univ Montpellier, MONTPELLIER; 27biostatistics; 28LYSARC, LYON; 29Maladies du Sang CHRU Lille, LILLE; 30CHU de Rennes, Université de Rennes, INSERM U1236, EFS, RENNES; 31Saint Louis Hospital, PARIS, France Background: Cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) are common immune-related toxicities associated with chimeric antigen receptor (CAR)–T-cell therapy. Their clinical manifestations can be severe and potentially life threatening. Aims: Here, we report the French experience of CAR-T toxicity, we specifically addressed the modifiable risk factors for toxicity and we assessed management toxicity in real-world population. Methods: We conducted a study in a large cohort of R/R aggressive B-cell lymphoma patients (pts) treated with commercial products. All data were collected through the French DESCAR-T registry. CRS/ICANS were graded prospectively (ASTCT grade scale). Regarding previously validated predictive score of CRS and ICANS in the literature, EASIX score (LDHxCreatinine/Platelets), modified-EASIX score (m-EASIX: CRPxCreatinine/Platelets) and simplified-EASIX score (s-EASIX: LDH/Platelets) (Pennisi et al., 2021) were assessed in our cohort. Results: A total of 705 pts were included for the analysis of toxicity with a median follow-up of 12 months (range: 0.2-39). Notably, 74 pts (11%) pts had an ECOG/PS ≥ 2 before lymphodepletion. CRS of any grade occurred in 587 pts (83.3%) including 62 (10.5%) with grade 3 or higher (grade 3+). The median time from the infusion to the onset of CRS was 2 days (range: 0-34); the median time to resolution was 6 days (range: 1-30). ICANS of any grade occurred in 289 pts (41%) including 78 (27%) with grade 3+. The median time from the infusion to the onset was 6 days (range: 0-379); the median time to resolution was 7 days (range: 1-100). Most patients (94.5%) with ICANS had previously experienced CRS. Only 89 pts (29.9%) required admission to intensive care unit. Tocilizumab was the most common treatment for toxicity (411 pts, 68%) and 272 pts (45%) received corticosteroids. Interestingly, 38% of patients with CRS grade 1, 53 % with grade 2 and 65% with grade 3+ developed ICANS subsequently. In multivariate analysis, the parameters which retained statistical significance to predict any CRS were Axi-cel ([OR] = 2.79, 95% CI 1.75-4.45; p <0.0001) and elevated LDH (>upper limit normal; [OR] = 1.69, 95% CI 1.03-1.63; p = 0.03). Bulky mass > 5 cm ([OR] = 2.95, 95% CI 1.60-5.45; p = 0.0005), age <65 years ([OR] = 0.42, 95% CI 0.20-0.86; p = 0.01) and higher s-EASIX score ([OR] = 1.38, 95% CI 1.15-1.67; p = 0.0005) increased the risk of developing grade 3+ CRS. Parameters which retained statistical significance to predict any ICANS were Axi-cel ([OR] = 3.95, 95% CI 2.57-6.06; p <0.0001), ECOG ≥2 ([OR] = 2.10, 95% CI 1.26-3.51; p= 0.004), age ≥65 ([OR] = 3.17, 95% CI 2.10-4.79; p <0.0001) and higher s-EASIX score ([OR] = 1.20, 95% CI 1.03-1.40; p= 0.01). Axi-cel ([OR] = 13.1, 95% CI 3.98-43.3; p <0.001), ECOG ≥2 ([OR] = 0.04, 95% CI 1.007-4.38; p= 0.04) and higher s-EASIX score ([OR] = 1.35, 95% CI 1.08-1.67; p= 0.0067) increased the risk of developing grade 3+ ICANS. No survival impact (OS and PFS) was detected for pts who experimented any grade or grade 3+ of ICANS or CRS (Figure 1). Importantly, cumulative dose and duration of tocilizumab or corticosteroids were not significantly associated with adverse PFS or OS. Image: Summary/Conclusion: In this large study, robust biological and clinical predictive factors of CRS and ICANS were identified. A propensity-score matched comparison of axi-cel and tisa-cel for toxicities will be performed for the EHA congress. Neither grade 3+ CRS, ICANS, nor Tocilizumab or corticosteroids use for CAR-T toxicity had negative impact on survival. S211: CLINICAL AND PATIENT-REPORTED OUTCOMES IN A PHASE 3 STUDY OF AXICABTAGENE CILOLEUCEL (AXI-CEL) VS STANDARD-OF-CARE IN ELDERLY PATIENTS WITH RELAPSED/REFRACTORY LARGE B-CELL LYMPHOMA (ZUMA-7) A. Sureda1,*, J. Westin2, F. L. Locke3, M. Dickinson4, A. Ghobadi5, M. Elsawy6, T. van Meerten7, D. B. Miklos8, M. Ulrickson9, M.-A. Perales10, U. Farooq11, L. Wannesson12, L. Leslie13, M. J. Kersten14, C. A. Jacobson15, J. M. Pagel16, G. Wulf17, P. Johnston18, A. P. Rapoport19, L. I. Gordon20, Y. Yang21, A. Peng21, L. Du21, J. T. Snider21, J. Shah21, M. Schupp21, P. Cheng21, C. To21, O. O. Oluwole22 1Hematology Department, Institut Català d’Oncologia-Hospitalet, IDIBELL, Universitat de Barcelona, Barcelona, Spain; 2The University of Texas MD Anderson Cancer Center, Houston; 3Moffitt Cancer Center, Tampa, United States of America; 4Peter MacCallum Cancer Centre, Royal Melbourne Hospital and the University of Melbourne, Melbourne, Victoria, Australia; 5Washington University School of Medicine, St Louis, United States of America; 6Division of Hematology, Department of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada; 7University Medical Center Groningen, on behalf of HOVON/LLPC, Groningen, Netherlands; 8Stanford University School of Medicine, Stanford; 9Banner MD Anderson Cancer Center, Gilbert; 10Memorial Sloan-Kettering Cancer Center, New York; 11University of Iowa, Iowa City, United States of America; 12Istituto Oncologico della Svizzera Italiana, Bellinzona, Switzerland; 13John Theurer Cancer Center, Hackensack, United States of America; 14Amsterdam UMC, University of Amsterdam, on behalf of HOVON/LLPC, Amsterdam, Netherlands; 15Dana-Farber Cancer Institute, Boston; 16Swedish Cancer Institute, Seattle, United States of America; 17University Medicine Göttingen, Göttingen, Germany; 18Mayo Clinic, Rochester; 19University of Maryland School of Medicine and Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore; 20Northwestern University Feinberg School of Medicine, Chicago; 21Kite, a Gilead Company, Santa Monica; 22Vanderbilt University Cancer Center, Nashville, United States of America Background: The median age at large B-cell lymphoma (LBCL) diagnosis is 66 years, and older patients with relapsed/refractory (R/R) LBCL are at risk of inferior outcomes, increased toxicity, and inability to tolerate second-line standard-of-care (SOC) treatment (Di M, et al. Oncologist. 2021). Further, second-line SOC treatment is often associated with poor health-related quality of life (Lin V, et al. J Clin Oncol. 2020;38:e20070). In the global Phase 3, randomized ZUMA-7 study, axi-cel, an autologous anti-CD19 CAR T-cell therapy, significantly improved event-free survival (EFS; hazard ratio [HR], 0.398, P<0.0001; median 8.3 vs 2 months, respectively) compared with second-line SOC in R/R LBCL (Locke FL, et al. N Engl J Med. 2022;386:640-654). Aims: Here we report results of a planned subgroup analysis of the ZUMA-7 study assessing outcomes, including patient-reported outcomes (PROs), of second-line axi-cel vs SOC in patients aged ≥65 years. Methods: Patients with ECOG performance status 0-1 and R/R LBCL ≤12 months after first-line chemoimmunotherapy were randomized 1:1 to axi-cel or SOC (2-3 cycles of platinum-based chemoimmunotherapy; patients with partial or complete response [CR] proceeded to high-dose therapy with autologous stem cell transplantation). PRO instruments, including the EORTC QLQ-C30 (Global Health and Physical Functioning) and the EQ-5D-5L visual analog scale (VAS), were administered at baseline (prior to treatment), Day 50, Day 100, Day 150, and Month 9, then every 3 months up to 24 months or time of EFS event, whichever occurred first. The quality-of-life analysis set included all patients who had a baseline PRO and ≥1 completed measure at Day 50, 100, or 150. A clinically meaningful change was defined as 10 points for each EORTC QLQ-C30 score and 7 points for EQ-5D-5L VAS score. Results: The data cutoff for this analysis was March 18, 2021 and included 51 axi-cel and 58 SOC patients with median ages of 70 years (range, 65-80) and 69 years (range, 65-81), respectively. At baseline, more axi-cel vs SOC patients had high-risk features, including second-line age-adjusted International Prognostic Index 2-3 (53% vs 31%) and elevated lactate dehydrogenase (61% vs 41%). EFS was superior with axi-cel vs SOC (HR, 0.276, P<0.0001), with higher CR rates (75% vs 33%). Grade ≥3 treatment-emergent adverse events (AEs) occurred in 94% and 82% of axi-cel and SOC patients, respectively, and Grade 5 treatment-related AEs occurred in 0 and 1 patient. In the quality-of-life analysis set comprising 46 axi-cel and 42 SOC patients, there were statistically significant and clinically meaningful differences in mean change of scores from baseline at Day 100 favoring axi-cel for EORTC QLQ-C30 Global Health (P<0.0001) and Physical Functioning (P=0.0019) and EQ-5D-5L VAS (P<0.0001). For all 3 domains, scores also favored (P<0.05) axi-cel over SOC at Day 150. The mean estimated scores numerically returned to or exceeded baseline scores earlier in the axi-cel arm (by Day 150) but never equaled or exceeded baseline scores by Month 15 in the SOC arm. Summary/Conclusion: Axi-cel demonstrated superiority over second-line SOC in patients ≥65 years with significantly improved EFS and a manageable safety profile. Axi-cel also showed meaningful improvement in quality of life over SOC, measured by multiple validated PRO instruments, with suggested faster recovery to pretreatment quality of life. The superior clinical outcomes and patient experience with axi-cel over SOC should help inform treatment choices in second-line R/R LBCL for patients ≥65 years. S212: PHASE I STUDY OF YTB323, A CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELL THERAPY MANUFACTURED USING T-CHARGE™, IN PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA M. Dickinson1,*, M. Kwon2, J. Briones3, U. Jäger4, K. Kato5, E. Bachy6, D. Blaise7, N. Boissel8, N. Shah9, M Frigault10, P. Riedell11, L. Shune12, T. Teshima13, X. Zhu14, E. Orlando14, L. Yi15, J. Davis16, E. Bleickardt16, I. Flinn17, P. Barba18, P. Barba18 1Peter MacCallum Cancer Centre and Royal Melbourne Hospital and the University of Melbourne, Melbourne, Australia; 2Department of Hematology, Hospital General Universitario Gregorio Marañón, Institute of Health Research Gregorio Marañón, Madrid; 3Hematology Department, Hospital Santa Creu i Sant Pau, Barcelona, Spain; 4Clinical Division of Hematology and Hemostaseology, Department of Medicine I, Vienna General Hospital – Medical University of Vienna, Vienna, Austria; 5Department of Hematology, Oncology and Cardiovascular Medicine, Kyushu University Hospital, Fukuoka, Japan; 6Hematology department, Hospices Civils de Lyon and Université Claude Bernard Lyon; 1, Lyon; 7Département d’Hématologie, Programme de Transplantation et de Thérapie Cellulaire, Centre de Recherche en Cancérologie de Marseille, Aix-Marseille University, Institut Paoli Calmettes, Marseille; 8Hematology Adolescent and Young Adult Unit, Saint-Louis Hospital, Paris, France; 9Medical College of Wisconsin, Milwaukee; 10Massachusetts General Hospital, Massachusetts; 11University of Chicago, Chicago; 12Division of Hematologic Malignancies and Cellular Therapeutics, University of Kansas Medical Center, Kansas City, United States of America; 13Department of Hematology, Hokkaido University Hospital, Sapporo, Japan; 14Novartis Institutes for BioMedical Research; 15Novartis Pharmaceuticals Corporation, Cambridge; 16Novartis Pharmaceuticals Corporation, East Hanover; 17Sarah Cannon Research Institute and Tennessee Oncology, Nashville, United States of America; 18Hematology Department, Hospital Universitari Vall d’Hebrón, Universitat Autònoma de Barcelona, Barcelona, Spain Background: CD19-directed CAR-T cell therapies (tx) demonstrate efficacy in patients (pts) with B-cell malignancies. However, despite positive outcomes with currently approved CAR-T cell tx, many patients fail to respond or relapse after initial response. YTB323 is an autologous CD19-directed CAR-T cell tx generated by the innovative T-Charge™ platform, which demonstrates high potency, preserves T-cell stemness in the final product, and takes <2 d to manufacture. Aims: T-Charge™ is expected to prolong CAR-T cell persistence and yield higher response rates and durability. Methods: These data from the ongoing Phase I, multicenter, dose-escalation study (NCT03960840) focus on safety and efficacy of YTB323 in adults with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). Eligible pts had measurable disease at enrollment, ECOG 0-1, and r/r DLBCL after ≥2 lines of prior tx. Pts received single-dose YTB323 at targeted dose levels (DL) 1 (2.5×106 CAR+ cells), DL2 (12.5×106 CAR+ cells), DL3 (25×106 CAR+ cells), or DL4 (40×106 CAR+ cells). Results presented here focus on DL1 and DL2. The primary endpoints are to identify a recommended dose for subsequent trials and characterize the safety and dose-limiting toxicities. Secondary endpoints include overall response rate (ORR) by local investigator assessment and cellular kinetics. Results: As of August 20, 2021, 20 pts with r/r DLBCL received YTB323: 4 at DL1 and 16 at DL2. Median age was 65 y, 65% received 2 prior lines of tx, and 35% had prior SCT. The median follow-up for pts in DL1 and DL2 was 20 and 7 months, respectively. Of 20 pts evaluable for safety, all experienced at least 1 adverse event (AE) of any Grade (Gr) and 80% at least 1 AE Gr ≥3. One pt (25%) experienced Gr 1 cytokine release syndrome (CRS) at DL1, and 5 (31%) pts experienced CRS at DL2, including 4 (25%) Gr 1 or 2 and 1 (6%) Gr 4 (Lee et al, 2014), which met protocol defined DLT criteria at DL2. Tocilizumab and corticosteroids were used for CRS management in 4 (80%) and 2 (40%) pts at DL2, respectively. Median time to CRS onset was 10 d (range, 1-17 d). Five pts (25%) had neurological events (NE), of which the majority were Gr 1. Median time to onset and resolution of NE was 7 and 12 d, respectively. Five pts died on trial (beyond 30 d); 3 pts died due to disease progression (1 at DL1, 2 at DL2), and 1 pt each due to sepsis (1 at DL1) and intestinal hemorrhage (1 at DL2). At DL1, ORR and complete response (CR) rate were both 75%. At DL2, ORR and CR rates were 81% and 75%, respectively. Of the 15 pts who received YTB323 at DL2 at least 3 mo prior to the data cutoff, CR rate at mo 3 was 73% (95% CI, 44.9%-92.2%) (Figure). Median time to peak YTB323 expansion was ~16 d and coincided with cytokine peak. YTB323 expansion (Cmax and AUC0-28d) with 12.5×106 CAR+ cells (DL2) was comparable to a median tisagenlecleucel dose of 312×106 CAR+ cells in pts with DLBCL, a 25-fold lower median dose (Awasthi R, et al. 2020). Limited data indicate that CAR expression was detectable by flow cytometry for at least 9 mo at DL2. T-Charge™ allowed preservation of CD4 and CD8 naive/stem memory T cells in the final product according to flow cytometry. Bulk RNAseq analysis demonstrated that YTB323 retained a naive stem-like gene signature. Image: Summary/Conclusion: YTB323 is a potent new CAR-T cell tx with distinct cellular kinetics, encouraging early efficacy results across DL1 and DL2, and a manageable safety profile. Updated results will be presented at the meeting along with a recommended dose for subsequent trials. S213: EFFICACY AND SAFETY OF HUMANIZED VERSUS MURINIZED CD19 AND CD22 CAR-T CELL COCKTAIL THERAPY FOR REFRACTORY/RELAPSED B-CELL LYMPHOMA L. Huang1 2, J. Li2, J. Yang2, X. Zhang2, M. Zhang2, J. He2, G. Zhang2, Y. Su2, W. Li2, H. Wang2, P. Lu1 2,* 1Tsinghua University School of Medicine-Lu Daopei Institute of Hematology, Beijing; 2Hebei Yanda Lu Daopei Hospital,., langfang, China Background: CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has demonstrated about 50~60% of response rate in relapsed or refractory (R/R) B-cell non-Hodgkin lymphoma. However, antigen-escape relapse has emerged as a major challenge for long-term disease control after CD19-targeted therapies. In our previous study, we described a dual-targeted of CD19 and CD22 CAR-T cell cocktail therapy, whichyielded 93.3% of complete remission (CR) rate in R/R B-cell acute lymphoblastic leukemia and could reduce the risk of CD19-negative relapse. Aims: A phase I/II clinical trial (NCT05206071) was designed to explore the safety and efficacy of CAR19/22 T-cell cocktail therapy for patients with R/R aggressive B-cell lymphoma. Methods: Eligible R/R aggressive B-cell lymphoma patients were enrolled from July 2020 toDecember 2021. All patients received fludarabine 30 mg/m2/d and cyclophosphamide 250 mg/m2/d for 3 consecutive days (day −5 to day −3) followed by cocktail CAR-T cell infusion. CAR-T-cell-related cytokine release syndrome (CRS) was graded to assess the safety. PET-CT was performed on day 28 and 3rd month to evaluate the response rate. Results: A total of 26 eligible patients received CAR19/22 cocktail infusion with a median age of 46 years old (4-75) and a median of 3 prior lines of therapies (2-5 lines). Of 26 patients, 4 were Burkitt lymphoma, 2 were follicular lymphoma (grade 3a), and the rest were diffuse large B-cell lymphoma. Besides, 7 patients were diagnosed with double-hit lymphoma and 4 patients were relapsed after previous CD19 CAR-T cell therapy. Fourteen patients received the murinized CAR-T cells at a median dose of 2.18×106/kg (1-3×106/kg) CAR19 and 1.75×106/kg (1-3×106/kg) CAR22, while 12 patients received the does of the humanized CAR-T cells consisting of 2×106/kg CAR19 and 5×105/kg CAR22. At the day 28 assessment, 12/12 (100%) patients received the humanized CAR-T cells achieved overall response, including 9/12 (75%) CR and 3/12 (25%) partial remission (PR), while the overall response rate (ORR) and CR rate of the murinized group was 85.71% (12/14) and 42.86% (6/14) respectively. Among of them, all 4 Burkitt lymphoma patients in both groups achieved CR. In 3 months evaluation, 7/10 (70%,2 cases received infusion for less than 3 months) patients in the humanized group maintained in CR, which was higher than the murinized group with CR rate of 5/14 (35.71%). (Figure 1A-B). At a median follow-up of 291 days (range, 31 to 544), patients in the humanized group had a favorable progression-free survival (PFS) than those in the murinized (1-year PFS of 67.9% vs. 26.8%), although there was no statistical significance (p=0.08) due to the limited number of patients enrolled (Figure 1E-F). Achieving CR on day 28 (HR: 0.21, 95% CI: 0.06-0.72; P=0.012) and maintaining CR till the 3rd month (HR: 0.18, 95% CI: 0.06-0.59; P=0.004) were found as independent prognostic factors associated with favorable PFS. Maintaining CR till the 3rd month (HR: 0.10, 95% CI: 0.01-0.61; P=0.012) and non-double hit (HR: 0.11, 95% CI: 0.02-0.76, P=0.024) predicted a longer overall survival (OS) (Figure 1G-L). Despite impressive CR rates wereachieved, majority of patients had mild CRS (grade 1-2) in both group. Only 2patients experienced grade 3 CRS, and 2 patients developed grade 3 neurotoxicity. Image: Summary/Conclusion: This clinical trial demonstrates promising efficacy and safety of CD19/CD22 CAR-T cocktail therapy for R/R aggressive B-cell lymphoma, and patients in the humanized group showed better results than those with murinized, although this not a randomized trial. S214: PHASE 1/2 STUDY OF ANBAL-CEL, NOVEL ANTI-CD19 CAR-T THERAPY WITH DUAL SILENCING OF PD-1 AND TIGIT IN RELAPSED OR REFRACTORY LARGE B CELL LYMPHOMA W. S. Kim1,*, S. J. Kim1, S. E. Yoon1, J. R. Kim2 1Hematology and Oncology, Samsung Medical Center, Seoul; 2Clinical Development, Curocell Therapeutics, DaeJeon, South Korea Background: Anbal-cel is a novel 2nd generation autologous CD19 CAR-T cell therapy which has been knock-downed for PD-1 and TIGIT using OVIS platform. Anbal-cel demonstrated the eradication of CD19 positive tumor cells in vitro and in vivo better than conventional CD19 CAR-T cells. The knock-down of PD-1 and TIGIT at CD19 CAR-T cells exerts the superior T-cell functionality by delaying the exhaustion of CAR-T cells. Aims: This phase 1 dose escalation part (NCT04836507) was to evaluate the safety (DLT, MTD), PK and preliminary efficacy (objective response rate and duration of response) in patients with r/r LBCL. Anbal-cel was manufactured at GMP facility with fresh leukapheresis product. Methods: Patient was administered as a single intravenous dose at dose level 1 (2x105 cells/kg), dose level 2 (7x105 cells/kg) or dose level 3 (2x106 cells/kg). Lymphodepletion with cyclophosphamide (500mg/m2) and fludarabine (30mg/m2) was performed for 3 days prior to Anbal-cel infusion. Results: As of Jan 17 2022, 9 patients with r/r DLBCL were infused with Anbal-cel; 4pts at DL1, 3pts at DL2 and 2pts at DL2. Median age was 54 (range 26-71); all patients received 2 or more prior lines of therapy and 44% (4/9) received ≥4 prior line of treatment before the study. 78% (7/9) patients were refractory to their last treatment. 67% (6/9) of patients were at IPI 3-4 and 44% (4/9) of patients had bulky disease. No patient experienced DLT during the study. Of the 9 patients, 5 (56%) experienced CRS; 4 (44%) were grade 1 or 2 and one patient experienced grade 3 CRS. Median time to onset of CRS was 7 days (range, 1-16) with median duration of 4 days (range, 0-18). One patient dosed at DL3 experienced grade 2 ICANS, time to onset of ICANS was 7 days and lasted for 13 days. This patient had prior CNS involvement history before the study. Most commonly reported grade 3/4 AEs were neutrophil count decrease (6/9, 67%), anemia (5/9, 56%), thrombocytopenia (2/9, 22%), platelet count decrease (2/9, 22%) and no infection was reported. Complete response rate (CRR) was 78% and complete responses were observed at the lowest dose level and from patients expressing less than 10% CD19 at IHC; 3 complete responses (CR) at DL1, 2 CRs at DL2 & DL3 respectively. Dose-dependent CRC01 expansion was observed; median Tmax was 15.4, 15.8, 11.0 days at DL1, DL2 & DL3 each; median Cmax was 18,003, 30,103, 53,688 copies/ug gDNA at DL1, DL2 & DL3 each; median AUC0-28day was 679,125, 1,110,108, 2,852,235 copies/ug gDNA at DL1, DL2 & DL3 respectively. Summary/Conclusion: Anbal-cel demonstrated promising efficacy and tolerable safety profile in this dose escalation study. Based on this phase 1 study, phase 2 patient enrollment will be commenced in Mar 2022 to evaluate the response rate, duration of response of CRC01 as well as safety. In addition, various biomarker studies are planned to investigate the differential mode of action of Anbal-cel during phase 2 study. S215: GEMSTONE-201: PRE-PLANNED PRIMARY ANALYSIS OF A MULTICENTER, SINGLE-ARM, PHASE 2 STUDY OF SUGEMALIMAB IN PATIENTS WITH RELAPSED OR REFRACTORY EXTRANODAL NATURAL KILLER/T CELL LYMPHOMA (R/R ENKTL) H. Huang1,*, R. Tao2, Y. Yang3, H. Cen4, H. Zhou5, Y. Guo6, L Zou7, J. Cao8, Y. Huang9, J. Jin10, L. Zhang11, H. Yang12, X. Xing13, H. Zhang14, Y. Liu15, K. Ding16, X. Zhu17, T. Fang17, H. Dai17, Q. Qi17, J. Yang17 1Sun Yat-sen University Cancer Center, Guangzhou; 2Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai; 3Fujian Cancer Hospital & Fujian Medical University Cancer Hospital, fuzhou; 4Guangxi Cancer Hospital and of Guangxi Medical University Affiliated Cancer Hospital, nanning; 5Hunan Cancer Hospital, changsha; 6Shanghai East Hospital, Tongji University School of Medicine, Shanghai; 7State Key Laboratory, Cancer Center, West China Hospital of Sichuan University, chengdu; 8Fudan University Shanghai Cancer Center, Shanghai; 9The Affiliated Cancer Hospital of Guiyang Medical University, guiyang; 10First Affiliated Hospital, College of Medicine, Zhejiang University, hangzhou; 11Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, wuhan; 12Zhejiang Cancer Hospital, hangzhou; 13Liaoning Cancer Hospital & Institute, Shenyang; 14Tianjin Medical University Cancer Institute & Hospital, tianjin; 15Henan Provincial Cancer Hospital, zhengzhou; 16Anhui Provincial Cancer Hospital, Hefei; 17CStone Pharmaceuticals (Su Zhou) Co., Ltd., Shanghai, China Background: R/R ENKTL is a rare and aggressive type of non-Hodgkin’s lymphoma. Responses to chemotherapy after failure of prior asparaginase-based regimen were not durable with a median OS of < 7 months (mos) and 1-year OS rate of < 20% (Lim et al, Ann Oncol 2017; Bellei et al, Haematologica 2018). The only targeted therapy approved in China for R/R peripheral T cell lymphoma (ENKTL included) showed an ORR of 18.8% and a CR rate of 6.3% (Shi et al, Ann Oncol 2015). Aims: Here, we present the primary analysis from GEMSTONE-201, the largest registrational study reported to date to evaluate an anti-PD-L1 mAb in R/R ENKTL. Sugemalimab (Suge) received breakthrough therapy designation from US FDA in 2021 for adult R/R ENKTL patients (pts) based on preliminary data of this study. Methods: Pts with ECOG PS of 0 or 1 and histologically confirmed ENKTL who failed prior asparaginase-based regimen were enrolled. Pts accepted suge at 1200 mg intravenously every 3 weeks, for up to 24 mos until progression, death, or withdrawal from study. The primary endpoint was ORR (CR+PR) assessed by independent radiological review committee (IRRC) per Lugano 2014 criteria. Key secondary endpoints included investigator-assessed ORR, CR and PR rate, DoR assessed by IRRC and investigators, and safety. Results: As of the data cutoff date, 10 November 2021, 80 pts were enrolled and treated (median follow-up of 13.4 mos). Median age was 48 years (range 29-74); 64% were males; 74% had ECOG PS of 1 at baseline; 68% had stage IV disease; about half (49%) received ≥ 2 lines of prior systemic therapy. The median duration of treatment was 5.2 mos (range 0.7-37.4); 23 pts remained on treatment. Among the 78 evaluable pts as per IRRC, ORR was 46.2% (95% CI: 34.8%, 57.8%); 29 (37.2%) pts achieved CR; median DoR was not reached (NR); 12-mo DoR rate was 86%. Investigator’s assessments in 79 evaluable pts were consistent with IRRC results, i.e., ORR of 45.6% (95% CI: 34.3%, 57.2%), 24 (30.4%) pts with CR, and median DoR of NR. The 1- and 2-year OS rates were 68.6% and 54.6%, respectively; median OS was NR (range 0.9-37.2+ mos). Of all pts, 96% (n = 77) had at least one AE. The most common AEs were pyrexia and WBC decreased (n = 24 each, 30%). Grade ≥ 3 AEs occurred in 31 (38.8%) pts. Suge-related AEs occurred in 61 (76%) pts and were mostly (60%) Grade 1 or 2. The most common immune-related AE assessed by sponsor was hypothyroidism (n = 13, 16%). SAEs occurred in 18 (23%) pts; 5 (6%) pts had suge-related SAEs which had all been resolved (1 with sequelae). Fatal AEs occurred in 5 (6%) pts and none were suge-related as assessed by investigators. Summary/Conclusion: Suge has demonstrated deep and durable anti-tumor activity in R/R ENKTL pts, with a high CR rate and a promising OS benefit trend comparing to historical data. Suge had a well-tolerated safety profile and no new safety signals were detected. Primary analysis indicates that suge could provide a new treatment option to R/R ENKTL pts. S216: CLINICAL ACTIVITY OF CC-99282, A CEREBLON E3 LIGASE MODULATOR (CELMOD) AGENT, IN PATIENTS (PTS) WITH RELAPSED/REFRACTORY NON-HODGKIN LYMPHOMA (R/R NHL) – RESULTS FROM A PHASE 1, OPEN-LABEL STUDY J.-M. Michot1,*, J. C. Chavez2, C Carpio3, S. Ferrari4, T. A. Feldman5, D. Morillo6, J. Kuruvilla7, A. Pinto8, V. Ribrag1, E Bachy9, T. J. Buchholz10, S. Carrancio11, W.-C. Chou12, C. Guarinos13, F. Wu14, S. Li15, P. Patah16, M. Pourdehnad17, L. Nastoupil18 1Département d’Innovation Thérapeutique et d’Essais Précoces (DITEP), Gustave Roussy Institute of Cancer, Villejuif, France; 2Department of Malignant Hematology, Moffitt Cancer Center, University of South Florida, Tampa, United States of America; 3Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), University Hospital Vall d’Hebron, Autonomous University of Barcelona (UAB), Barcelona, Spain; 4Hematology and Bone Marrow Transplant Unit, ASST Papa Giovanni XXIII, Bergamo, Italy; 5Lymphoma Division, John Theurer Cancer Center at Hackensack Meridian Health, Hackensack, United States of America; 6Department of Hematology, University Hospital Fundación Jiménez Díaz, Madrid, Spain; 7Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Canada; 8Hematology-Oncology & Stem Cell Transplantation Unit, National Cancer Institute, Fondazione G. Pascale, IRCCS, Naples, Italy; 9Department of Hematology, Hospices Civils de Lyon, Lyon, France; 10Early Clinical Development, Oncology, Bristol Myers Squibb, San Francisco; 11Oncogenesis (ONC) Thematic Research Center (TRC), Bristol Myers Squibb, San Diego; 12Early Development Predictive Sciences, Bristol Myers Squibb, Cambridge, United States of America; 13ONC TRC-Celgene Institute for Translational Research Europe (CITRE), Bristol Myers Squibb, Seville, Spain; 14Clinical Pharmacology & Pharmacometrics, Bristol Myers Squibb, Summit; 15Global Biometric Sciences, Bristol Myers Squibb, Berkley Heights; 16Early Clinical Development, Bristol Myers Squibb, Lawrenceville; 17Early Clinical Development, Hematology/Oncology and Cell Therapy, Bristol Myers Squibb, San Francisco; 18Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: CC-99282 is a novel, oral small molecule CELMoD® agent that co-opts cereblon to induce targeted degradation of Ikaros/Aiolos, transcription factors critical for development of B-cell malignancies. Compared with immunomodulatory drugs (IMiD®) and other CELMoD agents, CC-99282 had similar immunostimulatory effects and 10- to 100-fold stronger antiproliferative and apoptotic activity in preclinical models of diffuse large B-cell lymphoma (DLBCL). Aims: To evaluate the safety, tolerability, maximum tolerated dose, pharmacokinetics (PK), and preliminary efficacy of CC-99282 in pts with R/R NHL. Methods: CC-99282-NHL-001 (NCT03930953) is a 2-part multicenter first-in-human study comprising dose escalation of CC-99282 monotherapy (part A) and expansion with or without combination partners (part B). Part A includes pts with R/R DLBCL or follicular lymphoma (FL) who had progressed after ≥2 lines of therapy, including IMiD/CELMoD agents and chimeric antigen receptor T-cell (CAR-T) therapy, and pts with R/R DLBCL who received ≥1 line of standard therapy and are unfit for transplant. Pts receive oral CC-99282 0.2, 0.4, 0.6, or 0.8 mg once daily on 3 intermittent dosing schedules of 28-day cycles, with ≥3 pts per dosing cohort. Results: As of Oct 6, 2021, 50 pts were treated in part A (38 DLBCL, 12 FL; median age 66y; 58% male); 17 pts (34%) were ongoing and 26 pts (52%) discontinued due to progressive disease. Grade 3/4 CC-99282–related adverse events were reported in 32 pts (64%); the most common were neutropenia (29 pts [58%]), thrombocytopenia (4 pts [8%]), and anemia (4 pts [8%]). Correlation analysis showed that grade 4 neutropenia in the first month was more strongly associated with number of prior lines of alkylating agent and anti-CD20 therapy than with CC-99282 dose level or schedule. Neutropenia was manageable with CC-99282 dose modifications and granulocyte colony–stimulating factors. All dose-limiting toxicities were hematologic. The maximal recommended doses for schedules of interest were 0.6 mg on 7/14 days and 0.4 mg on 14/28 days. For doses ≥0.4 mg on schedules of interest, the overall response rate was 42% (15/36 evaluable pts; 6 FL, 9 DLBCL), with complete responses in 6 pts and partial responses in 9 pts. Responders included pts previously treated with CAR-T therapy and/or IMiD/CELMoD agents. Responses were durable, with median durations of 239 days (range 48–587; median follow-up [mFUP] 247 days [range 21–690]) and 112 days (range 63–414; mFUP 121 days [range 22–464]) for 7/14- and 14/28-day schedules, respectively. CC-99282 was absorbed rapidly and had a prolonged terminal half-life (median ~50 hours at doses ≥0.4 mg), with considerable distribution into the peripheral compartment. Increase in plasma CC-99282 and degradation of Ikaros/Aiolos in peripheral T cells occurred in a dose-dependent manner, and maximum degradation (>90%) occurred by day 4 of treatment at doses ≥0.4 mg. Within 2 weeks of initiating CC-99282, peripheral T-cell subsets showed a significant shift toward a more activated phenotype (P<0.05). Analysis of mutant circulating tumor DNA levels showed rapid reductions that correlated with response to CC-99282, suggesting strong tumor cell–intrinsic activity. Summary/Conclusion: CC-99282 monotherapy showed a manageable safety profile and demonstrated promising efficacy in heavily pretreated pts with R/R NHL. PK and pharmacodynamic data were consistent with robust and rapid CC-99282 antitumor activity. This study is ongoing, with patients actively being enrolled in the monotherapy and CC-99282+rituximab combination expansion cohorts. S217: PRELIMINARY ANALYSIS OF THE PHASE II STUDY USING TOLINAPANT (ASTX660) MONOTHERAPY IN 98 PERIPHERAL T-CELL LYMPHOMA AND 51 CUTANEOUS T-CELL LYMPHOMA SUBJECTS WITH RELAPSED REFRACTORY DISEASE. J.-M. Michot1, A. Mehta2, F. Samaniego3, E. Bachy4, P. L. Zinzani5, A. Prica6, G. P. Colins7, V Ribrag1,*, N. Wagner-Johnston8, D. El-Sharkawi9, O. A. O’Connor10, R. Wilcox11, L. Wang12, L. Wilson12, M. Sims13, J. A. Taylor12, H. N. Keer12, F. Foss14 1Department of Hematology, Institut Gustave Roussy, Villejuif, France; 2University of Alabama at Birmingham, Birmingham; 3The University of Texas MD Anderson Cancer Center, Houston, United States of America; 4Hematology Department, Lyon Sud Hospital, Lyon, France; 5Institute of Hematology “L. e A. Seràgnoli”, Bologna, Italy; 6Princess Margaret Cancer Centre, Toronto, Ontario, Canada; 7Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom; 8Johns Hopkins University, The Sidney Kimmel Comprehensive Cancer Center, Baltimore, United States of America; 9The Royal Marsden NHS Foundation Trust, Surrey, United Kingdom; 10University of Virginia, Charlottesville; 11University of Michigan Medical School, Ann Arbor; 12Astex Pharmaceuticals, Inc., Pleasanton, United States of America; 13Astex Pharmaceuticals, Cambridge, United Kingdom; 14Yale Cancer Center, New Haven, United States of America Background: There are limited treatment options for patients with Peripheral T-cell lymphoma (PTCL) and Cutaneous T-cell lymphoma (CTCL), especially when front line therapy has failed. Tolinapant (ASTX660) is a novel oral non-peptidomimetic, small-molecule antagonist of cellular/X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which also induces necroptosis in T-cell lymphoma models (Ferrari et al., Blood Advances, 2021). Tolinapant is being evaluated in a first-in-human ongoing Phase I/II study in subjects with advanced solid tumors and lymphoma (ClinicalTrials.gov NCT02503423). Phase I results were previously reported (Mita et al. Clin Cancer Res, 2020) and the recommended phase 2 dosing (RP2D) was established. Initial results for Phase II were previously reported at EHA 2019 (Mehta et al., EHA 2019, # PS1073). Aims: Here we report the preliminary efficacy and safety analysis for the Phase 2 PTCL and CTCL cohorts. Methods: This is a single-arm open-label Phase II study. To be eligible, subjects must have evidence of documented progressive disease and received at least two prior systemic therapies. Subjects received treatment with tolinapant at the RP2D 180 mg/day on Days 1 to 7, and 15 to 22 in a 28-day cycle. The primary endpoint is best overall response rate (ORR) as assessed by the investigator according to either the Lugano criteria (PTCL) or Global Assessment (CTCL). Adverse events (AEs) are assessed per CTCAE v4.03. The efficacy data set is based on subjects who had tumor evaluation at baseline and at least 1 post-treatment tumor evaluation visit, unless they died or stopped treatment earlier due to clinical progression or toxicity. The safety data set is based on all subjects that received at least one dose of tolinapant. Results: As of the data cut of January 5, 2022, there were 98 subjects with PTCL and 51 subjects with CTCL that received drug and 98 and 50 subjects that were evaluable respectively. The study is currently closed to enrollment with a minimum of 6 months follow-up on all subjects at the time of the data cut. Subject characteristics: median (range) age PTCL 62.5 (27, 82) and CTCL 62 (24,87), median number of previous therapies PTCL 3 (0-8) and CTCL 6 (1-10). Among all subjects, the most common related AEs of any grade (≥ 15%) were: lipase elevation (35%), amylase elevation (25%), rash (combined listings) (24%), ALT elevation (15%), and AST elevation (15%). Related AEs ≥ Grade 3 (≥ 5%) were: lipase elevation (15%), rash (combined listings) (9%), and amylase elevation (7%). Pancreatitis was identified in 2 subjects (1%) (both Grade 4). There were no related ≥ Grade 3 AEs for diarrhea, nausea or vomiting; for related Grade 2 AEs there was a 5% incidence of diarrhea and 1% incidence of nausea/vomiting. The ORR for PTCL is 22%, including 9 complete responses (CRs) and 12 partial responses (PRs). The ORR in CTCL is 26% including 2 CRs and 11 PRs. The median durability of response for PTCL is 133 (Q1-Q3; 69 - 280) days and for CTCL is 148 (Q1-Q3; 103 - 294) days. Pharmacodynamic and correlative analysis is ongoing with preliminary analysis suggesting an immunomodulatory antitumoral effect of tolinapant (Ferrari et al., Blood Advances, 2021). Summary/Conclusion: In this Phase II study, the novel oral agent tolinapant has shown meaningful clinical activity against PTCL and CTCL with a manageable safety profile. These results support the continued development of tolinapant for the treatment of R/R PTCL and CTCL. A drug combination study using tolinapant in R/R PTCL is being developed. S218: A PHASE I/II STUDY OF GOLIDOCITINIB, A SELECTIVE JAK1 INHIBITOR, IN REFRACTORY OR RELAPSED PERIPHERAL T CELL LYMPHOMA W.-S. Kim1,*, D.-H. Yoon2, Y. Song3, H. Yang4, J. Cao5, D. Ji5, Y. Koh6, H. Jing7, H.-S. Eom8, J.-Y. Kwak9, W.-S. Lee10, J.-S. Lee11, H.-J. Shin12, J. Jin13, M. Wang14, J. Li14, X. Huang14, X. Deng14, Z. Yang14, J. Zhu3 1Department of Hematology and Oncology, Samsung Medical Center; 2Department of Oncology - Hematologic Cancer & BMT center, Asan Medical Center, Seoul, South Korea; 3Department of Lymphoma, Peking University Cancer Hospital, Beijing; 4Department of Lymphoma, Zhejiang Cancer Hospital, Hangzhou; 5Department of Oncology, Fudan University Shanghai Cancer Center, Shanghai, China; 6Department of Hemato-Oncology Center, Seoul National University Hospital, Seoul, South Korea; 7Department of Hematology, Peking University Third Hospital, Beijing, China; 8Center for Hematologic Malignancy, National Cancer Center, Goyang; 9Department of Hemato-Oncology Center, Chonbuk National University Hospital, Jeonju; 10Department of Hemato-Oncology Center, Inje University Busan Paik Hospital, Busan; 11Department of Hematology & Medical Oncology, Seoul National University Bundang Hospital, Seongnam; 12Department of Hemato-Oncology Center, Pusan National University Hospital, Busan, South Korea; 13Department of Hematology, The First Affiliated Hospital of Zhejiang University, Hangzhou; 14Clinical Development, Dizal Pharmaceutical, Shanghai, China Background: Peripheral T cell lymphoma (PTCL) is a group of heterogeneous T cell lymphomas. Patients who relapse from or are refractory to 1st line therapy face dismal prognosis. The response rates to commonly used 2nd line agents such as histone deacetylase inhibitors are below 30%. Immunotherapies, such as anti-PD1 antibodies, may induce hyperprogression in certain PTCL subtypes. Hence, r/r PTCL patients urgently need better therapies. Aims: Preclinical data shows JAK/STAT pathway may mediate the pathogenesis of PTCL, making it a promising target. Golidocitinib (DZD4205) is an orally available, potent, JAK1 specific inhibitor, demonstrating profound anti-tumor activities in T lymphoma cells in vitro and tumor xenograft in vivo. Here we report the preliminary data from an ongoing phase I/II study (NCT04105010) of Golidocitinib in r/r PTCL. Methods: The study included two parts: Part A (dose escalation) and Part B (dose expansion). In Part A, patients with r/r PTCL were enrolled and received Golidocitinib at different doses (150 mg or 250 mg, QD) to determine the recommended phase II dose (RP2D). Evaluation of safety and efficacy were performed by investigators per CTCAE and Lugano criteria, respectively. Part B is a single-arm, pivotal study, where patients with r/r PTCL will receive Golidocitinib at the RP2D till disease progression or intolerance. Results: As of May 31, 2021, a total of 51 patients enrolled in Part A and received Golidocitinib at 150 mg (n = 35) or 250 mg (n = 16). Patient characteristics: median age (range): 61.0 years (29-79); median prior systemic therapies (range): 2 lines (1-8). Ten patients (19.6%) had undergone hematopoietic stem cell transplantation. Fifteen patients (29.4%) had bone marrow involvement at baseline. Histological subtypes included PTCL-NOS (41.2%), AITL (39.2%), ALCL ALK- (7.8%), NKTCL (7.8%), and MEITL (3.9%). At the data cut-off (DCO), 49 patients completed at least one post-treatment Lugano assessment, of whom 21 (42.9%) achieved tumor response, including 11 complete responses (CRs, 22.4%) and 10 partial responses (20.4%). Tumor response was observed in various subtypes, including AITL (13/20), PTCL-NOS (5/19), ALCL ALK- (2/4) and NKTCL (1/4). At the DCO, the median duration of response (DoR) was not reached, and the longest DoR was > 14 months. Forty-eight patients (94.1%) experienced treatment emergent adverse events (TEAEs), of whom 30 (58.8%) experienced ≥ grade 3 TEAEs. Per investigators’ assessment, 20 patients (39.2%) experienced ≥ grade 3 TEAEs possibly related to the drug. The most common (≥ 10%) ≥ grade 3 TEAEs were neutropenia (29.4%), thrombocytopenia (15.7%) and pneumonia (11.8%). The majority of TEAEs were reversible or clinically manageable with dose modifications. Image: Summary/Conclusion: Golidocitinib shows good safety and promising anti-tumor efficacy in r/r PTCL, indicating its potential as a therapeutic option for this unmet medical need. S219: FIRST CLINICAL STUDY OF THE ANTI-SIGNAL REGULATORY PROTEIN-ALPHA (SIRPΑ) ANTIBODY CC-95251 COMBINED WITH RITUXIMAB IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) NON-HODGKIN LYMPHOMA (NHL) P. Strati1,*, E. Hawkes2, N. Ghosh3, J. Tuscano4, Q. Chu5, M. A. Anderson6, A. Patel7, M. R. Burgess7, K. Hege7, S. Chhagan7, S. Boyanapalli7, T. Day7, F. Shen7, A. Mehta8 1The University of Texas MD Anderson Cancer Center, Houston, United States of America; 2Austin Health–Austin Hospital, Heidelberg, Australia; 3Levine Cancer Institute, Charlotte; 4University of California, Davis, Sacramento, United States of America; 5Cross Cancer Institute, Edmonton, Canada; 6Peter MacCallum Cancer Centre, Melbourne, Australia; 7Bristol Myers Squibb, Princeton; 8University of Alabama at Birmingham, Birmingham, United States of America Background: CD47 is a cell-surface ligand overexpressed in various malignancies that binds to SIRPα on effector macrophages to promote tumor cell evasion of phagocytosis. Blockade of this CD47–SIRPα interaction enhances phagocytosis mediated by tumor-targeting antibodies, such as rituximab (ritux). Agents targeting CD47 combined with ritux have demonstrated promising clinical activity in R/R NHL; however, the broad expression of CD47 potentially acts as an antigen sink, reducing anti-tumor activity, and leads to on-target, off-tumor toxicities, including hemolytic anemia. CC-95251 is a novel, fully human immunoglobulin G1 antibody (Ab) that binds to SIRPα on macrophages to potently block the CD47–SIRPα interaction. Aims: The primary objectives of this multicenter, open-label, phase 1, dose-escalation and dose-expansion study were to evaluate the safety and tolerability of escalating doses of CC-95251 + ritux and to define the maximum tolerated dose (MTD) and/or recommended phase 2 dose for the combination in patients (pts) with CD20+ R/R NHL (NCT03783403). Here, we report interim results. Methods: Pts were treated in 28-day cycles (C) with CC-95251 administered intravenously at doses of 3, 10, or 20 mg/kg every week (QW) and with ritux 375 mg/m2 on days (D) 1, 8, 15, and 22 of C1, D1 of C2–5, and D1 of every other cycle C6–24 until disease progression or unacceptable toxicity. Results: As of 5 August 2021, 17 pts had received ≥ 1 dose of CC-95251 + ritux. Median age was 67 (range 30–84) years. Pts had received a median of 3 (range 1–7) prior systemic therapies, with 100% of pts having a prior history of anti-CD20 Ab exposure. Enrolled tumor types included R/R diffuse large B-cell lymphoma in 13 (77%) pts, follicular lymphoma in 2 (12%), and mantle cell lymphoma and marginal zone lymphoma in 1 (6%) pt each. Twelve (71%) pts had disease refractory to any prior line of therapy (LOT), including 10 (59%) refractory to any anti-CD20 Ab-containing regimen, and 9 (53%) to their last LOT. Pts received a median of 4 (range 1–14) cycles of CC-95251. Median duration of treatment was 16.4 (range 2.1–53.1) weeks. There was 1 CC-95251 dose reduction, and 6 (35%) pts experienced ≥ 1 treatment-emergent adverse event (TEAE) leading to CC-95251 dose interruption. MTD has not been reached. Neutropenia and infection were the most common TEAEs for any grade (71% and 59% respectively) and grade ≥ 3 (59% and 29% respectively). Treatment-related adverse events of grade ≥ 3 included neutropenia in 9 pts (53%) and infection in 2 pts (12%); no treatment-related anemia or deaths were reported. Overall response rate in the efficacy evaluable population was 56% (9/16), with 4 (25%) pts achieving a complete response (CR). Of 9 pts refractory to any prior anti-CD20 Ab-containing regimen, 2 pts achieved a CR and 2 pts had stable disease. Median time to response was 7.9 weeks. Duration of response ranged 7.4–28.1 weeks with a CR ongoing in 1 pt. In this dose-escalation study, CC-95251 demonstrated dose-proportional increases in exposure at doses > 3 mg/kg QW and full receptor occupancy at doses ≥ 3 mg/kg. Summary/Conclusion: CC-95251 + ritux demonstrated a manageable safety profile and promising efficacy in pts with heavily pretreated CD20+ R/R NHL. The study is currently enrolling in the dose-expansion phase. S220: GLOFITAMAB INDUCES DURABLE COMPLETE REMISSIONS AND HAS FAVORABLE SAFETY IN PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA AND ≥2 PRIOR THERAPIES: PIVOTAL PHASE II EXPANSION RESULTS M. Dickinson1,*, C. Carlo-Stella2, F. Morschhauser3, E. Bachy4, P. Corradini5, G. Iacoboni6, C. Khan7, T. Wróbel8, F. Offner9, M. Trněný10, S.-J. Wu11, G. Cartron12, M. Hertzberg13, A. Sureda14, D. Perez-Callejo15, L. Lundberg15, J. Relf16, E. Clark16, K. Humphrey16, M. Hutchings17 1Peter MacCallum Cancer Centre, Royal Melbourne Hospital and The University of Melbourne, Melbourne, VIC, Australia; 2Humanitas University and IRCCS Humanitas Research Hospital, Milan, Italy; 3Hôpital Claude Huriez and CHU de Lille, Lille; 4Centre Hospitalier Lyon-Sud, Lyon, France; 5Università degli Studi di Milano and Fondazione Istituti di Ricovero e Cura a Carattere Scientifico (IRCSS) Istituto Nazionale dei Tumori, Milan, Italy; 6Vall d’Hebron University Hospital, Barcelona, Spain; 7Allegheny Health Network, Pittsburgh, PA, United States of America; 8Uniwersytet Medyczny we Wrocławiu, Wroclaw, Poland; 9Universitair Ziekenhuis Gent, Ghent, Belgium; 10Charles University Hospital, Prague, Czechia; 11National Taiwan University Hospital, Taipei, Taiwan; 12CHU de Montpellier, Montpellier, France; 13Prince of Wales Hospital and University of New South Wales, Sydney, NSW, Australia; 14Institut Català d’Oncologia Hospital, Barcelona, Spain; 15F. Hoffmann-La Roche Ltd, Basel, Switzerland; 16Roche Products Ltd, Welwyn Garden City, United Kingdom; 17Rigshospitalet, Copenhagen, Denmark Background: Glofitamab is a T-cell engaging bispecific antibody (Ab) with a novel 2:1 configuration that confers bivalency for CD20 (B cells) and monovalency for CD3 (T cells). In a Phase I/II study (NCT03075696), escalating glofitamab doses were highly active and well tolerated in patients with relapsed/refractory (R/R) B-cell lymphomas, with obinutuzumab pretreatment (Gpt) and Cycle (C) 1 step-up dosing providing effective cytokine release syndrome (CRS) mitigation. Aims: We present pivotal Phase II expansion results in patients with R/R diffuse large B-cell lymphoma (DLBCL) and ≥2 prior therapies. Methods: All patients had DLBCL (DLBCL not otherwise specified [NOS], high-grade B-cell lymphoma, primary mediastinal large B-cell lymphoma, or transformed follicular lymphoma) and had received ≥2 prior regimens, including ≥1 anti-(a) CD20 Ab and ≥1 anthracycline. Intravenous (IV) Gpt (1000mg) was given 7 days before the first glofitamab dose. IV glofitamab was then given as step-up doses on Day (D) 1 (2.5mg) and D8 (10mg) of C1 and at the target dose (30mg) on D1 of C2–12 (21-day cycles). The primary endpoint was complete response (CR) rate assessed by Independent Review Committee (IRC) using Lugano 2014 criteria. CRS was assessed using ASTCT criteria. All patients provided informed consent. Results: As of September 14, 2021, 107 patients had received ≥1 dose of study treatment (median age: 66 years [21–90]; Ann Arbor stage III–IV disease: 74%; IPI score ≥3: 54%; DLBCL NOS: 74%). Median prior therapies was 3 (2–7); 59% had ≥3 prior therapies and 35% had received prior CAR T-cells (CAR-Ts). Most patients were refractory to a prior aCD20 Ab-containing regimen (85%) and to their most recent regimen (85%). Many were refractory to their initial therapy (59%) and to prior CAR-Ts (32%). After a median follow-up of 9 months (0.1–16), overall response and CR rates by IRC were 50.0% and 35.2%, respectively. CR rates were consistent in patients with and without prior CAR-Ts (32% vs 37%). Median time to CR was 42 days (95% CI: 41–48). The majority of CRs (33/38; 87%) were ongoing at data cut. An estimated 84% of complete responders and 61% of responders remained in response at 9 months. At data cut, the projected 12-month overall survival rate was 48%, and 92% of complete responders were alive. These results are consistent with earlier Phase I data in 100 patients treated with target glofitamab doses ≥10mg (CR rate: 34%; estimated 20-month CR rate in complete responders: 72%). CRS occurred in 68% of patients, was primarily associated with the initial doses, and was mostly Grade (Gr) 1 (51%) or Gr 2 (12%); Gr 3 (3%) and Gr 4 (2%) events were uncommon. All but 2 CRS events were resolved at data cut. Glofitamab-related neurologic adverse events (AEs) potentially consistent with immune effector cell-associated neurotoxicity syndrome (ICANS) occurred in 3 patients (all Gr 1–2). No glofitamab-related Gr 5 (fatal) AEs occurred. Glofitamab-related AEs leading to discontinuation were uncommon (3 patients, 3%). Summary/Conclusion: Fixed-duration glofitamab induces durable complete remissions and has favorable safety in patients with R/R DLBCL and ≥2 prior therapies, including those with prior exposure to CAR-Ts. Glofitamab is a promising new therapy for patients with heavily pretreated and/or highly refractory DLBCL. S221: BORTEZOMIB TO R-DHAP COMPARED TO R-DHAP IN RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA ELIGIBLE TO STEM CELL TRANPLANTATION: FINAL RESULTS OF PHASE II RANDOMIZED FIL-VERAL12 A. Chiappella1,*, M. Balzarotti2, B Botto3, A. Castiglione4, F. Cavallo5, S. V. Usai6, M. Zanni7, C. Califano8, F. Re9, C. Ghiggi10, A. Olivieri11, P. Corradini12, A. M. Liberati13, S. Volpetti14, M. G. Michieli15, A Tucci16, G. Gaidano17, M. Tani18, F. Ciambelli19, G. Musuraca20, D. Vallisa21, F. Merli22, A. L. Molinari23, A. Tafuri24, V. R. Zilioli25, D. Marino26, C. Stelitano27, S. A. Pileri28, G. Ciccone4, U. Vitolo29 1Hematology, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milano; 2Hematology, Humanitas Cancer Center, Istituto Clinico Humanitas, Rozzano; 3Hematology, AOU Città della Salute e della Scienza; 4Unit of Clinical Epidemiology, AOU Città della Salute e della Scienza e CPO Piemonte; 5Hematology, Department of Molecular Biotechnologies and Health Sciences, Università degli studi e AOU Città della Salute e della Scienza, Torino; 6Hematology, Ospedale Businco, Cagliari; 7Hematology, AO SS. Antonio e Biagio e Cesare Arrigo, Alessandria; 8Oncology and Hematology, Presidio ospedaliero “A. Tortora”, Pagani; 9Hematology, Ospedale Policlinico San Martino S.S.R.L. - IRCCS per l Oncologia, Parma; 10Hematology, Ospedale Policlinico San Martino S.S.R.L. - IRCCS per l Oncologia, Genova; 11Hematology, AOU Ospedali Riuniti, Ancona; 12Chair of Hematology, Università degli Studi di Milano e Hematology, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milano; 13Oncology and Hematology, AO. S. Maria, Terni; 14Hematology, Azienda Sanitaria Universitaria Friuli Centrale, Udine; 15Oncology and Hematology, IRCCS Centro di Riferimento Oncologico, Aviano; 16Hematology, ASST Spedali Civili, Brescia; 17Hematology and Department of Translational Medicine, Università del Piemonte Orientale e AOU Maggiore della Carità, Novara; 18Hematology, Ospedale delle Croci, Ravenna; 19Oncology, AO S. Antonio Abate, Gallarate; 20Hematology, IRCCS Istituto Romagnolo per lo studio dei Tumori “Dino Amadori” – IRST S.R.L., Meldola; 21Hematology, Ospedale Guglielmo da Saliceto, Piacenza; 22Hematology, Azienda Unitа Sanitaria Locale-IRCCS - Arcispedale Santa Maria Nuova, Reggio Emilia; 23Hematology, Ospedale degli Infermi, Rimini; 24Hematology, AO Sant Andrea, Roma; 25Hematology, ASST Grande Ospedale Metropolitano Niguarda, Milano; 26Oncology, IRCCS Istituto Oncologico Veneto, Padova; 27Hematology, Grande Ospedale Metropolitano Bianchi Melacrino Morelli, Reggio Calabria; 28Haematopathology, Istituto Europeo di Oncologia IRCCS, Milano; 29Hematology, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy Background: The standard treatment in patients with diffuse large-B cell lymphoma (DLBCL) relapsed/refractory (R/R) after first line therapy is a cisplatin-containing regimen followed by consolidation with high-dose chemotherapy and autologous stem cell transplantation (auto-SCT) in responsive ones after induction. Bortezomib had proven activity in aggressive lymphomas. Aims: On these bases, the Fondazione Italiana Linfomi designed the FIL-VERAL12 trial, aimed at evaluating whether the addition of bortezomib to rituximab-cisplatin-cytarabine-dexametasone (BR-DHAP) increases complete response rate (CR, according to Lugano 2007 criteria) prior auto-SCT compared to standard R-DHAP. Methods: FIL-VERAL12 was a prospective, multicenter, two-arm randomized phase II trial (NCT01805557).The primary study endpoint was CR after 4 courses of R-DHAP or BR-DHAP, assuming a 30% CR for the standard arm and a 50% CR in experimental arm. Inclusion criteria were: patients aged 18-65 years eligible to high-dose therapy, with R/R DLBCL after first line chemoimmunotherapy. Patients were stratified by relapsed or refractory and randomized 1:1 to receive: a) the standard salvage therapy R-DHAP every 28 days for 4 cycles and b) subcutaneous 1.5 mg/ms bortezomib on days 1 and 4 of each 4-week cycle in addition to the same regimen. Results: From January 2013 to November 2018, 114 patients were screened, and 107 patients that fulfilled the inclusion criteria were enrolled into the trial and randomized to receive R-DHAP or BR-DHAP (54 patients in R-DHAP, 53 in BR-DHAP). Principal clinical characteristics were: median age 57 years (IQR: 48;62); stage III/IV 83 patients (78%); International Prognostic Index (IPI) risk >2 37 (35%). All patients received rituximab and anthracycline-based regimens as first line treatment. Considering the time at relapse, 53 patients (50%) were registered as relapsed (median time at relapse 10.8 months, IQR: 6.9;20.9) and 54 (50%) as refractory (0.9 months, IQR 0.52;1.3). 52 (49%) patients completed the planned 4 cycles of therapy; 55 did not, due to progressive disease in 42, adverse events or clinician decision in 13. At the end of the 4 courses, the pre-auto-SCT response was: CR 29 (27%), Partial Response (PR) 9 (17%); according to arm of randomization, the primary end point was not met, with CR 28% for R-DHAP and 26% for BR-DHAP (p-value 0.563). Fifty patients (44%) performed a consolidation with SCT, 24 in R-DHAP arm and 26 in BR-DHAP arm; auto-SCT was performed in 39 patients and allo-SCT in 11. The addition of bortezomib to standard R-DHAP did not impact the mobilization, with a median number of CD34+ collected of 6.43 x 10^6 cells CD34/kg (IQR: 4.40;9.11) in R-DHAP and 6.78 x 10^6 cells CD34/kg (IQR: 5.00;9.68) in BR-DHAP. Sixty patients died: 49 (82%) due to lymphoma, 1 due to toxicity, 3 due to transplant related mortality, 7 due to other causes. The incidence of adverse events was similar in the two arms, with grade 3-4 haematological toxicities in 96 patients (90%), g3-4 infection in 5 (5%), g3-4 neurotoxicity in 4 (4%). At a median follow-up of 50 months, 2-years PFS was 29% (95%CI: 19.94;41.83) and 41% (27.67;53.84) for R-DHAP and BR-DHAP, respectively; HR 0.65 (0.41;1.02) p 0.062; 2-years OS was 43% (28.98;56.30) and 52% (37.80;64.56) for R-DHAP and BR-DHAP, respectively; HR 0.74 (0.44;1.23) p 0.244. Summary/Conclusion: In the FIL-VERAL12 phase II randomized trial, the addition of bortezomib to R-DHAP did not improve the CR rate pre-auto-SCT of R/R DLBCL patients eligible to high-dose chemotherapy plus SCT; a numerically higher 2-year PFS rate was observed in BR-DHAP arm. S222: MATURE T AND NK CELL LYMPHOMAS CLASSIFIED ACCORDING TO 2016 WHO CLASSIFICATION. A REPORT OF THE INTERNATIONAL PROSPECTIVE T-CELL PROJECT 2.0. M. Federico1,*, Y. Stepanishyna2 3, T. Skrypets2 4, C. S. Chiattone5, M. H. Prince6, A. Pavlovsky7, A. Lymboussakis1, M. Manni1, M. Civallero1, C. A. de Souza8, E. A. Hawkes9, L. Fiad10, R. Nair11, I. Kriachok12, F. Hitz13, O. Kostina14, C. Tomuleasa15, A. Guarini16, S. Luminari1 17 1Surgical, Medical and Dental Department of Morphological Sciences related to Transplant, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia; 2TCP2 Trial Office, TCP2 Trial Office, Modena, Italy; 3Department of Bone Marrow Transplant, National Cancer Institute, Kiev, Ukraine; 4Clinical and Experimental Medicine (CEM), University of Modena and Reggio Emilia, Modena, Italy; 5Santa Casa Medical School, Santa Casa Medical School, Sao Paulo, Brazil; 6Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia; 7Haematology, Fundaleu, Buenos Aires, Argentina; 8Universidade de Campinas (UNICAMP), Universidade de Campinas (UNICAMP), Campinas, Brazil; 9School of Public Health and Preventive Medicine Monash University and Olivia Newton John Cancer Research Institute, Austin Health, Melbourne, Australia; 10Hospital Italiano La Plata, Hospital Italiano La Plata, Buenos Aires, Argentina; 11TATA Medical Center, TATA Medical Center, Kolkata, India; 12Department of Oncohematology, National Cancer Institute, Kiev, Ukraine; 13Department of Oncology/Haematology, Cantonal Hospital, St Gallen, Switzerland; 14North Estonia Medical Centre, North Estonia Medical Centre, Tallinn, Estonia; 15Ion Chiricuta Oncology Institute, Ion Chiricuta Oncology Institute, Cluj Napoca, Romania; 16Haematology and Cell Therapy Unit, IRCCS-Istituto Tumori ‘Giovanni Paolo II’, Bari; 17Hematology Unit, Azienda Unità Sanitaria Locale IRCCS, Arcispedale Santa Maria Nuova IRCCS, Reggio Emilia, Italy Background: Mature T and NK-cell lymphomas represent a heterogeneous group of rare lymphoid disorders arising from mature T cells of post-thymic origin. In 2018 the International T-cell non-Hodgkin’s Lymphoma Study Group launched the T-cell Project 2.0. Here we report the distribution of cases of PTCLs registered in the study, based on the diagnosis made locally according to the WHO 2016 classification, and some preliminary information on treatment and outcome. Aims: The aim of the study is a better understanding of this group of rare disorders, capturing a real-life snapshot of the evolving landscape of T-cell lymphoma biology, treatment strategies, and outcome. Methods: The T-cell Project 2.0 (ClinicalTrials.gov Identifier: NCT03964480) is a prospective, longitudinal, international, observational study of patients with PTCLs. Results: Between October 2018 and February 2022, 901 patients with newly diagnosed PTCL were registered by 94 active centers across 17 countries. Distribution of cases according to different subtypes is reported in Figure 1. Overall, the most frequent 6 subtypes account for 93% of cases, and the remaining 7% was represented by few cases of 12 different subtypes. Of note, only 9 cases have been classified according to entities not considered in the previous WHO 2008 classification. With respect to clinical presentation, the median age at diagnosis was 56 years (18-93), 57.4% of the patients were male, the presence of systemic symptoms was reported in 31% of cases, 8.9% had ECOG-PS 3-4, 71% advanced disease and 35.4% bone marrow involvement. At time of data lock, 831 cases had information on initial therapy. Overall, 731 patients (88%) were treated with combination chemotherapy and 125 (15%) were consolidated with high dose therapy and stem cell transplantation. After a median follow-up of 20 months, the 2-year PFS and OS were 49% and 57%, respectively. For patients with PTCL-NOS, AITL, ENKTL, ALCL ALK- and ALCL ALK+, a comparison in terms of PFS was performed for those enrolled in the TCP2 and in the previous TCP1. Interestingly, a statistically significant difference (p=0.009) was observed, with 2-year PFS of 50% and 45% for patients enrolled in the TCP2 and TCP1, respectively. Image: Summary/Conclusion: Regardless the difficulties linked to the COVD-19 pandemic, the TCP2 is recruiting very well and allow us in better understanding the outcome of patients with PTCL classified according to the 2016 WHO in the real world. Moreover, our data show that some improvement in the curability of patients affected by PTCL is emerging. S223: LATE CARDIOVASCULAR TOXICITY AFTER HIGH-DOSE CHEMOTHERAPY FOR LYMPHOMA: A DANISH POPULATION-BASED STUDY S. Husby1,*, J. Baech2, T. Trab2, J. Gørløv3, J. M. Jørgensen4, S. Gudbrandsdottir5, M. T. Severinsen2, K. Grønbæk3, T. S. Larsen6, P. Brown3, L. H. Jakobsen2, K Kragholm7, T. C. El-Galaly2 1Hematology, Aalborg University hospital, Copenhagen N; 2Hematology, Aalborg University hospital, Aalborg; 3Hematology, Rigshospitalet, Copenhagen N; 4Hematology, Aarhus University Hospital, Aarhus; 5Hematology, Zealand University Hospital, Roskilde; 6Hematology, Odense University Hospital, Odense; 7Cardiology, Aalborg University hospital, Aalborg, Denmark Background: Salvage chemotherapy followed by high-dose chemotherapy (HDT) and autologous stem cell transplantation (ASCT) are standard treatments for younger patients with relapsed/refractory aggressive diffuse large B-cell lymphoma (DLBCL) and Hodgkin’s lymphoma (HL) as well as for consolidation in 1st line treatment for mantle cell lymphoma (MCL) and peripheral T-cell lymphoma (PTCL). Congestive heart failure (CHF) is a known late complication to lymphoma therapy, but it is unclear how much HDT adds to CHF risk. Delineating late complications to HDT/ASCT is increasingly important due to the availability of alternatives such as CAR-T therapy. Aims: To investigate how HDT/ASCT treatment influences the risk of developing congestive heart failure in a population-based nationwide setting. Methods: Data were obtained from the nationwide Danish lymphoma registry and the Danish National Patient Register. DLBCL, MCL, HL, and PTCL patients treated with HDT between 2001 and 2017 and without CHF prior to HDT were included. Patients were compared to two populations: 1) A matched population of individuals from the general population without prior CHF matched on age and sex in a 1:5 ratio, and 2) A population of lymphoma patients (HL or DLBCL) in clinical remission two years after standard 1st line treatment without HDT. The latter group was chosen due to very low lymphoma-specific mortality. CHF was defined by ICD-10 outpatient clinic and hospital discharge diagnosis codes. The Aalen-Johansen estimator was used to compute the cumulative risks treating death as competing event. This analysis was conducted for CHF and cardiovascular diseases in general, separately (CVD; ischemic heart disease, atrial fibrillation/flutter, ventricular fibrillation/flutter, and CHF). Crude and adjusted cause-specific hazard ratios (HR) were obtained using Cox regression and adjustments were made for age and sex. Results: A total of 958 HDT patients were identified (35% DLBCL, 28% MCL, 21% PTCL, and 16% HL) with a median follow-up of 7.9 years. Almost all patients were previously treated with anthracycline (97.8%). The risk of both CHF and CVD was significantly increased in patients treated with HDT compared to a matched background population (p<0.001; Fig. 1a, 1b). The risk of CHF was more pronounced in HDT-treated males compared to females (10-year incidence 7.8% vs 3.8%, respectively, Fig. 1c and Fig. 1d); however, the risk was significantly increased for both sexes (p<0.001 for both). Multivariable Cox regression adjusted for sex and age confirmed the increased risk of CHF for HDT patients. The risk was increased both when compared to the matched background population (adjusted HR 6.40) and to lymphoma patients not treated with HDT (adjusted HR 2.54). This is intriguing since HDT patients are generally considered more fit than unselected lymphoma patients. Comparison of comorbidities between the groups is ongoing. In the HDT group, the major risk factors for CHF were older age at the time of HDT (HR 1.20 per decade), and male sex (HR 2.15). Image: Summary/Conclusion: The risk of CHF and CVD in general is significantly increased for HDT treated patients compared to both a matched background population and patients treated with 1st line therapy without consolidating HDT, despite likely selection of healthy patients to HDT. Major risk factors were male sex and age. These findings are important when considering the possible use of new therapies such as CAR-T cell therapy which have a different safety profile. S224: PRELIMINARY RESULTS OF A PHASE II STUDY OF ORELABRUTINIB IN COMBINATION WITH ANTI-PD-1 MONOCLONAL ANTIBODY IN REFRACTORY OR RELAPSED PRIMARY CNS LYMPHOMA Y. Zhang1,*, W. Wang1, D. Zhao1, Y. Zhang2, W. Zhang1, D. Zhou1 1Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College; 2Department of Hematology, Beijing Longfu Hospital, Beijing, China Background: The survival outcomes of patients with relapsed/refractory (R/R) primary central nervous system lymphoma (PCNSL) remain extremely poor and there are no approved therapies or widely accepted “standard-of-care” approaches. Previous studies showed that Bruton tyrosine kinase (BTK) inhibitor and anti-programmed death protein-1(PD-1) monoclonal antibody have significant activities in R/R PCNSL, respectively. The combination of BTK inhibitor and anti-PD-1 monoclonal antibody demonstrated synergistic effects both in vivo and in vitro in diffuse large B cell lymphoma, but no clinical data is currently available for PCNSL. Orelabrutinib is a new-generation BTK inhibitor with high CSF concentration and sintilimab is a new-generation anti-PD-1 monoclonal antibody. Aims: To evaluate the safety and efficacy of orelabrutinib combined with sintilimab in patients with R/R PCNSL. Methods: The prospective, multicenter, single-arm phase II study (NCT04899427) enrolled immunocompetent adult patients with R/R PCNSL and eligible organ functions. The patients received once daily orelabrutinib (150mg) in combination with sintilimab (200mg on day 1 of each cycle) every 3 weeks per cycle up to two years or until disease progression, intolerable toxicity, or death. The primary objective was the overall response rate (ORR; defined as partial response [PR] or better) after 4 cycles. Other endpoints mainly included 1-year progression free survival (PFS) rate, time to response (TTR) and safety. Results: The data cut-off date was February 25, 2022. Thirteen patients were enrolled from March 2021 to January 2022, with a median follow-up of 7.0 (1.5-10.5) months. Median age was 61 yr (range 48 to 71) and 6 (46.1%) were male. The median lines of prior treatment were 2 (range 1 to 4). All the patients were high-dose methotrexate treated, seven of those (53.8%) were refractory to the last treatment. Ten patients completed 4 cycles of the experimental regimen while 3 patients ended of treatment in the first 2 cycles due to disease progression. The toxicities were quite mild, with one grade 3 adverse event (AE) of interstitial pneumonitis related pneumocystis carinii infection. No other Grade 3-4 hematological or non-hematological AE was reported. All patients were evaluable for response. The ORR was 61.5%, and 4 patients achieved complete remission (CR), 1 CRu, and 3 PR. The median TTR was 6 weeks (2 to 4 cycles). No relapses were observed in patients who achieved ORR after 4 cycles. Only 1 patient died of disease progression (Fig. 1). The estimated median1-year PFS rate was 67.7% (Fig. 2). Plasma and CSF sample from 4 of 13 patients were collected at 2 h after 15 days orelabrutinib administration. The median CSF concentration of orelabrutinib was 28.7ng/ml (rang 11.8 ng/ml to 52.7 ng/ml). The median CSF/plasma free ratio was 59.8%(rang 46.09% to 86.67%). Image: Summary/Conclusion: The combination of orelabrutinib and sintilimab showed a high ORR and a rapid onset of response in patients with r/r PCNSL with well-tolerated toxicities. Although preliminary, these results supported the use of BTK inhibitor plus anti-PD-1 monoclonal antibody. More clinical data will be updated from this ongoing study. S225: CIRCULATING TUMOR DNA IS A PROGNOSTIC BIOMARKER AT BASELINE AND IMPROVES THE ACCURACY OF INTERIM PET IN CLASSIC HODGKIN LYMPHOMA M. C. Pirosa1,*, A. Bruscaggin1, L. Terzi di Bergamo1, M. Salehi1, K. Pini1, V. Spina1, S. Bocchetta1, A. Condoluci1, G. Forestieri1, D. Piffaretti1, J. Marques De Almeida1, S. Annunziata2, F. Bergesio3, E. Borsatti4, P. Bulian5, S. Chauvie3, C. Marco6, D. T. Martina7, G. Bernhard8, M. Kurlapski9, A. Moccia10, R. Moia11, A. Rinaldi12, M. Rodari13, G. Romanowicz14, G. M. Sacchetti15, A. Stasia8, A. Stathis10, G. Stüssi8, I. Zangrilli16, L. M. Larocca17, A. Pinto18, A. Santoro7, F. Cavalli1, E. Zucca10, V. Gattei19, J. M. Zaucha9, C. Carlo-Stella7, S. Hohaus16, G. Gaidano11, L. Ceriani6, D. Rossi1 1Division of Experimental Hematology, Institute of Oncology Research, Bellinzona, Switzerland; 2Institute of Nuclear Medicine, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome; 3Department of Medical Physics, Santa Croce e Carle Hospital, Cuneo; 4Nuclear Medicine, IRCCS CRO; 5Clinical and Experimental Onco Hematology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy; 6Clinic of Nuclear Medicine and Molecular Imaging, Imaging Institute of Southern Switzerland, Bellinzona, Switzerland; 7Humanitas Cancer Center, Humanitas Clinical and Research Center, Milan, Italy; 8Division of Hematology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 9Department of Hematology and Transplantology, Medical University of Gdańsk, Gdańsk, Poland; 10Clinic of Medical Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 11Division of hematology, University of Eastern Piedmont, Novara, Italy; 12Genomics Facility, Institute of Oncology Research, Bellinzona, Switzerland; 13Unit of Nuclear Medicine, Humanitas Clinical and Research Center, Milan, Italy; 14Department of Nuclear Medicine, Medical University of Gdańsk, Gdańsk, Poland; 15Nuclear Medicine, “Maggiore della Carità ” Hospital, Novara; 16Department of diagnostic imaging, oncological radiotherapy and hematology, Fondazione Policlinico Universitario Agostino Gemelli IRCCS; 17Division of Pathology, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome; 18Hematology‐Oncology and Stem Cell transplantation Unit, National Cancer Institute, Fondazione “G. Pascale” IRCCS, Naples; 19Clinical and Experimental Onco Hematology Unit, IRCCS CRO, Aviano, Italy Background: Classic Hodgkin lymphoma (cHL) has few pre-treatment prognostic biomarkers. Circulating tumor DNA (ctDNA) is a metrics of tumor volume and inflammation, which are both prognostic in cHL, it improves the accuracy of treatment response assessment, which is an unmet need at the interim timepoint in cHL, and it allows to accurately genotype the disease, which has technical hurdles if done of the tumor biopsy in cHL. Aims: The study aims at addressing the following questions: i) is pre-treatment ctDNA load a metrics that captures both tumor and inflammation extents in a single, measurable and radiation-free test? Can the integration of ctDNA with interim PET (iPET) improve accuracy of the sole iPET in predicting treatment outcome? Can ctDNA identify molecular groups with phenotype- and outcome-associated signatures? Methods: IOSI-EMA003 (NCT03280394) is a prospective, observational, multi-center study. The study recruited adult patients with previously untreated cHL. Blood samples were collected during staging and disease response assessment at the same time of PET scan acquisition. PET scans were centrally and blindly reviewed by a panel of nuclear medicine physicians. ctDNA was genotyped and quantified by phased variant-enhanced-LyV4.0 ctDNA CAPP-seq assay. Interim molecular response (iMR) was defined as lack tumor reporters in cfDNA collected at the interim timepoint (sensitivity 10-4). Results: A total of 215 patients were recruited. Median follow-up was 30 months. The full analysis dataset was divided into training (N=135) and validation (N=80) cohorts by using a random sample procedure. In the training cohort, ctDNA load directly associated with inflammation (B symptoms, elevated ESR), but not with disease burden: total metabolic tumor volume (TMTV), GHSG stage. The optimized threshold of pretreatment ctDNA load to stratify PFS was 850 hGE/mL of plasma (Figure 1A). In multivariable analysis, pretreatment ctDNA levels remained prognostic for PFS when controlling for either GHSG stage, TMTV, B symptoms or ESR. The validation cohort confirmed that patients with higher pretreatment ctDNA levels had inferior PFS (Figure 1B). Among intermediate and advanced stage patients, the PFS of patients with positive iPET was significantly inferior (Figure 1C). iMR was achieved in 54% patients. Patients who did not achieved iMR had a lower PFS. iMR combined with iPET had a statistically significant superior accuracy for the anticipation of progression or relapse compared to iMR or iPET alone (Figure 1D). To identify molecular subgroups within cHL, we focused on fragmentation profiles of cfDNA. cfDNA fragmentome can comprehensively represent both genomic and chromatin characteristics. Pre-treatment cfDNA fragmentation patterns were characterized by a more prominent mononucleosomal fragments abundance in 51% of patients (mono-nucleosomal cluster), whereas 33% of patients had a more prominent shift towards submono-nucleosomal fragment lengths (submono-nucleosomal cluster) (Figure 1E). Patients belonging to the submono-nucleosomal cluster had different clinical and biological nuances, including advanced stage, B-symptoms, elevated ESR, higher TMTV and total lesion glycolysis, higher mutation load, higher circulating immune suppressive cytokines and chemoattractants for monocytes and Th-cells. Accordingly, patients with submono-nucleosomal cluster had a lower PFS. Image: Summary/Conclusion: ctDNA is a validated prognostic biomarker of cHL, can improve the accuracy of interim response assessment and provide the bases for a molecular classification of cHL. S226: LOSS OF NR4A1 IS ASSOCIATED WITH HIGHER EXPRESSION OF IMMUNE CHECKPOINT COMPONENTS AND REDUCED T CELL-MEDIATED LYMPHOMA CELL KILLING IN AGGRESSIVE LYMPHOMA K. Pansy1,*, K. Fechter1, A. Arra2, M. Szmyra1, S. Haingartner1, A. Ramsay3, H. Greinix1, C. Beham-Schmid4, P. Neumeister1, A. Deutsch1 1Division of Hematology, Medical University of Graz, Graz, Austria; 2Clinic for Experimental Pedriatics, Otto-von-Guericke-Universität, Magdeburg, Germany; 3Lymphoma Immunology research group, Kings ‘s College, London, United Kingdom; 4Institute of Pathology, Medical University of Graz, Graz, Austria Background: Aggressive lymphomas represent the most common type of lymphoid malignancies with a five-year survival rate of 60%. Despite effective initial treatment, one-third of all patients will experience a relapse, warranting more research to discover novel therapeutic strategies. We recently detected a significant lower expression of NR4A1 that correlates with a poor lymphoma-specific survival. Furthermore, ectopic expression of NR4A1 induces apoptosis in vitro and suppresses lymphoma growth in xenografts indicating its tumor suppressive properties. Aims: The aim of this study was to comprehensively study the function of Nr4a1 loss in lymphomagenesis. Methods: Therefore, we intercrossed the EµMyc lymphoma mouse model with the Nr4a1-/- mouse and monitored them until the onset of disease. Furthermore, we transplanted lymphoma cells of EµMyc Nr4a1-/- and EµMyc Nr4a1+/+ mice into immune-competent C57BL/6 mice and immune-deficient Fox Chase SCID beige mice. Furthermore, we determined the expression levels of immune checkpoint components in biopsies of our human diffuse large B cell lymphoma (DLBCL) cohort and correlated them to NR4A1 content. Finally, to investigate the immune-regulatory function of Nr4a1, we performed co-culture cytotoxicity assays using OVA257-264 peptide-pulsed EµMyc Nr4a1+/+ and EµMyc Nr4a1-/- lymphoma cells and Ctla4+/+ and Ctla4-/- OVA targeting CD8+ T cells derived from OT-I mice and measured T cell-mediated lymphoma cell lysis via flow cytometry, respectively. Results: We observed that the loss of Nr4a1 leads to an accelerated lymphomagenesis in vivo, concomitant with increased expression of immune checkpoint components of the Pd1-Pdl1-Pdl2- and Ctla4-Cd80-Cd86-axes. Immuno-competent, but not immune-deficient mice, transplanted with Nr4a1-deficient lymphoma cells exhibited rapid lymphoma development, reduced survival, and upregulation of the immune checkpoint components like in the primary model. Importantly, low NR4A1 expression correlated with high expression of immune checkpoint components in biopsies of our human DLBCL cohort largely resembling our mouse data. To unravel the impact of Nr4a1 on T cell-mediated lymphoma cell killing and on the regulation of the Ctla-4-Cd80-Cd86-axis of aggressive lymphomas, we performed co-culture cytotoxicity assays using OVA peptide-pulsed EµMyc Nr4a1+/+ and EµMyc Nr4a1-/- lymphoma cells and Ctla4+/+ and Ctla4-/- OVA targeting CD8+ T cells. In these experiments, we observed a massively diminished lymphoma cell killing in the EµMyc Nr4a1-/- setting, when we used Ctla4+/+ OT-1 CD8+ T cells. Interestingly, when we used Ctla4-/- OT-1 CD8+ T cells for the cytotoxicity assays, lymphoma cell killing was enhanced in the Nr4a1-/- setting compared to that using Ctla4+/+ OT-1 CD8+ T cells. Image: Summary/Conclusion: Our data suggest that Nr4a1 plays a critical role in regulating the licensing of immune evasion in aggressive lymphomas by regulating immune checkpoint expression. Thus, it might act as a promising target to restore anti-lymphoma immune responses. S227: SBNO2 IS A SPECIFIC DEPENDENCY OF STAT3-DRIVEN T-CELL MALIGNANCIES T. Brandstoetter1,*, B. Maurer1, J. Schmoellerl2, S. Kollmann1, J. Huuhtanen3 4, R. Grausenburger1, S. Mustjoki3 5, J. Zuber2, V. Sexl1 1Dept. of Biomedical Sciences at the Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna; 2Research Institute of Molecular Pathology, Vienna, Austria; 3Hematology Research Unit Helsinki, University of Helsinki and Helsinki University Hospital Comprehensive Cancer Center, Helsinki; 4Department of Computer Science, Aalto University, Espoo; 5Translational Immunology Research program, University of Helsinki, Helsinki, Finland Background: The transcription factor signal transducer and activator of transcription 3 (STAT3) plays important roles in regulating the survival, growth and differentiation of hematopoietic cells, particularly within the lymphoid lineage. Recently, gain-of-function mutations within the SH2 domain of STAT3 have been identified in patients suffering from T-cell large granular lymphocytic leukemia (T-LGLL). The most frequently found hyperactivating mutation represents STAT3Y640F. Aims: Transcription factors are notoriously difficult to target, hence a better understanding of STAT3-dependent transcriptional co-factors and their molecular targets is critical to identify novel therapeutic approaches. Methods: To unravel the molecular mechanisms behind STAT3Y640F-induced malignancies, we developed a murine in vitro model that recapitulates common transcriptional features that are found in CD8+ transformed T-LGLL cells from patients harbouring STAT3 mutations. Gene expression analyses and chromatin occupancy profiling of mutated STAT3Y640F identified a core set of direct transcriptional targets, which were interrogated for their functional relevance in genome-wide CRISPR/Cas9-based loss-of-function screens. Results: Expression of STAT3Y640F blocked differentiation of murine hematopoietic stem and progenitor cells (mHSPC), enhanced their self-renewal capacity and increased proliferation in comparison to wild type STAT3 expressing cells. Mechanistically, the STAT3Y640F mutation led to increased and prolonged activation of STAT3 signalling and decreased their dependence on cytokine stimulation. In contrast to mHSPC expressing wild-type STAT3, the Y640F-mutated variant resulted in a strong inflammatory gene expression signature that was characterized by overactivation of TNF alpha/Interferon gamma pathways, and is conserved in T-LGLL patients harbouring STAT3 mutations. Through genome-wide loss-of-function screens using CRISPR/Cas9 we identified that the transcriptional co-regulator strawberry notch homolog 2 (SBNO2) is an essential direct transcriptional target of STAT3Y640F in transformed mHSPC. SBNO2 was overexpressed in both, STAT3Y640F-transformed mHSPC (versus STAT3WT) and T-LGLL patients harbouring STAT3 mutations (versus T-LGLL patients with wild type STAT3). As seen with loss of STAT3-Y640F expression, loss of SBNO2 also impaired proliferation of transformed mHSPC. Strikingly, global analyses of gene dependency datasets revealed that various human T-cell malignancies that are driven by aberrant STAT3 signalling depend on SBNO2 expression. Summary/Conclusion: Together, our data show that the STAT3/SBNO2 axis could be a promising therapeutic intervention site in STAT3 driven T-cell malignancies. S228: ACTIVATED SUMOYLATION RESTRICTS MHC CLASS I ANTIGEN PRESENTATION TO CONFER IMMUNE EVASION IN LYMPHOMA U. M. Demel1,*, M. Böger1, S. Yousefian2, C. Grunert1, P. Hotz3, S. Haas2, S. Müller3, M. Wirth1, M. Schick1, U. Keller1 1Charité Universitätsmedizin Berlin; 2Max Delbrück Center for Molecular Medicine, Berlin; 3Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt, Germany Background: During the last decade, the implementation of immunotherapies has revolutionized cancer treatment. However, despite the striking success, often only subgroups of patients respond, and loss of the MHC-I antigen presentation machinery (APM) has been identified as a frequent cause of primary and acquired resistance to immunotherapies. Thus, strategies to restore these pathways may enhance the efficacy of immunotherapies. The post-translational protein modification SUMOylation (SUMO) is a crucial regulatory mechanism in the cellular stress response. Activated SUMOylation is regarded as a hallmark of aggressive cancers. Notably, a selective small molecule inhibitor of SUMOylation (SUMOi), subasumstat, is currently tested in clinical trials. Aims: We aimed to identify the implications of activated SUMOylation for lymphoma immune evasion, and to develop a pharmacological intervention strategy to improve cancer immunotherapies. Methods: A multi-OMICs approach was used to interrogate SUMO-driven immune evasion mechanisms in lymphoma. In vitro findings were informed by in vivo studies in lymphoma xenograft models and syngeneic murine tumor models. Single-cell RNA sequencing (CITE-seq) was applied to decipher in vivo SUMOi-driven changes in blood and immune cell subsets. Results: Starting from a targeted screening for SUMO-regulated immune evasion mechanisms, we identified an evolutionarily conserved pathway of MYC-induced SUMOylation, which attenuated the immunogenicity of lymphoma cells. While activated SUMOylation repressed the MHC-I antigen processing and presentation machinery (APM) and enabled lymphoma cells to evade CD8+ T-cell immunosurveillance, pharmacological inhibition of SUMOylation restored T-cell mediated tumor killing. Importantly, SUMOi amplified the activation of the STAT1 pathway in response to IFNg, which is one of the key regulators of cytokine-induced MHC-I expression. Beyond this, we identified a previously unknown mechanism of SUMO-mediated basal repression of MHC-I and established SAFB, a repressor of immune regulators, as a SUMO-regulated mediator of MHC-I suppression that mechanistically links SUMOylation to MHC-I repression. We thus showed that activated SUMOylation converted lymphoma cells into a less immunogenic state that was restored by pharmacological SUMO inhibition, enhancing the susceptibility of lymphoma cells to CD8+ T-cell mediated killing. Moreover, we revealed that activated SUMOylation was associated with lower abundance and activity of tumor-infiltrating T-cells in vivo, proposing SUMOi as a novel therapeutic strategy to enhance efficacy of immunotherapies. Moreover, by means of single cell RNA-seq analysis we accounted SUMOi with a key function in altering the global immune landscape. Summary/Conclusion: In summary, we show that activated SUMOylation allows tumor cells to evade anti-tumor immunosurveillance and establish SUMOi as rational therapeutic strategy for enhancing the efficacy of immunotherapies in lymphoma and other aggressive cancers. S229: CHARACTERISATION OF TUMOR MICROENVIROMENT REMODELLING IN THE PROGRESSION OF AITL WITH SINGLE-CELL RNA SEQUENCING AND IMAGING MASS CYTOMETRY H. Jin1,*, X. Lu1, L. Fan1, L. Cao1, W. Zhang1, J. Li1, Z. Wu1 1Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China Background: Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma (PTCL). AITL is an aggressive malignancy with a poor prognosis, and its clinical manifestations vary greatly among individuals. The current chemotherapy regimens based on anthracycline show limited efficacy, and there is no best rescue treatment for patients with relapsed and refractory (RR) AITL. In addition, the lack of optimal AITL models in vitro greatly limits the basic research on the mechanism of disease occurrence and progression, and also hinders the development of new drugs and preclinical trials. Aims: Our study aims to deeply analyze the tumor heterogeneity and microenviroment remodelling in the progression of AITL at single cell resolution, discovering key molecules of drug resistance and potential theraputic targets. Methods: We detected fresh lymph node samples from initail diagnosed (ID) and relapsed and refractory (RR) AITL patients using single-cell RNA sequencing (scRNA-seq), combined with imaging mass cytometry (IMC) and whole exome sequencing. scRNA-seq was performed to compare the differential transcriptome expression patterns in ID- and RR-AITL samples. IMC was performed to analyze the spatial position relationship and protein expression characteristics of different subgroups in the tumor microenvironment of AITL. Immunofluorescence staining was conducted to detect the expression of relative markers on tissue microarrays in patients with AITL. In addition, AITL patient-derived organoid model was established to study the regulatory role of YY1 and its inhibitors in relapsed and refractory AITL. Results: We found that RR-AITL samples exhibited significant differences in the tumor microenvironment compared with ID patients. ScRNA-seq analysis revealed that transcription factor YY1 was significantly highly expressed in follicular helper T cells (Tfh) of RR-AITL patients, which promoted the proliferation and drug resistance of AITL cells (Fig A-D), consistent with the results of IMC (Fig E,F). The proportion of CD8+ T cells in the RR-AITL sample was reduced, while the proportion of Treg was increased, as well as the depletion of T cells (Fig G). Furthermore, the stemness of B cells in RR-AITL was enhanced and exhibits significant malignant characteristics (Fig H). We also found decreased interaction in RR-AITL samples. B cells and myeloid subgroups may play important roles in the progression of AITL (Fig I,J). As shown in Fig K, EBV+ B cells exhibited a wider and more distribution in RR-AITL (Fig L). Interestingly, EBV+ endothelial cells were presented in both ID- and RR- samples, while the spatial analysis showed that the distance of EBV+ endothelial cells to B cells in RR-AITL was obviously greater than in ID group, and the distance of Tfh to B cells also showed similar result (Fig M,N). Moreover, for the first time, we established AITL patient-derived organoid models that can be stablely cultured in vitro. On this basis, we could further clarify the important roles of transcription factor YY1 in the drug resistance of AITL, evaluate the cytotoxic effect of YY1 inhibitor NP-001 on AITL tumor cells. Image: Summary/Conclusion: In conclusion, our study revealed the differences between initial diagnosed and relapsed /refractory AITL in terms of tumor microenvironment, single-cell transcriptomes, the spatial distributions of different clusters and their interactions features. YY1 may serve as an novel target for drug resistance for RR-AITL patients. These findings may provide a theoretical foundation for improving the clinical treatment of AITL. S230: HUMAN ΒETA-DEFENSIN 2 TREATMENT MODULATES THE INTESTINAL MICROBIOME AND THE ALLOGENEIC T CELL RESPONSE TO LIMIT ACUTE GRAFT-VERSUS-HOST DISEASE T. Rückert1,*, G. Andrieux2 3, M. Boerries2 3, N. M. Woessner4 5 6, S. Minguet4 5 7, S. Doetsch4 5, K. Aumann8, M Schiff1, L. M. Braun1, E. Haring1 4, B. A. Siranosian9, A. S. Bhatt9 10, P. Nordkild11, J. Wehkamp12, B. A. H. Jensen13, J. Duyster1 3, R. Zeiser1 3 14, N. Köhler1 14 1Department of Medicine I; 2Institute of Medical Bioinformatics and Systems Medicine, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg; 3German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Freiburg; 4Faculty of Biology; 5Signalling Research Centres BIOSS and CIBSS; 6Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg; 7Institute for Immunodeficiency, Center for Chronic Immunodeficiency (CCI); 8Institute of Surgical Pathology, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; 9Department of Genetics; 10Department of Medicine (Hematology, Blood and Marrow Transplantation), Stanford University, Stanford, United States of America; 11Defensin Therapeutics, Copenhagen, Denmark; 12Department of Internal Medicine I, University Hospital Tübingen, Tübingen, Germany; 13Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; 14CIBSS – Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy option for patients with hematological malignancies. Acute graft-versus-host disease (GVHD) is primarily driven by allogeneic donor T cells and remains a serious cause of morbidity and mortality post allo-HCT. In particular, acute GVHD of the gastrointestinal tract (GI GVHD) is associated with a high mortality rate and treatment options are limited. Human β-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide, which is induced by inflammatory stimuli and possesses both antimicrobial and immunomodulatory functions. Aims: In this study, we aimed to investigate the expression of beta defensins in human and murine GI GVHD and the therapeutic effect of recombinant hBD-2 on acute GVHD development in mouse models. Methods: To investigate the functional role of hBD-2 post allo-HCT, we employed established murine GVHD and graft-versus-leukemia (GVL) models and analyzed the allogeneic T cell response using transcriptome and kinome profiling. Shotgun metagenomic sequencing was used to examine the effect of hBD-2 on the intestinal microbiome. Furthermore, we studied beta-defensin expression in two independent acute GVHD patient cohorts and in mice with acute GVHD. Results: We found that expression of murine beta-defensin 4 (mBD-4), the murine orthologue for hBD-2, was reduced in the colon and ileum of mice developing acute GVHD. Oral treatment of mice with recombinant hBD-2 post allo-HCT reduced weight loss and acute GVHD severity and mortality. Furthermore, hBD-2 treatment affected the intestinal microbial composition, including a shift towards higher abundance of Bacteroides species in hBD-2 treated compared to vehicle treated mice. The changes in the microbiome resulted in reduced neutrophil infiltration of the ileum during acute GVHD induction in hBD-2 treated mice. Additionally, hBD-2 dampened pro-inflammatory Th1 cytokine production (TNF, IFN-gamma) by allogeneic T cells in vivo, while preserving the beneficial GVL effect in two different leukemia models. Mechanistically, oral hBD-2 treatment decreased alloreactive T cell infiltration and the expression of genes involved in T cell receptor (TCR) signaling in the ileum of mice with acute GVHD. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation and metabolism by reducing proximal TCR signaling. In patients with acute GI GVHD, intestinal hBD-2 expression was inadequately induced in response to inflammation at the mRNA and protein level when compared to healthy subjects and patients with ulcerative colitis. Summary/Conclusion: In conclusion, our study demonstrates that hBD-2 reduces acute GVHD severity, likely through its effects in shaping the intestinal microbiota, reducing neutrophil infiltration in the ileum and dampening allogeneic T cell responses. Both human and murine acute GVHD are characterized by a lack of intestinal beta-defensin induction and recombinant hBD-2 represents a potential novel therapeutic strategy to counterbalance endogenous hBD-2 deficiency. S231: GLUCOCORTICOID AND GLYCOLYSIS INHIBITORS COOPERATIVELY ABROGATE ACUTE GRAFT-VERSUS-HOST DISEASE Q. wen1, Z. xu1,*, Y. wang1, M. lv1, Y. song1, Z. lv1, T. xing1, L. xu1, X. zhang1, X. huang1, Y. kong1 1Peking University Institute of hematology, Beijing, China Background: Although glucocorticoids(GCs) are the standard first-line therapy for acute graft-versus-host disease(aGvHD), approximately 50% of aGvHD patients have no response to GCs. The role of T cell metabolism in murine aGvHD has been recently reported. Pharmacological inhibition of glycolysis by targeting 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3) ameliorates aGvHD. However, the question of whether glycolysis is required for the function of activated T cells and whether abnormal glycolysis is involved in the occurrence of human aGvHD need to be elucidated. Moreover, the issue as to whether GCs and metabolism regulators can cooperatively suppress T cell alloreactivity and ameliorate aGvHD remains to be elucidated. Aims: The study aimed to analyse the glucose metabolism profiles of activated T cells in aGvHD patients, and to evaluate the roles of PFKFB3-stimulated glycolysis in alloantigen-activated T cells and aGvHD induction. Furthermore, the study was performed to explore the issue of whether GCs could restore activated T cells by regulating glycolysis and the effect of GCs combined with glycolysis inhibitors on activated T cells. Methods: In this prospective nested case–control study, a total of 15 aGvHD patients at diagnosis and 15 matched non-aGvHD patients. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. Subsequently, to assess whether functional T cell activation is caused by the regulation of PFKFB3, we used lentivirus transduction for the genetic regulation of PFKFB3 in vitro and in a humanized xenogeneic aGvHD model. The effect of GCs and glycolysis inhibitors on activated T cells was further explored in vitro and in a humanized murine model of aGvHD and graft versus leukaemia (GVL). In addition, to evaluate the synergistic effect of GCs, 3PO or their combination, combination index (CI) studies were performed by using the Chou-Talalay method for drug interactions. Results: Increased glycolysis, which was characterized by elevated PFKFB3, as well as higher rates of glucose consumption and lactate production in aGvHD T cells. Importantly, in vitro treatment with glycolysis inhibitor 3PO improved the activity of T cells derived from aGvHD patients through down-regulating glycolytic activity of T cells. Genetic upregulation of PFKFB3 induced T cell proliferation and differentiation into proinflammatory T cells. In a humanized mouse model, PFKFB3-overexpressing T cells aggravated aGvHD, as characterized by high aGvHD clinical scores, pathological scores and rapid lethality. Importantly, our integrated data from patient samples in vitro showed that GCs combined with 3PO decreased proinflammatory T cells and T cell proliferation, reduced glucose consumption, lactate production and PFKFB3 expression compared with the single GCs group. Notably, the average CI values of GCs and 3PO were 0.364 for the synergistic inhibition of IFN-γ synthesis and 0.475 for the synergistic inhibition of proliferation, respectively. In a humanized murine model of aGvHD and GVL demonstrated that GCs and 3PO cooperatively reduced the alloreactivity of T cells and ameliorate aGvHD without a loss of GVL effects. Summary/Conclusion: Our data indicated that glycolysis is critical for T cell activation and the induction of human aGvHD. GCs combined with glycolysis inhibitors demonstrated synergistic effects on reducing T cell alloreactivity and ameliorating aGvHD by regulating T cell glycolysis in vitro and in vivo. Thus, GCs combined with glycolysis inhibitors promise to be a novel therapeutic strategy for aGvHD patients. S232: EXPANDING THE REPERTOIRE OF HLA CLASS I-RESTRICTED MINOR HISTOCOMPATIBILITY ANTIGENS FOR IMMUNE MONITORING AND MODULATION AFTER ALLOGENEIC STEM CELL TRANSPLANTATION K. Fuchs1,*, M. van de Meent1, W. Honders1, C. van Bergen1, J. F. Falkenburg1, M. Griffioen1 1Hematology, LUMC, Leiden, Netherlands Background: Allogeneic stem cell transplantation is given as curative treatment for hematological malignancies, but patients face the risk of relapse of their malignancy as well as Graft-versus-Host Disease. After transplantation, donor T cells recognize polymorphic peptides presented by HLA surface molecules on the patient’s cells. These polymorphic peptides, called minor histocompatibility antigens, are caused by genetic differences in single nucleotide polymorphisms (SNPs) between patient and donor. Dependent on whether the antigen is presented on tumor cells or healthy non-hematopoietic tissues of the patient, donor T cells may induce the favourable Graft-versus-Leukemia effect or Graft-versus-Host-Disease, respectively. Aims: The aim is to identify the dominant repertoire of minor histocompatibility antigens in seven common HLA class I molecules for immune monitoring and modulation after allogeneic stem cell transplantation. Methods: Donor T cells isolated from patients after allogeneic stem cell transplantation are tested for recognition of a new panel of 191 selected B cell lines, which are sequenced in the 1000 Genomes Project. This panel enables the inclusion of seven common HLA class I molecules and increases SNP coverage to 11 million (MAF > 0.01). SNPs that strongly associate with T cell recognition are subsequently validated to encode minor histocompatibility antigens. Results: Using an optimized approach for whole genome association scanning, more than 80 new minor histocompatibility antigens have been found that are presented by seven common HLA class I molecules. Antigens targeted in immune responses after transplantation were often shared between patients, and about 25-30% of antigens were translated in other reading frames than human proteins with known function. Furthermore, the extended repertoire was investigated for T cell frequency and tissue distribution of antigen-encoding genes, pointing towards several antigens as potential targets for immunotherapy. Summary/Conclusion: In conclusion, despite many SNP mismatches between patients and donors, our data demonstrate that the repertoire of minor histocompatibility antigens is confined. As the antigens were identified by an unbiased forward strategy (T cell-to-antigen), our collection provides relevant insight into the various sources of antigens in annotated and unannotated reading frames, which is essential for accurate antigen prediction by reverse strategies (antigen-to-T cell). Furthermore, we more than doubled the number of minor histocompatibility antigens which are fundamental to predict, follow or manipulate immune responses after allogeneic stem cell transplantation to improve clinical outcome of transplanted patients. S233: THERAPEUTIC GENE EDITING OF T CELLS CORRECTS CTLA4 INSUFFICIENCY. T. Fox1,*, B. Houghton1, L. Petersone1, N. Edner1, O. Preham1, E. Waters1, C. Hinze1, A. McKenna1, C. Williams1, A. Kennedy1, A. Pesenacker1, P. Genovese2, L. Walker1, S. Burns1, D. Sansom1, C. Booth1, E. Morris1 1University College London, London, United Kingdom; 2Boston Children’s Hospital, Boston, United States of America Background: Heterozygous mutations in CTLA4 result in an inborn error of immunity (IEI) (also known as primary immunodeficiency) with a severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic stem cell transplantation. The mutational landscape and requirement for tight regulation of CTLA4 make viral gene addition approaches unappealing. Gene editing strategies permit alteration of CTLA4 while retaining the endogenous gene control machinery. Aims: We set out to devise a CRISPR/Cas9/AAV6 gene editing strategy to correct CTLA4 insufficiency in T cells. Methods: We designed several homology directed repair (HDR) editing strategies that would correct the genetic defect. We first assessed correction of an individual point mutation. We then evaluated several universal strategies that enable correction of most disease-causing mutations with a single edit; the first that inserts the CTLA4 cDNA in exon 1, and a second that inserts the CTLA4 cDNA at the 3’end of the first intron of CTLA4. All AAV6 HDR donor templates included a GFP reporter gene to enable easy identification of the edited cells. Results: Superior editing efficiencies were obtained with the intronic approach compared to the other editing strategies and this strategy was then further evaluated. CTLA4 function and expression kinetics were assessed following editing using flow cytometry-based assays. Functional studies using CTLA4 transendocytosis (TE) assays, demonstrated restoration of CD80 and CD86 internalization in the edited CD4+ T cells. Following gene editing, transgene expression kinetics were comparable to healthy control CD4+ T cells. Gene editing of T cells isolated from patients with CTLA4 insufficiency restored CTLA4 expression and rescued transendocytosis of CD80 and CD86 in vitro. Using a similar approach, gene corrected T cells from CTLA4-/- mice engrafted in immunodeficient mice at clinically relevant frequencies and tail vein bleeds were performed 1, 3 and 4 weeks post adoptive transfer. In the mice which received the GFP+ edited cells, a stable population of GFP+ cells was detectable at all timepoints demonstrating in vivo persistence as well as genetic stability. All mice were sacrificed 4 weeks after cell transfer. To assess lymphoproliferation, the cellularity of peripheral lymph nodes and spleen weight were analyzed. Spleen and lymph node size, lymph node cell counts, and spleen weight were all significantly lower in mice which received the edited cells (n=5) compared to non-edited controls (n=5) and there was no significant difference in these parameters between mice who had received edited CTLA4-/- T cells and those who received WT T cells (n=4). Analysis of lymph node and spleen cells confirmed persistence of CTLA4 expression in the edited (GFP+) cells and revealed that a higher proportion of Treg than Tconv had been successfully edited. Summary/Conclusion: Together these data demonstrated that CTLA4 edited T cells survived in vivo, expressed CTLA4 and were able to control the clinical phenotype of CTLA4 insufficiency, providing a powerful proof-of-principle of our T cell GT approach. Our data provide proof-of-concept that gene editing can restore CTLA4 function in T cells demonstrating the potential of this approach to treat CTLA4 insufficiency. A similar approach could be used in other IEIs that are caused by multiple heterozygous mutations. S234: CLINICAL-GRADE MBIL21/4-1BBL EXPANDED NK CELLS EXHIBIT STRONGER COMPETENCE COMPARED WITH PRIMARY NK CELLS AGAINST HCMV INFECTION Q.-N. Shang1 2 3,*, X.-X. Yu1 2 3, Z.-L. Xu1 2, T.-T. Han1 2, J. Xie1 2, Z.-Y. Fan1 2, M. Zhao1 2, X.-H. Cao1 2, X.-F. Liu1 2 3, Y.-H. Chen1 2, M. Lv1 2, Y.-Q. Sun1 2, Y.-J. Chang1 2, Y. Wang1 2, L.-P. Xu1 2, X.-H. Zhang1 2, K.-Y. Liu1 2, X.-Y. Zhao1 2, X.-J. Huang1 2 3 1Peking University People’s Hospital; 2Peking University Institute of Hematology; 3Peking-Tsinghua Center for Life Sciences, Beijing, China Background: Cytomegalovirus (CMV) infection remains a common complication and leads to high mortality in subjects who undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT). Human NK cells are the first lymphocyte recovering after allo-HSCT. Previous studies indicated the early NK cell reconstitution may be protective against development of HCMV infection post HSCT. Our previous data also showed that rapid reconstitution of IFN-γ secreted NK cells at day 15 post transplantation predicted lower HCMV reactivation. However, little is currently known about whether and how NK cells are responsible for preventing HCMV infection post transplantation. NK cell adoptive transfer is a promising method for cancer immunotherapy. Ex vivo mbIL21/4-1BBL expanded NK cells exhibited high cytotoxicity against leukemia cells. Nevertheless, whether expanded NK cells owned higher anti-HCMV function compared with primary NK cells in vitro and in vivo were still unknown. Aims: To investigate whether expanded NK cells owned higher anti-HCMV function compared with primary NK cells in vitro and in vivo and the efficacy of adoptive NK cells infusion to patients post HSCT to prevent HCMV infection. Methods: NK cells were firstly ex vivo expanded by K562-mbIL21/4-1BBL feeder cells. We compared not only the cytotoxicity against HCMV infected fibroblast but also the competence of inhibiting HCMV propagation and reducing HCMV infection between ex vivo expanded NK cells and primary NK cells. Then primary NK cells and expanded NK cells were adoptive infused to HCMV-infected humanized mice to investigate the ability to eliminate HCMV infection in vivo. At last, 20 patients post HSCT were enrolled in our clinical trial to explore the safety and efficacy of adoptive infusion of K562-mbIL21/4-1BBL expanded NK cells to protect from HCMV infection. Results: 1. Most activating receptors, cell adhesion molecule receptors and chemokine receptors exhibited enhanced expression on expanded NK cells (figure 1a). Expanded NK cells showed stronger cytotoxicity against HCMV infected fibroblasts and enhanced abilities to inhibit HCMV propagation compared with primary NK cells in vitro (figure 1b). 2. Both expanded NK cells and primary NK cells showed the ability to migrate to the spleen, liver, and lung and persisted in these target organs in HCMV-infected humanized mice. On day 14 post infusion, expanded NK cells showed higher percentage in tissues than primary NK cells. Moreover, mice with expanded NK cell infusion exhibited more effective HCMV elimination compared with primary NK cell infusion. 3. The cumulative incidence of HCMV and refractory HCMV infection for patients in NK infusion cohort were significantly reduced compared with control cohort (figure 1c). Total HCMV persisting time was shortened (14 (5-24) days vs. 17 (7-56) days, p=0.013). What is more, the absolute number of NK cells was higher for patients in NK cell infusion group than that in control group. Higher quantitative reconstitution, more matured NK cell phenotypes and stronger function were determined in patients with expanded NK cell infusion than those in control group. Image: Summary/Conclusion: Based on in vitro and in vivo studies, our data demonstrated that adoptive NK cells infusion exhibited stronger activities compared with primary NK cells against HCMV infection. S235: A TWO-PART, SINGLE- AND TWO-ARM RANDOMIZED, OPEN-LABEL STUDY TO EVALUATE THE SAFETY, TOLERABILITY AND PHARMACOKINETICS OF THE S1P RECEPTOR MODULATOR KRP203 IN SUBJECTS WITH HEMATOLOGICAL MALIGNANCIES S. dertschnig1,*, J. finke2, D. heim3, U. schanz4, E. holler5, U. holtick6, G. socié7, T. teshima8, C. bucher9, J. passweg10 1Priothera SAS, Saint Louis, France; 2Heamtology, Oncology and SCT, University Freiburg, Freiburg, Germany; 3Hematology, University Hosptial Basel, Basel; 4University Hospital Zürich, Zürich, Switzerland; 5Klinik und Poliklinik Innere Medizin III, Unuversitätsklinikum Regensburg, Regensburg; 6Klinik I für Innere Medizin, Köln, Germany; 7Saint Louis Hospital, Paris, France; 8Hokkaido University, Faculty of Medicine, Sapporo, Japan; 9Universtity Hospital Basel; 10University Hosptial Basel, Basel, Switzerland Background: The success of allogeneic hematopoietic stem cell transplantation (HSCT) is limited by disease relapse. Alloreactive donor T cells have the potential to prevent relapse by the graft-versus-leukemia (GVL) response. The GVL response is essential, however, the same allo-T cells cause acute graft-versus-host disease (aGVHD). T cell trafficking out of lymphoid organs is a major step in raising peripheral immune responses and is regulated by sphingosine-1-phosphate (S1P) gradients. Pre-clinical data showed that pharmacological modulation of S1P receptor (S1PR) signalling by KRP203 sequesters T cells in lymphoid organs and prevents their egress to aGVHD target sites. In murine models, KRP203 efficiently reduced aGVHD. Because T cell function is not suppressed by KRP203, GVL was maintained, resulting in improved survival. Here, we show data of the first clinical trial investigating the safety and tolerability of KRP203 in patients with hematological malignancies undergoing HSCT. Aims: The aim of this study was to evaluate the safety, tolerability and pharmacokinitecs of a S1P receptor modulator towards a novel treatment approach that aims at reducing GvHD while maintaining Graft versus leukemia. Methods: This multicentric, phase Ib, prospective, open label, two-part study evaluated safety, tolerability and pharmacokinetics of KRP203 in intermediate to high-risk patients undergoing HSCT for hematological malignancies. Three treatment arms were investigated: 3mg KRP203+CsA/MTX, 1mg KRP203+CsA/MTX and 3mg KRP203+Tac/MTX. KRP203 was administered from day 1 until day 111 and HSCT was performed on day 11. Results: KRP203 was safe and well-tolerated in these fragile patients. Upon conditioning and KRP203 treatment absolute lymphocyte counts (ALC) were reduced to about 0.2x109/L, engrafted normally and during KRP203 exposure ALC stabilized at <0.7x109/L in all three treatment arms, well below pre-HSCT levels. The treatment effect on ALC counts resolved when KRP203 was stopped on day 111, resulting in blood ALC to pre-HSCT levels. CD4+ and CD8+ T cells, were reduced in peripheral blood in response to KRP203 treatment. CD4+ T cells remained below pre-HSCT level (<100 cells/µl) during the treatment period. CD8+ T cell counts recovered more rapidly, also during treatment. Overall survival probability was highest for subjects in the 3mdg KRP203+CsA arm with the first death reported on day 532. Overall survival at 1 year was 100% in the 3mg KRP203+CsA, 67% in the 1mg KRP203+CsA and 36% in the 3mg KRP203+Tac arm. The median time to relapse was 749 days. 2/6 subjects in 1mg KRP203+CsA, 4/10 subjects in 3mg KRP203+CsA and 4/7 subjects in 3mg KRP203+Tac arm relapsed. The median time to any aGVHD was 55 days. 22% of subjects presented with grade III-IV aGVHD. The median time to any chronic GVHD was 174 days. 57% of subjects presented with moderate and 9% with severe chronic GVHD. Summary/Conclusion: KRP203 is safe and well tolerated and shows promising early clinical outcomes with a limited number of relapses, acute and chronic GVHD. These data support the initiation of a phase 2b trial investigating KRP203 as an adjunctive and maintenance treatment as an adjunctive and maintenance treatment to increase leukemia free survival in adult AML patients undergoing allo-HSCT. S236: RANDOMIZED MULTICENTER PHASE III STUDY OF HAPLO VERSUS HLA-MATCHED UNRELATED DONOR (UD) ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION (ALLO HSCT) FOR PATIENTS OLDER THAN 55 YEARS S. Harbi1, J.-M. Boher2, E. Forcade3, P. Chevallier4, R. Peffault De Latour5, F. Malard6, S. Francois7, A. Charbonnier8, E. Hermet9, C. E. Bulabois10, A. Huynh11, A. Berceanu12, T. Cluzeau13, M. T. Rubio14, S. Furst1, R. devillier15, M. Mohty6, D. Blaise1,* 1hematology; 2DRCI, Institut Paoli Calmettes, Marseille; 3hematology, CHU, Bordeaux; 4hematology, CHU, Nantes; 5Transplant Unit, Hopital Saint Louis; 6hematology, Hopital Saint Antoine, Paris; 7hematology, CHU, Angers; 8hematology, CHU, Amiens; 9hematolgy, CHU, Clermont Ferrand; 10hematolgy, CHU, Grenoble; 11hematology, CHU, Toulouse; 12hematology, CHU, besancon; 13hematology, CHU, Nice; 14hematology, CHU, Nancy; 15hematolgy, Institut Paoli Calmettes, Marseille, France Background: In most situations where allo HSCT is wished a matched sibling donor (MSD) is lacking. The respective advantages of alternative donor such as UD or HaploD are yet to be precisely assessed notably in older population including the search and identification process. Aims: We performed a prospective, multicenter, open-label, randomized controlled trial (NCT02623309) comparing Haplo versus UD search strategies for allo HSCT in pts with hematological malignancies older than 55 years after the absence of MSD was established. Methods: The primary objective was the comparison of Chronic Graft-versus-Host Disease-free and Relapse-free survival (cGRFS) from time of randomization. Conditioning was RIC based on fludarabine (150 mg/m2), IV busulfan (260 mg/m2) for haplo HSCT with the adjunction of thiotepa (5 mg/kg) for UD-HSCT. Pts receiving Haplo-SCT and UD-HSCT received GVHD prophylaxis based on HD-PTCy (100 mg/kg) and ATG (5 mg/kg), respectively. Additionnal prophylaxis consisted in CSA and mycophenolate mofetil for all patients (pts). Results: 108 pts were enrolled, and 106 pts were analyzed with a median follow-up of 27 (14-34) months. Median age was 65 years (55-70). Diseases were myeloid malignancies in 84 pts (79%). DRI was low, intermediate and high in 5(5%), 59(55%) and 42(40%),respectively. 55 and 51 pts were assigned to Haplo and UD group respectively. In case an alternative donor was not identified in due time, a search for another donor was allowed. Of the 106 pts, 77 (73%) pts proceeded to transplant (40 (73%) and 37 (73%) pts in Haplo and UD group respectively). Fifteen (27%) pts in Haplo group did not because of progression (n=9), patient contraindication (n=5), absence of donor (n=1). Likewise 14 (27%) pts in UD group were not transplanted because of progression (n=8), patient contraindication (n=6). Overall median time from randomization to allo-SCT was 76 (21-179) and 95 (37-310) days in haplo and UD group respectively (P=NS). Thity-one (56%) and 26 (51%) pts actually were transplanted according to randomization while 9 (16%) and 11 (22%) pts were transplanted from a UD or a HaploD in haplo and UD search group respectively. Median time from randomization to allo-SCT was 81 (21-288) when the initial desired donor was found and 117 (38-310) days when another donor type was to be searched (p=0,04). In an intent-to-treat analysis from date of randomization, 2-year GRFS, PFS and OS did not differ between the two groups (Haplo vs UD search group: 29% vs 37%, p=0.22; 45 vs 49 %, p=0.56; 50 vs 59%, p=0.47). Overall 42 and 35 pts were actually transplanted from a haploidentical and unrelated donor, respectively whatever the randomization arm. With a median follow up of 24 months after transplant 2-year GRFS and PFS from transplant did not differ between Haplo and UD HSCT: 40% vs 34%, p=0.66; 48%vs 45%, p=0.78). No statistical difference was documented for G2-4 aGVHD, severe cGVHD, NRM and relapse probabilities. Summary/Conclusion: This trial from alternative donor search initiation time-point establishes for the first time that the search for a HaploD or UD conduct to the same outcomes in older pts. However, although 77% of the patients can achieved a transplant with either strategies, only half of the original pts will receive a transplant from the prospected donor type. Outcomes being similar, this may invite to initially perform search for both type of donors to avoid delay with secondary searches. S237: ORCA-T, AN ENGINEERED ALLOGRAFT, RESULTS IN HIGH GVHD-FREE AND RELAPSE-FREE SURVIVAL FOLLOWING MYELOABLATIVE CONDITIONING FOR HEMATOLOGICAL MALIGNANCIES E. Meyer1,*, A. Pavlova1, A. Gandhi2, R. Hoeg3, C. Oliai4, R. Mehta5, S. Srour5, J. McGuirk6, E. Waller7, N. Fernhoff8, M. S. Killian8, J. Mcclellan8, A. Putnam8, B. Shaw9, M. Abedi10, R. Negrin11 1BLOOD AND MARROW TRANSPLANTATION AND CELLULAR THERAPY, Stanford Hospital and Clinics, Stanford, CA; 2Department of Medicine, Division of Hematology/Medical Oncology, Oregon Health and Science University, Portland, OR; 3Department of Medicine, Division of Bone Marrow Trasnplant, University of California, Davis, Comprehensive Cancer Center, Davis, CA; 4Department of Medicine, Blood and Bone Marrow Transplant Program, University of California, Los Angeles, Los Angeles, CA; 5Department of Stem Cell Transplantation and Cellular Therapy, Division of Cancer Medicine, MD Anderson Cancer Center, Houston, TX; 6Department of Hematologic Malignancies and Cellular Therapeutics, University of Kansas Medical Center, Kansas City, KS; 7Bone Marrow and Stem Cell Transplant Center, Winship Cancer Institute of Emory University, Atlanta, CA; 8Orca Bio, Menlo Park, CA; 9Department of Medicine, Division of Hematology and Oncology, BMT Program, Medical College of Wisconsin, Wilwaukee, WI; 10Department of Medicine, Division of Bone Marrow Transplant, University of California, Davis Medical Center, Davis, CA; 11Department of Blood and Marrow Transplantation and Cellular Therapy, Stanford Hospital and Clinics, Stanford, CA, United States of America Background: Rates of graft versus host disease (GVHD) and non-relapse mortality (NRM) following myeloablative allogeneic hematopoietic stem cell transplant (MA-alloHSCT) remain unacceptably high. Strategies to reduce GVHD and NRM have been compromised by limited efficacy or increased risk of infection and relapse, emphasizing the need for new approaches that holistically improve outcomes. Orca-T is a high-precision, allogeneic investigational cell therapy product comprised of stem and immune cells that leverages highly purified, polyclonal donor regulatory T cells to control alloreactive immune responses, reducing the need for pharmacologic GVHD prophylaxis. Orca-T is produced in a central GMP facility and has been successfully scaled to clinical centers throughout the U.S. Aims: The aim of these studies was to evaluate the safety and efficacy of Orca-T in patients with hematologic malignancies. Methods: As of 28 February 2022, 138 patients with high-risk hematologic malignancies have received Orca-T in a single-center Phase 1-2 study (NCT01660607, n=41) and a multicenter Phase 1b study (NCT04013685, n=97) and have ≥ 100 days of follow-up. Informed consent was obtained from all transplant recipients and donors, and the studies received IRB approval from participating institutions. Orca-T was produced from G-CSF-mobilized peripheral blood (PB) from matched related donors (n=72), matched unrelated donors (n=62), or mismatched unrelated donors (MMUD, n=4). Median follow-up for recipients was 300 days (range: 27-1941). Median age was 49 years, and diagnoses included AML (43%), ALL (27%), MDS (10%), myelofibrosis (7%), and CML (6%). Patients received myeloablative conditioning (busulfan-based, n=109; TBI-based, n=27; BCNU, n=2) followed by GVHD prophylaxis with either single-agent tacrolimus (tac, n=127), sirolimus (n=7), or tac plus mycophenolate (n=4, MMUD). A contemporaneous CIBMTR-based control arm was obtained that consisted of patients with similar diagnoses who received myeloablative alloHSCT from a PB source followed by tac/methotrexate PPX. Results: Orca-T was successfully manufactured, distributed, and infused for all patients enrolled. Overall time from donor centers to recipient centers was under 60 hours in all cases. Median time to neutrophil engraftment was 13 days. The rates of grade ≥ 3 acute GVHD in the first 180 days and moderate to severe chronic GVHD through 1 year were low with Orca-T at 4% and 5%, respectively. NRM was infrequent at 4% through 1 year. Orca-T exhibited GRFS of 71% & OS of 90% at 1 year. No formal comparison to the CIBMTR cohort was performed, and a Phase 3 study has been initiated to confirm these findings. Longitudinal immune reconstitution data was collected and will be presented. Clinical data is summarized in Table 1. *MAGIC Criteria **NIH Consensus Grading Image: Summary/Conclusion: Results from patients treated with Orca-T, a high-precision Treg-engineered donor product, suggest a reduction in cGVHD, improved GRFS, and low toxicity relative to historic data. Orca-T manufacturing was accomplished with consistent and reliable cell manufacturing and distribution across a wide geographic area. A multicenter randomized-control trial phase 3 trial comparing Orca-T to SOC has been initiated. S238: MATCHED RELATED VERSUS UNRELATED VERSUS HAPLOIDENTICAL DONORS FOR ALLOGENEIC TRANSPLANTATION IN AML PATIENTS ACHIEVING FIRST COMPLETE REMISSION AFTER TWO INDUCTION COURSES: A STUDY FROM THE ALWP/EBMT A. Nagler1,*, M. Labopin2, S. Mielke3, J. Passweg4, D. Blaise5, T. Gedde-Dahl6, J. J. Cornelissen7, U. Salmenniemi8, I. Yakoub-Agha9, P. Reményi10, G. Socié11, G. Van Gorkom12, H. Labussière-Wallet13, X.-J. Huang14, M. Thérèse Rubio15, J. L Byrne16, C. Craddock17, L. Griskevicius18, F. Ciceri19, M. Mohty20 1Hematology Division, Sheba Medical Center, Ramat-Gan, Israel; 2EBMT ALWP office, Hôpital Saint-Antoine, Paris, France; 3Hematology, Karolinska University Hospital; 4Department of Cell Therapy and Allogenic Stem Cell Transplantation (CAST), Department of Laboratory Medicine (LabMED), Karolinska University Hospital and Institutet, Karolinska Comprehensive Cancer Center, Stockholm, Sweden; 5Programme de Transplantation & Therapie Cellulaire, Centre de Recherche en Cancérologie de Marseille, Paoli Calmettes, Marseille, France; 6Hematology Dept., Section for Stem Cell Transplantation, Oslo University Hospital, Rikshospitalet, Clinic for Cancer Medicine, Oslo, Norway; 7Department of Hematology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, Netherlands; 8Stem Cell Transplantation Unit, HUCH Comprehensive Cancer Center, Helsinki, Finland; 9CHU de Lille LIRIC, INSERM U995, Université de Lille, Lille, France; 10Haematology and Stem Cell Transplant, Dél-pesti Centrumkórház – Országos Hematológiai és Infektológiai Intézet, Budapest, Hungary; 11Hematology – BMT, Hopital St. Louis, Paris, France; 12Department of Internal Medicine, Division of Hematology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, Netherlands; 13Service Hematologie, Centre Hospitalier Lyon Sud, Lyon, France; 14Institute of Haematology, Xicheng District, Peking University People´s Hospital, Beijing, China; 15CHRU BRABOIS, Vandoeuvre les Nancy, Nancy, France; 16Nottingham University, Hucknall Road, Nottingham; 17Haematology, University Hospital Birmingham NHSTrust, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, United Kingdom; 18Haematology, Oncology & Transfusion Center, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania; 19Hematology and BMT, San Raffaele Scientific, Milan, Italy; 20EBMT ALWP office Paris, Saint Antoine Hospital, Paris, France Background: Transplantation (HSCT) outcome is significantly inferior in AML patients (pts) who required 2 rather than 1 induction course to achieve CR1, with a higher relapse (RI) and lower leukemia-free (LFS) and overall survival (OS) and lower graft-versus-host disease (GVHD)-free, relapse-free survival (GRFS). Aims: To allocate the best donor for HSCT in AML pts achieving CR1 after 2 inductions comparing matched related sibling donors (MSD) with 9-10/10 HLA compatible unrelated donors (UD) and non-T cell depleted haploidentical (Haplo) donors, respectively. Methods: This was a retrospective analysis including adult pts aged ≥18 years with AML undergoing HSCT while in CR1 achieved after 2 inductions during the period 2010-2020. Allografts were from MSD, UD or Haplo donors. Multivariate analysis (MVA) adjusting for potential confounding factors was performed using a Cox’s proportional-hazards regression model for main outcomes. Results: 1295 pts were included: MSD (n=428), UD 10/10 (n=554), UD 9/10 (n=135), Haplo (n=178). Median follow up was 48.4, 46.0,40.5 and 35.2 months, respectively. Median age was 52.1, 53.8, 52.2 and 49.5 years, respectively. The 4 groups did not differ with respect to pts’ gender, type of AML, cytogenetics, and performance status. Haplo transplants were performed more recently, with more BM grafts and lower frequency of pt CMV seropositivity. Post-transplant cyclophosphamide (PTCy) was the main anti GVHD prophylaxis in HSCT from Haplo donors, while in vivo T-cell depletion was more frequently used in transplants from UD. Myeloablative conditioning was less frequently used in transplants from UD and time from diagnosis to HSCT was shorter in transplants from MSD. Cumulative incidence of ANC >0.5x109/L at day 30 was 98.1%, 98.5%, 95.5% and 92.1%, respectively (global P value=0.03). Univariate analysis of 2- and 5-year outcomes after transplantation are given in Table 1. On MVA, acute (a) GVHD II-IV but not III-IV was higher in all groups compared to MSD: UD 9/10, HR=2.53; 95% CI 1.68-3.82, P<0.0001; UD 10/10, HR=1.96; 95% CI 1.42-2.69, P<0.0001 and Haplo HR=2.15; 95% CI 1.41-3.3, P=0.0004. Incidence of extensive chronic (c) GVHD was significantly higher in UD 9/10 (HR=2.52; 95% CI 1.55-4.11, P=0.0002) and UD 10/10 (HR=1.48; 95% CI 1.03-2.13, P=0.036) and cGVHD all grades was higher in UD 9/10 vs MSD (HR=1.77; 95% CI 1.26-2.49, P=0.0009), while it did not differ in Haplo transplants. Non-relapse mortality (NRM) was higher in HSCT from UD 10/10, 9/10 and Haplo donors compared with those from MSD (HR= 1.75; 95% CI 1.16-2.63, P= 0.007, HR =2.22; 95% CI 1.29-3.83, P= 0.004 and HR=2.53; 95% CI 1.47-4.34, P=0.0008), respectively. RI, LFS and OS did not differ significantly between donor types: RI HR=0.84, P=0.18; HR=0.9, P=0.59 and HR=0.76, P=0.17 in UD 10/10, UD 9/10 and Haplo vs MSD, respectively. LFS HR=1.02, P=0.87; HR=1.15, P=0.35 and HR=1.11, P=0.49 and OS HR=1.12, P=0.3, HR=1.27, P=0.14 and HR=1.33, P=0.081, respectively. Finally, GRFS was lower in HSCT from UD 9/10 (HR=1.56, 95% CI 1.20-2.03, P<0.001) but not in those from UD 10/10 (HR=1.13, P=0.22) and Haplo donors (HR=1.12, P=0.43) compared to MSD, respectively. Main cause of death in all groups was original disease, followed by infections, and GVHD. Image: Summary/Conclusion: In AML patients undergoing allogeneic transplantation in CR1 achieved after two induction courses which has been shown to be a poor prognostic factor, and those who do not have an MSD, HLA matched UD and Haplo donors are comparable alternatives. 40-50% of these pts achieved long term LFS. S239: COMPARABLE LONG-TERM OUTCOMES BETWEEN UPFRONT HAPLOIDENTICAL AND IDENTICAL SIBLING DONOR HSCT IN APLASTIC ANEMIA: A NATIONAL REGISTRY-BASED STUDY Z. xu1,*, L. xu1, D. wu2, S. wang3, X. zhang4, R. xi5, S. gao6, L. xia7, J. yang8, M. jiang9, X. wang10, Q. liu11, J. chen2, M. zhou3, X. huang1 1Peking university institute of hematology, Beijing; 2The First affiliated Hospital of Soochow University, Soochow; 3Guangzhou First People’s Hospital, Guangzhou; 4Xinqiao Hospital affiliated to Third Military Medical University, Chongqing; 5General Hospital of Lanzhou Military Region of PLA, Lanzhou; 6The First Hospital of Jilin University, Changchun; 7Xiehe Hospital; 16affiliated to Huazhong University of Science and Technology, Wuhan; 8Changhai Hospital affiliated to Second Military Medical University, Shanghai; 9The First affiliated Hospital of Xinjiang Medical University, Urumchi; 10Shandong Provincial Hospital, Jinan; 11Nanfang Hospital affiliated to Southern Medical University, Guangzhou, China Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a curative option for severe aplastic anemia (SAA), and transplantation from identical sibling donors (ISDs) has been recommended as a first-line treatment. Aims: Haploidentical donor (HID) transplantation for SAA has made great advances; thus, an increased role of HID-SCT in SAA should be considered if an ISD is unavailable. Methods: We performed a national registry-based analysis comparing long-term outcomes in the upfront HID or upfront ISD SCT setting. Results: A total of 342 SAA patients were enrolled, with 183 patients receiving HID SCT and 159 receiving ISD SCT. The estimated 9-year overall survival (OS) and failure-free survival (FFS) were 87.1±2.5% and 89.3±3.7% (P = 0.173) and 86.5±2.6% vs. 88.1±3.8% (P = 0.257) for patients in the HID and ISD SCT groups, respectively. Transplantation from HID or ISD SCT has greatly improved quality of life (QoL) levels post-HSCT compared to pre-HSCT. The occurrence of chronic graft versus host disease was the only identified adverse factor affecting each subscale of QoL. Physical and mental component summaries in adults as well as physical, mental, social, and role well-being in children were all similar between HID and ISD SCT at 5-year points. At the last follow-up, the proportion of returning to society was comparable between the HID and ISD groups, showing 78.0% versus 84.6% among children and 74.6% versus 81.2% among adults. A total of 342 SAA patients were enrolled, with 183 patients receiving HID SCT and 159 receiving ISD SCT. The estimated 9-year overall survival (OS) and failure-free survival (FFS) were 87.1±2.5% and 89.3±3.7% (P = 0.173) and 86.5±2.6% vs. 88.1±3.8% (P = 0.257) for patients in the HID and ISD SCT groups, respectively. Transplantation from HID or ISD SCT has greatly improved quality of life (QoL) levels post-HSCT compared to pre-HSCT. The occurrence of chronic graft versus host disease was the only identified adverse factor affecting each subscale of QoL. Physical and mental component summaries in adults as well as physical, mental, social, and role well-being in children were all similar between HID and ISD SCT at 5-year points. At the last follow-up, the proportion of returning to society was comparable between the HID and ISD groups, showing 78.0% versus 84.6% among children and 74.6% versus 81.2% among adults. Image: Summary/Conclusion: These data suggest the recommendation for upfront transplantation with a haploidentical donor for SAA patients in the absence of an ISD, especially in experienced transplant centers. S240: CD7 CHIMERIC ANTIGEN RECEPTOR T CELLS BRIDGING TO ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION IMPROVED DISEASE-FREE SURVIVAL IN REFRACTORY/RELAPSED T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA Z. Li1,*, X. Wang1, X. Wen1, T. Xu1, Y. Song1, T. Wu1 1BEIJING GOBROAD BOREN HOSPITAL, Beijing, China Background: Our previous clinical study has shown that the patients with refractory/relapsed T-cell acute lymphoblastic leukemia (r/r T-ALL) have achieved 90% complete remission (CR) with donor-derived CD7 chimeric antigen receptor T cells (CART), but some cases have developed donor-recipient mixed chimerism and remarkable pancytopenia (Pan J. et al. JCO 2021). It is crucial to quickly bridge to allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematopoietic reconstitution. Aims: In current study, the safety and efficacy of allo-HSCT in r/r T-ALL after CD7-CART are investigated. Methods: From February 2018 to December 2021, total 57 patients with r/r T-ALL who underwent allo-HSCT in our hospital were included. The median age was 13(4-69) years old. Extramedullary disease (EMD) was found in 32 patients (56.1%). Somatic gene mutations (NOTCH 18, IL7R 3, JAK 2, FBXW7 2, WT1 2, NRAS 2) were detected in 17 patients. Eighteen patients who were resistant to chemotherapy received CD7-CART (CART group) before transplant, and all patients except one achieved CR. Thirty-nine r/r T-ALL patients were managed with chemotherapy before transplant (non-CART group), and 25 patients achieved CR (CR arm), 14 patients were in non-remission (NR) (NR arm) before transplantation. Conditioning regimens in CART group were either busulfan/fludarabine (n=12) or TBI/fludarabine (n=6). Conditioning regimens in non-CART group were either busulfan/fludarabine (n=11) or TBI/ fludarabine (n=28). ATG was used for haploidentical or unrelated transplants. Cyclosporine, mycophenolate mofetil and short-term methotrexate were employed for GVHD prophylaxis. Results: In CART group, the median time of neutrophils and platelets engraftment were 15 (10-22) and 18 (8-50) days. With the median follow-up 283 (14-496) days, 13 patients survived and 5 patients died (infection 4, cerebral hemorrhage 1). Three of 18 (16.7%) patients relapsed (CD7- 1, CD7 + 2) after transplantation and have been receiving treatment so far. One-year overall survival (OS) and disease-free survival (DFS) were 77.8% and 61.2%. Transplantation-related mortality (TRM) was 16.6%. In non-CART group, the median time of neutrophils and platelets engraftment were 15 (10-22) and 15 (8-28) days. Total 25 patients survived and 14 cases died (relapse 3, infection 4, GVHD 1, multiple organ failure 6). Fifteen of 39 (38.5%) patients relapsed. In CR arm, with the median follow-up 637(9-1357) days, one-year OS and DFS were 71.0% and 59.2%. In NR arm, with the median follow-up 517 (7-513) days, one-year OS and DFS were 59.2% and 40%. TRM was 28.2%. Summary/Conclusion: Donor-derived CD7-CART followed by allo-HSCT has achieved similar survival (OS, DFS) and lower relapse rate and TRM compared with chemotherapy followed by allo-HSCT in r/r T-ALL. CD7-CART may result in better disease control which will translate into less disease recurrence after transplant. S241: NON-RESTRICTIVE DIET DOES NOT INCREASE GASTROINTESTINAL INFECTIONS AND FEBRILE NEUTROPENIA IN PATIENTS WITH NEUTROPENIA AFTER STEM CELL TRANSPLANTATION: DATA FROM A MULTICENTRE, RANDOMIZED TRIAL F. Stella1,*, V. Marasco2, G. Levati2, A. Guidetti1, A. Chiappella2, G. Perrone2, M. Pennisi2, C. Tecchio3, N. Mordini4, G. Ferrara2, G. Gobbi2, L. Saracino2, C. Carniti2, P. Corradini1 2 1Dept. of Oncology and Hematology, Università degli studi di Milano; 2Division of Hematology and Bone Marrow Transplant, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan; 3Department of Medicine, University of Verona, Verona; 4Division of hematology, Azienda Ospedaliera S. Croce e Carle, Cuneo, Italy Background: Infections represent a major cause of morbidity and mortality during neutropenia, especially after hematopoietic stem cell transplantation (HSCT). Multiple preventive measures, including low microbial diet, have been adopted to reduce these complications. However, the effect of a protective diet (PD) in this setting has never been explored prospectively and evidence-based results lack. Conversely, there is evidence that PD could negatively affect the quality of life and that a prolonged fasting duration can increase the incidence of acute gastrointestinal graft-versus-host disease (aGVHD) in allo-HSCT recipients. Aims: To prospectively compare infection and aGVHD rates in patients receiving a non-restrictive diet (NRD) compared to PD after HSCT. Methods: Since July 2016 we conducted a multicentre randomized interventional study comparing the use of a PD (Arm A) vs NRD (Arm B) in hematological patients aged≥18 years hospitalized to receive autologous or allogenic HSCT. Stratification for allo-HSCT patients was planned. Patients received the assigned diet during the entire period of neutropenia. For PD, foods cooked >80°C and/or thick peel fruit were considered diet-specific. For the NRD, raw fruit and vegetables (adherent to hospital hygiene standards) were considered diet-specific. The primary objective was to demonstrate the lack of significant differences in incidence of infections grade >2 (according to CTCAE 4.0) and deaths during neutropenia between the two arms. Secondary endpoints included assessment of gastrointestinal infections and fever of undetermined origin (FUO), change in body weight, length of hospitalization, overall survival estimated at 30 days and cumulative incidence of aGVHD within 100 days after allo-HSCT. Results: Overall, 162 patients were analyzed at interim analysis, 80 patients in PD group and 82 in NRD group. The two arms were well balanced in terms of: sex, age, disease type, number of previous therapeutic lines, disease status at enrollment, antimicrobial prophylaxis, and reason for hospital admission. Moreover, 32 patients received an allo-HSCT, 17 in the PD and 15 in the NRD group respectively. Detailed patients’ characteristics are summarized in Table 1. We did not observe a significant difference in terms of infections in the two randomized arms; infections grade >2 or death were reported in 35 (43.7%) patients in arm A and in 34 (41.5%) patients in arm B [relative risk RR=1.05, confidence interval (CI)95%=0.76-1.4]. The number of patients developing gastrointestinal infections and FUO during hospitalization was 8 (10%) vs 8 (9.8%) [RR=1.01, CI95%=0.57-1.53] and 32 (40%) vs 28 (34.1%) [RR=1.13, CI95%=0.82-1.54] in arm A and arm B, respectively. No differences in weight variations from admission to discharge were observed comparing arm A and arm B (mean 4.15kg vs 3.66kg, p=0.3). Average hospitalization length in the two arms was respectively 20.6 vs 21.5 days (p=0.4). No deaths were reported at day+30. For 32 allo-HSCT recipients, aGVHD grade ≥2 incidence at day +100 was 5% vs 1.2% in arm A and arm B, respectively (RR 1.65, CI95%=0.77-2.19). Image: Summary/Conclusion: Results of this multicentre prospective trial show similar rate of infections and deaths between patients receiving a PD versus a NRD during neutropenia after HSCT. These data suggest that the use of NRD could be considered for transplanted patients without risks of more infective events. S242: EARLY INFUSION OF MSCS IN THE PROPHYLAXIS OF GVHD AFTER HAPLO-HEMATOPOIETIC STEM-CELL TRANSPLANTATION R. Huang1,*, T. Chen1, S. Wang2, J. Wang3, Y. Su4, J. Liu5, Q. Wen1, P. Kong1, C. Zhang1, L. Gao1, L. Gao1, X. Zhang1 1Medical Center of Hematology, Xinqiao Hospital of Army Medical University, Chongqing, 2920th Hospital of Joint Logistics Support Force of People’s Liberation Army of China, Sichuan; 3The Affiliated Hospital of Guizhou Medical University, Guiyang; 4The General Hospital of Western Theater Command PLA, Sichuan; 5The Third Xiangya Hospital of Central South University, Hubei, China Background: After the efficiency of repeated infusion umbilical cord-derived mesenchymal stromal cells (UC-MSCs) in preventing chronic graft versus host disease (GVHD) from 100 days after haploidentical hematopoietic stem-cell transplantation (haplo-HSCT) was proved in the last trial, we design a sequel clinical trial to further explore the early repeated infusion of UC-MSCs in GVHD prophylaxis. Aims: To evaluate the efficacy of repeated infsion of MSCs in GVHD prophylaxis from D45 after haplo-HSCT. Methods: 128 qualified subjects aged between 18 and 60 with haplo-HSCT in five transplant centers in Mid-West of China were enrolled and randomly assigned equally into the MSCs group and the control group. Patients in the MSCs group received 4 rounds infusion of UC-MSCs (1×106 cells/kg per two weeks from 45 days after transplantation). We investigated the incidence of GVHD and relapse free survival (GRFS), the incidence and severity of aGVHD and cGVHD and the accumulative relapse rate. Results: The one-year GRFS in the MSCs group is 65.6% in the MSCs group, which is significantly higher than the control group (43.7%, P= 0.0129). The accumulative incidence of III-IV aGVHD and severe cGVHD of patients in the MSCs group significantly decreased, which is 3.1% and 6.3%, while the control group is 25.6% and 18.8% (P=0.0089 and P=0.0260). The MSCs infusion did not influence the accumulative relapse rate, which is 10.9% in both groups. Image: Summary/Conclusion: In this trial, we proved that early repeated infusion of UC-MSCs could effectively improve the GRFS rate for patients after haplo-HSCT by decreasing the incidence and severity of GVHD without increasing the incidences of relapse for these patients. S243: ANTIBODY AND T-CELL RESPONSE SIX MONTHS AFTER INITIATION OF BNT162B2 VACCINATION IN PATIENTS WITH MULTIPLE MYELOMA OR CHRONIC LYMPHATIC B-CELL LEUKEMIA COMPARED TO HEALTHY CONTROLS L. D. Heftdal1 2 3,*, L. Pérez-Alós4, S. R. Ham2, J. R. Madsen4, K. Fogh5, C. C. Kronborg2, A. P. Vallentin1 3, R. B. Hasselbalch5, S. R. Ostrowski6 7, R. Frikke-Schmidt7 8, E. Sørensen6, L. Hilsted8, H. Bundgaard7 9, K. Iversen5 7 10, P. Garred4 7, S. D. Nielsen2 7 11, K. Grønbæk1 3 7 1Department of Hematology; 2Department of Infectious Diseases, Rigshospitalet, Copenhagen University Hospital; 3Biotech Research and Innovation Centre, University of Copenhagen; 4Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Rigshospitalet, Copenhagen University Hospital, Copenhagen; 5Department of Cardiology, Herlev and Gentofte Hospital, Copenhagen University Hospital, Herlev; 6Department of Clinical Immunology, Section 2034, Rigshospitalet, Copenhagen University Hospital; 7Department of Clinical Medicine, University of Copenhagen; 8Department of Clinical Biochemistry; 9Department of Cardiology, Rigshospitalet, Copenhagen University Hospital, Copenhagen; 10Department of Emergency Medicine, Herlev and Gentofte Hospital, Copenhagen University Hospital, Herlev; 11Department of Surgical Gastroenterology and Transplantation, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Background: The ongoing COVID-19 pandemic has resulted in more than 419 million cases and more than 5.9 million deaths. Previous studies have indicated inferior responses to SARS-CoV-2 vaccination across different hematological diseases. Through this prospective cohort study, we examined the development and durability of anti-receptor binding domain (RBD) IgG after two doses of BNT162b2 in 179 patients with either multiple myeloma (MM) or Chronic Lymphatic B-cell Leukemia (B-CLL) six months after vaccination and compared to immunocompetent controls. Aims: We aimed to investigate the durability of immune responses to COVID-19 vaccination in patients with MM or B-CLL compared to healthy controls, and to identify risk factors for humoral non-response, including type of diagnosis. Methods: We measured anti-receptor binding domain (RBD) IgG after two doses of BNT162b2 in 179 patients (MM: n=78, B-CLL: n=101) and 179 age and sex matched healthy controls up to six months after first vaccination. Anti-RBD IgG levels and neutralizing capacity of antibodies were measured at first and second dose of BNT162b2 and two and six months after first dose. Humoral response was defined as anti-RBD IgG > 225 AU/mL with a neutralizing index ≥ 25%. Humoral non-response was defined as the absence of a humoral response. T-cell responses were assessed six months after the first dose using an ELISA-based interferon-gamma release assay. A positive T-cell response was defined as IFN-ɣ release > 200 mIU/mL. Data on diagnoses were obtained through medical records, and data on vaccination status were obtained from the Danish Vaccination Register. Results: In patients with MM or B-CLL, the geometric mean concentration (GMC) of anti-RBD IgG increased from baseline 1.49 AU/mL (95% CI: 1.21-1.84) to three weeks after the first vaccine dose 15.10 AU/mL (95% CI: 9.39-24.29) and after receiving the second dose 1179.60 AU/mL (95% CI: 727.78-1919.85). From two to six months after first vaccine there was a significant decline in the GMC of anti-RBD IgG to 252.75 AU/mL (95% CI: 159.17-403.43). The mean neutralizing capacity in patients with MM or B-CLL was lower than in controls at all time points after the first vaccine dose. Six months after first vaccine dose, 79 of 179 (44.1%) patients with MM or B-CLL had a positive humoral response, while this was the case for 170 of 179 controls (95.0%), p<0.001. Having MM or B-CLL was significantly associated with risk of humoral non-response. This was most pronounced in B-CLL patients who had an age and sex adjusted risk ratio (RR) of 12.25 (95% CI: 6.42-23.38, p< 0.001) of humoral non-response compared to healthy controls. For MM patients the RR was 4.65 (95% CI: 2.21-9.80, p< 0.001). T-cell response was assessed in a subset of 48 patients with MM (n=28) or B-CLL (n=20) and 26 controls, six months after first vaccine dose. A total of 21 (43.8%) patients with MM (12/28) or B-CLL (9/20) and 14 (53.8%) controls had a positive T-cell response (p =0.56). Seven of 20 (35.0%) patients with MM or B-CLL who did not develop a humoral response, developed a T-cell response (MM: 3/8, B-CLL: 4/12), while 14 of 28 (50.0%) patients with MM or B-CLL who developed a humoral response developed a T-cell response (p =0.46, MM: 9/11, B-CLL: 5/8). In healthy controls 14 of 25 (56.0%) people who developed a humoral response also developed a T-cell response. Summary/Conclusion: Humoral response to BNT162b2 was impaired in patients with MM or B-CLL compared to healthy controls. Both patients with MM and B-CLL were at higher risk of humoral non-response compared to healthy controls. S244: EFFECT OF ANTI-SPIKE NEUTRALIZING MONOCLONAL ANTIBODIES ON COVID-19 PROGRESSION AND TIME TO VIRAL CLEARANCE IN PATIENTS WITH HEMATOLOGICAL MALIGNANCIES AND SARS-COV-2 INFECTION: THE GIMEMA EXPERIENCE V. Marasco1,*, A. Guidetti1 2, A. Piciocchi3, A. Candoni4, M. Bocchia5, R. Bruna6, P. Musto7, A. Visentin8, M. Turrini9, A. Tucci10, C. Selleri11, E. Crea3, P. Fazi3, F. Passamonti12, P. Corradini1 2 1Hematology and Bone Marrow Transplantation, Istituto Nazionale Dei Tumori di Milano; 2School of Medicine, Università degli Studi di Milano, Milan; 3GIMEMA Foundation, Rome; 4Dipartimento di Medicina Specialistica, University of Udine, Udine; 5Hematology Unit, University of Siena, Azienda Ospedaliero Universitaria Senese, Siena; 6Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont and Ospedale Maggiore della Carità, Novara; 7Department of Emergency and Organ Transplantation, “Aldo Moro” University School of Medicine and Unit of Hematology and Stem Cell Transplantation, AOU Consorziale Policlinico, Bari; 8Hematology and Clinical Immunology unit, Department of Medicine, University of Padova, Padova; 9Hematology, Ospedale Valduce, Napoli; 10Department of Hematology, ASST Spedali Civili di Brescia, Brescia; 11Hematology, Ospedale San Giovanni di Dio e Ruggi D’Aragona, Salerno; 12Department of Medicine and Surgery, niversity of Insubria and ASST Sette Laghi, Ospedale di Circolo of Varese, Varese, Italy Background: Patients with hematological malignancies (HM) infected with SARS-CoV-2 have a higher risk of developing severe coronavirus disease (COVID-19) with consequent death, due to immune system impairment. Anti-spike Neutralizing Monoclonal Antibodies (nMoAbs) are indicated for the treatment of paucisymptomatic COVID-19 patients, but evidence of safety and efficacy among HM subjects is still lacking. Aims: To assess the efficacy of different nMoAbs approved by Agenzia Italiana del Farmaco (AIFA) on HM patients affected by paucisymptomatic SARS-COV-2. Methods: Multicenter retrospective observational study at ten sites in Italy, which enrolled consecutive patients with SARS-CoV-2 infection and treated with nMoAbs from February 2020 to December 2021. Only HM subjects on treatment or in disease remission within 6 months from treatment discontinuation with paucisymptomatic SARS-COV-2 infection were included. nMoAbs approved by AIFA include Bamlanivimab, Bamlanivimab/Etesevimab, Casirivimab/Imdevimab, Sotrovimab, and Regdanvimab. The primary endpoint was to assess the time to SARS-CoV-2 molecular swab negativization. A comparison to an historical control not receiving nMoAbs was assessed. Secondary endpoints consisted in evaluation of hospitalization rate due to COVID-19, including intensive care unit (ICU) admission rate due to respiratory failure, and safety assessment. Results: Overall 51 HM patients (median age 62 years; 35% women) were evaluated. Seventeen of them had non-Hodgkin lymphomas, 9 multiple myeloma, 6 chronic lymphocytic leukemia, 6 acute myeloid leukemia, 3 Hodgkin lymphoma, 2 acute lymphoblastic leukemia, 2 myeloproliferative neoplasm, 1 Waldenstrom macroglobulinemia and 5 had other HM diagnosis. Thirty-six patients were on active treatment, whereas 11 had completed their therapies within 6 months from nMoAbs administration, for 4 patients data were missing. In 7 subjects the last treatment was chemotherapy, in 19 immunotherapy with or without chemotherapy, in 9 target therapy, in 4 autologous stem cell transplantation, in 2 allogeneic stem cell transplantation, for 4 patients data were missing. Detailed description of patients’ characteristics is reported in table 1. Twenty-six patients were treated with Bamlanivimab/Etesevimab, 17 with Casirivimab/Imdevimab, 3 with Bamlanivimab, and 2 with Sotrovimab, for 3 patients data were missing. Median time to SARS-CoV-2 molecular swab negativization was evaluable in 41 subjects and was 17 days (min 5, IQR 12-26, max 174). This result compared well with the previous finding of 28 days reported in an historical group of HM patients not treated with nMoAbs. We did not find any subpopulation, according to age, diagnosis, period of infection or type of nMoAbs who achieved a major benefit from nMoAbs treatment. The rate of hospitalization due to COVID-19 progression was 19% (10/51), with an extremely low percentage of patients requiring ICU admission due to sever COVID-19 (2%,1/51). Most frequent side effects included chills (8%), diarrhea (6%), headache (2%), nausea (2%) and vomiting (2%). Image: Summary/Conclusion: Among paucisymptomatic SARS-CoV-2 positive HM patients on active treatment or in disease remission within 6 months from treatment discontinuation, the administration of nMoAbs substantially reduced the time to swab negativization compared to an historical control of HM subjects. This treatment was also able to reduce the rate of hospitalization and death due to COVID-19 progression in this high risk group. S245: ALTERATION OF BONE MARROW NICHE BY ALLOGENEIC IMMUNE REACTION AFTER HSCT M Kimura1,*, N. Asada2, M. Matsuda1, M. Abe1, W. Kitamura1, K.-I. Matsuoka1, H. Fujiwara2, M. Ono3, W. Ziyi3, T. Mukai4, Y. Morita5, Y. Maeda1 1Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science; 2Hematology and Oncology, Okayama University Hospital; 3Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama; 4Immunology and Molecular Genetics; 5Rheumatology, Kawasaki Medical School, Kurashiki, Japan Background: Allogeneic stem cell transplantation (allo-SCT) has been considered as a curable therapy for hematologic diseases. Post-transplant myelosuppression is one of the life-threatening complications. Hematopoietic stem cell (HSC) functions are tightly regulated by a specialized microenvironment called “niche” in the bone marrow (BM) and we have identified differential contributions of perivascular stromal cells as HSC niche (Asada et al. Nat Cell Biol 2017). Recent studies indicate that BM niche can be targeted by allo-immune reaction but it remains unclear how the perivascular niche cells are involved in the mechanism of HSC dysfunction after allo-SCT. Aims: In this study, we aim to investigate the mechanisms of the impairment of the niche cells by allo-immune reactions, and to explore a targetable pathway for myelosuppression after allo-SCT. Methods: To evaluate the alterations in niche cells after allo-SCT, we utilized allo-SCT mice models (C3H/HeJ into C57BL/6, C57BL/6 into B6D2F1). Hematopoietic cells, including HSCs and mature cells, and perivascular stromal cells in BM were analyzed by flow cytometry (FACS) after transplantation. In imaging studies, we visualized perivascular stromal cells using Nestin-GFP (Nes-GFP), Myh11-CreER/tdTomato, LeptinR-Cre/tdTomato transgenic mice and observed them with confocal laser scanning microscopy. The gene expression analysis of niche factors was performed by real-time PCR. To assess the landscape of transcriptional remodeling in BM after allo-SCT, we performed single-cell RNA sequencing (scRNAseq) of BM cells. Results: FACS analyses showed that Nes-GFP+ stromal cells were dramatically reduced, and HSC recovery was severely impaired in allo-SCT mice compared to control mice at day 21 after SCT. Imaging studies also revealed that perivascular stromal cells are morphologically damaged in allo-SCT mice. Nes-GFP+ stromal cells in allo-SCT mice presented a decreased expression of niche factors, including Cxcl12 and Scf, which are essential for HSC maintenance, indicating the dysfunction of niche cells. We confirmed the impaired niche function in allo-SCT mice by tandem transplantation analysis in vivo, in which the recovery of syngeneic HSCs was also impaired in damaged microenvironment in allo-SCT mice. Prevention of allo-reactive T cell expansion by post-transplant cyclophosphamide ameliorated the niche cell reduction along with improved recovery of HSCs. These results indicate that niche impairment caused by allo-immune reaction leads to HSC dysfunction. The scRNAseq of BM highlighted the elevated expression levels of inflammation response genes, including Cxcl9 and Cxcl10, and MHC classⅡ, together with the deceased expression of niche factors in niche cells in allo-SCT mice. These alterations seemed to be induced by proinflammatory cytokines such as interferon-gamma (INF-γ) derived from allo-T cells, as the increased number of T cells and high levels of INF-γ in BM were observed in allo-SCT mice. Additionally, evaluation of T cell localization by using imaging techniques of BM revealed that allo-T cells reside significant closer to LepR+ niche cells in allo-SCT mice. Since activation of CXCR3, which is a receptor for chemokines Cxcl9 and Cxcl10, has been shown to induce chemotaxis of activated T cells, we treated allo-SCT mice with AMG487, an antagonist of CXCR3, resulted in an improved recovery of both perivascular niche cells and hematopoiesis in allo-SCT mice. Summary/Conclusion: Collectively, allo-immune reaction severely damages perivascular HSC niche cells, leading to impaired recovery of hematopoiesis after allo-SCT. S246: A HUMAN BONE MARROW ORGANOID FOR DISEASE MODELLING AND DRUG SCREENING IN BLOOD CANCERS A. Khan1 2,*, M. Colombo2, J. Reyat1, G. Wang2 3, A. Rodriguez-Romera2, W. X. Wen2 3, L. Murphy2, B. Grygielska1, C. Mahoney4, A. Stone5, A. Croft4, D. Bassett6, G. Poologasundarampillai7, A. Roy8, S. Gooding8, J. Rayes1, K. Machlus5, B. Psaila2 1Institute of Cardiovascular Sciences, University of Birmingham, Birmingham; 2MRC Weatherall Institute of Molecular Medicine; 3Centre for Computational Biology, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford; 4Rheumatology Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom; 5Vascular Biology Program, Boston Children’s Hospital, Department of Surgery, Harvard Medical School, Boston, United States of America; 6Healthcare Technologies Institute, School of Chemical Engineering; 7School of Dentistry, Institute of Clinical Sciences, University of Birmingham, Birmingham; 8MRC Weatherall Institute of Molecular Medicine, Department of Paediatrics and National Institute of Health Research (NIHR) Oxford Biomedical Research Centre, University of Oxford, Oxford, United Kingdom Background: Blood cancers are the most common cancer affecting children, and among the top 10 most common cancers in adults, in whom they remain largely incurable. In the era of advanced “omics”, a major bottleneck to the development of novel therapies is target prioritization and validation. There is a pressing need for experimental platforms that model the cellular and molecular complexity of the bone marrow niche, to enable mechanistic studies into the role of the bone marrow microenvironment in the initiation and propagation of blood cancers. Aims: Organoid technologies have been transformative in other settings, but an organoid with adequate homology to native haematopoietic tissues has not yet been reported. We therefore sought to develop organoids that emulate the cellular, molecular and spatial architecture of human bone marrow. Methods: A directed-differentiation protocol was devised in which human iPSCs formed mesodermal aggregates before hydrogel embedding and commitment to vascular and hematopoietic lineages. Multi-modal imaging and single cell RNA-sequencing were used to confirm cellular, molecular and architectural homology to native human hematopoietic tissues. Results: We optimized hydrogel compositions and cytokine cocktails to support the differentiation of a 3D bone marrow perivascular niche. The resulting organoids contained distinct haematopoietic stem/progenitor cells (HSPCs), stromal and myeloid cellular subtypes including pro-platelet forming megakaryocytes and lumen-forming vasculature. 3D whole-organoid imaging revealed a vascular network with sinusoid-like vessels invested with MSCs/fibroblasts (Fig1A), and the extravasation of haematopoietic cells into vessel lumens. Megakaryocytes closely associated with vessels, and extended pro-platelet protrusions into the vasculature with remarkable similarity to native bone marrow. scRNAseq analysis confirmed transcriptional homology to human bone marrow cellular subtypes, and highlighted strong cell-cell communication networks and haematopoietic support from organoid stroma. We first explored the utility of the organoids to model myelofibrosis, an exemplar malignancy that involves cancer-induced bone marrow remodelling and where there is a need for improved models for target prioritization and novel therapies. Treatment of organoids with TGFβ resulted in a dose-dependent increase in hallmarks of fibrosis (Fig 1B), which was effectively reversed using pharmacological agents, enabling drug screening. Given their homology to native bone marrow, we hypothesized that the organoids may support engraftment of primary human cells. Indeed, cells from healthy donors and patients with blood cancers efficiently engrafted throughout the organoids. After 14 days, organoids engrafted with myelofibrosis but not healthy cells showed dramatic fibrotic remodelling (Fig 1C). The organoids also supported primary cells from other myeloid and lymphoid blood cancers, including those notoriously difficult to maintain in vitro – e.g. myeloma cells engrafted into organoids were >90% viable at 10 days following seeding, while rapidly dying within 48 hrs without organoid support. The ability to study primary cells ex vivo overcomes a huge hurdle to translational research in the myeloma field. Image: Summary/Conclusion: This platform is an enabling technology for the interrogation of disease mechanisms in haematological disorders. Target identification and screening using a species-specific, 3D model that can incorporate primary patient cells will reduce dependence on animal models and may accelerate translational research. S247: EMERGENCY GRANULOPOIESIS INDUCES A LYMPHOID TO MYELOID BIAS SWITCH IN A SUBSET OF HEMATOPOIETIC STEM CELLS K. Vanickova1 2,*, P. Danek1, M. Milosevic3, M. K. Adamcova1 4, N. Rai5, F. Matteini6 7, M. C. Florian6 7 8, J. D’Armiento9, J. Rohlena3, K. Rohlenova3, M. Alberich-Jorda1 4 1Department of Hemato-oncology, Institute of Molecular Genetics of the CAS, Prague; 4; 2Faculty of Science, Charles University; 3Institute of Biotechnology of the Czech Academy of Sciences; 4Childhood Leukaemia Investigation Prague, Department of Pediatric Haematology and Oncology; 2nd Faculty of Medicine, Charles University in Prague, University Hospital Motol, Prague, Czechia; 5Department of Pediatrics, Columbia University, New York, United States of America; 6Stem Cell Aging Group, Regenerative Medicine Program, The Bellvitge Institute for Biomedical Research (IDIBELL); 7Program for Advancing the Clinical Translation of Regenerative Medicine of Catalonia, P-CMR[C], Barcelona; 8Center for Networked Biomedical Research on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain; 9Department of Anesthesiology, Columbia University, New York, United States of America Background: Granulocytes represent the first line of defense against bacteria and fungi, thus their production needs to be adapted to specific demands. While daily production of granulocytes is sustained by steady-state granulopoiesis, upon infection this program switches to emergency granulopoiesis (EG), to ensure enhanced and accelerated granulocytic production. Recently, it was shown that unperturbed hematopoiesis is sustained by multipotent progenitors, however, during stress conditions, hematopoietic stem cells (HSCs) become active and responsible for coping with stress situations. Yet, whether and how HSCs play a role during EG is unknown. Aims: The aim is to understand the role of HSCs at early stages of acute infection, and to determine the mechanisms that drive emergency granulopoiesis at the stem cell level. Methods: The methods used in the project include single cell RNA sequencing, qRT-PCR, ELISA, whole-mount bone marrow microscopy, flow cytometry sorting and analysis, stem cell in vitro cultures, Wnt10b knockout mouse model and murine LPS administration. Results: To understand the contribution of HSCs to the EG response, we performed single cell RNA sequencing analysis of sorted murine HSCs (Lin-c-Kit+Sca-1+CD48-CD150+) 4 hours after lipopolysaccharide (LPS) stimulation, which mimics a bacterial infection and activates EG in vivo. Strikingly, we observed radical changes in the HSC cluster composition between PBS control and LPS treated mice. Interestingly, these changing population dynamics were marked by alterations in HSC lineage bias, demonstrating that under EG there is an expansion of the myeloid-bias HSCs at expenses of the lymphoid-bias HSCs. Remarkably, we identified Procr (CD201) as a specific cell surface marker for this lymphoid to myeloid transition, allowing us to distinguish, sort and track the switch from CD201+ lymphoid-bias HSCs (present in PBS control) towards CD201- myeloid-bias HSCs (present upon LPS stimulation). Further, the abundant CD201- myeloid-bias HSC clusters were characterized by an inflammatory gene signature. Particularly interesting, was the elevated expression of Wnt10b. We verified that Wnt10b is upregulated in murine primary bone marrow cells upon LPS stimulation in vitro using qRT-PCR. Further, we demonstrated that Wnt10b is released in the bone marrow following LPS treatment in vivo using ELISA and whole-mount microscopy. We showed that Wnt10b activates the canonical Wnt/β-catenin signaling pathway and promotes myeloid differentiation of Lin-c-Kit+Sca-1+ cells in vitro. In addition, our preliminary data suggests that the EG response is impaired in Wnt10b KO mice following LPS stimulation in vivo. Summary/Conclusion: In conclusion, we observed that HSCs actively take part in early stages of EG by activating an inflammatory signature associated with the switch from lymphoid to myeloid bias, therefore ensuring sufficient production of myeloid cells to fight the infection. Acknowledgements: This work was partially supported by a GACR grant 22-18300S and institutional funding from the IMG CAS (RVO 68378050). S248: THE ENDOSOMAL ADAPTOR PROTEIN MYCT1 CONTROLS ENVIRONMENTAL SENSING IN HUMAN HSCS J. Aguade-Gorgorio1,*, Y. Jami-alahmadi1, M. Kardouh1, I. Fares1, V. Calvanese2, H. Johnson1, M. Magnusson3, J. Shin4, H. Goodridge4, J. Wohlschlegel1, H. Mikkola1 1UCLA, Los Angeles, United States of America; 2University College London, London, United Kingdom; 3Lund University, Lund, Sweden; 4Cedars Sinai, Los Angeles, United States of America Background: Hematopoietic stem cells (HSC) integrate diverse environmental cues to maintain the balance between quiescence, self-renewal and differentiation throughout life. However, the molecular programs governing HSC stemness become dysregulated during culture, compromising HSC self-renewal and engraftment ability. Despite recent advances in optimizing culture conditions for human HSC, our ability to expand or modify functional human HSCs in culture for therapeutic use is still limited. Our data uncovered MYCT1 (Myc target 1) as a critical human HSC regulator that is selectively expressed in undifferentiated HSPCs and endothelial cells (EC) but becomes drastically downregulated during culture, concomitantly with loss of engraftment potential. Aims: We aimed to understand the molecular programs controlled by MYCT1 that are essential for maintaining human HSC function. Methods: We analyzed multiple RNAseq gene expression data sets of cultured and uncultured human HSPCs from developmental and postnatal hematopoietic tissues to identify genes associated with self-renewing HSCs. To assess the importance of MYCT1 expression in human HSC function we transduced fetal liver (FL) and cord blood (CB) HSPCs (CD34+CD38-CD90+ and GPI80+ in FL) with lentiviral shRNA vectors. We quantified the expansion of control or knockdown (KD) HSPCs at distinct time points, and determined engraftment ability at 6, 12, and 24 weeks after transplanting equal numbers of sorted HSPCs into conditioned immunodeficient mice. To define MYCT1 localization and interactome we performed subcellular fractionation, immunofluorescence, and immunoprecipitation coupled with high-sensitivity mass spectrometry in endothelial cells and the HSC-like KG1 cell line. We assessed endocytosis by quantifying the internalization of fluorescently labeled dextran and transferrin by FACS in human HSPCs and ECs after MYCT1 KD or overexpression (OE). To determine the signaling and cellular consequences of MYCT1 loss we performed phospho-proteomics, western blot, and single cell RNAseq in ECs and/or human HSPCs. HSPC proliferation was quantified after MYCT1 KD or OE by CFSE dilution assay and monitoring of single cell divisions for 5 days. Results: MYCT1 KD experiments revealed that MYCT1 is critical for human HSPC ex vivo expansion and engraftment after transplantation. We determined that MYCT1 is a membrane-associated protein localized in endosomes, where it interacts with vesicle trafficking components and signaling receptors with critical functions in HSC biology. MYCT1 KD in human ECs and HSPCs led to hyperactivation of endocytosis, a crucial regulatory step determining the responsiveness to extracellular cues and governing cell fate decisions. MYCT1 KD then led to widespread dysregulation of signaling pathways, including a hyperactivation Akt, and cellular functions, including defective proliferation, whereas MYCT1 overexpression had the opposite effect Summary/Conclusion: Our data suggest that MYCT1 governs human HSC stemness and fate decisions by controlling the sensing of extracellular signals and their downstream signaling through endocytosis. As MYCT1 expression is downregulated in cultured HSPC, this work suggests that the inability to properly sense microenvironmental signals in cultured HSCs is a key mechanism contributing to culture-associated HSC dysfunction that will need to be overcome to improve transplantability of ex vivo expanded human HSCs. S249: EX VIVO HEMATOPOIETIC STEM CELL (HSC) EXPANSION USING BONE-LINING REINVIGORATING MESENCHYMAL STROMAL CELLS (RMSCS) S. Sood1 2,*, L Klein1, P. Albert1, F. Pilz1, N. Schmitt3, D. Nowak3, M. Essers1 2 1Inflammatory Stress in Stem Cells; 2HI-STEM, DKFZ, Heidelberg; 3Department of Hematology and Oncology, Heidelberg University, Mannheim, Germany Background: Bone marrow transplants (BMTs) have highlighted the Hematopoietic Stem Cell (HSC) potential to restore a new functional hematopoietic system in diseased recipients. However, a major roadblock for the clinical application and translational research on HSCs is our limited potential for ex vivo HSC expansion. Aims: To develop an approach for improved ex vivo HSC expansion using novel bone-lining reinvigorating Mesenchymal Stromal Cells (rMSC)-based culture system. Methods: Using a functional approach, we developed a robust pipeline for the fluorescence-activated cell sorting (FACS)-based isolation and ex vivo expansion of rMSCs from both murine and patient-individualized human samples. We tested the long-term HSC expansion potential in the rMSC-based co-culture system, phenotypically using FACS based proliferation analysis and functionally using limiting dilution and colony forming assays. Further, we performed transplantation experiments to study the hematopoietic reconstitution ability of the ex vivo expanded HSCs. Moreover, we expanded a single HSC over multiple cell divisions using our culture system to demonstrate bona fide HSC self-renewal potential of the rMSC-based system. Results: We propose a potent ex vivo HSC expansion system based on novel bone lining-derived reinvigorating Mesenchymal Stromal Cells (rMSCs). Both bulk- and single-HSCs expanded long-term using the rMSC co-culture system maintained phenotypic stemness over multiple cell differentiation cycles and possessed functional bone marrow reconstitution capabilities upon transplant. Notably, our rMSC co-culture system outperformed existing alternatives for HSC expansion including systems using stromal cells, non-cellular coating factors, or different medium compositions. Further, our results highlight the reliable isolation and robust culture of human rMSCs using our experimental strategy, and the potential to also utilize the rMSC co-culture system for the ex vivo expansion of human HSCs. Summary/Conclusion: We could demonstrate that our rMSC-based system for HSC expansion can play a pivotal role in research to reduce the number of mice used for ex vivo experiments. Moreover, our data also shows that the rMSC-based co-culture can be used for human HSC expansion, opening possibilities of applying this potent system for research on numerous diseases including immunodeficiencies and leukaemia. S250: CHOLINERGIC SIGNALS PROMOTE THE QUIESCENCE OF NORMAL OR LEUKAEMIC STEM CELLS THROUGH THE ACTIVATION OF ALPHA 7-NICOTINIC RECEPTOR IN BONE MARROW MESENCHYMAL STROMAL CELLS C. Fielding1 2 3,*, A. García-García1 2 3, C. Korn1 2 3, S. Gadomski1 2 3 4 5, Z. Fang1 2 3, C. Kapeni1 2 3, J. L. Reguera6, J. A. Pérez-Simón6 7, B. Göttgens1 3, S. Méndez-Ferrer1 2 3 8 9 1Department of Haematology, University of Cambridge; 2National Health Service Blood and Transplant, Cambridge Biomedical Campus; 3Wellcome-MRC Cambridge Stem Cell Institiute, University of Cambridge, Cambridge, United Kingdom; 4Skeletal Biology Section, National Institute of Dental and Craniofacial Research, Bethesda; 5NIH-OxfordCambridge Scholars Program in partnership with Medical University of South Carolina, Charleston, United States of America; 6Department of Haematology, University Hospital Virgen del Rocio, 41013 Sevilla, Spain; 7National Health Service Blood and Transplant, NIH-OxfordCambridge Scholars Program in partnership with Medical University of South Carolina, Charleston, United States of America; 8Instituto de Biomedicina de Sevilla (IBiS/CSIC); 9Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, 41013 Sevilla, Spain Background: Our recent research has suggested that the sympathetic nervous system, a master regulator or homeostasis and stress responses, regulates the activity of hematopoietic stem cells (HSCs) in different bone marrow niches. We recently showed that chemotherapy or irradiation, which are routinely used for cancer treatment or as conditioning regimens for HSC transplantation, increase the release of cholinergic signals by sympathetic neurons and osteolineage cells. As a result, not all HSCs become activated (which could cause their exhaustion), but some remain quiescent, thereby retaining their full potential. Cholinergic signals activate α7 nicotinic receptor (α7nAChR) in bone marrow mesenchymal stromal cells (BMSCs), which in turn preserve stem cell quiescence under proliferative stress (Nat Commun 13:543). In addition, α7nAChR signalling exhibits anti-inflammatory properties (PMID:32595942). However, whether this signalling pathway influences acute myeloid leukaemia (AML) development and response to treatment is unclear. Aims: Investigate the influence of cholinergic signals on leukaemogenesis and therapy response in AML. Methods: Analysis of bone marrow (BM) from mouse models lacking sympathetic cholinergic innervation or α7nAChR in BMSCs, transplanted with BM cells from an inducible MLL-AF9+ AML model (Cancer Cell 15:791) and treated with cytarabine. Flow cytometry, histology, FACS, qRT-PCR. Results: We confirmed reduced density of sympathetic noradrenergic innervation in the BM of AML mice; however, interestingly, the cholinergic innervation, which also has sympathetic origin (Cell Stem Cell, in press) was preserved. To investigate the role of these fibres in AML development, we utilised a mouse model lacking sympathetic cholinergic innervation due to loss of the neurotrophic receptor GFRα2. AML developed faster in cholinergic-neural-deficient mice, compared with WT mice, transplanted with iMLL-AF9+ BM cells. This was associated with reduced quiescence of leukaemia-initiating cells (LIC) upon acute cytarabine treatment of mice lacking cholinergic skeletal innervation. This effect was more pronounced after repeated cytarabine treatment, leading to increased proliferation of LICs and augmented leukaemia burden at relapse. Given that α7nAChR transduces the cholinergic regulation of normal HSCs by BMSCs, we utilised LepR-Cre;Chrna7f/f mice to conditionally delete this signalling pathway in leukaemic mice. Reproducing the observations in mice lacking cholinergic innervation, α7nAChR deletion in BMSCs accelerated AML development, and repeated treatment with cytarabine further increased LIC proliferation, spleen infiltration and leukaemia burden after relapse. Summary/Conclusion: These results suggest that cholinergic signals increase the quiescence of normal or leukaemic stem cells through the activation of a7-nicotinic receptor in BMSCs and might influence AML response to chemotherapy. S251: DISRUPTION OF SUCCINATE RECEPTOR SIGNALING PROMOTES MYELOPROLIFERATION AND ACUTE MYELOID LEUKAEMIA V. Cuminetti1,*, J. Konieczny1, A. Bernal1, A. Villatoro1, H. Taman2, M. Ristic1, N. Villaplana-Lopera3, R. H. Paulssen2, G. Giovinazzo4, A. Vik5, P. Garcia3, L. Arranz1 1IMB, Stem Cells Ageing and Cancer; 2Genomics Support Center Tromsø, UiT The Arctic University of Norway, Tromsø, Norway; 3Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom; 4Pluripotent Cell Technology Unit, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain; 5Department of Hematology, University Hospital of North Norway, Tromsø, Norway Background: Unlike healthy haematopoietic stem cells (HSC), leukaemia stem cells (LSC) have high oxidative phosphorylation and hence targeting the tricarboxylic acid (TCA) cycle seems a promising therapeutic strategy in acute myeloid leukemia (AML) treatment. Accumulation of the TCA cycle oncometabolite succinate is associated with tumourigenesis, but its potential involvement in HSC function and leukaemia is unkown. Succinate stabilizes the oxygen-sensing transcription factor hypoxia-inducible factor (HIF)-1-α and activates interleukin (IL)-1β production in macrophages. Succinate may also be exported and has cell extrinsic effects through its receptor, succinate receptor-1 (SUCNR1), which may be pro- or anti-inflammatory. Aims: To investigate the in vivo role and molecular mechanisms of succinate and its receptor in normal and malignant haematopoiesis. Methods: To study the effect of succinate receptor on haematopoiesis, we characterized the immunophenotype of Sucnr1-KO mice. To dissect the role of Sucnr1 on healthy haematopoiesis via cell-autonomous effects, we transplanted fl/Sucnr1/fl control or Mx1-Cre fl/Sucnr1/fl bone marrow (BM) cells into wild-type (WT) C57BL/6J mice, which were later injected with pIpC. To study the role of Sucnr1 through non-cell autonomous effects, we transplanted WT BM into Nes-Cre ERT2 fl/Sucnr1/fl recipients or fl/Sucnr1/fl controls, which were induced with tamoxifen. We characterized the effects of Sucnr1 deletion at the molecular level by bulk and single-cell RNA-sequencing (scRNA-seq), and aimed to uncover the molecular mechanisms mediating the effects of Sucnr1 ablation using in vivo tageting with drugs. To study the effect of succinate on leukaemic progression, we used two different mouse models, i.e. Mx1-Cre Nras G12D and MLL-AF9 knock-in (KI). In human AML, we quantified SUCNR1 expression in peripheral blood mononuclear cells (PBMC) from AML patients versus healthy volunteers, and treated xenograft recipients of CD34+ AML cells with succinate or vehicle. Results: In vivo deletion of Sucnr1 induced myeloid bias and expansion of the haematopoietic stem and progenitor cell (HSPC) compartment. In vivo deletion of Sucnr1 from Nestin+ stromal cells promoted expansion of long-term HSC (LT-HSC) versus intact recipients. Conversely, in vivo deletion of Sucnr1 from the haematopoietic system induced expansion of multipotent progenitors (MPP) versus intact haematopoietic cells. RNA-seq in HSPC from Sucnr1-KO versus WT mice showed changes in genes associated with myeloid output, and pinpointed related pathways potentially involved. scRNA-seq confirmed expansion of LT-HSC in Sucnr1-KO versus WT mice. In vivo treatments targeted signalling pathways at least partially responsible for the haematopoietic abnormalities in Sucnr1-KO. We found higher content of succinate in the BM extracellular fluid of leukaemic Mx1-Cre Nras G12D . Succinate injection to xenograft recipients of CD34+ AML cells increased leukaemic output versus vehicle, promoted myeloid bias in Mx1-Cre Nras G12D mice and reduced survival in mice transplanted with BM of MLL-AF9 leukaemic donors. PBMC from AML patients expressed lower level of SUCNR1 versus healthy donors and BM nucleated cells from Mx1-Cre Nras G12D also expressed less Sucnr1 than their healthy counterparts. Summary/Conclusion: We show a novel role for succinate and its receptor in the haematopoietic system and pinpoint this pathway as a novel means of communication within the BM HSC microenvironment, whose disruption may be involved in myeloid malignancy. These findings may help design new therapeutic strategies for AML patients. S252: M2-POLARIZED MACROPHAGES CONTROL LEUKEMIC STEM CELL FATE BY PROMOTING METABOLIC REPROGRAMMING I. Weinhäuser1 2 3,*, D. A. Pereira-Martins1 2 3, J. R. Hilberink1, L. Y. Almeida2 3, D. R. A. Silveira4, L. Quek4, S. M Hogeling1, J. M. Mota5, E. Ammatuna1, A. R. Lucena-Araujo6, G. A. Huls1, E. M. Rego7, J. J. Schuringa1 1Experimental Hematology, University medical centre groningen, Groningen, Netherlands; 2Center for Cell Based Therapy, University of Sao Paulo; 3Internal Medicine, Medical School of Ribeirao Preto, Ribeirao Preto, Brazil; 4Myeloid Leukaemia Genomics and Biology Group, Kings College London, London, United Kingdom; 5Medical Oncology Service, Sao Paulo State Cancer Institute, Sao Paulo; 6Department of Genetics, Federal University of Pernambuco, Recife; 7Center for Cell Based Therapy, University of Sao Paulo, Sao Paulo, Brazil Background: Being a crucial part of the tumor microenvironment (TME) in solid tumors, tumor-associated macrophages are often associated with poor prognosis (Bruni et al. Nat Rev Cancer 2020). Yet, in acute myeloid leukemia (AML) the role of macrophages remains unclear. Aims: Here, we evaluated the impact of M2 macrophages in AML using a patient derived xenograft (PDX) model. Methods: To do so, we first injected 1x105 peripheral blood (PB) derived M2 macrophages into NSGS mice and next transplanted notoriously difficult to engraft primary Acute Promyelocytic Leukemia (APL) cells (1 x 106 cells - n=7 different patient samples) via the retro-orbital vein. Results: As a result, mice with co-injected human M2-macrophages developed full-blown leukemia with increased spleen weight in comparison with the control. Perhaps even more strikingly, ex vivo culture of APL and other favorable AML subtypes such as inv (16) or NPM1 mutant leukemic cells on M2-macrophages for 48h was sufficient to “train” these cells to engraft and induce fatal leukemia. Maintenance of self-renewal was shown in a secondary transplant assay and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays (LSC frequency: Control: 1/6x106 cells vs M2 pre-culture: 1/7.2x104 cells). To better understand the biological changes induced on leukemic blast when exposed to M2 macrophages, we performed an RNA sequencing analysis comparing AML/APL samples at diagnosis to cells that were “trained” (48h) on M2-macrophages or on MS5 mesenchymal bone marrow stromal cells, as a control. Gene ontology and gene set enrichment analysis on the genes up regulated upon M2 co-culture were significantly enriched for oxidative phosphorylation (OxPhos) signatures. Evaluation of functional respiration using seahorse measurements, confirmed the increase of oxygen consumption rate (OCR, basal and maximum) in primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5. The increase of basal and maximum OCR suggested enhanced mitochondrial metabolism, which prompted us to determine whether macrophages, similar to MSC cells, can transfer mitochondria to primary AML cells (van der Vlist et al. Neuron 2022). Flow cytometry analysis revealed an efficient mitochondrial transfer from M2 macrophages to leukemic blasts, which was more efficient compared to mitochondrial exchange from MS5 cells. Treatment with the CPT1A inhibitor Etomoxir (50 µM), prevented the gain in functional respiration and enhanced proliferation of AML cells when those were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures. These results suggest increased fatty acid oxidation to drive the OXPHO-like state in AML. Summary/Conclusion: Overall, we revealed that M2 macrophages can support leukemic growth of favorable AML subtypes that are commonly difficult to engraft in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter the biology of AML cells transforming a non-engraftable cell into a cell with high leukemic potential. In vitro we show that AML blast cells cultured on M2 macrophage adapt a more OxPhos-like state linked to intrinsic changes associated with increased uptake of mitochondria. In summary, our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions. S253: THE SHEDDASE DOMAIN OF ADAM10 AUGMENTS THE INTERACTION OF LEUKEMIA CELLS WITH THE BONE MARROW NICHE IN VIVO AS SHOWN BY RECONSTITUTING PDX LEUKEMIA CELLS WITH CRISPR-CAS9-INDUCED KNOCKOUT J. P. Schmid1 2,*, E. Bahrami1, M. Becker1, A. K. Jayavelu3, A.-K. Wirth1, V. Jurinovic1 4 5, R. Öllinger6 7 8, R Rad2 6 7 8, B. Vick1 2, M. Mann3, T. Herold1 5, I. Jeremias1 2 4 1Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU); 2German Cancer Consortium (DKTK), partner site Munich, Munich; 3Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried; 4Department of Pediatrics, University Hospital, Ludwig Maximilian University (LMU); 5Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich; 6Center for Translational Cancer Research (TranslaTUM), TUM School of Medicine, Technische Universität München; 7Department of Medicine II, Klinikum rechts der Isar, Technische Universität München; 8Institute of Molecular Oncology and Functional Genomics, Technische Universität München, Munich, Germany Background: Tumor-microenvironment interactions are critically important determinants contributing to leukemia formation and maintenance. Interrupting the leukemia-bone marrow interaction represents an attractive therapeutic approach in acute leukemia (AL). Functional genomics significantly increases our understanding of the vulnerabilities and gene dependencies of individual tumors. Aims: Here, we developed a CRISPR-Cas9 screening approach for functional analysis of surface molecules in patient-derived xenograft (PDX) AL models in vivo. Methods: Size of CRISPR library was determined by genetic barcoding. Stable expression of fluorescently labelled Cas9 and sgRNA constructs in two PDX samples. Enrichment of double positive cells by MACS and injection into NSG mice. Gene depletion analysis using MAGeCK algorithm to screen and functional competitive in vivo assays to validate the candidates. Characterization of the ADAM10 KO or inhibitor (GI254023X) treated cells for engraftment capacity by homing assay, frequency of leukemic stem cells by competitive limiting dilution transplantation assay (LDTA), sensitivity towards routine chemotherapy by in vivo competitive chemotherapy trials in both lineages. Rescue assay by reconstitution of ADAM10 variants in functional competitive in vivo assays. Results: When running a customized CRISPR-Cas9 screen targeting about 100 cell surface candidates in two AL PDX samples, several sample-specific, but also commonly depleted candidates were identified. CRISPR screen findings were confirmed on the level of single molecules, using a competitive molecular in vivo approach and testing the PDX cells with and without knockout in the same mouse. These experiments validated an essential function for the two well-known depleted candidates CXCR4 and ITGB1 in both PDX models in vivo. Of note, various members of the Solute Carrier Family (SLC) were among the list of drop-out candidates. ADAM10 was identified as a commonly depleted candidate in both PDX models. In vivo competitive experiments confirmed the essential role of ADAM10 in PDX models from 6 additional patients with either acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia (AML), indicating a broad essential role of ADAM10 in both, ALL and AML, independent from their oncogenic-driver mutations and chromosomal abnormalities. Moreover, treating PDX cells with an ADAM10 chemical inhibitor resulted in significantly reduced engraftment capacity into the bone marrow (BM), indicating a role for ADAM10 in the early engraftment and homing process in the BM microenvironment. Knockout of ADAM10 reduced the frequency of leukemia stem cells, indicating that a relevant fraction of stem cells depends on ADAM10. ADAM10 KO ALL and AML PDX samples showed increased sensitivity towards routine chemotherapy treatments, indicating that inhibition of ADAM10 sensitizes AL towards conventional chemotherapy. When ADAM10 knockout cells were reconstituted with different recombinant ADAM10 variants, PDX in vivo experiments revealed that wildtype ADAM10 rescued the phenotype, while an ADAM10 variant lacking the enzymatic domain did not, highlighting the importance of the sheddase activity for ADAM10 function in leukemia maintenance. Summary/Conclusion: In summary, we established CRISPR-Cas9 drop-out screens in PDX models in vivo as technology to explore patient-specific tumor dependencies. Our data revealed a yet unknown function of ADAM10 to maintain patient leukemic cells in the bone marrow microenvironment niche. ADAM10 thus represents an attractive future therapeutic target for the treatment of acute leukemia. S254: CD4+ T CELL-DERIVED IL21 REGULATES STEM CELL FATE IN ACUTE MYELOID LEUKEMIA V. Rubino1 2 3,* 1Department for BioMedical Research, University of Bern; 2Department of Medical Oncology, Inselspital, Bern University Hospital; 3Graduate School of Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland Background: Acute myeloid leukemia (AML) is characterized by poor prognosis for patients, due to the very high rate of relapse, which occurs even after chemotherapy-induced complete remission. Therapy-insensitive AML cells, so-called leukemia stem cells (LSCs), are thought to be the main driver of relapse. In fact, high LSCs number and active LSC self-renewal programs are related to poor therapy response and adverse outcome in AML. Therefore, eliminating LSCs is an urgent, yet unmet, medical need. LSCs properties are crucially regulated and maintained also through interactions with the bone marrow microenvironment, which includes immune cells. Despite evidences of mutual interactions between AML cells and immune cells, their interplay is still poorly understood. A better understanding of these interactions might provide novel methods to effectively eradicate LSCs and achieve durable AML cure. Aims: The aim of this study was to investigate the role of IL21/IL21R signaling in the so far unexplored context of AML. Our aims involved identification of IL21 source in murine and human AML and elucidation of the mechanisms how IL21/IL21R signaling regulates LSCs properties. Methods: We used a combination of flow cytometry, ELISA and qRT-PCR to determine protein and mRNA expression of both IL21 and IL21R in primary AML samples and analyzed the correlation between IL21 serum levels with patients’ survival and complete remission rate. Colony-forming assays and short-term cultures were used to determine the in vitro effect of IL21 on primary normal and leukemic stem cells. In addition, patient-derived xenografts were employed to study IL21 effect on stem cell frequency and AML engraftment capacity. We used various syngeneic murine AML models (e.g. MLL/AF9 and MLL/ENL) to study the functional role of IL21/IL21R signaling in vivo. We performed RNA-Seq, together with analysis of symmetric cell division, expression of differentiation markers, NF-kB signaling, stem cell frequencies by limiting-dilution secondary transplantation experiments to investigate the mechanisms how IL21 regulates LSCs. Results: In human AML, serum IL21 was identified as an independent positive prognostic biomarker for overall survival (OS). Furthermore, high dose chemotherapy was more effective in patients with high levels of IL21 at diagnosis, as illustrated by a significantly higher rate of complete remission (CR rate: 81 vs 59 %) and prolonged OS (median survival: 915 vs. 364 days, HR: 1.8). Functionally, IL21 inhibited cell growth and clonogenic potential of primary AML stem and progenitor cells but not normal hematopietic stem cells (HSCs). In murine AML models, IL21/IL21R signaling in LSCs reduced stem cell frequency, prolonged survival of the mice and resulted in the downregulation of stemness-related and the up-regulation of differentiation-promoting pathways. In both human and murine AML, CD4+ T cells were identified as the main source of IL21. Image: Summary/Conclusion: In this work, we have identified IL21/IL21R signaling pathway as an important regulator of cell fate in human and murine LSCs, but not HSCs. We found that IL21 is a positive prognostic marker for OS and that higher serum IL21 levels correlate with better survival and higher rate of complete remission in patients that undergo chemotherapy. Our findings therefore suggest that promoting IL21/IL21R signaling on LSCs may be a novel approach to decrease stemness and increase differentiation in AML. S255: EFFICACY AND SAFETY OF TISAGENLECLEUCEL IN PEDIATRIC AND YOUNG ADULT PATIENTS (PTS) WITH RELAPSED OR REFRACTORY (R/R) MATURE B-CELL NON-HODGKIN LYMPHOMA (NHL): THE PHASE II BIANCA STUDY V. Minard-Colin1,*, J. Buechner2, F. Locatelli3, B. Gonzalez Martinez4, B. J. Vormoor5, S. Cooper6, J. Krueger7, S. Napolitano8, A. Attarbaschi9, A. Baruchel10, S. Ghorashian11, M. L. Hermiston12, H. Hiramatsu13, M. Ifversen14, S. John15, S. L. Khaw16, T. A. O’Brien17, C. L. Phillips18, C. Diaz de Heredia19, D. Tomizawa20, K. Vettenranta21, A. S. Wayne22, S. Newsome23, R. Awasthi24, S. Redondo25, A. Masood24, S. L. Maude26, B. Burkhardt27 1Department of Pediatric and Adolescent Oncology, Gustave Roussy, Université Paris-Saclay, Villejuif, France; 2Department of Pediatric Hematology and Oncology, Oslo University Hospital, Oslo, Norway; 3Department of Pediatric Hematology and Oncology, IRCCS Ospedale Bambino Gesù Children’s Hospital, Sapienza, University of Rome, Rome, Italy; 4Hospital Universitario La Paz, Madrid, Spain; 5Prinses Maxima Centrum, Utrecht, Netherlands; 6Johns Hopkins Comprehensive Cancer Center, Baltimore, MD, United States of America; 7Division of Haematology/Oncology/Bone Marrow Transplantation, Department of Paediatrics, The Hospital for Sick Children, Toronto, ON, Canada; 8Hematology-Oncology and Bone Marrow Transplantation Unit, Pediatric Department, and Monza and Brianza Foundation for Children and their Mums, San Gerardo Hospital, Lombardia, Monza, Italy; 9St. Anna Kinderspital and Children’s Cancer Research Institute, Vienna, Austria; 10University Hospital Robert Debré (APHP) and Université de Paris, Paris, France; 11Great Ormond Street Hospital for Children NHS Trust Haematology Department, London, United Kingdom; 12University of California San Francisco Benioff Children’s Hospital, San Francisco, CA, United States of America; 13Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 14Department of Pediatrics and Adolescent Medicine, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark; 15University of Texas Southwestern Medical Center, Dallas, TX, United States of America; 16Children’s Cancer Centre, Royal Children’s Hospital and Murdoch Children’s Research Institute, Parkville, VIC; 17Kids Cancer Centre, Sydney Children’s Hospital, Randwick, NSW, Australia; 18Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States of America; 19Hospital Universitari Vall d’Hebron, Vall d’Hebron Institut de Recerca, Barcelona, Spain; 20Children’s Cancer Center, National Center for Child Health and Development, Tokyo, Japan; 21University of Helsinki, Helsinki, Finland; 22Children’s Hospital Los Angeles, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States of America; 23Novartis Pharma AG, Basel, Switzerland; 24Novartis Pharmaceutical Corporation, East Hanover, NJ, United States of America; 25Novartis Farmaceutica S.A., Madrid, Spain; 26Division of Oncology, Center for Childhood Cancer Research and Cancer Immunotherapy Program, Children’s Hospital of Philadelphia and Department of Pediatrics, Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 27Pediatric Haematology and Oncology, University Hospital Muenster, Muenster, Germany Background: Chimeric antigen receptor (CAR)-T cell therapy (tx) targeting CD19 is approved for treatment of adult r/r large B-cell lymphoma (LBCL) and pediatric r/r B-cell acute lymphoblastic leukemia (ALL). Pediatric and young adult pts with r/r mature B-NHL have dismal prognoses, especially those with Burkitt lymphoma (BL), and limited clinical benefit from available tx. Aims: Herein we report primary efficacy and safety outcomes from the global, multicenter, open-label, single-arm Phase II BIANCA trial (NCT03610724). Methods: Pts ≤25 y and ≥6 kg at screening with histologically confirmed CD19+ r/r mature B-NHL after ≥1 prior lines of tx were eligible following successful leukapheresis. Pts received optional bridging chemotherapy and fludarabine/cyclophosphamide or cytarabine/etoposide for lymphodepletion prior to a single intravenous injection of tisagenlecleucel (target dose range, 0.2-5x106/kg bodyweight [pts ≤50 kg] or 0.1-2.5x108 [pts >50 kg] CAR+ viable T cells). The primary endpoint was overall response rate (ORR=complete response [CR]+partial response [PR]) by local investigator assessment in the efficacy analysis set (EAS), which excludes pts with pre-infusion CR. Secondary endpoints included progression-free survival (PFS), overall survival (OS), safety, and CAR-T cell kinetics. Results: As of October 10, 2021, 33/34 pts enrolled received tisagenlecleucel (EAS, N=28). Median age was 13 y (range, 3-22 y), 70% were male, 55% had BL, and 45% had LBCL. Pts received a median of 2 prior tx (18% received prior stem cell transplant [SCT]), 85% had stage III/IV disease at initial diagnosis, 15% were primary refractory, 30% were refractory, and 55% had relapsed/progressed at study entry. Median time from enrollment to infusion was 35 d (range, 7-62 d). Median time from tisagenlecleucel infusion to data cutoff was 16 mo (range, 6-30 mo). Bridging chemotherapy was given to 94%. ORR was 32% (95% CI: 15.9-52.4); 7% had a CR. Subgroup analysis suggested that pts with BL had a lower ORR than pts with LBCL (20% vs 46%). Median PFS was 2.5 mo (95% CI: 1.1-2.9); median OS was 11.4 mo (95% CI: 3.4-not estimable); 12-mo estimated PFS and OS were 23% (95% CI: 8.9-40.3) and 47% (95% CI: 26.6-65.0), respectively. Of the 18 BL pts, 7 (39%) are alive post infusion (1 received allogeneic SCT, 1 surgery, 3 other tx, and 2 no other tx). There were no tx-related deaths; most tx-related grade (gr) ≥3 (94%) and serious (52%) adverse events occurred ≤8 w post infusion. A neurologic event (NE) occurred in 27%; 15% had a gr ≥3 NE, and none had gr 5 NE. Cytokine release syndrome (CRS; per Lee 2014 criteria) occurred in 70%, 9% had gr 3 CRS, and there was no gr ≥4 CRS. Median time to CRS onset and duration were 6 d (range, 1-27 d) and 5 d (range, 1-13 d), respectively. Only 1 pt died ≤30 d post infusion, due to disease progression. Geometric mean maximal expansion (Cmax) across all pts was 5730 copies/μg, and median time to Cmax was 12.8 d (range, 2.5-21.9 d) by qPCR. Median CAR-T cell persistence in pts with CR or PR was 182 d (range, 20.9-562 d). Image: Summary/Conclusion: Tisagenlecleucel demonstrated efficacy in pediatric and young adult pts with r/r mature B-NHL and comparable safety to that recorded in adults with DLBCL. The OS is encouraging; however, optimal positioning of tisagenlecleucel in the treatment of pediatric r/r B-NHL requires further exploration, especially for BL, which is highly aggressive and challenging to treat in the relapse setting. S256: TABELECLEUCEL FOR EBV-DRIVEN POST-TRANSPLANT LYMPHOPROLIFERATIVE DISEASE FOLLOWING ALLOGENEIC HEMATOPOIETIC CELL OR SOLID ORGAN TRANSPLANT AFTER FAILURE OF RITUXIMAB ± CHEMOTHERAPY (ALLELE) S. Prockop, MD1,*, A. Beitinjaneh, MD2, S. Choquet, MD3, S. Dahiya, MD4, R. Dinavahi, MD5, R. Farah, MD6, L. Gamelin, MD, PhD5, A. Ghobadi, MD7, A. Mehta5, P. Nayak, MD8, R. Reshef, MD9, G. Satyanarayana, MD10, P. Stiff, MD11, W. Ye, PhD8, K. M. Mahadeo, MD, MPH12 1Boston Children’s Hospital/Dana Farber Cancer Institute, Boston; 2University of Miami/Jackson Memorial Hospital, Miami, United States of America; 3Sorbonne Université, Hôpital de la Pitié-Salpêtrière, Paris, France; 4University of Maryland School of Medicine, Baltimore; 5Atara Biotherapeutics, Thousand Oaks; 6UPMC Hillman Cancer Center, Pittsburgh; 7Washington University, Division of Oncology, St. Louis; 8Atara Biotherapeutics, South San Francisco; 9Columbia University Medical Center, New York; 10Ingram Cancer Center, Vanderbilt Health, Nashville; 11Loyola University Medical Center, Chicago; 12MD Anderson Cancer Center, Houston, United States of America Background: Tabelecleucel is an investigational, off-the-shelf, allogeneic Epstein–Barr Virus (EBV)-specific T-cell immunotherapy being explored in patients (pts) with EBV-driven post-transplant lymphoproliferative disease (EBV+ PTLD). Median overall survival (OS) after rituximab (R) ± chemotherapy (C) failure is 0.7 months in EBV+ PTLD post allogeneic hematopoietic cell transplant (HCT) (Sanz ASH 2021) and 4.1 months post solid organ transplant (SOT) after R+C failure (Dharnidharka ASH 2021), demonstrating an urgent need for therapies in this ultra-rare disease. Tabelecleucel has shown promising outcomes (Prockop JCI 2020). Additionally, an investigator-assessed objective response rate (ORR) of >60% with >80% 2-year OS (Prockop EBMT 2021, Prockop ATC 2021, Prockop ASH 2021) was seen in pts with relapsed/refractory (R/R) EBV+ PTLD, with either complete or partial response to tabelecleucel. Safety data in >180 pts with tabelecleucel treated EBV+ PTLD demonstrate tumor flare reaction (TFR) as the only identified risk (Atara Biotherapeutics, Data on file). Aims: Report data from the ongoing Phase 3 ALLELE study (NCT03394365). Methods: ALLELE is a multicenter, open-label study investigating tabelecleucel in pts with EBV+ PTLD post HCT after R failure (n=33) and post SOT after R±C failure (n=33). Pts receive tabelecleucel at 2 x 106 cells/kg on Days 1, 8 and 15 in 35-day cycles. Response is evaluated by investigator and by independent oncologic response adjudication (IORA; primary assessment) using Lugano Classification with LYRIC modification. Efficacy endpoints include ORR, duration of response (DOR), time to response (TTR), and OS. Pts are assessed post treatment for up to 5 years for survival. Results: As of May 2021, 38 pts (14 HCT, 24 SOT) were evaluable by IORA and had the opportunity for 6 months follow-up. Median age was 52.9 (range, 3.2–81.5) years, 44.4%/47.2% of pts were high/intermediate risk per PTLD-adapted prognostic index, and median number of lines of prior systemic treatment was 1 (range, 1–5). R/R HCT and SOT pts received a median (range) of 3 (1–5) and 2 (1–6) cycles of tabelecleucel, respectively. ORR was 50.0% (19/38, 95% CI: 33.4, 66.6) overall, 50.0% (7/14, 95% CI: 23.0, 77.0) in HCT, and 50.0% (12/24, 95% CI: 29.1, 70.9) in SOT (Table). Overall, median TTR was 1.1 (0.7–4.7) months, 11 of 19 responders had DOR > 6 months, and median DOR was not reached (Table). Median OS was 18.4 (95% CI: 6.9, NR) months overall, not yet reached for HCT, and 16.4 (95% CI: 3.5, NR) months for SOT. 1-year survival rates were 61.1% (95% CI: 42.9, 75.0) overall, 66.8% (95% CI: 32.4, 86.6) for HCT, and 57.4% (95% CI: 35.2, 74.5) for SOT. Median OS was NR (95% CI: 16.4, NR) for responders and 5.7 (95% CI: 1.8, 12.1) months for non-responders. Responders had a higher 1-year survival rate vs non-responders: 89.2% (95% CI: 63.1, 97.2) vs 32.4% (95% CI: 12.1, 54.9) (Table). Serious treatment emergent AEs (TEAEs) and fatal TEAEs were reported in 57.1% and 7.1% of HCT and 62.5% and 16.7% of SOT pts respectively. No fatal TEAE was treatment related. There were no reports of TFR, infusion reactions, cytokine release syndrome, marrow rejection, or transmission of infectious diseases, and no events of graft vs host disease or organ rejection reported as related to tabelecleucel. Image: Summary/Conclusion: Tabelecleucel Phase 3 data show clinically meaningful outcomes and promising ORR and OS in a pt population with poor survival and no approved therapies. Tabelecleucel was well tolerated without evidence of safety concerns typically observed with other adoptive T-cell therapies. S257: A PHASE 1, FIRST-IN-HUMAN, DOSE-ESCALATION CLINICAL TRIAL OF MEMORY-ENRICHED CD30-CAR T-CELL THERAPY FOR THE TREATMENT OF RELAPSED OR REFRACTORY HODGKIN LYMPHOMA AND CD30+ T-CELL LYMPHOMA A. C. Caballero1,*, L. Escribà-Garcia1, R. Montserrat-Torres1, E. Escudero-López1, P. Pujol-Fernández1, C. Ujaldón-Miró1, I. Garcia-Cadenas1, A. Esquirol1, R. Martino1, J. Sierra1, C. Alvarez-Fernández1, J. Briones1 1Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Background: Up to 30% of Hodgkin lymphoma (HL) patients are refractory or relapse (R/R) after first treatment and their prognosis is poor. We have developed a refined CD30-CAR-T (HSP-CAR30) targeting a proximal epitope within the CD30 molecule to overcome soluble CD30 and generated products enriched in memory T-cells to ensure efficient engraftment, persistence and enhancement of antitumor efficacy (Alvarez-Fernandez et al, 2021). Here, we report the results of our Phase 1 study evaluating HSP-CAR30 for the treatment of R/R HL and CD30+ T-cell non-Hodgkin lymphoma (T-NHL) (NCT04653649). Aims: Primary endpoints were to assess safety of HSP-CAR30 and to establish maximum tolerated dose (MTD) recommended for the following Phase 2. Secondary objectives include best response rates after infusion. Methods: We conducted a phase 1 dose-escalation study in 11 patients with R/R HL or CD30+ T-NHL. HL patients were R/R to treatments including chemotherapy, brentuximab and anti-PD-1 antibodies, while T-NHL patients were R/R to at least 2 chemotherapy treatments. T-cells were transduced with a lentivirus encoding a second-generation 4-1BB costimulated CAR, containing a scFv directed against an epitope from the proximal non-cleavable part of CD30 protein. Three cell-dose levels were evaluated: DL1 (3x106/kg), DL2 (5x106/kg) and DL3 (10x106/kg) CAR30+ T-cells. Results: From February 2021 to December 2021, 11 patients (9 HL and 2 CD30+ T-NHL) were enrolled and underwent leukapheresis. Of these, 10 patients received HSP-CAR30: 3 patients at DL1, 3 at DL2 and 4 at DL3. Demographic characteristics and baseline disease features are summarized in the Table. Median age was 49.9 years (range 21–65). Median number of prior lines of treatment was 4.6 (range 3–7). One patient did not received treatment due to lack of T-cell expansion. All patients received LD before infusion, fludarabine/bendamustine in HL patients (n=8) and fludarabine/cyclophosphamide in T-NHL (n=2). Mean HSP-CAR30 expression was 94.79±3,38% (±SD). Memory T-cell subset comprised 93.07±4,8% (±SD) in CD4+ and 91.64±4,9% (±SD) in CD8+. Mean time to HSP-CAR30 cell peak level across all doses was 29 days (range 6–98). CAR+ T-cells were detectable by flow cytometry up to 11 months after infusion. HSP-CAR30 infusion was well tolerated; there were no dose limiting toxicities (DLTs). Relevant adverse events are shown in the Table. Grade 1 cytokine release syndrome (CRS) was observed in 6 (60%) patients. No patient developed neurotoxicity. Self-limited skin rash was seen in 4 (40%) patients. One patient with history of cytomegalovirus (CMV) infections had CMV pneumonia. Another patient developed pulmonary tuberculosis. At data cutoff (February 21st, 2021), the median follow-up was 204 days (60–351). Best objective response was 100%, including 5 (50%) patients with complete response (CR), all with HL (DL1=1; DL2=3; DL3= 1). Three patients have died of progressive disease (2 T-NHL and 1 HL). There were no non-relapse mortality events. Median PFS and median overall survival (OS) was not reached. Six-month PFS for HL patients was 75%. Image: Summary/Conclusion: This is the first European academic CART clinical trial evaluating a T-cell memory-enriched CART 30. Our Phase 1 study provides evidence for feasibility and safety of HSP-CAR30. Additionally, HSP-CAR30 has shown promising efficacy in heavily treated HL patients that is being explored in a phase 2 trial already started. S258: LISOCABTAGENE MARALEUCEL (LISO-CEL) AS SECOND-LINE THERAPY FOR R/R LARGE B-CELL LYMPHOMA (LBCL) IN PATIENTS NOT INTENDED FOR HSCT: PRIMARY ANALYSIS FROM THE PHASE 2 PILOT STUDY A. Sehgal1,*, D. Hoda2, P. A. Riedell3, N. Ghosh4, M. Hamadani5, G. C. Hildebrandt6, J. E. Godwin7, P. Reagan8, N. Wagner-Johnston9, J. Essell10, R. Nath11, S. R. Solomon12, R. Champion13, E. Licitra14, S. Fanning15, N. Gupta16, R. Dubowy17, A. D’Andrea18, L. Wang17, L. I. Gordon19 1University of Pittsburgh Medical Center, Hillman Cancer Center, Pittsburgh; 2Intermountain Healthcare, Loveland Clinic for Blood Cancer Therapy, Salt Lake City; 3University of Chicago Comprehensive Cancer Center, Chicago; 4Levine Cancer Institute, Atrium Health, Charlotte; 5BMT & Cellular Therapy Program, Medical College of Wisconsin, Milwaukee; 6Markey Cancer Center, University of Kentucky, Lexington; 7Providence Cancer Center, Earle A. Chiles Research Institute, Portland; 8University of Rochester Medical Center, Rochester; 9Johns Hopkins Hospital, Baltimore; 10Oncology Hematology Care, Cincinnati; 11Banner MD Anderson Cancer Center, Gilbert; 12Northside Hospital Cancer Institute, Atlanta; 13Norton Cancer Institute, Louisville; 14Astera Cancer Care, East Brunswick; 15Prisma Health, Greenville; 16Stanford Cancer Genetics Clinic, Palo Alto; 17Bristol Myers Squibb, Seattle, United States of America; 18Celgene, a Bristol-Myers Squibb Company, Boudry, Switzerland; 19Northwestern University, Feinberg School of Medicine, Robert H. Lurie Comprehensive Cancer Center, Chicago, United States of America Background: Patients with relapsed or refractory (R/R) LBCL after first-line treatment who are unable to undergo high-dose chemotherapy (HDCT) and hematopoietic stem cell transplantation (HSCT) have poor outcomes and limited treatment options. Aims: PILOT (NCT03483103) evaluated liso-cel, an autologous, CD19-directed chimeric antigen receptor (CAR) T cell product, as second-line treatment in patients with R/R LBCL not intended for HSCT. Methods: Eligible patients were adults with R/R LBCL after first-line treatment who were not deemed candidates for HDCT and HSCT by their physician and met ≥ 1 frailty criteria as follows: age ≥ 70 years, Eastern Cooperative Oncology Group performance status (ECOG PS) of 2, diffusing capacity for carbon monoxide ≤ 60%, left ventricular ejection fraction < 50%, creatinine clearance < 60 mL/min, or alanine aminotransferase/aspartate aminotransferase > 2 × the upper limit of normal. Bridging therapy was allowed. Patients received lymphodepletion with cyclophosphamide and fludarabine, followed 2–7 days later by liso-cel infusion at a target dose of 100 × 106 CAR+ T cells. Cytokine release syndrome (CRS) was graded per Lee 2014 criteria. Neurological events (NE) were defined as investigator-identified neurological adverse events related to liso-cel and graded using the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. The primary endpoint was objective response rate (ORR) per independent review committee; all patients had ≥ 6 months of follow-up from first response. Results: Of 74 patients who underwent leukapheresis, 61 received liso-cel and 1 received nonconforming product (ie, product wherein one of the CD8 or CD4 cell components did not meet one of the requirements to be considered liso-cel). Common reasons for preinfusion dropout included death and loss of eligibility (5 each). For liso-cel–treated patients, median age was 74 years (range, 53–84; 79% ≥ 70 years) and 69%, 26%, and 5% met 1, 2, and 3 frailty criteria, respectively; 26% had ECOG PS of 2 and 44% had Hematopoietic Cell Transplantation-specific Comorbidity Index score ≥ 3. After first-line treatment, 54% were chemotherapy refractory, 21% relapsed within 12 months, and 25% relapsed after 12 months; 51% of patients received bridging chemotherapy. Median (range) on-study follow-up was 12.3 months (1.2–26.5). ORR and complete response rate were 80% and 54%, respectively (Table). Median duration of response and progression-free survival were 12.1 months and 9.0 months, respectively. Median overall survival has not been reached. The most frequent treatment-emergent adverse events (TEAE) were neutropenia (51%), fatigue (39%), and CRS (38%), with grade 3 CRS in 1 patient (2%) and no grade 4/5 CRS events. Any-grade NEs were seen in 31% (n = 19) of patients; grade 3 NEs occurred in 5% (n = 3) of patients and no grade 4/5 NEs were reported. Seven percent (n = 4) received tocilizumab only, 3% (n = 2) received corticosteroids only, and 20% (n = 12) received both tocilizumab and corticosteroids for treatment of CRS and/or NEs. Overall, grade ≥ 3 TEAEs occurred in 79% (n = 48) of patients, with grade 5 TEAEs in 2 patients (both due to COVID-19). Two patients (3%) had grade 3/4 infections and 15 (25%) had grade ≥3 neutropenia at Day 29. Image: Summary/Conclusion: In the PILOT study, liso-cel as second-line treatment in patients with LBCL who met ≥ 1 frailty criteria and for whom HSCT was not intended demonstrated substantial and durable overall and complete responses, with no new safety concerns. S259: DUAL ANTIGEN TARGETING WITH CO-TRANSDUCED CD19/22 CAR T CELLS FOR RELAPSED/REFRACTORY ALL S. Ghorashian1,*, G. Lucchini2, R. Richardson3, K. Nguyen3, C. Terris3, J. Yeung3, J. Chu2, L. Williams2, K. Ko2, C. Walding4, K. Watts5, S Inglott1, S. Adams1, E. Gravett1, K. Gilmour6, A. Lal7, S. Kunaseelan7, B. Popova7, A. Lopes7, Y. Ngai7, E. Kokalaki8, K. Rao2, R. Chiesa2, J. Silva2, K Mullanfiroze2, A. Lazareva2, D. Bonney5, R Wynn5, M. Pule8, R. Hough4, P. Amrolia2 1Haematology; 2Bone Marrow Transplant, Great Ormond St Children’s Hospital; 3Molecular and Cellular Immunology, UCL Great Ormond St Institute of Child Health; 4Haematology, University College London Hospital NHS Trust, London; 5Blood and Marrow Transplant, Royal Manchester Children’s Hospital, Manchester; 6Cell Therapy and Immunology, Great Ormond St Children’s Hospital; 7Cancer Research UK & UCL Cancer Trials Centre; 8Autolus Ltd, London, United Kingdom Background: CD19 negative escape is a major cause of relapse after CD19 CAR T cell therapy for relapsed/refractory (r/r) paediatric ALL and dual targeting of CD19/CD22 may overcome this. We have previously shown that AUTO1, a fast off rate autologous CD19 CAR T cell therapy was highly active in ALL with a favorable safety profile and excellent persistence (Ghorashian et al Nat.Med. 2019). Building on these properties, we developed AUTO1/22 in which autologous T cells are co-transduced with 2 different lentiviral vectors encoding our existing CD19 CAR and a novel CD22CAR designed to recognise targets with low antigen density. AUTO1/22 was evaluated in a Phase I study in children/young adults with r/rALL (NCT02443831). Aims: To determine the safety/biological efficacy of AUTO1/22 Methods: Patients with r/r B-ALL age < 25 ywho were ineligible for/relapsed after Tisagenlecleucel were recruited. Following fludarabine/cyclophosphamide lymphodepletion, patients received 1x106 /kg CAR+ T cells. The presence of CAR T cells in the blood/bone marrow (BM) was assessed by flow cytometry + qPCR and BM MRD was assessed by IgH qPCR + flow cytometry. Primary end-points were incidence of grade 3-5 toxicity and the proportion of patients achieving MRD negative remission. Results: Ten patients have been treated and 8 are evaluable with >1 month follow-up. The median age was 12 years and patients had a median of 3.5 prior lines of therapy (range 2-6). Five of 8 patients had relapsed post allogeneic SCT, 4 had received prior Blinatumomab/Inotuzumab and 3 had relapsed after prior Tisagenlecleucel. Prior to lymphodepletion, 2 patients had >5% BM disease, 5 had MRD between 10-2 and 10-5 and 1 was BM MRD negative. CAR T cell products had a central memory phenotype with predominance of CD19/22 double positive cells (median 59.3%) and balanced populations of CD19 and CD22 single positive cells (16% and 10.9% respectively).Cytokine release syndrome (CRS) occurred in 7/8 patients (grade 1 n=2, grade 2 n=5) requiring Tocilizumab in 3 cases, but severe (≥ grade 3) CRS was not seen and no patients required ICU admission for CRS. Grade 1-2 ICANS was observed in 3 patients. One patient had delayed grade 4 leucoencephalopathy (MRI/brain biopsy were more indicative of fludarabine toxicity than CAR T related) and has ongoing neurological recovery. 7 patients had grade 3-4 cytopenia persisting beyond/recurring after day 28, requiring a CD34+ stem cell top up in 1 case. 5/8 patients had CD19CAR T cells and 3/8 patients had CD22CAR T cells detectable at last follow-up. 7 of 8 evaluable patients (88%) achieved MRD negative CR/CRi at 1 month post-infusion. One patient did not respond with CD19+ CNS relapse + MRD level BM disease at day 28. Of the 7 responding patients, 1 had frank CD19+CD22+ BM and extramedullary relapse at 3 months and 1 had emergence of MRD level disease at 10.5 months post infusion, in both cases associated with loss of CAR T cells. One other patient had early loss of CAR T cells with B cell recovery but ongoing MRD negative CR at 3 months post-infusion and remains in MRD negative CR on maintenance chemotherapy. Overall, at a median follow-up of 4.8 months, 5/8 patients remain in MRD negative CR at last follow-up. Summary/Conclusion: We demonstrate that dual CD19/22 targeting CAR T cells generated by co-transduction show an acceptable safety profile, with robust expansion/persistence and early efficacy in a heavily pre-treated cohort. To date with limited follow-up we have not observed antigen negative relapse but longer follow up is needed. S260: A MATCHED COMPARISON OF TISAGENLECLEUCEL AND AXICABTAGENE CILOLEUCEL CAR T CELLS IN RELAPSED OR REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA: A REAL-LIFE LYSA STUDY FROM THE FRENCH DESCAR-T REGISTRY E. Bachy1,*, S. Le Gouill2, P. Sesques3, R. Di Blasi4, M. Guillaume5, G. Cartron6, D. Beauvais7, L. Roulin8, F. X. Gros9, M. T. Rubio10, P. Bories11, J. O. Bay12, C. Castilla Llorente13, S. Choquet14, R.-O. Casasnovas15, M. Mothy16, S. Guidez17, M. Joris18, M. Loschi19, S. Carras20, J. Abraham21, A. Chauchet22, L. Drieu La Rochelle23, J. Zerbit24, O. Hermine25, T. Gastinne26, J. J. Tudesq6, E. Gat27, F. Broussais27, C. Thieblemont28, R. Houot5, F. Morschhauser7 1Hematology, Hospices Civils de Lyon, Pierre Bénite; 2Institut Curie, Paris; 3Hospices Civils de Lyon, Pierre Bénite; 4AP-HP, Paris; 5CHU Rennes, Rennes; 6CHU Montpellier, Montpellier; 7CHU Lille, Lille; 8CHU Mondor, Paris; 9CHU Bordeaux, Bordeaux; 10CHU Nancy, Nancy; 11CHU Toulouse, Toulouse; 12CHU Clermont Ferrand, Clermont Ferrand; 13GUSTAVE ROUSSY CANCER CAMPUS GRAND PARIS, Villejuif; 14CHU Pitié Salprétrière, Paris; 15CHU Dijon, Dijon; 16CHU Saint Antoine, Paris; 17CHU Poitiers, Poitiers; 18CHU Amiens, Amiens; 19CHU Nice, Nice; 20CHU Grenoble, Grenoble; 21CHU Limoges, Limoges; 22CHU Besancon, Besancon; 23CHU Tours, Tours; 24APHP Cochin; 25CHU Necker, Paris; 26CHU Nantes, Nantes; 27LYSARC, Lyon; 28CHU Saint Louis, Paris, France Background: Axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) have both demonstrated impressive clinical activity in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). In a previous propensity score matching (PSM) analysis (Bachy et al., ASH 2021) allowing for balanced comparison between axi-cel and tisa-cel outcomes, we reported on a prolonged progression-free survival (PFS) but a higher toxicity associated with axi-cel compared with tisa-cel. No OS difference was observed but the follow-up was short (6 months). Aims: We aim at reporting on PSM analysis with longer follow-up and with additionnal patients treated with axi-cel or tisa-cel. Methods: All patients treated in France with axi-cel or tisa-cel from the 1st July 2018 to the 1st October 2021 and included in the DESCAR-T registry were considered. Propensity score matching (PSM) was used to create a balanced covariate distribution between a cohort of patients treated with tisa-cel and a cohort of patients treated with axi-cel. An exhaustive list of covariates was used for PSM: age, sex, LDH level, C reactive protein (CRP), time between last treatment and infusion, Eastern Cooperative Oncology Group (ECOG) performance status (PS), Ann Arbor stage, number of prior lines of treatment before CAR-T, bridging and response to bridging, prior stem cell transplant (SCT) either autologous or allogeneic, bulk assessed at lymphodepletion, centre, histological diagnosis. PSM was performed considering a 1:1 matching without replacement and with optimal matching applying a calliper width of the propensity score set at 0.1. Inverse probability of treatment weighting (IPTW) was used as another approach to further validate PSM analysis. Results: 809 patients from 25 French centres with R/R DLBCL after at least 2 lines of previous therapy had a commercial CAR-T order with axi-cel or tisa-cel and were registered in DESCAR-T. Out of 809 patients with a CAR-T order, 60 were not infused due to progression or death between leukapheresis and lymphodepletion and 20 did not proceed to lymphodepletion for other reasons. Finally, 729 proceeded to lymphodepletion and CAR-T infusion. In the 1:1 matched population (N=418, 209 patients treated with tisa-cel and 209 patients treated with axi-cel), the best ORR/CRR was 66/42% versus 80/60% for patients treated with tisa-cel compared with axi-cel, respectively (P<0.001 for both ORR and CRR comparisons). After a median FU of 11.7 months (95% CI, 10.5-12.0 months) the 1-yr PFS was 33% for tisa-cel and 47% for axi-cel (HR=1.65, 95% CI 1.26-2.18, P=0.0003). OS was also significantly longer following axi-cel infusion than following tisa-cel infusion (1-yr OS 63% versus 49%; HR=1.58, 95% CI, 1.13-2.21; P=0.0072). Similar findings were found using IPTW statistical approach. Grade 1-2 CRS were significantly more frequent with axi-cel than tisa-cel (P=0.004) but no significant difference was observed for grade 3 or more CRS (9% versus 5% for tisa-cel and axi-cel respectively, P=0.130). Regarding ICANS, both all grades and severe (i.e. grade ≥3) ICANS were significantly more frequent with axi-cel than tisa-cel. 48% of patients experienced ICANS after axi-cel infusion compared to 22% after tisa-cel infusion. 29 patients (14%) presented a grade ≥3 ICANS with axi-cel compared with 6 (3%) only with tisa-cel. Image: Summary/Conclusion: In conclusion, our matched-comparison study supports a higher efficacy but also a higher toxicity of axi-cel compared with tisa-cel in third or more treatment line for R/R DLBCL. S261: SAFETY AND PRELIMINARY EFFICACY FINDINGS OF AUTO4, A TRBC1-TARGETTING CAR, IN RELAPSED/REFRACTORY TRBC1 POSITIVE SELECTED T CELL NON-HODGKIN LYMPHOMA K. Cwynarski1,*, E. Tholouli2, G. Iacoboni3, T. Menne4, D. Irvine5, L. Wood6, N. Balasubramaniam7, J. Shang8, M Zhang8, K. Duffy9, B. Huber10, M. Vinson11, W. Brugger9, M. Pule12 13 1Haematology, University College London, London; 2Manchester Royal Infirmary, Manchester, United Kingdom; 3VHIO Vall d’Hebron Hospital, Barcelona, Spain; 4Freeman Hospital Newcastle, Newcastle; 5University of Glasgow, Glasgow; 6Cancer Clinical Trials Unit, University College London Hospitals; 7Cancer Clinial Trials Unit, University College London, London, United Kingdom; 8Autolus Therapeutics, Rockville, United States of America; 9Clinical Development, Autolus Therapeutics, London, United Kingdom; 10Autolus Therapeutics, Munich, Germany; 11Autolus Therapeutics, London, United Kingdom; 12Research & Development, Autolus Therapeutics; 13University College London, London, United Kingdom Background: Peripheral T cell lymphomas (PTCL) are typically aggressive, treatment resistant and associated with poor prognosis. Clinical application of immunotherapy is limited by a lack of target antigens that discriminate malignant from normal T cells. Unlike B cell depletion, pan–T cell aplasia is prohibitively toxic. We recently described a targeting strategy based on the mutually exclusive expression of T cell receptor beta-chain constant domains 1 and 2 (TRBC1 and TRBC2) (Maciocia, PM. et al, Nat Med 2017) which can spare a proportion of the normal T cell compartment. Aims: Here we describe early clinical findings of AUTO4, a TRBC1 directed autologous CAR T cell therapy, tested against relapsed/refractory (r/r) TRBC1+ PTCL. Methods: NCT03590574 is multi-centre, single-arm study of AUTO4 with a phase I dose escalation component and a phase II expansion cohort. Here we report the initial findings of the phase I component. Biopsies from patients >18 years of age were screened for TRBC1-positive PTCL using next-generation sequencing. Four flat dose levels were explored: 25 x 106, 75 x 106, 225 x 106, and 450 x 106 CAR T cells administered as a single dose. CAR T-cell products are generated using a semi-automated closed process. Patients received lymphodepletion with fludarabine (30mg/m2 x4, day-6 to day-3) and cyclophosphamide (500mg/m2 x2 on day-6 and day-5) (Flu/Cy) prior to AUTO4 infusion on Day 0. Primary endpoints were incidence of Grade 3 to 5 toxicity occurring within 60 days of AUTO4 infusion and the frequency of dose limiting toxicities within 28 days of AUTO4 infusion (DLT period). Overall response (CR+PR) rate post AUTO4 infusion by PET-CT (Lugano 2014 criteria) was a secondary endpoint. Results: As of 09-FEB-2022, n=64 patients consented for screening of TRBC1-positive PTCL. N=24 samples were TRBC1-positive; 7 patients were screen failures including 1 patient who died during screening. 11 products were manufactured; one patient relapsed prior to AUTO4 infusion, and one patient screen failed after product manufacture. To date 9 patients have been treated with AUTO4. The median age in these 9 patients was 57 years (range 34 to 63 years). The T-cell lymphoma subtypes treated were PCTL-NOS (n=4), ALCL (n=1), and AITL (n=4). Two patients had prior stem cell transplantation. The median number of prior treatment lines was 3 (range 1-5). After lymphodepletion with Flu/Cy, 3 patients received 25 × 106 CAR T cells, 2 patients received 75 × 106 CAR T cells, 1 patient received 225 × 106 CAR T cells and 3 patients received 450 × 106 CAR T cells. No patient experienced any dose limiting toxicities. The median (range) number of CD3+ T-cells/µl in blood prior to lymphodepletion and at the end of the DLT period (Day 28) was 204 (94-698) and 123 (19-458), respectively. 3 patients (33%) experienced CRS (1 patient with Grade 1, 1 patient with Grade 2, and 1 patient with Grade 3). None of the patients experienced neurotoxicity/ICANS. The most common treatment-emergent adverse events were cytopenias (anemia and neutropenia). Of the 9 patients treated, 5 patients had achieved complete metabolic responses (CMR) by PET-CT at Month 1, one patient remains with a PR 6 months post AUTO4 infusion, and 3 patients did not respond. All 3 patients at the 450x106 cell dose achieved a CMR at Month 1. Summary/Conclusion: AUTO4 has a tolerable safety profile in patients with r/r TRBC1+ peripheral T-cell lymphoma. Early data shows encouraging response rates. Updated data and longer follow up will be presented. S262: THE COBALT-LYM STUDY OF CTX130: A PHASE 1 DOSE ESCALATION STUDY OF CD70-TARGETED ALLOGENEIC CRISPR-CAS9–ENGINEERED CAR T CELLS IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) T-CELL MALIGNANCIES S. P. Iyer1,*, R. A. Sica2, P. J. Ho3, B. Hu4, J. Zain5, A. Prica6, W.-K. Weng7, Y. H. Kim8, M. S. Khodadoust9, M. L Palomba10, F. M. Foss11, K. Tipton12, E. L. Cullingford12, Q. He12, A. Sharma12, S. M. Horwitz10 1Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston; 2Department of Oncology, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, United States of America; 3Institute of Haematology, Royal Prince Alfred Hospital, Camperdown, Australia; 4Division of Hematology and Hematologic Malignancies, Huntsman Cancer Institute, Salt Lake City; 5Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, United States of America; 6Princess Margaret Cancer Centre, Toronto, Canada; 7Division of Blood and Marrow Transplantation and Cellular Therapy; 8Department of Dermatology; 9Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford; 10Memorial Sloan Kettering Cancer Center, New York; 11Department of Dermatology, Yale School of Medicine, New Haven; 12CRISPR Therapeutics, Cambridge, United States of America Background: Overall survival (OS) in a subset of patients (pts) with T-cell lymphoma (TCL) has improved with front-line combination chemotherapy; however, R/R TCL pts continue to have very limited treatment options. For pts with R/R peripheral (PTCL) and transformed cutaneous TCL (CTCL), median OS is 1-2.5 and <5 yrs, respectively. Adapting autologous chimeric antigen receptor (CAR) T cell therapy for TCL continues to be challenging due to poor function of donor T cells, fratricide effect, and risk of infusing transduced malignant CAR T cells into pts. CTX130TM is a first-in-class, CD70-targeting allogeneic (allo) CAR T therapy that may allow for CAR T therapy in pts whose own T cells are not ideal to manufacture auto CAR T cells. CD70 is a co-stimulatory protein with temporally limited expression on activated lymphocytes and is highly expressed in many TCLs. CTX130 is modified with CRISPR/Cas9-editing to eliminate expression of: 1) T-cell receptor (TCR) by TCR alpha constant disruption, 2) major histocompatibility complex class I expression by β2-microglobulin disruption, and 3) CD70 to mitigate fratricide and enhance performance. Aims: Investigate safety and efficacy of CTX130 in pts with R/R TCL. Methods: COBALTTM-LYM (NCT04502446) is an open-label, multicenter, global study evaluating the safety and efficacy of CTX130 in pts ≥18 y with CD70+ (≥10% by immunohistochemistry) R/R TCL (PTCL or CTCL). Pts with PTCL and CTCL must have received ≥1 or ≥2 prior lines of systemic therapy, respectively. Pts received lymphodepleting chemotherapy (LDC) with fludarabine 30mg/m2 and cyclophosphamide 500mg/m2 for 3 days, followed by CTX130. Pts were treated with CTX130 at doses from 3x107 (dose level [DL]1) to 9x108 (DL4) CAR+ T cells. Pts could receive a second course of CTX130 if response was not achieved but had experienced clinical benefit or disease progression. The primary endpoint is safety (incidence of dose limiting toxicities [DLTs]). Key secondary endpoints include overall response rate (ORR, by Lugano and ISCL criteria for PTCL and CTCL, respectively), disease control rate (DCR; ≥stable disease [SD]), duration of response and OS. Results: As of 6 Dec, 2021, 17 pts with TCL were enrolled; 15 received CTX130, were evaluable for a Day 28 assessment and are included in the analysis. Among the pts who received CTX130, median age was 67 y, 7 pts had PTCL, and 8 pts had CTCL. Median CD70 expression was 90% (range, 20-100%). Median follow up was 3.1 months. At DL ≥3, ORR was 71%, CR rate was 29% and DCR was 100% (Table). Responses were observed in PTCL (75% ORR at DL≥3) and CTCL (67% ORR at DL≥3) and across disease compartments. CTX130 had an acceptable safety profile across all DLs. There were no instances of graft versus host disease, tumor lysis syndrome, hemophagocytic lymphohistiocytosis, or infusion reactions with LDC or CTX130. There were no DLTs, no Grade (Gr) ≥3 cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS); Gr 1-2 CRS and ICANS were 47% and 20%. There was no increase in frequency or severity of CRS in pts who received second infusions of CTX130. 1 pt (7%) experienced a Gr ≥3 infection. There was a sudden death in 1 pt with William’s syndrome in the context of a lung infection. Image: Summary/Conclusion: We have observed clinically meaningful responses, including CRs with CTX130, the first CAR T directed against the novel target, CD70. CTX130 has an acceptable safety profile in pts with heavily pretreated R/R TCL and will be investigated further in an expansion phase of the study. S263: PHASE I OPEN-LABEL SINGLE ARM STUDY OF GPRC5D CAR-T CELLS (ORICAR-017) IN PATIENTS WITH RELAPTSED/REFRACTORY MULTIPLE MYELOMA (POLARIS) M. Zhang1, Y. Hu1, X. Ding2, Y. Tang2, Y. Yang2, S. Chen2, H. Huang1,* 1Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou; 2Oricell Therapeutics Co., Ltd, Shanghai, China Background: G-protein–coupled receptor class 5-member D (GPRC5D), a type-C 7-pass transmembrane receptor protein, is predominantly expressed on malignant plasma cell phenotype, including most malignant plasma cells from pts with multiple myeloma (MM). The autologous GPRC5D-directed CAR-T cell (OriCAR-017), owning to an additional proprietary Ori element, is the standard second generation CAR-T cell with improvement in expansion and durability of CAR-T cells post-transfusion. Here we report initial results from the phase I open-label single arm study of GPRC5D CAR-T Cells (OriCAR-017) in pts with Relapsed/Refractory (R/R) MM (NCT05016778). Aims: The primary objective in the dose escalation phase was to evaluate safety and tolerability of OriCAR-017. Secondary endpoints included efficacy and pharmacokinetics (expansion and persistence of OriCAR-017. Methods: Key enrolment criteria included adults with measurable MM, R/R or intolerant to established MM therapies, prior BCMA-targeted therapy allowed. Patient received lymphodepleting chemotherapy with Fludarabine 30mg/m2 daily and cyclophosphamide 300mg/m2 daily for 3 days followed by a single infusion of OriCAR-017. The trial followed a standard 3 + 3 design with the following dose cohorts: 1×106/kg, 3×106/kg and 6×106/kg CAR+ T cells. Results: Eleven pts with R/R MM were enrolled and underwent apheresis during June 9, 2021 and February 25, 2022. Nine of them have completed OriCAR-017 infusion to date. 2 pts were suspended infusion due to rapid disease progression. Of the 9 pts infused, median age of 65 years (range: 41-71) and a median of 6 (range 3-17) prior lines of therapy; 4 (44.4%) prior BCMA CART therapy. 3 (33.3%) pts with extramedullary plasmacytoma ≥ 1 and 7 (77.8%) pts with bone marrow plasma cells ≥60% at baseline. Five out of 6 pts had high-risk cytogenetic profiles, including 2 pts with del(17p). No dose-limiting toxicities occurred. The most common treatment-related AEs were hematological toxicities. All pts experienced Cytokine Release Syndrome (CRS, with 8(88.9%) pts in G1, 1 (11.1%) patient in G2), no G3/4 CRS was observed. All CRS cases were rapidly relieved after conventional CRS intervention, including tocilizumab and steroids. No neurologic toxicities were reported to date. All 9pts median follow up time was 118 days (range33—220). A 100.0% ORR were observed with 3 (33.3%) pts achieved CR/sCR, 3 pts (33.3%) were VGPR, 3 pts were PR (33.3%). 4 pts relapsed from BCMA CAR-T therapy had responses with 1 sCR, 2 VGPR, 1 PR. All 9 pts were Minimal Residual Disease (MRD) negative in the bone marrow by flow cytometry (sensitivity: 10-5) at day 28 after infusion, and 6pts continued MRD negative at month 3 and 2 pts at month 6 after infusion. Robust OriCAR-017 expansion in peripheral blood by using qPCR in 3 dose cohorts, median peak was 349866.1copies/ml (range 60837.5 – 2240358.5), median time to peak expansion was 10 days (range 7-14). 8 out of 9 patients detected CAR + cell in bone marrow at D28 after infusion with median 498048.3copies/ml (range 495.4- 2358538.6). Image: Summary/Conclusion: In this phase I study, OriCAR-017 was safe and showed impressive efficacy among previously heavily treated R/RMM pts. Majority of AEs were transient, manageable, and reversible. 100% ORR and 100% MRD negative rate, along with favorable safety evidenced support OriCAR-017 could be a competitive therapy for pts with R/RMM. Furthermore, pts who had relapsed from BCMA CAR-T therapy may still benefit from OriCAR-017 treatment. S264: PRELIMINARY ANALYSES OF A NON-GENE-EDITING ALLOGENTIC CAR-T IN CD19+ RELAPSED OR REFRACTORY NON-HODGIN’S LYMPHOMA X. Wang1, L. Xue1,*, S. Li1, Q. Fan1, K. Liu2, R. Jin2, X. Yang1, T. Wang2, L. He2, J. Li2 1Department of Hematology, The First Affiliated Hospital of USTC, Hefei; 2Fundamenta Therapeutics Inc., Suzhou, China Background: A novel non-gene-editing allogeneic CAR-T platform was developed on the base of intracellular retention of membrane proteins, and named ThisCART (This = TCR and/or HLA-I intracellular sequestered). CD19-directed ThisCART (ThisCART19A) cells are readily produced with a single lentiviral vector, encoding both a CD19-targeting CAR and an anti-CD3 single chain antibody with the KDEL peptide fused to its C-termini (Figure 1). ThisCART19A cells, deficient of surface TCR/CD3 complexes in extended culture, induced no xegoneic GvHD reaction in murine model. Similar to conventional CD19-directed CAR-T, they exhibited potent CD19-specific cytotoxicities both in vitro and in vivo. Aims: To evaluate the safety, pharmacokinetics and clinical activity of ThisCART19A in patients with refractory or relapsed CD19 positive B cell malignancies. Methods: A single-center, open-label, single-arm study (NCT04384393) is ongoing to evaluate the safety, pharmacokinetics and clinical activity of ThisCART19A in patients with refractory or relapsed CD19 positive B cell malignancies, including acute or chronic lymphoblastic leukemia, non-Hodgkin’s lymphoma. Standard or enhanced lymphodepletion with fludarabine and cyclophosphamide was followed by infusion of ThisCART19A at a dose range of 0.2-60x10 6 cells/kg. The pharmacokinetics of ThisCART19A cell was assessed by flow cytometry or quantitative PCR. Results: As of January 30th, 2022, eight evaluble subjects with aggressive or advanced non-Hodgkin’s lymphoma received ThisCART19A infusion (Table 1). Subject #3 had received two subsequent autologous CAR-T therapies and relapsed after each treatment. Adverse event profile is acceptable: no ≥3 grade of CRS or ICANS and no evidence of GvHD reaction. Two subjects experienced reversible Hemophagocytic lymphohistiocytosis with decreased lymphocytes and neutrophils. Dose-dependent CAR-T expansion was observed in six subjects pretreated with enhanced lymphodepletion. Lack of CAR-T expansion in the other two is likely due to the standard lymphodepletion or quick disease progress. PET/CT (≥28 days) scan demonstrated one partial response and five complete responses. Among these five, three remain in remission with one in continued follow-up over 8 months. Table 1 Patient baseline characteristics. Patients No. Pt1 Pt2 Pt3 Pt4 Pt5 Pt6 Pt7 Pt8 Age/Sex 41/F 54/M 42/M 58/F 44/M 40/M 67/F 51/F Pathological type DLBCL MCL DLBCL DLBCL tFL-DLBCL FL DLBCL MZL Ann Arbor/ IPI/FLIPI IV B/4 IV A/3 IV B/3 III B/4 IV B/3 IV A/4 IV B/3 IV B/1 Since Initial Diagnosis 3 yrs 1 yr 4 yrs 1 yr 2 yrs 1 yr 1 yr 3yrs Extranodal disease Yes No Yes Yes No Yes Yes Yes Lines of prior therapies 4 4 4 5 7 5 2 3 Prior auto CAR T No No Auto CART CD19 Auto CART CD20 No No No No No R/R Refractory Refractory Relapsed Refractory Relapsed Refractory Relapsed Relapsed Best Response PR CR CR CR CR CR PD PD Image: Summary/Conclusion: ThisCART19A cells demonstrated potent CD19-dependent cytotoxicity and no xenogentic GvHD in murine models. In this preliminary analysis on evaluable lymphoma subjects, ThisCART19A exhibited an acceptable safety and an encouraging clinical response profiles, both comparable to those for marketed autologous products. S265: RADIOMICS AND ARTIFICIAL INTELLIGENCE FOR IDENTIFICATION AND MONITORING OF SILENT CEREBRAL INFARCTS IN SICKLE CELL DISEASE: FIRST ANALYSIS FROM THE GENOMED4ALL EUROPEAN PROJECT M. P. Boaro1, R. Biondi2, N. Biondini2, A. Collado Gimbert3, E. F. JM4, V. Pinto5, N. Romano6, V. Voi7, G. B. Ferrero8, M. Casale9, M. Cirillo10, G. Palazzi11, F. Cavalleri12, G. L. Forni5, G. Reggiani1, S. Perrotta13, M. Manu Pereira14, S. Zazo15, K. Marias16, M. De Montalembert17, P. Bartolucci18, E. van Beers19, F. Alvarez20, F. Cremonesi21, T. Sanavia22, P. Fariselli22, G. Castellani23, R. Manara24, R. Colombatti1,* 1Woman’s and Child’s Health, University of Padova, Padova; 2Of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy; 3Servei d’Oncologia i Hematologia Pediàtriques, Hospital Universitari Vall d’Hebron; 4Radiology, Hospital Universitari Vall d’Hebron, Barcelona, Spain; 5Centro della Microcitemia, Anemie Congenite e Dismetabolismo del Ferro; 6Department of Diagnostic and Interventional Neuroradiology, Ospedali Galliera, Genova; 7Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale San Luigi Gonzaga, Orbassano; 8Dipartimento di Scienze Cliniche e Biologiche, University of Torino, Torino; 9Donna, del Bambino e di Chirurgia Generale e Specialistica, Università̀ degli Studi della Campania “Luigi Vanvitelli”; 10Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli, Napoli; 11Dipartimento Integrato Materno Infantile, Azienda Ospedale-Università di Modena e Reggio Emilia; 12Neurological Department, University of Modena, Modena; 13Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica, Università̀ degli Studi della Campania “Luigi Vanvitelli”, Napoli, Italy; 14Vall d’Hebron Research Institute, Barcelona; 15Information Processing and Telecommunications Center, Universidad Politécnica de Madrid, Madrin, Spain; 16Computational Medicine Laboratory, Institute of Computer Science, FORTH, Heraklion, Greece; 17Department of Pediatrics, Hopital Necker, Paris; 18Red Cell Genetic Disease Unit, Université Paris Est, Institut Mondor de Recherche Biomédicale (IMRB), Creteil, France; 19Universitair Medisch Centrum Utrecht, Utrecht, Netherlands; 20Universidad Politecnica Madrid, Madrid, Spain; 21Datawizard s.r.l, Roma; 22Medical Sciences, Division of Gastroenterology and Hepatology, Città della Salute e della Scienza di Torino, University of Turin, Torino; 23Department of Physics, University of Bologna, Bologna; 24Neuroscience, University of Padova, Padova, Italy Background: The use of Artificial Intelligence (AI) for personalized medicine has recently guided improvements in the diagnostic pathway of many diseases. The EU Project GENOMED4ALL: “Genomics and Personalized Medicine for All through Artificial Intelligence in Haematological Diseases” aims at using European data of patients affected by Sickle Cell Disease (SCD) to find correlation between -omics data – and phenotype, seizing the opportunity to improve diagnostics through AI. Silent Cerebral Infarcts (SCIs) are a significant cause of morbidity in SCD: they affect 25% of children by the age of 6 and 40% by the age of 18 with consequences on cognition, schooling, working capacity and quality of life. Hence, one of the aims of the SCD clinical case in GENOMED4ALL is the use of radiomics – quantitative method for the evaluation and interpretation of medical images- and AI firstly to develop an automatic and uniform identification and characterization of SCI on MRIs, secondly, to correlate imaging data with other types of omics data in order to predict risk of recurrence Aims: The first phase of the GENOMED4ALL project aimed at collecting anonymized MRI data available in Centers of the EuroBloodNet Network to develop an algorithm that could identify SCI on MRI from different sources, distinguishing them from other lesions Methods: MRI protocol included 3D-T1, FLAIR and DWI sequences. Neuroradiological reports were checked for consistency. A stepwise segmentation (identification of lesion volume), pre-processing and extraction of MRIs was performed utilizing different open-source software: Lesion Segmentation Tools and UNet for segmentation, Freesurfer for brain extraction. To optimize SCI identification, the best software for segmentation was selected Results: Six SCD expert centers participated in the first phase with 501 MRIs: 225 were classified as abnormal by the local neuroradiologists due to presence of SCI; 70% were pediatric MRIs. Different instruments were used in the centers: Philips 1.5 T (n.2), Siemens 1.5 T and 3 T (n.2), GE 3T (n.2). A stepwise procedure allowed optimization of SCI identification, with distinction between SCI and non-clinically significant background (periventricular areas) or other white matter hyperintensities (transient glial maturation). As shown in Figure 1, to segment SCI in FLAIR MRI, we used a pre-trained UNet [Li H, 2018] winner of the MICCAI challenge. Firstly, we extracted the brain registering the MNI152 on the T1 image. Then we applied the brain mask and a threshold taking only the largest component. The UNet performed the segmentation of hyperintensities. We removed the regions surrounded by less than 90% of White Matter or near the brain ventricles to remove non-SCI region (refinement). We then enlarged the remaining ones. To date, segmentation on 303 exams from different centers showed very few false positive and some false negatives highlighting the need to take into account different technical characteristics (various equipment in different centers) Image: Summary/Conclusion: SCD is a rare systemic disorder with extreme phenotypic variability. Radiomics and AI offer the opportunity to seize the potential of big dataset analysis to understand natural history and optimize diagnostics. SCI can be detected automatically from different datasets. Four more European Centers will add their MRI in the second phase, in order to increase variability and allow correlation of detailed Radiomics features with clinical-hematological variables and other omics data S266: OXYGEN GRADIENT EKTACYTOMETRY-DERIVED BIOMARKERS ARE ASSOCIATED WITH THE OCCURRENCE OF ACUTE COMPLICATIONS IN SICKLE CELL DISEASE M. Rab1,*, C. Kanne2, C. Boisson3, J. Bos1, B. van Oirschot1, M. Houwing4, C. Renoux5, R. Schutgens6, M. Bartels6, A. Rijneveld7, E Nur8, M. Cnossen4, P. Joly5, R. Fort9, P. Connes3, R. van Wijk1, V. Sheehan2, E. van Beers6 1Central Diagnostic Laboratory-Research, University Medical Center Utrecht, Utrecht, Netherlands; 2Department of Pediatrics, Emory University School of Medicine, Atlanta, United States of America; 3Laboratory LIBM EA7424, University of Lyon; 1, Lyon, France; 4Department of Pediatric Hematology, Erasmus University Medical Center, Rotterdam, Netherlands; 5Laboratory of Biochemistry and Molecular Biology, Hospices Civils de Lyon, Lyon, France; 6Van Creveldkliniek, University Medical Center Utrecht, Utrecht; 7Department of Hematology, Erasmus University Medical Center, Rotterdam; 8Department of Hematology, Amsterdam University Medical center, Amsterdam, Netherlands; 9Department of Internal Medicine, Hospices Civils de Lyon, Lyon, France Background: Sickle cell disease (SCD) is a monogenetic disorder with a highly complex pathophysiology. There is an unmet need for robust reproducible biomarkers that can assess red blood cell (RBC) function and predict disease severity and complications. Aims: To explore the association between oxygen gradient ektacytometry-derived biomarkers, and blood viscosity with incidence of major (acute) SCD-related complications. Methods: Oxygen gradient ektacytometry measures RBC deformability continuously while the sample is gradually deoxygenated and subsequently reoxygenated, and identifies the oxygen tension at which sickling occurs. We examined associations between the occurrence of acute chest syndrome, cerebral infarction and vaso-occlusive crisis (VOC) and known biomarkers such as fetal hemoglobin, blood viscosity as well as exploratory oxygen gradient ektacytometry-derived biomarkers in an adult cohort of 50 individuals with SCD (HbSS or HbS/βo-thalassemia) and a pediatric cohort consisting of 177 children with SCD (HbSS or HbS/βo-thalassemia). A substantial number of subjects were on hydroxyurea therapy (64% of adults and 89% of children). Subjects that received a blood transfusion less than three months prior to measurements were excluded from the study. A logistic regression analysis was performed; odds ratios were adjusted for age and hydroxyurea therapy. Results: In the adult cohort, for every 10 mmHg increase in Point of Sickling (PoS, the pO2 tension where RBCs start to sickle, reflecting sickling tendency) the likelihood of >1 acute complication increased; the adjusted odds ratio (aOR) was 3.00 (p=0.015). For every 0.1 increase in EImax (reflecting RBC deformability at normoxia), the aOR was 0.33 (p=0.035, Table 1). In the pediatric cohort, for every 10 mmHg increase in PoS, the likelihood of >1 acute complication increased; the aOR was 1.65 (p=0.006). For every 0.1 increase in EImin (reflecting RBC deformability at hypoxia), the aOR was 0.50 (p=0.007). Fetal hemoglobin and blood viscosity levels were not associated with likelihood of multiple acute complications. However, fetal hemoglobin was associated with reduced likelihood of VOC in adults (aOR of 0.32 for every 10% increment, p=0.010) but not in children (aOR of 0.68, p=0.231, data not shown). In the adult cohort higher EImax was associated with reduced likelihood of VOC (aOR 0.31, p=0.029). There was a trend found for an association between higher PoS and greater likelihood of VOC (aOR 2.22, p=0.050), and no association for EImin. In the pediatric cohort only EImin was associated with VOC (aOR 0.68, p=0.036). Image: Summary/Conclusion: These findings indicate that oxygen gradient ektacytometry generates novel clinically relevant biomarkers and provide a rationale for further development of these biomarkers in the evaluation of novel therapies, as part of clinical care, or clinical trial endpoints. In particular, in assessment of treatment strategies that do not target HbF induction, such as pyruvate kinase activators, voxelotor and l-glutamin, oxygen gradient ektacytometry can generate relevant biomarkers. S267: PROPERDIN-BLOCKING ANTIBODIES ATTENUATE COMPLEMENT ALTERNATIVE PATHWAY ACTIVATION TRIGGERED BY CELL-FREE HEME IN SICKLE CELL DISEASE MODELS T. Trovati Maciel1, R. Cofiell2, C. Carvalho1, S. Allali1, R. Rignault1, A. Bennet3, Y. Dai3, P. Tamburini4, C. Gasteyger5, O. Hermine1, S. K. Kim2,* 1Laboratory of Cellular and Molecular Mechanisms of Hematological Disorders and Therapeutical Implications, Imagine Institute, Inserm, Paris, France; 2Translational Research, Alexion AstraZeneca Rare Disease, New Haven, CT; 3Clinical Development and Translational Sciences, Alexion AstraZeneca Rare Disease, Boston, MA; 4Research, Alexion AstraZeneca Rare Disease, New Haven, CT, United States of America; 5Clinical Development and Translational Sciences, Alexion AstraZeneca Rare Disease, Zurich, Switzerland Background: Complement activation may play an important role in sickle cell disease (SCD) pathophysiology. Heme has been identified as a trigger of complement activation in SCD patients and is present in excess during severe vaso-occlusive crises. Heme is also a DAMP (damage associated molecular pattern) and induces thrombo-inflammation through tissue factor expression. ALXN1820 is a bi-specific VHH nanobody that simultaneously binds human albumin and properdin, thereby effectively and selectively inhibiting alternative pathway (AP) activation and formation of C3 and C5 convertases. The safety of ALXN1820 has been assessed in non-human primates and is currently being tested in a phase 1 study in healthy participants. Aims: 1) To assess if complement activation and heme are functionally linked; 2) To investigate the therapeutic potential of ALXN1820 in in vitro SCD models and in in vivo SCD models using a mouse properdin-blocking monoclonal antibody (mAb), 14E1. Methods: In vitro, complement inactivated C3b (iC3b) and C5b-9 deposition on red blood cells (RBCs) from patients with SCD and complement iC3b and C5b-9 deposition on the endothelial cell line HMEC-1 were assessed by flow cytometry after exposure to heme +/- ALXN1820. In vivo, in Townes SCD mice, vaso-occlusion and hyper-hemolysis were induced by intravenous heme injection (50 µmol/kg) or hypoxia-reoxygenation (8% O2 for 3h then 21% O2 for 1h) in the presence and absence of 14E1 (40 mg/kg), a mouse-specific properdin inhibitor. Vaso-occlusion was measured by staining RBCs with immunofluorescence-labeled Ter-119 antibodies on lung and liver sections. C3b and C5b-9 deposition on SCD RBCs was assessed by flow cytometry. Markers of hemolysis (bilirubin, lactate dehydrogenase, free hemoglobin, free heme) were quantified in plasma using commercial assays. Results: In RBCs from patients with SCD, cell-free heme triggered marked iC3b and C5b-9 deposition (Table). Deposition was blocked in the presence of ALXN1820 by >95% for iC3b and by >85% for C5b-9. HMEC-1 cells exposed to heme showed marked deposition of iC3b and C5b-9. In the presence of ALXN1820, deposition was blocked by >70% for iC3b and >85% for C5b-9. Pretreatment of mice with 14E1 markedly ameliorated vaso-occlusion and reduced both C3b and C5b-9 deposition on RBCs, and hemolysis biomarkers. Image: Summary/Conclusion: These data strengthen the hypothesis that in SCD, cell-free heme is a potent trigger of complement AP activation, which can be blocked by targeting properdin. Investigating the efficacy and safety of anti-properdin ALXN1820 in patients with SCD is warranted as a novel approach to treatment of acute and chronic SCD complications. S268: SAFETY, TOLERABILITY, AND PHARMACOKINETIC/PHARMACODYNAMIC RESULTS FROM PHASE 1 STUDIES OF GBT021601, A NEXT-GENERATION HBS POLYMERIZATION INHIBITOR FOR TREATMENT OF SICKLE CELL DISEASE C. Brown1,*, C. Key2, I. Agodoa3, J. Olbertz3, K. Duchin3, A. Barth3, E. Lisbon3 1Aflac Cancer and Blood Disorder Center of Children’s Healthcare of Atlanta and Department of Pediatrics, Emory School of Medicine, Atlanta; 2ICON plc, San Antonio; 3Global Blood Therapeutics, South San Francisco, United States of America Background: Sickle cell disease (SCD) is caused by polymerization of sickle hemoglobin (HbS). Voxelotor is a first-in-class HbS polymerization inhibitor approved by the United States Food and Drug Administration for the treatment of SCD in adult and pediatric patients aged ≥4 years, and by the European Medicines Agency for the treatment of hemolytic anemia due to SCD in adult and pediatric patients aged ≥12 years as monotherapy or in combination with hydroxycarbamide. GBT021601 is a next-generation HbS polymerization inhibitor with improved pharmacokinetic (PK) properties that stabilizes Hb in the oxygenated state, thus inhibiting polymerization. GBT021601 has the potential to achieve higher Hb occupancies at lower doses than voxelotor, potentially reducing treatment burden and improving clinical outcomes for patients with SCD. Aims: To explore the safety, tolerability, PK, and pharmacodynamics of GBT021601 in healthy volunteers and patients with SCD. Methods: Two studies are in progress: (1) GBT021601-011: a phase 1, randomized, double-blind, placebo-controlled, parallel-group study of single ascending doses (SADs) and multiple ascending doses (MADs) of GBT021601 in healthy volunteers aged 18 to 55 years; and (2) GBT021601-012: a phase 1, single-arm, intrapatient, single-dose and MAD study in patients with SCD (homozygous HbS) aged 18 to 60 years. In the GBT021601-011 SAD phase, participants (n=8) were randomized 6:2 to receive a single oral dose of GBT021601 (50 to 2200 mg) or placebo. In the GBT021601-011 MAD phase, participants (n=10) were randomized 7:3 to 14 days of GBT021601 (3 days of loading doses followed by 11 days of once-daily maintenance doses) or placebo. Data from the 50 mg MAD cohort are reported in this analysis. In the GBT021601-012 study, patients with SCD (N=6) received a single 100 mg dose of GBT021601, followed by an 8-week washout, then 2 continuous MADs: once-daily maintenance doses of 50 mg (5 weeks) then 100 mg (3 weeks). Each MAD phase participant received 2 days of loading doses. Results: In the GBT021601-011 50 mg MAD cohort, blinded safety data demonstrated that GBT021601 was well tolerated. Adverse events (AEs) were predominantly grade 1 and not related to treatment. Drowsiness, headache, and constipation were the most frequent AEs. Mean (SD) Hb occupancy of GBT021601, determined via PK analysis, was 25.8% (3.9%). In GBT021601-012, patients with SCD completed treatment with no AEs leading to study discontinuation. Three vaso-occlusive crises (grades 2 and 3) occurred in 2 patients and were deemed unrelated to GBT021601. After dosing for 8 weeks, the mean (SD) Hb occupancy achieved was 32.6% (9.4%), and the mean (SD) increase in Hb was 2.3 (0.9) g/dL. Markers of hemolysis decreased, with a mean (SD) change in absolute reticulocytes of –37.4% (17.3%), reticulocyte percentage of –54.0% (11.3%), indirect bilirubin of –6.6% (119.3%), and lactate dehydrogenase of –33.1% (15.4%). The terminal elimination half-life of GBT021601 from blood averaged about 10 days. Ektacytometry scans demonstrated improvements in red blood cell (RBC) health, and blood smears showed increased counts of RBCs with improved morphology (Figure); no changes in erythropoietin levels were observed. Image: Summary/Conclusion: Multiple daily doses of GBT021601 were well tolerated in both studies. In patients with SCD, the GBT021601 100 mg maintenance dose led to a mean Hb occupancy >30%, increased Hb, reduced markers of hemolysis, and improved RBC health. These data highlight the potential of GBT021601 and support its further development. S269: MACROPHAGE FUNCTIONAL AND METABOLIC REWIRING THROUGH PPAR/PGC1 MODULATION IMPROVES HEME/IRON-DRIVEN DEFECTIVE EFFEROCYTOSIS AND PROMOTES TISSUE DAMAGE RESOLUTION IN SICKLE CELL DISEASE R. Sharma1, S. Z. Vance1, A. Antypiuk1, D. Manwani2, F. Vinchi1,* 1Iron Research Laboratory, New York Blood Center; 2Pediatric Hematology, The Children’s Hospital at Montefiore, New York, United States of America Background: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is contributed by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme, which is released from oxidized hemoglobin upon hemolysis and acts as damage-associated molecular pattern to stimulate macrophage pro-inflammatory phenotypic switching through TLR4 signaling activation and ROS production. Heme-induced M1 pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to organ damage typically associated with this disease. Aims: While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties and whether this exacerbates tissue injury remain unexplored. Macrophage functions as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages, to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme strongly alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme and iron excess drives defective efferocytosis of apoptotic neutrophils in vitro, in cultured bone-marrow-derived macrophages, and in vivo, in mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the transcription factor PPARγ and its coactivator PGC1α. These changes lead to reduced expression of efferocytic receptors and impaired mitochondrial biogenesis as well as mitochondrial dysfunction, and is associated with metabolic skewing. This results in limited recognition and engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4/10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. We further demonstrate that impaired phagocytic capacity and tissue damage are improved by hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment in SCD mice through phenotypic and functional macrophage rewiring. Interestingly, defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients’ plasma and rescued by hemoglobin/heme scavenging via haptoglobin and hemopexin and PPARγ/PGC1α modulation via PPARγ agonist or IL-4. Summary/Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events. S270: LONGER-TERM ANALYSIS OF EFFICACY OF LUSPATERCEPT VERSUS PLACEBO IN PATIENTS WITH TRANSFUSION-DEPENDENT BETA-THALASSEMIA ENROLLED IN THE BELIEVE STUDY M. D. Cappellini1,*, A. T. Taher2, J. B. Porter3, K. H. Kuo4, T. D. Coates5 6, E. Voskaridou7, G. L. Forni8, S. Perrotta9, A. Khelif10, A. Lal11, A. Kattamis12, A. Piga13, O. Hermine14 15, N. Holot16, F. Lersch17, J. K. Shetty17, S. Vodala16, J. Zhang16, D. Miteva17, V. Viprakasit18 1Fondazione IRCCS Ca’ Granda Policlinico Hospital, University of Milan, Milan, Italy; 2Department of Internal Medicine, American University of Beirut Medical Center, Beirut, Lebanon; 3University College London, University College London Hospitals, London, United Kingdom; 4Division of Medical Oncology and Hematology, Department of Medicine, University Health Network, Division of Hematology, Department of Medicine, University of Toronto, Toronto, ON, Canada; 5Children’s Center for Cancer and Blood Diseases, Children’s Hospital Los Angeles; 6USC Keck School of Medicine, Los Angeles, CA, United States of America; 7Thalassemia and Sickle Cell Center, Laiko General Hospital, Athens, Greece; 8Centro della Microcitemia e Anemie Congenite e del Dismetabolismo del Ferro, Ospedale Galliera, Genoa; 9Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica, Università della Campania, Luigi Vanvitelli, Naples, Italy; 10Farhat Hached Teaching Hospital, Sousse University, Sousse, Tunisia; 11University of California San Francisco Benioff Children’s Hospital, Oakland, CA, United States of America; 12First Department of Pediatrics, National and Kapodistrian University of Athens, Athens, Greece; 13University of Turin, Turin, Italy; 14Department of Hematology, Hôpital Necker, Assistance Publique Hôpitaux de Paris, University Paris Cité; 15INSERM U1163 and CNRS 8254, Imagine Institute, Université Sorbonne Paris Cité, Paris, France; 16Bristol Myers Squibb, Princeton, NJ, United States of America; 17Celgene International Sàrl, a Bristol-Myers Squibb Company, Boudry, Switzerland; 18Siriraj Hospital, Mahidol University, Bangkok, Thailand Background: Ineffective erythropoiesis and anemia are hallmarks of β-thalassemia. Luspatercept, a first-in-class erythroid maturation agent, has shown efficacy in the phase 3 BELIEVE (NCT02604433) trial in patients (pts) with β-thalassemia requiring regular red blood cell transfusions (RBCT). However, longer-term analyses of efficacy are yet to be reported. Aims: To observe and report the longer-term effect of luspatercept treatment on transfusion burden and liver iron concentration (LIC). Methods: Pts ≥18 years of age with β-thalassemia or hemoglobin E/β-thalassemia (compound β-thalassemia mutation and/or multiplication of α-globin genes allowed) requiring regular RBCT (6–20 RBC units/24 wk before randomization with no transfusion-free period >35 d) were randomized 2:1 to luspatercept 1.0 mg/kg (titration up to 1.25 mg/kg allowed) or placebo subcutaneously every 3 wk. Response was defined as RBCT burden reduction of ≥33% (≥33% response) or ≥50% (≥50% response) from baseline (BL; ≥2 units) during a rolling 12- or 24-wk interval. LIC was determined by MRI. Results: As of Jan 5, 2021, 125/224 (55.8%) pts randomized to luspatercept completed 144 wk of treatment and 6 (2.7%) pts completed 192 wk. The main reasons for treatment discontinuation were pt withdrawal (23.7% luspatercept vs 11.6% placebo), adverse events (10.3% vs 1.8%), and lack of efficacy (1.8% vs 7.1%). More pts receiving luspatercept vs placebo had ≥33% or ≥50% response during any rolling 12- or 24-wk interval up to the cutoff date (all P<0.0001); the number of responders receiving luspatercept was higher compared with previous data cuts (Table). The median (95% CI) longest duration of response for ≥33% and ≥50% responders (rolling 12-wk interval) in the luspatercept group increased from the primary data cut to 114.0 (107.0–137.0) and 99.0 (95.0–104.0) d, respectively (Table); median (95% CI) longest duration of response for placebo pts was 90.0 (86.0–94.0) and 86.0 (84.0–103.0) d, respectively. The median (range) total duration of response for ≥33% and ≥50% responders (rolling 12-wk interval) in the luspatercept group was 586.0 (84–1300) and 357.5 (84–1267) d, respectively, and 171.0 (84–627) and 169.0 (84–485) d in the placebo group. The 6 pts who completed 192 wk of luspatercept required on average 6.41 (SD 4.32) fewer RBC units during wk 145–192 compared with BL. Overall mean change from BL in transfusion window for ≥50% responders (any 24-wk interval) was increased by 9.88 d (SD 22.04, n=51). More pts receiving luspatercept (27/224 [12.1%]) than placebo (2/112 [1.8%]) achieved RBCT independence (RBC-TI) ≥8 wk (P=0.0015), an increase from previous data cuts (Table). Median (95% CI) longest durations of RBC-TI were 72.0 (62.0–103.0) d for luspatercept and 71.5 (62.0–NA) for placebo, though the placebo result was driven by 2 pts. Longer-term treatment with luspatercept resulted in a decreasing trend in LIC compared with BL for ≥33% responders (rolling 12-wk interval) with a mean (SD) change from BL in LIC at wk 144 (n=25) of −3.73 (9.08) mg/g dry weight. Image: Summary/Conclusion: Continued treatment with luspatercept resulted in more pts experiencing reduced RBCT burden and longer duration of response compared with previous data cuts. RBC units transfused decreased over the longer-term treatment period and the time between transfusions increased compared with BL for ≥50% responders. Longer-term luspatercept treatment also resulted in more pts experiencing RBC-TI ≥8 wk and a potential decreasing trend in LIC; LIC analysis is ongoing. These data indicate that pts continue to benefit from longer-term luspatercept treatment. S271: BONE MARROW TFR2 DELETION IMPROVES THE THERAPEUTIC EFFECT OF ACTIVIN LIGAND TRAP RAP-536 IN Β-THALASSEMIC MICE E. Tanzi1 2, S. M Di Modica1, J. Bordini3, V. Olivari1 4, M. Pettinato1, A. Pagani1, A. Campanella3 4, L. Silvestri1 4, A. Nai1 4,* 1Division of Genetics and Cell Biology, Ospedale San Raffaele; 2University of Milan Bicocca; 3Division of Experimental Oncology, Ospedale San Raffaele; 4Vita-Salute San Raffaele University, Milan, Italy Background: β-thalassemia is a disorder due to mutations in the β-globin gene, characterized by ineffective erythropoiesis (IE), anemia, and iron overload, whose treatment still requires improvement. Luspatercept, an activin receptor-ligand trap that promotes terminal erythroid differentiation downregulating the TGF-β pathway is a recently approved drug for treatment of transfusion dependent β-thalassemia (TDT) and in phase 3 trial for non TDT (NTDT). However, its precise mechanism of action remains to be elucidated and the possible connection with the erythropoiesis driving cytokine erythropoietin (EPO) is still controversial. In addition, Luspatercept is only partially effective in correcting anemia, thus the identification of strategies for enhancing its efficacy would be of clinical benefit. The second transferrin receptor (TFR2) balances red blood cells (RBC) production with iron availability, contributing to the activation of the iron hormone hepcidin in the liver and acting as an EPO receptor partner in erythroid cells. We have demonstrated that bone marrow (BM) Tfr2 deletion increases erythroblast EPO sensitivity causing erythrocytosis in wild-type mice (Nai et al, Blood 2015) and improving IE, anemia, RBCs morphology and iron-overload in NTDT mice (Artuso et al, Blood 2018). Aims: As we have previously demonstrated that the combined targeting of different pathophysiologic cues of the disease results in a synergistic benefit on thalassemic erythropoiesis and anemia (Casu, Pettinato, et al, Blood 2020), here we investigate whether BM Tfr2 genetic inactivation improves the efficacy of Luspatercept in a NTDT model (Hbb th3/+ ). Methods: Hbb th3/+ mice with BM Tfr2 deletion (Tfr2 BMKO /Hbb th3/+ ) and Hbb th3/+ controls were treated with RAP-536 (Celgene, BMS group), a murine analogue of Luspatercept, 10mg/kg, twice a week for 8 weeks. Complete blood count was evaluated every two weeks. At sacrifice, blood and organs were collected for full phenotypic analysis. Results: As expected, BM Tfr2 deficiency strongly ameliorated anemia and IE of Hbb th3/+ mice (hemoglobin, Hb=13.48±0.19g/dL vs 8.65±0.24g/dL in controls, P<0.000001). RAP-536 moderately increased RBC count, Hb and hematocrit after 2 weeks of treatment, and the beneficial effect was maintained until the end of the protocol with a similar efficacy in Hbb th3/+ (Hb=9.54±0.33g/dL) and Tfr2 BMKO /Hbb th3/+ mice (Hb=14.48±0.50g/dL), without significant changes in MCV, MCH and reticulocyte count. Splenomegaly was not affected by RAP-536 in Hbb th3/+ mice, despite an amelioration of both spleen and BM IE, with reduced proportion of immature erythroblasts and increased mature RBCs. Erythroid differentiation was further improved in RAP-536-treated Tfr2 BMKO /Hbb th3/+ mice both in the spleen and in the BM, leading to a significant reduction of spleen size, to values of wt animals. Hepatic and serum iron levels were reduced in mice lacking Tfr2, likely because of high erythroid consumption, while RAP-536 did not affect iron homeostasis in both genotypes. Summary/Conclusion: BM Tfr2 deletion and RAP-536 treatment had additive effect in correcting IE and anemia in β-thalassemic mice. Since RAP-536 has comparable therapeutic efficacy in Hbb th3/+ and Tfr2 BMKO /Hbb th3/+ animals, it improves erythropoiesis through mechanism(s) other than Tfr2 deletion, thus independent of EPO stimulation. Overall, our results add a piece of information on the mechanism of action of Luspatercept and suggest that the concomitant TFR2 targeting might represent a promising option for boosting the therapeutic efficacy of activin receptor ligand-traps. S272: SAFETY AND PRELIMINARY PHARMACODYNAMIC EFFECTS OF THE FERROPORTIN INHIBITOR VAMIFEPORT (VIT-2763) IN PATIENTS WITH NON-TRANSFUSION-DEPENDENT BETA THALASSEMIA (NTDT): RESULTS FROM A PHASE 2A STUDY A. Taher1,*, A. Kourakli-Symeonidis2, A. Tantiworawit3, P. Wong4, P. Szecsödy5 1American University of Beirut Medical Center, Beirut, Lebanon; 2University of Patras – Rio Regional University Hospital, Rio, Greece; 3Chiang Mai University – Faculty of Medicine, Chiang Mai; 4Naresuan University Hospital, Mueang Phitsanulok, Thailand; 5Vifor Pharma AG, Glattburg, Switzerland Background: NTDT is characterized by an imbalanced α/β-globin chain ratio, ineffective erythropoiesis, increased intestinal iron absorption and iron overload. Vamifeport inhibits ferroportin-mediated iron export into plasma and has been shown to decrease serum iron and transferrin saturation (TSAT) levels in relevant animal disease models and healthy volunteers. Aims: The primary objective of this multicenter, Phase 2a, double-blind, randomized, placebo-controlled study (NCT04364269) was to assess the safety and tolerability of vamifeport (given once [QD] or twice [BID] daily) versus placebo in patients with NTDT over a 12-week treatment period. A secondary objective was to assess preliminary efficacy of vamifeport versus placebo with respect to pharmacodynamic effects on iron parameters (serum iron, ferritin, hepcidin and TSAT) in these patients. Methods: Adults (18–65 years) with documented NTDT (including β-thalassemia intermedia) who provided informed consent, were included. NTDT was defined as receipt of <5 units of red blood cells during the 24 weeks prior to randomization. Patients also required a mean baseline hemoglobin of ≤11 g/dL on two consecutive measurements ≥1 week apart within 6 weeks prior to randomization. Use of iron chelation therapy (ICT) within 4 weeks prior to randomization was not permitted; patients with TSAT <30%, serum ferritin <150 ng/mL and/or liver iron content ≤1 mg/g (<300 ng/mL/3 mg/g, respectively, for those recently on ICT) were also excluded. Patients received vamifeport at a dose of 60 mg if their body weight was 40–59 kg or 120 mg if their weight was 60–100 kg. Results: Twenty-five patients were included (vamifeport QD n=9, BID n=12, placebo n=4; median age 42.0, 31.0 and 40.5 [range 18–61] years, respectively). Overall, 64% were male, 56% had a body weight <60 kg and 16% had received prior ICT. At baseline, mean (standard deviation [SD]) serum iron concentrations were 27.2 (11.6) µmol/L and mean TSAT was 76.2 (27.6)% for the population as a whole. Mean (SD) serum ferritin concentrations were 746.7 (1431.7) µg/L. There were no differences between groups in treatment-emergent adverse event (TEAE) rates (vamifeport QD 66%, BID 58%; placebo 75%). All TEAEs were mild or moderate intensity, and each was reported only once. There were no deaths or serious AEs (1 patient receiving vamifeport QD had an acute hemolytic event); one TEAE (increased creatine kinase and transaminase levels, not drug-related) led to drug discontinuation in the vamifeport BID group. There were also no clinically relevant changes in any assessed safety parameter. Serum iron concentrations decreased 3 h after first vamifeport dose (Day 1) in all vamifeport-treated patients (mean [SD] decrease: QD -11.3 [7.2] µmol/L; BID -17.0 [9.6] µmol/L) and were maintained below baseline levels at 3–4 h post vamifeport administration at each visit throughout the remaining treatment period. Mean (SD) TSAT also decreased at Day 1 (3 h post dose; QD -32.6 [19.6]%; BID -46.8 [22.6]%) and were below baseline levels 3–4 h post-dose at each subsequent visit in the vamifeport groups (Figure). There were no clinically meaningful changes from baseline in serum iron or TSAT levels in the placebo group, or in ferritin or hepcidin levels in any treatment group. Image: Summary/Conclusion: In this 12-week Phase 2a study, vamifeport had a favorable safety and tolerability profile and showed promising target engagement and pharmacodynamic effects on serum iron and TSAT levels versus placebo in adults with NTDT. S273: EVIDENCE OF NONINFERIORITY OF MITAPIVAT VERSUS SPLENECTOMY IN MURINE HEREDITARY SPHEROCYTOSIS A. Matte’1,*, A. Recchiuti2, E. Federti1, P. A. Kosinski3, V. Riccardi1, I. Iatcenko1, L. Dang3, A. Iolascon4, R. Russo4, C. Brugnara5, A. Janin6, C. Leboeuf6, N. Mohandas7, L. De Franceschi1 1Dept of Medicine, University of Verona, Verona; 2Dept of Medical, Oral, and Biotechnological Science, “G. d’Annunzio” University of Chieti, Chieti, Italy; 3Agios Pharmaceuticals, Cambridge, United States of America; 4Dept. of Molecular Medicine and Medical Biotechnology and CEINGE, University of Naples Federico II, Naples, Italy; 5Dept of Laboratory Medicine, Boston Children’s Hospital - Harvard Medical School, Boston, United States of America; 6University Diderot of Paris, Paris, France; 7Laboratory of Red Cell Physiology, New York Blood Center, New York, United States of America Background: Hereditary spherocytosis (HS) is the most common cause of inherited red cell membranopathy, due to mutations in genes encoding membrane or cytoskeletal proteins, including band 3, ankyrin, spectrin or band 4.2. Loss of membrane cohesion results in surface area loss and generation of spherocytic red cells with decreased cellular deformability and reduced red cell survival due to splenic sequestration. Relative PK deficiency has been observed in HS red cells (Andreas O et al. BJH 187: 386, 2019). Splenectomy is the gold standard treatment of anemia in patients with HS. However, the benefits of splenectomy in mild HS is still controversial and its cost/effectiveness might be underestimated. Using band 4.2-/- mice, a well-established model of HS (Peters LL et al JCI 103: 1527, 1999), we recently show that mitapivat, an oral PK activator, ameliorates anemia, reduces chronic hemolysis and improves RBC features in 4.2-/- mice (Matte A et al. EHA 2021). Aims: To understand whether the oral pyruvate kinase activator, mitapivat, is not less efficacious than splenectomy in treatment of anemia in 4.2-/- mice. Methods: Splenectomized 4.2-/- mice (2.5 months of age) were either treated with vehicle or mitapivat at dosages of 100 mg/kg/day over 4 months. An unsplenectomized group of 4.2-/- mice treated with and without mitapivat was also analyzed. The following parameters were evaluated: hematologic parameters, markers of hemolysis, erythropoiesis and iron homeostasis. Results: In band 4.2-/- mice, splenectomy resulted in (i) amelioration of anemia with reduced reticulocyte count (Hb 13.3 ±0.5 g/dL, n=3 vs 11.9±0.6 g/dL, n=8; P<0.05; retics: 4.9±0.5%, n=3 vs 10.0±1.4%, n=8 P<0.05) and reduced hemolytic indices (LDH, total bilirubin) when compared to non-splenectomized 4.2-/- mice. The decreased hemolysis was associated with reduction in serum erythropoietin (EPO). Mitapivat was non-inferior to splenectomy for amelioration of anemia (Hb 13.4±0.8 g/dL, n=11 vs 11.9±0.6 g/dL, n=8), reduction in reticulocyte count (retics: 7.8±1.4%, n=11 vs 10.0±1.4%, n=8 P<0.05) and EPO levels (401±123 U/L, n=3). Noteworthy, in splenectomized 4.2-/- mice, mitapivat further improved Hb (Hb: 14.1±0.3 g/dL, n=3), reduced reticulocyte counts (retics: 3.6±0.2%, n=3) and EPO levels when compared to vehicle treated splenectomized 4.2-/- mice. Image: Summary/Conclusion: Our data indicate that mitapivat was at least as effective as splenectomy in treatment of anemia in a murine model of HS. S274: A NOVEL SUBTYPE OF ANEMIA CAUSED BY MUTATIONS IN TFRC GENE S. Colucci1 2,*, V. Venturi3, F. Nicole1 2, D. Jové Solavera3, M. Zimon4, P. Richter-Pechanska1, G. Hernandez3 5, S. Unal6, F. Gumruk6, A. Diaz-Conradi7, L. Romero-Cortadellas3, X. Ferrer-Cortès3 5, M. Olivella8, M. Erlacher9, C. Niemeyer9, T. Wiesel10, R. Pepperkok4, M. D. Fleming11, A. E. Kulozik1 2, M. Sanchez3 5, M. U. Muckenthaler1 2 1Department of Pediatric Oncology, Hematology, and Immunology, Heidelberg University; 2Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany; 3Department of Basic Sciences, Iron metabolism: Regulation and Diseases, Universitat Internacional de Catalunya (UIC), Sant Cugat del Vallès, Spain; 4European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 5BloodGenetics S.L. Diagnostics in Inherited Blood Diseases, Esplugues de Llobregat, Spain; 6Department of Pediatrics, Hacettepe University Faculty of Medicine, Ankara, Turkey; 7Hospital de Nens de Barcelona, Barcelona; 8Biosciences Department, Faculty of Sciences and Technology (FCT), University of Vic - Central University of Catalonia, Vic, Spain; 9Department of Pediatrics and Adolescent Medicine, Medical Center, University of Freiburg, Freiburg; 10Vestische Kinder- und Jugendklinik Datteln, University Witten/Herdecke, Datteln, Germany; 11Department of Pathology, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America Background: Anemia affects ~25% of the world population and is frequently caused by iron deficiency as a consequence of malnutrition or inflammation. Only in rare cases anemia is due to mutations in genes responsible for balancing iron homeostasis. Among these, loss of function variants of MT2 cause iron-refractory iron-deficiency anemia (IRIDA); a disease characterized by inappropriately high levels of hepcidin. Hepcidin is a hepatic hormone that limits dietary iron uptake and iron release from intracellular stores and thus renders IRIDA patients resistant to iron therapies. In mice, a form of anemia resistant to iron supplementation is further caused by mutations in Transferrin Receptor 1 (TfR1; Tfrc gene). TfR1 internalizes iron-bound transferrin, a process fundamental for erythropoiesis. However, up to date, the sole human report describing a TfR1 mutation (p.Tyr20His) showed that it impairs the immunological compartment rather than erythroblast maturation. Here, we report pediatric patients with previously undescribed mutations in TFRC, diagnosed with microcytic hypochromic anemia, partially resistant to iron therapies. Aims: Identification of novel disease alleles that cause anemia. Methods: Whole exome sequencing (WES) of DNA of patients with anemia. Functional characterization of novel mutations identified in TFRC gene was assessed in HeLa cells overexpressing TfR1 wild-type and mutated by western blotting and wide-field microscopy. Results: A 4-year old boy manifested symptoms of anemia and the analysis of hematological parameters revealed low levels of hemoglobin, MCV and MCH. Plasma iron, transferrin saturation and hepcidin were within physiological ranges. Unexpectedly, soluble TfR1 (sTfR1) levels were almost undetectable and the bone marrow smear revealed increased erythropoiesis and iron deficiency in this compartment. The condition of hypochromic microcytic anemia was persistent over years and could not be corrected by oral iron therapy. WES analysis identified a TFRC_c.941C>T homozygous mutation, encoding for TfR1P314L. Relatives carrying the same mutation in heterozygosity were not anemic. This indicates that TFRC_c.941C>T mutation causes a recessive form of anemia. Overexpression of a construct expressing the TfR1P314L mutant in HeLa cells showed a reduction in total and secreted TfR1, thus reflecting upon the observation of very low sTfR1 levels in the blood of the patient. We further show that lower TfR1 levels are likely due to a faster degradation rate of TfR1P314L compared to TfR1WT. In addition, the TfR1P314L mutated protein showed impaired internalization of fluorescently labeled transferrin, most likely explaining the reduced iron levels observed in the bone marrow. In addition to this index patient, 3 additional children of 2 independent families were identified with TFRC mutations and hypochromic microcytic anemia. The genetic analysis detected a TFRC_c.967C>G heterozygous mutation encoding for TfR1P323A and a TFRC_c.934G>A homozygous mutation, encoding for TfR1G312R. All three TFRC mutations are highly conserved across species and are located in the same extracellular loop of TfR1. Summary/Conclusion: We identified a novel subtype of juvenile hereditary hypochromic microcytic anemia that is partially resistant to iron therapies and caused by missense mutations in the TFRC gene. Reduced endocytosis of iron-bound transferrin may contribute to insufficient iron supply for erythropoiesis and resistance to iron supplementation. In the clinical practice, our findings suggest to include TFRC sequencing analysis for patients with unexplained anemia. S275: ALTERATION OF IRON HOMEOSTASIS THROUGH GENETIC AND PHARMACOLOGIC MODULATION OF FERROPORTIN MODIFIES MDS PATHOPHYSIOLOGY IN A PRECLINICAL MOUSE MODEL S. Z. Vance1, A. Antypiuk1, R. Sharma1, F. Dürrenberger2, V. Manolova2, F. Vinchi1,* 1Iron Research Laboratory, New York Blood Center, New York, United States of America; 2Vifor (International) Ltd, St. Gallen, Switzerland Background: Patients with myelodysplastic syndromes (MDS) are prone to develop iron overload as a consequence of ineffective erythropoiesis and chronic transfusion therapy. Although iron overload is a common feature in MDS, it remains unclear whether and how iron excess is detrimental for MDS pathophysiology. Aims: This study aimed at characterizing altered iron homeostasis in a preclinical MDS mouse model, understanding the molecular mechanisms underlying iron toxicity and unraveling the potential therapeutic value of pharmacologic iron restriction in this disease condition. Methods: To this end, we took advantage of complementary approaches and analyzed the effect of iron overload obtained through genetic activation of the iron exporter ferroportin (FPNC326S), and iron restriction achieved through the administration of the FPN inhibitor Vamifeport for 3 months in NUP98-HOXD13 MDS mice. Mice were analyzed at 6 months of age. Results: MDS mice develop anemia and low white blood cell counts in the peripheral blood associated with a significant iron overload phenotype, hallmarked by low hepcidin levels, elevated serum iron and transferrin saturation, non-transferrin-bound iron (NTBI) formation and tissue iron deposition. FPNC326S MDS mice show an aggravated iron phenotype, with further elevated NTBI and tissue iron accumulation and still inappropriately low hepcidin levels. Vamifeport administration in MDS mice reduced serum iron and NTBI levels and prevented tissue iron loading. While iron excess in FPNC326S MDS mice did not improve erythropoiesis and hematologic parameters in the peripheral blood, iron restriction by Vamifeport significantly ameliorated red blood cell maturation and hemoglobin levels in MDS mice. The improved ineffective erythropoiesis was associated with reduced oxidative stress and apoptosis in erythroid progenitors. Unexpectedly, we observed a major role of the iron status in myeloid expansion in MDS. The number of immature myeloid blasts and myeloid progenitors as well as inflammation markers in the bone marrow of MDS mice were aggravated by iron overload in FPNC326S MDS mice and attenuated by iron restriction in Vamifeport-treated MDS mice compared to control MDS animals. Myeloid bias, monitored as percentage bone marrow myeloid cells, was exacerbated by iron overload and alleviated by iron restriction. Overall, this translated into a faster and delayed transition to AML, respectively. Finally, iron showed a profound impact on the stem cell pool in MDS. Iron overload aggravated and iron restriction alleviated the formation of reactive oxygen species, accumulation of DNA damage and levels of the apoptosis marker annexin V in hematopoietic stem and progenitor cells (HSPCs). By reducing the intracellular labile iron pool, Vamifeport improved HSPC quality and survival, partially rescuing the stem cell pool size in MDS mice. The improved stem cell pool, reduced myeloid bias and attenuated anemia resulted in a significant survival increment in iron-restricted compared to control MDS mice. Summary/Conclusion: Our results show for the first time in a preclinical mouse model of MDS that iron excess driven by ineffective erythropoiesis has pathological implication in transfusion-independent MDS. These effects are likely aggravated by transfusions causing additional iron overload. Finally, iron restriction achieved through pharmacologic FPN inhibition significantly improves MDS pathophysiology, uncovering the therapeutic potential of early prevention of NTBI formation in MDS. S276: TRANSFERRIN RECEPTOR 2 TARGETING AMELIORATES DIFFERENT FORMS OF ANEMIA CAUSED BY CHRONIC INFLAMMATION V. Olivari1 2,*, M. R. Lidonnici3, E. Tanzi1 4, S. M. Di Modica1, F. Tiboni3, L. Silvestri1 2, G. Ferrari2 3, A. Nai1 2 1Regulation of Iron Metabolism Unit - Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute; 2Vita-Salute San Raffaele University; 3Gene Transfer into Stem Cell Unit, SR-Tiget, IRCCS San Raffaele Scientific Institute; 4University of Milan Bicocca, Milan, Italy Background: Anemia is a common complication of chronic inflammation. It is caused by an increased production of the iron-regulatory hormone hepcidin, that leads to iron restriction, and by decreased erythrocytes sensitivity to erythropoietin (EPO) stimulation. Anemia of Chronic Kidney Disease (CKD) is a form of anemia of inflammation, in which progressive renal damage, besides causing systemic inflammation and iron deficiency, impairs erythropoietin (EPO) production. Current EPO-based treatments may cause off-target effects on long term and often require iron supplementation, mainly for CKD. Transferrin Receptor 2 (TFR2) modulates iron homeostasis in the liver, contributing to the transcriptional activation of hepcidin, and acts as a brake of EPO signaling in erythroid cells. Its selective deletion in the bone marrow (BM, Tfr2 BMKO ) enhances erythropoiesis both in wild-type and thalassemic mice. Aims: We investigated whether Tfr2 targeting might represent a therapeutic option for different forms of anemia, able to enhance erythropoiesis in chronic inflammation and simultaneously increase iron availability in CKD. Methods: Chronic sterile inflammation was induced by weekly subcutaneous injection of turpentine oil (100ul/20g body weight for 3 weeks) in Tfr2 BMKO mice. Mice were sacrificed 2 or 14 days after the last injection. CKD was induced feeding Tfr2 BMKO mice an adenine-rich diet for 8 weeks. The same protocol was applied also in germline Tfr2 deficient (Tfr2-ko) animals to simultaneously inactivate erythroid and hepatic Tfr2. Complete blood count was periodically determined, and a complete phenotypic analysis was performed at sacrifice. Results: Chronically inflamed Tfr2 BMKO mice maintained red blood cell count and hemoglobin levels higher than controls for the entire 5-week-long timespan, despite comparable inflammation. In CKD mice, BM-specific Tfr2 inactivation enhanced erythropoiesis and delayed anemia until iron availability was sufficient: indeed, hemoglobin came back to control values at the end of the protocol. The drop in hemoglobin levels was prevented in Tfr2-ko mice, in which iron supply is increased. Renal damage was comparable among the experimental groups, excluding a differential effect of the diet. Of note, BM-specific Tfr2 deletion boosted erythropoiesis and activated EPO-EPOR pathway without affecting EPO levels both in CKD and turpentine mice. Summary/Conclusion: We proved that erythroid Tfr2 targeting ameliorates hematological parameters in murine models of anemia of inflammation and anemia of CKD. The beneficial effect is achieved enhancing erythroid EPO responsiveness, without affecting EPO levels per se. For this reason, an approach based on erythroid TFR2 targeting would avoid the side effects of the available therapies. Moreover, we demonstrated that the concomitant inhibition of hepatic and erythroid Tfr2 in anemia of CKD would represent a therapeutic opportunity to simultaneously increase iron availability and erythroid responsiveness in a balanced manner. Overall, our results prove that TFR2 targeting may represent a tunable therapeutic approach for different forms of anemia. S277: HYPOFERREMIA CAUSED BY FERROPORTIN DOWNREGULATION IS REGULATED BY NFКB THROUGH HDAC-DEPENDENT MECHANISMS O. Marques1,*, N. Horvat1, M. C. Lai2, L. Zechner1, S. Colucci1, R. Sparla1, S. Zimmermann3, C. J. Neufeldt4, S. Altamura1, J. Zaugg2, K. Mudder1, J. D. Knopf5, M. K. Lemberg5, M. Pasparakis6, M. W. Hentze2, M. U. Muckenthaler1 1Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Universitatsklinikum Heidelberg; 2European Molecular Biology Laboratory; 3Department of Medical Microbiology and Hygiene; 4Department of Molecular Virology, Universitatsklinikum Heidelberg; 5ZMBH, University of Heidelberg; 6CECAD, University of Cologne, Heidelberg, Germany Background: Anemia of Inflammation (AI) is a highly prevalent comorbidity in patients with chronic inflammatory disorders. The underlying pathophysiology is partly explained by hypoferremia that results from iron sequestration in macrophages and the reduction of dietary iron uptake due to decreased iron export mediated by Ferroportin (Fpn). In response to inflammation, Fpn levels are decreased by hepcidin-mediated post-translational and/or transcriptional mechanisms, depending on the type of inflammatory pathway activated. Despite the fact that Fpn transcriptional repression is frequently observed in inflammatory conditions, the regulatory mechanisms involved remained unidentified. Aims: To gain mechanistic insight how Fpn mRNA levels are regulated in response to inflammation. Methods: We conducted a pharmacological and RNAi screen, targeting mediators of TLR2/6 signalling, in bone marrow-derived macrophages (BMDMs) stimulated with the TLR2/6-specific ligand FSL1. Changes in chromatin accessibility were evaluated by chromatin-accessibility Real-Time Polymerase Chain Reaction (CHART-PCR) and Assay for Transposase-Accessible Chromatin using sequencing (ATACseq). Data obtained in cell-based assays were validated in mice injected with FSL1. Results: The screen in BMDMs together with validation experiments demonstrated that the FSL1 induced TLR2 response represses Fpn mRNA levels via MYD88 and TRAF6 signaling transducers. While FSL1 can activate independent JNK, p38 and NFκB signaling branches, we show that Fpn transcriptional repression is specifically dependent on alternative NFκB signaling involving the transcription factor RELB. This contrasts our findings for TLR4 stimulation by LPS, where reduced Fpn mRNA expression depends on classical NFκB signaling, involving the NFкB subunits RELA and p50. Using ATACseq we identified Fpn genomic regions that are altered by inflammatory stimuli. These data show chromatin condensation around the Fpn transcription start site (TSS) upon FSL1 or LPS treatment. Since chromatin condensation is mainly promoted by histone deacetylases (HDACs), we next tested whether Fpn mRNA reduction upon inflammatory cues could be counteracted by treatment with HDAC inhibitors (HDACi). We demonstrate that pre-treatment with the pan-HDACi SAHA (Suberoylanilide Hydroxamic Acid) prevented chromatin remodelling at the Fpn locus and Fpn transcriptional repression in response to inflammation and RELB overexpression, suggesting that the effect of HDACs on Fpn responses occurs downstream of NFкB. In line with these data, NFκB pharmacological inhibition also prevented Fpn chromatin remodeling in the vicinity of the TSS. The findings obtained in BMDMs were extended in mice treated with SAHA prior to FSL1 injection, showing reduced hypoferremia and attenuation of splenic Fpn mRNA repression, while serum hepcidin levels remained unaltered. Summary/Conclusion: These results identify NFκB as an important repressor of Fpn transcription in response to inflammatory signals involving chromatin remodeling in a genomic region near Fpn’s TSS. Importantly, HDAC inhibition attenuates Fpn mRNA repression as well as the resulting hypoferremia in mice. Our findings generate molecular insight about how hypoferremia is generated and demonstrate that Fpn, like hepcidin, is controlled by HDACs. S278: EVALUATION OF THE MAIN REGULATORS OF SYSTEMIC IRON HOMEOSTASIS IN PYRUVATE KINASE DEFICIENCY A. Zaninoni1, R. Russo2 3, R. Marra2 3, I. Andolfo2 3, E. Fermo1, A. P. Marcello1, D. Consonni4, B. Eleni Rosato2 3, S. Martone2 3, B. Fattizzo1, W. Barcellini1, A. Iolascon2 3, P. Bianchi1,* 1Hematology Unit - Physiopathology of Anemias Unit, Foundation IRCCS Cà Granda Policlinico Milan, Milan; 2Department of Molecular Medicine and Medical Biotechnology; 3CEINGE – Advanced Biotechnologies, University Federico II, Naples; 4Epidemiology Unit, Foundation IRCCS Cà Granda Policlinico Milan, Milan, Italy Background: Iron loading anemias are characterized by ineffective erythropoiesis and iron overload. This group of anemias includes thalassemia syndromes, congenital dyserythropoietic anemias (CDA), and some forms of congenital hemolytic anemias. Among them pyruvate kinase deficiency (PKD) has been shown to develop iron overload also in absence of transfusions suggesting dyserythropoietic features. Moreover, severe forms can be misdiagnosed as CDA due to bone marrow abnormalities and ineffective erythropoiesis further supporting these evidences. The hormone erythroferrone (hERFE) is produced by erythroblasts in response to erythropoietin (EPO), and acts by suppressing hepcidin, thereby increasing iron absorption and mobilisation for erythropoiesis demand. The ERFE-hepcidin axis seems to play a crucial role in the pathogenesis of these disorders; an increased erythroferrone release by immature erythroid cells results in hepcidin suppression and secondary iron overload that could finally results in ineffective erythropoiesis and anemia. Aims: To investigate the pathophysiological basis of iron overload in PKD. Methods: We analysed the levels of hERFE, EPO, hepcidin, and soluble transferrin receptor (sTFR) in a large group of 41 PKD patients equally distributed by gender, age and severity. The results were analysed in comparison with two groups of patients affected by hemolytic anemia with overt dyserythropoiesis (42 patients with CDA type II) and with congenital hemolytic anemia due to RBC membrane defects (51 patients with hereditary spherocytosis [HS]), respectively. Results: Demographic, hematologic, and biochemical features of the three groups of patients are reported in the Figure 1A. Among the PKD patients, 18/41 were <18 yrs, median Hb level at the time of the study was 9.05g/dL (range 5.5-14.5), 12 underwent splenectomy, 28 ever received at least three transfusions their life, 14 of them transfusion dependent (>6 tx/yrs). Mean ferritin levels at the time of the study were 546 ng/ml (range 59-4990), 15/41 patients requiring chelation therapy for iron overload developed also in absence of transfusions. As expected, CDAII patients showed decreased hepcidin levels (3.74 ng/mL; n.v. 17.25, P<0.001) associated with increased erythropoietin (62.7 IU/L, n.v. 6.5, P=0.01) and hERFE (24.8 ng/mL, n.v. 1, P<0.0001). On the contrary, HS showed increased hepcidin, with less marked increased of ERFE (9.9 ng/mL, P=0.02) and EPO (36.4 IU/L, P=0.005). In PKD patients we observed decreased hepcidin levels (7.15 ng/mL, P=0.03)), increased hERFE (18 ng/mL, P<0.0001) and EPO (75.6 IU/L, P=0.009). Instead, sTFR was equally increased in the three groups of patients (Figure 1B). Interestingly, by comparing the three groups of patients, PKD showed dyserythropoietic features as evidenced by the observation of intermediate values between HS and CDAII of hepcidin (P=0.007 PKD v CDAII and P=0.0002 PKD vs HS), hEFRE, and sTFR (Figure 1C). Image: Summary/Conclusion: This study provides the first analysis of the main regulators of systemic iron homeostasis in PK deficiency compared either with the model of a structural RBC defect (HS) or with the typical model of dyserythropoietic anemia with ineffective erythropoiesis, such as CDAII. These data provide evidence of the dyserythropoietic features of PK deficiency, underlining the need of accurate diagnosis and paving the way of novel therapeutic approaches in PK deficiency. S279: INCREASED RISK OF INFECTIONS IN INDIVIDUALS WITH HEMOCHROMATOSIS C282Y HOMOZYGOSITY AND IN INDIVIDUALS WITH HIGH OR LOW PLASMA IRON OR TRANSFERRIN SATURATION: A PROSPECTIVE STUDY OF 138252 INDIVIDUALS M. Mottelson1 2 3,*, A. Glenthøj1 2, B. Nordestgaard2 3 4 5, C. Ellervik4 6 7 8, S. Bojesen2 3 4 5, J. Helby1 3 1Department of Hematology, Copenhagen University Hospital - Rigshospitalet; 2Department of Clinical Medicine, University of Copenhagen; 3The Copenhagen General Population Study; 4Department of Clinical Biochemistry, Copenhagen University Hospital - Herlev and Gentofte; 5The Copenhagen City Heart Study, Copenhagen University Hospital – Bispebjerg and Frederiksberg, Copenhagen; 6Department of Production, Researh, and Innovation, Region Zealand, Sorø, Denmark; 7Department of Laboratory Medicine, Boston Children’s Hospital; 8Department of Pathology, Harvard Medical School, Boston, MA, United States of America Background: Plasma iron and transferrin saturation are measures of iron available in the blood stream. Plasma ferritin indicate body iron stores. Levels of plasma iron, transferrin saturation and ferritin are increased in hemochromatosis C282Y homozygous individuals. Aims: We tested the hypothesis that high and low plasma iron and transferrin saturation are associated with increased risk of infections observationally and genetically through hemochromatosis C282Y homozygosity. Methods: We studied 138,252 general population individuals. Plasma iron and transferrin saturation were measured in 136,656 and 136,599 individuals, respectively. 132,542 individuals were genotyped for the C282Y hemochromatosis variant. Individuals were followed prospectively for up to 28 years for hospital admissions and emergency room visits due to infectious diseases using the National Patient Register which covers all danish hospitals. Results: During follow-up 31,199 individuals were hospitalized due to an infection. After multivariable adjustment including other risk factors for infections, individuals with the 5% lowest and 5% highest plasma iron levels had hazard ratios for any infection of 1.20 (95% CI:1.12-1.28;P<0.001) and 1.14 (95% CI:1.07-1.22;P<0.001) respectively, when compared to individuals with plasma iron levels between the 25th-75th percentile. Findings for transferrin saturation were similar. Hazard ratio for any infection in hemochromatosis C282Y homozygous individuals was 1.40 (95% CI:1.16-1.69; P<0.001) when compared to wildtype individuals. Risk of sepsis was especially high in C282Y homozygotes (hazard ratio 1.69;95% CI:1.05-2.73; P=0.03). Risk of any infection was increased even in C282Y homozygotes with normal levels of plasma iron, transferrin saturation or ferritin. Summary/Conclusion: High and low plasma iron and transferrin saturation were associated with increased risk of infections. Hemochromatosis C282Y homozygotes had increased risk of infection even when they had normal levels of plasma iron, transferrin saturation or ferritin. Current hemochromatosis treatment strategy centralized around ferritin normalization may not reduce risk of infection satisfactorily in patients with hereditary hemochromatosis genotype. S280: B-CELL MALIGNANCIES TREATED WITH TARGETED DRUGS AND SARS-COV-2 INFECTION. A EUROPEAN HEMATOLOGY ASSOCIATION SURVEY (EPICOVIDEHA) M. S. Infante1, J. Salmanton-García2,*, A. Fernandez-Cruz3, F. Marchesi4, Z. RÁČIL5, M. Hanakova6, O. Jaksic7, B. Weinbergerova8, C. Besson9, R. Duarte10, F. Itri11, T. VALKOVIĆ12, A. Busca13, A. Guidetti14, A. GLENTHØJ15, G. Collins16, V. Bonuomo17, U. Sili18, G. Seval19, M. Machado20, A. Lopez-Garcia21, R. Cordoba22, O. Blennow23, G. Abu-zeinah24, S. Lemure25, A. Kulasekararaj26, I. Falces-Romero27, C. Cattaneo28, O. A. Cornely29, J.-A. Hernandez-Rivas30, L. Pagano31 1Hematology Department, University Hospital Infanta Leonor, MADRID, Spain; 2Internal Medicine, University of Cologne, Excellence center for Medical Mycology, Cologne, Germany; 3Internal Medicine, Hospital Universitario Puerta de Hierro, Majadahonda, Spain; 4hematology and stem cell transplant Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy; 5Institute of Hematology and Blood transfusion, Prague, Czechia; 6Hematology Department, Institute of Hematology and Blood transfusion, Prague, Czechia; 7Hematology Department, University Hospital Dubrava, Zagreb, Croatia; 8Internal Medicine, Masaryk University and University Hospital Brno, Brno, Czechia; 9Hematology Department, Centre Hospitalier de Versailles, Versailles, France; 10hematology and stem cell transplant Unit, Hospital Universitario Puerta de Hierro, MADRID, Spain; 11Hematology Department, San Luigi Gonzaga Hospital, Orbassano, Italy; 12Croatian Cooperative Group for Hematological Diseases (CROHEM), University Hospital Centre, Rijeka, Croatia; 13Stem Cell Transplant Center, AOU Cittá della Salute e della Scienza, Turin; 14Hematology Department, Fondazione IRCCS Istituto Nazionale, Milan, Italy; 15Hematology Department, Rigshospitalet University Hospital, Copenhagen, Denmark; 16Hematology Department, NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom; 17Hematology Department, University Hospital of Verona, Verona, Italy; 18Hematology Department, Marmara University Hospital, Istanbul; 19Hematology Department, Ankara University Hospital, Ankara, Turkey; 20Clinical Microbiology and Infectious diseases, Hospital Universitario Gregorio Marañon; 21Hematology Department, Fundación Jimenez Diaz University Hospital; 22Hematology, Fundacíon Jimenez Diaz, Madrid, Spain; 23Infectious diseases, Karolinska University Hospital, Stockolm, Sweden; 24Hematology Department, Weil Cornell Medicine, New York, United States of America; 25Hematology Department, CHU de Montpellier, Montpellier, France; 26Hematology Department, King’s College Hospital, London, United Kingdom; 27Hematology, University Hospital La Paz, MADRID, Spain; 28Hematology, ASST-Ospedali Civili, Brescia, Italy; 29Internal Medicine, University of Cologne, Cologne, Germany; 30Hematology, University Hospital Infanta Leonor, Madrid, Spain; 31Hematology, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Roma, Italy Background: Patients with lymphoproliferative diseases (LPD) appear particularly vulnerable to SARS-CoV-2 infection, partly because of the effects of the anti-neoplastic regimens (chemotherapy, signaling pathway inhibitors, and monoclonal antibodies) on the immune system. The real impact of COVID-19 on the life expectancy of patients with different subtypes of lymphoma and targeted treatment is still unknown. Aims: The aim of this study is to describe and analyse the outcome of COVID-19 patients with underlying LPD treated with targeted drugs such as monoclonal antibodies (obinutuzumab, ofatumumab, brentuximab, nivolumab or pembrolizumab), BTK inhibitors (ibrutinib, acalabrutinib), PI3K inhibitors (idelalisib), BCL2 inhibitors (venetoclax) and IMIDs, (lenalidomide). Methods: The survey was supported by EPICOVIDEHA registry. Adult patients with baseline CLL or non-Hodgkin Lymphoma (NHL) treated with targeted drugs and laboratory-confirmed COVID-19 diagnosed between January 2020 and January 2022 were selected. Results: The study included 368 patients (CLL n=205, 55.7%; NHL n=163, 44.3%) treated with targeted drugs (Table 1). Median follow-up was 70.5 days (range 19-159). Most used targeted drugs were ITKs (51.1%), anti-CD20 other than rituximab (16%), BCL2 inhibitors (7.3%) and lenalidomide (7.9%). Of note, only 16.0% of the patients were vaccinated with 2 or more doses of vaccine at the onset of COVID-19. Pulmonary symptoms were present at diagnosis in 244 patients (66.2%). Severe COVID-19 was observed in 47.8 % patients while 21.7% were admitted to to intensive care unit (ICU), being 55 (26.8%) CLL patients and 25 (15.3%) NHL patients. More comorbidities were reported in patients with severe-critical COVID-19 compared to those with mild- asymptomatic infection (p=0.002). This difference was relevant in patients with chronic heart diseases (p=0.005). Overall, 134 patients (36.4%) died. Primary cause of death was COVID-19 in 92 patients (68.7%), LPD in 14 patients (10.4%), and a combination of both in 28 patients (20.9%).Mortality was 24.2% (89/368) at day 30 and 34.5%(127/368) at day 200. After a Cox multivariable regression age >75 years (p<0.001, HR 1.030), active malignancy (p=0.011, HR 1.574) and admission to ICU (p<0.00, HR 4.624) were observed as risk factors. Survival in patients admitted to ICU was 33.7% (LLC 38.1%, NHL 24%). Mortality rate decreased depending on vaccination status, being 34.2% in not vaccinated patients, 15.9-18% with one or two doses, decreasing to 9.7% in patients with booster dose (p<0.001). There was no difference in OS in NLH vs CLL patients (p=0.344), nor in ITKs vs no ITKs treated patients (p=0.987). Additionally, mortality rate dropped from the first semester 2020 (41.3%) to last semester 2021 (25%). Image: Summary/Conclusion: - Our results confirm that patients with B-malignancies treated with targeted drugs have a high risk of severe infection (47.8%) and mortality (36.4%) from COVID-19. - Presence of comorbidities, especially heart disease, is a risk factor for severe COVID-19 infection in our series. - Age >75 years, active malignancy at COVID-19 onset and ICU admission were mortality risk factors. - COVID-19 vaccination was a protective factor for mortality, even in this population with humoral immunity impairment. - The learning curve in the management of the infection throughout the pandemic and the development of COVID-19 treatments showed benefit in this particularly vulnerable population. S281: PRIOR EXPOSURE TO IMMUNOSUPPRESSIVE AGENTS AND COMORBIDITIES ARE ASSOCIATED WITH WORSE OUTCOMES OF SARS-COV2 INFECTION IN PH-NEG CHRONIC MYELOPROLIFERATIVE NEOPLASMS: RESULTS OF EPICOVIDEHA SURVEY M. Marchetti1,*, J. Salmanton-García2, S. El-Ashwah3, M. V. Sacchi1, F. Marchesi4, Z. Ráčil5, M. Hanakova6, G. Zambrotta7, L. Verga7, F. Passamonti8, F. Itri9, J. Van Doesum10, S. Martìn-Pérez11, A. López-García12, J. Dávila-Valls11, R. Cordoba13, G. Abu-Zeinah14, G. Dragonetti15, C. Cattaneo16, V. Bonuomo17, L. Prezioso18, A. Glenthøj19, F. Farina20, R. Duarte21, O. Blennow22, O Cornely23, L. Pagano24 1Hematology & TMO Unit, Azienda Ospedaliera SS Antonio e Biagio e Cesare Arrigo, Alessandria, Italy; 2Department I of Internal Medicine, Excellence Center for Medical Mycology (ECMM), University of Cologne and University Hospital, Faculty of Medicine, Cologne, Germany; 3Oncology Center, Mansoura University, Mansoura, Egypt; 4Hematology and Stem Cell Transplant Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy; 5Institute of Hematology and Blood Transfusion, Prague and Brno Hospital; 6Institute of Hematology and Blood Transfusion, Prague Hospital, Prague, Czechia; 7Hematology Unit, Azienda Ospedaliera San Gerardo; University Bicocca-Milan, Monza; 8Division of Hematology, Department of Medicine and Surgery, University of Insubria, Varese; 9Hematology Unit, Hospital San Luigi Gonzaga, Orbassano, Italy; 10University Medical Center Groningen, University of Groningen, Groningen, Netherlands; 11Hospital Nuestra Señora de Sonsoles, Ávila; 12Health Research Institute IIS-FJD; 13Fundacion Jimenez Diaz University Hospital, Madrid, Spain; 14Division of Hematology and Oncology, Weill Cornell Medicine, New York, United States of America; 15Hematology Unit, Fondazione Policlinico Universitario Agostino Gemelli - IRCCS, Rome; 16Hematology Unit, ASST-Spedali Civili, Brescia; 17Department of Medicine, Section of Hematology, Azienda Ospedaliera Universitaria Integrata, Verona; 18Hematology and Bone Marrow Unit, University Hospital, Parma, Italy; 19Department of Hematology, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark; 20Hematology & TMO unit, IRCCS Ospedale San Raffaele, Milan, Italy; 21Hospital Universitario Puerta de Hierro, Majadahonda, Madrid, Spain; 22Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden; 23Dpt I of Internal Medicine, ECMM, CECAD, Clinical Trials Centre Cologne (ZKS Köln), Center for Molecular Medicine Cologne (CMMC), University of Cologne, Faculty of Medicine and University Hospital Cologne, German Centre for Infection Research (DZIF), Cologne, Germany; 24Hematology Unit, Fondazione Policlinico Universitario Agostino Gemelli - IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy Background: Philadelphia-negative chronic myeloproliferative neoplasms (MPN) typically incur high rates of thrombosis and infections and cytoreductive drugs may modulate such risks. Aims: The present analysis aims at assessing the severity and outcomes of MPN facing coronavirus disease 2019 (COVID-19). Hence, we aimed to assess the impact of immunosuppressive agents and comorbidity burden in COVID-19 outcome. Methods: The EPICOVIDEHA registry is an online survey (www.clinicalsurveys.net) that has collected since April 2020 until January 2022 5,445 cases of COVID-19 in individuals with baseline haematological malignancies (Salmanton-García et al, 2021 Hemasphere) The survey is promoted by the European Hematology Association - Infectious Diseases Working Party (EHA-IDWP) and has been approved centrally by the Institutional Review Board and Ethics Committee of Fondazione Policlinico Universitario A. Gemelli – IRCCS – Università Cattolica del Sacro Cuore, Rome, Italy (Study ID: 3226). Results: Overall, 308 patients (5.6%) with MPN were observed for a median of 102 days (IQR: 21-223, range 22-97) after COVID-19 diagnosis. Median age at infection was 69 years (IQR: 58-77, range 22-97) and at least one comorbidity was reported from most of the individuals (62.6%, n = 193). A large portion of patients had a history of cardiopathy (n=109, 35.4%), diabetes (n=40, 15.9%), or chronic pulmonary disease (n=44, 14.3%). Myelofibrosis (MF) (n=140, 45.4%) was the most prevalent baseline malignancy, with 18 MF patients (12.9%) reporting 3 or more comorbidities. Out of the whole cohort, 72 patients (42.8% of MF) received immunosuppressige therapies including steroids, immunomodulatory drugs (IMiDs) or JAK-inhibitors. Hospitalization and consecutive admission to intensive care unit was required for 187 (60.7%) and 45 (24%) patients, respectively. At multivariate logistic regression, hospital admission was predicted by age ≥70 years (OR 2.809; 95% CI 1.651-4.779), exposure to immunosuppressive therapies (OR 2.802; 95% CI 1.5380-5.103) and comorbidity burden. During the study follow-up (median 101 days; range 21-222) 84 patients deceased after a median time of 14 days (IQR: 8-49, range 0-457) since COVID-19 diagnosis. The fatality rate (FR) decreased from 40.3% (50 out of 124) in the first two quarters of year 2020 to 15.8% (3 out of 19) in the first two quarters of year 2021 (p<0.05). Death was principally attributable to COVID-19 in 58 patients (69.0%) and contributable by COVID-19 in 15 (17.9%). FR was particularly high (54 out of 140, 38.6%) in MF patients and in patients receiving immunosuppressive agents (32 out of 86, 37%). Moreover, FR increased from 13.0% in individuals with no comorbidity to 36.0% and 62.1% in those with ≥2 or ≥3 comorbidities, respectively. More specifically, three comorbidities independently increased the FR: chronic cardiopathy (HR 1.653; 95%CI 1.017-2.687), chronic pulmonary disease (HR 1.847; 95% CI 1.097-3.109), and diabetes mellitus (HR 1.712; 95% CI 1.006-2.914). A heavy comorbidity burden, namely 3 or more comorbidities (HR 2.956; 95% CI 1.403-6.227), advanced age, namely ≥70 years (HR .809; 95% CI 1.651-4.779), myelofibrosis (HR 2.501; 95% CI 1.384-4.519), and ICU admission (HR 2.669; 95% CI 1.641-4.342) independently predicted FR. Image: Summary/Conclusion: COVID-19 infection led to a particularly dismal outcome in patients exposed to immunosuppressive agents and in those with chronic heart or pulmonary diseases, or diabetes. These data allow to tailor future strategies for preventing severe COVID-19 in MPN patients. S282: A RANDOMIZED CONTROLLED CLINICAL TRIAL DEMONSTRATES THAT PLASMA FROM CONVALESCENT AND VACCINATED DONORS IMPROVES OUTCOME OF COVID-19 IN PATIENTS WITH HEMATOLOGICAL DISEASE, CANCER OR IMMUNOSUPPRESSION C. Müller-Tidow1,*, M. Janssen1, U. Schäkel1, J. Gall2 3, A. Leo4, P. Stelmach1, J. Krisam5, L. Baumann5, J. Stermann5, U. Merle6, M. Zeier7, M. A. Weigand8, L. Bullinger9, J.-F. Schrezenmeier9, M. Bornhäuser10, N. Alakel10, O. Witzke11, T. Wolf12, M. J. Vehreschild12, S. Schmiedel13, M. M. Addo13, F. Herth14, M Kreutner15, P.-R. Tepasse16, B. Hertenstein17, M. Hänel18, A. Morgner18, M. Kiehl19, O. Hopfer19, M.-A. Wattad20, C. C. Schimanski21, C Celik21, T. Pohle22, M. Ruhe22, W. V. Kern23, A Schmitt1, M. Schmitt1, P. Dreger1, H.-M. Lorenz1, M. Souto-Carneiro1, N. Halama24, S. Meuer4, H.-G. Kräusslich25, B. Müller25, R. Bartenschlager26, J. Klemmer1, K. Kriegsmann1, R. F. Schlenk1 2 3, C. M. Denkinger27 1Department of Internal Medicine V, Heidelberg University Hospital; 2NCT-Trial Center, National Center of Tumor Diseases, Heidelberg University Hospital; 3German Cancer Research Center; 4Institute for Clinical Transfusion Medicine and Cell Therapy Heidelberg; 5Institute for Medical Biometry and Informatics, Ruprecht-Karls University Heidelberg; 6Department of Internal Medicine IV, Heidelberg University Hospital,; 7Department of Nephrology, University of Heidelberg, Heidelberg; 8Department of Anaesthesiology, Heidelberg University Hospital, Heidelberg, Heidelberg; 9Department of Hematology, Oncology and Tumor Immunology, Charité University Medicine, Campus Virchow Clinic, Berlin; 10Department of Internal Medicine I, University Hospital and Faculty of Medicine Carl Gustav Carus of TU Dresden, Dresden; 11Department of Infectious Diseases, West German Centre of Infectious Diseases, University Hospital Essen, University Duisburg-Essen, Essen; 12Department of Internal Medicine, Infectious Diseases, University Hospital Frankfurt, Goethe University Frankfurt, Frankfurt am Main; 13I. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg; 14Pneumology and Critical Care Medicine, Thoraxklinik, University of Heidelberg; 15Center for Interstitial and Rare Lung Diseases, Thoraxklinik, University of Heidelberg, German Center for Lung Research (DZL), Heidelberg; 16Department of Medicine B, Gastroenterology and Hepatology, University Hospital Münster, Münster; 17Medical Department I, Klinikum Bremen-Mitte, Bremen; 18Department of Internal Medicine III, Klinikum Chemnitz gGmbH, Chemnitz; 19Department I of Internal Medicine, Frankfurt (Oder) General Hospital, Frankfurt (Oder); 20Department of Hematology, Oncology, Palliative Care and Stem Cell Transplantation, Klinikum Hochsauerland GmbH, Meschede; 21Department of Internal Medicine II, Klinikum Darmstadt GmbH, Darmstadt; 22Department of Internal Medicine I, Klinikum Herford, Herford; 23Department of Medicine II, Division of Infectious Diseases and Travel Medicine, University Medical Centre Freiburg, Freiburg; 24Department of Medical Oncology, National Center for Tumor Diseases, Heidelberg University Hospital; 25Department of Infectious Diseases, Virology, Heidelberg University Hospital; 26Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital; 27Division of Tropical Medicine, Department of Infectious Diseases, Heidelberg University Hospital, Heidelberg, Germany Background: Therapy options are limited for COVID-19 patients with hematological disease, cancer, immunosuppression or advanced age. Even though no benefit was observed for convalescent plasma in unselected patients with COVID-19, retrospective data suggest that it could be effective in patients unable to mount a sufficient immune response upon SARS-CoV-2 infection. Plasma from vaccinated donors has not been systematically assessed for COVID-19 treatment. Aims: We conducted a randomized clinical trial to address plasma efficacy in patients at high risk for an adverse outcome. Methods: COVID-19 patients with confirmed SARS-CoV-2 infections and oxygen saturation <=94% were randomized (NCT05200754). Patients received convalescent or vaccinated SARS-CoV-2 plasma in two bags (238 - 337 ml plasma each) from different donors on day 1 and 2 (PLASMA) or standard of care (CONTROL). Randomization was stratified according to four clinical patient groups, hematological/solid cancer (group-1), treatment or disease associated immunosuppression (group 2), high risk disease by standard parameters (group-3) or age >=75 years (group-4). Mechanically ventilated patients were not eligible. Plasma was obtained from donors with high level neutralizing activity (titer >=1:80) either after SARS-CoV-2 infection (convalescent) or after vaccination with at least two doses of mRNA vaccines (vaccinated). Crossover for the control group was allowed at day 10. The primary endpoint was time to improvement as two points on a seven-point ordinal scale or live discharge from the hospital (IMPROVEMENT) with prespecified analyses of subgroups (Janssen M, et al. Trials 2020 Oct 6;21(1):828). Results: A total of 133 patients were randomized with 68 receiving PLASMA with a median age of 68 years (range 36-95) or CONTROL (n=65, of which n=10 (15.4%) crossed over at day 10) with a median age of 70 years (range 38-90). The distribution of the four predefined groups was group-1, n=53; group-2, n=18; group-3, n=35; and group-4, n=27. The intention to treat analysis revealed a non-significant shorter time to IMPROVEMENT for patients in PLASMA (median 12.5 days, 95%-CI [10; 16]) compared to patients in CONTROL (median 18 days, 95%-CI [11; 28]), hazard ratio 1.24, 95% confidence interval [0.83; 1.85], p=0.29). Overall, 27 patients died (PLASMA, n=12; CONTROL, n=15; p=0.80). Predefined subgroup analysis revealed a clinically significant benefit in patients with hematological malignancies, other cancers or immunosuppression (group-1, group-2, n=71). With a median time to improvement of 13 days (95%-CI [9; 19]) for PLASMA and 32 days (95%-CI [17; 57]) for CONTROL(HR 2.03, 95%-CI [1.17; 3.6], p=0.01). A sensitivity analysis revealed that IMPROVEMENT appeared to be seen even earlier with vaccinated (median 10 days, 95%-CI [8; 14]) compared to convalescent SARS-CoV-2 plasma (median 13 days, 95%-CI [6; 38]) and CONTROL. Within group-1 and group-2, six patients in PLASMA (18.2%) and 10 in CONTROL (28.6%) died. No significant differences in improvement were observed in group-3 and group-4 with a HR of 0.72 (95%-CI [0.41; 1.28], p=0.26). Within group-3 and group-4, six patients in PLASMA (18.8%) and five in CONTROL (16.7%) died. No previously unknown side effects of plasma therapy emerged within the trial. Image: Summary/Conclusion: Plasma from convalescent and particularly vaccinated donors improved outcome of COVID-19 patients with an underlying hematological disease /cancer or other reasons of impaired immune response. Plasma did not improve outcome in immune-competent patients with other risk factors and/or older age. S283: ERN-EUROBLOODNET EUROPEAN REGISTRY OF PATIENTS AFFECTED BY RED BLOOD CELL DISORDERS AND COVID-19 P. Velasco1,*, F. Longo2, A. Piolatto2, E. J. Bardón-Cancho3 4 5, B. Ponce-Salas6, P. Flevari7, E. Voskaridou8, B. J. Biemond9, E Nur9 10, P. Delaporta11, T. Besse-Hammer12, A. Ruiz-Llobet13, S. Raso14, A. Spasiano15, M. E. Guerzoni16, D. Beneitez-Pastor17 18 19 20, L. Dedeken21, A. Pepe22, R. Rosso23, J. B. Kunz24, M. de Montalembert25, S. Campisi26, A. Glenthøj27, P. Gonzalez Urdiales28 29, F. S. Benghiat30, M.-A. Azerad31, C. J. Saunders32, T. Ferreira Faria33, T. Casini34, S. Bagnato35, A. Van de Velde36, V. Labarque37, E. Bertoni38, A. Van Damme39, M. D. Diamantidis40, R. Russo41 42, E. Stiakaki43, A Quota44, S. Christou45, M. J. Teles46 47 48 49 50, I. Lafiatis51, J.-L. Kerkhoffs52, M. Argüello Marina53, M. Lorite54, A. Rodriguez55 56, A. Iolascon41 42, A. T. Taher57, R. Colombatti58, N. Roy59, M. D. M. Mañú Pereira60 1pediatric oncology and hematology, Hospital Vall d’Hebron, Barcelona, Spain; 2Department of Clinical and Biological Sciences, University of Torino, Orbassano (To), Italy; 3Pediatric Hematology Unit, Hospital General Universitario Gregorio Marañón; 4Facultad de Medicina, Universidad Complutense de Madrid; 5Instituto Investigación Sanitaria Gregorio Marañón; 6Onco-Hematología Infantil, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 7Center of Excellence in Rare Haematological Diseases-Haemoglobinopathies, Laiko General Hospital; 8Center of Excellence in Rare Haematological Diseases-Haemoglobinopathies, Laiko General Hospital, Athens, Greece; 9Department of Hematology, Amsterdam University Medical Centers; 10Department or Blood Cell Research, Sanquin Research, Amsterdam, Netherlands; 11First Department of Pediatrics, National and Kapodistrian University of Athens, ‘AghiaSophia’ Children’s Hospital, Athens, Greece; 12URC, CHU Brugmann, Brussels, Belgium; 13Paediatric Haematology Department, Hospital Sant Joan de Déu, Universitat de Barcelona, Barcelona, Spain; 14Department of Hematology and Rare Diseases, V Cervello, Azienda Ospedaliera Ospedali Riuniti Villa Sofia-Cervello, Palermo; 15Malalttie Rare del Globulo Rosso, AORN A. Cardarelli, Naples; 16Pediatric Unit,Department of Medical and Surgical Sciences of Mothers, Pediatric Unit,Department of Medical and Surgical Sciences of Mothers, Modena, Italy; 17Translational ResearchGroup in Rare Anemia Disorders, Vall d’Hebron Institut de Recerca; 18Hematology Department, Vall d’Hebron Hospital Universitari; 19Experimental Hematology Group, Vall d’Hebron Institute of Oncology (VHIO); 20Vall d’Hebron Hospital Universitari, ERN-EuroBloodNet, Barcelona, Spain; 21Hôpital Universitaire des Enfants Reine Fabiola (ULB), Brussels, Belgium; 22Fondazione Toscana G. Monasterio per la ricerca medica e di sanità pubblica, Pisa; 23Thalassemia and Heamoglobinophaties, Policlinico Universitario G. Rodolico, Catania, Italy; 24University Hospital, Heidelberg, Germany; 25Hôpital Necker, Paris, France; 26Ospedale Umberto I, Siracusa, Italy; 27Department of Hematology, Rigshospitalet, Copenhagen, Denmark; 28Pediatric oncology and hematology department, Hospital Universitario Cruces; 29Instituto de investigaciónBiocruces Bizkaia, Barakaldo, Spain; 30CUB Erasmus Hospital, Brussels; 31Hematology, CHU Liège Citadelle site, Liège, Belgium; 32Hospital dos Capuchos, Centro Hospitalar e Universitário Lisboa Central, Lisbon; 33Pediatric Department, Hospital Fernando Fonseca, Amadora, Portugal; 34Pediatric OncoHematology, Meyer Children Hospital, Florence; 35Thalassemia, Ospedale Civile, Lentini, Italy; 36Hematology, University Hospital Antwerp, Edegem; 37Pediatric Hemato-Oncology, University Hospitals Leuven, Leuven, Belgium; 38Oncohaematology and BMT Unit, ASST Spedali Civili di Brescia, Brescia, Italy; 39Paediatric Hémato-oncology, Cliniques Universitaires Saint-Luc, Brussels, Belgium; 40Thalassemia and Sickle Cell Disease Unit, Department of Hematology, General Hospital of Larissa, Larissa, Greece; 41Department of Molecular Medicine and Medical Biotechnology, University Federico II; 42CEINGE - Advanced Biotechnologies, Naples, Italy; 43Pediatric Hematology-Oncology Department, Universityof Crete, University Hospital of Heraklion, Heraklion, Greece; 44UOSD Thalassemia, V.Emanuele Hospital, Gela, Italy; 45Thalassemia Clinic, Archbishop Makarios III Hospital, Nicosia, Cyprus; 46Clinical Pathology, Centro Hospitalar e Universitário de São João; 47Clinical Hematology, Centro Hospitalar e Universitário do Porto; 48Anemia Working Group Portugal; 49Institute for the HealthResearch and Innovation (i3S); 50Hematology Working Group of the Portuguese Society of Clinical Pathology, Porto, Portugal; 51General Hospital of Mytilini, Mytilini, Greece; 52Hematology, HagaZiekenhuis, The Hague, Netherlands; 53Hematology, Hospital Príncipe de Asturias, Madrid; 54Department of pediatric Hematology, Hospital Son Espases, Palma de Mallorca; 55Clinical Pharmacology Service, Hospital Universitari Vall d’Hebron; 56Vall d’Hebron Institut de Recerca, Barcelona, Spain; 57Division of Hematology and Oncology, Department of Internal Medicine, American University of Beirut Medical Center, Beirut, Lebanon; 58UOC Pediatric Hematology Oncology, University of Padova, Padova, Italy; 59Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom; 60Translational Research in Child and Adolescent Cancer, Vall d’Hebron Institut de Recerca, Barcelona, Spain Background: Patients with red blood cell disorders (RBCD), are likely to be at increased risk of complications from SARS-Cov-2 (Covid-19), but evidence in this population is scarce due to its low frequency and heterogeneous distribution. Aims: ERN-EuroBloodNet, the European Reference Network in rare hematological disorders, established a European registry to determine the impact of COVID-19 on RBCD patients and identify risk factors predicting severe outcomes. Methods: The ERN-EuroBloodNet registry was established in March 2020 by VHIR based on Redcap software in accordance with the Regulation (EU) 2016/679 on personal data. The local Research Ethics Committee confirmed that the exceptional case of the pandemic justifies the waiver of informed consent. Eligible patients had confirmed RBCD and COVID-19. Data collected included demographics, diagnosis, comorbidities, treatments, and COVID-19 symptoms and management. For analysis of COVID-19 severity, two groups were established 1) Mild: asymptomatic or mild symptoms without clinical pneumonia and 2) Severe: pneumonia requiring oxygen/respiratory support and/or admission to intensive care unit. Continuous variables were compared using the Wilcoxon rank-sum test or Kruskall Wallis test, while categorical variables were analyzed using the Chi-square test or Fisher’s Exact test. Relevant factors influencing disease or severity were examined by the logistic regression adjusted for age. Results: As of February 25, 2022, 42 medical centers from 10 EU countries had registered 428 patients: 212 Sickle cell disease (SCD), 186 Thalassemia major and intermedia (THAL). The mean age of SCD was lower (22y) than of THAL (39.4y). Splenectomy and comorbidities were higher in THAL (51.4% and 61,3%) than in SCD (16,3% and 46,8%) (p<0.001, p=0.004). Age and BMI correlated with COVID-19 severity, as described in the general population (p=0.003, p<0.001). Fig 1 shows age distribution and COVID-19 severity by disease severity groups. The mean age for severe COVID-19 was lower in patients with severe SCD (SS/SB0 vs SC/SB+: 23y vs 67.5y) and THAL (major vs intermedia: 43.5 vs 51.3y) (p<0.001). Potential risk factors such as elevated ferritin, current chelation or history of splenectomy did not confer additional risk for developing severe COVID-19 in any patient group. Only diabetes as a comorbidity correlated with severity grade in SCD (p=0.01) and hypertension in THAL (p=0.009). While severe COVID-19 infection in SCD was associated with both ACS (p<0.001) and kidney failure requiring treatment (p<0.001), this was not predicted by a history of previous ACS or kidney disease in steady state. Overall, 14,6% RBC patients needed oxygen/respiratory support, 4% were admitted to ICU with an overall mortality rate of 1%, much lower than reported in other similar cohorts. Image: Summary/Conclusion: Results obtained so far show that severe COVID-19 occurs at younger ages in more aggressive forms of SCD and THAL. Current preventive approaches focus on age over disease severity. Our data highlights the risk of severe COVID-19 infection in some young patients, particularly those with SS/SB0 SCD, suggesting that immunization should be considered in this pediatric group as well. Results between similar sized cohorts of RBCD patients vary between each other and those presented here, highlighting the importance of collecting all of these small cohorts together to ensure adequate statistical power so that definitive risk factors can be reliably identified and used to guide management of patients with these rare disorders in the light of the ongoing pandemic. S284: RISK FACTORS AND OUTCOMES OF PATIENTS WITH HAEMATOLOGICAL MALIGNANCY AND COVID-19: ONGOING REDUCTION IN MORTALITY DURING THE 3RD WAVE OF THE PANDEMIC J. Maddox1,* 1Haematology, South Tees NHS Trust, Middlesborough, United Kingdom Background: It was established early in the COVID-19 pandemic that patients with cancer, in particular haematological malignancy, had worse outcomes than non-cancer patients. There is a lack of recent data to describe how mortality in patients with haematology malignancy has changed with widespread vaccination and several effective treatments now being available. Aims: Based in a large NHS Trust in the North-East of England, our aim was to identify all local patients with haematological malignancy who had contracted COVID-19 since the start of the pandemic. We then examined these patients in more detail to ascertain risk factors for mortality, and how this has changed over the course of the pandemic. Methods: We included patients with an active diagnosis of haematological malignancy, or those who had received potentially curative treatment within the past 3 years. We excluded patients with pre-malignant conditions. Data up to the end of November 2021 found 213 eligible patients. Nearly all patients identified in the 1st wave of the pandemic were identified from hospital testing, reflecting the lack of availability of widespread testing in the community. More recently, patients identified in the community have predominated. Results: Overall mortality following COVID-19 infection was 21.6% at 4 weeks and 27.7% at 8 weeks after COVID-19 diagnosis. Mortality was highest in wave 1 (March – June 2020), decreasing in wave 2 (Sept 20 – March 2021) and again in wave 3 (May 2021 – current), with 4-week mortality figures of 44%, 26% and 8% respectively. It should be noted that widespread community testing was not available early in the pandemic so the recorded cases in wave 1 were sicker patients needing hospital care. Removing pillar 2 data still shows a reduction in 4-week mortality over the course of the pandemic, from 46% (wave 1) to 39% (wave 2) and 8% (wave 3). Although official COVID-19 mortality figures only include deaths within 4 weeks of a confirmed infection, we note that the Kaplan-Myer mortality curve did not level out until 6-8 weeks after initial infection (figure 1). Overall mortality at 8 weeks was therefore higher at 61% (wave 1), 31% (wave 2) and 11% (wave 3). The main risk factor for mortality was patient age, with 8-week mortality in age groups <60yr, 60-69yr, 70-79yr, 80-89yr and 90+yr being 2%, 26%, 30%, 51% and 86% respectively. Although men comprised the majority of detected cases (62%), mortality at four and eight weeks was near identical between sexes. Although disease subgroups were relatively small, we found the highest COVID-related mortality in patients with CLL (43%) and MDS (42%). It is perhaps not surprising as these two disease groups also had the highest mean patient ages. Patients receiving chemotherapy had no significant increase in 8-week mortality compared to those not receiving chemotherapy, 30% vs. 27% (p=ns). The presence of neutropenia was however a risk factor for mortality. Patients who were neutropenic at the time of infection had an 8-week mortality of 43%, compared to 25% in those who were not neutropenic (P=0.04). Image: Summary/Conclusion: Overall, COVID-19 related mortality in patients with haematological malignancy has significantly declined over the course of the pandemic. The main risk factor for death is increased patient age, with neutropenia also being a risk factor. S285: INHIBITION OF COMPLEMENT C1S WITH SUTIMLIMAB IN PATIENTS WITH COLD AGGLUTININ DISEASE (CAD): 2-YEAR FOLLOW-UP FROM THE CARDINAL STUDY A. Röth1,*, W. Barcellini2, S. D’Sa3, Y. Miyakawa4, C. M. Broome5, M. Michel6, D. J. Kuter7, B. Jilma8, T. H. A. Tvedt9, I. C. Weitz10, T. Sourdille11, J. Wang11, D. S. Vagge12, K. Kralova13, F. Shafer14, M. Wardecki15, M. Lee14, S. Berentsen16 1Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 3UCLH Centre for Waldenström’s Macroglobulinemia and Related Conditions, University College London Hospitals NHS Foundation Trust, London, United Kingdom; 4Thrombosis and Hemostasis Center, Saitama Medical University Hospital, Saitama, Japan; 5Division of Hematology, MedStar Georgetown University Hospital, Washington DC, United States of America; 6Henri-Mondor University Hospital, Assistance Publique-Hôpitaux de Paris, UPEC, Créteil, France; 7Division of Hematology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States of America; 8Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria; 9Section for Hematology, Department of Medicine, Haukeland University Hospital, Bergen, Norway; 10Keck School of Medicine of USC, Los Angeles, CA; 11Sanofi, Cambridge, MA, United States of America; 12IQVIA, Bangalore, India; 13Sanofi, Paris, France; 14Sanofi, Bridgewater, NJ, United States of America; 15Sanofi, Warsaw, Poland; 16Department of Research and Innovation, Haugesund Hospital, Haugesund, Norway Background: CAD is a rare chronic autoimmune hemolytic anemia characterized by classical complement pathway (CP)-mediated hemolysis. Sutimlimab is a first-in-class humanized monoclonal antibody that selectively inhibits C1s of the C1 complex, preventing CP activation, while leaving the alternative and lectin pathways intact. One-year interim follow-up from the CARDINAL study (NCT03347396) have previously demonstrated that sutimlimab resulted in sustained improvements in hemolytic markers and quality of life. Aims: To report 2-year sutimlimab efficacy and safety from the CARDINAL Part B extension. Methods: CARDINAL was a Phase 3, open-label, single-arm study with a 26-week treatment period (Part A) and a 2-year extension (Part B) after the last patient (pt) finishes Part A. Sutimlimab was administered through intravenous infusions on Days 0 and 7, followed by biweekly dosing. Efficacy data through Week 131, the last data recording within the 2-year Part B period, are reported here. Efficacy endpoints included change from baseline in hemolytic markers, pharmacodynamic (PD) markers and blood transfusions. Quality of life (QOL) was assessed using the Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue Scale. Safety was recorded until end-of-study visit 9 weeks after their last dose; endpoints included incidence of treatment-emergent adverse event (TEAE) and serious TEAE (TESAE). Descriptive statistics, frequency, and percentage were used to analyze outcomes. Results: Of the 24 pts enrolled in Part A, 22 completed Part A and entered Part B, with 19 (86.4%) pts completing Part B. Sutimlimab treatment improved mean (SD) hemoglobin (Hb) levels within one week; mean Hb remained >11 g/dL from Week 5–131 (baseline: 8.64 (1.67) (Figure). Mean total bilirubin was normalized from Week 3–131 (Figure). Mean FACIT-Fatigue scores improved within 1 week and remained ≥5 from Week 1–123 (Figure), consistent with a clinically meaningful change. Improvements in Hb, bilirubin, and FACIT-Fatigue correlated with normalization of C4 and near-complete inhibition of CP activity. Normalization of mean absolute reticulocyte count was observed alongside normalized haptoglobin levels and reductions in LDH. From Week 26–131, 15 (68.2%) pts remained transfusion-independent. All 22 pts experienced ≥1 TEAE; 12 (54.5%) pts experienced ≥1 TESAE. Serious infections were reported in 7 (31.8%) pts, including one pt with sepsis due to streptococcus pneumoniae. No meningococcal infections were reported. Three pts discontinued the study due to AEs (cyanosis and klebsiella pneumoniae (n=1); vitreous hemorrhage (n=1); cyanosis and gastrointestinal symptoms including erosive gastritis (n=1)). No pts developed systemic lupus erythematosus, serious hypersensitivity or anaphylaxis. Two pts died during the study (klebsiella pneumoniae (n=1); exacerbation of CAD (n=1) in a patient with a femoral neck fracture and complex medical history including myelodysplastic syndrome, approximately 1.5 months after receiving the last dose of sutimlimab). Image: Summary/Conclusion: Sutimlimab, a first-in-class selective anti-C1s classical complement pathway inhibitor, maintained mean Hb levels >11g/dL, achieved sustained normalization of mean bilirubin, haptoglobin and reticulocyte count. Sutimlimab continued to improve FACIT-Fatigue scores, with no newly identified safety concerns at 2 years of treatment. This study has demonstrated that sutimlimab is an effective and well-tolerated long-term therapy for the management of chronic CAD through continued upstream inhibition of the classical CP. S286: LONG-TERM EFFICACY AND SAFETY RESULTS FROM AN ONGOING OPEN-LABEL PHASE 2 STUDY OF PARSACLISIB FOR THE TREATMENT OF AUTOIMMUNE HEMOLYTIC ANEMIA (AIHA) W. Barcellini1,*, I. Murakhovskaya2, L. Terriou3, F. Pane4, A. Patriarca5, K. Butler6, S. Moran6, S. Wei6, U. Jäger7 1Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2Albert Einstein College of Medicine/Montefiore Medical Center Bronx, New York, United States of America; 3Univ. Lille, Inserm, CHU Lille, Centre de Référence des Maladies Autoimmunes Systémiques Rares du Nord et Nord-Ouest de France (CeRAINO), INFINITE – Institute for Translational Research in Inflammation, Lille, France; 4University of Naples “Federico II”, Naples; 5University of Eastern Piedmont and AOU “Maggiore della Carità”, Novara, Italy; 6Incyte Corporation, Wilmington, United States of America; 7Medical University of Vienna, Vienna, Austria Background: AIHA is a rare condition caused by autoantibody-mediated hemolysis of red blood cells. Few therapies beyond steroids and rituximab are available. Aims: To report updated results from an ongoing multicenter, phase 2, open-label study of the phosphoinositide 3-kinase-δ inhibitor parsaclisib in patients (pts) with AIHA (NCT03538041). Methods: Pts ≥18 years old with warm AIHA (wAIHA), cold agglutinin disease (CAD), or mixed-type AIHA; hemoglobin (Hgb) 7–10 g/dL; and failure of ≥1 standard therapy were eligible. After informed consent, pts were treated with oral parsaclisib for 12 wk at a starting dose of 1.0 mg once daily (QD; cohort 1) or 2.5 mg QD (cohort 2). Increases to 2.5 mg QD were allowed in cohort 1 after 6 wk if no clinical response was achieved and/or transfusion was required; reductions to 1.0 mg QD were permitted in cohort 2 for tolerability issues. Concomitant corticosteroids (≤20 mg/d prednisone) were allowed. After 12 wk of treatment, pts responding on parsaclisib could continue into an extension period. Primary endpoints were efficacy (proportion of pts with complete response [CR; Hgb ≥12 g/dL] or partial response [PR; Hgb 10–12 g/dL or ≥2 g/dL increase from baseline, inclusive of CRs] at any visit from Wk 6–12) and safety (treatment-emergent adverse events [TEAEs]). Results: As of Aug 5, 2021, 25 pts enrolled and received parsaclisib (cohort 1, n=10 [8 with dose increase]; cohort 2, n=15); 20 pts (80%) completed 12 wk of treatment. Sixteen (64%), 6 (24%), and 3 (12%) pts had wAIHA, CAD, and mixed AIHA, respectively. Mean (SD) age was 61.6 (17.0) years; 14 pts (56%) were female and 23 (92%) were White. Mean (SD) Hgb at baseline was 8.9 (0.8) g/dL, and 9 pts (36%) had transfusions in the past year. Mean parsaclisib exposure was 334 (range, 7–819) days. Overall, 8 pts (32%) achieved CR and 16 (64%) had PR at any visit from Wk 6–12. Among pts with wAIHA (n=16), 14 pts (88%) completed 12 wk of treatment; 8 (50%) and 12 (75%) achieved CR and PR, respectively, at any visit from Wk 6–12. Seventeen pts entered the extension period (12 with wAIHA). Increase in Hgb was sustained in the total cohort and among pts with wAIHA during the initial 12-wk treatment and extension periods (Figure). During the 12-wk treatment period, TEAEs occurred in 21 pts (84%) overall. Six pts (24%) had grade (Gr) ≥3 TEAEs, with only neutropenia occurring in >1 pt (n=2 [8%]); 2 pts (8%) had serious AEs (SAEs). Seven pts (28%) had treatment-related AEs, with only pruritic rash reported in >1 pt (n=2 [8%]). Three pts discontinued during the 12-wk treatment period (AE [n=1; thrombocytopenia], lack of efficacy [n=1], withdrawal by subject [n=1]), and 2 pts were of unknown status. During the extension period, 15/17 pts (88%) experienced ≥1 TEAE. Gr ≥3 AEs and SAEs were each reported in 9 pts (53%). Six pts (35%) had treatment-related AEs, with diarrhea and rash reported in >1 pt (n=2 [12%] each). One Gr 3 TEAE (psoriasis) and 3 SAEs (diarrhea [Gr 2], cytomegalovirus reactivation [Gr 2], psoriasis [Gr 3]) were deemed treatment related. Two (12%) pts had TEAEs leading to parsaclisib discontinuation. One fatal TEAE (acute respiratory failure in the extension period) was deemed unrelated to parsaclisib. Image: Summary/Conclusion: Parsaclisib was generally well tolerated and resulted in Hgb improvements as early as Wk 2 that increased over 12 wk of treatment and were sustained through the extension period. Parsaclisib may be an effective oral treatment for AIHA, and a randomized, controlled phase 3 trial in wAIHA is now recruiting (NCT05073458). S287: FACTORS INFLUENCING AUTOLOGOUS LYMPHOCYTE COLLECTIONS FOR CHIMERIC ANTIGEN RECEPTOR (CAR) T-CELLS – THE ROLE OF T-CELL SENESCENCE V. Vucinic1,*, T. Tumewu1, M. Brückner1, M. Jentzsch1, F. Ramdohr1, R. Buhmann2, Y. Remane3, S. Hoffmann1, M. Janz4, O. Penack5, G. Vogtmann1, E. Ruschpler1, L. Bullinger5, U. Keller4, M. Cross1, S. Schwind1, M. Herling1, G.-N. Franke1, E. Bach1, H. Reinhard2, U. Platzbecker1 1Medical Clinic and Policlinic for Hematology and Celltherapy; 2Institute for Transfusion Medicine; 3Pharmacy, University Leipzig, Leipzig Medical Center, Leipzig; 4University of Berlin, Campus Benjamin-Franklin; 5University of Berlin, Campus Virchow-Klinikum, Berlin, Germany Background: The apheresis of autologous CD3+ lymphocytes is the first pivotal step in the production process of chimeric antigen receptor T-cells. A range of factors like previous cytotoxic therapies or disease status can influence the quality of collection but may also impact the fitness of T-lymphocytes. Repeated T-cell activation leads to progressive loss of expression of CD27 and CD28, receptors shown to be uniformly present on naïve CD4+ cells (van Leewen et al, J. Immunol, 2004). Aims: To evaluate factors influencing both collection of autologous lymphocytes and the subsequent manufacturing of tisagenlecleucel with a special focus on T-cell senescence, defined as loss of CD27 and CD28. Methods: Between February 2019 and October 2021, 59 collections were performed for subsequent CAR-T cell therapy with tisagenlecleucel in 51 patients with relapse/refractory (r/r) diffuse large B-cell lymphoma and one patient with refractory acute lymphoblastic leukemia. The median age was 60.5 (range 17-80) years and 40 (77%) patients were male. The collections were performed on a Spectra Optia cell-separator by processing a median 3x total blood volume. Results: The target numbers of CD3+ cells (>0.55 x109) were successfully collected within a single collection day in all patients but one. The median yield of CD3+ cells was 5.0 (0.4-31.9 x109, with yields for CD3+CD4+ and CD3+C8 + 20.1 (range 2.4-102.8) x108 and 27.4 (range 2.41-266.8) x108, respectively. The median CD4:CD8 ratio was 0.7 (0.08-5.3). The yields for CD3+CD27+CD28+ and CD3+CD27-CD28- cells were 25.9 (range 1.1-93.0) x108 and 10.9 (range 0.2-183.8) x108, respectively. We observed no difference in median collected CD3+ cell yields in patients with > vs ≤ 3 prior therapy lines (p=0.29), with or without prior treatment with bendamustine (p=0.42) or bone marrow infiltration (p=0.95). 56 collections underwent further manufacturing, with production according to specification in 38 (68%) collections. 18 collections resulted in production failure, 13 (72%) due to insufficient proliferation of T-cells, 1 (6%) because of microbiological contamination and 4 (22%) due to undeterminable test results for mycoplasms. The manufacturing failures did not associate with age >60 (p=0.16), sex (p=0.99), bone marrow infiltration (p=0.47) or >3 prior treatment lines (p=0.78). However, we did notice a trend regarding prior treatment with bendamustine (p=0.07). Importantly, the collections resulting in production failure had significantly lower CD3+/CD27+/CD28+ counts of 16.1 (range 1.1-52.3) vs 33.2 (range 3.3-93.5) x108 (p<0.01), as well as a trend for lower CD3+/CD4+ counts with 15.9 (range 3.0-43.9) vs 24.5 (range 2.4-102.7) x108 (p=0.05). However, there were no differences in CD3+/CD8+ counts (p=0.19), CD4:CD8 ratio (p= 0.77) or CD3+/CD27-/CD28- cells (p=0.81; Figure 1). Image: Summary/Conclusion: In our real-life cohort of lymphoma patients intended to undergo treatment with tisagenlecleucel, adequate numbers of CD3 cells could be collected in most cases irrespective of the number of prior therapies. We could observe a negative prognostic role of T-cell senescence on the manufacturing process. Further analyses will be required to determine measures appropriate for further optimization and improvement of collection outcomes. S288: A 9 YEAR REVIEW OF BLOOD TRANSFUSION PRACTICE AND ADHERENCE TO NICE GUIDELINES AT A DISTRICT GENERAL HOSPITAL, UK N. Clayden1,*, E. O’Donovan1 1Haematology, Surrey and Sussex Healthcare Trust, Surrey, United Kingdom Background: Blood transfusions are common medical practice in the UK, with approximately 2.1 million blood products being issued by UK blood services in 2020. The Serious Hazard of Transfusion (SHOT) scheme reported that the risk of death and serious harm related to transfusions was 1 in 53,193 and 1 in 15,142 in 2020, respectively. Multiple complications can occur as a result of blood transfusions, with the most common cause of death being Transfusion Associated Circulatory Overload (TACO). Although low risk, there has been an increase in deaths related to blood transfusions from 17 deaths in 2019, to 39 deaths in 2020, in the UK, with 81.6% of adverse reactions and events being due to preventable errors. This stresses the importance of safe transfusion practice. Aims: To review the rates of blood transfusions from 2013 to 2021, and compare our adherence to NICE guidance. We analysed rates of transfusion, number of red blood cells (RBC) units per transfusion, haemoglobin levels and the incidence of iron deficiency anaemia (IDA) prior to transfusion. Methods: Data was collected retrospectively from patients’ records and the transfusion laboratory database at Surrey and Sussex healthcare Trust. Adults (>16 years old) who received inpatient or outpatient RBC transfusions from 2013 to 2021 were included (n = 53,941). We looked in further detail at the RBC transfusions from the first 2 weeks of December from 2014 to 2021 (n=546). Results: Figure 1 shows that there has been a significant decline in the number of RBC transfused from 2014 to 2021; an average gradient of -36.25 units per year (R2= 0.72). There has also been a decline in the average number of units per blood transfusion, with a downward trendline (gradient -0.15 RBC units/year, p= 0.001). There has been a significant increase in the percentage of single unit transfusions from 14% in 2014 to 65% in 2021; the trendline has a gradient of +7% / year (p=0.0009). The average haemoglobin at initiation of transfusion has remained relatively unchanged (69-78g/L), which is in line with NICE guidance. There is no improvement in transfusion of patients with IDA; defined as a low transferrin saturation (<20%). The percentage of these patients being transfused varying from 43% to 79%, with no significant trend over the years from 2014 to 2021 (p=0.71). Image: Summary/Conclusion: The total number of RBC transfused has significantly decreased over 9 years. However, there is a slight rise from August 2020, which may have been a result of expansion of the hospital bed base from 697 to 800 between years 2018-2021. It also may have been impacted by the COVID-19 pandemic. There is a decrease in the amount of RBC units transfused per patient. We are also encouraged to see a significant increase in the number of single unit transfusions, in line with NICE Guidance. We found there was no improvement in the number of patients with IDA being transfused (average 64%). This indicates that, depending on the clinical scenario, some patients may be receiving unnecessary blood transfusions. They may benefit from receiving iron replacement as an alternative treatment, thereby minimising exposing patients unnecessarily to the risks associated with blood transfusion. Appropriate management of IDA needs further work in the trust, and we have initiated an IV iron service over the last 18 months to improve this. Limitations of this study include using two weeks of the year to extrapolate for each year and data may have been skewed over the last two years due to the COVID-19 pandemic. S289: MOTIVATORS AND BARRIERS TO BLOOD DONATION AMONG POTENTIAL DONORS OF AFRICAN AND CAUCASIAN ETHNICITY H. Fogarty1 2,*, M. Sardana3, L. Sheridan4, P. Chieng3, S. Kelly3, N. Ngwenya2, C. Sheehan2, K. Morris5, E. Tuohy2 1Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland; 2Department of Haematology, St James’s Hospital; 3School of Medicine; 4School of Pharmacy, Royal College of Surgeons in Ireland; 5Irish Blood Transfusion Service, Dublin, Ireland Background: Blood donors of minority ethnicity, especially those of African origin, are under-represented in many high-income Western countries, including in Ireland. Conversely, the rising number of patients with Sickle Cell Disease (SCD) in many European countries has resulted in increased demand for blood transfusion. Given the high risk of alloimmunization among haemoglobinopathy patients, extended red blood cell matching is now recommended. To increase the probability of a phenotypic match, donors and recipients should share the same racial and/or ethnic background. Hence, the discrepancy between the number of African blood donors and the number of SCD patients is creating a strain on the blood supply chain and recruitment of African donors has become a major priority for transfusion services worldwide. Aims: A key step towards enhancing recruitment of blood donors is to first understand perceived barriers and facilitators to blood donation, which may differ across different demographic categories, including ethnicity. This study aims to explore 1) Barriers and 2) Motivators to blood donation and 3) Awareness of SCD among potential donors of diverse ethnic backgrounds in Ireland. Methods: Following ethical approval, patients attending the National Sickle Cell Disease and Thalassemia service at St James’s Hospital, Dublin, were invited to share an anonymous, online, 33-item survey within their local communities to achieve snowball-sampling. Inclusion criteria were adults aged ≥18 years who had not previously donated blood in Ireland. Participants were asked to state their age, sex and ethnicity. Responses for each survey item were recorded on a four-point Likert scale: Strongly Agree, Agree, Disagree or Strongly Disagree. Survey data were collected from August-December 2021. Statistical analysis was conducted using GraphPad Prism 9.1 (GraphPad Software Inc., USA). Results: 387 respondents completed the survey, including 311 non-donors (median age 25 years, 67% female). Ethnic backgrounds included: African (59%), Caucasian (25%), Asian (8%), Hispanic/Latino (3%), Middle Eastern (3%) and Multiracial (2%). The most common barrier overall was lack of information on blood donation, identified by 60%. African respondents were 3 times more likely to report lack of information (OR 3.1 CI 1.8-5.3, p<0.0001) and 5 times more likely to report malaria-related barriers than Caucasians (OR 5.1 CI 2.1-12.3, p=0.0002). Conversely, Caucasians were twice as likely to report fear of needles/pain (OR 2.1, CI 1.1-3.7, p=0.02). Motivators also varied by ethnicity, with African respondents 9 times more likely to donate to help someone within their own community (OR 9.3 CI 4.6-18.3, p<0.00001) and 5 times more likely to donate for religious motivators (OR 5.5 CI 2.9-10.4 p<0.0001) than Caucasians. Awareness of SCD was 5 times higher among African respondents (92% versus 66%, OR 5.3 CI 2.7-10.5, p<0.0001). Summary/Conclusion: This study enhances our understanding of ethnic differences in barriers and motivators to blood donation. While some barriers are shared across ethnic groups, including lack of information, notable differences exist between Caucasian and African respondents. Given that many European countries experience difficulties in the provision of phenotypically matched blood for SCD patients, these data provide valuable insights to inform future campaigns aimed at recruitment of African donors. At a time when the importance of diversity and inclusivity are increasingly recognized in society, enhancing ethnic diversity in blood donors may reflect a positive force for healthcare and social equality. S290: ATRA CAN CORRECT DEFECTIVE HIF-1Α/S1P AXIS-MEDIATED CYTOSKELETAL REORGANIZATION IN PROPLATELET FORMATION OF ITP Q.-S. Huang1 2,*, J. Xue1 2, F.-Q. Liu1 2, Q. Chen1 2, G.-C. Zhang1 2, X.-Y. Sun1 2, C.-C. Wang1 2, L.-P. Yang1 2, Y.-Y. Li3 4 5, Q.-F. Wang3 4 5, J. Peng6, M. Hou6, X.-J. Huang1 2 7 8, X.-H. Zhang1 2 7 8 1Peking University People’s Hospital; 2National Clinical Research Center for Hematologic Disease; 3Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China; 4China National Center for Bioinformation; 5University of Chinese Academy of Sciences, Beijing; 6Department of Hematology, Shangdong Key Laboratory of Immunochematology, and Shandong Provincial Clinical Medicine Research Center for Hematology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan; 7Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation; 8Collaborative Innovation Center of Hematology, Beijing, China Background: Bone marrow physiological hypoxia plays a crucial role in haematopoietic stem cell homeostasis. Hypoxia inducible factor (HIF) is central to mediating the cellular response to hypoxia. HIF-1α expression was decreased in the bone marrow of patients with immune thrombocytopenia (ITP), and HIF-1α activation was shown to enhance megakaryopoiesis in mice. Recent studies suggest that “inside-out” signalling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that HIF-1α/S1P axis-mediated cytoskeletal reorganization was defective in the PPF of ITP. All-trans retinoic acid (ATRA), which has been shown to be a promising treatment option for ITP patients in our clinical studies (Blood, 2021; Lancet haematology, 2017; Lancet haematology, 2021), could restore cytoskeletal reorganization and correct impaired PPF. Aims: This study aimed to explore the role of hypoxia inducible factor-1α (HIF-1α) in proplatelet formation (PPF) and the underlying mechanisms of ATRA treatment in ITP patients. Methods: Thirty consecutive patients with newly diagnosed ITP and 30 healthy donors were included in our study. MKs were isolated from bone marrow samples. Targeted and untargeted metabolomic profiling through metabolomic analysis was performed to explore the relationship between the metabolome and ITP. Confocal microscopy and transmission electron microscopy were used to observe the PPF and cytoskeleton structure of ITP MKs. An ITP mouse model was established to observe the therapeutic effects of ATRA in the PPF. Results: In the present study, we observed that MKs displayed altered cytoskeletal reorganization and impaired proplatelet formation (PPF) in ITP patients. Targeted and untargeted metabolite profiling revealed a decreased sphingosine-1-phosphate (S1P) level in ITP. Downregulated sphingosine kinase 2 (SPHK2) expression in MKs accounted for the low level of S1P in ITP. S1P is essential for S1P receptor 1 (S1PR1) and Rac1 activation, Src family kinases (SFKs) activity, and subsequent cytoskeletal reorganization and PPF regulation. Moreover, we demonstrated that HIF-1α mediated SPHK2 activation and S1P production. Decreased HIF-1α levels were found in the MKs of patients with ITP, contributing to impaired PPF. We then investigated the effect of ATRA on PPF in ITP patients. ATRA upregulated HIF-1α and SPHK2 expression, increased S1P production and corrected impaired PPF in vitro. In an ITP mouse model, ATRA alleviated thrombocytopenia and restored cytoskeletal reorganization. ATRA corrected impaired PPF by upregulating HIF-1α expression. The exposure of ITP MKs to selective RARα (AM580) or RARγ (BMS961) agonists did not change PPF. However, the treatment of ITP MKs with a selective RARβ agonist (CD2314) significantly increased PPF. Furthermore, we found that the effect of ATRA on enhancing PPF in ITP MKs was reversed in the presence of an RARβ antagonist (CD2665). These data suggest that impaired PPF in ITP MKs is corrected by ATRA in a RARβ-dependent manner. Summary/Conclusion: Together, our data show that the HIF-1α/S1P axis mediates altered cytoskeletal reorganization and impaired PPF in ITP and suggest that ATRA correction of impaired PPF is a potential mechanistic explanation for the clinical efficacy of ATRA in ITP. S291: PHASE I/II STUDY OF RILZABRUTINIB, AN ORAL BRUTON TYROSINE KINASE INHIBITOR, IN PATIENTS WITH IMMUNE THROMBOCYTOPENIA: LONG-TERM FOLLOW-UP D. J. Kuter1,*, M. Efraim2, Z. Kaplan3, J. Mayer4, P. Choi5, A. G. Jansen6, V. McDonald7, R. Baker8, R. Bird9, M. Garg10, J. Gumulec11, M. Kostal12, T. Gernsheimer13, W. Ghanima14, M. Yao15, A. Daak16, N. Cooper17 1Hematology Division, Massachusetts General Hospital, Harvard Medical School, Boston, United States of America; 2Multiprofile Hospital for Active Treatment Sveta Marina EAD, Varna, Bulgaria; 3Monash Medical Centre, Clayton, Australia; 4Department of Internal Medicine, Hematology and Oncology, Masaryk University Hospital, Brno, Czechia; 5The Canberra Hospital, Garran, Australia; 6Erasmus MC, University Medical Center, Rotterdam, Netherlands; 7Barts Health NHS Trust, The Royal London Hospital, London, United Kingdom; 8Perth Blood Institute, Murdoch University, Perth; 9Princess Alexandra Hospital, Woolloongabba, Australia; 10Leicester Royal Infirmary, Leicester, United Kingdom; 11Department of Hematooncology, University Hospital Ostrava and Faculty of Medicine University of Ostrava, Ostrava; 12Fourth Department of Internal Medicine and Hematology, Faculty of Medicine, University Hospital of Hradec Kralove, Hradec Kralove, Czechia; 13University of Washington Medical Center, Seattle, United States of America; 14Østfold Hospital Foundation, Gralum, Norway; 15Biostatistics, Sanofi US Services Inc., Bridgewater; 16Sanofi Genzyme, Cambridge, United States of America; 17Department of Medicine, Hammersmith Hospital, London, United Kingdom Background: Immune thrombocytopenia (ITP) is an autoimmune disease associated with autoantibody-mediated platelet destruction and impaired platelet production, resulting in thrombocytopenia and high bleeding risk. In a global phase I/II trial (NCT03395210) of heavily pretreated patients with long-standing disease, the Bruton tyrosine kinase inhibitor rilzabrutinib showed a rapid and durable platelet count increase and was well tolerated. We present results for patients initiating rilzabrutinib 400 mg BID who are continuing in the long-term extension (LTE). Aims: Assess if the efficacy and safety of rilzabrutinib 400 mg BID continue to be maintained in LTE patients on rilzabrutinib ± concomitant medication. Methods: Patients with 2 baseline platelet counts <30×109/L were required to have responded to ≥1 prior ITP therapy but at baseline were unable to maintain an adequate response to prior/concomitant therapies. The main treatment period was 24 weeks; patients who achieved platelet counts ≥50×109/L at ≥50% of the visits during the last 8 weeks of the main period were permitted to continue 400 mg BID in the LTE. Primary endpoints were safety and efficacy (≥2 consecutive platelet counts ≥50×109/L and increased ≥20×109/L from baseline without requiring rescue medication). Stable doses of concomitant ITP medication (thrombopoietin-receptor agonists [TPO-RA] and corticosteroids [CS]) were allowed for patients with inadequate platelet response. Patients who received rescue medication discontinued from the study. All patients provided informed consent. Results: At baseline, the 45 patients initiating 400 mg BID in the main period had a median age of 49 years, median duration of ITP of 6.1 years, median platelet count of 15×109/L, and a median of 4 unique prior therapies (24% prior splenectomy). A total of 15 patients (33%) received rilzabrutinib monotherapy, and 30 had concomitant therapy (TPO-RA n=13 [29%], CS n=12 [27%], TPO-RA + CS n=5 [11%]). Primary platelet response was achieved by 18 patients (40%): 6 on monotherapy and 12 on concomitant medication. Platelet counts of ≥50×109/L, ≥30×109/L, and ≥20×109/L from baseline were maintained for a median of 71%, 95%, and 87% of weeks, respectively. As of 21Jan2022, 16/60 patients in the main study population had proceeded to LTE, of whom 11 were ongoing. Five LTE patients (31%) continued on rilzabrutinib monotherapy and 11 used concomitant medication (TPO-RA n=2 [13%], CS n=7 [44%], TPO-RA + CS n=2 [13%]; Figure); 1 patient used rescue medication. Median platelet count at LTE entry was 87×109/L. In patients on rilzabrutinib monotherapy, the median platelet count was 68×109/L, which was sustained at 6 months of follow-up (Table). In all LTE patients, platelet counts ≥50×109/L, ≥30×109/L, and ≥20×109/L from baseline were maintained for a median of 88%, 100%, and 97% of weeks, respectively. In patients on rilzabrutinib ± concomitant medication, results were consistent at 3 and 6 months of follow-up (Table). Three patients (19%) experienced related treatment-emergent adverse events (TEAEs), including 1 grade 2 upper respiratory tract infection. Three patients (19%) discontinued rilzabrutinib due to a TEAE. Six patients had ≥1 bleeding event, none were treatment-related; no related serious adverse events or deaths. The average ITP-BAT bleeding scale score at 6 months showed no increase in bleeding in the LTE. Image: Summary/Conclusion: With extended treatment duration, rilzabrutinib 400 mg BID showed durable clinical efficacy and was well tolerated in patients on rilzabrutinib ± concomitant medication. S292: SUSTAINED RESPONSE OFF TREATMENT IN ELTROMBOPAG-TREATED PATIENTS WITH ITP WHO ARE REFRACTORY OR RELAPSED AFTER FIRST-LINE STEROIDS: PRIMARY ANALYSIS OF THE PHASE II TAPER TRIAL N. Cooper1,*, W. Ghanima2, N. Vianelli3, D. Valcárcel4, I. Yavaşoğlu5, A. Melikyan6, E. Yañez Ruiz7, J. Haenig8, J. Lee9, J. Maier8, N. Zolkin10, F. Zaja11 1Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, Hammersmith Hospital, London, United Kingdom; 2Department of Medicine, Østfold Hospital Trust, Kalnes, Norway; 3Scientific Institute for Research, Hospitalization and Healthcare (IRCCS), Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 4Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), University Hospital Vall d’Hebron, Barcelona, Spain; 5Department of Hematology, Adnan Menderes University, Aydin, Turkey; 6National Research Center for Hematology, Moscow, Russia; 7Hematology-Oncology Unit, Department of Internal Medicine, School of Medicine, Universidad de la Frontera, Temuco, Chile; 8Novartis Pharma AG, Basel, Switzerland; 9Novartis Pharmaceuticals Corporation, East Hanover, NJ, United States of America; 10IQVIA, St. Petersburg, Russia; 11Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy Background: Corticosteroids (CSs) are the standard 1st-line treatment for primary immune thrombocytopenia (ITP); however, long-term CS use is associated with high relapse rates and considerable toxicity. Treatments that achieve a sustained response and reduce the need for long-term CS use are needed. Eltrombopag (EPAG) is a thrombopoietin receptor agonist (TPO-RA) indicated in Europe for the treatment of patients (pts) aged ≥1 year with primary ITP lasting ≥6 months who are refractory to other treatments (eg, CSs). Evidence suggests that a proportion of pts treated with TPO-RAs achieve sustained responses that are maintained after TPO-RA tapering and discontinuation; however, much of the evidence is retrospective with only a few prospective studies investigating sustained response off treatment (SRoT). Aims: TAPER (NCT03524612), a Phase II, open-label, prospective, single-arm study, aims to determine whether EPAG can induce SRoT in pts with ITP after 1st-line CS failure. Methods: Adult (≥18 years) pts with ITP who did not respond to or had relapsed after initial CS therapy, with platelet counts <30×109/L and assessed as needing treatment, were included. Patients received a 50 mg/day starting dose of EPAG (25 mg/day for East/Southeast Asian pts; 12.5 mg/day for Japanese pts in Japan), which could be increased up to 75 mg/day (50 mg/day in Japan) if needed. The primary endpoint was the number (%) of pts with SRoT by Month 12; SRoT was defined as achieving a complete response (CR, ie, platelet count ≥100×109/L), then maintaining a stable platelet count (no counts <70×109/L) for 2 months, followed by successful EPAG tapering and discontinuation with platelet counts ≥30×109/L and no bleeding events or rescue therapy. Patients with SRoT at Month 12 were followed for a further year. Secondary outcomes included SRoT duration and platelet count changes from baseline. Data to Month 12 are presented. Results: N=105 pts were enrolled. The median (interquartile range [IQR]) age was 46 (30-65) years; 61% were female. In the 1st 12 months, median (IQR) duration of exposure to EPAG was 5.6 (2.3-11.9) months and median (IQR) EPAG dose was 57.1 (37.5-69.0) mg/day. Overall, 89 pts (85%) achieved CR at least once and 65 pts (62%) maintained a platelet count ≥70×109/L for 2 months after CR. EPAG tapering and discontinuation was achieved in 44 pts (42%). The primary endpoint was met, with 32 pts (30.5% [95% confidence interval, 21.9-40.2]; P<0.001 [H1: P>15%; alpha: 0.05]) achieving SRoT until Month 12 (Fig. 1); SRoT was maintained from last dose to Month 12 for a median (IQR) of 33.3 (25.7-45.3) weeks. The median (IQR) absolute increase in platelet counts from baseline at Month 12 was 77.0×109/L (35.0-145.0). All-grade adverse events (AEs) occurred in 92/105 (88%) pts (grade ≥3 AEs: 33/105 [31%]). Treatment-related AEs occurred in 37 (35%) pts (8 [7.6%] grade ≥3). The most common all-grade AEs were headache (21% of pts with ≥1 event [grade ≥3: 1% of all pts]), thrombocytopenia (17% [10.5%]), and petechiae (11% [1%]). There were 4 deaths (none were considered treatment related): 3 were on-treatment (central nervous system hemorrhage [n=1], intracranial hemorrhage [n=1], metastases to peritoneum [n=1]) and 1 death (malignant neoplasm) occurred 238 days after last dose. Image: Summary/Conclusion: Data from the TAPER study indicate that a significant proportion of pts experience sustained response following tapering and discontinuation of EPAG, even after a relatively short duration of exposure. Overall EPAG was well tolerated with no unexpected AEs. S293: RISK OF FRACTURES IN ADULT PATIENTS WITH PRIMARY AND SECONDARY IMMUNE THROMBOCYTOPENIA: A DANISH NATIONWIDE COHORT STUDY N. Mannering1 2,*, D. L. Hansen1 2, G. Moulis3 4, W. Ghanima5 6, A. Pottegård7, H. Frederiksen1 2 1Department of Hematology, Odense University Hospital; 2Clinical Institute, University of Southern Denmark, Odense, Denmark; 3Department of Internal Medicin; 4Clinical Investigation Center 1436, Team PEPSS, Toulouse University Hospital, Toulouse, France; 5Department of Hematology, Østfold Hospital; 6Institute for Clinical Medicine, University of Oslo, Oslo, Norway; 7Department of Public Health, University of Southern Denmark, Odense, Denmark Background: The mainstay of first line treatment in immune thrombocytopenia (ITP) has remained high-dose corticosteroids for decades. Although steroid treatment is recommended to be tapered quickly in ITP, previous data shows that during the first six weeks of primary ITP treatment the mean accumulated steroid dose was 2-3 g of prednisolone. In addition, patients may be exposed to repeated courses of corticosteroids during relapses due to its rapid effect on platelet count. Corticosteroids is a well-known risk factor for bone demineralization and osteoporosis that may lead to fractures, but it is unknown if patients with ITP suffer an increased risk of fractures. Aims: In this study we investigated incidences of fractures in primary (pITP) and secondary ITP (sITP) compared to the general population. Methods: Incident patients with ITP ≥18 years were identified in the nationwide Danish health registries during 1980-2016 by using the first registration of the designated codes 287.10 (ICD-8) or D.69.3 (ICD-10). Prevalent ITP and other thrombocytopenic conditions were excluded. Secondary ITP was classified when one or more associated diagnoses were registered any time before or up to 30 days after ITP diagnosis. Each patient with ITP was age-sex matched with up to 40 comparators from the general population. Date of the first ITP registration marked start date of follow-up, and comparators were assigned the same start date. Incident fractures were identified using designated fracture registrations, and divided into five groups: hip and femoral, humeral, antebrachial, axial, and any of the before mentioned. All individuals were followed from start date until the first of the following: fracture, death, emigration, or end of study. Using these data, we calculated rates, incidence-rate-ratios (IRR) and cumulative incidences for fractures in patients vs. comparators after 1, 5 and 10 years, and end of study period. Results: We identified 4,789 patients with pITP, 654 patients with sITP, and 217,370 comparators. Median age was 59.6 years for primary ITP and 57.3 for secondary ITP. Women constituted 54% of primary ITPs, and 64% of secondary ITP patients. IRR in pITP was 1.15 [95% CI 1.04; 1.27] for any fracture, and 1.34 [1.09; 1.62] for axial fractures. For sITP, overall IRR was 1.37 [1.06; 1.74] for any fracture, and 1.65 [1.16; 2.29] for hip – and femoral fractures. IRR was significantly elevated for any fracture during the first 5 years after diagnosis of pITP, but equalized hereafter. The IRR of hip – and femoral fractures in the first year after diagnosis was 2.04 [1.41; 2.86] in pITP, and 2.30 [1.32; 3.74] for axial fractures, however these differences also equalized over time. For sITP, the risk of any fracture was particularly elevated during the first year with an IRR of 2.65 [1.39; 4.63], as well as the risk of distal antebrachial fracture with an IRR of 3.16 [1.33; 6.46]. During year 2-5, IRR for hip – and femoral fracture was 1.92 [1.04; 3.26]. All remaining IRR-estimates were not statistically significant. Cumulative incidence proportions showed similar trends (Figure) with largest differences during first years after diagnosis, but equalizing over time. Image: Summary/Conclusion: The incidence of some fractures in ITP is increased during the first years after diagnosis compared to the general population. Clinical attention and action should be directed upon this matter. Data on comorbidity, sub grouped cumulative incidences, and additional risk estimates will be presented with the abstract. S294: LONG-TERM SAFETY AND EFFICACY OF CAPLACIZUMAB FOR ACQUIRED THROMBOTIC THROMBOCYTOPENIC PURPURA (ATTP): THE POST-HERCULES STUDY M. Scully1,*, J. de la Rubia2 3, K. Pavenski4 5, A. Metjian6, P. Knöbl7, F. Peyvandi8 9 10 11, S. Cataland12, P. Coppo13, J. A. Kremer Hovinga14, R. De Passos Sousa15, F. Callewaert16, S. Gunawardena17, J. Lin17 1Department of Haematology, University College London Hospitals, London, United Kingdom; 2Hematology Department, Internal Medicine, School of Medicine and Dentistry, Catholic University of Valencia; 3Hospital Doctor Peset, Valencia, Spain; 4Departments of Medicine and Laboratory Medicine, St. Michael’s Hospital; 5University of Toronto, Toronto, ON, Canada; 6Division of Hematology, Department of Medicine, University of Colorado–Anschutz Medical Center, Denver, CO, United States of America; 7Department of Medicine; 1, Division of Hematology and Hemostasis, Medical University of Vienna, Vienna, Austria; 8Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 9Angelo Bianchi Bonomi Hemophilia and Thrombosis Center; 10Fondazione Luigi Villa; 11Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy; 12Department of Internal Medicine, Ohio State University, Columbus, OH, United States of America; 13Department of Hematology, Reference Center for Thrombotic Microangiopathies (CNR-MAT), Saint-Antoine University Hospital, AP-HP, Paris, France; 14Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland; 15Sanofi, Lisbon, Portugal; 16Sanofi, Diegem, Belgium; 17Sanofi, Cambridge, MA, United States of America Background: The efficacy and safety of caplacizumab (CPLZ) for patients with acquired thrombotic thrombocytopenic purpura (aTTP; also known as immune-mediated TTP) was demonstrated in the Phase 3 HERCULES trial, with a 28-day follow-up period after end of treatment. Aims: To evaluate long-term safety and efficacy of CPLZ in patients with aTTP, and safety and efficacy of repeated CPLZ use for aTTP recurrence. Methods: In post-HERCULES (NCT02878603), patients who completed the HERCULES trial were invited to attend twice-yearly visits over 3 years. Patients could receive open-label CPLZ with therapeutic plasma exchange (TPE) and immunosuppressive therapy (IST) in case of aTTP recurrence. Safety was assessed during the overall study period in the intention-to-observe (ITO) population (all enrolled patients; n=104). TTP-related events (TTP-related mortality, recurrence, or major thromboembolic events) were assessed in patients without a recurrence during HERCULES or prior to the start of post-HERCULES (efficacy ITO population, n=78). Safety and efficacy outcomes were also evaluated during recurrences (recurrence population, n=19), including patients treated at least twice with CPLZ (received CPLZ in HERCULES or twice in post-HERCULES; repeat-use population, n=9). All patients provided informed consent. Results: Of 104 patients who enrolled, 75 had been treated with CPLZ with TPE+IST during HERCULES (CPLZ group) and 29 had been treated with TPE+IST only (placebo group). Incidence of adverse events (AEs) and serious AEs during the overall study period was similar between groups. Recurrence occurred in 11/75 patients (15%) in the CPLZ group and 8/29 (28%) in the placebo group. In the efficacy ITO population, TTP-related events occurred in 4/49 patients (8%) randomized to CPLZ versus 11/29 (38%) randomized to placebo; in patients randomized to CPLZ, relapse rates were similar between patients with (10%) versus without (7%) prior rituximab use. A total of 19 patients had ≥1 recurrence; 13 were treated with CPLZ (6/13 received concomitant rituximab during the first recurrence period). The first recurrence episode was resolved (12/13) or resolving (1/13) for all patients treated with CPLZ, including the 9 patients with repeat CPLZ use. All second recurrences (6/6) were resolved. One patient who was not treated with CPLZ in either HERCULES or post-HERCULES died as an outcome of recurrence. For the first recurrence period, the median (min, max) time to initial platelet count ≥150x109/L with subsequent stop of TPE within 5 days was 7.0 (3, 24) days for patients treated with CPLZ (n=13) and 10.0 (5, 15) days for patients not treated with CPLZ (n=5); duration of TPE was 5.0 (2, 19) days (n=13) and 5.5 (4, 7) days (n=4), respectively; length of hospital stay was 7.0 (4, 23) days (n=11) and 10.0 (9, 11) days (n=3), respectively. The safety profile of CPLZ for recurrence was consistent with that observed in HERCULES (Table). In the repeat-use population (n=9), bleeding events were reported in 5/9 patients (56%) for the first recurrence; 2 patients had a serious treatment-related treatment-emergent AE: genitourinary bleeding (in 1 patient during the first recurrence) and gastrointestinal bleeding (in 1 patient during the second recurrence). Image: Summary/Conclusion: The long-term safety profile of patients treated with CPLZ in combination with TPE+IST was generally similar to those who received IST+TPE only, with no observed increases in aTTP recurrence. Repeat use of CPLZ was efficacious, with no new safety concerns. S295: CLINICAL RELEVANCE OF SPLEEN VOLUME AND PLATELET COUNT WITH BLEEDING EVENTS IN PATIENTS WITH ACID SPHINGOMYELINASE DEFICIENCY (ASMD) M. Fournier1, J. Msihid1, A. Willemze2, F. Laredo3, R. Pulikottil-Jacob4,* 1Sanofi, Chilly-Mazarin, France; 2Sanofi, Amsterdam, Netherlands; 3Sanofi, São Paulo, Brazil; 4Rare Disease, Health Economics and Value Assessment, Sanofi, Berkshire, United Kingdom Background: Acid Sphingomyelinase Deficiency (ASMD) is an ultra-rare, lysosomal storage disease with a broad spectrum of clinical manifestations. Within that spectrum, ASMD type B and type A/B are often referred to as chronic ASMD. In Type 1 Gaucher disease, increased bleeding tendency is attributed to thrombocytopenia secondary to hypersplenism and/or bone marrow infiltration noticed in patients with splenomegaly and thrombocytopenia. However, ASMD patients have a bleeding tendency that is not proportionally attributed to any association or explanation; hence further research is needed to study the cause of bleeding among patients with ASMD. Aims: To characterize the association between bleeding events (BE), spleen volume (SV), and platelet count by retrospectively analyzing data from a previously published prospective, multicenter, longitudinal study (MSC12840 [SPHINGO-001-00]) in patients with chronic ASMD (N=59; age: 7–64 years; 30 were < 18 years; male: 53%; up to 3 visits per patient with 6–11 years follow-up, 2001–2012). Methods: This study retrospectively screened medical charts of 59 adults and pediatrics for BE specified under the hematopoietic body system and their respective SV and platelet count. SV (in multiple of normal, MN) and platelet count (in 103/UL) were measured at baseline, year 1, and end of study [median years of observation (range), 10.2 years (4.7–11.1)]. As reported in the medical history captured at each visit, BE were selected with a series of keywords indicating hemorrhage, bleed, or transfusion post-medical review. To characterize their association with BE, SV and platelet counts were considered at the visit prior to the observation of the event. A general linear mixed model was performed using SV as a continuous or a class variable (<6, 6–15, and >15 MN). The associations between platelet counts (below 100 x 103/UL) and SV were also explored. Results: Overall, 59 patients were enrolled, 50 patients completed year 1 visit, and 32 patients completed the final visit (9 died, 9 discontinued, and 9 were lost to follow-up). Twenty-six patients reported BE across the two observed periods, i.e., baseline to 1 year and 1 year to final study visit. No BE was reported with SV <6 MN at any period; however, there was a trend for a higher event rate in patients with higher SV, per visit (Table 1). A higher SV of +5 MN or +10 MN was associated with a 1.8-times (odds ratio [95% confidence interval]: 1.85 [1.05, 3.29]) and 3.4-times (3.45 [1.10, 10.82]) increased risk of BE, respectively. Severe splenomegaly (>15 MN) was associated with a higher and non-significant risk of BE vs. moderate splenomegaly (6–15 MN; 2.54 [0.58, 11.08]). In addition, patients with low platelet counts (<100 x 103/UL) had higher SV, per visit (Table 1). The p-value for the chi-square test for association between platelet count and SV across visits was 0.006; it was 0.058, 0.127, and 0.302 at baseline, year 1, and end of the study, respectively. There was a moderate negative correlation (r = –0.47) between platelet count and SV across visits. Image: Summary/Conclusion: This analysis highlighted the association between SV and BE among patients with chronic ASMD. Since a statistically significant association between SV and thrombocytopenia was observed across visits, hypersplenism appears to be a contributor for the pathophysiology of bleedings among these patients. These associations help better understand the clinical relevance of SV measurements in ASMD. Prospective validation is warranted to confirm the results of these analyses. S296: IN VITRO ANALYSIS OF THE IMPACT OF INHIBITORS ON THE PROCOAGULANT EFFECTS OF BYPASS AGENTS OR HEMOSTATIC FACTORS IN COMBINATION WITH EMICIZUMAB A. Dos Santos Ortas1,*, E. García Arias-Salgado1, E. Monzón Manzano1, P. Acuña1, M. T. Álvarez Roman1, M. Martín Salces1, M. I. Rivas Pollmar1, E. García Pérez1, M. Gutiérrez Alvariño1, A. Gonzalez Ceberino1, S. García Barcenilla1, N. Butta1, V. Jiménez Yuste1 1HOSPITAL UNIVERSITARIO LA PAZ, MADRID, Spain Background: Emicizumab is a non-replacement treatment used in hemophilia A patients (PwHA). However, patients on prophylaxis with emicizumab exhibit mild-moderate bleeding phenotypes, thus Factor (F) VIII or bypassing agents (BPAs) are required to control breakthrough or perioperative bleeds. Increments of FIX levels, the main limiting factor for the FIXa-emicizumab-FX complex formation, also produce an increase of the emicizumab procoagulant effects similar to those obtained with FVIII or BPAs. Aims: To elucidate the impact of FVIII inhibitors in the efficacy of this hemostatic response, we compared the in vitro procoagulant effects of BPAs and FIX products in samples from emicizumab-treated patients with or without inhibitors. Methods: Blood from 21 patients on prophylaxis with emicizumab, 8 with inhibitor, was collected in tubes with CTI (Corn Trypsin Inhibitor) to prevent activation of the contact pathway. Spiking with therapeutic doses of 100 IU/dl octocog-alfa (rFVIII), BPAs [1 µg/ml eptacog alfa activated (rFVIIa); 5 U/dl activated prothrombin complex concentrate (aPCC)] or 100 IU/dl of plasma-derived FIX (pFIX), two standard-half-life (SHL) rFIX (nonacog-alfa and nonacog-gamma), and one extended-half-life (EHL) rFIX (albutrepenonacog-alfa) were tested. Clotting time (CT) was evaluated by Rotational Thromboelastometry (ROTEM) with whole blood, and Thrombin Peak by thrombin generation test (TGT) in plasma using low tissue factor concentration. Results: Emizicumab-treated samples showed prolonged CT values (Fig.1A) and lower thrombin peak levels (Fig.1B) than healthy controls and no significant differences were observed between samples with and without inhibitors. Addition of 100 IU/dl of all FIX products normalized the CT values similarly to the addition of the same dose of rFVIII. Nonacog-alfa was more efficient producing similar procoagulant effects than the addition of 1μg/ml rFVIIa or 5 U/dl aPCC. Reduction of CT was similar in samples from PwHA without or with inhibitors in all assayed conditions (Fig.1A). Higher thrombin peak values were also obtained by the addition of all the factors or BPAs tested by TGT but the procoagulant effect of all the recombinant FIX was significantly higher in samples with inhibitors (Fig.1B). Image: Summary/Conclusion: Procoagulant effect of BPAs with emicizumab was similar with or without inhibitors whereas thrombin generation increment produced by rFIX was enhanced in the presence of inhibitors. These results provide a rationale for investigating the concomitant use of rFIX and emicizumab in PwHA with inhibitor in breakthrough bleeding and surgery. Funding: ISCIII-FEDER (PI19/00631); CLS Behring; Catedra UAM-Roche; and Investigator-Initiated Reserach grant (no. IISR-2019-104361) from Baxalta GmbH, a wholly owned subsidiary of Takeda Pharmaceutical Comany Limited. S297: ASSESSING THE COST-EFFECTIVENESS OF LONG-TERM PROPHYLAXIS STRATEGIES IN VON WILLEBRAND DISEASE M. Wilson1, G. Castaman2, G. Escolar3, W. Miesbach4, S. Santos5, S. Yan6,* 1RTI Health Solutions, Research Triangle Park, NC, United States of America; 2Center for Bleeding Disorders, Careggi University Hospital, Florence, Italy; 3Department of Hematopathology, Centre Diagnostic Biomedic, Hospital Clinic, Barcelona, Spain; 4Haemophilia Centre, Medical Clinic II, Institute of Transfusion Medicine, Goethe University Hospital, Frankfurt am Main; 5CSL Behring, Hattersheim am Main, Germany; 6CSL Behring, King of Prussia, PA, United States of America Background: Von Willebrand disease (vWD) is the most common inherited bleeding disorder. Patients with vWD may experience excessive bleeding events resulting in morbidity, reduced quality of life, and a substantial economic burden. Management strategies include von Willebrand factor concentrates either as on-demand treatment (ODT) of bleeds or long-term prophylaxis (LTP) to prevent bleeds. According to recent treatment guidelines, long-term prophylaxis is recommended in individuals with severe and frequent bleeds. Aims: To assess the cost effectiveness of ODT and LTP treatment strategies in vWD patients with low, medium and high annual bleed rate (ABR), using pdVWF/FVIII 2,4:1 products (2,4:1 product A in the United Kingdom (UK) and 2,4:1 product B in Sweden). Methods: A Markov structure considering risk of joint surgery and bleed rate was used to estimate the life years (LYs), quality-adjusted life years (QALYs), and costs of vWD treatment over a lifetime horizon. Treatment options included ODT or LTP with pdVWF/FVIII 2,4:1 or pdVWF/FVIII 1:1 in the UK and in Sweden. Product dosing for LTP and for ODT were obtained from each product’s summary of product characteristics or assumed equal to similar products where data are not available. Bleed risks (major and minor bleed) were estimated for each product in ODT and LTP using published prophylaxis studies and were used to estimate costs of product needed and to determine the probability of requiring joint surgery over time based on the progression of Pettersson Score. Resource use (inpatient and outpatient) costs for bleeds and joint surgery were obtained from standard country-specific costing sources. All costs are shown in 2021 GBP or Swedish Kr for the UK and Swedish analyses, respectively. Health-state utility weights and disutilities of bleeds were obtained from published literature. Annual discount rates for costs and outcomes were 3.5% for the UK and 3% for Sweden. One-way and probabilistic sensitivity analyses were conducted to evaluate the impact of parameter uncertainty. Each analysis considered a low, medium, and high ABR patient population. Results: In the base case analyses (medium ABR) over a lifetime horizon, pdVWF/FVIII 2,4:1 LTP regimens were cost-effective compared with ODT. For pdVWF/FVIII 2,4:1 in the UK, LTP was both cost-saving (-£831,206 incremental cost) and more effective (6.14 incremental QALY) than ODT. Costs of bleed events more than offset the costs of prophylaxis. In Sweden with pdVWF/FVIII 2,4:1, LTP was also dominant (-8,841,901 kr; 6.52 QALY) compared with ODT. In comparisons of LTP regimens, pdVWF/FVIII 2,4:1 was dominant versus pdVWF/FVIII 1:1 in both the UK (-£2,307,370; 1.56 QALY) and in Sweden (-21,939,947 kr; 1.42 QALY). In comparison of ODT regimens, pdVWF/FVIII 2,4:1 was less expensive than pdVWF/FVIII 1:1 in the UK (-£1,250,369; equal QALY) and in Sweden (-7,923,741 kr; equal QALY) due to differences in product costs. Results were similar for comparisons in the high ABR populations. In probabilistic sensitivity analyses, pdVWF/FVIII 2,4:1 was dominant in over 95% of simulations in the UK and in over 85% of simulations in Sweden, and was cost-effective in 95% or more of simulations in both countries. Summary/Conclusion: These results suggest that LTP with pdVWF/FVIII 2,4:1 is a cost-effective strategy compared with ODT, in both the UK and Sweden, for medium and high ABR vWD patients. pdVWF/FVIII 2,4:1 was shown to be cost-effective compared with pdVWF/FVIII 1:1 in the LTP setting, as well as less costly for patients treated ODT for both the UK and Swedish settings, respectively. S298: CS585 IS A FIRST-IN-CLASS COMPOUND TARGETING THE IP RECEPTOR FOR PREVENTION OF THROMBOSIS WITHOUT INCREASED RISK OF BLEEDING S. Lambert1, R. Adili1, P. Yalavarthi1, N. Rhoads1, B. Dahlof2 3, A. White4, N. Bergh2 3, M. Holinstat1 5,* 1Pharmacology, University of Michigan Medical School, Ann Arbor, United States of America; 2Cereno Scientific; 3Institute of Medicine, University of Gothenburg, Gothenburg, Sweden; 4Medicinal Chemistry; 5Internal Medicine, University of Michigan Medical School, Ann Arbor, United States of America Background: Uncontrolled platelet activation leads to the formation of occlusive thrombi resulting in myocardial infarction, stroke, VTE and critical limb ischemia. There is an unmet need for more efficacious anti-thrombotics with less bleeding in this area. Our group recently identify a novel oxidized lipid in the blood that potently and selectively inhibits platelet activation through activation of the prostacyclin receptor. Aims: Develop a first-in-class antiplatelet drug, CS585, that potently and selectively inhibits platelet reactivity and thrombosis without altering hemostasis and bleeding risk. Methods: We developed a mimetic of the oxidized lipid shown to selectively bind to and activate the prostacyclin receptor. Using human platelets, we assessed the ability of CS585 to inhibit platelet activation by assessing 1)aggregometry, 2)adhesion under arterial flow, and 3) granule secretion and integrin activation using flow cytometry. We additionally assessed thrombosis in vivo in 2 different mouse models as well as bleeding. Finally, we assessed potential off-target effects using thromboelastography (TEG). Results: CS585 potently inhibited both collagen and thrombin-induced platelet aggregation. This inhibition was confirmed by measuring inhibition of αIIbβ3 activation, α-granule secretion, and dense-granule secretion by flow cytometry. Selectivity was confirmed in human platelets by demonstrating a full reversal of inhibition when the IP receptor was pharmacologically blocked or genetically eliminated in IP receptor knockout mice. In vivo inhibition of injury-induced thrombosis in the small (laser-induced cremaster thrombosis model) and large (FeCl3-induced carotid artery thrombosis model) vessels by CS585 was demonstrated and no increased risk for bleeding was observed using the tail vein bleeding model. Finally, TEG experiments in human blood spiked with CS585 demonstrated no delay or decrease in clot strength confirming the tail vein bleeding assay experiments in the mouse. Summary/Conclusion: We have shown for the first time that CS585, a first-in-class analog of an oxidized lipid in the blood potently and selectively activates the prostacyclin receptor resulting in inhibition of human and mouse platelet activation and thrombosis without an increased risk of bleeding. This discovery represents a new class of inhibitors for prevention of platelet activation and thrombosis and protection from myocardial infarction, stroke, VTE, and critical limb ischemia. S299: ROLE OF RED BLOOD CELL MEMBRANE-DERIVED PARTICLES ON ENDOTHELIAL DAMAGES DURING ONSET STAGE OF DELAYED HEMOLYTIC TRANSFUSION REACTION N.-P. Kim-Anh1 2,*, L. Kiger2, X. Decrouy3, L. Bencheikh1 2, A. Habibi4, F. Pirenne1 2 5, L. Roumenina6, P. Bartolucci2 4 5 1EFS recherche, Etablissement Français du sang; 2Team Pirenne; 3Imaging platform, INSERM U955 IMRB; 4Sickle cell referral center, Hôpital Henri-Mondor; 5Université Paris Est Créteil, Créteil; 6INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, Sorbonne Universités, Université de Paris, Paris, France Background: Delayed hemolytic transfusion reaction (DHTR) is a life-threatening complication of red blood cell (RBC) transfusion in sickle cell disease (SCD) patients, leading to the increase of plasma hemoglobin (Hb) and heme which are responsible for endothelial damage and organs failure. By the development of an in vitro model reproducing endothelial damages at the early phase of DHTR (Nguyen et al, ASH 2018), our previous study suggested that RBC membrane-derived particles released during the onset stage of DHTR could be also involved in DHTR physiopathology. Aims: In this study, we aim to determine the mechanism of endothelial damages induced by these RBC particles. Methods: Human Umbilical Vein Endothelial Cells (HUVECs) cultured in flow condition for 24 hours were perfused by either serum only (decomplemented or not) or serum containing either whole hemolysate (sonicated RBCs) or different hemolysate components or RBCs membranes (ghosts) under shear stress 1 dyne/cm2. Annexin V and Eculizumab were used for complement studies. Endothelial activation and damages were assessed by membrane CD54 (PECAM-1, Platelet endothelial cell adhesion molecule 1), CD106 (VCAM-1, Vascular Cell Adhesion Molecule-1) and actin network staining. The whole blood adhesion assays and platelets aggregation study on hemolysate-preconditioned HUVECs were performed in continuous flow. Confocal microscopy was used to study interaction between HUVECs and hemoglobin (Hb) and RBC membrane-derived particles. Results: Our fluidic model reproduced a hemolysate-induced pro-inflammatory phenotype and damages of HUVECs via NFkB signaling pathway in a TLR-4 independent manner. The most deleterious effects were observed in whole hemolysate conditions in which, an important quantity of RBC large-size particles (> 2 μm) mostly positive for C3 was detected. These effects on HUVECs were significantly reduced after their elimination by 14000 g centrifugation (Figure 1). Noteworthy, the suspension of ghosts induced a similar activation on HUVECs compared to whole hemolysate suggesting the major role of RBC membrane-derived particles in early hemolysis-induced endothelial damages. Confocal microscopy for intracellular compartment of hemolysate-treated HUVECs demonstrated that while the CD235a+ Hb+ particles were only found in whole hemolysate conditions, the Hb was detected similarly in all conditions tested. Selective blocking of RBCs particles and Hb endocytosis did not affect hemolysate-induced HUVECs activation. These observations demonstrate that in the very early stage (≤4H) of intravascular hemolysis, erythrocytes large-size particles can be internalized in HUVECs, but their internalization is not necessary for endothelial activation effect. Complement inactivation of serum or treatment of RBC particles by either Annexin V or Eculizumab could reduced their effects on endothelial activation suggesting a complement-dependent mechanism Image: Summary/Conclusion: This study suggested for the first time the major role of RBCs large-size particles on endothelial damages at the onset of DHTR in a complement-dependent manner justifying the complement blocking treatment in certain DHTR patients. Furthermore, we have also demonstrated the endocytosis of free Hb by HUVECs, but its pathological impact is still unclear. This endocytosis would facilitate the Hb clearance during a massive hemolysis? These new understandings will allow a better and earlier management to preserve endothelial functions. S300: HEALTH-RELATED QUALITY OF LIFE IN TRANSPLANT-INELIGIBLE REAL-LIFE MULTIPLE MYELOMA PATIENTS TREATED WITH BORTEZOMIB-MELPHALAN-PREDNISONE (VMP) VS. LENALIDOMIDE-DEXAMETHASONE (RD) M. D’Agostino1,*, S. Bringhen1, R. Ria1, F. Ciceri1, A. P. Falcone1, M. Michieli1, M. Grasso1, F. Pane1, M. Quaresima1, F. Cattel2, M. Mirabile1, F. Fioritoni1, M. T. Petrucci1, V. Cotugno2, A. Capra1, S. Pezzatti1, M. L. Mosca Siez1, M. Cantonetti1, G. Margiotta Casaluci1, P. Bertazzoni1, R. Floris1, M. Offidani1, G. Pietrantuono1, A. Evangelista3, M. Boccadoro1, A. Larocca1 1European Myeloma Network, (EMN), Italy; 2S.C. Farmacia Ospedaliera, A.O.U. Città della Salute e della Scienza di Torino, Torino, Italy; 3Unit of Clinical Epidemiology, A.O.U. Città della Salute e della Scienza di Torino and CPO Piemonte, Torino, Italy Background: Multiple Myeloma (MM) is a chronic disease, and patients (pts) receive long-lasting treatment. Thus, the impact on health-related quality of life (HRQoL) may be burdensome, especially in elderly pts. Bortezomib-melphalan-prednisone (VMP) and lenalidomide-dexamethasone (Rd) represented standard-of-care treatments for transplant-ineligible (NTE) newly diagnosed (ND)MM pts before the introduction of daratumumab upfront. No prospective data are available comparing HRQoL in pts receiving VMP vs Rd in a randomized fashion. Aims: We conducted an analysis of Patient Reported Outcomes (PROs) in the context of a randomized multicenter phase IV trial (Real MM Trial, NCT03829371; funded by the Italian Medicines Agency AIFA - Independent Research), to compare HRQoL differences associated with VMP vs Rd treatment in an unselected real-life MM population. Methods: NTE NDMM pts were randomized to receive 9 VMP cycles vs continuous Rd according to standard practice. Pts gave written informed consent and were enrolled regardless of performance status, comorbidities, renal function, or baseline laboratory values. PROs were collected and analyzed using the validated EORTC QLQ-C30 scales and the EQ-5D-5L visual analog scale (VAS) instruments. PROs were collected at baseline, every 3 months during the first year, and every 6 months thereafter. The PROs analyses included pts from the interim analysis (median follow-up: 14.0 months) who had at least a baseline and a follow-up questionnaire available. Data through month 12 are reported. Change in HRQoL from baseline in VMP vs Rd pts was analyzed in a linear mixed model adjusted for International Myeloma Working Group (IMWG) frailty score and cytogenetic risk. Results are presented as least squares (LS) mean change from baseline. Results: At the data cut-off (17-12-2021), 104 pts (56 in the VMP arm and 48 in the Rd arm) had available PROs and were eligible for the analysis. Overall, 46% of pts had >75 years and 40% were frail. No differences in terms of baseline characteristics and response rates were found in the VMP vs Rd arms. Mean baseline values of PROs reflected the deep impairment of pts’ HRQoL at MM diagnosis and were similar in the two arms. After the start of treatment, the different QLQ-C30 scales were analyzed. Global Health Status (GHS) was significantly worse with VMP vs Rd at 3 months (-3.3 vs +9.0; P=0.002), while from the 6-month time point onwards no differences can be found due to an improvement in GHS in the VMP arm (Figure). The physical functioning scale was worse in the VMP vs Rd arm at 3 (-7.8 vs +0.04; P=0.070) and 6 months (-8.2 vs +3.2; P=0.013), with no differences in later time points. The role functioning scale behaved similarly (-5.9 vs +5.2 at 3 months with VMP vs Rd; P=0.038). VMP also increased fatigue (+9.4 vs -2.7; P=0.015), nausea (+8.6 vs +2.8; P=0.04), and appetite loss (+12.0 vs -3.0; P=0.007) at 3 months, with an improvement in the symptoms thereafter. In both arms, pain decreased after treatment. No clear differences in the other QLQ-C30 scales were observed. The EQ-5D-5L VAS scale showed a significantly worse score in the VMP vs Rd arm at 3 months (-7.9 vs +1.7; P=0.005) and at 12 months (-0.6 vs +10.3; P=0.017). Image: Summary/Conclusion: A comparison of PROs in real-life NTE NDMM pts treated with VMP vs Rd showed a worse HRQoL in the VMP arm early in the treatment course that improved thereafter. At 3 months, after treatment start, VMP was associated with worse GHS, physical functioning, role functioning, fatigue, nausea, appetite loss, and EQ-5D-5L VAS scores. S301: GERMAN AMLCG-SURVIVORSHIP STUDY: QUALITY OF LIFE AND LIFE SATISFACTION IN AML LONG-TERM SURVIVORS E. Telzerow1,*, D. Görlich2, C. Sauerland2, A. S. Moret1, M. Rothenberg-Thurley1, F. H. A. Mumm1, S. Amler2 3, W. E. Berdel4, B. Wörmann5, U. Krug6, J. Braess7, P. Heussner8, W. Hiddemann1, K. Spiekermann1, K. H. Metzeler9 1Department of Medicine III and Comprehensive Cancer Center (CCC Munich LMU), University Hospital, LMU Munich, Munich; 2Institute of Biostatistics and Clinical Research, University of Münster, Münster; 3Current adress: Friedrich Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald; 4Department of Medicine A, Hematology and Oncology, University of Münster, Münster; 5Charité University Hospital Berlin, Berlin; 6Department of Medicine; 3, Hospital Leverkusen, Leverkusen; 7Department of Oncology and Hematology, Hospital Barmherzige Brüder, Regensburg; 8Departement of Internal Medicine, Hospital Garmisch-Partenkirchen, Garmisch-Partenkirchen; 9Department of Medicine; 1, Hematology and Cell Therapy, University Hospital Leipzig, Leipzig, Germany Background: An increasing proportion of patients with acute myeloid leukemia (AML) become long-term survivors. Somatic and psycho-social outcomes in survivors are therefore becoming increasingly important, but little is known about the long-term effects of the disease and its treatment. Aims: The primary aim of this study was to compare quality of life (QoL, measured by the FACT-G questionnaire) and general and health-related life satisfaction (gLS/hLS, measured by the FLZ-M questionnaire) of AML-LTS with normative data of German adults who were not diagnosed with AML. Methods: We designed a comprehensive analysis of AML survivorship outcomes including psycho-social well-being and somatic health status and conducted a questionnaire-based study collecting data from AML long term survivors (AML-LTS). This report focuses on overall and health-related quality of life. Somatic morbidity in AML-LTS is reported separately (Moret et al.). Results: 427 former AML patients who had been enrolled in AMLCG trials (AMLCG-1999, AMLCG-2004, AMLCG-2008) or the AMLCG patient registry, participated in this study between 5 and 18.6 years (y) after their initial AML diagnosis (median, 11.3y). Median age of AML-LTS was 61y (range 28y-93y), and 56% were female. Thirty-eight percent of participants had been treated with chemotherapy alone, while 62% received at least one allogeneic stem cell transplant (alloHSCT). A relapse occurred in 24% of the participants. Unexpectedly, age- and sex-normalized quality of life and general life satisfaction summary scores were significantly higher in AML-LTS (p<.001) compared to adults without the diagnosis of AML. Raw score points of AML-LTS on the FACT-G summary scale also were higher than in age and sex-matched normal adults by a median of 4.7 points (95% CI: 2.82 – 7.2) – a differences that likely is clinically not relevant, considering an established cutoff for clinical relevance of 7 raw score points. No difference between AML-LTS and normal adults was found for health-related life satisfaction (hLS). Using the cutoff for clinical importance (i.e., 7 points below age- and sex-matched population norm), 26.1% of participants reported relevant impairment of overall QoL. To identify factors potentially associated with poor overall QoL, we constructed a logistic regression model including pre-specified cofactors (age, sex, time since initial diagnosis, relapse and alloHSCT) and additional covariables that associated with QoL in univariate analyses (Figure 1). We found that participants with no children, lower educational level, shorter time since diagnosis and altered financial situation reported significantly lower QoL. No influence was found for disease- and treatment related factors including treatment (alloHSCT vs. no alloHSCT), previous relapse, or de novo vs secondary or therapy-related AML. Image: Summary/Conclusion: Unlike previous studies of AML survivorship, our large cohort included a diverse spectrum of patients regarding age, time since diagnosis, and treatment modalities, which allows for new insight into long-term QoL. Our study establishes that overall QoL in AML long-term survivors is comparable to the general population, with further improvement from five years post diagnosis onwards. Importantly, disease- and treatment-related factors, such as prior relapse or alloHSCT, are not associated with overall QoL. However, we were able to identify risk factors for worse QoL, delineating a subgroup of patients that may still have a need for targeted psycho-social interventions five or more years after an AML diagnosis. S302: COST-EFFECTIVENESS OF KTE-X19 IN PATIENTS WITH RELAPSED/REFRACTORY MANTLE CELL LYMPHOMA M. Marchetti1,*, C. Visco2 3 1Hematology & TMO Unit, Azienda Ospedaliera SS Antonio e Biagio e Cesare Arrigo, Alessandria; 2University of Verona; 3Section of Hematology, Department of Medicine, Azienda Ospedaliera Universitaria Integrata, Verona, Italy Background: Relapsed/refractory (R/R) mantle-cell lymphoma (MCL) post BTK-i has a poor prognosis when treated with currently available treatments with median overall survival between 6 to 15 months. In the ZUMA-2 study, KTE-X19, a chimeric antigen receptor (CAR) T-cell therapy, displayed very high response rate (93% overall objective response rate) with 83% of patients in the study alive at 12 months (Wang et al 2020). An indirect comparison of overall survival from ZUMA-2 with patients receiving standard of care (SOC) treatment reported propensity-adjusted HR 0.32-0.49 (Hess et al 2021). Based on such data, international scientific societies (NCCN, ESMO) recommend the use of CAR-T for R/R MCL patients and marketing authorization of KTE-X19 was granted by FDA and EMA. Aims: The present study aimed to estimate the long-term clinical benefits and overall healthcare costs of KTE-X19 in R/R MCL patients post BTK-i from the perspective of the Italian HealthCare System, compared to current standard therapy for these patients in Italy: rituximab-bendamustine-cytarabine (R-BAC). Methods: A partitioned-survival model was used to extrapolate overall survival, progression-free survival, quality-adjusted survival and healthcare costs of R/R MCL patients over a lifetime horizon, assuming that patients who had not relapsed within 5 years experienced long-term remission. R-BAC (6 cycles) was used as baseline SOC, however a mix of treatments was tested as sensitivity analysis. The source of safety and survival data for KTE-X19 was the ZUMA-2 trial (median potential follow up 28.8 mo), while data from retrospective real-world studies, including SCHOLAR-2, were used for SOC. The healthcare costs for patients assigned to the CAR-T strategy included bridging chemotherapy (36.8% of the patients), hospitalization (median length 21.2 days, of which 23% in intensive care), and the price of KTE-X19. The healthcare costs of patients assigned to SOC included in addition to R-BAC treatment costs, the cost for allogeneic hematopoietic stem cell transplant (€179,418) for 31% of patients (McCullogh 2020). Life years (LYs), quality-adjusted life years (QALYs) and costs were estimated by discounting future outcomes (3% per year). Uncertainty analysis included one-way and probabilistic sensitivity Results: Over a lifetime horizon, mean estimated survival was 8.85 years for KTE-X19 and 1.56 years for R-BAC, while discounted QALYs were 6.40 for KTE-X19 versus 1.20 for R-BAC. Discounted lifetime costs were €411,403 for KTE-X19 versus €74,415 for R-BAC, which corresponds to a cost of €64,798 per QALY gained and €46,264 per LY gained. Detailed results are reported in the table below. The ICERs for KTE-X19 versus SCHOLAR-2 based and meta-analysis based comparisons were €57,915, and €56,010, respectively. The most influential model parameters were patients age and quality of life values, KTE-X19 acquisition cost, and the proportion of patients receiving allo-SCT in the comparator arm, and model time horizon. Probabilistic sensitivity analysis showed that at a willingness-to-pay of €70,000 per QALY, the probability of KTE-X19 to be cost-effective was 65%. Image: Summary/Conclusion: This analysis shows that KTE-X19 may be a cost-effective alternative to R-BAC for patients with RR-MCL pre-exposed to BTK-i and chemoimmunotherapy, from an Italian third-party payer perspective S303: EFFECT OF PEGCETACOPLAN ON QUALITY OF LIFE IN COMPLEMENT-INHIBITOR NAÏVE PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA: RESULTS FROM THE PHASE 3 PRINCE STUDY D. Gomez-Almaguer1,*, R. Wong2, T. Dumagay3, M. Al-Adhami4, J. Savage4, Z. Hakimi5, A. Bogdanovic6 1Hematology Service, Hospital Universitario “Dr José Eleuterio González,” Universidad Autónoma de Nuevo León, Monterrey, Mexico; 2Sir Y.K. Pao Centre for Cancer & Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong; 3Department of Cellular Therapeutics, Makati Medical Centre, Makati, Philippines; 4Apellis Pharmaceuticals, Inc., Waltham, United States of America; 5Swedish Orphan Biovitrum AB, Stockholm, Sweden; 6Clinic of Hematology, Clinical Center of Serbia, Faculty of Medicine, University of Belgrade, Belgrade, Serbia Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening disease characterized by complement-mediated hemolysis and thrombosis. Historically, PNH was treated with C5 inhibitors (eculizumab [ECU)/ravulizumab). Despite their proven efficacy in the control of intravascular hemolysis (IVH), up to 72% of patients experience persistent hemolysis leading to suboptimal hemoglobin (Hb) levels and 36% require transfusions despite ECU treatment which results in a significant impact on the quality of life (QoL), including persistent fatigue. Pegcetacoplan (PEG) is an FDA/EMA-approved C3 complement-inhibitor that provides broad hemolysis control (including IVH and extravascular hemolysis) in patients with PNH. Aims: This analysis evaluates QoL measures in the Phase 3 PRINCE study (NCT04085601), a multicenter, randomized, open-label, controlled study evaluating the efficacy and safety of PEG compared to control treatment (CTRL; excluding complement-inhibitors) in complement-inhibitor naïve patients with PNH. Methods: PRINCE has been described previously and was superior for the coprimary endpoints of hemoglobin (Hb) stabilization (avoidance of >1 g/dL decrease in Hb from baseline), change from baseline in lactate dehydrogenase at 26 weeks and PEG-treated patients saw clinically meaningful increases in the FACIT-Fatigue scores (Wong SR, et al. Blood 2021; 138 [Supplement 1]: 606). Secondary endpoints specific to QoL measures included the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 Scale (EORTC QLQ-C30), Linear Analog Scale Assessment (LASA) and The Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue). The EORTC QLQ-C30 contains 30 questions comprising 5 functional, 9 individual symptoms, and one global health status/QoL item(s), where a ≥10-point change in score is considered clinically meaningful. The LASA consists of 3 sections asking to rate the perceived level of functioning (scale 0-100, where a higher score corresponds to a better QoL) and contains specific domains for activity level, ability to carry out daily activities, and overall QoL. The FACIT-Fatigue is a 13-item Likert scaled instrument (scale 0-51), where a ≥3-point increase is considered clinically meaningful. Results: Relevant baseline characteristics for PRINCE are reported in Table A. Mean baseline scores of the EORTC QLQ-C30 functional and symptom scales were generally similar between groups (except for role and cognitive functioning and insomnia; Table B). After 26 weeks of PEG-treatment, patients displayed clinically meaningful improvements in most of the functional scales and the global health status/QoL at Week 26 as indicated by a ≥10-point score increase from baseline (Table B). Improvements in the symptom scales, including fatigue and dyspnea scales, were also observed in the PEG group at Week 26 as indicated by a reduction in the symptom score close to the general population norms (general population norm: fatigue: 24.1, dyspnea: 10.9). Mean total LASA scores increased in the patients receiving PEG treatment for 26 weeks (Table B). FACIT-Fatigue scores increased for PEG-treated patients to near the general population norm [43.6], supporting the gains shown in the EORTC QLQ-C30-Fatigue scale. Image: Summary/Conclusion: Substantial and clinically relevant improvements in QoL were consistently observed with PEG across both EORTC QLQ-C30, total LASA and FACIT-Fatigue scores and clinically significant improvements in fatigue and dyspnea are highly relevant to the symptomology of PNH. S304: SUTIMLIMAB, A COMPLEMENT C1S INHIBITOR, PROVIDES SUSTAINED IMPROVEMENTS IN PATIENT-REPORTED OUTCOMES IN PATIENTS WITH COLD AGGLUTININ DISEASE (CAD): 2 YEAR FOLLOW-UP FROM THE CARDINAL STUDY A. Röth1,*, C. M. Broome2, W. Barcellini3, T. Henrik Anderson Tvedt4, Y. Miyakawa5, S. D’Sa6, D. Cella7, F. Joly8, J. Wang9, T. Sourdille9, F. Shafer10, M. Wardęcki11, I. C. Weitz12 1Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2Division of Hematology, MedStar Georgetown University Hospital, Washington, DC, United States of America; 3Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 4Section for Hematology, Department of Medicine, Haukeland University Hospital, Bergen, Norway; 5Thrombosis and Hemostasis Center, Saitama Medical University Hospital, Saitama, Japan; 6UCLH Centre for Waldenström’s Macroglobulinemia and Related Conditions, University College London Hospitals NHS Foundation Trust, London, United Kingdom; 7Department of Medical Social Sciences, Center for Patient-Centered Outcomes, Institute for Public Health and Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States of America; 8Sanofi, Chilly-Mazarin, France; 9Sanofi, Cambridge, MA; 10Sanofi, Bridgewater, NJ, United States of America; 11Sanofi, Warsaw, Poland; 12Keck School of Medicine of USC, Los Angeles, CA, United States of America Background: CAD is a rare chronic autoimmune hemolytic anemia characterized by classical complement pathway (CP)-mediated hemolysis, anemia, fatigue, and poor quality of life (QOL). Sutimlimab is a first-in-class humanized monoclonal antibody that selectively inhibits C1s of the C1 complex, preventing CP activation, while leaving the alternative and lectin pathways intact. One-year interim follow-up from the CARDINAL study (NCT03347396) have previously demonstrated continuous classical CP inhibition with sutimlimab resulted in rapid, sustained improvements in all patient-reported outcomes (PROs) measures evaluated. Aims: To report sutimlimab effect on PROs at 2 years, from the CARDINAL Part B extension. Methods: CARDINAL was a Phase 3, open-label, single-arm study with a 26-week treatment period (Part A) and a 2-year extension (Part B) after the last patient finishes Part A. Sutimlimab was administered through intravenous infusions on Days 0 and 7, followed by biweekly dosing. PRO data through Week 135, the last data recording within the 2-year Part B time period, are reported here. Efficacy endpoints included hemolytic markers and the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) as a measure of QOL. Exploratory QOL endpoints included mean change from baseline in EuroQol 5-dimension 5-level (EQ-5D-5L) scores, 12-Item Short Form Health Survey (SF-12), Patient Global Impression of Change (PGIC) and Patient Global Impression of Fatigue Severity (PGIS). PRO measures were evaluated every 3 months and conducted in the following order: FACIT-Fatigue, PGIS, PGIC, SF-12 and EQ-5D-5L. Descriptive statistics, frequency, or percentage were used to analyze outcomes. Results: Overall, 24 patients enrolled and 22 finished Part A and entered Part B, with 19 (86.4%) patients completing Part B. The mean (SD) FACIT-Fatigue score at baseline was 32 (11) and improved by 7 (8) within 1 week of sutimlimab treatment; the mean change score from baseline remained ≥5 from Week 1 to Week 135 (minimum score of 38 (9) at Week 123, maximum score of 44 (5) at Week 7), consistent with a clinically meaningful improvement. Efficacy for the EQ-5D-5L visual analogue scale (VAS) was sustained over 2 years; the mean (SD) change from baseline in EQ-5D-5L visual analogue scale (VAS) score (n=15/22) at Week 135 was 17.1 (21.6) (Figure A). The majority of evaluable patients (n=13/15) indicated an improved disease state compared to baseline on the PGIC at Week 135. No or mild fatigue was reported in patients (80%, n=12/15) on completing PGIS at Week 135, compared with one-third at baseline (n=2/6). The mean increase in SF-12 physical and mental component scores (n=6/22) from baseline to Week 123 were 4.7 (6.9) and 3.8 (14.1) (Figure B), consistent with the clinically important changes of 3.9 and 2.8 respectively. Image: Summary/Conclusion: From this CARDINAL follow-up extension study, sutimlimab has shown to produce rapid and sustained improvements in FACIT-Fatigue and other PRO measures evaluated up to 2 years, demonstrating the continued meaningful impact of sutimlimab on patient QOL. Posters P305: COMPREHENSIVE TRANSCRIPTIONAL AND CYTOGENETIC PROFILING IMPROVES CLASSIFICATION AND DETECTION OF RISK-STRATIFYING MARKERS IN THE B-OTHER PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA Ž. Antić1,*, P. Chouvarine1, J. Lentes1, C. Schröder1, J. Alten2, A. Möricke2, M. Brüggemann3, E. Carrillo-de Santa Pau4, T. Illig1, T. Laguna4, D. M. Schewe2,5, M. Stanulla6, M. Tang1, M. Zimmermann6, M. Schrappe2, B. Schlegelberger1, G. Cario2, A. K. Bergmann1 1Institute of Human Genetics, Hannover Medical School, Hannover; 2Berlin-Frankfurt-Münster ALL Study Group Germany (BFM-G), Department of Pediatrics; 3Department of Hematology, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Germany; 4Computational Biology Group, Precision Nutrition and Cancer Research Program, IMDEA Food Institute, Madrid, Spain; 5University Children’s Hospital, Medical Faculty, Otto-von-Guericke-University-Magdeburg, Magdeburg; 6Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy. Improvements in the genetic-based risk stratification and treatment adaptations have resulted in an increased overall survival, reaching 90% in the contemporary treatment protocols. However, in 25-30% of patients, known as B-other, no recurrent genetic aberrations relevant for the risk stratification and treatment personalization can be detected using conventional cytogenetic methods. Therefore, identification and implementation of new risk-stratifying markers in the current treatment protocols may aid further improvement in the management of children with BCP-ALL. Aims: Our aim was to investigate the applicability of the integrated use of the whole transcriptome RNA sequencing and conventional cytogenetic methods in the routine diagnostic of the B-other BCP-ALL cases. Methods: We performed RNA sequencing and analyzed gene fusions, expression profiles, and mutations in diagnostic samples of 174 children with B-other ALL. In order to further refine genetic classification and validate findings obtained with RNA sequencing, we integrated our results with the findings obtained using immunophenotyping, FISH, karyotyping, arrayCGH and Sanger sequencing. Results: Our analysis unraveled the presence of risk-stratifying fusion transcripts, in the cases in which these alterations were not detectable using conventional cytogenetic methods. These included six cases with cryptic KMT2A rearrangements and one of each with TCF3-PBX1 and TCF3-HLF fusion genes. In addition, we were able to detect 10 cases with recently described fusions involving ZNF384 gene (ZNF384r), four cases with NUTM1 rearrangements and three cases with fusions involving MEF2D gene. Gene expression-based clustering unraveled a subset of B-other cases which cluster together with the known subtypes, indicating the presence of previously described ZNF384r-like, ETV6-RUNX1-like and BCR-ABL1-like subtypes. Furthermore, we identified 27 previously unassigned B-other cases co-clustering together with 13 DUX4-positive ALL cases. This finding suggests that gene expression-based clustering can identify the cases with fusions involving DUX4 gene, known to be cryptic to most of cytogenetic and NGS approaches. We further assessed the ability of the analysis pipeline to detect fusion transcripts in the samples with <50% of tumor blasts. In five tested cases, with BCR-ABL1 or ETV6-RUNX1 fusions, our analysis pipeline was able to detect the presence of fusion transcripts, with high reliability, even in the samples with down to 7% of tumor blasts. Finally, we assessed the applicability of using commonly available EDTA tubes and RNA stabilizing PAXgene tubes for bone marrow sampling, RNA isolation and whole transcriptome sequencing. We assessed the quality of isolated RNA, library complexity and ability of our analysis pipeline to detect relevant fusion transcripts. PCA analysis of matched samples stored in either EDTA or PAXgene tubes showed high similarity in gene expression, while the correlation between TPM expression values and decay constants indicates that genes over-abundant in EDTA samples are not dominated by slow-degrading transcripts. These findings suggest that for short-term storage EDTA tubes are a viable alternative to the RNA stabilizing PAXgene tubes. Summary/Conclusion: Taken together, our findings demonstrate the applicability of whole transcriptome sequencing for personalized diagnostics in pediatric ALL, including the tentative classification of the B-other cases that are difficult to diagnose using conventional methods. P306: PAX5 DEFICIENCY AND GERMLINE SUSCEPTIBILITY TO PEDIATRIC LEUKEMIA F. Auer1,*, A. Escudero2, M. Sipola3, A. Viitasalo3, M. Morcos1, U. A. Friedrich4, A. Pandyra5, A. Borkhardt5, M. Takagi6, J. Hauer1 1Department of Pediatrics, Technical University Munich, Munich, Germany; 2Institute of Medical and Molecular Genetics (INGEMM) La Paz University Hospital, Madrid, Spain; 3Institute of Biomedicine, School of Medicine, University of Eastern Finland, Kuopio, Finland; 4Pediatric Hematology and Oncology, Department of Pediatrics, University Hospital “Carl Gustav Carus”, TU Dresden, Dresden; 5Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-Heine University Duesseldorf, Medical Faculty, Duesseldorf, Germany; 6Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Background: Somatic mutations affecting the PAX5 gene are common in pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, germline PAX5 mutations are extremely rare. While it is known that PAX5 acts as an indispensable master regulator of proliferation and differentiation in B-cells, the pre-leukemic setting mediated by reduced PAX5 levels is still poorly understood. Aims: To investigate the genetic and immunological effect of inherited PAX5 germline mutations in humans and to characterize the effect of Pax5 heterozygosity on B-cell development utilizing single-cell RNA Sequencing in mice. Methods: Aiming to understand the etiology of childhood BCP-ALL in the setting of reduced Pax5 levels, we used 9-color FACS staining and scRNA/bulk RNA-Sequencing, as well as whole exome Sequencing technology. Results: Here, we describe two new families with inherited PAX5 germline variants affecting amino acid 183 and B-ALL development, including one family with a novel amino acid substitution p.Gly183Arg. Exome analysis of both families together with one family harboring PAX5 p.Gly183Ser previously reported by Auer et al., (Leukemia, 2014) yielded no additional common germline variants that could explain the incomplete penetrance in the observed leukemia development. A study of the respective B-cell differentiation comparing PBMCs from unrelated non-carriers, healthy carriers, and patients revealed a significant decrease of intermittent B-cells and memory B-cells in carriers and patients compared to non-carriers (Student’s T-test; p<0.05 and p<0.01, respectively). Finally, immunological studies performed using bone marrow samples from a patient and an unrelated control showed a reduction in the number of mature B cells, suggesting a block of the B-cell differentiation at the pre-B stage in the patient. In line, a comprehensive flow cytometry analysis of the B-cell development in the bone marrow of healthy Pax5 heterozygous (Pax5 +/-) mice, showed a robust and age independent enrichment of the pre-BII population (B220+CD19+IgM-CD25+) compared to their Wildtype (WT) littermates (Student’s t-test; p=0.003). Bulk RNA-Sequencing of sorted preB-II cells (3 WT vs. 3 Pax5 +/- mice) revealed deregulations in major B-cell receptor (BCR) signaling components including downregulation of Cd79a/b, Lyn, MTor and various ITIMs. Subsequent scRNA-Sequencing (4 WT vs. 4 Pax5 +/- mice, pool of 20,000 cells hashtag labeled, with 50,000 reads/cell and VDJ-rearrangement analysis) confirmed a delayed BCR assembly and showed a skewing of light chain rearrangements towards the lambda isotype in Pax5 +/- mice. Summary/Conclusion: Overall, our data provide evidence for B-cell dysregulations mediated by reduced Pax5 levels in the germline of mice and men. Ultimately these characterizations will help to advance the understanding of molecular mechanisms and susceptible populations associated with B-ALL. In turn, these findings are important to address the far more frequent somatic PAX5 mutations with the goal to prevent or treat a significant proportion of childhood leukemias. P307: DEL(17)(Q11) IS TYPICAL MARKER OF IMMATURE T-ALL OF ADULTS, WITH NF1, UTP6, AND SUZ12 HAPLOINSUFFICIENCY, GENOME INSTABILITY, AND GENE DOWNREGULATION V. Bardelli1,*, V. Pierini1, S. Arniani1, E. Mvridou1, C. Matteucci1, A. G. Lema Fernandez1, M. Moretti1, L. Elia2, F. Giglio3, F. Forghieri4, M. Cerrano5, N. Fracchiolla6, M. Delia7, S. Sica8, C. Mecucci1, R. La Starza1 1Hematology and Bone Marrow Transplantation Unit, Department of Medicine and Surgery, University of Perugia, Perugia; 2Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome; 3Hematology and Bone Marrow Transplantation Unit, IRCCS Ospedale San Raffaele, Milan; 4Section of Hematology, Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, Modena; 5Department of Molecular Biotechnology and Health Sciences, Division of Hematology, University of Turin, Turin; 6Hematology, Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan; 7Hematology and Stem Cell Transplantation Unit, AOUC Policlinico of Bari, Bari; 8Section of Hematology, Department of Radiological and Hematological Sciences, Catholic University of the Sacred Heart, Rome, Italy Background: T-ALLs derive from the accumulation of multiple genetic and epigenetic events. Some deregulate T-cell transcription factors and define specific genetic subtypes, while the large majority are involved in pivotal cellular processes, such as signaling, cell cycle, apoptosis, proliferation, ribosome biogenesis, and epigenetic modulation. Genomic imbalances, and in particular deletions, are the most frequent cytogenetic rearrangements in T-ALL. Whether cryptic or large, they often share loss regions - the common deletion region (CDR) - where they map putative suppressor genes that undergo haploinsufficiency or inactivation, thus driving the leukemogenic process. Aims: Our study focused on T-ALL cases harboring interstitial del (17q) with the aim of assessing the incidence and distribution of this cytogenetic marker, and to detect associated clinical and molecular features. Methods: The study was carried out on 378 adult (=223) and pediatric (=155) T-ALL, including 279 males and 99 females. The genetic background was investigated by integrated molecular-cytogenetics (FISH and SNPa),1 RNA microarrays (Affymetrix), targeted sequencing (custom card by Sophia Genetics, Arrow Diagnostics), and RTq-PCR. Data analysis was carried out referring to available databases (UCSC, NCBI, Data of Genomic Variants, Biocarta, KEGG, Reactome). Results: Cases were classified according to genetic rearrangements as HOXA (=104), TAL/LMO (=74), TLX1/NKX2.1 (=50), TLX3 (=42), BCL11B-a (=8), SPI.1 (=2); 98 were undetermined. Interstitial monoallelic del(17q) was detected 31/378 cases and was mainly found in cases with HOXA related abnormalities (8/104) or cases undetermined (17/98). There were 27 adults and 4 children (p<0.0001). In 19/24 with available flow cytometry the diagnosis was consistent with ETP/near ETP ALL (p<0.001). All cases shared a common deleted region (CDR) of about 650kb, that invariably involved SUZ12, UTP6, and NF1 which were expressed at a significantly lower level than in T-ALL without del(17q). RNA microarray showed that 148 differentially expressed genes (DEG), 67 up- and 81 down- regulated, distinguished del(17q) T-ALL cases from T-ALL without. Among up-regulated there were MEF2C, MN1, and IGFBP7, which have been all associated with an immature phenotype, and MAF and RUNX2 transcription factors. Instead, among down-regulated, there were putative suppressors involved in genome stability, i.e. BRCA1, FANCI, BRIP1, XRCC2, and CHEK1. Accordingly, cell cycle, homologous recombination, DNA replication, and Fanconi anemia pathways, were significantly deregulated in T-ALL with del(17q), and a significant association with deletions of RB1 (37%)(p<0.001) and TP53 (20%)(p=0.017) emerged. In addition, del(17q) cases had a higher number (≥5) of copy number abnormalities (p<0.001). Other recurrent alterations affected JAK/STAT members and/or modulators (56% of cases). Summary/Conclusion: Del(17q) was a recurrent marker in immature T-ALL of adults and characterized 19% of cases undetermined at molecular-cytogenetic level. It caused the monoallelic loss/haploinsufficiency of three genes, i.e. NF1, UTP6, and SUZ12, that restricted the CDR. Our study provided evidence that del(17q) identified a major leukemogenic mechanism, through the cooperation of: 17q genes loss of function, JAK/STAT constitutive activation, and altered cell cycle. Interestingly, RUNX2 and IGFBP7, high-risk markers in T-ALL,2,3 were aberrantly expressed in this subset of T-ALL. 1La Starza R, et al. J Mol Diagn 2020 2Matthijssens F, et al. JCI 2021 3Bartram I, et. al., BMC Cancer 2015 P308: NOVEL COPY NUMBER ABERRATION-BASED CLASSIFICATION METHODS REFINE RISK ASSESSMENT IN PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA G. Bedics1,*, B. Egyed2, L. Kotmayer1, A. Benard-Slagter3, K. de Groot3, A. Bekő1, L. L. Hegyi1, S. Krizsán1, G. Kriván4, D. J. Erdélyi2, G. Kovács2, B. Kajtár5, L. Pajor5, Á. Vojcek6, G. Ottóffy6, A. Ujfalusi7, C. Kiss8, I. Szegedi8, K. Bartyik9, G. Péter10, E. Sebestyén1, Z. Jakab11, A. Matolcsy1, S. Savola3, C. Bödör1, D. Alpár1 1HCEMM-SU Molecular Oncohematology Research Group, Department of Pathology and Experimental Cancer Research; 22nd Department of Pediatrics, Semmelweis University, Budapest, Hungary; 3Department of Oncogenetics, MRC Holland, Amsterdam, Netherlands; 4Central Hospital of Southern Pest - National Institute of Hematology and Infectious Diseases, Budapest; 5Department of Pathology; 6Department of Pediatrics, University of Pécs Clinical Centre, Pécs; 7Department of Laboratory Medicine; 8Department of Pediatric Hematology-Oncology, Institute of Pediatrics, Faculty of Medicine, University of Debrecen, Debrecen; 9Department of Paediatrics and Paediatric Health Care Center, Faculty of Medicine, University of Szeged, Szeged; 10Hemato-Oncology Unit, Heim Pál Children’s Hospital; 11Hungarian Childhood Cancer Registry, Hungarian Pediatric Oncology Network, Budapest, Hungary Background: The genomic landscape of pediatric acute lymphoblastic leukemia (ALL) is heterogeneous with distinct copy number aberrations (CNAs) being detectable in vast majority of the patients. A subset of these alterations provides prognostic and/or predictive information; therefore, various CNA-based patient classifiers were introduced in the past. Aims: We applied a next-generation sequencing (NGS) based method to comprehensively screen for recurrent, disease-relevant CNAs in a cohort of Hungarian patients, allowing us to establish novel patient risk stratification approaches. Methods: Diagnostic bone marrow samples from 261 children with B-ALL and 22 matching samples drawn from 21 patients at first or second relapse were investigated by digital multiplex ligation-dependent probe amplification (digitalMLPA) using the ALL-specific D007 probemix. DigitalMLPA libraries were sequenced on an NGS platform. Whole chromosome gains and losses, as well as subchromosomal CNAs were simultaneously profiled. Survival rates were estimated using the Kaplan-Meier method and compared by log-rank tests in R version 4.1.2. Results: In total, 1,400 CNAs including numerical chromosomal aberrations and subchromosomal CNAs were detected in 93.5% of the diagnostic samples. On average, 5.36 CNAs were observed per patient with a mean of 2.45 subchromosomal alterations. Subtype-defining aberrations were identified in 36.0% of the patients with hyperdiploidy and iAMP21 detected in 32.2% and 3.8% of the cases, respectively. Numerous CNAs in disease-relevant genes responsible for cell cycle control, lymphoid development, signaling, or tumor suppression were identified. Considering all affected exons and an upstream region, 10 distinct patterns of IKZF1 deletion were observed. Unbiased co-segregation analysis revealed 15 positive and 3 negative correlations between the various CNAs, e.g. common co-occurrence of iAMP21 and CDKN2A/B deletion, and negative correlation between ETV6 and BTG1 deletions. Comparative analysis of diagnostic and matching relapse samples revealed characteristic temporal changes of copy number profiles with additional aberrations (65%), as well as both emerging and disappearing CNAs (30%) detected at relapse as compared with diagnosis. One patient did not show CNAs in the analyzed samples (5%). Prognostic subgroups determined based on cytogenetic findings were combined (i) with IKZF1del/IKZF1plus status and (ii) with CNA subgroups defined by a comprehensive digitalMLPA profiling. The combined genetic classification methods identified 3 and 4 patient subgroups, respectively, with significantly different 5-year progression-free survival rates. Summary/Conclusion: Comprehensive and highly optimized CNA profiling with digitalMLPA revealed subtype-defining gross-chromosomal changes and additional disease-relevant subchromosomal CNAs in a large cohort of Hungarian patients treated with ALL IC BFM protocols. Comparative scrutiny of matching diagnostic and relapse samples from 21 patients unveiled two different patterns of temporal evolution. Two novel risk stratification approaches have been established by combining cytogenetic data with digitalMLPA-based copy number profiling, with one of those laying more emphasis on CNA data than seen in previously introduced classifications, and the other one combining cytogenetic data with IKAROS status for the first time. DigitalMLPA offers a fast, reliable and standardizable DNA copy number analysis which is easily implementable in the diagnostic workflow of pediatric ALL. P309: IMAGE-BASED HIGH-CONTENT DRUG SENSITIVITY SCREENING IDENTIFIES CONVENTIONAL, EMERGING, AND POTENTIAL NOVEL THERAPEUTIC TARGETS IN HIGH-RISK ADULT ACUTE LYMPHOBLASTIC LEUKEMIA H. Bell1,*, M. Singh1, H. Blair1, A. Poll1, E. Law1, O. Heidenreich2, F. van Delft1, A. Moorman1, J. Lunec3, J. Irving1 1Wolfson Childhood Cancer Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom; 2Prinses Maxima Centrum, Utrecht, Netherlands; 3Paul O’Gorman Building, Newcastle University, Newcastle upon Tyne, United Kingdom Background: Despite concerted efforts to optimize and intensify treatment approaches in adult acute lymphoblastic leukemia (ALL) more than half of patients still succumb to their disease within 5 years of diagnosis, emphasizing the limits of current therapy and the critical requirement for more effective and safer therapeutics. Patient-derived xenograft (PDX) ALL models represent reliably predictive preclinical models and allow in vivo amplification of patient material in quantities sufficient for high-throughput screening applications. Aims: To identify novel and effective therapies for high-risk ALL using an in vitro co-culture model of PDX ALL blasts and mesenchymal stem cells (MSCs) by high-content screening of >1200 pharmacologically-active small-molecule drug compounds in various stages of clinical development. Methods: PDX leukemic cells derived from seven patients with high-risk ALL, including relapsed Philadelphia-positive B-ALL, low hypodiploid B-ALL, and ABL-class T-ALL, were used for the drug screen. ALL cells were co-cultured with hTERT-immortalized MSCs to support survival of the ALL blasts during a 96 hour short-term culture with the drug library compounds (final concentration, 1mM) in 384-well format. Drug sensitivity was assessed using an automated, high-throughput, fluorescence image-based microscopy pipeline which identifies and discriminates live ALL cells and MSCs using random forest machine learning algorithms based on cellular nuclear staining. Results: Drug sensitivity profiles revealed a wide diversity of responses to the library compounds reflected by different “hit” rates (4.5-7.4%), where “hit” is defined as compounds reducing cell survival by more than 50%. Despite this heterogeneity in response, 59 unique compounds were identified with broad anti-leukemic activity; that is, “hits” in 4/7 PDX samples. Concurrent image analysis of MSCs in the co-culture system revealed most hit compounds did not affect MSC numbers relative to vehicle-treated controls (n=50/59), indicating a possible therapeutic window for targeting leukemic cells specifically. Several hit compounds (n=14) were drugs or from drug classes which form part of conventional ALL treatment regimens — including anthracyclines, glucocorticoids, and nucleoside analogues — validating the screening approach for successful identification of anti-leukemic compounds. Drug classes with recently recognized and emerging preclinical anti-leukemic activity were also identified including cyclin-dependent kinase (CDK) inhibitors (n=5), cell cycle-related kinase inhibitors (n=6), bromodomain and extra-terminal domain (BET) inhibitors (n=4), histone deacetylase family inhibitors (n=3), and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (n=3). Nine hit compounds elicited significant reductions in cell survival consistently across the panel of PDX samples (7/7), providing some evidence towards anti-leukemic activity independent of cytogenetic background. These included pan-CDK family inhibitors alvocidib, CGP60474, and roscovitine-derived CR8. Additionally, the selective CDK (-1, -2, -9) inhibitor AZD 5438 exhibited anti-leukemic activity in all samples (n=6/7), bar the Philadelphia-positive sample. Summary/Conclusion: We provide evidence that high-content drug screening in a co-culture system is an effective strategy to identify clinically-relevant novel and emerging therapeutic targets in high-risk ALL. Our analyses promote a strong rationale for exploring the targeting of CDKs as a potential pan-effective approach in high-risk adult ALL. P310: BH3 PROFILING IDENTIFIES SELECTIVE BCL-2 DEPENDENCE OF ADULT EARLY T-CCELL PROGENITOR AND TYPICAL ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS K. Bhatia1, A. Mahesh1, E. Olesinski2, J. Garcia2, N. Jain3, W. Y. Jen4, M. Ooi Gaik Ming5, A. Letai2, M. Konopleva3, S. Bhatt1,* 1Department of Pharmacy, National University of Singapore, Singapore, Singapore; 2Department of Medical Oncology, Dana-Farber Cancer Institute, MA; 3Department of Leukemia, University of Texas M.D. Anderson Cancer Center, Houston, Texas, United States of America; 4Department of Haematology-Oncology, National University of Singapore; 5Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore Background: T-cell acute lymphoblastic leukemia (ALL) results from malignant transformation of immature T-cells, accounting for 10-15% of childhood and 20-25% of adult ALL cases. Although long-term survival rates for standard-risk childhood T-cell ALL (T-ALL) have shown striking improvements to 90%, response outcomes in adults remain much lower. Among T-ALL, a high-risk subtype originating from clonal expansion of recently immigrated thymocytes, defined as early T-cell precursor (ETP) leukemias, has a significantly worse outcome in adults. Aims: BH3 mimetics, small molecule antagonists of anti-apoptotic BCL-2 family proteins, have shown remarkable clinical success in the treatment of hematological malignancies. We previously showed that pediatric ETP-ALL, is dependent on BCL-2, while typical T-ALL is dependent on BCL-XL. These findings importantly led to the clinical trial of venetoclax in combination with hyperCVAD in relapsed/refractory (R/R) T-ALL patients. However, whether adult patients with T-ALL also show the selective pattern of anti-apoptotic dependence related to the differentiation stage of T-cell has not been studied. Methods: BH3 profiling, and cell cytotoxicity assay. Results: We investigated the survival dependencies of adult ETP-ALL and T-ALL on BCL-2 family proteins using 42 primary samples collected from 3 centers (Dana-Farber, MD Anderson, and National Cancer Institute Singapore). All 15 ETP-ALL samples showed robust mitochondrial depolarization response to the BAD peptide as compared to the HRK peptide and DMSO control using BH3 profiling assay, suggesting primary dependence on pro-survival BCL-2 protein. We next performed cell death assays by exposing ETP-ALL primary tumors to short-term treatment with venetoclax and navitoclax and found that consistent with BCL-2 dependence, adult ETP-ALL can be efficiently targeted for apoptosis via venetoclax. We then asked if there is a difference in dependencies between adult ETP-ALL and T-ALL. In ETP-ALL, cytochrome c release was caused by the BAD peptide alone, while in T-ALL, both BAD and HRK peptides induced cytochrome c release. This suggests that ETP-ALL primary tumors are primarily BCL-2 dependent, while T-ALL primary tumors are co-dependent on BCL-2 and BCL-XL. Further, this dependence endorses mechanistic efficacy of venetoclax and navitoclax, where venetoclax serves to antagonize BCL-2, and navitoclax serves to antagonize BCL-XL and to a lesser extent BCL-2, through BIM displacement. Image: Summary/Conclusion: We hereby validated that BH3 profiling predicted remarkable on-target cytotoxicity of venetoclax in adult ETP-ALL and T-ALL. In addition, some T-ALL with enriched HRK signaling and BCL-XL dependence revealed that these patients can benefit from navitoclax. These findings provide pre-clinical evidence for venetoclax and navitoclax as a potentially efficacious combination therapy of BH3 mimetics for adults with T-ALL. P311: IMBALANCES IN HOMOCYSTEINE METABOLISM AND LEUKEMIA N. Bouayed Abdelmoula1,* 1Genomics of Signalopathies at the service of Medicine, Medical University of Sfax, Sfax, Tunisia Background: The correlation between imbalances in homocysteine metabolism and leukaemia is a matter of debate. While the effects of polymorphisms of key homocysteine metabolism enzymes on homocysteinemia are confirmed, their direct causality in the genesis of leukaemias or their role as disease markers (diagnostic or prognostic) remains controversial. It has been reported in several studies that the C677T and A1298C polymorphisms of the MTHFR may play a role in the pathophysiology of acute leukaemias and various other types of signalopathies such as cancers. Homocysteine could indeed modulate the expression of certain tumour suppressor genes and induce epigenetic alterations. Aims: In this work, the aim was to identify this correlation using a molecular study of MTHFR polymorphisms in a Tunisian cohort with acute lymphoblastic leukemia. Methods: The molecular study involved a cohort of 41 Tunisian children with acute lymphoblastic leukemia. Genomic DNA was extracted from blood samples. Amplification of the regions of interest of the MTHFR gene at exons 4 and 7 was performed by PCR using primers designed in silico. Genotyping of the 677C>T and 1298 A>C polymorphisms was performed by PCR-RFLP of the amplicons using two restriction enzymes HinfI and MboII. Results: MTHFR polymorphisms were detected in 39% of the children. The frequency of polymorphic alleles corresponded to 15.85% for the A1298C polymorphism and 4.87% for the C677T polymorphism. In fact, The C677T and A1298C MTHFR polymorphisms were detected in respectively 4 and 11 patients as heterozygous. Only one patient harbored homozygous A1298C MTHFR polymorphism. Only two cases were heterozygotes for both polymorphisms. Summary/Conclusion: Imbalances in MTHFR homocysteine metabolism associated with the polymorphisms do not appear to be a risk factor for ALL in the study cohort. P312: EMPLOYING WHOLE GENOME OPTICAL MAPPING TO CHARACTERIZE THE LANDSCAPE OF STRUCTURAL VARIANTS IN B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA D. Brandes1,2,*, T. Brozou1, S. Soura1, A. Bergmann3, U. Fischer1,4, A. Borkhardt1,4, R. Wagener1,4 1Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Germany and University Hospital Duesseldorf; 2Duesseldorf School of Oncology (DSO), Duesseldorf; 3Institute of Human Genetics, Hannover Medical School (MHH), Hannover; 4German Cancer Consortium (DKTK), partner site Essen/Duesseldorf, Duesseldorf, Germany Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent pediatric cancer. The most common subtypes are ETV6::RUNX1+ BCP-ALL, which harbor a reciprocal translocation between chromosome 12 and 21 leading to a juxtaposition of ETV6 to RUNX1, and high hyperdiploid (HHD) BCP-ALL harboring 51-67 chromosomes. In large-scale genomic studies, the mutational landscape of somatic single nucleotide variants and indels as well as copy number gains and losses based on array CGH have been well described in both subtypes. However, a comprehensive analysis of somatic structural variants (SVs) is still lacking which may reveal significant new insights on tumor development, progression, and treatment options in BCP-ALL. Aims: We aimed to primarily employ the novel technology whole genome optical mapping (WGOM) to unmask the landscape of structural variants in BCP-ALL. Firstly, we determined the concordance of chromosomal abnormalities identified by WGOM in comparison to standard techniques. Superiorly, we explored the landscape of somatic aberrations in ETV6::RUNX1+ and HHD tumors to identify recurrent alterations and novel SVs affecting genes not yet related to BCP-ALL pathogenesis. Methods: We extracted ultra-high molecular weight (UHMW) DNA of 13 ETV6::RUNX1+ and 18 HHD BCP-ALL with a tumor cell content of 25-96% at diagnosis. Additionally, we isolated DNA of fibroblasts or remission material of each pediatric patient as matching non-tumor control. Informed consent was obtained from all participants. DNA was fluorescently labeled by a sequence specific enzyme and subsequently linearized and imaged using a Saphyr instrument (Bionano Genomics). Using this approach, we generated comprehensive datasets of 31 matched tumor/non-tumor pairs, allowing the identification of somatically acquired SVs >500bp. In addition, corresponding karyotype, fluorescence in situ hybridization and SNP-array data of the leukemia samples were integrated and compared to WGOM data. Results: We validated 95% of 233 events observed by standard techniques with WGOM. All hallmark alterations including ETV6::RUNX1 fusion and high hyperdiploidy were identified by WGOM. In addition, we detected a median of 11 (1-36) somatically acquired events in leukemic cells with ETV6::RUNX1 fusion, compared to 3 (1-10) events in HHD cases. WGOM identified recurrent somatic structural aberrations occurring in at least three BCP-ALL cases, including focal deletions affecting ETV6 (9/31), PAX5 (5/31), ARPP21 (3/31), CD200/BTLA (3/31) and RAG2 (3/31). We detected subtype specific recurrent focal deletions disrupting ATF7IP (7/13) and BTG1 (5/13) in ETV6::RUNX1+ subclones whereas deletion of the CDKN2A/B locus (3/18) was only observed in HHD BCP-ALL. Interestingly, we discovered novel recurrently deleted regions on 12q24.11 (GPN3, 4/31) and Xq25 (STAG2, 3/31) in all BCP-ALL cases as well as focal loss of 19q13.11 (UBA2, 3/13) specific for ETV6::RUNX1+ subclones using WGOM. Moreover, complex three-way translocations in ETV6::RUNX1+ tumors were discovered by WGOM, including t(6;8;12)(p12.3;q24.23;q24.21) that leads to the disruption of MED13L and HMGCLL1. Summary/Conclusion: Our study showed that WGOM detects SVs as translocations and aneuploidies in BCP-ALL as good as conventional methods with a 95% concordance. However, WGOM allows a higher resolution and more precise localization of chromosomal breakpoints compared to standard techniques. Hence, we identified novel recurrent somatically acquired SVs including a deletion on Xq25 affecting STAG2, which might play an important role in pediatric BCP-ALL leukemogenesis. P313: RECURRENT PATHOGENIC GERMLINE CHEK2 VARIANTS IN PEDIATRIC PATIENTS WITH HEMATOLOGICAL MALIGNANCIES T. Brozou1,*, R. Wagener1, U. Fischer1, A. Borkhardt1 1Department of Pediatric Hematology, Oncology and Clinical Immunology, Uniklinik Dusseldorf, DUSSELDORF, Germany Background: CHEK2 is a tumor-suppressor gene encoding CHEK2 kinase that plays a crucial role in DNA repair and cell cycle arrest as part of the ATM-CHEK2-p53 pathway. Predisposing germline variants in CHEK2 are associated with breast cancer and other adult-type malignancies such as prostate, gastric and thyroid cancer. Only few studies have examined the occurrence of germline CHEK2 variants in adults with leukemia, but the association between inherited CHEK2 variants and hematological malignancies in childhood has not been studied. Aims: Our aim is to understand the frequency and the impact of germline variants in the cancer predisposing CHEK2 gene in pediatric hematological malignancies. Methods: We analyzed parent-child WES data of 150 children (≤18yrs) with hematological malignancies (n=107 leukemia, n=43 lymphoma) and mined the data for CHEK2 variants. We performed functional analysis of the identified pathogenic variants to analyze the effect of the alteration on protein function. Results: We detected in 15/150 children with hematological malignancies a CHEK2 germline variant (Figure 1A). The majority of those patients had a leukemia (n=11 BCP-ALL, n=1 T-ALL, n=1 CML, n=2 Hodgkin Lymphoma). Overall, eight different CHEK2 variants including three truncating variants were detected. Three variants were recurrent (p.Ile157Thr n=5, p.Ile160Met n=3, p.Thr367Metfs*15 n=2). According to the classification of the American College of Medical Genetics and Genomics 7/8 variants were predicted to be pathogenic or likely pathogenic. For two of these variants an impaired function had been already described in the literature. To test the functional consequences of the five remaining variants, we overexpressed these, in comparison to two control CHEK2 variants, in U2OS cells. Four out of seven variants (n=1 truncating, n=1 in-frame deletion, n=2 missense) led to loss of protein expression (Figure 1B) and loss of CHEK2-Thr68 phosphorylation upon irradiation induced DNA damage. Five out of fifteen patients carried a CHEK2 variant impairing its function. Interestingly, 4/5 patients with a pathogenic variant had a BCP-ALL and 1/5 had a CML that rapidly progressed to CD10+, CD19+ lymphoid blast crisis while under imatinib therapy (400 mg/m2). Figure 1: (A) Schematic representation of the CHEK2 protein indicating the localization of the identified variants. The length of the bars correlates to the number of patients with the respective CHEK2 variant and the color of the bars to the variant type (green: missense, red: frameshift indels, orange: splicing). (B) Western blot analysis of HA-tagged CHEK2 protein variants ectopically expressed in U2OS cells. GAPDH was used as a loading control. CHEK2 variants Arg145Trp and Glu161 del were included as controls in our experiments. (C) Bar blot depicting the quantification of the CHEK2 variant protein expression analyzed by Western blotting of three independent experiments. Each experiment is represent by a dot, the bar indicates the mean of all three experiments. Image: Summary/Conclusion: We identified heterozygous CHEK2 variants in the germline of 10% of children with hematological malignancies by WES-based screening of 150 cases. Expression studies revealed that two of the variants identified in our cohort were associated with reduction/loss of CHEK2 expression. We conclude that reduced CHEK2 expression/phosphorylation upon DNA damage may be an important germline-encoded host factor predisposing to the development of BCP-ALL in children. P314: THERAPEUTICALLY TARGETING THE UNIQUE BARCODE OF MLL/AF4 R. Cameron1,*, L. Swart1, M. Rasouli1, O. Heidenreich1 1Prinses Maxima Centrum, Utrecht, Netherlands Background: Patients harbouring MLL (KMT2A)-rearrangements with AF4 remain a high-risk subgroup in acute lymphoblastic leukaemia cases, with currently <40% event free survival in infants (<1 year). Occurring in >70% of infant cases, improved therapies are essential. The presence of the MLL/AF4 is vital for transformation and maintenance of the leukaemia, making it an ideal therapeutic target. Small interfering RNAs (siRNA) can silence disease driving genes. Fusion genes such as MLL/AF4 present with a unique sequence across the breakpoint that is only present in the cancer cells, potentially allowing for an on-target. We therefore designed an siRNA targeting the fusion site within MLL/AF4. Since siRNA alone has been shown to have poor pharmacokinetic (PK) and pharmacodynamic (PD) properties, we aimed to improve efficacy chemically modifying siRNA and encapsulating them in lipid nanoparticles. Aims: The aim of this study is to develop a lipid nanoparticle delivery system to encapsulate a therapeutic siRNA targeting the MLL/AF4 breakpoint and explore the impact of this in t(4;11) cell lines. Methods: Cells expressing the MLL/AF4 fusion were treated with an MLL/AF4 (siMA6) or control (siMM) siRNA through electroporation or encapsulation by various LNP formulations. Cellular uptake and on target efficiency were investigated in tissue culture using the t(4;11)-positive cell line SEM. Results:: Electroporations were used to optimise chemically modified siRNA for improving PK and PD of siMA6, this data suggested that >200 nM was required to achieve 60% knockdown (KD) within 24 hours. Following this we tested to reduced concentrations of siRNA to investigate if LNPs improve delivery and efficacy. Cells were given 2ug/ml of siMA6 encapsulated in either cholesterol and PEG rich LNP (LNP-chol) or sphingomyelin rich LNP (LNP-SM) and compared. Uptake of LNP-SM is slower compared to LNP-chol within the first 3 hours, 60% and 90% retrospectively. However, by 6 hours, only a 5% difference with LNP-SM achieving 90% compared to 95% in LNP-chol. Surprisingly, functional studies suggest that consequential effects are enhanced in LNP-SM. Sequentially dosed cells with LNP-chol shows a knockdown ranging from 25%-65% in both MLL/AF4 and downstream target genes when compared to the control over 9 days of sequential dosing. A 10-15% reduction in proliferation and viability was seen at day 9. When treated with LNP-SM these consequences were enhanced, KD of MLL/AF4 is reduced 40-60% and downstream genes such as PROM1 and ANGPT1 to >70% by day 9. Furthermore, proliferation of the cells is impaired, reflected in substantial decrease in viable cells by 1.5-fold and notable increase was observed in the G1 and subg1 populations of the cells cycle. Surprisingly, both LNP-chol and LNP SM exhibited a strong impact on self-renewal with a reduction of ≥50% and ≥60% retrospectively by day 9. Summary/Conclusion: This study has demonstrated the advantage of targeting fusion genes with a therapeutic siRNA. We further prove the impact of using an LNP delivery system to improve therapeutic potential of siRNA. This allowed use to more clinically relevant concentrations to achieve a 60% of MLL/AF4 and downstream target genes. Notably, using an LNP-SM formulation enhanced activity of siMA6, showing a substantial impact on cell proliferation, viability, and capacity to self-renew. The next important step of this project is to test PK properties in-vivo and validate the promising findings observed in-vitro. These encouraging results could lead to improved treatments for these ALL patients with a clinically poor prognosis P316: THE ROLE OF TET2 IN (PRE)-LEUKEMIC T-ALL: TET2 TO THE RESCUE? S. De Coninck1,2,3,*, P. Van Vlierberghe1,2,3, S. Goossens2,4, B. Lintermans1,2,3, J. Roels1,2,3 1Biomolecular Medicine; 2Cancer Research Institute Ghent (CRIG); 3Center for Medical Genetics; 4Diagnostic Sciences, Ghent University, Ghent, Belgium Background: T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of pediatric and 25% of adult ALL patients. Therapy has been intensified over the last decades, leading to gradual improvements in survival. However, 10-15% of pediatric and 50% of adult T-ALL cases relapse and ultimately die because of refractory disease. During malignant transformation of T-cells, a clonal expansion of immature thymic cells is selected through the gradual accumulation of advantageous epigenetic changes and genetic mutations. Long-lived preleukemic stem cells (pre-LSCs) were uncovered as an initiating event in various blood-born cancers, including in mouse models of T-ALL. Advanced next-generation sequencing technology allowed for the identification of these predisposing mutations in preleukemic myelodysplastic syndrome patients before they progress towards acute myeloid leukemia. Substantial enrichment for mutations in regulators of DNA methylation were identified, including loss of TET2. TET2 is an enzyme that catalyzes DNA demethylation and Tet2 knockouts are prone to develop lymphoid malignancies, including T-cell lymphoblastic leukemia/lymphoma. In T-ALL, the role of TET2 remains to be investigated. We hypothesize that TET2 acts as a tumor suppressor in T-ALL, involved in a premalignant clonally expanded pre-LSC population. Aims: We aimed to evaluate the tumor suppressor role of TET2 in the development of preleukemic stem cells that ultimately give rise to T-ALL, and to gain a better understanding of the molecular mechanisms involved in this process of malignant T cell transformation. Methods: We evaluated the expression of TET2 in RNAseq data of primary T-ALL samples and compared this with their normal T cell counterparts. Next, we used a spontaneous T-ALL mouse model, CD2-LMO2tg mice. Already at young age, preleukemic CD2-Lmo2tg thymocytes gain aberrant self-renewal capacity, which allows clonal expansion of an immature thymocyte precursor subpopulation. This T-ALL mouse model was crossed with a R26-Tet2 knock-in mouse model that allows conditional (CD2-icre) Tet2 expression in all lymphocytes, at an early differentiation stage, before T-cell commitment. Results: In T-ALL, TET2 mutations are sporadically identified. However, gene expression profiling of primary T-ALL samples identified a large subset of T-ALLs characterized by a profound loss of TET2 expression compared to healthy T cell precursors (Fig. 1A). These low TET2 levels occur in patients from different T-ALL subtypes and were confirmed in a subset of T-ALL cell lines. To further explore the relevance of TET2 silencing and its potential implications in clonal expansion, preleukemic CD2-Lmo2tg mice were sacrificed at different time points (8 and 24 weeks). Gene expression levels in premalignant T-cells were compared with age matched wild-type control mice. A gradual decrease of Tet2 expression, with a co-occurrent increase of Tet2 promotor methylation, was detected in preleukemic CD2-Lmo2tg mice (Fig. 1B-C), which confirms the important role of Tet2 in pre-LSC. To support the tumor suppressor role of Tet2 in T-ALL, an aging cohort of CD2-Lmo2tg with and without Tet2 overexpression was built. Survival analysis showed that CD2-LMO2tg mice with Tet2 overexpression have an increased latency of T-ALL (Fig. 1D, p=0,03). Image: Summary/Conclusion: Tet2 plays an important role in pre-LSC and can act as a tumor suppressor in a spontaneous T-ALL mouse model, which supports the possible tumor suppressor role of TET2 in a subgroup of T-ALL patients. Loss of TET2 in these patients might be a good predictor for response to DNA methylation inhibitors. P317: TARGETING HYPERACTIVE PDGFR-B IN T-ALL AND T-LBL S. De Coninck1,2,3,*, P. Van Vlierberghe1,2,3, S. Goossens2,4, R. De Smedt1,2,3, B. Lintermans1,2,3 1Biomolecular Medicine; 2Cancer Research Institute Ghent (CRIG); 3Center for Medical Genetics; 4Diagnostic Sciences, Ghent University, Ghent, Belgium Background: T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. General prognosis of T-ALL has been improving over time. However, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains dismal. To this end, we have screened a T-ALL/T-LBL patient cohort for copy number variations and identified a novel MYH9-PDGFR-β fusion in a T-LBL case, driving aberrant activation of platelet derived growth factor β (PDGFR-β). Moreover, RNA sequencing data of an independent T-ALL patient cohort showed overexpression of PDGFR-β in the TLX subgroup and in immature T-ALL patients. PDGFR-β is a transmembrane glycoprotein dimer molecule that functions as a receptor tyrosine kinase. Ligand binding results in receptor dimerization and subsequent activation via autophosphorylation, causing downstream activation of various signaling pathways such as PI3K/AKT, MAPK/ERK, JAK/STAT and Notch. As constitutively active PDGFR-β is observed in a variety of malignancies, multi-target kinase inhibitors are already used in the clinic. However, they are not specific and often result in adverse side-effects. CP-673451 is a novel and selective PDGFR inhibitor. Mechanistically, CP-673451 inhibits autophosphorylation of dimeric PDGFR-β and in this way prevents phosphorylation of downstream PI3K/AKT and GSK-3α/β. Previous studies showed growth inhibition of multiple tumor xenografts (lung and colon carcinomas) mainly due to a direct antitumor effect. Aims: We aim to assess whether specific PDGFR-β inhibitors can be used as a novel and targeted therapy in T-ALL/T-LBL treatment or if they can be of added value to the current chemotherapy regimen for T-ALL/T-LBL patients. In the future, we would like to investigate the transforming capacity of the MYH9-PDFR-β fusion in an in vitro pro-T cell culture system. Methods: T-ALL cell lines were screened for PDGFR-β levels and cell lines were selected with both high (CTV-1, SEM) and low (MOLT-16, Jurkat, Loucy) PDGFR-β levels. CP-673451 was tested on the selected cell lines. Cell viability was measured using an ATP assay (CellTiter Glo) and downstream signaling pathways were quantified with flow cytometry and western blot. Additionally, spleen cells from PDGFR-βhigh and PDGFR-βlow T-ALL PDX models were treated ex vivo using CP-673451. Results: Both PDGFR-βhigh cell lines and PDGFR-βhigh PDX models showed sensitivity to CP-673451 treatment in nanomolar range, whereas PDGFR-βlow models did not (Fig. 1A-B). Further characterization of CP-673451 treatment showed inhibition of PDGFR-β autophosphorylation both on western blot and flow cytometry. Downstream, phosphorylation levels of STAT3, STAT5 and GSK-3β were decreased upon PDGFR-β inhibition (Fig. 1C). Dephosphorylation of GSK-3β results in activation of this kinase, leading to phosphorylation of MCL1, promoting degradation of this antiapoptotic protein. Interestingly, high MCL1 levels are associated with lowered sensitivity to glucocorticoids, core components of T-ALL/T-LBL treatment. To this end, combination therapy of PDGFR-β inhibition with glucocorticoids can result in synergistic effects. Next, we will conduct in vivo studies to verify if specific PDGFR-β inhibitors could be advantageous in the treatment of PDGFR-βhigh T-ALL/T-LBL cases. Image: Summary/Conclusion: In this study, we have identified MYH9-PDGFRB as a novel rare fusion activating PDGFR-β in a T-LBL patient sample. Moreover, in vitro evaluation in T-ALL cell lines and ex vivo treatment of T-ALL PDX models showed therapeutic benefits from PDGFR-β inhibition by CP-673451. P318: A DUAL ROLE FOR PSIP1 IN T-ALL L. Demoen1,2,*, F. Matthijssens1,2, Z. Debyser3, S. Goossens2, P. Van Vlierberghe1,2 1Center for Medical Genetics; 2Cancer Research Institute Ghent, Ghent University, Gent; 3Department of Pharmaceutical and Pharmacological Sciences, Katholieke Universiteit (KU) Leuven, Leuven, Belgium Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer characterized by the diffuse infiltration of malignant T-cell progenitors in the bone marrow. Currently, the cure rates have augmented to 80% for pediatric and 60% for adult T-ALL cases due to modern intensified chemotherapy treatments. These cure rates sadly come at the cost of severe side effects and current therapy still provides a poor outcome for primary therapy-resistant or relapse cases. Therefore, there is an urgent need for the identification of key drivers and pathways in T-ALL to develop targeted and less toxic therapies. PSIP1, a histone mark reader, was primarily found as a dependency factor in MLL-rearranged AML, which was dispensable for normal hematopoietic development, making it an interesting therapeutic target. Nevertheless, the identification of inactivating mutations and deletions in T-ALL patients and lower expression in all T-ALL subtypes, with exception of the TLX1-subtype, compared to normal T-cell subsets, is rather an indication that PSIP1 can also serve as a tumor suppressor in certain contexts. Aims: In pursuance of determining the role of PSIP1 in tumor initiation and maintenance in T-ALL, we studied the effect of loss of PSIP1 by utilizing several different T-ALL mice models and human cell lines. Methods: To identify the role of PSIP1 during tumor initiation, we crossed a conditional Psip fl/fl knock-out mouse model with two different spontaneous T-ALL mouse models. On the other hand, to unravel the consequences of losing PSIP1 expression during tumor maintenance a proliferation competition assay was performed and RNA-seq profiling was used to identify downstream targets of PSIP1. Furthermore, tamoxifen-inducible Psip fl/fl mouse cell lines were created from Lmo2-driven T-ALL mouse tumors to test the loss Psip1 during tumor maintenance both in vitro and in vivo. Results: A conditional Psip fl/fl knock-out mouse model was crossed with two different spontaneous T-ALL mouse models, namely the Notch1 independent Lck-Cretg/+ Ptenfl/fl model and the Notch1 dependent CD2-Lmo2tg/+ transgenic, which can also give rise to ETP-like murine T-ALL tumors. The loss of Psip1 was shown to significantly accelerate T-ALL development in both models, with a p-value of 0.0156 and 0.0002 (see figure) respectively. Interestingly, in contrast with the role of PSIP1 loss in tumor initiation, the loss of PSIP1 expression impaired cell proliferation in five different human T-ALL cell lines. Further, tamoxifen-inducible Psip1 knock-out mouse cell lines were established and are being used to further confirm the dependency role of Psip1 during tumor maintenance. Lastly, COX20, an assembly factor of the cytochrome c oxidase in the mitochondria, was found to be a downstream target of PSIP1 upon knock-down and could explain the impairment of proliferation upon the loss of PSIP1 in these cell lines. Image: Summary/Conclusion: Altogether, these data indicate that PSIP1 can exert a dual role in the context of T-ALL, either as a tumor suppressor gene during tumor initiation or as a dependency factor in tumor maintenance. Its dispensability for normal hematopoiesis and its dependency role during tumor maintenance, illustrate the potential of PSIP1 as a therapeutic target in T-ALL. P319: THE TESTICULAR NICHE OF ACUTE LYMPHOBLASTIC LEUKEMIA – MOLECULAR AND CELLULAR FACTORS FOR THE PREFERENTIAL MIGRATION AND SURVIVAL T. Skroblyn1, J. J. Joedicke1, M. Pfau2, K. Krüger1, J.-P. Bourquin3, S. Izraeli4, C. Eckert2,*, U. E. Höpken1 1Max-Delbrück-Center for Molecular Medicine; 2Paediatric Oncology/Haematology, Charité - Universitätsmedizin Berlin, Berlin, Germany; 3University Children’s Hospital, Zurich, Switzerland; 4Schneider Children’s Medical Center of Israel, Petach Tiqva, Israel Background: The testis is the second most frequent (30%) non-hematological extramedullary site of relapse in pediatric acute lymphoblastic leukemia (ALL) treated according to European protocols. Testicular relapses usually occur late (> 6 months after completion of frontline treatment), and boys with a ETV6::RUNX1 fusion gene positive ALL have a significantly higher risk of a testicular relapse. In adult ALL, only about 1% of patients have a testicular relapse. Event-free survival is between 40%-80% depending on timing of relapse, involvement of the contralateral testis and of the bone marrow. The surgical removal of the clinically-involved testis or high-dose irradiation are the only current treatment options for testicular relapses to achieve a long-term event-free survival, but it impacts long-term quality of life. The molecular mechanisms regulating leukemic cell migration, growth, and survival in the testis have not been sufficiently assessed. Aims: We aimed at investigating our hypothesis that the testis is a frequent source of extramedullary relapse in pediatric ALL because of the physiologically strong CXCL12 gradient. This allows circulating leukemia cells to migrate into the testis during pre-puberty and to survive for potentially longer periods because of the specific immune suppressive microenvironment at this unusual anatomical site. Methods: To dissect the pre-pubertal cellular requirements and molecular pathways contributing to testicular leukemic cell dissemination and survival, we combined analysis of primary human leukemias with a patient derived xenograft (PDX) mouse model. We analyzed chemokine receptor expression profiles of pediatric B-ALL samples from patients with different relapse sites, studied the crosstalk of leukemia cell-stroma in co-cultures, and established a pediatric B-ALL PDX mouse model with testicular involvement. Results: The CXCL12-CXCR4 was identified as the driving force for B-ALL cell migration and survival in the testicular leukemic niche. Analysis of primary pediatric patient samples revealed that CXCR4 was the only chemokine receptor being robustly expressed on B-ALL cells both at the time of diagnosis and relapse. One prerequisite for leukemic cell infiltration in the testis mice was high surface expression of CXCR4 on PDX-ALL cells, and CXCL12 secretion from testicular stroma. In affected patient testes, leukemic cells localized within the interstitial space in close proximity to testicular macrophages. Another requirement for migration and survival of leukemia cells in the testis was pre-pubertal age of the recipient mice. Leukemic cell interactions with specific testis macrophage subpopulations, isolated from affected testes, altered their phenotype towards a pro-tumorigenic M2-like phenotype. The blockage of CXCR4-mediated functions by anti-CXCR4 antibody treatment reduced testicular infiltration of PDX-ALL cells. Summary/Conclusion: Collectively, a pre-pubertal condition together with high CXCR4 expression are factors affecting the leukemia permissive testicular microenvironment. CXCR4 could be proposed as a promising target for therapeutic prevention of testicular relapses in childhood B-ALL. P320: TCF3-PBX1 LEUKEMIA INVADES IPSC-DERIVED CEREBRAL ORGANOIDS P. Gebing1,*, S. Hänsch2, L. Lenk3, D. Schewe3,4, A. Borkhardt1,5, U. Fischer1,5, S. Bhatia1,5 1Department of Pediatric Oncology, Hematology and Clinical Immonology, Medical Faculty, Heinrich Heine University Düsseldorf; 2Center for Advanced Imaging - Cai, Heinrich Heine Universität Düsseldorf, Düsseldorf; 3Department of Pediatrics I, ALL-BFM Group, Christian-Albrechts University Kiel and University Medical Center, Kiel; 4Department of Pediatrics, Otto-von-Guericke-University, Megdeburg; 5German Cancer Consortium (DKTK), Düsseldorf, Germany Background: Infiltration to the central nervous system (CNS) remains a major clinical challenge to this day in treating childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The relevant biological mechanisms underlying the invasion of the CNS, are not yet understood and are mainly investigated using 2D cell culture and in vivo mouse models1. Differences between species exist, particularly in cell types and architecture between human and murine CNS, as well as the lack of specificity of infiltration between leukemia subtypes. Human brain organoids potentially address some of these drawbacks and may therefore present a complemental approach to study CNS invasion. Aims: We aimed to establish human iPSC-derived cerebral organoids2 as a 3D model for CNS invasion. Applying these organoids in coculture with primary patient-derived xenograft (PDX) BCP-ALL cells (TCF3-PBX1), we aimed to investigate CNS-specific engraftment while using cord-blood-derived CD34-positive hematopoietic stem cells as a healthy control. Methods: Brain organoids were derived from induced pluripotent cell lines (iPSCs) of different donors and differentiated for 30 and 60 days. Intermediate progenitor, as well as matured neuronal cell types, were controlled by immunofluorescent staining of their respective markers (PAX6/Nestin, TUJ1/MAP2) which assemble in the complex neuroepithelium of cerebral organoids. We cocultured CFDA-SE stained TCF3-PBX1-positive patient-derived leukemia cells and cell lines with cerebral organoids. The initial co-cultivation was designed to investigate when/if infiltration could occur. Furthermore, a limiting dilution was introduced to determine the lowest cell concentration required for invasion. 3D imaging of cleared organoids was performed to analyze invasion. Results: Patient-derived TCF3-PBX-leukemic xenografted cells and cell lines invade cerebral organoids after 14 days of coculture. We found that this is independent of the iPSCs donors (testing in different batches an iPSC donors). The invasiveness was determined by quantifying single and larger colonies of cells engrafted into the organoids. TCF3-PBX1 revealed high invasiveness (P-value: 0.0006) compared to normal cord-blood-isolated CD34+ hematopoietic stem cells (HSCs). In addition, as few as 50 cells (TCF3-PBX1) per organoid were sufficient to invade the organoid tissue. Interestingly, the shRNA-downregulation of CD79a/Igα in TCF3-PBX1 (697) cells, impeded CNS infiltration to cerebral organoids (P-value: 0.0006), annealing their results in murine models1. Image: Summary/Conclusion: Human iPSC derived brain organoids display some key specificity with regards to of BCP-ALL CNS infiltration. Although key features (vasculature, leptomeninges etc.) which could strengthen the model, have not yet been successfully developed for brain organoids, they may present a promising 3D culture system to adopt in CNS infiltration studies. References: 1. Lenk, L. et al. Commun. Biol. 4, 73 (2021). 2. Lancaster, M. A. et al. Nature 501, 373–379 (2013). P321: PERTURBATIONS IN STIMULUS-INDUCED CYTOKINE RESPONSES IN CHILDREN WITH BCP-ALL N. Ruechel*1,, M. Oldenburg*1, S. Janssen*2, A. Pandyra1, L. Wei3, D. Hein1, V. Jepsen1, U. Fischer1, J. Hauer4, F. Auer4, A. Beer5, O. Adams6, C. Mackenzie7, M. Jaeger8, M. Netea9, A. Borkhardt*1, K. Goessling*1 1Pediatric Oncology, Hematology and Clinical Immunology, University Hospital Duesseldorf, Duesseldorf; 2Algorithmic Bioinformatics, Department of Biology and Chemistry, Justus Liebig University, Gießen; 3Department of Molecular Medicine II, Medical Faculty, Heinrich-Heine University, Duesseldorf; 4Department of Pediatrics and Children’s Cancer Research Center (CCRC), Technical University (TU), Muenchen; 5Pediatric Hematology and Oncology, Department of Pediatrics, University Hospital Carl Gustav Carus, Dresden; 6Institute for Virology, Medical Faculty, University Hospital; 7Institute of Medical Microbiology and Hospital Hygiene, University Hospital Duesseldorf, Duesseldorf, Germany; 8Radboudumc, Nijmegen, Department of experimental internal medicine, Radboucumc; 9Radboudumc, Nijmegen, Department of experimental internal medicine, Radboudumc, Nijmegen, Netherlands Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common subtype of childhood leukemia with a peak during early childhood when children are exposed to viral infections. About 5% of all newborns carry a preleukemic clone, but only about 0,2% of them develop BCP-ALL later in life. It has been assumed that a dysregulated, altered immune response underlies the outgrowth of a pre-leukemic clone in infection-triggered B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Aims: We investigated whether children with BCP-ALL differ in their cytokine response from healthy, age-matched, non-leukemic children, after immune cells were challenged with viral, fungal or bacterial stimuli. Methods: We set up a functional experimental platform with 73 stimulus-cytokine pairs to comprehensively characterize the immune profile of pediatric patients with BCP-ALL with the two most common genetic subtypes, either carrying the ETV6/RUNX1 (E/R) gene fusion or the high-hyperdiploid (HD) karyotype in comparison with an age-and gender-matched healthy control cohort without BCP-ALL and their healthy parents (in total n = 101 individuals). Children were in stable first remission, at least two years after the end of therapy and had a fully reconstituted immune system. Blood was taken of patients and controls in parallel and peripheral blood mononuclear cells (PBMCs) were isolated using a density gradient centrifugation. Cells had been stimulated with various fungal, bacterial and viral pathogens, such as Candida albicans, Staphylococcus aureus, Influenza virus, Respiratory Syncytial virus and toll-like receptor ligands (TLR), such as lipopolysaccharide (TLR4), R848 (TLR7/8) or CpG (TLR9); subsequently cytokines (IFNa, IL-1b, IL-1Ra, IL-10, IL-12p70, IL-17, IFNg) had been measured in the cell culture supernatant. Results: Cytokine base line level did not differ between the groups, while the challenge with various infectious antigens resulted in an overall elevated cytokine production of the BCP-ALL patients with E/R fusion (n=11) (p < 0.01, Mann-Whitney-Wilcoxon test two-sided with Benjamini-Hochberg correction). By deciphering the characteristics of the immune response, a remarkable difference for the anti-viral immune response (p < 0.01) was identified, while the anti-bacterial and anti-fungal response did not differ: The IFNa-response induced by Influenza virus and R848 and the IL-17-response induced by R848 differed most prominently, although not significantly after correction for multiple testing. Further analysis of the cytokine profile of the E/R parents compared with an age- and gender-matched healthy control group revealed an overall significantly elevated cytokine response (p < 0.05) after stimulation, but the different sub-features, such as the fungal, bacterial and viral immune responses were not different. Most strikingly, we could identify this pattern specifically for the E/R cohort. Comparing the cytokine responses of the HD patients (n=8) with the healthy controls (n=22) did not result in any significant differences. Image: Summary/Conclusion: Our study suggests that an altered pro-inflammatory anti-viral cytokine response pattern in children with E/R-BCP-ALL is specific to those children, who later developed the BCP-ALL. It is still present years after chemotherapy and possibly contributes to the outgrowth of a pre-leukemic clone, consistently generated in utero during fetal hematopoiesis. P322: THERAPEUTIC EFFECT OF TARGETING CASEIN KINASE II / WDR5 AXIS IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA Q. Han1, C. Song2,3, Z. Ge1,* 1Department of Hematology, Zhongda Hospital，School of Medicine, Southeast University, Institute of Hematology Southeast University, Nanjing, China; 2Hershey Medical Center, Pennsylvania State University, Hershey; 3Division of Hematology, The Ohio State University Wexner Medical Center, the James Cancer Hospital, Columbus, United States of America Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignance with limit treatment choices and extremely poor prognosis. WD repeat domain 5 (WDR5) is essential for the methyltransferase activity of MLL1 complex and plays an important role in various biological functions. We reported that targeting Casein Kinase II (CK2)/Ikaros axis by CK2 inhibitor CX-4945 has therapeutic efficacy in B-ALL. However, the effect of WDR5 inhibitor and CX-4945 in T-ALL remains unknown. Aims: This study is to explore the synergistic effect of WDR5 inhibitor OICR-9429 and CK2 inhibitor CX-4945 and the potential underlying mechanisms of this novel drug combination in T-ALL. Methods: The expression profile and clinical significance of WDR5 and ATAD2 in T-ALL were explored by analyzing the transcriptome data of T-ALL patients from the GEO database. CCK-8 assay was performed to analyze the synergistic effect of OICR-9429 combined CX-4945 in CEM and MOLT4 cell lines. Cell cycle effect of inhibitors were detected by flow cytometry after 72h cultured. RNA-seq was performed after CEM cells were treated with OICR-9429, CX-4945, and vehicle control for 72 hours. Differential expression genes (DEGs) and pathways were analyzed by R-software. WDR5 and ATAD2 were knockdown by shRNA in CEM and MOLT4 cells. qPCR and Western Bolt were performed to test the DEGs detected by RNA-seq. Results: WDR5 is significantly over-expressed in T-ALL patients compared with healthy controls in two independent cohorts and is associated with shorter overall survival (P<0.01). Bioinformatic analysis revealed that the cell cycle was the most significantly altered pathway between WDR5 high and low expression patients (P<0.001) (Figure 1A). In vitro assay showed that OICR-9429 and CX-4945 alone exhibited an anti-proliferation effect on T-ALL cells after being treated for 72h. In addition, we found that OICR-9429 and CX-4945 had a significant synergistic effect on cell proliferation arrest in CEM (Figure 1B) and MOLT4 cells compared with a single drug (P<0.05). Cell cycle assay revealed that OICR-9429 (Figure 1C) and CX-4945 induced a G1-phase arrest in CEM and MOLT4 cells and this effect was significantly increased upon OICR-9429 and CX-4945 combination (P<0.01) (Figure 1D). The anti-proliferation and cell cycle arrest effects were also exhibited in WDR5 knockdown CEM and MOLT4 cells (P<0.05). Pathway analysis of the DEGs showed that the cell cycle pathway was obviously altered after OICR-9429 and CX-4945 treated (P<0.001) (Figure 1E). In addition, CX-4945 strongly down-regulated WDR5 (Figure 1F) expression in both transcriptome level and protein level, and knockdown of WDR5 increased the anti-leukemic effect of CX-4945 (P<0.01) (Figure 1G), suggesting that WDR5 was the downstream target of CK2 and CX-4945 in T-ALL. Moreover, RNA-seq data showed that ATAD2, an important oncogene was suppressed upon treatment of OICR-9429, CX-4945 (Figure 1F), and WDR5 knockdown (P<0.001). Moreover, the combination of OICR-9429 and CX-4945 strongly induced ATAD2 downregulation compared with either single drug (P<0.001) (Figure 1H). Likewise, we found that ATAD2 is up-regulated in T-ALL patients and associated with poor prognosis and cell cycle pathway abnormal activation (P<0.001). ATAD2 knockdown significantly inhibited cell proliferation and induced a G1-phase cell cycle arrest in T-ALL cells (P<0.01) (Fig 1I). Image: Summary/Conclusion: Our data showed that OICR-9429 and CX-4945 have synergistic efficacy in T-ALL. Dual targeting WDR5 and CK2 may be a potential therapeutic approach for T-ALL through the CK2-WDR5-ATAD2 axis. P323: STREAMLINING PRECLINICAL IN VIVO TREATMENT TRIALS BY MULTIPLEXING GENETICALLY LABELLED PDX MODELS OF SEVERAL PATIENTS IN A SINGLE MOUSE K. Hunt1,*, D. Amend1, R. Ludwig1,2, B. Vick1,2, A. K. Wirth1, T. Herold1,3, I. Jeremias1,2,4 1Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), Munich; 2German Cancer Consortium (DKTK), partner site Munich; 3Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich; 4Department of Pediatrics, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany Background: Better treatment options are intensively needed for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Before application in clinical trials, novel therapies require preclinical testing which is resource intensive. In previous work, we had shown that multiplexing several PDX cell populations within a single mouse in competitive in vivo experiments gives identical results to studying each population in separate mice (Liu et al., Biomarker Research 2020). Aims: Here, we aimed at transferring multiplexing to preclinical in vivo treatment trials. We aimed to increase the efficiency of preclinical drug testing in vivo by simultaneously testing several samples in parallel in a single mouse, using a competitive in vivo approach. Methods: From primary patient material, we had established patient derived xenograft (PDX) mouse models of ALL and AML which allow serial transplantation in immunodeficient NSG mice. We designed and cloned 5 different lentiviral constructs to label 5 individual samples with both a unique fluorochrome as well as a connected unique genetic barcode, for later detection by flow cytometry or next generation sequencing, respectively. In addition, luciferase was recombinantly expressed in all PDX models to allow repetitive monitoring of leukemia growth and treatment response using bioluminescence in vivo imaging (BLI). Up to 5 PDX samples, either ALL or AML, were multiplexed and aliquots injected into groups of mice (n=4-6). For post mortem analysis, PDX cells were reisolated from murine bone marrow and spleen and absolute tumor burden of PDX cells was quantified by flow cytometry of exactly 1/10 of the bone marrow and spleen. Determining fluorochrome composition allowed quantifying the proportion that each of the 5 samples contributed to the entire tumor load. In case of treatment, a response rate for each individual samples was determined by comparing cell numbers in treated versus control mice. Results: From our pool of established PDX samples, we selected 5 ALL or 5 AML samples with different molecular alterations, but similar in vivo growth behaviour. Each PDX model was labelled by its individual fluorochrome and genetic barcode, while all models expressed luciferase. Tumor growth as well as the ratio between the samples was repetitively analysed over the entire course of leukemia outgrowth. The composition of the injection mixture was optimized such that each sample accounted for roughly 20% of the total leukemic burden at the time point of start of treatment. Mice containing multiplexed PDX cells from 5 different patients with ALL were treated for 2 weeks with either verum treatment or with solvent as control. In a study using the BCL-2 inhibitor Venetoclax in PDX ALL models, we were able to distinguish between sensitive and resistant PDX samples. While two samples showed a drastic decrease in tumor burden, three samples showed no or only a mild response. This effect could be observed both in cells isolated from bone marrow as well as spleen. Variances between mice were rather small, with few exceptions for some treated mice. With this approach, we were able to define the effect of a single drug on up to five distinct samples in parallel, reducing resources by factor 5. Image: Summary/Conclusion: Taken together, we established a multiplex protocol for in vivo therapy trials that allows simultaneous testing of up to 5 PDX samples in competitive in vivo trials. The approach reduced the required number of experimental mice by a factor 5, in line with the 3R concept. In the future, our approach might rationalize in vivo drug trials. P324: PHARMACOLOGIC INHIBITION OF DYRK1A RENDERS HIGH-RISK KMT2A-R ALL SENSITIVE TO VENETOCLAX C. Hurtz1,*, G. Wertheim2, J. Chukinas3, R. Bhansali4, S. Swaminathan5, J. Crispino6, J. Shi7, S. Tasian3, M. Carroll8 1Fels Institute for Personalized Medicine, Temple University; 2Department of Pathology and Laboratory Medicine, University of Pennsylvania; 3Oncology, CHOP, Philadelphia; 4Hematology Oncology, Upenn, Pehiladelphia; 5Systems Biology, City of Hope, Monrovia; 6Experimental Hematology, St. Jude Children’s Research Hospital, Memphis; 7Cancer Biology; 8Medicine, Upenn, Philadelphia, United States of America Background: KMT2A-rearranged (R) ALL is a high-risk disease with a frequency of 70% in infants and 10% in children and adults with ALL and is associated with chemoresistance, relapse, and poor survival. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. Aims: Determine if pharmacologic DYRK1A inhibition may be a novel treatment strategy for patients with KMT2A-R ALL. Methods: To identify novel targets in KMT2A-R leukemia, we performed a domain-specific kinome-wide CRISPR screen and identified multiple kinases required for cell growth. We focused on DYRK1A as it met the following three criteria: 1) Growth inhibition upon kinase targeting was greater in KMT2A-R leukemic cells than in non-KMT2A-R cells, 2) DYRK1A was not found to be common essential gene assessed through the Cancer Dependency Map, 3) Small molecule inhibitors are available. Results: We analyzed multiple ChIP-Seq experiments and identified that KMT2A-fusions directly bind to the DYRK1A promoter. Our RT-PCR and Western blot analyses demonstrate that KMT2A-R ALL cells treated with a menin inhibitor to disrupt the transcriptional activity of the KMT2A-R complex, downregulate DYRK1A, indicating direct regulation of DYRK1A by the KMT2A-fusion. We further observed that pharmacologic inhibition of DYRK1A with EHT1610 induced leukemic cell growth inhibition in vitro and in vivo, demonstrating that DYRK1A could be a new therapeutic target in KMT2A-R ALL cells. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which surprisingly resulted in the upregulation of MYC and hyperphosphorylation of the RAS/MAPK target ERK. Given that ERK hyperactivation stops B cell proliferation during early B cell development to allow them to rearrange their B cell receptor, we hypothesized that cell cycle inhibition upon ERK hyperactivation remains as a conserved mechanism of cell cycle regulation in KMT2A-R ALL. Strikingly, combining DYRK1A inhibition with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibitor does not induce apoptosis and cells restart cell proliferation after EHT1610 withdrawal we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. However, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt’s Lymphoma. Given the increased expression of MYC following DYRK1A inhibition we performed a new Western blot analysis and validated increased expression of BIM in our KMT2A-R ALL cell lines after EHT1610 treatment. To test if targeting the interaction of BIM with BCL2 will induce an apoptotic effect when combined with EHT1610, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, the combination of DYRK1A inhibition with BCL2 inhibition synergistically killed KMT2A-R ALL cells and significantly reduced the leukemia burden in vivo. Image: Summary/Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo validation is needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. P325: A KINASE-DEAD VARIANT OF CDK6 IS ASSOCIATED WITH REDUCED TUMORIGENIC POTENTIAL AND A DISTINCT TRANSCRIPTIONAL REWIRING IN AN ALL MODEL T. Klampfl1,*, S. Nebenführ1, M. Zojer1, F. Bellutti1, V. Sexl1, K. Kollmann1 1Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria Background: The cell cycle kinase Cdk6 is a major regulator of cell cycle progression from G1 to S phase. Cdk4/6 kinase inhibitors are approved for the treatment of breast cancer and show promising results in pre-clinical studies of other cancers, including hematological malignancies. Cdk6 was also shown to exhibit kinase-independent functions including transcriptional regulation of gene expression. Aims: A systematic understanding of the kinase independent functions of Cdk6 should allow for a better comprehension of the mechanism of action of Cdk4/6 inhibitors. Methods: To model kinase-independent functions of Cdk6 in Acute Lymphoblastic Leukemia (ALL), mouse pre/pro B-cell lines were generated. Whole bone marrow from mice carrying a kinase-dead mutant of Cdk6 (Cdk6-K43M), Cdk6 knock-out (Cdk6-KO) or wildtype Cdk6 (Cdk6-WT) were retrovirally transduced with the BCR-ABL1p185 fusion gene. The resulting cell lines were analyzed in-vivo in transplant settings as well as on the molecular level where RNA-seq and ChIP-seq were performed. Results: Tail vain injection of Cdk6-K43M, Cdk6-KO and Cdk6-WT pre/pro B-cell lines into NSG mice led to the development of lymphoid malignancies albeit with different latencies depending on genotype. Mice injected with Cdk6-KO lines showed a significantly longer survival than mice injected with Cdk6-WT lines. The injection of Cdk6-K43M cells led to an intermediate phenotype with significantly shorter survival than Cdk6-KO, but significantly longer survival than Cdk6-WT. Subcutaneous injection of the cell lines also allowed for the formation of tumors, where tumor weight was largest in mice injected with Cdk6-WT cells, intermediate in the case of Cdk6-K43M cells and lowest in the case of Cdk6-KO cells. Therefore, while kinase-dead Cdk6 had reduced tumorigenic potential compared to Cdk6-WT, loss of kinase function alone exhibited stronger tumorigenic potential than the complete Cdk6 KO. Differential gene expression analysis between cell lines of the three genotypes revealed common alterations associated with Cdk6-K43M and Cdk6-KO compared to Cdk6-WT. Some of these changes could directly be attributed to the loss of kinase function, including for example the downregulation of E2F target genes. Additionally, changes specific for either Cdk6-K43M or Cdk6-KO were observed. As Cdk6-K43M retains kinase-independent functionality, which includes transcriptional regulation, we investigated the DNA binding profile of transcriptional complexes containing Cdk6-K43M. ChIP-seq analysis revealed that such complexes bind the DNA at largely similar sites as complexes containing Cdk6-WT. The majority of genes differentially expressed between Cdk6-WT and Cdk6-K43M were associated with transcriptional complexes harboring Cdk6. Summary/Conclusion: Pre/pro B-cell lines carrying kinase-dead Cdk6 (Cdk6-K43M) showed intermediate tumorigenic potential between lines with Cdk6-WT and Cdk6-KO. While Cdk6-K43M is associated with the DNA at similar sites as Cdk6-WT, there are distinct transcriptional changes associated with this mutation. The further elucidation of these alterations is expected to provide a better understanding of both, kinase independent functions of Cdk6 as well as the effects of its pharmacological inhibition. P326: PROGNOSTIC UTILITY OF NON-HOMOLOGOUS END JOINING DNA REPAIR PATHWAY GENES IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA S. KUMARI1,*, J. SINGH1, M. Arora2, S. Bakhshi3, J. K. Palanichamy2, A. Sharma3, P. Tanwar1, A. R. Singh1, I. Qamar4, A. Chopra1 1Lab Oncology Unit; 2Department of biochemistry; 3Department of medical oncology, All India Institute of Medical Sciences, New Delhi; 4School of Biotechnology, Gautam Buddha University, Gerater Noida, India Background: T-ALL is a molecularly heterogeneous malignancy characterized by transformation and differentiation blockage at different stages of T cell development. Efficient DNA repair has shown to enable leukemic cells to survive the damaging effects of therapeutics, therefore, understanding of DNA repair alterations in leukemia may provide novel druggable targets and prognostic biomarkers. Notably, it is important to determine that whether the variable ability of T cell to repair DNA damage also translates into the corresponding molecular profiles of T-ALL. Several components of different DNA repair pathways have been recently identified to play critical role in ALL pathophysiology and drug resistance. However, the clinical significance of major DNA double strand break repair pathway Non-Homologous End Joining (NHEJ) in T cell acute lymphoblastic leukemia remains to be studied in detail. Aims: This study aims to assess the importance of DNA damage response pathways in T-cell acute lymphoblastic leukemia cancer (ALL). Methods: We assessed the gene expression pattern of 21 genes involved in NHEJ pathwayincluding six core genes, accessory genes and other regulators such as MRE complex genes in 207 T-ALL patients. In order to determine the changes in expression pattern of NHEJ genes during the course of T-ALL, gene expression was assessed at different time points, including clinical sample at diagnosis, minimal residual diseaseand at relapse. Real time PCR was used to assess the expression of these genes. We further determined the association of NHEJ pathway gene expression with clinical and molecular features. Results: We observed higher expression of TOX, WRN, NHEJ1, APLF, TDP2 and reduced expression of XRCC4, POLL, PRKDC, APTX, PNKP, XRCC5, DCLRC1C, MRE11, XRCC6, NBN in diagnostic T-ALL samples compared to normal bone marrow. Further, PRKDC, RAD50, and XRCC6 displayed lesser expression in prednisolone resistant group compared to sensitive group. At minimal residual disease, XRCC4, POLL, WRN, POLM, APTX, PNKP, XRCC5, DCLRC1C, XRCC6, NBN, TDP1 exhibited higher expression and TOX, LIG4, MRE11 exhibited reduced expression compared to diagnostic samples. XRCC4, PRKDC, NHEJ1, PNKP, XRCC5, DCLRC1C, MRE11, XRCC6, NBN and TDP1 exhibited higher expression in relapse samples compared to diagnostic samples. The clinical significance of expression of NHEJ genes was explored in detail. Low expression of XRCC4, PRKDC, RAD50, XRCC5, XRCC6 and TDP1 gene was associated with immature immunophenotype (IPT) as compared to cortical and mature IPT.Patients with ETP-ALL IPT more frequently had lesser expression of XRCC4, PRKDC, RAD50, LIG4, APTX, MRE11, XRCC6andgene compared to other IPT groups. In diagnostic samples, higher XRCC4 expression was associated with better overall survival (OS) and even free survival (EFS). Additionally, low WRN expression was associated with poor EFS and relapse free survival (RFS). Interestingly, high DCLRC1C expression was associated with favorable OS, EFS and RFS. Summary/Conclusion: We observed a distinct expression profile of NHEJ genes among molecularly heterogeneous groups of T-ALL patient’spossibly explaining different extent of DNA repair among patient with different immunological groups and in response to therapy. Interestingly, this depicted a clear change in the DNA repair responses in the leukemia patients before and post chemotherapy. Gene expression emerged as the predictor of OS, EFS and RFS in T-ALL patientsthat might be helpful in the improvement of the treatment outcome in T-ALL therapy P327: QUANTITATIVE EXPRESSION PROFILING OF SURFACE ANTIGENS ON PERIPHERAL BLOOD LEUKOCYTE SUBSETS AND CHILDHOOD T-ALL CELLS USING A STANDARDIZED FLOW CYTOMETRY WORKFLOW: A HCDM CDMAPS INITIATIVE D. Kužílková1,*, J. Puñet-Ortiz2, P. M. Aui3, J. Fernandez2, F. Karel1, P. Engel2, M. C. van Zelm3,4, T. Kalina1 1CLIP (Childhood Leukemia Investigation Prague), Department of Pediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, Prague, Czech Republic and University Hospital Motol, Prague, Czechia; 2Department of Biomedical Sciences, University of Barcelona, Barcelona, Spain; 3Department of Immunology and Pathology, Central Clinical School, Monash University; 4Department of Allergy, Immunology and Respiratory Medicine, Central Clinical School, Monash University and Alfred Hospital, Melbourne, Australia Background: Human Leukocyte Differentiation Antigen (HLDA) workshops are organized by the Human Cell Differentiation Molecules (HCDM) consortium to test and validate the reactivity of particular antibody clones to specific targets. Thereby the consortium provides the scientific community validated antibody clones reactive to particular cluster of differentiation (CD) markers. Although this approach has been used since the 1980s, quantitative profiling of CD markers at “single-cell“ level and benchmarking of reagents are currently lacking. Aims: We aimed to develop a flow cytometric procedure allowing CD marker expression profiling in a standardized way in time and place Methods: First, we developed and titrated two antibody panels with a free position in the phycoerythrin (PE) channel. The panels enable identification of 27 innate and adaptive leukocyte cell populations present in peripheral blood. The panels were custom dried in 96-well plates, and the Quantibrite™ PE Beads were used for quantification of the PE signal. Subsequently, we developed a high content framework to evaluate the titration of PE conjugated monoclonal antibodies using fluorescently barcoded cell lines and peripheral blood cells. The selected titer and critical antibody information (such as clone, catalogue number, vendor, gene and CDname etc.) were centrally stored in an inventory table, which was expanded into an experiment master table (EMT) following inclusion of experimental details (e.g. the position of individual mAbs in 96-well plate, experiment name, operator etc.). The EMT was used to generate an experimental protocol with automated calculation of reagent amounts and volumes. Post acquisition, the fcs files were annotated using all relevant experimental information from the EMT table. Results: To validate our approach, we quantified protein expression of four selected CD markers (CD11b, CD31, CD38 and CD40) with well-known expression pattern on peripheral blood leukocytes that showed high reproducibility across centers. We also performed benchmarking of four anti-CD3 clones, of which the titration curves revealed variable performance: from high-performance TB3 clone, through intermediate-performance UCHT1 and SK7 clones to low-performance MEM-57 clone. Three out of four anti-CD3 clones showed similar pattern of staining, whereas the MEM-57 clone showed decreased intensity on CD4 and CD8 T cells while retaining comparable intensity to the other clones for TCRγδ+ T cells. Our pilot results on childhood T-ALL patient samples (n=7) revealed potential targets for minimal residual disease monitoring. Summary/Conclusion: In summary, we optimized a procedure for quantitative expression profiling of surface antigens on subsets of blood leukocyte and proved its feasibility with inter-laboratory comparison in three different laboratories. The presented workflow enables (i) to map the expression patterns of HLDA-approved antibody clones to CD markers, (ii) to benchmark new antibody clones to established CD markers, (iii) to define new clusters of differentiation in future HLDA workshops and (iv) mapping of childhood T-ALL cells. Acknowledgement: The reagents were kindly provided by Exbio and BioLegend. The work was financially supported by project NU20-05-00282 of the Czech Republic Ministry of Health P328: DYNAMIC EVOLUTION OF TCF3-PBX1 LEUKEMIAS AT THE SINGLE-CELL LEVEL UNDER CHEMOTHERAPY PRESSURE M. Kusterer1, M. Lahnalampi2,*, M. Voutilainen2, G. Gentile1, S. Pennisi1, J. Norona1, G. Greve1, M. Lübbert1, R. Sankowski3, M. Prinz3, S. Killmer4, M. Salvat Lago4, B. Bengsch4, M. Cleary5, V. Zachariadis6, M. Enge6, O. Lohi7, M. Heinäniemi2, J. Duque-Afonso1 1Department of Hematology/Oncology/Stem Cell Transplantation, University of Freiburg Medical Center, Freiburg, Germany; 2Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland; 3Department of Neuropathology; 4Department of Gastroenterology, University of Freiburg Medical Center, Freiburg, Germany; 5Department of Pathology, Stanford University, Standford, United States of America; 6Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden; 7Tampere Center for Child, Adolescent, and Maternal Health Research, Tampere University, Tampere, Finland Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. While research has been focused on high-risk patients, the biology of low-to-intermediate-risk patients has so far been inadequately investigated and can lead to overtreatment with severe side effects and under treatment with risk of relapse. The translocation t(1;19) codes for chimeric fusion protein TCF3-PBX1, which is associated with intermediate risk ALL. Using our previous generated TCF3-PBX1 conditional knock-in mice, we established a model to study in vivo chemotherapy resistance. Aims: We hypothesize that chemotherapy and microenvironment play a crucial role in development of resistance in TCF3-PBX1 leukemias by influencing transcriptional regulation, activation of signaling pathways and the hierarchical structure of leukemic cells. In this project we aim to characterize dynamic changes of TCF3-PBX1 leukemia cells in different tissues under chemotherapy pressure. Methods: Recipient C57/BL6 healthy mice were sub-lethally irradiated, transplanted with mouse TCF3-PBX1 leukemia cells and treated with vehicle (n=15), prednisolone (n=13) and daunorubicin (n=15) for 20 days. Mice were monitored for signs of disease regularly and circulating GFP+ TCF3-PBX1 leukemia cells were assessed by flow cytometry. Sick mice were sacrificed, leukemia cells were isolated from five different organs (bone marrow, spleen, lymph nodes, spinal cord and brain) and characterized by immunophenotyping, sanger sequencing, bulk RNA sequencing (bulk RNAseq) of GFP+ sorted cells, single cell RNA sequencing and mass cytometry (CYTOF). Results: All mice transplanted with TCF3-PBX1 leukemia cells and treated with vehicle succumbed to disease with a median survival of about 70 days. We optimized chemotherapy drug concentration and transplanted cell dose, so 60% of mice treated with prednisolone or daunorubicin survived at least 150 days. Leukemic infiltration was showed by histological stainings in analyzed tissues including central nervous system (CNS) (spinal cord, brain) and GFP+ leukemia cells were quantified by flow cytometry. No major differences were observed in immunophenotype of TCF3-PBX1 leukemia and variant allele frequency (VAF) of the known PTPN11 mutation in analyzed tissues or depending on in vivo treatments. Bulk RNAseq of FACS-sorted GFP+ TCF3-PBX1 leukemia cells were clustered and CNS cells separated from other tissues. scRNAseq revealed additional heterogeneity within each tissue based on signaling pathway and cell cycle activity. Interleukin signaling, regulation of apoptosis pathways, and signaling by the B cell receptor (BCR) were regulated based on pathway analysis in CNS compared to other tissues. Hence, we elucidated the hierarchical structure of hematopoietic cells, interaction of leukemic cells with the microenvironment, and changes in signaling response to chemotherapy in vivo treatment by mass spectrometry (CyTOF) to validate the identified altered signaling pathway activities at protein level. Summary/Conclusion: We have developed a mouse model in order to characterize in vivo chemotherapy resistance depending on niche. Global transcriptomics and phospho-proteomics at the single-cell level might elucidate novel mechanisms of chemotherapy resistance suitable for pharmacological therapies. P329: UNRAVELING THE ROLE OF GATA3 IN EARLY HUMAN T CELL DEVELOPMENT AND EARLY T CELL PRECURSOR ALL (ETP-ALL) N. Lambrechts1,2,*, K. L. Liang1,2, T. Putteman1, J. Roels1,3, J. Van Hulle1, T. Taghon1,2 1Diagnostic Sciences, Ghent University; 2Cancer Research Institute Ghent; 3Biomolecular Medicine, Ghent University, Ghent, Belgium Background: ETP-ALL is a subtype of T cell acute lymphoblastic leukemia (T-ALL) and can be identified by a characteristic immunophenotype and gene expression profile. Treatment of relapsed ETP-ALL remains challenging using conventional chemotherapy and little targeted therapies are available. A better understanding in the molecular mechanisms involved in the leukemogenesis and maintenance of ETP-ALL could lead to improved and more targeted treatments. An estimated 9% of ETP-ALL patients carry mutations in GATA3, a transcription factor known to regulate T cell commitment by inducing T lineage genes and inhibiting genes important for other lineages such as NK and B cells. Although these mutations have been described to be loss-of-function mutations, the exact mechanisms through which they contribute to ETP-ALL remain to be elucidated. Aims: We aim to gain insights into the molecular mechanisms through which GATA3 regulates normal human early T cell development and how disruption of GATA3 function, as a result of mutation, can contribute to the emergence of ETP-ALL. Methods: We have studied the effect of the most prevalent GATA3 mutation (c.827G>A, p.R276Q) in ETP-ALL on normal human T cell development using in vitro differentiation cultures. We examined the impact of GATA3 p.R276Q on gene expression, DNA binding and chromatin accessibility through RNA-, CUT&Tag- and ATAC-sequencing respectively, starting from primary CD34+CD1a- early human T cell progenitors isolated from thymus or the ETP-like cell line PER117. Results: While overexpression of wild-type GATA3 in thymic progenitor T cells induces an acceleration of development towards the CD4+CD8ab+ double positive stage, overexpression of the mutant form results in a delay and an accumulation of CD4 immature single positive cells in OP9-DL1 co-cultures. Remarkably, the inhibition of NK cell development, which is an important function of wild-type GATA3, is maintained by GATA3 R276Q. This indicates that GATA3 R276Q is not merely a loss-of-function mutation. RNA-, CUT&Tag- and ATAC sequencing after overexpression in human CD34+CD1a- immature thymocytes or the ETP-ALL-like cell line PER117 revealed that most key regulatory effects are maintained by GATA3 R276Q compared to its wild-type counterpart, including induction of T cell genes and inhibition of stem cell and NK cell genes, binding to its target regions by recognizing GATA motifs and exerting a pioneering function by opening and closing chromatin. Summary/Conclusion: Continuous GATA3-R276Q has a detrimental effect on T cell development in vitro. Since the inhibition on NK cell development is maintained, these findings suggest that this decision is uncoupled from the induction of T cell lineage commitment. Many important effects on RNA expression, DNA binding and chromatin accessibility are maintained by the R276Q mutant compared to wild-type GATA3, pointing out that this mutation not purely acts as a loss-of-function. The exact mechanisms through which GATA3 R276Q contributes to ETP-ALL still remain to be elucidated, but our results show that inhibition of T cell development most likely is one piece of the puzzle that leads to malignant transformation. P330: TARGETING BTN2A1 BY A UNIQUE ACTIVATING MAB IMPROVES ANTI-TUMOR FUNCTIONS OF VΓ9VΔ2 T CELLS A.-C. Le Floch1,*, C. Imbert1, A. de Gassart2, A. Le Roy1, L. Gorvel1, N. Vey3,4, A. Anastasio1, A. Briantais1, N. Boucherit1, C. E. Cano2, D. Olive3,4 1Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068; 2ImCheck Therapeutics; 3Institut Paoli-Calmettes; 4Aix-Marseille Université UM105, CNRS UMR 7258, Marseille, France Background: Vγ9Vδ2 T cells are new promising cytotoxic effectors in cancer immunotherapy. In acute myeloid leukemia and in non-Hodgkin lymphomas, Vγ9Vδ2 T cells-based immunotherapy has shown encouraging results both in preclinical models and in early phase clinical trials. But very few data are currently available on susceptibility of acute lymphoblastic leukemia (ALL) cells to Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cells are activated by phosphoantigens bound to BTN3A1 on target cells. BTN3A targeting agonist antibody ICT01 is being developed in a multicentric Phase 1 and 2 study called EVICTION by Imcheck Therapeutics. Recently the biology of Vγ9Vδ2 T cells has recently undergone a new paradigm with the identification of BTN2A1 as the direct ligand for Vγ9 chain of γδ TCR. BTN2A1 seems to be mandatory for Vγ9Vδ2 T cell activation but its precise role in modulating functions of Vγ9Vδ2 T cells remains unknown. Aims: Here, we show we show that Vγ9Vδ2 T cells exert cytolytic functions against ALL cell lines and primary ALL blasts and that Vγ9Vδ2 T cell cytotoxic activity is enhanced after treatment with a unique agonist mAb targeting BTN2A1 called 107G3B5. Mechanistically, anti-BTN2A1 enhances interactions between Vγ9Vδ2 T cells and target cells and improves the binding of Vγ9Vδ2 TCR on target cells. Methods: 15 hematological cancer cell lines and PBMC from 17 adults ALL patients at diagnosis (7B-ALL, 7T-ALL and 3Ph+ ALL) were tested in functional assays. We quantified the relative surface expression of BTN2A (using 107G3B5 and 7.48 mAbs) and BTN3A (using 20.1 and 108.5 mAbs) on cell lines and primary ALL blasts. In parallel, allogenic Vγ9Vδ2 T cells functions against cell lines and primary ALL blasts were evaluated. ALL samples were also tested for their expansion capacities after 14 days of PBMC culture in presence of Zoledronate. Interactions of Vγ9Vδ2 T cells with target cells was investigated using a 3D imaging in real-time by holo-tomographic microscopy. Binding assays were realized using a recombinant tetramerized Vγ9Vδ2 TCR. Results: We showed that Vγ9Vδ2 T cells exert spontaneous cytotoxicity against hematological cell lines and primary ALL blasts with a heterogeneous susceptibility depending on the target. We demonstrated that anti-BTN2A1 agonist mAb (107G3B5) significantly enhanced Vγ9Vδ2 T cells mediated apoptosis, in comparison to control condition. This effect was increased over time even for the less spontaneously susceptible cells. We confirmed these observations with autologous Vγ9Vδ2 T cells expanded from 4 ALL patients at diagnosis for which effector functions were increased after treatment with 107G3B5. In live microscopy, 107G3B5 enhanced interactions between target cells cocultured during 24h with Vγ9Vδ2 T cells. Finally, we observed that 107G3B5 strongly increased binding of a recombinant Vγ9Vδ2 TCR to target cells. Summary/Conclusion: Here, we demonstrated that targeting BTN2A1 led to improve Vγ9Vδ2 T cell antitumor response at molecular, cellular and functional levels. Our results highlighted that Vγ9Vδ2 T cells exert cytolytic functions against ALL cells, both in allogenic and autologous setting and demonstrated that BTN2A1 targeting with our unique agonist mAb could potentiate effector activities of Vγ9Vδ2 T cells against ALL blasts. These results indicate that the sensitization of leukemic cells can be induced by activation BTN3A as reported previously as well as with BTN2A1 mAbs. These findings could be of great interest for the design of innovative Vγ9Vδ2 T cells-based immunotherapy strategies for treating ALL that could be extended to other cancer types. P331: T-CELL RECEPTOR REPERTOIRE DIVERSITY AND L-ASPARAGINASE HYPERSENSITIVITY IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA S. Lee1,*, Z. Li1, E. Lim2, E. Chiew2, A. M. Tan3, H. Ariffin4, J. J. Yang1, A. Yeoh2 1St. Jude Children’s Research Hospital, MEMPHIS, United States of America; 2National University of Singapore; 3KK Women and Children’s Hospital, Singapore, Singapore; 4University of Malaya Medical Centre, Kuala Lumpur, Malaysia Background: Asparaginase is an indispensable component of therapy of acute lymphoblastic leukemia (ALL). Treatment with asparaginase is associated with a plethora of adverse effects, amongst which the occurrence of hypersensitivity is one of the most common. Hypersensitivity affects the ability to maintain dose intensity which in turn results in poorer outcomes, especially if there is no suitable replacement. The basis of L-asparaginase hypersensitivity is undoubtedly immune-mediated. T-cell recognition of antigens is arguably one of the most important facets for establishing an immune response, of which a crucial component is the repertoire and diversity of T-cell receptors (TCR). The exact aspects of T-cell immunity underpinning association with asparaginase allergy has yet to be characterized. Aims: To evaluate the associations of TCR repertoire and its diversity with asparaginase hypersensitivity in children with ALL. Methods: We longitudinally profiled the TCR repertoires in 67 children with ALL treated on the frontline Ma-Spore ALL 2010 trial, all of whom receive native L-asparaginase in induction, consolidation, and reinduction. Only patients who developed grade 2 or higher reactions (CTCAE 3.0) were included in this study. These children had bone marrow cells sampled at 3 time-points – diagnosis, week 5 (post-induction), and week 12 (post-consolidation). We performed DNA-based TCR-sequencing on N=180 samples, and evaluated TCR characteristics to determine their associations with asparaginase hypersensitivity. Results: 12 out of 67 children (17.9%) had allergy in our cohort, representing 16 episodes of varying severity (Figure 1A). First, we evaluated the association of TCR-repertoire diversity early on in treatment with the occurrence of subsequent allergy. We found that a higher TCR diversity at all time-points was significantly associated with late allergy (P=0.03 for Shannon’s entropy, P=0.01 for inverse Simpson’s index, Figure 1B). This TCR diversity was characterized by a higher proportion of infrequent clones occurring <0.5% (P=0.008, Figure 1C). Patients with allergy had significantly lower proportion of shared clonotypes (P=0.003, Figure 1D). Examining the dynamic changes of the TCR repertoire between diagnosis and week 5 for each patient, we found that patients with allergy had a much less similar clonotypic set between timepoints compared to non-allergic patients (P=0.003, Figure 1E). In fact, patients who demonstrated a higher variability in their clonotypes between timepoints (i.e. similarity coefficient of <0.05) had an 8.1-fold risk of allergic event (95% CI 1.7 – 39.1, P=0.001, Figure 1F). Evaluating the TCR-repertoire before and after an allergy, we found that there was convergence of TCR towards a common antigen (Figure 1G), where clonotypes became more closely related after an allergy (Figure 1H). Finally, we found that allergic patients had a lower proportion of public clonotypes (i.e. public clonotypic sequences found on VDJDB database) compared to non-allergic patients (P=0.02, Figure 1I), supporting the “hygiene hypothesis” as a predisposing factor in development of allergy. Image: Summary/Conclusion: Higher TCR-repertoire diversity is associated with the risk of L-asparaginase hypersensitivity. This more diverse immunological mileau is characterized by infrequent, dissimilar, and less shared clonotypes, which increases the chance of a reactive clonotype matching to the allergen. Understanding the immunological basis of T-cell response in allergy may help in developing strategies to mediate this toxicity, to further improve outcomes in children with ALL. P332: MIR-625-5P IS NOVEL CANDIDATE ONCOMIR IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA IMPLICATED IN REGULATION OF APOPTOSIS VIA REPRESSION OF HARAKIRI N. Maćkowska-Maślak1,*, M. Drobna-Śledzińska1, R. Jaksik2, M. Kosmalska1, M. Witt1, M. Dawidowska1 1Institute of Human Genetics Polish Academy of Sciences, Poznan; 2Institute of Automatic Control Silesian University of Technology, Gliwice, Poland Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL, arising from T-cell precursors. miRNAs are non-coding RNAs, contributing to leukemogenesis by involvement in key cellular functions such as proliferation, apoptosis, and signaling. We previously found in miRNA transcriptome analysis that miR-625-5p is overexpressed in T-ALL patients, thus is a potential oncomiR in T-ALL. Here, we confirm the implication of miR-625-5p in the regulation of apoptosis in T-ALL cells in vitro. We show that it acts via repression of HARAKIRI (HRK). HRK was originally identified as a proapoptotic gene induced by reduced levels of cytokine in hematopoietic cells and repressed by the expression of death-repressor proteins. Although its proapoptotic function is well described, little is known about the post-transcriptional mechanisms that may participate in HRK inactivation. Aims: The aim of this study was to investigate the mechanism of miR-625-5p oncogenic action via unraveling its targetome and functional effects in T-ALL in vitro. Methods: T-ALL JURKAT cells (with high endogenous miR-625-5p level) were transduced for stable inhibition of this miRNA. To evaluate the influence of miR inhibition on growth of T-ALL cells, we used flow cytometry GFP competition assay and CCK8 proliferation assay. To identify the genes mediating the effect of miR-625-5p, we subjected JURKAT cells to Ago2-RIP-seq. We used magnetic beads coated with anti-AGO2 antibody to immunoprecipitate RNA bound to RISC complexes. Ago2-IP and total RNA fractions were sequenced (polyA RNA-seq, 150 bp reads, 60M PE reads/sample, Illumina NovaSeq 6000). Transcripts depleted in Ago2-IP fraction upon miR inhibition were screened for the presence of miR-625-5p binding sites. Luciferase assay was performed to confirm direct interaction between hsa-miR-625-5p and 3’UTR of HRK gene. The changes of HRK protein level upon miR-625-5p inhibition were evaluated via Western Blot. To estimate the apoptosis rate in T-ALL cells we used two flow cytometry assays based on Annexin V and Caspase 3/7 staining. Results: Inhibition of miR-625-5p decreased the growth of JURKAT cells. In RIP-seq analysis we identified 384 transcripts depleted in RISC by at least 20% upon miR-625-5p inhibition and having at least one putative 3’UTR binding site for this miRNA. Overrepresentation analysis performed for these genes indicated their involvement in the apoptotic process. Among transcripts depleted in RIP-seq with predicted binding sites for miR-625-5p, we found HRK gene, previously reported to have proapoptotic activity. We postulate that repression of this gene by overexpressed miR-625-5p contributes to decreased apoptosis and to growth advantage of T-ALL cells. To further confirm that miR-625-5p affects growth of T-ALL cells via negative regulation of apoptosis, we performed apoptotic assays in JURKAT cells and showed that inhibition of this miRNA increased apoptosis rate, in line with our hypothesis on the oncogenic potential of miR-625-5p. We confirmed the direct interaction between miR-625-5p and HRK 3’UTR by luciferase assay. Additionally, in Western blot a subtle (approaching statistical significance) increase of HRK protein upon miR-625-5p inhibition was observed, supporting our notion that this miRNA is indeed a negative regulator of HRK. Image: Summary/Conclusion: miR-625-5p is a novel candidate oncogenic miRNA in T-ALL, implicated in the negative regulation of apoptosis. Post-transcriptional repression of the proapoptotic HRK gene is a putative mechanism contributing to oncogenic properties of this miRNA when overexpressed in T-ALL cells. P333: MULTI-COHORT GENE EXPRESSION RESOURCE FOR AUTOMATIC SUBTYPING OF B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA V.-P. Mäkinen1,*, J. Rehn1, J. Breen2, D. Yeung1, D. White1 1South Australian Health and Medical Research Institute; 2University of Adelaide, Adelaide, Australia Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and comprises multiple distinguishable genomic subtypes. RNA sequencing provides a functional snapshot that we propose will provide rapid diagnostic information for most ALL subtypes and deep biological insight to guide diagnostic efforts of atypical cases. Aims: The aims of this study were to elucidate the predictive associations between mRNA-seq profiles and independently verified genomic lesions, and to develop easy-to-interpret transcriptome-wide biomarkers for ALL subtyping in the clinical setting. Methods: A total of 1,279 ALL patients were included in the discovery dataset from six publicly available North American cohorts and 767 Australian ALL patients were included as an external validation cohort. Additional publicly available mRNA-seq data (n = 1,160) were used for quality control modelling. A novel batch correction method was introduced and applied to adjust for cohort differences. Three machine learning models (random forest, projections to latent structures and nearest Euclidean neighbor) were trained with the discovery set and validated in the Australian dataset. Results: Out of 18,503 genes with usable expression, 11,830 (64%) were confounded by cohort effects and excluded. There were no substantial differences between the machine learning methods in the validation set; we chose the nearest neighbor technique as the simplest alternative. Six ALL subtypes (ETV6-RUNX1, KMT2A, DUX4, PAX5 P80R, TCF3-PBX1, ZNF384) that covered 32% of patients were robustly detected by mRNA-seq (PPV ≥ 87%). Five other frequent subtypes (CRLF2, hypodiploid, hyperdiploid, PAX5 alterations and Ph-positive) were distinguishable in 40% of patients, although overlapping transcriptional profiles led to lower accuracy (52% ≤ PPV ≤ 73%). Based on these findings, we developed the Allspice R package that predicts ALL subtypes and driver genes from unadjusted mRNA-seq read counts as encountered in real-world settings. Image: Summary/Conclusion: We demonstrated high diagnostic accuracy for a fully automated mRNA-seq method for a third of ALL patients and additional contribution to diagnostic efforts for patients with inconclusive or mixed genomic characteristics. P334: CHARACTERIZATION OF A DUX4-R INHIBITOR AS A POSSIBLE TREATMENT FOR ACUTE LYMPHOBLASTIC LEUKEMIA S. Mara1,*, V. Runfola1, M. Pannese1, C. Caronni1, R. Giambruno1, D. Campolungo1, C. Ghirardi1, D. Gabellini1 1Division of Genetics and Cell Biology, IRCCS San Raffaele Hospital, Milan, Italy Background: Acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer and the major cause of cancer-related death before the age of 20. In up to 10% of cases, the disease is caused by translocation of the Double Homeobox 4 (DUX4) transcription factor to the immunoglobulin heavy chain (IGH) locus, giving rise to rearranged DUX4 (DUX4-r), maintaining the DNA binding domain (dbd) but with a rearranged C-terminus. Contrary to wild type DUX4, which induces apoptosis, DUX4-r acquires transforming ability in cellular and animal models. Through proteomics, we identified a potential DUX4-r inhibitor (iD) for its ability to bind directly to DUX4-r dbd. Aims: The goal of my project is to test the antileukemic potential of iD in cellular and animal models of DUX4-r leukemia. Methods: Human and murine cell lines and primary murine bone barrow progenitor cells are lentivirally-transduced to induce the expression of DUX4-r alone or in combination with iD, and the effects on proliferation, transformation, and B lymphoid differentiation potential are being evaluated. To establish a B-ALL model, DUX4-r PDX cells have been expanded in NSG mice. These cells have been transduced with GFP or iD fused to GFP (GFP-iD) and injected into new NSG recipients for expansion. Serial xenograft transplantation of the purified transduced cells have been performed to test iD-dependent effect on PDX cells expansion and leukemia latency in mice. Human markers and GFP expression were monitored over time in peripheral blood (PB) to assess disease progression. Results: We confirmed that DUX4-r can transform NIH-3T3 murine fibroblasts and inhibits B-cell differentiation of primary murine bone marrow progenitor cells in vitro. We found that iD decreases DUX4-r dependent transactivation. Moreover, lentiviral expression of iD in B-ALL NALM6 cells (carrying an endogenous DUX4-r translocation) reduces the levels of DUX4-r targets and impairs cell proliferation compared to transduced control cells. Preliminary results show that PDX cells expressing GFP-iD expand less and have a delayed PB engraftment compared to GFP control cells. Moreover, we noticed that in iD cohort only contaminant GFP-ve PDX cells take over in vivo despite purification by FACS sorting of transduced cells. Summary/Conclusion: So far, iD expression is associated with reduced proliferation of human DUX4-r leukemia cells and reduced DUX4-r targets expression. Pre-clinical validation of iD could identify effective therapeutic approaches for the treatment of DUX4-r B-ALL patients. P335: GENOMIC DETERMINANTS OF THERAPY RESPONSE IN ETV6-RUNX1 LEUKEMIA L. Oksa1,*, S. Moisio2, K. Maqbool3,4, H. Foroughi3,4, R. Kramer2, A. Nikkilä1, V. Zachariadis5, M. Enge5, K. Vepsäläinen6, J. Duque-Afonso7, J. Hauer8, V. Wirta3,4, O. Lohi1,9, M. Heinäniemi2 1Tampere Center for Child, Adolescent, and Maternal Health Research, Faculty of Medicine and Health Technology, Tampere University, Tampere; 2The Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland; 3Clinical Genomics facility, Science for Life Laboratory; 4Department of Microbiology, Tumor and Cell biology; 5Department of Oncology-Pathology, Karolinska Institute, Stockholm; 6Department of Pediatrics, Kuopio University Hospital, Kuopio, Sweden; 7Department of Hematology/Oncology/Stem Cell Transplantation, Faculty of Medicine, University of Freiburg Medical Center, Freiburg; 8School of Medicine; Department of Pediatrics, Technical University of Munich, Munich, Germany; 9Tays Cancer Centre, Tampere University Hospital, Tampere, Finland Background: Acute lymphoblastic leukemia (ALL) is the most common cancer in children, with about 3500 children diagnosed yearly in Europe. A quarter of cases harbor the ETV6-RUNX1 (E/R) fusion gene. E/R leukemias are usually classified into low risk group. However, a fraction of patients still encounters disease recurrence. The increased relapse risk has been linked with a non-optimal initial therapy response as measured by minimal residual disease (MRD). Aims: The aim of this study is to identify genetic differences between E/R cases stratified by MRD and to identify potential mediators of poor therapy response. Methods: We analyzed the genetic landscape of E/R leukemia in a discovery cohort of 35 patients treated with the NOPHO2008 protocol using whole genome sequencing (WGS). Targeted sequencing was included in validation, including additional cohorts. Patients were classified into three categories (slow, intermediate or fast responder) based on MRD at the end of induction therapy (day 29). Variant callers available in the Balsamic workflow were used to detect DNA alterations and subclonal events analyzed using Battenberg and FastClone tools. Results were compared against published recurrent ALL mutations, bone marrow single cell RNA-sequencing and genome-wide CRISPR-screens in the E/R+ cell line REH, with vincristine, cytarabine, methotrexate, L-asparaginase, maphosamide, daunorubicin and 6-mercaptopurine sensitivity. Nalm-6 screen data was used for evaluating glucocorticoid response. Results: We found comparable number of single nucleotide variants (SNVs), indels and structural variants (SVs) between the MRD-categories. However, based on mutation signature analysis, a significant negative correlation of MRD-level at day 15 was found with Signature 2 (APOBEC), while day 29 MRD correlated positively with Signature 3 (failure of DNA double-strand repair). Interestingly, the signature related to AID/APOBEC family has been reported with oncogenic role in T-ALLs, where APOBEC3 family genes display high expression during thymocyte development. We used single cell RNA-seq profiles from healthy bone marrow to study the relevance of APOBEC3 expression during precursor B-cell development, and showed that APOBEC3B is highly expressed in the dividing hematopoietic precursor, pro-B and especially pre-B cell stages. Well-known ALL-related driver genes did not differ between MRD categories, with the exception of KRAS. Instead of activating mutations, we found prevalent deletions in the fast responder category. To focus specifically on drug response modulating genes, we focused on genes that significantly increased or decreased sensitivity in genome-wide CRISPR screens (top 5% screen hits). Overall, each case harbored multiple DNA alterations in these genes. Moreover, fast responder cases had increased number of hits in sensitizing genes. Summary/Conclusion: Combining MRD analysis with WGS studies carries the potential to reveal crucial information about sensitizing and resistance mutations and underlying cellular mechanisms. The found DNA variants that associate with MRD-status can be utilized in the design and implementation of personalized therapy in E/R+ ALL. P336: A CHEMOTRANSCRIPTOMIC SCREENING IDENTIFIES THE REVERSAL OF GLUCOCORTICOID RESISTANCE IN NOTCH1 MUTATED T-ALL L. Pagliaro1,*, L. Moron Dalla Tor1, V. Federica2, P. Andrei3, L. Monica3, S. Kleissle4, M. Neuenschwander5, A. Gherli1, E. Cerretani2, A. D’Antuono1, E. Simoncini1, A. Montanaro1, G. Roti1 1Medicine and Surgery, University of Parma, Parma; 2University of Ferrara, Ferrara; 3University of Parma, Parma, Italy; 4Max Delbrueck Center for Moleculare Medicine in the Helmholtz Association (MDC); 5Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Background: Gain-of-function NOTCH1 mutations are the most common genetic abnormality in T-cell Acute Lymphoblastic Leukemia (T-ALL), accounting for 55-60% of the cases. Consequently, modulators of the Notch pathway, such as γ-secretase inhibitors (GSI), would be expected to have clinical efficacy (Rao, Cancer Res 2009). However, their application was limited by an excess of toxicity due to the suppression of wild-type (WT) NOTCH1 proteins in normal tissue (Deangelo, JCO 2006; Doody, Alzherimer’s Res Ther 2015). In the past, we identified the Sarco-Endoplasmic Ca2+ ATPase (SERCA) as a gatekeeper of the oncogenic Notch1 signaling. Thapsigargin (TG), a potent SERCA inhibitor (SI), possesses an anti-NOTCH1-leukemia activity both in vitro and in vivo by preferentially targeting mutated NOTCH1 proteins over WT ones (Roti, Cancer Cell 2013). Aims: Since SIs display a favorable therapeutic index by targeting mutated NOTCH1 proteins, development of new SIs is under preclinical development (Marchesini, Cell Chem Biol 2020). Thus, is important to establish molecular mechanisms portending resistance in order to develop effective therapeutic strategies to overcome or prevent drug resistance. Methods: We established drug-resistant clones (R) from parental ALL/SIL T-ALL cell line using a stepwise increase in treatment dose with TG. We quantified the abundance of differentially expressed (DE) genes (P adj<0.05) in the sensitive or resistant cell line by RNA-seq and identified enriched pathways in the insensitive SI cell line. In parallel, we screened a small molecule library of nearly 2500 bioactive compounds (from the European Chemical Biology Library provided by EU-OPENSCREEN) in ALL/SIL and ALL/SIL R. Compound hits were marked by their ability to inhibit each cell line or both. Confirmatory experiments were completed in multiple T-ALL preclinical models. Results: ALL/SIL R cells displayed a mutation (c.G770T -> p.Gly257Val) in the ATP2A2 gene occurring between the Asp254-Leu260 in the third SERCA2 transmembrane (TM) helix. This variation avoids the efficient TG binding to the catalytic domain, resulting in a diminished inhibitory effect. These cells showed an increase of TG IC50 >150-fold compared to the parental line while remaining partially sensitive to CAD204520, an inhibitor that binds SERCA in a pocket different from the one occupied by TG (Marchesini, Cell Chem Biol 2020). This result suggests that part of the resistance mechanism is transcriptionally mediated and virtually common to several SI. In SI-R cells, transcriptional analysis of 6241 DE genes revealed enrichment in steroids synthesis and response pathways, including cholesterol and lipid metabolism. Consistently, among small molecules targeting the steroid hormone receptors subfamily 3, glucocorticoids (GC) scored among the top hits in ALL/SIL R. While naïve cells were resistant to GC, R cells showed an enhanced sensitivity at low nanomolar concentrations of several GC including dexamethasone, clobetasol and fluticasone. This effect is at least in part mediated by the GC receptors since the R cells displayed an elevated GC receptors protein level, as showed in western blotting and RT-PCR, and the effect on viability were reversed by RU486 co-treatment. Consequently, the association of SI plus GC displayed a synergistic effect in multiple preclinical models, including steroid-resistant cell lines, primary leukemia cells, and PDX models. Summary/Conclusion: These findings suggest that SERCA-Ca2+ modulation mediates GC and steroid signaling and that innovative SI can pharmacologically modulate glucocorticoid resistance in T-ALL. P337: LONG TERM INFECTION-DRIVES CHANGES IN THE BONE MARROW MICRO-ENVIRONMENT OF PAX5+/- MICE PREDISPOSED TO B CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA W. Liu1, O. Stencel2, Z. Lu2, C. Xu1, P. Lang1, F. Auer3, J. Hauer3, U. Fischer2, A. Borkhardt2, A. Pandyra2,* 1Department of Molecular Medicine II; 2Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-Heine University, Düsseldorf; 3Department of Pediatrics, Technical University of Munich, Munich, Germany Background: Pax5 heterozygosity (Pax5 +/-) in mice mimics germline or somatic Pax5 dysregulation (resulting in reduced Pax5 levels) observed in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). While a link between general non-specific infectious exposure and BCP-ALL has been demonstrated in the Pax5 +/- mouse model, the effects of a specifically tractable infection are poorly understood. In particular, little is known about potential interactions of pre-leukemic early progenitor B cell populations and immune cells present in the bone marrow microenvironment (BME) during an infection. Aims: We aimed to characterize the short-term and long-term effects of a viral infection in the BME of the Pax5 +/- mouse model employing the Lymphocytic choriomeningitis virus (LCMV). LCMV is a non-cytopathic virus that has been extensively utilized to investigate virus-induced immunopathology, effector responses and immune tolerance. Methods: Pax5 +/- mice backcrossed to the C57BL/6J background (N10) were infected with LCMV. Using flow cytometry, ELISA and plaque assay, innate and late adaptive immune responses in infected Pax5 +/- and WT (Pax5 +/+) were assessed. Pre-leukemic early progenitor B cell populations, antigen-specific CD8+ T cells and regulatory T cells (Treg) were evaluated in the BME. This was complemented by short-term ex vivo assays where B cells were stimulated with differing immune stimuli. Results: When we investigated immune responses in a short-term ex vivo culture system using the bone marrow of Pax5 -/+ and WT mice, we found differences in the induction of the interleukin 7 receptor (IL-7r) on immature Pro-B cells as well as the production of cytokines (IL-6 and TNF-▫▫) in response to the toll-like receptor (TLR7) agonist R848 and the lymphocytic choriomeningitis virus (LCMV) Docile strain. When we infected Pax5 -/+ and WT mice with LCMV-Docile, we found that innate (early interferon production) and adaptive immune responses (tetramer positive CD8+ T cells and early viral titers) were not intrinsically impaired in Pax5 -/+ mice. However, at day 90 post-infection, LCMV-specific T cells were present within the BME and tetramer specific CD8+ T cells raised against the LCMV nucleocapsid protein (np 396) were increased in the BM of Pax5 -/+ infected mice (Figure 1A). Furthermore, when we evaluated Major Histocompatibility Complex Class II (MHC-II) expression on different B cell subsets in the BME of infected and uninfected mice, we found that there was a significant upregulation in all immature B-cell subsets (Pro-B, PreBI-II) in response to infection in the WT but not in the Pax5 -/+ infected mice. Pax5 -/+ mice were susceptible to LCMV infection resulting in shorter long-term survival compared to infected WT mice (Figure 1B). Image: Summary/Conclusion: This study demonstrates that the BME environment as well as survival of pre-leukemic Pax5 -/+ mice is differently affected by LCMV infection supporting the observations that immune dysregulation could contribute to the emergence of the leukemic clone1. 1Supported by the Carreras foundation project no. DJCLS07R/2019 P338: BRANCHED CHAIN AMINO ACID TRANSAMINASE 1 ASSOCIATES WITH THE KU70/KU86 HETERODIMER AND MODULATES THE DNA DAMAGE RESPONSE IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA CELLS L. Di Martino1,*, V. Tosello2, S. Dalla Santa1, A. Papathanassiu3, G. Arrigoni4, P. van Vlierberghe5, E. Piovan1,2 1Surgery, Oncology and Gastroenterology, University of Padova; 2Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, Padova, Italy; 3Ergon Pharmaceuticals, LLC, Washington DC, United States of America; 4Department of Biomedical Sciences, University of Padova, Padova, Italy; 5Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Background: T-cell acute lymphoblastic leukemia (T-ALL) is a biologically and clinically heterogeneous disease mostly associated with NOTCH1 mutations that characterize over 60% of cases. Therapy-resistant or refractory T-ALL remains a major clinical challenge. Branched Chain Amino Acid Transferase 1 (BCAT1), a cytosolic aminotransferase converts BCAAs into their corresponding branched-chain α-keto acids and vice versa. Aberrant expression of BCAT1 has been demonstrated in different tumor models, including acute leukemia. However, its biological role in T-ALL remains to be elucidated. Aims: The purpose of this study was to dissect the functional role of BCAT1 in T-ALL, with particular emphasis on a putative link between BCAT1 and NOTCH1 in promoting T-ALL initiation and progression. Methods: We evaluated BCAT1 expression using RNAseq data from 264 T-ALL samples (ALL0434 protocol). BCAT1 transcript and protein levels were determined in T-ALL cell lines (n=13) and patient derived xenografts (PDX; n=12). Metabolomics analysis (using 13C6 labeled Leucine) was performed following NOTCH1 inhibition. Further, five PDX samples were treated with a γ secretase inhibitor (DBZ) to dampen NOTCH signaling. BCAT1 promoter occupancy by NOTCH1 was determined by Chromatin Immunoprecipitation (ChIP) coupled with qPCR. Tandem affinity purification (TAP) and mass spectrometry (MS) analysis were used to identify BCAT1 interacting proteins from CUTLL1 T-ALL cells. Kinetics of the DNA damage response (DDR) was evaluated in BCAT1 knock-down cells. Drug combination experiments using the DNA damaging agent (etoposide) and a specific BCAT inhibitor (ERG245) were performed in vitro and in vivo in PDX T-ALL models. Results: We found variable BCAT1 expression levels amongst the different T-ALL molecular subgroups, with higher expression levels in TLX1 (p<0.001), HOXA-TLX3 (p<0.05) and NKX2-1 (p<0.01) subtypes. Considering recurrent genetic alterations, we found BCAT1 expression to be significantly higher (p<0.01) in NOTCH1 mutated cases compared to un-mutated samples. Interestingly, NOTCH1 mutated PDX samples also showed higher BCAT1 expression compared to un-mutated cases. Metabolic studies disclosed that BCAAs oxidation is decreased following NOTCH1 inhibition (reduction in 3-methyl-2 oxovaleric acid). Further, inhibition of NOTCH1 signaling with DBZ decreased BCAT1 expression in numerous T-ALL models. ChIP analysis and luciferase reporter assays suggest that NOTCH1 may directly regulate BCAT1 expression. Functionally, TAP followed by MS analysis of BCAT1 interacting proteins disclosed that BCAT1 may be implicated in non-metabolic processes such as DNA replication and repair and rRNA processing. Indeed, we found Ku70/Ku86 proteins to interact with BCAT1. Evaluation of the DDR following DNA damage, disclosed that BCAT1 deficient cells present an accentuated but defective DDR which translates into marked DNA damage leading to pronounced cell death. Consistently, a marked synergistic therapeutic response was found between the DNA damaging drug etoposide and the BCAT specific inhibitor ERG245 in numerous T-ALL models. Summary/Conclusion: NOTCH1 may directly regulate BCAT1 expression in T-ALL cells. BCAT1 is involved in sustaining genomic integrity following DNA damage through its interaction with the Ku70/Ku86 heterodimer, known to mediate classical non-homologous end-joining. Our results identify BCAT1 as a novel therapeutic target and suggest that the combination between DNA double stand break inducing agents (such as etoposide) and a BCAT inhibitor could be particularly useful in NOTCH1-mutant T-ALL cases. P339: BOOSTING THE EFFICACY OF CD20-TARGETING IMMUNOTHERAPY IN B CELL ACUTE LYMPHOBLASTIC LEUKEMIA M. Poprzeczko1,*, K. Domka1,2, A. Pastorczak3, K. Fidyt2, L. Komorowski1, M. Winiarska2, A. Dabkowska2, K. Siudakowska2, A. Wojciechowska2, E. Patkowska4, M. Firczuk1,2 1Laboratory of Immunology, Mossakowski Medical Research Institute Polish Academy of Sciences; 2Department of Immunology, Medical University of Warsaw, Warsaw; 3Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Lodz; 4Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland Background: CD20 is a B cell-specific surface protein that appears at the stage of pre-B cells during B cell development. Currently, monoclonal antibodies (mAb) against CD20 are commonly used for immunotherapy of mature B-cell malignancies. B cell precursor acute lymphoblastic leukemia (BCP-ALL) originates from immature B cells in a bone marrow. Overall, the effects of treatment with high-dose multi-agent chemotherapy are good. However, for relapsed/refractory patients, novel treatment options (mainly immunotherapy-based) targeting B cell-specific antigens are already used in clinics and further optimized in preclinical models. Recent clinical trials conducted in Philadelphia (Ph) chromosome-negative BCP-ALL patients with CD20 expression revealed that the outcome of young adults may be improved by a combination of chemotherapy with rituximab, the anti-CD20 mAb. However, in most patients, the expression of CD20 on BCP-ALL blasts is heterogeneous and may be insufficient for effective treatment with CD20 mAb. Also, the regulation of CD20 in BCP-ALL is not well defined. Aims: The aim of this study is to identify drugs that are able to upregulate CD20 in BCP-ALL, in order to improve the efficacy of anti-CD20 mAbs in preclinical models of the disease. Methods: We tested the impact of 38 anti-cancer drugs used for the treatment of BCP-ALL as well as tested in clinical trials on the CD20 surface levels after 48hrs of incubation. For the in vitro studies, we employed cell lines and primary cells representing selected high-risk subtypes (Ph-positive, Ph-like, B-other). The levels of CD20 were determined by flow cytometry. Next, the potential of selected drugs to activate the anti-CD20 mAb, rituximab, was tested in a functional assay, Antibody-Dependent Cellular Cytotoxicity (ADCC) with Human Peripheral Blood Mononuclear Cells (PBMC) or primary NK cells, isolated from healthy donors. Results: Based on the initial screening of cell lines, we selected 10 agents which increased the level of CD20: three histone deacetylases inhibitors, one inhibitor of MDM2 protein (p53 signaling pathway), one apoptosis inducer, three kinase inhibitors, one mitosis inhibitor, and one antimetabolic agent. Importantly, we found that many tyrosine kinases inhibitors and some retinoids significantly decreased CD20 levels in BCP-ALL cell lines. Some of these findings were also confirmed in primograft BCP-ALL blasts co-cultured with OP9 murine stromal cells. Furthemore, we found that five out of ten drugs enhance antibody-dependent cytotoxicity of rituximab, in functional assay. Moreover, two of the tested drugs increased phagocytosis of primograft BCP-ALL cells by primary macrophages differentiated from human peripheral blood monocytes. Summary/Conclusion: In summary, we identified agents upregulating CD20 levels in BCP-ALL cells in vitro. More importantly, some of the selected drugs improved RTX-mediated effector mechanisms such as ADCC and immunophagocytosis. Further studies aiming to elucidate the mechanisms of CD20 upregulation and to validate our in vitro findings in murine models are ongoing. P340: IN VITRO AND IN VIVO EFFICACY OF A NOVEL KINASE INHIBITOR TARGETING JAK2 GENE REARRANGEMENTS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA M. Quadri1,*, C. Saitta1, S. Palamini1, C. Palmi1, A. Biondi1, G. Cazzaniga1, G. Fazio1 1Centro di Ricerca Fondazione M. Tettamanti, Monza, Italy Background: Although risk-based treatment is curative for 85% of children with B-cell precursor acute lymphoblasticleukemia (BCP-ALL), relapse remains a leading cause of mortality, urging the need of novel molecular targets. The JAK/STAT alterations represent about 7% of the ‘Philadelphia-like’ cases. JAK2 gene encodes for a non-receptor tyrosine kinase fundamental for hematopoiesis and itsmutations have been widely studied, whereas JAK2 fusion genes are still poorly characterized. Aims: This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients, developing a target strategy in preclinical models. Methods: We applied RNA Next Generation Sequencing to find JAK2 fusion genes in a cohort of high risk BCP-ALL pediatric patients. Fusions were validated by RT-PCR and/or FISH. In vivo expansion of patients’ cells has been carried out in NSG mice. After drug treatments with JAK2 inhibitors, phosphoflow and apoptosis assays were done. Results: We identified 10 pediatric cases carrying a JAK2 fusion with different partners, where PAX5 gene was the only recurrent.After in vivo expansion of cells from 3 cases, carrying PAX5::JAK2, ATF7IP::JAK2 and ZEB2::JAK2, we demonstrated that JAK2 signaling was activeat basal level, through phosphorylation of JAK2 Y1007-1008 compared to cases wild type for JAK2 and CRLF2 (+70%, two-tailed P value 0.03); and also compared to P2RY8::CRLF2 rearrangements and JAK2 mutation(+40%, two-tailed P value 0.16). The JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated. We targeted JAK2using CHZ868, a new class-II tyrosine kinase inhibitor (TKI) (Novartis, Basel, CH). After 30 minutes and till48h, we appreciated a mean inhibition of -62% of Y1007-1008 JAK2 residues in PAX5::JAK2, -22% in ATF7IP::JAK2 and -35% in ZEB2::JAK2. Contemporarily, we observed a decrease of pS727-STAT3 (-35-50%) and pY694-STAT5 (-15-50%) and the significant reduction of phosphorylation on PI3K pathway,downregulating PDPK1, AKT, 4pEBP1 and pS6. After 48h monotherapy treatment by CHZ868, we detected decrease in cell viability (20-75% at IC50). In combination with dexamethasone, a further decrease of viability was observed. In the PAX5::JAK2 case, we also performed treatments with BIBF1120/Nintedanib, LCK inhibitor (activated downstream to PAX5 fusions) and we observed a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, ruxolitinib caused autophagy as observed by higher levels of LC3-II compared to untreated cells (+45%, p<0.01), with reduction of apoptosis. Indeed, active caspase 3 increased after ruxolitinib and chloroquine (autophagy inhibitor) combination(+20% vs ruxolitinib alone, p<0.01). Instead, CHZ868 alone or in combination with chloroquine does not induce autophagy, with no effect on bothLC3-II and active caspase 3 levels. Finally, we demonstrated the in vivo efficacy of CHZ868 in PAX5::JAK2, ATF7IP::JAK2 and ZEB2::JAK2 patient-derived-xenografts. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in BM(-43-85%), spleen(-72-89%), CNS (-13-62%) and PB (-46-80%).Moreover, CHZ868 in vivo treatment significantly reduced the phosphorylation of pJAK2 (-18-46%), pSTAT5 (-23-71%) and pAKT(Ser473) (-18-34%). Summary/Conclusion: CHZ868 is a promising drugfor the treatment of JAK2 fusionsBCP-ALL. Further studies will include combination with standard chemotherapy drugs, by reducing the intensity and toxicity of chemotherapy. P341: A NOVEL BISPECIFIC T CELL ENGAGER (UMG2-CD3) IS EFFECTIVE AGAINST CORTICAL-DERIVED ACUTE LYMPHOBLASTIC LEUKEMIA C. Riillo1,*, D. Caracciolo1, K. Grillone1, N. Polerà1, F. M. Tuccillo2, P. Bonelli3, G. Juli1, S. Ascrizzi1, F. Scionti4, M. Arbitrio5, M. Lopreiato1, M. A. Siciliano6, S. Sestito6, G. Talarico7, E. Galea7, M. C. Galati7, M. Rossi7, A. Ballerini8, M. Gentile9, M. T. Di Martino1, P. Tagliaferri1, P. Tassone1,10 1Clinical and Experimental Medicine, Università Magna Graecia, Catanzaro; 2Istituto Nazionale Tumori “ Fondazione Pascale” - IRCCS; 3Istituto Nazionale Tumori “ Fondazione Pascale” - IRCCS, Napoli; 4Institute of Research and Biomedical Innovation (IRIB), Italian National Council (CNR), Messina; 5Institute of Research and Biomedical Innovation (IRIB), Italian National Council (CNR); 6Università Magna Graecia; 7Pugliele-Ciaccio Hospital, Catanzaro; 8biovelocITA srl, Milano; 9Annunziata Hospital, Cosenza, Italy; 10College of Science and Technology,Temple University, Philadelphia, United States of America Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived by T cell precursors and characterized by poor prognosis.The immunotherapy has revolutionized the outcome of B cell acute lymphoblastic leukemia (B-ALL), but the absence of tumor-specific T cell antigen hampers its efficacy in T-ALL. Therefore, the development of novel immune-therapeutic options for the management of this orphan disease is eagerly awaited. CD1a is a glycoprotein expressed on cortical T-ALL and only on healthy thymocytes and Langerhans cells. Taking into account its safe pattern of expression, CD1a might represent a valuable therapeutic target for thetreatment of T-ALL. Aims: With the aim to provide an effective immune-therapeutic strategy for T-ALL, we developed a bispecific T cell engager (BTCE) derived from a novel UMG2 mAb that recognize a previously uncharacterized CD1a epitope. Methods: To evaluate the specificity of UMG2 binding to CD1a epitope, HEK293T cells which do not express CD1a endogenously, have been used. In this regards, cells were transfected with a plasmid that encode for CD1a or with an empty vector and UMG2 reactivity has been evaluated by flow cytometry. To assess the unicity of UMG2 mAb binding, a competitive binding assay between UMG2 and commercially available CD1a antibodies has been performed. The UMG2 expression profile on peripheral blood cells from healthy donors and on a panel of cortical T-ALL cells has been evaluated. To develop a UMG2-CD3 construct, an asymmetric 2 + 1 UMG2-CD3 bispecific T cellengager (BTCE) has been generated by using knobs into hole technology. UMG2-CD3 T cell-redirected cytotoxicity has been evaluated on HEK293T wild type, on HEK293T-CD1a+ and on patient-derived T-ALL cells, co-cultured with peripheral blood mononuclear cells (PBMCs), CD4/CD8 depleted and CD56 enriched lymphocytes at different E:T ratio.Moreover, T cell activation has been assesed by flow cytometry. UMG2-CD3 anti-tumor activity against a CD1a+ T-ALL cells has been evaluated in vivo. For this purpose,Hu-PBMCs NSG mouse model has been generated and tumor growth has been assessed by fluorescent imaging probe via IVIS system. Results: UMG2 mAb recognizes a novel CD1a epitope and does not compete with any of the commercially available anti-CD1a mAbs with the exception of a partial competition with NA1/34-HLK clone. A strong UMG2 reactivity has been observed on T-ALL cells, while no binding has been found on normal blood cells. A concentration-dependent T cell cytotoxicity on CD1a+ T-ALL cells co-cultured with PBMCs in the presence of UMG2-CD3 has been observed. Minimal UMG2-CD3 residual anti-tumor activity has been observed in CD4/CD8 depleted and CD56 enriched lymphocytes. CD56 depleted and Fc-blocked BMCs were able to induce an anti-T-ALL activity comparable to total PBMCs, demonstrating that UMG2-CD3 could not recruit monocytes and NK cells through Fc-FcyR interaction. Moreover, the concentration-dependent increase of i) T cell proliferation, ii) cytotoxic degranulation marker (CD107a), iii) expression of cell surface activation markers (CD25, CD69),and iv) pro-inflammatory cytokine secretion (IL-2, TNF-α, IFN-γ) has been observed in the presence of UMG2-CD3. Importantly, in an in vivo immune-humanized NSG mice model,UMG2-CD3 was able to significantly inhibit tumor growth and then conferring the survival advantage of treated animals. Summary/Conclusion: All our findings, demonstrated that UMG2-CD3 BTCE represents a promising immune-therapeutic agent against T-ALL to be further investigated for clinical translation. P342: BASELINE GENE EXPRESSION ANALYSIS OF RELAPSED ACUTE B-LYMPHOBLASTIC LEUKEMIA PATIENTS TREATED WITH INOTUZUMAB OZOGAMICIN C. Sartor1,*, I. Vigliotta2, V. Robustelli1, G. Cristiano1, J. Nanni1, L. Zannoni1, S. Parisi2, S. Paolini2, G. Marconi1,3, G. Martinelli3, A. Curti2, M. Cavo1,2, C. Terragna2, C. Papayannidis2 1Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Istituto di Ematologia Seràgnoli; 2IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna; 3Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, 47014, Meldola (FC), Italy Background: Despite introduction of novel agents, relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) carries dismal outcome. Inotuzumab ozogamicin (InO) is a humanized anti-CD22 monoclonal antibody conjugated to calicheamicin approved for R/R B-ALL able to obtain high CR rates but of short duration. Mechanisms underlaying InO resistance are largely unknown. Aims: To characterize the baseline differentially expressed genes in a series of R/R B-ALL patients in relation with patient response to InO in order to individuate potential pathways involved in resistance. Methods: Gene expression profile of 18 R/R B-ALL patient samples was analyzed with RNA-seq, before InO exposure. All patients received at least 1 InO course. Patient population was divided in poor/non-responders (NR) and responders (R), based on InO response. R were defined as patients with duration of response (DoR) ≥3 months after bone marrow (BM) complete remission (CR) achievement. NR were defined either refractory or with a DoR <3 months after CR achievement. The list of significant (p<0.05) differentially expressed genes (DEGs) in NR, as compared to R whose absolute fold change (FC) was ≥2, was analyzed. P values were corrected with the Benjamini-Hochberg algorithm (false discovery rate; FDR). Gene expression results were analyzed with QIAGEN Ingenuity Pathway Analysis (IPA). Results: Eighteen R/R BM samples of CD22-positive B-ALL were analyzed with RNA-seq. Eight patients were defined as NR and 10 patients as R. Patient disease characteristics in NR and R groups were homogeneous for age, sex, number of Philadelphia (Ph)-positive ALL patients, duration of 1st remission, number of previous therapy lines including hematopoietic stem cell transplantation (HSCT) and Blinatumomab. At the time of InO therapy all patients had morphological BM relapse and all were CD22-positive. Median CD22 expression percentage on leukemic blasts was 100% (range 100-100) in NR patients and 100% (range 70-100) in R patients (p=0.177). Two patients in R group had a CD22 expression blast percentage of 70% and 76%. Median CD22-fluorescent intensity (CD22-FI) in NR as compared to R was 75.38 (IQR 59.58, 89.51) and 136.51 (IQR 114.38, 151.57) respectively, with significantly lower values in NR group (p = 0.04). Overall, 370 genes were differentially expressed (p<0.05) in NR, as compared to R patients, of which 32 were significantly differentially expressed (corrected p < 0.05, FC ≥2). Thirty-one were down- and 1 was up-regulated in NR vs R. DEGs were involved in basophil differentiation, carbon dioxide transport, erythrocyte and myeloid cell development, heme metabolic and porphyrin-containing compound biosynthetic processes, cation and cellular ion homeostasis. Both IPA upstream regulator and regulator effect analysis identified the serine/threonine homeodomain-interacting protein kinase-2 (HIPK2) as predicted downregulated. The inhibition of HIPK2 was predicted to be the causal upstream condition of the under-expression of six DEGs from the set (FECH, ANK1, SCL4A1, EPOR, GATA1, HBZ) with activation Z score of -2.449 and p value of overlap = 1.02E-09. No difference in terms of HIPK2 expression was appreciated in the two groups, suggesting downregulation at post-transcriptional level. Image: Summary/Conclusion: A unique pattern of gene expression signature based on HIPK2 downregulation was identified in poor responders to InO, providing potentially important insights in mechanisms of resistance. HIPK2 downregulation needs to be further validated. CT and CP contributed equally P343: GENOMIC CHARACTERISATION OF B-OTHER ALL IN UKALL2003 PATIENTS BY NEXT GENERATION SEQUENCING C. Schwab1,*, R. Cranston1, S. Ryan1, E. Butler1, E. Winterman1, Z. Hawking1, M. Bashton1, J. Murray1, A. Enshaei1, J. Gibson1, A. Vora2, A. Moorman1, C. Harrison1 1Leukaemia Research Cytogenetics Group, Newcastle University, Newcastle upon Tyne; 2Department of Haematology, Great Ormond Street Hospital, London, United Kingdom Background: Incorporating genetics into risk stratification for the treatment of childhood acute lymphoblastic leukaemia (ALL) has contributed to increased survival rates. About 30% of patients (B-other-ALL) harbour none of the known major chromosomal changes. Recently, we estimated that up to two thirds of B-other-ALL can be classified into novel subtypes using FISH and MLPA alone. We showed that ABL-class fusions and ERG deletions were linked to prognosis (Schwab et al, BJHaem, 2022). Aims: To characterise B-other-ALL in the UKALL2003 trial using next generation sequencing (NGS), to evaluate the added benefits of these techniques and validate the prognostic significance of B-other-ALL subtypes. Methods: B-other-ALL patients were tested using whole genome sequencing (WGS) (n=158) and bespoke targeted NGS (t-NGS) for the detection of abnormalities in 64 genes commonly mutated or rearranged in B-ALL (n=180), 42 patients had both WGS and t-NGS. Data were integrated with results from FISH, MLPA and cytogenetics. Results: A representative cohort of 347 B-other-ALL was classified into subgroups, including six with ≥20 cases (Figure). Among patients tested by WGS, 92% (n=146/158) were classified compared to 75% (n=104/138) tested by t-NGS. NGS-based approaches allowed for the detection of PAX5 P80R, IKZF1 N169Y and ZEB2 H1038R and fusion partner genes. Identification of DUX4-r was not possible using t-NGS. PAX5alt was the most frequently observed (n=90), including dic(9;20) (n=27), dic(9;12) (n=11), PAX5 fusions (n=21), PAX5 mutation (n=11) and PAX5-ITD (n=12). Additionally, 11 patients were observed with PAX5 P80R mutations, classified as a separate subgroup due to the associated gene expression signature. PAX5alt had an outcome similar to B-other-ALL overall (overall survival (OS) at 10yrs 82% vs 86%). There was no significant difference in outcome between PAX5 abnormalities, although an association with age was observed: dic(9;20) was more commonly observed in children aged 1-4 (p< 0.001), while both dic(9;12) and PAX5 P80R were seen in older children aged 10-15 years (p=0.005). One subgroup with DUX4 rearrangements and/or ERG deletions (DUX4-r/ERG-d) (n=78) had DUX4 involvement confirmed by WGS in 57 patients. In the remaining 21 patients ERG-d were identified by t-NGS and/or MLPA. ERG-d is exclusive to DUX4-r patients, thus can be used as a surrogate marker. In line with other studies, patients with DUX4-r had a lower relapse rate (RR) (5%) and improved OS (96%) compared to other subgroups. Patients classified as ETV6-RUNX1-like (n=21) were characterised by multiples abnormalities of ETV6, including rearrangements (n=17) and/or deletion (n=12) as well as rearrangements (n=7) and/or deletions (n=6) of IKZF1. An ETV6-RUNX1-like gene expression signature was confirmed by RNA-Seq (n=6). No relapses or deaths were reported within this subgroup. As previously reported, ABL-class fusions (n=25) were associated with an inferior outcome compared to other subgroups (RR=63% and OS=50%). Patients with rearrangements of CRLF2 (n=52) and ZNF384 (n=37) had similar outcomes to the cohort overall. Image: Summary/Conclusion: A combination of techniques has classified a representative cohort of B-other-ALL patients from UKALL2003 into clinically relevant subgroups. The identification of DUX4-r, subgroup defining mutations and fusion partner genes demonstrates the value of NGS-based approaches. We have confirmed the good and poor prognostic associations of DUX4-r and ABL-class fusions, respectively, and identified ETV6-RUNX1-like subgroup to have a good prognosis. P344: THE DUAL BCL-2 AND BCL-XL INHIBITOR AZD4320 SHOWS ON-TARGET ACTIVITY IN ALL AND ACTS SYNERGISTICALLY WITH MCL-1 INHIBITION M. C. Wichert1, A. Niedermayer1, S. Enzenmüller1, K.-M. Debatin1, L. H. Meyer1, F. Seyfried1,* 1Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany Background: Targeting anti-apoptotic BCL-2 family proteins by BH3-mimetics has become a promising treatment strategy in acute lymphoblastic leukemia (ALL). Heterogeneous activity of the selective BCL-2 inhibitor venetoclax has been observed, but other anti-apoptotic proteins including BCL-XL promote venetoclax insensitivity. In clinical trials, targeting BCL-XL has previously resulted in a dose-limiting decrease of platelets. AZD4320 was developed as a dual inhibitor of BCL-2 and BCL-XL, and its dendrimer conjugate (AZD0466) was recently reported to demonstrate anti-tumor activity in hematological cancer models, while showing only a transient thrombocytopenia. Aims: In this study, the anti-leukemia activities of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of MCL-1-selective AZD5991 were evaluated and compared to the effects of other BH3-mimetics (BCL-2-selective venetoclax, BCL-XL-selective A-1331852 and MCL-1-selective S63845). The on-target activity of the inhibitors was functionally characterized and combination effects were analyzed. Methods: Cell viability assays were performed in ALL cell lines and patient-derived xenograft (PDX) samples analyzing half maximal effective concentrations (EC50). Protein complexes and expression of apoptosis regulators were analyzed by immunoprecipitation and immunoblotting. Dynamic BH3 profiling using synthetic BH3-peptides was performed to determine the dependency of ALL cells on BCL-2 family proteins. Combination effects were assessed by dose-response matrix analyses. Results: First, we determined the efficacy of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of the MCL-1 inhibitor AZD5991 for cell death induction in seven B-cell precursor (BCP) ALL cell lines and in a series of 13 PDX samples, showing heterogeneous responses. Interestingly, sensitivities of individual samples to both compounds were not associated with each other. However, we found a significant correlation of sensitivity to the dual inhibitor (AZD4320) with BCL-2 inhibition (venetoclax; N=13; rs=0.56; p=0.049), but no association with BCL-XL inhibition (A-1331852). Analyzing activities of all five BH3-mimetics including venetoclax, we found lowest EC50 values for AZD4320, indicating particular sensitivity for this dual inhibitor as compared to the single inhibitors (p<0.001). Investigating dependencies of ALL cells on BCL-2 family proteins by dynamic BH3 profiling, we found a shift towards MCL-1-dependence upon exposure to AZD4320, while AZD5991 induced an increased combined dependence on BCL-2 and BCL-XL, indicating potential synergistic activity of triple inhibition. Using co-immunoprecipitation analyses, we found that the exposure of ALL cells to AZD4320 reduced binding of both BCL-2 and BCL-XL to BIM, confirming on-target activity. Moreover, AZD5991 reduced binding of BIM to MCL-1. Accordingly, combined treatment with both inhibitors results in the release of BIM and downstream apoptosis signaling. Assessing combinatorial treatment in a primary PDX sample using multi-dose matrix analyses of both inhibitors revealed a positive mean Bliss synergy score of +4.8 indicating synergistic activity. Importantly, the highest synergism was found at low concentrations of both inhibitors, suggesting efficacy at moderate concentrations, which could potentially be achieved in vivo. Summary/Conclusion: In summary, our study demonstrates sensitivity, on-target activity and synergism of the dual BCL-2 and BCL-XL inhibitor AZD4320 with inhibition of MCL-1, thereby providing strong evidence for further clinical evaluation in ALL. P345: AN IMMUNE SCREEN IDENTIFIES 5-NONLOXYTRYPTAMINE AS A NOVEL ANTI-CANCER AGENT CAPABLE OF IMPROVING ANTI-TUMOR IMMUNITY IN COMBINATION WITH ANTI-PD1 THERAPY P. Stachura1,*, W. Liu1, A. Wlodarczyk2, C. Xu1, S. Bhatia2,3, P. A. Lang1, A. Borkhardt2,3, A. A. Pandyra2,3 1Department of Molecular Medicine II; 2Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-Heine-University Dusseldorf; 3German Cancer Consortium (DKTK), Partner site Essen/Düsseldorf, Dusseldorf, Germany Background: Exploiting the immune system, particularly CD8+ T cells to eliminate tumors has revolutionized the treatment landscape. However, despite the evident successes of CD8+ T cell-targeting immunotherapies such as anti-PD1 checkpoint inhibitors, many patients fail to respond especially those with poorly immunogenic tumors. Additional obstacles to successful immunotherapy responses include therapy-induced toxicity, lack of bio-markers of response, an immunosuppressive environment leading to T cell dysfunction and exhaustion and poor immune infiltration highlighting a need for novel combinatorial approaches to augment immunotherapeutic activity. Aims: We aimed to uncover novel therapeutic agents capable of augmenting the anti-tumoral responses of CD8+ T cells in ex vivo systems and in vivo syngenetic poorly immunogenic tumor models. Methods: To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen, using Lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells that were combined tumor cells expressing the LCMV gp33 (B16) peptide CTL epitope and incubated with an NIH 770 compound library. Hits that increased tumor-cell killing when combined with LCMV-primed splenic T cells were further validated ex vivo. The key candidate was assessed in vivo in C57BL/6J syngeneic tumor models. Immunocompromised NSG mice as well as antibody depletion were used validate CD8+ T cell dependence. In vitro and in vivo immunomodulatory effects were deciphered using flow cytometry, immunoblot, CRISPR/Cas9 and histological analyses. Results: We identified 5-Nonyloxytryptamine (5-NL), a serotonin receptor (HTR) agonist, as increasing the ability of T cells to target tumor cells. In vitro, 5-NL induced apoptosis in melanoma and childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) at low micromolar levels. 5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts’ immune system, specifically CD8+ T cells as their depletion abrogated the phenotype (Figure 1A-B). 5-NL’s pro-immune effects were attributed to the upregulation of antigen presenting machinery in melanoma, BCP-ALL and other poorly immunogenic tumors with low MHC-I/HLAA-C expression (Figure 1C). Importantly, the upregulation of MHC-I/HLAA-C occurred without concomitant increases in PD-L1 expression and was linked to upregulation of cAMP Response Element-Binding Protein (CREB) in vitro and in vivo. As demonstrated through CRISPR/Cas9 HTR1D knockout, the immunomodulatory and pro-apoptotic effects of 5-NL occurred independently of receptor target signaling. 5-NL was successfully combined with an anti-PD1 antibody in vivo for maximal tumor growth inhibition. Figure 1: The serotonin agonist 5-Nonyloxytryptamine (5-NL) was identified as potentiating T cell mediated anti-tumor immunity. (A) C57BL/6J or (B) CD8 depleted C57BL/6J mice were subcutaneously injected with 5 x 105 gp33 expressing tumor cells. 7 days post-tumor injection, mice were randomized and treated daily 6.25 mg/kg of 5-NL or vehicle for five consecutive days and tumor volume was measured (n = 9-12). H2-Db (MHC-I, murine cell lines) and HLAA-C (human cells lines) were assessed by FACS analysis following treatment with 5-NL for 24 hours (n = 3-9). Error bars in all experiments indicate SEM; *P < 0.05 as determined by a Student´s t-test (unpaired, 2 tailed). Image: Summary/Conclusion: This study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors and increasing their responsiveness to immunotherapies. P346: THE ROLE OF CYTOKINES, TNF, TGFΒ1 AND NEUTROPHIL-TO-LYMPHOCYTE RATIO IN THE COURSE OF THE ACUTE LEUKEMIA V. Barilka1,*, V. Matlan2, S. Prymak3, O. Shalay4 1Laboratory of Immunology and Cytogenetics of Blood Neoplasms, SI “Institute of Blood Pathology and Transfusion Medicine of National Academy of Medical Science of Ukraine”; 2Associate professor; 3•Department Oncology and Medical Radiology, Lviv National Medical University named Danylo Galytsky; 4Head of the •Laboratory of Immunology and Cytogenetics of Blood Neoplasms, SI “Institute of Blood Pathology and Transfusion Medicine of National Academy of Medical Science of Ukraine”, Lviv, Ukraine Background: Lymphocytes (Lym) and mature segmental neutrophils (Neu) together with blasts are most commonly detected in the peripheral blood (PC) of patients (pta) with acute leukemia (AL). Besides, Neu, Lym and blast cells are an important source of transforming growth factor β1 (TGFβ1), tumor necrosis factor (TNF) production. Accumulation of TNF, TGFβ1 in the plasma of AL pts is considered an unfavorable sign in AL. However, the role of TNF, TGF β1 in the relationship with neutrophil-to-lymphocyte ratio (NLR) in the course of AL is not definitively established. Aims: Determine the role of cytokines, TNF, TGF β1 in the relationship with NLR to assess the course of AL. Methods: 42 adult AL pts were examined before treatment, who at the time of the examination had blast cells in the peripheral blood (52.54 ± 5.63 x 109 / L). The NLR was calculated from peripheral blood cell counts obtained at the time of diagnosis of AL by dividing the Neu count by the Lym count. The median NLR (0.99) was used to dichotomize patients into high- NLR and low- NLR groups. The concentration of TNF, TGF β1 was determined by biological methods using sensitive cell lines L929 and CCL 64, respectively. Statistical processing of the results was estimated by the value of p ˂ 0, 005. The correlation between the indicators was estimated by the value of r. Results: There were 25 people in the low-NLR group. Median age 66 years (36 - 81 years). Among the pts were 9 people with ALL and 16 people with AML. Number of blasts 55, 83 ± 6.38 x 109 / L; New count was 11.73 ± 2.95 109 / L, Lym count - 21.27 ± 3.64 109 / L. The NLR index is 0.53 ± 0.09. In this group, 88% of pts died in the short term without achieving remission, the remaining 12% of pts had short-term remission. The concentration of TNF in plasma was 1.20 ± 0.13 ng / ml, TGF β1 - 4.02 ± 0.48 ng / ml, which in both cases was higher than in healthy individuals (p˂0.001 and p˂0.05, respectively). TNF levels were positively correlated with Lym (r = 0.30) and blasts (r = 0.40), suggesting a possible role for TNF in neoplastic clone survival. There were 17 people in the high-NLR group, including 10 pts with ALL and 7 people with AML who achieved remission. The median age is 38 years (from 16 to 68 years). The number of blasts was 33.20 ± 9.67 x 109 / L and was significantly lower than in the low-NLR group (p˂0.05); New count was 22.23 ± 0.10 x 109 / L; Lym count - 22.83 ± 3.04 109 / L. The NLR was 1.24 ± 0.16. In this group, 94% of pts had remission. Plasma TNF and TGF concentrations were lower than in the low-NLR group, apparently due to lower blast content. However, no statistical difference in cytokine concentration between the compared groups was found. Summary/Conclusion: More favorable prognostic features in AL could be NLR, the median of which is equal to or greater than 0.99. Under these conditions, a decrease in the number of blast cells, the concentration of cytokines TNF and TGF β1 in the plasma and the achievement of remission were observed. The data obtained may indicate a prognostic role of Neu and Lym blood subpopulations in the course of AL and in the relationship with cytokines, TNF and TGF β1, may be potential targets for the correction of treatment of AL pts. P347: COOPERATION OF TLX3 AND FLT3-ITD IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA IS ENHANCED BY TLE4 INACTIVATION Q. Van Thillo1,2,3,*, L. Lauwereins1,2,3, S. Demeyer1,2,3, S. Provost1,2, N. Mentens1,2, C. de Bock4, J. Cools1,2,3 1Center for Human Genetics, KU Leuven; 2Center for Cancer Biology, VIB; 3Leuvens Kankerinstituut, KU Leuven - UZ Leuven, Leuven, Belgium; 4Children’s Cancer Institute, UNSW, Sydney, Australia Background: One fifth of T-cell acute lymphoblastic leukemia (T-ALL) cases is defined by the aberrant expression of TLX3. Being similar to the closely related TLX1, its expression is mainly associated with mutations in the JAK/STAT pathway (Liu et al., Nat Genetics 2017), which have been shown to cooperate with TLX1 in driving T-ALL (Vanden Bempt et al., Cancer Cell 2018). However, it is not known whether TLX3 has the same oncogenic role as TLX1. Additionally, TLX3 expression is also regularly found with FLT3-ITD mutations, which are virtually absent in TLX1-positive cases (Liu et al., Nat Genetics 2017), suggesting there are some important functional differences between TLX3 and TLX1. Aims: To set up a model of cooperation between TLX3 and FLT3-ITD and to elucidate the oncogenic role of TLX3 in T-ALL. Methods: We performed in vivo bone marrow transplant (BMT) assays by transducing lineage-negative cells derived from C57BL/6 mice with the respective oncogenes using retroviruses. Pro-T cell growth curves were performed in the absence of interleukin-7 (Il7) as described previously (Van Thillo et al., Nat Communications 2021). Fluorescence activated cell sorting (FACS) staining was performed on the MACSQuant VYB (Miltenyi Biotec) or Fortessa (BD). RNA was prepared at the KU Leuven Genomics Core and 3’ prime end sequencing (QuantSeq) was performed. Differential gene expression analyses were conducted using the DeSeq2 R package (v1.22.0). Results: The combination of TLX3 and FLT3-ITD in a constitutive BMT assay resulted in a rapid leukemia with an immature myeloid immunophenotype (CD11b+, Gr1-, CD4-, CD8-). The leukemia cells were positive for both FLT3-ITD (GFP) and TLX3 (mCHERRY), indicating cooperation between the two oncogenes in vivo. Similarly, in our in vitro pro-T-cell model, the TLX3 and FLT3-ITD double-positive cells outcompeted the single-positive cells after withdrawal of Il7. Next, we performed RNA-sequencing in CreER (estrogen receptor) pro-T cells 24 hours after activation of TLX3 expression by adding tamoxifen. This showed both up- and downregulation of many genes, including Tle4 among the downregulated genes. Interestingly, TLE4 expression was also lowest in the TLX3-subgroup of patients (Liu. et al. Nat Genetics 2017). TLE proteins are transcriptional co-repressors that can interact with the engrailed-homology 1 (Eh1) domain of transcription factors (Riz et al. Biochem Biophys Res Commun 2009). Therefore, we inserted a point mutation in the Eh1 domain of TLX3, resulting in a phenylalanine to glutamic acid substitution at position 18, to impair this interaction. Strikingly, TLX3(F18E) in combination with FLT3-ITD led to a proliferative advantage in pro-T cells after the omission of Il7 compared to wild-type TLX3. On top of that, cells with TLX3(F18E) and FLT3-ITD were able to grow in the absence of stem cell factor (Scf). Summary/Conclusion: We demonstrate that TLX3 cooperates with FLT3-ITD in vivo and in vitro in a pro-T-cell context. TLX3 downregulates the co-repressor Tle4 and inhibiting the interaction between TLX3 and Tle4 reinforces the proliferative capacity of pro-T cells in the absence of growth factors. P348: MRK-560 AND DEXAMETHASONE ARE SYNERGISTIC IN THE TREATMENT OF T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA C. Vandersmissen1,2,3,*, C. Prieto Fernàndez1, O. Gielen1, K. Jacobs1, I. Govaerts1, J. Maertens4, H. Segers5, J. Cools1 1Human Genetics, KU Leuven; 2Cancer biology, VIB; 3Leuvens Kanker Institutie (LKI), KU Leuven - UZ Leuven; 4Microbiology, Immunology and Transplantation, KU Leuven; 5Oncology, KU Leuven - UZ Leuven, Leuven, Belgium Background: NOTCH1 activating mutations are found in the majority of T-cell acute lymphoblastic leukemia (T-ALL) cases and inhibition of NOTCH1 activation is therefore a potential therapeutic option in T-ALL. Gamma-secretase inhibitors have been shown to block NOTCH1 activation, but are also associated with dose-limiting toxicity, preventing their use for T-ALL treatment. We have recently shown that a more selective inhibition of the gamma-secretases with PSEN1-selective γ-secretase inhibitors, such as MRK-560, retains a strong anti-leukemia effect with minimal gastrointestinal toxicities. Aims: We wanted to investigate if MRK-560 would show synergy with currently used chemotherapeutic drugs, such as dexamethasone, vincristine and doxorubicin, and if it could be safely combined with chemotherapy for treating T-ALL patients. Methods: Dose response curves were performed in DND-41 and SUPT-1 T-ALL cell lines, which were treated with DMSO/MRK-560 alone or in combination with different concentrations of chemotherapy. Apoptosis assay was conducted with annexin-V/PI staining. Human primary T-ALL samples were transduced with a luciferase vector to obtain luciferase positive xenografts in immunodeficient NSG mice that could be used for in vivo treatment. Mice were treated for 3 weeks (5 days on – 2 days off) with vehicle, dexamethasone (5mg/kg, IP), MRK-560 (15mg/kg, IP) or the combination. Leukemia progression was followed by weekly blood sampling and bioluminescent imaging after injection of luciferin. Results: Treatment with MRK-560 increased the sensitivity to dexamethasone in NOTCH1-dependent T-ALL cell lines (DND-41 and SUPT-1) and we documented a strong synergy between these drugs. The combination between MRK-560 and doxorubicin/vincristine showed only an additive effect. Furthermore, we demonstrated that treatment with MRK-560 and dexamethasone increased apoptosis 2.5 fold compared to single dexamethasone treatment. Dexamethasone acts by activating the glucocorticoid receptor (NR3C1) which activates several target genes including BIM, an inducer of apoptosis. Mechanistically, we found that mRNA and protein levels for NR3C1 and BIM were significantly more upregulated after combination treatment compared to single drug treatment. This was associated with strong downregulation of HES-1 levels. These data indicate that MRK-560 synergizes with dexamethasone via downregulation of HES-1 (downstream of NOTCH1), resulting in enhanced glucocorticoid signaling with higher BIM expression levels and apoptosis. Finally, we also studied the synergy between MRK-560 and dexamethasone in patient-derived xenograft models of T-ALL. Bioluminescent imaging showed significant reduction in leukemia progression in combination treatment compared to single treatments. Furthermore, combination treatment (41 days) significantly prolonged survival compared to single MRK-560 (34 days), dexamethasone (30 days) or placebo (16 days) treatment. Summary/Conclusion: We have shown that the PSEN1-selective gamma-secretase inhibitor MRK-560 can be safely combined with chemotherapy for the treatment of T-ALL patients thereby demonstrating a synergistic effect between MRK-560 and dexamethasone. Mechanistically, MRK-560 acts in a similar way as broad spectrum gamma-secretase inhibitors, but does not show gastrointestinal toxicity. P349: CLONAL ARCHITECTURE DISSECTING AT SINGLE-CELL RESOLUTION REVEALS MECHANISMS OF CHEMOTHERAPY RESISTANCE AND RELAPSE IN T CELL ACUTE LYMPHOBLASTIC LEUKEMIA J. Zhang1,*, Y. Duan1, Y. Zhang1, X. Zhu1 1Institute of Hematology & Hospital of Blood Diseases,CAMS &PUMC, Tianjin, China Background: T-lineage acute lymphoblastic leukemia (T-ALL) comprises approximately 10-15% of pediatric ALL cases with distinct feature in biology and largely inferior outcome compared to B-ALL. Growing evidence has reflected pivotal roles of clonal evolution in T-ALL recurrence, but bulk sequencing may not serve as the perfect model to reliably infer clonal heterogeneities and their immunomodulatory milieu during leukemia development. In this study, single-cell sequencing was applied to uncover leukemic clonal relationships with relapse throughout chemotherapy in T-ALL at a more accurate resolution. Aims: We performed bulk and single cell multi omics analysis for diagnostic and relapse T-ALL, with the aim to dissect mechanisms of chemotherapy resistance and relapse in T cell acute lymphoblastic leukemia. Methods: We performed bulk whole-exome sequencing for sorted CD7+ BMMCs from 5 pairs of diagnosis-relapse (Dx_Rel) samples, revealing a series of well-reported hotspot mutations in T-ALL. To dissect clonal diversities within and across the 5 Dx_Rel T-ALL pairs, we carried out high-throughput droplet-based 5’-single-cell RNA-seq (scRNA-seq) and paired T cell receptor sequencing (scTCR-seq). Results: By performing unsupervised clustering of scRNA- seq profiles encompassing 10 samples, we identified 23 distinct T-lineage clusters (Cluster 0-22) based on the two-dimensional UMAP visualization. In 2 out of 5 patients, diffusion map of T-lineage sub-clusters between diagnostic and relapsed samples appeared to be almost identical, while distinct shifts from diagnosis to relapse in the compositions have been observed in the other 3 out of 5 patients. We sought to further deconvolute the clonal architecture trajectory for T-ALL Dx_Rel pairs. We observed that dominant diagnostic clones of 4 patients diminished or vanished at relapse, sparing newly emerged subclones predominantly substituted at relapse. We clearly depicted two distinct patterns of evolutionary trajectories in these 4 Dx_Rel pairs by comprehensively mapping hierarchical TCR clonotypes onto leukemic clonotypes at single cell levels. Specifically, in T956 and T723, we observed significant outgrowth of incidental diagnostic sub-clones at relapse, whereby surrogate TCR repertoires correspondingly enumerated, suggestive of dynamic shifts in dominant clone over continuous chemo-exposure. Whereas in T593 and T856, expanding clones at relapse were showed up with completely different gene signatures from the diagnostic ones, but dominant clones at diagnosis and relapse were surprisingly presented with identical TCR repertoires. This was undoubtedly informative of leukemic “clonal drift” within which hypothetical intrinsic transformation happened to the same subclones over persistent chemotherapy. Image: Summary/Conclusion: Collectively, our presented study accurately distinguished leukemic cells from normal T cells in T-ALL at a single-cell resolution. By tracking transcriptomic profiles within and across Dx_Rel T-ALL pairs, we further identified distinct clonal evolutionary patterns, which may determine diversified fates of leukemic clones in response to therapeutic pressures, extending significant implications for future precise chemotherapies. P350: BLINATUMOMAB AND DONOR LYMPHOCYTE INFUSION (DLI) FOR MOLECULAR RELAPSE AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION IN PEDIATRIC PATIENTS F. Muriano1,*, F. Cacace2, V. Caprioli2, M. R. D’amico2, G. De Simone2, G. Giagnuolo3, G. Menna3, F. P. Tambaro2 1Hematology, AOU Federico II; 2BMT UNIT; 3Hematology, AORN Santobono Pausilipon, Naples, Italy Background: Detection of measurable residual disease (MRD) after Hematopoietic Stem Cell Transplantation (HSCT) is an early predictor of frank relapse. The role of DLI as a pre-emptive treatment to prevent relapse is still debated. Blinatumomab, a CD3/CD19 bispecific T-cell engager molecule, is approved in adult patients with MRD and in children with relapsed or refractory acute lymphoblastic leukemia (ALL). Aims: We evaluated the safety and feasibility of blinatumomab plus DLI to treat MRD-positive pediatricALLafter HSCT. Here, we report our experience of three patients with post-transplant MRD-positive ALL who received blinatumomab and DLI. Methods: Three pediatric patients with high-risk B-ALLwere treated according to AEIOP ALL 2009 and AIEOP ALL 2017 protocols and referred for allo-HSCT. Patients became MRD-positive after allo-HSCT and received blinatumomab with DLI.Patients’ characteristics are summarized in the Table. Results: Patient 1 developed CNS relapse during maintenance, then received salvage therapy according to the INTREALL HR 2010 and 1 course of blinatumomab, achieving MRD-positive CR1.Patient 2, a 16-year old female, had secondary ALL and achieved CR1 after 1 course of chemotherapy and subsequently received 2 courses of blinatumomab, due to chemotherapy-associated toxicity and achieved MRD-undetectable CR1. Patient 3, a 17-year old male was primary refractory to 2 lines of chemotherapy and blinatumomab, received 2 courses of inotuzumab and achieved an MRD-positive complete remission (CR).All patients proceeded to allo-SCT and were MRD-positive at 8, 31 and 14 weeks post-allo-SCT, respectively. All patients discontinued immunosuppressive therapy and received 2, 1 and 4 courses of DLI with blinatumomab, respectively. Blinatumomab was started one week after each DLI, was given at a dose of 15-28cg/kg for a total of 4 weeks each course. Only patient 3 experienced Grade 2 liver GVHD. None of the patients experienced serious adverse events requiring blinatumomab discontinuation. Patient 3 developed isolated CNS relapse after 4 courses of combined DLI plus blinatumomab; patient 2 achieved and remains in MRD-undetectable CR with current follow up and a patient 1 achieved MRD-negative CR after two courses of combined treatment and CNS relapse. Image: Summary/Conclusion: The role of cellular therapy with DLI is still debated in ALL. Most post-transplant relapses are associated withloss of surface HLA bythe leukemia cells, hence DLI alone has limited efficacy in patients with post HSCT relapse. Blinatumomab directs T cells to bind CD19 present on malignant B cells and engages CD3 on T cells causing activation and inducing cytotoxicity against ALL blasts. We, therefore, tested the efficacy of blinatumomab plus DLI in 3 MRD-positive patients with ALL after allogeneic HSCT. The use of blinatumomab allowed recruitment of fit donor-derived T lymphocytes (not exposed to immune suppressive agents) against ALL B cells. This hypothesis is supported by the fact thatpatient 3, who received blinatumomab pre-HSCT and had disease progression, probably due to lack of T cells showed by flow cytometry, achieved MRD-undetectablestatus after receiving DLI plus blinatumomab post-transplant. Twopatients reached stable MRD-undetectablestatusand 2 subsequently developed CNS relapse; none received CNS prophylaxis post-HSCT. We hypothesize that blinatumomab plus DLI can clear the hematologic disease, but it is not effective in preventing CNS relapse. Although very interesting, this hypothesis should be confirmed in a larger number of patients. P351: DOSE-ADJUSTED EPOCH + INOTUZUMAB OZOGAMICIN (DA-EPOCH-INO) IS SAFE AND ACTIVE IN ADULTS WITH RELAPSED/REFRACTORY (R/R) B LYMPHOBLASTIC LEUKEMIA (B-ALL): INITIAL RESULTS OF A PHASE I TRIAL R. Cassaday1,2,3,*, K.-L. Garcia1, T. Gooley2, C. Martino3, M.-E. Percival1,2,3, A. Halpern1,2,3, V. Oehler1,2,3, J. Abkowitz1,2,3, R. Walter1,2,3, C. Ghiuzeli1,3, E. Estey1,2,3 1University of Washington; 2Fred Hutchinson Cancer Research Center; 3Seattle Cancer Care Alliance, Seattle, United States of America Background: Despite new drugs, survival with R/R B-ALL remains poor. Combination therapies may improve outcomes, but none are proven superior, and toxicities may pose limits. InO has been added to low-intensity chemotherapy, but the risks/benefits of such combinations are poorly defined. As we recently found DA-EPOCH to be effective for untreated ALL, we here tested the addition of InO to DA-EPOCH (#NCT03991884). Aims: The primary objective was to estimate the maximum tolerated dose (MTD) of InO when added to DA-EPOCH. Methods: Eligible adults had R/R CD22+ B-ALL with ≥5% blasts in blood/marrow or ≥1 site of extramedullary disease [EMD] ≥1.5 cm in diameter. No history of sinusoidal obstructive syndrome (SOS) or chronic liver disease was allowed. Other key eligibility criteria included bilirubin ≤1.5x upper limit of normal (ULN), AST/ALT ≤2.5x ULN, creatinine ≤1.5x ULN, QTc ≤500 msec, and ECOG 0-2. All patients (pts) gave written informed consent. DA-EPOCH was given on Days 1-5 with G-CSF. Three dose levels (DL) of InO given on Days 8 and 15 were studied in 28-day cycles: DL1, 0.3 and 0.3 mg/m2; DL2, 0.6 and 0.3 mg/m2; and DL3, 0.6 and 0.6 mg/m2 (respectively). In Cycles 2+, EPOCH was dose-adjusted based on the hematologic nadir from the prior cycle. Up to 4 cycles were permitted. Hepatic prophylaxis with ursodiol and CNS-directed therapy with intrathecal chemotherapy was recommended. MTD was the dose of InO yielding a true dose-limiting toxicity (DLT) rate of 33%. Initial dose-escalation plan for InO was a 6 + 6 design, but after 5 pts enrolled, this was modified to Bayesian Optimal Interval Design (BOIN) with target accrual of 24 pts. Secondary objectives were descriptions of treatment-related adverse events (TRAEs) per NCI CTCAE v5; efficacy by rate of complete remission without or with incomplete hematologic recovery (CR/CRi) and response at EMD sites per NCCN; measurable residual disease negativity (MRD-) by multiparameter flow cytometry (MFC) and high-throughput sequencing (HTS; clonoSEQ) of bone marrow aspirate; and survival. Results: To date, 17 pts have been enrolled (Table). All are evaluable for DLT. 5 pts were treated at DL1; then per BOIN, 3 pts at DL2, and 9 pts at DL3. Median number of cycles given was 2 (range, 1-3). There have been 4 DLTs: 1 at DL2 (20%; grade 4 sepsis) and 3 at DL3 (33%; grade 4 hyponatremia, grade 4 SOS, and prolonged pancytopenia). The single case (6%) of SOS occurred after allografting and is ongoing. There have been no treatment-related deaths. Grade 3+ non-heme TRAEs were seen in 13 pts (76%). Those seen in >1 pt were infections (5 pts; 8 events), neutropenic fever (5 pts; 5 events), oral mucositis (3 pts; 4 events), and increased ALT (2 pts; 3 events); all these events resolved. Other hepatic toxicity was generally mild. Of 10 pts who received ≥2 cycles, 5 (50%) escalated the dosing of EPOCH, while 2 (20%) de-escalated. 17 pts are evaluable for response. Of 13 with >5% blasts, 12 (92%; 90% confidence interval 68-100%) achieved CR/CRi, 10 (77%) after 1 cycle. MRD- by MFC was achieved in 9 (75%); 1 had prior InO. In the 5 pts with EMD, 4 (80%) responded including 3 CRs. To date, 6 pts (35%) have undergone allograft post-study. Median follow-up of survivors is 7 mo, with 7 pts in ongoing remission beyond 6 mo. Image: Summary/Conclusion: DA-EPOCH-InO has a manageable toxicity profile with infrequent DLTs and only 1 case of SOS in heavily pre-treated adults with B-ALL. Nearly all pts with >5% blasts at enrollment achieved CR, with most being MRD- by MFC. Responses in EMD were common. Enrollment is ongoing. Updated follow-up and responses by HTS will be presented. P352: SYMPTOMATIC VTE INCIDENCE IN ADULT ALL TREATED WITH PEG-ASPARAGINASE COMPARED TO THE NATIVE L-ASPARAGINASE IN WITH AN ASPARAGINASE-BASED PROTOCOL R. Chen1,*, J. Seki1, L. Liu1, E. Atenafu1, K. Yee1, A. Schuh1, V. Gupta1, S. Chan1, A. Schimmer1, D. Maze1, M. Minden1, H. Sibai1 1University of Toronto, Toronto, Canada Background: Venous thromboembolism (VTE) is a well-known complication in adults receiving aspraginase (ASNase)-based chemotherapy for adults in acute lymphoblastic leukemia, including the ASNase-intensive Dana-Farber Cancer Institute (DFCI) 91-01 protocol. Previous studies have shown an VTE incidence of up to 41% in patients receiving the DFCI protocol and up to 29% with prophylactic anticoagulations. Since 2019, native L-asparaginase is no longer available in the Canada such that pegylated (PEG)-ASNase has gradually replaced native e.coli ASNase (L-ASNase) as part of the DFCI protocol at our centre. Aims: The object of this study is to evaluate the incidence of VTE between patients who received PEG-ASNase vs L-ASNase, with emphasis on the highest risk periods of induction and intensification phases. Methods: A single centred retrospective cohort study of 249 adult patients with Philadelphia chromosome negative (Ph-) between 2011 to 2021 were conducted. There were 178 patients received L-ASNase between 2011 to 2019 and 71 patients received PEG-ASNase between 2018-2021. Of note, all patients received enoxaparin for DVT prophylaxis during the intensification phase. Results: During the induction phase, 19 out of 178 (10.6%) patients who received L-ASNase during induction developed VTE, whereas 21 out of 71 (29.6%) patients with PEG-ASNase developed VTE (p-value = 0.0002, OR = 3.079, 95 CI 1.43-6.64), with multivariable analysis after adjusting for age, sex, weight, and central line types. Similarly, during the intensification phase, 19 out of 135 (14.1%) patients had VTE on L-ASNase while 10 out of 34 (29.4%) patients on PEG-ASNase developed VTE (p-value =0.014, OR= 2.94, 95% CI 1.21-7.13) after adjusting for age, sex, weight, and central line types. Of note, PICC line was noted to be associated with higher incidence of CVC lines with during induction (p-value= 0.021, OR= 2.52, 95% CI 1.15-5.51). Image: Summary/Conclusion: PEG-ASNase is associated with higher incidence of VTE compared to L-ASNase, both during induction and intensification, despite the administration of prophylactic anticoagulation. Further investigations and strategies on reducing VTE risk associated with PEG-ASNase is needed in adult ALL patients who receive an asparaginase-based protocol. P353: FORTY MONTHS UPDATE OF THE GIMEMA LAL2116 (D-ALBA) PROTOCOL AND ANCILLARY LAL2217 STUDY FOR NEWLY DIAGNOSED ADULT PH+ ALL S. Chiaretti1,*, R. Bassan2, A. Vitale1, L. Elia1, A. Piciocchi3, P. Viero2, F. Ferrara4, M. Lunghi5, F. Fabbiano6, M. Bonifacio7, N. Fracchiolla8, P. Di Bartolomeo9, P. Fazi3, M. S. De Propris1, M. Vignetti3, A. Guarini10, A. Rambaldi11, R. Foà1 1Translational and Precision Medicine, Sapienza University of Rome, Rome; 2Hematology, Ospedale dell’Angelo, Venice; 3GIMEMA Data Center, Fondazione GIMEMA – Franco Mandelli Onlus, Rome; 4Hematology, Ospedale Antonio Cardarelli, Naples; 5Translational Medicine, University of Eastern Piedmont, Novara; 6Hematology, Hospital Villa Sofia-cervello, Palermo; 7Medicine, Verona University, Verona; 8Fondazione IRCCS, Ca’ Granda-Ospedale Maggiore Policlinico, Milan; 9Hematology, Transfusion Medicine and Biotechnologies, Ospedale Civile, Pescara; 10Molecular Medicine, Sapienza University of Rome, Rome; 11Hematology and Bone Marrow Transplant Unit, ASST Papa Giovanni XXIII, Bergamo, Italy Background: The outcome of adult Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients greatly improved since the introduction of tyrosine kinase inhibitors (TKIs). A further improvement was achieved with a chemo-free induction/consolidation strategy based on the sequential administration of dasatinib followed by blinatumomab - the D-ALBA GIMEMA LAL2116 trial - with overall survival (OS) and disease-free survival (DFS) rates of 95% and 88% at 18 months (Foa et al, NEJM 2020). Aims: To provide an updated follow-up of the D-ALBA trial and to document the long-term management of previously enrolled patients. Methods: In the D-ALBA trial, after the primary endpoint, patients were followed for 12 months. Data on subsequent treatment and survival are being collected in the ancillary study GIMEMA LAL2217. Results: As reported, 63 patients were enrolled (median age 54 years, 24-82; no upper age limit); the median follow-up is 40 months (0.9-62.5). Since enrollment in the D-ALBA trial, 9 relapses have been documented: 4 hematologic (1 major protocol deviation and 1 in an 82-year old woman who rapidly went off study), 4 at CNS and 1 nodal; median time to relapse was 4.4 months (1.9-25.8). Six deaths were recorded (3 post-allogeneic transplant) in 1st complete hematologic remission (CHR). Of the 58 patients who started blinatumomab, 29 continued treatment with TKI: 21 with dasatinib (85% after receiving all 5 cycles of blinatumomab), 2 switched to imatinib due to intolerance (all after the 5 cycles of blinatumomab) and 5 switched to ponatinib for a molecular minimal residual disease increase or medical decision (66% had received all 5 cycles of blinatumomab). Twenty-nine patients were allografted, including 6 patients in 2nd CHR: 9 from a sibling, 13 from an unrelated donor, 6 from a haploidentical donor and 1 from a cord blood. Six transplants were performed in 2nd CHR. Before transplant, 8 patients received 2 and 3 cycles of blinatumomab, respectively, 5 4 cycles and 6 5 cycles (2 patients were allografted after 1 cycle). Among grafted patients, 6 deaths occurred, 3 of which in cases transplanted in 2nd CHR; thus, the transplant-related mortality in 1st CHR is 10%. When considered in a covariate model, transplant did not impact on both OS and DFS; similar results were obtained also considering allograft only in 1st CHR. It must, however, be underlined that the allografted population was enriched in cases who did not achieve a molecular response (23 pts). Further investigation is ongoing on this aspect. In the updated follow-up, the estimated 48 months OS is 78% (95% CI: 66-92%) and the DFS is 75% (95% CI: 64-87%). DFS was significantly better in patients achieving a molecular response upon induction than in those who did not (100% vs 67%, p=0.016, Fig. 1A), with no events recorded in the group of complete molecular responders and positive-not-quantifiable cases. Furthermore, we confirmed the inferior DFS for patients carrying an IKZF1 plus genotype compared to that of cases with no IKZF1 deletions/IKZF1 deletions alone (85% vs 47%, p=0.012, Fig. 1B). Image: Summary/Conclusion: In the updated analysis of the D-ALBA trial and the ancillary GIMEMA LAL2217 study, with a median OS of 40 months, we confirm the reported very favorable outcome. Among the few relapsed cases, we observed a rather high incidence of CNS involvements. These results represent the scientific rationale for the ongoing phase 3 GIMEMA LAL2820 trial, where dasatinib has been substituted by ponatinib, CNS prophylaxis has been increased, and transplant allocation is based on genomic features and minimal residual disease monitoring. P354: REAL-WORLD ANALYSIS OF THE RISKS OF THROMBOTIC AND BLEEDING EVENTS IN PATIENTS RECEIVING PEGYLATED ASPARAGINASE FOR TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA B. Mautner1, B. Brewer2, J. Mort1, E. Pierce1, F. Ghamsari1, S. Dibenedetto1, E. Morse1, L. Wells1, A. Morris1, C. Allen3, I. Patel4, D. D’Souza4, C. Jackson5, M. Perciavalle6, B. Yelvington6, R. Miller6, K. Abernathy6, K. Walsh6, H. Wolfe7, S. Locke7, D. Reed5, V. Duong4, B. Dholaria6, T. LeBlanc7, R. Shallis3, M. Keng1, B. Horton2, F. El Chaer1,1,* 1Hematology and Oncology; 2Public Health Sciences, Division of Translational Research and Applied Statistics, University of Virginia, Charlottesville; 3Hematology and Oncology, Yale University, New Haven; 4Hematology and Oncology, University of Maryland Medical Center, Baltimore; 5Hematology and Oncology, Wake Forest Baptist Medical Comprehensive Cancer Center, Winston-Salem; 6Hematology and Oncology, Vanderbilt University Medical Center, Nashville; 7Hematology and Oncology, Duke Cancer Institute, Durham, United States of America Background: The prognosis of younger adult patients with acute lymphoblastic leukemia (ALL) has improved with the adoption of pediatric-inspired chemotherapy regimens. PEGylated asparaginase (PEG-Asp) is essential to these regimens but is associated with an increased risk of coagulopathy, venous thromboembolism, and depletion of antithrombin III (AT3). There are limited data evaluating the risk of clinical coagulopathy (thrombosis and bleeding) and the optimal antithrombotic interventions in these patients are unknown. Aims: We aimed to assess the risk of thrombotic and bleeding events in patients with ALL treated with PEG-Asp and compare the treatment modalities with either AT3 + fibrinogen repletion vs the addition of prophylactic anticoagulation (AC) with either subcutaneous heparin 5000 TID or subcutaneous lovenox 30-40mg/day. Methods: We performed a multicenter retrospective analysis of patients ≥ 18 yo who received PEG-Asp for B- or T-ALL as part of multiagent chemotherapy from 2015-2020 treated across 6 US academic institutions. Categorical variables were summarized using means and standard deviations. The former was compared using Fisher’s exact test; the latter was compared using either the Wilcoxon text (for two groups) or the Kruskal-Wallis (for more than two groups) as appropriate. One way ANOVA test was used to compare continuous variables. Results: Our study included 129 treated patients who met eligibility criteria for induction and 73 patients who underwent post-induction therapy. During induction, the majority of the patients (90%) had AT3 and fibrinogen repletion, while only 10% received additional prophylactic anticoagulation. The rates of any thrombotic events were lower in the AT3/cryoprecipitate-only treatment group (18% vs 50%, p=0.015). Venous thromboembolism accounted for the majority of the thrombotic events (91% and 100% in the AT3/cryoprecipitate vs AT3/cryoprecipitate + prophylactic anticoagulation, respectively). Bleeding rates were numerically higher in patients who received AT3/cryoprecipitate + prophylactic anticoagulation (33% vs 14%, p=0.18). The rates of thromboses or bleeding events were not different when either prednisone or dexamethasone were included in the induction treatment regimen. During the post-induction phase, the rates of thrombotic and bleeding events were similar across all groups. The median time to thrombotic event was 11 days after the last PEG-Asp administration during induction, and 123 days during post-induction. Image: Summary/Conclusion: Although the increased thrombotic risk in adult patients receiving PEG-Asp is well-characterized, rates of reported thromboses vary widely. Studies examining bleeding risk associated with PEG-Asp regimens are even scarcer and with more variable outcomes. In our study, there was both a statistically significant increase in the rate of any thrombotic event as well as a trend towards increased bleeding in patients receiving AT3/cryoprecipitate + prophylactic anticoagulation versus those receiving AT3/cryoprecipitate alone. More investigation into the relative thrombotic and bleeding risks associated with PEG-Asp is needed to determine optimal antithrombotic interventions in adult patients with ALL. P355: MINI-HYPER-CVD PLUS INOTUZUMAB OZOGAMICIN, WITH OR WITHOUT BLINATUMOMAB, IN OLDER ADULTS WITH NEWLY DIAGNOSED B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA: UPDATES FROM A PHASE II TRIAL F. Haddad1,*, H. Kantarjian1, N. Short1, F. Ravandi1, N. Jain1, W. Macaron1, T. Kadia1, Y. Alvarado1, N. Daver1, G. Borthakur1, C. DiNardo1, M. Konopleva1, W. Wierda1, J. Jacob1, E. Roy1, C. Loiselle1, A. Milton1, J. Rivera1, R. Garris1, E. Jabbour1 1Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Blinatumomab (Blina) and inotuzumab ozogamicin (INO) improve the outcomes of patients (pts) with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). These drugs may allow for use of less chemotherapy and improve remission durations and overall survival (OS) in older pts with newly diagnosed B-ALL. Aims: To evaluate the combination of low-intensity chemotherapy and INO with or without Blina, in older pts with newly diagnosed B-ALL. Methods: Pts ≥60 years with newly diagnosed Philadelphia chromosome (Ph)-negative B-ALL were eligible to receive mini-hyper-CVD for up to 8 cycles. Initially, INO was given at 1.3-1.8mg/m2 on day (D) 3 of cycle 1 and 0.8-1.3mg/m2 on D3 of cycles 2-4. Rituximab (if CD20+) and prophylactic IT chemotherapy were given for the first 4 cycles. POMP maintenance was given for up to 3 years. Starting with pt #50, the protocol was amended and INO was administered in fractionated doses with each of the 4 cycles of hyper-CVD (0.6 mg/m2 on D2 and 0.3 mg/m2 on D8 of cycle 1; 0.3 mg/m2 on D2 and 8 of cycles 2-4) followed by 4 cycles of blina. The cumulative doses of INO given before and after this amendment were 4.3 mg/m2 and 2.7 mg/m2, respectively. Maintenance was also amended to include 12 cycles of POMP and 4 cycles of blina (1 cycle of blina after every 3 cycles of POMP). Results: 80 pts have been treated with a median age of 68 years (range, 60-87); 30 pts (38%) were ≥70 years. 6 pts were in complete remission (CR) at enrollment and unevaluable for morphological response. Pt characteristics are summarized below (Figure 1A). The overall response rate (ORR) among 74 evaluable pts was 99% (CR, n=66; CRi, n=7). MRD negativity by flow cytometry was achieved in 61/76 pts (80%) after 1 cycle and 74/79 pts (94%) overall. The 30-day and 60-day mortality rates were 0% and 3%, respectively. Among 79 pts who achieved remission, 33 (42%) are still in ongoing remission without stem cell transplant (SCT), 31 (39%) died in remission, 11 (14%) relapsed without SCT, and 4 (5%) proceeded to SCT in first remission (1 of whom subsequently relapsed). Notably, 9 pts (11%) later developed MDS/AML, 7 of whom had a TP53 mutation. Overall, 6 pts (8%) developed VOD, 1 after allogeneic SCT. With a median follow-up of 55 months (range, 3-119), the 5-year continuous remission and OS rates were 76% and 47%, respectively (Figure 1B). Pts aged 60-69 years had better outcomes compared with pts ≥70 years (5-year OS rates: 59% vs 27%, respectively; P=0.04) and as did those without poor cytogenetics (i.e. complex, KMT2A-rearranged, low-hypodiploidy/near-triploidy) compared with poor cytogenetics (5-year OS rates: 54% and 25%, respectively; P=0.02). Deaths in remission were more frequent in pts ≥70 years compared with those 60-69 years (70% vs 35%; P=0.005). The 5-year OS rates for pts age 60-69 without poor-risk cytogenetics (n=37), age 60-69 with poor-risk cytogenetics (n=13), age ≥70 without poor-risk cytogenetics (n=24), and age ≥70 with poor-risk cytogenetics (n=6) were 69%, 39%, 36% and 0% respectively (Figure 1C). Presence of a TP53 mutation did not significantly impact OS (P=0.22). Image: Summary/Conclusion: Reduced-intensity chemotherapy plus INO, with or without blina, resulted in an ORR of 99% and a 5-year OS rate of 47% in older pts with newly diagnosed Ph-negative B-ALL. Outcomes were particularly favorable in those 60-69 years of age without poor-risk cytogenetics. Pts ≥70 years were at a higher risk of death in remission. A chemotherapy-free approach, with INO plus blina only, is therefore now being explored in this population. P356: SUBGROUP ANALYSES OF KTE-X19, AN ANTI-CD19 CHIMERIC ANTIGEN RECEPTOR (CAR) T-CELL THERAPY, IN ADULT PATIENTS WITH RELAPSED OR REFRACTORY B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (R/R B-ALL) IN ZUMA-3 B. D. Shah1,*, R. D. Cassaday2, J. H. Park3, R. Houot4, O. O. Oluwole5, A. C. Logan6, N. Boissel7, T. Leguay8, M. R. Bishop9, M. S. Topp10, D. Tzachanis11, K. M. O’Dwyer12, M. L. Arellano13, Y. Lin14, M. R. Baer15, G. J. Schiller16, M. Subklewe17, M. Abedi18, M. C. Minnema19, W. G. Wierda20, D. J. DeAngelo21, P. Stiff22, D. Jeyakumar23, J. Dong24, S. Adhikary24, L. Zhou24, P. C. Schuberth24, B. Kharabi Masouleh24, A. Ghobadi25 1Moffitt Cancer Center, Tampa, FL; 2University of Washington, Fred Hutchinson, Seattle Cancer Care Alliance, Seattle WA; 3Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 4CHU Rennes, University Hospital Rennes, Inserm & EFS, Rennes, France; 5Vanderbilt University Cancer Center, Nashville, TN; 6UCSF Medical Center, San Francisco, CA, United States of America; 7Hôpital Saint-Louis, Paris; 8Service d’hématologie clinique et thérapie cellulaire Hôpital du Haut-Leveque CHU de Bordeaux, Bordeaux, France; 9The University of Chicago Medicine, Chicago, IL, United States of America; 10Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany; 11University of California San Diego, San Deigo, CA; 12Wilmot Cancer Institute of University of Rochester, Rochester, NY; 13Winship Cancer Institute of Emory University, Atlanta, GA; 14Mayo Clinic, Rochester, MN; 15University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD; 16David Geffen School of Medicine at UCLA, Los Angeles, CA, United States of America; 17Ludwig-Maximilians-Universität München, Munich, Germany; 18University of California Davis Comprehensive Cancer Center, Sacramento, CA, United States of America; 19University Medical Center Utrecht, on behalf of HOVON/LLPC, Utrecht, Netherlands; 20The University of Texas MD Anderson Cancer Center, Houston, TX; 21Dana-Farber Cancer Institute, Boston, MA; 22Loyola University Chicago Stritch School of Medicine, Maywood, IL; 23University of California Irvine Medical Center, Irvine, CA; 24Kite, a Gilead Company, Santa Monica, CA; 25Washington University School of Medicine, St Louis, MO, United States of America Background: Brexucabtagene autoleucel (KTE-X19) is approved in the US for the treatment of adult patients with R/R B-ALL based on the positive results of the ZUMA-3 study. In the pivotal Phase 2 portion of ZUMA-3, the overall complete remission (CR) rate (CR + CR with incomplete hematologic recovery [CRi]) was 71% (N=55; median follow-up 16.4 months; Shah et al. Lancet 2021). Aims: To assess outcomes in ZUMA-3 by prior number of therapy lines, prior blinatumomab, prior allogeneic stem cell transplant (alloSCT), and subsequent alloSCT. Methods: Eligible adults had R/R B-ALL and received a single infusion of KTE-X19 following leukapheresis and conditioning chemotherapy. Outcomes assessed by independent review are reported in all patients treated at the pivotal dose (1×106 CAR T cells/kg). Results: As of 23 July 2021, the median follow-up among all treated patients in Phase 2 (N=55) was 26.8 months (range, 20.7-32.6). At baseline, 10 patients (18%) had 1 prior line of therapy, and 45 patients (82%) had ≥2 prior lines. The overall CR/CRi rates (95% CI) for patients with 1 or ≥2 prior lines of therapy were 90% (55-100) and 67% (51-80), respectively. In patients with 1 or ≥2 prior lines of therapy, the median (95% CI) duration of remission (DOR) was 4.7 months (1.8-not estimable [NE]) and 14.6 months (9.4-NE), and the median (95% CI) overall survival (OS) was not reached (NR) (2.1-NE) and 25.4 months (14.2-NE), respectively. Grade ≥3 cytokine release syndrome (CRS) occurred in 10% and 27% of patients with 1 or ≥2 prior lines of therapy, and Grade ≥3 neurologic events occurred in 30% and 24%, respectively. Among the 25 patients (45%) who had prior blinatumomab at baseline, the overall CR/CRi rate (95% CI) was 60% (39-79), and 80% (61-92) in the 30 patients (55%) without prior blinatumomab. The median (95% CI) DOR was 19.1 months (1.3-NE) and 10.3 months (5.2-NE) in patients with or without prior blinatumomab, and the median (95% CI) OS was 14.2 months (3.2-26.0) and NR (18.6-NE), respectively. In patients with or without prior blinatumomab, rates of Grade ≥3 CRS were 24% and 23%, and rates of Grade ≥3 neurologic events were 20% and 30%, respectively. The overall CR/CRi rate in the 23 patients (42%) who had prior alloSCT vs the 32 patients (58%) who did not was 70% (47-87) vs 72% (53-86), respectively. In patients with vs without prior alloSCT, the medians (95% CI) for DOR were 14.6 months (8.7-23.6) vs NR (4.7-NE), and the medians (95% CI) for OS were 25.4 months (14.2-NE) vs NR (9.0-NE), respectively. Grade ≥3 CRS occurred in 17% and 28% of patients with or without prior alloSCT, and Grade ≥3 neurologic events occurred in 26% and 25%, respectively. For patients who achieved CR/CRi and did (n=10) or did not (n=29) proceed to subsequent alloSCT post–KTE-X19 infusion, the median (95%CI) DOR was NR (NE-NE) and 14.6 months (8.7-23.6), and the median (95% CI) OS was NR (7.6-NE) and 26.0 months (18.6-NE), respectively. A pooled analysis of all Phase 1 and 2 patients treated at the pivotal dose (N=78) was newly conducted, with a median follow-up of 29.7 months (range, 20.7-58.3). The similar results observed in this expanded data set as shown in the Table further support the subgroup outcomes described above in Phase 2 patients. Image: Summary/Conclusion: Adults with R/R B-ALL benefitted from KTE-X19, with manageable safety, regardless of prior exposure to blinatumomab or prior alloSCT, though survival appeared better in patients without these prior therapies and in earlier lines of therapy, with limited patient numbers in some subgroups. roups. P357: MULTICENTER RETROSPECTIVE ANALYSIS OF CLINICAL OUTCOME OF ADULT PATIENS WITH MIXED-PHENOTYPE ACUTE LEUKEMIA (MPAL) DIAGNOSED AND TREATED IN THE LAST TEN YEARS. A CAMPUS-ALL STUDY. D. Lazzarotto1,*, I. Tanasi2, A. Vitale3, M. Piccini4, M. Dargenio5, F. Giglio6, F. Forghieri7, N. Fracchiolla8, M. Cerrano9, E. Todisco10, C. Papayannidis11, M. Leoncin12, M. Defina13, F. Guolo14, C. Pasciolla15, M. Delia16, P. Chiusolo17, A. Mulè18, A. Candoni1, M. Bonifacio2, G. Pizzolo2, R. Foà3 1Clinica Ematologica, Azienda Sanitaria Universitaria Friuli Centrale, Udine; 2Dipartimento di Medicina, Sezione di Ematologia, Università di Verona, Verona; 3Ematologia, Dipartimento di Medicina Traslazionale e di Precisione, “Sapienza” Università di Roma, Roma; 4SOD Ematologica, AOU Careggi, Firenze; 5S.C. Ematologia, Ospedale Vito Fazzi, Lecce; 6Unità di Ematologia e Trapianto di Midollo Osseo, IRCCS Ospedale San Raffaele, Milano; 7S.C. Ematologia, Azienda Ospedaliero Universitaria di Modena, Modena; 8U.O. Ematologia, IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milano; 9S.C. Ematologia 2, AO Città della Salute e della Scienza, Torino; 10Divisione di Ematoncologia, Istituto Europeo di Oncologia, Milano; 11Dipartimento di Oncologia e Ematologia, Policlinico S. Orsola-Malpighi, Bologna; 12U.O. di Ematologia, Ospedale dell’Angelo, Mestre; 13UOC Ematologia, Azienda Ospedaliero Universitaria Senese, Siena; 14Ematologia e Terapie Cellulari, IRCCS Policlinico San Martino, Genova; 15U.O. di Ematologia, IRCCS Istituto Tumori Giovanni Paolo II; 16U.O. Ematologia con Trapianto, Azienda Ospedaliero-Universitaria Consorziale, Policlinico di Bari, Bari; 17Servizio di Ematologia, Policlinico Gemelli, Roma; 18Divisione di Ematologia ad indirizzo oncologico, A.O. Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy Background: Mixed phenotype acute leukemia (MPAL) is a very rare disease in adults. Most data derive from small retrospective series, some including also pediatric patients. Generally, treatment is similar to that of acute lymphoblastic leukemia (ALL), but the outcomes in adults and the role of allogeneic stem cell transplantation (AlloSCT) are not well defined. Aims: Purpose of this retrospective study was to provide data on a large cohort of adult patients diagnosed and treated in 18 Italian hematology centers in the last 10 years. Methods: Seventy-seven adult patients diagnosed with MPAL according to the EGIL and WHO criteria in the last 10 years (2011-2021) and treated with curative intent were included in the study. Endpoints were the CR rate after induction, overall survival (OS), disease-free survival (DFS) and rate of AlloSCT. Results: Forty-eight of the 77 (62%) patients had B/myeloid MPAL, 27 (35%) had T/myeloid MPAL and 2 (3%) had B/T/myeloid MPAL. Median age at diagnosis was 49 years (range 17 - 77); it was lower in patients with T/myeloid MPAL than in B/myeloid MPAL cases (39 vs 54 years, P=0.04). Extramedullary involvement was detected in 26/77 (34%) of patients (mainly lymph nodes and spleen), but none had a central nervous system involvement. Cytogenetic analysis was normal in 40% of patients; 36% of cases had a complex karyotype, 24% had other cytogenetic abnormalities (including 8% with a BCR-ABL1 rearrangement). FLT-ITD mutation was detected in 14% of patients. No differences were found between B/myeloid and T/myeloid MPAL cases. Thirty/77 (39%) of patients were treated with an acute myeloid leukemia (AML)-like induction regimen, while 47 (61%) were treated with an ALL-like induction. Globally, CR rate after induction was 62% and 42% of these cases were also MRD negative. Patients treated with an AML-like induction had a higher rate of refractory disease after induction (33% vs 10%, P=0.02). The death during induction (DDI) rate was similar in the 2 groups (7% vs 8%). In univariate analysis an ALL-like therapy was the only variable associated with a better CR rate (P=0.0447). Patients refractory to AML-like induction were mainly salvaged with a subsequent AlloSCT or ALL-like therapy followed by an AlloSCT, and 60% of them achieved a CR. The median OS and DFS of the entire cohort were respectively 41.9 and 37.6 months, with an OS and DFS at 5 years of 43% and 39%, respectively. Age, type of induction therapy, karyotype, presence of FLT3-ITD mutation, extramedullary involvement and type of MPAL (B/myeloid vs T/myeloid) were tested in univariate analysis for OS and DFS. Age below 60 years was the only variable associated with a better OS (median OS of 67 months vs 26 months, P=0.0138), while no variable impacted on DFS. AlloSCT was performed in 50 patients (65%), 80% of them (40 patients) as part of the frontline treatment (36/40 patients transplanted in CR1). Among transplanted patients, we observed a 5-year OS of 54% (median not reached), that improved to 69% in patients transplanted as part of the frontline treatment. Summary/Conclusion: This study describes one of the largest cohorts of adult patients with MPAL. These data outline that this disease responds better to ALL-like induction therapy than to AML-like therapy and that consolidation therapy should include, whenever possible, an AlloSCT. Prospective studies are needed to uniform the therapeutic approach and to identify variables capable of predicting outcome and to guide therapy. P358: COMPREHENSIVE DIAGNOSTICS OF ACUTE LYMPHOBLASTIC LEUKEMIA BY WHOLE TRANSCRIPTOME RNA SEQUENCING J. Li1,* 1SINO-US Diagnostics, Tianjin, China Background: Acute lymphoblastic leukemia (ALL) has genetic heterogeneity, which is helpful to clinical diagnosis, prognosis stratification and treatment guidance. Gene aberrations are structurally diverse and are currently analyzed by different assays. Aims: This study aims to establish a single and comprehensive platform for ALL diagnosis by whole transcriptome RNA sequencing (WTRS), which can detect fusion, mutations and expression. Methods: We analyzed the data of 70 ALL patients by WTRS. Results: We found that the fusion coincidence rate of WTRS was 100% (19 / 19) comparing with quantitative real-time polymerase chain reaction (qPCR). At the same time, six rare fusion forms were detected, which increased the diagnosis rate of fusion gene from 27% (19 / 70) to 36% (25 / 70). The expression of CRLF2 gene in 5 cases was consistent with that of qPCR. Seven cases with IKZF1 gene skipping identified by qPCR were consistent with the results of WTRS, and a rare IKZF1 deletion subtype was detected. Of the 51 genes associated with ALL, 100% (103/103) gene mutation at the RNA level by WRTS is consistent with the results at the DNA level by targeted DNA sequencing (except 3 splice site mutations and 4 mutations with poor coverage). Summary/Conclusion: In conclusion, the standardization of experimental operation and improvement of biological information pipline of WRTS are very important factors in its use in standard diagnostic procedures. WRTS has the potential to provide accurate comprehensive diagnostic information for clinic and further improve the ALL diagnostic rate. P359: IMPROVED OVERALL SURVIVAL AND MRD CLEARANCE WITH BLINATUMOMAB VS CHEMOTHERAPY AS PRE-TRANSPLANT CONSOLIDATION IN PEDIATRIC HIGH-RISK FIRST-RELAPSE B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL) F. Locatelli1,*, G. Zugmaier2, C. Rizzari3, J. Morris4, B. Gruhn5, T. Klingebiel6, R. Parasole7, C. Linderkamp8, C. Flotho9, A. Petit10, C. Micalizzi11, Y. Zeng4, R. Desai12, W. Kormany4, C. Eckert13, A. Möricke14, M. Sartor15, O. Hrusak16, C. Peters17, V. Saha18,19, L. Vinti1, A. von Stackelberg20 1IRCCS Ospedale Pediatrico Bambino Gesù, Sapienza, University of Rome, Rome, Italy; 2Amgen Research (Munich), Munich, Germany; 3MBBM Foundation, ASST Monza, University of Milano-Bicocca, Monza, Italy; 4Amgen Inc., Thousand Oaks, United States of America; 5Universitaetsklinikum Jena, Jena; 6Universitätsklinikum Frankfurt am Main, Frankfurt, Germany; 7Azienda Ospedaliera di Rilievo Nazionale Santobono Pausilipon, Napoli, Italy; 8Medizinische Hochschule Hannover, Hanover; 9Universitätsklinikum Freiburg, Freiburg, Germany; 10Hopital Armand Trousseau, APHP.Sorbonne Université, Paris, France; 11Istituto Pediatrico di Ricerca e Cura a Carattere Scientifico G Gaslini, Genova, Italy; 12IQVIA Inc., Durham, United States of America; 13Charité Campus Virchow-Klinikum Pädiatrie m.S. Onkologie/Hämatologie, Berlin; 14Univ.-Klinikum Schleswig-Holstein, Kiel, Germany; 15Westmead Hospital, Sydney, Australia; 16Charles University, Motol University Hospital, Prague, Czechia; 17St Anna Children’s Hospital, Vienna, Austria; 18Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom; 19Tata Translational Cancer Research Centre, Tata Medical Center, Kolkata, India; 20Charite Universitaetsmedizin CVK Berlin, Berlin, Germany Background: Children with high-risk first-relapse B-ALL have a poor prognosis. We previously reported that, as compared with chemotherapy, treatment with blinatumomab resulted in prolonged event-free survival (EFS) and overall survival (OS) and higher rates of minimal residual disease (MRD) remission in a phase 3 trial (JAMA 2021;325:843-54; ASH 2021:Abs 1231). Aims: Here we provide follow-up (FU) data as of Sep 2021, with a focus on survival and MRD response. Methods: In this phase 3 trial (Amgen NCT02393859), children >28 days and <18 years old with high-risk first-relapse B-ALL were randomized 1:1 after induction and 2 cycles of consolidation to receive either a third intensive multidrug chemotherapy consolidation or blinatumomab (15 μg/m2/day, 4 weeks, continuous IV infusion). Study enrollment required M1 (<5% blasts) or M2 (≥5% and <25% blasts) bone marrow (BM) at randomization. Patients with complete remission (CR, ie, M1 BM) after treatment could undergo allogeneic hematopoietic stem cell transplant (alloHSCT). EFS, the primary endpoint, was calculated from randomization to whichever occurred first of relapse or M2 BM after CR, failure to achieve a CR at the end of treatment, second malignancy, or death (any cause). Survival outcomes and response rates were analyzed by baseline MRD, ie, MRD at enrollment prior to chemotherapy or blinatumomab. Parents or a legally acceptable representative provided written informed consent. Results: Enrollment was stopped based on the interim analysis for benefit of blinatumomab (7/17/19 primary analysis). The following results reflect an updated analysis as of Sep 2021. Between Nov 2015 and Aug 2019, 111 patients were randomized: 54 (49%) to blinatumomab and 57 (51%) to chemotherapy at 47 centers in 13 countries. Baseline characteristics were comparable in both arms as previously reported. After a median FU of 44 months, EFS was significantly higher with blinatumomab vs chemotherapy (at 4 years: 59% vs 27%, stratified log-rank p<0.001, hazard ratio [HR]: 0.35, 95% CI: 0.20-0.61). Blinatumomab also showed a strong benefit for the secondary endpoint of OS (at 4 years: 77% vs 49%, stratified log-rank p=0.002, HR: 0.34, 95% CI: 0.17-0.69). EFS, OS, and MRD remission (<10-4 blasts) were all improved with blinatumomab, both overall and by baseline MRD subgroup (< or ≥10-3) (Table 1a-b, Figure). Further, patients with and without extramedullary disease both had a benefit with blinatumomab. AlloHSCT in second CR was performed in 51/54 patients in the blinatumomab arm and in 39/57 patients in the chemotherapy arm. No new safety signals were identified. Second relapses occurred in 16/54 (30%) patients in the blinatumomab arm and 34/57 (60%) patients in the chemotherapy arm. In the blinatumomab arm, these second relapses were extramedullary in 5/54 patients, CNS in 5 (9.3%), 2 of which were solely CNS. In the chemotherapy arm, these second relapses were extramedullary in 8/57 patients, CNS in 3 (5.3%), 2 of which were solely CNS. CD19-negative relapse was seen in 3 patients in the blinatumomab arm (3/54) and 1 patient in the chemotherapy arm (1/57) prior to any subsequent CD19-directed therapy (ie, after study therapy). Image: Summary/Conclusion: In children with high-risk first-relapse B-ALL, treatment with 1 cycle of blinatumomab vs chemotherapy before alloHSCT resulted in superior EFS and improved OS and MRD response rates, all independent of baseline MRD. The incidence of CD19-negative relapse after blinatumomab was low. P360: EFFICACY AND SAFETY OF DARATUMUMAB IN PEDIATRIC AND YOUNG ADULT PATIENTS WITH RELAPSED/REFRACTORY T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA OR LYMPHOBLASTIC LYMPHOMA: RESULTS FROM PHASE 2 DELPHINUS STUDY A. Vora1,*, T. Bhatla2, D. Teachey3, F. Bautista4,5, J. Moppett6, P. Velasco Puyó7, C. Micalizzi8, C. Rossig9, N. Shukla10, G. Gilad11,12, F. Locatelli13, A. Baruchel14, C. M. Zwaan4,15, E. A. Raetz16, N. Bandyopadhyay17, L. Lopez Solano18, R. M. Dennis18, R. Carson18, L. E. Hogan19 1Department of Haematology, Great Ormond Street Hospital for Children, London, United Kingdom; 2Department of Pediatrics, Children’s Hospital of New Jersey at Newark Beth Israel Medical Center, Newark, NJ; 3Division of Oncology, Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 4Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands; 5Department of Pediatric Oncology and Hematology, Hospital Niño Jesús, Madrid, Spain; 6Paediatric Haematology, Bristol Royal Hospital for Children, Bristol, United Kingdom; 7Servicio de Oncología y Hematología Pediátricas, Vall d’Hebron Hospital, Barcelona, Spain; 8Clinical Experimental Haematology Unit, IRCCS Istituto Giannina Gaslini, Genova, Italy; 9University Children’s Hospital Münster, Münster, Germany; 10Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 11Department of Pediatric Hematology-Oncology, Schneider Children’s Medical Center of Israel, Petach Tikva; 12Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 13Department of Pediatric Hematology and Oncology, IRCCS Ospedale Pediatrico Bambino Gesù, Sapienza University of Rome, Rome, Italy; 14Pediatric Hematology and Immunology Department, Robert Debré University Hospital (APHP and Université de Paris), Paris, France; 15Department of Pediatric Oncology, Erasmus MC-Sophia Children’s Hospital, Rotterdam, Netherlands; 16New York University Langone Medical Center, New York, NY; 17Janssen Research & Development, LLC, Raritan, NJ; 18Janssen Research & Development, LLC, Spring House, PA; 19Department of Pediatrics, Stony Brook Children’s, Stony Brook, NY, United States of America Background: Approximately 15-20% of pediatric patients with T-cell acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LL) will be refractory to/relapse after frontline treatment; relapsed disease is associated with poor outcomes. In a phase 2 study, 2 of 7 (28.6%) patients with T-cell ALL in first relapse achieved a complete response (CR) using the vincristine, prednisone, PEG-asparaginase, and doxorubicin (VPLD) reinduction backbone. Daratumumab (DARA), a human IgGκ monoclonal antibody targeting CD38 approved for treating multiple myeloma, has shown preclinical efficacy in ALL models. Aims: Here we report the initial results of DARA plus VPLD in pediatric and young adult patients with relapsed/refractory T-cell ALL or LL enrolled in the phase 2, open-label DELPHINUS study. Methods: Eligible patients were aged 1-30 y, had T-cell ALL or LL in first relapse or refractory to 1 prior induction/consolidation regimen, and had a performance status ≥70. DARA (16 mg/kg IV QW) was given with VPLD in Cycle 1 and with methotrexate, cyclophosphamide, cytarabine, and 6-mercaptopurine in Cycle 2. Patients received age/risk-adjusted intrathecal therapy. Response was measured at the end of each cycle by local bone marrow morphology. The primary endpoint for ALL patients was CR rate in pediatric patients at the end of Cycle 1. Patients achieving CR after Cycles 1 or 2 could proceed to allogeneic HSCT off study. Overall response rate (ORR) was defined as CR or CR with incomplete hematological recovery (CRi) at any time before start of subsequent therapy or HSCT. Minimal residual disease (MRD) negativity (<0.01%) at any time before disease progression, start of subsequent therapy, or HSCT was centrally reviewed by flow cytometry. Results: Twenty-four pediatric (age 1-17 y) and 5 young adult (age 18-30 y) ALL patients and 10 LL patients (age 1-30 y) received ≥1 DARA dose. Median (range) age was 10.0 (2-25) y (ALL) and 14.5 (5-22) y (LL); median (range) time from initial diagnosis to first study treatment was 2.0 (0.1-6.1) y (ALL) and 0.8 (0.5-6.0) y (LL). Pediatric ALL patients received a median (range) of 2 (1-3) treatment cycles; young adult ALL patients and LL patients each received 2 (1-2). Among pediatric ALL patients, 10 (41.7%) patients (90% CI, 24.6-60.3) achieved CR at the end of Cycle 1. ORR was 83.3% (CR, 13 [54.2%] patients; CRi, 7 [29.2%] patients) in pediatric and 60.0% (all CR) in young adult ALL patients and 40.0% (all CR) in LL patients. Ten (41.7%) pediatric ALL patients achieved MRD negativity. All pediatric ALL patients had a grade 3/4 TEAE. No pediatric ALL pt discontinued DARA primarily due to AEs and 1 (4.2%) died due to TEAEs (brain edema and hepatic failure) attributed to study treatment but unrelated to DARA. Summary/Conclusion: The addition of DARA to VPLD in pediatric and young adult patients with relapsed/refractory T-cell ALL or LL showed initial activity, generating improved response rates compared to those achieved with backbone therapy alone, with a manageable safety profile. P361: EARLY MEASURABLE RESIDUAL DISEASE ANALYSIS IS ONLY PREDICTIVE OF SURVIVAL OUTCOMES AFTER TWO CYCLES OF TREATMENT IN ADULTS WITH B-LYMPHOBLASTIC LEUKEMIA RECEIVING HYPER-CVAD INDUCTION. R. Nedumannil1,2,*, D. Ritchie3,4, A. Bajel3,4, A. P. Ng3,5, S. J. Harrison3,4, D. Westerman1,4 1Department of Pathology, Peter MacCallum Cancer Centre, Melbourne; 2Department of Diagnostic Haematology, Royal Melbourne Hospital, Parkville; 3Department of Clinical Haematology, Peter MacCallum Cancer Centre and The Royal Melbourne Hospital, Melbourne; 4Sir Peter MacCallum Department of Oncology, The University of Melbourne; 5Immunology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia Background: Measurable residual disease (MRD) monitoring using multi-parametric flow cytometry (MFC) has a well-established role in B-lymphoblastic leukemia (B-ALL). However, the optimal time-point (TP) for early MRD testing and the associated prognostic impact remain undefined in adult B-ALL patients receiving Hyper-CVAD induction chemotherapy, a dose-intensive regimen comprising alternating A cycles (hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone) and B cycles (methotrexate and cytarabine). Aims: To evaluate the utility of MRD analysis by MFC after one cycle (TP1) in comparison to that after two cycles (TP2) of induction treatment in adult B-ALL patients receiving frontline Hyper-CVAD chemotherapy. Methods: A retrospective chart review was performed of consecutive patients older than 18 years with newly diagnosed B-ALL who received frontline Hyper-CVAD induction chemotherapy at two Australian tertiary referral centres from 2010-2020. Patient inclusion required data regarding remission and MRD statuses after each 21-day cycle of induction treatment (defined as two [A and B] cycles of Hyper-CVAD chemotherapy). Two flow cytometry panels (a two tube 8-color panel and a single tube 10-color panel) were used; samples were analyzed using Kaluza Analysis Software (Beckman Coulter) on FACSCanto II (BD Biosciences) and Navios EX (Beckman Coulter) cytometers, respectively. The sensitivity of these assays was 0.001%-0.01%. Clinicopathological and survival data [pertaining to overall survival (OS) and event-free survival (EFS)] were collected from review of patient records. Results: 49 of 76 (64%) adult B-ALL patients received frontline Hyper-CVAD chemotherapy and had available MFC MRD assessments at each of the two TPs. 19 patients (39%) had Philadelphia chromosome-positive (Ph+) disease and received concomitant tyrosine kinase inhibitor (TKI). Median times to TP1 and TP2 relative to start of treatment were 21 and 45 days, respectively. At TP1, 40 patients (82%) achieved complete remission (CR), amongst whom 12 (30%) were MRD negative. At TP2, the majority of patients (48, 98%) achieved CR, of whom 23 (48%) were MRD negative. Among those who achieved CR after Hyper-CVAD induction, 25 (52%) received Hyper-CVAD consolidation chemotherapy ± TKI while 24 (50%) proceeded to alloSCT in first CR. After cycle 1A, patients with residual morphological disease compared to those who attained CR had a median OS of 10 months and 44 months, respectively (HR, 6.15; 95% CI, 1.36-27.94; P = 0.019), while the median EFS was 58 months and not reached, respectively (HR, 1.31; 95% CI, 0.33-5.17; P = 0.423). MRD negativity at TP1 was not associated with a significant improvement in either OS (P = 0.426) or EFS (P = 0.335) when compared to patients with MRD positivity. In contrast, MRD negativity at TP2 was associated with significantly higher OS (P = 0·005) and EFS (P = 0.047) over patients with MRD positivity. Multivariate analysis demonstrated that KMT2A-rearrangement and MRD positivity at TP2 were the only factors (including Ph+ status) that correlated with worse EFS and OS. Summary/Conclusion: In the absence of residual morphologic disease, MRD analysis after one cycle of Hyper-CVAD induction chemotherapy does not correlate with survival outcomes when compared to MRD testing after two cycles of Hyper-CVAD in adult B-ALL patients. Although validation with larger studies is required, initial MRD testing after two cycles of Hyper-CVAD induction (i.e. after day 42) promotes appropriate resource utilization and ensures no loss in the prognostic power of early MRD monitoring in adult B-ALL. P362: MINIMAL-LOSS-CYTOMETRY FOR EVALUATION OF CEREBROSPINAL FLUID IN PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA. SINGLE CENTER STUDY. M. Nováková1,2,*, I. Janotová2, E. Vodičková3, A. Houdková3, J. Hanzalová4, M. Vášková1,2, D. Kužílková1,2, E. Mejstříková1,2, L. Šrámková2, J. Starý2, O. Hrušák1,2 1CLIP-Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University; 2Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol; 3Department of Clinical Hematology, University Hospital Motol; 4Department of Immunology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czechia Background: Central nervous system (CNS) involvement at diagnosis of acute lymphoblastic leukemia (ALL) represents a risk factor for CNS relapse. Currently, examination of the cerebrospinal fluid (CSF) is performed and evaluated quantitatively using a counting chamber and qualitatively using cytomorphological analysis of cytospin. Recently, flow cytometry (FC) evaluation of CSF was shown to provide more reliable results and to predict relapse risk (de Haas et al., 2021, Thastrup et al., 2020). Independently of these studies, we applied a lyse-no wash technique aiming at minimizing cell loss during preparation and at improved cell concentration estimate. Aims: Our aim was to compare our findings with morphological criteria and published data and correlate them with clinical outcome in pediatric patients with ALL. Methods: We included pediatric patients with newly diagnosed ALL (04/2014 – 01/2022), whose CSF examination was performed in CLIP laboratory (n=208). Patients were classified as B cell precursor ALL (BCP ALL; 85%) and T ALL (15%) and treated according to following protocols: AIEOP-BFM ALL 2009 (n=118), AIEOP-BFM ALL 2017 (n=71), EsPhALL(n=8), Interfant06 (n=5), off-protocol (n=6). During follow up, 14 patients relapsed (5 CNS incl. combined, 9 non-CNS). CSF (drawn into tube with no fixative agent and processed within 2 hours) was concentrated by centrifugation and incubated with combination of antibodies (CD20/CD10/CD45/CD34/CD19/CD3/Syto41 in BCP ALL and CD4/CD99/CD5/CD3/CD7/CD16 + 56/CD8/CD45/Syto41 in T ALL). Residual erythrocytes were lysed with NH4Cl solution, followed by immediate acquisition on BD FACS Lyric or BD LSRII cytometers. Blasts were quantified to initial CSF volume. Cluster of 20 events was required to assign samples as FC positive (FC+). In parallel, CNS status was concluded according to morphological criteria, being classified as CNS1 (no blasts), CNS2a/b (≤5 WBC/μL with blasts), CNS2c/CNS3 (≥5 WBC/μL with blasts) according to treatment protocol guidelines. Continuous variables were compared with the Mann–Whitney test, survival data were analyzed using Mantel-Cox test. P values<0.05 were considered as significant. Results: Using FC, we identified atypical blasts in 50 out of the 208 analyzed samples (24%). Median of analyzed CSF volume was 367µL (range 143-1033µL), which enabled median sensitivity 0.054 events/µL (range 0.02-0.14ev/µL). Mean blast concentration was 0.47ev/µL (range 0-25ev/µL) in all samples and 2 ev/µL (range 0.01-25ev/µL, median 0.53ev/µL) in FC+ samples. We observed significantly higher blast concentration in patients with T ALL, in patients with CNS2 and CNS3 and those who subsequently relapsed. Patients with FC+ had significantly lower relapse-free survival than FC- patients (55% vs 94%), which provided better separation than CNS status (68% vs.85% for CNS2/3 vs. CNS1, respectively). Image: Summary/Conclusion: Using fresh CSF sample with lyse-no wash protocol enabled us to detect approximately 10x higher blast count (0.24ev/µL in FC+CNS1 and 1.25ev/µL in FC+CNS2/3) than was recently published (0.015ev/µL in FC+CNS1 and 0.073ev/µL in FC+CNS2/3 in Thastrup et al., 2020), and thus it better correlates to cytomorphological data. Interestingly, the proportion of FC+ patients in our cohort is comparable to (24% vs 25%), showing that although in our method we lose fewer cells, we identify the same proportion of FC+ patients. The presented study shows a higher impact of FC positivity on relapse risk than described previously. The disadvantage of our method is a need for a fresh sample, which is investigated locally. Supported by UNCE/MED/015, NU20J-07-00028. P363: TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA DURING COVID-19 E. Paramonova1,*, E. Zhelnova1, E. Misyurina1, E. Baryakh1, E. Karimova1, E. Zotina1, E. Grishina1, D. Gagloeva1, M. Lysenko1 1hematology, moscow city clinical hospital 52, Moscow, Russia Background: During the coronavirus pandemic, the risk of severe COVID-19 and mortality are higher in certain groups, in particular in patients with oncohematological diseases. Acute lymphoblastic leukemia (ALL) is a special group of oncohematological diseases in which mortality in the era of COVID-19 has increased 2-3 times. Currently, there is no consensus on the treatment of ALL during coronavirus infection. Aims: To determine the basic principles and features of the management of patients with ALL during COVID-19. Methods: 46 patients with ALL and COVID-19 (men 52.2%, women 47.8%) aged 18-74 years (median-44.5) were treated at the Moscow City Clinical Hospital 52 on 01.04.20-01.11.21. B-ALL was 58.7% (27 patients), T-ALL - 34.8% (16 patients), biphenotypic - 4.3% (2 patients), not defined - 2.2% (1 patient), Ph-positive ALL - 17.4% (8 patients). The status of the disease of patients upon admission to the hospital differed: debut of ALL - 20 patients (43.5%), remission - 16 patients (34.8%), relapse and refractory course - 10 patients (21.7%). All patients were treated COVID-19 in accordance with the current guidelines for the prevention, diagnosis and treatment of COVID-19 (interleukin 6 inhibitor, anticoagulant and antibacterial therapy, glucocorticoids (GCs), human immunoglobulin (IG) against COVID-19). According to vital indications and with stabilization of the patient’s condition, 18 patients (39.1%) received chemotherapy (CT). Results: There were no deaths in the group of patients with remission of ALL. In patients with the debut of ALL, mortality was 45% (9 patients), in relapse and refractory course - 50% (5 patients) (p=0.005). Mortality in the group who did not receive CT was 35.7%, and in the group who received CT - 22.2%. 6 patients with Ph-positive ALL (75.0%) continued therapy with tyrosine kinase inhibitors (TKI). According to the protocol for the treatment of ALL, full doses of GCs (100%) and anthracyclines (ATC) (100%) were used, lumbar punctures (LP) and intrathecal (IT) injections of CT (100%) were continued. Due to the high risk of thrombotic complications in COVID-19 and asparaginase therapy, anticoagulant therapy was performed (100%). Prevention of pneumocystis pneumonia (PCP) (89.1%), antifungal (37.0%) and antibacterial (87.0%) therapy were carried out in the treatment of COVID-19. With the persistence of COVID-19 and the absence of antibodies to COVID-19, 2 patients received repeated transfusion of human IG against COVID-19. Summary/Conclusion: During the COVID-19 pandemic, patients in remission of ALL coronavirus infection are treated and controlled. Treatment of COVID-19 in patients with ALL is carried out according to general protocols for the treatment of COVID-19, taking into account the peculiarities of nosology (agranulocytosis, high risk of PCP and fungal infection with long-term therapy of GCs, persistence of COVID-19). When the patient’s condition is stabilized, the issue of CT should be decided individually in each case, taking into account all the risks of ALL and COVID-19. During CT, use full doses of GCs, ATC. In patients with mild and moderate COVID-19, continue LP and IT injections of CT, therapy with TKI. P364: DONOR-DERIVED CD19 CAR-T CELLS AND CHEMOTHERAPY PLUS DONOR LYMPHOCYTE INFUSION IN THE TREATMENT OF RECURRENT CD19+ B-ALL AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION X. Tan1, C. Zhang1, L. Gao1, L. Gao1, P. Kong1, Q. Wen1, S. Lou2, X. Zhang1,* 1Medical center of hematology, Xinqiao hospital, Army medical university; 2Department of hematology, the second affiliated hospital, chongqing medical universtiy, Chongqing, China Background: Donor derived CD19 car T cells plus donor lymphocyte infusion is rarely used in the treatment of recurrent CD19 positive B-ALL after allogeneic hematopoietic stem cell transplantation (allo-HSCT) Aims: To compare the efficacy of donor derived CD19 car T cells and chemotherapy plus donor lymphocyte infusion in the treatment of recurrent CD19 positive B-ALL after allogeneic hematopoietic stem cell transplantation (allo-HSCT) Methods: We retrospectively analyzed 43 patients with B-ALL who relapsed after allo-HSCT from March 2016 to November 2019，including 22 patients treated with donor derived CD19-CAR-T cells (CAR-T group) and 21 patients treated with chemotherapy plus donor lymphocyte infusion (Chemo-DLI group). Results: The rates of complete remission (77.3%) in the CAR-T group were significantly higher than those in the Chemo- DLI group (38.1%); The 1- and 2-year leukemia-free survival(LFS) in the CAR-T group and Chemo-DLI group were 54.5% vs 9.5%, 45.5% vs 4.8%, respectively. The 1- and 2-year overall survival(OS) in the CAR-T group and Chemo-DLI group were 68.2% vs 19%, 63.6%vs 9.5%, respectively. Six patients (28.6%) in the Chemo-DLI group developed grade II-IV acute graft-versus-host disease (aGVHD). Two patients (9.1%) with grade I-II aGVHD were found in the CAR-T group. The incidence of CRS in the CAR-T group was 86.4%, the grade I-II CRS was59.1%, the grade III CRS was 27.3%. There was no grade 4 CRS. Two subjects developed ≤grade-2 immune effector cell-associated neurotoxicity syndrome (ICANS). Summary/Conclusion: Compared with chemotherapy plus DLI, donor-derived CD19 CAR-T is more effective and safe in the treatment of recurrent B-ALL after allogeneic HSCT. P365: INCORPORATION OF NELARABINE (NEL), PEGYLATED ASPARAGINASE (PEG) AND VENETOCLAX (VEN) IN THE FRONTLINE THERAPY OF ADULT PATIENTS WITH T-ACUTE LYMPHOBLASTIC LEUKEMIA/T-LYMPHOBLASTIC LYMPHOMA (T-ALL/LBL) F. Ravandi1,*, E. Jabbour1, N. Jain1, T. Kadia1, B. Gautam1, M. Konopleva1, W. Wierda1, J. Burger1, G. Issa1, A. Maiti1, H. Balkin1, M. Kelly1, R. Garris1, P. Kebriaei1, A. Ferrajoli1, G. Garcia-Manero1, Y. Alvarado1, N. Short1, H. Kantarjian1 1The University of Texas - MD Anderson Cancer Center, HOUSTON, United States of America Background: NEL is effective for patients (pts) with relapsed T-ALL/LBL but its incorporation into the front-line therapy has not been investigated. In children, addition of NEL to the induction regimen improved disease-free survival (DFS) but not overall survival (OS) (Dunsmore KP, JCO, 2020). VEN has shown preclinical and clinical activity in relapsed T-ALL/LBL. Aims: We investigated addition of NEL and PEG into the hyperCVAD (HCVAD) regimen in adult pts with T-ALL/LBL. We then explored the safety and efficacy of addition of VEN to this regimen. Methods: Pts with previously untreated or minimally pre-treated T-ALL/LBL were eligible if they had an ECOG PS ≤3, creatinine ≤ 2 mg/dL, bilirubin ≤ 2 mg/dL and ALT/AST ≤ 4 x ULN. Pts received 8 cycles of HCVAD (cycles 1,3, 5, 7) alternating with high dose ara-C and methotrexate (MTX) (cycles 2, 4, 6, 8) at approximately 3 weeks intervals. 2 cycles of NEL (650 mg/m% daily x 5) were initially administered after the 8 cycles (cohort 1) but later moved to after cycle 4 (cohort 2) and then PEG (1500 IU/m2 capped at 3750 IU) was added on day 5 of NEL cycles (cohort 3). More recently, VEN 400 mg daily on the first 7 days of each of 8 cycles of therapy was added (cohort 4) and this was later adjusted to be given for 7 days on the induction cycle and reduced to 3 days per cycle in pts with ETP-ALL or with persistent MRD with no VEN after cycle 1 in all other pts (cohort 5) Pts with ETP-ALL were referred for allogeneic stem cell transplant in CR. After the completion of the intensive phase, all pts in all cohorts received 30 cycles of maintenance with monthly POMP and early intensification with NEL/PEG on cycles 6 and 7 and late intensification with MTX/PEG in cycle 18 and HCVAD on cycle 19. All pts received 8 intrathecal doses of MTX alternating with ara-C and mediastinal radiation was considered in pts with balky mediastinal disease. Results: Between 8/2007 and 12/2021. 120 pts were enrolled in the 5 study cohorts sequentially (cohort 1= 30, 2= 49, 3=17, 4=16, 5=8)(Table). 74 pts had T-ALL, 45 T-LBL and 1 biphenotypic T/myeloid leukemia. Median age was 35 yrs (range 18-78), 93 pts were male (78%) and 106 pts (88%) had PS 0-1. In pts with T-ALL, median bone marrow blast % was 81% (range, 20-97%). 6 pts (5%) had CNS disease. Cytogenetics was diploid in 77 pts (64%), abnormal in 34 (28%), and unavailable in 9 pts (7.5%). Pts were further characterized as thymic (n=53), early T-cell precursor (ETP; n=23), near ETP (N=19) mature (n=11), not otherwise specified (NOS; n=11), and early-non-ETP (n=3). 25 pts had received 1-2 prior cycles of therapy prior to enrollment with 18 having achieved CR. 30-day mortality was 0%. 106 pts (88%) had ≥ one grade 3/4 non-hematological treatment-emergent adverse events (TEAE) with the most frequent grade 3/4 TEAEs being neutropenic infections in 92 pts (77%) and elevated liver enzymes in 25 pts (21%). Overall response rate (CR/CRi/PR) was 97% with CR/CRp in 95/102 pts (93%), PR in 4 (4%), and no response in 3 (3%). The median no. of cycles to response (including negative PET scans) was 1 (range 1-10). CR/CRp rate in T-ALL was 88% and T-LBL was 100%. At a median follow-up of 48 months (mo; 2-168), 79 pts (66%) are alive with a median PFS and OS of 135 and 135 mo (Figure) and 73 pts (61%) remain in CR. Image: Summary/Conclusion: Addition of NEL/PEG cycles to the HCVAD regimen is feasible and may be beneficial. Addition of VEN to each cycle is associated with significant myelosuppression. Lower doses of VEN may benefit selected pts. P366: BLINATUMOMAB ADDED TO PREPHASE AND CONSOLIDATION THERAPY IN NEWLY DIAGNOSED PRECURSOR B-ALL IN ADULTS. A PHASE II HOVON TRIAL A. Rijneveld1,*, P. Gradowska2, M. Bellido3, O. de Weerdt4, A. Gadisseur5, D. Deeren6, L. van der Wagen7, Y. Jauw8, R. Fijnheer9, D. van Lammerren10, D. Selleslag11, S. Halkes12, B. Biemond13, D. Breems14, I. Moors15, G. van Sluis16, M. Bakkus17, C. Homburg18, V. Janda19, V. van der Velden20, J. Cornelissen21 1Hematology; 2HOVON data Center, Erasmus MC, Rotterdam; 3hematology, Univerisity Medical Center Groningen, Groningen; 4Hematology, Sint Antonius Hospital, Nieuwegein, Netherlands; 5Hematology, Universitair ziekenhuis Antwerpen (UZA), Antwerp; 6Hematology, AZ Delta, Roeselare, Belgium; 7Hematology, University Medical Center, Utrecht; 8Hematology, Amsterdam UMC, VU University Medical Center, Amsterdam; 9Hematology, Meander Medical Center, Amersfoort; 10Hematology, Haga Teaching Hospital, The Hague, Netherlands; 11Hematology, St Jan hospital, Brugge, Belgium; 12Hematology, Leiden University Medical Center, Leiden; 13Hematology, Amsterdam UMC, Amsterdam Medical Center, Amsterdam, Netherlands; 14Hematology, ZNA Stuivenberg, Antwerpen; 15Hematology, Universiteits ziekenhuis Gent, Gent, Belgium; 16Hematology, Isala Clinic, Zwolle, Netherlands; 17Hematology, University Hospital Brussels, Brussels, Belgium; 18Immunocytology, Sanquin Diagnostic Services, Amsterdam, Netherlands; 19Immunology, UZ Gent, Gent, Belgium; 20Immunology; 21Hematology, Erasmusm University Medical Center, Rotterdam, Netherlands Background: The bispecific antibody blinatumomab (blina) is approved for patients(pts) with relapsed/refractory precursor B-cell ALL (ALL). It has also shown to be highly effective in MRD+ pts in first line treatment. TABLE Pt characteristics Total, N(%) 71(100%) Age(y), median(range) 53(18-70) Age (y), N(%) ≤40 22(31%) 40-60 29(41%) >60 20(28%) BM blasts (%), N(%) ≤50% 4(6%) >50% 62(87%) Unknown 5(7%) Karyotype, N(%) Ph+ 26/71(37%) KMT2A 2/67(3%) Complex 23/67(34%) Hypodiploidy 5/67(7%) Aims: To evaluate whether blina added in upfront therapy to prephase and after consolidation (cons)-1 would increase MRD negativity measured by qPCR or flowcytometry at a cut-off of <10-4 (primary endpoint). Secondary endpoints included CR, EFS, OS, adverse events and treatment-related mortality. Methods: Pts, 18-70 years(y) old, with newly diagnosed CD19+ ALL (incl. Ph+), were included. Treatment was based on a pediatric inspired protocol (HOVON 70, Rijneveld et al., 2011) with reduced doses of anthracyclines, MTX, etoposide and PEG-ASP for pts ≥40y old. Prephase consisted of 10 days steroids, from day 5 combined with 14 days blina in the standard step-up dosing schedule. After cons-1 and after intensification 2, two 4-week blina courses were added irrespective of MRD. The protocol was amended twice due to toxicity. First, in 2018 the first PEG-ASP administration was omitted. Second, in 2021 doxorubicin, dexamethasone and PEG-ASP were reduced during intensification 1. Rituximab (if CD20+), prophylactic ITs and imatinib (if Ph+) were standard. AlloHSCT was offered to intermediate and high-risk pts. Trial was registered with ClinicalTrials.gov, identifier NCT03541083. Results: Seventy-one pts were enrolled. Pt characteristics are presented (Table). Fifteen pts discontinued treatment before blina cons-1 due to refractory disease (n=8), toxicity (n=7) or death (n=2). In the total study population, 55/71 pts (77%) achieved CR after (blina) cons-1. Among pts still on treatment after cons-1, 55/56 (98%) pts achieved CR, 50/55 (91%) reached MRD negativity. After prephase, CR was already reached in 63% and MRD negativity in 53%. Blina related AEs in prephase were as expected (83% of pts had ≥1 AE and 10% had ≥1 SAE (hepatotoxicity 3 pts, pain lymph node 1, CRS 1, pneumonia 1, renal insufficiency 1)). CRS was observed in 35% of pts, 32% of whom experienced grade 3 and no grade ≥4. During prephase 5 pts discontinued blina; during blina cons-1, 4 pts stopped blina. With a median follow-up of 17,6 months, the estimated 2-y EFS was 64% standard error (SE) ± 7% (≤60y 71% SE ± 9% and >60y 47% SE ± 12%). Overall, 14 (20%) pts died. OS after 2y was 73% SE ± 7% (≤60y 82% SE ± 8% and >60y 52% SE ± 14%) (Figure 1). For pts with Ph+ ALL, 2-y EFS was 88% SE ± 6%and OS also 88% SE ± 7%. For Ph- ALL, 2-y EFS and OS were 53% SE ± 9% and 68% SE ± 9%, resp. Among pts who reached CR on protocol (n=60), 5 (8%) had relapse, 6 (10%) died and 6 (10%) discontinued treatment due to toxicity. Until now, 22 pts proceeded to alloHSCT and 11 with maintenance. Image: Summary/Conclusion: Blina can safely be added to prephase of an intensified pediatric schedule for newly diagnosed ALL up to 70y of age, albeit with dose reductions for PEG-ASP, doxorubicin and dexamethasone. The combination increases CR and MRD negativity rate. The early addition of blina resulted in very early achievement of MRD negativity with 53% after prephase and 91% after blina cons-1. Further reductions of chemotherapy should be explored (especially for Ph+) if these results are maintained with longer follow-up. P367: LONG TERM OUTCOMES OF NEWLY DIAGNOSED CRLF2 REARRANGED B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA J. Senapati1,*, E. J. Jabbour1, N. J. Short1, F. Ravandi1, P. Kebriaei1, T. M. Kadia1, G. Borthakur1, N. Pemmaraju1, R. S. Garris1, D. Bansal1, S. Konoplev1, S. Wang1, W. Wang1, G. Tang1, K. P. Patel1, M. Konopleva1, N. Jain1 1Leukemia, MD Anderson Cancer Center, Houston, United States of America Background: CRLF2 rearranged B cell acute lymphoblastic leukemia (B-ALL), a subtype of Philadelphia (Ph) -like ALL, constitutes a high-risk subset of B-ALL with poor outcomes with chemotherapy. Targeted therapies such as blinatumomab (blina) or inotuzumab (ino) may improve treatment outcomes for these patients (pts). Aims: To study the outcomes of newly diagnosed CRLF2 rearranged B-ALL treated with hyper CVAD based regimens and the impact of targeted therapies like inotuzumab and blinatumomab Methods: We retrospectively analyzed pts with newly diagnosed B-ALL (diagnosed between 01/2001 and 12/2021) at our center who had documented CRLF2 overexpression. Initial therapy, including use of ino and blina in CR1 were noted. Outcomes measures included CR/CRi, MRD response, relapse free survival (RFS) and overall survival (OS). RFS was censored at allogeneic stem cell transplantation (ASCT). Results: A total of 76 pts with a median age of 38 years (yrs) (range, 18-80) were identified, of which 70% were males and 81% were of Hispanic ethnicity. All pts had overexpression of CRLF2 documented by flow cytometry or gene expression profile. A subset of pts (n=37) had a concomitant CRLF2 FISH performed with all confirming CRLF2 rearrangement. Baseline disease parameters, treatment and outcomes are detailed in Table 1. Sixty-five pts (85%) received Hyper-CVAD based induction therapy [HCVAD-based (n=51); mini-CVD-based (n=14)] and 11 (15%) received augmented BFM. We focus on the outcomes of pts treated with HCVAD/mini-HCVD (n=65); among these pts, 24/65 (37%) received blina in CR1 during consolidation at a median of 3.6 months after starting induction therapy, 22/65 (34%) received ino in CR1 (in C1 as part of mini-HCVD-ino, n=14; in C2 as part of HCVAD, n=8). A total of 14/65 (22%) received both ino and blina in CR1; 32/65 (49%) received ino and/or blina. The median follow-up was 39 months (mos). CR/CRi rate after C1 was 52/65 (80%) with 25/47 (53%) MRD evaluable pts and 25/65 (38%) overall achieving MRD-neg post C1. The median RFS and OS was 17.6 and 26.6 mos, respectively (Fig. 1). Among the 32 pts who received ino and/or blina, the median RFS and OS was 17.6 mos and 38.8 mos respectively. CR/CRp rate after C1 among pts who received mini-HCVD-ino in C1 was 100% (14/14) with 79% MRD-neg. On landmark analysis to the time to blina initiation, blina treated pts had similar RFS and trended for improved OS. A total of 19/65 (29%) had ASCT in CR1; all were MRD-neg prior to ASCT. Landmark analysis for OS based on the time of ASCT (6 mos) in CR1 favored ASCT in CR1 (47.2 vs. 17.6 mos, p=0.04). Table 1: Disease and treatment parameters Parameters N (%) or median [range] (N=76) Age, yrs 38 [18-80] Gender, male 53 (70) Ethnicity Hispanic 62 (81) Baseline parameters WBC (x 109/L) 17 [1-602] Platelets (x 109/L) 39 [3-195] PB blasts (%) 72 [0-98] BM blasts (%) 89 [29-98] CNS positive 10 (80) Cytogenetics and molecular Diploid karyotype 28 (37) CRLF2 mutation (n=31) 4 (13) JAK2 mutation (n=67) 25 (37) JAK1 mutation (n=31) 9 (29) Frontline regimen Augmented BFM 11 (15) HCVAD 51 (67)  Chemotherapy alone 33/51 (65)  with ino alone 2/51 (4)  with blina alone 10/51 (20)  with ino + blina 6/51 (12) Mini-HCVD-ino 14 (18)  with ino alone 6 (43)  with ino + blina 8 (57) Image: Summary/Conclusion: Despite improvements in treatment options, CRLF2 overexpressed B-ALL continue to have inferior outcomes. Earlier initiation of targeted therapies might improve outcomes. P368: A PHASE II STUDY OF INOTUZUMAB OZOGAMICIN FOR THE TREATMENT OF MEASURABLE RESIDUAL DISEASE-POSITIVE B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA J. Senapati1,*, H. Kantarjian1, N. Short1, Y. Alvarado1, J. Burger1, N. Jain1, M. Konopleva1, F. Ravandi1, C. DiNardo1, L. Masarova1, K. Sasaki1, P. A. Thompson1, A. Ferrajoli1, J. O. Jacob1, E. D. Mayor1, A. M. Milton1, C. Loiselle1, R. S. Garris1, E. J. Jabbour1 1Leukemia, MD Anderson Cancer Center, Houston, United States of America Background: Persistence or re-emergence of measurable residual disease (MRD) in B-cell acute lymphoblastic leukemia (B-ALL) is strongly associated with shorter relapse-free survival. Inotuzumab ozogamicin (INO) is an anti-CD22 antibody-drug conjugate with the potential to eradicate MRD in B-ALL. Aims: We aimed to evaluate the efficacy of INO in clearing MRD in patients (pts) with persistent or recurrent MRD positivity after treatment with conventional chemotherapy. Methods: This is a single arm phase II trial of pts with B-ALL in complete remission (CR) who did not achieve MRD negativity (MRD-ve) or had MRD-positive (MRD+ve) relapse after at least 3 months (mos) from the start of frontline therapy (i.e. CR1) or 1 month from the start of any salvage therapy (i.e. ≥ CR2). Eligibility was defined by MRD+ve at ≥ 0.01%. MRD-ve was defined as undetectable MRD by flow cytometry at a minimum sensitivity of 10-4 for Philadelphia (Ph) negative (Ph-) B-ALL and undetectable MRD by both flow and PCR at 10-4 for Ph positive (Ph+) B-ALL. INO was given at a dose of 0.6 mg/m2 on D1 and 0.3 mg/m2 on D8 of C1 and 0.3 mg/m2 on D1 and 8 of subsequent cycles (up to 6 total cycles, given every 21-28 days) along with ursodiol prophylaxis. Pts with Ph+ ALL received concomitant TKI, the choice of which was decided by the treating physician. Results: Between 11/2018 and 1/2022, 20 pts with MRD+ve B-ALL, with a median age of 40 years (range, 19-68) were treated. Twelve pts (60%) had Ph+ B-ALL, 11 of whom received concurrent ponatinib and 1 dasatinib. Eight pts were Ph- (including 3 Ph-like ALL). Fourteen pts (70%) were in CR1, and 6 pts (30%) were in ≥ CR2 (3 in CR2, 2 in CR3 and 1 in CR4). Eleven pts (55%) had received prior blinatumomab (blina) and 4 pts (20%) had prior stem cell transplantation (SCT). The median BCR-ABL1 level in Ph+ B-ALL was 0.58% (range, 0.001-17.5%) and median MRD level by flow cytometry in Ph- B-ALL was 0.2% (range, 0.05-1.24%). Twelve pts (60%) became MRD-ve (responders), 10 after cycle 1 and 2 after cycle 2. In the Ph+ group, 6 pts (50%) responded; another 3 pts attained MMR as best response. Six of 8 (75%) Ph- pts responded. The median number of cycles were 3 (range, 1-6). Seven of 9 pts (78%) without prior blina exposure became MRD-ve, compared with 5/11 (45%) pts with prior blina exposure (p=0.22). Patient disposition is shown in Fig. 1A. Amongst the 12 responders, 5 pts were consolidated with ASCT after a median of 3 cycles (range, 1-3) of INO, all in MRD-ve CR pre-ASCT. Three responders (25%) relapsed (including 1 relapse after ASCT). At a median follow-up of 17 mos, 15 pts (75%) are alive, 8 of whom are in continued MRD-ve CR. The estimated 18 mos RFS and OS were 63% and 73% respectively, for the whole group (Fig. 1B), and the 18 mos OS was 87% and 55% for responders and non-responders, respectively. Outcomes were similar between pts with Ph+ ALL vs. Ph- ALL, prior blina exposure vs. no prior exposure, and pts in CR1 vs. ≥ CR2. INO was overall well-tolerated with no grade 4 non-hematological toxicities. Grade 3 non-hematological toxicities included elevated transaminases in 1 pt and veno-occlusive disease in 1 pt without history of prior ASCT. Ten pts (50%) had grade 3-4 hematological toxicities (primarily neutropenia). Image: Summary/Conclusion: Low-dose, fractionated INO is a well-tolerated option for MRD eradication in Ph+ and Ph- B-ALL pts, including in those with prior blina exposure. P369: A PHASE II STUDY OF MINI-HYPER-CVD PLUS VENETOCLAX IN PATIENTS WITH PHILADELPHIA CHROMOSOME-NEGATIVE ACUTE LYMPHOBLASTIC LEUKEMIA J. Senapati1,*, H. Kantarjian1, N. Short1, M. Konopleva1, F. Ravandi1, N. Jain1, P. A. Thompson1, N. Pemmaraju1, W. G. Wierda1, G. Borthakur1, T. M. Kadia1, G. Garcia-Manero1, M. Yilmaz1, J. Thankachan1, M. Zhao1, C. Loiselle1, M. T. Talley1, M. I. Kwari1, R. S. Garris1, E. J. Jabbour1 1Leukemia, MD Anderson Cancer Center, Houston, United States of America Background: Venetoclax (VEN), an orally active Bcl-2 antagonist, has shown activity in acute lymphoblastic leukemia (ALL) in preclinical models and in combination with navitoclax (BCL-XL inhibitor) in early clinical studies. Combining VEN with other active agents in ALL might synergize this antileukemic effect. Aims: We aimed to evaluate the efficacy and tolerability of VEN added to mini-hyper CVD (mHCVD) chemotherapy in patients (pts) with relapsed/refractory (R/R) ALL. Methods: Pts ≥18 years of age with R/R Philadelphia chromosome negative (Ph-) B- or T-cell ALL/lymphoblastic lymphoma (LBL) were eligible. Pts were required to have a PS of ≤3, bilirubin ≤1.5 mg/dl, AST/ALT ≤3 x ULN and creatinine ≤2 mg/dl. Treatment consisted of mHCVD for up to 8 cycles. VEN was given at a dose of 400 mg daily on D1-14 of C1 and on D1-7 of C2-8. Rituximab (if CD20+) and prophylactic IT chemotherapy x 8 doses were given in C1-C4. Pts with T-cell ALL received an additional 2 cycles of nelarabine (650 mg/m2 daily on D1-5) and peg-asparaginase (1,500 IU/m2 [capped at 3750 IU] on D5), without VEN, during consolidation and another 2 cycles during maintenance. Responding pts received vincristine and prednisone maintenance with VEN daily on D1-14 of each 28-day cycle for up to 2 yrs. Results: From 6/2019 to 2/2021, 20 pts with R/R ALL were treated, 15 (75%) with B-ALL, 4 (20%) with T-ALL (including 1 ETP-ALL) and 1 (5%) with T-LBL. The median age was 45 yrs (range, 20-70). The median lines of prior therapy was 2 (range 1-5). Of the 15 B-ALL pts, 6 had previously received both blinatumomab (blina) and inotuzumab (INO), 7 had received blina without INO, and 2 had received neither. Overall, 11 pts (55%) had undergone prior stem cell transplantation (SCT). Pt disposition is shown in Fig. 1A. One pt was in measurable residual disease (MRD)-positive CR at trial enrollment; all others had active disease and were evaluable for response assessment. Pts received a median of 2 cycles of mHCVD-VEN (range, 1-6). Overall, 12 of 19 evaluable pts (63%) responded (9 CR, 3 CRi), 9 (75%) of whom after 1 cycle. Response in B-ALL was 9/15 (60%) and in T-ALL was 3/4 (75%). Six of the 12 responders (50%) attained MRD negativity (MRD-ve). Of the 12 responders, 4 (33%) underwent SCT, 3 of whom subsequently relapsed and 1 of whom is in continued MRD-ve CR. All of the 8 responders (67%) who did not undergo SCT, subsequently relapsed and died, with a median duration of response of 2.1 mos. Six of the 7 non-responders have died. With a median follow up of 15 months (mos), 5 pts are alive with a median RFS and OS of 6.2 and 7.1 mos respectively (Fig. 1B. The median OS in responders was 8.8 mos (Fig. 1C). Fig.1: (A) Patient disposition and survival, (B) OS and RFS for the entire cohort, (C) OS of responders and non-responders The therapy was generally well-tolerated. There were no grade 4-5 adverse events. Four pts had a grade 3 related event (hyperbilirubinemia and mucositis in 1 pt and transaminitis, fatigue and tumor lysis syndrome in 1 pt each). One pt required VEN dose reduction due to cytopenias. The median times to neutrophil and platelet recovery in C1 in responding pts were 18 and 27 days, respectively. Image: Summary/Conclusion: Low dose combination chemotherapy with VEN in a population of heavily pretreated R/R ALL was well-tolerated and resulted in a response rate of 63%. This study has now been amended to add navitoclax in an attempt to further improve outcomes. P370: EARLY RESULTS OF A SAFETY AND EFFICACY STUDY OF ALLOGENEIC TRUUCAR™ GC502 IN PATIENTS WITH RELAPSED/REFRACTORY B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (R/R B-ALL) S. Li1,*, Z. Yuan1, L. Liu1, Y. Li1, S. Martina2, J. Liu2, Z. Li2, X. Wang2, J. He2, W. Zhao2, L. Shen2, X. Zhang3, S. Wang1 1920th Hospital, Kunming; 2Gracell Biotechnologies Ltd, Shanghai; 3The Second Affiliated Hospital of Army Medical University, Chongqing, China Background: CD19 targeted autologous CAR-T therapies have been approved for the treatment of r/r B-ALL and greatly improved outcome. However, some patients may not be eligible to receive autologous CAR-T. TruUCAR™ GC502 is an allogeneic, universal CAR-T product with CD19/CD7 dual directed CAR. Preclinical data of GC502 were reported at ASH 2021 (Abstract 148500). Aims: Here, we report early clinical results from a phase I open-label, non-randomized, prospective investigator initiate trial (IIT) of GC502 in r/r B-ALL patients to evaluate safety and preliminary efficacy Methods: GC502 is manufactured using leukopaks from HLA-unmatched healthy donors. It contains a 4-1BB based CD19/CD7 dual directing CAR, a T cell enhancer, and genetically disrupted TRAC and CD7 loci to avoid GvHD and fratricide. Patients (pts) with r/r B-ALL were enrolled and treated with one of two different formulations (A or B) in escalating dose levels ranging from 1.0x107 (DL1) to 1.5x107 (DL2) cells/kg. Prior to infusion of GC502, pts received a Flu/Cy based lymphodepletion regimen. Adverse events, disease response and expansion kinetics were evaluated in this study. Results: At date cut off of Feb. 22, 2022, 4 pts (15-34 yrs) had been enrolled into the investigator initiate study of GC502 (NCT05105867). All of patients were heavily pretreated, and had received either autologous or donor derived CD19 or CD19-CD22 targeted CAR-T in prior lines of therapy. Baseline marrow blast levels ranged from 19.5% to 92% (median 48.1%). 1 pt had extramedullary (EM) involvement. At data cut-off all pts had received a single dose of GC502: 1 pt at DL 1 1.0x107cells/kg in formulation A and 3 patients at DL 2 1.5x107cells/kg – out of which 2 were treated with formulation B. At day 28 post CAR-T infusion, 3 out of 4 response evaluable patients had achieved CR/CRi; 1 pt with EM achieved PR at month 1 and subsequently received allo-HSCT on day 39. TEAEs presented as Gr 3 febrile neutropenia (4/4), Gr 4 thrombocytopenia (1/4) and Gr 3 anemia (3/4). All TEAE resolved after treatment with SOC. Non-hematological TEAE presented as Gr≥3 γ-GT increase (3/4), Gr≤3 AST increase (2/4) and Gr≤3 ALT increase (3/4). 2 pts received formulation A and experienced Gr 3 CRS with a duration of 7 and 10 days respectively (CRS was graded according ASTCT Consensus Grading). CRS presented as Gr 2 in the 2 patients with formulation B with a duration of 9 and 15 days respectively. CRS in all pts was manageable and resolved after treatment with Ruxolitinib, SOC and supportive care. No ICANS or aGvHD were observed. The pt treated in DL 1 did not show adequate GC502 cellular expansion. Peak expansions of GC502 in peripheral blood were observed between week 1-2 in DL 2. Median peak CAR copies were 149,945 copies/ug DNA (range 10,849-195,400). Summary/Conclusion: TruUCAR™ GC502 demonstrated promising early results with a manageable safety profile. Robust CAR-T cell expansion was observed in DL2 at 1.5x107cells/kg in heavily pretreated r/r B-ALL patients, including those previously treated with CD19 or CD19-CD22 CAR-T therapies. The study is ongoing and continues accruing patients. P371: HYPER-CVAD WITH SEQUENTIAL BLINATUMOMAB, WITH OR WITHOUT INOTUZUMAB OZOGAMICIN, IN ADULTS WITH NEWLY DIAGNOSED PHILADELPHIA CHROMOSOME-NEGATIVE B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA N. Short1,*, H. Kantarjian1, F. Ravandi1, M. Yilmaz1, T. Kadia1, P. Thompson1, X. Huang2, M. Konopleva1, A. Ferrajoli1, N. Jain1, K. Sasaki1, Y. Alvarado1, G. Borthakur1, C. Dinardo1, M. Ohanian1, W. Macaron1, S. Kornblau1, M. Zhao1, M. Kwari1, C. Loiselle1, R. Delumpa1, A. Milton1, J. Rivera1, S. Lewis1, R. Garris1, E. Jabbour1 1Department of Leukemia; 2Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Blinatumomab and inotuzumab ozogamicin (INO) are highly effective in relapsed/refractory B-cell acute lymphoblastic leukemia (ALL); blinatumomab is also the only approved therapy for eradication of persistent or recurrent measurable residual disease (MRD) after initial ALL therapy. We hypothesized that early incorporation of blinatumomab and INO in patients (pts) with newly diagnosed Philadelphia chromosome (Ph)-negative B-cell ALL would lead to deeper and more durable responses, reduce relapses, and improve survival. Aims: We evaluated the efficacy and safety of hyper-CVAD with sequential blinatumomab, with or without INO, in pts with newly diagnosed Ph-negative B-cell ALL. Methods: Pts 14-59 years of age with newly diagnosed Ph-negative B-cell ALL, including pts who had received no more than 1 prior cycle of chemotherapy, were eligible. Pts were required to have a performance status of ≤3, total bilirubin ≤2 mg/dl, creatinine ≤2 mg/dl, and no significant CNS pathology (with the exception of CNS leukemia). Pts received hyper-CVAD alternating with high-dose methotrexate and cytarabine for up to 4 cycles, followed by 4 cycles of blinatumomab at standard doses. Pts with CD20+ disease (≥1% cells) received 8 doses of ofatumumab (2000 mg) or rituximab (375 mg/m2). Eight doses of prophylactic IT chemotherapy were given. Maintenance was with alternating blocks of POMP (given in maintenance cycles 1-3, 5-7, 9-11, and 13-15) and blinatumomab (given in maintenance cycles 4, 8, and 12). Those with high-risk disease features started blinatumomab after 2 cycles of hyper-CVAD. Beginning with pt #39, INO at a dose of 0.3 mg/m2 on day 1 and 8 was added to the 2 cycles of MTX/Ara-C and to 2 cycles of blinatumomab consolidation (4 total cycles with INO). Results: As of February 2022, 58 pts have been treated (38 without INO and 20 with INO). Pt characteristics are summarized in Table 1. Median age was 34 years (range, 17-59 years). 13 pts were in CR at enrollment. Among 45 pts with active disease at study entry, 100% achieved CR, with 80% achieving CR after the first cycle. MRD negativity by 6-color flow cytometry was achieved in 32/42 evaluable pts (76%) after 1 cycle and 55/58 evaluable pts (95%) overall. Two of the 3 pts who did not achieved MRD negativity were later found to have a NUP214::ABL1 fusion. The median duration of follow-up is 26 months (range, 3-61 months). Overall, 5 pts (9%) did not undergo stem cell transplant (SCT) and subsequently relapsed, 18 pts (34%) underwent SCT in first remission (including 2 additional pts who relapsed post-SCT), 2 pts (3%) died in CR, and 33 pts (57%) remain in continuous remission without SCT. All relapses occurred in pts with ≥1 poor-risk feature(s), and no relapses have occurred beyond 2 years from the start of treatment. For the entire cohort, the 3-year continuous remission and OS rates were 84% and 85%, respectively (Figure 1). No relapses or deaths have occurred in the INO group, and the estimated 1-year OS rate is 100%. Treatment was overall well-tolerated. One pt discontinued blinatumomab due to a related adverse event (grade 2 encephalopathy and dysphasia). No pts discontinued INO due to toxicity, and no cases of veno-occlusive disease have been observed. Image: Summary/Conclusion: Hyper-CVAD with sequential blinatumomab, with or without INO, is highly effective as frontline treatment of Ph-negative B-cell ALL, with an overall MRD negativity rate of 95% and a 3-year OS rate of 85%. Early outcomes with the addition of INO to this regimen are encouraging, with no relapses or deaths observed to date. P372: DISMAL OUTCOMES OF PATIENTS WITH RELAPSED/REFRACTORY B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA AFTER FAILURE OF BOTH INOTUZUMAB OZOGAMICIN AND BLINATUMOMAB W. Macaron1,*, E. Jabbour1, M. Konopleva1, F. Ravandi1, N. Jain1, G. Issa1, T. Kadia1, K. Sasaki1, P. Kebriaei1, M. Yilmaz1, P. Thompson1, K. Takahashi1, H. Abbas1, W. Wierda1, R. Garris1, H. Kantarjian1, N. Short1 1Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: The development of novel monoclonal antibodies such inotuzumab ozogamicin (INO) and blinatumomab (Blina) results in superior response rates and survival in patients (pts) with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL). However, outcomes of pts after failure of both INO and Blina are not well-established, and effective treatment options for these pts are limited. Aims: To determine response rates to subsequent therapies and overall survival (OS) in pts with R/R Philadelphia chromosome (Ph)-negative B-cell ALL after failure of both INO/Blina. Methods: We conducted a retrospective analysis of outcomes in adult pts with Ph-negative B-cell ALL who relapsed or were refractory to both INO/Blina, at least one of which given as salvage therapy. Results: The baseline characteristics of the 65 pts included are shown in Table 1. Of 29 pts with paired flow cytometry for CD19 and CD22 prior to receipt of either INO/Blina, 4 pts (14%) lost CD19 expression and 3 pts (10%) lost CD22 expression at time of INO/Blina failure. After INO/Blina failure, 54 pts (83%) received subsequent therapy, with a median of 2 therapies (range, 1-6). Of 53 evaluable pts, 16 pts (30%) achieved CR/CRi with the first salvage therapy received after INO/Blina failure (CR=9%, CRi=21%), and 22 pts (42%) achieved CR/CRi with at least one salvage therapy (CR=17%, CRi=25%). Of the 22 responders, 16 pts (73%) achieved measurable residual disease (MRD) negativity by flow cytometry (sensitivity=0.01%) at best response. Nine responses (41%) were achieved with an investigational drug/regimen and 13 (59%) were achieved with commercial agent(s). Among 54 pts who received at least one subsequent therapy, 12 pts (22%) received CAR T-cells, and 9 pts (17%) proceeded to hematopoietic stem cell transplant (HSCT). With a median follow-up of 22.3 months, the median OS from the time of INO/Blina failure for the entire population was 3.8 months, and the 1-year OS was 22% (figure 1A). Median relapse-free survival (RFS) was 3.5 months, with a 1-year RFS of 12%. Median duration of response (DOR) to subsequent therapy was 5.0 months, with a 1-year DOR of 17%. Median OS from INO/Blina failure in pts who received salvage therapy compared to those who did not were 4.7 months and 1.4 months, respectively (figure 1B). In the 9 pts who underwent HSCT, 2 (22%) are still alive and in continuous remission. The median OS from time of HSCT was 13.4 months, and the 1-year post-HSCT OS was 59%. In the 12 pts who received CAR-T cells as a subsequent therapy, 6 (50%) responded and 2 were bridged directly to HSCT. The median OS from time of CAR T-cell therapy was 3.9 months, and the 1-year OS was 36%. Outcomes of pts who received both INO and Blina as salvage therapy (n=50), excluding those who received 1 of these agents in the frontline or MRD setting, were also analyzed. Median OS from the time of INO/Blina failure was 2.6 months. Thirty-nine (78%) of these 50 pts received subsequent therapy after INO/Blina failure. Of 38 evaluable pts, 15 pts (39%) achieved CR/CRi with at least one subsequent salvage therapy (CR=13%, CRi=26%). Median RFS and OS in pts who underwent subsequent therapy were 5.9 months and 3.9 months, respectively. Image: Summary/Conclusion: This study highlights the very poor outcomes of pts with R/R B-cell ALL after failure of both INO/Blina. For pts who respond to subsequent salvage therapy, a consolidative approach with SCT may improve outcomes. These data provide a historical reference for expected outcomes that may serve as a benchmark for the evaluation of novel drugs and combinations in the setting of INO/Blina failure. P373: CIRCULATING ENDOTHELIAL PROGENITOR CELLS AND METABOLIC FACTORS IN CHILDHOOD CANCER SURVIVORS E. Athanasopoulos1,*, G. Martimianaki1, P. Iordanis1, M. Stratigaki1, N. Katzilakis1, E. Stiakaki1 1Department of Pediatric Hematology-Oncology and Laboratory of Blood Diseases and Childhood Cancer Biology, University Hospital of Heraklion, Medical School, University of Crete, Heraklion Crete, Greece Background: Circulating Endothelial Progenitor cells (cEPCs) participate in the regulation and maintenance of vascular integrity, balancing the coagulation–anticoagulation mechanisms, as well as the immune response. Obesity and hypertension constitute late effects of chemotherapy in children and numerous studies have shown a decrease in cEPCs number, underlining poor vascular repair capability. Aims: The determination of cEPCs in children treated for Acute Lymphoblastic Leukemia (ALL), Lymphomas (LYM) and solid tumors (ST) and the study of their levels in correlation with patients’ Body Mass Index (BMI) and blood pressure (BP) in different post-treatment time points. Methods: Peripheral blood from children with ALL (n=166), LYM (n=37), ST (n=109) and children without malignancies (Control, n=191) were studied. The cEPCs population was determined by Flow cytometry based on CD34, CD45, CD133, VEGFR2 expression. The BMI and the blood pressure (BP) values were registered and the corresponding percentiles calculated. Patients were divided into three groups: <1 year post-treatment, ≤1-3 years post-treatment, and ≥3 years after completion of treatment. Statistical analysis was performed using t-test (Holm-Sidak) and 2way ANOVA (Tukey’s multiple comparisons test). Results: In ALL group decreased levels of CD34+VEGFR2+ and CD34+CD133+VEGFR2+ were estimated in contrast with the LYM, ST and Control group (p=0,0144, 0,0262 respectively). Decreased levels of both populations were observed immediately after treatment completion in ALL, while in the post treatment period the levels gradually increased. On the contrary, in the LYM and ST group, at completion of treatment higher level of cEPCs were measured which gradually decreased (p= 0,0405 & 0,0026 respectively). The obesity was correlated with higher levels of cEPCs in the ST and LYM compared to the ALL group. The hypertensive patients had elevated cEPCs. Image: Summary/Conclusion: Conditions causing vascular damage such as obesity and hypertension are correlated with increased cEPCs in patients treated for ALL, LYM and ST. The effect of treatment seems to have a different impact in these groups, possibly not only related with the biology of the disease but with the differences of treatment protocols as well. The increase of cEPCs during the 1st post-treatment year probably reflects an effort to repair vascular damage caused by the treatment. Further investigation is needed to clarify these results. P374: BLINATUMOMAB AS CONSOLIDATION FOR ADULT B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA. A REAL-WORLD STUDY I. Urbino1,*, E. Lengline2, M. Cerrano1, F. Rabian2, R. Kim3, M. Sebert2, R. Itzykson2, E. Audisio1, L. Ades2, H. Dombret2, E. Raffoux2, D. Ferrero4, E. Clappier3, N. Boissel2 1Hematology Department, AOU Città della Salute e della Scienza di Torino, Torino, Italy; 2Hematology Department; 3Hematology Laboratory, Saint-Louis Hospital - Université de Paris, Paris, France; 4Department of Molecular Biotechnology and Health Sciences, Division of Hematology, University of Torino, Torino, Italy Background: Blinatumomab is a CD3/CD19 bispecific T-cell engager approved for the treatment of relapsed/refractory (R/R) or minimal residual disease (MRD)-positive B-cell precursor acute lymphoblastic leukemia (B-ALL). Recent phase 3 studies in children and young adults with B-ALL in first relapse suggested that blinatumomab used as consolidation after chemotherapy salvage could be more beneficial than given as single agent in overt relapse. Whereas current strategies aim to demonstrate the benefit of blinatumomab in first line, the optimal use of blinatumomab in adult patients with first B-ALL relapse still deserves to be further explored Aims: To evaluate the efficacy of blinatumomab given as consolidation in adults with B-ALL in a real-world setting Methods: We retrospectively included 115 consecutive patients with B-ALL treated with blinatumomab from April 2012 until June 2021 at Saint-Louis Hospital, Paris, France (n=100), and at AOU Città della Salute e della Scienza, Turin, Italy (n=15). Patients included in clinical trials were excluded. Patients were divided in three subgroups: 68 patients treated in 1st complete remission (CR1), 31 patients treated in 2nd CR after chemotherapy-base salvage therapy (CR2), and 16 patients treated in overt relapse (R/R). Patients in CR1 received blinatumomab for MRD persistence (n=59/68, 87%) or due to inability to receive standard consolidation (n=9/68, 13%, off-label use). The number of blinatumomab cycles along with the use of chemotherapy and/or of allogeneic hematopoietic stem cell transplant (alloHSCT) after blinatumomab was up to physician choice. Results: The median age of patients was 37 years (range, 15-84). Among the 115 patients, 24% (n=28) were Philadelphia (Ph)-positive. Age, sex, baseline disease features and genetic risk categories did not differ between subgroups. After blinatumomab a complete MRD-response was achieved in 83% of CR1 and 86% of CR2 patients (p=.99). A complete remission was reached by 9/15 R/R patients (60%). In the 3 subgroups, the median number of blinatumomab cycles given was 2 (range, 1-6). Forty-six patients (42%) treated in CR (41% CR1, 45% CR2) and 4 R/R patients (25%) were bridged to allo-HSCT in continuous CR after blinatumomab. With a median follow up of 3.1 years, 3-year DFS was 68% in CR1 and 67% in CR2 patients (p=0.41); 3-year OS was 80% in CR1 and 71% in CR2 patients (p=0.32). In R/R patients, 3-year DFS and OS were 13% and 20% respectively. Considering patients who received blinatumomab in CR (CR1+CR2), univariate analysis showed that higher MRD levels both before and after blinatumomab were associated with shorter DFS, while only MRD response to blinatumomab was associated with OS. Both pre- and post-blinatumomab MRD levels retained significance in bivariate analysis for DFS Image: Summary/Conclusion: The present study underlines the efficacy of blinatumomab in consolidation after chemotherapy-based salvage, showing comparable outcomes between patients treated in CR2 and CR1. In CR2 patients, promising DFS and OS were observed as compared to historical cohorts of patients treated with chemotherapy alone or with blinatumomab in overt relapse. Our data suggest that blinatumomab should be preferably used in consolidation rather than as salvage therapy in patients with B-ALL in first relapse P375: CHALLENGES IN MANAGEMENT OF ACUTE LYMPHOBLASTIC LEUKAEMIA IN A RESOURCE CONSTRAINED SETTING IN LOWER-MIDDLE-INCOME COUNTRIES (LMICS) T. Vaid1,*, R. Dhawan1, M. Aggarwal1, P. Kumar1, J. Dass1, G. VIswanathan1, T. Seth1, S. Tyagi1, M. Mahapatra1 1Hematology, All India Institute of Medical Sciences, Delhi, India Background: Improvement in supportive care, introduction of more intensive chemotherapy regimens and strategic use of allogenic stem cell transplant in some patients has resulted in significant improvement in outcomes of patients with acute lymphoblastic leukemia (ALL). However, this improvement is largely restricted to the developed world. We explore these challenges encountered in treating ALL in one of the largest government hospitals in India. Aims: This study aims to assess the baseline characteristics, outcomes and challenges in management of acute lymphoblastic leukaemia in a resource constrained setting in lower-middle-income countries (LMICs) Methods: Consecutive patients diagnosed with acute lymphoblastic leukaemia in the department of Hematology from 1 January 2017 to 31 December 2019 were included. Pediatric patients were treated with standard or augmented BFM protocols depending on risk stratification. Adolescent and yound adults (AYA) were treated with pediatric inspired protocols or GMALL like protocols. Adult patients were treated with GMALL like protocol. Results: Of the 273 patients diagnosed with ALL, only 197 (72%) were able to get admitted for treatment at our institute. Median time from diagnosis to admission was 12.5 days (range: 0 -190 days) and the median distance of the patient’s permanent residence and our institute was 217 km (range 3-2400km). Baseline characteristics of these 197 patients are mentioned in Table 1. Of the 197 patients treated at our centre, only 165 (83.7%) were able to receive induction chemotherapy. Of the remaining 32 patients, 26 (13.2%) died before treatment could be initiated and 6 (3%) were lost to follow up. Patients who died prior to starting chemotherapy had significantly higher incidence of baseline infection and organ dysfunction compared to those receiving induction. Neutropenic fever was present in 50.56% patients at the time of admission. Focus of infection was lungs in 68.5%, sinusitis in 6.8%, skin & soft tissues in 5.6%, neutropenic enterocolitis in 2.2%, pyelonephritis in 1.1%, splenic abscess in 1.1% & bacteraemia with no obvious focus in 14.7% patients. 58.6% of the patients developed a new episode of neutropenic fever during induction chemotherapy. Post induction, a cumulative of 70 episodes of neutropenic fever and other infections were noted in 57 patients, of these 19 patients needed hospitalization. At the end of induction, 64.5% (n=127) patients achieved complete remission. Induction mortality was 27.9% (n=55) and 4.1% (n=8) patients were lost to follow up. Primary refractory disease was noted in 3.5% (n=7) patients. After a follow up period of 4 years, only 22.8% (n=45) patients were alive and on regular therapy, 32% (n=63) patients died & 25.4% (n=50) patients were lost to follow up. CNS relapse & medullary relapse was noted in 7.1% (n=14) & 9.1% (n=18) patients respectively. 4-year event free survival was 57% for both standard risk and high risk paediatric ALL patients respectively, 44% & 35% for standard risk and high risk AYA patients respectively & 20% and 27% for standard risk and high-risk adult ALL patients. Image: Summary/Conclusion: ALL patients in our study had a high incidence of infection and organ dysfunction at baseline that complicated therapy. With limited access to healthcare, poor socio-economic status and high incidence of infection, developing countries face a unique set of challenges. Outcomes of ALL patients in our cohort are at odds with those reported from the developed world. P376: CD38: A FUNCTIONING TARGET IN RELAPSED/REFRACTORY ACUTE LYMPHOBLASTIC LEUKEMIA. LIMITATIONS IN TREATMENT AND DIAGNOSTICS. B. Vakrmanova1,*, M. Novakova1, P. Riha2, M. Zaliova1, E. Fronkova1, E. Mejstrikova1, L. Reznickova Rezkova1, J. Stary2, O. Hrusak1, L. Sramkova2 1Department of Paediatric Haematology and Oncology, CLIP, Second Faculty of Medicine and Motol University Hospital; 2Department of Paediatric Haematology and Oncology, Second Faculty of Medicine and Motol University Hospital, Prague, Czechia Background: The prognosis of relapsed T-acute lymphoblastic leukemia (ALL) is dismal and there is a need for new treatment options. Daratumumab, a monoclonal kappa chain antibody against CD38 is routinely used in multiple myeloma treatment. CD38 is also expressed in malignant cells of most cases with pediatric ALL. Accordingly, daratumumab can be used experimentally in treatment of relapsed ALL but data about efficacy of such a treatment is limited. The loss of CD38 described in myeloma patients can be one of the reasons of treatment failure. Aims: Does daratumumab provide a benefit in relapsed ALL? Can we observe the inability of anti-CD38 mAbs to bind to leukemia cells after daratumumab treatment as described in myeloma patients? Methods: We treated five patients with relapsed ALL with daratumumab between 10/2019 – 10/2021 (four of them for a first relapse of T-ALL and one for a second CD19neg relapse of B cell precursor (BCP) ALL). In three patients, daratumumab was used in combination with chemotherapy, in one patient, chemotherapy was early discontinued for toxicity and the remaining patient received it in monotherapy due to clinical condition. Blast positivity of CD38 by a diagnostic monoclonal antibody (mAb) at relapse was confirmed in all patients before treatment started. Results: Three of five patients responded to daratumumab plus chemotherapy and were in second complete remission (CR2) and underwent stem cell transplantation (SCT). CD38 expression on blasts after daratumumab was not evaluated due to blast elimination in these cases. Two of these patients relapsed with CD38pos ALL 5 and 7 months after SCT, respectively. Third patient is in CR2 one year after SCT. In two of five patients (one with T-ALL and one with a CD19neg BCP ALL) blasts were still detected by flow cytometry after daratumumab. In patient 4 disease progressed under daratumumab treatment. Under the progressing disease patient died 52 days after the relapse. In the other patient, the amount of blasts decreased by one log after the first course of treatment. Nevertheless, the blasts remained on the same log level. Therefore, daratumumab treatment was stopped and replaced by inotuzumab and subsequent SCT, which led to a molecular CR3. We could not detect binding of a diagnostic anti-CD38 (clone T16, HIT2) in neither of the two cases early after the start of daratumumab treatment. We then tested whether the lack of anti-CD38 binding could be caused by steric hindrance with daratumumab molecules as had been described in myeloma patients. We proved our patients had blasts CD38 positive on mRNA level and intracellularly (patient 4). We also detected daratumumab directly on the blasts by detecting its kappa chain on the blasts (patients 4). Moreover, adding daratumumab to blasts of a patient freshly diagnosed with CD38pos BCP ALL blocked anti-CD38 antibody (clone T16) binding. Collectively, the data showed that like in myeloma, daratumumab may block binding of some diagnostic antibodies. We are currently testing the feasibility of multiepitope CYT-38F2 antibody in children with ALL on daratumumab treatment. Summary/Conclusion: In conclusion, daratumumab can lead to CR2 in relapse/refractory ALL, however, the effect is often temporary. Daratumumab administration may result in weeks lasting inability of anti-CD38 mAbs to bind to leukemia cells. Supported by Ministry of Health of the Czech Republic, grants nr. NU20-03-00284 and NU20J-07-00028. P377: GENETIC CHARACTERISTICS AND CD7-CAR-T THERAPY OF TRAD::MYC TRANSLOCATION POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS T. Wang1,2, J. Ni3, H. Zhang3, S. Hui1, T. Liu3, X. Zhang4, G. Zhang4, X. Ma3, X. Wang1, Y. Zhang1, Q. Zhang1, H. Liu1,2,* 1Division of Laboratory Medicine, Hebei Yanda Lu Daopei Hospital, Langfang; 2Beijing Lu Daopei Institute of Hematology, Beijing; 3Division of Laboratory Medicine, Hebei Yanda Lu Daopei Hospital, Langfang; 4department of chemotherapy, Hebei Yanda Lu Daopei Hospital, Langfang, China Background: TRAD::MYC is a recurrent but rare translocation in leukemia and lacks relevantreports. Aims: To investigate the genetic characteristics of acute lymphoblastic leukemia (ALL) with TRAD::MYC translocation and the clinical outcome of chimeric antigen receptor T cells (CAR-T) therapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: A retrospective analysis was performed of 5,450 patients with ALL admitted to our hospital from Apr. 1, 2016, to Dec. 31, 2021. Cases positive for t(8;14)(q24.1;q11.2) in karyotype analysis were selected, TRAD::MYC translocation and CDKN2A/B gene deletion were confirmed by fluorescence in situ hybridization (FISH). The TRAD::MYC positive cases were further screened for a panel of 36 common leukemia fusion genes (FGs), RNA-seq for comprehensive analysis of FGs, and mutation screening of a panel of 86 leukemia driver genes. Results: A total of 12 cases were TRAD::MYC positive, 9 males and 3 females, aged 3-28 years (median 15 years), 9 children (<18 years old) and 3 adults. The diagnosis included 11 T-ALL and 1 B-ALL, and 7 cases (63.6%) manifested high white blood cells (180-486.18×109/L) at the onset. Nine cases had karyotype results at the initial onset, of which three were positive for t(8;14)(q24.1;q11.2), and the rest 6 acquired this translocation at relapse. In the 3 cases with no karyotype results at the initial onset, t(8;14)(q24.1;q11.2) was detected in relapse specimens. Of the 12 cases, 11 showed additional karyotype abnormalities in t(8;14)(q24.1;q11.2) positive specimens. FISH confirmed the involvement of TRAD and MYC genes corresponding to t(8;14)(q24.1;q11.2) in all 12 cases, and CDKN2A/B gene loss was detected in 4 (33%) cases. All cases were screened for fusion genes, and RNA-seq was performed in five cases. Six cases were positive for driver FGs, including 3 STIL::TAL1, 1 KMT2A::AF9 in the B-ALL case, 1 CTCF::NFATC3, and 1 ZC3HAV1::ABL2. Six cases (50%) were positive for PTEN mutation and 3 positive for FBXW7 mutation, other mutated genes including NOTCH1, TP53, CCND3, etc. Eleven cases were followed up for 6-67 months (median 11 months), and 8 died during the follow-up period. Eight cases (72.7%) experienced central nervous system (CNS) leukemia, and 3 had mediastinal invasion. All patients relapsed after chemotherapy, and the leukemia cells proliferated rapidly. Six cases received CD7-CAR-T therapy, and all achieved disease remission after CAR-T cell infusion, of which 1 died from secondary severe lung infection and 2 died from relapse. Four cases underwent allo-HSCT, including 2 salvage transplantation and 2 bridging transplantation after CD7-CAR-T regimen. All 3 cases that remained alive received CD7- CAR-T therapy, 2 of which were bridged to allo-HSCT. Image: Summary/Conclusion: Recurrent t(8;14)(q24.1;q11.2)/TRAD::MYC have a low incidence, mainly in T-ALL cases, but can also be seen in B-ALL. More than half of cases acquired the translocation at relapse and in some cases accompanied with other driver FGs in T-ALL such as STIL::TAL1, KMT2A fusions, and ABL family gene fusions. TRAD::MYC translocation confers aberrant MYC expression and hyperproliferative of tumor cells. Half cases were also accompanied by PTEN mutation, which further adversely affected the prognosis. In these cases, the response to conventional chemotherapy is extremely poor and prone to early relapse, and CNS infiltration is common. CD7-CAR-T regimen bridging to allo-HSCT might be a promising therapeutic strategy for better survival in this subgroup, and the long-term outcomes remain to be further investigated. P378: THE PROGNOSIS FACTORS OF CAR-T THERAPY IN PATIENTS WITH RELAPSED/REFRACTORY B-ALL Y. Wang1, X. Hu1,*, D. Zhang1, Q. Gao1, X. Zhai1, H. Wang1, Y. Gao1, Y. Miao1, Y. Guo1, W. Zhang1, X. Ru1, X. Li2, F. Guan2 1Department of Hematology, Shaanxi Provincial People’s Hospital, Xi’An; 2School of Medicine, Northwest University, Xi’An, China Background: CAR-T therapy showed good clinical efficacy on relapsed/refractory B-ALL (R/R B-ALL) patients. However, some latest studies revealed that most patients tend to relapse during long-term follow-up after CAR-T infusion. Few studies have examined the factors associated with the prognosis of R/R B-ALL patients. Aims: The aim of the study is to investigate the factors associated with the long-term survival of R/R B-ALL patients after CAR-T therapy. Methods: Thirty-eight R/R B-ALL patients were included in the study. Patients received CAR-T cells infusion from May 2015 to August 2018 in Shaanxi Provincial People’s Hospital. Peripheral blood mononuclear cells were collected, and CD3+ cells were sorted. CAR-T cells were prepared by lentivirus. These cells were transfused 3 to 5 days after lymphocyte removal. Bone marrow smears, flow cytometry, and QT-PCR were used to assess the development of disease between 2 and 4 weeks after infusion. The endpoint of follow-up was the time of death or Feb 15, 2022. Cox regression models were used to analyse prognosis factors. Results: The median age of the 38 patients was 25 years (6-59 years), including 21 males and 17 females. 26.3% (10/38) of patients had leukemic cells higher than 30% in their bone marrow smears. There were 34.21% (13/38) of patients with Ph(+), 34.21% (13/38) with extramedullary disease (EMD), 10.52% (4/38) with MLL-AF4 fusion gene (+), and 5.26% (2/38) had received HSCT prior to CAR-T therapy. Within 4 weeks of CAR-T cells infusion, 86.84% (33/38) of patients achieved MRD (-). 13 patients were disease-free by Feb 15, 2022. For all the 38 patients, the median OS and DFS were 19 months and 16.5 months. We found that a higher proportion of leukemic cells in bone marrow, MRD (+) after CAR-T infusion and the presence of the MLL-AF4 fusion gene could predict a worse prognosis. We found that sex, Ph (+), and HSCT after CAR-T infusion is not associated with the long-term survival of patients. Patients who had received maintenance therapy after CAR-T therapy showed better OS (49 months vs 9 months, P<0.001) and DFS (49 months vs 6 months, P<0.001) compared to those who received no maintenance therapy. In addition, we unexpectedly found that patients with EMD showed better OS (49 months vs 15 months, P=0.019) and DFS (49 months vs 12 months, P=0.045) than those without EMD. Summary/Conclusion: R/R B-ALL patients could achieve long-term survival with CAR-T cell therapy. The proportion of leukemic cells in bone marrow, MRD(+), and the presence of the MLL-AF4 fusion gene could predict a worse prognosis of R/R B-ALL patients. We found that maintenance treatment after CAR-T therapy could significantly improve patients’ long-term survival and disease-free time, while HSCT showed no significant benefit to long-term prognosis. P379: MUTATIONAL LANDSCAPE IN THERAPY-RELATED ACUTE LYMPHOBLASTIC LEUKEMIA K. D. Hofer1,*, E. Haralambieva2, C. Fritz2, M. Roncador1, C. Ruetsche3, M. Buehler2, J. Tchinda4, U. Schanz1, C. C. Widmer1,5 1Department of Medical Oncology and Hematology; 2Institute of Pathology; 3Hematology, University Hospital Zurich; 4Oncology Laboratory, University Children’s Hospital Zurich, Zurich; 5Hematology, University Hospital Basel, Basel, Switzerland Background: There is increasing evidence that therapy-related acute lymphoblastic leukemia (t-ALL) with a reported incidence of 3% to 9% is a distinct entity, which is associated with inferior survival compared with de novo ALL (dn-ALL). It appears that this results from a poor cytogenetic predisposition, but data are still very limited due to the rarity of the disease and the mutational landscape has not been adequately explored, as molecular analyses are sparse in this unique patient cohort. Aims: This study attempted to investigate the molecular landscape of patients with an initial diagnosis of ALL and a prior history of malignancy with consecutive genotoxic therapy and to evaluate the cytogenomic results and survival. Methods: Data were collected from 131 adult patients with an initial diagnosis of ALL treated at the Department of Medical Oncology and Hematology, University Hospital Zurich, Switzerland. Patients with prior treatment of another malignancy with alkylating agents, topoisomerase inhibitors, radioactive iodine ablation (for thyroid cancer) and/or irradiation were classified as t-ALL. Results: Within our ALL cohort, 14% of the patients met the criteria for t-ALL. The median latency between genotoxic therapy and diagnosis of t-ALL was 6.5 years, with breast cancer being the most common neoplasm. KTM2A rearrangement was not significantly more common in t-ALL than in dn-ALL (15.7% vs 11%, p = 0.15), a finding which was not as expected from the literature. In therapy-related myeloid neoplasms large chromosomal deletions, such as del(5q) and del(7q) are known to be associated with alkylating agents and topoisomerase II inhibitors. In 12% of our t-ALL patients deletions in chromosome 7 were found, but none in chromosome 5. On the other hand, a high proportion of Philadelphia chromosome (Ph) 36.8% was present in the t-ALL group. On a molecular level, the most frequently observed mutation was KMT2D, followed by myeloid neoplasm-associated mutations of CDKN2A, KRAS and DNTM3A, but no TP53 mutation was found. KRAS mutation was never present in combination with Ph+, but was associated with KMT2A rearrangement. Outcome was particularly poor in Ph+ t-ALL patients compared to Ph+ dn-ALL, an effect that was mitigated by allogeneic stem cell transplantation. Image: Summary/Conclusion: In addition to KMT2A rearrangement as a high-risk genomic subtype, a high proportion of Ph+ was observed in the t-ALL group. Ph+ t-ALL showed a different mutational landscape than KTM2A-mutated patients, suggesting that the development of t-ALL is driven by multiple pathways with particularly bad outcome for patients with Ph+. Although there is overlap in the molecular profile of t-ALL and myeloid neoplasms, no TP53 mutation was found in our t-ALL cohort. The heterogeneity of the genetic aberrations in ALL render molecular genome sequencing of somatic variants an important step to advance our understanding of the disease, especially in those patients without KTM2A rearrangement but Ph positivity. Our findings help further define therapy-associated ALL as a distinct entity, but also highlight the need for a better molecular understanding of its specific disease evolution. P380: EX VIVO IMMUNE ACTIVATION WITH THE MACROPHAGE-TARGETING IMMUNOTHERAPY, ANTI-CLEVER-1 ANTIBODY BEXMARILIMAB, IN ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME S. Aakko1,2,*, A. Ylitalo3, H. Kuusanmäki2, M. Björkman1, J. Mandelin1, J. Jalkanen1, M.-L. Fjällskog1, C. Heckman2, M. Hollmén3, M. Kontro2,4 1Faron Pharmaceuticals Ltd, Turku; 2Institute for Molecular Medicine Finland, HiLIFE, University of Helsinki, Helsinki; 3MediCity Research Laboratory, University of Turku, Turku; 4Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland Background: Despite the recent approvals of targeted therapeutic agents for acute myeloid leukemia (AML), the treatment options remain few and for myelodysplastic syndrome (MDS), even fewer. The potential of immunotherapies in these myeloid malignancies remains under investigation. To this end, bexmarilimab, an anti-Clever-1 antibody, aims to harness the therapeutic potential of macrophages. Clever-1 (STAB1) is a scavenger receptor, expressed on the surface of a subpopulation of immunosuppressive macrophages. By inhibiting Clever-1, bexmarilimab demonstrates potential to turn the anti-inflammatory macrophages to pro-inflammatory and activate CD8+ T cells (Virtakoivu R. et al. 2021. Clin. Cancer Res) along with promising anti-tumor activity against solid tumors in patients with multiple lines of previous treatment (Bono P. et al. 2019. Ann. Oncol.). As Clever-1 is expressed in myeloid cells and high STAB1 levels associate with poor survival in AML (Lin S.Y. et al. 2019. Mol. Ther. Nucleic Acids), bexmarilimab may possess therapeutic potential in AML and MDS. Aims: The aim of this study was to profile Clever-1 expression in AML and MDS and to test in preclinical models bexmarilimab’s growth inhibitory and immunomodulatory potential, as a single agent and in combination with azaciditine and venetoclax. Methods: AML cell lines (n=11) and frozen mononuclear cells, extracted from AML (n=27) and very high risk MDS (n=4) patient bone marrow (BM) aspirates provided by the Finnish Hematology Registry and Biobank, were used in this study. Samples were treated for 48h with bexmarilimab alone, or in combination with azacytidine and/or venetoclax. Flow cytometry was used to detect different myeloid and TBNK cell populations along with Clever-1, HLA-DR, PD-(L)1 and Granzyme B protein expression. Results: Our results confirm Clever-1 protein expression in AML cell lines with high STAB1 level and most importantly, in AML and MDS patient-derived BM monocytes and blasts. AML samples from FAB M4/M5 subtypes exhibited highest Clever-1 levels, along with a FAB M2 sample collected prior to allogeneic stem cell transplantation and consequent rapid relapse. Clever-1 expression correlated negatively with monocyte MHC class II molecule, HLA-DR, expression, and BM T cell frequency, in line with the immunosuppressed state associated with high Clever-1. Ex vivo treatment of the primary AML cells with bexmarilimab resulted in a notable, 5-10x fold increase of monocyte HLA-DR in samples with low basal HLA-DR and high Clever-1. The combination of azacitidine with bexmarilimab augmented HLA-DR induction by 20-110% (mean 44%). On the other hand, combination with bexmarilimab overcame the HLA-DR suppressing effect of venetoclax. The BM TBNK cell populations and thus bexmarilimab’s effect on the effector cells, showed great inter-sample variability. FAB M1/M2 AML showed increase of activation markers, such as Ki67, CXCR3 and Granzyme B after ex vivo bexmarilimab treatment in CD8+ T cells. Furthermore, bexmarilimab reduced PD-1 expression in NK- and CD8+CXCR3+ T cell populations of FAB M0-M2 AML and MDS-EB2. Summary/Conclusion: Our results confirm that Clever-1 is expressed in AML and MDS patient BM blasts and monocytes. Ex vivo treatment with bexmarilimab, alone or in combination with azacytidine or venetoclax, demonstrated increased HLA-DR expression, indicating enhanced antigen presentation capability. Furthermore, NK and CD8+ T cells showed decreased PD-1 and increase of activation markers. These results confirm the therapeutic potential of bexmarilimab in myeloid malignancies and await further validation in clinical trials. P381: DNA METHYLATION PROFILING OF MESENCHYMAL STROMAL CELLS ISOLATED FROM FEMURAL HEAD BONE MARROW VERSUS BONE MARROW ASPIRATES: RELEVANCE FOR AML STUDY BASED CONTROLS A. Abdul-Aziz1,*, C. Weigel2, A. Kovacs2, Y.-Z. Wu2, J. Byrd1, E. Hertlein1, C. Oakes2 1Department of Internal Medicine, University of Cincinnati, Cincinnati; 2Department of Internal Medicine, The Ohio State University, Columbus, United States of America Background: Bone marrow mesenchymal stromal cells (BMSCs) are important precursors for multiple differentiated cell types. Alterations in BMSCs contribute to several disease states, including acute myeloid leukemia (AML) and other hematologic cancers. As BMSCs are used as controls for research and obtained from various bone tissue locations with active hematopoiesis, it is important to understand the developmental capacity and potential differential phenotypes of BMSCs derived from various anatomical sites. Epigenetic regulation plays an essential role in cell lineage specification and development; carefully controlled transcription factor (TF) activity and other chromatin-associated proteins modify the genomic landscape to generate stable, cell type-specific global patterns of gene expression. Aims: We examined genome-wide DNA methylation patterns in BMSCs derived from either femoral head bone marrow or bone marrow aspirates of the iliac crest of healthy donors to identify the optimal control cell population for hematological malignancy research. Methods: Hip-derived BMSCs were obtained from femur heads obtained during hip replacement surgeries (n=19). All patients provided informed consent in accordance with an IRB-approved protocol and the Declaration of Helsinki. Aspirate-derived BMSCs (n=7) were expanded from bone marrow mononuclear cells obtained from a commercial source (AllCells). For genome-wide DNA methylation analyses, the EPIC/850K array platform was used following manufacturer’s protocols (Illumina). Analyses included data obtained from the Gene Expression Omnibus (GEO, GSE79695 and GSE124390) generated from 450K (n= 12) and 850K array data (n=10), respectively. Overlapping probes between the arrays that passed QC were used for downstream analysis. Sex chromosomes and non-CpG probes were removed. The data was reduced to 5,000 most-variable CpGs followed by clustering analysis as described in results. Results: Unsupervised clustering using principal component analysis (PCA) and hierarchical clustering revealed two groups that segregated perfectly between hip-derived versus aspirate-derived BMSCs. There were no observable differences in aspirate-BMSCs generated by our group and those obtained from GEO. Two group supervised analysis (hip versus aspirate) revealed 4,568 CpGs (probes) hypomethylated in hip-derived samples (q<0.05, difference>20%) and 1,433 probes hypermethylated in hip-derived samples (q<0.05, difference>20%). DNA sequence motif enrichment analysis using the HOMER software revealed that hypomethylation in hip-derived BMSCs involved activation of CRE, HOX and AP-1 family TFs. We also observed parallel hypomethylation of the promoters of CREB5, genes from the HOXB and HOXC clusters, and several genes of the MAPK pathway. Hypermethylation in hip-derived BMSCs conversely involved RUNX motifs, corresponding with hypermethylation of the RUNX3 gene. We also observed robust DNA methylation changes at the promoters of other lineage-specifying genes including TBX3, TBX5, PITX2, NR2F2, and EN1. Pathway analysis of genes demonstrating promoter hypomethylation in hip-derived samples revealed strong enrichment for adipocyte progenitors and development. Summary/Conclusion: These findings demonstrate that hip-derived BMSCs are phenotypically distinct from aspirate-derived BMSCs, which may affect their potential use in both therapeutic and research settings where stem-like non-primed BMSCs are required. Our study highlights the importance of using proper selection of control cells to study the role of BMSCs in hematologic malignancies. P382: PTK2B MEDIATES TYROSINE KINASE INHIBITOR DRUG RESISTANCE IN AML ASSOCIATED WITH ALTERED MIGRATION AND ADHESION PROPERTIES C. Allert1,2,*, S. Zimmermann2,3, S. Göllner1, D. Heid1,4,5, M. Bruckmann1, M. Janssen1, B. Besenbeck1, J. Krijgsveld2,3, C. Müller-Tidow1,2,4, M. F. Blank1,3,4 1Medical Department V Hematology, Oncology and Rheumatology, Heidelberg University Hospital; 2University of Heidelberg Medical Faculty; 3Division Proteomics of Stem Cells and Cancer, German Cancer Research Center (DKFZ); 4Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory (EMBL); 5European Molecular Biology Laboratory EMBL, Heidelberg, Germany Background: Tyrosine kinase inhibitor (TKI) therapy is a well-established therapy approach in FLT3-mutated AML. However, TKI resistance occurs frequently while the underlying mechanisms remain incompletely understood. One concept of chemotherapy-resistance in leukemia is cell adhesion mediated drug resistance (CAM-DR). Aims: In this study we aimed to investigate the role of CAM-DR at an early stage of therapy resistance in FLT3-ITD mutated AML. Methods: We characterized FLT3-ITD mutated AML cell lines upon shorter and longer TKI exposure. Emerging TKI resistance was assessed by transcriptome and total proteome analyses, as well as nascent proteomics and MS-based protein stability/degradation assays. Further, we investigated the impact of emerging TKI-resistance on cell adhesion and migration. After identification of a highly expressed protein in TKI resistant cells with implications in cell adhesion, i.e. PTK2B, we investigated the effects of available inhibitors of this protein on AML cell lines in the presence or absence of other TKIs or chemotherapeutic agents and confirmed the effects in primary AML patient samples (n=6). Results: We discovered that protein tyrosine kinase 2b (PTK2B) was induced during early TKI resistance development. Interestingly, PTK2B, a member of the focal adhesion kinase superfamily, was upregulated at the transcriptional and translational level. PTK2B was also post-translationally stabilized in TKI resistant cells. Of note, PTK2B was highly expressed in leukemic stem cells (LSCs) from FLT3-ITD mutated patients. The upregulation of PTK2B was accompanied by increased migration of TKI-resistant cells with concomitant loss of cell adhesion, suggesting altered niche interactions. Notably, treatment with a PTK2B inhibitor, such as PF-431396, reverted the resistance-associated phenotypes. Enhanced migration was abolished upon PF-431396 treatment. Significantly altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon treatment of resistant cells with a PTK2B inhibitor. The FAK/PTK2B inhibitors PF-431396 and Defactinib synergized with several TKIs, such as midostaurin or gilteritinib, and commonly used chemotherapeutic agents, such as daunorubicin, in FLT3-ITD mutated AML cells. Synergism was restricted to FLT3 mutated cells. This synergism was significantly more pronounced in TKI-resistant cells and the addition of a PTK2B inhibitor could overcome TKI resistance to various extent. Also in primary AML patient samples, Defactinib showed lower IC50s specifically for FLT3-ITD mutated patients. Summary/Conclusion: These data establish that FLT3-ITD AML resistance towards TKI depends on PTK2B which alters leukemia cell adhesion and migration. PTK2B inhibitors might be synergistic with TKI in FLT3-ITD AML therapy. PTK2B inhibition could thus prevent the outgrow of drug-resistant clones at an early stage and may improve the outcome of FLT3-ITD mutated AML. P383: USING CF-DNA AS STARTING MATERIAL FOR MRD STUDIES BY NGS IMPROVES THE SENSITIVITY OF THE METHOD AGAINST CTCS. N. Álvarez Sánchez-Redondo1,*, S. Dorado2, A. Martín3, L. Rufián1, V. Garrido1, A. Giménez1, L. Moreno1, Y. Heredia4, J. Martínez-López5, S. Barrio6, R. Ayala7 11. Department of Translational Hematology, Research Institute Hospital 12 de Octubre (i+12), Hematological Malignancies Clinical Research Unit H120-CNIO, CIBERONC; 23.Computer Science and 4.Altum Sequencing Co., Madrid, Spain. Engineering Department. Carlos III University, Madrid and; 34.Altum Sequencing Co., Madrid, Spain.; 44. Altum Sequencing Co., Madrid, Spain.; 51.Department of Translational Hematology, Research Institute Hospital 12 de Octubre (i+12), Hematological Malignancies Clinical Research Unit H120-CNIO, CIBERONC, and 2.Complutense University of Madrid; 61.Department of Translational Hematology, Research Institute Hospital 12 de Octubre (i+12), Hematological Malignancies Clinical Research Unit H120-CNIO, CIBERONC, and 4.Altum Sequencing Co., Madrid, Spain.; 71.Department of Translational Hematology, Research Institute Hospital 12 de Octubre (i+12), Hematological Malignancies Clinical Research Unit H120-CNIO, CIBERONC, and 2.Complutense University of Madrid., Madrid, Spain Background: The quantification of Minimal Residual Disease (MRD) has become a relevant marker as it can detect acute myeloid leukemia (AML) patients with a high risk of relapse. Although the reference MRD method in AML is to quantify tumor cells present in bone marrow (BM), the study of circulating tumor cells (CTC) in peripheral blood (PB) is also being incorporated into clinical protocols. However, cfDNA as an MRD biomarker in AML has not yet been validated. Keywords: AML, MRD, Liquid Biopsy, NGS. Aims: The purpose of the study is to evaluate the use of cfDNA by NGS test as a new method for MRD quantification in AML and compare it application with the use of PB CTCs in the follow-up of the disease. Methods: Three AML patients treated at the Hospital Universitario 12 de Octubre and with cfDNA available have been included. The mutational profile was defined by NGS (Ion Torrent System) at diagnosis using a panel of 42 genes involved in myeloid pathologies. Then, somatic mutations were selected, and MRD quantification was performed in liquid biopsy combining whole blood cells (WBC) and cfDNA. Both fractions were informatically differentiated by including molecular tags in the first PCR amplification. A total of 18 follow-up samples were analyzed with a threshold for EMR positivity set at 1x10^ (-4) as we previously described (Onecha E et al. Haematologica 2019). An average of 66ng of cfDNA and 660ng of gDNA from WBC were used. The results were compared with those obtained by multiparametric flow cytometry (MFC) in BM. Results: In the first patient, four variants affecting the genes CBL (VAF =7.4%), MPL (50%), JAK2 (51.5%) and ETV6 (10,6%) were detected at diagnosis in BM. The Leukemia-associated variant affecting ETV6 was selected as an MRD biomarker and detected in cfDNA (0.86%) post-consolidation 1. On this time-point (TP), the result of MRD by MFC on BM was also positive (0.002%), being negative in CTC-PB by NGS. The Non-leukemia-associated variants (JAK2, MPL, CBL) were also followed-up. Of interest, those alterations showed constant VAF levels in pre-transplant remission. After transplant, the VAF of JAK2 and MPL correlated with the donor chimerism studied by conventional methods after transplantation. On the other hand, CBL VAF was below the threshold sensitivity limit (0.01%) during all follow-ups. This result that CBL was also associated with leukemia, likely affecting a treatment-sensitive subclone. In patient 2, variants affecting IDH2 (92%), RUNX1 (43%), and DNMT3A (49%) genes were detected by at diagnosis. IDH2 mutation was selected as MRD biomarker, showed a VAF of 9.4% at relapse, correlating with the blast count of 10% defined by the morphology study of BM. Of note, in this TP, MRD-BM by MFC was 0.011%. In patient 3, the SF3B1 (43%) and EPAS1 (49%) mutations were detected by NGS in BM at diagnosis. The leukemia-associated variant (SF3B1) was observed in the cfDNA sample (0,0003) at the first follow-up sample but was negative at the following TPs. MRD by CTCs and MFC-BM were also negative. This patient has not relapsed to date (follow-up time six months after diagnosis). Image: Summary/Conclusion: MRD quantification based on the use of cfDNA by NGS offers promising results and, in the future, could be a good option for disease monitoring or early detection of relapse. The results of the whole series will be presented at the meeting. P384: THE ROLE OF THE MANNOSE RECEPTOR C-TYPE 2 (MRC2) IN LEUKEMIC STEM CELL MAINTENANCE IN AML M. Arnone1,*, E. Görsch1, S. Pöschel1, P. Godavarthy1, A. Stanger1, S. Rudat1, C. Lengerke1 1Innere Medizin 2, University Hospital Tuebingen, Tuebingen, Germany Background: Therapy-resistant leukemia-initiating cells, so called leukemic stem cells (LSC), are responsible for disease relapse and a major cause of death in patients with acute myeloid leukemia (AML). Effective identification and targeting of LSCs are thus important goals. Our previous RNA sequencing analyses revealed that the MRC2 gene, which encodes a transmembrane receptor known for its ability to bind and degrade collagen as well as for its pro-migratory and -invasion functions, is enriched in LSCs. Aims: We aim to gain a better understanding of the function(s) of MRC2 in AML in general and LSCs in particular. Methods: We used flow cytometry to characterize MRC2 expression in primary AML samples and healthy cells from the hematopoietic system. Imaging flow cytometry experiments were performed to study the role of MRC2 in gelatin uptake. Results: We found that mRNA expression of the MRC2 gene was increased in the LSC compartment of 5 AML patients compared to their non-LSC counterparts. Analyzing MRC2 cell surface presentation by flow cytometry, we found that AML cell lines (n=10) displayed the highest levels of MRC2 expression followed by healthy CD34+ cord blood (CB) cells (n=7), while primary AML patient samples (n=21) were characterized by an intermediary MRC2 cell surface presentation with the lowest levels of MRC2 presentation in differentiated healthy hematopoietic cells. Interestingly, high MRC2 expression correlated with increased clonogenic activity as only sorted MRC2+ but not MRC2- primary AML cells from the same sample were able to form colonies in a methylcellulose assay; and siRNA-mediated knockdown of MRC2 in the bulk of primary AML cells led to a reduction in colony formation when compared to control siRNA transfected cells. Furthermore, using CRISPR/Cas9 we generated a MRC2 knock-out AML cell line and observed in in vitro assays that MRC2 expression is required for migration, invasion and adhesion to collagen and laminin 421. Finally, we could show by imaging flow cytometry (ImageStream) that AML cells lacking MRC2 expression were unable to internalize fluorescently-labelled gelatin. Summary/Conclusion: Together, our results suggest that AML cells benefit from MRC2 expression due to its multiple functions involving migration, invasion and the interaction with extracellular matrix components. Additionally, MRC2 has a potential role in LSC biology such as the maintenance of stemness-associated properties. Further in vivo studies will improve our understanding of MRC2, which could then inform us on novel therapeutic approaches. P385: DETECTION OF KMT2A PARTIAL TANDEM DUPLICATIONS IN ACUTE MYELOID LEUKEMIA PATIENTS BY NGS A. Artigas-Baleri1,*, G. Oñate2, A. Brell2, J. Esteve3, S. Vives4, M. Tormo5, M. Arnan6, A. Garcia7, R. Coll8, A. Sampol9, J. Bargay10, F. Vall-llovera11, O. Salamero12, J. Nomdedéu2, M. Pratcorona2 1Hematology, Insitut de Recerca de Hospital de la Santa Creu i Sant Pau; 2Hematology, Hospital de la Santa Creu i Sant Pau; 3Hematology, Hospital Clínic, Barcelona; 4Hematology, Institut Català d’Oncologia, Hospital Germans Trias i Pujol, José Carreras Leukemia Research Institute, Badalona; 5Hematology, Hospital Clínico Universitario, Biomedical Research Institute INCLIVA, Valencia; 6Hematology, Insitut Català d’Oncologia, Hospital Duran i Reynals, Institut Biomèdica de Bellvitge (IDIBELL), L’Hospitalet de Llobregat; 7Hematology, Hospital Universitari Arnau de Vilanova, Lleida; 8Hematology, Institut Català d’Oncologia, Hospital Josep Trueta, Girona; 9Hematology, Hospital Son Espases; 10Hematology, Hospital Son Llàtzer, Palma de Mallorca; 11Hematology, Mutua Terrassa, Terrassa; 12Hematology, Hospital Vall d’Hebron, Barcelona, Spain Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic disease consisting of an aberrant proliferation and differentiation of hematopoietic stem cells in the bone marrow. In the last years the identification of cytogenetic and molecular biomarkers has improved the AML classification considering prognosis, and allowing a better risk-adapted treatment strategy. Regarding molecular biomarkers, NPM1, RUNX1 and ASXL1 mutations, FLT3-ITD (internal tandem duplications), CEBPAbi or KMT2A-PTD (partial tandem duplications) have been shown to have clinically significant prognostic value and they define specific AML disease subtype. Our study focuses on the detection of PTD in KMT2A which is involved in the regulation of gene expression during hematopoiesis. KMT2A-PTDs are found in 5-10% of normal karyotype patients and have been associated with poorer disease-free and overall survival rates. Mainly, PTDs in this gene span exons 2 to 8-10 and their detection is based on long-distance genomic PCR with extracted DNA from bone marrow. This is a time-consuming technique, and that is probably the main reason to explain why this marker is not widely performed at the time of diagnosis. Aims: We aimed to analyze the potentiality of Next Generation Sequencing (NGS) in the identification of KMT2A-PTD, a technique that is being implemented in many laboratories and could be a good strategy to detect this marker. Methods: This study included patients from CETLAM group (Grupo Cooperativo de Estudio y Tratamiento de las Leucemias Agudas y Mielodisplásicas) with a diagnosis of AML in 2021 (from January to October) and age≤70. The study of KMT2A-PTDs was carried out with DNA samples through two different techniques: PCR and NGS. DNA was extracted from bone marrow with the AllPrep DNA/RNA Mini Kit (Qiagen®). For the PCR amplification we used both custom 8-exon forward primer and 2-exon reverse primer. NGS was performed by hybridization capture technology (Nonacus®) using a custom panel which includes 43 genes. MiSeq (Illumina®) system was used for sequencing and Datagenomics for analysing the results. Hybridization capture allows the comparison of the number of reads per position between samples to identify structural variants as PTDs. This approach permits to know exactly which exons have been duplicated. Results: We included a total of 72 AML patients who were genotyped for KMT2A-PTD with both PCR and Hybridization capture NGS. We identified 4 and 7 patients with a partial tandem duplication in KMT2A gene by PCR and NGS, respectively. The four KMT2A-PTDs identified with PCR were also detected by NGS. In contrast, three PTDs were observed only with NGS. In two of them the PTD spanned exons 2 to 8 (Figure 1), while in the other it spanned exons 4 to 8. The first two PTDs were confirmed with the classic long-distance genomic PCR but performed with cDNA, skipping the presence of a large non-coding DNA in the rearrangement which could not be amplified by conventional PCR. To confirm the third PTD a new reverse primer in exon 4 was designed. All three NGS observed KMT2A-PTDs could be verified. Image: Summary/Conclusion: Our results show that Hybridization capture NGS is a good technique to detect KMT2A-PTDs, with a better sensibility than PCR. NGS is a cost-effective method as it is becoming an essential tool for AML classification at diagnosis. P386: CELL FATE DETERMINING TRANSCRIPTION FACTORS AS VULNERABILITIES IN CHILDHOOD AND ADULT MLL-REARRANGED AND T(8;16) ACUTE MYELOID LEUKEMIA S. Arza Apalategi1,*, B. Heuts2, S. Bergevoet1, S. van Heeringen3, J. Jansen1, J. Martens2, B. van der Reijden1 1Laboratory Medicine, Laboratory of hematology, Radboudumc; 2Molecular Biology; 3Molecular Developmental Biology, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands Background: The MLL-AF9 protein contributes to acute myeloid leukemia (AML) development through transcriptional activation of oncogenic gene programs. Remarkably, MLL-AF9 AML consists of two subgroups, one with high oncogenic MECOM expression and dismal outcome; the other with a unique gene expression profile lacking MECOM expression and a favourable outcome. How the differences between these two clinically and biologically different subgroups are brought about in the context of the same MLL-AF9 genetic background is currently unknown. Aims: To identify vulnerabilities in AML beyond the MLL-AF9 oncofusion gene we determined cell fate determining transcription factors pertinent to the two clinically and biologically different subgroups. Methods: Transcription factors key to cell fate were determined by first analysing differential transcriptional start sites of genes and gene enhancers by genome-wide “Cap Analysis Gene Expression (CAGE)”-sequencing of three primary MECOM positive and negative MLL-AF9 AML samples. Sequencing data were subsequently analysed using a new bioinformatic tool that builds gene regulatory networks based on differential gene expression and enhancer activities to predict key transcription factors in cell fate determination. For predicted factors AML cohort-wide gene expression patterns, transforming potential and downstream gene programs were determined. Results: Bioinformatic analyses of CAGE-seq data identified the transcription factor MECOM as top hit for cell fate determination in MECOM+ MLL-AF9, confirming the power of this approach. In the group lacking MECOM expression we identified HMX3 as most influential transcription factor. HMX3 is essential for neuronal development and not expressed in any healthy blood cell type. In a large adult and childhood AML cohort (totalling 630 cases), high HMX3 expression was observed in 4% and 11% cases, respectively. HMX3 positive cases clustered tightly together in unsupervised clustering analyses, and 80% of cases were positive for MLL-rearranged (MLL-AF9, AF10, ENL) and t(8;16) AML. None were positive for other known recurrent AML mutations except for RAS mutations. In line with these observations, MLL-AF9 positive human cell lines expressed HMX3 (eg. THP1 and MOLM13). HMX3 silencing in THP1 and MOLM13 cells caused pronounced cell cycle arrest and apoptosis evidenced by PI, Annexin and 7AAD staining in flow cytometry. Forced lentiviral HMX3 expression in primary human GCSF-mobilized CD34+ cells resulted in an over 70% clonogenic growth inhibition compared to control transduced cells. To identify relevant gene programs we silenced HMX3 in MOLM13 cells followed by RNA-sequencing. Gene ontology analyses of over 3000 differentially expressed genes (p<0.01) suggested a role for HMX3 in cell cycle progression, sister chromatid separation, and immune response suppression (p<0.05). Summary/Conclusion: Bioinformatic analyses of CAGE-sequencing of primary leukemia samples predicted the transcription factors MECOM and HMX3 key to poor and good outcome MLL-AF9 AML, respectively. High HMX3 expression associated with a unique gene expression profile and associated strongly with MLL-rearranged and t(8;16) AML. HMX3 only was unable to transform healthy human immature blood cells but appeared essential for survival of MLL-AF9 leukemia cell lines, potentially by positively regulating cell cycle progression and immune evasion. Insight into the factors that activate HMX3 expression in AML could provide potential targets for therapies in MLL-rearranged and t(8;16) AML. P387: UNRAVELING LEUKEMIC STEM CELLS MITOCHONDRIAL DEPENDENCY IN PEDIATRIC ACUTE MYELOID LEUKEMIA M. Benetton1,*, A. Da Ros1, G. Borella1, G. Longo1, C. Tregnago1, F. Locatelli2, M. Pigazzi1 1Department of Women’s and Children’s Health, Haematology-Oncology Clinic and Laboratory, University of Padova, Padova; 2Department of Pediatric Hematology and Oncology, IRCCS Ospedale Pediatrico Bambino Gesù and Sapienza University of Rome, Roma, Italy Background: Relapse still represents an unsolved problem for children with acute myeloid leukemia (AML), therefore more effective therapeutic strategies are needed for a complete blasts eradication. Leukemic stem cells (LSCs) have been proposed as the therapy-resistant reservoir of cells responsible for failure of antiproliferative chemotherapy and disease recurrence in AML due to their self-renewal capacity and quiescence allowing evasion from chemotherapy. Thus, the identification of LSCs-specific vulnerabilities is needed for more efficacious treatments. Aims: We aim to dissect novel LSCs characteristics to unravel mechanisms of chemoresistance and disease recurrence, and characterize biological properties of LSCs that can be targeted to completely eradicate AML. Methods: To study LSCs derived from primary pediatric AML samples, we based on reactive oxygen species (ROS) content since it was shown that functionally defined LSCs have relatively low levels of ROS. We isolated the lowest 20% of cells fluorescing with CellROX probe (ROS low) and the highest 20% (ROS high) by cell sorting, and analyzed cell cycle, engraftment in mice and RNA sequencing to identify deregulated pathways. Results: After isolating ROS low and ROS high cells, we first assessed classical stemness features. By colony-forming assay, we observed that ROS low and ROS high cells have similar colony-forming capacity, but only ROS low cells preserve this capacity after a second replating whereas the ROS high population does not (p=0.002). By BrdU assay, we found that ROS low cells are relatively dormant, being more in G0/G1 cell cycle phase (41.9%) than the ROS high counterpart (7.9%, p=0.001), also confirmed by CFSE staining, revealing that ROS low cells proliferate more slowly (at day 4, 31% undivided cells in ROS low vs 8% in ROS high). According to our previously published data, we confirmed that ROS low cells express higher levels of CDK6-AS1 (RQ=1.38) and CDK6 genes (RQ=2.69; RQ=1 for ROS high) and that, being more quiescent, are characterized by lower levels of mitochondrial membrane potential by TMRE and lower content of mitochondrial ROS, byproducts of mitochondrial activity, by MitoSOX staining. We also verified that ROS low cells have a lower mitochondrial mass by MitoTracker and TOMM20 staining (mitochondria to nucleus ratio 1.18 vs 1.35 for ROS high, p=0.0005), suggesting that mitochondrial mass and functions might be involved in quiescent cells properties. However, engraftment experiments did not reveal different capacity of ROS low or ROS high cells in recreating leukemia in NSG mice, neither in terms of human CD45+ cells in mice peripheral blood (23.3% vs 41.9% respectively, p=0.12) nor bone marrow infiltration (73% vs 84.8%, p=0.44) at 8 weeks post-engraftment, opening for further dissection of the presence of LSCs within ROS high cells. By RNA sequencing, we found that the top 150 differentially expressed genes between ROS low and ROS high cells are involved in focal adhesion (logP=-13.51), regulation of hemopoiesis (logP=-12.31), cell response to stress (logP=-8.73) and cytokine production (logP=-8.67). Further analyses will confirm the role of these pathways in maintaining ROS low LSCs. Moreover, we identified 5 differentially expressed surface antigens that are under investigation as putative ROS low LSCs markers. Summary/Conclusion: In conclusion, the ROS low strategy permits to identify a quiescent AML subpopulation with peculiar mitochondrial features and transcriptome allowing to pinpoint novel molecular pathways that might be targeted to enhance LSCs clearance. P388: PREFERENTIAL SYNERGISTIC EFFECT OF INDISULAM IN COMBINATION WITH VENETOCLAX AND AZACYTIDINE IN AML CELLS WITH SF3B1 MUTATIONS. P. Bernardo1,2,*, M. Pereira1, B. Galvão1, J. Desterro1,3, A. Nunes3, M. D. J. Frade3, J. Lobato3, M. G. da Silva3, M. Lemos4, M. Silva4, P. Ribeiro4, S. Matos5, N. Custódio1, M. Carmo-Fonseca1 1Instituto Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa; 2Serviço de Hematologia Clínica, Hospital da Luz Lisboa; 3Serviço de Hematologia Clínica, Instituto Português de Oncologia de Lisboa Francisco Gentil; 4Serviço de Hematologia Clínica, Centro Hospitalar Lisboa Central - Hospital de St. António dos Capuchos; 5GenoMed - Diagnósticos de Medicina Molecular SA, Lisbon, Portugal Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis characterized by a high rate of relapse after conventional combination chemotherapy, highlighting the need for improved therapeutic interventions. Genes involved in pre-mRNA splicing, namely SF3B1, U2AF1 and SRSF2, are frequently mutated in AML.Recent studies suggest that pharmacological targeting of the splicing machinery results in preferential lethality of splicing mutant AML cells, potentially providing a novel strategy for treatment of this disease. Existing anti- cancer sulfonamides have been shown to interfere with splicing and induce preferential killing of splicing mutant AML cell lines. A particular sulfonamide drug, indisulam, was previously studied in cancer clinical trials where it was found to be safe but demonstrated limited efficacy, most likely because neither the mechanism of action nor potential biomarkers of response were known. Aims: We investigated, in this study, the efficacy of indisulam, either alone or in combination with ABT-199 (venetoclax) and azacitidine, in cultured primary human AML cells. Methods: Bone marrow cells were collected from adults newly diagnosed with AML, non-treated. Cells were cultured and incubated for 48 and 72 hours with indisulam at a range of nanomolar concentrations. Additionally, cells were incubated with ABT-199 (10nM) and azacitidine (1μM), alone or in combination with indisulam. Cell viability and cell death were measured. For mutation profiling, a targeted myeloid gene panel was used. Results: We found that treatment with indisulam resulted in lethality of primary AML cells harboring mutations in SF3B1, but had minor or no effect on AML cells with other mutations including mutations in splicing factors U2AF1 and SRSF2. In SF3B1-mutated cells, a clear synergistic effect was further observed when indisulam was combined with ABT-199 and azacitidine. The correlation between efficacy of indisulam and synergy with ABT-199 and azacitidine in AML cells with different mutations will be shown and discussed. Summary/Conclusion: Our results indicate that indisulam is preferentially active and synergistic with venetoclax and azacitidine in SF3B1-mutated cells from newly diagnosed, non-treated AML patients. P389: BISPECIFIC ANTIBODIES ACTIVATE AUTOLOGOUS LEUKEMIA-REACTIVE T CELLS IN ACUTE MYELOID LEUKEMIA D. Böhm1,*, D. Primo2, J. Ballesteros2, K. Wennerberg1 1BRIC - Biotech Research & Innovation Centre, University of Copenhagen, Copenhagen, Denmark; 2Vivia Biotech, Tres Cantos, Madrid, Spain Background: Acute myeloid leukemia (AML) is a highly immunosuppressive disease with a high risk of relapse. This makes the finding of effective immunotherapy-based treatments important, but also difficult. T cell-engaging bispecific antibodies (TCEs) are designed to bind to both T cells and target cells, activate the T cells and thus induce an immune response against the leukemia cells without employing the MHC-T cell receptor (TCR) interaction. Several therapies of this kind are under development for treatment of AML. Surprisingly, both in vitro and in vivo, a subset of these TCE immunocoached T cells (ICTs) can subsequently target autologous AML cells and mediate efficient TCR-MHC-dependent AML cell killing in the absence of the TCE. This points to that a) a portion of the T cells present in an AML are leukemia-reactive but have been immunosuppressed - a suppression that can be reversed by the addition of a TCE, b) that this anti-AML T cell reactivation may play an important part of the mechanism of action of AML-targeted TCEs and c) that by understanding this reactivation mechanism, we will be able to design improved AML immunotherapies in the future. Aims: To understand the mechanisms by which a TCE induces reactivation of leukemia-reactive T cells (LRTs) in AML. Methods: T cells in biobanked AML patient bone marrow samples were activated with either an anti-CD3 x anti-CD123 TCE or a conventional anti-CD3 antibody for 5 days before isolating the ICTs. ICT assay: ICTs were washed to remove the TCE, and isolated ICTs were co-cultured with additional autologous bone marrow cells at different E:T ratios. Killing efficiency and T cell expansion were determined over several days using flow cytometry. Trogocytosis assay: ICTs were co-cultured with stained additional autologous AML cells for 1 h and T cell populations of interest were isolated using FACS. T cell repertoires were analyzed by targeted RNAseq of the TCR. Results: ICTs generated with a TCE kill autologous AML cells efficiently without further addition of the TCE. Adding additional TCE at this stage also does not increase the AML killing efficiency of the ICTs. ICTs generated with an activating anti-CD3 antibody show a similar AML killing efficiency and a similar trogocytotic behavior as TCE ICTs. Most of the TCR clones present in the AML bone marrow pre-stimulation were also represented among the ICTs and trogocytotic ICTs (ICTs that are directly targeting the AML cells after either TCE or anti-CD3 stimulation; Figure 1), suggesting that the bulk of T cells in an AML bone marrow represent heterogeneous immunosuppressed LRTs which can be equally well reactivated by a TCE or an anti-CD3 antibody. Trogocytotic ICTs generated from both types of T cell activation have a similar TCR distribution. ICTs derived from the bone marrow target autologous AML cells more efficiently than ICTs from peripheral blood. Image: Summary/Conclusion: Similarities between TCE ICTs and CD3 ICTs in regards to TCR distributions and killing efficiencies suggest that the TCE reactivates leukemia-reactive T cells via a CD3-dependent mechanism rather than a more complex proximity mechanism. Our data further suggests that T cells in the AML bone marrow are highly heterogeneous and leukemia targeted to a very high degree. In summary, AML bone marrow ICTs are targeting and killing leukemic cells efficiently in a polyclonal fashion and our results highlight that boosting this mechanism can leverage the clinical impact of AML TCEs and be utilized in other novel AML immunotherapy approaches. P390: ACUTE MYELOID LEUKEMIA SUPPRESSION BY PALBOCICLIB AND PONATINIB IN PATIENT-DERIVED XENOGRAFT D. Busa1,*, M. Culen1,2, T. Loja3, I. Jeziskova1,2, A. Folta1,2, J. Mayer1,2,3 1Department of Internal Medicine - Hematology and Oncology, Masaryk University; 2Department of Internal Medicine - Hematology and Oncology, University Hospital Brno; 3Central European Institute of Technology, Brno, Czechia Background: Palbociclib, a CDK4/6 inhibitor approved for breast cancer treatment, and ponatinib, a BCR/ABL1 inhibitor with a multi-kinase activity approved for chronic myeloid and acute lymphoid leukemia, were previously shown to be effective in vitro against different cancer types including acute myeloid leukemia (AML). Aims: To test palbociclib and ponatinib, along with reference drugs – venetoclax, azacitidine, cytarabine+doxorubicine (chemotherapy) on 2 primary AML samples in a patient-derived xenograft model. Methods: Two newly diagnosed AMLs (AML #1: myelomonocytic AML, intermediate cytogenetic risk; AML #2: AML with myelodysplasia-related changes, poor cytogenetic risk) were xenotransplanted into NOD SCID gamma mice. Treatment was initiated at detection of approximately 5-20% hCD45+ cells in mouse peripheral blood (PB). Chemotherapy (cytarabine+doxorubicine, AraC/Dox) was administered as a 5 + 3 regimen. Azacitidine was administered subcutaneously, five days per week in 3 cycles – 1 week on, 1 week off. Palbociclib, ponatinib, venetoclax, and vehicle were administered orally for 3 weeks, 5 days per week. Gene sequencing was performed using targeted 37 gene Archer Variantplex Core Myeloid Kit panel. Results: From the reference treatments, significant AML reduction and prolonged overall survival (OS) was achieved with venetoclax in both AMLs, and with chemotherapy in AML #1. No statistically significant effect was observed with azacitidine, but interestingly in AML #2 the azacitidine treatment induced the longest remission in the whole study, almost 10 weeks (<1% hCD45+ cells in PB) in 2/4 mice. Of the tested drugs, palbociclib showed the same effect as venetoclax, with significantly reduced AML and prolonged OS in both AMLs. Ponatinib led to prolonged OS only in AML #1 (Fig. 1A-D). Overall, none of the treatments led to complete AML eradication and all PDX groups gradually relapsed. No phenotype changes were observed in the relapsed cells in palbociclib and ponatinib groups compared to vehicle (SSClowCD45dim blasts, SSCdimCD45high monocytes, CD34+ and CD34+CD38- cells). Azacitidine group showed altered monocyte, CD34+, and CD34+CD38- fractions in AML #1. Azacitidine, chemotherapy, and venetoclax showed altered blast and monocyte fraction in AML #2 and venetoclax also altered CD34+ fraction in AML #2. Mutational status in AML #1 remained unchanged (FLT3-TKD and NRAS mutated) in all PDX groups at relapse. In AML #2, KRAS mutation from the primary tumor remained conserved, whereas all PDXs lost an IDH1 mutation and gained a WT1 mutation. This indicates that mutation selection was induced by the xenotransplantation rather than treatment. In addition, a second WT1 mutation was gained only in the palbociclib group. Treatment toxicity manifested by weight decrease after the end of treatment was only seen with chemotherapy and was accompanied by early mouse mortality in 3/4 mice for AML #2. Image: Summary/Conclusion: Palbociclib demonstrated in vivo AML suppression that was comparable to venetoclax and superior to chemotherapy. Effect of ponatinib was only limited. This encourages further investigation of treatment efficacy in different AML subtypes and in combination with other drugs. Funding: MUNI/A/1330/2021 P391: FLT3-ITD MEASUREMENT AT DIAGNOSIS AND FOR THE ASSESSMENT OF MINIMAL RESIDUAL DISEASE AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA: CDNA VS. DNA D. Carbonell1,2,*, M. Chicano1,2, A. Cardero-Illán1, G. Rodriguez-Macías1, P. Muñiz1,2, R. Bailén1,2, G. Oarbeascoa1,2, I. Gómez-Centurión1,2, J. Anguita1,2, M. Kwon1,2, J. L. Díez-Martín1,2,3, I. Buño1,2,4,5, C. Martínez-Laperche1,2 1Hospital General Universitario Gregorio Marañón; 2Instituto de Investigación Sanitaria Gregorio Marañón; 3Department of Medicine; 4Department of Cell Biology, Complutense University of Madrid; 5Genomics Unit, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain Background: DNA is standardly preferred to cDNA for the analysis of FLT3- internal tandem duplication (ITD). Although its screening is mandatory at diagnosis in clinical routine, its use as minimal residual disease (MRD) marker after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains controversial due to the poor sensitivity of its detection method and its apparent instability during the disease. Aims: To compare FLT3-ITD mutation analysis in DNA and cDNA samples at diagnosis and to prove the usefulness of its expression measurement as MRD marker after allo-HSCT. Methods: Forty-six diagnosis samples from 46 patients and 80 samples from 34 patients who underwent an allo-HSCT (34 pre-HSCT samples, 34 at day 30 after infusion and 12 before relapse) were analyzed. DNA was purified using a Maxwell RSC Blood DNA Kit (Promega), RNA was isolated using TRIzol reagent (Invitrogen) and cDNA synthesis was performed using the First Strand cDNA Synthesis kit (Roche). FLT3-ITD was analyzed in both DNA and cDNA samples by fragment analysis using an ABI3130xl DNA sequencer (Applied Biosystems). Fragment analysis was analyzed through Peak Scanner Software (Thermo Fisher). Allelic ratio (AR) quantification was performed by dividing the area under the curve of the mutant allele by the area under the curve of the wild type allele. Every clone was analyzed as an event, comparing its DNA ratio with its cDNA ratio. Results: Median AR of the 58 FLT3-ITD clones at diagnosis were 0.54 [0-9.47] in DNA samples and 0.63 [0.012-13] in cDNA samples, (Wilcoxon test, p<0.001, Figure A). In six patients, FLT3-ITD AR was <0.5 in DNA and ≥0.5 in cDNA, changing their prognosis based on ELN algorithm. One of them was not candidate for chemotherapy and of the five remaining, four were refractory or relapsed after intensive treatment. In terms of sensitivity, cDNA was better than DNA, quantifying higher AR values in most cases, optimizing the detection of minor clones and the prognostic classification. In respect of HSCT samples, seven patients were positive for FLT3-ITD mutation pre-HSCT in cDNA and two in DNA. Of the seven patients which were positive by cDNA, four relapsed after HSCT. The three patients who did not relapse had FLT3-ITD AR lower than 0.05. As for the two patients who tested positive in the DNA samples, only the one who also tested positive for cDNA relapsed. Despite the small sample size, these results reveal that the analysis of pre-HSCT cDNA samples could have a predictive value for relapse. No patient was positive on day 30 after HSCT, suggesting that it may not be useful, whether the DNA or cDNA sample is studied. However, the analysis at this time point could be crucial in those patients who may relapse early. On respect of the last sample before relapse (a median of 22 days before relapse, range 7-85), of the 12 cases, three were positive for FLT3-ITD mutation in DNA samples and nine were positive in cDNA samples. Great differences were observed between AR of both type of samples (Wilcoxon test, p<0.001, median 0 [0-0.04] vs. 0.15 [0-0.57], Figure B). In three patients FLT3-ITD cDNA measurement did not anticipate the relapse, due to the loss of the mutation or because the day on which the sample was analyzed was too early to detect the mutation, issues that can be solved by complementing with other MRD markers. Image: Summary/Conclusion: In conclusion, cDNA fragment analysis of FLT3-ITD by capillary electrophoresis is an easy-to-implement technique that could be a great alternative approach in AML patients at diagnosis and in allo-HSCT monitoring. P392: PHARMACOLOGICAL INHIBITION OF SYK CONFERS ANTI-PROLIFERATIVE AND NOVEL ANTI-TUMOR IMMUNE RESPONSES IN AML L. A. Carvajal1,*, B. Robinson2, Y. Kosaka2, T. Jacob2, J. Lee2, T. Hood1, K. Baker2, A. Kaempf2, S. N.-A. Amara1, J. Pucilowska2, E. Lind2, C. Tognon2, J. Tyner2, P. Kumar1, T. Vu2, J. DiMartino1 1Kronos Bio, Inc., San Mateo; 2Oregon Health & Science University (OHSU), Portland, United States of America Background: Spleen tyrosine kinase (SYK) acts as a key integrator of signals from cell surface receptors containing an immunoreceptor tyrosine-based activation motif to boost cellular proliferation. In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network linked to NPM1, HOXA9 and MEIS1. The selective, orally bioavailable SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity and tolerability in HOXA9/MEIS1-driven AML. ENTO is currently being investigated in a global Phase 3 trial, AGILITY (NCT05020665), in combination with intensive induction/consolidation chemotherapy in patients (pts) with treatment-naive NPM1-mutated (NPM1m) AML. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency and selectivity to ENTO but with more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in pts with relapsed or refractory FLT3-mutated AML (NCT05028751). Aims: To investigate the effects of ENTO and LANRA in T-cell responses in AML. Methods: All pt-derived bone marrow (BM) and peripheral blood samples were obtained in accordance with IRB (OHSU#004422) approval. Results from gene expression profiling studies (RNA sequence [RNA-seq]) in bone marrow/peripheral blood mononuclear cells (MNCs) derived from 152 pts with AML, for which matching NPM1 status and ex vivo sensitivity to ENTO was available, previously published by Tyner et al (2018), were reanalyzed using trimmed mean of M values normalization and differential expression using edgeR. ENTO/LANRA sensitivity was assessed ex vivo after 72 hours in culture at 37°C by MTS assay. T-cell functional assays were performed by culturing MNCs with anti-CD3 and treating with ENTO or LANRA, singly and in combination with anti-PD1. CD3+ T-cell proliferation as measured by Ki67 expression and pSYK activation were assessed by quantitative single-cell immunofluorescence microscopy. Six formalin-fixed, paraffin-embedded BM biopsies from newly diagnosed AML pts were assessed for spatial expression of mRNA using a digital spatial profiling method. Results: In AML, mutations in NPM1 with co-expression of HOXA9/MEIS1 at baseline predicted anti-proliferative activity to ENTO in ex vivo drug sensitivity studies. Accordingly, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. Pathway analysis of archival RNA-seq data from pts enrolled in the Leukemia and Lymphoma Society BEAT AML Master Protocol (NCT03013998 [BAML-16-001-S6]) revealed gene expression signatures at baseline associated with the observed sensitivity to ENTO, including significant enrichment in the expression of genes associated with leukemogenesis, myeloid differentiation and immune regulation. This was supported by T-cell functional studies, which demonstrated that ENTO and LANRA, singly as well as in combination with anti-PD1, could restore T-cell proliferation in primary AML pt samples that exhibited suppression. Lastly, Cancer Transcriptome Atlas analysis on archival BM biopsies obtained from these AML pts showed a strong correlation between immune checkpoint response and gene expression associated with immune pathway signaling. Summary/Conclusion: Inhibition of SYK is a promising therapeutic approach in NPM1m AML. Our studies illustrate that ENTO and LANRA may restore T-cell proliferation in a subset of AML pts with dysfunctional T-cell responses, suggesting a novel mechanism of action. Additional studies are required to fully understand the mechanism of SYK-mediated antitumor immune responses in AML. P393: SYNERGISTIC EFFECT OF EZH2 INHIBITOR WITH BCL2 INHIBITOR BY ENHANCING PIK3IP1 EXPRESSION IN ACUTE MYELOID LEUKEMIA C. Yang1, C. Song2,3, Z. Ge1,* 1Department of Hematology, Zhongda Hospital，Medical School of Southeast University, Institute of Hematology Southeast University, Nanjing, China; 2Hershey Medical Center, Pennsylvania State University Medical College, Hershey; 3Division of Hematology, The Ohio State University Wexner Medical Center, the James Cancer Hospital, Columbus, United States of America Background: Acute Myeloid Leukemia(AML) is a highly heterogeneous blood cancer with poor outcomes. PI3K Interacting Protein 1(PIK3IP1) binds to the p110 catalytic submit and downregulate PI3K activity. EZH2 is the catalytic subunit of polycomb repressive complex 2(PRC2). Inhibition of EZH2 activity is a therapeutic vulnerability in cancer cells. Combination of the BCL2 inhibitor Venetoclax(Ven) with hypomethylating agents has been approved for treatment in AML with substantial benefits, however, most patients develop resistance. Thus, a more efficacious treatment by targeting the drug resistance paradigm based on BCL2 inhibitor is required. Aims: To investigate the anti-leukemic activity of the combination of the EZH2 inhibitor DZNeP with BCL2 inhibitor Ven and to evaluate the association of PIK3IP1 with clinical characteristics of patients with AML. Methods: EZH2 and PIK3IP1 mRNA level was examined by qPCR in newly-diagnosed AML patients and healthy control from Feb. 1, 2016, to Jan. 30, 2022, at our institute with approval of the Ethics Committee. Cell Counting Kit-8 assay(CCK8) was used for cell proliferation and cytotoxicity in U937 and MV-4-11 AML cells treated with vehicle control, EZH2 inhibitor(DZNeP), Ven, and Combination (Ven+DZNeP). Synergistic effect was analyzed with Calcusyn. RNA-seq was performed with total RNA isolated from U937 cells treated with 2μM DZNeP for 48 hours. Apoptosis was measured by cell staining with Annexin V+propidium iodide (PI) following flow cytometry analysis. Lentiviral PIK3IP1 shRNA knockdown was performed in AML cells. Gene expression was analyzed by qPCR and Western blot. Results: EZH2 mRNA level was significantly increased in 53 newly diagnosed patients with AML compared to 20 healthy bone marrow control(p<0.0001)(Fig.1A). Both DZNeP and Ven had a dose-dependent and time-dependent effect on cell proliferation arrest in AML cells. DZNeP significantly sensitized the effect of Ven on cell proliferation arrest compared to single drug-only control. CalcuSyn analysis showed the synergistic effect of the Ven+DZNeP(Fig.1B). The total apoptosis rate increased significantly in the Ven+DZNeP group(Fig.1C). RNAseq analyses showed that PIK3IP1 is dramatically upregulated(Fig.1D). GSEA enrichment analyses showed that the Differentially Expressed Genes mainly upregulated apoptosis and downregulated cell cycle pathway(Fig.1E). Moreover, the PIK3IP1 mRNA level was significantly decreased in the aforementioned AML cohort compared to the control(P<0.005)(Fig.1F). The AML patients were divided into two PIK3IP1High expression groups(n=29) and PIK3IP1Low expression groups(n=24). PIK3IP1low expression is significantly associated with a lower overall response rate (96.55% vs 79.17%, p=0.047), higher relapse or death rate(58.33% vs 24.14%, p=0.029). A moderately negative correlation between EZH2 and PIK3IP1 was observed(r=-0.376, p=0.006)(Fig.1G). In addition, qPCR data showed that PIK3IP1 is significantly upregulated by DZNeP or Ven+DZNeP(Fig. 1H). PIK3IP1 is efficiently knocked down in U937 and MV-4-11(Fig.1I), and PIK3IP1 knockdown significantly blocked the combination of DZNeP and Ven mediated cell proliferation arrest and apoptosis(Fig.1J-K). Image: Summary/Conclusion: The combination of EZH2 inhibitor DZNeP and BCL2 inhibitor Venetoclax exhibited a strong synergistic cell proliferation arrest and apoptosis of AML cells in PIK3IP1 dependent manner. PIK3IP1low expression is associated with oncogenic markers and poor outcomes in AML. Our results highlight the likelihood of further in vivo pre-clinical study for the potential clinical trial of the novel combination in AML patients. P394: INSIGHTS INTO PATIENT-DERIVED XENOGRAFT MODELS FOR DEK-NUP214 LEUKEMIA F. Charles Cano1,*, A. Kloos1, T. Fangmann1, N. Kattre1, R. Schottmann1, K. Döhner2, A. Ganser1, M. Heuser1 1Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Medizinische Hochschule Hannover, Hannover; 2Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany Background: Acute myeloid leukaemia (AML) with the translocation DEK-NUP214 (t(6;9)(p23;q34)) is a rare subtype present in 1% of adult and 2% of paediatric patients and is recognized by the world health organization (WHO) as a unique subgroup of AML. These patients, typically, are diagnosed with de novo AML and have poor clinical outcome and resistance to chemotherapy. DEK-NUP214 is the sole cytogenetic abnormality in the majority of these patients at the time of diagnosis, suggesting its central role in disease development. This subgroup of leukaemia remains poorly characterized. There is only one cell line (FKH-1) and only very few reported DEK-NUP214 patient derived xenograft (PDX) models. The major limitation is the lack of serially transplantable PDX models, which are required for pharmacologic studies. The development of DEK-NUP214 PDX models will aid in the understanding of the molecular pathogenesis of this disease. Aims: To establish patient-derived xenograft models that recapitulate DEK-NUP214 leukaemia. Methods: Five patient samples were selected based on their cytogenetic profile and 1x106 cells were injected intravenously into irradiated NSG-SGM3 (NSGS) mice (n=3) per sample. Engraftment of human cells in peripheral blood by flow cytometry and blood counts was monitored monthly. Engrafted cells were used for serial transplantations. Cytospin preparations of peripheral blood, spleen and bone marrow of the PDX samples as well as the patient samples were stained with Wright-Giemsa stain. The DEK-NUP214 fusion transcript was confirmed by Sanger sequencing. Mutational analysis was performed with a custom myeloid sequencing panel. Results: Three patient samples showed a mean of 12.5±6.0 (PDX1), 0.68±0.23 (PDX2) and 4.9±2.5% (PDX3) engraftment of human CD45+CD33+ cells at 20-24 weeks in peripheral blood and were transplanted into secondary mice. The characteristics of the patients at diagnosis, whose samples engrafted, were 19, 53 and 51 years old, respectively, with a median of 52.8 x109/L (range 34.1-116 x109/L) white blood cells (WBC), 7.4 g/dl (range 6-9.4 g/dl) of haemoglobin (Hb), and 38 x109/L (range 26-61 x109/L) platelets (Plt). The average circulating blasts were 56.7±18% in peripheral blood and blasts in bone marrow were 84.7±4.7%. PDX models at the 1st transplantation (Tx) had an increased WBC and decreased Hb at the time of sacrifice. Average spleen weights were 196±24.1, 59.7±3.18, and 103±35.4 g, respectively. The morphological analysis revealed large blasts with partially granulated cytoplasm and few vacuoles similar to the patients’ cells. The engrafted cells showed a myeloid phenotype with expression of CD38, CD33 and CD14, with higher engraftment in bone marrow than spleen. Secondary transplants from PDX1 showed 12.5% of hCD45+ cells at 16 weeks in peripheral blood, with average spleen weight of 168 g, with a similar immunephenotype as primary mice, and were transplanted into tertiary mice. Mutational analysis showed that the PDX samples retained the same mutations as observed in the patient sample (FLT3-ITD for PDX1 and PDX2, and WT1 for PDX1) and the fusion transcript as well. Summary/Conclusion: We have successfully established AML PDX models for DEK-NUP214 leukaemia that reflect the morphological, immunophenotypic and molecular genetics of this disease subtype. This will enable pharmacologic studies in this rare disease and will allow a better understanding of the involved pathways. P395: PHARMACEUTICAL TARGETING OF RAS IN ACUTE MYELOID LEUKAEMIA D. Coleman1,*, L. Strate1, J. Griffin1, T. Rabbitts2, P. Cockerill1, C. Bonifer1 1University of Birmingham, Birmingham; 2The Institute of Cancer Research, London, United Kingdom Background: Acute Myeloid Leukaemia is characterised by a small number of co-occurring driver mutations which combine to make a diverse collection of disease subtypes. Mutations in genes associated with cell signalling are a key group of driver mutations and lead to the constitutive activation of these proteins and their associated signalling cascades. RAS mutations occur in 6% of AML and confer a proliferative advantage. However, 36% of AMLs include mutations which lead to activated RAS such as FLT3 mutations and are associated with a worse outcome after induction chemotherapy1. Inhibitors which bind to mutated and activated members of the RAS protein family to block the interaction between the protein and its downstream targets have been developed2 which allow us to investigate the effects of pan-RAS inhibition in Acute Myeloid Leukaemia. These Pan-RAS inhibitors inhibit RAS-RAF and RAS-PI3K signalling leading to reduced phosphorylation of ERK and AKT. Aims: 1. We have used novel specific and panRAS RAS inhibitors designed in the Rabbitts lab followed by system-wide analyses to identify the specific gene regulatory network of mutant RAS AML and identify RAS-responsive genes 2. We have interrorgated key transcription factor nodes with RNAi Methods: Primary cells were co-cultured on primary hMSCs for perturbation experiments. Fresh blood or bone marrow samples from patients with AML were purified and sorted for CD34 or CD117, these samples were profiled by RNA-seq an ATAC-seq. Data from multiple samples with RAS mutations was integrated and compared to data from healthy CD34+ PBSCs to establish the gene regulagtory network. Results: In vitro panRAS inhibitors cause cytotoxicity in primary AML samples with RAS activating mutations whilst cells with WT RAS exhibit reduced sensitivity. An exception to this finding are cells with FLT3-ITD mutations, which were also sensitive to RAS inhibition. This is of particular interest as patients treated with FLT3 inhibitors can relapse with a RAS mutant subclone. To understand the role of RAS mutations in AML we have constructed a gene regulatory network from the genomic and transcriptomic profiling of primary cell samples from patients which has allowed us to identify a core network of transcription factors. The direct association of aberrant RAS signalling with these transcription factors has been determined through perturbation experiments using RAS inhibitors, and by targeting key transcription factor nodes with RNAi. These include transcription factors ETV5 and KANK1, both of which have been linked to RAS signalling in solid tumour models3,4. Summary/Conclusion: These experiments will identify targets of the RAS pathway essential for the survival of AML cells. 1. Ball, B.J., et al. (2019), “RAS Mutations Are Independently Associated with Decreased Overall Survival and Event-Free Survival in Patients with AML Receiving Induction Chemotherapy.” Blood, 134(Supplement_1): 18-18. 2. Cruz-Migoni, A., et al. (2019), “Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds.” Proceedings of the National Academy of Sciences 116(7): 2545-2550. 3. Mus, L.M., et al. (2020), “The ETS transcription factor ETV5 is a target of activated ALK in neuroblastoma contributing to increased tumour aggressiveness.” Sci Rep 10(218) 4. Jonathan R. Dry, et al. (2010), “Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)”. Cancer Res; 70(6): 2264–2273 P396: DYSREGULATED DNAM-1 AND KIR2DL1 EXPRESSION IN NATURAL KILLER CELLS CORRELATES WITH POOR OUTCOME IN ACUTE MYELOID LEUKEMIA A. F O Costa1,2,*, L. O Marani1, I. A Lopes1, L. S Binelli1, P. S Scheucher1, J. L S Schiavinato1, M. I. A Madeira1, K. B B Pagnano3, B. K Duarte3, A. B. F Glória4, E. M Rego5, F. Traina1, R. Welner2, L. L Figueiredo-Pontes1 1Hematology Division, Department of Medical Images, Hematology, and Clinical Oncology, Ribeirao Preto Medical School at University of Sao Paulo, Ribeirao Preto, Brazil; 2Division of Hematology and Oncology, Department of Medicine, UAB Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, United States of America; 3Hemocentro, University of Campinas, Campinas; 4Hematology Division, Hospital das Clinicas da Universidade Federal de Minas Gerais, Belo Horizonte; 5Internal Medicine, Medical School of the University of Sao Paulo, Sao Paulo, Brazil Background: Although improvements have been made in understanding the physiopathology of Acute Myeloid Leukemia (AML), most patients still relapse leading to poor long-term prognosis and cure rates. Natural Killer (NK) cells are regulated by opposing signals from receptors that activate and inhibit effector function and are known to mediate anti-leukemic immunosurveillance in AML, but the mechanisms underlying the control of hematopoietic neoplasms by these cells remain unclear. Aims: We hypothesized that FLT3-ITD mutation, a molecular marker of adverse prognosis, is associated with phenotypically and functionally impaired NK cells due to imbalanced expression of inhibitory and activating receptors during AML. Methods: We analyzed the expression of DNAM-1 and KNG2D activating and NKG2A, KIR2DL1 inhibitory receptors on CD56+, CD56bright CD16- and CD56dim CD16+ NK subsets and its correlation with FLT3-ITD mutation in 70 AML patients and 12 healthy bone marrows (NBM) by flow cytometry. Seven marrows from AML patients in complete remission after induction chemotherapy (AML-CR) were used to evaluate NK cell and receptor recovery. Mann Whitney or Kruskal-Wallis tests (P < 0.05) were performed using GraphPad Prism (V8.0.2). Results: A decreased frequency of all NK cell subsets was found in AML (CD56+, P=0.03; CD56bright, P=0.04, CD56dim, P<0.01). Decreased expression of NKG2D (P<0.01) and a tendency of decreasing DNAM-1 (P=0.38) was found in CD56+ NK in AML, recovering to values closer to normal in AML-CR. Expression of NKG2D was decreased in both CD56bright and CD56dim NK subsets (P=0.03 and P<0.01), while DNAM-1 was found to be considerably decreased in CD56dim cytotoxic NK (P=0.02). FLT3 mutated patients showed even lower expression of DNAM-1 (P=0.02) and NKG2D (P=0.13) in CD56+ NK. Even though the latter was not statistically significant, we observed a decreasing pattern in these patients, that was confirmed by an important decrease of both activating receptors uniquely in the CD56dim subset (DNAM-1, P=0.03; NKG2D, P=0.05), which are known to contain the most cytotoxic activity. As for the inhibitory receptors, our cohort demonstrated a trending increase in NKG2A expression on all NK subsets, although not significant, and no difference was found in FLT3 mutated patients. Meanwhile, KIR2DL1 expression was higher in CD56+, CD56bright and CD56dim NK cells in AML (P<0.01; P=0.06 and P<0.01), while AML-CR showed frequencies comparable to healthy In FLT3-ITD mutated patients, CD56dim NK had increased expression of KIR2DL1 (P=0.03), strongly correlating with an imbalance between activation and inhibition of NK cell activity. Summary/Conclusion: Our data indicate that CD56dim NK show an exhaustion immunophenotype in FLT3-ITD mutated AML, with high expression of KIR2DL1 inhibitory receptor and low expression of both NKG2D and DNAM-1 activating receptors and that this imbalance likely leads to impaired cytotoxicity, compromised anti-leukemic activity and could be contributing to worst disease outcome, which will be confirmed through functional assays. Closing these gaps in knowledge informs the fundamental characteristics of NK dysfunction during leukemia and is of significant interest for targeting, therapeutic interventions and NK cell-mediated immunotherapy. P397: PROLACTIN RECEPTOR CONFERS CHEMORESISTANCE DUE TO SENESCENCE ENTRANCE IN ACUTE MYELOID LEUKEMIA L. Cuesta-Casanovas1,2,*, J. M. Cornet-Masana1, J. M. Carbó1, J. Delgado-Martínez1,3, L. Clément-Demange4, A. Banús-Mulet5, F. Guijarro6,7, J. Esteve5,6,7,8, R. M. Risueño1 1Leukemia Stem Cell, Josep Carreras Leukemia Research Institute, Badalona; 2Faculty of Bioscience, Autonomous University of Barcelona; 3Faculty of Pharmacy, University of Barcelona; 4Leukos Biotech, Barcelona; 5Josep Carreras Leukemia Research Institute, Badalona; 6Faculty of Medicine, University of Barcelona; 7Department of Hematology, Hospital Clínic; 8Institut d’Investigacions Biomèdiques August Pi I Sunyer, Barcelona, Spain Background: Acute myeloid leukaemia (AML) is a hematopoietic neoplasm characterized by frequent relapse because of chemotherapy resistance, which confers poor outcome. For this reason, the study of new therapeutic approaches that completely eradicate the disease is an urgent need. A previous in silico analysis performed in our laboratory demonstrated that PRLR signalling pathway is overactivated in leukemic stem cells (LSCs) in comparison to hematopoietic stem cells (HSCs), suggesting a relationship between PRLR and AML resistance and progression. Aims: The main objective was to decipher the role of PRLR and its signalling pathway in the transformation events associated with the initiation and maintenance of AML. Methods: mRNA expression of PRLR and Stat5a was analysed from public repositories. Surface PRLR expression was assessed by flow cytometry and total protein expression by western blot. Stat5 activation (phosphorylation status) and induced gene regulation were determined in AML cells ectopically expressing the wildtype PRLR (PRLRwt) and the dominant negative isoform (PRLRsh) and treated with its natural ligand (PRL) or an antagonist (G129R-PRL), by western blot and luciferase reporters. Senescence status was analysed by SA-β-galactosidase staining by microscopy and quantified using ImageJ software. Cytarabine resistant cells (AraC-R) were obtained by treating them during several weeks with increasing doses of cytarabine. PRLR expression was silenced using the CRISPR-Cas9 technology. Clonogenicity was studied in AML samples and healthy linage-depleted cord blood cells treated with PRL and hPRL-G129R and seeded in a semisolid medium in presence of instructive cytokines. Results: AML patient cells express higher levels of PRLR in terms of mRNA, protein surface expression and total protein in comparison to healthy donors. Stat5 mRNA was overexpressed in AML, and PRL induced its phosphorylation and activation in presence of PRLR wt isoform, while the dominant negative did not respond to PRL. The antagonist of the receptor did not induce any effect. Stat5 mRNA expression was increased at relapse, and so it was the PRLR surface and total protein when AML cells acquire chemoresistance. Moreover, PRLR wildtype cells showed greater levels of SA-β-galactosidase, an indicative of the entrance in senescence. This effect was reverted when PRLR was knocked-down using CRISPR-Cas9 technology. The inhibition of PRLR with an antagonist or its downregulation with the CRISPR-Cas9 technology decreased clonogenicity capacity of AML patient cells, without affecting healthy donor cells. Summary/Conclusion: PRLR expression is higher in AML patient samples, and correlates with chemoresistance and refractoriness, making the receptor an adequate biomarker for AML diagnosis and follow-up. PRLR-PRL signalling promotes the activation of Stat5, which is increased in AML samples, especially at relapse, suggesting an important role in AML progression and chemoresistance, a process related with increasing levels of PRLR surface expression. Moreover, the suppression of PRLR expression sensitizes the cells to chemotherapy. Those results demonstrate for the first time that PRLR suppression increases the sensitivity to chemotherapy and decreases the clonogenicity capacity of AML cells, suggesting the use of antagonists of the receptor as new therapeutic strategies against AML. P398: DEVELOPMENT AND CHARACTERIZATION OF PRECLINICAL IN VIVO MODELS FOR THE IDENTIFICATION AND TESTING OF NEW THERAPEUTIC APPROACHES FOR PEDIATRIC ACUTE MYELOID LEUKEMIA A. Da Ros1,*, V. Indio2, E. Porcù1, G. Borella1, M. Benetton1, C. Tregnago1, G. Longo1, S. Cairo3, B. Michielotto1, S. Bresolin1, B. Buldini1, A. Pession4, F. Locatelli5, M. Pigazzi1 1Department of Women’s and Children’s Health, Haematology-Oncology Clinic and Laboratory, University of Padova, Padova; 2Department of veterinary medical science, University of Bolgna, Bologna, Italy; 3XenTech, Evry, France; 4Pediatric Unit, IRCCS Azienda Ospedaliera-University of Bologna, Bologna; 5Department of Pediatric Hematology and Oncology, IRCCS Bambino Gesù Children’s Hospital, Sapienza University of Rome, Rome, Italy Background: In pediatric acute myeloid leukemia (AML) chemotherapy is the standard of care, but >25% of patients still relapse and after a disease recurrence the survival probability is extremely low (<50%). To ameliorate patients’ outcome there is an urgent need to discover new treatments. Nevertheless, pediatric drug development is extremely reduced by the need of a better understanding of the adverse event profile of adult cancer indications in children, by the lack of pediatric-specific formulations, and by the reduced number of pediatric AML patients that can be included in clinical trials. Thus, robust preclinical AML models to faithfully predict new drug efficacy is urgently needed to advance new drugs in clinical setting. Aims: This study aims to generate and characterize AML patient derived xenografts (PDXs) and accelerate the evaluation of innovative medicines for AML. Methods: We generated PDXs from primary AML samples by inoculating blasts in NSG mice and, when engrafted, in 3 consequent mice recipients (namely P0, P1 and P2-PDX). We characterized AML by immunophenotipic profile and by RNA and whole-exome sequencing (WES). According to somatic mutations and copy number alterations we determined AML clonal compositions. We selected drugs and performed drug testing in vivo, after the expansion of P3-PDXs. Results: We generated 22 AML-PDXs representing high-risk AML subtypes for genetic characterization harboring NUP98-NSD1, or NPM1-MLF1, or CBFA2T3-GLIS2, or FUS-ERG or KMT2A somatic translocations, or FLT3-ITD mutation. We monitored the AML associated immunophenotype in PDXs finding it was similar to that of the original AML. By WES we detected a consistent number of variants in each patients’ AML (ranging from 28 to 69), confirming an high AML intra-tumoral heterogeneity. Furthermore, we did not find any mutation recurrence among models, underlining an high inter-tumoral heterogeneity. In all models we tracked clonal evolution from patients’ AML to P2-PDX highlighting that most of the variants were maintained, with very few variants acquired during model development. Monitoring clonal dynamics we recognize a specific “founder” clone characterized by an average of 30 variants which are maintained up to P2 at the same allelic frequency, other small clones with average of 10 variants increasing the allelic frequencies in P2 and, in a restricted number of models, we observed that some clones were lost. By WES and transcriptome analysis we highlighted druggable mutations and pathways allowing the selection of novel targeted drugs. We screened their efficacy in vitro alone or combined with chemotherapic (Arabinoside) and biological agents (Venetoclax) by using AML ex vivo cells and mesenchymal stromal cells in a 3D co-culture system for exploring their synergy in reducing AML proliferation. Four selected drugs are under evaluation in AML-PDX models. Summary/Conclusion: We have created a series of paired AML and xenograft models for advancing pediatric AML therapeutics. Our models represent a concrete perspective for both, the identification of new variants and pathways involved in AML progression, and the possibility to perform novel drug screenings useful to increase AML drug portfolio. P399: UNRAVELING THE TUMOR SUPPRESSIVE ROLE OF STAT3Β IN ACUTE MYELOID LEUKEMIA S. Edtmayer1,*, A. Witalisz-Siepracka1, B. Zdársky1, T. Eder2, V. Poli3, F. Grebien2, D. Stoiber1 1Department of Pharmacology, Physiology and Microbiology, Karl Landsteiner University of Health Sciences, Krems an der Donau; 2Institute of Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria; 3Department of Molecular Biotechnology and Health Sciences, University of Torino, Turin, Italy Background: Signal transducer and activator of transcription 3 (STAT3) is a mediator of cytokine signaling existing in two alternatively spliced isoforms known as the full-length isoform STAT3α (770aa) and the C-terminally truncated isoform STAT3β (722aa). Initially STAT3β was described as the dominant negative form of STAT3α. It gained more attention as it turned out to initiate transcription independent of STAT3α and being capable to rescue embryonic lethality in absence of STAT3α. In acute myeloid leukemia (AML) patients, STAT3 has been reported to be constitutively activated thereby promoting proliferation and blast survival. However, most of those studies did not consider the two distinct isoforms of STAT3. Recently, our group identified STAT3β as a novel tumor suppressor and favorable prognostic marker in AML, however the underlying mechanism in leukemic cells remained elusive. Aims: With this study we aim to provide in-depth knowledge on the role of STAT3 isoforms in AML development and disease outcome. Methods: By using murine AML cell lines lacking either STAT3α or STAT3β, we aim to elucidate the STAT3 isoform-specific impact on cellular processes shaping AML development and progression. To gain better understanding we use AML in vivo models and in vitro assays in combination with next-generation sequencing (NGS) approaches to identify molecular mechanisms explaining the tumor suppressive function of STAT3β in AML. In brief, fetal liver-derived stem cells, isolated from STAT3α and STAT3β deficient mice on C57BL/6J background, were transduced with a retrovirus introducing the human fusion-oncogene MLL-AF9, and intravenously transplanted into immunocompromised mice. Results: Animals transplanted with leukemic cells lacking STAT3β showed accelerated disease progression and poorer overall survival confirming its tumor suppressive properties. Furthermore, absence of STAT3β favored leukemic infiltration and migration. Additionally, RNA sequencing revealed that lack of STAT3β in leukemic cells leads to a dysregulation of crucial genes driving leukemogenesis. Summary/Conclusion: Understanding the mechanisms behind the tumor suppressive property of STAT3β has potential translational impact and is crucial to identify novel therapies and preventative strategies. Further validation and ChIP sequencing will provide novel direct target genes of STAT3β which could serve as prognostic or therapeutic molecules. P400: PROTEOGENOMIC CHARACTERIZATION OF 5-AZACYTIDINE EFFECTS ON ACUTE MYELOID LEUKEMIA IMMUNOPEPTIDOME G. Ehx1,*, N. Nandita2, C. Durette2, J. Courtois1, M.-P. Hardy2, K. Vincent2, J.-P. Laverdure2, J. Lanoix2, F. Baron1, P. Thibault2, C. Perreault2 1GIGA-I3: Hematology, University of Liege, Liege, Belgium; 2IRIC, University of Montreal, Montreal, Canada Background: Hypomethylating agents like 5-azacytidine (AZA) are licensed for the treatment of acute myeloid leukemia (AML) patients ineligible for allogeneic hematopoietic cell transplantation. While previous reports suggested that AZA promotes the recognition of AML blasts by cytotoxic T cells, the mechanism behind this improved recognition is not fully understood. Specifically, AZA is assumed to promote the expression of transcripts repressed by genomic methylation, namely cancer-testis antigens (CTA) and endogenous retroelements (ERE), resulting in the presentation of immunogenic MHC-I-associated peptides (MAPs, collectively referred to as the immunopeptidome) derived from the translation of these transcripts. However, the presentation of such MAPs after AZA treatment has not been firmly demonstrated thus far. Aims: Our study aims to characterize how AZA treatment shapes the identity of MAPs presented by AML cells. Methods: Four different AML cell lines, THP-1, OCI-AML3, SKM-1, and MOLM-13 were treated with a non-cytotoxic dose of AZA for 3 days. Their transcriptome has been characterized by high-coverage RNA sequencing (RNA-seq) and their immunopeptidome by shotgun mass spectrometry (MS). To identify MAPs deriving from unannotated genomic regions, we have designed a cutting-edge proteogenomic pipeline using the RNA-seq data to build personalized MS databases that enabled the identification of non-canonical MAPs such as ERE-derived MAPs. Results: Paired transcriptomic comparisons between treated and untreated cells showed that AZA induces a large-scale gene upregulation (87% differentially-expressed transcripts were upregulated). Among them, 38% were EREs and 6% were CTAs, suggesting that AZA-induced MAPs have greater chances of deriving from EREs than from CTAs. However, we could not identify a single ERE-derived upregulated MAP among the immunopeptidome of AZA-treated AML cells while multiple CTA-derived MAPs (0.4 - 0.7% of the upregulated MAPs) were presented at greater levels by AZA. Because a GO-term analysis of the upregulated protein-coding genes evidenced a robust innate immune response in AZA-treated cells, we conclude that AZA-induced enhanced CTL recognition is more dependent on CTA- than on ERE-derived MAP presentation and that ERE enhanced expression could rather trigger the observed innate immune response. An in-depth analysis of the immunopeptidome showed that MAPs having an altered presentation following AZA treatment derived only partially from transcripts whose expression was affected by AZA. The analysis of the other AZA-induced MAPs showed that they resulted preferentially from the activity of the constitutive proteasome (vs the immunoproteasome) and derived preferentially from proteins having less aromatic residues (and therefore more prone to misfolding). Because the degradation of misfolded proteins by the constitutive proteasome can indicate that AZA induces an unfolded protein response, we have examined whether AZA stimulates autophagy, a process frequently triggered in association with such stress. Accordingly, flow cytometry assays on AML cells treated with AZA for 24h evidenced a robust autophagy induction. Summary/Conclusion: Altogether our results show that AZA promotes the presentation of CTA-derived rather than ERE-derived MAPs and that autophagy induction could enable the survival of AML cells to AZA-induced proteotoxic stress. Our findings suggest that autophagy inhibitors could synergize with AZA in AML therapy. P401: NEXT-GENERATION FLOW CYTOMETRY TO DETECT MEASURABLE RESIDUAL DISEASE IN ACUTE MYELOID LEUKEMIA USING A 19-COLOR FULL SPECTRUM FLOW CYTOMETRY PANEL H. Fokken1,*, N. Kattre1, A. Kloos1, M. Heuser1, T. Kacprowski2,3, M. Tobias4, A. Schwarzer1,5 1Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover; 2Division Data Science in Biomedicine, Peter L. Reichertz Institute for Medical Informatics of TU Braunschweig and Hannover Medical School; 3Braunschweig Integrated Centre of Systems Biology (BRICS), TU Braunschweig, Braunschweig; 4Department of Pediatric Hematology; 5Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany Background: The detection of residual leukemic cells (“measurable residual disease”/MRD) has dramatic prognostic impact on relapse incidence and patient survival in AML and has become key to guide therapeutic decision making. MRD can be measured by PCR, next-generation sequencing, and conventional multicolor flow cytometry (Flow-MRD, 8-10 colors), respectively. Flow-MRD relies on the aberrant expression of surface marker combinations on leukemic blasts (leukemia-associated immunophenotypes - LAIPs), which are not found on normal hematopoietic cells. Hence, the challenge of Flow-MRD is to detect and track small populations of leukemic cells against the complex and heterogenous background of human bone marrow cells. With the development of next-generation Spectral Flow Cytometers, it is now possible to stain cells with 20-30 colors at once which would greatly increase the number of LAIPs that can be tracked and increase sensitivity of Flow-MRD when the number of available cells is limited. We hypothesized that spectral Flow-MRD allows for monitoring leukemic disease burden with unprecedented resolution. Aims: To establish a one-tube full spectrum MRD detection assay and a semi-automated computational workflow for improved detection of minimal residual disease in AML. Methods: We established a 19-color full spectrum flow cytometry panel with one tube containing CD45, CD34, CD117, CD13, CD33, HLA-DR, CD2, CD4, CD5, CD7, CD11b, CD14, CD15, CD19, CD22, CD38, CD56, CD64 and CD133. Limit of detection and quantification, linearity, intra-/inter-assay precision, and robustness were determined via spike-in assays. We measured normal bone marrow of healthy donors and MRD samples from AML patients. Data were analyzed with GemStone using its unique highly automated approach to characterizing normal cells in bone marrow based on a template model designed specifically for data generated with this panel. Results were then explored with the Cen-Se dimensionality reduction algorithm (Bagwell et al. J Biom Biostat (2019)) and subsequently compared to expert gating of data generated on the same samples using a well-established 5-Tube conventional flow cytometry panel. Results: We defined reference values for known leukemia-associated immunophenotypes using normal bone marrow. Expert analysis of data simultaneously analyzed with the full-spectrum panel and the 5-tube conventional flow cytometry panel yielded highly correlating results for the proportions of granulocytes, lymphocytes, monocytes, and immature blasts in measured bone marrow with r2 values greater than 0.90. In addition, quantitative and qualitative MRD results correlated very well when comparing semi-automated analysis of our full-spectrum panel using GemStone and expert analysis of the 5-tube conventional flow cytometry panel. Summary/Conclusion: We have developed an integrated pipeline for full-spectrum flow cytometry that improves the applicability and performance of flow-cytometry-based MRD detection in AML. P402: MUTATIONS IN BONA FIDE ONCOGENES KRAS AND DNMT3A ARE NOT RELATED TO DEPENDENCY IN ESTABLISHED TUMORS, IN PDX MODELS OF ACUTE LEUKEMIA IN VIVO Y. Gao1,*, M. Ghalandary1, M. Becker1, D. Amend1, M. Rothenberg-Thurley2, K. Metzeler2,3, I. Jeremias1,4,5 1Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU); 2Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, Ludwig Maximilians University (LMU), Munich; 3Department of Hematology and Cell Therapy, University Hospital Leipzig, Leipzig; 4German Cancer Consortium (DKTK), Partner Site; 5Department of Pediatrics, University Hospital, Ludwig Maximilians University (LMU), Munich, Germany Background: The role of oncogenic mutations during the process of tumor formation was intensively studied in genetically engineered mouse models, while much less is known about their function in established tumors of patients. Aims: To bridge the gap, we studied frequently mutated genes for their dependency in established tumors in vivo, using genetic engineering in patient-derived xenograft (PDX) models. Methods: Primary tumor cells from seven patients with acute myeloid leukemia (AML) were transplanted into immunocompromised NSG mice. PDX cells were lentivirally transduced to express the Cas9 protein and a sgRNA. The customized sgRNA library was designed and cloned using our CLUE platform (www.crispr-clue.de; Becker et al., Nucleic Acids Res. 2020). PDX cells transgenic for Cas9 and the CRISPR/Cas9 sgRNA library were transplanted into NSG mice, grown until advanced AML disease and sgRNA distribution measured by next-generation sequencing. From dropout hits in PDX in vivo screens, single molecules were validated by fluorochrome-guided competitive in vivo experiments, comparing cells with and without knockout in the same mouse and determining cell distributions by flow cytometry gating on the different recombinant fluorochromes. Human AML cell lines were studied in vitro for comparison. Results: We cloned a small customized sgRNA library targeting 34 genes recurrently mutated in AML and tested the library in two PDX AML models in vivo. In hit validation experiments, knockout of NPM1 abrogated in vivo growth in all PDX AML models tested, reproducing the known common essential function of NPM1. KRAS proved an essential function in PDX AML models both with and without an oncogenic mutation in KRAS, suggesting that patients suffering tumors both with and without KRAS mutation might benefit from treatment inhibiting KRAS. DNMT3A belongs to the genes most frequently mutated in AML, but most AML cell lines tested in vitro did not depend on DNMT3A and knockout of DNMT3A did not reduce, but rather increased AML cell line growth in vitro. In contrast, in PDX models in vivo, we found a clear essential function for DNMT3A in certain AML samples and PDX in vivo results were discordant to cell line in vitro data. These data highlight the complementary use of PDX models to study gene dependencies. To our surprise, the dependency of AML cells on DNMT3A was unrelated to the presence or absence of a hot spot mutation in DNMT3A. Summary/Conclusion: We conclude that both KRAS and DNMT3A harbor an essential function in certain patients established AML tumors in vivo, although independently from the presence of absence of a mutation in the gene. Warranting verification in additional patient samples, oncogenes and tumor entities, our data might indicate re-consider basic principles of decision making in Molecular Tumor Boards. P403: PROGNOSTIC SIGNIFICANCE OF LONG NON-CODING RNA GAS5 AND MICRORNA-222 EXPRESSION PROFILES IN YOUNGER AML PATIENTS Ð. Pavlović1, N. Tošić1, B. Zukić1, I. Marjanović1, Z. Pravdić2, N. Suvajdžić Vuković2,3, S. Pavlović1, V. Gašić1,* 1Institute of Molecular Genetics and Genetic Engineering; 2Clinic of Hematology; 3Faculty of Medicine, Belgrade, Serbia Background: Acute myeloid leukemia (AML) is a heterogeneous malignant disease, that accounts for 80% of all acute leukemias in adults. The treatment protocol and prognosis for AML are based on the classification of the patients into risk groups based on the pretreatment karyotype analysis. However, nearly half of the patients don’t have any detectable cytogenetic changes. Therefore, there is a constant need to find new prognostic markers, as well as markers that can be used as targets for therapeutics that could improve the treatment. Recently, using high-throughput technologies, the search for new biomarkers has pointed towards non-coding RNAs, especially long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs). Numerous studies have shown lncRNA GAS5 expression level dysregulation in multiple cancers, but it was poorly investigated in AML. Additionally, miR-222 expression levels have shown significant influence on cancers in general, including AML. Since GAS5 acts like a molecular sponge for miR-222, co-expression profiles of these non-coding RNAs could be novel prognostic markers in AML. Aims: We have investigated the expression level pattern of lncRNA GAS5 in younger AML patients and examined its potential influence on disease outcome. GAS5 expression levels were also investigated in AML patients with normal karyotype, while taking into account miR-222 expression levels, in order to examine their potential role as dual biomarkers. Methods:: 94 bone marrow samples of younger (<65 years) AML patients have been collected. Diagnostics and stratification were based on cytomorphology, immunophenotyping and cytogenetic analysis. All patients received the same induction chemotherapy. Along with them, 14 healthy controls were analysed. The AML patient group with normal karyotype (AML-NK) had 39 patients. GAS5 and miR-222 expression levels were analysed using Real-Time PCR. Relative quantification was performed using the ddCt method, respective healthy controls were used as the calibrator for each non-coding RNA. The ROC curve analyses found the optimal cut-off value to discriminate between Gas5high and GAS5low. While the cut-off value for dividing miR-222 expression levels into miR-222high and miR-222low was performed using the value of the median level of expression among healthy controls. Results: Our results showed that GAS5 expression level in AML patients was lower compared to healthy controls (p < 0.01). Lower GAS5 expression on diagnosis was related to an adverse prognosis, since higher levels of lactate dehydrogenase was detected in the GAS5low group compared to the GAS5high group (p < 0.01). The disease-free survival (DFS) and the overall survival (OS) were significantly lower in the GAS5low group, compared to the GAS5high group, but survival analysis failed to confirm this finding. In the AML-NK group patients had higher expression of miR-222 compared to healthy controls (p < 0.01). A synergistic effect of GAS5low/miR-222high status on disease prognosis was not established. Summary/Conclusion: This is the first study focused on examining the lncRNA GAS5 and miR-222 expression pattern in AML patients. Its initial findings indicate potential prognostic significance of GAS5 expression and the need for further investigation of these two non-coding RNAs, their potential roles in leukemogenesis, and the prognosis of AML patients. P404: PROGNOSTIC IMPACT OF CEBPA-MUTATIONAL SUBGROUPS IN ADULT AML – RESULTS OF A LARGE METAANALYSIS IN MORE THAN 1000 CEBPA-MUTANT PATIENTS J.-A. Georgi1,*, S. Stasik1, M. Kramer2, C. Röllig1, T. Haferlach3, P. Valk4, D. Linch5, T. Herold6, N. Duployez7, F. Taube1, U. Platzbecker8, H. Serve9, C. Baldus10, C. Müller-Tidow11, C. Haferlach3, M. Meggendorfer3, S. Koch3, E. L. Boertjes4, R. K. Hills12, A. Burnett13, G. Ehninger2, K. Metzeler8, M. Rothenberg-Thurley6, A. Dufour6, H. Dombret14, C. Pautas15, L. Fenwarth7, M. Bornhäuser1, R. Gale5, C. Thiede1,16 1Medizinische Klinik und Poliklinik I, Universitätsklinikum Dresden; 2AvenCell Europe GmbH, Dresden; 3MLL Münchner Leukämielabor GmbH, München, Germany; 4Erasmus University Medical Center, Rotterdam, Netherlands; 5Department of Haematology, UCL Cancer Institute, London, United Kingdom; 6Labor für Leukämiediagnostik, Medizinische Klinik und Poliklinik III, Universitätsklinikum München, München, Germany; 7Institut de Recherche contre le Cancer de Lille, Centre Hospitalier Universitaire de Lille, Lille, France; 8Klinik und Poliklinik für Hämatologie, Zelltherapie und Hämostaseologie, Universitätsklinikum Leipzig, Leipzig; 9Medizinische Klinik 2, Universitätsklinikum Frankfurt, Frankfurt am Main; 10Klinik für Innere Medizin II, Universitätsklinikum Schleswig-Holstein, Kiel; 11Klinik für Hämatologie, Onkologie und Rheumatologie, Universitätsklinikum Heidelberg, Heidelberg, Germany; 12Nuffield Department of Population Health, Oxford University, Oxford; 13Department of Haematology, Cardiff University, University Hospital of Wales, Cardiff, United Kingdom; 14Institut de Recherche Saint-Louis, Université de Paris, Hôpital Saint-Louis, Paris; 15Service d’Hématologie Clinique, Hôpital Henri Mondor, Créteil, France; 16AgenDix GmbH, Dresden, Germany Background: CEBPA double mutations (dm) are included in the current AML WHO-classification as a favorable-risk independent entity, but recent data suggest this might be limited to those approximately 90% of patients (pts) with in-frame mutations in the basic leucine zipper (bZIP) domain, and that bZIP single allele mutations (sm) might also be favorable. Aims: To investigate further, we performed a meta-analysis involving detailed sequencing data from 1010 CEBPA-mutant pts. Methods: Primary pt data was obtained from six study groups: 98 from ALFA, 104 AMLCG, 191 HOVON, 200 MLL, 240 SAL, 177 UK(MRC/NCRI). All pts were treated with curative intention using age and risk-adapted strategies. CEBPA sequences were classified by localization (bZIP vs transactivation domains [TAD] 1 + 2), allelic status (sm vs dm), and type of mutation (in-frame [if] or frameshift [fs] insertions/deletions [InDel] or missense [ms]). Data for GATA2, WT1, FLT3, NPM1 co-mutations were available for most pts. Results: Overall, 546 (54%) had dm and 464 (46%) sm CEBPA (289 TAD1/2; 175 bZIP). To study the impact of the different mutations, we generated 8 groups: dmCEBPA pts with 1/more bZIPInDel-if (Gr1), bZIPInDel-fs (Gr2) or bZIPms (Gr3) mutation or 2 TAD mutations (Gr4), as well as the corresponding sm groups (Gr5-8). Of note, dm/sm pts with bZIPInDel-if mutations (Gr1 + 5) were significantly younger and predominantly had de novo AML (Tab.1) whereas Gr2-4 and Gr6-8 pts were older with higher incidence of sAML. GATA2 and WT1 mutations were more common in Gr1 + 5 pts; mutations in NPM1, spliceosome (SF3B1, U2AF1, SRSF2 and ZRSR2) and DNA-methylation (ASXL1, DNMT3A, TET2, IDH1/2) associated genes were confined to the other groups. Pts in Gr1 showed the highest rate of CR1 (Tab.1), better RFS and OS (Fig. 1), whereas dmCEBPA pts in Gr2-4 did significantly worse. Outcome of Gr5 pts was less favorable than Gr1, but still better than for the other subgroups (Gr2-4 and 6-8). In a combined analysis of Gr2-4 and Gr6-8, prognostic factors according to ELN2017 identified three clearly separated subgroups, with the favorable subgroup predominantly associated with mut. NPM1; Fig. 1B). The prognostic impact of Gr1 for CR, OS and RFS was confirmed in multivariable analyses taking into account the individual study groups. dmCEB P AbZIPInDel-fs dmCEBPAbZIPInDel-if n=425Gr1 dmCEBPAbZIPInDel-fs n=26Gr2 dmCEBPAXbZIPmsnn=35Gr3 dmCEBPANNTADNNNn=60Gr4 smCEBPAbZIPInDel-if n=66Gr5 smCEBPAbZIPInDel-fs n=55Gr6 smCEBPAXbZIPmsnn=54Gr7 smCEBPANNTADN.Nn=289Gr8 p.value BPANDN289 Median age, years 42 60 57 64 47 59 52 58 <.001 AML type, % <.001 de novo 98 81 94 88 92 96 90 90 sAML/tAML 2 20 6 13 8 4 10 10 FLT3-ITD mut ,.% 11 8 9 7 8 29 9 32 <.001 NPM1 mut , % 1 12 9 13 3 29 15 44 <.001 GATA2 mut , % 39 8 12 9 33 10 12 4 <.001 WT1 mut , % 20 4 15 3 13 4 6 8 <.001 Spliceosome mut., % 2 29 6 40 5 39 25 25 <.001 Methylation mut., % 25 83 69 71 35 74 46 63 <.001 CR1, % 94.3 73.9 78.1 73.1 92.1 77.6 79.6 73.5 <.001 Median RFS, months 152 9 15 16 64 9 16 22 <.001 Median OS, months 215 16 40 22 126 70 30 41 <.001 Image: Summary/Conclusion: The results of this large metaanalysis confirm our previous results that only dmCEBPA with InDel-if mutations show a specific biology and very favorable prognostic implications, and further supports the notion that the subgroups with other dm pts differ substantially and have a significantly worse outcome. SmCEBPA bZIPInDel-if mutant pts and those with other CEBPA mutations and favorable ELN2017 genotype also show an improved outcome. P405: INHIBITION OF CKS1-DEPENDENT PROTEOSTASIS REVEALS VULNERABILITIES IN LEUKAEMIC STEM CELLS WITH CONCOMITANT PROTECTION OF HEALTHY HAEMATOPOIETIC STEM CELLS W. Grey1,2,*, A. Rio-Machin3, P. Casado-Izquierdo3, E. Gronroos2, S. Ali2, J. Miettinen4, F. Bewicke-Copley3, A. Parsons4, C. Heckman4, C. Swanton2, P. Cutillas3, J. Gribben3, J. Fitzgibbon3, D. Bonnet2 1York Biomedical Research Institute, The University of York, York; 2The Francis Crick Institute; 3Bart’s Cancer Institute, London, United Kingdom; 4HiLIFE, Helsinki, Finland Background: Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low two-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols to deplete LSCs, and the significant toxicity towards healthy hematopoietic cells. Whilst much work has been done to identify genetic and epigenetic vulnerabilities in AML LSCs, little is known about protein homeostasis – so called “Proteostasis” – in drug resistance and relapse. Aims: - Characterisation of proteostatic targeting in AML and haematopoiesis. - Identify proteostatic vulnerabilities in LSCs. Methods: This study used a range of primary patient AML samples, highthroughput drug screening, in vivo transplantation models and proteomics to demonstrate mechanism of action and functionality of a small molecule inhibitor of CKS1-dependent protein degradation. Results: We tested a range of proteostatic targeting agents against primary, poor risk, AML patient samples and demonstrate a selective vulnerability of both AML blasts and LSCs to inhibition of CKS1-dependent protein degradation in vitro and in vivo using a small molecule inhibitor. Mechanistically, inhibition of CKS1 leads to hyperactivation of RAC1, increased NADPH production and critical levels of intracellular ROS, resulting in death of LSCs. Conversely, CKS1 inhibition has the opposite effect on healthy haematopoiesis. Healthy haematopoietic stem and progenitor cells (HSPCs) display increased quiescence upon inhibition of CKS1, a phenotype which confers chemoprotection of healthy HSPCs during clinical chemotherapy protocols (Cytarabine + Doxorubicin, 5 + 3). A phenotype conserved by intestinal stem and progenitor cells. Summary/Conclusion: Together these findings demonstrate a key vulnerability of AML-LSCs to CKS1-inhibition, while it preserves healthy stem cells. This offers the prospect of both depleting LSCs in vivo and bringing back clinically unfit patients into the pool for intensive chemotherapeutic treatment. P406: ENHANCED SIGNIFICANCE OF FLT3-ITD RESIDUAL DISEASE DETECTION ON TREATMENT OUTCOME IN ACUTE MYELOID LEUKEMIA T. Grob1,*, M. Sanders1, C. Vonk1, F. Kavelaars1, M. Rijken1, D. Hanekamp1, P. Gradowska1, J. Cloos2, Y. Fløisand3, M. V. Marwijk Kooy4, M. Manz5, G. Ossenkoppele2, L. Tick6, V. Havelange7, B. Löwenberg1, M. Jongen-Lavrencic1, P. Valk1 1Hematology, Erasmus Medical Center, Rotterdam; 2Hematology, Amsterdam University Medical Center, Amsterdam, Netherlands; 3Hematology, Oslo University Hospital, Oslo, Norway; 4Hematology, Isala Hospital, Zwolle, Netherlands; 5Hematology, University Hospital Zurich, Zurich, Switzerland; 6Hematology, Maxima Medisch Centrum, Eindhoven, Netherlands; 7Hematology, Cliniques Universitaires Saint-Luc, Brussels, Belgium Background: The prognostic significance of FLT3-internal tandem duplications (FLT3-ITD) in acute myeloid leukemia (AML) in relation to the allelic mutational burden and other concurrent gene mutations at diagnosis remains subject of scientific controversy. Increasing evidence indicates that treatment outcome prediction can be improved by assessing the kinetics and depth of response during therapy by detection of minimal residual disease (MRD). Until now, FLT3-ITD MRD detection by RQ-PCR has been hampered by the variety in patient specific FLT3-ITD (i.e. sequence, position and length) and is currently not recommended because of potential instability of FLT3-ITD clones during relapse. However, systematic studies evaluating the applicability of FLT3-ITD MRD detection with next-generation sequencing (NGS) in AML are currently lacking. Aims: Here, we set out to compressively investigate the impact of NGS-based FLT3-ITD MRD detection on treatment outcome in a cohort of newly diagnosed patients with AML in the context of current prognostic factors at diagnosis and other MRD measurements during therapy, including mutant NPM1 and multiparameter flow cytometry (MFC). Methods: In 176 de novo FLT3-ITD AML patients who were treated in HOVON-SAKK multicenter prospective phase III clinical trials, NGS was performed at diagnosis and in complete remission (CR) following two cycles of standard induction chemotherapy. The NGS libraries were paired-end sequenced (2×221-bp) with custom primers on an Illumina MiSeq according to manufacturer’s recommendation (Illumina, San Diego, CA). We used our in-house data analysis pipeline for variant calling. The primary endpoints of the study were relapse and overall survival. The Cumulative Incidence of Relapse (CIR) was estimated with competing-risks regression analyses according to the method of Fine & Gray. The Cox proportional hazard model was used to calculate overall survival estimates. Results: NGS-based FLT3-ITD MRD was present in 43 of 176 (24%) AML patients with a median variant allele frequency of 0.01% (range 3.1x10-4% to 3.10%). Presence of FLT3-ITD MRD was associated with increased risk of relapse (4-year risk of relapse, 79% FLT3-ITD MRD vs. 34% no FLT3-ITD MRD; P<0.001) and inferior overall survival (4-year rate of overall survival, 27% FLT3-ITD MRD vs. 57% no FLT3-ITD MRD; P<0.001). In multivariate analysis, detection of FLT3-ITD MRD in CR confers independent prognostic significance for relapse (hazard ratio, 4.00; P<0.001) and overall survival (hazard ratio 2.78; P<0.001). Strikingly, FLT3-ITD MRD exceeds the prognostic value of most generally accepted clinical and molecular prognostic factors, including FLT3-ITD allelic ratio at diagnosis and MRD assessment by NGS-based mutant NPM1 detection or MFC. Image: Summary/Conclusion: NGS-based detection of FLT3-ITD MRD in CR identifies AML patients with a profound risk of relapse and death that outcompetes the significance of most accepted prognostic factors at diagnosis and during therapy, and furnishes support for FLT3-ITD as a clinically relevant biomarker for dynamic disease risk assessment in AML. P407: SYNERGISTIC EFFECT OF A NOVEL COMBINATION OF HDACI CHIDAMIDE WITH CLADRIBINE ON CELL PROLIFERATION ARREST AND APOPTOSIS IN ACUTE MYELOID LEUKEMIA BY TARGETING MYC/HDAC2 SIGNALING S. Gu1, C. Song2,3, Z. Ge1,* 1Department of Hematology, Zhongda Hospital, School of Medicine, Southeast University, Institute of Hematology Southeast University, Nanjing, China; 2Hershey Medical Center, Pennsylvania State University Medical College, Hershey; 3Division of Hematology, The Ohio State University Wexner Medical Center, the James Cancer Hospital, Columbus, American Samoa Background: Chidamide (CHI), a novel Histone deacetylase inhibitor (HDACi), which is now involved in the salvage treatment of relapsed/refractory acute myeloid leukemia in a clinical trial. Cladribine (2-chloro-2’-deoxyadenosine (2-CdA)), is a purine nucleoside antimetabolite analog, which resists degradation by adenosine deaminase and therefore accumulates to cytotoxic levels. The anti-leukemia effects of either single drug have been reported by inducing cell apoptosis through suppressing multi-oncogenic pathways in human tumor cells. However, whether and how the combination of the two drugs exerts the synergistic anti-tumor effects are still undetermined in AML. Aims: This study is to investigate the synergistic therapeutic effects of 2-CdA in combination with CHI in AML. Methods: Cell Counting Kit-8 (CCK-8) cell proliferation assay, Annexin V apoptosis assay, Propidium Iodide cell cycle assay, and Western Blot (WB) were performed in U937 AML cells in presence of CHI only, 2-CdA only, CHI+ 2-CdA, and vehicle control for 48h. RNA-seq was performed in U937 cells treated with CHI and 2-CdA; the differentially expressed genes (DEGs) were identified by R studio, and pathway enrichment of the DEGs was analyzed with Metascape online tool. Co-Immunoprecipitation (co-IP) was used to examine the interaction of cMYC with HDAC1/2. Data were expressed as the mean ± standard deviation. T-tests statistical analysis was employed for determining the significant difference between groups. A synergistic effect was determined by the combination indices (CIs) with CompuSyn software. Results: The 50% inhibiting concentration (IC50) of CHI and 2-CdA on U937 cell proliferation is 9.358±1.448μmol/L and 0.024±3.071μmol/L, respectively. The combination of various doses of CHI with either IC50 or 1/2 IC50 of 2-CdA significantly decreases the survival rate of U937 cells compared to single drug control (Fig.1a). The CI analysis showed the high synergistic therapeutic effects of CHI with ether dose of 2-CdA (Fig. 1b). The combination of (8mM) CHI with (0.02mM) 2-CdA ignificantly elevated the apoptosis rate and induced the G0/G1 cell phase arrest of AML cells compared with either single drug alone (p<0.0001, Fig. 1c, d). Moreover, the expression of apoptotic markers, caspase-3, PARP-1, and caspase-9 is elevated, and the cell cycle marker p21 proteins were significantly upregulated, but Cyclin-dependent kinase CDK2 and Cyclin E2 down-regulated in the combination group compared to either single drug control (p<0.05, Fig. 1e). To further understand the underlying mechanism of the synergy, RNA-seq analysis was performed in U937 cells treated with either CHI or 2-CdA, respectively. The overlapped altered DEGs were identified, and cMYC is dramatically down-regulated. The pathway analysis of the altered DEGs showed the enrichment of MYC pathways further indicated the critical role of MYC (Fig. 1f). It is reported that cMYC is associated with the histone deacetylase complex. Indeed, the co-IP assay showed an interaction of MYC with HDAC2, not HDAC1 in U937 (Fig. 1g). These data suggested the role of cMYC/HDAC2 signaling in the synergistic effect of the combination of CHI with 2-CdA in the disease. Image: Summary/Conclusion: The combination of Chidamide and Cladribine has a synergistic effect on cell proliferation arrest, apoptosis, and cell cycle arrest in AML cells through MYC/HDAC2 pathway. Our data provide experimental evidence for the novel potential combination of AML therapy. More in vivo pre-clinical studies will be explored for future clinical trials. P408: NRAS MUTATIONS ARE INVOLVED IN ACUTE MYELOID LEUKAEMIA DRUG RESISTANCE F. Healy1,*, V. Marensi1, J. Woolley1, D. MacEwan1 1Department of Pharmacology and Therapeutics, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United Kingdom Background: Acute Myeloid Leukaemia is the most common adult leukaemia, typically associated with poor prognosis. While rates of response to initial cytarabine-based induction chemotherapy are high, the majority of patients will relapse even with haematopoietic stem cell transplant. Thus, the addition of targeted therapies to induction regimens are becoming more widespread, particularly the use of midostaurin and gilteritinib. Even so, resistance is emerging to FLT3 inhibitors, and can often be attributed to NRAS mutations. Aims: Here, we use over-expression and CRISPR-Cas9 to assess the involvement of NRAS mutations in FLT3-inhibitor resistance cell line models (MOLM-13, FLT3-ITD heterozygous and MV4-11, FLT3-ITD homozygous) that had been previously generated in our lab. Methods: Drug sensitivity was determined by quantification of apoptosis 72 hours post-treatment (Annexin-V/Propidium Iodide staining). NRAS mutational hotspots associated with increased activation and drug resistance were analysed by Sanger Sequencing. NRAS over-expression and knock-out cell lines were generated using either transfection (HEK293T) or lentiviral transduction (MOLM-13 and MV4-11). Signalling dynamics driving resistance were determined by immunoblotting. Results: Mutations arose in the MOLM-13-resistant and MV4-11 resistant cell lines at common NRAS mutational hotspots (Q61 and G12, respectively). NRAS Q61K over-expression in the parental MOLM-13 cell line decreased sensitivity to the FLT3 inhibitors gilteritinib, quizartinib, as well as cytarabine, relative to the parental cell line. However, they remained more sensitive to FLT3 inhibition and cytarabine than the resistant cells. In contrast, NRAS-Q61K over-expression caused ~3-fold decrease in sensitivity to trametinib, the MEK inhibitor that acts downstream of NRAS, relative to both the MOLM-13 and MOLM-13-resistant cell lines (Table 1). MOLM-13-NRAS-Q61K over-expression cells showed a growth advantage compared to parental cells in normal culture. In MV4-11-NRAS-Q61K cells, preliminary mechanistic analysis has shown a decrease in ERK phosphorylation. Conversely, CRISPR-Cas9-mediated NRAS knockout in HEK293T appeared to increase ERK activity. Image: Summary/Conclusion: NRAS mutations contribute to a FLT3-inhibitor-resistant phenotype, confer a growth advantage in FLT3-ITD+ AML and may act independently of ERK. We have engineered the MOLM-13-resistant and MV4-11-resistant cell lines to express Cas9 under a doxycycline-inducible promoter, to permit stable reversion of the respective mutations. Future work will use these cells to create a CRISPR-mediated reversion of the NRAS mutations which have arisen in the resistant cell lines, to better understand the role of NRAS mutations in FLT3-ITD+ AML. We will better characterise the impacts of a host of NRAS mutations within AML using over-expression and knockout cell lines, and assess drug sensitivity and subsequent pathway re-wiring. P409: NPM1 MUTATED SAMPLES ARE ESPECIALLY VULNERABLE WHEN TARGETING RAC1 IN PATIENT-DERIVED AML CELLS IN VITRO A. Hemsing1,2,*, K. P. Rye2, K. J. Hatfield3, H. Reikvam1,2 1Department of Medicine, Haukeland University Hospital; 2Department of Clinical Science, University of Bergen; 3Department of Immunology and Transfusion Medicine, Haukeland University Hospital, Bergen, Norway Background: Ras-related C3 botulinum toxin substrate 1 (Rac1) is a GTPase signaling molecule known to be overexpressed in many solid cancers and leukemia. Rac1 plays a part in several pathways regulating, e.g., cell migration, adhesion, and cell proliferation. Recent studies have revealed activation and widespread implication of the Rac1 molecule in acute myeloid leukemia (AML), and in vitro Rac1 inhibition in AML cell lines induces apoptosis and improves chemosensitivity. Aims: We studied the in vitro effect on proliferation, apoptosis, and cytokine release of five RAC1 inhibitors in a cohort of heterogeneous, consecutive patient-derived AML cells at diagnosis. We aimed to map the antileukemic effect in a relatively large AML cohort to identify potential subgroups of AML patients that could be particularly vulnerable to RAC1 inhibition. Methods: 79 patient-derived AML cell samples from diagnosis were thawed after preservation in liquid nitrogen and cultured at standard in vitro methods with the Rac1-inhibitors ZINC69391, ITX3, EHOP-16, 1A-116, and NSC23766. Cell proliferation was measured by the 3H-thymidine incorporation assay, and apoptosis was measured by the Annexin V/Propidium Iodine apoptosis assay for flow cytometry. Assessment of cytokine profiles in culture media was done with Luminex multiplex immunoassay. Results: Measured IC50 values for each compound were as follows; ZINC69391 23 µM (95% CI 15 -29 µM), ITX3 24 µM (15 – 40µM), EHOP-16 3 µM (1,8 – 3,9 µM), 1A-116 10 µM. (8-11 µM) and NSC23766 12 µM (9-15 µM). All five Rac1 inhibitors resulted in an overall antiproliferative effect, tested at dosage around IC80. Using bioinformatical classification, we observed that high versus intermediate/low antiproliferative effect was more common in NPM1 mutated (Fischer’s exact test, p=0.002) and CD34 negative (p=0.008) patient samples. The differences were insignificant regarding the presence of FLT3 mutation or according to cytogenetics, FAB classification, gender, and age. All five compounds did induce significant apoptosis and necrosis compared to untreated control by Wilcoxon signed-rank test (p< 0.0001) at dosages around IC40 and IC80. For NSC23766 only, the presence of NPM1 mutation was associated with more severe apoptosis and necrosis (p=0.01). The presence of FLT3 mutation or CD34 negativity or positivity did not influence the depth of apoptosis and necrosis. Presence of NPM1 mutation was associated with reduced viability after treatment with EHOP-16 (p=0.025), ITX3 (p=0.047) and NSC23766 (p=0.005). CD34 negativity was significantly associated with reduced viability after treatment with NSC23766 (p=0.006). Finally, Rac1 inhibition significantly reduced the cytokine release of several cytokines crucial for leukemogenesis, with the strongest effects observed for Rac1 inhibitors 1A-116 and NSC23766 (Figure 1, Euclidean clustering). Image: Summary/Conclusion: Our results suggest potent and significant antiproliferative and antiapoptotic effects of Rac1 inhibition in primary AML samples. Interestingly, patients harboring the NPM1 mutation seem significantly more vulnerable to such inhibition. Our findings support further research regarding the role of Rac1 and its pharmacological inhibition in AML. P410: LPIN1 REGULATES LIPID HOMEOSTASIS AND MAINTAINS STEM CELL ENRICHED POPULATIONS IN AML K. Huber1,2,*, S. Garg3, L. He1,4, A. Pouya1, C. Rohde1,4, C. Lüchtenborg5, C. Arnold4,6, J. Zaugg4,6, S. Raffel1, C. Müller-Tidow1,4, B. Brügger5, C. Pabst1,4 1Medizinische Klinik V für Hämatologie, Onkologie und Rheumatologie, Universitätsklinikum Heidelberg, Heidelberg; 2Medizinische Klinik II für Hämatologie und Onkologie, Universitätsklinikum Schleswig-Holstein, Kiel, Germany; 3Dana-Farber Cancer Institute, Boston, United States of America; 4Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory (EMBL); 5Heidelberg University Biochemistry Center (BZH); 6European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Background: Leukemic stem cells (LSCs) often escape chemotherapy treatment due to the acquisition of stem cell like properties such as slow cell cycle progression, which makes them main drivers of cytologic relapse in acute myeloid leukemia (AML). Identification of novel therapeutic targets to specifically eradicate residual chemoresistant AML cells therefore represents a key challenge in AML therapy. Since LSCs show altered metabolic features, which include dependency on low ROS levels and oxidative phosphorylation of amino acids, targeting LSC metabolism has evolved as a promising approach. Recently the phospholipid phosphatidylserine was shown to be required for LSC stemness and differentiation. However, the role of the main phospholipid synthesis pathway in which the phosphatidic acid phosphatase (PAP) lipin1 catalyzes a rate limiting step remains largely unexplored. Aims: We investigated the role of the phosphatidic acid phosphatase LPIN1 in AML and healthy hematopoietic cells. Methods: We applied a gene depletion approach using shRNAs and CRISPR/Cas9 on AML cell lines, primary AML samples and cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) to monitor the effects of LPIN1 depletion on proliferation, differentiation, colony forming capacity and bone marrow engraftment in mice. To gain insights into the mechanisms of action, we combined RNA-sequencing (RNA-seq) with lipidomics assays. Results: Here we show that regulation of lipid homeostasis by lipin1 is required for HSPC and LSC function. RNA-seq data analysis of healthy hematopoietic cells revealed highest LPIN1 expression in the immature CD34 positive compartment, which steadily decreases during myeloid differentiation. In normal karyotype (NK) AML LPIN1 was utmost expressed in an AML subpopulation characterized by high LSC frequency and co-positivity for the LSC markers CD34 and GPR56. Knock down (KD) of LPIN1 in CD34 positive HSPCs and patient derived xenograft (PDX) leukemic cells hampered proliferation and reduced the CD34 positive LSC compartment in vitro. In xenotransplantation assays LPIN1 KD reduced bone marrow engraftment of healthy HSPC and human AML cells. Furthermore, in vivo differentiation of CD34 positive cells into B-cells was almost completely abrogated upon LPIN1 KD. In the leukemia setting, LPIN1 KD particularly reduced the LSC-enriched CD34+GPR56+ co-positive fraction. While RNA-seq of the AML cell line OCI-AML3 and CD34 positive cells showed only subtle gene expression changes in phospholipid metabolism upon LPIN1 KD, lipidomics analyses revealed significant differences in the composition of phospholipids upon LPIN1 KD. Summary/Conclusion: Our data establish LPIN1 as an essential enzyme maintaining lipid homeostasis and stem cell functions in the hematopoietic system. Identification of phospholipid entities with differential impact on healthy versus leukemic cells might help refine LSC targeted therapies. P411: SF3B1 MUTATIONS IN AML ARE STRONGLY ASSOCIATED WITH MECOM REARRANGEMENTS AND MAY BE INDICATIVE OF AN MDS PRE-PHASE S. Huber1,*, S. Hutter1, M. Meggendorfer1, G. Hoermann1, W. Walter1, C. Baer1, W. Kern1, T. Haferlach1, C. Haferlach1 1MLL Münchner Leukämie Labor GmbH, München, Germany Background: Mutations in the splicing factor gene SF3B1 are most frequent in myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN) but are also recurrently found in acute myeloid leukemia (AML). Splicing factor mutations have been suggested to have additive value if incorporated into the current risk classification of AML (van der Werf et al., Blood Advances 2021). Aims: Explore the genetic landscape of SF3B1 mutated AML and determine the prognostic impact of SF3B1 mutations. Methods: We analyzed 735 patients diagnosed with AML based on cytomorphology, cytogenetics, and molecular genetics according to WHO classification 2017. Reads from amplification-free whole genome sequencing (WGS, median coverage 103x, Illumina, San Diego, CA) were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and variants were called using Strelka Somatic Variant Caller v2.4.7. Results: We identified SF3B1 mutations in 41 of 735 (6%) AML patients. SF3B1 mutations were found in AML with recurrent genetic abnormalities (24/471; 5%), AML with myelodysplasia-related changes (MRC; 11/158; 7%) and AML not otherwise specified (NOS; 6/106; 6%). When focussing on SF3B1 mut cases, AML-MRC (11/41; 27%) and AML with GATA2::MECOM (10/41; 24%) were most frequent. 9 additional cases showed another MECOM rearrangement (-rear) resulting in 46% (19/41) of patients with SF3B1 mutations accompanied by a MECOM-rear. A prior history of MDS or MDS/MPN was documented in 20% (8/41) of SF3B1 mut patients. Thereof, 63% (5/8) harbored a MECOM rearrangement and 25% RUNX1 mut. On average, SF3B1 mut patients harbored 2.85 mutations (including SF3B1). The most frequent additional mutations of SF3B1 mut patients were RUNX1 (9/41; 22%) and NRAS (8/41; 20%), and NPM1, TET2, or DNMT3A mutations or FLT3-ITD were detected in 15% (6/41) each. SF3B1 showed an average variant allelic frequency (VAF) of 41%. Notably, the VAF of SF3B1 in AML with NPM1 or RUNX1 was higher than the VAFs of NPM1 or RUNX1 in 9/11 (82%) cases, suggesting that SF3B1 preceded these mutations. In 16/41 (39%) cases molecular follow-up data was available. The SF3B1 mutation persisted during the entire disease course in 8 cases (including 1 with relapse). In particular, in two of these AML patients with mutated NPM1, the SF3B1 mutation remained detectable by NGS, despite complete hematologic remission and undetectable NPM1 mutation by high-sensitive qPCR. In all 5 cases with relapse the SF3B1 mutation re-occurred at relapse. In 4 patients, the VAF of SF3B1 decreased, similar to accompanying mutations. In contrast to MDS, SF3B1 mutations did not affect the overall survival (median OS: 16.9 months in SF3B1 mut; 5.7 months in unmutated group; p=0.822) in the total AML cohort. When stratified for AML sub-entities, the prognosis of the SF3B1 mut AML seems to be dominated by the sub-entity. Image: Summary/Conclusion: Within AML, SF3B1 mutations are enriched in AML patients with GATA2::MECOM and AML-MRC. This association with poor risk subtypes dominates the prognosis of SF3B1 mut AML. The high VAF of SF3B1 mutations and the persistence of SF3B1 mutations in AML patients in complete remission suggest the early acquisition of SF3B1 mutations in a pre-leukemic clone in a number of patients. While an antecedent MDS or MDS/MPN has been documented in some cases of SF3B1 mut AML, it might be unidentifiable in others. Alternatively, SF3B1 mut clonal hematopoiesis of indeterminate potential (CHIP) may represent a relevant pre-phase of SF3B1 mut AML. P412: LOW EXPOSURE, EXTENDED DOSING MIMICKING CLINICAL EXPOSURES OF ORAL AZACITIDINE SYNERGIZES WITH VENETOCLAX IN PRECLINICAL AML MODELS D. Jeyaraju1,*, M. Alapa1, A. Polonskaia1, A. Risueno Perez2, R. Hurren3, X. Wang3, M. Gronda3, A. Ahsan1, C. Wang1, P. Subramanyam4, J. Sriganesh4, A. Anand4, M. Bysani Reddy4, K. Ghosh5, C. Kyriakopoulos2, N. Lailler6, C. Hartl6, D. Lopes de Menezes7, A. Schimmer3, P. Hagner1, A. Gandhi1, A. Thakurta1 1Translational Medicine, Bristol Myers Squibb, Summit, United States of America; 2BMS Center for Innovation and Translational Research Europe, Bristol Myers Squibb, Seville, Spain; 3Princess Margaret Cancer Center, Toronto, Canada; 4BBRC; 5Bristol Myers Squibb, Bangalore, India; 6Rancho Biosciences, San Diego; 7Bristol Myers Squibb, San Francisco, United States of America Background: Acute myeloid leukemia (AML) has a high incidence of relapse despite the advancement of novel therapies. AML patients that are ineligible for intensive chemotherapy are typically administered injectable azacitidine (AZA) at a dose of 75 mg/m2 for 7 days in combination with BCL-2 inhibitor venetoclax (VEN) as induction therapy. Injectable-AZA dose was determined primarily through safety, tolerability, and bioavailability studies based on the CALGB and AZA-001 trials and not through a dose escalation study. Thus, it is not known if similar efficacy and combinability of AZA with VEN could be achieved at lower exposures and extended duration with oral-AZA dosing. The approval of oral-AZA in AML maintenance therapy, albeit at a lower cumulative exposure conferred at doses of 300 mg for an extended duration of 14 days of a 28-day dosing cycle, creates opportunities to answer these questions. Aims: To investigate the mechanism of action and combinability of oral-AZA with VEN in pre-clinical models. Methods: For in vitro cell-line based work, 1 day x 1mM AZA and 5days x 0.2mM AZA were used to mimic injectable-AZA like and oral-AZA like regimens respectively. For in vivo studies we modeled injectable-AZA and oral-AZA doses with 3mg/kg, qd x 5 days and 1 mg/kg, qd x 15 days respectively (intraperitonially in both cases, modified from Vu et al., Nat. Commun. 2020). VEN was administered orally at 100 mg/kg, qd x21 days. CRISPR screen was conducted with OCI-AML2-Cas9 cells using the Cellecta sgRNA library in the presence of AZA (121nm (IC50) x 5 days). Results: To identify synthetic lethal targets of oral-AZA, we conducted a genome-wide CRISPR screen. Among others, BCL-2 emerged as a synergistic hit. During in vitro validation of the CRISPR screen data, VEN synergized (anti-proliferative effect) with oral-AZA like exposure only when added after 5 days (0.2mM /day) of repeated AZA dosing. On the other hand, injectable-AZA like exposure synergized with VEN in 24 hours suggesting that the Oral-AZA/VEN synergy was driven more through epigenetic changes and not by stress response as has been reported for injectable-AZA. To further validate the CRISPR screen findings, we utilized in vivo AML xenograft mouse (injected with human AML cell lines) treated with an oral-AZA like or injectable-AZA like regimen with/without VEN. Mouse survival data demonstrated that both AZA regimens combined with VEN with equivalent efficacy. The persistence of leukemic stem cells (LSCs) is one of the reasons for relapse in AML. In vitro, oral-AZA like exposure resulted in decreased numbers of CD34+ CD38- LSCs. In an in vivo primary AML xenograft model using intrafemoral engraftment, oral-AZA like exposure was effective in reducing engraftment of leukemic cells from the right to the left femur (10-fold reduction). Analysis of cells at the site of injection (right femur) revealed induction of differentiation from a granulocyte-monocyte progenitor (GMP) to a megakaryocyte-erythrocyte progenitor (MEP) or common myeloid progenitor (CMP) state in response to oral-AZA. Summary/Conclusion: Collectively, our pre-clinical in vitro and in vivo data in AML models indicates that oral-AZA can combine effectively with VEN. In addition, oral-AZA can effectively eliminate LSCs and induce differentiation of immature cells. Through the CRISPR screen, our work identifies potential novel combination partners for oral-AZA. P413: THE POTENTIAL USE OF AN ORAL HYPOMETHYLATING AGENT, OR-2100, AS A COMBINATION THERAPY FOR MYELOID MALIGNANCIES K. Kamachi1,2,*, H. Ureshino1,2, K. Kawasoe1,2, N. Yoshida-Sakai1,2, Y. Yamamoto1, Y. Fukuda-Kurahashi2,3, Y. Kurahashi1,2,3, T. Watanabe2, S. Kimura1,2 1Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine; 2Department of Drug Discovery and Biomedical Sciences, Saga university, Saga; 3OHARA Pharmaceutical Co.,Ltd., Shiga, Japan Background: The combination therapy of hypomethylating agents (HMAs), azacytidine (AZA) and decitabine (DAC), with venetoclax (VEN), a selective BCL-2 inhibitor, have revolutionized the outcome of elderly patients with acute myeloid leukemia (AML), which have demonstrated the potential effect of HMAs as a combination therapy with molecular targeting agents for myeloid malignancies. However, there are a few studies on the combined effect of HMAs with tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and the mechanism by which HMAs enhance the efficacy of molecular targeting agents remain poorly understood. We have developed OR-2100 (OR21), an oral HMA, a 5’-O-trialkylsilylated DAC. OR21 is as effective as DAC, but less myelosuppressive, in xenograft mouse models of solid and hematological malignancies (Hattori et al. Clin Epigenetics 2019, Watanabe et al. Blood 2020, Ureshino et al. Mol Cancer Ther 2021, Kamachi et al. Cancer Letters 2022). We investigated the efficacy and mechanism of OR21 in combination with VEN or TKIs for myeloid malignancies, AML, and CML. Aims: To investigate the efficacy and mechanism of the combination therapy of OR21 for myeloid malignancies. Methods: We investigated the efficacy and mechanism of OR21 in combination with VEN against AML and with TKIs against CML, in vitro and in vivo. Results: In AML, OR21 plus VEN synergistically inhibited cell growth, increased apoptosis in AML cell lines as similar to AZA and DAC, and significantly prolonged survival in a xenograft mouse model using HL60 cell line (p<0.05). Gene expression analysis using AML cell lines (HL60, KG1a) showed that OR21 plus VEN significantly downregulated VAMP7 which regulates mitophagy/autophagy to maintain mitochondrial homeostasis compared to VEN monotherapy. Consistently, OR21 plus VEN enhanced apoptosis by increasing ROS accumulation and attenuated mitophagy/autophagy response triggered by decreased mitochondrial membrane potential. The addition of mitophagy inducers, such as rapamycin and p62-mediated mitophagy inducer, to OR21 plus VEN decreased ROS accumulation and apoptosis, indicating OR21 enhanced ROS accumulation via attenuating VEN-induced mitophagy/autophagy pathway. In CML, OR21 plus TKIs (imatinib, dasatinib, ponatinib) synergistically inhibited cell growth, increased apoptosis in CML cell lines as similar to AZA and DAC, and significantly suppressed the colony-forming capacity of bone marrow CD34+ stem/progenitor cells in CML patients. OR21 plus imatinib significantly suppressed tumor growth compared to imatinib monotherapy in a xenograft mouse model using K562 cell line (p<0.05). In phosphoproteomic, transcriptome and methylome analysis (K562, KBM5), OR21 augmented imatinib-induced apoptosis by dephosphorylating of STAT and Src family proteins by upregulating several tumor suppressor genes, including PTPN6, through demethylation of their promoter CpG regions. Image: Summary/Conclusion: OR21 enhanced the anti-tumor effect in combination with the molecular agents for myeloid malignancies with different mechanisms: OR21 increased ROS accumulation in combination with VEN, and dephosphorylated BCR-ABL1 downstream protein kinases by demethylating effect in combination with TKIs. With its advantage as an oral HMA, OR21 is expected to have future clinical trials as a combination therapy for myeloid malignancies. P414: RNA MODIFICATION-FOCUSED CRISPR-CAS9 SCREEN REVEALS POTENTIAL THERAPEUTIC TARGETS MEDIATING CHEMORESISTANCE IN AML M. Kienhöfer1,2,3,*, C. Pauli1,2, S. Delaunay2, C. Rohde1,3,4, M. F. Blank1,2,4, D. Heid1,4, M. Frye2, C. Müller-Tidow1,3,4,5 1Department of Medicine V, Hematology, Oncology and Rheumatology, University Hospital Heidelberg; 2German Cancer Research Center (DKFZ); 3University of Heidelberg Medical Faculty; 4Molecular Medicine Partnership Unit, European Molecular Biology Laboratory–Heidelberg University Hospital; 5National Center for Tumor Diseases (NCT), Heidelberg, Germany Background: Acquired therapy resistance is one of the main causes of death in Acute Myeloid Leukemia (AML). RNA-modifying proteins (RMP) are a diverse group of enzymes responsible for the deposition and the removal of over 170 chemical modifications, impacting key features of RNA biology. The RMPs represent a new focus for target therapies. Aims: The objective of the current study is to uncover how RNA-modifying proteins affects resistance to treatment in AML. Methods: A CRISPR-Cas9-based screening approach was utilized to study RNA-modifying proteins in an AML cell line and a derived cell line resistant to cytarabine. The library pool targeted 150 RNA modifying enzymes, the 250 most abundant expressed snoRNAs in AML, as well as a group of control genes and non-targeting sgRNAs. A lentiviral system was used to introduce the CRISPR system. Multiplicity of infection (MOI) was held under 0.3 to guarantee one gene knockout per cell. Post-infection, the cell lines were divided into a control and a treatment group. The treatment group was stimulated with increasing doses of cytarabine over 22 days. Samples were analyzed via NGS. Results: In the CRISPR screen, we observed that many of the gene knockouts led to cell depletion. Overall, 37 genes knockouts were depleted in the control group of both cell lines without additional drug treatment, suggesting that the related genes are required for survival and/ or proliferation. Further, 28 RNA-modifying enzymes and 15 snoRNAs were altered upon drug treatment in at least the parental or the resistant cell line. Interestingly, 15 of those 28 enzymes show additional effects on proliferation. Based on this data, we chose a methyltransferase responsible for specific modification on tRNAs for further analysis. Single knockout of the enzyme sensitized different AML cell lines for cytarabine treatment and showed impairment of proliferation as well as the ability to form colonies. Summary/Conclusion: RNA modifications promote several hallmarks of cancer. This study reports the involvement of specific RNA-modifying enzymes for therapy resistance and proliferation in AML. However, it remains unclear whether these enzymes are dysregulated in leukemic cells and therefore could display a targetable vulnerability. In addition, further studies of the affected cellular pathway could contribute to a better understanding of the underlying resistance mechanisms. P415: TARGETED 3RD-GENERATION (NANOPORE) SEQUENCING REVEALS MANY NOVEL CIRCULAR RNAS OF THE APOPTOSIS-RELATED BCL2L12 GENE, EXPRESSED IN HUMAN CELLS FROM ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES K. Xenou1, C. Sotiropoulou1, P. Karousi1, N. Machairas1, A. Scorilas1, V. Pappa2, S. Papageorgiou2, C. Kontos1,* 1Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens; 2Second Department of Internal Medicine and Research Unit, University General Hospital “Attikon”, Athens, Greece Background: Circular RNAs (circRNAs) are non-coding RNAs generated via back-splicing. They are indirect – yet pivotal – regulators of gene expression, as many of them bind microRNAs (miRNAs) and render them unavailable to exert their direct regulatory functions. They also interact with chromatin, altering its structure and affecting transcription of several genes. Lastly, though considered as non-coding, few of them have also been shown to encode short, functional polypeptides. Due to their multifaceted role and the deregulation of their expression in cancer, circRNAs are related to the development and progression of malignancies, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). BCL2L12 is a prominent member of the apoptosis-related BCL2 family, with an already established role in AML. Moreover, BCL2L12 mRNA expression has been associated with chemoresistance of malignant bone marrow blasts. However, the identity and role of the circRNAs produced by alternative back-splicing of BCL2L12 primary transcripts in MDS and AML has not been investigated, so far. Aims: We sought to determine the identity of BCL2L12 circRNAs expressed in MDS and AML cell lines and to explore their putative interactions with miRNAs having an established role in these disease entities. Methods: MDS-L cells [MDS with del(5q) and complex karyotype], NB-4 and HL-60 cells (acute promyelocytic leukemia with and without PML-RARA, respectively), U-937 and THP-1 cells (acute monocytic leukemia), KASUMI-1 cells (AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1) and MOLM-13 cells (AML with t(9;11)(p21.3;q23.3); KMT2A-MLLT3) were propagated. Total RNA was extracted from these seven cell lines; 2μg of each RNA extract were reverse-transcribed using random hexamers. Next, nested PCR with divergent primers was performed, starting from each exon of the BCL2L12 gene; thus, only cDNAs from BCL2L12 circRNAs were amplified. After having mixed and purified all nested-PCR products of each cell line cDNA, nanopore sequencing libraries were built. Following nanopore sequencing and basecalling in a MinION Mk1C 3rd-generation sequencer, sequencing reads were aligned and corrected using Minimap2 and TranscriptClean, respectively. circRNA identification was performed using an in-house–built, PERL-based algorithm; miRDB was used for prediction of their interactions with miRNAs. Results: We discovered 83 novel BCL2L12 circRNAs, 9 of which were common between MDS-L cells and at least one AML cell line. The exon structure of these novel circRNAs showed a remarkable diversity, comprising exons also encountered in messenger RNAs (mRNAs) of BCL2L12 as intronic regions. This phenomenon was mostly observed in MDS-L cells. Several BCL2L12 circRNAs in NB-4 and HL-60 cells exhibited complete intron retention; additionally, a novel exon was present at a remarkable frequency in NB-4 cells. Moreover, several BCL2L12 circRNAs are predicted to sponge miRNAs with a key role in AML (miR-7-5p, miR-125b-5p and miR-182-5p) and/or MDS (miR-150-5p). Summary/Conclusion: Numerous circRNAs are produced through alternative back-splicing of the primary BCL2L12 transcripts. The circRNA expression profile of this gene differs among the MDS and AML cell lines, as well as among the six AML cell lines. Intronic regions of this apoptosis-related gene are included in several circRNAs, augmenting the repertoire of miRNAs predicted to be bound by BCL2L12 transcripts. This phenomenon was more common in the MDS cell line, followed by both acute promyelocytic leukemia cell lines. Our data suggest an important regulatory role for this gene in MDS and AML subtypes. P416: CLONAL HEMATOPOIESIS IS COMMON IN AML LONG-TERM SURVIVORS AND MAY ASSOCIATE WITH DIABETES AND SECONDARY NEOPLASIAS, BUT NOT OTHER HEALTH OUTCOMES S. M. Krauß1,*, E. Telzerow2, A. S. Moret2, D. Richter3, M. Rothenberg-Thurley2, D. Görlich4, M. C. Sauerland4, W. E. Berdel5, B. Wörmann6, U. Krug7, J. Braess8, P. Heussner9, W. Enard3, W. Hiddemann2, K. Spiekermann2, U. Platzbecker1, K. H. Metzeler1 1Dept. Hematology and Cell Therapy, University Hospital Leipzig, Leipzig; 2Dept. of Medicine III, University Hospital, Ludwig-Maximilians University; 3Dept. Biology II, Ludwig-Maximilians University, Munich; 4Institute of Biostatistics and Clinical Research; 5Dept. of Medicine, Hematology and Oncology, University of Münster, Münster; 6Deutsche Gesellschaft für Hämatologie und medizinische Onkologie, Berlin; 7Dept. of Medicine 3, University Hospital Leverkusen, Leverkusen; 8Dept. of Oncology and Hematology, Hospital Barmherzige Brüder, Regensburg; 9Psycho-Oncology, Hospital Garmisch-Partenkirchen, Garmisch-Partenkirchen, Germany Background: Clonal hematopoiesis (CH), the outgrowth of hematopoietic stem cells and their progeny with somatically acquired gene mutations, is a common phenomenon in elderly individuals without known hematological disorders. CH has been linked to an increased risk of developing myeloid neoplasia and cardiovascular diseases, resulting in higher all-cause mortality. More specifically, clonal hematopoiesis of indeterminate potential (CHIP) has been defined by hematopoietic clones with a variant allele frequency (VAF) ≥2% in the absence of hematologic disease. CH is also known to be common in patients with acute myeloid leukemia (AML) briefly after therapy, but prevalence and relevance of CH in AML long-term survivors (AML-LTS) has not been investigated so far. Aims: We aimed to study CH in a cohort of 380 AML-LTS using a targeted next generation sequencing (NGS) assay utilizing single-molecule Molecular Inversion Probes (smMIPs), and to analyze determinants of CH and its effects on somatic health outcomes. Methods: We employed smMIP-technology, which follows a hybridization-capture protocol for NGS library preparation to selectively enrich and amplify hundreds of genomic target loci covering 82 regions in 24 CH and AML-related genes in a single reaction. Unique molecular identifiers (UMIs) within probes enabled computational correction of sequencing errors to significantly increase precision of variant calls. Results: We successfully established a UMI-based smMIP NGS assay as well as a custom computational analysis pipeline to enable the reliable detection of variants ≥0.5% VAF. In whole blood specimens from 380 AML-LTS we detected CH in 61.4% of individuals, including CHIP (VAF ≥2%) in 35.8%. Stratification by treatment group, i.e. chemotherapy/autologous hematopoietic stem cell transplantation only (chemo) vs. allogeneic hematopoietic stem cell transplantation (alloSCT), revealed a significantly lower prevalence of CHIP in the alloSCT group (26.0% vs. 52.1%; p<.001). Concordantly, median VAF of CH mutations after alloSCT was significantly lower than in the chemo group (1.2% vs. 1.8%; p=.005). CHIP prevalence increased with age in the chemo group, from 21.7% (<50 years) to 70.0% (≥70 years), whereas it was independent of patient age in the alloSCT group (Figure A). Conversely, CHIP prevalence was independent from time since start of therapy in the chemo group, but increased with time after transplantation in the alloSCT group, from 18.4% (0-5 years) to 46.2% (16-18 years; Figure B). In both treatment groups, the known CHIP driver genes DNMT3A and TET2 were most frequently mutated. Notably, PPM1D (p<.001) and TP53 (p<.001) were more frequently mutated in the chemo group (Figure C). We did not detect significant differences in complete blood counts of AML survivors according to CH status. While most somatic comorbidities showed no clear association with CH status, prevalence of diabetes and development of secondary cancer after leukemia therapy were increased in patients with CH (VAF <2%), and more so in those with CHIP (Figure D). Image: Summary/Conclusion: We report a high prevalence of CH in AML long-term survivors, particular in those treated with chemotherapy without alloSCT. CHIP prevalence increases with age in patients treated with chemotherapy, and with time elapsed after transplantation in patients who underwent alloSCT. We reveal distinct mutation spectra for both groups. Although associations between CH and somatic morbidities seem to be weak overall, we found associations with diabetes and the development of secondary cancer after leukemia treatment, which warrant further exploration. P417: A SINGLE AMINO ACID AT THE PNT DOMAIN OF ERG MEDIATES ITS LEUKEMOGENIC ACTIVITY THROUGH INTERACTION WITH THE NCOR-HDAC3 CO-REPRESSOR COMPLEX. E. Kugler1,2,*, S. Madiwale2, D. Yong3, Y. Birger2,4, J. A. I. Thoms5,6, D. B. Sykes7,8, M. Yassin9, N. Aqaqe9, A. Rein2,4, H. Fishman2, I. Geron2, C.-W. Chen10,11,12, B. Raught13, Q. Liu10, M. Milyavsky9, J. Pimanda5,6, G. G. Privé3,13, S. Izraeli2,4 1Institute of Hematology, Davidoff Cancer Center, Rabin medical center, Petah Tikva; 2Department of Human Molecular Genetics and Biochemistry, Sackler Medical School, Tel Aviv University, Tel Aviv, Israel; 3Department of Biochemistry, University of Toronto, Toronto, Canada; 4The Rina Zaizov Pediatric Hematology and Oncology Division, Schneider Children’s Medical Center, Petach Tikva, Israel; 5Adult Cancer Program, Lowy Cancer Research Centre; 6Faculty of Medicine, UNSW Sydney, Sydney, Australia; 7Center for Regenerative Medicine, Massachusetts General Hospital, Boston; 8Harvard Stem Cell Institute, Cambridge, United States of America; 9Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 10Department of Systems Biology, Beckman Research Institute, City of Hope, Duarte; 11Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston; 12City of Hope Comprehensive Cancer Center, Duarte, United States of America; 13Princess Margaret Cancer Centre, University Health Network, Toronto, Canada Background: The ETS transcription factor ERG is essential for the maintenance of hematopoietic stem cells. However, it has also been implicated as an oncogene in the development of acute leukemia. Our studies and those of others have demonstrated that ERG directly contributes to the initiation and maintenance of lymphoid and myeloid acute leukemia subtypes. Nevertheless, ERG co-factors critically involved in leukemogenesis remain largely uncharacterized. Aims: Here we report a critical role for the conserved amino-acid proline at position 199, at the 3’ end of the PNT domain, for ERG’s leukemogenic activity. Methods: A genomic and proteomic analysis has been used to study the role of P199 in the leukemogenic pathway of ERG. Results: We specifically demonstrate in functional and gene expression assays that P199 is required for ERG-induced myeloid differentiation restriction and for self-renewal and maintenance of murine hematopoietic stem and progenitor cells (HSPC). As P199 is located within the PNT domain (protein interaction domain), we attempted to identify key protein interactions associated with leukemia progression induced by ERG. To this end, we used proximity ligation-mass spectrometry (BioID). In HEK293 cells, proximity maps of WT-ERG and a mutated form of ERG at position 199 (P199L-ERG) were compared. A total of 240 putative protein interactors with a significance analysis of interactome were identified. The hits were highly enriched with chromatin modifiers. Most significantly, the mutation severely impaired ERG’s interaction with components of the NCoR-HDAC3 complex, resulting in a 40% reduction in the number of spectral counts in comparison with wild-type ERG. ChIP sequencing for histone markers conducted on ER-Hoxb8 cells (transformed murine GMPs) demonstrated a decrease in H3K27ac signature at over 1500 unique sites in cells transduced with WT-ERG compared to those transduced with the mutant. Interestingly, these sites were predominantly associated with enhancers of genes expressed in mature myeloid cells. Next, we addressed the role of HDAC3 in ERG’s transcriptional activity. RNA sequencing of human ER-Hoxb8 cells treated with BRD3308 (an HDAC3 inhibitor) for 48 hours was performed. Interestingly, leukemia-associated pathways (Hoxa9-Meis1, CBFA2T3 and repression of myeloid differentiation genes) were ranked at the leading edge of gene sets that were significantly perturbed after HDAC3 inhibition. Moreover, we tested the significance of the ERG/NCoR-HDAC3 interaction in models of human leukemia. We demonstrated that ERG-dependent human AML cells as SKNO1 (AML1-ETO) and TF1 (CBFA2T3-ABDH12) were more sensitive to HDAC3 inhibition (RGFP966) as compared to ERG independent cells. Furthermore, RGFP966 exhibited a significant in-vivo antileukemic effect as measured by the burden of disease in the bone marrow of NSG mice transplanted with SKNO1 cells. By using the CRISPR-dCAS9 system we were able to show that leukemia development is abrogated in mice transplanted with SKNO1 cells expressing a reduced level of HDAC3. Remarkably, this effect was translated into a significant survival advantage. Summary/Conclusion: Taken together, our findings indicate that the aberrant overexpression of ERG maintains HSPCs in an undifferentiated state and promotes AML development. We suggest that the interaction between ERG and the NCoR-HDAC3 complex has an important role in the leukemogenic process, and that HDAC3 inhibition could be beneficial in AML characterized by high ERG expression. P418: MODELLING AND TARGETING ACUTE MYELOID LEUKAEMIA CELLS IN THE BONE MARROW PROTECTIVE NICHE E. E. Ladikou1,2,*, K. Sharp1, T. A. Burley1, E. Kennedy1, T. Chevassut2, C. Pepper1, A. G. Pepper1 1Clinical and Experimental Medicine, Brighton and Sussex Medical School; 2Department of Haematology, Brighton and Sussex University Hospital Trust, Brighton, United Kingdom Background: AML is a therapeutic challenge due to its aggressiveness and biological heterogeneity. Although 80% of patients initially achieve a complete remission, the long-term disease-free survival is poor. One issue contributing to disease relapse, is persistence of disease in the protective niche of the Bone Marrow microenvironment (BMME). Here, AML cells are surrounded by other cell types that promote their survival, which enables them to evade therapeutic destruction and promotes the emergence of drug resistance. Clinical trials of single agent mobilising drugs, like Plerixafor, have yielded promising results but they are not curative. So, the development of combinatorial therapies that simultaneously target different components of AML cell adhesion may release more AML cells into the circulation, where they can be targeted by standard therapies. Aims: a) To develop a multi-cellular, co-culture system to recapitulate the adhesive BMME, b) To test rational drug combinations and identify targets with the potential to mobilise anchored AML cells and c) To perform paired transcriptomic and phenotypic analysis of persistently adhered versus mobilised AML cells to detect novel targets. Methods: We have developed a novel 96-well plate multi-cellular, co-culture system using stromal cells (HS5), endothelial cells (HUVEC), osteoblasts (hFOB 1.19) and AML cells (OCI-AML3 and KG1a). Multiple mobilisation drugs, alone and in combination, were tested and AML cell release was quantified by flow cytometry. Drugs tested to date include: Plerixafor (CXCR4 inhibitor), Natalizumab (anti-α4-integrin antibody), ONO-7161 (novel CXCR4 inhibitor), purified anti-CD44 and anti-E-Selectin. Phenotypic and transcriptional comparisons were made between the persistently adherent and mobilised AML cells. Results: The most adhesive physiological “BMME mix” was identified as HS5, HUVEC and hFOB 1.19 in equal 1/3 proportions (p=0.0001). Co-culture with AML cells resulted in 74% and 68% adherence of KG1a and OCI-AML3, respectively. Phenotypic analysis of adhered vs non-adhered AML cells identified CD44, CXCR4, CD49d and CD38 as important markers of optimal BMME adhesion. Subsequently, our novel co-culture system was used as a drug testing platform to assess clinically available agents. Plerixafor and Natalizumab increased detachment by 1.3-fold (p=0.0132) and 1.2-fold (p=0.015) respectively. In contrast, ONO7161 and anti-E-selectin had little effect. The most promising candidate, anti-CD44, resulted in a 2-fold and 1.6-fold detachment of KG1a (p=0.004) and OCI-AML3 (p=0.027), respectively. However, even in the presence of the maximum dose of anti-CD44 (5µg/mL)), 46% of KG1a and 48% of OCI-AML3 cells remained persistently adhered. Synergy experiments with anti-CD44 in combination with either Plerixafor or Natalizumab yielded no better release than anti-CD44 alone. Summary/Conclusion: We have developed a novel, multi-cellular co-culture system that recapitulates the adhesive BMME. We are using this as a drug testing platform for AML to identify the most potent release agents and test the effects of cytotoxic drugs on those most persistently adhered. To-date, anti-CD44 is the most effective release agent in our model, but it still failed to release all AML cells. Transcriptomic analysis of the persistently adherent AML cells should enable us to delineate the critical biological interactions required for BMME adhesion. This will enable us to identify rational new targets to effectively remove chemo-resistant AML cells from the protective BM niche. P419: IDENTIFICATION OF NOVEL THERAPEUTIC OPTIONS FOR VENETOCLAX-RESISTANT AML CELLS THROUGH DRUG REPURPOSING A. Ladungova1,2,*, D. Busa3, Y. Lodhi1,4, J. Hyl3, M. Culen3,5, M. Smida1,3,5 1Central European Institute of Technology, Masaryk University; 2National Centre for Biomolecular Research, Faculty of Science, Masaryk University; 3Department of Internal Medicine - Hematology and Oncology, Faculty of Medicine, Masaryk University; 4Department of Biology, Faculty of Medicine, Masaryk University; 5Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czechia Background: Acute myeloid leukemia (AML) is a malignant disease derived from the bone marrow precursors of myeloid lineage. Treatment options are rather limited, primarily based on chemotherapy, and often result in disease progression. Recently, venetoclax-based therapies have transformed the frontline regimens of elderly patients and patients unfit for intensive chemotherapy. Despite its promising outcomes in clinical studies, multiple resistant subclones evolved during the treatment acting as a barrier in disease regression. Understanding the venetoclax-resistance mechanisms and detecting the major determinants could reveal previously unrecognized novel perspectives for therapeutic strategies to improve patients’ outcomes. Moreover, performing high-throughput screenings with clinically approved drugs could reveal novel treatment options for resistant subclones of AML. Aims: Our project aims 1) to identify novel FDA-approved drugs effective for treating venetoclax-resistant AML cells through a high throughput screening, 2) to validate the most effective compounds, prove their specificity, and 3) to investigate the molecular background mechanisms underlying the observed sensitivity. Methods: Two different models mimicking the microenvironment of venetoclax-resistance formation have been established: 1.) Patient-derived mouse xenograft (PDX) model. AML PDX model was generated by using AML patient sample transplanted into an immunocompromised murine host and treated with different regimens of venetoclax; 2.) Venetoclax-resistant cell lines (MOLM-13, OCI-AML3, HL-60). Generation of the venetoclax-resistant AML cell lines was achieved through the chronic administration of the compound, inhibiting the cell viability to 80-90% in multiple rounds with gradually increasing concentrations until the cells become fully resistant. Both models are next used for the high-throughput screening of 859 approved drugs, covering broad chemical space. The standard concentration (1 μM) of drugs in the library is automatically added to cells on 384-well-plates with a programmed liquid handling system epMotion (Eppendorf). Cell viability is determined after 72 hours by a Cell-titer Glo assay. Compounds with the strongest effect upon resistant AML cell viability are validated through 10-point dose-response curves with 2-fold-dilutions. Moreover, the investigation of molecular mechanisms responsible for the sensitization of resistant AML cells is planned by using various cell biology and molecular biology techniques. Results: Treating the PDX mouse model with venetoclax for 6 weeks resulted in the generation of resistant cells. Importantly, these cells also acquired additional resistance to topoisomerase inhibitors, DNA-damaging agents and many other compounds. On the contrary, both 6-week as well as 3-week venetoclax treatments generated cells exquisitely sensitive to the iron chelator deferoxamine mesylate and a purine nucleoside analogue clofarabine. In addition, we generated a venetoclax-resistant MOLM-13 cell line with 1500-fold decreased sensitivity and have subjected it to the drug screening to correlate its response with the PDX model. Summary/Conclusion: The outcomes of this research project point out the importance of novel treatment options to overcome venetoclax-resistance in AML cells. Further validations and investigation of the molecular mechanisms are planned for the top-performing drugs to strengthen the translational potential for AML therapy. This project was supported by the grants number MUNI/A/1330/2021, MUNI/A/1419/2021 and by OPRDE (No.CZ.02.2.69/0.0/0.0/19_073/0016943). P420: FORCING LEUKAEMIC STEM CELL TO LEAVE QUIESCENT STATE BY INHIBITING HEDGEHOG, NOTCH AND WNT/BETA-CATENIN SIGNALLING PATHWAYS D. Láinez-González1,*, J. Serrano-López1, P. Llamas-Sillero2, J. M. Alonso-Dominguez2 1Experimental Hematology Lab, Instituto de Investigación Sanitaria Fundación Jiménez Díaz; 2Department of Hematology, Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain Background: Acute myeloid leukaemia (AML) is a clonal neoplasm with a high relapse rate. It is thought that this relapse is caused by chemoresistance of leukaemic stem cells (LSC) due to their quiescent state. Numerous signalling pathways have been described that regulate quiescence. Key players in this setting are the Hedgehog (Hh), Notch and WNT/β-Catenin pathways. Current chemotherapeutic agents target the cell population in the proliferative phase of the cell cycle. Therefore, cells in quiescence are not affected by drugs such as cytarabine (AraC). Theoretically, forcing quiescent cells into the proliferative phase would help to eradicate the LSC population and avoid relapses. Aims: To force quiescent LSC to enter cell cycle by inhibiting Hedgehog, Notch or/and Wnt/Beta-Catenin. Methods: Three acute myeloid leukemia cell lines, HL60, OCI-AML3 and KASUMI1, and 13 primary samples were used to study cell cycle and cytotoxicity. They were treated during 24-48 hours with Glasdegib, Nirogacestat and PRI-724 which target Smoothened (Hedgehog), Gamma-Secretase (Notch) and Beta-Catenin (Wnt) proteins respectively. Studies of drug combination were also performed to inhibit different signaling pathways at the same time. Cytarabine was employed during 48-72 hours in cell lines or during 24-48 hours in primary samples. Cytotoxicity was analyzed by WST8 assay in cell lines and flow cytometry in primary samples by using KI67 and Annexin V. Cell cycle was study by flow cytometry using KI67 and 7AAD, and in primary samples CD45, CD34 and CD38 antibodies were used to detect LSC. Cell lines experiments were triplicated. Results: In HL60, double inhibition during 48h of Hedgehog and Notch with 15 nM Glasdegib + 5 nM Nirogacestat reduced quiescent state in 67.27% compared with DMSO. Furthermore, this proposed treatment in combination with 109.1 nM AraC decreased 27-70% viability compared with AraC monotherapy at 72h. Similar results were obtained in OCI-AML3, in which the proposed treatments reduced quiescent phase compared with control, but no changes were observed in viability. On the other hand, in KASUMI1 there were no differences either in cell cycle or viability assay. In primary CD34+ samples, when Hedgehog and Notch were inhibited during 48h, quiescent state decreased in 33.06%, but no effects in viability were observed. Image: Summary/Conclusion: The number of primary samples should be increased to shed light on the efficacy of this interesting therapeutic strategy. Nevertheless, inhibition of Hh, Notch and WNT pathways reduce the quiescent population either measuring the disease as a whole or looking at the LSC population. Differences observed between different AML cell lines regarding increase of the citotoxicity might be explained by AML genetic heterogeneity. P421: LOWER VΓ9VΔ2 T CELLS RELATIVE FREQUENCIES PREDICT POORER SURVIVAL IN NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA A.-C. Le Floch1,2,*, F. Orlanducci1,2, A. Le Roy1,2, J.-F. Hamel3, N. Ifrah4, P. Cornillet-Lefebvre5, J. Delaunay6, C. Récher7, E. Delabesse8, A. Pigneux9, M.-C. Béné10, N. Vey11, A.-S. Chretien1,2, D. Olive1,2 1Equipe Immunité et Cancer, Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR7258, Institut Paoli-Calmettes, Aix-Marseille Université, UM105; 2Plateforme d’immunomonitoring, Institut Paoli-Calmettes, Marseille; 3Département de Biostatistiques, CHU d’Angers, Université d’Angers; 4Département d’Hématologie, CHU d’Angers, Université d’Angers, Inserm, CRCINA, Angers; 5Laboratoire d’Hématologie, CHU de Reims, Reims; 6Département d’Hématologie, CHU de Nantes, Nantes; 7Département d’Hématologie; 8Laboratoire d’Hématologie, CHU de Toulouse, Institut Universitaire du Cancer de Toulouse Oncopôle, Université Toulouse III Paul Sabatier, Toulouse; 9Département d’Hématologie et Thérapie Cellulaire, CHU de Bordeaux, Bordeaux; 10Laboratoire d’Hématologie, CHU de Nantes, Nantes; 11Département d’hématologie, CRCM, INSERM U1068, CNRS UMR7258, Institut Paoli-Calmettes, Aix-Marseille Université UM105, Marseille, France Background: Major role of γδ T cells has recently emerged in anti-tumor immunity. Across several tumor subtypes, increased relative frequencies of γδ T cells had the best prognostic value in comparison to other immune subsets. In acute leukemias, patients with increased γδ T cells reconstitution after allogenic stem cell transplantation had better outcomes. The less studied putative prognostic impact of Vγ9Vδ2 T cells abundance in newly diagnosed (ND) AML relies almost on inference of transcriptomic data or post induction frequencies and has not been assessed with respect to other confounding factors. Aims: The aim of this study was to determine the predictive and prognostic impact of blood Vγ9Vδ2 T cells frequencies at diagnosis, as measured using phenotypic analysis, taking into account biological and clinical parameters in patients treated for AML with induction. Methods: Proportion of Vγ9Vδ2 T cells among lymphocytes has been assessed by flow or mass cytometry on PBMCS from AML patients at diagnosis, obtained from the Department of Hematology of Institut Paoli Calmettes or from the tumor bank of the FILO group. Correlations between clinical outcome, patient or disease characteristics, and Vγ9Vδ2 T cells frequencies have been analyzed. For some of the samples, we characterized immune alterations displayed by Vγ9Vδ2 T cells, or performed Vγ9Vδ2 T cell expansion and degranulation assays of autologous Vγ9Vδ2 T cells. Results: 198 patients were included in this study. Mean age was 56 years [18.9-81.5], 79 (39.9%) and 119 (60.1%) patients had respectively a favorable or an intermediate ELN classification. Mean CR rate was 81.3%. Analysis of overall survival (OS) and relapse free survival (RFS) based on Vγ9Vδ2 T cell frequencies among lymphocytes, highlighted that the 0.8% threshold was the most significant. Vγ9Vδ2 T cells relative frequencies were considered low for values below this threshold. By univariate analysis, patients with lower Vγ9Vδ2 T cells at diagnosis had significantly lower 5-year OS and RFS (HR = 1.54, 95%CI = 1.03-2.31, p = 0.028 and HR = 1.69, 95%CI = 1.09-2.62, p = 0.015). These results were confirmed in multivariate analysis (HR = 1.55 [1.04-2.30], p = 0.030 and HR = 1.64 [1.06, 2.53], p = 0.025). Phenotypic alterations found in patients with lower Vδ2 included a loss of some cytotoxic Vγ9Vδ2 T cell subsets and a decrease in BTN3A expression on the surface of blasts. Finally, in vitro, samples irrespective of their Vγ9Vδ2 T cells numbers did expand and displayed similar functional effects against target cells after stimulation with zoledronate or anti-BTN3A 20.1 agonist mAb. Summary/Conclusion: This study confirmed the prognostic value of Vγ9Vδ2 T cells predominance among lymphocytes, in ND AML patients treated with induction. Vγ9Vδ2 T cells frequencies could be easily included in algorithm of treatment decision. Our findings contribute also to a strong rationale to elaborate consolidation protocols targeting enhancement of Vγ9Vδ2 T cells response, such as anti-BTN3A agonist antibodies that are under development (Marabelle et al. ESMO 2021). P422: OMIPALISIB, A DUAL PI3K/MTOR INHIBITOR, TARGETS MITOCHONDRIA AND IMPAIRS OXIDATIVE PHOSPHORYLATION IN ACUTE MYELOID LEUKEMIA C.-Y. Tseng1,*, C.-Y. Kuo1, Y.-H. Fu1,2, H.-A. Hou3, H.-F. Tien3, L.-I. Lin1 1Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan; 2Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, United States of America; 3Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Background: Mitochondria play a central role in cell metabolism. Recent studies explored mitochondrial alterations contributing to metabolic vulnerability in myeloid leukemia cells may considered as therapeutic targets. We previously reported that omipalisib, a PI3K/mTOR dual inhibitor, could significantly inhibit the growth of myeloid leukemia cells and impair their mitochondrial respiration. However, the precise mechanism of the mitochondria dysfunction in response to omipalisib is still not fully studied. Aims: To explore the mechanism of the mitochondria dysfunction in response to omipalisib. Methods: OCI-AML3 and THP-1 myeloid leukemia cell lines were used in this study. Omipalisib (GSK2126458) and another PI3K/mTOR dual inhibitor gedatolisib (PF-05212384) were used. Flow cytometry was used for mitochondrial analysis. RNA-seq was performed by using an Illumina NovaSeq 6000 platform. Differentially expressed genes (DEGs) between control and omipalisib (or gedatolisib) were identified by EBseq with a threshold of p ≤ 0.05. The mRNA, nuclear and mitochondrial DNA quantification were measured by using QuantStudio 3 Real-Time PCR Systems. The parameters of mitochondrial respiration were analyzed by using the XFe 24 extracellular flux analyzer. Results: We demonstrated the anti-proliferative effect of omipalisib and gedatolisib on a panel of AML cell lines with different genetic backgrounds. OCI-AML3 cell lines had significant response to omipalisib and gedatolisib with IC50 of 17.45 nM and 153.38 nM, respectively. Subsequently, the transcriptome analysis of 50 nM omipalisib or 200 nM gedatolisib-treated OCI-AML3 cells were analyzed. Compared to DMSO-treated, a total of 1199 (595 upregulated and 604 downregulated) and 326 (218 upregulated and 108 downregulated) DEGs were identified in the omipalisib-treated cells and the gedatolisib-treated cells, respectively. Gene set enrichment analysis (GSEA) indicated that both omipalisib and gedatolisib could suppress “E2F targets”, “Myc targets”, and “G2M checkpoint”, and trigger “TNFα signaling via NFκB”, and “Inflammatory response”. Of them, we found that omipalisib had more significant effect to inhibit “Oxidative phosphorylation” (NES: -2.01) than gedatolisib did (NES: -1.40), especially mitochondrial biogenesis and amino acid metabolism-related pathways. Omipalisib had an inhibitory effect on the mitochondrial basal respiration rate and maximal respiration. Further analysis revealed quantitative reverse-transcription PCR analysis revealed that both omipalisib and gedatolisib could potently suppress serine synthesis (PHGDH, PSAT1, PSPH), glycine synthesis (SHMT1/2), and tetrahydrofolate cycle-related genes (MTHFD1/2) at the transcriptional level. On the other hand, rather than gedatolisib, omipalisib could significantly inhibit expression of glycolysis-related genes (GLUT1, HK2, PKM2, and LDHA) as well as mitochondrial biogenesis-related genes (PPARGC1B, TFAM, and NDUFS6). In addition, omipalisib could strongly decrease mtDNA and mitochondrial mass. These results suggest that omipalisib could suppress the mitochondrial biogenesis through alternative mechanisms, including but not limited to PI3K-AKT-mTOR signaling. Summary/Conclusion: Rather than gedatolisib, omipalisib could significantly inhibit mitochondrial biogenesis and suppress the respiration of mitochondria in myeloid leukemia cells. Our results indicate that omipalisib could suppress cell proliferation not only through PI3K-AKT-mTOR signaling but also via impairing mitochondrial biogenesis. This information may be potentially suitable for future clinical applications. P423: ADAPTING CRISPR CAS9 DROPOUT SCREENS TO IN VIVO PDX MODELS OF ACUTE LEUKEMIAS R. Ludwig1,2,*, D. Amend1, E. Bahrami1, I. Jeremias1,2,3 1Apoptosis in Hematopoietic Stem Cells, German Research Center for Environmental Health (HMGU), Munich; 2German Cancer Consortium (DKTK), partner site Munich; 3Department of Pediatrics, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany Background: Acute leukemias require better treatment and targeted therapies represent interesting future therapeutic options. Such therapies precisely inhibit molecules with essential function, also called vulnerabilities or dependencies, so that leukemia cells die once the target is inhibited. Aims: Here, we aimed at identifying therapeutic targets on a patient individual level and in the surrounding of a living organism. Towards this aim, we established CRISPR Cas9 dropout screens in PDX models of acute leukemias in vivo. Methods: Primary patient acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) samples were transplanted into immunocompromised NSG mice and re-passaged to develop serially transplantable PDX models. To overcome limited transduction efficiency inherent to PDX leukemia cells, lentiviruses were used. Results: We first elaborated on the maximum library size to be used in PDX models in vivo. As only a limited number of PDX cells home into mice, library size is limited to ensure the coverage of the library. We used, screening sgRNAs as genetic barcodes in Cas9 negative samples to determine the maximum library size upon next generation sequencing (NGS); a regular distribution of all sgRNAs recovered from mice at the end of the experiments indicated a suitable library size. We found that PDX ALL models tolerated a larger library compared to PDX AML models that might reflect lower intra-sample heterogeneity and higher leukemia stem cell frequency in ALL compared to AML. To perform knockout screens, a split-construct for Cas9 was used to reduce plasmid size and optimize lentiviral transduction efficiency into PDX cells; concomitantly split-GFP was used to enrich cells expressing both Cas9 split plasmids by flow cytometry. A customized library consisting of 146 target genes combined with positive and negative control genes was designed at 5 sgRNAs per gene using the CLUE platform (www.crispr-clue.de) (Becker et al., Nucleic Acids Res. 2020) and cloned into a lentiviral vector backbone. The sgRNA library vectors include either a puromycin-resistance cassette or an H-2Kk surface marker, each combined with a BFP fluorescent marker. This allows to determine transduction efficiency by FACS-based detection of BFP and to enrich the transduced PDX cells either by puromycin selection or by H-2Kk-MACS selection. Input samples were collected after library transduction and enrichment to >90% CRISPR/Cas9 sgRNA library positive cells, while remaining cells were injected into immunocompromised NSG mice, grown in vivo and re-isolated at advanced disease stage. Analysis of input and endpoint samples using NGS enabled to determine the frequency of sgRNAs and bioinformatically compare the abundance of different sgRNAs between the control and the samples of interest using MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout). A significant sgRNA dropout was characterized by a p-value below 0.05 and a FDR below 0.1. The screening experiments led to the top 10 depleted dropout genes for ALL and AML PDX samples. Besides individual dropouts of the different samples, also shared dropouts were detected. Especially sample overlapping dropouts might represent interesting starting points for new therapeutic options. Summary/Conclusion: In summary, we have established a CRISPR Cas9 screening pipeline, which allows investigating therapeutic targets on a patient-individual level in ALL PDX models and, for the first time, in AML PDX models in vivo. P424: INVESTIGATING GPR56 FUNCTION IN HUMAN IPSC-DERIVED HEMATOPOIETIC CELLS USING A TET-ON MODEL OF CONSTITUTIVE ACTIVATION L. Mackintosh1,*, A. Maglitto1, C. Rodriguez Seoane1, S. A. Mariani1, A. G. Rossi1, E. Dzierzak1 1Centre For Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom Background: GPR56 (ADGRG1) is a widely expressed adhesion G-coupled protein receptor in humans and mice which is implicated to play a role in both haematopoietic development and Acute Myeloid Leukaemia (AML). In AML, there exists a low frequency subpopulation of leukaemic stem cells (LSCs) which, similar to their benign Hematopoietic Stem Cell (HSC) counterparts, exhibit stem cell behaviours. High expression of GPR56 forms part of the molecular signature of these LSCs, and AML cases with high levels of GPR56 at diagnosis have poorer outcomes. Our hypothesis is that GPR56 activity underlies some of the stem cell behaviours seen in both HSCs and LSCs. Aims: To characterise the effects of GPR56 receptor activation so as to better understand its role in AML and hematopoietic development. Methods: We developed a Tet-On human inducible stem cell line (M1) transfected with a construct encoding a truncated GPR56 protein. This constitutively active GPR56 receptor form is overexpressed following doxycycline administration. Flow cytometry-based cell cycle and apoptosis studies were performed on the M1 line and wild type Sci55 (WT) iPSCs +/- doxycycline exposure. IncuCyte® transmigration studies utilising an SDF-α gradient were performed to examine cell migration. Finally, the M1 cells were subjected to a hematopoietic differentiation protocol and assessed for CD34+ CD38- compartment size at day 12 of differentiation. Results: GPR56 activation alters cell cycle progression with significant accumulation of M1 cells in the G0/1 and G2/M phases of the cell cycle compared to WT (p <0.005). GPR56 activation also causes a significant reduction in apoptotic cells in dox-treated M1 cells (p <0.005), despite doxycycline being pro-apoptotic in WT cells (p <0.05). GPR56 activation reduces the number of transmigrating M1 cells in response to an SDF-α gradient (p <0.05). Importantly, GPR56 activation during differentiation increases the CD34+ CD38- compartment size in M1 cells treated with doxycycline at day 12 (p<0.05) as compared to untreated cells Summary/Conclusion: GPR56 activation results in accumulation of undifferentiated M1 iPSCs in the G0/1 and G2/M phases, suggestive of cell cycle checkpoint arrest, and exerts a powerful anti-apoptotic effect. GPR56 activation also reduces cell transmigration in response to SDF-α, suggesting receptor involvement in cell adhesion and migration. Finally, GPR56 activation during hematopoietic differentiation results in an increased yield of CD34+ CD38- cells, suggesting that GPR56 activity may influence the proportion of immature cells arising during haematopoietic differentiation. These results are of interest in relation to normal HSC functions and may indicate the mechanisms through which overexpression of GPR56 (with increased activation) in LSCs contributes to poorer outcomes in AML. Future work will include identification and characterisation of the signalling pathways downstream of the GPR56 receptor to better understand this receptor’s role in the haematopoietic system in health and disease. P425: ROLE OF LYSOSOMAL-ASSOCIATED GENE LAPMT4B IN LEUKEMIA J. Delgado-Martínez1,2,*, J. M. Cornet-Masana1, L. Cuesta-Casanovas1,3, J. M. Carbó1,4, L. Clément-Démange4, R. M. Risueño1 1Josep Carreras Leukaemia Research Institute; 2Faculty of Pharmacy, University of Barcelona; 3Faculty of Bioscience, Autonomous University of Barcelona; 4Leukos Biotech, Leukos Biotech, BARCELONA, Spain Background: Despite the efforts to find druggable targets in acute myeloid leukemia (AML), successful therapeutic approaches are needed in this disease. Due to the particular characteristics of lysosomes in cancer, the clinical relevance of key lysosomal genes’ expression was searched in public repositories. LAPTM4B gene expression was found to be directly correlated with a worse clinical outcome in AML. Aims: To decipher the role of the lysosome-associated gene LAPTM4B in AML will be the main objective of this study. Methods: Generation of AML cells overexpressing both LAPTM4B isoforms (35kDa -long- and 24kDa -short) were obtained by lentiviral transduction. Sensitivity to chemoresistance was measured by flow cytometry. To determine the role of LAPTM4B in the leukemia regeneration capacity, conditioned NSG mice were transplanted with both LAPTM4B isoforms-expressing AML cells and the engraftment was analysed by flow cytometry. Activation of key secondary messengers was measured by luciferase response elements and Western Blot. Results: LAPTM4B overexpression directly correlated with a higher resistance to cytarabine. LAPTM4B short isoform presented an increased clonogenic potential versus the long isoform. Interestingly, a competitive bone marrow transplantation assay confirmed this effect in terms of in vivo engraftment capacity. Moreover, LAPTM4B-mediated activation was able to induce MAPK signalling in the leukemic cell context. Summary/Conclusion: LAPTM4B overexpression not only increases the regeneration potential but also confers resistance to cytarabine, suggesting an effect in the maintenance of leukemia. In summary, this study will increase the knowledge of the role of lysosomes in leukemogenesis, and additionally, it will help to identify new lysosome-related biomarkers. P426: A SINGLE-CENTER RETROSPECTIVE ANALYISIS OF THE MUTATIONAL PROFILE IN ACUTE MYELOID LEUKEMIA PATIENTS Y. Martínez Díez1,*, A. Franganillo Suárez1, J. Cornago1, L. Solan2, R. Ayala3, J. Martinez3, P. Llamas1, J. L. Lopez Lorenzo1 1Hematology; 2Hospital Fundación Jiménez Díaz; 3Hematology, Hospital 12 de Octubre, Madrid, Spain Background: Acute myeloid leukemia (AML) is a clonal disorder originating from specific genetic aberrations in myeloid precursor cells. Next generation sequencing (NGS) analyses provide clinicians with a deeper knowledge of AML pathogenesis and is being implemented as a routine test at diagnosis due to its relevance in management and prognosis, following the characterization of molecular subgroups. Here we provide a summary of our experience in a single centre (Fundación Jiménez Díaz University Hospital (FJDH), Madrid, Spain), describing the molecular profile of our AML patients, studied as part of a national Spanish project lead by the PETHEMA group, and an analysis on which alterations could be significant to overall survival (OS). Aims: Analyse prognosis associated to molecular profile of new cases of AML Methods: AML new diagnoses and relapses in FJDH from 2018 to 2020 were retrospectively evaluated. The samples were referred to a central laboratory (12 de Octubre University Hospital, Madrid, Spain) where NGS custom panels were run. Other significant variables were analysed. Results: 66 AML patients were evaluated with a mean age of 60yo (22-93). 191 mutations were detected and most NGS were run at diagnosis 57 (86.4%). Most of the sample received an intensive treatment 42 (63.6%), however only 25 (37.9%) patients achieved CR, 2 (3%) CRi, 6 (9.1%) +MRD, 4 (6.1%) PR and 26 (39.4%) were refractory. 11 (16.7%) patients relapsed. Median OS was 13 months (CI 95% 7.9 – 18.1). Survival analysis confirmed significant differences between the 3 ELN prognostic groups and some of the genetic alteration (Figure 1). In this series there was high incidence of secondary and adverse prognosis AML, as expected from the high expression of ASXL1 (12.1%) and TP53 (15.2%) mutations. DNMT3A mutations were most frequent (25.8%), followed by NPM1 (22.8%). We also found IDH mutations (28.8%), which though less common could also be expected in adverse prognosis AML. Our sample size was big enough that prognostic differences within each ELN classification group were observed, as well as related to treatment. There was statistical significance in OS between patients with wildtype vs mutated TP53 (8.9 vs 22.8; p=0.037) or JAK2 (3.8 vs 22.5, p=0.001), as supported by literature. Interestingly, we also found DNMT3A mutated AML had a worse OS (24.1 vs 12.7; p=0.023). This may support recommending HSCT in DNMT3A-mutated AML, as argued for TP53- and JAK2-mutated AML. Although our OS graphs inclined towards favourable prognosis in the presence of NPM1 mutations (27.9 vs 19.9; p=0.12) and adverse prognosis with ASXL1 (11.1 vs 22.5; p=0.055), the statistical analysis did not show significance in either case, probably due to small sample size. RUNX1 did not seem to play a role in prognosis (19.1 vs 20.4; p= 0.741) which might be because most patients underwent HSCT, although current studies seem to suggest RUNX1 mutations are independent to prognosis. Similarly, FLT3-ITD mutations did not confer worse outcomes (25.5 vs 20.1; p=0.464). This may be due to the mutation often being absent in disease relapse or progression, sometimes in relation to targeted therapies. In our sample IDH mutations did not confer changes to OS (18.2 vs 21.9; p=0.707), though this could be explained by the limited availability of targeted therapies in our country. Image: Summary/Conclusion: We found high incidence of high risk AML. Most of our data agree with the published literature. Of note, RUNX1 doesn’t seem to play an important role in prognosis. However, DNMT3A mutations alone and mainly as a group with TET2 and ASXL1 were related to a bad outcome. P427: THE SINGLE CELL T CELL LANDSCAPE OF AML PATIENTS POST ALLOGENEIC STEM CELL TRANSPLANTATION A. Mathioudaki1,2,*, X. Wang3, R. Huth3, J. Zaugg4, C. Pabst3,4 1Molecular Medicine Partnership Unit, European Molecular Biology Laboratory (EMBL); 2Faculty of Biosciences, Heidelberg University; 3Department of Medicine V, Hematology, Oncology and Rheumatology, University Hospital Heidelberg; 4Molecular Medicine Partnership Unit, EMBL Heidelberg, Heidelberg, Germany Background: In Acute myeloid leukemia (AML), chemotherapy may lead to long-term remission, but allogeneic stem cell transplantation (alloSCT) often remains the only curative therapeutic approach, particularly in AML with high-risk genetics. However, not every patient responds to alloSCT. Several hypotheses have been associated with therapy failure, such as the incapability of T cells to recognize and eliminate residual leukemia stem cells (LSCs), which thus escape the graft-versus-leukemia (GVL) effect. However, the role of the T cells’ repertoire in therapy outcome still remains unclear. Aims: Here, we investigated the impact of the bone marrow (BM) T cell composition on the therapy outcome of AML patients after alloSCT. Methods: We performed single-cell RNA sequencing (scRNA-seq) of T cells and hematopoietic stem and progenitor cells (HSPCs) isolated from BM aspirates 100 days post alloSCT of three patients in complete remission (CR) versus three suffering relapse (REL). We investigated the prognostic impact of putative T-cell biomarkers in therapy outcome, using flow cytometry analysis on an independent cohort. Results: Using scRNA-seq, we identified 21 T cell and 9 HSPC populations and observed differences in T cell population abundances between REL and CR. In particular, we observed an enrichment of specific CD8+ T cell subsets in the CR group, while certain CD4+ T cell subsets, such as regulatory T cells were enriched in REL patients. Furthermore, we observed an increased T cell cytotoxicity signature in the CR group, that potentially contributes to better clinical outcome. Within the genes upregulated in the CR condition, we identified surface molecules linked to T cell cytotoxicity. Subsequent flow cytometry analysis corroborated a link between these markers and better clinical outcome. Our data suggest that the early bone marrow T cell signature post alloSCT might reflect GVL-effects required to achieve long-term survival. Image: Summary/Conclusion: Understanding the role of T cell composition on therapy response may offer novel approaches for both predicting prognosis as well as developing new therapeutic strategies. P428: SYK INHIBITION DRIVES DEEP RESPONSES IN A BIOMARKER GUIDED SUBSET OF AML ALONE AND IN RATIONAL COMBINATIONS M. McKeown1,*, L. A. Carvajal1, M. A. Day1, C. Noe1, P. Kumar1, J. DiMartino1, D. Saffran1 1Kronos Bio, Inc., San Mateo, United States of America Background: Spleen tyrosine kinase (SYK) acts as a key integrator of signals from cell surface receptors containing an immunoreceptor tyrosine-based activation motif to boost cellular proliferation. In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network linked to NPM1, HOXA9 and MEIS1. The selective, orally bioavailable SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity and tolerability in HOXA9/MEIS1-driven AML. ENTO is currently being investigated in a global Phase 3 trial, AGILITY (NCT05020665), in combination with intensive induction/consolidation chemotherapy in patients with treatment-naive NPM1-mutated (NPM1m) AML. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency and selectivity to ENTO but with more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). Aims: To assess the potential of biomarker-guided responses to ENTO and LANRA in mutationally defined subsets of AML and its activity in combination with other targeted agents in translationally relevant models. Methods: Full kinome profiling was run at fixed 1uM concentration followed by dose response IC50 determination for top hits. AML and lymphoma cell lines were tested for antiproliferative effects using CellTiter Glo (CTG) at 5 days. AML patient-derived ex vivo models were tested in microtiter plate viability assays with CTG or in methylcellulose colony viability assays for the NPM1m/FLT3-ITD model used in combination studies. Results: Kinase selectivity profiling found >10-fold higher selectivity of ENTO and LANRA for SYK vs other kinases with improved potency and selectivity compared with the approved first-generation agent, fostamatinib. SYK inhibition with either agent showed robust anti-proliferative activity in a panel of AML and lymphoma cell lines with varying mutational backgrounds. In addition, synergy was observed when ENTO or LANRA was combined with a Menin inhibitor in MLLr, FLT3-ITD cell lines. LANRA and ENTO showed strong anti-proliferative activity in NPM1m AML patient samples validating NPM1m as a patient selection biomarker for ENTO and LANRA. In an analysis of a large public dataset of ex vivo patient samples, the presence of NPM1m and/or FLT3-ITD was significantly predictive of response to ENTO. In an ex vivo model of NPM1m/FLT3-ITD AML, consistent sub-micromolar response to LANRA was observed. Combinations with AML standard-of-care and investigational agents, including azacytidine, showed at least additive effects. Strong synergy with the JAK inhibitor ruxolitinib was consistent mechanistically with a reporter model that demonstrated the ability of LANRA to block activation of STAT5 in response to proleukemic paracrine signaling. Finally, robust synergy across a range of LANRA concentrations was found when paired with venetoclax and gilteritinib. These results prompted further testing with an in vivo, patient-derived xenograft. Summary/Conclusion: SYK is a promising therapeutic target for subsets of AML patients defined by specific mutations. The highly predictive response for SYK inhibition with concurrent NPM1m/FLT3-ITD mutations and synergy with gilteritinib observed in patient derived models is supportive of its clinical evaluation in combination with a FLT3 inhibitor. Our work supports the rationale for an ongoing Phase 1b/2 study of LANRA in combination with gilteritinib in patients with R/R FLT3-mutated AML. P429: CLONALLY RESOLVED SINGLE-CELL MULTI-OMICS IDENTIFIES LEUKEMIA SURFACE ANTIGENS A. K. Merbach1,*, S. Beneyto-Calabuig2, J.-A. Kniffka1, C. Szu-Tu2, C. Rohde1, M. Antes1, M. Janssen1, A. Waclawiczek3, J. J. M. Landry4, V. Benes4, J. Anna5, M. Brough5, B. Besenbeck1, J. Felden1, S. Bäumer6, M. Hundemer1, T. Sauer1, C. Pabst1, M. Scherer2, S. Raffel1, L. Velten2, C. Müller-Tidow1 1Department of Medicine, Hematology, Oncology and Rheumatology, University Hospital Heidelberg, Heidelberg, Germany; 2Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain; 3Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ) and DKFZ-ZMBH Alliance; 4Genomics Core Facility, European Molecular Biology Laboratory (EMBL); 5Institute of Human Genetics, University Hospital Heidelberg, Heidelberg; 6Department of Medicine A, Hematology and Oncology, University Hospital Muenster, Münster, Germany Background: At the time of diagnosis, AML is a highly heterogeneous disease, usually consisting of various epigenetically and genetically distinct pre-leukemic, as well as leukemic subclones of various maturation stages that coexist in a competitive, or even cooperative manner. This clonal heterogeneity and, in particular, the strong resemblance of leukemic cells with their healthy counterparts presents a significant challenge in the identification of cancer specific gene expression and surface markers, both prerequisites for the development of targeted therapies. Existing approaches to add clonal resolution to droplet-based single-cell RNAseq data use qualitative information available from calls of individual mitochondrial or genomic single nucleotide variants as well as copy number variants. These single cell mutational calls, however, cannot be taken at face value, but need to be analysed through appropriate statistical methods, which are currently missing. Aims: Here we aimed at developing a novel method, CloneTracer, to add clonal resolution to new or existing human single-cell transcriptomic or multiomic datasets to ultimately allow identification of leukemia surface antigens through intra-sample comparison of cancerous and healthy cells of matched differentiation state. Methods: CloneTracer amplifies mitochondrial and selected nuclear SNVs, infers copy number variation, and estimates clonal hierarchies and identities through a Bayesian model that accounts for the noise properties of single-cell RNA-seq data. Results: Through combination of clonal identities with information on surface markers at single cell resolution, CloneTracer identified novel leukemia markers that enabled quantification of the leukemic burden in clinical specimens and enrichment for leukemic or healthy cells by FACS. Image: Summary/Conclusion: In summary, our data illustrate the benefits of clonally resolved single cell multi-omics for identifying cancer specific surface antigens and potential druggable targets by intra-patient comparisons. P430: MOLECULAR CHARACTERIZATION OF CLINICAL RESPONSE IN NEWLY-DIAGNOSED ACUTE MYELOID LEUKEMIA PATIENTS TREATED WITH IVOSIDENIB + AZACITIDINE COMPARED TO PLACEBO + AZACITIDINE S. de Botton1,*, S. Choe2, D. Marchione2, P. Montesinos3, C. Recher4, S. Vives Polo5, E. Zarzycka6, J. Wang7, G. Bertani8, M. Heuser9, R. Calado10, A. Schuh11, S.-P. Yeh12, J. Hui2, S. Pandya2, D. Gianolio2, S. Daigle2, C. DiNardo13, H. Dohner14 1Institut Gustave Roussy, Villejuif, France; 2Servier Pharmaceuticals LLC, Boston, United States of America; 3Hospital Universitari i Politècnic La Fe, València, Spain; 4Institut Universitaire du Cancer de Toulouse Oncopole, Toulouse, France; 5Hospital Universitario Germans Trias i Pujol-ICO Badalona, Josep Carreras Research Institute, Badalona, Spain; 6Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk, Poland; 7Institute of Hematology & Hospital of Blood Disease, Tianjin, China; 8ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 9Hannover Medical School, Hannover, Germany; 10Ribeirão Preto School of Medicine, University of São Paulo, São Paulo, Brazil; 11Princess Margaret Cancer Centre, Toronto, Canada; 12China Medical University, Taichung, Taiwan; 13The University of Texas M.D. Anderson Cancer Center, Houston, United States of America; 14Ulm University Hospital, Ulm, Germany Background: Acute myeloid leukemia (AML) is a disease with a dynamic mutational landscape; 6–10% of patients (pts) have somatic mutations in isocitrate dehydrogenase 1 (IDH1), which can drive oncogenesis. Ivosidenib (IVO) is a potent oral targeted inhibitor of mutant IDH1 (mIDH1). IVO 500 mg QD + azacitidine (AZA) 75 mg/m2 SC or IV for 7 days in 28-day cycles was shown to significantly improve event-free survival (HR=0.33 [95% CI 0.16, 0.69], p=0.0011), median overall survival (24.0 vs 7.9 months), and complete remission + partial hematologic recovery rates (CR/CRh; 52.8% vs 17.6%) vs placebo + AZA in the double-blind phase 3 AGILE study (NCT03173248) in pts with newly diagnosed IDH1-mutated AML (ND-AML). Aims: To assess the impact of IVO+AZA on IDH1-mutation clearance (IDH1-MC) and baseline co-mutation analysis from AGILE. Methods: Genomic DNA from bone marrow mononuclear cells (BMMCs) or peripheral blood mononuclear cells (PBMCs), and/or bone marrow aspirate (BMA) were used for molecular studies. IDH1-MC analysis on BMMCs was performed by BEAMing digital PCR (limit of detection 0.02%-0.04%). BMA, BMMCs and PBMCs were utilized for co-mutational analysis by next-generation sequencing, ACE Extended Cancer Panel (detection limit 2%). All patients gave written informed consent. Results: 146 pts were randomized: 72 to IVO+AZA; 74 to placebo+AZA. Median (range) baseline mIDH1 variant allele frequency in BMMCs was 36.7% (3.1–50.5%) in the IVO+AZA arm and 35.5% (3.0–48.6%) in the placebo+AZA arm. Updated IDH1-MC data (October 2021) from 47 IVO+AZA and 32 placebo+AZA treated pts with at least 1 on-treatment sample demonstrated IDH1-MC in 21/35 (60%) IVO+AZA pts achieving CR/CRh vs 4/11 (36%) placebo+AZA pts. In CR/CRh pts with time points available after IDH1-MC, suppression of the mIDH1 was durable and IDH1-MC maintained in all subsequent samples in 17/17 (100%) IVO+AZA treated pts and 1/3 (33%) placebo+AZA pts. Further analysis of baseline co-mutations on 120 pts (IVO+AZA: n=58; placebo+AZA: n=62) showed that DNMT3A, SRSF2, and RUNX1 were the most frequent in both treatment arms. Importantly, comparison of CR/CRh and non CR/CRh responses by cohort did not identify any single gene or pathway associated with an inferior outcome in IVO+AZA pts compared to placebo+AZA pts (p<0.05, Fisher’s Exact test). Several genes (DNMT3A, RUNX1, SRSF2, STAG2) and pathways (Differentiation, Epigenetics, Splicing) were associated with improved outcomes with IVO+AZA, including the RTK pathway, which was previously reported to be associated with primary resistance to IVO monotherapy. Further analysis of patient subgroups, including R132 variants (i.e., R132C vs R132S), will be presented. Summary/Conclusion: These data suggest that improved clinical outcomes with IVO+AZA are associated with sustained clearance of the mIDH1 clone including pts with disease that harbor mutations implicated in resistance to IVO monotherapy (e.g., with RTK pathway mutations). P431: THE ROLE OF SMARCA4 IN HEMATOPOIESIS AND ACUTE MYELOID LEUKEMIA F. Modemann1,2,*, L. Ramke1, J. Muschhammer1, L. Behrmann1, V. Thaden3, M. Schoof3,4, C. Göbel3, S. Neyazi3,4, C. Bokemeyer1, J. Wellbrock1, U. Schüller3,4,5, W. Fiedler1 1Department of Oncology and Hematology, University Medical Center Hamburg-Eppendorf; 2Mildred Scheel Cancer Career Center, University Medical Center Hamburg-Eppendorf; 3Research Institute Children’s Cancer Center Hamburg; 4Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf; 5Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Background: SMARCA4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a) is the central ATPase containing enzyme in the SWI/SNF- (switch/sucrose non-fermentable) complex, which influences gene transcription by regulating chromatin accessibility. In solid tumors, SMARCA4 mainly possesses tumor suppressive features. However, in the setting of acute myeloid leukemia (AML), SMARCA4seems to play a different role, acting as a proliferation stimulus for myeloid blasts. Aims: The aim was to investigate the role of SMARCA4 in normal hematopoiesis and in pathogenesis and maintenance of AML to evaluate the prognostic and potential therapeutic relevance of this protein. Methods: We screened 152 bone marrow samples of newly diagnosed adult AML patients compared to 29 healthy bone marrow samples for SMARCA4 expression by using RT-qPCR. SMARCA4 expression was correlated to overall and relapse free survival. We further analyzed proliferation and colony-formation capacity of AML cell lines with lentivirally transduced shRNA-mediated SMARCA4 knockdown or SMARCA4 overexpression induced by lentiviral transduction. Two mouse models were generated: Firstly, to evaluate the impact of SMARCA4 knockout on normal hematopoiesis, a transgenic mouse model with Cre-recombinase mediated conditional knockout of SMARCA4 under the control of an Mx-1 promoter was developed. Blood counts, bone marrow smear, colony formation assays, and RNA expression profiles were analyzed. Secondly, a xenograft mouse model was generated by transplantating the AML cell line MV4-11 with SMARCA4 knockdown or overexpression in NSG mice to analyze overall survival. Results: Expression of SMARCA4 was significantly higher in adult AML samples compared to healthy CD34+ cells (p<0.0001). High SMARCA4 expression was associated with worse overall (p=0.001) and relapse free survival (p=0.0073). SMARCA4 knockdown in AML cell lines resulted in significantly lower proliferation rate in three different AML cell lines compared to controls, whereas SMARCA4 overexpression led to higher proliferation rates. Overall survival of mice transplanted with SMARCA4 overexpressing MV4-11 cells was significantly worse than in the control group, whereas mice transplanted with SMARCA4 knockdown MV4-11 cells lived significantly longer. Transgenic SMARCA4 knockout mice showed significantly lower leukocyte, reticulocyte, and thrombocyte counts and showed lower colony forming capacity compared to Cre-negative control mice. Bone marrow smears showed aplasia of all cell lines in SMARCA4 knockout mice. RNA-sequencing revealed 749 upregulated and 520 downregulated genes with a log fold change ≥ +/- 1 in the bone marrow of SMARCA4knockout mice compared to Cre-negative control mice. Relevant pathways according to REACTOME data base performing over representation analysis included genes of generation of second messenger molecules, cell surface interactions at the vascular wall, and immunoregulatory interactions between lymphoid and non-lymphoid cells. Image: Summary/Conclusion: SMARCA4 is upregulated in primary AML cells and is an unfavorable prognostic marker in AML patients. SMARCA4 overexpression in AML cell lines leads to higher proliferation rate and to a worse overall survival in transplanted mice. In normal hematopoiesis, SMARCA4 knockout leads to decreased cell growth in bone marrow, highlighting the dependence on SMARCA4 for proliferation of hematopoietic (stem) cells. P432: RISK STRATIFICATION IN ACUTE MYELOID LEUKEMIA: EXTERNAL VALIDATION OF THE STELLAE-123 GENE EXPRESSION MODEL AND COMPARISON WITH CURRENT PROGNOSTIC SCORES A. Mosquera Orgueira1, A. Peleteiro Raindo1,*, J. A. Diaz Arias1, M. Cid Lopez1, R. Abal Garcia1, B. Antelo Rodriguez1, M. S. Gonzalez Perez1, J. C. Vallejo Llamas1, M. M. Perez Encinas1 1Hematology, CHUS, Santiago de Compostela, Spain Background: The identification of recurrently mutated genes and cytogenetic anomalies has provided high prognostic and therapeutic significance in patients diagnosed with AML. Currently, some of this information has been incorporated into risk stratification guidelines, such as those of the European Leukemia Net (ELN). However, these guidelines are based on cytogenetic analyses and a limited number of mutations, and they don’t consider the genomic complexity of AML. Recently, we presented a new prognostic score based on gene expression analysis (Stellae-123) which achieved high discriminative power in survival prediction of AML patients, particularly among those bearing high-risk mutations. It is necessary to evaluate this signature in new AML cohorts and to test its performance compared with standard risk stratification scores. Aims: To validate the prognostic precision and discriminative power of the Stellae-123 gene expression signature in external cohorts and to compare its performance with standard prognostic scores: ELN-2017 in adult patients and clinical risk score in pediatric patients. Methods: RNA-seq data from 2 adult cohorts and one pediatric AML cohort was retrieved. The BeatAML (N=334) and the AMLCG-2008 (N=199) cohorts consisted of adult AML patients, whereas the TARGET AML cohort (N=144) was composed solely of pediatric patients. Gene expression estimates (FPKM) were normalized between cohorts using ComBat. Common genes were retrieved, and we selected those included in the Stellae-123 predictor, reaching a total of 69 genes. Random forests were built to predict survival in the largest cohort (BeatAML). The precision of the predictors was evaluated using cross-validated time-dependent AUCs derived from cox models. In the particular case of BeatAML, gene expression survival models were based on out-of-bag predictions in order to reduce the risk of overfitting during the training phase of the model. Results: We initially built a survival predictor using random forests based on the expression of the 69 genes incorporated in Stellae-123. This predictor was trained in the BeatAML cohort, and external validation was performed on the remaining two cohorts. The model achieved c-indexes of 0.635, 0.645 and 0.598 in the BeatAML, AMLCG-2008 and TARGEL AML cohorts, respectively. We then evaluated the precision of this signature to predict survival at 6 months, 1 year and 2 years after diagnosis using cross-validated cox models. The results indicated that this model achieved greater precision than the ELN-2017 and the pediatric clinical risk score in the majority of evaluated time points (Figure 1). Since age is a variable deeply associated with survival in AML, we evaluated the performance of the models including this covariate. We observed clear improvements in AUCs for the BeatAML and TargetAML, but not for the AMLCG-2008 cohort, a finding which is probably related to the fact that these were fit adult patients recruited in a clinical trial. Notably, we also observed an improved performance of the gene expression signature plus age model compared with the ELN-2017 and pediatric clinical risk scores plus age models, particularly in the prediction of survival within the first year after diagnosis. Image: Summary/Conclusion: The Stellae-123 gene expression signature can predict overall survival in AML with greater precision than the ELN-2017 and the pediatric clinical risk score. Therefore, gene expression profiling emerges as a powerful tool to optimize patient risk stratification. There is a growing need to standardize these tests for clinical use. P433: ADAPTER HOLIDAYS MAINTAIN IN-VITRO ADCAR-T CELL FUNCTIONALITY AGAINST AML D. Nixdorf1,2,*, M. Sponheimer1,2, D. Berghammer2, N. Zieger1,2, L. Rohrbacher1,2, S. Dapa3, C. M. Seitz4, K. Brandstetter5, M. von Bergwelt-Baildon1, H. Leonhardt5, J. Mittelstaet6, A. Kaiser3, V. Bücklein1,2, M. Subklewe1,2 1Department of Medicine III; 2Laboratory for Translational Cancer Immunology, LMU, Munich; 3R&D Department, Miltenyi Biotec GmbH, Bergisch Gladbach; 4Department of General Pediatrics, University Children’s Hospital Tuebingen, Tuebingen; 5Department of Biology II, LMU, Munich; 6R&D Department, Miltenyi Biotech GmbH, Bergisch Gladbach, Germany Background: The rise of immunotherapies has sparked hope for improving the treatment options of acute myeloid leukemia (AML). However, bispecific antibodies (AB) and CAR-T cells demonstrated only moderate efficacy in clinical trials. The heterogeneous nature of the disease requires more flexible, potent and safer approaches to make T-cell based treatments available for AML patients. We have recently developed a novel, highly modular adapter CAR-T cell (AdCAR) platform capable of targeting multiple antigens via biotin-labeled adapter molecules (AMs) in the context of a specific linker (Seitz et al., 2021). Aims: Utilizing an AdCAR-T cell approach to overcome well described obstacles in AML we evaluated several key parameters of our platform, including cytotoxicity, AM internalization and serial targeting capability. Furthermore, we highlight the possibility of intermittent (int.) AM dosing regimens (AM holidays) to delay AdCAR-T cell dysfunction due to continuous (cont.) stimulation. Methods: The cytotoxic capacity of AdCAR-T against AML cell lines and primary AML (pAML) samples was evaluated in co-culture assays utilizing Fab-based AMs. Specific lysis was assessed by FC after 48-72 h. Internalization kinetics of AMs were assessed via surface staining of AML cells with AMs and secondary staining with anti-Biotin-PE AB. The % surface bound AMs over time at 37 °C vs 4 °C was analyzed. For serial targeting experiments the AMs were either added once, replenished every 3 days, or changed to AMs with different target specificity. To mimic AM holidays in vitro, cont. vs int. AM-exposed AdCAR-T were co-cultured with OCI-AML-3 (21 d vs 7 d AM exposure, followed by 7 d AM absence). Cytotoxic capacity, T cell proliferation and cytokine secretion were assessed. Results: AdCAR-T cells elicited high cytotoxicity against several AML cell lines utilizing AMs of different specificity (AMs for CD33, CD123, CLL-1; IC50 = 2.1, 0.6, 1.9 ng/ml). Against pAML similar data were obtained in an E:T and AM-concentration dependent manner. We demonstrated feasibility of serial AM usage by performing long-term pAML/AdCAR-T co-cultures. In comparison to one-time or low-dose cont. CD33 AM exposure, which led to an outgrowth of pAML cells after 12 d, increasing AM concentrations and/or switching the target antigen improved AdCAR-T cell efficacy. We further observed receptor-mediated endocytosis of AMs (reduction of MV4-11 surface bound CD33 AM after 6 h = 81%) and postulate that AM internalization is a common form of antigen sink in AM-based T-cell recruiting therapies. Internalization of AMs reduces AM half-life and decreases cytotoxic capacity of AdCAR-T cells. While limited AM levels can be overcome by repetitive dosing, AM internalization potentially also contributes to faster off-switch dynamics. Importantly, we observed that cont. AdCAR-T cell stimulation leads to T cell dysfunction (Fig 1) in in vitro long-term efficacy studies. Strikingly, these effects can be mitigated by implementing int. AM dosing regimens, shown by significantly increased T cell proliferation (fold change d14: 1.9 vs 3.5), cytotoxic capacity (% specific lysis d14: 43 vs 64) and IL-2 production (202 vs 823 pg/ml; all mean+/-SEM). Image: Summary/Conclusion: Taken together, AdCAR-T cells provide flexibility in target antigen choice, safety through short half-life of the AMs and long-term functionality by mitigating AdCAR-T cell dysfunction through AM holidays. Future trials need to consider the high risk of CAR-T cell exhaustion upon cont. stimulation and AdCAR-T cells might pave the way for rational design of more sophisticated treatment schedules. P434: MITOCONDRIAL MCL1 REGULATES LEUKEMIC CELLS METABOLISM VIA DIRECT INTERACTION WITH HEXOKINASE II. METABOLIC SIGNATURE AT ONSET PREDICTS OVERALL SURVIVAL IN AMLS’ PATIENTS N. I. Noguera1,2,*, G. Catalano1,2, A. Zaza1,2, C. Banella3, T. Ottone2,4, E. Pelosi5, S. Travaglini1,2, M. Divona6, M. I. Del Principe1, F. Buccisano1, L. Maurillo1, E. Amatuna7, U. Testa5, A. Venditti1, M. T. Voso1,2 1Tor Vergata University; 2Fondazione Santa Lucia, Rome; 3Meyer Children’s University Hospital, Florence; 4Tor Vergata University, Roma; 5Istituto Superiore di Sanità; 6Policlinico Tor Vergata, Rome, Italy; 7University Medical Center Groningen, Groningen, Netherlands Background: Metabolic reprogramming is a basic feature in cancer. Leukemia associated cellular reprogramming, metabolic heterogeneity at onset and metabolic clonal evolution, driven by therapy, are essential insights that still remain unclear Aims: We compared Acute Myeloid Leukemias’ (AMLs) cells metabolism to hematopoietic progenitors’’ and normal maturing bone marrow cells’, to acquire useful prognostic information and to uncover actionable therapeutic targets. Methods: Methods We analyzed fresh primary blast from 19 AML patients, hematopoietic progenitors obtained from normal CD34+ cells differentiated to promyelocytes and granulocyte and the MV4-11, OCI-AML2 and OCI-AML3 cell lines, using a Seahorse Bioscience XFe96 analyzer. We silence MCL1 by siRNA and evaluated the interaction between HK2, MCL-1 and VDAC by co-immunoprecipitation and confocal microscopy. We assessed expression of MCL-1 and HK2 by western blot; HK2 by q-RTPCR. Results: Primary AML blast cells feature a lower spare respiratory capacity (SRC) (p=0.02) and lower glycolytic capacity (p=0.02) as compared to early progenitors/precursors (EP/P) from cultured CB CD34+ cells at day 7 of culture (N7, mostly promyelocytes) (Figure 1 a and b). Primary AML blast depend principally on fatty acids (p<0.05); they display a great flexibility, switching to glucose or glutamine to meet their energetic needs (Figure 1c). Consistent with the high adaptability of AML cells, and the emergence of resistance to therapy. We could define two populations (cut off value 10 pmol/min/x105 cells): one with higher (22±12 pmol/min/x105 cells) and on with lower (3±2 pmol/min/x105 cells) p<0.0001 levels of proton leak. The cases with higher proton leaks levels presented a reduced, extremely short, overall survival (p=0.048). We defined two SRC populations (cut off value 80 pmol/min/x105 cells): higher (124±47 pmol/min/x105 cells) and lower (52±25 pmol/min/x105cells) levels (p=0.0001). Interestingly the cases with higher SRC showed a trend of reduced overall survival. Associating high proton leak levels with high SRC the significance increases to p= 0.007. Considering the Basal OXPHOS of the AML patient’s cells, we observed two populations higher (54±12 pmol/min/x105 cells) and lower (16±6 pmol/min/x105cells) levels (p=0.0001). In patients with both high SRC plus high basal OXPHOS, the overall survival shortage is significant (p=0.002), indicating that an high SRC associated to higher basal respiration in AML blast cells confers greater aggressiveness and resistance to the therapy (Figure 1d). Patients with high mitochondrial respiration had a significantly higher myeloid cell leukemia 1 protein (MCL1) expression. We ascertained that MCL1 directly binds to Hexokinase II (HK2) on the outer mitochondrial membrane (OMM) affecting its stability. Image: Summary/Conclusion: We demonstrate that high proton leak and high mitochondrial respiration at onset, arguably with the concourse of MCL1/HK2 action, is significantly linked with a shorter overall survival in AMLs’ patients. Our data describe a new function of MCL1 protein in AMLs’ cells, forming a complex with HK2 co-localized to the voltage dependent anion channel (VDAC) on the OMM, thus promoting glycolysis and OXPHOS, ultimately conferring metabolic plasticity and promoting resistance to therapy. The lower spare respiratory capacity and lower glycolytic capacity of AML patients’ blast respect to normal early hematopoietic precursors suggest a therapeutic window to use glycolytic and mitochondrial inhibitors in resistant AML patients. P435: ANALYSIS OF MYELOBLAST IMMUNOPHENOTYPE PROFILE IN ACUTE MYELOID LEUKAEMIA AS PREDICTOR FOR CLINICAL OUTCOME M. Sobas1, M. Nowak1,*, D. Szymczak1, I. Andrasiak2, T. Wróbel1 1Department of Haematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical Univeristy; 2Independent Researcher, -, Wrocław, Poland Background: The European LeukemiaNet (ELN) classification, is currently widely accepted risk stratification of acute myeloid leukaemia (AML). ELN stratification is based on genetic abnormalities and divides AML in low, intermediate and high risk groups. However, the outcome of AML patients, even within one classified group may be different. Flow cytometry analysis of blast cells play an essential role in diagnosis of AML, but the prognostic value of particular antigen expression is still unknown. Aims: The aim of this study was to investigate the prognostic impact of the immunophenotype of blast cells. Methods: We performed a retrospective analysis on 146 AML patients (aged 21-65) who were diagnosed and treated with intensive chemotherapy between 2017-2020. Allo-HSCT recipients were labelled TPL1, the others - TPL0. Patients with acute promyelocytic leukaemia were excluded from the study. Quantitative value (%) of particular antigen (CD2, CD4, CD7, CD9, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD25, CD33, CD34, CD36, CD38, CD41, CD45, CD56, CD61, CD64, CD65, CD71, CD73, CD96 CD117, CD123, HLA-DR) expression was collected. ELN classification at diagnosis, cytogenetic, mutational status (FLT3-ITD, FLT3-TKD, NPM1, CEBPA, CBFB-MYH11, RUNX1-RUNX1T1) and outcome (complete remission (CR), primary resistance, relapse after achieving CR were recorded). Categorical variables were presented as frequencies with percentages, median and interquartile range (IQR) were used to describe continuous variables. Non-normally distributed variables were analysed using Mann Whitney test and Kruskal-Wallis test. The Kaplan-Meier method and Cox proportional-hazards model were used to estimate time-to-event variables and determine hazard ratio (HR). All statistical tests were two-tailed with the significance level set at α=0,05. The study was performed in accordance with the Declaration of Helsinki. Results: The baseline characteristics of the patients are presented in a table. The median age at diagnosis was 54 (aged 21-65). CR rate was 73%. The median follow up of 146 AML was 443 days. The median value of HLA-DR of relapsed (n=31, 21%) vs non-relapsed AML (n=115, 79%) was higher: 100 (90-100) vs 90 (67-99); p=0.006. There was no relationship between the HLA-DR expression (%) and ELN risk (p=0.705) nor mutational status (p>0.05). The primary resistance was more common in patients with higher CD34 and lower CD38 levels. The median value of CD34 in resistant AML vs those who achieved CR was 93% (22-99) vs 47% (0-91); p=0.017 and the median value of CD38 was 61% (31-93) vs 93% (83-98); p=0.011. The median CD34 value was significantly higher in the adverse vs favourable ELN-group: 79% (18-96) vs 2% (0-69), respectively (p=0.001) while the median CD38 value was significantly higher in the favourable vs adverse ELN-group, 96% (88-99) vs 86% (58-96), respectively, (p=0.005). Improved EFS was reported for decrease of HLA-DR (HR=1.01; p=0.049) and increase of CD38 (HR=0.99; p=0.046) among TPL0 patients. These variables are borderline during OS prediction for TPL0 - HLA-DR (HR=1.01; p=0.081) and CD38 (HR=0.99; p=0.051). Image: Summary/Conclusion: Together with genetic mutations, immunophenotyping could be an equivalent component of risk assessment. The levels of CD34 and CD38 turned out to modify the first line treatment response and correlate with the ELN stratification. Higher percentage of CD34 and lower of CD38 may characterise refractory to standard intensive therapy AML, while higher HLA-DR may proceed relapse. Further, analyses, on prospective trials, should be conducted to confirm these correlations. P436: TARGETED DELIVERY OF T22-PE24-H6 TO CXCR4 POSITIVE CELLS FOR ACUTE MYELOID LEUKEMIA TREATMENT Y. Núñez Amela1,2,*, A. Garcia-León1,2, A. Falgàs1,2,3, N. Serna3,4,5, L. Sánchez-García3,4,5, A. Garrido2,6, J. Sierra2,6, A. Gallardo1,7, U. Unzueta1,2,3,5, E. Vázquez3,4,5, A. Villaverde3,4,5, R. Mangues1,2,3, I. Casanova1,2,3 1Biomedical Research Institute Sant Pau (IIB-Sant Pau), Barcelona; 2Josep Carreras Leukaemia Research Institute (IJC), Badalona; 3CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Madrid; 4Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona; 5Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Cerdanyola del Vallès; 6Department of Hematology, Hospital de la Santa Creu i Sant Pau; 7Department of Pathology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Background: Acute myeloid leukemia (AML) is a malignant heterogeneous group of hematological diseases that originates from a hematopoietic progenitor with altered maturation capacity. Recently, considerable advances have been made in the development of targeted therapies for distinct molecularly defined subtypes. Although many AML patients initially respond to current treatment, the majority of them relapse. Therefore, new targeted therapies are needed to overcome drug resistance and improve treatment effectiveness. CXCR4 is a chemokine receptor that is expressed in over 20 cancer types, both hematologic cancers and solid tumors. CXCR4 receptor and its ligand CXCL12 are key mediators of the interactions between AML cells and the bone marrow (BM) microenvironment. Moreover, CXCR4 is highly expressed in around 50% of AML patients. Thus, we have generated a nanoparticle (T22-PE24-H6) that selectively delivers the exotoxin A from Pseudomonas aeruginosa to CXCR4+ leukemic cells. Aims: To assess the selectivity of T22-PE24-H6 cytotoxicity in CXCR4+ AML cell lines as well as in BM samples from AML patients. In addition, we evaluated the antineoplastic effect of the nanoparticle in a CXCR4+ AML disseminated mouse model. Methods: OCI-AML-3, MONO-MAC-6 and HEL cell lines were characterized for CXCR4 expression using flow cytometry and immunohistochemistry (IHC). The in vitro antineoplastic effect of T22-PE24-H6 was determined by cell viability assays. Competition assays with the CXCR4 antagonist AMD3100 were performed to demonstrate the CXCR4-dependent cytotoxicity of the nanoparticle. In vivo effect was assessed in NSG mice intravenously injected with 1x106 luminescent MONO-MAC-6 cells. Animals were treated with 5 μg T22-PE24-H6 or Buffer (166 mM NaCO3H pH 8) daily for 10 doses. Bioluminescence signal (BLI) was monitored to quantitatively measure and evaluate in vivo disease progression. Animal tissue samples were collected after mice euthanasia and used to analyze AML dissemination and toxicity. CXCR4 expression levels and T22-PE24-H6 effect were also determined in BM samples from 10 newly-diagnosed AML patients. Results: T22-PE24-H6 nanoparticle demonstrated a potent in vitro anticancer effect in MONO-MAC-6 cell line. The specific cytotoxic activity of the nanoparticle was CXCR4 dependent as demonstrated by performing competition assays with AMD3100. Furthermore, T22-PE24-H6 intravenous administrations showed significant BLI reduction in a CXCR4+ AML disseminated mouse model compared to buffer-treated mice. In addition, no differences between groups in mouse body weight, biochemical parameters or histopathological changes in normal tissues were detected. Finally, T22-PE24-H6 causes a significant cell viability reduction in AML patient samples with high CXCR4 expression. In contrast, the nanoparticle has no effect in AML patient samples with low expression of CXCR4. Summary/Conclusion: T22-PE24-H6 selectively kills AML cell lines and BM samples from AML patients with CXCR4 overexpression. The nanoparticle also induces a high antineoplastic effect in a CXCR4+ AML disseminated mouse model without detectable systemic toxicity. These data strongly support that T22-PE24-H6 may obtain therapeutic benefit for AML patients with high CXCR4 expression. P437: SURVIVAL-BASED 4-GENE PROGNOSTIC INDEX IMPROVES THE ELN-RISK CLASSIFICATION IN ACUTE MYELOID LEUKEMIA C. A. Ortiz Rojas1,2,3,*, D. A. Pereira-Martins1,2,3,4, C. C. Bellido More5, J. R. Hilberink4, J. L. Coelho-Silva1,3, I. Weinhäuser1,2,3,4, L. Yamamoto de Almeida1,3, V. M. de Deus Wagatsuma1,3, C. Hassibe Thomé1, G. Aguiar Ferreira1, E. Ammatuna4, G. Huls4, J. J. Schuringa4, E. Magalhães Rego1,2,3 1Center for Cell-Based Therapy, University of Sao Paulo, Ribeirão Preto; 2Hematology Division, LIM31, Faculdade de Medicina, University of Sao Paulo, Sao Paulo; 3Department of Medical Imaging, Hematology, and Oncology, Ribeirão Preto Medical School, University of Sao Paulo, Ribeirão Preto, Brazil; 4Department of Hematology, Cancer Research Centre Groningen, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands; 5Department of Pediatrics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirão Preto, Brazil Background: The European Leukemia-Net (ELN) criteria have been routinely used for risk categorization in acute myeloid leukemia (AML), classifying patients into favorable, intermediate and adverse-risk groups based on the presence of specific chromosomal and molecular alterations (Döhner et al., 2017). Despite this classification, high heterogeneity in the disease course is still a clinical barrier since leukemia can progress differently than expected. In this sense, the identification of markers that could improve the risk prediction is an unmet medical need. Aims: We decide to perform a multicohort analysis of transcriptomic data to identify a gene signature consistently associated with prognosis in AML patients. Methods: Publicly available data of five AML cohorts treated with intensive chemotherapy were included in this study: TCGA, n=122; BeatAML, n=230; HOVON (GSE6891), n=392; NTUH (GSE71014), n=104; German (GSE37642), n=136. Also, we retrospectively included 38 patients diagnosed with AML, treated with intensive chemotherapy at the University Medical Center Groningen, in order to validate our gene signature by qRT-PCR. Univariate and multivariate Cox regression and area under the ROC curve (AUC) was applied separately to each gene in each cohort, in order to filter genes associated with prognosis. Prognostic indexes (PIs) models were generated by weighted sum using the pooled β coefficient. Then, the best PI was evaluated in its ability of improving ELN-risk classification. Results: We found seven genes whose expression levels were associated with overall survival. After modeling possible PIs, we selected the PI that generates a better model in combination with ELN classification by using the Akaike’s Criterion Information (AIC) estimator. Thus, we selected a 4-gene prognostic index (4-PI) composed of CYP2E1, DHCR7, IL2RA, and SQLE genes. Survival stratification by 4-PI resulted in 5-year survival rates of 44.7 vs. 7.8% in the TCGA cohort, 28.5 vs. 0% in the BeatAML cohort, 47.7 vs. 26.6% in the HOVON cohort, 41.5 vs. 9.1% in the German cohort, and 88.7 vs. 44.1% in the NTUH cohort. Next, after correction for confounders variables, the 4-PI showed to be independently associated with prognosis, with an HR=3.94, 2.1, and 1.78 for TCGA, BeatAML, and HOVON cohorts, respectively. Among the patient molecular features, we found that inv(16) and dmCEBPA cases were more frequent in the low 4-PI, while TP53 alterations were frequent in patients with a higher 4-PI. Next, we evaluated the utility of the 4-PI in recategorizing patients from the ELN-risk subgroups in a best-fitted risk category. In the BeatAML cohort, ELN2017-adverse patients represented 36.9% (n=85) of patients, with a median OS of 12.2 months, but in the integrated risk, the adverse group increased to 38.7% (n=89) with a median OS of 10.3 months. We found similar results after integration of the ELN2010 with our 4-PI in the TCGA and HOVON cohorts. This power of stratification was maintained after applying the revised ELN proposed by Herold et al., 2020. Next, in our validation cohort, when we integrated the ELN2017 with the 4-PI, 14 patients were recategorized from the intermediate to the adverse group, improving the difference between them (median OS=33.8 vs. 7.6 months). Finally, we found a consistent enrichment of cholesterol metabolism related-pathways in cases with high 4- PI in all the cohorts. Summary/Conclusion: We conclude that our 4-PI outperforms currently used prognosis predictors in AML and its incorporation into ELN categorization can improve risk stratification. P438: ARYL HYDROCARBON RECEPTOR SIGNALLING IN NORMAL HAEMATOPOIETIC (HSC) AND LEUKEMIA STEM CELLS (LSC): DIFFERENTIAL EFFECT OF AHR ANTAGONIST ON LSC COMPARED TO HSC A. Oryshchuk1,*, R. Desai1, P. Kakadiya1, S. Bohlander1 1Molecular medicine and pathology, University of Auckland, Auckland, New Zealand Background: Acute myeloid leukaemia (AML) is a devastating disease with poor prognosis. It is driven by leukemia stem cells (LSCs), which are able to survive treatment and drive relapse. This highlights the importance of characterizing LSCs. LSC-targeted treatment strategies are limited because there are no known surface markers that are specific for LSCs in every leukaemia. Targeting a signalling pathway that is altered in all LSCs could be a novel, promising treatment strategy. The aryl hydrocarbon receptor (AHR) pathway was shown to be critical for the self-renewal properties of normal haematopoietic stem cells (HSCs). Aims: We aimed to study the consequences of manipulating the AHR pathway in vivo in a murine leukaemia model. Methods: We used the following methods: murine bone marrow transplantation leukemia model (MBMTLM) - CALM-AF10 with limiting dilution assay (LDA); competitive repopulation assay (CRA) with LDA (CD45.2 donors into CD45.1 recipient mice), transcriptome analysis (sequencing with the Illumina platform and analisys with the DESeq2). Treatment of the cells was performed at various experiments with the AHR agonist, ITE, and an AHR antagonist, CH-223191 (a vehicle control is DMSO). Results: We used a murine retroviral transduction bone marrow transplantation leukemia model (MBMTLM)) with the CALM-AF10 minimal fusion gene (CAMF) as the oncogenic driver, to study the effect of AHR agonist (ITE) and an AHR antagonist (CH-223191) treatment on LSCs. Interestingly, both AHR agonist and antagonist treatment affected LSC frequency and survival of mice transplanted with treated cells. The LSC frequency as measured by limiting dilution transplantation assays (LDA) was 3.7 times lower after agonist treatment (1:163, blue survival curve in fig. 1, A) compared to a vehicle control (1:44, DMSO, black survival curve)). Very interestingly, after antagonist treatment the LSC frequency was so low that could not be measured as none of the mice transplanted with antagonist-treated cells in the LDAs developed leukaemia (n=9, CH-223191 (red curve in fig. 1, A)). Of note, our CAMF MBMTLM has a very high LSC frequency of about 1:4 in cells that were not cultured after thawing. We performed RNA-Seq on 90 samples to determine changes in gene expression patterns after treatment of healthy bone marrow cells and CAMF leukemia cells with the AHR agonist and antagonist. Preliminary differential expression analysis with DESeq2 revealed differential expression of genes involved in apoptosis and proliferation in leukaemia cells treated with CH-223191. As leukemia did not develop in recipients of cells treated with the antagonist CH-223191, genes differentially regulated in these cells might be critical and specific to LSC function and will be candidate targets for LSC-specific treatments. To confirm that AHR antagonist treatment affects LSCs differently than normal HCS, we performed competitive repopulation assay (CRA). Our preliminary data (n = 12) show that treatment of HSCs with CH-223191 results in a steady increase in the engraftment over a 47-week period following transplantation (coral bars, fig. 1, B), as opposed to the grafts from the ITE-treated arm (blue bars), vehicle or untreated HSCs (black and green bars, respectively). Image: Summary/Conclusion: In summary, our data show that the LSCs react differently than normal HSCs to changes in AHR signalling. These differences suggest it might be possible to specifically inhibit LSCs while sparing normal HSCs, thus paving the way to develop strategies to treat leukemia based on manipulating AHR signalling. P439: ASSOCIATING EX VIVO DRUG SENSITIVITY WITH METABOLIC STATUS IDENTIFIES EFFECTIVE COMBINATION STRATEGIES IN ACUTE MYELOID LEUKEMIA D. A. Pereira-Martins1,2,3,*, E. Griessinger1, I. Weinhauser1,2, J. L. Coelho-Silva2,3, D. R. Silveira4, L. Quek4, A. Erdem1, J. R. Hilberink1, E. V. de Paula5, S. T. Olalla Saad5, E. Ammatuna1, G. Huls1, E. M. Rego2, A. R. Lucena-Araujo3, J. J. Schuringa1 1Department of Experimental Hematology, Cancer Research Centre Groningen, University Medical Center Groningen, Groningen, Netherlands; 2Center for Cell Based Therapy, University of Sao Paulo, Ribeirao Preto; 3Department of Genetics, Federal University of Pernambuco, Recife, Brazil; 4Myeloid Leukaemia Genomics and Biology Group, School of Cancer and Pharmaceutical Sciences, King’s College London, London, United Kingdom; 5Hematology and BloodTransfusion Center, University of Campinas, Campinas, Brazil Background: Metabolic reprogramming is a hallmark of cancer, and acute myeloid leukemia (AML) is no exception. Yet, we and others have shown that clear heterogeneity exists in metabolic programming between genetically distinct AML subtypes and altered function of mitochondria (mt) might be linked to chemoresistance in some patients. Aims: Here, we evaluated the mitochondrial function of AML cells in more detail. Methods: Retrospective analysis of the mtDNA content (mtDNAc) in a large cohort of Brazilian patients with de novo AML (03 centers, n=482, age:18-65y) treated with the 3 + 7 scheme, and sex- and age-paired healthy donors (HD, n=308), revealed higher mtDNAc in 34% of the AML samples when compared to the highest value of mtDNAc in the HD group. Results: High mtDNAc was independently associated with increased relapse risk. These findings were confirmed in a validation cohort of AML patients treated with the 3 + 7 scheme at the UMCG in the Netherlands (n=92, age:18-65y). Clustering analysis using label-free quantified proteome of sorted CD34+ cells from AML samples (n=30) associated high mtDNAc with oxidative phosphorylation (OxPhos) metabolism and L-GMP leukemia. Flow cytometry analysis for Hematopoietic progenitors (defined by CD34, CD38, CD123, and CD45RA gated inside the CD45+ cells) and mitochondrial markers (Mitotracker Green/DeepRed for mitochondrial mass/potential, respectively) in a set of primary AML samples (n=32), showed an enhanced proportion of L-GMP cells and a positive correlation with the mitochondrial potential (r=0.51) in AMLs with high mtDNAc. Consistently, oxygen consumption rate (OCR) and lactate production measured by Seahorse was increased in primary AML blasts (n=11) and AML cell lines (n=14) with high mtDNAc, suggesting increased energetic metabolism. Multiomics analysis of AML cell lines (n=19) revealed a positive correlation between mtDNAc and mitochondrial metabolism and apoptosis regulation. To evaluate the role of mtDNAc during drug-induced apoptosis, we treated primary AML blasts (n=67) with cytarabine (AraC, 100 nM) for 48h. We observed a negative correlation between mtDNAc and apoptosis rate (r=-0.75). Moreover, the reduction of mtDNAc in AML blasts (n=16) using shRNA targeting the POLG gene resulted in mixed phenotypes. POLG silencing impaired cell proliferation and induced monocytic differentiation for AMLs with high mtDNAc, while promoted cell proliferation in mtDNAclow AMLs, suggesting a completely distinct dependency on OxPhos for these 2 groups of AMLs. Pharmacological treatment of 9 AML cell lines with ETC complex I inhibitors (CIi; rotenone, 50 nM and metformin, 1 mM) in combination with AraC (10-500 nM) and Venetoclax (VEN, 10-1000 nM), resulted in a reduction of mtDNAc and functional respiration in AML cells. Combination therapy of CIi with AraC resulted in increased cell death in OxPhos - but not in glycolysis-driven AML cells. In contrast, the combination of CIi+VEN resulted in enhanced cell death in all AML cells, regardless of their metabolic state. Since metformin is an FDA-approved drug, we assessed the metformin-induced apoptosis, in combination with AraC or VEN in 64 primary AML samples ex vivo. Metformin treatment resulted in a reduction of basal OCR of AML blasts (n=7). The metformin-induced apoptosis rate positively correlated with mtDNAc (r=0.89), displaying additive effects with AraC- and VEN-induced apoptosis in AML samples. Summary/Conclusion: Overall, our findings suggest that mtDNA content can be used to identify patients with a higher risk of chemoresistance and supports the idea of repurposing metformin into AML therapy. P440: THE COMBINATION VENETOCLAX PLUS MCL1 OVERCOMES DRUG RESISTANCE IN ACUTE PROMYELOCYTIC LEUKEMIA D. A. Pereira-Martins1,2,*, N. I. Noguera3, T. Ottone3,4, A. Diepstra5, C. Ortiz1, I. Weinhauser1,2, G. Huls2, J. J. Schuringa2, E. M. Rego1, E. Ammatuna2 1Center for Cell Based Therapy, University of Sao Paulo, Ribeirao Preto, Brazil; 2Department of Experimental Hematology, Cancer Research Centre Groningen, University Medical Center Groningen, Groningen, Netherlands; 3Department of Biomedicine and Prevention, University of Tor Vergata; 4Neuro-Oncohematology, Santa Lucia Foundation, I.R.C.C.S., Rome, Italy; 5Department of Pathology and Medical Biology, Cancer Research Centre Groningen, University Medical Center Groningen, Groningen, Netherlands Background: Although acute promyelocytic leukemia (APL) is the most favorable subtype of acute myeloid leukemia, it remains a life-threating disease for high-risk patients. Despite progress in APL with arsenic (ATO) plus retinoic acid (RA) as front-line treatment, approximately 10-15% of patients will acquire resistance to ATO and/or RA and relapse. Relapse after ATO/RA based therapy remains very challenging, and novel drug schemes to overcome resistance are urgently needed. Analysis of bone marrow (BM) biopsies of 15 consecutive APL patients at diagnosis, revealed a homogeneous staining for BCL2 in all cases. These results suggest BCL2 inhibition could be a therapeutic option for resistant APL. In vitro treatment with the BCL2 inhibitor Venetoclax (VEN), resulted in increased MCL1 expression, suggesting a resistance mechanism to this therapy. Aims: Here, we investigated the effects of VEN+MCL1 inhibitor S64315 in APL cells resistant to ATO/RA, and evaluated the effect of the combination therapy in vivo, using a cell line derived xenograft (CLDX) model for APL with the ATO-resistant associated mutation PML A216V. Methods: Primary leukemic blasts from APL mice (n=4) and patients (age, 25-52y; n=10) were treated with VEN (250-500nM), ATO, ATRA (1µM), and S64315 (10-50nM) monotherapies and combinations and evaluated for cell survival. In addition, we evaluated the transcriptome of 16 APL patients (age, 25-74y; 7 males) enrolled in the TCGA study regarding the BCL2 expression. NB4/NB4R2 (RA-resistant) cell lines were transduced with PML A216V. Additionally, U937 cells were transduced with the inducible PML-RARA system. After induction of PML-RARA expression with Zinc for 0-24h, BCL2 protein levels were evaluated by western blot. Additionally, RA/ATO treated (1µM) NB4 cells for 6-72h were evaluated for BCL2/MCL1 levels. For the CLDX model, NB4 PML A216V cells were transplanted into NSG mice, and after chimerism detection (hCD45+) in the peripheral blood, mice were equally distributed into 4 groups of treatment: vehicle, VEN (25mg/kg/day), S64315 (20mg/Kg/day) and combination (n=5/group) for 21 consecutive days. Results: Gene set enrichment analysis using the transcriptome of APL patients with BCL2 high associated these patients with the terms “EPPERT_HSC_LSC” and “17_LSC”. These results suggest that APL patients with high BCL2 expression differ in their metabolic and proliferative state compared to patients with BCL2 low. Functionally, the induction of PML-RARA expression in U937 cells resulted in increased BCL2 expression in a time-dependent manner (peak at 6h). Contrarily, treatment with ATO/RA resulted in decreased BCL2 levels with upregulation of the MCL1 protein. BH3 profile revealed that ATO/RA treated NB4 cells displayed an increase in the MCL1 anti-apoptotic dependency. Additionally, in vitro and ex vivo treatment of APL cells (cell lines and primary blasts) revealed synergic activity of S64315, but not the BCL2 inhibitor (VEN), in combination with ATO. Furthermore, treatment of ATO resistant NB4 cells (by gradual exposure to ATO or by expression of the PML A216V) revealed sensitivity of those cells to the combination VEN+S64315. Finally, in vivo CLDX model revealed limited effect of VEN and S64315 monotherapy, while the combination was able to effectively reduce the leukemic burden, resulting in increased overall survival (vehicle:15 vs Combo:42 days). Summary/Conclusion: The combination of BCL2 and MCL1 inhibition is a potential effective treatment in ATO/RA resistant APL patients and could be clinically explored in this category of patients with very poor prognosis. P441: DIFFERENCES IN GENE EXPRESSION PATTERN AND IMMUNOPHENOTYPE OF BONE MARROW MULTIPOTENT MESENCHYMAL STROMAL CELLS IN PATIENTS AT THE ONSET OF ACUTE LEUKEMIA AND AFTER ACHIEVING REMISSION A. Sadovskaya1,*, N. Petinati2, N. Kapranov2, N. Drize2, A. Vasilieva2, O. Gavrilina2, E. Parovichnikova2 1Faculty of biology, Department of Immunology, Lomonosov Moscow State University; 2NATIONAL MEDICAL RESEARCH CENTER FOR HEMATOLOGY, Moscow, Russia Background: The stromal microenvironment plays a key role in both maintaining normal hematopoiesis and assisting malignant cells during leukemia development. In patients with acute leukemia, the properties of multipotent mesenchymal stromal cells (MSCs) are changed both at the onset of the disease due to interaction with tumor cells and upon reaching remission due to chemotherapy. As a result, the ability of MSCs to maintain normal hematopoiesis is reduced. A detailed study of these changes may allow the development of rehabilitation measures for restoring the stromal microenvironment of patients. Aims: The aim of this work was to study general changes in gene expression pattern and immunophenotype of MSCs in patients with acute leukemia at the onset of the disease and in remission. Methods: The study included MSCs obtained from the bone marrow (BM) of 15 patients: 4 with ALL (1 male, 3 female, median age 26.5) and 11 AML (4 male, 7 female median age 33), BM of 30 donors of appropriate age was used as control. All donors and patients signed informed consent. MSCs were cultured by standard methods. Immunophenotypes were studied by flow cytometry. Relative expression levels (REL) of several genes were investigated by real time PCR. Results: The study of the mean fluorescence intensity (MFI) of MSC markers revealed a significant decrease in MFI of CD274 (PD-L1) in primary patients compared to healthy donors and its absence on the cells of patients in remission. At the onset both MFI of CD54 (ICAM-1) and the proportion of CD54+ MSCs were decreased. In remission these parameters decreased even more, so the treatment reduced the adhesive capacity of MSCs. The expression of CD105 was reduced at the onset of the disease and increased after the end of treatment, but did not normalize. In patients at the onset of the disease and in remission, compared with healthy donors, the expression of IL1R1, PDGFRβ, ANG1 was reduced; expression of IL6, PPARγ, JAG1, was increased (see Figure). Decreased level of IL8 and SOX9 normalized in remission. The decrease in growth factor (GF) receptors PDGFRβ and IL1R1 (IL1β may act as a GF for BM cells) in patients may be a result of negative feedback loops: GFs typically act as auto- and paracrine regulators, and malignant cells often overexpress various GF. The fact that these genes’ expression does not return to normal in remission may be a result of damage from treatment. IL1β expression in patients was not different from normal, but its increase in remission compared to onset implies stroma restoration processes. Another important effect is the alteration in MSCs’ interactions with stem cells: JAG1 product helps to ensure stem cells’ quiescent state and bond with stroma. In contrast, there is an increase in IGF1 expression at the onset, which can lead to stem cell activation. Alterations in the RELs of differentiation markers (SOX9, SPP1, PPARγ) indicate changes in the predisposition of MSCs to differentiation. PPARγ is a multifunctional nuclear receptor, and can inhibit proliferation. PPARγ upregulation may once again be a result of negative feedback networks preventing MSCs’ uncontrolled proliferation in GF-rich microenvironment. Image: Summary/Conclusion: The data suggest dramatic alterations in hematopoietic and stromal cells interaction at the onset of acute leukemia that were not undone by achieving remission. This is supported by decreased levels of endoglin and ICAM-1 and alteration of gene expression pattern. The materials supported by grants from the Russian Foundation for Basic Research project 19-29-04023 were used. P442: SPINK2 PROTEIN EXPRESSION QUANTIFIED BY IMMUNOHISTOCHEMISTRY REFINES RISK STRATIFICATION AND PREDICTS THERAPY OUTCOMES IN AML H. A. Pitts1,*, C. K. Cheng1, J. S. Cheung1, M. K. H. Sun1, M. H. Ng1 1Blood Cancer Cytogenetics and Genomics Laboratory, Department of Anatomical & Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, SAR, China Background: Suboptimal prognostication, therapy refractoriness and relapse contribute to the poor outcome of Acute Myeloid Leukemia (AML) patients. Leukemic stem cells (LSC) are considered crucial drivers of relapse and therapy resistance. Clinicopathological characterization of LSC-associated genes with convenient assessment tools such as immunohistochemistry (IHC) is needed. Our initial screening of AML datasets for LSC-associated genes identified Serine Protease Inhibitor Kazal type 2 (SPINK2) with high expression in AML, particularly in LSC fractions. However, in-depth analysis of its clinicopathological associations and prognostic utility in AML are lacking. Aims: To assess SPINK2 expression in adult AML with IHC, and determine its clinicopathological associations, prognostic impact and association with therapy response Methods: We studied the expression of SPINK2 by IHC in 172 non-M3 adult AML patients (median age:52yrs, range: 18-86yrs) treated at the Prince of Wales Hospital. The majority (90.8%) were de novo type and 72.3% had intermediate-risk(IR) cytogenetics, with 88.5% receiving Daunorubicin + Ara-C (DA 7 + 3) induction. SPINK2 expression was assessed by 3 hematopathologists in a double-blinded manner and quantified by a composite score (range: 0-16). Clinical data was collected for all cases. Results: SPINK2 staining in leukemic blasts was consistently cytoplasmic, and its protein expression strongly correlated with mRNA levels by qPCR in a subset of 128 patients (r=0.716, P<0.0001). The cohort was dichotomized at the median score 3, since this cut-off showed strongest association with poor event-free survival (EFS) and overall survival (OS). High SPINK2 (SPINK2 hi) patients accounted for 77/172 (44.8%) cases, and low SPINK2 (SPINK2 lo) patients for 95/172(55.2%). SPINK2 hi associated with IR cytogenetics (P=0.014), normal karyotype (P=0.019) and NPM1 mutation (P<0.001), while SPINK2 lo with t(8;21) and CEBPA double-mutation (both P<0.001). Survival and treatment-response analyses were performed on a subgroup of 137 patients who received DA 7 + 3 based induction. SPINK2 hi patients had lower CR rates (73.8% vs 88.3%,P=0.028), higher 6-month relapse rates (31.8% vs 9.1%,P=0.004), lower 5-yr RFS (25.8% vs 46.8%,P=0.004), EFS (16.6% vs 37.2%,P<0.001) and OS (25.3% vs 51.2%,P<0.001). SPINK2 status also identified high risk patients of the IR cytogenetic subgroup. Of these, SPINK2 hi patients had lower CR rates (68.6% vs 90.0%,P=0.013), higher 6-month relapse rates (31.4% vs 6.9%,P=0.018), lower 5-yr RFS(27.0% vs 44.6%,P=0.018), EFS(15.4% vs 38.3%,P<0.001) and OS(22.9% vs 51.5%,P=0.002). In this subgroup, median SPINK2 score was higher in patients requiring ≥2 inductions to achieve CR vs. patients requiring 1 course (7 vs 2,P=0.009). Multivariate analyses in whole and IR cytogenetics cohorts showed the poor prognostic effect of SPINK2 hi on EFS and OS independent of age, cytogenetic risk, mutations and achievement of CR1, including stem cell transplantation given in CR (Table 1). In our NPM1 mut subgroup (N=41), SPINK2 hi status associated with poor EFS (HR:3.3,P=0.04) and OS (HR: 4.0,P=0.007) independent of age and FLT3-ITD with high allelic ratio. Incorporation of SPINK2 with FLT3 status refined ELN2017 risk definition, whereby 15/22 favorable-risk patients could be reclassified as intermediate-risk. Image: Summary/Conclusion: High SPINK2 protein expression by IHC predicted increased chemoresistance and early relapse risk in AML, and was an independent adverse prognostic biomarker, particularly in patients with intermediate-risk cytogenetics and NPM1 mutations. P443: IMPACT OF PHARMACOGENETIC VARIANTS ON CYTARABINE AND ANTHRACYCLINE EFFECT ON OUTCOMES IN ADULT AML PATIENTS Z. Pravdic1,*, M. Virijevic1,2, M. Mitrovic1,2, N. Pantic1, V. Gasic3, S. Pavlovic3, N. Sabljic1, D. Pavlovic3, I. Marjanovic3, N. Suvajdzic Vukovic1,2 1Clinic of Hematology, UCCS; 2Faculty of Medicine, University of Belgrade; 3Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia Background: Acute myeloid leukaemia (AML) is primarily treated with combination of cytarabine and anthracyclines. Despite high remission rates, the 5-year overall survival (OS) is <50% and <20% for<60-year-old a ≥60-year-old patients, respectively. This grim clinical outcome could be partly explained by the patients’ genetic variability of proteins involved in metabolic paths of cytarabine and anthracyclines. Pharmacogenetic variants of the main cytarabine membrane transporter SLC29A1 (solute carrier family 29 member 1), DCK (Deoxycytidine kinase) the first enzyme in cytarabine stepwise activation process, genes coding main anthracycline efflux pump ABCB1 (ATP binding cassette) and genes coding glutathione S-transferases GSTM1 and GSTT1, main cytosolic detoxifiers od anthracycline-induced oxidative stress, are shown to influence clinical outcome in AML patients. Aims: to evaluate: 1) the effects of variants in pharmacogenes SLC29A1, DCK, ABCB1, GSTM1 and GSTT1, on complete remission (CR) and relapse rate (RR), disease free survival (DFS) and overall survival (OS), 2) the influence of demographic, laboratory and AML-related parameters on CR, RR, DFS and OS. Methods: 100 newly-diagnosed consenting adults (18-62 years old) diagnosed with AML, except acute promyelocytic leukaemia in Clinic of Haematology UCCS from January 2015 to January 2018, were included in retrospective cohort. Patients received one or two inductions ‘’3 + 7’’ cycles. In patients achieving CR either three consolidation cycles with high/intermediate doses of cytarabine or allogenic SCT in selected patients were performed. Demographic, standard laboratory and AML-related parameters (blood/bone marrow blast percentage, cytologic, flow cytometry and genetic markers enabling ELN risk stratification were collected from patients’ health records. Variants SLC29A1 rs9394992, DCK rs12648166, ABCB1 rs2032582 and GSTM1 and GSTT1 gene deletions were detected by methodology based on PCR, fragment analysis and direct sequencing. The study was approved by the Ethics Committee of the UCCS. The methods of descriptive and analytic statistics were used, while survival analysis was done by the Kaplan-Meier method using the Log-Rank test. Results: 100 patients (53 males) were included in the study, with the median age of 51 (range 18-62). Median laboratory parameters were: WBC 15.5x109/L (range 1-348.8), Hgb 97g/L (range 20-166), Plt 53.5 (range 1-422), LDH 249U/L (1-4169). CD34 was positive in 56% patients. According to ELN 15, 55 and 30 patients were classified in favorable, intermediate and adverse risk group, respectively. CR was achieved in 57 patients (41 after the first and 16 after the second induction cycle). A total of 34% of patients relapsed. Median DFS was 5.3 months (range 0.25-61.5), while median OS was 6 months (range 0.3-75). There was a significant difference in median DFS between CC, CT and TT genotype of SLC29A1: 13, 8 and 1.75 months, respectively (p=0.00037). Median DFS was significantly decreased in null genotype of GSTM1 (9.5 (null) vs 18 months (non-null); p=0.028). Significant difference in OS within ABCB1 genotypes TT, GG, GT and AG+AT was registered: 13, 7, 6.5 and 3 months, respectively (p=0.0036). Other parameters did not differ significantly. Summary/Conclusion: Our results, regarding impact of variants of SLC29A1, GSTM1 on DFS and impact of variant ABCB1 on OS in AML patients, are in line with previous studies. Although further studies, with larger cohorts, are needed, these pharmacogenetic variants show a potential to become prognostic markers in AML. P444: IDENTIFICATION OF FUNCTIONAL GENETIC DEPENDENCIES IN CEBPA-MUTATED ACUTE MYELOID LEUKEMIA L. Proietti1,*, E. Heyes1, T. Eder1, T. Brandstoetter2, G. Manhart1, V. Sexl2, F. Grebien1 1Institute for Medical Biochemestry; 2Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria Background: C/EBPα is a key myeloid transcription factor that regulates the switch between proliferating uncommitted cells and terminally differentiated cells. The CEBPA gene is mutated in 10-15% of AML patients. CEBPA lesions are divided into C-terminal mutations that abrogate DNA binding and N-terminal frameshift mutations, which cause expression of an N-terminally truncated isoform, termed p30. A large fraction of AML patients carries biallelic CEBPA mutations. In these patients, the shorter C/EBPα p30 variant is the only functional isoform of C/EBPα. While we and others have identified molecular mechanisms underlying the development and maintenance of CEBPA-mutated AML, a comprehensive analysis of vulnerabilities that is associated with this disease subtype is lacking. Aims: We aimed to characterize the functional genetic dependencies of AML with biallelic CEBPA mutations. Methods: We generated an immortalized AML cell line by serial transplantation of fetal liver cells carrying biallelic CEBPA mutations (CNC, C ebpa N-terminal/C-terminal) followed by prolonged culture of leukemic cells. After stable expression of Cas9, we performed a genome-scale CRISPR/Cas9 loss-of-function screen in CNC cells and compared the results to genome-wide screening results from the HPC7 hematopoietic multipotent progenitor cell line, which were used as a representative of the healthy, untransformed state. Results: Transplantation of CNC cells into C57BL/6 mice induced an aggressive Mac1+/Gr1+/c-Kit+ leukemia within 7 weeks, similar to the initial model (Bereshchenko et al, 2009). This immuno-phenotype was preserved upon prolonged in vitro culture. Analysis of the CRISPR/Cas9 screen in CNC cells identified 2488 genes whose mutational inactivation was associated with reduced fitness - 438 of these genes are “core” essential, as they have been annotated to be required for the survival of both cancer and normal cells. In HPC7 cells, the loss of 1674 genes caused cell depletion, including 388 core essential genes. Intersection of screening results from CNC and HPC7 cells revealed 1290 commonly depleted genes, of which 380 were core essential. We identified 308 genes that were exclusively required for the proliferation and survival of CNC cells. This list of high-confidence candidates is enriched for genes involved in mRNA metabolism, DNA repair and signaling pathways. We are currently in the process of validating prioritized target genes using a variety of approaches. Summary/Conclusion: This approach allows the acquisition of deeper insights into the molecular mechanisms underlying the development and maintenance of leukemia with CEBPA mutations. Multi-layered validation of high confidence candidates will be performed in vivo and in vitro to identify leukemia-specific vulnerabilities that can serve as starting points for rational targeting strategies. P445: CRYPTIC TRANSLOCATION T(5;11)(Q35;P15) RESULTING IN NUP98::NSD1 GENE FUSION IN ADULTS WITH DE NOVO ACUTE MYELOID LEUKEMIA (AML) S. Ransdorfova1,*, J. Markova1, M. Valerianova1, J. Brezinova1, M. Onderkova1, I. Mendlikova1, L. Lizcova2, L. Pavlistova2, K. Svobodova2, S. Izakova2, A. Jonasova3, C. Salek1, Z. Racil1, Z. Zemanova2 1Institute of Hematology and Blood Transfusion; 2Center of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and First Faculty of Medicine, Charles University; 31st Medical Department, General University Hospital and First Faculty of Medicine, Charles University, Prague, Czechia Background: The NUP98::NSD1 fusion, a product of the cryptic translocation t(5;11)(q35;p15.5), is a recurrent genetic change in cytogenetically normal patients with AML. It occurs most frequently in children (16%) and young (2%) AML patients, very rarely in adult patients. The coexistence of internal tandem duplication of FLT3 gene (FLT3::ITD) found in more than 70% of NUP98::NSD1 positive patients is always associated with a poor prognosis and results in high frequency of induction failure. Aims: The aim of this study was to determine the incidence of the NUP98::NSD1 fusion gene in adults with AML and NUP98 rearrangement. Methods: We examined the bone marrow cells of 268 newly diagnosed AML patients using conventional karyotyping in combination with FISH (Abbott, MetaSystems) and mFISH/mBAND (MetaSystems). We used RT-PCR followed by direct sequencing to detect fusion genes. We processed the PCR product by ExoSAP-IT and directly sequenced on ABI Prism 310 genetic analyzer using the Big Dye Terminator kit v. 3.1. Results: In nine out of 268 cases we identified rearrangement of the 11p13-15 region. We confirmed t(5;11)(q35;p15.5) with NUP98::NSD1 gene fusion in four of them (4/268; 1.5%). Sequence analyses proved the NUP98-NSD1 transcript, arising from fusion of NUP98 exon 12 with exon 6 in NSD1 gene, in all four patients (2M/2F; FAB M4/M5b; age 42, 54, 64 and 64 years). FLT3::ITD mutation was detected in all of them. Specific primers and a probe have been designed to monitor minimal residual disease during therapeutic treatment of the patients. Out of 4 patients, two died (median OS 9 months) and two patients are alive (4 and 1 year after allogenic bone marrow transplantation). Summary/Conclusion: Our study demonstrated the occurrence of t(5;11)(q35;p15.5) with NUP98::NSD1 gene fusion also in adults over 60 years of age. We confirmed the NUP98::NSD1 fusion gene in 1.5% adults AML patients. With respect to the poor prognosis of the patients with NUP98::NSD1 fusion gene, we suggest pre-screening of NUP98 gene rearrangement using FISH in cytogenetically normal AML patients with confirmed FLT3::ITD mutation. Supported by MH CZ-DRO-VFN64165, DRO-UHKT00023736. P446: USE OF MIRNA EXPRESSION PATTERNS REVEALS ACTIVIN RECEPTOR 2 PATHWAY IN MEDIATING CHEMO-RESISTANT AML P. Reichelt1,*, S. Bernhart2, F. Wilke1, U. Platzbecker1, M. Cross1, G. Behre3 1Department of Hematology and Cell Therapy, University Hospital Leipzig; 2Interdisciplinary center for bioinformatics Leipzig, Leipzig; 3Clinic for Internal Medicine I, Municipal Hospital Dessau, Dessau, Germany Background: Acute myeloid leukemia (AML) is a highly aggressive and heterogeneous disease and the most prevalent type of acute leukemia in adults. Standard chemotherapy using cytarabine and idarubicin/daunorubicin usually achieves remission, but this is commonly followed by chemoresistance and relapse. The treatment of chemoresistant AML remains a major challenge that will require personalized approaches. Aims: With this study, we aim to establish NGS microRNA expression profiling and pathway analysis to identify pathways that are regulated differentially between chemo-sensitive and -resistant AML as potential targets for targeted therapy. Methods: MicroRNA expression profiles were analyzed by NGS (next generation sequencing) of chemo-resistant and -sensitive subclones of AML cell line HL60. Bioinformatical identification of candidate deregulated pathways was performed using miRTarbase and stringdb. Pathway activity in cell sublines was assessed by protein expression, proliferation and flow cytometric analysis, using western blotting, phosphlow and MTS assays. Paired samples of AML pre- and post- relapse were used to validate gene expression changes by qPCR and analyses were extended to the publically available dataset of the AML patient cohort of the cancer genome atlas (TCGA). Results: Our microRNA based bioinformatic approach predicted 27 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways to be represented differentially between resistant and sensitive cells (FDR<0.05). Prominent among these were significant changes in signaling pathways of the TGFβ ligand family. Cell culture experiments and protein expression analysis confirmed both a higher sensitivity to TGFβ induced growth arrest and increased abundance of TGFβ signaling proteins in chemo-resistant HL60 cells. Consistent differences in TGFß pathway gene expression were also detected in AML patients of the TCGA dataset. Furthermore, our data suggest a shift of the predominantly expressed isoform of activin receptor 2B to activin receptor 2A in chemo-resistance. Analysis of paired bone marrow samples from AML patients at diagnosis and first relapse showed the latter to be associated with higher expression of TGFβ receptors and the TGFβ target protein CDKN2B as well as significant upregulation of activin receptor 2A. Image: Summary/Conclusion: Here we show how miRNA screening approaches can be used to predict deregulated cellular signaling pathways as potential targets for the therapy of chemoresistant AML. MicroRNA based prediction of targetable pathways combined with database screening identified the activin receptor 2A/B as a potential modulator of chemoresistance. P447: BCL2-INHIBITION TARGETS LEUKEMIC STEM CELLS IN ACUTE MYELOID LEUKEMIA INDEPENDENT OF MONOCYTIC BLAST POPULATIONS S. Renders1,2,*, A.-M. Leppä1, A. Waclawiczek1, C. Reyneri1, M. Janssen2, E. Donato1, J. Unglaub2, C. Pabst2, R. Schlenk2, M. Hundemer2,3, S. Raffel2, T. Sauer2, C. Müller-Tidow2, A. Trumpp1 1DKFZ/ Hi-Stem; 2Department of Hematology, University Hospital Heidelberg, Heidelberg; 3MVZ Hämatologische Diagnostik, Heppenheim, Germany Background: Treatment with Hypomethylating agents (HMA) in combination with the BCL-2 inhibitor Venetoclax (VEN) has recently become the standard of care for AML patients unsuitable for intensive induction chemotherapy and shows superior results to treatment with Azacitidine (AZA) alone (DiNardo et al., 2020, NEJM). However, upfront resistance and relapse remain major obstacles. It has recently been proposed that monocytic differentiation associated with increased expression of antiapoptotic-protein MCL-1 predicts resistance to AZA/VEN treatment in AML (Pei et al., 2020 Cancer Discovery). However, other studies did not find impaired outcome in patients with monocytic AMLs treated with HMA/VEN (DiNardo et al., 2021, Lancet Oncology; Stein et al., 2021 Blood Advances). Aims: We aimed to understand apoptotic dependencies linked to differentiation states in AML subpopulations in combined with clinical data to understand and treatment effects and resistance upon HMA/VEN therapy. Methods: We performed retrospective analysis of >50 patients at Heidelberg University hospital to identify risk factors of treatment failure. For functional investigation, we studied 24 AML cell lines and generated Xenografts in NSG mice of 12 AML patients to study mature and leukemic stem cell (LSC)-like primary AML subpopulations. We characterized these samples further by RNA sequencing and BCL-2 family level assessment. To functionally validate our findings, we performed ex vivo drug treatment and subpopulation specific BH3 profiling. Results: In our clinical cohort the only factors associated with HMA/VEN resistance were previous myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) and complex karyotype, but not monocytic differentiation. The CD64+CD11b+, mature blast population making up >50% of leukemic cells in monocytic and <20% in primitive samples showed high levels of resistance to AZA/VEN therapy in both primitive and monocytic leukemias but did not engraft when transplanted into NSG mice, arguing they do not contain LSCs. In contrast, we found immature GPR56+ LSCs to be sensitive to AZA/VEN treatment irrespective whether they were derived from monocytic or primitive primary AMLs. LSCs from monocytic and primitive AMLs initiated disease in NSG mice, highlighting that targeting LSCs is essential to treat AML. Next, we investigated expression of BCL2, MCL1 and BCL-xL levels in the same primary patient samples and observed high MCL1 expression in monocytic AML samples. However, MCL1 expression predominantly derived from the mature population whereas LSCs expressed robust levels of BCL2 but comparatively low levels of MCL1, independent of whether monocytic or primitive AMLs were analyzed. Additionally, transcriptomes of LSCs from either monocytic or primitive samples did not display significant differences. We next performed BH3 profiling and found LSC to be dependent on BCL2 and mature blasts on MCL1 in all samples studied. In line, ex vivo HMA/VEN treatment specifically eliminated LSCs. We further monitored blast clearance in longitudinal patient samples and found robust loss of LSC within hours in patients, while mature blasts persisted for days. Summary/Conclusion: We find that LSCs, independent of the overall mature blast content, are sensitive to HMA/VEN treatment and highlight that genetic risk factors and disease history are relevant predictors of treatment failure. P448: PROGNOSTIC IMPACT OF SOMATIC CEBPA BZIP DOMAIN MUTATIONS IN ACUTE MYELOID LEUKEMIA F. G. Ruecker1,*, A. Corbacioglu1, F. Theis1, M. Christopeit2, U. Germing3, G. Wulf4, M. Abu Samra5, L. Teichmann6, M. Lübbert7, M. W. Kühn8, M. Bentz9, J. Westermann10, L. Bullinger10, V. I. Gaidzik1, E. Jahn1, M. Gröger1, S. Kapp-Schwoerer1, D. Weber1, F. Thol11, M. Heuser11, A. Ganser11, H. Döhner1, K. Döhner1 1Department of Internal Medicine III, University Hospital of Ulm, Ulm; 2Medical Clinic, Department of Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tübingen, Tübingen; 3Department of Hematology, Oncology and Clinical Immunology, Heinrich Heine University, Düsseldorf; 4University Medical Center Göttingen, Göttingen; 5Department of Internal Medicine IV, University Hospital of Gießen, Gießen; 6Department of Medicine and Polyclinic III, Bonn University Hospital, Bonn; 7Department of Hematology, Oncology and Stem Cell Transplantation, University of Freiburg Medical Center, Freiburg; 8Department of Hematology, Medical Oncology and Pneumology, University Medical Center Mainz, Mainz; 9Department of Internal Medicine III, Hospital of Karlsruhe, Karlsruhe; 10Department of Hematology, Oncology and Tumor Immunology, Charité University Medicine, Berlin; 11Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Hannover, Germany Background: Mutations of CEBPA (CEBPA mut) are present in ~5-10% of newly diagnosed adult acute myeloid leukemia (AML), and approximately half of the patients (pts) exhibit biallelic mutations (CEBPA bi). CEBPA bi defines a distinct entity within the 2016 WHO classification and is categorized as favorable in the 2017 risk stratification by the European LeukemiaNet. CEBPA mut can be divided into basic leucine zipper domain (bZIP) or transcription activation domains (TAD) mutations, respectively. Recent studies have demonstrated CEBPA bZIP mutations, in particular in-frame mutations (CEBPA bZIP-inf), to be associated with favorable outcome, regardless of mono- or biallelic status. Aims: To evaluate the prognostic impact of CEBPA bZIP, in particular CEBPA bZIP-inf mutations in AML. Methods: Investigating a cohort of 454 intensively treated CEBPA mutated AML pts entered into the AMLSG BiO Registry study (NCT01252485). Results: Of the 454 pts, 223 had CEBPA bi and 231 monoallelic CEBPA mutations (CEBPA sm) affecting bZIP in 78 pts or TAD in 153 pts. Genotypes differed significantly with regard to clinical and genetic features: CEBPA bi pts were younger than CEBPA smbZIP and CEBPA smTAD (median age in yrs: 52 vs 59 vs 60; P<.001), had a higher rate of de novo AML (97% vs 81% vs 84%; P<.001), lower platelet counts (median G/l 36 vs 57 vs 52; P<.001), higher peripheral blood (PB) blast counts (median 71% vs 60% vs 44%; P<.001), and showed an inverse correlation with FLT3 internal tandem duplication (FLT3-ITD) and NPM1 mutation (NPM1 mut) (6% vs 21% vs 35% and 0% vs 26% vs 50%; P<.001 each). Outcome analysis revealed a significant improved overall (OS) and event-free survival (EFS) for CEBPA bi with no difference between CEBPA smbZIP and CEBPA smTAD (5-year OS: 62% vs 42% vs 51%; P<.001, 5-year EFS: 47% vs 27% vs 43%; P<.022). Subgroup specific analysis within CEBPA smbZIP revealed an improved outcome for CEBPA bZIP-inf pts (n=46) (5-year OS: 52% vs 29%; P=.001, and 5-year EFS: 34% vs 18%; P=.018). To further address the impact of CEBPA bZIP-inf, pts were categorized as CEBPA bZIP-inf (n=250), irrespective of the allelic status, vs all others (CEBPA other) (n=204). CEBPA bZIP-inf pts were younger (median age in yrs: 52 vs 62; P<.001), had a higher rate of de novo AML (97% vs 81%; P<.001), lower platelet counts (median G/l 36 vs 53; P<.001), higher white blood cell (WBC) (median G/l 25.4 vs 16.1; P=.029), and higher PB blast counts (median 72% vs 46%; P<.001); FLT3-ITD and NPM1 mut were less common in CEBPA bZIP-inf pts (9% vs 30% and 3% vs 44%; P<.001 each). CEBPA bZIP-inf exhibited a significant improved OS (5-year OS: 60% vs 48%; P<.001) and EFS (5-year EFS: 47% vs 37%; P=.007); in multivariate Cox models for OS and EFS, including allogeneic hematopoietic cell transplantation (HCT) in first complete remission as time-dependent covariate, age (HR: 1.50; P<.001), WBC (HR: 1.40; P=.018), and adverse cytogenetics (HR: 2.33; P=.010) were unfavorable factors for OS, whereas NPM1 mut (HR: 0.63; P=.040), HCT (HR: 0.46; P=.010), and CEBPA bZIP-inf (HR: 0.59; P=.007) revealed as favorable. For EFS, age (HR: 1.19; P=.002), WBC (HR: 1.30; P=.028), and FLT3-ITD (HR: 1.83; P=.003) were unfavorable, whereas NPM1 mut (HR: 0.65; P=.028) and HCT (HR: 0.33; P<.001) were favorable. Summary/Conclusion: In this cohort of 454 CEBPA mutated adult AML pts, CEBPA bZIP-inf was associated with specific clinical and genetic characteristics. Furthermore, CEBPA bZIP-inf pts had a significantly superior outcome irrespective of the allelic status. This study confirms recent findings suggesting a prognostic role of this mutation type. FGR and AC contributed equally P449: NOVEL, FIRST-IN-CLASS, SOMATIC IDH2 DELETION-INSERTION VARIANT C.516_518DELINSTGC CONFERS BORDERLINE PHENOTYPE AND DOES NOT SHOW SUSCEPTIBILITY TO ENASIDENIB IN-VITRO L. Ruhnke1,*, D. M. Poitz2, S. Herold3, S. Dressler4, C. Dill1, M. Peitzsch2, U. Oelschlägel1, J. Frimmel1, D. Kunadt1, T. Kretschmann1, H. Altmann1, F. Stölzel1, C. Röllig1, T. Chavakis2, G. Baretton3, K. Schäfer-Eckart4, M. Bornhäuser1 1Department of Internal Medicine I; 2Institute of Clinical Chemistry and Laboratory Medicine; 3Institute of Pathology, University Hospital Dresden, Dresden; 4Department of Internal Medicine V, Nuremberg Hospital North, Paracelsus Medical University, Nuremberg, Germany Background: Mutations in IDH2 are among the most common genetic alterations in patients with acute myeloid leukemia (AML). So far, only substitution variants (also known as single nucleotide variants (SNVs)) - in particular affecting the hotspots at codon Arg140 and Arg172 - have been identified. . Aims: Here we report and functionally characterize a novel, first-in-class, somatic IDH2 deletion-insertion (delins) variant affecting codons Arg172/His173 identified in a patient diagnosed with AML with myelodysplasia related changes (AML-MRC). Methods: The novel delins variant was identified performing amplicon-based targeted next generation sequencing (NGS), Sanger sequencing was used for verification. For functional evaluation high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS), global DNA methylation and DNA hydroxymethylation assays and in-vitro drug testing was performed; AML patients without evidence of IDH1 or IDH2 mutations and patients with IDH2 Arg172 mutation served as negative and positive controls, respectively. Results: A 63-year-old male patient (unique patient number (UPN) 1) was diagnosed with AML-MRC. Further work-up revealed an aberrant karyotype (47,XY,+11/ 46,XY,der(7)t(7;11)(q31;q12)/ 46,XY), a partial tandem duplication of KMT2A (KMT2A-PTD) as well as variants in IDH2, DNMT3A and NRAS (Figure 1.) (no CBFB::MYH11, PML::RARA or RUNX1::RUNX1T1 fusion, no ASXL1, CEBPA, FLT3, NPM1, RUNX1 or TP53 mutation) (Figure 1A). He received induction therapy with liposomal daunorubicin/cytarabine, achieved CR and underwent allogeneic hematopoietic cell transplantation. Of note, the IDH2 variant identified was a novel three base-pair deletion-insertion variant (c.516_518delinsTGC) leading to the replacement of amino acids arginine and histidine at codon 172 and 173 for serin and alanine (p.Arg172_His173delinsSerAla), variant allel frequency was 44%. The novel delins variant could be validated performing Sanger sequencing; parallel Sanger sequencing of buccal saliva DNA ruled out germline origin (Figure 1B). Since there are no valid in-silico prediction tools for delins variants, we decided to assess the potential pathogenicity of the novel alteration. First, we applied LC-MS/MS to evaluate 2-hydroxyglutarate (2-HG) levels as compared to BM-MNCs obtained from IDH1/IDH2 WT AML patients (UNP2-4) and IDH2MUT AML patients (UPN5-10). In line with previous reports, 2-HG levels were increased in patients with IDH2 mutations as compared to their IDH1/IDH2 WT counterparts (1000-fold). However, in UPN1 only slightly elevated 2-HG levels were found (10-fold) (Figure 1C); no differences in isocitrate or ketoglutarate levels were seen (data not shown). Similar results were obtained when performing DNA methylation and DNA hydroxymethylation studies. While the percentage of methylated DNA in UPN1 was comparable to the amount of 5-methylcytosine (5-mC) seen in the IDH2 mutated group (UNP5-10), 5-hydroxymethylcytosine (5-hmC) levels in UPN1 tended towards those observed in the IDH2 WT cohort (UPN2-4) (data not shown). Finally, we performed in-vitro drug testing to evaluate response to the IDH2 inhibitor (IDH2i) enasidenib. Interestingly, enasidenib did not affect granulocytic differentiation of BM-MNCs as assessed by CD11b, CD15 and CD16 flow cytometry (Figure 1D). Image: Summary/Conclusion: Here, we report a novel, somatic delins variant in IDH2, which, albeit affecting the known Arg172 hotspot, does not show susceptibility to the IDH2i enasidenib in-vitro. Our report argues for functional characterisation of novel variants to ensure a valid biomarker-driven therapy in AML patients. P450: PRECLINICAL AND CLINICAL SIGNS OF RVU120 EFFICACY, A SPECIFIC CDK8/19 INHIBITOR IN DNMT3A MUTATION POSITIVE AML AND HR-MDS U. Pakulska1, N. Angelosanto2, M. Mikula3, H. Nogai2, R. Dudziak2, C. Abboud4, M. Obacz1, K. Goller1, M. Cybulska3, M. Mazan1, M. Kozakowska1, T. Rzymski1,* 1R&D; 2Clinical Department, Ryvu Therapeutics, Kraków; 3Maria Skłodowska-Curie Institute - Cancer Center, Warsaw, Poland; 4Washington University School of Medicine in St. Louis, St Louis, United States of America Background: CDK8 and its paralog CDK19 regulate transcription as a part of mediator complex, that links enhancers with core promoters. AML likewise many other cancers highjack CDK8 and CDK19 to maintain undifferentiated state and prevent apoptotic cell death. First CDK8/CDK19 inhibitor RVU120 has reached clinical development phase Ib (NCT04021368) in AML and HR-MDS patients. Aims: It is now critical to establish relationship between preclinical and clinical efficacy results, and molecular characteristics in order to identify actionable markers predicting response to CDK8/CDK19 inhibitors. Methods: Association of molecular profiles with responses to RVU120 has been performed on genetically annotated AML PDCs, followed by flow cytometry and bioinformatic analysis. PDCs were implanted intravenously into NSG-SGM3 mice and after disease onset animals were treated orally with RVU120. Profiling of transcriptional response to RVU120 in DMNT3A mutant cells has been performed by RNA-seq. The First in Human study of RVU120 is currently active enrolling R/R AML or HR-MDS patients, in a dose escalation design aimed at exploring safety/tolerability and identify the RPD2. RVU120 is administered orally, each other day for 7 total doses per 21 days cycle, until disease progression/unacceptable toxicity. Response to the study drug is assessed according to Dohner 2017 criteria. Results: Screening of AML PDC against RVU120 indicated high anti-cancer efficacy in >40% of tested samples. Correlation of efficacy with genetic profile of samples indicated specific enrichment of DNMT3A or NPM1 mutants in responder group. These results were further corroborated in disseminated PDX AML model, showing complete clearance of DNMT3A and NPM1 mutation- positive blasts and recovery of murine BM in animals treated with RVU120. Transcriptomic analysis of DNMT3A and NPM1 positive AML cells indicated distinct transcriptomic profiles, characterized by high expression of homeobox genes, involved in determination of cell faith. Immunophenotyping of responder cells indicated elimination of lineage committed blast cells. At the date of this abstract submission, 13 patients have been enrolled into CLI120-001 trial, including 2 patients with DNMT3A mutations. Notably, first R/R AML patient that achieved CR was positive for DNMT3A and NPM1 mutations. At study entry this patient was progressing with pancytopenia, 65% BM blasts and extramedullary localization of leukemia. BM showed a complete clearance of blasts at the end of 1st cycle of 75mg dose, followed by hematological recovery and signs of monocytic differentiation. Importantly, monocytic differentiation is observed as a pharmacodynamic effect of RVU120 in preclinical studies. Skin leukemia lesions improved gradually up to a CR in cycle 7. Second DNMT3A mutation patient with HR-MDS, escalated from 50 to 75 mg dose from cycle 7, continues treatment at the cycle 24 with SD and erythroid hematological improvement. Summary/Conclusion: AML PDCs with DNMT3and NPM1 mutations show differential sensitivity to RVU120 treatment both in vitro and in vivo. Anti-cancer efficacy of RVU120 was associated with transcriptomic reprogramming and lineage commitment. Preliminary evidence of clinical response to RVU120 has been also shown in R/R AML and HR-MDS patients positive for DNMT3A mutations. Further molecular studies in greater number of patients under RVU120 treatment are ongoing and will provide evidence for predictive markers of response to RVU120 in AML. P451: PRMT2: AN ANTI-INFLAMMATORY EPIGENETIC FACTOR INVOLVED IN ACUTE MYELOID LEUKEMIA AGGRESSIVENESS C. Sauter1,*, J. Simonet1, C. Fournier1,2, M. Mounier1,3, M. Rebourgeon1, L. Brignoli1, A. Aznague1,2, A. Largeot1, Y. Hérault4, G. Sauvageau5, F. Guidez1, M. Callanan1,2, J.-N. Bastie1,6, L. Delva1, R. Aucagne1,2 1UMR 1231 Inserm, Équipe Epi2THM, Équipe Labex LipSTIC, Université de Bourgogne Franche-Comté; 2Unité d’Innovation en Génétique et Épigénétique en Oncologie (IGEO), Plateforme CRISPR Genomics (CRIGEN), CHU François-Mitterrand; 3Registre des hémopathies malignes de Côte d’Or, Université de Bourgogne Franche-Comté, Dijon; 4Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département of de Génomique Fonctionnelle, UMR 7104 CNRS, UMR 964 Inserm, Université Louis Pasteur, Collège de France et Institut Clinique de la Souris (ICS), Illkirch, France; 5Laboratoire de Génétique Moléculaire des Cellules Souches, Institut de Recherche en Immunologie et Cancer, Université de Montréal, Montréal, Canada; 6Service d’Hématologie Clinique, CHU François-Mitterrand, Dijon, France Background: Protein Arginine Methyltransferases (PRMTs) are epigenetic factors involved in several cellular processes including regulation of gene expression through methylation of histone tails. Overexpression or overactivity of the two major PRMTs (1 and 5) have been previously identified in Acute Myeloid Leukemia (AML) patients and their biological or pharmacological inhibition leads to a decreased leukemia progression in mice (PRMT1, 4, 5). Among the nine members of this protein family, PRMT2 has been less well characterized. It has been shown to be overexpressed in glioblastoma and is responsible for the H3R8me2a epigenetic mark leading to transcription activation of target genes. In addition, PRMT2 has also been demonstrated to have a role in the inflammatory pathway but its effect is not fully understood yet. Moreover, it is well known that severe chronic inflammation can contribute to the development of certain cancers. Exacerbated inflammatory responses and aberrant myeloproliferation are thus interconnected. Aims: We are investigating the role of PRMT2 in the development of AML and we aim to discover its precise role in the control of inflammatory processes. Methods: We used a PRMT2 knockout mouse model (Prmt2KO), AML patient cohorts of Dijon (Dijon university hospital, France) and Montréal (IRIC, QC, Canada). We also generated a knockout human AML HL-60 cell line using the CRISPR-Cas9 system and a HL-60 cell line overexpressing PRMT2 through the use of a Citrine-tagged fusion protein. Results: Prmt2KO mice do not exhibit difference in bone marrow progenitor or mature cell populations compared to wild type mice, indicating that PRMT2 has little or no influence in the hematopoietic stem cell maintenance. In contrast, we observed that Prmt2KO bone marrow-derived macrophages (BMDM) are more sensitive to lipopolysaccharides (LPS) stimulation and express higher levels of pro-inflammatory cytokine mRNA, suggesting a role of PRMT2 in the negative regulation of the inflammatory processes. Analysis of datasets from a cohort of 371 AML patients (Guy Sauvageau, Leucegene project, IRIC, Montréal, QC, Canada) reveals that patients with a lower PRMT2 expression display a worse survival rate compared to patients with a higher PRMT2 expression. Gene Set Enrichment Analysis of this cohort show that PRMT2 is involved in the inflammatory response signaling pathway, thus confirming our hypothesis. In a cell model we show that PRMT2 knockout cells exhibit higher levels of phosphorylated STAT3 protein compared to wild type cells after LPS stimulation. PRMT2 could therefore be involved directly or indirectly in the STAT3 pathway by preventing its phosphorylation and activation. Ongoing transcriptomic and epigenetic studies could provide new clues for better understanding the role of PRMT2 in an inflammatory context in AML and the association between inflammation and leukemogenesis. Summary/Conclusion: Taken together, our data suggest that PRMT2 has an anti-inflammatory effect in mice and AML patients with a low PRMT2 expression inducing a higher inflammatory phenotype, thus resulting to worse overall survival. PRMT2 could be involved in the STAT3 signaling pathway by preventing its activation leading to increased inflammation. P452: THE INTERACTOME OF CDK6 IN DIFFERENT ONCOGENIC CONTEXTS L. Scheiblecker1,*, S. Nebenfuehr1, M. Zojer1, E. Doma1, V. Sexl1, K. Kollmann1 1Institute for Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria Background: The mammalian cell cycle is controlled by a complex signaling network that is frequently deregulated in cancer. Cyclin-dependent kinases (CDKs) are the key players in governing cell cycle progression. CDK6 and its close homologue CDK4 are responsible for driving the cell cycle from G1 to S-Phase. In complex with their regulatory subunits, the D-type cyclins, CDK4/6 phosphorylate the retinoblastoma protein family members (Rb) to induce G1 to S-Phase progression and DNA replication. CDK6, and not CDK4, plays an important role in hematopoiesis and is frequently found overexpressed in hematologic malignancies. In the recent years, CDK6 has been shown to regulate transcription by interacting with chromatin in a kinase dependent and independent manner. This function is crucial in leukemia as well as hematopoietic stem cells. Aims: To better understand the transcriptional role of CDK6 in transformed and untransformed cells we will analyze the interactome of CDK6. We hypothesize that different oncogenes lead to the expression of different CDK6-dependent transcriptional programs and to different CDK6-containing protein complexes on DNA level. We aim to identify common and leukemia subtype-specific interaction partners of CDK6 that will represent novel therapeutic vulnerabilities in combination with CDK6 inhibition. Methods: To characterize the interactome of CDK6 we transformed the murine hematopoietic progenitor cell line HPC7 with the BCR-ABL or the MLL-AF9 oncogene that give rise to myeloid leukemia. We isolated CDK6-containing protein complexes by immunoprecipitation followed by mass spectrometry. To extend our data and to get a specific set of interaction partners involved in the transcriptional role of CDK6, we performed a nuclear fractionation prior to immunoprecipitation and mass spectrometry. This enabled us to focus on interaction partners solely in the nucleus. Results: We obtained proteins interacting with CDK6 on a global cell level as well as specific for the nucleus that represent co-factors of CDK6-mediated transcriptional regulation. By intersecting the proteomics data from transformed and untransformed HPC7 cells, we detected interaction partners of CDK6 that are common for all tested leukemia entities, specific for leukemic subtypes and interaction partners which just occur in untransformed cells. As a next step, the determined proteins will be subjected to pathway analysis to reveal CDK6 dependent signaling networks. These proteins might serve as valuable targets for potential combinatorial treatments together with CDK6 inhibition. Summary/Conclusion: Combining our results with next generation sequencing data will allow us deciphering the distinct transcriptional signatures regulated by CDK6 in different leukemia entities. Our insights will provide a better understanding of malignancies with enhanced CDK6 expression and may help to develop novel treatment strategies by interfering with CDK6 interaction partners. P453: CDK6 DEGRADATION IS IMPEDED BY P16INK4A AND P18INK4C IN AML B. Schmalzbauer1,*, T. Thondanpallil1, G. Heller2, C.-M. Sperl1, I. M. Mayer1, V. M. Knab1, S. Nebenfuehr1, M. Zojer1, A. C. Mueller3, F. Fontaine3, T. Klampfl1, V. Sexl1, K. Kollmann1 1Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna; 2Department of Medicine I, Division of Oncology, Medical University of Vienna; 3CeMM – Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Background: Acute myeloid leukemia (AML) is a highly aggressive disease that comprises a heterogeneous group of genetically distinct subtypes. AML is initiated by leukemic cells, which are able to self-renew and give rise to malignant myeloid blasts. A key regulator and prognostic biomarker for AML is the cell cycle regulator Cyclin-dependent kinase 6 (CDK6) which represents a novel therapeutic target for the treatment of certain subtypes of AML. CDK4/6 kinase inhibitors are extensively studied in several cancer types but the beneficial effects may be limited by primary and secondary resistance mechanisms. Pharmacological CDK6 degraders, which eliminate kinase -dependent and -independent effects, represent an alternative therapeutic option. The exact mechanism and efficacy of CDK6 degraders in AML subtypes remain unknown. The involvement of p16INK4A in Palbociclib resistance and the role of INK4 proteins in AML disease progression require a systematic investigation of INK4 proteins in the context of CDK6 degradation in distinct AML subtypes. Aims: We aim to elucidate CDK6 degrader efficacy in two CDK6-dependent AML subtypes harboring either MLL-AF9 or AML1-ETO. We hypothesize an involvement of INK4 proteins on CDK6 degrader efficacy. Understanding the exact mechanisms of the CDK6 degrader will help to determine AML subtypes that would benefit from CDK6-targeted therapy. Methods: We took advantage of a novel hematopoietic progenitor cell model (HPCLSKs) to study CDK6 degrader efficacy in a genetically defined setting of AML subtypes. We analyzed INK4 RNA expression of human AML patients and the HPCLSK cells using RNA-Seq and Microarray data sets. We analyzed cell proliferation and cell cycle changes using flow cytometry. CDK6 Co-immunoprecipitation followed by Mass Spectrometry was performed to screen for CDK6 interaction partners that are enriched upon CDK6 Degrader treatment. These findings were validated via immunoblotting. Human AML cell lines were used to further validate the results in a different in vitro system and to translate them to human settings. Results: We show that efficacy of the CDK6 specific protein degrader varies among AML subtypes and depends on low expression of the INK4 proteins p16INK4A and p18INK4C. INK4 protein levels are significantly elevated in MLL-AF9+ compared to AML1-ETO+ cells, contributing to the different CDK6 degradation efficacy. We demonstrate that CDK6 complexes containing p16INK4A or p18INK4C are protected from pharmacological degradation and that INK4 levels define the proliferative response to CDK6 degradation. These findings define INK4 proteins as predictive marker for CDK6 degradation – targeted therapies in AML. Summary/Conclusion: We here identify p16INK4A and p18INK4C as dominant CDK6 binding partners that counteract pharmacological protein degradation in AML. Our data highlight the need for novel CDK6 specific therapies targeting CDK6 sites not competing with INK4 or CIP/KIP binding to overcome therapeutic limitations. P454: VENETOCLAX AND GILTERITINIB SYNERGIZE IN FLT3 WILDTYPE ACUTE MYELOID LEUKEMIA BY SUPPRESSION OF MCL-1 VIA COMBINED AXL AND FLT3 TARGETING C. Schmidt1,*, M. Janssen1,2, P.-M. Bruch1,2, M. F. Blank1,2,3, C. Rohde1,2, A. Waclawiczek4, S. Renders1,4, D. Heid1,2, S. Göllner1, L. Vierbaum1, K. Weidenauer1, S. Herbst1, M. Knoll1, C. Kolb1, B. Besenbeck1, M. Fabre5,6, M. Gu5,6, R. Schlenk1, F. Stölzel7, M. Bornhäuser7, C. Röllig7, U. Platzbecker8, C. Baldus9, H. Serve10, T. Sauer1, S. Raffel1, C. Pabst1,2, G. Vassiliou5,6,11, B. Vick12,13,14, I. Jeremias12,13,14,15, A. Trumpp4, J. Krijgsveld3,16, C. Müller-Tidow1,2, S. Dietrich1,2 1Department of Medicine V, Hematology, Oncology and Rheumatology, University hospital Heidelberg; 2Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory (EMBL); 3Division Proteomics of Stem Cells and Cancer; 4Division of Stem Cells and Cancer, German Cancer Research Centre (DKFZ), Heidelberg, Germany; 5Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton; 6Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom; 7Department of Medicine I, University hospital Carl Gustav Carus, Dresden; 8Medical Clinic and Policlinic I, Hematology and Cellular Therapy, Leipzig University Hospital, Leipzig; 9Department of Hematology and Oncology, University hospital Schleswig-Holstein, Campus Kiel, Kiel; 10Department of Medicine II, Hematology-Oncology, Goethe University Hospital Frankfurt, Frankfurt am Main, Germany; 11Department of Hematology, University of Cambridge, Cambridge, United Kingdom; 12Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München; 13German Research Center for Environmental Health (HMGU); 14German Consortium for Translational Cancer Research (DKTK); 15Department of Pediatrics, Dr. von Hauner Children’s Hospital, Ludwig Maximilians University München, Munich; 16Medical Faculty Heidelberg, Heidelberg University, Heidelberg, Germany Background: BCL-2 inhibition has been shown to be effective in older patients with acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, treatment resistance and relapse represent major clinical challenges. Thus, there is an unmet need to overcome resistance to current venetoclax-based regimens. Aims: In order to establish an alternative treatment approach for AML in elderly patients, we aimed at identifying a highly synergistic drug combination partner for venetoclax. Methods: We performed a high-throughput drug screening to identify the most effective combination partner for venetoclax in AML. Overall, 64 anti-leukemic drugs were screened in 31 primary high-risk AML samples in the presence or absence of venetoclax. We calculated drug synergies, validated top venetoclax combination partners in AML cell lines and primary patient samples and performed mechanistic analyses. Results: Established anti-leukemic drugs exhibited only weak synergism with venetoclax in high-risk AML samples. In contrast, when targeting distinct pathways such as FLT3 signaling, we observed increased synergistic activity. Gilteritinib exhibited highest synergy with venetoclax in FLT3 wildtype AML, with especially high synergy values in TP53 mutated samples. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability and abolished colony-formation potential in FLT3 wildtype as well as venetoclax-azacitidine resistant cell lines and primary patient samples. Proteomics revealed increased levels for proteins involved in FLT3 wildtype signaling in specimens with low in-vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159 and proteasomal degradation. Compared to other FLT3 inhibiting drugs, gilteritinib showed highest synergism with venetoclax due to the additional inhibition of AXL that reinforced the downregulation of MCL-1. Gilteritinib and venetoclax proved efficacious in an FLT3 wildtype PDX model with TP53 mutation. Furthermore, the drug combination reduced leukemic burden in four patients with venetoclax-azacitidine refractory AML, who received gilteritinib and venetoclax as an individual treatment approach. Summary/Conclusion: In summary, our results identified gilteritinib as a potential therapy combination partner for venetoclax in patients with FLT3 wildtype AML. P455: BREAKING THE PUMP: TARGETING THE SODIUM-POTASSIUM PUMP AS A THERAPEUTIC STRATEGY IN ACUTE MYELOID LEUKEMIA C. Schneider1,2,*, H. Spaink1, G. Alexe1,2, N. V. Dharia1,2, D. Khalid1, S. Scheich3,4, B. Haeupl4,5, T. Oellerich4,5, K. Stegmaier1,2 1Pediatric Oncology, Dana-Farber Cancer Institute, Boston; 2The Broad Institute of MIT and Harvard, Cambridge; 3Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, United States of America; 4Department of Medicine II, Department for Hematology/Oncology, Goethe University, Frankfurt; 5German Cancer Research Center and German Cancer Consortium, Heidelberg, Germany Background: Acute myeloid leukemia (AML) is a heterogenous hematologic malignancy with poor overall survival despite intensive cytotoxic chemotherapy and newer targeted agents. With a 5-year survival rate of only 29% in adults with AML, new therapeutic strategies are needed. Aims: In order to identify novel, selective targets for AML, we used the Cancer Dependency Map, which provides genome-scale CRISPR/Cas9 depletion screen data in hundreds of cancer cell lines. Here, we identified ATP1B3 as a context-specific dependency that could be therapeutically exploited. ATP1B3 is the smaller glycoprotein subunit (beta) of the sodium-potassium pump (Na/K-ATP pump). Together with the catalytic alpha subunit ATP1A1, it forms a heterodimer located in the plasma membrane, regulating the electrochemical gradient through the transport of Na and K ions across the membrane. The Na/K-ATP pump is of vital importance in the maintenance of cellular homeostasis and membrane potential and because of its function as a receptor and signal transducer. While the ATP1A1 carries out ion transport and enzymatic activity, making it a common essential gene (a gene which ranks among the most depleted genes in at least 90% of screened cell lines), ATP1B3 is not essential in all cancer cells. This beta subunit seems to be crucial for intracellular transport and stabilization of the alpha subunit in the membrane. The beta subunit has 4 paralogs, showing similar expression patterns among tissues, with one exception, ATP1B1. Methods: We used a diverse panel of functional genomic based assays, validating our finding using CRISPR/Cas9 knockout and overexpression constructs for the Na/K-ATP pump beta subunit paralogs. In an approach to analyze protein-protein interactions we generated BioID constructs for a mass spectrometry-based analysis. For an in vivo study we used a bioluminescent imaging (BLI)-based orthotopic mouse model of AML, carrying non-targeting, ATP1B1 or ATP1B3 knockout guides. Results: By using CRISPR/Cas9 knockout we could validate ATP1B3 as a selective dependency and performing competitive growth assays we showed that loss of ATP1B3 in ATP1B1 low expressing AML cells leads to synthetic lethality. The absence of both paralogs of the beta subunit results in the loss of their common essential binding partner ATP1A1 in hematologic malignancies, while higher expression of ATP1B1 can stabilize ATP1A1 under the loss of ATP1B3 in solid tumors or in ATP1B1 overexpressing AML cells. Next, we validated our findings in vivo. In a BLI-based orthotopic mouse model of AML, we found that the AML model with ATP1B3 knockout showed lower leukemia burden and normal spleen weight in comparison to a non-targeting or ATP1B1 knockout control. To understand the specific role of ATP1B3 we produced BioID (proximity-dependent biotin identification) constructs for ATP1B1 and ATP1B3. In ongoing studies, through this mass spectrometry-based analysis of protein-protein interactions, we will gain a greater insight into the protein partners of the Na/K-ATP pump. Summary/Conclusion: Taken together, we identified ATP1B3 as a selective dependency in AML. We propose that the elimination of ATP1B3 leads to the destabilization of the sodium-potassium pump when ATP1B1 levels are low, making it a potential tumor-selective therapeutic target for AML and other hematologic malignancies with low expression of ATP1B1. P456: A LONG READ SEQUENCING AND CRISPR-CAS9 BASED APPROACH FOR RAPID COPY-NUMBER ALTERATION AND STRUCTURAL VARIATION DETECTION IN HEMATOLOGIC MALIGNANCIES J. Schrezenmeier1,2,*, J. E. Straeng1, O. Blau1, A. Elashy1, A. Lazarides1, S. Skambraks3, E. Jahn3, B. Gillißen1, C. Eckert4, A. Nogai1, I.-W. Blau1, K. Giannopoulos5, M. Heuser6, K. Döhner3, L. Bullinger1, A. Dolnik1 1Hematology, Oncology and Tumorimmunology, Charité-Universitätsmedizin Berlin; 2Berlin Institute of Health at Charité – Universitätsmedizin Berlin, BIH Biomedical Innovation Academy, Berlin; 3Department of Internal Medicine III, University of Ulm, Ulm; 4Pediatric Hematology and Oncology, Charité-Universitätsmedizin Berlin, Berlin, Germany; 5Department of Experimental Hematooncology, Medical University of Lublin, Lublin, Poland; 6Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany Background: Current standard karyotyping methods are labor-intensive and have severe limitations in terms of resolution and duration to results. For hematologic malignancies, but not restricted to, this represents a relevant obstacle in translating molecular findings into time critical treatment decisions. Aims: To develop a sequencing based approach for rapid and scalable cytogenetics that can be applied to a wide spectrum of hematologic malignancies to detect clinically relevant molecular markers within 48hrs Methods: For copy number alteration detection, whole genome sequencing was performed on a GridION sequencer (Oxford Nanopore Technologies, ONT, using the SQK-LSK109 kit) in a cohort of 53 hematologic neoplasms, including acute myeloid leukemia (AML, n=18), chronic lymphocytic leukemia (CLL, n=8), multiple myeloma (MM, n=17) and pediatric acute lymphocytic leukemia (ALL, n=10) samples. For the identification of balanced alterations, transcriptome sequencing data were generated using ONT (SQK-DCS109 kit), and we applied an in-house developed analysis pipeline based on minimap2 alignment followed by Blast with an ensuing filtering algorithm based on orientation, inter-read distance and length filtering. For targeted sequencing of structural aberrations, we developed a CRISPR-Cas9 based tiling approach using sgRNA pools (Integrated DNA Technologies, IDT). This approach allows to cover AML relevant targets such as t(8;21), the KMT2A (MLL) breakpoint area, and the FLT3-ITD region as well as lymphoma relevant regions such as the immunoglobulin heavy chain locus (library preparation was performed using SQK-CS9109 kit). Results: For copy number profiling a median whole-genome coverage of 2.6 was reached (range 0.27-7 fold). ONT sequencing and conventional karyotyping approaches showed a high concordance with Pearson correlation coefficients >0.95 for copy number alteration comparisons between conventional cytogenetics and ONT sequencing results for all investigated disease entities. Regarding the detection of structural aberrations transcriptome sequencing and fusion gene analysis using the above described analysis and filtering pipeline we could e.g. reliably detect the t(9;22) translocation from the K562 cell line and the t(8;21) translocation from Kasumi-1. Additionally, using this method we were able to correctly identify the primary AML sample harboring a t(8;21) translocation that was among the n=12 AML patient samples. In addition to an RNA-based work-flow, we established a CRISPR-Cas9 based DNA enrichment approach for the detection of recurrent structural aberrations from specified genomic DNA loci. The enrichment of genomic regions of interest using sgRNA libraries led e.g. to a median coverage of 44 reads (range 5-136-fold) of the t(8;21) region-of-interest in primary AML samples, which allow the detection of the exact coordinates of the breakpoints. Summary/Conclusion: Long read based sequencing based copy number alteration and structural variation detection based on combination of different long read sequencing approaches like low-coverage whole genome sequencing, transcriptome sequencing and CRISPR-Cas9 based sequencing of genomic loci of interest represents a highly promising tool for high resolution and high speed cytogenetics that has the potential to overcome many limitations of conventional cytogenetics. P458: TRANSCRIPTIONAL AND TRANSLATIONAL SIGNATURES OF TRIPLE MUTATED DNMT3AKO/FLT3ITD/NPM1C MICE SHOW ALTERED RNAS PROCESSING AND CELL CYCLE PROGRESSION A. Scialdone1,*, D. Sorcini1, A. Stella1, V. Tini1, A. Marra1, C. Rompietti1, F. De Falco1, E. Dorillo1, F. M. Adamo1, E. C. Barcelos1, R. Arcaleni1, M. P. Martelli1, P. Sportoletti°1, B. Falini°;° co-last author1 1Medicine and Surgery, University of Perugia, Perugia, Italy Background: The top ranking recurrently mutated genes in AML are NPM1, FLT3 and DNMT3A 1,2. Co-occurrence of these three mutations is associated to a unique AML subset characterized by high peripheral blood and bone marrow blast counts, normal cytogenetic and very poor EFS and OS3. Nevertheless, the cooperative leukemogenic mechanisms of these three mutations have not been deeply explored and a successful targeting treatment of AML patientsDNMT3Amut/ FLT3 -ITD/ NPM1 c still represents an unmet clinical need. Aims: To dissect how DNMT3A loss of function, FLT3 Internal Tandem Duplication (ITD) and NPM1 mutations (NPM1 c) cooperate in driving AML onset and progression through a picture of the transcriptome and the translatome of triple mutated AML cells. Methods: Mx1-Cre-NPM1 c and/or Mx1-Cre-DNMT3A KO and FLT3 ITD mutant mice were crossed to obtain the following lines: DNMT3A KO/FLT3 ITD/NPM1 c, FLT3 ITD/DNMT3A KO and FLT3 ITD/NPM1 c. Peripheral blood (PB) was periodically taken and analysed, previous anesthesia, while bone marrow (BM) and spleen were harvested two months after pIpC induction or at conclamate disease (n=7 each group). To investigate the mechanisms underlying the triple mutated mice phenotype, we looked BM leukemic samples from all groups both at transcriptional level by RNA sequencing (RNAseq) and at translational level (protein synthesis capacity of AML blasts) by Ribosome footprinting (Riboseq)4. Unique Differentially Expressed Genes (DEGs) (filtered by p-value<0.05 and FC >1.5) belonging to FLT3 ITD/DNMT3A KO/NPM1 c vs FLT3 ITD/DNMT3A KO were identified by Venn diagram and used in enrichment analysis on Reactome database and MSigDB (C6-oncogenic signature). Results: The FLT3ITD/DNMT3A KO/NPM1c, DNMT3A KO/FLT3 ITD and FLT3 ITD/NPM1 c displayed a fully penetrant leukemic phenotype. As expected, FLT3ITD/ DNMT3A KO/NPM1 c showed a more aggressive disease characterized by higher WBC counts, more pronounced blasts infiltration of the spleen and shorter survival (>2 fold vs double mutants). The FLT3ITD/DNMT3A KO/NPM1 c mice displayed 1429 (842 UP and 587 DOWN) DEGs compared to FLT3 ITD/DNMT3A KO mice. The enrichment analysis suggested that DNMT3A and NPM1 mutations, in a constitutive FLT3 ITD expression context, cooperate to activate transcriptional programs sustaining the proliferation and maintenance of blasts through concerted upregulation of a number of pathways, including the Anaphase-Promoting Complex (APC), which regulates cell cycle progression, the m-TOR-mediated autophagy inhibition, the Rho-GTPases effectors (with Rac1 overexpression), the Hippo-mediated self renewal, the HOXA9 signature and TP53-regulated apoptosis. Interestingly, genes involved in transport of mature transcripts to cytoplasm and nuclear RNAs metabolism were also upregulated. In keeping with these RNAseq findings, the Riboseq data showed that both the quote of processed transcripts (15 vs 9%) and the percentage of processed pseudogenes were more abundant in triple vs double mutated mice. On the other hand, the long intergenic noncoding RNAs (lincRNA) fraction was reduced in triple mutants. This finding may reflect dysfunctional remodeling of tumor microenvironment, contributing to AML cells immune escape in the BM5. Image: Summary/Conclusion: The leukemic DNMT3A KO/FLT3 ITD/NPM1 c genotype is characterized by unique transcriptional and translational signatures. The information about deregulated pathways and altered protein synthesis establishes the rational basis for designing new therapeutic approaches in this poor prognosis AML genotype. P459: NUC-7738 REGULATES BETA-CATENIN SIGNALLING RESULTING IN REDUCED PROLIFERATION AND SELF-RENEWAL OF AML CELLS A. M. Shahid1,*, M. Elshani1, D. J. Harrison1 1School of Medicine, University of St Andrews, St Andrews, United Kingdom Background: NUC-7738, a phosphoramidate transformation of 3’deoxyadenosine (3’dA), is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine triphosphate (3’-dATP) directly in cells, bypassing key cancer resistance mechanisms of transport, activation and breakdown. NUC-7738 is currently in a Phase I/II clinical study to assess safety and determine the recommended dose in patients with advanced solid tumors and lymphoma. We have recently shown in cell lines a potential therapeutic role for NUC-7738 in patients with acute myeloid leukemia (AML), whereby NUC-7738 induced myeloid cell differentiation and mitochondrial-mediated apoptosis. AML leukemia stem cells (LSCs) are required for the initiation and maintenance of the disease. Activation of the Wnt/β-catenin pathway is required for the survival and development of LSCs and therefore, targeting β-catenin is a potential therapeutic strategy. Aims: The aim of this study was to determine whether NUC-7738 regulates β-catenin and the expression of its target genes in AML cells Methods: AML cell lines KG1a, OCI-AML3, HL-60 and U937 were treated with NUC-7738 for 48 or 72 hours. LSCs were determined by the expression of surface markers CD34, CD38 and CD123 via flow cytometry. Self-renewal was assessed by performing the colony forming assay using MethoCult Enriched media. Cell proliferation was determined by the Alamar Blue assay. Immunoblotting was performed for β-catenin, PI3K-p110, phosphorylated AktSer473 and phosphorylated GSK3βSer9 Results: NUC-7738 reduced PI3K-p110, phosphorylated AktSer473 and phosphorylated GSK3βSer9 resulting in reduced β-catenin expression. NUC-7738 decreased the percentage of CD34+ CD38- CD123+ (LSCs) from 15% to 7% and significantly reduced the total number and size of leukemic colonies. NUC-7738 inhibited cell proliferation in U937, OCI-AML3 and HL-60 cells, with IC50’s of 1.55 µM, 7 µM and 16 µM, respectively. Summary/Conclusion: Self-renewal pathways, including Wnt/β-catenin, are key for the initiation and long-term maintenance of LSCs. Through the ability to reduce PI3K-p110, phosphorylated AktSer473, and phosphorylated GSK3βSer9 and suppress β-catenin signalling, NUC-7738 exploits a key developmental process of AML, supressing the expansion and survival of LSCs. These results indicate that targeting LSCs with NUC-7738 may offer a successful therapeutic strategy for treating patients with leukemia. P460: RECOMBINANT SLIT2 INHIBITS ACUTE MYELOID LEUKEMIA CELL PROLIFERATION IN VITRO AND IN VIVO L. Araujo de Albuquerque Simoes1,2,3,4,*, I. Weinhäuser2,5, D. A Pereira-Martins2,5, C. A. Ortiz Rojas2, T. Mantello Bianco2, R. D. C. Cavaglieri6, E. Magalhães Rego2,3,4,7 1Center of Translational Research in Oncology, ICESP - Faculty of Medicine, USP, Sao Paulo; 2Center for Cell Based Therapy, Fundação Hemocentro de Ribeirão Preto, Ribeirão Preto; 3Medical Research Laboratory on Molecular Hematology (LIM31), University of São Paulo; 4Hematology, Faculty of Medicine, University of Sao Paulo, Sao Paulo, Brazil; 5Department of Experimental Hematology, University Medical Center Groningen, Groningen, Netherlands; 6Medical Research Laboratory on Molecular Hematology (LIM31), Faculty of Medicine, University of Sao Paulo; 7Center of Translational Research in Oncology, ICESP - Faculty of Medicine, USP, Sao Paulo, Brazil Background: The SLIT/ROBO axis has been shown to play a key role in many physiological processes such as organogenesis, axon guidance and angiogenesis. Additionally, several studies conducted in solid tumors observed frequent hypermethylation of either Slit or Robo at their promoter site. In the context of leukemia, Golos et al., 2019 showed that the low expression of SLIT2 was associated with lower overall survival in adults with acute myeloid leukemia (AML). Furthermore, it was demonstrated by our group that the knockdown of SLIT2 in Acute Promyelocytic Leukemia (APL) cells leads to an increase of cell proliferation in vitro and a more aggressive course of the disease in vivo (Weinhäuser et al., 2020). Aims: Given the evidence of the relevance of SLIT2 in APL, we opted to transfer our gained knowledge to a more challenging leukemia subset and decided to study the role of SLIT2 in AML. Methods: We first evaluated the methylation pattern of SLIT2 in AML patients compared to healthy donors by analyzing publicly available datasets (GSE58477, normal karyotype blasts: 62, healthy CD34+: 10; GSE63409, LSC: 14, HSC: 5), followed by the assessment of the level of SLIT2 in the bone marrow (BM) plasma of AML patients and healthy donors by ELISA. Additionally, to functionally assess the biological role of SLIT2, we treated AML cell lines (KASUMI1, MV4-11, and MOLM13) with recombinant SLIT2 (50ng/mL) in vitro. We performed the knockdown of SLIT2 in AML cell lines (THP-1 and OCI-AML3) and evaluated their proliferation capacity as well. Moreover, AML cells were treated with decitabine and recombinant SLIT2. Finally, we evaluated the anti-leukemic effects of SLIT2 in vivo. To do so NSGS mice were transplanted with luciferase-transduced MV4-11 cells and the animals were either treated with vehicle (control group) or recombinant SLIT2 (25 ng/g of body weight) two days after transplant for a period of one week. Results: Our analysis indicated increased methylation at the SLIT2 promoter site in AML patients compared to healthy CD34+. In accordance with our analysis, we detected decreased level of SLIT2 protein in the bone marrow (BM) plasma of AML patients (1.43 ng/mL) when compared to healthy donors (3.51 ng/mL) (p<0.05). The functional role of SLIT2 was determined with the treatment of KASUMI1, MV4-11 and MOLM13 cells with recombinant SLIT2 in vitro. Our results showed that SLIT2 treatment reduced the cell proliferation and colony formation capacity and induced cell cycle retention in the G1 phase for all AML cell lines. Contrarily, the knockdown of SLIT2 promoted increased cell proliferation in THP-1 and OCI-AML3 cell lines. Moreover, we observed increased induction of decitabine-induced apoptosis when MV4-11 and MOLM13 cells were treated in the presence of recombinant SLIT2. Finally, the engraftment and progression of the disease of NSGS mice transplanted with luciferase-transduced MV4-11 cells were monitored by the detection of luciferase bioluminescent signals. Our results showed, that SLIT2 treatment was able to significantly delay the progression of AML in vivo. Mice treated with SLIT2 presented improved overall survival (vehicle 15d: CI 13-16d; SLIT2 19d: 95% CI: 16-22d. P value = 0.0320), decrease leukemic infiltration in the BM and spleen, reduced spleen size (vehicle: 125 mg; SLIT2: 100 mg P value= 0.04), indicating lower disease burden when compared to the control group. Summary/Conclusion: In conclusion, our results suggest that SLIT2 has tumor suppressive functions in AML in vitro and in vivo highlighting the therapeutic potential with low cytotoxicity of SLIT2 in AML. P461: MYELOID KINOME INHIBITOR HM43239 OVERCOMES ACQUIRED RESISTANCE IN ACUTE MYELOID LEUKEMIA MODELS R. Bejar1,*, S. J. Baek2, N. Abbasi1, A. Krasny1, R. Sinha1, H. Zhang1, S. Howell3, W. Rice1 1Aptose Biosciences Inc., San Diego, United States of America; 2Hanmi Pharmaceutical Company, Seoul, South Korea; 3Moores Cancer Center, University of San Diego Health, San Diego, United States of America Background: Oral HM43239 is in development for the treatment of acute myeloid leukemia (AML) because of its capacity to potently inhibit kinases that drive myeloid malignancies, including diverse forms of the FLT3, SYK, JAK and c-KIT kinases. Wildtype FLT3 is overexpressed in most AML patients, and approximately 30% of newly diagnosed adult AML patients harbor internal tandem duplications (ITDs) or point mutations in the tyrosine kinase domain (TKD). These mutations drive aberrant activation of downstream proliferation pathways and are associated with a high risk of relapse. Likewise, the c-KIT alternative receptor kinase, as well as the SYK and JAK1/2 intracellular kinases, mediate oncogenic signaling in AML that can promote drug resistance to certain FLT3 inhibitors. HM43239 was developed to overcome shortcomings of other FLT3 inhibitors. It inhibits a broad set of mutant and wildtype forms of FLT3, while simultaneously disrupting downstream SYK, JAK/STAT5, ERK, and other rescue signaling pathways. This rationale supports the development of HM43239. Aims: Evaluate the activity of HM43239, an orally active drug, as a FLT3-focused myeloid kinome inhibitor in human AML models. Methods: Biochemical kinase assays were performed by RBC and Carna. The effects of HM43239 on cell proliferation (IC50), growth rate (GR50) and concentration at half-maximal effect (Growth Effective Concentration; GEC50) were determined using the MTS assay with vehicle controls. Cell-based inhibition of target phosphorylation was assessed by Western blot and flow cytometric analyses. The AML cell lines tested included MV-4-11, MOLM-13, MOLM-14 and BAF3/ITD. In vivo efficacy was assessed using the MOLM-14 FLT3-Mutated xenograft model. Results: HM43239 inhibited the enzymatic activities of FLT3-WT, -ITD, and -D835Y variants with IC50 values of 1.1, 1.8 and 1.0 nM, respectively. Binding K d values for FLT3-WT, -ITD, -D835Y, -D835H, -ITD/D835V and -ITD/F691L were 0.58, 0.37, 0.29, 0.4, 0.48, and 1.3 nM, respectively. HM43239 killed AML cells that harbor the FLT3-ITD mutation (MV-4-11, MOLM-13 and MOLM 14) with IC50, GR50 and GEC50 values ranging from 4 to 10 nM. In cell lines expressing wild type FLT3 (KG1, HEL92.1, SKM-1, and THP-1) the values for these parameters ranged from 0.05 nM to 3 µM. Western blot and flow cytometric analyses of MV-4-11, MOLM-14 cells and Ba/F cells transfected with target proteins revealed that the phosphorylation of FLT3 and SYK was reduced by 50-90% at HM43239 concentrations of 10 – 100 nM, and that these concentrations produced >80% reduction in phosphorylation of ERK and JAK/STAT5 that function downstream of FLT3. HM43239 inhibited FcγR-induced SYK and JAK/STAT5 activation in KG-1a (FLT3-WT) cells that upregulate RAS signaling which is a mechanism of acquired resistance to gilteritinib. In murine xenograft studies, HM43239 was more effective than gilteritinib in both SC and orthotopic FLT3-F691L and FLT3-ITD/F691L mutant MOLM-14 models. Summary/Conclusion: HM43239 inhibits wild type and mutant forms of FLT3 at low nM concentrations and demonstrates in vivo efficacy at doses that are well tolerated. Its ability to also inhibit SYK and, by reducing the activity of these upstream kinases, to also impair the activity of EKR1/2 and JAK/STAT5 that participate in rescue pathways, makes this a particularly interesting molecule with the potential of offsetting the development of resistance that is common with other FLT3 inhibitors. A Phase 1/2 trial of HM43239 in patients with AML is open and accruing patients (NCT03850574). P462: IDENTIFICATION OF TARGETED THERAPIES DIRECTED TO ACUTE MYELOID LEUKEMIA MINIMAL RESIDUAL DISEASE N. van Gils1,*, F. Kessler1, M. Broux2, F. Denkers1, S. Demeyer2, J. Cools2, D. C. De Leeuw1, J. Janssen1, L. Smit1 1Hematology, UMC Amsterdam, location VUmc, Cancer Center Amsterdam, Amsterdam, Netherlands; 2Center for Human Genetics, KU Leuven, Leuven, Belgium Background: Despite good responses to intensive polychemotherapy in the initial treatment phase, the biggest challenge in the treatment of acute myeloid leukemia (AML) is persistence of residual therapy-resistant cancer cells (measurable residual disease, MRD) that can develop into recurrence. Leukemia cells with stem cell features (“leukemic stem cells”, LSCs) residing within MRD are thought to be at the origin of relapse. Current knowledge gaps on residual leukemic cell persistence and ways to target MRD are hampering progress in development of treatments successfully preventing relapse and increasing AML cure rates. Aims: To identify transcriptional and epigenetic characteristics and vulnerabilities of AML MRD, guiding the design of “targeted” combination treatment strategies that successfully eliminate relapse-initiating cells in individual AML patients. Methods: We developed in vivo patient-derived xenograft (PDX) NOD/SCID/IL2Rγc-deficient (NSG) mouse models mimicking MRD by 1) intravenous (IV) injection of primary AML diagnosis samples and, as soon as leukemic cells were detectable in the peripheral blood, treatment with combination chemotherapy, and 2) IV injection of AML patient samples at the stage of MRD in NSG mice. Moreover, we selected samples from individual AML patients at different time points, diagnosis, MRD and relapse, and purified by flow cytometry the leukemic cells using the leukemia-associated immunophenotype (LAIP) expressed on the AML cells. To elucidate the mutational landscape, transcriptional program and chromatin accessibility involved in development and persistence of MRD, we performed DNA-, RNA- and the assay for transposase-accessible chromatin (ATAC)-sequencing on the purified leukemic cells. Results: We revealed that in patient AML samples at the stage of MRD there is clonogenic capacity of leukemic stem/progenitors (Figure 1A). Treatment with combination chemotherapy significantly reduced leukemic burden in the bone marrow of NSG mice (Figure 1B top). Interestingly, the number of CD34+CD38- leukemic cells in AML1 was hardly affected by chemotherapy, whereas treatment significantly reduced the number of these cells in AML2 (Figure 1B bottom), indicating that leukemic CD34+CD38- cells residing within different patients have a heterogeneous response to polychemotherapy. Injection of AML MRD from several patients resulted in leukemia engraftment (example Figure 1C). For one case, marked as MRD-negative by flow cytometry, we even observed that MRD could initiate high leukemia load in the mice (Figure 1D), while after injection of diagnosis and relapsed AML from the same patient no engraftment was observed. Together, our results implicated that AML cells after therapy, even if the patient is marked as MRD-negative, could have high leukemia-initiating potential, which could have been induced by chemotherapy. Using RNA-sequencing we identified and compared the gene expression profile and chromatin accessibility of leukemic cells at diagnosis, MRD and relapse from patients samples. We identified several genes upregulated in AML MRD cells as compared to diagnosis AML. Moreover, we showed increased chromatin accessibility, indicating open chromatin structure and potential transcriptional activity, at several loci in MRD compared to diagnosis AML, giving opportunities to therapeutically target MRD. Image: Summary/Conclusion: We generated AML MRD PDX mouse models, and identified transcriptional and epigenetic characteristics of AML MRD that might guide the design of “targeted” combination treatment strategies potentially eliminating relapse-initiating cells. P463: RE-PURPOSING OF GENE SIGNATURES IN AML UNCOVERS NOVEL ENERGETICS-ASSOCIATED MOLECULAR SUBTYPES P. Strain1,*, E. E. Scanlon1, G. Jellema2, R. D. Kennedy1,2, K. I. Mills1, J. K. Blayney1 1Patrick G Johnston Centre for Cancer Research, Queens University Belfast, Belfast; 2Almac Diagnostics, Almac, Craigavon, United Kingdom Background: AML is a highly heterogeneous disease with great diversity in clinical features and patient response to treatments. Despite recent improvements in disease understanding, treatments have remained unchanged for 30 years. This presents the need to characterise the disease more fully and characterise its underpinning molecular subtypes. The re-use of published and validated prognostic and predictive gene signatures e.g. Cancer Hallmarks presents an invaluable in silico opportunity to uncover the biological mechanisms underpinning treatment response in AML. Aims: To develop an automated statistical analysis pipeline combined with machine learning techniques to derive robust patient clusters representative of novel molecular AML subtypes significantly associated with clinical variables and survival outcomes. Methods: Analysis was carried out using a primary dataset (AML-OHSU: 451 AML patient samples) and a validation dataset (TCGA-LAML: 200 AML patient samples). Both datasets had been processed using Almac’s claraT platform, a software-driven solution which provides a comprehensive overview of tumour profiles using gene expression signatures. An automated analytical pipeline was developed using Consensus Clustering (CC), a method that determines the number and membership of potential clusters, using different combinations of 8 distances and 7 linkages within a dataset. Robustness was tested via bootstrapping. This pipeline was used to stratify patients via a dimension reduction approach whereby clustering was performed on 210 gene signatures categorised by 10 different hallmarks of cancer. Analysis was performed to identify clinical associations within robust clusters that were linked to differences in survival. Results: The automated clustering pipeline analysed a total of 1,314 stable clusters across 10 cancer hallmarks in the AML-OHSU dataset. Stable clusters were subsequently processed via log rank analysis (OS right-censored at 60 months) identifying 134 stable clusters with significant differences (p-value <0.05) in survival outcome. Stable clusters with significant survival differences were tested against 32 clinical categorical variables present in the AML-OHSU dataset. The results were filtered for a significant threshold (chi square p-value <0.05 and BH p-value <0.2). Here we found gene signatures representative of the Energetics hallmark, incorporating 22 signatures, to be one of the most frequently clustered throughout our results, ranking highest where K=3. A significant difference in overall survival probability (Log rank p-value: 0.033) was found between clusters (Energetics, K=3). Patients in the poorest survival cluster were characterised by a refectory induction response to treatment, having the lowest number of fusions, a low frequency of NPM1 mutations and a high proportion of patients above the age of 65. To validate energetics results from the AML-OHSU dataset, CC was again performed using gene signatures from the TCGA dataset that were representative of the energetics hallmark. A significant difference between overall survival probability (Log rank p-value: 0.019) was again found between stable clusters of the energetics hallmark (K = 3). Summary/Conclusion: We have demonstrated that a novel analytical pipeline developed here to analyse Hallmark-related gene signatures can aid in the discovery of new molecular subtypes in AML associated with prognosis. We have subsequently validated these in an independent dataset. The Energetics hallmark has not, to our knowledge, been linked with AML prognosis before, and may suggest novel biology linked to treatment response. P464: COMPARED TO CLASSICAL CYTOGENETICS OPTICAL GENOME MAPPING (OGM) DETECTS MULTIPLE ADDITIONAL STRUCTURAL CHROMOSOMAL ABERRATIONS IN PEDIATRIC ACUTE MYELOID LEUKEMIA (AML) J. Suttorp1,*, J. L. Lühmann2, D. Steinemann2, D. Reinhardt1, N. von Neuhoff1, M. Schneider1 1Clinic of Pediatrics III, University Hospital Essen, Essen; 2Department of Human Genetics, Hannover Medical School, Hannover, Germany Background: AML is the 2nd most frequently diagnosed blood cancer in children affecting approximately 130 patients annually in Germany. Pediatric AML is a heterogeneous malignancy that has multiple genetic aberrations. This is relevant for classification, prognosis and selection of optimal therapy. To detect these alterations established procedures (karyotyping, FISH, RNA-based techniques) are used with well-known limitations (resolution of karyotyping and FISH, respectively; cell cultivation is necessary to generate metaphases; cost-intensiveness; necessity of experienced and intensively trained staff). OGM is an emerging technique addressing these limitations. Based on extraction of ultra-high weight DNA followed by fluorescent labelling at the sequence CTTAAG a barcode-like pattern of the genomic DNA is created. The DNA strands labelled in this way are stretched and drawn as single ultra-long fragments through nanochannels. The results are translated into molecule maps which are compared to the reference genome (resolution down to 500 bp) allowing the identification of genetic alterations. This method combines high resolution with picturing almost whole chromosomes without cell cultivation. Aims: The aim of this study was to test to what extend OGM might supplement classical cytogenetics (CCG) to detect risk defining genetic aberrations at initial diagnosis in pediatric patients with AML. Methods: We analyzed 24 specimen of pediatric AML, MPAL and bilineage leukemia patients by OGM starting from stored frozen material (bone marrow, peripheral blood) obtained at initial diagnosis. Results of OGM were compared with karyotyping and FISH. Primer walking and breakpoint spanning PCR were used to validate newly detected aberrations by OGM in selected cases. Applying breakpoint-spanning PCR, validated aberrations were used as markers for assessment of minimal residual disease (MRD) in selected cases during AML treatment. Results: Overall, we detected discrepant results between CCG and OGM in 17/24 (70%) cases including 9 cases in which karyotyping detected aberrations which were not found by OGM. However, these aberrations were not relevant for risk stratification. In general, focusing on genetic aberrations important for risk stratification the results between CCG and OGM were concordant in 23/24 (96%) cases. In a single case OGM was able to detect a high-risk aberration which was missed by CCG. In total, 33 previously unknown genetic alterations were identified by OGM. Out of these four newly detected variants (2 deletions, 2 translocations) were validated by PCR. The translocations t(2;12) and t(8,12) both affected gene ETV6 on chr. 12 with different fusion partners (chr. 2: AC064875.1; chr. 8: NSMCE2). In both translocations sequences at the breakpoints were determined. Two deletions affecting genes RNF157 on chr. 17 and MEF2B on chr. 19 were validated. These aberrations had not been described before in pediatric AML. In 2 cases - both without a previously identified MRD marker – the newly detected translocations by OGM were used for MRD monitoring. Summary/Conclusion: OGM considerably expands the range of techniques to optimize the diagnosis of pediatric AML and has much potential to omit limitations of classical cytogenetics. Furthermore, the results will contribute to a better understanding of the leukemogenesis of pediatric AML. In addition, OGM offers the possibility to identify new aberrations which can serve as patient specific MRD markers especially in cases without a previously known MRD marker. P465: A LIPID NANOPARTICLE DELIVERY SYSTEM FOR TARGETING THE LEUKAEMIC FUSION GENE RUNX1/ETO BY SIRNA L. Swart1,*, P. Derevyanko1, A. van Oort1, M. Ashtiani1, A. Krippner-Heidenreich1, A. Koekman2, C. Seinen2, H. Issa3, H. Blair4, R. Schiffelers2, O. Heidenreich1,4 1Princess Maxima Center for Pediatric Oncology; 2Department of Central Diagnostic Laboratory Research, University Medical Center Utrecht, Utrecht University, Utrecht, Utrecht, Netherlands; 3Department of Pediatrics, University Hospital Frankfurt, Frankfurt (Main), Germany; 4Wolfson Childhood Cancer Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom Background: The leukaemic fusion gene RUNX1/ETO initiates and drives leukaemogenesis and, thus, constitutes an ideal cancer-specific target. Direct and exclusive targeting of leukaemic fusion proteins using RNA interference is an attractive concept, but has proven challenging because of the unfavourable pharmacokinetic properties of siRNA. To address this challenge, we have developed lipid nanoparticle (LNP) formulations that interfere with the expression in leukaemic cell lines and in primary cells. To further harness their use in patient-derived AML cells, we are exploring and optimizing targeted LNPs for effective siRNA delivery to primary leukaemic cells. Aims: To develop and validate a delivery system for the therapeutic application of fusion gene-specific siRNAs. Methods: LNPs encapsulating siRE (active siRNA targeting the RUNX1/ETO fusion transcript) and siMM (mismatch siRNA control with two nucleotides swapped in the sense strand) were prepared using microfluidic mixing. Ligand or fluorescent dyes were conjugated to polyethylene glycol (PEG) following a copper-free click chemistry approach. LNPs were characterized by dynamic light scattering (DLS) and cryo electron microscopy (EM). Functional experiments with patient-derived t(8;21) cells were performed in co-culture with mesenchymal stromal cells under conditions that preserved and expanded immature VLA-4 expressing leukaemic populations as determined by an 11 antibody panel and flow cytometry analysis. Transcript and protein levels of RUNX1/ETO and target genes were determined by bulk and single cell (sc) RNA-seq, real-time polymerase chain reaction and western blotting. Results: To monitor uptake of 50 – 60 nanometers sized LNPsiRNA we labelled the surface with Cy3 and a ligand by a PEG post-insertion approach. To improve uptake and tissue retention, we chose a modified short peptide ligand harbouring an LDV motif with high affinity towards the very late antigen-4 (VLA-4) that is widely expressed across haematopoietic cells. Decoration of the LNPs with the LDV resulted in an enhanced uptake in t(8;21) cell lines within the first 8 hours. While undecorated LNPs were hardly taken up by patient-derived material, the decoration with LDV resulted in an efficient uptake and a concomitant knockdown of RUNX1/ETO transcript and protein after a single dose. Sequential treatment leads to longer-lasting reduction of RUNX1/ETO transcript and associated target genes (LAPTM5, CCND2, C/EBPA and ANGPT1) and loss of RUNX1/ETO protein. Combined multiparameter flow analysis and scRNAseq of patient cells show that RUNX1/ETO depletion causes massive shifts in cell populations and an almost complete loss of immature populations (CD34+ CD38- CD117+ CD45RA+) associated with a gain in mature (CD15+) populations, indicating a release from the differentiation block caused by the fusion protein. Gene Set Enrichment Analysis (GSEA) of scRNAseq data confirm the role of RUNX1/ETO in promoting stemness and cell cycle progression and highlight a key function of this fusion protein in coordinating metabolism in support of leukaemic propagation. Currently, we are performing in vivo biodistribution and efficacy studies using orthotopic mouse models. Summary/Conclusion: Decoration of LNPs with an LDV motif enhances uptake of fusion transcript-specific siRNAs and enables efficacious knockdown of RUNX1/ETO in patient-derived cells. The biologic consequences validate RUNX1/ETO as a highly promising and leukaemia-specific target and highlight the LNP-mediated delivery of siRNAs as a promising new approach for the treatment of fusion gene-driven leukaemias. P466: AMPLIFIED EPOR/JAK2 GENES DEFINE A UNIQUE SUBTYPE OF ACUTE ERYTHROID LEUKEMIA J. Takeda1,*, K. Yoshida1, M. Nakagawa1, Y. Nannya1, A. Yoda1, R. Saiki1, Y. Ochi1, L. Zhao2, R. Okuda1, X. Qi1, T. Mori1, A. Kon1, K. Chiba3, H. Tanaka4, Y. Shiraishi3, M.-C. Kuo5, C. Kerr6, Y. Nagata6, D. Morishita7, N. Hiramoto8, A. Hangaishi9, H. Nakazawa10, K. Ishiyama11, S. Miyano4, S. Chiba12, Y. Miyazaki13, T. Kitano14, K. Usuki9, N. Sezaki15, H. Tsurumi16, S. Miyawaki17, J. Maciejewski6, T. Ishikawa8, K. Ohyashiki18, A. Ganser19, M. Heuser19, F. Thol19, L.-Y. Shih5, A. Takaori−Kondo20, H. Makishima1, S. Ogawa1 1Department of Pathology and Tumor Biology; 2Institute for the Advanced Study of Human Biology, Kyoto University, Kyoto; 3Division of Genome Analysis Platform Development, National Cancer Center Research Institute; 4M&D Data Science Center, Tokyo Medical and Dental University, Tokyo, Japan; 5Division of Hematology−Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan; 6Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, United States of America; 7Chordia Therapeutics Inc., Kanagawa; 8Department of Hematology, Kobe City Medical Center General Hospital, Kobe; 9Department of Hematology, NTT Medical Centre Tokyo, Tokyo; 10Department of Hematology, Shinshu University Hospital, Matsumoto; 11Department of Hematology, Kanazawa University, Kanazawa; 12Department of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba; 13Department of Hematology, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki; 14Department of Hematology, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka; 15Department of Hematology, Chugoku Central Hospital, Hiroshima; 16Department of Hematology, Gifu University, Gifu; 17Division of Hematology, Tokyo Metropolitan Ohtsuka Hospital; 18Department of Hematology, Tokyo Medical University, Tokyo, Japan; 19Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; 20Department of Hematology / Oncology, Kyoto University, Kyoto, Japan Background: Acute erythroid leukemia (AEL) is a rare subtype of acute myeloid leukemia (AML) characterized by erythroid predominant proliferation and classified into two subtypes with pure erythroid (PEL) and erythroid/myeloid (EML) phenotypes based on the degree of erythroid hyperplasia. Despite an intensive mutational analysis, the mechanism of erythroid hyperplasia in AEL is still poorly understood and so are feasible therapeutic targets. Aims: To understand the mechanism of erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL. Methods: We analyzed a total of 124 adult AEL cases, where whole genome/exome sequencing of 35 cases were followed by targeted-capture sequencing in all cases. RNA sequencing was also performed in 23 cases. The mutational profile of AEL cases was compared to that of 409 cases with non-erythroid AML (non-AEL). Patient-derived xenograft (PDX) mouse models of AEL with JAK2 and/or EPOR amplification were established from 6 AEL patients with these abnormalities. These models were tested for their response to JAK1/2 inhibitor. Results: In accordance with a recent report, AEL cases were classified into 4 genomic groups (A-D), which are characterized by biallelic TP53 mutations and complex karyotype (Group-A), mutated NPM1 (Group-B) and STAG2 (Group-C), and other mutations in histone modifiers and transcription factors (Group-D). In particular, all but one PEL cases belonged to Group-A. Also found in non-AEL cases, these group-defining lesions in AEL were uniquely associated with focal gains/amplifications of EPOR, JAK2, and/or ERG/ETS2 loci (Group-A), PTPN11 mutations (Group-B), and KMT2A-PTD (Group-C), which might be responsible for the AEL phenotype. Highly enriched in PEL cases (7/13), EPOR/JAK2 focal gains/amplifications were implicated in their extreme erythroid hyperplasia and associated with particularly poor prognosis, even compared to other Group-A cases, who had shorter survival than those in other groups. As expected, JAK2/EPOR-amplified cases showed upregulated STAT5 signaling compared to non-AEL cases, which however, was also observed in other AEL cases, suggesting that upregulated STAT5 signaling is a hallmark of AEL. Based on these findings, we tested the effect of JAK2 inhibition on cell growth of EPOR/JAK2-amplified AEL cells in in vitro culture and xenograft model. Of interest, ruxolitinib-mediated JAK2 inhibition resulted in significantly suppressed in vitro cell growth of EPOR/JAK2-amplified AEL cells and prolonged overall survival in 4 PDX models with phospho-STAT5 downregulation, although other 2 models were resistant to JAK2 inhibition with persistent STAT5 activation. Summary/Conclusion: AEL is a heterogenous subgroups of AML characterized in common by upregulated JAK/STAT5 signaling. Gains/amplifications of JAK2/EPOR are frequent in TP53-mutated cases, particularly those with the PEL phenotype, and could be exploited as potential therapeutic targets using JAK2 inhibitors. P467: FUNCTIONAL CHARACTERIZATION OF ABERRANT GATA1 PROTEIN COMPLEXES IN NORMAL AND MALIGNANT HUMAN ERYTHROBLASTS S. Tauchmann1,*, F. Otzen Bagger2, T. Bock3, R. Sivalingam1, T. Eder4, E. Heyes4, A. Fagnan5, M. von Lindern6, T. Mercher5, F. Grebien4, J. Schwaller1 1Childhood Leukemia, University of Basel, University Children`s Hospital Basel, Department Biomedicine, Basel, Switzerland; 2University of Copenhagen, Center of Genomic Medicine, Copenhagen, Denmark; 3Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland; 4Institute for Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria; 5INSERM U1170, Equipe Labellisée Ligue Contre le Cancer, Institut Gustave Roussy, Université Paris-Saclay, Université de Paris, Paris, France; 6Department Hematopoiesis, Sanquin Research, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands Background: Terminal differentiation of erythroid progenitor cells is controlled by the master transcription factor GATA1 acting in activating and repressive protein complexes. In acute erythroid leukemia (AEL), cell differentiation is impaired leading to accumulation of immature erythroblasts. Previous work in mouse models and primary human cells suggested impaired function of abundantly expressed GATA1 protein in AEL cells. Interestingly, GATA1 overexpression induced or enhanced terminal erythroid differentiation of the human AEL cell line K562 or immortalised HUDEP2 erythroblasts, respectively. Aims: We hypothesize that impaired terminal erythroid differentiation in AEL might be the consequence of aberrant GATA1 protein interactions. Methods: We compared the proteomes of three human AEL cell lines (F36P, K562, KMOE2) and primary AEL patient cells with HUDEP2 cells and primary human erythroblasts (hEBST) that were capable of in vitroterminal erythroid differentiation, using a tandem mass tag(TMT)-based approach (n=3/type). In parallel, we compared the GATA1 interactomes by immunoprecipitation (IP) followed by mass spectrometry (MS) (n=3/type). In addition, we performed a targeted CRISPR screen in Cas9-expressing K562 cells in which we monitored growth kinetics upon mutational disruption of candidate genes and simultaneously assessed changes in erythroid differentiation by measuring CD71 and CD235a surface markers. Results: Unsupervised hierarchical clustering displayed a clear separation of AEL proteomes from those of normal erythroblasts with 386 proteins higher expressed in the first and 623 proteins more abundant in the latter group (logFC≥2;q<0.05). GATA1-IP-MS analysis of nuclear lysates from all six cell types identified 1616 proteins, of which 126 were differentially associated with GATA1 depending on cell state. 54 proteins preferably interacted with GATA1 in the AEL group, whereas 72 proteins were more enriched in “normal erythroblasts” (q<0.5). GATA1 interactomes in hEBST and HUDEP2 cells highly overlapped and were more similar to GATA1 partners identified in F36P and KMOE, than those of K562 and primary AEL cells. Integrative analysis revealed 118 proteins that were differentially expressed and/or precipitated by GATA1-IP, of which 49 were enriched in malignant and 69 proteins in normal erythroblasts (q<0.5). To identify if GATA1-associated proteins are involved in erythroid differentiation we functionally characterized them in a CRISPR screen in K562 cells. 4 patterns were observed: inactivation of cluster 1 genes such as ZEB2 or IKZF1 did not change cell differentiation or growth, loss of cluster 2 genes like RPS21 or RANGAP1 did not change differentiation, but impaired cell growth/survival. Disruption of cluster 3 genes like SND1, or EIF5A resulted in an increase in CD71 and/or CD235a expression without significant changes in cell growth, and deletion of cluster 4 genes including SMC1A or ATP1A1 caused significant signs of differentiation together with cell depletion over time. Overall, knockout of 41 genes caused significantly increased CD71 and/or CD235a surface expression, indicating that these GATA1-interacting proteins affect erythroid differentiation. Summary/Conclusion: Our work suggests that impaired erythroid differentiation of AEL cells involves aberrant GATA1 protein complexes. Ongoing gain- and loss of function validation experiments of selected candidate genes and chromatin assays will identify novel regulators of terminal differentiation in normal and malignant erythropoiesis. P468: MRD AS A BIOMARKER FOR RESPONSE TO DONOR LYMPHOCYTE INFUSION AFTER AL-LOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA K. Teich1,*, M. Stadler1, R. Gabdoulline1, C. Wienecke1, B. Heida1, P. Klement1, K. Büttner1, L. Venturini1, M. Wichmann1, W. Puppe2, C. Schultze-Florey1, G. Beutel1, M. Eder1, A. Ganser1, M. Heuser1, F. Thol1 1Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation; 2Department of Virology, Hannover Medical School, Hannover, Germany Background: Donor lymphocyte infusions (DLIs) can be employed for acute myeloid leukemia (AML) patients as therapeutic or prophylactic treatment after allogeneic hematopoietic cell transplantation (alloHCT). Application of DLI after alloHCT may induce graft-versus-host disease (GVHD). Therefore, biomarkers that predict the effect of DLIs may be useful to guide DLI use. Molecular measurable residual disease (MRD) is prognostic for the response to standard chemotherapy as well as for targeted therapies. Aims: To evaluate MRD as a prognostic marker for DLIs after alloHCT. Methods: Patients with AML or myelodysplastic syndrome (MDS) aged ≥18 years undergoing DLI at Hannover Medical School between 1998 and 2018 with available PB and/or BM DNA samples were included. Patients were excluded if they had no detectable mutations at diagnosis, had a second alloHCT in the observed time or if MDS patients had <10% bone marrow blasts at diagnosis. DNA libraries were prepared using the TruSight Myeloid Panel or Nextera Flex for enrichment (Illumina) and sequenced on a MiSeq device. Error-corrected sequencing of patient specific mutations was performed 30 days (Follow Up 30 days, FU30) and 90 days (FU90) after the first DLI. All mutations detected at diagnosis were used for MRD monitoring. Marker positivity was defined as variant allele frequency above the limit of detection as not all patients were in complete remission (CR), in CR with incomplete hematological recovery (CRi) or in morphologic leukemia free state (MLFS). Results: The median time from alloHCT to DLI was 11.3 months (range 3.1 to 74.4 months). Of 78 patients, 24 patients were marker negative and 54 patients were marker positive before DLI (of the marker positive patients 23 were in CR/CRi/MLFS). As expected, marker negative patients before DLI had a significantly better overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) than marker positive patients. From the 54 marker positive patients before DLI, 27 patients were in CR/CRi at FU90, 22 patients did not reach CR/CRi and five patients died before FU90. OS, EFS and RFS were significantly improved in patients that were in CR/CRi at FU90. CR/CRi status at FU90 correlated strongly with remission status before DLI (p<0.001). Next, the prognostic effect of MRD in CR/CRi patients at FU90 (n=27) was analyzed. 18 patients reached MRD negativity at FU90 and nine patients stayed MRD positive. Baseline characteristics such as patient sex, cytogenetic risk group, age at diagnosis, the median time between alloHCT and DLI or use of prophylactic vs. therapeutic DLIs were similar between MRD negative and positive patients. Treatment intensity until FU90 was similar between the two groups. Remission status before DLI was not associated with MRD negativity at FU90 (p=0.782). There was no significant difference in OS (p=0.593), EFS (p=0.229) and RFS (p=0.226) between FU90 MRD negative and positive patients. Exclusion of DTA (DNMT3A, TET2, ASXL1) mutations as MRD markers affected the number of patients in CR/CRi at FU90 (20 patients MRD negative and five patients MRD positive), while patient characteristics and outcome did not differ between the two groups. Summary/Conclusion: Patients that achieve CR/CRi until FU90 after DLIs have a significantly better outcome than the remaining patients. However, the MRD status in patients in CR/CRi at FU90 had no prognostic effect in the patient cohort investigated here. P469: CHEMORESISTANCE IN ACUTE MYELOID LEUKEMIA: A SINGLE-CELL RNA SEQUENCING APPROACH C.-L. Teng1,*, P.-L. Cheng1, T.-H. Hsiao2, C.-H. Chen3 1Division of Hematology/Medical Oncology; 2Department of Medical Research, Taichung Veterans General Hospital, Taichung; 3National Institute of Cancer Research, National Health Research Institutes, Zhunan, Taiwan Background: Our previous study demonstrated that myc, mitochondrial oxidative phosphorylation, mTOR, and stemness were independently responsible for chemoresistance in AML. Aims: The current study aimed to further identify the potential mechanisms of chemoresistance of the “7 + 3” induction in AML using a single-cell RNA sequencing (scRNA-seq) approach. Methods: Thirteen untreated de novo AML patients were enrolled and stratified into the complete remission (CR) (n=8) and the non-CR (n=5) groups. We used scRNA-seq to analyze the genetic profiles of 28950 AML cells from these 13 patients. A previously published bulk RNA-seq dataset validated our results. Results: Chemoresistant AML cells had more premature accumulation in early hematopoiesis. The hematopoietic stem cell-like cells from the non-CR group expressed more leukemic stem cell markers (CD9, CD82, IL3RA and, IL1RAP) than those from the CR group. Besides, chemoresistant progenitor cells had impaired myeloid differentiation due to early hematopoiesis arrest. Importantly, the AML cells analyzed by the scRNA-seq and bulk RNA-seq harbored comparable myeloid lineage cell fraction, which internally validated our results. Using the TCGA database, our analysis demonstrated that AML patients with a higher expression of chemoresistant genetic markers (IL3RA and IL1RAP) had a worse overall survival (p < 0.01 for IL3RA; p < 0.05 for IL1RAP). Image: Summary/Conclusion: The AML cells responsive and resistant to the “7 + 3” induction had diverse cell origins according to specific genetic biomarkers using a scRNA-seq approach. Besides, hematopoiesis arrest occurred earlier in chemoresistant AML cells. This result provided an insight into a more understanding of chemoresistance in AML. P470: NGS-BASED MINIMAL RESIDUAL DISEASE DETECTION IN PERIPHERAL BLOOD SHOWS GOOD PROGNOSTIC VALUE FOR OS AND EFS IN PATIENTS WITH ACUTE MYELOID LEUKEMIA RECRUITED IN THE UNIFY TRIAL C. Thiede1,2,*, C. Schuster2, C. Krippendorf2, G. Koenen3, T. Medts3, P. Marques Ramos3, L. Gou4, P. Montesinos5, W. Fiedler6, R. Müller7, J. Krauter8, S. Sica9, J. Westermann10, M. Levis11, R. Stone12, J. Sierra13, H. Döhner14 1Medizinische Klinik und Poliklinik, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden; 2AgenDix, Gesellschaft für angewandte molekulare Diagnostik mbH, Dresden, Germany; 3Novartis Pharma AG, Basel, Switzerland; 4Novartis Pharmaceuticals Corporation, East Hanover, NJ, United States of America; 5Hospital Universitario i Politecnico La Fe Valencia, Valencia, Spain; 6Med. Klinik, Universitätsklinikum Hamburg Eppendorf, Hamburg, Germany; 7Klinik für Medizinische Onkologie und Hämatologie, Universitäts Spital Zürich, Zürich, Switzerland; 8Städtisches Klinikum Braunschweig, Braunschweig, Germany; 9Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma, Italy; 10Charité University Medical Center, Berlin, Germany; 11The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD; 12Dana-Farber Cancer Institute, Boston, United States of America; 13Hospital Santa Creu i Sant Pau, Universitat Autonoma of Barcelona, Jose Carreras Leukemia Research Institute, Barcelona, Spain; 14Universitätsklinikum Ulm, Ulm, Germany Background: Assessment of measurable residual disease (MRD) to evaluate the depth of remission at the time of achieving morphological complete remission (CR) or CR with incomplete blood count recovery (CRi) by multiparameter flow cytometry (MFC) or quantitative polymerase chain reaction (qPCR) has been shown to be predictive of outcome in patients (pts) with acute myeloid leukemia (AML), and is recommended according to European LeukemiaNet guidelines. However, accurate MRD assessment is still limited by lack of suitable markers in all pts (qPCR) and/or limited specificity/sensitivity (MFC). Next generation sequencing (NGS) holds promise to overcome some of these limitations and allows versatile and sensitive MRD assessment in almost all AML pts. However, data on NGS-MRD in this setting are still limited. Aims: To further extend the experience with NGS-based MRD assessment, we prospectively collected samples to explore prognostic implications of MRD as detected by NGS at CR/CRi in the context of a randomized, placebo-controlled phase 3 study (UNIFY; NCT03512197), investigating midostaurin added to conventional 7 + 3 based chemotherapy in FLT3wt pts. Following the recommendation by an independent DMC, the UNIFY study was stopped in Sep 2019 due to futility. The current subset analysis was performed to investigate the impact of NGS-MRD results on long-term outcomes. Methods: Peripheral blood mononuclear cell (PBMC) or bone marrow mononuclear cell (BMMC) samples were collected from pts treated in the UNIFY study. NGS-MRD was performed on genomic DNA using targeted NGS-error-controlled sequencing method (Ion-Torrent S5XL) with a sensitivity between 0.1 and 0.001%. Targets for MRD detection were identified by NGS of BM samples taken at diagnosis (Archer Myeloid Panel) covering 74 genes frequently mutated in myeloid disease. MRD positivity was defined as any mutation call >0.1%, excluding mutations in genes associated with clonal hematopoiesis, ie., DNMT3A, TET2 and ASXL1. Results: Total 731 BMMC (from 305 out of the total 501 pts) and 564 PBMC samples (from 204 pts) were collected and analyzed, of which 467 were paired (from 175 pts). The 10 most frequently used genes for MRD monitoring were NRAS, IDH2, RUNX1, SRSF2, TP53, PTPN11, IDH1, BCOR, CEBPA and GATA2. Analyses in paired samples showed numerically higher MRD+ rates in BMMC vs. PBMC (57% vs 46% MRD+ at the end of induction, respectively). No differences in MRD rates were seen between treatment arms, and data was pooled for analysis. A high correlation of variant allele frequency (Pearson’s r≥0.84) and a high level of concordance in MRD calls were seen between BMMC and PBMC (91% overall percent agreement). For pts who fulfilled the criteria of a successful induction treatment, detection of MRD in either BMMC or PBMC in CR/CRi pts at the end of induction was associated with significantly lower event free survival (EFS) and overall survival (OS) compared to MRD− status (1-year EFS of 26% vs 72% in BMMC and 34% vs 71% in PBMC; 1-year OS of 73% vs 93% in BMMC and 67% vs 94% in PBMC) (Figure). Preliminary multivariate analysis, controlling for age and sex, confirmed the independent prognostic value of NGS-MRD for OS. Additional analysis is ongoing. Image: Summary/Conclusion: Taken together, these data indicate that MRD by NGS at the time of CR/CRi in both BMMC and PBMC, is prognostic for EFS and OS. Although the results should be interpreted with caution due to the premature termination of the study and limited follow-up, these further support the use of molecular MRD assessment to predict outcome in pts with AML. P471: ERYTHROID/MEGAKARYOCYTIC DIFFERENTIATION BIAS IN BONE MARROW OF AML/MDS PATIENTS AFTER DECITABINE TREATMENT F. Tiso1,*, A. O. de Graaf1, L. I. Kroeze2, B. A. van der Reijden1, S. M. C. Langemeijer3, K. M. Hebeda2, J. H. Jansen1 1Department of Laboratory Medicine, Laboratory of Hematology; 2Department of Pathology; 3Department of Hematology, Radboudumc, Nijmegen, Netherlands Background: The hypomethylating agent Decitabine is used mainly in AML patients who are not eligible for standard induction chemotherapy or as a bridge therapy before a stem cell transplantation. Decitabine is an analogue of cytidine deoxynucleoside and it is incorporated into the DNA as decitabine triphosphate. In the DNA decitabine triphosphate inhibits the methylation of the DNA, leading to the reactivation of tumour suppressor genes which have been epigenetically silenced, induces cell differentiation and also apoptosis. Aims: We studied early signs of morphological changes in bone marrow biopsies (BMB) of AML/MDS patients after treatment with Decitabine and correlated these with the mutational background. Methods: We investigated 45 patients with AML/MDS who were treated with Decitabine. Bone marrow biopsies were morphologically examined, and we assessed the mutational status of the BMB. Sequencing was performed using a panel of 27 commonly mutated genes in myeloid malignancies, using a targeted error corrected approach. Results: We observed a striking erythroid/megakaryocytic differentiation bias after treatment in 16/45 patients, whereas the other patient group (n=29) showed predominant myeloid differentiation. Patients developing this erythroid/megakaryocytic bias showed a trend towards a higher frequency of mutations in genes encoding for spliceosome components (U2AF1, SF3B1 and SRSF2), in particular U2AF1 was found significantly (p-value=0.05) more mutated. Furthermore, this group carried a significantly (p-value=0.03) lower number of mutations in genes encoding for proteins involved in signalling and kinase pathways (ETNK1, FLT3, CBL, JAK2, KIT, KRAS, MPL, BRAF, NRAS) compared to the group with predominant myeloid differentiation. Summary/Conclusion: A bias towards erythroid/megakaryocytic differentiation was observed in the bone marrow of one third of the patients after treatment with Decitabine. This bias was over-represented in patients with mutations in spliceosome component encoding genes, particularly U2AF1. Patients with myeloid predominant Decitabine response harboured an increased number of mutations in signalling and kinase proteins encoding genes. P472: THE FLT3-ITD RECEPTOR CAN PROMOTE PROTEIN FOLDING IN THE ENDOPLASMIC RETICULUM THROUGH THE OXIDOREDUCTASE ERO1 M. Turos Cabal1,*, A. M. Sánchez Sánchez1, N. Puente Moncada1, C. Rodriguez1, F. Herrera2, V. Martin1 1Morphology and Cellular Biology, University of Oviedo, Oviedo, Spain; 2Department of Chemistry and Biochemistry, University of Lisbon, Lisbon, Portugal Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy and the most common myeloid tumour in adults. Internal tandem duplications (ITD) in FLT3 receptor appear in 30% of cases and is associated with poor disease outcome. This receptor is synthesized and processed in the endoplasmic reticulum (ER) before being transported to the plasma membrane, where it is activated by ligand binding. Upon activation, it regulates cell differentiation, proliferation, and survival. Mutations in FLT3, such as ITD mutations, not only constitutively activate this receptor, but also prevent its correct processing in the endoplasmic reticulum. Therefore, it is mostly retained in this organelle. Aims: We decided to study if the aberrant accumulation of FLT3-ITD affects the functioning of the ER. Methods: Experiments were performed on wild-type and FLT3-ITD AML cell lines, as well as on stably transfected FLT3 and FLT3-ITD Ba/F3 cell lines. Protein aggregation, reactive oxygen species (ROS) and glutathione (GSH) levels were determined by Thioflavin T, DCFH-DA and Thioltracker Violet fluorescence, respectively. Protein detection was performed by Western Blot. Cell viability and death was analysed by cell count and Trypan Blue exclusion. Results: Protein folding in the ER generates reactive oxygen species (ROS). Antioxidant molecules such as GSH are needed to maintain redox homeostasis. We found that FLT3-ITD cell lines have lower levels of protein aggregates, as well as ROS and GSH. This data could suggest that mutant cell lines have more efficient protein folding mechanisms. In fact, among the ER proteins that are involved in the protein folding process, we found the oxidoreductase ERO1 to be increased in FLT3-ITD cell lines. Supporting our findings, we saw that FLT3-ITD cell lines were less sensitive to protein folding blocking agents, DTT and 2-mercaptoethanol. This was reflected by a slight decrease in cell proliferation compared to wild-type cell lines. We confirmed that the decrease in cell viability in FLT3-ITD cell lines was not accompanied by cell death, as opposed to wild-type cell lines. Under ER stress conditions, such as an accumulation of misfolded proteins in the ER, the unfolded protein response (UPR) is activated. We decided to study if there were differences in the basal levels of UPR by western blot. We found FLT3-ITD cell lines to have lower levels of UPR, reflected by lower protein expression of the active forms of PERK and IRE (p-PERK and p-IRE respectively), together with the protein BiP, the major chaperone found in the ER and binds to misfolded proteins. Altogether, our data suggest that FLT3-ITD cell lines have less ER stress, reflected by lower levels of chaperones and UPR proteins, possibly because they rely on more efficient protein folding mechanisms. To confirm the importance of protein folding process in acute myeloid leukemia, we tested the effect of ERO1 inhibitor (EN460), and found it to have a greater impact in FLT3-ITD cell lines. In order to determine a possible relation between the ITD mutation in the FLT3 receptor and ERO1, we used stably transfected Ba/F3 cell lines carrying the normal or mutated FLT3 receptor. We found ERO1 to be overexpressed on FLT3-ITD Ba/F3 cell lines. Summary/Conclusion: Current AML treatments are mainly based on the administration of FLT3 inhibitors, with limited success. Our data suggest that FLT3-ITD cell lines, with greater resistance to treatments, have more efficient protein folding mechanisms. This process should be studied in more detail to determine its potential as a therapeutic target for AML treatment. P473: DEVELOPMENT OF POTENTIAL BIOMARKERS FOR IRAK4 INHIBITOR EMAVUSERTIB IN HUMAN ACUTE MYELOID LEUKEMIA A. Ugolkov1,*, M. Pilichowska2, C.-C. Li1, R. C. Hok1, M. Samson1, M. Lane1, R. Von Roemeling1, R. Martell1 1Curis, Inc., Lexington, MA; 2Tufts Medical Center, Boston, MA, United States of America Background: Acute myeloid leukemia (AML), the second most common form of leukemia in adults, remains a highly fatal disease. Interleukin-1 receptor-associated kinase 4 (IRAK4) has been demonstrated as a potential therapeutic target in human AML. IRAK4-mediated activation of NF-kappaB signaling pathway could play a critical role in NF-kappaB-regulated survival and chemoresistance of cancer cells. The results of ongoing Phase 1 study demonstrated clinical activity of IRAK4 inhibitor emavusertib (CA-4948) in patients with relapsed/refractory AML and high-risk MDS. To support the development of companion diagnostic for emavusertib, we developed an immunohistochemical (IHC) assay and explored expression of potential biomarkers in bone marrow (BM) samples obtained from AML patients. Aims: The objective of the present study was to analyze expression of IRAK4, NF-kappaB p-p50 and NF-kappaB p-p65 by IHC staining in human AML with further evaluation of these molecules as potential biomarkers for emavusertib therapy in AML patients. Methods: We used IHC staining and immunoblotting to determine the expression of IRAK4, NF-kappaB p-p50 S337 and NF-kappaB p-p65 S536 proteins in human leukemia cell lines and clinical AML samples. Exploratory biomarker evaluations were performed using serial sections of formalin-fixed paraffin-embedded BM clot samples obtained from 19 AML patients. Eight BM AML samples were purchased from Analytical Biological Services and 11 BM samples were obtained at the screening from evaluable patients with relapsed/refractory AML enrolled in CA-4948-102 clinical trial. Results: Using IHC staining, we found IRAK4 nuclear expression in blasts in 9/19 AML cases. To the best of our knowledge, this is the first report showing nuclear accumulation of IRAK4 in cancer cells. In support of our findings in clinical AML samples, we detected IRAK4 protein expression in nuclear lysates prepared from leukemia cell lines THP-1, HL-60 and K562. Using AML BM samples, we found that IRAK4 nuclear expression in blasts was significantly correlated with activation of NF-kappaB as determined by nuclear accumulation of NF-kappaB p-p50 and p-p65 in 9/19 cases. Cytoplasmic expression of IRAK4 was detected in 2/19 cases whereas IRAK4, NF-kappaB p-p50 and p-p65 expression was not detectable in 8/19 cases of AML. Clinical response data will be presented in the context of these novel findings. Summary/Conclusion: Our findings uncovered a previously unknown nuclear expression of IRAK4 in leukemia cells. Our results demonstrated co-expression of nuclear IRAK4, NF-kappaB p-p50 and p-p65 in blasts suggesting a potential novel mode of interaction between IRAK4 and NF-kappaB in human AML. Although the role of nuclear IRAK4 in leukemia cells remains to be investigated, our preliminary findings revealed new perspectives into emavusertib treatment stratification and demonstrate the possibility of discovering novel biomarkers through IHC analysis of clinical samples. Additional accrual is ongoing to better define emavusertib biomarkers in AML patients. P474: PIEZO1 AND THE THERAPEUTIC POTENTIAL OF MECHANORECEPTORS IN ACUTE MYELOID LEUKAEMIA M. Velasco Estevez1,*, A. Fernandez1, A. Otero-Sobrino1, P. Aguilar-Garrido1, M. A. Navarro-Aguadero1, R. Sanchez2, A. Gimenez Sanchez2, V. Garrido Garcia2, L. Carneros Blanco2, J. Martinez-Lopez2, M. Gallardo1 1H12O-CNIO Haematological Malignancies Group, Clinical Research Unit, Centro Nacional de Investigaciones Oncologicas (CNIO); 2Grupo de Hematología Traslacional, Hospital 12 de Octubre de Madrid, Madrid, Spain Background: Mechanotransduction is the process through which cells sense the mechanical input in and out the cell, and translate it into biochemical signals adapt and respond. In the past few years, it has been observed the important contribution of mechanical input in cell differentiation, fate, stemness, senescence and cancer, amongst others. Piezo1 is a mechanoreceptor discovered in 2010 and constitutes a cation channel activated through stretch and shear flux leading to a calcium influx in the cell that activates a myriad of different pathways. Interestingly, despite being a mechanoreceptor, Piezo1 can also be activated through a small molecule, Yoda-1, and blocked by a peptide, GsMTx4, allowing the pharmacological study of this receptor. Mutations in PIEZO1 lead to pathologies as Xerocitosis hereditaria and it has been linked to erythroid formation. However, the expression and role of Piezo1 in haematological malignancies is yet to be explored Aims: We aim to analyse the expression of Piezo1 in Acute Myeloid Leukaemia (AML) and healthy donors, and investigate the putative role of Piezo1 in cell differentiation and fate as well as its therapeutic potential for AML. Methods: Mouse embryonic stem cells (mESC) were cultured and treated with Yoda-1 and GsMTx4. PIEZO1 expression in AML and healthy donors was analysed through RNAseq databases (Bloodspot, GEPIA2 and MILE study) and corroborated by ICC staining of human samples. Lastly, mononuclear cells in viability from AML or healthy donors were cultured in vitro for dose-response curves to Yoda-1 and GsMTx4 and in methylcellulose to analyse the phenotype and proliferation under those treatments. Both imaging and immunophenotyping with flow cytometry were performed in those samples. Results: When analysing the expression of PIEZO1 in AML, it was seen that Piezo1 was higher in AML compared to healthy donors, and interestingly, there was a much higher expression in AML inv(16), same chromosome as PIEZO1 is (Figure 1A). We observed that activation of Piezo1 in mESCs led to a higher expression of stemness marker Oct3/4 (Figure 1B), suggesting that it is involved in the stemness state of haematopoietic stem cells. Furthermore, we observed a dramatic effect of both Yoda-1 and GsMTx4 in leukaemia haematopoietic cells (LSCs) from AML patients grown in methylcellulose, where GsMTx4 caused an increment in the cell number and shifted the cell fate towards the erythroid lineage, while Yoda-1 decreased the number of cells and ablated the erythroid differentiation. Interestingly, such effects were barely significant in cells from healthy donors, showing a wide window for treatment (Figure 1C-D). Image: Summary/Conclusion: Our study shows that Piezo1 changes with cell differentiation and it could be a mechanism through which stem cells direct their fate. Furthermore, Piezo1 is highly expressed in AML and its blockage leads to a shift towards the erythroid differentiation while its activation blocks cell proliferation and differentiation, which such effects being much more significant in AML samples than healthy donors, suggesting a potential as therapeutic target. This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295), ISCIII Miguel Servet (CP19/00140) and IF-Marie Sklodowska Curie Actions grant (MAtChinG – 101027864). P475: CONTROL LEUKEMIA BY INDUCING ANTI-CANCER IMMUNE REACTIVITY IN VIVO? POTENTIAL OF A DC-TRIGGERED MECHANISM G. Filippini Velazquez1,*, D. Amberger2, L. Klauer2, E. Rackel2, M. Atzler2, S. Ugur2, C. Plett2, A. Rabe3, C. Kugler2, A. Rank1, M. Inngjerdingen3, T. Baudrexler2, B. Eiz-Vesper4, C. Schmid1, H. Schmetzer2 1Department of Hematology and Oncology, Section for Stem Cell Transplantation, Augsburg University Hospital, Augsburg; 2Medical Department III, Department for Hematopoietic Cell Transplantation, Munich University Hospital, Munich, Germany; 3Institute of Clinical Medicine, Department of Immunology, University of Oslo, Oslo, Norway; 4Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, Hannover, Germany Background: There are virtually no treatment options for therapy-refractory or relapsed AML/MDS and high rates of relapse in successfully treated patients. The combination of the (clinically approved) immune-modulatory compounds GM-CSF+ Prostaglandine (PGE)1, the combination referred to as KIT-M converts myeloid blasts into dendritic cells of leukemic origin (DCleu). After stimulation with DCleu, antileukemic (T)cells are activated. Aims: Kit-M treatment may be an attractive tool for immunotherapy in myeloid leukemia. Methods: Generation of DC from leukemic whole blood (WB) samples. Mixed lymphocyte culture (MLC) followed by functional antileukemic assays. Results: 1. ex vivo : Treatment of 65 leukemic WB samples with KIT-M does not induce blast proliferation, but triggers generation of mature DC/DCleu and reduces tolerogenic DC. Kit treated WB activates the adaptive and innate immune system after MLC (Tcell proliferation, antitumor-supportive Tcells (TCRgd,Tb7), memory cells (Tcm,Tb7cm) and downregulates immunosuppressive Tcells (Treg4 and 8). Moreover leukemia-specific (interferon g (g) and/or degranulating (deg)) adaptive (g-degT4,T8,TCRgd,Tb7,Tcm) and innate cells (g-degNK,NKb7,CIKb7) are increased and regulatory cells (g-degTreg4) downregulated. In addition, blast lysis is increased vs control. Ex vivo achieved blast lysis correlates positively with frequencies of mature DC/DCleu, leukemia-specific T3,T4,T8,TCRgd,Tb7 and NK cells and negatively with Treg4 and 8. Blast lysis does not correlate with age, sex, ELN risktype, blast counts, or response to chemotherapy. 2. In vivo - rats: Kit-M treatment of 3 leukemically diseased (vs 3 control) rats (followed by sacrification after treatment) leads to reduced blasts and Tregs in blood and spleen and increased DCleu and memory-like Tcells. 3. In vivo - human: Kit-M therapy was offered to a 72 year old pancytopenic male as an individual salvage attempt (applied as continuous infusion), after discussion with the ethical commitee, the patient’s information about the experimental nature of the treatment and his written consent. The treatment was well tolerated and the patient improved clinically. Neutrophils in WBC increased from 10% to 50%, thrombocytes reached 100 G/l after 24 days. Immune monitoring showed a continuous increase of proliferating and non-naïve Tcells, NK, CIK- and NKT-, TH17 cells, Bmem-cells and DC in PB. The production of IFNg producing T-, CIK and NKT-cells was demonstrated, suggesting an in vivo production/activation of (potentially leukemia-specific) cells. Immune stimulatory effects decreased after discontinuation of therapy. After 4 weeks of treatment, the patient was discharged in good clinical condition. Unfortunately, at two weeks from discharge, AML progressed and the patient died few days later. Summary/Conclusion: Treatment of WB ex vivo with Kit-M leads to activation of adaptive and innate (leukemia-specific) immune reactive cells (and downregulated suppressive mechanisms) via a DC/DCleu triggered mechanism – resulting in significantly improved blast lysis compared to controls (independent of patients‘ risk classification, MHC, age or sex). In vivo treatment of leukemically diseased rats or humans was well tolerated, led to an increase of platelets and granulocytes and stable (low) blast counts in PB – probably mediated by a (leukemia specifically) DC/DCleu activated immune system. A dose defining clinical trial in carefully selected patients to confirm clinical safety and underscore clinical efficacy is being prepared. P476: LOSS OF CHD4 IN ACUTE MYELOID LEUKAEMIA GIVES RISE TO INCREASED DNA DAMAGE REPAIR DEFECTS AND ALTERED CELL CYCLE PROGRESSION D. Venney1,*, Y. Atlasi2, A. Mohd-Sarip2, K. Mills1 1Haematology; 2Queens University Belfast, Belfast, United Kingdom Background: Acute Myeloid Leukaemias (AMLs) are an array of blood cell disorders arising from alterations within myeloid precursors. Cancer genome studies highlighted alterations within the nucleosome remodelling and deacetylation (NuRD) complex occur in over 20% of all cancers. The NuRD complex controls nucleosome assembly and chromatin accessibility; CHD4 is a core subunit of the NuRD which is involved in a multitude of functions including epigenetic regulation, cell cycle progression and DNA replication and repair. CHD4 is linked to PARP dependent DNA damage response (DDR) pathway by rapid but transient accumulation at sites of damage, preventing transcription which enables homologous recombination (HR). Aims: We aim to explore the function of CHD4-NuRD in AML using a CHD4 knockout (CHD4-/-) isogenic cell line models created using CRISPR/Cas9 to assess the mechanism of function of CHD4 within AML disease progression and evaluate if there is targetable therapeutic potential. Methods: CRISPR-Cas9 was used to generate isogenic CHD4-/- models in two representative AML cell lines. CHD4-/- models underwent functional studies; proliferation, cell cycle and protein expression assays were used to phenotype models before inducing DNA damage at a clinically relevant dose and reassessing cell response. A high throughput drug screen was performed to identify DNA damage compounds that showed significant sensitivity in CHD-/- models and validation was assessed using single compound treatments and co-culture assays. Results: Analysis of publicly available TCGA AML transcriptomic data set (N=161), found that AML patients (N=82) who express below median CHD4 levels have a poorer prognostic outcome suggesting a link between stalled transcription enabling HR-based DDR and patient survival (P=0.047). Analysis of the intermediate cytogenetic risk group showed patients with a low CHD4 expression had a worse outcome; which was comparable to the adverse risk group patients (P=0.032), indicating that loss of CHD4 may contribute to a more aggressive phenotype AML in this group. Phenotype analysis of CHD4-/- models showed DNA damage markers 53BP1 and PARP expression increases significantly, highlighting the role of CHD4 within the DDR. This increased DNA damage response was accompanied by reduced cellular proliferation in CHD4-/- cells. Accordingly, cell cycle analysis showed a decrease in cells entering the G2/M phase alongside an increase in cells in G1 and S phase suggesting a potential cell cycle checkpoint deficiency causing stunted proliferation in CHD4-/- cells. Of note, we did not observe a significant compensatory increase in CHD3-NuRD components expression in the absence of CHD4 suggesting there is no compensation for the loss of CHD4. We measured behaviour of CHD4-/- cells to stress-inducing conditions; in response to irradiation and chemotherapy drugs commonly used in AML. CHD4-/- cells display much stronger reduction in cellular proliferation post-2Gy irradiation compared to control while cell cycle analysis showed further cellular arrest in G2/M. Furthermore, a drug screen (160 DNA damaging compounds) showed an increased sensitivity of CHD4-/- cells to compounds targeting DNA/RNA synthesis pathways, including Tubercidin and Cytarabine, indicating that loss of CHD4 results in hyper-increase in damage. Summary/Conclusion: Our data has identified a role of CHD4 in AML progression mediated through loss of cell cycle progression control and an elevated response to DNA damage. Therefore, targeting cells with lower CHD4 expression with compounds targeting DNA/RNA synthesis may have an impact on patient outcome. P477: PHENOTYPICALLY-DEFINED STAGES OF LEUKEMIA ARREST PREDICT MAIN DRIVER MUTATIONS SUBGROUPS, AND OUTCOME IN ACUTE MYELOID LEUKEMIA F. Vergez1,*, L. Largeaud1, S. Bertoli2, M.-L. Nicolau-Travers1, J.-B. Rieu1, I. Vergnolle1, E. Saland1, A. Sarry2, S. Tavitian2, F. Huguet2, M. Picard2, J.-P. Vial3, N. Lechevalier3, A. Bidet3, P.-Y. Dumas4, A. Pigneux4, I. Luquet1, V. Mansat-De Mas1, E. Delabesse1, M. Carroll5, G. Danet-Desnoyers5, J.-E. Sarry6, C. Recher2 1Laboratory of Hematology; 2Department of Hematology, CHU Toulouse, Toulouse; 3Laboratory of Hematology; 4Department of Hematology, CHU Bordeaux, Bordeaux, France; 5Stem Cell and Xenograft Core, UPENN, Philadelphia, United States of America; 6UMR1037, INSERM, Toulouse, France Background: Normal blood cell maturation is organized according to a functional hierarchy at the top of which are multipotent hematopoietic stem cells (HSC). Immunophenotypically, human HSC are enriched in a population of Lin-, CD34+, CD38-, CD90+, CD45RA- cells. Upon differentiation, HSC give rise to multipotent progenitors (MPP), which retain the ability to produce all blood lineages but have lost their self-renewal capacity. The classical model of hematopoiesis postulates that MPP are then orientated toward either the lymphoid or myeloid lineage, developing into common myeloid progenitors (CMP) or lymphoid-primed multipotent progenitors (LMPP) which can still produce defined myeloid cell types. Along the myeloid pathway, CMP can differentiate into either granulocyte-monocyte progenitors (GMP) or megakaryocyte-erythroid progenitors (MEP). GMPs finally differentiate into granulocyte progenitors (GP) or monocyte progenitors (MP). Aims: In this study, we hypothesized that the stages of leukemic arrest (SLA) in myeloid maturation can be defined by flow cytometry by analogy with the stages of hematopoiesis and they correlate with the oncogenic events implicated in the mechanism of differentiation block. Methods: Six stages of leukemia differentiation-arrest categories based on CD34, CD117, CD13, CD33, MPO and HLA-DR expression by flow cytometry were identified in 2 independent fully annoted cohorts of 2087 and 1209 AML patients. Next-generation sequencing was performed in 409 AML patients. Results: HSC/MPP-like AMLs display low proliferation rate, inv(3) or RUNX1 mutations, and high leukemic stem cell frequency as well as poor outcome, whereas GMP-like AMLs have CEBPA mutations, RUNX1-RUNX1T1 or CBFB-MYH11 translocations, lower leukemic stem cell frequency, higher chemosensitivity and better outcome. NPM1 mutations correlate with most mature stages of leukemia arrest together with TET2 or IDH mutations in GP-like AML or with DNMT3A mutations in MP-like AML. Image: Summary/Conclusion: AML immunophenotyping can establish a new SLA classification that strongly correlates with cellular behaviour of the leukemic bulk, predicts main genetic subgroups early at diagnosis and outcome after intensive chemotherapy. Each SLA is defined by specific oncogenic events whose penetrance may be dependent on the differentiation stages of hematopoiesis and their gene expression. Identifying disrupted gene pathways specific for each SLA should therefore form the basis for targeted therapies aimed at inducing AML differentiation. P478: THE GERMLINE VARIANT GFI1-36N PROMOTES GENETIC INSTABILITY IN LEUKEMIC CELLS AND OPENS NEW THERAPEUTIC APPROACHES FOR ABOUT 15% OF ALL AML PATIENTS J. Vorwerk1,*, D. Frank1, K. Sun1, F. Neumannn2, M. Heuser3, D. Kunadt4, W. E. Berdel1, J.-H. Mikesch1, C. Schliemann1, G. Lenz1, A. K. Jayavelu5,6,7,8,9, C. Khandanpour1 1Department of Medicine A, University Hospital Münster; 2Fluorescence Microscopy Facility Münster, Institute of Medical Physics and Biophysics, University of Münster, Münster; 3Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover; 4Department of Internal Medicine I, University Hospital Dresden, Dresden; 5Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Munich; 6Clinical Cooperation Unit Pediatric Leukemia, German Cancer Research Center (DKFZ); 7Department of Pediatric Oncology, Hematology, and Immunology; 8Hopp Children’s Cancer Center Heidelberg (KiTZ); 9Molecular Medicine Partnership Unit, European Molecular Biology Laboratory (EMBL), Heidelberg University, Heidelberg, Germany Background: The zinc finger protein Growth Factor Independence 1 (GFI1) acts as a transcriptional repressor regulating differentiation of myeloid and lymphoid cells. In a single-nucleotide polymorphism (SNP) of GFI1, GFI1-36N, the amino acid serine (S) is replaced by asparagine (N) at position 36. GFI1-36N has a prevalence of 7% in healthy Caucasians and 15% in acute myeloid leukemia (AML) patients, hence predisposing to AML. We have shown previously that this is particularly since the GFI1-36N protein differs from the GFI1-36S variant regarding its ability to induce epigenetic changes leading to a derepression of oncogenes. Now we have evidence that GFI1 is implicated in DNA repair as AML blast cells of patients carrying the GFI1-36N SNP feature more frequently complex-aberrant karyotypes and more mutations leading to an inferior prognosis. Aims: Therefore, in this work, we explored whether presence of GFI1-36N alters DNA repair activity and whether this can be targeted therapeutically. Methods: We analyzed the association between GFI1-36N and chromosomal aberrations in human AML samples. As a murine model of human AML, we knocked in the human GFI1-36S or GFI1-36N variant into the murine Gfi1 gene locus and retrovirally expressed MLL-AF9 to induce AML. We then performed γ-H2AX immunofluorescence and flow cytometry to quantify the number of DNA double-strand breaks (DSBs), confocal immunofluorescence microscopy to detect DNA repair foci of key enzymes of non-homologous end joining (NHEJ) and homologous recombination (HR) as well as proteomics and immunoblotting to determine the level of DNA damage response enzymes and cell cycle kinases. Cell viability assay and colony-forming unit assay were used to examine the response of GFI1-36S and GFI1-36N leukemic cells to palbociclib treatment. Results: A complex aberrant karyotype was found twice as often in patients carrying the GFI1-36N SNP compared to GFI1-36S controls (38% vs. 20%, p ** = 0.0080). Murine GFI1-36N-MLL-AF9 cells showed 43 times more genetic and cytogenetic aberrations than GFI1-36S-MLL-AF9 controls (p *** = 0.0003). Upon exposure to irradiation, murine GFI1-36N leukemic cells featured more DSBs and delayed repair. Regarding the two main DSB repair pathways, we did not see a significant difference in the ability of GFI1-36N or GFI1-36S leukemic cells to perform NHEJ, but a reduced HR capacity in presence of GFI1-36N. On a molecular level, GFI1-36N was associated with less foci of the HR key enzyme RAD51, reduced protein levels of the DNA damage response enzymes CHK1 and CDK2, higher levels of the cell cycle propagating CDKs, and reduced levels of the CDK inhibiting CDKNs. As especially CDK4 and CDK6 protein levels were significantly increased in GFI1-36N leukemic cells, we subsequently treated malignant cells with the established CDK4/6 inhibitor palbociclib. We observed that GFI1-36N leukemic cells were more susceptible to this treatment, indicated by 49% less CFUs compared to GFI1-36S leukemic controls (p ** = 0.0063). Image: Summary/Conclusion: The findings suggest that the GFI1-36N SNP increases cell cycling and reduces DNA repair activity. This could promote emergence of chromosomal aberrations and hence contribute to genomic instability of leukemic cells (Figure 1). On a therapeutic level, GFI1-36N could possibly be a marker for a specific subset of AML patients that is sensitive to CDK4/6 inhibitors. P479: DNA METHYLATION PROFILING REFINES THE PROGNOSTIC CLASSIFICATION OF ACUTE MYELOID LEUKEMIA PATIENTS TREATED WITH INTENSIVE CHEMOTHERAPY S. Vosberg1,*, A. Ohnmacht2, C. Moser3, A. Arneth2, V. Jurinovic3, K. H. Metzeler4, M. C. Sauerland5, D. Görlich5, W. E. Berdel6, J. Braess7, S. Amler8, U. Krug9, W. Hiddemann3, K. Spiekermann3, C. C. Oakes10, M. P. Menden2, T. Herold3, P. A. Greif3 1Department of Internal Medicine, Medical University of Graz, Graz, Austria; 2Institute of Computational Biology, Helmholtz Zentrum Munich, Neuherberg; 3Department of Medicine III, University Hospital LMU Munich, Munich; 4Department of Hematology, Cellular Therapy, and Hemostaseology, University of Leipzig, Leipzig; 5Institute of Biostatistics and Clinical Research; 6Department of Medicine, Hematology and Oncology, University of Münster, Münster; 7Department of Oncology and Hematology, Hospital Barmherzige Brüder, Regensburg; 8Friedrich-Loeffler-Institut, Greifswald-Insel Riems; 9Department of Medicine III, Hospital Leverkusen, Leverkusen, Germany; 10Department of Internal Medicine, The Ohio State University, Columbus, United States of America Background: Multiple studies have shown that DNA methylation is frequently altered in acute myeloid leukemia (AML). Aims: Here we propose a prognostic classifier for the survival of AML patients that describes DNA Methylation-based Risk Assessment (“DMRA”), trained and validated using a total of 538 adult AML patients from two independent cohorts, all treated with intensive cytarabine-based chemotherapy with curative intent, and compared it to the current ELN 2017 classification. Methods: Patients from the first cohort were either treated within one of two consecutive clinical trials of the German AMLCG study group or documented within the AMLCG registry. DNA methylation profiles of 377 diagnostic AML samples were generated using Illumina Infinium MethylationEPIC BeadChip arrays, selected based on their chemotherapy regimen (i.e. 7 + 3, S-HAM, HAM(-HAM), or TAD-HAM). The clinical annotation includes overall survival (OS) and the ELN risk assessment. Our DMRA classifier was trained on the most variable CpG sites genome wide, weighted upon their relevance to OS based on ridge regression. Three prognostic subgroups (“low”, “medium” and “high” risk) were identified using cutoffs by optimizing the prediction error to avoid over- and underfitting. We validated our approach by 10-fold cross-validation within AMLCG data, ensuring that samples are either used for training or validation at the same time, as well as using an independent cohort of diagnostic AML samples, selected for cytarabine-based “standard chemotherapy” (n=161, BeatAML). Results: Overall, the three risk groups defined by DMRA reveal more distinct survival characteristics between each other as compared to between ELN risk groups (Figure 1A,B), with 3-year OS of 64% / 44% / 31% by ELN vs. 64% / 39% / 15% by DMRA. Of note, nearly half of patients were assigned to a different risk group using DMRA as compared to ELN (n=240/538, 45%, Figure 1C). Importantly, we were able to define more precise risk groups within ELN subgroups, with significant differences in OS. Within ELN favorable AML, 22% of patients (n=44/203) were classified with increased risk (3-year OS 70% vs. 44%, p=0.003 “low” vs. “medium”, log-rank test, Figure 1D). Within ELN intermediate AML, 33% of patients (n=44/135) were classified as low risk AML (3-year OS 58% vs. 38%, p=0.03 “low” vs. “medium”, Figure 1E). Within ELN adverse AML, 71% of patients (n=142/200) were classified with reduced risk (n=27 “low risk”, n=115 “medium risk”, 3-year OS 38% vs. 37% vs. 13%, p=0.0003 “low” vs. “high”, p=0.0002 “medium” vs. “high”, Figure 1F). Image: Summary/Conclusion: Based on this approach, we were able to distinguish clinically relevant prognostic subgroups of AML patients within well described (cyto-) genetic risk groups. Patients with favorable (cyto-) genetics but high-risk epigenetics might benefit from alternative consolidation treatment strategies, such as allogeneic stem cell transplantation. On the other hand, patients with adverse (cyto-) genetics but low-risk epigenetics might be considered for consolidation chemotherapy instead of stem cell transplantation in first complete remission. Finally, patients with both, adverse (cyto-) genetics and high-risk epigenetics, must be considered as very poor risk subgroup with very short OS (median 228 days). Our results demonstrate that DNA methylation profiling is a feasible and robust approach for risk stratification in AML, able to refine current risk assessment based on (cyto-) genetics alone. We propose that DNA methylation-based risk assessment may serve as a promising complement for current standards in prognostic classification of AML. P480: NKG2D-MEDIATED ANTI-TUMOR IMMUNITY CONTRIBUTES TO THE FAVORABLE PROGNOSIS IN APL H. Wang1,*, X. Zhang1, S. Gong1, H. Du1, N. Mei1 1First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a poor prognosis. Acute promyelocytic leukemia (APL) is a unique subtype of AML, with PML-RARA fusion caused by t(15;17)(q22;q12) translocation, and is now considered a highly curable disease. Exploring the difference between APL and non-APL AML, may shed new insight on the treatment of AML. NKG2D is an activating receptor on NK cells that recognizes ligands on the surface of tumor cells and plays a crucial role in tumor cell killing. Prior studies have demonstrated that one mechanism of AML immune evasion is downregulation of NKG2D ligands (NKG2DL) or secretion of receptor-blocking soluble ligands. Whereas, reports on NKG2DL expression in APL are still lacking. In this study, we investigate the differences of NKG2DL expression on APL and non-APL cells. Aims: Exploring the difference between APL and non-APL AML, may shed new insight on the treatment of AML. Methods: Bone marrow samples were derived from de novo APL and non-APL AML patients, diagnosed according to French-American-British (FAB) criteria in The First Affiliated Hospital of Xi’an Jiao Tong University. Flow cytometry was performed with the anti-NKG2DL mAbs (MIC-A/B-PE, ULBP-1-APC, ULBP-2/5/6-PerCP). Leukemic blasts were identified using the CD45/SSC gating procedure. Non-APL AML patients were divided into high or low group based on the median expression levels of NKG2DL for survival analysis. Results: Totally 46 patients comprising of 23 APL and 23 non-APL AML were enrolled in this study. There was no significant difference in the baseline characteristics between the two groups. All non‐APL AML cells had low or no NKG2DL surface expression, while most APL cells expressed high NKG2DL levels. In particular, just two APL patients was negative for the NKG2DL but positive for CD34. The median percentage of NKG2DL among non-APL AML cells and APL cells were 1.2% vs 20.3% in MIC-A/B (P<0.01), 1.1% vs 54.8% in ULBP-1 (P=0.017), and 3.8% vs 36.35% in ULBP-2/5/6 (P<0.01). (Fig.1) 23 non-APL AML patients were enrolled for study inclusion; one patient dropped out due to cerebral infarction. The median follow-up time was 17.2 weeks. The high ULBP-2/5/6 group showed a superior overall survival rate compared to the low ULBP-2/5/6 group (P < 0.01), but there was no significant difference between high and low MIC-A/B groups (P = 0.31) or between the high and low ULBP-1 groups (P = 0.52). Image: Summary/Conclusion: NKG2DL was decreased significantly on the surface of non-APL AML cells compared with APL cells. These results may indicate that NKG2D-mediated anti-tumor immunity contributes to the favorable prognosis in APL, while immune escape is an underlying mechanism for unfavorable prognosis in non-APL AML. NKG2DL may be used as a potential biomarker to predict prognosis of patients with non-APL AML, and upregulation of its expression could improve the clinical efficacy. Further studies with larger sample size and longer follow-up are required to confirm our conclusion. P481: MP0533, A NEW MULTISPECIFIC DARPIN CD3 ENGAGER TARGETING THREE TUMOR ASSOCIATED ANTIGENS, INDUCES SPECIFIC T-CELL ACTIVATION AND AML TUMOR KILLING IN VIVO A. Croset1,*, M. Bianchi1, M. Franchini1, T. Lekishvili1, Y. Kaufmann1, A. Auge2, M. Hänggi1, W. Ali1, C. Zitt1, S. Wullschleger1, M. Matzner1, C. Reichen1, S. Fischer1, Y. Grübler1, A. Eggenschwiler1, T. Looser1, R. Watson1, P. Spitzli1, V. Kirkin1, D. Steiner1, A. Goubier1 1Molecular Partners AG, Schlieren, Switzerland; 2Molecular Partners AG, Molecular Partners AG, Schlieren, Switzerland Background: AML treatment options are generally focused on chemotherapy followed by allogeneic hematopoietic stem cell transplantation. However, many patients are ineligible for these options and often receive palliative treatments with poor long-term survival and there is an urgent need for improved therapeutic solutions. Newer therapies, including T cell engagers (TCE) and chimeric antigen receptor (CAR) T cells that target specific molecules overexpressed on leukemic blasts and stem cells are promising alternative treatment options for AML. However, achieving efficacy and low toxicity remains challenging. MP0533 is a multi-specific, half-life extended, T cell engaging DARPin in development for treatment of patients with AML and higher risk myelodysplastic syndrome (HR-MDS). MP0533 consists of a chain of 6 covalently linked DARPin domains. Three of these domains engage CD33, CD123 and CD70 on the target cells, utilizing an avidity driven approach to ensure that binding occurs preferentially when 2 or more TAAs are present. A fourth DARPin domain targets CD3 for T-cell engagement. Lastly 2 additional HSA binding DARPin domains prolong half-life in circulation. In vitro, MP0533 has demonstrated, in both allogeneic and autologous settings, single to double digit pM potency against AML cell lines and AML primary cells expressing any combination of at least 2 of the 3 targeted TAAs and low cytokine release. Aims: To evaluate and confirm that the previously established in vitro and ex vivo efficacy and safety profiles of MP0533 are also observed in an in vivo human PBMC xenograft mouse model. Methods: NXG mice were injected intraperitoneally with hPBMC from healthy donors and xenografted with MOLM-13 cells subcutaneously two days after hPBMC injection. Therapeutic treatment was initiated eight days after tumor implantation once tumors were established. DARPins were injected intravenously three times per week for two weeks. Several readouts such as T cell activation and human immune cell infiltration in tumors were performed by flow cytometry three days after first treatment. Additionally, cytokine and chemokine release were analyzed in serum four hours after the first treatment injection and in tumor supernatant 3 days after the first treatment injection. Results: MP0533 induced AML tumor killing in vivo (P value <0.0001 at day 18 compared to PBS). Three days after the first injection, MP0533 recruited human immune cells in the tumor (around 10% of hCD45+ cells detected by FACS, P value = 0.0044 compared to PBS) and induced T cell activation (around 46% of CD4+/CD25+/CD69+ cells and 38% of CD8+/CD25+/CD69+ cells detected by flow cytometry, P value <0.0001 compared to PBS) which triggered cytokines and chemokines release in the tumor such as IL-6, INFγ and TNFα. However, MP0533 didn’t induce significant cytokine release, including at the highest tested dose, in mice serum four hours after the first injection. Summary/Conclusion: We were able to generate a multi-specific CD3 engaging DARPin molecule with tailored affinities towards different TAAs showing significant efficacy in vivo that induced T cell activation without significant cytokine/chemokine release. P482: NOVEL BISPECIFIC INNATE CELL ENGAGER AFM28 FOR THE TREATMENT OF CD123 POSITIVE ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME J.-J. Siegler1,*, N. Schmitt2, J. Pahl1, T. Haneke1, I. Kozlowska1, S. Sarlang1, A. Beck2, S. Knackmuss1, P. Ravenstijn1, U. Reusch1, J. Medina-Echeverz1, J. Endell1, T. Ross1, D. Nowak2, C. Merz1 1Affimed GmbH, Im Neuenheimer Feld 582, Heidelberg; 2Department of Hematology and Oncology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Background: Novel treatments for patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (HR-MDS) with relapsed or refractory (R/R), or measurable residual disease are urgently needed. Targeting of the surface antigen CD123 expressed on both leukemic blasts and stem cells (LSCs) of most patients allows for depletion of both compartments, which is considered a prerequisite for deep anti-tumor responses and induction of prolonged remission. AFM28 is a novel bispecific Innate Cell Engager (ICE®) designed for bivalent high-affinity binding of CD16A on natural killer (NK) cells, redirecting effector cell cytotoxicity to CD123+ tumor cells. Allogeneic NK cell therapy is emerging as a next-generation treatment with demonstrated activity in R/R AML and a very benign safety profile. Retargeting of NK cells with AFM28 may be a particularly effective treatment strategy in patients with CD123+ AML and HR-MDS. Aims: 1. Preclinical characterization of AFM28 pharmacology and toxicology in vitro and in vivo to support clinical development. 2. Assessment of binding and activation of NK cells by AFM28 to test suitability for combination development with allogeneic NK cell therapy. Methods: Flow cytometry (FC) was used to assess binding and retention of AFM28 on NK cells, NK cell activation (degranulation, IFNγ expression) and depletion of leukemic cells from AML and MDS patient specimens. Quantification of secreted cytokines in leukocyte cultures employed bead-based luminex methodology. In vivo efficacy data were acquired by BLI measurement from a murine AML model. Cynomolgus data were acquired from non-GLP dose range finding and a GLP toxicology study using standard methods (ELISA, IHC, FC). Results: AFM28 induced NK cell activation at low picomolar concentrations and mediated efficacious depletion of CD123+ tumor cell lines and primary leukemic cells, including cells with low CD123 expression. NK cell activation was induced more potently than by an effector-enhanced IgG1 and was accompanied by more pronounced degranulation and IFNγ production. Similarly, AFM28 induced depletion of leukemic cells in patient bone marrow samples, without lysis of CD34+/CD123− cells, suggesting sparing of healthy hematopoietic progenitors. Potent anti-tumor activity was confirmed in vivo in a murine tumor model. In preclinical toxicology models, AFM28-induced target cell depletion was associated with minimal release of inflammatory cytokines, including IL-6, TNFα and IFNγ from leukocytes in vitro, and only low-level release of IL-6 in cynomolgus monkeys, despite efficacious depletion of CD123+ basophils and plasmacytoid dendritic cells (pDCs). Characterization of NK cell binding revealed high-avidity interaction, long cell surface retention, and potent induction of NK cell-mediated tumor cell lysis with no impact on NK cell viability. Summary/Conclusion: These data demonstrate that AFM28 exhibits potent pharmacological activity in vitro and in vivo and efficaciously induces NK cell-mediated depletion of primary leukemic cells. Further, toxicology data suggest good tolerability with a low risk of cytokine release syndrome. Together, these findings support the clinical investigation of AFM28 as a potential novel treatment of CD123+ AML and HR-MDS. A first-in-human trial investigating safety and activity of single agent AFM28 in patients with R/R AML is currently in preparation. In addition, these data support the combination of AFM28 with allogeneic NK cell therapy, which holds potential as an effective, novel treatment strategy. P483: STAT3 DRIVES IMMUNE EVASION OF MLL-AF9-DRIVEN ACUTE MYELOID LEUKEMIA B. Zdarsky1, A. Witalisz-Siepracka1,*, S. Boigenzahn1, K. Fiedler1, D. Stoiber1 1Department of Pharmacology, Physiology and Microbiology, Division Pharmacology, Karl Landsteiner University of Health Sciences, Krems, Austria Background: Acute myeloid leukemia (AML) is a heterogenous disease with poor prognosis. Although the treatment opportunities in AML expanded in recent years, relapse still remains a major issue and novel therapies aiming to eradicate minimal residual disease are needed. Natural killer (NK) cell immunotherapy is put forward as a potential strategy fulfilling these unmet clinical needs. Importantly, NK cell function of AML patients is largely diminished due to different mechanisms of tumor-mediated immune suppression and escape. Signal transducer and activator of transcription 3 (STAT3) is a member of the JAK-STAT signaling pathway driving transcriptional responses downstream of many cytokines. To escape the immune surveillance, tumor cells tend to downregulate NK cell responses and STAT3 was proposed to drive this process in solid cancers. Aims: We aim to investigate the function of STAT3 in AML evasion from NK cell surveillance. Methods: Using CRISPR/Cas9 we generated STAT3-deficient human AML cell lines carrying the MLL-AF9 oncogene. The cell lines were characterized via flow cytometry, Western blot and qPCR and used as target cells for a human NK cell line in cytotoxicity assays. Results: Using publicly available databases, we show that STAT3 expression negatively correlates with several NK cell-activating ligands in AML patients. Moreover, when studying a panel of human AML cell lines with a range of STAT3 expression levels, we observed a trend for the most efficient NK-cell mediated killing in cell lines expressing the lowest levels of STAT3. AML cells lacking STAT3 showed no major impairment in survival and proliferation under homeostatic conditions but were less resistant to different types of cellular stress. Interestingly, loss of STAT3 significantly enhanced killing by NK cells. The effect was also supported by elevated expression of NK-activating ligands in STAT3-deficient cells. We are currently testing if STAT3 inhibition recapitulates the observed effect. Summary/Conclusion: Our in vitro data indicate that targeting STAT3 in AML might be a strategy to enhance NK cell-mediated killing. We plan to extrapolate our findings into in vivo xenograft systems with adoptive transplants of primary human NK cells. The study will provide important insights into AML evasion from NK cell-mediated surveillance and potentially give a novel rationale for STAT3 inhibition in AML. P484: WHOLE EXOME SEQUENCING (WES) CHARACTERIZES THE MUTATIONAL LANDSCAPE OF BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM (BPDCN) H. Witte1,*, A. Kuenstner2, J. Schwarting3, V. Bernard4, H. Merz4, N. von Bubnoff3, H. Busch2, A. Feller4, N. Gebauer3 1Hematology and Oncology, German Armed Forces Hospital Ulm, Ulm; 2Medical Systems Biology Group, University of Luebeck; 3Hematology and Oncology, UKSH Campus Lübeck; 4Haematopathology, Haematopathology Luebeck, Luebeck, Germany Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and clinically aggressive hematologic malignancy. Due to novel technologies pathogenetic concepts of BPDCN underlie a dynamic process. However, its genomic features including novel therapeutic vulnerabilities remain sparsely characterized. Aims: The present study aimed to illustrate the mutational landscape of BPDCN. Methods: To delineate the mutational landscape of BPDCN, whole-exome sequencing (WES) of 47 cases was performed. Significantly mutated genes, oncogenic drivers and perturbed pathways were compared with data from acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML). Results: Median age was 73 years (range 26 – 91) and 72.3% were male. Primary involvement of the skin and the bone marrow was present in 28 (59.6%) and 15 (34.9%) cases, respectively. A median of 98 mutations per sample was detected resulting in a tumor mutational burden (TMB) of 2.57 mutations/Mb/sample. WES and consecutive gene set enrichment analysis revealed an accumulation of oncogenic mutations in epigenetic regulators of gene-expression (95.7% of cases; TET2, DNMT3A, KMT2D, SETD2, IDH2), RTK-RAS (93.6%; NRAS, MET, EGFR), NOTCH (76.6%; NOTCH2, CREBBP, EP300) and WNT signaling pathways (59.6%; CTNNB1, MED12). Moreover, WES identified a subset of shared myeloid drivers across the spectrum of BPDCN, AML and CMML. In the era of precision oncology, exome profiling revealed several potential therapeutic targets such as NOTCH2, NRAS, EGFR or EZH2. Summary/Conclusion: To the best of our knowledge, we provide genomic profiling in the largest WES cohort in BPDCN to date. Current results confirmed previous genomic features associated with myeloid pathogenesis, identified additional significantly mutated drivers and novel potential therapeutic vulnerabilities. P485: TARGETING HISTONE LYSINE-SPECIFIC DEMETHYLASES BY JIB-04 IMPROVES RESPONSE OF AML CELLS TO VENETOCLAX TREATMENT K. Wohlan1,*, D. Fan2,3, J. Su2,3, A. G. Guzman1, M. A. Goodell1,4,5,6,7, R. E. Rau8 1Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, United States of America; 2Institute of Biomedical Big Data; 3School of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, China; 4Program in Developmental Biology; 5Stem Cells and Regenerative Medicine Center, and Center for Cell and Gene Therapy; 6Interdepartmental Program in Integrative Molecular and Biomedical Sciences; 7Department of Molecular and Human Genetics, Baylor College of Medicine; 8Department of Pediatrics, Baylor College of Medicine, Texas Children’s Hospital, Houston, United States of America Background: Acute myeloid leukemia (AML) is a heterogeneous, hematologic malignancy that is associated with a poor prognosis. Available treatment options are limited, especially for older patients and others who are ineligible for intensive chemotherapy. The BCL2-specific inhibitor venetoclax has proven efficacy in AML treatment particularly in combination with hypomethylating agents (HMA) or low-dose cytarabine. However, data from a clinical phase II study indicated a 30% failure rate after venetoclax+HMA treatment of newly diagnosed patients (PMID: 30361262), suggesting additional options are needed for patients who do not respond to available treatments or develop resistance. Compared to normal bone marrow histone lysine-specific demethylases are more highly expressed in AML, suggesting they are potential targets for therapeutic intervention. JIB-04 is a potent small molecule inhibitor for multiple members of the KDM 4, 5 and 6 families with KDM5A being the most sensitive. Combined pharmacological inhibition of KDM6 and KDM4 family members has been shown to reduce proliferation and increase apoptosis of primary AML cells of various subtypes (PMID: 29111428). Knockdown of KDM4 and KDM5 proteins leads to proliferation inhibition, cell cycle arrest, and apoptosis in AML cells (PMID: 34017391; PMID: 29602065). Together, these data highlight that KDM4, KDM5, and KDM6 proteins play an important role in the survival of AML cells. Aims: Describe the efficacy of JIB-04 alone and in combination with venetoclax for the treatment of AML. Identify the molecular changes due to JIB-04 in AML cells of varying genetic subtypes. Methods: We treated AML cell lines, patient AML blasts, and CD34+ cells from healthy donors with JIB-04, venetoclax, or the combination for 72 hours. Apoptosis was quantified by annexinV/propidium iodide staining. Cell proliferation of AML cell lines treated with JIB-04 was evaluated using MTT assay and transcriptome profiling was done by RNA-Seq. Results: Treatment of AML cell lines with low nanomolar doses (IC50 range from 8-60nM) of JIB-04 resulted in inhibition of proliferation and apoptosis. The combination of JIB-04 with venetoclax showed a strong synergistic effect in all AML cell lines including AML cell lines with relative resistance to venetoclax (Figure 1A). Moreover, patient AML blasts also showed a synergistic response to the drug combination compared to single-drug treatments (Figure 1B). In contrast, viability of normal CD34+ hematopoietic cells was not affected by JIB-04 treatment at a dose of 100nM that leads to apoptosis in AML cells, indicating a therapeutic window. Changes in the transcriptome after JIB-04 treatment were analyzed in AML-OCI3 and MOLM-13 cells. In both cell lines, genes related to the mTOR pathway and stress response were downregulated, indicating possible mechanisms reducing proliferation and increasing apoptosis. Further experiments will be performed to confirm the identified regulated genes as druggable targets in AML that improve venetoclax response. Image: Summary/Conclusion: While JIB-04 alone shows some anti-leukemic effect, this study highlights the potential therapeutic benefit of combining JIB-04 and venetoclax in AML. This novel drug combination synergistically inhibits cell growth and induces apoptosis in AML cells with various subtypes. In vivo studies are currently underway to show the efficacy of this drug combination in patient derived xenograft models. P486: CD9 MARKS DIFFERENTIATION AND ANTI-TUMOR IMMUNITY IN PEDIATRIC ACUTE MYELOID LEUKEMIA Y. Xu1,*, K. Y. Y. Chan1, S. P. Fok1, C. K. Li1, K. T. Leung1 1Department of Paediatrics, The Chinese University of Hong Kong, Shatin, Hong Kong Background: Despite intensive treatment regimes, the clinical outcomes for children with acute myeloid leukemia (AML) remain suboptimal, underscoring the need to decipher the underlying pathology and translate into therapeutic modalities. Emerging evidence suggest myeloblasts could evolve multiple machineries to evade immune patrol and hinder immunotherapies. Aims: We recently reported the importance of CD9 in pediatric ALL but its role in AML remains unknown. In this disease context, we pursued to characterize its prognostic significance, elucidate its regulation and function, and identify its role in leukemia immunosurveillance. Methods: Patients were stratified based on CD9 status for comparison of long-term survival. Epigenetic control of CD9 was investigated by ChIP-sequencing and confirmed by HDACi treatment. Impact of CD9 on leukemia aggressiveness was measured by competition and colony formation assays. Influence of CD9 on leukemia progression was evaluated in xenograft models, coupled with global transcriptome profiling of AML samples. Immunomodulatory effect of CD9 was dissected by single-cell transcriptomics and validated by flow cytometry. Immune-related CD9 interactors were identified by IP-MS, followed by proof-of-function experiments in an immune-reconstituted mouse model. Results: The expression of CD9 on blasts of AML patients (12.2%, n=82) was significantly lower than those of ALL patients (90.4%, n=219, P<0.001) or stem cells from normal bone marrow donors (48.4%, n=22, P=0.014). Among AML cases, the blasts of 32 patients (39%) were CD9+. The 5-year relapse-free survival rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P=0.018). A marked decrease of H3K9/27Ac occupancy in the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold, P<0.05), and strongly correlated with CD9 repression (r=0.585-0.719, P<0.01). Exposure of CD9- AML cell lines (n=8) or samples (n=9) to panobinostat significantly elevated CD9 mRNA and protein expression (3.1-32.2-fold, P<0.05), and restored activating histone acetylation marks (4.1-41.6-fold, P<0.05). Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation (P<0.01) and colony formation (P=0.002). NOD/SCID mice receiving CD9+ AML exhibited a drastic reduction of leukemic load by 70.7-91.8%. Molecular expression analysis revealed decreased stemness (NES: -1.7, P=0.01) and increased monocyte (NES: 1.8, P=0.034) gene signatures in CD9+ patient samples. Concordantly, a profound up-regulation of CD9 (9.4-51.1-fold, P<0.01) was observed in PMA-mediated monocyte/macrophage differentiation but not in ATRA-mediated neutrophil differentiation of myeloblasts. Mechanistically, CD9 promoted basal and IFNγ-induced MHC-I/II expression (P<0.01) through the JAK2/STAT5 axis. Inter-patient comparisons of bone marrow samples (n=31) revealed a higher MHC-I expression in CD9+ AML (P<0.001). Interestingly, CD9 physically bound to MHC-I/II, formed an immune complex and regulated intracellular trafficking. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML (MV4-11 and MOLM-13), concomitant with a robust bone marrow infiltration of cytotoxic T cells. Summary/Conclusion: Our data provided molecular, cellular and clinical evidence showing the plausible function of CD9 as a key driver intertwining differentiation and immune recognition in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy. P487: SMALL NON-CODING RNAS ASSOCIATE WITH CHROMATIN TO MAINTEIN PROPOGATION OF ACUTE MEYLOID LEUKEMIA H. Yun1,2,*, F. Zhou1,2, C. Rohde1,2, J. Zoller1, X. Yu1,2, S. Göllner1, C. Müller-Tidow1,2,3 1Department of Medicine V, Hematology, Oncology and Rheumatology, Heidelberg University Hospital; 2Molecular Medicine Partnership Unit, European Molecular Biology Laboratory (EMBL); 3National Center for Tumor Diseases (NCT), Heidelberg, Germany Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the clonal expansion of myeloid progenitors without terminal differentiation. The interplay between genome and epigenome alterations drives aberrant gene network linking to leukemia transformation. Altered transcription may occur at three-dimensional chromatin level and be orchestrated by a combination of multiple regulatory factors. Although some RNA molecules, especially non-coding RNAs, were reported to modulate transcription in leukemia cells, the regulatory role of genome-wide RNAs on transcription through chromatin association remains largely unexplored. Aims: To identify novel RNA molecules as chromatin-association factors critical for leukemogenesis through modulating transcription Methods: We performed in situ Mapping of RNA-Genome Interactome (iMARGI) in a human acute leukemia cell line – MV4-11 cells. Chromatin association of candidate RNAs was verified using Chromatin Isolation by RNA Purification (ChIRP) by means of biotinylated oligonucleotides for target capture. A series of ChIP-seq assays were performed in multiple AML cell lines to profile chromatin occupancy by Fibrillarin (FBL), a catalytic component of C/D box ribonucleoproteins (snoRNPs), as well as to observe the remodelling of chromatin states marked by H3K27ac and H3K9me3 upon shRNA-mediated knockdown of FBL. Loss-of-function experiments using antisense oligonucleotides were performed on candidate chromatin-associated RNAs in AML cell lines, AML primary samples, and CD34+ healthy bone marrow cells. Results: In total, about 52.1 million RNA-chromatin interactions were identified out of deep sequencing of four iMARGI libraries. Whereas the vast majority (50.5 million, 97%) of RNA-chromatin interactome were intra-chromosome, a small fraction (1.6 million, 3%) occurred inter chromosome. Though protein-coding mRNAs predominate the chromatin-associated RNA species, other non-coding RNAs were also captured. Intriguingly, different from protein-coding mRNAs and long intergenic non-coding RNAs (lincRNAs) which mainly form intra-chromosome interactions, two small non-coding RNA classes, small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs), were found with prevalent frequency (62% and 92%, respectively) of inter-chromosome associations. SNORD3A (U3) and SNORD118 (U8) were C/D box snoRNAs ranked top by the number of their associated interactions in MV4-11 cells as well as in other cell types. Chromatin association of U3 and U8 was verified using ChIRP, with the observation of unique chromatin binding for both U3 and U8, and of specific binding motifs of transcription factors at their binding sites. ChIP-seq revealed that the snoRNP FBL occupied only a small set of genes (n=118), and shRNA-mediated knockdown of FBL had a minimal impact on H3K27ac and H3K9me3, suggesting that snoRNA-chromatin interactions may function independently on FBL-associated snoRNPs. ASO-mediated loss of U3 or U8 significantly impairs leukemia cells proliferation and colony forming capacity in multiple AML cell lines and primary AML blast samples, whereas only mildly affects CD34+ healthy cells. Summary/Conclusion: We identified chromatin-associated snoRNAs U3 and U8 which were essential for leukemia maintenance and may serve as therapeutic targets for leukemia eradication. P488: THZ1, A COVALENT CDK7 INHIBITOR, INDUCES APOPTOSIS IN ACUTE MYELOID LEUKEMIA CELLS AND EXERTS SYNERGISTIC ANTILEUKEMIA ACTIVITY WITH AZACITIDINE S. Zhang1,*, H. Liu1 1Beijing Hospital, Beijing, China Background: Acute myeloid leukemia (AML) is a malignant blood cancer that develops mainly in elderly adults and has dismal clinical outcomes. Azacitidine (AZA) is still the preferred treatment for elderly patients unfit for intensive chemotherapy. However, the remission rate of azacitidine monotherapy is dismal, which necessitates the multidrug combination therapy. THZ1, a CDK7 inhibitor, has been proven to be effective in various cancers by regulating transcription and the cell cycle and inducing cell death. Here, we investigated the antitumor effect of THZ1 monotherapy and its combination treatment with AZA. Aims: To investigate the antitumor activity of the CDK7 inhibitor - THZ1 in acute myeloid leukemia (AML). Methods: We first examined the cell viability of THZ1 against THP-1, MOLM-13, OCI-AML3 cell lines using CCK-8 assays. We used flow cytometry to detect apoptosis after cells were stained with Annexin V-FITC/Propidium Iodide (PI), and western blot to detect the expression levels of proteins. We detect cell cycle by flow cytometry after cells were stained with 50 µg/ml PI and then analyzed with ModFit LT 5.0. RNA-sequencing analysis was performed to explore the potential mechanism of THZ1 in AML cells. In the double-drug combination experiment, we used CCK-8 assays to detect the effect of drugs on the viability of the three cell lines, used CompuSyn software to calculate the combination index (CI), which indicates additive effects (CI = 1.0), synergism (CI < 1.0), and antagonism (CI > 1.0). Results: THZ1 decreased viability and induced apoptosis in THP1, MOLM-13, and OCI-AML3 cells in a dose- and time-dependent manner. Besides, THZ1 inhibited phosphorylation of Ser2, Ser5, and Ser7 residues of RNA Pol II CTD and induced cell cycle arrest at G0/G1 phase in AML cells. RNA-sequencing analysis revealed that THZ1 induced changes in the expression of genes involved in apoptosis, the cell cycle, and DNA repair. Combined treatment with AZA and THZ1 showed a synergetic effect (CI < 1.0). Western blot analysis of apoptosis markers, including cleaved caspase3 and PARP1, demonstrated that THZ1 increased the expression of these markers and potentiated AZA-related apoptosis. Compared with monotherapy, combined treatment with AZA and THZ1 resulted in more Annexin V positive cells detected by flow cytometry, which further verified the synergistic effect of the two drugs. Western blot results indicate that combined treatment with AZA and THZ1 downregulates MCL1 at the protein level without an apparent decrease in BCL2 protein expression. Image: Summary/Conclusion: Our data demonstrate that the CDK7 inhibitor THZ1 induces the apoptosis of AML cells and exerts synergistic antileukemia activity with azacytidine, which provide the rationale for combination treatment with AZA and THZ1 for AML. P489: THE LONG NON-CODING RNA LINC01547 PROMOTES PROLIFERATION, CLONOGENICITY, AND CELL CYCLE PROGRESSION IN ACUTE MYELOID LEUKEMIA S. M. N. Zimmermann1,2,3,*, M. F. Blank1,2,4, D. Heid1,4,5, M. Bruckmann1, C. Müller-Tidow1,3,4, J. Krijgsveld2,3 1Department of Medicine V, Hematology, Oncology and Rheumatology, University Hospital Heidelberg; 2Division Proteomics of Stem Cells and Cancer, German Cancer Research Center (DKFZ); 3University of Heidelberg Medical Faculty; 4Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory (EMBL); 5European Molecular Biology Laboratory EMBL, Heidelberg, Germany Background: Non-coding sequences account for about 98% of the human genome, and it has become increasingly apparent that non-protein coding transcripts drive numerous physiological and pathological cellular processes. Long non-coding RNAs play important roles in cancer, but functions in leukemogenesis and leukemic maintenance, as well as underlying mechanisms, remain to be elucidated. Aims: In this study, we characterize the functional and mechanistic implications of the long non-coding RNA LINC01547 in acute myeloid leukemia (AML). Methods: We generated single-cell derived, biallelic LINC01547 knock-out clones in Kasumi-1 cells using the CRISPR/Cas9 system with paired sgRNAs. Knock-out and control cells were assessed for differences in proliferation and clonogenicity by cell growth and methylcellulose assays. Alterations in proliferation, cell cycle progression, apoptosis, and global protein synthesis were subsequently evaluated in flow cytometry-based assays. Finally, we characterized the impact of LINC01547 loss on RNA and protein homeostasis by RNA sequencing, as well as nascent and steady-state proteomics. Results: Bioinformatic analyses of expression data suggested a role for LINC01547 in AML. LINC01547 was highly expressed in AML (TCGA data), and its expression is associated with worse overall survival in AML. Biallelic deletion of LINC01547 suppressed AML cell growth and colony formation capacity. Similarly, EdU-based proliferation assays showed significantly reduced proliferation upon loss of LINC01547. Overall protein synthesis, assessed by O-propargyl-puromycin (OPP) assays, and apoptosis, analyzed by annexin V assays, were not significantly affected. Transcriptome and steady-state proteome analyses revealed that depletion of LINC01547 impacted the expression of multiple mRNAs and proteins. An overall weak correlation between differential expression on the transcript and the protein level indicated that LINC01547 might mediate its effects through both, transcriptional and post-transcriptional mechanisms. In line, mass-spectrometric analysis of nascent and steady-state proteomes upon LINC01547 depletion showed numerous alterations on the translational and total protein level, whereas the respective mRNAs often remained unaffected. Especially proteins involved in cell cycle regulation exhibited reduced translation rates upon loss of LINC01547. Consistently, cell cycle assays showed that depletion of LINC01547 slowed cell cycle progression and led to accumulation of cells in G0/G1 phase. Overlapping our transcriptome, total proteome, and nascent proteome data sets, we identify potential downstream targets through which LINC01547 presumably exerts its newly uncovered pro-oncogenic function. Summary/Conclusion: We demonstrate that the previously uncharacterized long non-coding RNA LINC01547 has wide-ranging functions in AML. Its overexpression in AML and negative correlation with overall survival are substantiated by its uncovered implications in cell proliferation, clonogenicity, and cell cycle progression. In-depth characterization and overlap of its impact on transcriptome, nascent proteome and total proteome reveal numerous genes and pathways affected by LINC01547 depletion. Further assessment of LINC01547 and its downstream targets will improve our understanding of the mechanisms underlying leukemogenesis and potentially reveal novel leverage points in AML therapy. P490: SNORNA-DERIVED RNAS (SDRNAS) ARE IMPORTANT DRIVERS OF LEUKEMOGENESIS AND LEUKEMIC MAINTENANCE IN AML R. Zinz1,2,*, C. Rohde1,3, D. Heid1,3,4, M. Bruckmann1, M. Bornhäuser5, C. Röllig5, U. Platzbecker6, C. Baldus7, H. Serve8, F. Zhou1, C. Pabst1,3, C. Müller-Tidow1,2,3, M. F. Blank1,3,9 1Medical Department V, Hematology, Oncology and Rheumatology, Heidelberg University Hospital; 2University of Heidelberg Medical Faculty; 3Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory (EMBL); 4European Molecular Biology Laboratory (EMBL), Heidelberg; 5Medical Clinic and Policlinic I, University Hospital Carl Gustav Carus and Medical Faculty of the TU Dresden, Dresden; 6Department of Hematology, Cellular Therapy and Hemostaseology, Leipzig University Hospital, Leipzig; 7Medical Department II, Hematology/Oncology, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel; 8Department of Medicine II, Hematology/Oncology, University Hospital Frankfurt, Goethe-University Frankfurt, Frankfurt; 9Division Proteomics of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Background: Small non-coding RNAs play important roles in leukemogenesis. The canonical function of small nucleolar RNAs (snoRNAs) is to facilitate 2’-O-methylation of rRNA via a target site-specific sequence and thus promote ribosomal biogenesis and function. Recently, we have shown that a subset of small non-coding RNAs, i.e. C/D box snoRNAs are overexpressed in acute myeloid leukemia (AML) and required for AML1/ETO-driven leukemia initiation and maintenance. Depletion of snoRNP protein components or even single snoRNAs can potently inhibit AML cell growth or decrease clonogenicity, and the levels of many snoRNAs positively correlate with leukemic stem cell frequency in AML patients. Over the past years, it has been shown that snoRNAs can be further processed into shorter snoRNA-derived RNA fragments (sdRNAs), but the expression patterns of sdRNAs in AML, as well as mechanistic and functional implications, remain widely elusive. Aims: In this study, we investigated sdRNA expression patterns, functions and mechanisms in AML and healthy hematopoiesis. Methods: We prospectively characterized snoRNA and sdRNA expression in 96 AML patient samples, as well as in healthy hematopoietic stem and progenitor cells and differentiated white blood cells by small RNA sequencing. mRNA sequencing was performed to investigate connections between snoRNAs, sdRNAs and the transcriptome. Further, we depleted AML cells of different snoRNA loci by CRISPR/Cas9-based knock-out and performed lentiviral rescue overexpression of the respective sdRNA or full-length snoRNA. Results: We discovered that more than 120 snoRNAs are further processed into sdRNA fragments in AML. Single snoRNAs often evolve into multiple sdRNA isoforms. Interestingly, sdRNA expression varied significantly between different AML patients. Correlation of sdRNA and full-length snoRNA expression with patient characteristics, clinical outcome and mutational status revealed numerous implications. Those include e.g. a distinct pattern of enriched sdRNAs in NPM1-mutated patients. Expression of several sdRNAs was associated with poor overall, event-free and relapse-free survival in AML. We identified sdRNAs that were overexpressed in AML as compared to HSCs and found characteristic expression patterns among differentiated white blood cells. Notably, ratios of sdRNAs and their host snoRNA were not stable across different samples, suggesting an active mechanism regulating sdRNA processing and thus their downstream pathways. Gene set enrichment analyses based on mRNA and sdRNA correlations suggested distinct cell type specific properties. Forced expression of several sdRNAs rescued the reduced clonogenic potential observed upon depletion of the respective host snoRNA locus. This effect was often identical or even more pronounced than the rescue with the respective snoRNA precursor. Of note, overexpression of distinct sdRNAs in AML cell lines enhanced clonogenic potential. Summary/Conclusion: SnoRNA-derived sdRNAs are a common feature of AML with characteristic expression patterns determined by mutation status and clinical features. Targeting certain sdRNAs or factors involved in sdRNA processing or function might constitute promising novel therapeutic leverage points in AML. P491: PREDICTIVE FACTORS OF DIFFERENTIATION SYNDROME IN PATIENTS WITH ACUTE PROMYELOCYTIC LEUKEMIA M. Ben Salah1,*, M. Bchir1, R. Berred1, R. Kharrat1, L. Aissaoui1, Y. Ben Abdennebi1, R. Ben Lakhal1, H. Ben neji1, B. Meddeb1 1Hematology department, Tunis El-Manar University, Faculty of medicine of Tunis, Aziza Othmana Hospital, Tunis, Tunisia Background: Differentiation syndrome (DS) may be a life-threatening complication in patients with acute promyelocytic leukemia (APL) treated with all-trans retinoic acid (ATRA) and anthracycline chemotherapy. Identifying high risk patients enables a more efficient follow up and an optimal prophylaxis strategy. Aims: The aim of this study was to analyze the incidence, characteristics, and the risk factors of DS occurring during induction treatment in APL patients treated with ATRA and anthracycline. Methods: We conducted a retrospective single-center study, including patients diagnosed with APL between 2010 and 2019 in our department and treated with ATRA and chemotherapy according to PETHEMA protocols LPA99 and LPA2005. Obesity, renal failure and a high risk (according to the Sanz score) represented an indication for a DS prophylaxis prescription, using whether prednisone (LPA99) or dexamethasone (LPA2005). A univariate and a multivariate analysis were conducted, in order to identify predictive factors of DS. Results: Ninety patients were included in our study, with an average age of 34 years old. According to Frankel’s diagnostic criteria, 16 patients (18%) experienced a DS, with a mean age of 39 years [13-71 years], and a sex ratio of 0.6. Half of this group was classified as high risk according to the Sanz score, 44% as intermediate risk, and 6% as low risk. A severe differentiation syndrome was described in 7 patients (44%). The syndrome occurred at a median of 4 days after the start of induction therapy [2-26 days]. A Prophylaxis had been prescribed for 10 patients (62.5%): 8 were classified as high risk, and 2 had obesity. In order to identify the risk factors of DS, the following parameters were analyzed: age, sex, performance status, Body Mass Index, clinical presentation at diagnosis, treatment protocol, Sanz score, creatinine level, LDH level, hemoglobin level, white blood cell (WBC) count, platelets, cytological type, PML-RARa variant, CD2, CD15, CD34, CD56 expression, fibrinogen level, prothrombin time, presence of disseminated intravascular coagulopathy (DIC), occurrence of bleeding and thromboembolic complications, and corticosteroid prophylaxis. The univariate analysis identified a statistically significant difference between the group of patients with DS and the one without, for the following factors: WBC greater than 50G/L (31,3% versus 8,1%; p=0,02), the presence of DIC at diagnosis (81.3% versus 18.7%; p=0.01), the expression of CD34 (p=0.017), the LDH level (a median of 307 versus 590; p=0.026), and the treatment protocol LPA 99(11 patients (68.8%) treated according to LPA99 versus 5 (31,3%) treated according to LPA2005; p=0.003). Upon multivariate analysis, WBC count greater than 50 G/L (p=0,007), the presence of DIC at diagnosis (p=0,03), and the treatment protocol LPA99 (p=0,02), remained independent risk factors of DS (Table1). Image: Summary/Conclusion: Differentiation syndrome is a significant complication secondary to the ground-breaking treatment of APL. It is necessary to accurately identify high risk patients in order to elaborate effective risk-adapted prophylaxis and treatment. P492: TAMIBAROTENE IN COMBINATION WITH VENETOCLAX AND AZACITIDINE IN PREVIOUSLY UNTREATED ADULT PATIENTS SELECTED FOR RARA-POSITIVE AML WHO ARE INELIGIBLE FOR STANDARD INDUCTION THERAPY (SELECT AML-1) E. Stein1,*, S. de Botton2, A. Pigneux3, C. McMahon4, B. Ball5, G. Borthakur6, A. Eghtedar7, S. Kambhampati8, J. Tache9, E. Wang10, H. Kelley11, A. Volkert11, K. Baker11, Q. Kang-Fortner11, G. Hodgson11, C. Madigan11, E. Warlick11, D. Roth11, M. Kelly11, D. Pollyea4 1Department of Medicine, Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, United States of America; 2Institut Gustave Roussy, Paris; 3CHU de Bordeaux - Hôpital Haut-Lévèque, Bordeaux, France; 4University of Colorado, Aurora; 5City of Hope, Duarte; 6Department of Leukemia, Division of Cancer Medicine, MD Anderson Cancer Center, Houston; 7Colorado Blood Cancer Institute, Sarah Cannon Research Institution, Denver; 8HCA Midwest Research Medical Center, Sarah Cannon Research Institution, Kansas City; 9BRCR Global, Plantation; 10Roswell Park, Buffalo; 11Syros, Cambridge, United States of America Background: RARA-positive (RARA+) AML is a novel genomically defined patient subset with an actionable biological target for treatment with tamibarotene, an oral and selective RARα agonist (McKeown 2017). RARA+ patients can be selected by a blood-based biomarker test, with approximately 30% of newly diagnosed (ND) AML patients being RARA+ (Vigil 2017). As a biologically targeted agent for patients with RARA overexpression, tamibarotene has the potential to provide benefit irrespective of mutation or cytogenetic risk classification. In RARA+ ND AML patients ineligible for standard induction therapy, tamibarotene plus azacitidine (aza) led to a CR/CRi rate of 61% and a rapid onset of response (de Botton 2020). Approximately one-third of patients with ND unfit AML do not respond to front-line standard of care venetoclax (ven)/aza (DiNardo 2020). Translational data suggest RARA positivity enriches for monocytic features reported to be associated with ven resistance (Fiore 2020, Pei 2020). This data suggests the RARA biomarker selects for patients who may respond to tamibarotene and may be less likely to respond to ven/aza. Given that tamibarotene plus aza has been generally well tolerated, with no increase in myelosuppression compared to single agent aza (de Botton 2020), it is anticipated that tamibarotene can be administered safely in combination with ven/aza. Aims: This is a Phase 2, open-label, multi-center study in the U.S. and France comparing the clinical activity of tamibarotene/ven/aza to ven/aza in treatment-naive RARA+ AML patients ineligible for standard induction chemotherapy. The primary objectives are to characterize the safety of the combination and to compare the CR/CRi rate of tamibarotene/ven/aza vs. ven/aza, with secondary objectives to compare CR rate, CR/CRh rate, duration of response, and time to response. The overall response rate using tamibarotene/ven/aza following ven/aza treatment failure will be explored. Clinical activity will be characterized by ELN criteria (Dohner 2017). Methods: This 3-part trial includes a safety lead-in, randomized efficacy study, and salvage arm. Following the safety lead-in, approximately 80 patients will be randomized 1:1 to receive tamibarotene/ven/aza or ven/aza. In the salvage arm, tamibarotene will be added for study patients randomized to ven/aza who experience progressive disease, relapse, or treatment failure. Patients will be treated with aza at 75 mg/m2 IV/SC daily on days 1-7, ven on days 1-28 per VENCLEXA USPI, followed by tamibarotene at 6 mg twice per day by mouth on days 8-28 of each 28-day cycle. Results: Response rates and 95% exact binomial confidence intervals will be calculated by treatment group. Summary/Conclusion: The SELECT AML-1 trial (NCT04905407) opened in July 2021 with ongoing enrollment. P493: SINGLE CENTER EXPERIENCE OF VENETOCLAX (VEN) IN COMBINATION WITH FLAG-IDA IN PATIENTS (PTS) WITH NEWLY DIAGNOSED (ND) AND RELAPSED/REFRACTORY (R/R) ACUTE MYELOID LEUKEMIA (AML) Y. Abaza1,*, T. Khan1, S. Dinner1, O. Frankfurt1, J. K. Altman1 1Northwestern University, Chicago, United States of America Background: Despite the high complete remission (CR) rates achieved with intensive chemotherapy in AML, relapse rates remain high. This underscores the need for novel regimens capable of inducing deeper remissions via eradicating measurable residual disease (MRD). Recently, FLAG-IDA + VEN was shown to be an effective regimen in AML inducing high rates of MRD negative CR in both ND-AML and R/R AML allowing patients to be successfully bridged to allogenic stem cell transplantation (allo-SCT) (DiNardo 2021). Aims: Assess the clinical activity of FLAG-IDA + VEN in pts with high-risk AML Methods: We conducted a single-center retrospective study to assess the clinical activity of FLAG-IDA + VEN in a high-risk pt population. Standard cytogenetic (CN) testing was conducted and molecular data was obtained using a 40-gene next generation sequencing platform. Responses were based on the modified International Working Group criteria and included CR, CR with incomplete platelet recovery (CRp), morphologic leukemia-free state (MLFS), and partial remission (PR). Results: From March 2020 to January 2022, 17 pts with AML (10 ND and 7 R/R) were treated using FLAG-IDA + VEN (Table 1). Median age was 48 years (range, 21-68) with male predominance (76%). Among the 10 pts with ND-AML (5 de novo, 3 AML-MRC, 2 treated-secondary), 3 had extramedullary disease (EMD). The most common ELN cytogenetic risk groups were adverse (N=2), complex (N=4), and KMT2A-rearranged (N=2). Five pts (50%) harbored a RAS mutation. Median time to initiation of therapy was 5 days (range, 2-14). Five pts [KMT2A-r: 2; complex CN: 2; diploid: 1] achieved CR/CRp including 1 pt with EMD. Median time to count recovery (ANC ≥ 500 and platelets ≥ 50,000) was 21 days (range, 21- 42). Number of cycles of consolidation given to date were 1, 1, 1, 2, and 5 cycles, respectively, and none of these pts have received allo-SCT yet. MRD status was available for 3 pts of which 2 achieved MRD negativity using flow cytometry (<10-3) and one remained MRD positive (NPM1 PCR) after induction. With median follow-up of 4.7 months (range, 1.6-7.6), 4 pts remain alive in CR/CRp and 1 pt [complex CN, del 17p, TP53 mutant (VAF: 90%)] relapsed after 2 cycles of therapy with no response to salvage decitabine (DAC) plus VEN and died due to progressive disease (PD). Of the remaining 5 nonresponders, 2 had treated-secondary AML, 2 with adverse CN [inv (3), -(7)], and 2 pts had EMD. All 5 pts died due to PD including 3 pts who had no response to salvage therapy. Among the 7 pts with R/R AML, 6 were in salvage 1 and 1 pt received prior allo-SCT. Responses were observed in all patients: 4 CR, 1 CRp, 1 MLFS, and 1 PR. Median time to count recovery in R/R AML pts was 33 days (range, 24-78). Of the 5 pts who achieved CR/CRp, 4 were bridged to allo-SCT and 1 pt died in CR after 3 cycles of consolidation due to sepsis. Of the 4 SCT recipients, 2 pts relapsed 2 months post-SCT (1 died due to PD, 1 alive receiving salvage therapy) and 2 remain alive in CR for 4+ and 9+ months, respectively. The 2 pts who achieved MLFS and PR died due to PD and fungal pneumonia after 5 and 1.5 months of starting therapy, respectively. After median follow-up of 5.3 months (range, 0.7-13.2), median overall survival for the entire population was 6.2 months and the median duration of response for all pts who achieved CR/CRp was not reached. Image: Summary/Conclusion: FLAG-IDA plus VEN is a feasible and active regimen in AML. Longer follow-up and larger multicenter studies are needed to confirm the efficacy of this regimen. P494: MYELOID LEUKEMIA IN PATIENTS WITH INFLAMMATORY BOWEL DISEASES: A RETROSPECTIVE COHORT STUDY A. Abomhya1,*, A. Razzaq2, S. Lukose1 1Internal Medicine, The Brooklyn Hospital Center, Brooklyn, United States of America; 2St. George’s University School of Medicine, West Indies, Grenada Background: Inflammatory bowel diseases include ulcerative colitis and Crohn’s disease and affect around 3.1 million adults in the United States (US). The data on the risk of myeloid leukemia among patients with IBD are limited and there has been only a couple of studies that evaluated the incidence of myeloid leukemia in this patient population. Aims: This study aimed to estimate the prevalence of myeloid leukemia in patients with IBD. Secondary outcomes included mortality, length of stay, all-cause 30-day non-elective readmission rate, and total cost of hospitalization. Methods: This is a retrospective cohort study for patients with IBD in the US. We queried the Nationwide Readmission Databases 2016-2018 using ICD-10-CM codes to identify all adult patients admitted for IBD. Patients with a comorbid diagnosis of myeloid leukemia including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), Myeloid sarcoma, and Unspecified Myeloid leukemia were identified. Figure 1 summarizes the case selection process. Median and IQR were used to describe Continuous variables, and proportions were used with categorical variables. Comparison between groups was performed by Mann Whitney test for continuous variables and Chi-Square test for Categorical variables. Multivariate regression analysis was performed to study the impact of comorbid myeloid leukemia on inpatient mortality and non-elective readmissions. Statistical analyses were performed using SPSS Version 25 (IBM Corporation, Armonk, NY, USA). Results: We extracted 365,152 index hospitalization records for IBD, 1052 (0.3%) had myeloid leukemia. Six hundred sixteen patients had acute myeloid leukemia (AML), 341 patients had chronic myeloid leukemia, 10 patients had myeloid sarcoma and 104 had unspecified myeloid leukemia. IBD patients with myeloid leukemia were older (64; Interquartile Range (IQR): 52-73 vs. 56; IQR: 38-70, P <0.001), more common to be males (50.8% vs. 49.2%, P <0.001) compared to IBD patients without myeloid leukemia. Having myeloid leukemia was associated with increased length of stays in days (7; Interquartile Range (IQR): 3-23 vs. 3; IQR: 2-6, P <0.001), increased median total charges ($74,413; IQR: $31,700 - $259,676 vs. $32,586; IQR: $17,827 - $62,260, P <0.001). On multivariate analysis; having myeloid leukemia was associated with increased mortality (Odds ratio (OR): 6.544; 95% confidence interval (CI): 5.318-8.052, P <0.001) and higher odds of all-cause 30-day non-elective readmission (OR: 1.4; 95% CI: 1.155-1.697, P= 0.001). Image: Summary/Conclusion: In our nationwide cohort of IBD patients, 0.3% had myeloid leukemia. Given the associated mortality and morbidity, we recommend considering a hematological consult for IBD patients with leukocytosis or leukopenia for further evaluation and appropriate management. More research studies are needed to investigate the pathogenesis of myeloid leukemia in IBD patients. P495: PHASE 2 STUDY OF ASTX727 (DECITABINE/CEDAZURIDINE) PLUS VENETOCLAX IN PATIENTS WITH RELAPSED/REFRACTORY ACUTE MYELOID LEUKEMIA (AML) OR PREVIOUSLY UNTREATED, ELDERLY PATIENTS UNFIT FOR CHEMOTHERAPY T. Abuasab1,*, Y. Alvarado1, G. Issa1, R. Islam1, N. Short1, M. Yilmaz1, N. jain1, L. Masarova1, S. Kornblau1, E. Jabbour1, N. Pemmaraju1, G. Montalban-Bravo1, S. Pierce1, C. DiNardo1, T. Kadia1, N. Daver1, M. Konopleva1, G. Garcia-Manero1, F. Ravandi1 1Department of Leukemia, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Houston, United States of America Background: ASTX727, is an oral formulation of the fixed dose combination of decitabine and cytidine deaminase inhibitor cedazuridine (35 mg/100 mg). Aims: To investigate whether a total oral therapy regimen of ASTX727+venetoclax (ven) is feasible and safe. Methods: Pts aged ≥18 years (yrs) with relapsed/refractory AML (R/R) or pts with AML aged ≥ 75 or 18 -74 with comorbid conditions prohibiting intensive chemotherapy were eligible to participate (frontline-FL). Other eligibility criteria included adequate renal and hepatic function and an ECOG performance status (PS) of≤2. ASTX727 is administered daily on days 1‐5 of each treatment cycle and ven on days 1‐28 of the 1st cycle after a dose ramp up of 100-200-400 mg over 3 days (with tumor lysis prophylaxis precautions and with ven dose adjustments as needed. A bone marrow exam is performed on day 21±3 of 1st cycle and ven is held if blasts <5% to allow count recovery. Cycles are repeated every 4-8 weeks depending on count recovery and Ven is administered for 21 days in subsequent cycles, with dose adjustments as necessary depending on count recovery. Results: Between March 2021 and January 2022, 28 pts (15 FL and 13 R/R) have been treated on the study. The median age is 75 yrs (range, 47-90) with FL cohort 81 and R/R cohort 72. 9 FL pts (60%) were ≥80 and 5 (30%) 70-80 years. In R/R cohort 9 pts (69%) were 70-80 yrs. The median PS is 2 (range 0-3) and in the R/R cohort, the median number of prior treatments is 2 (range, 1-4). In the FL cohort 5 (33%) had normal and 6 (40%) a complex karyotype; 3 had other. In the R/R cohort, 15% had normal, 46% complex karyotype and 31% other. Mutations of note in the FL cohort were RUNX1 (33%), ASXL1 (33%), DNMT3A (7%), TET2 (40%) and TP53 (20%). The overall response (ORR) including complete response (CR), CR with incomplete count recovery (CRi) and morphological leukemia free state (MLFS) in the FL cohort is 61% (4 CR, 4 CRi, 1 MLFS and 3 non-responders). 3 pts received only one day of therapy for severe adverse events unrelated to therapy (1 due to ischemic stroke, 1 septic shock and 1 debilitation) and were not evaluable for response. In the R/R cohort, the ORR rate was 45% (2 CR, 2 CRi, 2 MLFS with 5 non-responders and 2 not evaluable). The median number of cycles received is 2 (range, 1-5) for both cohorts. With a median follow-up of 5 months, the median survival for the FL cohort has not been reached (range, 0.6 – 7.3) and is 7.2 (range, 0.8-7.3) months for the R/R cohort. Grade 3 or higher adverse events directly attributable to therapy were mainly myelosuppression-related and included neutropenic infections in 3 (11%) and elevation of liver enzymes in 1 (4%) pt. Image: Summary/Conclusion: The combination of ASTX727+ven is feasible, particularly in the advanced elderly population, and demonstrates significant efficacy in pts unfit for chemotherapy both in the FL and R/R settings. P496: CLINICAL CHARACTERISTICS OF SECONDARY MYELOID NEOPLASMS IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE T. Abuasab1,*, S. F. Mohadam1, H. Hwang2, X. Wang2, K. Sasaki1, M. Yilmaz1, T. Kadia1, C. DiNardo1, N. Daver1, N. Pemmaraju1, G. Borthakur1, F. Ravandi1, G. Garcia-Manero1, K. Takahashi1 1Department of Leukemia; 2Department of Biostatistics, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Houston, United States of America Background: Inflammatory Bowel Disease (IBD) comprises two major disorders: ulcerative colitis (UC) and Crohn’s disease (CD). Patients (pts) with IBD are at an increased risk of cancer secondary to long-standing intestinal inflammation and secondary to immunosuppressive therapies, including but not limited to colorectal cancer, hepatobiliary tract cancer, Hodgkin, and non-Hodgkin lymphomas. However, very little data is available regarding the risk of secondary myeloid neoplasm (MNs) in pts with IBD except for the well-known association between azathioprine with therapy related MNs. Aims: To describe the clinical characteristics of secondary MNs in patients with IBDs. Methods: Retrospective chart review of patients with MNs who were previously treated for IBDs was performed. A descriptive statistic was performed to define the demographics, clinical and biological characteristics of the pts included in the study. Results: Between 2012 and 2020, 43 pts were identified to have developed a secondary MN during or after the treatment of IBDs. Sixty-three percent (27/43) were female, and 37% (16/43) were male, with most of the pts being of white ethnicity (35/43, 81%). Seventy percent (30/43) of the secondary MNs arose after the therapy for CD, whereas 30% (13/43) arose after UC therapy. The median age at the time of MN diagnosis was 59 years (yrs) (range, 23-83) and latency from IBD diagnosis was 16 years (range, 0-56). Twenty-five pts were on active treatment for IBD at the time of MN diagnosis (9 on biological agent, 11 on mesalamine, and 2 on azathioprine), whereas 18 were in remission. In addition, 8 pts (19%) had secondary cancer before the MN diagnosis (2 pts lymphoma, 2 pts skin cancer, ovary Ca, uterine Ca, schwannoma, and adrenal tumor) and 3 of them received therapy (chemotherapy and/or radiation therapy). Seventy four percent (32/43) of MN diagnoses were Acute myeloid leukemia (AML), 9 (21%) pts had myelodysplastic syndrome (MDS), and one each for MDS/myeloproliferative neoplasm (MPN) and chronic myelomonocytic (CMML). Seventy percent of the patients had cytogenetic abnormalities:13 pts (30%) with complex karyotype and 6 pts (14%) with core-binding factor (CBF) abnormalities (5 with inv16 and 1 with t [8;21]). The most common somatic mutations included: TP53 (N = 10, 23%), FLT3 (N = 7, 16%), RAS (N = 8, 19%), TET2 (N = 7, 16%), and DNMT3A mutations (N = 6, 14%). Interestingly, all the pts with CBF-AML had prior history of CD, two of them treated with anti TNF-α treatment. The median overall survival (OS) was 2.17 yrs for the whole cohort, and there was no difference in OS between MNs arose from CD and UC (2.35 and 2.08 yrs for CD and UC, respectively). Seventeen patients underwent allogenic stem cell transplantation, which resulted in remission of IBDs in 13 pts (76%). Image: Summary/Conclusion: We described the clinical characteristics of secondary MNs after IBD therapy. Secondary MNs after IBD therapy included higher than expected prevalence of core-binding factor AML, which was strongly associated with prior history of CD. This observation raises an important question about the association between CD and CBF-AML. Of interest, allogeneic stem cell transplantation led to remission of both MNs and IBDs, highlighting the role of allo SCT in autoimmune diseases. P497: PROGNOSTIC IMPACT OF RAS AND C-KIT MUTATIONS (SINGLE VS. MULTIPLE) IN CORE-BINDING FACTOR ACUTE MYELOID LEUKEMIA TREATED WITH FLUDARABINE, CYTARABINE, G-CSF (FLAG) BASED REGIMEN T. Abuasab1,*, J. Senapati1, T. Kadia1, F. Ravandi1, C. DiNardo1, N. Pemmaraju1, M. Ohanion1, Y. Alvarado1, H. Kantarjian1, G. Borthakur1 1Department of Leukemia, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Houston, United States of America Background: Core-binding factor acute myeloid leukemia (CBF-AML), including inv (16) and t (8;21) AML, represent 15%-20% of all adult AML patients (pts) and is generally recognized as favorable AML subgroups. Despite being favorable, up to 30-50% of patients with CBF-AML eventually relapse. Both RAS (particularly multiclonal) and KIT mutations in CBF-AML have been associated with poor outcomes, but our initial analysis for patients treated with a fludarabine-based regimen did not support a negative impact of these mutations. In addition, we showed that the optimal reduction of disease defining transcripts (RUNX1-RUNX1T1 and CBFB-MYH11) by PCR at end of induction and end of consolidations, has the most impact on the relapse free survival of these patients. Aims: We studied the prognostic impact, in a consecutive cohort of CBF-AML patients treated uniformly, of kinase mutations (RAS and KIT) and the number (Single vs. multiple) of mutations in addition to the impact of mutation status on attainment of optimal PCR reductions at most informative pre-defined time points; end of induction and end of treatment. Methods: Pts aged ≥ 18 years (yrs) with de novo CBF-AML, treated with FLAG based regimen with idarubicin or gemtuzumab ozogamicin between April 2013 and December 2019 at our center were included in this analysis. RAS and KIT mutations were tested by next generation sequencing. PCR for disease-specific transcripts was monitored at the end of C1 and end of therapy (EOT). Optimal response was considered as PCR transcripts < 0.1% post C1 and <0.01% at EOT. Results: A total of 91 pts with CBF AML were evaluated with a median age of 52 yrs (range, 22-79), 41 of whom were females (45%). Fifty-three pts (58%) had Inv 16 and the rest had t (8;21). Twenty-six pts (29%) had FLT3 mutation (ITD and/or TKD), 32 pts (35%) had RAS mutation (19 pts with NRAS, 3 pts with KRAS, and 10 pts with both mutation), and 30 pts (33%) had KIT mutation. Out of 32 pts with RAS mutation, 16 (50%) had single mutation and16 (50%) had multiple RAS mutations, while of 30 pts with KIT mutation, 25 pts (83%) had single KIT mutation, and 5 pts (17%) had multiple KIT mutations. On regression analysis of presence of KIT or RAS mutation and their impact on end of induction and end of treatment PCR, only KIT mutation was associated with lower odds (0.33, 95% CI 0.11-0.9) of optimal EOT response. Amongst mutated pts, the presence of single versus multiple KIT or RAS mutation did not affect optimal PCR responses. At a median follow up of 53.1 months (mos), the median relapse free survival (RFS) for the whole cohort was 27.7 mos (range,1.7-93.6). RAS mutated pts had longer RFS compared to non-mutated pts (not reached vs. 43.78 mos, p=0.02); however, KIT mutation did not affect RFS. In addition, the number of the RAS or KIT mutation (One mutation vs. multiple mutations) also did not impact RFS. Image: Summary/Conclusion: KIT mutation might negatively affect attainment of optimal PCR responses at end of FLAG based therapy, but it lacked significant impact on relapse free survival. Single versus multiple KIT or RAS mutations also do not seem to have an impact on therapy response. P498: CLINICAL AND BIOLOGICAL MARKERS ASSOCIATED WITH LONG-TERM SURVIVAL FOR PATIENTS WITH ACUTE MYELOID LEUKEMIA (AML) IN REMISSION AFTER CHEMOTHERAPY IN THE QUAZAR AML-001 TRIAL OF ORAL AZACITIDINE A. H. Wei1,*, H. Döhner2, H. Sayar3, F. Ravandi4, P. Montesinos5, H. Dombret6, D. Selleslag7, K. Porkka8, J.-H. Jang9, B. Skikne10, C. Beach10, T. Prebet10, G. Zhang10, A. Risueño11, M. Ugidos Guerrero11, W. L. See10, D. Menezes10, G. J. Roboz12 1Department of Clinical Haematology, Alfred Hospital, and the Australian Centre for Blood Diseases, Monash University, Melbourne, Australia; 2Department of Internal Medicine III, Ulm University Hospital, Ulm, Germany; 3Indiana University Cancer Center, Indianapolis; 4Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America; 5Hospital Universitario y Politécnico La Fe, Valencia, Spain; 6Hematology, Hôpital Saint-Louis, Assistance Publique – Hôpitaux de Paris (AP-HP), and Institut de Recherche Saint-Louis, Université de Paris, Paris, France; 7AZ Sint-Jan Brugge-Oostende AV, Bruges, Belgium; 8HUS Comprehensive Cancer Center, Hematology Research Unit Helsinki and iCAN Digital Precision Cancer Center Medicine Flagship, University of Helsinki, Helsinki, Finland; 9Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 10Bristol Myers Squibb, Princeton, United States of America; 11BMS Center for Innovation and Translational Research Europe (CITRE, a Bristol-Myers Squibb Company), Seville, Spain; 12Weill Cornell Medicine and New York Presbyterian Hospital, New York, United States of America Background: In the randomized, phase 3 QUAZAR AML-001 trial, oral azacitidine (Oral-AZA) significantly prolonged overall survival (OS) compared with placebo (PBO) (median OS 24.7 vs 14.8 months [mo], respectively) in older patients (pts) with AML in first remission after intensive chemotherapy (IC). At an updated data cutoff performed in Sep 2020, 34.9% of pts in the Oral-AZA arm and 24.4% of pts in the PBO arm remained alive at ≥3 y from randomization. Aims: Assess clinical and biological variables associated with long-term survival (LTS) in QUAZAR AML-001. Methods: In all, 472 pts were randomized 1:1 to receive Oral-AZA 300 mg or PBO QD ×14d/28d within 4 mo of achieving first complete remission (CR) or CR with incomplete blood count recovery (CRi) after IC. The primary endpoint was OS, time from randomization until death, withdrawal of consent, or loss to follow-up. The LTS cohort comprised pts who were alive ≥3 y from randomization as of Sep 2020 and the non-LTS cohort included pts who died or were censored before 3 y. Variables assessed for association with LTS were diagnostic (Dx [pre-IC]) features (AML subtype, cytogenetic risk, NPM1 and FLT3 mutations [mut]); pre-study treatment (Tx) variables (response to IC [CR/CRi], receipt of consolidation, number of consolidation cycles); baseline (BL) demographic and disease characteristics, hematologic parameters (red blood cells [RBCs], hemoglobin, platelets, and leukocyte subsets), and measurable residual disease (MRD) status; and post-BL variables (MRD response [conversion from MRD+ at BL to MRD– on study], timing of MRD– [MRD– response on-study vs BL MRD–], and receipt of transplant after Tx discontinuation [D/C]). Associations of LTS with bone marrow immune parameters (CD3, CD4, and CD8 T-cell counts, and expression of PD-1/TIM-3 T-cell exhaustion markers) were investigated in a subset of pts (n=108). Variables were compared within Tx arms (LTS vs non-LTS) in univariate analyses with P values corrected for multiple testing. A logistic multivariable regression analysis of the effects of prognostic BL covariates on LTS was performed. Results: The LTS cohort included 83/238 pts (34.9%) in the Oral-AZA arm and 57/234 pts (24.4%) in the PBO arm. Within both arms, factors significantly associated with LTS were intermediate (Int)-risk cytogenetics and NPM1 mut at Dx, and MRD response (MRD+ to MRD–) on study (Figure). MRD response rate was 2-fold higher with Oral-AZA vs PBO (37% vs 19%, respectively), and while early attrition was more common in the PBO arm, most MRD responses occurred within 6 mo. Factors significantly associated with LTS only in the PBO arm were BL (post-IC) MRD– status and receipt of transplant after Tx D/C. No significant associations were observed between BL hematological or immune parameters and LTS. The multivariable analysis (MVA) confirmed Oral-AZA Tx as independently significantly predictive of LTS vs PBO. Other covariates significantly associated with LTS in MVA were Int-risk cytogenetics and NPM1 mut at Dx, and MRD– status at BL. Image: Summary/Conclusion: Oral-AZA Tx was significantly associated with LTS vs PBO. In univariate analysis, Int-risk cytogenetics and NPM1 mut at Dx, and MRD response on-study, were significantly prognostic of LTS in both arms, whereas MRD– status at BL (post-IC) was associated with LTS only in the PBO arm. P499: THE PHASE 1B OMNIVERSE TRIAL OF ORAL AZACITIDINE IN COMBINATION WITH VENETOCLAX FOR TREATMENT OF RELAPSED/REFRACTORY OR NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA (TRIAL IN PROGRESS) A. H. Wei1,*, H. E. Carraway2, L. Taningco3, E. Laille3, J. Gong3, T. Prebet3, D. Lopes de Menezes3, F. Ravandi4 1Department of Clinical Haematology, Alfred Hospital, and the Australian Centre for Blood Diseases, Monash University, Melbourne, Australia; 2Leukemia Program, Taussig Cancer Institute, Cleveland Clinic, Cleveland; 3Bristol Myers Squibb, Princeton; 4Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: For older patients (pts) with acute myeloid leukemia (AML) who are ineligible for standard intensive chemotherapy (IC) regimens, traditional lower-intensity AML treatment (Tx) options include low-dose cytarabine (LDAC) or hypomethylating agents (HMAs; azacitidine [AZA] and decitabine), which are generally well tolerated but associated with suboptimal outcomes vs IC [Vey 2020]. Therefore, there remains a need for less toxic and more efficacious lower-intensity AML Tx regimens, especially agents that can be administered in the outpatient setting, which may increase convenience and reduce resource utilization. Venetoclax (VEN) is an oral, selective, small-molecule BCL2 inhibitor that has demonstrated considerable activity when combined with LDAC or HMAs in older pts with AML. In a recent phase 3 trial, VEN + injectable AZA significantly improved response and survival vs AZA alone in IC-ineligible pts with newly diagnosed (ND) AML [DiNardo 2020]. In the USA, VEN is approved in combination with an HMA or LDAC for Tx of pts with ND AML ≥ 75 years (y) of age or who cannot use IC due to comorbidities. Oral-AZA (CC-486) is approved for pts with AML in first remission (CR1) after IC who are ineligible for transplant or other curative therapies. In the randomized, phase 3 QUAZAR AML-001 trial of Oral-AZA in pts with AML in CR1 after IC, Oral-AZA 300 mg QD for 14 days (d)/28d Tx cycle was generally well tolerated and associated with significantly improved overall survival vs placebo. Gastrointestinal symptoms were the most common adverse events reported with Oral-AZA [Wei 2020]. Oral-AZA has also shown clinical activity in pts with active AML [Savona 2015]. As incorporation of AZA into DNA is S-phase-restricted, extended Oral-AZA dosing regimens (> 7d/28d Tx cycle) increase drug incorporation into cycling tumor cells to prolong epigenetic activity throughout the Tx cycle [Laille 2015]. An all-oral VEN + Oral-AZA combination regimen allows for outpatient administration and thus improves pt adherence [Eek 2016]. Aims: Describe the study design and objectives of the OMNIVERSE trial of Oral-AZA + VEN in older pts with ND AML or pts with AML relapsed/refractory (R/R) to prior Tx. Methods: OMNIVERSE (NCT04887857) is a multicenter, open-label, 2-part phase 1b trial (Figure). The key objectives of the trial are to assess safety and determine the maximum tolerated dose of Oral-AZA + VEN in pts with R/R AML (WHO 2016 criteria) ineligible to receive further IC (part I), and then in pts with ND AML ≥ 75 y of age, or those ≥ 18–74 y of age ineligible for IC or HSCT due to comorbidities (part 2). Main eligibility criteria include an ECOG performance status of 0–2 (ECOG 3 is allowed for pts ≥ 18–74 y of age with comorbidities) and an unfavorable cytogenetic risk profile for pts with ND AML. The initial dose of Oral-AZA is 300 mg QD × 14d/28d cycle, with de-escalation to 200 mg × 14d/28d cycle allowed for dose-limiting toxicities. VEN 400 mg is taken orally QD in continuous 28d cycles (or for 21d/cycle for dose level –2). A modified toxicity probability interval-2 design is used to evaluate the planned dose levels. The sample size is dependent on the dose levels included in the study (≤ 18 pts/part). Results: N/A Image: Summary/Conclusion: Study enrollment began in 2021. The trial is ongoing at clinical sites in the United States and Australia. P500: A REAL WORLD MULTI CENTRE STUDY OF CPX-351 REVEALS NO DIFFERENCE IN OVERALL SURVIVAL WHEN COMPARED WITH FLAG-IDA AND 3 + 7 IN HIGH RISK AML. C. Andrews1,*, I. AlNabhani1, T. Young1, E. G. Atenafu1, S. E. Assouline2, J. M. Brandwein3, S. M. Chan1, S. Chow4, D. Khalaf5, V. Gupta1, D. D. H. Kim6, D. Maze1, M. M. Minden1, T. Murphy1, D. Sanford7, A. D. Schimmer1, A. C. Schuh1, J. Sibai1, K. Yee1, H. Sibai1 1Division of Medical Haematology and Oncology, Princess Margaret Cancer Centre, Toronto; 2Division of Hematology, Sir Mortimer B. Davis-Jewish General Hospital, Montreal; 3Division of Hematology, University of Alberta, Edmonton; 4Odette Cancer Centre, Sunnybrook Health Science Centre, Toronto; 5Division of Hematology, Juravinski Cancer Centre, McMaster University, Hamilton; 6Hans Messner Allogeneic Blood and Marrow Transplant Program, Princess Margaret Cancer Centre, Toronto; 7Leukemia/Bone Marrow Transplant Program of British Columbia, University of British Columbia, Vancouver, Canada Background: CPX-351 is a liposomal formulation of daunorubicin and cytarabine in a fixed synergistic ratio of 5:1. It has been approved by both the FDA and EMA for use in high-risk AML including therapy related AML (t-AML) and AML with myelodysplastic related change (AML-MRC). However, this is little data of CPX-351 in a real-world setting. There is also no data comparing CPX-351 to FLAG-IDA which is used in our institution front line for high-risk AML Aims: The objective of our multi-centre study was to assess outcomes of CPX-351 and then compare these outcomes in patients who received FLAG-IDA and 3 + 7 for high risk AML(tAML and AML-MRC). Methods: Patients aged 18 and over who were treated with induction chemotherapy and who met the WHO criteria for t-AML and AML-MRC were included in the study. Retrospective data was collected from 10 centres throughout Canada for CPX-351. The Princess Margaret Cancer Centre database was used to collect data for patients who received induction with FLAG-IDA and 3 + 7. Targeted sequencing was performed on DNA samples using the TruSight Myeloid Sequencing Panel. Overall survival (OS) and progression free survival (PFS) rates were calculated using the Kaplan-Meier method. Results: 76 patients treated with CPX-351 were identified with baseline characteristics seen on Table 1. Targeted sequencing was performed on 72% of patients (55/76) and the average number of mutations was 2(0-7). RUNX1 was the most commonly mutated gene found in 22% (12/55), followed by ASXL1 mutated in 19% (10/55) and SRSF2 in 15%. Assessing treatment responses, 53% (38/76) of patients achieved a complete remission (CR) or a complete remission with incomplete recovery (CRi). The median CR duration was short at 7.3 months. Median follow up was 7.78 months (range 0.2 to 20 months). OS was 57% at 12 months and 38% at 18 months. PFS was 40% at 12 months and 23% at 18 months. There were no differences in OS when stratified by ELN risk (p=0.46), adverse risk cytogenetics (p=0.1485), or poor risk mutations such as RUNX1 (p=0.73), ASXL1 (p=0.47) or TP53(p=0.53).Patients who received an ASCT had significant improvement of OS of 82% at 18 months compared to those who did not receive a transplant of 15% at 18 months (Fig 2; p=0.0001). Patients who received 3 + 7 and FLAG-IDA for the same indications as CPX-351 (tAML and AML-MRC) were identified. Baseline characteristics are shown in Table 1. 69% of patients receiving FLAG-IDA and 67% of those receiving 3 + 7 had adverse ELN risk stratification. CR rates were highest with FLAG IDA with 75% achieving a CR or CRi. 68% of patients who received CPX-351 proceeded to ASCT compared with 59% with FLAG-IDA and 57% with 3 + 7. OS at 18 months was 42% for FLAG-IDA and 39% for 3 + 7. There was no statistical difference in OS when comparing CPX-351 to FLAG-IDA and 3 + 7 (p=0.855) (Figure 1). Similarly, there was no difference in PFS when comparing all 3 inductions (p=0.26). Patients who received a ASCT had significant improvement of OS of 59% at 18 months compared to those who did not receive a transplant of 24% at 18 months (p=<0.0001)irrespective of what induction was used. Image: Summary/Conclusion: High risk AML remains an unmet clinical need which is supported by our study which reveals no difference in OS or PFS when treated with either CPX-351, FLAG-IDA or 3 + 7. Unsurprisingly, ASCT improved OS across all patients irrespective of induction type. Further study with a prospective randomised control trial is required. P501: CLI120-001 PHASE1B DOSE ESCALATION STUDY OF RVU120 IN PATIENTS WITH AML OR HIGH RISK MDS SAFETY AND EFFICACY DATA UPDATE C. Abboud1, Z. Jan Maciej2, G. Borthakur3, S. Solomon4, B. Howard5, T. Bradley6, E. Mouhayar7, N. Angelosanto8,*, H. Nogai8, A. Glasmacher9, R. Dudziak8, K. Brzozka10, T. Rzymski10, P. Littlewood11, E.-L. Maranda12 1Division of Oncology, Washington University In Saint Louis, Saint Louis, United States of America; 2Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk, Poland; 3Department of Leukemia, MD Anderson Cancer Center, Houston; 4Blood and Marrow Transplant Leukemia and Immunotherapy Unit, Northside Hospital, Atlanta; 5Sarah Cannon Research Institute, Sarah Cannon, Nashville; 6Division of Hematology, University of Miami Health System, Miami; 7Department of Cardiology, MD Anderson Cancer Center, Houston, United States of America; 8CLINICAL DEVELOPMENT, Ryvu therapeutics, Krakow, Poland; 9Dep. of Internal Medicine III, University Clinic Bonn, Bonn, Germany; 10Research & Preclinical Development; 11DMPK Department, Ryvu Therapeutics, Krakow; 12Deaprtment of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland Background: CDK8 and its paralog CDK19 have central roles in maintenance of cancer cell viability and undifferentiated state for a variety of tumor types. (Dannappel et al. 2019; Rzymski et al. 2015; Philip et al. 2018). RVU120 (SEL120), a novel CDK8/CDK19 kinase inhibitor with significant efficacy in preclinical AML models, has shown clinical efficacy in a currently ongoing phase Ib trial in patients with relapsed/refractory (R/R) AML or HR-MDS (NCT04021368). This paper provides update with new available data on disease evaluation from ongoing patients and further enrolment into next cohort level 85 mg. Aims: The primary objective of the study is to determine preliminary safety profile, dose limiting toxicities (DLTs), maximum tolerated dose (MTD) and the recommended phase 2 dose of RVU120 as a single agent. Secondary objectives include PK, antileukemic activity and exploratory PD characterization. Methods: The study comprises at least 7 dose escalating cohorts. The first 3 cohorts followed an accelerated scheme, 1 patient enrolled/cohort from 10 to 50 mg dose levels, from cohort 4 (75 mg) to 7 (100 mg) doses onwards a 3 + 3 design is followed. Data from each cohort is evaluated by a data review committee (DRC). RVU120 is administered orally every other day, for a total of 7 doses, in a 3-week treatment cycle until disease progression/unacceptable toxicity. Adverse events are graded according to NCI-CTCAE v.5.0. DLTs are assessed at completion of C1. Disease evaluation is performed according to Dohner 2017 and Cheson 2006 response criteria for AML and MDS respectively. PK parameters are calculated by non-compartmental analysis. Pharmacodynamic (PD) activity is assessed by flow cytometry measure of pSTAT5 Ser725 levels, that are highly dependent on the activity of CDK8 and CDK19 in AML/MDS cells. Results: At data cut off of 23rd Feb22, 13 pts have been enrolled, median age 73 years and median 2 previous lines of therapy, ECOG PS 2 in 4 pts, 1 in 7, 0 in 2. No DLTs were observed, all 14 Serious Adverse Events, including 1 COVID19 death and 1 pancreatitis, were not related to study drug (G1 fever, G2 Upper Respiratory Infection, G3: pseudomonas sepsis; urinary tract infection; febrile neutropenia; lung infection, pain, hemoptysis, pleural effusion, G5 pneumonitis, death NOS, pancreatitis). Cohort 1 pt, 10 mg dose level, and cohort 2 pt, 25 mg, showed stable (SD) and progressive disease (PD) respectively at the end of C1. Cohort 3 pt, an 81 YO male HR-MDS, escalated from 50 to 75 mg dose from C7, is SD at C24D13 with Erythroid Hematological Improvement on C5, C7, C10, C18. Cohort 4, 75 mg dose pt, a 62 YO male with AML DNMT3A pos, relapsing after Ven/Dec, achieved CRi at the end of C1 and CR in C7, and progressed at the end of C8. Two out of the remaining 4 pts treated at 75 mg reached SD (1 still ongoing at C3D15 and another died on C3D20 while on SD), 1 pt died of COVID-19 pneumonitis on C1D18, 1 pt with AML secondary to MPN was SD at C2 and progressed on C4. Two pt were treated at 110 mg (cohort 5), 1 not evaluable died for pancreatitis and 1 was SD at the end of C1. 2 pt entered cohort 6, 85 mg, and will be evaluable at the end of March 2022. Summary/Conclusion: Preliminary results from the first 6 cohorts have shown a favorable safety and a predictable PK profile of RVU120. Meaningful PD activity and clinical efficacy were observed at 50 and 75 mg doses. Enrollment is currently ongoing at 85 mg cohort P502: CLINICAL IMPLEMENTATION OF GERMLINE GENETIC TESTING FOR HEMATOLOGIC DISORDERS S. Ansar1,*, J. Malcolmson2,3, K. M. Farncombe4, K. Yee1, R. H. Kim1,5,6, H. Sibai1 1Division of Medical Oncology and Hematology; 2Familial Cancer Clinic, Princess Margaret Cancer Centre, University Health Network; 3Department of Molecular Genetics, University of Toronto; 4Toronto General Hospital Research Institute, University Health Network; 5Division of Clinical and Metabolic Genetics, The Hospital for Sick Children; 6Department of Medicine, University of Toronto, Toronto, Canada Background: Up to 18% of adult hematology patients (pts) suspected of having an inherited predisposition are found to have a germline mutation leading to a hereditary hematologic disorder (HHD). Timely identification of these disorders leads to heightened surveillance for malignancies, specific treatment regimens, donor selection, transplant conditioning regimens, cascade testing in family members and supportive care. Unlike solid tumour hereditary cancer syndromes, the investigations in HHD are more complex and there is no formal consensus of referral criteria or genetic testing criteria. We describe our initial experience in this patient population at our centre, the Princess Margaret Cancer Centre (PM). We have established a workflow to initiate germline genetic testing in pts under suspicion for a HHD. This involves a collaboration with a genetics clinic to 1) capture pts based on their personal/family history of cancer and suggestive genetic findings identified on bone marrow/blood, 2) procure a skin biopsy for fibroblast culture and germline DNA extraction and 3) provide follow-up counselling and management for positive results. Aims: Our goal is to evaluate the positivity rate of germline mutations in adult hematology pts who were referred for genetics assessment. Methods: The PM is the largest leukemia Centre in Canada, and has seen over 3000 adult pts with a malignant disorder from 2015-2021 (AML:1597, MDS:472, ALL:286, MPN:664 and marrow failure:22). We performed a retrospective chart review of all adult hematology pts referred to cancer genetics service for HHD workup during this time period. Results: 116 pts (66 male, 50 female) were suspected for a HHD and referred for germline genetic testing on fibroblast DNA. 52 pts (45%) were ≤40y old and 64 pts (55%) >40y old (age range 18-86y, median 53y). 71 pts (61%) were referred with a positive family history of hematologic disorders (HD). In addition, 61 pts presented with a myeloid malignancy (46 with MDS, AML, or CML, and 15 with a MPN), 42 presented with a lymphoid malignancy (ALL, CLL, or lymphoma), 8 presented with bone marrow failure, and 5 presented with other HD. 40 pts were found to have a germline genetic mutation, 7 of which were associated with carrier status. In total, 33 (28.4%) referred pts were found to have at least one actionable germline mutation. This corresponds to a positive genetic result found in 30% of pts referred with MDS/AML, 33% of pts referred with a lymphoid malignancy, and 18% of pts referred with bone marrow failure, MPN, or other HD. Additionally, 18 (55%) of these pts had a positive family history of HD, while 15 (45%) presented with no family history of HD. Of these 33 positive cases, 13 (11.2% of all referrals) occurred in a gene associated with a hematologic malignancy and resulted in a HHD diagnosis. 20 pts were found to have a mutation in a gene associated with a non-hematologic hereditary cancer syndrome. After the implementation of a HHD genetics workflow in 2018, we saw an increase in the total number of referrals to a genetics clinic over the past seven years, with 25 pts (6 positive cases) referred between 2015-2018, and 91 pts (27 positive cases) referred between 2019-2021. Image: Summary/Conclusion: Our overall positivity rate was 28.4% which includes mutations in any hereditary cancer gene, and 11.2% for HHDs. Our high pickup rate across all age groups and the increase in the number of referrals to genetics service suggests that more pts with HD, including older pts, would benefit from consultation with specialized centres that are experienced in the evaluation and treatment of these disorders. P503: MRD MONITORING DURING INTENSIVE CHEMOTHERAPY IN PEDIATRIC AML- DATA OF THE AML-BFM GROUP E. Antoniou1,2,*, S. Hahn1,2, S. Sendker1,2, N. von Neuhoff1,2, D. Reinhardt1,2, M. Schneider1,2 1Clinic of pediatrics III, University hospital of Essen; 2AML-BFM study group, Essen, Germany Background: During the past decades, minimal residual disease (MRD) has been established as a diagnostic tool, providing critical prognostic information in pediatric acute myeloid leukemia (AML). To measure initial treatment response, immunophenotyping was used by most cooperative study groups. However, known limitation of sensitivity and specificity supported the continuous evaluation of qPCR-based MRD detection in pediatric AML. Aims: To determine the prognostic role of MRD monitoring by RT-qPCR in the pediatric AML. Methods: A total of 238 pediatric AML patients were monitored for MRD by reverse-transcriptase quantitative PCR (RT-qPCR) assay from September 2012 to December 2020. Since the inclusion to this study is based on the presence of quantifiable genetic markers, the patient group is strongly biased. The treatment was conducted according to the AML-BFM study 2004 and 2012 as well as the register 12 and 17. All measurements were performed in bone marrow samples or in peripheral blood at initial diagnosis. MRD monitoring included the following aberrations: t(8;21), inv(16), inv(11), NPM1, FLT3-ITD, t(15;17), t(5;11), t(9;11), t(6;9), WT1, t(1;11), t(11;11), t(1;22), t(11;12), t(17;19), t(3;5), t(4;10), t(11;19), t(2;11), t(6;11), t(X;11), t(6;8), t(8;16) and t(8;22). Results: The patients had a mean age at diagnosis of 9.2 years (range 10 days to 18.6 years) and 51 % were females. They were distributed to the following stratification groups: standard risk (56 %), intermediate risk (27 %) and high risk (17 %). The event free survival was 71 % (±3 %) and the OS reached 90 %(±2 %). In core binding factor acute myeloid leukemia (CBF-AML) (n=91) a positive MRD level persists in the majority of cases. Following the 3rd treatment block 64% of cases remained MRD-positive (MRD threshold ≥1x10-5). However, MRD monitoring showed no prognostic relevance as relapses were detected in 18 % (6/33) and 17 % (10/58) of MRD-positive and negative cases, respectively. For patients with NPM1 mutations (n=26) MRD-positivity and -negativity (threshold of ≥1x10-6) showed a similar relapse rate of 10 %. This data are in contrast to reports in adults (Ivey et al. NEJM 2016). In patients with an AML and t(9;11) MRD-positivity after first and second induction indicated a significant higher risk of relapse. After first induction MRD-positive cases (n=19) had a relapse rate of 32 % compared to 13 % for negative cases (n=24). After second induction the relapse rates were 63 % and 22 % for MRD-positive (n=8) and negative (n=27) cases, respectively. Summary/Conclusion: MRD monitoring is a powerful tool to improve risk group stratification in pediatric AML. Despite its high sensitivity and specificity, the prognostic informativeness of MRD monitoring through RT-qPCR varies widely among different genetic subgroups. In CBL-AML or NPM1-positive AML, which are prognostically more favorable, persisting MRD-positivity alone was not associated with an increase in the risk of relapse. In contrast, MRD-positivity in AML with t(9;11) appears to be highly informative. Further studies with larger number of patients are needed to further define AML subgroups and thresholds for MRD-positivity in the pediatric AML. P504: CLINICAL MUTATIONAL ANALYSIS BY NEXT-GENERATION SEQUENCING AND REAL-LIFE VALIDATION OF THE REVISED 2017 EUROPEAN LEUKEMIANET GENETIC RISK STRATIFICATION IN AML PATIENTS C. Aparicio Pérez1,*, F. Salas Hernandez1, A. C. Gonzalez Teomiro1, F. Jimenez Najar1, I. Fernandez Camacho1, C. Martin Calvo1, J. Sánchez García1,2, J. Serrano López1,2 1hematology, Reina Sofia University Hospital; 2IMIBIC, Córdoba, Spain Background: Recently, a census of mutated genes in AML has been described but their value in clinical practice is not fully elucidated. The revised 2017 European LeukemiaNet (ELN) recommendations for genetic risk stratification of AML have been widely adopted, but have not yet been validated in large cohorts of AML patients in real life. Aims: The objective of this work is to analyze the mutations detected by Next Generation Sequencing (NGS) and to study whether the application of ELN 2017 scale improves prognostic risk stratification with respect to ELN 2010 and Medical Research Council (MRC). Methods: We included 112 adult AML patients diagnosed in our center between Jun2017 and Dec2021 and who were studied at diagnostic using the spanish PLATFOLMA PETHEMA. The prognostic risk was established according to MRC, ELN2010 and ELN2017 classification. Baseline demographic, disease characteristics, treatment procedures and mutations by functional groupsare summarized in Table 1 NPM1 and FLT3-ITD were determined by melting curve analysis and standard PCR-EC technique according to Thiede et al (Blood 2002) in ABI 3130 Analyzer (Thermofisher). For NGS, the commercial panel Myeloid SolutionTM (Sophia Genetics) KAPA Kit amplification libraries and sequencing on ILUMINA Myseq platform were used. Variant analysis was performed using DDM software (Sophia Genetics). Results: The majority of the patients (97.1%) had at least one mutation at diagnosis detected by NGS. The median number of mutations was 2.36 (0-6). Grouped by functional groups, the most frequent were those related to DNA methylation (44.6 %) and signaling/kinase pathways (37.5%). The most prevalent were FLT3 (29 %), IDH1/IDH2 (27%) and TP 53 (19.6%) followed by NPM1 and RUNX-1 (17%). Patients older than 60 years, presented higher percentage of unfavorable ASXL1 (18.6%), RUNX1 (20%) and TP53 (25.7%) mutations with respect to younger (4.8%, 11.9% and 9.5 %) being these differences statistically significant in ASXL 1 and TP 53 (χ2 p=0.044 and p=0.047 respectively). NPM1 (28.6%) and FLT3 (40.5%) mutations were significantly more frequent in younger patients (χ2 p=0.017 and p=0.013 respectively), than in >60 years (10% and 17.1%, respectively). Re-stratifying according to ELN 2017, the 53% of patients categorized within the intermediate risk group according to MRC change prognostic group: 28.9% became redefined as unfavorable risk and 24.1% as favorable. In the overall series the overall survival analysis shows statistically significant differences taking into account either ELN 2017 or ELN 2010 and MRC (T.logrank p=0.003, p=0.018 and p=0.012, respectively). However, according to ELN 2017 there are greater differences between intermediate and unfavorable groups than in the other classifications. Statistically significant differences in overall survival according to ELN 2017 (T.logrank p=0.028) are also found in patients receiving intensive treatment. The estimated overall survival at two years is 72.%, 52.1% and 46.8% in the favorable, intermediate and unfavorable group, respectively. Image: Summary/Conclusion: - NGS proves its usefulness by detecting more clinically relevant alterations than conventional cytogenetic techniques and PCR, and stratifies a larger group of patients as favorable and unfavorable. - Our results validate the prognostic significance of the ELN2017 classification in real life, both in the overall series and in candidates for intensive QT. - ELN 2017 establishes greater survival differences between the intermediate and unfavorable group than ELN 2010 and MRC redefining the intermediate group. P505: SAFETY AND EFFICACY OF CASEIN KINASE 1Α AND CYCLIN DEPENDENT KINASE 7/9 INHIBITION IN PATIENTS WITH RELAPSED OR REFRACTORY AML: A PHASE 1, FIRST-IN-HUMAN STUDY OF BTX-A51 B. J. Ball1,*, G. Borthakur2, A. Stein1, K. W. Chan3, D. Thai3, E. M. Stein4 1Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte; 2Leukemia, MD Anderson Cancer Center, Houston; 3BioTheryx, San Diego; 4Hematologic Malignancies, Memorial Sloan Kettering Cancer Center, New York, United States of America Background: Inactivation of p53 and overexpression of Mcl1 are common mechanisms that cancer cells use to evade apoptosis. BTX-A51 is a novel, oral, direct inhibitor of casein kinase 1α (CK1α), cyclin dependent kinase 7 (CDK7), and CDK9. CDK7 and CDK9 phosphorylate RNA polymerase II (Pol II) to enable transcriptional initiation and elongation, particularly at large clusters of transcriptional enhancers termed super-enhancers (SE). Preclinical studies have demonstrated that BTX-A51 robustly increased p53 protein levels via CK1α inhibition and Mdm2 downregulation while preferentially decreasing SE transcription of key oncogenes such as Myc and Mcl1, enabling selective apoptosis of leukemia cells. Here, we report the interim results of the phase 1, first-in-human (FIH) study of BTX-A51 in patients (pts) with R/R AML. Aims: 1. To evaluate the safety and preliminary efficacy of BTX-A51 in patients with R/R AML 2. To determine the pharmacokinetics and pharmacodynamics of BTX-A51 Methods: This is an open-label, multi-center, FIH Phase 1 study. The study utilizes a hybrid accelerated titration with single pt cohorts and a Bayesian optimal interval design to assess 9 potential dosing cohorts. Key eligibility criteria include age ≥ 18 years, R/R AML or R/R high-risk MDS, ECOG ≤ 2, adequate kidney and liver function, WBC ≤ 25K/uL. PD studies include digital droplet PCR from PB to quantify target genes and measurement of macrophage inhibitory cytokine level by ELISA from serum at serial timepoints. Results: As of 25 January 2022, 30 pts (28 with AML; 2 with HR-MDS) enrolled at dose levels between 1 and 42mg; 2 pts remain on treatment. Monotherapy doses between 1 and 42 mg were administered orally 3 days/week (wk) (3 wk in a 28-day cycle) and at 21 mg (4 wk in a 28-day cycle). Baseline characteristics include median age 75 years, median number of prior therapies 3, 97% received prior treatment with venetoclax, 97% had prior HMA, and 43% had prior induction failure. The most common treatment-emergent AEs (TEAEs) were nausea (60%), vomiting (52%), hypokalemia (48%), diarrhea (36%), and hypotension (36%). The most common Grade 3 or higher TEAEs were anemia (28%), febrile neutropenia (28%), platelet count decreased (24%), and hypokalemia (20%). The only DLTs were grade 3 hepatic failure at the 42 mg dose and grade 3 alkaline phosphatase elevation at the 21 mg dose. All events resolved after holding study drug. Plasma PK of BTX-A51 was roughly dose-proportional between 1 and 42 mg with accumulation based on AUC between Day 1 and Day 5. Estimated half-life was between 18 and 55 hours. Among the 30 pts with R/R AML and MDS, CR/CRi rate was 10% (3/30) with 1 pt at the 11 mg and 2 pts at the 21 mg dose levels attaining CRi. Bone marrow (BM) blast reduction >50% occurred in 4 patients including the 3 responders, all at the 11 and 21mg dose levels. All 4 pts with >50% BM blast reduction had RUNX1 mutations; 9 pt with RUNX1 enrolled in the trial. The median duration of response for pts achieving CR/CRi was approximately 1.5 month. Responses were not observed in MDS pts. PD data will be provided in the full presentation. Based on the clinical data from dose escalation, the RP2D is 21 mg administered 3 days/wk for 4 wk of a 28-day cycle. Image: Summary/Conclusion: In this FIH study, BTX-A51 demonstrated an acceptable safety profile and promising antileukemic activity in pts with heavily pretreated R/R AML. The 21 mg dose administered 3x/wk for 4 wk was identified as the RP2D. RUNX1 mutations were enriched among responders and pts attaining >50% BM blast reduction. P506: ACHIEVEMENT OF MEASURABLE RESIDUAL DISEASE CLEARANCE IS A STRONGER PREDICTOR OF PATIENT OUTCOME THAN TREATMENT INTENSITY IN NEWLY DIAGNOSED PATIENTS WITH ACUTE MYELOID LEUKEMIA A. Bazinet1,*, T. Kadia1, N. Short1, G. Borthakur1, S. Wang2, S. Loghavi2, J. Jorgensen2, K. Patel2, C. DiNardo1, N. Daver1, Y. Alvarado1, F. Haddad1, S. Pierce1, M. Andreeff1, E. Jabbour1, M. Konopleva1, H. Kantarjian1, F. Ravandi1 1Leukemia; 2Hematopathology, University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Modern therapy for acute myeloid leukemia (AML) can consist of either intensive or low-intensity regimens selected based on patient (pt) age/comorbidities. Measurable residual disease (MRD) has emerged as a strong independent prognostic factor in AML. It is unknown whether pts who achieve similar levels of MRD clearance experience comparable outcomes irrespective of treatment intensity. Aims: To establish the relative prognostic contribution of treatment intensity and MRD status on overall survival (OS) and relapse-free survival (RFS) in newly diagnosed AML pts who have achieved a first response. Methods: We conducted a retrospective chart review to identify non-CBF, non-APL AML pts treated at our institution between 2010 and 2021 with intensive (IA; int/high-dose cytarabine + anthracycline-based, without venetoclax) or low-intensity (Lo + VEN; low-dose cytarabine/hypomethylating agent-based, with venetoclax) regimens who had achieved CR/CRi/MLFS and undergone MRD testing by multiparameter flow cytometry at time of first response. Differences in baseline pt characteristics were evaluated using the Chi-square or Wilcoxon-Mann-Whitney tests for categorical and continuous variables, respectively. The Kaplan-Meier method was used to estimate median OS and RFS, with pts censored at the time of stem cell transplantation (SCT). Multivariate analysis was performed using a Cox proportional hazards model. Results: We identified 635 pts meeting inclusion criteria. Baseline characteristics are shown in Table 1. Compared to the IA-treated pts (n=385), pts treated with Lo + VEN (n=250) were significantly older, more likely to be male, less likely to achieve MRD clearance, and more likely to be classified as adverse risk by ELN 2017. The SCT rate in the Lo + VEN cohort was half that of the IA cohort (25% vs 50%). Median OS was 51m, 24.6m, 15m, and 9.9m in the IA MRD(-), Lo + VEN MRD(-), IA MRD(+), and Lo + VEN MRD(+) groups, respectively. Median RFS was 27.8m, 15.4m, 7.3m, and 5.2m in the IA MRD(-), Lo + VEN MRD(-), IA MRD(+), and Lo + VEN MRD(+) groups, respectively. Pts within the same MRD category (+ or -) had significantly higher OS and numerically higher RFS if treated with IA versus Lo + VEN. When the analysis was restricted to pts aged ≥ 60 years (n=56 in IA, n=239 in Lo + VEN; Fig. A-B), the differences within MRD categories did not reach statistical significance (median OS NR in IA MRD(-) vs 24.6m in Lo + VEN MRD(-), p=0.08; median OS 13m in IA MRD(+) vs 10.6m in Lo + VEN MRD(+), p=0.50; median RFS NR in IA MRD (-) vs 15.4m in Lo + VEN MRD(-), p=0.07; median RFS 25.1m in IA MRD(+) vs 5.3m in Lo + VEN MRD(+), p=0.24). The SCT rate was 29% (87/295) in the age ≥ 60 cohort. Given the confounding effect of unbalanced pt characteristics between the IA and Lo + VEN cohorts, we performed a multivariate analysis on the full population (n=635) taking into consideration the effects of age, treatment intensity, MRD status, and ELN category on OS and RFS (all significant by univariate analysis). In the multivariate analysis for OS, ELN adverse risk was the strongest predictor (HR 2.09, p<0.001), followed by MRD(+) status (HR 1.68, p<0.001), while treatment intensity and age were not significantly predictive. For RFS, ELN adverse risk (HR 2.31, p<0.001) and MRD(+) status (HR 1.98, p<0.001) were also the only two significant predictors. Image: Summary/Conclusion: In newly diagnosed AML patients, MRD status at time of first response and ELN risk are stronger predictors of pt outcome than intensity of therapy received. P507: AGREEMENT BETWEEN REAL-WORLD PHYSICIAN RESPONSE ASSESSMENT AND THE 2017 EUROPEAN LEUKEMIANET (ELN) CRITERIA IN ACUTE MYELOID LEUKEMIA (AML): A COMPARATIVE ANALYSIS P. A. Patel1,*, F. Hoff1, A. J. Belli2, E. Hansen2, C. Anderson2, A. Barcellos2, L. L. Fernandes2, H. Foss2, M. He2, M. Schulte2, C.-K. Wang2 1University of Texas Southwestern Medical Center, Dallas, TX; 2COTA, Inc., New York, NY, United States of America Background: Real-world data (RWD) is a valuable resource to understand the experience of oncology patients treated outside a clinical trial. Increasingly, RWD is being used in support of regulatory decision-making to evaluate outcomes compared to what is observed in clinical trials. In the real-world setting, physicians often do not utilize the specific inclusion/exclusion criteria and objective response criteria that are implemented in clinical trials. Rather, a descriptive assessment of response and clinical benefit is often used. Additionally, response assessment frequency may not adhere to the requirements from clinical trials. Subsequently, it is critical that real-world, physician-assessed responses are investigated to understand the concordance with objective criteria in order to contextualize endpoints reported using RWD. Aims: To evaluate the concordance and performance of physician-assessed response in the COTA real-world database to derived response using the 2017 ELN criteria as the established standard for response assessment in clinical trials. Methods: A total of 879 patients meeting the following criteria were identified in the COTA database: diagnosed with AML on or after April 1st, 2017, age ≥18 years at diagnosis, received systemic treatment after diagnosis, and had at least 28 days of follow up or a post-therapy bone marrow assessment. The COTA database is a USA-based dataset composed of longitudinal, de-identified data on the diagnosis, clinical management, and outcomes of patients with cancer. Physician-assessed response was manually captured as documented in electronic health records (EHRs). Derived response was defined as 2017 ELN response based on bone marrow findings and complete blood count (CBC) results available in the EHR. Rates of agreement and disagreement between physician-assessed and derived response to first line treatment were calculated. Overall response rate (ORR) was estimated by both response definitions. Results: The study population (n=879) had a median age of 67 years and were predominately white (78%), treated in the community setting (65%), and adverse risk per ELN criteria (42%). Overall agreement between response categories was 65.1% with the highest agreement among CR (61.7%). Agreement by response category is shown in Table 1. In a selected group of patients with all required bone marrow and lab values to derive ELN response (n=435), the ORR (95% confidence interval) was 80.5% (76.4, 84.1) for derived responders and 79.1% (75, 82.8) for physician-assessed responders. Image: Summary/Conclusion: In our real-world cohort of patients with AML, agreement between derived response per 2017 ELN criteria and physician-assessed response was 65.1%. Among patients with derivable ELN response, the ORR calculations were similar by both response definitions. These findings are significant in showing that responses assessed by physicians treating patients with AML in the real-world setting are comparable to those derived via 2017 ELN criteria. Of interest, discordance in derived response vs. physician-assessed response was most common in the setting of CRi and MLFS. These two response types require the incorporation of CBC results and bone marrow cellularity. Future research will investigate the outcomes of patients with discordant response assessments and incorporate additional response categories including CR with partial hematologic recovery (CRh) and CR without minimal residual disease (CR MRD-). P508: REAL LIFE EXPERIENCE USING FRONT-LINE CPX-351 FOR THERAPY-RELATED AND AML-MRC: RESULTS FROM THE SPANISH PETHEMA REGISTRY. T. Bernal1,*, G. Rad2, A. de Laiglesia3, C. Benavente4, A. Garcia Noblejas5, D. Garcia Belmonte6, R. Riaza7, O. Salamero8, A. Foncillas9, A. Roldán10, V. Noriega Concepcion11, J. Perez de Oteyza12, J. M. Bergua Burgues13, S. Lorente de Uña14, A. de la Fuente Burguera15, M. J. Garcia Perez16, J. L. Lopez Lorenzo17, P. Martinez18, C. Alaez19, M. Callejas20, C. Martinez Chamorro21, J. Rifon Roca22, L. Amador Barciela23, M. Lopez24, K. Gomez Correcha25, E. Lavilla Rubiera26, M. L. Amigo27, F. Vall-Llovera28, A. Garrido29, M. Garcia Fortes30, D. de Miguel Llorente31, A. Aules Leonardo32, C. Cervero33, R. Coll Jorda34, M. Perez Encinas35, M. Polo Zarzuela4, D. Martinez Cuadron36, P. Montesinos36 1Hematology, Hospital Universitario Central Asturias, ISPA, IUOPA; 2Hematology, Hospital Universitario Central Asturias, ISPA, Oviedo; 3Hematology, Hospital Puerta de Hierro; 4Hematology, Hospital Clinico San Carlos; 5Hematology, Hospital La Princesa; 6Hematology, Hospital Universitario Sanitas La Zarzuela; 7Hematology, Hospital Universitario Severo Ochoa, Madrid; 8Hematology, Hospital Vall d´Hebron, Barcelona; 9Hematology, Hospital Infanta Leonor; 10Hematology, Hospital Infanta Sofia San Sebastian de los Reyes, Madrid; 11Hematology, Complejo Hospitalario de A Coruña, A Coruña; 12Hematology, Hospital Madrid Norte San Chinarro, Madrid; 13Hematology, Hospital San Pedro Alcantara, Caceres; 14Hematology, Hospital Vithas Xanit Internacional, Malaga; 15Hematology, M.D. Anderson Cancer Center, Madrid; 16Hematology, Complejo Hospitalario Torre Cardenas, Almeria; 17Hematology, Fundacion Jimenez Diaz; 18Hematology, Hospital Doce de Octubre; 19Hematology, Hospital Universitario La Moncloa; 20Hematology, Hospital Universitario Principe de Asturias; 21Hematology, Hospital Universitario Quiron Pozuelo, Madrid; 22Hematology, Clinica Universitaria de Navarra, Pamplona; 23Hematology, Complejo Hospitalario Pontevedra, Pontevedra; 24Hematology, Hospital General de Valencia, Valencia; 25Hematology, Hospital Juan Ramon Jimenez, Huelva; 26Hematology, Hospital Lucus Augusti, Lugo; 27Hematology, Hospital Universitario Morales Messeguer, Murcia; 28Hematology, Hospital Mutua Tarrasa; 29Hematology, Hospital de la santa Creu i San Pau, Barcelona; 30Hematology, Hospital Universitario Virgen de la Victoria, Malaga; 31Hematology, Hospital Universitario de Guadalajara, Guadalajara; 32Hematology, Hospital Miguel Servet, zZragoza; 33Hematology, Hospital Virgen de la Luz, Cuenca; 34Hematology, ICO Girona, Hospital Universitario Dr Josep Trueta, Girona; 35Hematology, Hospital Universitario Santiago de Compostela, Santiago de Compostela; 36Hematology, Hospital Universitari i Politecnic La Fe, Valencia, Spain Background: Acute Myeloid Leukemias arising after cytotoxic therapy (t-AML) or with myelodysplasia-related changes (AML-MRC) share adverse risk features and poor outcomes after standard 3 + 7 chemotherapy. In a randomized clinical trial, CPX-351 has shown superior overall survival (OS) compared to standard anthracycline-cytarabine schedule in these AML subtypes. Aims: to evaluate the effectiveness of CPX-351 treatment in a real-world setting. Methods: adult t-AML and AML-MRC patients who have been treated upfront with CPX-351 in 35 Spanish centers between 2018 and 2021. All patients were included in the PETHEMA registry (NCT02606825). Primary end-point was OS. Secondary end points were complete remission with or without hematological recovery (CR/CRi), proportion of minimal residual negativity (MRD), rate of allogeneic hematopoietic stem cell transplant (HSCT) and safety. Results: CPX-351 was administered to 74 patients as first induction chemotherapy. Median age was 67 (63-71) years, with 40% (30/74) of female patients. ECOG performance status score was 0-1 in 85% (53/62) patients. A diagnosis of t-AML was present in 30% (22/74) patients. Hypomethylating agents were used for MDS or CMML phase in 15%. Hematopoietic Transplant Comorbidity Index was low, intermediate and high in 14% (10/74), 43% (32/74) and 43% (32/74) patients. ELN17 risk classification was favourable, intermediate, adverse and indeterminate in 9% (7/74), 38% (28/74), 46% (34/74) and 5% (4/74). CR/CRi was obtained in 43% (32/74) patients after first cycle with CPX-351. Three additional patients achieved Morphological Leukemia Free Status. A second induction with CPX-351 was administered in 5 patients with 100% CR (CR/CRi after 2 cycles 37/74, 50%). MRD was evaluated in 97% (36/37) of CR/CRi patients, being low (<0.1%) in 47% (17/36) of them. In univariate analysis, no factor was associated with response. However, 71% (5/7) response rate was observed in the favourable ELN17 genetic risk group compared to 48% (32/67) in the non-favourable. HSCT was performed in 27% (20/74) patients, in 1st CR/CRi in 70% (14/20), MLFS in 15% (3/20) and active disease in 10% (2/20). With median follow up of 11.9 months (7.2-23.6) from HSCT, median OS was not reached (95% CI 9.5-NR months (Figure). In univariate analysis no factors were significantly associated with OS after HSCT. Median time to absolute neutrophil count ≥0.5 x109/L and platelets ≥50 x109/L were 30.5 (25-35) and 30 (26-39) days, respectively. Deaths within the first 30 days after induction occurred in 12% (9/74) patients. Causes of early death: infection in 5 patients, haemorrhage in 3 and respiratory failure in 1. With 6.1 (1.8-14) months of median follow up, median OS was 10.5 (6.9-NR) months, with significant differences between HSCT and non-HSCT patients [NR, (95% CI: 12.7-NR), vs. 6 months, (95% CI: 4-NR), P<0.001]. In univariate analysis, factors associated with lower OS were non-favourable ELN17 [8.9 vs 23.5, P=0.04]; ECOG≥2 [0.7 vs 12., P=0.002]; age ≥65 [7.9 vs NR, P=0.007] and male [6.6 vs NR, P= 0.007]. In multivariate analysis, age above 65 years [HR 1.6, P<0.001], male [HR -0.9, P=0.02] and ECOG≥2 [HR 1.9, P<0.001] were associated with OS. Image: Summary/Conclusion: Our real-life cohort was comparable to the target population of the pivotal trial including patients diagnosed with sAML (t-AML, AML-MRC and ELN17 high risk AML), obtaining similar outcomes than the CPX-351 phase 3 trial arm. To optimize treatment outcomes CPX-351 should be preferably offered to t-AML and AML-MRC patients who are suitable for HSCT. P509: CLONAL HEMATOPOIESIS-ASSOCIATED MUTATIONS AS MEASURABLE RESIDUAL DISEASE MARKERS IN ACUTE MYELOID LEUKEMIA PATIENTS FOLLOWING ALLOGENEIC STEM CELL TRANSPLANTATION L. Bischof1,*, J. Ussmann1, D. Brauer1, D. Backhaus1, L. Herrmann1, G.-N. Franke1, V. Vucinic1, K. H. Metzeler1, U. Platzbecker1, S. Schwind1, M. Jentzsch1 1Hematology, Leipzig University Hospital, Leipzig, Germany Background: Clonal hematopoiesis (CH)-associated mutations (mut) are frequent in acute myeloid leukemia (AML), appear early in leukemogenesis, and often persist in remission after chemotherapy. Following allogeneic hematopoietic stem cell transplantation (HSCT), which replaces the patients’ hematopoiesis, CH mut may present useful measurable residual disease (MRD) markers. Aims: To evaluate patient specific CH-associated mut as MRD markers in AML patients (pts) after allogeneic HSCT. Methods: We analyzed 31 AML pts with CH-associated mut present at diagnosis (DNMT3A: n=17; SRSF2: n=9; IDH2: n=7; ASXL1: n=4; TET2: n=2, and JAK2: n=1), 23 pts had 1, 7 had 2, and 1 patient had 3 CH mut. All received a myeloablative (36%), reduced-intensity (48%), or non-myeloablative (16%) HSCT (median age 58, range 32-72 years). 84% were transplanted in complete remission (CR) or CR with incomplete recovery (CRi). European Leukemia Net (ELN) risk was 23% favorable, 37% intermediate, and 40% adverse. Mut-specific digital droplet PCR assays were developed using a competitive probe-approach. MRDpos was defined as variant allele frequency (VAF) ≥2% for ASXL1 & ≥0.05% for all other analyzed mut. 209 samples with a median of 6 (range 1-13) blood or bone marrow samples per patient were available after HSCT with a median of 1.2 years follow-up time for pts alive. Results: Prior to HSCT, 89% of pts remained CH-associated mut positive in CR/CRi. 11 pts relapsed after HSCT (35%), all with the same CH-associated mut present at diagnosis (median VAF at relapse 15.6, range 0.2-41.2%). Of those, 6 pts had at least 1 MRD sample available prior to relapse. In 5 pts, impeding relapse was predicted by a CH-associated mut MRD conversion with a median VAF of 0.27 (range 0.06-1.0)% at a median of 103 (range 14-199) days prior to relapse. The lead time was longest for a DNMT3A mut patient (199 days) & shortest for a SRSF2 mut patient (14 days). For the patient relapsing without prior CH MRD positivity, the last available sample was taken 169 days before relapse & was CH MRDneg. 20 pts retained remission during follow-up. Of those, 17 pts remained CH MRDneg in all samples (median samples per analyzed mutation 6, range 1-13). 2 pts had 2 MRDpos samples directly after HSCT but became MRDneg in subsequent samples. 1 patient received Enasidenib maintenance after HSCT with fluctuating CH-MRD values after HSCT (SRSF2 & IDH2, VAFs <0.05-0.34% & <0.05-0.14%, respectively). Of the pts with MRD samples within the first year after HSCT available, pts with at least 1 MRDpos sample after HSCT had a significantly higher cumulative incidence of relapse (CIR, P=.008, Figure 1A) & shorter relapse-free survival (RFS, P=.02, Figure 1B). 8 pts had concurrent NPM1 mut (n=4) or RUNX1 mut (n=4), which showed concordant MRD results in 7 pts (4 remained MRDneg, 3 concurrently converted MRDpos). 1 patient suffered a NPM1 MRDneg relapse which was predicted by 2 re-detected CH-associated mut (DNMT3A mut & IDH2 mut, 112 & 199 days prior to relapse, respectively, Figure 1C). Image: Summary/Conclusion: Prior to HSCT most CH-associated mut remain detectable in pts in CR/CRi. However, pts with at least one CH MRDpos sample following allogeneic HSCT had higher CIR & shorter RFS. All relapsing pts were positive for the same CH-associated mut present at diagnosis. 5 of 6 showed increasing VAFs prior to hematologic relapse & the majority of pts retaining remission remained CH MRDneg. While CH-associated mut in chemotherapy treated AML pts have limited MRD value, these mut are feasible MRD markers after HSCT & may inform relapse preventing therapy decisions. P510: THE IMPACT OF POST-REMISSION GRANULOCYTE COLONY-STIMULATING FACTOR USE IN THE PHASE 3 STUDIES OF VENETOCLAX COMBINATION TREATMENTS IN PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA C. DiNardo1,*, K. Pratz2, P. Panayiotidis3, X. Wei4, V. Vorobyev5, Á. Illés6, I. Kim7, V. Ivanov8, G. Ku9, C. L. Miller10, M. Zhang10, F. Tatsch10, J. Potluri10, X. Schmidt10, C. Recher11 1Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston; 2Abramson Cancer Center, University of Pennsylvania, Philadelphia, United States of America; 3Haematology Clinic and BMT Unit, NKUA, Laiko General Hospital, Athens, Greece; 4The Affiliated Cancer Hospital of Zhengzhou University/Henan Cancer Hospital, Zhengzhou, China; 5Department of Hematology, S. P. Botkin City Clinical Hospital, Moscow, Russia; 6Department of Hematology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary; 7Seoul National University Hospital, Seoul, South Korea; 8Almazov National Medical Research Centre, Saint Petersburg, Russia; 9Genentech, South San Francisco; 10AbbVie, Inc., North Chicago, United States of America; 11Centre Hospitalier Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse Oncopole, Université de Toulouse 3 Paul Sabatier, Toulouse, France Background: In the randomized Phase 3 VIALE-A (NCT02993523) and VIALE-C (NCT03069352) trials, venetoclax (Ven) + azacitidine (Aza) and Ven + low-dose cytarabine (LDAC), respectively, improved outcomes vs low intensity chemotherapy in patients (pts) with newly diagnosed acute myeloid leukemia (AML) ineligible for intensive chemotherapy (IC). Evidence is limited on the use of granulocyte colony-stimulating factor (G-CSF) and impact on outcomes in this pt population. Aims: To explore outcomes in pts with post-remission G-CSF use in the VIALE-A and VIALE-C studies. Methods: In VIALE-A, pts received Ven 400 mg or placebo (PBO) daily plus Aza 75 mg/m2 on d 1−7 (28-d cycles). In VIALE-C, pts received Ven 600 mg or PBO daily plus LDAC 20 mg/m2 on d 1−10 (28-d cycles). G-CSF use was given per institutional practice. Here, pts who achieved a best response of complete remission (CR) or CR with incomplete hematologic recovery (CRi) were analyzed by post-remission G-CSF use. Results: In VIALE-A, 190/286 pts (66%) treated with Ven+Aza and 41/145 (28%) pts treated with PBO+Aza achieved CR/CRi; 93 (49%) and 10 (24%) pts with CR/CRi received G-CSF post-remission (median time to first post-remission G-CSF use [range], 36 d [2−483] and 35 d [4−127]), respectively. In VIALE-C, 69/143 pts (48%) treated with Ven+LDAC and 9/68 (13%) pts treated with PBO+LDAC achieved CR/CRi; 30 (43%) and 2 (22%) received G-CSF post-remission (median time to first post-remission G-CSF use [range], 30 d [2−459] and 229 d [169−289]), respectively. In VIALE-A and VIALE-C, baseline demographics were generally similar in Ven-treated pts regardless of post-remission G-CSF use (Table). In VIALE-A, median duration of response (mDOR) for CR/CRi (95% CI) was not reached (NR; 17.5−NR) and was 12.9 mo (7.9−17.3) in the Ven+Aza+G-CSF and Ven+Aza+non-G-CSF groups, respectively (Table); DOR at 12 mo was 67% and 53%. In VIALE-C, mDOR was 10.8 (4.9−17.8) and 11.8 mo (5.9−NR) in the Ven+LDAC+G-CSF and Ven+LDAC+non-G-CSF groups, respectively; DOR at 12 mo was 45% and 49%. In VIALE-A, median overall survival (mOS [95% CI]) was NR (NR−NR) with Ven+Aza+G-CSF and was 21.1 mo (15.2−NR) with Ven+Aza+non-G-CSF; 12-mo OS rates were 83% and 71%. In VIALE-C, mOS was 20.8 (11.9−NR) with Ven+LDAC+G-CSF and 13.7 mo (10.8−NR) with Ven+LDAC+non-G-CSF; 12-mo OS estimates were 68% and 57%. Among 164 evaluable pts in VIALE-A, measurable residual disease response (MRD<10−3) was achieved by 67, of whom 38 (57%) had post-remission G-CSF. In VIALE-C, MRD<10−3 was achieved by 9 of 64 evaluable pts, of whom 6 (67%) had post-remission G-CSF. In VIALE-A, post-remission Gr ≥3 neutropenia and febrile neutropenia (FN) rates were 33% (n=31) and 39% (n=36) with Ven+Aza+G-CSF, and 29% (n=28) and 20% (n=19) with Ven+Aza+non-G-CSF, respectively. Median durations of post-remission Gr ≥3 neutropenia and FN in VIALE-A were 12.5 d and 8 d (Ven+Aza+G-CSF), and 16 d and 10.5 d (Ven+Aza+non-G-CSF). In VIALE-C, post-remission Gr ≥3 neutropenia and FN rates were 53% (n=16) and 23% (n=7) with Ven+LDAC+G-CSF, and 51% (n=20) and 8% (n=3) with Ven+LDAC+non-G-CSF, respectively. Median durations of post-remission Gr ≥3 neutropenia and FN in VIALE-C were 15 d and 6 d (Ven+LDAC+G-CSF), and 12.5 d and 29 d (Ven+LDAC+non-G-CSF). Image: Summary/Conclusion: In VIALE-A and VIALE-C, G-CSF was frequently used per institutional practice post-remission in responding patients to manage neutropenia. Overall, there was a trend towards shorter durations of Gr ≥3 neutropenia and FN with post-remission G-CSF use, without evidence of negative impact on DOR or OS. P511: RISK FACTORS AND INCIDENCE OF CARDIAC EVENTS IN A LARGE COHORT OF 525 ADULT PATIENTS WITH NEWLY DIAGNOSED NON-M3 ACUTE MYELOID LEUKEMIA B. Boluda1,2,*, A. Solana-Altabella2,3, I. Cano1,2, D. Martínez-Cuadrón1,2, E. Acuña-Cruz1,2, L. Torres-Miñana1,2, R. Rodríguez-Veiga1,2, D. Martínez-Campuzano1, R. García-Ruiz1, P. Lloret1, P. Asensi1, A. Serrano1, A. Osa-Sáez4, J. Agüero-Ramón-Llín4, M. Rodríguez-Serrano4, F. Buendía-Fuentes4, J. E. Megías-Vericat3, B. Martín-Herreros1,2, E. Barragán1, C. Sargas1,2, M. Salas5, M. Wooddell5, C. Dharmani5, M. A. Sanz1,2, J. De La Rubia1, P. Montesinos1 1Hematology, Hematology Department, Hospital Universitari i Politècnic La Fe, València, Spain; 2Instituto de Investigación Sanitaria La Fe; 3Pharmacy; 4Cardiology, Hospital Universitari i Politècnic La Fe, Valencia, Spain; 5Daiichi Sankyo, Inc., Basking Ridge, United States of America Background: The incidence of cardiac morbidity and mortality in acute myeloid leukemia (AML) population is not well known. AML patients (pts) receive potentially cardiotoxic drugs such as anthracyclines, QTc prolonging drugs such as FLT3 inhibitors, azoles, among others. Cardiac events can emerge in pts with previous cardiac diseases, or due to the toxicity of chemotherapy and other concomitant medications. So far, no previous studies have provided a holistic view of cardiac issues in a real-world series of AML pts. Aims: To estimate the cumulative incidence (CI) of cardiac events in a large series of unselected pts with AML in a single Spanish institution, and to identify risk factors associated with their development. Methods: Between January 2011 and June 2020, 571 pts were consecutively diagnosed with AML in Hospital La Fe, Valencia. The median follow-up of alive patients was 33 months. Clinical records were reviewed from diagnosis to death/last follow-up. Cardiac events were coded according to CTCAE. There were 525 treated pts, 285 (54%) with intensive chemotherapy and 240 (46%) with non-intensive therapy. Results: Among 46 untreated pts, 7 (15%) died due to cardiac events. Median age of 525 treated pts was 65 years, 331 (58%) male, and 73 (13.9%) had prior relevant cardiac comorbidities; 77 (14.7%) had FLT3-ITD mutation and 38 (7.2%) received front-line FLT3 inhibitors. Overall, 488 cardiac events were recorded, 19 (3.6%) pts had fatal cardiac events and 288 (54.9%) had any grade of non-fatal cardiac events. The 9-years CI of grade 1-2 QTcF prolongation was 11.2%, 2.7% pts had grade 3 QTcF prolongation >500ms, and no patient had grade 4 o 5. The 9-years CI of grade 1-2 cardiac failure was 1.3%, grade 3-4 15%, and grade 5 2.1%. The 9-years CI of grade 1-2 arrhythmia was 1.9%, grade 3-4 9.1%, and no pts had grade 5. Among 307 pts with cardiac events, 38 (12%) pts developed the first event before starting treatment, 132 (43%) during first cycle, 31 (10%) during consolidation, 55 (18%) during non-intensive further cycles, 14 (5%) during hematopoietic cell transplantation and 37 (12%) during follow-up. In the univariate analysis, pts with prior cardiac antecedents had increased incidence of fatal cardiac events compared with pts with no prior cardiac disease (CI at 9 years 20.1% vs 4.9%, p<0.001). Pts older than 65 years of age (64.4% vs 49.9%, p<0.001), prior cardiac disease (73% vs 54.6%, p=0.004) or included in clinical trial (65.2% vs 50.8%, p<0.001) had increased CI of non-fatal cardiac events. Multivariate analysis showed that prior cardiac disease was associated with increased incidence of fatal cardiac events [Hazard Ratio (HR) 1.9, p<0.001]. Age >65 (HR 2.2, p<0.001), prior cardiac disease (HR 1.4, p=0.02) and non-intensive chemotherapy (HR 1.8, p=0.004) were associated with increased CI of non-fatal cardiac event. Median overall survival (OS) in the overall cohort was 11.4 months (9.6-13.4 months, IC 95%). Among 285 intensive therapy pts, median OS was 22 months, 34 months in pts without cardiac events (n=125), 43 months with grade 1-2 (n=52), 15 months with grade 3-4 (n=98), and 5.2 months with grade 5 (n=10) (p<0.001). Image: Summary/Conclusion: We observed a high CI of cardiac events (58.5%) in a real-world series of patients undergoing AML therapy, associated with significant mortality due to cardiotoxicity (3.6%), and decreased OS. Prior history of cardiac comorbidity was associated with an increased risk of fatal cardiac events. Older age, cardiac comorbidities, and non-intensive therapies were related to non-fatal events. P512: PRELIMINARY RESULTS OF VEN-A-QUI STUDY: A PHASE 1-2 TRIAL TO ASSESS THE SAFETY AND EFFICACY OF THE COMBINATION OF AZACITIDINE OR LOW-DOSE CYTARABINE WITH VENETOCLAX AND QUIZARTINIB IN NEWLY DIAGNOSED J. M. Bergua-Burgues1,*, R. Rodríguez-Veiga2, I. Cano2, F. Vall-llovera3, A. García-Guiñon4, J. Gómez-Estruch5, M. Colorado6, I. Casas-Avilés1, J. Esteve-Reyner7, M. V. Verdugo8, F. Ramos9, M. Valero10, E. Acuña-Cruz2, B. Boluda2, L. Torres-Miñana2, J. Martínez-López11, E. Barragán2, R. Ayala11, D. Martínez-Cuadrón2, P. Montesinos2 1Hospital San Pedro de Alcántara, Cáceres; 2Hospital Universitari i Politècnic La Fe, Valencia; 3Hospital Universitari Mútua Terrassa, Barcelona; 4Hospital Arnau de Vilanova, Lleida; 5Hospital Clinico Universitario Virgen de la Arrixaca, Murcia; 6Hospital Marqués de Valdecilla, Santander; 7Hospital Clinic Barcelona, Barcelona; 8Hospital de Jerez, Jerez de la Frontera; 9Hospital Clinico de León, León; 10Hospital Arnau de Vilanova, Valencia; 11Hospital 12 de Octubre, Madrid, Spain Background: Venetoclax (VEN) combined with Azacitidine (AZA) or Low Dose Cytarabine (LDAC) has emerged as new therapeutic option for unfit acute myeloid leukemia (AML) patients (pts), but primary resistance is observed in roughly 40% of them, while relapses occur in the vast majority. We speculate that adding a FLT3-ITD inhibitor could improve the complete remission (CR) and overall survival (OS) rates in this setting. Aims: To explore the safety and efficacy of VEN-AZA or VEN-LDAC regimens in combination with Quizartinib (QUI) (VEN-A-QUI trial; EUDRACT2020-000406-28). Methods: The target population comprised newly diagnosed pts aged ≥ 60 years old unfit for intensive treatment, including those with secondary AML, with or without prior exposure to AZA. The Phase I consisted in two arms, one with AZA (Arm A) and the other with LDAC (Arm B) plus VEN combined with QUI to establish the recommended phase 2 dose (RP2D) of both triplets. Phase 1 scheme was based in 3 + 3 cohorts of patients observing cycle 1 dose limiting toxicities. Once established the RP2D the phase 2 comprised randomized 1:1 assignment of 60 patients (48 FLT3 wild type and 12 FLT3-ITD mut) to VEN-AZA-QUI vs. VEN-LDAC-QUI, comparing the CR/CRi rate of both arms. Secondary objectives were to evaluate the CR/CRi after cycle 1 and 4, compare OS and RFS between both triplets, quality of life, medical resources, exploration of biomarkers, and immune recovery. Results: Data cut-off for preplanned interim analysis included 57 pts screened and 45 enrolled, 16 in phase 1 and 29 in phase 2. Median age was 76,5 years (range 67-87), males/females (28/23). Previous MDS or MPN was present in 28 pts (59%); and 22 (48%) had previous treatment with AZA for MDS or MPN phase. We included 16 pts in phase 1, 9 with AZA and 7 with LDAC. RP2D of QUI was 60 mg in AZA arm and 40 mg in LDAC arm. No DLT was observed in arm B, and in arm A a brain hemorrhage after more than 40 days of thrombocytopenia at dose of 60 mg. The safety committee recommended performing an early (day 14-21) bone marrow assessment in cycle 1, leading to VEN interruption in case of aplastic morphology with grade 4 neutropenia or thrombocytopenia. No grade ≥3 related non-hematological adverse events (AEs) were noted during phase 1. The most frequent non-hematological serious AEs during phase 1 were infections (n=23), and gastrointestinal (n=20). No grade 3 QTc prolongation was observed. Objective responses were CR+CRh+CRi 7 pts (44%), PR 1 (6%), death 4 (25%), and resistance/progression 4 (25%). Twenty-nine pts (4 with FLT3-ITD mut) were enrolled in the phase 2 (15 in AZA and 14 in LDAC Arm). A median of 1 cycle (range 1-4) was administered at data cut-off, with best response among 24 evaluable pts: CR+CRh+CRi 10 (42%), MLFS in 3 (12%), PR 5 (21%), death 4 (17%), and resistance/progression 2 (8%). The overall response (CR+CRh+CRi+MLFS) was 54%. The more frequent non-hematological AEs were infections (n=35) and gastrointestinal (n=31). Two cardiac failures, 1 chest pain and 1 atrial fibrillation were noted in phase 2 (all of them unrelated to VEN or QUIZ). No grade 3 QTc prolongation was observed. Summary/Conclusion: This interim report shows an overall response rate of 54% using triplets (VEN-AZA-QUI or VEN-LDAC-QUI) for newly diagnosed unfit AML pts. However, substantial toxicity and early death cases were observed. Of note, 59% of enrolled pts had secondary AML, and 48% was exposed to AZA before inclusion. Final analyses with more pts and follow-up will clarify the efficacy and tolerability of these triplets. P513: CPX-351 TREATMENT FOR ACUTE MYELOID LEUKEMIA IN ENGLAND: REAL-WORLD OUTCOMES IN ADULTS AGED <60 YEARS VERSUS ≥60 YEARS A. Legg1,*, R. Muzwidzwa2, A. Lambova2, K. Styles2, P. Doubleday2, G. Medalla1 1Jazz Pharmaceuticals, Oxford; 2IQVIA Inc., London, United Kingdom Background: CPX-351 (Vyxeos® liposomal) is a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio. Since 2018, NICE and the EMA have recommended the use of CPX-351 for adults with newly diagnosed, therapy-related acute myeloid leukemia (t-AML) or AML with myelodysplasia-related changes (AML-MRC) due to prior myelodysplastic syndrome (MDS)/chronic myelomonocytic leukemia (CMML) or de novo AML with myelodysplasia-related cytogenetic changes. Aims: To compare the characteristics and overall survival (OS) of younger (18–60 years) versus older (≥60 years) adults with AML who were treated with CPX-351 in England. Methods: This retrospective study utilized the Cancer Analysis System database available through England’s National Cancer Registration and Analysis Service. A diagnosis of t-AML or AML-MRC between 01/01/2013 and 31/03/2021 was determined either directly using International Classification of Diseases for Oncology, Third Edition (ICD-O-3) codes or indirectly using nonspecific ICD-O-2, ICD-O-3, or ICD-10 AML codes in combination with prior systemic anticancer therapy or radiotherapy (t-AML) or a prior diagnosis of MDS/CMML (AML-MRC). OS was estimated from the diagnosis date or landmarked from the hematopoietic cell transplantation (HCT) date. Results: Overall, 211 patients with AML received CPX-351; their median (IQR) age was 65 (59, 70) years, 133 (63%) were male, 187 (89%) were White, 87 (41%) had secondary AML (t-AML or prior MDS/CMML), 33 (16%) had other AML-MRC, and 91 (43%) had unspecified de novo AML. Of the 60 (28%) patients aged <60 years, 12 were aged 18–44 years and 48 were aged 45–59 years. Of the 151 (72%) patients aged ≥60 years, 97 were aged 60–69 years and 54 were aged ≥70 years. The cutoff date for OS was 31/08/2021, with a median (IQR) follow-up of 11.3 (4.7, 20.1) months. The estimated median OS (95% CI) was 12.9 (10.5, 17.5) months overall, 18.5 (11.0, not estimable) months for adults aged <60 years, and 11.2 (8.5, 15.9) months for adults aged ≥60 years, with 2-year survival of 35%, 44%, and 32%, respectively (Figure). Early mortality at 30 days was 6% overall, 3% for adults aged <60 years, and 7% for adults aged ≥60 years. Among adults aged <60 years, HCT was reported for 29/60 (48%) patients, including 29/50 (58%) with ≥3 months of follow-up. Among adults aged ≥60 years, HCT was reported for 55/151 (36%) patients, including 55/115 (48%) with ≥3 months of follow-up. When landmarked from the HCT date, median OS was not reached in either age group (Figure). In a treatment patterns analysis, after receiving CPX-351, 14/60 (23%) adults aged <60 years and 63/151 (42%) aged ≥60 years died without salvage therapy, and 19/60 (32%) adults aged <60 years and 37/151 (25%) aged ≥60 years were alive without receiving subsequent therapy by the end of the study period. The most common salvage therapy after CPX-351 was FLAG-based chemotherapy (aged <60 years: n=12/60; aged ≥60 years: n=17/151). Image: Summary/Conclusion: This study provides real-world survival outcomes in younger and older adults with AML who were treated with CPX-351 in England. These results suggest CPX-351 is an effective treatment for patients with AML aged <60 and ≥60 years in a real-world setting that bridged many patients to HCT, with promising post-HCT outcomes. P514: V-FAST MASTER TRIAL: PRELIMINARY RESULTS OF TREATMENT WITH CPX-351 PLUS MIDOSTAURIN IN ADULTS WITH NEWLY DIAGNOSED FLT3-MUTATED ACUTE MYELOID LEUKEMIA J. McCloskey1,*, V. Pullarkat2, G. Mannis3, T. L. Lin4, S. A. Strickland5, A. T. Fathi6, H. P. Erba7, S. Faderl8, D. Chakravarthy8, Y. Lutska9, V. Chandrasekaran9, R. S. Cheung8, M. Levis10 1John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ; 2City of Hope Comprehensive Cancer Center, Duarte, CA; 3Stanford University Medical Center, Palo Alto, CA; 4University of Kansas Medical Center, Kansas City, KS; 5Vanderbilt-Ingram Cancer Center, Nashville, TN; 6Massachusetts General Hospital Cancer Center/Harvard Medical School, Boston, MA; 7Duke University School of Medicine, Durham, NC; 8Jazz Pharmaceuticals, Palo Alto, CA; 9Jazz Pharmaceuticals, Philadelphia, PA; 10Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, United States of America Background:: CPX-351 (Europe: Vyxeos® liposomal; United States: Vyxeos®), a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio, is approved for newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes in adults in Europe and patients aged ≥1 year in the United States. In a phase 3 study in older adults with newly diagnosed, high-risk/secondary AML, CPX-351 significantly improved overall survival and remission rates versus conventional 7 + 3, with a comparable safety profile. Preclinical data suggest CPX-351 may have synergistic activity with targeted agents, including the FLT3 inhibitor midostaurin. Aims: Herein, we report preliminary results for the cohort of adults treated with CPX-351 + midostaurin in the V-FAST (Vyxeos – First Phase Assessment with Targeted Agents) trial. Methods: V-FAST is an open-label, multicenter, multi-arm, nonrandomized, phase 1b master trial (NCT04075747) to evaluate the safety and preliminary efficacy of CPX-351 combined with targeted agents (midostaurin, venetoclax, enasidenib). Eligible adults in the CPX-351 + midostaurin cohort were aged 18 to 75 years, had newly diagnosed AML with a FLT3 internal tandem duplication (ITD) or tyrosine kinase domain (TKD) mutation, were fit for intensive chemotherapy, and had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2. The dose-exploration phase (3 + 3 design) determined a recommended phase 2 dose of CPX-351 100 units/m2 (daunorubicin 44 mg/m2 + cytarabine 100 mg/m2) on Days 1, 3, and 5 + midostaurin 50 mg twice daily on Days 8 to 21. There were no dose-limiting toxicities, and additional patients were enrolled in the expansion phase at this dose. Results: A total of 23 patients received CPX-351 + midostaurin and had sufficient data to be included in the analysis (cutoff date: 20 January 2022). Patient baseline characteristics are shown in the Table. Treatment-emergent adverse events (TEAEs) in ≥40% of patients included febrile neutropenia (78%), nausea (65%), increased alanine aminotransferase (57%), leukopenia (57%), thrombocytopenia (57%), headache (43%), and hyponatremia (43%). All patients experienced a grade 3/4 TEAE, primarily hematologic events. Nonhematologic grade 3/4 TEAEs in ≥2 patients included pneumonia (17%), lung infection (13%), and hyperglycemia (9%). There were no grade 5 TEAEs and no deaths on or before Day 60. Complete remission was achieved by 18/22 (82%) patients with an evaluable remission assessment after the first induction cycle. Image: Summary/Conclusion: Preliminary results from the V-FAST trial suggest the combination of CPX-351 + midostaurin is feasible, with a manageable safety profile and promising remission rates in adults with newly diagnosed AML who have a FLT3 mutation. P515: LOWER-INTENSITY CPX-351 + VENETOCLAX FOR PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA WHO ARE UNFIT FOR INTENSIVE CHEMOTHERAPY G. L. Uy1, V. A. Pullarkat2,*, P. Baratam3, R. K. Stuart3, R. B. Walter4, E. S. Winer5, S. Faderl6, V. Chandrasekaran6, Q. Wang6, D. Chakravarthy6, R. S. Cheung6, T. L. Lin7 1Washington University School of Medicine, St. Louis, MO; 2City of Hope Comprehensive Cancer Center, Duarte, CA; 3Medical University of South Carolina, Charleston, SC; 4Fred Hutchinson Cancer Research Center, Seattle, WA; 5Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; 6Jazz Pharmaceuticals, Palo Alto, CA; 7University of Kansas Medical Center, Kansas City, KS, United States of America Background: CPX-351 (Europe: Vyxeos® liposomal; United States: Vyxeos®) is a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio. CPX-351 is approved for newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes in adults in Europe and patients aged ≥1 year in the United States who are candidates for intensive chemotherapy (IC). However, the appropriate dosage of CPX-351 in patients unfit for IC may be different from the label dosage. Venetoclax (VEN; BCL-2 inhibitor) + low-dose cytarabine has demonstrated efficacy in unfit patients with AML, and drug synergism/additivity in preclinical studies provided a rationale for combining CPX-351 + VEN clinically. Aims: Our study evaluates the safety and efficacy of lower-intensity CPX-351 + VEN in adults with newly diagnosed AML who are unfit for IC. Methods: This is an ongoing, open-label, phase 1b study (NCT04038437). Patients who achieve at least partial remission after 1 or 2 cycles may receive up to 4 similar cycles in the dose-exploration phase (DEP) or up to 8 similar cycles in the expansion phase (EP). Patients are assessed for response (morphology, measurable residual disease [MRD]) and monitored for safety and survival. Results: The data include 31 patients enrolled by 15 September 2021, with a data cutoff of 2 December 2021: 4 patients in the DEP at dose level 1 (CPX-351 20 units/m2 on Days 1 and 3 + VEN 400 mg on Days 2 to 21 of each cycle), 7 patients in the DEP at dose level 2 (CPX-351 40 units/m2 + VEN 400 mg), and a total of 20 patients in the DEP and EP at dose level 1b (CPX-351 30 units/m2 + VEN 400 mg), which was established as the recommended phase 2 dose. Patients were considered unfit for IC based on age ≥75 years (n=15) or health (Eastern Cooperative Oncology Group performance status of 2 to 3 and/or comorbidities [n=16]). Median age was 74 years (range: 60, 90); 65% were male; 77% had de novo AML; 58% had poor-risk disease; and 23% had a TP53 mutation. Nonhematologic treatment-emergent adverse events (TEAEs) in ≥20% of patients were diarrhea (26%), cough (23%), dyspnea (23%), and nausea (23%). Hematologic grade ≥3 TEAEs were reported in 17 (55%) patients; no nonhematologic grade ≥3 TEAE was reported in >10% of patients. There were no deaths by Day 30; mortality at Day 60 was 13%, with deaths due to myocardial infarction unrelated to therapy (n=1), worsening lung infection (n=1), and disease progression (n=2). Median (interquartile range) recovery times were 30 days (22, 34.5) to neutrophils ≥500/μL and 21 days (21, 27) to platelets ≥50,000/μL. Complete remission (CR) or CR with incomplete neutrophil or platelet recovery (CRi) was achieved by 16/28 (57%) patients with an evaluable remission assessment. All 16 of these patients achieved remission (CR or CRi) after the first treatment cycle. MRD negativity was achieved by 12/16 (75%) patients with CR or CRi, primarily after Cycle 1 (Cycle 1: n=8; Cycle 2: n=2; Cycle 3: n=1; Cycle 4: n=1). Survival data are not yet mature. Summary/Conclusion: Lower-intensity CPX-351 + VEN was generally well tolerated in adults with newly diagnosed AML who are unfit for IC and showed promising initial efficacy, with CR or CRi in the majority of patients. P516: ADULT LANGERHANS CELL HISTIOCYTOSIS WITH THYROID GLAND INVOLVEMENT: CLINICAL PRESENTATION, GENOMIC ANALYSIS AND OUTCOME H. Cai1,2,*, T. Liu1, H. Cai1,2, M. Duan1,2, J. Li1,2, D. Zhou1,2, X. Cao1,2 1Department of Hematology; 2State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Background: Langerhans cell histiocytosis (LCH) is a rare, clonal disorder derived from CD1a-positive and CD207-positive immature myeloid dendritic cells. The clinical presentations and outcomes of LCH are extremely variable. Thyroid gland involvement is rare, and solitary thyroid gland involvement with LCH is extremely rare, with most cases presenting as part of multisystem disease.Until now, little information exists on the genomic analysis of LCH with thyroid gland involvement with or without coexisting papillary thyroid carcinoma. Aims: To evaluate the characteristics and treatment outcomes of adult Langerhans cell histiocytosis (LCH) patients with thyroid involvement. Methods: We retrospectively described the clinical, biological, and genomic characteristics of a series of 36 LCH patients with thyroid involvement in our centre between January 2001 and December 2021. Results: We retrospectively described the clinical, biological, and genomic characteristics of a series of 36 LCH patients with thyroid involvement in our centre between January 2001 and December 2021. At the time of diagnosis, only 1 patient was classified as having single-system LCH, and 35 patients were classified as having multisystem (MS) LCH. Three patients had coexisting papillary thyroid carcinoma. Patients with thyroid gland involvement had higher frequencies of pituitary (88.6% vs. 53.4%, P<0.001), liver (45.7% vs. 20.7%, P=0.003) and lymph node (54.3% vs. 31.6%, P=0.012) involvement and a lower frequency of bone (45.7% vs. 72.0%, P=0.003) involvement than patients without thyroid gland involvement. Sixteen patients had abnormal thyroid function, including 9 patients with primary hypothyroidism, 1 patient with central hypothyroidism, and 6 patients with subclinical hypothyroidism. BRAF V600E, BRAF N486_P490 and MAP2K1 mutations were detected in 14.3%, 57.1% and 7.1% of patients, respectively. After a 43-month median follow-up, none of the patients died, and 15 patients experienced reactivation. The median event free survival was 37.5 months. Two of 6 patients with subclinical hypothyroidism had normal thyroid function, and 12 patients still had hypothyroidism after treatment. Summary/Conclusion: As the largest adult LCH cohort with thyroid gland involvement to date, we found that patients with thyroid gland involvement had different clinical characteristics, genetic profiles and outcomes than patients without thyroid gland involvement. P517: PROSPECTIVE MULTICENTRIC STUDY ON INFECTIOUS COMPLICATIONS AND CLINICAL OUTCOME IN 230 UNFIT AML PATIENTS TREATED IN FIRST-LINE WITH HYPOMETHYLATING AGENTS ALONE OR IN COMBINATION WITH VENETOCLAX. A. Candoni1,*, M. Piccini2, D. Lazzarotto1, V. Bonuomo3, M. Dargenio4, M. Riva5, L. Mellillo6, C. Papayannidis7, M. Stulle8, G. Dragonetti9, M. I. Del Principe10, C. Cattaneo11, C. Pasciolla12, R. De Marchi13, M. Delia14, M. C. Tisi15, M. E. Zannier1, G. Nadali3, M. Sciumè16, A. Spadea17, C. Sartor7, D. Griguolo8, E. Buzzatti10, C. M. Basilico18, C. Sarlo19, A. L. Piccioni20, R. Cairoli5, A. Olivieri21, L. Pagano9 1Division of Hematology and Stem Cell Transplantation, ASUFC, University of Udine, Udine; 2Division of Hematology, Azienda Ospedaliero-Universitaria Careggi, Florence; 3Division of Hematology, AOUI, Policlinico GB Rossi, Verona; 4Division of Hematology, Ospedale Vito Fazzi, Lecce; 5Dipartimento di Ematologia ed Oncologia, Niguarda Cancer Center ASST Grande Ospedale Metropolitano, Milano; 6Division of Hematology, Lecce and IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Lecce; 7Institute of Hematology and Medical Oncology “L. and A. Seragnoli”, University of Bologna, Bologna; 8Division of Hematology, ASUGI, Trieste; 9Division of Hematology, Polo Onco-Ematologico, Fondazione Policlinico A. Gemelli-IRCCS, Università Cattolica del Sacro Cuore; 10Hematology, Fondazione Policlinico Tor Vergata, Roma; 11Section of Hematology, Spedali Civili, Brescia; 12Haematology Unit, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari; 13Onco Hematology, Department of Oncology, Veneto Institute of Oncology IOV, IRCCS, Padova; 14Hematology and Bone Marrow Transplantation Unit, Azienda Ospedaliero-Universitaria Consorziale Policlinico-University of Bari, Bari; 15Cell Therapy and Hematology, San Bortolo Hospital, Vicenza; 16U.O. Oncoematologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milano; 17Hematology and Stem Cell Transplant Unit, IRCCS Regina Elena National Cancer Institute, Roma; 18Division of Hematology, ASST Sette Laghi, Ospedale Circolo e Fondazione Macchi, Varese; 19Hematology and Stem Cell Transplantation Unit, University Campus Bio-Medico; 20Dipartimento di Ematologia, Azienda Ospedaliera San Giovanni Addolorata, Roma; 21Division of Hematology, Azienda Ospedaliero-Universitaria Ospedali Riuniti di Ancona, Ancona, Italy Background: The hypomethylating agents (HMAs) are an important therapeutic option for older patients (pts) with AML and have become the backbone for combination regimens (eg, with Venetoclax). However, there are very limited real-life prospective studies regarding clinical outcome of these pts, including infectious complications and infection related mortality (IRM) during treatment. Aims: To investigate the infectious complications and clinical outcome in AML patients treated with HMAs± Venetoclax (V) outside of clinical trials. Methods: The recruitment of this prospective multicentric study (CE-Id-study:2908) has been completed on December 31, 2020. We enrolled 230 AML pts with a median age of 75 years (range 25-94); 157 pts (68%) had >2 relevant comorbidities. Of the 230 cases, 132 (57%) received a first-line therapy with a combination of HMAs+V while 98 (43%) were treated with HMAs monotherapy (azacitidine or decitabine). A total of 1550 cycles of HMAs have been administered (680/1550 with HMAs+V). Results: The best response achieved, with HMAs treatment, was: CR in 44% of cases (57,6% with HMAs+V and 25,5% with HMAs alone, P=0,0001), PR in 17% and SD in 14% of cases (ORR 61%; 72% in HMAS+V and 46% in HMAs alone, P=0,0007). The microbiological or radiological proven infectious complications (almost one) occurred in 160/230 (70%) of pts, mainly pneumonia (in 42% of pts) and/or bacteremia/sepsis (one or more events in 29% of pts). Febrile neutropenia (one or more episodes) occurred in 38% of pts and 14 cases of Covid-19 (6%) were reported. After a median follow-up of 9 months (1-24) from the start of HMAs therapy, 144 (63%) pts died and 86 (37%) were alive. The 1 yr OS probability was 46% with a median OS of 10,3 months (11 months in HMAs+V and 9 months in HMAs alone; P=ns). The primary causes of death were: progression of AML (42%), Infection (26%-37/144), Infection+AML (24%), other causes (8%). The IRM was 26% and 19/144 (13%) pts died of infectious complication while in CR/PR (16 in HMAs+V group and only 3 in HMAs group; P=0,005). Data on antibiotic prophylaxis, hospitalization, drug-doses modulation, are available and analyzed in this study. Summary/Conclusion: The results of this real-life, multicentric, prospective study, confirm a higher CR rate in pts treated with HMAs+V compared to HMAs alone (P=0,0001). However, we found a high rate of infectious complications and IRM (26%) with a higher infection related deaths in patients in CR/PR who were treated with HMAs+V (P=0,005). Findings from this study highlight the critical relevance of infection prevention in reducing infectious mortality, which adversely impacts the OS of this frail AML population. P518: PEDAL/EUPAL INTERNATIONAL COLLABORATION TO IMPROVE THE OUTCOME OF CHILDREN WITH RELAPSED OR REFRACTORY ACUTE MYELOID LEUKEMIA V. Ceolin1,2,3,*, S. Ishimaru1, F. Bautista1,4, B. Goemans1, E. A. Kolb5, L. Di Laurenzio6, T. M. Cooper7, M. Bakker1, J. Wahlstrom8, G. Sunkersett8, G. Ku9, C. Zwaan1,3,4, D. Reinhardt10, G. Nichols6 1Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands; 2Regina Margherita Children’s Hospital, University of Turin, Turin, Italy; 3Sophia Children’s Hospital, Erasmus University MC, Rotterdam, Netherlands; 4ITCC consortium, Paris, France; 5Nemours Centers for Childhood Cancer Research & Cancer and Blood Disorders, Wilmington; 6The Leukemia & Lymphoma Society, New York; 7Seattle Children’s Hospital, Washington; 8AbbVie One Oncology; 9Genentech Inc, San Francisco, United States of America; 10University Hospital of Essen, Essen, Germany Background: The prognosis of children with acute myeloid leukemia (AML) has increased stepwise over the last decades, and the overall survival (OS) is currently in the 75-80% range. However, at relapse, OS is approximately 40-50%. Over the last decade, many novel drugs have been developed specifically for use in adults with AML and few have been authorized for children. Given the medical need, effective and less toxic therapies are urgently also needed for children to overcome the limitations of current intensive chemotherapy. Aims: The Leukemia & Lymphoma Society (LLS) Pediatric Acute Leukemia (PedAL) program is a strategic initiative that aims to overcome the obstacles in treating children with acute leukemia via a Transatlantic collaboration. The aim is to run a program of early and late phase clinical trials evaluating the safety and the efficacy of new agents in pediatric leukemia, eventually setting a new standard of treatment for relapsed AML. In Europe this will be implemented as a master clinical trial with sub-trials. The European Pediatric Acute Leukemia (EuPAL, including United Kingdom) consortium was set up to coordinate the European efforts for the PedAL initiative. Methods: In Europe, the current project consists of a registry protocol (EuPAL2021 Registry) and a Master protocol with sub-trials (ITCC-101). In North America patients will be enrolled in the PedAL Screening Protocol - APAL2020SC. Any patient with known or suspected relapsed or refractory AML, who is less than 22 years of age will be eligible for inclusion in the registry in Europe or in the screening protocol in North America, to provide confirmation of relapse and target expression using standardized methodologies. In Europe, the EuPAL 2021 Registry will lead to harmonization of diagnostics in country/collaborative group-specific centralized laboratories where the analysis will be carried out as part of standard of care. In North America, Australia and New Zealand APAL2020SC will utilize commercial laboratories (Hematologics, Foundation Medicine). The ITCC-101 PedAL/EuPAL Master protocol is a complex clinical trial with a stratification approach to allocate these patients to sub-trials, according to relevant biomarkers and disease related information. After confirmation of relapse, patients may be enrolled in one of the open sub-trials, provided the detailed eligibility criteria are met. Each sub-trial will effectively run as one study performed on a global scale, and data will be entered in one database. Sub-trials may not start at the same time and may be added by a substantial amendment to the master. In North America each sub-trial will be added as an amendment to the LLS Investigational New Drug. For the first wave of sub-trials, Investigational Medicinal Product prioritization was mainly achieved through international collaboration at the Pediatric Forum for AML organized by ACCELERATE Strategy Forum in April 2020. Results: The first sub-trial will be APAL2020D, an open-label Phase III randomized multicenter trial to assess if venetoclax combined with FLA+GO (fludarabine, high-dose cytarabine, and gemtuzumab ozogamicin) will improve overall survival compared to FLA+GO, with the aim to establish the standard treatment for the 2nd relapsed AML patients (NCT05183035) and will open in Q2/Q3 2022. Summary/Conclusion: The PedAL/EuPAL international collaboration will determine, using biology-based selection markers for treatment stratification, the standards of care for AML in 1st and 2nd relapse and define the safety and efficacy parameters necessary for the efficient evaluation of novel targeted therapies. P519: TRANSFUSION INDEPENDENCE AMONG NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA PATIENTS RECEIVING VENETOCLAX-BASED COMBINATIONS VS OTHER THERAPIES: RESULTS FROM THE AML REAL WORLD EVIDENCE (ARC) INITIATIVE O. Wolach1, J. S. Garcia2, P. Vachhani3, J. Zeidner4, C. Lai5, Y. Moshe6, M. Xavier7, S. Lee8, T. Zuckerman9, D. A. Pollyea10, S. Abedin11, E. C. Chen2, S. Bathini3, W. Edwards7, C. N. Bui12, W. W.-H. Cheng12, A. Svensson12, A. Guérin13, J. Maitland13, E. Ma14, M. Montez14, M. Grunspan15, A. D. Goldberg16,* 1Rabin Medical Center, Petah Tikya, Israel; 2Dana-Farber Cancer Institute, Boston; 3O’Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham; 4University of North Carolina, Lineberger Comprehensive Cancer Center, Chapel Hill; 5University of Pennsylvania Perelman Center for Advanced Medicine, Philadelphia, United States of America; 6Tel Aviv (Sourasky) Medical Center, Tel Aviv, Israel; 7Scripps-MD Anderson, San Diego; 8Janssen Research and Development, Springhouse, United States of America; 9Rambam Health Care Campus, Haifa, Israel; 10University of Colorado School of Medicine, Aurora; 11Medical College of Wisconsin, Milwaukee; 12AbbVie Inc, North Chicago, United States of America; 13Analysis Group Inc, Montreal, Canada; 14Genentech, South San Francisco, United States of America; 15AbbVie Inc, Hod-Hasharon, Israel; 16Memorial Sloan Kettering Cancer Center, New York, United States of America Background: Venetoclax (VEN), a novel BCL-2 inhibitor, is FDA approved in combination with hypomethylating agents (azacitidine or decitabine) or low-dose cytarabine for the treatment of newly-diagnosed (ND) acute myeloid leukemia (AML) in adults ≥75 years or those who have comorbidities that preclude use of intensive induction chemotherapy. This study is part of the ongoing AML Real world evidenCe (ARC) Initiative which aims to provide insight into the use and relative efficacy of VEN-based combinations among ND patients (pts) with AML. Aims: To describe transfusion independence (TI) among ND AML pts treated with VEN-based combinations vs non-VEN regimens in clinical practice. Methods: The ARC Initiative is a multicenter chart review study of adult pts with AML who received VEN ≥April 2016 (VEN cohort) or non-VEN regimens ≥May 2015 (control cohort). Pts in the VEN cohort were matched 1:1 to pts in the control cohort based on age category (<60; 60-74; ≥75) and European Leukemia Net (ELN) risk classification. Outcomes, including TI, hematopoietic cell transplantation (HCT), and best response, were assessed during first-line therapy over a transfusion observation period defined as time from initiation of therapy to the earliest amongst progression, initiation of second-line therapy, admission to hospice, death, or last visit at study site. TI was defined as no red blood cell (RBC) or platelet (PLT) transfusions during any consecutive ≥56-day period during the transfusion observation period. Kaplan-Meier (KM) analyses were conducted to assess the TI rate at 3 months after treatment initiation. Interim descriptive results are presented from data cutoff of Sept 2021; data will be updated for the meeting. Results: A total of 119 VEN and 119 matched control pts with ND AML who had available information on transfusions were included in this analysis (Table 1). Among pts in the control cohort, 65.5% received a high intensity regimen (e.g., cytarabine+daunorubicin [37 pts, 31.1%], CPX-351 [16 pts, 13.4%]) and 34.5% received a low intensity regimen (decitabine [24 pts, 20.2%], azacitidine [15 pts, 12.6%]). Of pts tested for genetic mutations, common mutations were TP53 (VEN: 23.1%; control: 15.8%), RUNX1 (VEN: 17.1%; control: 17.5%), and IDH1/IDH2 (VEN: 12.8%; control: 14.0%). During a median transfusion observation period of 4.0 months in the VEN cohort and 3.6 months in the control cohort, 87.4% of VEN and 93.3% of control pts received ≥1 RBC or PLT transfusion. Pts in the VEN cohort received a median of 2.0 RBC and/or PLT transfusions per month and 5.0% received HCT; pts in the control cohort received a median of 3.5 RBC and/or PLT transfusions per month and 13.4% received HCT (all p <0.05). Among the 103 pts in the VEN cohort and 99 pts in the control cohort who had ≥56 days of follow-up, 61.2% of VEN pts and 50.5% of control pts achieved TI during first-line therapy (p=0.17), and 60.6% of VEN pts and 55.8% of control pts with response data achieved response (p=0.59). The KM rate of achieving TI at 3 months was 45.0% in the VEN cohort and 35.9% in the control cohort (log-rank p=0.12). Pts in the VEN and control cohort achieved TI after a median of 2.3 and 2.8 months, respectively (p=0.08). Image: Summary/Conclusion: Interim ARC initiative results show that pts receiving VEN-based combinations receive statistically fewer transfusions and are trending towards achieving TI more frequently and reaching TI more quickly after initiation of treatment compared to matched control pts receiving low and high intensity therapies. Further analyses are planned to understand the real-world outcomes of ND pts with AML. P520: PHASE 1/2 STUDY OF SEL24/MEN1703, A FIRST-IN-CLASS DUAL PIM/FLT3 KINASE INHIBITOR, IN PATIENTS WITH IDH1/2-MUTATED ACUTE MYELOID LEUKEMIA: THE DIAMOND-01 TRIAL. G. Martinelli1,*, A. Santoro2, C. Gambacorti-Passerini3, S. Vives Polo4, S. R. Solomon5, S. Mukherjee6, E. Lech-Maranda7, M. Yair Levy8, A. Wierzbowska9, M. Calbacho-Robles10, G. Marconi11, M. Benedetta Giannini11, I. Cano12, L. Torres Miñana13, E. Acuña-Cruz12, N. Angelosanto14, A. Galleu15, S. Baldini15, S. Blotta15, F. Ravandi16, P. Montesinos17 1IRCCS Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori”-IRST S.r.l., Meldola; 2Department of Oncology, IRCCS, Humanitas Research Hospital; 3Department of Hematology, University of Milano-Bicocca, Milan, Italy; 4Institut Català d’Oncologia-Hospital Germans Trias i Pujol, Badalona, Spain; 5Northside Hospital Cancer Institute, Atlanta; 6Taussig Cancer Institute, Cleveland Clinic, Cleveland, United States of America; 7Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland; 8Texas Oncology-Baylor Charles A. Sammons Cancer Center, Dallas, United States of America; 9Department of Hematology, Medical University of Lodz, Lodz, Poland; 10Department of Hematology, Ramón y Cajal University Hospital, Madrid, Spain; 11IRCCS Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” - IRST S.r.l., Meldola, Italy; 12Department of Hematology; 13Department of Hematology, La Fe University Hospital, Valencia, Spain; 1413Clinical Development-Hematology, Ryvu Therapeutics, Krakow, Poland; 15Hematologic Malignancies - Global Oncology TA, Menarini Group, Florence, Italy; 16Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America; 17La Fe University Hospital, Valencia, Spain Background: Mutations in the FLT3 tyrosine kinase are the most frequent mutations that occur in adults with acute myeloid leukemia (AML) and can co-occur with mutations in IDH1 or IDH2 (collectively IDHm) in up to 30% of cases. SEL24/MEN1703 is an orally available, first-in-class, dual PIM/FLT3 kinase inhibitor. Preliminary results from the phase 1/2 first-in-human DIAMOND-01 trial (NCT03008187) evaluating single-agent SEL24/MEN1703 showed activity in adults with relapsed/refractory (R/R) IDHm AML, where 3 of 8 IDHm patients responded. Aims: Here we report the first safety and efficacy results from an additional expansion cohort of the DIAMOND-01 trial in 20 patients with R/R IDHm AML. Methods: Patients with IDHm R/R AML and no standard therapeutic options were eligible. The recommended dose of 125 mg SEL24/MEN1703 was given orally, once daily for 14 days over a 21-day cycle until disease progression or unacceptable toxicity. The primary endpoint was safety, and adverse events (AEs) were graded according to NCI CTCAE v4.03. The secondary endpoint was antileukemic activity including overall response rate (ORR). Results: As of 10 January 2022, 14 patients were enrolled in the IDHm cohort. Median age was 68 years (range 37–79). Four patients had AML secondary to myelodysplastic syndrome and 7 patients had intermediate cytogenetic risk. Median number of prior lines of treatment was 2 (range 1–3). Seven patients had IDH2, 1 had IDH1/2, and 4 had IDH1 mutations. Concomitant mutations in FLT3-ITD were detected in 2 patients. Median duration of treatment was 2 cycles (range 1–8). Safety data (N=12) showed that serious treatment-emergent AEs (TEAEs; reported in ≥5% patients) were pneumonia (33%), skin infection, and clostridial gastroenteritis (8% each). These events were all unrelated to study drug. Drug-related TEAEs were liver injury and hyponatremia (8% each). The drug-related liver injury occurred in a patient who was concomitantly receiving other drugs with known hepatotoxic potential. Grade ≥3 TEAEs (≥10% patients) were pneumonia (33%) and asthenia (17%), both unrelated to study drug. No differentiation syndrome was observed. Of the 7 patients who completed ≥1 treatment cycle and had ≥1 postbaseline assessment or clear disease progression, ORR was 28.6%; 1 patient achieved a complete response with incomplete blood count recovery (CRi) at cycle 3 and underwent hematopoietic stem cell transplant, 1 patient had a partial response at cycle 4 (confirmed at cycle 7 and still on treatment), 4 had disease progression, and 1 discontinued for an AE (not drug related). Among the 7 remaining patients, 3 discontinued before completion of cycle 1 without progression or response, while 4 patients were ongoing and have not yet had any postbaseline assessment. Summary/Conclusion: Preliminary results in the IDHm cohort confirm that SEL24/MEN1703, a first-in-class, orally available, dual PIM/FLT3 inhibitor, has a manageable safety profile and single-agent activity in patients with R/R IDHm AML. Updated results will be presented at the congress. P521: TREATMENT OF BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM IN PEDIATRIC PATIENTS WITH TAGRAXOFUSP, A CD123-TARGETED THERAPY N. Pemmaraju1,*, B. Cuglievan1, J. Lasky2, A. Kheradpour3, N. Hijiya4, A. S. Stein5, S. Meshinchi6, C. Mullen7, E. Angelucci8, L. Vinti9, T. I. Mughal10,11, A. Pawlowska12 1Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston; 2Cure 4 The Kids Foundation, Las Vegas; 3Department of Pediatric Hematology and Oncology, Loma Linda University Children Hospital, Loma Linda; 4Division of Pediatric Oncology, Hematology, and Stem Cell Transplantation, Columbia University Irving Medical Center, New York; 5Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte; 6Department of Pediatrics, University of Washington School of Medicine, Seattle; 7Division of Pediatric Hematology/Oncology, Department of Pediatrics, Golisano Children’s Hospital, University of Rochester, Rochester, United States of America; 8Hematology and cellular therapy unit, IRCCS Ospedale Policlinico San Martino, Genova; 9Department of Paediatric Haematology/Oncology, Cell and Gene Therapy, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy; 10Division of Hematology-Oncology, Tufts University Medical School, Boston; 11Stemline Therapeutics Inc, New York; 12Department of Pediatrics, City of Hope National Medical Center, Duarte, United States of America Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy derived from plasmacytoid dendritic cell precursors that overexpress CD123, the interleukin-3 receptor alpha. Though primarily a disease of the elderly, BPDCN has been documented in infants and children in 10–20% of all cases. The rarity of BPDCN in the pediatric population makes collecting robust safety and efficacy data challenging and there is a paucity of data published for pediatric patients (pts). A first-in-class, CD123-directed therapy, tagraxofusp (TAG, SL-401) is the only approved treatment for patients with BPDCN. A multicohort prospective study (NCT02113982) demonstrated a tolerable safety profile and a durable overall response rate of 75% (median duration 24.9 months) in first-line (1L) pts. TAG is approved by the US FDA for the treatment of BPDCN in pts over the age of 2 years with newly diagnosed and relapsed or refractory (R/R) BPDCN; it is also approved by the EMA for 1L treatment of adult pts. Herein, we present a case series of pediatric pts with BPDCN treated with TAG, to provide further insight into the efficacy and safety of this therapy in this understudied patient population. Aims: To expand on the body of data regarding TAG therapy in pediatric pts with BPDCN. Methods: Pediatric case reports of BPDCN were collected from centers across the US and Europe. TAG was administered according to local institutional guidelines 1L, or R/R. Data was collated and summarized descriptively. Analyses included tumor response, survival, and safety (adverse events [AEs] and laboratory values). Results: Eight pediatric pts with BPDCN were treated with TAG and included in this analysis. Median age was 15.5 years (range 2 – 21 years), and 7 of the 8 pts were female. Five pts were treatment naïve, and 3 pts were R/R. Four pts had skin only disease. Two pts had skin, bone marrow (BM), and lymph node (LN) involvement; 1 of these also had central nervous system (CNS) involvement. One patient had BM and LN, but no skin involvement, and 1 patient had BM only disease. A 2-year-old patient received 7 mcg/kg TAG in combination with venetoclax and azacitidine at second relapse; all other pts received 12 mcg/kg throughout all treatment cycles. The number of TAG cycles administered ranged from 1 to 4 at the time of data cutoff. A decrease in albumin occurred in 2 pts, and 2 pts had a rise in transaminases, all of which were manageable. One patient experienced headaches, hot flashes, fatigue, and mouth sores. One patient experienced grade 2 capillary leak syndrome in cycle 1, which was treated and resolved, and the patient went on to receive subsequent cycles. No AEs were seen in 3 pts. Three 1L pts achieved a complete response, including 1 patient with extensive disease (skin, bone marrow, and CNS) who also received intrathecal therapy. One 1L and 1 R/R patient achieved stable disease (no progression after 4 TAG cycles each), and 1 patient with R/R BPDCN progressed while on TAG. Five pts treated with TAG were bridged to a stem cell transplant (n=3 1L, n=2 R/R; Table 1). Median survival data will be presented (6 of 8 pts remain alive). Image: Summary/Conclusion: These cases expand the knowledge base of BPDCN treatment in pediatric pts. TAG was well tolerated in all pts, with a manageable safety profile that was similar to that reported for adults in the pivotal study. Treatment with TAG demonstrated promising efficacy, with responses that enabled 5 of 8 pediatric pts, including 3 1L and 2 R/R, to be bridged to stem cell transplant. P522: ABSOLUTE LYMPHOCYTE COUNT IS AN INDEPENDENT SURVIVAL PREDICTOR IN PATIENTS WITH ACUTE MYELOID LEUKEMIA TREATED WITH INTENSIVE CHEMOTHERAPY G. Cristiano1,*, J. Nanni1, L. Zannoni1, C. Sartor1, S. Parisi2, S. Paolini2, C. Papayannidis2, M. Cavo1,2, A. Curti2 1Hematology, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna; 2Hematology, IRCCS Azienda ospedaliero-universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy Background: Absolute lymphocyte count (ALC) is known to be an independent prognostic factor for overall survival (OS) in patients receiving autologous transplantation for Hodgkin’s and non-Hodgkin’s lymphomas, multiple myeloma and breast cancer. In acute myeloid leukemia (AML) patients, enhanced ALC recovery after intensive chemotherapy (IC) has been associated with superior OS and leukemia-free survival (LFS). However, few if any data correlated ALC recovery with novel therapies as well as with updated prognostic factors, such as European Leukemia Net (ELN) risk-classification. Aims: Our study aims at evaluating the predictive value of ALC recovery in AML patients treated with IC by analysing its impact through different patient subgroups according to therapy response, type of chemotherapy regimen and the use of allogeneic stem cell transplantations (HSCT). Methods: We evaluated 148 newly diagnosed adult AML patients treated with IC at Bologna Seràgnoli Hematology Institute since 2007. We defined 4 ALC time-points (TPs): at 15, 21 and 28 days from the start of induction chemotherapy and before consolidation therapy (CC). ALC cut-off was established at 500/mm3. In addition to the assessment of the single TP, patients were also grouped in those who obtained ≥500/mm3 ALC in all 4 TPs and those who had <500/mm3 ALC in at least one TP. Median follow-up was 51,4 months. Results: When ALC recovery was assessed at the single TP, only ALC-CC recovery showed a statistically significant association with better OS and LFS. However, significant outcome differences were observed among CR patients (76,3%), subdivided in those who achieved ALC in all TPs vs others. A trend toward better correlation of ALC with OS was observed in patients who received “3 + 7-based” regimen (30,8%;) versus those who were treated with “fludarabine-based” regimen (69,1%;). Better outcome was observed in “3 + 7” patients who had ALC recovery in all TPs and at day 15 post-chemotherapy. Of note, in the “3 + 7” group, FLT3-ITD positive patients, who achieved early ALC recovery at day 21 had better outcomes. We, then, analysed the impact of post-chemotherapy ALC recovery in patients who received HSCT (66,3%) vs those who did not (33,6%) (Fig.1). As expected, transplanted patients had globally better outcome than patients who received only chemotherapy. In patients who received HSCT the impact of ALC recovery after chemotherapy was minimal. In contrast, patients who had ALC <500/mm3 ALC in at least one TP and did not undergo HSCT had the worst outcome. Interestingly, patients who were not transplanted but had ≥500/mm3 ALC in all 4 TPs had a similar outcome as patients who received HSCT. Image: Summary/Conclusion: Our study indicates ALC recovery after IC as a promising survival predictor in AML patients, especially in those who do not undergo HSCT. In the path that aims to investigate the complex interaction between leukemia and the immune system, our study may provide the clinical rationale for the construction of an immunological score to be integrated in the current disease-centered risk-classification system. Future studies in larger cohorts addressing the impact of ALC recovery in association with the use of novel agents and minimal residual disease status are highly warranted. P523: MIDOSTAURIN PLUS INTENSIVE CHEMOTHERAPY IN FLT3 MUTATED AML. “REAL LIFE” DATA VERSUS THE RATIFY STUDY A. De La Fuente1,1,*, M. Diaz Beya2, P. Beneit3, C. Botella4, A. Fernandez Moreno5, A. Sampol6, M. Arnan Sangerman7, A. Yeguas Bermejo8, M. D. L. L. Amigo9, J. Labrador10, A. Garcia Guinon11, A. Garrido12, J. Serrano13, S. Vives Polo14, M. Garcia Fortes15, M. J. Sayas16, J. M. Bergua17, M. T. Olave18, F. Vall LLovera19, J. Bargay20, M. Pereiro Sanchez21, R. Garcia Boyero22, A. Diaz Lopez23, M. Tormo24 1Hematology, MD Anderson CC Madrid, Madrid; 2Hematology, H. U. Clinic IDIBAPS, Barcelona; 3Hematology, H. U. San Juan de Alicante; 4Hematology, H. General de Alicante, Alicante; 5Hematology, H. U. Central de Asturias, Oviedo; 6Hematology, H. U. Son Espases, Palma de Mallorca; 7Hematology, ICO Hospitalet, L’Hospitalet de Llobregat, Barcelona; 8Hematology, H. U. Salamanca, Salamanca; 9Hematology, H. U. Morales Meseguer, Murcia; 10Hematology, H. U. de Burgos, Burgos; 11Hematology, H. U. Arnau de Vilanova, Lleida, Lleida; 12Hematology, Hospital de la Santa Creu i Sant Pau. Instituto de investigación Jose Carreras, Barcelona; 13Hematology, H. U. Reina Sofia, Cordoba; 14Hematology, ICO H. Germans Trias i Pujol, Barcelona; 15Hematology, H. Virgen de la Victoria, Malaga; 16Hematology, H. U. Doctor Peset, Valencia; 17Hematology, H. U. san Pedro de Alcantara, Caceres; 18Hematology, H. U. Lozano Blesa, Zaragoza; 19Hematology, H. U. Mutua Terrassa, Barcelona; 20Hematology, H. U. Son LLatser, Palma de Mallorca; 21Hematology, C. H. U. Orense, Orense; 22Hematology, H. G. U. de Castellon, Castellon de la Plana; 23Hematology, Fundacion MD Anderson Madrid, Madrid; 24Hematology, 20Clinico Universitario Instituto de Investigación Sanitaria INCLIVA, Valencia, Spain Background: FLT3 mutation is associated with adverse prognosis in AML (ELN2017, Dohner et al, Blood 2017) and Intensive chemotherapy (IC) has been the standard of treatment for FLT3 mutated AML for decades. Midostaurin (Midos) has been approved in combination with IC for FLT3-mutated AML based on an improvement in overall survival noted in the RATIFY phase 3 trial (Stone et al, N Engl J Med 2017) Aims: The aims of this study are to analyze safety and effectiveness of Midos plus IC in FLT3 AML, to validate RATIFY results in a “real-world” setting and to identify risk factors. Methods: We carried out a retrospective multicenter study (MDA-AML-2018-06) in 27 Spanish centers. Inclusion criteria: age >18 years, FLT3-mutated AML diagnosis according to WHO criteria and start of treatment with midostaurin in combination with IC between June 2016 and June 2021. We evaluated the response according to 2017 ELN criteria, toxicity according to CTCAE v4.0 and overall survival (OS) by Kaplan-Meier. Statistical analysis was performed using SPSS program version 20.0. Results: A total of 175patients (pt) were included (93 female), median age 53 (18-76), of them 111were younger than 60yrs and 110pts had ECOG<2. ELN2017 stratification was favorable in 53pt; Intermediate: 72pt; adverse in 26pt. Median WBC count 44.500/µL (0.4-395.000). A total of 133pts had FLF3-ITD mutation and of them 74pt with ratio ≥0.5. Safety: Total Midos cycles 548 (median 2), 20 pt (11.5%) suffered QT prolongation, 26pt (14,8%) Midos interruption and 10pt (5.7%) Midos reduction. There were no deaths related to Midos. Effectiveness: 144 (81.4%) pt achieved CR after Induction1or2, of them 76pt were consolidated with alloSCT, 24pt received maintenance and 41pt proceed to W&W. Median OS for the whole population was not reached and the 24months OS was 68%. In our experience ELN2917 classification and ECOG<2 identify groups with differences in OS (p0.019 and p0.04 respectively). Age <60vs≥60 resulted in no differences for OS. We observed significant differences for maintenance versus W&W (p=0.001) in favorable and intermediate risk patients. Comparing maintenance vs alloSCT we observe no differences in the Intermediate ELN2017 group. Image: Summary/Conclusion: Our experience confirms safety and effectiveness of Midos plus IC as first line in FLT3 AML patients with similar 2yOS as the previous reported in the RATIFY trial. Interesting that in our experience age 60 and above resulted in no differences for OS. P524: PHASE 1B/2 STUDY ON SAFETY, PK, PD, AND PRELIMINARY EFFICACY OF THE SELECTIVE SYK INHIBITOR LANRAPLENIB IN COMBINATION WITH THE FLT3 INHIBITOR GILTERITINIB, IN FLT3-MUTATED R/R AML (KB-LANRA 1001) E. M. Stein1,*, A. Patel2, L. C. Michaelis3, G. Schiller4, R. Swords5, L. A. Carvajal6, G. Bray6, J. DiMartino6, M. Y. Levy7 1Memorial Sloan Kettering Cancer Center, New York; 2The University of Chicago Medical Center, Chicago; 3Medical College of Wisconsin, Milwaukee; 4University of California Los Angeles (UCLA), Los Angeles; 5Oregon Health and Science University, Portland; 6Kronos Bio, Inc., San Mateo; 7Texas Oncology - Baylor Charles A. Sammons Cancer Center, Dallas, United States of America Background: Spleen tyrosine kinase (SYK) is a key driver of lymphoid and myeloid cell signaling pathways and has been implicated in the pathogenesis of acute myeloid leukemias (AML) defined by dysregulated expression of HOXA9 and MEIS1 transcription factors. SYK also cooperates with internal tandem duplication (ITD)-mutated FMS-like tyrosine kinase 3 (FLT3) to drive leukemogenesis. Combined pharmacologic inhibition of SYK and FLT3 results in robust antileukemic effects in preclinical models of FLT3 ITD-driven AML. Lanraplenib (LANRA) is an oral, selective SYK inhibitor. Studies demonstrate dose-dependent reductions in viability of leukemic cells ex vivo from newly diagnosed FLT3 ITD-mutated AML patients (pts) as well as additive reductions in viability when combined with the selective FLT3 inhibitor, gilteritinib. Once daily (QD) LANRA was well-tolerated at doses up to 50mg for 7 days and 30mg for up to 12 weeks in healthy volunteers and pts with autoimmune disorders. Aims: The KB-LANRA 1001 trial will investigate the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of LANRA when combined with gilteritinib in relapsed or refractory pts with FLT3-mutated AML. We will also evaluate the rate of complete responses (CR) and CR with partial hematologic recovery (CRh), duration of response (DoR), event-free (EFS) and overall survival (OS). Methods: KB-LANRA 1001 is a global, multicenter trial of LANRA in combination with standard dose gilteritinib (120mg QD) in FLT3-mutated AML pts aged ≥18 with at least 1 prior therapy. Those who have been treated with midostaurin or other multikinase inhibitors are eligible, though pts who failed to achieve at least a partial response or who relapsed following prior exposure to gilteritinib or other next-generation FLT3 inhibitor monotherapy are excluded. Both medications will be administered daily in sequential, 28-day cycles until progression/relapse, intolerance, or failure to achieve at least a partial remission after 6 cycles. The Phase 1b component will define the maximum tolerated and/or recommended Phase 2 dose (MTD/RP2D) of LANRA when added to gilteritinib in accordance with a 3 + 3 dose escalation design. PK and PD parameters will be characterized for LANRA monotherapy and when co-administered with gilteritinib. Four LANRA doses are planned for evaluation (20, 40, 60 and 90mg). All decisions regarding dose escalation including declaration of the MTD will be made by a dose-escalation committee based on the prospectively defined definition of dose-limiting toxicities and other safety metrics. The Phase 2 component will further evaluate safety, PK, PD and preliminary efficacy of the combination at the LANRA RP2D. For both trial components, response assessments will be conducted on Day 1 of Cycles 2 and 3 and every 3 cycles thereafter until 2 consecutive assessments indicate CR or CRh. A 2-stage design will be employed to test the null hypothesis that the rate of CR/CRh for the combination is ≤23%, as reported for gilteritinib monotherapy, versus the alternative hypothesis of CR/CRh ≥46%, among pts treated at the LANRA RP2D. All pts will be followed for relapse, EFS and OS. Key exploratory endpoints will be assessments of measurable residual disease among pts who achieve CR/CRh, correlations with selected baseline biomarkers that may predict efficacy outcomes and assessment of LANRA PD properties (including target engagement) when administered alone and in combination with gilteritinib. Results: Trial in progress. Summary/Conclusion: Accrual is ongoing. P525: PHASE 3, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY OF THE EFFICACY AND SAFETY OF ENTOSPLETINIB ADDED TO INTENSIVE INDUCTION AND CONSOLIDATION CHEMOTHERAPY IN NEWLY DIAGNOSED NPM1-MUTATED AML J. C. Byrd1,*, J. E. Cortes2, M. D. Minden3, T. Oellerich4, E. M. Stein5, J. Elder6, P. Kumar7, G. Bray7, J. DiMartino7, W. Stock8 1University of Cincinnati, Cincinnati; 2Georgia Cancer Center at Augusta University, Augusta, United States of America; 3Princess Margaret Cancer Centre, Toronto, Canada; 4Frankfurt Cancer Institute, Frankfurt am Main, Germany; 5Memorial Sloan Kettering Cancer Center, New York; 6PharPoint Research, Inc., Durham; 7Kronos Bio, Inc., San Mateo; 8University of Chicago Medicine, Chicago, United States of America Background: Spleen tyrosine kinase (SYK) is a component of both lymphoid and myeloid cell signaling pathways and is implicated in the pathogenesis of a subset of acute myeloid leukemia (AML) defined by dysregulated expression of HOXA9 and MEIS1 transcription factors. Entospletinib (ENTO) is an oral, selective SYK inhibitor that is acceptably tolerated when administered with intensive induction and consolidation in newly diagnosed AML patients (pts). In a Phase 2 study, following induction with cytarabine and daunorubicin (7 + 3) plus ENTO, higher rates of complete response (CR) or CR with incomplete hematologic recovery (CRi) occurred in pts with rearrangements of the KMT2A gene (MLL-r) and mutations of the nucleophosmin 1 (NPM1) gene, both of which are associated with aberrant expression of HOXA9 and MEIS1, compared to pts without these mutations. In an exploratory analysis, pts with HOXA9/MEIS1 expression levels above the median experienced superior overall survival (OS) compared to pts with expression levels below the median. Aims: We hypothesize that the addition of ENTO to intensive induction/consolidation in newly diagnosed pts with NPM1-mutated AML will improve the CR rate without evidence of measurable residual disease (MRD-negative CR) post-induction and duration of event-free survival (EFS). Methods: AGILITY is a global, multi-center, double-blind, placebo-controlled trial of ENTO in combination with cytarabine plus daunorubicin or idarubicin induction (7 + 3) and age-adjusted high-dose cytarabine (HiDAC) consolidation in newly diagnosed AML pts aged 18–74 who are candidates for intensive induction and harbor a documented NPM1 mutation. Pts with co-mutated FLT3 are excluded. Pts will be stratified based on age (<60 vs ≥60 years) and anthracycline administered during induction (daunorubicin vs idarubicin). Approximately 180 pts will be randomized to receive 7 + 3 induction and HiDAC consolidation with ENTO (400mg orally twice daily) or with placebo. Pts with <5% leukemic blasts after induction cycle 1 will proceed to the first cycle of consolidation; pts with ≥5% residual blasts will undergo a second induction cycle. Pts who do not achieve CR after the last cycle of induction plus ENTO or placebo will be designated as induction treatment failures (ITF) for purposes of EFS estimation. Pts who achieve or remain in CR after 2 chemotherapy cycles (either 2 induction cycles or 1 induction and 1 consolidation cycle) will be evaluated for MRD in bone marrow based on enumeration of mutant NPM1 alleles using a molecular assay. Pts may receive ≤3 cycles of consolidation with HiDAC and ENTO or placebo per their original randomized treatment assignment beyond chemotherapy cycle 2. All pts will be followed for relapse and survival. The primary endpoint is the rate of MRD-negative CR (<0.01% mutant NPM1 alleles). A key secondary endpoint is EFS, defined as time from randomization to earliest occurrence of ITF, relapse from CR, or death from any cause. OS will be evaluated. Key exploratory endpoints will be the correlation between recurring genomic mutations and response or progression and longitudinal assessment of peripheral blood for detection of NPM1-m alleles among pts who achieve MRD-negative CR post-induction. Results: Trial in progress. Summary/Conclusion: Accrual is ongoing. P526: REAL-WORLD COMPARISON OF DIFFERENT TREATMENT MODALITIES FOR FAVORABLE RISK ACUTE MYELOID LEUKEMIA: STANDARD DOSE ANTHRACYCLINE VS HIGH DOSE ANTHRACYCLINE VS ADDITION OF GEMTUZUMAB OZOGAMICIN J. Mort1, B. Brewer2, B. Mautner1, S. Dibenedetto1, E. Pierce1, E. Morse1, L. Wells1, F. Ghamsari1, A. Morris1, S. Clark3, J. Reid3, I. Patel4, C. Jackson5, M. Perciavalle6, B. Yelvington6, R. Miller6, K. Abernathy6, H. Wolfe7, S. Locke7, J. Zeidner3, D. Reed5, V. Duong4, B. Dholaria6, T. LeBlanc7, M. Keng1, B. Horton2, F. El Chaer1,* 1Hematology and Oncology; 2Public Health Sciences, Division of Translational Research and Applied Statistics, University of Virginia, Charlottesville; 3Hematology and Oncology, University of North Carolina, Chapel Hill; 4Hematology and Oncology, University of Maryland Medical Center, Baltimore; 5Hematology and Oncology, Wake Forest Baptist Medical Comprehensive Cancer Center, Winston-Salem; 6Hematology and Oncology, Vanderbilt University Medical Center, Nashville; 7Hematology and Oncology, Duke Cancer Institute, Durham, United States of America Background: The standard remission induction regimen for medically fit patients with acute myeloid leukemia (AML) consists of a backbone of cytarabine & an anthracycline (“7 + 3” therapy). However, the choice & dose of the anthracycline varies between institutions & practitioner preference, particularly in AML with favorable risk cytogenetics. Gemtuzumab ozogamicin (GO) may improve outcomes in this patient population but is associated with increased toxicities. Aims: We aimed to compare the outcomes of 3 cohorts of newly diagnosed AML with favorable cytogenetics who received induction 7 + 3 induction therapy: 1. Standard dose (45 or 60 mg/m2 daunorubicin) (SD); 2. Higher dose (90 mg/m2 daunorubicin or 12 mg/m2 idarubicin) (HD) of anthracycline, 3. 7 + 3 with fractionated GO. Methods: We performed a multicenter retrospective analysis of medically fit patients ≥ 18 yo with favorable risk AML determined using ELN 2017 guidelines from 2015-2020 treated across 6 US academic institutions. Categorical variables were summarized using means and standard deviations. The former was compared using Fisher’s exact test; the latter was compared using either the Wilcoxon text or the Kruskal-Wallis as appropriate. One way ANOVA test was used to compare continuous variables. Kaplan-Meier curves were generated to provide visual summaries of survival and time-to-event data. Results: Our study included 189 treated patients who met eligibility criteria with a median follow up of 2 years. Patients’ characteristics and outcomes are summarized in Table 1. Sixty-nine (37%) received induction with SD, 100 (53%) received HD, and 20 (11%) received GO. Patients in the SD and HD group were younger (median age 50 and 52 yo, respectively) and the majority (>94%) had de novo AML. The rates of composite complete remission in the SD, HD, and GO groups were 66.7%, 70% and 75%, respectively (p=0.761). RFS and OS were similar between all groups (p=0.4 and 0.3, respectively). The rate and duration of cytopenias at day 40 after induction, major bleeding, infections, admission to the intensive care unit, new onset cardiomyopathy (ejection fraction<50%), liver dysfunction and acute renal injury were similar among all groups. No patients developed veno-occlusive disease. Table 1: Participant Characteristics and Outcomes by Treatment Regimen GON = 20 HDN = 100 SDN = 69 P-value Age in years (standard deviation) 57.75 (9.32) 49.68 (13.67) 51.55 (13.83) 0.038 de novo AML, N (%) 19 (95.0) 94 (94.0) 65 (94.2) 1.000 Disease status after induction 0.812  CR, N (%) 14 (70.0) 66 (66.0) 40 (58.0)  CRi, N (%) 0 (0.0) 2 (2.0) 2 (2.9)  Persistent, N (%) 1 (5.0) 4 (4.0) 2 (2.9) Composite CR, N (%) 15 (75.0) 70 (70.0) 46 (66.7) 0.761 Admission to ICU, N (%) 4 (20.0) 17 (17.0) 11 (15.9) 0.969 Infections, N (%) 17 (85.0) 74 (74.0) 62 (89.9) 0.082 Major bleeding, N (%) 3 (15.0) 6 (6.0) 4 (5.8) 0.299 VOD, N (%) 0 (0.0) 0 (0.0) 0 (0.0) Transaminitis, N (%) 4 (20.0) 19 (19.0) 19 (27.5) 0.538 Acute renal injury, N (%) 3 (15.0) 19 (19.0) 15 (21.7) 0.962 Cardiomyopathy, N (%) 4 (20.0) 8 (8.0) 5 (7.2) 0.523 Cytopenias at D40 after induction, N (%) 0 (0.0) 7 (7.0) 6 (8.7) 0.542 Image: Summary/Conclusion: Our analysis demonstrated no differences in RFS and OS in patients with AML treated with either GO, HD or SD. In addition, the rates of complications secondary to chemotherapy were similar among all groups. However, a measurable residual disease analysis is ongoing for further characterization of disease response. P527: PHASE 1 TRIAL OF PEGCRISANTASPASE IN COMBINATION WITH VENETOCLAX IN ADULTS WITH RELAPSED OR REFRACTORY ACUTE MYELOID LEUKEMIA (R/R AML) - SAFETY, EFFICACY AND PK/PD IN THE FIRST TWO COHORTS Y. Liu1, O. M. Bah1, D. Bollino1, K. Caprinolo1, J. Zarrabi1, S. Philip1, R. G. Lapidus1, E. T. Strovel2, Z. N. Singh3, M. E. Kallen3, Y. Ning3, R. Koka3, S. Niyongere1, V. H. Duong1, M. R. Baer1, M. Graveno1, A. Emadi1,* 1University of Maryland Greenebaum Comprehensive Cancer Center; 2Pediatrics; 3Pathology, University of Maryland School of Medicine, Baltimore, United States of America Background: Glutamine depletion induced by Erwinia asparaginases including pegcrisantaspase (PegC) downregulates the mTOR/p70S6K pathway and inhibits 4EBP1 phosphorylation. We recently found that the combination of PegC with the Bcl-2 inhibitor venetoclax (Ven) has strong in vitro and in vivo activity in poor-prognosis AML, mediated by enhancement of eIF4E-4EBP1 interaction on the cap-binding complex and decreasing protein synthesis. We hypothesized that Ven-PegC treatment will be safe and effective in the treatment of R/R AML. Here we report the results from the first two cohorts of a phase 1 trial of Ven-PegC in adults with R/R AML. Aims: The primary objective is to estimate the maximum tolerated dose and to determine the regimen-limiting toxicities (RLT) of Ven-PegC. The exploratory endpoints include plasma amino acids levels 1- and 2-weeks post-PegC administration. Methods: This is an open-label phase 1b clinical trial (NCT04666649) of daily oral Ven in combination with biweekly out-patient IV PegC in 28-day cycles that will be administered to 4 cohorts (Table 1A) based on a standard 3 + 3 design. Results: Eight patients (pts) were enrolled into the first two cohorts; 7 received ≥ 1 dose of PegC and one dropped out before receiving study drugs. Pts’ characteristics are shown in Table 1B. Median age was 66 (59-76) years; two (29%) were female; six (86%) had adverse-risk AML by ELN classification; and four (57%) had complex karyotypes. Pharmacokinetic (PK) and pharmacodynamic (PD) data are available for 5 pts; all pts achieved a nadir plasma asparaginase activity > 0.1 IU/mL during cycle 1 (3/3 in cohort 1 within 3 weeks and 2/2 in cohort 2 within 10 days). Mean plasma amino acid levels (µmol/L) at baseline were: asparagine (Asn) 29 (17-38), glutamine (Gln) 421 (384-465), glutamate (Glu) 59 (22-103), serine (Ser) 66 (49-80) and glycine (Gly) 203 (166-207). All pts achieved an Asn level of 0 by day 7; Asn remained undetectable throughout the study. Plasma Gln (µmol/L) reduction from baseline occurred in 4/5 (80%) pts within cycle 1: pt4 423→273 (Day15), pt5 446→86 (Day28), pt6 467→216 (Day22), pt7 388→156 (Day8), pt10 420→395 (Day10). Plasma Glu levels increased in all pts, indicating conversion of Gln to Glu by PegC. Interestingly, plasma levels of Ser increased 1.5-2-fold at the nadir of plasma Gln (Figure 1A-C, maximum amino acid changes after Ven-PegC). Seven pts from cohorts 1 (4 pts) and 2 (3 pts) who received at least one dose of Ven-PegC were included in analysis of toxicity and efficacy, with data cutoff February 26, 2022 (Table 1B). No RLT has been observed. In cohort 1, pt2 died before completing cycle 1 due to septic shock and delay in seeking medical care, pt4 died of AML progression. In cohort 2, pt7 had progressive disease after cycle 1, pt9 opted for comfort care before evaluation of response, and pt10 died of AML progression, In cohort 1, pt6 achieved measurable residual disease (MRD)-negative complete remission with partial hematologic recovery (CRh) and proceeded to allogeneic hematopoietic stem cell transplant (allo-HSCT) after 3 cycles of Ven-PegC, and bone marrow blasts in pt5 decreased from 78% to 9% after cycle 1. These two pts had the lowest plasma Gln levels (103 [pt6, cycle2 day29] and 86 [pt5, cycle1 day28]). Image: Summary/Conclusion: No RLT has been observed following Ven-PegC in cohorts 1 or 2. The Ven-PegC combination at the lowest dose of PegC administered, produced an MRD negative CRh in one patient and a transient substantial reduction in bone marrow blast count in another, both associated with decreases in plasma Gln levels. The study is ongoing. P528: A MIRNA SIGNATURE RELATED TO STEMNESS IDENTIFIES HIGH-RISK PATIENTS IN PEDIATRIC ACUTE MYELOID LEUKEMIA E. Esperanza Cebollada1,2,*, S. Gomez Gonzalez3, N. Vega García1,2, C. Vicente Garcés1,2, M. Torrebadell1,2,4, S. Rives2,4,5, J. L. Dapena2,5, A. Català2,4,5, M. Camós1,2,4 1Hematology Laboratory, Hospital Sant Joan de Déu; 2Leukaemia and other Pediatric Hemopathies. Developmental Tumors Biology Group; 3Developmental Tumor Biology Laboratory, Institut de Recerca Hospital Sant Joan de Déu, Esplugues de Llobregat, Barcelona; 4Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid; 5Pediatric Hematology and Oncology Department, Hospital Sant Joan de Déu, Esplugues de Llobregat, Barcelona, Spain Background: MicroRNAs (miRNAs) regulate gene expression and may function as tumor suppressors or cancer promoters. MicroRNAs regulating hematopoietic stem cells (HSC) could be prognostic biomarkers in leukemia, particularly in KMT2A-rearranged (KMT2A+) patients, in whom few mutations have been described. Aims: To analyze the role of miRNAs involved in HSC self-renewal and stemness pathways (NOTCH, WNT/beta-catenin, HOX, and FLT3) in a pediatric acute lymphoblastic leukemia (ALL) and myeloid acute leukemia (AML) cohort of patients. Methods: We analyzed by quantitative PCR the expression of 89 selected miRNAs. To calculate miRNAs ∆∆CT expression differences between paired subgroups we used the lmFit function of the Limma R package, and the false discovery rate method to adjust the p-values with a 0.05 cut-off. Results: We studied 110 patients (38% female, median age 6.1 years (range 0-17.4)), including 70 ALL, 39 AML, and one undifferentiated leukemia. Our series was primed with KMT2A+ cases of all lineages (n=28). Globally, in the unsupervised hierarchical cluster analysis (HCA), patients were separated by lineage. Based on the sub-branching, we tried to correlate the clusters with clinic-biological variables. We found no association with age, sex, central nervous system infiltration, or leukocyte count. However, Kaplan-Meier analysis revealed that some clusters had significantly different event-free survival (EFS) (p=0.014): poor (clusters P1 and P4), good (P2), and intermediate (P3). Of note, clusters with good and poor survival branched off again according to lineage (P1 included most of AML cases and P4 most of ALL patients). We then studied survival according to lineage. Within ALL patients, we saw a group with a non-significant worse outcome (P4ALL), as compared with the remaining patients (P2ALL and P3ALL). In contrast, in AML patients, the HCA distinguished a group of 14 patients (36%, P1AML) with significantly worse outcome: OS at 5 years of 64.3%±12.8 vs. the remaining AML patients, all showing excellent survival (grouped in P2AML, 90.5%±6.4), p=0.028. Notably, differences in EFS at 5 years were even higher: 43%±13.2 (P1AML) vs. 86.5%±7.2 (P2AML), p=0.003. P1AML included cases from all molecular categories (RUNX1::RUNX1T1, CBFB::MYH11, KMT2A+, and cases without recurrent genetic alterations). Of note, no PML::RARA cases fell in this cluster. We identified 24 miRNAs (23 underexpressed and 1 overexpressed) differentially expressed in P1AML patients. The 24 miRNAs signature included underexpressed miRNAs known to act as tumor suppressors regulating cell proliferation or apoptosis (hsa-miR-223-3p, hsa-miR-193a-3p, hsa-miR-181a-5p, hsa-miR-181b-5p, hsa-miR-181c-5p, hsa-miR-708-5p, hsa-miR-34b-5p, and hsa-miR-22-3p). The only miRNA overexpressed, hsa-miR-9-5p, promotes proliferation and inhibits apoptosis, functioning as oncomiR in KMT2A-rearranged AML cases. The remaining miRNAs (hsa-miR-199b-5p, hsa-miR-222-5p, hsa-miR-373-3p, hsa-miR-195-5p, hsa-miR-151a-5p and hsa-miR-1290, hsa-miR-20b-5p and hsa-miR-17-5p, hsa-miR-24-3p, hsa-miR-128-3p, hsa-miR-21-5p, hsa-miR-331-5p, hsa-miR-30b-5p, hsa-let-7g, and hsa-let-7i-5p), among their pleiotropic functions, regulate cellular stemness and proliferation. Our results suggests that within each molecular category, a miRNA signature could distinguish those patients with activated stemness and proliferation pathways and a more aggressive leukemia. Image: Summary/Conclusion: We obtained a distinctive signature of 24 miRNAs capable of distinguishing pediatric AML patients with excellent or poor outcome. P529: FIRST EUROPEAN REAL-WORLD EVIDENCE PROSPECTIVE REGISTRY OF FIRST-LINE ADULT PATIENTS WITH BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM TREATED WITH FIRST-IN-CLASS CD123-TARGETED THERAPY TAGRAXOFUSP U. Platzbecker1,*, E. Angelucci2, P. Montesinos3, R. M. Lemoli4, A. Spyridonidis5, J. Casariego6, T. I. Mughal7,8, M. Mohty9 1Medical Clinic and Policlinic of Hematology, Cell Therapy and Hemostaseology, University Hospital Leipzig, Leipzig, Germany; 2Hematology and Cellular Therapy Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy; 3Department of Hematology, Hospital Universitari i Politècnic La Fe, València, Spain; 4Clinic of Hematology, Department of Internal Medicine, IRCCS San Martino Hospital, University of Genoa, Genoa, Italy; 5Department of Internal Medicine, BMT Unit, University of Patras, Patras, Greece; 6Aldebaran Research & Development SL, Madrid, Spain; 7Division of Hematology-Oncology, Tufts University School of Medicine, Boston; 8Stemline Therapeutics, New York, United States of America; 9Department of Hematology and Cellular Therapy, Hôpital Saint-Antoine, Sorbonne University, Paris, France Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy derived from plasmacytoid dendritic cells that overexpress interleukin-3 (IL-3) receptor alpha (CD123). It is a highly aggressive disease characterized by skin and bone marrow involvement and is associated with a poor prognosis (median overall survival 8–14 months). Tagraxofusp (TAG, SL-401) is a first-in-class CD123-targeted therapy comprising human IL-3 fused to a truncated diphtheria toxin payload. It is currently the only approved first-line (1L) treatment for adult patients (pts) with BPDCN in Europe. As of July 2021, 65 pts with BPDCN have received TAG in a clinical setting outside the US as part of an expanded access program. Collecting real-world clinical data from TAG is important to increase understanding of clinical outcomes, especially in pts commonly underrepresented in clinical trials. We describe the first real-world evidence prospective registry in pts with BPDCN receiving TAG. It has been designed to meet requirements outlined in the marketing authorization of the European Commission for further evaluation of the efficacy and safety of TAG in pts with 1L or relapsed/refractory BPDCN. Aims: To investigate effectiveness and safety of TAG in pts with 1L BPDCN. Methods: This is a noninterventional, single-arm, post-authorization study (EudraCT: 2021-001684-24) in adult pts with BPDCN prescribed TAG under real-world routine clinical practice conditions (12 mcg/kg intravenously [IV] once daily on days 1–5 of a 21-day cycle, per the summary of product characteristics recommendation). A minimum of 80 pts will be included in approximately 40–55 sites in Europe, estimated over 4 years. Eligible pts have a diagnosis of BPDCN and are to be treated (or have recently started treatment) with TAG monotherapy as 1L treatment, per physician’s decision. Primary endpoints are rate of complete response (CR), defined as CR + clinical CR (CR with residual skin abnormality not indicative of active disease) after 3 months of treatment, and safety of TAG including the incidence and severity of capillary leak syndrome (CLS). Secondary endpoints include number of pts bridging to SCT, progression-free survival, overall survival, best overall response, duration of response, TAG dose interruptions/administration of IV albumin supplementation in pts with CLS or CLS symptoms, as well as safety and incidence and severity of adverse events of special interest (which include CLS and hepatic, renal, and cardiac events). Quarterly data collection is anticipated, as well as collection at screening, enrollment, early discontinuation, and study end (Figure). Safety data collection will end 18 months after the last enrolled pt’s first visit (LPFV). Interim analyses will be performed annually; an effectiveness interim analysis is scheduled at 20 months post-LPFV. Analyses will be performed using descriptive statistics. Survival data will be summarized using the Kaplan-Meier method. Subgroup analyses for effectiveness and safety may be performed, on the basis of gender, age, or baseline Eastern Cooperative Oncology Group performance status. Enrollment is planned to start by June 2022. Taking the registry as the basis, upon study finalization a complementary comparative analysis of effectiveness and safety data from the TAG registry with a retrospective clinical cohort will be undertaken using appropriate methodology, such as propensity score matching or inverse probability treatment weighting. Results: This is a TiP and there are no results at this time. Image: Summary/Conclusion: This is a TiP and there are no results at this time. P530: TAGRAXOFUSP IN BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM WITH/WITHOUT CENTRAL NERVOUS SYSTEM INVOLVEMENT AND INTRATHECAL CHEMOTHERAPY AS PRIMARY TREATMENT OR PROPHYLAXIS: AN ITALIAN EXPERIENCE. G. Rivoli1,*, G. Beltrami1, A. Raiola1, A. Dominietto1, M. Riggi2, E. Angelucci1 1Hematology and cellular therapy unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy; 2Stemline Therapeutics Switzerland GmbH, Zug, Switzerland Background: The precise frequency of central nervous system involvement (CNS+) in patients (pts) with blastic plasmacytoid dendritic cell neoplasm (BPDCN) is currently unknown. Recent small series of clinical experience suggest the incidence to be about 10% at baseline and 30% at first relapse. CNS is a ”sanctuary” for any systemic treatment and can lead to disease recurrence even after the achievement of a complete response (CR) at the medullary and skin level. Thus, it is essential to ensure clearance (or prophylaxis) of the disease including the meningeal compartment. Intrathecal (IT) chemotherapy is a valid complementary tool aimed at eradication of the disease, with modest systemic side effects; it is currently indicated as both prophylaxis and primary treatment. Combining IT chemotherapy with systemic treatment has made it possible to obtain long remissions in pts with BPDCN even in the presence of CNS+. Tagraxofusp (TAG), a first-in-class CD123-targeted therapy, was approved by the European Medicines Agency in 2021 for treatment of BPDCN in first-line adult pts. Aims: Data from 5 pts with BPDCN with/without CNS+ treated with TAG were retrospectively collected in our center in Geneva. The aim was to evaluate if CNS treatment/prophylaxis impacts prognosis and efficacy of systemic treatment with TAG. Methods: A diagnosis of BPDCN was confirmed by hematopathology with biomarkers, including CD123, CD4, and CD56, and presence of CNS involvement by the characteristic morphology and “8-color flow cytometry” for CD123. Pts received TAG intravenous infusions at 12 mcg/kg once daily on days 1–5 of a 21-day cycle. TAG was repeated for 1–4 cycles, depending on the level of response and potential bridge to hematopoietic stem cell transplant (HSCT). Hospitalization was mandatory for the first cycle; the following cycles were administered in an outpatient setting if no severe complications occurred during the first cycle. IT chemotherapy (methotrexate 12.5 mg, dexamethasone 4 mg, and cytarabine 50 mg) was administered at each cycle at the same doses until negative cerebrospinal fluid (CSF) was observed in CNS+ pts. After 4 treatment cycles, response was evaluated, including CSF examination. Results: TAG was administered in 5 first-line pts with BPDCN; 3 were CNS+ (1 clinically evident case and 2 with cytofluorometric positivity only). All pts received IT chemotherapy, both as a primary-intention treatment in the 3 CNS+ pts, and as prophylaxis in the 2 CNS-negative pts. Table 1 reports the main pt characteristics. Pts 2, 3, and 5 with CNS+ achieved clearance of the disease and none reported CNS relapse. In pts receiving CNS prophylaxis, pt 1 received TAG plus HSCT and is in CR after 29 months; pt 4 received high-dose chemotherapy, achieved a good partial remission after TAG, and remains alive after 4 months with HSCT shortly planned. No pts developed CNS adverse events following IT chemotherapy. Image: Summary/Conclusion: These preliminary results confirm the feasibility of IT chemotherapy with systemic TAG. Baseline disease and CNS involvement did not appear to predispose pts to different efficacy results or treatment-related adverse events. IT chemotherapy effectively cleared and controlled CNS involvement. IT prophylaxis should be considered in pts with BPDCN who receive CD123-targeted therapies. P531: AN OBSERVATIONAL, MULTICENTER, RETROSPECTIVE ANALYSIS OF PATIENTS WITH BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM TREATED WITH TAGRAXOFUSP IN THE EUROPEAN EXPANDED ACCESS PROGRAM M. Herling1,*, E. Deconinck2, M. Anant3, D. Manteigas4, M. Riggi3, E. Angelucci5 1Department of Hematology, Cell Therapy and Hemostaseology, University Hospital Leipzig, Leipzig, Germany; 2Regional University Hospital of Besançon, Besançon, France; 3Stemline Therapeutics Switzerland GmbH, Zug, Switzerland; 4Cmed, West Sussex, United Kingdom; 5Hematology and Cellular Therapy Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare aggressive hematologic malignancy, with poor prognosis (median age 67 yr). It is characterized by clonal expansion of plasmacytoid dendritic tumor cells expressing specific markers including the interleukin-3 receptor alpha (CD123). Primary sites are skin and bone marrow, followed by peripheral blood, lymph nodes, viscera, and central nervous system. Tagraxofusp (TAG), a CD123-targeted therapy, was approved by the European Medicines Agency on 07.01.2021. In 08.2019, a Global Expanded Access Program (EAP) was implemented to provide access to patients (pts) prior to regulatory authorization of TAG in real-world practice. Aims: We conducted a European multicenter non-interventional, retrospective analysis of BPDCN pts treated with TAG. Main objectives were rates of complete response, and incidence and severity of capillary leak syndrome (CLS). Secondary outcomes included rate of pts bridged to stem cell transplantation, progression-free survival, and overall survival, safety of TAG measured by the incidence and severity of adverse events (AEs), and number of TAG doses administered in each cycle. Methods: The main inclusion criterion was diagnosis of BPDCN, confirmed by hematopathology with established marker panels (including CD123). The physician who signed the supply form of TAG in each participating center informed the pts on their treatment. Training of the physicians, nurses, and pharmacists was mandatory before delivering the treatment. The analysis included all pts enrolled in the European EAP from 08.2019 to 12.2021. Pts received TAG intravenous infusions, at 12 mcg/kg once daily on days 1–5 (up to day 10 allowed) of a 21-day cycle. Hospitalization was required only for the first cycle (subsequent cycles were allowed to be administered in an outpatient setting). Results: Overall, 76 adult (median age 64 yr, range 21–85 yr) and 4 pediatric pts (1, 4, 14, and 16 yr) were included across 57 European centers: Germany 18 centers, France 17, Italy 9, Switzerland 5, United Kingdom 4, Spain 3, and Austria 1. Most pts were male (78%), representing real-world distribution. Sixty-three pts received first-line TAG and 17 pts as second or further line of treatment. The median number of cycles (based on the number of treatments delivered for the TAG EAP supply of each 21-day cycle) was 2.5 (range 1–8) in first line and 2.6 (range 1–13) cycles in second-line and further pts, respectively. No deaths due to CLS were reported. The analysis is still ongoing at the time of abstract finalization; complementary data on safety, efficacy, number of pts transplanted, and time-related parameters will be reported at the meeting. Summary/Conclusion: The is the largest retrospective analysis of real-world clinical practice outside of a clinical trial in BPDCN pts treated with TAG. The EAP was carefully followed in all 57 centers with initial training provided to the multidisciplinary teams before treatment initiation. This is thought to have positively affected prevention and management of CLS and other grade 3–4 AEs; no death related to CLS occurred. The preliminary results confirm the feasibility and safety of TAG, allowing the administration also in elderly pts, with manageable safety. In fact, AEs mainly occurred in cycle 1 in an inpatient setting, with similar rates and severity in older vs younger pts. Baseline characteristics did not appear to predispose pts to different treatment-related AEs. P532: ACUTE MYELOID LEUKEMIA (AML): UNICENTRIC REPORT ON 1029 PATIENTS DIAGNOSED IN TERTIAL REFERAL CENTER A. Fricke1,*, K. Nachtkamp1, K. Döhner2, B. Hildebrandt3, B. Betz3, M. Rudelius4, M. Seidl5, C. Zahner1, G. Kobbe1, A. Kündgen1, P. S. Jäger1, B.-N. Baermann1, U. Germing1 1Department of Hematology, Oncology and Clinical Immunology, University Hospital Düsseldorf, Düsseldorf; 2Department of Internal Medicine III, University Hospital Ulm, Ulm; 3Institute of Human Genetics, University Düsseldorf, Düsseldorf; 4Pathological Institute, Ludwig-Maximilians-University Hospital Munich, Munich; 5Pathological Institute, University Hospital Düsseldorf, Düsseldorf, Germany Background: Since 2016 patients with AML can be diagnosed according to the WHO classification proposals. The ELN genetic risk classification is a tool for treatment planning. Aims: In order to validate the WHO and the ELN classification, we collected data from 1029 patients diagnosed and / or treated in our institution from 2005 to 2021. Methods: Patients were followed up until December 2021. Diagnoses were based on cytomorphology, histomorphology, flow cytometry, karyotyping, and molecular genetics performed at the University of Düsseldorf and partly at the University of Ulm. Treatment intensity was defined as follows: no treatment at all, best supportive care (BSC) (including low dose Ara-C and Hydroxyurea), Hypomethylating agents (HMA), induction and consolidation, or allogeneic hematopoietic stem cell transplantation (HSCT). Results: Median age at diagnosis was 64 years (ys) (19 – 94). 46% were females. There were 270 patients with recurrent genetic abnormalities (26.5%), 346 with AML-MRC (34%), 121 with tAML (11.9%), 243 with AML-NOS (23.9%), 3 with myeloid sarcoma (0.3%), 4 with blastic plasmacytoid dendritic neoplasm (0.4%), 20 with biphenotypic AML (2%), and in 22 patients the classification was not possible. 17.7% of patients belong to the good risk category of ELN classification, 21.6% to the intermediate risk category and 30% to the adverse risk group. In 30.7%, the classification was not possible due to missing data. 274 patients were treated with HSCT as most intensive therapy (28%), 348 patients with induction and consolidation (35.6%). 209 patients received HMA (21.4%). 127 patients were treated with BSC (13%) and in 20 patients (2%) no therapy was administered. In 51 patients, AML was diagnosed at our laboratory, but treatment took place outside of our department. The median survival time of the entire group was 18 months with 39% of the patients alive by the end of 2021. Median survival was 21 months in females as compared to 16 months in males (p = 0.04). Patients aged over 65 ys had a median survival of 7 months as compared to 134 months in patients aged less than 65 ys (p < 0.0005). There was no difference in overall survival in patients younger than 50 ys of age. Patients with recurrent genetic abnormalities did not reach median survival whereas median survival of patients with AML-MRC, tAML, and NOS was 11, 12, and 14 month (p < 0.0005). The latter three categories did not differ in terms of median survival (p = 0.23). Within the group of AML with recurrent genetic abnormalities, only the patients with t(6;9) and inv(3) reached median survival whereas all other categories had a favorable outcome. Patients of the good risk ELN group did not reach the median survival time whereas median survival of the intermediate and adverse risk groups was 27 and 12 months (p < 0.0005). Patients who underwent HSCT did not reach median survival. Patients treated with induction and consolidation had a median survival time of 123 months compared to 8 months median survival in patients who got HMA (p < 0.0005). In a multivariate analysis including intensity of therapy, age at diagnosis, and ELN group, only intensity of treatment and ELN group were independently influencing overall survival. Summary/Conclusion: The WHO and the ELN classification could be assessed correctly in about 70% of patients only, primarily due to the missing examination of somatic mutations. The ELN classification together with the treatment intensity have major impact on outcome. Patients who neither receive induction and consolidation nor HSCT are facing an unfavorable outcome, even if they are treated with HMA. P533: SINGLE-CENTER PHASE 2 STUDY OF PD-1 INHIBITOR COMBINED WITH DNA DEMETHYLATION AGENT + CAG REGIMEN IN PATIENTS WITH HIGH-RISK ACUTE MYELOID LEUKEMIA: INTERIM ANALYSIS X.-N. Gao1,*, Y.-F. Su1, Y. Jing2, J. Wang1, L. Xu1, L.-L. Zhang2, A. Wang1, Y.-Z. Wang1, Y.-F. Li2, D.-H. Liu1 1Senior Department of Hematology, the Fifth Medical Center; 2Department of Hematology, the First Medical Center, Chinese PLA General Hospital, Beijing, China Background: The expression of PD-L1 is increased in acute myeloid leukemia (AML) cells. However, blocking the immune checkpoint alone has limited efficacy as a single agent in highly proliferative leukemia cells. Here we report the results of an interim analysis of an ongoing single-arm phase 2 trial (no. NCT04541277) of PD-1 inhibitor combined with DNA demethylation agent + CAG regimen in patients with high-risk AML. Aims: To assess safety and response to Tislelizumab + Azacytidine/Decitabine + CAG regimen after minimum 1 cycle of therapy in patients who failed at least one prior induction course of therapy or developed relapse (Cohort 1), and patients with persistent minimal residual disease (MRD)-positivity after consolidation treatment (Cohort 2). Methods: Patients must have failed at least one prior induction course of therapy or have a relapse or have a persistent MRD-positive disease after consolidation treatment. Other eligibility criteria included in ECOG performance status ≤ 2, normal organ function, and no autoimmune diseases requiring systemic immunosuppression. The patients received Azacytidine 75 mg/m2 subcutaneously daily, day 1-7 or Decitabine 20 mg/m2 intravenously daily, day 1-5 plus CAG regimen (cytarabine 100 mg intravenously every 12 hours, day 1-5; aclarubicin 20 mg intravenously daily, day 1-5; and concurrent use of G-CSF 5 mg/kg/day subcutaneously) with Tislelizumab 200 mg beginning on the next day after chemotherapy was stopped and every 4 weeks thereafter. This study was approved by the Ethics Committee of the Chinese PLA General Hospital, and signed informed consents were obtained from all patients. Results: Efficacy: Fifteen patients has been enrolled (Table 1). Fourteen (93.3%) are evaluable for response, 7 (46.7%) completed 2 cycles and 8 (53.3%) completed 1 cycle: Of the 12 patients in the previous treatment failure cohort, 7 achieved complete remission (CR)/CR with incomplete hematologic recovery (CRi) (1/7) (Table 1B), 3 partial remission (PR), and 2 non-remission. Of the 2 patients in the persistent MRD-positivity after consolidation treatment cohort, 1 achieved MRD negativity and 1 had no response. The 8-week mortality were 18.8%: 2 patients died of rapidly progressive disease concomitant with lung infection at 34 days and 56 days after treatment, respectively. One died of relapse of disease at +4 months after received HLA-haploidentical peripheral blood stem cell transplantation (PBSCT). One died of acute graft-versus-host disease of the gastrointestinal tract at +35 days after received a HLA-haploidentical PBSCT in NR status at transplant. With a median follow-up of 5.4 months (1.1 -12.7), the median overall survival (OS) is not reach with an estimated 1-yr OS of 59.6% (95%CI, 17.8%-85.6%). The median OS for responders (CR/CRi+PR) is not reach vs. 1.9 months for non-responders (p=0.083). The median event-free survival (EFS) is 6.5 months with an estimated 1-yr EFS of 58.3% (95%CI, 7.7%-89.3%). Toxicity: Grade 2 immune-related adverse events (IRAEs) were observed in 2 (13.3%) patients, included in 1 patient with grade 2 skin rash which resolved with steroids, and the other with grade 2 peripheral sensory neuropathy. All patients developed treatment-related grade 3 or 4 hematologic toxicities. Two patients died of lung infection at 34 days and 56 days after treatment in the setting of PD. Image: Summary/Conclusion: Tislelizumab + Azacytidine/Decitabine + CAG regimen is safe and effective for high-risk AML patients. IRAEs are mild, low-grade. Special attention needs to be paid to patients who have an increased risk of developing graft-versus-host disease. P534: AZACITIDINE PLUS VENETOCLAX FOR THE TREATMENT OF RELAPSED AND FIRST-LINE AML PATIENTS S. Garciaz1,*, M.-A. Hospital1, A.-S. Alary2, C. Saillard1, Y. Hicheri1, B. Mohty1, J. Rey1, E. D’incan1, A. Charbonnier1, V. Ferdinand1, V. Maisano1, L. Lombardi1, A. Ittel3, M.-J. Mozziconacci3, V. Gelsi-Boyer3, N. Vey1 1Hematology; 2Molecular biology; 3Biopathology, Institut Paoli-Calmettes, MARSEILLE, France Background: Venetoclax (VEN) belongs to a novel BH3-mimetic class of small molecules that selectively targets BCL-2, activating the apoptosis effectors BAX and BAK to drive mitochondrial outer membrane permeabilization, cytochrome c release and cell death. Combination of VEN and the hypomethylating agent azacitidine (AZA) has deeply changed the paradigm of treatment of newly diagnosed (ND) AML patients ineligible for high dose chemotherapy because of older age or comorbidities. There is scarce evidence for the utilization of VEN-AZA for relapsed or refractory (R/R) AML, a category of patients classically associated with an extremely poor outcome. Aims: The objective of our study was to describe a R/R AML cohorts of AML patients treated with VEN-AZA in our institution and to compare the clinical and molecular characteristics predicting response in R/R AML versus ND AML Methods: This retrospective study included consecutive patients treated with VEN-AZA for R/R AML and ND AML. Patients received AZA at standard dose of 75 mg/m2 QD for seven days and VEN was administrated either at 400 mg or at 100 mg when associated with strong CYP3P450 A3 inhibitors after three days ramp up. Response was determined using the ELN 2017 criteria. The ORR was defined as the combination of complete response (CR), CR with incomplete hematologic recovery (CRi), and morphologic leukemia-free state (MLFS). Results: We compared the outcome of 39 R/R AML and 38 concomitant ND AML patients treated in our institution between Jan. 2020 and Dec. 2021. The median age was 69 (22-86) and 73 (61-81) in the R/R and ND groups, respectively. Thirty-five percent of patients had MRC-AML. Adverse cytogenetics was found in 36% of patients in the R/R group and 59% of patients in the ND group. Most frequent mutations were ASXL1, RUNX1, TET2, IDH1/2 and TP53 found in 33%, 33%, 28%, 24% and 22% of patients, respectively. Overall response rate was lower in R/R AML (37% versus 56%) including 13% CR, 8% CRi, 3% PR and 13% MLFS in the R/R AML group and 32% CR, 13% CRi and 13% MLFS in the ND AML group. Adverse cytogenetics was associated with treatment failure only in the R/R group (Relative Risk=0.10, p=0.005). ASXL1, IDH1/2 and SFSR2 mutations were associated with a trend in a higher response rate in the R/R group. Median overall survival (OS) were 5.9 months in the R/R group and 9.4 months in the ND group. In the R/R group, median OS were 2.2 months in the adverse cytogenetics group versus 8.7 months in the intermediate cytogenetics group (p=0.02). Median leukemia-free survival of responding patient was not different between the two groups (9 months), indicating that VEN-AZA can be efficient as a salvage treatment for selected R/R AML patients. Summary/Conclusion: We described one of the largest series of R/R AML patients treated wiht VEN-AZA. By a doing a direct comparison between R/R AML and ND AML treated concomitantly, we found that adverse cytogenetics was associated with treatment failure only in the R/R group suggesting that this subgroup of patients should not be treated with VEN-AZA. Further analyses including more patients are needed to determine which subgroup may benefit from the VEN-AZA as a salvage treatment. P535: UPDATES FROM ITALIAN MULTICENTER REAL-LIFE EXPERIENCE ON CPX-351 THERAPY IN YOUNG PATIENTS (<60 YEARS OLD). B. Garibaldi1,*, M. Franciosa2, F. Pilo3, D. Menotti4, V. Cardinali5, L. Brunetti4, E. A. Martino6, E. Vigna6, I. Tanasi7, A. Duminuco1, C. Maugeri8, M. S. Parisi8, P. F. Fiumara8, E. Mauro8, M. Gentile6, M. P. Martelli5, D. Capelli4, C. Romani3, S. Galimberti2, G. A. Palumbo1,8, F. Di Raimondo8, C. Vetro8 1Postgraduate School of Hematology, University of Catania, Catania; 2Division of Haematology, A.O.U. Pisana, U.O. Ematologia, Pisa; 3U.O.C. Ematologia e Trapianto di cellule staminali emopoietiche, A.O. Brotzu, Cagliari; 4SOD clinica ematologica, Ospedale Umberto I di Ancona, Ancona; 5Division of Haematology, A.O. S. Maria della Misericordia, Perugia; 6Division of Haematology, A.O. di Cosenza, Cosenza; 7Division of Haematology, A.O.U. Policlinico ‘G.B. Rossi’, Verona; 8Division of Haematology, A.O.U. Policlinico “G.Rodolico” - S.Marco, Catania, Italy Background: CPX-351 has been indicated as a valid treatment approach in acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MRC) and therapy-related AML (t-AML). Data on young patients are limited. Aims: Explore CPX-351 efficacy in younger patients (pts) (<60 years old) in real-life. Methods: Since September 2019 we treated 32 pts from 7 Italian centres with CPX-351. Median age was 55 (range 32-59) and ECOG range 0-2. Our cohort consisted in 6 pts with t-AML and 26 pts with MRC-AML (10 morphologic, 11 secondary to MDS, 3 secondary to MDS/MPN, 6 with MRC cytogenetics features including 2 secondary to MDS and 2 morphologic AML-MRC). 2 pts (both with AML-MRC) harboured FLT3-ITD and 1 patient FLT3-TKD, 4 pts IDH1 and 3 pts TP53 mutations. NPM1 was negative in all pts. 10 pts had complex karyotype (CK) and 5 pts received prior treatment with hypomethylating agents for MDS. Results: All pts underwent induction therapy with intravenous administration of CPX-351 at day 1, 3 and 5 except 1 patient who received two doses due to pneumonia. 2 pts (6%) died during induction. Most frequent complication was febrile neutropenia (46%). At disease re-evaluation, we obtained 16 (50%) Complete Remission (CR), 5 (16%) CR with incomplete hematological recovery (CRi), 1 (3%) Morphological Leukemia Free State (MLFS), 4 (12,5%) Partial Remission (after reinduction converted to 2 CR, 1 CRi, while 1 patient was refractory) and 4 (12,5%) refractory pts who switched to salvage therapies except for one who died due to disease progression. Overall response rate (ORR) was 68% after induction and 78% after reinduction. All 25 responding pts underwent consolidation, except one that proceeded directly to allogenic stem cell transplantation (HSCT). 16 out 25 responding pts (64%) underwent HSCT. CR rate in patient with CK was 60%. At induction, median days of severe neutropenia (defined as neutrophils ≤ 500/uL) and thrombocytopenia (defined as platelets ​​≤20.000/uL) were respectively 26 and 22. Regarding subgroup analysis by driving mutations, the 4 IDH1 pts obtained a CR and 3 of them received HSCT. 2 out of 3 TP53 pts were refractory and the other one obtained a CR followed by HSCT. About the 2 FLT3-ITD pts, one died during induction and the other one obtained CR after reinduction and then underwent HSCT. After a median follow-up of 19 months (mo), 6 pts out of the 25 who reached CR/CRi (24%) relapsed and 20 out of 32 (63%) pts are alive. Median relapse-free survival (RFS) for responding pts was not reached. RFS was inferior for t-AML compared to AML-MRC (7 mo vs not reached median, p=0.032). RFS was negatively influenced by CK (median RFS 7 mo for CK vs not reached if no CK, p=0.009) and performance status (PS) (median RFS not reached for PS 0 vs 15 months for PS 1 vs 7 mo for PS 2, p=0.017). Median overall survival (OS) was 20 mo. Pts responding to induction did not reach median OS, while refractory pts had a median OS of 9 months. Noteworthy, OS was not influenced by cytogenetic risk, subtype of AML (if MRC or t-AML) or mutational status. Pts undergoing HSCT at first CR, did not reach median OS, while not transplanted pts had a median OS of 17 mo. COX multivariate analysis showed that reaching CR was the only significative parameter for OS (HR 8, 95% CI 1.18-54). Summary/Conclusion: CPX-351 is active in young pts with t-AML and AML-MRC with an ORR higher than that reported in the pivotal study (78% vs 47%) and high rate of bridge to transplant at first remission (56%) also considering the different age range. CK and PS 1-2 negatively impacted RFS. OS was influenced by response to induction and HSCT. P536: VALUE OF MEASURABLE RESIDUAL DISEASE BY MULTIPARAMETRIC FLOW CYTOMETRY IN NON-PROMYELOCYTIC ACUTE MYELOID LEUKEMIA. REAL WORLD EVIDENCE A. D. Gimenez Conca1,*, J. Gonzalez2, M. M. Rivas3, A. Navickas4, I. Fernandez5, I. Rey6, H. Dick7, S. Cranco8, M. Moirano9, L. Ferrari5, M. Clavijo10, A. Suero11, R. Ramirez12, A. L. Basquiera13, N. Carnelutto14, M. L. Rapan15, A. Jorge Alberto1, C. Belli16 1Hospital Italiano de Buenos Aires; 2Hospital Durand, Buenos Aires; 3Hospital Austral, Pilar; 4Hospital El Cruce, Florencio Varela; 5Fundaleu; 6Hospital Ramos Mejia, Buenos Aires; 7Hospital Italiano de La Plata, La Plata; 8Instituto Alexander Fleming, Buenos Aires; 9Hospital San Martin, La Plata; 10Hospital Aleman; 11Hospital Cesar Milstein, Buenos Aires; 12Instituto Conciencia, Neuquen; 13Hospital Privado, Cordoba; 14Hospital de Clínicas; 15Sanatorio Sagrado Corazon; 16Academia Nacional de Medicina, Buenos Aires, Argentina Background: Measurable residual disease (MRD) allows recognition of a group of patients with acute myeloid leukemia (AML) with a higher risk of relapse. There are, however, remaining open questions, since clinical studies applied variable techniques, cutoff values and time points. Multiparameter flow cytometry (MFC) is the most accessible method in Argentina, since real time PCR assessment is not available for the majority of the institutions. We designed, therefore, this real-life study using decentralized assessment of MRD by MFC to solve the role of positive MRD at different times during treatment with standard chemotherapy. Aims: Our aim was to assess the impact of positive MRD at the end of induction and after first consolidation (C1) as a prognostic factor for relapse-free survival (RFS) and overall survival (OS) in patients with non-promyelocytic AML. Methods: The registry of the Argentinian Group of Acute Leukemia (GALA) from the Argentine Society of Hematology (SAH) includes data from 20 centers. We retrospectively selected an analytical cohort with all patients with AML, treated with an intensive regimen and in complete remission (CR) after induction, from January 2010 to March 2021. The OS and RFS were estimated with Kaplan-Meier method and its comparison was evaluated by log-rank, censoring patients at the time of bone marrow transplantation (BMT). The cut-off point for MRD analysis was 0.1% evaluated at the end of induction and after C1. A subanalysis of patients with unknown and intermediate cytogenetic risk (CRM modified according to available molecular data of FLT3, NPM1 and CEBPA) was performed. Results: Of the 1,160 patients enrolled in the registry, 493 met the inclusion criteria and 20 were excluded due to lack of MRD data at the end of induction. Among the 473 patients analyzed: 238 were women, the median age was 49.5 years [IQR 36.6-59.7]), 184 (38.9%) were intermediate risk and 51 (10.8%) with not evaluable cytogenetic and molecular data. With a median follow-up of 18.9 months, 158 (33.4%) relapsed and 130 (27.5%) died. The median (Me) OS for patients with post induction negative MRD (MRDneg) was 61.2 (CI95% not evaluable) versus 25.9 months (CI95% 2.5-49.2) with positive MRDpos, p =0.03. The RFS were 20.4 (CI95 8-32.8) and 12.4 (CI95% 6.5-18.3), p=0.06, for MRDneg and MRDpos respectively. Regarding the evaluation of MRD post C1, the Me OS was not reached for patients with MRDneg, while it was 22.1 months (CI95% 9.3-34.8) among patients with MRDpos, p=0.03. Their respective RFS were 30.3 (CI95% not evaluable) and 14.4 months (CI95% 0-30.7), p=0.03, respectively. In the subanalysis carried out in the group with intermediate or unknown cytogenetic risk post C1, the Me OS was not reached in MRDneg versus 17.5 months (CI95% 9.3-25.7) in MRDpos, p=0.007. In addition, a median RFS of 20.4 months (CI95% 0.7-40.1) vs 7.4 (CI95% 3.9-10.9), p=0.003, among those with MRDneg and MRDpos, respectively. Summary/Conclusion: The assessment of MRD by MFC showed predictive power at the different moments of follow-up evaluated. It was more evident after consolidation, with statistically significant impact in OS and RFS. The post-consolidation MRD subanalysis in the group with IR or non-evaluable cytogenetics showed promising results. This real-practice data is consistent with those published recently. Post-C1 MRD could be a useful tool in clinical practice deciding the best post-induction strategy in intermediate-risk patients. A further detailed study is required to confirm our preliminary results. P537: SURVIVAL AFTER ALLOGENEIC TRANSPLANTATION FOR ACUTE MYELOID LEUKEMIA IN ADULTS IN DENMARK FROM 2000 TO 2020: A POPULATION-BASED COHORT STUDY L. K. Gjærde1,2,*, L. H. Jakobsen3,4, C. Juhl-Christensen5, G. Olesen5, I. Petruskevicius5, M. T. Severinsen3, C. W. Marcher6, K. Theilgaard-Mönch1, N. S. Andersen1, L. S. Friis1, B. Kornblit1, S. L. Petersen1, I. Schjødt1, H. Sengeløv1,2 1Department of Hematology, Rigshospitalet; 2Department of Clinical Medicine, University of Copenhagen, Copenhagen; 3Department of Hematology, Aalborg University Hospital; 4Department of Mathematical Sciences, Aalborg University, Aalborg; 5Department of Hematology, Aarhus University Hospital, Aarhus; 6Department of Hematology, Odense University Hospital, Odense, Denmark Background: Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative treatment of acute myeloid leukemia (AML), but survival is challenged by the risk of relapse and treatment-related/non-relapse mortality (NRM). In the recent two decades, older patients (>50 years of age) and patients with comorbidities have been able to receive HCT due to the introduction of non-myeloablative conditioning in Denmark in 2001. Aims: We aimed to investigate trends in overall survival (OS) and other HCT-related outcomes in a population-based cohort study of all adults (≥18 years) who received a first HCT for AML in Denmark between 2000–2020. Methods: Indications for HCT comprised intermediate- or adverse-risk (including secondary/treatment-related) AML in 1st complete remission (CR) and all patients in ≥2nd CR. The total effect of calendar year of HCT on transplant outcomes was tested in a Cox model for OS and Fine-Gray competing risks models for NRM, relapse, grade II–IV acute graft-versus-host disease (GvHD) and chronic GvHD. Time-specific OS estimates and cumulative incidences of HCT outcomes were derived using the pseudo-observation framework. Cure fractions (the fraction of patients who experienced no excess mortality compared to the Danish general population matched on calendar year, age and sex) were estimated in a mixture cure model. Results: From 2000 to 2020, 659 adults received a first HCT for AML (95% de novo AML; 5% secondary/therapy-related AML) in Denmark. Median (min–max) age at HCT was 56 (19–74) years, going from 44 years in 2000–2005 to 59 years in 2016–2020. 63% of patients were transplanted in 1st CR, 35% in ≥2nd CR, and 2% after primary induction failure. Non-myeloablative conditioning was performed in 60% of patients, going from 26% in 2000–2005 to 65% in 2016–2020. Median follow-up was 7.5 years. Main causes of death during follow-up were relapse (56%), infection (11%), and organ failure (10%). OS at 2- and 5-years for all patients was 66% (95% CI: 63–70%) and 57% (95% CI: 53–61%), respectively. OS differed over time (p = 0.02), decreasing from 2000 to 2010 from when it increased until 2020 (Figure Panel A). This change was mainly driven by corresponding changes in NRM over time (p < 0.001, Figure Panel B) rather than significant increases in the relapse rate over time (p = 0.16, Figure Panel C). Adjusting for age did not influence the trends notably. For acute GvHD, the risk decreased over time (p = 0.004), ranging from a 1-year cumulative incidence of 42% (95% CI: 26–62%) in 2000 to 26% (95% CI: 19–34%) in 2020, whereas the risk of chronic GvHD remained stable (p = 0.49), with a 2-year cumulative incidence of 49% (95% CI: 45–53%) in the full cohort. Cure fractions differed over time (p = 0.05) decreasing from 63% (95% CI: 41–78%) in 2000 to 46% (95% CI: 38–54%) in 2010, and increasing again to 68% (95% CI: 52–80%) in 2020. Image: Summary/Conclusion: Survival after HCT for AML in adults in Denmark decreased in the first decade after introducing non-myeloablative conditioning regimens for older or comorbid patients, mainly because of higher NRM, but survival improved again in the later decade. The risk of acute, but not chronic, GvHD decreased over time. The latest improvements in peri-HCT supportive care and post-HCT maintenance treatments may further improve survival in the coming decade. P538: TREATMENT PATTERNS AND CLINICAL OUTCOMES IN ACUTE PROMYELOCYTIC LEUKEMIA: REAL-WORLD DATA V. B. Goli1,*, H. Jain1, L. Nayak1, J. Thorat1, B. Bagal1, A. Rajendra1, S. P G2, D. Shetty3, H. Jain3, P. Tembhare2, N. Patkar2, M. Sengar1 1Medical Oncology; 2Pathology; 3Cytogenetics, TATA MEMORIAL HOSPITAL, MUMBAI, India Background: All-trans retinoid acid (ATRA) and arsenic trioxide (ATO) have changed the treatment paradigm of acute promyelocytic leukemia (APML). Current therapeutic strategies are based on the risk stratification at diagnosis and molecular response at the end of consolidation.We report our experience of treatment patterns and clinical outcomes of newly diagnosed APML patients. Aims: The primary objective of our study was to evaluate event-free survival in all risk categories of APML.Secondary objectives include disease-free survival, overall survival, rates of complete remission (CR) at the end of induction, rates of molecular CR at the end of consolidation. Methods: We included treatment-naïve APML patients age>14 years who were treated between May 2014 to May 2018.The diagnosis of APML was established by peripheral blood/bone marrow morphology and flow cytometry (FCM) and confirmed by fluorescent in situ hybridization (FISH) and reverse-transcriptase polymerase chain reaction (RT-PCR) for PML-RARA.The details of patients were retrieved from electronic medical records.Patients with low/intermediate risk were treated with ATO-ATRA based induction and consolidation,where as patients with high risk were treated with varying combination of ATO/ATRA/chemotherapy with or without maintenance. Results: We registered 149 patients during the study period.The median age was 37 years(range 15-72 years, male 56.6%, female-37%).93 patients (62.4%) were stratified as low risk and 56 patients (37.5%) as high risk based on Sanz’s score.The induction therapy for high risk APML was single agent ATO (42.8%),ATO/ATRA/anthracycline (26.7%),ATO/anthracycline (25%),and ATO/ATRA (5%).The consolidation for high risk APML includes ATRA/daunorubicin (50%),ATO/ATRA (26.8%),HIDAC (4%) and APL 2000 in one patient.All low-risk APML patients received ATO-ATRA in induction and consolidation.Anthracycline was added to three patients in induction to decease white cell count.The proportion of deaths that occurred in first week include 6.5% in low risk and 9% in high risk group.The median follow up was 42 months.The hematological CR at the end of induction was 85%.(128/149 patients,expired during induction-14,lost to follow up-7).The rate of end of consolidation PCR negativity was 83%.Differentiation syndrome occurred in 75% of patients.For the overall patient population, the 3-year probability for OS, EFS, DFS was 88.1% (95% C.I, 82.9 93.6), 75.9% (95% C.I, 68.9-83.5), 81.4% (95% C.I, 75-88.4) respectively. The 3-year survival probability for OS, EFS, and DFS for low risk APML was 90% (95% C.I, 84.1-96.4), 80.1% (95% C.I, 72-89.1), 84.5% (95% C.I, 76.9-92.7) respectively whereas for high risk APML was 84.7% (95% C.I, 75.3-95.2), 68.6% (95% C.I, 56.9-82.9), 76.1% (95% C.I-65-89) respectively.The total number of relapses were 11.The median time to relapse for low risk and high risk was 23months(18-32months) and 20months(range 5-39months) respectively. Image: Summary/Conclusion: Treatment with ATO and ATRA based regimen are effective in real world setting.The emphasis should be to reduce the early death rate due to bleeding and infectious complications. P539: PROGNOSTIC RELEVANCE OF MINIMAL RESIDUAL DISEASE IN THERAPY RELATED AND SECONDARY ACUTE MYELOID LEUKEMIA RECEIVING CPX-351 OR FLUDARABINE-BASED INDUCTION. F. Guolo1,2,*, P. Minetto1, C. Riva1,2, F. Parodi2, M. Miglino1,2, E. Tedone3, N. Colombo3, E. Carminati4, C. Nurra4, A. Cagnetta1, M. Cea1,2, R. M. Lemoli1,2 1Clinic of Hematology, Department of Internal Medicine (DiMI), IRCCS Ospedale Policlinico San Martino; 2University of Genoa; 3Flow Cytometry Unit, Department of Pathological Anatomy; 4Molecular Biology Unit, Department of Pathological Anatomy, IRCCS Ospedale Policlinico San Martino, GENOVA, Italy Background: Minimal residual disease (MRD) assessment retains high prognostic value in Acute Myeloid Leukemia patients (AML) undergoing intensive induction therapy. Widely available MRD techniques include multicolor flow cytometry (MFC), RT-PCR for recurrent genetic lesion and, for patients lacking specific markers, RT-PCR for the pan- leukemic marker WT1. However, most of the data on the prognostic value of MRD come from trials including younger patients treated with conventional 3 + 7 regimen. Furthermore, AML arising from a previous myelodisplastic syndrome (s-AML) and therapy-related AML (t-AML) are usually under-represented in trial involving younger patients. Few data are, therefore, available on the kinetics and the prognostic value of MRD in elderly s- AML and t-AML patients; especially in the context of more modern frontline treatment such as CPX-351. Aims: We evaluated MRD in a cohort of elderly s-AML or t-AML patients receiving induction therapy either with an age-adjusted fludarabine-containing regimen (FLAI3) or CPX-351, in order to compare the probability of achieving MRD negativity, to assess the prognostic value of MRD and to define the best time-points for MRD assessments. Methods: A total of 151 elderly (>60 year, median age 68, range 60-77) patients were analyzed in this study. Patients were treated between January 2005 and January 2020 in our Center, either with CPX-351 (n=50) or fludarabine- high dose cytarabine-idarubicin (FLAI), with (n=72) or without (n =29) gemtuzumab-ozogamicin (GO). MRD was analyzed in all patients achieving hematological complete remission (CR) with both MFC and WT1 expression levels. All patients were affected by s-AML or t-AML, defined according to the WHO 2016 criteria. Results: After induction, CR was achieved in 95 patients (59%). CR rate was 40/50 in patients treated with CPX-351 (80)% significantly higher when compared to patients receiving FLAI (55/101, 54.5%, p<0.05). The addition of GO to FLAI did not increase CR rate. Among CR patients, a total of 51 (53.7%) and 53 patients (55.8%) achieved MRD negativity, with MFC or WT1-based methods, respectively. MFC MRD negativity probability was higher among patients receiving CPX-351 as induction therapy (MFC MRD negativity rate of 26/40, 65% and 25/55, 45% in CR patients who received CPX-351 or FLAI, respectively, p<0.05). Adding GO to FLAI did not improve MRD negativity probability. The most informative timepoint was after first cycle. WT1-based MRD assessment led to similar results. In multivariate analysis, MRD showed significant prognostic value for Overall Survival (OS) in all treatment group (2-year OS of 34% and 77% in patients with or without residual MFC MRD after induction, respectively, p<0.05). Similarly, consolidation with allogeneic stem cell transplantation (HSCT) was correlated with higher OS. Notably, 12/40 (30%) CR patients treated with CPX 351 underwent HSCT. Summary/Conclusion: In conclusion MRD assessment retains a strong prognostic value also in s-AML and t-AML patients. The evaluation of MRD with both methods lead to similar conclusions and allowed us to obtain MRD data from virtually all AML patients treated in the selected time period. CPX-351 treatment resulted in higher CR rate with deeper MRD responses, if compared to FLAI3 and allowed a significant number of elderly AML patients to proceed to HSCT. P540: PEVONEDISTAT, AZACITIDINE AND VENETOCLAX FOR PATIENTS WITH RELAPSED/REFRACTORY ACUTE MYELOID LEUKEMIA– A PHASE I STUDY G. S. Guru Murthy1,*, S. Kaufmann2, A. Saliba2, A. Szabo3, L. Michaelis1, S. Abedin1, L. Runaas1, K. Carlson1, S. Maldonado-Schmidt4, A. Hinman4, A. Thomas4, A. Baim4, M. Litzow2, E. Atallah1 1Hematology-Oncology, Medical College of Wisconsin, Milwaukee; 2Hematology-Oncology, Mayo Clinic, Rochester; 3Biostatistics; 4Clinical trials, Hematology-Oncology, Medical College of Wisconsin, Milwaukee, United States of America Background: Outcomes of patients with relapsed/refractory acute myeloid leukemia (RR-AML) have remained poor. Venetoclax therapy in RR-AML is associated with lower complete remission (CR) as compared to newly diagnosed AML. Preclinical studies suggest overexpression of anti-apoptotic MCL-1 as a mechanism of BCL-2 inhibitor resistance (Konopleva et al. Cancer Cell 2006). Pevonedistat is a first in class inhibitor of Nedd8 activating enzyme that induces pro-apoptotic NOXA leading to neutralization of MCL-1 and apoptosis. In preclinical AML models, combination of pevonedistat and venetoclax showed synergistic effect (Knorr KL et al. Cell Death Differ. 2015). Aims: To assess the safety and outcomes of adding pevonedistat to azacitidine and venetoclax in patients with RR-AML. Methods: This is a phase I multicenter study (NCT04172844) with 3 + 3 design to determine the safety and recommended phase 2 dose (RP2D) of pevonedistat, venetoclax and azacitidine in RR-AML. Patients aged 18 years or above with morphologically documented RR-AML, ECOG performance status 0-2 and adequate organ function were eligible. Exclusion criteria were isolated extramedullary relapse, hematopoietic stem cell transplantation (HSCT) within 100 days of enrollment, and active acute graft versus host disease. Previous therapy with hypomethylating agent (HMA) or venetoclax was not an exclusion. Treatment included azacitidine (75 mg/m2 daily x 7 days), venetoclax (400 mg daily x 28 days), and pevonedistat in escalating doses (10-20 mg/m2 IV days 1,3,5 of each cycle) in 28-day cycles. Pevonedistat was given at 10 mg/m2 dose in cohort 1, 15 mg/m2 in cohort 2 and 20 mg/m2 in cohort 3. Results: Sixteen patients with RR-AML participated in the study (15 evaluable for response). Median age was 73 years (61-91), 37.6% had secondary/therapy related AML, 56.3% received prior venetoclax/HMA and 18.1% had relapse after prior allogeneic HSCT. Most common grade 3 or higher adverse events included neutropenia (44%), thrombocytopenia (38%), febrile neutropenia (25%), anemia (25%), and sepsis (19%). There was 1 dose limiting toxicity (DLT) in cohort 1 (atrial fibrillation), but no subsequent DLT despite planned dose escalation. Pevonedistat 20 mg/m2 was established as the RP2D. The rate of CR/CRi/morphological leukemia free state (MLFS) was 40% (CR/CRi 33%) for the overall cohort and 85.7% in venetoclax/HMA naïve RR-AML with a median time to response of 1 cycle. Among patients achieving CR/CRi, 60% attained minimal residual disease negativity by flow cytometry. Correlative studies with BH3 mimetic profiling showed variable baseline sensitivity to BH3 mimetics and sequential western blot assays showed upregulation of PUMA (for two patients in CR) and NOXA (for one patient with MLFS). Higher levels of DNMT1 were seen in patients with CR or MLFS. One patient with CR also had evidence of leukemic stem cell/progenitor cell sensitivity to pevonedistat at 50-100 nM. Summary/Conclusion: Combining pevonedistat with venetoclax and azacitidine is safe and tolerable in patients with RR-AML. Dose escalation yielded encouraging efficacy with pevonedistat 20 mg/m2 as the RP2D. P541: OUTCOMES AND MANAGEMENT OF PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA PRESENTING WITH HYPERLEUKOCYTOSIS F. Haddad1,*, K. Sasaki1, T. Abuasab1, S. Venugopal1, D. Rivera Delgado1, A. Bazinet1, R. Babakhanlou1, K. Kim1, J. Senapati1, F. Ong1, S. Desikan1, N. Short1, N. Pemmaraju1, G. Borthakur1, C. DiNardo1, N. Daver1, E. Jabbour1, G. Garcia-Manero1, F. Ravandi1, T. Kadia1 1Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Patients with acute myeloid leukemia (AML) presenting with hyperleukocytosis have higher mortality rates and inferior outcomes. Analyzing the factors associated with mortality and survival in AML patients with a white blood cell count (WBC) ≥100 x 109/L can guide management and improve early mortality. Aims: To analyze the outcomes of patients with AML presenting with hyperleukocytosis and establish a treatment algorithm for the management of those patients. Methods: In a retrospective analysis, we screened patients who presented to our institution for newly diagnosed AML over the period of 10 years and identified those with WBC ≥100 x 109/L. Logistic regression models estimated odds ratios (OR) for 4-week mortality and Cox proportional hazard models estimated hazard ratios (HR) for overall survival (OS). Results: We identified 129 patients with newly diagnosed AML and hyperleukocytosis. Median age was 65 years (range, 23-86 years) and 66% of patients had ECOG performance status (PS) <2. Median WBC was 146 x 109/L (range, 100-687) and 78 patients had clinical leukostasis (CL) including renal failure in 31 patients (24%), new onset hypoxia in 29 patients (23%), headache in 19 patients (15%), chest pain in 9 patients (7%) and neurological symptoms in 3 patients (2%). FLT3 and RAS pathway mutations were found in 63% and 27% of patients, respectively. 29 of 129 patients (22%) had poor-risk cytogenetics. Compared with patients without evidence of CL, those with CL were less likely to have good PS (ECOG PS <2, 58% vs 82%; P = 0.006), had higher 4-week mortality (16% vs 2%; P = 0.015) and 8-week mortality (19% vs 6%; P = 0.038). Cytoreduction consisted of hydroxyurea in 124 patients (96%), cytarabine in 69 patients (54%) and leukapheresis in 31 patients (24%). Patients who underwent leukapheresis were less likely to receive cytarabine compared with those who did not (35% vs 59%; P = 0.024); and tended to have more CL compared with patients who did not (74% vs 56%; P = 0.093). 30 patients had tumor lysis syndrome (TLS). TLS risk did not increase with WBC and was not associated with the cytoreductive modality used. 11 patients had intracranial hemorrhage (ICH): 9 patients (82%) with WBC ≥150 x 109/L and 18% with WBC <150 x 109/L (P = 0.048). No association was observed between the incidence of ICH and the cytoreductive therapy. 4-week and 8-week mortality rates were 10% and 14%, respectively. After a median follow-up of 49.4 months (95% CI, 26.2-72.6), median OS was 14.3 months (95% CI, 7-21.6), with 2-year OS of 40%. Median OS was 12.3 months (95% CI, 7.4-17.2) compared with 29 months (95% CI, 2.1-55.6) in patients with or without CL, respectively (P = 0.007). Median OS was 9.9 months (95% CI, 7.5-12.2) and 21.3 months (95% CI, 10.7-31.8) among those who did or did not undergo leukapheresis, respectively (P=0.003). Median OS was 42 months (95% CI, 14.2-69.8) in patients younger than 65 years compared with 8 months (95% CI, 6-10) in those 65 years and older. Image: Summary/Conclusion: Older age, poor-risk cytogenetics, TLS and disseminated intravascular coagulation (DIC) were associated with early mortality and inferior OS in patients with hyperleukocytosis. Careful monitoring of those patients with prompt cytoreduction and management of complications may help improve their outcomes. P542: REAL WORLD OUTCOMES FOR PATIENTS WITH ACUTE PROMYELOCYTIC LEUKEMIA IN BRITISH COLUMBIA (BC): EXCELLENT OUTCOMES WITH LOW EARLY DEATH RATE AND HIGH OVERALL SURVIVAL IN A POPULATION BASED STUDY R. Henderson1,*, Y. Eissa1, R. Stubbins1, Y. Abou Mourad1, S. Chung1, D. Forrest1, K. Hay1, F. Kuchenbauer1, S. Nantel1, T. Nevill1, M. Power1, J. Rodrigo1, C. Roy1, D. Sanford1, K. Song1, H. Sutherland1, C. Toze1, J. White1, S. Narayanan1 1Leukaemia/Bone Marrow Transplant Programme, Vancouver General Hospital, Vancouver, Canada Background: Acute promyelocytic leukaemia (APL) therapy has significantly improved with the use of All trans retinoic acid (ATRA) and Arsenic Trioxide (ATO) for low risk disease (Lo-Coco 2013). Treatment of high risk disease remains more challenging with a higher risk of early mortality & greater concern of relapse. All patients (pts) with APL in British Columbia, Canada are treated by the Leukemia/BMT Program at Vancouver General Hospital, Vancouver. Since 2014, all pts have received ATRA/ATO based therapy based on the Le-Coco study. Pts with high risk disease (WCC > 10 x 109/L at presentation) also receive a 3 day course of doxorubicin (60mg/m2) for induction only. Aims: We sought to review treatment outcomes since initiating these treatment protocols with emphasis on drug toxicities, dose modifications & management of high risk & elderly patients in order to describe real world outcomes. Methods: Ethics was approved by the board of the BCCA and UBC. Data was collected by chart reviews, hospital databases & collated on a central file. Demographics, risk stratification, treatment received, adverse events & survival was collected & analysis performed on Microsoft excel v16.57 & SPSS Statistics v28.0.1.1. Kaplan Meier curves were performed for PFS & OS, student t and chi squared tests were performed to assess differences between subgroups. Results: We identified 67 pts with APL during the study period. 40% were male 60% female, the median age was 61 yrs. (21-83yo). 36(54%) pts were > 60 yrs at diagnosis. The median duration of follow up was 2.9 yrs. All had molecular confirmation of disease. 16(24%) pts had high risk disease at diagnosis. At time of censor overall survival was 97% and PFS 94%. 55(82%) pts had a documented toxicity. Common toxicities included differentiation syndrome (DS) in 27(40%) pts, hepatotoxicity 17 (25%), QTc prolongation 13(19%) & neurological effects 9 (13%). Infections were documented in 40(59%) pts including bacterial infections in 25 patients (37%), culture negative febrile neutropenia in 12 (20%), viral infections in 5 (7%) & fungal infections in 1 (1.5%). Clinically significant bleeding was documented in 10 (15%) of patients, 5 of which were CNS/retinal bleeds. DS was more common in high risk disease though this difference was not statistically significant (p=0.365). 8(30%) pts with DS needed ICU admission. There were 2 early deaths, in the cohort, both in pts > 75 yo with high risk disease due to multi-organ failure and bleeding within 5 days of presentation. Dose modifications were assessed in 64 pts (2 deaths & one lost to follow up). 37 (58%) were described as having had a dose modification. 13 (20%) had a reduction in treatment dose, 16(25%) had doses/days of drug omitted. 6(9%) had reductions in dose and omissions. The most common reasons for dose modifications were DS 12(19%), QTc prolongation 10 (15%) & hepatotoxicity 8 (12%). Pts over 60yo were more likely to have a dose modification than pts below 60yo (73% vs 43% p = 0.014). 65 pts diagnosed achieved morphological remission & 64 a molecular remission at the end of planned therapy. 2 relapses occurred in our cohort, 1 had a concomitant cytogenetic abnormality incorporating a Tp53 deletion. The second initially received ATRA only induction due to age & comorbidity. Summary/Conclusion: This real world, population based data for patients with APL in BC confirms high response rates, survival and relatively low early death rate and acceptable toxicity even in older patients with high risk disease and comorbidities. In our cohort appropriate dose modifications did not impact remission or relapse rates. P543: TARGETING SAMHD1 WITH HYDROXYUREA IN FIRST-LINE CYTARABINE-BASED THERAPY OF NEWLY DIAGNOSED ACUTE MYELOID LEUKAEMIA: RESULTS FROM THE HEAT-AML TRIAL M. Jädersten1,*, I. Lilienthal2, N. Tsesmetzis2, M. Lourda2, S. Bengtzén3, A. Bohlin3, C. Arnroth4, T. Erkers4, B. Seashore-Ludlow4, G. Giraud5, G. S. Barkhordar6, S. Tao7, L. Fogelstrand8, L. Saft4, P. Östling4, R. Schinazi7, B. Kim7, T. Schaller9, G. Juliusson10, S. Deneberg1, S. Lehmann11, G. Rassidakis4, M. Höglund11, J.-I. Henter2, N. Herold2 1Department of Hematology, Karolinska University Hospital; 2Childhood Cancer Research Unit; 3Centre for hematology and regenerative medicine; 4Department of Oncology-Pathology, Karolinska Institutet, Stockholm; 5Department of Immunology, Uppsala University, Uppsala; 6Department of Clinical Genetics and Genomics, Sahlgrenska University Hospital, Gothenburg, Sweden; 7Department of Pediatrics, Emory University, Atlanta, United States of America; 8Department of Laboratory Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; 9Department of Infectious Diseases, University Hospital Heidelberg, Heidelberg, Germany; 10Department of Hematology, Skåne University Hospital, Lund; 11Department of Medical Sciences, Uppsala University, Uppsala, Sweden Background: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-CTP levels through targeted inhibition of SAMHD1. Aims: To evaluate feasibility, safety, and efficacy of adding hydroxyurea to standard AML-directed therapy according to national guidelines. To perform translational studies including SAMHD1-staining on bone marrow sections, pharmacokinetics and drug-sensitivity analysis on leukemic cells ex vivo. Methods: This phase-1 trial (EudraCT-number: 2018-004050-16) was run at two sites (Karolinska University Hospital and Uppsala University Hospital, Sweden). Eligibility criteria included age >18 years, newly diagnosed non-promyelocytic AML, and fitness for intensive chemotherapy. Patients with CBF-AML eligible for treatment with gemtuzumab-ozogamicin were excluded. Treatment comprised 2 to 4 cycles of ara-C 1000 mg/m2 i.v. b.i.d. on day 1-5 during all 4 cycles and daunorubicin 60 mg/m2 i.v. q.d. on day 1-3 during cycles 1 and 2, and on day 1-2 during cycle 3. Patients with FLT3-mutated AML received midostaurin 50 mg b.i.d. on day 8-21 of each cycle. Risk-adapted allo-HSCT was performed at the discretion of the treating haematologist. The dose of hydroxyurea was escalated in a 3 + 3 design: 500 + 500 mg (level 1), 1000 + 500 mg (level 2), and 1000 + 1000 mg (level 3), each dose being given 1 hour prior to start of the ara-C infusion b.i.d. on day 1-5. Here we report the results of the first 9 patients in the run-in phase 1 part of the study. The phase 2 part will include an additional 60 patients. Recruitment is ongoing, utilizing the highest dose of hydroxyurea. Expression of SAMHD1 was assessed using a double-immunostaining method (SAMHD1/CD68), an autostainer system (BenchMark Ultra, Ventana, Rotkreuz, Switzerland) and previously validated protocols20. CD68+/SAMHD1+ histiocytes (macrophages) served as internal controls in all bone marrow biopsies assessed. Drug sensitivity analysis was performed on AML mononuclear cells utilized a high-throughput system evaluating >500 cytotoxic agents including combinations of ara-C and hydroxyurea in different doses. Results: All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow-cytometry or RT-qPCR) had an MRD level <0.1% after two cycles of chemotherapy. Six of nine patients underwent hematopoietic stem cell transplantation. With a median follow-up of 13.2 months, no relapse has been observed. No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in ara-CTP levels (1.5-fold; P=0.04) in the 6 patients receiving single doses of 1000 mg hydroxyurea. Drug-sensitivity analysis indicated an additive effect of ara-C and hydroxyurea on leukemic cells ex vivo. There was no apparent correlation between expression of SAMHD1-expression and efficacy; all patients had deep responses. Image: Summary/Conclusion: The high rate of complete remission and MRD negativity together with the pharmacokinetic and ex vivo evidence suggest that the efficacy of cytarabine-based AML treatment can be enhanced by addition of hydroxyurea as a targeted inhibitor of SAMHD1. Importantly, orally administered hydroxyurea may provide a safe, inexpensive, and broadly accessible strategy to improve outcome in AML. These results will have to be validated in a larger patient cohort. P544: MOLECULAR PREDICTORS OF RESPONSE AND SURVIVAL IN TREATMENT-NAÏVE PATIENTS WITH ACUTE MYELOID LEUKEMIA FOLLOWING VENETOCLAX AND HYPOMETHYLATING AGENTS I. Johnson1,*, K. McCullough2, F. Farrukh3, A. Al-Kali3, H. Alkhateeb3, K. Begna3, A. Mangaonkar3, M. Litzow3, W. Hogan3, M. Shah3, M. Patnaik3, A. Pardanani3, A. Tefferi3, N. Gangat3 1Department of Medicine; 2Divison of Hematology; 3Mayo Clinic, Rochester, United States of America Background: Venetoclax (Ven) in combination with hypomethylating agents (HMA) is FDA-approved for elderly/unfit AML patients. Limited data exists on molecular predictors of response and survival. In a prior study, response was superior (CR/CRi >80%) with NPM1, IDH1/2, DNMT3A mutations, inferior with TP53, RUNX1, FLT3/ITD, RAS and prolonged survival with NPM1 and IDH2 mutations (2 yr OS 71.8%/79.5%) (DiNardo, Blood, 2020). Aims: Our objective was to determine the impact of mutations on response and survival in treatment-naïve AML patients receiving Ven+HMA. Methods: Treatment-naïve AML patients receiving Ven+HMA outside a clinical trial at the Mayo Clinic were included. Molecular studies were performed by next-generation sequencing. Response was assessed according to the 2017 European LeukemiaNet (ELN) criteria. Standard statistical analyses were performed using JMP Pro (Version 16.0.0). Results: i) Patient characteristics 103 AML patients (median age 74 years, 67% male, 62% de novo) received upfront Ven+HMA. ELN cytogenetic risk included favorable (6%, n=6), intermediate (50%, n=52) or adverse (44%, n=45). Mutations involved TP53 in 25 patients (25%), TET2 in 24 (23%), IDH1/IDH2 in 20 (19%), RUNX1 in 19 (19%), ASXL1 in 18 (18%),), SRSF2 in 18 (18%), K/NRAS in 15 (15%), NPM1 in 13 (13%), DNMT3A in 13 (13%), FLT3-ITD in 10 (10%) patients. ii) Predictors of response 40 (39%) patients achieved complete remission (CR), 20 (19%) CR with incomplete hematological recovery (CRi), resulting in CR/CRi in 60 (58%) patients. In univariate analysis, presence of ASXL1 mutation was associated with favorable response (CR/CRi 83% vs 53%, p=0.01), secondary AML (CR/CRi 49% vs 65%, p=0.09), adverse karyotype (49% vs 67%, p=0.11), presence of TP53 (32% vs 67%, p=0.002) and FLT3-ITD mutations (30% vs 61%; p=0.06) predicted inferior response. In multivariable analysis, including the aforementioned variables, presence of ASXL1 mutation (83% vs 53%; OR 4.5) and absence of TP53 (67% vs 32%; OR 3.3) and FLT3-ITD mutations predicted favorable response (61% vs 30%; OR 6.4). Moreover, in ASXL1 mutated patients, CR/CRi was not impacted by presence of TP53 mutation (100% vs 81%) whereas in ASXL1 unmutated patients, presence of TP53 mutation predicted inferior response (26% vs 63%; p=0.001). Presence of NPM1 (CR/CRi; 69% vs 57%, p=0.41), IDH1/2 (70% vs 55%; p=0.23), DNMT3A (54% vs 59%; p=0.73), RUNX1 (58% vs 58%; p=0.1), RAS (60% vs 58%, p=0.88) did not impact response. iii) Predictors of survival After a median follow up of 6.6 months, 68 patients died and 9 underwent allogeneic stem cell transplant. Median overall survival (mOS) was 8.5 months and longer in transplanted patients (not reached vs 8.4 months, p=0.08). Age-adjusted analysis in 94 patients not transplanted, identified CR/CRi (p<0.0001), NPM1 (p=0.009), IDH1/2 mutations (p=0.02) as favorable, and TP53 (p=0.01), ASXL1 mutations (p=0.17) adverse karyotype (p=0.05) unfavorable risk factors for survival. Despite higher CR/CRi, ASXL1 mutated patients had shortened mOS due to higher relapse. Multivariable analysis confirmed the negative survival impact of not achieving CR/CRi, ASXL1 mutation and adverse karyotype. Accordingly, a three-tiered model was generated by using the three variables (Figure 1). Image: Summary/Conclusion: Our observations differ from those of DiNardo, et al. (Blood, 2020). The current study identifies presence of ASXL1 mutation and absence of TP53 and FLT3-ITD mutations as predictors of favorable response. Additionally, we propose a novel three-tiered survival model based on response, ASXL1 mutation and karyotype. P545: CHARACTERISTICS AND OUTCOME OF PATIENTS WITH ACUTE MYELOID LEUKEMIA AND TRISOMY 19 S. Kayser1,2,*, D. Martínez-Cuadrón3,4, R. Rodriguez-Veiga5, M. Hänel6, M. Tormo7, K. Schäfer-Eckart8, C. Botella9, F. Stölzel10, T. Bernal del Castillo11, U. Keller12, C. Rodriguez-Medina13, G. Held14, M.-L. Amigo15, C. Schliemann16, M. Colorado17, M. Kaufmann18, M. Barrios Garcia19, S. W. Krause20, M. Görner21, E. Jost22, B. Steffen23, A. D. Ho24, C. Baldus25, H. Serve26, U. Platzbecker1, C. Müller-Tidow24, C. Thiede27, M. Bornhäuser28, P. Montesinos4,5, C. Röllig10, R. F. Schlenk24,29,30 1Medical Clinic and Policlinic I, Hematology and Cellular Therapy, University Hospital Leipzig, Leipzig; 2National Center of Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg, Germany; 3Hematology Department, 3HemaHospital Universitari i Politècnic, La Fe, Valencia; 4CIBERONC, Instituto Carlos III, Madrid; 5Hematology Department, Hospital Universitari i Politècnic, La Fe, Valencia, Spain; 6Klinikum Chemnitz, Chemnitz, Germany; 7Hematology Department, Hospital Clínico Universitario, INCLIVA Research Institute, University of Valencia, Valencia, Spain; 8Hospital Nord, Nürnberg, Germany; 9Hospital General, Alicante, Spain; 10Department of Medicine I, University Hospital Carl-Gustav-Carus, Dresden, Germany; 11Hospital Central de Asturias, Asturias, Spain; 12Department of Hematology, Oncology and Tumor Immunology, Charité-University Medical Center, Campus Benjamin Franklin, Berlin, Germany; 13Hematology Department, Hospital Universitario de Gran Canaria Doctor Negrín, Las Palmas de Gran Canaria, Spain; 14Westpfalz Klinikum, Kaiserslautern, Germany; 15Hospital General Universitario Morales Meseguer, Murcia, Spain; 16University Hospital Muenster, Münster, Germany; 1716Hospital Universitario Marqués de Valdecilla, Santander, Spain; 18Robert Bosch Hospital Stuttgart, Stuttgart, Germany; 19Department of Hematology, 18DepartmentHospital Regional Universitario de Málaga, Malaga, Spain; 20Department of Internal Medicine 5 – Hematology/Oncology, 19Department of Internal Medicine 5 – HeUniversity Hospital of Erlangen, Erlangen; 21Klinik für Hämatologie, Onkologie und Palliativmedizin, Klinikum Bielefeld Mitte, Bielefeld; 22University Hospital Aachen, Aachen; 23Department of Internal Medicine II, Department oUniversity Hospital of Frankfurt Main, Frankfurt am Main; 24Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg; 25Department of Internal Medicine II, University Hospital of, Kiel; 26Department of Internal Medicine II, 22Department of InterUniversity Hospital of Frankfurt Main, Frankfurt am Main; 27Department of Medicine I, 9DepartmUniversity Hospital Carl-Gustav-Carus; 28Department of Medicine I, 9DepUniversity Hospital Carl-Gustav-Carus, Dresden; 29NCT Trial Center, National Center of Tumor Diseases, German Cancer Research Center (DKFZ); 30Department of Medical Oncology, National Center for Tumor Diseases (NCT), Heidelberg University Hospital, Heidelberg, Germany Background: Trisomy 19 is a recurrent but rare cytogenetic abnormality reported in patients with acute myeloid leukemia (AML). The prognostic significance of this abnormality in AML patients is not clear. Prognosis of AML patients with trisomy 19 seems to be poor as compared to that of patients with intermediate-risk cytogenetics. Allogeneic hematopoietic stem cell transplantation (allo-HCT) may improve survival if applied early in first complete remission (CR). Aims: To characterize AML patients with trisomy 19 and compare outcomes according to different treatment strategies. Methods: We retrospectively studied 97 AML patients with trisomy 19 (median age at diagnosis, 57 years; range, 17-83 years) treated between 2001 and 2019 within 2 study groups (SAL & PETHEMA). Standard statistical methods were applied. Results: Median white blood cell count at diagnosis was 6.7/nl (range, 0.1-151/nl) and platelets 48.5/nl (range, 4-307/nl). Type of AML was de novo in 66 (68%), secondary after myelodysplastic syndrome/ myeloproliferative neoplasm in 16 (16%), and therapy-related in 9 (9%) patients (missing, n=6; 6%). Thirty-five (36%) patients were female. Cytogenetic analysis revealed trisomy 19 as the sole abnormality in 10 (12.5%), additional abnormalities in a non-complex karyotype in 8 (8%) and a complex karyotype in 79 (81.5%) patients. Most frequent additional cytogenetic abnormality was trisomy 8 (n=46); in 17 patients karyotypes were characterized by trisomies only. A total of 65 patients (67%) had NPM1 and FLT3-ITD mutation testing. Of those, only 3 (5%) and 1 (2%) harbored NPM1 and FLT3-ITD mutations, respectively. Only 4 (8%) of 51 patients were CEBPA mutated. Ninety-two patients (95%) were treated intensively and 4 (4%) received non-intensive therapy (missing, n=1, 1%). In intensively treated patients early death rate was 10% (n=9); CR was achieved in 52% (n=48) and 35% (n=35) patients were refractory. Factors associated with response to intensive induction therapy were trisomy 19 as sole abnormality or within a karyotype characterized by trisomies only (OR, 5.64; 95%-CI, 1.71-18.63; P=0.005) and age at diagnosis (10 years difference OR, 0.57; 95%-CI, 0.40-0.80; P=0.001). One of 4 patients treated non-intensively achieved a CR. An allo-HCT was performed in 34 (35%) patients, of whom 19 patients were transplanted in first CR after induction therapy. Type of donor was matched-related in 12 and matched-unrelated in 22 patients. Median follow-up of the whole cohort was 6.4 years (95%-CI, 2.91-8.97 years). Five-year overall (OS) and relapse-free survival rates were 20% (95%-CI, 13-31%) and 26% (95%-CI, 16-43%). OS rates were significantly higher in intensively treated patients with trisomy 19 as sole abnormality or within a karyotype characterized by trisomies only (P=0.05). An Andersen-Gill model including allo-HCT as a time dependent covariable on OS revealed as significant parameters trisomy 19 as sole abnormality or within a karyotype characterized by trisomies only (HR, 0.47; 95%-CI, 0.25-0.89; P=0.021) and age at diagnosis (10 years difference; HR, 1.29; 95%-CI, 1.10-1.52; P=0.002), whereas allo-HCT had no beneficial impact (HR, 1.45; 95%-CI, 0.81-2.59; P=0.21). Summary/Conclusion: Patients with trisomy 19 are very heterogeneous in particular with respect to cytogenetic and molecular abnormalities. In our cohort, patients with trisomy 19 as sole abnormality or within a karyotype characterized by trisomies only had a high CR rate and better clinical outcome. In the cohort of intensively treated patients, allo-HCT did not improve OS. P546: EFFECTS OF CHEMOTHERAPY DOSE REDUCTIONS IN OVERWEIGHT AND OBESE PATIENTS WITH ACUTE MYELOID LEUKEMIA – A DANISH NATIONWIDE COHORT STUDY D. Kristensen1,2,*, L. B. Nielsen1,2, L. H. Jakobsen1,3, T.-C. C. Kristensen1, T. C. El-Galaly1,2, A. S. Roug1,2,4, M. T. Severinsen1,2 1Department of Hematology, Clinical Cancer Research Center, Aalborg University Hospital; 2Department of Clinical Medicine; 3Department of Mathematical Sciences, Aalborg University, Aalborg; 4Department of Hematology, Aarhus University Hospital, Aarhus, Denmark Background: The majority of chemotherapeutic agents are dosed according to a body weight derived variable. Studies in solid cancers have shown that overweight patients frequently receive dose reduction (DR) of chemotherapy, despite no evidence corroborates increased toxicity of full dosing. Rather, DR to ≤95% of actual weight-based dose, has been shown to result in shortened overall survival (OS). Consequently, the American Society of Clinical Oncology does not recommend up-front dose reduction based on body mass index (BMI) or body surface area (BSA) in overweight patients. Current evidence regarding DR and outcome among overweight patients with acute myeloid leukemia (AML) receiving induction chemotherapy (IC) is limited. Aims: The purpose of this study was to investigate the association between DR and outcome in overweight patients with AML. Methods: We utilized the Danish National Acute Leukemia Registry to conduct a retrospective cohort study. Overweight (BMI≥25) AML patients aged 18-75 years and treated with IC between 2000-2012 were included. We defined DR as ≤95% of actual BSA-based chemotherapy dose. Relative risks (RR) for DR, complete remission (CR) rates, and 30- and 90-day mortality were modeled, and OS and relapse-free-survival (RFS) were calculated and compared using the 5-year restricted mean survival time difference (Δ5y-RMST). Results: The study population included 536 overweight AML-patients of whom 54 patients (10.1%) were categorized as DR (mean reduction 11.2%). Risk factors for DR in univariate analysis were increasing BMI (30-34.9: RR, 2.52 [95% CI, 1.32-4.71]; ≥35: RR, 4.66 [95% CI, 2.37-8.91]), increasing BSA (2.0-2.2: RR 4.61 [95% CI, 1.96-12.6]; ≥2.2: RR 15.21 [95% CI, 6.75-40.67]), therapy-related AML (RR 2.85 [95% CI, 1.12-7.24]) and favorable risk cytogenetics (RR 2.20 [95% CI, 1.02-4.33]). No significant differences were observed for rates of CR, 30- and 90-day mortality between patients receiving DR and non-DR IC. Dose reduction did not affect median RFS (DR, 14.5 [95% CI, 9.0 to 41.7] months; non-DR, 15.0 [12.3 to 19.3]) with an adjusted Δ5y-RMST of 0.2 (-8.4 to 8.8) months nor median OS (DR, 17.0 [11.9-45.5] months; non-DR, 17.5 [14.8-20.5]) with an adjusted Δ5y-RMST of 0.8 (-5.7 to 7.3) months (figure panel A+C). We constructed a case-matched cohort matched on age, sex, AML subtype and BMI (figure panel B+D) and performed a sensitivity analysis using ≤90% cut-off to define DR which led to the same conclusions. Image: Summary/Conclusion: This study demonstrates that ~10% of Danish overweight AML-patients treated with IC are dose reduced ≥5% compared to full BSA-based doses. Risk factors were increasing BMI and BSA in addition to therapy-related AML and favorable cytogenetic risk. Importantly, our results suggest that IC dose reduction does not adversely impact AML outcomes including 30- and 90-day mortality, rates of CR, RFS and OS. However, we encourage future prospective clinical studies to address this question with specific and uniform standards or protocol specifications for dose reduction or dose capping in overweight and obese patients with AML. P547: CHARACTERISTICS AND OUTCOMES OF PATIENTS WITH ACUTE MYELOID LEUKAEMIA TREATED WITH VENETOCLAX COMBINATION THERAPY: REAL-WORLD EXPERIENCE IN BOTH FRONTLINE AND RELAPSED/REFRACTORY SETTINGS H. P. J. Lam1,2,*, S. Leong1,2, Z. Kirkham1,2, A. Wilson1, R. Burt1, R. Sellar1, A. Fielding1, R. Gupta1, E. Payne1, M. Mansour1, P. Kottaridis1, A. Khwaja1, J. O’Nions1,2 1Department of Haematology; 2NIHR UCL Clinical Research Facility, University College London Hospitals NHS Foundation Trust, London, United Kingdom Background: Venetoclax (Ven) in combination with hypomethylating agents, such as azacitidine (Aza) and low dose cytarabine (LDAC) has been shown to be effective therapy in acute myeloid leukaemia (AML) and has become standard of care for newly-diagnosed patients unfit for intensive chemotherapy (DiNardo et al., 2020; Wei et al., 2019; Pollyea et al., 2020). Efficacy has also been shown in the relapsed/refractory (R/R) setting in more limited data sets (Báez-Gutiérrez et al., 2021; Pollyea et al., 2020, Stahl et al., 2020; DiNardo et al., 2019). Ven combination therapy has become widely used in newly-diagnosed patients in the UK since its approval during the COVID-19 pandemic as an alternative to intensive chemotherapy and subsequently for patients unfit for intensive therapy. Aims: We describe the characteristics and outcomes of patients with AML or high risk myelodysplastic syndrome (HR-MDS) receiving Ven combinations in frontline and R/R settings to provide real-world insight into their use in UK clinical practice. Methods: A retrospective analysis was performed of all patients with AML or HR-MDS who received Ven combination therapy at University College London Hospital between April 2020 and September 2021. Patient demographics, treatment history and bone marrow results were obtained from electronic health care and laboratory records. Disease stratification and response assessments were made as per European LeukemiaNet (ELN) criteria (Döhner et al., 2017). Results: At the time of analysis, 95 patients received Ven combinations (61 as frontline treatment and 34 for R/R AML), with a median follow up of 14 months. The majority of patients in both groups had adverse risk ELN classification (70.5% of frontline patients, 64.7% of R/R) and received Ven-Aza (100% frontline and 91.1% R/R) (Table 1). The median ages were 72 and 59 years respectively. The incidence of composite CR/CRi was 70.5% in the frontline setting, with median duration of response (DoR) of 8.3 months and overall survival (OS) of 7.1 months. In R/R AML, the CR/CRi rate was 64.7%, median DoR 10.5 months and median OS 9.8 months. Four out of the 43 patients who achieved CR/CRi (9.3%) following frontline treatment and 9 of the 22 R/R (40.9%) patients proceeded to allogeneic stem cell transplant (alloSCT) post induction. The median survival for all patients who underwent alloSCT is not reached in this analysis. The highest CR/CRi rates were observed in intermediate risk patients (90.9% in frontline treatment, 71.4% in R/R), with lower rates in both favourable (80% and 66.7%) and adverse risk patients (65.1% and 59.1% respectively). The presence of NPM1 and IDH1/2 mutations were associated with high CR/CRi rates in both the frontline (85.7% and 84.6% respectively) and R/R groups (100% and 81.8%), with below average response rates seen in TP53 mutated AML (62% in frontline, 40% in R/R). Notable responses were seen in patients with RUNX1 mutations in both settings (77.8% frontline, 66.6% R/R). Image: Summary/Conclusion: Our data describes real world effectiveness for venetoclax combinations as both frontline and salvage therapy in UK clinical practice, similar to that seen in clinical trials. This further contributes to our understanding of these therapies, in particular their use as a viable treatment option in R/R patients and as a bridge to alloSCT, and highlights the importance of further characterisation of genetic predictors of response to inform treatment decisions in real-world practice. P548: BEMCENTINIB COMBINED WITH LOW-DOSE CYTARABINE IS EFFICACIOUS AND WELL TOLERATED IN RELAPSED AML PATIENTS UNFIT FOR INTENSIVE CHEMOTHERAPY. UPDATES FROM THE ONGOING PHASE II TRIAL (NCT02488408) S. Loges1,2,3,*, M. Heuser4, J. Chromik5, G. Sutamtewagul6, S. Kapp-Schwoerer7, M. Crugnola8, N. Di Renzo9, R. Lemoli10, D. Mattei11, I. Ben-Batalla1,2,3, J. Waizenegger1,2,3, L.-M. Rieckmann1,2,3, M. Janning1,2,3, C. D. Imbusch2, N. Beumer1,2,12, D. Micklem13, C. Gorcea-Carson14, G. Lawson14, J. Nautiyal14, S. Deharo14, W. Fiedler15, Y. Alvarado-Valero16, B. Gjertsen17 1DKFZ-Hector Cancer Institute, University Medical Center Mannheim, Mannheim; 2Division of Personalized Medical Oncology (A420), German Cancer Research Center (DKFZ), Heidelberg; 3Department of Personalized Oncology, University Hospital Mannheim and Medical Faculty Mannheim, University of Heidelberg, Mannheim; 4Hematology, Haemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover; 5University Hospital Frankfurt, Frankfurt, Germany; 6University of Iowa Hospitals and Clinics, Iowa City, United States of America; 7University Hospital of Ulm, Ulm, Germany; 8University of Parma, Parma; 9Haematology and SCT Unit, Vito Fazzi Hospital, Lecce; 10University of Genoa, Genoa; 11ASOS. Croce e Carle, Cuneo, Italy; 12Faculty of Biosciences, Heidelberg University, Heidelberg, Germany; 13BerGenBio ASA, Bergen, Norway; 14BerGenBio Ltd, Oxford, United Kingdom; 15University Medical Center Hamburg, Hamburg, Germany; 16The University of Texas M.D. Anderson Cancer Center, Houston, United States of America; 17Haukeland University Hospital, Bergen, Norway Background: The new standard of care (SOC) in newly-diagnosed AML patients (pts) unfit for intensive chemotherapy (IC) due to age or co-morbidities yields favourable efficacy. However, beyond 1st line, these pts have limited treatment options, with a dismal mOS of 2.4 months at relapse, highlighting a significant unmet need for new treatments in this pt population. Bemcentinib (BEM) is a first-in-class, orally bioavailable, highly selective AXL-inhibitor. AXL (a receptor tyrosine kinase) represents an important target in AML, as its expression on AML cells and stem cell compartment is associated with poor prognosis, resistance to chemotherapy and decreased antitumor immune response. Aims: The ongoing BGBC003 PhII trial studied the safety and efficacy of the BEM+LDAC combination in relapsed (REL) AML pts unfit for IC; in addition, a comprehensive translational biomarker analysis was pursued. Here, we present preliminary efficacy and safety data and include initial translational multiomics data from bone marrow mononuclear cells (BMMNC). Methods: Pts received BEM at 200 mg/OD with 3 loading doses at 400 mg/OD and LDAC at SOC schedule. Study endpoints included overall survival (OS), objective response (OR), clinical benefit rate (CBR) (OR+unchanged [UC]) and exploratory biomarker analyses. Longitudinal BMMNC samples (n=32) from 13 pts underwent scRNA-seq and CITEseq (Chromium 10x genomics; TotalSeq, Biolegend). Pts were stratified by best response: CR, CRi, PR for Responders; UC, PD for Non-Responders. Cell type annotations were inferred from expression of known markers at RNA and protein level and for identification of malignant cells copy number variation was included. Results: As of 13 Dec 2021, 22 REL AML pts (10/22 in 1st relapse) were treated with BEM+LDAC. Median age was 75.5yrs [66-86], median prior lines of therapy 1 [1-8] and adverse cytogenetic risk in 7/22 (32%). Only 19/22 pts were evaluable for efficacy. The overall REL population (n=19) demonstrated a composite CR (CRc=CR+CRi) of 26% (5/19) and CBR of 79% (14/19); AML pts in 1st relapse (10/19) showed a CRc of 30% (3/10) and CBR of 70% (7/10) whereas REL pts with time on treatment (ToT) > 3 months (11/19) achieved CRc of 45% (5/11) and CBR 91% (9/11); median ToT of 12.9 months for CR/CRi pts; 1 pt remains on treatment. Median OS was 6.2 months in the overall REL pts (n=19), 7.4 months in the AML pts in 1st relapse (10/19) and 11.3 months in REL pts with ToT >3 months. Late onset responses suggest an immunological mechanism of action (iMOA) and may contribute to a longer ToT and survival. CITEseq analysis identified differences in the immune compartment, underscoring an iMOA associated with response to BEM+LDAC. Furthermore, increased gene set enrichment of TNFα signalling via NfkB at screening in malignant blasts of responders emphasizes the influence of the TNFα signalling pathway as an interesting field of further research. The safety profile of BEM+LDAC in the REL AML pts (n=19) is comparable with the known safety profile of LDAC. TRAEs of ≥G3 observed in ≥10% of pts were anaemia (33% BEM+LDAC; 22% BEM), and ECG QT prolonged (11% BEM+LDAC; 11% Bem). No G5 TRAEs reported. Summary/Conclusion: BEM+LDAC is efficacious and well tolerated in REL AML pts unfit for IC, with promising survival benefit compared to historical data. Translational research showed activation of CD8+T cells and B/Plasma cells in response to treatment, indicating that BEM elicits activation of two major adaptive immune cell populations responsible for anti-AML immune responses. BEM+LDAC warrants further evaluation in this population. P549: ANALYSIS OF THE CLINICAL SIGNIFICANCE AND PROGNOSTIC IMPACT OF TET2 SINGLE NUCLEOTIDE POLYMORPHISM I1762V IN PATIENTS WITH ACUTE MYELOID LEUKEMIA Y. Li1, X. Lyu1,* 1Central Lab, Henan Cancer Hospital, Zhengzhou, China Background: Acute myeloid leukemia (AML) is a malignancy that originates from myeloid hematopoietic stem/progenitor cells in the bone marrow. The currently recognized cause of AML is chromosomal abnormalities or genetic mutations in myeloid hematopoietic stem/progenitor cells induced by various physicochemical factors such as benzene, pesticides, ionizing radiation, and chemotherapeutic drugs. With the development of high-throughput sequencing technology, a variety of acquired somatic mutations are used for the diagnosis, treatment and prognostic assessment of AML. Also, genetic susceptibility due to many single nucleotide polymorphisms (SNPs) is thought to be important in the development of AML. The TET2 gene encodes a methylcytosine dioxygenase that catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine and contributes to the epigenetic regulation of myelopoiesis. Mutations in the TET2 gene lead to dysmethylation and myeloid transformation. The TET2 SNP I1762V (rs2454206AG/GG, c.5284A>G, p.Ile1762Val) has an allele frequency of approximately 21% in the Chinese population. Some studies have shown that pediatric AML patients carrying mutations at this locus have a better prognosis. In this study, we investigated the clinical significance of the TET2 SNP locus I1762V in AML patients and its impact on prognosis through a comprehensive analysis of high-throughput sequencing results from 413 AML patients. Aims: This study aimed to explore the clinical significance and effect on the prognosis of TET2 Single nucleotide polymorphism I1762V in patients with acute myeloid leukemia (AML). Methods: Target sequencing on 58 hematological tumor-related genes in bone marrow samples of 413 AML patients was performed using the high-throughput sequencing method. TET2 I1762V and other somatic mutations were annotated and compared with patients’ clinical information and prognosis. Results: I1762V was detected in 154 patients with AML, which was significantly different from the normal population in NyuWa Chinese Population Variant Database (χ2=72.4, P<0.001). I1762V was not related to sex, age, and karyotype of AML patients (P>0.05). The proportion of NPM1 gene mutations and KIT gene mutations in patients with I1762V was significantly higher than the others(P < 0.001). NPM1 mutations and KIT mutations were mutually exclusive. The survival analysis results showed that the OS and PFS of AML patients with I1762V were significantly higher than those of wild type patients (HR=0.55, P < 0.05), while the OS and PFS in AML patients with DNMT3A mutation (with or without I1762V mutation) were lower than those of wild type patients (HR=1.79, P < 0.05). Summary/Conclusion: TET2 SNP I1762V is an factor affecting the prognosis of AML patients, which can be used to guide the treatment and evaluate the prognosis of AML. TET2 SNP I1762V influenced the concomitant or reciprocal mutations in AML patients; I1762V was closely associated with the prognosis of AML patients. The findings demonstrate that the TET2 SNP locus I1762V is an important guide for the diagnosis, treatment and prognostic assessment of AML patients. P550: A PHASE 3, RANDOMIZED TRIAL OF MAGROLIMAB IN COMBINATION WITH VENETOCLAX AND AZACITIDINE IN PREVIOUSLY UNTREATED PATIENTS WITH ACUTE MYELOID LEUKEMIA INELIGIBLE FOR INTENSIVE CHEMOTHERAPY (ENHANCE-3) N. G. Daver1,*, K. Liu2, S. Werneke2, E. Rustia2, G. Ramsingh2, P. Vyas3 1The University of Texas MD Anderson Cancer Center, Houston; 2Gilead Sciences, Inc., Foster City, United States of America; 3Department of Medicine, University of Oxford Radcliff, Oxford, United Kingdom Background: Acute myeloid leukemia (AML) is an aggressive clonal hematopoietic malignancy of myeloid cells associated with limited outcomes for patients who are ineligible for intensive chemotherapy due to age or comorbidities. Magrolimab (Hu5F9-G4) is an antibody blocking CD47, a “don’t eat me” signal on cancer cells, resulting in tumor phagocytosis by macrophages. A triplet regimen of magrolimab + azacitidine + venetoclax has shown promising activity in patients with AML. Aims: To evaluate the efficacy, safety, and tolerability of magrolimab + azacitidine + venetoclax in previously untreated patients with AML who are ineligible for intensive chemotherapy. Methods: This is a phase 3, randomized, double-blind, placebo-controlled, multicenter study. Approximately 432 patients will be randomized 1:1 to receive magrolimab + azacitidine + venetoclax (experimental arm) or placebo + azacitidine + venetoclax (control arm) (Figure). Randomization will be stratified by age, cytogenetic risk group, and geographic region. Patients are eligible if they are ≥75 years of age or 18 to 74 years of age with specific comorbidities. Receipt of prior antileukemic therapy for AML is not allowed. Magrolimab will be administered intravenously with an initial 1 mg/kg priming dose on days 1 and 4 to mitigate on target anemia. The dose will then be escalated to 15 mg/kg on day 8, then to 30 mg/kg on days 11 and 15, then weekly for 5 doses, followed by every other week beginning 1 week after the fifth weekly 30 mg/kg dose. Placebo will be dosed at the same frequency as magrolimab. Venetoclax will be administered orally at 100 mg on day 1, 200 mg on day 2, and 400 mg daily starting on day 3 and thereafter. Azacitidine will be given intravenously or subcutaneously at 75 mg/m2 on days 1 to 7, or days 1 to 5 and 8 to 9 of 28-day cycles. Patients will remain on study until disease progression, relapse, loss of clinical benefit, or unacceptable toxicities, or until other discontinuation criteria are met. The dual primary endpoints are complete remission (CR) rate within 6 treatment cycles and overall survival. Secondary endpoints include duration of CR, transfusion independence, and event-free survival. Results: Trial in progress. Image: Summary/Conclusion: Patient enrollment is ongoing. Clinical trial information: NCT05079230. P551: A PHASE 3, RANDOMIZED, OPEN-LABEL STUDY EVALUATING THE SAFETY AND EFFICACY OF MAGROLIMAB IN COMBINATION WITH AZACITIDINE IN PREVIOUSLY UNTREATED PATIENTS WITH TP53-MUTANT ACUTE MYELOID LEUKEMIA N. G. Daver1,*, P. Vyas2, M. P. Chao3, G. Xing3, C. Renard3, G. Ramsingh3, D. A. Sallman4, A. H. Wei5 1The University of Texas MD Anderson Cancer Center, Houston, United States of America; 2Weatherall Institute of Molecular Medicine, MRC Molecular Hematology Unit, University of Oxford, Oxford, United Kingdom; 3Gilead Sciences, Inc., Foster City; 4Moffitt Cancer Center, Tampa, United States of America; 5Department of Haematology, Alfred Hospital and Monash University, Melbourne, Australia Background: Magrolimab (Hu5F9-G4) is an antibody blocking CD47, a macrophage immune checkpoint and “don’t eat me” signal on cancer cells, that eliminates leukemia stem cells by inducing tumor phagocytosis. Hypomethylating agents synergize with magrolimab by inducing “eat me” signals on leukemic blasts, thereby enhancing phagocytosis. Magrolimab with azacitidine (AZA) has shown encouraging activity in frontline acute myeloid leukemia (AML) unfit for intensive chemotherapy and myelodysplastic syndrome. In TP53-mutant (TP53m) AML, it demonstrated an objective response rate of 69%, complete response (CR) rate of 45%, and median overall survival (OS) of 12.9 months. The TP53 gene mutation is observed in approximately 10-15% of newly diagnosed AML and is associated with poor survival. The published first-line median OS in TP53m AML is 4-7 months, regardless of whether patients are treated with intensive chemotherapy or non-intensive approaches, such as a hypomethylating agent with venetoclax (VEN). AML patients harboring the TP53 gene mutation represent a significant unmet medical need. Aims: To evaluate the efficacy, safety, and tolerability of magrolimab+AZA vs physician’s choice of VEN+AZA or “7 + 3” chemotherapy in patients with previously untreated TP53m AML. Methods: This is a phase 3, randomized, open-label, multicenter study. Approximately 346 patients will be randomized (1:1) to magrolimab+AZA (experimental arm) or physician’s choice of VEN+AZA or 7 + 3 chemotherapy, based on patient fitness. Randomization will be stratified by appropriateness for non-intensive vs intensive therapy, geographic region (US vs non-US sites), and age (<75 vs ≥75 years). Patients are eligible if they are ≥18 years old with histologically confirmed AML with no prior antileukemic therapy, with at least one TP53 gene mutation that is not benign or likely benign (confirmed by central laboratory) or biallelic 17p deletions (based on a locally evaluated karyotype/fluorescence in situ hybridization report). During the first 28-day cycle, patients randomized to magrolimab+AZA will receive magrolimab intravenously (IV) at a priming dose of 1 mg/kg on days 1 and 4, 15 mg/kg on day 8, and 30 mg/kg on days 11, 15, and then weekly for 5 doses, followed by every other week beginning 1 week after the fifth weekly 30 mg/kg dose. For patients randomized to the VEN+AZA arm, VEN and AZA will be administered according to labeled indications. Patients treated with 7 + 3 chemotherapy will receive 1-2 induction cycle(s) with IV daunorubicin or idarubicin on days 1-3 and cytarabine 100 mg/m2 or 200 mg/m2 on days 1-7 followed by up to 4 consolidation cycles with high-dose cytarabine (3000 mg/m2). Patients receiving magrolimab+AZA or VEN+AZA will remain on treatment until disease progression, relapse, loss of clinical benefit, unacceptable toxicities, or stem cell transplant. Patients can undergo stem cell transplantation per investigator decision. The primary endpoint is OS in patients appropriate for non-intensive therapy, and the key secondary endpoint is OS in all patients. Results: Trial in progress. Summary/Conclusion: Patient enrollment is ongoing. Clinical trial information: NCT04778397. P552: TREATMENT OUTCOMES IN NEWLY DIAGNOSED, UNTREATED PATIENTS WITH TP53-MUTATED ACUTE MYELOID LEUKEMIA: A SYSTEMATIC LITERATURE REVIEW AND META-ANALYSIS N. G. Daver1,*, S. Iqbal2, C. Renard2, R. J. Chan2, K. Hasegawa2, H. Hu2, P. Tse3, J. Yan3, M. J. Zoratti3, F. Xie3, G. Ramsingh2 1The University of Texas MD Anderson Cancer Center, Houston; 2Gilead Sciences, Inc., Foster City, United States of America; 3McMaster University, Hamilton, Canada Background: TP53 mutations are present in 10% to 15% of patients with acute myeloid leukemia (AML) and are associated with resistance to therapy and poor outcomes. Currently available frontline therapies for TP53-mutated AML include intensive chemotherapy (IC), hypomethylating agents (HMAs), and venetoclax combined with HMA (VEN+HMA). Aims: To describe response and survival outcomes associated with IC, HMAs, and VEN+HMA in newly diagnosed, untreated patients with TP53-mutated AML. Methods: EMBASE and MEDLINE were systematically searched to identify prospective and retrospective studies that reported complete remission (CR), CR with incomplete hematologic recovery (CRi), median overall survival (OS), event-free survival (EFS), or duration of response (DOR) outcomes in patients with TP53-mutated AML treated with IC, HMAs, or VEN + HMA. Screening and data extraction were conducted independently and in duplicate by 2 reviewers. Response outcomes (CR and CRi) were pooled using random-effects models, and the median of medians method was used for time-related outcomes (OS, EFS, DOR). Results: From the 3006 abstracts identified (May 20, 2021), 17 publications (12 studies: 6 randomized controlled trials, 2 single-arm trials, and 4 retrospective studies) satisfied the inclusion criteria (Table). The percentage of patients achieving CR was highest with IC (43%; 95% CI, 30%-56%; N=133), followed by VEN+HMA (33%; 95% CI, 22%-47%; N=54;) and HMA (21%; 95% CI, 7%-49%; N=14). The CRi rate was highest with VEN+HMA (20%; 95% CI, 12%-33%; N=54), followed by IC (6%; 95% CI, 1%-20%; N=35). No studies reported CRi outcomes for HMA alone. The rates of CR/CRi were 49% (95% CI, 37%-60%; N=121) for VEN+HMA, 46% (95% CI, 39%-53%; N=200) for IC, and 13% (95% CI, 2%-48%; N=28) for HMA alone. The median OS was similarly low across the 3 treatments: 6.5 months for IC (95% CI, 5.1-8.5 months; N=155), 6.2 months for VEN+HMA (95% CI, 5.2-7.2 months; N=73), and 6.1 months for HMA (95% CI, 4.9-7.2 months; N=34). The median EFS was 3.7 months (95% CI, 1.6-5.7 months; N=133) with IC. The median DOR was 3.5 months (N=35) with IC and 5.0 months (95% CI, 3.5-6.5 months; N=54) with VEN+HMA. No studies reported EFS for HMA alone or VEN+HMA or DOR for HMA alone. Table. Outcomes in newly diagnosed, untreated adult patients with TP53-mutated AML treated with IC, HMA, and VEN+HMA ICstudies Total, N Outcome(95% CI) HMAstudies Total, N Outcome(95% CI) VEN+HMAstudies Total, N Outcome(95% CI) CR rate 2 133 0.43(0.30-0.56) 1 14 0.21(0.07-0.49) 2 54 0.33(0.22-0.47) CRi rate 1 35 0.06(0.01-0.2) 0 - - 2 54 0.20(0.12-0.33) CR/CRi rate 4 200 0.46(0.39-0.53) 2 28 0.13(0.02-0.48) 4 121 0.49(0.37-0.60) Median OS, months 3 155 6.5(5.1-8.5) 2 34 6.1(4.9-7.2) 2 73 6.2(5.2-7.2) Median EFS, months 2 133 3.7(1.6-5.7) 0 - - 0 - - Median DOR, months 1 35 3.5(not reported) 0 - - 2 54 5.0(3.5-6.5) Summary/Conclusion: CR, CR/CRi, DOR, and median OS were modest across all treatments for newly diagnosed, untreated patients with TP53-mutated AML. Limitations of this analysis include low study and patient numbers for some interventions, the lack of direct trial comparisons, and that CR/CRi outcomes were not reported separately for DOR. The findings of this review highlight the significant unmet need for this very difficult-to-treat population. P553: A PHASE 2, OPEN-LABEL, MULTIARM, MULTICENTER STUDY TO EVALUATE MAGROLIMAB COMBINED WITH ANTILEUKEMIA THERAPIES FOR FIRST-LINE, RELAPSED/REFRACTORY, OR MAINTENANCE TREATMENT OF ACUTE MYELOID LEUKEMIA P. Vyas1,*, N. G. Daver2, M. P. Chao3, G. Xing3, C. Renard3, G. Ramsingh3, A. H. Wei4, D. A. Sallman5 1Weatherall Institute of Molecular Medicine, MRC Molecular Hematology Unit, University of Oxford, MRC Molecular Haematology Unit and Oxford Biomedical Research Centre, University of Oxford and Oxford University Hospitals, Oxford, United Kingdom; 2The University of Texas MD Anderson Cancer Center, Houston; 3Gilead Sciences, Inc., Foster City, United States of America; 4Department of Haematology, Alfred Hospital and Monash University, Melbourne, Australia; 5Moffitt Cancer Center, Tampa, United States of America Background: Magrolimab (Hu5F9-G4) is an antibody blocking CD47, a macrophage immune checkpoint and “don’t eat me” signal on cancer cells. Magrolimab induces tumor phagocytosis and eliminates leukemia stem cells. Chemotherapy and hypomethylating agents synergize with magrolimab by inducing “eat me” signals on leukemic blasts, thereby enhancing phagocytosis. Magrolimab+azacitidine (AZA) has shown encouraging clinical efficacy with an objective response rate (ORR) of 63% and a complete remission (CR) or CR with incomplete hematologic recovery (CRi) rate of 54% in first-line acute myeloid leukemia (AML). Newly diagnosed AML patients (pts) who are ineligible for intensive chemotherapy (IC) are incurable despite the progress made with AZA+venetoclax (VEN; median overall survival [OS] 14-18 months), while AML pts with relapsed/refractory (R/R) disease after intensive regimens have a dismal prognosis. Furthermore, for pts in remission, maintenance therapy with oral AZA has improved OS and prevented relapse, although relapse rates remain high. Magrolimab combinations are being evaluated in each of these settings to improve the standard of care. Aims: To evaluate the safety, efficacy, and tolerability of magrolimab combined with antileukemia therapies in first-line pts ineligible for IC (cohort 1), R/R pts after IC (cohort 2), and pts in CR/CRi with presence of minimal residual disease (MRD) after IC (cohort 3). Methods: This is an open-label, multiarm, multicenter study that includes 3 safety run-ins with corresponding phase 2 cohorts. Pts in cohort 1 must be ≥75 years or 18-74 years with comorbidities that preclude IC. Pts in cohort 2 must have R/R AML after initial IC. Pts in cohort 3 must have achieved a CR/CRi with MRD positivity, as assessed by flow cytometry, after IC, and not be candidates for hematopoietic stem cell transplant. Each cohort will initially enroll 6 pts for 1 cycle (28 days) to assess dose-limiting toxicities and determine the recommended phase 2 dose (RP2D). Pts in cohort 1 will receive a combination of magrolimab, VEN, and AZA. Pts in cohort 2 will receive magrolimab in combination with mitoxantrone, etoposide, and cytarabine (MEC). Pts in cohort 3 will receive magrolimab with oral CC-486. All will receive magrolimab on the same schedule. In cycle 1, magrolimab will be administered intravenously (IV) as priming and ramp-up doses at 1 mg/kg on days 1 and 4, 15 mg/kg on day 8, 30 mg/kg on days 11, 15, and then weekly for 5 doses, followed by every other week beginning 1 week after the fifth weekly 30 mg/kg dose. For cohort 1, AZA (75 mg/m2) will be administered IV/subcutaneously on days 1-7 or 1-5, 8, and 9 of each cycle. VEN, MEC, and CC-486 will be administered according to labeled indications. After completion of the safety run-in, additional pts will be enrolled in cohort 1 (n=40), cohort 2 (n=30), and cohort 3 (n=40) for phase 2. In phase 2, pts in cohorts 1 and 3 will receive study treatment at RP2D until disease progression, unacceptable toxicity, or loss of clinical benefit. Pts in cohort 2 will receive magrolimab with MEC for up to 3 cycles followed by magrolimab alone for a total of 12 magrolimab cycles. In cohorts 1 and 2, the primary efficacy endpoint is the CR rate. Secondary endpoints of interest include OS, ORR, and MRD-negative CR/CRi rates. The primary efficacy endpoint in cohort 3 is the MRD-negative CR rate. Results: Trial in progress. Summary/Conclusion: Patient enrollment is ongoing. Clinical trial information: NCT04778410. P554: GILTERITINIB VERSUS SALVAGE CHEMOTHERAPY FOR RELAPSED/REFRACTORY FLT3-MUTATED ACUTE MYELOID LEUKEMIA: A PHASE 3, RANDOMIZED, MULTICENTER, OPEN-LABEL TRIAL IN ASIA J. Wang1,*, B. Jiang2, J. Li3, L. Liu4, X. Du5, H. Jiang6, J. Hu7, M. Yuan8, T. Sakatani9, T. Kadokura9, M. Takeuchi9, S. Izuka9, L. Girshova10, J. Tan11, S. Bondarenko12, L. L. L. Wong13, A. Khuhapinant14, E. Martynova15, N. Hasabou16, R. Tiu16 1State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin; 2Department of Hematology, Peking University International Hospital; 3Department of Hematology, Peking Union Medical College Hospital, Beijing; 4Department of Hematology, Shanghai Tongren Hospital, Shanghai; 5Department of Hematology, Guangdong Provincial People’s Hospital, Guangzhou; 6Department of Hematology, Peking University Peoples Hospital, Beijing; 7Fujian Institute of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian; 8Astellas Pharma, Inc., Beijing, China; 9Astellas Pharma, Inc., Tokyo, Japan; 10Almazov National Medical Research Centre, St. Petersburg, Russia; 11Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak, Sarawak, Malaysia; Department of Hematology, Ampang Hospital, Selangor, Malaysia; 12Clinical Research Institution of Pediatric Hematology and Transplantation under the Name of Raisa Gorbacheva, State Medical University, St. Petersburg, Russia; 13Hematology Unit, Queen Elizabeth Hospital, Kota Kinabalu, Sabah, Malaysia; 14Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; 15Krasnoyarsk Regional Clinical Hospital, Krai, Russia; 16Astellas Pharma, Inc., Northbrook, United States of America Background: Due to poor prognosis, treatment options for patients (pts) with relapsed/refractory to therapy (R/R) FLT3-mutated (FLT3 mut+) acute myeloid leukemia (AML) are needed globally. The phase 3 ADMIRAL trial showed superior survival benefit and favorable safety for pts with R/R FLT3 mut+ AML receiving gilteritinib vs salvage chemotherapy (SC); randomized, controlled trials of treatments in a mostly Asian population are lacking. Aims: We evaluated the efficacy and safety/tolerability of gilteritinib vs SC in Asian pts with R/R FLT3 mut+ AML after first-line therapy. Methods: In this phase 3, open-label COMMODORE (NCT03182244) trial, adult pts in China, Russia, Singapore, Thailand, and Malaysia with R/R FLT3 mut+ AML were randomized 1:1 to gilteritinib 120 mg orally per day or SC (low-dose cytarabine; mitoxantrone/etoposide/intermediate-dose cytarabine; or fludarabine/high-dose cytarabine/granulocyte colony-stimulating factor) over continuous 28-day cycles. Pts had ECOG score ≤2; pts with acute promyelocytic leukemia, BCR-ABL–positive leukemia, active central nervous system disease, or secondary AML were excluded. Primary endpoint was overall survival (OS); key secondary efficacy endpoints were event-free survival (EFS) and complete remission (CR). Other endpoints were duration of remission, composite CR (CRc), and safety/tolerability. OS and EFS were analyzed with stratified Cox proportional hazard models; response rates were analyzed with the Cochran-Mantel-Haenszel test. Subgroup analyses were planned. Interim analysis results are presented. Results: As of June 30, 2020, 234 pts were randomized (gilteritinib, n=116; SC, n=118). Median age was 51.5 and 49.5 years in the gilteritinib and SC groups, respectively; most pts had not received prior FLT3 inhibitors (87.9% and 93.2%). Baseline FLT3 mutations in the gilteritinib vs SC groups were: FLT3-ITD (91.4% vs 83.1%), FLT3-TKD (6.0% vs 11.9%), and both FLT3-ITD and FLT3-TKD (2.6% vs 5.1%). Median OS follow-up duration was 11.1 mo for gilteritinib and 6.9 mo for SC. Median OS was longer with gilteritinib (9.0 mo) vs SC (4.7 mo; HR 0.549 [95% CI: 0.379, 0.795]; P=0.00126); 1-year survival rate was 33.3% and 23.2%, respectively. OS benefit was seen with gilteritinib vs SC across most subgroups (Figure). Pts on gilteritinib had longer EFS than pts on SC (median EFS 2.8 vs 0.6 mo; HR 0.551 [95% CI: 0.395, 0.769]; P=0.00004). More pts had CR on gilteritinib (16.4%) vs SC (10.2%; P=0.17690); CRc rates were 50.0% and 20.3% (P<0.00001). Grade ≥3 adverse events (AEs) with gilteritinib (97.3%) vs SC (94.2%) were comparable; serious AE rates were higher for gilteritinib (73.5%) vs SC (61.5%). Adjusted for treatment exposure, AE rates were lower with gilteritinib (grade ≥3, 55.56 events/pt-year [E/PY]; serious, 6.19 E/PY) than SC (grade ≥3, 164.00 E/PY; serious, 12.40 E/PY). Most common AEs for gilteritinib were anemia (76.1%), thrombocytopenia (46.9%), pyrexia (41.6%), and increased blood lactate dehydrogenase (41.6%); for SC, most common AEs were anemia (64.4%), decreased white blood cell count (41.3%), and thrombocytopenia (38.5%). AEs leading to death occurred in 22 (19.5%) and 15 (14.4%) pts receiving gilteritinib or SC, respectively. Image: Summary/Conclusion: Gilteritinib significantly prolonged OS and EFS vs SC in pts with R/R FLT3 mut+ AML in Asia. Safety/tolerability adjusted for treatment exposure was favorable for gilteritinib vs SC. COMMODORE results further validate/affirm the clinical efficacy/safety data from ADMIRAL, reinforcing the significant benefit of gilteritinib in R/R FLT3 mut+ AML. P555: A PHASE 1B/2 STUDY OF THE CD123-TARGETING ANTIBODY-DRUG CONJUGATE PIVEKIMAB SUNIRINE (IMGN632) IN COMBINATION WITH VENETOCLAX (VEN) AND AZACITIDINE (AZA) FOR PATIENTS WITH CD123-POSITIVE AML N. G. Daver1,*, P. Montesinos2, A. Aribi3, G. Martinelli4, J. Altman5, G. Roboz6, E. S. Wang7, P. W. Burke8, D. Jeyakumar9, R. B. Walter10, D. J. DeAngelo11, H. P. Erba12, A. Advani13, L. Gastaud14, X. Thomas15, E. Todisco16, N. Pemmaraju1, L. Mendez17, A. de la Fuente18, G. Gaidano19, A. Curti20, N. Boissel21, C. Recher22, C. Schliemann23, P. Vyas24, C. M. Sloss25, J. Wang25, K. A. Malcolm25, P. A. Zweidler-McKay25, K. L. Sweet26 1Department of Leukemia, The University of Texas MD Anderson, Houston, United States of America; 2Hospital Universitari i Politècnic La Fe, Valencia, Spain; 3Gehr Family Center for Leukemia Research, City of Hope, Duarte, United States of America; 4Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology “L. e A. Seràgnoli”, Bologna, Italy; 5Division of Hematology and Oncology, Northwestern University Feinberg School of Medicine, Chicago; 6Division of Hematology & Medical Oncology, Weill Cornell Medicine/New York Presbyterian Hospital, New York; 7Roswell Park Comprehensive Cancer Center, Buffalo; 8Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan, Ann Arbor; 9University of California Irvine, Chao Family Comprehensive Cancer Center, Orange; 10Fred Hutchinson Cancer Research Center, Seattle; 11Department of Medical Oncology, Harvard Medical School, Dana-Farber Cancer Institute, Boston; 12Division of Hematologic Malignancies and Cellular Therapy, Duke University, Durham; 13Taussig Cancer Institute, Cleveland Clinic, Cleveland, United States of America; 14Medical Oncology Department, Antoine Lacassagne Hospital, Nice; 15Department of Hematology, Hospices Civils de Lyon, Lyon-Sud Hospital, Lyon, France; 16Division of Onco-Hematology, European Institute of Oncology IRCCS, Milano, Italy; 17Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States of America; 18MD Anderson Cancer Center Madrid, Madrid, Spain; 19Division of Hematology, Department of Translational Medicine, Università del Piemonte Orientale, Novara; 20IRCCS Azienda ospedaliero-universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy; 21Université de Paris, Service Hématologie, Hôspital Saint-Louis, Paris; 22Service d’Hématologie, CHU de Toulouse - Institut Universitaire du Cancer Toulouse Oncopole, Toulouse, France; 23Department of Medicine A, Hematology, Oncology and Pneumology, University Hospital Muenster, Muenster, Germany; 24MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford NIHR Biomedical Research Centre, and Oxford University Hospitals NHS Trust, Oxford, United Kingdom; 25ImmunoGen, Inc., Waltham; 26Department of Malignant Hematology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, United States of America Background: Overexpression of CD123 occurs in multiple hematological malignancies, including acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute lymphoblastic leukemia (ALL) and others. With limited expression on normal hematopoietic progenitor cells, the CD123 antigen is an attractive target for new therapeutics. IMGN632 is a CD123-targeting antibody-drug conjugate (ADC) comprising a novel anti-CD123 antibody coupled, via a peptide linker, to a unique DNA-alkylating cytotoxic payload of the IGN (indolinobenzodiazepine pseudodimer) class. In preclinical models of AML, IMGN632 exhibited potent anti-leukemia activity, with a wide therapeutic index. Confirming preclinical expectations, encouraging single-agent activity has emerged for IMGN632 in the ongoing Phase I trial in patients with CD123-positive BPDCN and AML (Daver. Blood (2019) 134 (Supplement_1):734.). Preclinical data have demonstrated increased activity with the addition of IMGN632 to AZA alone and to AZA+VEN in multiple AML xenograft and PDX models, leading to improved survival in these models (Kuruvilla. Blood (2019) 134 (Supplement_1):1375.). We have reported compelling activity (ORR 59% and CCR rate 38%) of the IMGN632 triplet in relapsed or refractory AML patients (Daver. ASH 2021 Abstract #372). The safety profile of the triplet included rates of cytopenias similar to those observed with AZA+VEN; additional low-grade adverse events included infusion-related reactions, dyspnea, fatigue, gastrointestinal toxicities, electrolyte imbalances, and pneumonia. Aims: This Phase 1b/2 study is designed to determine the safety, tolerability, and preliminary anti-leukemia activity of IMGN632 when administered in combination with AZA and VEN to patients with relapsed and frontline CD123-positive AML. Methods: Dose escalation for the IMGN632+AZA+VEN triplet is complete. The Recommended Phase 2 Dose levels are: IMGN632 45 mcg/kg given on day 7 with 14 days of VEN, and either AZA 50 OR 75 mg/m2 for 7 days of a 28-day cycle. Results: N/A Summary/Conclusion: Phase 2 expansion cohorts for patients with untreated/frontline and relapsed AML are enrolling to further characterize the safety profile and assess the antileukemic activity. NCT04086264 P556: CHANGES IN HEALTH-RELATED QUALITY OF LIFE IN PATIENTS WITH NEWLY-DIAGNOSED ACUTE MYELOID LEUKEMIA RECEIVING IVOSIDENIB + AZACITIDINE OR PLACEBO + AZACITIDINE A. Schuh1,*, S. de Botton2, C. Recher3, S. Vives Polo4, E. Zarzycka5, J. Wang6, G. Bertani7, M. Heuser8, R. Calado9, S.-P. Yeh10, J. Hui11, S. Pandya11, D. Gianolio11, C. Chamberlain11, H. Dohner12, P. Montesinos13 1Princess Margaret Cancer Centre, Toronto, Canada; 2Institut Gustave Roussy, Villejuif; 3Institut Universitaire du Cancer de Toulouse Oncopole, Toulouse, France; 4Hospital Universitario Germans Trias i Pujol-ICO Badalona, Josep Carreras Research Institute, Badalona, Spain; 5Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk, Poland; 6Institute of Hematology & Hospital of Blood Disease, Tianjin, China; 7ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 8Hannover Medical School, Hannover, Germany; 9Ribeirão Preto School of Medicine, University of São Paulo, São Paulo, Brazil; 10China Medical University, Taichung, Taiwan; 11Servier Pharmaceuticals LLC, Boston, United States of America; 12Ulm University Hospital, Ulm, Germany; 13Hospital Universitari i Politècnic La Fe, València, Spain Background: Ivosidenib (IVO) is a potent, targeted inhibitor of mutant isocitrate dehydrogenase 1 (mIDH1) that is approved for acute myeloid leukemia (AML). IVO plus azacitidine (AZA) demonstrated clinical benefit compared with placebo and AZA in the AGILE study (NCT03173248). Aims: To assess the impact of IVO+AZA versus placebo+AZA on health-related quality of life (HRQoL) using data from the AGILE study. Methods: In the double-blind, placebo-controlled phase 3 AGILE study, patients (pts) were randomized 1:1 to IVO 500 mg QD + AZA 75 mg/m2 SC or IV for 7 days in 28-day cycles, or placebo+AZA. HRQoL was a secondary endpoint assessed using two validated questionnaires: the European Organisation of Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30) and the EuroQol 5-dimension 5-level questionnaire (EQ-5D-5L). Questionnaires were administered pre-dose on cycle (C) 1 Day (D) 1, on C1D15, C2D1, C2D15, and on D1 of every odd cycle thereafter until the end of treatment. Score change from baseline across visits for all subscales of EORTC QLQ-C30 was analyzed with mixed models. A 10-point threshold in EORTC QLQ-C30 subscale score was used to evaluate clinically meaningful changes from baseline or differences between arms. Two-sided nominal p-values are reported. All patients gave written informed consent. Results: At baseline, 69 and 68 pts out of 72 receiving IVO+AZA completed the EORTC QLQ-C30 and EQ-5D-5L, respectively, and 66 pts out of 74 receiving placebo+AZA completed both. Mean baseline HRQoL scores were similar between treatment arms (EORTC QLQ-C30 global health status [GHS/QoL] score was 56.3 and 53.2 in the IVO+AZA and placebo+AZA arms, respectively). There was an initial decline in HRQoL (EORTC QLQ-C30 GHS/QoL) in both arms for ~4 months, consistent with time to response, and which was generally not clinically meaningful (Table). IVO+AZA was associated with preserved or improved HRQoL compared to baseline for most subscales of the EORTC QLQ-C30 from C5 to C19 (after which no placebo+AZA HRQoL data were available), and at most timepoints for EQ-5D-5L VAS scores and index values. EORTC QLQ-C30 subscales with clinically meaningful improvements from baseline at most timepoints from C5 to C19 in the IVO+AZA arm included GHS/QoL (Table), fatigue, pain and appetite loss. In contrast, there were few clinically meaningful improvements from baseline in placebo+AZA pts. GHS/QoL scores were significantly improved (p≤0.05) for IVO+AZA versus placebo+AZA at C2D1, C2D15, C7 and C9 (Table), and differences between treatment arms were clinically meaningful at C2D1, C2D15, C7, C9, C13, C15 and C19 (Table). Likewise, improvements in EORTC QLQ-C30 fatigue, appetite loss, nausea and vomiting, diarrhea, cognitive functioning and social functioning favored IVO+AZA over placebo+AZA at multiple timepoints. Image: Summary/Conclusion: Data from the AGILE study show that patients with mIDH1 AML receiving treatment with IVO+AZA tended to report maintenance or improved HRQoL from cycle 5 through to cycle 19 compared with placebo+AZA. P557: HEMATOLOGIC IMPROVEMENTS WITH IVOSIDENIB + AZACITIDINE COMPARED WITH PLACEBO + AZACITIDINE IN PATIENTS WITH NEWLY-DIAGNOSED ACUTE MYELOID LEUKEMIA H. Dohner1,*, P. Montesinos2, S. Vives Polo3, E. Zarzycka4, J. Wang5, G. Bertani6, M. Heuser7, R. Calado8, A. Schuh9, S.-P. Yeh10, A. de la Fuente11, C. Cerchione12, S. Daigle13, J. Hui13, S. Pandya13, D. Gianolio13, C. Recher14, S. de Botton15 1Ulm University Hospital, Ulm, Germany; 2Hospital Universitari i Politècnic La Fe, València; 3Hospital Universitario Germans Trias i Pujol-ICO Badalona, Josep Carreras Research Institute, Badalona, Spain; 4Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk, Poland; 5Institute of Hematology & Hospital of Blood Disease, Tianjin, China; 6ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 7Hannover Medical School, Hannover, Germany; 8Ribeirão Preto School of Medicine, University of São Paulo, São Paulo, Brazil; 9Princess Margaret Cancer Centre, Toronto, Canada; 10China Medical University, Taichung, Taiwan; 11MD Anderson Cancer Center Madrid, Madrid, Spain; 12Hematology Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy; 13Servier Pharmaceuticals, Boston, United States of America; 14Institut Universitaire du Cancer de Toulouse Oncopole, Toulouse; 15Institut Gustave Roussy, Villejuif, France Background: Ivosidenib (IVO) is a potent oral targeted inhibitor of mutant isocitrate dehydrogenase 1 (mIDH1). IVO plus azacitidine (AZA) significantly improved event-free survival (EFS), overall survival and complete remission + partial hematologic recovery rates compared with placebo + AZA, in patients (pts) with newly diagnosed IDH1-mutant acute myeloid leukemia (AML) in the Phase 3 AGILE trial (NCT03173248). Aims: To report blood count recovery results from the AGILE trial. Methods: Pts were randomized 1:1 to IVO 500 mg QD + AZA 75 mg/m2 SC or IV for 7 days in 28-day cycles (n=72), or placebo+AZA (n=74). Red blood cell (RBC)/platelet transfusion history were assessed at screening and follow-up. Bone marrow (BM) and peripheral blood samples were obtained at screening, and during weeks 9, 17, 25, 33, 41, 53, and every 24 weeks thereafter, and at end of treatment and during EFS follow up. Samples were analyzed at each local site according to ICSH guidelines. All patients gave written informed consent. Results: In the IVO+AZA and placebo+AZA arms, 4.2% and 5.5% of pts, respectively, received concomitant granulocyte colony-stimulating factor. Hemoglobin levels steadily increased from baseline at a similar rate in both treatment arms. Mean platelet count recovered from baseline values in the IVO+AZA and placebo+AZA arms (71.0 and 92.6 x 109/L, respectively) as early as week 9 of treatment (171.1 and 155.1 x 109/L, respectively) and continued to steadily increase thereafter in the treated population. In pts receiving IVO+AZA, mean neutrophil counts rapidly increased from baseline (0.99 x 109/L) to week 2 (2.05 x 109/L) and week 5 (4.07 x 109/L), and then generally stabilized to within the normal range to study end (last available cycle value; ~2.0 x 109/L). Mean neutrophil counts initially declined with placebo+AZA before slowly recovering to near-normal levels after 36-40 weeks. The increased blood counts were accompanied by a rapid decrease in the mean BM blast percentage from 54.8% at baseline to 12.0% and 7.2% at week 9 and 17, respectively, in IVO+AZA treated patients and were maintained for 149 weeks. The decline in BM blasts was slower in the placebo+AZA arm (53.7%, 34.6% and 19.6% at baseline, week 9 and week 17, respectively). Among patients who were RBC/platelet transfusion-dependent at baseline (~54.0% in both groups), 46.2% in the IVO+AZA group achieved RBC/platelet transfusion independence compared with 17.5% in the placebo+AZA arm (1-sided p=0.0032). Additionally, fewer adverse events of febrile neutropenia (28.2% vs 34.2%) and infections (28.2% vs 49.3%) were reported in the IVO+AZA arm compared to the placebo+AZA arm. Summary/Conclusion: IVO+AZA demonstrated a significant clinical benefit compared with placebo+AZA and this sub-analysis demonstrated a rapidly improved recovery of blood counts and a reduced dependence on RBC and/or platelet transfusion. Moreover, rates of febrile neutropenia and infections were reduced with IVO+AZA. P558: GERMAN AMLCG-SURVIVORSHIP STUDY – SOMATIC LONG-TERM CONSEQUENCE OF AML AND ITS THERAPY: FROM HEART TO KIDNEY. A. S. Moret1,*, E. Telzerow1, C. Sauerland2, M. Rothenberg-Thurley1, F. H. A. Mumm1, J. Braess3, B. Wörmann4, U. Krug5, W. E. Berdel6, W. Hiddemann1, K. Spiekermann1, P. Heußner7, K. Kraywinkel8, D. Görlich2, K. H. Metzeler9 1Department of Medicine III and Comprehensive Cancer Center (CCC Munich LMU), University Hospital, LMU Munich, Munich; 2Institute of Biostatistics and Clinical Research, University of Münster, Münster; 3Department of Oncology and Hematology, Hospital Barmherzige Brüder, Regensburg; 4Charité University Hospital Berlin, Berlin; 5Department of Medicine 3, Hospital Leverkusen, Leverkusen; 6Department of Medicine A, University of Münster, Münster; 7Hospital Garmisch-Partenkirchen, Garmisch-Partenkirchen; 8Department of Epidemiology and Health Monitoring, Robert Koch Institute, Berlin; 9Department of Hematology, Cell Therapy and Hemostaseology, University Hospital Leipzig, Leipzig, Germany Background: As outcomes of patients with acute myeloid leukemia (AML) have improved, the fraction of patients surviving long-term are increasing. Information on somatic and psycho-social health consequences of AML and its treatment is sparse. Aims: Our aim was to perform a multi-dimensional analysis of health outcomes in AML long-term survivors (AML-LTS). This report focuses on somatic morbidity; overall and health-related quality of life are reported separately (Telzerow et al.). Methods: We conducted a cross-sectional study including AML survivors who had been enrolled in clinical trials or the patient registry of the AML-CG study group and were alive ≥5 years after initial diagnosis. Data concerning somatic health status were collected through patient questionnaires, assessment by the patients’ physicians, and medical and laboratory reports. An age- and sex-matched control cohort was derived from German population-based health surveys (Robert Koch Institute, DEGS1 survey; n=6013). Results: Data on somatic health status was available for 355 survivors, aged 28-93 years, who participated 5 to 19 years after their AML diagnosis. Thirty-eight percent of survivors were treated with chemotherapy with or without an autologous transplant, whereas 62% had undergone allogeneic transplantation (alloSCT). We compared the prevalence of somatic diseases between AML-LTS and the general German population (DEGS1 sample), focusing on cardiovascular diseases and risk factors due to their relevance for overall morbidity and mortality, and their known association with survivorship in other cancers. Using age- and sex-adjusted multivariate models (Figure A), we found that AML survivors had a 2-fold higher risk of type 1/2 diabetes (DM 1/2), and a 3.5-fold increased risk of congestive heart failure (CHF) compared to the general population. Prevalence of hypertension, coronary artery disease (CAD) and myocardial infarction were similar between the groups. To identify factors associated with development of CHF among AML-LTS, we constructed multivariate models incorporating patient- and treatment-related covariables. We found an increased risk of CHF for AML-LTS who have had AML relapse (OR, 3.16; 95% CI: 1.46 – 6.83; P=0.004) and, in trend, for patients with sAML or tAML (OR 2.19; 95%CI: 0.92 – 5.22, P=0.076). Disease- or treatment-related factors did not significantly associate with any of the other comorbidities we studied. Furthermore, AML-LTS had significantly impaired kidney function, as assessed by the glomerular filtration rate (eGFR) estimated using the CKD-EPI-formula. Figure B displays the median eGFR according to age groups for AML-LTS and DEGS1. For each age group AML-LTS had significantly lower eGFR in comparison to DEGS1. We did not find an association between kidney function and time since diagnosis (p=.5) and no differences between AML-LTS treated by chemotherapy or alloSCT (p=.5), survived a relapse (p=.9) or were diagnosed with de novo-AML vs. sAML/tAML (P=.6). Image: Summary/Conclusion: Compared to the German general population, AML-LTS had increased risks of CHF and diabetes, but not hypertension or CAD. We identified AML relapse as a risk factor for developing of CHF, suggesting that cumulative chemotherapy exposure might be causally involved. Notably, AML-LTS who had undergone alloSCT did not have increased risks of CHF, CAD, hypertension or diabetes, compared to survivors treated with chemotherapy only. In addition, AML-LTS had a higher prevalence of impaired renal function. We did not identify treatment- or disease-related factors that might cause the lower kidney function in AML-LTS. P559: HUMORAL RESPONSE TO MRNA-BASED COVID-19 VACCINE IN PATIENTS WITH MYELOID MALIGNANCIES A. Mori1,*, M. Onozawa2, S. Tsukamoto1, T. Ishio1, E. Yokoyama1, K. Izumiyama1, M. Saito1, H. Muraki3,4, M. Morioka1, T. Teshima2, T. Kondo1 1Blood Disorders Center, Aiiku Hospital; 2Department of Hematology, Hokkaido University Faculty of Medicine; 3Sapporo Clinical Laboratory Inc.; 4Division of Laboratory, Aiiku Hospital, Sapporo, Japan Background: The end of the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease-19 (COVID-19), is not foreseen. Vaccination using two subtypes of mRNA-based vaccines, BNT162b2 or mRNA-1273, is an effective public health measure to reduce the risk of infection and severe complications from COVID-19. However, COVID-19 vaccine response data for patients with myeloid malignancy, who are at severe risk in case of infection, has not emerged. Aims: We investigated the antibody titers of COVID-19 in patients with myeloid malignancies who received two doses of mRNA-based COVID-19 vaccine. Methods: Previously treated, currently treated, and newly diagnosed 46 patients with acute myeloid leukemia (AML) and 23 patients with myelodysplastic syndrome (MDS) were included in this study. Anti-spike SARS-CoV-2 antibody titers were measured at 3 months after the second vaccination and compared them to those in healthy controls. Results: Seroconversion rates for AML and MDS were 94.7% and 100%, without significant difference from healthy controls (100%). In AML patients, the median antibody titers of patients in complete remission (CR) (816.5 [interquartile range (IQR): 250.0-2063.5] U/ml vs 1023.0 [640.0-1535.0] U/ml, P=0.668), especially those who were under treatment-free observation in CR (1630.0 [806.0-2454.0] U/ml vs 1023.0 [640.0-1535.0] U/ml, P=0.1220), were comparable to those in healthy controls. On the other hand, even in CR, the antibody titer in AML patients under maintenance therapy was significantly lower than that in patients under treatment-free observation (154.0 [126.0-289.0] U/ml vs 1630.0 [806.0-2454.0] U/ml, P=0.0003). Among the AML patients in CR, patients receiving maintenance treatment had a significantly lower median absolute lymphocyte count (0.81 [0.71-1.46] x 109/l vs 1.58 [1.29-1.93] x 109/l, P=0.0094) and a significantly lower median absolute neutrophil count (1.45 [1.15-1.64] x 109/l vs 3.45 [2.68-4.28] x 109/l, P<0.0001) than that in patients under treatment-free observation. Significantly lower antibody titers were associated with current active treatment (92.2 [37.5-216.3] U/ml vs 1630.0 [806.0-2454.0] U/ml, P<0.0001), AML with myelodysplasia-related changes (50.8 U/ml [39.9-109.1] vs 816.5 [283.0-1935.3] U/ml, P=0.0022), advanced age more than the median age of 68 years (195.0 [43.3-743.0] U/ml vs 1630.0 [806.0-3391.0] U/ml, P=0.0002), and vaccine subtypes of BNT162b2 (285.0 [127.8-1045.3] U/ml vs 3037.0 [2198.50-4537.0] U/ml, P=0.0002) in AML patients and with current active treatment (41.0 [10.7-227.5] U/ml vs 623.5 [173.8-1613.3] U/ml, P=0.0233), subtypes of excess blasts (11.1 [4.8-34.1] U/ml vs 212.0 [81.7-600.0] U/ml, P=0.0293), and high and very high risk of the revised international prognostic scoring system in MDS patients (9.0 [3.4-41.0] U/ml vs 169.0 [48.5-327.0] U/ml, P=0.0380). Image: Summary/Conclusion: This is one of the first studies on the effect of COVID-19 vaccines focusing on patients with AML and MDS, and there are many new findings. The response to COVID-19 vaccine appears to be related to disease and treatment status. Myeloid malignancies may have less impact than lymphoid malignancies on the vaccine response. AML patients under treatment-free observation in CR could be expected to have a vaccine effect that is comparable to that in healthy individuals. In contrast, since the response to vaccination might be insufficient in AML patients undergoing maintenance therapy, maintenance therapy should be continued with strict measures for prevention of infection even after vaccination. P560: ANALYSIS OF GENE MUTATION SPECTRUM AND CORRELATION BETWEEN GENETIC ABNORMALITIES IN 207 PATIANTS WITH T(8; 21) AML Y. Nie1,*, Y. Li1, H. Zhang1, H. Li1, J. Zhang1, C. Wang1, Z. Chen1, S. Yang1, Y. Lin1, K. Ru1 1Sino-us diagnostics, Tianjin, China Background: The core binding factor (CBF) involves a subgroup of AML which is composed of a and b subunits encoded by RUNX1(AML1) and CBFβ genes respectively, and CBF is responsible for regulating multiple hematopoietic genes of myeloid differentiation. AML with abnormal CBF accounts for 12%-15% of adult AML, including t(8;21)(q22;q22) and inv (16)(p13q22), which produce RUNX1-RUNX1T1 (also known as AML1-ETO) and CBFβ-MYH11 fusion gene respectively. Next generation sequencing(NGS) revealed that AML with t (8; 21) had other somatic gene mutations at the same time. In addition to the known genetic abnormalities of RAS and tyrosine kinase signaling pathways, the genes such as epigenetic regulation showed recurrent genetic abnormalities. Some of these gene abnormalities, such as KIT, JAK2 and FLT3-ITD mutations, have been reported in many studies and result in poor prognosis. However, due to the relatively small sample or detected genes size, there are some differences in the research conclusions. This study analyzed the molecular correlation test results of 207 patients with t (8; 21) AML, and conducted a comprehensive retrospective analysis by 56 gene targeted sequencing, so as to clarify the gene distribution and related pathways involved in t (8; 21) AML patients in Chinese population, as well as the correlation between different gene mutations and chromosome abnormalities. Aims: To evaluate the gene mutational profile and its correlation with chromosomal abnormalities in acute myeloid leukemia (AML) patients with t (8; 21). Methods: The data of 207 patients with t(8;21) AML was retrieved from Sino-us diagnostics lab from June 2015 to October 2021. We analyzed the gene mutational spectrum and characterized the pathways of 56 AML-associated genes by NGS technology. Results: Among these 207 patients with t(8;21) AML, the incidence of KIT(53%) gene mutation was the highest, followed by NRAS (18%), ASXL2 (17%), FLT3 (12%), and EZH2 (10%). Most mutations were enriched in signal transduction, chromatin modification, and transcriptional regulation pathways. According to their median VAF, we found that DNA methylation genes, such as IDH1(50.8%), IDH2(49.0%), TET2 (43.8%) and DNMT3A (37.2%) or Chromatin remodeling genes, like ASXL1 (42.1%), ASXL2 (37.3%) are present in the majority of the cells. Meanwhile, alterations in RAS/RTK signaling genes like FLT3 (23.1%), KIT (32.5%) and the RAS GTPases NRAS (8.6%) and KRAS (13.3%) have the lower VAF. In addition, most KIT mutations (121/154) were located in exon 17, mainly on the SNV with D816 or N822. According to the correlation analysis, we found that IDH1/2 and complex karyotype (CK) or ZBTB7A mutation, ASXL2 and CCND2 mutation, t (x; 8; 21) abnormality and JAK2 or FLT3 mutation may co-mutate. On the contrary, KIT and NRAS mutation were mutually exclusive. Image: Summary/Conclusion: From the patients with t(8;21) AML, most gene mutations occurred in pathways involving signal transduction, chromatin modification, or transcriptional regulation. DNA methylation or Chromatin remodeling genes may occur in the early stage of leukemogenesis while RAS/RTK signaling genes may indicated later events. The coexistence or mutual exclusion between genes and chromosomes may provide a basis for further definitive diagnosis and therapeutic regimen. P561: VIALE-T: A RANDOMIZED, OPEN-LABEL, PHASE 3 STUDY OF VENETOCLAX IN COMBINATION WITH AZACITIDINE AFTER ALLOGENEIC STEM CELL TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA C. Craddock1,*, U. Platzbecker2, M. Heuser3, V. Pullarkat4, S. Chaudhury5, D. Wu6, S. Addo7, B. Chyla7, Q. Jiang7, P. Lee7, J. E. Wolff7 1University of Birmingham, Birmingham, United Kingdom; 2University Hospital in Leipzig, Leipzig; 3Hannover Medical School, Hannover, Germany; 4City of Hope, Duarte, CA; 5Ann & Robert H. Lurie Children’s Hospital of Chicago, Northwestern Feinberg School of Medicine, Chicago, United States of America; 6The First Affiliated Hospital of Soochow University, Soochow, China; 7AbbVie Inc, North Chicago, United States of America Background: Acute myeloid leukemia (AML) is an aggressive malignancy and is the most common and second most common form of acute leukemia in adults and children, respectively. The combination of the highly selective BCL-2 inhibitor venetoclax and the hypomethylating agent azacitidine was shown to be safe and effective in clinical trials (DiNardo et al. Blood. 2019;133:7-17; DiNardo et al. N Engl J Med. 2020;383:617-629) and is approved by the United States Food and Drug Administration and European Medicines Agency for the treatment of patients with AML who are not eligible to receive intensive chemotherapy. Following allogeneic stem cell transplantation (alloSCT), most patients do not receive antileukemic therapy; however, an unmet need remains as disease relapse and graft-versus-host disease (GvHD) commonly occur posttransplant. In addition to the antileukemic effect of venetoclax shown in clinical studies, preclinical studies suggest venetoclax may mitigate the risk of GvHD. VIALE-T is a Phase 3, randomized, open-label trial in progress (NCT04161885) evaluating the safety and efficacy of venetoclax in combination with azacitidine versus best supportive care (BSC) as maintenance therapy following alloSCT in patients with AML. Aims: N/A Methods: This Phase 3 study consists of 2 parts (Figure). Key inclusion criteria include diagnosis of AML; plans to receive alloSCT or have received alloSCT within the past 30 days; bone marrow blasts <10% before pretransplant conditioning and <5% posttransplant; have received myeloablative, or reduced intensity, or nonmyeloablative pretransplant conditioning protocols. Grafts are allowed from various sources (bone marrow, peripheral blood stem cells, cord blood cells). Patients must be ≥18 years old for Part 1 and ≥12 years old for Part 2. Additionally, patients must meet key laboratory values for absolute neutrophil count (Part 1, ≥1500/µL; Part 2, ≥1000/µL), platelet count (Part 1, ≥80,000/µL; Part 2, ≥50,000/µL), bilirubin ≤3 times the upper limit of normal, and creatinine clearance >30 mL/min. Patients who have received venetoclax and had no history of disease progression while receiving venetoclax are eligible. Part 1 evaluates dose levels of venetoclax combined with azacitidine to determine the recommended Phase 3 dose (RP3D), which will be confirmed in approximately 12 additional patients enrolled in the Safety Expansion Cohort. Part 2 will be a randomized, open-label evaluation of the RP3D of venetoclax combined with azacitidine and BSC versus BSC only in adults and children aged 12 years or older. All venetoclax-treated patients will receive antibiotic prophylaxis during Cycle 1. The primary endpoint for Part 1 is the frequency of dose-limiting toxicities. The primary endpoint for Part 2 is relapse-free survival as assessed by an independent review committee. Key secondary endpoints for Part 2 include overall survival, GvHD-free relapse-free survival, and the rate of patients without higher grade GvHD at 90 days after randomization. Enrollment into the Safety Expansion Cohort will be completed in 2021. Part 2 will enroll approximately 400 patients across approximately 175 participating study sites in 17 countries, with recruitment beginning in 2022. Results: N/A Image: Summary/Conclusion: N/A P562: LEMZOPARLIMAB (LEMZO) WITH VENETOCLAX (VEN) AND/OR AZACITIDINE (AZA) IN PATIENTS (PTS) WITH ACUTE MYELOID LEUKEMIA (AML) OR MYELODYSPLASTIC SYNDROMES (MDS): A PHASE 1B DOSE ESCALATION STUDY J.-Z. Hou1,*, N. G. Daver2, D. A. Stevens3, T. Yamauchi4, Y. Moshe5, C. Y. Fong6, A. Marzocchetti7, R. Adamec8, M. Patel8, S. Lambert9, K. Y. Wu8, C. Röllig10 1UPMC Hillman Cancer Center, Pittsburgh, PA; 2Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX; 3Norton Cancer Institute, Louisville, KY, United States of America; 4University of Fukui Hospital, Fukui, Japan; 5Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 6Department of Clinical Haematology, Austin Health, Heidelberg, Victoria, Australia; 7AbbVie S.r.l., Rome, Italy; 8AbbVie Inc., North Chicago, IL; 9AbbVie Inc., South San Francisco, CA, United States of America; 10Universitätsklinikum Carl Gustav Carus an der TU Dresden, Dresden, Germany Background: Despite treatment advances, pts with AML or higher-risk MDS who are ineligible for standard intensive treatments still have poor survival, highlighting the need for novel treatments. Overexpression of CD47 is common in leukemic stem cells and AML blasts and correlates with poor clinical outcomes. Lemzo is an anti-CD47 antibody with red blood cell–sparing properties. Treatment with ven plus aza has shown favorable safety and efficacy in older/unfit pts with AML and higher-risk MDS. Blocking CD47 is hypothesized to hypersensitize AML cells to the antitumor activity of ven and aza. This study will evaluate the safety and dose-limiting toxicities (DLTs) of lemzo with ven + aza for pts with treatment-naïve AML, as well as lemzo with aza ± ven for treatment-naïve higher-risk MDS. Aims: N/A Methods: This phase 1b, open-label, dose-escalation study (NCT04912063) is enrolling adults with: (1) treatment-naïve AML with adverse cytogenetic/molecular risk not suitable for induction therapy, with an Eastern Cooperative Oncology Group Performance Status (ECOG-PS) 0–2 (aged ≥75 years) or 0–3 (aged ≥18–74 years) excluding acute promyelocytic leukemia; or (2) treatment-naïve higher-risk MDS (Revised International Prognostic Scoring Score >3) with <20% bone marrow blasts, ECOG-PS 0–2, and no immediately planned stem cell transplant. For each 28-day cycle, aza is administered subcutaneously or intravenously (IV) daily for 7 days within the first 9 days (7-0-0 or 5-2-2 schedule); ven is administered orally daily on days 1–28 (AML, following dose ramp-up) or days 1–14 (ven-containing MDS cohorts). Lemzo is administered IV at a schedule that is to be determined in this study. Dose escalation has Bayesian optimal interval design and may be expanded to investigate alternate dosing for lemzo. Dose expansion will initiate at recommended phase 2 dose. Treatment discontinuation criteria are unacceptable toxicity, progressive disease, lack of partial/complete remission or clinical benefit within 6 cycles, or at physician’s discretion. Pts who discontinue study treatment without progression will continue with posttreatment follow-up; pts who progress will enter survival follow-up. The primary endpoints are DLTs of lemzo. Secondary endpoints for both cohorts include best overall responses of complete remission, duration of response, event-free survival, and overall survival. Exploratory biomarker endpoints are included. Safety assessments include adverse event (AE, graded per National Cancer Institute Common Terminology Criteria for AEs v5.0) monitoring, physical examinations, vital signs, electrocardiograms, and laboratory tests. Response rates will be analyzed with estimates and 95% confidence intervals based on exact binomial distribution. Time-to-event endpoints will be analyzed using Kaplan–Meier methodology. Results: N/A Summary/Conclusion: N/A P563: A RANDOMIZED, DOUBLE-BLIND, 2-ARM, MULTICENTER, PH3 STUDY OF VENETOCLAX & ORAL AZACITIDINE VS. ORAL AZACITIDINE AS MAINTENANCE THERAPY FOR PTS WITH AML IN FIRST REMISSION AFTER INTENSIVE CHEMOTHERAPHY V. Ivanov1,*, S.-P. Yeh2, J. Mayer3, L. Saini4, A. Unal5, M. Boyiadzis6, D. M. Hoffman7, K. Kang7, S. N. Addo7, W. L. Mendes7, A. T. Fathi8 1Almazov National Medical Research Centre, Saint Petersburg, Russia; 2China Medical University Hospital, Taichung City, Taiwan; 3Fakultni Nemocnice Brno, Lískovec, Czechia; 4London Health Sciences Center, Ontario, Canada; 5Erciyes University Medical School, Kayseri, Turkey; 6Genentech Inc., South San Francisco, CA; 7AbbVie Inc, North Chicago, IL; 8Massachusetts General Hospital, Boston, MA, United States of America Background: Acute myeloid leukemia (AML) is an aggressive, heterogenous hematologic malignancy with poor prognosis. For eligible patients, standard treatment includes intensive chemotherapy during induction, followed by consolidative chemotherapy or stem cell transplantation. Despite current therapies, the prevention of relapse is still a major therapeutic challenge and an unmet need for patients in remission. Venetoclax (Ven) is a selective, potent, oral BCL-2 inhibitor that induces apoptosis in AML cells. Ven in combination with Azacitidine (Aza) leads to prolonged overall survival (OS) and rapid, durable remissions in treatment-naïve AML patients ineligible for intensive chemotherapy. The QUAZAR AML-001 (NCT01757535) trial showed that the median OS in patients receiving maintenance oral Aza was superior to patients receiving placebo following upfront induction and consolidation; this benefit was also observed for patients who achieved either complete remission (CR) or CR with incomplete blood count recovery (CRi). VIALE-M is a randomized, double-blind, two-part study (NCT04102020) to determine the recommended Phase 3 dose (RPTD) of Ven in combination with oral Aza (CC-486) that can be safely administered as maintenance therapy, and to evaluate the safety and efficacy of Ven in combination with oral Aza + BSC compared to placebo and oral Aza + BSC in patients with AML who have achieved CR or CRi after intensive induction and consolidation. Aims: N/A Methods: The study is enrolling patients aged ≥18 years with newly diagnosed AML per WHO 2016 classification, with confirmed CR or CRi following intensive induction and consolidation therapies, from > 200 sites worldwide. Patients must have achieved first CR or CRi (after induction) ≤120 days of first dose of study drug or ≤75 days past last dose of last consolidation cycle, have intermediate or poor risk cytogenetics per NCCN 2016 categorization, ECOG ≤2, and no history of acute promyelocytic leukemia or active central nervous system involvement with AML. For the dose finding part of the study, patients will receive Ven QD for up to 24 cycles and oral Aza QD on D1-14 of each 28-day cycle for up to 24 cycles to determine the RPTD. For safety expansion, patients will receive Ven QD for up to 24 cycles and oral Aza QD on D1-14 of each cycle for up to 24 cycles at the RPTD. For the randomization part, patients will be randomized 1:1 to receive placebo or Ven QD at the RTPD and oral Aza QD on D1-14 of each cycle for 24 cycles. In all parts, treatment may discontinue due to relapse/unacceptable toxicity, or continue beyond 24 cycles under investigator’s discretion. For the randomization portion, the primary endpoint is relapse-free survival; secondary outcomes include OS, minimal residual disease conversion, and improvement in quality of life. Results: N/A Summary/Conclusion: N/A P564: FINAL RESULTS OF THE QOLESS AZA-AMLE RANDOMIZED TRIAL TO EVALUATE THE EFFICACY OF 5-AZA FOR POST-REMISSION THERAPY OF ACUTE MYELOID LEUKEMIA IN ELDERLY PATIENTS E. N. Oliva1,*, P. Salutari2, F. Di Raimondo3, G. Reda4, D. Capelli5, G. Iannì6, G. Tripepi7, C. Alati1, C. Mammì8, M. G. D’Errigo8, P. Niscola9, C. Selleri10, P. Musto11, E. Vigna12, A. Volpe13, N. Cascavilla14, M. C. Cannatà8, D. Mannina15, A. Candoni16 1U.O.C. di Ematologia, Grande Ospedale Metropolitano Bianchi Melacrino Morelli, Reggio di Calabria; 2U.O. di Ematologia, Ospedale Civile di Pescara, Pescara; 3U.O.C. di Ematologia, Policlinico Catania, Catania; 4U.O.C. di Ematologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano; 5Clinica di Ematologia, Ospedali Riuniti di Ancona, Ancona; 6Dielnet SRL; 7Ifc-CNR; 8U.O.S.D. Genetica Medica, Grande Ospedale Metropolitano Bianchi Melacrino Morelli, Reggio di Calabria; 9U.O. di Ematologia, Ospedale Sant’Eugenio, Roma; 10Dipartimento di Oncoematologia, AOU San Giovanni di Dio e Ruggi d’Aragona, Salerno; 11Department of Emergency and Organ Transplantation, “Aldo Moro” University School of Medicine, and Unit of Hematology and Stem Cell Transplantation, AOUC Policlinico, Bari; 12U.O. di Ematologia, Ospedale L’Annunziata, Cosenza; 13U.O. di Ematologia, Azienda Ospedaliera San Giuseppe Moscato, Avellino; 14U.O. Ematologia, Ospedale Casa Sollievo della Sofferenza IRCCS, San Giovanni Rotondo; 15U.O.C. di Ematologia, Azienda Ospedaliera Papardo, Messina; 16Clinica Ematologica, Centro Trapianti e Terapie Cellulari, Azienda Sanitaria Universitaria Integrata di Udine, Udine, Italy Background: Elderly patients in complete remission (CR) after first-line therapy for acute myeloid leukemia (AML) relapse within months unless additional therapy is given. In elderly patients with AML in CR after intensive chemotherapy, the HOVON 97 study reported on the efficacy and safety of azacitidine (5-Aza) post-remission therapy and the randomized QUAZAR trial showed that CC-486 as maintenance therapy was associated with significantly longer overall and relapse-free survival than placebo. We report final results of the QoLESS AZA-AMLE Phase III, randomized, open label, Italian multicentre, trial to evaluate the efficacy of 5-Aza for post-remission therapy of elderly AML patients compared to best supportive care (BSC). Aims: The primary endpoint is disease-free survival (DFS, from CR to relapse/death at 2 and 5 years. Secondary endpoints are post-remission hospitalizations, overall survival (OS) at 2 and 5 years and changes in quality of life (QoL). Methods: Patients aged ≥ 61 years with untreated AML, “de novo” or evolving from MDS, fit for intensive chemotherapy but ineligible for stem cell transplantation received induction chemotherapy consisting of 2 courses of 3 + 7 (Daunorubicin 40 mg/m2 daily days 1-3 and cytarabine 100 mg/m2 intravenous infusion days 1-7. Cases in CR received cytarabine 800 mg/m2 3-hour infusion twice daily for 3 days. Patients in CR were randomized to receive BSC or 5-Aza 50 mg/m2 for 7 days every 28 days and dose increase after 1st cycle to 75 mg/m2 for further 5 cycles, followed by cycles every 56 days for 4.5 years. QoL was assessed with QOL-E version 3 and EORTC QLQ-C30 version 3.0. Results: 149 patients were enrolled to finally randomize 54 patients alive and in CR (Table 1). After randomization, no patients died before relapse, 7 cases completed the study while 43 subjects relapsed: at 2 years, 22 cases in BSC (median DFS: 6.0 months, 95% CI: 0.2–11.7) versus 18 in the 5-Aza arm (median DFS: 10.8 months, 95% CI: 1.9-19.6; p=0.20); at 5 years, 1 subject on BSC withdrew consent and 23 subjects relapsed (median DFS: 6.0 months, 95% CI: 0.2–11.7) while 2 subjects on 5-Aza withdrew consent, 1 discontinued for bladder cancer and 20 relapsed (median DFS: 10.8 months, 95% CI: 1.9-19.6; p=0.23). Age modified the effect of the allocation arm on DFS. Data adjustment for cytogenetic risk further amplified the efficacy of 5-Aza in patients aged >68 years, both at 2 (HR: 0.24, 95% CI: 0.08-0.69, P=0.008) and 5 years (HR: 0.28, 95% CI: 0.10-0.76, P=0.012). The effect of 5-Aza versus BSC tended to be statistically significant on DFS at 2 (P=0.053) and 5 years (P=0.068) in Cox models including cytogenetic risk and MRD as covariates. Kaplan-Meier analyses of the effect of 5-Aza on DFS (at 2 and 5 years) showed that patients aged >68 years and positive MRD tended to have a benefit in terms of DFS at both 2 and 5 years (P=0.056). In the 5-Aza arm, 5 subjects out of 27 (19%) were hospitalised whereas no patient was hospitalised in the BSC arm (P=0.023). QoL was similar in both arms after randomization. Twenty grade 3-4 adverse events, mainly neutropenia, occurred in 5-Aza versus 1 in BSC arm (p=0.002). Image: Summary/Conclusion: 5-Aza is well tolerated in elderly patients with AML who have achieved CR following standard chemotherapy. MRD remains an important predictor of DFS in the long-term, independent of 5-Aza maintenance. No statistically significant differences were observed between the two arms in terms of DFS, but after data adjustment it is suggested that patients aged >68 years and positive MRD may benefit from 5-Aza maintenance. P565: IMPROVED OUTCOME OF PATIENS WITH ACUTE MYELOID LEUKEMIA HARBORING FLT3 MUTATION IN THE ERA OF TARGETED THERAPY G. Oñate1,2,*, M. Pratcorona1, A. Garrido1, A. Artigas1, A. Bataller3, M. Tormo4, M. Arnan5, S. Vives6, R. Coll7, O. Salamero8, F. Vall-Llovera9, A. Sampol10, A. Garcia11, M. Cervera12, S. Garcia Avila13, J. Bargay14, X. Ortin15, J. Esteve3, J. Sierra1 1Hematology, Hospital de la Santa Creu i Sant Pau; 2Universitat Autònoma de Barcelona; 3Hematology, Hospital Clínic, Barcelona; 4Hematology, Hospital Clínico Universitario, Biomedical Research Institute INCLIVA, Valencia; 5Hematology, Institut Català d’Oncologia, Hospital Duran i Reynals, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, Hospitalet de Llobregat; 6Hematology, ICO-Hospital Germans Trias i Pujol, Badalona; 7Hematology, ICO-Hospital Josep Trueta, Girona; 8Hematology, Hospital Universitari Vall d’Hebron, VHIO; 9Hematology, Hospital Universitari Mútua Terrassa, Barcelona; 10Hematology, Hospital Son Espases, Palma de Mallorca; 11Hospital Universitari Arnau de Vilanova, Lleida; 12ICO-Hospital Joan XXIII, Tarragona; 13Hematology, Hospital del Mar, Barcelona; 14Hospital Son Llàtzer, Palma de Mallorca; 15Hematology, Hospital Verge de la Cinta, Tortosa, Spain Background: In recent years, the treatment of AML is rapidly evolving due to the advances in targeted therapy, risk-adapted protocols and MRD-guided decisions. The prognosis of patients harbouring FLT3-ITD differs according to its allelic ratio and the presence of NPM1 co-mutation (NPM1mut). Nonetheless, FLT3-ITD prognostic impact and allogeneic hematopoietic cell transplantation (alloHCT) indication in this setting might be redefined by the widespread use of FLT3 inhibitors. Since 2012, chemotherapy eligible patients are treated following a risk-adapted and MRD-guided protocol in the CETLAM group (AML-12). Since 2016, targeted therapy with midostaurin was progressively incorporated in the protocol for FLT3-mutated (FLT3mut) patients. Aims: To analyse the outcome of FLT3mut AML treated with a risk-adapted protocol in two different time periods, mainly differentiated by the advent of midostaurin. Methods: All adult patients with de novo AML and available FLT3 status treated with the intensive protocol AML-12 (clinicaltrials.gov #NCT04687098) in the Spanish CETLAM centres from 2012 to 2020 were retrospectively analysed. Molecular testing of NPM1, and FLT3-ITD and its allelic ratio were studied as previously described. Patients were divided into two study cohorts: the early (2012-2015) and the late cohort (2016-2020). All patients received the same chemotherapy protocol except for the addition of midostaurin since 2016 in most FLT3mut patients. Complete remission (CR), overall survival (OS), event-free survival (EFS) and risk of relapse (RR) followed ELN criteria. OS and EFS were studied with the Kaplan-Meier method and log-rank test, cumulative incidence and Grey test were used to estimate relapse risk (CIR) and non-relapse mortality (NRM). Results: A total of 906 patients were selected, 390 from the early and 516 from the late cohort. Median follow-up was 38 months; FLT3mut was present in 227 cases (25%) and constitute the focus of this study. There were not differences on main variables between cohorts (age, cytogenetic risk, ELN category, frequency of NPM1 mutation or FLT3-ITD allelic ratio). TKD mutations were only available since 2015. Midostaurin was administered to 62% of FLT3mut patients from the late cohort: 63 with ITDs (20 low and 43 high ratio) and 20 TKDs. CR rates were similar between cohorts. AlloHCT in first CR (CR1) was performed in 60% (early) and 67% (late) patients (p=ns). A higher RR was observed in the early group in comparison to the late cohort (2-year RR 42±11% vs 28±10%; p=0.014), without differences in NRM (Figure 1B), translating into an improved EFS (median EFS 9.4 vs. 26 months, p=0.014) and OS in the late cohort (median OS: 15 months vs. non-reached, p=ns, Figure 1A). Among NPM1mut AML patients, FLT3-ITD and ELN 2017 categories retained its prognostic value in the early cohort, with a worse OS, EFS, and higher RR among FLT3-ITD vs wt patients (HR for OS 1.9 95% CI 1.15-3.2, p=.011; figure 1C) and FLT3mut ELN-intermediate vs favourable patients (OS p<0.001; figure 1D). On the contrary, in the late cohort, FLT3-ITD and FLT3 high allelic ratio lost their adverse prognostic impact, with comparable RR, EFS, and OS regardless of FLT3-ITD status (Figure 1E and F). Image: Summary/Conclusion: This real-life study demonstrated an improved outcome among FLT3mut AML patients treated in the most recent period, probably reflecting the beneficial effect of midostaurin on relapse prevention in this context. Future analyses should revise the role of alloHCT in CR1 for non-favourable FLT3mut AML patients treated with FLT3 inhibitors who achieve MRD eradication. P566: IMPACT OF FRONTLINE INDUCTION APPROACH ON POST-STEM CELL TRANSPLANT (SCT) OUTCOMES IN OLDER ADULTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA (AML) F. Ong1,*, F. Ravandi1, U. Popat2, T. M. Kadia1, N. Daver1, C. DiNardo1, M. Konopleva1, G. Borthakur1, E. J. Shpall2, B. Oran2, G. Al-Atrash2, R. Mehta2, E. J. Jabbour1, M. Yilmaz1, G. C. Issa1, G. Garcia-Manero1, A. Maiti1, H. A. Abbas1, R. E. Champlin1, N. J. Short1 1Leukemia; 2Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, HOUSTON, United States of America Background: A hypomethylating agent plus venetoclax (VEN) is standard of care for older, unfit patients (pts) with newly diagnosed AML. However, the optimal induction regimen for older pts with AML who are adequately fit for SCT is not well-established. Aims: We investigated the post-SCT outcomes of older adult pts with newly diagnosed AML based on the intensity of the induction regimen received. Methods: This is a retrospective analysis of pts ≥60 years (yr) of age with newly diagnosed AML who received allogeneic SCT after achieving first remission (CR, CRi or MLFS) between 9/2012 and 7/2021 at our cancer center. Out of 127 pts, 44 pts received intensive chemotherapy (IC) without VEN as induction therapy, 36 pts received low-intensity therapy (LIT) without VEN, and 47 pts received LIT with VEN. Overall survival (OS), relapse-free survival (RFS), cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) after SCT were compared among the groups. Results: The baseline characteristics of the groups are shown in Table 1. Pts in the IC cohort was significantly younger (median age of 63 yrs) than pts who received LIT with or without VEN (median age of 68 yrs, p<0.01). Pts in the IC group were more likely to have ECOG PS of 0 (34%) as compared to those who received LIT (14%, p=0.07). Most pts had adverse ELN cytomolecular risk on presentation (43% in IC, 50% in LIT without VEN, and 55% in LIT with VEN). The median numbers of cycles prior to SCT were 3 in all groups. Pts in LIT without VEN group were more likely to receive matched unrelated donor (75%) than those in the LIT with VEN (49%) and IC groups (50%) and were less likely to receive a haploidentical SCT (3% vs. 17% and 14%, respectively). Pts in LIT with VEN group were more likely to receive reduced-intensity conditioning (92%) prior to SCT, compared with 53% and 58% in the IC and LIT without VEN groups, respectively (p<0.01). HLA matching was well-balanced in 3 groups. The majority of pts were in CR/CRi prior to SCT (100% in the IC group, 95% in the LIT without VEN group, and 92% in the LIT with VEN group), while the rest of pts achieved MLFS. MRD negativity prior to SCT was achieved in more pts treated with IC (77%) or LIT with VEN (66%), compared with LIT without VEN (49%). For the entire cohort, the median follow-up was 37 months (mos) post-SCT. Post-SCT outcomes are shown in Figure 1. The 2-yr OS was highest in the LIT with VEN group (73%) when compared with the IC or LIT without VEN groups (58% and 44%, respectively); OS was statistically superior with LIT with VEN compared with LIT without Ven (p=0.02) and there was a trend towards superior OS compared with IC (p=0.17). 2-yr RFS was also superior in the LIT with VEN group (62%) as compared with the IC or LIT without VEN groups (54% and 42%, respectively). 2-yr CIR was highest in LIT without VEN group (36%), and was similar in the LIT with VEN (19%) and IC groups (18%). 2-yr NRM was highest in IC group (27%) as compared with the LIT with VEN (11%) and LIT without VEN groups (22%) (p=0.02 for IC vs. LIT with VEN). Image: Summary/Conclusion: LIT with VEN resulted in comparable—and possibly superior—outcomes compared with IC in SCT eligible patients ≥60 yrs with newly diagnosed AML. Despite the older age and worse PS of the LIT with VEN group, their post-SCT NRM was lower than those who received IC, which translated to numerically superior OS. These results support LIT with VEN as an effective and tolerable induction strategy in older SCT-eligible pts with newly diagnosed AML. P567: CENTRAL NERVOUS SYSTEM INVOLVEMENT IN ADULT PATIENTS WITH ACUTE MYELOID LEUKEMIA – INCIDENCE AND OUTCOME M. Virijevic1,2,*, I. Djunic1,2, M. Mitrovic1,2, N. Pantic1, Z. Pravdic1, N. Sabljic1, A. Novkovic3, M. Cvetovic2, J. Rajic1, A. Vidovic1,2, M. Todorovic-Balint1,2, N. Suvajdzic-Vukovic1,2 1Clinic of Hematology, University Clinical Center of Serbia; 2Faculty of Medicine, University of Belgrade; 3Department of Hematology, Clinical Hospital Center “Zemun”, Belgrade, Serbia Background: Examination of central nervous system (CNS) involvement is not routine diagnostic in adult patients with acute myeloid leukemia (AML). Therefore, its impact on the disease course is not well defined. There are studies showing that CNS involvement in AML is associated with a worse outcome, whereas others did not confirm such impact on long-term survival. Aims: To determine the incidence of CNS involvement, its impact on the disease free and overall survival, and to define risk factors for CNS involvement. Methods: This single center, retrospective study involved 645 adult patients with nonpromyelocytic AML, diagnosed in period of 2013. and 2021. In patients with the presence of CNS symptoms, with increased leukocyte count (≥30x109/L), FLT3 mutation, monocyte phenotype, CD56 positivity, a lumbar puncture was performed. All cerebrospinal fluid (CSF) samples were examined by flow cytometry (FCM). Treatment of CNS+ include intrathecal CT: cytarabine 100 mg. Assessment of remission of CNS+ was performed after 8 i.t. CT. The following parameters were estimated as risk factors for CNS+ at diagnosis of AML: age, WBC (<30x109/L vs. ≥30x109/L), ECOG PS, HCT CI score, elevated lactate dehydrogenase (LDH), leukemia-related parameters (cytogenetics (including ELN risk stratification), flow cytometry). The methods of descriptive and analytical statistics were used. Univariate and multivariate Cox Proportional Hazards models were used to identify risk factors. Results: CNS involvement examination was performed in 272 patietns. A total of 55/272 (20.2%) patients had proven CNS involvement, while 217/272 (79.8%) were without neuroleukemia. Complete remission was achieved by 168/272 (61.7%) of patients, but there was no statistically significant difference between CNS positive and CNS negative patients (p=0.619). Patients with CNS involvement at diagnosis had a significantly higher frequency of hyperleukocytosis (≥30x109/L), age ≤50 years, French–American–British M5 subtype, expression of CD56 and CD15 antigen. Multivariate analysis identified CD56+ as the most important risk factor for CNS+ at diagnosis in AML patients: p=0.003, HR 2.271; 95% confidental interval (CI)= 1.320-3.908. There were no statistically significant differences in 5-year overall survival and disease free survival between CNS positive and CNS negative patients. Summary/Conclusion: Our study showed a high incidence of CNS involvement in AML patients, compared to studies in which the incidence was significantly less (3.3%). Our study confirmed the accuracy of the factor we used to assess CNS involvement. There was no difference in the outcome between patients with CNS involvement and those without. P568: REAL-LIFE EXPERIENCE OF TREATMENT REFRACTORY/RELAPSED ACUTE MYELOID LEUKEMIA PATIENTS WITH VENETOCLAX COMBINATION THERAPY H. Pomares Marin1,*, J. Villarreal Hernández2, M. Condom Esteve2, S. Vives Polo3, M. Cervera Calvo4, R. Coll Jorda5, C. Maluquer Artigal1, G. Ibarra Fernández3, A. Torrent Catarineu3, M. Galiano Barajas1, A. M. Sureda Balari1, M. Arnan Sangerman1 1Servei d’Hematologia, Institut Català d’Oncologia, Hospital Duran i Reynals, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat; 2Servei d’Hematologia, Institut Català d’Oncologia, Hospital Duran i Reynals, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat; 3Servei d’Hematologia, Institut Català d’Oncologia, Hospital Germans Trias i Pujol, Institut de Recerca contra la Leucèmia Josep Carreras, Universitat Autònoma de Barcelona, Badalona; 4Servei d’Hematologia, Institut Català d’Oncologia, Hospital Universitari Joan XXIII, Tarragona; 5Servei d’Hematologia, Institut Català d’Oncologia, Institut d’Investigació Biomèdica de Girona (IDIBGI), Universitat de Girona, Girona, Spain Background: Venetoclax, a BCL-2 specific inhibitor, used in combination with azacitidine in patients with newly-diagnosed acute myeloid leukemia (AML) who were ineligible for intensive chemotherapy treatment, has shown high response rate and overall survival compared with azacitidine monotherapy (Di Nardo et al. N Engl J Med 2020; 383:617-629). Moreover, Venetoclax in combination with hypomethylating agents or with low-dose cytarabine is being explored in other settings being frequently used in relapsed/refractory (R/R) AML. Aims: We performed a retrospective study of patients with R/R AML receiving venetoclax combinations in the Catalan Institute of Oncology (ICO) in order to determine the efficacy and safety of the combination. Methods: We analyze 60 patients diagnosed with R/R AML at 4 hospitals belonging to ICO in Spain, treated with venetoclax (400mg/24h; initial daily dose of 100mg with a 3-day ramp-up to target dose of 400mg) in combination with hypomethylating agents (Azacitidine 75mg/m2 7/28 days or Decitabine 20mg/m2 5/28 days) or low-dose cytarabine (20mg/m2 10/28 days) from May 2019 until December 2021. Event was defined as death, refractoriness to treatment or progressive disease. Results: Characteristics of our cohort are described in Table 1. Notably, 31 (52%) patients had high-risk AML according to ELN 2017 classification. Fourteen (23%) patients received venetoclax in combination with decitabine, 34 (57%) patients with azacitidine, and 12 (20%) patients with low-dose cytarabine. The median number of cycles received was 3 (range 1-28), with a median of one cycle to achieve the best response (range: 1-4). Early mortality in the first 30 days was 15% (9 patients), 5 due to progression disease, 2 due to infection and 2 due to clinical worsening. Overall response rate after first cycle (Complete response (CR) + complete response without haematological recovery (CRi) + partial response (PR)) was 58%. Three patients were treated in a molecular-MRD positive status, and all of them achieved molecular response. Six of 19 patients (32%) were transitioned to allogeneic stem cell transplantation (alloSCT). The median event-free survival and overall survival was 5.03 months and 7.2 months, respectively. Response to treatment after 3-4 cycles, discriminate two groups of patients with an OS of 12.66 months in those patients who achieved CR or PR vs 2.4 months in non-responders (p0.000). Patients relapsed after alloSCT (7 patients) presented a poor outcome (median OS was 1.6 months). Image: Summary/Conclusion: Our study showed that real-world experience of treating patients with R/R AML with venetoclax in combination with hypomethylating agents or low-dose cytarabine is feasible as salvage treatment in patients relapsed to hypomethylating agent and as bridge therapy to alloSCT with a rapid response rate. Moreover, rapid responses shown with the combination, allow us to identificate those patients who may benefit from this approach. P569: GILTERITINIB AND QUIZARTINIB IN RELAPSED/REFRACTORY (R/R) ACUTE MYELOBLASTIC LEUKEMIA (AML) WITH FLT3 MUTATIONS: A REAL-LIFE EFFECTIVENESS AND SAFETY STUDY D. Quintela1,*, M. Morgades1, A. Serrano2, M. Cervera3, A. Balerdi4, M. Díaz-Beyá5, M. Arnan6, A. Garrido7, R. Coll8, M. Tormo9, J. López-Marin10, B. Merchan11, S. Garcia11, M. Casado12, A. Sampol13, J. Esteve5, D. Martínez-Cuadrón2, J. Sierra7, M. Á. Sanz2, J. M. Ribera1, P. Montesinos2, S. Vives1 1Clinical Hematology, ICO-Hospital Universitari Germans Trias i Pujol, Institut de Recerca Josep Carreras, Badalona; 2Clinical Hematology, Hospital Universitari i Politècnic La Fe, Valencia; 3Clinical Hematology, ICO-Hospital Joan XXIII, Tarragona; 4Clinical Hematology, Hospital Universitario Cruces, Barakaldo; 5Clinical Hematology, Hospital Clínic de Barcelona, Barcelona; 6Clinical Hematology, ICO-Hospital Duran i Reynals, L’Hospitalet del Llobregat; 7Clinical Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona; 8Clinical Hematology, ICO-Hospital Josep Trueta, Girona; 9Clinical Hematology, Hospital Clínico Universitario de Valencia, Valencia; 10Clinical Hematology, Hospital General Universitario de Alicante, Alicante; 11Clinical Hematology, Hospital del Mar, Barcelona; 12Clinical Hematology, Hospital Universitario de Badajoz, Badajoz; 13Clinical Hematology, Hospital Universitari Son Espases, Palma, Spain Background: Patients with R/R AML have poor prognosis. FLT3 mutations worsened the prognosis but offer a targeted therapy. FLT3 inhibitors have demonstrated efficacy as monotherapy in FLT3-positive R/R AML. Aims: The objective of the present study was to analyze the efficacy and safety of Gilteritinib and Quizartinib in R/R FLT3-mutated AML in real-life context. Methods: Between December 2016 and April 2021, 22 patients were treated with Gilteritinib and 5 patients with Quizartinib in 13 centers of the Spanish PETHEMA and CETLAM groups. Results: The median age was 62 years (range 25; 81) and 56% were women. Most patients presented ITD FLT3 (80%), 56% were refractory and 44% were relapsed (CR1 duration ≥6 months in 60%). The 1st line therapy was intensive chemotherapy (78%, 38% with midostaurin) or HMA (22%). A total of 13/27 patients (48%) had previously received a FLT3 inhibitor (midostaurin=6, sorafenib=2, quizartinib=2, midostaurin + crenolanib vs placebo=2 and midostaurin + quizartinib=1). Seventy percent of the patients received >1 line of treatment prior to Gilteritinib/Quizartinib (the most frequent was FLAG-IDA). Compared with diagnosis, patients in R/R had poorer functional status (ECOG<2 88% vs. 73%) but lower leukocyte counts, LDH level and FLT3 ITD ratio (21.5 x109/L vs 4.7x109/L, 563 U/L vs 330 U/L and 0.60 vs 0.42, respectively). Three patients displayed clonal evolution in R/R with acquisition of cytogenetic alterations in patients with altered karyotype at diagnosis and appearance of alterations in patients with normal karyotype. The ORR was 63% (21% CR, 21% CRi and 21% PR) and the median OS was 5.8 (95%CI, 3.8; 7.9) months. Time to achieve better response was 1.8 months (range 0.9;4.1). Eight patients received the FLT3 inhibitor as a bridge to SCT, and six of them were finally transplanted. Gilteritinib dose was 120mg/day, increasing to 200mg/d in 41% of patients (due to lack of response after 28 days). Toxicity was observed in 69% of patients (g3/4 in 30%). Febrile neutropenia was the most frequent toxicity (35%) followed by hepatotoxicity (28%) and QTc prolongation (16%). One patient died of toxicity attributed to Gilteritinib (febrile neutropenia). Summary/Conclusion: Treatment with Gilteritinib and Quizartinib as monotherapy is an effective and tolerable option for patients with R/R FLT3-mutated AML in real-life, with similar response rates and toxicity to those reported in Phase 3 trials, despite the fact that the study population from our series was more heterogeneous. P570: REAL-WORLD EFFICACY OUTCOMES OF VENETOCLAX PLUS AZACITIDINE VS INTENSIVE CHEMOTHERAPY FOR INDUCTION THERAPY IN ADULT PATIENTS WITH ACUTE MYELOID LEUKEMIA A. M. Zeidan1,*, D. A. Pollyea2, U. Borate3, A. Vasconcelos4, R. Potluri5, D. Rotter5, Z. Kiendrebeogo5, L. Gaugler4, G. Bonifacio4, T. Prebet4, C. Chen4 1Yale University School of Medicine, New Haven, CT; 2University of Colorado School of Medicine, Aurora, CO; 3Oregon Health & Science University, Portland, OR; 4Bristol Myers Squibb, Princeton, NJ; 5SmartAnalyst Inc., New York, NY, United States of America Background: Intensive chemotherapy (IC) is commonly used to achieve remission in patients with newly diagnosed acute myeloid leukemia (AML), although the relapse rate remains high. The combination of venetoclax, a BCL2 inhibitor, plus intravenous or subcutaneous azacitidine (ie, VEN-AZA), is FDA- and EMA-approved for the treatment of patients with newly diagnosed AML aged ≥ 75 years or with comorbidities that preclude the use of IC. Because of the challenging induction and maintenance decisions for some patients, it is important to understand the real-world patterns of use and outcomes of VEN-AZA vs IC. Aims: To compare US real-world outcomes for the use of VEN-AZA vs IC, particularly in older populations. Methods: This retrospective analysis was performed on a cohort of patients from the US-based Flatiron Health electronic health record-derived de-identified database, who had newly diagnosed AML and received induction therapy with VEN-AZA or IC between 21 Nov 2018 and 01 Jun 2021. Using an intention-to-treat analysis (including patients with less than 2 months of follow-up), patient groups were propensity score-matched in a 1:1 ratio using nearest neighbor matching with calipers set to 0.01 to reduce bias. Matched-patient assessments included morphological complete remission, overall survival (OS; defined as from first day of therapy to death), and relapse-free survival (RFS; from first day of therapy to relapse or death). Univariate analyses were used to investigate possible treatment-associated OS and RFS benefit in patient subgroups, including those with high-risk mutations. Results: Of 7484 patients in the Flatiron database with newly diagnosed AML, 1188 met the selection criteria and 436 were propensity score-matched resulting in 218 per treatment group. In the matched VEN-AZA vs IC cohorts, patients had a mean age of 69.9 vs 69.0 y and Charlson Comorbidity Index of 0.5 vs 0.7, respectively; 39% vs 38% were female. The rates of patients with complete remission (CR) and those with stem cell transplant (SCT) were significantly lower with VEN-AZA vs IC at 46% vs 62% (P=0.001) and 16% vs 31% (P<0.001), respectively. Fewer VEN-AZA– vs IC-treated patients received an early posttreatment bone marrow biopsy, with respective rates of 31% vs 69% (1 mo) and 57% vs 86% (2 mo). Median OS for VEN-AZA vs IC in the matched population was 13.0 vs 17.2 mo (P=0.263), respectively; it was 11.0 vs 15.6 mo (P=0.410) with censoring for SCT. Median RFS was numerically shorter for VEN-AZA than for IC (9.5 vs 11.0 mo; P=0.689); when including censoring for SCT, RFS was longer for VEN-AZA (7.5 vs 5.7 mo; P=0.027). Among subgroups, including those with high-risk mutations, no significant factor was found to be associated with OS in the matched patients (Figure). The frequency of known patients with high-risk mutations in RUNX1, ASXL1, and TP53 was similar for both treatment groups. Similar subgroup results were observed for RFS. Image: Summary/Conclusion: Rates of CR and SCT were significantly higher with IC. OS was nominally longer with IC vs VEN-AZA, with and without censoring for SCT; differences were not statistically significant. None of the subgroups tested, including patients with high-risk mutations, showed a significant OS association. Longer follow-up time may contribute to better characterized outcomes achieved with IC vs VEN-AZA in routine clinical practice. These data support more investigation on strategies to improve survival outcomes, including the impact of maintenance therapy. P571: ORAL AZACITIDINE VS MIDOSTAURIN AS MAINTENANCE TREATMENT FOR FLT3 MUTANT ACUTE MYELOID LEUKEMIA IN COMPLETE REMISSION: AN INDIRECT TREATMENT COMPARISON E. N. Oliva1,*, C. Chen2, S. N. Rahim2, B. Seuro3, A. Vasconcelos2, L. Gaugler2, B. Skikne2, T. Prebet2, H. Cameron3, C. Beach2, G. Thompson2 1Grande Ospedale Metropolitano Bianchi Melacrino Morelli, Reggio Calabria, Italy; 2Bristol Myers Squibb, Princeton, NJ; 3Eversana, San Diego, CA, United States of America Background: Patients (pts) with FLT3-mutant (mut) acute myeloid leukemia (AML) have a high risk of relapse, poor prognosis, and limited treatment (tx) options. A trend toward improved survival with oral azacitidine (Oral-AZA) vs placebo (PBO) as maintenance treatment (MT) was observed in pts in first complete remission (CR) after intensive chemotherapy (IC) induction ± consolidation in an exploratory analysis of the phase 3 QUAZAR AML-001 study (QUAZAR; NCT01757535). Midostaurin (MIDO) plus chemotherapy was FDA-approved as induction and consolidation tx for pts with FLT3-mut AML based on results of the phase 3 RATIFY study (NCT00651261); absence of randomization prior to MT makes inferences about the effectiveness of MIDO as MT difficult, although MIDO was EMA-approved for MT. The relative efficacy of MT with Oral-AZA vs MIDO has not been compared directly in clinical trials. Aims: To compare the survival outcomes of MT with Oral-AZA vs MIDO in pts with FLT3-mut AML who achieved first CR after IC (± consolidation tx) by performing an indirect tx comparison (ITC) of data from QUAZAR and RATIFY. Methods: An ITC of overall survival (OS) and relapse-free survival (RFS) comparing Oral-AZA vs MIDO was performed, in which data from QUAZAR pts with FLT3-mut AML who had achieved a CR with full blood count recovery (matching RATIFY eligibility criteria) were compared with efficacy outcomes from the RATIFY MT phase (Larson RA, et al. Leukemia 2021). Primary analyses utilized the anchored Bucher method, which assumes the relative tx effect is comparable across studies with similar effect modifiers. Time-varying methods (parametric and spline models) were also explored to account for violation of the proportional hazards (PH) assumption. Results: Of 472 pts in the intent-to-treat population of QUAZAR, 56 pts with FLT3-mut AML achieving a CR (Oral-AZA, 24; PBO, 32) were included in the primary analysis population, as were all 205 RATIFY MT-phase pts (MIDO, 120; PBO, 85). In the QUAZAR and RATIFY populations, respectively, mean age was 66.4 and 42.7 y, 55% and 50% were female, 36% and 29% had point mutations in the FLT3 tyrosine kinase domain, 0% and 57% had favorable cytogenetic risk, and 0% and 5% underwent stem cell transplantation during first CR. Bucher ITCs of both OS and RFS appeared to favor Oral-AZA over MIDO, but were not statistically significant (Table). To assess age imbalance (QUAZAR pts were ≥ 55 y; RATIFY pts, 18–59 y), an exploratory analysis with QUAZAR pts (n = 9; age-matched to RATIFY pts) showed OS and RFS results with Oral-AZA vs PBO were similar to those of all QUAZAR pts. Evidence suggests the PH assumption had been violated in some of these analyses, so best fitting parametric models were explored. Landmark OS and RFS rates (generalized gamma model) were found to be higher with Oral-AZA than MIDO (Table). Similar results were obtained using a spline model. Image: Summary/Conclusion: Despite more favorable prognostic factors among pts in RATIFY (eg, younger age), the Bucher ITC analysis showed a trend toward longer OS and RFS with Oral-AZA vs MIDO as MT for pts with FLT3-mut AML. Analyses that accounted for the PH assumption violation also indicated a survival benefit for Oral-AZA vs MIDO. Limitations included trial differences and small sample size. More research is needed to support this observation. P572: VENETOCLAX AND HYPOMETHYLATING AGENT COMBINATIONS FOR THE TREATMENT OF ADVANCED MYELOPROLIFERATIVE NEOPLASMS AND ACUTE MYELOID LEUKEMIA WITH EXTRAMEDULLARY DISEASE K. Sanber1,*, K. Ye2, H.-L. Tsai1, M. Newman3, A. Ambinder1, A. DeZern1, T. Jain1 1Department of Oncology; 2School of Medicine; 3Department of Pharmacy, Johns Hopkins University, Baltimore, United States of America Background: Progression of myeloproliferative neoplasms (MPN) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), into accelerated phase (AP) or blast phase (BP) disease is associated with poor prognosis and decreased responsiveness to intensive chemotherapy regimens that are typically used in young and fit patients with acute myeloid leukemia (AML). Although the combination of a hypomethylating agent (HMA) and venetoclax (HMA/venetoclax) is being increasingly utilized to treat patients with advanced MPN or MDS/MPN as well as AML with extramedullary disease (EMD), outcomes in these subgroups of patients is not well characterized as they were largely excluded from the pivotal VIALE-A trial. Aims: We sought to evaluate the outcomes associated with HMA/venetoclax in disease groups that were not well-represented in the pivotal phase III VIALE-A trial. Methods: We performed an IRB-approved, retrospective chart review of patients who received HMA and venetoclax (for at least 14 days) between 1/1/2016 and 5/1/2021 at Johns Hopkins University. Composite complete response included patients who attained a complete molecular response (CMR), complete cytogenetic response (CCR) or acute leukemia response-complete (ALR-C) for patients with advanced MPN or MDS/MPN based on consensus guidelines (Mascarenhas et al., 2012), as well as complete response (CR) and CR with incomplete hematologic recovery (CRi) for patients with AML and EMD based on the ELN 2017 criteria (Dohner et al., 2017). Results: The composite CR rate and median overall survival (OS) were: 42.9% and 9.7 months for the overall population (n=35), 42.9% and 10.2 months for the BP cohort (n=21), 60% and 9.0 months for the AP cohort (n=5), 33.3% and 8.1 months for the AML with EMD (n=9). A summary of patient outcomes and their mutational landscape is represented in Figure 1. Five out of 15 patients who achieved a composite CR went on to receive an allogeneic bone marrow transplant. Patients with advanced MDS/MPN had numerically higher composite CR rate (70.0% versus 31.3%; P= 0.1054) and median OS (11.9 months versus 5.1 months; P= 0.1585) compared to patients with advanced MPN. Patients with a mutation in the SRSF2 gene had a higher composite CR rate (80% versus 20%; p= 0.0082) and median OS (10.9 months versus 8.0 months; P= 0.2614) compared to those with a wild-type SRSF2 gene. On the other hand, there was a trend towards lower composite CR in AML/blast phase patients who had EMD compared to those without EMD (33% vs 67%; P=0.1414). Image: Summary/Conclusion: The composite CR rate (42.9%) and median OS (9.7 months) for the overall patient population in our study are lower than those reported in the VIALE-A trial (42.9% versus 66.4% and 9.7 months versus 14.7 months, respectively). This is not unexpected since advanced MPN and extramedullary AML are known to have a worse prognosis than de novo AML. While limited by a small sample size, our study suggests that HMA/venetoclax may be associated with more favorable outcomes in patients with advanced MDS/MPN compared to those with advanced MPN. Mutations in the SRSF2 gene may also be associated with more favorable outcomes. These findings will need to be confirmed in larger studies and their biological basis will need to be further investigated. P573: PHARMACOKINETIC EXPOSURE EQUIVALENCE AND PRELIMINARY EFFICACY AND SAFETY FROM A RANDOMIZED CROSS OVER PHASE 3 STUDY OF AN ORAL HYPOMETHYLATING AGENT DEC-C COMPARED TO IV DECITABINE IN AML PATIENTS K. Geissler1, Z. Koristek2, T. Bernal del Castillo3, J. Novák4, G. Rodriguez Macias5, S. K. Metzelder6, A. Illes7, A. Nagy8, J. Mayer9, M. Arnan10, M.-M. Keating11, J. Krauter12, M. Lunghi13, N. Stefano Fracchiolla14, U. Platzbecker15, V. Santini16, Y. Sano17,*, A. Oganesian17, H. Keer17, M. Lübbert18 1Clinic Hietzing, Vienna, Austria; 2University Hospital Ostrava, Ostrava, Czechia; 3Hospital Universitario Central de Asturias, Oviedo, Spain; 4Department of Haematology, 3rd Faculty of Medicine, Charles University and Faculty Hospital Kralovske Vinohrady, Prague, Czechia; 5Hospital General Universitario Gregorio Marañón, Madrid, Spain; 6Philipps-Universität Marburg, Marburg, Germany; 7Division of Haematologoy, Department of Internal Medicine, Faculty of Medicine, University of Debrecen, Debreccen; 81st Department of Internal Medicine, University of Pécs, Pécs, Hungary; 9Fakultní Nemocnice, Brno, Czechia; 10Hematology Department, Servei d’Hematologia, Institut Català d’Oncologia, Hospital Duran i Reynals, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain; 11Queen Elizabeth II (QEII) Health Sciences Centre, Halifax, Nova Scotia, Canada; 12Städtisches Klinikum Braunschweig, Braunschweig, Germany; 13Azienda Ospedaliero-Universitaria Maggiore della Carità Novara, Novara; 14Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milano, Italy; 15University Hospital Leipzig, Leipzig, Germany; 16MDS Unit, AOU Careggi, DMSC, University of Florence, Firenze, Italy; 17Astex Pharmaceuticals, Inc., Pleasanton, United States of America; 18Universitaetsklinikum Freiburg, Freiburg, Germany Background: Parenterally administered hypomethylating agents (HMAs), decitabine (DEC) and azacitidine (AZA), are approved in Europe for adult patients with acute myeloid leukemia (AML) who are not candidates for standard induction chemotherapy as single agent or in combination with venetoclax. ASTX727 (DEC-C) is a fixed dose combination (FDC) tablet of 35 mg DEC and 100 mg cedazuridine, a novel cytidine deaminase inhibitor (CDAi). In clinical trials with myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemia (CMML) patients, DEC-C provides DEC exposures that are equivalent to IV DEC at the approved dose of 20 mg/m2 daily×5 and is approved as INQOVI® in the US, Canada, and Australia. Aims: To demonstrate DEC exposure bioequivalence of oral DEC-C and IV-DEC and generate clinical data using DEC-C in AML patients. Methods: The ASCERTAIN study was a randomized cross over design. Patients were randomized 1:1 to either Sequence A: DEC-C (35 mg DEC/100 mg cedazuridine) in Cycle 1 followed by IV-DEC at 20 mg/m2 in Cycle 2, or Sequence B: receiving IV-DEC in Cycle 1 followed by DEC-C on Cycle 2 to compare PK [primary endpoint Area Under the Curve (AUC) equivalence over 5 days of dosing]. All patients received DEC-C from Cycle 3 onwards until treatment discontinuation to assess safety and clinical efficacy. Patients were eligible as per the EMA-approved decitabine label (newly diagnosed AML who are not candidates for standard induction chemotherapy). Clinical responses were assessed according to modified International Working Group (IWG) 2003 response criteria. Results: 89 patients were randomized, of whom 87 were treated. The median age of patients was 78.0 years (range, 61 to 92) with 31 (35.6%) males and 56 (64.4%) females. Cytogenetic risk classification was poor-risk in 33 (37.9%) and intermediate-risk in 45 (51.7%) patients. For the primary endpoint, preliminary PK data was available from 69 patients who successfully completed PK assessments for both IV DEC and DEC-C cycles, and the DEC AUC0-24 (h*ng/mL) 5-Day geometric mean estimate was 904 for DEC-C and 907 for IV-DEC resulting in an oral/IV geometric LSM AUC ratio of 99.64% (90% CI of 91.23-108.8%). Safety findings were consistent with those anticipated for IV-DEC (related Grade ≥ 3 AEs in more than 10% were thrombocytopenia, anemia, febrile neutropenia, neutropenia, and pneumonia). As of the data cutoff date (10 SEP 2021), median follow up was 7.95 months (IQR 6.11-11.86). Of the 77 patients who had ≥6 months of follow up or discontinued treatment, the best response was complete response (CR) in 17 (22.1%, 95% CI: 13.4, 33.0%). In addition, 4 patients (5.2%) had CR with incomplete blood cell count recovery (CRi), with 1 patient (1.3%) who had CR with incomplete platelet recovery (CRp), resulting in composite response rate [CR + CRp] of 23.4% [18/77 patients, 95% CI: 14.5, 34.4%]. These results obtained with DEC-C are consistent with those observed for IV DEC. Based on preliminary and limited study follow-up with ~46% censored observations, the median survival was approximately 7.9 months (95% CI: 5.9, 13.0). Summary/Conclusion: This randomized phase 3 study in AML patients not candidates for standard induction chemotherapy demonstrates that the oral FDC of DEC-C (35mg/100 mg) resulted in an equivalent DEC AUC exposure to IV-DEC at 20 mg/m2 over 5 days. In addition, safety findings and preliminary clinical activity is also consistent with published data from IV-DEC, suggesting that DEC-C has the potential to be an oral alternative to the standard IV decitabine Daily×5 regimen. P574: CPX-351 COMBINED WITH HEMATOPOIETIC CELL TRANSPLANTATION WITH REGULATORY AND CONVENTIONAL T CELL IMMUNOTHERAPY FOR HIGH-RISK ACUTE MYELOID LEUKEMIA S. Sciabolacci1,*, L. Ruggeri1, V. Cardinali1, L. Brunetti1, S. Tricarico1, S. Saldi2, F. Marzuttini1, M. Griselli1, G. Perta1, V. Viglione1, G. Cimino1, A. Osmani1, M. Caridi1, R. Sembenico1, A. Terenzi1, T. Zei1, R. Iacucci Ostini1, M. F. Martelli1, B. Falini1, A. Velardi1, C. Aristei2, C. Mecucci1, A. Carotti1, M. P. Martelli1, A. Pierini1 1Division of Hematology and Clinical Immunology; 2Department of Surgical and Biomedical Science, Perugia General Hospital, Perugia, Italy Background: Patients with newly diagnosed acute myeloid leukemia with myelodysplasia-related changes (MRC-AML) or therapy-related AML (t-AML) have an extremely poor prognosis. CPX-351, a dual-drug liposomal encapsulation of cytarabine and daunorubicin, improved significantly rates of remission and overall survival (Lancet JE et al, JCO 2018). Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative strategy for such patients, but incidence of post-transplant relapse is still unacceptably high. We developed a novel HSCT strategy that combines an age-adapted myeloablative irradiation-based conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy (Treg/Tcon HSCT). HLA-haploidentical Treg/Tcon HSCT ensured low relapse rate (4%) and resulted in an unprecedented chronic graft versus host disease/relapse free survival (CRFS, 75%) in AML patients (Pierini et al., Blood Adv. 2021). Aims: We report preliminary outcomes of the combination therapy with CPX-351 and Treg/Tcon HSCT for MRC-AML and t-AML at our center. Methods: We included in the study all patients with MRC-AML or t-AML who were referred at our center from September 2019 to January 2022 and could be treated with CPX-351. Treg/Tcon HSCT consisted of a myeloablative conditioning regimen with total marrow/lymphoid irradiation, thiotepa, fludarabine and cyclophosphamide followed by an infusion of 2x106/Kg donor Tregs on day -4, 1x106/Kg Tcons on day -1 and a megadose (>6x106/Kg) of purified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GvHD prophylaxis was given post-transplant (clinicaltrials.gov: #NCT03977103). Results: Seventeen patients with a median age of 57.5 years (46-72) were treated with CPX-351: 13 with newly diagnosed AML (11 MRC-AML and 2 t-AML) and 4 patients who relapsed after a previous HSCT and who acquired novel cytogenetic abnormalities (2 complex karyotypes, CK). 9/13 patients with newly diagnosed AML had karyotype abnormalities: 4 monosomy 7, 1 CK, 1 t(3;3), 1 isolated del(20q) and 2 (both t-AML) t(11;20) which resulted in NUP98-TOP1 fusion. Nine patients achieved complete remission (CR), 2 partial response (PR) and 2 had persistent disease. No patient experienced grade>3 extrahematologic toxicity. Ten/13 patients underwent a first HSCT (8 in CR, 2 in PR). Eight underwent Treg/Tcon HSCT (3 from HLA-matched, 5 from HLA-haploidentical donor), while 2 unmanipulated haploidentical HSCT followed by post-transplant cyclophosphamide (PT-Cy). All patients achieved full-donor engraftment. No patients experienced non-relapse mortality (NRM). Three/10 patients had a grade ≥ III acute GvHD; all of them are alive and off immunosuppressive therapy. At a median follow up of 16.2 months (range 9-29 months), 9/10 patients are alive and in CR with no chronic GvHD. No relapse occurred in the 8 patients who underwent Treg/Tcon HSCT, while one patient who underwent HSCT followed by PT-CY relapsed. The 4 patients who were treated after one previous HSCT received one single induction cycle with CPX-351, achieved CR and experienced no grade>3 extra-hematologic toxicity. All of them could undergo a second HSCT. NRM occurred in 1 patient (septic shock), while 3 patients are alive to date. Summary/Conclusion: CPX-351 allowed most of MRC-AML and t-AML patients to reach HSCT in CR despite high risk genetics and even when used in patients who relapsed after a 1st HSCT. Combination of CPX-351 with Treg-Tcon HSCT is safe and exert powerful antileukemic activity. Thus, it appears to be a promising strategy to ensure high rate of CRFS in very high-risk AML. P575: IMPACT OF MOLECULAR RESPONSE AND CHEMOTHERAPY REGIMEN ON OUTCOMES IN CORE BINDING FACTOR AML J. Senapati1,*, F. Haddad1, F. Ravandi1, T. Kadia1, C. DiNardo1, N. Daver1, N. Pemmaraju1, Y. Alvarado1, M. A. Brandt1, H. Kantarjian1, G. Borthakur1 1Leukemia, MD Anderson Cancer Center, Houston, United States of America Background: Fludarabine based induction/consolidation regimens result in excellent outcomes in core binding factor acute myeloid leukemia (CBF-AML) Attainment of early polymerase chain reaction (PCR) based optimal minimal residual disease (MRD) responses positively impact long term outcomes and provide early decision timepoints for alternative therapies including stem cell transplantation (SCT). Aims: We studied factors that affected early and late optimal PCR responses (OPR) and survival in patients (pts) of CBF AML who were treated with fludarabine based regiemns (fludarabine, cytarabine and GCSF) with idarubicin (FLAG-IDA) or gemtuzumab (FLAG-GO). Methods: This is a prospective study of pts with N/D CBF-AML treated at our center on FLAG based regimens. Pts received up to 7 cycles of therapy with appropriate dose reductions based on age or toxicity. PCR for the disease defining transcripts (CBFB::MYH11 or RUNX1::RUNX1T1) were monitored after C1, C3 and end of therapy (EOT). Based on our earlier report (Boddu et. al., Leukemia 2018) OPR was defined as PCR transcript <0.1% after C1 and <0.01% for later time points. Results: Between 4/2007 to 12/2019, 174 pts, with a median age of 51 years (range, 19-78) were included. A total of 109 pts (63%) received FLAG-IDA, while the rest got FLAG-GO therapy. This was mostly due to withdrawal of approval of GO in the US for a period of time. Median number of cycles received was 6 (range, 1-7) Baseline parameters are described in Table 1. PCR data is available for 94% pts after C1, 90% after C3 and 92% at EOT. EOT was considered the timepoint at which pt stopped further FLAG based therapy beyond C1. The percent of patients achieving OPR at each time point by regimen are: 68% vs. 41% (p=0.002) after C1, 75% vs. 39% (P< 0.0001) after C3 and 92% vs. 55% (p<0.0001) at EOT for FLAG-GO and FLAG-IDA respectively. On univariate analysis, FLAG-GO regimen favored attainment of post C1 OPR over FLAG-IDA (OR, odds ratio=3.1, 95%CI 1.6-6.2) while age, baseline BM blast, cytogenetic subtype of CBF, ACA and kinase mutations did not. The benefit of FLAG-GO in attaining OPR after C1 was maintained even on a multivariate (MV) model. For EOT OPR, FLAG-GO (OR= 8.5, 95% CI 3.4-26) fared better over FLAG-IDA. Absence of KIT mutation (OR=2.4) and attainment of post C1 OPR (OR= 11.3) were the other relevant factors. On MV analysis, only chemo arm and post C1 OPR remained significant. At a median follow up of 92 months (mos), the RFS was 124 mos and OS not reached (NR) for the whole cohort (RFS 68 mos and OS 102 mos for FLAG-IDA treated pts vs. both NR for FLAG-GO treated pts, p=0.004 and 0.04 respectively). Across the 2 groups there was no difference in RFS or OS between pts who attained early or late OPR. However, on stratifying serial PCR responses with therapy arms, even in pts with early OPR (after C1), RFS was better (NR) in FLAG-GO arm vs 101 mos for FLAG-IDA arm (p= 0.03), though OS was similar, likely reflecting efficacy of salvage therapies in this subset of AML. In a Cox proportional hazards model that included age, therapy arm, kinase mutations, cumulative therapy cycles, post C1 and EOT PCR responses; only younger age and EOT OPR favorably affected RFS; however FLAG-GO treated pts had lower hazards for RFS at any level of PCR response compared to FLAG-IDA treated pts (Fig. 1). Image: Summary/Conclusion: In CBF AML, FLAG-GO regimen leads to higher rate of earlier and end of therapy OPR than FLAG-Ida and substantially better RFS. At any combination of PCR response, FLAG-GO treated patients had lower hazards for relapse than FLAG-IDA treated pts. P576: A PHASE I/II STUDY OF MILADEMETAN (DS3032B) IN COMBINATION WITH LOW DOSE CYTARABINE WITH OR WITHOUT VENETOCLAX IN ACUTE MYELOID LEUKEMIA J. Senapati1,*, J. Ishizawa1, H. A. Abbas1, S. Loghavi1, G. Borthakur1, G. C. Issa1, S. I. Dara1, R. Pourebrahim1, N. Daver1, E. J. Jabbour1, S. M. Kornblau1, N. Pemmaraju1, G. Garcia-Manero1, F. Ravandi1, M. Muftuoglu1, J. Khoury1, C. DiNardo1, M. Andreeff1 1Leukemia, MD Anderson Cancer Center, Houston, United States of America Background: In TP53 wild type (WT) acute myeloid leukemia (AML), inhibition of MDM2 increases p53 protein expression and mediates antileukemic effects. MDM2i monotherapy in AML has shown only modest responses; combining MDM2i with anti-leukemic therapies with additive or synergistic pre-clinical activity and non-overlapping toxicities might potentiate therapeutic benefit. Aims: To evaluate safety and efficacy of milademetan (DS-3032b), an MDM2i, with low dose cytarabine (LDAC) ± venetoclax (VEN) in patients (pts) with AML. Methods: This open label, single arm Phase I/II trial (NCT03634228) included pts with AML and WT TP53. The phase I utilized a 3 + 3 design to identify safety and tolerability of the combination and determine the recommended combination dose to proceed to the expansion phase (phase II). Pts included were adult (≥18 years), ECOG PS ≤ 3, with adequate renal and hepatic function. Key exclusion criteria included acute promyelocytic leukemia, prior therapy with MDM2i, or stem cell transplantation (SCT) in the preceding 60 days. Doses of milademetan and combination therapy are provided in Table 1. Toxicity was graded according to the NCI-CTCAE v.5 and responses according to the ELN 2017 AML response criteria. Results: A total of 21 pts were screened; 16 pts were enrolled and treated in phase I. Median age was 70 years (range, 23-80) and 11 (69%) were women. Median number of prior therapies was 3 (range, 1-7), with 6 pts (37%) who had undergone prior SCT. Two pts (12.5%) were newly diagnosed with treated secondary AML; having received 3 and 2 lines of therapy for prior MDS. Milademetan combination therapy was reasonably well tolerated at all dose levels, the most common adverse events being gastrointestinal. One pt had an attributable grade 3-4 toxicity (diarrhea), which was considered a dose limiting toxicity. Pts received a median of 1 cycle (range, 1-4). Two pts (12.5%) achieved an overall response (CRp, 12.5%, 1 each at dose level 1 and 2). At a median follow up of 10.5 mos (range, 0.6-17), 5 pts are alive (31%), all on subsequent lines of therapy. Ten of 11 deaths occurred in non-responding pts and were attributed to disease progression or infection. One responding pt died of infection in cycle 3. Given the minimal response rates at all dose levels, the phase II expansion portion of the trial was not conducted. p53 immunohistochemistry was performed on baseline bone marrow (BM) samples from 11 pts, all of whom had WT expression pattern. BM samples of 1 amongst 7 pts evaluated post C1 demonstrated acquisition of mutant p53. We performed CyTOF analysis of sequentially collected samples to interrogate signalling pathways, the p53-MDM2 axis, and the abundance of pro/anti-apoptotic molecules. We observed that CD34+ leukemia blasts expressed high levels of Bcl-2 and were eliminated after therapy in a patient with CRp while Bcl-2 low non-malignant monocytic cells preferentially enriched. Comparative single-cell proteomic analysis in responders and non-responders are underway to identify proteomic correlates of favourable response vs. therapy resistance. Image: Summary/Conclusion: The combination of milademetan with LDAC ± venetoclax was reasonably well tolerated but showed only minimal clinical responses at all 3 dose levels in a heavily pre-treated AML population. Further studies are needed to decipher the most appropriate combination regimen and pt population to benefit from milademetan. P577: 20% BLAST CRITERIA FOR DIAGNOSING AML/MDS: IS IT TIME TO MOVE BEYOND? V. Sheth1,*, R. Walter2,3, C. Shaw3, E. Estey2,3, M.-B. Percival2,3 1Deprtment of hematology and Bone marrow transplant, UAB, Birmingham; 2Division of hematology, University Washington; 3Clinical research division, Fred Hutch, SEATTLE, United States of America Background: Classification of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) is driven by an arbitrary blast percentage cut off (20%, using the 2016 WHO criteria), which subsequently affects treatment options (including enrollment in clinical trials) without taking genomic and biological characteristics into consideration. It has recently been posited that AML and MDS with excess blasts (EB) exist on a continuum based on biological features rather than blast percentages, and their outcomes post treatment are very similar. Aims: We therefore hypothesized that the IPSS-R (MDS prognostication using hematologic parameters and cytogenetic features) should predict outcomes for WHO-defined AML, and similarly, ELN2017 (AML prognostication using molecular and cytogenetic features) should predict outcomes for WHO-defined MDS-EB (5-19% blasts). Methods: We retrospectively analyzed 1034 adult patients from the UW/FHCRC registry database, who received treatment for AML or MDS-EB between the years 2005-2018. WHO-defined AML occurred in 777 patients (67%) and WHO-defined MDS-EB in 257 (23%). Median follow up was 5 years. Outcomes were relapse-free survival (RFS) and overall survival (OS). Results: Median age of patients in AML group was 63 years (range 18-91), and in MDS group was 60 years (range 22-85). The majority of the patients in AML cohort had intermediate (37%) or high-risk disease (37%) using ELN2017 classification; many were reclassified to very high risk by IPSS-R (54%). Most of the patients in MDS cohort were very high risk (54%) using IPSS-R; many were reclassified to ELN high risk (46%). For AML patients, classification using the IPSS-R was highly predictive of OS for AML in univariate (p=0.001) and multivariate analysis (reference low risk/intermediate risk) (p=0.001, HR-2.1, 95% confidence interval (CI) 1.1-2.9 for very high risk and p=0.2, HR-1.4, 95%CI 0.76- 1.9 for high risk in AML), as well as RFS in univariate (p=0.001) and multivariate analysis (reference low risk/intermediate risk) (p=0.001, HR-2.08, 95%CI 1.2-2.8 for very high risk and 0.2, HR-1.4, 95%, CI 0.2-1.8 for high risk in AML). Similarly, ELN 2017 was predictive of OS in MDS cohort in univariate (p=0.025), and multivariate analysis (reference ELN adverse (p=0.005, HR-0.1, 95%CI 0.02-0.5, ELN favorable risk), as well as RFS in univariate (p=0.043) and multivariate analysis (reference ELN adverse) (p=0.019, HR-0.25 for ELN favorable risk, 95%CI 0.04- 0.8). In the subgroup of AML patients who achieved CR/CRi, IPSS-R was predictive of RFS in univariate analysis (p=<0.001), and multivariate analysis (ref low/int) (p=0.001, HR=2.08 for very high risk). Similarly, in the subgroup of MDS patients who achieved CR/CRi, ELN 2017 was predictive of RFS (p=<0.001), but this effect was not sustained in multivariable analysis. Summary/Conclusion: Data from our cohort suggest that IPSS-R can be predictive of survival outcomes in WHO-defined AML patients and ELN 2017 can similarly be predictive in WHO-defined MDS patients. Therefore, we suggest that diagnostic criteria for AML and MDS should include more biological and genomic features rather than a blast percentage cut-off, using tools such as the recently presented IPSS-molecular for MDS. Clinical trials of new therapies should include all patients having 35% blasts (MDS-EB and AML). This study, however, carries a potential drawback of retrospective classification and lack of complete molecular data through comprehensive targeted next generation sequencing panel in patients treated at earlier time periods P578: GILTERITINIB FOR RELAPSED/REFRACTORY FLT3 MUTATED ACUTE MYELOID LEUKEMIA A REAL-WORLD, MULTI-CENTER, MATCHED ANALYSIS S. Shimony1,2,3, J. Canaani3,4, E. Kugler2,3,*, B. Nachmias5, R. Ram6,7, A. Frisch8, C. Ganzel9, V. Vainstein5, Y. Moshe3,10, S. Aumann5, M. Yeshurun2,3, Y. Ofran9, P. Raanani2,3, O. Wolach2,3 1Leukemia, Dana Farber Cancer Institute, Boston, United States of America; 2hematology, Rabin medical center, Petah Tikva; 3Sackler Medical School, Tel Aviv University, Tel Aviv; 4Leukemia, Chaim Sheba Medical Center, Ramat Gan; 5hematology, Hadassah Medical Center and Hebrew university Faculty, Jerusalem; 6BMT Unit; 7Sackler Medical School, Tel Aviv Sourasky Medical Center, Tel Aviv; 8Department of Hematology and Bone Marrow Transplantation, Rambam Health Care Campus, and Rappaport Faculty of Medicine - Technion, Haifa; 9hematology, Shaare Zedek Medical Center, and Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem; 10hematology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Background: Patients with FLT3-mutated relapsed or refractory (R/R) acute myeloid leukemia (AML) have a dismal prognosis. Gilteritinib is a FLT3 tyrosine kinase inhibitor (TKI) recently approved for patients with R/R AML. Aims: We aimed to characterize real-world data regarding gilteritinib treatment in FLT3-mutated R/R AML and to compare outcomes with matched FLT3-mutated R/R AML patients treated with chemotherapy-based salvage regimens. Methods: Survival analysis was performed by Kaplan Meyer with log rank test to compare between subgroups. Cox proportional hazards regression models were fitted to predict effect of covariates on overall survival (OS) and event free survival (EFS). All statistical analyses used 2-sided p value with a threshold of 0.05. Results: Twenty-five patients from six academic centers were treated with gilteritinib for FLT3-mutated R/R AML. The median age was 58 (IQR1-3 47-73) years, 80% (n=20) were treated with a prior intensive induction regimen and 40% of them received prior TKI therapy. After a median time of seven months post gilteritinib initiation (range 1-34), 12 patients (48%) achieved complete response (CR) with gilteritinib (figure 1A). The estimated median overall survival (OS) of the entire cohort was eight (CI 95% 0-16.2) months and was significantly higher in patients who achieved CR compared to those who did not (16.3 months, CI 95% 0-36.2 vs. 2.6 months, CI 95% 1.47-3.7; p value=0.046, figure 1B). In a multivariate cox regression analysis, achievement of CR was the only predictor for longer OS (HR 0.33 95% CI 0.11-0.97, p=0.044). Prior TKI exposure did not affect OS but was associated with better event-free survival (HR 0.15 95% CI 0.03-0.71, p=0.016). An age and ELN-risk matched comparison between patients treated with gilteritinib and intensive salvage revealed similar response rates (50% in both groups); median OS was 9.6 months (CI 95% 2.3-16,8) vs. 7 months (CI 95% 5.1-8.9) in gilteritinib and matched controls, respectively (p = 0.869, figure 1C). Image: Summary/Conclusion: In the real-world setting gilteritinib is effective, including in heavily pre-treated, TKI exposed patients. P579: A PHASE 4 STUDY OF FRACTIONATED GEMTUZUMAB OZOGAMICIN ON QT INTERVAL AND SAFETY IN PATIENTS WITH RELAPSED/REFRACTORY CD33-POSITIVE ACUTE MYELOID LEUKEMIA P. Montesinos1,*, V. Kota2, J. Brandwein3, P. Bousset4, R. J. Benner5, E. Vandendries6, M. F. McMullin7 1Department of Hematology, Hospital Universitario i Politècnico la Fe, Valencia, Spain; 2Department of Medicine: Hematology and Oncology, Medical College of Georgia, Augusta University, Augusta, United States of America; 3Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada; 4Pfizer Inc., Paris, France; 5Pfizer Inc., Groton; 6Pfizer Inc., Cambridge, United States of America; 7School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast, United Kingdom Background: Gemtuzumab ozogamicin (GO) is a CD33-directed antibody-drug conjugate indicated for treatment of relapsed/refractory (R/R) acute myeloid leukemia (AML). Fractionated dosing of GO has demonstrated an enhanced safety profile without hampering efficacy. Aims: To assess the effect of fractionated GO as monotherapy on corrected QT (QTc) interval, safety, pharmacokinetics (PK), and immunogenicity of GO in patients (pts) with R/R AML. Methods: Pts aged ≥12 y with R/R AML were enrolled in a single-arm, open-label, phase 4 study (NCT03727750) and received a fractionated dose of GO 3 mg/m2 on days 1, 4, and 7 of each cycle, up to 2 cycles. Pts receiving ≥1 dose of GO were evaluable for QTc, safety, and PK. The primary endpoint was mean change from baseline in QTc. Secondary endpoints included PK parameters, adverse events (AEs), including veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS), incidence of anti-drug antibodies (ADAs)/neutralizing antibodies (NAb), overall survival (OS), and response rate of complete remission (CR)/CR with incomplete hematologic recovery (CRi). This analysis includes data from pts aged ≥18 y. Results: Of 51 adult pts enrolled (median age 67 y), 50 (98%) pts received ≥1 dose of GO during cycle 1; 9 (18%) pts were treated with GO during cycle 2. The upper limit of the 2-sided 90% CI for least squares mean differences in QTc according to Fridericia’s formula (QTcF) was <10 msec for all timepoints during cycle 1 (Table). Most pts had a QTcF ≤450 msec (90%) and a maximum increase from baseline QTcF ≤30 msec (94%). No pts had a post-baseline QTcF >480 msec or a change from baseline >60 msec. Treatment-emergent AEs (TEAEs) occurred in 98% of pts; 54% were grade 3/4. The most common (>10%) grade 3/4 TEAEs were febrile neutropenia (36%) and thrombocytopenia (18%). One pt experienced grade 3 atrial fibrillation and supraventricular tachycardia, unrelated to GO. No pts experienced grade 4 or higher cardiac conduction TEAEs. VOD/SOS was not reported; however, one pt experienced treatment-related grade 5 capillary leak syndrome associated with pleural effusion, ascites, hyperbilirubinemia, and endothelial syndrome. The overall incidence of ADAs and NAbs were 12% and 2%, respectively. ADAs were persistent in 5/6 pts (83%). Of the 6 pts with treatment-induced ADAs, 2 experienced pyrexia (grade 1 and grade 3) within 24 h of the infusion. Among ADA-negative pts (n=44), infusion-related reactions were observed in 7 (16%) pts. These were grade 1/2, except for 1 instance of grade 3 urticaria. Median (95% CI) OS was 2.8 (1.7–4.2) mo. Disease progression was the most common cause of death in 35/45 (78%) pts. A CR/CRi was observed in 5/51 (10%) pts. Image: Summary/Conclusion: Fractionated dosing of GO (3 mg/m2 per cycle) is not predicted to pose a clinically significant safety risk for QT prolongation in pts with R/R AML, with the upper limit of the 2-sided 90% CI for the least squares mean change from baseline falling below the 20 msec threshold of clinical concern for oncology drugs. TEAEs reported in this study are consistent with the known safety profile of GO, and the presence of ADAs does not appear to be associated with any potential safety concerns. Response rates were lower than previously reported in the MyloFrance 1 study, which included less heavily pretreated patients (i.e., patients only in first relapse; Taksin et al, Leukemia, 2007). P580: THE USE OF LEUKAEMIA Q-FUSION GENE SCREENING ASSAY (Q30) IN THE DIAGNOSTIC EVALUATION OF ACUTE MYELOID LEUKAEMIA (AML) S. Tayabali1,*, R. Baker1,2, E. Nacheva1,2, J. O’Nions1,2, R. Gupta1,2, A. J. Wilson1,2, K. Xu1,2 1University College London Hospital; 2UCLH Specialist Integrated Haematology Malignancy Diagnostic Service, Health Services Laboratories, London, United Kingdom Background: AML is a clinically heterogenous disease characterised by chromosomal and genetic abnormalities which guide risk stratification and management. Hence timely cytogenetic and molecular profiling is essential to initiate appropriate treatment. Approximately 30% of AML cases have fusion genes. We use a combination of fluorescence in situ hybridization (FISH) to rapidly screen for common chromosomal abnormalities and fusion genes, and chromosomal microarray (CMA) to assess copy number variations across the whole genome. However, cytogenetic analysis is resource intensive and may take several days which can delay patient management. The QuanDx© Leukaemia Q-Fusion Genes Screening Kit (Q30) uses multiplex reverse transcription real-time PCR (RT-qPCR) to allow simultaneous detection of 30 fusion genes with 140+ breakpoints within hours. The use of Q30 may allow earlier risk stratification and treatment before FISH and CMA results are available. Aims: To evaluate the Q30 Kit to detect rearrangements on bone marrow and peripheral blood specimens from newly diagnosed AML patients; and compare concordance of Q30 and FISH in identifying leukaemia fusion genes. Methods: We retrospectively reviewed FISH, CMA and Q30 data for new AML cases diagnosed in the specialist integrated haematological malignancy diagnostic service (SIHMDS) at University College London Hospital over a 2 year period: 01 Jan 19 to 31 Dec 20. Results: 114 cases were included. The median age was 59 [range 19 to 91]. Male 59 (52%), Female 55 (48 %). ELN Cytogenetic Risk Group (n;%) Favourable 19 (17%) Intermediate 69 (61%) Adverse 26 (23%) Type of AML (n;%) De novo 105 (92%) Secondary 7 (6%) Therapy Related 2 (2%) 30 (26%) of 114 patients had a detectable fusion gene by Q30 (Figure 1). 25/30 were confirmed by FISH either on our standard panel or subsequently using targeted probes. The 5/30 fusion genes detected by Q30, but not by FISH were: - t(3;5)(q25;q34) NPM1-MLF1 (n=1) - t(9;22)(q34;q11) BCR-ABL1 with a high cycle threshold (Ct) value (n=3) - t(16;21)(p11;q22) TLS-ERG (n=1) The discordant results were due to high Ct values (detecting low level gene fusions below the limit of FISH sensitivity), or a lack of commercially available fusion probes. Image: Summary/Conclusion: The ability to rapidly risk-stratify newly diagnosed AML patients allows earlier initiation of targeted therapies. We found that Q30 is highly sensitive and showed 100% concordance in identifying fusions associated with good cytogenetic risk AML in our cohort. Q30 can also identify high risk fusion genes including DEK-CAN, MLL-AF6 and other high risk MLL translocations included in the panel which are rare and for which FISH fusion probes are not routinely used. These may have been otherwise undetected, but can subsequently be confirmed by FISH, and importantly allow the generation of molecular measurable residual disease (MRD) assays to assess treatment response. We conclude that Q30 is a rapid, simple and sensitive adjunct to conventional FISH in the diagnosis of AML and, in combination with CMA, may preclude the requirement for conventional and time consuming G-banding analysis. P581: PHASE II STUDY OF LOWER-INTENSITY FRONTLINE THERAPY FOR NEWLY DIAGNOSED PATIENTS WITH AML WHO ARE UNFIT OR OTHERWISE NOT ELIGIBLE FOR FRONTLINE CLINICAL TRIALS S. Venugopal1,*, E. Jabbour1, N. Pemmaraju1, G. Montalban-Bravo1, K. S. Chien1, N. Daver1, N. Jain1, J. Burger1, Y. Alvarado1, A. Maiti1, C. D. DiNardo1, G. Borthakur1, R. Malla1, G. Garcia-Manero1, F. Ravandi1, H. Kantarjian1, T. Kadia1 1Leukemia, The University of Texas, MD Anderson Cancer Center, Houston, United States of America Background: Pts with newly diagnosed (ND) AML frequently present with abnormal organ function, poor performance status (PS), concurrent active malignancies, and active infections. These factors often preclude these pts from enrollment on frontline clinical trials, since standard eligibility routinely exclude them. Aims: Therefore, we designed a lower-intensity regimen of cladribine plus low-dose cytarabine (LDAC) alternating with decitabine (DAC) with less stringent inclusion criteria in ND pts unfit or otherwise ineligible for existing clinical trials. Methods: Pts >18 years with untreated AML and ineligible for other frontline AML clinical trials were enrolled (NCT01515527). Eligibility criteria included either creatinine ≥2mg/dL; or total bilirubin ≥2 mg/dL; or ECOG PS of 3 or 4; or ineligible for participation in a higher priority protocol. Pts with active concurrent malignancies and ongoing infection related to their AML could be enrolled. Induction was cladribine 5 mg/m2 IV on D1-5, Cytarabine 20 mg SQ twice daily on D 1-10, followed by consolidation with cladribine 5 mg/m2 IV on D1-3, Cytarabine 20 mg SQ twice daily on D 1-10 alternating with decitabine 20 mg/m2 IV, daily on D 1-5. The primary objective was 60-d survival rate. Results: A total of 25 pts have been enrolled. The median age was 73 years (range, 52-82) with 76% of the cohort older than 70 years. Six pts (24%) had concurrent active malignancy, 4 (16%) had baseline creatinine >2mg/dL, and 9 (36%) had PS ≥3. 76% of them were adverse risk per ELN 2017. Baseline characteristics are shown in figure. Among 25 evaluable pts, 17 (68%) achieved a composite complete response (CRc) including 10 (40%) CR and 7 (28%) CR with incomplete count recovery (CRi). Among responders, 6 pts (35%) achieved MRD neg by flow. Of 7 pts with no response, all were ELN adverse risk. Median cycles to response was 1 (range: 1 - 4). 30- and 60 d mortality was 8% and 16%, respectively, including 2 pts (8%) who died due to pseudomonal sepsis on D8, and the other due to pneumonia on D11. At a median follow up of 9.4 months (range, 0.4- 19.9 m),median OS (6-mo OS% - 61 %), and EFS was 8.3 mo each (6-mo RFS% - 56 %) with a 60-d OS and EFS rate of 83% each, with a median RFS of 5.8 mo (2-mo RFS%-66%; 6-mo RFS% - 49 %). In this challenging patient population, this lower-intensity regimen was well tolerated, with an acceptable toxicity profile. Image: Summary/Conclusion: In an unfit patient population with a high comorbidity burden, that were ineligible for other clinical trials, induction therapy with Cladribine plus LDAC was feasible and effective in newly diagnosed pts with AML. The regimen produced high rates of response, encouraging EFS, OS, and low early mortality in a cohort predicted to have a high risk of early death. Treating this pt population on a clinical trial is feasible and can allow pts to achieve remission and move on to effective post-remission therapy. P582: FIRST RESULTS OF A PHASE II STUDY (STIMULUS-AML1) INVESTIGATING SABATOLIMAB + AZACITIDINE + VENETOCLAX IN PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA A. M. Zeidan1,*, J. Westermann2, T. Kovacsovics3, S. Assouline4, A. C. Schuh5, H.-J. Kim6, G. Rodriguez Macias7, D. Sanford8, M. R. Luskin9, E. M. Stein10, K. Malek11, J. Lyu12, M. Stegert11, J. Esteve13 1Yale University and Yale Cancer Center, New Haven, United States of America; 2Charité-University Medical Center Berlin, Campus Virchow Clinic, Berlin, Germany; 3University of Utah and Huntsman Cancer Institute, Salt Lake City, United States of America; 4Jewish General Hospital, McGill University, Montreal; 5Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada; 6Catholic Hematology Hospital, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea; 7Hospital General Universitario Gregorio Marañón, Madrid, Spain; 8BC Cancer, University of British Columbia, Vancouver, Canada; 9Dana-Farber Cancer Institute, Harvard Medical School, Boston; 10Memorial Sloan Kettering Cancer Center, New York, United States of America; 11Novartis Pharma AG, Basel, Switzerland; 12Novartis Institutes for BioMedical Research, Shanghai, China; 13Hospital Clínic, Barcelona, Spain Background: The BCL-2 inhibitor venetoclax (VEN) in combination with a hypomethylating agent (HMA) has improved outcomes for patients with newly diagnosed (ND) acute myeloid leukemia (AML) who are unfit for intensive chemotherapy (IC; DiNardo CD, N Engl J Med, 2020); however, responses are often transient. Sabatolimab is a novel immuno-myeloid therapy targeting TIM-3, an immune regulator expressed on immune cells and myeloid leukemic progenitors but not on normal hematopoietic stem cells. Sabatolimab + HMA has shown promising durable responses in a Phase Ib study in patients with ND-AML and myelodysplastic syndrome (Wei AH, ASH 2021; NCT03066648). Treatment with sabatolimab + VEN + azacitidine (AZA) may also induce different pathways of cancer cell elimination. Aims: In this first presentation of the study, we report findings from the dose-escalation part of STIMULUS-AML1 (NCT04150029), a Phase II, single-arm study of sabatolimab + AZA + VEN in adult patients with AML. Methods: Adult patients with ND-AML ineligible for IC were enrolled. In dose-escalation part 1 (safety run-in), patients received either 400 mg (Cohort 1) or 800 mg (Cohort 2) sabatolimab every 4 weeks (Q4W) plus 400 mg VEN once a day every day, plus 75 mg/m2 AZA on Days (D) 1-7, or D1-5+D8-9, or D1-6+D8 of a 28-day cycle (Zeidan A, ESH-AML 2022). For the safety run-in part, the primary endpoint was assessment of the incidence of dose-limiting toxicities (DLTs) between Cycle 1 D8 and the end of Cycle 2. Results: As of the Sep 6, 2021 data cutoff, 18 patients have been treated in part 1 of the study with sabatolimab + AZA + VEN (Cohort 1, n=5; Cohort 2, n=13). Baseline demographics are shown in Table 1. Treatment was ongoing for 9 (50%) patients (Cohort 1, n=1; Cohort 2, n=8). Reasons for study treatment discontinuation included adverse events (AEs; n=3), progressive disease (n=3, including a case of chloroma), planned stem cell transplant (n=2), and physician decision (n=1). Only 1 DLT (elevated troponin T/asymptomatic myocarditis) was reported in Cohort 2 and no DLTs were reported in Cohort 1. Table 2 summarizes AEs observed in >20% of patients in the study. Consistent with AEs often observed during AZA + VEN therapy (DiNardo CD, N Engl J Med, 2020), the 5 most common AEs regardless of relationship to study treatment were constipation (all grades [gr]: 39%; gr ≥3: 0%) and hematologic events, including anemia (33%; 28%), decreased platelets (33%; 33%), neutropenia (39%; 39%), and febrile neutropenia (gr: 50%). Treatment-related AEs gr ≥3 in >25% of all patients included neutropenia (39%), decreased platelet count (33%), and decreased neutrophil count (28%). Treatment-related AEs led to study treatment discontinuation in 3 patients (platelet count decreased [n=2], troponin level increased/asymptomatic myocarditis [n=1]). AEs led to sabatolimab dose interruption in 6 patients, all in Cohort 2. No sabatolimab dose reduction was observed. Fourteen patients had dose interruption of VEN due to AEs. Five patients had dose reduction of VEN (none due to AEs). Serious AEs were reported in 14 (78%) patients; the most common was febrile neutropenia (44%). No other serious AEs were reported in >1 patient. Efficacy data will be presented at the EHA meeting. Image: Summary/Conclusion: Safety and tolerability of sabatolimab + AZA + VEN were comparable at 2 dose levels (400 and 800 mg) of sabatolimab and were overall comparable to the reported safety profile of VEN + AZA doublet therapy. These findings supported initiation of the expansion cohort of STIMULUS-AML1 at a sabatolimab 800 mg dose. P583: PATIENTS AT HIGH RISK OF RELAPSE POST-TRANSPLANT: A PHASE 1 STUDY DESIGN WITH A NOVEL TREATMENT STRATEGY USING THE ESTIMAND FRAMEWORK R. Zeiser1,*, C. Schmid2, G. Al-Atrash3, Y. Xu4, H.-J. Weber5, L. Eldjerou6, S. Weber7, L. Widmer7, C. Craddock8 1Department of Medicine I, Medical Center, University Freiburg, Freiburg; 2Department of Hematology and Oncology, Augsburg University Hospital and Medical Faculty, Augsburg, Germany; 3Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston; 4Oncology Analytics, Novartis Pharmaceutical Corporation, East Hanover, United States of America; 5Oncology Analytics, Novartis Pharma AG, Basel, Switzerland; 6Clinical Development, Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 7Advanced Exploratory Analytics, Novartis Pharma AG, Basel, Switzerland; 8Centre for Clinical Haematology, University Hospitals Birmingham NHS Foundation Trust, Birmingham, United Kingdom Background: The risk of disease relapse in patients (pts) allografted for acute myeloid leukemia (AML) is high and remains the main cause of transplant failure. The great majority of pts destined to relapse post-allogeneic stem cell transplant (SCT) will do so within the first 12 months and survival after early relapse is dismal. Therefore, strategies to prevent relapse are urgently required. A potent graft-versus-leukemia (GvL) effect is exerted after SCT, and strategies that may augment a GVL effect have the potential to reduce relapse risk post-SCT. Siremadlin (HDM201) is a novel investigational MDM2 inhibitor with single-agent (SA) anti-AML activity. MDM2 inhibitors, including siremadlin, possess potent immunomodulatory effects (IME) in murine solid tumor and AML models. The post-allogeneic SCT setting is ideal for investigating the IME of MDM2 inhibition on enhancing GvL effect. Here we present a phase Ib/II proof-of-concept study designed to assess safety and preliminary efficacy of a treatment strategy with siremadlin as monotherapy and, subsequently, in combination with donor lymphocyte infusions (DLIs) for AML in post-SCT setting using the estimand framework (ICH E9 (R1) addendum). Aims: The objective of the planned treatment strategy is to enhance GvL post-SCT by siremadlin as monotherapy and in combination with DLIs. Due to the risk of Graft-vs-Host Disease (GvHD) with DLI early after SCT, siremadlin will be given as priming monotherapy to prevent early relapse prior to starting siremadlin/DLI combination therapy. SA siremadlin maintenance after the combination therapy is intended to prolong and enhance the GvL reaction to eradicate residual leukemic blasts. Methods: The estimand framework mandates a careful definition of the experimental intervention according to the clinical question of interest. In this trial, the experimental intervention follows a treatment strategy: Patients (pts) with AML in complete remission (CR) post-SCT but at high risk for relapse based on pre-SCT risk factors will start with siremadlin priming monotherapy. Pts who tolerate the siremadlin dose with no evidence of GvHD are eligible to start siremadlin/DLI combination therapy. After combination therapy, pts may continue with SA siremadlin maintenance. Depending on the treatment journey, pts may receive only a part of the intervention’s sequence (e.g., pts in siremadlin priming monotherapy who are not eligible for DLI combination will continue the priming part). The clinical questions addressed in this trial are to determine the recommended dose (RD) of siremadlin across all parts of the treatment strategy and to assess preliminary efficacy regardless of the parts of the treatment strategy pts received. The doses of interest were preselected based on SA studies and in pts who received siremadlin after SCT. Results: The proposed approach is tailored to investigate safety and efficacy of the treatment strategy. The RD of siremadlin will be determined over 2 parts: A Bayesian logistic regression model will guide dose finding of siremadlin during priming (Cycle 1) and a Bayesian time-to-DLT model will assess the siremadlin dose for combination. Efficacy will be assessed by proportion of pts remaining in CR for at least 6 months. Image: Summary/Conclusion: In clinical practice, treatment decisions follow a strategy. The estimand framework guided the design of a clinical study in AML post SCT to conceptualize the assessment of a treatment strategy. Accordingly, safety and efficacy of the treatment strategy as a whole will be assessed taking into account different patient journeys. P584: OVERALL SURVIVAL WITH INTENSIVE CHEMOTHERAPY (IC) VS NON-IC IN PATIENTS (PTS) WITH NEWLY DIAGNOSED (ND) AML FROM THE CONNECT® MYELOID DISEASE REGISTRY INELIGIBLE FOR RANDOMIZED CLINICAL TRIALS (RCT) H. Erba1,*, D. Pollyea2, M. Sekeres3, G. Garcia-Manero4,5, K. Seiter5, I. DeGutis6, P. Kiselev6, A. McBride6, E. Yu6, G. Roboz7,8 1Duke University, Durham; 2University of Colorado, Aurora; 3Sylvester Comprehensive Cancer Center, University of Miami Health System, Miami; 4The University of Texas MD Anderson Cancer Center, Houston; 5New York Medical College, Valhalla; 6Bristol Myers Squibb, Princeton; 7Weill Cornell Medicine; 8New York-Presbyterian Hospital, New York, United States of America Background: Pts with AML in RCTs often do not reflect the population seen in clinical practice due to strict eligibility criteria. Aims: To evaluate patient outcomes of IC vs venetoclax (VEN)-containing regimens based on eligibility criteria from a recent RCT (the VIALE-A trial) in a broad cohort of real-world pts with AML from the Connect® Myeloid Disease Registry (NCT01688011). Methods: Pts were stratified into 3 groups based on the non-IC phase 3 VIALE-A trial eligibility criteria: 1) “eligible” pts who would have met all VIALE-A inclusion criteria; 2) “unfit” pts who would have been ineligible for VIALE-A due to ≥ 1 of the following: abnormal liver/kidney function, high ECOG, recent prior malignancy, comorbidities score ≥ 2 by ACE-27, hepatic grade ≥ 1, AIDS grade ≥ 1; 3) “fit” pts who would have been ineligible for a VEN-based regimen in VIALE-A because they would have qualified for IC (defined as: ≤ 74 y of age, low ECOG, no apparent cardiovascular/renal comorbidities, and did not meet any criteria in #2). Baseline characteristics were summarized by eligibility group. Overall survival by group was estimated using the Kaplan–Meier method. Induction regimens were categorized as IC (any regimen containing 7 + 3, MEC, CLAG, FLAG) or VEN-based. Hazard ratios (HR) for induction regimens among each group were estimated using Cox models adjusted for age, ELN risk, ECOG, frailty score, and comorbidity index. Results: Of 734 enrolled pts with AML (Dec 2021), most were male (61%) and white (84%); median age 71 y (range, 55–97). Only 26% of pts (n = 192) were eligible for a non-IC RCT, 45% (n = 327) were ineligible due to unfitness, and 29% (n = 215) were ineligible due to overall fitness. The main reason for non-IC RCT ineligibility was high overall comorbidity grade (n = 265 [36%]). At baseline, fit pts intended to undergo transplant more often compared with unfit pts. Median duration of overall survival for eligible, unfit, and fit pts was 14, 10, and 22 months, respectively (HR [95% CI], eligible vs unfit, 0.02 [−0.19 to 0.22], P = 0.8735; eligible vs fit, 0.57 [0.33–0.81], P < 0.0001; unfit vs fit, 0.55 [0.33–0.77], P < 0.0001). Among unfit pts, those receiving IC had significantly longer overall survival compared with pts receiving a VEN-based regimen (median overall survival, 14 vs 6 months, respectively; HR, 0.51 [95% CI, 0.27–0.98]; P = 0.042; Figure). Unfit patients who received IC went on to transplant more frequently than those who received VEN-based therapies (16% [n = 17] vs 1% [n = 1], respectively). Eligible pts who received IC (n = 31) tended to have shorter median overall survival (13 months) vs pts who received VEN-based therapies (n = 27; 23 months; HR, 1.45, 95% CI, 0.66–3.17; not sig.). Among fit pts, median overall survival was 19 months for those receiving IC (n = 69) but could not be estimated for pts receiving VEN-based therapies due to small sample size (n = 10). Image: Summary/Conclusion: The majority of pts with ND AML in the Connect® Myeloid Disease Registry would have been ineligible for a non-IC RCT due to being too fit or unfit. Among pts ineligible for an RCT due to unfitness, there was an association with increased overall survival in pts receiving IC vs those receiving VEN-based therapies, and pts in the IC group were more likely to receive a transplant. This analysis suggests that RCTs may be excluding pts who appear unfit but can potentially tolerate IC and experience improved survival outcomes. P585: VENETOCLAX IN COMBINATION WITH INTENSIVE TREATMENT PROTOCOLS FOR PATIENTS WITH HIGH-RISK ACUTE MYELOID LEUKEMIA – A MULTICENTER REAL-WORLD ANALYSIS O. Wolach1,*, A. Frisch2, L. Shargian1, M. Yeshurun1, A. Apel3, V. Vainstein4, Y. Moshe5, S. Shimony1, O. Amit5, Y. Bar On5, Y. Ofran6, P. Raanani1, B. Nachmias4, R. Ram5 1Hematology, Rabin Medical Center, Petah Tikva; 2Hematology, Rambam Health Care Campus, Haifa; 3Hematology, Shamir Medical Center, Zriffin; 4Hematology, Hadassah Medical Center, Jerusalem; 5Hematology, Tel Aviv Sourasky Medical Center, Tel Aviv; 6Hematology, Shaare Zedek Medical Center, Jerusalem, Israel Background: The addition of venetoclax, a selective BCL2-inhibitor, to low intensity therapies was shown to improve the survival of patients with acute myeloid leukemia (AML) that are ineligible for intensive induction chemotherapy. Based on the encouraging anti-leukaemic properties of venetoclax and safety profile, several studies are currently exploring venetoclax in combination with high-intensity chemotherapy in high-risk AML scenarios such as ELN adverse-risk AML and in relapsed and refractoy (R/R) AML. The addition of venetoclax to established intensive chemotherapy protocols in these trials are generally reported to be associated with enhanced efficacy, frequently at the cost of significant hematological and infectious toxicities that requires dose modifications of both chemotherapy and venetoclax dosing Aims: To assess the safety and efficacy of venetoclax in combination with intensive treatment protocols in a ‘real-world’ setting. Methods: A multicenter retrospective cohort study in six academic centers in Israel. The decision to treat a patient with venetoclax in addition to an intensive treatment protocol as well as the choice of protocol and dosing were at discretion of the treating physician. Results: Twenty-nine patients were included in analysis (median age 53.4 years). Most patients were treated for R/R AML (n=27, 93%) with a median of one (0-5) previous lines of therapy and 48% of patients (n=14) having prior allogeneic hematopoietic-cell transplantation (HCT). Most patients were treated with venetoclax in combination with FLAG-IDA protocol (n=25, 86%). Median follow-up was 6.3 (0.7-23.7) months. Platelet and neutrophil recovery were observed at a median of 31 (95%CI 17.6-38.3) and 24 (95%CI 20-28) days, respectively. Infectious complications were common (blood stream infections, 41% and invasive fungal infections, 28%) and 30-day mortality was 14%. Composite complete remission (CRc) was 72% for the entire cohort and 91% in patients treated for post-HCT relapse. Incidences of relapse-free and overall survival at 12 months were 90% (95% CI 76%-98%) and 55% (95% CI 21%-63%), respectively. Image: Summary/Conclusion: The addition of venetoclax to intensive therapy is effective in high-risk AML, especially in post-HCT relapse setting. Prophylaxis and surveillance for infections are crucial. P586: IADADEMSTAT COMBINATION WITH AZACITIDINE SHOWS ENCOURAGING SAFETY AND EFFICACY DATA IN ELDERLY AND UNFIT AML PATIENTS O. Salamero1,*, T. Somervaille2, A. Molero1, E. Acuña-Cruz3, J. A. Perez-Simon4, R. Coll5, M. Arnan Sangerman6, B. Merchan7, A. Perez8, I. Cano3, R. Rodriguez-Veiga3, M. Arevalo9, S. Gutierrez9, C. Buesa9, F. Bosch8, P. Montesinos3 1Dep. of Hematology, Vall d’Hebron University Hospital. Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain; 2The Christie Hospital NHS Foundation Trust, Cancer Research Manchester Institute, Manchester, United Kingdom; 3Hospital Universitario y Politécnico La Fe, Valencia; 4Hospital Universitario Virgen del Rocio; Instituto de Biomedicina de Sevilla (IBIS)/CSIC/U. de Sevilla, Sevilla; 5ICO Hospital Dr Josep Trueta, Girona; 6Institut Català d’Oncología (ICO), Hospital de Bellvitge, Hospitalet de Llobregat; 7Hospital del Mar; 8Vall d’Hebron University Hospital. Vall d’Hebron Institute of Oncology (VHIO), Barcelona; 9Oryzon Genomics SA, Cornella de Llobregat, Spain Background: Acute Myeloid Leukemia (AML) is an hematological malignancy with highest incidence and lower survival rates in elderly. Previously reported ORR with hypomethylating agents such as azacitidine alone is less than 30%. Recently, combinations of hypomethylating agents with venetoclax have shown improved response ratios. However, refractoriness or relapse is still a challenge for most patients (pts), particularly in the elderly and the high-risk subpopulations. It has been shown that LSD1 is involved in malignant transformation in AML. Iadademstat (iada) is a potent and selective LSD1 inhibitor that has been administered to more than 100 oncology pts in Ph1 and Ph2 trials, showing manageable toxicity and signs of preliminary activity. Aims: ALICE (EudraCT 2018-000482-36) is a Phase IIa study to assess the safety, tolerability and the recommended Phase II dose (RP2D) of iada (PO 5d ON, 2d OFF every week in 28d cycles) in combination with azacitidine (sc., d1-5 and 8-9 every cycle) for the treatment of adult patients diagnosed with AML, as per WHO 2016 classification, who have not received prior treatment and are ineligible for intensive chemotherapy. We present an update of the ongoing study six months after completion of recruitment. Methods: Two doses of iada are studied in the trial: 60 and 90µg/m2/d. Besides the safety and tolerability primary endpoints, secondary endpoints investigate anti-leukemic activity including ORR according to ELN recommendations, time to response (TTR) and duration of responses (DoR). Additional assessments include residual detectable disease status, overall survival and PK/PD determinations. Results: At EHA 2021, ALICE data from 27 pts were reported. In October 2021, recruitment of the targeted 36 pts was completed. Patient baseline characteristics are shown in Table 1. Main safety events by the end of 2021 included 348 adverse reactions (AR). The most frequently reported AR was platelet reduction, observed in 44% of pts, however, Grade ≥3 thrombocytopenia was already present at baseline in 61% of pts (Table 1). Only three Grade 3-4 nonhematological treatment-related ARs were observed in two pts (asthenia, dysgeusia and weight decrease). Among the 71 serious AR reported, only 2 were considered as potentially related to iada, corresponding to a differentiation syndrome and a fatal intracranial hemorrhage, previously presented at EHA 2021. By the time this abstract is written, among the pts intended to treat (34), 27 have had at least 1 bone marrow evaluation after cycle 1 and are therefore evaluable for efficacy. 78% of them (21 of 27) achieved an objective response; of which, 62% were CR/CRi. Moreover, 80% of the CRi pts also achieved partial hematology recovery (CRh). Among CR/CRi pts, 5 out of the 8 already assessed by MRD presented no residual detectable disease by flow cytometry. DoR is encouraging with 77% of the CR/CRi lasting more than 6 months, with a mean DoR increasing as the study progresses, with the longest CR to date above 1,100 days. Iada at 90 µg/m2/d is the confirmed RP2D for the combination, showing deep responses with manageable toxicity. Image: Summary/Conclusion: ALICE data confirms that the combination of iada with azacitidine gives robust, fast and durable responses in unfit, previously untreated, AML patients. Considering iada’s efficacy, pharmacologic properties, its manageable toxicity and low anticipated drug-drug interactions, iada combinations may offer additional therapeutic options for AML patients in first line or in the R/R setting. P587: COVALENT-101: A PHASE 1 STUDY OF BMF-219, A NOVEL ORAL IRREVERSIBLE MENIN INHIBITOR, IN PATIENTS WITH RELAPSED/REFRACTORY ACUTE LEUKEMIA, DIFFUSE LARGE B-CELL LYMPHOMA, AND MULTIPLE MYELOMA F. Ravandi-Kashani1,*, A. Kishtagari2, H. Carraway3, G. Schiller4, E. Curran5, B. Yadav6, A. Cacovean6, S. Morris6, T. Butler6, J. Lancet7 1Department of Leukemia, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston; 2Vanderbilt-Ingram Cancer Center, Nashville; 3Cleveland Clinic Foundation, Cleveland; 4University of California Los Angeles, Los Angeles; 5University of Cincinnati Medical Center, Cincinnati; 6Biomea Fusion, Inc, Redwood City; 7Department of Malignant Hematology, Moffitt Cancer Center, Tampa, United States of America Background: Menin, a protein involved in transcriptional regulation, impacting cell cycle control, apoptosis, and DNA damage repair, plays a direct role in oncogenic signaling in multiple cancers. Inhibition of menin is therefore a novel approach to cancer treatment. Preclinical data of BMF-219, a highly selective, orally bioavailable, small-molecule irreversible inhibitor of menin, show sustained potent abrogation of menin-dependent oncogenic signaling in vitro and in vivo. BMF-219 exhibited a strong anti-proliferative effect on various menin-dependent acute myeloid leukemia (AML) cell lines, diffuse large B-cell lymphoma (DLBCL) cell lines representing Double/Triple Hit Lymphoma (DHL/THL) & Double Expressor Lymphoma (DEL), and multiple myeloma (MM) cell lines with diverse mutational backgrounds. BMF-219 also showed high potency ex vivo in patient samples from MLL-rearranged and NPM1-mutant AML, THL and MYC-amplified DLBCL, and bone marrow mononuclear cells from treatment-naive and relapsed/refractory (R/R) MM. Aims: COVALENT-101 (BF-MNN-101: NCT05153330) is a prospective, open-label, multi-cohort, non-randomized, multicenter Phase I study evaluating the safety, tolerability, and clinical activity of escalating doses of once daily oral BMF-219 in patients with R/R acute leukemia (AL), DLBCL and MM who have received standard therapy. The primary objective of the study is to determine independently for each cohort/indication, the optimal biological dose (OBD)/ recommended Phase 2 dose (RP2D) of BMF-219 oral monotherapy. Key secondary objectives include further evaluation of safety and tolerability, characterization of the pharmacodynamics and pharmacokinetics of BMF-219, and assessment of its antitumor activity based on best overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), and time to progression (TTP) per disease-specific response criteria as assessed by the investigator. Food-effect studies will be performed in DLBCL and MM patients at certain dose levels. Methods: Utilizing an accelerated titration design, doses of BMF-219 will be escalated in single-subject cohorts independently for each indication until 1 subject experiences either a ≥ Grade 2 related adverse event or dose limiting toxicity (DLT). At that point, the cohort will switch to a classical “3 + 3” design. Treatment will continue in 28-day cycles until progression or intolerability. Expansion cohorts for each indication will enroll patients to obtain further safety and efficacy data. Patients with R/R AL, R/R DLBLC who received ≥ 2 but ≤ 5 therapies, and R/R MM who received ≥ 3 therapies and have either failed or are ineligible for any standard therapies are eligible. Patients must have ECOG PS ≤ 2, and adequate organ function. Key exclusion criteria include known CNS disease involvement, prior menin inhibitor therapy, and clinically significant cardiovascular disease. Results: The study is ongoing. Summary/Conclusion: The enrollment commenced in January 2022. P588: IMPROVED OUTCOME OF RELAPSED/REFRACTORY ACUTE MYELOID LEUKEMIA BY ADDITION OF LIPOSOMAL ADRIAMYCIN TO CLAG CHEMOTHERAPY H. Yao1,*, C. Zhang1, J. Li2, X. Yin3, J. Rao1, X. Tan1, T. Chen1, L. Gao1, P. Kong1, X. Zhang1 1Medical Center of Hematology, Xin Qiao Hospital, Chong Qing; 2Hematology, Changsha Central Hospital Affiliated to Nanhua University, Hu Nan; 3Department of Hematology,the 923rd Hospital of the Joint Logistics Support Force of the People′Liberation Army of China, Guang Xi, China Background: Prognosis of refractory and relapsed acute myeloid leukemia (r/r AML) is challenge and no standard of treatment exists. Cladribine based regimen in treatment of r/r AML proved to be effective,while the total rate of response is limited. Aims: To explore whether the addition of liposomal adriamycin can improve the response. Methods: We registered a clinical trial, which is CLAG plus liposomal adriamycin (CLAG+PLD) regimen for re-induction of r/r AML (ChiCTR1800017569), to observe the response, overall survival (OS) and disease free suivival (DFS) of the patients. In addition to that, CLAG alone as salvage therapy (no PLD group) for RR-AML in our medical center were chosen as historical comparison Results: To observe the response, overall survival (OS) and disease free suivival (DFS) of the patients. In addition to that, CLAG alone as salvage therapy (no PLD group) for RR-AML in our medical center were chosen as historical comparison.Of the 55 patients who were evaluable for remission after CLAG +PLD treatment(PLD group), 27（49.1%）achieved CR and negative of MRD, 12（21.8%）achieved CR and positive of MRD, 5（9.1%）achieved partial response, and 11（20%）no response. In Contrast, no PLD group has inferior response rate,with the corresponding data is 24（46.6%）, 6（10.9%）,10（18.2%）,and 13（23.6%）respectively (p= 0.008).As for the 2-year OS and DFS, the median OS and DFS for PLD group has not reached and 10 months for noPLD group (p= 0.023 and p= 0.045 respectively). The safety observation did not find increasing toxicity in PLD group.In subgroup analysis of PLD group, the response between FLT3-ITD mutated group and wild group showed significant difference (p=0.006). Moreover, hematopoietic stem cell transplantation (HSCT) group exert superiority in both OS and DFS compared to no HSCT group with 2-year of follow-up（p＜0.0001 and p＜0.00001 respectively）. Image: Summary/Conclusion: In conclusion, CLAG + PLD is a prospective salvage regimen for r/r AML which brings improved response rates, 2-year OS and DFS and similar toxicities compared to CLAG. P589: AGE AND AGE-ADAPTED RISK FACTORS HIGHLY INFLUENCE SURVIVAL PROBABILITY IN AML PATIENTS – A RETROSPECTIVE ANALYSIS K. Nachtkamp1, C. Zahner1,*, A. Fricke1, T. Ulrych1, F. I. Schulz1, A. Kündgen1, P. S. Jäger1, G. Kobbe1, U. Germing1 1Klinik für Hämatologie, Onkologie und Klinische Immunologie, Uniklinik Düsseldorf, Düsseldorf, Germany Background: Advanced age is strongly associated with inferior overall survival in patients with acute myeloid leukemia (AML). Despite advances in modified treatment regimen and genetic analyses of underlying mutations the prognosis for patients above the age of 65 remains poor. Aims: The aim was to determine patient-related risk factors and disease-associated variables. Methods: 915 AML patients (498 < 65 years, 417 ≥ 65 years) treated at the University Hospital of Düsseldorf were analyzed. Patients were diagnosed between 2008 and 2020 comprising 484 males and 431 females. Median age was 63 years. Patients’ survival was calculated using Kaplan-Meier-plots and multivariate regression analyses were performed to identify prognostic factors for survival. Results: Age itself proved to be an independent risk factor for survival: 6.9 months within the group of patients ≥ 65 years, 166 months in patients < 65 years (p < 0.001). In patients ≥ 65 years aberrant cytogenetic findings and an inferior ECOG status were more frequent at the time of diagnosis. Comparison of survival within the same cytogenetic risk group yielded significant differences between the two age-stratified cohorts: Bearing cytogenetic aberrations associated with a favorable prognosis in the group with patients aged over 65 was associated with a median survival of 18 months whereas patients younger than 65 did not reach estimated median survival time (p < 0.001). When comparing patients with the same ECOG-status, advanced age was the most decisive prognostic factor for survival. This was highly significant in patients with ECOG status 0. In the group with higher ECOG status an influence of age was notable but of lower significance. In patients with pathologic lung function tests age turned out to be significant for survival: 26 months (≥ 65 years) vs. not reached (< 65 years) (p = 0.008). In patients with pathologic cardiac ultrasound findings results were similar with 26 months (≥ 65 years) vs. not reached (< 65 years) (p = 0.001). Whether AML was de novo or secondary affected survival probability only in the group aged below 65 (166 months de novo AML, 69 months secondary AML). Summary/Conclusion: Age has significant influence on patient- and leukemia-related factors, thus modulating the impact of other parameters on the prognosis. We were able to confirm the following independent negative predictive parameters on survival in AML patients: increasing age ≥ 65 years, high-risk genetic aberrations, comorbidities (lung, heart) and higher ECOG-status. P590: PRELIMINARY SAFETY AND EFFICACY OF BGB-11417, A POTENT AND SELECTIVE B-CELL LYMPHOMA 2 (BCL2) INHIBITOR, IN PATIENTS (PTS) WITH ACUTE MYELOID LEUKEMIA (AML) J. Shortt1,*, S. Y. Tan2, P. Cannell3, T. F. Ng4, C. Y. Fong5, S. Ramanathan6, R. Rajagopal7, S. Leitch8, R. Gasiorowski9, C. Grove10, D. Lenton11, P. Tan12, C. DiNardo13, M. T. Ling14, S. Cheng14, Y. Liu14, M. Co14, W. Y. Chan14, D. Simpson14, A. H. Wei12,15 1School of Clinical Sciences, Monash University and Monash Health, Clayton; 2St Vincent’s Hospital Melbourne, Fitzroy, Victoria; 3Fiona Stanley Hospital, Murdoch, Western Australia; 4Gold Coast University Hospital, Southport, Queensland; 5Austin Health, Heidelberg, Victoria; 6The Saint George Hospital-Kogarah, Kogarah, New South Wales, Australia; 7Middlemore Hospital; 8North Shore Hospital, Auckland, New Zealand; 9Concord Repatriation General Hospital, Concord West, New South Wales; 10Linear Clinical Research & Sir Charles Gairdner Hospital, Nedlands, Western Australia; 11Orange Health Service (Central West Cancer Care Centre), Orange, New South Wales; 12One Clinical Research, Nedlands, Western Australia, Australia; 13University of Texas MD Anderson Cancer Center, Houston, TX; 14BeiGene (Shanghai) Co., Ltd., Shanghai, China and BeiGene USA, Inc., San Mateo, CA, United States of America; 15Department of Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Victoria, Australia Background: BCL2, a key regulator of apoptosis promoting cancer cell survival, is aberrantly expressed in many hematologic malignancies. The BCL2 inhibitor, venetoclax, is standard of care for the treatment of newly diagnosed AML in adults unfit for induction chemotherapy. Although venetoclax-based treatments have improved outcomes for pts with AML, there are concerns surrounding disease resistance after continued use and adverse events (AEs) such as gastrointestinal toxicities and neutropenia. Clinical findings have shown that 20-30% of pts using venetoclax are refractory to treatment, and a majority of pts who initially achieve remission ultimately relapse (N Engl J Med. 2020;383(7):617-629). The highly selective investigational BCL2 inhibitor, BGB-11417, demonstrated more potent antitumor activity than venetoclax in preclinical studies (Cancer Res. 2020;80[suppl, abstr]:3077). Aims: To present preliminary safety and efficacy results of BGB-11417 in combination with azacitidine in pts with AML enrolled in BGB-11417-103 (NCT04771130). Methods: BGB-11417-103 is an ongoing phase 1b/2, global, multicenter, dose-escalation and expansion study evaluating the combination of BGB-11417 and azacitidine in pts with AML (either treatment-naïve [TN] unfit for intensive induction chemotherapy or relapsed/refractory [R/R]), myelodysplastic syndrome, or myelodysplastic syndrome/myeloproliferative neoplasm. Pts who received prior azacitidine or BCL2 inhibitors were excluded. For pts with AML, the 10-day regimen consisted of BGB-11417 at 40 mg (Cohort 1), 80 mg (Cohort 2), or 160 mg (Cohort 3) in combination with azacitidine (75 mg/m2 x 7 days). In Cycle 1, a 4-day ramp-up of BGB-11417 was utilized starting at 1/8 of the target dose. The window to assess dose-limiting toxicity (DLT) was up to Day 28 for nonhematologic toxicities and Day 42 or initiation of Cycle 2 for hematologic toxicities. Treatment-emergent AEs were graded per Common Terminology Criteria for AEs v5.0. Responses were assessed using the 2017 European LeukemiaNet criteria (Blood. 2017;129(4):424-447). All pts gave informed consent. Results: As of 10 January 2022, 27 pts with AML were treated with the combination therapy (6 in Cohort 1; 15 in Cohort 2; 6 in Cohort 3; Table). Median age was 80 yrs (TN; 18 pts) and 70 yrs (R/R AML; 9 pts); 44% of pts had adverse karyotype. Median prior line of therapy for R/R AML was 1 (range 1-2). At a median study follow-up of 2.1 months and median duration of treatment of 1.84 months (range 0.3-7.6), 2 of 23 evaluable pts experienced DLTs (Grade 4 neutropenia and Grade 4 thrombocytopenia at the 80 mg x 10 day dose level), which did not meet the safety stopping protocol criteria. Laboratory tumor lysis syndrome (TLS) was observed in 1 pt with a known history of chronic kidney disease treated with 160 mg x 10 day; pt was asymptomatic and TLS resolved within 4 days. Most common nonhematologic AEs were constipation (37%) and azacitidine injection-site reaction (33%). Most common Grade ≥3 hematologic AEs were neutropenia (44%), thrombocytopenia (41%), and anemia (37%). No pts required dose reductions of BGB-11417. Majority (n=17; 63%) of pts are continuing study treatment. Ten pts discontinued due to AEs (n=3), proceeding to transplant (n=3), consent withdrawal (n=2), or disease progression (n=2). Preliminary CR/CRh rates for TN and R/R were 56% and 44%, respectively. Seven of the 9 CRs were achieved by end of Cycle 1. Image: Summary/Conclusion: Preliminary results show that a 10-day regimen of BGB-11417 plus azacitidine resulted in a majority of CR observed by the end of Cycle 1 and was well tolerated in pts with AML. P591: PRELIMINARY RESULTS OF A PHASE I STUDY OF CLIFUTINIB, A HIGHLY SELECTIVE, POTENT ORAL FLT-3 INHIBITOR, IN PATINETS WITH FLT3-MUTATED RELAPSED OR REFRACTORY ACUTE MYELOID LEUKEMIA W. Yu1, X. Wei2, Z. Ge3, Y. Li4, Z. Jiang5, L. Yang6, L. Lin7, Y. Yao8, X. Deng9, X. Du10, Y. Li11, T. Chen12, X. Feng13, J. Zhou14, M. Hou15, R. Fu16, J. Lan17, X. Hu18, S. Huang19, J. Wang20, X. Du21, H. Yang22, H. Yang23, H. Wang1, L. Zheng24, Z. Wang24, B. Liu24, N. Kang24, Y. Zhuang24, Y. Zhang24, J. Jin1,* 1The First Affiliated Hospital of College of Medicine, Zhejiang University, Hangzhou; 2Henan Cancer Hospital, Zhengzhou; 3Zhongda Hospital, Southeast University, Nanjing; 4Zhujiang Hospital of Southern Medical University, Guangzhou; 5The First Affiliated Hospital of Zhengzhou University, Zhengzhou; 6The Second Hospital of Shanxi Medical University, Taiyuan; 7​Hainan General Hospital, Haikou; 8Baoji Central Hospital, Baoji; 9Weihai Municipal Hospital, Weihai; 10Guangdong General Hospital; 11Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou; 12Huashan Hospital, Shanghai; 13The Affiliated Hospital of Qingdao University, Qingdao; 14Wuhan Tongji Hospital, Wuhan; 15Qilu Hospital of Shandong University, Jinan; 16Tianjin Medical University General Hospital, Tianjin; 17Zhejiang Provincial People’s Hospital, Hangzhou; 18Beida Medical Treatment Luzhong Hospital, Zibo; 19Xi’an Gaoxin Hospital, Xian; 20Aerospace Center Hospital, Beijing; 21The Second People’s Hospital of Shenzhen, Shenzhen; 22Shunde Hospital of Southern Medical University, Foshan; 23The 1st Affiliated Hospital of He’nan University of Science and Technology, Luoyang; 24Sunshine Lake Pharma Co., Ltd, Dongguan, China Background: An internal tandem duplication (ITD) in the juxtamembrane domain of FLT3 on chromosome 13q12, which accounts for 30% of patients with Acute Myeloid Leukemia (AML), is associated with poor prognosis, including a lower complete remission (CR) rate, a reduced duration of remission (DOR), a higher relapse rate, and a decreased overall survival. Clifutinib, a highly selective oral FLT-3 inhibitor, demonstrated potent activity against FLT3-ITD mutated human cell lines, both in vitro and in vivo. Aims: This present study (NCT04827069) is a first-in-human study of Clifutinib, including dose-escalation and dose-expansion phases, with a purpose to evaluate the safety, tolerability, pharmacokinetics (PK) and antitumor activity in Chinese patients with relapsed/ refractory (R/R) FLT3-mutated AML. Here we report the preliminary results of the study. Methods: AML patients aged≥18 years with refractory to ≥ 2 cycles of standard induction chemotherapy, or relapsed after achieved remission from prior treatments were enrolled. Previous use of FLT3 inhibitors was allowed. The primary endpoints included safety and tolerability, including dose limiting toxicity (DLT), adverse event (AE). Secondary endpoints were PK and anti-leukemic effect [CR/ CRh (Complete Remission with Partial Hematologic Recovery) rate, Composite Complete Remission (CRc) rate, DOR, and overall survival (OS)]. Clifutinib was administered orally on an empty stomach at 10~70 mg daily, 28 consecutive days as a treatment cycle. Resumption of Clifutinib was not allowed after hematopoietic stem cell transplantation (HSCT). Modified 3 + 3 and accelerated titration design was utilized. Results: As of December, 2021, fifty-seven patients were enrolled, including one at 10 mg, one at 20 mg, 6 at 55 mg, 34 at 40 mg, 15 at 70mg dose groups. The median age was 52 years, and one third of the subjects received prior FLT-3 inhibitors treatments. Two subjects discontinued from the study due to AEs. Eighteen subjects were evaluable for DLT determination. Only one subject in the 55 mg group experienced DLT (Grade 3 QT prolongation), and MTD had not been reached. Fifty-three subjects were evaluable for safety analysis. The most common treatment-related AEs of any grade included platelet count decreased (69.8%), decreased white cell count (69.8%), neutrophil count decreased (60.4%), anemia (43.3%), lymphocyte count decreased (32.1%). Plasma concentrations of Clifutinib increased with increasing dose, with tmax between 2 and 6 hours. Accumulation was observed following repeat dosing, with estimated t1/2 values of 68.3 to 114.2 hours based on accumulation index. Approximately dose-linear PK parameters were observed over the dose range for both single and repeat dosing. Of 53 FLT3-ITD positive, evaluable subjects, CR/CRh rate was 17.0% (9/53), and CRc rate was 45.3% (24/53). Among subjects administered with Clifutinib 40 mg daily, the CR/CRh rate was 18.2% (6/33) with 3 patients achieved CR, median DOR of CR/CRh was 5.7 months, the CRc rate was 48.5% (16/33), and median OS was 7.4 months. Among patients experienced only one prior regimen, the CR/CRh rate of 40 mg dose group was 25% (4/16), the CRc rate was 50% (8/16), and the median OS was 13.0 months. Summary/Conclusion: Preliminary results of this phase 1 study demonstrated that Clifutinib has an acceptable safety profile and promising antitumor activity, especially at the dose of 40 mg daily. A confirmatory phase 3 study is planned to further evaluate the efficacy and safety of Clifutinib at 40 mg daily in FLT3-mutated R/R AML. P592: GILTERITINIB IN COMBINATION WITH VENETOCLAX, LOW DOSE CYTARABINE AND ACTINOMYCIN D FOR FLT3 MUTATED RELAPSED OR REFRACTORY ACUTE MYELOID LEUKEMIA A. Žučenka1,*, R. Pileckytė1, K. Maneikis1, V. Vaitekėnaitė1, L. Kevličius1, L. Griškevičius1 1Hematology, Oncology, Transfusion Medicine Center, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania Background: A second generation FLT3 inhibitor Gilteritinib has become a standard of care for FLT3 mutated relapsed or refractory acute myeloid leukemia (FLT3m R/R AML). However, remission duration and overall survival remain unsatisfactory. Preliminary results of doublet Gilteritinib + Venetoclax and triplet Gilteritinib + Venetoclax + Hypomethylator regimens are encouraging. Herein, we report the quadruplet regimen consisting of Gilteritinib, Venetoclax, Low Dose Cytarabine and Actinomycin D (ACTIVE + G) for the treatment of FLT3m R/R AML in the clinical practice setting. Aims: To evaluate the efficacy and safety of the ACTIVE + G regimen. Methods: This was an observational, retrospective study. The patients were at least 18 years of age and had FLT3m R/R AML. All patients provided informed consent for treatment and data collection. The ACTIVE + G regimen consisted of Venetoclax 600mg/d p/o from day 1 up to day 28, Cytarabine 20mg/m2 s/c on days 1-10, Actinomycin D 12.5 µg/kg i/v on days 1-3 (on days 1-2 for patients ≥65 years) and Gilteritinib 120mg/d p/o starting from either day 4 or day 10 and continued up to day 28. Indications for stopping Venetoclax and Gilteritinib before day 28 were life-threatening infections or faster hematological recovery in responding patients. A second ACTIVE + G cycle was administered in non-responders without evidence of progressive disease after Cycle 1 or in responders with positive measurable residual disease (MRD). Responders after ACTIVE + G could proceed to either allogeneic stem cell transplantation (alloSCT) or maintenance therapy with Venetoclax, Low Dose Cytarabine and Gilteritinib. We evaluated baseline characteristics, composite CR (CRc = CR + CRi + CRp), overall response (ORR = CRc + MLFS), MRD negativity rates (<0.1% by multiparameter flow cytometry), overall survival (OS), relapse-free survival (RFS), grade 3-5 non-hematological toxicities and day 30 and day 60 mortality rates. Results: Fifteen patients had been treated with ACTIVE + G, of whom 8 (53%) were female. The median age was 66 years (34-87), median ECOG was 2 (0-3). FLT3-ITD mutation was confirmed in 80% (12/15) of cases and 20% (3/15) had FLT3-TKD. The most common co-mutations were NPM1 (53%, 8/15), DNMT3A (27%, 4/15), IDH1 (20%, 3/15) and IDH2 (20%, 3/15). Three patients (20%) had adverse cytogenetics. The median number of previous treatment lines was 2 (1-5). Twelve patients (80%) had received prior intensive chemotherapy, 3 patients (20%) had prior Venetoclax exposure and 9 patients (60%) had been previously treated with FLT3 inhibitors (Midostaurin – 7, Sorafenib – 1, Gilteritinib – 1). Three patients (20%) had relapsed after alloSCT. The majority of patients (80%, 12/15) had received 1 cycle of ACTIVE + G, 3 patients (20%) had been treated with 2 cycles. The CRc and the ORR were 67% (10/15) and 93% (14/15), respectively. MRD negativity was confirmed in 50% (5/10) of CRc cases. The median OS and RFS were 8.6 and 12.9 months, respectively. The most common non-hematological grade 3-5 adverse events were febrile neutropenia (73%, 11/15), sepsis/bacteremia (53%, 8/15), pneumonia (33%, 5/15) and secondary hemophagocytosis (13%, 2/15). Day 30 and day 60 mortality rates were 13% (2/15) and 20% (3/15), respectively. Image: Summary/Conclusion: A quadruplet regimen ACTIVE + G demonstrated high efficacy in this small group of R/R FLT3m AML patients irrespective of their prior exposure to FLT3 inhibitors or Venetoclax. The main toxicities were infectious complications attributable to prolonged myelosuppression. Prospective clinical trials are needed to verify our results. P593: MULTIDIMENSIONAL ANALYSIS OF THE B CELL RECEPTOR OFFERS INSIGHT INTO THE ONTOGENETIC RELATIONSHIP OF MONOCLONAL B-CELL LYMPHOCYTOSIS WITH CHRONIC LYMPHOCYTIC LEUKEMIA A. Agathangelidis1,*, C. Galigalidou2, A. Iatrou2, L. Zaragoza-Infante2, L. Scarfò3, M. C. Maniou2, P. Ranghetti3, N. Pechlivanis2, V. Junet4, A. Skaftason5, M. Tsagiopoulou2, F. Psomopoulos2, R. Rosenquist5, P. Ghia6, A. Chatzidimitriou2, K. Stamatopoulos2 1Department of Biology, Section of Genetics and Biotechnology, National and Kapodistrian University of Athens, Athens; 2Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece; 3Division of Experimental Oncology, Università Vita-Salute San Raffaele/Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale San Raffaele, Milan, Italy; 4Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Barcelona, Spain; 5Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 6Division of Experimental Oncology, Università Vita-Salute San Raffaele/Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale San Raffaele, Milan, Greece Background: The clonotypic B-cell receptor immunoglobulin (BcR IG) is key to the pathogenesis of CLL. Against that, however, scant information exists regarding the role of BcR IG in early stages of CLL ontogenesis, i.e. monoclonal B cell lymphocytosis (MBL). Aims: Here, we aimed to address this issue through the integrative analysis of: (i) the BcR IG gene repertoire, (ii) the transcriptome, and, (iii) the BcR IG reactivity profile, of both the high and the low-count MBL subtypes (HC-MBL and LC-MBL, respectively), complemented by comparisons to CLL. Methods: The study group comprised 17 individuals with LC-MBL and 14 with HC-MBL. “CLL-like” cells were flow-sorted. BcR IG NGS data was annotated with IMGT/HighV-QUEST and TRIPR. Clonotypes were defined as IG gene rearrangements with identical IG variable gene and CDR3 amino acid sequence. Stereotypy analysis in MBL cases focused on abundant clonotypes, which were compared against data from >39,000 CLL cases. Intraclonal diversification was assessed through graph networks of all distinct nucleotide variants of each clonotype. Transcriptome profiling was performed on polyA-selected RNA from 16 LC-MBL and 8 HC-MBL cases; comparisons were undertaken against RNAseq data from 74 CLL patients. Count normalization, differential gene expression and splice event analysis were performed with DESeq2, Limma and SGSseq, respectively; gene enrichment analysis was performed with Enrichr (p<0.05). BcR IG reactivity profiling was performed for 6 LC-MBL cases, 10 HC-MBL cases and 9 CLL cases against 123 autoantigens using a protein microarray; validation tests were performed with ELISA. Results: Clonality assessment revealed a monoclonal profile for most HC-MBL cases (86%), whereas LC-MBL cases were mainly oligoclonal (53%). Stereotypy analysis revealed a stronger immunogenetic connection of HC-MBL to CLL, since 19/47 abundant clonotypes (40%) from HC-MBL were assigned to CLL stereotyped subsets as opposed to only 19/167 (11%) from LC-MBL. Intraclonal diversification analysis was undertaken for: (i) 26 monoclonal cases (from HC-MBL or LC-MBL), (ii) 10 oligoclonal LC-MBL cases and 73 monoclonal CLL cases. Significant differences (p<0.05) were evident between oligoclonal MBL vs monoclonal MBL vs CLL; the former showed wide branching of variants due to distinct somatic hypermutations (SHM). In contrast, monoclonal MBL and CLL displayed progressively higher levels of variant connectivity due to the presence of shared SHMs, likely reflecting stronger antigenic pressure. Comparative transcriptomic analysis of either type of MBL vs CLL disclosed significant (p<0.05) down-regulation of genes in the Notch, AMPK and BcR signaling pathways. Moreover, LC-MBL showed overexpression of genes implicated in antigen processing and presentation and OXPHOS compared to either HC-MBL or CLL. In addition, both MBL types were characterized by virtual absence of alternative splicing, contrasting CLL where splicing events were identified in 14 genes of the BcR signaling pathway. Finally, BcR IG reactivity analysis revealed different patterns among individual cases, however all examined cases from MBL and CLL consistently bound a set of autoantigens, including myelin, centromere protein B, β2-microglobulin, C-reactive protein and the nucleosome. Summary/Conclusion: In conclusion, we highlight important differences between MBL and CLL stemming from the attributes of the BcR IG and the related signaling pathway. Overall, the reported findings allude to different maturation processes that likely contribute to shaping a distinct BcR IG signaling capacity in MBL, particularly LC-MBL, vs CLL. P594: INCREASED FREQUENCIES OF IGH LOCUS SUICIDE RECOMBINATION POINTS ON CHRONIC LYMPHOCYTIC LEUKEMIA WITH LOW RATE OF AID RELATED SOMATIC MUTATIONS, MYC OVEREXPRESSION AND SHORT TELOMERES I. Al Jamal1,2,*, M. Parquet1, H. Boutouil1, K. Guiyedi1, D. Rizzo1,3, M. Dupont1,3, M. Boulin1,3, S. Aoufouchi4, S. Al Hamaoui2, N. Makdissy2, J. Feuillard1,3, N. Gachard1,3, S. Peron1 1Centre National de la Recherche Scientifique (CNRS), UMR 7276/INSERM U1262,Limoges University, Limoges, France; 2Faculty of Sciences, GSBT Genomic Surveillance and Biotherapy Team, Lebanese University, Tripoli, Lebanon; 3Limoges University Hospital Center, Biological Hematology Laboratory, Limoges; 4UMR 9019 Genome Integrity and Cancers, Villejuif, France Background: In normal B-cells, Activation Induced-cytidine deaminase (AID) is the key enzyme for class switch recombination (CSR) and IGHV somatic hypermutation (SHM). AID is also implicated in another IgH rearrangement, the Locus Suicide Recombination (LSR). LSR occurs in activated B-cells and recombines the IgH locus between the switch µ (Sµ) region and one 3’a2 regulatory region (3’a2RR) of the IgH locus. LSR results in the complete deletion of the cluster of IgH constant genes. When LSR hits the active IgH locus, it induces the loss of BCR expression and the death of the concerned B cell. Chronic lymphocytic leukemia (CLL) is an indolent non-Hodgkin B-cell lymphoma. Tumor CLL B-cells weakly express a B cell receptor (BCR) on the surface which is composed, in the vast majority of cases, of immunoglobulins (Ig) of the mu (µ) and delta (δ) isotypes and Ig class-switched CLL are rare, raising the question of abnormalities in the Ig gene recombination machinery in this B-cell cancer. Aims: Searching for abnormalities of IgH locus recombination in CLL, we investigated CSR and LSR in CLL. Methods: In this study, we used different molecular biology technics as high throughput Sequencing (HTS) to analyze CSR and LSR junctions, IgHV, and PIM-1 mutation. Quantitative reel time PCR (quantification of AID, cMYC and IgH locus transcripts). Samples are from CLL patients (N=47) with more than 98% blood tumor cell infiltration and controls consisted in healthy volunteers (HV) (N=9). Bioinformatics uses CSReport tool. Statistics are from graph pad or R. Results: CSR levels were lower in CLL than in HV samples contrariwise to LSR that was found at comparable levels in both HV and CLL groups (Fig. A, B). Moreover, some patients exhibited increased LSR counts. Because distribution of LSR counts was bimodal with a valley at 27, that value being also the mean of LSR counts in HVs, we separated CLL patients in two groups so called LSR-High and LSR-Low with that threshold of 27 LSR count per sample. We analyzed the diversity of the LSR junctions using Shannon Index. LSR junction diversity was significantly higher in LSR-High than in LSR-Low CLLs and HVs. This result is in opposition with the IgHV mutation rate since LSR-High CLLs exhibited a stronger homology to IgHV reference sequences (unmutated CLLs). We also found that LSR-High CLLs exhibited a marked lowest rate of AID off-target PIM1 mutations (Fig. C). AID expression was low and at comparable levels in both LSR-Low and LSR-High CLLs. Because diversity of LSR junctions in LSR-High CLLs is evocative of an on-going process, we analyzed the expression of productive and non-productive transcripts of the constant part of IgH locus, which we found increased in LSR-High when compared to LSR-Low CLLs. We also observed shorter telomere lengths and c-Myc overexpression in the LSR-High group. Both shorter telomeres and c-Myc overexpression are very likely to reflect an increased number of DNA replication cycles in the history of the CLL tumor cell (Fig. D, E). Consistently, Kaplan Meyer curves of Treatment Free Survival (TFS) show that TFS of LSR-High CLLs was significantly shorter than the one of LSR-Low counterpart, an indication of a more rapidly evolving CLLs (Fig. F). Image: Summary/Conclusion: Altogether, these results indicate the accessibility of IgH locus and the proliferation in CLL patients with high rate and increased diversity of LSR junctions could be increased in Myc dependent manner resulting in shorter survival and pointing on an AID independent mechanism of IgH recombination. P595: DISCOVERY OF NOVEL CIRCULAR RNAS (CIRCRNAS) OF THE APOPTOSIS-RELATED BAX AND BCL2L12 GENES WITH A MULTIFACETED ROLE IN CHRONIC LYMPHOCYTIC LEUKEMIA, USING CUTTING-EDGE NANOPORE SEQUENCING TECHNOLOGY P. Artemaki1,*, N. Machairas1, P. Karousi1, V. Pappa2, A. Scorilas1, S. Papageorgiou2, M. Batish3, C. Kontos1 1Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens; 2Second Department of Internal Medicine and Research Unit, University General Hospital “Attikon”, Athens, Greece; 3Department of Medical and Molecular Sciences, University of Delaware, Newark, DE, United States of America Background: Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key indirect regulators of gene expression, with deregulated expression and established involvement in several human malignancies. Many circRNAs have been shown to bind microRNAs (miRNAs), thus making miRNAs unavailable to regulate protein-coding gene expression. Moreover, some of them have been described to interact with chromatin and affecting its structure. Despite their pivotal roles in the development and progression of cancer, only three studies have investigated their expression and role(s) in chronic lymphocytic leukemia (CLL), so far. The products of BCL2 and its homologues, including BAX and BCL2-like 12 (BCL2L12), are implicated in CLL, as apoptosis regulators. Moreover, expression levels of both BAX and BCL2L12 mRNAs are deregulated in CLL and are associated with patients’ response to therapy and overall survival. However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their regulatory potential. Aims: We sought to further elucidate the contribution of both BAX and BCL2L12 in CLL by unraveling the identity of their circRNAs in a B-cell leukemic cell line (EHEB) and by studying in silico their potential interactions with miRNAs having an already established role in CLL. Methods: EHEB cells were propagated according to ATCC instructions. After total RNA extraction, 2μg of total RNA were reverse-transcribed using random hexamers. Next, nested PCR with divergent primers was performed, starting from each exon of BAX and BCL2L12 genes; thus, only cDNAs from BAX and BCL2L12 circRNAs were amplified, respectively. After having mixed and purified all nested-PCR products, nanopore sequencing libraries were built according to an optimized protocol. Following nanopore sequencing and basecalling in a MinION Mk1C 3rd-generation sequencer, sequencing reads were aligned and corrected using Minimap2 and TranscriptClean, respectively. circRNA sequences were identified using an in-house–built, PERL-based algorithm. A single-molecule resolution fluorescent in situ hybridization (FISH) method called circFISH was used to visualize the circRNA distribution in the cells. Lastly, miRDB was used to predict interactions of the most abundant BAX and BCL2L12 circRNAs with miRNAs. Results: We discovered 18 and 21 novel circRNAs produced by BAX and BCL2L12 genes, respectively, in this leukemic cell line. The exon structure of these novel circRNAs showed remarkable diversity, also comprising genomic regions that have previously been considered as intronic, as they are not included in messenger RNAs (mRNAs). Interestingly, one of the two most abundant BAX circRNAs is composed of a single exon, also spanning two introns. Similar intron retentions have been observed in a few BCL2L12 circRNAs. The gamut of miRNAs predicted to be bound by novel circRNAs includes miR-484, miR-181a-2-3p and miR-214-3p. Particularly, the inhibition of miR-214-3p is associated with elevated apoptosis in leukemic B-cells. Summary/Conclusion: Alternative splicing and back-splicing mechanisms of the primary BAX and BCL2L12 transcripts produce multiple distinct circRNAs in this B-cell CLL cell line. Interestingly, one of the two most abundant circular transcripts of BAX is a circRNA spanning three exons and two intervening introns of this gene. Lastly, several miRNAs are predicted to be sequestered by circRNAs of either of these apoptosis-related genes, including miR-181 family members that have been associated with chemosensitivity. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL. P596: CLINICAL IMPACT OF TP53 DISRUPTION IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS TREATED WITH A BCR INHIBITOR. A CAMPUS CLL EXPERIENCE R. Bomben1,*, F. M. Rossi1, F. Vit1, T. Bittolo1, A. Zucchetto1, R. Papotti2, E. Tissino1, F. Pozzo1, M. Degan1, E. Zaina1, I. Cattarossi1, P. Nanni1, R. Marasca3, G. Reda4, L. Laurenti5, J. Olivieri6, A. Chiarenza7, L. Ballotta8, A. Cuneo9, M. Gentile10, F. Morabito11, A. Tafuri12, F. Zaja13, R. Foà14, F. Di Raimondo15, G. Del Poeta16, V. Gattei1 1Clinical and Experimental Onco-Heamtology Unit, Centro di Riferimento Oncologico IRCCS, Aviano; 2International PhD School in Clinical and Experimental Medicine, University of Modena and Reggio Emilia; 3Hematology Unit, Department of Oncology and Hematology, Azienda-Ospedaliero Universitaria (AOU) of Modena, Policlinico, Modena; 4Division of Ematologia, Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico di Milano, Milano; 5Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Universitaria Policlinico A Gemelli di Roma, Roma; 6Clinica Ematologica, Centro Trapianti e Terapie Cellulari “Carlo Melzi” DISM, Azienda Ospedaliera Universitaria S. Maria Misericordia, Udine; 7Division of Hematology, Policlinico, Department of Surgery and Medical Specialties, University of Catania, Catania; 8Dipartimento Clinico di Scienze Mediche, Chirurgiche e della Salute, University of Trieste, Trieste; 9Hematology Section, Department of Medical Sciences, University of Ferrara, Ferrara, Ferrara; 10Hematology, Unit AO of Cosenza; 11Biothecnology Research Unit, AO of Cosenza, Cosenza; 12Department of Clinical and Molecular Medicine and Hematology, Sant’Andrea - University Hospital - Sapienza, University of Rome, Rome; 13Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste; 14Hematology, Department of Translational and Precision Medicine, ‘Sapienza’ University, Roma; 15Division of Hematology, Policlinico, Department of Surgery and Medical Specialties, University of Catania, Catania; 16Division of Haematology, University of Tor Vergata, Roma, Italy Background: In chronic lymphocytic leukemia (CLL), the presence of TP53 aberration (mutations and/or deletion) predicts an increased risk of relapse and death after chemo-immunotherapy (CIT). Although TP53 deletion and mutations mostly co-occur and are considered equal prognosticators, the clinical impact of isolated or concomitant mutations and deletions remains unclear in the context of targeted agents. Aims: To investigate the clinical relevance of isolated or concomitant TP53 aberrations in a large cohort of CLL patients treated with BCR inhibitors. Methods: In the framework of an institutional Italian multicenter working group on CLL (Campus CLL), a retrospective analysis of 229 CLL patients (51 treatment naïve, and 178 relapsed/refractory) treated with ibrutinib was carried out. All patients referred to a single institution for del17p analyses by FISH (167-kb 17p13 orange probe, MetaSystems) and TP53 mutations by NGS, both analyses carried out on CD19-purified (>85% pure) CLL samples within 6 months prior the start of ibrutinib treatment. The median follow-up from ibrutinib treatment was 36.3 months (95% CI 29.5-41.5 months). Overall survival (OS) and progression free survival (PFS) were computed from the start of ibrutinib treatment. Results: In the CLL cohort, 76 patients with del17p showed a trend for inferior OS respect to wt cases (P=0.0662, Fig.1A), while a significant correlation was found with PFS (P=0.0162). With regard to TP53 mutations, 296 TP53 mutations were found in 126 patients (range of mutations/patients 1-11). As with CIT, TP53-mutated patients, irrespective of VAF (Fig.1B), experienced a significantly worse OS and PFS than wt cases also by univariate analyses (P=0.0160, and P=0.0378, respectively). The combination of del17p with TP53 mutation data identified 95 cases with no TP53 aberrations, 8 cases del17p only, 58 cases TP53-mutated only, and 68 cases bearing both del17p deletion and TP53 mutation (Fig.1CD). Only patients with concomitant TP53 mutations and del17p experienced significantly shorter OS and PFS compared to TP53wt cases (P=0.0122, and P=0.0076, respectively; Fig.1CD). Conversely, patients presenting a single aberration showed no significant differences compared to wt patients (Fig.1CD). The simultaneous presence of TP53 mutations and del17p remained an independent predictor factor both for OS and PFS by multivariate analysis, together with the previous therapy lines (0-1 vs. >1), and anemia. The evolution of TP53-mutated clones was assessed by longitudinal NGS analysis of sequential PB samples collected from 38 patients (16 relapsed, and 22 on treatment) corresponding to 127 TP53 mutations. Among relapsed cases, 7 showed a prominent expansion of the TP53-mutated clone, 8 remained stable, and the remaining case displayed an evident decrease (Fig.1E). In non-relapsed patients, 3 cases presented an increase of TP53 mutations, 13 remained stable, and 6 showed a reduction (Fig.1F), with no significant difference compared to relapsed cases (P=0.0623, c2 test). BTK and PLCG2 mutations were found in 9/16 (56%) relapsed cases and in 3/22 (14%) patients still on ibrutinib (P=0.0492, c2 test). Of note, only 3/7 relapsed cases that presented a positive selection for TP53 mutations showed the presence of BTK mutations at the time of relapse. Image: Summary/Conclusion: This retrospective study indicates that only the concomitant presence of TP53 mutations and deletion is an independent negative prognostic factor for OS and PFS in patients with CLL on ibrutinib treatment; the simultaneous investigation of del17p and TP53 mutations may lead to a more precise risk assessment. P597: HIGH DOSE IRON IMPAIRS MALIGNANT B-CELL VIABILITY IN CHRONIC LYMPHOCYTIC LEUKEMIA J. Bordini1,*, C. Lenzi2, L. Toscani1, P. Ranghetti1, E. Perotta1, L. Scarfò2, P. Ghia2, A. Campanella2 1Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute; 2Division of Experimental Oncology, Vita-Salute San Raffaele University, Milan, Italy Background: Identification of new strategies to improve Chronic Lymphocytic Leukemia (CLL) outcome is fundamental since the disease remains incurable. Malignant B-cells show high levels of reactive oxygen species (ROS), which favors adaptation and survival but also enhances sensitivity to pro-oxidant therapeutics. Redox homeostasis is affected by iron balance since iron excess works as pro-oxidant, and cancer cells rearrange iron trafficking proteins to promote iron uptake. This adaptation might be exploited by exposing malignant cells to iron excess to cause ROS generation, lipid peroxidation, and eventually ferroptosis. We previously demonstrated the feasibility of this strategy in multiple myeloma and prostate cancer pre-clinical models. Aims: We aim at exploiting high dose iron to negatively affect malignant cell survival and increase therapeutic efficacy of current target therapies (BTK and BCL2 inhibitors). We aim at dissecting the molecular mechanisms underlying iron toxicity and at testing whether iron can improve immune dysfunctions. Methods: CLL cell lines (MEC-1, MEC-2 and PCL-12), patients peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) and B lymphocytes purified from patients PB were treated with 300 µM ferric ammonium citrate (FeAC) alone or in combination with the BTK inhibitor ibrutinib (IBR, 5-10 µM) or the BCL2 inhibitor venetoclax (VNTX, 1.25-2.5 nM). We evaluated cell viability by annexin PI staining, lipid peroxidation and ferroptosis by measuring malondialdehyde (MDA) levels and antioxidant gene expression levels by qRT-PCR. We additionally evaluated T cell activation by measuring CD25 expression. MEC-1 xenografts generated in immunodeficient RAG2-/-yc-/- mice were treated with 20 mg/Kg ferric carboximaltose. Results: In vitro, iron treatment impaired cell proliferation in all CLL cell lines analyzed, inducing accumulation of MDA and cell death (p<0.01). In vivo, iron supplementation reduced the amount of MEC-1 cells in bone marrow and spleen of xenograft mice as compared to control saline-treated mice. In patients primary samples, iron induced cell death of leukemic lymphocytes (CD19+CD5+) either as purified cells or within bulk PBMC (p<0.01). Moreover, combination of iron with IBR or VNTX induced cell death at a higher extent compared to each single drug or to iron alone (p<0.01). Leukemic cells in BMMC samples were also iron sensitive and the effect became more evident upon combination with IBR or VNTX (p<0.05). Neither iron nor iron-drug combinations induced cell death in non-malignant cells. Iron also increased CD25 expression in CD8+ T cells and we are currently exploring whether this might indicate that iron can also improve cytotoxic mediated immune response against leukemic cells. As single patient analysis revealed heterogeneity of response, we investigated whether we might predict iron sensitivity by analyzing the antioxidant gene expression profile. All CLL cell lines analyzed showed a lower basal expression of NFE2L2, HMOX1, and GPX4 antioxidant genes than typical iron-resistant cell lines, such as prostate cancer PC-3 cells, suggesting that differences in antioxidant gene expression levels may mediate iron response and that this association is worth to be explored in CLL primary samples to explain heterogeneous iron response. Summary/Conclusion: Our pre-clinical studies suggests that exploiting iron toxicity might be a valuable strategy to improve current target therapies in CLL. Additional studies aimed at distinguishing iron sensitive patients from poor responders will improve the translational potential of this therapeutic approach. P598: EBI3/IL-27 DEPLETED MICROENVIRONMENT FAVOURS TUMOUR PROGRESSION IN CHRONIC LYMPHOCYTIC LEUKAEMIA I. C. Botana1,*, G. Pagano1, W. Marina1, P. M. Roessner2, B. Qu3, A. Ramsay4, B. Stamatopoulos5, M. Seiffert2, J. Paggetti1, E. Moussay1 1Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg; 2Molecular Genetics, German Cancer Research Centre (DKFZ), Heidelberg; 3Biophysik, Medizinische Fakultät der Universität des Saarlandes, Homburg, Germany; 4School of Cancer and Pharmaceutical Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom; 5Clinical Cellular Therapy Research Laboratory (LTCC), Institut Jules Bordet, Brussels, Belgium Background: Chronic Lymphocytic Leukaemia (CLL) is the most common adult leukaemia in western countries. Unfortunately, CLL remains an incurable disease, evidencing the urgency to develop novel effective treatments for this malignancy. CLL cells are known to be highly dependent on interactions with non-malignant cells in their tumour microenvironment (TME) for survival and proliferation. Such dependency highlights the potential of immunotherapy as a therapeutic approach in CLL. Recently, several pro-inflammatory cytokines have emerged as a powerful tool to reactivate the immune system in a wide range of malignancies. IL-27 is a member of the IL-12 family of heterodimeric cytokines reported to have pleiotropic functions during cancer development in different malignancies. Based on the existing literature, we wondered whether IL-27 affects the development and progression of CLL. Aims: The goal of this study is to elucidate the role of IL-27 in CLL development and progression. Methods: We used a constitutive knockout mouse model of the EBI3 subunit of IL-27 to abrogate IL-27 expression in vivo. We performed adoptive transfer (AT) of TCL1 derived CLL cells into Ebi3-/- and WT mice, as well as generated the transgenic mouse model Eµ-TCL1-Ebi3-/-.The splenic TME of these mice was characterized using flow cytometry. In addition, in vivo neutralization of IL-27 using a blocking antibody was conducted. Immunodeficient Rag2-/- mice were also injected with TCL1-derived CLL cells and either WT or Ebi3-/- CD3+ T cells to assess the role of T-cells in leukaemia control in the presence and absence of IL-27. Finally, the transcriptional differences among WT and Ebi3-/- T cells were assessed using bulk-RNA sequencing, and their impact on T cell cytotoxicity was investigated both in humans and mice using in vitro killing assays. The serum levels of IL-27 in CLL patients and sick mice were quantified via ELISA and compared to those of healthy controls. Results: A strikingly enhanced CLL development and survival reduction was observed when CLL was AT into Ebi3-/- mice, as well as in the transgenic Eµ-TCL1-Ebi3-/- mice when compared to controls (A). Consistently, flow cytometry analysis revealed an increasingly immunosuppressive splenic TME in the absence of EBI3, characterized by an enrichment of highly activated and immunosuppressive Tregs and a terminally exhausted CD8+ T cell subset. The aforementioned results were recapitulated in an IL-27 neutralization experiment (B). Using Rag2-/- mice, we demonstrated that T cells from EBI3-/- mice were less efficient in controlling CLL development (C). RNA sequencing revealed major changes in CD8+ T cells transcriptional program, in particular a decreased expression of crucial transporters (D). Finally, in vitro killing assays showed an increased cytotoxicity of both human and murine T cells in the presence of IL-27, while serum protein quantification showed a decrease in the levels of IL-27 in both CLL patients (E) and sick mice (F) when compared to their healthy counterparts. Image: Summary/Conclusion: Overall, our results demonstrate the antitumor role of IL-27 in CLL development and progression by promoting T-cell-mediated anti-tumor immunity, as well as establish this cytokine as a potential immunotherapeutic agent in CLL. P599: RARE GERMLINE VARIANTS IN ATM INFLUENCE THE PATHOGENESIS OF CLL B. L. Lampson1, A. Gupta1, S. Tyekucheva2, Z. Wang2, N. Wojciechowska3, C. J. Shaughnessy1, P. O. Baker1, S. M. Fernandes1, A. S. Kim3, J. R. Brown1,* 1Medical Oncology; 2Data Science, Dana-Farber Cancer Institute; 3Pathology, Brigham and Women’s Hospital, Boston, United States of America Background: Germline missense variants of unknown significance (VUS) are increasingly identified in cancer-related genes by next generation sequencing. The ATM gene on chromosome 11 is a tumor suppressor gene that is frequently deleted or mutated in CLL, and also carries over 1,000 germline missense VUS. Aims: We sought to evaluate whether germline ATM variants are more frequent in chronic lymphocytic leukemia (CLL) compared to other hematologic malignancies and whether they influence the clinical characteristics of CLL. We sought to determine whether one of the most common VUS has an impact on ATM function. Methods: In our hematologic malignancy clinic, we identified 3,128 patients (including 825 CLL patients), who underwent clinical-grade sequencing of the entire coding region of ATM between 2014 and 2019. We evaluated the frequencies of germline ATM variants in different hematologic neoplasms. In patients with CLL, we determined whether these variants affect CLL-associated characteristics such as somatic 11q deletion. Finally we generated an ATML2307F knock-in cell line to characterize the functional impact of the L2307F VUS on response to etoposide treatment or irradiation. Results: Germline ATM variants are present in 24% of patients with CLL, significantly higher than in patients with other lymphoid malignancies (16% prevalence), myeloid disease (15%), or no hematologic neoplasm (14%). CLL patients with germline ATM variants are younger at diagnosis and twice as likely to have 11q deletion. The ATM variant p.L2307F is present in 3% of CLL patients and is associated with a three-fold increase in rates of somatic 11q-deletion. CLL in patients with two other common ATM VUS, p.S707P and p.F858L, was also significantly enriched for ATM-aberrant disease. Cell-based assays evaluating response to etoposide treatment or irradiation demonstrated that ATM p.L2307F shows hypomorphic function. Summary/Conclusion: Germline ATM variants cluster within CLL and affect the phenotype of the CLL that develops, implying that some of these variants (such as ATML2307F) have functional significance and should not be ignored. Further larger studies are needed to determine whether these variants affect the response to therapy or account for some of the inherited risk of CLL. P600: HIGH-DEPTH RNA-SEQUENCING IDENTIFIES CD8+ STAT3 MUTATED T-LGLL PATIENTS AS A DISTINCT BIOLOGICAL ENTITY WITHIN THE DISEASE HETEROGENEITY G. Calabretto1,*, A. Binatti2, A. Teramo1, G. Barilà3, A. Buratin4, V. R. Gasparini1, C. Vicenzetto1, E. Gaffo5, V. Trimarco6, L. Trentin1, M. Facco1, F. Vianello1, G. Semenzato1, R. Zambello1, S. Bortoluzzi2 1Department of Medicine - University of Padua, Veneto Institute of Molecular Medicine; 2Department of Molecular Medicine, University of Padua, Padua; 3Hematology Unit, Ospedale dell’Angelo, Mestre-Venezia; 4Department of Molecular Medicine and Department of Biology; 5CRIBI; 6Department of Medicine, University of Padua, Padua, Italy Background: T-Large granular lymphocyte leukemia (T-LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of cytotoxic T-LGL. We recognized a most common CD8+ subtype (CD8+ T-LGLL) and a CD4+ variant (CD4+ T-LGLL). STAT3 and STAT5B activating mutations, the main genetic lesions, are linked to the immunophenotype of the leukemic clone, T-LGLL clinical manifestations and disease prognosis. To schematize the biological heterogeneity of the disease, 4 main T-LGLL subsets can be identified: CD8+ STAT3M, CD8+ WT, CD4+ STAT5BM and CD4+ WT (M, mutated; WT, wild-type). Although STAT3 and STAT5b may regulate the transcription of genes involved in disease severity, the relationship between T-LGL immunophenotype (CD8+ or CD4+), STATs mutational status and gene expression profiles (GEP), including non-coding-RNAs, has never been investigated. Aims: The aim of the study was to disclose T-LGLL transcriptome unbalances accounting for disease pathogenesis and clinical manifestations, by investigating GEP of leukemic T-LGL from each disease subtype and healthy controls (CTR). Methods: High-depth RNA-seq was performed with a HiSeq3000 (Illumina) on RNA extracted from immuno-magnetically purified T-LGL of 20 T-LGLL patients (stratified in the 4 groups) and 5 CTR; 150 paired-end reads were generated (315 million reads/sample, on average). RNA-seq data were analyzed for reads alignment and transcripts quantification by CircComPara pipeline. Differentially expressed genes (DEGs) were assessed using DESeq (adj. p<0.01) and validated by RT-qPCR. Results: Unsupervised multi-dimensional scaling analysis of GEP clearly separated T-LGLL from CTR samples. Of note, CD8+ STAT3M patients, characterized by a symptomatic disease and reduced survival, resulted as a distinct biological entity with respect to the other T-LGLL subgroups, that showed similar transcriptomic features. In detail, we found 1612 DEGs in CD8+ STAT3M and 1707 DEGs in the other T-LGLL cases, with the majority being down-regulated as compared to CTR. Of these, 641 genes were commonly dysregulated in all T-LGLL subgroups, although a more marked dysregulation was observed in presence of STAT3 mutations. 971 genes were differentially expressed uniquely in CD8+ STAT3M cases, with 571 being differentially expressed also versus other T-LGLL subgroups. Overall, dysregulated genes included tumor suppressors, protein kinases and phosphatases, cytokines and receptors. Of note, two non coding-RNAs with a known oncogenic role in other malignancies were up-regulated in LGLL samples. Next, Gene Set Enrichment Analyses (GSEA) disclosed pathway dysregulation. In CD8+ STAT3M cases proteasome and Interferon signaling emerged among the most aberrantly activated signaling pathways with respect to CTR, whereas the cell cycle checkpoints pathway resulted to be activated as compared to the other T-LGLL subgroups. Considering CD4+ STAT5BM T-LGLL, we observed the upregulation of a JAK/STAT axis negative regulator, reported to be down-expressed in other STAT5B-related disorders characterized by poor prognosis. This transcriptional signature might account for the peculiar indolent clinical course of STAT5BM T-LGLL patients. Summary/Conclusion: We identified new genetic players involved in T-LGLL pathogenesis and disease severity, showing a peculiar GEP of CD8+ STAT3M cases, which are the most symptomatic and treatment-requiring patients. Most importantly, the identified DEGs are disclosing novel potential therapeutic targets to design innovative RNA-based therapies for T-LGLL patients. P601: TP53 ANALYSIS AND REPORTING IN CHRONIC LYMPHOCYTIC LEUKAEMIA: ARE LABORATORIES IN COMPLIANCE WITH THE EUROPEAN RESEARCH INITIATIVE ON CLL (ERIC) RECOMMENDATIONS? A. Cartwright1,*, S. Scott1, L. Whitby1 1UK NEQAS for Leucocyte Immunophenotyping, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom Background: The presence of TP53 aberrations, specifically del(17p) and/or TP53 nucleotide variants, in CLL are associated with adverse clinical outcomes and resistance to immunochemotherapy. As such, analysis of TP53 has become part of routine clinical diagnostics prior to initial and subsequent treatments, ensuring appropriate risk stratification and therapeutic intervention. Deletions of TP53 are identified by fluorescent in situ hybridisation, however, TP53 variants can be identified through various molecular techniques, including Sanger sequencing and next-generation sequencing (NGS). ERIC recommendations outline the requirements for testing, analysis and reporting of TP53 variants in CLL. Aims: The aim of this study was to evaluate laboratory approaches to analysis and reporting of TP53 variants in CLL in line with ERIC recommendations. Methods: The UK National External Quality Assessment Service for Leucocyte Immunophenotyping has provided external quality assessment for use of NGS gene panels in CLL since 2018. Samples are manufactured from cell line material and one sample per annum is issued for participating laboratories to evaluate using their in-house methodology. Between 2018-2021, three samples were formulated to contain TP53 variants and distributed. Laboratories were requested to report methodology and variants of clinical significance, in line with HGVS nomenclature at both the DNA and protein level. Additionally, details of assay limit of detection (LOD), reference sequences and interpretation databases utilised were also requested. Data returns were then evaluated relating to TP53 variant analysis and reporting. Results: In total, 34 (100%) participating laboratories reported the use of either amplicon or capture based NGS methods, with assay LOD ranging from 1-15%, with a median LOD of 5%. All participating laboratories sequenced the ‘minimum’ exons indicated in the guidelines (exons 4-10), with 91.2% (31) laboratories sequencing ‘optimal’ exons 2-11. Furthermore, 97.1% (33) laboratories utilised the recommended Locus Reference Genomic sequence LRG_321t1 (NM_000546), with one laboratory reporting use of LRG_321t3 (NM_001126114) variant transcript. The IARC TP53 locus-specific database for variant interpretation was utilised by 70.6% (24) laboratories and despite recommendations, use of dbSNP for filtering of polymorphic/neutral variants was undertaken by 76.5% (26) laboratories. Across the 3-sample period, 97.1% (33) laboratories reported correct DNA HGVS nomenclature and 58.8% (20) laboratories reported correct protein HGVS nomenclature. Of the 14 laboratories reporting inaccurate protein HGVS nomenclature, 11 (78.6%) reported minor nomenclature issues precluding full compliance. Summary/Conclusion: In this study, there is a high level of consensus amongst laboratories in the reporting and analysis of TP53 variants, except for protein nomenclature usage. Guidelines serve to promote harmonisation and standardisation of processes within laboratories and ERIC recommendations have achieved this in relation to TP53 variants in CLL, ensuring that patients are appropriately stratified for correct therapeutic interventions. Whilst there is currently a high level of consensus seen in analysis and reporting, the recommendations are likely to evolve as evidence and understanding of the genetic landscape in CLL therapeutics increases and further technological advances are made. As such, laboratories will need to ensure continued adoption to reflect new data reporting methods, treatments and standards of care for CLL patients. P602: DISTINCT IMMUNE-RESPONSE PROFILE OF RICHTER TRANSFORMATION: HIGH EXPRESSION OF IL-10, LAG-3 AND OTHER IMMUNE CHECKPOINT MOLECULES Q. Chen1,*, A. Behdad2, S. Ma2, M. Schipma2, Y.-H. Chen2 1Pathology; 2Northwestern University, Chicago, United States of America Background: Richter transformation (RT) is an aggressive progression of chronic lymphocytic leukemia (CLL), often to diffuse large B cell lymphoma (DLBCL), with poor prognosis and limited response to standard therapy. Our prior studies have shown differences in immune checkpoint molecule expression between RT and CLL; in particular, expression of programmed cell death 1 (PD1) in lymphoma cells and its ligands (PDL1) in background immune cells. Significantly, we have shown high PD1 expression in RT predicts its clonal-relatedness to CLL. Aims: To investigate immune-response pathway dysregulation in RT by gene expression profiling. To discover immune pathways and molecules that may serve as targets for immune therapy. To discover biomarkers that may help identifying patients that will benefit from checkpoint inhibitor therapy. Methods: We studied 16 patients diagnosed with RT and 18 with CLL. We performed a 400-gene targeted NGS gene expression profiling on RT and CLL cases using the Oncomine immune-response assay from ThermoFisher. Data analysis (RNA-seq with STAR and DESeq2) was done by aligning quality-sufficient reads to the human genome and counting reads for each gene. Gene normalization and differential expression were then calculated, with adjusted p-values <0.05 determining statistically significant differentially expressed genes. Next, a pathway analysis was performed (Metascape) to identify significant pathways among the differentially expressed genes. We also performed PD1 and LAG3 immunohistochemical stains in all cases. Results: Gene profiling analysis revealed significant differences in expression levels of 89 immune-response genes between RT and CLL cases. The top upregulated genes in RT comparing to CLL included several checkpoint pathway genes: LAG-3, HAVCR2/TIM3, and CD274/PD-L1. Several immune modulation/immune suppressive genes were significantly upregulated, in particular IL-10 and TGFb1. Other top upregulated genes included those involved in innate immunity (S100A9, S100A8, IGSF6), and in cytokine signaling (CCR1, IL-1B, IL-18). In addition to immune-response genes, MYC, TP63 and several cell proliferation pathway genes were upregulated in RT. The top down-regulated genes in RT compared to CLL included CD79B (B-cell receptor signaling), CD160 (checkpoint pathway), KLF2 and CD40LG (T-cell regulation), CIITA (IFNg signaling) and SELL (cell adhesion). To further confirm these findings we performed LAG3 IHC. LAG3 was highly expressed by large neoplastic B-cell in PD1-positive RT and only in scattered large cells in proliferation centers of CLL, and largely negative in PD1-negative RT cases. Average positive neoplastic cells were 67% in PD1+ RT (n=12), 2.8% in PD1- RT (n=4), and 10.8% in CLL (n=18) (P<0.05). Summary/Conclusion: Using a targeted NGS gene profiling analysis, we demonstrate a significant difference in expression levels of multiple immune-response genes in RT vs CLL. In particular, we found upregulation in immune modulation genes and checkpoint pathway genes PD1, LAG3 and TIM3, and immunosuppressive cytokine IL-10. Overexpression of LAG3 in large neoplastic B-cells of RT, specifically in cases clonally-related to CLL is an important finding that may serve as a diagnostic marker. Checkpoint molecules LAG3 and TIM3 can serve as new immune-therapy targets for the treatment of RT. IL-10 blockade has been shown to enhance T-cell immunity, and may be a potential therapeutic target. P603: GUT MICROBIOME IN CHRONIC LYMPHOCYTIC LEUKEMIA SHOWS DEPLETION OF SHORT-CHAIN FATTY ACIDS PRODUCING BACTERIA T. Faitova1,*, M. Jørgensen2, R. Svanberg1, C. da Cunha-Bang1, E. E. Ilett2, C. MacPherson2, C. Niemann1,3 1Department of Hematology; 2PERSIMUNE Centre of Excellence, Rigshospitalet, Copenhagen University Hospital; 3Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark Background: Recent studies have shown extensive crosstalk between our immune system and gut microbiome (GM). The host immune system plays a vital role in the maintenance of GM homeostasis by 1) establishing a balance between eliminating invading pathogens and promoting the growth of beneficial microbes, 2) producing short-chain fatty acids (SCFA), the main source of nutrition for the colon cells, and 3) modulating the immune system by cytokine production. Mounting evidence shows that the GM of patients with high rates of infection are characterized by an imbalance of bacteria, inducing proinflammatory states and reduced capacity for SCFA synthesis. As chronic lymphocytic leukemia (CLL) is, among others, also characterized by high rate of infectious complications and an altered immune system, it is warranted to explore composition of the CLL microbiome. Aims: We aim to investigate the hypothesis that deviation of the GM from homeostasis, i.e. loss of ‘health promoting’ gut microbes and/or overgrowth of pathogenic bacteria, distinguishes patients with CLL from the background population. Methods: Feces samples of patients with CLL were collected, immediately fixated and frozen within 72 h; total genomic DNA was sequenced. Feces samples of healthy controls were chosen to match the CLL population with respect to age, demographic data, sample collection method, and sequencing platform. Taxonomical profiling was done using an in-house bioinformatics pipeline. Results: A total of 61 CLL patients and 30 healthy individuals were included in the study. We observed reduced GM alpha diversity, and depletion of bacterial members of Lachnospiraceae and Ruminococcaceae families among the CLL patients when compared to healthy individuals. Our data show that members of the Lachnospiraceae family (Anaerostipes hadrus, Coprococcus comes, Blautia spp., Dorea spp.), and 3 members of Ruminococcaeae family (Ruminococcus torques, Ruminococcus bromii, Faecalibacterium prausnitzii) were among the most differentially abundant bacterial species between the microbiomes of healthy individuals and CLL. Their mean proportions were shown to be significantly higher in healthy microbiome samples. As further differentially abundant species we observed Bacteroides sp. and Alistipes finegoldii, which both demonstrated significantly higher mean proportions in CLL microbiomes (Fig 1). Image: Summary/Conclusion: To sum up, the CLL microbiomes in comparison to healthy controls demonstrated lower enrichment of Lachnospiraceae and Ruminococcaceae families, the major SCFAs-producing bacterial taxa reported to have a protective effect against inflammation. This supports the notion that proinflammatory risk factors identified in other cohorts with GM dysbiosis are also present within CLL patients. As CLL represents an antigen driven malignancy with immune dysfunction, we hypothesize that GM dysbiosis could both be implicated in the pathogenesis of CLL, through antigenic drive, and contribute to the distortion of the immune system in CLL. Notably, both immune dysfunction and treatment (e.g. antimicrobials) may influence the CLL microbiome itself and therefore confound cross-sectional clinical studies. We therefore plan to investigate the interaction between microbiome and CLL development in the TCL1 mouse model of CLL. Also, identification of potential mechanistic links between the GM and the microenvironment in CLL should be investigated, e.g. extravesicular vesicles or cytokines released from or impacted by the GM. In addition, modulation of the microbiome in animal models may help to establish causal connections between the GM and CLL development. P604: CATALASE EXPRESSION IN LEUKEMIA CELLS IS CONTROLLED BY GENETIC AND EPIGENETIC MECHANISMS M. Galasso1,*, E. Dalla Pozza1, R. Chignola2, S. Gambino1, C. Cavallini3, A. Pilatone1, F. M. Quaglia4, O. Lovato3, I. Dando1, G. Malpeli5, M. Krampera4, M. Donadelli1, M. G. Romanelli1, M. T. Scupoli1,3 1Department of Neurosciences, Biomedicine and Movement Sciences; 2Department of Biotechnology; 3Research Center LURM, Interdepartmental Laboratory of Medical Research; 4Department of Medicine, Section of Hematology; 5Department of Surgery, Dentistry, Pediatrics, and Gynecology, University of Verona, Verona, Italy Background: Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in Western countries. It is an incurable disease characterized by an extremely variable clinical course and response to treatment. We have recently shown that high catalase (CAT) expression identifies a more aggressive clinical behavior in CLL. However, molecular mechanisms controlling catalase expression in leukemia cells are still poorly characterized. The rs1001179 single nucleotide polymorphism (SNP) as well as DNA methylation in the CAT promoter have been shown to regulate CAT expression in various cell types. Aims: With the aim to characterize regulatory mechanisms underlying differential expression of catalase in CLL, in this study we investigated the role of rs1001179 SNP and CpG Island II methylation encompassing this SNP in the regulation of CAT expression. Methods: Peripheral blood mononuclear cells (PBMC) from 55 CLL patients and 50 healthy donors (HD) were used for the study. CAT mRNA levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). DNA samples were genotyped for the rs1001179 SNP using Restriction Fragment Length Polymorphism (RFLP)-PCR. Bioinformatic analysis was used to predict transcription factor (TF) binding to SNP sequences. Chromatin Immunoprecipitation (ChIP)-qPCR was performed to validate predicted TF binding sites. Methylation levels of CpG sites within the CAT promoter was determined by pyrosequencing of bisulfite-converted DNA. Results: The rs1001179 SNP genotyping showed that CLL cells harboring the T allele exhibited a significantly higher catalase expression compared with cells bearing the CC genotype. Interestingly, bioinformatic analysis predicted that the SNP provide distinct binding sites for various TF, including ETS-1 and GR-β. ChIP assay confirmed that CAT promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-β, but not to the other analyzed TFs. In addition, CLL cells exhibited lower methylation levels within the CAT promoter compared with HD B cells, in line with the higher catalase mRNA and protein levels expressed by CLL versus HD B cells. Moreover, methylation levels negatively correlated with CAT expression in CLL cells. Remarkably, inhibition of methyltransferase activity in leukemic cells induced a significant increase of CAT mRNA and protein levels, thus showing that DNA methylation could control catalase expression in CLL. Moreover, expression of DNA methyl transferase 1 (DNMT1) resulted significantly reduced in CLL cells compared with HD B cells and inversely correlated with CAT expression in CLL, thus suggesting that differences in methylation levels underlying catalase expression could be driven by the DNMT1 enzyme. Finally, modeling analysis showed that the CT/TT genotypes exhibited a lower methylation and a higher CAT expression level, suggesting that the rs1001179 T allele and methylation cooperate in inducing CAT gene expression. Summary/Conclusion: Our data show that SNP as well as methylation of the catalase promoter are involved in controlling catalase expression in CLL. The key advance of this study is to provide new insights into the regulatory mechanisms underlying differential expression of catalase in leukemic cells, which is of clinical relevance in CLL. Moreover, our study may form the basis for future challenges aimed at developing combinatorial therapies targeting catalase regulatory pathways. P605: T-CELL PROLYMPHOCYTIC LEUKEMIA: MOLECULAR CHARACTERIZATION OF A COHORT OF 18 PATIENTS AND EVALUATION OF BORTEZOMIB AS A POSSIBLE THERAPEUTIC STRATEGY V. R. Gasparini1,*, A. Martines2, G. Barilà3, A. Teramo1, G. Calabretto1, C. Vicenzetto1, S. Carraro4, V. Trimarco4, L. Trentin4, M. Facco4, G. Semenzato1, L. Bonaldi2, R. Zambello1 1Department of Medicine, University of Padova and Veneto Institute of Molecular Medicine; 2Immunology and Molecular Oncology Unit, Veneto Institute of Oncology, IOV-IRCCS, Padova; 3Hematology Unit, Ospedale dell’Angelo, Mestre-Venezia; 4Department of Medicine, University of Padova, Padova, Italy Background: T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive mature T-cell malignancy characterized by a progressive clinical course and resistance to chemotherapy, whose pathogenesis is still largely unknown. Recurrent somatic mutations were detected within the JAK/STAT axis (i.e., JAK3 and STAT5b genes). T-PLL patients present with progressive lymphocytosis, splenomegaly and lymphadenopathy. New therapeutic strategies are needed to overcome the overall survival of two years observed in T-PLL patients even after treatment with alemtuzumab and consolidation with allogeneic stem cell transplantation. Ex-vivo drug sensitivity and resistance tests suggested proteasome inhibitors as novel candidates for the treatment of T-PLL. Aims: The investigation of the molecular features of T-PLL aimed at understanding whether JAK/STAT genetic lesions might have a role in the clinical management of patients. Ex-vivo treatment with Bortezomib (Bz), a proteasome inhibitor, was performed to test the affordability of this compound as a new option for T-PLL therapy. Methods: Sanger sequencing was performed on peripheral blood mononuclear cells (PBMC) of 18 T-PLL patients to detect the presence of somatic mutation in the hotspot regions of JAK3 and STAT5b genes. Patients were recruited at the Padova University Hospital. Immunophenotypic characterization of the leukemic clone was performed in all cases, while 11 patients underwent cytogenetic analysis upon mitogen stimulation. PBMC of 10 patients were cultured for 48h with different concentrations (1.3, 2.6, 5.2 and 7.8nM) of Bz according to literature. Annexin staining and western blot analyses were performed at both 24h and 48h to assess the efficacy of the treatment. Results: At least one mutation within the JAK/STAT pathway was detected in 39% (7/18) of patients. Consistent with the literature’s data, N642H variant was the most recurrent STAT5b mutation. Conversely, the most frequent variant in JAK3 gene was N564S, which has never been detected in T-PLL and not listed in the COSMIC database. Cytogenetic analysis revealed a complex karyotype in 9/11 (82%) patients with specific primary T-PLL aberrations: inv(14)(q11q32) or variant rearrangements in 78% (7/9) and t(X;14)(q28;q11) in 2 cases. Known secondary alterations such as those involving chromosome 8 and deletion of 11q22 were found in 89% (8/9) and 67% (6/9) of cases, respectively. Analysis of the clinical data did not reveal a correlation with the patient’s mutational status. Ex-vivo treatment with Bz induced apoptosis of the leukemic clone in a dose and time dependent manner. Compared to the untreated condition (16%), Bz concentration of 5.2nM increased the mortality of 1.7 (27%) and 4 (63.5%) times at 24h and 48h, respectively. The apoptosis was confirmed also at protein levels by a trend of caspase 9 and PARP cleavages upregulation at 48h. No difference in the sensitivity to treatment with Bz was observed between mutated and wild-type patients. Summary/Conclusion: No correlations between recurrent lesions within the JAK/STAT axis and clinical outcome were found. This points against a pathogenetic role of these mutations in T-PLL, conversely from other leukemias where STAT mutations are regarded as prognostic markers. This hypothesis is further sustained by the same response to the treatment with Bz. Since the treatment of T-PLL mostly remains an unmet clinical need, the finding of an increased apoptosis in Bz-treated cells paves the way for a deeper study on molecular mechanisms involved in the apoptosis of the leukemic clone. P606: META-ANALYSIS OF LOW ALLELIC BURDEN C481 BTK-MUTATIONS CALLS FOR CAUTION IN IBRUTINIB RESISTANCE EVALUATION M. H. Hansen1,2,*, S. R. Veyhe1,2, N. Abildgaard1,2, C. G. Nyvold1,2, H. Frederiksen2 1Hematology-Pathology Research Laboratory, Research Unit for Hematology and Research Unit for Pathology; 2Department of Hematology, Odense University Hospital, Odense, Denmark Background: Introduction of the orally administered tyrosine kinase inhibitor ibrutinib, a drug approved for relapsed or refractory mantle cell lymphoma in late 2013 and chronic lymphocytic leukemia in 2014, has changed treatment algoritms for B-cell malignancies and improved progression-free survival. Ibrutinib irreversibly targets Bruton’s tyrosine kinase (BTK), thus interrupting B-cell receptor signaling. However, it is known that mutations in the BTK gene, located on the X chromosome, can mediate acquired resistance, specifically modifying cysteine residue 481 to serine (C481S, X:101356176, GRCh38). Aims: Despite this knowledge, it is still unknown how low mutational burden can contribute to general resistance towards ibrutinib treatment. Thus, we performed a meta-analysis based on previous cardinal studies to investigate the collective and gender-specific distribution of variant allele frequencies. Methods: We implemented meta-analysis of single-nucleotide variant calls from seven studies involving whole-exome sequencing (Woyach et al. 2014 and 2017, Maddocks et al. 2015, Ahn et al. 2017, Kanagal-Shamanna et al. 2019, Gángó et al. 2020, Bödör et al. 2021) on mutational profiling concerning ibrutinib resistance and CLL. Four of these studies contained gender specific data. Computational analyses, statistics, and visualization were performed in Wolfram Language, R, and GraphPad Prism. Results: From the available data included in these studies, we were able to base our analyses on 226 non-BTK mutations and 170 BTK mutations within a variant allele frequency (VAF) range of 0.0001–0.9, of which 93 mutation candidates from males (VAF < 0.9) and 77 from females (VAF < 0.44) were observed. Extrapolative analysis of allele frequency distribution from all other variants (excl. BTK) in the studies revealed an exponential increase in the number of low burden variants below 10% VAF (R2>0.99). These variants were not significantly different between genders (PMann-Whitney=0.24, nmale=130, nfemale=73). In contrast, a significant difference was observed between male variant allele frequencies (PMann-Whitney=0.005) and females owing to BTK hemizygosity in males. However, this significance persisted when female BTK mutations were transformed to reflect a single copy (VAF/2, PMann-Whitney=0.015, ΔVAF=0.05) for gender comparison and even when observations for males were alternatively transformed to reflect two copies (2*VAF, PMann-Whitney<0.001, ΔVAF=0.19). The previously discovered threshold of 10% was set as the detection limit. Summary/Conclusion: Defining specific molecular contributions in drug resistance is imperative for the correct use of targeted therapy. It has been questioned how the low allelic burden of BTK mutations plays a role. Therefore, it is crucial to investigate the lower limit of detection. The meta-analysis suggests that variants below a certain threshold, here empirically 10%, may in many cases be false-positive variants, in agreement with other reports involving WES in general, introduced by erroneous base calling, PCR errors, etc. Although intriguing, it remains to elaborate whether the haploinsufficiency of BTK in males skew in the degree of ibrutinib resistance. P607: A NOVEL LYMPH NODE-MIMICKING 3D CULTURE SYSTEM DISPLAYS LONG-TERM T CELL-DEPENDENT CLL PROLIFERATION AND SURVIVAL M. Haselager1,2,3,4,*, E. Perelaer1, A. Kater2,3,4,5, E. Eldering1,2,3,4 1Experimental Immunology, Amsterdam University Medical Center; 2Lymhoma and Myeloma Center Amsterdam, LYMMCARE; 3Cancer Center Amsterdam; 4Amsterdam Infection & Immunity Institute; 5Hematology, Amsterdam University Medical Center, Amsterdam, Netherlands Background: Primary chronic lymphocytic leukemia (CLL) cells, despite originating from a proliferative disease, rapidly undergo apoptosis in vitro in the absence of microenvironmental survival signals1. No current system permits long-term expansion of CLL cells in vitro due to difficulties of mimicking a physiological microenvironment. The lymph node (LN) is the critical site of in vivo CLL proliferation and is also thought to play a key role in the development of resistance to targeted agents2,3. Developing an in vitro culture system for CLL with pathophysiological relevance will greatly benefit studies of CLL proliferation, microenvironment, clonal outgrowth and relevant drug screening. Aims: To design an in vitro CLL culture system that incorporates key aspects of the CLL LN; contribution of non-CLL cells and cytokines, and induction of drug resistance and prolonged proliferation of CLL cells. Methods: Primary CLL cells were cultured in ultra-low attachment (ULA) plates in parallel to standard 2D cultures. PBMCs were cultured with or without T cells (in a specific CLL:T cell ratio to mimic the LN composition), or PBMCs were co-cultured with primary lymph node fibroblasts. CLL cells were either stimulated directly with a B cell cocktail (BCC) consisting of IL-2, IL-15, IL-21 and CpG and/or indirectly via a T cell stimulation of anti-CD3/CD28. Results: Compared to 2D cultures, 3D cultures showed several important CLL LN features. First, significantly increased CLL proliferation independent of IGHV mutation status, which was abrogated in the absence of T cells, or by JAK inhibitors. Notably, treatment with BTK inhibitors significantly inhibited CLL proliferation and resulted in disintegration of the 3D spheroid architecture. Second, co-culture with LN-derived stromal cells further increased CLL proliferation, reaching a maximum of 8 generations. Third, 3D cultures could be expanded approximately 3-4-fold over a course of 6 weeks using the 3D model. Fourth, when PBMCs were stimulated with BCC, spheroids developed proliferation center-like structures after 4 weeks of culture where T cells localized together with enrichment of Ki-67+ CLL cells. Finally, either B or T cell stimulation resulted in an induction of venetoclax resistance, showing that T cells are able to confer drug resistance to CLL cells in this model. Summary/Conclusion: We present a 3D culture system that underlines the role of T cells in sustained CLL proliferation, permitting investigation of CLL cells in the context of a protective niche consisting of multiple cell types, thereby opening up new avenues for clinically useful applications. P608: DISTINCT P53 PHOSPHORYLATION PATTERNS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS ARE REFLECTED IN CIRCUMJACENT PATHWAYS’ ACTIVATION UPON DNA DAMAGE M. Pesova1,2,*, V. Mancikova1,2, R. Helma1,2, S. Pavlova1,2, V. Hejret1, P. Taus1, J. Hynst1, K. Plevova1,2,3, J. Kotaskova1,2,3, J. Malcikova1,2, S. Pospisilova1,2,3 1Central European Institute of Technology (CEITEC), Masaryk University; 2Department of Internal Medicine - Hematology and Oncology, Faculty of Medicine of Masaryk University and University Hospital; 3Institute of Medical Genetics and Genomics, Faculty of Medicine, Masaryk University, Brno, Czechia Background: Presence of defects in the TP53 gene represents a clinically relevant biomarker in chronic lymphocytic leukemia (CLL). Besides gene mutations and deletions, the functionality of p53 protein can be disrupted by other mechanisms, such as aberrant phosphorylation. Aims: To uncover how p53 phosphorylations affect its functions in CLL cells. Methods: P53 phospho-patterns were analyzed in 71 TP53-intact primary CLL samples using Zn2+-Phos-tag™ SDS-PAGE after induction of p53 protein by DNA damage (fludarabine and doxorubicin treatments). Differences in gene expression were studied using RNA sequencing and compared to CLL cells carrying the mutated TP53 gene. The ability of p53 to activate transcription of its target genes (BAX, BBC3, CDKN1A, GADD45A) was assessed by qPCR. The presence of gene mutations was analyzed using a targeted NGS panel. Results: While fludarabine induced a consistent phospho-pattern, two distinct p53 phosphorylation profiles were identified upon doxorubicin treatment. Profile I featured heavily phosphorylated p53 protein, while only low phosphorylation occurred in profile II. On the transcriptomic level, samples from the profile II were less capable of activating p53 target genes upon doxorubicin treatment, thus resembling TP53-mutant cells, whereas proper p53 signaling was triggered in profile I. Consistently, induction of the studied downstream effector genes CDKN1A, BAX, BBC3 and GADD45 after doxorubicin treatment was lower in profile II than in profile I samples but still higher than in TP53 mutants. The differences between the two phoshoprofiles were also observed in untreated cells: the expression of miR-34 in profile II showed an intermediate pattern between profile I and TP53-mutant cells. Furthermore, profile II samples had significantly higher basal p53 protein levels than profile I samples, where p53 was generally detectable only upon DNA damage. The PROGENy analysis of the basal activity of selected cancer-related pathways in untreated cells pointed to the different regulation of the hypoxic pathway: the activity of hypoxic pathway in profile II samples was in between high levels found in TP53 mutants and low levels in profile I. Finally, we have found ATM locus/gene defects more frequently in profile II. Summary/Conclusion: Our study suggests that wt-TP53 CLL cells with less phosphorylated p53 show TP53-mutant-like behavior after DNA damage. Hypophosphorylation of p53 protein and the corresponding lower ability to respond to DNA damage are linked to ATM locus defects and to the higher basal activity of the hypoxia pathway. The project was supported by GACR 19-15737S, MZCR-RVO 65269705, MUNI/A/1330/2021 and AZV NU21-08-00237. P609: CLINICOBIOLOGICAL CHARACTERISTICS AND TREATMENT EFFICACY OF NOVEL AGENTS IN CHRONIC LYMPHOCYTIC LEUKEMIA WITH IGLV3-21R110 P. Hengeveld1,2,*, Y. E. Ertem1, J. Dubois3, C. Mellink4, A.-M. van der Kevie-Kersemaekers4, L. Evers3, K. Heezen1, P. M. Kolijn1, O. Mook4, M. M. Motazacker4, K. Nasserinejad5, S. Kersting6, P. Westerweel2, C. Niemann7, A. Kater3, A. Langerak1, M.-D. Levin2 1Department of Immunology, Erasmus MC, Rotterdam; 2Department of Internal Medicine, Albert Schweizer Ziekenhuis, Dordrecht; 3Department of Hematology; 4Department of Human Genetics, Amsterdam UMC, Amsterdam; 5Department of Hematology, Erasmus MC, Rotterdam; 6Department of Hematology, Haga Ziekenhuis, Den Haag, Netherlands; 7Department of Hematology, Rigshospitalet, Copenhagen, Denmark Background: The composition of the clonotypic B cell receptor (BCR) is of key importance in chronic lymphocytic leukemia (CLL). Recently, a novel immunogenetically defined CLL subset was described, characterized by a clonotypic BCR using the immunoglobulin lambda (IGL) IGLV3-21*01/IGLV3-21*04 gene with a distinctive G110R somatic hypermutation (IGLV3-21R110). IGLV3-21R110 CLL accounts for approximately 20% of all CLL and is associated with an adverse prognosis. However, the clinicobiological profile and predictive impact of IGLV3-21R110 CLL in the context of novel therapies remains incompletely characterized. Aims: To characterize the cytogenetic, immunogenetic and mutational landscape of IGLV3-21R110 CLL and to assess the predictive impact of this genotype in the context of novel therapies. Methods: We characterized the light chain genotype, clinicobiological features and response to therapy of patients enrolled in the HOVON-139/GIVE trial and the Dutch sub-cohort of the HOVON-141/VIsion trial. The HOVON-139/GIVE phase-II trial evaluated first-line minimal residual disease (MRD)-guided duration of treatment with obinutuzumab and venetoclax in CLL patients unfit for treatment with chemoimmunotherapy. The HOVON-141/VIsion phase-II trial evaluated MRD-guided ibrutinib and venetoclax combination treatment in relapsed or refractory (R/R) CLL patients. Results: The IGLV3-21R110 genotype was present in 16/65 patients (25%) in the first-line cohort and in 32/129 patients (25%) in the R/R cohort. Loss of 13q14 and 11q22 were enriched in IGLV3-21R110 patients, compared to other patients (del13q14: 79% vs. 57%, P=0.009; del11q22: 34% vs. 18%, P=0.03). In contrast, the IGLV3-21R110 genotype and trisomy 12 or loss of 17p13 were mutually exclusive (trisomy 12: 0% vs. 12%, P=0.008; del17p13: 0% vs. 12%, P=0.01). Mutations in the SF3B1 and ATM genes were significantly more common in IGLV3-21R110 patients, compared to other patients (SF3B1: 49% vs. 21%, P=0.0008; ATM: 36% vs. 18%, P=0.02). IGHV and IGHD gene usage of IGLV3-21R110 patients was markedly skewed. The IG heavy-chain complementarity determining region 3 (HCDR3) was shorter in IGLV3-21R110 patients, compared to other patients (median HCDR3 aa length 13 [range: 9-17] vs. 19 [range 8-30], P<0.0001). Whereas the IGHV SHM imprint of IGLV3-21R110 patients was centered around the 98% cutoff between unmutated and mutated IGHV, the range was wider in other patients (range 95.1%-99.7% vs. 85%-100%, P=0.01). When comparing patients with IGLV3-21R110 CLL versus all other light chains, were no differences regarding MRD (%uMRD<10-4 in peripheral blood, HOVON-139/GIVE after 12 cycles: 87% vs. 98%, P=0.15; HOVON-141/VIsion after 15 cycles: 52% vs. 61%, P=0.11) and progression-free survival (24-month PFS, HOVON-139/GIVE: 100% vs. 94%, P=0.52; HOVON-141/VIsion: 91% vs. 92%, P=0.74) in either trial. Summary/Conclusion: We have characterized the clinicobiological features of the largest cohort of IGLV3-21R110 patients reported thus far. We demonstrate that CLL with IGLV3-21R110 is typified by a distinct cytogenetic, mutational and immunogenetic profile. There was no evidence for a predictive impact of the IGLV3-21R110 genotype on the efficacy of the novel therapies with venetoclax and ibrutinib employed in the HOVON-139/GIVE and HOVON-141/VIsion trials. Our results suggest that novel targeted therapies may mitigate the adverse risk profile of IGLV3-21R110 CLL. To determine whether these patients should preferentially receive novel therapies, characterization of the predictive impact of IGLV3-21R110 in the setting of chemoimmunotherapy is warranted. P610: SYNERGISTIC ACTIVITY OF FTO INHIBITOR FB23-2 WITH IBRUTINIB IN XENOGRAFT MURINE MODEL OF CHRONIC LYMPHOCYTIC LEUKEMIA X. Hu1,*, H. Wang2, Y. Han1, X. Zhang1, Z. Tian1, Y. Zhang2, X. Wang1 1Department of Hematology, Shandong Provincial Hospital，Cheeloo College of Medicine, Shandong University; 2Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China Background: Several studies have documented that FB23-2, as the selective inhibitor of RNA N6-methyladenosine (m6A) demethylase FTO, exerts crucial role in dampening tumorigenesis. Indeed, FTO deactivation was proved to render resistant leukemia cells sensitive to targeted agents, which suggests a potential combination therapy strategy of FB23-2. Aims: The present study was aimed to identify the synergistic utility of FB23-2 combination with ibrutinib in CLL. Methods: A disseminated leukemia xenograft murine model was established to explore the efficacy of FB23-2 in CLL. Leukemia burden of CLL mice was quantified by luciferase intensity using bioluminescence imaging. Bone marrow and spleen infiltration of leukemic cells was assessed by flow cytometry. And cell survival was determined by cell counting kit-8. Results: To investigate the combinatory effect of FB23-2 and ibrutinib, cell viability assay was performed firstly. FB23-2 displayed potent synergistic activity with ibrutinib in promoting cytotoxicity in CLL cells (Figure 1A). Consistently, FTO silencing mediated by lentivirus exhibited exquisite sensitivity to ibrutinib in CLL cells (Figure 1B). Besides, CLL primary cells and MEC-1 cells with treatment of FB23-2 and ibrutinib simultaneously triggered increased cell apoptosis compared to single agent (Figure 1C). Further underlying cooperative mechanism investigation demonstrated an aberrant activation of DNA damage pathway after serial dilution of FB23-2 treatment. Hence, enhanced expression of p-ATM, p-chk2 and p-H2AX was detected in FTO knockdown and ibrutinib co-treatment group, indicating FTO inhibition inducing ibrutinib-sensitivity via regulating DNA damage (Figure 1D). In addition, we investigated the efficacy of FB23-2 alone and corporation in CLL xenograft murine model. Compared to ibrutinib alone, combination treatment with FB23-2 significantly prolonged the lifespan of CLL mice (median survival time 20 versus 22.5 days, respectively, p=0.02, Figure 1E). FB23-2+ibrutinib group showed an obviously declined proportion of CLL cells compared to ibrutinib-treated alone both in bone marrow(20.93%±2.95% versus 37.17%±4.22%, p=0.02) and spleen (23.58%±6.03% and 54.58%±9.01%, p=0.03, Figure 1F-G). Similar results were obtained with three treated group relative to control group (all p<0.05). Moreover, FB23-2 combined with ibrutinib diminished leukemia burden and splenomegaly in CLL mice (Figure 1H-I). As shown in Figure 1J, leukemia involvement and malignant proliferation were more evident in control group, confirming the potent anti-leukemic activity of FB23-2. Image: Summary/Conclusion: Taken together, our investigation provides pre-clinical evidence for the utility of FB23-2 in CLL, especially combinatory benefit with ibrutinib. FB23-2 represents a promising strategy to novel treatment optimum in CLL. P611: NEXT-CLL: A NEW NGS-BASED METHOD FOR RAPID ASSESSMENT OF IGHV MUTATIONAL STATUS IN CHRONIC LYMPHOID LEUKEMIA E. Bourbon1, K. Chabane2, A.-S. Michallet3, E. Ferrant1, I. Mosnier2, S. Poulain4, M. Giraud5, A. Bouvard2, H. Ghesquieres6, S. Hayette2, P. Sujobert2, S. Huet2,* 1Hematology clinics; 2Laboratory of hematology, Hospices Civils de Lyon, pierre-benite; 3Hematology clinics, Centre Leon Berard, Lyon; 4Laboratory of hematology; 5Bio-informatics, CHRU de Lille, Lille; 6Hematology clinics, Hospices Civils de Lyon, Lyon, France Background: Chronic Lymphocytic Leukemia (CLL) is a highly heterogeneous disease both in terms of biological landscape and clinical course. Current guidelines recommend IGHV mutation status (MS) determination prior to treatment initiation in order to guide the first-line therapeutic choice between chemoimmunotherapy and novel agents. Currently, the IGHV MS is determined in most routine laboratories by low-throughput Sanger sequencing that allows for the detection of the dominant clone in the majority of CLL cases. However, this technic remains technically challenging despite best practice guidelines, does not provide any insight into subclonal architecture and intraclonal diversity, and has a general failure rate ranging from 10 to 20%. Besides, commercially available NGS solutions have drawbacks (costs) and constraints (as they necessitate mandatorily a MiSeq sequencing machine and are not feasible on other devices) that restrict their use by any laboratory in routine practice. Aims: In this study, we aimed at providing a ready-to-use and rapid strategy to evaluate the IGHV gene MS using NGS in routine practice. We present a new method called Next-CLL, allowing rapid assessment on any NGS device available in routine diagnostic laboratories. Methods: Peripheral blood samples were obtained from 80 patients with typical CLL at diagnosis. Genomic DNA (gDNA) and complementary DNA (cDNA) were used. In order to fully cover the IGHV rearranged gene sequence, 4 mutliplex PCR reactions were designed: [LEADER-FR2R/JHc], [FR1c-JHc], [FR1-JHc] and [FR2-JHc], producing overlapping amplicons. Final products from the 4 PCR reactions were mixed and purified, subjected to A-tailing and ligated to Illumina-single indexed adapters. The library was then sequenced (2x150 bp) on a NextSeq 500 (Illumina). A reassembly of IGHV overlapping amplicons was performed using the BBTools Suite. The outputs were then uploaded on the Vidjil plateform for IGHV clonal analysis. The MS was calculate using the IMGT web interactive tool following ERIC recommendations. IgHV MS was assessed in parallel by Sanger sequencing or the commercial NGS assay IdentiClone® Assay (Invivoscribe) as reference. Gene usage, % identity to the germline and stereotype subsets were compared with the results obtained from Next-CLL. Results: Next-CLL identified a productive clone in 100% of cases (n=80), compared to PCR with Sanger sequencing (15% of failure). All cases that failed using the Sanger sequencing were subjected to a second NGS technique (Invivoscribe) that confirmed the results from Next-CLL, ruling out any possibility of false positive cases. Next-CLL showed 100% concordance with the reference techniques for IGHV gene identification and allowed assessment of the MS from the leader sequence, as recommended by the ERIC consortium. Only 2 cases showed discordant IGHV MS, close to the 98% identity cut-off, between Next-CLL and Sanger sequencing (the latter being unable to capture the leader sequences), thus providing 97% of concordance between the 2 techniques (Figure 1). Moreover, 100% concordant results were obtained between gDNA and cDNA (n=32 cases analysed for both) using Next-CLL. Image: Summary/Conclusion: Our new method Next-CLL allows assessment of the IGHV MS by NGS with an easy and rapid workflow, providing clinically useful information from the IGHV leader sequence and allowing analysis of intra-clonal diversity (not possible with the standard Sanger sequencing). Importantly, it might be used both on gDNA and cDNA and on any sequencing machine, on the contrary to existing commercially available kits. P612: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)-DERIVED T-CELLS HAVE DISTURBED FATTY ACID METABOLISM, POSSIBLY CONTRIBUTING TO T-CELL DYSFUNCTION C. F. Jacobs1,2,3,4,*, H. Simon-Molas1,2,3,4, N. Zelcer5, A. P. Kater2,3,4,6, F. S. Peters1,2,3,4 1Experimental Immuno-Hematology; 2Hematology, Amsterdam University Medical Center; 3Cancer Center Amsterdam; 4Amsterdam institute for Infection & Immunity; 5Medical Biochemistry, Amsterdam University Medical Center; 6Lymphoma and Myeloma Center Amsterdam, Amsterdam, Netherlands Background: T-cells from CLL patients are dysfunctional and display reduced activation, proliferation and cytotoxicity upon in-vitro activation. Whilst the role of glucose metabolism in activated T cells is extensively studied, the contribution of other energy sources is less clear. Fatty acid (FA) and lipid uptake in T cells involves the scavenger membrane transporter CD36. CD36-mediated lipid transport has been identified to (metabolically) modulate intratumoral T cells (both Treg and cytotoxic CD8+ T cells). However, lipid metabolism and a potential role for CD36 has not been studied in the context of T cell dysfunction in CLL Aims: To examine the role of FA metabolism and the fatty acid transporter CD36 in acquired T-cell dysfunction in CLL. Methods: Untreated CLL patients with a white blood cell count (WBC) greater than 20×109 cells/L and, as control, age-matched healthy donors (HD) were studied. T cells were analyzed by flow cytometry, confocal microscopy or extracellular flux analysis (Seahorse), either directly after thawing or after a 2 day culture with or without αCD3/αCD28 antibodies. Results: Analysis of CD36+ vs CD36- CD4 and CD8 T-cells from HD revealed that CD36+ cells had higher long-chain fatty acid (LCFA) and glucose uptake, and higher expression of the glucose transporter GLUT-1 and the rate-limiting mitochondrial FA transporter CPT1α (Fig. 1A). The cells expressing CD36 were also more likely to increase their CD25 expression and mitochondrial mass upon stimulation, indicating a metabolic and functional advantage potentially conferred by the surface expression of CD36. The absolute amount and percentage of T cells (CD4 and CD8) expressing FA transporter CD36 was lower in CLL T-cells as compared to HD (Fig. 1B). The levels of LCFA uptake and expression of CPT1α were comparable, indicating that the lower amount of CD36 might successfully cater to the FA demands of resting CLL T cells(Fig. 1C). However, while stimulation of HD T-cells markedly upregulated CD36 abundance, LCFA uptake and CPT1α, concomitantly with the activation marker CD25, CLL-derived T-cells failed to mount a similar response (Fig. 1D). This lack of induction of CD36 expression was also observed in CLL-derived T cells that increased CD25, which implies that this response is independent of T-cell activation. The transcription factors peroxisome proliferator-activated receptors (PPAR) α and γ are important regulators of FA metabolism. We observed reduced expression of these transcription factors in resting CLL T cells. However, distinct from CD36, CLL T cells were indistinguishable from HD T-cells in their ability to increase levels of both PPARα and PPARγ upon stimulation, indicating that alternative mechanisms might be implicated in the regulation of CD36 and FA metabolism in CLL T cells. Image: Summary/Conclusion: Collectively, our results show that disturbances in FA metabolism are present on multiple levels in CLL T cells and that lack of LCFA uptake into CLL T-cells could represent a limiting step in FA metabolism. We also highlight CD36+ cells as a metabolically distinct T-cell subtype that is largely absent in CLL T-cells, independently of their activation status. We are currently investigating strategies to increase CD36 expression and FA metabolism in general in CLL T cells, which could ameliorate CLL T cell dysfunction and provide targets to improve autologous T cell therapies. P613: TLR9 SIGNALLING IS A POTENTIAL TUMOUR ESCAPE MECHANISM FOLLOWING BTKI THERAPY FOR THE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKAEMIA. E. Kennedy1,*, S. Mitchell1, T. A. Burley1, T. Liloglou2, R. Johnston3, I. Ashworth1, D. Bak-Blaz3, K. Chamberlain3, E. Ladikou1, S. Mackay4, C. Pepper1, A. G. Pepper1 1Department of Clinical and Experimental Medicine, Brighton and Sussex Medical School, Falmer; 2Molecular and Clinical Cancer Medicine, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool; 3Brighton and Sussex University Hospitals NHS Trust, Brighton; 4Cell Biology and Drug Discovery and Design Groups, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom Background: CLL cell trafficking is fundamental to CLL progression and an important focus when identifying novel therapeutic targets. Toll-like receptor 9 (TLR9) is an intracellular pattern recognition receptor, and a potential contributor to CLL cell chemotaxis and tumour maintenance. TLR9 recognises unmethylated CpG motifs within bacterial/viral/mitochondrial DNA, and TLR9 activation promotes an NFkB and STAT3-driven activation/migratory phenotype in primary CLL cells. We have previously shown cell-free unmethylated DNA to be up to 28-fold higher in CLL patient plasma, relative to healthy controls. Aims: To investigate TLR9 activation as a potential mechanism of resistance to current B-cell receptor (BCR)-targeted therapeutics. Methods: Migration assays were performed using primary CLL cells stimulated -/+ 1μM ODN 2006 (TLR9 agonist), -/+ 1μM ibrutinib (BTK-inhibitor), 2μM ODN INH-18 (TLR9 antagonist) or -/+ 2.5-10μM CW15337 (NIK inhibitor). CLL cells migrated through 5μM pore membranes towards a CXCL12-gradient. Results: TLR9 activation induced a dichotomous migratory response in CLL patient samples. Following stimulation with ODN 2006, 25/42 (60%) patient samples showed an increase in CLL cell migration (‘Responders’) and 17/42 (40%) showed either no change or a decrease in CLL cell migration (‘Non/Reverse Responders). Interestingly, IGHV-mutated (M-CLL) samples were almost exclusively ‘Responders’ (i.e., 14/17 [82%]) and IGHV-unmutated (U-CLL) samples were equally likely to be ‘Responders’ or ‘Non/Reverse Responders’ (i.e., 11/25 [44%] vs 14/25 [56%] respectively). Whilst there was no difference in the expression levels of TLR9 in M-CLL vs U-CLL cells, U-CLL samples expressed significantly higher basal levels of CD69 (B-cell activation marker), and their TLR9-induced migratory response negatively correlated with their basal migration. We therefore hypothesised that U-CLL ‘Non/Reverse Responders’ may have reached maximal stimulatory capacity through BCR-signalling alone, rendering them unresponsive to further activation. To test this hypothesis, we simulated BCR and TLR9 stimulation in M-CLL and U-CLL cells using mathematical modelling. Our simulations showed M-CLL cells (with low basal BCR-activation) to induce NFkB signalling in response to TLR9 activation and U-CLL cells (with high basal BCR-activation) to be unresponsive. Importantly, when simulating treatment with a Bruton’s Tyrosine Kinase inhibitor (BTKi), U-CLL cells gained sensitivity to TLR9 activation. These results have since been verified in vitro using BTKi-treated patient samples, where we found a subset of U-CLL ‘Non/Reverse Responders’ to become ‘Sensitised’ to TLR9 activation in the presence of ibrutinib. In the ‘Responder’ subgroup, ODN INH-18 (TLR9 antagonist) and ibrutinib inhibited CLL cell migration synergistically. Together, these data implicate TLR9 signalling as a tumour escape mechanism following BTKi therapy. Since both the BCR and TLR9 signalling pathways culminate in NFkB activation, we are currently investigating components of NFκB signalling as potential novel therapeutic targets. Preliminary data using the NIK selective inhibitor ‘CW15337’ shows CLL cell migration to be inhibited with an IC50 of <1μM. Summary/Conclusion: CLL patient plasma contains high levels of TLR9 ligand (unmethylated DNA), which may promote CLL cell trafficking and BTKi resistance in select subgroups. We believe that targeting downstream NFκB signalling has the potential to inhibit both BCR and TLR9 signalling, and to increase treatment efficacy in TLR9 ‘Responder’ and BTKi ‘Sensitised’ patients. P614: SUBCLONAL ARCHITECTURE OF CHROMOSOMES REVEALED BY SINGLE-CELL ANALYSIS OF GENE EXPRESSION IN A PATIENT WITH CLONAL EVOLUTION OF RELAPSING/REFRACTORY CLL J. Kotaskova1,2,3,*, T. Kurucova2, K. Reblova1,2,3, M. Valisova1,2, K. Zavacka1,2, A. Mareckova1, M. Bohunova1, S. Pavlova1,2, J. Malcikova1,2, V. Navrkalova1,2, M. Jarosova1,3, M. Doubek1,2,3, K. Plevova1,2,3, S. Pospisilova1,2,3 1Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University; 2Central European Institute of Technology, Masaryk University; 3Institute of Medical Genetics and Genomics, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czechia Background: Chronic lymphocytic leukemia (CLL) manifests by remarkable intraclonal heterogeneity of genomic defects, especially in patients with relapsed/refractory disease, often connected with complex karyotype. In cancer cells, chromosomal changes are often multiple and complex. Aberrations can be present only in subclones and often underlie disease refractoriness. Using the bulk analysis such as WGS, WES, or genomic array to precisely determine the co-occurrence of aberrations in the individual cell provides limited information. Aims: To perform an in-depth, single-cell RNA sequencing (scRNAseq) study of subclonal changes associated with relapsed/refractory CLL. Methods: A bulk analysis of DNA from separated malignant cells using comprehensive NGS panel LYNX (PMID 34082072) was performed on a representative CLL case at three time-points (TPs): at dg (TP1, month 0, Rai II), before first therapy (TP2, month 19) and during relapse (TP3, month 59). Simultaneously, the transcriptome was analyzed using scRNAseq (Chromium system, 10x Genomics) in 2330 cells (TP1), 2397 cells (TP2), and 1902 cells (TP3). A detailed data analysis was carried out with the Seurat R package. In addition, we used the InferCNV tool to detect the chromosomal aberrations in every tested cell. We identified multiple chromosomal changes and compared the findings with the results of bulk DNA analysis using a genomic array (ThermoFisher Scientific) and FISH. Results: We detected a number of aberrations that were already present at TP1, including del13q, del11q, mutations in NOTCH1, RPS15, ZMYM3, and minor mutations in TP53 and ATM. At TP2, additional changes comprised mutations in PIM1, XPO1, and del(3q). Between TP2 and TP3, the patient underwent several treatment lines (FCR, BR, COP) without an apparent therapeutic effect. The cells from TP3 carried complex changes, including additional mutations in BIRC3 and RB1. Unsupervised clustering of scRNAseq gene expression data defined major clones bearing aberrations (losses on chromosomes 1, 9, 13, 17, 18). Moreover, these clones were composed of cells with additional aberrations defining a number of separate subclones. The proportion of subclones was typically under the detection limit of the genomic array and showed extreme intraclonal heterogeneity of the relapsed/refractory CLL case. Summary/Conclusion: We showcase how genomic alterations influence the expression of affected genes. Such fact can be exploited to reconstruct chromosomal disruptions in individual cells using scRNAseq and to uncover the subclonal architecture of the disease. Grants: MH-CZ_AZV_NU20-08-00314, MH-CZ_AZV_NV19-03-00091, MEYS-CZ_MUNI/A/1330/2021, MH-CZ_RVO_65269705. P615: BCL2 RESISTANCE MUTATIONS IN A REAL-WORLD COHORT OF PATIENTS WITH VENETOCLAX-TREATED CHRONIC LYMPHOCYTIC LEUKAEMIA L. Kotmayer1,*, T. László1, R. Kiss1, L. L. Hegyi1, G. Mikala2, P. Farkas3, A. Balogh3, T. Masszi3, J. Demeter4, J. Weisinger3, H. Alizadeh5, L. Gergely6, A. Sulák7, M. Egyed8, M. Plander9, P. Pettendi10, D. Lévai11, T. Schneider11, Z. Pauker12, A. Masszi11, R. Szász6, C. Bödör1, D. Alpár1 1Department of Pathology and Experimental Cancer Research, Semmelweis University; 2South-Pest Central Hospital – National Institute of Hematology and Infectious Diseases; 3Department of Internal Medicine and Hematology; 4Department of Internal Medicine and Oncology, Semmelweis University, Budapest; 51st Department of Internal Medicine, Clinical Centre, University of Pécs, Pécs; 6Division of Hematology, Department of Internal Medicine, University of Debrecen, Debrecen; 72nd Department of Internal Medicine and Cardiology Center, University of Szeged, Szeged; 8Kaposi Mór University Teaching Hospital of County Somogy, Kaposvár; 9Markusovszky University Teaching Hospital, Szombathely; 10Hetényi Géza Hospital and Clinic of County Jász-Nagykun-Szolnok, Szolnok; 11National Institute of Oncology, Budapest; 12Borsod-Abaúj-Zemplén County Hospital and University Teaching Hospital, Miskolc, Hungary Background: The Bcl-2 inhibitor venetoclax has transformed the therapeutic landscape of therapy refractory and treatment naïve chronic lymphocytic leukaemia (CLL). Despite the remarkable response rates in both patient groups, a subset of CLL patients receiving venetoclax treatment experience disease progression, frequently associated with pathogenic variants of the BCL2 gene. Apart from the most common hotspot G101V and D103Y mutations, pathogenic variants affecting several different loci of the coding region of BCL2 have been described, however data and international recommendations on the sensitive detection and monitoring of these mutations are still absent to date. Aims: Our aim was to develop a droplet digital PCR (ddPCR) based assay for the detection of BCL2 G101V and D103Y mutations and to reveal further resistance mutations emerging at relapse by performing targeted ultra-deep next-generation sequencing (NGS). Methods: Peripheral blood samples from 71 patients treated with venetoclax-rituximab or first line venetoclax-obinutuzumab combination were collected from 11 Hungarian oncohematological centres. Genomic DNA was extracted from peripheral blood mononuclear cells and leukemic cell purity was assessed by flow cytometry. Fractional abundance of the most common hotspot BCL2 mutations G101V and D103Y was assessed using the QX200 digital droplet PCR system. Following ddPCR analyses, targeted ultradeep NGS was performed on samples obtained at relapse using a custom-designed panel covering the whole coding regions of BCL2, BTK, PLCG2 and TP53 genes. Fractional abundance of BCL2 mutations identified by NGS was assessed retrospectively in serial peripheral blood samples by ddPCR using custom-designed, allele-specific assays. Results: Lower quantitative limit of detection of the BCL2 ddPCR assays was tested and could ubiquitously be established as 0.01%. With a median follow up of 14 months, BCL2 G101V and D103Y mutations were detected in 8.5% (6/71) and 9.9% (7/71) of the patients, respectively, with 90% (9/10) of them experiencing relapse during the follow-up period. Three patients harbored both resistance mutations as detected by ddPCR. BCL2 G101V and/or D103Y were observed in 43.5% (10/23) of the cases showing signs of disease progression. In four cases, emergence of the mutations predated the first clinical signs of relapse with a median 4 months. In samples obtained at relapse, targeted ultradeep NGS uncovered two additional BCL2variants (V156D and A113G), which were successfully backtracked and detected by ddPCR. Both V156D and A113G were detected in patients previously found to harbor G101V or D103Y, with no further BCL2 resistance mutations identified in G101V and D103Y wild type cases. Summary/Conclusion: In a real-world cohort of CLL patients receiving venetoclax in combination therapy, comprehensive and sensitive screening of BCL2 mutations can identify molecular mechanisms of venetoclax resistance in nearly half of the patients experiencing relapse. In patients harboring multiple BCL2 mutations, convergent evolution of the CLL subclones may contribute to the driver mechanisms of resistance, justifying the comprehensive approach for the detection of these variants. In secondary venetoclax resistant cases displaying wild type BCL2, further molecular screening methods are required to reveal alternative genetic or non-genetic reasons for disease progression. P616: THE LOSS OF IΚBΕ INHIBITOR ACCELERATES DISEASE DEVELOPMENT IN CHRONIC LYMPHOCYTIC LEUKEMIA C. Lenzi1,*, J. Bordini2, A. Pseftogkas1, A. Morabito2, G. Tsiolas3, F. E. Psomopoulos3, A. Campanella1, M. Frenquelli2, P. Ghia1 1Division of Experimental Oncology, Università Vita-Salute San Raffaele; 2Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy; 3Center for Research and Technology Hellas-CERTH, Thessaloniki, Greece Background: Chronic lymphocytic leukemia (CLL) is an indolent B cell malignancy in which B lymphocytes accumulate in peripheral blood (PB), bone marrow (BM) and spleen (SP), as a result of a complex intertwining between microenvironmental stimuli and genetic defects. Molecular studies revealed recurrent mutations in a large number of genes, each present with distinct frequency in different subgroups of patients. Among these genes, NFKBIE, a negative regulator of the NF-κB pathway, has been found mutated in a relevant proportion of patients with CLL, mainly those belonging to the clinically aggressive subset #1, suggesting that a reduced expression of NFKBIE may contribute to CLL aggressiveness through constitutive NF-κB activation independent of BcR signaling. Aims: We aimed at studying the effects of NFKBIE in the pathogenesis of CLL using functional inactivation of the gene both in vitro and in vivo. Methods: We engineered the MEC-1 CLL cell line using CRISPR/Cas9 technology to generate NFKBIE- knock-out (KO) cell lines. We analyzed the effect of NFKBIE ablation studying in vitro cell proliferation and migration, gene expression, BcR signalling pathway, and response to treatment with the BTK inhibitor Ibrutinib. We also evaluated the relevance of NFKBIE manipulation in vivo by injecting the engineered cells into RAG2-/- γc-/- immunodeficient mice. In addition, we generated NFKBIE-/wt mice and crossed them with Em-TCL1 mice, a validated CLL mouse model, to generate immunocompetent Em-TCL1-NFKBIE-/wt. We analyzed disease expansion by flow cytometry, measuring the amount of malignant B cells in the PB, SP and BM. Results: MEC-1 cells carrying NFKBIE ablation showed faster proliferation in vitro as compared to controls (p<0.05). RNAseq analyses indicated de-regulation of migratory pathway that was functionally confirmed by migration experiments with the specific chemokines (i.e. CCL19, CCL21 and SDF1). In particular, the percentage of migrated KO cells was significantly higher than that of control cells, either in the presence or the absence of each of the three chemokines. As predicted by the function of the gene, biochemical analyses in KO cells revealed the increase of cRel and p50 protein levels and the increase of p65 phosphorylation, all NF-κB transcription factors indicating unabated cellular activation. Interestingly, in vitro culture of the cells in the presence of Ibrutinib showed that NFKBIE KO cells are more resistant to apoptosis induction than control cells (p<0.05). The effect of NFKBIE manipulation was also more evident in vivo. Xenografts generated with MEC-1 NFKBIE KO cells in RAG2-/- γc-/- immunodeficient mice showed an increased disease expansion in the SP and BM (p<0.05) and a shorter survival (p<0.05) than mice injected with control cells. In immunocompetent Em-TCL1 mice, which develop the CLL-like disease with aging, preliminary analysis at 3, 6 and 9 months of age showed higher malignant cells expansion in PB of Em-TCL1-NFKBIE-/wt compared with control Em-TCL1 mice. Summary/Conclusion: The loss of NFKBIE function appears to be associated with a more aggressive disease in all CLL models tested, both in vitro and in vivo, accelerating disease development under CLL pro-tumorigenic conditions. In addition, it appears to be potentially involved in the onset of resistance to the BTK inhibitor Ibrutinib, thus providing an alternative mechanism that may explain drug failure in the resistant patients who do not carry BTK/PLCG2 mutations. P617: FACING LIMITED DATASET IN HEMATOLOGICAL MALIGNANCIES: OPTIMIZATION OF MACHINE LEARNING MODELS WITH THE GENERATION OF NEW TRAINING VARIABLES AND HYPER-PARAMETERS SELECTION. R. BIZOÏ1,*, L. Mauvieux2,3 1DBSYS; 2Laboratoire d’hematologie, Hopital Hautepierre; 3IRFAC, INSERM U1113, Strasbourg, France Background: There is a growing interest in the application of Machine-Learning (ML) techniques in medical research. However, the datasets in this field concern a limited number of individuals. Our goal was to implement technologies and measures for estimating learning performance in a small dataset of B-cell chronic lymphoid malignancies with extreme individuals (outliers) and a modality imbalance for the target variable. Aims: We postulated that a limited set of antigen expression could decipher the different diagnosis of chronic B-cell malignancies (CBCM). In this aim we retrospectively studied the flow cytometry results obtained at the diagnosis for 673 patients explored in our center: 212 CLL/lymphocytic lymphoma, 160 lymphoplasmacytic lymphoma (LPL, including Waldenstrom Macroglobulinemia), 76 mantle cell lymphoma (MCL), 169 marginal zone lymphoma (MZL), 35 Hairy cell lymphoma (HCL) and 27 follicular lymphoma. We focused on median fluorescence intensity (MFI) values of CD148, CD180 and CD200 and three categorical “positive or negative” markers (CD5, CD23, FMC7). Methods: We used the Scikit-Learn python library and LightGBM to test several ML models and develop techniques for generating new variables and selecting hyper-parameters. The treatments on the dataset were the following: 1. The three quantitative dimensions were normalized using Box-Cox transformation for LightGBM, LogisticRegression, RandomForest, KNN and GaussianNaiveBayes algorithms. 2. 70% of the data for training and 30% for testing were randomly separated. 3. Outlier removal and diagnostic dimension, modality balancing treatments were performed only on the learning sample using different unsupervised methods: localOutlierFactor, IsolationForest, OneClassSVM and EllipticEnvelope. 4. In order to avoid overfitting, the number of individuals of the least frequent modalities were balanced using (Synthetic Minority Over-sampling Technique) SMOTE algorithm. 5. For a better accuracy of classification algorithms, a discretization was set on the quantitative dimensions (CD148, CD180 and CD200 MFI). Thus all the values of the three dimensions were merged into one and this dimension was discretized into 96 quantiles. 6. New variables were created, that allowed a better learning for the LightGBM and RandomForest classification algorithms. Polynomial transformation and a principal component analysis were used for Logistic Regression. 7. Algorithms hyperparameters of ML were optimized in order to improve performance using GridSearchCV. The selection of the best ML models was done using cross-validation. Results: The models were used for the prediction of CLL, LPL, MCL and MZL diagnosis. The results for each of the classifiers are summarized in Table 1. The global accuracy of the prediction using only 6 antigenic markers was very stable, ranging from 80 to 85% for the different algorithms. The F1-score, that represents the harmonic mean of the precision and recall of prediction (TP/(TP+ ½* (FP + FN)) was the best for CLL (f1 = 91,4%) and the lowest values were obtained for MCL and LPL. Image: Summary/Conclusion: In conclusion, the methodology presented here allows an optimization of machine learning algorithms for limited set of data. Using only the results of six flow cytometry stainings, that can be analyzed in a single tube, high diagnosis prediction rates were obtained. These results, that need to be confirmed in different series, open the way to innovating diagnosis assistance tools in the field of hematological malignancies. These methods can also be applied to other diagnosis issues. P618: SECOND-GENERATION CD19 TARGETING TRI-SPECIFIC KILLER ENGAGER DRIVES ROBUST NK CELL FUNCTION AGAINST B CELL MALIGNANCIES J. Miller1,*, B. Kodal1, P. Hinderlie1, D. Vallera2, G. Berk3, V. Bachanova1, M. Felices1 1Medicine; 2Radiation Oncology, University of Minnesota, Minneapolis; 3GT Biopharma, Brisbane, United States of America Background: Chronic Lymphocytic Leukemia (CLL) involves uncontrollable clonal expansion of CD5+/CD19+ B lymphocytes. While patients can sometimes coexist with CLL for years, eventually there is progression to bulky adenopathy and pancytopenia from marrow suppression requiring therapy. In recent years, targeted therapies, including utilization of the inhibitors for B cell receptor (BCR) signaling and for B cell lymphoma 2 (Bcl-2) protein, have been effective treatment options. However, these treatments are associated with limitations such as development of drug resistance and the lower treatment efficacy ratio in high-risk patients. Meanwhile, CAR-T cell immunotherapy failed to produce effective treatment outcomes against CLL, mostly due to defects in the effector T cells leading to product failures, as well as being associated with high levels of off-target cytotoxicity. Aims: Thus, approaches to CLL treatment are necessary and NK cell immunotherapy presents a viable and non-toxic option to this problem. Methods: Our group previously demonstrated the immunotherapeutic potential of utilizing first-generation CD19-targeting 161519 Tri-Specific Killer Engager (TriKE®) to drive natural killer (NK) cell cytotoxicity against B-cell malignancies. We have now produced a novel second-generation CD19 TriKE, ‘CAM161519’, utilizing a humanized camelid anti-CD16 VHH single domain antibody (sdAb) ‘CAM16’, a wild-type IL-15 component, and anti-CD19 tumor antigen scFv all linked via short peptide linkers. The TriKE primarily works by generating a cytolytic bridge between NK cells and CD19 expressing B cell malignancies while also providing an expansion and survival signal to the NK cells. Using flow-based assays and imaging based cytolytic assays we explored the capability of CAM161519 to target a variety of B cell lines (Raji, Daudi, and Nalm6) and primary B cell malignancies from patients with CLL and B cell acute lymphoblastic leukemia (B-ALL). Expanded NK cells were incubated with primary CLL targets from 6 different patients and no treatment (NT), rhIL-15 (30 nM), Rituximab (10 ug/mL), or CAM16159 (30 nM). NK cell degranulation and IFNg production was evaluated after 5 hours of co-culture (Figure). CLL killing was evaluated in a similar co-culture, but with a staining mix containing CD5 and CD19, to identify CLL tumor cells in order to determine the percent CLL targets killed normalized to incubation of CLL cells with NK cells alone (Figure Image). Results: The CAM161519 robustly activated NK cell degranulation (CD107a) and inflammatory cytokine production (IFNg) against B cell lines. The activation was specific as it was strongly reduced against CD19 knockout Nalm6 cells. The TriKE also induced specific NK cell proliferation. IncuCyte imaging based cytolytic assays demonstrated strong killing activity against Raji targets. When evaluating activity against primary targets, CAM161519 induced enhanced NK cell degranulation, cytokine production, and cytotoxicity using flow cytometry-based assays against primary CLL (Figure Image) and B-ALL targets when compared to IL-15 treatment. CAM161519 also rescued functionality of NK cells from CLL patients against their autologous CLL cells. Image: Summary/Conclusion: Taken together, these data are indicative of the potential for clinical translation of the CAM161519 TriKE in the CLL and B-ALL settings alone or in combination with cellular products. P619: TARGETING TRANSLATION BY DISRUPTING THE NEWLY IDENTIFIED PROHIBITIN-EIF4F COMPLEX AS A NOVEL THERAPEUTIC STRATEGY IN CHRONIC LYMPHOCYTIC LEUKEMIA A. Largeot1, V. Klapp1, S. Gonder1,*, E. Viry1, M. Szpakowska2, D. Perez Hernandez2, G. Dittmar2, A. Chevigné2, L. Ysebaert3, B. Stamatopoulos4, L. Desaubry5, J. Paggetti1, E. Moussay1 1Department of Cancer Research, Luxembourg Institute of Health, Luxembourg; 2Department of Infection and Immunity, Luxembourg Institute of Health, Esch sur Alzette, Luxembourg; 3Service d’Hématologie, IUCT-Oncopole, Toulouse, France; 4Laboratory of Clinical Cell Therapy, ULB-Research Cancer Center (U-CRC), Jules Bordet Institute Université Libre de Bruxelles, Brussels, Belgium; 5Regenerative Nanomedicine Laboratory (UMR1260), Faculty of Medicine, FMTS, INSERM-University of Strasbourg, Strasbourg, France Background: CLL is the most common type of leukemia in adult, and despite great advance in the standard of care in the last decades, there is still no cure available. CLL cells are dependent on their microenvironment for proliferation and survival. Microenvironmental stimuli are associated with an increase in translation globally but also at the level of specific transcripts, including Myc (Yeomans et al., 2016, Blood). Aims: Here, we tested the targeting of translation initiation in CLL as a novel therapeutic strategy and identified prohibitin as partner of the translation initiation machinery. Methods: In order to target translation, we used the FL3 molecule, a synthetic flavagline, which is a known inhibitor of translation initiation in other types of cancers (Boussemart et al., 2014, Nature). This drug binds to the scaffold proteins prohibitins (PHBs), but the mechanism for translation inhibition is still unclear. PHBs are crucial partners for the activation of the Ras-Raf-MEK-ERK pathway, ultimately leading to eIF4E (a factor of the eIF4F translation initiation machinery) phosphorylation and to activation of translation. Results: We confirmed the increase in translation in human primary CLL cells upon stimulation (A). In addition, we showed that in the Eµ-TCL1 murine model, CLL cells have a higher translation rate compared to normal B cells and T cells. In vitro treatment of human CLL cells (either cell lines or primary patient samples) with FL3 leads to a decrease in translation rate. It is associated with a strong decrease of cell viability (B) and apoptosis induction, even at a low nanomolar dose. By western blot, we showed that in CLL, contrary to other cancer types, FL3 does not prevent RAF1 and ERK phosphorylation. However, eIF4e phosphorylation is impaired. This observation strongly suggests a direct impact of the drug through its molecular target PHBs on the translation initiation machinery. By co-immunoprecipitation, proximity-ligation assay (C) and Nanoluciferase experiments, we demonstrated the interaction between PHBs and the members of the eIF4F complex. In addition, gene silencing of PHB by shRNA leads to a decrease in translation. This indicates for the first time a direct role of PHBs in translation, and allows a better understanding of FL3’s mechanism of action. We thus propose that PHB is necessary for the correct assembly of the eIF4F complex. Interestingly, we showed that the loss of eIF4e phosphorylation is neither responsible for the impairment of translation nor for the decrease in cell proliferation. By pulse SILAC experiments, we determined the proteins that are subjected to increased translation upon TLR stimulation, and to decreased translation upon FL3 treatment. Among others, oncogenes such as c-MYC or ETS-1 (D) have been identified. In vivo treatment of mice with FL3 after TCL1 adoptive transfer leads to a significant delay in CLL progression and an increased survival (E-F), demonstrating the relevance and possible impact of this molecule. Finally, high expression of translation initiation-related genes correlated with poor survival and unfavorable clinical parameters in CLL patients (G). Image: Summary/Conclusion: To conclude, we identified translation initiation as a potential therapeutic target in CLL. The use of the inhibitor of translation FL3, efficiently impairs proliferation of CLL cells in vitro and controls CLL development in vivo. Moreover, we start to unveil the mechanism of action of this molecule, by the identification of a novel interactor of the translation initiation machinery, namely prohibitins. P620: DYNAMICS OF GENOMIC ABERRATIONS IN RELATION TO DISEASE ACTIVITY IN UNTREATED PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA V. Navrkalova1,2,*, J. Kotaskova1,2, K. Plevova1,2, T. Reigl2, M. Valisova1, M. Bohunova1, M. Zenatova1, L. Radova2, A. Panovska1, S. Pospisilova1,2 1Internal Medicine - Hematology and Oncology, University Hospital Brno and Masaryk University; 2Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czechia Background: Chronic lymphocytic leukemia (CLL) manifests remarkable clinical variability linked with extensive heterogeneity of genomic defects. Despite the progress with novel therapeutic agents, some patients still face relapse and disease persistence due to subclonal populations, which harbor various somatic defects, growth dynamics, and therapy sensitivity. Clonal evolution of genomic defects was observed mainly in treated CLL patients, whereas progression and therapy need occur at some point during the disease course suggesting clonal dynamics even in untreated patients. Aims: To describe impact of clonal dynamics in gene mutations and chromosomal defects on clinical course of the disease in paired samples of untreated CLL patients. Methods: 200 samples from 100 CLL patients were analyzed by well-established comprehensive NGS panel LYNX (PMID 34082072) in two time points; at the diagnosis and before first therapy. Variants in 70 genes associated with B-cell malignancies, copy-number alterations (CNAs) and copy-neutral loss of heterozygosity (CN-LOH) across the whole genome were evaluated by a dedicated in-house bioinformatic pipeline. Clinical data were correlated with obtained results. Results: The surveillance period median between two sampling points was 36 months (range 7-174). One third of patients was still untreated during the follow-up (median 86 months, range 21-271), and the time to first treatment among 68 patients was 38 months (range 11-141). At the baseline, the most frequent defects were mutations in NOTCH1 (15 %), SF3B1 (15 %), ATM (12 %) genes, and 13q- (56 %), 11q- (19 %) and tri12 (15 %). We observed common clonal and subclonal co-occurrence of genomic defects in the pretreatment period. In sequential samples, we detected a change in the composition of genomic aberrations. Most alterations were stable (59 % mutations, 70 % chromosomal defects), but we observed dynamic changes in the rest with the predominance of growing allelic frequency and occurrence of new defects. Unsupervised clustering according to the presence of individual genomic defects with the emphasis on their dynamic changes and CLL driver attribute enabled to distinguish three clusters. The correlation with clinical data showed significant differences among clusters in IGHV status, CLL activity (categorized according to therapy need), and an increase of absolute lymphocyte count (ALC) per month. Obviously, the most adverse was cluster 1 with unmutated IGHV, rapid increase in ALC, and dynamics in CLL activity. Cluster 2 seemed to be intermediate and cluster 3 indolent. Groups also significantly differed in the presence and evolution of 11q-, 13q-, ATM mutations, tri12, chr13 LOH, 3p-, 2p+, chr9 LOH. Importantly, stable defects of chr13 were the most abundant in cluster 3, stable tri12 and expansions of 13q- in cluster 2, dynamics defects including 11q-, ATM mutations, 3p-, 2p+ and LOH 9 in cluster 1. Summary/Conclusion: In untreated CLL patients, clonal stable genomic defects prevail. However, common subclonal alterations and dynamic changes were observed in a considerable proportion of patients. After unsupervised clustering, a group of patients showing the most adverse clinical disease course harbored ATM defects, 3p-, 2p+, and chr9 LOH, possibly standing behind fast progression and therapy need. Hence, detailed monitoring of clonal evolution can help understand the heterogeneity in the CLL activity in the pretreatment period. Supported by AZV NV19-03-00091, MZ-CR RVO 65269705, MUNI/A/1330/2021. P621: SUBCLONAL EVOLUTIONARY TRAJECTORIES IN IGLV3-21R110 CHRONIC LYMPHOCYTIC LEUKEMIA L. Paschold1,*, D. Simnica1, R. Benitez Brito2, C. Schultheiss1, T. Zhang1, C. Dierks1, M. Binder1 1Department of Internal Medicine IV, Oncology/Hematology, Martin-Luther-University Halle-Wittenberg, Halle, Germany; 2Department of Hematology, Inselspital, University of Bern, Bern, Switzerland Background: There is a large body of evidence supporting the importance of antigenic interactions of the B cell receptor (BCR) with self or non-self antigens that make this receptor and its signaling machinery one of the most important drivers in many types of non-Hodgkin lymphoma. This has led to a new pathobiological understanding of these diseases as well as novel therapeutic strategies. Chronic lymphocytic leukemia (CLL) is a prototype disease in which BCR-BCR self-interactions induce autonomous signaling. While virtually all CLL BCR are prone to autonomous signaling induced by such contacts, the IGLV3-21R110 molecular interaction found in 10% of CLL patients defines a distinct clinical subset of patients that show a very aggressive clinical course despite novel treatments. In these cases, a single amino acid replacement from G to R located at the boundary of variable and constant region of the BCR is an important tumor promotor driving neoplastic B cell growth. Aims: It has been unclear how the R110 point mutation is generated. We set out to investigate the trajectory of the malignant CLL B cell clone and subclonal heterogeneity in IGLV3-21R110 cases in order to gain insight into the molecular pathogenesis of this unique amino acid exchange. Methods: We screened an initial CLL cohort of 127 cases by flow cytometry using an anti-IGLV3-21R110 antibody. Positive cases were subjected to immunosequencing of the light chain locus via amplicon based next-generation sequencing and characterized for typical chromosomal deletions and driver gene mutations. Results: We identified twelve CLL cases with IGLV3-21R110. Of these, we found a higher frequency of mutations in ATM (42%) and SF3B1 (25%) than in unselected CLL cohorts, which is in line with findings reported by others. As expected, the repertoire of healthy B cells in the blood of IGLV3-21R110 CLL patients contained IGLV3-21 light chains with the wild-type G110 residue, indicating that the R110 exchange is of acquired nature. Interestingly, in half of the IGLV3-21R110 CLL cases we found additional clones with IGLV3-21 and R110, including cases with divergent J gene cassettes originating from different pre-B cells. This indicates that the R110 exchange may occur multiple times within independent B cell clones in the same patient. Due to the unique location at the variable-constant boundary and the high correlation of the IGLV3-21R110 patient subset with mutations of genes involved in DNA repair (ATM/SF3B1), R110 is likely generated as a result of incorrect V-J recombination facilitated by the error-prone-environment. Summary/Conclusion: Our work indicates that the G110R point mutation results from incorrect V-J rearrangements in the error-prone environment that may be induced by SF3B1 and ATM mutations associated with this CLL subset. We found evidence for a branching light chain evolution that leads to a striking convergence of separate clones on the identical light chain amino acid exchange in the same patient. This strongly emphasizes the role of this mutation as a tumor promotor involved in malignant transformation of this subset of CLL. P622: DOUBLE-HIT TRAF3 DELETION AND MUTATION IDENTIFIED BY HIGH-THROUGHPUT SEQUENCING DEFINE A NEW INDEPENDENT PROGNOSTIC FACTOR IN CHRONIC LYMPHOCYTIC LEUKEMIA C. Perez-Carretero1,2,*, M. Hernández-Sánchez1,2, M. Quijada-Álamo1,2, T. González1,2, A. Rodríguez-Sánchez1,2, M. Martín-Izquierdo1,2, J. Matías-Martín1, A. Rubio3, J. Dávila4, A. García de Coca5, J. Galende6, P. Jimenez-Montero7, J.-A. Queizán7, I. González-Gascón y Marín8, J.-Á. Hernández-Rivas8, R. Benito1,2, J.-M. Hernández-Rivas1,2, A.-E. Rodríguez-Vicente1,2 1Department of hematology, Hospital Universitario de Salamanca; 2Universidad de Salamanca, IBSAL, Centro de Investigación del Cáncer, IBMCC-CSIC, Salamanca; 3Department of hematology, Hospital Miguel Servet, Zaragoza; 4Department of hematology, Hospital Nuestra Señora de Sonsoles, Ávila; 5Department of hematology, Hospital Clínico, Valladolid; 6Department of hematology, Hospital El Bierzo, Ponferrada; 7Department of hematology, Hospital General, Segovia; 8Department of hematology, Hospital Universitario Infanta Leonor, Madrid, Spain Background: Chromosome 14q abnormalities involving the IGH gene are present in 5-15% of chronic lymphocytic leukemia (CLL) patients. Besides IGH translocations, interstitial 14q32.33/IGH deletions have been reported at lower frequencies (2%), and their clinical implications and underlying molecular mechanisms remain unknown. In other B-cell malignancies such as multiple myeloma, TRAF3 gene, proximal to the IGH locus, may be encompassed in the deletion, with mutations in the remaining allele. However, the molecular status of TRAF3 in CLL and their impact in prognosis have not been studied in depth. Aims: To analyze the genetic landscape of CLL patients with IGH deletion and evaluate the molecular status of TRAF3 to elucidate their impact in CLL pathogenesis and prognosis. Methods: A total of 897 CLLs were analyzed by FISH with the IGH break-apart probe and the 4-probe CLL panel (ATM, CEP12, D13S319 and TP53) (Vysis). 324 untreated CLLs (54 with IGH deletion (del-3’IGH) and 270 as the control group) were sequenced by targeted-NGS, with a custom panel including 54 CLL-related genes (Nextseq platform, Illumina). Copy number variations (CNVs) of TRAF3 were assessed by a NGS approach and validated by SNP Array 6.0. Results: FISH analyses identified a total of 54 out of 897 CLLs (6%) with a 300 kb deletion of the centromeric side of IGH (del-3’IGH CLLs). Moreover, our results showed that 76% of del-3’IGH CLLs had additional FISH abnormalities, being the most frequent the loss of 13q (42%), +12 (22%), del(11q) (11%) and 17p- (9%). NGS analyses showed that the most recurrently mutated genes in del-3’IGH CLL were NOTCH1 (30%), ATM (20%) and TRAF3 (13%). Notably, the mutational frequency of TRAF3 mutations was significantly higher in del-3’IGH CLLs than in the control group (13% vs. 0.5%, p<0.001). Given the surprisingly incidence of TRAF3 mutations in this subgroup, we further assessed its CNV status, noticing that TRAF3 loss was enriched in del-3’IGH CLLs when compared to the control group (11/54, 20% vs. 4/270, 1.5%, p<0.001). By integrating mutational and CNV information, we observed that 7 out of 11 patients with a TRAF3 loss harbored mutations in this gene. The deletion was present in a higher percentage of cells than the mutation (mean: 70.5% vs. 15.6%), which may suggest that TRAF3 mutation appears as a subclonal event, leading to a biallelic inactivation of this gene. Our results showed a novel biallelic alteration in CLL, reminding the double null event that occurs in other gene drivers such as TP53 and ATM. Of note, this double-hit on TRAF3 contributed to a shorter time to first treatment (TFT) in del-3’IGH CLL patients (5 vs. 54 months, p<0.001). Furthermore, TRAF3 biallelic inactivation had a similar TFT than TP53 or ATM-altered patients with an adverse prognosis (median TRAF3: 5 vs. 59 months, p<0.001, ATM: 2 vs 59, p<0.001 and TP53: 12 vs 59, p=0.002), being also an independent risk factor in the multivariate analyses in the CLL cohort (n=324) (HR=0.21, 95% CI=0.05-0.85, p=0.029) (Table 1). Image: Summary/Conclusion: Our work demonstrates that TRAF3 mutations and deletions are highly enriched in CLL patients with del-3’IGH, resulting in the biallelic inactivation of this gene. The presence of this double-hit on TRAF3 worsen the prognosis of CLL patients, being and independent prognostic factor with similar impact on TFT than the biallelic loss of TP53. Funding: PI21/00983; FI19/00191 (CPC); CD19/00222 (MHS); FEHH (MQÁ); USAL (RBS) P623: IBRUTINIB REGULATES MIR-181A/B VIA C-FOS IN CHRONIC LYMPHOCYTIC LEUKEMIA A. Ramassone1,*, F. Palmiero1, A. Tomasso2, I. Innocenti2, L. Laurenti2, R. Visone3 1Center for Advanced Studies and Technology, “G. d’Annunzio” University, Chieti; 2Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma; 3Department of Medicine, Dentistry and Biotechnology, “G. d’Annunzio” University, Chieti, Italy Background: B-cell receptor (BCR) signalling controls B-cell survival and proliferation, and its deregulation has a pathogenic role in Chronic Lymphocytic Leukemia (CLL), a B-cell malignancy. Currently, several BCR signalling inhibitors are used in therapy, among them the Ibrutinib. We previously reported that the BCR-signalling inhibition mediated by Ibrutinib leads to miR-181a/b activation; those miRNAs are significant biomarkers of CLL progression, and their downregulation is associated to a poor prognosis; furthermore, low levels of miR-181a/b impair several anti-apoptotic proteins, contributing to cell survival. BCR signalling also controls c-Fos/AP-1 pathway in pre-B cells. Recently, we suggested the involvement of c-Fos in miR-181b expression, however the mechanism by which this regulation is exerted is still not explored. Moreover, the involvement of c-Fos in miR-181a/b transcription during Ibrutinib is still not clear. Aims: Assess the impact of Ibrutinib on c-Fos expression; assess the involvement of the transcription factor c-Fos in miR-181a/b transcription; assess the impact of miR-181a/b in CLL cells survival during Ibrutinib treatment. Methods: To assess the impact of Ibrutinb on c-Fos transcription, we evaluated its expression in CLL cells treated with Ibrutinib or vehicle. To assess the involvement of c-Fos in miR-181a/b transcription, we evaluated i) the expression of the miRs and of their primary transcripts in CLL cells overexpressing c-Fos, and ii) the ability of c-Fos to bind miR-181a/b host gene promoter by luciferase, ChIP and EMSA assays. To assess the impact of the miRs on CLL cells survival during Ibrutinib treatment, we performed MTT assay in CLL cells after miR-181a/b silencing and Ibrutinib treatment. Results: Considering that c-Fos was found to be controlled by BCR signalling in pre-B cells, we evaluated whether BCR signalling affect c-Fos also in CLL cells: we found that c-Fos expression increases in primary CLL cells after Ibrutinib treatment. Since miR-181a/b was up-regulated after Ibrutinib treatment in vitro and in vivo in CLL cells, and considering that a possible involvement of c-Fos in miR-181b expression was already suggested, we sought to identify if c-Fos was a direct regulator of miR-181a/b expression. We confirmed that c-Fos overexpression affects miR-181b levels in CLL patients, but not miR-181a; however, the dysregulation of the miR shows heterogeneity among patients. Indeed miR-181b expression increases in 3 and decreases in 2 of 7 patients. To better understand how c-Fos impact on miRs expression, we analysed both miR-181a and miR-181b at the transcriptional level in CLL and lymphoblastoid cell lines, finding that c-Fos negatively regulates the pri-miR-181a/b. To clarify the mechanism by which c-Fos regulates miR-181a/b transcription, we analysed the miRs host gene (MIR181A2HG) promoter. Since MIR181A2HG promoter shows two consensus sequences for the transcription factor c-Fos, we studied whether c-Fos binds these regions. We found that c-Fos binds at MIR181A2HG promoter by recognizing the two consensus sequences. Accordingly, after their deletion, the ability of c-Fos to recognize the promoter was overcome. Finally, since miR-181a and miR-181b are involved in cell death, we assessed whether those miRNAs participate in CLL cells death induced by Ibrutinib: we found that the silencing of both miRNAs improves CLL cell survival during Ibrutinib treatment. Summary/Conclusion: c-Fos is a downstream target of BCR signalling and a direct regulator of miR-181a/b transcription in CLL. MiR-181a/b are effectors of Ibrutinib-induced CLL cells death. P624: CHROMOTHRIPSIS IN PATIENTS WITH CLL AND COMPLEX KARYOTYPE: PATTERNS OF ABERRATIONS AND PROGNOSTIC VALUE S. Ramos-Campoy1,2,*, A. Puiggros1,2, J. Kamaso1,2, S. Beà3, S. Bougeon4, M. J. Larráyoz5, D. Costa3, H. Parker6, G. M. Rigolin7, M. L. Blanco8, R. Collado9, I. Ancín10, R. Salgado11, M. Moro12, T. Baumann3, E. Gimeno1,13, C. Moreno8,14, M. J. Calasanz5, A. Cuneo7, J. C. Strefford6, F. Nguyen-Khac15, D. Oscier16, C. Haferlach17, J. Schoumans4, B. Espinet1,2 1Molecular Cytogenetics Laboratory, Pathology Department, Hospital del Mar; 2Translational Research on Hematological Neoplasms Group, Cancer Research Program, Institut Hospital del Mar d’Investigacions Mèdiques (IMIM); 3Hematopathology Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBERONC, Barcelona, Spain; 4Oncogenomic Laboratory, Hematology Service, Lausanne University Hospital, Lausanne, Switzerland; 5Department of Genetics, Universidad de Navarra, Pamplona, Spain; 6Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom; 7Hematology Section, St. Anna University Hospital, Ferrara, Italy; 8Department of Hematology, Hospital Universitari de la Santa Creu i Sant Pau, Barcelona; 9Department of Hematology, Consorcio Hospital General Universitario, Valencia; 10Department of Hematology and Hemotherapy, Hospital Universitario Cruces, Bilbao; 11Department of Hematology, Fundación Jiménez Díaz, Madrid; 12Department of Hematology, Hospital Universitario Central de Asturias, Oviedo; 13Applied Clinical Research in Hematological Malignances, Cancer Research Program, Institut Hospital del Mar d’Investigacions Mèdiques (IMIM), Barcelona; 14Josep Carreras Leukemia Research Institute, Badalona, Spain; 15Hematology Department and Sorbonne University, Hôpital Pitié-Salpêtrière, APHP, INSERM U1138, Paris, France; 16Department of Molecular Pathology, Royal Bournemouth Hospital, Bournemouth, United Kingdom; 17MLL Munich Leukemia Laboratory, Munich, Germany Background: Chromothripsis is a unique catastrophic event found in 1-5% of chronic lymphocytic leukemia (CLL) patients, associated with poor prognostic factors and short survival (Edelmann et al, 2012; Puente et al, 2015; Leeksma et al, 2021). It is characterized by the presence of multiple genomic rearrangements in one chromosome, resulting in oscillating switches between 2-3 copy number states by chromosomal microarrays (CMA) or next-generation sequencing (Stephens et al, 2011). Optical genome mapping (OGM) is a new method based on the imaging of long DNA molecules (>250kb) labelled at specific motifs, generating a unique pattern. This allows mapping of the genomic location of each molecule and detection of structural and numerical chromosomal abnormalities with high resolution and sensitivity. Aims: 1. To analyze the genome complexity associated with chromothripsis and the patterns of rearrangements detected by OGM; 2. To determine the prognostic impact of chromothripsis in a cohort of CLL patients enriched in complex karyotypes (CK). Methods: A total of 33 patients with chromothripsis detected by CMA were included: 11 with chromothripsis-like patterns (≥7 switches), 22 with classical chromothripsis (≥10 switches). Nine of the cases were studied with OGM (Bionano Genomics). Clinico-biological features and time to first treatment (TTFT) were analyzed. Results were compared with a cohort of 129 patients showing CK without chromothripsis (control group) (Ramos-Campoy et al, 2021). Results: All patients with chromothripsis showed a high genomic complexity (30/33 with CK by chromosome banding analysis and 3/33 classified as complex by CMA [range: 10-24 CNV, being at least three of them >5Mb (range: 3-6)]. Forty-six chromothriptic events were identified. These affected 1-4 chr/patient, but a single chromosome was involved in 25/33 patients (76%). Chromothripsis was distributed throughout all chromosomes, most frequently affecting 3 (n=5), 6 (n=5) and 13 (n=5). The events were mainly multiple losses (25/46) or alternated gains and losses (19/46). Size and coordinates of CNVs detected by OGM and CMA were highly concordant. OGM detected multiple rearrangements cryptic by CMA (median: 9/chromothriptic event). Two patterns of chromothripsis-related rearrangements were identified: one clustered in the chromothriptic region (3/9) and the other involving both chromothriptic and non-chromothriptic chromosomes (range: 1-6 chr) (6/9). One patient showed 36 rearrangements throughout the genome, suggesting the coexistence of chromothripsis and chromoplexy, characterized by the presence of multiple chained translocations (Figure). Patients with chromothripsis showed a higher frequency of TP53 del/mut (70% vs 39%; p=0.001) and a shorter TTFT (15m vs 2m; p=0.013) than the control group. No differences were observed for IGHV and del(11q) status. When stratifying patients based on TP53 status, the presence of chromothripsis was not associated with shorter TTFT. In the multivariate analysis including TP53, genomic complexity by CMA and chromothripsis, only TP53 was statistically significant (HR=1.6; p=0.029). Image: Summary/Conclusion: 1. Chromothriptic events involve multiple loci in CLL patients; 2. OGM allows not only the detection of CNV but also the identification of two rearrangement patterns associated with chromothripsis (clustered or involving non-chromothriptic chromosomes) whose clinical impact should be further explored. 3. In CLL patients with CK, the adverse prognosis of chromothripsis is associated with a higher frequency of TP53 del/mut. P625: SINGLE-CELL RNA SEQUENCING REVEALS THE HETEROGENEITY OF TUMOR CELLS AND IMMUNE MICROENVIRONMENT IN RICHTER SYNDROME AND ACCELERATED CLL Y. Sha1,2,*, Y. Miao1,2, S. Qin1,2, R. Jiang1,2, W. Wu1,2, Y. Xia1,2, L. Wang1,2, L. Fan1,2, W. Xu1,2, H. Jin1,2, H. Zhu1,2, J. Li1,2 1Department of Hematology, The First Affiliated Hospital of Nanjing Medical University; 2Department of Hematology, Pukou CLL Center, Pukou division of Jiangsu Province Hospital, Nanjing, China Background: Richter Syndrome (RS) is defined as development of aggressive lymphoma arising from chronic lymphocytic leukemia/Small lymphocytic leukemia (CLL/SLL). Accelerated CLL/SLL (aCLL/SLL) is pathologically defined as a highly proliferative form of CLL/SLL (either expanded proliferation center or Ki-67>40%). The underlying mechanism behind transformation from indolent CLL/SLL to aggressive DLBCL remains largely unknown. The biological difference between RS DLBCL and aCLL/SLL has not been explored yet. Aims: To explore intra- and inter-tumor and immune microenvironment (TME) heterogeneity of RS and aCLL/SLL. To disclose the molecular mechanism underlying transformation from CLL/SLL to DLBCL. Methods: Single-cell RNA sequencing (scRNA-seq) of paired lymph node (LN) and peripheral blood (PB) was done among 2 RS (P1, P2) and 2 aCLL/SLL (P3, P4) patients (pathologically diagnosed in LN, Fig. A) and bioinformatics analysis was conducted. Results: Identification of cell type in 8 samples showed great heterogeneity (Fig. B). Single-cell trajectory analysis was done using “monocle” inferred tumor development and differentiation of each patient. Monoclonal B cells with high Ki-67 expression and high proportion of G2M phase cells were enriched in the end of pseudotime trajectories (Fig. C). Further analysis of CD5, CD20 and CD23 expression alongside pseudotime trajectories were also conducted, indicating that our trajectories could depict the transformation from indolent CLL/SLL to aggressive DLBCL. Therefore, cell states were clustered according to the trajectories. P1 and P2 were clustered as seven groups respectively (P1A-G, P2A-G) and P3 were clustered as three groups (P3A-G). Notably, although minor proportion of proliferating cells were enriched in the end of P4 pseudotime trajectories, clusters could not be split in P4 patients. All cell states groups were mapped to UMAP analysis and the proportion of G2M phase was highest in state G of RS patients. Single-sample GSEA (ssGSEA) analysis was done to disclose the pathway activation in transformation process and found that pathways including cell cycle, TCA cycle, spliceosome, MYC targets, unfolded protein response, epigenetics, DNA repair, glycolysis and oxidative phosphorylation were gradually activated alongside trajectories from state A-G in RS. Moreover, pathways including NOTCH, P53, PI3K-AKT, Antigen processing and presentation, apoptosis, hypoxia and etc. were found significantly upregulated in RS (Fig. E). Further analysis of immune microenvironment of RS and aCLL/SLL showed that exhaustion T cells were significantly enriched in RS lymph node compared with aCLL/SLL, indicating the immune surveillance escape mediated by exhaustion T cells might prompt DLBCL transformation. PDCD1 (PD-1), CTLA4, LAG3, HAVCR2 (TIM3), TIGIT expression were significantly upregulated in exhaustion T cells of RS patients, suggesting potential treatment target of RS for immune checkpoint inhibitors (Fig. F). Image: Summary/Conclusion: Our study was the first one using scRNA-seq to disclose the tumor heterogeneity and immune microenvironment of RS and aCLL/SLL in single-cell dimension. Evolution trajectories from indolent CLL/SLL to aggressive DLBCL were constructed and pathway activation in the process of transformation were further explored. Moreover, immune microenvironment analysis of RS and aCLL/SLL showed that exhaustion T cells were significantly enriched in RS lymph node and immune checkpoints including PDCD1 (PD-1), CTLA4, LAG3, HAVCR2 (TIM3), TIGIT were significantly upregulated in exhaustion T cells of RS patients, suggesting potential treatment target of RS. P626: FUNCTIONAL SCREENING OF PI3K INHIBITORS STRATIFIES RESPONDERS TO IDELALISIB AND INDICATES DRUG CLASS ACTIVITY IN IDELALISIB-REFRACTORY CLL S. Skånland1,*, Y. Yanping1, P. Athanasiadis1, L. Karlsen1, A. Urban1, I. Murali2, S. Fernandes2, A. Hilli3, K. Taskén1, F. Bertoni4, A. Mato5, E. Normant6, J. Brown2, G. Tjønnfjord1, T. Aittokallio1 1Oslo University Hospital, Oslo, Norway; 2Dana-Farber Cancer Institute, Boston, United States of America; 3Diakonhjemmet Hospital, Oslo, Norway; 4Università della svizzera italiana, Bellinzona, Switzerland; 5Memorial Sloan Kettering Cancer Center; 6TG Therapeutics, New York, United States of America Background: The phosphatidylinositol 3-kinase inhibitors (PI3Ki) idelalisib and duvelisib are approved for relapsed chronic lymphocytic leukemia (CLL). While patients may show an initial response to these therapies, development of treatment resistance or intolerance remains clinical challenges. Prediction of individual treatment responses based on clinically actionable biomarkers is needed to overcome these challenges. Aims: 1. To characterize functional responses to 10 PI3Ki in CLL 2. To study PI3Ki drug class activity in idelalisib-refractory CLL 3. To investigate whether ex vivo drug sensitivity can predict in vivo treatment responses Methods: CLL cells from patients that were treatment naïve (n=7), idelalisib refractory (n=9), or on idelalisib treatment (longitudinal samples from n=6 patients) were screened against 10 PI3Ki (buparlisib, compound 7n, copanlisib, duvelisib, idelalisib, nemiralisib, pictilisib, pilaralisib, umbralisib, ZSTK474), both alone and in combination with the B-cell lymphoma 2 (Bcl-2) antagonist venetoclax. Two idelalisib-resistant lymphoma cell lines were used as reference. Treatment effects on cell signaling were profiled using phospho flow and effects on cell viability were monitored with detection of cleaved caspase-3 and using the CellTiter-Glo assay. Results: Pan-PI3Ki were most effective in inhibiting PI3K signaling both after 30 min and 24h exposure. Inhibition of cell signaling correlated with induction of apoptosis. Treatment with a PI3Ki induced cell death in 72h CellTiter-Glo assays, although to varying extent. The pan-PI3Ki were overall most potent in inhibiting CLL cell viability. Interestingly, pan-PI3Ki exhibited sustained activity both in idelalisib-resistant lymphoma cell lines and in CLL cells from idelalisib-refractory patients. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Notably, CLL cells from idelalisib-refractory patients remained sensitive to idelalisib+venetoclax combination. Ex vivo drug testing on CLL cells from a patient who presented with relapsed disease after sequential treatment with FCR, ibrutinib, idelalisib and venetoclax revealed sensitivity to PI3Ki+venetoclax treatment. The patient consequently started treatment with idelalisib+venetoclax. She obtained an initial partial response, but then suffered intolerable adverse effects and the therapy was stopped. Protein profiling of the patient’s CLL cells collected at 5 time-points before, during and after the combination regimen showed that B-cell signaling was reduced by the treatment, but again significantly increased when the therapy was stopped. Increased phosphorylation of Bruton’s tyrosine kinase (BTK) and extracellular signal-regulated kinase (ERK) at the latest time-point was mirrored by increased sensitivity to venetoclax combined either with a BTK or MEK inhibitor. Unfortunately, the patient passed away and the efficacy of these alternative therapies could not be tested. To more systematically assess the predictive value of ex vivo drug sensitivity data, we analyzed drug responses to 73 combinations on CLL cells from patients treated with idelalisib. CLL cells collected at baseline from patients who obtained a long-term response to idelalisib showed significantly higher drug sensitivity scores than CLL cells from patients who obtained a short-term response. Summary/Conclusion: Our findings indicate PI3Ki drug class activity in idelalisib-refractory CLL, and suggest that ex vivo drug sensitivity may guide precision medicine and predict treatment responses. These results warrant further testing in larger cohorts and in clinical trials. P627: COMBINED DRUG AND CRISPR/CAS9 SCREENING REVEALS SPECIFIC TARGETS FOR SF3B1- AND NOTCH1-MUTATED CLL CELLS L. Dostálová1,2, A. Ladungová1,3, D. Škrnová1, N. Varma Gottumukkala1,2, Y. Lodhi1,2, M. Smida1,4,* 1CEITEC Masaryk University; 2Department of Biology, Faculty of medicine, Masaryk university; 3National Centre for Biomolecular Research, Faculty of Science, Masaryk university; 4Department of internal medicine - Oncology and hematology, University hospital Brno and Medical faculty of Masaryk university, Brno, Czechia Background: Chronic lymphocytic leukemia (CLL) is genetically heterogeneous disease with a plethora of mutations occurring in relatively low fraction of patients. Mutations in SF3B1 and NOTCH1 genes are among the most frequent recurrent mutations found in 10-20% CLL cases. The presence of these mutations is considered as a negative prognostic factor, yet, there is no specific treatment available to achieve more prominent response. Aims: The aim of this study was to use the combination of CRISPR/Cas9 screening and high-throughput drug screening on cells with introduced SF3B1 or NOTCH1 mutations in order to reveal unique vulnerabilities specific to the cells carrying these frequent CLL mutations. Methods: Using CRIPSR/Cas9-based homology directed repair, we have introduced specific mutations most recurrent within the hotspots of SF3B1 (K700E) and NOTCH1 (P2514fs) into CLL-derived cell line HG3. These two knock-in cell line variants were then subjected to a drug screening with a library of 859 approved drugs, enriched with oncology-indicated drugs. Cell viability in response to 10 uM library concentration upon 72h was assessed by Cell-Titer Glo and evaluated against the wild-type counterparts. Simultaneously, genome-wide CRISPR/Cas9 knockout screen was performed in these cell lines to identify genes whose deletion is selectively lethal to SF3B1 or NOTCH1-mutated cells. Results: The CRISPR/Cas9 dropout screen has revealed several interesting genes that are in synthetic lethal relationship with the introduced point mutations. In particular, SPDYE1, a gene involved in cell cycle regulation, and LUC7L3, involved in splicing and cAMP regulatory element binding, were found to be synthetically lethal with the NOTCH1 mutation. On contrary, SNUPN, responsible for transport of spliceosomal snRNPs, and UQCRC1, involved in cytochrome c regulation and oxidative phosphorylation, were found to be essential for SF3B1-mutated cells. Drug screening using library of approved drugs demonstrated general hypersensitivity of both the mutant cell lines and the wild-type controls to the inhibitors of proteasome and HDAC enzymes. In addition, exquisite sensitivity of NOTCH1-mutant and SF3B1-mutant cell lines, contrary to their wild-type counterparts, was revealed towards the inhibitors of various hormone receptors or inhibitors of 20S proteasome. Summary/Conclusion: The combination of diverse screening approaches may reveal novel vulnerabilities specific for individual mutations frequently recurring in CLL patients. Thorough validations of the identified hits is ongoing, together with the analyses into the possible mechanisms behind these unique targets. This research was partly financed by the grant MUNI/A/1330/2021, MUNI/IGA/1516/2020 and MUNI/A/1419/2021. P628: HIGH THROUGHPUT IMMUNOGENETIC EVIDENCE FOR EXTENSIVE IN VIVO CLASS SWITCH RECOMBINATION EVENTS IN CLL E. Sofou1,2,*, A. Agathangelidis3, A.-M. Antoniadou1, E. Georgiou2, M. Papaioannou4, N. Pechlivanis1, F. Psomopoulos1, K. Stamatopoulos1, A. Chatzidimitriou1 1Institute of Applied Biosciences, Centre for Research and Technology Hellas; 2Laboratory of Biological Chemistry, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki; 3Department of Biology, School of Science, National and Kapodistrian University of Athens, Athens; 41st Department of Internal Medicine, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece Background: In vivo Class Switch recombination (CSR) has previously been reported for a fraction of patients with chronic lymphocytic leukemia (CLL) expressing unmutated IGHV genes (U-CLL) and high mRNA levels of activation-induced cytidine deaminase compared to patients expressing mutated IGHV genes (M-CLL). This observation is certainly intriguing, however inherently limited due to the low-sensitive, Sanger-based approach used for the immunogenetic characterization. Aims: Here, we sought to overcome the aforementioned limitation and obtain more insight into in vivo CSR in CLL using a highly-sensitive, next generation sequencing (NGS) methodology. Methods: We studied 73 patients (48 U-CLL, 25 M-CLL) sampled at diagnosis. Case selection was based on the dominant expression of the IgM isotype, as determined by flow cytometry. IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified for the μ, γ, and α transcripts utilizing IGHV Leader and IGHC-specific primers. All obtained PCR products (n=215) were subjected to paired-end NGS. High-quality, productive IGHV-IGHD-IGHJ gene rearrangements (n=23,217,561; median= 97,932/sample) were annotated with IMGT/HighV-QUEST. Clonotypes were defined as rearrangement sequences expressing the same IGHV gene and identical complementarity-determining region 3 (VH CDR3) amino-acid (AA) sequence. Clonotype variants were defined as different nucleotide sequences clustered in the same clonotype. Results: The IGH gene repertoire of μ transcripts was clearly monoclonal in all cases, showing a dominant CLL clonotype (DCC) with a median frequency of 93.5% for either U-CLL or M-CLL. Subsequently, the IGH gene repertoires of γ and α transcripts were scanned for the presence of the DCC. In U-CLL, 46/48 cases expressed switched transcripts with median frequencies of 14% (range 0.01-97.2%) and 21.3% (range 0.1-95.3%) for γ and α transcripts, respectively. In M-CLL, 24/25 patients expressed switched transcripts albeit at significantly lower levels than U-CLL (p=0.01) (median frequencies: 0.2% and 13.6%; ranges 0.03-94% and 0.04-91.2% for the γ and α transcripts, respectively). Interestingly, within U-CLL, cases belonging to stereotyped subset #1 (n=21) and its satellite subset #99 (n=2) exhibited significantly higher levels of switched transcripts (median frequencies: 37.9% and 37.6% for the γ and α transcripts, respectively) than all other cases (p=0.04 for γ transcripts, p=0.002 for α transcripts). Analysis of somatic hypermutation (SHM) in the DCC within the repertoires of μ, γ and α transcripts of U-CLL cases revealed the presence of shared SHMs (even among cases with 100% IGHV gene identity) but also distinct SHMs shared by the γ and α clonotypic transcripts but not the IgM, indicative of common selection pressure post-CSR. Interestingly, M-CLL patients displayed less intraclonal diversity in the γ and α clonotypic transcripts, despite the significant burden of SHM within the IgM DCC. Summary/Conclusion: In conclusion, our data reveal that the B cell receptor IGH gene repertoire is highly dynamic and interconnected in both U-CLL and M-CLL, albeit considerably more pronounced in the former. This is especially the case for patients assigned to the clinically aggressive stereotyped subsets #1 and #99, prompting speculations regarding the precise nature of the respective affinity maturation process. Shared SHMs between clonotype variants only among clonotypic γ and α transcripts rather than the μ transcripts indicate that CSR likely precedes SHM, particularly in U-CLL. P630: TARGETING NEGATIVE REGULATION OF MAPK SIGNALING IN CHRONIC LYMPHOCYTIC LEUKEMIA M. Stumpf1,*, V. Ecker1, P. Giansanti2, J. Lu3, T. Zenz4, I. Ringshausen5, B. Küster6, J. Ruland1, M. Buchner1 1TranslaTUM - Central Institute for Translational Cancer Research, Technische Universität München, München; 2Bavarian Biomolecular Mass Spectrometry Center (BayBioMS), Technical University of Munich, Freising; 3European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 4Department of Medical Oncology and Hematology, University Hospital and University of Zurich, Zurich, Switzerland; 5Wellcome Trust/MRC Cambridge Stem Cell Institute and Department of Haematology, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, United Kingdom; 6Chair of Proteomics and Bioanalytics, Technical University of Munich (TUM), Freising, Germany Background: The MAPK-ERK pathway is a key signaling cascade for cellular homeostasis. Following MAPK activation, feedback loops increase the production of pathway inhibitors, such as dual specificity phosphatases (DUSPs), to restrict MAPK signal. DUSPs can inhibit tumor growth by preventing ERK activation and may act as oncogenes by helping tumor cells in adjusting to elevated MAPK levels. Despite mounting evidence that MAPK signaling is critical for CLL formation and progression, little is known about the role of MAPK negative feedback via the DUSP family in CLL. Aims: In this study, we investigated the significance of DUSP1 and DUSP6’s negative regulation of MAPK in CLL and evaluated their usage as possible therapeutic targets in drug resistant disease. Methods: We examined the expression levels of DUSP1 and DUSP6 in primary CLL patients using RNAseq and immunoblot analysis. To determine their functional relevance, we employed the small molecule inhibitor BCI on primary murine and human (drug resistant) CLL cells in vitro and in vivo and generated genetic knockout MEC-1 CLL cells, to validate the inhibitor effects. In addition, we investigated the downstream effects of DUSP1/6 inhibition on CLL signaling events using global phosphoproteomics in primary CLL and MEC-1 cells. Finally, using particular inhibitors in in vitro experiments, we confirmed the importance of discovered pathway activations. Results: To evaluate a potential role of negative feedback regulation of MAPK signaling in CLL, we first analyzed mRNA expression levels of DUSP1 and DUSP6 in CLL samples (n=210). Both negative regulators were expressed in all of the CLL samples studied, with DUSP6 levels being upregulated in cases with activating mutations in the KRAS or BRAF genes (12.1±17 RNAseq counts for BRAF/KRASwt cases vs. 46.7±19 RNAseq counts for BRAF/KRAS mutated cases). Furthermore, after microenvironmental stimulation, DUSP levels increased in CLL lymph nodes compared to peripheral blood (DUSP1: log2-fold change of 2.6, p=0.0094; DUSP6: log2-fold change of 1.8, p=2.2*10e-7; Herishanu et al, 2011) and stromal coculture. Notably, increased DUSP6 mRNA expression was linked to a poor clinical outcome, as evidenced by a shorter time to treatment (p=0.0109; Log-rank Test) and overall survival (p=0.0075; Log-rank Test). We next used the dual-specific DUSP1/6 inhibitor BCI to block the phosphatase activity and found that this causes cell death in CLL cells, but not or only mildly affected the survival of healthy donor B cells or other T- and B-cell lines. In the TCL1-driven CLL mouse model, administration of the DUSP1/6 inhibitor is effective in lowering CLL cell progression (>50% reduction of total CLL cell count in the spleen; n=5; p=0.0029). We generated genetic knockout lines of DUSP1 or DUSP6, which inhibited CLL cell expansion, to confirm the inhibitor’s specificity. We next used phosphoproteomics to explore the signaling consequences of DUSP1/6 inhibition in CLL, and discovered that DUSP1/6 inhibition caused acute stimulation of BCR/MAPK signaling. Subsequently, the DNA damage response (DDR) pathway is activated, resulting in the induction of apoptotic cell death. Finally, we tested whether DUSP1/6 inhibition is efficacious in drug-resistant CLL by using BCI to treat therapy-naive and ibrutinib refractory CLL samples, and we discovered that DUSP1/6 inhibition is extremely successful in targeting treatment-resistant CLL. Summary/Conclusion: Conclusion: Based on these results, we propose transient DUSP1/6 inhibition as a novel and unexpected concept for eliminating drug-resistant CLL cells. P631: A PCR-BASED ASSAY FOR IGLV3-21R110 SCREENING CONFIRMS ITS PROGNOSTIC VALUE IN AN INDEPENDENT COHORT OF 613 PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA. C. Syrykh1,2,*, N. Russiñol1, T. Baumann3,4, M. Kulis1,5, M. Alcoceba5,6, M. González5,6, E. Colado7, Á. R. Payer7, M. Aymerich1,3,5, M. J. Terol8, S. Ruiz-Gaspà1, A. López-Guillermo1,3,5,9, J. I. Martín-Subero1,5,9,10, D. Colomer1,3,5,9, J. Delgado1,3,5,9, E. Campo1,3,5,9, F. Nadeu1,5 1Institute d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 2Department of Pathology, Institut Universitaire du Cancer, CHU de Toulouse, Toulouse, France; 3Hospital Clinic of Barcelona, Barcelona; 4Hospital Universitario 12 de Octubre; 5Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid; 6Biología Molecular e Histocompatibilidad, IBSAL-Hospital Universitario, Centro de Investigación del Cáncer-IBMCC (USAL-CSIC), Salamanca; 7Servicio de Hematología y Hemoterapia, Hospital Universitario Central de Asturias, Oviedo; 8Servicio de Hematología, Hospital Clínico Universitario, INCLIVA, Universidad de Valencia, Valencia; 9Universitat de Barcelona; 10Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Background: Recent studies have shown that expression of the immunoglobulin lambda light chain IGLV3-21 gene carrying a point mutation in the amino acid 110 (named IGLV3-21R110) defines a subset of chronic lymphocytic leukemia (CLL) with an intermediate epigenetic subtype and poor prognosis (Maity et al. PNAS, 2020; Nadeu et al. Blood, 2021). This mutation is found in up to 6.5% of patients with CLL at diagnosis and up to 18% of cases enrolled in clinical trials. In addition, IGLV3-21R110 seems to have prognostic value independently of the IGHV gene somatic hypermutation (SHM) status, stereotype subset #2, and disease stage, which makes it a promising prognostic biomarker. However, further studies on independent cohorts are needed to support its application in clinical practice. Aims: To develop a rapid detection method of the IGLV3-21R110 mutation and to assess its prognostic significance in a cohort of 613 previously unpublished CLL patients. Methods: A multiplex IGLV3-21R110 mutation-specific polymerase chain reaction (msPCR) assay was established in a cohort of 12 patients (including 6 IGLV3-21R110 mutated) and validated in 159 cases (including 7 mutated; Nadeu et al. Blood, 2021). This msPCR assay was applied on an independent cohort of 613 CLL patients (575 CLL, 38 monoclonal B-cell lymphocytosis). Clinical analyses were performed for time to first treatment (TTFT) and overall survival (OS) from time of diagnosis considering only cases diagnosed as CLL. Results: A msPCR approach was designed integrating two forward primers aligning to distinct regions of the IGLV3-21 gene and two R110-specific reverse primers matching the IGLJ1 and IGLJ2/3 genes, respectively. A third pair of primers targeting exon 9 of FBXW7 was used as an internal control. PCR conditions were set up on a cohort of 12 cases and subsequently validated on 159 previously published cases with 100% concordance. We then applied this msPCR assay to a cohort of 613 previously unpublished CLL patients and identified the R110 mutation in 22 (3.6%) cases, including 75% mutated IGHV (M-CLL) and 25% unmutated IGHV (U-CLL). Moreover, 84.6% of IGLV3-21R110 cases carried non-stereotyped immunoglobulin genes while the remaining stereotyped IGLV3-21R110 cases were subset #2. Ten of the IGLV3-21R110 cases were classified according to the epigenetic subtypes and all belonged to the intermediate CLL (i-CLL) subgroup. Clinically, M-CLL patients carrying the IGLV3-21R110 as well as i-CLL with IGLV3-21R110 had a shorter TTFT compared to M-CLL and i-CLL lacking IGLV3-21R110, respectively, and similar to U-CLL/naïve-like CLL (p<0.005). Multivariable analyses including IGLV3-21R110, disease stage, and IGHV gene SHM status or epigenetic subtypes confirmed the independent prognostic value of IGLV3-21R110 on TTFT (p<0.005). IGLV3-21R110 had also a prognostic impact on OS since M-CLL carrying the IGLV3-21R110 had shorter OS similar to that of U-CLL (Figure). Finally, we combined these novel data with those of our previously published 489 CLL cohort (Nadeu et al. Blood, 2021) (N total=1102). Clinical analyses of the whole cohort confirmed the independent prognostic value of IGLV3-21R110. Image: Summary/Conclusion: We have developed a reliable, easy-to-use msPCR assay suitable for IGLV3-21R110 screening in large cohorts. By applying this msPCR on a cohort of 613 CLL patients, our results corroborate the relevance of IGLV3-21R110 testing to improve the risk stratification of CLL patients in the clinical practice, especially within M-CLL and i-CLL subtypes. P632: ORAL AND GUT MICROBIAL DIVERSITY CORRELATES WITH PROGNOSTIC FEATURES IN CHRONIC LYMPHOCYTIC LEUKEMIA M. Szelest1,*, M. Paziewska1, M. Kiełbus1, M. Masternak1,2, J. Zaleska1, E. Wawrzyniak3, E. Kalicińska4, P. Jabłonowska4, T. Wróbel4, A. Wolska-Washer5,6, J. Z. Błoński3,6, K. Giannopoulos1,2 1Department of Experimental Hematooncology, Medical University of Lublin; 2Department of Hematology, St John’s Cancer Centre, Lublin; 3Department of Hematology, Medical University of Lodz, Lodz; 4Department of Haematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University, Wroclaw; 5Department of Experimental Hematology, Medical University of Lodz; 6Copernicus Memorial Hospital, Lodz, Poland Background: For the last years it has been speculated that immunologic and inflammatory factors, including antigen stimulation, could be involved in the pathogenesis of chronic lymphocytic leukemia (CLL). With increasing evidence that the activation of cellular proinflammatory signaling pathways driven by somatic mutations and an increased release of proinflammatory cytokines is associated with CLL development, it seems relevant to investigate the role of chronic inflammation in the pathogenesis of this disease. Recent studies have revealed that the intestinal microbiota could modulate the activity of immune system through creating an imbalance between cell proliferation and apoptosis. Thus, the relationship of microbial dysbiosis and specific taxon abundances with CLL biology and clinical outcome should be examined. Aims: As it was reported that the activity of human microbiome is involved in cancer development and host response against anticancer therapy, the aim of this study was to investigate the stool and oral microbiome alterations in CLL patients with respect to selected prognostic and predictive markers. Methods: The study included 92 patients with untreated CLL. Microbiota composition of 85 stool samples and 76 oral samples from these cases were determined by 16S rRNA next-generation sequencing and analyzed with respect to the Binet stage, IGHV and TP53 mutation status, CD38 and ZAP-70 expression, and cytogenetic aberrations. The species richness and evenness were analyzed using Shannon and Chao1 indexes, respectively. The DADA2 tool was used to classify sequences into appropriate taxonomic groups. The differences in taxonomic profiles and species diversity in oral and stool samples with distinct prognostic features were investigated using the Wilcoxon or Kruskall-Wallis tests. Results: Alpha-diversity analysis indicates that there were no significant differences in richness and evenness between probes with distinct CLL features in both fecal and oral samples. However, at phylum level, an increase in Proteobacteria, a common feature of gut dysbiosis, was observed in stool samples from patients with stage Binet C compared to stage Binet A (p=0.042), and in oral samples with del11q compared to samples with no del11q (p=0.021). The abundance of Bacteroidota was higher in fecal samples collected from patients with stage Binet A in comparison to stage Binet B (p=0.051) and C (p=0.009). Moreover, Bacteroidota was relatively more abundant in oral samples from patients with stage Binet A in comparison to stage Binet C (p=0.008). Bacteroidota was significantly less abundant in stool samples from patients with unmutated IGHV compared to samples with mutated IGHV (p=0.029). Stool samples from patients with CD38+ exhibited decreased abundance of Bacteroidota and increased abundance of Firmicutes in comparison to CD38- cases (p=0.031 and p=0.022, respectively). Furthermore, fecal samples from patients with del6q showed a higher abundance of Firmicutes and lower abundance of Bacteroidota compared to samples with no del6q (p=0.006 and p=0.018, respectively). Additionally, a relative increase in Actinobacteriota and Firmicutes was found in oral samples with tri12 in comparison to samples with no tri12 (p=0.039 and p=0.016, respectively). Image: Summary/Conclusion: Our findings provide new insights into oral and gut microbial diversity which is related to distinct prognostic features in CLL and might point to the inflammatory-related modulation of the clinical course of disease. P633: ACTIVE CHROMATIN REGULATORY LANDSCAPE OF STEREOTYPED SUBSETS IN CHRONIC LYMPHOCYTIC LEUKEMIA REVEALS A DISTINCTIVE SIGNATURE IN SUBSET #8 M. Tsagiopoulou1,2,*, V. Chapaprieta3, N. Russiñol3, N. Pechlivanis1, N. Papakonstantinou1, N. Stavroyianni4, F. Psomopoulos1, E. Campo3, K. Stamatopoulos1, J. I. Martin-Subero3 1Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece; 2CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain; 3Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain; 4Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece Background: Chronic lymphocytic leukemia (CLL) patients with stereotyped B cell receptors have been characterized from the perspective of genetic and DNA methylation alterations. Recent studies have characterized the chromatin landscape of non-stereotyped CLLs and normal B cells (Beekman, et al. Nat Med. 2018). Aims: To characterize the active chromatin regulatory landscape of 4 major stereotyped subsets as compared to IGHV status-matched non-stereotyped CLLs. Methods: We used chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) with an antibody for the H3K27ac (a bona fide histone mark for regulatory element activation) in sorted CLL cells from 15 normal B cell subpopulations and 44 CLL, including 18 cases from stereotyped subsets #1, #2, #4, and #8. To understand their functional impact, we integrated our data with 16 RNA-seq data considering the 3D chromatin configuration (Hi-C data: GM12878 and a representative U-CLL case) (Figure 1) Results: Unsupervised principal component analysis (PCA) of H3K27ac data revealed different profiles in CLL as a whole, regardless of stereotypy status, vs normal B cells (PC1, 25%). Of note, PC2 (14%) revealed a clear separation between subset #8, a highly aggressive CLL subtype with the highest risk of Richter’s transformation amongst all CLL, vs other CLLs or normal B cells, suggesting that subset #8 may have a specific acetylation pattern. Indeed, supervised differential acetylation analysis (FDR<0.01) uncovered a signature of 903 Differentially Acetylated Regions (DAR) in subset #8 vs U-CLL (Figure 1) and no differences for the other subsets. This signature was unrelated to +12, highly prevalent (~60%) in subset #8, or IgG expression, a hallmark of subset #8. Hierarchical clustering analysis of this signature revealed 6 clusters of regions with different chromatin dynamics highlighting the de novo active cluster1 (n=197 regions) in subset #8 vs other CLLs or normal B cells. These differentially acetylated regions (DARs) were enriched (FDR<0.05) in binding sites of several differentially expressed TFs in #8 vs U-CLL, e.g. members of SOX, GATA, and CEBP families as well as MEF2A and HNF1A, which showed the highest enrichment. Next, we used the topologically associating domains (TAD) resulting from Hi-C data and RNAseq data to identify the potential target genes of the 197 de novo DARs in subset #8. We identified the target genes in 78/197 (39.6%) DARs that were linked to 112 genes, as some active regulatory regions were associated with more than one gene. Among them, the DAR located in chr9:137085994-137086793 bp was related to the expression levels of 8 genes (LCNL1, CLIC3, FBXW5, AJM1, MAMDC4, CCDC183, TRAF2 and an un-annotated transcript) within the same TAD. This region also contained 6 additional H3K27ac peaks with higher signal in subset #8, though not reaching significance. This finding suggests deregulation of a broad region in chromosome 9 leading to the coordinated upregulation of 8 transcripts. Concerning the activated genes, TRAF2 is a remarkable example since TRAF family members (TRAF6, -4, -1) are found overexpressed in U-CLL and here we report an active region in subset #8 leading to TRAF2 overexpression. Image: Summary/Conclusion: We report a subset #8-specific chromatin acetylation signature containing de novo activated regions, linked to the upregulation of target genes, in some cases including blocks of adjacent genes. This study further supports the distinct biological nature of subset #8 and may help to understand the aggressive clinical behavior of this distrinctive CLL variant. P634: MHC-BASED LARGE-SCALE SCREENING FOR ANTI-TUMOR T CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA REVEALS CD8+ T CELLS WITH SPECIFICITY AGAINST THE CLONOTYPIC B-CELL RECEPTOR IMMUNOGLOBULIN A. Vardi1,2,*, Y. Basavaraju3, A. Agathangelidis2,4, N. Wulff Pedersen3, M. Karipidoy2, A.-L. Schaap-Johansen3, A. Fylaktou5, N. Stavroyianni1, M. Iskas1, A. Anagnostopoulos1, A. Chatzidimitriou2, P. Marcatili3, S. R. Hadrup3, K. Stamatopoulos2 1Hematology Department & HCT Unit, G.Papanikolaou Hospital; 2Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece; 3Department of Health Technology, Technical University of Denmark, Lyngby, Denmark; 4Department of Biology, School of Science, National and Kapodistrian University of Athens, Athens; 5Department of Immunology, National Peripheral Histocompatibility Center, Thessaloniki, Greece Background: Chronic lymphocytic leukemia (CLL) is currently incurable, indicating a need for novel strategies towards disease eradication, including reinvigoration of anti-tumor immune responses. T cells in CLL appear selected by restricted antigens, with evidence suggesting that the selecting epitopes may lie within the clonotypic B-cell receptor immunoglobulins (BcR IG). Aims: We previously performed ad hoc prediction of putative T-cell class I neoepitopes contained within the clonotypic BcR IG of CLL patients. Here, we performed major histocompatibility complex (MHC)-based large-scale screening to identify autologous CD8+ T cells recognizing the predicted neoepitopes. Methods: We evaluated 653 peptides derived from the clonotypic BcR IG of 25 CLL patients (median 21 predicted neoepitopes/patient, across 13 MHC-I alleles). Considering the MHC-I typing of each patient, we constructed patient-specific peptide-MHC multimers labeled with a unique DNA barcode plus a fluorochrome (PE). We generated MHC-specific multimer libraries which we then mixed with respective autologous T-cell enriched peripheral blood mononuclear cells (PBMCs). Duplicate samples/patient were analyzed. In addition, known viral peptide-MHC multimers labeled with a different fluorochrome (APC) as well as three MHC-matched healthy donor PBMCs were used as controls. PE- and APC-positive multimer-binding CD8+ T cells were sorted for each cell sample; subsequently, DNA barcode amplification and sequencing was performed. Sequencing data were processed (Barracoda software) to obtain the number of clonally reduced barcode reads assigned to a given sample and peptide-MHC specificity. The number of clonally reduced reads for a given pMHC specificity was used to estimate the frequency of T cells specific for a given epitope, based on the average number of T cell receptor–MHC multimer interactions detected in the total MHC multimer-binding T cell pool in a given cell sample. Peptides with a log fold change >2 were considered as significant [log fold change: (number of DNA barcode reads associated with a specific pMHC)/3x (number of total barcode reads derived from the same sample)]. Results: Overall, 3 peptide-MHC multimers were recognized by CD8+ T cells: (i) VTVADTAVYY (peptide A) and (ii) INLNPSLKRR (peptide B), both within the context of the A03*01 allele, and (iii) YSFTSYWINW (peptide C) within the context of the A24*02 allele. Peptide A derived from the IGHV4-34 FR3 region of a somatically hypermutated clonotypic BcR IG, containing a single A to V somatic hypermutation (SHM) at position 96. Peptide B derived from the IGHV4-39 CDR2-FR3 junction of a somatically hypermutated clonotypic BcR IG, containing 3 SHMs (T to I at position 65, Y to L at position 67 and S to R at position 74). Peptide C derived from the IGHV5-10-1 CDR1-FR2 junction of a clonotypic BcR IG assigned to stereotyped subset #1, containing the sole SHM of the IG (S to N at position 40). The immunogenicity of these peptides was further corroborated by the fact that they were recognized not only by the autologous T cells of the patient from whom the peptide derived, but also by T cells of other patients as well as a healthy donor sharing the respective MHC allele. Summary/Conclusion: In conclusion, we offer proof of concept that the targeted SHM which shapes the CLL BcR IG repertoire may produce idiotypic targets for T cell-based therapy or peptide vaccine design. Characterization of the epitope-binding T cells is currently underway by our group, aiming to provide further insight on how these cells could be recruited into effective anti-tumor responses. P635: TARGETING THE ONE-CARBON METABOLISM AS NOVEL THERAPEUTIC STRATEGY IN CHRONIC LYMPHOCYTIC LEUKEMIA E. Viry1,*, A. Largeot1, S. Gonder1, S. Chemlal1, N. Kiweler2, J. Meiser2, T. Helleday3, E. Moussay1, J. Paggetti1 1Tumor Stroma Interactions, Department of Cancer Research; 2Cancer Metabolism Group, Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg; 3Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institute, Solna, Sweden Background: Chronic Lymphocytic Leukemia (CLL), the most common type of leukemia in adults, is characterized by the clonal expansion of CD5+ CD19+ B cells. CLL progression is highly dependent on complex interactions with non-malignant cells of the tumor microenvironment. Despite great advance in the standard of care in the last decades, there are still unmet medical needs (long-life treatment, resistance…). Altered cellular metabolism has emerged as a hallmark of cancer by sustaining the uncontrolled growth of cancer cells. The one-carbon pathway is a major driver for tumor proliferation, providing building blocks for biosynthesis of nucleotides through pyrimidines (dTTP) and purines synthesis, redox metabolism (GSH), and energy balance by suppling ATP and NADPH to cells (A). Understanding the metabolic reprogramming occurring in cancer, notably in CLL, may provide insights to support the development of novel therapies. Aims: We aim to get insights into one-carbon metabolism regulation in CLL, identify new therapeutic targets and find new treatment options for CLL patients. Methods: Through a collaboration with a biotech company that develops inhibitors of the one-carbon metabolism, we tested new MTHFD1/2 inhibitors, which we believe, are promising in the context of CLL. The cytotoxic activity of the inhibitors was evaluated on a panel of murine and human CLL cell lines and primary cells, and also on others B cell malignancies such as Mantle Cell Lymphoma (MCL), Diffuse Large B-Cell Lymphoma (DLBCL), and Multiple Myeloma (MM). The molecular mechanisms sustaining the cytotoxic activity were evaluated by performing metabolic tracing, rescue experiments and CRISPR/Cas9 KO. Results: We showed a strong expression of both MTHFD1 and 2 in all the cancer cell lines tested and in stimulated primary human CLL cells. A higher expression of both enzymes was also observed in primary CLL cells, isolated from the Eµ-TCL1 murine model of CLL compared to normal B cells. In vitro treatment with the inhibitors efficiently reduced cell viability of CLL cell lines and primary cells, as for MCL and DLBCL cell lines, at low nanomolar dose while no effect was observed on MM cell lines (B). This effect is associated with a blockade of cell proliferation. Using 13C-serine isotope tracing, we could showed that the inhibitors significantly reduced ATP production from serine in CLL cells, probably resulting in altered nucleotides biosynthesis. By performing rescue experiments with thymidine or hypoxanthine, we confirmed that the cytotoxic effect of the inhibitors on CLL cells is mediated through a defect in pyrimidine synthesis (C). Interestingly, we demonstrated that the resistant MM cell lines overexpressed the SHMT1 enzyme, and could be sensitized to MTHFD1/2 inhibition by combination therapy with SHMT1/2 inhibitor (D). Taken together these results suggest that SHMT1 may be a key player in the response to MTHFD1/2 inhibition. Furthermore, we have previously reported that CLL induces a dramatic remodeling of tumor microenvironment, notably leading to the accumulation of highly immunosuppressive Tregs (Wierz et al., Blood, 2018). We showed that both enzymes are overexpressed in those highly immunosuppressive Tregs, and that MTHD1/2 inhibitors are able to reduce the viability of ex vivo polarized Tregs. Image: Summary/Conclusion: MTHFD1/2 inhibitors display a high cytotoxic activity in CLL and other B-cell malignancies by impairing cell proliferation through a defect in pyrimidine synthesis. These inhibitors also exhibit cytotoxic effect on activated Treg cells ex vivo, reinforcing its therapeutic potential for CLL treatment. P636: REAL WORLD OUTCOME WITH IBRUTINIB IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA FROM THE GERMAN REALITY STUDY M. Welslau1,*, R. Schlag2, B. Heinrich3, L. Vornholz4, S. Buchholz4, B. Marquard4, S. Dörfel5, G. Anke6 1Oncology and Hematology, MVZ at Medical Clinic Aschaffenburg-Alzenau, Aschaffenburg; 2Oncology and Hematology, Oncological Practice, Würzburg; 3Oncology and Hematology, Oncological Practice, Augsburg; 4Medical & Scientific Affairs, Janssen-Cilag GmbH, Neuss; 5Oncology and Hematology, Onco Center Dresden, Dresden; 6Hematological and Oncological Diseases, MVZ Potsdam, Potsdam, Germany Background: Chronic lymphocytic leukemia (CLL) is one of the most common forms of leukemia, characterized by abnormal survival and proliferation of mature B cells in peripheral blood, bone marrow, and lymph nodes. Ibrutinib is a once-daily Bruton’s tyrosine kinase inhibitor (BTKi) with a significant benefit in both progression free survival (PFS) and overall survival (OS) demonstrated in multiple phase 3 trials for patients (pts) with previously untreated (1L) or relapsed/refractory (R/R) CLL. In 2014, ibrutinib was approved by the European Medicines Agency and made available to CLL pts in Germany. Prior to the REALITY study, limited data were available on the effectiveness of ibrutinib-treated pts with CLL in routine clinical practice in Germany. Aims: REALITY was a prospective, multicenter, non-interventional study to evaluate the effectiveness of ibrutinib as the 1st, 2nd, or ≥3rd line of treatment (cohorts C1, C2, and C3 respectively) in pts with CLL during the first 2 years of observation period in a real-world setting. Methods: 302 pts were enrolled in the three cohorts (C1: 104 pts; C2: 90 pts; C3: 108 pts) and treated in 57 study sites in Germany from January 2017 to July 2021. Written informed consent was obtained from all pts. Key study endpoints include retention rate (defined as ratio of pts on ibrutinib treatment to the number of pts at risk), start of next therapy, PFS, OS, and safety. The results of this study are descriptive, and no formal hypotheses was pre-specified. Results: Median age at time of ibrutinib treatment initiation was 74.0 years for the 1L pts in C1, and 72.5 and 73.0 years in the R/R cohorts of C2 and C3, respectively. In cohort C1 (1L), pts with genetic high risk features del(17p) and TP53 mutation were more frequent compared to C2 and C3. In C1 del(17p) was reported in 34/51 (66.7%) pts and TP53 mutations in 31/49 (63.3%) pts. In the R/R cohorts C2 and C3 del(17p) was reported in 11/57 (19.3%) pts and 13/65 (20.0%) pts, respectively. TP53 mutations were reported in 14/48 (29.2%) pts in C2 and 21/40 (52.5%) pts in C3. The primary study endpoint was retention rate, 77.9% pts continued to be on treatment after 1 year in C1 (1L). In C2 and C3 (R/R), the 1-year retention rates were 74.4% and 58.3% respectively. In C1, with median follow-up of 30.6 months, median PFS was not reached (NR). The 1-year PFS and OS in the C1 cohort (1L) were 91.3% and 96.2%, respectively. Only 8 pts initiated next line treatment in C1 after 1 year. In C2, with a median follow-up of 31.5 months, median PFS was NR. The 1-year PFS and OS were 85.6% and 93.3%, respectively. 11 pts initiated next line treatment in C2 after 1 year. In C3 median follow-up was 30.9 months and median PFS was NR. The 1-year PFS and OS were 83.3% and 93.5%, respectively. 26 pts initiated next line treatment in C3 after 1 year. Detailed results after 1 and 2 years of observation are summarized in Table 1. No new safety signals for ibrutinib were observed. Image: Summary/Conclusion: This first, comprehensive data from clinical practice of ibrutinib-treated pts with CLL in Germany showed that ibrutinib is a highly effective treatment option, especially as a 1st line treatment. The effectiveness and safety profile were consistent with clinical trials. P637: FRONTLINE THERAPY CLL WITHOUT 17P DEL/P53 ABERRATIONS: SYSTEMATIC REVIEW AND BAYESIAN NETWORK META-ANALYSIS G. Caridà1, D. Ciliberto1, G. Pelaia2, N. Staropoli1, M. A. Siciliano3, N. D. Calandruccio1, R. Toscano4, M. Rossi5,6,* 1Oncology Unit; 2Pediatric Unit, “Magna Graecia” University, Catanzaro; 3Oncology Unit, “S. Giovanni di Dio” Hospital, Crotone; 4Oncology Unit, AO “S. Francesco di Paola”, Paola (CS); 5Department of Experimental and Clinical Medicine, Magna Græcia University, Catanzaro, Italy, “Magna Graecia” University; 6Hematology Unit, AO “Pugliese Ciaccio”, Catanzaro, Italy Background: Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia, with a median age at diagnosis of 72 years. IgVh unmutated CLL without 17p del/p53 aberrations (UN-only CLL) still carry a poorer prognosis as compared to IgVh mutated (MU-only) cases. Although many treatment options are currently available for the front-line therapy, the best choice and treatment sequencing stratified on efficacy and tolerability for UN/MU-only CLL remains to be defined. Aims: We performed a systematic review and Bayesian network meta-analysis to define best frontline therapy and treatment sequence for UN/MU-only CLL. Methods: Analysis of the literature was performed through the main databases. Due to different therapeutic approaches to front-line therapy of CLL, we chose to conduct a Bayesian Network Meta-Analysis (NMA), allowing to rank (from best to worst) multiple treatments in a single analysis. We generated a rank for Progression Free Survival (PFS), Overall Response Rate (ORR), Minimal Residual Disease (MRD), assessed by flow cytometry on peripheral blood, and tolerability. PFS was evaluated in unselected population for IgVh, and in UN/MU-only CLL patients. Results: Fifteen clinical trials, with a total of 7958 patients, have been included, while 16 different treatments were considered to build four different network meta-analyses to identify the best treatment in PFS, ORR, MRD, and tolerability: Acalabrutinib (ACA); ACA + Obinutuzumab (ACA-O); Alemtuzumab; Bendamustine + Rituximab; Chlorambucil; Chlorambucil + O; Chlorambucil + Ofatumumab; Chlorambucil + Rituximab; Fludarabine + Cyclophosphamide + Rituximab; Fludarabine + Cyclophosphamide; Ibrutinib (IBR); IBR + O; IBR + Rituximab; Lenalidomide, Venetoclax + IBR (VEN-IBR), Venetoclax + Obinutuzumab + Ibrutinib (VEN-O-IBR), Venetoclax + O (VEN-O). ACA-O scored the best in PFS in an unselected population (SUCRA 100%, probable the best 99.9%), UN- and MU- only CLL patients. Our analysis showed that VEN-O ranked the first for ORR (SUCRA value 100%, probably the best 100%). VEN-O ranked the first also for MRD (SUCRA 90.4%, probably best 36.2%) versus the second VEN-O-IBR (SUCRA 84.0%, probably best 35.9); in the direct comparison, the difference between VEN-O and VEN-O-IBR is not statistically significant (Odds Ratio 1.44, CrI 0.00,563.57). Finally, ACA monotherapy is the most tolerable therapy (SUCRA value 100%, probably the best 100%), whereas IBR-O turned to be the most toxic treatment (SUCRA value 0%, probably the best 1%). Interestingly, IBR monotherapy ranked the 9th in the network, whereas all combination regimens including Obinutuzumab carried a high risk of toxicity. Image: Summary/Conclusion: To our knowledge, this is the first network meta-analysis that compares at the same time PFS, tolerability, ORR, and MRD. Our PFS analysis showed that ACA-O was the best treatment in both MU/UN-only patients. Furthermore, ACA turned out to be the most tolerated regimen, whereas the BTKis-O based regimens significantly carried a high toxicity profile as compared to BTKis alone, counterbalancing the advantage of O-addition in terms of ORRs, where O-based regimens achieved the higher rank, when combined either with VEN or BTKis. ACA-O was the best treatment for PFS, although with a worse toxicity profile as compared to ACA monotherapy. VEN-O scores as the best in ORR and MRD. The combination ACA-O has the highest probability to be the best treatment for prolonging the PFS in untreated, UN/MU-only CLL patients. ACA monotherapy is a valuable alternative considering both the safety and efficacy profile. P638: THE RISK OF SECOND PRIMARY MALIGNANCIES AND CAUSE-SPECIFIC MORTALITY AMONG PATIENTS WITH T-CELL PROLYMPHOCYTIC LEUKEMIA IN THE UNITED STATES: A POPULATION-BASED STUDY. K. Chamarti1,*, M. Mannem1, B. Bakhati1 1Department of Internal Medicine, Texas Tech University Health Sciences Center at Permian Basin, Odessa, United States of America Background: T cell prolymphocytic leukemia (T-PLL) is a rare lymphoid malignancy, compromising 2% of mature lymphocytic leukemias. The T-PLL follows an aggressive clinical course with median survival of 7.5 months with conventional chemotherpay. In the era of targeted therapy with anti-CD52 antibody for patients with T-PLL, there is data lacking on the spectrum of second primary malignancies (SPMs) and cause-specific mortality. Aims: In this current study we aim to identify the risk of SPM and cause specific mortality in patients with T-PLL in the era of targeted therapies with anti-CD52 antibodies using a national registry from the United States. Methods: The incidence of SPMs and cause-specific mortality among two-month survivors of T-PLL, was estimated using the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER)-13 registries. We included patients from 1992-2018 with ICD-O-3 code 9834/3 from the registry. We estimated the risk of SPMs using standardized incidence ratios (SIRs) and cause-specific mortality by standardized mortality ratios (SMRs) and absolute excess risk (AER)/10,000 population. Results: We included a total of 314 patients with T-PLL in this study. The median age at diagnosis was 68.5 years (range 1-85 years). The median follow-up duration was 12 months (range 0-196 months). A total of 13 (4%) patients developed 14 SPMs at the last follow-up. The SPM risk for the cohort was significantly elevated (SIR 2.54, 95% confidence interval [CI] 1.39-4.27) compared with general population. The median latency period for SPM development was 28 months (range 8-108 months). The SIR was significant among males (SIR 2.96, 95% CI 1.42-5.44), but not in females (SIR 1.88, 95% CI 0.51-4.82). The SIR according to age groups was significant in patients with age < 60 years (SIR 3.84, 95% CI 1.25-8.97), but not in age > 60 years (SIR 2.14, 95% CI 0.98-4.06). Specific SPMs with the highest risk included nasopharyngeal cancers (SIR 200.87, 95% CI 5.09-1,119.20) and multiple myeloma (SIR 33.39, 95% CI 6.89-97.58). At last follow-up, 155 (49%) patients had died, of which 89% were due to malignant neoplasms. There was statistically significant risk of mortality due to all malignant cancers combined (SMR 58.35, 95% CI 49.03-68.94), largely due to mortality from lymphocytic leukemia (SMR 2587, 95% CI 2021-3264, AER 1757), non-Hodgkin lymphoma (SMR 100, 95% CI 45.88-190.45, AER 220), stomach and duodenal ulcers (SMR 73.74, 95% CI 1.87-410.84, AER 24.43), uterine cancer (SMR 66.55, 95% CI 1.68-370.78, AER 24.39), and pneumonia and influenza (SMR 11.88, 95% CI 2.45-34.71, AER 68.04). The risk of death from lymphocytic leukemia was highest within the first year of diagnosis of T-PLL and continued to decline slowly thereafter, although it remained statistically significantly elevated more than other causes of death (SMR 1yr=3405, AER 2666; SMR 1-5 yrs=2623, AER 1715; SMR 5-10 yrs=1015, AER 516). Summary/Conclusion: Our study reveals that the risk of developing SPMs remains elevated in patients with T-PLL compared to the general population. Additionally, men are at an increased risk for SPMs compared to women. Despite therapeutic advances in T-PLL, hematologic neoplasms (including lymphocytic leukemia) have remained the leading cause of death in the last three decades. Further studies are needed to better define the healthcare needs of T-PLL patients beyond initial treatment to reduce the burden of mortality from T-PLL and other SPMs. P639: OUTCOMES FOR PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA PREVIOUSLY TREATED WITH A COVALENT BTK AND BCL2 INHIBITOR IN THE UNITED STATES: A REAL-WORLD DATABASE STUDY A. R. Mato1,*, Y. Chen2, A. C. Girvan2, L. M. Hess2, L. Bowman2, P. Abada2, H. Konig2, R. A. Walgren2 1Memorial Sloan Kettering Cancer Center, New York; 2Eli Lilly and Company, Indianapolis, United States of America Background: Covalent BTK inhibitors (cBTK) and B-cell lymphoma 2 inhibitors (BCL2i) have transformed outcomes in patients with chronic lymphocytic leukemia (CLL). Although these agents are highly effective, they are not curative, and a proportion of patients will require additional therapy. No prospective randomized clinical trials have evaluated the efficacy of therapies following treatment with these two modern drug classes. Aims: We therefore evaluated treatment outcomes in patients with CLL following treatment with at least these two drug classes using a real-world dataset from the United States (US). Methods: De-identified patient-level electronic medical record data linked to claims data in the US from ConcertAI were utilized for this study. Eligible patients were ≥18 years old, diagnosed with CLL between December 2011 and October 2020. To ensure a minimum of 1 year follow-up for all patients, data were available through July 2021. Patients were required to have completed treatment with at least one cBTKi and a BCL2i during the 1st-3rd lines of therapy. Time-to-event analyses utilized the Kaplan-Meier method. Results: 382 patients (median age 73.4 years, male 64.4%) met eligibility criteria. Of these, 228 (59.7%) received subsequent therapy following BTK inhibitor and BCL2 inhibitor regimens, while the remaining 154 (40.3%) did not receive further treatment. Among those who received further treatment, the most common therapies included retreatment with a venetoclax-containing regimen (n=112, 49.1%). Of note, only 2.6% (n=6) of patients received immediate post-dual failure therapy with a PI3K inhibitor in this setting. Among those who received subsequent therapy (n=228), the median time from the start of the immediate post-cBTKi/BCL2i therapy to discontinuation or death was 5.5 months (95% CI: 3.5-6.9). For the entire cohort of patients with CLL who discontinued BTKi and BCL2i regimens (n=382), the median time from the end of the last of BTKi/BCL2i therapy to discontinuation of subsequent therapy or death was 5.6 months (95% CI: 4.3-6.0). Summary/Conclusion: Patients with CLL who have been previously treated with both a cBTKi and BCL2i experience poor outcomes as observed by duration of treatment and time to treatment discontinuation of subsequent therapy. This population represents a growing area of unmet medical need for patients with CLL. P640: PIRTOBRUTINIB, A HIGHLY SELECTIVE, NON-COVALENT (REVERSIBLE) BTK INHIBITOR IN COMBINATION WITH VENETOCLAX±RITUXIMAB IN RELAPSED/REFRACTORY CLL: RESULTS FROM THE BRUIN PHASE 1B STUDY L. E. Roeker1,*, A. R. Mato1, J. R. Brown2, C. C. Coombs3, N. N. Shah4, W. G. Wierda5, M. R. Patel6, K. L. Lewis7, M. Balbas8, J. Zhao8, N. C. Ku8, J. F. Kherani8, D. E. Tsai8, B. Nair8, C. Y. Cheah7 1Memorial Sloan Kettering Cancer Center, New York; 2Dana-Farber Cancer Institute and Harvard Medical School, Boston; 3University of North Carolina at Chapel Hill, Chapel Hill; 4Medical College of Wisconsin, Milwaukee; 5MD Anderson Cancer Center, Houston; 6Florida Cancer Specialists/Sarah Cannon Research Institute, Sarasota, United States of America; 7Linear Clinical Research and Sir Charles Gairdner Hospital, Perth, Australia; 8Loxo Oncology at Lilly, Stamford, United States of America Background: Covalent Bruton tyrosine kinase inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia (CLL), but patients (pts) discontinue these agents due to resistance or intolerance. Pirtobrutinib is an oral, highly selective, non-covalent (reversible) BTKi with promising efficacy and safety in heavily pretreated relapsed/refractory (R/R) CLL pts, regardless of BTK C481 mutation status. Recent clinical studies reported on the safety and efficacy of time-limited venetoclax and covalent BTKi combination regimens. Aims: We evaluated the safety and efficacy of pirtobrutinib combined with venetoclax ± rituximab in pts with R/R CLL. Methods: BRUIN is a phase 1/2 global, multicenter study (NCT03740529) of pirtobrutinib in pts with advanced B-cell malignancies. The phase 1b portion evaluated the safety of pirtobrutinib at a continuous dose of 200 mg QD from Cycle 1, Day 1 plus venetoclax starting on Cycle 2, Day 1 with a standard 5-week dose ramp to 400 mg QD (PV) and PV plus rituximab at 375 mg/m2 on Cycle 1, Day 1, then 500 mg/m2 on Day 1 of Cycles 2-6 (PVR). Prior BTKi was allowed; prior venetoclax was not permitted. Objectives included safety and overall response rate (ORR) of each combination. Results: As of 27 SEP 2021, 15 pts received PV and 10 pts received PVR. Median age was 66 years (range, 39-78). Median prior lines of therapy was 2 (range, 1-4). The majority of pts in both cohorts had received prior chemotherapy (56%, n=14), CD20 monoclonal antibody (72%, n=18), and/or covalent BTKi (68%, n=17). No dose-limiting toxicities were reported. Safety profiles were generally similar across both cohorts. The most common treatment-emergent adverse events (TEAE) of any grade, regardless of attribution, were neutrophil count decrease (36%), nausea (32%), fatigue (32%), diarrhea (28%), and constipation (24%). The only Grade ≥3 TEAE to occur in more than 2 pts was neutrophil count decrease (36%, n=9). One pt experienced Grade 4 clinical tumor lysis syndrome with resultant acute kidney injury during venetoclax dose escalation, which resolved with supportive measures. No pts discontinued treatment due to AEs. For the 22 pts with efficacy data available as of 03 NOV 2021, median duration of follow-up was 9 months (range, 3.9-15) and the ORR was 95.5% (95% CI, 77-100). All responding pts except 1 remain on therapy (PVR responder discontinued due to death unrelated to study treatment). As all responses were ongoing and early, and MRD analysis was not yet performed. Summary/Conclusion: Pirtobrutinib combined with venetoclax ± rituximab was well tolerated and had a safety profile consistent with known drug class findings and no clear additive toxicities in pts with R/R CLL. Early results demonstrate promising efficacy with combination therapy. P641: RETREATMENT WITH VENETOCLAX AFTER VENETOCLAX, OBINUTUZUMAB +/- IBRUTINIB: POOLED ANALYSIS OF 13 PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) TREATED IN GCLLSG TRIALS P. Cramer1,*, M. Fürstenau1, A. Giza1, S. Robrecht1, E. Tausch2, C. Schneider2, C.-M. Wendtner3, M. Hoechstetter3, J. Schetelig4, S. Böttcher5, P. Dreger6, A.-M. Fink1, P. Langerbeins1, O. Al-Sawaf1, K. Fischer1, S. Stilgenbauer2, B. Eichhorst1, M. Hallek1 1Department I of Internal Medicine and German CLL Study Group; Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), University of Cologne, Cologne; 2Department III of Internal Medicine, University Hospital Ulm, Ulm; 3Department of Hematology, Oncology, Immunology, Palliative Care, Infectious Diseases and Tropical Medicine, Klinikum Schwabing, Munich; 4Department I of Internal Medicine, University Hospital Carl Gustav Carus, Dresden; 5Department III of Internal Medicine, University Hospital Rostock, Rostock; 6Department V of Internal Medicine, University Hospital Heidelberg, Heidelberg, Germany Background: Fixed duration venetoclax (V)-containing therapies are commonly used in CLL, either combined with obinutuzumab (Ven-G) for treatment-naïve or with rituximab for relapsed/refractory disease. Current clinical trials also combine Ven-G with ibrutinib (GIVe) or acalabrutinib (GAVe) to increase efficacy and possibly shorten treatment duration with minimal residual disease (MRD)-guided discontinuation strategies. Aims: There is only little evidence regarding the best sequence of currently available therapies and if time-limited therapies can be repeated at progression. Therefore, we pooled the patients (pts) with two consecutive V-containing treatment lines from GCLLSG trials, to study efficacy and safety of a re-treatment with V-based regimens. Methods: Thirteen pts with two sequential V-containing lines (V1 and V2) were identified within the following 5 multicenter phase-II or -III trials: 3 pts each received V1 in CLL2-BAG and CLL2-GIVe, 2 Ven-G in CLL13 (excluded from time-to event analyses due to unavailable data) and 1 in CLL14; all were subsequently treated in CLL2-BAAG (V2). Four additional pts received V1 and V2 in CLL2-BAG. Results: At the start of V1, the median age was 58 (range 49-65) years and 11 pts (92%) had a high/very high CLL-IPI, all 13 pts had an unmutated IGHV, 8 (62%) a del(17p) and/or TP53mut and 6 (46%) a complex karyotype. Four pts had already received a median of 2.5 (range 1-4) prior treatment lines (chemo(immuno)therapies only). V1 was Ven-G in 10 pts, including 4 pts with a bendamustine debulking, and GIVe in 3 pts. Median duration of V1 was 13 (range 11-29) months for all pts and 14 (range 13-26) months in the 9 pts with a MRD-guided discontinuation strategy who achieved uMRD, one pt failed to achieve uMRD in bone marrow and had to stop after approximately 2.5 years as per protocol. Three pts received V1 with a fixed duration of approximately one year. V2 was GAVe in 9 pts, including 2 with a bendamustine debulking, and Ven-G or Ven in 2 each, all with a MRD-guided discontinuation. Thus far, 5 pts have stopped V2: two pts due to uMRD and one each due to adverse events (allergic skin reactions), planned allogeneic stem cell transplantation and end of study treatment after approximately 2.5 years. Median treatment duration was 16 (range 8-30) months for V2, which is already longer than with V1 (see above) but 8 pts still continue V2. All pts experienced adverse events (AEs) during V1 and V2, AEs CTC °III/IV occurred in 11 and 8 pts during V1 and V2, respectively and serious AEs in 6 and 4, respectively. Hematological AEs °III/IV occurred in 6 and 2 pts during V1 and V2, tumor lysis syndromes °III in 2 and 3 pts and infections °III/IV in 2 each. All pts responded to V1 and V2, uMRD in PB was achieved at the end of V1 in 12/13 pts (92%) and thus far with V2 in 9/13 pts (69%). Median time between end of V1 and start of V2 was 29 (range 15-55) months. Median progression-free survival from start of V1 was 42 months and after V2 no progressions occurred so far at a median follow up time of 19 (range 8-33) months. All pts are alive, see also Fig. 1. Image: Summary/Conclusion: In this pooled analysis of 13 pts with two consecutive time-limited V-containing therapies, which includes mainly pts with adverse risk factors and a short remission duration, V-based re-treatment appeared to be safe and efficacious: no increased rate of AEs was seen so far and all pts responded with at least 2/3 even achieving uMRD again. P642: IMPACT OF TYPE 2 DIABETES ON SURVIVAL AND TREATMENT IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA E. Curovic Rotbain1,2,3,4,*, K. Rostgaard2,4, M. A. Andersen1,4,5, N. Vainer1, C. Da Cunha-Bang1, H. Hjalgrim1,2,4,6, H. Frederiksen3,7,8, S. Slager9, C. Utoft Niemann1,6 1Department of Hematology, Rigshospitalet; 2Hematology Group, Danish Cancer Society Research Center, Copenhagen; 3Department of Hematology, Odense University Hospital, Odense; 4Department of Epidemiology, Statens Seruminstitut; 5Department of Clinical pharmacology, Bispebjerg and Frederiksberg Hospital; 6Department of Clinical Medicine, Copenhagen University, Copenhagen; 7Departemnt of Clinical Research, University of Southern Denmark; 8Academy of Geriatric Cancer Research (AgeCare), Odense University Hospital, Odense, Denmark; 9Department of Health Sciences Research, Mayo Clinic, Rochester, United States of America Background: Because of a generally advanced age at diagnosis of chronic lymphocytic leukemia (CLL), this group of patients is often burdened by comorbid conditions. Comorbid conditions are associated with increased mortality in CLL, due to causes both related and unrelated to CLL. Diabetes is one of the most common comorbid conditions with a prevalence of 8-21% at time of CLL-diagnosis, and the prevalence of diabetes is projected to double in the general population within the next two decades. While many comorbid conditions may have limited potential for improvement of management, treatment for type 2 diabetes (T2D) is rapidly evolving with new treatment combinations displaying survival benefits. Aims: To assess the impact of T2D on time to treatment and mortality for patients with CLL from time of diagnosis and from time of first-line treatment. Methods: In Denmark, all citizens have access to free health care services. Contacts with the health care system are registered by the unique personal identification number, enabling linkage of data on person level across national registers. We followed all patients registered with newly diagnosed CLL in the Danish CLL Register or the Danish Cancer Register in the period 2002-2018 from 1 month after CLL-diagnosis until death or end of follow-up. Data on causes of death were obtained through the Danish National Register of Causes of Death. T2D was defined as having an International Classification of Diseases-10 diagnosis of T2D (E11) in the Danish National Patient Register or ≥1 prescription for non-insulin anti-diabetic drugs (ATC code A10B) in the Danish Prescription Register. Fine-Gray and Cox proportional hazards regressions were fitted, and corresponding Kaplan-Meier curves computed. Results: In total, 7 446 patients with CLL were identified, of whom 802 (11%) had T2D at CLL diagnosis. T2D was associated with longer time to first treatment (hazard ratio (HR) 0.81, 95% confidence interval (CI) [0.67-0.99]) and higher mortality (HR 1.58, 95% CI [1.42-1.76]) from time of CLL diagnosis. Patients with T2D had an increased mortality due to causes both related (HR 1.15, 95% CI [0.99-1.34]) and unrelated (HR 2.07, 95% CI [1.67-2.55]) to CLL. Among 1 487 patients receiving first-line treatment, 164 (11%) had T2D. Upon first-line treatment, patients with T2D had shorter event-free survival (HR 1.40, 95% CI [1.07-1.79]) and higher mortality (HR 1.22, 95% CI [0.95-1.56]) when compared with patients with CLL without T2D. Treatment with fludarabine, cyclophosphamide, and rituximab (FCR) was not associated with a superior survival compared with bendamustine and rituximab (BR) in patients with T2D (HR 2.58, 95% CI [0.61-10.86]). Summary/Conclusion: T2D is associated with an inferior prognosis in CLL. CLL patients with T2D had a lower probability of receiving CLL-treatment yet higher mortality. While the latter may be related to surveillance bias, further investigation in to causes is required. Though not statistically significant, analyses suggest that among patients with CLL and T2D, BCR treatment might be superior to FCR. P643: RISK FACTORS FOR UNFAVORABLE OUTCOME OF HOSPITALIZED PANTIENTS WITH CONCURENT CHRONIC LYMPHOCYTIC LEUKEMIA AND COVID-19 – EXPERIENCE OF THREE SERBIAN UNIVERSITY CENTERS Z. Cvetkovic1,2,*, O. Markovic2,3, K. Markovic4, A. Novkovic1, A. Divac3, N. Vukosavljevic4, M. Tanasijevic1, T. Bibic1 1Department of Hematology, Clinical Hospital Center Zemun; 2University of Belgrade, Medical Faculty; 3Department of Hematology, Clinica Hospital Center Bezanijska kosa; 4Department of Hematology, Clinical Hospital Center Zvezdara, Belgrade, Serbia Background: Vulnerability of patients (pts) with chronic lymphocytic leukemia (CLL) and their susceptibility to Covid-19 infection is documented in several studies with reported case fatality rates (CFRs) up to 40%, but there is still paucity of data on identifying risk factors of their adverse outcome. Aims: To evaluate demographic, patient-related, CLL-related and Covid-19 related risk factors in hospitalized pts with concurrent CLL and Covid-19. Methods: Total of 81 CLL pts were identified in medical records of three University centers in Belgrade: Clinical Hospital Center (CHC) Zemun, CHC Bezanijska kosa and CHC Zvezdara dedicated to treatment of Covid-19 pts during pandemic (from 15 March 2020 to 31 December 2021). Results: For all 81 pts CFR was 32.1%. Age (median age 68 yrs;range 45-90 yrs) and sex (apparent male prevalence: 61 male and 20 female; M:F=3.05) had no influence on outcome. Pts with Charlson comorbidity index >4 (29/81;35.8%) had significantly higher CFR (38% vs 9.5%, p=0,025). Concerning CLL-directed treatment: 26/81(32.1%) pts were on active treatment (5 pts were on Bruton tyrosine kinase inhibitor, 21pts receiving imunochemotherapy), 11/81(13.6%) pts were in remission on previous lines of therapy, while 44/81(54.3%) pts were treatment naive. CLL treatment history had no impact on CFR, as well as anemia (Hb<100g/l) that was present in 29/81(35.8%)pts, hipogammaglobulinemia (21/81;26%pts) and hiperferritinemia>450ng/mL (50/81;61.7%pts). Of evaluated laboratory parameters, high levels of lactate-dehydrogenase (LDH>2xUNL:6/81;7.4%pts), D-dimer (>1000ng/mL:36/81;44.4%pts), and C-reactive protein (CRP>100mg/L: 31/81;38.3%pts) proved to be associated with adverse outcome; p-values 0.002, 0.039 and <0.001, respectively. According to Covid-19 clinical course, the severe Covid-19 score had 35(43,2%)pts, and critical 19(23.5%)pts. Covid-19 infection was treated according to current National guidelines. Corticosteroids were administrated to 81.5% of pts, antiviral agents to 38.3%, IL-6 receptor inhibitor to 11.1%, antiviral monoclonal antibodies to 7.4% and intravenous immunoglobulin to 19.8% of pts. None of listed therapeutic approaches had impact on CFRs. Antibiotics were administrated to 43/81 (53.1%) of pts with documented or highly suspected concomitant bacterial infection (procaltitonin level>0.5ng/mL and/or chest X-Ray image corresponding to bacterial pneumonia), and the bacterial coinfection had adverse impact on CFR (51.2% vs.10.2%; p<0.001). Significantly higher mortality was documented in pts who needed supplemental oxygen (58/81;71%) (CFR 43.1 vs.4.3%; p<0.001), and intensive care unit (ICU) admission (25/81-30.9%; 19/25 needed mechanical ventilation) (CFR 88% vs.7.1%;p<0.001). In multivariate analysis, bacterial coinfection and ICU admission proved to be the most significant adverse parameters influencing outcome (p=0.012). Summary/Conclusion: Our study proved the dismal outcome of CLL pts with concurrent Covid-19. That could be mainly attributed to the high proportion of bacterial coinfections reflecting their frailty and sucessibility to both viral and bacterial infections. P644: THE LIFE EXPECTANCY OF PATIENTS WITH HAIRY CELL LEUKEMIA VERGES UPON THE LIFE EXPECTANCY OF THE GENERAL POPULATION: A POPULATION-BASED STUDY IN THE NETHERLANDS A. Dinmohamed1,2,3,*, C. Maas1,2, O. Visser4, J. Doorduijn5, R. Mous6, R. Raymakers6, A. Kater3, W. Posthuma1,7,8 1Research and Development, Netherlands Comprehensive Cancer Organisation (IKNL), Utrecht; 2Public Health, Erasmus MC, Rotterdam; 3Hematology, Amsterdam UMC, Amsterdam; 4Registration, Netherlands Comprehensive Cancer Organisation (IKNL), Utrecht; 5Hematology, Erasmus MC Cancer Institute, Rotterdam; 6Hematology, UMC Utrecht, Utrecht; 7Internal Medicine, Reinier de Graaf Gasthuis, Delft; 8Hematology, Leiden University Medical Center, Leiden, Netherlands Background: At present, no other therapeutic strategy has substantially outperformed purine nucleoside analogues (PNAs)—introduced around the late 1980s and early 1990s—to manage hairy cell leukemia (HCL). The most recent population-based study in HCL showed that 10-year relative survival of HCL patients diagnosed in the Netherlands during 2001-2015 was 97%, 95%, and 83% in the age groups <60, 60-69, and ≥70 years, respectively (Dinmohamed AG et al. Blood; 2018). Given the improved longevity of HCL patients, relative survival rates fall short to inform on longevity across the entire patients’ life span. Aims: Studies estimating the life expectancy of HCL patients are hitherto lacking. Therefore, we assessed trends in the life expectancy of HCL patients from a historical and contemporary perspective. Methods: We selected all HCL patients diagnosed between 1989 and 2019—with follow-up for survival up to January 1, 2021—from the Netherlands Cancer Registry (N=1,828; median age, 60 years; interquartile range, 50-70 years; 78% males). We estimated the loss in expectation of life (LEL)—i.e., the difference between the life expectancy of patients and an age-, sex-, and period-matched group from the general population—using flexible parametric relative survival models. The LEL is interpreted as the average number of life years lost due to an HCL diagnosis. The LEL can vary markedly across ages because life expectancy is age-dependent. Therefore, the proportional LEL (PLEL) was estimated. These survival measures were presented by year of diagnosis for four ages at diagnosis (i.e., 40, 50, 60, and 70 years), stratified by sex. Results: The life expectancy of HCL patients increased gradually across all ages between 1989-2019, irrespective of sex (Fig A). It is noteworthy that HCL patients diagnosed in 1990 lost comparatively few life-years due to their diagnosis, ranging from a LEL of 1.7 to 3.9 life-years lost depending on age and sex (Fig B). Estimates for patients diagnosed in 2019 ranged from 0.8 to 1.9 life-years lost (Fig B). Over time, the decrease in LEL was most pronounced for younger patients (Fig B). For example, a 50-year-old male diagnosed with HCL in 1990, on average, has a LEL of 3.8 years (95% confidence interval [CI]: 2.1-5.5), whereas a male with a HCL diagnosis in 2019, on average, has a LEL of 1.0 years (95% CI, 0.3-1.7). The corresponding estimates for a 70-year-old male HCL patient were 2.3 (95% CI, 1.4-3.1) and 1.1 (95% CI, 0.6-1.7), respectively. The PLEL estimates also portrays that outcomes in HCL patients improved over time across all ages (Fig C). Nevertheless, there was a persistent age differential in the PLEL over time. More specifically, younger patients consistently have more remaining life-years than older patients, reflected in lower PLEL estimates in younger patients. Of note, the life expectancy estimates of female patients should be interpreted with caution by considering the wideness of the 95% CIs due to the comparative rarity of HCL in females to estimate the life expectancy accurately. Image: Summary/Conclusion: The life expectancy of HCL patients verges upon the life expectancy of the general population. This encouraging finding was already objectified around the early-1990s when PNA therapy was introduced for HCL management. Thereafter, the life expectancy gradually increased over time. Novel therapeutic strategies may reduce the minimal excess mortality encountered in contemporary diagnosed patients, particularly among the elderly. P645: THE INFLUENCE OF ACTIVE CARDIAC MONITORING WITH REMOTE CONTROL ON THE SURVIVAL OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA, RECEIVING IBRUTINIB. E. Emelina1,*, G. Gendlin1, I. Nikitin1 1Hospital Therapy №2, Pirogov Russian National Research Medical University, Moscow, Russia Background: Survival of chronic lymphocytic leukemia (CLL) patients receiving ibrutinib (Ib) is influenced by many factors, among which cardiovascular disease, both in history and during treatment with Ib, is of great importance. Aims: To assess the impact on 5-year overall survival (5-OS) of patients CLL treated with Ib two options for dynamic monitoring by a cardiologist: standard cardiac monitoring (SC) and active cardiac monitoring with remote control (ACM). Methods: We observed in dynamics for 5 years’ period 217 patients with CLL, constantly receiving therapy with Ib 420 mg/day. In the ACM group (n=89), in addition to the standard examination, every week or more often using messengers, we did active medical monitoring of the patient’s symptoms and well-being, assessment of blood pressure and heart rate, control of cardioprotective medicines taken, correction of the therapy, calling patients for examination and additional examination. The remaining 128 patients were examined in dynamics, but did not support remote control, constituting the SC group. The age of patients in the ACM group and in the SC group did not differ and was 66.0 (60.0–70.0) years and 66.0 (59.0–74.0) years, respectively (p = 0,39). There were slightly more men in the SC group (68.8%) than in the active cardiac monitoring group (53.9%), p = 0.026. The proportion of patients with CLL with the number of pretreatment lines from 0 to 2 (median -1) in the ACM group was 52.7%, in the SC group it was 58.1%; with the number of lines from 3 to 12 (median - 4) in the AKM group - 47.3%, in the SC group - 41.9% (p = 0.53). The AKM and SC groups did not differ in the results of the Geriatric 8 scale, Charlson index, and echocardiography parameters at visit 1. In the ACM group, there were more patients with cardiac problems: with arterial hypertension (AH) (p < 0.0001) and atrial fibrillation (p < 0.0001), receiving anticoagulants (p < 0.0001), a comparable number of patients with coronary artery disease. Results: In the ACM group, 70 out of 77 (90.9%) patients with CLL and AH achieved a stable level of target blood pressure values, in contrast to the SC group - 26 out of 66 (39.9%), p < 0.0001. Significantly more events requiring cardiac surgery (stenting of the coronary arteries, installation of a pacemaker) were detected in the ACM group – 12, versus 0 in the SC group (p = 0.0004). In the ACM group, despite a more pronounced cardiac comorbidity, 5-OS was significantly better, than in the SC group in both men (p < 0.0001) and women (p < 0.0001) with CLL and in patients older than 70 years old (p = 0.0004). 5-OS was also better in the ACM group than in the SC group in patients with CLL with a median number of previous lines of therapy equal to 1 (p<0.0001) and in patients with a median number of chemotherapy lines equal to 4 (p<0.0001), in patients with genetic abnormalities (p=0.004) and pretreated with fludarabine and/or anthracyclines (p < 0.0001). Summary/Conclusion: Early detection and correction of cardiovascular complications/events, achievement of stable target blood pressure values, constant monitoring of cardioprotective treatment in the ACM group explain the statistically highly significant differences in 5-OS in patients with CLL who are on constant Ib therapy. Conducting active cardiomonitoring with remote control makes it possible to achieve higher rates of total 5-OS in patients with CLL receiving Ib. P646: EFFECTIVENESS AND SAFETY OF VENETOCLAX IN COMBINATION WITH RITUXIMAB (VENR) IN R/R CLL PATIENTS WITH OR WITHOUT RISK-ASSOCIATED GENETIC MARKERS – DATA FROM THE OBSERVATIONAL VERVE STUDY I. Schwaner1,*, H. Hebart2, C. Losem3, T. Wolff4, K. Famulla5, J. Huelsenbeck5, B. Schmidt6, T. Nösslinger7, D. Rossi8 1Onkologie Kurfürstendamm, Berlin; 2Kliniken Ostalb, Mutlangen; 3MVZ Onkologie und Hämatologie, Neuss; 4Onkologie Lerchenfeld, Hamburg; 5AbbVie Deutschland GmbH & Co KG, Wiesbaden; 6Hämatologisch-Onkologische Gemeinschaftspraxis, München, Germany; 7Medizinische Abteilung für Hämatologie und Onkologie, Hanusch Krankenhaus, Wien, Austria; 8Institute of Oncology Research, Bellinzona, Switzerland Background: Treatment with Venetoclax in combination with Rituximab (VenR) has shown promising efficacy and good tolerability in clinical trials1 However, there is limited real-world data on VenR in CLL patients with or without genetic markers, known to be associated with unfavorable outcomes. Aims: The non-interventional observational study VeRVe aims to investigate safety and effectiveness of VenR for treatment of CLL under real-world conditions in Germany, Austria, and Switzerland. In this analysis we focus on safety and effectiveness of VenR in CLL patients with or without TP53 mut and/or del(17p) as well as IGHV status. Methods: CLL patients included in this study required therapy and were eligible for VenR treatment according to local label2. Study documentation is possible at baseline, weekly during ramp-up, monthly until the end of month 6 and quarterly afterwards up to a maximum of 3 years. Response assessment can be documented at end of ramp-up, after 3, 12, 24 and 36 months. Results: At the time of analysis (November 4th, 2021), 100 patients were enrolled in the VenR arm of the VeRVe study and received at least one dose of Ven. 86 of them did also receive at least one dose of rituximab. 28 patients were female, 32 had aberrant TP53 (TP53 mut and/or del(17p)) and 40 were IGHV unmutated. TP53, del(17p) and IGHV status was unknown for 20, 18 and 42 patients, respectively. The median age was 72 y in the population with TP53 aberration and 71 y in the population with wild-type (wt) TP53. Within the TP53 aberrant vs the TP53 wt group 48% vs 67% had received 1, 48% vs 33% had received 2 or more prior lines of therapy. At least 1 comorbidity was reported for 85% of TP53 aberrant and 81% of TP53 wt patients. After 12 months of therapy, the best ORR was 88% in the total, 90% in the TP53 aberrant and 91% in the TP53 wt population. Thereby, CR/CRi was achieved in 54% of TP53 aberrant and 44% of TP53 wt patients. When subdivided according to IGHV status, the best ORR was 88% in IGHV mutated and 91% in IGHV unmutated patients. 29% of IGHV mutated and 62% of IGHV unmutated patients reached CR/CRi as best ORR within 12 months. However IGHV status was not documented for 42 patients. Estimated PFS-rates after 1 year were 88% in the total population and 86% and 90% in the TP53 aberrant and TP53 wt population, respectively (figure). Estimated OS-rates after 1 year were 90% in the total population, 93% in the TP53 aberrant population and 90% in the TP53 wt population. After a median observation time of 451 days (range 1-1016), CTCAE grade ≥3 AEs were reported in 57% (n=57) of all patients, 61% (n=20) in TP53 aberrant patients and 64% (n=27) in the TP53 wt patients. SAEs were reported in 36% (n=36) of all, 42% (n=14) in TP53 aberrant and 40% (n=17) in the TP53 wt patients. TLS occurred in 10 patients (10%) of the overall population. 2 of them where clinical TLS (both in the group TP53 wt). Image: Summary/Conclusion: In conclusion, patients with and without TP53 aberrations were comparable regarding age and burden of comorbidities, but in the group with TP53 aberrations slightly more patients had a high number of prior therapy lines. However, both populations responded to VenR treatment with comparably high best ORR, estimated PFS-rates and estimated OS-rates. This was also the case for IGHV mutated vs unmutated patients, however patient number was low for this analysis, because the IGHV status was often not documented in this real-world study. VenR treatment was well tolerated in the overall population and all subgroups and no new safety signals were detected. P647: SAFETY AND EFFECTIVENESS OF VENETOCLAX MONOTHERAPY IN R/R CLL PATIENTS WITH OR WITHOUT RISK-ASSOCIATED GENETIC MARKERS – DATA FROM THE OBSERVATIONAL VERVE STUDY I. Schwaner1,*, H. Hebart2, C. Losem3, T. Wolff4, K. Famulla5, J. Huelsenbeck5, B. Schmidt6, T. Nösslinger7, D. Rossi8 1Onkologie Kurfürstendamm, Berlin; 2Kliniken Ostalb, Mutlangen; 3MVZ Onkologie und Hämatologie, Neuss; 4Onkologie Lerchenfeld, Hamburg; 5AbbVie Deutschland GmbH & Co KG, Wiesbaden; 6Hämatologisch-Onkologische Gemeinschaftspraxis, München, Germany; 7Medizinische Abteilung für Hämatologie und Onkologie, Hanusch Krankenhaus, Wien, Austria; 8Institute of Oncology Research, Bellinzona, Switzerland Background: In chronic lymphocytic leukemia (CLL), the association of genetic markers such as del(17p), TP53 and IGHV status with disease prognosis is well established. In clinical trials, treatment with Venetoclax (Ven) monotherapy has shown promising efficacy and good tolerability in all subgroups1,2. However, there is limited real-world data on Ven usage in specific genetic subgroups. Aims: The non-interventional observational study VeRVe aims to investigate safety and effectiveness of Ven for treatment of CLL in a real world setting in Germany, Austria, and Switzerland. Here, we present VeRVe data focusing on Ven monotherapy in CLL patients with or without the genetic risk factors TP53 mut and/or del(17p) as well as IGHV status. Methods: CLL patients included in this study required therapy and were eligible for Ven monotherapy treatment according to local label3. Study documentation is possible at baseline, weekly during ramp-up, monthly until the end of month 6 and quarterly afterwards up to a maximum of 2 years. Response assessment can be documented at end of ramp-up, after 3, 12 and 24 months. Results: On Nov 4th, 2021, 77 patients were enrolled in the VeRVe study and received at least one dose of Ven monotherapy, 29 of which were female. 40 of them had TP53 aberrations (TP53 mut and/or del(17p)) and 27 were IGHV unmutated. For 14 patients TP53 mut status, for 10 patients del(17p) and for 40 patients IGHV status was not documented. At start of therapy, the median age of patients with TP53 aberrations was 73 years and 71 years for patients with wildtype (wt) TP53. In the population with TP53 aberrations, 40% had received 1, 28% 2 and 32% 3 or more prior therapies. In the population with wild-type (wt) TP53, 12% had received 1, 40% 2 and 48% 3 or more prior therapies. 80% of patients with TP53 aberration and 72% of patients with wt TP53 had at least 1 comorbidity. After 12 months of therapy, the best ORR (CR/CRi) was 74% (34%) in the total population. Thereby, the best ORR (CR/CRi) was 76% (43%) in patients with TP53 aberrations and 69% (27%) in patients without TP53 aberrations. When subdivided according to IGHV status, the best ORR (CR/CRi) was 83% (33%) in IGHV mutated and 85% (43%) in IGHV unmutated patients (see figure). Moreover, estimated PFS-rate after 1 year was at 80% in the total population. Thereby, estimated PFS-rate was at 78% in patients with TP53 aberrations and at 83% in patients with wt TP53. Estimated OS-rate after 1 year was 82% in the total population, 77% in patients with TP53 aberration and 84% in patients with wt TP53. With a median observation time of 294 days (range 0-1130), CTCAE grade AEs ≥3 were reported in 56%(n=43) of the total population, 53% (n=21) in the TP53 aberrant and 64% (n=16) in the TP53 wt population. SAEs were reported in 37% (n=39) of the total population, 45% (n=18) in the TP53 aberrant and 32% (n=8) in the TP53 wt population. Overall, 5 patients had a TLS with 3 of them being clinical TLS. No new safety signals were detected. Image: Summary/Conclusion: In summary, patients with TP53 aberration were slightly more pre-treated and suffered from co-morbidities more frequently than patients with wt TP53. Nevertheless, best ORR, estimated PFS-rates and OS-rates were comparably high in both populations. IGHV status was often not determined in this real-world study. For the available data, best ORR in IGHV mutated and IGHV unmutated patients were comparable. Surprisingly, CR/CRi rates were highest in patients with TP53 aberration or IGHV unmutated patients. Treatment was well tolerated in the overall population and all subgroups. P648: T CELL PHENOTYPIC CHANGES IN PARTICIPANTS OF A MULTIPHASE OPTIMIZATION STRATEGY (MOST) BEHAVIORAL INTERVENTION OF HEALTHY DIET AND EXERCISE IN PATIENTS (PTS) WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) M. Gordon1,*, J. Crane2, C. Y. Lee3, S. Fares3, K. Basen-Engquist3, M. Markofski4, A. Ferrajoli5, E. LaVoy4 1Cancer Medicine, University of Texas MD Anderson Cancer Center; 2Biology, University of Houston; 3Behavioral Science, University of Texas MD Anderson Cancer Center; 4Health and Human Performance, University of Houston; 5Leukemia, University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Clinical outcomes in pts with CLL are influenced by disease biology, host immune response and comorbidities (Roessner et al, Leukemia, 2020 and Rotbain et al, Leukemia, 2021). Intensive physical exercise can modulate T cell function and slow leukemic progression (Sitlinger et al, Blood Adv., 2020). Here we report baseline and 4 month follow up of T cell phenotype in CLL pts after participation in a MOST exercise program. Aims: Assess the association of comorbidities and T cell subsets in pts with CLL participating in an organized exercise program. Methods: Pts with CLL treated at MD Anderson were randomized to one of 16 combinations of 5 evidence-based behavioral interventions. Pts with baseline and follow up data were included. T cell phenotype was identified by multicolor flow cytometry assessing CD3, CD4, CD8, HLADR and PD1. Difference in baseline and pre/post intervention T cell subset was assessed by t-test and Mann-Whitney. A p-value <.1 was considered significant. Results: Median age was 63 years in the 24 pts with CLL. TP53 aberrancy was present in 8%, unmutated IGHV in 46% and 63% had moderate-severe comorbidities (CLL comorbidity index [CI] score, 1-2). Median prior lines of therapy were 1, 85% were receiving a BTK inhibitor (ibrutinib n=7, acalabrutinib n=2 and zanubrutinib n=2); 50% were untreated. Baseline T cell phenotype showed that a CLL-CI score of 1-2, compared to 0 was associated with lower HLADR+CD4+ T cells (p=.07). CLL-CI score of 1 vs 0 was associated with high PD1+CD8+ T cells (p=.03) and PD1+CD4+ T cells (p=0.06; Fig 1). By individual comorbidity category only upper gastrointestinal comorbidities (n=6) were associated with T cell phenotype, lower HLADR+CD8+ T cells (p=.04). Pts ≥65 years had lower HLADR+CD4+ (p=.04) and HLADR+PD1+CD4+ (p=.09) T cell populations. Advanced Rai stage (2-4) was associated with higher CD8+ T cells (p=.03). Treatment, IGHV and Beta 2 microglobulin were not associated with T cell phenotype. T cell phenotype at 4 months showed that a CLL-CI score of 1-2 vs 0 was associated with lower HLADR+CD4+ T cells (p=.06). Both PD1+CD4+ and PD1+CD8+ T cells were higher in pts with a CLL-CI score of 1 vs 0 (p=.09 for both). Pts with vascular comorbidities had lower CD8+ (p=.09) and higher HLADR+PD1+CD4+ (p=.09) T cells. Endocrine comorbidities were associated with lower HLADR+CD4+ (p=.08) and lower HLADR+PD1+CD4+ (p=.09) T cells. Advanced Rai stage was associate with increased HLADR+CDC8+ T cells (p=.02). Treatment and age did not significantly impact T cell phenotype. Image: Summary/Conclusion: Comorbidities are associated with shorter survival and more rapid progression of CLL (Gordon et al, CCR, 2021 and Rotbain et al, Blood adv., 2022). But the mechanism(s) underlying these observations are not well described. High HLADR+PD1+CD4+ T cells are associated with CLL progression (Elston et al, BJH, 2020). We hypothesized that comorbidities, measured by the CLL-CI, could influence T cell function and thus lead to CLL progression. CLL-CI score was associated T cell phenotype in this study. Pts with comorbidities had higher PD1 expression at baseline. Interestingly, after participation in the exercise intervention, pts with vascular disease had higher HLADR+PD1+CD4+ T cells while those with endocrine comorbidities had lower HLADR+PD1+CD4+ T cells. This suggest that pts with obesity and diabetes may particularly benefit from participation in behavioral interventions by lowering the % of HLADR+PD1+CD4+ T cells. P649: A FIRST-IN-HUMAN PHASE 1 TRIAL OF NX-2127, A FIRST-IN-CLASS ORAL BTK DEGRADER WITH IMID-LIKE ACTIVITY, IN PATIENTS WITH RELAPSED AND REFRACTORY B-CELL MALIGNANCIES A. Mato1,*, A. Danilov2, M. R. Patel3, M. Tees4, I. Flinn5, W. Ai6, K. Patel7, M. Wang8, S. M. O’Brien9, S. Nandakumar10, M. Tan10, E. Meredith10, M. Gessner10, S. Y. Kim10, A. Wiestner11, W. G. Wierda8 1Memorial Sloan Kettering Cancer Center, New York; 2City of Hope National Medical Center, Duarte; 3Florida Cancer Specialists/Sarah Cannon Research Institute, Sarasota; 4Colorado Blood Cancer Institute, Denver; 5Sarah Cannon Research Institute and Tennessee Oncology, Nashville; 6University of California San Francisco Medical Center, San Francisco; 7Swedish Cancer Institute, Seattle; 8MD Anderson Cancer Center, Houston; 9Chao Family Comprehensive Cancer Center, University of California, Irvine; 10Nurix Therapeutics, Inc., San Francisco; 11National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, United States of America Background: Bruton’s tyrosine kinase inhibitors (BTKi) have received regulatory approvals and are standard of care for patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and WaldenstrÖm macroglobulinemia (WM). However, BTKi-resistant disease remains a clinical challenge with limited options for subsequent therapy. Immunomodulatory drugs (IMiDs, e.g., lenalidomide) are approved as monotherapy for follicular lymphoma (FL), MZL, and MCL, in combination with other therapies for diffuse large B-cell lymphoma (DLBCL) and have shown synergy with BTK-targeted therapy. Dual activity of BTK protein degradation with IMiD-like activity offers a unique approach to overcome known resistance to BTKi. NX-2127 is an oral small molecule that induces BTK degradation via recruitment of cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NX-2127 has shown preclinical activity similar to IMiDs by catalyzing the ubiquitination of Ikaros (IKZF1) and Aiolos (IKZF3), ultimately leading to increased T-cell activation. NX-2127 was shown to degrade both wild-type (WT) and C481-mutated (ibrutinib-resistant) BTK protein in vitro. Robust BTK degradation was also shown in non-human primate studies. Further, NX-2127 demonstrates potent tumor growth inhibition in BTK-dependent mouse xenograft tumor models expressing either WT or ibrutinib-resistant C481S BTK-mutant protein. This dual activity of BTK degradation and IMiD-like activity offers a promising treatment for patients who have failed prior therapy. Aims: The primary objectives are to evaluate safety and tolerability and to determine the maximum tolerated dose (Phase 1a), and to evaluate the early clinical activity of NX-2127 in expansion cohorts (Phase 1b). Methods: NX-2127-001 is a first-in-human, dose escalation (Phase 1a) and cohort expansion (Phase 1b) study designed to evaluate the safety, tolerability, and preliminary efficacy of NX-2127 in adult patients with relapsed/refractory B-cell malignancies with once daily oral dosing. Dose escalation will proceed using a modified Fibonacci design with 1 patient per cohort, proceeding to a standard 3 + 3 design based on protocol specified criteria. There will be up to 5 expansion cohorts in Phase 1b enrolling patients with CLL/SLL, DLBCL, FL, MCL, MZL, and WM. Key eligibility criteria include ≥2 two prior lines of therapy (≥1 prior for WM); measurable disease; and an Eastern Cooperative Oncology Group performance status of 0 or 1. Approximately 130 patients (30 in Phase 1a, 100 in Phase 1b) will be enrolled and treated until disease progression or unacceptable toxicity. The primary endpoint are dose-limiting toxicities and maximum tolerated dose (Phase 1a), objective response (Phase 1b), and safety (Phase 1a and Phase 1b) of NX-1607. Secondary endpoints (Phase 1a and Phase 1b, unless otherwise indicated) include pharmacokinetics, pharmacodynamics, duration of response, progression-free survival, overall survival (Phase 1b), safety (Phase 1b), and complete response rate/complete response rate with incomplete marrow recovery. Results: The Phase 1a part of this study is currently enrolling in the United States. Summary/Conclusion: Accrual is ongoing. Clinical trial information: NCT04830137. P650: A FIRST-IN-HUMAN PHASE 1 TRIAL OF NX-5948, AN ORAL BTK DEGRADER, IN PATIENTS WITH RELAPSED AND REFRACTORY B-CELL MALIGNANCIES K. Linton1,*, G. P. Collins2, D. El-Sharkawi3, R. Mous4, F. Forconi5, M. Tan6, S. Nandakumar6, E. Meredith6, K. L. Jameson6, S. G. Injac6, J. Doorduijn7 1The University of Manchester, Manschester; 2Oxford University Hospitals NHS Trust, Oxford; 3Royal Marsden NHS Foundation Trust, Sutton, United Kingdom; 4UMC Utrecht Cancer Center, University Medical Center, Utrecht, Netherlands; 5University Hospital Southampton NHS Trust, Southhampton, United Kingdom; 6Nurix Therapeutics, Inc., San Francisco, United States of America; 7Erasmus MC Cancer Institute, Rotterdam, Netherlands Background: Bruton’s tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) signaling pathway and chronic activation of BTK-mediated BCR signaling is a hallmark of many B-cell malignancies. BTK-targeted therapy has been shown to be safe and effective in a variety of B-cell lymphomas, including chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM). However, BTK inhibitor (BTKi) resistant disease remains a clinical challenge with limited options for salvage therapy and overall poor patient outcomes. BTK degradation may be effective in patients who have developed resistance to BTKi or in B-cell indications where treatment with BTKi has been less effective. NX-5948 is an oral, small molecule that induces BTK degradation through the recruitment of cereblon, an adaptor protein of the E3 ubiquitin ligase complex, and does not induce degradation of other cereblon neosubstrates. NX-5948 degrades both wild type BTK and BTKi resistant mutants (C481) and inhibits ibrutinib-resistant tumor cell line growth at concentrations where ibrutinib and acalabrutinib are inactive. NX-5948 potently inhibits tumor growth in xenograft models that contain either wild type BTK or BTKi-resistant mutations. Further, NX-5948 crosses the blood-brain barrier, thus positioning it as a therapeutic agent in patients with primary central nervous system lymphoma (PCNSL) and other lymphomas that have CNS involvement. Aims: The primary objectives are to evaluate safety and tolerability, determine the maximum tolerated dose (MTD; Phase 1a only), and the early clinical activity in each expansion cohort (Phase 1b only) of NX-5948 in patients with relapsed and refractory B-cell malignancies. Methods: NX-5948-301 is a first-in-human, dose escalation (Phase 1a) and cohort expansion (Phase 1b) study designed to evaluate the safety, tolerability, and preliminary efficacy of NX-5948 in adult patients with relapsed and refractory B-cell malignancies. NX-5948 will be given orally daily and dose escalation will proceed using a standard 3 + 3 design. There will be up to 5 expansion cohorts in Phase 1b composed of patients with the following tumor types: (a) CLL/SLL with a C481 mutation; (b) CLL/SLL without a C481 mutation; (c) aggressive subtypes diffuse large B-cell lymphoma (DLBCL) and MCL; (d) indolent subtypes FL, MZL, and WM; and (e) primary/secondary CNS lymphoma. Other key eligibility criteria include 2 or more prior lines of therapy (only 1 prior for WM and PCNSL); measurable disease; and an Eastern Cooperative Oncology Group performance status of 0 or 1. Up to 130 patients (30 in Phase 1a, 100 in Phase 1b) will be enrolled and treated until disease progression or unacceptable toxicity. Results: Enrollment in this study has begun in Europe. Summary/Conclusion: Accrual is ongoing. Clinical trial information: NCT05131022. P651: VENETOCLAX-BASED TREATMENT OF PATIENTS WITH RICHTER SYNDROME: OUTCOMES FROM A MULTICENTER RETROSPECTIVE STUDY P. Hampel1,*, S. Parikh1, W. Wierda2, A. Ferrajoli2, N. Jain2, J. Burger2, T. Call1, Y. Wang1, S. Kenderian1, W. Ding1, P. Thompson2 1Hematology, Mayo Clinic, Rochester; 2Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, United States of America Background: Patients (pts) with chronic lymphocytic leukemia (CLL) who develop Richter Syndrome (RS) have a poor prognosis with median overall survival (OS) often reported as less than 12 months (Tsimberidou JCO 2006; Rogers BJH 2018), using chemoimmunotherapy (CIT) regimens typically administered to de novo large cell lymphoma. Venetoclax (ven) has demonstrated single-agent activity in RS with an overall response rate of 43% (Davids JCO 2017). Ven combined with DA-EPOCH-R achieved a median OS of 19.6 months and the highest complete response (CR) rate (50%) reported in pts with RS (Davids Blood 2022). These results support a synergistic effect when compared to CR rates of 20% and 0% with EPOCH-R and ven monotherapy, respectively. Aims: To evaluate the outcomes of pts with RS treated with ven-based treatment, outside clinical trials, including novel-novel combinations and CIT combinations. Methods: We analyzed pts with RS treated with a ven-based regimen at MDACC (n=37) or Mayo Clinic (n=18) between 12/2013 and 8/2021. Patient and disease characteristics from the time of ven-based treatment start were ascertained. Retrospective response assessment was as per Lugano 2014 guidelines. Toxicity was recorded per iwCLL 2018 guidelines (hematologic toxicity) or CTCAE v5.0 (non-hematologic toxicity). OS and progression-free survival (PFS) were analyzed using the Kaplan-Meier method, with and without censoring for allogeneic hematopoietic stem cell transplantation (alloSCT). No formal statistical comparisons were made between different treatment groups. Results: Fifty-five pts were identified with a median age of 66 years (range 43-83 years); 31% were female. High-risk CLL disease characteristics were frequently identified: 92% unmutated IGHV, 42% del(17p), 57% TP53 mutation, and 62% complex karyotype. The median number of prior CLL-directed therapies was 2 (range 0-7), including prior chemotherapy (42%), prior Bruton tyrosine kinase inhibitor (BTKi; 38%), and prior ven (22%); 62% of pts were previously untreated for RS. Median follow-up from the start of ven-based RS treatment was 9 months (mo). The most common ven-based combination regimens achieved overall response and CR rates as follows: 50%/40% with intensive CIT + ven (n=20), 40%/30% with BTKi + ven +/- CD20 antibody (n=20), 60%/50% with R-CHOP + ven (n=10). Among the remaining 5 patients, 2 received ven monotherapy and 3 received varied ven-based combination regimens. Nine pts proceeded to subsequent alloSCT. The median PFS for the total cohort when censored at alloSCT was 4.0 mo; PFS uncensored for alloSCT was 4.4 mo (Figure 1A). Median PFS by treatment group (Figure 1B) was as follows: 3.7 mo for intensive CIT + ven, 3.9 mo with BTKi + ven +/- CD20 antibody, not reached with R-CHOP + ven. The median OS for the total cohort was 9 mo. The estimated median OS by treatment group (Figure 1C) was 8.4 mo with intensive CIT + ven, 10.6 mo with BTKi + ven +/- CD20 antibody and not reached with R-CHOP + ven. Grade 3-4 neutropenia and thrombocytopenia were more common with intensive CIT + ven (71%; 69%) and R-CHOP + ven (77%; 67%) compared to BTKi + ven +/- CD20 antibody (35%; 25%). Febrile neutropenia occurred in 42%, 33%, and 25% of pts in these three groups, respectively, with 37%, 33%, and 30% experiencing a grade 3-4 infection. Image: Summary/Conclusion: In the notoriously difficult-to-treat RS patient population, ven-based combination regimens achieve high response rates, including some with prolonged duration, particularly with the use of R-CHOP + ven. A prospective clinical trial evaluating this combination is ongoing (NCT03054896). P652: MEASURING MINIMAL RESIDUAL DISEASE BEYOND 10-4 THROUGH IGHV LEADER-BASED NEXT GENERATION SEQUENCING IMPROVES PROGNOSTIC STRATIFICATION IN CHRONIC LYMPHOCYTIC LEUKEMIA P. Hengeveld1,2,*, M. van der Klift1, P. M. Kolijn1, F. Davi3, F. Kavelaars4, E. de Jonge5, S. Robrecht6, J. Assmann1, L. van der Straten1,2, M. Ritgen7, P. Westerweel2, K. Fischer6, V. Goede8, M. Hallek6, M.-D. Levin2, A. Langerak1 1Department of Immunology, Erasmus MC, Rotterdam; 2Department of Internal Medicine, Albert Schweizer Ziekenhuis, Dordrecht, Netherlands; 3Department of Hematology, Pitié-Salpêtrière Hospital, Paris, France; 4Department of Hematology; 5Department of Clinical Chemistry, Erasmus MC, Rotterdam, Netherlands; 6Department I. of Internal Medicine, Center for Intergrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne; 7Department of Medicine II, University Hospital of Schleswig Holstein, Kiel; 8Division of Oncogeriatrics, St Marien Hospital, Cologne, Germany Background: The sensitivity of conventional techniques for reliable quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10-4. Measuring MRD <10-4 could help to further distinguish between CLL patients with durable remission and those at risk of early relapse. Aims: To develop an academically sourced IGHV leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL. Methods: The IGHV leader primer set developed by the EuroClonality-NGS working group was used to amplify all IGH rearrangements present in an end of treatment (EOT) DNA pool. The IGH clonal target was amplified in a single-step PCR and subsequent sequencing was performed on the Illumina MiSeq platform. To calculate MRD depth, NGS output was annotated through in the ARResT/Interrogate immunoprofiler. Technical validation of the IGHV leader-based assay was performed on contrived MRD samples, created by serial dilution of pretreatment CLL DNA pooled PBMC DNA from healthy donors. Clinical validation of the IGHV leader-based assay was performed on EOT samples from the CLL11 trial (NCT01010061). Results: In 176/183 (96%) measurements on contrived samples, the CLL-specific rearrangement was detected. The limit of detection and limit of quantitation were estimated at 3.4 [95% CI 1.9-16.0] and 3.8 [95% CI 1.7-8.2] malignant cells per assay, respectivelly. The limit of blank was found to be 0. Linearity was established in the MRD 10-2-10-5 range (r=0.94 [95%CI 0.91-0.96]). Of note, when provided with sufficient DNA input, MRD could even be detected down to MRD 10-6. There was high inter-assay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative PCR (r=0.92, [95%CI 0.86-0.96]) and droplet digital PCR (r=0.93, [95%CI 0.88-0.96]). In a cohort of 67 patients from the CLL11 trial, using 5μg DNA input and MRD 10-5 as a cut-off, undetectable MRD (uMRD) was associated with superior progression-free survival (PFS) and time to next treatment. Importantly, deeper MRD measurement allowed for additional stratification of patients with MRD <10-4 but ≥10-5. Patients with MRD in this range had a significantly shorter PFS, compared to patients with uMRD <10-5, but significantly longer, compared to patients with MRD ≥10-4 (median PFS: ≥10-4, 10.4 months; <10-4 but ≥10-5, 27.5 months; uMRD <10-5, NR, 4-year PFS rate: ≥10-4, 66.7%; <10-4 but ≥10-5, 25.0%; uMRD <10-5, 7.2%, P<0.001) (Figure 1). Image: Summary/Conclusion: We here present an academically-developed, IGHV leader-based NGS assay for the detection and quantification of MRD in CLL beyond MRD 10-4. The assay has high sensitivity and is quantitative and linear up to MRD 10-5 when using 5μg DNA input. Measurement to MRD 10-6 is feasible, conditional on sufficient DNA input. The deeper MRD measurements enabled by the IGHV leader-based NGS assay resulted in improved stratification of CLL patients following treatment. P653: OUTCOMES FOLLOWING TREATMENT WITH A COVALENT BTK AND BCL2 INHIBITOR AMONG PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)/SMALL LYMPHOCYTIC LYMPHOMA (SLL): A REAL-WORLD STUDY OF A LARGE U.S. DATABASE T. A. Eyre1,*, L. Hess2, T. Sugihara3, R. A. Walgren2, P. B. Abada2, H. Konig2, J. Pagel4, L. E. Roeker5, A. Mato5 1Oxford University Hospitals, NHS Foundation Trust, Oxford, United Kingdom; 2Eli Lilly and Company, Indianapolis; 3Syneos Health, Austin; 4Loxo Oncology, Stamford; 5Memorial Sloan Kettering Cancer Center, New York, United States of America Background: Covalent Bruton tyrosine kinase inhibitors (cBTKi) and B-cell lymphoma 2 inhibitors (BCL2i) have improved the outcomes for patients with CLL/SLL. Although these agents are highly effective, they are not curative, and over time patients will require additional therapy. Real-world data of patient outcomes post cBTKi and BCL2i exposure in CLL/SLL are limited to small retrospective studies, and the optimal treatment in this setting is unknown. Aims: This study was designed to address this data gap by evaluating the characteristics and clinical outcomes about patients with CLL/SLL after treatment with both cBTKi and BCL2i. Methods: Eligible patients were adult patients in the Flatiron Health Electronic Health Record (EHR)-derived de-identified database diagnosed with CLL/SLL between December 2011 and October[LMH1] 2020. Follow-up data were available through October 2021 at the time of analysis. Patients were required to have at least one record of receiving both a cBTKi and a BCL2i. Time-to-event analyses using Kaplan-Meier method included duration of therapy, time to next treatment discontinuation, transformation or death, and overall survival (OS) from the time of cBTKi/BCL2i discontinuation. Results: 339 patients (median age 66 years, interquartile range 59, 73; male 69.6%) met eligibility criteria. Most patients received cBTKi and BCL2i as early lines of therapy: 40.4% received cBTKi in first line and BCL2i in second line; an additional 21.8% received cBTKi in the second line and BCL2i in third line. Transformation was recorded among 24 (7.1%) patients during the study period. Of all eligible patients, 215 had discontinued both cBTKi and BCL2i and could be evaluated for post-cBTKi/BCL2i outcomes. A total of 47 patients (21.9% of the 215 who discontinued therapy) died before receiving subsequent therapy, 116/215 (54%) received subsequent therapy, and the remaining 52/215 (24.2%) were alive without additional treatment after cBTKi/BCL2i exposure. The median time from discontinuation of cBTKi/BCL2i (whichever agent was last) to the immediate next treatment discontinuation, transformation, or death was 4.6 months (95% confidence interaval [CI]: 3.0-6.9). Among those who received subsequent therapy, the most common immediate next line of therapy included additional BCL2i-based therapy (venetoclax re-treatment with or without other agents, n=71/116; 61.2%) or anti-CD20 antibody-based therapy (with or without other agents, 73/116 (62.9%). Fewer patients received PI3K inhibitor-based treatment (n=14/116; 12.1%) or additional cBTKi therapy (n=19/116; 16.4%) at any time after discontinuation of the initial cBTKi/BCL2i. As shown in the Figure, for those who received subsequent therapy (n=116), the median duration of the immediate post-cBTKi/BCL2i therapy was 4.8 months (95% CI: 3.0-7.3). Among the 215 who discontinued cBTKi/BCL2i therapy, median OS from discontinuation was 14.1 months (95% CI: 11.7-21.0). Image: Summary/Conclusion: Patients with CLL/SLL who have been previously treated with both a cBTKi and BCL2i experience poor outcomes as observed by time to next treatment discontinuation, duration of subsequent therapy and overall survival. There remains a need for more effective therapies for patients with CLL/SLL after progression on cBTKi/BCL2i. P654: IBRUTINIB PLUS VENETOCLAX IN PATIENTS WITH COMPLEX KARYOTYPE AND CHRONIC LYMPHOCYTIC LEUKEMIA A. Petrenko1,2,*, M. Kislova1, E. Dmitrieva1, T. Novikova3, M. Kislitsyna3, T. Obukhova3, E. Nikitin1,2, V. Ptushkin1,2 1Moscow City Center of Hematology, Botkin hospital; 2Russian Medical Academy of Continuous Medical Education; 3Karyology laboratory, National Medical Research Center for Hematology, Moscow, Russia Background: In the largest study of Baliakas et al. (2019) the presence of at least 5 abnormalities, was associated with dismal clinical outcome, independently of the somatic hypermutation status and TP53 status. The presence of 3 or 4 aberrations is defined as clinically relevant in the absence of TP53. Studies by Kittai (2021) and Al-Sawaf (2020) showed the impact of karyotypic complexity on survival in patients with chronic lymphocytic leukemia (CLL) treated with ibrutinib or venetoclax. The complex karyotype (CK) is a topic that is being intensively researched, both in the aspect of increasing karyotypic complexity stratification and clonal evolution. Optimal therapy for patients with CLL has not yet been developed. The combination therapy of ibrutinib and venetoclax was superior to chlorambucil and obinutuzumab in terms of undetectable minimal residual disease (MRD) responses according to data from the GLOW trial (Tunir, 2021). The importance of achieving a complete response with undetectable MRD as the goal of therapy in CLL was proposed (Montserrat, 2005). Aims: The aim of our study is to evaluate the effectiveness of therapy with ibrutinib and venetoclax in combination for the patients with CLL and CK. Methods: This ambilinear observational study included patients with CLL with high genetic complexity (high-CK), defined as >=5 aberrations or CK (>=3 aberrations) in combination with a 17p deletion (CK+del17p). The first retrospective cohort included patients treated with ibrutinib monotherapy (Imono) to progression or intolerable toxicity since May 2015. The second prospective cohort included patients receiving ibrutinib in combination with venetoclax (IVen) since July 2019. Venetoclax therapy was started at the 3rd month of ibrutinib (from the escalation phase). Combination therapy was continued until a complete response, defined as three consecutive PET-CT-negative and MRD-negative results 3 months apart. If this criterion was not achieved at 24th month of therapy, venetoclax was discontinued and ibrutinib continued indefinitely. Results: Seventy-nine patients are included in the study. Twenty-nine patients in the first cohort and 50 patients in the second cohort. The characteristic is presented in Table. At the current follow-up periods, there were no significant differences in PFS and OS regarding a follow-up period <= 24 months (with the exception of death from COVID-19, since patients were not observed at parallel time intervals). In the group of patients treated with Imono, the majority of patients achieved partial remission or partial remission with lymphocytosis by 12 months. In 21 patients from Iven group, with a median follow-up of 7.4 months, a complete remission was achieved (72.4%); of these, 8 had unmeasurable MRD. Four patients did not complete the escalation period. There was a significant difference in the median MRD response achieved between 3 (log10>10) and 12 (log10<0,1) months in IVen group (p=0,03). In 2 patient from the IVen group progression of the disease was noted. Image: Summary/Conclusion: Combination therapy with ibrutinib and venetoclax is an effective oral regimen for high-risk patients with complex karyotype disorders. PFS in both groups is currently not significantly different, which is obviously due to the short follow-up period. Patients receiving the IVen regimen achieve a significantly better response, which paves the way for allogeneic transplantation in these patients. P655: RACIAL AND SOCIOECONOMIC DISPARITIES AMONG PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA IN THE US: ANALYSIS OF SURVEILLANCE, EPIDEMIOLOGY, AND END RESULTS PROGRAM DATA A. Kittai1,*, Y. Huang1, J. Fisher1, S. Bhat1, M. Grever1, E. Paskett1, K. Rogers1, J. Woyach1 1The Ohio State University, Columbus, United States of America Background: Therapy for chronic lymphocytic leukemia (CLL) has changed dramatically over the past 20 years. With the cost of new therapies and rapid practice changes, it is unclear if patients are benefiting equally from this progress. We assessed Surveillance, Epidemiology, and End Results (SEER) program data to determine how race and socioeconomic status (SES) affect survival for patients with CLL. Aims: Aim 1 - Determine the prognostic significance of race on overall survival for patients with CLL. Aim 2 - Determine the prognostic significance of socioeconomic status on overall survival for patients with CLL. Methods: CLL cases reported to 18 SEER Program registries from 2006 – 2018 were included. Patient characteristics such as age at diagnosis, sex, year of diagnosis, race, and SES as determined by rural/urban census tract residence (RUCA), and neighborhood (as represented by the Yost Index, a composite measure of 7 variables assessing different aspects of the SES of a census tract) were collected and analyzed. Multivariable cox regression (MVA) was used to determine adjusted odds of survival. Two separate databases were utilized, one which included data to 2018, and another which contained SES data but only had data available to 2016. Results: 46,605 cases from 2009 – 2018 were identified without SES data. The median age was 70 years, 60% were male, and there was an even distribution of patients diagnosed with CLL annually from 2009 – 2018. Of the cases with race reported, 89.9% were white, 7.3% Black, 2.4% Asian/Pacific islander, and 0.3% American Indian/Alaska Native. After a median follow up of 47 months, the median 3-, 5-, and 10-year overall survival (OS) was 79.5%, 69.5%, and 48.8%, respectively. MVA showed Black race (HR 1.5, 95% CI 1.4 – 1.6) as the strongest independent prognostic variable for worse OS controlling for year of diagnosis, suggesting race was a significant factor in OS in the era of modern therapies. Using the linked RUCA and Yost tertiles for SES, 47,867 cases of CLL from 2006 – 2016 were analyzed. Like the prior analysis, median age was 70 years, 60% were male, and there was an even distribution of patients diagnosed with CLL annually from 2006 – 2016. Of the cases with race reported, 90.4% were white, 7.1% Black, 2.3% Asian/Pacific islander, and 0.3% American Indian/Alaska Native. Per the Yost index, 24.8%, 33.8%, and 41.4% of patients were in Groups 1, 2, and 3, respectively. Per RUCA categories, 8.9% and 91.1% of patients were residents of rural and urban areas, respectively. MVA showed American Indian/Alaska Native, and Black race as independent prognostic variables for worse OS, and Yost group 2 and 3, representing higher SES, were found to be significant independent prognostic variables for improved OS (Table 1). In this analysis, race remained an independent variable for worse OS after controlling for SES. Image: Summary/Conclusion: Black race and low SES are prognostic of OS in CLL. Further research is needed to determine whether this is due to access to therapy, quality of care, social determinants of heath, or disease biology. P656: INCIDENT VENOUS THROMBOEMBOLISM (VTE) IN PATIENTS WITH MONOCLONAL B-CELL LYMPHOCYTOSIS (MBL) AND CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) – A POPULATION-BASED STUDY A. Koehler1,*, M. Moise1, T. Call1, K. Rabe2, S. Achenbach2, D. Crusan2, K. Bailey2, T. Petterson2, W. Ding1, S. Kenderian1, J. Leis3, Y. Wang1, E. Muchtar1, P. Hampel1, S. Schwager1, S. Slager2, N. Kay1, A. Ashrani1, S. Parikh1 1Hematology; 2Biostatistics, Mayo Clinic, Rochester; 3Hematology, Mayo Clinic, Scottsdale, United States of America Background: Data on risk of VTE after MBL/CLL diagnosis is limited;it is unclear if MBL/CLL truly confers increased risk of VTE as seen in other malignancies. Aims: To determine incidence of VTE after MBL/CLL diagnosis and compare it to age- and sex-matched VTE incidence in the general population. Methods: We identified all individuals with newly diagnosed, untreated MBL or CLL between 1998-2021 within a 27-county region surrounding Rochester, MN. Patient demographics and CLL-specific characteristics were extracted from the Mayo Clinic CLL Database and the electronic health record (EHR). Incident VTE after diagnosis of MBL/CLL was identified by querying a) the Mayo Clinic CLL Database; b) EHR for VTE-specific ICD-9 and ICD-10 diagnosis codes; and c) all radiographic studies performed during longitudinal care of these patients. Risk of VTE was estimated using Cox proportional hazards model with CLL/SLL status as a time-dependent variable. The unadjusted cumulative risk of VTE was estimated using Kaplan-Meier methods in SAS 9.4. Rate of incident VTE among age- and sex-matched population residing in Olmsted County, MN from 2001-2015 was pulled from the Rochester Epidemiology Project. Age- and sex-specific VTE risk associated with MBL/CLL (standardized incidence rate ratio; SIR) was estimated by dividing observed post-MBL/CLL diagnosis VTE incidence rate by expected VTE incidence rate in the general population. We performed an exact binomial test in the statistical package R (version 4.1.2) to evaluate whether the proportion of VTE cases after MBL/CLL diagnosis (versus those occurring in the general population) was consistent with their respective proportion of person-years at-risk for VTE. Results: We identified 946 patients with newly diagnosed MBL/CLL, of whom 42 (4%) reported a prior history of VTE and were excluded from this analysis. Of the 904 evaluable subjects, 293 had MBL and 611 had CLL. The median age was 69 years (range, 28 - 96 years) and 587 (65%) were male. After a median follow-up of 6 yrs (range: 1 day-23 yrs), 70 of 904 patients developed VTE after MBL/CLL diagnosis; 43 (61%) with deep vein thrombosis (DVT), 24 (34%) with pulmonary embolism (PE), and 3 (4%) with both DVT and PE as the first event. Risk of VTE was similar in CLL compared to MBL [HR (95% CI) = 0.90 (0.49-1.65)]. The 5-year and 10-year cumulative risk of VTE was 4.9% and 11.5%, respectively. Forty-seven (68%) patients had an identifiable provoking factor apart from MBL/CLL including second active malignancy (n=21), surgery (n=9), recent hospitalization (n=6), travel (n=4), trauma (n=3), line-associated VTE (n=3), and immobility (n=1). The age-adjusted VTE incidence rates for females and males with newly diagnosed MBL/CLL were 1275 and 1228 per 100,000 person-years, respectively. In contrast, the age-adjusted VTE incidence rates for females and males residing in Olmsted County, MN were 193 and 218 per 100,000 person-years, respectively. The overall VTE incidence rate in females and males after MBL/CLL diagnosis was 6.0 (95% CI: 4.0-8.8) and 5.7 (95% CI: 4.1-7.7) higher, respectively, compared to the general population; the risk of VTE was highest in 18–49-year-old MBL/CLL patients (SIR: 16.2, 95% CI: 3.3-47.6) (Figure 1). Image: Summary/Conclusion: In this large population-based cohort study, 1 in 12 patients with MBL/CLL developed VTE after a median follow-up of 6 yrs. Patients with MBL/CLL demonstrated a 6-fold increased risk of VTE compared to the age- and sex-matched general population. Importantly, nearly 3 in 4 MBL/CLL patients had additional provoking factors at time of VTE, most notably second active malignancy. P657: REAL-WORLD TREATMENT PATTERNS OF PATIENTS DIAGNOSED WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IN THE UNITED STATES (US) X. Yang1,*, E. Zanardo2, D. Lejeune3, E. De Nigris1, E. Sarpong1, N. Lema2, M. Farooqui1, F. Laliberté3 1Merck & Co., Inc., Kenilworth; 2Analysis Group, Inc., Boston, United States of America; 3Groupe d’analyse, Ltée, Montréal, Canada Background: CLL represents the most common type of leukemia among adults in the US, but current data on the treatment patterns of patients in clinical practice is limited. Standard of care frontline therapy consists of various types of targeted therapy or chemoimmunotherapy (CIT), but it is reserved for patients with advanced-stage disease. Aims: This study aims to describe real-world treatment patterns among patients diagnosed with CLL in the US, with a focus on ibrutinib therapy. Methods: A retrospective database analysis was conducted using the Optum Clinformatics DataMartTM database (01/2007–07/2020). Patients with ≥2 medical encounters with a diagnosis code for CLL or small lymphocytic lymphoma (SLL) on different dates were selected (earliest date was defined as the index date). At least 12 months of continuous enrollment pre-index date (baseline period) and ≥18 years of age as of the index date were required. Patients with a diagnosis for mantle cell lymphoma or with evidence of anticancer therapy (antineoplastic, radiation, or cell therapy) during baseline were excluded. A subset of patients with ≥1 pharmacy claim for ibrutinib was identified. An adapted algorithm developed from previously published studies was used to identify lines of therapy (LOTs). Treatment patterns, including duration of therapy (DOT) and regimens were reported. DOT spanned from LOT initiation up to discontinuation of all agents in the LOT, a switch to another LOT, or the addition of a new agent. Median time to the first line (1L) and from 1L to second line (2L) were evaluated using a Kaplan-Meier analysis (KM) to account for censoring. Results: Among 23,087 patients with CLL, the median age was 74 years and 43% were female. Of these, 1,387 were treated with ibrutinib (median age: 75; female: 38%). Patients had a mean Charlson comorbidity index score of 1.9. Analysis of treatment patterns (Table 1) showed that 7,192 patients (31%) were treated with ≥1 LOT (mean ± standard deviation [SD] DOT of 1L: 1.6 ± 2.0 years), and 10% of patients were treated with ≥2 LOT (mean ± SD DOT of 2L: 1.2 ± 1.7 years). Ibrutinib was used by 907 patients in 1L (DOT in 1L: 1.0 ± 1.1 years) and 617 in subsequent LOTs (DOT in 2L+: 0.9 ± 1.1 years). Median time from index date to 1L initiation was 8.1 years. Of the patients who used antineoplastics therapies in 1L, a majority used CLL-related ones (79%). Most treated patients received targeted therapy (37%; rituximab: 19% and ibrutinib: 12%) and CIT (32%; bendamustine + rituximab: 13% and cyclophosphamide + fludarabine + rituximab: 5%). In 2L, 16% of patients used ibrutinib, 12% a bendamustine + rituximab regimen, and 4% chlorambucil. Median time from 1L initiation to 2L initiation was 4.4 years. Proportion of patients treated with CIT tended to diminish with subsequent LOT, whereas the proportion treated with ibrutinib tended to increase. Image: Summary/Conclusion: This real-world long-term data shows that patients receive their 1L many years after a first CLL diagnosis, with a median time to treatment of ~8 years. Among US patients with CLL, rituximab, bendamustine + rituximab, and ibrutinib were identified as the most used 1L regimens, and the latter two as the most used 2L regimens. As novel therapies are increasingly used, and genetic testing becomes more available, further research will be needed to evaluate the changes in the way CLL is treated and its effects on clinical outcomes. P658: COMPARISON OF CHARACTERISTICS AND OUTCOME OF COVID-19 INFECTION IN PATIENTS WITH LYMPHOPROLIFERATIVE DISEASE AND IN PATIENTS FROM THE GENERAL POPULATION - EXPERIENCE FROM THREE UNIVERSITY CENTERS O. Markovic1,2,*, A. Divac2, Z. Cvetkovic1,3, D. Stanisavljevic4, K. Markovic5, I. Bukurecki2, T. Bibic3, N. Stanisavljevic1,2, J. Bila1,6, M. Zdravkovic1,7 1University of Belgrade, Medical Faculty; 2Department of Hematology, CHC Bezanijska kosa; 3Department of Hematology, CHC Zemun; 4Institut of Statistics, University of Belgrade, Medical Faculty; 5Department of Hematology, CHC Zvezdara; 6Clinic of Hematology, Clinical Center of Serbia; 7Department of Cardiology, CHC Bezanijska kosa, Belgrade, Serbia Background: Patients with lymphopproliferative diseases (LPD) and covid-19 have poor outcome as consequence of inadequate humoral and cellular immunity due to the hematological disease itself but also due to the administered chemotherapy which further increases the risk of complications and mortality. Aims: The aim of this study is to analyze demographic and clinical characteristics, laboratory parameters, the presence of comorbidities, laboratory parameters, disease status, as well as outcome of the patients with COVID-19 and lymphoproliferative disease and compare them with characteristics of covid-19 infection in patients from general population (GP). Methods: This is a prospective multicenter observational study conducted in the following 3 University centers in period from 15 March 2020 to 31 October 2021. The study included hospitalized patients diagnosed with COVID-19 infection: 161 patients with LPD and 162 patients from the GP. Statistical analysis included demographic statistics, the χ2 test, the Mann-Whitney test, Kaplan-Meier method for analysis of survival and multivariate logistic regression model for analysis of risk factors for mortality. Results: In the LPD group, there were 54 patients (33.54%) with chronic lymphocytic leukemia (CLL), 72 patients (44.72%) with Non-Hodgkin lymphoma/Hodgkin lymphoma (NHL/HL) and 35 patients (21.74%) with multiple myeloma (MM). Ninety-six (59,63%) patients were on active treatment and 14(8.7%) patients were newly diagnosed. The LPD and GP group differed significantly in relation to age (66 vs. 54 years), gender (male: 60.2% vs. 75.3%), presence of comorbidities (109, 67.7% vs. 81, 50%) patients, covid score (mild 22.5% vs. 1.9%, moderate 80, 50.3% vs. 121, 74.7%), and severe/critical 44(27.1%) vs. 38(23.4%) patients. Group of patients with LPD had also significantly lower level of hemoglobin, lowest value of lymphocytes, platelets, higher level of CRP, ferritin, D-dimer (on admission and maximal values) and LDH with respect to group of patients from GP. Mortality rate was higher in LPD group of patients than in GP group (45, 28% vs. 26, 16%) patients. Among the LPD group, the highest mortality rate was observed in patients with MM (16, 45.71%) patients, followed by CLL (15, 27.9%) patients and NHL/HL group (14, 19.4%) patients. Independent factors related to survival are high value of D dimer, anemia (hemoglobin <100g/l) and moderate/critical COVID score in LPD group, while maximal value of CRP, anemia, leucocytosis and age (>60 years) in GP group. Summary/Conclusion: Our study showed significant difference in the characteristics and outcome in covid-19 between patients with LPD and patients from GP. Patients with LPD are older, they have significantly higher inflammatory parameters and more frequent presence of comorbidities compared to patients from GP. Independent factors related to survival in the LPD group are high values of D dimer, moderate/critical COVID score and anemia, while maximal values of CRP, anemia and older age are identified in the GP group. P659: IBRUTINIB IN OVER-EIGHTIES PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: A MULTICENTER ITALIAN COHORT G. Reda1, V. Mattiello1,*, A. M. Frustaci2, A. Visentin3, F. R. Mauro4, I. Innocenti5, M. Gentile6, D. Giannarelli7, A. Noto1, R. Cassin1, L. Laurenti5, A. Tedeschi2 1Hematology Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 2Department of Hematology, Niguarda Cancer Center, ASST Grande Ospedale Metropolitano Niguarda, Milano; 3Hematology and Clinical Immunology unit, Department of Medicine University of Padua, Padova; 4Hematology, Department of Translational and Precision Medicine, ‘Sapienza’ University, Rome; 5Dipartimento Scienze Radiologiche ed Ematologiche, Divisione di Ematologia Fondazione Policlinico universitario A Gemelli, Roma; 6Azienda Ospedaliera of Cosenza, Cosenza; 7Biostatistic Unit, Scientific Directorate, Fondazione Policlinico Universitario A. Gemelli, IRCCS, Roma, Italy Background: Ibrutinib is a first-in-class covalent inhibitor of Bruton’s tyrosine kinase that has changed the treatment paradigm of chronic lymphocytic leukemia (CLL) in both treatment-naive (TN) and relapsed/refractory (R/R) setting. Clinical trials, in selected populations showed that the BTK inhibitor is manageble even in the elderly. Nevertheless, few studies are focused on the role of ibrutinib in unselected patients aged ≥80 years. Aims: We aimed to assess the activity and safety of ibrutinib in a cohort of patients with CLL aged ≥80 years at therapy start. The primary endpoint was safety evaluation. Key secondary endpoints were overall (ORR), complete (CR) and partial (PR) response rates according to iwCLL 2018, median progression free survival (PFS) and overall survival (OS). Methods: Sixty consecutive patients (age ≥80 years) diagnosed with TN (20 patients) or R/R CLL (40 patients) who started ibrutinib were enrolled in this multicenter study from six Italian sites; data were retrospectively analyzed. Pre-existing cardiovascular risk factors were present in 66.7% of patients; 23.3% had experienced a cardiovascular event prior ibrutinib initiation. (Table 1) Results: After a median follow up of 27 months, at least one adverse event (AE) occurred in 68.3% patients, leading to treatment discontinuation in 23.3%. The most common grade ≥3 events were infections (23%) and neutropenia (6%). Cardiovascular events occurred in 31.6% of patients, with an incidence of atrial fibrillation (AF) and arterial hypertension both increasing over time and reaching 16% at 24 months. Although no significant increase in incidence of cardiovascular events was noted among patients with concomitant cardiovascular risk factors or previous events, baseline higher left atrial diameter at echocardiography was predictive of AF occurrence. OS did not differ between patients experiencing AF or not. During treatment, two patients experienced sudden cardiac death. Bleeding was the most frequent AE, occurring in 36.6% of patients, with a median time to event of 24 months and predominantly in patients assuming concomitant antiplatelet or anticoagulant drugs. A total of 23 infective events was registered, mostly in the first 12 months, leading to drug permanent discontinuation in 5 patients. Of note, the infectious rate was higher in del(17p) CLL (p=0.05). The obtained ORR was 88.3%, with 21.7% of patients achieving CR and 66.7% PR; median PFS was 51.8 months (95% CI: 47.4-56.2). No significant difference in PFS was observed comparing TN to R/R patients (p=0.83), or IGHV mutated and unmutated patients (p=0.45). Furthermore, no difference emerged comparing PFS data of patients with TP53 dysfunction (del17 and/or TP53 mutation) to patients without TP53 dysfunction (p=0.13). Patients achieving a response during ibrutinib experienced a prolonged PFS compared to patients achieving SD (p<0.0001). Drug withholding for more than 7 days due to ibrutinib-related toxicities affected patients’ outcome, translating in a shorter PFS with a trend to statistical significance (p=0.07). Median overall survival was 53.2 months (95% CI: 43.3-63.0). Image: Summary/Conclusion: A high proportion of patients had an overall response to ibrutinib and the risk-benefit profile was favourable, providing further evidence for use of ibrutinib in this subset of elderly and unselected patients. Safety profile remains consistent with literature data with no emergent adverse events, thus making ibrutinib an attractive therapeutic possibility even in patients with advanced age and multiple comorbidities. P660: SEROLOGIC RESPONSE TO THE SECOND AND THIRD DOSE OF THE SARS-COV-2 VACCINE IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: RESULTS OF A PROSPECTIVE, CENTRALIZED, MULTICENTER STUDY. F. R. Mauro1,*, D. Giannarelli2, C. Galluzzo3, A. Visentin4, A. M. Frustaci5, P. Sportoletti6, C. Vitale7, G. Reda8, M. Gentile9, L. Levato10, R. Murru11, D. Armiento12, C. Ielo13, R. Maglione13, E. Crisanti13, A. Cipiciani14, V. Mattiello15, V. Gianfelici10, L. Barabino16, R. Amici17, M. Coscia18, A. Tedeschi19, L. Trentin20, S. Baroncelli17 1Department of Translational and Precision Medicine-Sapienza University of Rome, Italy, Sapienza University; 2Design and Analysis of Clinical Trials Unit, Scientific Directorate, Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Italy; 3National Center for Global Health, Istituto Superiore di Sanità, Rome; 4Hematology and Clinical Immunology Unit, Department of Medicine University of Padua, Padova, University of Padua, Padova; 5Hematology, ASST Grande Ospedale Metropolitano Niguarda, Milan; 6Institute of Hematology-Centro di Ricerca Emato-Oncologica (CREO), University of Perugia, Perugia; 7Department of Molecular Biotechnology and Health Sciences, University of Torino and Division of Hematology, A.O.U. Città della Salute e della Scienza di Torino, Rome; 8Hematology Unit, Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore, University of Milan, Milan, Italy., Milan; 9Hematology and Oncology Department, Biotechnology Research Unit, Cosenza, Italy., Cosenza; 10Department Hematology-Oncology, Azienda Ospedaliera Pugliese-Ciaccio, Catanzaro; 11Hematology and Stem Cell Transplantation Unit, Ospedale Oncologico A. Businco, ARNAS “G. Brotzu”, Cagliari; 12Hematology, Stem Cell Transplantation Unit, University Campus Bio-Medico; 13Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome; 14Institute of Hematology-Centro di Ricerca Emato-Oncologica (CREO), University of Perugia, Perugia; 15Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore, University of Milan, Milan; 16Hematology and Stem Cell Transplantation Unit, Ospedale Oncologico A. Businco, ARNAS “G. Brotzu”, Cagliari; 17National Center for Global Health, Istituto Superiore di Sanità, Rome; 18Department of Molecular Biotechnology and Health Sciences, University of Torino and Division of Hematology, A.O.U. Città della Salute e della Scienza di Torino, Torino; 19ASST Grande Ospedale Metropolitano Niguarda, Milan; [e-mail], Hematology and Clinical Immunology Unit, Department of Medicine, University of Padua, Padova, Italy Background: Patients with chronic lymphocytic leukemia (CLL) show high infection-related morbidity and mortality due to variable degree of humoral and cellular immune deficiency. High Covid-related mortality and reduced response to the SARS-Cov-2 vaccine have been reported in this patient population. Aims: We carried out a prospective multicenter study to define the rate of CLL patients with an appropriate immune response after the mRNA SARS-CoV2 vaccine (Pfizer-BioNTech; Moderna). Methods: Two-hundred patients with CLL received the first dose of the SARS-CoV-2 vaccine between February and August 2021. Centralized assessment of the anti-SARS-Cov-2 IgG levels (Sero Index, Kantaro Quantitative SARS-CoV-2 IgG Antibody, RUO-R&D System) was performed at the Istituto Superiore di Sanità of Rome, Italy. The median follow-up of this study is 10.7 months (range 1-12.9). Results: The median age of patients was 70 years, the median IgG level was 635 mg/dl, 61% of patients were IGHV unmutated, and 34% showed TP53 disruption. The majority of patients, 83.5%, were previously treated. Prior treatment included chemoimmunotherapy in 20 (10%) patients, ibrutinib–based therapy in 72 (36%; front-line, 21%; advanced line, 15%), venetoclax-based therapy in 75 (37.5%; front-line, 13.5%; advanced line, 24%). Overall, 135 (77.5%) patients had been previously treated with rituximab, 33 (16.5%) of them within 12 months before vaccination. We assessed the serologic response after the second dose of the SARS-CoV2 vaccine in 195 patients while five were excluded from the analysis (positive test before vaccination, 3 patients; lost to the follow-up, 1; Richter syndrome, 1). Adequate levels of anti-SARS-Cov-2 IgG were detected in 76/195 (39%) patients. Age (<70 vs.≥ 70 years; p <0.0001), CIRS value (<6 vs. ≥6; p=0.005), beta-2 microglobulin (<3.5 vs. ≥ 3.5mg/dl; p=0.04), IgG levels (<550 vs. ≤ 550 mg/dl; p <0.0001), prior treatment (p=0.0001), number of prior treatments (0 + 1 vs. ≥ 2; p=0.002) and the time between prior rituximab and vaccination (>12 vs. ≤12 month; p=0.001) showed a significant impact on the humoral response. In multivariate analysis only age (OR: 0.92 [95% CI: 0.92-0.97] p=0.0001), IgG levels (OR: 0.28 [95% CI: 0.13-0.58] p<0.001), and the time between prior rituximab and vaccination (OR: 0.10 [95% CI: 0.03-0.37] p=0.001), revealed a significant and independent impact on response. When the analysis was restricted to patients who received targeted therapy, in addition to the younger age (OR: 0.96 [95% CI: 0.92-0.99] p=0.04), higher IgG levels at baseline (OR: 0.31 [95% CI: 0.12-0.79] p=0.014), longer time between the start of ibrutinib or venetoclax-based therapy and vaccination (<18 vs.≥18 months; OR: 0.17 [95% CI: 0.06-0.44], p <0.0001) showed a favorable and independent impact on response. Ninety-three% (182/195) of patients received a third dose of the vaccine. A significant increase in the rate of serologic responses, 51.5% (85/165 evaluated patients, p=0.019), was observed after the booster dose. Moreover, a response was detected in 25% (26/103 evaluated patients) of previously seronegative patients. Summary/Conclusion: In this prospective, multicenter, centralized study, we recorded an effective immune response to the SARS-CoV-2 vaccine in about a third of patients with CLL. Younger age, higher IgG levels, no prior treatment, or stable disease after targeted therapy that suggest preserved immunocompetence were associated with a greater likelihood of achieving an effective immune response. A booster dose of the SARS-CoV-2 vaccine proved beneficial also in previously seronegative patients. P661: POPULATION-WIDE PATTERNS OF CARE IN CHRONIC LYMPHOCYTIC LEUKEMIA IN AUSTRALIA: AN ANALYSIS OF THE PHARMACEUTICAL BENEFITS SCHEME DATASET C. Tam1,*, F.-L. Zhao2, R. Gauba2, K. Yang3, S. Azmi3, S. C. Li4 1Peter MacCallum Cancer Centre, St. Vincent’s Hospital, and University of Melbourne, Melbourne; 2BeiGene AUS PTY Ltd., Sydney, Australia; 3BeiGene USA, Inc., San Mateo, United States of America; 4University of Newcastle, Callaghan, Australia Background: The treatment landscape in patients with chronic lymphocytic leukemia (CLL) is changing with the approvals of Bruton’s tyrosine kinase inhibitors (BTKis) in Australia. Aims: We sought to understand the practice impact of the introduction of publicly funded novel agents for the treatment of CLL. The objective of this study was to describe the evolving treatment patterns of Australian patients with CLL over the last 10 years using population-wide prescription records. Methods: Patients who initiated a treatment for CLL between 01/01/2011 and 07/31/2021 were extracted from the Services Australia 10% Pharmaceuticals Benefits Scheme (PBS) dataset. This dataset includes the dispensing records for 10% of the Australian population and captures all publicly funded treatments in Australia. The index date was defined as the commencement of any drug for the treatment of CLL. First-line (1L) therapy was defined as the first treatments prescribed for CLL. A patient was defined as relapsed/refractory (R/R) if they had commenced a drug which was in a different therapeutic category, or if they re-started the same regimen after a gap of more than 180 days. Descriptive analyses were conducted to examine the use of treatment regimens for the overall 10-year population by line of therapy. Analyses by calendar year were also performed to assess changes in treatment patterns. Results: Overall, 803 patients with CLL were identified. The majority of patients were male (65%) and age > 60 years (77%), with most being aged 70-79 years (33% of total). Many patients were receiving comedications at baseline, including antihypertensives (47%), antipsychotics or antidepressants (17%), and/or anticoagulants (13%). In the overall population (2011-2021), the majority of patients had received 1L treatment with fludarabine-cyclophosphamide-rituximab (FCR, 49%), chlorambucil ± CD20 (27%), or CD20 monotherapy (17%). The most commonly used regimens in R/R patients at any subsequent episode of treatment included CD20 monotherapy (56%), BTKi (41%) or FCR (33%). A trend in adoption of novel agents was observed throughout the years following their PBS listing. Analysis by calendar year showed that from 2011 to 2020 use of FCR in 1L decreased from 78% to 10%; and use of BTKis in R/R increased from 0% to 62%. Summary/Conclusion: CLL treatment patterns have significantly changed in Australia since the introduction of the BTKis (e.g., ibrutinib, acalabrutinib). The use of FCR in 1L CLL has decreased and use of BTKis in R/R patients has increased. P662: PATIENT-REPORTED OUTCOMES FROM A PHASE 3 RANDOMIZED STUDY OF ZANUBRUTINIB VERSUS BENDAMUSTINE PLUS RITUXIMAB (BR) IN PATIENTS WITH TREATMENT-NAÏVE (TN) CLL/SLL P. Ghia1,*, G. Barnes2, K. Yang2, C. Tam3,4,5,6, P. Hillmen7, T. Robak8, J. Brown9, B. Kahl10, T. Tian2, A. Szeto2, J. Paik2, M. Shadman11,12 1Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milano, Italy; 2BeiGene USA, Inc., San Mateo, United States of America; 3Peter MacCallum Cancer Centre, Melbourne; 4University of Melbourne, Parkville; 5St Vincent’s Hospital Melbourne, Fitzroy; 6Royal Melbourne Hospital, Parkville, Australia; 7St James’s University Hospital, Leeds, United Kingdom; 8Medical University of Lodz, Lodz, Poland; 9Dana-Farber Cancer Institute, Boston; 10Washington University School of Medicine, St. Louis; 11Fred Hutchinson Cancer Research Center; 12Medicine, University of Washington, Seattle, United States of America Background: Zanubrutinib is an oral, highly selective, next-generation Bruton tyrosine kinase (BTK) inhibitor. In cohort 1 of the phase 3 SEQUOIA trial (BGB-3111-304; NCT03336333), efficacy and safety of zanubrutinib and BR were compared in adult patients (pts) with TN chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) without del(17p). Aims: Here, we report the health-related quality of life (HRQoL) outcomes from an interim analysis of the SEQUOIA trial. Methods: Patient-reported outcomes (PROs) were secondary endpoints and assessed using the EORTC QLQ-C30 and EQ-5D-5L VAS. Patients completed these questionnaires at baseline, every 12 weeks for 96 weeks, and then every 24 weeks until disease progression, death, or withdrawal from study. The PRO endpoints included global health status (GHS), physical and role functions, and symptoms of fatigue, pain, diarrhea, and nausea/vomiting, measured by QLQ-C30, with critical clinical cycles of Weeks 12 and 24. Descriptive analyses were performed on all questionnaire responses, and a mixed model for repeated measures was performed on the PRO endpoints at Weeks 12 and 24. Results: Baseline demographics and disease characteristics between the zanubrutinib (n=241) and BR (n=238) arms were similar. Across all pts, adjusted completion rates for PROs (# of pts who completed the questionnaire at each cycle divided by # of pts expected to complete the questionnaires) were high (~80%) at Weeks 12 and 24. Compared with pts who received BR, pts treated with zanubrutinib experienced greater improvements in HRQoL at Weeks 12 and 24 as reported on the QLQ-C30 (Table). By Week 24, significant improvements were observed with zanubrutinib vs BR in GHS, physical functioning, role functioning as well as greater reductions in diarrhea, fatigue, and nausea/vomiting. Per EQ-5D-5L VAS, comparable improvements from baseline between zanubrutinib and BR in the health status were observed at Weeks 12 (4.3 vs 3.5) and 24 (4.5 vs 4.9), respectively. Image: Summary/Conclusion: In the SEQUOIA trial, zanubrutinib was associated with significant improvements in HRQoL in pts with TN CLL/SLL without del(17p), as indicated by the PRO endpoints of the GHS, physical and role functions, and greater reductions in symptoms of fatigue, diarrhea and nausea/vomiting compared with BR. P663: HEALTH-RELATED QUALITY OF LIFE OUTCOMES ASSOCIATED WITH ZANUBRUTINIB VS IBRUTINIB MONOTHERAPY IN PATIENTS WITH RELAPSED/REFRACTORY (RR) CLL/SLL: RESULTS FROM THE RANDOMIZED PHASE 3 ALPINE TRIAL P. Hillmen1,*, J. Brown2, N. Lamanna3, S. O’Brien4, C. Tam5,6,7,8, L. Qiu9, K. Yang10, G. Barnes10, K. Wu10, T. Salmi11, B. Eichhorst12 1St James’s University Hospital, Leeds, United Kingdom; 2Medical Oncology, Dana-Farber Cancer Institute, Boston; 3Herbert Irving Comprehensive Cancer Center, Columbia University, New York; 4Chao Family Comprehensive Cancer Center, University of California, Irvine, United States of America; 5Peter MacCallum Cancer Centre, Melbourne; 6University of Melbourne, Parkville; 7St Vincent’s Hospital Melbourne, Fitzroy; 8Royal Melbourne Hospital, Parkville, Australia; 9Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China; 10BeiGene USA, Inc., San Mateo, United States of America; 11BeiGene Switzerland GmbH, Basel, Switzerland; 12Department of Internal Medicine, University of Cologne, Center for Integrated Oncology Aachen, Cologne, Germany Background: In the phase 3 ALPINE trial (BGB-3111-305; NCT03734016), efficacy and safety of zanubrutinib, a highly selective, next-generation Bruton tyrosine kinase inhibitor (BTKi), were compared with the first-generation BTKi, ibrutinib, in adult patients with RR chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Aims: This abstract, based on the interim analysis of the ALPINE trial, describes the effects of zanubrutinib monotherapy and ibrutinib monotherapy on the health-related quality of life (HRQoL). Methods: Health-related quality of life was examined by patient-reported outcomes (PROs) measures assessed by EORTC QLQ-C30 and EQ-5D-5L at baseline, Cycle 1, and then every 3rd cycle until end of treatment. Key PRO endpoints included global health status (GHS), physical and role functions, and fatigue, pain, diarrhea, and nausea/vomiting. Descriptive analysis on all the scales was conducted as was a mixed model repeated-measure (MMRM) analysis of the longitudinal QLQ-C30 data. Data presented are from key cycles (7 and 13), corresponding to 6 and 12 months of treatment, respectively. Results: In the intent-to-treat population (N=652; zanubrutinib, n=327; ibrutinib, n=325), adjusted completion rates were high (>85%) in both arms at Cycles 7 and 13. On the QLQ-C30, estimated mean treatment differences and 95% CI in key PRO endpoints demonstrated treatment differences, in favor of zanubrutinib, in GHS, physical functioning, and fatigue in Cycle 7, and diarrhea in Cycle 13 (Table). Mean change from baseline (SD) in EQ-5D-5L VAS showed consistently more improvement with zanubrutinib compared with ibrutinib at both Cycle 7 (8.4 [18.2] vs 4.0 [16.6]) and Cycle 13 (6.8 [18.8] vs 5.2 [17.5]). Image: Summary/Conclusion: In the ALPINE trial, patients with RR CLL/SLL who received zanubrutinib monotherapy reported improvements in key HRQoL endpoints compared with patients who received ibrutinib monotherapy. P664: GRADING QUALITY OF EVIDENCE AND STRENGTH OF RECOMMENDATIONS FOR THE TREATMENT OF RELAPSED/REFRACTORY CLL. S. Molica1,*, C. Patti2, P. Sportoletti3, A. Chiarenza4, A. M. Giordano5, F. Chiurazzi6, N. Di Renzo7, P. Musto8, F. Pane9, F. Di Raimondo4, L. Trentin10, F. R. Mauro11, D. Giannarelli12 1Department Hematology, Hull University Teaching Hospitals NHS Trust, Hull, UK, Hall, United Kingdom; 2hematology, Ospedale Cervello, Palermo; 3Hematology Unit, University Perugia, Perugia; 4Hematology Unit, Ferrarotto Hospital, Catania; 55Department of Emergency and Organ Transplantation (D.E.T.O.), Hematology Section, University of Bari, Bari; 6Department of Clinical Medicine and Surgery, Hematology Unit, Federco II Medical School Napoli, Napoli; 7Hematology Unit, Presidio Ospedaliero Vito Fazi, Lecce; 8Department of Emergency and Organ Transplantation (D.E.T.O.), Hematology Section, University of Bari, Bari; 9Department of Clinical Medicine and Surgery, Hematology Unit, Federico II Medical School Napoli, Napoli; 10Hematology and Clinical Immunology Unit, Department of Medicine, University of Padua, Padua; 11Hematology, Department of Translational and Precision Medicine, Sapienza University Rome; 12Biostatistic Unit, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Rome, Italy Background: Changes in the treatment landscape of CLL have complicated the decision-making processes, especially in the context of relapsed/refractory (R/R) disease. In this setting current guidelines do not provide clear information on the sequencing of targeted agents (TAs). Aims: With this in mind a series of evidence-based recommendations were produced to support pts, clinicians, and other health care professionals in their decisions about the management of R/R CLL. Methods: A multidisciplinary panel that included specialists in methodology, CLL management, and hematology, performed a systematic review of the literature using the databases MEDLINE, EMBASE, and the Cochrane Library, then participated in a virtual consensus conference held at the end of December 2021. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used to formulate recommendations. Specifically, the strength of recommendations was determined by the balance between desirable and undesirable consequences of alternative management strategies, quality of evidence, variability in values, preferences, and resource used. Results: Firstly, the panelists prioritized 10 scientific questions in the context of 4 specific scenarios of R/R CLL: (1) relapse after frontline chemo-immunotherapy (CIT), (2) progression of disease (PD) while receiving ibrutinib (IBR), (3) progression after treatment with venetoclax (VEN), and (4) impact of adherence to IBR or VEN on the clinical outcome. The following recommendations were produced: 1- A TA should be offered to all pts relapsing after first-line CIT (strong recommendation based on high-quality evidence). In absence of head-to-head trials comparing a BTKi vs an anti-BCL2 inhibitor, the panel suggests a patient-oriented choice consisting of either continuous BTKi (i.e., IBR or acalabrutinib) or time-limited (TL) VEN-based therapy in both standard or high genomic risk (conditional recommendation based on moderate-quality evidence). Remarks: TL VEN-based regimen is more cost-effective than continuous IBR. 2- The panel recommends switching to VEN for those pts who experience PD whilst on a BTKi (in absence of a valid alternative the recommendation becomes strong even if the quality evidence is moderate). There is no evidence to recommend the combination of VEN plus rituximab or an alternative covalent BTKi over VEN single agent (conditional recommendation based on low certainty in the evidence of effects). Noncovalent BTKi pirtobrutinib needs validation in phase 3 studies. 3- The panel recommends utilizing a BTKi monotherapy instead of re-challenging VEN for pts who have progressed after completing VEN-based therapy (conditional recommendation based on low certainty in the evidence of effects). 4- Finally, panelists agreed that the impact of adherence to IBR or VEN on PFS in R/R pts is supported by a low quality of evidence (conditional recommendation based on low certainty in the evidence of effects). Summary/Conclusion: These evidence-based consensus recommendations strongly support the use of a TA in R/R CLL patients. The choice between TL and continuous TA is patient-oriented with the remark that a TL approach is cost-effective within current pricing structures. A cross-over approach to an alternative TA is suggested in pts progressing on BTKi or BCL2i. However, these recommendations are guided by the fundamental principle that optimal patient care involves ongoing discussions between clinicians and pts, continuously addressing goals of care and the relative risk-benefit balance of treatment. P665: CARDIAC ADVERSE EVENTS AND SECOND PRIMARY MALIGNANCIES WITH ACALABRUTINIB IN CHRONIC LYMPHOCYTIC LEUKEMIA -A SYSTEMATIC REVIEW AND META-ANALYSIS OF RANDOMIZED CONTROLLED TRIALS K. Parmar1,*, D. Pawar1, K. Bharathidasan1, G. DelRioPertuz1, L. Tijani1 1Texas Tech University Health Sciences Center, Lubbock, United States of America Background: Cardiac adverse events were the most common cause of treatment discontinuation in patients with chronic lymphocytic leukemia (CLL) on ibrutinib. Acalabrutinib is a new, selective, irreversible Bruton’s tyrosine kinase inhibitor (BTKi) that is approved for the treatment of CLL. Patients with CLL are inherently immunodeficient and are at increased risk of second primary malignancies (SPM). Additionally, BTKi have been studied in association with risk for secondary malignancies. The risk of cardiac adverse events and SPM with acalabrutinib in patients with CLL. Aims: We conducted this systematic review and meta-analysis to estimate risk of cardiac adverse events and SPM with acalabrutinib in patients with CLL. Methods: A comprehensive literature search was performed in PubMed, EMBASE, Clinical trials.gov and meeting abstracts till January 31,2021. All randomized controlled trials comparing acalabrutinib-based regimens with a control group in patients with CLL, with cardiac adverse events and SPM reported as adverse effects were included. Pooled risk ratios (RR) with 95% confidence intervals (CI) were calculated using the Mantel-Haenszel method of the fixed-effect model. Heterogeneity of effect size was quantified using the I2 statistic. Statistical analysis was performed using the Review Manager, version 5.3. Results: Three phase III RCT with total of 1362 patients were included for our analysis (Figure). Byrd et al., compared acalabrutinib with ibrutinib. The ASCEND trial compared acalabrutinib with investigator’s choice chemotherapy, either idelalisib plus rituximab or bendamustine plus rituximab. In the ELEVATE -TN study, the comparator arm included the combination of chlorambucil and Obinutuzumab. Acalabrutinib was used at a dose of 100 mg twice daily in all 3 studies. All but ELEVATE -TN study included patients previously treated with CLL. Atrial fibrillation was the most common cardiac adverse event across all trials. Non melanoma skin cancer was the most common SPM across all trials. There was no increase in any grade cardiac adverse events with acalabrutinib when compared to control arm (pooled RR 1.06; 95%CI 0.84-1.34). There was no increased risk of atrial fibrillation (pooled RR 0.85; 95%CI 0.57-1.26), ventricular arrythmias and cardiac arrest (Figure). There was significant increase in hypertension (HTN) in the patients on the acalabrutinib containing regimens (pooled RR 1.78; 95%CI 1.24-2.56; P= 0.002); however, there was no increase in grade 3+ HTN. There was a decrease in the SPM with acalabrutinib as compared to the control group (pooled RR 0.67; 95%CI 0.46-0.96; P= 0.03) (Figure). There was also significant decrease in non-melanoma skin cancer in the acalabrutinib as compared to the control group (pooled RR 0.59; 95%CI 0.37-0.93; P 0.02). There was no significant difference in the genitourinary cancer, lung cancer and hematologic cancer. Image: Summary/Conclusion: There is no increase in risk of atrial fibrillation with the acalabrutinib based regimens as compared to the non-acalabrutinib – based therapies; however, there was a significantly increased risk of HTN. There is lower risk of SPM and non-melanoma SPM in the acalabrutinib based regimens than the control arm. P666: ACALABRUTINIB ± OBINUTUZUMAB VS OBINUTUZUMAB + CHLORAMBUCIL IN TREATMENT-NAIVE CHRONIC LYMPHOCYTIC LEUKEMIA: 5-YEAR FOLLOW-UP OF ELEVATE-TN J. P. Sharman1,*, M. Egyed2, W. Jurczak3, A. Skarbnik4, K. Patel5, I. W. Flinn6, M. Kamdar7, T. Munir8, R. Walewska9, L. M. Fogliatto10, Y. Herishanu11, V. Banerji12, G. Follows13, P. Walker14, K. Karlsson15, P. Ghia16, A. Janssens17, F. Cymbalista18, E. Ferrant19, W. G. Wierda20, V. Munugalavadla21, T. Yu21, M. H. Wang21, J. A. Woyach22 1Willamette Valley Cancer Institute and Research Center, Eugene, OR, United States of America; 2Somogy County Mór Kaposi General Hospital, Kaposvár, Hungary; 3Maria Skłodowska-Curie National Research Institute of Oncology, Krakow, Poland; 4Novant Health Cancer Institute, Charlotte, NC; 5Swedish Cancer Institute, Center for Blood Disorders and Stem Cell Transplantation, Seattle, WA; 6Sarah Cannon Research Institute and Tennessee Oncology, Nashville, Tennessee; 7University of Colorado Cancer Center, Aurora, CO, United States of America; 8Haematology, Haematological Malignancy Diagnostic Service (HMDS), St. James’s Institute of Oncology, Leeds; 9Cancer Care, University Hospitals Dorset, Bournemouth, United Kingdom; 10Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil; 11Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 12Departments of Internal Medicine, Biochemistry & Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba and CancerCare Manitoba, Winnipeg, Canada; 13Department of Haematology, Addenbrooke’s Hospital NHS Trust, Cambridge, United Kingdom; 14Peninsula Health and Peninsula Private Hospital, Frankston, Melbourne, Australia; 15Skåne University Hospital, Lund, Sweden; 16Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milano, Italy; 17University Hospitals Leuven, Leuven, Belgium; 18Bobigny: Hématologie biologique, Hopital Avicenne, Université Sorbonne Paris Nord, Bobigny; 19Hospices Civils de Lyon, Centre Hospitalier Lyon Sud, Service d’Hématologie Clinique, Pierre-Bénite, France; 20Department of Leukemia, Division of Cancer Medicine, MD Anderson Cancer Center, Houston, Texas; 21AstraZeneca, South San Francisco, CA; 22The Ohio State University Comprehensive Cancer Center, Columbus, OH, United States of America Background: For ELEVATE-TN (NCT02475681), we previously reported superior efficacy of acalabrutinib (A) ± obinutuzumab (O) vs O + chlorambucil (Clb) in patients with treatment-naive (TN) chronic lymphocytic leukemia (CLL) at 28.3 and 46.9 months median follow-up. Aims: To report the updated efficacy and safety results from the ELEVATE-TN study after a median follow-up of 5 years. Methods: Patients were randomized to A+O, A, or O+Clb after informed consent was obtained. Patients who progressed on O+Clb could cross over to A monotherapy. Investigator-assessed (INV) progression-free survival (PFS), INV overall response rate (ORR), overall survival (OS), and safety were evaluated. Results: A total of 535 patients (A+O, n=179; A, n=179; O+Clb, n=177) had a median age of 70 years; 63% had unmutated IGHV and 9% had del(17p). At a median follow-up of 58.2 months (range, 0.0–72.0; data cutoff Oct 1, 2021), median PFS was not reached (NR) (hazard ratio [HR]: 0.11) for A+O and A (HR: 0.21) vs 27.8 months for O+Clb (both P<0.0001). The estimated 60-month PFS rates were 84% (A+O), 72% (A), and 21% (O+Clb). In patients with unmutated IGHV, the median PFS was NR for A+O and A vs 22.2 months among patients in the O+Clb arm (HR: 0.06 [A+O] and 0.12 [A]; both P<0.0001); estimated 60-month PFS rates were 82% (A+O), 72% (A), and 6% (O+Clb). In patients with del(17p), the median PFS was NR and 64.1 months for A+O and A vs 17.7 months for O+Clb (HR: 0.21, P=0.0031 [A+O]; HR: 0.29, P=0.0130 [A]); estimated 60-month PFS rates were 75% (A+O), 71% (A), and 27% (O+Clb). Median OS was NR in any treatment arm, and significantly longer in the A+O vs O+Clb arms (HR: 0.55; P=0.0474); estimated 60-month OS rates were 90% (A+O), 84% (A), and 82% (O+Clb). ORR was significantly higher with A+O (96%; 95% CI 92–98) and A (90%; 85–94) vs O+Clb (83%; 77–88; P<0.0001 [A+O], P=0.0499 [A]). Complete response (CR)/CR with incomplete hematologic recovery (CRi) rates were higher with A+O (29%/3%) vs O+Clb (13%/1%); 13%/1% had CR/CRi with A; CR increased since the interim analysis (previously 21% [A+O] and 7% [A]). Adverse events (AEs) and treatment exposure are shown in the Table. Treatment is ongoing in 65% (A+O) and 60% (A) of patients; the most common reasons for treatment discontinuation were AEs (17% [A+O], 16% [A], 14% [O+Clb]) and progressive disease (6%, 10%, 2%, respectively). Crossover from O+Clb to A occurred in 72 (41%) patients; 25% of these patients discontinued A (10% due to AEs and 11% due to progressive disease). Image: Summary/Conclusion: After 5 years of follow-up, efficacy and safety of A+O and A monotherapy were maintained, with significantly longer OS in the A+O arm compared with O+Clb. P667: LONG-TERM EFFICACY OF ACALABRUTINIB-BASED REGIMENS IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA AND HIGHER-RISK GENOMIC FEATURES: POOLED ANALYSIS OF CLINICAL TRIAL DATA M. Davids1,*, J. Sharman2, P. Ghia3, J. Woyach4, W. Jurczak5, T. Siddiqi6, P. Miranda7, M. Shahkarami8, T. Yu8, U. Emeribe7, J. Byrd9 1Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts; 2Willamette Valley Cancer Institute and Research Center, Eugene, Oregon, United States of America; 3Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milano, Italy; 4The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, United States of America; 5Maria Skłodowska-Curie National Research Institute of Oncology, Krakow, Poland; 6City of Hope Comprehensive Cancer Center, Duarte, California; 7AstraZeneca, Gaithersburg, Maryland; 8AstraZeneca, South San Francisco, California; 9Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America Background: Chronic lymphocytic leukemia (CLL) has a highly variable disease course; patients (pts) with higher-risk genomic features typically have poor response to chemoimmunotherapy, and pts with del(17p) also have short, suboptimal response to venetoclax + obinutuzumab. Thus, novel agents such as Bruton tyrosine kinase inhibitors (BTKi), including the highly selective covalent BTKi acalabrutinib (A), are preferred treatments in this higher-risk population based on demonstrated effectiveness in higher-risk subgroups in individual studies. Aims: To assess long-term efficacy of A-based regimens in pts with treatment-naive (TN) or relapsed/refractory (R/R) CLL and higher-risk genomic features. Methods: Data were pooled from CLL pts with higher-risk genomic features treated with A ± obinutuzumab (O) in 3 clinical studies in TN CLL (ELEVATE-TN, CL-001 [TN cohort], CL-003) and 3 in R/R CLL (ASCEND, ELEVATE-RR, CL-001 [R/R cohort]). Higher risk was defined as del(17p) and/or TP53 mutation [del(17p)/TP53m], unmutated IGHV (uIGHV), or complex karyotype (CK, ≥3 chromosomal abnormalities). Efficacy analyses (progression-free survival [PFS], overall survival [OS], response rates) focus on del(17p)/TP53m and uIGHV; safety analyses also include pts with CK. Results: 801 pts (TN 313; R/R 488, median 2 prior therapy lines [range 1–10]) were included; 64 (20%) TN and 219 (45%) R/R pts had del(17p)/TP53m, among whom 44 (69%) and 170 (78%) also had uIGHV. Overall, 288 (92%) TN and 425 (87%) R/R pts had uIGHV and 47 (15%) and 160 (33%) had CK. Median pt age was 68 (TN) and 66 (R/R) y. For A-based regimens combined (A and A+O), overall response rate (ORR) was 91% (n=58/64; complete response [CR]: 20% [n=13/64]) in TN pts with del(17p)/TP53m and 96% (n=276/288; CR 16% [n=47/288) in TN pts with uIGHV. At 47.3 mo median follow-up (range 1.0–82.0), median PFS was not reached (NR) in TN pts with del(17p)/TP53m with A-based regimens; PFS rates at 48 mo suggest similar efficacy with A and A+O in TN pts with del(17p)/TP53m (76% and 77%, respectively) (Fig 1A). Median PFS was NR among TN pts with uIGHV with both A and A+O; PFS rates at 48 mo with A and A+O were 84% and 86%, respectively (Fig 1B). Median OS was NR with A and A+O in TN pts with del(17p)/TP53m or uIGHV; OS rates at 48 mo were similar for A and A+O in pts with del(17p)/TP53m (89% for both) and in pts with uIGHV (92% and 95%). In the R/R cohort (A monotherapy only), ORR was 86% (n=184/214; CR 5% [n=11/214]) in pts with del(17p)/TP53m and 87% (n=356/408; CR 7% [n=29/408]) in pts with uIGHV. At 44.0 mo median follow-up (range 0–88.0), median PFS was 38.6 mo and 46.9 mo and PFS rates at 36 mo were 54% and 65% in pts with del(17p)/TP53m and uIGHV, respectively (Fig 1C). Median OS was 60.6 mo and NR in pts with del(17p)/TP53m and uIGHV, respectively; OS rates at 36 mo were 73% and 82%. Among all higher-risk pts, incidences of grade ≥3 treatment-related AEs were 30% (TN) and 35% (R/R). Discontinuations due to treatment-related AEs occurred in 4% (TN) and 6% (R/R) of pts; 60% and 27%, respectively, remained on treatment at data cutoff. Image: Summary/Conclusion: In this pooled analysis of clinical trial data in 801 CLL pts with higher-risk genomic features, efficacy of A-based regimens led to high PFS and OS rates at a median follow-up of nearly 4 y. The safety profile in this analysis was similar to the overall safety profile of acalabrutinib. Our results demonstrate the long-term benefit of A-based regimens in CLL pts with higher-risk genomic features, regardless of line of therapy. P668: ACALABRUTINIB VS RITUXIMAB PLUS IDELALISIB OR BENDAMUSTINE IN RELAPSED/REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA: ASCEND RESULTS AT ~4 YEARS OF FOLLOW-UP P. Ghia1,*, A. Pluta2, M. Wach3, D. Lysak4, M. Simkovic5, I. Kriachok6, A. Illes7, J. de la Serna8, S. Dolan9, P. Campbell10, G. Musuraca11, A. Jacob12, E. Avery13, J. H. Lee14, G. Usenko15, M. H. Wang16, T. Yu17, W. Jurczak18 1Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milano, Italy; 2Department of Hematological Oncology, Oncology Specialist Hospital, Brzozow; 3Department of Hemato-Oncology and Bone Marrow Transplantation, Medical University of Lublin, Lublin, Poland; 4Fakultní Nemocnice Plzen, Pilsen; 5University Hospital Hradec Kralove, Charles University, Hradec Kralove, Czechia; 6National Cancer Institute, Kiev, Ukraine; 7University of Debrecen, Faculty of Medicine, Department of Hematology, Debrecen, Hungary; 8Hospital Universitario 12 de Octubre, Madrid, Spain; 9Saint John Regional Hospital, University of New Brunswick, New Brunswick, Canada; 10Barwon Health, University Hospital Geelong, Geelong, Victoria, Australia; 11Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola, Italy; 12The Royal Wolverhampton NHS Trust, Wolverhampton, United Kingdom; 13Nebraska Hematology Oncology, Lincoln, Nebraska, United States of America; 14Gachon University Gil Medical Center, Incheon, South Korea; 15City Clinical Hospital No. 4 DCC, Dnipro, Ukraine; 16AstraZeneca, South San Francisco, California; 17AstraZeneca, South San Francisco, California, United States of America; 18Maria Sklodowska-Curie National Institute of Oncology, Krakow, Poland Background: Acalabrutinib (acala) is a next-generation, highly selective, covalent Bruton tyrosine kinase (BTK) inhibitor approved for patients (pts) with chronic lymphocytic leukemia (CLL). In the primary analysis of ASCEND (median follow-up 16.1 mo), acala showed superior efficacy with an acceptable tolerability profile vs idelalisib (Id) plus rituximab (R) (IdR) or bendamustine (B) plus R (BR) in pts with relapsed/refractory (R/R) CLL (Ghia et al. J Clin Oncol. 2020;38:2849-2861). Aims: We report the results of the ASCEND study at ~4 years of follow-up. Methods: In this multicenter, randomized, open-label, phase 3 study (NCT02970318), pts with R/R CLL received oral (PO) acala 100 mg BID until progression or unacceptable toxicity or investigator’s (INV) choice of IdR (Id: 150 mg PO BID until progression or unacceptable toxicity; R: 375 mg/m2 x1 then 500 mg/m2 IV [8 total infusions]) or BR (B: 70 mg/m2 IV; R: 375 mg/m2 x1 then 500 mg/m2 IV [6 cycles]). Progression-free survival (PFS), overall survival (OS), overall response rate (ORR), and safety were assessed. Results: A total of 310 pts (acala, n=155; IdR, n=119; BR, n=36) were randomized (median age 67 y; del(17p) 15%, unmutated IGHV 74%, Rai stage 3/4 42%). At median follow-up of 46.5 mo (acala) and 45.3 mo (IdR/BR), acala significantly prolonged INV-assessed PFS vs IdR/BR (median not reached [NR] vs 16.8 mo; P<0.0001); 42-mo PFS rates were 62% for acala vs 19% for IdR/BR. In pts with del(17p), median PFS was NR (acala) vs 13.8 mo (IdR/BR; P<0.0001). In pts with unmutated IGHV, median PFS was NR (acala) vs 16.2 mo (IdR/BR; P<0.0001). Median OS was NR in both arms; 42-mo OS rates were 78% (acala) vs 65% (IdR/BR). ORR was 83% (acala) vs 84% (IdR/BR) (ORR + partial response with lymphocytosis: 92% [acala] vs 88% [IdR/BR]). AEs led to drug discontinuation in 23% of acala, 67% of IdR, and 17% of BR pts. Events of clinical interest (acala vs IdR/BR) included all-grade atrial fibrillation/flutter (8% vs 3%), all-grade hypertension (8% vs 5%), all-grade major hemorrhage (3% vs 3%), and grade ≥3 infections (29% vs 29%). Image: Summary/Conclusion: At ~4 years of follow-up, acala maintained efficacy compared with standard-of-care regimens and a consistent tolerability profile in R/R CLL. P669: FIXED-DURATION (FD) IBRUTINIB + VENETOCLAX FOR FIRST-LINE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)/SMALL LYMPHOCYTIC LYMPHOMA (SLL): 3-YEAR FOLLOW-UP FROM THE PHASE 2 CAPTIVATE STUDY FD COHORT C. Moreno1,2,*, W. G. Wierda3, P. M. Barr4, T. Siddiqi5, J. N. Allan6, T. J. Kipps7, L. Trentin8, R. Jacobs9, S. Jackson10, A. Tedeschi11, S. Opat12, R. Bannerji13, B. J. Kuss14, L. J. Croner15,16, E. Szafer-Glusman15,16, C. Zhou16, A. Szoke16, J. P. Dean16, P. Ghia17, C. S. Tam18 1Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona; 2Josep Carreras Leukemia Research Institute, Barcelona, Spain; 3Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX; 4Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY; 5City of Hope National Medical Center, Duarte, CA; 6Weill Cornell Medicine, New York, NY; 7UCSD Moores Cancer Center, La Jolla, CA, United States of America; 8University of Padova, Padova, Italy; 9Levine Cancer Institute, Charlotte, NC, United States of America; 10Middlemore Hospital, Auckland, New Zealand; 11ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 12Monash University, Clayton, VIC, Australia; 13Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United States of America; 14Flinders University and Medical Center, Bedford Park, SA, Australia; 15AbbVie, North Chicago, IL; 16Pharmacyclics LLC, an AbbVie Company, South San Francisco, CA, United States of America; 17Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy; 18Peter MacCallum Cancer Center & St. Vincent’s Hospital and the University of Melbourne, Melbourne, VIC, Australia Background: CAPTIVATE (PCYC-1142) is a multicenter phase 2 study of first-line ibrutinib + venetoclax in CLL. The primary analysis evaluating FD treatment with ibrutinib + venetoclax was previously presented (Ghia et al., ASCO 2021). Aims: To present 3-year follow-up results from the FD cohort of CAPTIVATE. Methods: Patients aged ≤70 years with previously untreated CLL/SLL received 3 cycles of ibrutinib then 12 cycles of ibrutinib + venetoclax (ibrutinib 420 mg/day orally; venetoclax ramp-up to 400 mg/day orally). Responses were investigator assessed per iwCLL 2008 criteria. Undetectable minimal residual disease (uMRD; <10-4) was measured by 8-color flow cytometry. Serious AEs (SAEs) deemed related to ibrutinib reported >30 days after last dose of study drug were collected. Results: 159 patients were enrolled (median age 60 years), including patients with high-risk features of del(17p)/TP53 mutation (17%), unmutated IGHV (uIGHV; 56%), and complex karyotype (19%). 147 (92%) and 149 (94%) patients completed treatment with ibrutinib and venetoclax, respectively. With 1 year of additional follow-up since primary analysis, median time on study was 39 months (range 1-41). ORR was 96% and was consistent (96%-97%) in patients with high-risk features (Table). The primary endpoint of complete response (CR) including CR with incomplete bone marrow recovery (CRi) rate in patients without del(17p) (n=136) increased nominally from 56% (95% CI, 48-64) to 58% (95% CI 50-66); in all patients, CR rate increased from 55% (95% CI 48-63) to 57% (95% CI 50-65). In patients achieving CR, 93% had durable responses lasting ≥12 months post-treatment. Of patients with uMRD in peripheral blood at 3 months post-treatment, 66/85 (78%) evaluable patients maintained uMRD through 12 months post-treatment. At 36 months, PFS was 88% (95% CI 82‒92) and OS was 98% (95% CI 94-99); similar rates were seen in patients with high-risk features (Table). All patients are off treatment; no new SAEs of any kind have occurred since the primary analysis. Available data on relevant mutations in BTK, PLCɣ2, or BCL-2 at time of PD will be presented. As of January 2022, 12 patients were retreated with single-agent ibrutinib after progressive disease (treatment duration range 3-29 months); of evaluated patients, 7/9 had partial responses and 2/9 had stable disease. Image: Summary/Conclusion: Fixed duration ibrutinib + venetoclax continues to provide deep, durable responses and clinically meaningful PFS, including in patients with high-risk disease features, representing an all-oral, once-daily, chemotherapy-free FD regimen for previously untreated patients with CLL/SLL. With an additional 1 year of follow-up, no OS events or SAEs occurred. Manageable safety profile is unchanged as previously reported. To date, successful single-agent ibrutinib retreatment responses are observed. P670: ABSENCE OF BTK, BCL2, AND PLCG2 MUTATIONS IN RELAPSING CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AFTER FIRST-LINE TREATMENT WITH FIXED-DURATION IBRUTINIB (I) PLUS VENETOCLAX (V) L. J. Croner1,2,*, J. N. Allan3, N. Jain4, J. F. Seymour5, K. Eckert2, L. W. K. Cheung1,2, E. Szafer-Glusman1,2 1AbbVie, North Chicago, IL; 2Pharmacyclics LLC, an AbbVie Company, South San Francisco, CA; 3Weill Cornell Medicine, New York, NY; 4MD Anderson Cancer Center, Houston, TX, United States of America; 5Peter MacCallum Cancer Center & Royal Melbourne Hospital, Melbourne, VIC, Australia Background: CAPTIVATE (PCYC-1142; NCT02910583) is a multicenter phase 2 study of first-line I+V in patients (pts) with CLL in 2 cohorts: Minimal Residual Disease (MRD) and Fixed Duration (FD). We previously reported that fixed-duration I+V provides deep, durable responses (Ghia et al, ASCO 2021; Wierda et al, J Clin Oncol 2021). Clinically-relevant mutations in BTK, PLCG2, and BCL2 have previously been reported in pts with progressive disease (PD) following single agent BTK or BCL2 inhibitor therapies. Such resistance-associated mutations may occur with increasing frequency after prolonged drug exposure. Fixed duration I+V is hypothesized to decrease the likelihood of developing resistance mutations and facilitate extended clinical benefit. Aims: We sought to ascertain presence or absence of resistance mutations at the time of PD in samples from pts treated with fixed-duration I+V in CAPTIVATE. Baseline genomic risk features and potentially prognostic mutations in CLL-related genes of interest were also evaluated in pts with and without subsequent PD. Methods: The analysis was conducted in 191 pts aged ≤70 y with previously untreated CLL who completed fixed-duration I+V, defined as 3 x 28d cycles of I then 12 cycles of I+V (I 420 mg/d orally; V ramp-up to 400 mg/d orally); this included pts from the FD cohort, and pts from the MRD cohort with confirmed undetectable MRD who received an additional cycle of I+V and were randomized to placebo. Samples from pts at the time of PD were evaluated for resistance mutations by targeted next-generation sequencing (NGS) using a custom-designed high-read-depth 100-gene panel (limit of detection 1% VAF, negative predictive value ≥95% assuming population prevalences <20%, for BTK, PLCG2, or BCL2 variants) in CD19-enriched peripheral blood (PB) or bone marrow. Mutations in additional genes of interest (Table) were evaluated by NGS using the Personalis ACE panel in baseline PB samples. Genomic risk features (Table) were also assessed at baseline. Mutations and genomic risk features evident at baseline were compared across PD and non-PD pts. Results: Baseline mutations in genes of interest and genomic risk features in 190 pts who completed fixed-duration I+V and with available baseline data are shown in the Table. Rates of mutations were similar between pts with (n=30) and without (n=160) subsequent PD. Of 30 pts with PD events (occurring between 16 and 53 mos after start of treatment), 22 (73%) had baseline mutations in genes of interest and 27 (90%) had ≥1 baseline genomic risk feature. Pts with PD generally had more baseline genomic risk features than pts without PD (median 3 vs 2; Wilcoxon P=0.02; analysis restricted to FD cohort pts to avoid bias). To date, 26 of 30 PD patient samples have been evaluated for mutations; no resistance-associated variants in BTK, PLCG2, or BCL2 were detected. Image: Summary/Conclusion: Earlier work has demonstrated a promising safety profile and efficacy of fixed-duration I+V in first-line CLL. Importantly, the current results show that no resistance-associated mutations in BTK, PLCG2, or BCL2 were identified in the pts with PD evaluated to date after first-line, fixed-duration I+V. The absence of such mutations at PD suggests that fixed-duration I+V treatment may offer the possibility of effective subsequent retreatment options with I and/or V and extend clinical benefit. P671: TREATMENT SEQUENCES AND OUTCOMES OF PATIENTS WITH CLL TREATED WITH TARGETED AGENTS IN REAL-WORLD SETTINGS A. R. Mato1,*, B. S. Manzoor2, C. C. Coombs3, N. Lamanna4, H. H. Tuncer5, J. R. Brown6, L. E. Roeker1, M. Shadman7, J. N. Allan8, C. Ujjani7, B. Eichhorst9, L. Leslie10, I. Fleury11, H. Alhasani2, J. Rhodes12, B. T. Hill13, P. M. Barr14, A. Skarbnik15, M. S. Davids6, R. Bannerji16, M. Fuldeore2, F. Lansigan17, A. Schuh18, L. Pearson19, C. P. Fox20, I. Pivneva21, L. Popadic22, A. Guerin21, T. A. Eyre18 1Memorial Sloan Kettering Cancer Center, New York; 2AbbVie, Inc., North Chicago; 3Lineberger Comprehensive Cancer Center, Chapel Hill; 4Columbia University, New York; 5The Cancer Center at Lowell General Hospital, Lowell; 6Dana-Farber Cancer Institute, Boston; 7Fred Hutchinson Cancer Research Center, Seattle; 8Weill Cornell Medicine University Medical Center, New York, United States of America; 9University of Cologne, Cologne, Germany; 10John Theurer Cancer Center, Hackensack, United States of America; 11Hôpital Maisonneuve-Rosemont, Montreal, Canada; 12Northwell Health Cancer Institute, Lake Success; 13Cleveland Clinic, Cleveland; 14Wilmot Cancer Institute, Rochester; 15Novant Health, Charlotte; 16Rutgers Cancer Institute of New Jersey, New Brunswick; 17Dartmouth-Hitchcock Medical Center, Lebanon, United States of America; 18Churchill Hospital, Oxford, United Kingdom; 19Tufts Medical Center, Boston, United States of America; 20Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; 21Analysis Group, Inc., Montreal, Canada; 22Analysis Group, Inc., New York, United States of America Background: Treatment (tx) options for patients (pts) with chronic/small lymphocytic leukemia (CLL/SLL) have expanded following regulatory approval of targeted agents. However, real-world evidence regarding pt outcomes and tx sequences is evolving. Aims: We assessed tx trends, sequences and outcomes following the introduction of targeted agents. Methods: The CLL Collaborative Study of Real-World Evidence (CORE), a retrospective, international, observational study (23 centers) for CLL/SLL pts provided data for this analysis. Pts were diagnosed with CLL/SLL, treated in the relapsed/refractory (R/R) setting after 2/12/2014. Tx sequences following targeted agents (ie, BTKi: acalabrutinib, ibrutinib; BCL2i: venetoclax), clinical response (physician-assessed in medical charts and reported as overall response rate [ORR]), and progression-free survival (PFS) were assessed. Results: Of the 1,624 study pts, in 1L 48.8% initiated CT/CIT, 40.6% targeted agents (BTKi: 32.6%; BCL2i: 4.5%), and 10.6% other therapies. We observed 1L CT/CIT use decreased starting 2014 (pre-2014: 58.1%; 2014–2016: 46.0%; 2017–2019: 24.6%; 2020–2021: 0%), while 1L targeted agent use increased over time (pre-2014: 30.5%; 2014–2016: 43.1%; 2017–2019: 67.0%; 2020–2021: 94.7%). Post-targeted agent introduction in 2014, the most frequent tx sequence has changed from 1L CT/CIT→BCRi in 2014–2016 to 1L BCRi→BCL2i in 2017–2021 (Figure 1). Across lines and years of tx, among 650 R/R CLL/SLL pts who initiated therapy >2/12/2014, 536 pts (82.5%) received BTKi-based regimens and 214 pts (32.9%) received BCL2i-based regimens. Of the 536 pts receiving BTKi-based regimens (ibrutinib alone: 454 [85.5%]; acalabrutinib alone: 21 [4.0%]), majority was in 2L and 3L+ (291 pts (54.3%); 167 pts (31.2%), respectively). Of the 457 pts with response assessed, 343 pts (75.1%) achieved ORR in the line of therapy (LOT) with BTKi-based regimen. The median PFS for pts receiving BTKi-based regimen was 36.4 months (95% confidence interval [CI]: 33.1, 41.9). 236 pts (44.0%) receiving a BTKi-based regimen had a subsequent LOT; of the 177 pts with response assessed, 120 pts (67.8%) achieved ORR. The most common subsequent tx were BCL2i-based (N=113; ORR: 76.2%) or another BTKi-based (N=41; ORR: 54.8%) regimens. Of the 214 pts receiving BCL2i (venetoclax alone: 129 [60.3%]), majority was in 2L and 3L+ (77 pts [36.0%] and 132 pts [61.7%], respectively). Of the 164 pts with response assessed, 127 pts (77.4%) achieved ORR in the LOT with BCL2i-based regimen. The median PFS for pts receiving BCL2i was 26.2 months (95% CI: 20.0, 38.7). 47 pts (22.0%) receiving BCL2i had a subsequent LOT; of the 37 pts with response assessed, 27 pts (73.0%) achieved ORR. The most common subsequent tx were BTKi-based (ibrutinib-based regimen: 14 [29.8%]; acalabrutinib-based regimen: 5 [10.6%]; ORR: 76.9%) or another BCL2i-based (venetoclax-based re-tx: 8 [17.8%]; ORR: 80.0%) regimens. Image: Summary/Conclusion: 1L CT/CIT use is declining since 2014 and 1L targeted agent use is now the standard of care in real-world practice. Of currently available targeted therapies, ibrutinib was the most commonly used. These results demonstrate sequences of BTKi use following venetoclax (venetoclax→BTKi) are effective, as well rechallenging with venetoclax (venetoclax→venetoclax), providing support for either strategy in the R/R CLL/SLL management. These results provide clinicians with valuable insights regarding pt outcomes associated with different tx sequences currently considered in modern clinical practice. Updated pt level data and survival analyses are underway. P672: REAL-LIFE EFFICACY AND SAFETY OF VENETOCLAX MONOTHERAPY IN RELAPSED/ REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA - INTERIM ANALYSIS OF MULTICENTRIC STUDY VERONE L. Ysebaert1,*, X. Troussard2, V. Levy3, R. Le Calloch4, R. Guieze5, S. Leprêtre6, A.-S. Michallet7, V. Leblond8, P. Feugier9, J. Ramier10, A. Delmer11 1Hematology Unit, IUCT Oncopole, Toulouse; 2Hematology Unit, CHU Caen, Caen; 3Hematology Unit, Hopital Avicenne, Bobigny; 4Hematology Unit, CH Cornouaille, Quimper; 5Hematology Unit, CHU Clermont-Ferrand, Clermont-Ferrand; 6Hematology Unit, Henri Becquerel Cancer Research Center, Rouen; 7Hematology Unit, Leon Berard Cancer Reseach Center, Lyon; 8Hematology Unit, Hôpital Pitié-Salpêtrière, Paris; 9Hematology Unit, CHRU Nancy, Nancy; 10AbbVie France, Rungis; 11Hematology Unit, CHU Reims, Reims, France Background: The efficacy and safety of venetoclax (VEN), the first oral inhibitor of BCL-2, has been demonstrated in clinical trials in patients with chronic lymphocytic leukemia (CLL). However, few real-life data in France is available today on this treatment. Aims: VERONE is an observational ongoing study, it aims to describe the efficacy and tolerance of VEN in a real-life situation, as well as its use in current medical practice. Methods: This longitudinal non-interventional study is conducted in adults starting treatment with VEN for CLL, with a follow-up over 48 months. This second interim analysis was performed in patients treated with monotherapy until progression, 43 months after the first inclusion. The overall response rate (ORR, assessed by investigator) and progression-free survival (PFS) were estimated 1 and 2 years after the first intake of VEN in patients with at least one follow-up visit in the study (evaluable population). Results: From March 2018 to September 2021, 196 patients included in 64 centers started VEN as monotherapy for CLL (tolerance population), 195 met the selection criteria (analysis population) and 113 could be evaluated (evaluable population). In the analysis population (n = 195), patients and CLL characteristics at baseline were: median age 74.0 years (33.0-91.0), male 68.2%, ECOG ≥2 18.5%, ≥1 comorbidities 87.7%, median time since diagnosis 8.3 years (0.0-29.4), 37.3% of 169 genetic examinations was TP53 and/ or del (17)p, median number of previous treatments 2 (1-9) (chemoimmunotherapy 87.2%, BCR inhibitors (ibrutinib, idelalisib) 70.8%). Before the first intake of VEN, preventive measures against tumor lysis syndrome (TLS) were taken in the majority of cases (hospitalization 85.2%, hydration 85.2% and anti-hyperuricemia prophylaxis 66.3%). The median duration of the titration period was 5.0 weeks, and 77.0% of patients reached the recommended dose of 400mg at the time of analysis. In the 113 evaluable patients, the overall response rate was 91.2% [95%CI 84.5%-95.1%] at 1 year and 92.9% [95%CI 86.7%-96.4%] at 2 years. After a median follow-up of 23.2 months, the PFS rate was 81.8% [95%CI 72.5%-88.2%] at 1 year and 71.1% [95%CI 58.0% 80.7%] at 2 years. The median duration of treatment was 15.5 months. In terms of safety, 85.2% of the 196 patients presented adverse events [AEs, neutropenia (46.4%) and diarrhea (24.5%)], including 64.3% of AEs of grade ≥3 (including 32.7% neutropenia). Permanent discontinuation of treatment due to AEs related to VEN was observed in 20.4% of patients. 4 AEs grade 5 related to VEN was observed in 3 patients (deterioration of general condition, ascites, hepatic failure, death). At inclusion, the risk level of developing TLS, assessed in 149 patients, was low (28.2%) moderate (69.8%) or high (2.0%). During the dose escalation phase, 19 patients (9.7%) developed TLS and 4 (2.0%) stopped VEN treatment. Summary/Conclusion: This real-life study confirms the efficacy of VEN in heavily treated patients with a safety profile little different from that observed in selected populations in clinical trials. P673: DEPLETION AND RECOVERY OF NORMAL B-CELLS DURING AND AFTER TREATMENT WITH CHEMOIMMUNOTHERAPY, IBRUTINIB OR VENETOCLAX. A. Rawstron1,*, N. Webster2, A. Pitchford3, S. Dalal2, A. Bloor4, R. de Tute1, A. Hockaday3, S. Jackson3, D. Cairns3, N. Greatorex3, D. Allsup5, T. Munir6, P. Hillmen2 1HMDS, Leeds Cancer Centre; 2Experimental Haematology, University of Leeds; 3Leeds Cancer Research UK Clinical Trials Unit, Leeds Institute of Clinical Trials Research, Leeds; 4Haematology, The Christie NHS Foundation Trust, Manchester; 5Haematology, Hull York Medical School, Hull; 6Haematology, Leeds Cancer Centre, Leeds, United Kingdom Background: Infectious complications are a major cause of morbidity and mortality in Chronic Lymphocytic Leukaemia (CLL). Therapeutic approaches that deplete CLL cells also affect normal B-cells. Optimal treatment would result in eradication of CLL cells and recovery of normal immune function. FLAIR (ISRCTN01844152) is a phase III trial for previously untreated CLL comparing ibrutinib plus rituximab (IR) with fludarabine, cyclophosphamide and rituximab (FCR) and subsequently amended to also compare ibrutinib plus venetoclax (I+V) and ibrutinib alone (I) with FCR. Measurable residual disease (MRD) and normal B-cell levels were assessed at multiple timepoints. Aims: To assess the depletion of normal B-cells during treatment and recovery after end of treatment. Methods: Participants aged under 75 years with <20% TP53-deleted cells were initially randomised to FCR or IR and subsequently to FCR, IR, I+V or I with the IR arm closed after randomisation of 771 participants to FCR/IR. FCR was given for 6 cycles, while treatment in the IR, I and I+V arms continued for up to 6 years except in participants attaining <0.01% MRD who continued treatment for the time taken to achieved MRD <0.01% and then stopped if MRD remained <0.01%. Month (M) 24 was earliest permitted stopping point. MRD flow cytometry was performed according to ERIC guidelines (panel: CD19/5/20/43/79/81+ROR1, acquisition of 0.5–2.2 million cells, BD Biosciences Lyric). Additional analysis of normal B-cell subsets was performed in a cohort of >500 patients (panel: CD19 to identify B-cells, CD20/5/79b+ROR1 and CD3 to exclude CLL & contaminating cells, with CD27/ 38/IgD/IgM to characterise normal B-cell subsets using a Coulter Cytoflex LX). Results: Normal B-cells were undetectable during FCR treatment and only rarely detectable until 12 months after last FCR cycle. Circulating normal B-cells were reduced in number or undetectable in participants receiving ibrutinib-containing regimens with greater depletion in the I+V and IR arms relative to I monotherapy. B-progenitors persist through FCR treatment but were depleted during I, I+R or I+V treatment. Normal B-cell levels at 24 and 36 months after randomisation, with time off-treatment if applicable, are shown in Figure 1. In the ibrutinib-containing arms (IR, I, and I+V), there was a trend towards fewer COVID-associated SAE at any time point for participants with detectable B-cells at 24M (4/181, 2.2%) compared to those with no detectable B-cells (14/344, 4.1%) and COVID-associated SAEs were not observed in FCR-treated participants who had recovered any level of normal B-cells by 24M (0/215). However, the data on COVID infections are limited and there was no apparent association between normal B-cell levels at 24M with the proportion of participants experiencing an infectious SAE overall. Assessment of normal B-cell subsets during ibrutinib-based treatment demonstrated a mix of naïve and memory B-cells. Serological response to COVID infection/vaccination in this cohort is currently being performed. Participants stopping I+V treatment at 24-30 months post-randomisation due to MRD eradication showed rapid recovery of normal naive B-cells within 6-12 months after end of treatment in the vast majority (>95%) of evaluable cases. Image: Summary/Conclusion: Normal circulating B-cells are depleted during treatment with rituximab but can persist at a low level during I, IR or I+V treatment. Most patients in remission after treatment with FCR or I+V show recovery of normal B-cells at 12 months of stopping treatment. P674: UPDATED RESULTS OF A PHASE 1B STUDY OF IBRUTINIB (IBR) PLUS OBINUTUZUMAB (OBIN) IN PATIENTS WITH RELAPSED OR REFRACTORY (R/R) CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) C. Ryan1,*, D. Brander2, P. Barr3, S. Tyekucheva4, Y. Ren4, L. Hackett1, M. McDonough1, K. Pena Del Aguila1, M. McEwan1, M. Collins1, J. Abramson1, E. Jacobsen1, A. LaCasce1, D. Fisher1, J. Brown1, M. Davids1 1Medical Oncology, Dana-Farber Cancer Institute, Boston; 2Duke University Cancer Center, Durham; 3University of Rochester Wilmot Cancer Institute, Rochester; 4Data Science, Dana-Farber Cancer Institute, Boston, United States of America Background: Combination ibr + obin was approved in frontline CLL based on results of iLLUMINATE; however, less data are available on this regimen in R/R CLL. Though active in R/R CLL, ibr monotherapy in RESONATE led to 4-year (yr) PFS/OS of ~45%/65%, leaving room for improvement. We previously reported initial safety and efficacy results of this phase 1b study investigating ibr + obin in R/R CLL (Davids et al., EHA 2020). Here, we report updated results, with median follow-up now of 41.5 months (mos). Aims: To assess longer-term safety and efficacy of ibr + obin in patients (pts) with R/R CLL. Methods: We conducted a phase 1b, investigator-initiated study of ibr + obin in R/R CLL (NCT02537613). The primary objective was to assess safety of 3 different drug sequencing regimens in cycle 1; the main secondary objective was to assess efficacy. Pts were randomized 1:1:1 to either receive obin 1 mo before ibr, ibr 1 mo before obin, or start concomitantly. Both drugs were given at the approved doses and schedule, with 6 total cycles of combination followed by ibr monotherapy until progression or unacceptable toxicity. Notable eligibility criteria: requiring treatment (tx) for R/R CLL/SLL by 2008 iwCLL criteria, ECOG PS ≤2, no prior ibr or obin. Assessments: toxicities by CTCAE v4, efficacy by 2008 iwCLL criteria, MRD by 4-8 color flow (10-4 sensitivity). Serum concentrations of 21 cytokines were assessed pre-tx and after 1 week of combination tx in a representative subset of 27 pts (n=9 per arm) by bead-based multiplex immunoassay (Eve Technologies, Calgary). Results: 52 pts were accrued, and all received ≥1 cycle of combination tx. Median age: 67 yrs (range 33-84); 77% male; median # prior tx: 1 (range 1-6); 25% del(17p) or TP53 mut; 27% del(11q); 50% unmutated IGHV; 27% bulky LAD (nodes ≥5 cm). As of 14 Feb 2022: median follow-up 41.5 mos (range 2.3-73.3). 27 pts (52%) remain on tx. All-grade heme toxicities: thrombocytopenia (88%; 23% Gr3/4), neutropenia (71%; 37% Gr3/4), febrile neutropenia (8%, all Gr3/4), anemia (73%; 8% Gr3). Notable non-heme toxicities: bruising (58%, all Gr1/2), arthralgia (38%, all Gr1/2), diarrhea (37%, all Gr1/2), transaminitis (35%; 4% Gr3), ≥Gr3 infection (17%, all Gr3), and cardiotoxicities: hypertension (46%; 10% Gr3), atrial fibrillation (21%; 10% Gr3). 1 pt had sudden cardiac death after 11 mos on study. 7 pts required ibr dose-reduction due to toxicity. The best ORR is 96%, including 50% CR and 46% PR (Fig. 1A). All 3 pts who previously progressed on venetoclax responded (1 CR, 2 PR). By ITT, 10/52 (19%) achieved a best response of undetectable MRD in bone marrow (BM, 44 pts tested), and 14/52 (27%) in peripheral blood (PB, 34 pts tested). 4-yr PFS and OS are 74% and 93% (Fig. 1B-C), with 3 deaths (n=1 each: sudden death, MDS, Richter’s syndrome). Pts who achieved BM and PB undetectable MRD had a significantly larger decrease in circulating CCL4 (p=0.02) and CXCL13 levels (p=0.01), respectively, comparing baseline to 1 week of combination tx (Fig. 1D). Image: Summary/Conclusion: Ibr + obin is a highly active combination in R/R CLL, achieving 50% CR and 4-yr PFS/OS of 74%/93%, suggesting a potential positive impact of obin (though possibly also reflecting an earlier relapse population than RESONATE). Responses were observed in all 3 pts who had progressed after venetoclax, including 1 CR. With ~3.5 yrs median follow-up, and some pts on tx over 6 yrs, cardiotoxicity was consistent with prior ibr studies, and no new safety concerns have emerged. Our data support continued exploration of combination BTK inhibitor plus obin in R/R CLL. P675: TREATMENT OF HYPERTENSION IN PATIENTS RECEIVING BTK INHIBITORS: A MULTICENTER RETROSPECTIVE STUDY M. Shadman1,2,*, J. Voutsinas3, B. Fakhri4, S. Khajavian3, S. Spurgeon5, D. Stephens6, A. Skarnbik7, A. Mato8, C. Broome9, A. Gopal1,2, S. Smith1, R. Lynch1, M. Rainey10, S. Kim5, O. Barrett-Campbell11, E. Hemond11, M. Tsang12, D. Ermann6, N. Malakhov13, D. Rao8, M. Shakib Azar3, B. Morrigan3, A. Chauhan14, T. Plate15, T. Gooley1, K. Ryan16, F. Lansigan11, B. Hill10, G. Pongas15, S. Parikh17, L. Roeker8, J. Allan18, R. Cheng2, C. Ujjani1 1Fred Hutchinson Cancer Research Center / University of Washington; 2University of Washington; 3Fred Hutchinson Cancer Research Center, Seattle; 4University of California San Francisco (UCSF), San Francisco; 5Oregon Health and Science University (OHSU), Portland; 6University of Utah, Salt Lake City; 7Novant Health, Charlotte; 8Memorial Sloan Kettering Cancer Center, New York; 9Georgetown University, Washington DC; 10Cleveland Clinic, Cleveland; 11Dartmouth-Hitchcock Medical Center, Lebanon; 12UCSF, San Francisco; 13New York-Presbyterian Hospital, Weill Cornell Medicine, New York; 14Georgia Cancer Center at Augusta University, Agusta; 15Sylvester Comprehensive Cancer Center, University of Miami, Miami; 16Astra Zeneca, Gaithersburg; 17Mayo Clinic, Rochester; 18Weill Cornell Medicine, New York, United States of America Background: Hypertension (HTN) is an adverse event (AE) associated with the use of ibrutinib and less commonly with second generation BTK inhibitors (BTKis) as shown by a significant lower rate of HTN with acalabrutinib compared to ibrutinib in the ELEVATE-RR trial. Nevertheless, HTN is considered an AE of BTKis as a class. Aims: We aimed to identify a class or combination of antihypertensive drugs (anti-HTNs) that may be most effective in pts with HTN while taking a BTKi. Methods: In this multicenter retrospective real-world study, we included consecutive pts with lymphoid malignancies taking BTKis and anti-HTNs with at least 3 months of follow-up. Eligible pts were stratified into 2 groups: 1) Pts with pre-existing HTN before starting BTKi (pre-HTN) and 2) Pts who developed incident HTN after starting BTKis (de novo HTN). The anti-HTN drugs were categorized as follows: 1) Angiotensin-converting-enzyme inhibitors and Angiotensin receptor blockers (ACEi/ARB); 2) Beta-blockers (BB); 3) Calcium channel blockers (CCB); 4) Hydrochlorothiazide (HCTZ) and 5) non-HCTZ diuretics (other-DU). Timepoints for mean arterial pressure (MAP) measurements and start/end time of each anti-HTN were documented in relation to the index date (first date of concurrent use of BTKi and anti-HTN drug). Generalized estimating equations were used to assess the association between time varying MAPs and the individual drug categories, as well as the number of medication categories. Results: We included 196 pts (118 pre-HTN and 78 de novo HTN) from 12 centers. The majority of pts had CLL (94%) on ibrutinib (90%) for first (49%), second (25.5%) or later (25.5%) lines of therapy. The median age was 67 (IQR 37-88), most pts were male (72%), and caucasian (94.4%). The median duration of follow-up was 38.9 months (IQR 3.5-110). Pre-BTKi comorbidities included diabetes (17.3%), coronary disease (11.7%), cerebrovascular accidents (2.6%) and chronic kidney disease (12.8%). BTKi dose was reduced in 53 pts (27%) with 9 (4.6%) having dose reduction because of HTN. Twelve pts (6.2%) switched to other BTKis and 5 pts (2.6%) did so for HTN. In the pre-HTN cohort, a trend for statistically significant BP reduction was only achieved when 4+ anti-HTN drugs were used [Est -4.81; 95% CI (-9.93, 0.307); p = 0.07]. In this cohort, concurrent use of HCTZ and BB showed a significant trend for a lower BP [Est -5.05; 95% (-10.0, -0.06); p= 0.04]. In de novo HTN cohort, treatment with 3+ anti-HTN drugs was associated with a statistically significant reduction in the BP [Est -5.70; 95% CI (-9.94, -1.46); p =0.008]. In de novo cohort combination of HCTZ and ACEi/ARB was associated with a significant MAP reduction [Est -5.47; 95% CI (-10.9, -0.001); p= 0.05]. When the 2 cohorts were combined, treatment with 4+ drugs [Est -6.27; 95% CI (-11.8, -0.69); p = 0.02] and treatment with HCTZ and ACEi/ARB combination [Est -5.05; 95% CI (-9.61, -0.48); p = 0.03] or HCTZ and BB [Est -5.05; 95% CI(-9.61,-0.48); p= 0.03] were associated with MAP reduction. Gender or race did not modify these associations. Image: Summary/Conclusion: Treatment of HTN in pts taking Ibrutinib is challenging and requires utilization of multiple anti-hypertensive drugs both in pre-HTN and de novo HTN pts. Among different anti-HTN classes, we observed a statistically significant MAP reduction in pts with pre-HTN on HCTZ and BB combination and in pts with de novo HTN on HCTZ and ACEi/ARB combination. These findings need to be further assessed in pts taking newer generation BTKi and confirmed in large prospective studies. P676: LOW-DOSE FCR COMPARED TO BR IN PREVIOUSLY UNTREATED PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA WITHOUT TP53 ABERRATIONS: A REAL – WORLD RETROSPECTIVE ANALYSIS BY THE CZECH CLL STUDY GROUP. L. Smolej1,*, Y. Brychtová2, E. Cmunt3, M. Oršulová2, P. Vodárek1, M. Dostál1, P. Turcsányi4, R. Urbanová4, A. Panovská2, J. Zuchnická5, J. Mihályová5, D. Lysák6, M. Brejcha7, H. Móciková8, K. Klásková8, M. Šimkovič9, M. Špaček3, M. Doubek10 14th Department of Medicine - Hematology, University Hospital and Faculty of Medicine, Hradec Králové; 2Department of Internal Medicine – Hematology and Oncology, University Hospital, Brno; 3First Department of Internal Medicine - Hematology, General University Hospital, Prague; 4Department of Hemato - oncology, University Hospital, Olomouc; 5Department of Hematooncology, University Hospital, Ostrava; 6Department of Hematology - Oncology, University Hospital, Pilsen; 7Department of Hematology, Hospital Agel, Nový Jičín; 8Department of Internal Medicine - Hematology, University Hospital Královské Vinohrady, Prague; 94th Department of Medicine - Hematology, University Hospital, Hradec Králové; 10Department of Internal Medicine - Hematology, University Hospital, Brno, Czechia Background: Low – dose fludarabine, cyclophosphamide, and rituximab (LDFCR) regimen showed promising activity in treatment-naïve elderly / comorbid patients (pts) with chronic lymphocytic leukemia (CLL) (Smolej et al., Br J Haematol. 2021). However, no data are available regarding comparison to bendamustine and rituximab (BR), which has been one of the predominant first - line options for CLL patients ineligible for full – dose FCR. Aims: To perform a historical comparison of the efficacy and safety of LDFCR and BR regimens used as the first - line therapy of CLL within routine practice. Patients with 17p deletion and/or TP53 mutation were not included in this analysis because chemoimmunotherapy is no longer indicated in this extremely unfavourable subgroup due to very low activity. Methods: The analysis included 237 pts treated with LDFCR and 320 pts treated with BR (median age, 69 vs 70 years; males, 70 vs 61%; median CIRS score, 6 vs 7; Rai stage III/IV, 57 vs 59%; bulky lymphadenopathy > 5cm, 35 vs 35%; unmutated IGHV, 74 vs 70%, deletion 11q, 28 vs 26%) treated between March 2009 and December 2019 at 17 centers cooperating within the Czech CLL Study Group. LDFCR consisted of fludarabine 20 mg/m2 orally or 12 mg/m2 on days 1-3, cyclophosphamide 150mg/m2 orally or iv on days 1-3, and rituximab 500 mg/m2 D1 (375 mg/m2 in Cycle 1). BR included bendamustine 90 mg/m2 iv on days 1-2 and rituximab 500 mg/m2 D1 (375 mg/m2 in Cycle 1). Median follow – up was 98 months for LDFCR and 57 months for BR. Results: Distribution of demographic data and prognostic factors was comparable between LDFCR and BR groups. The overall response rate / complete remissions were similar for LDFCR vs BR (84/46% vs. 88/51%, p=n.s.). The median progression – free survival (PFS) was 30 vs 34 months (hazard radio [HR] 0.90; p=n.s.); PFS was markedly longer in pts with mutated IGHV and absence of del 11q (LDFCR vs BR, median 58 vs 59 months, HR 0.84; p=n.s.; Fig. 1a). Median overall survival (OS) was 73 vs 77 months for LDFCR vs BR (HR 1.09; p=n.s.) but markedly longer (median 116 months vs not reached) in patients with mutated IGHV without del 11q (HR 0.90; p=n.s.; Fig. 1b). Grade ≥ 3 neutropenia occurred in 55% (LDFCR) vs 51% (BR) pts but serious infections developed in 15% vs 20% pts only. Image: Summary/Conclusion: Our data indicate that both low-dose FCR and BR are well – tolerated regimens with similar efficacy and safety and represent a suitable first - line treatment alternative for elderly / comorbid CLL patients with favourable biological prognosis. Detailed results including multivariate analysis will be presented. P677: CLINICAL EFFICACY AND TOLERABILITY OF VENETOCLAX PLUS RITUXIMAB IN RELAPSED OR REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA – REAL WORLD ANALYSIS OF POLISH ADULT LEUKEMIA STUDY GROUP A. Soboń1,*, J. Drozd-Sokołowska2, E. Paszkiewicz-Kozik3, L. Popławska3, M. Morawska4,5, J. Tryc-Szponder6, L. Bołkun7, J. Rybka8, K. Pruszczyk1, A. Juda9, A. Majeranowski10, E. Iskierka-Jażdżewska11, P. Steckiewicz12, K. Wdowiak13, B. Budziszewska1, K. Jamroziak2, I. Hus1, E. Lech-Marańda1, B. Puła1 1Department of Hematology, Institute of Hematology and Transfusion Medicine; 2Department of Hematology, Transplantation and Internal Medicine, Medical University of Warsaw; 3Department of Lymphoid Malignancies, Maria Sklodowska-Curie National Research Institute of Oncology, Warszawa; 4Experimental Hematooncology Department, Medical University of Lublin & Hematology Department; 5St. John’s Cancer Center, Lublin; 6Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznań; 7Department of Hematology, Medical University of Białystok, Białystok; 8Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University, Wrocław; 9Department of Hematology and Bone Marrow Transplantation, Medical University of Lublin, Lublin; 10Department of Hematology and Transplantology, Medical University of Gdańsk, Gdańsk; 11Department of Hematology, Medical University of Łódź, Copernicus Memorial Hospital, Łódź; 12Department of Hematology, Holy Cross Cancer Center, Kielce; 13Department of Internal Diseases and Oncological Chemotherapy Faculty of Medical Sciences, Medical University of Silesia, Katowice, Poland Background: The introduction of venetoclax into clinical practice has improved the outcome of patients with relapsed/refractory chronic lymphocytic leukemia (RR-CLL). The results of the MURANO trial published in March 2018 showed significantly longer progression-free survival (PFS) and overall survival (OS) in RR-CLL patients treated with venetoclax and rituximab (VEN-R) comparing to bendamustine and rituximab (BR) and resulted in the approval of VEN-R in the therapy of RR-CLL in the European Union and the United States. It should be noted that the results of registration studies often do not correspond with the data from real-life observations. Aims: To study the clinical efficacy and safety profile of VEN-R treatment in RR-CLL patients outside clinical trials. Methods: We performed retrospective analysis of RR-CLL patients treated with VEN-R in hematology centers of the Polish Adult Leukemia Study Group (PALG) from 2019 to 2021. Results: Clinical data of 117 RR-CLL patients treated with VEN-R were collected. Median patient age upon initiation of VEN-R therapy was 67 years (range 33 – 84 years). Seventy-two patients (61.5%) were men. Median Cumulative Illness Rating Scale (CIRS) was 6 (range 2 - 16). Patients were treated with a median of 2 (range 1–9) previous lines of therapy, whereas 32 patients (27.4%) had relapsed following the first line of treatment. Overall, 25 patients (21.4%) had 17p deletion, whereas TP53 mutation was identified in 13 patients (11.1%). The median follow-up was 9.96 months (range 0.27 - 29.13). The overall response rate (ORR) was 95.2%. Seventeen patients (14.5%) achieved complete remission (CR), 83 (70.9%) partial remission (PR), while in 5 patients (4.3%) disease progression was noted. In the patients with 17p deletion (n=22) or TP53 mutation (n=11), CR and PR were observed in 4 (12.1%) and 29 (87.9%) patients, respectively. The median PFS in the whole cohort was 20.8 (95% CI 18.43 - not reached) months and the median OS was not reached. In our study none of the analyzed clinico-pathological factors had significant impact on ORR, PFS and OS. During the follow-up time four (3.4%) cases of Richter transformation were diagnosed. There were 18 deaths recorded during the course of observation; 3 (16.7%) due to disease progression and 7 (38.9%) due to COVID-19 infection. The others were due to infections other than SARS-CoV-2 (n=3, 16.7%) and the cause of death could not be specified in five cases (27.8%). Eighty-three patients (70.9%) remain on treatment, while treatment was discontinued in thirty-four cases (29.1%). Reasons for therapy discontinuation included patient’s death (52.9%), treatment-related cytopenias (17.6%), disease progression (14.7%), Richter’s transformation (11.8%), autoimmune hemolytic anemia (5.9%), diarrhea (2.9%) and infections (8.8%). In one case treatment discontinuation was due to consent withdrawal and one patient was lost to follow-up. The following adverse events of VEN-R treatment were reported during the study: all grade neutropenia (71.8% with grade 3/4 in 55.6%), anemia (51.3%), thrombocytopenia (47%), pneumonia (9.4%), neutropenic fever (6.8%), autoimmune hemolytic anemia (4.3%), immune thrombocytopenic purpura (1.7%), diarrhea (4.3%) and in one case exacerbation of heart failure was observed. Summary/Conclusion: In this retrospective analysis the outcomes of treatment with the VEN-R regimen in real-life setting were worse than those reported in the MURANO trial. P680: SARCOPENIA EVALUATED BY CT SCAN IS ASSOCIATED WITH SHORTER SURVIVAL IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA TREATED WITH TARGETED THERAPIES. A PROSPECTIVE STUDY. A. Visentin1,*, F. Crimì2, A. Corso2, R. Angelone2, C. A. Cavarretta1, A. Cellini1, F. Angotzi1, V. Ruocco1, S. Pravato1, E. Quaia2, L. Trentin1 1Hematology and Clinical Immunology unit, Department of Medicine, University of Padova; 2Radiology Institute, Department of Medicine, University of Padova, Padova, Italy Background: Chronic lymphocytic leukemia (CLL) usually affects elderly patients with comorbidities. Although drugs targeting BTK, BCL2 and PI3K have been proved to be better tolerated than chemoimmunotherapy and to improve the survival of CLL patients, comorbidities, assessed by Cumulative Illness Rating Scale (CIRS) score, are still associated with a worse outcome. Indexes of sarcopenia, subcutaneous or visceral adipose tissues assessed by CT scan, are emerging as survival markers in cancer patients. However, CT scan is not recommended by iwCLL guideline nor we known the clinical impact of body composition in CLL. Aims: The aim of this study was to assess the impact of muscle and adipose tissue indexes in patients with CLL treated with targeted agents. Methods: We performed a single center prospective study of CLL patients treated with BTK, BCL2 and PI3K inhibitors out of clinical trials. Inclusion criteria were diagnosis of CLL and need of treatment according to iwCLL guideline, creatinine clearance >30ml/min and CT scan performed at baseline. For each patient, with NIH ImageJ software, we extracted the areas of subcutaneous fat, visceral fat and the skeletal muscle from an unenhanced axial CT image at vertebra L3. The values obtained (cm2) were divided by the square of patient’s height (m2), obtaining the subcutaneous adipose tissue index (SATI), the visceral adipose tissue index (VATI) and the skeletal muscle index (SMI). Overall survival (OS) was calculated as months from starting targeted therapies to death (event) or last available follow-up (censored). Survival curves with Log-rank test. Results: We included 118 patients, 70 (59%) were males, mean age was 71.6±8.6 years, 74 (63%) were at Rai stage III-IV, 50 (42%) had a CIRS≥6, 96 (81%) unmutated IGHV, 41 (35%) 17p13 deletion, 39 (33%) TP53 mutation. The median number of therapies was 3 (range 1-9). Sixty-six patients (56%) received ibrutinib, 27 (23%) idelalisib+rituximab and 25 (21%) venetoclax±rituximab, including 17 (14%) front line therapies (13 ibrutinib, 4 venetoclax). We observed that SMI values correlated with SATI (p=0.035) and VATI (p<0.0001). SMI, SATI and VATI were higher in males vs females, median 48.4 vs 42.2 (p=0.007), 61.0 vs 67.9 (p=0.0087) and 70.0 vs 50.3 (p=0.0113)(Fig 1A). Using these thresholds, low-SMI, suggestive of sarcopenia, and low SATI correlated with a more advance number of previous therapies (p=0.027 and p=0,037). Low-SMI was more common in patients with CIRS≥6 (p=0.039). SMI, SATI and VATI did not correlated with age, stage and biological markers. After a median follow-up of 32 months the median OS for the whole cohort was 46.7 months. We confirmed that CIRS≥6 correlated with a worse OS in our study (p<0.0001, Fig. 1B). The 3-year OS was 53% and 66% for patients with low-SMI vs high-SMI, and the median OS was 40.3 months for low-SMI but not reached for high-SMI patients (p=0.0199, Fig. 1C). SATI and VATI did not correlate with OS. Combining data of CIRS≥6 and low-SMI, 40 (34%) patients, 47 (40%) and 31 (26%) displayed 0, 1 and 2 markers (Fig. 1D). The 3-year OS was 80%, 56% and 42% for patients at score 0, 1 and 2. The median OS was not reached for score 0 patients, but decreased from 42 to 23 months for patients at score 1 and 2 (p=0.0006, Fig. 1E). Image: Summary/Conclusion: In this prospective study we found that that basal CT scan identifies sarcopenic CLL patients with low SMI featured by an adverse prognosis even if treated with targeted oral drugs, in particular when combined with the CIRS score. The prognostic and predictive impact of basal CT scan in CLL deserves further investigation. P681: EUROFLOW STANDARDIZATION TECHNIQUE AND NORMALIZATION PROCEDURES IN LONGITUDINAL FLOW CYTOMETRIC EXPRESSION ANALYSIS OF CD20 IN CLL PATIENTS RECEIVING ANTI-CD20 DIRECTED THERAPY P. J. Walter1,*, A. Schilhabel1, P. Cramer2, J. von Tresckow3, S. Kohlscheen1, K. Fischer2, B. Eichhorst2, S. Böttcher4, M. Brüggemann1, M. Kneba1, M. Hallek2, M. Ritgen1 1Laboratory for Specialized Hematological Diagnostics, Medical Department II, University Hospital Schleswig-Holstein, Kiel; 2Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, German CLL Study Group, University Hospital Cologne, Cologne; 3Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen; 4Medical Department III Hematology, Oncology, Palliative Care, Center for Inner Medicine, University Hospital Rostock, Rostock, Germany Background: CD20 expression is still a controversial issue regarding its prognostic value for therapy outcome. While up to 50% of patients relapse within four of years after anti-CD20 directed treatment and constitute a need for a salvage or relapse/refractory treatment, only few publications address the longterm influence on CD20 expression assessed by flow cytometry. The technical complexity of the method and factors affecting the variability of measurements of samples at different time points and from different sources/patients are well recognized by the field and addressed by different approaches as standardization of instruments, automated gating and alignment of population or normalization. Aims: In order to evaluate long term influence of anti-CD20 directed therapy on CD20 expression in patients with chronic lymphocytic leukemia who showed only weak MRD responsiveness to therapy in different clinical trials, we aimed to establish a normalization approach based on the instrument standardization to reduce the proportion of technical variance within data. Methods: Mean fluorescence intensities (MFI) of peak seven of standardized fluorescence beads from daily quality control and instrument standardization of flow cytometers according to the EuroFlow protocol were used to establish a normalization approach. Proof of prinicple was performed using the residual peaks covering the MFI range provided by the beads. To assess different sources of variance ANOVA of a 3x3x3 experiment evaluating marker expressions on different cell populations in the blood of healthy donors using Quantibrite-PE beads was performed addressing technical variation (day, instrument) and biological variation. CD20 and CD19 MFI on CLL cells were retrospectively assessed from MRD measurements of peripheral blood samples of patients enrolled in the phase II GCLLSG trials CLL2-BIG (n=57), -BAG (n=58), -BIO (n=58), -BCG (n=22), combining facultative Bendamustin (B) debulking with either Obinutuzumab (G), Ofatumumab (O), Venetoclax (A), Ibrutinib (I) or Idelalisib (C). Results: Long term shifts of fluorescence intensities, and coefficients of variation were reduced 2 to 10 times across the MFI range covered by the fluorescence beads without distorting the longitudinal course of instrument performance. Most of the variance in assessments of molecules equivalent soluble fluorochrome from MFIs of NK cell and B cell markers in peripheral blood of healthy donors were related to inter-donor variations (p<0.002). Normalized MFIs for CD19 and CD20 on CLL cells do not significantly differ from the measured values in the Mann-Whitney-U test (p>0.85). Higher CD20 expression at therapy start seems to be correlated to strong MRD response solely in the CLL2-BIO trial (p=0.036, Fig. 1). Prognostic factors TP53-, IGHV-, and NOTCH1- mutation did not seem to correlate with the observed CD20 expression differences. Therapy strata showed differences (statistically not significant) in the proportion of firstline and r/r treated patients. Figure 1 Pre-treatment CD20 MFI on CLL cells of patients categorized according to treatment and variable (strong, medium, weak, and no) MRD response. Image: Summary/Conclusion: Following standardized staining and instrument monitoring, it should be possible to robustly assess longitudinal biological variations of marker expression based on MFI values. In a cross trial comparison Obinutzumab showed highest proportion of patients with strong MRD response independent from initial CD20 expression, whereas strong response to Ofatumumab seems to correlate with higher CD20 expression levels. P682: NEMTABRUTINIB (MK-1026), A NON-COVALENT INHIBITOR OF WILD-TYPE AND C481S MUTATED BRUTON TYROSINE KINASE FOR B-CELL MALIGNANCIES: EFFICACY AND SAFETY OF THE PHASE 2 DOSE-EXPANSION BELLWAVE-001 STUDY J. Woyach1,*, I. W. Flinn2, F. T. Awan3, H. Eradat4, D. Brander5, M. Tees6, S. A. Parikh7, T. Phillips8, W. Wang9, N. M. Reddy10, M. Z. Farooqui10, J. C. Byrd11, D. M. Stephens12 1Division of Hematology, The Ohio State University, Columbus, Columbus; 2Sarah Cannon Center Research Institute, Nashville, Nashville; 3Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas; 4Department of Hematology-Oncology, David Geffen School of Medicine at UCLA, Los Angeles; 5Duke Cancer Institute, Duke University Medical Center, Durham; 6Colorado Blood Cancer Institute, Denver; 7Division of Hematology, Mayo Clinic, Rochester; 8Division of Hematology and Oncology, University of Michigan Rogel Cancer Center, Ann Arbor; 9Veristat, LLC, Southborough; 10Merck & Co., Inc., Kenilworth; 11Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati; 12Division of Hematology and Hematologic Malignancies, University of Utah Huntsman Cancer Institute, Salt Lake City, United States of America Background: For patients (pts) with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and certain B-cell neoplasms, resistance to Bruton tyrosine kinase inhibitors (BTKi) develops primarily through mutations at the cysteine binding site (C481) or PLCγ2 mutations. Nemtabrutinib (MK-1026, formerly ARQ-531) is a non-covalent, potent inhibitor of both wild type and C481-mutated BTK. Aims: In the phase 1/2 dose-escalation and dose-expansion BELLWAVE-001 study (NCT03162536), the preliminary recommended phase 2 dose (RP2D) of nemtabrutinib was determined to be 65 mg once daily. The efficacy and safety of nemtabrutinib in patients (pts) with CLL/SLL and B-cell non-Hodgkin lymphoma (NHL) were also evaluated at a higher dose during the dose-expansion phase. Methods: In this open-label, single-arm phase 2 study, 9 expansion cohorts were initiated following determination of preliminary nemtabrutinib RP2D. Approximately 10-25 eligible pts were enrolled into Cohort A (relapsed/refractory (r/r) CLL/SLL, with ≥2 prior therapies including covalent BTKi, with documented C481 mutation), Cohort B (r/r CLL/SLL progressed on/intolerant to a BTKi, with ≥2 prior therapies without C481 mutation), Cohort C (Richter transformation with ≥1 prior therapy), Cohort D-H (follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, high-grade B cell lymphoma with known MYC, BCL-2 or BCL-6 translocations, and Waldenström macroglobulinemia [WM], respectively, who received ≥2 prior therapies), Cohort I (food-effect cohort including pts with B-cell NHL, CLL/SLL, and WM). Primary end point was ORR (per iwCLL criteria, by investigator) for participants with CLL/SLL. Secondary end points included DOR and safety and tolerability. Results: Among 118 pts enrolled, 44 had B-cell NHL, 68 CLL/SLL, and 4 WM. Of these, 94 (79.6%) were treated at the preliminary RP2D, including 51 (54.3%) participants with CLL/SLL. Pts with CLL/SLL had a median (range) number of prior therapies of 4 (1-18), 43 (84%) had prior BTKi therapy, 12 (23%) had del17p, 30 (59%) had IGHV unmutated status, and 32 (63%) had C481S BTK mutation. At data cut-off (April 7, 2021), median (range) follow-up was 4.6 mo (0.1-26.7) for all treated pts. ORR was 57.9% (22/38; 1 CR, 21 PR/PRL) per iwCLL criteria in the efficacy-evaluable population of pts with CLL/SLL treated at preliminary RP2D. Median duration of response was not estimable [NE] (range, 8.3 mo-NE). Among all treated participants, 114 (97%) had a treatment-emergent adverse event (TEAE), with 78 (66%) having a drug-related TEAE, and 9 (8%) having a drug-related TEAE that led to discontinuation. Common TEAEs (≥ 20%) included fatigue (33%), constipation (31%), dysgeusia (28%), cough (25%), nausea (25%), pyrexia (25%), dizziness (23%), hypertension (23%), peripheral edema (22%), diarrhea (21%), and arthralgia (20%). Grade ≥3 TEAEs occurred in 80 (68%) participants. Grade 5 TEAEs occurred in 7 (6%) participants and included death following disease progression 3 (3%), sepsis 1 (1%), dyspnea 1 (1%), and respiratory failure 2 (2%). Common drug-related TEAEs (≥10%) included dysgeusia (15%), nausea (11%), fatigue (11%), and decreased neutrophil count (10%). Grade 3-4 drug-related TEAEs occurred in 31 (26%) participants. No drug-related TEAEs led to death. Summary/Conclusion: Nemtabrutinib has promising antitumor activity with a manageable safety profile in pts with CLL/SLL exposed to multiple lines of therapy, including in those who had progression of disease on prior covalent BTKi. Further evaluation of nemtabrutinib in B-cell malignancies is ongoing. P683: A FIRST-IN-HUMAN PHASE 1 STUDY OF ORAL LOXO-338, A SELECTIVE BCL2 INHIBITOR, IN PATIENTS WITH ADVANCED HEMATOLOGIC MALIGNANCIES (TRIAL IN PROGRESS) W. Jurczak1,*, A. J. Alencar2, G. S. Guru Murthy3, M. Hoffmann4, J. M. Pagel5, V. Patel5, J. M. Pauff5, P. L. Zinzani6, S. Le Gouill7, A. R. Mato8, L. E. Roeker8 1Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland; 2Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, Miami, FL; 3Division of Hematology/Oncology, Medical College of Wisconsin, Milwaukee, WI; 4Hematologic Malignancies and Cellular Therapeutics, University of Kansas Cancer Center, Overland Park, KS; 5Loxo Oncology at Lilly, Stamford, CT, United States of America; 6Institute of Hematology Seràgnoli, University of Bologna, Bologna, Italy; 7Institut Curie Hospital, Paris, France; 8Memorial Sloan Kettering Cancer Center, New York, NY, United States of America Background: B-cell lymphoma 2 (BCL2) is a key regulator of apoptosis and provides protection from cell death in many hematological malignancies. LOXO-338 is a novel, orally bioavailable small molecule inhibitor of BCL2, designed to achieve selectivity over BCL-xL and thus avoid dose-limiting thrombocytopenia associated with BCL-xL inhibition. In preclinical studies, LOXO-338 showed a favorable pharmacological profile, selectively inhibited BCL2, and was well-tolerated in vivo. LOXO-338 also demonstrated dose-dependent tumor growth inhibition in various murine xenograft models, and showed improved efficacy in combination with pirtobrutinib, a highly selective, non-covalent (reversible) BTK inhibitor (Brandhuber et al. Cancer Res 2021;81,13 Suppl:1258). Aims: To determine the recommended phase 2 dose of LOXO-338 and evaluate the safety profile of LOXO-338 as monotherapy and in combination. Methods: LOXO-BCL-20001 is a global, open-label, multi-center, first-in-human phase 1 study of oral LOXO-338 in patients with advanced hematologic malignancies who have received standard therapy (NCT05024045). The study will be conducted in 2 parts. Part 1 will evaluate LOXO-338 as monotherapy, and will explore different dosing strategies. Part 2 will evaluate LOXO-338 in combination with pirtobrutinib. Part 1 dose escalation portion of the study will follow an i3 + 3 design. Each cycle will be 28 days (4 weeks). Eligible patients include those with CLL/SLL, mantle cell lymphoma, and Waldenstrӧm macroglobulinemia (WM) who received standard therapy. Patients with other B-cell non-Hodgkin lymphomas (NHL) who failed standard therapy or, in the opinion of the investigator, have no known available options to provide benefit for the patient’s condition, are also eligible. Patients must have recovered from prior treatment-related adverse events. Patients with active or suspected Richter transformation, transformed low grade lymphoma, Burkitt or Burkitt-like lymphoma, and multiple myeloma are also eligible. Patients with AL amyloidosis are eligible in monotherapy dose expansion; all other patients are eligible in both monotherapy and combination dose expansion. Patients must not have progressed while receiving prior BCL2 inhibitor; those with WM or AL amyloidosis must not have received a prior BCL2 inhibitor. Key exclusion criteria include history of CNS involvement, stem cell transplant or CAR-T therapy <60 days, concurrent anticancer therapy, and clinically significant cardiovascular disease. The primary objective of part 1 is to determine the recommended phase 2 dose of oral LOXO-338 in patients who were previously treated for CLL/SLL and other B-cell NHL, administered alone or in combination with pirtobrutinib. Determining antitumor activity in patients with WM and AL amyloidosis is an additional primary objective of part 1 monotherapy. Secondary objectives include determining the safety profile and tolerability, PK properties and antitumor activity of LOXO-338 in combination with pirtobrutinib. Antitumor activity will be evaluated based on overall response rate (ORR), progression-free survival (PFS), time to progression (TTP) and duration of response (DOR) based on disease-specific response criteria per investigator assessment. Results: N/A Summary/Conclusion: N/A P684: PHASE 1 STUDY OF LP-108, A SELECTIVE BCL-2 INHIBITOR, IN PATIENTS WITH RELAPSED OR REFRACTORY B-CELL NON-HODGKIN LYMPHOMA W. Xu1,*, R. Feng2, L. Wang1, Y. Song3, H. Zhu1, L. Fan1, X. Guo2, X. Wei2, N. Lin3, C. Xu4, Z. Wang5, X. Xiao5, Y. Shen5, Y. Chen6, Y. Chen6, F. Tan5, S. P. Anthony6, J. Li1 1Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing; 2Department of Hematology, Nanfang Hospital of Southern Medical University, Guangzhou; 3Department of Lymphoma, Beijing Cancer Hospital, Beijing; 4Phase I Unit, Nanfang Hospital of Southern Medical University; 5Department of Clinical Development, Guangzhou Lupeng Pharmaceutical Co., Ltd., Guangzhou, China; 6Department of Clinical Development, Newave Pharmaceutical Inc., Pleasanton, United States of America Background: Targeting BCL-2 family proteins is a well-recognized therapeutic approach. Globally approved BCL-2 inhibitor venetoclax has shown efficacies in certain hematologic malignancies (HMs) but requires weekly ramp-up to the target dose to mitigate the risk of tumor lysis syndrome (TLS). LP-108 is a novel, highly potent, orally bioavailable, and selective BCL-2 inhibitor that has shown promising preclinical antitumor activity in various HMs. Aims: To present preliminary results from an ongoing phase 1 study of LP-108 under daily dose ramp-up in patients with relapsed or refractory (r/r) B-cell non-Hodgkin lymphoma (NHL) (NCT04356846). Methods: This is an open-label, multicenter, phase I study to assess the safety, pharmacokinetics (PK) and preliminary efficacy of LP-108 in Chinese patients with r/r B-cell NHL, consisting of a dose-escalation phase and a dose-expansion phase. Adult r/r B-cell NHL patients requiring therapy are eligible if they had received at least one prior line of treatment (except BCL-2i). LP-108 is orally administered once daily in 28-day cycles until disease progression, unacceptable toxicity, or per investigator discretion. All patients provided and signed informed consent. Results: As of February 18, 2022, 19 patients have been enrolled and were evaluable for safety, with disease types of MCL (n = 7), CLL/SLL (n = 7), DLBCL (n = 2), FL, WM, and MALT (n = 1 each), and median age of 61 years (range, 39 to 83). Patients had received a median of 3 prior lines of therapy (range, 1~6), of which 13 (68%) patients were previously BTKi exposed. No dose-limiting toxicity (DLT) has been observed in patients who were treated with LP-108 up to 600 mg, thus the maximum tolerated dose (MTD) was not identified. With median duration of treatment of 55 days (range, 4~472), treatment-related adverse events (TRAEs) were reported in 17 patients (89.4%), most of which were grade 1 to 2 in severity. TRAEs of any grade occurred ≥20% of patients were neutropenia (36.8%), leukopenia (31.5%), ALT elevation (31.5%), AST elevation (26.3%), hyperphosphatemia (21%), hyperuricemia (21%), and diarrhea (21%). Grade 3 or 4 TRAEs were reported in 7 patients (36.8%), including neutropenia, thrombocytopenia, lymphopenia, laboratory TLS, and rash. Serious TRAEs included rash, thrombocytopenia, and laboratory TLS, each occurring in 1 patient. No patient discontinued treatment due to toxicity, and no clinical TLS was observed. No deaths occurred during treatment or within 28 days of the end of treatment. At doses ≥200 mg, 3 of 10 efficacy evaluable patients achieved an objective response (1 CR and 2 PR), 4 patients had stable disease (2 with substantially reduced lymphomegaly), and 3 patients had progressive disease. Rapid reduction in ALC was observed in CLL patients (pending imaging assessment) during ramp-up and the first cycle of treatment at doses as low as 20mg. At data cutoff, 11 patients remain on treatment and 8 patients discontinued treatment due to disease progression or patient decision. The PK profile showed that plasma exposure proportionally increased with doses ranging from 20 to 600 mg (mean AUC24h range, 1.26~46.5 h*μg/ml, mean Cmax range, 0.087~2.67 μg/ml). Plasma concentrations peaked approximately 4 to 10 hours after dosing, and the average terminal half-life of LP-108 was approximately 11.8 hours (range, 6.9~16.6). Image: Summary/Conclusion: LP-108 showed favorable safety profile up to 600 mg with daily dose ramp-up schedule, and antitumor activity in patients with r/r B-cell NHL. Dose escalation beyond 600 mg and further dose expansion are ongoing. P685: REAL WORLD EVIDENCE OF IMPACT OF ATRIAL FIBRILLATION ON CLINICAL AND ECONOMIC OUTCOMES IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA K. Yang1, T. Liu2, B. Tang1, A. Chanan-Khan3,* 1Beigene USA, San Mateo; 2Beigene USA, Emeryville; 3Mayo Clinic, Jacksonville, United States of America Background: While the incidence of atrial fibrillation (AF) in chronic lymphocytic leukemia (CLL) has been increasing, the implications of AF in real-world CLL patients remain understudied. Aims: This study aimed to assess the impact of AF on clinical and economic outcomes in CLL patients. Methods: This retrospective observational study used the US IBM MarketScan® commercial claims/Medicare supplement database (2017-2020) to identify newly diagnosed CLL patients (≥18 years) and incidence of AF after index date, defined as the first CLL diagnosis date. Patients were followed for ≥3-months pre-index, and from index to last follow-up or death. In addition to patient characteristics, clinical outcomes (incidence of heart failure [HF], bleeding and stroke) and economic outcomes (costs and healthcare resource utilization [HRU]) were compared between CLL patients with AF (CLL+AF) and CLL patients without AF (CLL-AF). HRU was evaluated by inpatient, outpatient, ER, and pharmacy visits. Multivariable regression analyses were conducted to examine the association between AF and clinical outcomes. Results: Among a total of 16,800 newly diagnosed CLL patients included in the study, 20% developed AF. CLL+AF were significantly older than CLL-AF (median: 77 vs 62 years; P < .001). Compared to CLL-AF, CLL+AF also had significantly more comorbidities at baseline, as shown by higher Charlson comorbidity index (CCI) (median: 1.0 vs 3.0; P < .001) and more patients with previous AF history (0.3% vs 8.4%; P < .001). Further assessing clinical outcomes in CLL+AF vs CLL-AF, significantly higher incidence of HF (26.3% vs 3.0%; P < .001), bleeding (12.2% vs 4.8%; P < .001) and stroke (6.6% vs 1.2%; P < .001) were observed. For HRU, CLL+AF were reported to have significantly higher rates of ER visits (29.4% vs 12.9%; P < .001) and hospitalizations (42.2% vs 14.5%; P < .001) than CLL-AF. In CLL+AF, the average total AF-related costs were $13,520.21 within 30 days after AF diagnosis, and $22,304.82 within 60 days after AF diagnosis. Controlling for demographics and comorbidities, multivariable regressions reported statistically significant associations between AF and HF, as well as AF and stroke (Table). Image: Summary/Conclusion: This real-world study reported significantly higher incidence of HF, bleeding and stroke incurred by CLL patients who developed AF compared with those who did not. The presence of HF, bleeding and stroke further increased HRU and costs. These findings highlight the importance of better disease management and treatment selection to prevent AF in CLL patients. P686: A PHASE 1 FIRST IN-HUMAN STUDY OF BGB-16673, A BRUTON TYROSINE KINASE PROTEIN DEGRADER, IN PATIENTS (PTS) WITH B-CELL MALIGNANCIES (TRIAL IN PROGRESS) C. S. Tam1,2,3,*, C. Cheah4,5,6, D. A. Stevens7, K. By8, X. Chen8, B. Tariq8, G. S. Vosganian8, J. Huang8, M. Alwan9 1Peter MacCallum Cancer Centre, Melbourne, Victoria; 2University of Melbourne; 3Royal Melbourne Hospital, Parkville, Victoria; 4Department of Haematology, Sir Charles Gairdner Hospital and Pathwest Laboratory Medicine, Nedlands, Western Australia; 5Medical School, University of Western Australia, Crawley, Western Australia; 6Linear Clinical Research, Nedlands, Western Australia, Australia; 7Norton Cancer Institute, Louiseville, KY; 8BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA, United States of America; 9Perth Blood Institute, West Perth, Western Australia, Australia Background: Bruton tyrosine kinase (BTK) functions downstream of the B-cell antigen receptor (BCR) and plays a critical role within the BCR signaling pathway and the pathogenesis of several B-cell malignancies. BTK inhibitors (BTKi) have revolutionized management of B-cell malignancies. However, resistance caused by BTK mutations, which can abrogate BTKi binding capacity or BTK scaffold function, or cause kinase hyper activation, may limit therapeutic options in subsequent lines of therapy. Re-challenging patients with agents that can overcome the resistance due to BTK mutations may provide a novel treatment option. BGB-16673 is an investigational, orally available agent with preclinically demonstrated BTK degradation activity against both wild type and mutant forms commonly identified in pts who have progressed on BTKi. Aims: In the dose-escalation part of the study, we aim to assess the safety and tolerability of BGB-16673 in selected relapsed or refractory (R/R) B-cell malignancies, to characterize its pharmacokinetic (PK) and pharmacodynamic (PD) profiles, to determine a recommended phase 2 dose (RP2D) and to evaluate anti-tumor activity. In the dose-expansion part of the study, we aim to evaluate the safety, tolerability, PK, PD, and anti-tumor activity under the RP2D in pts with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL). Methods: BGB-16673-101 (NCT05006716) is a phase 1 open-label, dose-escalation and -expansion study evaluating BGB-16673 in adult pts with R/R B-cell malignancies. Key inclusion criteria include confirmed diagnosis of R/R B-cell malignancy (including marginal zone lymphoma, follicular lymphoma, MCL, CLL/SLL, and Waldenström macroglobulinemia [WM]), Eastern Cooperative Oncology Group performance status score of 0 to 2, and adequate organ function. Key exclusion criteria include current or history of central nervous system involvement, autologous stem cell transplant (ASCT) within 3 months or chimeric antigen receptor T cell therapy or allogeneic SCT within 6 months of the first dose of BGB-16673 or requiring treatment with strong inhibitors or inducers of CYP3A. All pts will be followed for safety and tolerability, including treatment-emergent adverse events that occur during treatment and up to 30 days after treatment discontinuation, or until the initiation of another anti-cancer therapy, whichever occurs first. The totality of the available safety, efficacy, PK, and PD data from the dose-escalation part will be used by the Safety Monitoring Committee to determine the RP2D. The dose-expansion part will commence subsequent to RP2D determination. Responses will be evaluated per the 2014 Lugano Classification, the 2018 International Workshop on CLL guidelines response assessment with modification for treatment-related lymphocytosis, or the 6th International Workshop on WM consensus criteria. Additional efficacy analyses will include progression-free survival and overall survival. All pts will give informed consent. Results: This is a trial in progress; safety and tolerability results of BGB-16673 are expected. Summary/Conclusion: BGB-16673-101 is the first in-human study of the BTK degrader BGB-16673. P687: A PHASE 1 STUDY WITH THE NOVEL B-CELL LYMPHOMA 2 (BCL2) INHIBITOR BGB-11417 AS MONOTHERAPY OR IN COMBINATION WITH ZANUBRUTINIB (ZANU) IN PATIENTS (PTS) WITH B-CELL MALIGNANCIES: PRELIMINARY DATA S. Opat1,2,*, C. Y. Cheah3,4,5, M. Lasica6, E. Verner7,8, P. J. Browett9, H. Chan10, J. D. Soumerai11, E. González Barca12, J. Hilger13, Y. Fang13, J. Huang13, D. Simpson13, C. S. Tam14,15,16 1Monash Health, Clayton; 2Monash University, Clayton, Victoria; 3Sir Charles Gairdner Hospital and Pathwest Laboratory Medicine, Nedlands; 4Medical School, University of Western Australia, Crawley; 5Linear Clinical Research, Nedlands, Western Australia; 6St. Vincent’s Hospital Melbourne, Fitzroy, Victoria; 7Concord Repatriation General Hospital, Concord; 8University of Sydney, Sydney, New South Wales, Australia; 9Auckland City Hospital; 10North Shore Hospital, Auckland, New Zealand; 11Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, United States of America; 12Institut Català d’Oncologia-Hospitalet, IDIBELL, Universitat de-Barcelona, Barcelona, Spain; 13BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA, United States of America; 14Peter MacCallum Cancer Centre, Melbourne; 15University of Melbourne, Parkville; 16Royal Melbourne Hospital, Parkville, Victoria, Australia Background: BCL2 is aberrantly expressed in many hematologic malignancies and promotes tumorigenesis by enabling cells to evade apoptosis. BCL2 inhibitors have an established role in the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and acute myeloid leukemia. The currently approved BCL2 inhibitor, venetoclax, is associated with mild gastrointestinal toxicities, neutropenia, and the development of BCL2 mutations leading to resistance. BGB-11417 is a highly selective inhibitor of BCL2 with superior potency to venetoclax in human acute lymphoblastic leukemia, mantle cell lymphoma (MCL) cell lines, and xenograft model of diffuse large B-cell lymphoma (DLBCL). BGB-11417 has favorable pharmacokinetics with excellent bioavailability and selectivity for BCL2. Toxicology studies have shown a broad therapeutic index and an improved safety profile. Aims: BGB-11417-101 is an ongoing first-in-human phase 1/1b dose-escalation and expansion study (NCT04277637) evaluating the safety, tolerability, maximum tolerated dose (MTD), and recommended phase 2 dose of oral BGB-11417 as monotherapy or in combination with the BTK inhibitor ZANU, in pts with B-cell malignancies. Methods: In separate monotherapy and combination therapy dose-escalation cohorts, pts with relapsed/refractory (R/R) B-cell malignancies received escalating doses of BGB-11417 (40, 80, 160, 320, or 640 mg once daily [QD]) with a weekly or daily ramp-up to intended target dose. Pts in the combination therapy cohorts received ZANU (320 mg QD or 160 mg twice daily) 8-12 weeks before BGB-11417. Dose-limiting toxicity for each dose cohort was evaluated by a Bayesian logistic regression model during dose ramp-up through day 21 at intended dose. Adverse events (AEs) were reported per Common Terminology Criteria for AEs v5.0. Results: As of 17 Dec 2021, 58 pts received BGB-11417 (32 monotherapy; 26 combination). Of the pts receiving monotherapy, 26 with R/R non-Hodgkin lymphoma (NHL; 17 DLBCL, 6 follicular lymphoma, and 3 marginal zone lymphoma) received BGB-11417 ≤640 mg and 6 with R/R CLL/SLL received ≤160 mg. Of the pts receiving combination treatment, 19 with R/R CLL/SLL received BGB-11417 ≤160 mg and 7 with R/R MCL received ≤80 mg. MTD has not yet been reached. Median follow-up was 3.9 months (range, 0.1-20.4). AEs across all dose levels are listed in the table. Only 2 grade ≥3 AEs (1 neutropenia, 1 autoimmune hemolytic anemia) were reported in combination cohorts. Twenty pts discontinued treatment (17 disease progression; 1 AE; 2 other reasons). One high-risk pt with CLL on monotherapy had laboratory tumor lysis syndrome (TLS) that resolved with no intervention (laboratory TLS <2%). Efficacy data are early: most pts had reduction in sum of product of perpendicular diameters; 2 pts with NHL (monotherapy) achieved responses (1 complete response). Pts with CLL/SLL had notable reductions in absolute lymphocyte count at doses as low as 1 mg; 2 responses (partial response or better) were seen with monotherapy and 12 responses with combination (partial response with lymphocytosis or better). Image: Summary/Conclusion: These preliminary data show promising efficacy for BGB-11417 and an improved safety profile, particularly in combination cohorts. Grade ≥3 neutropenia was uncommon. BGB-11417 is tolerable up to doses of 640 mg as monotherapy and up to 160 mg in combination with ZANU. Dose escalation continues as an MTD has not yet been reached in any dose-escalation cohort. Enrollment continues, with data for Waldenström macroglobulinemia and treatment-naïve CLL/SLL cohorts forthcoming. P688: VIRTUAL SCREENING PROTOCOL TO IDENTIFY TYROSINE KINASE INHIBITORS WITH A POTENTIAL TO INTERACT WITH P-GLYCOPROTEIN AND TO ACT AS MDR CHEMOSENSITIZERS: A VALIDATION STUDY E. Beleva1,2,3,*, I. Tsakovska1, P. Alov1, T. Pencheva1, I. Lessigiarska1, I. Pajeva1 1QSAR and Molecular Modeling, Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia; 2Clinical Oncology, Medical University - Plovdiv, Plovdiv; 3Faculty of Mathematics and Informatics, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria Background: Resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) has been attributed to both BCR-ABL-dependent pathways - acquisition of point mutations and BCR-ABL-independent ones – drug efflux mediated by ATP-binding cassette proteins. Primary multidrug resistance (MDR) has been mostly attributed to aberrant expression of P-glycoprotein (Pgp). Therefore research efforts have been directed to improving anticancer drug efficacy by co-administration of TKIs as Pgp modulators. Recently novel SRC family kinase (SFK) inhibitors based on the pyrazolol[3,4-d]-pyramidine scaffold were shown to inhibit Pgp in two MDR cancer cell lines with the proforms outlining a stronger potential to modulate Pgp [1]. Aims: In this study we aimed: (i) to validate the in-house developed virtual screening (VS) protocol for TKIs with a potential to interact with Pgp; (ii) to compare the Pgp binding potential of the novel SFK inhibitors (SI306, SI221, pro-SI306, pro-SI221) with that of widely explored TKIs (core group), including those used for treatment of CML - imatinib, nilotinib and dasatinib. Methods: Molecular modelling was performed in MOE software [2]. Two VS protocols were explored in the study: (A) docking without pharmacophore filter and with triangle matching placement; (B) an in-house developed VS protocol based on pharmacophore filter and docking with pharmacophore placement. The following docking settings were applied: (i) rigid receptor / flexible ligand mode; (ii) London dG scoring function applied at both the placement stage and the rescoring stage. VS protocol B is based on an in-house library of 43 TKIs collected from the scientific literature and DrugBank database. The library includes structural identificators of the compounds and experimental data proving their P-gp binding. It is freely available at http://biomed.bas.bg/qsarmm/Tyrosine_kinase_inhibitors.The VS protocols were applied to predict the Pgp binding affinity of the SFK inhibitors SI306, SI221, pro-SI306, and pro-SI221 as well as the core group drugs. Results: The results are presented in Table 1. The pro-forms showed higher interaction energies in the docking simulations compared to their parent ones in agreement with the experimental results pointing to the pro-forms having a stronger potential to modulate Pgp (Table 1) [1]. Further the comparison with the reference compound Dasatinib shows that except for SI221, the novel SFK inhibitors have a similar or even higher potential to interact with P-gp. The comparison with the core group of drugs outlines the following order of predicted P-gp affinity: (i) for VS protocol A: pro-SI306>pro-SI221>Nilotinib; (ii) for VS protocol B: pro-SI221>Lapatinib>pro-SI306>Tepotinib>Nilotinib. In both cases pro-SI306 and pro-SI221 are among the top-ranked P-gp ligands. Image: Summary/Conclusion: In summary, the previously developed VS screening protocol based on pharmacophore filtering and docking was validated on novel SFK inhibitors that were predicted as Pgp ligands in agreement with the experimental results. Further, the VS results outlined their similar or even higher affinity to Pgp compared to Dasatinib, which was used as reference compound due to its dual BCR-ABL and SRC inhibitory activity. Therefore they are suitable lead structures in the design of Pgp modulators able to restore sensitivity to TKIs in resistant tumour cell lines. The presented protocols as well as the investigated compounds might be useful for virtual screening/drug design of TKIs that act as MDR chemosensitizers in a combined anti-cancer therapy. P690: GENE MUTATIONAL PROFILE ASSOCIATED WITH TREATMENT FAILURE OR PROGRESSION IN CHRONIC MYELOID LEUKEMIA N. Estrada1,2,*, B. Xicoy3, M. Cabezón3, S. Marcé3, A. Senin4, A. Angona5, E. Alonso4, M. Ratia4, M. E. Plensa6, J. Buch7, X. Ortín8, L. Zamora3 1Myeloid Neoplasms, Josep Carreras Leukaemia Research Institute; 2Germans Trias i Pujol Health Science Research Institute (IGTP); 3Institut Català d’Oncologia (ICO)-Hospital Germans Trias i Pujol, Josep Carreras Leukaemia Research Institute, Badalona, Barcelona; 4Institut Català d’Oncologia - Hospital Duran i Reynals, Hospitalet de Llobregat; 5Institut Català d’Oncologia - Hospital Universitari Doctor Josep Trueta, Girona; 6Hospital de Mataró, Mataró; 7Hospital de Calella and Institut Català d’Oncologia-Girona, Girona; 8Hospital de Tortosa Verge de la Cinta, Tortosa, Spain Background: Approximately one third of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKI) fail to achieve optimal response due to resistance. It is known that BCR-ABL1 kinase domain (KD) mutations are associated with TKI resistance. However, only half on non-responsive patients carry BCR-ABL1 KD mutations. Other resistance mechanisms independent of BCR-ABL1 KD mutations, such as mutations in myeloid-related genes, may participate in the lack of response. Moreover, resistance to TKI therapy confers a much higher risk of progression to advanced disease stages of CML, such as accelerated phase (AP) and blast crisis (BC), and consequently, a worse prognosis. Aims: The main objective of this study was to explore the impact of mutations in myeloid-related genes in TKI resistance and progression to AP/BC by targeted deep-sequencing (TDS). Methods: The mutation profile study was done by TDS using a 32 myeloid-related gene panel (Myeloid Solution Capture Kit, Sophia Genetics, Switzerland). The platform Sophia DDM (Sophia Genetics) was used for data analysis. Regarding variant filtering, sequencing and mapping errors were eliminated and intronic and synonymous variants were filtered out. Variants present with a minor allele frequency (MAF) >1%, according to population databases (ExAc, 1000 genomes), were considered polymorphic changes without clinic relevance. COSMIC, VARSOME and Franklin databases as well as in silico functional predictors (SIFT, PolyPhen2 and MutationTaster) were used for variant interpretation. Variants were also filtered according to the variant allele frequency (VAF): all variants with VAF ≥5% were reported, as well as hot-spot variants with VAF 2-5% and at least 25 reads. Only variants described as pathogenic or potentially pathogenic were reported. Finally, BCR-ABL1 mutations were analyzed by Sanger in resistant patients. Results: TDS was performed in a total of 78 samples from 67 patients. Across patients with available sample at diagnosis, 25% (16/64) of patients harbored at least one mutation. Overall, 11 (17.2%) patients had 1 mutation, 3 (4.6%) had 2 mutations, 1 (1.6%) patient had 3, and 1 (1.6%) patient had 4 mutations (Figure 1A). Most frequently mutated genes at diagnosis were ASXL1 (14.1%), DNMT3A (6.3%), JAK2 (3.1%) and TET2 (3.1%), followed by ETV6, EZH2, IDH2, SETBP1, SRSF2, TP53 and WT1, all of them at a frequency of 1.6% (Figure 1B). Of the 67 CML patients included, 15 (22.4%) had resistance to first-line TKI and 5 of them (33%) progressed to AP/BC. From the 15 resistant patients, TDS was performed in 12 cases at diagnosis, in 5 at time of resistance, and at progression in 4/5 patients. At diagnosis, 6 (50%) resistant patients harbored 1 mutation in ASXL1 (3/12), DNMT3A (2/12) or TET2 (1/12) and 1 patient harbored 2 mutations (ASXL1 and WT1). Only 9 of 52 (17.3%) non-resistant patients harbored ≥1 mutation at diagnosis; most frequently mutated gene was ASXL1 (5/9). At time of resistance, 5 patients had BCR-ABL1 mutations by Sanger technique and 3 patients harbored cancer-related gene mutations by TDS (ASXL1 in 2 patients). At progression, all patients harbored at least 1 mutation by TDS, except one patient in which only 2 mutations in BCR-ABL1 KD were detected. Image: Summary/Conclusion: ASXL1 mutation was the most frequent mutation in CP at diagnosis and ASXL1 and ABL1 were the most common variants at resistance and progression. Acquisition of mutations was detected during CML progression. Acknowledgments: this work was supported by a grant from Incyte (EU-ES-H-18021) P691: MODELLING OF IMMUNE RESPONSE IN CHRONIC MYELOID LEUKEMIA PATIENTS SUGGESTS POTENTIAL FOR TREATMENT REDUCTION PRIOR TO CESSATION E. Karg1, C. Baldow1, T. Zerjatke1, R. E. Clark2, I. Roeder1,3, A. C. Fassoni4, I. Glauche1,* 1Institute for Medical Informatics and Biometry (IMB), TU Dresden, Dresden, Germany; 2Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom; 3National Center for Tumor Diseases (NCT), Partner Site Dresden, Dresden, Germany; 4Instituto de Matemática e Computação, Universidade Federal de Itajubá, Itajubá, Brazil Background: Chronic myeloid leukemia (CML) is a malignancy driven by the characteristic BCR-ABL1-fusion oncogene, while the BCR-ABL1 transcript is used as a marker for diagnosis and monitoring. The introduction of tyrosine kinase inhibitors (TKI) marked a substantial progress in CML therapy. Current efforts focus on the discontinuation of TKI therapy for well-responding patients as the long-term drug administration is associated with side effects and high economical costs. Many studies reported that about 50% of patients can achieve sustained treatment free remission (TFR) while 50% present with molecular disease recurrence, typically within two years of stopping. It has been speculated that the immune system (IS) plays a major role in the control of residual disease levels and influences the individual recurrence behaviour. The recently published DESTINY trial (Clark et al., Lancet Haematol, 2019; NCT 01804985) showed that TKI dose reduction prior to cessation can proactively increase the fraction of patients to remain in sustained TFR. However, there has been no systematic investigation to evaluate how dose reduction regimens can further improve the success of TKI stop trials. We have previously shown that mathematical models of CML can correctly describe patient time courses after TKI stop (Hahnel et al., Cancer Res, 2020). We classified patients into different groups according to their predicted immune system configuration and showed that one class of patients required complete CML eradication to achieve TFR, while other patients were able to control residual leukemia levels after treatment cessation. While a prospective stratification of the patients is difficult to achievable, we aimed to investigate whether a further optimized dose reduction scheme prior to stopping could further improve TFR. Aims: We aim to use a mathematical description of patient time courses to investigate different TKI dose reductions schemes prior to therapy cessation and evaluate them with respect to the total amount of drug used and the expected TFR success. Methods: Our analysis is based on a cohort of 72 patients from the DESTINY trial (Clark et al., Lancet Hematol, 2019; NCT 01804985). We applied an established mathematical model (Hahnel et al., Cancer Res, 2020) and obtain individual fits for all patients using a genetic algorithm. Applying a re-sampling approach we derive estimates of recurrence times and fractions for different dose reduction schemes in which the timing and the amount of dose reduction are varied in a systematic manner. Results: We use computer simulations to show that TKI dose reduction prior to treatment cessation appears as an equally efficient approach to achieve high levels of CML patients remaining in TFR compared to full dose treatment for the same duration. Our model simulations confirm clinical findings that the overall time of TKI treatment is a major determinant of TFR success, while at the same time indicating that lower dose TKI treatment is sufficient for many patients. Our model results indicate that a stepwise dose reduction prior to TKI cessation (e.g. 12 month post MR4 at 100% TKI, 12 more months at 50% and 12 more months at 25%) does not limit the overall success rate of TFR, while it substantially reduces the amount of total administered TKI and thereby decreases side effects as well as overall treatment costs. Summary/Conclusion: Our findings illustrate the potential of dose reduction schemes prior to treatment cessation and suggest corresponding and clinically verifiable strategies that are applicable to many CML patients. P692: IMMUNE FACTORS MAINTAINING TREATMENT-FREE REMISSION IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA AFTER DISCONTINUATION OF IMATINIB P. Kwaśnik1,*, J. Zaleska1, D. Link-Lenczowska2, M. Zawada2, B. Ochrem2, G. Bober3, E. Wasilewska4, E. Mędraś5, I. Hus6,7, M. Szarejko8, W. Prejzner8, O. Grzybowska-Izydorczyk9, A. Klonowska-Szymczyk9, T. Sacha2, K. Giannopoulos1,10 1Department of Experimental Hematooncology, Medical University of Lublin, Lublin; 2Department of Haematology, Jagiellonian University Hospital in Krakow, Krakow; 3Department of Hematooncology and Bone Marrow Transplantation, Medical University of Silesia, School of Medicine in Katowice, Katowice; 4Department of Hematology, Medical University of Bialystok, Bialystok; 5Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation, Medical University of Wroclaw, Wroclaw; 6Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw; 7Department of Clinical Transplantology, Medical University of Lublin, Lublin; 8Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk; 9Department of Hematology, Medical University of Lodz, Copernicus Memorial Hospital, Lodz; 10Department of Hematology, St John’s Cancer Center, Lublin, Poland Background: CML treatment aims to achieve long-term remission without treatment (TFR). The mechanisms that are responsible for CML relapses remain elusive. It is suggested that maintaining TFR is not directly related to the total disposing of the gene transcript BCR-ABL1, but it might be a result of the immune surveillance restoration in CML. Aims: To determine immune factors responsible for elimination or control over residual CML cells, which may be crucial for achieving long-term relapse-free survival (RFS) and TFR after imatinib discontinuation. Methods: Peripheral blood mononuclear cells from 63 CML patients were analyzed using flow cytometer analysis at two-time points: at discontinuation of imatinib and 3 months after withdrawal. The major populations of the immune system that have a potential effect on the maintenance of the patient in remission were assessed. BCR-ABL1 transcript were analyzed using two molecular biology methods: standard real-time quantitative polymerase chain reaction (RQ-PCR) and droplet digital PCR (ddPCR). Results: We found a significant increase in the percentage of HLA-DR+ cells (median 1.25 vs 1.90%, p<0.0001), mDC (median 0.20 vs 0.28%, p<0.01), pDC (median 0.10 vs 0.15%, p<0.01) and a significant decrease in PD-1 expression on mDC cells (median 35.81 vs 32.23%, p=0.03) in 3 months after treatment discontinuation. There was a significantly reduced level of NKT cells (median 14.45 vs 12.80%, p=0.02), a significantly increased PD-1 expression on CD161+ cells (median 21.02 vs 21.28%, p=0.02) and a significantly decrease of PD-1 expression on CD56brightCD16- cells (median 2.31 vs 2.08%, p=0.04). Analyses of T cells showed a significant decrease in frequencies of CD4+CD25+ T cells (median 18.01% vs 13.42%, p<0.0001), CD4+CD25+FOXP3+ (median 3.00 vs 2.89%, p=0.02) and a significantly lower expression levels of PD-1 on CD4+ cells (median 21.59 vs 19.34%, p=0.02) after 3 months compared to moment of withdrawal. We found a weak negative correlation between age and CD19+ cell depletion (p<0.01, R=-0.39) and a moderate positive correlation between age and PD-1 expression on CD19+ cells (p<0.001, R=0.43) at the moment of withdrawal. In addition, we found a weak positive correlation between age and HLA-DR+ cell count (p=0.01, R=0.32) and a weak negative correlation with CD19+ cell count (p=0.04, R=-0.26) in 3 months after stopping treatment. Significantly higher levels of NK cells: CD56dimCD16+ (median 8.93 vs 14.23%, p<0.01) and CD56brightCD16- (median 0.33 vs 0.52%, p=0.02) were found in men compared to women 3 months after withdrawal. Analysis of the BCR-ABL1 transcript by both RQ-PCR and ddPCR methods in relation to the immune subpopulations showed significant differences between the percentage of HLA-DR+ cells and the depth of the molecular response: in RQ-PCR greater than or equal to MR4.0 at the moment of withdrawal vs loss of MR4.0 after 3 months (median 1.24 vs 1.87, p=0.03); greater than or equal to MR4.0 at the moment of withdrawal vs greater than or equal to MR4.0 after 3 months (median 1.24 vs 1.92, p<0.001). Summary/Conclusion: The demonstrated changes in the percentage of populations of immune cells show the importance of immunocompetent cells’ involvement in maintaining the patient in TFR. The characterization of the immune system, which probably plays the most important role in achieving long-term relapse-free response and maintaining remission without treatment, may help define the group of patients who could safely discontinue imatinib. Funding: NCN 2018/31/B/NZ6/03361 P693: DIAGNOSTIC ROLE OF CD26+ LEUKEMIC STEM CELLS IN CHRONIC MYELOID LEUKEMIA S. Tiwari1,*, J. Dass1, G. Vishwanathan1, R. Dhawan1, M. Agarwal1, P. Kumar1, T. Seth1, S. Tyagi1, M. Mahapatra1 1Hematology, All India Institute of Medical Sciences, New Delhi, India Background: Diagnosis of CML consists of persistent leucocytosis with the demonstration of the Philadelphia (Ph) chromosome abnormality, the t(9;22)(q34;q11), by routine cytogenetics, fluorescence in situ hybridization (FISH) or by molecular studies such as RT-PCR.These conventional methods suffer from a longer turn around time, higher costs and requirement of labs with adequate expertise. CD26 is a highly specific marker expressed in CML stem cells, which is not present on normal hematopoietic stem cells or on Leukemia stem cells (LSCs) of other myeloid neoplasms. A flow-cytometry based assessment of CD26+LSCs may prove to be an optimal biomarker for the diagnosis and monitoring of CML patients. Aims: 1)Assessment of CD 26 expression in suspected cases of CML-chronic phase 2)Correlation of CD 26+ stem cells with clinico-pathological parameters at baseline and its kinetics on tyrosine kinase inhibitor (TKI) treatment on further follow-up at 12 months. Methods: Suspected patients of CML-CP were included in the study. Peripheral blood were utilized for flowcytometric assessment of CD26+ LSCs. The results were correlated with conventional diagnostic tests of CML. Patients with myeloid neoplasms other than CML who proved negative for BCR-ABL1 by RT-PCR and normal HSCs donors treated with granulocyte-colony stimulating factors (G-CSF), were included as negative controls. All samples were processed using standard stain -lyse-wash method within 24 hr of collection. To reach a sensitivity of 10−5, acquisition of at least 1.0 × 106 cells was performed on FACS Canto II flow cytometer data a FACS DIVA 8.0.3 software (BD, Biosciences).CD26 expression was evaluated by a sequential gating strategy. After exclusion of doublets and debris CD45 dim to intermediate/CD34+/CD38- population was gated sequentially and then the expression of CD 26+ was studied on this population. All the samples were simultaneously assessed for BCR-ABL1 transcript by standard RT-PCR analysis. Patients on treatment were also followed up at 12 months to detect levels of CD26+ LSCs and correlated with BCR -ABL1 levels by qPCR. Results: A total of 86 patients of suspected cases of CML were included in the study from June 2020 to Dec 2021.The On flowcytometry analysis, CD 26 expression was present in 75 cases. All the cases with positive CD26+ LSCs were found to be positive on RT-PCR for BCR-ABL transcript. Eleven cases which were scored negative for CD26+ LSCs were also found to be negative for any BCR-ABL transcript. These negative cases included 8 cases of primary myelofibrosis, 2 cases of chronic myelomonocytic leukemia and 1 case of Polycythaemia Vera. The median percentage of CD26+ LSCs was 39.8% (Range-0.5-100). There was no significant correlation of the CD26 levels with age (p value=0.96), sex (p value=0.7), percentage of blasts (p value = 0.88), total leucocyte counts (p value=0.32), basophils (p value = 0.19), Sokal score (p value = 0.99) and ELTS score (p value = 0.99). Follow-up data was available for 7 patients and at 12 months all of these patients had achieved major molecular remission on TKIs. Two patients scored negative for CD26+LSCs while five patients showed significant fall in the level of CD 26+ LSCs however it was still detectable by flowcytometry (p value= 0.04). Summary/Conclusion: Flowcytometry assessment of CD26 + LSCs is a rapid and cheap diagnostic tool for diagnosis of CML with high diagnostic efficacy. Falling levels on TKIs warrants further research to prove it as a surrogate for monitoring response by conventional methods. P694: INVESTIGATING THE ROLE OF CELL ADHESION MOLECULES IN TUNNELING NANOTUBES FORMATION IN CHRONIC MYELOID LEUKEMIA MICROENVIRONMENT L. Turos-Korgul1,*, A. Zieminska1, L. Parker2, K. Piwocka1 1Laboratory of Cytometry, Nencki Institute of Experimental Biology PAS, Warsaw, Poland; 2Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, United States of America Background: The cross-talk and interactions between leukemia cells and their microenvironment play an important role in the development of drug resistance. Tunneling nanotubes (TNTs) represent a novel type of direct cell-cell communication way allowing for direct transfer of different types of cargo between distant cells. Our previous studies (Kolba et al., 2019) have shown that these structures are formed between bone marrow stromal HS-5 cells and chronic myeloid leukemia (CML) K562 cells. Significantly, TNT-mediated transfer of membrane vesicles from stromal to leukemic cells resulted in the stroma-mediated protection of CML cells from imatinib-induced apoptosis. Despite the efforts made to understand the molecular mechanisms regulating TNTs formation, they still remain unclear - especially in the leukemia microenvironment. We have shown that TNTs are formed between stromal and leukemic cells, following direct cell-cell interaction and cell dislodgement. Thus, we have focused our attention on cell adhesion molecules, as potential key players in tunneling nanotubes formation and TNT-mediated vesicles transfer between leukemic and stromal cells. Aims: The aim of presented studies was to elucidate the role of CD44 and ITGβ1 - one of the most important cell adhesion molecules, in TNTs formation between leukemic and stromal cells and TNT-mediated exchange of vesicles. Methods: To examine this, we established the HS-5 stromal cell line with silenced CD44 protein expression. Then, we co-cultured control (wt) or CD44-silenced HS-5 cells with leukemic K562 cells. For ITGβ1 studies, we used blocking anti-ITGβ1 antibody. Using confocal microscopy, three types of TNTs in co-cultures were counted: homotypic TNTs formed between stromal cells, homotypic TNTs formed between CML cells and TNTs formed between stromal and CML cells (heterotypic). Moreover, the TNT-mediated transfer of cellular vesicles from stromal to leukemic cells was assessed in these co-cultures using flow cytometry. Since CD44 expression increases in CML cells after co-culture with stroma, we examined its potential role in the drug resistance in CML acceptor cells. To do this, rhodamine-123 efflux assay was used. Results: We found that both, tunneling nanotubes number and uptake of cellular vesicles by leukemia, decreased after co-culture with CD44-silenced stromal cells and after blocking with anti-ITGβ1 antibody. Flow cytometry analyses of rhodamine-123 efflux showed significantly increased efflux pump activity in leukemic acceptor cells. Summary/Conclusion: Our data suggest that CD44 and ITGβ1 can play an important role in formation of tunneling nanotubes between leukemic and stromal cells and TNT-mediated cellular vesicles exchange. Moreover, CD44 presented in acceptor cells is involved in the development of drug resistance in leukemic cells, by promoting activity of efflux transporters. P695: PROGNOSTIC IMPACT OF ASXL1 MUTATIONS IN CHRONIC PHASE CML A. Bidikian1,*, H. Kantarjian1, E. Jabbour1, N. Short1, F. Ravandi1, G. Issa1, K. Sasaki1 1Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America Background: While the clinical impact of mutations in the ABL1 gene on response to therapy in chronic phase chronic myeloid leukemia (CP-CML) is well established, less is known about mutations in genes frequently involved in myeloid malignancies. Aims: To characterize the clinical impact of mutations in non-ABL1 genes in CP-CML. Methods: We identified 66 patients (pts) with CP-CML where targeted next generation sequencing was performed at our institution. This panel included 81 genes recurrently mutated in myeloid malignancies. Event-free survival (EFS) was measured from treatment initiation to the time of an event defined as loss of hematologic response, loss of major cytogenetic response (MCyR), transformation to accelerated or blast phase and lack of MCyR after 12 months of treatment. Failure-free survival (FFS) was measured similarly with the additional events of drug discontinuation due to lack of response or adverse effect. We subsequently performed univariate and multivariate analyses for survival. Results: The median age of pts was 61 years (18 – 80 years) and 48% were female. The most common first line therapy in these patients was Dasatinib (52%), followed by Imatinib (19%), Dasatinib + Venetoclax (16%), Nilotinib (8%), Bosutinib (3%) and Ponatinib (2%). The distribution of evaluable patients to low, intermediate and high Sokal risk groups was 22%, 62% and 16%, respectively. Non-ABL1 mutations were found in 22/66 patients (33%), with 5 (8%) of pts with at least 2 mutations. Most common mutations involved ASXL1 (8/66 pts; 12%), DNMT3A (4/66 pts; 6%) and RUNX1 (3/66 patients; 5%). Mutations in ASXL2, CALR, EZH2, GNAS, IDH2, SF3B1, SMC3, SRSF2, STAG2 and SUZ12 were detected at least once. Unlike mutations in other genes, mutated ASXL1 was associated with worse EFS compared with wild-type with a median of 33 vs 88 months (P = 0.009) (Figure 1A) and worse FFS with medians of 32 vs 58 months, respectively (P = 0.05) (Figure 1B). There was no difference in overall survival by mutational status (Figure 1C). In a multivariate analysis including all baseline prognostic variables, mutated ASXL1 was independently associated with worse EFS with a hazard ratio of 5.94 (95% CI 1.25 – 28.21; P = 0.03). There was no difference in rates of cytogenetic and molecular responses by mutational status. However, time to achievement of response was longer in patients with mutated ASXL1 albeit not statistically significant. (Figure 1D) Image: Summary/Conclusion: Mutations in ASXL1 are associated with delayed responses and worse outcomes when detected in chronic phase CML. P696: SUBTHERAPEUTIC TKI DOSES FOR THE MAINTENANCE OF CML PATIENTS WITH INTOLERANCE OR REFUSAL TO DISCONTINUE: A SINGLE CENTER FEASIBILITY STUDY. A. Borrero1,*, A. Segura1, S. Sanchez1, R. Stuckey1, J. F. López1, C. Bilbao1, M. T. Gomez casares1 1HOSPITAL UNIVERSITARIO DE GRAN CANARIA DOCTOR NEGRIN, LAS PALMAS DE GRAN CANARIA, Spain Background: Discontinuation has become a common practice in patients with CML treated with TKI who have maintained a deep molecular response. 50-60% of patients successfully discontinue treatment without relapsing (treatment-free remission, TFR), which implies that 40-50% will have to remain with TKI treatment for life. Many studies have been carried out on TKI treatment with reduced doses, often used to manage adverse events and TKI intolerance. Such studies have shown that most patients maintain MMR and many even achieve it when reduced doses are administered from diagnosis. There are fewer cases reported of patients who receive subtherapeutic doses, i.e. doses lower than those considered in the TKI data sheet. Reasons why subtherapeutic doses may be employed in clinical practice include the patient’s refusal to discontinue or a failed discontinuation attempt (loss of TFR), as a maintenance strategy, or in those patients with intolerance. Although its use is likely, information on patient outcome is scarce. Aims: We investigated the feasibility of the use of subtherapeutic doses in CML patients. We studied the reason for the dose reduction and the impact of the dose reduction on molecular response. Methods: In this observational, retrospective, single-center study, we searched the medical records of all patients diagnosed with CML in chronic phase at our hospital between 2003 and 2020. Patients were identified who were treated with TKIs at subtherapeutic doses. Subtherapeutic doses were defined as doses lower than those considered in the TKI data sheet: dasatinib 20 mg, imatinib 100 mg or 200 mg, nilotinib 150 mg, bosutinib 100 mg. Results: Medical records were searched and 13 CML patients who received infratherapeutic TKI doses were identified, 7 men (53.9%) and 6 women (46.2%) with a mean age of 75.5 years. Of these patients, 7 (53.8%) received imatinib (6 with 200 mg doses and 1 with 100 mg), 1 (7.7%) received bosutinib 100 mg and 5 (38.5%) received dasatinib 20 mg. The mean follow-up time from the start of the dose reduction was 60.5 months and 40.7 months from the last reduction. None of the patients presented TKI resistance or progression during the follow-up period. 3 patients reduced the dose as a practical alternative for fear of discontinuation; 2 patients discontinued, but after relapse they remained with the minimum dose to maintain MMR; the rest of the patient reduced doses because of intolerance. All patients presented MMR at last follow-up and 92.3% deep MMR (≥MR4). Summary/Conclusion: Our results show that TKI doses can be optimized on an individual basis to infratherapeutic levels and MMR or better can be maintained. Although only a small series of patients was studied, no patients developed TKI resistance or progression while receiving infratherapeutic doses during an average period of 5 years. For intolerant patients who received infratherapeutic doses, adverse events were resolved and quality of life improved. The administration of infratherapeutic TKI doses could also be a good option for patients who relapse after a discontinuation attempt or for those who do not want to discontinue, as a form of “maintenance” therapy. The use of infratherapeutic doses, rather than a full TKI discontinuation, would still provide economic savings. Moreover, it would be interesting to evaluate if long-term use of infratherapeutic doses can prevent the development of serious off-target TKI toxicities, such as vascular events. Studies with larger patient cohorts are needed to validate the feasibility of this clinical practice. P697: PONATINIB IN A REAL-LIFE SETTING: A RETROSPECTIVE ANALYSIS FROM THE MONITORING REGISTRIES OF THE ITALIAN MEDICINES AGENCY (AIFA) M. Breccia1,*, P. P. Olimpieri2, S. Celant2, O. M. Olimpieri2, F. Pane3, A. Iurlo4, V. Summa2, P. Corradini5, P. Russo2 1Department of Translational and Precision Medicine, Sapienza University; 2Italian Medicines Agency, AIFA, Roma; 3Federico II University, Napoli; 4Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 5Università degli Studi di Milano & Divisione Ematologia, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milano, Italy Background: The efficacy and safety of ponatinib, a third-generation tyrosine kinase inhibitor (TKI), are mainly reported in sponsored clinical trials. Aims: The aim of this study, based on a retrospective analysis from the monitoring Registries of the Italian Medicines Agency (AIFA wMRs), is to provide real-world data on daily practice management, treatment modifications and outcome of a large cohort of chronic myeloid leukemia (CML) patients treated with ponatinib. Methods: AIFA wMRs is an administrative database whose main scope is monitoring the appropriateness of drug prescription in Italy. Information collected through the wMRS included demographic (place of birth, age, and sex) and clinical data (BCR-ABL levels, mutations and chromosomal abnormalities if evaluated), drug prescription and administration data (date of prescription and administration, dose administered, dose changes, occurrence of adverse events as yes/no dichotomic variable, reasons for treatment interruption and/or discontinuation) and response to treatment (response assessment was requested every 90 days). Subjects who had not been administered ponatinib for ≥ 120 days were adjudicated as “discontinued” even in the absence of the “end of treatment form”. Time to treatment discontinuation (TTD) was defined as the time occurring between the date of first administration and the date of treatment discontinuation for any cause, including death. Results: Overall, 666 CML subjects were eligible for analysis: 515 patients had chronic phase (CP) CML, 50 accelerated phase (AP) and 101 blast phase (BP). Median age at baseline was 58.7 years, male prevalence (57.1%). Median time from diagnosis to start of ponatinib was 2.35 years: 259 (38.9%) subjects had received 2 lines of treatment, 260 (39.0%) 3 lines and 147 (22.1%) 4 or more lines. Mutational status was available for 58.3% of patients (n=388), with a T315I mutation reported in 46 (6.9%) patients and other mutations (the most represented being E255K, F317L, Y253H and V299L) in 99 (14.9%) subjects. At baseline, 181 patients (27.2%) had arterial hypertension, 33 (5.0%) a history of arterial and/or venous thromboses, 32 (4.8%) a pre-existing ischemic heart disease, and 13 (2.0%) a history of congestive heart failure. Overall, 593 patients (89.0%) were evaluable for best response. Ten cases (1.7%, 3 AP/BP and 7 CP) did not achieve molecular response, whereas 58 patients (8.7%, 26 AP/BP patients and 32 CP) reached a BCR/ABL1 ratio between 1% and 10% IS. A MR2 (less than 1%IS) was obtained by 82 subjects (12.3%, 20 AP/BP and 62 CP patients); 128 patients (19.2%, 12 AP/BP and 116 CP patients) achieved a molecular response ranged between MR3 (0.1%) and MR4 (0.01% IS) and 266 (39.9%, 44 AP/BP and 222 CP patients) a deeper molecular response (< 0.01% IS). With a median follow-up of 14.4 months, 136 subjects (20.4%) required at least one dose reduction due to adverse events; 309 patients (46.4%) had their dose decreased in the absence of any evidence of side effects. Treatment discontinuation occurred in 144 patients (21.6%): intolerance (7.4%), primary resistance (3.5%) and acquired resistance (5.6%). Kaplan-Meier estimated TTD was 47.2 months (CI 95% 39.3 – NA) and 7.3 months (CI 95% 4.6 – 11.8) for CP and AP/BP patients, respectively. The probability of treatment discontinuation did not significantly differ for ponatinib in second, third or subsequent line of therapy (p=0.58). Summary/Conclusion: This real-life investigation shows that ponatinib dose reductions were mainly performed in the absence of reported toxicity rather than owing to the occurrence of adverse reactions. P698: BOSUTINIB DOSE OPTIMIZATION IN THE SECOND-LINE TREATMENT OF ELDERLY CML PATIENTS: EXTENDED 3-YEAR FOLLOW-UP AND FINAL RESULTS OF THE BEST STUDY F. Castagnetti1,2,*, M. Bocchia3, E. Abruzzese4, I. Capodanno5, M. Bonifacio6, G. Rege Cambrin7, M. Crugnola8, G. Binotto9, C. Elena10, A. Lucchesi11, M. Bergamaschi12, F. Albano13, L. Luciano14, F. Sorà15, F. Lunghi16, F. Stagno17, M. Cerrano18, A. Iurlo19, A. R. Scortechini20, S. Leonetti Crescenzi21, R. Spadano22, E. Trabacchi23, M. Lunghi24, G. Spinosa25, D. Ferrero26, D. Rapezzi27, M. Ladetto28, L. Nocilli29, G. Gugliotta1, M. Iezza1, M. Cavo1,2, G. Saglio30, F. Pane14, G. Rosti11 1Istituto di Ematologia “Seràgnoli”, IRCCS Azienda Ospedaliero-Universitaria di Bologna; 2Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, BOLOGNA; 3Hematology Unit, University of Siena, Siena; 4Hematology Unit, S. Eugenio Hospital, Tor Vergata University, Roma; 5Hematology Unit, Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia, Reggio Emilia; 6Department of Medicine, Section of Hematology, University of Verona, Verona; 7Internal Medicine Unite, San Luigi Gonzaga Hospital, Orbassano (TO); 8Hematology Unit, Azienda Ospedaliero-Universitaria di Parma, Parma; 9Department of Medicine, Hematology and Clinical Immunology, University Hospital of Padova, Padova; 10Department of Hematology Oncology, Foundation IRCCS Policlinico San Matteo, University of Pavia, Pavia; 11Hematology Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola (FC); 12Hematology Unit, Policlinico S.Martino-IRCCS, Genova; 13Hematology Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari; 14Hematology Unit, Federico II University, Napoli; 15Hematology Unit, Policlinico A. Gemelli, Università Cattolica del Sacro Cuore, Roma; 16Hematology and Bone Marrow Transplant Unit, San Raffaele Scientific Institute IRCCS, Milano; 17Hematology Section and BMT Unit, AOU Policlinico V. Emanuele, Catania; 18Hematology Unit, AOU Città’ della Salute e della Scienza, Torino; 19Hematology Unit, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano; 20Hematology Unit, A.O.U. Ospedali Riuniti, Ancona; 21Hematology Unit, San Giovanni - Addolorata Hospital, Roma; 22Hematology Unit, Casa Sollievo Della Sofferenza, San Giovanni Rotondo (FG); 23Hematology and BMT Unit, Department of Hematology and Oncology, Guglielmo da Saliceto Hospital, Piacenza; 24Hematology Unit, AOU Maggiore della Carità, Novara; 25Hematology Unit, Azienda Ospedaliero Universitaria Ospedali Riuniti, Foggia; 26Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino; 27Hematology Unit, AO S. Croce e Carle, Cuneo; 28Hematology Unit, Ospedale SSAntonio e Biagio e Cesare Arrigo, Alessandria; 29Hematology Unit, Azienda Ospedaliera Papardo, Messina; 30Divisione Universitaria di Ematologia e Terapie Cellulari, A.O. Ordine Mauriziano, Torino, Italy Background: The median age of CML patients requiring a second-line treatment is older than 60 years and many of these patients have relevant comorbidities. Bosutinib (BOS) is a second-generation TKI, with similar efficacy to dasatinib and nilotinib, but more favorable toxicity profile in the elderly, because of lower incidence of cardiovascular (CV) adverse events (AEs). BOS safety profile may be an added value in this setting, but a fixed initial dose of 500 mg OAD may be higher than necessary. Aims: All TKIs have been approved in CML at a specific initial dose, with dose reductions only in case of toxicity. The aim of our study was to evaluate the efficacy of low-dose BOS in the second line treatment of elderly CML patients, with subsequent dose increase only in patients without optimal molecular response at given timepoints (dose optimization). Methods: A prospective phase 2 single-arm study has been designed by the GIMEMA CML Working Party (NCT02810990). Study design: all patients, ≥ 60 yrs old, started BOS 200 mg for 2 weeks (“run-in” period), then the dose was increased to 300 mg; after 3 months, pts with BCR-ABLIS transcript ≤ 1% continued 300 mg, while in pts with transcript > 1% the dose is furtherly increased to 400 mg, in absence of relevant toxicity. The primary endpoint was the rate of MR3 at 12 months (core phase), but all pts were followed for additional 2 years (extension phase) to describe the long-term efficacy and outcome. Results: Sixty-three pts have been enrolled. Median age: 73 yrs (range 60-90). Reasons for switching to BOS: intolerance 65%, resistance 35%. First-line TKI: imatinib 83%, DAS 11%, NIL 6%. The rate of pts in MR3 at 12 mos was 59% (already reported). Median follow-up 38 mos (range 35-59). Maximum BOS dose: 400 mg, 21%; 300 mg, 73%; 200 mg, 6%. The probabilities of achieving or maintaining MR3, MR4 and MR4.5 by 36 mos were 78%, 54% and 46%, respectively (excluding pts with response at baseline, 73%, 50% and 44%, respectively); the probabilities were lower in the resistant cohort (64%, 36% and 18%, respectively). Overall, 24%, 33% and 11% of pts had 1 log, 2 logs or ≥ 3 BCR-ABLIS transcript logs reduction from baseline (68% of pts had a molecular improvement from baseline). At 36 mos, 36 pts (57%) were still on BOS (33 pts in optimal response, 3 pts in MR2), while 27 pts (43%) discontinued the study drug. Events leading to permanent treatment discontinuation: 7 CML unrelated deaths, 9 AEs (transaminase increase in 5 pts, skin rash, myalgia, GI toxicity and renal failure in 1 patient each), 9 unsatisfactory responses (without progression to advanced phases), 1 treatment-free remission attempt, 1 other reason. The overall survival probability was 81%. Pts with CV AEs: acute coronary syndromes, 6 pts; pericarditis, 2 pts; peripheral arterial thrombosis, 1 pt (all pts had CV risk factors). No pleural effusions were observed. The incidence of GI toxicity was lower than reported elsewhere. Thirty-six pts were still on BOS at the last contact: 6% on 400 mg, 50% on 300 mg, 44% on 200 mg. Image: Summary/Conclusion: A progressive dose increase, based on molecular response, in elderly CML patients treated with second-line bosutinib produced high response rates; few patients required a dose increase to 400 mg, while most pts remained on 300 mg or less. Not unexpectedly, given the old age, unrelated CV AEs and deaths occurred, but the study drug was generally well tolerated. An initial use of low dose TKIs, with dose optimization in case of absence of optimal response, is a promising strategy in elderly patients. P699: REAL-LIFE OUTCOMES OF PONATINIB TREATMENT IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) OR PHILADELPHIA CHROMOSOME-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (PH+ALL): 5-YEAR-DATA FROM A BELGIAN REGISTRY T. Devos1,2,*, V. Havelange3, K. Theunissen4, S. Meers5, F. S. Benghiat6, A. Gadisseur7, G. Vanstraelen8, H. Vellemans9, B. Bailly10, N. Granacher11, P. Lewalle12, A. De Becker13, K. Van Eygen14, M. Lejeune15, M. Janssen16, A. Triffet17, I. Vrelust18, D. Deeren19, D. Mazure20, M. Beck21, H. Sebti21, D. Selleslag22 1Department of Hematology, University Hospitals Leuven; 2Department of Microbiology and Immunology, Laboratory of Molecular Immunology (Rega Institute), Leuven; 3UCL Saint-Luc, Woluwe-Saint-Lambert; 4Jessa Ziekenhuis, Hasselt; 5Algemeen Ziekenhuis Klina, Brasschaat; 6Hôpital Erasme, Bruxelles; 7Universitair Ziekenhuis Antwerpen, Edegem; 8CHR Verviers, Verviers; 9CHU UCL Namur, Site Godinne, Yvoir; 10Hôpital de Jolimont, Haine-Saint-Paul; 11Ziekenhuis Netwerk Antwerpen Stuivenberg, Antwerpen; 12Institut Jules Bordet, Université Libre de Bruxelles, Bruxelles; 13Universitair Ziekenhuis Brussel, Jette; 14Algemeen Ziekenhuis Groeninge, Kortrijk; 15Centre Hospitalier Universitaire de Liège (Sart-Tilman), Liège; 16Ziekenhuis Oost-Limburg, Genk; 17Centre Hospitalier Universitaire Charleroi Vésale, Charleroi; 18Algemeen Ziekenhuis Sint-Elisabeth, Turnhout; 19Algemeen Ziekenhuis Delta, Roeselare; 20Universitair Ziekenhuis Gent, Gent, Belgium; 21Incyte Biosciences Benelux B.V., Amsterdam, Netherlands; 22Algemeen Ziekenhuis Sint-Jan Brugge, Brugge, Belgium Background: Ponatinib is a third-generation tyrosine kinase inhibitor (TKI) indicated for adult patients with chronic, accelerated or blast phase CML resistant or intolerant to nilotinib or dasatinib or with Ph+ALL resistant or intolerant to dasatinib or for patients with the T315I mutation. Real-world data on ponatinib treatment are limited but are important to evaluate the effectiveness and safety of ponatinib and optimize its use in daily practice. Aims: To report 5-year-Belgian registry data on ponatinib use in CML and Ph+ALL patients in routine clinical practice. Methods: This ongoing prospective multi-center registry (NCT03678454) includes ≥18-year-old patients with CML or Ph+ALL eligible for ponatinib treatment per product label. Data on demographics, effectiveness (major molecular response [MMR] rate) and safety were collected for patients enrolled from 01-March-2016 onwards and are presented here up to 17-May-2021. Results: In this interim analysis, 77 patients from 21 hospitals were enrolled (50 CML, 27 Ph+ALL). Of the 28 patients (39%; 14 CML, 14 Ph+ALL) with mutations in the BCR-ABL1 kinase domain, 17 (8 CML, 9 Ph+ALL) had the T315I mutation at entry. Median age at ponatinib start was 61 years for CML and 56 years for Ph+ALL patients. 88% of CML and 96% of Ph+ALL patients had received ≥2 prior TKIs. Potential pre-existing risk factors for TKI cardiovascular toxicity were observed: history of cardiovascular disease (27 patients), hypertension (23), smoking (15), diabetes (13), hypercholesterolemia (8), hyperlipidemia (6). The most frequently reported risk factor was history of cardiovascular disease (36% CML, 33% Ph+ALL). Median follow-up was 545 (14-3190) days for CML and 210 (26-2933) days for Ph+ALL patients. Reasons for starting ponatinib therapy were intolerance to previous TKIs (26 CML, 11 Ph+ALL), relapse on/refractoriness to previous TKIs (11 CML, 8 Ph+ALL), T315I mutation (6 CML, 6 Ph+ALL) and disease progression (7 CML, 2 Ph+ALL). Ponatinib starting doses were: 45 mg/day (66%), 30 mg/day (10%), 15 mg/day (22%) in CML patients (1 patient started with 15 mg every 2 days) and 45 mg/day (67%), 30 mg/day (15%), 15 mg/day (19%) in Ph+ALL patients. MMR was achieved in 58% of CML and 52% of Ph+ALL patients. The median time-to-MMR was 170 days in CML and 86 days in Ph+ALL patients. Of the 37 patients who started ponatinib due to intolerance to previous TKIs, 59% (15 CML, 7 Ph+ALL) achieved MMR. Adverse reactions (ARs) were reported in 63 patients (82%); the most common were rash (23% of all), constipation (18% of CML) and headache (11% of Ph+ALL patients). Seventeen patients developed 26 serious AR. Serious cardiovascular ARs were reported in 7 patients (coeliac artery stenosis [in 2 patients], ischemic stroke [1], worsening hypertension [1], hypertension [1], deep venous thrombosis [1], possible transient ischemic attack [1]). Eight deaths were recorded, none related to ponatinib treatment. 56% of CML and 24% of Ph+ALL patients discontinued ponatinib treatment due to an AR. Summary/Conclusion: This real-world Belgian registry over 5 years supports the use of ponatinib in CML and Ph+ALL patients resistant or intolerant to previous TKIs or carrying the T315I mutation. Results obtained in routine clinical practice are in concordance with clinical trials, including PACE, in which CML patients had durable and clinically meaningful responses to ponatinib. Funding: Incyte Biosciences Benelux BV Acknowledgements: Medical writing support was provided by Adina Truta (Modis) P700: TREATMENT-FREE REMISSION (TFR) AFTER DASATINIB IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE (CML-CP) AND DEEP MOLECULAR RESPONSE (DMR): FINAL 5-YEAR RESULTS OF DASFREE N. P. Shah1,*, V. García-Gutiérrez2, A. Jiménez-Velasco3, S. Saussele4, D. Rea5, F.-X. Mahon6, M. Y. Levy7, M. T. Gómez-Casares8, M. J. Mauro9, O. Sy10, P. Martin-Regueira10, J. H. Lipton11 1UCSF School of Medicine, San Francisco, United States of America; 2Servicio Hematología y Hemoterapia, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid; 3Hospital Universitario Carlos Haya, Malaga, Spain; 4Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany; 5Hôpital Saint-Louis, Paris; 6Institut Bergonié Cancer Center, Université Bordeaux, Bordeaux, France; 7Baylor Charles A. Sammons Cancer Center, Dallas, United States of America; 8Hospital Universitario de Gran Canaria Dr. Negrín, Las Palmas de Gran Canaria, Spain; 9Memorial Sloan Kettering Cancer Center, New York; 10Bristol Myers Squibb, Princeton, United States of America; 11Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada Background: Patients (pts) with CML-CP who have a DMR are eligible to discontinue treatment (Tx) to attempt TFR. DASFREE (NCT01850004) is a single-arm, open-label, phase 2 trial evaluating dasatinib discontinuation in pts with a stable DMR. At the 2-year (y) follow-up, 46% of pts remained in TFR; in a multivariate analysis, longer duration of prior dasatinib, first-line (1L) dasatinib, and older age were significantly associated with TFR maintenance (Shah et al. Leuk Lymphoma 2019). Aims: To report the final 5-y results assessing TFR maintenance after dasatinib discontinuation. Methods: Eligible pts were aged ≥18 y, received dasatinib Tx as 1L or subsequent therapy for ≥2 y, and had a DMR (MR4.5 or BCR-ABL ≥0.0032% on the international scale, confirmed during pre-screening and with 2 central lab assessments 3 months [mo] apart) for ≥1 y prior to study entry. Pts discontinued dasatinib and were monitored for up to 5 y. All pts provided written informed consent. If loss of major molecular response (MMR) occurred, pts restarted dasatinib at the same dose as at enrollment. A univariate analysis was conducted to identify baseline correlates of maintaining TFR. Rate of TFR maintenance (event-free survival) in the extended follow-up, rate of MMR recapture after relapse, identification of predictive factors for TFR maintenance, and safety were all key secondary or exploratory endpoints. Results: A total of 84 pts discontinued dasatinib. At a minimum follow-up of 60 mo (database lock, Dec 2021), 44% (n=37) of pts remained in TFR and the remaining 56% (n=47) had lost MMR and restarted Tx. No relapses occurred later than 39 mo after discontinuation. Baseline characteristics were balanced between pts who remained in TFR versus those who restarted Tx, except for age (65% of pts who remained in TFR vs 85% who restarted Tx were aged <65 y). Among enrolled pts, 24 discontinued the study early (4 due to drug-related adverse events [AEs]; none due to death); 60 discontinued as planned per protocol (end of study). Patients who discontinued from the study were evaluated for response regained before discontinuation. All evaluable pts (n=46; one patient withdrew from the study 1 month after restarting Tx and did not undergo molecular assessment) who restarted Tx regained MMR and MR4.5 in a median (range) time of 1.9 mo (0.9–3.7) and 3.3 mo (1.5–29.6), respectively. The 5-y TFR rate was 43.8% (95% CI, 33.1–54.4; Figure). A univariate analysis of baseline correlates identified older age and 1L dasatinib as significantly associated with maintaining TFR. Musculoskeletal and connective tissue disorders (any grade) were experienced by 39% of pts (n=33). The most common any-grade AEs were arthralgia (18%) and hypertension (13%). There were no deaths due to CML. Withdrawal events (n=15) were experienced by 9 pts (11%) after a median (range) of 3.7 (<1–18) mo from dasatinib discontinuation, with no new events beyond 18 months of study follow-up; 10 events resolved in a median (range) of 5.98 (<1–70) mo. Image: Summary/Conclusion: Discontinuation of dasatinib after 1L therapy and beyond is a viable option for pts with CML-CP in sustained DMR. About half of the pts who discontinued dasatinib maintained TFR at 5 y. Re-treatment after relapse was successful; all evaluable pts who lost MMR regained MMR and MR4.5 after therapy was reinitiated. The overall safety profile was consistent with the known safety profile of dasatinib and with the 2-y results. P701: COVID-19 IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN TREATMENT-FREE REMISSION: DISEASE SEVERITY AND IMPACT ON TFR STATUS S. Saußele1, N. Evans2, F. E. Nicolini3, E. Chelysheva4, D. Réa5, F. Castagnetti6, S. Claudiani7, R. Rodrigues8, C. Kok9, T. Hughes2,9,* 1III. Med. Klinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany; 2International CML Foundation, London, United Kingdom; 3Hematology department, Centre Léon Bérard and INSERM1052, Lyon, France; 4National Research Center for Hematology, Moscow, Russia; 5Hematology department, Saint-Louis Hospital, Paris, France; 6Istituto di Ematologia “Seràgnoli”, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 7Department of Haematology, Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom; 8Departamento de Hematologia, Instituto Português de Oncologia de Lisboa - IPO, Lisbon, Portugal; 9Precision Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, Australia Background: Severe SARS-CoV-2 infections associated with high mortality rates are reported in a higher percentage of patients (pts) with hematologic malignancies compared to general population. In chronic myeloid leukemia (CML), pts with uncontrolled disease have a higher mortality risk. The impact of SARS-CoV-2 infection on CML pts in treatment-free remission (TFR) has not been studied so far. In particular, as immune control of residual disease may be important for TFR, the concern is that the infection could induce loss of TFR. Aims: To evaluate the outcome of SARS-CoV-2 infection in CML pts in TFR and assess any impact on maintenance of TFR. Methods: From March 2020 to December 2021, the CANDID study organized by the international CML Foundation has collected data on COVID-19 positive CML pts worldwide. Details on the registry were presented recently (Pagano ASH 2021). For this sub-analysis on pts in TFR additional information were collected including; molecular remission status (BCR::ABL1 ratios) before, during and after SARS-CoV-2 infection covering at least 6 months. For molecular analyses, BCR::ABL1 ratios were classified according to Cross et al (Leukemia 2015). In addition, ratios of 0% without indication of sensitivity were allocated as MR4 i.e. 0.01%IS. PCR outlier results were identified using the ROUT method by nonlinear regression with a maximum false discovery rate (FDR) of 1% (Motulsky et al 2006). Time to molecular relapse (MR) was measured from the date of COVID-19 diagnosis to the date of MR defined as loss of major molecular remission (MMR, BCR::ABL1 >0.1%IS) or the date of last molecular test. Molecular relapse-free survival (MRFS) and overall survival (OS) were estimated with the Kaplan-Meier method. The statistical difference between groups was performed using log-rank test. Results: By December 2021, 1050 COVID-19 positive CML pts were registered. 95 pts were in TFR at the time point of SARS-CoV-2 infection of which 89 (93.68%) recovered and 6 deceased (6.32%). Median age of TFR pts was 57 years, male were 51 (53.68%). Median time from CML diagnosis to reporting date was 13 years (range 3.7-27.0 years). TFR duration was 2.83 years in median (range 0.5 months – 10.1 years) including 19 pts with a duration < 1 year. From the 89 recovered TFR pts, 74 pts completed the 6-month follow up (83%), a further 6 pts with molecular follow-up of 3-5 months after COVID-19 diagnosis were still in TFR, 9 pts were lost to follow-up. Of 74 pts with complete reports, 69 pts remained in TFR (93%) and 5 pts lost TFR. For 71 pts, PCR results were obtained before, during and after infection. With the ROUT method 10 pts demonstrated outlier PCR tests, 61 pts demonstrated stable PCR results. There was no statistically significant difference in PCR results before and during/after infection (p>0.2). MRFS for these 71 pts 15 months after COVID-19 diagnosis was 86%. Probability of TFR loss was higher in pts with a TFR duration < 6 months compared to pts with TFR duration >6 months (27% vs 10%, Fig 1A). Additionally, there were no statistically differences in hospitalization rate (16% vs 23%, p=0.12) and severity of COVID-19 symptoms (12.6% vs 12%, p=0.87) comparing TFR and TKI treated pts. OS of COVID-19 positive TFR pts did not differ from COVID-19 positive pts on TKI therapy (HR 1.1, CI 0.47-2.54) (Fig 1B). Image: Summary/Conclusion: In this sub-analysis of the CANDID study, CML pts in TFR had similar severity and survival to CML pts who were on TKI therapy and there was no evidence of an increased risk of TFR loss after SARS-CoV-2 infection. P702: FIRST INTERIM RESULTS OF THE PROSPECTIVE STUDY OF DOSE REDUCTION AND DISCONTINUATION OF TYROSINE KINASE INHIBITORS (READIT-2020) IN CHRONIC MYELOID LEUKEMIA PATIENTS WITH DEEP MOLECULAR RESPONSE M. Gurianova1,*, O. Shukhov1, E. Chelysheva1, A. Petrova1, A. Bykova1, I. Nemchenko1, E. Kuzmina1, N. Tsyba1, L. Gavrilova2, E. Stepanova1, A. Kohno1, A. Turkina1 1National Research Centre for Hematology, Moscow; 2Republican Clinical Hospital №4, Saransk, Russia Background: The feasibility of the one-step tyrosine kinase inhibitors (TKI) dose reduction before treatment-free remission (TFR) phase in chronic myeloid leukemia (CML) patients (pts) has been confirmed in several trials.There are no data of the prospective two-step dose reduction trials of different TKIs followed up with TFR observation.The experience of resuming TKIs at reduced doses after the molecular relapse is limited as well. Aims: To analyze the interim data of the survival without loss of major molecular response (MMR,BCR::ABL1≤0,1%) after TKI stop in CML pts taking reduced-dose TKI therapy and the probability of MMR and deep MR (DMR, BCR::ABL1≤0.01%) recovery after resuming TKIs at reduced doses (Clinicaltrials.gov NCT 04578847). Methods: The prospective trial had 2 phase:1)TKI dose reduction phase for at least 12 months (mo),2)TFR phase for at least 24 mo.TKI dose reduction consisted of 2 steps each lasting for 6 mo.The inclusion of pts was possible at any step if they met the key inclusion criteria:CML in chronic phase,age ≥18 years (y),TKI therapy duration≥ 3 y,MMR and DMR duration≥2 y and ≥1 y.The inclusion into TFR was done after the dose reduction phase in pts with a DMR duration≥2 y and ≥MR4.5 at the time of TKI stop.In case of MMR loss the TKI dose was increased by +1 level from the dose at which the MMR loss developed. Results: A total of 103 CML pts were included from Dec.2019 till Dec.2021 at different trial phases (Tab 1). Female 60%, Median (Me) age at diagnosis and at inclusion 45 y (23-74 y) and 51 y (23-74 y),respectively;ELTS score 61%:17%:1%, 21% for low,intermediate,high and unknown risk group.A history of at least one TKI stop was in 29 pts. Me TKI duration was 7 y (3-19,7 y);Me duration of MMR and DMR was 3,3 y (2-126 y) and 2,5 y (1,2-10,5 y), respectively. At baseline 69 (67%) pts received imatinib (IM) and 34 (33%) pts-second-generation (2G)TKI (Tab 1). The reasons for 2GTKI switch were treatment failure (n=12),drug toxicity (n=25),others (n=1). Sixty-four pts completed the 1st dose reduction step lasting for 6 mo. There was no MMR loss after the 1st dose reduction step. Four pts lost DMR on IM 300 mg,but three pts restored DMR in 3 mo at the same dose thereafter. Fifty-seven pts completed the 6 mo 2nd step of dose reduction.Two pts lost MMR on IM 200 mg.Nine pts lost DMR (without MMR loss):6 pts on IM 200 mg,1 pt on dasatinib (DAS) 25 mg and 2 pts on nilotinib (NIL) 200 mg. Forty-nine pts were included in TFR phase,16 pts (32,6%) had a history of at least one TKI stop.Eight pts had a history of resistance to TKI therapy.Me follow-up after TKI discontinuation was 10 mo (1-24 mo). The survival without MMR loss was 61% and 41% in pts with 1st and 2nd TKI stop,respectively (Fig 1). The survival without MMR loss was 54% after 12 mo in total group. TKI therapy was resumed at reduced doses after MMR loss:IM 200 mg(n=12),NIL 200 mg (n=4),DAS 25 mg (n=2);BOS 200 mg (n=3).The probability of recovery of MMR and DMR was 86% and 83% after 6 mo resumption of TKIs at reduced doses (Fig 2). Image: Summary/Conclusion: The 2-step dose reduction of TKIs before TFR phase is a promising approach for CML pts.Use of the reduced TKI doses in pts with DMR lasting for≥1 y is a safe treatment option under the strict molecular monitoring. The 61% survival rate without MMR loss in CML pts having the 1st TKI discontinuation after the dose reduction phase is encouraging.The high probability of MMR and DMR recovery after resuming TKIs at reduced doses allows to consider this possibility in case of the molecular relapse. P703: ASSOCIATION BETWEEN BARIATRIC SURGERY AND OUTCOMES IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA TREATED WITH ORAL TYROSINE KINASE INHIBITORS F. Haddad1,*, H. Kantarjian1, E. Jabbour1, N. Short1, A. Bidikian1, J. Ning2, L. Xiao2, N. Pemmaraju1, K. Marx1, F. Ravandi1, K. Sasaki1, G. Issa1 1Leukemia; 2Statistics, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Bariatric surgery is widely used in patients (pts) with morbid obesity with great success in reduction of associated metabolic complications. However, it can also alter the pharmacokinetics of oral medications. Oral tyrosine kinase inhibitors (TKIs) represent the mainstay of chronic myeloid leukemia (CML) therapy. The impact of bariatric surgery on CML treatment outcomes is largely unknown. Aims: To evaluate the clinical impact of bariatric surgery on outcomes of CML pts treated with TKIs. Methods: In a retrospective analysis, we screened pts with CML treated at our institution and identified those who had any type of bariatric surgery including gastric bypass, gastric sleeve or gastric banding. We then compared their responses and outcomes to a control cohort of pts without history of bariatric surgery, using propensity score matching for Sokal Risk and body mass index (BMI) at a 2:1 ratio. In addition to cytogenetic and molecular responses, we assessed times to achieving responses and BCR::ABL1 halving times to investigate differences in response dynamics. We assessed comorbidities using the Charlson Comorbidity Index. Event-free survival (EFS) was measured from treatment start to loss of response, progression, or death, whereas failure-free survival (FFS) additionally accounted for therapy discontinuation for other reasons such as intolerance. Overall survival (OS) was measured from treatment start date to death or censored at last follow-up. Univariate and multivariate analyses (MVA) were used to assess the association between characteristics and survival outcomes. Results: We identified 28 pts with CML and a history of bariatric surgery (22 pts had their surgery before the diagnosis of CML) and 56 pts as a matched control. Their baseline characteristics are summarized below. Among pts with bariatric surgery, 61% required >1 TKI throughout the course of their disease due to intolerance or resistance, compared with 25% of the control group (P=0.002). BCR::ABL1 halving time was higher in the bariatric surgery group vs control (26 days vs 13 days; P<0.0001), suggesting slower response dynamics. The median time to achieve complete cytogenetic response (CCyR) and major molecular response (MMR) was significantly longer in pts with history of bariatric surgery: 6 months vs 3 months (P<0.0001) and 12 months vs 6 months (P=0.001), respectively. Bariatric surgery was associated with inferior EFS (10-year: 31% vs 69%; P=0.009) and FFS (10-year: 16% vs 54%; P<0.0001) compared with control, with a trend for worse OS (10-year: 65% vs 85%; P=0.09) (Figure 1). We subsequently conducted univariate and MVA and assessed the impact of bariatric surgery and covariates such as sex, BMI, Sokal risk, TKI used, age and use of proton pump inhibitors. Bariatric surgery was the only independent predictor for the risk of treatment failure (hazard ratio [HR] 5.95, 95% CI 2.38-14.89; P=0.0001) or EFS (HR 3.6, 95% CI 1.38-7.91; P=0.007) in the MVA but did not independently affect the risk of death. The Charlson Comorbidity Index was the only independent predictor for the risk of death (HR 1.65, 95% CI 1.10-2.48; P=0.02). Image: Summary/Conclusion: Bariatric surgery is associated with slower responses to TKIs and higher rates of treatment failure. There is an unmet need to design treatment strategies for these patients. Although not readily available in the clinical setting, studies measuring drug level in these patients are needed to assess which TKI has a better bioavailability, which in turn could translate to improved outcomes. P704: ASCIMINIB PROVIDES DURABLE MOLECULAR RESPONSES IN PATIENTS (PTS) WITH CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE (CML-CP) WITH THE T315I MUTATION: UPDATED EFFICACY AND SAFETY DATA FROM A PHASE I TRIAL T. Hughes1,*, J. E. Cortes2, D. Réa3, M. J. Mauro4, A. Hochhaus5, D.-W. Kim6, K. Sasaki7, F. Lang8, M. C. Heinrich9, M. Breccia10, M. Deininger11, Y.-T. Goh12, J. J. Janssen13, M. Talpaz14, V. Gómez García de Soria15, P. le Coutre16, S. Kapoor17, S. Cacciatore18, F. Polydoros18, N. Agrawal18, F.-X. Mahon19 1SA Pathology and South Australian Health and Medical Research Institute, University of Adelaide, Adelaide, Australia; 2Georgia Cancer Center, Augusta, United States of America; 3Hôpital Saint-Louis, Paris, France; 4Memorial Sloan Kettering Cancer Center, New York, United States of America; 5Universitätsklinikum Jena, Jena, Germany; 6Uijeongbu Eulji Medical Center, Geumo-dong, Uijeongbu-si, South Korea; 7The University of Texas MD Anderson Cancer Center, Houston, United States of America; 8Goethe University Hospital, Frankfurt, Germany; 9Portland VA Health Care System and OHSU Department of Medicine, Division of Hematology and Oncology, Knight Cancer Institute, Portland, United States of America; 10Policlinico Umberto I-Sapienza University, Rome, Italy; 11Versiti Blood Research Institute, Milwaukee, United States of America; 12Singapore General Hospital, Bukit Merah, Singapore; 13Amersterdam University Medical Centers, Amersterdam, Netherlands; 14University of Michigan Rogel Cancer Center, Ann Arbor, United States of America; 15Hospital Universitario La Princesa, Madrid, Spain; 16Charité - Universitätsmedizin Berlin, Berlin, Germany; 17Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 18Novartis Pharma AG, Basel, Switzerland; 19Institut Bergonié, Bordeaux, France Background: In pts with CML, the BCR::ABL1 T315I mutation is associated with poor clinical outcomes and confers resistance to previously approved ATP-competitive tyrosine kinase inhibitors (TKIs). Until recently, ponatinib (PON) was the only TKI available for these pts, but its use may be limited by associated cardiovascular events. In the primary analysis of the phase I trial X2101, asciminib—the 1st BCR::ABL1 inhibitor to Specifically Target the ABL Myristoyl Pocket (STAMP)—demonstrated efficacy and a favorable safety profile in heavily pretreated pts with CML with T315I. These results supported the FDA approval of asciminib as a new treatment option for pts with CML-CP with T315I (NCCN 2021). We report updated efficacy and safety data in these pts (data cutoff: January 6, 2021). Aims: Provide updated safety and efficacy data for pts with CML-CP with T315I treated with asciminib monotherapy 200 mg twice daily (BID) after added exposure. Methods: Pts with CML-CP with T315I were enrolled if treated with ≥1 prior TKI and no other effective therapy was available, provided informed consent, and received asciminib 200 mg BID. Results: 48 pts with T315I were included; 2 (4.2%) pts had additional BCR::ABL1 mutations at baseline. Eight (16.7%), 15 (31.3%) and 25 (52.1%) pts received 1, 2, and ≥3 prior TKIs, respectively. At data cutoff, treatment was ongoing in more than half (27 [56.3%]) of pts; the predominant reason for treatment discontinuation was physician’s decision (11 [22.9%]), mainly due to lack of efficacy. Of the 48 pts, 45 were evaluable (BCR::ABL1 IS >0.1% at baseline) for major molecular response (MMR); 3 were excluded for BCR::ABL1 atypical transcripts. Among evaluable pts, 19 (42.2%) achieved MMR by wk 24 and 22 (48.9%) by wk 96; 19 were still in MMR at the cutoff date. Evaluable pts included 26 PON-pretreated and 19 PON-naive pts; 34.6% and 68.4%, respectively, achieved MMR by the cutoff date (Table). The probability of pts maintaining MMR for ≥96 wks was 84% (95% CI, 68.1-100.0). Thirteen (28.9%) and 11 (24.4%) pts achieved MR4 and MR4.5, respectively. Twenty (54.1%) and 23 (62.2%) of 37 pts with BCR::ABL1 IS >1% at baseline achieved BCR::ABL1 IS ≤1% by wk 48 and 96, respectively. The median duration of exposure was 2.08 (range, 0.04-4.13) yrs with more than half (27 [56.3%]) of pts receiving treatment for ≥96 wks; the median daily dose intensity was 398.3 (range, 179-400) mg/day. The safety/tolerability profile of asciminib remained favorable after ≈9 months of added follow-up (Table). The most common (≥5%) grade ≥3 adverse events (AEs) were lipase increase (18.8%, all asymptomatic elevations), thrombocytopenia (14.6%), and vomiting, ALT increase, abdominal pain, hypertension, anemia, neutropenia, and neutrophil count decrease (6.3% each). Arterial occlusive events occurred in 4 (8.3%) pts; none led to dose adjustment/interruption/discontinuation. AEs leading to discontinuation were reported in 2 new pts since the previous data cutoff; both pts discontinued and died due to COVID-19. These were the only study deaths reported in this pt population. Image: Summary/Conclusion: Asciminib monotherapy 200 mg BID exhibited a sustained, favorable safety profile after added exposure with no new safety signals in pts with CML-CP with T315I—a population with high unmet medical need. The clinical efficacy of asciminib is demonstrated by the high proportion of pts achieving durable MMR and BCR::ABL1 IS ≤ 1%. The updated analysis confirms asciminib as a treatment option for pts with CML-CP with T315I, including those for whom treatment with PON has failed. P705: MULTI-CENTER CHART REVIEW STUDY EXAMINING TREATMENT PATTERNS AND CLINICAL OUTCOMES AMONG PATIENTS WITH CHRONIC PHASE (CP) CHRONIC MYELOID LEUKEMIA (CML) TREATED IN THIRD-LINE (3L) OR LATER IN FRANCE F. E. Nicolini1, G. Etienne2, F. Huguet3, M. Gu4, C. Bouvier1, A. Yocolly5, R. Favier6, M. Trancart6, L. Huynh4,* 1Centre Leon Berard, Lyon; 2Institut Bergonié, Bordeaux; 3Institut Universitaire de Cancérologie de Toulouse, Toulouse, France; 4Analysis Group, Inc., Boston, MA; 5Novartis Services, Inc., East Hanover, NJ, United States of America; 6Novartis Oncologie, Rueil-Malmaison, France Background: The standard of care for CML has substantially evolved over a relatively short period of time; pharmacologic inhibition of ABL1 with adenosine triphosphate (ATP)-competitive tyrosine kinase inhibitors (TKIs) remains the cornerstone of modern treatment. Clinical guidelines for CP-CML recommend that patients who have failed ≥2 prior TKIs switch to an alternative 2nd or 3rd generation TKI. However, up to 65% of these patients have prior exposure to ATP-competitive TKIs and history of either TKI intolerance or resistance. Aims: This retrospective, multi-center, chart review study aimed to characterize the disease burden, treatment patterns, and clinical outcomes of CP-CML patients who have failed ≥2 TKIs. Methods: De-identified demographic and clinical data for adult patients diagnosed with CP-CML treated in 3L or later at three reference centers in France were abstracted from medical charts using electronic case report forms. Descriptive statistics were summarized for patient characteristics, clinical outcomes during 3L treatment, and adverse events. Molecular data were standardized and expressed in the international scale (IS). The cumulative incidence of patients achieving molecular response (major molecular response [MMR, 0.01% IS ≤0.1%] or deep molecular response [MR4.0, 0.0032% < BCR–ABL1IS ≤0.01% or MR4.5, BCR–ABL1IS ≤0.0032%]) were summarized at specific time points. Progression-free survival (PFS) and overall survival (OS) from 3L initiation were examined using the Kaplan–Meier (KM) method. Results: Medical data for 157 CP-CML patients were assessed; the mean follow-up was 66.9 months, the median age at 3L initiation was 62.1 years, 56% were male, and 90% had major BCR-ABL1 rearrangement (among the 89 patients with mutation status assessed, 7 [8%] had BCR-ABL1 T315I mutation, 3 [3%] had M244V, and 3 [3%] had F359I). According to EUTOS long-term survival score, 40% of patients had low risk, 25% had intermediate risk, and 11% had high risk of death due to CML. Cardiovascular diseases were present in 55% of patients at 3L; mean systolic and diastolic blood pressure were 140.9 and 78.9 mmHg, respectively. Mean ± SD low-density lipoprotein cholesterol was 141.0 ± 54.0 mg/dl. Median duration of 3L therapy was 17.0 months. TKIs received in 3L were dasatinib (32%), nilotinib (19%), imatinib (18%), ponatinib (17%), and bosutinib (14%). Treatment-free remission was initiated by 16 (10%) patients. In patients with documented responses, 42% patients achieved MMR, 27% achieved MR4.0, and 14% achieved MR4.5 at 12 months. Although median PFS and OS were not reached as of data collection, 19 (12%) patients died due to disease progression (n=7), toxicity (n=1), or other reasons (n=10) and 1 patient had an unknown cause of death. Approximately 50% of patients discontinued treatment; 37% had ≥4 lines of treatment and 16% had ≥5 lines. The primary reasons for discontinuation (not mutually exclusive) were intolerance (54/78 [69%]), resistance (18/78 [23%]), and signs of ineffectiveness (14/78 [18%]). AEs were documented for 139/157 patients (89%). The mean number of AEs per patient was 2.7; infections (18%) and asthenia (13%) were the more commonly documented AEs. Summary/Conclusion: CP-CML patients continue to experience a substantial disease burden and poor prognosis after 3L treatment with available TKIs, underscoring the need for novel therapies that are well tolerated and can achieve durable responses. P706: ASCIMINIB USE IN CML: THE UK EXPERIENCE A. Innes1,2,*, V. Orovboni3, S. Claudiani1, F. Fernando1, A. Khan1, J. Byrne4, P. Gallipoli5, M. Copland6, G. Horne6, C. Arnold7, A. Collins8, N. Cunningham7, A. Danga9, R. Frewin10, P. Garland11, G. Hannah12, S. Hassan13, S. Makkuni14, K. Rothwell15, L. Foroni2, C. Hayden3, J. Apperley2, D. Milojkovic1 1Department of Haematology, Hammersmith Hospital, Imperial College Healthcare NHS Trust; 2Centre for Haematology, Faculty of Medicine, Department of Immunology and Inflammation, Imperial College London; 3SIHMDS (Molecular Laboratory), Hammersmith Hospital, Imperial College Healthcare NHS Trust, London; 4Nottingham University Hospitals NHS Trust, Nottingham; 5Cancer Research UK Barts Centre, London; 6Institute of Cancer Sciences, Paul O’Gorman Research Centre, Gartnavel General Hospital, Glasgow; 7Belfast City Hospital, Belfast; 8Norfolk and Norwich University Hospital NHS Foundation Trust, Norwick; 9The Hillingdon Hospital, London; 10Gloucestershire Hospital NHS Trust, Gloucester; 11Princess Royal University Hospital; 12King’s College Hospital NHS Foundation Trust, London; 13Queen’s Hospital, Romford, London; 14Mid and South Essex NHS Foundation Trust, Essex, London; 15Calderdale and Huddersfield NHS Foundation Trust, Huddersfield, United Kingdom Background: Asciminb is an allosteric BCR-ABL1 inhibitor that has been available for patients with chronic myeloid leukaemia (CML) in the United Kingdom under a managed access program (MAP) from Novartis since 2016. Aims: The aim of this project was to share real-world experience of asciminib use in the UK. Methods: Using a national survey proforma (REC reference: 21/HRA/2157), we collated baseline, outcome and toxicity data on 44 patients treated with asciminib in the UK. Responses were assess using BCR-ABL1 IS values and categorised by ELN criteria. Intolerances were reported using CTCAEv5.0 criteria. Results: The median age at treatment was 58 [22-88] years. Vascular comorbidities were seen in 22 (50%) patients, the most frequent being hypertension (n=12, 27%), peripheral vascular disease (n=6, 14%), AF (n=6, 14%) and ischaemic heart disease (n=6, 14%). Chronic kidney disease was present in 7 (16%) patients. All but one patient was in chronic phase at the time of treatment, but tyrosine kinase domain mutations (TKDM) were common prior to treatment initiation (T315I in 11/44 (25%), other TKDM in 8/44 (18%)). The median number of prior TKIs were 4 [2-5], with 44% of prior lines stopped for resistance, and 56% for intolerance. The most recent TKI was discontinued for resistance in 14 (32%) and intolerance in 30 (68%). Eight (18%) patients had developed pleural effusions on a previous TKIs. Most patients had previously received ponatinib (n=30, 68%) which has been discontinued for resistance in 8 (27%) and intolerance in 22 (73%), with 7 (16%) vascular events on treatment. Five (11%) patients had received prior allogeneic stem cell transplant. At the time of data cut-off, 25 (57%) patients remained on treatment with a median treatment duration of 20 [3-51] months. Reason for treatment discontinuation was resistance (n=7, 16%), intolerance (n= 7, 16%), pregnancy (n=1, 2%), treatment-free remission (n=1, 2%) and non-compliance (n=1, 2%). Two patients died whilst on treatment of unrelated causes. The median daily dose delivered was 240mg [20-400] in patients with T315I-TKDM and 80mg [20-80] in others. While 11 patients had no significant response (including those with early treatment discontinuation for intolerance), 23 (53%) patients achieved MR3 or better, with 14 (32%) achieving MR4 or better. Of 9 patients in MR3 or better at asciminib initiation, 3 (33%) did not tolerate treatment, 2 (22%) maintained their response, and 4 (44%) deepened their response by one category or more. The presence of a non-T315I TKDM was associated with a lower incidence of achieving MR3, with 1 of 6 (16%) evaluable patients in this group achieving MR3 or better, compared with 22 of 31 (71%) evaluable patients with no TKDM or a T315I-only TKDM (p=0.04) (Table1). Haematological toxicity was seen in 17 (38%) patients, and was grade 3-4 in 6 (14%) (predominantly anaemia and thrombocytopaenia). Non-haematological toxicity was reported in 19 (43%) patients (most commonly fatigue (n=4), insomnia (n=3), bone pain (n=2), back pain (n=2), nausea (n=2), fluid retention (n=2)), with only 2 (4%) patients experiencing grade 3-4 toxicity (fatigue in both cases). No worsening of baseline vascular co-morbidity or renal dysfunction was noted. One patient developed a new pleural effusion on the 200mg bd dose, which did not recur on dose reduction. Image: Summary/Conclusion: These data show that asciminib is a well-tolerated, effective treatment for patients who are resistant to or intolerant of multiple TKIs, many of whom often have a number of co-morbidities including vascular disease. P707: DOSE MODIFICATION DYNAMICS OF PONATINIB IN PATIENTS WITH CHRONIC-PHASE CHRONIC MYELOID LEUKEMIA (CP-CML) FROM THE PACE AND OPTIC TRIALS J. Apperley1,*, H. Kantarjian2, M. Deininger3, E. Abruzzese4, J. Cortes5, C. Chuah6, D. J. DeAngelo7, J. DiPersio8, A. Hochhaus9, J. Lipton10, F. Nicolini11, J. Pinilla-Ibarz12, D. Rea13, G. Rosti14, P. Rousselot15, M. Mauro16, N. Shah17, M. Talpaz18, A. Vorog19, X. Ren19, E. Jabbour2 1Imperial College London, London, United Kingdom; 2The University of Texas MD Anderson Cancer Center, Houston, TX; 3University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, United States of America; 4S. Eugenio Hospital, Tor Vergata University, Rome, Italy; 5Georgia Cancer Center at Augusta University, Augusta, GA, United States of America; 6Singapore General Hospital, Duke-NUS Medical School, Singapore, Singapore; 7Dana-Farber Cancer Institute, Boston, MA; 8Washington University School of Medicine, St. Louis, MO, United States of America; 9Universitätsklinikum Jena, Jena, Germany; 10Princess Margaret Cancer Centre, Toronto, Ontario, Canada; 11Centre Léon Bérard, Lyon, France; 12H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, United States of America; 13Hôpital Saint-Louis, Paris, France; 14IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola (FC), Italy; 15Hospital Mignot University de Versailles Saint-Quentin-en-Yvelines, Paris, France; 16Memorial Sloan Kettering, New York, NY; 17University of California San Francisco, San Francisco, CA; 18Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI; 19Takeda Development Center Americas, Inc., Lexington, MA, United States of America Background: Ponatinib, a potent oral, third-generation tyrosine kinase inhibitor (TKI), is FDA approved for treatment of patients with relapsed/refractory CML. In the pivotal Phase 2 PACE (Ponatinib Ph+ ALL and CML Evaluation) trial (NCT01207440), patients with resistant/intolerant CP-CML demonstrated deep, lasting responses to ponatinib 45 mg once daily. The Phase 2 OPTIC (Optimizing Ponatinib Treatment in CP-CML) trial (NCT02467270) prospectively evaluated a response-based dose-reduction strategy in an attempt to optimize the dose schedule of ponatinib in patients with CP-CML resistant to second-generation (2G) BCR::ABL1 TKI therapy or with a T315I mutation. Aims: Since the unique dosing strategies in the PACE and OPTIC trials provide the opportunity to closely evaluate the dose and schedule of ponatinib, we conducted an in-depth analysis of dosing dynamics between the 2 trials and compare efficacy and safety outcomes. Methods: Adults with resistant/intolerant CP-CML from the PACE and OPTIC trials were enrolled. In PACE, patients received an initial dose of ponatinib 45 mg once daily, in OPTIC, patients were randomly assigned (1:1:1) to an initial oral dose of ponatinib 45 mg, 30 mg, or 15 mg once daily. In PACE, proactive dose reductions were mandated in ≈2 years from initiation of first patient (in 2013) as arterial occlusive events (AOEs) emerged as notable adverse events (AEs). OPTIC was designed to incorporate a mandatory response-based dose-reduction strategy; patients in the 45-mg and 30-mg cohorts in OPTIC achieving ≤1% BCR::ABL1 IS reduced their dose to 15 mg once daily; doses also were reduced to manage AEs. This analysis includes data from patients with CP-CML in PACE and from the 45-mg starting dose cohort in OPTIC. Efficacy outcomes include ≤1% BCR::ABL1 IS, progression-free survival (PFS), and overall survival (OS). Safety outcomes include treatment-emergent adverse events (TEAEs) and treatment-emergent AOE (TE-AOE) rates. Results: Together, 364 patients with CP-CML had ≥1 prior 2G TKI or had a T315I mutation and received a starting dose of ponatinib 45 mg (PACE, n=270; OPTIC, n=94). Percentages of patients receiving ≥2 or ≥3 prior TKIs were 93% and 60% in PACE and 99% and 53% in OPTIC, respectively. Percentages of patients with vascular disorders were 44% in PACE and 32% in OPTIC. Median follow-up was 57 months (PACE) and 32 months (OPTIC). ≤1% BCR::ABL1 IS response by 24 months was 52% in PACE and 56% in OPTIC, 2-year PFS was 68% in PACE and 80% in OPTIC, and 2-year OS was 86% in PACE and 91% in OPTIC. Median time to ≤1% BCR::ABL1 IS response was 5.6 months in PACE and 6 months in OPTIC. Median duration of response was not reached in either trial. Median time to dose reduction for AEs was 2.85 months in PACE and 3.64 months in OPTIC and dose reductions due to AEs occurred in 82% of patients in PACE and 46% in OPTIC. Median time on therapy was 12.6 months in PACE and 19.5 months in OPTIC. Per 100-patient years, exposure-adjusted TE-AOEs were 15.8 events at 0 to <1 year in PACE and 7.6 events at 0 to <1 year in OPTIC. Individual dosing dynamics by safety and efficacy will be presented. Summary/Conclusion: The response-based dose-reduction strategy in OPTIC resulted in comparable or better efficacy outcomes, fewer dose reductions related to AEs, fewer exposure-adjusted TE-AOEs, and longer median time on therapy compared with PACE. These results further demonstrate the benefit of the response-based dosing regimen used in OPTIC. This abstract is an encore from the American Society of Hematology 2021 Annual Meeting. P708: CANADIAN AND RUSSIAN EXPERIENCES OF ASCIMINIB IN CHRONIC MYELOID LEUKEMIA (CML) PATIENTS WHO FAILED MULTIPLE LINES OF TYROSINE KINASE INHIBITOR (TKI) THERAPY. F. Khadadah1,*, A. G. Turkina2, E. Lomaia3, E. V. Morozova4, O. A. Shukhov2, A. Petrova2, T. Chitanava3, J. J. Vlasova5, E. Kuzmina2, I. Nemchenko2, E. Y. Chelysheva2, A. Bykova2, M. Gurianova2, A. Xenocostas6, L. Busque7, K. Jamani8, S. Cerquozzi8, P. Kuruvilla9, R. Kaedbey10, B. Leber10, S. Assouline11, D. D. H. Kim1 1Princess Margaret Cancer Centre, Toronto, Canada; 2National Medical Research Center for Hematology, Moscow; 3Almazov National Medical Research Centre; 4. Raisa Gorbacheva Memorial Research Institute of Children’s Oncology, Hematology and Transplantation; 5Raisa Gorbacheva Memorial Research Institute of Children’s Oncology, Hematology and Transplantation, Saint-Petersburg, Russia; 6London Health Sciences Centre, Victoria Hospital, London; 7Maisonneuve-Rosemont Hospital, Montreal; 8Tom Baker Cancer Centre, Calgary; 9William Osler Health System, Brampton; 10Juravinski Cancer Center, Hamilton; 11Jewish General Hospital, Montreal, Canada Background: Asciminib (ASC) is a novel, first in class ‘specifically targeting the ABL myristate pocket (STAMP) inhibitor. Recent update of the ASCEMBL phase 3 data showed a superior cumulative incidence of major molecular rate (MMR) of 33.2% with ASC over bosutinib (BOS) at 18.6% by 48 weeks and higher MR4 rate (BCR-ABL1 ≤0.01%) of 14.0% with ASC over 6.6% with BOS at 48 weeks in CML patients (pts) in chronic phase (CP) who have failed at least two lines of TKI therapy. Aims: Canadian and Russian groups individually reported their real-world experiences of ASC therapy under the Managed Access Program in heavily pre-treated CML patients. Methods: Data was collected, updated and merged from 80 CML pts treated with ASC between Nov 2018 - Dec 2021 (n=57 in Russia; n=23 in Canada). Median age was 57 years (range 20-92). The median number of previous TKIs was 4 (range 2-6); Imatinib (n=74, 93%), dasatinib (n=64, 80%), nilotinib (n=58, 73%), bosutinib (n=51, 64%), ponatinib (n=35, 44%), and others (n=13, 16%). Median duration from diagnosis to ASC treatment was 92 mo (months) (range 11-310). 27 (34%) pts had a past history of clinically significant cardiovascular disease or high risk profile for cardiovascular disease. Of the 80 pts, 27 (34%) had a preexisting T315I mutation and 18 (23%) had a non-T315I mutation. Pts failed previous TKI therapy due to A)resistance or suboptimal response (n=59; 74%) and B)intolerance (n=21; 26%). BCR-ABL qPCRs were monitored at each institution. Achievement of MMR and molecular response of 4 log reduction (MR4) was assessed at 6 and 12 mo. Results: With a median of 9 mo of follow-up (range 1-38), MMR was noted in 15/68 (22%) and 14/36 pts (39%) evaluated at 6 and 12 mo, respectively. MR4 was noted in 11/68 (16%) and 9/36 pts (25%) at 6/12 mo. The cumulative incidence of MMR and MR4 was 32.6% (20.9-44.8%) and 15.9% (7.9-26.3%) at 12 mo. The probability of freedom from treatment failure (FTF) at 12 mo was 42.4% (27.6-56.4%), based on the ELN 2013 failure criteria for 2nd line therapy or beyond. In the overall population (n=80), FTF was significantly associated with reason for switch to ASC (p=0.006), and there was a trend toward association of FTF with disease phase (p=0.08) and ponatinib therapy (p=0.12), but not with ABL1 kinase domain mutation (p=0.402), additional cytogenetic abnormality (p=0.908), nor cardiovascular risk profile (p=0.345). When analysis was restricted to the patients in CML-CP (n=65), a median duration of FTF was calculated as 11.6 mo, while the 12 mo FTF rate was 46.8% (28.9-62.8%). The patients previously treated with ponatinib (n=25) showed a 27.7% (8.4-51.4%) FTF rate at 12 mo, which was lower than that in those naïve to ponatinib (n=40), 61.7% (34.9-80.2%; p=0.023). Those switched to ASC for intolerance (n=21) showed higher 12 mo FTF rate of 81.2% (41.5 -95.2%) compared to 29.4% (11.3-50.2%) in those with resistance (n=44; p=0.008). Fifteen pts discontinued ASC due to treatment failure (n=11), progression of disease to blast phase (n=3) or grade 4 thrombocytopenia (n=1). No cardiovascular event was noted. Image: Summary/Conclusion: This combined Canadian and Russian real-world experience of ASC study includes heavily pretreated CML pts including 15 pts in advanced phase. The MMR and MR4 rates were comparable in those in CP, while it was lower in non-CP pts. The 12 mo FTF rate of 42.4% was comparable to the one reported from the ASCEMBL study, 57.7%, considering that this group includes more patients who had heavily pre-treated with advanced disease. *AGT and FK contributed equally as co-first author. P709: CLINICAL OUTCOME OF ASCIMINIB TREATMENT IN A REAL-WORLD MULTI-RESISTANT CML PATIENT POPULATION. C. C. Kockerols1,*, J. J. Janssen2, N. M. Blijlevens3, S. K. Klein4, L. G. van Hussen-Daenen5, G. N. van Gorkom6, W. M. Smit7, P. van Balen8, B. J. Biemond9, M. J. Cruijsen10, M. F. Corsten11, P. A. te Boekhorst12, H. R. Koene13, G. L. van Sluis14, J. J. Cornelissen12, P. E. Westerweel1 1Internal Medicine, Albert Schweitzer Hospital, Dordrecht; 2Hematology, Amsterdam University Medical Center, location VUMC, Amsterdam; 3Hematology, Radboud University Medical Center, Nijmegen; 4Hematology, University Medical Center Groningen, Groningen; 5Hematology, University Medical Center Utrecht, Utrecht; 6Hematology, Maastricht University Medical Center, Maastricht; 7Hematology, Medisch Spectrum Twente, Enschede; 8Hematology, Leiden University Medical Center, Leiden; 9Hematology, Amsterdam University Medical Center, location AMC, Amsterdam; 10Hematology, Catharina Hospital, Eindhoven; 11Hematology, Meander Medical Center, Amersfoort; 12Hematology, Erasmus University Medical Center, Rotterdam; 13Hematology, St.-Antonius Hospital, Nieuwegein; 14Hematology, Isala, Zwolle, Netherlands Background: Asciminib is a first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) tyrosine kinase inhibitor (TKI). Asciminib was found to have superior efficacy compared to bosutinib in patients (pts) intolerant or refractory to ≥2 currently registered TKI with good tolerability. While awaiting its EMA approval, asciminib is made available by Novartis to pts with high clinical need in an early access programme (EAP) in The Netherlands. Aims: We aimed to assess treatment outcomes in this nationwide patient cohort. Methods: Dutch hematologists who treated CML pts in the asciminib EAP were asked to provide clinical data of consenting pts. Pts were included if ≥18 years and treated in this asciminib EAP, thus outside clinical trials. Treatment failure was defined as progression to advanced stage disease (AP/BC), not reaching a complete cytogenetic response or BCR-ABL1 <1%IS (CCYR/MR2.0) (=primary) or losing previously achieved responses (=secondary). Event-free survival (EFS) was defined as time from asciminib start until allogeneic stem cell transplantation (SCT), progression to AP/BC or death; whatever came first. Responses were assessed with the cumulative incidence competing risk (CICR) method considering the competing event of TKI discontinuation for any cause. Results: All hematologists agreed to participate and 53 out of 56 pts from 14 medical centers consented and were included. Most pts (72%) had been exposed to ≥3 TKIs prior to asciminib. The majority (79%) started with asciminib because of multi-resistance (with or without intolerance) including 15 pts with primary ponatinib-failure, 15 pts harboring the T315I mutation and 3 pts with compound mutations. Two pts were treated with asciminib post-SCT and were excluded from response analyses. Median asciminib exposure was 7 months (IQR 3-16). Frequently reported adverse events (AE) were cytopenia and folliculitis, classified as probably related to asciminib and manageable generally without dose modification. Only one pt definitely discontinued asciminib due to persistent anemia, but in the context of also a failing response. Two vascular events were reported, one recurrent TIA therefore classified as unrelated and one NSTEMI classified as possibly related to asciminib. Sixteen pts discontinued asciminib, mostly because of treatment failure (primary n=8, secondary n=2) or proceeding to SCT (n=5). Six out of 7 pts with AP/BC discontinued asciminib within 6 months because of treatment failure/SCT. The CI of maintaining or reaching CCYR/MR2.0 and MMR by 6 months for CP pts were 60% and 44%, respectively. Of note, only one out of 10 pts in CP with primary ponatinib-failure reached MR2.0 and none of these patients reached MMR. However, CP pts with secondary ponatinib-failure responded well with CCYR/MR2.0 or better in 78% (7/9). During asciminib treatment 5 pts progressed to advanced stage disease, 9 pts eventually underwent SCT and 7 pts died of whom 6 with a CML-related cause. Overall survival and EFS were 87% and 72%, with a median follow-up time of 10 and 8 months, respectively. Image: Summary/Conclusion: Our results show that asciminib is a well-tolerated novel treatment option for CML with adequate response rates, even in a heavily pretreated multi-resistant patient cohort. Most frequently reported AEs were manageable cytopenia and folliculitis. No patient discontinued asciminib solely due to intolerance. The CI of maintaining or reaching MR2.0/CCYR at 12 months was 57%. In our cohort, prior primary ponatinib-failure generally heralded subsequent asciminib treatment failure, both in the presence and absence of a T315I-mutation. P710: CLINICAL STUDY ON THE RELATIONSHIP BETWEEN NON-ABL1 KINASE REGION MUTATION AND TKI DRUG RESISTANCE AND DISEASE PROGRESSION IN CHRONIC MYELOID LEUKEMIA W. xianwei1, L. xiaodong1,* 1Central Lab, Henan Cancer Hospital, Zhengzhou, China Background: The emergence of small molecule tyrosine kinase inhibitors (The Tyrosine Kinase Inhibitors, TKI) has gradually transformed chronic myelocytic leukemia (CML) from a fatal malignant disease into a chronic disease, while ABL1 kinase Kinase Domain (KD) mutation is the main reason for poor efficacy or resistance to TKIs. In recent years, with the clinical application of Next Generation Sequencing (NGS) technology, it has been found that CML patients are often accompanied by non-ABL1 kinase region gene mutations, but whether these accompanying mutations are related to the efficacy of TKIs and disease progression in CML? still uncertain. This study retrospectively analyzed the correlation between CML with non-ABL1 KD mutation and TKI efficacy and disease progression, in order to provide basis and scientific guidance for further accurate diagnosis and treatment of CML patients. Aims: To investigate the relationship between CML associated with non ABL1 Kinase Domain (KD) mutation and TKI efficacy and disease progression. Methods: 108 blood tumor related genes in 120 patients with CML were detected by Next-generation sequencing (NGS). The non ABL1 KD mutations and their relationship with relevant clinical information and ABL1 KD mutation were analyzed. Results: Non ABL1KD mutations were detected in 58 patients (48%), and the mutation proportion of patients with additional chromosome abnormalities (63%) was significantly higher than that of patients without additional chromosome abnormalities (34%) (P=0.003). In the analysis of mutation proportion in patients with different stages of CML, the mutation proportion in blastic phase(73%), accelerated phase (67%) and chronic phase (42%) decreased gradually (P=0.031). The proportion of WT1 mutation and transcriptional regulation related mutations in patients with AP CML (WT1+: 17%, P=0.027; Transcriptional regulatory mutations 42%, P=0.001) were higher than those in CP. In the analysis of TKI treatment response, the proportion of mutations detected in the best response group (30%), warning group (52%) and drug resistance group (61%) increased gradually (P=0.018). The proportion of RUNX1 mutation in TKI treatment failure group (17%) was higher than that in warning group (2%) (P=0.029). The proportion of ABL1 KD mutation in CML patients with non ABL1 KD mutation (60%) was significantly higher than that in patients without non ABL1 KD mutation (36%) (P=0.022). Summary/Conclusion: Non ABL1 KD mutations are closely related to TKI efficacy and disease progression, while WT1 and RUNX1 mutations may be new molecular targets for accurate diagnosis of CML. P711: KINETICS OF CITED2 GENE EXPRESSION IN CHRONIC MYELOID LEUKEMIA PATIENTS B. Atef1,*, S. El-Ashwah1, L. M. Saleh2, H. Gawish3, M. Nasr1 1Hematology Unit, Internal Medicine Department; 2Hematology Unit, Clinical Pathology Department; 3Diabetes & Endocrinology Unit, Internal Medicine Department, Faculty of Medicine Mansoura University, Mansoura, Egypt Background: In chronic myeloid leukemia (CML), BCR-ABL1 oncoprotein induces overexpression of STAT5 and its target genes. Some of these genes are involved in regulating the leukemic stem cells (LSCs) cell cycle. CITED2 gene controls the balance between the proliferation and the quiescence states of the LSCs that resist tyrosine kinase inhibitors (TKIs). Recently, studies reported that adding peroxisome proliferator‐activated receptor γ agonists (approved for type 2 Diabetes mellitus treatment) to TKI therapy may decrease the transcription of STAT5 and its target genes, eliminate the quiescent LSCs and ameliorate the patients’ response. Aims: The aim of the study is to clarify the value of combining the glitazones with the TKIs in the treatment of denovo CML CP patients as regard the patients’ response and effect of this combination therapy on the expression of CITED2 gene. Methods: In a context of a clinical trial (approved by the ethical Committee of the Faculty of Medicine Mansoura University), eligible denovo patients of CML chronic phase (N=26) were treated with imatinib 400 mg combined and pioglitazone 15 mg (off label use) daily for 6 months after having their consent. Responders continued on same TKI and their sequential BCR/ABL Q-PCR was monitored. The non-responder patients were switched to another line and excluded. Pre and post-treatment samples were collected to assess the expression levels of CITED2 gene. Results: The study group included 15 males (57.7%) and 11 females (42.3%) with a mean age of 43.2 years. Their median BCR-ABL gene expression before treatment was 79% (range = 0.63-380) that decreased to 3.65% (range = 0.05-20) and 0.750 % (range = 0.002-46) on 3rd and 6th months post-treatment. Among the study group 100% of the patients achieved hematological response (HR) at 3 months. At 6 months 65.4% of the patients had response more than CCyR and 23% of them had MMR at 6 months respectively. The most frequent complains among patients were increase body weight and edema (p value < 0.001). The median of CITED2 gene pre-treatment and post-treatment expression levels were: 276.3 (range= 1-241221.7) and 2.6 (range= 0.006-86475.3) with a significant decrease in expression (p=0.005) after the combined treatment. Summary/Conclusion: Pioglitazone in combination with imatinib improved the patients’ response and downregulate the expression of the CITED2 overexpressed gene. Longer follow up is planned and needed to confirm whether it can effectively induce more deep molecular response among the patients or not. P712: ASCIMINIB ITALIAN MANAGED ACCESS PROGRAM: EFFICACY PROFILE IN HEAVILY PRE-TREATED CML PATIENTS M. Breccia1,*, A. V. Russo Rossi2, B. Martino3, E. Abruzzese4, M. Annunziata5, G. Binotto6, A. Ermacora7, C. Fava8, L. Giaccone9, V. Giai10, A. P. Nardozza11, P. Coco11, A. Gozzini12, L. Levato13, A. Lucchesi14, L. Luciano15, M. Maria Cristina16, G. Rege-Cambrin17, M. Santoro18, B. Scappini19, A. R. Scortechini20, P. Sportoletti21, E. Trabacchi22, F. Castagnetti23 1Department of Translational and precision medicine-Az, Policlinico Umberto I-Sapienza University, Rome; 2Hematology and Transplantation Unit, University of Bari, Bari; 3Azienda Ospedaliera “Bianchi Melacrino Morelli”, Reggio Calabria; 4Hematology, Sant Eugenio Hospital, Rome; 5Division of Hematology, AORN Cardarelli, Naples; 6Hematology Unit, University of Padova, Padova; 7Department of Internal Medicine, Pordenone General Hospital, Pordenone; 8Clinical and Biological Sciences, University of Turin; 9Department of Molecular Biotechnology and Health Sciences, University of Turin, Struttura Semplice Dipartimentale Allogeneic Stem Cell Transplant, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino; 10Division of Haematology, Città della Salute e della Scienza, Turin; 11Novartis Oncology, Medical Department, Novartis Farma SpA, Origgio; 12Department of Cellular Therapies and Transfusion Medicine, AOU Careggi, Florence; 13Department Hematology-Oncology, Azienda Ospedaliera Pugliese-Ciaccio, Catanzaro; 14Hematology Unit, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola; 15Hematology Unit, AUOP Federico II, Naples; 16Hematology Department, San Bortolo Hospital, Vicenza; 17Medicina Interna e Ematologia, Ospedale San Luigi, Università di Torino, Orbassano; 18Hematology, University Hospital Paolo Giaccone, Palermo; 19Hematology Unit, Azienda Ospedaliero-Universitaria Careggi, Florence; 20Division of Hematology, Azienda Ospedaliero Universitaria Ospedali Riuniti Ancona, Ancona; 21Institute of Hematology, Centro Ricerche Emato-Oncologiche, Ospedale S. Maria della Misericordia, University of Perugia, Perugia; 22Hematology Unit and BMT Center, Ospedale G. Saliceto, Piacenza; 23Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy Background: Chronic myeloid leukemia (CML) patients who have experienced failure and/or intolerance to multiple lines of treatment have limited therapeutic possibilities. Novel treatment options are needed to achieve sustained responses and increased tolerability in this subset of patients. Asciminib, unlike all approved tyrosine kinase inhibitors (TKIs) that bind to the ATP site of the BCR-ABL1 oncoprotein, is an allosteric, first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor with a new mechanism of action that has shown efficacy in heavily pre-treated patients after two TKIs or more. Aims: We share our experience on asciminib use in CML patients outside of clinical trials. Methods: We describe retrospective data from 34 chronic phase CML patients in their 3rd or later line, treated with asciminib between April 2019 and October 2021 in 22 Italian institutions. The drug was granted by Novartis under a Managed Access Program (MAP). BCR::ABL1/ABL ratio was expressed as % IS in all centers. Efficacy was analyzed by comparing the response registered at the latest time point (34 pts evaluable) or at 3 months (27 pts evaluable – we excluded those patients for whom molecular analysis was not assessed) vs the response at baseline. Patient recruitment, dosing regimen and safety were recorded according to the MAP recommendations. The safety information was extracted from Argus database, a Novartis database where all adverse events related to Novartis compounds are stored. Results: Median time of asciminib treatment was 8.3 months (3.3–33.2) for the whole cohort. Patients’ characteristics are displayed on Fig 1. Twenty-five patients (73.5%) were pretreated with at least 3 or more TKIs and 50% have reported to have ≥3 comorbidities. Approximately 59% of patients received prior ponatinib. Switch to asciminib occurred for resistance in 21 pts (61.8%) and intolerance in 10 pts (29.4%). Ten patients (29.4%) harbored mutations in ABL kinase domain, and 5 (14.7%) have a T315I mutation. All the T315I patients had a previous ponatinib exposure. Nineteen out of 27 (70.4%), 12/27 (44.4%) and 6/27 (22.2%) achieved or maintained respectively Molecular Response of 2 log reduction (MR2), Major Molecular Response (MMR) and Deep Molecular Response (DMR) at three months’ time point. Twenty-four out of 34 (70.5%), 14/34 (41.7%) and 8/34 (23.5%) achieved or maintained respectively MR2, MMR, DMR response at the last follow up. After 3 months and at the last follow up, 18/27 (66.7%) and 20/34 (58.8%) of patients, respectively, showed an improvement of previous baseline response. Approximately 37% and 35%, without MMR response at baseline, reached at least an MMR at three months and last follow up, respectively. We also observed that ponatinib treated patients showed a reduced probability of reaching MR2, MMR and DMR responses compared to ponatinib naive patients at both 3 months and last follow up time points. Seventy-five percent of patients with a T315I mutation showed a response improvement, respect to baseline, already after 3 months on asciminib. The safety profile of asciminib, based on the information spontaneously reported by physicians, remained consistent with that already reported, with no new safety findings. Image: Summary/Conclusion: Asciminib has shown a promising efficacy profile and tolerability in a setting of CML patients resistant and/or intolerant to 2 or more TKIs and with a high comorbidity burden. Our results confirmed what observed in sponsored trials and in the other world-wide programs, strongly suggesting a possible role for this new agent in the future therapeutic scenario. P713: REAL-WORLD MANAGEMENT OF PATIENTS WITH CHRONIC MYELOID LEUKAEMIA (CML): FIRST ANALYSIS FROM THE UK NATIONAL REGISTRY FOR CML G. Nesr1,*, R. Szydlo1, B. Braithwaite2, S. Frackelton2, P. Mathew2, M. Hashmi2, F. Fernando1, A. Innes3, S. Claudiani1, A. Khan1, A. Rao4, L. Foroni1, D. Milojkovic3, J. F. Apperley1, R. E. Clark5 1Centre for Haematology, Imperial College London, London; 2The Royal Liverpool University Hospitals, Liverpool; 3Department of Clinical Haematology, Imperial College Healthcare NHS Trust; 4Department of Clinical Haematology, Great Ormond Street Hospital for Children, London; 5Department of Molecular & Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom Background: Analysing data emerging from population based cancer registries (PBCR) informs current practice. The UK National Registry for chronic myeloid leukaemia (CML) patients (ptn) is a web-based PBCR, initially established as a regional registry in 2010, then expanded nationally in 2015. Aims: To understand real world management of UK CML ptn. Methods: The registry utilizes an online interface to capture baseline and yearly follow up data from contributing centres (CC). Data are securely transferred using the N3 backbone of the NHS network. Ptn are consented for enrolment at their respective centres. A “treatment episode” is defined as the duration during which a ptn received a specific tyrosine kinase inhibitor (TKI). The 2013 version of the ELN guidelines was used to define treatment milestones. For each milestone timepoint, treatment switches were counted if they occurred before the next milestone. Results: Demographic and baseline disease characteristics data were available for 748 ptn from 43 CC (Figure 1). Of those, 79%, 75% and 94% had available karyotypes, FISH for BCR-ABL1, and RT-PCR for BCR-ABL1, respectively, at diagnosis and/or follow up. Follow up and treatment data were available for 570 and 534 ptn, respectively. Median follow up duration was 4.7 years (range: 0.1-23.7). Median age at diagnosis was 53.3 years (range: 14.6-95.1) and 53% were males. Karyotype and RT- PCR for BCR ABL1 at diagnosis were available for >75% of ptn in 40% of CC. Molecular monitoring data were available for 540 ptn: 342 (63%) had a median testing frequency of ≤ 3 months, 167 (31%) between 3-6 months, 20 (4%) between 6-12 months, and 11 (2%) of > 12 months. Imatinib (IM), nilotinib (NIL), dasatinib (DAS), bosutinib and ponatinib were prescribed as 1st or subsequent line therapy in 46%, 23%, 21%, 14%, and 7% of the treatment episodes, respectively. IM remained the most frequent 1st-line treatment after NICE approval of 2GTKI in newly diagnosed ptn. The choice of 1st line TKI (IM vs 2G) was not influenced by the Sokal, Euro, EUTOS or ELTS scores. Of ptn started on 1st line IM, NIL, and DAS; 94%, 63% and 100% received the licensed doses (400 mg OD, 300 mg BD, and 100 mg OD), respectively. Optimal responses at 3,6 and 12 months were achieved in 73%, 63%, and 51% of pts. Failure at 3 months was not evaluable as the registry did not capture sequential FBCs. Of those with a suboptimal response at 3 months, 15% changed treatment. At 6 months 28% and 50% of ptn with suboptimal and failure responses, respectively, switched their TKI. At 12 months, 10% and 28% of ptn in the suboptimal and failure categories changed TKI. Between 1-6 treatment switches, for failure and/or intolerance, occurred in 46% of ptn. Treatment switches on IM were more common for resistance than intolerance and vice versa for 2G and 3G TKI. Of all ptn who switched TKI, 40% underwent a further TKI switch. Kinase domain mutation analysis was requested in 36% and 33% of ptn resistant to their TKI at 6 and 12 months, respectively. Five-year overall survival (OS) probability was 93% for CP-CML ptn. Ptn achieving CCyR had a significantly higher OS probability than those who did not (p<0.001 by log rank test). However, differences in OS probability were not statistically significant for ptn attaining deeper responses (p<0.5 for MR3, p<0.076 for MR4, p<0.141 for MR4.5, and p<0.084 for MR5). Image: Summary/Conclusion: Our results highlight the lack of conformance to the current guidelines in various aspects of CML management, the excellent OS survival achievable with currently available TKIs and the importance of achieving CCyR for better OS. P714: EFFECT OF TYROSINE KINASE INHIBITORS ON MALE FERTILITY IN PATIENTS WITH CHRONIC PHASE CHRONIC MYELOID LEUKAEMIA G. Nesr1,*, S. Claudiani1, D. Milojkovic2, I. Caballes2, S. Lovato1, J. Channa3, J. F. Apperley1 1Centre for Haematology, Imperial College London; 2Department of Clinical Haematology, Imperial College Healthcare NHS Trust; 3Section of Investigation Medicine, Imperial College London Faculty of Medicine, London, United Kingdom Background: With the current availability of 3+ generations of tyrosine kinase inhibitors (TKIs) and the resultant survival benefit, chronic phase chronic myeloid leukaemia (CP-CML) is considered a chronic disease for most patients. Consequently, haematologists are faced more with questions regarding the effects of treatment on fertility and pregnancy outcomes. Whilst the teratogenic effect of TKIs is well established, data on their effect on fertility, and especially male fertility, are scarce. Semen analysis parameters (sperm number, motility and morphology) remain the cornerstone in assessing male fertility, despite its limitations in comprehensively assessing sperm functions. Aims: To compare semen analysis parameters before and after TKI therapy in CP-CML patients treated in the haematology department of Imperial College Healthcare NHS Trust (ICHNT). Methods: In collaboration with the Andrology Department, newly diagnosed men with CP-CML are offered sperm banking before starting cytoreductive or TKI therapy, and sperm analyses are routinely undertaken. Patients treated with TKIs who had sperm banking at diagnosis were invited for repeat semen analysis. Treatment history and semen analysis parameters were collected from patients’ paper/electronic medical records. Median and range were used to summarize continuous non-parametric data. Paired sample t-test was used to compare continuous semen analysis parameters before and after TKI therapy. Double-sided p value of < 0.05 was considered as statistically significant. SPSS version 25 software was used for statistical analysis. Results: Twenty-five patients were identified to have a semen analysis performed around the time of starting TKI therapy and were approached for repeat testing. Of those, 13 refused to participate, 1 was an international patient no longer residing in the UK and 1 stopped TKI therapy for treatment free remission trial. Therefore, 10 patients were finally included. Median age at diagnosis was 35.6 years (range: 22.9-45.4) and patients were diagnosed between 2001 to 2014. Six and 2 patients received hydroxyurea and interferon α, respectively, before TKI therapy. Six patients received 1 TKI (imatinib (IM), n = 4; nilotinib (NIL), n = 1; bosutinib (BOS), n = 1), 2 received 2 TKIs (IM followed by NIL), and 2 received 3 TKIs (IM, DAS, NIL and IM, NIL, DAS). Switches were for failure and/or intolerance. Median duration of TKI therapy at the time of repeat semen analysis was 5.9 years (range: 3.3-16.5). Patients 6 and 10 had the initial semen analysis after 736 and 475 days, respectively, from commencing TKI therapy and all remaining patients had it before starting. All patients had a deep molecular response at the time of the repeat semen analysis. The differences between sperm concentration, % progressive motility and % total motility were all non-statistically significant (p = 0.457, 0.535, and 0.574, respectively). In 2 patients the semen parameters were below the reference ranges in both occasions. Morphology (% normal forms) was less than the reference 4% in the repeat samples of 5 patients (Table 1). Image: Summary/Conclusion: In our cohort, no significant differences were found in sperm concentration or motility before and after treatment with different TKIs for a prolonged period. The relevance of the finding of a low % of normal forms, in the presence of normal sperm concentration and motility, in some of the patients in the semen sample after TKI therapy is not clear. Studies with larger sample size would be needed to confirm or refute our findings. P715: MULTICENTER OBSERVATIONAL STUDY OF PONATINIB IN CML PATIENTS IN ARGENTINA. REAL-WORLD EXPERIENCE. M. J. Mela Osorio1, M. V. Osycka2, B. Moiraghi2, M. A. Pavlovsky1, A. I. Varela2, I. Fernandez1, F. Sackmann Massa1, L. Ferrari1, I. Giere1, C. Sighel1, M. Riddick3, C. Pavlovsky1,* 1HEMATOLOGY, FUNDALEU; 2HEMATOLOGY, Hospital General de Agudos Jose Maria Ramos Mejia; 3HEMATOLOGY, Instituto de Investigaciones Fisicoquímica Teóricas y Aplicadas (INIFTA, Facultad de Ciencias Exactas, Universidad Nacional de La Plata- CONICET). Departamento de Matemática, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Buenos Aires, Argentina Background: Ponatinib is a 3rd generation tyrosine kinase inhibitor (TKI) indicated for adults with resistant or intolerant chronic phase (CP), accelerated phase (AP) or blast phase (BP) chronic myeloid leukemia (CML) as well as in patients carrying the T315I mutation. There is a lack of data on its use in the real-world setting. Aims: We aimed to evaluate treatment patterns, outcomes and cardiovascular toxicity in CML patients treated with ponatinib in Argentina. Methods: This retrospective study included all patients aged ≥ 18 years with CP, AP and BP CML who initiated ponatinib in routine clinical practice across 2 medical centers in Argentina from 2013 to 2021. Demographic, efficacy and safety data were collected from patient medical charts. Continuous variables were dichotomized. Association was evaluated through Fisher exact test. Results: A total of 56 patients were analyzed, 53 in CP, 2 in AP and 1 in BP. One patient (1.8%) received ponatinib in first-line, 19 (34%) in second-line (2L), 35 (62.4%) in 3L, and 1 (1.8%) in ≥4L. Prior cardiovascular (CV) risk factors were recorded: hypertension 8/56 14%, body mass index ≥25kg/m2 32/56 57%, hyperlipidemia (>220mg/dl) 13/56 23.2%, smokers 10/56 18% and diabetes mellitus 4/56 7%. Median age at ponatinib start was 43.5 years (range, 18–73). Of 38 evaluated patients, 23 (60.5%) had a confirmed ABL1 mutation, including 19 (50%) with the T315I mutation. Starting doses of ponatinib were 45 mg (34%), 30 mg (53.5%), or 15 mg (12.5%). The median follow-up was 130 months (range, 13-324) and median treatment duration was 38 months (range, 1–105). At baseline, 51 patients (91%) with CP CML had less than CCyR and 2 (3.5%) were in CCyR. For 3 patients (5.3%), assessment was not available. At 3 months, 15/51 (29%) evaluable patients with CP CML were in CCyR; at 6 months, 17/44 (38.6%) evaluable patients with CP CML were in CCyR and at 12 months, 19/34 (56%) evaluable patients with CP CML were in CCyR. Additionally, 21 (37.5%) and 9 (16%) patients with CP CML achieved a major molecular response (MMR) or a deep molecular response (MR4.0–MR5.0) at least once during follow-up, respectively. Progression-free survival rates estimated for patients with CP CML at 12 and 24 months were 90.3% (95% CI, 82.6–98.8%) and 86.2% (95% CI, 77.3–96.3%), respectively. Corresponding overall survival rates were 100%, and 96.1% (95% CI, 90.9–100%). Serious arterial thrombotic adverse events (AT-AEs) occurred in 8 patients (14%), 60% of these were coronary artery disease, 30% cerebrovascular and 10% peripheral arterial disease. AT-AEs events occurred after a median time on ponatinib of 5 months (range, 2-48), the majority of the events were severe but resolved, however two were fatal. Patients who developed an AT-AE after starting ponatinib were older than 60 years (p=0.0174), and had higher cholesterol levels (p=0.03979). No associations were found regarding obesity (p=0.403), previous TKI lines (p>0.5), smoking history (p>0.5) or ponatinib starting dose ≤30mg vs >30mg (p=0.4248). Summary/Conclusion: This is the first analysis of ponatinib treated patients in real-world in Argentina and demonstrates that ponatinib has a favorable efficacy and safety profile in patients with CML treated in standard clinical practice. Cardiovascular toxicity of ponatinib in routine clinical practice is increased. Older (>60y) and dyslipemic patients were at higher-risk of occlusive events. Prompting aggressive control of CV risk factors as well as reducing doses at optimal time-points may help optimize the use of ponatinib during daily practice. P716: OUTCOMES AND PATTERNS OF TREATMENT IN CHRONIC MYELOID LEUKEMIA, A GLOBAL PERSPECTIVE BASED ON A REAL-WORLD DATA GLOBAL NETWORK A. Sanz1,*, R. Ayala1, G. Hernández2, N. López1, D. Gil1, R. Gil1, R. Colmenares1, G. Carreño-Tarragona1, J. M. Sánchez Pina1, R. A. Alonso1, N. García Barrio3, D. Pérez-Rey2, L. Meloni4, M. Calbacho1, M. Pedrera-Jiménez3, P. Serrano-Balazote3, J. de la Cruz5, J. Martínez-López1 1Hematology, Hospital 12 de Octubre; 2Biomedical Informatics Group, Universidad Politécnica de Madrid; 3Data Science Group, Research Institute imas12, Madrid, Spain; 4Trinetx, LLC, Cambridge, United States of America; 5Research Institute imas12, Hospital 12 de Octubre, Madrid, Spain Background: The natural history of chronic myeloid leukemia (CML) experienced a major change in the early 2000s with the development of the first BCR-ABL1 specific tyrosine kinase inhibitors (TKIs); however, there is limited supporting evidence from electronic health records (i.e., real-world data [RWD]). TriNetx is a global health research platform that provides researchers access to electronic medical records from healthcare organizations (HCOs) for conducting research studies. It allowed us to analyze trends in survival in CML patients throughout the years from a global perspective, as well as examine relevant comorbidities and long-term toxicities. Aims: The objective of this study is to better define the current survival landscape of CML and the long-term toxicities, so that we can have a better and updated understanding of the disease and the actual clinical practice in local, regional and global settings. Methods: We analyzed data from more than 7500 patients diagnosed with CML from four research networks (Hospital 12 de Octubre (H12O), the EMEA network, the US network, the Global Network) covering more than 16 million subjects. Propensity score matching for age and gender was applied, comparing samples equal in size (CML and controls). This study was conducted with anonymized data accessed via the TriNetX platform, which provided diagnoses, procedures, medications, laboratory values, and genomic information from approximately 6,000 CML patients treated with TKIs from 57 health care organizations. Results: Survival at 20 years was not significantly different between patients with CML treated with TKIs and non-cancer patients (63·4% vs 69·05%, HR 1.095 (0·924 - 1·298) p = 0·295, Figure 1). No significant change in survival probability was seen comparing patients treated 2001-2010 and 2011-2020 (HR 1·113 (0·858 - 1·443), p = 0.42) or comparing imatinib with second-generation TKIs (imatinib vs dasatinib HR 1·017 (0·895 - 1·157), log-rank p = 0·80; imatinib vs nilotinib HR 1·101 (0·925 - 1·310). When comparing the occurrence of second malignancies, an increased incidence was seen in CML patients (64·4% vs 17·2%, OR 8·72 (7·955, 9·549)). In the case of cardiovascular diseases, CML patients again showed a higher incidence than non-CML patients (74·2% vs 60·5%, p<0·001, OR 1·879 (1·729, 2·041)). No specific TKI was associated with the increased risk of any of these conditions. Regarding TKI usage and switching patterns, dasatinib was the most common second-line treatment for both H12O and the US, but not in the EMEA where it was nilotonib. Ponatinib assumed a larger role in third-line and beyond. Image: Summary/Conclusion: From this large real-world analysis, we conclude that in the TKI era, the CML population has the same survival as a non-cancer population. However, the main TKI toxicities and comorbidities remained high in the CML population, and despite them not seeming to impact survival, it could affect the quality of life. Therefore, strategies to stop lifelong TKI use should be prioritized to reduce adverse events. P717: BOSUTINIB IN NEWLY DIAGNOSED CHRONIC MYELOID LEUKEMIA: GASTROINTESTINAL, LIVER, EFFUSION AND RENAL SAFETY CHARACTERIZATION IN THE BFORE TRIAL J. E. Cortes1,*, D. Milojkovic2, C. Gambacorti-Passerini3, V. García-Gutiérrez4, M. J. Mauro5, E. Leip6, S. Purcell7, A. Viqueira8, T. H. Brümmendorf9 1Georgia Cancer Center, Augusta, United States of America; 2Hammersmith Hospital, London, United Kingdom; 3University of Milano-Bicocca, Monza, Italy; 4Universitario Ramón y Cajal, Ramón y Cajal Health Research Institute, Madrid, Spain; 5Memorial Sloan Kettering Cancer Center, New York; 6Pfizer Inc, Cambridge, United States of America; 7Pfizer Ltd, London, United Kingdom; 8Pfizer SLU, Madrid, Spain; 9Universitätsklinikum RWTH Aachen, Aachen, Germany Background: Bosutinib is approved for the treatment of Philadelphia chromosome–positive (Ph+) chronic myeloid leukemia (CML) resistant/intolerant to prior therapy and newly diagnosed Ph+ chronic phase (CP) CML. Efficacy and safety of bosutinib vs imatinib in patients with newly diagnosed CP CML was assessed in the phase 3 BFORE trial (NCT02130557). Aims: The safety profile of bosutinib after 5 years follow-up, with a focus on gastrointestinal, liver, effusion and renal treatment-emergent adverse events (TEAEs), was characterized. Methods: Patients who received ≥1 dose of bosutinib (n=268) or imatinib (n=265) 400 mg/day in BFORE were included. Adverse events (AEs) of special interest were analyzed by selecting prespecified MedDRA terms to generate TEAE clusters. This analysis is based on the final database lock: June 12, 2020. Results: Median duration of treatment was 55 months for patients receiving bosutinib or imatinib; respective median (range) dose intensity was 393.6 (39–583) vs 400.0 (189–765) mg/d. Any grade TEAEs occurred in 98.9% and 98.9% of bosutinib- vs imatinib-treated patients. The most common newly occurring TEAEs (any grade) after 12 months were increased lipase (9.0%) with bosutinib, and diarrhea (8.3%) with imatinib. In bosutinib- vs imatinib-treated patients, 25.4% vs 14.3% had AEs leading to permanent treatment discontinuation; the majority discontinued in year 1 (14.2% vs 10.6%). The most frequent AEs leading to discontinuation were increased ALT (overall, 4.9%; year 1, 4.5%) with bosutinib vs thrombocytopenia (overall, 1.5%; year 1, 1.5%) with imatinib. Gastrointestinal, liver, effusion and renal TEAEs, respectively, occurred in 79.9%, 44.0%, 6.0% and 10.4% (maximum grade 3/4 [G3/4]: 9.0%, 26.9%, 1.1% and 2.2%) of bosutinib- vs 61.5%, 15.5%, 2.3% and 9.8% (G3/4: 1.1%, 4.2%, 0.4% and 0.8%) imatinib-treated patients. One grade 5 renal TEAE occurred in the bosutinib arm and was not considered related to treatment. Cumulative rates per treatment year are shown in the Table. The most common gastrointestinal TEAEs were diarrhea (bosutinib vs imatinib: 75.0% vs 40.4% [G3/4: 9.0% vs 1.1%]) with bosutinib, and nausea (37.3% vs 42.3% [G3/4: 0% vs 0%]) with imatinib. In both arms, the most common liver, effusion and renal TEAEs, respectively, were increased ALT and/or AST (34.0% vs 8.3% [G3/4: 22.0% vs 2.3%]), pleural effusion (5.2% vs 1.9% [G3/4: 0.7% vs 0.4%]) and increased blood creatinine (6.7% vs 8.3% [G3/4: 0.4% vs 0.4%]). Gastrointestinal, liver, effusion and renal TEAEs infrequently led to treatment discontinuation (1.9%, 7.8%, 0.7% and 0.7% vs 1.1%, 0.8%, 0% and 0.4%). Image: Summary/Conclusion: The safety profiles of bosutinib and imatinib in BFORE were distinct, with no new safety signals identified after 5 years of follow-up. The onset of TEAEs occurred primarily during year 1 (e.g., gastrointestinal and liver), with an increased incidence of some TEAEs (e.g., effusion and renal) in later years. Discontinuations due to AEs generally occurred early into treatment, with low rates of discontinuation due to gastrointestinal, liver, effusion and renal AEs. These long-term safety results further support the use of first-line bosutinib as a standard of care in patients with CP CML. P718: ASCIMINIB MANAGED-ACCESS PROGRAM (MAP) IN RUSSIA O. Shukhov1,*, A. Turkina1, E. Lomaia2, E. Morozova3, A. Petrova1, T. Chitanava2, Y. Vlasova3, E. Chelysheva1, E. Kuzmina1, I. Nemchenko1, A. Bykova1, M. Gurianova1, A. Kohno1, E. Parovichnikova1 1National Medical Research Center for Hematology, Moscow; 2Almazov National Medical Research Centre; 3Raisa Gorbacheva Memorial Research Institute of Children’s Oncology, Saint-Petersburg, Russia Background: Asciminib is aa ABL kinase inhibitor that works by Specifically Targeting the ABL Myristoyl Pocket (STAMP) that has shown efficacy and a good safety profile in phase I and III studies in patients with Ph-positive leukemia failing prior TKIs. In Russia, asciminib is available under the Novartis approved Managed Access Program (MAP). Aims: to present interim results of the use of aciminib in clinical practice under the MAP program in Russia. Methods: From September 2019 to January 2022, 68 patients (pts) with CML were enrolled in the MAP program from three Russian clinics. We analyzed therapy results from 50 pts. Eleven pts who received asciminib for less than 3 months and 7 pts who underwent bone marrow transplantation were excluded from the analysis. Pts recruitment, dosing regimen, response monitoring, and toxicity control were performed according to the MAP treatment plan. Complete cytogenetic response (CCyR), major molecular response (MMR) and deep molecular response (MR4) were assessed by cumulative incident function (CIF) with Gray’s test for comparison responses in subgroups. Multivariate analysis was performed to find independent factors for MMR. Differences were considered significant if the p ≤ 0.05 Results: Baseline characteristics: female 66%; Ме age 53 years (range 26-81); median duration of CML before asciminib 8 years (range 1-24); 42 pts were in chronic phase (CP) CML, 7 and 1 pts had a history of accelerated phase (AP) and myeloid blast crisis (MBC), respectively. Twenty nine (58%) pts had BCR::ABL1 mutations, 20 pts (40%) had BCR::ABL1 T315I, 8 (16%) pts had at least two mutations. Eight (16%) pts had additional chromosomal abnormalities (ACAs). Among the thirty three (66%) pts who received ≥4 TKIs, 20 (40%) had a history of ponatinib treatment. Me follow-up period was 11 months (range 4-30), 7(14%) pts discontinued asciminib (5 due to resistance, 2 due to progression). One-year survival rate without treatment discontinuation was 92%. In 30 (60%) pts, The initial dose was 40 BID in 30 (60%) pts and 200 mg BID in 20 (40%) pts The 2-year overall survival was 96%, two (4%) pts progressed to advanced phase and died. CIF of CCyR, MMR and MR4 at 12 month was 37.5% (CI:24-58), 32% (CI:20-52) and 14% (CI:7-31), respectively. Univariate analysis was performed for the following factors: history of advanced phases, initial dose of asciminib, presence of BCR::ABL1 mutations and ACAs, best molecular response on previous TKIs, molecular response at the time of asciminib starting, history of ponatinib treatment, number of prior TKIs and duration of TKI therapy before asciminib. Best molecular response to previous TKIs, molecular response at the time of asciminib starting, number of TKIs prior to asciminib, and history of previous ponatinib treatment were identified as predictors of MMR at 12 months (Table 1). Best molecular response on previous TKIs (≤1% vs 1-10% vs ≥10%) was found to be independently significant factor due to multivariate analysis (p=0,0072, hazard ratio 7,6 (1,7-33)) Twenty two (44%) patients experienced adverse events (AEs) of any grade and 8 (16%) had AEs of grade 3-4 (Table 2), reported during treatment, regardless of the connection with the drug. No one stopped treatment because of toxicity. No differences in toxicity were found between doses of 80 and 400 mg/day. Image: Summary/Conclusion: Asciminib shows promising results for the treatment of highly pre-treated CML patients and should be considered as an therapeutic options for pts with failure to previous TKIs P719: EFFICACY AND SAFETY OF BOSUTINIB IN LATER-LINE PATIENTS WITH CHRONIC MYELOID LEUKEMIA: A SUB-ANALYSIS FROM THE PHASE 4 BYOND TRIAL C. Gambacorti-Passerini1,*, T. H. Brümmendorf2, T. Ernst3, E. Leip4, S. Purcell5, A. Viqueira6, F. J. Giles7, G. Rosti8, A. Hochhaus3 1University of Milano-Bicocca, Monza, Italy; 2Universitätsklinikum RWTH Aachen, Aachen; 3Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany; 4Pfizer Inc, Cambridge, United States of America; 5Pfizer Ltd, London, United Kingdom; 6Pfizer SLU, Madrid, Spain; 7Developmental Therapeutics Consortium, Chicago, United States of America; 8IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola, Italy Background: Bosutinib is approved for patients with Philadelphia chromosome-positive chronic myeloid leukemia (CML) resistant or intolerant to prior therapy and newly diagnosed patients in chronic phase. The efficacy and safety of bosutinib in patients with CML resistant or intolerant to prior tyrosine kinase inhibitors (TKIs) was evaluated in the phase 4 BYOND trial (NCT02228382; Gambacorti-Passerini et al, Blood, 2021). Aims: The efficacy and safety of bosutinib in resistant and intolerant patients with CML treated with 2 (3L) and 3 (4L) prior TKIs was characterized. Methods: The BYOND trial included 163 patients with CML resistant or intolerant to prior TKIs. A sub-analysis of 48 patients treated with 2 (3L) and 3 (4L) prior TKIs, categorized by resistance or intolerance to the last received TKI, was performed. This sub-analysis is based on the final November 23, 2020 database lock. Results: There were 18 and 30 patients resistant or intolerant to the last received TKI who entered the study without complete cytogenetic response (CCyR) or major molecular response (MMR), respectively. Median (range) treatment duration was 10.6 months (1.6–48.5) vs 28.3 months (0.2–48.6) and median (range) dose intensity was 447.1 mg/d (131.3–520.4) vs 288.8 mg/d (79.7–500.0) for resistant vs intolerant patients. Prior TKIs included imatinib (88.9% vs 100.0%), dasatinib (88.9% vs 83.3%), and nilotinib (66.7% vs 63.3%) for resistant vs intolerant patients. Overall, 61.1% vs 66.7% of resistant vs intolerant patients discontinued bosutinib, mostly commonly due to adverse events (AEs) in 27.8% vs 16.7% patients; 16.7% vs 6.7% discontinued bosutinib due to insufficient clinical response. Rates of CCyR and MMR are shown in the Table. Among responders (resistant vs intolerant patients), median (range) time to CCyR was 5.1 months (2.8–8.8) vs 3.0 months (2.7–6.1); median (range) time to MMR was 5.8 months (2.8–9.4) vs 3.2 months (2.8–9.3). In resistant vs intolerant patients, any grade treatment-emergent AEs (TEAEs) were reported by 100.0% vs 96.7% patients; grade 3/4 TEAEs were reported by 72.2% vs 83.3% patients. Grade 3/4 TEAEs >10% in resistant patients were thrombocytopenia (22.2%) and neutropenia (11.1%), and in intolerant patients were increased alanine aminotransferase (26.7%), diarrhea (23.3%), pleural effusion (13.3%), and rash (13.3%). Image: Summary/Conclusion: This sub-analysis of resistant and intolerant patients without baseline CCyR or MMR shows bosutinib was active in heavily pretreated patients with resistance or intolerance to the last received TKI. Despite a difference between resistant and intolerant patients with CML, efficacy outcomes, though lower than the overall BYOND population, are encouraging, and safety was generally consistent with previous reports. P720: PROACTIVE DASATINIB DOSE REDUCTION IN THE ALLG CML 12 DIRECT STUDY BASED ON TROUGH LEVELS MINIMISE TOXICITY AND PRESERVE EFFICACY D. Yeung1,2,*, A. Grigg3, N. Shanmuganathan1, A. Soltenbeck4, D. White2, S. Branford5, N. Viiala6, P. Rowlings7, A. Mills8, J. Shortt9, C. Tiley10, D. Ross11, D. Kipp12, R. Harrup13, I. Cunningham14, J. Kwan15, R. Eek16, H. Mutsando17, K.-S. Tan18, K. Burbury19, M. Wright20, T. Hughes1,2, On Behalf of the ALLG21 1Haematology, Royal Adelaide Hospital; 2Precision Medicine, SAHMRI, Adelaide; 3Haematology, Austin Hospital; 4Statistical Revelations, Melbourne; 5Molecular Pathology, SA Pathology, Adelaide; 6Haematology, Liverpool Hospital, Sydney; 7Haematology, Calvary Mater Hospital, Newcastle; 8Haematology, Princess Alexandra Hospital, Brisbane; 9Haematology, Monash Medical Centre, Melbourne; 10Haematology, Gosford Hospital, Gosford; 11Haematology, Flinders Medical Centre, Adelaide; 12Haematology, Barwon Health, Geelong; 13Haematology, Royal Hobart Hospital, Hobart; 14Haematology, Concord Hospital; 15Haematology, Westmead Hospital, Sydney; 16Haematology, Border Medical Oncology, Albury; 17Haematology, Toowoomba Hospital, Toowoomba; 18Nephrology, Princess Alexandra Hospital, Brisbane; 19Haematology, Peter MaCallum Cancer Centre, Melbourne; 20Haematology, Finoa Stanley Hospital, Perth; 21ALLG, Melbourne, Australia Background: Dasatinib treatment leads to excellent molecular responses in chronic phase chronic myeloid leukemia (CP-CML) but at 100mg leads to a substantial risk of pleural effusions, which may be higher in the elderly. Rousselot et al (BJH 2021) suggested this risk may be mitigated through dose modifications as guided by trough levels (Cmin). Aims: The CML12 (DIRECT) study, run by the ALLG, is a single arm phase II study, aiming to minimise dasatinib related toxicity whilst preserving efficacy, through dose adaptation as guided by dasatinib trough levels. We report here the primary end point - cumulative incidence of pleural effusion (PEf) at 24 months (mos) and key efficacy secondary end points. Methods: DIRECT enrolled newly diagnosed CP-CML pts. The first 34 patients (pts) were aged >60 years (yrs), after which the protocol was amended to include pts aged >18 yrs at the recommendation of the trial management committee. All pts started on dasatinib 100mg QD, with Cmin taken at 7, 28, 56 & 90 days, then every 3 mos hence. Pts sequentially dose reduced to 70mg, then to 50mg QD, for Cmin >3nM, within the first 2 mos, prior to assessment of early molecular response at 3 mos. Doses <50mg QD were permitted temporarily only for toxicity management. Chest x-ray and transthoracic echocardiograms were performed at baseline & 24 mos. Results: Eighty pts were enrolled from 14 centres, with a median follow up of 36 mos (range 24-60). Median age was 64 yrs (range 21-86) and 46% were female. The ELTS score was low and intermediate in 54% and 34% respectively, high in 5% and missing in 8%. Cmin and treatment assigned at various timepoints are detailed in Table 1. Older patients had higher Cmin, particularly prior to dose adjustments. The majority of pts were dose reduced to 50mg QD. Fifteen cases of pleural effusion (PEf) occurred, 5 within the first mo, and 12 in total by 24 mos; 3 were asymptomatically detected on chest x-ray. The cumulative incidence of PEf by 24 mos was 15% overall (95% CI 8-25%, Fig 1); with values of 7.6%, 14.3% and 45.5% respectively in patients <60 yrs, 60-75 yrs and ≥75 yrs. The Cmin prior to the PEf was >3nM in 8/15 pts. At 24 mos, 59 (74%) of pts remained on dasatinib. Reasons for discontinuation were intolerance / adverse events (AE) (n=15, 19%); treatment failure (n=4, 5%); death (n=1, 1%) and consent withdrawn (n=1, 1%). AEs leading to discontinuation were PEf (n=6, 7.5%) pulmonary hypertension (n=4, 5%; 2 reported as SAEs), pericardial effusion (n=2, 2.5%) and cardiac failure (n=2, 2.5%). There were no cases of peripheral vascular disease or strokes. An early molecular response (BCR::ABL1 <10% IS at 3 mos) was achieved in 77 pts (96%). Cumulative incidence of MMR (<0.1%) by 12 and 24 mos were 75% (95% CI 66-84%) and 92% (95% CI 86-97%), respectively; MR4.5 by 24 and 48 mos were 48% (95% CI 38-60%) and 76% (95% CI 64-90%), respectively. No patient progressed to AP/BC on or off study. Three pts have died of non CML-related causes (infection n=2; biliary carcinoma n=1). Image: Summary/Conclusion: High rates of MMR/MR4.5 were achieved, despite significant dose reductions, according to target drug levels over the first 2 months. Toxicity profile was also broadly favourable. However, such adaptive approaches had limited capacity to influence early development of PEf. A strategy using lower starting doses, with escalation for failure to achieve molecular targets, may further minimise toxicity, especially in the elderly. P721: PREDICTIVE SCORING SYSTEMS FOR MOLECULAR RESPONSES IN PERSONS WITH CHRONIC MYELOID LEUKEMIA RECEIVING INITIAL IMATINIB THERAPY X. Zhang1,*, Z. Li1, M. Zhang1, P. G. Robert2, X. Huang1, Q. Jiang1 1Peking University People’s Hospital, Beijing, China; 2Centre for Hematology Research, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom Background: Nowadays more patients have benefited from tyrosine kinase inhibitors (TKIs) therapy to achieve a stable major molecular response (MMR) and even deep molecular responses (DMR, at least MR4), which prevented disease progression and allows for pursuing treatment-free remission (TFR). However, there were still lacking of robust prediction models for molecular responses including MMR or DMR to TKI-therapy. Aims: Develop and validate predictive scoring systems for MMR and MR4 in persons with newly-diagnosed chronic phase chronic myeloid leukemia (CML) receiving imatinib. Methods: Data from 1364 consecutive subjects were randomly-assigned 2:1 to training (n=909) and validation (n=455) datasets. Co-variates significantly associated with MMR and MR4 in multi-variable analyses in the training dataset were used to develop a predictive scoring system based on standardized regression coefficients (beta [β]) and the model tested in the validation dataset. Time-dependent area under the receiver-operator characteristic curves (AUROC), calibration plots and decision curve analyses (DCAs) were used to evaluate predictive accuracy. Results: In the training dataset, male sex (β=-0.350, HR=0.7 [95%CI: 0.6, 0.8]), WBC ≥ 130 ×10E+9/L (β=-0.538, HR=0.6 [0.5, 0.7]), hemoglobin < 110 g/L (β=-0.490, HR=0.6 [0.5, 0.8]) and EUTOS long-term survival (ELTS) risk score (intermediate-risk: β=-0.464, HR=0.6 [0.5, 0.8]; high-risk: β=-0.743, HR =0.5 [0.3, 0.7]) were significantly-associated with lower cumulative incidence of MMR. Each co-variate was assigned 1 point in the predictive scoring system for MMR except WBC ≥ 130 ×10E+9/L and ELTS high-risk assigned 2 points. Using the predictive scoring system, subjects were classified into (1) low- (score=0-1; n=429, 47%), (2) intermediate- (score=2-4; n=340, 38%); and (3) high- (score=5-6; n=140, 15%) risk subgroups. 5-year cumulative incidences of MMR were 86% (84, 89%), 61% (55, 65%) and 34% (27, 43%) (p for trend <0.001; Figure A). Comparable 5-year cumulative incidences in validation dataset were 86% (82, 90%), 69% (64, 74%) and 31% (22, 40%) (p for trend<0.001; Figure B). AUROC values were 0.77 and 0.74 in the training and validation datasets. Similar analyses for MR4 in training dataset identified male sex (β=-0.282, HR=0.8 [0.6, 0.9]), WBC ≥ 120 ×10E+9/L (β=-0.747, HR=0.5 [0.4, 0.6]), hemoglobin<110 g/L (β=-0.496, HR=0.6 [0.5, 0.8]) and ELTS risk score (intermediate-risk: β=-0.509, HR=0.6 [0.4, 0.8]; high-risk: β=-1.212, HR=0.3 [0.1, 0.6]) were significantly-associated with lower cumulative incidence of MR4. Male sex was assigned 1 point; ELTS intermediate-risk and hemoglobin < 110 g/L, 2 points; WBC ≥ 120 ×10E+9/L, 3 points; and ELTS high-risk, 4 points in the predictive scoring system for MR4. Subjects in the training dataset were classified into (1) low- (score=0-2; n=416, 46%), (2) intermediate- (score=3-7; n=348, 38%); and (3) high- (score=8-10; n=145, 16%) risk cohorts. 5-year cumulative incidences of MR4 were 71% (70, 73%), 35% (31, 39%) and 16% (10, 22%) (p for trend<0.001, Figure C). Similar 5-year cumulative incidences in validation dataset were 66% (61, 71%), 45% (38, 52%) and 21% (12, 30%) (p for trend<0.001, Figure D). AUROC values were 0.76 and 0.72. Calibration plots and DCAs showed good performance for predicting MMR and MR4. Image: Summary/Conclusion: We developed the predictive scoring systems for MMR and MR4 in persons with chronic phase CML receiving imatinib. These data may help physicians make an appropriate therapy decision. P722: IS THE SECOND-GENERATION TYROSINE KINASE INHIBITOR A BETTER THERAPY THAN IMATINIB FOR PERSONS WITH CHRONIC MYELOID LEUKEMIA PRESENTING IN ACCELERATED-PHASE? X. Zhang1,*, S. Yang1, Y. Liu1, Q. Jiang1 1Peking University People’s Hospital, Beijing, China Background: Although most patients with chronic myeloid leukemia (CML) were presenting in the chronic phase at diagnosis, approximately 5% presented with features of accelerated phase. The features of accelerated phase were variably defined by different criteria, such as MD Anderson and European LeukemiaNet (ELN) criteria most often used. Regardless of these criteria used to defined accelerated phase, only scant data were available regarding the prognostic co-variates for outcome. Moreover, evidence that patients could benefit more from the second generation (2G-) tyrosine kinase inhibitor (TKI) than imatinib as initial therapy was still extremely limited. Aims: Explore whether patients with chronic myeloid leukemia (CML) presenting in accelerated-phase could benefit from initial second-generation tyrosine kinase inhibitor (2G-TKI) therapy. Methods: We interrogated data from consecutive patients with CML presenting in accelerated phase at diagnosis by the MD Anderson criteria (n = 325) or ELN criteria (n = 275). Propensity score matching (PSM) analysis was used to compare cytogenetic and molecular responses and outcomes in those receiving initial imatinib versus a 2G-TKI. COX multi-variable regression model was performed to identify the prognostic co-variates. Results: In the cohort defined by MD Anderson criteria, PSM analysis showed that patients receiving initial 2G-TKI (n = 89) had higher probability of achieving a CCyR (p = 0.01), MMR (p = 0.04) and MR4.5 (p = 0.004) than those receiving imatinib (n = 156), however, probabilities of FFS, PFS and survival were similar. Multi-variable analyses showed that lower platelets and higher blasts in blood or bone marrow were significantly-associated with poor FFS, PFS and/or survival (Figure A). In an attempt to determine whether choice of therapy contributed to the differences in responses and outcomes, platelets < 600 × 10E+9/L and blood / bone marrow blasts ≥ 5% were identified as the prognostic categorical co-variates by ROC analyses. The entire cohort were classified into low- (possessing none of these co-variates, n = 57), intermediate (possessing any 1 of these co-variates, n = 127) or high-risk (possessing all of these co-variates, n = 61) subgroups. There were significant differences in probabilities of outcomes among these risk subgroups (all p values < 0.001, Figure B). In each risk subgroup, patients receiving initial 2G-TKI had the similar outcomes to those receiving imatinib except higher incidences of responses including CCyR, MMR and MR4.5. The same results were observed in the cohort defined by ELN criteria. Image: Summary/Conclusion: Initial 2G-TKI lead to similar outcomes as imatinib in patients with CML presenting in accelerated-phase except higher therapy responses. P723: LOSS OF THE NK MOTIF AND ANKYRIN REPEAT DOMAIN 1 (KANK1) LEADS TO LYMPHOID COMPARTMENT DYSREGUATION IN MICE M. Almosailleakh1,*, S. Narcisi1, C. Roger Michel Côme1, H. Luche2, K. Grønbæk1,3 1Biotechnology Research and innovation Center, University of Copenhagen, Copenhagen, Denmark; 2Centre d’Immunophénomique, Parc Scientifique et Technologique de Luminy, Marseille, France; 3Department of Hematology, Regishospitalet, Copenhagen, Denmark Background: We identified a novel germline loss of heterozygosity (LOH) mutation encompassing the NK Motif And AnKyrin Repeat Domain 1 (KANK1) gene in a young Myelodysplastic syndrome (MDS) patient and his healthy father, with no additional MDS-related mutations or chromosomal abnormalities. Clinical investigation suggested development of MDS following severe infection, likely triggered by an autoimmune mechanism. KANK1 protein plays a role in the control of cytoskeleton formation by regulating actin polymerization. A t(5;9) translocation resulting in a fusion of the platelet-derived growth factor receptor beta gene (PDGFRB) and KANK1 was detected in a patient with myeloid neoplasm (MN) characterized with severe thrombocythemia. The protein is thought to have a tumour suppressor function, as its expression is downregulated or missing in several tumor tissues, while over-expressing the protein was reported to inhibit the proliferation of tumor cells in solid cancer models. In addition, defects in cytoskeletal proteins are reported to contribute to immunological dysfunction and disruption of normal immune processes. Aims: To investigate the role of KANK1 in the regulation of normal hematopoiesis and the development of myeloid disorders. Methods: We generated a new genetically modified KANK1 (Kank1 -/-) knockout mouse model and analysed the phenotypic and developmental changes in hematopoiesis in young and aged mice. Results: We confirmed the loss of Kank1 mRNA and protein expression by q-PCR and targeted protein mass-spectrometry respectively. The mice are viable and fertile, and reproduced according to mendelian ratio. Over the two years observation period, Kank1 -/- mice did not exhibit any signs of disease, and their peripheral blood parameters remained within the normal range. Flow cytometry analysis of bone marrow (BM) cells from 17-20 weeks kank1 -/- old mice showed minor increase in CD3+ T-cell population, while the expression of the double positive B220/CD19 B-cells population was reduced in Kank1 -/- compared to Kank1 +/+. Comprehensive flow cytometry analysis of spleen and thymus cells from young (n=8 each, 6-7 weeks old) and aged (n=8, 68-71 weeks old) Kank1 -/- mice showed further dysregulation of the lymphoid compartment when compared to Kank1 +/+. In young mice, a slight decrease of Treg cells is noticed in the spleen, coupled with a slight decrease of activated CD4 in the peripheral blood. In the spleen of aged Kank1 -/- mice, the proportions of macrophages and Treg cells were decreased, while the central memory CD4+ T-cells were increased. Total protein analysis from BM cells of Kank1 -/- (n=3) compared to Kank1 +/+ mice (n=3) showed a significant reduction (FC >0.5, adj.p. <0.05) in proteins associated with cytoskeleton formation such as Myosin1 (MYH1), Tropomysin-1 (TDM1) and Annexin (ANXA2), and extracellular matrix components such as Collagena(CO1A1) and Matrix metalloproteinase9 (MMP9). We also detected a significant increase in expression of the DNA licensing factor minichromosome maintenance complex component 7 (MCM7), and the mitochondrial membrane associated transporter ATP binding Cassette Subfamily B Member 10 (ABCBA). Summary/Conclusion: Dysregulation of the immune system has been widely recognized to play a role in the development of MDS. Based on our results, we propose a role for KANK1 in regulating the development of immune cells in the mouse through it invovlement in cytoskeleton regulation. In vivo infectious challenge experiments using our model will futher elucidate the importance of KANK1 in the development of MDS. P724: THE BONE MARROW MESENCHYMAL CELL-DERIVED EXTRACELLULAR MATRIX IS SIGNIFICANTLY ALTERED IN PATIENTS WITH MELODYSPLASTIC SYNDROMES A. Bains1,*, L. Behrens Wu2, M. Richter1, V. Magno3, S. Rother4, M. Cross1, C. Werner3, M. Wobus2, U. Platzbecker1 1Medical Clinic and Policlinic I, Hematology and Cellular Therapy, University Hospital Leipzig, Leipzig; 2Department of Medicine I, University Hospital Carl Gustav Carus; 3Leibniz Institute of Polymer Research, Technische Universität, Dresden, Dresden; 4Center for Molecular Signaling, Saarland University School of Medicine, Homburg (Saar), Germany Background: Myelodysplastic syndromes (MDS) are characterised by ineffective hematopoiesis and peripheral cytopenia in one or more hematopoietic lineages. MDS is driven by genetic and epigenetic lesions in the hematopoietic clone itself, but also by changes in the bone marrow microenvironment. Here, the mesenchymal stromal cells (MSCs) regulate hematopoietic cell behaviour through cell-cell contact, paracrine signalling and metabolic support. The MSC-derived Extracellular matrix (ECM) plays a key role in coordinating both physical and biochemical interactions but the involvement of ECM in MDS has yet to be determined. Aims: We aimed to characterise the relevant parameters of the mesenchymal cell-derived ECM generated by MSCs from healthy donor and MDS patient bonemarrow. Methods: MSC from low risk (LR) and high risk (HR) MDS patients and age-matched healthy donors were cultured for 10 days in DMEM Glutamax medium supplemented with 10% FBS on glass slides pre-coated sequentially with poly-octadecene-alt-maleic anhydride (POMA) and human fibronectin. The confluent monolayers were washed in PBS, decellularized by washing with 20mM ammonia and were subject to DNase treatment. The resulting ECM were stored for up to 4 weeks at 4oC. Matrix structure was analysed by scanning electron microscopy (SEM) and atomic force microscopy (AFM), glycosaminoglycan (GAG) content by staining with peanut and wheat germ agglutinin and collagen composition by immunostaining with antibodies to collagens 1 and 4. Total collagen content was quantified by Sircol assay. In parallel, the expression of matrix-related genes was measured by real time PCR of RNA extracted from the MSCs. Results: In SEM, the ECM network generated by MSCs derived from both low risk (LR) and high risk (HR) MDS was thicker and of a higher density than that produced by MSCs from healthy donors. AFM analysis showed ECM of MDS-derived MSCs also had a significantly reduced rigidity. An increase in overall collagen content in MDS-derived ECM was detected by sircol assay and confirmed by immunostaining for the most prevalent collagen subtypes 1 and 4. Lectin staining revealed disease stage-specific differences in GAG composition: The levels of both GAGs carrying N-acetyl glucosamine and those carrying N-acetyl galactosamine sugars were increased in ECM from LR-MDS, while ECM from HR-MDS retained high levels of N-acetyl glucosamine but carried only low levels of N-acetyl galactosamine GAGs. Finally, the pattern of hyaluronan synthase (HAS) gene expression differed markedly between MSCs from healthy donor, LR- and HR-MDS. Specifically, HAS1 was strongly increased in LR-MDS and remained significantly above control levels in HR-MDS, while HAS3 was down-regulated specifically in HR-MDS. Consistent with this, increased levels of low molecular weight hyaluronic acid was observed in ECM from LR-MDS Summary/Conclusion: We report both structural and compositional differences between the MSC-derived ECM from healthy donors, LR-MDS and HR-MDS. The thicker and softer structure of the MDS matrix may be both a consequence and a driver of changes in the MSC phenotype. Increase in the major collagens 1 and 4 are consistent with an increase in overall ECM, but it remains unclear whether the decreased rigidity of ECM in MDS is due to differences in protein cross-linking or in the levels of hydrated glycosaminoglycans. MDS-associated changes in hyaluronic acid expression suggest that MSC-derived HA may contribute to the inflammatory bone marrow state characteristics of LR-MDS. P725: METABOLIC PROPERTIES OF MESENCHYMAL STROMAL CELLS IN MYELODYSPLASTIC SYNDROMES E. Balaian1,2, K. Sockel1,2, M. Cross3, A. Funk4, M. Bornhäuser1,5, T. Chavakis4,5, M. Wobus1,2,* 1Medical Department 1, University Hospital Carl Gustav Carus Dresden, Dresden; 2German Cancer Consortium (DKTK), Partner Site Dresden and German Cancer Research Center (DKFZ), Heidelberg; 3Medical Department I - Hematology and Cell Therapy, University of Leipzig Medical Center, Leipzig; 4Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus Dresden, Dresden; 5National Center for Tumor Diseases, Partner Site Dresden and German Cancer Research Center (DKFZ), Heidelberg, Germany Background: The crosstalk between the mutant hematopoietic stem and progenitor cells and the bone marrow microenvironment plays an important role in the pathogenesis of myelodysplastic syndromes (MDS). Mesenchymal stromal cells (MSC) represent one of the key cellular elements in this context. The functional properties of MDS-derived MSCs including impaired hematopoietic support, as well as increased senescence and defective osteogenic differentiation have been extensively studied. Additionally, the detrimental effect of high erythropoietin (Epo) concentration on MDS-MSCs has been demonstrated by our group. However, whether these deficiencies are associated with cellular metabolic changes has not been hitherto addressed. Aims: We aimed to assess the basal metabolic characteristics in MDS-derived MSCs and the effect of Epo on their energetic profile in vitro. Methods: MSC from bone marrow aspirates of patients with MDS (low risk n=3, MDS del5q n=1, chronic myelomonocytic leukemia n=2, high risk n=3) or age-adjusted healthy donors (n=3) were isolated by Ficoll density centrifugation and plastic adherent culture. Metabolic measurements were performed before and after incubation with Epo (50 IU/ml) for 2 days. Seahorse X96 Flux Analyzer (Seahorse Biosciences, Billerica, MA, USA) was used to analyse glucose metabolism by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Metabolite concentrations were measured in the culture supernatants using NMR spectroscopy. Results: MSC from healthy donors demonstrated a higher OCR/ECAR ratio compared to MDS counterparts (6.19±4,71 vs 1.14±0.28, p=0.005), suggesting enhanced glycolysis engagement of MDS-MSCs. This was confirmed by increased levels of lactate in the culture medium from MDS-MSCs after 48 hours of cultivation (healthy: 3.78±0.28 mM vs MDS: 6.75±2.35 mM, p=0.049), whereas the concentrations of pyruvate, alanine and acetate did not differ. Treatment with Epo resulted in a significant increase of the OCR/ECAR ratio (from 1.14±0.28 to 3.25±1.75, p=0.003) in MDS-MSCs, whereas changes in healthy MSCs were not significant (from 6.19±4,71 to 3.81±1.86, p=0.46). Summary/Conclusion: In contrast to healthy MSCs which rely mainly on mitochondrial respiration, MDS-MSCs preferentially utilize the glycolytic pathway under normoxic conditions in vitro. Epo treatment leads to increased mitochondrial respiration in MDS-MSCs, but not in healthy counterparts. Whether this effect has a causative link to the disturbed function of MDS-MSCs remains to be clarified. P726: MYELOID-DERIVED SUPPRESSOR CELLS AS POTENTIAL TARGETS FOR IMMUNE THERAPY IN CCUS AND LOW RISK MDS S. Bentivegna1,*, M. O. Holmström2, M. H. Andersen2, K. Grønbæk1,3 1Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen; 2National Center for Cancer Immune Therapy (CCIT-DK), Department of Oncology, Copenhagen University Hospital, Herlev; 3Department of Hematology, Rigshospitalet Copenhagen University Hospital, Copenhagen, Denmark Background: Myeloid-derived suppressor cells (MDSC) are powerful immune suppressing cells that infiltrate the bone marrow (BM) microenvironment in myelodysplastic syndrome (MDS) and suppress both a potential immune response against the malignant HSPC and the normal hematopoiesis (Chen et al., 2013). Different groups reported an expansion of MDSC in MDS, and an increased number of MDSC is associated with disease progression (Chen et al., 2013; Kittang et al., 2016). MDSC mediate their immunoregulatory effects releasing immunosuppressive cytokines, such as TGFβ, and enzymes such as indoleamine 2,3-dioxygenase (IDO) and arginase (ARG) (Mondanelli et al., 2017). The presence of self-reactive T cells that specifically target immunosuppressive cells in both healthy donors and cancer patients has previously been described (Andersen, 2017). These cells, termed anti-regulatory T cells (anti-Tregs), recognize epitopes derived from immunosuppressive proteins such as IDO, ARG, which are expressed by MDSC (Andersen, 2018). We speculate that the stimulation of anti-Tregs cells using peptide-based vaccines can lead to the depletion of the immunosuppressive cells in the BM, and it will help to prevent disease progression in patients with clonal cytopenia of undetermined significance (CCUS) or MDS. Aims: The aim of this work was to characterize the immune cell composition of blood and BM samples from CCUS/MDS patients and investigate the presence of circulating anti-Treg cells in blood samples and their reactivity towards immunogenic peptides. Methods: Using flow cytometry we analyzed blood and BM samples from 20 patients (pts) with CCUS/MDS (10 CCUS and 10 low risk-MDS) and 6 elderly healthy controls (EHC). The presence of anti-Tregs was investigated in 7 pts, by ELISPOT assay on patient T cells that had been stimulated with epitopes derived from the immunosuppressive proteins ARG, IDO or TGFβ. Results: Although there were no significant differences in the percentage of MDSC in BM samples between controls and pts, the proportion of IDO+ and ARG+ MDSC in pts was significantly higher compared to EHC. In addition, there was a significantly higher percentage of CTLA-4+ and PD-1+ T cells present in the BM samples from pts compared to EHC. Interestingly, in the ELISPOT assay, 4 out the 7 pts showed a significant response towards the ARG peptide, 2 showed a response towards IDO peptide and in 4 of the patients there was a significant response towards the TGFβ peptide. Overall, of the 7 pts analyzed, 6 harbored T cells specific to one or more of the immunosuppressive proteins ARG, IDO and TGFβ (figure 1). Image: Summary/Conclusion: There is an increased proportion of activated MDSC in BM in pts with CCUS/MDS compared to EHC. As the immune checkpoint molecules are expressed after T cell activation, the increased percentage of CTLA-4+ and PD-1+ cells in pts BM suggests that there is a T cell immune response in the BM of pts with CCUS/MDS. The ELISPOTs showed that circulating anti-Tregs are present in CCUS/MDS patients. These results suggest, that the tumor specific immune response in patients with CCUS/MDS is present, albeit suppressed by (amongst others) MDSC. We speculate that therapeutic cancer vaccination targeting immunosuppressive cells such as MDSC by vaccination using e.g. IDO, ARG or TGFβ derived epitopes will incite an anti-regulatory immune response, which may break the local immune suppression in the BM and allow effector immune cells to exert killing of transformed cells. P727: NK-CELL IMMUNOEDITING BY MESENCHYMAL STROMAL CELLS IN MDS V. Bisio1,2,*, B. Schell1,2, L.-P. Zhao1,2, E. Lereclus1,2, M. Boy1,2, A. Bonaud1,2, A. Caignard1,2, A. Toubert1,2,3, P. Fenaux4, M. Espéli1,2, K. Balabanian1,2, L. Adès4, N. Dulphy1,2,3 1Université de Paris, Institut de Recherche Saint Louis, EMiLy, Inserm U1160; 2OPALE Carnot Institute, The Organization for Partnerships in Leukemia, Hôpital Saint-Louis; 3Laboratoire d’Immunologie et d‘Histocompatibilité, Assistance Publique des Hôpitaux de Paris (AP-HP), Hôpital Saint-Louis; 4Université de Paris, Department of Hematology, AP-HP, Paris, France Background: Disorders of the immune system contribute to the pathophysiology and progression of myelodysplastic syndrome (MDS). Cytotoxic Natural Killer (NK) lymphocytes have been extensively studied for their anti-leukemic activity. However, whether the dysplastic bone marrow (BM) in MDS can directly imprint NK cells and hence alter immunosurveillance is still controversial. Extensive characterizations of MDS patients uncovered deep perturbations in the phenotype and function of cytotoxic lymphocyte subsets and of the stromal compartment, in particular mesenchymal stromal cells (MSC), linked with disease predisposition, initiation and progression. However, few studies have been undertaken to characterize the interactions established within the MDS immune microenvironment between immune and stromal cells. Aims: Recent studies on MDS BM ecosystem suggest a possible symbiotic relationship between cancer, immune and stromal cells. Our project thus aims to underpin the influence of the pathological MSC from MDS BM over NK cells to create a leukemia-permissive microenvironment allowing disease development and progression. Methods: We performed an extensive in vitro characterization of the NK and MSC interactions by co-culture system. Healthy donor (HD) NK cells were co-cultured either with MSC isolated from HD or MDS patient BM samples and were analyzed by high-parametric flow-cytometry, qRT-PCR, secretome and imaging assays. Results: Analyses of supernatants from MSC/NK co-cultures unveiled altered secretion of soluble factors related to inflammation and BM homing such as IL6, CCL2 and CXCL10 in the pathologic condition compared to the healthy one. MDS MSC did not support efficiently NK-cell viability. Moreover, NK cells cultured with MDS MSC were less efficient at eliminating cancer cells compared to NK cells cultured with HD MSC. Finally, metabolic analyses uncovered an altered NK cell energetic profile dictated by the different MSC that could contribute to their poor survival and reduced cytolytic function. Summary/Conclusion: Altogether, our results suggest that MSC from MDS BM edit NK cells to promote an immunosuppressive environment facilitating MDS escape and progression. Further analyses are ongoing to underpin the pivotal role of immune-microenvironment interactions in the treatment response. We expect this study to identify new biomarkers to forecast disease progression and open an avenue for new combination therapies targeting the tumour microenvironment. P728: GENE EXPRESSION PROFILE REVELS T-LYMPHOCYTES ACTIVATION THROUGH CELL CYCLE PROGRESSION AND MITOCHONDRIAL METABOLISM REGULATION IN MYELODYSPLASTIC SYNDROMES WITH DEL(5Q) AFTER LENALIDOMIDE TREATMENT M. del Rey1,*, L. A. Corchete2, T. González1, F. López-Cadenas3, E. Lumbreras1, S. M. Toribio1, B. Xicoy4, J. Sánchez-García5, T. Bernal6, G. F. Sanz7, P. Fenaux8, J. M. Hernández-Rivas9, M. Diez-Campelo10 1Cancer Research Center - University of Salamanca; Instituto de Investigación Biomédica de Salamanca (IBSAL); 2Instituto de Investigación Biomédica de Salamanca (IBSAL); Hematology Department, Hospital Clínico Universitario de Salamanca; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC); 3Hematology Department, Hospital Clínico Universitario de Salamanca, Salamanca; 4Hematology Department, Institut Català d’Oncologia-Hospital Germans Trias i Pujol; Josep Carreras Leukemia Research Institute, Universitat Autònoma de Barcelona, Badalona; 5Hematology Department, Hospital Reina Sofía, Córdoba; 6Hematology Department, Hospital Universitario Central de Asturias, Oviedo; 7Hematology Department, Hospital Universitario y Politécnico La Fe, Valencia, Spain; 8Hôpital Saint-Louis, Paris, France; 9Hematology Department, Hospital Clínico Universitario de Salamanca; Cancer Research Center - University of Salamanca; Instituto de Investigación Biomédica de Salamanca (IBSAL); 10Hematology Department, Hospital Clínico Universitario de Salamanca; Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain Background: Lenalidomide is a potent drug whose mechanisms of action in del(5q) clone from Myelodysplastic Syndromes (MDS) have been extensively described. Lenalidomide also induces immune modulation independent of del(5q) and has multiple effects on T-cell signaling; however, the molecular and cellular determinants that mediate this immunomodulatory function remain elusive. Aims: To better understand the immunomodulatory activity of lenalidomide in MDS patients with del(5q) by in vivo sequential gene expression profile studies of T lymphocytes in response to the treatment. Methods: Our study was conducted in patients included in the Sintra-REV Clinical Trial (phase III). Sequential study was carried out in 26 samples from 13 paired patients. Seven out 13 were treated with lenalidomide and achieved a major erythroid and cytogenetic response. Peripheral blood (PB) samples were collected before and one month after treatment in treated patients and at the same time points for non-treated patients. CD3+ cells were collected from PB samples and total RNA was isolated. SureSelect Strand Specific RNA library (Agilent Technologies) was applied to study changes in RNA levels. Raw reads were aligned against the Human genome GRCh37 using the STAR aligner. Counts were assigned to Ensembl gene IDs through HTseq using its UNION version. Differential gene expression was determined with DESeq2, considering as statistically significant those genes with FDR < 0.05. Pathway over-representation analysis (ORA) was conducted in the Webgestalt suite. Results: 332 genes were differentially expressed in CD3+ lymphocytes one month after lenalidomide treatment in our cohort of patients; of note, none of them were observed after one month in non-treated patients. The ORA revealed significant differences in the gene expression profile of sixteen cytokines (e.g. IL10, TNFSF10, IFNG, IL6 and MBP6). These genes could contribute to activation of an antileukemic immune response, help to correct the anemia as well as attenuate inflammation signaling in MDS patients with del(5q). The ORA also showed an enrichment of genes involved in cell cycle (35 genes). The most represented up-regulated genes related to this pathway were: cyclines (CCNB1, CCNB2, CDK1), centromere genes (CENPE, CENPM, CENPU), kinesin family members (KIF18A, KIF23, KIF2C) and genes involved in cell division (CDC6, CDC7, CDC25A). It has been described that lenalidomide inhibits CDC25A selectively in the del(5q) clone resulting in G2/M arrest and apoptosis. By contrast, our study showed that this gene was upregulated in T-lymphocytes promoting cell cycle and proliferation of these cells. In addition, 456 genes were differentially expressed between non-treated and lenalidomide treated samples; 30 of them were related to mitochondrial metabolism (e.g HIBCH, ECHS1, DECR1, ACSM1, HADH, COQ3, COQ5, SDHAF3, SDHAF4) and mitochondrial translation (e.g. MRPL15, MRPL24, MRPL48, MRPL51, MRPS33). Thereby, T lymphocytes adaptive responses as well as sustained killing by cytotoxic T cells could be mediated by mitochondria in treated patients. Summary/Conclusion: To our knowledge, this is the first report describing RNA expression profiles in PB CD3+ lymphocytes collected from lenalidomide-treated del(5q) patients, contributing to overall understanding of lenalidomide action. A better understanding of the molecular targets that mediate the immunomodulatory activity is needed to harness the full potential of this agent. Functional experiments trying to test the new biological mechanisms described above are currently conducted. P729: DEREGULATION OF NONCODING RNAS DERIVED FROM KDM GENES IS ASSOCIATED WITH AZACITIDINE RESPONSE IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES AND ACUTE MYELOID LEUKEMIA M. Dostalova Merkerova1,*, A. Hrustincova1, I. Trsova1, Z. Krejcik1, D. Kundrat1, J. Klema2, K. Szikszai1, A. V. Le2, J. Cermak3, A. Jonasova4, M. Belickova1 1Department of Genomics, Institute of Hematology and Blood Transfusion; 2Department of Computer Sciences, Czech Technical University; 3Laboratory of Anemias, Institute of Hematology and Blood Transfusion; 4First Department of Medicine, General University Hospital, Prague, Czechia Background: Prediction of response to azacitidine (AZA) treatment is an important challenge in the management of higher-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To date, no clinical, cytogenetic, or molecular markers of AZA treatment outcome have been validated to support clinical decisions. Moreover, little is known about how AZA efficiency can be affected by various noncoding RNAs (ncRNAs), such as long ncRNAs (lncRNAs) and circular RNAs (circRNAs). KDM genes encode demethylases of histone lysine residues (e.g., KDM1 mediates demethylation of H3K4 and H3K9, and KDM4 acts on H3K9, H3K36, and H1K26). KDMs are often differentially expressed in leukemia and they cooperate with transcription factors to activate or repress gene expression. Aims: Our aims were to provide biological insight into the contribution of ncRNAs to AZA mechanisms of action and to propose novel disease biomarkers that would be able to predict future response to AZA treatment. Based on our results, we particularly focused on expression and function of KDM-related ncRNAs. Methods: Whole transcriptome RNA sequencing (RNA-seq) was performed in CD34+ bone marrow cells from 26 patients with MDS or AML with myelodysplasia-related changes (AML-MRC) before AZA treatment (11 responders and 16 nonresponders), and 9 healthy controls. Expression of protein coding genes (PCGs), lncRNAs and circRNAs was bioinformatically processed. Results: Overall, differential expression analysis between AZA responders and nonresponders identified significant deregulation of 202 PCGs, 34 lncRNAs, and 21 circRNAs. Surprisingly, machine learning data showed that predictive potential of ncRNAs was stronger than that of PCGs. Within the data, deregulation of several KDM-related ncRNAs was observed, e.g., CTC-482H14.5 (antisense RNA to KDM4B gene, downregulated in AZA responders), hsa_circ_0003889 (circRNA processed from KDM1A, downregulated in AZA responders), and hsa_circ_0001580 (circRNA processed from KDM1B, upregulated in AZA responders). By contrast with these noncoding transcripts, the levels of corresponding PCGs (namely KDM1A/1B/4B) were not altered between AZA responders and nonresponders, suggesting that the deregulated ncRNAs play specific roles independent of their host genes. Further, the best predictor genes were defined by machine learning using RNA-seq data and pathway analysis linked these markers to cellular processes related to MDS/AML. Interestingly, CTC-482H14.5 was identified as one of the strongest predictors of AZA treatment response. Expression of this KDM-related ncRNA was coregulated with expression of DNMT1, EZH2, and multiple MCM genes, associating this transcript with epigenetic regulation and recombinational repair pathways. Summary/Conclusion: Several ncRNAs processed from KDM genes seem to be closely related to the responsiveness of MDS/AML-MRC patients to AZA treatment. In this study, novel functions were predicted for these ncRNAs, pointing to possible mechanisms by which their deregulation could affect AZA treatment response. Acknowledgements: Supported by GA CR (No. 20-19162S) and MH CZ-DRO (UHKT, 00023736). P730: GENETIC LANDSCAPE OF SOMATIC MYELOID MUTATIONS IN THE PRESENCE OF RARE TERT VARIANTS AND THEIR RELATION WITH MYELOID NEOPLASIA A. Ferrer1,2,*, A. Mangaonkar1, T. Lasho1, M. Wylam3, C. Finke1, J. Fernandez1, R. He4, D. Viswanatha4, M. Patnaik1,2 1Division of Hematology; 2Center for Individualized Medicine; 3Division of Pulmonary and Critical Care Medicine; 4Department of Hematopathology, Mayo Clinic, Rochester, United States of America Background: Genetic variants in TERT, the gene that encodes telomerase reverse transcriptase, can result in accelerated telomere attrition. Impaired telomere maintenance is implicated in the pathogenesis of myelodysplastic syndromes (MDS), with mechanisms remaining to be elucidated. Aims: To explore the clinical and genetic associations of TERT variants in an unselected cohort of patients screened with a myeloid mutation next generation sequencing panel due to clinical suspicion of a myeloid neoplasia. Methods: Using a research protocol approved by the Mayo Clinic Institutional Review Board, we performed a retrospective chart review to identify a cohort of unselected individuals within all historical data at our institution that had undergone somatic mutational analysis for suspected myeloid neoplasia using our insitutional amplicon based next generation sequencing panel (42 genes, including exons 2 to 16 of TERT, with a read depth of at least 250X). TERT variants were classified into those with CADD score >20 and <20 in line with previous literature to identify variants within the top 1% according to their predicted deleteriousness. Additionally, we identified within our cohort individuals that received a diagnosis of MDS, clinical and survival information. Results: We identified 55 different TERT variants (46 missense, 3 in canonical splice sites, 4 synonymous and 2 small inframe deletions) in 148 individuals tested from April of 2016 to November of 2021 (Fig. 1A&B). The five most recurrent variants were c.1234C>T (48 patients), c.1323_1325del (29 patients), c.3164C>T (5 patients), c.604G>A (4 patients) and c.969G>A (4 patients), all with CADD<20. None of these patients were diagnosed with a bona fide telomere biology disorder and no clinical information about telomere length status was available. All variants were considered germline since their allele frequency was near 50%, although no germline confirmation was performed. All variants were clinically classified as uncertain significance according to the American College of Medical Genetics guidelines. Of the 148 total patients, 17 (12%) carried TERT variants with CADD>20 that were distributed thorough all the gene domains with no clustering or presence of hotspots (Fig.1A&B). 59% (10 patients) of CADD>20 variants carriers were diagnosed with MDS: 5 patients with <5% bone marrow blasts (4 with normal karyotype) and 5 patients with 5-19% blasts, all with complex karyotype. This percentage was significantly higher compared to CADD<20 variant carriers (34%, 49 patients, p=0.048 by Chi-square test, Fig. 1C). However, no major differences were found between the two groups in terms of age at diagnosis (median 70 vs 73 years old, p = 0.2305) or AML-free survival (median 10 vs 15.5 months since diagnosis, p=0.4674). Similarly, the percentage of patients presenting additional somatic variants in other myeloid-related genes was similar between CADD<20 and CADD>20 TERT variant carriers (60% vs 64%). The two most frequenly mutated genes in CADD>20 carriers were TP53 (22%), TET2 (18%), RUNX1 (9%), U2AF1 (9%) and BCOR (9%) while in the CADD<20 carriers were TET2 (14%), ASXL1 (11%), SRSF2 (9%), TP53 (7%) and JAK2 (6%). Our data showed that TP53 pathogenic variants were significantly enriched in CADD>20 TERT variant carriers compared to CADD<20 carriers. (22.7% vs 7.4%, p=0.0169, Fig. 1D). Image: Summary/Conclusion: Carriers of TERT variants with CADD score of >20 were more likely to be associated with a diagnosis of MDS and with somatic TP53 mutations in comparison to those with CADD score <20, findings that we plan to validate functionally and prospectively. P731: INTERPLAY BETWEEN INFLAMMATION AND EPIGENETICS IN TUMOR REPROGRAMMING OF MESENCHYMAL STROMAL CELLS (MSCS) IN MYELODYSPLASTIC SYNDROMES (MDS) C. Giallongo1,*, I. Dulcamare2, D. Tibullo3, D. Pieragostino4, M. C. Cufaro4, M. A. Amorini5, G. Lazzarino6, A. Romano7, G. Scandura2, T. Zuppelli2, M. Di Rosa8, A. Duminuco2, G. Broggi9, R. Caltabiano9, R. Floresti10, R. Motterlini11, F. Di Raimondo7, G. A. Palumbo1 1Department of Medical, Surgical Sciences and Advanced Technologies G.F. Ingrassia; 2Division of Hematology, AOU Policlinico; 3Department of Biomedical and Biotechnological Sciences, Section of Biochemistry, University of Catania, Catania; 4Department of Innovative Technologies in Medicine and Dentistry, ‘‘G. d’Annunzio’’, University of Chieti-Pescara, Chieti; 5Department of Biomedical and Biotechnological Sciences, Section of Biochemistr, University of Catania, Catania; 6UniCamillus - Saint Camillus International University of Health Sciences, University of Rome, Rome; 7Department of General Surgery and Medical-Surgical Specialties; 87Department of Biomedical and Biotechnological Sciences, Human, Histology and Movement Science Section; 9Department of Medical and Surgical Sciences and Advanced Technologies “G.F. Ingrassia”, Anatomic Pathology, University of Catania, Catania, Italy; 10University Paris-Est Créteil, INSERM, IMRB, University Paris-Est; 11University Paris-Est Créteil, INSERM, IMRB, University Paris Est, Paris, France Background: The alteration of hematopoiesis in MDS patients is deeply associated with microenvironment alterations, in particular in MSCs. Stromal cells are epigenetically reprogrammed to function in cooperation with leukemic cells and propagate the disease as a “tumor unit”. Notably, dysfunctions of MDS-MSCs persist following expansion ex vivo suggesting a hereditable epigenetic dysregulation which endures despite removal of disease-associated microenvironment factors. Aims: Here, we investigated the role of histone H2A variant MacroH2A1 (mH2A1) in promoting pro-tumorigenic inflammation and metabolic reprogramming of MSCs. Methods: MSCs were collected from BM of MDS patients (n=18) and matched healthy controls (n=8; HC). HS-5 cells were used as model of healthy MSCs. Real time, western blot, immunohistochemistry and immunofluorescence were used. Proteomic and metabolic analysis were performed. Results: MDS patients had significant higher signature of mH2A1 in bone marrow slides compared to age-matched control. Investigating mH2A1 expression in stromal cells, MDS-MSCs had increased levels of expression in respect of HC-MSCs. In accordance with the aberrant inflammation described in MDS microenvironment, MDS-MSCs showed higher levels of the sum of NO2+ and NO3+ associated to increased GSH and NADP+/NAPH. TLR4 also was upregulated and positively correlated to mH2A1 expression. To better investigate the relationship between mH2A1 and TLR4 in MDS-MSCs, we induced mH2A1 overexpression in HS-5 cells (mH2A1-OE) by mH2A1-CT-MYC plasmid. Our data showed that mH2A1-OE upregulated TLR4 and increased NFkB nuclear translocation compared to cells transfected with empty vector (CTL). Proteomic analysis confirmed upregulation of intracellular serine protease inhibitors (SerpinB2, B8, B6) strongly induced during inflammation and important for the maintenance of TLR4 activation. Moreover, proteomic approach identified upregulation of several proteins associated to hypermethylation of DNA and histones in mH2A1-OE. In particular, S-adenosylhomocysteine hydrolase which regulates the concentration of S-adenosylhomocysteine (SAH), a strong inhibitor of methyltransferase reactions and of the methyl donor S-adenosyl-methionine (SAM), resulted overexpressed. HPLC analysis showed higher SAM/SAH ratio associated to a significant reduction of SAH in mH2A1-OE, confirming the increase of the methylation index. In addition, the higher levels of CBX3, a suppressive epigenetic mark which recognizes histone H3K9me3, contributes to the maintenance of the heterochromatin. The higher levels of H3K9me3 in mh2A1-OE were confirmed by western blot analysis. Overexpression of mH2A1 also induced metabolic reprogramming with the acquisition of a more glycolytic metabolism characterized by decreased levels of NAD+/NADH, upregulation of LDHA and MCT4. As LDHA could translocate into the nucleus, we evaluated if it used a noncanonical enzymatic activity in mh2A1-OE. Data showed increased nuclear localization of LDHA producing histone H3K79 hypermethylation. Nuclear LDHA was also observed in MDS-MSC. Finally, we treated ex-vivo HC- and MDS-MSCs with azacytidine founding a significant reduction both of mH2A1 and TLR4 associated to lower levels of nuclear NFKB. Summary/Conclusion: Our data provide a key role of mH2A1in driving the crosstalk between inflammation, metabolism and epigenetic signatures in MDS-MSC. Future experiments will define the contribution of mH2A to ineffective hematopoiesis and leukemic evolution. P732: TH17/TREG IMBALANCE IN LOW RISK MDS PATIENTS M. Boada1, N. Trias2, A. Brugnini2, C. Guillermo1, S. Grille1,2,* 1Catedra de Hematologia; 2Laboratorio de Citometria y Biologia Molecular. Depto. Básico de Medicina, Hospital de Clinicas, Montevideo, Uruguay Background: Myelodysplastic Syndromes (MDS) are a heterogeneous group of clonal haematopoietic stem/progenitor cells (HSPC) disorders characterized by ineffective haematopoiesis, peripheral cytopenia, and risk of acute myeloid leukemia (AML). MDS pathogenesis is complex and depends on the interaction between HSPC and microenvironment. Many studies have highlighted the role of different immune cells in immune dysregulation leading to pathogenesis of MDS and progression of disease to AML. Immune response differs between risk groups favouring a pro-inflammatory profile in low risk, whereas in high-risk patients there is a trend towards immunosuppressive environment. Regulatory T cells (TRegs) and T helper 17 cells (Th17) are CD4 T cell functional subsets and their balance (TH17/TReg) is essential for maintaining immune haemostasis Aims: To study T cell subsets and cytokines plasma levels in low risk MDS patients and healthy controls. Methods: This is a case control study conducted in Hospital de Clinicas Montevideo, Uruguay. Patient inclusion criteria were Low Risk MDS (R-IPSS <4.5) and control group were healthy blood donors. We assessed peripheral blood T cell subsets by multiparametric flow cytometry using the following monoclonal antibodies: CD45, CD3, CD4, CD8, CXCR3, CCR6, CCR4, CD25 and CD127. Subpopulations were defined as reported by Maecker et al (1). Cytokine’s plasma levels were determined by Cytometric Bead Array (BD). We studied pro-inflammatory, TH1, TH2 and TH17 cytokines (IL-2, IL-4, IL-6, IL-10, INFg, TNFa, and IL-17). The protocol was authorized by the Ethics Committee of the institution and all subjects signed an informed consent. Results: We included 24 MDS patients and 19 controls with a median age of 68.5 (47-78) and 48 (32-55) years respectively. Male:Female ratio was 1.3:1 in controls and 0.9:1 in patients (p˃0.05). According to R-IPSS we included 13 (30.2%) Very Low Risk, 11 (25.2%) Low risk and 19 (44.2%) Intermediate patients. MDS patients showed lower proportion of Treg cells (p<0.001) and higher TH17/Treg ratio (p<0.001) than controls (Figure 1). There were no statistical differences in total T CD4+ cells and TH1/TH2 ratio. IL-2, IL-6 and IFN-γ levels were higher in patients than controls. Median levels of IL-2 were 0.2 (0.1-4.5) pg/mL in patients and 0.1 (0.0-2.6) pg/mL in controls, p=0.002. Median levels of IL-6 were 9.4 (0.5-10.5) pg/mL in patients and 0.5 (0.2-2.9) pg/mL in controls, p=0.001. Median levels of IFN-γ were 2.2 (1.1-3.3) pg/mL in patients and 0.1 (0.0-2.6) pg/mL in controls, p=0.003. There was no statistical difference in IL-4, IL-10, TNFa, and IL-17A between groups. Image: Summary/Conclusion: Our results showed that TH17/Treg balance is impaired, revealing a predominant proinflammatory state in in MDS Low risk patients. This occurs in concordance with elevated pro-inflammatory cytokines (IL-2, IL-6 and IFN-γ). These findings agree with previous reports that evidenced TH17/Treg ratio was higher in low risk MDS patients when compared with high risk. However, those studies highlighted the importance of TReg expansion and its prognostic impact in high risk. Evidence comparing TH17/TReg balance between low risk MDS patients and controls is scant. We believe that these results support the importance of a pro-inflammatory response in low-risk patients. 1. Maecker HT et al. Standardizing immunophenotyping for the Human Immunology Project. Nature Reviews Immunology. 2012;12(3):191-200. doi:10.1038/nri3158 P733: DHX9 HELICASE DYSFUNCTION CONTRIBUTES TO DISEASE PROGRESSION IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES N.-F. Huang1, C.-K. Chang1, Q. He1, L.-Y. Wu1, Z. Zhang1, X. Li1, F. Xu1,* 1Department of Hematology, ShangHai Jiaotong University Affiliated Sixth People’s Hospital, ShangHai, China Background: DHX9 is a member of the DEAH (Asp-Glu-Ala-His) helicase family, and participates in regulating DNA replication and RNA processing. DHX9 dysfunction promotes tumorigenesis in several solid cancers. However, the role of DHX9 in myelodysplastic syndromes (MDS) is still unknown. Aims: to investigate the role of DHX9 in the pathogenesis of myelodysplastic syndromes. Methods: DHX9 expression was analyzed in 120 MDS patients and 42 non-MDS benign controls by quantitative PCR. Clinical significance of changes in DHX9 expression was investigated in disease progression and survival. Lentivirus-mediated DHX9 knockdown experiments were performed to investigate its biological function. The mechanistic involvement of DHX9 in MDS development was explored in cell functional assays, gene microarray and pharmacological intervention. Considering the importance of DHX9 in regulation of R-loop, we also investigated the role of DHX9 and R-loop in MDS. Results: We found that overexpression of DHX9 is frequent in MDS patients, and associated with poor survival and high risk of AML transformation. Cell functional assays showed that DHX9 is essential for the maintenance of malignant proliferation of leukemia cells, and knockdown of DHX9 results in increased cell apoptosis and growth inhibition. Gene expression profile and bioinformatics analysis reveal that DHX9 may be involved in PI3K-AKT signaling pathways. Validating experiments show that DHX9 is indispensable for the activation of PI3K-AKT signaling as knockdown of DHX9 inactivates the PI3K-AKT signaling, reduces the expression of anti-apoptosis genes such as CCND2, MYC and BCL2. In addition, we found that DHX9 knockdown leads to R-loop accumulation in MDS cells, and induces R-Loop-dependent DNA damage through ATR-Chk1 activation. It suggests that overexpressed DHX9 could counteract cell growth defect caused by R-Loop which is quite common in MDS patients especially in those with splicing genes mutations. Summary/Conclusion: Our data suggest that DHX9 contributes to disease progression by activation of PI3K-AKT signaling and correcting R-loop-mediated growth defect in MDS, and may serve as a novel prognostic marker for AML transformation and therapeutic target in MDS. P734: DOWN-REGULATION OF RING1 AND YY1 BINDING (RYBP) LEADS TO EPIGENETIC OVEREXPRESSION OF ANTI-APOPTOTIC PROTEIN BCL2 IN MYELODYSPLASTIC SYNDROMES F. Xu1,*, X. Li1, Q. He1, L.-Y. Wu1, Z. Zhang1, C.-K. Chang1 1Department of Hematology, ShangHai Jiaotong University Affiliated Sixth People’s Hospital, ShangHai, China Background: Excessive activation of oncogenes plays a crucial role in apoptosis resistance and disease progression to leukaemia in myelodysplastic syndromes (MDS). RYBP is a member of BCOR complex for epigenetic transcription repression, and also considered as an apoptosis-associated protein. However, the role of RYBP is still unclear in the myelodysplastic syndromes. Aims: To investigate the role of RING1 and YY1 binding (RYBP) in the myelodysplastic syndromes. Methods: The expression of RYBP mRNA and its clinical significance was analyzed in MDS patients. Lentivirus-mediated RYBP knockdown and overexpression were performed to investigate its biological function. The mechanism of RYBP in MDS development was investigated by cell functional assays, gene microarray and chromatin immunoprecipitation. Results: In this study, we found that reduced RYBP expression is observed in MDS patients and associated with high AML transformation. Overexpression of RYBP inhibits cell proliferation, and results in an increased cell apoptosis along with p53 activation in myeloid cell lines. However, knockdown of RYBP promotes cell proliferation, induces apoptosis resistance to etoposide and decitabine, and simultaneously reduces H2AK119ub1 level. Gene expression profile analysis identifies BCL2 as a possible target of RYBP. Further functional analysis shows that depletion of RYBP leads to transcription activation of BCL2 through removal of H2AK119ub1 at the promoter region of BCL2 gene. BCL-2 specific inhibitor ABT-199 significantly induces cell apoptosis in RYBP-depleted cells, and restores the apoptosis sensitivity to etoposide and decitabine. Summary/Conclusion: Reduced RYBP expression is frequent in MDS patients and associated with high AML transformation. RYBP inhibition leads to epigenetic overexpression of anti-apoptotic protein BCL2 in MDS, which contributes to apoptotic resistance of clonal cells and disease progression. BCL2 inhibitor may be considered as a candidate agent in advanced MDS. P735: THE ROLE OF SF3B1 MUTATIONS IN MYELODYSPLASTIC SYNDROMES S. Huber1,*, S. Hutter1, M. Meggendorfer1, G. Hoermann1, W. Walter1, C. Baer1, W. Kern1, T. Haferlach1, C. Haferlach1 1MLL Münchner Leukämie Labor GmbH, München, Germany Background: SF3B1 is one of the most commonly mutated genes in patients with myelodyplastic syndromes (MDS), associated with ring sideroblasts (RS) and an indolent disease course. Malcovati et al. proposed MDS with mutated SF3B1 fulfilling certain criteria as distinct MDS entity (Malcovati et al., Blood, 2020). Aims: Study the SF3B1 mutation in MDS with respect to the genomic landscape, AML transformation rate and clinical outcome. Methods: We analyzed 734 patients diagnosed with MDS based on cytomorphology, cytogenetics and molecular genetics and classified according to WHO classification 2017. Amplification-free WGS was performed with a median coverage of 103x (Illumina, San Diego, CA). Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and variants were called using Strelka Somatic Variant Caller v2.4.7. Results: SF3B1 mutations were identified in 231 of 734 (31%) MDS patients and were mainly found in MDS-RS (171/200; 86%; MDS-RS-SLD: 43/51, 84%; MDS-RS-MLD: 128/149; 86%). In addition, 13% (37/300) of MDS with excess blasts (MDS-EB1/2) and 20% of MDS with isolated del(5q) harbored SF3B1 mutations. In the total cohort SF3B1 mutations were associated with better outcome (median OS: 79 vs. 53 month; p<0.0001). Within the different MDS entities SF3B1 mutations were favorable in MDS-RS-SLD (median OS: 106 vs. 25 month; p=0.009), MDS-RS-MLD (median OS: 82 vs. 64 month; p=0.049) and MDS-EB-2 (median OS: 129 vs. 25 month; p=0.011), but were associated with shorter survival in MDS with isolated del(5q) (median OS: 69 vs. 79 month; p=0.044). 144/231 (62%) SF3B1 mutated samples fulfilled the criteria proposed by Malcovati et al. (SF3B1ent). These cases were associated with longer survival compared to SF3B1 mutated samples not falling into the proposed SF3B1 entity (SF3B1nent). Within SF3B1ent 47% (67/144) did not harbor any additional mutation resulting in an average of 1.8 mutations (including SF3B1) in this group. SF3B1nent patients showed on average more mutations (MDS with isolated del(5q): 1.9; MDS-EB: 2.7; MDS-RS: 3.1). The most frequent additional mutations in all SF3B1 mutated patients were TET2 (29%), DNMT3A (16%) and ASXL1 (9%). Regarding cytogenetic abnormalities, 69/231 (30%) SF3B1 mutated samples showed aberrant karyotypes (SF3B1ent: 27/144, 19%; SF3B1nent: 42/87, 48%). Of SF3B1 mutated patients 7% (15/231) progressed to AML compared to 15% (75/503) of SF3B1 wildtype patients (median follow-up: 112 months). Notably, 80% of SF3B1nent showed AML transformations in comparison to 20% of SF3B1ent (median follow-up: 122 and 112 months). Within the 15 SF3B1 mutated patients progressing to AML 47% showed additional RUNX1 and 27% DNMT3A or TET2 mutations at MDS diagnosis. In AML-transforming patients with a low SF3B1 variant allelic frequency (VAF, n=2) additional spliceosome mutations (SRSF2 or ZRSF2) were identified at the time of diagnosis. Two other SF3B1 mutated patients showing disease progression to AML harbored MECOM rearrangements when MDS was diagnosed. During progression one patient gained a MECOM rearrangement and two patients other chromosomal aberrations present in the AML state. Image: Summary/Conclusion: SF3B1 mutations occur in several MDS entities, are associated with good clinical outcome and mainly co-occurred with TET2 mutations. Patients showed a better prognosis (longer OS, lower AML transformation rate) when categorized into the proposed SF3B1 entity. AML transformation of SF3B1 mutated MDS patients is determined by the entire genomic landscape indicating RUNX1 as potential driver. P736: BCOR MUTATION REGULATES MYELODYSPLASTIC SYNDROMES PROGRESSION THROUGH REPRESSING AUTOPHAGY FLUX MEDIATED BY HDAC6 ACTIVATION C. Jianan1, Y. dongqin2, J. jiacheng1, W. LINGYUN1,* 1xueye, shanghai no.6 hospital; 2xueye, shanghai no.8 hospital, shanghai, China Background: Past studies have revealed that mutations in bcor are involved in various human cancers including MDS and AML. Autophagy plays a critical role in cancer progression. However, the role of bcor mutations in autophagy and MDS progression remains subject to debate. Aims: This study investigated the role of bcor mutations in autophagy and MDS progression. Methods: Cell lines with bcor p483L stable overexpression were established with a recombinant lentiviral vector. The effects of bcor mutations on cell proliferation, cell cycle progression, and cell apoptosis were detected by CCK8 assay, colony formation assay, and flow cytometry. We detected changes in autophagy levels by Western blot (WB), quantitative polymerase chain reaction (qPCR), and most importantly, transmission electron microscopy. The function of histone deacetylase 6 (HDAC6) was inhibited by Tubasatin A （TBA）, and We detected the consequent changes in the functional phenotype of the cells by WB and qPCR. The interactions between related proteins were analyzed by a co-immunoprecipitation assay. To test cell lines for mitochondrial dysfunction, bcor p483L mutant cells, negative control cells, and wild-type cells were double-stained with MitoTracker Green and MitoTracker Red, or single-stained with MitoSOX followed by fluorescence-activated cell sorting (FACS) analysis. The changes in pyroptosis level caused by bcor p483L mutation were detected by WB and qPCR. Results: The cell proliferative ability of bcor p483L mutant cells was repressed compared to the negative control cells. Besides, bcor p483L mutation could promote cell apoptosis and block the cell cycle progression to the G2 phase. Notably, bcor p483L mutation repressed autophagic flux in cultured cells, as corroborated by decreased expression of autophagy marker genes (e.g., ATG7, ATG10) and reduced numbers of autophagosomes or autolysosomes, which were accompanied by upregulation of HDAC6, downregulation of FoxO1 and ac-FOXO1. HDAC6 was required for inhibiting the deacetylation of FoxO1. Enhanced HDAC6 expression and repression of FOXO1 were required for bcor p483L -mutation-deduced autophagy. Depression of HDAC6 by TBA was sufficient for increasing the expression of FOXO1, ac-FOXO1, and inducing autophagic flux. Notably, we examined the direct interaction between HDAC6 and FOXO1, FOXO1 and ATG7. Bcor p483L mutated cells accumulated dysfunctional, non-respiring mitochondria and increased mitochondrial ROS production compared to negative control cells. And we found upward adjustment of pyroptosis level and DNA damage. Image: Summary/Conclusion: Bcor p.P483L mutation could disrupt BCOR protein structure and function, therefore reducing the transcriptional repressive effect of BCOR on HDAC6. HDAC6 was upregulated and decreases the acetylation of FOXO1, further repressing autophagy. We further verified the protein: protein interaction between HDAC6 and FOXO1, FOXO1 and ATG7. Defects in the clearance of damaged mitochondria by bcor p483L-mutation-regulated autophagy contribute to the accumulation of cellular reactive oxygen species（ROS）, thus promoting cell apoptosis and repressing cell proliferation. The escalation in pyroptosis levels was found after bcor mutations and may be partly due to increased ROS levels. We also found increased gene damage and increased likelihood of gene mutations in bcor p483L mutant cells, which would promote MDS transformation into AML. These findings indicate a completely novel BCOR-HDAC6-FOXO1-ATG7 axis in the regulation of autophagy and MDS progression, providing potential possibilities for MDS treatment. P737: FUNCTIONAL ROLES OF DDX41 MUTATIONS IN THE DEVELOPMENT OF MYELOID MALIGNANCIES A. Kon1,*, M. Nakagawa1, K. Kataoka2, H. Makishima1, M. Nakayama3, H. Koseki4, Y. Nannya1, S. Ogawa1 1Dept. Pathology and Tumor Biology, Kyoto University, Kyoto; 2Division of Molecular Oncology, National Cancer Center Research Institute, Tokyo; 3Department of Technology Development, Kazusa DNA Research Institute, Chiba; 4Laboratory for Developmental Genetics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan Background: DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~3-7 % of sporadic cases with MDS/AML, in which a germline protein-truncating allele is compounded by a somatic missense mutation affecting the helicase domain (p.R525H) in the remaining allele in typical cases. However, little is known about the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms. Aims: This study aimed to clarify the molecular pathogenesis of DDX41 mutated myeloid neoplasms. Methods: We generated mice models carrying conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in (KI) alleles in various combinations. By crossing these mice with Rosa26-CreERT2 transgenic mice, we obtained heterozygous Ddx41 KO (Ddx41 +/-), homozygous Ddx41 KO (Ddx41 -/-), heterozygous Ddx41 R525H mutation (Ddx41 R525H/+), and Ddx41 R525H/KO ( Ddx41 R525H/-) mice, in which expression of the mutant allele was induced by tamoxifen administration. We then investigated the effect of these alleles on hematopoietic cells. Results: First, we assessed cell intrinsic effects of these Ddx41 alleles, in noncompetitive transplantation experiments. Shortly after tamoxifen administration, most of the recipient mice that were reconstituted with bone marrow (BM) from Ddx41 -/- or Ddx41 R525H/- mice died within a month after CreERT2 induction due to severe BM failure (BMF). By contrast, the mice transplanted with BM from Ddx41 +/- mice showed significantly reduced WBC counts and myeloid skewing in long-term observation. We also assessed the reconstitution capacity of BM cells from different Ddx41 mutant mice using peripheral blood (PB) chimerism after competitive transplantation with Ddx41 +/+ cells. The PB donor chimerism of Ddx41 -/- and Ddx41 R525H/- mice-derived cells was markedly reduced compared to that of Ddx41 +/+ cells. In contrast, Ddx41 +/- and Ddx41 R525H/+ mice-derived cells showed a slightly reduced PB chimerism compared to Ddx41 +/+ mice-derived cells. Of interest, when Ddx41 R525H/--derived BM cells were co-transplanted with Ddx41 +/--derived BM cells, some of the recipient mice showed impaired hematopoiesis, suggesting the presence of a non-cell autonomous effect of Ddx41 R525H/- cells on Ddx41 +/- cells. Transcriptome analysis of stem cells (Kit+Sca-1-Linlow cells) from different Ddx41 mutant mice revealed significant changes in gene expression and splicing patterns in many genes in all mutant mice, with larger changes for Ddx41 R525H/- than Ddx41 +/- and Ddx41 R525H/+ cells. Notably, Ddx41 R525H/--derived stem cells exhibited a significant upregulation of genes involved in innate immunity, including an upregulation of cGAS-STING innate immunity pathways, as well as an enhanced Trp53 pathway, whereas there was a downregulation of genes related to RNA metabolism and ribosome biogenesis. Summary/Conclusion: Our results suggest that monoallelic Ddx41 loss leads to age-dependent impaired hematopoiesis, while compound biallelic loss-of-function and R525 alleles showed early development of severe BM failure, where activated innate immunity and impaired ribosome functions may play important roles. The role of DDX41 mutations in myeloid neoplasms, however, need further evaluation. P738: EXPRESSION OF TRANSPOSABLE ELEMENTS IN CD34+ CELLS IN MYELODYSPLASTIC SYNDROMES Z. Krejcik1,*, A. Hrustincova1, I. Trsova1, D. Kundrat1, K. Szikszai1, M. Belickova1, J. Cernak2, A. Jonasova3, M. Dostalova Merkerova1 1Department of Genomics; 2Laboratory of Anemias, Institute of Hematology and Blood Transfusion; 3First Department of Medicine, General University Hospital, Prague, Czechia Background: Myelodysplastic syndromes (MDS) are a heterogenous group of malignant hematopoietic stem cell (HSC) disorders characterized by genomic instability resulting in aberrant differentiation of HSCs, peripheral blood cytopenia, and a tendency toward leukemic transformation. Transposable elements (TEs) are common in the human genome and, with regard to their ability to change the position in the genome, may represent one of the sources of this instability. Although TE dysregulation has previously been reported in different hematological malignancies, e.g., in acute myeloid leukemia (AML), information about TEs in MDS is rare. In the past, Colombo et al. demonstrated suppressed TE expression in higher risk MDS in a small cohort of patients. They proposed this suppression as a mechanism for immune escape in MDS/AML because the higher TE expression can potentially trigger immune-mediated cell clearance of cancer cells via “viral mimicry” pathways. Aims: Our aims were to identify, quantify and compare TE expression in different stages of MDS on a larger cohort of patients to provide a deeper insight into their occurrence and possible roles in MDS. Methods: We prepared, sequenced, and analyzed rRNA-depleted RNA libraries of CD34+ HSCs in a cohort of 75 MDS patients, and 13 healthy controls, using the SalmonTE tool for the TE detection. Results: Comparison between healthy controls and MDS patients revealed significantly upregulated class I LTR (Long Terminal Repeat) retrotransposons (p = 0.0006, logFC = 0.040), especially then ERV1 (Endogenous Retrovirus) class (p = 0.0002, logFC = 0.052), in MDS patients. Detailed analysis revealed 11 significantly (FDR < 0.05) differentially expressed TEs, nine upregulated (ERV1: HERV-Fc1, MER51E, LTR27E, MER65C, HUERS-P1, PABL_A, ERV3: MER54B, CR1: X5A_LINE, SINE (Short Interspersed Element): MIR3) and only two downregulated (ERV1: LTR71A, LTR26E) in the group of MDS patients. After stratification of patients into lower (IPSS-R score ≤ 3.5, n = 48) and higher risk (IPSS-R score ≥ 4, n = 27) categories, we observed significant dysregulation of non-LTR retrotransposons in general, and in particular of SINE (p = 6.2x10-14, logFC = -0.21) and LINE1 (Long Interspersed Element) (p = 3x10-7, logFC = 0.06) clades. Detailed differential expression analysis revealed that 18 out of 21 most significantly (FDR < 0.001) deregulated TEs were suppressed in higher risk MDS, namely AluY, AluYb8, AluYd8, AluYe2, AluSq4, AluYa1, AluYa5, AluYb3a2, AluYc1, AluYbc3a, AluYe5, AluYf2, SVA_B and SVA_F (all SINE), L1HS (LINE1), LTR12E and LTR1A2 (both ERV1); and only three TEs were upregulated (HARLEQUIN, LTR24C, PABL_AI (all ERV1)). Summary/Conclusion: To our knowledge, this is the first report on TE expression and MDS-risk specific dysregulation in a larger cohort of MDS patients, revealing their importance in the pathogenesis of MDS. Significant suppression of TEs observed in higher risk MDS patients supports the previously formulated potential mechanism of immune escape in these MDS patients. Acknowledgements: Supported by AZV CR (NU20-03-00412) and MH CZ-DRO (UHKT 00023736). Reference: Colombo et al. Suppression of transposable elements in leukemic stem cells. Sci Rep. 2017. 1;7(1):7029. doi: 10.1038/s41598-017-07356-9. P739: THE EPI-GENOMIC LANDSCAPE OF MONOSOMY 7 IN ADULT MDS/AML A. G. Lema Fernandez1,*, C. Nardelli1, V. Di Battista1, M. Quintini1, F. Pellanera1, C. Matteucci1, V. Pierini1, B. Crescenzi1, M. Moretti1, V. Bardelli1, P. Gorello1, C. Mecucci1 1Department of Medicine, University of Perugia, Perugia, Italy Background: Monosomy 7 is one of the most frequent aneuploidies in myeloid malignancies often occurring in the context of high-risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) and associated to poor prognosis. Despite several studies have focused on the pathogenetic role of genes mapping at chromosome 7q, its molecular landscape remains undetermined. Aims: The aim of this study was to characterize biological features of adult MDS/AML with isolated monosomy 7 by investigating the epi-genomic landscape. Methods: Epi-genomic characterization included karyotype, Fluorescent In Situ Hybridization (FISH), Single Nucleotide Polymorphism Array (SNPa), Targeted Genomic Sequencing, RNA sequencing and multiplex Enhanced Reduced Representation Bisulfite Sequencing (mERRBS). This comprehensive approach was used in analyzing a cohort of three adult MDS/AML cases with isolated monosomy 7 against a cohort of three non-neoplastic cytopenias and against dic(1;7)(q10;p10) cases previously characterized by our group (Leukemia, 2019). Results: Karyotype identified the presence of isolated monosomy 7 in all cases, interphase-FISH showed clonality as accounting for 65-85% of the total bone marrow population. Targeted sequencing showed multiple variants affecting myeloid genes (mean 5.3 events; from 2 to 9 per case) but did not identify a common mutational marker. Gene expression profile (FDR<0.1, log2FC>I1I) provided us with a specific signature differentiating monosomy 7 from both controls [632 differentially expressed genes (DEGs): 405 up and 227 down] and dic(1;7) [1.706 DEGs: 832 up and 874 down]. In keeping with monosomy, a strong gene dosage effect at chromosome 7 genes emerged. Functional pathways analysis specifically identified the presence of active stemness programs in monosomy 7, through deregulation of mTOR pathway, Rho-GTPases and hematopoietic stem cell markers. Deregulation of genes involved in apoptosis escape, included in TP53 and heat-shock proteins signaling, supported the undifferentiated phenotype. Interestingly, PTCH1 tumor suppressor downregulation was a differential feature of monosomy 7 with respect to the other groups and it was validated in an independent cohort of MDS/AML with isolated monosomy 7. Global methylation further delineated specific boundaries between monosomy 7 and dic(1;7) cases showing a methylation gradient that defined monosomy 7 as the most hypermethylated group. Private localization of differentially methylated regions in monosomy 7 emerged at intergenic enhancers, enriched for homeo-domain transcription factors binding sites, and at CTCF binding sites linked to promoters. A subset of hypermethylated enhancers in monosomy 7 was in accordance with repression of their respective target genes, almost totally located on chromosome 7 (ARPC1A, CCZ1, IKZF1, PMS2, RBAK, RBM33, RHEB, RNF216P1, ZNF12 and ZNF853). Their downregulation was related not only to the chromosome loss but also to the effect of enhancer hypermethylation in the retained chromosome, supporting a combined cytogenetic and epigenetic mechanism in determining biallelic deregulation. Among these deregulated genes, IKZF1 and its target genes accounted for > 6% of the whole typical expression profile. Summary/Conclusion: In conclusion our results first identified a specific signature of DNA methylation and an active epi-genomic program reminiscent of stem cells in MDS/AML with monosomy 7, providing new insights to the knowledge of biological targets in this aggressive aberration. P740: IMPACT OF MAGROLIMAB IN COMBINATION WITH AZACITIDINE ON RED BLOOD CELLS IN PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROME (HR MDS) J. Y. Chen1,*, L. Johnson2, K. M. McKenna2, T. S. Choi2, J. Duan2, D. Feng2, J. M. Tsai3, N. Garcia-Martin4, K. Sompalli2, R. Maute2, P. Vyas4, R. Majeti5, C. H. M. Takimoto2, J. Liu1, G. Ramsingh2, M. P. Chao2, J.-P. Volkmer2, I. L. Weissman5 1Stanford University School of Medicine, Stanford; 2Gilead Sciences, Inc., Foster City; 3Brigham and Women’s Hospital, Boston, United States of America; 4Molecular Hematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; 5Ludwig Cancer Center and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, United States of America Background: Magrolimab is a monoclonal antibody that blocks CD47, a “don’t eat me” signal expressed on cancer cells, to escape immune surveillance and macrophage-mediated clearance. Prior preclinical studies have shown that CD47 is critical to red blood cell (RBC) homeostasis, with CD47 deficiency decreasing RBC half-life. Fc-mediated opsonization also depletes RBCs, raising concerns that potential on-target anemia could result from the use of anti-CD47 agents via multiple mechanisms. Nonetheless, several clinical trials have demonstrated that magrolimab can be safely administered as monotherapy, with an initial lower “priming” dose yielding transient anemia with compensatory reticulocytosis and no anemia observed at subsequent higher maintenance doses. However, the mechanism underlying this observed protection has not been fully defined. Aims: To describe manageable anemia in patients with HR MDS treated with magrolimab in combination with azacitidine (AZA) in a phase 1 clinical trial (NCT03248479) and further investigate the underlying mechanisms in preclinical models. Methods: In a multicenter prospective study, complete blood counts (CBCs), peripheral blood, and bone marrow (BM) were collected at prespecified time points from patients with HR MDS (n=57) treated with magrolimab in combination with AZA. CBCs were measured, and blood and BM samples were analyzed by flow cytometry for expression of CD47 on RBCs and white blood cells (WBCs). Magrolimab was initially administered as a priming dose (1 mg/kg) followed by an initial weekly maintenance dose (30 mg/kg) before transitioning to maintenance dosing every 2 weeks. AZA 75 mg/m2 was administered on days 1-7 of each 28-day cycle. Preclinical modeling studies were conducted with intact and Fc-deficient anti-mouse CD47 (MIAP410) and anti-human CD47 (magrolimab) antibodies in murine models, including C57BL/6J B-hSIRPA/hCD47 mice. Results: Combination treatment with magrolimab and AZA resulted in tolerable anemia that correlated with rapid, near-complete loss of CD47 in RBCs but not WBCs. The initial 1-mg/kg priming dose was sufficient for this CD47 loss, which persisted with subsequent 30-mg/kg maintenance doses. Both findings are consistent with prior clinical observations of magrolimab monotherapy in patients with solid tumors and magrolimab in combination with rituximab in patients with lymphoma. Our preclinical studies with mouse models revealed that CD47 removal is mechanistically independent of previously described RBC antigen modulation mechanisms and cellular compartments. Instead, this CD47 loss requires anti-CD47 cross-linking between RBCs and non-RBCs. Summary/Conclusion: Overall, these results support the idea that on-target magrolimab-mediated anemia is mitigated by a near-complete loss of RBC CD47. Patients with HR MDS treated with magrolimab in combination with AZA had tolerable anemia with the use of priming and maintenance doses. P741: DDX41 MUTATIONS DEFINE A UNIQUE SUBTYPE OF MYELOID NEOPLASMS. H. Makishima1,*, R. Saiki1, Y. Nannya2, S. Korotev3, C. Gurnari4, J. Takeda1, Y. Momozawa5, S. Best6, P. Krishnamurthy6, T. Yoshizato1, Y. Atsuta7, Y. Shiozawa1, Y. Iijima-Yamashita8, K. Yoshida1, Y. Shiraishi9, Y. Nagata1, N. Kakiuchi1, M. Onizuka10, K. Chiba9, H. Tanaka9, A. Kon1, Y. Ochi1, M. Nakagawa1, R. Okuda1, T. Mori1, H. Itonaga11, Y. Miyazaki11, M. Sanada8, H. Tsurumi12, S. Kasahara13, C. Müller-Tidow14, A. Takaori-Kondo15, K. Ohyashiki16, T. Kiguchi17, F. Matsuda18, J. Jansen19, C. Polprasert20, S. Miyano9, L. Malcovati21, T. Haferlach22, M. Kubo5, M. Cazzola21, A. Kulasekararaj6, L. Godley3, J. Maciejewski4, S. Ogawa1 1Department of Pathology and Tumor Biology; 2Kyoto University, Kyoto, Japan; 3Departments of Medicine and Human Genetics, Section of Hematology/Oncology, The University of Chicago, Chicago; 4Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, United States of America; 5Laboratory for Genotyping Development, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan; 6King’s College Hospital NHS Foundation Trust, London, United Kingdom; 7Department of Healthcare Administration, Nagoya University Graduate School of Medicine; 8Department of Advanced Diagnosis, Clinical Research Center, Nagoya Medical Center, Nagoya; 9Division of Genome Analysis Platform Development, National Cancer Center Research Institute, Tokyo; 10Department of Hematology and Oncology, Tokai University School of Medicine, Isehara; 11Department of Hematology, Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki; 12Department of Hematology, Gifu University; 13Department of Hematology, Gifu Municipal Hospital, Gifu, Japan; 14Heidelberg University Hospital, Heidelberg, Germany; 15Department of Hematology, Kyoto University, Kyoto; 16Department of Hematology, Tokyo Medical University, Tokyo; 17Chugoku Central Hospital, Fukuyama; 18Center for Genomic Medicine, Kyoto University, Kyoto, Japan; 19Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center, Nijmegen, Netherlands; 20Department of Medicine, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Bangkok, Thailand; 21Department of Molecular Medicine, University of Pavia, Pavia, Italy; 22MLL, Munich Leukemia Laboratory, Munich, Germany Background: DDX41 was identified as a causative gene for late-onset familial myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While DDX41 is thought to be one of the most frequent targets of germline mutations responsible for sporadic cases with AML/MDS and other myeloid neoplasms (MN), the entire spectrum of pathogenic DDX41 variants and their effect size therein are still to be elucidated. The clinical and genetic pictures of DDX41-mutated myeloid neoplasms have not fully been investigated in a large cohort of patients, either. Aims: In this study, we enrolled a total of 9,081 sporadic and familial cases with different MNs from different ethnicities, in which germline and somatic mutations in DDX41 and other major driver genes in AML/MDS were analyzed using next generation sequencing. Methods: To estimate the size of the risk of MN development associated with pathogenic germline DDX41 variants, we calculated their enrichment in 3,672 MN cases as compared with a control cohort (n=20,238). We also estimated the penetrance of pathogenic DDX41 germline variants for the development of MNs. Results: We identified 292 cases with pathogenic or likely pathogenic DDX41 mutations with conspicuous ethnic diversity, of which 159 (54%) accompanied somatic mutations as well, while the remaining 53 cases had somatic mutations alone. Among 292 germline variants, 197 were truncating variants, whereas only 2.6% of somatic mutations were truncating (P<0.0001). Pathogenic germline mutations were significantly more frequent in Asian than in Caucasian cases (odds ratio (OR)=1.64), consistent with a significantly higher frequency of pathogenic germline DDX41 variants in the former cases. We observed a significant enrichment of 5 DDX41 variants in myeloid neoplasms, including p.A500fs, p.E7*, p.Y259C, p.E256K, and p.S363del, compared with 20,238 control cases, with a mean odds ratio of 10.6. To estimate the penetrance of NMs associated with pathogenic germline variants, we calculated cumulative incidence of MN in a cohort of 384 first-degree relatives (kin-cohort) of DDX41-mutated NM cases. Penetrance of pathogenic DDX41 germline variants was only negligible under 40 years of age, but rapidly elevated thereafter, reaching as high as 38.5% at the age of 85, confirming late onset of DDX41-mutated MNs. DDX41 variants were significantly more common in higher risk MDS (7.1%) and secondary AML (9.7%), compared with other myeloid neoplasms (1.9%). Patients with MDS having both germline and somatic mutations were more likely to classified in refractory anemia with excess blasts (RAEB), compared with those with germline or somatic alone (P=0.029). DDX41 variants were significantly associated with lower WBC and hypocellular bone marrow. Most frequently co-occurring mutations included those in ASXL1, SRSF2, TET2, CUX1, and DNMT3A, of which only CUX1 mutations were statistically significant. Among MDS cases, DDX41 mutations were associated with a significantly higher incidence of leukemic evolution (P=0.006) than DDX41-unmutated cases, which was virtually confined to those with truncating DDX41 variants. However, despite this, DDX41-mutated cases had a better OS than un-mutated cases, particularly when treated with hypomethylating agents (HMAs) (P<0.001). Summary/Conclusion: In summary, DDX41-mutated myeloid neoplasms define a distinct entity of myelodysplasias associated with higher incidence of leukemic evolution and better responsiveness to hypomethylating agents. P742: TRANSCRIPTOMIC ANALYSIS OF ISOLATED CD4+ AND CD8+ T CELLS REVEALS DIFFERENCES IN TCR REPERTOIRE IN PATIENTS WITH HIGH RISK MDS COMPARED TO AML. K. Katsiki1, A. Tasis1, A. Filia1, E. Lamprianidou1, K. Liapis1, I. Kotsianidis1, I. Mitroulis1,* 1Department of HEmatology, Democritus University of Thrace, Alexandroupolis, Greece Background: Myelodysplastic syndrome (MDS) is a group of clonal disorders of hematopoietic progenitor cells, which often progresses towards acute myeloid leukemia (AML). Even though MDS is a clonal hematopoietic disease, bone marrow microenvironment and more specifically inflammation has been linked to its progression. Among other cell types, CD4+ and CD8+ T cells are major players in the regulation of the microenvironment in malignancies. Aims: In this study, we aimed to investigate the molecular signature of bone marrow CD4+ and CD8+ T cells from patients with high-risk MDS, AML and chronic myelomonocytic leukemia (CMML). Methods: RNA sequencing was performed on isolated CD4+ and CD8+ T cells from the bone marrow of six patients with high risk MDS (RAEB-II), six patients with CMML and four patients with AML, using cell sorting. Except from the analysis of gene expression, we performed TCR repertoire analysis using VDJtools, an open-source Java/Groovy-based framework that can analyze immune repertoire sequencing (RepSeq) data and allows applying a diverse set of post-analysis strategies. To do so, RNA sequencing data were used as input for the V-(D)-J junction mapping software and analysis was performed on the MiXCR platform. The output of MiXCR wereused as input for the VDJtools after running a convert routine with -S mixcr argument to prepare datasets in a format for VDJtools analysis. Results: Analysis of CD8+ T cells from patients with high risk MDS and AML identified 38 differentially expressed genes. We observed that the expression of the TCR variants TRAV20 and TRAV13-1 was increased in patients with AML compared to high risk MDS (Figure 1A). Further analysis of the TCR repertoire revealed that the diversity of TCR was higher in CD8+ T cells from patients with AML (Figure 1B), whereas multidimentional scaling analysis revealed a clear separation of the two groups (Figure 1C). To the same direction, clustering of the usage of TRAV-TRAJ further confirmed this finding (Figure 1D). On the other hand, we did not observe any difference in the TCR diversity of the variant usage when we compared CD8+ T cells from patients with CMML and AML (Figure 1E-F), or in the comparison between CD4+ T cells from patients with high risk MDS and AML (Figure 1G-I). Image: Summary/Conclusion: CD8+ T cells are a major cell population that exerts anti-tumour properties in cancer. Herein, we compared the TCR repertoire of CD8+ and CD4+ T cells in the bone marrow of patients with high risk MDS and AML and observed that despite the common features of these two disorders, there are significant differences in the TCR repertoire specifically in CD8+ T cells, especially concerning TCR diversity. These findings imply that there is more active T cell response in the bone marrow in AML compared to MDS. However, it is not clear to what degree this T cell response targets AML neo-antigens. P743: THE PIVOTAL ROLE OF GLUTAMINOLYSIS IN MYELODYSPLASTIC SYNDROME (MDS): A NOVEL STRATEGY FOR THE TARGETED THERAPY OF MDS S. Okabe1,*, Y. Tanaka1, A. Gotoh1 1Department of Hematology, TOKYO MEDICAL UNIVERSITY, Tokyo, Japan Background: Myelodysplastic syndromes (MDS) is indicated by bone marrow failure and increased risk of transformation to acute myelogenous leukemia (AML). MDS is more common with advancing age and a combination of comorbidities is associated with significantly worse overall survival. Moreover, iron overload (IOL) starts to develop in MDS patients by transfusion in many cases. Because the outcome of MDS is poor, alternative therapeutic strategies are required to improve the survival of MDS and AML patients. Aims: Glutamine is the most abundant amino acid in blood. Glutaminase (GLS) is the initial enzyme in glutamine metabolism and glutaminolysis contributes to tumor growth. Because GLS is frequently activated in various types of cancer, GLS inhibitor could suppress MDS and AML cells in combination with BCL2 inhibitor. Methods: In this study, we investigated whether GLS was involved in MDS progress. We also investigated the efficacy of GLS inhibitor (CB-839 or IPN-60090) and BCL2 inhibitor, venetoclax by using MDS and AML cell line, SKM-1, MDS-L, MOLM-14, THP-1, MV4;11 and osteoblastic cell line, MC3T3-E1. Results: We first investigated the relationship between GLS expression and MDS patients by microarray gene expression data from the online Gene Expression Omnibus (GEO). In mammalian cells, there are two paralogous GLS genes, GLS1 and GLS2. GLS1 expression was increased in refractory anemia with excess of blasts (RAEB)-2 cells compared to normal control cells (GSE19429). In contrast, GLS2 expression was not changed. High GLS1 expression is associated with poor prognosis in AML patients. Deprivation of glutamine in culture medium revealed that cellular growth inhibition. We next evaluated the effect of GLS inhibitor (CB-839 or IPN-60090) or venetoclax on proliferation of MDS and AML cell lines. 72 h treatment of MDS and AML cells were inhibited by GLS inhibitor or venetoclax in a dose dependent manner. Cellular cytotoxicity and caspase 3/7 activity was also increased. Glutamine is converted by GLS into glutamate and related nicotinamide adenine dinucleotide phosphate (NADP) production. Intracellular NADPH and NADH were reduced by GLS inhibitor. Co-treatment with GLS inhibitor and venetoclax was superior effect than single drug alone. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased. The bone marrow is a relatively hypoxic microenvironment. Gene expression of GLS1 is increased under hypoxia condition. The proteasome 20S activity and phosphorylation of nuclear factor-kappa B (NF-κB) were increased. Efficacy of venetoclax is reduced under hypoxia condition. Co-treatment with GLS inhibitor and venetoclax overcome in hypoxia mediated drug resistance. GLS1 shRNA transfected cells reduced cellular proliferation. Cell cycle analysis showed G1 arrest and increased sensitivity of venetoclax. Because MDS patients with IOL have reduced overall survival and poorer outcomes, we next examined IOL by using osteoblastic cell line, MC3T3-E1. The cellular proliferation was reduced by ferric ammonium citrate (FAC). Intracellular ROS was increased by FAC. GLS inhibitor treatment protected FAC mediated cell death. Summary/Conclusion: Targeting of glutaminolysis and BCL2 inhibition combine to enhance therapeutic efficacy and has been proposed as a novel strategy high-risk MDS and AML. We also provide the promising clinical relevance as a candidate drug for treatment of MDS and AML patients. P744: UNBALANCED TRANSLOCATION DER(1;7)(Q10;P10) AS A DISTINCT SUBTYPE IN MYELODYSPLASTIC SYNDROMES R. Okuda1,*, Y. Ochi1, K. Chonabayashi2,3, N. Hiramoto4, M. Sanada1,5, H. Handa6, S. Kasahara7, S. Sato8, N. Kanemura9, T. Kitano10, M. Watanabe3, W. Kern11, M. Creignou12, Y. Shiraishi13, M. Watanabe14, K. Usuki15, S. Imashuku16, E. Hellstrom-Lindberg12, T. Haferlach11, S. Chiba17, N. Sezaki18, L.-Y. Shih19, Y. Miyazaki8, Y. Yoshida2, T. Ishikawa14, K. Ohyashiki20, Y. Atsuta21, Y. Shiozawa1, S. Miyano13,22, H. Makishima1, Y. Nannya1, S. Ogawa1,12,23 1Department of Pathology and Tumor Biology; 2Center for iPS Research and Application; 3Department of Hematology and Oncology, Kyoto University, Kyoto City; 4Department of Hematology, Kobe City Medical Center General Hospital, Kobe City; 5Department of Advanced Diagnosis, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya City; 6Department of Hematology, Gunma University Graduate School of Medicine, Gunma; 7Department of Hematology, Gifu Municipal Hospital, Gifu; 8Japan Adult Leukemia Study Group, Japan Adult Leukemia Study Group, Japan; 9Department of Internal Medicine, Gifu University, Gifu; 10Department of Hematology, Kitano Hospital, Osaka, Japan; 11MLL Munich Leukemia Laboratory, MLL Munich Leukemia Laboratory, Munich, Germany; 12Department of Medicine, Karolinska Institute, Stockholm, Sweden; 13Laboratory of Sequence Data Analysis, The University of Tokyo, Tokyo; 14Department of Hematology, Hyogo Prefectural Amagasaki General Medical Center, Amagasaki; 15Department of Hematology, NTT Medical Center Tokyo, Tokyo; 16Department of Laboratory Medicine, Uji-Tokushukai Medical Center, Uji; 17Department of Hematology, University of Tsukuba, Tsukuba; 18Department of Hematology, Chugoku Central Hospital, Hiroshima, Japan; 19Division of Hematology-Oncology, Chang Gung University, Taoyuan, Taiwan; 20Department of Hematology, Tokyo Medical University, Tokyo; 21The Japanese Data Center for Hematopoietic Cell Transplantation, The Japanese Data Center for Hematopoietic Cell Transplantation, Japan; 22Laboratory of DNA Information Analysis, The University of Tokyo, Tokyo; 23Institute for the Advanced Study of Human Biology, Kyoto University, Kyoto City, Japan Background: der(1;7)(q10;p10) (der(1;7)) is an unbalanced translocation between chromosomes 1 and 7 found in 1.5-6% of patients with myelodysplastic syndromes (MDS), depending on different ethnicity, where the common consequence is +1q and -7q. Previous studies have noted a higher incidence of RUNX1 mutation and better prognosis for der(1;7) cases compared to -7/del(7q) MDS cases. While previous studies have described several features of der(1;7), the understanding of der(1;7)+ MDS/AML is largely limited due to their small study size. Aims: To elucidate the clinical and genetic characteristics of der(1;7) with integrated omics analysis in a large cohort of myeloid neoplasms. Methods: We enrolled 148 cases with der(1;7) and analyzed their mutation profiles and clinical features using whole exome and/or targeted-capture sequencing of major driver genes of myeloid neoplasms and RNA sequencing. Distinct features of der(1;7) were investigated by comparing the results with those from an additional 3,238 non-der(1;7) cases with different myeloid neoplasms comprising -7/del(7q), +1q, and other cases. Results: 148 der(1;7) cases comprised 72.3% MDS, 23.7% AML, and 3.4% MDS/MPN cases. Compared to -7/del(7q) AML cases, der(1;7) AML cases were more likely to be AML with dysplasia or MDS-derived AML and therapy-related AML (84.4% vs 53.2% and 9.1% vs 1.9%, respectively) (p<0.001). Targeted-capture sequencing of der(1;7) cases revealed that 91.9% of cases harbored at least one copy number change or mutation. The frequency of genetic mutation and copy number alteration for der(1;7) cases substantially differed compared to -7/del(7q), +1q, and other cases. der(1;7) cases had frequent mutations affecting RUNX1 (37.8%), EZH2 (18.9%) and ETNK1 (18.2%), which were less common in -7/del(7q) cases (OR=5.58, OR=6.52, and OR=6.23, respectively). By contrast, TP53 mutations were less frequent in der(1;7) cases (OR=0.021). Copy number analysis revealed a frequent co-occurrence of +8 and del(20q) with der(1;7) cases (18.9% and 37.4%), while del(5q), which frequently co-occurred with del(7q), was not found in der(1;7) cases. Prognosis of der(1;7) MDS cases tended to be better than -7/del(7q) cases (HR=0.71, p=0.098), but poorer than +1q (HR=1.36, p=0.11) and others cases (HR=1.8, p<0.001). The most frequent cause of death in der(1;7) cases was infection (45.5%), followed by disease progression (36.4%). This high frequency of infection as a cause of death was unique in der(1;7) as compared to -7/del(7q) (infection 13.9% and disease progression 72.3%) and others (infection 10.8% and disease progression 76.9%). Finally, RNA-sequencing analysis between der(1;7) MDS cases and non-der(1;7) MDS cases showed unique expression profiles in der(1;7). 1,463 differentially expressed genes were identified, of which 895 were down-regulated and 573 were up-regulated. GSEA analysis of major pathways showed down-regulation of those related to cell cycles (including E2F targets), innate immunity, and TNFα signaling via NFκB. Gene ontology analysis also revealed down-regulation of cell cycle-related pathways, further supporting the significance of the down-regulated cell cycles in der(1;7) MDS cases, compared to non-der(1;7) cases. Summary/Conclusion: der(1;7)-positive MDS and AML represent a distinct subtype of myeloid neoplasms, characterized by unique patterns of co-mutations/copy-number lesions and gene expression profiles showing down-regulation of the cell-cycle pathway. P745: ASSESSMENT OF MIRNA BIOGENESIS GENES VARIANTS IN MDS DEVELOPMENT AND PROGRESSION D. Bug1,*, A. Tishkov1, I. Moiseev2, Y. Porozov3,4, N. Petukhova1 1Bioinformatics Research Center; 2R.M. Gorbacheva Scientific Research Institute of Pediatric Hematology and Transplantation, Pavlov University, St Petersburg; 3World-Class Research Center “Digital Biodesign and Personalized Healthcare”, I.M. Sechenov First Moscow State Medical University, Moscow; 4Department of Computational Biology, Sirius University of Science and Technology, Sochi, Russia Background: Experimental studies demonstrated that knockout of miRNA processing enzymes Dicer and Drosha is associated with the development of phenotype similar to myelodysplastic syndrome (MDS). DICER1 inherited syndrome is associated with cancer predisposition. There is a limited number of clinical studies evaluating the spectrum of mutations in the microRNA processing genes in patients with hematological malignancies and the pathogenic significance of these mutations is unknown. Thus, we conducted a bioinformatics analysis of sequencing data from our data set of MDS patients and available results in the public databases. Aims: We aim to identify DICER1 and DROSHA variants in hematological malignancies that lead to the protein function alteration based on bioinformatics analysis. Methods: Data on 35 previously sequenced high-risk MDS patients (Moiseev IS et al., PLoS One. 2021 Mar 17;16(3):e0248430) was analyzed, and the list of DICER1 and DROSHA variants was enriched with disease-specific coding variants retrieved from COSMIC database, and 3’-UTR-variants - from dbSNP. All collected variants were subjected to bioinformatics analysis. We used 3D-structures of Drosha and Dicer for molecular dynamics (MD) simulations for 200 and 300 ns, respectively (Schrödinger Suite 2020-4, Schrödinger, LLC, New York, NY, USA, 2020). The IntaRNA and MicroSNiPer algorithms were used to calculate interaction energies of 3’-UTRs hybridizing with the most abundant miRNAs in MSCs to evaluate noncoding variants. Results: The prevalence of mutations in DICER1 and DROSHA was 54% and 17%, respectively, predominantly located in 5’-UTR and 3’-UTR regions. Detected allele frequencies and prevalence suggested their association with minor clones within the bone marrow niche. MD simulation demonstrated apparent structural alterations with consequent protein dysfunction. RMSD results and calculated total energy of studied proteins were higher for all mutants compared to wild-type Drosha (at least 30% RMSD shift). All mutations were shown to be located in close proximity of the miRNA binding site, all were destabilizing for protein structure alone with consequent alterations in bonds and protein-miRNA binding. The most significant structural shift and protein destabilization was detected for DICER1 variants F508C and T993R characterized by local transformation of bonds interplay. The conformational changes in the protein due to Dicer variants were studied in 300 ns: differences in average RMSD values were within ~ 1Å between WT protein and mutants. The most significant changes were observed at the local area for I445S, F508C, and T993R characterized by shift of molecular bonds and alteration of protein stability parameters. A list of SNPs that exhibit the highest repression of DICER1 and DROSHA was obtained (Table 1). The most prominent SNPs (rs1479981622, rs536092006, rs1296755923, rs1207839989) had less than 0.03% population allele frequency which is in concordance with their possible pathogenicity. Image: Summary/Conclusion: We identified potential coding and non-coding “hot spots” for future analysis. Also the study shows that 3’-UTR SNPs of DICER1, DROSHA have more potential to impact MDS progression than coding variants. Simultaneously, we found low frequency of reports for these mutations in the available databases, indicating bias and absence of these genes in the targeted sequencing panels. This builds the foundation for future research of DICER1 and DROSHA clinical role in hematological malignancies. P746: COMBINATION OF RAS/MAPK MODULATOR AND AZACITIDINE AFFECTS HISTONE MARKS AND IMPACTS THE INNATE IMMUNE SIGNALING PATHWAY IN THE MDS-L CELL LINE R. Rai1,*, F. Patel1, J. Feld1, S. Melana1, S. Navada1, R. Odchimar-Reissig1, E. Demakos1, E. P. Reddy2, A. Horowitz2, L. Silverman1 1Division of Hematology and Medical Oncology; 2Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, United States of America Background: Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective hematopoiesis, and a propensity to transform into acute myeloid leukemia. MDS patients exhibit chronic activation of the innate immune response and a hyperinflammatory microenvironment (Barryero L, et al. Blood, 2018). Azacitidine (Aza), as a single agent has an overall response rate (ORR) of only 50% (Silverman LR, et al., JCO 2002; Fenaux P et al., Lancet Oncol. 2009). The response to Aza is not durable and all patients relapse with worsening bone marrow failure. Aza combined with a multi-kinase inhibitor of RAS and MAPK pathways, rigosertib (RAS/MAPK), reverses the bone marrow failure state and demonstrates an ORR of 54% in patients who failed a prior HMA based therapy. (Navada SC, et al. EHA Library 2019). This represents a critical observation in overcoming the epigenetic clinical resistance phenotype, though the mechanism is still elusive. Aberrant RAS/MAPK signaling has also contributed to MDS pathophysiology. Aims: To investigated the impact of RAS modulation combined with Aza on the innate immune signaling pathway. Methods: We used the MDS-L cell line as a model to limit the heterogeneity observed in MDS patients. The cells were treated with, Aza, RAS/MAPK and RAS/MAPK-Aza for 48 hrs and further analyzed by qPCR and western blot. We studied the expression of several histone proteins and various pattern recognition receptors (TLRs, RIG-I, MDA5, STING), their intermediate adaptor molecules myeloid differentiation factor 88 (MYD88), mitochondrial antiviral signaling (MAVS) gene; and interferon regulatory factor (IRF)-3 and -7 by qPCR. Results: We found an increase in H3K9ac protein expression with RAS/MAPK and RAS/MAPK-Aza; Aza and RAS/MAPK alone each had similar effects on H3K4me3, however, its expression was markedly upregulated with RAS/MAPK-Aza. Effects on H3K36me3 were comparable in all treated cells. We observed marked effects on the repression marks H3K9me3 and H3K27me3 by RAS/MAPK and RAS/MAPK-Aza combination. Furthermore, we observed that Aza, RAS/MAPK and RAS/MAPK-Aza significantly inhibit the expression of TLR1, 2 and 6. However, TLR-3, 9, RIG-I, MDA5, STING, MAVS, MYD88, and IRF-3, 7 were significantly inhibited by RAS/MAPK and RAS/MAPK-Aza. Summary/Conclusion: We find that the RAS/MAPK modulator rigosertib has histone modifying effects alone and when combined with Aza. The combination has effects on histone modification of both activator and repressor marks and is associated with down-regulation of the innate immune signaling pathway. In the clinic RAS/MAPK modulation combined with Aza reverses bone marrow failure in MDS. Further studies are underway to determine the correlation of the histone modification and innate immune signaling changes, as well as the role of RAS/MAPK modulation, to determine how these mechanisms contribute to the improvement in hematopoiesis in MDS patients. P747: LR-MDS IS CHARACTERIZED BY DIFFERENTIAL EXPRESSION OF INFLAMMASOME-RELATED GENES IN SPECIFIC CELL POPULATIONS C. Rolfs1,*, M. Schneider1, M. Trumpp1, L. Fischer2, S. Winter2, S. Uxa1, V. Menger1, K. Nenoff1, A. S. Kubasch1, K. Metzeler1, K. Sockel2, C. Thiede2, L. Hofbauer3, A. Brüderle4, J. Woo5, M. Cross1, U. Platzbecker1 1Department of Hematology and Cell Therapy, University Hospital Leipzig, Germany, Leipzig; 2Medical Clinic and Policlinic I; 3Department of Medicine III, University Hospital Carl Gustav Carus, Dresden, Germany; 4Novartis Oncology; 5Translational Clinical Oncology, Novartis Institutes for BioMedical Research, Basel, Switzerland Background: Myelodysplastic Syndromes (MDS) are a group of hematopoietic neoplasms characterized by clonal expansion of hematopoietic stem and progenitor cells (HSPC) and peripheral blood cytopenia. MDS can develop from clonal hematopoiesis of indeterminate potential (CHIP) and progress through low-risk (LR) to high-risk (HR)-MDS and further to acute myeloid leukemia (AML). Recent findings emphasize the role of sterile inflammation in the bone marrow (BM) as a driver of neoplastic progression. Our previous analysis of whole BM mononuclear cells (BM-MNC) has revealed distinct expression patterns of inflammasome-related genes, including IL1B and IL18, to be associated with LR-MDS subgroups and clinical features. This raises the prospect of interrupting disease evolution at an early stage by targeted, personalized anti-inflammatory therapy. The development and stratification of such therapies will ultimately require more detailed knowledge of the variety and consequences of inflammation networks in MDS bone marrow. Aims: Our objective was to assess inflammasome-related gene expression levels in individual cell types in the BM of CHIP, LR-MDS and HR-MDS individuals as well as non-CHIP controls in order to determine a) which cell types contribute to the expression of inflammasome-related genes and b) whether specific disease states are associated with consistent patterns of cell-specific gene expression. Methods: Cryopreserved BM-MNC from 3 non-CHIP controls, 3 CHIP individuals, 14 LR-MDS and 5 HR-MDS patients were obtained from the MDS registry and the BoHemE Study (NCT02867085) at the University Hospitals in Dresden and Leipzig. Hematopoietic stem and progenitor cells (HSPC), monocytes, monocytic myeloid-derived suppressor cells (M-MDSC), polymorphonuclear MDSC (PMN-MDSC), B lymphocytes, T lymphocytes and CD45- cells (control) were sorted from thawed BM-MNC on a BD FACS Jazz. RNA was isolated and the gene expression of IL1B, IL18, S100A9, IRAK4, NLRP3, PYCARD, CASP1 and NLRC4 was assessed by RT-qPCR. Results: The majority of the inflammasome-related genes, including IL1B, NLRP3, NLRC4 and S100A9 were expressed in all disease states predominantly by monocytes and M-MDSC (p<0.001). However, IL18 was expressed at the highest level in HSPC. Consistent to our previous analysis of whole BM-MNC, we found an association between cell-specific gene expression and LR-MDS genetics, with HSPC-derived IL18 mRNA being highest in SF3B1-mutated LR-MDS, while monocytic IL1B mRNA was highest in del(5q) LR-MDS. Furthermore, an apparent progression in expression from non-CHIP through to LR-MDS was detected, with mRNA levels of IL1B and NLRP3 in monocytic cells and PYCARD and CASP1 in HSPC increasing from non-CHIP to CHIP between 1.5- and 7.9-fold. From CHIP to LR-MDS, mRNA levels of CASP1 and NLRC4 in monocytic cells and PYCARD both in HPSC and in monocytic cells increased by 1.8- to 2.1-fold. Summary/Conclusion: We identify features of inflammasome-related gene expression in individual cell populations and disease states reflecting the progression from non-CHIP through CHIP to MDS. Our analysis suggests strongly that high resolution studies of gene expression in specific populations or single cells will resolve a spectrum of MDS-related inflammation states relevant to prognosis and personalized therapy. Therefore, we are currently performing RNA-Seq from sorted cell populations in order to extend our analysis beyond the core inflammasome reported here. P748: SINGLE CELL GENOTYPING OF MATCHED BONE MARROW AND PERIPHERAL BLOOD CELLS IN TREATMENT NAIVE AND AZA-TREATED MDS AND CMML A. Schnegg-Kaufmann1,2,*, J. Thoms2, G. S. Bhuyan3, H. Henry2, L. Vaughan2, K. Rutherford4, P. Kakadia5, E. Johansson6, T. Failes7, A. Greg7, J. Koval8, R. Lindeman9, P. Warburton10, A. Rodriguez-Meira11, A. Mead11, A. Unnikrishnan3, S. Bohlander12, E. Pappaemmanuil13, O. Faridani2, C. Jolly2, F. Zanini3, J. Pimanda14 1Department of Haematology and Central Haematology Laboratory, Inselspital, Bern, Switzerland; 2School of Medical Sciences, Lowy Cancer Research Centre; 3Prince of Wales Clinical School, Lowy Cancer Research Centre, UNSW Sydney, Sydney, Australia; 4Computational Oncology Service, Department of Epidemiology & Biostatistics, Center for Computational Oncology, Memorial Sloan Kettering Cancer Center, New York, United States of America; 5University of Auckland, Auckland, New Zealand; 6Flow Cytometry Facility, Mark Wainwright Analytical Centre; 7Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Sydney; 8Ramaciotti Centre for Genomics, UNSW Syndey, UNSW; 9Department of Haematology, Prince of Wales Hospital, Sydney; 10Department of Haematology, Wollongong Hospital, Wollongong, Australia; 11Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; 12Leukaemia and Blood Cancer Research Unit, Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand; 13Computational Oncology Service, Department of Epidemiology & Biostatistics, Center for Computational Oncology, Memorial Sloan Kettering Cancer Center, New York, United States of America; 14School of Medical Sciences, Lowy Cancer Research Centre, UNSW, Sydney, Sydney, Australia Background: Somatic mutations in hematopoietic stem cells (HSCs) are a central pathogenic event in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) where they induce proliferative advantages and impaired differentiation with subsequent cytopenias in peripheral blood (PB). Patients with high-risk disease who are ineligible for allogenic stem cell transplantation are treated with hypomethylating agents, including 5-azacytidine (AZA). AZA treatment can improve PB counts and delay progression to Akute Myeloid Leukemia. However, AZA response does not require eradication of mutated HSCs and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. Aims: In this project, we aimed to determine the distribution of individual subclones and even wild-type cells within the HSPC-compartment and the contribution of these subclones and wild-type cells to effective hematopoiesis in patients with MDS or CMML. Methods: We used a combination of index sorting and single cell genotyping to characterize the somatic mutations of individual stem- and progenitor cells and matched high-turnover circulating cells in three patients, one treatment naïve and two AZA-treated. Using this approach, myeloid driver mutations previously detected in a traditional approach were tracked in HSC/multipotent progenitors (MPP), MDS stem cells MDS-SC, progenitor cells (common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), megakaryocyte erythroid progenitor (MEP)), as well as in monocytes, neutrophils and naïve B cells (nBC). Results: Patient H198302 and Patient H198303 both had CMML and had been treated with AZA for ~10 years with complete response. Patient H198304 had MDS-EB1 and has never been treated with hypomethylating agents. For each patient, the figure shows the proportions of cells across the haematopoietic hierarchy carrying zero, one, two, three, or four mutations in the specified alleles. Only very small numbers of HSPCs carrying no mutation could be detected. In all three patients, most stem and progenitor cells carried a majority of tracked mutations, with no major differences between stem cells, progenitor cells and MDS-stem cells. In one patient, H198304, MDS-stem cells had a higher proportion of cells with 3 or 4 mutations detected. Overall, in all three patients, a similar frequency of mutations was observed in differentiated monocytes and neutrophils with a substantial proportion of the circulating cells derived from highly mutated progenitors. One notable exception were nBCs in Patient H198304, which were mostly wild-type, suggesting that the small wildtype HSC population is the predominant origin of nBCs in this individual. Analysis of additional lymphoid populations in PB from this patient revealed that naïve T, but not NK cells were also predominantly wild-type, suggesting specific impairment of B- and T-lineage maturation in the mutated cells. Image: Summary/Conclusion: In conclusion, attrition of highly mutated cells during myeloid maturation was not observed in any of the three patients, irrespective of HMA therapy. This suggests that in vivo, highly mutated stem and progenitor cells retain the capacity to differentiate to mature myeloid and in some patients mature lymphoid cells and contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment. P749: PROGNOSTIC SIGNIFICANCE OF SERIAL CIRCULATING TUMOR DNA STATUS POST TREATMENT IN MYELODYSPLASTIC SYNDROMES AND ACUTE MYELOID LEUKEMIA H. Tong1,*, X. Zhou1, W. Lang1, C. Mei1, Y. Ren1, L. Ma1, G. Xu1, L. Xu1, Y. Li1 1department of hematology, The first affiliated hospital Zhejiang university school of medicine, Hangzhou, China Background: A major advance in our understanding of Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) biology has been the implementation of next generation sequencing (NGS) which influenced diagnostic, prognostic, and therapeutic decisions in myeloid malignancies. Circulating tumor DNA (ctDNA) sequencing, as as a novel and minimally invasive measure, was reported to exhibit excellent correlations with matched bone marrow NGS in MDS and AML. However, the clinical relevance of dynamic ctDNA monitor during active treatment is unknown. Aims: In this study, we assessed the role of ctDNA as a biomarker to monitor therapeutic response and clonal evolution in MDS and AML. Methods: Thirty-one MDS and AML patients who had both bone marrow NGS and ctDNA at baseline were included for concordance analysis. Twenty-seven patients who had at least two serial ctDNA assessment with at least one month interval were included for dynamic ctDNA analysis. Targeted deep sequencing was performed on bone marrow and ctDNA using a customized panel of 165 genes known to be recurrently mutated in MDS and acute myeloid leukemia. Results: Thirty out thirty-one patients were identified with ctDNA mutation at baseline. Diagnostic ctDNA and matched bone marrow DNA exhibited excellent correlations with variant allele frequencies (VAFs) (R2=0.721, p<0.001). For 27 patients who had ctDNA mutation in baseline and treated with HMA or chemotherapy, the average mutation VAFs at the end of treatment was lower in patients achieving a CR/CRi versus those with mCR, mLFS or SD (2.7% vs 24.1%, respectively, p<0.001). As expected, mutation VAFs from all patients with treatment failure did not decrease during treatment. (Figure 1). Similarly, the average decrease in mutation VAFs from pretreatment to end of treatment was greater in patients achieving a CR versus those who did not (87.7% vs 35.5%, respectively, p= 0.001). ctDNA negative post treatment was associated with longer PFS (median PFS not reached (NR) vs. 5.6 (95% CI 4.08-7.13) months; P=0.009)(Fig 2A) and OS (median OS NR vs. 12.0 (95% CI 9.08-15.0) months; P=0.023) (Fig 2B). Seven of ten MDS patients who transformed to AML were identified with lately acquired subclones harboring FLT3 or NF1, mutations involving in signaling pathway. Moreover, new subclones were detected 0.5 to 4 months prior to AML transformation in 4 cases, indicating that genetic progression can predate morphological progression. Image: Summary/Conclusion: ctDNA status post treatment was relevant to clinical response and was of prognostic significance for disease progress and relapse. Dynamic ctDNA changes revealed complex patterns of clonal structure of MDS and AML in response to treatment. ctDNA sequencing could be an attractive, prospective measure for disease monitoring. P750: CLONAL HEMATOPOIESIS AND EPIGENETIC AGE ACCELERATION IN ELDERLY DANISH TWINS M. Tulstrup1,2,3,4,*, M. Soerensen2,5, J. W. Hansen1,3,4, J. Weischenfeldt3,4,6, K. Grønbæk1,3,4, K. Christensen2,5,7 1Department of Hematology, Rigshospitalet, Copenhagen; 2The Danish Twin Registry, University of Southern Denmark, Odense; 3Biotech Research and Innovation Centre; 4The Danish Stem Cell Centre (DanStem), University of Copenhagen, Copenhagen; 5Department of Clinical Genetics, Odense University Hospital, Odense; 6Finsen Laboratory, Rigshospitalet, Copenhagen; 7Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark Background: Clonal hematopoiesis of indeterminate potential (CHIP) is commonly ocurring in the elderly and associated with increased morbidity and mortality. CHIP is associated with accelerated epigenetic age (Robertson et al. Curr Biol 29, R786–R787, 2019; Nachun et al., Aging Cell, 20, e13366, 2021) and especially with intrinsic measures of epigenetic age, i.e. measures which reflect cell-type independent effects of aging on DNA methylation, as opposed to extrinsic measures that reflect age-related changes to the immune-cell composition of the peripheral blood. Furthermore, Nachun et al. reported that the combined measure of Hannum and GrimAge acceleration modifies the effect of CHIP mutation status, resulting in strongly increased risk of all-cause mortality in individuals with both CHIP and accelerated Hannum age and GrimAge age. Aims: To replicate and extend these findings in elderly Danish twins, especially investigate gene-specific effects on extrinsic vs. intrinsic estimators and the effect of shared environmental and genetic factors in intra-twin pair analyses. Methods: Blood samples collected from elderly Danish twins (mean age 79 years, range 73–90) in 1997 were analyzed for CHIP mutations using a custom Illumina TruSeq next-generation sequencing panel targeting 21 og the most commonly mutated genes in CHIP. DNA methylation profiling was performed on the Illumina HumanMethylation450K BeadChip. Associations between CHIP and epigenetic age were investigated using linear regression (individual-level analyses) and conditional logistic regression (intra-pair analyses). The interaction between CHIP and epigenetic age acceleration was analysed using Cox proportional hazards regression. Results: Of 308 individuals (154 twin pairs), 116 carried a CHIP mutation. The strongest association with CHIP in individual-level analyses was seen for the Intrinsic Epigenetic Age Acceleration (IEAA) estimator (CHIP carriers 1.40 years older [95% CI: -0.01–2.81]), and in intra-pair analyses Hannum age showed the strongest association (OR 1.1 [1.01–1.19]). In mutation-specific analyses, TET2 mutations were associated with the extrinsic Hannum age estimator in both individual-level (3.12 years [1.07–5.16]) and intra-pair analyses (OR 1.11 [0.99–1.24]). DNMT3A mutations were associated with IEAA in individual-level (1.82 years, [0.07–3.57]) but not intra-pair analysis (OR 1.03, 0.92–1.14). Analyses of logit-transformed VAF were consistent with these results. Individuals with both CHIP and accelerated Hannum and GrimAge estimates did not have an increased mortality risk in our cohort (HR 1.02, [0.70–1.46]). Summary/Conclusion: TET2 mutations were predominantly associated with extrinsic epigenetic age acceleration and the effect remained when controlling for shared factors between co-twins. Conversely, DNMT3A mutations were associated with intrinsic epigenetic age acceleration, and the intra-pair analysis indicated that this association may be due to a shared genetic and/or environmental background. The association between TET2 mutations and extrinsic epigenetic aging indicates that TET2 mutated clones may be drivers of immunosenescence, while the association between DNMT3A mutations and intrinsic epigenetic aging may be due to increased stem cell proliferation rates in individuals with DNMT3A mutations. The CHIP-age acceleration interaction reported by Nachun et al was not replicated, indicating that this mortality model should be used with caution, especially in this age group. P751: CHARACTERIZING CIRCULAR RNA EXPRESSION IN MYELODYSPLASTIC SYNDROME E. Wedge1,2,3,*, C. R. M. Côme2,3, J. W. Hansen1,2,3, J. S. Jespersen2,3,4, M. Dahl1,2,3, C. Schöllkopf1, K. Raaschou-Jensen5, B. Porse2,3,4, J. Weischenfeldt2,3,4, L. S. Kristensen6, K. Grønbæk1,2,3 1Department of Hematology, Rigshospitalet; 2Biotech Research and Innovation Center (BRIC); 3Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), Faculty of Health and Medical Sciences, University of Copenhagen; 4The Finsen Laboratory, Rigshospitalet, Copenhagen; 5Department of Hematology, Odense University Hospital, Odense; 6Department of Biomedicine, Aarhus University, Aarhus, Denmark Background: Circular RNA (circRNA) research is an expanding field, reflecting a recognition that circRNAs may have various roles in cancer pathology. CircRNAs are formed when back-splicing events create covalently closed loop structures from pre-mRNA molecules (Kristensen LS, Nat Rev Genet, 2019). CircRNA expression is cell-type specific, also during hematopoietic differentiation (Nicolet BP, Nucleic Acids Res, 2018), but their biological significance in myeloid cancers is still largely unknown. Aims: We aimed to profile circRNA expression in myelodysplastic syndrome (MDS), along with the related diseases chronic myelomonocytic leukemia (CMML) and clonal cytopenia of undetermined significance (CCUS), in order to identify disease-specific expression patterns and relevant candidates for biological or biomarker exploration. Methods: Seventy-one patients and eight healthy age-matched controls were included. Bone marrow aspirate underwent FACS sorting to obtain CD34+ stem and progenitor cells. RNA was extracted and total RNA libraries were created using enzymatic ribosomal RNA removal. The RNA was sequenced on a NovaSeq 6000 (mean 270 million paired-end reads per sample, read length 150 bp). Bioinformatic pipelines find_circ and CIRI2 were used to identify and quantify reads originating from the back-splicing junctions of circRNAs. Detection by both pipelines was required, plus presence in at least 10% of the samples. DESeq2 was used for normalization and comparison of groups. CircRNAs with a mean expression of >10 normalized reads were considered highly abundant and this subset was the focus of analysis. Results: In total, 8,651 unique circRNAs passed the filtering criteria, and the top 826 were highly abundant. Global circRNA expression showed upregulation in CCUS relative to healthy controls (p<0.001) and MDS relative to CCUS (p<0.001) (Figure A). We found that 110 unique circRNAs were significantly upregulated in MDS relative to healthy controls, whilst none were significantly downregulated (Figure B). In addition, high or very high IPSS-R score was associated with significantly higher circRNA expression than lower scores (p<0.001). We used LASSO regression to identify 14 circRNAs which may be related to clinical outcomes, and used these to calculate a Myeloid Circ Score (MCS). Patients were designated ‘high MCS’ or ‘low MCS’ with a cut-off at the median. A high MCS was associated with poorer progression-free survival (PFS) with a hazard ratio (HR) of 33.5 (95%CI 7.8-143.5, p<0.001) and poorer overall survival (OS) with a HR of 16.5 (95%CI 3.8-71.5, p<0.001) in the whole cohort. The same could be seen in analysis of MDS patients only (PFS HR 14.2 (95%CI 3.2-62.7, p<0.001), OS HR 8.5 (95%CI 1.9-38.3, p=0.005)). This a greater hazard ratio than that of the IPSS-R in this cohort (Very high/high/Intermediate IPSS-R relative to low/very low IPSS-R; HR for PFS of 4.27 (95%CI 1.7-11.1, p=0.003), and HR for OS of 2.5 (95%CI 0.9-7.3, p=0.09)). Image: Summary/Conclusion: We have observed associations between overall circRNA abundance and disease severity in myeloid cancer, including a global upregulation along the spectrum of disease from healthy to CCUS to MDS, and upregulation in high risk MDS compared to lower risk MDS. CircRNAs may be directly implicated in disease biology or be reflective of other changes at the cellular level. The Myeloid Circ Score has potential in risk stratification but requires validation in independent cohorts and further investigation in more readily available tissues such as bulk bone marrow or peripheral blood. P752: TARGETING S100A9 IN THE MYELODYSPLASTIC INFLAMMATORY BONE MARROW MICROENVIRONMENT BY TASQUINIMOD IMPROVES THE SUPPORTIVE FUNCTION OF MESENCHYMAL STROMAL CELLS M. Wobus1,2,*, E. Balaian1,2, M. Lissner1, R. Towers1, U. Oelschlägel1, R. Wehner2,3, T. Chavakis4, M. Törngren5, H. Eriksson5, E. Bondesson5, U. Platzbecker6, M. Bornhäuser1,2, K. Sockel1 1Department of Medicine 1, University Hospital TU Dresden, Dresden; 2German Cancer Consortium (DKTK), Partner Site Dresden, and German Cancer Research Center (DKFZ), Heidelberg; 3Institute of Immunology; 4Institute of Clinical Chemistry and Laboratory Medicine, University Hospital TU Dresden, Dresden, Germany; 5Active Biotech AB, Lund, Sweden; 6Medical Clinic and Policlinic 1, Hematology and Cellular Therapy, Leipzig University Hospital, Leipzig, Germany Background: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. An aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species (ROS). Tasquinimod (TASQ, Active Biotech) is a novel, small molecule inhibitor of S100A9 which has been investigated in several solid tumor entities and is currently under investigation in a phase Ib/IIa trial of patients with relapsed/refractory multiple myeloma. So far, little is known about its effects on myeloid malignancies. Aims: We aimed to investigate the role of S100A9 in cellular models of MDS and the in vitro potential of TASQ to target S100A9 within the MDS microenvironment. Methods: Mesenchymal stromal cells (MSCs) from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) ± TASQ (10µM). Subsequently, TLR4 downstream and proinflammatory signaling was analyzed by Western blot and real-time PCR. ROS levels were determined with a CellROX Flow Cytometry Kit (Thermo Fisher). Moreover, MSC differentiation and colony-forming unit-fibroblast (CFU-F) capacity were tested. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium. Results: Exposure of MDS and healthy MSCs to S100A9 induced TLR4 downstream signaling as demonstrated by increased expression of IRAK1 and NF-kB-p65 as well as gasdermin, an inductor of pyroptosis. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation. In line with this, OXPHOS and ROS levels were decreased after addition of TASQ. Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1β and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50% both at the mRNA and protein level. Moreover, addition of TASQ to MDS MSC cultures resulted in a significantly higher CFU-F number as well as abolished blocked the excessed adipogenic differentiation. In co-cultures with HSPCs, we observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs, which could be restored by TASQ pre-treatment. Summary/Conclusion: We provide evidence that TASQ mitigates the pathological inflammasome activation in the myelodysplastic BM niche in vitro by inhibition of the S100A9-mediated TLR4 signalling as well as its effects on NF-kB-p65 transcription and PD-L1 expression, resulting in improved hematopoietic support by MSCs, which suggests a beneficial effect in cytopenic MDS patients. P753: DYSFUNCTIONAL BONE MARROW ENDOTHELIAL PROGENITOR CELLS ARE INVOLVED IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES T. Xing1,2,*, Z.-S. Lyu1,2, C.-W. Duan3, H.-Y. Zhao1, S.-Q. Tang1, Q. Wen1, Y.-Y. Zhang1, M. Lv1, Y. Wang1, L.-P. Xu1, X.-H. Zhang1, X.-J. Huang1,2, Y. Kong1 1Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University People’s Hospital; 2Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing; 3Key Laboratory of Pediatric Hematology and Oncology Ministry of Health and Pediatric Translational Medicine Institute, Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of medicine, Shanghai, China Background: Myelodysplastic syndromes(MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective haematopoiesis and immune deregulation. Emerging evidence has shown important roles of the bone marrow(BM) microenvironment in regulating haematopoiesis and immune balance. As an important component of BM microenvironment, murine studies and our previous studies(Blood2016, EbioMedicine2020, CEI2021) reported endothelial progenitor cells(EPCs) modulate the physiology and regeneration of haematopoietic stem cells(HSCs) and differentiation of effector T cell subsets. Some evidences suggest that EPCs demonstrate inferior supporting ability to normal HSCs in MDS. However, the dual supporting abilities of BM EPCs to normal HSCs and malignant cells in patients with different stages of MDS remain unclear. In addition, the immunomodulatory ability of BM EPCs in MDS needs to be elucidated. Aims: To determine the number, the functions and the abilities of haematopoiesis and immune regulation of BM EPCs in patients with different stages of MDS. Moreover, to explore the correlation between dysfunctional BM EPCs and different stages of MDS and underlying mechanisms of dysfunction. Methods: In this study, patients with lower-risk MDS(N=15), higher-risk MDS(N=15) and de novo acute myeloid leukaemia(AML)(N=15) and healthy donors(HDs)(N=15) were enrolled. The number of primary BM EPCs were detected by flow cytometry. The functions of BM EPCs were evaluated by DiI-Acetylated low-density lipoprotein uptake and FITC-UEA-1 binding assay, tube formation and migration assays. In order to assess the gene expression profiles of BM EPCs, RNA sequencing and gene set enrichment analysis were performed. The supporting abilities of BM EPCs from MDS patients to normal HSCs, leukaemia cells and T cells were assessed by in vitro coculture experiments(coculture with HSCs and T cells from HDs and HL-60 cells, respectively). Results: Increased but dysfunctional BM EPCs were found in MDS patients compared with HDs, especially in patients with higher-risk MDS. RNA-seq indicated the changes of haematopoiesis- and immune-related pathways in MDS BM EPCs. In vitro coculture experiments verified that BM EPCs from HD, lower-risk MDS, and higher-risk MDS to AML exhibited a progressively decreased ability to support normal HSCs, manifested as elevated apoptosis rates and intracellular reactive oxygen species(ROS) levels and decreased colony-forming unit plating efficiencies of HSCs. Moreover, BM EPCs from higher-risk MDS patients demonstrated an increased ability to support leukaemia cells, characterized by increased proliferation, leukaemia colony-forming unit plating efficiencies, whereas decreased apoptosis rates and apoptosis-related genes of HL-60 cells. Furthermore, BM EPCs induced normal T cell differentiation towards more immune-tolerant cells, like regulatory T cells, in higher-risk MDS patients in vitro. In addition, the levels of intracellular ROS and the apoptosis ratio were increased in BM EPCs from MDS patients than HDs, especially in higher-risk MDS patients, which may be underlying mechanisms of dysfunctional BM EPCs. Summary/Conclusion: The current study demonstrated that increased but dysfunctional BM EPCs in MDS patients. Moreover, the dysfunctions of BM EPCs were more severe in higher-risk than lower-risk MDS patients, manifested as more supportive ability to leukaemia cells but worse ability to normal HSCs. Our data indicated that repair of dysfunctional BM EPCs may be a promising therapeutic approach for MDS patients. P754: INITIAL RESULTS OF PHASE I/II STUDY OF AZACITIDINE IN COMBINATION WITH QUIZARTINIB FOR PATIENTS WITH MYELODYSPLASTIC SYNDROME AND MYELODYSPLASTIC/MYELOPROLIFERATIVE NEOPLASM WITH FLT3 OR CBL MUTATION T. Abuasab1,1,*, E. Jabbour1, N. Short1, M. Konopleva1, K. S. Chien1, S. Fareed Mohamed1, N. Daver1, R. Kanagal-shamanna2, G. Garcia-Manero1, G. Montalban-Bravo1 1Department of Leukemia; 2Department of Hematopathology, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Houston, United States of America Background: FMS-like tyrosine kinase 3 (FLT3) mutations occur in about 1% of patients (pts) with newly diagnosed myelodysplastic syndrome (MDS) and up to 19% at time of failure of hypomethylating agent (HMA). In addition, Casitas B-lineage Lymphoma mutations (CBL) are observed in 10% of pts with MDS/MPN and up to 13-15% of pts with CMML (mainly proliferative type). Preclinical studies suggest that CBL-mutant cells are dependent on FLT3 signaling. Aims: The primary objective is to determine the safety, tolerability, and maximum tolerable dose (MTD) of quizartinib. Secondary objectives include evaluation of overall response per IWG 2006 criteria, in addition to overall survival (OS), duration of response, leukemia-free survival (LFS), relapse-free survival (RFS). Methods: We designed a phase I/II study of azacitidine in combination with quizartinib for pts with newly diagnosed or previously treated MDS or MDS/MPN with detectable FLT3 and/or CBL mutations. The study included an initial phase I dose escalation portion followed by a phase II dose expansion. Dose escalation was planned to evaluate 3 dose levels of quizartinib (30, 40 and 60mg, respectively) administered on days 1-28 of every cycle in combination with azacitidine (75 mg/m2/day) on days 1-5 each 28-day cycle. Results: As of February 25, 2022, a total of 10 pts have been enrolled (9 in dose escalation, 1 in dose expansion): 7 pts with CMML, two with MDS with excess blast and one with atypical chronic myeloid leukemia (aCML). A total of 8 pts (80%) had normal karyotype, one ptn (10%) had complex karyotype and one (10%) had monosomy 7. Six pts had FLT3 mutations and 4 pts CBL mutations. Pts characteristics are summarized in Table 1. Nine out of 10 pts were evaluable at the time of the analysis. Median number of cycles administered was 5 (range 2-10). All pts have achieved response so far with 8 pts (89%) having achieved marrow CR (mCR), including hematological improvement (HI) in 1 patient out of 3 pts with baseline cytopenias evaluable for HI. One patient had hematologic improvement-erythropoietic (HI-E) with control of leukocytosis and thrombocytosis to normal levels. During cycle 1, responses were associated with recovery of platelets to above 50 and 100x109/L by day 28 of therapy in 67% and 56% of pts, respectively. In addition, absolute neutrophils count (ANC) was recovered to 1 x109/L by day 28 in all pts except of one patient at day 52. With a median follow up of 9.87 months (range 2.5-18), the median response duration is 6.3 months (range 1.6-14.3) with a median OS of 17.34 months (range 2.5 – 18). A total of 4 pts (44%) have stopped the treatment: 1 patient due to disease relapse and one due to disease progression after both received 6 cycles of treatment and two proceeded to allogenic stem cell transplantation after achieving response. No dose-limiting toxicities have been observed to date up to the maximum dose of 60 mg of quizartinib. Two pts required dose reductions of Quizartinib, one due to cytopenias and the other one due to concomitant posaconazole administration. Regarding the grade 3 or more adverse events (AEs): 3 pts (33%) had severe anemia/thrombocytopenia, 2 pts (22%) had skin infection, one patient (11%) had cardiac arrythmia with Mobitz II AV block and one patient (11%). Image: Summary/Conclusion: Preliminary data suggest azacitidine in combination with quizartinib for pts with MDS and MDS/MPN with FLT3 or CBL mutations have an acceptable toxicity profile and associated with promising responses. Further follow-up with a larger patient cohort are required to emphasize the safety and efficacy. P755: HYPERFERRITINEMIA IS A PREDICTIVE BIOMARKER OF POOR CLINICAL OUTCOMES IN CMML L. E. Aguirre1,*, S. Ball1, A. Jain1, N. Al Ali1, D. Sallman1, A. Kuykendall1, K. Sweet1, J. Lancet1, E. Padron1, R. Komrokji1 1Malignant Hematology, Moffitt Cancer Center, Tampa, United States of America Background: CMML is a heterogenous disease exhibiting features innate to MPN and MDS. Increasing evidence supports a close interplay between systemic inflammation and risk of myeloid malignancies, notably for those with history of infection or autoimmune disease. CMML has been associated with inflammation and end-organ damage related to CKD and CVD. Analysis of gene signatures from CMML-derived monocytes has shown them to be highly proinflammatory. High ferritin may serve as a practical biomarker of disease activity to help identify pts at higher risk of poor outcomes. Aims: We aimed to identify whether hyperferritinemia correlated with worse outcomes in CMML and to characterize relevant baseline clinical and molecular features associated with this clinical phenotype. Methods: Retrospective data was collected from a database of pts with CMML treated at Moffitt Cancer Center. Pts were stratified in 2 cohorts based on ferritin levels (<1000 or ≥1000 ng/mL). Hyperferritinemia was defined as ferritin >1000 as seen at diagnosis or during follow-up. Kaplan–Meier was used to estimate OS. Cox regression was used for multivariate analysis. Results: Between August 1995 and October 2020 729 pts with CMML were identified. Median age at diagnosis was 71 (17-95). Out of 571 pts with available ferritin levels 29% (n=168) developed hyperferritinemia vs 71% (n=403) who did not. mOS was 32.4 mos (95%CI 30-35 mos). Pts with higher ferritin tended to present with CMML-2 (p=0.001) and harbor a proliferative phenotype (p=0.01). They presented with higher marrow cellularity (mean 83%, p =0.08), PLT (mean 177k, p= 0.038), and lower Hb (mean 9.5, p<0.05). There was no association with % circulating IMC, monocytes, WBC or ANC at baseline. Hyperferritinemia was associated with more profound fibrosis (p=0.007), cytopenias (p<0.05), % peripheral blasts (p<0.05), RBC and PLT transfusion dependence (p<0.05). Pts with hyperferritinemia had higher risk disease per IPSS-R, CPSS and all CMML models (p<0.05); and had higher rates of AML transformation (p<0.05). Pts were also more likely to require treatment earlier (within 3 yrs of diagnosis) (p<0.05). ASXL1 (p=0.002), EZH2 (p=0.003), and SETBP1 (p=0.019) mutations were more common among pts who developed hyperferritinemia. Conversely, TET2 (p=0.001), CBL (p=0.028) and SRSF2 (p=0.003) mutations were less common. mOS for pts with hyperferritinemia was 23.9 mos (95%CI 19.9-27.9 mos), much lower than for those with ferritin <1000 (mOS 40.5 mos, 95%CI 35.4-45.5 mos) (p<0.05). In multivariate analysis, hyperferritinemia was a significant independent covariate for OS after adjusting for CPSS, transfusion dependence and disease phenotype (dysplastic vs proliferative) (HR= 0.69; 95%CI 0.53-0.89; p=0.005). Summary/Conclusion: Almost 1/3 of pts with CMML will develop hyperferritinemia. This is associated with more aggressive disease and rates of AML transformation leading to dismal outcomes. ASXL1, EZH2, and SETBP1 MTs confer a higher risk of hyperferritinemia. Our findings indicate that hyperferritinemia is an independent prognostic biomarker that may serve as a surrogate representative of disease biology and comorbidities in CMML. P756: ALLOGENEIC STEM CELL TRANSPLANTATION IN MYELODYSPLASTIC SYNDROME/MYELOPROLIFERATIVE NEOPLASM (MDS/MPN) OVERLAP SYNDROMES. A SINGLE CENTER EXPERIENCE AND DISSECTION OF MUTATIONAL PROFILE A. Avendaño Pita1,*, M. Martín Izquierdo1, S. Muntión Olave1, T. Jímenez Solas1, M. García Antúnez1, L. Eva1, M. Del Rey1, S. Toribio Castelló1, A. Yeguas Bermejo1, T. González1, J. M. Hernández-Rivas1, A. Á. Martín López1, M. Cabrero1, E. Pérez López1, A. Cabero1, M. Baile1, L. Vázquez1, L. López Corral1, F. Sánchez-Guijo1, D. Caballero Barrigon1, M. Cortés Rodríguez1, M. Díez Campelo1 1Hematology, University Hospital of Salamanca / IBSAL, Salamanca, Spain Background: MDS/MPN (myelodysplastic syndrome/myeloproliferative neoplasm) overlap syndromes are myeloid malignancies for which allogeneic hematopoietic stem cell transplant (allo-HSCT) is potentially curative in the absence of disease-modifying therapies. Aims: To describe clinical, biological and molecular characteristics at diagnosis and transplant outcomes of our series trying to detect witch parameters may have impact on survival. Methods: We retrospectively reviewed 35 consecutive patients who underwent to allo-HSCT from 1999 to 2021 as MDS/MPNS overlap syndrome. Descriptive statistics and survival analysis were performed trough jamovi (Version 2.2.). Results: Clinical, biological and molecular baseline characteristics are summarized in image. Median age at allo-HSCT were 58 (27-70) with a median time from diagnosis to allo-HSCT of 8 months (1-128). Twenty patients (57%) were referred in complete remission (CR). Preferred graft source were peripheral blood in 34 patients (97.1%). Regarding to conditioning regimen, 26 patients (74.3%) received reduced intensity, 7 patients (20%) received myeloablative and 2 patients (5.7%) sequential conditioning. Eighteen patients (51.4%) had matched sibling donor, 8 patients (22.8%) had matched unrelated donor, 3 patients (8.6%) mismatched unrelated donor and 6 (17.1%) haploidentical donor. All patients reached neutrophil graft and 31 patients (88.6%) reach platelet graft with a median time of 19 (12-29) and 14 (10-36) days respectively. On day +100, 29 patients (82.9%) were in CR, 4 patients (11.4%) did not reach response and 2 (5.7%) were not evaluable. Twenty three patients (65.7%) developed acute graft versus host disease (GVHD), in 19 cases (54.3%) grades II-IV, and 15 patients (42.9%) developed chronic GVHD, in 8 cases (22.9%) moderate-severe grade. Median follow-up were 13 months (2-153) with 17 months (2-153) for chronic myelomonocitic leukemia (CMML) and 11.5 months (2-133) for atypical chronic. myeloid leukemia (aCML). Overall survival (OS) at 1, 3, and 5 years were 69.7%, 54.9% and 42.5% respectively. Sixteen patients (45.7%) relapsed with a median time of 7.5 months (0-85). Among relapsed patients, 5 patients (31.3%) were not treated, 3 patients (19.8%) received donor lymphocyte infusion, in 5 patients (31.3%) we administered hypometilating agents and salvage chemotherapy in 2 (12.5%) remaining patients reaching second allo-HSCT in two cases. Non-relapse mortality were 31.4%, in most cases due to infection. Transplant related mortality (TRM) at day +100 and global TRM were 5.7% and 14.3% respectively. At last follow-up, 13 patients (37.1%) remain alive, 11 (31.4%) in CR and 2 (5.7%) in relapsed situation. On the univariate analysis, harboring signal transduction mutation (p = 0.008), mutational global burden by variant allelic frequency (VAF) at diagnosis greater than 30% (p = 0.045) were asociated with worse outcomes. On the other hand, reaching CR at day +100 (p <0.0001) and developing chronic GVHD (p = 0.0001) seems to improve survival. If we look at the CMML subgroup, we found that new CMML transplant score significantly stratifies outcomes (p = 0.006). Image: Summary/Conclusion: Once again, poor results are confirmed for this MDS/MPN overlap syndromes with only a third of the patients being long term survivors. Molecular data offered by NGS may help to refine prognostic scores and to stablish transplant referral. Mutations in signaling pathways seems to confer poor prognosis as a late event in disease course and provide more aggressive clinical behavior. P757: RESULTS OF A PHASE 1 STUDY OF AZACITIDINE COMBINED WITH VENETOCLAX FOR TREATMENT-NAIVE AND RELAPSED HIGH-RISK MYELODYSPLASTIC SYNDROME AND CHRONIC MYELOMONOCYTIC LEUKEMIA A. Bazinet1,*, E. Jabbour1, H. Kantarjian1, K. Chien1, C. DiNardo1, M. Ohanian1, N. Daver1, R. Kanagal-Shamanna2, T. Kadia1, K. Takahashi1, L. Masarova1, N. Short1, Y. Alvarado1, P. Thompson1, G. Montalban-Bravo1, M. Yilmaz1, F. Ravandi1, M. Konopleva1, M. Andreeff1, S. Kornblau1, N. Pemmaraju1, D. Hammond1, H. Schneider1, B. Mirabella1, G. Garcia-Manero1 1Leukemia; 2Hematopathology, University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Patients (pts) with higher-risk myelodysplastic syndromes (HR-MDS) who are ineligible for allogeneic stem cell transplant (SCT) have limited treatment options beyond the hypomethylating agents (HMAs) azacitidine (AZA) and decitabine (DAC). Responses to HMA monotherapy are time-limited and prognosis after HMA-failure is dismal. Venetoclax (VEN) is an oral small molecule BCL-2 inhibitor with the potential to prolong/deepen responses when combined with an HMA. Aims: To evaluate the safety/tolerability, recommended phase 2 dose (RP2D), and preliminary efficacy of VEN in combination with AZA for the treatment of HR-MDS/CMML. Methods: This was a single-center dose-escalation phase 1 study with “3 + 3” design. Key inclusion criteria were a diagnosis of MDS or CMML by WHO 2016, age ≥ 18 yrs, int-2 or high risk by IPSS, and bone marrow (BM) blasts of 5-19%. Pts could be treatment-naïve or relapsed/refractory, defined as failure after 4+ cycles of HMA. Prior VEN exposure was not permitted. Treatment consisted of AZA 75 mg/m2 on D1-5 and escalating doses of VEN on D1-7 or 1-14 (Table 1) every 28D. VEN dosing was adjusted for pts on concomitant CYP3A inhibitors. The primary objective was safety/tolerability and determination of the RP2D. Secondary objectives were response rates, overall survival (OS), and progression-free survival (PFS). Adverse events (AEs) were graded as per the CTCAE v5.0 and responses assessed using the modified IWG 2006 criteria. All pts provided informed consent. The study was registered on ClinicalTrials.gov (NCT04160052). Results: Data cutoff was February 19th, 2022. 23 pts (13 HMA-naïve MDS, 4 HMA-naïve CMML, 4 HMA-failure MDS, 2 HMA-failure CMML) were enrolled. The median age was 68 years (range 58-84) and median BM blasts 11% (range 6-19%). IPSS was int-2 in 78% of pts and high in 22% of pts. 8/23 (35%) had complex cytogenetics, 6/23 (26%) had TP53 mutations, and 5/23 (22%) had therapy-related MDS/CMML. A single dose-limiting toxicity (DLT) occurred at dose level +2 (BM aplasia and delayed count recovery). The maximum tolerated dose (MTD) was not reached. The most common grade 3/4 AEs were neutropenia (39%), thrombocytopenia (39%), anemia (13%), and infections: pneumonia (30%), neutropenic fever (17%), diverticulitis (9%), sepsis (9%), cellulitis (4%), and splenic abscess (4%). The RP2D was established at AZA 75 mg/m2 on D1-5 plus VEN 400 mg on D1-14. 3 deaths occurred on study, all from sepsis. 30 and 60-day mortality were 4% and 9%, respectively. Other reasons for discontinuing therapy included progression of MDS/CMML (n=6), transformation to AML (n=4), SCT (n=4), and social reasons (n=1). The ORR by intention-to-treat (ITT) analysis was 82% (14/17; 3 CR, 5 mCR+HI, 6 mCR) in the HMA-naïve cohort and 100% (6/6; 6 mCR) in the HMA-failure cohort. A median of 3 cycles (range 1-11) were given. Responses occurred after a median of 1 cycle (range 1-2) and lasted a median of 4.5m (range 1.2m to 21.2m). At a median follow-up of 13.2m, median OS was not reached (1-year OS 68.6%) in the HMA-naïve cohort and 8.3m (1-year OS 22%) in the HMA-failure cohort. Median PFS was 13.1m (1-year PFS 51.5%) in the HMA-naïve cohort and 6.8m (1-year PFS 0%) in the HMA-failure cohort. Image: Summary/Conclusion: AZA combined with VEN is tolerable and yields a high ORR in HR-MDS/CMML, with or without prior HMA. Rates of myelosuppression and infection are high and careful monitoring is required. The RP2D is AZA 75 mg/m2 on D1-5 plus VEN 400 mg on D1-14, every 28D. The phase 2 dose expansion component of this study is ongoing. P758: INHIBITION OF ATR WITH CERALASERTIB (AZD6738) FOR THE TREATMENT OF PROGRESSIVE OR RELAPSED MYELODYSPLASTIC SYNDROMES AND CHRONIC MYELOMONOCYTIC LEUKEMIA: A PHASE IB/II STUDY A. Brunner1,*, Y. Liu2, L. Mendez3, J. Garcia2, P. Amrein1, D. Neuberg2, E. Dean4, S. Smith4, R. Stone2, A. Fathi1, M. Walter5, T. Graubert1, M. Jacoby5 1Massachusetts General Hospital; 2Dana-Farber Cancer Institute; 3Beth-Israel Deaconess Medical Center, Boston, United States of America; 4AstraZeneca, Cambridge, United Kingdom; 5Washington University School of Medicine, St. Louis, United States of America Background: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are enriched for mutations in the spliceosome complex, including the genes SF3B1, SRSF2, ZRSR2, and U2AF1. Ceralasertib (AZD6738) is a selective and potent inhibitor of Ataxia Telangiectasia and Rad3 Related (ATR) kinase. We have previously shown that cells with splicing factor (SF) mutations have increased R loops, which require ATR for resolution and preferentially undergo apoptosis after ATR inhibition. Aims: We hypothesized that inhibition of ATR may be useful in patients with MDS or CMML, particularly those who harbor SF gene mutations. Methods: This 2-part study evaluated ceralasertib monotherapy in adult patients with MDS or CMML progressing on or not responsive to front-line therapy (DNMTI for HR MDS, ESA for LR MDS). Enrollment cohorts included SF mutant (SF3B1, SRSF2, U2AF1, or ZRSR2) and SF wildtype (wt). Part 1 enrolled 3-6 patients at de-escalating dose levels: 160mg BID days 1-14 of 28 d cycles, 120mg BID d1-14, and 80mg BID d1-14. DLTs were assessed the first 30d and de-escalation would occur for DLTs in >1/6 patients. Higher-risk MDS (HR-MDS, IPSS-R > 3.5) and CMML patients were enrolled first; after a dose was deemed safe, lower-risk MDS (LR-MDS, IPSS-R 3.5, transfusion-dependent post/ineligible for ESA or severe neutropenia/thrombocytopenia) was evaluated. Part 2 enrolled up to 20 SF mut patients and 20 SF wt patients for efficacy. A Simon 2-stage design mandated that at least 1/10 patients in each group needed to respond (by IWG 2006 criteria) to enroll the final 10 patients. Results: At the data cut-off (12/15/21) 32 patients have been enrolled with 30 evaluable: 22 with HR-MDS or CMML, and 8 with LR-MDS. The median age was 73 (range 43-88) and predominantly male (26/30). 21 patients harbored SF mut disease while 9 had SF wt MDS/CMML. The most frequently seen ≥grade 3 adverse events (AEs) for higher-risk patients (n=20) included anemia (n=6), febrile neutropenia (n=4), neutropenia (n=5), thrombocytopenia (n=6). In LR-MDS (n=8), ≥grade 3 AEs included thrombocytopenia (n=2), and neutropenia (n=2). 5 deaths occurred within 30d of study treatment; 1 patient with active CMML had an intracranial bleed with thrombocytopenia, possibly related to study treatment, while 4 deaths were attributed to underlying disease (3 with progression to AML, 1 MDS patient with sepsis). No DLTs were observed in the HR or LR groups and 160mg BID days 1-14 of 28 days was deemed the RP2D. Responses were assessed across the cohort as well as according to SF mutant status (Table). A total of 8 patients had an IWG response (BOR: CR, n=1; mCR, n=3; HI, n=4), including 6/21 SF mut patients (ORR 29%), and 2/9 SF wt patients (ORR 22%). The responses in SF wt patients included one mCR (5% to 1% marrow blasts in a patient with DNMT3A and ETV6 mut and del(chr7)), and one HI-P (82 BL to 234 max in a patient with DNMT3A, IDH2, ETV6 mut and del(chr7)). Responses in SF mut included CR (n=1), mCR (n=2, one with associated HI-N), SD with HI (n=3). An additional 3 SF mut patients had improvements in blood counts but not lasting 8 weeks (ANC n=2, platelets n=1) and one with platelet improvement but a baseline not meeting IWG criteria (plt avg=108k prior to C1D1). Image: Summary/Conclusion: Ceralasertib is an oral ATR inhibitor with preliminary activity in patients with progressive/refractory MDS and CMML at doses of 160mg BID on days 1-14 of a 28 day cycle. The study is ongoing and will evaluate additional dosing schedules. P759: CORRELATION ANALYSIS OF DNA METHYLATION LEVEL AND CLINICAL CHARACTERISTICS IN JMML PATIENTS Y. Cai1,*, J. Zhang1, M. Yi1, X. Liu1, X. Zhang1, Y. Wan1, L. Chang1, L. Zhang1, X. Chen1, Y. Guo1, Y. Zou1, Y. Chen1, Y. Zhang1, W. Yang1, X. Zhu1 1Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China Background: As a rare, aggressive pediatric myeloproliferative disease, juvenile myelomonocytic leukemia (JMML) encompassed both biological features of myelodysplastic syndrome and myeloproliferative neoplasm. Studies have shown that the methylation level in JMML patients is closely related to prognosis, and patients with high methylation level have poor prognosis. Aims: This study aimed to find clinical indicators that were associated with different methylation levels and prognosis. Methods: The clinical information of 24 JMML patients with DNA samples admitted to our center from December 2013 to May 2020 was retrospectively analyzed, and the DNA methylation level of their whole genome was detected. Results: The median age of onset was 14.5 months (0.1-153 months) among the 24 cases, including 17 males and 7 females. At diagnosis, the median WBC count was 27.1×109/L (6.2-98.1×109/L), and the median platelet count was 38×109/L (10-277×109/L). Chromosome karyotype abnormalities were found in 12.5% (3/24) of patients. Next-generation sequencing results showed that 79.2% (19/24) patients had at least one Ras pathway-related classical gene mutation, and 41.7% (10/24) patients had two or more somatic mutations. Genomic DNA methylation levels were divided into three groups: 11 cases in the hypomethylation group, 2 cases in the moderate methylation group, and 11 cases in the hypermethylation group. The concordance rate of our methylation subgroup compared with the international consensus classification was 91.7%. There were significant differences in age, platelets, PTPN11 gene mutation and the number of somatic mutations ≥2 in different methylation groups (P<0.05). Correlation analysis showed that hypermethylation level was significantly correlated with PTPN11 gene mutation and ≥2 somatic mutations (P<0.001). Image: Summary/Conclusion: JMML patients with high methylation level in the DNA genome at diagnosis were older and with lower platelet levels, and hypermethylation were significantly correlated with high-risk prognostic factors such as PTPN11 gene mutation and ≥2 somatic mutations. P760: PATTERNS OF HYPOMETHYLATING AGENT FAILURE IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES K. Chien1,*, K. Kim1, Z. Li2, R. Kanagal Shamanna3, F. Ong1, G. Montalban Bravo1, T. Kadia1, E. Jabbour1, N. Pemmaraju1, D. Hammond1, N. Short1, F. Ravandi1, Y. Alvarado1, S. Pierce1, X. Q. Dong1, H. Kantarjian1, G. Garcia-Manero1 1Leukemia; 2Biostatistics; 3Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Hypomethylating agents (HMA), such as azacitidine and decitabine, are the current standard-of-care for patients (pts) with myelodysplastic syndrome (MDS). Though HMA improve cytopenias and delay progression of disease, most pts eventually response to these agents with poor survival outcomes afterward. This poses significant treatment challenges after HMA failure (HMA-F) and an increased risk of transformation to acute myeloid leukemia (AML). Aims: Here, we assess the clinicopathologic characteristics and outcomes of pts with HMA-F MDS. Methods: We retrospectively evaluated all pts with MDS seen at a single tertiary cancer center from July 2017 to July 2021 and identified those who were previously untreated and later developed HMA-F. Pt characteristics, laboratory values, and bone marrow (BM) data, including cytogenetics and next generation sequencing (NGS), were assessed at both diagnosis and the time of HMA-F. Genomic DNA was extracted from whole BM aspirate samples and subject to 28- (n=12) or 81-gene (n=131) target PCR-based sequencing using a NGS platform at diagnosis and an 81-gene NGS panel (n=85) at the time of HMA-F. Survival data was updated in January 2022. Results: Out of 799 untreated MDS pts, 147 pts (32%) developed HMA-F with a median follow-up time of 44.0 months (mo) from diagnosis. Baseline pt characteristics and subsequent treatment are summarized in Table 1. The median age was 71 with therapy-related MDS (t-MDS) in 37%. At diagnosis, most pts had higher-risk MDS with IPSS-R high (29%) or very high (39%) disease, and complex karyotype was seen in 44%. The median number of mutations identified was 2 with TP53 (44%) and ASXL1 (21%) the most frequently discovered mutations. Regarding HMA therapy, 67% received HMA monotherapy and 33% received HMA in combination with various agents. The median number of cycles of HMAs received was 7 (range: 1-37). At the time of HMA-F, pts presented with higher BM blasts (6% vs 10%, p<0.001) and lower platelet counts (63 K/µL vs 36 K/µL, p<0.001) than at diagnosis. The median number of cytogenetic abnormalities increased from 1 to 2 with no change in the median number of mutations from diagnosis to HMA-F, but both had significant increases by paired t-test (p<0.001). The median time from diagnosis to HMA-F was 10.8 mo, and the median overall survival (mOS) from HMA-F was 6.8 mo (95% CI: 5.4, 9.0). Survival was well-stratified by both IPSS-R at diagnosis (14.6 mo in lower-risk MDS vs 5.4 mo in higher-risk MDS, p=0.001) and at HMA-F (11.4 mo in lower-risk MDS vs 3.5 mo in higher-risk MDS, p<0.001). TP53 mutations at diagnosis (HR 3.02, p<0.001) and t-MDS (HR 2.50, p<0.001) were associated with shorter mOS. In pts with higher-risk MDS, initial response to HMA therapy (7.2 mo in responders vs 2.7 mo in non-responders, p=0.011) and stem cell transplantation at HMA-F (4.7 mo in non-transplanted vs not reached in transplanted, p<0.001) were associated with improved survival. Progression to acute myeloid leukemia (AML) was seen in 72 pts (49%), and the mOS from the time of AML diagnosis was 3.4 mo. Image: Summary/Conclusion: Pts with HMA-F MDS have high-risk features, including poor cytogenetics and adverse mutations, and are often therapy-related. At the time of HMA-F, patients present with higher BM blasts, worsening thrombocytopenia, and more cytogenetic abnormalities and mutations. Unfortunately, after failure of HMA, pts have frequent transformation to AML and dismal overall survival. Further understanding of the underlying biology of HMA-F MDS is warranted with an urgent need for therapeutic interventions after failure of HMA therapy. P761: MONOCYTOSIS IN PRIMARY CARE AND HEMATOLOGICAL MALIGNANCIES M. Christensen1,2,*, V. Siersma2, M. Kriegbaum2, B. Lind3, J. Samuelsson4, K. Grønbæk1, L. Granfeldt5, C. L. Andersen1 1Department of Hematology, Rigshospitalet; 2Institute for Public Health, University of Copenhagen, Copenhagen; 3Department of Clinical Biochemistry, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark; 4Department of Hematology, Universitetssjükhuset, Lindköping, Sweden; 5Department of Hematology, Odense University Hospital, Odense, Denmark Background: Monocytosis (blood monocyte count > 0.8 x109/L) is relatively often observed in routine blood work from primary care, however, the significance is not always apparent as monocytosis may be present in a spectrum of diseases spanning from mild infections to chronic myelomonocytic leukemia. Aims: We aimed to examine the predictive value of monocytosis in primary care by relating monocyte count to subsequent 3-year incidence of hematological malignancy. Methods: We included 663.184 adult patients from primary care in the greater Copenhagen Area who had a complete blood cell count (CBC) performed at any time between 2000-2016. For patients with multiple samples registered, a random sample was selected and samples predating the index CBC by 3 months were assessed to account for sustained monocytosis. Using the extensive Danish health data registers, we collected data on incident hematological malignancy for 3 years following the CBC. We modelled the association between monocyte count and hematological malignancies as well as all-cause mortality using multiple logistic regression analysis. Results: Monocytosis was observed in 4,6 % of CBCs. We saw an increased risk of myeloid malignancies with the most noticeable being an odds ratio (OR) for CMML > 100 with monocytosis > 1.0 x 109/L. Sustained monocytosis increased this risk to OR >140. The incidence was, however, still low with only 0.1% of patients with monocytosis developing CMML within 3 years. Risk of lymphoproliferative and M-component related diseases were also increased, however, other relevant cell line were affected as well in these cases. Summary/Conclusion: Monocytosis is a common finding in primary care, but even with monocytosis > 1 x 109/L hematological malignancies are rare. Optimized referral to secondary care remains a challenge and if the clinical presentation leaves doubt of the diagnosis, repeated measurements help demask if malignancy is the cause of the monocytosis. P762: NONLINIEAR IMPACT OF CLINICAL FEATURES ON THE SURVIVAL OUTCOME OF MYELODYSPLASTIC SYNDROMES PATIENTS F. Darbaniyan1,*, G. Montalban Bravo2, K. S. Chien2, R. Kanagal Shamanna3, K. sasaki2, H. Yang2, K. A. Soltysiak2, Z. Li1, K.-A. Do1, G. Garcia-Manero2 1Biostatistics; 2Leukemia; 3hematopathology, MD Anderson Cancer Center, Houston, United States of America Background: Myelodysplastic syndromes (MDS) are a group of hematopoietic stem-cell disorders with heterogenous prognosis. The revised international prognostic scoring system (IPSS-R) (Greenberg et al., Blood, 2012) is the most widely used score for prognostication. However, the allocation of IPSS-R risk categories remains imprecise for some patients leading to unsatisfactory therapy decisions. Newer molecular prognostic models (Nazha et al., JCO, 2021 and Bernard et al., ASH, 2021) have added mutation data to improve survival predictions for MDS patients. Aims: Our aims were to investigate the nonlinear association of normalized clinical data in combination with molecular and cytogenetic data and to improve the prognostication potential of IPSS-R for patients with MDS. Methods: A multivariable fractional polynomials (MFP) algorithm was applied on clinical data of 943 MDS patients and nonlinear associations between continuous covariates and survival outcomes were efficiently modeled within Cox regression. Selected transformed variables were picked and the potential overfit was avoided by a closed test procedure for function selection (Royston et al., John Wiley & Sons, 2008). The performance of the model was evaluated in an independent set of 436 MDS patients and the accuracy was assessed using a concordance (c)index and the area under the Receiver Operating Characteristic curve (AUC) at each time point. Mutation data was obtained in a subset of 695 patients using next-generation sequencing, evaluating a panel of 81 genes at the time of diagnosis. Results: An MFP-model consisting of cytogenetics per IPSS-R and WHO subtypes, continuous forms of age and hemoglobin, and non-linear transformations of platelets and bone marrow blast percentage yielded a c-index performance of 0.82 vs 0.78 for IPSS-R and a significant AUC upgrade at all time points in the training set (Figure a). In comparison with the IPSS-R, absolute neutrophil count was not identified as an independent prognostic covariate and common features were treated in a non-linear fashion. Validation of the model in an independent set of 438 patients yielded a higher c-index (0.72 vs 0.68) with an improved AUC over time compared to the IPSS-R (Figure b). Based on MFP risk score, patients were then clustered into 5 different risk categories and compared with IPSS-R classifications. Our data showed no significant difference in time to death between IPSS-R groups for very low risk and low risk or between low risk and intermediate risk (Figure c); however, MFP classification showed 5 significantly different subgroups in predicting time to death for MDS patients (Figure d). We further investigated the effect of molecular data on disease prognostics in a subset of 695 patients sequenced using an 81 gene panel. In addition to the selected clinical covariates, occurrence of TP53, SRSF2, and ZRSR2 mutations were independently associated with lower survival, while the presence of TET2 mutation showed a depression effect on TP53 mutation towards a better outcome. As SF3B1 was not selected in the final model, we further evaluated its potential interactions and identified its correlation with cytogenetics and platelets, which predominated in the MFP model. This might be due to heterogeneity within the data set (e.g., 25% co-occurrence of SF3B1 with TP53 mutation) and needs to be investigated in larger cohorts. Image: Summary/Conclusion: We have established a nonlinear model that yields improved risk assessment compared to IPSS-R criteria. P763: VENETOCLAX WITH AZACITIDINE IN RELAPSE/REFRACTORY HIGHER RISK MYELODYSPLASTIC SYNDROME: UPDATED PHASE 1 RESULTS S. P. Desikan1,*, G. Montalban-Bravo1, M. Ohanian1, N. Daver1, T. Kadia1, S. Venugopal1, K. Chien1, H. Kantarjian1, G. Garcia-Manero1 1Leukemia, MD Anderson, houston, United States of America Background: Patients with higher risk (HR) MDS status post HMA failure have poor survival, 4-6 months. [Lancet Oncology Apr 2016] The combination of azacitidine(Aza) and venetoclax(Ven) has produced encouraging results in the frontline settings for patients with HR disease.(Garcia et.al ASH 2019) Aims: The primary objective is to establish the safety of azacitidine and venetoclax in HR MDS after HMA failure. The secondary objectives include rates of CR, marrow response, transfusion independence, duration of response, and overall survival. Methods: In this phase 1 study, patients 18 years and older with HMA failure (relapse or progression after 4 cycles), adequate organ function and performance status are being enrolled. Patients who had received prior Ven were excluded. All patients received Aza 75mg/m2 on D1-5. A 3 + 3 design was utilized with Ven. A 3 + 3 design was utilized with Ven with dose levels 0, 1, 2 receiving 100mg, 200mg, and 400mg respectively. Patients are currently being enrolled in the expansion cohort(400mg). Response was determined based on IWG06 criteria. Results: Baseline characteristics are in Table 1. Fifteen patients have been enrolled with a median age of 78[67 – 81]. The median blast% on enrollment was 9%[6 – 17] with a median ANC, Hgb, and PLT of 1.67 K/µL, 7.7mg/dL, and 39 K/µL. When stratified based on cytogenetics, 1 (7%) had good risk, 7 (47%) patients had intermediate risk, 2 (13%) had poor risk, and 5(33%) had very poor risk. When stratified based on IPSS-R criteria: 2(13%) had intermediate risk, 5(33%) had high risk, and 8(54%) had very high risk disease. ASXL1 and TP53 mutations were enriched, occurring in 46% and 53% of patients. Four(33%) patients had Grade ¾ cytopenias (either thrombocytopenia or neutropenia) relating to this combination. Three patients required dose reductions. No tumor lysis syndrome has occurred to date. Two patients recently started treatment and could not be included for response assessment. Among the 13 patients evaluable, the ORR is 62%(n=8). The median number of cycles to response was 1[1-4]. Responses consisted of 1 CR, 4 marrow responses with either platelet or neutrophil recovery, and 3 marrow responses without count recovery. Three patients achieved transfusion independence. Five patients had NR with 4 having TP53 mutations. The median OS was 8.5 months with a 30 day and 60 day mortality of 8% and 23% respectively. Early mortality was related to infection in 2 patients and progression in another. Image: Summary/Conclusion: With a response rate of 62% and an OS of 8.5 months, this combination may have benefit in HR RR MDS. TP53 mutations and higher cytogenetic risk confer poor prognosis. P764: RESPONSE AND SURVIVAL OUTCOMES WITH HYPOMETHYLATING AGENTS IN AN ARGENTINEAN COHORT OF 113 PATIENTS WITH CHRONIC MYELOMONOCITIC LEUKEMIA J. Gonzalez1,*, A. Perusini2, F. Russo3, L. Fuentes4, T. Deluca5, A. L. Basquiera6, A. Navickas7, L. Kornblihtt8, N. Pintos9, E. Nucifora2, M. Iastrebner10, A. Enrico5, J. Arbelbide2, C. Belli11 1Hematology, Hospital Agudos Carlos G Durand; 2Hematology, Hospital Italiano de Buenos Aires; 3Hematology, Hospital Paroissien; 4Hematology, Instituto Alexander Femming, CABA; 5Hematology, Hospital Italiano de La Plata, La Plata; 6Hematology, Hospital Privado de Cordoba, Cordoba; 7Hematology, Hospital El Cruce, Florencio Varela; 8Hematology, Hospital de Clinicas Jose de San Martin; 9Hematology, Sanatorio Mendez; 10Hematology, Sanatorio Sagrado Corazon; 11Genetic, IMEX-CONICET/ANM, CABA, Argentina Background: Hypomethylating agents (HMA) are the first line option for high-risk patient and lower-risk patient with transfusion dependence, or according to worsening clinical features. Data regarding HMA efficacy in CMML has been largely retrospective, or from MDS studies that included CMML patients, with overall response rates (ORR) ranging from 40% to 50% and true CR rates being <20%. Local data is scarce. Aims: To examine the influence of prognostic factors at diagnosis and during the follow-up in the outcome and response to HMA therapy in a CMML cohort from Argentine. Methods: We performed a retrospective analysis of 113 CMML patients from the Argentine Registry of MDS promoted by the Argentine Society of Hematology who were treated between January-07/February-22. Statistical analysis included Kaplan-Meier survival analysis, Cox proportional hazard and Chi2/Fisher’s exact test. Results: The median age at diagnosis was 67 years (IQR 58-74) being 71.7% >60 years old, 18.8% a Charlson’s Index (CCI)>2, 81 (72%) were males, and 59 (52%) CMML-0, 25 (22%) CMML-1 and 29 (26%) CMML-2. At treatment initiation, 70.5% showed hemoglobin <10g/dL, 22.1% platelet counts <30,000/µL, 13.1% poor karyotypes and 91.2% at high risk according to the CPSS, IPSS, IPSS-R or Bournemouth scoring systems. During the follow-up, median 20 months (IQR 8.6-42.7), 45.1% evolve to AML and 75.2% died. Regarding HMA therapy, the median time to treat was 2.4 months, 78.8% patients received AZA and 22.2% DAC, the median number of cycles of 7 (IQR 4-12) during a median period of 8.1 months (m). The median overall survival (OS) of the cohort was 25.5m (95%CI 18.9-32.1), since treatment initiation 17.1m (95%CI 13.3-20.8), and after cessation 5.3m (95%CI 4.3-6.3). Most of parameters and scoring systems analyzed were useful to predict outcome from diagnosis or from treatment initiation to last follow up. Cox regression analysis revealed that CCI>2 (HR 2.7, 95%CI 1.5-5.0, p=0.001), WHO classification (ref. CMML-0, CMML-1, HR 2.1, 95%CI 1.1-4.1, p=0.026; CMML-2, HR 3.4, 95%CI 1.6-6.8, p=0.001), lower hemoglobin level (<10g/dL, HR 1.9, 95%CI 1.1-3.5, p=0.023), platelet count <30.000/µL (HR 3.5, 95%CI 2.0-6.0, p<0.001), time to treat <6m (HR 4.7, 95%CI 2.4-9.1, p<0.001) and the presence of blast in PB (HR 1.7, 95%CI 1.0-2.8, p=0.05) and were independently associated with a reduced OS. Almost all parameters, with the exception of lower Hb levels, sustained their independency since treatment initiation. A total of 99 patients were evaluated for response to treatment with an overall response rate (at 4-6 cycles) of 59.6% (CR/mCR/PR: 36.4%, HI: 23.2%, SD: 14.1%). The median overall survival of responders was 44.8m, similar to those with SD (33.2m, p=0.246), afterward grouped, with 8.3m in non-responders (p<0.001). We also evaluated whether prognostic factors were useful to predict response. Only the WHO 2016 proposal (CMML-0 61.6% vs 34.6%, CMML-1 21.9% vs 30.8%, CMML-2 16.4 vs 34.6%, p=0.046) and the absence of peripheral blasts (61.1% vs 36.0%, p=0.037) were associated with statistically different rates of response, with a tendency to treat later ≥6m (37.0% vs 15.4%, p=0.05). Summary/Conclusion: The present series represents the first experience in Latin America evaluating HMA agents in CMML patients. Our results highlight the adverse impact of several parameters, including the severity of the thrombocytopenia <30,000/µL on the outcome of CMML patients under HMA. However, clinical parameters are limited to predict the response rate. P765: UPDATED ENROLLMENT AND RESULTS FROM THE PHASE 1 SUBSTUDY OF IVOSIDENIB IN PATIENTS WITH IDH1-MUTANT RELAPSED/REFRACTORY MYELODYSPLASTIC SYNDROME (R/R MDS) C. D. DiNardo1,*, J. M. Foran2, J. M. Watts3, E. M. Stein4, S. de Botton5, A. T. Fathi6, G. T. Prince7, R. M. Stone8, P. A. Patel9, G. J. Roboz10, M. L. Arellano11, H. P. Erba12, A. Pigneux13, P. Baratam14, R. K. Stuart14, X. Thomas15, I. R. Lemieux16, X. Bai16, S. M. Kapsalis16, G. Garcia-Manero1, D. A. Sallman17 1MD Anderson Cancer Center, University of Texas, Houston; 2Mayo Clinic, Jacksonville; 3Sylvester Comprehensive Cancer Center, University of Miami, Miami; 4Memorial Sloan Kettering Cancer Center, New York, United States of America; 5Institut Gustave Roussy, Villejuif, France; 6Massachusetts General Hospital, Harvard Medical School, Boston; 7Johns Hopkins Hospital, Baltimore; 8Dana-Farber Cancer Institute, Boston; 9University of Texas Southwestern Medical Center, Dallas; 10Weill Cornell Medicine and The New York Presbyterian Hospital, New York; 11Winship Cancer Institute, Emory University School of Medicine, Atlanta; 12Duke University, Durham, United States of America; 13Centre Hospitalier Universitaire de Bordeaux Haut-Lévêque, Pessac, France; 14Medical University of South Carolina, Charleston, United States of America; 15Centre Hospitalier Universitaire de Lyon-Sud, Lyon, France; 16Servier Pharmaceuticals LLC, Boston; 17Moffitt Cancer Center, Tampa, United States of America Background: Isocitrate dehydrogenase 1 (IDH1) is mutated in ~3% of patients with MDS, increasing the risk of transformation to acute myeloid leukemia (AML). Ivosidenib (IVO), an oral, potent, targeted inhibitor of the mutant IDH1 (mIDH1) enzyme, is FDA approved for mIDH1 R/R AML and mIDH1 newly diagnosed AML in patients ≥75 years old or with comorbidities precluding the use of intensive induction chemotherapy. Promising safety and efficacy findings in the first-in-human, open-label, dose escalation and expansion study of IVO in patients with mIDH1 advanced hematologic malignancies (NCT02074839) led to the FDA granting Breakthrough Therapy designation to IVO in mIDH1 MDS. 12 patients with R/R MDS received IVO 500 mg once daily (QD). Median age was 72.5 (range 52–78) years and all had received prior MDS treatment. The investigator-assessed overall response rate (ORR) was 75%, with median response duration of 21.4 months. This substudy enrolled additional patients with mIDH1 R/R MDS; therefore, we report updated results. Aims: To further evaluate safety, tolerability, clinical activity, and pharmacokinetics/pharmacodynamics of IVO in patients with mIDH1 R/R MDS. Methods: This substudy of the single-arm study of IVO evaluated patients with mIDH1 R/R MDS after documented failure or relapse following prior standard therapy including intensive chemotherapy and hypomethylating agents. Patients must have given informed consent, have high disease burden based on IPSS or IPSS-R risk at baseline, and an Eastern Cooperative Oncology Group performance status score of 0–2. IVO 500 mg QD was given orally on days 1–28 of 28-day cycles. Results: As of 08May2021, 16 patients with R/R MDS were enrolled: 3 of the 16 who received 500 mg IVO QD were women; median age was 73.5 (range 52–78) years and 44% were ≥75 years of age. As of the data cut, 5 (31%) patients remained on treatment and free from leukemic transformation and 11 (69%) had discontinued including 6 for disease progression, 1 for allogeneic stem cell transplantation, and 1 owing to an adverse event (AE) of sepsis (the only fatal AE; reported by investigator as not related to IVO). Differentiation syndrome was observed in 2 patients (grade 2). Electrocardiogram QT prolonged was reported in 2 patients (grade 1 and 2). The Table outlines AEs. Complete response (CR) was achieved by 7 of 16 patients (44%; 95% CI, 20%, 70%), partial response (PR) by 1 (6%), and marrow CR by 5 (31%), giving an ORR of 81% (95% CI, 54%, 96%). At 12 months, the Kaplan-Meier estimate of duration of CR+PR was 60.0%. 3 patients experienced CRs lasting 24.0, 63.7, and 65.4 months, which remain ongoing. 11 of 16 (69%) patients achieved hematologic improvement in ≥1 lineage. Among 7 patients who were transfusion dependent at baseline, 5 (71%) became independent of red blood cell or platelet transfusions for 56 or more consecutive days on treatment. Further translational data are being processed. Image: Summary/Conclusion: IVO monotherapy induced durable remissions and transfusion independence in patients with mIDH1 R/R MDS, with a manageable safety profile. These results contribute to IVO’s potential as an effective, oral, targeted treatment for patients with mIDH1 R/R MDS. P766: CLINICAL OUTCOMES IN PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROMES RECEIVING HYPOMETHYLATING AGENTS: A LARGE POPULATION-BASED ANALYSIS A. M. Zeidan1,*, E. S. Mearns2, C. Ng2, A. Shah2, N. Lamarre3, A. Yellow-Duke2, N. Alrawashdh2, B. Yang4, W. Cheng5, C. N. Bui5, A. Svensson5 1Yale Cancer Center, Yale University School of Medicine, New Haven; 2Genentech, Inc., South San Francisco; 3Real World Data Analytics, Genesis Research, Hoboken; 4Roche Diagnostics, Santa Clara; 5AbbVie, North Chicago, United States of America Background: Myelodysplastic syndromes (MDS) are a spectrum of hematopoietic neoplasms characterized by variable degrees of cytopenias and risk of progression to acute myeloid leukemia (AML). Although the hypomethylating agents (HMA) azacitidine (AZA) and decitabine (DEC) are widely used to treat higher-risk (HR)-MDS in the United States (US), questions remain about their longer-term clinical benefits. Aims: To understand the survival outcomes and progression-related events of patients (pts) treated with AZA or DEC for HR-MDS in the US. Methods: Older adults (≥66 years old) diagnosed with refractory anemia with excess blasts (RAEB), an established proxy for HR-MDS, between January 2009 and December 2017, were included from the Surveillance, Epidemiology, and End Results (SEER)-Medicare linked database. The index date was date of HMA initiation. Pts who received AZA or DEC, had ≥12 months (mo) of continuous enrollment prior to the index date, and who did not receive a stem cell transplant (SCT) prior to the index date were included. Overall survival (OS) from the index date to death was estimated using Kaplan–Meier methodology. Baseline platelet and red blood cell (RBC) transfusion dependence (TD) were defined as ≥2 transfusion episodes in the 8 weeks prior to the index date; baseline transfusion use (TU) was defined as one transfusion episode in the 8 weeks prior to the index date. A competing risk analysis was used to assess the incidence of progression to AML, with death as the competing event. Results: Of 973 pts with HR-MDS treated with HMA, 75.8% received AZA and 24.2% received DEC. Median time from diagnosis to HMA initiation was 1.2 mo (interquartile range [IQR]: 0.6–2.6). Median treatment duration was 5.7 mo (IQR: 2.3–11.5) overall (5.8 mo [IQR: 2.6–11.8] for AZA and 5.0 mo [IQR: 1.9–11.0] for DEC). At baseline, 13.8% of pts had platelet TD/TU, 29.9% had RBC-TD and 27.0% had RBC-TU. Only 6.4% of pts received SCT during follow-up. Median follow-up from the index date until death or censoring was 13.8 mo (IQR: 6.2–25.3). Median OS (mOS) was 13.9 mo (95% confidence interval: 12.9–15.0). There was no significant difference in mOS for pts treated with AZA vs DEC (13.4 vs 15.2 mo, respectively; p=0.22). Pts with RBC-TD/TU had shorter mOS vs those with RBC-transfusion independence (TI) (10.7 vs 18.6 mo, respectively; p<0.0001). Similarly, pts with platelet TD/TU had shorter mOS vs those with platelet TI (8.4 vs 14.9 mo, respectively; p<0.0001). There were no statistically significant differences in OS by the route of AZA administration, insurance category, county metropolitan status, or county poverty level. Overall, 38.0% of HMA-treated pts progressed to AML; median time from HMA initiation to AML progression was 8.2 mo (IQR: 3.5–15.1). Cumulative incidences of AML and death were 25.7% and 30.7%, respectively, at Year 1 and 34.3% and 45.8%, respectively, at Year 2, with the rates of progression slowing over time. Summary/Conclusion: Compared with previously published literature from the SEER-Medicare database (Zeidan Br J Haematol 2016; mOS: 11 mo [AZA] and 12 mo [DEC] in pts diagnosed with HR-MDS between 2004 and 2011), pts with HR-MDS treated with HMA have improved mOS, but OS remains substantially worse vs the landmark AZA-001 trial (Fenaux Lancet Oncol 2009; mOS: 24.5 mo). The incidence of death and AML progression was highest in the first year after initiation of therapy. SCT rates remain very low among pts with HR-MDS, further emphasizing the urgent need for novel treatment options for pts with HR-MDS. P767: CLINICAL SIGNIFICANCE OF ROUTINE HIGH-RESOLUTION STRUCURAL VARIANT PROFILING IN MYELODYSPLASTIC SYNDROMES H. Yang1, G. Garcia-Manero1, K. Sasaki1, G. Montalban-Bravo1, Z. Tang1, Y. Wei1, T. Kadia1, K. Chien1, D. Rush1, H. Nguyen1, A. Kalia1, M. Nimmakayalu1, C. Bueso-Ramos1, H. Kantarjian1, L. J. Medeiros1, R. Luthra1, R. Kanagal-Shamanna1,* 1THE UNIVERSITY OF TEXAS M.D. ANDERSON CANCER CENTER, Houston, United States of America Background: Structural Variant Profiling (SVP) using high-resolution technologies, in combination with NGS-based mutation analysis, can inform critical cytogenomic data needed for improving the prognostication and outcomes in patients (pts) with MDS. Optical Genome Mapping (OGM) is a novel non-sequencing-based technique for genome-wide high-resolution SVP for all types of SVs: copy number alterations, structural rearrangements, inversions and partial tandem duplications (PTD). Aims: The goal was to evaluate the clinical implications of SVP on prognosis and risk-stratification in a large series of consecutive treatment naïve MDS pts. Methods: All patients (pts) underwent conventional chromosomal banding analysis (CBA), OGM and targeted 81-gene panel NGS. We compared the cytogenetic [based on comprehensive cytogenetic scoring system (CCSS)] and R-IPSS risk groups between data generated by CBA and OGM respectively and correlated with outcomes. For OGM, CCSS was calculated from the predicted karyotype generated by consolidatied alignment of multiple individual SVs. Results: There were 101 newly diagnosed MDS pts (71 men; 29 women). The median age was 72 (25-92). The distribution of CCSS groups [Very Good (3%), Good (43%), Intermediate (22%), Poor (10%), Very Poor (21%)] and R-IPSS risk categories [Very low (5%), low (30%), Intermediate (27%), High (17%), Very high (20%)]. Two patients had incomplete karyotype. By generating predicted karyotypes from OGM data, there were 224 clinically significant unique aberrations. Of these, 94 (40%) aberrations (seen across 34 patients) were not detected by CBA. These included SVs affecting KMT2A, MECOM, TP53, NUP98 among others. Comparison of CCSS cytogenetic groups between OGM and CBA showed that OGM data upgraded the CCSS cytogenetic-risk in 12 pts (resulting in R-IPSS upgrade in 9 pts) and downgraded in 7 pts (R-IPSS downgraded in 6 pts). Together with the results for 2 patients with non-informative karyotype, OGM modified the CCSS for 21% pts, and R-IPSS category for 17 (17%) pts. Since CCSS is influenced by the number of alterations, there were patients where OGM data, though clinically important, did not change the CCSS/R-IPSS category. To capture this, we defined an “actionable SV” as (1) prognostic, not recognized in the CCSS criteria (KMT2A-PTD, TP53 allele status) or (2) potential eligibility for a clinical trial, such as rearrangements in KMT2A, MECOM, or NUP98. Well-established SVs defined in CCSS were ignored. Based on this, 13 (13%) pts had an “actionable alteration” (9 with a positive finding). When both the CCSS change and actionable alterations were combined, OGM results provided informative data in 28 (28%) patients. Next, we evaluated the impact of CCSS-CBA and CCSS-OGM groups using outcome data. Over a median follow-up duration of 34 months, the median OS was 86 months. Using Harrell’s concordance index, OGM had a slightly improved prediction of prognosis for both CCSS [0.7 vs. 0.665] and R-IPSS categories [0.705 vs. 0.69] compared to CBA. By UVA, the following parameters associated with outcome: SF3B1 mutation [p=0.030; HR 0.343; CI 0.130-0.901], TP53 mutation [p=0.001; HR 3.232; CI 1.582-6.601], CCSS score by CBA [p=0.001; HR 1.690; CI 1.250-2.286] and CCSS by OGM [p<0.001; HR 2.051; CI 1.488-2.827]. By MVA analysis, CCSS by OGM [p<0.001; HR 1.956; CI, 1.367-2.798], but not CCSS by CBA), BM blast percentage [p=0.026; HR 1.079; CI, 1.009-1.153] and TP53 mutation [p=0.040; HR 2.335; CI, 1.038-5.250] associated with prognosis. Summary/Conclusion: High-resolution SVP by OGM improved the prognostic risk stratification in MDS. P768: GUADECITABINE (SGI-110) VS. TREATMENT CHOICE (TC) IN RELAPSED/REFRACTORY(R/R) MYELODYSPLASTIC SYNDROME (MDS), RESULTS OF A GLOBAL, RANDOMIZED, PHASE 3 STUDY. G. Garcia-Manero1,*, S. Bart2, J. K. McCloskey3, P. Fenaux4, D. Selleslag5, G. Reda6, D. Valcárcel7, V. Santini8, J. Mayer9, B. Xicoy10, H. Yamaguchi11, M. Lübbert12, Y. Miyazaki13, H. Keer14, Y. Hao14, M. Azab14, H. Döhner15 1The University of Texas MD Anderson Cancer Center, Houston; 2Fred Hutchinson Cancer Research Center, Seattle; 3John Theurer Cancer Center at Hackensack University Medical Center, Hackensack, United States of America; 4Hôpital Saint Louis, Paris, France; 5Algemeen Ziekenhuis Sint-Jan Brugge-Oostende, Brugge, Belgium; 6Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy; 7Hospital Universitari Vall d’Hebrón, Barcelona, Spain; 8MDS Unit AOU Careggi, DMSC University of Florence, Florence, Italy; 9Fakultní Nemocnice Brno, Brno, Czechia; 10Institut Català d’Oncologia-Hospital Universitari Germans Trias i Pujol, Badalona, Spain; 11Nippon Medical School Hospital, Tokyo, Japan; 12Universitaetsklinikum Freiburg, Freiburg, Germany; 13Nagasaki University Hospital, Nagasaki, Japan; 14Astex Pharmaceuticals, Inc., Pleasanton, United States of America; 15Ulm University Hospital, Ulm, Germany Background: Guadecitabine (G) is a next-generation hypomethylating agent (HMA), administered as a small-volume subcutaneous (SC) injection, designed with the potential to overcome pharmacokinetic resistance to first-generation HMAs (decitabine and azacitidine). Preliminary guadecitabine phase 2 data in r/r MDS showed an overall survival of almost 12 months (Garcia-Manero,G, et al; Lancet Haematol http://dx.doi.org/10.1016/S2352-3026 [19]30029-8) leading to the ASTRAL-3 study. Aims: Compare overall survival of guadecitabine to that of TC consisting of low-dose cytarabine (LDAC), intensive chemotherapy (IC), or best supportive care (BSC). Secondary objectives included multiple standard assessments of response and safety. Methods: Subjects with r/r MDS or Chronic Myelomonocytic Leukemia (CMML) who had progressed or failed to respond after 6 full cycles of standard HMA therapy were randomized 2:1 between guadecitabine (60 mg/m2) SC Days 1-5 every 28 days vs. a pre-selected TC (BSC, IC, or LDAC). Stratification was by disease, disease burden (>10% bone marrow blasts or not, geography, and pre-selected TC option). Primary endpoint was overall survival (OS) and the study was designed with ~90% power to detect a hazard ratio of 0.68 (approximately 2.8 month difference in median survival). Results: 417 MDS/CMML subjects were randomized to G or TC (G:277, TC: 140). Demographics and disease status were reasonably balanced across the groups (see Table 1 below) as was percentage of commonly associated genetic mutations (TET2, DNMT3A, SF3Baa, RUNX1, and TP53). Median guadecitabine exposure was 4 cycles and 3 cycles for TC. Neither overall survival (G: median 9.1 months, TC: median 8.3 months, p=0.61, HR:0.94 with 95% CI: [0.74-1.19]) nor leukemia-free survival (LFS) (G: median 5.7 months, TC: median 5.9 months; p=0.38) demonstrated a significant difference between the two groups and subgroup analyses did not suggest a difference between guadecitabine and any of the different TC options or different genetic mutations. Transfusion dependence for 8 weeks was similar with 32.1% and 22.4% of the G group and 37.9% and 20.0% of the TC group being platelet transfusion or red blood cell transfusion independent, respectively. Safety was consistent with known profiles of the respective agents with the G group having a slightly higher overall incidence of adverse events (AE) (99.3% vs 92.6% for TC) and grade 3 or higher AE (92.2% vs 70.5% for TC). The AEs with the highest incidence in subjects who received guadecitabine were febrile neutropenia (38.5 vs 18.9% for TC), pneumonia (34.4% vs 18.9% for TC), neutropenia (34.1% vs 15.6% for TC), and thrombocytopenia (32.2% vs 21.3% for TC). Image: Summary/Conclusion: This large, global, randomized phase 3 study did not demonstrate superiority of guadecitabine over Standard TC in MDS/CMML patients who were refractory or relapsed following full course of prior HMA treatment. P769: MUTATION PROFILES AND RISK STRATIFICATION IN HYPOCELLULAR MYELODYSPLASTIC SYNDROME K. Kim1,*, K. Chien1, O. Faustine1, G. Montalban-Bravo1, R. Kanagal-Shamanna2, T. Kadia1, E. Jabbour1, Y. Alvarado1, K. Sasaki1, X. Q. Dong1, S. Pierce1, C. Bueso-Ramos2, H. Kantarjian1, G. Garcia-Manero1 1Department of Leukemia; 2Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Hypocellular myelodysplastic syndrome (hMDS) is a subset of MDS, defined by marrow cellularity less than 30% in age under 70 and less than 20% in age 70 or more. The clinicopathologic features of hMDS have been previously studied, but genetic data and clinical impact of hMDS remain unclear. Aims: We conducted a comparative study of hMDS to normo-/hypercellular MDS (non-hMDS) with clinical characteristics and mutations and further stratified the risk by mutation profile. Methods: We conducted a retrospective review using a single tertiary cancer center registry of untreated MDS patients from 2020 to 2021. Mutation data was obtained by 28- or 81-gene next-generation sequencing (NGS) panels. We analyzed clinicopathological and mutation data comparing hMDS to non-hMDS pts and conducted risk stratification in hMDS using mutation profiles. Results: In a total of 1899 patients, 174 (9%) patients had hMDS. 1352 (71%) pts had mutation profiles that were further analyzed; 56% and 44% patients had 81-gene and 28-gene NGS panels performed, respectively. The median follow-up time was 19.5 months. Thrombocytopenia (p=0.016) and neutropenia (p<0.0001) were more common in hMDS than in non-hMDS. By IPSS-R cytogenetic scores, hMDS had scores of 3 than non-hMDS (20% vs. 9%, p<0.001), but non-hMDS had more cytogenetic scores of 4 (27% vs. 19%, p=0.018). The distribution of other IPSS-R cytogenetic scores or IPSS-R risk classifications was similar between the 2 groups. hMDS patients were more likely to be age<65 (68% vs. 47%, p<0.001) and have therapy-related MDS (t-MDS) (44% vs. 32%, p=.001). hMDS patients also had a median overall survival (mOS) of 29.2 months, which was similar to that observed in non-hMDS pts (HR 1.04, p=0.702). This finding was maintained after multivariate adjustment including age, IPSS-R, t-MDS, and certain high-risk mutations (TP53, ASXL1, RUNX1, RAS pathway). Hypocellularity did not affect survival in either de novo or t-MDS. AML transformation rates (20% in both groups) were similar. Interestingly, pts with hMDS had lower rates of high-risk mutations (38% vs. 51%, p=0.009) with less RUNX1 (p=0.079) and ASXL1 (p=0.010) mutations while the frequency of TP53 and RAS pathway mutations were comparable. In t-MDS, hMDS pts had less frequent TP53 (p=0.005) or U2AF1 (p=0.087) mutations. In de novo MDS, hMDS patients had less RUNX1 (p=0.036), SRSF2 (p=0.014), and TET2 (p=0.019) mutations. In hMDS, mutations in the RAS pathway (HR 3.64, p=0.007) or in TP53 (HR 2.47, p=0.002) were associated inferior mOS, while TET2 mutations (HR 0.29, p=0.036) correlated with superior mOS. hMDS also had worse mOS in RAS pathway-mutated MDS (HR 2.62, p=0.043). Based on mutation data and multivariate selection models, patients were stratified into 3 groups: (1) pts with TET2 or SF3B1 (only available 81-gene panels) mutations, (2) pts with TP53 mutations or RAS pathway mutations, and (3) pts without TET2/SF3B1/TP53/RAS mutations. The TET2/SF3B1 group showed better mOS (HR 0.34, p=0.041) compared to the TP53/RAS group showing inferior mOS (HR 4.64, p<0.001) (shown in figure). The mOS difference observed with the TET2/SF3B1 group did not carry over into non-hMDS pts (p=0.889). Image: Summary/Conclusion: Our study showed that hMDS patients have similar outcomes with non-hMDS pts with unique molecular profiles. hMDS pts have less frequent high-risk mutations with distinct risk stratification. Improving risk stratification tools in hMDS by incorporating genomic data, such as TP53/RAS pathway and TET2/SF3B1 mutations, would be a study of interest. P770: A COMPARATIVE STUDY OF LEUKEMIC TRANSFORMATION IN THERAPY-RELATED AND DE NOVO MYELODYSPLASTIC SYNDROME AFTER HYPOMETHYLATING AGENT FAILURE K. Kim1,*, F. Ong1, Z. Li2, R. Kanagal-Shamanna3, G. Montalban-Bravo1, T. Kadia1, E. Jabbour1, N. Pemmaraju1, D. Hammond1, N. Short1, F. Ravandi1, Y. Alvarado1, S. Pierce1, X. Q. Dong1, H. Kantarjian1, G. Garcia-Manero1, K. Chien1 1Department of Leukemia; 2Department of Biostatistics; 3Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Therapy-related myelodysplastic syndrome (t-MDS) is subset of myelodysplastic syndrome (MDS), known for having dismal survival outcomes. The majority of patients (pts) with t-MDS have similar clinical courses with de novo MDS patients after hypomethylating agent failure (HMA-F) that often results in transformation to acute myeloid leukemia (AML). Aims: We conducted a study to identify unique clinicopathologic features of t-AML from t-MDS compared to secondary AML (sAML) from de novo MDS after HMA-F. Methods: We identified sAML patients after HMA-F from all untreated MDS pts in a tertiary cancer center registry from 2017-2021. Clinical characteristics and pathologic data including cytogenetic (CG) and next generation sequencing (NGS) information were collected at the time of MDS diagnosis (dx), HMA-F, and AML dx. Results: 71 (48%) MDS pts with AML transformation after HMA-F were included in the study with a median follow-up time of 15.4 months (mo). At the time of MDS dx, 31 (43%) pts had t-MDS: 97%, 58% and 29% had a history of chemotherapy, radiation, and prior stem cell transplant (SCT), respectively. t-MDS patients were more likely to present with age<65 (48% vs. 28%, p=0.070) and thrombocytopenia (33,000 vs. 75,500, p=0.004) when compared to de novo MDS pts. The majority of t-MDS pts had very high risk disease by IPSS-R (67% vs. 39%, p=0.031), while de novo MDS pts frequently had high risk disease (50% vs. 20%, p=0.013). 90% of t-MDS pts had adverse CG (IPSS-R CG score 3-4) at MDS dx, compared to 46% in de novo MDS pts (p=0.004). NGS at MDS dx showed more frequent TP53 mutations in t-MDS than in de novo MDS (73% vs 36%, p=0.003). Interestingly, ASXL1 (36% vs. 1%, p=0.001), RUNX1 (9% vs. 0%, p=0.004), STAG2 (21% vs. 0%, p=0.013), TET2 (13% vs. 0%, p<0.001), and SRSF2 (18% vs. 0%, p=0.027) were more frequently found in de novo MDS (Table). t-MDS had worse overall survival (OS) from MDS dx (HR 1.82, p=0.026). The median OS from MDS dx was 19.4 mo and 14.3 mo in de novo MDS and t-MDS, respectively. The number of HMA cycles received before HMA-F was 6 (range: 1-13) in t-MDS and 5 (range: 1-23) in de novo MDS. At the time of HMA-F, t-MDS pts presented with more anemia (p=0.005) and thrombocytopenia (p=0.064). The number of CG clones were similar, but the number of CG abnormalities were higher in t-MDS pts (p=0.025). Among pts with subsequent AML transformation after HMA-F, the median time from HMA-F to AML was 1.5 mo in t-MDS to AML, compared to 4.6 mo in de novo MDS to AML. At time of AML dx, 48 (68%) pts had mutation panels available. All TP53 mutation status stayed the same in t-MDS pts at the time of MDS and AML dx. Other changes in mutations were shown in Table. Though the median OS from AML transformation was short in all pts, t-MDS pts had inferior outcomes (2.4 mo vs. 5.0 mo, HR 1.70, p=0.043). The initial response rate to salvage therapy was comparable between t-MDS and de novo MDS after AML transformation (50% and 56%, p=0.761), but more pts underwent SCT with de novo MDS compared to t-MDS (10% vs. 3%, p=0.383). Image: Summary/Conclusion: Transformation to AML after HMA-F results in poor outcomes, but pts with t-MDS have worse survival with distinct CG and mutation profiles. Further understanding of the pathogenesis of AML transformation in both t-MDS and de novo MDS after HMA-F is warranted with improved therapy options an area of unmet need. P771: PROGNOSTIC CHARACTERISTICS OF PATIENTS WITH MDS WITH ABERRATIONS OF CHROMOSOME 5. DATA FROM THE DÜSSELDORF MDS REGISTRY F. Schulz1, J. Kostova1,*, B. Hildebrandt2, K. Nachtkamp1, C. Strupp1, N. Gattermann1, M. Lübbert3, S. Blum4, S. Parmentier5, K. Götze6, J. Lipke7, F. Beier8, W.-K. Hofmann9, U. Germing1 1Department of Hematology, Oncology and Clinical Immunology; 2Institute of Human Genetics, University Hospital Düsseldorf, Düsseldorf; 3Department of Medicine I: Hematology, Oncology, and Stem-Cell Transplantation, University Medical Center Freiburg, Freiburg, Germany; 4Department of Hematology and Oncology, Lausanne University Hospital, Lausanne; 5Oncology and Hematology, Tumor Center, St. Claraspital, Basel, Switzerland; 6Department of Internal Medicine III, University Hospital Klinikum Rechts der Isar, Munich; 7Gefos Dortmund, Dortmund; 8Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, University Hospital Aachen, Aachen; 9Department of Hematology and Oncology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Background: Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with a wide spectrum of biological and clinical features. Up to 20% of MDS patients present with aberrations of chromosome 5, either as isolated del(5q) or combined with other aberrations. Patients with a medullary blast count < 5% and isolated del(5q), or with one additional aberration except anomalies of chromosome 7, are diagnosed as MDS del(5q), a defined entity according to the WHO 2016 classification. They have a favorable prognosis compared to other types of MDS, although profound anemia, chronic transfusion dependency and the risk of disease progression into AML present clinical challenges. Aims: We analyzed the prognostic impact of karyotype complexity in MDS del(5q). Methods: From the Düsseldorf MDS registry we retrieved 499 patients with MDS or CMML according to WHO 2016, who showed any kind of del(5q). Biological and clinical parameters such as karyotype, IPSS-R and type of treatment were evaluated. Survival and progression to AML was calculated using Kaplan-Meier-plots. Cox regression analysis was performed to identify disease-related prognostic factors. Patients were followed up until Dec 31th 2021. Results: 58% of the patients were female, median age was 67 years. Median medullary blast count was 4% (0-19%), and median survival in the entire group was 28 months. For assessing the natural course of disease, we screened for patients with non-intensive treatment only (best supportive care, lenalidomide, low-dose Ara-C, hydroxyurea). Separation by medullary blast count (< 5%, 5-9% or ≥ 10%) was associated with significantly different median survival times, ranging from 63 months in the first group to 3 months in patients with ≥ 10% medullary blasts (p < 0.001). Survival was also affected by the cytogenetic risk group according to IPSS-R. Patients in the low-risk group (median survival 72 months) differed significantly from patients in the very high risk group (median survival 5 months) (p < 0.001). A similar trend was observed according to karyotype complexity represented by number of aberrations apart from del(5q). Median survival of patients with an isolated del(5q) was 86 months, while patients with a complex karyotype (del(5q) and ≥3 additional aberrations) had a median survival of 5 months. Biological risk factors were assessed using Cox regression analyses. Considering overall survival as endpoint, medullary blast percentage, cytogenetic risk group, and complexity of the karyotype had a significant impact on survival (p < 0.001). In the entire patient cohort, as well as among patients receiving non-intensive treatment, the rate of AML progression was significantly influenced by karyotype complexity (p < 0.001). While the median time to AML progression in both isolated del(5q) and del(5q) combined with one aberration had not been reached at the end of the observation period, the median time to AML progression for patients with del(5q) as part of a complex karyotype was 31 months in the entire cohort and 14 months in the low-intensity treatment group. Image: Summary/Conclusion: Both the IPSS-R cytogenetic risk group and the complexity of the 5q- karyotype showed prognostic impact in patients with del(5q). This was true for overall survival as well as risk of AML progression. Regardless of therapy or medullary blast count in patients with del(5q), the risk of AML progression increased dramatically with increasing complexity of the 5q- karyotype. P772: PROGNOSTIC IMPACT OF DISEASE-RELATED RISK FACTORS AND TREATMENT APPROACHES IN PATIENTS WITH ADVANCED MYELODYSPLASTIC SYNDROMES (MDS) C. Lesch1,*, A. Kündgen1, K. Nachtkamp1, A. Kasprzak1, C. Strupp1, M. Rudelius2, N. Gattermann1, G. Kobbe1, B. Hildebrandt3, U. Germing1 1Department of Haematology, Oncology and Clinical Immunology, Heinrich-Heine University Hospital, Düsseldorf, Duesseldorf; 2Institute of Pathology, Ludwig-Maximilians-University of Munich, Munich; 3Institute of Human Genetics, Heinrich- Heine University Hospital, Düsseldorf, Duesseldorf, Germany Background: Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell disorders. Treatment approaches depend on the type of MDS according to the WHO classification and on risk assessment according to the international prognostic scoring system (IPSS-R). For patients with advanced MDS, as defined by an excess of blasts, therapeutic options include hypomethylating agents and allogeneic stem cell transplantation. Aims: To determine the prognostic impact of disease-related risk factors and treatment approaches in 432 patients with MDS EB1 and EB2. Methods: Patients included in this analysis were classified as having MDS EB1 (5-9% marrow blasts) or MDS EB2 (10-19% marrow blasts) at diagnosis. All therapeutic procedures prior to the development of acute myeloid leukemia (AML) were documented. Patients were followed up until 31st December 2021, or until death. Therapeutic approaches were categorized as best supportive care (BSC) only, low-dose chemotherapy, immunomodulatory treatment, hypomethylating agents (HMA), intensive chemotherapy (IC) and allogeneic stem cell transplantation (alloSCT). The mentioned therapy regimes were weighted in different categories according to their intensity, with BSC defined as the therapy with the lowest intensity and alloSCT as the one with the highest (BSC < low-dose-chemotherapy < immunomodulatory treatment < HMA < IC < allo SCT). Results: MDS EB1 was diagnosed in 182 patients (42,1%), and MDS EB2 in 250 patients (57,9%). Distribution among IPSS-R risk groups was as follows: 2% low risk, 26.3% intermediate risk, 38% high risk, and 33,7% very high risk. Median life expectancy in the entire cohort including patients with all types of treatment, was 22 months. Progression to AML occurred in 186 (43%) of the patients. Patients receiving supportive care only (42.6%) had a median survival of 13 months. Patients treated with a hypomethylating agent (18%) showed an overall survival of 26 months, which was significantly better compared with best supportive care (p=0.002). Patients undergoing induction chemotherapy (6%) had a median survival of 21 months, comparable with HMA treatment. Patients receiving low-dose chemotherapy (4.6%) had a median survival of 9 months. The longest median survival (131 months) was seen in patients undergoing alloSCT. Multivariate Cox regression analysis showed that treatment category (p<0.00005), together with cytogenetic risk category according to IPSS-R (very low, low and intermediate versus high versus very high) (p<0.0005), and hemoglobin (<≥10g/dl) (p=0.003) were the only parameters influencing survival independently, whereas WHO type, gender, ANC, and platelets did not show independent influence on survival. Summary/Conclusion: Stratification according to cytogenetic risk groups is very important for the prognostic assessment of MDS patients. Assignment to the cytogenetic group high risk and very high risk has greater prognostic impact than the medullary blast count. Even though hypomethylating agents can improve overall survival compared to best supportive care, long-term survival can only be achieved through allogeneic stem cell transplantation. Therefore, all patients eligible for intensive treatment should be evaluated for this treatment option at the time of diagnosis. Furthermore, in recognition of the fact that clonal evolution may occur in MDS patients, consideration should be given to repeating the chromosome analysis as the most important prognostic factor at regular intervals. P773: THE ELEVATION OF RED BLOOD CELL DISTRIBUTION IS AN INDEPENDENT PROGNOSTIC FACTOR FOR JUVENILE MYELOMONOCYTIC LEUKEMIA W. Liang1,*, C. Liu2, J. Zhang2, M. Yi2, Y. Cai2, A. Zhang2, L. Liu2, L. Zhang2, X. Chen2, Y. Zou2, Y. Chen2, Y. Guo2, Y. Zhang2, X. Zhu2, W. Yang2 1Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China; 2Department of Pediatrics, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China Background: Juvenile myelomonocytic leukemia (JMML) is an overlapping myeloproliferative and myelodysplastic disorder of infants and early children. The existing clinical and laboratory prognostic features are inadequate to predict a worse outcome alone. New prognostic indicators are urgently needed since the great clinical heterogeneity. Red cell distribution width (RDW) is a parameter of the complete blood count (CBC) measured automatically by haematology analyzers to reflect the heterogeneity of erythrocyte size. In recent years, RDW has been suggested to have a high negative value for outcomes in various cancers. However, the role of RDW at diagnosis in patients with JMML is still unclear. Aims: Our aim is to investigate the prognostic role of RDW in children with JMML. Methods: We conducted a single center retrospective study to investigate the prognostic role of RDW in children with JMML. Seventy-seven patients were enrolled from January 1, 2008 to November 31, 2019. Survival rates were calculated and compared using Kaplan-Meier methods and log-rank tests. Univariate and Multivariate analysis was carried out according to the Cox proportional hazard regression model. Results: The mean red cell distribution width standard deviation (RDW-SD) for patients is 53.3(39.3-73.7) fl. Cox proportional hazard models showed that patients with RDW-SD >50.50fl at diagnosis were significantly associated with poor overall survival (OS) (HR = 5.22, CI = 1.50-18.21, P=0.010). The multivariate analysis demonstrated that the RDW-SD count was the independent risk factor for OS. Patients with PTPN11 mutation and RDW elevation displayed the worst survival rate, in comparison with other subgroups (P=0.00013). Summary/Conclusion: The results suggested that the elevated level of RDW is an independent prognostic factor for JMML. Furthermore, the PTPN11 mutation and a high RDW level may involve a synergy during the natural course of JMML. P774: CLINICAL OUTCOMES ASSOCIATED WITH AZACITIDINE MONOTHERAPY FOR TREATMENT-NAIVE PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROME (HR MDS): A SYSTEMATIC LITERATURE REVIEW AND META-ANALYSIS K. Hasegawa1,*, A. H. Wei2, G. Garcia-Manero3, N. G. Daver3, N. Rajakumaraswamy1, S. Iqbal1, R. J. Chan1, H. Hu1, P. Tse4, J. Yan4, M. J. Zoratti4, F. Xie4, D. A. Sallman5 1Gilead Sciences, Inc., Foster City, United States of America; 2Department of Clinical Haematology, The Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Australia; 3The University of Texas MD Anderson Cancer Center, Houston, United States of America; 4McMaster University, Hamilton, Canada; 5Moffitt Cancer Center, Tampa, United States of America Background: Azacitidine (AZA) is a standard of care for patients with HR MDS; however, the number of large data sets describing outcomes of AZA monotherapy in HR MDS is limited. Aims: To aggregate clinical outcomes data associated with AZA monotherapy for treatment-naive patients with HR MDS. Methods: CENTRAL, EMBASE, and MEDLINE were searched to identify interventional, prospective, and retrospective observational studies using AZA monotherapy in treatment-naive HR MDS (defined as intermediate-2 or high risk by International Prognostic Scoring System [IPSS] or intermediate to very high risk by Revised IPSS). Inclusion of observational studies was limited to those with >20 patients in the AZA monotherapy arm. Responses according to International Working Group (IWG) 2000 or IWG 2006 criteria—including complete remission (CR) rate, overall response rate (ORR; defined as CR, marrow CR [mCR], partial remission, and hematologic improvement), overall survival (OS), duration of response (DOR), and time to response (TTR)—were extracted. All articles were reviewed by 2 independent abstractors. Response rates were synthesized using random-effects models; median of medians was used for OS, DOR, and TTR. Results: Of 3250 abstracts identified, 34 publications describing 16 studies met inclusion criteria: 5 randomized controlled trials (RCTs), 3 prospective studies, and 8 retrospective studies. None of the response endpoints were reported in all studies (Table). The pooled CR rates, reported in 2 RCTs (N=55), 1 prospective study (N=27), and 3 retrospective studies (N=509), were 14%, 11%, and 16%, respectively. The pooled CR rate across all studies (N=591) was 16% (95% CI, 13%-19%). The mCR rate from 1 RCT was 19% (95% CI, 10%-34%; N=42), and the pooled mCR rate from 2 retrospective studies was 18% (95% CI, 13%-26%; N=126). The pooled ORRs ranged from 44% to 55% across RCTs, prospective studies, and retrospective studies. The pooled median OS was 16.7 months in 3 RCTs (N=161), 16.5 months in a single prospective study (N=34), and 14.4 months in 5 retrospective studies (N=1472). The pooled median OS across all studies (N=1667) was 16.4 months (95% CI, 12.0-17.3 months). The pooled median DOR was 10.1 months (95% CI, 9.1-11.0 months), and the median TTR was 4.6 months (95% CI, 3.0-9.0 months). Table: Outcomes in treatment-naive patients with HR MDS treated with AZA monotherapy Endpoint RCTs Total N Outcome(95% CI) Prospectivestudies Total N Outcome(95% CI) Retrospective studies Total N Outcome(95% CI) All studies Total N Outcome(95% CI) CR rate, % 2 55 14(6-31) 1 27 11(4-29) 3 509 16(13-20) 6 591 16(13-19) mCR rate, % 1 42 19(10-34) 0 - - 2 126 18(13-26) 3 168 19(13-25) ORR, % 3 159 50(42-57) 1 27 44(27-63) 4 967 55(42-67) 8 1153 52(44-60) Median OS, months 3 161 16.7(9.5-26.3) 1 34 16.5(10.4-21.9) 5 1472 14.4(11.6-17.3) 9 1667 16.4(12.0-17.3) Median DOR, months 1 20 9.1(6.0-NR) 0 - - 1 405 11.0 (NA) 2 425 10.1(9.1-11.0) Median TTR, months 1 42 4.6(2.0-6.5) 0 - - 2 493 6.0 (3.0-9.0) 3 535 4.6(3.0-9.0) NA, not available; NR, not reached. Summary/Conclusion: These findings provide evidence that benefit with AZA monotherapy in treatment-naïve patients with HR MDS is limited. Opportunity exists for novel therapies to increase CR rates and prolong survival. P775: CLINICAL OUTCOMES AND SOCIOECONOMIC FACTORS IN CHRONIC MYELOMONOCYTIC LEUKEMIA IN THE UNITED STATES USING THE NATIONAL CANCER DATABASE: 2004-2017 N. Mclaughlin1,*, G. Ruan2, K. Wudhikarn3, A. Al-Kali2, A. Tefferi2, N. Gangat2, R. Go2, M. Patnaik2, M. Shah2 1Division of Internal Medicine; 2Division of Hematology; 3Mayo Clinic, Rochester, United States of America Background: Chronic myelomonocytic leukemia (CMML) is a rare myeloid malignancy characterized by proliferation of dysplastic monocytic cells and carries a risk for leukemic transformation. Previously, we described the incidence and outcomes of CMML at the population level (2004-2015) using the Surveillance, Epidemiology, and End Results Program (SEER) and National Cancer Database (NCDB) (Ruan et al., ASH 2020). We studied population-based outcomes in CMML using the NCDB from 2004-2017. Aims: Our aim was to understand the impact of social determinants of health on outcomes in CMML. Secondly, we studied if the outcomes of CMML have improved. Methods: The NCDB is a nationwide oncology outcomes database for >1500 cancer programs in the US and Puerto Rico, capturing 70% of all newly diagnosed cases of cancer. We queried the NCDB using the ICD-O-3 diagnosis code 9945/3 from 2004-2017. Only patients (pts) with diagnostic confirmation via histopathologic examination were included. Percent without high school degree (HSD) reflects the education level within the pt’s zip code, which was stratified into 4 quartiles. Median household income was estimated by matching the pt’s zip code at time of diagnosis against US Census 2000 data, which was stratified into 4 quartiles. Hazard ratios (HR) with confidence intervals (CI) were calculated using a Cox proportional hazards model. Overall survival (OS) was estimated using the Kaplan-Meier method. Variables significant in univariable analysis were included in a multivariable analysis. Results: We identified 7143 CMML pts in the NCDB. The median age at diagnosis was 72 years (interquartile range [IQR] 65-81) and 4386 (61%) were males. With a median follow up of 6.1 years (IQR 3.6-9.4), the median OS was 1.4 years (IQR 0.4-3.8). 1600 (23%) had private insurance, 4862 (70%) had Medicare, 298 (4%) had Medicaid, and 129 (2%) were uninsured. In univariate analysis, private insurance had an improved OS compared to patients with Medicaid (HR 1.2, 95% CI 1.06-1.4; p value 0.007), Medicare (HR 1.5, 95% CI 1.4-1.6; p value < 0.001), and other government insurance (HR 1.5, 95% CI 1.1-2.0; p value 0.004). Pts living in a zip code with percent of adults without an HSD of < 14% had an improved OS compared to zip codes with > 29% (HR 1.2, 95% CI 1.1-1.3; p value < 0.001). Median household income by zip code of ≥ $46,000 had an improved OS compared to < $30,000 (HR 1.2, 95% CI 1.1-1.4; p value < 0.001). Receiving treatment at an academic program had an improved OS compared to community cancer programs (HR 1.2, 95% CI 1.1-1.3; p value < 0.001), comprehensive community cancer programs (HR 1.3, 95% CI 1.3-1.4; p value < 0.001), and integrated network cancer programs (HR 1.3, 95% CI 1.2-1.4; p value < 0.001). Finally, pts diagnosed in 2012-2017 had an improved OS compared to pts diagnosed in 2004-2011 (HR 0.8, 95% CI 0.8-0.9; p value < 0.001). Variables significant in univariate analysis were included in multivariate analysis (Table 1). When education level and median household income per zip code were included in multivariate analysis, education level remained statistically significant and income did not. Image: Summary/Conclusion: CMML continues to have a poor prognosis, but OS has improved in recent years. Socioeconomic factors including insurance status, level of education and median household income per zip code, and facility type appear to be associated with outcomes. On multivariable analysis, age < 65, private insurance (vs government insurance), living in a zip code with increased percent HSD, and treatment at an academic program had improved outcomes. P776: A PHASE 2, OPEN-LABEL, ASCENDING DOSE STUDY OF KER-050 FOR THE TREATMENT OF ANEMIA IN PATIENTS WITH VERY LOW, LOW, OR INTERMEDIATE RISK MYELODYSPLASTIC SYNDROMES S. Tan1,*, A. Arbelaez2, L. Chee3, C. Y. Fong4, D. Hiwase5, G. Kannourakis6, J. Kwan7, J. Liang8, A. Puliyayil9, H. Rose10, D. Ross11, T.-C. Teh12, D. Westerman13, J. Wight14, W. Feng15, J. Lachey15, A. McGinty15, H. Natarajan15, C. Rovaldi15, S. Cooper15 1St Vincent’s Hospital, Melbourne; 2Tweed Hospital, Tweed Heads; 3Royal Melbourne Hospital and Peter MacCallum Cancer Centre, Melbourne; 4Austin Health, Hiedleberg; 5Royal Adelaide Hospital, Adelaide; 6Ballarat Oncology, Ballarat; 7Westmead Hospital, Westmead, Australia; 8Middlemore Hospital, Auckland, New Zealand; 9Albury Wodonga Health, Albury-Wodonga Regional Cancer Centre, Auckland; 10Barwon Health, Geelong; 11Flinders Medical Centre, Adelaide; 12Box Hill Hospital, Eastern Health, Victoria; 131. Peter MacCallum Cancer Centre. 2. University of Melbourne, Melbourne; 14Townsville University Hospital, James Cook University, Douglas, Australia; 15Keros Therapeutics, Lexington, United States of America Background: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis; many patients develop high transfusion burden (HTB) requiring ≥4 units RBCs every 8 weeks and have high unmet medical need. KER-050, an investigational modified activin receptor type IIA inhibitor, is designed to target TGF-β ligands to promote differentiation of erythroid and megakaryocyte lineages to improve anemia and thrombocytopenia. Aims: Evaluate safety, tolerability, pharmacodynamics (PD), efficacy of KER-050 in MDS in an open-label Phase 2 study. Methods: IPSS-R lower-risk anemic MDS patients are enrolled, including non-transfused, low-transfusion burden (LTB) and HTB patients. In Part 1 base study, ascending dose cohorts receive KER-050 subcutaneously q4w for 4 doses until a recommended Part 2 dose is determined. Safety endpoints include incidence of adverse events (AEs); erythroid efficacy endpoints (≥8 weeks duration) include rates of transfusion independence (TI) and reduction in RBC transfusions by ≥4 units (IWG 2006 HI-E) in HTB patients. Safety-set includes patients receiving ≥1 dose. PD/efficacy results are reported in efficacy evaluable (EE) patients who contributed ≥8 weeks of HGB and transfusion data. New analyses presented here describe response and markers of erythropoiesis and thrombopoiesis in HTB patients. Results: At data cut-off (25-Oct-2021), median follow-up was 124 days (range 10-217). 24 patients received ≥1 dose of KER-050 across 4 dose levels (0.75mg/kg to 3.75mg/kg q4W). Of these, 16 were EE (8 not EE due to: duration of treatment (n=6), death (n=1), study discontinuation (n=1)). Median time from diagnosis was 2.2 years (range 0.2 – 8.6), 14 (58.3%) were HTB. Of HTB patients, 7 were RS+, 6 were on iron chelators. No DLTs were reported in the safety-set. 5 (20.8%) reported grade ≥3 treatment-emergent AEs (TEAEs), 5 (20.8%) reported SAEs (none related), 4 (16.7%) developed grade 1 or 2 treatment-related AEs. Most frequent TEAEs (≥10%) were diarrhea, dyspnea, fatigue, nausea, anemia, headache. 2 discontinued study drug: 1 participant decision, 1 death. 1 required dose modification due to unrelated TEAE; none required dose modification for increased HGB or thrombocytosis. Results from 16 EE cohort 1-3 patients were presented at ASH 2021 (Poster #3675), 8 (50%) achieved HI-E or TI. New analyses focused on HTB patients found that 6 of 9 HTB, EE patients achieved HI-E and 4 achieved TI for ≥8 weeks (Panel A). Increases in reticulocytes (RETs) (Panel B) and sTfR and decreases in serum ferritin (Panel C) in these HTB patients were observed. Mean maximum RETs increase was 56.87 x109/L, range 15.7-142.5x109/L with mean maximum ferritin reduction of 29.7%, range -17 to 65% and mean maximum sTfR increases of 51%, range 29-90%. Increases in platelets were observed in HTB patients achieving HI-E or TI (Panel D). Maximum increase from baseline was 98 x109/L (mean), range 33-179 x109/L. Image: Summary/Conclusion: KER-050 was generally well-tolerated as of data cut-off date. HI-E and TI ≥8 weeks have been observed in HTB patients treated with KER-050. Observed PD effects in RETs, sTfR and ferritin support the proposed mechanism of action of increasing erythropoiesis. Increases in platelets have been observed in HTB patients achieving HI-E or TI which supports the potential of KER-050 as a treatment for multilineage cytopenias in difficult-to-treat HTB patients. Dose escalation is ongoing in this Phase 2 study of anemic patients with MDS; updated data from Part 1 with safety, PD and efficacy data from cohorts 4 and 5 will be presented for the first time at the meeting. P777: RUNX1 VARIANTS WITH HIGH VARIANT ALLELE FREQUENCY IN MYELOID NEOPLASMS. GERMLINE OR NOT? N. J. Nitschke1,2,*, M. Krogh3, K. Raaschou-Jensen4, M. T. Severinsen5, A. S. Roug6, J. S. Jespersen2, J. W. Hansen1,2, J. Weischenfeldt2, M. K. Andersen7, K. Grønbæk1,2 1Department of Hematology, Rigshospitalet; 2Biotech Research and Innovation Center (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen; 3Department of Hematology, Sjælland University Hospital, Roskilde; 4Department of Hematology, Odense University Hospital, Odense; 5Department of Hematology and Clinical Cancer Research, Aalborg University Hospita, Aalborg; 6Department of Clinical Medicine, Aarhus University Hospital, Aarhus; 7Department of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark Background: RUNX1 is a transcription factor frequently mutated in myeloid neoplasms e.g. 10% of acute myeloid leukemia (AML) patients, but RUNX1 variants detected in tumor tissue at a variant allele frequency (VAF) close to 50% can potentially be germline. As pathogenic germline RUNX1 variants are causative for RUNX1 Familial Platelet Disorder with Associated Myeloid Malignancies it is crucial to distinguish between somatic and germline variants. Previous studies in AML patients estimate the frequency of germline RUNX1 variants to be approximately 3%. The frequency of RUNX1 germline variants in lower risk myeloid neoplasms (MN) such as myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), clonal cytopenia of undetermined significance (CCUS) and idiopathic cytopenia of undetermined significance (ICUS) remains unknown. Aims: To estimate the frequency of RUNX1 germline variants in MN. Methods: Patients referred to a hematological department in Denmark with suspicion of MN (excluding myeloproliferative disorders) were included in the study. At first visit, a skin biopsy was drawn and frozen dry at -80 °C. RUNX1 variants were identified in either blood or bone marrow using 4 different amplicon based next generation sequencing panels. Variants detected with a VAF > 40 % were further classified as somatic or germline by bi-directional sanger sequencing of germline DNA purified from skin biopsies. Results: Among 595 patients, 85 (14%) had variants in RUNX1 and 28 patients (5%) had variants with a VAF above 40%. High VAF RUNX1 variants were either missense, frameshift, nonsense, or splice-site variants. A majority of the variants were located in the runt homology domain (RHD). We observed a higher proportion of patients with AML and a lower proportion of patients with CCUS in patients with a RUNX1 variant compared to patients without a RUNX1 variant, and this tendency was enhanced if the RUNX1 variants were present at VAF > 40%. Among 22 patients with a high VAF RUNX1 variant and available germline DNA two variants (9%) were classified as germline (NM_001754.5; c.668A>G, c.649G>A, minimal allel frequency reported in gnomAD; 0.012%, 0.003%). The c.668A>G variant was detected in a patient with a low platelet count and CCUS diagnosed at age 24. The c.649G>A variant was detected in a patient with a normal platelet count and MDS diagnosed at age 64. Both germline variants were missense variants located in the region between the RHD and the transactivating domain (TAD) and both were predicted to be pathogenic in-silico by several prediction tools. Unfortunately, family history wasn’t available for any of the two patients with germline variants. Pathogenic variants co-occurring at similar VAF as RUNX1 were detected in 17 out of 20 patients with a somatic RUNX1 variant, but not in any of the patients with germline variants (Figure 1). Pathogenic variants in SRSF2, TET2 and ASXL1 were frequently observed to co-occur with high VAF RUNX1 variants. Image: Summary/Conclusion: RUNX1 variants with a VAF above 40% were classified as germline in two out of 22 MN patients (9%). The variants were detected in patients with MDS and CCUS indicating that germline testing should be considered not only in AML, but also in other MN with high VAF of a RUNX1 variant. Furthermore, a high VAF RUNX1 variant without co-occurring variants with similar VAF in other genes should increase the consideration for germline testing. P778: LONG-TERM UTILIZATION AND BENEFIT OF LUSPATERCEPT IN PATIENTS (PTS) WITH LOWER-RISK MYELODYSPLASTIC SYNDROMES (LR-MDS) FROM THE MEDALIST TRIAL P. Fenaux1,*, V. Santini2, R. S. Komrokji3, A. Zeidan4, G. Garcia-Manero5, R. Buckstein6, D. Miteva7, K. Keeperman8, N. Holot8, J. Zhang8, J. A. Nadal7, B. Rosettani7, A. Yucel8, U. Platzbecker9 1Service d’Hématologie Séniors, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris and Université Paris 7, Paris, France; 2MDS Unit, AOU Careggi, University of Florence, Florence, Italy; 3Moffitt Cancer Center, Tampa; 4Department of Internal Medicine, Yale School of Medicine and Yale Cancer Center, New Haven; 5Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America; 6Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, Canada; 7Celgene International Sàrl, a Bristol-Myers Squibb Company, Boudry, Switzerland; 8Bristol Myers Squibb, Princeton, United States of America; 9Medical Clinic and Policlinic 1, Hematology and Cellular Therapy, University Hospital Leipzig, Leipzig, Germany Background: Luspatercept has been shown to improve anemia in the phase 3 MEDALIST trial of pts with LR-MDS ineligible/intolerant or refractory to erythropoiesis-stimulating agents (ESAs). Aims: To report the long-term clinical value of luspatercept treatment (Tx) in pts from the MEDALIST study including dosing, duration of Tx (DOT) and response, baseline characteristics of pts receiving luspatercept in a long-term follow-up (LTFU) study, and rates of progression to acute myeloid leukemia (AML) and high-risk MDS (HR-MDS). Methods: Eligible pts were ≥18 y of age, had LR-MDS requiring regular red blood cell (RBC) transfusions, and were ineligible/intolerant or refractory to ESAs. Pts were randomized 2:1 to subcutaneous luspatercept or placebo every 3 wk for 24 wk. The primary endpoint was RBC transfusion independence (RBC-TI) ≥8 wk during wk 1–24. MEDALIST pts who continued to receive luspatercept due to ongoing clinical benefit were eligible for enrollment into the LTFU study, a phase 3b rollover study to evaluate the long-term safety of luspatercept in pts who participated in other luspatercept studies. DOT was calculated as (Tx end date − date of first dose) + 1) / 7. Duration of response was determined by Kaplan–Meier analysis. Total person-years for pts at risk of HR-MDS/AML progression was calculated from LR-MDS diagnosis to HR-MDS/AML diagnosis, or to last HR-MDS/AML follow-up date for pts who did not progress. Results: As of Jan 15, 2021, the median (95% confidence interval [CI]) DOT was 11.70 (8.97–16.33) mo for luspatercept pts and 5.52 (5.52–5.59) mo for placebo pts. Median (interquartile range [IQR]) DOT for placebo pts who transitioned to LTFU was 24.0 (24.0–25.3) wk and 24.0 (24.0–39.0) wk for placebo pts who discontinued prior to LTFU. The median (IQR) DOT for luspatercept pts who transitioned to LTFU was longer than for those pts who discontinued (190.0 [86.0–211.0] wk vs 31.0 [24.0–64.1] wk). Baseline characteristics more frequently observed in luspatercept pts who transitioned to LTFU than in pts who discontinued include younger age, female sex, and lower Eastern Cooperative Oncology Group performance status score, transfusion burden, erythropoietin, and serum ferritin levels (Table A). In MEDALIST, 106/153 (69.3%) pts receiving luspatercept and 64/76 (84.2%) receiving placebo escalated to the maximum dose of 1.75 mg/kg. During the entire Tx phase, RBC-TI ≥8 wk was observed in 74/153 (48.4%) and 12/76 (15.8%) luspatercept and placebo pts, respectively, with a median (95% CI) cumulative duration of response of 80.7 (53.71–154.14) wk and 21.0 (10.86–NE) wk, respectively. During the entire Tx period, RBC-TI ≥16 wk was observed in 48/153 (31.4%) and 6/76 (7.9%) luspatercept and placebo pts, respectively (Table B). Among luspatercept pts, 13/153 (8.5%) progressed to HR-MDS/AML during the entire Tx period, compared with 5/76 (6.6%) placebo pts. The total person-years for pts randomized to luspatercept at risk of progressing to HR-MDS/AML was 401.7 y vs 190.9 y for placebo. Image: Summary/Conclusion: Pts receiving luspatercept had an extended period of clinical benefit and >50% of pts continued to receive luspatercept for >1 y, the majority of whom underwent dose escalations to achieve an optimal response. Pts experienced durable responses with luspatercept, with a median cumulative duration of RBC-TI response of approximately 20 mo. Pts receiving luspatercept also remained on Tx longer than placebo regardless of participation in the LTFU study, suggestive of luspatercept’s clinical benefit with significant durability. P779: OPTIMIZING TREATMENT OUTCOMES FOR CHILDREN WITH HIGHER-RISK MYELODYSPLASTIC SYNDROME UNDERGOING ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION Y. Ren1,*, F. Liu1, X. Chen1, X. Li1, L. Liu1, W. Yang1, Y. Guo1, X. Zhu1 1Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China Background: Myelodysplastic syndrome (MDS) are rare clonal hematopoietic disorders in children. Risk stratification system for adults is unfit for children. Refractory anemia with excess blasts(RAEB), acute myeloid leukemia with myelodysplasia-related changes(AML-MRC), monosomy 7 and complex karyotype always indicate poor prognosis. Hypomethylating agent (HMA) improved survivals of high-risk MDS in adults, and decitabine as part of conditioning regimen for allogenetic hematopoietic stem cell transplantation(allo-HSCT) was associated with favorable outcomes. However, these findings have not been tested in pediatric cohorts broadly. Aims: To evalute the efficacy and safety of hypomethylating agents as a part of pre-transplant treatments and contidioning regimens in children with higher-risk MDS. Methods: Patients from 1 to 14 years of age who were diagnosed with higher-risk MDS and underwent allo-HSCT consecutively between February 2019 and August 2020 in Pediatric Blood Diseases Center were enrolled. All identified patients were retrospectively reviewed with a collection of clinical data, laboratory test results, and outcome. Results: We reviewed 18 pediatric patients with higher-risk MDS, including 7 with RAEB, 8 with AML-MRC, and 3 with refractory cytopenia of childhood with monosomy 7. Regardless of initial diagnosis, monosomy 7 is the most common cytogenetic aberration (8/18, 44.4%). Next-generation sequencing (NGS) was performed on 15 specimens and the most frequent detection of mutation gene was SETBP1, followed by ETV6. Before transplantation, ten patients received monotherapies or combined chemotherapies with HMA, two patients received AML-like chemotherapies, and nine of those achieved bone marrow complete remission. The stem cell sources came from matched sibling donor (n=1), haploidentical donor (n=11), and unrelated cord blood (n=6), respectively. Low-dose decitabine-containing myeloablative conditioning regimens were administered in all cases. About 50% of patients (n=9) experienced Grade II- IV acute graft versus host diseases post-transplant. The cumulative incidence of transplantation-related mortality (TRM) was 11.1%. During a median follow-up of 10.5 (range, 2–36) months after transplantation, 15 of 18 patients were alive. At time of last follow-up, all survivors had resolution of hematologic disorder without relapse. Summary/Conclusion: In summary, administration of hypomethylating agents to allogeneic SCT resulted in excellent outcomes in children with higher-risk MDS. P780: COMBINATION THERAPY OF ELTROMBOPAG AND CYCLOSPORINE A CAN IMPROVE HEMATOPOIESIS IN PATIENTS WITH LOWER RISK MYELODYSPLASTIC SYNDROME L. Song1,*, C. Chang1 1Hematology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid disorders characterized by ineffective hematopoiesis and cytopenias, with variable risks of progression to acute myelogenous leukemia (AML). Peripheral blood cytopenias (thrombocytopenia, leucopenia, and anemia) are the major sources of morbidity and mortality for lower-risk MDS (LR-MDS) patients. Among the cytopenias, thrombocytopenia is a severe complication of ineffective hematopoiesis and is associated with poor overall survival.Eltrombopag (EPAG) is an orally administered small-molecule, nonpeptide thrombopoietin (TPO) receptor agonist, C-mpl is a TPO receptor expressed on megakaryocytes, HSCs, and progenitor cells. The interaction between EPAG and megakaryocytes regulates platelet production, maturation, and release through the binding of C-mpl on megakaryocytes. Aims: The above studies provided a rationale for the use of the combination of EPAG and CysA in the treatment of LR-MDS. In this study, we investigated the efficacy and safety of the combination of EPAG and CysA in LR-MDS in a non-randomized phase II investigator-initiated trial (IIT). Methods: Herein, a non-randomized phase II investigator-initiated trial was conducted to investigate the efficacy and safety of the combination of EPAG and cyclosporine A (CysA) in LR-MDS. EPAG dose was escalated from 50 mg/day to a maximum of 100 mg/day. The dose of CysA is 3–5 mg/kg/d to maintain serum trough concentration at 200 mg/mL. Results: All the 31 patients were evaluated for response at the primary endpoint. Among them, 21 (67.7%) patients achieved treatment response in at least one eligible lineage with an objective response rate (ORR), 4 (12.9%) patients achieved a CR, 5 (16.1%) patients achieved bi-lineage responses (HI-PE R), 11 (24.3%) patients achieved platelet lineage responses (HI-P R), and 1 (3.2%) patient achieved erythroid lineage response (HI-E R). The median time to response was 8 (range, 2–24) weeks. Of the 21 patients eligible for an erythroid lineage response (RBC transfusion-dependent or Hb <90 g/L),10 patients (47.6%) had responded by the endpoint and the platelet response rate was 64.5% (20/31). The median increase in Hb levels and platelets was from 60.4±18.2 g/L to 68.0±21.2 g/L (P=0.015) and from 12.3±11.2×109/L to 32.5±37.3×109/L (P=0.003). The median DORs of HI-E and HI-P were not achieved, but the response rates of erythroid and platelet lineages in 20 months were 80.0% and 70.6%, respectively. The RBC and platelet transfusion-dependent rate decreased from 42.9% (9/21) to 28.6% (6/21) (X2=0.933，P=0.334) and from 58.0% (18/31) to 25.8% (8/31) (X2=6.624, P=0.010). respectively. The median time to response is 8 weeks. All 31 patients were evaluated for toxicity, and the treatment was found to be well-tolerated. The most commonly reported adverse events (AEs) of any grade were nausea (6/31,19.4%), elevated liver transaminases (4/31,12.9%), and headache (3/31,9.7%); no severe AEs had been reported until the cutoff date. Summary/Conclusion: In conclusion, our results indicated that the addition of Epag to CysA is well-tolerated and is an effective treatment for patients with low to intermediate-1 risk MDS. These response rates are better than CysA monotherapy, especially HI-P. Also, the hematological responses are durable, although continued Epag administration might be necessary. Further large prospective and controlled studies are warranted to define the role of this combination treatment in LR-MDS in comparison to CysA P781: CLINICAL OUTCOMES AND HEALTHCARE RESOURCE UTILIZATION (HCRU) IN PATIENTS WITH LR-MDS REINITIATING ESAS FOLLOWING PREVIOUS ESA TREATMENT G. Garcia-Manero1,*, R. K. Matsuno2, A. McBride3, T. D. Brown2, D. Idryo2, R. Broome2, A. Herriman2, T. Johnson2, K. Wilkinson2, S. Walters2, A. Schrag2, C. Johanson2, M. Izano2, S. Mukherjee4 1MD Anderson Cancer Center, Houston; 2Syapse, San Francisco; 3Bristol Myers Squibb, Princeton; 4Cleveland Clinic, Cleveland, United States of America Background: The median survival for patients with lower-risk myelodysplastic syndromes (LR-MDS) is estimated to be 5–10 years. Erythropoiesis-stimulating agents (ESAs) are utilized as first-line treatment for anemia in LR-MDS; however, real-world treatment patterns, clinical outcomes, and healthcare resource utilization (HCRU) for patients with LR-MDS who reinitiated ESAs following previous discontinuation are not well-established. Aims: To examine treatment patterns, clinical outcomes, and HCRU in patients with LR-MDS who reinitiated treatment with ESAs. Methods: This was a retrospective study of patients with LR-MDS in a network of US community health systems who initiated ESAs as first-line therapy between January 1, 2016 and June 30, 2019 and were followed through June 30, 2021. Patients were required to have discontinued (had a ≥ 3-week gap between treatment with epoetin alfa, or a ≥ 6-week gap between treatment with darbepoetin alfa) and subsequently reinitiated ESA therapy at least once during follow-up. Outcomes included failure to achieve transfusion independence (TI), progression to acute myeloid leukemia (AML), overall survival (OS), number and type of health system visits, and medication use. TI was defined per IWG 2006 criteria in the subset of patients who had received ≥ 1 transfusion in the 8 weeks prior to ESA initiation. Results: There were 108 patients with confirmed LR-MDS who initiated ESA-based therapy. Patients had a median age of 79 years at diagnosis (interquartile range [IQR] 73–85), were predominantly male (58%), White (97%), and overweight or obese (52%). The median follow-up period was 17.0 months (IQR 7.2–34.7). The most used ESA was darbepoetin alfa (60%), followed by epoetin alfa (38%) and epoetin alfa-epbx (2%). Of the 33 patients who received a baseline transfusion, 52% did not achieve TI; 53% and 43% failed to achieve TI at 6 and 12 months, respectively. The majority of patients were repeatedly treated with ESAs (n = 61, 56%). Among these patients, 2% progressed to AML and 48% died during follow-up; median OS was 45 months. The median number of health system visits per patient-month was 3.2 (IQR 0.6–4.9); 89%, 62%, and 72% of pts had ≥ 1 outpatient, emergency department, or inpatient visit respectively, with median length of hospitalization of 12 days (IQR 4.8–27.8). Most patients (54%) received an antibiotic at least once during follow-up; overall, use of immunosuppressive therapy (2%) or iron chelation (3%) was rare. Summary/Conclusion: In this real-world study of community practice in the USA, reinitiation of ESA treatment after prior discontinuation in LR-MDS was prevalent. In these patients, poor clinical outcomes and frequent health system visits were observed. This study highlights the need to consider alternative treatment options for patients with LR-MDS. P782: IMMUNOPHENOTYPIC AND MOLECULAR GENETIC MARKERS OF UNFAVORABLE PROGNOSIS IN PATIENTS WITH MYELODYSPLASTIC SYNDROME H. Trubkina1,*, I. Iskrou2, V. Smolnikova3, T. Lebedeva3, I. Lendzina1 1hematology department №3, State Institution “Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology”; 2Department of Clinical Hematology and Transfusiology, Belarusian Medical Academy of Postgraduate Education; 3clinical laboratory STCM, State Institution “Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology”, Minsk, Belarus Background: Therapy is selected based on risk, need for transfusion of blood components, percentage of bone marrow blasts, and cytogenetic profile. In low-risk groups, the goal of treatment is to reduce the need for replacement therapy and prevent transformation to higher-risk or AML. In higher risk groups, the goal is to prolong survival. Currently, there are no approved criteria for selecting therapy for patients with MDS based on the classifications and prognostic scales used, treatment regimens for progression of MDS or its refractory forms. Aims: The aim of the study was to identify unfavorable prognostic immunophenotic and molecular genetic markers of blast cells in patients with high-risk myelodysplastic syndrome Methods: The prospective cohort study included 68 patients with newly diagnosed MDS in the period from January 2017 to December 2021. The patients underwent a primary diagnostic complex of MDS, including immunophenotyping and molecular genetic research of bone marrow aspirate. The age of patients is 25-82 years, the median age is 64 years. According to the IPSS classification included: high risk (10 pats), intermediate-2 (26 pats), intermediate-1 (26 pats), low risk (6 pats). According to the IPSS-R classification included: very high risk (23 pats), high risk (17 pats), intermediate (14 pats), low risk (12 pats), very low risk (2 pats). The following molecular genetic markers were found: complex aberrations >3 (10 pats), trisomy 8 chromosome (8 pats), absence of molecular genetic disorders (27 pats), isolated 5q chromosome (13 pats), any changes of the 7 chromosome (8 pats), deletion of the 20 chromosome (3 pats), deletion of the 11 chromosome (1 pats), TP53 (2 pats). To explore immunophenotic and molecular genetic markers we use three state “illness-death” model. We consider the following states: diagnosis, transformation into leukemia; death. Results: We identified high-risk immunophenotypic and molecular genetic markers - · for transition “diagnosis-death”: - CD38 <50% HR 3.7 (1.2-11.5 CI; p = 0.022), - CD13> 50% HR 8.7 (1.1-67.8 CI; p = 0.04), - complex aberrations >3 HR 5.8 (1.4-24.6 CI; p=0,015); - · for transition “diagnosis-transformation”: - CD71 ≥65% HR 4.1 (1.35-12.4 CI; p = 0.013); - CD13> 75% HR 2.8 (1.1-7.1 CI; p = 0.034), - complex aberrations >3 HR 2.8 (0,86-9,0 CI; p=0,087); - · for transition “transformation-death“: - CD25 <5% HR 6.3 (1.4-28 / 4 CI; p = 0.017), - CD 33 <50% HR 6.6 (1.3-34.7 CI; p = 0.026)), - complex aberrations >3 HR 0,22 (0,06-0,84 CI; p=0,027), - trisomy 8 chromosome HR 7.9 (0.71-88 CI; р=0,093). Figure 1 – The probability of various conditions depending on the time since the diagnosis. State 1 - estimation of the probability of being in the state «diagnosis-transformation» State 2 - estimation of the probability of being in the state «transformation-death» State 3 - estimation of the probability of being in the state «diagnosis-death» Image: Summary/Conclusion: The selection of groups of patients with identified immunophenotypic and molecular genetic markers of a high risk of transformation into acute leukemia and a high risk of death (“diagnosis-death”: CD38<50%, CD13>50%, complex aberrations>3; “diagnosis-transformation”: CD71≥65%, CD13>75%, complex aberrations>3; “transformation-death“: CD25<5%, CD33<50%, complex aberrations>3, trisomy 8 chromosomes) requires consideration of a new approach to therapy. P783: SYSTEMIC THERAPY UTILIZATION AND HEMATOLOGIC OUTCOMES IN LOWER-RISK MYELODYSPLASTIC SYNDROMES (LR-MDS): FINDINGS FROM A REAL-WORLD MEDICAL RECORD REVIEW STUDY IN THE US, UK, AND EUROPE (EU) M. Diez-Campelo1,*, A. Yucel2, R. Goyal3, R. C. Parikh3, S. Dhuliawala3, M. Jimenez3, M. Sluga-O’Callaghan4, C. Hughes5, D. Tang2, U. Germing6 1Hematology Department, Institute of Biomedical Research of Salamanca, University Hospital of Salamanca, Salamanca, Spain; 2Bristol Myers Squibb, Princeton, NJ; 3RTI Health Solutions, Research Triangle Park, NC, United States of America; 4RTI Health Solutions, Lyon, France; 5Bristol Myers Squibb, Summit, NJ, United States of America; 6University Hospital of Dusseldorf, Dusseldorf, Germany Background: Patients (pts) with LR-MDS experience a mean life-year loss of 6 years, and the treatment focus is improvement of quality of life and prolongation of life expectancy. Aims: This retrospective medical record review assessed real-world data to determine prevailing LR-MDS treatment patterns and hematologic outcomes. Methods: Eligible pts (≥18 years of age) with LR-MDS (Jul 1, 2013–Sep 30, 2018) were identified by participating hematologist-oncologists in the US, Canada (CAN), UK, and EU (France, Germany, Spain). Study measures and hematologic outcomes were descriptively assessed (data abstraction Jun–Nov 2021). Aggregated analyses used pooled US/CAN and UK/EU data sets for the overall population and pt subgroups treated with clinician-defined first-line (1L) and second-line (2L) erythropoiesis-stimulating agents (ESAs). Results: Medical record data were abstracted by 114 US/CAN (n=174 pts; median age 60.6 years; 68.4% male) and 166 UK/EU clinicians (n=319 pts; median age 67.1 years; 72.4% male). Median follow-up duration from MDS diagnosis was 60.6 months (US/CAN) and 67.6 months (UK/EU). Approximately half of the pts had “low” risk status (49.4% US/CAN; 53.9% UK/EU) categorized by the Revised International Prognostic Scoring System at initial diagnosis. The most common karyotype abnormality was del(5q) (27.6% US/CAN; 20.4% UK/EU). SF3B1 mutation was recorded for 7.5% US/CAN and 8.2% UK/EU pts. Almost all pts had received ≥1 line of systemic treatment for managing MDS-associated anemia (US/CAN: 165 [94.8%] 1L, 77 [44.3%] 2L; UK/EU: 294 [92.2%] 1L, 133 [41.7%] 2L). In 1L, ESA-based regimens were the most common treatment with median duration of therapy of ≥2 years (US/CAN: 142 [86.1%], of which the majority (80.3%) was ESA monotherapy; UK/EU: 264 [89.8%], comprising 82.2% ESA monotherapy). Treatment characteristics and outcomes are shown in the Table. Among pts who were transfusion dependent (TD) before 1L ESA, a very small proportion achieved transfusion independence (TI) or red blood cell transfusion reduction ≥50%, which was maintained for a median of <1.5 years. Among the 1L ESA-treated pts who were TD, TI was achieved by 5.7% (US/CAN) and 12.9% (UK/EU). In 2L ESA-treated pts (n=23 US/CAN; n=46 UK/EU), almost all were previously treated with a 1L ESA-containing regimen (95.7% US/CAN; 84.8% UK/EU). The most common reasons for reinitiating ESA in 2L (n=22, 15.5% US/CAN; n=39, 14.8% UK/EU) were overall health status (40.9% US/CAN; 33.3% UK/EU), treatment efficacy (40.9% US/CAN; 48.7% UK/EU), and compliance with national guidelines (13.6% US/CAN; 46.2% UK/EU). Among the pts who reinitiated ESA in 2L, the most common reasons for discontinuation of 1L ESA were completion of planned course of therapy (45.5% US/CAN; 28.2% UK/EU), pt decision (40.9% US/CAN; 20.5% UK/EU), progressive disease (36.4% US/CAN; 51.3% UK/EU), and adverse events (13.4% US/CAN; 15.4% UK/EU). Lenalidomide was used in 1L therapy among 8.5% pts in US/CAN and 5.8% in UK/EU. Among those with del(5q) mutation, ESA was used in 1L therapy among 83.3% pts in US/CAN and 87.7% in UK/EU. Image: Summary/Conclusion: ESA-based regimens were the most common treatment for anemia management in pts with LR-MDS; >1/3 of pts were TD before initiating 1L ESA therapy. A considerable proportion of 1L ESA-treated pts reinitiated ESA in 2L despite the majority having discontinued 1L treatment due to planned therapy completion, pt decision, or progressive disease. This real-world study suggests that there is an unmet need in addressing anemia burden with ESAs, and novel and effective treatments are needed. P784: A PHASE I/II STUDY OF VENETOCLAX IN COMBINATION WITH ASTX727 (DECITABINE/CEDAZURIDINE) IN TREATMENT‐NAÏVE HIGH‐RISK MYELODYSPLASTIC SYNDROME (MDS) OR CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) S. Venugopal1,*, H. kantarjian1, A. Maiti1, N. Short1, G. Montalban-Bravo1, Y. Alvarado1, K. S. Chien1, R. Kanagal-Shamanna2, N. Pemmaraju1, N. Daver1, T. Kadia1, G. Borthakur1, E. Jabbour1, G. Garcia-Manero1 1Leukemia; 2Hematopathology, The University of Texas, MD Anderson Cancer Center, Houston, United States of America Background: In MDS, hypomethylating agents (HMA) remain the standard of care. ASTX727, an oral fixed dose combination of HMA decitabine (35mg) and cytidine deaminase inhibitor cedazuridine (100mg), was recently approved in the US for the treatment of MDS and CMML. Venetoclax (Ven), an orally bioavailable BCL-2 inhibitor in combination with azacitidine has shown preliminary clinical activity in treatment-naïve, higher risk MDS Aims: Based on this data, we designed a study to evaluate a total-oral regimen of Ven+ASTX727 combination in pts with higher risk MDS or CMML Methods: This single arm Phase I/II study of orally administered ASTX727 in combination with Ven (NCT04655755) is enrolling patients ≥18 years with treatment‐naïve, higher risk MDS (intermediate-2- or high-risk categories) per IPSS or CMML with excess blasts ≥5%. The primary objective is to determine the safety and tolerability (phase 1) and overall response rate (ORR, defined as CR+mCR) (phase 2) of Ven+ASTX727 combination. ASTX727 is administered orally daily on D1‐5 and Ven is administered orally daily on D1‐14 of 28-d cycles. 3 dose levels of venetoclax in combination with ASTX727 will be tested. To mitigate tumor lysis syndrome, pretreatment white blood cell count should be less than 10 × 109/L. Cytoreduction is allowed. The safety population includes all patients who received any dose of Ven+ ASTX727, and the efficacy population includes patients who have a valid baseline and post-baseline disease assessment and had received at least one dose of the study drug Results: 15 pts have been enrolled to date (Figure). The median age is 72 years (range 54-94) with 12 pts aged ≥65 years. These patients had a median bone marrow blast count of 12% (range 6-15%) and harbored a median number of 4 (range 1-9) mutations. Most pts had adverse risk mutations such as ASXL1 (67%) and RUNX1 (47%). No DLTs were observed in the initial 6 patient safety lead-in. There were no deaths during the 30-day and 60-day window. No tumor lysis syndrome was observed. The ORR was 87% with 3 pts achieving CR (20%) and 10 pts achieving marrow CR (67%). All pts achieved a response within 1 cycle among which 4 pts, including one with TP53 mut,proceeded to hematopoietic stem cell transplant. 2 pts had stable disease at the end of 1 cycle. At a median follow up of 8.8 months, the median duration of response was not reached (range 0.9-11.1 months), and the median overall survival was not reached (range 1.0-12.1 months). Image: Summary/Conclusion: Ven+ASTX727 combination appears safe and demonstrates preliminary efficacy in pts with higher risk MDS or CMML with excess blasts. Total-oral regimen of Ven+ASTX727 combination appears to be a promising strategy for high-risk MDS or CMML pts and may alleviate the burden of chronic, long-term parenteral HMA treatment P785: CURRENT CARDIOVASCULAR DISEASE RISK PREDICTION MODELS ARE NOT APPLICABLE IN MDS PATIENTS: PRELIMINARY RESULTS OF A PROSPECTIVE OBSERVATIONAL SINGLE-CENTRE COHORT STUDY C. Misidou1, E. Gkolia2, C. Kymparidou1, V. Papadopoulos1, M. Papoutselis1, K. Liapis1, I. Mitroulis1, G. Vrachiolias1, S. P. Deftereos2, D. Stakos3, I. Kotsianidis1,* 1Department of Hematology, Democritus University of Thrace Medical School; 2Department of Radiology, University Hospital Of Alexandroupolis; 3Cardiology Department, Democritus University of Thrace Medical School, Alexandroupolis, Greece Background: Cardiovascular disease (CVD) is a leading cause of death in lower-risk Myelodysplastic syndrome (LR-MDS) patients and a shared pathobiology between MDS and CVD has been postulated. However, current evidence emanates only from retrospective cohorts suffering from crucial limitations. Aims: Prospective annotation can overcome these limitations and delineate the role of MDS as an independent CVD risk factor. For this reason we conducted a prospective observational single-centre cohort study in LR-MDS patients. Methods: Patients underwent thorough evaluation for CVD every 6 months. Cerebrovascular, peripheral and coronary vascular beds were assessed for atherosclerosis by ultrasound and coronary artery calcium (CAC), respectively. Cardiac structure and function was assessed by echocardiography. Multi-Ethnic Study of Atherosclerosis (MESA), Framingham (FRS) risk scores and serum markers of myocardial injury (Serum high-sensitivity troponin T, hsTnT), stress (N-terminal pro–B-type natriuretic peptide, NT-proBNP), and systemic inflammation (high-sensitivity C-reactive protein, hsCRP) were also estimated at each visit. Chi-square, Wilcoxon signed-ranks tests and Pearson’s correlation coefficient were used as appropriate. Multiple linear regression was applied for modeling the relationship between a dependent and one or more independent variables. The level of statistical significance was set to P=0.05. Results: Table 1 presents baseline characteristics of 24 patients included so far. There were no correlations of CAC, MESA and FRS with MDS subtype and IPSS-R risk category when adjusting for MDS-CI, preexisting CVD, age, sex, and transfusion dependence. In sharp contrast to the general population (Okwuosa, TM et al. JACC 2011), CAC score was not associated with FRS and was disproportionately increased in low and very low FRS risk patients (Figure 1). Likewise, the number of affected vascular beds (AVB) did not correlate with FRS (Figure 2), further suggesting that the current CVD risk prediction tools are inaccurate in MDS and arguing for an intrinsic tendency of MDS towards CVD development. In 9 out of 24 patients a second evaluation at 6 months was completed. All 9 patients increased significantly CAC (p=0.004) and MESA (p=0.003) scores at 6 months, while 5/9 patients also increased the number of AVB. The average increase of CAC score (189.77 +/-212.89) was markedly higher than expected in the age-matched general population (Budoff, MJ et al. JACC 2013) and was not associated with FRS at baseline, while it was also independent of MDS subtype and IPSS-R risk category after adjusting for MDS-CI, preexisting CVD, age, sex, and transfusion dependence. Finally, baseline NT-proBNP levels correlated strongly with CAC score (Figure 3) in line with numerous evidence showing that NT-proBNP can serve as a predictor of CVD risk in both MDS and non-MDS individuals. Image: Summary/Conclusion: To our knowledge this is the first study in LR-MDS patients assessing longitudinally all critical parameters and providing objective measurements of subclinical atherosclerosis in all vascular beds. Our preliminary results indicate that the established CVD prediction models do not operate in LR-MDS patients and reinforce the notion that MDS represents an independent factor for CVD development. Accurate assessment of CVD risk and prompt detection of subclinical cardiovascular disease in MDS patients will assist clinical decisions on the introduction of intensive monitoring and preventive use of CVD-specific interventions in these individuals. P786: ANTI-PD-1 ANTIBODY (SINTILIMAB) PLUS DECITABINE AS FIRST LINE TREATMENT FOR PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROME（MDS）: PRELIMINARY RESULTS FROM A SINGLE-ARM, OPEN-LABEL, PHASE II STUDY J. wang1,*, S. li1, H. Jiang1, Y. Chang1, X. Zhao1, J. Jia1, X. Zhu1, L. Gong1, X. Liu1, W. Yu1, X. Huang1,2 1Peking University People’s Hospital, Peking University Institute of Hematology. National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation; 2XiaoJun Huang, Correspondence.Peking University, People’s Hospital, Peking University Institute of Hematology, 11 Xizhimen South Street, Beijing, China Background: Hypomethylating agents (HMAs) are the preferred treatment for untreated patients (pts) with higher-risk MDS, but the survival of pts after HMAs treatment is poor. It has been demonstrated that the PD-1/PD-L1 expression was upregulated by HMAs in MDS pts, providing a strong rationale for combining HMAs with PD-1 antibody for MDS treatment. Aims: To evaluate safety and efficacy of Sintilimab plus decitabine for pts with higher-risk MDS in a single-arm, open-label,phase 2 study(ChiCTR2100044393). Methods: Adult pts with higher-risk MDS by the IPSS-R were enrolled. Patients received decitabine 20mg/m2 intravenously daily for 5 days and sintilimab 200mg IV starting on the first and 22nd day of a cycle every 42 days until unacceptable toxicity, relapse, or progression, for a maximum of 8 cycles. The primary endpoint was overall response rate (CR+PR+mCR). Simon’s optimal two-stage design was employed.If five or more pts in stage I (13 pts) achieved ORR, the study would enroll 34 additional pts in stage II (47 pts).Secondary endpoints included safety, and survival outcomes. The relationship between expression levels of immune-checkpoint and efficacy of the combination therapy, as well as other potential biomarkers were also explored via genomic profiling. Results: At data cut-off (February 22, 2022), 21 pts were enrolled with a median follow-up time of 6.5 months. The median age of pts was 64 years (range 30-83), and the other characteristics are summarised in Table 1.The ORR was 62%, with six pts reaching complete response (CR),four pts reaching marrow CR and three pts achieved marrow CR+ hematologic improvement (HI). In addition,four pts reaching HI,so the overall improvement rate was 81%. The most common grade 3 treatment-emergent adverse events(TEAEs)(>10%) were febrile neutropenia (76.2%) and pulmonary infection (38.1%). No grade 4 TEAEs and treatment-related deaths occurred. A total of 12 pts （57.1%）experienced immune-related adverse event, including rash (28.6%), pneumonia (9.5%), hypothyroidism (9.5%), elevated serum bilirubin (4.8%) and transpeptidase (4.8%), which were all resolved by glucocorticoid. In these 21 pts, the most frequently mutated genes are ASXL1（28.6%）， RUNX1 (14.3%) and TET2(14.3%). Updated biomarker data will be presented during the EHA meeting. Image: Summary/Conclusion: To the best of our knowledge, this is the first study to evaluate the efficacy and safety of sintilimab plus decitabine in pts with untreated higher-risk MDS. The preliminary results demonstrate that the combination therapy is relatively safe with anti-tumor activity. P787: SABATOLIMAB (MBG453) COMBINATION TREATMENT REGIMENS FOR PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROMES: THE MYELODYSPLASTIC SYNDROMES STUDIES IN THE STIMULUS IMMUNO-MYELOID CLINICAL TRIAL PROGRAM A. M. Zeidan1,*, A. Al-Kali2, U. Borate3, T. Cluzeau4, A. E. DeZern5, J. Esteve6, A. Giagounidis7, K. Kobata8, R. Lyons9, U. Platzbecker10, D. A. Sallman11, V. Santini12, G. F. Sanz13, M. A. Sekeres14, A. H. Wei15, Z. Xiao16, M. Van Hoef17, C. Nourry-Boulot17, I. Sadek18, F. Ma18, A. Iordan17, J. Sabo18, G. Garcia-Manero19 1Yale University and Yale Cancer Center, New Haven; 2Mayo Clinic, Rochester; 3Oregon Health & Science University, Portland, United States of America; 4Centre Hospitalier Universitaire de Nice, Nice, France; 5Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins, Baltimore, United States of America; 6Hospital Clinic, Barcelona, Spain; 7Marien Hospital, Düsseldorf, Germany; 8None, Los Angeles; 9The US Oncology Network, San Antonio, United States of America; 10University Hospital Leipzig, Leipzig, Germany; 11H. Lee Moffitt Cancer Center & Research Institute, Tampa, United States of America; 12University of Florence, Florence, Italy; 13Hospital Universitari i Politécnic La Fe; and CIBERONC, Instituto de Salud Carlos III, Valencia; Madrid, Spain; 14Sylvester Comprehensive Cancer Center, University of Miami, Miami, United States of America; 15Department of Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Australia; 16Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin, China; 17Novartis Pharma AG, Basel, Switzerland; 18Novartis Pharmaceuticals Corporation, East Hanover; 19MD Anderson Cancer Center, Houston, United States of America Background: Patients (pts) with higher-risk myelodysplastic syndromes (HR-MDS) unfit for hematopoietic stem cell transplant (HSCT) have poor outcomes with single-agent hypomethylating agent (HMA) therapy. Novel treatments with durable responses and survival benefit are needed. TIM-3 regulates innate and adaptive immunity and is expressed on leukemic stem cells (LSCs) and blasts but not normal hematopoietic stem cells. Sabatolimab is a novel immuno-myeloid therapy that binds to TIM-3 on immune cells, facilitating immune activation and phagocytosis of leukemic cells. Sabatolimab also binds to TIM-3 on leukemic cells, potentially impeding self-renewal of LSCs. Sabatolimab + HMAs had promising efficacy and response durability in HR-MDS in early-phase (ph) trials, with few significant immunologic adverse events (AEs; Brunner AM. ASH 2021. Oral 244). Aims: The STIMULUS immuno-myeloid clinical trial program investigates the safety, efficacy, and durability of sabatolimab-based combination therapies in patients with myeloid malignancies. Exploration of sabatolimab’s mechanism of action and identification of potential biomarkers predictive of response are planned. This abstract summarizes the design of 4 ongoing STIMULUS trials in previously untreated adult pts with HR-MDS ineligible for intensive chemotherapy or HSCT. Methods: STIMULUS-MDS1 (NCT03946670) is an international, randomized, double-blind, placebo-controlled, ph II trial evaluating sabatolimab + HMA in pts with very-high, high-, or intermediate-risk (vH/H/IR)-MDS who have completed enrollment (N=127). Primary endpoints are complete remission (CR) and progression-free survival (PFS). STIMULUS-MDS2 (NCT04266301) is an international, randomized, double-blind, placebo-controlled, ph III trial of sabatolimab + azacitidine (AZA) in pts with vH/H/IR-MDS or chronic myelomonocytic leukemia-2; the study has completed recruiting (N=530). Primary endpoint is overall survival (OS). Key secondary endpoints are time to definitive deterioration of fatigue; red blood cell transfusion-free interval; and improvement of fatigue, physical, and emotional functioning. STIMULUS MDS-US (NCT04878432) is a US-based, open-label, single-arm, ph II trial of sabatolimab + HMA of investigator’s choice (AZA intravenous [IV] or subcutaneous, decitabine IV, or oral decitabine/cedazuridine) in pts with vH/H/IR-MDS. Target enrollment is 90 pts. Primary endpoint is safety. Secondary endpoints include CR, PFS, leukemia-free survival (LFS), and OS. STIMULUS-MDS3 (NCT04812548) is an international, open-label, single-arm, ph II trial that explores triplet therapy of sabatolimab + AZA + BCL-2 inhibitor venetoclax (VEN) in pts with vH/HR-MDS. Part 1 is a safety run-in comprising 2 safety cohorts with ~6 pts receiving sabatolimab 400 mg + AZA + VEN and ~12 pts receiving sabatolimab 800 mg + AZA + VEN. Primary endpoint of part 1 is safety. If (both 400- and 800-mg doses) sabatolimab + AZA + VEN are safe, the trial will move to an expansion cohort (~58 pts) of sabatolimab 800 mg Q4W + AZA + VEN. Primary endpoint is CR. Secondary endpoints include CR + mCR rate, overall response rate (CR + mCR + PR + hematologic improvement), OS, PFS, LFS, and event-free survival. The STIMULUS immuno-myeloid clinical trial program is investigating the efficacy and safety profiles and the quality-of-life improvements for sabatolimab-based combination therapies in pts with myeloid malignancies. The STIMULUS-MDS trials aim to gain insight into the durable benefit of sabatolimab combination therapies in pts with HR-MDS. Results: None. Image: Summary/Conclusion: None. P788: FACTORS DRIVING TREATMENT DECISION IN PATIENTS WITH INTERMEDIATE-RISK MYELODYSPLASTIC SYNDROME (MDS): A RETROSPECTIVE ANALYSIS FROM THE GRUPO ESPAÑOL DE SÍNDROMES MIELODISPLÁSICOS SPANISH MDS REGISTRY M. Díez-Campelo1,2,*, L. Hernandez-Donoso3, E. Sasse3, S. Colicino3, J. Curto4, A. Molero Yordi5, M. Tormo Díaz2,6,7, M. Arnan8, G. Sanz9,10, M. Díaz-Beyá11, M. T. Cedena Romero12, A. Jerez13, D. Valcárcel Ferreiras2,5 1Hospital Universitario de Salamanca, Salamanca; 2Grupo Español de Síndromes Mielodisplásicos (GESMD), Valencia, Spain; 3Novartis Pharma AG, Basel, Switzerland; 4MFAR Clinical Research SL, Madrid; 5Vall d’Hebron Institute of Oncology (VHIO), University Hospital Vall d’Hebron, Barcelona; 6Hospital Clínico Universitario Valencia; 7INCLIVA Biomedical Research Institute, Valencia; 8Institut Català d’Oncologia, Hospital Duran i Reynals, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona; 9Hospital Universitario y Politécnico La Fe, Valencia; 10CIBERONC, Instituto de Salud Carlos III, Madrid; 11Hospital Clinic Barcelona, Barcelona; 12Hospital Universitario 12 de Octubre, Madrid; 13Hospital Universitario Morales Meseguer, Murcia, Spain Background: Current treatment (tx) options for patients (pts) with MDS are limited, although multiple potential txs are in development. The use of the Revised International Prognostic Scoring System (IPSS-R) for diagnosis of MDS supports tx decisions and prognostic assessment. Pts with very low/low-risk (vL/LR) MDS usually receive supportive care (SC) to manage symptoms, whereas those with very high/high-risk (vHR/HR) MDS commonly receive active txs such as hypomethylating agents (HMAs), allogeneic hematopoietic stem cell transplantation (alloSCT), or chemotherapy (CT). There is limited agreement on whether active tx or standard SC should be used for pts with intermediate-risk (IR) MDS. Aims: We aimed to identify factors possibly associated with the real-world use of tx in pts with IR-MDS in the Spanish MDS registry. Methods: This was a retrospective, observational study of data from pts registered in the Grupo Español de Síndromes Mielodisplásicos (GESMD) from January 2008-June 2020. Adult pts with MDS (WHO 2008 classification) who signed an informed consent and had a minimum follow-up of 6 months in the registry were included. Pts with unavailable data on IPSS-R risk or HMA tx were excluded. We collected demographic, clinical, and laboratory data, and tx used, including azacitidine (AZA), CT, alloSCT, SC (eg, transfusions, growth factors) and other tx (eg, immunosuppressors, androgens). Univariate analysis was performed to evaluate the association of potential risk factors, using categorical and continuous variables, with the tx use. We focused our analysis on AZA and SC in pts with IR-MDS ineligible for alloSCT. Odds ratio, 95% confidence interval, and exploratory P value were reported for comparison between tx use. Results: Overall, 4604 pts (median age: 76 y; male: 61%; IPSS-R score vHR: 432, 9.4%; HR: 487, 10.6%; IR: 761, 16.5%; LR: 1795, 39%; vLR: 1129, 24.5%) were included. Pts with IR-MDS were 63% male and similar in number by score and median age at diagnosis compared with the total study population: 3.5 (n=268, 73 y), 4.0 (n=270, 75 y) and 4.5 (n=223, 75 y). Most common treatments received were AZA (n= 241, 31.7%) and SC (n=250, 32.9%); a minority received CT and/or alloSCT (n=84, 11%), while 186 pts (24.4%) did not receive any. AZA was the only HMA used in this population. The median time from diagnosis to start of AZA in this group was 3 months. When comparing AZA with SC use in pts with IR-MDS, univariate analysis (Table 1) showed that gender, age at diagnosis, year of diagnosis, bone marrow and peripheral blood blasts, transfusion dependency, and hematologic parameters at diagnosis (eg, hemoglobin, platelet, and monocyte counts) were associated with tx selection. A multivariate analysis is ongoing for deeper understanding of these data. Image: Summary/Conclusion: These preliminary data show several factors significantly associated with tx use, suggesting that in some pts with IR-MDS, AZA is more likely to be used versus SC when these factors are considered. It is possible that selection bias, misclassification and confounding may have occurred as the registry only collects routine data according to local practices, with variability in the quality and completeness of data across participating centers. Other factors not captured, such as pt characteristics, comorbidities, access to health care, and social/familial support, could have also influenced the tx decision. Further analyses are ongoing to identify the relationship of multiple factors at once and assert the strength of these associations while removing the effect of confounders. P789: ANALYSIS OF THE CLINICAL FEATURES, PROGNOSIS AND GENE MUTATIONS IN PRIMARY MYELODYSPLASTIC SYNDROME PATIENTS WITH MYELOFIBROSIS G. Xu1,*, S. Zhu1, H. Tong1 1Hematology, Hospital, Hangzhou, China Background: Bone marrow(BM) fibrosis in myelodysplastic syndrome (MDS-MF) is observed approximately in 10–50% of primary MDS patients in the previous reports. BM biopsy is essential for the diagnosis and evaluation of MDS-MF patients and they differ from MDS without MF in terms of clinical performance, prognosis and survival. Although MDS-MF is associated with an adverse prognosis in the view of most hematologists, MDS-MF is not independently listed as a subtype of MDS according to the 2016 World Health Organization classification, indicating the need for further investigation. In this study, we retrospectively analyzed 417 newly diagnosed primary MDS cases in our hospital and to compare the clinical characteristics, prognosis and gene mutations of the two group patients with or without myelofibrosis. Aims: This study aims to analyze the clinical features, prognosis and characteristics of the gene mutations of the primary myelodysplastic syndrome with myelofibrosis (MDS-MF) patients, ultimately to improve the cognition of MDS-MF. Methods: From January 1, 2013, to February 1, 2020, 417 newly diagnosed primary MDS patients in the first affiliated hospital of Zhejiang University with bone marrow (BM) biopsy examination were included. They were divided into two groups according to whether BM associated with fibrosis (2005 European Myelofibrosis Network criteria), the MDS-MF group(MF grades 1–3）and the MDS without MF group (MF grade 0）.The clinical features, prognosis and gene alterations were analyzed between the two groups retrospectively. Results: ① MF was confirmed in 46.3％ cases of all the MDS patients（193/417）, of which 66.3％（128/193）were MF-1, 25.9％（50/193）were MF-2 and 7.8% were MF-3 respectively. There is no statistically significant difference in the proportion of subtypes patients according to the 2016 who classification (P=0.342). ②Compared with those without MF, MDS-MF were significantly associated with worse OS(P=0.035) and worse PFS(p<0.001). Subgroup analysis showed that both OS and PFS of either the MDS-MF1-2 group or the MDS-MF 3 group were also significantly worse than those of the MDS without MF group（P＝0.013 and <0.001 respectively）. ③The clinical features of the two groups were statistically significant differences in the higher number of BM blast cell(P=0.006), higher prevalence of death (P=0.031), higher rate of Leukemic transformation (P<0.001), higher level of serum lactate dehydrogenase (LDH) (p<0.001), higher level of β2-microgram (p=0.003) and higher level of CRP in the peripheral blood (P=0.004) in the MDS-MF group than in the MF0 group. ④Among all the patients,337 were finished Next-generation-sequencing (NGS) test. Using the same panel analysis, U2AF1 gene mutation was more frequently in the MDS-MF compared with those patients without MF (P=0.027). There was totally 14.2% (48/417) patients with the mutation of U2AF1, of which 19.7%（29/147) in MDS-MF group and 10.0%（19/190) in MDS without MF group. There was significant difference concomitant with U2AF1 mutation between the two groups(P=0.038). Image: Summary/Conclusion: MDS-MF has unique laboratory and clinical characteristics. MF is an independent risk factor for shorter overall survival (OS) and worse progression-free survival (PFS) in primary MDS. Evaluation of MF is very significant for prognosis judgment in primary MDS. The U2AF1 mutation may be a biomarker related to the poor prognosis of MDS-MF. However, the mechanism of U2AF1 in MDS-MF patients needs to be further explored. P790: THE EXPRESSION LEVEL OF WT1 MRNA IN PATIENTS WITH IDIOPATHIC CYTOPENIA OF UNDETERMINED SIGNIFICANCE (ICUS) PROGRESSING TO MYELODYSPLASTIC SYNDROME (MDS) X. Ye1,*, J. Huang1 1Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Background: Idiopathic cytopenia of undetermined significance (ICUS) is defined as patients with one or more unexplained cytopenia who do not meet diagnostic criteria for myelodysplastic syndrome (MDS) or another hematologic disorder. In the clinical course of ICUS, some patients eventually have typical MDS changes. Until now, there is no effective method to predict the development of the disease. Recent studies have shown that the expression level of WT1 mRNA in peripheral blood has important clinical significance in the diagnosis, occurrence, development and prognosis of MDS. At the same time, some studies have proved that there is a significant difference in the expression level of WT1 mRNA between MDS and hemocytopenia caused by other reasons. Aims: In order to clarify the clinical significance of WT1 mRNA expression in the progression of ICUS to MDS. Methods: Between April 2017 and January 2020, untreated adult patients with ICUS or lower-risk MDS were enrolled in the study. All patients detected the WT1 mRNA expression of bone marrow and peripheral blood at diagnosis. Then patients with ICUS were followed up. Every three months, the patients with ICUS detected the WT1 mRNA expression of peripheral blood, until the patient progressed to MDS. The results of WT1 transcript levels are depicted as WT1 (copies/mL)/c-ABL (copies/mL) as a percentage. Results: Total of 80 patients with ICUS and 40 patients with lower-risk MDS were enrolled. At diagnosis, the median expression level of WT1 mRNA in MDS group was 5.99 (0.08-79.27), and that in ICUS group was 0.50 (0.16-22.48). There was significant difference between them (P = 0.000). Then 80 patients with ICUS were followed up to January 2022. The median follow-up time was 30 months (11-54 months). 8 patients developed MDS, the progression rate was 10%, and the median progression time was 6.5 months (2-19 months). The median expression level of WT1 mRNA at the initial diagnosis in ICUS group without progressing to MDS was 0.44 (0.16-7.58), and that in progressing to MDS group was 9.44 (0.26-22.48). There was significant difference between the two groups (P = 0.031). The median expression level of WT1 mRNA was 19.185 (0.73-42.30) when MDS was diagnosed. The median maximum expression level of WT1 mRNA in peripheral blood of ICUS group without MDS was 0.98 (0.22-7.58). There was significant difference between the two groups (P = 0.007). Summary/Conclusion: The study clarified the WT1 mRNA expression status of ICUS patients. There were significant differences in WT1 mRNA expression level between patients with ICUS and lower-risk MDS, and the WT1 mRNA expression level of patients with lower-risk MDS was relatively high. The study confirmed that patients with high WT1 mRNA expression level in the patients with ICUS (WT1 mRNA expression level > 9.44) are very likely to progressing to MDS; Dynamic monitoring of WT1 mRNA expression in peripheral blood is an effective method for following-up after ICUS diagnosis. The progressive increase of WT1 mRNA expression is one of the indicators for researchers to judge the progress of ICUS to MDS. P791: COMMON VARIANTS IN COMPLEMENT PROTEINS, C3 AND CR1, ENHANCE COMPLEMENT ATTACK ON PNH ERYTHROCYTES AND INCREASE RISK FOR EXTRAVASCULAR HAEMOLYSIS A. J. Baral1,*, C. Mckinley2, R. Fellows1, T. Hallam1, T. Cox1, D. Payne3, S. Richards2, A. Pike4, R. Kelly4, M. Griffin4, P. Hillmen2, K. Marchbank1, D. Kavanagh5, D. Newton6, C. Harris1 1Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne; 2Division of Haematology and Immunology, Leeds Institute of Medical Research, University of Leeds; 3Haematological Malignancy Diagnostic Service, St. James’s University Hospital; 4Department of Haematology, St James University Hospital, Leeds; 5Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne; 6Division of Haematology and Immunology, Leeds Institute of Medical Research, University of Leeds, Leeds, United Kingdom Background: In paroxysmal nocturnal haemoglobinuria (PNH), absence of glycosylphosphatidylinositol (GPI) anchors leads to loss of the complement inhibitor, CD59, on PNH erythrocytes (E) causing complement-mediated intravascular haemolysis. Treatment with anti-C5 anitbody (eculizumab or ravulizumab) rescues PNH-E but loss of another GPI-anchored regulator, CD55, leads to accumulation of C3 fragments on E and extravascular haemolysis (EVH) due to C3-driven erythrophagocytosis. Therefore, approximately 30% of patients still require blood transfusion. Deposition of C3b and generation of downstream inactivation fragments such as iC3b and C3dg is controlled by binding of complement receptor type 1 (CR1) to C3b with factor I (FI), forming a trimolecular complex (TMC). We hypothesise that polymorphisms in C3 and CR1 influence the efficacy of this inactivation process and dictate propensity for EVH. Aims: To determine the combined effect of C3-S/F and CR1 density polymorphisms in susceptibility to EVH by the inactivation of C3b/iC3b on PNH-E. Methods: Forty-two eculizumab-treated PNH patients were genotyped for their single nucleotide polymorphism in C3 (rs2230199,S/F) and CR1 (rs11118133,H/L). C3 loading on PNH-E of patients were measured by flow cytometry as part of the routine analysis at the Leeds Haematological Malignancy Diagnostic Service. Lactate dehydrogenase, haemoglobin levels and reticulocyte count were also recorded. Soluble CR1 (sCR1) constructs were generated using recombinant technology and C3 variants were purified from human plasma using classical chromatography techniques. Interactions between C3b and CR1 and formation of the alternative pathway regulatory TMC (C3b:CR1:FI) were analysed using surface plasmon resonance (SPR). Healthy donors were genotyped for their CR1 density polymorphism and relative CR1 expression on E was quantitated by flow cytometry. Impact of CR1 density polymorphism on C3b inactivation was studied using C3b-coated streptavidin beads and solubilised E from CR1 H/H or H/L donors and C3b breakdown to iC3b/C3dg was measured using flow cytometry. Results: There was a trend for higher mean percentage of C3 loading in patients who have the C3-S/S allele (n=28, 30%) compared to patients with the C3-S/F allele (n=12, 18%) and a patient who had the C3-F/F allele (7%). Patients with high (H/H) CR1 expression (n=25, 20%) showed a trend for lower mean percentage of C3 loading on PNH-E than patients with intermediate (H/L) CR1 expression (n=17, 32%). In patients with the C3-S/S polymorphism, haemoglobin levels were significantly lower (p=0.0132) and higher numbers of patients (20 of 29) had at least one transfusion event. Using SPR, we demonstrated that CR1 bound C3b-F with a higher affinity than C3b-S. This led to higher levels of TMC formation with FI and more effective decay of C3-F convertase (C3b-F:Bb) by CR1. Solubilised E from a CR1-H/H donor converted iC3b more effectively to C3dg compared to E from CR1-H/L donors. Summary/Conclusion: These data indicate that both C3-S/F and CR1 density polymorphisms may influence C3b loading on PNH-E, with C3 fragments influencing EVH risk. Weaker binding of C3b-S to CR1 led to a decreased regulatory TMC formation with FI and slower decay of C3-S convertase (C3b-S:Bb). These data also indicate that E from individuals with lower CR1 expression convert the iC3b to C3dg more slowly. Overall, these data demonstrate mechanisms that enhance erythrophagocytosis in susceptible PNH patients by increased C3 loading on PNH-E and a decreased in removal of phagocytic opsonin, iC3b. P792: CRISPR/CAS9-BASED MODEL OF HETEROZYGOUS CXCR4 WT/R334X MUTATION TO STUDY CELLULAR PHENOTYPES IN WHIM SYNDROME K. Zmajkovicova1,*, S. Pawar1, S. Maier-Munsa1, A. Badarau2, A. G. Taveras3 1X4 Pharmaceuticals (Austria) GmbH; 2Former Employee at X4 Pharmaceuticals (Austria) GmbH, Vienna, Austria; 3X4 Pharmaceuticals, Boston, United States of America Background: WHIM syndrome is a phenotypically heterogenous primary immunodeficiency characterized by Warts, Hypogammaglobulinemia, Infections, Myelokathexis, neutropenia and lymphopenia. WHIM syndrome pathogenesis is causally linked to heterozygous gain-of-function (GOF) mutations in the C-terminus of the chemokine receptor CXCR4, a master regulator of immune cell trafficking and homeostasis, causing desensitization defects and hyperactivation of downstream signaling. The most frequently reported mutation in patients with WHIM syndrome is c.1000C>T, which results in a C-terminal truncation of the receptor at position R334 (R334X). Aims: In vitro assays using cell lines with exogeneous overexpression of CXCR4 WHIM variants can model the GOF cellular phenotypes of WHIM syndrome but may not entirely mimic the condition in patients, who typically have one wildtype (WT) CXCR4 allele. We aimed to characterize a cellular model of homozygous and heterozygous CXCR4 R334X in the endogenous locus, to better understand the pathogenic impact of harboring mutations in one or both alleles. Methods: CRISPR-Cas9 platform was used to establish a model of heterozygous mutations found in patients with WHIM syndrome. Jurkat cell line (with endogenous expression of WT CXCR4) was edited to harbor the c.1000C>T/R334X mutation in a single allele (WT/RX) or in both alleles (RX/RX). Unedited parental Jurkat cell line and Jurkat cells with edited silent mutations (WT/WT) were used as controls. Results: Upon stimulation with C-X-C chemokine ligand 12 (CXCL12), RX/RX cell lines displayed an internalization defect (65% of CXCR4 receptors remaining on the cell surface at 100 nM CXCL12) compared to the parental (24%) and WT lines (20%). The RX/WT-expressing cells had an intermediate phenotype (43%). We analyzed the signaling responses downstream of activated CXCR4, for which GOF phenotypes had been reported in R334X patient cells. Calcium mobilization in response to CXCL12 was enhanced in cells harboring the R334X mutation, reaching a 2.5- to 3-fold higher maximum effect (Emax) compared to cells with WT CXCR4. ERK activation downstream of CXCR4 reached a higher amplitude (8-fold increase over baseline) and duration after stimulation with CXCL12 in all lines expressing R334X compared to the parental and WT/WT cell lines (6-fold increase). Presence of a single mutant allele seemed to confer the full GOF phenotype in both signaling readouts in this cell line. Migration of cells toward CXCL12 was significantly enhanced in R334X-expressing cells. Mavorixafor, a CXCR4 antagonist currently in clinical trials for the treatment of patients with WHIM syndrome, was active in an unbiased manner on a spectrum of CXCR4-related functions (ligand binding inhibition, calcium mobilization, ERK activation and chemotaxis) with comparable biological activity and potency in cells expressing the WT and R334X CXCR4 receptor. Summary/Conclusion: We established the first model recapitulating the heterozygous CXCR4WT/R334X mutations found in patients with WHIM syndrome using the CRISPR/Cas9 platform. This cellular model recapitulates the functional defects found in immune cells from patients with WHIM syndrome. When stimulated with CXCL12, WT/RX -expressing cells appeared to display full GOF phenotype in downstream signaling assays compared to RX/RX-expressing cells. This observation is consistent with the dominant inheritance pattern of WHIM syndrome. The present study brings several novel insights in CXCR4WHIM biology and enriches the toolbox of models available for studying WHIM syndrome. P793: SCREENING OF NATURALLY OCCURRING CXCR4 VARIANTS FOR IDENTIFICATION OF NOVEL PATHOGENIC MUTATIONS FOR WHIM SYNDROME S. Pawar1,*, I. Wiest1, S. Maier-Munsa1, B. Maierhofer1, N. Sondheimer2, A. G. Taveras3, A. Badarau4, K. Zmajkovicova1 1X4 Pharmaceuticals (Austria) GmbH, Vienna, Austria; 2Division of Clinical and Biochemical Genetics, The Hospital for Sick Children, Toronto, Ontario, Canada; 3X4 Pharmaceuticals, Boston, United States of America; 4Former Employee at X4 Pharmaceuticals (Austria) GmbH, Vienna, Austria Background: WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare, autosomal-dominant primary immunodeficiency marked by neutropenia and lymphopenia. Classically, WHIM syndrome pathogenesis is causally linked to a variety of heterozygous gain-of-function mutations in the C-terminus of chemokine receptor CXCR4, a master regulator of immune cell trafficking and homeostasis. As of February 2022, 18 variants in CXCR4 have been implicated in WHIM syndrome, while a substantial number remain unexplored for their association with the disease. Using in vitro functional assays, we previously found that impaired receptor internalization and enhanced chemotaxis are conserved in all CXCR4 WHIM variants and that defective internalization correlates with neutropenia, the most penetrant phenotype of patients with WHIM syndrome. We used this approach to confirm functional defects in primary cells isolated from patients harboring a novel variant, suggesting that the in vitro profiling approach can potentially drive identification of pathogenic variants. Aims: We aimed to functionally characterize multiple previously uncharacterized variants of CXCR4 using in vitro assays. We also examined allele frequencies of these novel variants to estimate the potential number of individuals harboring these variants with a long-term goal of determining the actual prevalence of WHIM syndrome. Methods: The CXCR4-negative K562 cell line was used as a model system to express 53 novel CXCR4 variants identified in patient and population databases (ClinVar, Ensembl, CentoMD, GnomAD) and genetic screening initiatives (Invitae PATH4WARD) found in the C-terminus (hotspot of CXCR4 WHIM mutations) as well as throughout the protein. The effects of these mutations on CXCR4 internalization and chemotaxis were studied in cells stimulated with the C-X-C chemokine ligand 12 (CXCL12). The in vitro functional parameters were compared with known pathogenic CXCR4 variants and were used to assign a cumulative score (impaired internalization + enhanced chemotaxis) of functional defect severity. Results: Out of 53 selected variants, 42 were missense (ms), 6 were frameshift (fs), 3 were nonsense (ns), and 2 were transcript variants (tv). Eighteen of the screened variants led to a decreased internalization of CXCR4 in comparison to the wild-type (WT) receptor upon stimulation with CXCL12 while 20 variants demonstrated enhanced chemotaxis of cells toward CXCL12. Overall, 34 variants showed defects in at least one CXCR4-dependent marker. Correlation analysis and scoring of the functional parameters in cells expressing mutated or CXCR4 WT revealed 17 variants (9 ms, 5 fs, 2 ns, 1 tv) that co-segregated with the known pathogenic WHIM variants. Finally, white blood cell counts in 3 patients harboring novel variants showed that CXCR4 internalization defects in vitro correlated with the severity of neutropenia. Summary/Conclusion: This is the first study characterizing functional impairments in naturally occurring CXCR4 variants via a pipeline of assays intended to predict pathogenicity. Seventeen CXCR4 variants were identified (several outside the C-terminus of CXCR4) that caused in vitro functional defects resembling those exhibited by known WHIM variants. Many (10/17) of these newly identified variants are present at high frequencies (4.4 × 10-4–8.0 × 10-6) in genomic databases. Potential association of these variants with WHIM syndrome is under investigation and is likely to expand current estimates of WHIM syndrome prevalence. P794: DIFFERENTIATION POTENTIAL AND RELATIVE GENE EXPRESSION LEVELS ARE CHANGED IN CFU-F FROM BONE MARROW OF APLASTIC ANEMIA PATIENTS AT THE ONSET OF THE DISEASE A. Dorofeeva1,*, I. Shipounova1, A. Luchkin1, Z. Fidarova1, E. Mikhailova1 1National Research Center for Hematology, Moscow, Russia Background: Aplastic anemia (AA) is a heterogeneous blood system disease characterized by the bone marrow (BM) aplasia in and pancytopenia. There are non-severe (NAA), severe (SAA) and very severe (VSAA) forms of AA. The main cause of AA is believed to be the damage to the hematopoietic stem cells (HSC) by autoreactive cytotoxic T-lymphocytes. Another reason for the AA development may be a dysfunction of the BM stromal microenvironment. One of the ways of study the BM stroma is the analysis of colony-forming units of fibroblasts (CFU-F). Aims: To determine the CFU-F concentration in BM of AA patients, to assess the differentiation potential of individual colonies and the expression level of genes associated with proliferation, differentiation, immune response, and maintenance of HSC in CFU-F from BM of untreated AA patients. Methods: The study included 38 AA (15 with SAA, 4 with VSAA and 19 with NAA) patients in the debut. VSAA was combined with SAA for the analysis. The control group included 22 healthy donors. BM was aspirated at diagnostic punctures in patients and planned exfusion from donors after informed consent. CFU-F cultures were obtained according to the standard method. The colonies number was counted, and total RNA was isolated. Individual CFU-F colonies were obtained by plating 15000 BM mononuclear cells per well of a 96-well plate in aMEM with 20% FBS. Wells with individual clones had been analyzed further. The differentiation potential of CFU-F colonies was determined by the level of marker gene expression after incubation in a medium with inducers of osteogenic or adipogenic differentiation. Gene expression was determined by TaqMan RT-PCR. Data are presented as mean±standard error of the mean. Differences were considered significant at p<0.05 using an unpaired Student’s t-test. Results: CFU-F concentration in the BM of AA patients was not altered (33.58±9.12 per 106 BM mononuclear cells versus 34.43±8.39 in donors). The differentiation potential of CFU-F clones from SAA patients was skewed to osteogenesis when compared with donors and NAA patients. The proportion of CFU-F clones that failed to differentiate was decreased in SAA patients, while in NAA patients the proportion of such clones increased in comparison with healthy donors. These data may indicate a change in the ratio of stromal precursors of different maturity in the BM of patients with NAA and SAA compared with healthy donors (Table 1). CFU-F cultures of SAA patients significantly differed from those of healthy donors in the expression level of 10 genes (Table 2). The similar changes in 5 of these genes (PDGFRA, PDGFRB, ANG1, CFH, NES) were also found in NAA, but, with the exception of NES, the changes were not significant. Most of these genes are associated with the HSC maintenance, thus, the identified changes could be the result of BM aplasia. The expression level of the other 5 genes in CFU-F of NAA patients did not differ from that in donors (FGFR1, IL1B, PPARG, HLA-DR) or tended to change oppositely than in SAA (IL10). These genes are functionally associated with immune response and differentiation. Possibly, the activity of these genes prevents the development of more pronounced aplasia in NAA patients. Image: Summary/Conclusion: CFU-F of NAA and SAA patients differ from CFU-F of healthy donors by their differentiation potential and gene expression. Changes in CFU-F of SAA patients are more pronounced, which may be associated with deeper aplasia. This work was supported by the Russian Foundation for Basic Research, grant no. 19-015-00280. P795: CHARACTERIZATION OF ERCC6L2 SYNDROME PATIENTS’ TRANSCRIPTOME S. P. Douglas1,2,*, I. Kaaja1,2, T. H. Räisänen1,2, E. Pitkänen1,3, O. Kilpivaara1,2,4, U. Wartiovaara-Kautto1,5 1Applied Tumor Genomics Research Program, Faculty of Medicine; 2Department of Clinical and Medical Genetics, Medicum; 3Institute for Molecular Medicine Finland (FIMM), University of Helsinki; 4HUSLAB Laboratory of Genetics, HUS Diagnostic Center, Helsinki University Hospital; 5Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, University of Helsinki, Helsinki, Finland Background: Biallelic germline mutations in ERCC6L2 cause a bone marrow failure syndrome with a tendency for acquiring somatic TP53 mutations and possible progression to acute myeloid leukemia with erythroid characteristics. The disease and some functions of ERCC6L2 have only recently been characterized but a lot remains uncovered. ERCC6L2 affects DNA repair, but also plays a role in immunoglobulin class-switching and mitochondrial function. Although the biallelic ERCC6L2 mutation is in all the patient’s cells, it seems to affect the hematopoietic cells most drastically. However, we have seen differences also in the metabolism and growth of the ERCC6L2-deficient patients’ skin fibroblasts. Constitutional changes in RNA expression levels and altered metabolism due to ERCC6L2 deficiency may also affect the bone marrow niche and help us understand the disease more profoundly. Aims: We aimed to characterize ERCC6L2 syndrome on the transcriptome level using patient-derived samples (blood, skin fibroblasts, lymphoblastoid cell lines [LCLs]). Methods: 32 blood samples were collected from 15 ERCC6L2 patients (some in multiple time points), and 6 healthy controls. Skin fibroblast cell lines from 4 ERCC6L2 patients, and 3 healthy controls were grown in duplicates. In total 14 samples were grown in high-glucose (25mM) media until about 80% confluence and cells were harvested after six hours of changing the media. LCLs derived from EBV-transformed PBMCs from 6 ERCC6L2 patients and 4 healthy controls were grown in duplicates in two different media compositions (in total 20 samples). 3’ RNA sequencing was performed on RNA extracted from all patient samples. Differential expression analysis was done with DESeq2 and pathway enrichment analysis with PathfindR. Results: ERCC6L2-patients and controls differ significantly in their mRNA expression levels in multiple cell types and enriched pathways differ between cell types. In the patient blood samples, global genome nucleotide excision repair and transcription-coupled nucleotide excision repair (TC-NER) pathways are overrepresented compared to healthy controls. ERCC6L2 has been suggested to be involved both in TC-NER and non-homologous end joining of DNA double strand breaks. Also neddylation, which is a ubiquitin-like posttranslational modification affecting gene expression, is highly overrepresented. Cellular response to chemical stress is also upregulated, which demonstrates the reported sensitivity of ERCC6L2-deficient cells to some chemicals. The patients’ skin fibroblasts have different expression patterns compared to healthy controls in pathways related to cell differentiation, response to stimuli and extracellular matrix composition. These results support our previous observations about impaired fibroblast growth and metabolism. Expression changes in fibroblasts can tell us about effects that the germline ERCC6L2 mutation can have on extra-hematopoietic cells and tissues in the absence of somatic TP53 mutations. LCLs from ERCC6L2 patients’ cells differ from healthy controls by various immune system pathways: the enrichment of complement activation, phagocytosis, B-cell receptor regulation, but also in their response to metal ions. Summary/Conclusion: Multiple cell types of ERCC6L2 patients’ cells differ significantly from healthy controls’ cells in pathways related to DNA repair, stress response, cell morphology and immune function. These enriched pathways demonstrate the diverse effects the germline ERCC6L2 mutation has on the patients’ cells. P796: ENRICHED CD16+ MONOCYTES DRIVE T LYMPHOCYTES CELL RESPONSES IN APLASTIC ANEMIA M. Ge1,*, H. Wu1, J. Huo1, Y. Shao1, Y. Zheng1 1Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China Background: Monocytes, an important component of innate and acquired immunity, encompass three subsets, the classical monocytes (CM, CD14++CD16-), intermediate monocytes (IM, CD14++ CD16+) and nonclassical monocytes (NCM, CD14+CD16++). IM and NCM are collectively referred to CD16+ monocytes, which has been studied in many inflammatory diseases. Whereas, the distribution and function of monocytes in acquired aplastic anemia (AA) remain unclear. Aims: This study aims to determine the distribution of peripheral blood (PB) and bone marrow (B M) monocyte subpopulations in AA and expound the role of CD16+ monocytes on T lymphocytes function. Methods: The proportions of monocyte subsets in the PB and BM of AA patients and healthy donors (HD) were analyzed by flow cytometry. CD16+ monocytes were isolated by magnetic beads and co-cultured with CD3+ T cells, afterwards, the basic biological characteristics of T cells including proliferation, activation and differentiation etc. were evaluated. Results: Results showed that the percentage of CD16+ monocytes in PB and BM was significantly increased in AA patients compared with that in HDs, and the proportion of CM was decreased. However, there was no difference between patients with SAA and NSAA. CD16+ monocytes were further divided into two subgroups, which named IM and NCM. In contrast with HD, the two subgroups were both elevated in AA, mainly the IM. After treatment, NCM and IM were reduced and CM was increased in the patients of complete and partial remission. Then the effect of CD16+ monocytes on T cells function were elaborated. Using a co-culture model, we discovered that CD16+ monocytes in AA patients had stronger ability to promote the proliferation of CD3+ T, CD3+CD4+ T and CD3+CD8+ T cells than that in HDs (Fig 1A, b-d). Furthermore we found that AA-CD16+ monocytes played an important role in the over activation of CD3+CD4+ T and CD3+CD8+ T cells (Fig 1B, b-d). As expected, AA-CD16+ monocytes were extremely potent inducers of Th1、Th17 and cytotoxic T cells (Tc1) differentiation in vitro (Fig 1C, b-d), nevertheless, there was no difference on the effect of monocytes on Treg between AA and HD. Image: Summary/Conclusion: This study provided that the expanded CD16+ monocytes, including NCM and IM，had a major role in driving T cell responses in AA patients. P797: METABOLIC PROFILING IN ERCC6L2 AND SHWACHMAN DIAMOND SYNDROME I. Kaaja1,2,*, S. P. Douglas1,2, T. H. Räisänen1,2, K. Haimilahti3,4, A. I. Nieminen5, E. Pirinen3,6, U. Wartiovaara-Kautto1,7, O. Kilpivaara1,8 1Applied Tumor Genomics Research Program, Faculty of Medicine; 2Department of Medical and Clinical Genetics, Medicum, Faculty of Medicine; 3Clinical and Molecular Metabolism Research Program, Faculty of Medicine; 4Stem Cells and Metabolism Research Program, Faculty of Medicine; 5Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki; 6Research Unit for Internal Medicine, Faculty of Medicine, University of Oulu, Oulu; 7Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, University of Helsinki; 8HUSLAB Laboratory of Genetics, HUS Diagnostic Center, Helsinki University Hospital, Helsinki, Finland Background: Germline mutations in genes involved in DNA repair, telomere maintenance, and ribosome biogenesis are established causes of bone marrow failure (BMF) syndromes. Biallelic ERCC6L2 germline mutations cause BMF and accumulation of somatic TP53 mutations in bone marrow, contributing to the disease progressing into acute myeloid leukemia. ERCC6L2 is tentatively indicated in DNA repair and mitochondrial function, however, understanding its function is still in its infancy. Aims: To particularly understand the role of mitochondrial function in ERCC6L2 syndrome, we compared cellular metabolism in ERCC6L2 syndrome and Shwachman Diamond syndrome (SDS), a ribosomopathy also prone to somatic TP53 mutagenesis. Our aim was to discover any underlying alterations in mitochondrial function and to perceive any differences between the two syndromes that are similar in phenotype but different in genotype. Methods: To study the effect of the ERCC6L2 and SDBS germline mutations on cellular metabolism in isolation from the effect of accumulating somatic mutations in the bone marrow, we examined patient-derived skin fibroblasts (ERCC6L2 syndrome, n=2; SDS, n=2) and healthy controls (n=2). Mitochondrial function was studied with Seahorse XFe96 Analyzer where mitochondrial oxygen consumption rate (OCR) was measured under different glucose and glutamine concentrations. Furthermore, we inhibited mitochondrial pyruvate carrier (MPC) to examine the metabolic compensation for mitochondrial pyruvate deficiency via Seahorse Substrate Oxidation Stress Test. Mitochondrial DNA (mtDNA) amount was quantified in patient samples using qPCR. To further investigate the metabolism in each BMF syndrome, untargeted metabolomic screening of ERCC6L2 (n=4), SDS (n=3) and healthy control (n=3) fibroblasts in normal and low-glucose conditions was conducted by liquid chromatography mass spectrometer. We cross-referenced differentially expressed (DE) metabolites with DE genes (3’ RNAseq) in a matched set of samples to identify important biological pathways. Results: Our results indicate impaired mitochondrial respiration under low energy availability in both ERCC6L2 syndrome and SDS, as both BMF syndromes showed decreased maximal respiratory and reserve capacity in low-glucose conditions. Equal mtDNA amount was confirmed in patient and control cells suggesting that the decline was not related to changes in mitochondrial content. In ERCC6L2, but not in SDS cells, maximal respiratory and reserve capacity improved upon the addition of L-glutamine. In addition, we observed a lower glutamine concentration in both ERCC6L2 and SDS in low-glucose conditions. MPC inhibition lowered the respiration in SDS cells, but not in ERCC6L2 cells, indicating that pyruvate may be compensated by other substrates, such as glutamine oxidation, in ERCC6L2 but not in SDS cells. Together, these findings imply increased consumption of glutamine in ERCC6L2 cells and possibly impaired glutamine utilization in SDS cells. Metabolomic screening and integration of metabolomics data with transcriptomics detected subtle trends indicating changes in TCA metabolite and gene expression as well as in nicotinate/nicotinamide metabolism. Image: Summary/Conclusion: We show altered mitochondrial function in low-glucose conditions in two BMF syndromes, and propose glutamine-dependency as a factor for challenged mitochondrial function under low energy substrate availability in ERCC6L2 syndrome. Further studies on the metabolic behavior of the hematopoietic stem cell and its niche in BMF syndromes are needed to understand the role of metabolism in the disease progression. P799: SPATIAL ANALYSIS IDENTIFIES A SPECTRUM OF IMMUNE DYSREGULATION IN ACQUIRED BONE MARROW FAILURE CONDITIONS R. Koldej1,2,*, A. Prabahran1,2,3, C. W. Tan2,4, M. Davis2,4, D. Ritchie1,2,3 1ACRF Translational Research Laboratory, Royal Melbourne Hospital; 2Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne; 3Clinical Haematology, Royal Melbourne Hospital; 4The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia Background: Poor Graft Function (PGF), manifested by multilineage cytopenias with complete donor chimerism post allogeneic stem cell transplantation, and acquired Aplastic Anaemia (AA) are acquired bone marrow failure syndromes. Both are thought to result from T cell activation resulting in excessive interferon-γ (IFNγ) production, which in turn suppresses haematopoiesis. In AA, this can promote clonal stem cell selection and potentially progression to myeloid malignancy. Despite similarities in the clinical presentation and proposed mechanism, no studies to date have compared the immunobiology of the bone marrow (BM) microenvironment in these conditions. Aims: To examine the immune microenvironment of the BM in archival AA and PGF BM trephines and identify common immunopathologies. Methods: Archival BM trephines were sourced from patients with PGF (n=20), AA at Diagnosis (AA_DX) (n=15), AA at Progression (AA_PROG) to MDS/AML (n=15) and normal controls (NC) (n=20). 6 x 300µm regions identified by dual CD3/CD45 immunofluorescent staining were analysed per BM trephine using NanoString GeoMX™ digital spatial profiling for the expression of 57 proteins with an immunology focused panel. Data was analysed using a Limma-Voom bioinformatics pipeline. Results: Significant dysregulation was identified across multiple proteins. Overall, 22 proteins had significantly different expression in AA_DX samples (13 up, 9 down) compared to NC with AA_PROG and PGF having 8 (4 up, 4 down) and 14 (7 up, 7 down) respectively. As would be expected given the hypocellular BM, expression of CD45 was significantly reduced compared to NC across AA_DX (adj P = 5.413e-10), AA_PROG (adj P = 8.299e-8) and PGF (adj P = 8.634e-9) groups. When AA_DX patients were stratified into those who progressed (n=6) vs those who did not (n=9), or when AA_DX vs AA_PROG was compared in patients with matched samples (n=6) there were no significantly differentially expressed proteins identified. This suggests that while AA provides an immune microenvironment that is permissive to MDS/AML pathogenesis, it may not directly impact on clonal evolution. Total monocytes were unchanged by CD11c expression. However, AA and PGF samples exhibited upregulation of CD163 compared to NC (AA_DX adj P = 4.5e-6; AA_PROG adj P = 1.2e-5; PGF adj P = 1.58e-8) suggesting an increase in monocyte/macrophage lineage cells. Expression of CD66b was also downregulated in AA_DX (adj P = 8.459e-24), AA_PROG (adj P = 1.193e-10) and PGF (adj P = 2.244e-14) suggesting a reduction in non-classical monocytes, neutrophils or granulocytic MDSCs. Importantly, VISTA was downregulated across AA_DX (adj P = 6.84e-27), AA_PROG (adj P = 1.1e-5) and PGF (adj P = 1.58e-8) and STING was downregulated in AA_DX compared to NC (adj P = 6.139e-13) with a trend towards reduced expression in PGF. Decreased expression of these key immunoregulatory proteins may have wide ranging effects on BM resident macrophages, dendritic cells, MDSCs and T cells leading to reduced immunoregulation and increased T cell activation/IFNγ production, ultimately resulting in stem cell depletion. Data on cell specific expression is currently being obtained to refine this model. Summary/Conclusion: Spatial analysis demonstrated that patients with AA and PGF exhibit similar patterns of protein expression likely resulting in a BM immune microenvironment of decreased immunoregulation. AA_DX samples exhibited a greater degree of dysregulation of multiple markers compared to PGF suggesting that these diseases represent a spectrum of immune dysregulation. P800: BLOCKADE OF COMMON GAMMA CHAIN CYTOKINE SIGNALING WITH REGN7257, AN INTERLEUKIN 2 RECEPTOR GAMMA (IL2RG) MONOCLONAL ANTIBODY, PROTECTED MICE FROM GRAFT-VERSUS-HOST DISEASE AND IMMUNE APLASTIC ANEMIA A. Le Floch1,*, K. Nagashima2, T. Norton2, L. Perlee2, A. Murphy2, M. Sleeman2, J. Orengo2 1Inflammation and Immune Diseases; 2Regeneron, Tarrytown, United States of America Background: Pathogenic T-cell responses in T cell-mediated diseases can be driven by cytokines of the common gamma chain (γc) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γc cytokines signal through their corresponding receptors, expressed primarily on immune cells (including T cells), that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling. Aims: To understand the roles of γc cytokines in driving graft-versus-host disease (GVHD) and bone marrow failure disorders such as immune aplastic anemia (AA), we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and we tested its ability to suppress pathogenic T-cell responses in mouse models of T cell-mediated disease. Methods: We evaluated the efficacy of REGN7257 in a xenogeneic mouse model of GVHD, where immunodeficient NOD-scid-IL2RGnull mice were engrafted with human peripheral blood mononuclear cells (huPBMC). Mice were treated with REGN7257 either prophylactically or therapeutically, and monitored for weight loss and survival, peripheral human T-cell engraftment as well as serum pro-inflammatory cytokine levels over time. In a separate experiment, mice were sacrificed at day 49 post-huPBMC injection (after 4 weeks of antibody treatment) for tissue analysis (liver, lung, skin and bone marrow) of immune cell infiltration (T cells and macrophages), inflammation and/or fibrosis. Results: In a xenogeneic model of GVHD, both prophylactic and therapeutic γc cytokine signaling blockade with REGN7257 effectively protected mice from weight loss and resulted in improved survival, by reducing T-cell expansion in blood and production of pro-inflammatory cytokines in serum. Consistent with the classic pathology of GVHD, lungs, liver and skin of control mice were highly infiltrated by T cells, while γc cytokine signaling blockade strongly reduced T-cell infiltration, with reduced tissue levels of pro-inflammatory cytokines. Importantly, therapeutic γc cytokine signaling blockade in established GVHD (i.e. when mice already showed weight loss) provided similar benefit. Blockade of γc cytokine signaling also led to a reduction in the severity of chronic GVHD, with decreased macrophage infiltration in liver and associated hepatic fibrosis. In this xenogeneic model of GVHD, hemoglobin levels and platelet numbers in blood were both reduced, indicating anemia and thrombocytopenia, respectively, which are two complications associated with aplastic anemia. In addition to peripheral pancytopenia, recipient mice that were engrafted with huPBMC also showed severe marrow aplasia. Importantly, this phenotype of aplastic anemia was prevented by blockade of γc cytokine signaling. Summary/Conclusion: Blockade of γc cytokine signaling with REGN7257 protected against T cell-mediated pathology in a xenogeneic GVHD mouse model that uniquely presents hallmarks of both acute and chronic GVHD, with T-cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. These data provide evidence of γc cytokines as key drivers of T cell-mediated responses, offering a potentially novel strategy for the management of T cell-mediated diseases, such as GVHD and immune AA. P801: EVALUATION OF COMMON GAMMA CHAIN CYTOKINE SIGNALING BLOCKADE WITH REGN7257, AN INTERLEUKIN 2 RECEPTOR GAMMA (IL2RG) MONOCLONAL ANTIBODY, ON IMMUNE CELL POPULATIONS ACROSS SPECIES A. Le Floch1,*, K. Nagashima2, D. Birchard2, H. Pan2, C. Korgaonkar2, T. Norton2, L. Perlee2, A. Murphy2, M. Sleeman2, J. Orengo2 1Inflammation and Immune Diseases; 2Regeneron, Tarrytown, United States of America Background: The common γ chain cytokine receptor (γc; IL2RG) family of cytokines includes interleukin 2 (IL2), IL4, IL7, IL9, IL15, and IL21. This set of cytokines exhibits broad pleiotropic actions on both the innate and adaptive immune system with each cytokine sharing the IL2RG chain as part of its signaling receptor complex. Mutations in the IL2RG gene result in X-linked severe combined immunodeficiency (XSCID) in humans, whereby patients present with dramatically diminished numbers of T cells and NK cells, and dysfunctional B cells. Aims: Given the crucial role for γc cytokines in the development and function of lymphocytes, modulating their activities may offer therapeutic potential in a range of immune-mediated diseases. To understand the effects of γc cytokine blockade on immune cells including T-cell subsets, we utilized REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and tested its ability to suppress immune cell populations and their functions across species (mouse, cynomolgus monkey [CM] and human). Methods: We evaluated the effects of γc cytokine signaling blockade with REGN7257 on immune cell populations in Il2rg hu/hu mice and CM by flow cytometry. Mixed lymphocyte reaction assays were performed to look at the impact of γc cytokine signaling blockade on activation/proliferation of human T cells. To further analyze the contribution of γc cytokines to T-cell differentiation and function, we performed in vitro transcriptomic studies on human peripheral blood mononuclear cells stimulated with anti-CD3/CD28 beads. Results: γc cytokine signaling blockade with REGN7257 in Il2rg hu/hu mice efficiently reduced circulating B, NK and T cell populations, with no changes in blood neutrophil counts. In addition, the impact of γc cytokine signaling blockade on lymphocyte populations in vivo were investigated in CM in a single dose pharmacokinetic/pharmacodynamic study and a repeat-dose toxicology study. Inhibition of γc cytokine signaling with REGN7257 in CM led to similar phenotypic changes to that observed in Il2rg hu/hu mice, with decreases in peripheral T cells and NK cells, without impacting B cells, granulocytes, platelets or red blood cells. γc cytokine signaling blockade led to a reduction in both CD4+ and CD8+ T cell counts with effector memory T cells being the most impacted T-cell population studied. Furthermore, blockade of γc cytokine signaling led to reduction in activated and proliferating T cells in CM and demonstrated potent inhibition of allogeneic responses in mixed human lymphocyte reaction assays, by preventing T-cell activation and proliferation. Two-tailed gene set enrichment analysis identified 4 distinct patterns of significantly enriched gene sets that were impacted by γc cytokine signaling upon CD3/CD28-induced activation of human T cells: gene sets involved in inflammatory responses, differentiation and proliferation, activation and immune responses, as well as those related to adhesion and migration. These signatures were blocked with REGN7257 treatment. Summary/Conclusion: Taken together, these pharmacological observations highlight the major role for γc cytokine signaling in maintenance of lymphocyte populations (i.e. NK cells and T cells) in mouse and CM, but not other immune cell populations (i.e. granulocytes, platelets and red blood cells). Furthermore, our data highlight the importance of γc cytokines in driving functions of CM and human T cells, opening a potential new route for the management of T cell-mediated diseases. P802: PATTERNS OF T-CELL AUTOREACTIVITY DIFFER BETWEEN PEDIATRIC APLASTIC ANEMIA AND HEPATITIS-ASSOCIATED BONE MARROW FAILURE M. Nováková1,2,*, M. Svatoň1,2, A. Skotnicová1, M. Suková2, L. Řezníčková1,2, T. Valová1,2, D. Pospíšilová3, O. Fabri4, P. Švec4, J. Trka1,2, O. Hrušák1,2, J. Starý2, E. Mejstříková1,2, E. Froňková1,2 1CLIP-Paediatric Haematology and Oncology; 2Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague; 3Department of Pediatrics, Palacky University and University Hospital Olomouc, Olomouc, Czechia; 4Department of Pediatric Hematology and Oncology, National Institute of Children’s Diseases and Medical Faculty, Comenius University, Bratislava, Slovakia Background: Both idiopathic aplastic anemia (AA) and hepatitis-associated bone marrow failure (HABMF) are considered to be autoreactive T cell mediated diseases. This is based on indirect evidence, most often the success of immunosuppression or oligoclonality with T cell repertoire restriction. Aims: Our questions were: Does the composition of T cells and T-cell receptor beta (TRB) repertoire differ between HABMF, BMF without preceding hepatitis (nonHABMF) and normal bone marrow (BM)? Are there any shared clonotypes suggesting common antigenic stimulus? How does immunosuppressive therapy affect T-cells and does this correlate with outcome? Methods: In total 22 pediatric patients diagnosed in 2004-2021 with HABMF were included in the study. Sixteen HABMF patients were treated with immunosuppressive therapy (IST) as a frontline treatment, of which 8 patients did not respond. As a control group, we included 10 nonHABMF patients with AA (n=6) and refractory cytopenia of childhood (n=4), all treated with IST with poor response in 6 patients, as well as 10 samples of healthy donor BM grafts and 8 newborn peripheral blood (NBPB) samples. We performed flow cytometry immunophenotyping at diagnosis (D0) and on day 120 (D120) after initiation of IST in HABMF and nonHABMF patients. We performed sequencing of the TRB gene rearrangements according to the EuroClonality-NGS working group protocols using DNA isolated from these samples and normalized DNA input for TRB library preparation to the equivalent of 20 000 CD3+ cells per sample based on flow cytometry data, if possible. Whole exome sequencing (WES) was performed from diagnostic samples of HABMF patients. Results: Patients with HABMF had a significantly lower proportion of CD3+ T cells in their BM compared to the nonHABMF with the predominance of CD8+ T cells and their activation by expression of HLA-DR observed with flow cytometry. The analysis of TRB repertoire was performed in 22 D0 and 10 corresponding D120 samples of HABMF patients that had undergone IST and we compared the diversity and clonotype composition with the nonHABMF patients as well as the BM graft samples and NBPB. Expansion of individual TRB clonotypes (>5% of all reads) at D0 was observed more frequently in HABMF patients (9 out of 22) compared to the nonHABMF patients (1 out of 10). None of these expanded clonotypes were shared among more patients based on their nucleotide or amino acid sequence. There was no correlation of expanded T cell clone dynamics and response to IST. The diversity of TRB repertoire was reduced in the nonHABMF group after IST, however we did not observe any correlation between the clinical response to the IST and initial TRB diversity in either group of patients. WES did not reveal any pathogenic variants in genes associated with immune dysregulation. Summary/Conclusion: Both the proportion of T cells and T-cell composition in the BM differed between HABMF and nonHABMF patients at diagnosis. Although we observed an expected reduction in the CD3+ cells after IST both in the HABMF and nonHABMF group, there was no significant difference in the TRB repertoire diversity in the HABMF patients between D0 and D120. We did not observe any correlation between the repertoire diversity and clonotype evolution and the clinical response to IST. Our data further support the hypothesis that T-cells play a significant role in both AA and HABMF, but suggest that the pathogenetic mechanism of both entities is different. Supported by NV20-03-00284, NV19-05-00332, UNCE/MED/015, NU20J-07-00028 and GAUK 534120. P803: TELOMERE SHORTENING IN BONE MARROW MESENCHYMAL STEM CELLS OF ACQUIRED APLASTIC ANEMIA PATIENTS ASSOCIATE WITH ALTERED EXPRESSION OF GENES INVOLVED IN TELOMERE MAINTENANCE, DNA DAMAGE, AND SENESCENCE J. Srivastava1,*, P. Saxena1, N. Tripathy1, S. Nityanand1, C. P. Chaturvedi1 1Stem Cell Research Centre, Department of Hematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India Background: Telomeres are repetitive nucleotide sequences that protect the chromosome ends from DNA damage. Compelling data suggest that telomere attrition is associated with alteration in the DNA damage pathways, thereby inducing cell senescence. Idiopathic acquired aplastic anemia (AA) is a bone marrow (BM) failure disease characterized by pancytopenia and a hypoplastic fatty marrow. Several studies have reported shortening of telomere length (TL) in hematopoietic stem cells (HSCs) and lymphocytes in AA patients. The bone marrow mesenchymal stem cells (BM-MSC), which are the key cells of the BM niche, have garnered lots of interest as they are found to be functionally impaired in AA patients. However, a study highlighting telomere dysfunction in the BM-MSC of AA patients remains obscure. Therefore, we aims to study the telomere length shortening and its correlation with the alteration of genes involved in the telomere maintenance, DNA damage, and cellular senescence in AA patients. Aims: The study aims to evaluate the telomere length in BM-MSC of AA patients in comparison to that of controls. Further, it correlates the telomere length shortening with the alteration of genes involved in telomere maintenance, DNA damage, and cellular senescence in Bm-MSC of AA patients. Methods: BM-MSC were harvested from the BM of newly diagnosed idiopathic acquired AA patients (n=20) and healthy control (n=12) after informed consent. The telomere length and the expression of genes associated with telomere maintenance, DNA damage responses, and cell senescence of the BM-MSC of AA patients were analyzed by real-time quantitative-PCR (RT-qPCR). The correlation between telomere shortening and expression of telomere maintenance, DNA damage, and cellular senescence associated genes was done using Pearson’s correlation. Student’s t-test and Mann Whitney test were used to compare differences between groups. All the results were analyzed using GraphPad Prism software. The data was represented as mean ± standard deviation, and p-value <0.05 was considered significant. Results: Twenty patients with idiopathic acquired AA (11 non-severe aplastic anemia (NSAA) patients and 9 severe aplastic anemia (SAA)) patients and 12 healthy controls were included in the study. The TL was significantly shorter in the BM-MSC of AA patients [0.77 (Range: 0.4 – 1.56) as compared to that of healthy controls [1.40 (range: 0.71 – 3.22); p=0.002] (Figure 1). The TL in BM-MSC was not influenced by age (p=0.360), disease severity, and other hematological parameters. A significant alteration was observed in the expression of genes involved in telomere maintenance, DNA damage, and cell senescence with a positive correlation between the telomere length and telomere maintenance genes TRF2 (r=0.788; p=0.007), TPP1 (r=0.636; p=0.04) and TOP1 (r=0,676; p=0.03) as well as with gene involved in DNA damage responses CDKN1A (r= 0.667; p=0.03) and ATM (r=0.783; p=0.007). Image: Summary/Conclusion: This is the first study to highlight telomere shortening in the BM-MSC of AA patients. Furthermore, our study demonstrates that telomere shortening is in BM-MSC of AA patients is accompanied by altered expression of genes involved in telomere maintenance, DNA damage, and cellular senescence. These results shed new insight into that telomere attrition in the BM-MSC of AA patients could be attributable to DNA damage induced cell senescence or vice-versa. Overall, the findings of this study reveal that BM-MSC of AA patients potentially contributes towards the disease pathobiology. P804: EX VIVO TREATMENT WITH ELTROMBOPAG RESCUES HEMATOPOIETIC STEM CELLS FROM ANTI-THYMOCYTE GLOBULIN-RELATED DAMAGE DURING IMMUNOSUPPRESSIVE THERAPY IN PATIENTS WITH APLASTIC ANEMIA M. Vieri1,2,*, B. Rolles1,2, M. Crocioni1,2, S. Isfort1,2, J. Panse1,2, T. H. Brümmendorf1,2, F. Beier1,2 1Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation; 2Center for Integrated Oncology Aachen Bonn Cologne Düsseldorf (CIO ABCD), Uniklinik RWTH Aachen, Aachen, Germany Background: Immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG) and cyclosporine A is one first-line treatment option for patients (pts) with aplastic anemia (AA). One side effect of ATG is the release of interferon-gamma (IFN-γ), which is considered a major factor in the pathogenic autoimmune depletion of hematopoietic stem cells (HSC). Recently, eltrombopag (EPAG) was introduced for therapy of refractory AA pts due to its ability to improve HSC function. Clinical trials have evidenced that EPAG used alongside with IST leads to a higher response rate as compared to its later administration e.g. 2 weeks after ATG start. However, the potential underlying cause for this difference has not been clarified to date. Aims: We hypothesize that EPAG might protect HSC from negative effects of ATG-induced IFN-γ release. Methods: Sera of six pts with moderate to severe AA were collected pre-and post-ATG administration on days +1 and +2. Bone marrow (BM) of AA pts was collected and cryopreserved before ATG administration. In addition, we collected CD34+ cells isolated from five healthy donors (HD) undergoing HSC mobilization. HD-derived CD34+ or AA-derived BM cells were cultured in presence of pre as well as post-ATG serum from pts with AA, in combination with 5 µM EPAG and/or an IFN-γ neutralizing antibody (NAB; 2.5 µg/mL). After 48 h, colony forming unit (CFU) assay with cultured cells and intracellular staining of phospho-SMAD2/3 (known to be down-regulated in response of IFN-γ) were performed, while IFN-γ was measured via flow cytometry in collected sera. Results: Serum IFN-γ levels were 0.56 ± 0.6 pg/mL (mean ± standard deviation) post-ATG, whereas they were 0.015 ± 0.04 pg/mL pre-ATG (n= 6; p=0.08). When we tested effects of ATG sera on the clonogenic potential (CP) of HD HSC (n=5), a significant decrease of the number of CFU colonies (NoC) treated with post-ATG (148 ± 75) sera was found compared to pre-ATG treatment (201 ± 75, p= 0.032). This negative effect was mitigated by adding EPAG to the post-ATG sera (223 ± 100; p=0.036). To specifically analyze the potential suppressive role of IFN-γ, we added a IFN-γ NAB to the post-ATG serum (n=4), which also significantly increased colony formation (NoC 225.7 ± 33.4) compared to 140.1 ± 9.3 post-ATG without AB (p=0.014). Furthermore, phosphorylation of phospho-SMAD2/3 was found to be significantly lower in post-ATG (2052 ± 428, mean fluorescence intensity, n=4) compared to pre-ATG (3323 ± 32; p=0.03) treated cells. This effect was partially rescued by adding EPAG to the post ATG sera (2247 ± 627, p=0.239). Finally, we analyzed whether EPAG could rescue AA BM in presence of their matching serum, pre- and post- ATG treatment (n=6). As expected for AA BM, the NoC obtained was substantially reduced. Adding post-ATG serum resulted in further impairment of their CP (NoC: 2.0 ± 1.6) compared to the pre-ATG serum (6.0 ± 6.7; p=0.04). Again, addition of EPAG to the post-ATG serum was able to rescue AA BM cells’ CP from such effect (NoC: 5.6 ± 4.6; p=0.04). Summary/Conclusion: We provide evidence that treatment with ATG has an initial negative impact on the CP of HSC from HD and AA pts, likely mediated through acute release of IFN-γ. The addition of both IFN-y-NAB as well as of EPAG protects HSC from this effect. These results indicate a potential explanation for the superior response rates of EPAG given concurrently with ATG-based IST compared to later application and suggest that administration of EPAG should be started simultaneously with IST in order to maximize its beneficial effect in AA pts. P805: ALEMTUZUMAB IN RELAPSED SEVERE APLASTIC ANEMIA: LONG-TERM RESULTS OF A PHASE II STUDY N. Aggarwal1,*, A. L. Manley1, J. Durrani1, R. Shalhoub2, O. Rios1, J. Lotter1, B. Patel1, C. Wu2, N. Young1, E. Groarke1 1Hematology Branch; 2Office of Biostatistics Research, National Heart, Lung, and Blood Institute, Bethesda, United States of America Background: Aplastic anemia (AA) is characterized by pancytopenia and a hypocellular bone marrow from immune-mediated bone marrow destruction. Immunosuppressive therapy (IST) is a good alternative to hematopoietic stem cell transplantation (HSCT), but relapse occurs in about 30% of cases, necessitating further therapy. We investigated alemtuzumab, a humanized IgG1 monoclonal antibody targeting CD52. We initially reported on 25 relapsed patients with 56% response. Here, we present long-term results of a total 42 patients. Aims: Assess the efficacy and long-term outcomes in patients with relapsed severe AA (SAA) who received alemtuzumab. Methods: Participants met Camitta criteria for SAA, had received at least one course of antithymocyte globulin (ATG)-based IST, and had relapsed. Alemtuzumab was administered intravenously (IV) (n=28) or subcutaneously (SC) (n=14). The primary endpoint was hematologic response at 6 months, with blood counts no longer meeting SAA criteria. Secondary endpoints included robust response (platelet or absolute reticulocyte count >50x109/L at 6 months), relapse, clonal evolution to myelodysplasia and leukemia, response to therapy after relapse, and survival. Patients were assessed annually, and additional AA therapy and transfusion dependence was also recorded. Cumulative incidence curves were used to estimate the probability of relapse among responders and clonal evolution, with death as a competing risk. Overall survival probabilities were evaluated using Kaplan-Meier curves. This trial is registered at clinicaltrials.gov (NCT00195624). Results: Patients were enrolled over 9 years with a median follow-up of 6 years. Median age was 32 years and 57% were female. Five patients (12%) had neutrophil count <0.2x109/L prior to receiving alemtuzumab. At 6 months, 18 (42%) patients had achieved a response, and 12 (29%) attained a robust response. Response was achieved in 15 (54%) of those who received alemtuzumab IV, versus 3 (21%) who received SC. Of non-responders or those off study at 6 months (n=24), 2 achieved a late response by 1 year. Another relapse occurred in 8 patients, and cumulative incidence at median follow-up was 39%. Of those who were refractory to or relapsed (n=28) after alemtuzumab, 4 (15%) proceeded to HSCT, 12 (43%) to other medical AA therapies, and 10 (37%) to both. Subsequent treatments included eltrombopag (n=13), r-ATG (n=8) CSA (n=6), androgen (n=3) or other (n=4); response to further treatment occurred in 2 (29%) of relapsed and 5 (38%) of refractory patients, with response unknown in 7. Clonal evolution occurred in 9 patients; cumulative incidence at median follow-up was 23%. Of these, 4 were non-responders. Evolution was high risk (morphological MDS, AML, or chromosome 7 abnormality) in 7 patients. Overall survival was 67% at median follow-up (Figure 1). At last follow-up, 10 of the 18 responders (56%) had durable response and were both transfusion- and AA therapy-independent. Patients had prolonged immunosuppression after alemtuzumab, with CD4+ T cell counts <200 in 81% of assessed patients at 6 months, 67% at 1 year, and 42% at 2 years. Image: Summary/Conclusion: Alemtuzumab is effective for relapsed SAA. The rate of hematologic response was greater with IV rather than SC administration. Most responders achieved durable long-term hematologic improvement, and the rate of clonal evolution was similar to that seen in our SAA patients treated with rabbit ATG. Immunosuppression can persist for years following alemtuzumab therapy, requiring long-term monitoring and antimicrobial prophylaxis. P806: IMPACT OF MARROW HEMOPHAGOCYTOSIS IN DIAGNOSTIC DEFINITION AND PROGNOSTIC SIGNIFICANCE IN ADULT POPULATION C. Almeida1,*, T. Cardoso2, A. Roque1, S. Moreira2, J. Cascais Costa2, N. Silva2, L. Santos2, C. Geraldes1 1Hematology Department; 2Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal Background: Hemophagocytic lymphohistiocytosis (HLH) is associated with high mortality and is an entity difficult to diagnose. Bone marrow hemophagocytosis (BMHF) is the most easily accessible histomorphological criterion, but per si is neither specific nor sensitive for the diagnosis of HLH and its clinical significance is still not clear, mainly in adult population Aims: To evaluate the clinical and prognostic significance of BMHF. Methods: Retrospective analysis of adult patients with documentation of BMHF between 2007 and 2020. The identification of the patients was carried out by searching the database by the following keywords: “phagocytosis”, “phagocyte” and “histiocyte”. For the definition of HLH, the HLH-2004 and the revised HLH-2009 criteria were applied. HScore values ​​>169 were considered highly suggestive of HLH. Stratified models for each of the risk scores (HLH-2004 and HLH-2009) were compared using Akaike’s information criterion (AIC) and the Harrell’s concordance index (C-index). Results: Ninety-five patients (pts) were included, 63.2% males with median age of 64 (20-95) years old. Twenty-two pts (23.2%) presented ≥5 HLH-2004 criteria and 24 (25.3%) HLH-2009 criteria, with only 6 patients (27.3%) with simultaneous diagnosis by both criteria (Cohen’s kappa 0.0253). Forty-three (45.3%) patients presented ≥3 HLH-2004 criteria and 34 pts (35.8%) had Hscore>169. After a median follow-up of 24.3 months, the median overall survival (OS) was 39.4 months. OS was significantly lower in patients with ≥5 HLH-2004 (0.9 vs 86.5 M; HR 3.56; p<0.001), with ≥3 HLH-2004 criteria (3.17 vs NR; HR 2.95; p<0.001), with HLH-2009 criteria (5.5 vs 78.0 M; HR 1.89; p=0.034), and HScore>169 (1.45 vs 86.5; HR 3.08; p<0.001), compared with those that do not have. Excluding variables included in the above scores, the only clinical/analytical variable that was statistically significant for OS in univariate analysis was LDH>UNL (HR 1.02; p<0.001). After multivariate analysis for age, aetiology and LDH>ULN, ≥5 HLH-2004 (HR 4. p<0.001), HLH-2009 2009 (HR 1.87 p=0.045), ≥3 HLH-2004 (HR 3.06; p<0.001) and Hscore>169 (HR 3.39; p=0.001), the four scores retain significance for OS. In patients with BMHF, HLH-2004 criteria discriminated best between patients with poor and favourable OS than HLH-2009 (C-index 0.6364 vs 0.5601; AIC 400.24 vs 411.53). Concerning the aetiology, 51.6% was neoplastic (24.5% [n=12] with ≥5 HLH-2004 criteria), 13.7% infectious, 28.4% autoimmune and in 28,4% the trigger was not identified. Summary/Conclusion: In our cohort, HLH-2004 criteria had the best predictive value for OS, compared with HLH-2009 However, in the absence of the data need to calculate these scores, both ≥3 HLH-2004 criteria and Hscore>169 are independent predictors of OS, which, due to the deleterious prognosis of HLH, support their utility to start treatment earlier P807: APLASTIC ANEMIA FOLLOWING THE SARS-COV-2 VACCINE R. Babakhanlou1,*, T. Kadia1, K. Chien1, P. Thompson1 1Leukemia, MD Anderson Cancer Center, Houston, United States of America Background: There have been several reports describing the possible relationship between the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine and the development of hematological diseases, such as autoimmune hemolytic anemias (AIHA), paroxysmal nocturnal hemoglobinuria or immune thrombocytopenic purpura. Limited data is available on the relationship between the SARS-CoV-2 vaccine and the development of aplastic anemia (AA). We report a series of four cases of acquired AA developing in patients, who had received an mRNA-based SARS-CoV-2 vaccine. Aims: To study the temporal association of COVID-19 vaccination and acquired aplastic anemia Methods: We reviewed the cases of all patients who presented to MD Anderson Cancer Center between January 2021 and December 2021 for pancytopenia. We identified patients who were diagnosed with aplastic anemia after receiving two doses of the SARS-CoV-2 vaccine and summarized baseline characteristics and clinical course. Results: Four patients with newly diagnosed AA were identified. All patients were male with a median age of 56 years (range, 19-69). Two patients (50%) had received the Pfizer-BioNTech vaccine and two (50%) the Moderna mRNA-1273 vaccine. One of the patients had contracted COVID 8 weeks prior to his vaccination. Three patients suffered from chronic medical problems including type 1 diabetes, hyperlipidemia, and hypertension. None of those patients developed any symptoms after the first dose of the vaccine. Symptoms occurred on average three weeks after the second vaccine and have been outlined in Table 1. Most common symptoms were fatigue, bruising and bleeding, development of petechiae, dizziness and shortness of breath. Specific autoimmune workup was done and all patients received baseline bone marrow aspiration and biopsy with cytogenetics and next-generation sequencing. In all four patients cytogenetics showed a diploid karyotype with no chromosomal abnormalities. Flow cytometry and molecular diagnostics did not show any abnormalities. In two cases the bone marrow was hypocellular and in two cases the bone marrow was acellular. All patients received combination immunosuppressive therapy with steroids, horse anti-thymocyte globulin, cyclosporine and eltrombopag, three on a clinical trial and one off protocol. Patients were on therapy for a median of 26 weeks (16-48). One patient achieved complete remission at 12 weeks. One patient has remained transfusion dependent, while the remaining two patients became transfusion independent at six months. Image: Summary/Conclusion: Although we cannot prove a causative relationship, in rare circumstances, the SARS-CoV-2 vaccine could be associated with the development of AA. Additional epidemiologic and laboratory studies are planned to further evaluate any potential causative relationship. P808: CONGENITAL NEUTROPENIA PREVALENCE AMONG POLISH CHILDREN – SUMMARY OF A NATIONWIDE GENETIC SCREENING CAMPAIGN K. Bąbol-Pokora1,*, W. Dobrewa1, M. Bielska1, J. Madzio1, S. Janczar1, W. Młynarski1 1Department of Pediatrics Oncology and Hematology, Medical University of Lodz, Lodz, Poland Background: Severe congenital neutropenia (SCN) is a heterogenous group of rare primary immunodeficiency disorders, characterized by impaired maturation of neutrophil granulocytes. Patients with severe congenital neutropenia are prone to recurrent gingivitis, mouth and rectal ulcerations, and often life-threatening bacterial infections. They are also predisposed to myelodysplastic syndromes or acute myeloid leukemias. Nine types of severe congenital neutropenia have been distinguished so far with the most frequent pathogenetic defects in the ELANE gene. This study explores the distribution and mutation spectrum of disease-causing genetic variants associated with congenital neutropenia in the Polish pediatric cohort and the observed ten-fold increase in the referral rate following a nationwide information campaign. Aims: The aim of this study was to estimate prevalence of congenital and autoimmune neutropenia among Polish children and to improve the efficiency of recruiting patients with suspected SCN by launching the FIX-NET project with a nationwide advertising campaign. Methods: Molecular analyses were performed either by direct Sanger sequencing (78 patients recruited in 2008-2015) or by Targeted NGS (201 patients recruited in 2016-2021) using Illumina platform, focused on the 54 genes related to SCN and neutropenia associated syndromes. Overall 279 neutropenic patients were enrolled in the studies and written informed consent has been given. The following results describe the largest cohort of genetic variation associated with SCN in Poland. Results: We identified pathogenic changes in genes associated to severe congenital neutropenia or related syndromes among 66 patients, most of which were variants in the ELANE gene (44%). There were a number of patients with pathogenic changes in CXCR4 (12%) and SBDS (11%) genes, and also in the recently described dominant SCN-related genes – SRP54 (8%) and CLPB (5%). Novel mutations accounted for 30% of all reported variants. In addition among 32 patients we found changes in genes that cause neutrophil dysfunctions or may lead to autoimmune neutropenia, such as AIRE, FAS, or CTLA4. Summary/Conclusion: Our study shows the prevalence of congenital neutropenia in Poland in the largest pediatric neutropenic cohort described so far. It indicates that since the introduction of NGS to the diagnosis of primary and secondary neutropenia, more genetic changes have been detected, however, the greatest impact on increasing awareness among doctors, and thus improving the effectiveness of recruitment had the nationwide information campaign. P809: MODULATION OF ARACHIDONIC ACID PATHWAY BY IBUPROFEN RESULTS IN COMPLETE HEMATOLOGICAL RESPONSE IN GHOSAL HEMATODIAPHYSEAL DYSPLASIA WITHOUT NEED FOR CORTICOSTEROID N. Barrett1,*, C. McDonnell2,3, M. Cotter4 1Paediatric Haematology, Royal Hospital for Children, Glasgow, United Kingdom; 2Paediatric Endocrinology, CHI Temple Street; 3Discipline of Paediatrics, Univerisity of Dublin Trinity College Dublin; 4Paediatric Haematology, CHI Temple Street, Dublin, Ireland Background: Ghosal hematodiaphyseal dysplasia (GHDD) is a rare autosomal recessive disorder characterized by pancytopenia and increased bone density of the long bones. First described in families of Indian and Middle Eastern descent, subsequent studies identified the genetic lesion in this disease as due to loss of function mutations in the TBXAS1 gene encoding thromboxane synthase, a key component of the arachidonic acid pathway responsible for the formation of thromboxane A2 from prostaglandin H2. As well as pancytopenia, laboratory features include platelet aggregation defects and increase in osteoblastic activity, the latter being the basis of increased bone density in patients with Ghosal syndrome. Simultaneously, loss of thromboxane synthase activity may be associated with shunting of prostaglandin precursors away from production of thromboxane into production of pro-inflammatory prostaglandins. This may be the mechanism responsible for the observed marrow suppression seen in these patients. We describe a 3 year old female of non-consanguineous parents of Filipino origin who presented with marked pancytopenia. Next Generation Sequencing bone marrow failure gene panel revealed a novel homozygous mutation in TBXAS1. Subsequent DXA scanning revealed increased bone mineral density consistent with the diagnosis (Z -score +2.4 for lumbar spine (L1-4)) although clinical sequelae of bony disease were not apparent. Aims: While multiple case reports document that these patients have hematological response to long term corticosteroid therapy, we hypothesized that inhibition of cyclooxygenase (COX) upstream of thromboxane synthase in the arachidonic acid pathway may be effective in this condition. Methods: Patient was treated with ibuprofen at regular therapeutic doses (approximately 30 mg/kg/24 hours daily initially) and serial full blood counts were monitored. Results: The patient exhibited a rapid and complete response to ibuprofen with normalization of full blood counts. The dose of ibuprofen was reduced to 10 mg/kg/day, and hematologic response is maintained on low dose after one year.The treatment is well tolerated with no side effects. Image: Summary/Conclusion: Treatment of GHDD with ibuprofen is a novel approach that offers clear potential benefits in comparison to the known adverse effects profile of long term corticosteroids. Bone density scan and markers are in process to ascertain the impact of this treatment on bone density, although this may take longer to show beneficial effects. The treatment was easily tolerated and well managed by the patient and family at home with minimum disruption to her quality of life. P810: BONE MARROW FAILURE IN PATIENTS CARRYING VARIANTS ON CARD11 GENE. A. Beccaria1,*, A. Grossi2, F. Fioredda3, M. Lanciotti3, E. Palmisani3, P. Terranova3, M. Lupia3, G. Dell’Orso3, I. Ceccherini2, C. Dufour3, M. Miano3 1DOPO Clinic and Hematology Unit, Division of Pediatric Hematology and Oncology; 2Laboratory of Genetics and Genomics of Rare Diseases; 3Hematology Unit, Division of Pediatric Hematology and Oncology, IRCCS Istituto Giannina Gaslini, Genova, Italy Background: An accurate differential diagnosis in children with Marrow Failure (MF) is crucial for the clinical management of the disease. In addition to the classical congenital MF (cMF), the role of Inborn Errors of Immunity (IEI) have been recently highlighted as potential cause of the disease. CARD11 is a membrane protein acting as a key signaling scaffold which controls the antigen-induced lymphocyte activation during the adaptive immune response (NF-kB, JNK and mTOR pathway). Germline CARD11 mutations can result in gain/loss of function of the protein leading to different phenotypes (SCID, BENTA disease, atopy, CVID). Several heterozygous hypomorphic/dominant negative (DN) variants of CARD11 have demonstrated a loss of protein function, exhibiting high penetrance and variable expressivity. In particular, mutations in the GUK (Guanylate Kinase) domain seem to be involved in modulating the self-inactivating capacity of CARD11. No CARD11 variants have ever been described to be associated with MF. Herein, we report 4 patients with MF carrying variants on the CARD11 gene. Aims: To evaluate the clinical/immunological features and genetic profile of patients with MF carrying CARD11 variants and followed in our Centre. Methods: A restrospective review of clinical, immunological and genetic data was performed from patients’ records. Molecular analysis was conducted using Next-Generation Sequencing (NGS), targeted panels including genes involved in both cMF and IEI, or by Whole Exome Sequencing (WES). Filtering of variants was performed according to Mendelian disease segregation and zigosity (OMIM), in silico prediction of pathogenicity (ACMG criteria in “Varsome”), and minor allele frequency (reported in GnomAD). Results: Four children carrying variants in the CARD11 gene presented with MF as initial sign of the disease (n=2) or with immune cytopenias further evolving into MF (n=2). Their clinical/immunological features, genetic profile, treatment and follow-up are reported in Table1. Lymphocytes subsets analysis revealed a deficiency of B memory, Tregs and an increased of HLADR+ T cells in all patients. Two cases showed elevated CD4-CD8- TCR αβ+ T-cells (“double negatives T-cells”). All patients showed autoimmune stigmata and 2 of them also presented autoimmune hepatitis. No patients responded to first-line treatment. One responded to second-line treatment with Mychofenolate-mofetil (MMF) and periodic infusion of immunoglobulins. The remaining 3 patients underwent hematopoietic stem cell transplantation (SCT) from haploidentical-αβCD19 depleted transplant (n=2) or HLA-identical cousin (n=1) (Table1). Image: Summary/Conclusion: This report highlights novel phenotypic characteristics of patients carrying variants in CARD11 such as MF and autoimmune hepatitis, thus widening the clinical spectrum of the disease. This underlines the need for an enlarged molecular analysis in pediatric cases of MF and suggests that, in some patients, immune-mediated destruction of blood and marrow cells cooperate in generating the cytopenia. Three variants detected in our cohort are located in the GUK domain of the gene, close to other reported pathogenic dominant negative mutations leading to haploinsufficiency, suggesting interference with the CARD11’s ability to self-inactivate. Functional study will be necessary to confirm the pathogenic role of such variants. Although treatment with MMF may be considered, SCT represents the only curative option for such patients. P811: A SINGLE CENTER HISTORICAL CONTROL STUDY OF ELTROMBOPAG ADDED TO IMMUNOSUPPRESSIVE THERAPY FOR SEVERE APLASTIC ANEMIA IN CHILDREN B. yang1,*, L. fu1, H. li1, H. chen1, J. ma1 1Hematology Oncology Center, Beijing Children Hospital, Beijing, China Background: Severe aplastic anemia is caused by immune-mediated destruction of bone marrow. Standard immunosuppressive therapy (rabbit anti-thymocyte globulin combined with cyclosporine) is effective, but the effect needs to be improved. A thrombopoietin-receptor agonist, eltrombopag, added to standard immunosuppressive therapy led to clinically significant increases in blood counts. We combined eltrombopag and standard immunosuppressive therapy in pediatric severe aplastic anemia patients. Aims: To evaluate the efficacy and safety of eltrombopag added to immunosuppressive therapy for severe aplastic anemia in children. Methods: The clinical data of patients with severe aplastic anemia and received immunosuppressive therapy from March 2013 to July 2020 were collected and analyzed retrospectively. We conducted a historical control study, in which the patients treated alone with immunosuppressive therapy were used as the control group. We compared the cases between control group and eltrombopag group, included hematological response, effective time, relapse, clonal evolution, adverse reactions, event free survival (EFS) and overall survival (OS). Results: 1. A total of 115patients (60 males), median aged 5.77 years, median follow-up time was 45 months, were enrolled in this study. All patients were diagnosed with severe or very severe aplastic anemia, including 49 patients in the historical control group and 66 patients in the eltrombopag group. 2. The complete response rate (CRR) at 3 months was 49.0% in eltrombopag cohort and 8.2% in historical control. The overall response rate (ORR) at 3months was 63.6% and 30.3% respectively. The CRR at 6 months was 50.0% in eltrombopag cohort and 10.2% in control cohort. The ORR at 6months were 71.2% and 57.1% respectively. There were significant differences of the CRR between two groups, there was no significant difference between two groups.3. The median effective time of the control group and eltrombopag group was 90 days and 71 days respectively (P = 0.007), the median complete remission time was 360 days and 98 days respectively (P < 0.001). The median time of separated from granulocyte colony stimulating factor (G-CSF), Red Blood Cell transfusion and Platelet transfusion in the control group were 105 days, 46 days and 54 days respectively, and 68 days, 45 days and 45 days in the eltrombopag group. There were significant differences in the time of separated from G-CSF (P = 0.003), Red Blood Cell transfusion (P = 0.001) and Platelet transfusion (P < 0.001) in the two groups. 4.The relapse rate was 4.1% (n = 2) in the control group and 10.2% (n = 5) in the eltrombopag group. One patient (2.0%) in the control group developed clonal evolution and progressed to myelodysplastic syndrome.5. The common adverse reaction were transient and reversible hyperbilirubinemia (13.6%, n = 9), elevated liver enzymes (4.5%, n = 3) and hyperuricemia (1.5%, n = 1).6. The 1-year OS and 2-year or of the control group was 100% and 98.5% respectively, comparing to 95.9% and 97.0% in eltrombopag group. The 2-year EFS of the control group and the eltrombopag group was 79.6% and 74.2%, respectively, and the 3-year EFS were 73.5% and 72.7%, respectively. Summary/Conclusion: The addition of eltrombopag to standard immunosuppressive therapy was associated with markedly higher rates and effected more quickly of hematologic response among pediatric severe aplastic anemia patients than in a historical cohort and showed a reliable security. P812: LONG-TERM COMPLEMENT INHIBITION AND SURVIVAL OUTCOMES IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA: AN INTERIM ANALYSIS OF THE RAVULIZUMAB CLINICAL TRIALS A. Kulasekararaj1,*, R. Brodsky2, M. Griffin3, A. Kulagin4, M. Ogawa5, J. Wang5, A. Mujeebuddin5, J.-I. Nishimura6, R. Peffault de Latour7, J. Szer8, J. W. Lee9 1King’s College Hospital, National Institute of Health Research/Wellcome King’s Clinical Research Facility and King’s College London, London, United Kingdom; 2Division of Hematology, Johns Hopkins Medicine, Baltimore, United States of America; 3St James Hospital, NHS Teaching Hospitals, Leeds, United Kingdom; 4RM Gorbacheva Research Institute, Pavlov University, St Petersburg, Russia; 5Alexion, AstraZeneca Rare Disease, Boston, MA, United States of America; 6Osaka University Graduate School of Medicine, Suita, Japan; 7Hôpital Saint-Louis AP-HP, Paris, France; 8Peter MacCallum Cancer Centre and The Royal Melbourne Hospital, Melbourne, VIC, Australia; 9Department of Hematology, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, chronic, hematologic disorder characterized by uncontrolled terminal complement activation, intravascular hemolysis, thrombotic events and significant morbidity and mortality. Complement component 5 (C5) inhibitors, eculizumab and ravulizumab, are the current standard of care in patients with PNH. Eculizumab has been the mainstay of treating patients with PNH since approval by both US Food and Drug Administration (FDA) and European Medicines Agency (EMA) in 2007. Ravulizumab (approved in 2018 [FDA] and 2019 [EMA]) is a new treatment for patients with PNH, offering complete complement C5 inhibition throughout the 8-week (q8w) dosing interval. The ravulizumab clinical trial program has allowed for analysis into the long-term management of patients with PNH treated with C5 inhibitors and their associated clinical outcomes. Aims: To analyze patient survival using long-term data from the ravulizumab clinical trial program. Methods: This analysis utilized pooled long-term survival data across the ravulizumab phase 1b, 2 and 3 trials (103 [NCT02598583], 201 [NCT02605993], 301 [NCT02946463] and 302 [NCT03056040]) with up to 4 years of open-label extension. Complement C5 inhibitor (eculizumab/ravulizumab) and dose received varied during the initial treatment period. During open-label extension, patients received weight-based dosing of intravenous ravulizumab q8w. Deaths reported, patient survival over time and adverse events resulting in patient death were analyzed. Instances of patients that died owing to infection or sepsis were specifically analyzed to provide insight into the events leading to death. Results: This analysis reported 1479.0 patient-years of follow-up in 475 patients with PNH treated with ravulizumab. Of the 475 patients who received ravulizumab, 12 (2.5%) died during the initial treatment period and 4-year open-label extension with an overall incidence of 0.8 per 100 patient-years. Nine of these deaths occurred within the first 3 years of treatment. The remaining three patients died during the fourth (n = 2) and fifth (n = 1) years of treatment. Of the 12 deaths reported, six were attributed to infection or sepsis (Table 1). These patients were predominantly male (83.3%), and white or Asian (50.0%, respectively), and median (range) age was 61 (43–75) years. In addition, of these six patients, two had aplastic anemia and one patient had pancytopenia. Most (83.3%) of these patients were hospitalized owing to onset of infection; however, one patient (white male; 43 years old; ravulizumab to ravulizumab) was hospitalized owing to worsening of aplastic anemia. During hospitalization, this patient was diagnosed with acute respiratory infection and died owing to sepsis. One patient (Asian male; 62 years old; ravulizumab to ravulizumab) died owing to meningococcal sepsis (strain unknown). This patient was vaccinated against meningococcal groups A, C, Y and W-135, but had not received prophylactic antibiotics. Other causes of death included cardiac disorders (n = 1), neoplasms (n = 4; acute myeloid leukemia, metastatic urothelial carcinoma of the kidney, metastatic malignant neoplasm of the lung and non-small cell lung carcinoma) and respiratory disorders (n = 1). Image: Summary/Conclusion: This analysis reports the longest period of follow-up in 475 patients with PNH treated with ravulizumab. Overall, long-term ravulizumab treatment was associated with a low incidence of death and few patients died owing to infection. This analysis supports the long-term use of ravulizumab for PNH. P813: EFFICACY, TREATMENT ADMINISTRATION SATISFACTION AND SAFETY OF SUBCUTANEOUS RAVULIZUMAB THROUGH 1 YEAR IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA WHO RECEIVED PRIOR INTRAVENOUS ECULIZUMAB M. N. Yenerel1,*, F. Sicre de Fontbrune2, C. Piatek3, F. Sahin4, W. Füreder5, M. Ogawa6, I. Tomazos6, H. Doll7, A. Ozol-Godfrey6, J. R. Sierra6, J. Szer8 1Division of Hematology, Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey; 2Hematology and Bone Marrow Transplant Unit, Assistance Publique Hôpitaux des Paris, Saint Louis Hospital, Paris, France; 3Jane Anne Nohl Division of Hematology, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States of America; 4Department of Hematology, Ege University Bornova, Izmir, Turkey; 5Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria; 6Alexion, AstraZeneca Rare Disease, Boston, MA, United States of America; 7Clinical Outcomes Solutions, Folkstone, United Kingdom; 8Haematology at Peter MacCallum Cancer Centre and The Royal Melbourne Hospital, Melbourne, VIC, Australia Background: The efficacy of ravulizumab (intravenous [IV] formulation; administered every 8 weeks) for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH) has been demonstrated in several randomized trials (NCT02946463, NCT03056040, NCT03406507). In study 303 (NCT03748823), subcutaneous (SC) ravulizumab, administered weekly via an on-body delivery system, showed pharmacokinetic non-inferiority to IV ravulizumab in adult patients with PNH who were clinically stable on prior IV eculizumab treatment. Here, we report results from the first 1 year of SC treatment, starting at day 15 for patients who continued SC ravulizumab during the extension period (SC/SC) and day 71 for patients who switched from IV ravulizumab to SC ravulizumab (IV/SC). Aims: To evaluate the efficacy, treatment administration satisfaction and safety of SC ravulizumab through the first 1 year (day 351) of treatment in adult patients with PNH previously treated with eculizumab. Methods: Patients (≥ 18 years) with clinically stable PNH (lactate dehydrogenase [LDH] levels ≤ 1.5 × upper limit of normal [246 U/L]) and ≥ 3 months prior eculizumab treatment were enrolled in the study, which consisted of a screening period (day -1 to day -30), a 10-week randomized treatment period and an extension period of up to 172 weeks. During the randomized treatment period, patients were assigned (2:1 ratio) to receive either SC ravulizumab or IV ravulizumab; all patients received SC ravulizumab during the extension period. Efficacy endpoints included: change in LDH from baseline; incidence of breakthrough hemolysis; transfusion avoidance; and stabilized hemoglobin (avoidance of a ≥ 2 g/dL decrease in hemoglobin in the absence of transfusion). Treatment administration satisfaction was assessed via the Treatment Administration Satisfaction Questionnaire (TASQ), which has been validated in a PNH population. Safety, including adverse events (AEs), serious AEs (SAEs) and adverse device effects (ADEs), were also assessed up to the 1-year data cut-off. Results: In total, 128 patients received SC ravulizumab (SC/SC: n = 84; IV/SC: n = 44; mean [range] duration of SC treatment: 486.4 [37–709] days). Efficacy endpoints (SC/SC and IV/SC) remained stable over time through 1 year of SC ravulizumab treatment. Mean (standard deviation [SD]) percentage change in LDH from baseline to SC day 351 was 0.9% (20.5%). Breakthrough hemolysis events were infrequent: 5/128 patients (3.9%); no event was considered free C5-related. Transfusion avoidance was maintained in 83.6% of patients during SC treatment, and 79.7% achieved stabilized hemoglobin. Improvement in total TASQ score with SC ravulizumab (compared with baseline IV eculizumab) was apparent at the first post-SC treatment assessment (SC day 29) and maintained until data cut-off (Figure). The most common AEs (reported by ≥ 10% of patients, excluding ADEs related to device product issues) during SC treatment were headache (14.1%, all grade ≤ 2), COVID-19 (14.1%, one death) and pyrexia (10.9%); injection site reaction (4.7%) was the most common non-device related ADE. Treatment-emergent SAEs were experienced by 21.1% of patients through to data cut-off. Although many patients had ≥ 1 device issue ADE, full SC dose administration was achieved in 99.9% of attempts. ADE incidence decreased over time. Image: Summary/Conclusion: The SC method of administration provides an additional treatment option for patients with PNH receiving ravulizumab therapy. Patients may be switched from IV eculizumab or IV ravulizumab to SC ravulizumab without loss of efficacy. P814: CLINICAL CHARACTERISTICS AND GENE MUTATION ANALYSIS OF 148 CHILDREN WITH FANCONI ANEMIA IN CHINA L. Chang1,*, L. zhang1, W. An1, Y. wan1, Y. cai1, Y. Lan1, M. Ruan1, X. liu1, Y. Zou1, X. Zhu1 1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical Colleg, Tian jin, China Background: Fanconi anemia (FA) is a rare autosomal recessive, X-linked (FANCB) or autosomal dominant (FANCR/RAD51) disease of bone marrow failure. Pathogenesis is mainly caused by gene mutation related to the FA pathway. At present, at least 23 genes have been discovered to play a role in the FA pathway. The gene mutations of FA have been reported in more and more countries. In China, there no large-scale cases have been reported. Aims: In this study, we analyzed the clinical characteristics of 148 FA children from 20 provinces in China from October 2003 to October 2021. We explored the relationship between genotype and phenotype in 105 children with FA genotyping results. This is the largest series of subtyped Chinese Fanconi anemia patients to date, and the results will be helpful for future clinical management. Methods: A total of 148 Fanconi anemia patients from 140 families were diagnosed by clinical phenotype, family history, chromosome breakage test induced by mitomycin C, Single cell gel electrophoresis experiment, targeted - sequence, whole exon sequencing, and MLPA method from October 2003 to October 2021. Since there were no genetic test results for the children before 2010, only the 105 patients after 2010 had gene sequencing results, which allowed genotyping. DNA damage repair defects were detected by MMC-induced Single cell gel electrophoresis and chromosome breakage experiments. Targeted-seq was used to identify the FA mutations of the patients. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large fragment deletions in patients with negative results in the targeted-seq but conforming to the FA by MMC-induced chromosome breakage experiments test and clinical manifestations. We performed WES of DNA samples from five patients and their parents. Results: 1) The most common subtype of FA in China is FA-A, followed by FA-D2 and FA-P。2) The most common deformities in FA-A patients are finger deformities and skin pigmentation, while heart deformities are more common in FA-D2/B/G/I/P subtypes. 3) The common mutations of FANCA are exon23c.2101A>G, exon28c.2778 + 1G>A, exon34c.3348 + 1G>A, exon21c.1844delC, and exon30 C2941T>G, which are different from Korea and Japan. 4) Homozygous nonsense mutation of exon32 c.3188G>A and exon29 c.2851C>T in FA-A likely benign, but the homozygous splicing mutation in exon29c.2852 + 1G>T is harmful. 4) Children with splicing and Del mutations of FANCD1 gene had poor clinical prognosis, while children with homozygous mutations of exon10c. 1792A>G is likely benign. 5) Cases of initial diagnosis of MDS or poor disease progression tend to occur in FA-B/C/E/G/J/L/M and FA-S subtypes. 6)Chromosome 1, 3, 7, and 8 abnormalities and mutations of SF3B1 P53 are related to disease progression. Summary/Conclusion: We made a comprehensive description and analysis of the clinical characteristics and mutant genes of Children with FA in China. We proposed the mutation sites related to clinical prognosis and the cytogenetic abnormalities of disease progression, providing new data for our comprehensive understanding of FA. Our data will be useful for FA future menagement. P815: PHARMACOKINETIC AND PHARMACODYNAMIC SIMILARITY OF ABP 959 AND ECULIZUMAB REFERENCE PRODUCT: UNBOUND ECULIZUMAB AND CH50 FROM THE RANDOMIZED, DOUBLE-BLIND, SINGLE-DOSE STUDY IN HEALTHY VOLUNTEERS V. Hanes1, J. Pan1, D. Mytych1, V. Chow1,* 1Amgen Inc, Thousand Oaks, United States of America Background: ABP 959 is being developed as a biosimilar to eculizumab reference product (RP). ABP 959 and the RP are humanized recombinant IgG2/4κ mAbs which bind with specificity to the human complement C5, blocking its cleavage to C5a and C5b and preventing the generation of the terminal complement complex (TCC), thereby blocking cell lysis. Excessive activation or insufficient control of C5 is believed to play a role in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), and neuromyelitis optical spectrum disorder (NMOSD). Drug binding to C5 blocks this biological function and contributes to efficacy across these indications for which the RP is approved. Aims: The goal of this analysis is to further support the previously demonstrated pharmacokinetic (PK)/pharmacodynamic (PD) similarity of ABP 959 with eculizumab RP by providing additional data on similarity in PK exposure with respect to unbound eculizumab RP and 50% total hemolytic complement activity (CH50). Methods: This was a randomized, double-blind, single-dose, 3-arm, parallel-group trial in healthy adult male subjects, aged between 18 and 45 years, with a body mass index of 18 to 30.0 kg/m2, who were randomized following informed consent, to receive a 300-mg IV infusion of ABP 959, or FDA-licensed eculizumab RP (eculizumab US), or EU-authorized eculizumab (eculizumab EU). Serum samples for PK and PD evaluations were collected over 56 days. Primary objectives were to demonstrate PK and PD equivalence of ABP 959 versus eculizumab US and ABP 959 versus eculizumab EU, as assessed by area under the total serum concentration versus −time curve (AUC) from time 0 extrapolated to infinity (AUCinf) and an area between the effect curve (ABEC) of 50% total hemolytic complement activity (CH50). Secondary PK endpoints included AUC from time 0 to the time of the last observed quantifiable concentration (AUClast) and maximum observed concentration (Cmax). Pre-specified equivalence criterion for the primary PK and PD parameters was 90% confidence interval (CI) for geometric means (GM) ratio within 0.80 to 1.25. Other secondary endpoints included the safety, tolerability, and immunogenicity of the investigational products (IPs). Results: A total of 219 subjects were randomized (ABP 959, n=71; eculizumab US, n=74; eculizumab EU, n=74). As with PK and PD parameters of total eculizumab following a single 300 mg IV infusion of IP,1 PK/PD parameters for unbound eculizumab were similar between ABP 959 versus eculizumab US and ABP 959 versus eculizumab EU. The GM and GM coefficient of variation (%CV) of AUCinf (h.µg/mL) for unbound eculizumab was: 5072.1 (25) for ABP 959, 5527.6 (31) for eculizumab US, and 5070.3 (31) for eculizumab EU. The GM and %CV of ABEC of CH50 (%*h) was 17724.5 (38.75) for APB 959, 16549.4 (42.23) for eculizumab US, and 16361.1 (36.94) for eculizumab EU. The resulting 90 and 95% CI of GM ratios were within the bioequivalence criteria of 0.80 to 1.25. 1Eur J Haematol. 2020;105:66–74. Image: Summary/Conclusion: These additional clinical pharmacology results demonstrate bioequivalence of ABP 959 to eculizumab RP with regard to unbound eculizumab and CH50. These results further support the PK/PD similarity of ABP 959 with eculizumab RP. P816: TRANSIENT MONOSOMY 7 IN SAMD9/9L SYNDROMES: IS IT SAFE TO WATCH AND WAIT? M. Erlacher1,*, F. Andresen1, A. Yoshimi1, M. Sukova2, J. Stary2, B. de Moerloose3, M. Dworzak4, S. Polychronopoulou5, G. Göhring6, C. Kratz7, J. van der Werff ten Bosch8, M. Seidel9, B. Strahm10, M. Wlodarski10, C. Niemeyer10 1Division of Pediatric Hematology and Oncology, University Medical Center Freiburg, Freiburg, Germany; 2Department of Pediatric Hematology and Oncology, University Hospital Motol, Prague, Czechia; 3Department of Pediatric Hematology-Oncology, Ghent University Hospital, Ghent, Belgium; 4Department of Pediatrics, St. Anna Children’s Hospital and Children’s Cancer Research Institute, Vienna, Austria; 5Department of Pediatric Hematology/Oncology, Aghia Sophia Children’s Hospital, Athens, Greece; 6Department of Human Genetics; 7Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany; 8Department of Pediatric Onco-Hematology, Universitair Ziekenhuis Brussels, Brussels, Belgium; 9Division of Pediatric Hemato-Oncology, Medical University Graz, Graz, Austria; 10Division of Pediatric Hematology and Oncology, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany Background: Monosomy 7 is a non-random but poorly understood somatic event in myelodysplastic syndrome (MDS). Most enigmatic is the fact that monosomy 7 can disappear spontaneously. Such transient monosomy 7 was recently associated with germline mutations in sterile alpha-motif domain-containing protein 9 (SAMD9) and its paralog SAMD9-like (SAMD9L), both located on chromosome 7q. Mechanistically, gain-of-function (GOF) SAMD9/9L mutations inhibit proliferation and reduce survival in different cell types thus leading to bone marrow failure, immunodeficiency and non-hematological phenotypes. Hematopoietic cells are under a high selection pressure to inactivate this toxic gene and achieve this by multiple mechanisms: uniparental disomy, monosomy 7, del(7q) or somatic loss-of-function (LOF) mutations of the mutated allele. Multiple “rescued” clones exist in parallel within the bone marrow of patients, and dominance of an UPD or somatically mutated clone can displace the monosomy 7 clone and result in hematological remission. Aims: We recently identified SAMD9/9L mutations in 8% of children with MDS, and a strong association with monosomy 7 (EWOG-MDS; Sahoo et al, Nat Med, 2021). This retrospective analysis revealed transient monosomy 7 and spontaneous hematological remission in some infants. To prospectively study the propensity of patients to lose their monosomy 7 clone, EWOG-MDS elected to delay immediate hematopoietic stem cell transplantation (HSCT) in children <5 years of age diagnosed of refractory cytopenia (RCC) with germline SAMD9/9L mutation and monosomy 7. Methods: Patients with SAMD9 or SAMD9L mutation and monosomy 7, younger than 5 years of age, were closely monitored with bone marrow morphology, SAMD9/9L mutational analysis and cytogenetic studies every 3-4 month. HSCT was recommended in case of severe infections, persistent severe neutropenia, progression with additional cytogenetic aberrations or increase in blasts (MDS-EB), or persistence of monosomy 7 beyond the age of 5 years. Results: Eight patients diagnosed at a median age of 11 months (range 4-36 months) with SAMD9L (n=7) or SAMD9 (n=1) germline mutation were studied. Two (P5 and P8) were born small for gestational age, P5 presented with global developmental delay and cerebellar atrophy. With a median follow-up of 27 months, 6 of the 8 patients are alive. One patient succumbed to infection prior to HSCT (P4), another to transplantation-related toxicity (P8). HSCT was performed or is currently scheduled in 4 of the 8 patients; indications were severe neutropenia/infection (P5, P6), immunodeficiency (P7) or MDS-EB (P8). The monosomy 7 clone regressed in 3 patients (P1-P3) and was no longer detectable at 3 months (P1) or 1 year (P2) from diagnosis (P3 was lost to follow-up at 12 months). Somatic SAMD9L mutations were noted in P1 and P2, both of which experienced a complete hematological and cytogenetic recovery. In sum, we followed 8 patients with SAMD9/9L syndrome and monosomy 7. Transient monosomy 7 with hematological recovery was observed in 2 patients and accompanied by gain of somatic rescuing mutations. Summary/Conclusion: Transient monosomy 7 in young children with SAMD9/SAMD9L germline disorder and RCC with monosomy 7 is a rare event. Initiation of a watch and wait strategy instead of timely HSCT will require a stringent surveillance strategy including repetitive BM analyses for SAMD9/9L sequencing, cytogenetics and search for somatic oncogenic events. P817: SYNDROMES PREDISPOSING TO LEUKEMIA ARE A MAJOR CAUSE OF INHERITED CYTOPENIAS IN CHILDREN O. Gilad1,2,*, O. Dgany3, S. Noy -Lotan3, T. Krasnov3, J. Yacobovich4, R. Rabinowicz4, T. Goldberg5, A. Kuperman6, A. Abu-Quider7, H. Miskin8, N. Kapelushnik9, N. Mandel-Shorer10, S. Shimony11, D. Harlev12, T. Ben-Ami13, E. Adam14, C. Levin15, S. Aviner16, R. Elhasid17, S. Berger-Achituv17, L. Chaitman-Yerushalmi18, Y. Kodman4, N. Oniashvilli19, M. Hameiri- Grosman19, S. Izraeli4, H. Tamary1,3,20, O. Steinberg-Shemer1,3,20 1Sackler faculty of Medicine, Tel Aviv University, Tel Aviv; 2Department of Hematology-Oncology, Scneider Children’s medical center of Israe; 3Pediatric Hematology Laboratory, Felsenstein Medical Research Center; 4Department of Hematology-Oncology, Schneider Children’s Medical Center of Israel; 5Department of Hematology-Oncology, Scneider Children’s medical center of Israel, Petach Tikva; 6Blood Coagulation Service and Pediatric Hematology Clinic, Galilee Medical Center, Nahariya; 7Pediatric Hematology, Soroka University Medical Center, Ben-Gurion University, Beer Sheva; 8Pediatric Hematology Unit, Shaare Zedek Medical Center, Jerusalem; 9Goldschleger Eye Institute, Sheba Medical Center, Tel Hashomer; 10Department of Pediatric Hematology-Oncology, Rambam Healthcare Campus, Haifa; 11Institute of Hematology, Davidoff Cancer Centre, Rabin Medical center, Petach-Tikva; 12Pediatric Hematology-Oncology Department, Hadassah University Medical Center, Jerusalem; 13Pediatric Hematology Unit, Kaplan Medical Center, Rehovot; 14Pediatric Hematology-Oncology Department, Sheba Medical Center, Tel Hashomer; 15Pediatric Hematology Unit and Research Laboratory, Emek Medical Center, Afula; 16Department of Pediatrics, Barzilai University Medical Center, Ashkelon; 17Department of Pediatric Hemato-Oncology, Tel Aviv Medical Center; 18Genoox, Health Care Technology, Tel Aviv; 19Department of Hematology-Oncology, Schneider Children’s Medical Center of Israel; 20Department of Hematology-Oncology, Schneider Children’s Medical Center of Israel, Petach Tikva, Israel Background: Prolonged cytopenias are a non-specific sign with a wide differential diagnosis. Among inherited disorders, cytopenias predisposing to leukemia require a timely and accurate diagnosis to ensure appropriate medical management, including adequate monitoring and stem-cell transplantation prior to the development of leukemia. Aims: We aimed to define the types and prevalences of the genetic causes leading to persistent cytopenias in children. Methods: The study comprises children with persistent cytopenias, myelodysplastic syndrome (MDS), aplastic anemia, or suspected inherited bone marrow failure syndromes, who were referred for genetic evaluation from all pediatric hematology centers in Israel during 2016-2019. For variant detection, we used a custom-made targeted next-generation sequencing panel covering 226 genes known to be mutated in inherited cytopenias followed, if needed, by whole exome sequencing. Sanger sequencing was used for validation. Results: In total, 189 children with persistent cytopenias underwent a genetic evaluation. Pathogenic and likely pathogenic variants were identified in 59 patients (31.2%), including 47 with leukemia predisposing syndromes. Most of the latter (32, 68.1%) had inherited bone marrow failure syndromes, 9 (19.1%) had inherited thrombocytopenia predisposing to leukemia, and 3 (6.4%) had congenital neutropenia. Twelve patients had cytopenias with no known leukemia predisposition, including nine children with inherited thrombocytopenia and three with congenital neutropenia (not yet known to predispose to leukemia). Importantly only 3 had myelodysplastic syndrome. Thus, the majority of leukemia predisposition could not have been deduced from the morphological presence of MDS Summary/Conclusion: almost one-third of 189 children referred with persistent cytopenias had an underlying inherited disorder; 79.7% of whom had a germline predisposition to leukemia. Precise diagnosis of children with cytopenias should direct follow-up and management programs and may positively impact disease outcome. P818: NEXT GENERATION TARGETED SEQUENCING FOR ENHANCED GENOTYPING OF DIAMOND BLACKFAN ANEMIA IN ISRAEL T. Goldberg1,*, O. Steinberg-Shemer1,2,3, O. Dgany3, S. Noy-Lotan3, T. Krasnov3, O. Gilad1,2, J. Yacobovich1,2, H. Tamary1,2,3 1Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petah Tikva; 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; 3Pediatric Hematology Laboratory, Felsenstein Medical Research Center, Petah Tikva, Israel Background: Diamond Blackfan anemia (DBA), an erythroid-specific congenital bone marrow failure syndrome, is predominantly driven by mutations in one of several genes encoding ribosomal proteins (RP), leading to protein haploinsufficiency. In recent years, a number of patients with “DBA-like” syndrome were found to have mutations in non-RP genes including CECR1. Next generation sequencing (NGS) is rapidly replacing conventional Sanger sequencing approaches in the molecular diagnosis of patients with DBA and other inherited bone marrow failure syndromes. Targeted NGS panels enable high-resolution deep sequencing of all known genes associated with a specific clinical presentation such as congenital anemias. Aims: This study aims to use a targeted NGS panel specific for inherited anemias to genotype previously undiagnosed patients with clinical DBA in Israel. Enhanced genotyping efficiency provided by NGS may provide an opportunity to evaluate for genotype-phenotype correlations. Methods: A targeted NGS panel including over 260 genes was used as an adjunct to Sanger sequencing and multiplex ligation-dependent probe amplification for genotyping patients from the Israeli inherited bone marrow failure registry (I-IBMFR) with a clinical diagnosis of DBA. Over 90% of 48 patients with DBA from the Israeli registry had available DNA for genotyping. Results: Forty-eight patients with clinical DBA, registered to the I-IBMFR, have been followed for a median of 12.1 years. The majority of patients presented with anemia in the first year of life, with a median age at presentation of 3 months. Forty-four of these patients underwent genotyping. Pathogenic RP gene alterations were detected in 31 patients in 9 different RP genes; homozygous CECR1 mutations were found in 8 separate patients. Forty percent of these molecular diagnoses were detected by targeted NGS panel technology. More than half of the patients exhibited at least one congenital malformation. Cardiac anomalies were observed broadly in patients with small and large subunit RP gene mutations. Thumb anomalies were found exclusively in patients with RPL5 mutations; cleft lip and palate were reported only in patients with RPS10 and RPL11 mutations. Fewer than half of the patients given a steroid trial were responsive; patients with RPS19 mutations responded significantly better to steroid treatment than patients with RPL5 mutations. Just under half of the patients with RP gene mutations became red blood cell (RBC) transfusion dependent, while almost all of the patients with CECR1 mutations required RBC transfusions. Three patients, including two genetically undiagnosed patients, went into spontaneous remission after receiving chronic treatment. Summary/Conclusion: Use of targeted NGS has dramatically enhanced the efficiency of genotyping for patients with DBA and other inherited bone marrow failure syndromes, allowing for focused management of patients and discovery of novel genotype-phenotype correlations. Ongoing investment in registries and comprehensive genotyping are crucial in increasing our understanding of this rare disease and optimizing patient care. P819: TRANSPLANT, TREATMENT AND TRANSFUSION FREE (TTT-FREE) SURVIVAL AS RELEVANT CLINICAL ENDPOINT AFTER IMMUNOSUPPRESSIVE TREATMENT FOR ACQUIRED APLASTIC ANEMIA IN ADULTS E. Koster1, C. Halkes1,*, E. Bogers1, C. Hazenberg2, F. Heubel-Moenen3, S. Langemeier4, E. Meijer5, E. Nur5, M. Raaijmakers6, T. Snijders7, M. de Witte8, J. Tjon1, L. de Wreede9 1Hematology, Leiden University Center, Leiden; 2Hematology, University Medical Center Groningen, Groningen; 3Hematology, Maastricht University Medical Center, Maastricht; 4Hematology, Radboud Medical Center, Nijmegen; 5Hematology, Amsterdam Univeristy Medical Center, Amsterdam; 6Hematology, Erasmus Medical Center, Rotterdam; 7Hematology, Medical Center Twente, Enschede; 8Hematology, University Medical Center Utrecht, Utrecht; 9Department of Biomedical Data Sciences, Leiden University Center, Leiden, Netherlands Background: Acquired aplastic anemia (AA) is characterized by an aplastic bone marrow and pancytopenia. Adult patients with AA can be treated with immunosuppressive treatment (IST) consisting of ATGAM in combination with cyclosporin (CsA) or with an allogeneic hematopoietic stem cell transplantation (alloSCT). Patients <40 years with an HLA-identical sibling preferentially receive an alloSCT. Other patients are treated with IST as 1st line treatment. IST leads to transfusion independency in the majority of patients but it can take up to 6 months before this response occurs. Some patients need long-term treatment with CsA to maintain this response and durable efficacy is hampered by relapsing aplasia or the development of MDS, AML or PNH. While graft versus host disease and relapse-free survival (GRFS) is used as a favorable composite outcome after alloSCT, a widely accepted measurement for treatment success after IST is missing. Aims: In this study we examined Transplant, Treatment and Transfusion-free survival (TTT-free survival) as a relevant clinical outcome after standard IST with ATGAM and CsA in AA adult patients. We determined the 5-year TTT-free survival after standard IST in patients ≤40 years and >40 years. Methods: The Dutch Adult Aplastic Anemia Registry started in 2014 and contains detailed data of all consecutive AA patients who have been treated with ATGAM-based therapy in the participating hospitals, offering a unique possibility to evaluate real-life long-term efficacy and safety of 1st line IST treatment in AA. To determine TTT-free survival, a multistate model (MSM) was developed, to take into account that patients can have transient periods of treatment success and failure. Patients started in the dynamic state treatment & transfusion at time of the start of ATGAM. This state also included all other (2nd line) non-alloSCT treatments (for example Eltrombopag, Rabbit-ATG or Danazol). Other dynamic states patients could enter and leave during follow-up were treatment & no transfusion, transfusion & no treatment and no treatment & no transfusion (considered as TTT-free survival). Absorbing states, accessible from all dynamic states, were death, alloSCT for AA, treatment for MDS/AML and treatment for PNH. Results: ATGAM with CsA as 1st line treatment was started in 117 patients (median age 54 years, range 18-79). Overall survival after 5 year is 77% (95% Confidence Interval (95%CI) 67-86%) Figure 1 shows the results of the MSM. After 5 years, the TTT-free survival was 42% (95%CI: 33-55%) for the total cohort. 19% (95%CI: 12-31%) of the patients was transfusion independent but still needed medication at this time (mainly CsA or Eltrombopag). 15% (95%CI: 10-23%) had received an alloSCT as 2nd line treatment for AA, 5% (95%CI: 2-13%) had started treatment for MDS/AML and 2% (95%CI: 0-11%) had started treatment with complement inhibition for PNH. 5-year TTT-free survival was 60% (95%CI:44-82%) in patients aged ≤40 years (n=36), but only 33% (95%CI: 23-49%) in patients aged >40 years (n=81). Image: Summary/Conclusion: TTT-free survival can be used to evaluate treatment success after IST in AA patients, allowing achievement, loss and recovery of response. We showed that 5-year TTT-free survival is superior in patients ≤40 years compared to patients >40 years. This MSM can be used to predict outcomes in AA patients receiving IST and can help in the decision whether and when to offer a patient an alloSCT. P820: PREDICTING RESPONSE TO RABBIT ATG-BASED INTENSIVE IMMUNOSUPPRESSIVE THERAPY COMBINED WITH ELTROMBOPAG IN CHINESE ADULT PATIENTS WITH SEVERE APLASTIC ANEMIA R. Li1, Z. Liu1, X. Chen2, Q. Long2, Y. Yang3, S. Lin4, J. Jia5, G. He1,*, J. Li1 1Hematology, the First Affiliated Hospital of Nanjing Medical University; 2Hematology, the Second People’s Hospital of Nanjing, Nanjing; 3Hematology, the First Bethune Hospital of Jilin University, Changchun; 4Hematology, Zhejiang Province Hospital of Traditional Chinese Medicine, Hangzhou; 5Hematology, Peking University People’s Hospital, Beijing, China Background: The intensive immunosuppressive therapy (IST) consisted by antithymocyte immunoglobulin (ATG) and cyclosporin (CsA) combining with eltrombopag (E-PAG) is the initial therapy for adult patients with severe aplastic anemia (SAA). There is rabbit ATG (r-ATG) rather than horse ATG in the mainland of China and E-PAG is recommended with 75mg/d for East Asian population. Aims: To explore factors predicting the efficacy of E-PAG (75mg/d) plus r-ATG based IST for SAA patients in East Asia. Methods: From February 2018 to December 2020, we conducted a retrospective, multicenter study to analyze 58 adult SAA patients with rabbit ATG-based IST and E-PAG in the China Eastern Cooperation Group for Anemia (CECGA). The dosage of r-ATG was 3.5mg·kg-1·d-1 for 5 days with intravenous infusion and CsA was 3-5mg·kg-1·d-1 early in the regimen maintaining concentration at 150-200ng/ml, whose dosage could be modulated on the basis of drug concentration and severe adverse events (SAEs). E-PAG was recommended with 75mg/d at least 6 months. If or SAEs occurred or the count of platelet was higher than 200×109/L, E-PAG would be discontinued. Results: 58 patients with median age of 42.5 years (18 years to 74 years) were treated with r-ATG based IST plus E-PAG. Cohort 1 and cohort 2 included patients with or without a response at 6 months, whist the median age were 50.5 years (24 years to 74 years) and 38 years (18 years to 71 years), separately. The median time between diagnosis and treatment was 3 weeks (1 week to 106 weeks) and 3 weeks (1 week to 166 weeks), respectively. The response rates of E-PAG combining with IST at 3, 6 and 12 months after IST were 64%, 76% and 85% (44 of 52), while the complete response rates at 3, 6 and 12 months were 19%, 21% and 29% (15 of 52), separately. The baseline absolute lymphocyte count (ALC) (area under a curve (AUC)=0.706, 95%CI 0.522-0.890, P=0.057), red cell distribution width – coefficient of variation (RDW-CV) (AUC=0.722, 95%CI 0.494-0.950, P=0.040) and reticulocyte (Ret) percentage (AUC=0.798, 95%CI 0.640-0.956, P=0.006) were highly predictive of response at 6 months (Figure 1). The tipping values of Ret percentage, ALC and RDW-CV were 0.45%, 1.06×109/L and 11.75%, respectively. The sensitivity and specificity of Ret percentage were 81.6% and 66.7%; RDW-CV were 94.7% and 55.6%; ALC were 55.3% and 88.9%. The 2-year overall survival was 93%. Image: Summary/Conclusion: The baseline Ret percentage, RDW-CV and ALC were potential factors to predict a favorable effect in SAA patients of East Aisan. P821: LATE ONSET AND/OR LONG LASTING NEUTROPENIA IN CHILDHOOD: CLINICAL AND HEMATOLOGICAL CHARACTERISTICS AND OUTCOMES C. Kelaidi1,*, K. Antoniadi1, V. Tzotzola1, V. Papadakis1, M. Ampatzidou1, C. Pontikoglou2, I. Mavroudi2, M. Tzanoudaki3, G. Paterakis4, S. I. Papadhimitriou5, K. Tsitsikas1, A. Bountali1, K. Manola6, H. A. Papadaki2, S. Polychronopoulou1 1Pediatric Hematology-Oncology, Aghia Sophia Children’s Hospital, Athens; 2Haemopoiesis Research Laboratory, University of Crete, School of Medicine, Heraklion; 3Flow cytometric cell analysis, Laboratory of Immunology, Aghia Sophia Children’s Hospital; 4Flow Cytometry Analysis, Laboratory of Immunology; 5Laboratory of Hematology, Georgios Gennimatas General Hospital; 6Laboratory of Health Physics, Radiobiology & Cytogenetics, National Centre for Research Demokritos, Athens, Greece Background: In contrast with primary autoimmune neutropenia of infants and pre-school children, characterized by the presence of anti-polymorphonuclear antibodies (anti-PNN), benign course and spontaneous resolution at age 3-5 years, late onset (LO) and/or long lasting (LL) neutropenia of childhood is vaguely described. Aims: To address characteristics and outcomes in a cohort of children with LO/LL diagnosed and followed by our Department. Methods: Cross-sectional study of patients aged 0-16 years with LO (age >5 years, in accordance with Fioredda et al, 2020) or LL (duration >3 years) neutropenia (1.5 G/L) +/- leukopenia (Z-score ≤2 according to age and gender); with a first visit or continued follow-up in our Department of Pediatric Hematology-Oncology (T.A.O.) during the period 2015-2020. Patients with severe congenital neutropenia (SCN) were excluded. Detection of anti-PNN using GAT and GIFT was centralized. Results: From 1972 to 2021, 534 cases of neutropenia/leukopenia were recorded (5, 40, 82, 134, 273 during 1970-1979, 1980-1989, 1990-1999, 2000-2009, 2010-2021, respectively); 152 were included in the cross-sectional study. Sixty-one (40%) had no LO/LL (age<5 years, resolution within≤3 years), 77 (51%0 had LO and 14 (9%) had LL. Male/female ratio was 47% vs 52% vs 28% in no LO/LL vs LO vs LL, respectively (P=0.27), median age 1 vs 11.1 vs 3.1 years (P<10-4), median neutrophil count 0.5 vs 1.2 vs 0.6 G/L (P<10-4), and with concomitant leukopenia in 27% vs 53% vs 36% (P=0.009) or lymphopenia in 3% vs 10% vs 7% of cases (P=0.27). Anti-PNN were detected in 83% vs 42% vs 60% of patients no LO/LL vs LO vs LL, respectively (P=0.12). Familial neutropenia was observed in three pairs of siblings/cousins with LL, possible ethnic neutropenia in 7 patients, probable genetic predisposition to cancer in 4 patients LO/LL and a congenital metabolic syndrome in one patient LL. Infection, mostly cutaneous, occurred in 17% vs 24% vs 66% no LO/LL vs. LO vs. LL (P=0.02). Bone marrow examination was performed in 8 patients (4 LO, 4 LL) and showed mild hypoplasia in 2 (LO). Mild peripheral blood lymphocytic subsets abnormalities were observed in ¼ of the patients, particularly in LO, and mild reduction of serum immunoglobulin class levels in ¼ of the patients, without difference between no LO/LL and LO. Genetic typing performed in 9% of patients LO/LL (targeted pediatric MDS panel N=2, primary immunodeficiency N=2, SCN N=1, whole-exome sequencing N=2) showed a heterozygous TACI mutation in one patient with LL and frequent infections of moderate severity. In total, MDS, immunodeficiency and autoimmunity was noted in 1%, 5% and 4% of patients LO and immunodeficiency in 14% of patients LL. One patient LL developed B-cell precursor ALL. One patient LO with Evans syndrome received eltrombopag, without response, and MMF with response. All patients are alive. Neutropenia resolved spontaneously in 54% of patients. Two-year probability of unresolved neutropenia was 26% vs 78% vs 100% in patients no LO/LL, LO and LL, respectively (P<10-4). Summary/Conclusion: LO/LL neutropenia might be associated with bone marrow failure syndromes, immunodeficiency or autoimmunity. Long-term follow-up and investigations are needed in children with LO/LL neutropenia. Genetic typing in specialized reference networks, like EuNet-INNOCHRON or EWOG, might identify diagnostic subgroups of clinical relevance. P822: A PHASE 1 SINGLE ASCENDING DOSE STUDY OF CAN106, A LONG-ACTING ANTI-C5 COMPLEMENT MONOCLONAL ANTIBODY IN CLINICAL DEVELOPMENT FOR PNH AND OTHER COMPLEMENT-MEDIATED DISEASES C. M. KHOO1,*, X. SONG2, Q. WU2, G. F. COX3 1Department of Medicine, National University Hospital, Singapore, Singapore; 2Clinical Development and Medical Affair, CANbridge Pharmaceuticals Inc., Shanghai, China; 3Clinical Development and Medical Affair, CANbridge Pharmaceuticals Inc., Cambridge, MA, United States of America Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired genetic disorder caused by clonal mutations in the PIGA gene. Deficiency of PIGA protein renders erythrocytes susceptible to complement-mediated destruction, causing intravascular and extravascular hemolysis, which can lead to anemia, fatigue, thromboembolism, major organ damage, and death. Despite the approval of one anti-C3 and two anti-C5 therapies for PNH, high treatment costs limit market access in many countries. CAN106 is a novel anti-C5 monoclonal antibody designed for optimal pH-dependent binding to C5 and enhanced binding to FcRN to increase intracellular recycling and prolong its half-life. Aims: This first-in-human Phase 1 study aimed to evaluate the single-dose safety and tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of CAN106. Methods: This was a randomized, double-blind, placebo-controlled, single-ascending-dose study. We enrolled 31 healthy adult subjects at a single site in Singapore who received CAN106 (mg/kg) or placebo at one of the following 6 doses (mg/kg) and ratios: (0.25, n=1:0; 0.75, n=1:0; 2, n=3:2; 4, n=6:2; 8, n=6:2 or 12, n=6:2). The primary endpoint was the incidence of adverse events overall and by intensity, seriousness, type, and relatedness to study drug. Secondary endpoints were the pharmacokinetic characterization of CAN106, the pharmacodynamic effects on free C5 (target engagement) and CH50 (serum hemolytic activity), and the incidence of anti-drug antibodies. The original study duration of 196 days was shortened to 112 days based upon the observed half-life of CAN106 and the return of CH50 to normal values. Results: The mean age of subjects was 34 years, and 97% were male. All 31 subjects completed the study. Seven CAN106 subjects experienced 20 treatment-emergent adverse events (TEAEs), and three placebo subjects experienced five TEAEs. Most TEAEs were mild and not related to CAN106, and all resolved without any sequelae. Three CAN106 subjects experienced seven drug-related TEAEs at the two highest doses. Most were mild, none was serious, and all resolved without any sequelae. These events included a moderate infusion-related reaction that resolved upon discontinuing treatment; mild dizziness; and mild elevations in ALT, AST, hemoglobin, hematocrit, and RBC. CAN106 exposure (Cmax and AUC) was dose-proportional, linear, and had low inter-subject variability (<20% CV). The terminal elimination half-life was approximately 32 days. CAN106 led to dose-dependent reductions in free C5 and CH50 within 24 hours. At the 8 and 12 mg/kg doses, all subjects showed >99% reduction in free C5 and >90% inhibition of CH50, with the latter sustained for 2 to 4 weeks. Two subjects in the 4 mg/kg cohort tested positive for anti-drug antibodies at single time points (pre-dose and Day 28). Summary/Conclusion: CAN106 was safe and well-tolerated at single doses up to 12 mg/kg in healthy adult subjects. The dose-exposure relationship was linear, and the 32-day half-life in healthy subjects was similar to that of ravulizumab, which is dosed every 8 weeks in patients, suggesting the potential for an extended dosing interval. CAN106 led to rapid, dose-dependent reductions in free C5 and CH50, and at the two highest doses, achieved complete and sustained complement blockade (>90% inhibition of CH50) for up to 4 weeks. These promising results support further clinical development of CAN106 in PNH and other complement-mediated diseases. P823: THE INCIDENCE, OUTCOMES, AND RISK FACTORS OF SECONDARY POOR GRAFT FUNCTION IN HAPLOIDENTICAL HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR ACQUIRED APLASTIC ANEMIA F. lin1,*, T. han1, Y. zhang1, Y. cheng1, Z. xu1, X. mo1, F. wang1, C. yan1, Y. sun1, J. wang1, F. tang1, W. han1, Y. chen1, Y. wang1, X. zhang1, K. liu1, X. huang1, L. xu1 1Peking university institute of hematology, Beijing, China Background: Secondary poor graft function (sPGF) increases the risk of life-threatening complications after transplantation and is an obstacle to achieving better outcomes after hematopoietic stem cell transplantation (HSCT). Aims: We aimed to determine the incidence, clinical outcomes, and risk factors of sPGF in haploidentical HSCT (haplo-HSCT) for acquired aplastic anemia (AA) patients. Methods: We retrospectively reviewed 430 consecutive AA patients receiving haplo-HSCT in Peking University People’s Hospital between January 2006 and December 2020. Results: We reported the estimated 3-year cumulative incidence of sPGF was 4.64%, while no primary PGF occurred. The median time to sPGF was 121 days (range 30-626 days) after transplantation. To clarify the risk factors for sPGF, 17 sPGF cases and 382 without PGF were further analyzed. Compared to patients without PGF, the 2-year overall survival was significantly poorer for sPGF patients (67.7% vs 90.8%, p =.002). Twelve sPGF patients were alive until the last follow-up, and 7 achieved transfusion independency. The multivariable analyses revealed that a history of refractory cytomegalovirus viremia (CMViremia) (OR=7.038, p=.002) post-transplantation independently increased the risk of sPGF. There was weak evidence that a history of grade 3-4 acute graft-versus-host disease (aGvHD) increased the risk of sPGF (p=.092). Summary/Conclusion: In conclusion, sPGF can develop in 4.64% of AA patients after haplo-HSCT and significantly decreases survival. The independent hazard elements for sPGF were a history of refractory CMViremia and grade 3-4 aGvHD. We advocated better post-transplantation strategies to balance the risk of immunosuppression and viral reactivation for haplo-HSCT in AA patients. P824: HEMATOLOGIC FEATURES IN 102 FRENCH PATIENTS DIAGNOSED WITH GERMLINE MUTATIONS OF TELOMERES RELATED GENES AGED 15 YEARS OLD OR MORE F. Maillet1,*, J.-E. Galimard2, R. Borie3, C. Kannengiesser4, E. Lainey5, R. Peffault De Latour1, F. Sicre de Fontbrune1 1Hematology Department, Saint Louis Hospital; 2Statistical Unit, EBMT; 3Pulmonology Department; 4Genetics Department, Bichat Hospital; 5Hematology Department, Robert Debré Hospital, Paris, France Background: Short telomere syndrome (STS) is a group of genetic disorders characterized by germline mutations in telomeres related genes (TRG) leading to premature telomere shortening. In the recent years, the spectrum of STS diseases has widened and now includes heterogeneous associations of hematologic, pneumologic, hepatic and osteoarticular features, and cancer predisposition. STS clinical landscape remain unknown. We took advantage of a large cohort of patients to describe hematologic and extra-hematologic features of this rare disease. Aims: The aim of this study was to describe hematologic and extrahematologic features in patients with STS diagnosed at age 15 or more and followed in adult centers. Methods: All data were obtained from the French National reference Center for Aplastic Anemia. Between 2003 and 2021, we retrospectively included all patients 15 years old or older at diagnosis of STS, with pathogenic germline variant(s) in TRG, and at least one bone marrow assessment (bone marrow aspiration or biopsy). Overall survival (OS) was calculated from date of diagnosis to death or last follow-up using the Kaplan-Meier estimator. Results: One hundred and two patients were included: 89 (87%) were propositus and 13 were diagnosed after genetic counseling. The median follow-up after diagnosis was 3.6 years. The 4 years OS of the index cases was 63% (95% CI: 49 – 74). 62 (70%) and 6 (46%) patients were male, and median age at diagnosis was 42 years (IQR 28-56) and 27 years (IQR 21-48) for index cases and non-index cases, respectively. Median age at onset of first symptoms was 28 years (IQR 20-49) and 29 years (IQR 18-38), with a median duration from first symptoms to STS diagnosis of 5 years (IQR 2-11) and 6 years (IQR 0-7) for index cases and non-index cases, respectively. In index cases, TRG mutations involved TERT in 43 patients (49%), TERC in 24 patients (28%), RTEL1 in 11 patients (13%), DKC1 in 3 patients (3%), and in 1 patient each for NHP2, CTC1, PARN, TINF2, TERT plus TERC (0.9%). In index cases, mutations were heterozygous in 92%, homozygous in 6%, and X-linked in 2%. Among 68 patients with available telomere evaluation, the median telomere length was 6.2% (IQR 4.8-7.4%), whereas the median telomere length at first percentile was 7.6%. Telomere length was shorter than first percentile in 81.5% of patients. 69% of index cases had at least 1 relative with STS. Among index cases, 73 (82%) presented an hematological involvement. At diagnosis, the hematological presentation was BMF in 49 patients (70%), MDS in 17 (24%) and AML in 2 (3%), 1 CVID (1.5%), 1 lymphoma (1.5%) and not specified for 3 patients. During follow-up, evolution toward MDS was observed in 2 patients, and toward AML in 4. 14 patients received allo-HSCT (13.7%): 9 for BMF and 5 for MDS. Extra hematologic features among index cases: 49 patients (55%) had at least one clinical feature of dyskeratosis congenita. Pulmonary involvement was present in 55 patients (62%), of those 93% had diffuse interstitial pneumonia. 12 patients underwent lung transplantation. Hepatic involvement was present in 42 patients (47%), including nodular regenerative hyperplasia (29%), cirrhosis (33%) and portal hypertension (43%). 3 patients underwent liver transplantation. Cancer occurred in 7 patients: 4 solid cancers and 3 lymphomas. Summary/Conclusion: This study in STS patients allows a better understanding of the spectrum of this disease. Hematological presentation was the main feature, followed by lung and hepatic involvement. A long-term multidisciplinary close follow-up is mandatory in this population of patients. P825: FACTOR D INHIBITION WITH ORAL BCX9930 MONOTHERAPY LEADS TO SUSTAINED CONTROL OF HEMOLYSIS AND SYMPTOMS OVER 48 WEEKS IN SUBJECTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA NAÏVE TO C5 INHIBITORS A. McDonald1,*, J. L. R. Malherbe2, A. Kulasekararaj3, W. Füreder4, M. Griffin5, M. Cornpropst6, M. K. Farmer6, D. Kargl6, D. Collins6, W. Sheridan6, A. Risitano7 1Netcare Pretoria East Hospital, Moreleta Park; 2University of the Free State, Bloemfontein, South Africa; 3Kings College Hospital NHS Foundation Trust, London, United Kingdom; 4Medical University of Vienna, Vienna, Austria; 5Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom; 6BioCryst Pharmaceuticals, Durham, United States of America; 7AORN Moscati, Avellino, Italy Background: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolysis due to uncontrolled activity of the alternative pathway (AP) of complement. BCX9930 is an oral Factor D inhibitor that targets the rate-limiting enzyme of AP with the potential to prevent both intravascular hemolysis and extravascular hemolysis. We previously reported safety and effectiveness of BCX9930 at 22 weeks in subjects naïve to complement inhibitors (CI; McDonald et al. EHA 2021). Here, we report further long-term safety, tolerability, and effectiveness of BCX9930. Aims: This analysis evaluated effectiveness of BCX9930 in subjects completing at least 48 weeks of treatment at target dose of 400 mg BID or 500 mg BID. Additionally, safety data are presented for all subjects (n=16, including 6 subjects with an inadequate response to C5 inhibitors) enrolled in an open-label, dose-ranging study (NCT04330534) and extension study (NCT04702568). Methods: Study BCX9930-101 enrolled subjects with PNH with either no history of C5 inhibitor use or those with an inadequate response to C5 inhibitors (transfusion within 3 months of screening or Hb <10 g/dL). Subjects in each of three multiple dose cohorts received BCX9930 at 50 to 400 mg orally twice daily (BID) titrated to target dose of 400 or 500 mg BID. CI-naïve subjects deriving clinical benefit after 28 days of treatment could receive up to 48 additional weeks of BCX9930 before continuing into an extension study. Safety and effectiveness outcomes were evaluated monthly. Effectiveness endpoints were change from baseline (CFB) to 48 weeks in hemoglobin (Hb), red blood cell (RBC) transfusions, lactate dehydrogenase (LDH), absolute reticulocyte count (ARC), and % PNH Type II+III RBC clone size relative to PNH white blood cell (WBC) clone size. CFB in fatigue was assessed by Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue Scale (clinically important difference ≥3-point change, higher scores indicate less fatigue). Results: Ten CI-naïve adult subjects enrolled in BCX9930-101 and 9 completed at least 48 weeks of treatment. Mean (SD) age and time since PNH diagnosis were 29.2 (8.09) years and 2.6 (1.70), mean (SD) number of RBC units transfused in 12 months prior to study entry was 7.6 (8.2), and median (range) treatment exposure at 400 mg or 500 mg BID was 470 (350, 533) days. Mean (SEM) Hb CFB to Week 48 was 3.75 (0.89) g/dL (8.25 [0.60] g/dL to 12.0 [0.85] g/dL), and all 9 subjects were transfusion-free versus 2 of 9 in 12 months prior to study. At Week 48, mean (SEM) ARC CFB was – 57 (21.8) 103/µL (178 [20.7] vs. 121 [15.5] 103/µL), mean (SEM) % PNH RBC/WBC clone size increased from 51.9% (4.4%) to 93% (2.7%), and mean (SEM) LDH CFB was –70.5% (6.18%). Clinically meaningful improvements in FACIT-Fatigue scores were observed over 48 weeks, with mean (SEM) CFB of 5.3 (1.82) points (mean score 40.7 vs 46.0). The most common adverse events in all subjects (n=16) were headache (38%), self-limited rash (25%), and abdominal pain (19%). Summary/Conclusion: BCX9930 at doses up to 500 mg BID was generally well-tolerated. Notable improvements from baseline to 48 weeks were observed in Hb, LDH, ARC, and fatigue scores, with concomitant increases in PNH RBC clone size. Proximal AP inhibition of Factor D by BCX9930 demonstrated sustained relief of anemia and fatigue, and freedom from transfusions, supporting ongoing pivotal trials of BCX9930 monotherapy for treatment of PNH. P826: FACTOR D INHIBITION WITH ORAL BCX9930 LEADS TO SUSTAINED CONTROL OF HEMOLYSIS AND SYMPTOMS OVER 48 WEEKS IN SUBJECTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA INADEQUATELY CONTROLLED ON C5 INHIBITORS A. Kulasekararaj1,*, W. Füreder2, A. McDonald3, J. L. R. Malherbe4, S. Gandhi1, D. Collins5, M. Cornpropst5, S. Dobo5, M. K. Farmer5, D. Kargl5, W. Sheridan5, M. Griffin6, A. Risitano7 1Kings College Hospital NHS Foundation Trust, London, United Kingdom; 2Medical University of Vienna, Vienna, Austria; 3Netcare Pretoria East Hospital, Moreleta Park; 4University of the Free State, Bloemfontein, South Africa; 5BioCryst Pharmaceuticals, Durham, United States of America; 6Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom; 7AORN Moscati, Avellino, Italy Background: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolysis due to uncontrolled activity of the alternative pathway (AP) of complement. BCX9930 is an oral Factor D inhibitor that targets the AP rate-limiting enzyme with the potential to prevent both intravascular hemolysis and extravascular hemolysis. We previously presented safety and effectiveness data for BCX9930 at 11 weeks in subjects with an inadequate response (IR) to C5 inhibitors (C5 INH; Kulasekararaj et al. EHA 2021). Here, we report further long-term safety, tolerability, and effectiveness of BCX9930. Aims: This analysis evaluated effectiveness of BCX9930 in subjects completing at least 48 weeks of treatment at target dose of 400 mg BID or 500 mg BID. Additionally, safety data are presented for all subjects enrolled (n=16, including 10 subjects naïve to C5 INH) in an open-label, dose-ranging study (NCT04330534) and extension study (NCT04702568). Methods: Study BCX9930-101 enrolled subjects with PNH with either no history of C5 INH use or those with an IR to C5 INH (defined as having a transfusion within 3 months of screening or Hb <10 g/dL). For subjects with an IR to C5 INH treatment, BCX9930 was added to C5 INH and subjects were treated with 200 mg orally twice daily (BID) escalated to 400 mg BID, or 400 mg BID escalated to 500 mg BID. Subjects deriving clinical benefit could rollover into an extension study after 28 days, and C5 INH could be withdrawn. Safety and effectiveness outcomes were evaluated monthly. Effectiveness endpoints were change from baseline (CFB) to 48 weeks in hemoglobin (Hb), red blood cell (RBC) transfusions, lactate dehydrogenase (LDH), absolute reticulocyte count (ARC), and % PNH RBCs relative to PNH white blood cells (WBC). CFB in fatigue was assessed by Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue Scale (clinically important difference ≥3-point change, higher scores indicate less fatigue). Results: Six adult subjects with an IR to C5 INH enrolled in Study BCX9930-101 and rolled over into the open-label extension study, and 4 completed 48 weeks of treatment (3 of 4 on BCX9930 monotherapy at Week 48, mean [range] duration on monotherapy 107 [85, 129] days). Mean (SD) age and time since PNH diagnosis were 31 (9.42) and 7.7 (5.80) years, mean (SD) number of RBC units transfused in 12 months prior to study entry was 5.5 (4.4), and median (range) treatment exposure at 400 mg or 500 mg BID was 413.5 (337, 420) days. Mean (SEM) Hb CFB to Week 48 was 2.69 (0.97) g/dL (8.91 [0.56] g/dL to 11.60 [1.27] g/dL). 3 of 4 subjects were transfusion-free at Week 48 versus 1 of 4 in 12 months prior to study. At Week 48, Mean (SEM) ARC CFB was –63 (17.1) 103/µL (204 [15.5] vs. 141 [19.6] 103/µL), and mean (SEM) % PNH RBC/WBC clone size increased from 59% (4.4%) to 92% (3.8%,). Clinically meaningful improvements in FACIT-Fatigue scores were observed over 48 weeks, with mean (SEM) CFB of 10.7 (6.0) points (mean score 37.3 vs. 47.9). The most common adverse events for all subjects (n=16) were headache (38%), self-limited rash (25%), and abdominal pain (19%). Summary/Conclusion: BCX9930 at doses up to 500 mg BID was generally well-tolerated. Notable improvements were observed from baseline to 48 weeks in Hb, ARC, and fatigue scores, with concomitant increases in PNH RBC clone size. Proximal AP inhibition of Factor D by BCX9930 in combination with C5 INH (with 3 of 4 subjects transitioning to monotherapy) led to sustained relief of anemia, fatigue, and reduction of transfusion burden, supporting ongoing pivotal trials of oral BCX9930 monotherapy for treatment of PNH. P827: EFFICACY AND SAFETY OF ROMIPLOSTIM ADDED TO IMMUNOSUPPRESSIVE THERAPY AS A FIRST-LINE TREATMENT IN PATIENTS WITH APLASTIC ANEMIA: A PHASE 2/3 CLINICAL TRIAL H. Yamazaki1,*, J. W. Lee2, J. H. Jang3, M. Sawa4, M. Kizaki5, Y. Tomiyama6, K. Nagafuji7, K. Usuki8, J.-P. Gau9, Y. Morita10, J.-L. Tang11, H. Chang12, M. Noshiro13, A. Matsuda14, K. Ozawa15, K. Mitani16, Y. Kanda15, S. Nakao1 1Kanazawa University Hospital, Ishikawa, Japan; 2Seoul St. Mary’s Hospital​; 3Samsung Medical Center​, Seoul, South Korea; 4Anjo Kosei Hospital, Aichi; 5Saitama Medical Center, Saitama; 6Osaka University Hospital, Osaka; 7Kurume University Hopital, Fukuoka; 8NTT Medical Center Tokyo, Tokyo, Japan; 9Taipei Veterans General Hospital​, Taipei, Taiwan; 10Kindai University Hospital, Osaka, Japan; 11National Taiwan University Hospital​, Taipei; 12Chang Gung Medical Foundation- Linkou Branch​, Taoyuan, Taiwan; 13Kyowa Kirin Co.,Ltd., Tokyo; 14Saitama Medical University International Medical Center, Saitama; 15Jichi Medical University Hospital; 16Dokkyo Medical University Hospital, Tochigi, Japan Background: Romiplostim, a thrombopoietin (TPO) mimetic protein, has been shown to promote tri-lineage hematopoiesis in patients with acquired aplastic anemia (AA) refractory to immunosuppressive therapy (IST) or eltrombopag; however, its effectiveness in the combination therapy with antithymocyte globulin (ATG) plus cyclosporine (CsA) as a first-line treatment remains unknown. Aims: To clarify this issue, we conducted a phase 2/3 clinical trial to evaluate the efficacy and safety of romiplostim combined with rabbit ATG plus CsA in patients with AA requiring transfusions who had not been exposed to IST. Methods: This study was a multi-national, open-label study with romiplostim in IST-naïve adult patients with AA (NCT03957694). 10 µg/kg of romiplostim was started on day 1 of ATG therapy (2.5mg/kg/day for 5 days) and was weekly given for the first 4 weeks. The dose was adjusted from 5 to 20 µg/kg according to the prespecified dose adjustment procedure. Treatment lasted until Week 26. For those who intend to continue treatment, romiplostim administration was extended to Week 52. The primary endpoint was the hematological response rate (HRR) at Week 27 based on the response assessment criteria (Table 1). The secondary endpoints included the HRR at Week 14; the time to the hematological response; the proportion of patients who achieved transfusion independence or who showed a reduction of transfusion requirement among patients who had received a transfusion within 8 weeks prior to the first romiplostim administration. The bone marrow and cytogenetic analyses were performed prior to enrollment and at Week 27 and 53. Results: Twenty-six patients were screened, of which 17 (9 Japan, 7 Korea, and 1 Taiwan; 5 transfusion dependent non-severe AA, 6 severe AA (SAA) and 6 very SAA) were enrolled in the study. The median age was 44.0 years (range: 25-70). Two patients discontinued romiplostim before Week 27. The HRR (CR or PR) at Week 27 was 76.5% (95% CI: 50.10%, 93.13%), of which 6 (35.3%) achieved CR. The HRR at Week 14 was 41.2% (95% CI: 18.44%, 67.03%). The median day to achieve the first hematological response was 93.0 (range: 63-181). Of the 16 patients who depended on platelet transfusion before romiplostim administration, 14 (87.5%) achieved transfusion independence or showed a reduction of transfusion requirement at Week 27. In addition, of 16 who required erythrocyte transfusion at baseline, 13 (81.3%) achieved transfusion independence or showed a reduction of transfusion requirement at Week 27. The frequently reported adverse events (AEs) were constipation (41.2%) followed by headache (35.6%). The frequently reported drug-related AEs were headache (12.9%) followed by muscle spasms (9.7%), alanine aminotransferase increased, fibrin D dimer increased, malaise, and pain in extremity (each 6.5%). In bone marrow examination, increases of reticuline grade (grade 1 to 2) were observed in 3 patients at Week 27 and 1 patient at Week 53. No chromosomal abnormality or transformation into MDS and/or AML was observed. Image: Summary/Conclusion: This is the first clinical trial of romiplostim combined with rabbit ATG plus CsA in patients with acquired AA requiring transfusions who were previously untreated with IST. This regimen produced higher HRR at Week 27 (76.5%) than those of historical control received rabbit ATG plus CsA (approximately 50% at 6 months), with manageable AEs, and could therefore serve as a new first-line treatment option in patients with AA. P828: NORMALIZATION OF HEMATOLOGIC AND HEALTH-RELATED QUALITY OF LIFE MARKERS IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA TREATED WITH PEGCETACOPLAN AND BASELINE HEMOGLOBIN AT OR ABOVE 10 G/DL J. Panse1,*, N. Daguindau2, S. Okuyama3, R. Peffault de Latour4, P. Schafhausen5, N. Straetmans6, M. Al-Adhami7, T. Ajayi7, E. Persson8, M. Yeh7, R. Wong9 1Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, University Hospital RWTH Aachen, Aachen, Germany; 2Hematology Department, CH Annecy Genevois, Annecy, France; 3Hematology/Oncology Division, Denver Health Medical Center, Denver, United States of America; 4French Reference Center for Aplastic Anemia and Paroxysmal Noctural Hemoglobinuria, Assistance Publique - Hôpitaux de Paris; Université de Paris, Paris, France; 5Medical Clinic and Polyclinic II - Hematology, Oncology, University Medical Center of Hamburg-Eppendorf, Hamburg, Germany; 6Department of Hematology, Cliniques Universitaires Saint-Luc, Woluwe-Saint-Lambert, Belgium; 7Apellis Pharmaceuticals, Inc., Waltham, United States of America; 8Swedish Orphan Biovitrum AB, Stockholm, Sweden; 9Sir Y.K. Pao Centre for Cancer & Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare life-threatening disease characterized by chronic complement-mediated hemolysis and thrombosis. Patients living with PNH can experience anemia with associated fatigue, dyspnea, and require blood cell transfusions which can negatively impact quality of life (QoL). Pegcetacoplan (PEG), is a C3 complement-inhibitor approved by the FDA/EMA for adults with PNH. Aims: This post hoc analysis evaluated hemoglobin (Hb), lactate dehydrogenase (LDH), absolute reticulocyte count (ARC), and Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue normalization rates and safety after PEG treatment in a subgroup of patients with PNH and baseline Hb levels ≥10.0g/dL from the PADDOCK (NCT02588833), PEGASUS (NCT03500549), and PRINCE (NCT04085601) studies. Methods: PADDOCK evaluated PEG treatment (270-360mg/day subcutaneously [sc]) in complement-inhibitor naïve patients. PEGASUS enrolled patients with Hb levels <10.5g/dL at screening despite stable eculizumab (ECU) treatment (≥3 months). PEGASUS patients were randomized 1:1 to ECU or PEG (1080mg sc 2x weekly) during the randomized controlled period (RCP) through Week 16. Patients who received PEG during the RCP continued with PEG monotherapy (PEG-to-PEG) and ECU patients switched to PEG monotherapy (ECU-to-PEG) during the open-label period (OLP) through Week 48. PRINCE compared PEG treatment (1080mg sc 2x weekly) in complement-inhibitor naïve patients with PNH and Hb levels below the lower limits of normal (males: ≤13.6g/dL; females: ≤12.0g/dL) to control treatment (CTRL; excluding complement-inhibitors i.e., ECU/ravulizumab). The post hoc analysis included adult patients with PNH with baseline Hb levels ≥10.0g/dL and no transfusions within 14 days of the baseline measurement. Hb, LDH, and ARC normalization, as well as FACIT-Fatigue normalization (defined as increase to population norm) was evaluated after 16 weeks of PEG monotherapy for all three studies. Patients who withdrew or were lost to follow-up without providing efficacy data at the specified timepoints were classified as non-responders. Safety endpoints included incidences of adverse events (AEs). Results: Overall, 22 patients were included in the post hoc analysis: 5 PADDOCK, 9 PEGASUS, and 8 PRINCE patients. After 16 weeks of pegcetacoplan treatment, patients in all three studies showed improvements in Hb normalization, LDH normalization, and ARC normalization (Table 1). Additionally, improvements in FACIT-Fatigue normalization were demonstrated in PADDOCK, PRINCE, and PEGASUS (PEG-to-PEG) patients (Table 1). No incidences of serious AEs were reported in PADDOCK and PRINCE subgroups. One PEGASUS patient had a serious AE of breakthrough hemolysis (severe, unrelated to PEG), following an upper respiratory infection which resolved and did not lead to study discontinuation. Incidences of injection site reactions and infections and infestations occurred at similar rates between subgroups in all three studies (Table 1). No thrombotic events occurred in this patient population. Image: Summary/Conclusion: While this patient population had near normal Hb at baseline (median: 10.4g/dL), they also had elevated ARC and LDH prior to PEG therapy, suggesting ongoing hemolysis which was normalized in most patients after PEG treatment initiation. Overall, these results suggest PEG can be efficacious long-term in patients with less severe anemia, regardless of prior complement-inhibitor treatment, resulting in further clinical improvements in markers of hemolysis and QoL. The safety profile of PEG was consistent with results from previous studies. P829: A PHASE III RANDOMIZED CLINICAL TRIAL COMPARING SB12 (PROPOSED ECULIZUMAB BIOSIMILAR) WITH REFERENCE ECULIZUMAB IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA J. H. Jang1,*, R. Demichelis Gomez2, H. Bumbea3, L. Nogaieva4, L. L. L. Wong5, S. M. Lim6, J. Park7, Y. Kim8, S. Cho7 1Hematology-Oncology, Samsung Medical Center, Seoul, South Korea; 2Hematology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City, Mexico; 3Hematology, Bucharest Emergency University Hospital, Bucharest, Romania; 4Regional Treatment-Diagnostic Hematology Center, Communal Nonprofit Enterprise Cherkasy Regional Oncology Dispensary of Cherkasy Oblast Council, Cherkasy, Ukraine; 5Hematology Unit, Hospital Queen Elizabeth, Kota Kinabalu; 6Department of Medicine, Hospital Sultanah Aminah, Johor Bahru, Malaysia; 7Clinical Development; 8Biometrics, Samsung Bioepis, Incheon, South Korea Background: SB12, a proposed eculizumab biosimilar, is a humanized monoclonal antibody that blocks complement C5 cleavage, thereby inhibits terminal complement-mediated intravascular hemolysis. Aims: The randomized, double-blind, multicenter, cross-over study was aimed to demonstrate comparable clinical efficacy by evaluating the lactate dehydrogenase (LDH), safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of SB12 and reference eculizumab (ECU) in paroxysmal nocturnal hemoglobinuria (PNH) patients (NCT04058158). Methods: A total of 50 patients aged ≥ 18 years with a confirmed diagnosis of PNH and ≥ 1.5 upper limit of normal range (ULN) of LDH without previous exposure to a complement inhibitor were included. All patients provided written informed consents and were randomized (1:1) to treatment sequence I (TS1: SB12 to ECU, n=25) or II (TS2: ECU to SB12, n=25). Patient received 600 mg of SB12 (TS1) or ECU (TS2) intravenously every week for first 4 weeks (initial phase) and 900 mg for the fifth week, followed by 900 mg every 2 weeks thereafter (maintenance phase). The treatment was switched to ECU (TS1) or SB12 (TS2) at Week 26, and switched treatment was provided until Week 50. Primary endpoints were LDH level at Week 26 and time-adjusted area of under the effect curve (AUEC) of LDH from Week 14 to Week 26 and Week 40 to Week 52. Equivalence was declared for LDH level at Week 26 if the two-sided 95% confidence interval (CI) of the mean difference in between SB12 and ECU lied within the pre-defined equivalence margin of [−1.2 × ULN, 1.2 × ULN] = [−337.2, 337.2], where ULN = 281 U/L. Equivalence was declared for time-adjusted AUEC of LDH if the two-sided 90% CI of the ratio of geometric means between SB12 and ECU lied within the pre-defined equivalence margin of [0.77, 1.29]. Secondary endpoints were LDH profile over time and number of units of packed red blood cells (pRBCs) transfused throughout the study period, safety, PK, PD, and immunogenicity. Results: Of the 50 randomized patients, 49 patients received the study treatment and 46 patients completed the study. Baseline demographic and disease characteristic were comparable between the two treatment sequences. The 95% CI of mean difference in LDH level at Week 26 between SB12 and ECU (SB12 − ECU: 34.48, 95% CI [−47.66, 116.62]) lied within the pre-defined equivalence margin. The 90% CI of ratio of time-adjusted AUEC of LDH between SB12 and ECU (SB12/ECU: 1.08, 90% CI [0.95, 1.23]) lied within the pre-defined equivalence margin. The overall LDH profile during the study period was also comparable. The mean number of units of pRBCs transfused during Period 1 (TS1: 1.1 U; TS2: 0.9 U, respectively) and Period 2 (TS1: 1.1 U; TS2: 1.0 U, respectively) was comparable. Treatment-emergent adverse events (TEAEs) was reported in 72.3% for SB12 and 68.1% in ECU. Three patients in SB12 and 2 patients in ECU had serious TEAEs. None of three serious TEAEs reported in SB12 were related to the treatment. Two of three TEAEs (cellulitis; infusion site hypersensitivity) reported in ECU were treatment-related, and 1 death due to major adverse vascular event (portal vein thrombosis) not related to the treatment was reported in ECU. The mean eculizumab serum trough concentrations and the mean terminal complement activities in both treatment sequences were comparable. No patient developed anti-drug antibodies during the study period. Image: Summary/Conclusion: SB12 showed clinical equivalence to ECU measured by LDH. SB12 and ECU were comparable in terms of safety, PK, PD, and immunogenicity. P830: CROVALIMAB MAINTAINS CLINICAL BENEFIT OVER LONG-TERM TREATMENT IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA: RESULTS FROM THE COMPOSER TRIAL OPEN-LABEL EXTENSION A. Röth1,*, M. Egyed2, S. Ichikawa3, Y. Ito4, J. S. Kim5, Z. Nagy6, N. Obara7, J. Panse8, H. Schrezenmeier9, S. Sica10, J. Soret11, K. Usuki12, S.-S. Yoon13, K. Benkali14, M. Buri15, P. Lundberg15, H. Patel16, K. Shinomiya17, S. Sreckovic15, J.-I. Nishimura18 1Department of Hematology and Stem Cell Transplantation, West German Cancer Centre, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2Kaposi Mor Oktato Korhaz, Kaposvar, Hungary; 3Tohoku University Hospital, Miyagi; 4First Department of Internal Medicine, Hematology Division, Tokyo Medical University, Tokyo, Japan; 5Yonsei University College of Medicine, Severance Hospital, Seoul, South Korea; 6Semmelweis University, Budapest, Hungary; 7Department of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan; 8University Hospital RWTH Aachen, Aachen; 9Institute of Transfusion Medicine, University Hospital Ulm, Ulm, Germany; 10Fondazione Policlinico Universitario A. Gemelli-Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy; 11Centre d’Investigation Clinique, Hôpital Saint-Louis, Paris, France; 12NTT Medical Center Tokyo, Tokyo, Japan; 13Department of Internal Medicine, Seoul National University Hospital, Seoul, South Korea; 14Certara, Inc., Paris, France; 15F. Hoffmann-La Roche, Ltd., Basel, Switzerland; 16Genentech, Inc., South San Francisco, CA, United States of America; 17Chugai Pharmaceutical Co., Tokyo; 18Graduate School of Medicine, Osaka University, Osaka, Japan Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a potentially life-threatening acquired hematopoietic stem-cell disorder, resulting in hematuria, anemia, and acquired thrombophilia. In the Phase I/II COMPOSER trial (NCT03157635), patients (pts) with PNH who were treatment-naive or switched from eculizumab were treated with crovalimab, a novel anti-C5 monoclonal antibody designed with Sequential Monoclonal Antibody Recycling Technology (Röth et al. Blood 2020). Aims: To report the long-term efficacy, safety, pharmacokinetic (PK) and pharmacodynamic (PD) outcomes in pts with PNH treated with crovalimab in the open-label extension (OLE) period of the COMPOSER trial. Methods: COMPOSER consists of four sequential parts followed by an OLE period. Pts with PNH who were treatment-naive or switched from eculizumab in Parts 2, 3, and 4, who completed the primary treatment period through Week 20 and derived benefit from crovalimab treatment, were eligible to enter the OLE (Röth et al. EHA 2019). Long-term efficacy, safety, PK and PD data are presented for the OLE. Efficacy endpoints include change in lactate dehydrogenase (LDH), transfusion avoidance and hemoglobin stabilization (both assessed in 24-week intervals), and breakthrough hemolysis (BTH). PK and PD endpoints include assessment of serum concentrations of crovalimab, complement activity by liposome immunoassay (LIA), and free C5 over time. Results: Overall, 43 of 44 pts entered the OLE and at the clinical cutoff (Nov 1, 2021), 38 pts were ongoing. Mean normalized LDH was maintained at ≤1.5 × upper limit of normal (ULN) during the OLE, and 80% to 100% of evaluable pts achieved LDH ≤1.5 × ULN at each visit (Figure). Transfusion avoidance was achieved in 83% to 92% of pts over each 24-week interval in the OLE. Similarly, hemoglobin stabilization remained relatively stable during the OLE, with 79% to 88% of pts achieving hemoglobin stabilization across 24-week intervals. There was a total of five BTH events during the OLE across all pts (none leading to withdrawal) for an overall BTH rate of 0.05 events per pt-year (95% confidence interval: 0.01, 0.11). The incidence of adverse events (AEs) was reported cumulatively from baseline. Median treatment duration (range) for treatment-naive and switched pts on the study was 3.4 years (0.9-4.4) and 2.9 years (0.4-3.9), respectively. Any grade AEs occurred in 95% (42/44) of pts; none led to withdrawal from treatment. Severe AEs occurred in 17% (3/18) of treatment-naive pts and 19% (5/26) of switched pts. Serious AEs occurred in 32% (14/44) of pts (33% [6/18] in treatment-naive and 31% [8/26] in switched pts), of which 5% (2/44) were assessed as related to study treatment by the investigator. No meningococcal infections or deaths were reported. Crovalimab concentrations were stable and within the expected range over time. Terminal complement activity by LIA was near the lower limit of quantification (10 U/mL) and free C5 levels were maintained at low levels (<0.001 g/L) in most pts. PK/PD relationships between free C5 or LIA with crovalimab confirmed that crovalimab levels of ≈100 μg/mL resulted in complete inhibition of terminal complement activity. Image: Summary/Conclusion: Treatment-naive and switched pts with PNH treated with crovalimab in the COMPOSER trial maintained disease control over long-term follow-up. No new safety signals were identified, and complete terminal complement inhibition was maintained during the OLE. These data support the continued clinical development of crovalimab in PNH. P831: PATIENT-REPORTED OUTCOMES IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA TREATED WITH CROVALIMAB: RESULTS FROM THE COMPOSER TRIAL A. Röth1,*, J. Panse2, B. Gentile3, M. Buri4, H. Patel3, Z. Nagy5 1Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen; 2University Hospital RWTH Aachen, Aachen, Germany; 3Genentech, Inc., South San Francisco, CA, United States of America; 4F. Hoffmann-La Roche Ltd., Basel, Switzerland; 5Semmelweis University, Budapest, Hungary Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a potentially life-threatening acquired hematopoietic stem cell disorder, resulting in anemia, hemoglobinuria, and acquired thrombophilia. Patients with PNH can experience a variety of disease-related symptoms, particularly fatigue, that can negatively impact their day-to-day functioning and health-related quality of life. In the Phase I/II COMPOSER trial (NCT03157635), patients with PNH who were treatment-naive or switched from eculizumab were treated with crovalimab, a novel anti-C5 monoclonal antibody designed with Sequential Monoclonal Antibody Recycling Technology (Röth et al. Blood 2020). Aims: To explore the efficacy of crovalimab from the perspective of patients with PNH using patient-reported outcome (PRO) tools to assess fatigue, functioning, and global health status (GHS)/quality of life (QoL). Methods: COMPOSER included treatment-naive (Parts 2 and 4 Arm A [4A]) and switched (Parts 3 and 4 Arm B [4B]) patients who received different crovalimab dosing regimens for a 20-week treatment period (Figure). Paper PROs were completed at baseline and at scheduled visits during the treatment period (up to Week 10 for Part 2 and Week 20 for Parts 3 and 4). Fatigue was assessed with the Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue scale. Additional symptoms, functioning, and GHS/QoL were assessed with the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30). Results: The PRO completion rate at baseline was 100% of evaluable patients for Parts 2 (n=10), 3 (n=19), and 4A (n=8), and 86% for 4B (n=7). Completion rates remained ≥86% for all follow-up visits. Mean FACIT-Fatigue scores at baseline among treatment-naive patients in Parts 2 (29.7, standard deviation [SD]=13.7) and 4A (33.9, SD=8.7) were below or within the range for severe fatigue (30-34; Eek et al. J Patient Rep Outcomes 2021). Mean scores improved while on treatment with crovalimab, from baseline to Week 10 in Part 2 patients (8.8, SD=10.2) and to Week 20 in Part 4A patients (6.0, SD=6.1), exceeding the threshold for clinically meaningful improvement (5 points; Cella et al. ASH 2021). Switched patients in Parts 3 and 4B had less fatigue on average at baseline (37.3, SD=12.6; 37.0, SD=9.9, respectively) compared with treatment-naive patients, and showed largely minor changes at each follow-up. Similar results were observed for the EORTC QLQ-C30 Fatigue scale. Part 2 and Part 4A patients by Week 10 and 20, respectively, had mean fatigue severity levels comparable to (FACIT-Fatigue) or lower than (EORTC QLQ-C30 Fatigue) mean baseline values in switched patients. Mean EORTC QLQ-C30 scores in all functioning domains (i.e., physical, role, social, emotional, and cognitive) improved from baseline to Week 10 in Part 2 patients. Improvements in physical, role, and social functioning only were observed in Part 4A patients at all scheduled follow-up visits. Patients in both treatment-naive cohorts exhibited improvements in GHS/QoL. Switched patients, on average, exhibited limited change from baseline in EORTC QLQ-C30 scores, with mean improvements only observed for emotional functioning in Part 4B patients. Image: Summary/Conclusion: Treatment-naive patients experienced improvements from baseline in fatigue, functioning, and GHS/QoL whereas switched patients maintained baseline levels. These data provide supportive evidence for the treatment efficacy of crovalimab from the patient perspective. P832: CLINICAL OUTCOMES AND IMMUNE RESPONSES TO SARS-COV-2 VACCINATION IN PATIENTS WITH SEVERE APLASTIC ANEMIA R. Rajput1,*, X. Ma2, K. L. Boswell3, M. Gaudinski3, E. M. Groarke1, J. Lotter1, O. Rios1, I. Darden1, C. O. Wu2, R. A. Koup3, N. S. Young1, B. Patel1 1Hematology Branch, National Heart Lung and Blood Institute; 2Office of Biostatistics Research; 3Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland, United States of America Background: Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 has led to significant morbidity and mortality in patients with hematological disease. Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disorder that presents with pancytopenia and a hypocellular marrow due to immune-mediated destruction of hematopoietic stem cells (HSC). Patients are at high risk for infection due to neutropenia and treatment with immunosuppressive therapy (IST). Acute COVID-19 infection has been reported to cause worsening underlying marrow failure and death in unvaccinated SAA patients. Patients with SAA are often not vaccinated to protect against routine viruses due to concerns of ineffective protective specific antibody response to viral antigens and potential pathogenic global immune system activation. To date, the clinical impact and efficacy of COVID-19 vaccination in SAA has not been described. Aims: Assess the impact of SARS-CoV-2 vaccination on SAA disease status and to determine the humoral and cellular response to vaccination. Methods: SAA treated with IST including horse antithymocyte globulin (h-ATG), cyclosporine (CSA), and eltrombopag (EPAG) at the National Institute of Health with confirmation of SARS-CoV-2 vaccination between January and November 2021 were included. Current SAA disease status at the time of vaccination was recorded as complete response (CR), partial response (PR) and non-response (NR). Univariate analysis was performed using the Student paired t-test to compare available blood counts. Peripheral mononuclear blood cells (n=27) and serology (n=12) after completion of vaccination series were analyzed to assess cellular and humoral response, respectively. Results: Blood samples from fifty patients with SAA with median age of 42 years (9-78), 29 females, and 21 males, were studied. At time of vaccination, 15 patients (30%) were receiving CSA. Forty-seven of 50 (94%) of patients did not have any changes in disease status after vaccination. Among 40 patients with available data 3 months prior to and after completion of vaccination series, there was no significant difference in HGB (p=0.52) PLT (p=0.67), ANC (p=0.98), and ALC (p=0.42; Figure 1A-C). Relapse after vaccination, defined as progressive and substantial decline in blood counts, was noted in 3 (6%) cases. All 3 relapsed patients were deemed weak PR at time of vaccination (PLT < 50 K/μL) and were 6 months, 3 years, and 4 years from initial IST. Two of the 3 patients received an initial Pfizer inoculation and did not receive the second dose because of declining blood counts. Relapse occurred 4 weeks after completion of a full Moderna series in the third patient, who had demonstrated a decline in counts 6 months prior to vaccination. Vaccinated SAA patients all had adequate antibody production to the SARS-CoV-2 spike protein. They also exhibited a CD4+ Th1 dominant response, similar to healthy controls, even when receiving CSA at time of vaccination (Figure D). CD8+ responses to vaccination were observed, but patients with SAA appear to have qualitatively blunted responses compared to controls. Image: Summary/Conclusion: SARS-CoV-2 vaccination in SAA may result in transient decline in blood counts but true relapse is rare. Relapse may be more likely in patients whose blood counts are declining prior to vaccination. Appropriate humoral and cellular responses are seen in patients with SAA. A risk versus benefit assessment to vaccinate against SARS-CoV-2 in SAA may be warranted in patients with weak partial response to initial therapy or whom have clinical or laboratory evidence suggestive of active disease. P833: CATEGORIZING HEMATOLOGICAL RESPONSE TO PEGCETACOPLAN IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA: A POST HOC ANALYSIS OF THE PHASE 3 PRINCE STUDY DATA A. Risitano1,*, R. Wong2, M. Al-Adhami3, J. Savage3, R. Horneff4, R. Peffault de Latour5 1Hematology and BMT Unit, AORN, San Giuseppe Moscati, Avellino, Italy; 2Sir Y.K. Pao Centre for Cancer & Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong; 3Apellis Pharmaceuticals, Inc., Waltham, United States of America; 4Swedish Orphan Biovitrum AB, Stockholm, Sweden; 5French Reference Center for Aplastic Anemia and Paroxysmal Noctural Hemoglobinuria, Assistance Publique - Hôpitaux de Paris; Université de Paris, Paris, France Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare complement-mediated hematological disorder characterized by anemia, hemolysis, and bone marrow failure. Pegcetacoplan (PEG), an FDA/EMA-approved targeted C3 therapy for the treatment of PNH, can control intravascular and prevent extravascular hemolysis. A prior post hoc analysis of PEGASUS reported a higher proportion of patients presenting good or better hematologic responses to PEG versus eculizumab (Risitano A, et al., Blood, 2021;138 [Supplement 1]: 1104). The PRINCE study demonstrated efficacy/safety of PEG compared to control treatment (CTRL; supportive treatment of PNH without complement inhibitors) in complement-inhibitor naïve patients with PNH. Aims: This post hoc analysis categorized the hematologic response to PEG and CTRL at baseline and Week 26 in the PRINCE Trial (phase 3, multicenter, randomized, open-label, controlled trial [NCT04085601]). Methods: Adult complement-inhibitor naïve (no complement-inhibitor treatment within 3 months prior to screening) patients with PNH who had low hemoglobin (Hb) levels and high lactate dehydrogenase levels were included. Patients were randomized 2:1 to receive PEG or CTRL through Week 26. Hematologic response to PEG and CTRL was categorized as complete, good, partial, or minor at baseline and at Week 26 using the number of packed red blood cells transfusions needed in the last 6 months and Hb level at the specified timepoints (revised categorization criteria per Debureaux PE, et al. Bone Marrow Transplant. 2021; 56(10):2600-2602). Hematologic response categorization was performed according to the following criteria: Complete response: no transfusions, no anemia (≥12 g/dL); Good response: no transfusions, but with chronic mild anemia (10-12 g/dL); Partial response: no/occasional transfusions (≤2 units/6 months) and chronic moderate anemia (8-10 g/dL); Minor response: regular transfusions (≥3 units/6 months) and chronic moderate/severe anemia (<10 g/dL), or no/occasional transfusions (≤2 units/6 months) and chronic severe anemia (<8 g/dL). Any patients with missing data at Week 26 were evaluated using the most recent value within the 6 weeks prior. Statistical tests assessed significance for responses of ‘Good’ or ‘Complete’, collectively (Fisher’s Exact Test between PEG and CTRL groups at baseline; McNemar’s test within PEG group at baseline vs. Week 26). Results: In this study, 53 patients were randomized to PEG (n=35) or CTRL (n=18). At baseline, most patients had a partial or minor response to supportive therapy prior to randomization, with only 5.7% of patients in the PEG group and 0.0% in the CTRL group presenting a Good or Complete hematologic response (p= 0.5428). Hematologic response categorization for patients in the PEG group are shown for baseline and Week 26 (Figure). At Week 26, 80.0% of patients in the PEG group exhibited a Good or Complete response. The proportion of patients in the PEG group who demonstrated a Good or Complete response was significantly higher at Week 26 as compared to baseline (p<0.0001). One patient treated with PEG had missing data at Week 26 due to being lost to follow-up and was not evaluated. Image: Summary/Conclusion: This post hoc analysis of PRINCE trial data revealed that a substantial proportion of patients achieved good or complete hematologic response to PEG after 26 weeks. These findings further support the positive efficacy of PEG on hematologic parameters and provide evidence for the role of proximal complement inhibition on hematological and clinical improvements for patients with PNH. P834: REAL-WORLD EVIDENCE OF SAFETY AND EFFECTIVENESS OF ECULIZUMAB AND SWITCH TO RAVULIZUMAB IN A SWISS PATIENT POPULATION WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA A. Rovó1,*, L. Simeon2, M. Gavillet3, K. Samii4, N. Cantoni5, D. Heim6, G. Stüssi7, M. Bissig8 1Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, Bern; 2Department of Hematology and Central Hematology Laboratory, Cantonal Hospital Lucerne, Luzern; 3Service and Central Laboratory of Hematology, Department of Oncology and Department of Laboratory Medicine and Pathology, Lausanne University Hospital (CHUV), Lausanne; 4Hematology Division, Department of Oncology, Geneva University Hospitals, Geneva; 5Division of Oncology, Hematology and Transfusion Medicine, Kantonsspital Aarau, Aarau; 6Department of Hematology, University Hospital Basel, Basel; 7Clinic of Hematology, Oncology Institute of Southern Switzerland, Bellinzona; 8Department of Medical Oncology and Hematology, University Hospital of Zurich, Zurich, Switzerland Background: Eculizumab (ECU) a complement component 5 (C5) inhibitor, is the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH). Ravulizumab (RAV) is engineered from ECU with the main advantage of having an extended 8-week (vs 2-week with ECU) dosing interval, which decreases treatment burden markedly. Hence, RAV is expected to become the new standard of care to treat PNH. In Switzerland, RAV was approved for PNH treatment in 2020. Aims: The objective of this study was to evaluate data from the PNH Swiss Soliris and Ultomiris Reimbursement Registry (SSURR) to assess key parameters for safety and effectiveness of ECU and to assess the adoption of RAV for PNH treatment by physicians under real-world conditions. Methods: The SSURR was designed in 2012 as a prospective, longitudinal, multi-center registry study in Switzerland. Patients were recruited and followed in 9 centers. Health-related data were collected at the first consultation (baseline) and during follow-up visits. In this report, results are based on data collected from 23 February 2012 to 30 September 2021. Data were summarized using descriptive analysis. Ethics approval was obtained. All participants provided written informed consent. Results: In total, 56 patients with a median age of 51 (range 20-84) years were enrolled in the Registry, 52% were female. For 42 patients, the 12-month follow-up (FU) was available: 39 patients on ECU and 3 on RAV (switched from ECU). Median FU was 27 (range 12-108) months. Hemoglobin (Hb) mean was 63 ± 47 g/L at baseline (n=41), and Hb mean was 105 ± 25 g/L (n=26) at the last visit. The lactate dehydrogenase (LDH) decreased from a mean 1457 ± 706 U/L (baseline, n=42) to 403 ± 212 U/L after 12 months FU (n=40; 65% of patients < 1.5 × upper limit of normal [ULN]), and was for 88% of the patients < 1.5 × ULN at their last visit (n=24). Thrombosis events (TE) were reported in 33% of patients prior to treatment start (n=42) and 3 (7%) had thrombosis during FU of which 2 patients had no prior history of TE. Within 12 months prior to treatment start, 21 patients (50%) received one or more red blood cell (RBC) transfusions. At 12 months of FU, 91% (n=42) were RBC transfusion-free. RBC transfusions remained reduced (82%; n=27 at last visit). After 12 months FU, 19% (n=8) of the patients had withdrawn from the registry due to allogenic bone marrow transplantation, low PNH clone size, transformation to hypoplastic MDS, and 5 non-disease related reasons. Eight patients from the 56 enrolled received RAV as initial treatment. They were all C5-inhibitor treatment-naïve with a mean LDH level of 1481 ± 1432 U/L at inclusion, which decreased to 230 ± 62 U/L at the 6-month visit (n=4; all below 1.5 × ULN). Of the 56 enrolled patients with PNH, 43% were switched from ECU to RAV. These patients (58% female) had a median age of 55 (range 26-85) years and 75% of them had LDH < 1.5 × ULN at their last LDH measurement on ECU before RAV administration. There were 7 patients with a 6-month FU after switching at which 6 of them had a LDH below 1.5 × ULN. Summary/Conclusion: The current analysis provides real world evidence for the safety and effectiveness of ECU in the Swiss PNH patient population, demonstrated by pronounced reduction in LDH, low need for RBC transfusion and stable hemoglobin as well as reduced occurrence of thrombosis. The fast adoption of RAV demonstrates RAV may likely become the new standard of care for patients with PNH. In time, more data will become available for safety and effectiveness of RAV under real-world conditions. P835: GENETIC BACKGROUND AND CLINICAL CHARACTERISTICS OF CONGENITAL NEUTROPENIAS IN ISRAEL O. Steinberg-Shemer1,2,3,*, L. Yeshareem4, S. Noy-Lotan3, O. Dgany3, T. Krasnov3, G. Berger Pinto1, B. Bielorai2,5, A. A. Kuperman6,7, R. Laor8, N. Mandel-Shorer9,10, A. Ben Barak9, C. Levin10,11, A. Mahdi12, H. Miskin12, S. Revel-Vilk13,14, D. Levin15, M. Benish15, T. Zuckerman10,16, O. Wolach2,17, I. Pazgal2,18, O. Gilad1,2, T. Goldberg1, J. Yacobovich1,2, S. Izraeli1,2, H. Tamary1,2,3 1Hematology-Oncology, Schneider Children’s Medical Center of Israel, Petach Tikva; 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; 3Pediatric Hematology Laboratory, Felsenstein Medical Research Center; 4Pediatrics A, Schneider Children’s Medical Center of Israel, Petach Tikva; 5Pediatric Hematology-Oncology, Sheba Medical Center, Tel Hashomer; 6Blood Coagulation Service and Pediatric Hematology Clinic, Galilee Medical Center, Nahariya; 7Azrieli Faculty of Medicine, Bar-Ilan University, Safed; 8Hematology Service, Bnei Zion Medical Center; 9Department of Pediatric Hematology-Oncology, Ruth Rappaport Children’s Hospital, Rambam Healthcare Campus; 10Rappaport Faculty of Medicine, Technion-Institute of Technology, Haifa; 11Pediatric Hematology Unit and Research Laboratory, Emek Medical Center, Afula; 12Pediatric Hematology, Soroka University Medical Center, Ben-Gurion University, Beer Sheva; 13Pediatric Hematology/Oncology Unit, Shaare Zedek Medical Center; 14Faculty of Medicine, Hebrew University, Jerusalem; 15Pediatric Hemato-Oncology, Tel Aviv Medical Center, Tel Aviv; 16Hematology and Bone Marrow Transplantation Institute, Rambam Healthcare Campus, Haifa; 17Institute of Hematology, Davidoff Cancer Center, Rabin Medical Center; 18Comprehensive Center of Thalassemia, Hemoglobinopathies & Rare Anemias, Institute of Hematology, Beilinson Hospital, Rabin Medical Center, Petach Tikva, Israel Background: Congenital neutropenias comprise a heterogenous group of disorders causing low neutrophil counts with a variable clinical presentation. Patients may suffer from severe infections and have a high risk of myeloid transformation. To date, over 30 genes are known to cause congenital neutropenias, with a diverse relative frequency in different ethnicities. Understanding the spectrum of genetic causes of neutropenia in ethnically diverse populations and the related clinical presentation may direct the molecular work-up and clinical care. Israel is enriched with multiple ethnicities and high consanguinity. Aims: To evaluate the clinical and genetic spectrum of patients with congenital neutropenias in Israel. Methods: All patients diagnosed with congenital neutropenia from the Israeli Inherited Bone Marrow Failure registry were included. Data were extracted from the registry and patients’ charts. Sanger sequencing was first performed for ELANE or G6PC3, followed by less commonly mutated genes. In recent years, patients with wild-type ELANE/G6PC3 were referred for targeted next generation sequencing, followed by whole exome sequencing. Results: The cohort encompasses 66 patients, from 50 families, including 7 with cyclic neutropenia. Fifty patients (76%) were genetically diagnosed; 22 (33% of the cohort) had mutations in ELANE, 9 (13.6%) had homozygous G6PC3 mutations, 7 (10.6%) SBDS mutations, 6 (9%) patients had SRP54 mutations, two each had heterozygous GFI1 and homozygous JAGN1 mutations and one each had TAZ and SLC37A1 mutations. All patients with G6PC3 and GFI1 mutations were of consanguineous Muslim Arab origin. Extra-hematopoietic manifestations were common among patients with mutations in G6PC3. Most patients (82%) presented with severe infections and this was also the most common cause of death. Eight patients (12%) developed myeloid transformation; in 6 of those a somatic molecular work-up was performed, and truncating mutations in the granulocyte-colony stimulating factor were detected in 4. Eighteen (27%) patients underwent hematopoietic stem cell transplantation, most often due to insufficient response to treatment with granulocyte-colony stimulating factor. Summary/Conclusion: The prevalence of genes causing congenital neutropenias in Israel is unique, as it includes a relatively high frequency of patients with mutations in G6PC3 and an absence of patients with HAX1 mutations, thus directing the genetic work-up and the clinical care. Similar to other registries, a molecular diagnosis was not achieved for 24% of the patients, suggesting yet unknown genetic causes. P836: LONG-TERM DYNAMICS OF CLONAL HEMATOPOIESIS IN CHRONIC IDIOPATHIC NEUTROPENIA (CIN) G. Tsaknakis1,2,*, S. Papadakis1,2, P. Kanellou3, E. Boutakoglou1,2, I. Mavroudi1,2, C. Pontikoglou1,2, E. Papadaki1,2 1Haemopoiesis Research Laboratory, University of Crete School of Medicine; 2Department of Hematology, University Hospital of Heraklion; 3Department of Hematology, Venizeleion General Hospital, Heraklion, Greece Background: We have previously performed next generation sequencing (NGS) analysis of genes recurrently mutated in myeloid malignancies in a cohort of CIN patients. We have estimated for the first time the frequency of clonal hematopoiesis in these patients and found that clonal CIN patients have significantly higher risk to develop MDS/AML compared to non-clonal CIN patients. Aims: To conduct longitudinal follow-up NGS analyses of myeloid genes in CIN patients in order to assess clonal dynamics and evolution of clonal hematopoiesis in these patients. Methods: We have performed longitudinal NGS analyses in 53 patients with CIN diagnosis (mean absolute neutrophil counts -ANCs- 1320±460/μL; median 1500, range 200-1700/μL) according to previously published criteria i.e no evidence of any underlying condition related to neutropenia after an extended clinical/laboratory investigation including bone marrow biopsy, karyotype, immunophenotype. Genomic DNA was extracted from bone marrow or peripheral blood samples at baseline and follow-up timepoints, sequencing libraries were prepared and subjected to targeted NGS on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 myeloid genes. Results: Longitudinal NGS analysis in the patients was performed over a period of 4-173 months (median 31 months). The incidence of clonal hematopoiesis was similar at baseline (16/53 i.e. 30.2%) and follow-up (16/53 i.e. 30.2%) (Figure 1A). The mutation spectrum (i.e. absence of mutations, multiple mutated genes or presence of a single mutated gene) at baseline (Figure 1B) was comparable to that at follow-up (Figure 1C). In total, 28 mutations were detected in 17 CIN patients at one or both time-points. The variant allele frequencies (VAF) did not show a significant difference at baseline (9.68%) and follow-up (13.23%) and correlation analysis showed that VAFs at both points strongly correlated (r=0,59; P<0.01), with the majority of CIN patients showing a stable clone size (Figure 1D). We performed the same analysis for the most frequently mutated genes in our cohort of patients, namely DNMT3A, TET2, SRSF2 (Figure 1E-G). Overall, DNMT3A mutations did not show any positive selection over time, with a median increase in VAF of 1.78% (P=0.67) during the follow-up period (median period of 47.5 months, range 12-164). Similarly non-significant increases in VAF was seen for TET2 (0.49%) and SRSF2 (0.04%), within a median period of 26 months (range 22-111) and 55 months (range 12-98), respectively. One patient with newly developing clonal CIN and one patient with disappearance of clonal hematopoiesis were found at follow-up after 4 and 98 months, respectively. Two patients acquired a second mutation at follow-up. At baseline they carried mutations in SRSF2 and ZRSR2, respectively and at follow-up they both developed TET2 truncating mutations. No significant changes at baseline and follow-up in ANCs were observed in patients with clonal disease. Overall, four patients (7.55%) transformed to AML/MDS. Image: Summary/Conclusion: In the majority of CIN patients tested for clonal evolution over time, most mutant hematopoietic clones appeared to be remarkably stable, with limited VAF expansion over the follow-up period and no acquisition of new molecular alterations. Only two patients acquired an additional mutation over time and this propensity was associated with mutations in spliceosome genes. None of the individuals bearing DNMT3A or TET2 mutations acquired additional mutations. This study contributes to the better understanding of clonal hematopoiesis in CIN. P837: ELTROMBOPAG (EPAG) INDUCES HEMATOLOGIC RESPONSES FOR PATIENTS WITH POST-ALLOGENIC HEMATOPOIETIC STEM CELL TRANSPLANT POOR GRAFT FUNCTION (POST-ALLO HSCT PGF): RESULTS OF THE ELTION STUDY IN SPAIN C. Vallejo1,*, I. García2, F. Sanchez Guijo3, M. Cuesta4, C. Solano5, A. Sampol6, T. Torrado7, M. Vendranas8, M. Marigil8 1University Hospital Donostia, San Sebastian, Spain, University Hospital Donostia, San Sebastian, Spain, Santiago de Compostela; 2Department of Hematology, University Hospital Santa Cruz y San Pau, Barcelona; 3Department of Hematology, University Hospital of Salamanca, Salamanca; 4Department of Hematology, Carlos Haya University Hospital, Malaga; 5Department of Hematology, Hospital Clinico de Valencia, Valencia; 6Department of Hematology, University Hospital Son Espases, Palma de Mallorca; 7Department of Hematology, University Hospital Alvaro Cunqueiro, Vigo; 8Medical Affairs Department, Novartis, Barcelona, Spain Background: Persistent cytopenia due to poor graft function (PGF) is a life-threatening complication in patients undergoing allogeneic HSCT (allo-HSCT). Several therapeutic approaches have been tested in this subset of patients with poor clinical results. Aims: The objective of this multicenter open-label interventional prospective phase II Novartis study (ELTION, ClinicalTrials.gov id: NCT03718533), was to analyze efficacy and safety of EPAG in patients with post-allo-HSCT poor graft function. Methods: Adult patients diagnosed with PGF (defined as severe cytopenia after day +30 post-transplant, with two or more of the following: platelets <20000/µL -mandatory-, ANC <1000/µL, hemoglobin< 10 g/dL), and full donor chimerism, were eligible to enter the trial. Study treatment consisted of EPAG. at 150 mg/day administered up to 36 weeks; dose adjustments were contemplated as per protocol on an individual basis. The primary efficacy endpoint was the overall hematologic response (partial and complete), as determined by platelet, hemoglobin and neutrophil counts by 16 weeks after the initiation of EPAG. Results: Although the aim of the study was to include 33 patients, recruitment stopped prematurely due to the difficulties for hospital visits posed during COVID-19 pandemic, and eventually only 10 patients were included. The decision for this premature termination is not related to any safety concern related to the drug. Patient characteristics are shown in the Table 1 attached below. At EPAG. initiation, all 10 patients showed thrombocytopenia (<20000/µcL), 5 presented with anemia (Hgb <10 g/dL), and 4 had neutropenia (ANC <1000/µcL). Four patients discontinued EPAG before week 12 due to: disease progression/relapse (2 patients), protocol deviation (1 patient), and CMV infection (1 patient). In none of the cases, the event was related to study drug. At week 16, 4 patients (4/10, 40%) and at week 24, 5 patients, showed improvement in at least one of the 3 hematologic cell lines (partial response), respectively. Counts pre- and post-EPAG and global response in patients who stayed on treatment > 12 weeks are displayed below: Image: Summary/Conclusion: In our experience, EPAG worked well in subjects with PGF, an otherwise life-threatening condition for patients, and its use at 150 mg/day is safe and well tolerated in this setting. Our data suggest that eltrombopag might improve hematologic cell counts in patients with PGF, especially in those patients who remained on treatment at week 24, however further research is warranted to extend its applicability for larger cohorts P838: PEGCETACOPLAN RAPIDLY STABILIZES COMPLEMENT INHIBITOR NAÏVE PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA EXPERIENCING HEMOLYSIS WITH ACUTE HEMOGLOBIN DECREASES; PRINCE TRIAL POST HOC ANALYSIS R. Wong1,*, M. Al-Adhami2, J. Savage2, R. Horneff3, M. Yeh4, T. Dumagay5, D. Gomez-Almaguer6 1Sir Y.K. Pao Centre for Cancer & Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong; 2Apellis Pharmaceuticals, Inc., Waltham, United States of America; 3Swedish Orphan Biovitrum AB, Stockholm, Sweden; 4Apellis Pharmaceuticals, Waltham, Waltham, United States of America; 5Department of Cellular Therapeutics, Makati Medical Centre, Makati, Philippines; 6Hematology Service, Hospital Universitario “Dr José Eleuterio González”, Universidad Autónoma de Nuevo León, Monterrey, Mexico Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and life-threatening disease characterized by complement-mediated hemolysis and thrombosis. Pegcetacoplan (PEG) is the first FDA/EMA-approved C3 complement-inhibitor that provides broad hemolysis control in patients with PNH. Aims: This is the first report from the PRINCE trial on the efficacy of PEG in control arm rescue patients using a post hoc time-aligned analysis. Methods: PRINCE is a phase 3, randomized, open-label study, that evaluates the efficacy of PEG in improving lactate dehydrogenase (LDH) and hemoglobin (Hb) levels in patients with PNH. 53 adult (≥18 years old), complement-inhibitor naïve (no prior eculizumab/ravulizumab) patients with LDH ≥1.5x the upper limit of normal (ULN) (≥339 U/L) and Hb below the lower limits of normal (LLN) (males: ≤13.6 g/dL; females: ≤12.0 g/dL), were enrolled. Patients were randomized 2:1 to receive PEG (1080 mg subcutaneously twice weekly, n=35; PEG arm) or to the Control arm (trials were in regions where C5 inhibitors were not available, n=18) for 26 weeks. Patients in the Control arm had the option to escape to PEG treatment (Rescue arm) should they exhibit signs of hemolysis and acute Hb decline by ≥2 g/dL from their baseline. Key endpoints included both changes from baseline in LDH and Hb levels. Because patients could be rescued at any point, a post hoc analysis was performed to time-align the Rescue patients (n=11), based on the start of PEG dosing, with patients in the PEG arm, to compare changes in LDH and Hb levels over time by PEG exposure. Results: 61% (11/18) of Control arm patients experienced acute Hb decreases allowing rescue by PEG treatment. All Rescue patients had a pre-rescue LDH ≥2X ULN (mean 2424.0 U/L) and all had a rapid response to PEG treatment with a significant decrease in LDH level by Week 1 (mean 422.1 U/L) with 5/11 patients achieving ≤1.5X ULN; by Week 2 mean LDH levels were in the normal range (186.6 U/L) with 8 patients below ULN and 2 patients below 1.5X ULN, suggesting complete control of intravascular hemolysis. These results are similar to PEG arm patients (150.4 U/L) (Figure A). Seven of the eleven patients showed a significant increase in Hb level (Week 2 mean Hb for Rescue patients: 10.9 g/dL; PEG patients: 11.19 g/dL); four patients showed no change, potentially due to underlying bone marrow dysfunction (3 had a history of aplastic anemia). Three patients had a transfusion within 5 days of starting PEG treatment; 1 of them remained transfusion dependent after rescue and did not have a Hb change. The time-align plots (Figures A and B) show Rescue patients had rapid improvements in LDH to normal levels and Hb improvements similar to PEG arm patients. PEG demonstrated a favorable risk-benefit profile. No deaths or serious adverse events deemed related to PEG occurred and no drug discontinuations due to PEG-related adverse events were reported. The most common AEs were injection site reaction (PEG [including Rescue patients for all AEs], 30.4%, n=14; Control arm, 0.0%), hypokalemia (PEG, 13.0%, n=6; Control arm, 11.1%, n=2), dizziness (PEG, 10.9%, n=5; Control arm, 0.0%), and fever (PEG, 8.7%, n=4; Control arm, 0.0%). Image: Summary/Conclusion: PEG treatment was able to rapidly stabilize complement-inhibitor naïve patients experiencing acute hemolysis with substantial Hb decrease, and normalize LDH and raise Hb levels. The treatment effect of PEG on both LDH and Hb levels over time was largely similar between the PEG arm patients and Rescue patients. PEG represents a new effective therapeutic option with a favorable safety profile for PNH patients. P839: PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA TREATED WITH PEGCETACOPLAN SHOW IMPROVEMENTS IN D-DIMER NORMALIZATION AND DECREASE IN INCIDENCE OF THROMBOSIS I. Weitz1,*, M. Al-Adhami2, J. Min2, E. Persson3, M. Yeh2, J. Savage2, D. Dingli4 1Jane Anne Nohl Division of Hematology, Keck-USC School of Medicine, Los Angeles; 2Apellis Pharmaceuticals, Waltham, United States of America; 3Swedish Orphan Biovitrum AB, Stockholm, Sweden; 4Mayo Clinic, Rochester, United States of America Background: Thrombosis is the main life-threatening complication of paroxysmal nocturnal hemoglobinuria (PNH), a rare acquired hematologic disease. Pegcetacoplan (PEG) is a C3 complement-inhibitor approved by the FDA/EMA for treatment of adults with PNH. Aims: This post hoc analysis examined incidence of thrombosis, anti-thrombotic therapy (ATT), and D-dimer normalization in adult patients with PNH treated with PEG in the Phase 3 PEGASUS (NCT03500549) and PRINCE (NCT04085601) studies. Methods: PEGASUS enrolled adult patients with PNH with prior suboptimal response to eculizumab (ECU) despite stable ECU treatment (≥3 months) and hemoglobin (Hb) levels <10.5 g/dL at screening. For the randomized controlled period (RCP), patients were randomized 1:1 to ECU (n=39) or PEG (n=41; 1080 mg subcutaneously [sc] 2x weekly) for 16 weeks. Thereafter, ECU patients were switched to PEG monotherapy and PEG RCP patients remained on PEG during the open-label period (OLP, n=77) through Week 48. PRINCE compared PEG treatment (n=35; 1080 mg sc 2x weekly) in complement-inhibitor-naïve patients to patients receiving control treatment (CTRL; excluding complement-inhibitors i.e., ECU/ravulizumab; n=18). CTRL patients had the option to escape to the PEG group if Hb levels decreased ≥2 g/dL from baseline (n=11). Safety analyses in all trials included monitoring of thrombotic events. D-dimer normalization, defined as D-dimer level less than the upper limit of normal (0.5 µg/mL) was examined at baseline, Week 8, and the end of the study period (PEGASUS: 16 weeks [RCP] and 48 weeks [OLP], PRINCE: 26 weeks). Results: Prior to study entry, 31% (n=25) of PEGASUS patients (6 of these while on ECU therapy) and 9% (n=5) of PRINCE patients experienced at least one type of thrombosis. During the PEGASUS OLP, 2 of 77 patients experienced a thrombotic event, one in the setting of non-Hodgkin’s lymphoma and one during sepsis; neither event was deemed related to PEG. No patients in the PEGASUS RCP or PRINCE trial experienced a thrombotic event. Pooled analysis of completed clinical trials of PEG including PEGASUS and PRINCE indicated there were 2 cases of thrombosis in 164 patients comprising 130 patient-years (1.54 events per 100 patient-years), compared to 1.77 events per 100 patient-years for patients on ECU treatment before entry into PEGASUS. In PEGASUS, 34% (n=27) of patients were on ATT prior to study entry. During the PEGASUS RCP, concomitant ATT was observed in 37% (PEG, n=15) and 33% (ECU, n=13) of patients, and during the PEGASUS-OLP in 23% (n=18) of patients (PEG monotherapy). In PRINCE, concomitant ATT decreased from 21% (n=11) prior to the study to 7% (PEG, n=3) and 0% (CTRL, n=0) during the study. In PEGASUS, D-dimer normalization in the PEG arm increased from 73% (baseline, n=30) to 89% (Week 8, n=33) and was largely sustained at Week 16 and Week 48 (Table 1). D-dimer normalization in the ECU arm increased once ECU patients switched to PEG monotherapy, from 76% (Week 16, n=29) to 89% (Week 48, n=17) (Table 1). In PRINCE, D-dimer normalization in the PEG arm increased from 51% (baseline, n=18) to 67% (Week 8, n=18) and increased further to 68% (n=19) at Week 26 (Table 1). Image: Summary/Conclusion: Overall, these results demonstrate that PEG treatment can increase D-dimer normalization and reduces incidence of thrombotic events in patients with PNH who are complement-inhibitor naïve or remained anemic after ECU treatment, suggesting that PEG treatment is as effective as ECU in these outcomes. P840: A MATCHING-ADJUSTED INDIRECT COMPARISON OF THE EFFICACY OF PEGCETACOPLAN USING PRINCE TRIAL DATA VERSUS RAVULIZUMAB AND ECULIZUMAB IN COMPLEMENT-NAÏVE PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA R. Wong1, J. Fishman2,*, K. Wilson3, M. Yeh2, Z. Hakimi3, C. Yee4, L. Huynh4, M. S. Duh4 1Sir Y.K. Pao Centre for Cancer & Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong; 2Apellis Pharmaceuticals, Waltham, United States of America; 3Swedish Orphan Biovitrum AB, Stockholm, Sweden; 4Analysis Group, Boston, United States of America Background: Pegcetacoplan (PEG) is a PEGylated peptide targeting proximal complement protein C3 that has been shown to control both intravascular and extravascular hemolysis and recently approved by the FDA and EMA for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). PRINCE (NCT04085601), was a phase-3, randomized, open-label trial assessing the efficacy and safety of PEG compared to control (CTL) treatment, excluding complement inhibitors among treatment naïve patients with PNH. PEG demonstrated superior efficacy to CTL by improving hemoglobin (Hb) levels and reductions in lactate dehydrogenase (LDH) levels in patients with PNH. To date, no head-to-head clinical trials exist comparing PEG to complement inhibitors in treatment naïve patients. Aims: To assess the comparative effectiveness of PEG for complement-naïve patients with PNH against ravulizumab (RAV) and eculizumab (ECU) using matching-adjusted indirect comparison (MAIC) methodology to conduct cross-trial comparisons. Methods: Individual patient data from PRINCE as well as aggregated published results of RAV and ECU in the ALXN1210-PNH-301 (“301”) study (Lee et al. 2019) were used. To adjust for cross-study differences, propensity score weighting was developed using logistic regression analyses and used to balance demographic and clinical characteristics between the PEG arm and RAV and ECU arms from 301. Outcomes were compared at 26 weeks using unanchored MAIC between PEG versus RAV and PEG versus ECU and included mean and percent change in LDH levels from baseline, LDH normalization (defined as LDH levels dropping <1xULN [246 U/L] in the absence of transfusions during the randomized controlled period), Hb stabilization (defined as the avoidance of a ≥2 g/dL decrease in hemoglobin level in the absence of transfusions during the randomized controlled period), and transfusion avoidance. Weighted Wald tests and 95% confidence intervals were computed for comparisons of categorical and continuous outcomes. Results: A total of 34 patients were included in the PEG arm, 125 patients in the RAV arm, and 121 patients in the ECU arm. Prior to matching, significant differences existed between arms in the proportion of patients with White and American Indian or Alaska Native race, and mean LDH (all p<0.001). After matching the PEG to the RAV and ECU arms, separately, most patient characteristics were well balanced with a mean age of 43.8 years, 44.8 years, 46.2 years, respectively, 43.0-50.0% females, and a mean Hb of 9.4-9.6g/dL across arms; however, there were significant differences in Asian race and baseline LDH (all p<0.01). At week 26, patients in the PEG arm showed a significantly greater improvement in their LDH levels from baseline, with a mean of -523 U/L (-11.2%) compared to RAV and -578 U/L (-12.0%) compared to ECU (all p<0.001; Figure 1). Patients receiving PEG also showed significant improvements in LDH normalization (35.6% compared to RAV, p<0.001; 39.6% compared to ECU, p<0.001), Hb stabilization (28.2% compared to RAV, p<0.001; 31.7% compared to ECU, p<0.001), and transfusion avoidance (22.6% compared to RAV, p<0.001; 30.1% compared to ECU, p<0.001; Figure 1). Image: Summary/Conclusion: In the absence of head-to-head comparisons, this unanchored MAIC study shows that PEG is more efficacious versus RAV and ECU in complement inhibitors treatment naïve PNH patients in terms of significant improvements across all evaluated endpoints, Hb stabilization, LDH improvement, LDH normalization and transfusion avoidance. P841: EXTRACELLULAR VESICLE PROTEINS AS BIOMARKER OF OUTCOME IN MULTIPLE MYELOMA - A REAL-WORLD STUDY E. Arnault Carneiro1,*, B. Ferreira1,2,3, C. Pestana1,4, F. Barahona1,3, J. Caetano1,3,5, R. Lopes1,6, P. Lúcio5, M. Neves5, H. C. Beck7, A. S. Carvalho8, R. Matthiesen8, B. Costa-Silva9, C. João1,3,5 1Myeloma Lymphoma Research Group, Champalimaud Foundation, Lisbon; 2Hemato-Oncology Unit, Champalimaud Foundation, Odense; 3NOVA Medical School, NOVA Medical School; 4Centre of Statistics and its Applications, Faculty of Sciences, University of Lisbon; 5Hemato-Oncology Unit, Champalimaud Foundation; 6Faculty of Medicine, University of Lisbon, Lisbon, Portugal; 7Centre for Clinical Proteomics, Odense University Hospital, Odense, Denmark; 8Computational and Experimental Biology, CEDOC, NOVA Medical School; 9Systems Oncology Group, Champalimaud Foundation, Lisbon, Portugal Background: Multiple myeloma (MM) is a hematological malignancy characterized by clonal plasma cells accumulation within the bone marrow. MM diagnosis and prognosis rely on bone marrow biopsy. Given clonal plasma cell patchy distribution, bone marrow biopsies may not reflect disease heterogeneity whilst submitting patients to invasive procedures. Liquid biopsies, such as extracellular vesicles (EV) from peripheral blood, may overcome these limitations. In MM, there is evidence that EV intervene in key processes such as tumor progression and drug resistance. Most studies analyzing EV from MM patients focus on genome, and real-world studies on EV proteome are scarce. Aims: We characterized blood and bone marrow samples from the precursor stage, MGUS and from MM patients to determine whether EV content can discriminate between diagnosis and predict patient prognosis. These results were compared to healthy donor samples. Methods: After informed consent signed, a cohort of 102 patients and 19 healthy donors were followed in median time of 25 months. Blood and BM samples were collected at diagnosis, response and relapse of patients. EV were isolated by ultracentrifugation. Protein and EV particle concentrations were quantified. Proteomic analysis was performed by mass spectrometry (LC-MS/MS). Diagnostic and prognostic impacts of EV characteristics were investigated. A multivariable longitudinal logistic regression model was built to determine the association with common myeloma-related blood parameters. Results: We report a set of peripheral blood EV-proteins (PDIA3, C4BPA, BTN1A1, APRIL, PSMB8 and PDE8B) that have the potential to be used as new diagnosis biomarker for myeloma patients. Functional enrichment analysis revealed that proteins differentially expressed in patient vs healthy donors are strongly related to immune response, supporting increased immune dysfunctions in patients with active multiple myeloma. We identified that the level of EVcargo (protein/particle ratio) is significantly associated to patient overall survival. High EVcargo patients (>0.6 µg/108 particles) had a shorter overall survival compared to low EVcargo patients (≤0.6 µg/108 particles). High EVcargo is associated with high sFLC lambda, IgA immunoparesis and shorter time in response. The downregulation of IGHA1/IGHA confirmed patients immunoparesis. Upregulation of Ig lambda production proteins confirmed the increased presence of sFLC in the same patients. Also, we report an association between the progression free survival time and expression level at diagnosis of SERPINA2, RPS26 and UBBP4, suggesting their potential as prognosis biomarkers. Image: Summary/Conclusion: To our knowledge, this is the first report of EV protein content from a real-world MGUS and MM patient cohort followed for more than 2 years. Our findings show that PDIA3, BTN1A1, APRIL and complement proteins should be further explored in myeloma, as peripheral blood biomarkers of diagnosis. Circulating EVcargo is a promising prognostic biomarker for MM patients. Patients with high EVcargo had 12 times increased risk of dying and high EVcargo is associated with important prognostic features such as immunoparesis, sFLC or duration of treatment response. EVcargo is an indirect and more affordable measure to infer EV protein load compared to mass-spectrometry. Our results show that EV have the potential to be used as diagnosis biomarker in MGUS and MM patients and as an added approach to discriminate patients with poor survival. Our results substantiate the interest of EV as liquid biopsies in myeloma and future validation in independent clinical settings is urged. P842: NOVEL MULTIFUNCTIONAL TETRAVALENT CD38 NKP46 FLEX-NK ENGAGERS ACTIVELY TARGET AND KILL MULTIPLE MYELOMA CELLS L. Lin1, H.-M. Chang1, C. Nakid2, S. Frankel1, D. Wu3, J. Kadouche4, D. Teper4, O. Mandelboim5, J.-C. Bories2, A. Arulanandam1,*, W. Li1 1Cytovia Therapeutics, Natick, United States of America; 2INSERM U976 and Université de Paris, Paris, France; 3Lynx Biosciences, San Diego; 4Cytovia Therapeutics, Aventura, United States of America; 5Hebrew, University of Jerusalem, Jerusalem, Israel Background: Given that CD38 is a clinically validated target for natural killer (NK) cell mediated cytotoxicity in multiple myeloma (MM), we sought to leverage our FLEX-NKTM platform to create a NK engager antibody targeting CD38. FLEX-NKTM is a proprietary platform for production of tetravalent IgG1-like multifunctional NK engager antibodies with a novel FLEX-linker to allow for simultaneous binding of both the targeted cancer cells and NK cells via the activation receptor NKp46. Aims: Derive preclinical proof of concept with CD38 targeted NK-cell engager bispecific antibody in Mulitple Myeloma Methods: CYT-338 was expressed in CHOZEN cells and purified by step column chromatography. PB-NK cells were purified from healthy donor blood. PB-NK cytotoxicity against MM tumors was evaluated in the presence of CYT-338 or isotype control antibodies at a fixed E/T ratio. CYT-338 pharmacokinetics in mice was evaluated following a single intravenous injection. Anti-tumor efficacy of CYT-338 was evaluated in NSG-mice injected with PB-NK cells and MM cell line MM1S expressing luciferase. CYT-338 mediated fratricide was evaluated in purified PB-NK’s. CYT-338 cytokine release assessments and hemato-cytotoxicity was evaluated in-vitro in human PBMC assays. Results: CYT-338 showed dose dependent binding to CD38 expressing MM cell lines MM1S and KMS11 and no binding to a CD38 knock out MM.1S cell line. CYT-338 bound MM cell lines with ~ 2-fold higher mean fluorescence intensity than anti-CD38 monoclonal antibody (mAb) or daratumumab alone. Epitope mapping studies for our CD38 mAb binder using alanine scanning mutagenesis showed that amino acid S274 on CD38, critical for binding to daratumumab, is not important for CD38 binding, suggesting a distinct epitope detected by our CD38 binder. CYT-338 showed greater dose dependent NK cell redirected cytolysis, degranulation and cytokine production against MM1S cells compared to daratumumab. The CYT-338 pharmacokinetics study in mice showed a terminal half-life of 41 hrs. Preliminary in-vivo tumor model studies in NSG mice using PB-NK and CYT-338 injections showed tumor growth inhibition of MM1S tumor cells. Low NK cell fratricide was observed with CYT-338 while daratumumab showed significantly higher NK cell fratricide. In peripheral blood mononuclear cell hemato-toxicity studies, depletion of monocytes and NK cells were observed with daratumumab but no significant depletion was observed with CYT-338. Cytokine release assessment of CYT-338 in the human PBMC assay showed no evidence of cytokine release while high levels of cytokine release was observed with daratumumab, anti-CD28 (TGN1412) and CD3 antibody controls. Summary/Conclusion: These results suggest that the CYT-338 engager has a favorable NK cell engager profile for targeting CD38 expressing multiple myeloma distinct from daratumumab. P843: METABOLOMIC CHARACTERIZATION OF HUMAN MULTIPLE MYELOMA CELL LINE TO STUDY TUMOR RESISTANCE TO DIFFERENT CLASSES OF THERAPEUTIC AGENTS A. Steer1,*, D. Chemlal1, E. Varlet2, A. Machura1, A. Kassambara1, E. Alaterre2, G. Requirand3, N. Robert3, C. Hirtz4, H. De Boussac1, A. Bruyer1, J. Moreaux2 1Diag2Tec; 2Institute of Human Genetics, UMR 9002 CNRS-UM; 3Laboratory for Monitoring Innovative Therapies, Department of Biological Hematology, CHU Montpellier; 4Clinical Proteomics Platform, LBPC, IRMB, CHU, UM Montpellier, Montpellier, France Background: Multiple myeloma (MM) is the second most common hematological malignancy, characterized by the abnormal accumulation of plasma cells in the bone marrow. Although the latest treatments, including proteasome inhibitors, immunomodulating agents, or immunotherapy, have greatly improved patient survival, a residual subset of cells remains resistant to therapies and usually causes relapses. Aims: The drug resistance could be explained, among others, by the interactions with the microenvironment, MM’s high molecular heterogeneity, or the appearance of adaptive survival mechanisms after treatment exposure. However, a better understanding of the mechanisms involved in drug resistance remains of significant interest. Among the factors influencing the resistance of cancer cells, the “metabolic plasticity” of the tumor and, therefore, its ability to adapt to stress conditions is a mechanism increasingly studied in recent years in cancer. Although measuring mitochondrial metabolism has been identified as a major factor influencing response to treatments in several cancers, few studies have been documented in MM. Methods: Here, we characterized the metabolic profiles of a panel of 20 human MM cell lines (HMCL) representative of the molecular heterogeneity found in MM patients. This panel included 10 commercial HMCL and 10 HMCL derived in our laboratory. First, oxygen consumption rates (OCR), extracellular acidification rate (ECAR), and spare respiratory capacity (SRC) of our HMCL panel were assessed in a Mito Stress Assay using a Seahorse XFe96 analyzer. Results: Interestingly, the metabolic activities were shown very heterogeneous in HMCLs with a part of cell lines more dependent of the glycolysis activity and another part more dependent of the mitochondrial respiration. By integrating the HMCL’s metabolic profiles with their respective transcriptomic data (RNAseq), we defined a metabolomic score to classify the HMCL into different groups and represent their glycolysis level or mitochondrial activity. The score was calculated from the expression of 112 genes involved in the electron transport chain (Oxphos) or in glycolysis. For a given gene, the expression values were first log2-transformed and then normalized by subtracting the average. The score is computed as the difference between the Oxphos and glycolytic genes’ average expression. Interestingly, high significant correlations between the HMCL’s functional metabolic profiles and their calculated metabolomic score were identified. Furthermore, we investigated the potential prognostic value of this gene-based metabolomic score in the MMRF CoMMpass cohort (newly diagnosed MM patients, n=674) using the maxstat algorithm, which segregated the cohort into two groups with a significantly different outcome. We identified that 32% of patients with a high gene-based metabolomic score, were associated with poor overall survival (P = 3.1x10-6). We then validated this prognostic value in a second cohort of MM’s patients: the cohort of 206 patients (ArrayExpress public database under accession number E-MTAB-362). Moreover, we performed correlation analyses between the metabolic profiles of those HMCL and their respective response to the conventional treatments (IC50). We found a significant correlation between a high mitochondrial ATP production and the resistance to proteasome inhibitor (P = 0.023, n= 12). Summary/Conclusion: Altogether, we demonstrated that metabolomic deregulation could participate in drug resistance in MM. Inhibitors targeting metabolic activities may be of therapeutic interest to overcome drug resistance in MM. P844: CHARACTERIZATION OF MULTIPLE MYELOMA CELL LINES WITH ACQUIRED-RESISTANCE TO PROTEASOME INHIBITORS HIGHLIGHTS A LINK BETWEEN RESISTANCE AND METABOLIC DEREGULATION H. De Boussac1,*, A. Machura1, A. Steer1, A. Kassambara1, M. Gely1, D. Chemlal1, C. Gourzones2, G. Requirand3, N. Robert3, L. Vincent4, C. Herbaux4, A. Bruyer1, J. Moreaux5 1Diag2Tec; 2UMR 9002 CNRS-UM; 3Laboratory for Monitoring Innovative Therapies, Department of Biological Hematology; 4CHU Montpellier; 5Institute of Human Genetics, UMR 9002 CNRS-UM, Montpellier, France Background: Characterized by an abnormal clonal proliferation of malignant plasma cells, Multiple myeloma (MM) is the second most common hematological malignancy. Novels agents have significantly improved clinical outcomes, but most of the MM patients eventually relapse. A better understanding of the drug resistance mechanisms remains of significant interest to improve the treatment of patients Aims: We aimed to investigate the mechanisms involved in the resistance to proteasome inhibitors (PI). Methods: For that purpose, we have derived and characterized 6 Bortezomib (Btz)-resistant human myeloma cells lines (BR-HMCLs) from different molecular subgroups and still dependent on the addition of IL-6 including XG1BR t(11;14), XG2BR t(12;14), XG7BR, XG20BR, XG24BR t(4;14) and XG19BR t(14;16). These cell lines were cultured continuously with escalating concentrations of Btz during 12 months. Results: Interestingly, BR-HMCLs demonstrated significant resistance to Btz compared to their parental counterparts (CTR-HMCLs) (mean IC50: BR-HMCLs =5nM vs CTR-HMCL=2.3nM, p<0.05). In addition, these BR-HMCLs also showed significant cross-resistance to Carfilzomib (Cfz) and Ixazomib (Ixa) PIs (p<0.05 for Cfz or Ixa). Finally, no significant cross-resistance was observed with other therapeutic agents (melphalan, dexamethasone or IMIDs), indicating that the observed drug resistance mechanisms are especially related to PIs. We then used genomic approaches (whole genome sequencing and transcriptomic analyses) to understand the PIs resistance mechanisms acquired by MM cells. Remarkably, as observed in relapsed MM patients, among the 40 mutations identified in BR-HMCLs compared to the CTR-HMCLs, we identified a mutation residing in the Btz-binding pocket of the proteasome beta5-subunit (PSMB5), that reduces the PI binding capacity, thus preventing inactivation of the catalytic activity of the 20S proteasome. Further transcriptomic analyses on BR-HMCLs underlined significant deregulation of genes involved in cell metabolism and drug clearance that could allow the BR-cells to maintain metabolic homeostasis and survival in stringent redox conditions. Thus, in the BR-HMCL we identified an upregulation of enzymes directly involved in glycolytic and energy metabolism (ALDOC, ENO3, HK1, PDK1/3, PFKB3/4, PFKL, SLC2A1 (FC>1.5 p-value <0.05), but also a significant downregulation of 8 solute carrier protein (SLC) intake transporters together with a significant upregulation of xenobiotic receptors (FC> or < 1.5; p-value <0.05). We then tested the metabolomic of BR-HMCL using the Seahorse assay. This technology allows to analyze the glycolysis via the extracellular acidification rate (ECAR) and cell respiration via the mitochondrial oxidative phosphorylation based on the oxygen consumption rate (OCR). Following the increased gene expression of several glycolytic enzymes found in the BR-HMCLs, we observed that while CTR and BR displayed equivalent respiration and glycolytic activities, their treatment with Btz induces major metabolic modifications, including a significant increase of the glycolytic and mitochondrial activity of the BR-HMCL while it decreases it in the CTR-HMCL, suggesting a higher capacity of the BR-HMCL to respond to cell stress. Summary/Conclusion: Altogether, we developed acquired PIs resistant HMCLs that exhibit PSMB5 mutation as observed in patients, and we identified pathways linked to metabolism deregulation in these cell lines. These results make our PI-resistant models, an attractive preclinical model to test new therapeutic strategies to overcome PI resistance in MM. P845: IDENTIFICATION OF MULTIPLE MYELOMA BIOMARKERS VIA LIQUID BIOPSY BASED ON CELL-FREE DNA USING A CAPTURE-HYBRIDIZATION PANEL N. Buenache Cuenda1,*, A. Sánchez-delaCruz1, I. Cuenca Navarro1, L. Rufián Vázquez1, V. Garrido García1, A. Giménez Sánchez1, R. Alonso Fernández2, J. M. Sánchez Pina2, Y. Ruiz Heredia1, S. Barrio García1, I. Rapado Martínez1,2, R. Ayala Díaz1,2,3,4, J. Martínez-López1,2,3,4, J. M. Rosa-Rosa1 1Department of Translational Haematology, Research Institute Hospital 12 de Octubre (i+12) Haematological Tumors Clinical Research Unit H12O-CNIO; 2Department of Translational Haematology, Haematology Service, Hospital 12 de Octubre; 3Department of Medicine, Faculty of Medicine, Complutense University; 4CIBERONC, Madrid, Spain Background: Multiple myeloma (MM) is a haematological malignancy characterized by clonal proliferation of pathogenic CD138+ plasma cells (PPCs) in bone marrow (BM). In recent years, the treatment of MM has shown great evolution. However, most patients who achieve a complete response eventually relapse. For that, an early detection of PPCs could be very beneficial for MM patients, and therefore, a sensitive detection of this stage could allow early therapeutic interventions. Liquid biopsy using cell-free DNA (cfDNA) as a minimally invasive approach could be an alternative not only for the diagnosis but also for the detection of recurrences. Aims: The main objective of this study was the quantification of MM patient-specific biomarkers in cfDNA compared to PPCs and BM samples. Methods: Patients with active MM were treated with VRD, autologous stem cell transplantation and maintenance. We studied gDNA from 23 PPCs from BM aspirates, 16 gDNA from whole BM and 12 cfDNA from peripheral blood samples obtained at diagnosis. Capture panel was aimed to detect most common and relevant aberrations known in MM such as coding regions of 37 genes involved in MM progression and drug resistance, canonical IGH and IGK to capture IGH rearrangements and regions to cover traditional translocations. Capture libraries were generated with SureSelect Reagent kits (Agilent Technologies). We used 50 ng of genomic DNA for both PCCs and BM and for cfDNA a median of 10-200 ng input. Final libraries were run on an Illumina NextSeq 500 platform. A specific bioinformatics pipeline was applied: alignment to the hg38 genome, SNVs and Indels identification by combination of variant callers, and IGH/K rearrangements with MiXCR. Results: A total of 36 different mutations were identified in the PPCs with frequencies ranging from 0.011-0.570. Comparison between PPCs and cfDNA showed that 20 out of 23 (87%) mutations identified in the PPCs were observed in the cfDNA, whereas 32 out of 35 (91%) mutations were observed in BM. Moreover, 6 out of 11 (55%) mutations identified in the PPCs were observed in cfDNA and BM (Figure 1A). In 5 out of 5 patients at least 1 mutation of those evaluated in PPCs was detected in cfDNA. Interestingly, for the classical oncogenes (NRAS, KRAS and BRAF), 5 of 8 (63%) hotspot mutations were observed in cfDNA and 7 of 8 (88%) in BM. Using the ratio between the observed frequency in cfDNA/BM and that observed in PPCs, we estimated an average tumour burden of 6% in cfDNA and a mean clonality of ~30% in BM. In addition, 5 out of 5 translocations identified in PPCs were observed in BM and unexpectedly 1 out of 2 was observed in cfDNA. Regarding IGH/K rearrangements, 50% of the patients presented at least one rearrangement in the cfDNA fraction that was identified in the PPCs, and 93% in the BM fraction. Furthermore, the average reduction in frequency observed in cfDNA and BM was ~12% compared to the frequency seen in PPCs. Finally, 2 out of 5 patients showed the same IG rearrangement in the three fractions studied[jm1] (Figure 1B). Image: Summary/Conclusion: Most of molecular alterations identified in PPCs seemed to be present in peripheral blood of patients with MM, surprisingly, even translocation breakpoints could be seen in cfDNA. However, liquid biopsy monitoring is limited by signal dilution, as an approximately 1-log reduction in cfDNA was observed compared to PPCs, which should be considered in bioinformatic analyses. On the other hand, IGH/K rearrangements could be identified in fewer patients, but the signal reduction was only ~12%, confirming IG rearrangements as strong biomarkers for MM in cfDNA. P846: EXPRESSION SIGNATURE OF TP53 BIALLELIC INACTIVATION IDENTIFIES A GROUP OF MULTIPLE MYELOMA PATIENTS WITHOUT THIS GENETIC CONDITION BUT WITH DISMAL OUTCOME. C. De Ramón1,*, E. A. Rojas1, I. J. Cardona-Benavides1, M. V. Mateos1, L. A. Corchete1, N. C. Gutiérrez1 1Hematology, University Hospital of Salamanca, IBSAL, Cancer Research Center-IBMCC (USAL-CSIC), Salamanca, Spain Background: Cytogenetic abnormalities remain the most relevant prognostic factors, especially those related to TP53 gene. Biallelic inactivation of TP53, included in the definition of double-hit (DH) MM, entails an ominous prognosis, although is present in less than 5% of newly diagnosed MM (NDMM). However, ultra-high-risk MM, defined as those patients with a median survival less than 24 months, represents 15-20% of the MM population. While other high-risk cytogenetic abnormalities may account for this adverse prognosis, these kind of cytogenetic alterations are not present in all patients with such an unfavorable outcome. On the other hand, p53 can be deregulated by other mechanisms different from changes in DNA gene sequence, such as epigenetic regulation or altered expression of its regulators. Aims: To define the transcriptional signature of DH-TP53 and to find out if it was present in other patients who did not have biallelic inactivation of TP53. Methods: We analyzed RNA-seq, whole-genome and whole-exome sequencing data from 660 newly diagnosed MM (NDMM) patients from the MMRF (Multiple Myeloma Research Foundation) CoMMpass study to characterize the transcriptional signature of DH-TP53 MM. This gene signature was used to build a score based on a Spearman correlation coefficient and a scaled GINI index. Survival data were retrieved from the IA16 release and the analysis was performed using the Kaplan-Meier estimator and log-rank test. Multivariable Cox models were fitted in R. GSE4581 and GSE136400 gene expression series from GEO were used as validation cohorts. Results: TP53 biallelic inactivation, defined in this work as DH-TP53 group, was found in 23 out of 660 patients (3.5%) and was associated with an exclusive gene expression signature consisted of 78 genes. Based on these genes, we calculated the DH-TP53 score, which identified a subgroup of 50 patients that shared the same transcriptional profile (DH-TP53-like group). The prognosis of this group was particularly unfavorable with a median overall survival (OS) of 23 months; and a progression-free survival (PFS) of 15 months, even worse than that described for DH-TP53 patients (HR = 1.83 [95% CI, 1.00-3.35], p = 0.046). The prognostic value of the DH-TP53 was confirmed as an independent prognostic factor for PFS (HR 3.84 [95% CI 2.51-5.88], p < 0.001) and OS (HR 3.32 [95% CI, 2.31-4.77], p < 0.001) in the multivariable analysis. We also observed that survival for any of the cytogenetic abnormalities was significantly shortened in the DH-TP53-like group (p < 0.05). Furthermore, the prognostic value of the DH-TP53 score was externally validated using gene expression data obtained by microarray analysis Image: Summary/Conclusion: The expression signature of DH-TP53 MM was shared by other MM patients without TP53 biallelic inactivation (DH-TP53-like group). The DH-TP53 score identified an ultra-high-risk group of MM patients with a median overall survival below 24 months. In addition, this score refined the prognostic stratification of cytogenetic abnormalities, and was externally validated using expression data analyzed by microarrays. Funding: This study has been funded by ISCIII (PI16/01074 and PI19/00674) (co-funded by FEDER), Castilla y Leon (GRS 2058/A/19 and GRS 2331/A/21) and AECC (PROYE20047GUTI). CDR, EAR and IJCB were supported by the AECC (CLJUN18010DERA), the “Consejería de Educación de Castilla y León” and the ISCIII (FI20/00226), respectively. P847: MRD BY MASS SPECTROMETRY IN PERIPHERAL BLOOD AND NEXT GENERATION SEQUENCING IN BONE MARROW IN A PHASE 2 STUDY OF DARATUMUMAB, CARFILZOMIB, LENALIDOMIDE, AND DEXAMETHASONE FOR MULTIPLE MYELOMA B. Derman1,*, J. Rosenblatt2, D. Avigan2, A. Major1, M. Rampurwala1, D. Barnidge3, A. Stefka1, K. Jiang1, A. Jakubowiak1 1Section of Hematology/Oncology, University of Chicago, Chicago; 2Beth Israel Deaconess Medical Center, Boston; 3The Binding Site Group, Rochester, MN, United States of America Background: Measurable residual disease (MRD) as assessed by next generation sequencing (NGS) using bone marrow (BM) aspirate carries prognostic significance in multiple myeloma (MM). Though its analytical sensitivity reaches 10-6, MRD assessed from BM may be vulnerable to false negatives due to patchy or extramedullary disease, or due to inadequate aspirate sample. Mass spectrometry (MS) is a promising peripheral blood (PB) assay that may approximate current methods of MRD detection. Aims: In this phase 2 study evaluating the safety and efficacy of extended daratumumab, carfilzomib, lenalidomide, and dexamethasone (Dara-KRd) without autologous stem cell transplant (ASCT) in newly diagnosed MM, we are evaluating the concordance of MRD by NGS in BM and by MS in PB. Methods: Forty-one patients have been enrolled from two MM Research Consortium sites into this phase 2 study (planned enrollment 45 patients). All patients receive 24 cycles of Dara-KRd in 28-day cycles without ASCT. With optional stem cell collection for ASCT-eligible candidates after cycle 4. MRD by NGS was assessed from BM aspirates by the clonoSEQ® assay (Adaptive Biotechnologies) with a limit of detection <10-6. MRD by MS was evaluated using both an automated MALDI-TOF (EXENT) and liquid-chromatography-MS (LC-MS) by the Binding Site Group (assays under development). Paired PB MS and BM NGS samples were available for 13 patients at early (post-cycle 4) and 18 patients at later (cycles 8-24) timepoints. There were 44 paired NGS/EXENT samples (14 post-cycle 4) and 42 paired NGS/LC-MS samples (13 post-cycle 4). Cohen’s kappa test was used to assess concordance between MS and NGS samples. Results: For the early post-cycle 4 timepoint, there was low agreement between NGS (sensitivity threshold 10-6) and MS; there was 46% agreement (Cohen’s kappa -0.18) between NGS and EXENT and 54% agreement (Cohen’s kappa -0.15) between NGS and LC-MS. Of the discordant cases for NGS and EXENT, 4/7 (57%) were NGS-/EXENT+. Two of these 4 patients converted to EXENT- at C8. For discordant NGS/LC-MS cases, 5/6 (83%) were NGS-/LC-MS+. None converted to LC-MS-. The one NGS+/LC-MS- case was from a patient with kappa light chain MM. For the later timepoints (cycles 8-24), there was higher concordance between NGS and MS. There was 63% agreement (Cohen’s kappa 0.27) between NGS and EXENT and 59% agreement between NGS and LC-MS (Cohen’s kappa 0.13). Of the discordant cases for NGS and EXENT, 4/11 (36%) were NGS-/EXENT+ and 1 of these cases was followed by conversion to EXENT-. For discordant NGS/LC-MS cases, 9/12 (75%) were NGS-/LC-MS+. None converted to LC-MS-. Two of the three NGS-/LC-MS+ cases were from a patient with kappa light chain MM. With median follow-up of 10 months, there have been no progressions or deaths among these patients. The prognostic significance of persistent NGS+ or LC-MS+ patients cannot be determined at this time and requires longer follow-up. Image: Summary/Conclusion: MRD assessment by MS (EXENT and LC-MS) in PB and NGS in BM serve complementary roles. Early in treatment, MS positivity may represent false positives due to immunoglobulin recycling. EXENT from PB appears to more closely approximate the sensitivity of MRD by NGS at a sensitivity threshold of 10-5, while LC-MS from PB appears to reach and possibly exceed the sensitivity of MRD by NGS in BM at a sensitivity threshold of 10-6. The prognostic significance of persistent LC-MS positivity is unclear and requires longer follow-up. P848: TINOSTAMUSTINE (EDO-S101), AN ALKYLATOR AND HISTONE DEACETYLASE INHIBITOR, ENHANCES THE EFFICACY OF DARATUMUMAB IN PRECLINICAL MODELS OF MULTIPLE MYELOMA A. Diaz-Tejedor1,*, L. San-Segundo1, L. A. Corchete1, L. González-Méndez1, M. Martín-Sánchez1, M. Lorenzo-Mohamed1, M. González-Rodríguez1, M. Cruz-Hernández1, M. Sánchez-Blázquez1, N. C. Gutiérrez1, M.-V. Mateos1, M. Garayoa1, E. M. Ocio2, T. Paíno1 1Department of Hematology, Cancer Research Center-IBMCC; University Hospital of Salamanca-IBSAL, Salamanca; 2Department of Hematology, University Hospital Marques de Valdecilla (IDIVAL); University of Cantabria, Santander, Spain Background: Tinostamustine (EDO-S101) is a novel alkylating and deacetylase inhibiting molecule designed to improve drug access to DNA strands, induce DNA damage and counteract its repair in cancer cells. It has shown anti-myeloma (MM) activity in different preclinical models both in monotherapy and in combination with bortezomib. Aims: To evaluate whether tinostamustine enhances the anti-myeloma effect of daratumumab. Methods: Tinostamustine was provided by Mundipharma and daratumumab was obtained from pharmacy surplus of the University Hospital of Salamanca. Mechanisms of action of daratumumab were studied by flow cytometry (FCM) in MM cell lines pretreated with tinostamustine and cultured as specified: 1) with F(ab)2 fragments (direct apoptosis via crosslinking); 2) with 10% human serum (CDC assays); 3) co-cultures of MM and NK cells (ADCC assays). Expression of CD38 and NKG2D ligands (MICA and MICB) after tinostamustine treatment was evaluated by FCM, WB and qPCR. The effects of tinostamustine + daratumumab combination were also studied ex vivo in bone marrow (BM) samples from MM patients, and in vivo in two plasmacytoma models developed in CB17-SCID and NK-cell-humanized NSG mice. Results: Pretreatment of U266, JJN3, MM.1S, NCI-H929, RPMI-8226 and MOLP-8 cell lines with tinostamustine (1-2.5 μM) for 48 h increased CD38 expression. Tinostamustine-pretreated myeloma cells showed a higher level of daratumumab binding, as demonstrated by anti-human-IgG1-AF488 staining. Also, tinostamustine (1-2.5 μM) increased MICA and MICB expression in MM cell lines. In ex vivo cultures, tinostamustine increased expression of CD38 in 4 out of 10 patients and MICA in 3 out of 5 patients. Next, the influence of tinostamustine pretreatment (2.5 μM) on several daratumumab mechanisms was evaluated. In apoptosis via crosslinking, the percentage of apoptotic cells in presence of daratumumab was higher in tinostamustine-pretreated MOLP-8 cells vsDMSO-pretreated cells (90.57%±7.35 vs 60.36%±2.86; p<0.001). Likewise, pretreatment with tinostamustine increased the percentage of Annexin-V+/7AAD+ MOLP-8 cells in CDC assays with daratumumab, although not reaching statistical significance (93.22%±1.05 vs73.39%±11.93). In MM-NK co-cultures in absence of daratumumab (NK cell-mediated direct cytotoxicity), the percentages of apoptotic cells in tinostamustine-pretreated vs DMSO-pretreated cells were: 83.64%±5.28 vs 61.58%±6.81 in MOLP-8 cells (p<0.05); 54.27%±5.99 vs 34.24%±7.37 for RPMI-8226 (p<0.05); and 26.15%±4 vs 12.32%±1.97 in MM.1S (p<0.05). However, in the presence of daratumumab (ADCC) these percentages showed a similar tendency although not significant: 88.57%±3.77 vs 78.89%±5.57 in MOLP-8; 70.75%±5.69 vs 58.28%±7.52 for RPMI-8226; and 56.74%±7.25 vs 45.68%±7.1 in MM.1S. Ex vivo experiments with patients’ BM samples (n = 10) showed that simultaneous combination of daratumumab + tinostamustine significantly increased the percentage of eliminated MM cells compared to individual treatments, with an acceptable toxicity on healthy lymphocytes (Figure 1a). Finally, in the CB17-SCID model, tinostamustine treatment (24 h) followed by daratumumab administration controlled tumor growth significantly better than daratumumab alone (Figure 1b). The combination prolonged the median survival as compared to vehicle, and monotherapy with tinostamustine or daratumumab (Figure 1b). Similar results were obtained in the NSG model. Image: Summary/Conclusion: Our preclinical data demonstrate that tinostamustine increases the anti-myeloma effect of daratumumab presumably due to enhanced expression of CD38 and MICA. P849: DEPLETION OF DIS3 IN MULTIPLE MYELOMA LEADS TO PERTURBATION IN RNA METABOLISM, CELL CYCLE PROGRESSION AND MITOTIC CHECKPOINT. V. Favasuli1,*, I. Silvestris1, K. Todoerti2, D. Ronchetti1, S. Erratico3, D. giannandrea4, Y. Torrente5, R. Chiaramonte4, S. Fabbris1, N. Bolli1, L. Baldini1, A. Neri1, E. Taiana1 1Oncology and Hemato-oncology, University of Milano; 2Phatology and Molecular Oncology, IRCCS Istituto Nazionale dei Tumori; 3Novystem Spa, Novystem Spa; 4Department of Health Sciences; 5Stem Cell Laboratory, Unit of Neurology, University of Milano, Milan, Italy Background: MM is a genetically heterogeneous neoplasm in which the co-occurrency of multiple genomic events play a crucial role in tumor development, progression and drug resistance. DIS3 gene maps at 13q22 and encodes for a highly conserved ribonuclease involved in the RNA processing, quality control and degradation. Deletion of DIS3 gene is a common characteristic of MM patients (approximately 50% of cases); moreover, DIS3 mutations occur in about 11% of MM cases and are frequently (70%) associated with loss of the gene. DIS3 mutations along with del(13q) have been recently described by us as associated with a strong negative effect on clinical outcome. Aims: The purpose of this study is to shed light on the biological role and impact of DIS3 transcript in MM. Methods: LNA gapmeRs were used to silence DIS3 in HMCLs by gymnotic delivery in four human myeloma cell lines, two of which showing a monoallelic del(13q). qRT-PCR has been used to evaluate DIS3 silencing efficiency. Cell viability and cellular growth was evaluated by viable counts and by Cell Titer Glo assay. Modulation of cell cycle phases distribution was assessed by flow cytometry. Cell cycle synchronization was obtained using SynchroSet kit. Transcriptional analysis was performed using Affymetrix microarray. Protein expression was evaluated by Western Blot. Mitotic spindle organization was analyzed by immunofluorescence (IF). Results: DIS3 silenced HMCLs show a significant decrease of cellular growth right after 4 days from LNA gapmeR delivery. DIS3depleted cells showed a significant perturbation of cell cycle in all the HMCLs tested. Analysis of synchronized HMCLs revealed a more pronounced modulation of all cell cycle phases distribution confirming the involvement of DIS3 in cell cycle regulation. Accordingly, DIS3 silencing lead to an important modulation of cell cycle checkpoint proteins and mitotic spindle checkpoints. Confocal microscopy analysis performed in DIS3 silenced HMCLs revealed the presence of mitotic defects and microtubule organization abnormalities. Transcriptional profile analysis of DIS3-KD samples is associate to a strong modulation of cell cycle regulation and mitotic spindle related pathways, together with the expected, strong modulation of RNA degradation pathway. Summary/Conclusion: Our data indicate that DIS3 silencing in HMCLs leads to a cell cycle perturbation accompanied to an increase of mitotic defects. To the best of our knowledge, this is the first evidence in the context of the pathological role of DIS3 in MM. These results should be considered of interest considering that DIS3 abnormalities are a common feature of MM patients. Moreover, the transcriptional analyses may contribute to identify putative DIS3-related pathways and genes (with coding or non-coding potential) involved in tumorigenic mechanisms in MM and possibly implicated in the therapeutic settings. P850: THE HUMORAL AND CELLULAR RESPONSE TO SARS-COV-2 MRNA VACCINATION IN PATIENTS WITH MULTIPLE MYELOMA AND PRE-MALIGNANT MONOCLONAL GAMMOPATHIES: IMPACT OF OMICRON VARIANT P. Storti1,*, V. Marchica1, R. Vescovini1, V. Franceschi1, L. Russo1, V. Raimondi1, D. Toscani1, J. Burroughs Garcia1, N. T. Iannozzi1, B. Dalla Palma2, M. T. Giaimo1,2, L. Notarfranchi1, G. Donofrio1, N. Giuliani1,2 1Department of Medicine and Surgery, University of Parma; 2Hematology, “Azienda Ospedaliero-Universitaria”, Parma, Italy Background: Multiple myeloma (MM) patients have a high risk of infections with a possible reduced response to vaccines including anti-SARS-CoV-2 one. Currently, the impact the emerging new variants in MM patients underwent to anti-SARS-CoV-2 vaccination is still unknown. Aims: The humoral and cellular response to SARS-CoV-2 mRNA full vaccination and booster dose and the impact of new spike-mutated variants, including Omicron have been investigated in a cohort of MM patients and those with pre-malignant monoclonal gammopathies. Methods: We enrolled 38 COVID-19-naïve patients: 6 monoclonal gammopathies of undetermined significance (MGUS), 10 smoldering myeloma (SMM), 9 newly diagnosed MM (NDMM) and 13 relapsed MM (MMR). Peripheral blood (PB) samples were collected before the first dose and 14±2 days after the second dose (POST) of the mRNA BNT162b2 vaccine. In a subset of 16 patients with MM (MMD and MMR), PB samples were also collected after 14±2 days of a heterologous booster dose (BOOSTER) with mRNA-1273 vaccine. SARS-CoV-2 spike IgG antibodies (Abs) were measured by the COVID-SeroIndex Kantaro SARS-CoV-2 IgG test. The fraction of neutralizing Abs (NAbs) against SARS-CoV-2 spike of Wuhan-Hu-1 strain and five variants (alpha, beta, gamma, delta and Omicron) was assessed in the collected sera by a SARS-CoV-2 pseudovirus neutralization assay. Finally, the collected PB-mononuclear cells were stimulated, with overlapping peptides pools spanning SARS-CoV-2 spike glycoprotein and SARS-CoV-2 spike-specific CD4+ and CD8+ T cells were identified by intracellular cytokine staining for Interferon-gamma (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), and CD107a+ using flow cytometry assay. Results: The seropositivity rate for SARS-CoV-2 spike IgG Abs in the total cohort was 86.8% with 13.2% patients exhibiting no detectable Abs. We found that MMR patients had significantly lower SARS-CoV-2 spike IgG Abs and NAbs compared with MGUS, SMM and NDMM patients after full vaccination. All the analyzed variants, remarkably Omicron, had a significant negative impact on the neutralizing ability of the vaccine-induced Abs in all patients with MM and also with SMM. We also observed a variable spike-specific CD4+ and CD8+ T cell responses. We found that both SMM and NDMM patients had significantly reduced spike-specific IL-2+CD4+ T cell responses compared to MGUS patients. MM patients showed reduced IFN-γ+ and TNF-α+CD8+ T cells, compared to MGUS. In BOOSTER samples, MMR patients retained significantly lower anti-spike-IgG-Abs levels compared to MMD patients. After the booster dose, MMD patients lost the negative impact of the spike variants seen after full vaccination, due to a significant increase of the NAbs titers. On the other hand, MMR patients retained NAbs titers against Beta, Delta and Omicron variants significantly lower than against Whuan-Hu-1 spike. The booster dose increased SARS-CoV-2 spike-specific T cells in both MMD and MMR reaching the 100% of responder patients with an increased percentage of IL-2+CD4+ T cells compared to full vaccination. Summary/Conclusion: Our study underlines the negative impact of Omicron variants on the neutralizing ability of the vaccine-induced Abs in MM patients as well as in SMM after a full vaccination. Booster dose immunization improved spike humoral and cellular responses in MMD patients and in most, but not all, MMR patients suggesting these patients need to be considered still at risk of Omicron SARS-CoV-2 infection with a clinically relevant disease. P851: TARGETING GENE DEPENDENCIES IN MYC OVEREXPRESSING MULTIPLE MYELOMA L. Hasan Bou Issa1,*, L. Fléchon1, W. Laine1, A. Ouelkdite1, G. Escure2, S. Gaggero1, T. Ingegnere1, R. Sklavenitis Pistofidis3, I. M. Ghobrial3, T. Facon2, S. Mitra1, J. Kluza1, B. Quesnel1,2, S. Manier1,2 1Univ Lille, Canther, INSERM UMR-S1277 - CNRS UMR9020; 2CHU Lille, Department of Hematology, Lille, France; 3Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States of America Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by a proliferation of clonal plasma cells in bone marrow. MM progresses from precursor stages, named monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM), to the symptomatic myeloma, MM. MYC abnormalities play a critical role in the disease progression. However, MYC is not therapeutically targetable due to its nuclear localization and the short half-life of the protein. Aims: To overcome this, we hypothesized that the proliferative advantage promoted by MYC overexpression induces differential genomic dependencies on other signaling pathways thus creates vulnerabilities that can be exploited therapeutically. Methods: We searched for genetic vulnerabilities associated with MYC overexpression by leveraging genome-scale pooled short hairpin RNA screening data for 236 cancer cell lines from Project Achilles. We generated an isogenic model of MYC overexpression (OE) in U266 cell line using EF1A-C-MYC lentiviral vector. Then, we performed RNA-seq, quantitative proteomics by Tandem Mass Tag mass spectrometry (TMT-MS) and a drug screening with ~2000 compounds. For validation, we performed pharmacological inhibition of glutamine catabolism as well as shRNA-mediated GLS1 knockdown. To determine the functional mechanisms, we used capillary electrophoresis-mass spectrometry (CE-MS) and Agilent Seahorse XF analyzer. Results: Achilles analysis revealed main dependencies associated with MYC overexpression on glutamine metabolism and specifically GLS1 (glutaminase) and SLC1A1 (glutamine transporter). Using a screening of 2000 small molecules, we further observed that inhibitors of NAD synthesis and mTORC1, which rely on the intracellular glutamine pool, had preferential effect on the proliferation of U266/MYC. Both RNA-seq and quantitative proteomics showed no significant upregulation of glutaminolysis related genes, suggesting a non-oncogenic dependency. Our validation tests confirmed this metabolic dependency, MYC OE cell lines failed to proliferate in the context of glutamine starvation and showed higher sensitivity to CB-839 and V-9302 inhibiting GLS1 and SLC1A5, respectively. Using the Seahorse analyzer, we measured the oxygen consumption rate (OCR) induced by glutamine. U266/MYC cells possess the ability to oxidize glutamine at a higher rate compared to U266/Ctrl. Additionally, higher sensitivity of U266/MYC to CB-839 was observed on both baseline and FCCP-induced OCR highlighting the role of glutamine in controlling mitochondrial OXPHOS in MYC OE cells. To understand the differential metabolic rewiring in MYC OE cells, we performed metabolomic analysis and observed higher GLS1 activity in U266/MYC presented by elevated glutamine to glutamate flux. We also identified the enriched metabolic pathways under GLS1 inhibition. Notably, CB-839 in U266/MYC results in more pronounced changes in TCA cycle and energy debt. This effect was blunted by the co-incubation with a-ketoglutarate, in a rescue experiment. Interestingly GLS1 inhibition was not limited to this effect, but extended to redox balance. CB839 significantly decreased glutathione level in U266/MYC. Furthermore, the intracellular concentrations of the glutamate-dependent amino acids were more depleted under GLS1 inhibition in U266/MYC compared to U266/Ctrl. Summary/Conclusion: Combining these observations, we were able to identify vulnerabilities to glutamine metabolism in MYC overexpressing cells. These results may lead to developing new therapeutic strategies to target MYC in clinical practice. P852: NEOANTIGENS PREDICTED BY THE GNE NEOANTIGEN PIPELINE IN PATIENTS DIAGNOSED WITH DE NOVO MULTIPLE MYELOMA Y. Jiang1, C. Henneges2,*, S. Ruppert3, P. Kimes2, A. Amitai2, A. Qamra2, C. Dos Santos3, J. N. Paulson2, A. Bazeos1 1Product Development, Oncology, Roche Products Limited, Welwyn, United Kingdom; 2Product Development Data Sciences; 3Oncology Biomarker Development, gRED, Genentech, Inc., South San Francisco, United States of America Background: Neoantigens are tryptic peptides expressed and presented on MHC-I characteristic for specific tumor mutations. They enable immune surveillance against cancer and may be potential targets for individualized cancer immuno-therapy. Aims: Here we present neoantigen evaluations from reprocessing de novo Multiple Myeloma patients recruited into the CoMMpass study. Methods: Whole Exome/Genome Sequencing data as well as RNA-sequencing data was provided by the Multiple Myeloma Research Foundation CoMMpass (SM) Longitudinal Study (research.themmrf.org) [NCT01454297]. Data was processed with the GNE/Roche neoantigen pipeline applying Mutect/Strelka for mutation calling, RNAseq and hlahd for HLA haplotyping prior to predicting peptide binding affinity and rank using NetMHCpan 4.0. Only strong binding peptides (rank score <0.5) are displayed here and analyzed using the nested-model approach: the baseline model of primary therapy (PI, IMiD, both) was extended by a term for presence of individual neoantigens in a gene (prevalence >=5%) for modeling Progression-free (PFS) and Overall Survival (OS) and Number of Progression Events during Observation period. Neoantigens are reported if the Likelihood-Ratio-Test was statistically significant for the Cox PH model or a Poisson regression at the 5% level, or if the AIC was better than the reference model. We also report the c-Index and calibration slope, estimated on a predefined training:validation split. Results: In N=730 patients, we identified nine proteins with high affinity neoantigens in total (LRT p-value<0.05): MT-CYB, IGHJ5, MAGEC1, IGKV1_5, MT_ND1, IGLV3_1, KRAS, AC245369.1, and NRAS. All, except NRAS (p=0.055), had a statistically significant Risk-Ratio when included in the Poisson regression model. Of these, IGHJ5 and IGKV1-5 correlated with PFS; MT-ND1 and KRAS correlated with OS. HR and Risk-Ratios with 95% CI in Table. Neoantigens occur at different frequencies ranging from 4.5% to 31.1% in the study population. The most commonly shared neoantigens are from IGLV3-1 (31.1%). Mapping neoantigens to protein domains using the PFAM database, they are present in domains related to energy turn-over (PF00032, PF00033), RAS signaling (PF00071) or in the Ig-like domain (PF07686). Image: Summary/Conclusion: We identified nine neoantigens, including two pairs each affecting PFS and OS, that had an effect on the number of progression event rate. Most were associated with immuno-globulin chain mutations, four were found as mitochondrial mutations and or being part of signaling. More data is needed to determine the correlations of these neoantigens with clinical and cytogenetic risk factors in patients with multiple myeloma. In addition, further studies are needed to determine factors that affect the commonality of neoantigens in this population. P854: HEXABODY-CD38 INDUCES TROGOCYTOSIS AND EFFECTIVELY INDUCES COMPLEMENT-MEDIATED TUMOR CELL LYSIS AFTER TREATMENT WITH DARATUMUMAB OR ISATUXIMAB IN VITRO I. H. Hiemstra1,*, K. Santegoets1, M. L. Janmaat1, W. Ten Hagen1, S. Van Dooremalen1, B. E. De Goeij1, S. Bosgra1, A. K. Sasser2, E. C. Breij1 1Genmab BV, Utrecht, Netherlands; 2Genmab US, Inc, Plainsboro, NJ, United States of America Background: HexaBody-CD38 (GEN3014) is a next-generation CD38-specific IgG1 antibody with a hexamerization-enhancing mutation. HexaBody-CD38 is designed to induce strong anti-tumor activity in patients with CD38-expressing hematological malignancies through potent complement-dependent cytotoxicity (CDC) and other Fc-mediated effector functions. The safety and preliminary efficacy of HexaBody-CD38 are currently being evaluated in a first-in-human trial in relapsed/refractory multiple myeloma (MM) patients (NCT04824794). Trogocytosis has been suggested as effector mechanism of daratumumab: reduction of CD38 expression on tumor and immune cells is thought to reduce local immunosuppression and contribute to improved adaptive immune responses against MM cells (Krejcik, 2018, Oncotarget 9, 33621). Aims: The present study aimed to increase our understanding of the MoA of HexaBody-CD38, by studying its capacity to induce trogocytosis as well as its capacity to induce CDC in CD38+ tumor cells in the presence of daratumumab or isatuximab in vitro. Methods: Trogocytosis was evaluated as the amount of membrane transfer in absence of transfer of cytoplasm in a flow cytometry-based assay using Wien-133 cells as target cells and healthy donor monocytes as effector cells. Binding of HexaBody-CD38 to CD38+ cell lines SU-DHL-4, NCI-H929, and Wien-133 that were pre-treated with daratumumab or isatuximab was allowed for 15 min, 1 h, 4 h or 24 h, while daratumumab or isatuximab remained present at saturating concentrations. HexaBody-CD38 mediated CDC of daratumumab or isatuximab-opsonized Wien-133 cells, which were insensitive to CDC induction by daratumumab or isatuximab, was assessed by flow cytometry at 45 min, 4 h, or 24 h after adding human complement in the presence of daratumumab or isatuximab. Results: HexaBody-CD38 induced dose-dependent transfer of plasma membrane from CD38+ tumor cells to human monocytes (n=7). The mean EC50 for trogocytosis activity of HexaBody-CD38 was 8.79 ± 2.49 ng/mL (0.018 ± 0.014 nM). This was in the same range as EC50s of daratumumab and the HexaBody-CD38 parental antibody without E430G mutation, suggesting that the hexamerization mutation has limited impact on trogocytosis induction. HexaBody-CD38 was found to compete with daratumumab and isatuximab for binding to CD38. Binding of HexaBody-CD38 to CD38+ cells increased in time and with increasing concentration, generally faster and more extensive in the presence of isatuximab compared to daratumumab. At equimolar concentrations, near-complete displacement of isatuximab and daratumumab was observed after incubations ≥1 h and ≥4 h, respectively. Accordingly, after 24 h HexaBody-CD38 induced comparable maximum CDC of CD38+ cells in presence or absence of daratumumab. Comparable CDC activity in the presence or absence of isatuximab was already observed after 4 h. Summary/Conclusion: HexaBody-CD38 was shown to induce efficient monocyte-mediated trogocytosis of CD38+ tumor cells in vitro, suggesting HexaBody-CD38 may reduce CD38-associated immunosuppression in the tumor microenvironment. In addition, it has previously been reported that the main differentiating effector mechanism activity of HexaBody-CD38 compared to daratumumab and isatuximab is its increased capacity to induce CDC. Here we confirmed the CDC potency of HexaBody-CD38 even in the presence of saturating concentrations of daratumumab and isatuximab. This suggests that HexaBody-CD38 is capable of inducing CDC of CD38+ myeloma cells in patients who have received prior anti-CD38 mAb treatment with residual daratumumab or isatuximab present in their circulation. P855: SYNERGISTIC EFFICACY OF COMBINED SUMOYLATION AND PROTEASOME INHIBITION IN MULTIPLE MYELOMA U. Keller1,*, G. Heynen1, F. Baumgartner1, M. Heider2, U. Patra3, J. Braune1, P. Mertins4, S. Müller3, F. Bassermann2, J. Krönke1, M. Wirth1 1Hematology, Oncology and Cancer Immunology, Charite - Universitätsmedizin Berlin, Berlin; 2Medicine III, Technische Universität München, Munich; 3Biochemistry II, Goethe Universität Frankfurt, Frankfurt; 4Proteomics Core, Max-Delbrück Center for Molecular Medicine, Berlin, Germany Background: Multiple myeloma (MM) is a genetically and clinically heterogeneous neoplastic plasma cell disorder. Treatment regimens for MM include proteasome inhibitors (PIs) which are routinely combined with dexamethasone and chemotherapeutics or immunomodulatory drugs (IMiDs). While such regimens have resulted in significant improvement of disease control and survival, the development of drug resistance remains a major clinical problem. Relapsed/refractory MM (r/r MM) is associated with particularly poor prognosis. New therapeutic strategies to further improve the management of MM and to overcome drug resistance are therefore urgently needed. SUMOylation is a post-translational protein modification pathway closely related to ubiquitylation. Hyperactive SUMOylation (SUMO) signalling is involved in both cancer pathogenesis and cancer progression. A state of increased SUMOylation has been associated with aggressive cancer biology. Aims: We sought to identify stress response mechanisms associated with adverse prognosis and PI resistance. The overall goal was to develop novel therapy combinations targeting molecular vulnerabilities associated with advanced and/or PI-resistant MM. Methods: RNA-sequencing, unbiased mass spectrometry-based proteomics and bioinformatics analyses were used to interrogate the biology and in particular stress response pathways in MM. Cell culture finding were informed with primary MM patient data from gene expression studies and and survival data from clinical trials. Novel drug combination treatments were tested in MM cell-based models in vitro, in MM xenograft models in vivo, and in primary MM patient samples. Results: Here, we found that r/r MM is characterized by a state of activated SUMOylation, a ubiquitin-related post-translational protein modification pathway. High expression of the SUMO E1 activating enzyme (SAE1/UBA2) was associated with poor overall survival. Induced resistance to the second generation PI carfilzomib (CFZ) enhanced SUMO pathway activity. Accordingly, CFZ-pretreated patients showed enhanced SUMO pathway activity in the MM compartment. Treatment of MM cell lines with subasumstat (also TAK-981), a novel small-molecule inhibitor targeting the SUMO E1 activating enzyme, showed synergistic treatment efficacy with CFZ in both PI-sensitive and PI-resistant MM cell lines irrespective of the TP53 state. Combination therapy was effective in two murine MM xenograft models, where in vivo growth was significantly inhibited, and in patient-derived primary MM cells in vitro. Mechanistically, combinatorial treatment of subasumstat and CFZ enhanced genotoxic and proteotoxic stress and apoptosis. Summary/Conclusion: Our findings reveal activated SUMOylation as a therapeutic target in MM and point to combined SUMO/proteasome inhibition as a novel and potent strategy for the treatment of patients with MM. P856: A SINGLE-CELL FUNCTIONAL PRECISION MEDICINE LANDSCAPE OF MULTIPLE MYELOMA K. Kropivsek1,*, P. Kachel2, S. Goetze3,4,5, R. Wegmann1, Y. Severin1, B. D. Hale1, Y. Festl1, J. Mena1, A. van Drogen3,4,5, N. Dietliker2, J. Tchinda6, B. Wollscheid3,4,5, M. G. Manz2, B. Snijder1 1Department of Biology, Institute of Molecular Systems Biology, ETH Zurich; 2Department of Medical Oncology and Hematology, University Hospital Zurich; 3Department of Health Sciences and Technology, Institute of Translational Medicine; 4Swiss Multi-Omics Center, PHRT-CPAC, ETH Zurich, Zürich; 5Swiss Institute of Bioinformatics, Lausanne; 6Pediatric Oncology, Children’s Research Centre, University Children’s Hospital Zurich, Zürich, Switzerland Background: Multiple myeloma (MM) is a cancer of plasma cells, defined by complex genetics and extensive intra- and inter-patient heterogeneity. Despite improved patient survival driven by a plethora of treatment options, the disease remains incurable. Molecularly-guided precision medicine to individualize treatment strategies in MM has had limited success, in part due to the genetic and molecular complexity of the disease. Functional precision medicine, a complementary approach in which patient treatment is guided by the ex vivo drug response of patient cells, has not yet been evaluated for MM systematically. Aims: Our observational study has two aims: 1) To evaluate single-cell resolved functional precision medicine (also called Pharmacoscopy) as a way to tailor treatment for individual MM patients; 2) To identify new treatment options for, and improve our molecular understanding of, MM. Methods: In our observational clinical study, image-based ex vivo drug screening (Pharmacoscopy; PCY) measured the single-cell level responses to drug and immunotherapy combinations for 138 patient samples from 98 patients covering all stages of the disease. Single-cell morphologies are analyzed by deep convolutional neural networks, quantifying the cellular composition and morphological myeloma heterogeneity at time of sampling. PCY data is integrated with 1) sample-matched proteomics of purified cell subpopulations, 2) bone marrow serum cytokine profiling, as well as with 3) patient-matched genetics and clinical data. Lastly, ex vivo drug sensitivities are matched to clinical responses. Results: Our large-scale single-cell morphological characterization of Multiple Myeloma samples reveals three reoccurring cell community modes, which we call PhenoGroups (PGs). These PGs represent different disease stages, with both genetic and inflammatory associations, independently confirmed by orthogonal data. The ex vivo responses to 61 existing and novel therapeutic strategies (ranging from single drugs to quadruple drug combinations) reveal considerable differences in patients’ ex vivo responses to the same treatments. Integration with proteotype data discovers the comprehensive molecular network underlying variable drug sensitivities, recovering known regulators of drug response in myeloma and identifying new possible mechanisms of action. Furthermore, integration with genetics and clinical characteristics across patients recovers known biomarkers, e.g. proteasome inhibitor resistance for TP53 mutant patient samples, and proposes novel biomarkers for individualized patient treatments. The strongest predictor of ex vivo drug sensitivity is the sample composition as captured by the PhenoGroups, which we find is also reflected in the clinical course. Finally, longitudinal follow-up and Kaplan-Meier analyses confirm that Pharmacoscopy-measured drug responses are significantly predictive of the clinical response of patients (Figure 1). Image: Summary/Conclusion: Our single-cell functional precision medicine platform (Pharmacoscopy) combined with patient-matched OMICs provides molecular and clinical insights into existing and novel treatment strategies for MM patients. Furthermore, Pharmacoscopy is significantly predictive of clinical response in MM, warranting further clinical investigation. P857: BORTEZOMIB- AND CARFILZOMIB-RESISTANT MYELOMA CELLS SHOW INCREASED ACTIVITY OF ALL THREE ARMS OF UNFOLDED PROTEIN RESPONSE AND ENRICHMENT OF SIRTUIN SIGNALING PATHWAY T. Kubicki1,*, K. Bednarek2, M. Kostrzewska-Poczekaj2, M. Łuczak3, Z. Kanduła1, K. Lewandowski1, L. Gil1, M. Jarmuż-Szymczak2, D. Dytfeld1 1Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences; 2Institute of Human Genetics, Polish Academy of Science; 3Institute of Bioorganic Chemistry, Polish Academy of Science, Poznan, Poland Background: Proteasome inhibitors (PIs) are among most potent and widely-used class of drugs in multiple myeloma (MM) treatment. One of the main challenges in MM therapy is acquired resistance to drugs, PIs are no exemption from this phenomenon. Several theories were proposed to describe mechanisms responsible for resistance to most commonly used PIs – bortezomib and carfilzomib. One of the most promising explanations involves modulation of unfolded protein response (UPR) pathway. The pathway is initiated by conformational changes in three sensor proteins (PERK, IRE1α, ATF6) in response to overaccumulation of misfolded proteins. Recent study (Sarin et al, Leukemia 2020) suggests that MM cell lines used for in vitro experiments differ substantially in resemblance to patients’ tumors, partially explaining difficulties in translation of results from laboratory models to MM treatment. Here we evaluated MM1S cells, that are among best fitted to resemble actual MM patients’ biology. Aims: The study aimed at describing functional differences between PI-sensitive MM1S cells (MM1S WT) and their daughter cells, resistant to either bortezomib (MM1S/R BTZ) or carfilzomib (MM1S/R CFZ), as well as between both resistant cell lines. Methods: Resistant cell lines were generated by continuous culture with increasing concentrations of drugs. Subsequently proteomic profiling of sensitive and resistant cells was performed. To functionally analyze proteomic results proteasome activity and generation of reactive oxygen species (ROS) were measured. Finally, changes in UPR activation were assessed by Western blot analysis of key proteins involved in this pathway. Results: BTZ- and CFZ-resistant cell lines were successfully generated. IC50 values were 3-fold higher for the resistant cells (MM1S WT IC50=15.2 nM for BTZ, IC50=8.3 nM for CFZ; MM1S/R BTZ IC50=44.5 nM; MM1S/R CFZ IC50=23.0 nM). When exposed to different PI than during resistance generation period MM1S/R BTZ cells were resistant to CFZ (IC50=43.5 nM) whereas MM1S/R CFZ cells were similarly sensitive to BTZ as MM1S WT (IC50=24.0 nM). Unsupervised principal component analysis revealed that MM1S/R BTZ and MM1S/R CFZ differ significantly from MM1S WT cells and also from each other. Canonical pathway analysis showed similar pathways enriched in both comparisons – MM1S WT vs MM1S/R CFZ and MM1S WT vs MM1S/R BTZ, however important differences were present in terms of statistical significances of particular pathways (Figure). Sirtuin signaling was identified as the most enriched pathway in BTZ-resistant cells (top-4 in CFZ-resistant) and EIF2 signaling in CFZ-resistant cells. In functional studies, both PIs continued to block chymotrypsin-like proteasome activity in resistant cells. The relative reduction in the activity was similar for resistant and sensitive cells – 65% for MM1S/WT in BTZ, 45% for MM1S/R BTZ, 96% for MM1S/WT in CFZ, 77% for MM1S/R CFZ. Baseline activity of all 3 catalytical domains of proteasome was significantly higher in the resistant cells. MM1S/R BTZ cells generated lower amount of ROS in comparison to MM1S WT (64%), while there were no differences in similar comparison for MM1S/R CFZ cells. Both baseline and drug-induced activity of UPR was higher in PI-resistant cells then in MM1S WT and included all three arms of UPR - IRE1α/XBP1s, ATF6 and EIF2α/ATF4 (downstream effectors of PERK). Image: Summary/Conclusion: Contrary to some previous reports, performed mainly on other MM cell lines, PI-resistant MM1S cells show upregulation of UPR. This reflects heterogeneity of MM and prompts further studies on UPR role and its interplay with sirtuin signaling. P858: EPIGENETIC REGULATION OF CD155 ON MULTIPLE MYELOMA CELLS INFLUENCES ANTI-TUMOR T-CELL CYTOTOXICITY L. Martinez-Verbo1,*, P. Llinàs-Arias1, L. Villanueva1, C. A. García-Prieto1,2, D. Piñeyro1, G. Ferrer1,3,4, M. Esteller1,5,6 1Cancer Epigenetics, Josep Carreras Leukaemia Research Institute, Badalona; 2Barcelona Supercomputing Center, Barcelona, Spain; 3Feinstein Institutes for Medical Research, Northwell Health, New York, United States of America; 4Centro de investigación Biomédica en Red Cancer, Madrid; 5Institució Catalana de Recerca i Estudis Avançats; 6Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain Background: DNA methylation remains as one of the key molecular players regulating gene expression. In multiple myeloma (MM) and thanks to data available, we identified CD155 as a putative epigenetically regulated gene. Cytotoxic T-cells (CD8+) are crucial in the clearance of malignant cells and their activation is tightly regulated by immune checkpoint events (IC). Tumor cells take advantage of this system and escape immune recognition, avoiding T-cell attacks by inducing exhaustion. CD155 is involved in the CD8+ T-cell activation IC and it is of interest because could act as an inhibitory or stimulatory signal. We investigated the role of CD155 in MM and its importance in T-cell inhibition in-vitro, in relation to its epigenetic state and expression. Aims: To evaluate the role of CD155 methylation in MM cells and how it affects T-cell activation and tumor elimination. Methods: The methylation status of the promoter region of CD155 was studied in several MM cell lines from COSMIC database and experimentally validated in four (RPMI-8226, MM.1S, AMO-1 and KMS-12-BM). RPMI-8226 was selected as an unmethylated cell line for CD155 knock down model (depleted expression) and scramble model by lentiviral transduction. We analyzed if CD155 has an impact in cell proliferation and cell cycle with MTT assay and flow cytometry. Co-culture experiments were performed with the CD155 models and T-cells isolated from healthy donors for 48 hours and studied T-cell cytotoxicity by analyzing the remaining MM cells with flow cytometry. Finally, we analyzed CD155 expression and survival in MM from CoMMpass project public data. Results: We validated the in-silico methylation data by bisulfite sequencing and negative regulation by Western Blot and quantitative PCR in four different cell lines. Depletion of CD155 did not have a significant impact on cell growth, apoptosis or cell cycle. For instance, when we co-cultured the CD155 models with pre-activated T-cells, we detected more cytotoxicity towards CD155 depleted cells, whereas CD155 expressing cells resisted T-cell activity (p=0.02). In order to determine if the inhibition was mediated by interaction CD155-TIGIT, we added 10ug/ml of neutralizing αTIGIT and/or αPD1 antibody to the co-culture systems. While T-cells co-cultured with CD155 depleted cells in the presence of αTIGIT did not change their levels of cytotoxicity, expressing cells were eliminated more successfully. In the presence of αPD1, we saw a significant restoration of T-cell cytotoxicity against both models and antibody combination showed a synergic effect in expressing models’ co-culture whereas CD155 depleted levels remained independent of αTIGIT presence. Supporting these results, the data obtained from the CoMMpass project showed the MM patients (N= 793) with higher expression of CD155 had shorter overall survival than lower expressing ones (p<0.001). Summary/Conclusion: In MM cells, the expression of CD155 is regulated by the methylation status of its promoter region. Unmethylated MM cells expressing CD155 promote T-cell inhibition and its depletion resulted in an improvement of T-cell cytotoxicity activity against malignant cells. The addition of anti-TIGIT and anti-PD1 validated that the CD155 T-cell inhibition is mediated by the interaction with TIGIT, independently of PD1. These findings indicate that CD155 unmethylated MM cases are at higher risk warranting further investigation. P859: ANTI-CD38 NANOBODY JK36 ALLOWS RELIABLE MRD DETECTION IN DARATUMUMAB TREATED MULTIPLE MYELOMA PATIENTS E. Meseguer Martinez1,*, J. Marco Buades1, A. García Feria1, P. Ribas García1, M. J. Fernandez Llavador1, A. López Gabaldon1, S. Broseta Tormos1, E. Francés Aracil1, O. Cortés Ortega1, E. Donato Martínez1, M. Fernandez Zarzoso1, M. Panero Ruiz1, M. J. Cejalvo Andújar1, A. Tolosa Muñoz1, M. L. Juan Marco1, D. Ivars Santacreu1, E. Gómez Beltrán1, M. J. Sayas Lloris1 1Hematología y Hemoterapia, Hospital Universitario Doctor Peset, Valencia, Spain Background: The introduction of immunotherapies such as daratumumab, a CD38 monoclonal antibody has improved survival in multiple myeloma (MM), however a few considerations on minimal residual disease (MRD) assessment by flow cytometry have been raised and deserve our attention. In flow cytometry CD38 is frequently used as a gating marker of plasma cells (PCs), but daratumumab can hampering myeloma cell detection. A strategy to overcome this interference is to staining PCs with anti-CD38 nanobody JK36. Nanobodies are single variable domain antibody fragments (VHH) that often expose a long complementarity-determining region 3 (CDR3), feature that allows anti-CD38 nanobody JK36 recognizing a cryptic epitope not masked by anti-CD38 therapies. Aims: To compare the median fluorescence intensity (MFI) of CD38 multiepitope (ME) antibody and anti-CD38 nanobody in bone marrow samples from MM patients not treated with daratumumab. Secondly, to compare flow cytometric MRD data obtained using CD38-ME and anti-CD38 nanobody from daratumumab treated MM patients. Methods: Samples were obtained from 10 MM patients not treated with daratumumab to compare the MFI of CD38-ME vs CD38-nanobody at baseline and after incubation with daratumumab at a concentration of 10 nM for 60 min at room temperature. Marrow samples were stained using panel CD38/CD56/CD19/CD138/CD45 with variable CD38, CD38-ME vs anti-CD38 nanobody. Data were analyzed using Kaluza 2.1.1 software (Beckman Coulter). Moreover, 17 bone marrow samples were processed using bulk lysis and subsequently stained using the MM-MRD EuroFlow 8-color antibody combination panel and tubes 1 and 2 with variable CD38, CD38-ME vs anti-CD38 nanobody. Samples were acquired on CytoFlex flow cytometer (Beckman Coulter) and data were analyzed using Infinicyt 2.0 software (Cytognos). Statistical analysis was performed using SPSS version 24, verifying the assumption of normality of the variable, pairwise comparisons were carried out using the T-tests for related samples. Results: The MFI values of CD38 ME on samples from daratumumab not treated patients was higher than samples after incubation with daratumumab (p=0.003). For CD38 nanobody, no significant differences were observed for the MFI values of samples, independent of daratumumab incubation (p=0,139) (figure 1A). The percentage of loss of MFI were higher in the CD38 ME group (-36,32%) than in the CD38 nanobody group (-0,35%). Regarding 17 patients treated with anti-CD38 therapies, 14 were treated with daratumumab and 3 with isatuximab. Sixteen samples were MRD positive with a sensitivity of 10-5 or 10-6 by both approaches. As shown in figure 1B, CD38-ME and CD38 nanobody both showed high and comparable MFI on PCs from patients treated with anti-CD38 therapies. Image: Summary/Conclusion: The anti-CD38 nanobody JK36 construct has lower coefficient of variation than CD38 ME antibody. It is a viable alternative to CD38 ME antibody to gate PCs by flow cytometry allowing reliable MRD detection to identify PCs in the presence of anti CD38 biologics. Nanobody technology open new avenues in the detection capability of MRD flow cytometry studies in the era of immunotherapy. P860: THE DE NOVO DNA METHYLTRANSFERASE DNMT3B PLAYS AN IMPORTANT ROLE IN MM CELL GROWTH, CLONOGENICITY AND DRUG RESPONSE C. Muylaert1,*, L. Van Hemelrijck1, P. Vlummens1,2, A. Maes1, K. De Veirman1, E. Menu1, K. Vanderkerken1, E. De Bruyne1 1Department of Hematology and Immunology-Myeloma Center Brussels, VUB, Brussels; 2Department of Internal Medicine, Ghent University Hospital, Ghent, Belgium Background: Multiple myeloma (MM) is an incurable plasma cell malignancy due to the development of drug resistance (DR). In about half of the MM patients, modifications are observed in epigenetic modifiers (epiplayers) at the time of diagnosis and this frequency is further increased at relapse, indicating an important role for epiplayers in MM cell DR. However, so far, only for two epiplayers, MMSET and EZH2, a clear role in MM cell DR development has been established. With the aim to identify new, clinically relevant epiplayers involved in MM progression and relapse, we recently compared the RNASeq data from matched newly diagnosed and relapsed patients from the MMRF CoMMpass study. We found that the epiplayer DNMT3B is significantly increased in relapsed patients, suggesting a possible role for DNMT3B in MM relapse. Aims: The aim of this study is to explore the role of DNMT3B in MM cell biology and drug response. Methods: Publicly available gene expression profiling data of three independent cohorts of newly diagnosed MM patients (GSE4581, E-MTAB-372, MMRF-COMMPASS), one cohort of relapsed patients (GSE9782), BM plasma cells (E-MTAB-372), primary MM cells (E- MTAB-372), and human MM cell lines (HMCLs; E-TABM-1088 and E-TABM-937) was used. DNMT3B specific targeting was achieved by using the DNMT3B specific inhibitor Nanaomycin A and genetic inhibition using doxycycline inducible shRNA against DNMT3B. Viability and apoptosis were assessed using a CellTiter-Glo assay and an AnnexinV/7AAD staining respectively. In addition, BrdU incorporation and cell cycle analysis was evaluated to assess cell proliferation. Clonogenic capacity was evaluated by a colony formation assay. Results: We found that DNMT3B mRNA levels increase during disease progression and high DNMT3B mRNA expression correlates with a worse disease outcome in both newly diagnosed and relapsed patients, indicating a role for DNMT3B in MM progression and DR. In line, Nanaomycin A treatment (up to 72h) led to a significant and dose-dependent decrease in cell viability and proliferation and increase in apoptosis in the XG-2, XG-7 and AMO-1 HMCLs. We also validated the anti-MM activity of Nanaomycin A on human primary MM cells. Since DNMT3B is thought to play a significant role in cancer cell stemness, we next evaluated the effect on clonogenicity. A significant and dose-dependent decrease in the number of colonies was observed when low doses of Nanaomycin A were added to AMO-1 (100 and 200 nM) and XG-2 (30 and 50 nM) cells on the day of plating (day 0), but not when added 7 days after plating (day 7). In contrast, treatment with a high Nanaomycin A concentration (800 nM) significantly reduced the number of colonies at both timepoints, indicating that high Nanaomycin A doses are cytotoxic whereas low doses impair the proliferation and clonogenicity of the MM cells. The anti-clonogenic effect of DNMT3B targeting was confirmed by DNMT3B knockdown in AMO-1 cells. Finally, combining Nanaomycin A (100 nM) with bortezomib (Bz, 4 nM) resulted in a significant decrease in colony formation compared to either Nanaomycin A or Bz treatment alone. Summary/Conclusion: Taken together, our findings indicate that DNMT3B is a novel promising target to overcome or delay relapse in MM. DNMT3B targeting impairs MM cell proliferation and clonogenicity and sensitizes the cells to the proteasome inhibitor Bz. In the near future, the anti-myeloma activity of DNMT3B targeting will be validated in vivo using the 5TMM murine MM models and the underlying mechanisms will be further determined. P861: SIALOFUCOSYLATED STRUCTURES ENABLE PLATELET BINDING TO MYELOMA CELLS CONFERRING PROTECTION FROM NK-MEDIATED CYTOTOXICITY A. Natoni1,*, M. Cerreto1, M. S. De Propris1, M. T. Petrucci1, I. Del Giudice1, S. Intoppa1, M. L. Milani1, L. Kirkham-McCarthy2, R. Henderson3, D. Swan3, M. O’Dwyer2, A. Guarini4, R. Foà1 1Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy; 2Biomedical Sciences, School of Medicine, National University of Ireland Galway; 3Department of Haematology, Galway University Hospital, Galway, Ireland; 4Department of Molecular Medicine, Sapienza University, Rome, Italy Background: Multiple myeloma (MM) is a plasma cell disorder that develops in the bone marrow (BM), characterized by an unchecked proliferation and by the ability to disseminate in different parts of the skeleton. Several studies have highlighted a prominent role of platelets in the metastatic dissemination of cancer cells. Indeed, by binding to tumor cells, platelets promote adhesion to target organs and evasion from natural killer (NK)-mediated cytotoxicity. Importantly, tumor/platelet interactions are mostly mediated by P-selectin. We have previously shown that MM cells enriched for Sialyl Lewis X (SLex), a sialofucosylated structure recognized by selectins, efficiently home into the BM and become resistant to bortezomib in vivo (Natoni et al, 2017). Aims: We herein hypothesized that sialofucosylation participates in platelet recruitment on the surface of MM cells via P-selectin, forming a “cloak” that protects tumor cells from NK-mediated cytotoxicity. Methods: Platelets were purified from peripheral blood samples obtained from healthy individuals. MM cell lines and their derivative that have been enriched for SLex, which strongly bind to selectins, were co-cultured with platelets for 30 min and platelet binding was assessed by flow cytometry using the CD41/CD61 antibody. In some experiments, cells were incubated with platelets in the presence of an anti-P-selectin blocker antibody. To examine the NK-mediated cytotoxicity towards tumor cells, parental and SLex MM cells were co-cultured with platelets for 24 h and then incubated with autologous NK cells expanded in vitro for 7 days. NK-mediated cytotoxicity was assessed after 4h by flow cytometry using Annexin V/7-AAD staining. To examine platelet/MM binding in patients, BM samples from MM patients at different disease stages were analyzed by flow cytometry using a panel of antibodies to identify platelet binding to malignant plasma cells. Results: In MM cell lines, platelets bound exclusively to SLex enriched cells, suggesting that efficient binding requires sialofucosylation. This binding could be blocked by a P-selectin antibody, indicating that the interactions between MM cells and platelets are partially dependent on P-selectin. Platelets significantly decreased NK-mediated cytotoxicity in the SLex enriched (P=0.0025) but not in the parental MM cells, confirming the important role of sialofucosylation in platelet binding. Platelets also bound to malignant cells from primary BM aspirates. Interestingly, the binding to platelets occurred in SLex positive and negative cells, suggesting that in vivo non-sialofucosylated ligands may also be involved in platelet binding. Importantly, the SLex CD41/CD61 double positive population seems to accumulate in MM patients at relapse (median diagnosis vs relapse: 0.86 vs 3.03; P=0.0034). Summary/Conclusion: We herein report that MM cell lines enriched in SLex interact with platelets in vitro, and that this interaction partially protects MM cells from NK-mediated cytotoxicity. These results suggest that in vivo, platelets may promote immune evasion facilitating the dissemination of MM cells. Moreover, SLex positive primary malignant BM plasma cells bind platelets, particularly from patients at relapse, suggesting that in vivo these cells may accumulate as the disease progresses and becomes resistant to treatment. Finally, the interactions between platelets and MM cells could be disrupted by a P-selectin antibody, indicating a possible therapeutic target to restrict metastasis and re-sensitize MM cells to NK cells. P862: SERUM MASS SPECTROMETRY TO ANALYZE DISEASE RESPONSE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA RECEIVING ARI0002H, AN ACADEMIC BCMA-DIRECTED CAR T-CELL THERAPY I. Ortiz De Landazuri1,*, A. Oliver-Caldés2, M. Español-Rego1, M. T. Contreras3, A. Zabaleta4, C. Agulló3, N. Puig5, V. Cabañas6, V. González-Calle5, R. Jiménez2, S. Inogés4, P. Rodríguez-Otero4, B. Martin-Antonio7, J. L. Reguera8, A. López-Diaz de Cerio4, D. Benítez-Ribas1, L. G. Rodríguez-Lobato2, E. A. González1, L. Rosiñol2, J. Yagüe1, J. M. Moraleda6, Á. Urbano-Ispizua2, M. V. Mateos5, M. Juan1, B. Paiva4, M. Pascal1, C. Fernández de Larrea2 1Immunology Department; 2Hematology Department, Hospital Clínic de Barcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona; 3Clinical Biochemistry Department, Hospital Universitario de Salamanca, Salamanca; 4Clínica Universidad de Navarra, Centro de Investigacion Medica Aplicada (CIMA), IDISNA, CIBERONC, Pamplona; 5Hematology Department, Hospital Universitario de Salamanca, Salamanca; 6Hospital Clinico Universitario Virgen de la Arrixaca., Murcia; 7Instituto de Investigación Sanitaria, Fundación Jiménez Díaz, Madrid; 8Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS/CSIC/CIBERONC), University of Sevilla, Sevilla, Spain Background: ARI0002h is an academic BCMA-directed CAR T-cell that has been reported as effective in patients (pts) with relapsed/refractory multiple myeloma (RRMM). In these pts, next generation flow cytometry (NGF) in bone marrow (BM) allows the identification of deeper responses than serum protein immunofixation (IFE) after treatment. Aims: Here, we explore EXENT Quantitative Immunoprecipitation Mass Spectrometry (EXENT QIP-MS) as a highly-sensitive, serum-based, alternative monitoring technique and able to identify therapeutic monoclonal antibodies (t-mAb) interferences. Methods: Thirty-three RRMM pts with measurable disease in serum by IFE or free light chains were included. All of them received ≥2 prior regimens including a proteasome inhibitor, an immunomodulatory drug and daratumumab, and were refractory to the last one. These pts were treated with ARI0002h, 30 in the setting of the CARTBCMA-HCB-01 clinical trial (NCT04309981) and 3 as compassionate use. The M-protein (MP) was analyzed in serum by EXENT QIP-MS using IgG/A/M/κ/λ isotypic beads at three time points: before ARI0002h infusion, and 28 and 100 days post-infusion. Finally, 98 samples were analyzed by EXENT QIP-MS. Every patient had received daratumumab previously, but the last infusion date varied widely. The presence of disease in BM was also investigated by NGF (sensitivity ≥ 2x10-6) at 28 and 100 days post-infusion. The median follow-up of the pts was 15 months (range 5 to 20). Results: Before ARI0002h infusion, MP could be identified in 33 (100%) pts by EXENT QIP-MS. After 28 and 100 days post-infusion, MP was also detected by this method in serum in 24 (72.7%) and 18 (56.3%) pts, respectively. Investigating the ability of EXENT QIP-MS to identify the patient´s MP, interferences due to daratumumab and tocilizumab were successfully detected in serum with a IgG-kappa peak in the mass spectrum of 11693 and 11750 m/z, respectively. Before ARI0002h, daratumumab was identified in 10 pts (30.3%) and persisted even at day +100 in 5 of them (15.2%). Tocilizumab was detected at day +28 in 15 (71.4%) pts out of the 21 receiving it, persisting at day +100 in 3 of them (14.3%). Then, we compared the results obtained by EXENT QIP-MS and IFE at the two time-points analyzed (n=63). Results were concordant in 81% of samples and discordant in 19% (p<0.0001). All discordances were due to EXENT QIP-MS(+)/IFE(-) results, except in one case. In this patient, the serum was mistakenly considered as IFE(+) (IgG-kappa) owing to the interference of daratumumab. Discordant results were equally noted at day +28 and +100 (Fig.1). From 25 and 26 MRD-evaluable pts at day +28 and +100, 96% and 92% were MRD(-) in BM by NGF, respectively. NGF was discordant in all cases (EXENT QIP-MS(+)/NGF(-)), except in 1 patient at +28 days and 3 pts at +100 days, reflecting the MP persistence in the absence of BM disease. After 16 months, 75% of pts with EXENT QIP-MS(-)/NGF(-)/IFE(-) at day +28 were alive and without progression, and only 2 pts have relapsed from the disease, both after 12 months (14 and 16 months, respectively). Image: Summary/Conclusion: In this study including pts with RRMM and measurable disease, serum EXENT QIP-MS allowed the identification of the MP in all cases with high sensitivity. EXENT QIP-MS was also able to differentiate between the MP and t-mAb, which translated in a correct labeling of treatment response. As compared to both IFE in serum and NGF in BM, EXENT QIP-MS was able to identify residual disease in a higher proportion of cases. P863: GENERATION OF A FIRST-IN-CLASS INHIBITOR FOR THE MASTER ONCOREGULATOR HNRNP K IN MULTIPLE MYELOMA Á. Otero Sobrino1,*, J. Le Coq2, P. Aguilar-Garrido1, M. Á. Navarro Aguadero1, M. I. Albarrán3, J. Klett3, C. Blanco3, R. Fernández-Leiro4, J. Martínez-López5, M. Velasco1 1H12O-CNIO Haematological Malignancies Clinical Research Unit; 2Electron Microscopy Unit; 3Biology Section; 4Genome Integrity and Structural Biology Group, Spanish National Cancer Research Centre (CNIO); 5Grupo de Hematología Traslacional, Hospital 12 de Octubre de Madrid, Madrid, Spain Background: Haematological malignancies constitute a plethora of different neoplasms including leukaemia, lymphomas and myeloma. Despite all of them having robust first-line treatments and a myriad of therapeutic alternatives, some remain incurable due to the resistance and refractoriness of tumour cells. The heterogeneous nuclear ribonucleoprotein K, hnRNP K, is a master oncoregulator that binds C-rich tracks of nucleic acids and is implicated in multiple biological functions such as splicing, polyadenylation and translation. Indeed, hnRNP K contributes to treatment resistance and poor outcomes in haematological malignancies. Compared to other hnRNPs, hnRNP K has the unique structural characteristic of containing three K-homology (KH) domains that allows it to bind both ssDNA and RNA sequences, as well as a K-interactive (KI) region that binds to multiple critical proteins such as Src, Fyn or Lyn amongst others. Interestingly, hnRNP K regulates the p53/p21 and c-Myc pathways, and our group has characterised hnRNP K as a driver of leukaemia so far, thus proving its role in haematological neoplasms. Aims: We aim to develop a first-in-class inhibitor for hnRNP K and test its efficacy in circumventing resistance of haematological malignancies. Methods: We have produced full-length (FL) hnRNP K protein in-house and verified that it was properly folded and could bind RUNX1 RNA and mRNA sequence-based ssDNA, using Tycho and fluorescence anisotropy technologies. We then carried out a high-throughput screen of over 7,000 small molecules (both FDA and non-FDA approved) using AlphaLISA technology and validated both chemically and biologically the inhibition of hnRNP K with those identified compounds. Lastly, we have performed phenotypical analysis of the effects of the hnRNP K inhibitors in Multiple Myeloma cell line AMO1 and L363 overexpressing hnRNP K through CRISPR synergistic activation mediator (SAM). Results: We have successfully produced and purified the FL hnRNP K protein, with stable inflexion temperatures (Ti) as measured by Tycho. Furthermore, we corroborated that it maintained its ssDNA and RNA binding abilities through fluorescence anisotropy. Lastly, we set up the AlphaLISA system to screen a library of over 7,000 small molecules and identified circa 10 interesting hits, from which we validated the 5 best based on their inhibition capacity and specificity of hnRNP K. We verified the chemical properties of these small molecules. Lastly, we confirmed that they had a biological effect of decreasing the levels of hnRNP K downstream pathways as well as inducing changes in viability dose-response curves, using Multiple Myeloma cells (AMO1 and L363) genetically modified with CRISPR/SAM technology to overexpress hnRNP K. Likewise, we investigated the absence of such biological effect in hnRNP K-Knock Out (KO) AMO1 and L363 cells, obtained by CRISPR-Cas9. Image: Summary/Conclusion: We have identified a first-in-class hnRNP K inhibitor that specifically binds and blocks hnRNP K in modified Multiple Myeloma cell lines. This constitutes a novel and promising therapeutic approach that could help overcome the current drug resistance in haematological neoplasms currently found in the clinic. This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295), ISCIII Miguel Servet (CP19/00140) and IF-Marie Sklodowska Curie Actions grant (MAtChinG – 101027864). P864: CTPS1 IS A NOVEL THERAPEUTIC TARGET IN MYELOMA - SELECTIVE SMALL MOLECULE INHIBITION DELIVERS SINGLE AGENT ACTIVITY AND SYNERGISES WITH ATR INHIBITION C. Pfeiffer1,*, P. Beer2, H. Asnagli2, A. Bolomsky3, A. Grandits4,5, A. Schneller1, J. Huber1, N. Zojer1, M. Schreder1, A. Parker2, H. Ludwig1 1Department of Medicine I, Klinik Ottakring, Wilhelminen Cancer Research Institute, Vienna, Austria; 2Step Pharma, Saint-Genis-Pouilly, France; 3National Cancer Institute, Center for Cancer Research, Lymphoid Malignancies Branch, National Institutes of Health, Bethesda, United States of America; 4Department of Medicine I, Division of Oncology, Medical University of Vienna; 5Comprehensive Cancer Center, Vienna, Austria Background: Recent years have seen major improvements in the treatment of myeloma; however, relapse occurs in the majority of patients. Novel treatment approaches are moving away from cytotoxic chemotherapy towards targeted therapies with defined mechanisms of action. Malignant cells have an increased demand for key cellular components and a reliance on de novo synthesis pathways. CTP synthetase 1 (CTPS1) plays a pivotal role in pyrimidine production, by catalysing the rate limiting step in the de novo synthesis of CTP, which is required for DNA, RNA and phospholipids. Human genetic studies have identified an essential and non-redundant role for CTPS1 in lymphoid cell proliferation which is complemented by the homologous CTPS2 isoform outside the haemopoietic system. Aims: To evaluate the role of CTPS1 as a novel target in myeloma, elucidate the molecular consequences of CTPS1 inhibition and test rationally designed combination therapy. Methods: STP938 is a potent small molecule CTPS1 inhibitor with >1,300-fold selectivity for CTPS1 over CTPS2. The effects of STP938 on cell proliferation (metabolic activity, tetrazolium indicator), apoptosis (annexin V) and cell cycle (propidium iodide) were assessed in vitro using 12 myeloma cell lines. The role of CTPS1 was further assessed in CRISPR knock-out (KO) experiments. Activation of proteins in the replication stress and DNA damage response pathways was analysed by western blotting. Additive/synergistic effects of combining STP938 with two different ATR inhibitors were assessed on cell line proliferation and enumerated by Bliss score (Synergyfinder 2.0). Results: STP938 showed single agent activity against 6 of 12 cell lines tested, with IC50 values for sensitive lines ranging from 19 to 128 nM (Figure A). STP938 showed cytotoxic activity against sensitive lines, evidenced by induction of apoptosis. KO experiments confirmed that MM cell lines depend on CTPS1 for proliferation. Exposure to STP938 was associated with accumulation of cells in S phase, induction of a replication stress response as evidenced by activation (phosphorylation) of CHEK1, and induction of the DNA damage response pathway as evidenced by activation of CHEK2 (albeit to a lesser extent than activation of CHEK1). Importantly, S phase accumulation, induction of replication stress and induction of DNA damage response was observed in cell lines where STP938 did not produce significant anti-proliferative activity (Figure B). Given the ability of STP938 to activate CHEK1, STP938 was tested in combination with an ATR inhibitor, as ATR is the main upstream activator of CHEK1. The ATR inhibitor ceralasertib demonstrated additive or synergistic activity when combined with STP938 (Figure C). Notably, synergy between STP938 and ATR inhibition, along with induction of apoptosis, was observed in cell lines resistant to single agent STP938. Results were confirmed with a second ATR inhibitor (VE-821, Figure C). Image: Summary/Conclusion: Inhibition of CTPS1 by single agent STP938 showed anti-proliferative activity against 6 of 12 myeloma cell lines, and induced replication stress in all cell lines. Combined STP938 and ATR inhibition showed anti-proliferative activity in all cell lines, including those resistant to single agent STP938. These data suggest a model whereby myeloma cells with high background replication stress are sensitive to killing by single agent STP938, whereas cells with lower background replication stress are sensitised by STP938 to cell death induced by ATR inhibition (Figure D). STP938 will shortly enter clinical development for patients with late stage lymphoid neoplasms. P865: BIOMARKER ANALYSIS TO SUPPORT DOSE OPTIMIZATION OF IBERDOMIDE AS MONOTHERAPY AND IN COMBINATION WITH STANDARD OF CARE TREATMENTS FOR MULTIPLE MYELOMA FROM A PHASE 1/2 TRIAL M. Amatangelo1,*, Y. Cheng1, W. Pierceall1, N. W. van de Donk2, S. Lonial3, M. Wang1, J. Emerson1, K. Hong1, P. Maciag1, T. Peluso4, A. Gandhi1, A. Thakurta1 1Bristol Myers Squibb, Princeton, NJ, United States of America; 2Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Department of Hematology, Cancer Center Amsterdam, Amsterdam, Netherlands; 3Winship Cancer Institute, Emory University, Atlanta, GA, United States of America; 4Celgene International Sàrl, a Bristol-Myers Squibb Company, Boudry, Switzerland Background: Iberdomide (IBER), a novel cereblon E3 ligase modulator (CELMoD®) compound, induces ubiquitination and proteasome-dependent degradation of Ikaros and Aiolos proteins, resulting in tumoricidal and immunomodulatory activity in multiple myeloma (MM). IBER is being investigated for the treatment of relapsed/refractory MM (RRMM) in a phase 1/2 study (NCT02773030). Due to the recent focus by health authorities on dose selection for oncology products other than the maximum tolerated dose (MTD), a comprehensive analysis of pharmacodynamics (PD) and pharmacokinetics (PK) was implemented to support dose optimization of IBER. Aims: To inform dose selection of IBER as monotherapy, and in combination with dexamethasone (DEX; Iber+d) and with DEX and daratumumab (DARA; IberDd). Methods: PK samples were collected on treatment of cycles (C) 1–4 to estimate IBER exposure using population PK (area under the concentration curve over 24-h dosing interval [AUCτ]). Biomarkers included analysis of peripheral blood samples on C1 day (D)1 and mid-cycle through C4 for immunophenotyping, absolute neutrophil counts (ANC), and assessment of serum free light chain (sFLC), as a biomarker of tumor burden. Bone marrow samples were collected at screening and C2D15 for immunohistochemistry. Results: IBER AUCτ increased in a dose-proportional manner between 0.3 and 1.0 mg as monotherapy and 0.3 and 1.6 mg in combination with DEX, with moderate variability. Median time to maximum serum concentration of IBER was 2–4 h post dose with an elimination half-life of ~12 h. Comparable IBER exposure was observed between monotherapy and Iber+d. Reductions in Ikaros/Aiolos protein levels in tumor cells were observed at all dose levels in both cohorts with a >90% reduction at the 0.45-mg dose. However, a >50% decrease in sFLCs was observed only at doses ≥0.9 mg, and doses of 1.6 mg induced faster and deeper decreases vs lower doses. In the immune compartment, IBER treatment induced similar dose-/exposure-dependent PD changes as monotherapy and Iber+d, which appeared to saturate at higher doses. A >80% reduction in mature B cells and ~2-fold increase in proliferating/activated T and NK cells were observed at doses ≥1.0 mg. PD activity of IBER was not attenuated by prior refractoriness to immunomodulatory drugs (IMiD® agents) or anti-CD38 therapy. Based on these results, doses between 1.0 and 1.6 mg were tested in the IberDd cohort. PK and PD of IBER were similar with concomitant administration of DARA. In this cohort, a reduction in mature B cells and sFLCs was more consistently observed at 1.6 mg vs lower doses; however, higher IBER exposures were associated with more pronounced decreases in ANC when compared with Iber+d. This was clinically manageable and the MTD of IBER was not determined in any cohort. Summary/Conclusion: Similar PK and PD results were observed in patients treated with IBER monotherapy, Iber+d, and IberDd. Across all cohorts, a >20% difference in dose was needed for meaningful change in IBER exposure, immune PD began to saturate at doses ≥1.0 mg, and decreases in tumor burden were greatest at the 1.6-mg dose. Based on these results, IBER doses of 1.0, 1.3, and 1.6 mg were chosen for dose optimization of IberDd for RRMM. Taken together with data suggesting immune surveillance is a driver of maintenance therapy efficacy and a desire to increase IBER tolerability with long-term treatment, doses of 0.75, 1.0, and 1.3 mg were selected for dose optimization of IBER monotherapy in the newly diagnosed MM maintenance setting. P866: CORRELATIVE ANALYSIS TO DEFINE PATIENT PROFILES ASSOCIATED WITH MANUFACTURING AND CLINICAL ENDPOINTS IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS TREATED WITH IDECABTAGENE VICLEUCEL (BB2121) J. Rytlewski1,*, J. Fuller1, D. R. Mertz1, C. Freeman2, S. Manier3, N. Shah4, T. B. Campbell1 1Bristol Myers Squibb, Princeton, NJ; 2Moffitt Cancer Center, Tampa, FL, United States of America; 3CHU de Lille, University of Lille, Lille, France; 4University of California San Francisco, San Francisco, CA, United States of America Background: The anti-B-cell maturation antigen (BCMA) autologous chimeric antigen receptor (CAR) T cell therapy idecabtagene vicleucel (ide-cel; bb2121) has achieved deep and durable responses in relapsed/refractory multiple myeloma (RRMM) patients (Munshi, N Engl J Med 2021). Prior studies have described the correlation between patient characteristics and clinical outcomes from CD19 CAR T cell therapy (Finney, J Clin Invest 2019; Fraietta, Nat Med 2018). Aims: We sought to define patient profiles correlated with manufacturing and clinical endpoints in RRMM patients treated with ide-cel in clinical trials using unsupervised clustering and multivariate machine learning across multiple key variable domains. Methods: Clinical and manufacturing data were harmonized across 164 RRMM patients treated with ide-cel in KarMMa (NCT03361748) and KarMMa-2 cohort 1 (NCT03601078). Based on individual multivariate importance, 10 selected peripheral blood mononuclear cell (PBMC), drug product (DP), and in-process cell growth variables were used to define patient clusters. Random forest classifiers were generated to identify patient characteristics associated with manufacturing and clinical endpoints. Wilcoxon rank sum and Kruskal-Wallis tests were used to compare 2 and 3+ categorical groups; Cox proportional hazards were used to compare groups with time-to-event data. Results: Using an unsupervised method, 4 patient clusters were identified that represented 10%, 57%, 15%, and 18%, respectively, of the total 164 patients. Cluster 4 was the most favorable and was characterized by a higher frequency of T cells in PBMCs; increased T-cell size during manufacturing; higher DP T-cell transduction, potency, and vector copy number; and ultimately a > 3-fold higher CAR T cell yield compared with the least favorable cluster 1. Cluster 2 contained most patients and was associated with intermediate manufacturing endpoints. Patients in cluster 4 had a higher complete response rate, longer progression-free survival, and were defined by lower tumor burden, higher absolute lymphocyte count (ALC), and longer washout period after alkylator treatment, among others (Table). Image: Summary/Conclusion: The current study identified patient profiles in RRMM using accessible laboratory or medical history data that correlated with longitudinal outcomes. These findings may inform patients likely to achieve improved outcomes with CAR T cell therapy. P867: TUMOR PROFILING OF IDECABTAGENE VICLEUCEL (IDE-CEL; BB2121) PATIENTS IN KARMMA SHOWED COMPARABLE RESPONSES IN EXISTING MOLECULAR HIGH-RISK SUBSETS AND PRELIMINARY GENE SIGNATURE OF DURABLE RESPONSE N. Martin1,*, A. Xu**1, N. Stong**1, J. Rytlewski1, O. Finney2, T. Campbell1, W. Pierceall1, E. Flynt1, E. Thompson1, S. Kaiser1 1Bristol Myers Squibb, Princeton, NJ; 22seventy bio, Boston, MA, United States of America Background: Multiple myeloma (MM) tumors exhibit increasing prevalence of high-risk/resistance (HR) features with each successive relapse, leading to poorer outcomes in late-line patients (pts). This may arise primarily as malignancy heterogeneity increases, and with selective pressure from successive treatment regimens. Idecabtagene vicleucel (ide-cel; bb2121) was the first approved anti-BCMA CAR T therapy in late-line relapsed/refractory MM (RRMM), and CAR Ts represent a novel mechanism of action (MOA) in the MM treatment landscape. In the KarMMa study (NCT03361748), high incidences of overall responses (OR) to ide-cel were observed, including in the pt subgroup with HR cytogenetic features. Aims: Assess baseline multi-omics molecular profiles in KarMMa pt tumors to characterize known and novel molecular features and ide-cel outcomes. Evaluate post–ide-cel tumor composition by analyzing paired pt specimens (pretreatment and at relapse). Methods: Bone marrow aspirates were collected from KarMMa pts at pretreatment (n=97 RNA, n=68 DNA) and progression (n=64 RNA, n=33 DNA). RNA sequencing and whole genome sequencing was performed on CD138+ cells. Known molecular HR genomic (biallelic p53 inactivation, high cancer clonal fraction del17p, HR t(4,14), and cereblon mutation) and transcriptomic (MDMS8 gene signature) features were analyzed, and correlations with OR and progression-free survival (PFS) evaluated. BCMA mutation and copy number variation were evaluated. Differential gene expression and principal component analyses explored novel transcriptomic signatures and response. Results: Molecular HR features were identified in 44% (43/97) of pts at pretreatment and 48% (31/64) at progression. Some tumors had multiple HR features consistent with heterogeneity in late-line RRMM. Paired pre/post–CAR T samples did not show dominant enrichment for ≥ 1 HR features at progression. OR and PFS were similar in those with vs. without each molecular HR feature analyzed. One principal component (PC4) was correlated with PFS (p=0.002). The top weighted genes in PC4 may be a novel signature associated with durable ide-cel responses, and exploratory analyses of this signature are ongoing. Loss of one copy of BCMA was observed in 4% (3/68) and 12% (4/33) of pts at pretreatment and progression, respectively. Biallelic loss of BCMA at progression was observed in 6% (2/33) of pts, one of whom had single copy number loss pretreatment. Summary/Conclusion: Baseline multi-omic–based tumor HR features were not associated with OR. This finding suggests ide-cel activation and expansion remains a critical axis for OR that may not be substantially influenced by tumor intrinsic features. Biallelic loss of BCMA was not observed pretreatment and infrequently at progression, consistent with previous reports. Baseline HR features did not correlate with PFS, and a dominant selection for any one HR feature at progression was not noted. These observations were consistent with previous reports in non-molecularly defined HR subgroups from KarMMa and the hypothesis that CAR T MOA may have a broader spectrum of clinical activity across more diverse molecular subtypes of pts, especially existing HR subsets; this could be a consideration as CAR Ts are developed in earlier lines of therapy. A transcriptional signature was associated with more durable ide-cel responses, which we postulate may outline a distinct suboptimal pretreatment feature in an ide-cel context; further exploration of this preliminary signal is ongoing. **Authors contributed equally to this abstract. P868: A CIRCULATING SERUM MIRNAS-BASED MODEL TO PREDICT EARLY MORTALITY IN MULTIPLE MYELOMA PATIENTS TREATED WITH BORTEZOMIB-BASED REGIMENS. A. Puła1,2,*, P. Robak2,3, D. Jarych4, D. Mikulski2,5, I. Dróżdż6, J. Szemraj7, T. Robak1,2 1Department of Hematolgy, Medical University of Lodz; 2Copernicus Memorial Hospital; 3Department of Experimental Hematology, Medical University of Lodz; 4Laboratory of Virology, Institute of Medical Biology, Polish Academy of Sciences; 5Department of Biostatistics and Translational Medicine; 6Department of Clinical Genetics; 7Department of Medical Biochemistry, Medical University of Lodz, Lodz, Poland Background: Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow. Despite the progress made in the treatment of MM, some patients die within the first year of diagnosis. Numerous studies underline the role of miRNAs in the pathogenesis of MM and their potential role in prognosis. Aims: The aim of this study was to analyze the expression of selected miRNAs in the serum of MM patients treated with bortezomib-based regimens and determine their potential to predict early mortality. Methods: The study was conducted in prospectively-recruited patients with newly-diagnosed MM who were qualified for bortezomib-based treatment regimens. All were admitted to the Department of Hematology, Medical University of Lodz between 2017 and 2021. Venous blood samples were collected from the patients before treatment initiation, processed, and stored for further use. Total RNA, including miRNA, was isolated from serum, and reverse-transcribed to cDNA. The expression of 31 selected miRNAs was determined using a miRCURY LNA miRNA Custom PCR Panel. The miRNA selection was based on the results of our previous study (Robak et al., Cancers Cancers (Basel) 2020; 12(9): 2569). Three miRNAs were used for expression normalization: hsa-miR-23b-3p, hsa-miR-151a-5p and hsa-miR-152-3p. Clinical data, including patient characteristics on diagnosis, treatment regimen, response to treatment and follow-up were obtained from hospital records. Differential expression analysis, univariate and multivariate logistic regression models were performed using R version 3.6.3. Results: A total of 69 MM patients were included in the study with a mean age at diagnosis of 64.9 ± 11.0 years. Among them, 17 patients experienced death within 12 months of diagnosis. Patients with early mortality were significantly older (72.6 vs. 62.3 years, p=0.0005), and more frequently men (70.6% vs. 42.3%, p=0.0429). No differences were observed in R-ISS distribution or CRAB symptoms between the groups, nor were any significant differences between percentage myeloma infiltration in the bone marrow and various laboratory findings, including LDH, serum M protein, albumin, CRP, and uric acid. In the differential expression analysis, two miRNAs were significantly downregulated in early mortality group- hsa-miR-328-3p (fold change- FC: 0.72, p=0.0342) and hsa-miR-409-3p (FC: 0.49, p=0.0357). Similarly, hsa-miR-328-3p (OR 0.44, 95%CI: 0.20-0.97, p=0.0415) and hsa-miR-409-3p (OR 0.69, 95%CI: 0.48-0.98, p=0.0400) were found to have a protective effect against early mortality in univariate analyses. The multivariate logistic regression analysis found that miRNAs retained their significance when established clinical prognostic factors were included. The final model consisted of hsa-miR-409-3p (OR 0.61, 95%CI: 0.37-0.99, p=0.0480), hsa-miR-328-3p (OR 0.33, 95%CI: 0.13-0.87, p=0.0254), age (OR 1.13, 95%CI: 1.03-1.23, p=0.0096) and R-ISS 3 (OR 2.91, 95%CI: 0.63-13.47, p=0.1723). A receiver operating characteristics (ROC) analysis for the model yielded an area under the curve (AUC) of 0.863 (95%CI: 0.761-0.965). At the optimal cut-off value of 0.28, the sensitivity and specificity reached 88.2% and 77.5%, respectively. Image: Summary/Conclusion: hsa-miR-409-3p and hsa-miR-328-3p are independent factors related to early mortality in MM patients. Our results were used to generate a multiple regression model that may have the potential to predict early mortality. Further external validation of our model is necessary to confirm its clinical value. P869: PRODUCTION BY GUT MICROBIOTA AS PROGNOSTIC BIOMARKER IN MM A. Rodríguez-García1,*, R. Garcia-Vicente1, M. L. Morales1, R. Gómez-Gordo2, P. Justo1, C. Cuéllar1, J. Sánchez-Pina1, D. Gómez-Garre1, J. Martínez-López1, M. Linares3 1Department of Hematology, Hospital Universitario 12 de Octubre, Hematological Malignancies Clinical Research Unit H120-CNIO; 2Microbiota and Vascular Biology Laboratory, Hospital Clínico San Carlos-Instituto de Investigación Sanitaria San Carlos (IdISSC); 3Department of Biochemistry and Molecular Biology, Universidad Complutense de Madrid, Madrid, Spain Background: There is increasing evidence compelling that the microbiota has a strong impact on cancer. The presence of these microorganisms and their activity could have an impact on immune cells and the bone marrow, playing an important role in the progression of multiple myeloma (MM). The short-chain fatty acids (SCFAs) metabolites from gut microbiota have been linked to beneficial effect in the response of MM to treatment. Aims: In this work we explore whether microorganisms and metabolites of the gut microbiota are imbalanced in monoclonal gammopathies and their possible role in the development of MM. Methods: We collected 51 patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 12), smoldering (SMO) (n=7) and MM at diagnosis (MMdx) (n = 11), at relapse (MMrf) (n=7) and at complete remission (CR) (n = 7, 3 of them paired with their diagnostic stage). Furthermore, healthy patients (n = 8) were included as controls (C). After the extraction of the microbial DNA from fecal samples, the ribosomal subunit of the bacterial 16S gene was sequenced on the Illumina® MiSeq ™ and bioinformatic analysis and quantification of absolute abundance were carried out. The analysis of metabolites released by the gut bacteria was performed by the quantification of SCFAs acetate, butyrate and propionate on serum samples by MRM (LC-QQQ-MS). The effect of SCFAs on the proliferation of human MM tumor cell lines was evaluated using standard MTT assays. Results: Patients with MMrf showed the lowest estimated richness with significant differences in Alpha diversity measured by Pielou’s index (Fig 1A). Beta diversity clustered separately and was significant in patients with MM at complete remission compared to MMdx in paired samples (Fig. 1B). Then, we compare the taxonomic composition of the gut microbiota of patients at different stages of the disease, and we found significant differences in the relative abundance of some microorganisms. On one hand, the genus Butyricimonas decreased in MM and its preclinical stages compared to controls and the genus Blautia was lower at MMdx compared to MMcr patients. Both microorganisms are butyrate producers which suggests a possible beneficial role of these microorganisms. In contrast, some genus such as Lachnoclostridium were increased in MMdx (Fig. 1C). To delve into the functional profiles of the microbial communities, we predicted metabolic pathways based on taxonomic profiles and we observed an increased in pathways involving the SCFA propionate in MMcr compared to MMrf (Fig. 1D). The SCFA butyrate was found decreased in MMdx compared with MGUS patients in serum levels (Fig. 1E). The in vitro assays with MM cell lines showed that the SCFAs acetate, butyrate and propionate had an antiproliferative effect (Fig. 1E). Image: Summary/Conclusion: Our study showed differences in the abundance levels of gut microbiota both on the progression and the response of the MM disease. The presence of some microorganisms and their metabolites, such as SCFAs, suggests a beneficial role which could be exploited as biomarkers of prognosis. P870: DEFINING A NOVEL FUNCTION FOR THE POST-TRANSLATIONAL MODIFICATION UFMYLATION IN THE ADAPTIVE RESPONSE TO ARGININE DEPRIVATION IN MULTIPLE MYELOMA A. Romano1,*, G. Scandura1, C. Giallongo2, E. La Spina1, A. Barbato1, D. Tibullo3, K. Todoerti4, A. Neri4, G. A. Palumbo2, F. Di Raimondo2 1Dipartimento di Chirurgia e Specialità Medico Chirurgiche; 2DIPARTIMENTO DI SCIENZE MEDICHE, CHIRURGICHE E TECNOLOGIE AVANZATE; 3DIPARTIMENTO DI SCIENZE BIOMEDICHE E BIOTECNOLOGICHE, Università degli Studi di Catania, CATANIA; 4Department of Oncology and Hemato-oncology, University of Milan, Milano, Italy Background: Our previous work showed that arginine deprivation is associated to multiple myeloma (MM) bortezomib refractoriness, but the molecular basis of this adaptive response is still largely unknown. Aims: We designed an integrative transcriptional and metabolic study to explore the contemporary transcriptional and metabolic changes occurring upon arginine deprivation in four human myeloma cell lines (HMCLs, U266, NCI-H929, OPM2 and MM1.s). Methods: HMCLs were individually cultured with customized complete with different Arg concentrations, correspondent respectively to 100%, 25% and 10% of the arginine concentration in MM bone marrow. After 24-48 hours of culture, the cells were collected for global metabolomic analysis (Metabolon Inc) and transcriptome profiling by Illumina platform. Apoptosis and mitochondrial depolarization were measured by FACS. Protein expression was evaluated by western blot analysis. Results: In all tested HMCLs the progressive arginine deprivation (range 1124-0 µM) induced delay in cell cycle until proliferation arrest (in total lack of arginine in culture media), with increase of G0-G1 length, at specific timepoints for each cell line tested. However global protein synthesis was not affected and surprisingly, in all tested cell lines, the monoclonal component secretion in extracellular medium was preserved after 96 hours of arginine starvation. At 48 hours of arginine deprivation (range 1124-100 µM) GCN2 pathway was induced in HMCLs with consequent increase of basal levels of ATF4, p62, GABARAP and autophagy flux. After 48 hours of arginine starvation, coupled metabolomics and RNA-seq, disclosed an impairment in the pool of nucleotides and activation of salvage purine pathway, that could explain changes in cell cycle and proliferation. Arginine-deprived MM cells had higher levels of choline and phospho-ethanolamine with lower levels of glycerophosphorylcholine (GPC) compared to controls suggesting an effect of arginine-deprivation on membrane dynamics, associated to increased production of fatty acids, palmitate (16:0) and stearate (18:0), required for the generation of new membranes. Culture in arginine deficient media resulted in complete depletion of reduced glutathione (GSH) and oxidized glutathione (GSSG), with depletion of gamma-glutamylcysteine, indicating increased levels of oxidative stress. Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements, involving upregulation of genes required for the unfolded protein response, NF-kB response to TNF, p53 pathway and networks and proteosome degradation, with a minimal effect on metabolism features, except the upregulation of genes involved in lactate generation and degradation. In all cell lines, transcripts involved in the UFMylation pathway were affected, with reduction of UFSP2 and increase of UFMYlation targets RPL26, PKM2, ENO-1, PGK-1 and SQSTM1/p62. In a large series of primary samples (9N - 20MGUS - 33SMM - 170MM - 24PPCL - 12SPCL) and 18 MM cell lines, through bioinformatic analysis of previously published datasets (GENE1.0_BAv20_Neri-Gutierrez), we found that myeloma progression was associated to increased expression of UFMYlation targets ENO-1, PGK1 and DLD. Summary/Conclusion: Taken together, our data clearly indicate how arginine starvation can induce an heterogeneous adaptation processes occur in individual tumour clones. The ER stress adaptive response triggered by arginine deprivation promotes UFMylation activation, due to downregulation of UFSP2 and accumulation of RPN1/RPL26 targets. P871: ISB 1442, A FIRST-IN-CLASS CD38 AND CD47 BISPECIFIC ANTIBODY INNATE CELL MODULATOR FOR THE TREATMENT OF CD38 POSITIVE HEMATOLOGICAL MALIGNANCIES C. Grandclement1, C. Estoppey1, E. Dheilly1, M. Panagopoulou1, E. Martini1, V. Labanca1, S. De Angelis1, J. Frei1, A. Drake1, A. Rubod1, G. Gudi2, V. Udupa3, J. Koch Olsen4, R. Giovannini4, M. A. Doucey1, E. Feldman2, C. Konto2, A. Srivastava1, M. Perro1, S. Sammicheli1,* 1Ichnos Sciences, Epalinges, Switzerland; 2Ichnos Sciences, New York, United States of America; 3Glenmark Pharmaceuticals Limited, Mahape, India; 4Ichnos Sciences, La Chaux-de-Fonds, Switzerland Background: ISB 1442 is a first in class 2 + 1 biparatopic bispecific antibody targeting CD38 x CD47 using the BEAT® 2.0 (Bispecific Engagement by Antibodies based on the TCR) platform. The CD38 binding arm consists of bi-paratopic Fabs that strongly bind to CD38 in two different epitopes on tumor cells and do not functionally compete with daratumumab. The anti-CD47 arm comprises a single Fab arm designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes (including macrophages, monocytes and dendritic cells). With this design, the high affinity anti-CD38 Fab arms preferentially drive binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Aims: ISB 1442 was developed for the treatment of hematologic malignancies that express CD38, including multiple myeloma (MM), acute myeloid leukemia (AML), and T-acute lymphoblastic leukemia (T-ALL), with the intention of overcoming mechanisms of resistance to CD38-targeted therapies such as daratumumab and isatuximab, and minimizing hemagglutination/hemolysis on red blood cell (RBC) observed with anti-CD47 monoclonal antibodies (mAb) such as magrolimab. Methods: ISB 1442 was tested for its capacity in vitro to induce ADCP, ADCC and CDC relative to daratumumab and magrolimab across a broad range of MM, AML and T-ALL cell lines expressing different levels of CD38 and CD47. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells (ADCC), autologous macrophages (ADCP), and complement from human serum (CDC). In vivo, ISB 1442 was assessed in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice compared to daratumumab and magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. Results: In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing MM, AML and T-ALL tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. In the CDC, ADCC and MMoAK assays, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab in MM cell lines. In vivo, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. In monkeys, ISB 1442 showed more pronounced binding on RBC than magrolimab due to higher expression of CD38, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Summary/Conclusion: In summary, we report a novel approach for the treatment for CD38 positive hematologic malignancies by co-targeting CD38 and CD47 using a first in class multispecific antibody. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity in patients relative to anti-CD38 mAbs by overcoming primary and acquired tumor escape mechanisms of resistance. P872: DEL(1P32) REMAINS A POWERFUL PROGNOSTIC FACTOR IN A LARGE COHORT OF NDMM PATIENTS: AN UPDATE A. Schavgoulidze1,2,*, A. Perrot2,3, T. Cazaubiel4, X. Leleu5, S. Manier6, L. Buisson2,7, S. Maheo2,7, L. Do Souto Ferreira2,8, R. Lannes7,9, L. Pavageau2,7, C. Hulin4, J.-P. Marolleau10, L. Voillat11, K. Belhadj12, M. Divoux13, B. Slama14, S. Brechignac15, M. Macro16, A.-M. Stoppa17, L. Sanhes18, F. Orsini-Piocelle19, J. Fontan20, M.-L. Chretien21, H. Demarquette22, M. Mohty23, H. Avet-Loiseau2,7, J. Corre2,7 1Unit for Genomics in Myeloma, IUCT-Oncopole; 2CRCT INSERM U1037 and Paul Sabatier University; 3Hematology Department, University Hospital IUC-Oncopole, Toulouse; 4Hematology Department, University Hospital, Bordeaux; 5Hematology Department, University Hospital, Poitiers; 6Hematology Department, University Hospital, Lille; 7Unit for Genomics in Myeloma, University Hospital IUC-Oncopole; 8Unit for Genomics in Myeloma, University Hospital, Toulouse, France; 9Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States of America; 10Hematology Department, University Hospital, Amiens; 11Hematology Department, Hospital, Chalon-sur-Saône; 12Hematology Department, University Hospital, Créteil; 13Hematology Department, University Hospital, Nancy; 14Hematology Department, Hospital, Avignon; 15Hematology Department, University Hospital, Bobigny; 16Hematology Department, University Hospital, Caen; 17Hematology Department, Institut Paoli Calmettes, Marseille; 18Hematology Department, Hospital, Perpignan; 19Hematology Department, Hospital, Annecy; 20Hematology Department, University Hospital, Besançon; 21Hematology Department, University Hospital, Dijon; 22Hematology Department, Hospital, Dunkerque; 23Hematology Department, Saint-Antoine University Hospital, Paris, France Background: Multiple myeloma (MM) is the second hematological malignancy in the Western countries. Currently, the Revised International Staging System (R-ISS) is widely used to assess patients’ prognosis. Cytogenetic abnormalities (CA) affecting the chromosome 1, gain 1q and del(1p32), were not included in the new criteria of HR CA despite their relatively high frequencies (respectively 35% and 11%), due to insufficient data in the study. However, we have recently confirmed the significant impact on del(1p32) on myeloma‘s prognosis, being the second most pejorative abnormality, just after del(17p). Aims: The aim of this study is to update our data about the prognostic impact of del(1p32) on a large cohort of NDMM patients. Methods: Clinical data were obtained from 2551 NDMM patients, followed up for ≥ 36 months or having died or progressed within 36 months post treatment. Informed consent was obtained for all included patients. 1258 patients were treated by an intensive therapy. Follow-up duration was estimated using reverse Kaplan-Meier method. Overall survival (OS) and progression free survival (PFS) curves were estimated using the Kaplan-Meier method and were compared using the log-rank test. Tests were two-sided, and P < .05 was considered significant. All analyses were performed using R version 4.1.1. Results: We observed 12.4% of patients displaying del(1p32), which was the expected proportion. Median follow-up was 67.4 months. The OS of patients harboring del(1p32) was twice as short as the OS of patients without del(1p32) (60.4 and 123.9 months, respectively, P < 0.0001) (Figure 1). Likewise, PFS was significantly shorter in del(1p32) patients (18.1 and 29.1 months, respectively, P < 0.0001). These poorer outcomes were observed even in patients treated with an intensive therapy (del(1p32) vs. no del(1p32); PFS 26.5 vs. 37.0 months, P < 0.0001; OS 72.0 vs. 127.4 months, P < 0.0001). We observed higher frequencies of del(17p) and gain 1q in del(1p32) patients (21.3% and 53.2% respectively), compared to general MM population. To check if the poor survival is not only due to these higher levels of association, we have decided to focus on patients harboring del(1p32) without the main high-risk (HR) CA. HR CA were defined by the presence of del(17p), t(4;14) and/or gain 1q. Patients without HR CA had a lower PFS and OS when they carried del(1p32) (del(1p32) vs. no del(1p32); PFS 25.6 vs. 34.8 months, P = 0.0004; OS 83.0 vs. 136.1 months, P = 0.0002). It is now widely admitted that cumulating HR CA worsen the prognosis. Thus, we have assessed the effect of additional CA on the prognosis of del(1p32) patients. Additional CA were CA defined as HR CA above. As expected, the overall survival of del(1p32) patients significantly decreases when this abnormality was associated with other CA (OS: del(1p32) alone 83.0 months, del(1p32) with 1 HR CA 45.8 months, del(1p32) with 2 HR CA or more 36.5 months, P < 0.0001). Image: Summary/Conclusion: Here we have confirmed the pejorative impact of del(1p32) in multiple myeloma on the largest cohort of NDMM patients ever evaluated to our knowledge (316 del(1p32) patients). Our results demonstrate the importance of the detection of del(1p32) at diagnosis because of its huge impact on the prognosis, especially in the era of risk-adapted treatment strategy. The multivariate analysis is in progress. P873: CARFILZOMIB RESISTANCE IS ASSOCIATED WITH SIGNIFICANT DEREGULATION OF THE BH3 FAMILY PROTEINS A. Schneller1,*, A. Bolomsky1, N. Zojer1, M. Schreder1, H. Ludwig1 1Clinic Ottakring, Department of Medcin I, Wilhelminen Krebsforschung, Wien, Austria Background: Despite the remarkable clinical activity of PIs (Proteasome inhibitors) in MM (multiple myeloma), most patients ultimately relapse and become PI-resistant. Deregulation of the apoptosis pathway has been identified as a possible cause of drug resistance in tumor cells. The intrinsic apoptosis pathway is regulated by the BCL2 protein (BH3) family. Previous studies indicated a substantial role of the BH3 family in PI resistance and in response to PI. For instance, the sensitivity to the BCL-2 inhibitor venetoclax is increased in PI resistant AMO-1 cells and a deregulation of BH3 proteins Noxa and MCL1 was noted in MM cell lines exposed to bortezomib. These data suggest that mechanisms involved in PI resistance are closely interconnected with the intrinsic apoptosis pathway orchestrated via the BH3 family, revealing novel opportunities for therapeutic intervention. Aims: Here, we analyze the interplay between BH3 protein family members in PI resistant vs. sensitive cell lines aiming to decipher survival strategies of PI resistance. Methods: MM cell lines were exposed to serially increasing doses (max. 25nM) of carfilzomib for > 1 year, leading to the outgrowth of clones with acquired carfilzomib resistance. Analysis of BH3 protein expression and sensitivity to BH3 mimetics in PI resistant cell lines were performed via western blot and viability assays, respectively. Analysis of BH3 protein dependency was done using the flow cytometry-based BH3 profiling method. Thereby, cytochrome c release was measured upon exposure to peptides specifically binding anti-apoptotic BH3 proteins, allowing to identify the relevance of distinct anti-apoptotic BH3 proteins in the cell line of interest. Apoptosis induction of the cells was analyzed by flow cytometry. Results: Viability assays indicate that all 7 tested carfilzomib resistant MM cell lines developed a cross resistance to the MCL-1 inhibitors S63845 and AZD-5991. Decreased sensitivity to the BCL-2 inhibitor venetoclax as well as to the BCL-xL inhibitor A1331852 was observed in resistant KMS12PE cells. As previously shown, these effects are linked to MDR1 upregulation in carfilzomib resistant cells and reversible by MDR-1 inhibition. BH3 profiling indicates that under naïve conditions the changes in BH3 protein dependency could be observed in 3/7 carfilzomib resistant cell lines. Specifically, resistant KMS12PE cells exhibit a decreased sensitivity to the MCL-1 binding peptide, carfilzomib resistant OPM-2 cells show an increased sensitivity towards peptides binding to BCL-xL and A1 and resistant AMO-1 cells demonstrate a reduced sensitivity a peptide binding to BCL-xL, Bcl-2 and BCL-w. Protein expression analysis of carfilzomib sensitive vs. resistant cell line variants indicate alterations of BCL2 family members (MCL-1, BCL-xL, BCL-2, BAD, BID, BAX, BAK, BIM, NOXA and PUMA) in untreated conditions as well as under carfilzomib treatment (Figure 1). Notably, carfilzomib treatment upregulated BCL-xL in 5 and BAK in 7 out of 7 carfilzomib resistant cell line variants. Image: Summary/Conclusion: Our data show significant deregulation of the BH3 protein family in carfilzomib resistant cell lines highlighting the importance of the apoptosis signaling pathway in PI resistance. This is further emphasized by the coherent deregulation of BCL-xL and BAK throughout carfilzomib exposed carfilzomib resistant cell lines when compared to their sensitive cell line variants. Moreover, BH3 profiling indicates a change in dependency on anti-apoptotic BH3 protein in carfilzomib resistant vs. sensitive MM cell lines. P874: DECIPHERING PLASMA CELL HETEROGENEITY AND TUMOR MICROENVIRONMENT IN LIGHT-CHAIN AMYLOIDOSIS USING SINGLE CELL RNA-SEQUENCING A. Srivastava1,*, R. Kumari1, S. Adhikari1, P. Sergeev1, J. J. Miettinen1, M. H. Suvela1, K. Flanagan2, A. Slipicevic2, N. N. Nupponen2, F. Lehmann2, C. Heckman1 1Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland; 2Oncopeptides AB, Stockholm, Sweden Background: Immunoglobulin light-chain amyloidosis (AL) is a rare disease caused by plasma cell secretion of misfolded light chains that assemble as amyloid fibrils, which deposit on vital organs causing organ dysfunction. Similar to AL, multiple myeloma (MM) is also caused by atypical plasma cell (PC) expansion in the bone marrow. Although, much research has been done on MM to understand disease mechanisms and to develop effective treatments, knowledge related to AL is still limited. Notably, there is little information available regarding the associated tumor microenvironment (TME) of AL. As the TME plays an important role in disease development and treatment response, comprehensive investigation of the AL TME could improve disease diagnosis and treatment. Furthermore, information distinguishing PCs from AL and MM patients is relatively sparse, but could help determine if treatments developed for MM would also be applicable for the treatment of AL. Aims: The main objective of our study was to decipher the underlying disease mechanisms of the AL at cellular and molecular level by analyzing AL patient samples using single cell RNA sequencing (scRNAseq). To achieve this, we aimed to understand heterogeneity within AL vs MM plasma cells and the TME at the cellular and gene expression level. Methods: The Finnish Hematology Research Biobank provided 23 viably frozen bone marrow mononuclear cell (BM-MNC) samples from 22 AL patients and 24 samples from 23 MM patients. The BM-MNCs were thawed and sorted based on their CD138 expression and cell viability (7AAD dead cell marker). The viable CD138+ and CD138- cells were mixed in a ratio where CD138+ were enriched, but not exceeding 50%. The Chromium Single Cell 3’RNAseq run and library preparation were done using Chromium™ Single Cell 3’ Reagent version 3 chemistry. 10x Genomics Cell Ranger v3.0.1 pipelines were used for the initial data processing. Further, the quality control and data preprocessing was performed using Seurat 4.1.0. For cell type annotations to identify the clusters, specific cell type markers for the immune cells were obtained from sctype database, GSEA-MSigDB and manual curation. Results: From the integrated analysis of scRNAseq data from 22 AL patient samples, we identified a total of 27 distinct clusters of cells belonging to 10 unique cell types. Out of the 27 clusters, 10 clusters were identified as PC sub-populations. The proportions of PCs in the samples negatively correlated to the other major cell types such as NK cells, T cells, gamma delta T cells and monocytes. High transcriptional variability was observed between the PC clusters of the AL patients indicating a high degree of heterogeneity among the AL PCs. Analysis of the MM scRNAseq data resulted in 14 PC clusters, however variability between these clusters was not much distinct as compared to AL. A comparison between AL and MM PC clusters resulted in overlapping transcriptional profiles of 2 AL PC clusters with one MM PC cluster. 5 out of 10 clusters of PCs in AL patients were found to be associated with protein processing in the endoplasmic reticulum and apoptosis pathway. Summary/Conclusion: Within the AL TME, we observed negative correlation of major immune cell types with PCs, suggesting that immune cells may contribute to controlling tumor cell burden in AL. In addition, PC sub-populations in amyloidosis patients are very heterogeneous at the inter-individual level compared to MM patients further indicating that AL patients may benefit from more tailored treatment approaches. P875: ACTIVATION OF LNCRNA NEAT1 LEADS TO SURVIVAL ADVANTAGE OF MULTIPLE MYELOMA CELLS BY SUPPORTING A REGULATORY LOOP WITH DNA REPAIR PROTEINS: RATIONAL BASES FOR NEAT1 THERAPEUTIC TARGETING IN THE DISEASE E. Taiana1,2,*, D. Ronchetti1,2, V. K. Favasuli1,2, I. Silvestris1,2, N. Puccio1,2, K. Todoerti3,4, C. Bandini5,6, I. Craparotta7, L. Di Rito7, S. Erratico8,9, D. Giannandrea10, R. Piva5,6, M. Bolis7, Y. Torrente9, R. Chiaramonte10, N. Bolli1,2, L. Baldini1,2, A. Neri1,2 1Oncology and Hemato-oncology Department, University of Milan; 2Hematology, Fondazione Cà Granda IRCCS Policlinico; 3Pathology Department; 4Molecular Oncology 1 Department, IRCCS Istituto Nazionale dei Tumori, Milan; 5Molecular Biotechnology and Health Sciences Department, University of Turin; 6Città Della Salute e della Scienza Hospital, Turin; 7Oncology Department, Mario Negri IRCCS; 8Novystem Spa; 9Unit of Neurology, Fondazione Cà Granda IRCCS Policlinico; 10Health Sciences Department, University of Milan, Milan, Italy Background: Multiple myeloma (MM) is a fatal malignant proliferation of antibody-secreting bone marrow plasma cells characterized by marked genomic instability. Long non-coding RNA (lncRNA) represents the largest class of non-protein coding genes in the human genome. Their crucial role in solid tumor and hematological malignances, including MM, is well documented. NEAT1 is a highly expressed nuclear lncRNA, representing the core structural component of paraspeckle organelles. We previously demonstrated that NEAT1 silencing negatively regulates proliferation and viability of MM cells, both in vitro and in vivo, highlighting its pivotal role in DNA integrity maintenance, by regulating the homologous recombination. Despite the increasing information obtained by loss-of-function approaches, there is still a lack of information regarding possible oncogenic benefits acquired by MM cells upon NEAT1 overexpression. Aims: Taking advantage of the use of the CRISPR/Cas9 SAM genome editing approach, the present study aimed to shed light on the biological and molecular implication of NEAT1 overexpression in MM. Methods: We adopted the CRISPR/Cas9 Synergistic Activation Mediator editing system to transactivate NEAT1 in AMO-1 MM cell line. LNA-gapmeR antisense oligonucleotide technology was used to silence NEAT1 in MM cells by gymnotic delivery. Hypoxia was induced by culturing the cells into a modular incubator chamber flushed with a mixture of 1% O2, 5% CO2 and 94%N2 at 37 °C. NEAT1 expression was assessed by qRT-PCR and RNA-FISH. Cell cycle phases distribution and apoptotic cells percentage were assessed by flow cytometry. Clonogenic potential was evaluated by methylcellulose assay. Protein expression was investigated by WB and IF. Cell morphological analysis was performed after May-Grunwald Giemsa staining. Transcriptomic analysis was performed by RNA sequencing, following the TruSeq Stranded Total RNA protocol. Libraries with optimal quality and quantity were run on NextSeq 500. Statistical significance was determined by Student t test analysis; differences were considered significant when P-value was <0.05. Results: We performed a transcriptomic analysis of AMO-1 cells either transactivated or silenced for NEAT1. Our data revealed a NEAT1 pivotal role in the maintenance of DNA integrity, showing a significant deregulation of almost all the crucial cellular DNA repair mechanisms for both single and double strand breaks. In particular, we found that NEAT1 transactivation affects DNA repair mechanisms through the activation of a molecular axis including two fundamental kinase proteins, i.e. ATM and DNA-PKcs, and the direct target pRPA32. Furthermore, our data strongly confirmed the activation of this NEAT1-orchestrated molecular axis whether MM cells are exposed to nutrient starvation and hypoxia, suggesting its implication in conferring oncogenic and pro-survival properties to MM cells. The disruption of this oncogenic loop leads to MM cell death and the loss of pro survival advantages, thus suggesting that NEAT1 should be considered a crucial factor for MM cells survival. Summary/Conclusion: Overall, we provided novel important insights into the role of NEAT1 in MM pathobiology, demonstrating that its deregulation strongly correlates with adaptation to stress condition, often associated with late stages of the disease. Taken together, our results suggest that NEAT1 could represent Achilles’ heel for MM cells and should be considered a potential therapeutic target for MM treatment. P876: INCIDENTAL DISCOVERY OF PROBABLE PATHOGENIC GERMLINE VARIANTS IN MULTIPLE MYELOMA PATIENTS UNDERGOING SOMATIC GENOMIC PROFILING S. Thibaud1,*, T. Brander2, M. Balwani2, T. Mouhieddine1, D. Melnekoff2, O. Van Oekelen2, A. Lagana2, J. Houldsworth3, A. Chari1, J. Richter1, L. Sanchez1, S. Richard1, C. Rodriguez1, A. Rossi1, H. J. Cho1, S. Jagannath1, K. Onel4, S. Parekh1 1Hematology & Medical Oncology; 2Genetics and Genomic Sciences; 3Molecular Pathology, Icahn School of Medicine at Mount Sinai, New York; 4Sema4, Stamford, United States of America Background: Next-generation sequencing (NGS) is increasingly used to characterize the somatic mutational landscape of patients with multiple myeloma (MM) and guide precision medicine approaches. Somatic genomic profiling (SGP) in cancer patients can lead to the incidental discovery of pathogenic or likely-pathogenic germline variants (PGVs) in ~4% of cases (PMID 26787237). Our previous analysis of whole exome germline data from 895 MM patients in the MMRF CoMMpass dataset showed ~8% of MM patients carry a PGV in a hereditary cancer gene (HCG), and identified shared characteristics among PGV carriers including younger age at diagnosis, higher likelihood of having family history of hematologic malignancy, higher likelihood of t(11;14) MM, and favorable outcomes (Thibaud et al, ASH 2021). Aims: The present study aims to assess the rate of detection of probable PGVs (PPGVs) in MM patients undergoing targeted SGP. Methods: We retrospectively reviewed results of 1,614 commercial NGS panels performed by a single genetic testing laboratory on bone marrow aspirates of 1,095 MM patients treated at our institution since 2015 who were not enrolled in the MMRF CoMMpass study. We considered individual variants to be PPGVs when all of the following criteria were met: 1) variant found in a panel of 106 well-established HCGs (same panel used in our prior study, but excluding TP53 given high rate of somatic mutations in MM); 2) variant unambiguously listed as pathogenic or likely pathogenic (P/LP) in ClinVar, or variant classified as “conflicting interpretations of pathogenicity” but reported by ≥2 CLIA-certified labs as P/LP; 3) variant allele frequency (VAF) >30% (per PMID 33236764); 4) variant detected in all available test reports for a given patient (and VAF >30% in all available test reports). Well known founder variants (FV) in specific populations were labeled as PPGV-FVs. Results: 95 MM patients (8.7%) had a genetic variant meeting criteria to be classified as a PPGV. Two patients had 2 distinct PPGV. PPGV were most commonly detected in the following HCGs (Table 1): APC (n=22), CHEK2 (n=20), ATM (n=7), BRCA1 (n=6), MUTYH (n=6). Of all PPGV carriers, 47 (49%) carried a PPGV-FV. Median VAF for PPGVs was 48.2% (range 30-85%). 34 PPGV carriers had serial NGS tests available (range 2-5 reports); in these patients, VAF standard deviation across serial measurements was <5% in 89% of cases. Taken together, these findings indicate a high likelihood that PPGV detected in these MM patients while undergoing tumor sequencing are indeed incidentally-discovered PGV warranting referral to a genetic counselor for consideration of confirmatory testing. Image: Summary/Conclusion: SGP of bone marrow aspirate in MM patients incidentally detected PPGV in well-established HCG in 8.7% of cases. These findings are consistent with those of our prior study, which found an 8.6% prevalence of PGV in newly-diagnosed MM patients. In MM patients undergoing SGP, detection of a mutation involving a HCG and with a VAF >30% should prompt referral to genetic counseling for confirmatory testing, as PGV detection can have clear implications for patients and their families. P878: HIGH MUTATIONAL BURDEN OF CHROMATIN MODIFYING GENES DEFINES A RELATIVELY INDOLENT SUBGROUP OF PLASMA CELL DYSCRASIAS X. Wang1,*, B. Qiao2, Y. Yu1, J. Xu2, L. Zuo2, J. Chen1, H. Cai1, H. Han1, X.-X. Cao1, C. Sun2, J. Li1 1Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing; 2Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Background: Plasma cell dyscrasias (PCDs), including multiple myeloma (MM), light chain amyloidosis (AL), POEMS syndrome, and MGUS, present different clinical features. But as malignancies of plasma cells, they potentially have common mutational patterns associated with chemotherapy and prognosis, which haven’t been elucidated. Aims: We aimed to reveal the clinically relevant subgroups of MM, AL, POEMS, and MGUS defined by mutational patterns of bone marrow plasma cells (BMPCs). Methods: We conducted targeted gene sequencing including 370 genes of BMPCs from patients with MM (n=163), AL (n=121), POEMS (n=67) and MGUS (n=13). All the subjects provided informed consent. Results: Principal component analysis of the top 100 variable genes revealed 2 major subgroups among patients with PCDs (Fig. 1a). Approximately 25% of MM, AL, POEMS and MGUS, subgroup 1 was featured by the heavily mutated genes enriched in histone modifications and chromatin organization, such as KMT2D, EP400, RUNX1, SOX9, KDM5A, NCOR2, and ARID1B. Subgroup2 showed lower total mutational burden and was featured by mutations of proto-oncogene and B cell differentiation-associated genes, such as NCOR1, APOBEC4, KRAS, TP53, FGFR3, FAM46C, and MYC (Fig. 1b). The high mutational burden of chromatin-modifying genes was exclusively observed in subgroup 1 and was comparable among MM, AL, POEMS, and MGUS (Fig. 1c). Patients in subgroup 1 showed worse responses to the first-line CyBorD for MM and AL (Fig. 1d), but better responses to the lenalidomide-based VRD for MM and Rd for POEMS. Overall, patients in subgroup 1 demonstrated longer progression-free survival (PFS), which was observed in MM, AL (Mayo12, Stage I, II), and POEMS, respectively (Fig. 1e). Image: Summary/Conclusion: These results provide mechanistic insights into a relatively indolent subgroup among patients with MM, AL, POEMS, and MGUS, which is featured by a high mutational burden of chromatin-modifying genes. This subgroup can be identified by detecting the mutations of chromatin-modifying genes in clinical management. Overall longer PFS and better response to lenalidomide-based first-line therapy can be expected. P879: EXENT AND FLC MASS SPECTROMETRY FOR SEROLOGICAL IDENTIFICATION AND QUANTIFICATION OF MONOCLONAL IMMUNOGLOBULINS IN MULTIPLE MYELOMA S. Huhn1,*, A. M. Poos1, E. K. Mai1, M. Hänel2, H. J. Salwender3, J. Dürig4, I. W. Blau5, C. Scheid6, K. C. Weisel7, M. Munder8, O. Berlanga9, U. Bertsch1, H. Goldschmidt1, N. Weinhold1 1Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg; 2Department of Internal Medicine III, Clinic Chemnitz, Chemnitz; 3Asklepios Tumorzentrum Hamburg, AK Altona and AK St. Georg, Hamburg; 4Department for Hematology and Stem Cell Transplantation, University Hospital Essen, Essen; 5Medical Clinic, Charité University Medicine Berlin, Berlin; 6Department of Internal Medicine I, University of Cologne, Cologne; 7Department of Oncology, Hematology and BMT, University Medical Center Hamburg-Eppendorf, Hamburg; 8Third Department of Medicine, University Medicine Mainz of Johannes Gutenberg, Mainz, Germany; 9The Binding Site Group, Ltd, Birmingham, United Kingdom Background: Mass spectrometry has broad potential for screening and monitoring monoclonal gammopathies. The technology overcomes some of the limitations inherent to standard electrophoretic methods for identifying monoclonal immunoglobulins and is amenable to automation. Aims: Here we assess the performance of the EXENT assays for the quantification of M-proteins and whether free light chain evaluation by mass spectrometry (FLC-MS) offers added sensitivity during multiple myeloma monitoring. Methods: The MM5 trial compared two regimens of bortezomib-based induction therapy and of lenalidomide consolidation followed by lenalidomide maintenance in newly diagnosed multiple myeloma patients. We used mass spectrometry for identifying serum M-proteins in samples at baseline (n=434), post-induction (n=340), and post-consolidation (n=294). Briefly, polyclonal antibodies (anti-IgG,-IgA,-IgM,-total κ and -total λ [EXENT, The Binding Site, UK] and anti-free κ and -free λ [FLC-MS]) covalently attached to paramagnetic microparticles were separately incubated with serum, washed, treated to elute and reduce patient immunoglobulins, and spotted onto MALDI plates with HCCA matrix. Spectra were generated using the MALDI-TOF-MS system. Light chain mass spectra were obtained using EXENT software to yield immunoglobulin isotype, mass-to-charge ratio (m/z) and quantity (g/L). Results were compared to serum protein electrophoresis, immunofixation, and FLC ratios (Freelite®, The Binding Site, UK). Results: EXENT demonstrated improved sensitivity over immunofixation for identifying the M-protein at each time-point of the MM5 trial: 99% vs. 95% at baseline; 91% vs 80% post-induction; and 63% vs. 46% post-consolidation. Agreement between methods was 87%. The addition of FLC-MS to EXENT further improved sensitivity to 100%, 95.0%, and 72% at each respective time-point, whereas the combination of immunofixation and FLC ratios resulted in sensitivities of 100%, 90%, and 54%. Overall concordance between EXENT+FLC-MS and the combination of immunofixation and FLC ratios was 100% at baseline, 93% post-induction, and 73% post-consolidation. In all, 158 (11%) samples had an M-protein by EXENT+FLC-MS only, and 28 (2%) by the combination of immunofixation and FLC ratios. 18 and 92 patients achieved an IMWG complete response after induction and consolidation, respectively. EXENT identified an M-protein in 5 (28%), and 23 (25%) of these patients, whereas EXENT+FLC-MS was more sensitive and detected monoclonal proteins in 11 (61%), and 38 (41%) patients, respectively. There was acceptable quantitative agreement for M-protein measurements between EXENT and serum protein electrophoresis (n=631: Passing-Bablok fit: y=0.85x-0.58; coefficient of determination R2= 0.89; Bland-Altman relative bias (95%CI): -32.62% (-123.79-58.55%)). However, there was a substantial discordance in 285 samples <10g/L by serum protein electrophoresis (y=0.58x+0.54; R2=0.28; -44.84% (-179.29 – 84.60%)) compared to 346 samples >10g/L (y=0.85x-0.25; R2=0.82; -19.91% (95%CI: -97.32 – 23.56%)); suggesting overestimation by serum protein electrophoresis at lower levels. Summary/Conclusion: EXENT provides superior performance for the identification of M-proteins throughout monitoring. Sensitivity can be improved by incorporating FLC-MS assessments, thus providing a means for determining serological residual disease in patients at complete response by IMWG criteria. Quantification of the M-protein by mass spectrometry compares well to standard methods and offers accurate results at levels below the analytical threshold of confidence for electrophoretic approaches. P880: OVEREXPRESSION OF PFKFB4 PROMOTES ADAPTATION TO HYPOXIC MICROENVIRONMENT IN MULTIPLE MYELOMA J. Yang1,*, F. Wang1, B. Chen1 1Department of Hematology and Oncology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China Background: Multiple myeloma (MM) is a hematological malignancy with monoclonal proliferation of plasma cells, which is closely related to hypoxic bone marrow microenvironment. Previous studies have found that 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) is involved in glycolysis, cell cycle progression, autophagy, and metastasis in various tumor diseases and overexpression of PFKFB4 is associated with poor prognosis. However, the underlying mechanism of PFKFB4 in hypoxia adaptation in MM has not been reported. Aims: The aim of this study was to explore the role of PFKFB4 in MM cells adaptation to the hypoxic BM microenvironment and provide potential therapeutic target for clinical treatment. Methods: We analyzed expression profile GSE80140 and GSE80545 downloaded from National Center for Biotechnology Information-Gene Expression Omnibus (NCBI-GEO) database, and conducted cell transcriptome sequencing. 5MPN, a specific inhibitor of PFKFB4, was employed to evaluate the effect of PFKFB4 in hypoxia adaptation in MM by cell proliferation, apoptosis, and cycle assays, F-2,6-BP measurement, and Western blot. Results: MM cell lines (RPMI-8226 and U266) was found to express higher level of PFKFB4 under hypoxia (1% O2) compared to normoxia (20% O2). 5MPN inhibited MM cells proliferation, suppressed intracellular F-2,6-BP concentration, and induced cell cycle arrest in G1 phase. Inhibition of PFKFB4 function had no significant effect on inducing cell apoptosis. Western blot detected the expression of proliferation, apoptosis and cell cycle related proteins, and the results were consistent. Summary/Conclusion: Overexpression of PFKFB4 promotes MM proliferation and cell cycle progression. 5MPN effectively reserves the process, suggesting that PFKFB4 is a potential therapeutic target in MM. P881: CDK7 CONTRIBUTES TO METABOLIC REPROGRAMMING IN MM CELLS THROUGH C-MYC MEDIATED TRANSCRIPTIONAL CONTROL OF GLYCOLYTIC GENES Y. Yao1,2, J. Fong Ng1, W. D. Park3, D. Gramegna1, A. Samur1, M. Samur1, E. Morelli1, M. Chesi4, C. Mitsiades1, K. C. Anderson1, C. Lin3, N. Munshi1, M. Fulciniti1,* 1Dana Farber Cancer Institute, Boston, United States of America; 2Xuzhou Medical University, Xuzhou, China; 3Baylor College of Medicine, Houston; 4Mayo Clinic, Scottsdale, United States of America Background: We have recently elucidated the biological role of CDK7 and explore the functional consequence of its inhibition in MM using chemical and genetic approaches. We reported a specific vulnerability in MM cells upon disruption of the CDK7-driven molecular programs, supporting a model where cell cycle and transcriptional deregulation are counteracted by inhibition of CDK7 activity leading to cell cycle arrest and subsequent apoptosis in MM. Importantly, we established that CDK7 positively correlates with c-MYC and E2F1 transcriptional outputs in primary cells from MM patients, suggesting its regulatory role for both transcription factors. Aims: Bioinformatics analyses of RNA-seq data revealed that CDK7 kinase activity is required for the expression of many components of the glycolytic cascade; in contrast, most genes for OXPHOS were only weakly modulated by YKL-5-124. Interestingly, recent studies have identified CDK7 as an activator of glucose consumption in lung cancer cells, and autosomal dominant polycystic kidney disease. These observations prompted us to delve into the potential role of CDK7 in regulating glucose metabolism in MM cells. Methods: CDK7 inhibition was achieved with 1. selective covalent inhibitor YKL-5-124; 2. protein degradation dTAG system; 3. inducible KD/KO systems. The transcriptional networks were evaluated by integrated ChIP-seq and RNA-seq data analysis. Metabolic changes were evaluated by ECAR and OCR assessment using Seahorse analyzer. Results: To evaluate if CDK7 inhibition suppresses glycolysis we assessed control and treated cells during a glycolysis stress test with Seahorse analyzer using extracellular acidification rate (ECAR) as a measure of glycolytic activity. Compared to control, treated cells displayed a significant defect in glycolysis, glycolytic capacity, and glycolytic reserve in a c-MYC dependent manner. These differences in glycolytic activity upon YKL-5-124 treatment were not accompanied by changes in basal oxygen consumption rate (OCR). Similarly to the drug compound, CDK7 protein degradation using dTAG system diminished glycolytic activity, suggesting that CDK7 regulates glycolysis in MM model systems. Evaluation of tumors retrieved from mice after treatment revealed significant reduction in glycolysis rate. Cancer cells catabolize glucose to lactate in aerobic conditions: a sharp time dependent increase in extracellular lactate release was observed in MM cells over time which was suppressed by YKL-5-124 treatment via regulation of lactate dehydrogenase A (LDHA) expression and enzymatic activity. Together with impairment of aerobic glycolysis, YKL-5-124 treatment resulted in increased ROS levels and induction of apoptotic cell death. The cellular level of ROS is an important oxidative molecule causing endogenous DNA damage. We indeed observed induction of DNA damage as measured by investigation of γH2AX levels after YKL-5-124 treatment. To explore whether YKL-5-124 induced cell death by ROS-mediated mechanisms, we examined the effects of the thiol antioxidant N-acetyl-L-cysteine (NAC) on MM cells upon YKL-5-124 treatment. NAC treatment prevented ROS production, cell death, apoptosis and DNA damage accumulation induced by CDK7i, indicating a causative link between increased ROS levels upon CDK7i and tumor cell death. Summary/Conclusion: Our data demonstrate that CDK7 through its ability to link cell cycle, transcription and cellular metabolism affects MM cells at several levels, representing an attractive and therapeutically actionable molecular vulnerability in MM. P882: IDENTIFICATION OF M7G RNA METHYLATION REGULATORS AND M7G-RELATED GENES IN MULTIPLE MYELOMA M. Yu1,*, C. Li2, J. Xu3, M. Liu2, X. Cui2 1Shandong University of traditional Chinese Medicine; 2The Second Affiliated Hospital of Shandong University of traditional Chinese medicine; 3Affiliated Hospital of Shandong University of traditional Chinese medicine, Jinan, China Background: N7-methylguanosine (m7G) is a positively charged, essential modification at the 5’ cap of eukaryotic mRNA, impacting RNA processing and function, especially mRNA sublocation, translation, and splicing, which is crucial for the regulation of tumor progression. However, the m7G modification pattern of multiple myeloma (MM) is still unclear. Aims: To explore the impact of m7G regulators on MM prognosis and further conducted a comprehensive investigation on the differences in m7G-related genes among different m7G modification patterns. Methods: We analyzed the mRNA expression of m7G regulators in healthy donors (n = 9) and MM (n = 174) bone marrow plasma cells, investigated their prognostic values, and comprehensively evaluated the m7G modification patterns of 559 MM bone marrow samples. These data are from Gene Expression Omnibus (GEO) datasets. ConsensusClusterPlus R package (1.56.0) was used to group patients. Results: Compared with healthy donors, the expression of three m7G writers (METTL1, RNMT, and WBSCR22) was significantly upregulated in MM patients (Fig. 1A). The Kaplan-Meier (K-M) survival curve showed that three m7G regulators (METTL1, WDR4 and RNMT) were significantly correlated with survival, and their high expression was associated with shorter overall survival (OS) (Fig. 1B). Based on the expression levels of m7G regulators, MM patients with qualitatively distinct m7G modification patterns were classified into three different groups. The K-M curve analysis showed a significant survival difference among the subgroups, of which cluster A showed a worse outcome than clusters B and C (Fig. 1C), and the expression of m7G regulators was higher in cluster A (Fig. 1D). To further investigate the biological behaviors of these distinct m7G modification patterns, gene set variation analysis (GSVA) was performed. Compared with clusters B and C, cluster A was markedly enriched in cell proliferation-related pathways. Recent studies have found that the m7G cap in eukaryotic mRNAs is necessary to regulate sublocation, translation, and splicing. Therefore, we used the Pearson correlation coefficient to determine the potential m7G-related mRNAs. As a result, 88 interactions and 88 m7G-related mRNAs (absolute correlation coefficient >0.3, P <0.05) were identified. Further univariate Cox analysis revealed that 27 mRNAs were independent prognostic factors for MM patients, 20 of which were high-risk genes, with varying expression levels in each group (Fig. 1E and 1F). Among them, CDK4, a high-risk gene highly expressed in cluster A (Fig. 1G), is closely associated with cell proliferation by regulating the cell cycle, which is consistent with the GSVA results. As with other cancers, cell cycle dysregulation leads to the progression of MM, where CDK4/6 disorders are common. Image: Summary/Conclusion: This work showed that the high expression of m7G regulators was correlated with unfavorable prognostic outcomes in MM. The difference in m7G modification patterns may be an important factor in determining the prognosis of MM patients via causing disorders of the cell cycle. P883: TARGETING CALCINEURIN/NFATC1 SIGNALING SENSITIZES PROTEASOME INHIBITION TREATMENT IN MULTIPLE MYELOMA J. Zhang1,*, X. Luo1, T. Xia1, K. C. Cheng1, M. H. Ng1 1Blood Cancer Cytogenetics and Genomics Laboratory, Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China Background: Proteasome inhibitors (PIs) (e.g., Bortezomib) are key treatments to improve the clinical outcome of multiple myeloma (MM). However, disease relapse from the emergence of PI-resistant clones is the main problem. Insights into the mechanisms underlying PI-resistance will provide novel druggable targets to prevent relapse. It was well-recognized that PIs induce endoplasmic reticulum (ER) stress in MM cells. ER stress is associated with the Calcineurin/nuclear-factor-of-activated-T-cells (NFAT) signaling. Accumulating studies indicated the importance of Calcineurin/NFAT signaling in tumorigenesis and drug resistance. However, the involvement of Calcineurin/NFAT signaling in PI-resistance in MM has not yet been investigated. Aims: To dissect the role of the Calcineurin/NFAT axis in Bortezomib (BTZ) treatment response and explore the therapeutic potential by combining Bortezomib and Calcineurin/NFAT inhibitors in MM. Methods: We studied the expression pattern of PPP3CA and NFATC1 from MM datasets and MM cell lines and their association with BTZ responses in MM. The expression of the four principal NFAT members was detected by expression microarray and their activation status by western blotting in MM cell lines. We identified the most prominent NFAT member and assessed its activation under BTZ treatment. A lentivirus-transduction system was used for overexpressing genes, and siRNA transfection was applied to induce NFATC1 knockdown in a BTZ-resistant cell line ARH77. Flow cytometry and western blotting assessed the combinational effect of Calcineurin/NFAT inhibitors and BTZ on MM cells. Results: The expression level of PPP3CA, which encodes Calcineurin Catalytic subunit A (CnA), was correlated to the poor clinical outcome of recurrent MM patients (MMRF cohort). Calcineurin was highly expressed in ARH77, which is less sensitive to BTZ treatment than other MM cell lines. Among the four NFAT members, we found that NFATC1 and NFATC3 were the most highly expressed members in MM cell lines. However, NFATc1 was the only member suppressed by CsA but activated by the calcium channel agonist Ionomycin in ARH77. Consistent with Calcineurin, NFATc1 was highly expressed in ARH77, with reference to a confirmed NFATc1-positive cell line Raji and the nuclear-NFATc1 overexpressing HEK-293T. Additionally, BTZ activated CnA and NFATc1 time-dependently in ARH77 but failed to activate CnA and NFATc1 in the BTZ-sensitive cell line NCI-H929. Immunofluorescence showed that BTZ triggered the nuclear translocation of NFATc1. From GEO datasets, NFATC1 was overexpressed in patients less responsive to BTZ, but was unchanged in patients subjected to conventional therapies. The Calcineurin/NFATc1 was thus a potential mechanism of BTZ-resistance in MM. Depleting NFATc1 enhanced BTZ-induced DNA damage-related apoptosis in ARH77. The combinational treatment of Calcineurin/NFAT inhibitors (CsA and FK506) and BTZ decreased the nuclear/cytosolic ratio of NFATc1 and promoted apoptosis in two MM cell lines compared to the treatment of BTZ alone. Image: Summary/Conclusion: The activation of the Calcineurin/NFATc1 axis is an important mechanism that contributes to BTZ-resistance in MM. Targeting this axis confers translational potential for improving BTZ-based therapies. P884: POTENTIAL BENEFIT OF POST-TRANSPLANT DOXYCYCLINE IN AL ACHIEVING HEMATOLOGICAL RESPONSE AFTER AUTOLOGOUS STEM CELL TRANSPLANT – LONG TERM FOLLOW UP N. Abdallah1,*, A. Dispenzieri1, E. Muchtar1, F. Buadi1, P. Kapoor1, M. Lacy1, Y. Hwa1, A. Fonder1, M. Hobbs1, S. Hayman1, N. Leung1, D. Dingli1, R. Go1, Y. Lin1, W. Gonsalves1, M. Binder1, T. Kourelis1, R. Warsame1, R. Kyle1, S. Rajkumar1, M. Gertz1, S. Kumar1 1Hematology, Mayo Clinic, Rochester, United States of America Background: Systemic light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by multiorgan deposition of fibrils composed of misfolded immunoglobulin light chains. Current treatment strategies targeting the plasma cell clone do not result in immediate organ recovery, and thus effective fibril-directed therapies are needed. Doxycycline has been shown to disrupt amyloid fibril formation in preclinical models. In 2012, we reported initial results on the impact of posttransplant prophylaxis with doxycycline using data from 445 AL amyloidosis patients; prophylaxis with doxycycline was associated with improved survival compared to penicillin in those achieving hematologic response. Since then, few studies have evaluated doxycycline use in AL amyloidosis, mainly in combination with induction chemotherapy, with mixed results. We provide here updated results on outcomes of AL amyloidosis patients who received doxycycline prophylaxis post autologous stem cell transplantation (ASCT) in our institution. Aims: To evaluate the impact of doxycycline use for post-ASCT antimicrobial prophylaxis on survival in AL amyloidosis patients. Methods: We included 553 patients who underwent ASCT from July 1996 to June 2014. We excluded patients who died within 100 days of ASCT. Doxycycline 100 mg daily was substituted for penicillin prophylaxis in patients with penicillin allergy; since 2013, doxycycline has become the standard for prophylaxis in AL amyloidosis at our institution. Prophylaxis was typically continued for a year after ASCT. Antibiotic type was determined by review of medical records. The Kaplan-Meier method was used to estimate time to next treatment (TTNT) and overall survival (OS), both measured from the time of transplant; curves were compared using the log-rank test. Results: Median age was 59 and 60% were male. The median time from diagnosis to ASCT was 4 months; 90% had early ASCT (within 1 year of diagnosis) and 57% received upfront ASCT without prior induction. Penicillin was used for prophylaxis in 67% (372 patients); the rest received doxycycline. Hematologic and organ response rates were similar in the 2 groups. Median follow up from ASCT was 10.5 vs. 13.6 years in the doxycycline and penicillin groups, respectively. Among patients with early ASCT, TTNT was 5.5 (95%CI: 4.2-8.1) vs. 5.8 (95%CI: 4.7-6.8) years, respectively (P=0.95). Median OS was 12.0 (95%CI: 11.0-19.6) vs. 11.0 (95%CI: 9.6-12.7) years, respectively (P=0.17); 5- and 10-year OS rates were 81% vs. 75% and 62% vs. 54% in the doxycycline and penicillin groups, respectively. Among patients with day 100 hematologic response, there was a non-statistically significant trend towards improved OS with doxycycline, but no difference in those without hematologic response: median OS with doxycycline vs. penicillin was 21.1 vs. 14.6 years in patients with day 100 dFLC <4 mg/dL (P=0.18) Figure 1a. In patients with normal day 100 FLCr, median OS was NR vs. 12.8 years, respectively (P=0.10) Figure 1b. There was no difference between the 2 groups when OS was stratified by Mayo 2012 stage (1-2 vs 3-4), number of involved organs (1 vs. >1), ASCT period (before or after 2012), or pre-ASCT induction (Yes vs. No). Image: Summary/Conclusion: After 13 years of follow up we observed a trend towards improved OS with doxycycline as post-ASCT prophylaxis in patients who achieve a hematological response. Prospective studies are needed to elucidate whether doxycycline improves outcomes in AL amyloidosis and identify the optimal dose, duration, timing, and the subset of patients likely to benefit. P885: PROSPECTIVE ASSESSMENT OF MYELOMA TUMOUR BURDEN AND BONE DISEASE USING DW-MRI AND EXPLORATORY BONE BIOMARKERS G. Agarwal1,*, G. Nador1, S. Varghese1, H. Getu1, C. Palmer2, C. Rodgers3, C. Pereira2, G. Sallemi2, K. Partington4, N. Patel4, R. Soundarajan5, R. Mills5, R. Brouwer5, A. Shah6, D. Peppercorn6, C. Edwards2, M. Javaid2, S. Gooding1, K. Ramasamy1 1Department of Clinical Haematology, Oxford University Hospitals NHS Foundation Trust; 2The Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford; 3Department of Clinical Neurosciences, University of Cambridge, Cambridge; 4Department of Radiology, Oxford University Hospitals NHS Foundation Trust; 5University of Oxford, Oxford; 6Department of Radiology, Hampshire Hospitals NHS Foundation Trust, Hampshire, United Kingdom Background: Key clinical priorities for multiple myeloma (MM) are to reduce tumour burden and complications, of which lytic bone disease is the major cause of morbidity. To guide treatment, there is a pressing need for biomarkers that can accurately quantify MM tumour burden and associated bone disease. To this end, Diffusion-Weighted Magnetic Resonance Imaging (DW-MRI) and bone turnover markers are promising radiological and serum metrics, respectively, although their clinical value in prospective assessment is unclear. Aims: LOOMIS was a single-centre observational cohort study that evaluated the relative merits of DW-MRI and bone turnover markers in prospectively assessing tumour burden and bone disease when added to standard clinical assessment, in patients with MM and monoclonal gammopathy of undetermined significance (MGUS). Methods: A total of 67 patients were enrolled (14 newly diagnosed MM, 12 relapsed MM, 15 smouldering MM, 14 MGUS and 12 healthy volunteers) between March 2018 and March 2020. At baseline (and 6-month follow-up for MM/MGUS), participants had a DW-MRI scan and serum measurements of established (P1NP, CTX-1, ALP) and exploratory (DKK1, sclerostin, RANKL:OPG ratio) bone turnover markers. MM/MGUS patients additionally had standard myeloma bloods and Dual-Energy X-ray Absorptiometry (DXA) at each visit, as correlates of tumour burden and bone loss. DW-MRI scans were double reported by expert Radiologists for Apparent Diffusion Coefficient (ADC) measurements of lytic bone lesion(s), and Myeloma Response Assessment and Diagnosis System (MY-RADS) Response Assessment Category (RAC) scoring. Patients were classified by International Myeloma Working Group (IMWG) response criteria as a clinical correlate of therapy response. Results: On assessment of baseline tumour burden, there was no correlation between single lesion DW-MRI ADC and serum paraprotein [p>0.05]; however, there was moderate positive correlation between serum DKK1 and serum paraprotein [r=0.39, p=0.04]. At follow-up, radiological MY-RADS RAC scoring correlated with conventional IMWG response criteria [p=0.015]; additionally, longitudinal relative change in serum DKK1 differed between IMWG-defined therapy responders (37% decrease) and non-responders (15% increase) [p<0.01]. On baseline assessment of bone loss, there was moderate positive correlation between serum sclerostin and DXA bone mineral density at femoral neck [r=0.40, p<0.01] and lumbar spine [r=0.54, p<0.001]. At follow-up, there was a moderate negative correlation between longitudinal % change in the RANKL:OPG ratio and % change in DXA bone mineral density at femoral neck [r= -0.45, p<0.01]. Summary/Conclusion: Our prospective trial validates DW-MRI-based MY-RADS RAC scoring as a qualitative radiological tool to assess therapy response. Whilst previous work has supported single lytic lesion ADC measurements as a correlate of tumour volume, we did not find this; small sample size and prior chemotherapy in our study may have limited our ability to detect this. In comparison, serum bone turnover markers such as DKK1 may provide a more global measure to quantify myeloma burden both at baseline diagnosis and longitudinally with therapy. Additionally, our data highlight serum sclerostin and RANKL:OPG ratio as potential biomarkers to assess and monitor bone loss. Overall, our study highlights emerging radiological and serum biomarkers of tumour burden and associated bone loss in MM and MGUS, which merit further exploration alongside uniformly treated cohorts, to better understand their clinical utility. P886: AFRICAN AMERICAN PATIENTS WITH SMOLDERING MULTIPLE MYELOMA MAY HAVE A LOWER RISK OF PROGRESSION COMPARED TO WHITE PATIENTS T. Akhlaghi1,*, K. Maclachlan2, N. Korde2, S. Mailankody2, A. Lesokhin2, H. Hassoun2, S. X. Lu2, D. Patel2, U. Shah2, C. Tan2, A. Derkach3, O. Lahoud4, H. J. Landau4, G. L. Shah4, M. Scordo4, D. J. Chung4, S. A. Giralt4, S. Z. Usmani2, O. Landgren5, M. Hultcrantz2 1Department of Internal Medicine, Icahn School of Medicine, Mount Sinai Morningside and West; 2Myeloma Service, Department of Medicine; 3Department of Epidemiology and Biostatistics; 4Adult Bone Marrow Transplant Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York; 5Myeloma Service, Sylvester Comprehensive Cancer Center, University of Miami, Miami, United States of America Background: The incidence of multiple myeloma (MM) and its precursor stages is two to threefold higher in African Americans (AAs) compared to whites when adjusted for socioeconomic status, age, and sex. However, there is limited information on whether racial background affects the risk of progression from smoldering MM (SMM) to MM. Aims: To assess the effect of race on the progression from SMM to MM. Methods: Patients with SMM presenting to Memorial Sloan Kettering Cancer Center between the years 2000 and 2019 and who identified as either AA or white were included in this retrospective study. Baseline patient and disease characteristics were collected at the time of diagnosis including laboratory, imaging, and pathology reports. Differences in distributions of continuous and discrete characteristics were assessed by Kruskal-Wallis and chi-square tests. Time to progression (TTP) was assessed using the Kaplan-Meier method with log-rank test for comparisons. Univariate and multivariate Cox proportional hazard models were used to estimate effects of risk factors on TTP with hazard ratios (HR) and 95% confidence intervals (CI). Results: A total of 576 patients were included (70 were AA, 12%). Median follow-up time was 3 years in AAs and 4 years in whites. Differences in baseline characteristics between AAs and whites included median age (60 years in AAs [IQR 51-67] vs 64 years in whites [IQR 56-72], p = 0.01), median hemoglobin level (12.3g/dL in AA [IQR 11.8-13] vs 12.8g/dL in whites [IQR 11.8-13.9], p = 0.02), and immunoparesis including 1 or 2 uninvolved immunoglobulins (31% and 10% in AAs vs 56% and 27% in whites, p = 0.002). There was no difference in bone marrow plasma cell percentage (BMPC), M-spike, free light chain ratio, or Mayo-2018 SMM risk score. AA race was associated with a significantly decreased risk of progression in the univariate model (HR 0.57, CI 0.34-0.94). In the multivariate model adjusting for age, sex, and variables associated with an increased risk of progression in the univariate model (BMPC, M-spike, free light chain ratio, immunoparesis and low albumin), AA race remained associated with a decreased risk of progression (HR 0.39, CI 0.16-0.95). Overall, AA patients with SMM had a significantly (p = 0.027) longer median TTP (9.7 vs 6.2 years), and a lower 2-year (12.6% vs 20.1%) and 5-year (34% vs 44.6%) progression rate than whites. Because AA patients were younger at diagnosis, we stratified patients by age group, < 65 vs ≥65 years. In patients < 65 years, there was no difference in progression rate. In patients aged ≥65 years, AA patients continued to have a longer TTP than whites (9.8 vs 5.2 years, p = 0.02). Image: Summary/Conclusion: In our retrospective single institution experience, AA patients with SMM had a lower risk of progression to MM compared to whites. Both groups had similar Mayo-2018 risk scores, however, AA patients had a lower degree of immunoparesis at baseline. Future studies are needed to better understand if these differences are explained by differences in disease biology including genomic mechanisms, immune microenvironment, and systemic immune response. P887: IN-HOUSE ANTI BCMA CAR-T THERAPY FOR THE TREATMENT OF RELAPSED REFRACTORY MULTIPLE MYELOMA: PHASE 1 RESULTS. N. Asherie1,*, S. Kfir-Erenfeld1, B. Avni1, S. Grisariu1, M. Assayag1, T. Sharon1, E. Lebel2, C. J. Cohen3, M. E. Gatt2, P. Stepensky1 1Bone Marrow Transplantation; 2Hematology, Hadassah Medical Center, Jerusalem; 3The Laboratory of Tumor Immunology and Immunotherapy, Bar-Ilan University, Ramat Gan, Israel Background: B-Cell Maturation Antigen (BCMA) targeted CAR-T cell therapy is a treatment strategy showing great promise. Recently, anti-BCMA CAR-T cell therapy has been approved by the FDA for treatment of relapsed/refractory multiple myeloma. However, the major drawbacks of this treatment are the length of the manufacturing process, limited access and the excessive cost. Thus, there is an urgent need for development of in-house manufactured CAR-T cell treatments. Based on favorable pre-clinical results using our novel CAR-T-anti BCMA construct, in February 2021 we have launched a BCMA-targeted CAR-T cell based clinical trial treating relapsed refractory (R/R) multipe myeloma (MM) patients (NCT04720313). Aims: This phase I-II clinical trial is a dose escalation study. The first goal was to explore the safety of our product. The phase I first part of the trial consisted of three escalating cell doses ranging between 150- and 800x10^6 CAR+ cells. Methods: We enrolled 20 R/R MM patients having failed more than three lines of previous therapies, to receive our CAR-T-anti BCMA product at escalating doses of 150 x 106 (cohort 1, 6 patients), 450 x 106 (cohort 2, 7 patients) and 800 x 106 (cohort 3, 7 patients) cells. The median age was 62 (range: 44-75) years, with a median time of 55 months from initial disease diagnosis (range, 8-241) and a median number of 5.5 previous treatment lines (range, 3-13). Eighty percent had bone marrow (BM) involvement and the majority were categorized as ISS/R-ISS II. The vast majority of patients underwent previous autologous BM transplantation and were refractory to proteasome inhibitors, immunomodulatory agents (IMiD) and anti-CD38 antibody, while only 10 patients (50%) were previously exposed to an anti-BCMA agent (belantamab). Patients in cohort 1 had a higher rate of extramedullary disease, high LDH and massive BM involvement (>50% plasma cells in BM biopsy), while patients in cohort 3 had a higher ECOG performance score and a lower rate of cytogenetic high-risk disease. Results: At a median follow up of 156 days, overall response was 75% for the entire cohort with 2/6,1/7 and 1/7 patients not responding in cohorts 1, 2 and 3, respectively. Six patients achieved MRD negative CR (4 of them in cohort 3), while 3 achieved MRD positive (or MRD not evaluated) CR and 6 had achieved VGPR (4 of them in cohort 2). The median PFS for the entire study group was 166 days (range, 24-289); 75 days (range, 24-289), 181 days (range, 61-209) and not reached (range, 20-124) for cohort 1,2 and 3, respectively. The median duration of response (DOR) for the entire study group was 151 days (range, 32-259). The progression rate in patients exposed to belantamab, was higher (7/9, 78%) compared to the non-exposed patients (4/11, 36%). On the day of last follow up, 75% of patients were still alive. Eleven (55%) patients progressed. Four (25%) patients died due to disease progression. Ninety percent of patients developed cytokine release syndrome (CRS), mostly on the day of cell infusion (median day 0, range 0-21), while none suffered from grade 3-4 CRS. None of the patients developed immune effector cell-associated neurotoxicity syndrome (ICANS). Summary/Conclusion: The anti-BCMA CART - cell therapy developed at Hadassah Medical Center proves safe at the three cohorts, with manageable toxicities and evidence of initial short term favorable responses attesting to its efficacy in heavily pretreated R/R MM patients. These data are very encouraging and demonstrate the capability of single center local medical institution to develop, manufacture and deliver in-house CART. P888: REPLACING STEROIDS IN TRANSPLANT-INELIGIBLE MULTIPLE MYELOMA: THE PHASE 2 ISATUXIMAB-BORTEZOMIB-LENALIDOMIDE-DEXAMETHASONE REST STUDY F. B. Askeland1,2,*, E. Haukås3, T. S. Slørdahl4, D. Schjøll5, A. Lysen1, E. Hermansen6, F. Schjesvold1,2 1Oslo Myeloma Center, Department of Hematology; 2KB Jebsen Center for B Cell Malignancies, Oslo University Hospital, Oslo; 3Department of Cancer and Blood Diseases, Stavanger University Hospital, Stavanger; 4Department of Hematology, St. Olavs Hospital, Trondheim, Norway; 5Oslo Myeloma Center, Department of Hematology, Oslo University Hospital, Oslo; 6Department of Hematology, Zealand Iniversity Hospital, Roskilde, Denmark Background: Standard therapies for newly diagnosed multiple myeloma (NDMM) patient’s ineligible for autologous stem-cell transplantation (ASCT) have been lenalidomide-dexamethasone (Rd), bortezomib-lenalidomide-dexamethasone (VRd) or bortezomib-melphalan-prednisone (VMP). The benefit of adding an anti-CD 38 monoclonal antibody (mAb) to the standard treatments of VMP and Rd has been demonstrated in two large phase 3 studies (Facon, 2019. Mateos, 2018). Isatuximab is an anti-CD38 mAb approved in combination with pomalidomide-dexamethasone and carfilzomib-dexamethasone to treat relapsed/refractory MM, and has also shown clinical responses as combination therapy to treat NDMM (Trudel, 2019). Corticosteroids have been the backbone of most myeloma targeted therapies since the discovery of their effectiveness. Due to the continuous treatment paradigm, the cumulative dose of corticosteroids in patients is substantial. Steroids inflict reduction in both short- and long-term quality of life and increase patients’ receptiveness for infections. Infections are one of the main reasons for complications and death during active first line treatment, and the rate rises with age. Both steroid-free regimens and regimens with reduced corticosteroids have demonstrated improved safety and similar efficacy as regimens containing steroids. Aims: The study will evaluate isatuximab (Isa) in combination with bortezomib (V) and lenalidomide (R) with minimal dexamethasone (d) as first-line treatment in transplant-ineligible patients. The primary endpoint is the number of patients who achieve measurable residual disease negative (Euroflow NGF 10-5) complete response during and/or after 18 cycles of study treatment. Secondary endpoints include progression free survival, overall survival, overall response rate (ORR), safety evaluations and patient-reported outcome. Methods: The REST study is an academic, single arm, open-label, phase 2 study of NDMM patients ineligible for ASCT. 50 patients will be included and receive Isa-VRd (Isa:10 mg/kg IV Days 1, 8, 15, 22 during cycle 1, QW cycle 2-18; V: 1,3 mg/m2 SC Days 1,8,15 during cycle 1-8; R: 25 mg PO Days 1-21 during all cycles; d: 20 mg PO Days 1,8,15,22 only for the first 2 cycles), all 28-day cycles. Results: As of January 31, 2022, 17 patients have been enrolled. Baseline characteristics can be found in Table 1. At data-cutoff, the median number of cycles started by patients was 3.6 (range 1-8) and the median duration of exposure was 16 (range 3-28) weeks. 8 patients developed 14 grade ≥3 non-hematological adverse eventd (AE), including infections (n=7), fever (n=1), skeletal pain (n=2), acute renal failure (n=1), peripheral sensory neuropathy (n=1), diarrhea (n=1) edema (n=1). There were no AEs leading to definitive treatment discontinuation. At a follow up of 6 months, the ORR was 100% (15/15) and the ≥very good partial response rate was 53.3% (8/15 patients). Image: Summary/Conclusion: Isa-VRd in the transplant ineligible population has a tolerable safety profile. Isa-VRd is showing encouraging preliminary efficacy in NDMM ineligible for transplant. Enrollment and follow-up is ongoing. Acknowledgements: Sanofi sponsors the REST study. P889: WILL OUTCOME IMPROVE BY TREATING MULTIPLE MYELOMA PATIENTS AT MRD RELAPSE? THE REMNANT STUDY (RELAPSE FROM MRD NEGATIVITY AS INDICATION FOR TREATMENT) F. B. Askeland1,2,*, A.-M. Rasmussen1,2, A. Lysen1, E. Haukås3, A. Eilertsen4, M. Moksnes5, G. Tsykunova6, A. Vik7,8, B. D. Eiken9, J. H. Sørbø10, J. Rolke11, K. O. Sand12,13,14, R. F. Hallstensen15, L. Myrseth1, T. S. Slørdahl16,17, F. Schjesvold1,2 1Oslo Myeloma Center, Department of Hematology; 2KG Jebsen Center for B Cell Malignancies, Oslo University Hospital, Oslo; 3Department of Hematology and Oncology, Stavanger University Hospital, Stavanger; 4Department of Hematology, Akershus University Hospital, Oslo; 5Cancer and Hematology Center, Vestfold Hospital, Tønsberg; 6Department of Medicine, Haukeland University Hospital, Bergen; 7Department of Hematology, University Hospital of North Norway; 8Institute of Clinical Medicine, UiT, The Artic University of Norway, Tromsø; 9Department of Oncology, Hospital Østfold, Kalnes; 10Medical Department, Levanger Hospital, Levanger; 11Department of Hematology, Sørlandet Hospital, Kristiansand; 12Department of Medicine, Ålesund Hospital; 13Department of Reasearch and Innovation, Møre og Romsdal Hospital Trust; 14Department of Health Sciences in Ålesund, Norwegian University of Science and Technology, Ålesund; 15Department of Hematology, Nordland Hospital, Bodø; 16Department of Hematology, St. Olavs Hospital; 17Norwegian University of Science and Technology (NTNU), Trondheim, Norway Background: Although many new treatment modalities have become available, multiple myeloma (MM) is nevertheless considered an incurable disease. In recent years, the importance of achieving measurable residual disease (MRD) negativity is becoming more clearly defined in patients with MM. There is solid evidence that bone marrow MRD negativity is one of the strongest prognostic factors, and deeper responses correlate with increasingly favorable outcomes. The deepest and most durable responses are achieved in the first line (1.L) of treatment. Aims: The REMNANT study will evaluate if treating MRD relapse after 1.L therapy prolongs progression free survival (PFS) and overall survival (OS) compared to starting treatment at biochemical relapse/progressive disease (PD) in MM patients. Methods: The REMNANT study is an academic, multicenter, open-label, randomized phase II/III study of newly diagnosed (ND) MM patients eligible for autologous stem cell transplant (ASCT). Few exclusion criteria apply and patients with kidney failure and amyloidosis can be enrolled. 391 NDMM patients (age 18-75 years) eligible for ASCT will be enrolled in the phase II part of the study and receive standard of care (SOC) 1.L treatment according to Norwegian national guidelines; four pre-transplant induction and four post-transplant consolidation cycles of bortezomib, lenalidomide and dexamethasone (VRd). After induction, patients will undergo tandem or single transplant, depending on toxicity and response to first transplant. Following 1.L treatment 176 patients who are ≥ complete response (CR) and MRD negative (NGF Euroflow) will be enrolled in part 2 of the study. Patients will be randomized in a 1:1 ratio to receive second line treatment at MRD relapse in arm A or at PD in arm B. Randomization will be stratified by R-ISS stage at diagnosis and single versus tandem transplant. At loss of MRD negativity in arm A or at PD in arm B, second line treatment will be carfilzomib, dexamethasone and daratumumab until progressive disease. In part 2 we will compare different methods for measuring MRD; NGF Euroflow, Next Generation Sequencing (NGS), mass spectrometry and PET-CT. We will evaluate how the different methods compare when it comes to sensitivity and how they correlate to outcome (PFS and OS). The microbiota composition has shown to impact outcome in myeloma patients. Therefore, we will investigate how the microbiota at diagnosis differs in patients who achieve MRD negativity and those who still are MRD positive after treatment. Results: We will present updated results from part 1 of the study. As of February 16th 2022, 136 patients have been enrolled in part 1. Twenty-six patients have been evaluated for MRD status post consolidation. Thirteen patients (50%) were ≥CR and MRD negative and have been enrolled in part 2 of the study. In Table 1 baseline characteristics and results are presented. Image: Summary/Conclusion: Part 1 of the REMNANT study will clarify the overall response rate and depth of response measured by MRD, after SOC Norwegian 1.L treatment Part 2 will determine if treating patients at MRD relapse, when tumor burden is lower, will improve outcome. P890: THE OUTCOME OF SECOND PRIMARY MALIGNANCIES DEVELOPING IN MM PATIENTS I. Avivi1,*, D. H vesole2, J. Dávila Valls3, L. Usnarska-Zubkiewicz4, V. Milunovic5, B. B. Bogumiła Osękowska6, A. Kopińska7, M. Gentile8, B. P. MARTÍNEZ9, P. Robak10, E. crusoe11, R. LUIS GERARDO12, M. Gajewska13, V. Gergely14, M. Delforge15, Y. Cohen16, G. Alessandro17, C. peña18, C. Shustik19, G. Mikala20, K. Žalac21, A. H. Denis22, B. Peter23, K. Weisel24, J. martinez lopez25, A. Waszczuk-Gajda26, M. Krzystanski27, A. Jurczyszyn28 1Head of Hematology Division, Tel Aviv Medical Center, Tel AVIV, Israel; 2John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, United States of America; 3Complejo Asistencial de Avila, Complejo Asistencial de Avila, Avila, Spain; 4Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw, Poland; 5Division of Hematology, Clinical Hospital Merkur, Zagreb, Croatia; 6Pomeranian Medical University in Szczecin, Pomeranian Medical University in Szczecin, Szczecin; 7Department of Haematology, Oncology and Internal Diseases, University Clinical Centre, Medical University of Warsaw, Warsaw, Poland; 8Hematology Unit, AO Cosenza, Cosenza, Italy; 9(University Hospital of Salamanca, Cancer Research Center-IBMCC, Salamanca, Spain; 10Medical University of Lódź, Medical University of Lódź, Lódź, Poland; 11: Federal University Of Bahia, University Hospital and Rede D’or Oncologia, Bahina, Brazil; 12Clinic Barcelona Hospital Universitari, Clinic Barcelona Hospital Universitari, Barcelona, Spain; 13Department of Hematology and Bone Marrow Transplantation, Silesian Medical University, Katowice, Poland, Warsaw, Poland; 14Department of Internal Medicine and Haematology, Semmelweis University, Budapest, Hungary; 15Department of Hematology, UZ Leuven, Leuven, Belgium; 16Hematology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 17Hematology, Department of Medical Science, Surgery and Neuroscience, University of Siena, Siena, Italy; 18Sección Hematología, Hospital del Salvador, Santiago, Chile; 19Hematology Department, MUHC - McGill University Health Centre, montreal, Canada; 20Department of Hematology and Stem Cell Transplantation, South-Pest Central Hospital, Budapest, Hungary; 21Department of hematology of Clinics for Internal Medicine, University Hospital Center ‘‘Sestre Milosrdnice’’, Zagreb, Croatia; 22N Ireland Centre for Stratified Medicine, Ulster University, Londonderry, Ireland; 23Department of Medicine, Warren Alpert Medical School, Brown University, Rhode Island, United States of America; 24: University Medical-Center Hamburg-Eppendorf, Hamburg, Germany; 25Nieves Lopez-Muñoz Hospital, Madrid, Spain; 26Department of Hematology, Oncology and Internal Diseases, Warsaw Medical University, Warsaw; 27Department of Hematology; 28Department of Hematology, Jagiellonian University Medical College, crakow, Poland Background: The continuing improvement in response rates and overall survival (OS) of multiple myeloma (MM) patients exposed to the cumulative effect of multiple anti-MM agents including IMiDs, PIs, mAbs and cytotoxic chemotherapy, has resulted in an increased risk for second primary malignancies (SMPs). There is minimal detailed information regarding the clinical characteristics of patients who develop SPMs, risk factors predicting development, time to onset, SPM’s management and outcomes in patients treated with novel agents outside clinical trials. Aims: To investigate the characteristics and outcome of SPMs reported in MM patients and define impact of SPM diagnosis on MM management and outcome. Methods: A multicenter international ‘real-world’ retrospective analysis, reviewing MM databases of 25 participating centers and analyzing the characteristics and outcome of SPM reported in patients that were treated with novel-based induction. In addition, the impact of SPM diagnosis on MM management and outcome are reported. Results: Two hundred and one consecutive MM patients, experiencing SPM ≥ 6 months since initiation of first line therapy were included in the analysis. Clinical characteristics included the following: 64% were male, median age at 66±10.57 years, 10% with a prior history of cancer. 36% and 25% of evaluable cases presented with high ISS-3 and high-risk cytogenetics. 87(43%) underwent an autologous hematopoietic cell transplantation and 51(27%) received maintenance therapy. Median number of lines of therapy prior to SPM defection was 2 (1-7). With a median follow-up of 50± 45 months since MM diagnosis, 115 patients (57%) were diagnosed with solid cancers: colorectal cancer (n=20, 17.4%), lung (n=18, 15.6%), breast (n=14, 12%), prostate (n=8, 7%), melanoma (n=10, 9%), pancreas (n=10, 9%), bladder (n=7, 6%) and gastric (n=7, 6%), skin cancer (n= 34, 16.9%) and cancer of unknown origin (n=9, 4.5%). 46 patients (22.9%) developed hematological cancers, including lymphoma and MDS/AML; reported in 7(6%) and 27 (13.4%) cases respectively. 79% (n=158) were actively treated for SPM and 39.5% responded to therapy. Forty Eight percent (n=98) of the patients were concurrently receiving an anti-MM therapy at the time of SPM diagnosis. MM treatment remained unchanged in 27 patients, discontinued and not substituted with any other anti-MM therapy until MM progression in 59 patients and changed to a new anti-MM therapy in 12 patients. Within a median follow-up period of 112 months since MM diagnosis and 31 months since SPM detection, 50.5% (n-101) have died; 32 due to solid cancer (32/101, 28%), 11 due to hematological cancer (11/46, 24%) and 13 (6%) due to MM progression. Cause of death was not available for 45 cases. Median OS since MM diagnosis and since SPM detection were 63 months and 8.5 months, respectively (Figure 1). (Detailed analysis regarding causes of death and risk factors to be presented at the meeting). Image: Summary/Conclusion: With the continuing improvement in OS, a higher proportion of MM patients are likely to develop SPM. OS since SPM diagnosis is shortened, approaching 8.5 months only, suggesting that the SPMs, originating in this scenario, are generally aggressive. Surveillance for early detection and treatment of SPMs are warranted. Further studies are necessary to determine the factors associated with the development of SPM to avoid precipitating agents especially in patients with lower risk disease since these individuals should have extended survival that may be substantially shortened by the development of treatment-related SPM. P891: IXAZOMIB, POMALIDOMIDE AND DEXAMETHASONE (IXPD) IN RELAPSED OR REFRACTORY MULTIPLE MYELOMA (RRMM) CHARACTERIZED WITH HIGH-RISK CYTOGENETICS. IFM 2014-01 A. Bobin1, S. Manier2, J. De Keizer1, L. Karlin3, A. Perrot4, C. Hulin5, D. Caillot6, C. Mariette7, P. Lenain8, V. Richez9, M. Tiab10, C. Touzeau11, A. Jaccard12, O. Decaux13, C. Araujo14, K. Belhadj15, L. Benboubker16, C. Déal17, M. Macro18, L. Vincent19, B. Arnulf20, B. Bareau21, T. Braun22, C. Calmettes23, D. mamoun24, H. Zerazhi25, H. Demarquette26, P. Feugier27, C. Fohrer-sonntag28, S. Godet29, M.-O. Petillon2, H. Avet-Loiseau4, X. Leleu1,* 1CHU de Poitiers, Poitiers; 2chu de lille, lille; 3CHU de Lyon, Lyon; 4CHU de Toulouse, Toulouse; 5CHU de Bordeaux, Bordeaux; 6CHU de Dijon, Dijon; 7CHU de Grenoble, Grenoble; 8CHU de Rouen, Rouen; 9CHU de Nice, Nice; 10CH la Roche sur Yon, La Roche Sur Yon; 11CHU de Nantes, Nantes; 12CHU de Limoges, Limoges; 13CHU de Rennes, Rennes; 14CH de Bayonne, Bayonne; 15CHU de Créteil-APHP, Créteil; 16CHU de Tours, Tours; 17Intergroupe Francophone du Myélome, Paris; 18CHU de Caen, Caen; 19CHU de Montpellier, Montpellier; 20Hopital Saint Louis, Paris; 21Hopital Privé Sevigne, Cesson Sevigne; 22Hopital Avicenne, Bobigny; 23CH de Périgueux, Périgueux; 24CHU de Angers, Angers; 25CH de Avignon, Avignon; 26CH de Dunkerque, Dunkerque; 27CHRU de Nancy, Nancy; 28CHU de Strasbourg, Strasbourg; 29CHU de Reims, Reims, France Background: High risk (HR) cytogenetics remains of poor prognosis, particularly in the RRMM setting. IFM 2010-02, Pd in advanced HR RRMM, had demonstrated limited activity with no addition of a proteasome inhibitor (PI), and a good safety profile. We hypothesized that addition of Ixazomib (oral PI at increased dose density) to Pd (IxPd) in HR RRMM would improve convenience, thus adherence to treatment, and in parallel improve efficacy with no increased toxicity. Aims: We aimed to determine the efficacy and safety profile of the association ixazomib, pomalidomide and dexamethasone HR RRMM. Methods: Eligible patients had a RRMM in L2 refractory to lenalidomide but not to pomalidomide and ixazomib. HR was defined by presence of either del(17p) and/or t(4;14) at diagnosis or study entry. Patients received 17 induction cycles, 21-days long, consisting of ixazomib 3mg/day (d 1, 4, 8 and 11), pomalidomide 4mg/day (d1 to 14) and weekly dexamethasone, followed by a maintenance phase of 28-day cycles with ixazomib 4mg/day (d 1, 8 and 15) and pomalidomide 4mg/day (d 1 to 21), until progression. The primary endpoint was time to progression (TTP). The number of patients to be recruited was initially calculated based on an expected doubling of the median TTP obtained in IFM 2010-02 with Pd in either 2 HR RRMM population, but the number was recalculated due to difficulties in recruitment to demonstrate an expected doubling of the median TTP from 3 months, as a whole. No statistical comparison can be done across HR groups given that the study was powered to analyze the 2 groups as a whole and not separately with 26 patients. Results: Twenty-six patients were enrolled in the study. Median age was 72 years (Interquartile range: 67-78). Ten patients presented with del(17p) and twelve patients with t(4;14). All patients were refractory to lenalidomide. With a median follow-up time of 9.8 months, the median TTP was 9.7 months (95% confidence interval (CI) 4.4;-). The median OS was 12.2 months (CI95% 11.1;-). The ORR and ≥VGPR rate was 15 (58%) pts and 7 pts (27%) in the study as a whole. The ≥VGPR rate was 30% and 33% in del(17p) and t(4;14), respectively. No CR was observed with IxPd in our series. The most common adverse events (≥10% occurrence) were neutropenia (n=13), thrombocytopenia (n=12), asthenia (n=9), anemia (n=7), rash (n=4), diarrhea (n=3), dizziness (n=3), general physical health deterioration (n=3), muscle spasms (n=3), peripheral oedema (n=3), pyrexia (n=3). Ten SUSARs were declared with no modification to the safety profile of ixazomib and pomalidomide according to the independent data monitoring committee. Summary/Conclusion: The study IFM 2014-01/IxPd in HR L2 RRMM met its primary end point objective since the median TTP was superior to 3 months, the median TTP reported in IFM2010-02 as a whole, with the addition of Ixazomib to Pomalidomide and dexamethasone in this population characterized with a very poor outcome. This data confirms the importance of proteasome inhibitors in HR RRMM. This phase 2 study needs confirmation in a larger cohort. P893: CARFILZOMIB IN COMBINATION WITH LENALIDOMIDE AND DEXAMETHASONE (KRD) AS SALVAGE THERAPY FOR MULTIPLE MYELOMA PATIENTS: ITALIAN, MULTICENTER, RETROSPECTIVE EXPERIENCE OUTSIDE OF CLINICAL TRIALS E. A. Martino1,*, C. Conticello2, E. Zamagni3, V. Pavone4, S. Palmieri5, M. Musso6, P. Tacchetti7, A. Mele4, L. Catlano8, E. Vigna1, A. Bruzzese9, F. Mendicino1, C. Botta10, D. Vincelli11, G. Farina12, M. Barone13, C. Cangialosi14, K. Mancuso15, I. Rizziello15, S. Rocchi15, A. P. Falcone16, G. Mele17, G. Reddiconto18, B. Garibaldi19, E. Iaccino20, G. Tripepi21, F. Di Raimondo22, P. Musto23, A. Neri24, M. Cavo3, F. Morabito25, M. Gentile1 1Azienda Ospedaliera di Cosenza, COSENZA; 2University of Catania, Catania; 3Seràgnoli Institute of Hematology and Medical Oncology, University of Bologna, Bologna; 4Hospital Card. G. Panico, Tricase; 5Ospedale Cardarelli, Napoli; 6Dipartimento Oncologico, La Maddalena, Palermo; 7Istituto di Ematologia “Seràgnoli”, Bologna; 8AUOP “Federico II”, Napoli; 9Azienda Ospedaliera of Cosenza, Cosenza; 10Internal Medicine and Medical Specialties, University of Palermo, Palermo; 11Great Metropolitan Hospital “Bianchi-Melacrino-Morelli”, Reggio Calabria; 12AORN “Sant’Anna e San Sebastiano”, Caserta; 13”Tortora” Hospital, Pagani; 14A. O. Ospedali Riuniti Villa Sofia-Cervello, Palermo; 15IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna; 16IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo; 17Hematology Unit, Brindisi; 18Hospital Vito Fazzi, Lecce; 19Azienda Policlinico-OVE, University of Catania, Catania; 20University “Magna Graecia” of Catanzaro, Catanzaro; 21National Research Institute of Biomedicine and Molecular Immunology, Reggio Calabria; 22Hematology Section, University of Catania, Catania; 23”Aldo Moro” University School of Medicine, Bari; 24Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano; 25Biotechnology Research Unit, AO di Cosenza, Cosenza, Italy Background: Carfilzomib in combination with lenalidomide, and dexamethasone (KRd) have been approved for the treatment of relapsed and refractory multiple myeloma (RRMM) based on ASPIRE clinical trial. Efficacy and safety of the triplet outside clinical trial are still the object of investigation by many groups to confirm ASPIRE results in the setting of RRMM treated in real-life who don’t meet clinical trial restrictive inclusion criteria. Aims: The aim of the present study is to investigate the efficacy and safety of KRd regimen in the setting of RRMM treated in real-life. Methods: Here we report a retrospective multicenter analysis of 600 RRMM patients treated with KRd between December 2015 and December 2018 Results: The median age at KRd start was 64 years (range 33-85) and the median number of previous therapies was 2 (range 1-11). After a median number of 11 KRd cycles, the ORR was 79.9%. The median PFS was 22 months and the 2-year probability of PFS was 47.6%. At multivariate analysis advanced ISS (III), and high-risk cytogenetic were significantly associated with shorter PFS. The median OS was 34.8 months; the 2-year probability of OS was 63.5% At multivariate analysis creatinine clearance<30 ml/min, more than 1 line of previous therapy, and high-risk FISH were significantly associated with poor prognosis. After a median follow-up of 16 months (range 1-50), 259 withdrew from therapy. The main reason for discontinuation was progressive disease (81.8%). Seventy-four patients (12.3%) discontinued therapy for toxicity. The most frequent side effects were hematological (anemia 49.3%, neutropenia 42.7%, thrombocytopenia 42.5%) and cardiovascular (hypertension 14.5%, heart failure 2.5%, arrhythmias 3.6%). Summary/Conclusion: Our study confirms the safety and efficacy of KRd in the real-life setting of RRMM patients and encourages its use in clinical practice. P894: A PHASE II TRIAL TO EVALUATE THE EFFICACY OF DARATUMUMAB WITH DCEP IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS WITH EXTRAMEDULLARY DISEASE AFTER BORTEZOMIB BASED TREATMENT J. M. Byun1,*, C.-K. Min2, K. Kim3, S.-M. Bang4, J.-J. Lee5, J. S. Kim6, S.-S. Yoon1, Y. Koh1 1Seoul National University Hospital; 2Seoul St Mary’s Hematology Hospital; 3Samsung Medical Center, Seoul; 4Seoul National University Bundang Hospital, Seongnam; 5Chonnam National University Hwasun Hospital, Hwasun; 6Yonsei University College of Medicine, Seoul, South Korea Background: Extramedullary multiple myeloma (EMM) is an aggressive subentity of multiple myeloma (MM), characterized by the ability of a subclone to thrive and grow independent of the bone marrow microenvironment, resulting in a high-risk state associated with increased proliferation, evasion of apoptosis and treatment resistance. Despite improvement in survival for most patients with MM over recent decades, outcomes are generally poor when EMM develops. Thus, better understanding of the disease and more innovative therapeutic approaches are needed. Aims: To study the efficacy and safety of daratumumab in combination with dexamethasone, cyclophosphamide, etoposide and cisplatin (DCEP) Methods: This was a multi-center, prospective phase II study (ClinicalTrials.gov identifier: NCT04065308). A total of 33 patients older than 19 years with multiple myeloma according to IMWG, relapsed/refractory to bortezomib, and with EMM (short-axis ≥1cm by CT or PET-CT) were enrolled. As shown in the Figure, patients received 3 cycles of daratumumab in combination with DCEP, followed by daratumumab maintenance. The primary objective was complete response rate after 3 cycles of daratumumab with DCEP. The secondary objectives included: 1) overall response rates; 2) progression free survival (PFS); 3) overall survival; and 4) safety and toxicity profiles. Here we present the interim data of 24 patients. Results: The median age at MM diagnosis was 57 years (range 34-71) and 79.2% patients had EMM at diagnosis. There were 10 patients with t(4;14), 4 patients with del(17p) and 6 patients with t(14;16). There were 6 patients (25%) with ISS I disease, 10 (41.7%) with ISS II disease, and 8 (33.3%) with ISS III disease. The median number of prior lines of treatment was 3 (range 1-5), with all patients being exposed to bortezomib prior to enrollment. Also, 70.8% patients had prior exposure to carfilzomib, 87.5% to lenalidomide, and 50% to pomalidomide. There were 17/24 (70.8%) patients who completed 3 cycles: 3 showed CR; 2 showed VGPR; 11 showed PR and 7 with progressive disease as shown in the Figure. Six patients were able to complete the study protocol. For all patients the median PFS was 4 months, but for those who completed the study protocol the median PFS was not reached and all but 1 remained in remission until the data cut-off in 2022-Jan-31. Patients achieving PR or better response after cycle 3 showed significantly better PFS (6 vs 2 months, p<0.001) compared to those who didn’t. Most patients (22/24) underwent cyclophosphamide/etoposide/cisplatin dose reduction by 30% at cycle 1. One patient required further dose reduction at cycle 2. There was 1 patient who went off trial at D15 of cycle 1 according to attending physician’s decision and 2 patients who had to skip D22 daratumumab during cycle 1 due to thrombocytopenia. No patients required dexamethasone dose adjustments. During cycle 1, 12.5% showed anemia ≥grade 3, 25% showed thrombocytopenia ≥grade 3, 4.2% showed lymphopenia ≥grade 3, and 4 (16.7%) events of febrile neutropenia. During cycle 3, 5.3% showed anemia ≥grade 3, 10.5% showed thrombocytopenia ≥grade 3, while 15.8% showed lymphopenia ≥grade 3. The most common non-hematological adverse events was nausea (20.8%). Image: Summary/Conclusion: In conclusion, daratumumab in combination with DCEP showed overall response of 66.7% (CR 25%), and durable remission in 20.8% of the enrolled patients. P895: DARATUMUMAB, BORTEZOMIB, AND DEXAMETHASONE (D-VD) VERSUS BORTEZOMIB AND DEXAMETHASONE (VD) ALONE IN CHINESE PATIENTS WITH RELAPSED OR REFRACTORY MULTIPLE MYELOMA (RRMM): UPDATED ANALYSIS OF LEPUS W. Fu1,*, W. Li2, J. Hu3, G. An4, Y. Wang5, C. Fu6, L. Chen7, J. Jin8, X. Cen9, Z. Ge10, Z. Cai8, T. Niu11, M. Qi12, X. Gai13, Q. Li13, W. Liu14, W. Liu13, X. Yang14, X. Chen14, J. Lu15,16 1Shanghai Changzheng Hospital, Shanghai; 2The First Hospital of Jilin University, Jilin; 3Fujian Medical University Union Hospital, Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian; 4State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; 5Department of Hematology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin; 6The First Affiliated Hospital of Soochow University; 7The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Jiangsu; 8The First Affiliated Hospital of Zhejiang University, College of Medicine, Zhejiang; 9Peking University First Hospital, Beijing; 10Zhongda Hospital Southeast University, Jiangsu; 11Department of Hematology, West China Hospital Sichuan University, Sichuan, China; 12Janssen Research & Development, LLC, Spring House, PA, United States of America; 13Janssen Research & Development, LLC, Beijing; 14Janssen Research & Development, LLC, Shanghai; 15Peking University People’s Hospital, National Clinical Research Center for Hematologic Disease, Beijing; 16Collaborative Innovation Center of Hematology, Soochow, China Background: D-Vd prolonged progression-free survival (PFS) and induced deeper and more durable responses versus Vd alone in patients with RRMM in the global phase 3 CASTOR study (Weisel K, et al. ASH 2019. Abstract 3192). Results from a prespecified interim analysis of the phase 3 LEPUS study (median follow-up, 8.2 months) showed significant clinical benefit with D-Vd versus Vd in Chinese patients with RRMM and a safety profile that was generally consistent with that of CASTOR (Lu J, et al. Clin Lymphoma Myeloma Leuk 2021. 21[9]:e699-e709). Aims: To examine updated efficacy of D-Vd versus Vd in LEPUS at a median follow-up of 25.1 months. Methods: In the randomized, multicenter, phase 3 LEPUS study (NCT03234972), Chinese patients with RRMM and ≥1 prior line of therapy were randomized 2:1 to 8 cycles (21 days/cycle) of Vd (bortezomib 1.3 mg/m2 SC on Days 1, 4, 8, and 11; dexamethasone 20 mg PO/IV on Days 1, 2, 4, 5, 8, 9, 11, and 12) ± daratumumab (16 mg/kg IV once weekly in Cycles 1-3, every 3 weeks in Cycles 4-8, and every 4 weeks thereafter until disease progression). PFS was the primary endpoint. Results: A total of 211 patients were randomized (D-Vd, n = 141; Vd, n = 70), with a median age of 61 (range, 28-82) years. Patients received a median of 2 (range, 1-11) prior lines of therapy; 79.1% of patients previously received bortezomib, 26.5% were refractory to lenalidomide, and 64.0% were refractory to their last prior line of therapy. At a median follow-up of 25.1 months, D-Vd prolonged PFS versus Vd alone (median: 14.8 vs 6.3 months; hazard ratio [HR], 0.35; 95% confidence interval [CI], 0.24-0.51; P <0.00001). The PFS benefit of D-Vd versus Vd was maintained across subgroups, including patients with prior bortezomib (HR, 0.36; 95% CI, 0.25-0.53), patients with high-risk cytogenetics (HR, 0.41; 95% CI, 0.23-0.71), and patients who were refractory to last prior line of therapy (HR, 0.42; 95% CI, 0.27-0.65). Overall response rate (ORR; 84.7% vs 66.7%; P = 0.00314) and rates of very good partial response or better (71.5% vs 34.9%; P <0.00001) and complete response or better (40.1% vs 14.3%; P = 0.00016) were higher with D-Vd versus Vd. Median time to first response was numerically shorter (0.79 months vs 1.23 months) and median duration of response was numerically longer (16.8 months vs 9.0 months) with D-Vd versus Vd. The estimated 18-month overall survival rate was 78.8% in the D-Vd group and 65.8% in the Vd group. Summary/Conclusion: In this updated analysis of LEPUS, D-Vd continued to demonstrate substantial efficacy benefits versus Vd in terms of PFS, ORR, and depth of response. These data further support the use of D-Vd as a standard of care in Chinese patients with RRMM. P896: DEVELOPMENT AND VALIDATION OF A FRAILTY PREDICTION MODEL MORE SUITABLE FOR MULTIPLE MYELOMA IN CHINESE PATIENTS: THE TM FRAILTY SCORE. Y. Chen1, J. Gu1, B. Huang1, J. Liu1, X. Li1, J. Li1,* 1Department of Hematology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China Background: Frailty is associated with adverse clinical outcomes in patients with multiple myeloma (MM). Accurate evaluation of frailty is critical when choosing appropriate chemotherapy regimens for elderly patients. Currently, the IMWG geriatric assessment (GA) is widely used to assess tolerance to chemotherapy for elderly patients. However, the applicability of the IMWG GA in the Chinese MM population is limited. Aims: To developed a new frailty model that is more suitable for Chinese patients with MM. Methods: The applicability of the IMWG GA was evaluated in 167 consecutive patients diagnosed with MM from June 2019 to September 2021 at the First Affiliated Hospital of Sun Yat-sen University. Eight comprehensive geriatric assessment (CGA) domains were analyzed in 135 of these patients to screen items most closely associated with the outcomes, including grade ≥ 3 adverse events (AEs), treatment discontinuation, TTP, and OS. A new frailty prediction model was developed, and its fitness and predictive performance were compared to those of the IMWG GA. Results: The patients in the IMWG GA Frail group had a higher rate of grade ≥3 AEs, a higher rate of treatment discontinuation, and a greater risk of death than those in the Fit group (P<0.05). No significant differences were observed in patients in the Int-fit group who experienced chemotherapy AEs, treatment discontinuation, and TTP compared with the Fit group. By screening items in all 8 CGA domains, multivariate analysis confirmed that the Timed Up and Go test（TUG） and the Mini Nutritional Assessment Short-Form（MNA-SF） were independent prognostic factors for grade ≥3 AEs and OS. We found that the TUG and MNA-SF scores could stratify the risk of grade ≥ 3 AEs in the IMWG GA Int-fit group (P < 0.05). After MNA-SF was added to construct the IMWG GA Plus model, Harrell’s concordance index (C-index) of the IMWG GA Plus model for the prediction of grade ≥3 AEs increased from 0.662 to 0.701. The C-index for treatment discontinuation rose from 0.636 to 0.669. We further combined the TUG test and the MNA-SF to construct the TM frailty scoring system. The KM curve analysis showed that the TM frailty score could well distinguish the risk of AEs between the Fit and Frail groups (P<0.05). The C-index of the TM frailty score was 0.741, 0.690, and 0.702 for grade ≥ 3 AEs, treatment discontinuation, and OS, respectively, suggesting good predictive ability, which was higher than that of the IWMG GA (C-index=0.662, 0.636 and 0.631, respectively) and the IMWG GA Plus model (C-index=0.701, 0.656 and 0.618, respectively). Image: Summary/Conclusion: A novel scoring system—the TM frailty score—was developed by combining the TUG (T) and MNA-SF (M). The TM frailty score is more appropriate than the IMWG GA for evaluating frailty in the Chinese population. P897: UPDATED RESULTS FROM THE ONGOING PHASE 1 STUDY OF ELRANATAMAB, A BCMA TARGETED T-CELL REDIRECTING IMMUNOTHERAPY, FOR PATIENTS WITH RELAPSED OR REFRACTORY MULTIPLE MYELOMA A. Dalovisio1,*, N. Bahlis2, N. Raje3, C. Costello4, B. Dholaria5, M. Solh6, M. Levy7, M. Tomasson8, H. Dube9, M. Damore9, S. Jiang10, C. Basu10, A. Skoura11, E. Chan12, S. Trudel13, A. Jakubowiak14, M. Chu15, C. Gasparetto16, M. Sebag17, A. Lesokhin18 1Department of Hematology and Oncology, Ochsner Health, Jefferson, United States of America; 2Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada; 3Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston; 4Moores Cancer Center, University of California San Diego, La Jolla; 5Vanderbilt University Medical Center, Vanderbilt-Ingram Cancer Center, Nashville; 6Blood and Marrow Transplant Group of Georgia, Northside Hospital, Atlanta; 7Department of Medical Oncology, Baylor Scott and White Health, Dallas; 8Holden Comprehensive Cancer Center, University of Iowa, Iowa City; 9Oncology Research and Development; 10Early Clinical Development, Pfizer, San Diego; 11Early Clinical Development, Pfizer, Collegeville; 12Oncology Research and Development, Pfizer, South San Francisco, United States of America; 13Princess Margaret Cancer Centre, University Health Network, Toronto, Canada; 14Department of Medicine, University of Chicago Medical Center, Chicago, United States of America; 15Cross Cancer Institute, Edmonton, Canada; 16Department of Medicine, Duke University Cancer Institute, Durham, United States of America; 17Cedars Cancer Center, McGill University Health Center, Montreal, Canada; 18Division of Hematology and Oncology, Memorial Sloan Kettering Cancer Center/Weill Cornell Medical College, New York, United States of America Background: Elranatamab (PF-06863135), a bispecific molecule that engages B-cell maturation antigen (BCMA) on multiple myeloma (MM) and CD3 on T-cells, induces targeted proliferation and activation of T cells to redirect the immune response against MM. Aims: The Phase 1 study, MagnetisMM-1 (NCT03269136), aims to characterize the safety, pharmacokinetics (PK), pharmacodynamics and efficacy of elranatamab as a single agent or in combination with immunomodulatory agents for patients (pts) with relapsed or refractory MM. Methods: After informed consent, elranatamab was given subcutaneously weekly or every 2 weeks (Q2W) at doses from 80 to 1000µg/kg. A subset of pts received a single priming dose (600µg/kg or 44mg equivalent) followed 1 week later by the recommended Phase 2 dose (RP2D; 1000µg/kg or 76mg equivalent) thereafter. Treatment-emergent adverse events (TEAEs) were graded by Common Terminology Criteria for Adverse Events (v4.03) and cytokine release syndrome (CRS) by American Society for Transplantation and Cellular Therapy criteria. PK, cytokine and soluble BCMA profiling, and lymphocyte subset analyses were performed. Response was evaluated by International Myeloma Working Group (IMWG) criteria. Minimal residual disease (MRD) was assessed by next generation sequencing at a sensitivity of 1×10-5 in accordance with IMWG criteria. Results: A total of 55 pts received elranatamab monotherapy at doses ≥215µg/kg as of 1-Nov-2021. Median age was 64 years (range 42-80), 27% of pts were Black/African American or Asian, and 27% had high risk cytogenetics at baseline. Median number of prior lines of therapy was 6 (range 2-15), 91% were triple-class refractory, 22% received prior BCMA-targeted therapy, and 56% had prior stem cell transplant. The most common TEAEs (all causality) were CRS, neutropenia, anemia, injection site reaction, and lymphopenia. With a single priming dose and premedication, the incidence of CRS at the RP2D was 67% and divided equally between Grade 1 and 2, with no events greater than Grade 2. PK exposure was dose dependent, and elranatamab 1000µg/kg Q2W achieved exposure associated with anti-myeloma activity. Elranatamab therapy induced peripheral T-cell proliferation with a median time to response of 36 days (range 7-73), and the level of soluble BCMA decreased with disease response. With a median follow up of 8.1 months (range 0.3-21) and including only IMWG confirmed responses, overall response rate (ORR) was 64% (95% CI 50-75%), and 31% of pts achieved complete response or better. For responders (n=35), the probability of being event free at 6 months was 91% (95% CI 73-97%). Single-agent elranatamab induced durable clinical and molecular responses, and updated results including serial MRD assessment will be presented. Summary/Conclusion: Elranatamab demonstrates a manageable safety profile and achieves durable clinical and molecular responses for pts with relapsed or refractory MM. P898: COMPARISON OF TECLISTAMAB WITH BELANTAMAB MAFODOTIN IN PATIENTS WITH TRIPLE-CLASS EXPOSED RELAPSED/REFRACTORY MULTIPLE MYELOMA USING MATCHING-ADJUSTED INDIRECT TREATMENT COMPARISON M. Delforge1,*, S. Z. Usmani2, N. W. van de Donk3, A. L. Garfall4, P. Moreau5, A. Oriol6, A. K. Nooka7, L. Rosinol8, N. Bahlis9, P. Rodriguez-Otero10, T. Martin11, J. Diels12, S. Van Sanden12, L. Pei13, E. Ammann14, R. Kobos13, M. Slavcev14, J. Smit15, A. Londhe16, A. Krishnan17 1University of Leuven, Leuven, Belgium; 2Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 3Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 4Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 5University Hospital Hôtel-Dieu, Nantes, France; 6Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain; 7Winship Cancer Institute, Emory University, Atlanta, GA, United States of America; 8Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain; 9Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, AB, Canada; 10Clínica Universidad de Navarra, CIMA, CIBERONC, IDISNA, Pamplona, Spain; 11University of California, San Francisco, San Francisco, CA, United States of America; 12Janssen Pharmaceutica NV, Beerse, Belgium; 13Janssen Research & Development; 14Janssen Global Services, Raritan, NJ; 15Janssen Research & Development, Spring House, PA; 16Janssen Research & Development, Titusville, NJ; 17City of Hope Comprehensive Cancer Center, Duarte, CA, United States of America Background: Treatment options are limited for patients with relapsed/refractory multiple myeloma (RRMM) who are triple-class exposed (TCE) to immunomodulatory drugs, proteasome inhibitors, and anti-CD38 antibodies. While there is no standard of care for treatment of patients with TCE RRMM, belantamab mafodotin (belamaf) is a recently approved, novel therapeutic option. Teclistamab (tec; JNJ-64007957) is a B-cell maturation antigen × CD3 bispecific antibody currently being evaluated in the single-arm, phase 1/2 MajesTEC-1 study (NCT04557098) in patients with TCE RRMM who received ≥3 prior lines of therapy. Aims: Given the absence of a control arm in MajesTEC-1, the efficacy outcomes of patients treated with tec at the recommended phase 2 dose in MajesTEC-1 were compared with those treated with belamaf in the phase 2 DREAMM-2 study (NCT03525678). Methods: An unanchored matching-adjusted indirect comparison was conducted using individual patient-level data (IPD) from 150 patients treated with tec 1.5 mg/kg weekly in MajesTEC-1 (clinical cutoff of Sep 7, 2021), and published summary-level data from 97 patients treated with the approved dose of belamaf (2.5 mg/kg every 3 weeks) in DREAMM-2. After applying the DREAMM-2 eligibility criteria to patients from the intent-to-treat population of MajesTEC-1, IPD from MajesTEC-1 were weighted to match the aggregated DREAMM-2 baseline patient characteristics. Baseline characteristics of prognostic significance (such as cytogenetic profile, International Staging System stage, presence of extramedullary disease, number of prior lines of therapy, and refractory status) were adjusted for the analysis. Comparative efficacy of tec vs belamaf was estimated for overall response rate (ORR), complete response or better (≥CR) rate, duration of response (DOR), overall survival (OS), and progression-free survival (PFS). An odds ratio (OR) and 95% confidence interval (CI) derived from a weighted logistic regression analysis was used to compare the relative effects of tec vs belamaf for binary outcomes (ORR and ≥CR rate). A weighted Cox proportional hazards model was used to estimate time-to-event endpoints (DOR, OS, and PFS). Results: After adjustment, the effective sample size of the MajesTEC-1 cohort was 33. Baseline characteristics were balanced between MajesTEC-1 and DREAMM-2 after reweighting the MajesTEC-1 cohort. Tec-treated patients had improved outcomes compared with patients treated with belamaf: ORR (OR 2.05; 95% CI 0.92–4.57; P=0.0786), ≥CR rate (OR 2.13; 95% CI 0.80–5.65; P=0.1283), DOR (hazard ratio [HR] 0.19; 95% CI 0.05–0.73; P=0.0149), OS (HR 0.95; 95% CI 0.47–1.92; P=0.8897), and PFS (HR 0.63; 95% CI 0.34–1.15; P=0.1338). For most outcomes, the lack of statistical significance may be due to the reduced effective sample size following adjustment. Summary/Conclusion: In this comparative analysis, tec showed statistically improved DOR when compared with belamaf and numerically favorable results for other outcomes. This highlights the potential of tec as a treatment option for patients with TCE RRMM who received ≥3 prior lines of therapy. P899: REAL-WORLD ASSESSMENT OF TREATMENT PATTERNS AND OUTCOMES IN PATIENTS WITH LENALIDOMIDE-REFRACTORY RELAPSED/REFRACTORY MULTIPLE MYELOMA FROM THE OPTUM DATABASE B. Dhakal1,*, H. Einsele2, R. Potluri3, J. Schecter4, W. Deraedt5, N. Lendvai4, A. Slaughter6, C. Lonardi7, S. Nair5, J. He8, N. Joseph9, P. Cost8, S. Valluri8, F. Yalniz10, L. Pacaud10, K. Yong11 1Medical College of Wisconsin, Milwaukee, United States of America; 2Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Wuerzburg, Germany; 3SmartAnalyst Inc, New York; 4Janssen R&D, Raritan, United States of America; 5Janssen Pharmaceutica NV, Beerse, Belgium; 6Cilag GmbH International, Zug, Switzerland; 7Janssen, Buenos Aires, Argentina; 8Janssen Global Services, LLC, Raritan; 9Janssen Scientific Affairs, LLC, Horsham; 10Legend Biotech USA, Piscataway, United States of America; 11University College London Cancer Institute, London, United Kingdom Background: New treatment combinations using proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and anti-CD38 monoclonal antibodies have significantly improved survival outcomes in patients with multiple myeloma (MM). However, for patients who relapse and/or become refractory after exposure to PIs and IMiDs, selecting the next regimen remains a challenge. There are limited data characterizing treatments and outcomes in this difficult-to-treat population. Aims: To characterize treatment patterns and outcomes in difficult-to-treat patients with MM, stratified by their number of prior lines of therapy (PL). Methods: Data were derived from the Optum deidentified US claims and electronic health record (EHR) database. Patients with MM (index diagnosis in or after Jan 2011) were selected if they received 1-3 PL, including with a PI and an IMiD, and were refractory to lenalidomide. Subsequent treatments beginning in or after Jan 2016 were included to focus on contemporary therapies. Time zero (T0) was the date when the first subsequent therapy started after patient met inclusion criteria. Patient characteristics were assessed at T0. Time-to-event analyses were estimated using the Kaplan-Meier method (starting at T0) for overall survival (OS) and time to next treatment (TTNT). The log-rank test for trend was applied to compare Kaplan-Meier curves for OS by line of therapy. Results: The database included a total of 13,615 (claims) and 22,626 (EHR) patients with an index diagnosis of MM and ≥1 line of therapy. 1,028 (claims) and 1,416 (EHR) patients met inclusion criteria. Median age was 72 (claims) and 68 (EHR) years; 53.4% (claims) and 52.1% (EHR) were male. 12.2% (claims) and 4.9% (EHR) of patients received stem cell transplant prior to T0. Patients in both cohorts had significant comorbidities with mean Charlson Comorbidity Index 3.9 (claims) and 3.2 (EHR). Hypertension and renal failure were among the most prominent comorbidities. The most common subsequent treatment regimens based on hierarchy were daratumumab (22.1% claims; 17.4% EHR), carfilzomib (10.6% claims; 18.4% EHR), and pomalidomide based (12.3% claims; 8.7% EHR). Daratumumab/pomalidomide/dexamethasone was the most frequently used regimen (5.4% claims; 3.8% EHR). For claims data, median OS (months) was 36.7 (95% CI: 31.7-41.2). Median OS was not reached (42.3-NE) for patients with 1 PL (n=546), was 26.2 (21.7-35.7) for patients with 2 PL (n=380) and was 12.1 (7.6-25.0) for patients with 3 PL (n=102). Median TTNT (months) was 5.3 (4.9-5.9) overall, including 4.6 (4.3-5.1) for patients with 1 PL, 6.4 (5.3-7.6) for patients with 2 PL, and 6.0 (4.9-9.4) for patients with 3 PL. For EHR data, median OS was 34.0 (28.2-42.8), with median OS of 46.9 (42.8-NE) for patients with 1 PL (n=587), 28.2 (23.8-41.7) for patients with 2 PL (n=584), and 20.8 (14.9-29.8) for patients with 3 PL (n=245). Median TTNT was 5.8 (5.1-6.5) overall, with values of 5.1 (4.7-6.5) for patients with 1 PL, 6.2 (5.3-7.6) for patients with 2 PL, and 5.8 (4.4-8.2) for patients with 3 PL. Image: Summary/Conclusion: PI-exposed, lenalidomide-refractory patients with 1-3 PL were treated with various regimens. These patients have poor OS and progress quickly through current therapies, suggesting poor progression-free survival. OS is reduced with each successive line of therapy. This analysis highlights the need for new effective regimens for this patient population. P900: SALVAGE AUTOLOGOUS STEM CELL TRANSPLANT IN RELAPSED MULTIPLE MYELOMA: A SINGLE CENTRE EXPERIENCE S. Duarte1,*, A. Roque1,2, D. Mota1, J. Carda1,2, C. Geraldes1,2 1Clinical Hematology Department, University Hospital Center of Coimbra; 2Faculty of Medicine, University of Coimbra, Coimbra, Portugal Background: Multiple myeloma (MM) is an incurable disease and most patients (pts) will relapse during their lifetime. Although salvage therapy has improved with the advent of new drugs, salvage autologous stem cell (ASC) transplant (SAT) remains appropriate for pts relapsing after primary therapy that includes ASCT with initial remission duration >18 months (mo). Aims: Evaluate the outcome of SAT in MM in the new drugs era and efficacy and toxicity associated to this treatment. Methods: Single centre retrospective analysis of MM pts submitted to SAT between 2007-2021. Tandem ASCT excluded. Engraftment defined according to EBMT criteria. Results: Fifty-three pts submitted to SAT, 58.5% male, median age of 60 yo (37-71), 27.8% with international staging system score III. Median number of prior lines of therapy before SAT was 2 (2-4), 51 (96.2%) including proteasome inhibitors (PI), 38 (71.7%) immunomodulators (IMiD) and 13 (24.5%) daratumumab. Twenty-five (50%) pts submitted to maintenance therapy with thalidomide (Thal) after ASCT and 15 (30.6%) with lenalidomide (Len) after SAT. Pre-SAT response to therapy was complete remission (CR) in 9.6%, very-good partial response (VGPR) in 50% and PR in 40.4%. Most pts received conditioning with melphalan 200mg/m2 for both transplants (1st: 100%, 2nd: 96.2%). After SAT, median time to neutrophil engraftment was 12 days (6-21) and for platelet recovery was 13 days (3-26). Grade 3 oral and gastrointestinal mucositis observed in 14% and 6%, respectively, and febrile neutropenia in 91% of pts. SAT response evaluation showed CR in 28.3%, VGPR in 32.1% and PR in 37.7%. One patient showed progressive disease. No treatment related mortality (TRM) at 100 days post-SAT. Median PFS after 1st ASCT was 32 mo. Median time between 1st and 2nd ASCT was 46 mo (22-140). Median PFS and OS after SAT was 28 and 78 mo, respectively. Comparison between PFS post-ASCT and post-SAT was not significant (P=0.09). ISS stage with no impact on PFS (HR 1.99; p=0.263) and OS (HR 2.03; p=0.398), after SAT. No statistically significant difference in post-SAT PFS (HR 1.23 p=0.592) and OS (HR 1.84, p=0.312) between patients submitted to transplant before and after 2016, despite more intensive therapeutic protocols (100% triplets and 24.5% anti-CD38-based regimens in former period compared with duplets in the first). PFS of pts who reached CR post-SAT was significantly higher compared to pts in VGPR/PR (NR vs 22 mo, HR 2.97, p=0.044). However, CR post-SAT with no impact on OS (NR vs 118 mo, HR 1.27 p=0.71). Maintenance therapy with (Thal) post-ASCT with no impact on PFS (33 vs 32 mo, HR 1.24, p=0.47). Similarly, the impact of post-SAT maintenance with Len on PFS (51 vs 28 mo, HR 1.35, p=0.49) and OS (NR vs 73 mo, HR 4.40, p=0.157) was not significant. Pts with remission time duration after 1st ASCT ≥36 mo had median PFS and OS after SAT significantly longer (51 vs 22 mo, HR 2.41, p=0.024, and NR vs 71 mo, HR 6.24, p=0.015, respectively), compared to pts with post-ASCT remissions <36 mo. Univariate analysis failed to show other clinical and analytical variables with impact on PFS and OS. Summary/Conclusion: In our cohort, median PFS and OS after SAT was 78 mo and 28 mo, respectively. Pts with remissions after 1st ASCT ≥36 mo showed significantly longer PFS and OS. PFS was significantly improved in pts reaching CR after SAT. Post-SAT Len maintenance improved PFS and OS, however with no statistical significance. Severe toxicities were rare and never resulted in TRM. Overall, our centre experience shows that SAT is an effective and safe treatment in MM in the new drugs era. P901: POMALIDOMIDE, BORTEZOMIB, AND DEXAMETHASONE IN LENALIDOMIDE–PRETREATED MULTIPLE MYELOMA: A SUBANALYSIS OF OPTIMISMM BY FRAILTY A. Oriol1,*, M. Dimopoulos2, F. Schjesvold3,4, M. Beksac5, M. Yagci6, A. Larocca7, S. Guo8, Y. Mu8, K. Hong9, S. Dhanasiri10, P. Richardson11, K. Weisel12 1Hematology Department, Institut Català d’Oncologia, Barcelona, Spain; 2National and Kapodistrian University, Athens, Greece; 3Oslo Myeloma Center, Department of Hematology, Oslo University Hospital; 4KG Jebsen Center for B Cell Malignancies, University of Oslo, Oslo, Norway; 5Ankara Üniversitesi Tip Fakültes; 6Gazi University Medical Faculty, Ankara, Turkey; 7SSD Clinical trials in onco-ematologia e mieloma multiplo, Division of Hematology, University of Torino, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino, Torino, Italy; 8Evidera, Waltham; 9Bristol Myers Squibb, Princeton, United States of America; 10Celgene International Sàrl, a Bristol-Myers Squibb Company, Boudry, Switzerland; 11Dana-Farber Cancer Institute, Boston, United States of America; 12University Medical Center Hamburg-Eppendorf, Hamburg, Germany Background: Patients (pts) with multiple myeloma (MM) are likely to be older adults, and advanced age is associated with lower survival rates, in part due to comorbidities and frailty. Results from the phase 3 OPTIMISMM trial (NCT01734928) demonstrated that pomalidomide (P) in combination with bortezomib + dexamethasone (Vd) significantly improved progression-free survival (PFS) in lenalidomide (LEN)–pretreated pts with relapsed/refractory MM (RRMM) vs Vd, irrespective of age and Eastern Cooperative Oncology Group performance status (ECOG PS). Aims: Here, we report results from a post hoc analysis assessing outcomes of PVd vs Vd in pts with RRMM by frailty status. Methods: Pts with MM and 1–3 prior lines of therapy (including a LEN-containing regimen) who had been randomized 1:1 to PVd or Vd were assessed for frailty. Frailty scores were calculated using age (≤ 75 yr = 0; 76–80 yr = 1; > 80 yr = 2), the Charlson Comorbidity Index (≤ 1 = 0; > 1 = 1), and ECOG PS (0 = 0; 1 = 1; ≥ 2 = 2). Pts were classified as non-frail (NF; combined score: 0 or 1) or frail (F; combined score: ≥ 2). PFS, overall response rate (ORR), and safety outcomes were assessed by treatment group and frailty status. Results: In the intent-to-treat population (N = 559) (data cut-off Oct 26, 2017), 93/281 (33.1%) pts who received PVd and 93/278 (33.5%) who received Vd were frail. Baseline characteristics were similar between treatment groups within each frailty subgroup. Median PFS was longer with PVd vs Vd in NF (P = 0.001) and F (P = 0.006) subgroups (Table). ORR was higher with PVd vs Vd in NF (P < 0.001) and F (P < 0.001) subgroups. Incidence of grade ≥ 3 (G3) treatment-emergent adverse events (TEAEs) was higher with PVd vs Vd in NF (88.1 vs 61.3%) and F (96.8 vs 87.9%) subgroups; peripheral neuropathy, acute renal failure, and hypertension were the most common. Treatment discontinuation due to G3 TEAEs was greater with PVd vs Vd in NF (19.2 vs 18.5%) and F (30.1 vs 20.9%) subgroups. PVd had a longer median treatment duration vs Vd in NF (8.8 vs 5.7 mo) and F (8.9 vs 4.3 mo) subgroups. Image: Summary/Conclusion: Frail pts with RRMM who received PVd had longer PFS and higher ORR than pts who received Vd, consistent with the overall OPTIMISMM results. Frail pts experienced more G3 TEAEs and treatment discontinuations with PVd vs Vd, but treatment duration was longer with PVd vs Vd. P902: RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS. A MULTICENTER RETROSPECTIVE ANALYSIS OF ELIGIBILITY CRITERIA FOR CAR-T CELL THERAPY F. Fazio1,*, A. Di Rocco1, M. T. Giaimo2, T. Za3, N. Ciccone4, M. Sessa4, B. Gamberi5, V. Tomarchio6, L. De Padua7, V. Bongarzoni8, S. Mariani9, A. Rago10, A. Piciocchi11, F. Merli5, A. Cuneo4, V. De Stefano3, R. Foà1, M. Martelli1, M. T. Petrucci1 1Hematology - Azienda Policlinico Umberto I, Department of Traslational and Precision Medicine Sapienza University of Rome, Roma; 2Hematology - Azienda Ospedaliera Parma, University of Parma, Parma; 3Hematology - Fondazione Policlinico A. Gemelli IRCCS, Department of Radiological and Hematological Sciences, Catholic University, Roma; 4Hematology - Ospedale Sant’Anna, University of Ferrara, Ferrara; 5Hematology, AUSLL/IRCCS Santa Maria Nuova Hospital, Reggio Emilia; 6Hematology - Stem Cell Transplantation, University Campus Bio Medico, Roma; 7Hematology - Ospedale Fabrizio Spaziani, Frosinone; 8Hematology - Azienda Ospedaliera San Giovanni; 9Hematology - Ospedale Sant’Andrea, Sapienza University of Rome; 10UOSD Hematology ASLRoma 1; 11Italian Group For Adult Hematologic Diseases (GIMEMA), Roma, Italy Background: The overall survival (OS) of multiple myeloma (MM) patients (pts) has improved over the years due to the introduction of novel drugs, such as proteosome inhibitors (PI), immunomodulatory drugs (IMiDs) and anti- CD38 monoclonal antibodies (moAb). Nevertheless, the majority of pts continues to relapse and MM remains an incurable disease. No standard of care has been established for relapsed/refractory (RR) MM pts who have been exposed to the main anti-myeloma drugs. The outcome of pts failing standard of care regimens, which is now defined as triple-refractory, is poor, with a median progression-free survival (PFS) of 3-4 months and OS of 8-9 months. Novel therapeutic strategies are warranted to overcome the natural occurrence of relapse or therapy resistance in RRMM pts. Chimeric antigen receptor (CAR)-modified T cells are a promising new therapy approach for triple refractory RRMM. Specific CAR-T targets are being studied, but BCMA-directed CAR-T cells have so far provided the most convincing evidence of activity, with one product (idecabtagene vicleucel) recently approved by FDA and EMA. Aims: The primary endpoint of this observational and retrospective study was to define the clinical characteristics and outcome of a cohort of RRMM pts potentially eligible to CAR-T cell treatment according to the KarMMa trial criteria. Secondary endpoints were aimed at defining specific factors influencing CAR-T cell therapy eligibility and at identifying a real-life estimate of RRMM pts truly eligible for CAR-T cells. Methods: This is a cohort analysis on RRMM pts managed between January 2018 and July 2021 at 10 Italian hematology centers. At the time of data collection, 108 RRMM pts had underwent at least 3 prior therapy regimens; they had received a previous PI, IMiDs and a moAb, and were considered refractory to the last regimen. Results: Median age was 68 years (38-86); 63 (59%) pts were >65 years; 57 (53%) were male. Of 108 pts, 87 (80%) were ECOG 0-1 and 33 (35%) were ISS III. The majority of pts, 72 (67%), had undergone an autologous stem cell transplantation; 93 pts received >3 prior lines of therapy. Sixty-seven (62%) were triple-refractory and 41 (38%) were penta-refractory. Based on the KarMMa trial criteria, 49/108 pts (45%) would be defined as eligible and 59 (55%) not eligible for CAR-T cell therapy. Organ dysfunction such as impaired renal function, anemia, thrombocytopenia, neutropenia and a FEV <45% was the most common criteria for ineligibility (86%). Twenty-one pts (19%) were not eligible because of an ECOG >2. Of the 59 pts considered ineligible for CAR-T cell therapy, 46 (62%) presented ≥2 ineligibility criteria. After a median follow-up of 27 months (mo) (8-40.5), the median OS for the entire cohort was 21.1 mo (Fig. 1A). The median OS was 33 mo in eligible pts vs 8.3 mo in non-eligible pts (p=0.002) (Fig. 1B). The median PFS of the entire cohort was 8.7 mo (Fig. 1C) and the median PFS was 19.4 mo in eligible pts vs 6 mo (p=0.001) in non-eligible pts (Fig. 1D). Image: Summary/Conclusion: Despite the limits of a retrospective study and a limited cohort, our real-life analysis shows that heavily treated pts with RRMM are less likely to be eligible for CAR-T cell therapy. Considering the emergent role of quadruplet combined approaches for first- line therapy and given the therapeutic relevance of CAR-T cells for the management of RRMM pts previously exposed to PI, IMiDs and moAb, our data could help to better define pts who could benefit from CAR-T cells under the current indications, while waiting for an extension of this approach to earlier disease stages. P903: DARATUMUMAB PLUS BORTEZOMIB AND DEXAMETHASONE IN NEWLY DIAGNOSED PATIENTS WITH MAYO 2004 STAGE 3 LIGHT-CHAIN AMYLOIDOSIS: A PROSPECTIVE PHASE 2 STUDY Y.-J. Gao1,*, K.-N. Shen1, L. Chang1, J. Feng1, L. Zhang1, Y.-Y. Mao1, X.-X. Cao1, D.-B. Zhou1, J. Li1 1Department of Hematology, Peking Union Medical College Hospital, Beijing, China Background: Patients with systemic light-chain (AL) amyloidosis at the advanced cardiac stage exhibit extremely poor survival. Although daratumumab has shown superior outcome in treatment of AL amyloidosis according to the result of ANDROMEDA trial, whether these feeble patients can benefit from daratumumab therapy need further investigation. Aims: To prospectively explore the value of daratumumab plus bortezomib and dexamethasone in patients with AL amyloidosis at the advanced heart-stage. Methods: This is a phase 2, open-label, single center clinical trial planning to include 40 newly diagnosed patients with Mayo 2004 stage 3a and 3b AL amyloidosis at Peking Union Medical College Hospital (Beijing, China). Eligible patients should have measurable hematological disease (baseline dFLC >50mg/L). Initiation treatment includes daratumumab (intravenously at 16 mg/kg weekly during cycles 1-2, once every two weeks during cycles 3-6 and once every 4 weeks thereafter for up to 12 cycles), bortezomib (at 1.3mg/m2 subcutaneously weekly during cycles 1-6) and dexamethasone (20mg weekly during cycles 1-6). Each cycle consists of 4 weeks. Treatment responses are evaluated every week during the first cycle and at the end of each cycle since cycle 2. Termination of the therapy is considered when patients have progressed disease, serious side effects related to treatment or initiation of the second-line treatment. (ClinicalTrials.gov identifier: NCT04474938） Results: From 28th May, 2021 to 28th January, 2022, 38 patients were enrolled. Twenty-nine (76.3%) patients were male and the median age was 59 years (range 41-77). The median NT-proBNP was 10665 pg/ml (range 803 ~ >35000) and the median cTnI was 0.17ug/L (range 0.07-3.07). Twenty-one patients (55.3%) were stage 3b. Median dFLC was 265 mg/L (range 72-2966). Twenty-four patients (63.2%) had NYHA class III or IV heart function. The median number of organs involved was 2 (range 0-4). Fourteen (36.8%) patients had kidney involvement and nine (23.7%) patients had liver involvement. The median number of treatment cycles was 3.25 (range 0.25-9). For the best hematologic response, 34 of 38 patients (89.5%) reached ≥PR, including 18 patients (47.4%) with CR and 8 (21.1%) patients with VGPR. At the end of the first cycle, 8 patients (22.9%) achieved CR; 12 patients (34.3%) reached VGPR; 9 patients (25.7%) had PR and 6 patients (17.1%) were NR. The hematological ORR was 74.1% at 3 months (40.7% with CR and 18.5% with VGPR). The median time to the first hematologic remission was 7 days (range 7-21) and the median time to ≥VGPR was 14 days (range 7 days – 3 months). The cardiac response rate was 18.5% at 3 months and 23.5% at 6 months. After the median follow-up time of 4.5 months (range 0.7-8.6), the median OS was not reached. The 6-month survival rate of all patients was 76.6%, with 84.7% for stage 3a and 69.0% for stage 3b. The most common grade 3 or 4 adverse events were infection (n=8, 21.1%). Infusion reaction was recorded in 5 patients (13.2%) and only 1 patient was categorized as grade 3 reaction. All of them occurred during the first time of infusion. Other serious adverse effects included grade 3 diarrhea (n=3), pneumothorax (n=1), bone fracture (n=1), deep venous thrombosis (n=1), hematuresis (n=1), gastrointestinal bleeding (n=1), ischemic stroke (n=1) and intestinal obstruction (n=1). No patients withdrew due to adverse events. Image: Summary/Conclusion: Our results showed that daratumumab plus bortezomib and dexamethasone had favorable safety and potential advantage among patients with AL amyloidosis presenting severe cardiac involvement. P904: CILTACABTAGENE AUTOLEUCEL VS TREATMENTS FROM REAL-WORLD CLINICAL PRACTICE FOR TRIPLE CLASS EXPOSED PATIENTS WITH MULTIPLE MYELOMA: ADJUSTED COMPARISONS BASED ON CARTITUDE-1 AND THE EMMY FRENCH COHORT O. Decaux1,*, C. Hulin2, A. Perrot3, M. Macro4, L. Frenzel5, J. Diels6, N. J. Perualila6, F. Ghilotti7, B. Haefliger8, E. Goldsztajn9, S. Vernet10, J. Thevenon10, M. Willaime11, N. Texier11, J. M. Schecter12, D. Madduri12, C. Jackson12, S. Valluri13, P. Moreau14 1Internal medicine and clinical immunology, CHU Rennes, Rennes; 2Hematology and cell therapy, CHU Bordeaux, Bordeaux; 3Hematology, Oncopole, Toulouse; 4Hematology, CHU Caen, Caen; 5Hematology, Necker-enfants malades hospital, Paris, France; 6Health Economics, Market Access & Reimbursement, Janssen Pharmaceutica NV, Beerse, Belgium; 7Health Economics, Market Access & Reimbursement, Janssen Pharmaceutica NV, Cologno Monzese, Italy; 8Health Economics, Market Access & Reimbursement, Cilag GmbH International, Zug, Switzerland; 9Market Access & Reimbursement; 10Medical affairs, Janssen, Issy-les-Moulineaux; 11Biometry, Kappa Sante, Paris, France; 12Research and Development, Janssen R&D; 13Health Economics, Market Access & Reimbursement, Janssen, Raritan, United States of America; 14Clinical hematology, University Hospital Hotel-Dieu, Nantes, France Background: The prognosis of patients with triple-class (TC) exposed, relapsed and refractory multiple myeloma (RRMM) is poor. CARTITUDE-1 is an open-label, single arm, phase 1b/2 clinical trial performed to assess the safety and efficacy of ciltacabtagene autoleucel (cilta-cel), a novel chimeric antigen receptor T-cell therapy. Adjusted comparisons to other therapies used in real-world clinical practice (RWCP) help inform the relative efficacy of cilta-cel in TC exposed patients. The Epidemiology of the therapeutic management of Multiple MYeloma in France (EMMY) study is a retrospective study capturing information on the therapeutic management of patients from 71 centers in France, and can serve as an external control arm for CARTITUDE-1. Aims: To compare cilta-cel with treatments from RWCP in terms of overall response rate (ORR), very good partial response or better rate (≥VGPR), progression-free survival (PFS), and overall survival (OS). Methods: Adjusted comparisons were estimated using individual patient data (IPD) from CARTITUDE-1 (cut-off date July 2021) and reconstructed IPD from EMMY (cut-off date March 2021). Within EMMY, all treatment lines initiated after a patient met eligibility criteria (TC exposed patients in L4+ and ECOG<2) were used for analysis, so long as eligibility remained intact. Inverse probability weighting (IPW) was used to balance the cilta-cel and RWCP groups on prognostic baseline variables including refractory status, time to progression on prior line of treatment (LOT), number of prior LOTs, ECOG performance status, age, sex, years since MM diagnosis, average duration of prior LOTs, MM type, and prior transplant. Weights derived from propensity scores estimated with a multivariable logistic regression and were assigned to EMMY patients, such that the weighted EMMY cohort reflected the CARTITUDE-1 population. Balance was assessed using standardized mean differences (SMD). The relative efficacy of cilta-cel versus RWCP was assessed using relative response rates (RR) for binary outcomes and hazard ratios (HR) for time-to-event outcomes, using weighted logistic regression and proportional hazards modeling, respectively. Main analyses were performed in the population of infused patients in the cilta-cel group and an aligned population of EMMY RWCP patients that included those still progression-free after 52 days (mean time between apheresis and cilta-cel infusion). Similar analyses were performed for all enrolled patients. Results: 113 patients were enrolled in CARTITUDE-1 and 97 received cilta-cel infusions. 437 LOTs (238 patients) from EMMY were included in the RWCP external control arm and 309 LOTs (227 patients) were alive and progression-free after 52 days. More than 100 treatment regimens within the RWCP group were used. After reweighting, baseline traits were balanced between groups, with all SMD <0.20. ORR (RR 5.1, 95% CI: 3.7;7.1) and ≥VGPR (RR 14.5 [8.0; 26.1] were superior with cilta-cel vs. RWCP, as were both PFS (HR 0.15 [0.10; 0.22]) and OS (HR 0.21 [0.12; 0.37]). Results for the enrolled populations were consistent (see Table). Image: Summary/Conclusion: Adjusted comparisons between cilta-cel and RWCP in France showed superior efficacy on all outcomes for cilta-cel vs. RWCP, with 5.1 and 14.5-fold higher response rates for ORR and ≥VGPR, respectively, and reduced risks of disease progression/death and death by 85% and 79% with cilta-cel versus RWCP. These findings align with results from similar comparisons in other countries. Cilta-cel offers substantial clinical benefits for patients with triple class exposed RRMM. P905: IXAZOMIB-THALIDOMIDE-DEXAMETHASONE INDUCTION FOLLOWED BY IXAZOMIB OR PLACEBO MAINTENANCE IN NON-TRANSPLANT ELIGIBLE NEWLY DIAGNOSED MULTIPLE MYELOMA PATIENTS; LONG-TERM RESULTS OF HOVON-126/NMSG 21.13 K. Groen1,*, M. R. Seefat1, B. van der Holt2, F. H. Schjesvold3,4, C. A. Stege1, M.-D. Levin5, M. Hansson6,7, R. B. Leys8, J. Regelink9, A. Waage10, D. Szatkowski11, P. Axelsson12, T. H. Do13, A. Svirskaite14, E. van der Spek15, E. Haukas16, D. Knut-Bojanowska17, P. F. Ypma18, C. Blimark19, U.-H. Mellqvist20, N. W. van de Donk1, P. Sonneveld21, A. Klostergaard22, A. J. Vangsted23, N. Abdilgaard24,25, S. Zweegman1 1Hematology, Amsterdam UMC, Amsterdam; 2HOVON Data Center, Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, Netherlands; 3Hematology, Oslo Myeloma Center; 4KG Jebsen center for B Cell malignancies, University of Oslo, Oslo, Norway; 5Department of Internal Medicine, Albert Schweitzer Hospital, Dordrecht, Netherlands; 6Skane University Hospital, Lund; 7Sahlgrenska Academy, Göteborg, Sweden; 8Department of Internal Medicine, Maasstad Ziekenhuis, Rotterdam; 9Department of Internal Medicine, Meander Medisch Centrum, Amersfoort, Netherlands; 10IKOM, Norwegian University of Science and Technology, Trondheim; 11Førde Central Hospital, Førde, Norway; 12Skanes University Hospital, Lund, Sweden; 13Herlev Hospital, Herlev; 14Aalborg Hospital, Aalborg, Denmark; 15Department of Internal Medicine, Rijnstate, Arnhem, Netherlands; 16Stavanger University Hospital-Rogaland Hospital, Stavanger, Norway; 17NU-Hospital - Uddevalla Hospital, Uddevalla, Sweden; 18Department of Hematology, Haga Ziekenhuis, Den Haag, Netherlands; 19Sahlgrenska University Hospital, Gothenburg; 20Sodra Alvsborgs Sjukhus Boras, Boras, Sweden; 21Erasmus Medical Center Cancer Institute, Rotterdam, Netherlands; 22Aarhus University Hospital, Aarhus; 23Department of Hematology, Rigshospitalet, Copenhagen; 24Department of Hematology; 25Academy or Geriatric Cancer Research, Odense University Hospital, Odense, Denmark Background: In the HOVON 126/NMSG 21.13 trial non-transplant eligible newly diagnosed multiple myeloma (NTE-NDMM) patients were treated with 9 induction cycles of ixazomib, thalidomide and dexamethasone (ITd), followed by randomization between either ixazomib or placebo until progression or unacceptable toxicity. The overall response rate and PFS data have been previously published. Aims: We here present the long-term PFS2 and overall survival data. Methods: Patients were treated with 9 induction cycles (28 days) of ixazomib (4mg on day 1, 8 and 15), thalidomide (100mg on day 1-28) and dexamethasone (40mg on day 1, 8, 15 and 22), followed by maintenance with either ixazomib or placebo (4mg, both on day 1, 8 and 15, every 28 days). Patients were classified as fit, intermediate fit or frail, based on a modified IMWG frailty index which incorporated age, the Charlson Comorbidity Index (CCI) and the WHO performance as a proxy for (instrumental) Activities of Daily Living (iADL) (scoring WHO 0 as 0 points, WHO 1 as 1 point, and WHO 2-3 as 2 points). Results: From registration: 143 eligible patients were included in the study. After a median follow-up (FU) of 67.4 months (m), the median PFS was 14.3m (95% CI 11.5-16.8), median PFS2 was 34.6m (30.7-41.5) and median OS was 58.3m (50.5-65.0). There was no difference in PFS between frailty subgroups. In contrast, median PFS2 and OS were longer in fit patients (PFS2: 49.1m (34.6-74.1), OS: NR (66.6-NR)) versus intermediate-fit (30.1m (25.1-39.0); 51.2m (32.3-63.9) resp.) and frail patients (30.9m (24.0-42.3); 50.5m (32.9-59.4) resp.). From randomization: 78 (55%) patients were randomized, 39 patients in each arm. After a median FU of 60 months from randomization, there was no difference in PFS between the ixazomib-arm (median 9.5m; 95% CI 5.5-14.8) and the placebo-arm (8.4m; 3.0-13.8). Median PFS2 was 39.8m (28.8-60.0) for patients on ixazomib, as compared to 28.7m (22.8-43.2) for patients in the placebo arm, although this difference was not statistically significant. Median OS was not reached for the ixazomib arm and was 50.7m (41.3-58.1) for the placebo arm (HR 0.39; 95% CI 0.19-0.78, p=0.008). In both arms 32 (82%) patients received 2nd line treatment. With the caveat of low numbers and heterogeneous treatment regimens, more patients in the ixazomib arm received daratumumab-lenalidomide-dexamethasone (4 patients, 13%) and panobinostat-bortezomib-dexamethasone (6 patients, 19%), compared to the placebo arm (2 patients (6%) and 3 patients (9%) respectively). In order to explain the difference in OS, subsequent lines of therapy are currently being investigated. Image: Summary/Conclusion: With longer FU, we here confirm that ixazomib maintenance therapy did not improve PFS, compared to placebo. However, PFS2 tends to be longer and OS was superior in patients treated with ITd followed by maintenance with ixazomib versus placebo. P906: IXAZOMIB, DARATUMUMAB AND LOW DOSE DEXAMETHASONE IN FRAIL PATIENTS WITH NEWLY DIAGNOSED MULTIPLE MYELOMA (NDMM): RESULTS OF THE MAINTENANCE TREATMENT OF THE PHASE II HOVON 143 STUDY K. Groen1,*, M. Seefat1, K. Nasserinejad2, C. A. Stege1, E. van der Spek3, Y. M. Bilgin4, A. Kentos5, M. Sohne6, R. J. van Kampen7, I. Ludwig8, N. Thielen9, N. Durdu-Rayman10, N. C. de Graauw11, N. W. van de Donk1, E. G. de Waal12, M.-C. Vekemans13, G. J. Timmers14, M. van der Klift15, S. Soechit16, P. A. Geerts17,18, M. H. Silbermann19, M. Oosterveld20, I. Nijhof1,21, P. Sonneveld22, S. K. Klein23, M.-D. Levin24, S. Zweegman1 1Hematology, Amsterdam UMC, Amsterdam; 2HOVON Data Center, Department of Hematology, Erasmus MC Cancer Institute, Rotterdam; 3Department of Internal Medicine, Rijnstate Hospital, Arnhem; 4Department of Internal Medicine, Admiraal de Ruijter Hospital, Goes, Netherlands; 5Department of Hematology, Centre Hospitalier Jolimont, Haine-Saint-Paul, Belgium; 6Department of Internal Medicine/Hematology, St Antonius Hospital, Nieuwegein; 7Department of Internal Medicine, Zuyderland Medical Center, Sittard-Geleen; 8Department of Hematology, Bernhoven Hospital, Uden; 9Department of Internal Medicine, Diakonessenhuis, Utrecht; 10Department of Internal Medicine, Franciscus Hospital, location Vlietland, Schiedam; 11Department of Internal Medicine, Bravis ziekenhuis, Roosendaal; 12Department of Internal Medicine, Medisch Centrum Leeuwarden, Leeuwarden, Netherlands; 13Department of Hematology, St Luc Hospital, Bruxelles, Belgium; 14Department of Internal Medicine, Amstelland Hospital, Amstelveen; 15Department of Internal Medicine, Amphia Hospital, Breda; 16Department of Hematology, Reinier de Graaf Groep, Delft; 17Department of Internal Medicine, Deventer Hospital, Deventer; 18currently Isala, Zwolle; 19Department of Internal Medicine, Tergooi Hospital, Hilversum; 20Department of Internal Medicine, Canisius-Wilhelmina Hospital, Nijmegen; 21currently St Antonius Hospital, Nieuwegein; 22Department of Hematology, Erasmus MC Cancer Institute, Rotterdam; 23Department of Internal Medicine, Meander Medical Center, Amersfoort; 24Department of Internal Medicine, Albert Schweitzer Hospital, Dordrecht, Netherlands Background: Frail patients with newly diagnosed multiple myeloma (NDMM) have an inferior PFS and OS, a higher treatment discontinuation rate and more grade ≥3 non-hematologic toxicity, compared to fit patients. In order to improve their outcome we investigated a three-drug regimen with a presumed low-toxicity profile; ixazomib, daratumumab and low-dose-dexamethasone (Ixa-Dara-dex). This trial is registered at www.trialregister.nl as NTR6297. Aims: To present the long term follow up outcome, with an emphasis on the maintenance phase. Methods: NDDM patients, who were frail according to the IMWG-Frailty Index, were included. Patients defined frail based on age only (frail-age) were compared to frail patients based on other reasons (impairments (i)ADL and/or CCI≥2; frail-other) and patients frail based on age as well as other reasons (frail-both). After nine cycles of Ixazomib (4mg; days 1, 8, 15), daratumumab (16mg/kg iv; cycles 1-2: days 1, 8, 15, 22; cycles 3-6: days 1, 15; cycles 7-9: day 1) and low dose dexamethasone (on the days daratumumab was administered; cycle 1-2: 20mg; subsequent cycles 10mg), patients without progressive disease or excessive toxicity continued with maintenance treatment, consisting of 8-week cycles with ixazomib (4mg orally on days 1, 8, 15, 29, 36, 43), daratumumab (16mg/kg iv or 1800mg subcutaneously on day 1) with dexamethasone (10mg intravenously on day 1), until progression, for a maximum of two years. Results: Sixty-five frail patients were included. After a median follow-up of 39 months, the median progression free survival was 13.8m (95%CI: 9.2-17.7). The median PFS2 for all patients was 30.7m (22.2-39.1), 39.1 months in patients classified as frail-age, 24.5 months in frail-other patients and 26.6 months in frail-both patient (log-rank p=0.30). Median OS for all patients was 34.0m (24.0-41.2), not reached (frail age), 28.1m (frail-other) and 30.7 months (frail-both) (log-rank p=0.29) (Table 1). Thirty-two patients (49%) proceeded to the maintenance phase. During maintenance treatment, 6 patients (19%) had improvement of response: 1 SD to MR, 3 PR to VGPR, 1 VGPR to CR and 1 VGPR to sCR. The rate of VGPR or better improved from 41% to 50% during maintenance. During maintenance, 21/32 (66%) patients discontinued therapy, because of progressive disease (14/21; 67%), toxicity (2/21; 10%; infection and intracranial hemorrhage), non-compliance (1/21; 5%), intercurrent death (1/21; 5%) and other reasons (3/21; 14%; physician’s choice, dementia and patient condition). Hematologic ≥3 grade adverse events (AEs) during maintenance were limited: neutropenia 0%, anemia 3% and thrombocytopenia 9%. Non-hematologic ≥3 grade AEs occurred in 18 (56%) patients. Most common AEs were infections (9%), nervous system disorders (9%; cognitive disturbance, stroke and syncope) and gastro-intestinal complaints (6%). There were 2 (6%) second primary malignancies and one patient (3%) experienced grade 3 neuropathy. Image: Summary/Conclusion: Ixa-dara-dex maintenance treatment in frail patients was safe, and resulted in an improvement in response rate in 19% of patients. Patients frail based on age, had higher PFS, PFS2 and OS, as compared to other frail subgroups. P907: CARFILZOMIB, DEXAMETHASONE, AND DARATUMUMAB (KDD) VS CARFILZOMIB AND DEXAMETHASONE (KD) IN RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM): FRAILTY SUBGROUP ANALYSIS OF THE CANDOR STUDY H. Quach1,*, X. Leleu2, M.-V. Mateos3, S. Z. Usmani4, A. K. Nooka5, A. Goldrick6, R. Najdi6, N. Shu7, T. Facon8 1University of Melbourne, St Vincent’s Hospital, Melbourne, VIC, Australia; 2CHU de Poitiers, La Miletrie/INSERM CIC 1402, Poitiers, France; 3University Hospital Salamanca/ISAL, Salamanca, Spain; 4Memorial Sloan Kettering Cancer Center, New York; 5Winship Cancer Institute, Emory University, Atlanta; 6Amgen, Thousand Oaks, United States of America; 7Parexel, Chengdu, China; 8Hôpital Claude Huriez, Lille, France Background: Frailty scores based on age, comorbidities, and functional status can help identify frail patients (pts) at risk of higher rates of treatment-related toxicity or poor outcomes. An analysis of carfilzomib-treated pts in ASPIRE, ENDEAVOR, and ARROW found that efficacy and safety of carfilzomib regimens is consistent regardless of frailty status (Facon Blood Adv 2020). Aims: To compare efficacy and safety across frailty subgroups in pts with RRMM treated with KdD vs Kd in the phase 3 CANDOR study. Methods: This post hoc analysis used a planned interim readout (June 15, 2020) of CANDOR (NCT03158688). Pts were categorized as fit, intermediate (int), or frail based on a frailty score of 0, 1, or ≥2. Scoring used an algorithm based on the International Myeloma Working Group (IMWG) frailty index: the final score is based on age (0 if ≤75 years, 1 if 76-80 years, 2 if >80 years), modified Charlson Comorbidity Index (CCI [Facon Blood Adv 2020]) (0 if CCI ≤1, 1 if CCI >1), and Eastern Cooperative Oncology Group performance status (ECOG PS) (0 if ECOG PS=0, 1 if ECOG PS=1, and 2 if ECOG PS=2). Progression-free survival (PFS) was summarized descriptively, with hazard ratio (HR) and 95% CI estimated by stratified Cox proportional hazards model. Overall response rate (ORR) was summarized descriptively, with odds ratio (OR) and 95% CI estimated by Mantel-Haenszel method. Response and progression were assessed by the Onyx Response Computer Algorithm using IMWG criteria. Results: Frailty status was generally proportional between arms, with 27% and 35% classified as fit, 43% and 36% int, 25% and 27% frail, and 5% and 2% unknown for KdD and Kd, respectively. Although baseline characteristics of the overall population were generally balanced, more pts had prior transplant in the KdD vs Kd arm for int (65% vs 36%) and frail (41% vs 27%) subgroups, and in the frail subgroup fewer pts were lenalidomide exposed (37% vs 61%)/refractory (25% vs 46%) in the KdD vs Kd arm. Median duration of treatment for KdD vs Kd was 99 weeks (wks) vs 68 wks for fit pts, 82 wks vs 31 wks for int pts, and 51 wks vs 21 wks for frail pts. Median PFS in KdD vs Kd arms was not reached (NR) vs 17.6 months (HR 0.64, 95% CI 0.38–1.07) for fit, NR vs 11.1 months (HR 0.44, 95% CI 0.28–0.69) for int, and 18.5 vs 9.3 months (HR 0.66, 95% CI 0.38–1.14) for frail pts. ORR for KdD vs Kd arms was 89% vs 89% (OR 1.09, 95% CI 0.35–3.38) for fit pts, 87% vs 70% (OR 2.95, 95% CI 1.31–6.62) for int pts, and 75% vs 54% (OR 2.39, 95% CI 1.09–5.22) for frail pts (Figure). Any grade treatment-emergent adverse events (TEAEs) occurred in >95% of pts across arms and subgroups. Grade ≥3 TEAEs occurred in 87% (KdD) and 70% (Kd) of fit pts, 84% and 71% of int pts, and 91% and 90% of frail pts. Fatal TEAEs occurred in 4% and 2% of fit pts, 11% and 9% of int pts, and 16% and 8% of frail pts in the KdD and Kd arms. Carfilzomib was discontinued due to TEAEs in 25% vs 17% of fit pts, 22% vs 29% of int pts, and 35% vs 23% of frail pts. Among TEAEs of interest, acute renal failure occurred in 0 fit pts, 3% vs 14% of int pts, and 8% vs 5% of frail pts, and infusion reactions occurred in 16% vs 2% of fit pts, 13% vs 4% of int pts, and 14% vs 13% of frail pts in the KdD vs Kd arms (Table). Image: Summary/Conclusion: Consistent with previous findings of the efficacy and safety benefits of KdD, a PFS benefit with KdD vs Kd was observed across frailty subgroups, without increased toxicity. The clinically meaningful ORR benefit in the frail subgroup may help physicians evaluate treatment options in this challenging group. P908: FACTORS ASSOCIATED WITH THE CLINICAL PROGNOSTIC PERFORMANCE OF MINIMAL RESIDUAL DISEASE ASSESSMENT BY NEXT GENERATION FLOW CYTOMETRY IN MULTIPLE MYELOMA B. Nandakumar1, A. Baranwal1, M. Ebraheem1, H. Olteanu2, M. Shi2, S. Kumar1, W. Gonsalves1, D. Jevremovic2,* 1Hematology; 2Laboratory Medicine and Pathology, Mayo Clinic, Rochester, United States of America Background: Minimal Residual Disease (MRD) negativity (-) using next generation flow (NGF) cytometry with a minimum sensitivity of 10-5 is strongly associated with improved progression-free survival (PFS) and overall survival (OS). However, clinicopathologic factors affecting the prognostic performance of this assay are not clear at this time, and despite the achievement of MRD (-) status, disease progression is still readily observed in multiple myeloma (MM). Aims: We investigated quantitative non-clonal plasma cell factors measurable in the bone marrow sample that could predict disease progression in MRD (-) MM patients. Methods: We retrospectively reviewed all patients with MM who underwent a bone marrow biopsy at the Mayo Clinic, Rochester, USA from July 2017 to December 2020. MRD testing was done on bone marrow using the established Euroflow protocol with analytic sensitivity between 10-5 and 2x10-6. In addition to quantitative measurements of the number of clonal plasma cells, the number of hematogones, polyclonal plasma cells and mast cells were collected. The time to next therapy (TTNT) was defined as the time from the date of MRD testing to the date of starting a new treatment regimen due to disease progression. Patients who did not require change in treatment for MM were censored at the last known follow-up. Kaplan-Meier curves and log-rank method were used to compare TTNT. Results: A total of 1,142 NGF assessments, obtained in 783 different patients with MM, were found to have an MRD (-) result. The median age of patients at the time of collection was 63 years. The median absolute number of non-aggregate events captured was 8,586,360 (range: 840,904 - 9,975,075). For the entire sample cohort, the median number of polyclonal plasma cells were 1,874 (range: 0 - 121,221), the median number of hematogones were 70,307 (range: 0 - 6,486,739) and the median number of mast cells were 734 (range: 0 - 116,834). Of the 1,142 NGF assessments, follow-up data was available on 675 samples, of which 204 (30%) were associated with disease progression requiring a change in therapy. Number of polyclonal plasma cells and mast cells greater than their respective medians were considered elevated and were associated with disease progression (Χ2 p < 0.001 for polyclonal plasma cells and Χ2 p = 0.046 for mast cells). Patients with higher polyclonal plasma cell events were found to have significantly higher TTNT compared to those with lower plasma cell events (median NA vs. 31 months, p < 0.001). The median TTNT for patients with higher mast cell events was also significantly higher compared to those with lower mast cell events (median NA vs. 36 months, p = 0.002). Summary/Conclusion: The number of polyclonal plasma cells and mast cells in the bone marrow sample could be useful in predicting myeloma progression in patients with a MRD (-) bone marrow assessment by NGF. This observation may be related to the quality of the specimen (hemodilution), true biologic activity of polytypic plasma cells and mast cells, or both. P909: RENAL RESPONSE OF POMALIDOMIDE WITH BORTEZOMIB AND DEXAMETHASONE IN NEWLY DIAGNOSED MULTIPLE MYELOMA PATIENTS WITH RENAL IMPAIRMENT Y. Jian1, L. Chang2, M. Shi3, Y. Sun4, X. Chu5, H. Xue6, X. Shen7, J. Ma8, G. Jia9, Y. Feng10, Z. Xi11, Y. Zhao12, Y. Ma13, J. Xiao14, G. Ma15, Q. Wang16, W. Huang17, L. Bao18, Y. Dong19, H. Zhou20, C. Sun21, G. Su22, Y. Yan23, Q. Saiyin24, L. Su25, S. Gao26, W. Tian27, X. Sun28, H. Jing29, D. Gao30, W. Chen1, J. Li2, W. Gao1,* 1Beijing Chaoyang Hospital, Capital Medical University; 2Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing; 3The First Affiliated Hospital of Kunming Medical University, Hematology Research Center of Yunnan Province, Kunming; 4Chifeng Municipal Hospital, Chifeng; 5The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Qingdao; 6Affiliated Hospital of Hebei University, Baoding; 7Heping Hospital Affiliated To Changzhi Medical College, Changzhi; 8The First Affiliated Hospital of Zhengzhou University, Zhengzhou; 9The First Affiliated Hospital of Baotou Medical College, Inner Mongolia University of Secience and Technology, Baotou; 10The Third People’s Hospital Of Datong, Datong; 11Linfen People’s Hospital, Linfen; 12The First Affiliated Hospital, Harbin Medical University, Harbin; 13Second hospital of Shanxi Medical University, Taiyuan; 14Yantaishan Hospital Affiliated to Binzhou Medical University, Yantai; 15The Forth Hospital of Hebei Medical University, Shijiazhuang; 16The Second Affiliated Hospital of Nanchang University, Nanchang; 17The Fifth Medical Center of People’s Liberation Army (PLA) General Hospital; 18Beijing Jishuitan Hospital; 19Peking University First Hospital; 20Beijing Luhe Hospital, Capital Medical University, Beijing; 21Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan; 22Cangzhou Central Hospital, Cangzhou; 23Bayannur Hospital, Bayannur; 24Ordos Central Hospital, Ordos; 25Shanxi Cancer Hospital, Taiyuan; 26The First Hospital of Jilin University, Changchun; 27Shanxi Bethune Hospital, Taiyuan; 28The First Affiliated Hospital of Dalian Medical University, Dalian; 29Peking University Third Hospital, Beijing; 30The Affiliated Hospital of Inner Mongolia Medical University, Hohhot, China Background: Renal impairment (RI) is a common complication of multiple myeloma (MM), usually related to poor outcomes. Bortezomib-based regimens have been recommended as first-line therapy of MM with RI. Recently, pomalidomide also has shown significant efficacy and an acceptable safety profile in MM patients with RI. However, few prospective studies evaluated the value of combination of pomalidomide with bortezomib and dexamethasone (PVD) in these special patients. Aims: To evaluate the renal response, hematological response and safety of pomalidomide in combination with bortezomib and dexamethasone (PVD) as first-line therapy in newly diagnosed MM (NDMM) with RI. Methods: This study is an ongoing, prospective, open-label, multicenter, phase 2 study (ChiCTR2100043748). Eligible adult NDMM patients with MM-related RI (defined as estimated glomerular filtration rate (eGFR) < 40ml/min) who had measurable disease are enrolled in the study. Exclusion criteria included RI caused by other reasons than tubular nephropathy, such as renal amyloidosis, light chain deposition disease, etc. Patients received a maximum of 9 cycles of PVD therapy. For transplant-eligible patients, autologous stem cell transplantation (ASCT) was administrated after 3 to 6 PVD cycles. Primary endpoint was renal overall response rate (ORR) at 3 months. Secondary endpoints included best renal response, hematological response, minimal residual disease (MRD), progression-free survival (PFS), overall survival (OS) and safety. Both hematological response and renal response were assessed by International Myeloma Working Group (IMWG) criteria. Results: Between Feb 28, 2021 and Jan 21, 2022, sixty-one patients were enrolled across 28 Chinese hospitals, with a median age of 61 years (range: 38-79); 60.7% were male. Fifty-eight (95.1%) patients had cytogenetic information by FISH at diagnosis, fourteen (24.1%) of them had high-risk cytogenetic abnormalities (defined as t(4;14), t(14;16), and/or del(17p)). Median serum creatine level and eGFR were 349 μmol/L (range: 155-1718) and 12.9 mL/min (range: 2.5-36.3), respectively. Twelve (19.7%) patients received dialysis. Median number of PVD treatment cycles was 4 (range: 1-9). Six patients had received ASCT. Among 56 patients with evaluable hematological response, ORR was 91.1% (69.6% ≥VGPR, 41.1% CR). Forty-eight (78.7%) patients had got the primary endpoint with renal ORR at 3 months, which was 89.4% (48.9% ≥ renal-PR, 35.4% renal-CR). Median time to best renal response was 1.5 months (range: 0.3-11.0). Dialysis independence rate was 58.3% (7/12). Median time from first dose of PVD therapy to dialysis independence was 1 month (range: 0.5-2.0). Rapid reduction of serum free light chain (FLC) was seen at the first cycle of PVD therapy. Median reduction proportion of involved FLC at C1D8 was 77.5% (range: 0-98.5%), while at C1D22 was 91.7% (range: 0-99.9%). The most common adverse effects (incidence >10%) were infection (29.5%), myelosuppression (18.0%), peripheral neuritis (16.4%) and skin rash (11.5%). Median follow-up time was 6 months. Twelve patients (19.7%) discontinued PVD treatment, mainly due to toxicity and patients’ intention. Median PFS and OS has not been reached. Summary/Conclusion: PVD regimen is an effective and well-tolerated regimen for NDMM patients with RI, which offered high renal response (renal ORR at 3 months of 89.4%). P910: DOES MINIMAL RESIDUAL DISEASE OF STEM CELL COLLECTION HAVE PROGNOSTIC IMPACT ON PATIENTS WITH MULTIPLE MYELOMA? X. Jingyu1,*, Y. Wenqiang1, F. Huishou1, L. Jiahui1, L. Lingna1, D. Chenxing1, D. Shuhui1, S. Weiwei1, X. Yan1, Q. Lugui1, A. Gang1 1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Blood Diseases Hospital & Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China Background: Multiple myeloma (MM) is a kind of hematological malignancy involved in monoclonal plasma cells. In most clinical guidelines autologous stem cell transplantation (ASCT) is considered as the standard of care for transplant-eligible patients (TEMM) as long as patients achieving partial response (PR). However, it is unclear that under the circumstance of not achieving complete response (CR) weather it would lead to the presence of neoplastic plasma cells in the stem cell collection (SCC) and then result in negative impact on survival prognosis. Aims: Here, we evaluated the effect of the minimal residual disease (MRD) of SCC in the TEMM. Methods: We analyzed retrospectively clinical data of 90 patients with MM undergoing ASCT between January 1, 2013 to June 1, 2021 and MRD evaluation of both bone marrow (BM) and SCC were carried out at the same time. MRD was evaluated by multiparameter flow cytometry (MFC) with 10-4-10-5 sensitivity. The best response was defined as the deepest response during the follow-up. Here we defined the time from ASCT to disease progression or death as modified progression-free survival (mPFS) and the time from ASCT to death as modified overall survival (mOS). Results: A total of 90 patients met the inclusion criteria. The median age is 54 (37-69) and 62.2% were males. There were 25 (27.8%) patients presenting high-risk cytogenetic abnormalities by FISH, defined as the presence of at least one of t (4;14), t (14;16) or del (17p). Before ASCT 36.7% of patients achieved MRD negativity in BM and 76.7% in SCC. After the comparison among MRD-positivity status with different sensitivity and numbers of detectable MRD neoplastic plasma cells, we found that the percentage of patients with MRD positivity in SCC was much less than that in BM no matter the sensitivity (P <0.001). Neither mPFS (P=0.861, median mPFS, 40.83m vs. 34.17m for negativity vs. positivity) nor mOS (P=0.747, median mOS, 67.02m vs. 58.86m for negativity vs. positivity) was affected by MRD status in SCC. According to MRD status they were divided into 4 groups, namely MRD negativity in BM and SCC (Group A, 34.4%), MRD positivity in BM but negativity in SCC (Group B, 22.2%), MRD positivity in BM and SCC (Group C, 41.1%) as well as MRD negativity in BM but positivity in SCC (Group D, 2.3%). Having taken the inaccuracy of biopsy into consideration we excluded Group D. The median follow-up of the cohort was 26.8 months (15.1-105.1m). Patients among the three groups experienced similar mPFS (P=0.403, median mPFS, 41.07m vs. 34.17m vs. 40.83m, for Group A, B and C, respectively) and similar mOS (P=0.933, median mOS, 67.02m vs. 58.86m vs. 58.61m for Group A, B and C, respectively). Achievement of CR with the presence of MRD negativity was associated with prolonged mPFS and mOS compared with MRD-positive CR or ≤VGPR (P＜0.001, median mPFS, 55.88m vs. 23.03m vs. 27.10m, respectively; P=0.026, median mOS, 67.02 vs. 46.16m vs. 41.65m, respectively). Image: Summary/Conclusion: Our results demonstrated that neoplastic plasma cells in SCC have little impact on the survival prognosis in MM patients and it is sound to carry out ASCT when TEMM patients achieving PR. Also, MRD-negativity status can be considered more valuable on prognosis than CR. P911: SYMPTOM BURDEN AND ITS IMPACT ON DAILY LIFE AMONG PATIENTS WITH IDIOPATHIC MULTICENTRIC CASTLEMAN DISEASE (IMCD) – FINDINGS FROM AN INTERNATIONAL IMCD PATIENT SURVEY F. Shupo1, N. Mason2, E. Jones2, G. Wayi-Wayi1, M. Repasky3, M. Franklin4, J. Brazier4, N. Zibelnik1, S. Mukherjee5,* 1EUSA Pharma, Hemel Hempstead; 2BresMed Health Solutions, Sheffield, United Kingdom; 3Castleman Disease Collaborative Network, Paso Robles, United States of America; 4School of Health and Related Research, Sheffield, United Kingdom; 5Hematology and Medical Oncology, Cleveland Clinic Main Campus, Cleveland, United States of America Background: Idiopathic Multicentric Castleman disease (iMCD) is a rare lymphoproliferative disorder driven by proinflammatory hypercytokinemia. The presentation of iMCD is heterogeneous and can range from mild constitutional symptoms to chronic burdensome symptoms and in extreme cases life-threatening multiorgan failure. Consequently, disease-related symptoms in iMCD patients are likely to adversely impact daily life. To date, characterization of symptom burden and their impact on daily living in iMCD patients has not been systematically studied. Aims: We aimed to investigate, characterize, and map the symptoms and associated burden on daily life experienced by patients with various subtypes of iMCD. Methods: We developed an international patient-based online survey informed by clinical practice and published literature to elicit the burden of disease-related symptoms and effects of symptoms on daily life from a patient perspective. Eligible patients were > 18 years old with physician-confirmed diagnosis of iMCD-NOS (not otherwise specified), TAFRO (thrombocytopenia, anasarca, reticulin fibrosis of the bone marrow, renal dysfunction, and organomegaly) and POEMS—associated MCD (multicentric Castleman disease with polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes). This survey was shared with iMCD communities in Australia, Canada, the UK, and the US via the Castleman Disease Collaborative Network (CDCN). Burden of Illness (BOI) was quantitatively measured using a 5-point frequency Likert scale (from 0 ‘Does not affect my daily life’ to 4 ‘Very severely affects my daily life’), and mean impact scores (MIS) were calculated. Ethics approvals/waivers were attained for this one-time, cross-sectional, bespoke 47-question survey. Results: A total of 57 patient responses were collected during April–November 2021. On average, patients experienced 7.0 symptoms (range: 0─22) in the week prior to survey completion. Tiredness was the most frequently reported symptom (77%), followed by physical weakness (44%) and night sweats (40%). Individual symptoms were clustered into clinically relevant categories and frequency of symptom groupings reported by iMCD subtype (Fig 1). Constitutional (82%) and neuropsychiatric (68%) symptoms were most frequently experienced across all respondents in the week prior to survey completion. The average number of symptoms by iMCD subtypes were 7.1 by iMCD-NOS, 5.5 by TAFRO and 8.5 by POEMS-associated MCD. 91% of all patients with iMCD reported experiencing at least one symptom in the week prior to survey completion. When rating their most impacted aspects of daily life due to their symptoms (Table 1), patients with iMCD-NOS reported pain and discomfort (MIS 2.09) and personal relationships (MIS 2.08), patients with MCD-POEMS reported sexual functioning (MIS 3.40) and pain and discomfort (MIS 2.67), while patients with TAFRO reported sexual functioning (MIS 2.44) and ability to travel (MIS 2.22). Image: Summary/Conclusion: To our knowledge, this is the first study of its kind to characterize and map the BOI in iMCD patients assessed by symptom frequency, symptom burden (multiplicity of symptoms) and its adverse effects on different aspects of daily living. Through our ongoing work we hope to develop a symptom burden score/scale that captures the symptom severity and its impact on daily living which can then be incorporated as a patient reported outcome measure for shared treatment decision-making and response assessment in addition to the laboratory and radiologic parameters. P912: COVID19 SEVERITY AND THERAPEUTIC OPTIONS IN PATIENTS WITH MULTIPLE MYELOMA E. Karimova1,*, E. Zhelnova1, E. Baryakh1, K. Yatskov1, E. Zotina1, E. Grishina1, E. Paramonova1, D. Gagloeva1, E. Misyurina1 1hematology, Moscow city hospital №52, Moscow, Russia Background: Patients with multiple myeloma have higher risk of SARS-CoV-2 infection and higher risk of death than patients without MM Aims: Analysis of features of the course of COVID-19 in patients with MM. Methods: 89 pts with MM and COVID-19 were treated in the hospital from March 2020 to October 2021, 53 (60%) female, 36 (40%) male. The median age was 64 years (35-88 years). 32 pts (36%) were presented with active myeloma, 19 (21%) – R/R myeloma, 21 (24%) – CR, 17 (19%) - PR or VGPR. The majority of patients - 56 (63%) had III st. (Durie-Salmon). Kidney damage was presented in 33 pts (37%). The majority of patients had severe lung damage: CT 2 st. - 26 pts (30%), III st. - 35 (39%), IV st.-17 (19%). Patients received standard therapy for COVID19: etiotropic (antiviral, viral neutralizing MAB, convalescent plasma, immunoglobulin), pathogenetic (GCS, interleukin inhibitors, anticoagulants), therapy for secondary infectious complications. Results: IL inhibitor therapy was given 76 patients (85%), 35 (39%) required repeated administration of IL inhibitors. 58 pts (65%) received GCS therapy in various doses. Anticoagulant therapy was conducted in all patients, 51 pts (57%) received LMWH, 37 (42%) received continuous infusion of heparin, 1 patient received NOAC. The frequency of hemorrhagic complications on LMWH and heparin therapy was comparable (5.9% and 5.5%), mortality and the frequency of TEC in patients on heparin therapy were higher (49% and 13% versus 14% and 2% in patients on LMWH therapy, p<0.001 and p=0.034, respectively). It can be explained by the initial severity of patient’s condition, which required heparin therapy (30 pts - 81% had an initially high degree of lung damage - CT 3-4 st.) Secondary infectious complications were reported in 80% of patients: bacterial pneumonia in 71 pts (80%), sepsis in 24 (27%). Infectious endocarditis (2 pts), meningitis (2 pts), colitis (9 pts), colon gangrene complicated by peritonitis (1 patient) were less common. Fungal infections were verified in 19 pts (21%): 6 (7%) – mucosal candidiasis, 13 (14%) – invasive aspergillosis. Infection caused by herpes viruses was detected in 5 patients (6%). There was no significant connection between severity of COVID19, frequency of secondary infections and mortality and level of normal immunoglobulins. The median duration of hospitalization was 19 days versus 9 among patients without MM. 25 patients (28%) were hospitalized in the ICU. The overall mortality was 27% (24 pts), among this patients 71% (17 pts) had active or R/R myeloma and stage III of the disease. The mortality in the ICU was 88% (22 pts). The most frequent causes of fatality were secondary infectious complications in 92% (22 pts), progression of MM in 8% (2 pts). 27 patients (30%) required chemotherapy during COVID-19. Summary/Conclusion: Patient with R/R MM have the highest risk of mortality from COVID19. For patients with multiple myeloma the same COVID19 therapeutic options are effective as for non-immunodeficient patients. Patients with MM and COVID19 also have a high risk of secondary infectious complications. P914: EFFICACY AND SAFETY OF BELANTAMAB MAFODOTIN MONOTHERAPY IN PATIENTS WITH RELAPSED OR REFRACTORY LIGHT CHAIN AMYLOIDOSIS: A PHASE 2 STUDY BY THE EUROPEAN MYELOMA NETWORK E. Kastritis1,*, G. Palladini2, M. A. Dimopoulos1, A. Jaccard3, G. Merlini2, F. Theodorakakou1, D. Fotiou1, M. C. Minnema4, A. Wechalekar5, S. Gkolfinopoulos6, K. Manousou6, P. Sonneveld7, S. Schönland8 1Department of Clinical Therapeutics, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 2Amyloidosis Research and Treatment Center, University of Pavia, Pavia, Italy; 3Referral Center for AL amyloidosis, Limoges, France; 4Department of Hematology, University Medical Center Utrecht, Utrecht, Netherlands; 5Clinical Haematology, Cancer Division, University College London Hospital, London, United Kingdom; 6Health Data Specialists, Dublin, Ireland; 7Erasmus MC Cancer Institute, Rotterdam, Netherlands; 8University of Heidelberg, Heidelberg, Germany Background: Managing patients (pts) with relapsed/refractory (RR) light chain (AL) amyloidosis is challenging, as there is no current standard treatment, many available options are associated with low efficacy and toxicity, and options for daratumumab (DARA)- and bortezomib-exposed patients are limited. Belantamab mafodotin (belamaf), a multi-modal antibody-drug conjugate targeting BCMA, has shown efficacy and tolerability in heavily pretreated pts with RR multiple myeloma, including those refractory to DARA. Since clonal plasma cells in AL amyloidosis and MM are phenotypically similar, belamaf could be a novel treatment option in AL amyloidosis. Aims: To evaluate the efficacy and safety of belamaf monotherapy off-label in pts with RR AL amyloidosis. Methods: The ongoing prospective, open-label, multinational, phase 2, EMN27 study (NCT04617925) aims to enroll 36 adult pretreated pts with AL amyloidosis who require therapy. Pts at Mayo cardiac stage 3b are excluded. Belamaf monotherapy at 2.5mg/kg is administered by intravenous infusion every 6 weeks for a maximum of 8 cycles; dosing can be reduced to 1.92mg/kg for toxicity. Per study design, a safety analysis (after 6 pts received ≥1 treatment cycle) and an efficacy analysis (after 13 pts are enrolled) were planned. The safety analysis revealed no new safety signals, and pt accrual continued to 13 pts. The efficacy analysis is currently conducted; however, already 3 pts achieved complete response, or very good partial response (VGPR), or low difference of involved to uninvolved serum free light chains (dFLC) response and enrollment is continuing to include all planned pts. This descriptive analysis included pts initiating study treatment ≥3 months before the cut-off date (15/01/2022). Results: Of 11 pts included in the analysis, 4 (36.4%) continued treatment by the cut-off date, and 7 (63.6%) discontinued (disease progression: 5 [45.5%], death: 2 [18.2%]). The pts median age was 69.0 years (range 46.0–80.0), and most were males (7, 63.6%). At baseline, 3 (27.3%) and 8 (72.7%) pts had New York Heart Association class I and II symptoms, respectively; the median N-terminal pro-brain natriuretic peptide, high-sensitivity troponin T, and dFLC were 1,979 pg/mL (range 190.0–4,135.0), 41.6 pg/mL (range 11.0–80.8), and 34.2mg/dl (range 4.4–279.1), respectively. Except for the heart, commonly involved organs were the nervous system (4 pts, 36.4%) and the soft tissue (2 pts, 18.2%). The median number of previous AL amyloidosis treatments was 3.0 (range 1.0–7.0), including DARA. The median duration of belamaf therapy was 3.1 months (range 1.4–5.7). At a median follow up of 9.4 months (range 3.1–10.0), the overall response rate was 72.7% (8 pts; VGPR: 27.3% [3 pts] and partial response: 45.5% [5 pts]). Median time to first hematological response was 8.5 days (range 1.0–28.0) and to VGPR or better 15.0 days (range 8.0–15.0). The 3-month organ (heart, kidney, or liver) response rate was 36.4% (4 pts). All pts had ≥1 non serious adverse event (SAE). Four (36.4%) pts had ≥1 SAE, including 2 (18.2%) pts with a belamaf-related grade 2 and 4 visual impairment (1 [9.1%] pt each). Eight (72.7%) pts had ≥1 adverse event of special interest. Two (18.2%) pts had a fatal SAE (pneumonia and intestinal perforation, 1 [9.1%] pt each), both unrelated to belamaf. Summary/Conclusion: In this prospective study, belamaf monotherapy induced rapid, clinically meaningful responses with a manageable safety profile in heavily pretreated pts with RR AL amyloidosis. As the study progresses, additional data will be generated. P915: EFFICACY AND SAFETY OF DARATUMUMAB MONOTHERAPY IN NEWLY DIAGNOSED PATIENTS WITH STAGE 3B LIGHT CHAIN AMYLOIDOSIS: A PHASE 2 STUDY BY THE EUROPEAN MYELOMA NETWORK E. Kastritis1,*, M. C. Minnema2, M. A. Dimopoulos1, G. Merlini3, F. Theodorakakou1, D. Fotiou1, A. Huart4, K. Belhadj5, S. Gkolfinopoulos6, K. Manousou6, P. Sonneveld7, G. Palladini3 1Department of Clinical Therapeutics, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 2Department of Hematology, University Medical Center Utrecht, Utrecht, Netherlands; 3Amyloidosis Research and Treatment Center, University of Pavia, Pavia, Italy; 4Department of Nephrology and transplantation, Rangueil University Hospital, Toulouse; 5Lymphoid Malignancies Unit, Henri Mondor Hospital, Créteil, France; 6Health Data Specialists, Dublin, Ireland; 7Erasmus MC Cancer Institute, Rotterdam, Netherlands Background: Cardiac involvement and severity of cardiac dysfunction in light chain (AL) amyloidosis is a critical prognostic factor. Patients (pts) at Mayo cardiac stage 3b have a poor prognosis with a median overall survival (OS) of just 4 months and high rates of early death with current therapies; thus, there is a need for novel, non-toxic, effective treatments for these pts. Daratumumab (DARA), a human anti-CD38 antibody, has shown efficacy and tolerability in pts with AL amyloidosis. Aims: To evaluate the efficacy and safety of DARA monotherapy used off-label in newly diagnosed pts with stage 3b AL amyloidosis. Methods: The ongoing EMN22 phase 2, multinational, open-label study (NCT04131309) aims to enroll 40 newly diagnosed pts with stage 3b AL amyloidosis. Eligible adult pts have high-sensitivity troponin T (hsTnT) >54 pg/mL and N-terminal pro-brain natriuretic peptide (NT-proBNP) ≥8,500 pg/mL. DARA monotherapy, 16 mg/mL by intravenous infusion (09/2019–01/2020) and 1,800 mg by subcutaneous injection (01/2020 and thereafter), is administered weekly during cycles (C)1 and 2, every 2 weeks for C3–6, and every 4 weeks thereafter. Pts not achieving a hematological very good partial response (VGPR) or better by the end of C3 can receive additional weekly bortezomib and low dose dexamethasone (Vd). Treatment continues up to 2 years from initiation or until disease progression or initiation of a new therapy. Primary endpoint is OS rate at 6 months. This descriptive analysis included pts initiating treatment ≥6 months before the cut-off date (14/01/2022); the median (95% confidence interval [CI]) OS was obtained by Kaplan-Meier analysis. Results: Of 27 pts included, 8 (30%) continued study treatment by the cut-off date and 19 (70%) had discontinued. The pts median age was 68 (range 45–84) years, and most were male (16, 59%). At screening, 10 (37%) and 17 (63%) pts had New York Heart Association class II and IIIA symptoms, respectively; the median NT-proBNP was 15,512 pg/mL (range 8,816–72,522), hsTnT was 133 pg/mL (range 60–692), and the difference of involved to uninvolved free light chains was 406 mg/l (range 24–3,377). Beyond the heart, the median number of other organs involved was 2 (range 0–5), most commonly kidneys (14 pts, 52%) and peripheral nerves (11 pts, 41%). The median duration of DARA therapy was 7 months (range <1–24); seven (26%) pts received additional Vd. At a median observation time of 8 months (range <1–11), the overall response rate (ORR) was 67% (18 pts; complete response [CR]:19.0% [5 pts], VGPR:37% [10 pts], partial response:11% [3 pts]). The ORRs at 1, 2, and 3 months were 59% (16 pts), 63% (17 pts), and 63% (17 pts), respectively. Median time to first response was 7 days (range 6–114), and to VGPR or better 54 days (range 6–219). Median OS was 9 months (95% CI, 3–not reached). The 6- and 12-month median (95% CI) OS rates were 63% (42–78) and 49% (28–67), respectively. Twenty-five (93%) pts had ≥1 non-serious adverse event. Twenty (74%) pts had ≥1 serious adverse event (SAE), comprising 15 (56%) pts with ≥1 cardiac-related SAE and 11 (41%) pts with a fatal SAE. Six SAEs were treatment-related: 2 with DARA (grade 3 pneumonia, grade 5 sepsis); 3 with bortezomib (grade 2 fatigue, grade 2 fall, and grade 3 troponin I increase); and 1 with dexamethasone (grade 3 cardiac failure). Summary/Conclusion: Among pts with Mayo stage 3b AL amyloidosis, a subgroup with poor prognosis, DARA monotherapy induced rapid and deep hematological responses and no new safety signals; the median OS surpassed that reported previously. P916: COEXISTENCE OF ≥2 HIGH-RISK MOLECULAR ABNORMALITIES SUPERVENES THE PROGNOSTIC VALUE OF THE REVISED INTERNATIONAL STAGING SYSTEM FOR MYELOMA: REAL-WORLD DATA ANALYSIS FROM THE GREEK MYELOMA STUDY GROUP E. Katodritou1,*, D. Dalampira1, T. Triantafyllou1, M. Gavriatopoulou2, S. Delimpasi3, A. Pouli4, T. Papadopoulou1, E. Verrou1, A. Sevastoudi1, K. Tsirou1, L. Katsika1, G. Douganiotis1, N. Karampatzakis1, T.-E. Metallinou1, V. Palaska1, M. Kotsopoulou5, C. Lalayianni6, M.-C. Kyrtsonis7, E. Spanoudakis8, D. Maltezas5, A. Chatzivasili9, M.-A. Dimopoulos2, E. Terpos2, E. Kastritis2 1Hematology, Theagenio Cancer Hospital, Thessaloniki; 2Clinical Therapeutics, National and Kapodistrian University of Athens; 3Hematology and Bone Marrow Unit, Evangelismos General Hospital; 4Hematology, Agios Savvas Cancer Hospital; 5Hematology, Metaxa Cancer Hospital, Athens; 6Hematology and Bone Marrow Unit, George Papanicolaou General Hospital, Thessaloniki; 7First Department of Propaedeutic Internal Medicine, National and Kapodistrian University of Athens, Athens; 8Hematology, University Hospital of Alexandroupolis, Alexandroupolis; 9Hematology, Venizelion General Hospital, Heraclion, Greece Background: Revised International Staging System (R-ISS) has improved the prognostic value of ISS in multiple myeloma (MM). Recently, the Mayo Additive Staging System incorporated +1q21 to determine a 5-factor 3-tier system, providing an add-on value on R-ISS. The prognostic impact of the coexistence of ≥2 high-risk molecular abnormalities, including +1q21, compared to R-ISS, has not been validated adequately. Aims: The aim of this study was to evaluate the prognostic impact on survival of the coexistence of ≥2 high-risk molecular abnormalities, defined as Ultra High Risk (UHR) MM, in comparison with R-ISS and other established prognostic markers, in the real-world setting. Methods: We analyzed the data of 1352 consecutive newly diagnosed MM patients (M/F: 655/697, median age: 66, range: 29-87, IgG: 817, IgA: 363, light chain: 144, IgD: 9, IgM: 3, non-secretory: 16), treated between 2002-2021 and which had been tested for molecular abnormalities i.e. del17p, t(14;16), t(4;14) and +1q21 using fluorescence in situ hybridization. Patients with ≥2 high-risk features were classified as UHR. We compared the two groups for age, performance status, ISS, R-ISS, lactate dehydrogenase (LDH), albumin, hemoglobin (Hb), β2-microglobulin, estimated glomerular filtration rate (eGFR), 1st and 2nd line therapies and response rates. A Cox regression model was used to determine independent prognostic factors for overall survival (OS). Progression-free survival (PFS) and OS were plotted with Kaplan-Meier; a p<0.05 was considered as statistically significant. Results: One hundred sixteen patients (9%) were classified in the UHR group vs. 1236 (91%) in the non-UHR group; 106 patients had 2, and 10 patients had 3 molecular abnormalities. The most common combination of high-risk features was +1q21 plus t(4;14) (40%). Median age, sex, performance status, LDH and serum albumin, did not differ, whereas the UHR group had lower eGFR, higher β2-microglobulin and lower Hb (p<0.05). Early stage (ISS1/R-ISS1) was more frequent in the non-UHR group (p<0.05); 1st line treatment, including autologous transplantation (ASCT), and second line treatment were well balanced between groups (p<0.05); 66% of patients overall, received triplet/quadruplet combinations and 29% underwent ASCT upfront. Overall response rate after induction therapy was 86% and did not differ between groups (p<0.05). Complete response was lower in the UHR group (11% vs. 19%; p=0.03). After a median follow up of 49 months (95% CI: 44-54), 59% of patients were alive. Median PFS was significantly shorter for the UHR group (15.8 vs. 31.9 months, p<0.001; HR: 0.45, 95% CI: 0.36-0.58). Median OS was 28 months (95% CI:18-38) for patients in the UHR group vs. 69 months (95% CI: 61-77) for others (p<0.001). In the univariate analysis, age, anemia, eGFR, upfront ASCT, R-ISS and UHR myeloma were independent predictors for OS (p<0.05). In the multivariate analysis UHR myeloma was the strongest independent predictor for OS (p<0.001; HR: 0.42), supervening the prognostic value of R-ISS (R-ISS1 vs. R-ISS2 HR=0.58, R-ISS2 vs. R-ISS3 HR: 0.75). Additionally, UHR status singled out a distinct group within R-ISS2 patients with significantly worse OS (38 months, 95% CI: 28-48 vs. 64mo, 95% CI: 55-72) (p<0.001). Summary/Conclusion: According to our analysis of a large cohort of newly diagnosed MM patients UHR myeloma, was the strongest independent predictor for OS, supervening the prognostic value of R-ISS. Moreover, UHR status could serve as an additional prognostic marker for R-ISS2 patients, helping thus to optimize therapeutic approach of MM patients. P917: HOW COMORBID ARE OUR MYELOMA PATIENTS AND HOW MANY MAKE IT TO THE SECOND-LINE TREATMENT: REAL-WORLD DATA OF 251 MYELOMA PATIENTS TREATED IN THE HEMATOLOGICAL NETWORK OF THE OEGK F. Keil1,*, P. Attalla1 13rd Medical Department for Hematology and Oncology, Hanusch hospital, Vienna, Austria Background: Comorbid conditions have a negative impact on overall survival (OS) in multiple myeloma (MM) patients. Furthermore, a European multi-center study reported that only 61% of patients proceed to second-line treatment. Aims: The objective of this study was to determine the prevalence of common comorbidities and their impact on OS. Furthermore, we examined the mortality during first-line treatment and the percentage of patients proceeding to second-line treatment. Methods: This retrospective study was conducted in the hematology care network “Hämatologieverbund der Österreichischen Gesundheitskasse (OEGK)”, consisting of the Hanusch hospital and three outpatient centers, in Vienna, Austria. We included all patients who were observed and/or treated for multiple myeloma between 2012 and 2018 within the hematology care network. Follow-up was conducted until March 2021. Results: We analyzed 251 patients (median age 69 years). Prevalent concomitant conditions at diagnosis were hypertension (67.4%), further cardiovascular diseases excluding hypertension (23.1%), obesity (15.2%), diabetes (13.8%) and chronic lung disease (9.2%). For the evaluation of renal impairment, we used the CCI, which defines kidney disease as a creatinine >3mg/dl or the need for dialysis, as well as the chronic kidney disease (CKD) classification system. While 7.8% of patients fulfilled the CCI criteria for kidney disease, 39.5% had a CKD stage ≥3A. Comorbidity in general, as well as specific comorbid conditions had a negative effect on overall survival. Patients with a CCI score of 0 points at diagnosis showed a median OS of 8.7 years compared to 3.8 years and 2.6 years for patients with a score of 1 point and ≥2 points, respectively (p <0.001). We also observed a reduced OS of 4.9 years in patients with at least one creatinine clearance (CrCl) value <60 ml/min throughout the observation period compared to 16.4 years in patients with a CrCl consistently ≥60 ml/min (p <0.001). In total, 232 patients received a first-line treatment. During the observation period after first-line therapy, 146 patients proceeded to second-line treatment due to progress, 30 showed no sign of progress so far and 12 had an ongoing first-line maintenance therapy. 21 patients were referred from other centers for a second opinion and/or ASCT. These patients were still alive at the end of the observation period, but data about further treatment lines were missing. One patient eligible for second-line treatment refused further therapeutic measures. Furthermore, 22 patients died during first-line treatment or the subsequent treatment-free interval. Of these 22 patients, 11 died during active first-line treatment due to progression of myeloma or treatment-related toxicity. The other 11 patients died after completion of first-line treatment during the observation period due to secondary neoplasms (4) and infection (2). For the remaining five patients the cause of death was unknown. In total, 90.5% of patients who had received a first-line treatment were or still are eligible for second-line treatment. Summary/Conclusion: Comorbid conditions were prevalent in our population and exerted a major impact on mortality. In comparison to the CCI, the chronic kidney disease classification system showed superior sensitivity for detection of renal impairment, which is a good predictor of survival. In contrast to previously published data, a high proportion of patients were eligible for second-line treatment. Disease-specific mortality prevented only a small number of patients from receiving second-line treatment. P918: HIGH RESPONSES RATES WITH SINGLE AGENT BELANTAMAB MAFODOTIN IN RELAPSED SYSTEMIC AL AMYLOIDOSIS J. Khwaja1,*, J. Bomsztyk2, S. Mahmood2, B. Wisniowski2, R. Shah1, A. Tailor1, K. Yong1, R. Popat1, N. Rabin1, C. Kyriakou1, J. Sive1, S. Worthington1, A. Hart1, E. Dowling1, N. Correia1, C. Bygrave3, A. Rydzewski4, K. Jamroziak5, A. Wechalekar2 1Department of Haematology, University College London Hospital; 2National Amyloidosis Centre, University College London (Royal Free Campus), London; 3Department of Haematology, University Hospital of Wales, Cardiff, United Kingdom; 4Department of Internal Medicine, Nephrology and Transplantation Medicine, Central Clinical Hospital of the Ministry of Internal Affairs; 5Department of Hematology, Transplantation and Internal Medicine, Medical University of Warsaw, Warsaw, Poland Background: Systemic AL amyloidosis is an incurable relapsing plasma cell disorder. Despite therapeutic advances, there are no approved treatments for relapse disease. Treatment is often challenging due to underlying organ dysfunction. Belantamab mafodotin is an antibody-drug conjugate targeting B-cell maturation antigen with approval for relapsed refractory myeloma. In multiply pre-treated myeloma, the DREAMM-2 phase II trial showed an overall response rate of 32% for those with 2.5 mg/kg dose administered every three weeks with 2/3rd patients reporting keratopathy. A small case series of 6 patients with relapsed AL amyloidosis (Zhang et al, ASH 2021) was recently reported and a phase 2 trial is recruiting for patients with refractory amyloidosis (NCT04617925). Aims: We report our initial results using Belantamab monotherapy for the treatment of patients with AL amyloidosis with relapsed disease. Methods: Data for consecutive patients who were administered Belantamab at a specialist referral centre, National Amyloidosis Centre, University College London, was analysed. Results: Eleven patients were included 8 male, 3 female. Median age at Belantamab initiation was 65 (range 42-74) years. Eight patients had λ AL-type and three κ AL-type. At diagnosis, median involved free light-chain concentration was 534 (range 73-7181) mg/l. A median of two organs involved at baseline (range 1-3): 4 had cardiac involvement (half Mayo stage 2; half Mayo stage 3a) and 8 had renal involvement. The median prior lines of therapy was 3 (range 2-5) with all exposed to prior immunomodulatory drugs, proteasome inhibitors and 73% to anti-CD38 antibody treatments. Thirty-six percent had relapsed after melphalan-conditioned autologous stem cell transplantation. A median of 3 cycles of belantamab were delivered (range 1-8). The most frequent adverse event was ocular toxicity which was experienced in 8 patients (grade 1-3), necessitating dose modification of the three-weekly schedule. One patient developed transient grade 1 dyspnoea and liver dysfunction. No patients developed cytopenias, unlike previous reports (Zhang et al, 2021), nor infections beyond COVID (2 patients mild with no hospital admissions). The majority of the cohort required dose reduction either at initiation (patient 4, due to end stage renal failure; patient 11, post-renal transplant) or during therapy (n=5; three to 1.9mg/kg, two to 1.25mg/kg) due to ocular toxicity. Only one patient remained on the standard dose of 2.5mg/kg for ≥3 cycles. Ocular toxicity improved after treatment interruption (drug intervals 4-6 weeks) and no patients required complete treatment cessation. One patient is too early to assess response. Haematological responses (PR or better) were seen in 7 patients with 3 complete responses and two very good partial responses (VGPR) which are ongoing. Both renal patients (patients 4 and 11) commenced a dose of 1.25mg/kg and sustained a VGPR with no additional toxicity. Patient 3 had a 42% reduction in sFLC after two doses but then a prolonged gap due to keratopathy and has lost the response. There were no cardiac or renal toxicities observed. Image: Summary/Conclusion: Belantamab mafodotin demonstrates significant activity in patients with heavily pre-treated AL amyloidosis with 70% achieving a ≥PR. Apart from keratopathy requiring dose modification, no other substantial toxicity was observed. Two patients with renal impairment (stage V CKD and ESRD) and one patient post-renal transplant tolerated treatment with no additional toxicity. Belantamab mafodotin shows promise in treatment of relapsed AL and needs further prospective trials. P919: SIGNIFICANCE OF IHC AND BIOCHEMICAL MARKERS OF BONE METABOLISM FOR PREDICTING OSTEODESTRUCTIVE SYNDROME IN PLASMA CELL PROLIFERATION Z. Kozich1,*, V. Martinkov1, M. Zhandarov1, J. PUGACHEVA1, S. Mihno1, N. Klimkovich2 1The Republican Research Center for Radiation Medicine and Human Ecology, Gomel; 2Belarusian Medical Academy for Postgraduate Education, Minsk, Belarus Background: Multiple myeloma (MM) is a malignant disease of a lymphoid nature accompanied by the proliferation of tumor plasma cells, in its development passing through the stage of monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SM). One of the main manifestations of MM is the lesion of the skeleton bones, which may already manifest at the stage of MGUS and SM and further it can lead to a decrease in the quality of life of patients. Our work is devoted to the study of the role of markers that contribute to the detection of the progression of the destructive syndrome at the stage of MGUS and SM. Aims: To study significance of IHC and biochemical markers of bone metabolism for predicting osteodestructive syndrome in MGUS and SM. Methods: All patients underwent aspiration and BM biopsy for cytological and histopathological assessment of PC infiltration. An immunological study of blood serum and the determination of biochemical markers of bone metabolism of serum were also performed. All patients underwent CT and MRI of the whole body. The diagnosis of MGUS was based on international criteria: the presence of less than 10% of clonal plasma cells in the bone marrow aspirate, the concentration of M-protein in the blood serum <30 g/l. Among patients with MM, a group without osteodestructive lesions of the skeleton bones was identified. Statistical processing of the results was carried out using the Statistica 6.1 software package. Differences were considered statistically significant at p<0.05. Results: The study included 132 patients (63 MM patients and 68 MGUS patients), who did not differ in age at the time of diagnosis, p = 0.089, the median age was 64.0 years (25% and 75% - 56.0 to 69, 0) and 61.0 years (25% and 75% - 53.0 and 66.0), respectively. In the MG group, female patients predominated (70.1%), they were significantly more common than in the MM group (53.1%), p=0.045. Damage to the skeleton bones during primary diagnosis (including SP) was detected in 37.4% (68) of cases. Bone tissue destruction was more common in males (p=0.029). Changes in bone tissue for patients with MGUS and SM detected by MRI in most cases are presented as a diffuse lesion, foci of bone tissue rarefaction without obvious foci of destruction or SP. The presence of destructive lesions was more frequently detected in MGUS patients with subsequent progression to MM (p<0.002). Time to progression was about 14 months on average (range from 3 to 25 months). When analyzing the obtained results, in MGUS patients, an excess of the β CrossLaps level in serum occurred in 7.8% of cases. At the stage of MGUS, 25.3% of patients (according to the level of osteocalcin) and 16.1% (according to the level of VAR) had disorders in the processes of bone tissue formation. Bone tissue destruction was detected more often in patients with IgM secretion (p=0.014), the ratio of immunoglobulin light chains κ/λ <0.1 and >10 and more than 10% of CD 138+ plasma cells in immunohistochemical studies. Summary/Conclusion: At the stage of MGUS, disturbances in the processes of bone tissue remodeling occur, which is accompanied by the appearance of deviations in the level of biochemical markers (osteocalcin, β CrossLaps). Therefore, these markers have the potential to be used to identify individuals at increased risk of developing a destructive syndrome, and in conjunction with other risk factors (IgM secretion of more than 10% of CD 138+ plasma cells on immunohistochemistry) and to identify patients at increased risk of progression during MM. P920: EFFECTIVENESS AND SAFETY OF SELINEXOR-BASED REGIMEN IN THE TREATMENT OF RELAPSED AND REFRACTORY MULTIPLE MYELOMA: A MULTICENTER REAL-WORLD STUDY FROM CHINA L. Kuang1, B. Fang2, W. Chen3, A. Liu3, C. Li4, L. Bao5, C. Fu6, J. Chen7, H. Li7, Y. Pang8, A. Liao9, Y. Liang10, Y. Wei11, J. Li1,* 1First Affiliated Hospital of Sun Yat-sen University, Guangzhou; 2Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Zhengzhou; 3Beijing Chao-yang Hospital, Capital Medical University, Beijing; 4Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan; 5Beijing Jishuitan Hospital, Beijing; 6The First Affiliated Hospital of Soochow University, Suzhou; 7Sichuan Provincial Hospital of University of Electronic Science and Technology of China, Chengdu; 8The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou; 9Shengjing Hospital of China Medical University, Shenyang; 10Sun Yat-sen University Cancer Center; 11Nanfang Hospital, Southern Medical University, Guangzhou, China Background: Selinexor is the first approved oral selective nuclear exportin inhibitor. Clinical trials have shown that selinexor-based regimens improved the survival of RRMM patients. However, there are limited reports on the effectiveness and safety of selinexor in the real world. Aims: To evaluate the effectiveness and safety of selinexor-based regimens in the treatment of patients with relapsed and refractory multiple myeloma (RRMM) in real world settings. Methods: The clinical data of 53 patients with RRMM who were treated with selinexor-based regimens in 11 centers in China from July 2020 to December 2021 were retrospectively analyzed, and the response, survival and side effects were evaluated. Results: The median age of the 53 patients with RRMM at the time of treatment with the selinexor-based regimens was 60 years (range: 41-79). The median time since initial diagnosis was 30 months (range: 1-156). The median number of prior lines of therapy was 4 (range: 1-11), including 22 (41.5%) cases who had received autologous hematopoietic stem cell transplantation in the past, 8 cases (15.0%) who had been exposed to CART, 34 cases (64.1%) who were double-class refractory (proteasome inhibitors and immunomodulators), and 20 cases (33.7%) who were triple-class refractory(proteasome inhibitors, immunomodulators and daratumumab). The number of patients with high-risk cytogenetics [17p-/t (14;16)/t (4;14)/1q21] at onset was 24 (66.7%), and 20 (55.6%) at the time of the lastest relapse. The responses were evaluable in 47 patients, and the overall response rate (ORR, ≥PR) was 44.7%, including 3 cases with CR (6.4%), 4 cases with VGPR (8.5%), and 14 cases with PR (29.8%). The median follow-up time was 2.75 months (range: 0.14-17.5). In the responders, the median time to first response was 1.07 months (range: 0.25-2.71), and the median duration of response (DOR) was 7.75 months (95% CI: 4.705-10.795). The median progression-free survival (PFS) was 6.107 months (95%CI: 2.856, 9.358), and the median overall survival (OS) had not been reached. The major grade 3-4 adverse effets were hematological toxicity, and the incidences of grade 3-4 neutropenia, lymphopenia, and thrombocytopenia were 41.2%, 37.9%, and 53.0%, respectively. The incidence of dose reductions of selinexor due to myelosuppression was 7.1%. Among the non-hematological adverse effects, the most common grade 3-4 adverse effects were nausea and vomiting (38.7%), fatigue (18.6%), and infection (18.6%). Image: Summary/Conclusion: In the real world, the selinesor-based regimens have good effectiveness and safety profile in the treatment of RRMM. P921: UPDATED EFFICACY AND SAFETY RESULTS OF TECLISTAMAB, A B-CELL MATURATION ANTIGEN X CD3 BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA FROM MAJESTEC-1 J. Martínez-López1,*, P. Moreau2, S. Z. Usmani3, A. Garfall4, N. W. van de Donk5, J. F. San-Miguel6, A. Oriol7, A. Chari8, L. Karlin9, M.-V. Mateos10, R. Popat11, A. K. Nooka12, S. Sidana13, D. Trancucci14, R. Verona15, S. Girgis15, C. Uhlar15, T. Stephenson15, A. Banerjee15, A. Krishnan16 1Haematological Malignancies Clinical Research Unit, Hospital 12 de Octubre Universidad Complutense, CNIO, CIBERONC, Madrid, Spain; 2Hematology Clinic, University Hospital Hôtel-Dieu, Nantes, France; 3Levine Cancer Institute/Atrium Health, Charlotte, NC; 4Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 5Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 6Clínica Universidad de Navarra, CIMA, CIBERONC, IDISNA, Pamplona; 7Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Barcelona, Spain; 8Mount Sinai School of Medicine, New York, NY, United States of America; 9Service d’Hématologie Clinique, Centre Hospitalier Lyon Sud, Pierre-Bénite, France; 10University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca, Spain; 11University College London Hospitals, NHS Foundation Trust, London, United Kingdom; 12Winship Cancer Institute, Emory University, Atlanta, GA; 13Stanford University School of Medicine, Stanford, CA; 14Janssen Research & Development, Raritan, NJ; 15Janssen Research & Development, Spring House, PA; 16City of Hope Comprehensive Cancer Center, Duarte, CA, United States of America Background: Teclistamab (JNJ-64007957), a bispecific antibody targeting both B-cell maturation antigen (BCMA) and CD3 receptors, mediates T cell activation and subsequent lysis of BCMA-expressing myeloma cells. In the multi-cohort, open-label, phase 1/2 MajesTEC-1 (NCT03145181) study, the safety and efficacy of teclistamab in patients with relapsed/refractory multiple myeloma (RRMM) who previously received ≥3 lines of therapy (LOT) are being investigated. In phase 1, weekly subcutaneous dose of teclistamab 1.5 mg/kg, preceded by step-up doses of 0.06 and 0.3 mg/kg, was identified as the recommended phase 2 dose (RP2D). Initial results from phase 1/2 showed that teclistamab at the RP2D was well tolerated and provided encouraging efficacy in patients with no prior exposure to an anti–BCMA-targeted treatment. Aims: We report updated efficacy and safety results from MajesTEC-1 in patients treated at the RP2D, including additional patients and longer follow-up. Methods: MajesTEC-1 included patients aged ≥18 years with documented MM (as per the International Myeloma Working Group [IMWG] criteria) who had received ≥3 prior LOT including a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody. Patients previously exposed to BCMA-targeted therapy were not eligible in Phase 1. Patients received teclistamab at the RP2D. Overall response rate (ORR, assessed per the IMWG 2016 criteria) was the primary endpoint. CTCAE v4.03 (cytokine release syndrome [CRS] and ICANS graded per ASTCT guidelines) was used for grading adverse events (AEs). Results are based on a Sep 7, 2021 data cutoff for safety and a Nov 9, 2021 data cutoff for efficacy (N=165). Results: The median age was 64 y (range 33–84), 58% were male, and patients had received 5 (range 2–14) median prior LOT; 100% of patients were triple-class exposed, 78% were triple-class refractory, 70% were penta-drug exposed, and 30% were penta-drug refractory. ORR was 64% (95% CI 56–72), with 30% of patients achieving a complete response or better. Durable responses were observed, which deepened over time. The 12-month duration of response (DOR) rate was 66% (95% CI 49–79); median DOR was not reached. A reduction in soluble BCMA was observed in the first cycle of treatment in a majority of patients who responded to teclistamab. Neutropenia (65%; grade 3/4: 57%), anemia (50%; grade 3/4: 35%), thrombocytopenia (38%; grade 3/4: 21%), and lymphopenia (34%; grade 3/4: 32%) were the most common hematologic AEs. Infections occurred in 104 patients (63%; grade 3/4: 35%). The most common nonhematologic AE was CRS in 72% patients (grade 3, 0.6%; no grade 4/5). The median (range) time to CRS onset was 2 days (1–6) and median duration was 2 days (1–9). A total of 9 ICANS events (all grade 1/2; all resolved) were reported in 5 (3%) patients, of which 7 ICANS events were concurrent with CRS (all resolved). No dose reductions due to AEs were required, and no treatment-related deaths were reported. Summary/Conclusion: The deep and durable responses achieved with teclistamab in patients with highly refractory MM were reaffirmed with data from ~9 months of follow-up. No new safety signals were identified. Additional data with longer follow-up, including subgroup analyses and progression-free survival, will be presented. P922: HEALTH-RELATED QUALITY OF LIFE WITH TECLISTAMAB, A B-CELL MATURATION ANTIGEN X CD3 BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA FROM MAJESTEC-1 R. Popat1,*, P. Moreau2, S. Z. Usmani3, A. Garfall4, M.-V. Mateos5, J. F. San-Miguel6, A. Oriol7, A. K. Nooka8, L. Rosinol9, A. Chari10, L. Karlin11, A. Krishnan12, N. Bahlis13, T. Martin14, B. Besemer15, J. Martínez-López16, M. Delforge17, J. Fastenau18, K. S. Gries18, N. W. van de Donk19 1University College London Hospitals, NHS Foundation Trust, London, United Kingdom; 2Hematology Clinic, University Hospital Hôtel-Dieu, Nantes, France; 3Levine Cancer Institute/Atrium Health, Charlotte, NC; 4Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 5University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca; 6Clínica Universidad de Navarra, CIMA, CIBERONC, IDISNA, Pamplona; 7Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Barcelona, Spain; 8Winship Cancer Institute, Emory University, Atlanta, GA, United States of America; 9Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain; 10Mount Sinai School of Medicine, New York, NY, United States of America; 11Service d’Hématologie Clinique, Centre Hospitalier Lyon Sud, Pierre-Bénite, France; 12City of Hope Comprehensive Cancer Center, Duarte, CA; 13Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, CA; 14University of California San Francisco, San Francisco, CA, United States of America; 15University of Tuebingen, Tuebingen, Germany; 16Haematological Malignancies Clinical Research Unit, Hospital 12 de Octubre Universidad Complutense, CNIO, CIBERONC, Madrid, Spain; 17Universitaire Ziekenhuizen Leuven, Leuven, Belgium; 18Janssen Research & Development, Raritan, NJ, United States of America; 19Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands Background: Because patients with multiple myeloma (MM) have impaired health-related quality of life (HRQoL), it is important that patient-reported outcomes (PROs) are assessed in addition to clinical outcomes. Teclistamab (JNJ-64007957) is a B-cell maturation antigen (BCMA) x CD3 bispecific antibody designed to mediate T cell-induced lysis of BCMA-expressing MM cells. In the phase 1/2 MajesTEC-1 trial, initial results from the pivotal cohort (patients who received ≥3 prior lines of therapy [LOT], including a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody) showed that teclistamab was well tolerated with encouraging efficacy. Aims: We report PROs of the pivotal cohort from MajesTEC-1. Methods: MajesTEC-1 included patients aged ≥18 years with documented relapsed/refractory multiple myeloma (RRMM) as per the International Myeloma Working Group diagnostic criteria, progressive/measurable disease, and previous exposure to ≥3 prior LOT. Patients who had received prior anti-BCMA treatment were ineligible. Patients were given weekly subcutaneous teclistamab at the recommended phase 2 dose (1.5 mg/kg with step-up doses of 0.06 and 0.3 mg/kg). PRO assessments were performed at screening and at every even treatment cycle; results for cycles 2-8 are reported here. HRQoL was assessed by EORTC QLQ-C30 (range: 0–100; with higher scores indicating better global health status [GHS] but greater symptom severity [symptom scales]) and EuroQol 5-dimensional descriptive system (using the visual analog scale [VAS] with “0” indicating worst imaginable health state and “100” indicating best imaginable health state). Mixed-effects model with repeated measures was used to determine treatment effect. Meaningful improvement was defined as the proportion of patients with a change of ≥10 points. Kaplan-Meier estimate was used to determine time to worsening. Results: The analysis included 110 patients, with median follow-up duration of 7.8 months. The PRO compliance rates were high at baseline (85–90%) and through treatment cycles 2–8 (80–94%). Overall HRQoL was improved with teclistamab, as shown by improved GHS scores (cycle 2–8) and reduced pain (-4.2 [cycle 2] to -15.1 [cycle 8]; Table), with no overall changes in physical functioning and fatigue. Meaningful improvements from baseline to cycle 8 were observed in GHS (50% of patients), physical function (35%), pain (65%), and fatigue (73%). Meaningful improvement in overall health (VAS) was also observed in 50% of patients. Median time to improvement from baseline was ~1.5 months with longer time to improvement seen for nausea/vomiting and fatigue. Across all symptoms, median time to worsening ranged from 2 months to not estimable. Image: Summary/Conclusion: Teclistamab provided rapid, clinically meaningful improvements in HRQoL in patients with RRMM, which are consistent with clinical outcomes in MajesTEC-1. P923: TANDEM AUTOLOGOUS STEM CELL TRANSPLANTATIONS STILL BENEFIT FOR MYELOMA PATIENTS: A POOLING META-ANALYSIS C.-H. Lee1,*, C.-L. Ho1 1Hematology and Oncology, Tri-Service General Hospital/National Defense Medical Center, Neihu Dist, Taiwan Background: Although high-dose therapy and autologous stem cell transplant combined with novel agents continues to be the hallmark of first-line treatment in newly diagnosed transplant-eligible multiple myeloma patients, disease progression remains an issue. To deepen the treatment response and prolong disease free survival, tandem autologous stem cell transplant is one of treatment choice. Compared to toxicity of sub-sequent pharmacotherapy or allogeneic transplant, tandem ASCT is well-tolerated. However, the efficacy of tandem ASCT is not fully defined yet. Thus, we aimed to assess effectiveness of tandem ASCT. Aims: To evaluate the true effect of tandem autologous/autologous stem cell transplantation for myeloma patients in current era. Methods: Eligible randomized controlled trials published before Dec 2021 were retrieved from databases. Myeloma patients received tandem autologous stem cell transplantation was included. Systematic review and pairwise meta-analysis were performed. We calculated the hazard ratio (HR) with 95% confidence intervals (CIs). Results: Ten articles, involving 3,123 patients, met the selection criteria. All studies demonstrated acceptable quality. The mean age was 58.2 year-old with mean 36.2% high risk cytogenetics feature. Eight trials were international multi-centers phase III RCTs. Tandem autologous stem cell transplantation compared to single ASCT was associated with a significantly superior progression free survival (random-effect HR 0.89; 95% CI 0.82-0.97; I2 = 0.0%), but no significance difference regard to overall survival (random-effect HR 0.98; 95% CI 0.94–1.03; I2 = 0%). For high risk myeloma subgroup, 4 trials compared tandem ASCT versus lenalidomide, tandem ASCT demonstrated superior progression free survival (random-effect HR 0.87; 95% CI 0.82–0.91; I2 = 0%) Image: Summary/Conclusion: In conclusion, tandem ASCT are still to be considered as an acceptable treatment options in general myeloma patients. High-risk cytogenetics is frequently observed in newly diagnosed myeloma with worsens outcome after single autologous, whereas a tandem autologous transplant strategy may overcome onset poor prognosis. P924: TREATMENT EFFICACY OF PROGRESSION FREE SURVIVAL FOR REFRACTORY/RELAPSED MULTIPLE MYELOMA IN GERIATRIC PATIENTS: A NETWORK META-ANALYSIS C.-H. Lee1,*, C.-L. Ho1 1Hematology and Oncology, Tri-Service General Hospital, Neihu Dist, Taiwan Background: In recent 10 years, there were many studies explored the treatment efficacy of refractory/relapse multiple myeloma (R/R MM). Emerging evidences proved combination of new treatment modalities is significant better than traditional therapy. However, very few studies focus on geriatric patient who are more fragile and may not be able to tolerate treatment. Aims: Our aim was to synthesize all efficacy evidence, enabling an integrated comparison of all current treatment options in geriatric patients with R/R MM. Methods: We performed a systematic literature review to identify all publicly available randomized controlled trials (RCT) evidence. We searched Embase, PubMed, Cochrane Central Register of Controlled Clinical Trials, and the Web site www.ClinicalTrials.gov. Geriatric Patients with a diagnosis of R/R MM (including patients in second-line or more than second-line treatments) received subsequent active treatment were included. Geriatric patients should be set at least age 65 or above as a cut-off level. Data was extracted from subgroup analysis of each study. The evidence was synthesized using a Bayesian network meta-analysis. Hazard ratio (HR) was adopted. Results: In total, 4,966 citations were retrieved from the databases; 60 full texts were screened, of which 47 were excluded. In total, 13 RCTs were identified for quantitive analysis, including 14 treatment options (figure). Total 4337 geriatric patient were enrolled for network meta-analysis, twelve trials set cut-off level of age about 65, one set age 75 as a cut-off level. All trials had good quality. The triple combination therapy of daratumumab, lenalidomide and dexamethasone (DaraLenDex) was identified as the best treatment option in patients with R/R MM (figure). It was most favorable in terms of (1) HR for progression free survival (0.15; 95% credible interval (CI): 0.09 to 0.25) with significance and (2) probability of being best (96% of the cumulative ranking). Image: Summary/Conclusion: To the best of our knowledge, this is the first NMA on R/R MM that includes all regimens currently evaluated in randomized trials in elderly R/R MM. Our findings suggest that a 3-drug regimen containing the lenalidomide-dexamethasone backbone, preferentially combined with anti-MM mAbs daratumumab or elotuzumab, has the highest probability of being ranked as the best treatment in this setting, underlying the role of immunotherapy in MM. Prospective randomized trials are eagerly awaited to clarify the optimal sequencing of treatments for MM. We emphasize that it remains essential to conduct phase III RCTs to obtain more direct head-to-head evidence. Until such evidence becomes available, our results are highly important for informed decision making in everyday clinical practice P925: IMPACT OF PRIOR TREATMENT EXPOSURE ON THE EFFECTIVENESS OF IXAZOMIB-LENALIDOMIDE-DEXAMETHASONE IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS TREATED IN ROUTINE CLINICAL PRACTICE (THE INSURE STUDY) H. C. Lee1,*, K. Ramasamy2, M. Macro3, F. E. Davies4, R. Abonour5, F. van Rhee6, V. T. Hungria7, N. Puig8, K. Ren9, J. Silar10, V. Enwemadu11, D. Cherepanov9, D. M. Stull11, X. Leleu12 1MD Anderson Cancer Center, Houston, United States of America; 2Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom; 3CHU de Caen, Caen, France; 4Perlmutter Cancer Center, NYU Langone, New York; 5Indiana University School of Medicine, Indianapolis; 6University of Arkansas for Medical Sciences, Little Rock, United States of America; 7Clinica São Germano and Santa Casa Medical School, São Paulo, Brazil; 8Hospital Universitario de Salamanca Hematología, Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain; 9Takeda Development Center Americas, Inc. (TDCA), Lexington, United States of America; 10Institute of Biostatistics & Analyses, Ltd, Brno, Czechia; 11Takeda Pharmaceuticals U.S.A., Inc., Lexington, United States of America; 12Pôle Régional de Cancérologie, Department of Hematology, CHU La Milétrie-Poitiers, Poitiers, France Background: Results from INSURE, a pooled, global analysis of 3 observational studies, show that the effectiveness of ixazomib-lenalidomide-dexamethasone (IRd) used to treat relapsed/refractory multiple myeloma (RRMM) in routine clinical practice is comparable to its efficacy seen in the TOURMALINE-MM1 trial (median progression-free survival [PFS], 19.9 vs 20.6 months [mos]), with no new safety concerns (Leleu ASH 2021 #2701). Data on effectiveness outcomes following retreatment with agents used in earlier lines of therapy (LoTs) are limited, but may be of particular value for MM pts previously treated with lenalidomide (LEN) or proteosome inhibitors (PIs). Aims: To characterize the impact of prior exposure & refractoriness to LEN or PIs on the effectiveness & safety of IRd in RRMM. Methods: INSURE is a pooled analysis of data from 3 studies: INSIGHT MM, UVEA-IXA, & REMIX. INSIGHT MM is a prospective, global study of 4307 MM patients (pts) from 15 countries. UVEA-IXA is a multicenter, longitudinal, retrospective cohort study of 309 RRMM pts receiving ixazomib (IXA)-based therapy via an early-access program in Europe. REMIX is a retrospective/prospective study of 198 RRMM pts treated with IRd via a compassionate-use program in France. INSURE included adult RRMM pts who had received IRd in ≥2nd LoT. Primary outcomes were PFS & time-to-next therapy (TTNT). Secondary outcomes included: duration of treatment (DOT), overall survival (OS), overall response rate (ORR), & safety (discontinuations due to adverse events [AEs] were reported separately for each study). In this prespecified analysis pts were grouped by prior LEN or PI exposure (naïve, exposed, or refractory). Results: 562 pts were included: 391/100/71 were LEN-naïve/exposed/refractory & 37/408/117 were PI-naïve/exposed/refractory. In LEN-naïve/exposed/refractory pts, median age was 69/68/68 years (yrs; 24/14/18% >75 yrs) & 18/13/22% had an Eastern Cooperative Oncology Group performance status (ECOG PS) ≥2 (missing pts excluded from %); pts had received a median of 1/2/3 LoT(s) prior to IRd. In PI-naïve/exposed/refractory pts, median age was 71/68/69 yrs (38/22/15% >75 yrs) & 6/16/27% had an ECOG PS ≥2 (missing pts excluded from %); pts in all 3 subgroups had received a median of 2 LoTs prior to IRd. The Table shows effectiveness outcomes. Median DOT was 15.3/15.6/4.7 mos & median PFS was 21.6/25.8/5.8 mos in LEN-naïve/exposed/refractory pts. Median DOT & PFS in PI-naïve/exposed/refractory pts were 20.4/15.2/7.6 mos & not reached/19.7/12.9 mos, respectively. OS data were immature. The proportions of LEN-naïve/exposed/refractory pts in INSIGHT (n=114/39/28) & UVEA-IXA (n=161/15/19) who discontinued a study drug due to AEs were: IXA, 32/28/25% & 19/7/11%; LEN, 22/28/18% & 16/7/11%; dexamethasone (DEX), 18/21/14% & 11/0/11%, respectively. The proportions of PI-naïve/exposed/refractory pts in INSIGHT (n=9/130/42) & UVEA-IXA (n=18/125/52) who discontinued a study drug due to AEs were: IXA, 44/28/31% & 22/17/15%; LEN, 33/22/21% & 17/16/12%; DEX, 33/18/17% & 17/10/8%, respectively. Data for discontinuations due to AEs were not available for REMIX. Additional safety data will be presented. Image: Summary/Conclusion: IRd appeared to be effective in RRMM pts in routine clinical practice regardless of prior LEN or PI exposure, with better outcomes seen in LEN- &/or PI-non-refractory vs -refractory pts. PFS outcomes in the naïve/exposed populations are comparable to those reported in TOURMALINE-MM1. Pts with prior LEN or PI exposure can achieve clinical benefit when retreated suggesting that prior exposure should not preclude use of these agents in later LoTs. P926: DARATUMUMAB, BORTEZOMIB AND DEXAMETHASONE FOR TREATMENT OF PATIENTS WITH RELAPSED OR REFRACTORY MULTIPLE MYELOMA AND SEVERE RENAL IMPAIRMENT: RESULTS FROM THE PHASE 2 GMMG-DANTE TRIAL L. Leypoldt1,*, M. Gavriatopoulou2, B. Besemer3, H. Salwender4, M.-S. Raab5, A. Nogai6, C. Khandanpour7, V. Runde8, M. Zago9, P. Martus10, H. Goldschmidt11, C. Bokemeyer1, M. Dimopoulos2, K. Weisel1 1Department of Hematology, Oncology and Bone Marrow Transplantation with Section of Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2Department of Clinical Therapeutics, School of Medicine, National and Kapodistrian University of Athens, Alexandra General Hospital, Athens, Greece; 3Department of Hematology, Oncology, Immunology, Rheumatology and Pulmonology, University Hospital of Tuebingen, Tuebingen; 4Asklepios Tumorzentrum Hamburg, AK Altona and AK St. Georg, Hamburg; 5Department of Internal Medicine, Heidelberg University Hospital, Heidelberg; 6Department of Hematology, Oncology and Tumor Immunology, Charite Medical School, Berlin; 7Department of Medicine A, Hematology, Oncology and Pneumology, University Hospital Münster, Münster; 8Department of Hematology/Oncology, Wilhelm-Anton Hospital Goch, Goch; 9Center for Clinical Trials, University Hospital of Tuebingen; 10Department of Clinical Epidemiology and Applied Biostatistics, Eberhard Karls University of Tuebingen, Tuebingen; 11Internal Medicine V and National Center for Tumor Diseases (NCT), University Hospital Heidelberg, Heidelberg, Germany Background: Renal function impairment is one of the main characteristics of multiple myeloma (MM) leading to diagnosis and defining treatment requirement in first-line and subsequent relapse. However, limited data exist on safety and efficacy of anti-MM regimens in patients (pts) with severe renal impairment and those requiring hemodialysis as pts are mostly excluded from clinical trials and specific trials in this indication are rare. Proteasome inhibitors as bortezomib (BTZ) are known to represent one of the preferable drug classes in patients with renal impairment due to rapid response, lack of dose adjustment and renoprotective effects. Daratumumab (DARA) is an anti-CD38 monoclonal antibody which enhances response rates and efficacy when added to standard of care regimens, both in newly diagnosed and relapsed and refractory (RR)MM. The combination of BTZ, dexamethasone (DEX), and DARA (DVd) has been shown to be efficacious and safe in RRMM in the phase 3 study CASTOR (Palumbo et al., NEJM 2016). In the CASTOR trial, pts with a GFR < 20 ml/min were excluded. Aims: Here, we present results from the primary analysis of the investigator-initiated multicenter GMMG-DANTE (NCT02977494) trial investigating DVd in RRMM pts with severe renal impairment including pts on hemodialysis. Methods: RRMM pts with measurable disease who had received at least 1 prior line of therapy and a GFR < 30 ml/min or undergoing hemodialysis were eligible. Pts received DVd in the approved schedule with 8 cycles (21 d/cycle) of BTZ (1.3 mg/m2, SC) on Days 1, 4, 8, and 11 and DEX (20 mg, PO or IV) on days 1, 2, 4, 5, 8, 9, 11, and 12 + DARA (16 mg/kg, IV) given weekly for cycles 1-3, Q3W for cycles 4-8, and Q4W thereafter. 36 patients were planned to be included into the trial. The trial was closed prematurely after 22 patients due to inferior recruitment. The primary endpoint was overall response rate (ORR). Key secondary endpoints were progression-free survival (PFS), overall survival (OS) and safety. Response assessment was performed using the IMWG criteria. Results: 22 pts from 6 German and one Greek site were included from 2017-2020. Analyzed population included 21 patients. Median age was 70 years. Median GFR was 21.0 ml/min, 8 patients were under hemodialysis. Median number of prior lines was 2 (range 1-10). All patients started DVd treatment. For one patient it was not possible to obtain a response. ORR was 67% (14/20) with 6 pts (29%) showing a partial response (PR), 6 pts (29%) a very good partial response (VGPR) and 2 pts (10%) a complete response (CR). Median treatment duration was 24 weeks (range 2-106). After a median follow-up of 28 months, median PFS was 10.4 months, median OS was not reached (OS after 24 months until 39 months plateau 0.504). The most frequent toxicity ≥ grade 3 was hematologic, with anemia in 23.8%, thrombocytopenia in 23.8% and neutropenia in 9.5%. Main non-hematologic adverse events (AEs) ≥ grade 3 were infections (23.8%) with mainly pneumonias. Polyneuropathy all grade was described in 52.4%. Summary/Conclusion: In this phase II prospective trial we demonstrated that DVd shows relevant efficacy in RRMM pts with severe renal impairment and can be safely administered. Efficacy is comparable to the one reported in patients with no severe renal impairment. Toxicity does not differ from previously reported data on DVd. Acknowledging the generally reported impaired outcome of MM pts showing severe renal impairment, PFS and OS underline the importance of effective MM regimens which can be safely administered in a difficult to treat population. P927: CLINICAL CHARACTERISTICS, SURVIVAL OUTCOMES AND PROGNOSIS IN 123 IMMUNOGLOBULIN D MULTIPLE MYELOMA PATIENTS: A RETROSPECTIVE SINGLE-CENTER J. Liu1,*, H. Fan1, W. Yan1, J. Xu1, L. Li1, L. Qiu1, G. An1 1Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China Background: Immunoglobulin D (IgD) myeloma is a rare subtype of multiple myeloma (MM), and has been considered to have a more aggressive course and poor outcome. With the rapid progress of multiple myeloma treatment, including the application of proteasome inhibitors and immunomodulatory drugs and the promotion of autologous stem cell transplantation (ASCT), the prognosis of MM patients has been greatly improved. Considering the development of MM treatment, the survival outcome and prognostic factors of IgD MM patients are now controversial. Aims: To evaluate the clinical characteristics, survival outcomes and prognosis of IgD myeloma patients in the era of novel therapy. Methods: We retrospectively analyzed the clinical characteristics of 123 IgD MM patients and compared them with other MM subtypes. At the same time, we also discussed the survival outcomes and prognostic factors of IgD myeloma patients under different treatment modes. Results: Male to female ratio was 2.2, median age was 55 years (29-79 years)， with preponderance of lambda light chains (89.4%). IgD myeloma more likely presented advanced stage when diagnosed: higher frequencies of ISS stage Ⅲ, R-ISS stage Ⅲ, renal dysfunction, high lactate dehydrogenase (LDH) level, high β2MG level and high bone marrow plasmacytosis. Additionally, the genetics of IgD myeloma patients were also different from other subtypes myeloma. Complex karyotypes were more common in IgD myeloma patients (23.9% vs. 14.8%, p=0.017). Cytogenetic abnormalities demonstrated by FISH could be found in 86% (80/93) of the IgD myeloma patients. The frequencies of t (11;14) (38.6% vs. 13.6%, p=0.000) and 1q21 amplification (72.0% vs. 53.9%, p=0.001) were much higher in IgD MM patients than other subtypes myeloma, while deletion of 13q (34.4% vs. 45.7%, p=0.032) and high risk cytogenetic abnormalities (HRCA) (23.0% vs. 37.9%, p=0.009) were less. 71 IgD MM patients received induction therapy based on ‘novel therapies’, 26 on ‘traditional therapies’. Compared with ‘traditional therapies’, ‘novel therapies’ significantly improved the prognosis of IgD patients (PFS 16.9 vs. 29.1 months, P=0.008; OS 37.6 vs. 70.8 months, P=0.002). Further analysis of the ‘novel therapies’ group found that, in non-transplant patients, the prognosis of the IgD and non-IgD patients was similar, but in transplant patients, IgD MM patients showed worse outcomes compared to non-IgD MM patients (figure). Multivariate analysis of 71 IgD patients who received ‘novel therapies’ identified that R-ISS stage Ⅲ, complex karyotypes and deletion of 13q were independent adverse factors for OS, whereas complex karyotypes and thrombocytopenia were independent adverse factors for PFS. Image: Summary/Conclusion: In summary, our study compared the clinical characteristics of IgD and non-IgD MM patients, IgD myeloma patients are younger, have higher tumor burden and advanced disease stage. In addition, their genetics are significantly different from non-IgD MM: IgD MM patients have more complex karyotypes, amplification of 1q21 and t (11;14), while less deletion of 13q and HRCA. Meanwhile, we found that although new drugs combined with ASCT significantly improved the prognosis of MM patients, the improvement of IgD MM is less obvious than that of non-IgD MM patients, suggesting more targeted drugs need to be developed for patients with IgD MM. Finally, we identified that R-ISS stage Ⅲ, complex karyotypes and deletion of 13q were independent adverse factors for OS, indicating that R-ISS is still useful way to predict the outcomes of patients with IgD myeloma. P928: REAL-LIFE ANALYSIS OF THE MULTIPLE MYELOMA PATIENT’S SURVIVAL IN A LARGE COHORT OF PATIENTS N. Lopez-Muñoz1,*, G. Hernandez-Ibarburu2, J. M. Sánchez-Pina1, R. A. Alonso1, C. Cuellar1, M. Calbacho1, R. Ayala1, R. Iñiguez1, N. García Barrio3, E. Vera1, D. Pérez-Rey2, L. Meloni4, M. Pedrera-Jiménez3, P. Serrano-Balazote3, J. De La Cruz5, J. Martínez-López1 1Hospital 12 de Octubre; 2Biomedical Informatics Group, Universidad Politécnica de Madrid; 3Data Science Group, Research Institute imas12, MADRID, Spain; 4Trinetx, LLC, Cambridge, United States of America; 5Research Institute imas12, MADRID, Spain Background: Multiple myeloma (MM) constitutes approximately 10% of hematological malignancies, with a median age at diagnosis of 65 years. Patient survival has improved considerably over the last 20 years with the introduction of new drugs. In 1999, the first immunomodulatory drug, thalidomide, was approved, followed by lenalidomide in 2005. In 2003, proteosome inhibitors such as bortezomib were introduced and, in 2015, anti-CD38 monoclonal antibodies such as daratumumab. Aims: By relying on the TriNetx platform, a global health research platform, we have analyzed the impact on the outcome of the introduction new drugs for MM in the last 20 years. We also analyzed the difference patterns of treatments between networks. Methods: First, we retrospectively selected patients diagnosed with symptomatic multiple myeloma between 1999 and 2019 in a tertiary care hospital in Spain, Hospital 12 de Octubre (H12O) in Madrid. Then, we analyzed data from more than 7500 patients diagnosed with CML from research networks: EMEA and the US; covering more than 16 million subjects. We compared the time of survival since the MM diagnosis in three groups depending of the age at MM diagnosis (less than 65, between 65 and 75, and more than 75 years old) over three periods of time: 1999-2009, 2010-2014 and 2015-2019 (2020 was excluded because of pandemic impact). Kaplan-Meier analysis was used for analyzing overall survival (OS) and differences between groups were tested for statistical significance using the log-rank test. Also, we analyzed the patterns of treatment along this time. Results: Approximately, a total 760 patients were included in the study. In the H12O, median OS was 45.1, 45.2, 65.1 months and not reached for the 1999-2009, 2010-2014 and 2015-2019 periods, respectively (p=0.001). Among the group of patients younger than 65 years old, the median OS was 64 months (47.5-81.4; 95%) among patients diagnosed in the period 1999-2009; 80 months (36.1-124; 95%) in 2010-2014; and median not reached for the 2014-2019 (p<.001). Similar results were found in a US cohort, including approximately 7000 patients, between the years 2010-2014 vs 2014-2019 (59.71% vs 64.20%, p<0.0001, HR 1.154 (1.074- 1.241) (figure 1). The same results were observed for the 65-75 years cohort, median OS was 62.6 months (50.9-74.3; 95%) for the 1999-2009 period, 70.6 months (33.1-108.1; 95%) between 2010 and 2014, and not reached for the 2014-2019 period (p<.001). The same occurs among more than 3000 patients from the US base between the years 2010-2014 vs 2014-2019 (53.96% vs 57.73%, p=0.016, HR 1.099 (1.018 - 1.187) (figure 2). Otherwise, patients older than 75 years have a median OS of 31.1 months and do not show a statistically significant difference regardless on the year of MM diagnosis (p=.18). Nor were clinically significant results obtained between the EMEA and US cohorts. On the other hand, the first-line treatment used in patients was analysed, and in both the EMEA and US cohorts, the use of bortezomib (46% vs 47% in the EMEA cohort; 62% vs 70% in US cohort), lenalidomide (43% vs 57% in EMEA cohort, 15% vs 18% in US cohort) and daratumumab (0% vs 4% in EMEA cohort, 2% vs 4% in US cohort) increases over the years comparing the 2010-2014 vs 2015-2019 time cohorts. This could explain the improved survival in more recent times. Summary/Conclusion: The introduction of new agents for the treatment of MM has transformed the natural history of the disease, achieving long survival times in younger patients. Thus, it is essential to continue to advance and develop new therapies. P929: IXAZOMIB AND DARATUMUMAB WITHOUT DEXAMETHASONE (I-DARA) IN ELDERLY FRAIL RELAPSING MYELOMA (RRMM) PATIENTS: RESULTS OF THE PHASE 2 STUDY IFM 2018-02 OF THE INTERGROUPE FRANCOPHONE DU MYELOME (IFM). M. Macro1,*, C. Touzeau2, C. Mariette3, S. Manier4, S. Brechignac5, L. Vincent6, B. Hebraud7, O. Decaux8, S. Schulmann9, C. Lenoir10, P. Godmer11, A. Farge12, L. Peyro Saint Paul13, J.-J. Parienti13, X. Leleu14 1HEMATOLOGY IHBN, University Hospital, CAEN; 2HEMATOLOGY, University Hospital, Nantes; 3HEMATOLOGY, University Hospital, Grenoble; 4Hematology, University Hospital, Lille; 5Hematology, University Hospital, Paris; 6Hematology, University Hospital, Montpellier; 7Hematology, University Hospital, Toulouse; 8Hematology, University Hospital, Rennes; 9Hematology, University Hospital, Nancy; 10Hematology, Private Hospital, Bordeaux; 11Hematology, Community Hospital, Vannes; 12Hematology; 13Clinical Research, University Hospital, CAEN; 14Hematology, University Hospital, Poitiers, France Background: Frail patients with multiple myeloma have an inferior outcome, especially in the relapse setting. This adverse prognosis is mainly related to a high discontinuation rate due to treatment (Tx) related adverse events. Aims: The aim of this phase 2 study is to evaluate efficacy and tolerability of Ixazomib-Daratumumab (I-Dara) without Dexamethasone in elderly frail patients with relapsed myeloma (RRMM) (NCT03757221). Methods: Ixa-Dara naïve RRMM patients received oral Ixazomib (4 mg: days 1, 8, 15), IV Daratumumab (16 mg/kg; days 1, 8, 15, 22, cycles 1-2; days 1, 15, cycles 3-6; days 1, cycles 7+) and IV Methylprednisolone before Daratumumab (100 mg at day 1, 8, cycle 1 and then 60 mg). They were enrolled after 1 or 2 prior therapy if their frailty score was ≥ 2 by IMWG score. The primary endpoint was ≥ very good partial response rate (VGPR) at one year. Secondary endpoints included overall response rate (ORR), progression free survival (PFS), overall survival (OS) & toxicity according to NCI-CTCAE version 5 Results: Sixty-three patients were screened and 55 enrolled between 03/2018 and 09/2021. Patient were at first (n = 36) or second relapse (n = 19). Thirty-three patients (60%) were previously exposed to bortezomib, 37 (67%) were previously exposed to lenalidomide (Len) and 20 (36 %) were refractory to Len. Median age was 82 (72-93). All patients had a frailty score ≥2 and 13 (24 %) had a 3 or 4 frailty score. In 41 patients ISS at diagnosis was stage I (n = 11), II (n = 18) or III (n = 12). Seventeen (36%) patients harbored high-risk (HR) cytogenetic, including t(4;14) (n = 8) or del17p (n = 10). The median duration of Tx among 28 pts with ongoing Tx was 10 months [5-32] at data cutoff (February, 2). The median duration of Tx among 27 pts who stopped Tx was 6 months [0-18]: 18 had progressive disease. Nine patients died during the study: Daratumumab-related bronchospasm (D1C1); Ixazomib-related overdose (C2); sepsis (n = 4), progressive disease (n = 3). Regarding toxicity, 27 pts had a ≥grade 3 AE (49%). The most common grade 3-4 toxicities were thrombocytopenia (n = 9), other cytopenias (n = 4), infection (n = 8), hypertension (n = 3) and gastrointestinal disorders (n = 3). Fourteen out of 28 were SAE including 5 infections, 1 bronchospasm, 1 acute respiratory failure and 2 ixazomib overdoses. Overall response rate, including minimal response, was 86 % with a ≥VGPR rate of 32 % in the whole group. In Len refractory patients the ORR was 82 % and ≥VGPR 41%, in HR cytogenetic patients ORR was 85 % and ≥VGPR 46%. With a median follow-up of 11.6 months median PFS is 16 months and median OS NR (76% estimated at one year). Image: Summary/Conclusion: In this elderly frail population Ixa-Dara is a feasible combination with favorable efficacy profile even in Len refractory and HR cytogenetic patients. Early toxicity remains a concern in this population eventhough more manageable with Dara SC. P930: ISATUXIMAB, LENALIDOMIDE, BORTEZOMIB AND DEXAMETHASONE AS INDUCTION THERAPY FOR NEWLY-DIAGNOSED MULTIPLE MYELOMA PATIENTS WITH HIGH-RISK CYTOGENETICS: A SUBGROUP ANALYSIS FROM THE GMMG-HD7 TRIAL E. K. Mai1,*, U. Bertsch1,2, R. Fenk3, D. Tichy4, B. Besemer5, J. Dürig6, R. Schroers7, I. von Metzler8, M. Hänel9, C. Mann10, A. M. Asemissen11, B. Heilmeier12, E. Nievergall1, S. Huhn1, K. Kriegsmann1, N. Weinhold1, S. Luntz13, T. A. W. Holderrried14, K. Trautmann-Grill15, D. Gezer16, M. Klaiber-Hakimi17, M. Müller18, C. Khandanpour19, W. Knauf20, C. Scheid21, M. Munder22, T. Geer23, H. Riesenberg24, J. Thomalla25, M. Hoffmann26, M. S. Raab1, H. J. Salwender27, K. C. Weisel11, H. Goldschmidt1,2 1Department of Internal Medicine V, University Hospital Heidelberg; 2National Center for Tumor Diseases, Heidelberg; 3Department of Hematology, Oncology and Clinical Immunology, University Hospital Düsseldorf, Düsseldorf; 4Division of Biostatistics, erman Cancer Research Center (DKFZ) Heidelberg, Heidelberg; 5Department of Internal Medicine II, University Hospital Tübingen, Tübingen; 6Department for Hematology and Stem Cell Transplantation, University Hospital Essen, Essen; 7Medical Clinic, University Hospital Bochum, Bochum; 8Department of Medicine, Hematology/Oncology, University Hospital Frankfurt, Goethe University, Frankfurt am Main; 9Department of Internal Medicine III, Clinic Chemnitz, Chemnitz; 10Department for Hematology, Oncology and Immunology, University Hospital Gießen and Marburg, Marburg; 11Department of Oncology, Hematology and BMT, University Medical Center Hamburg-Eppendorf, Hamburg; 12Clinic for Oncology and Hematology, Hospital Barmherzige Brueder Regensburg, Regensburg; 13Coordination Centre for Clinical Trails (KKS) Heidelberg, Heidelberg; 14Department of Oncology, Hematology, Immuno-Oncology and Rheumatology, University Hospital Bonn, Bonn; 15Department of Internal Medicine I, University Hospital Dresden, Dresden; 16Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen; 17Clinic for Hematology, Oncology and Palliative Care, Marien Hospital Düsseldorf, Düsseldorf; 18Clinic for Hematology, Oncology and Immunology, Klinikum Siloah Hannover, Hannover; 19Medical Clinic A, niversity Hospital Münster, Münster; 20Center for Hematology and Oncology Bethanien, Frankfurt am Main; 21Department of Internal Medicine I, University Hospital Cologne, Cologne; 22Department of Internal Medicine III, University Hospital Mainz, Mainz; 23Department of Internal Medicine III, Diakoneo Clinic Schwäbisch-Hall, Schwäbisch-Hall; 24Hematology / Oncology Center, Bielefeld; 25Hematology / Oncology Center, Koblenz; 26Medical Clinic A, Clinic Ludwigshafen, Ludwigshafen; 27Asklepios Tumorzentrum Hamburg, AK Altona and AK St. Georg, Hamburg, Germany Background: The multicenter phase III trial GMMG-HD7 (NCT03617731) demonstrated superior minimal residual disease (MRD) negativity rate after induction therapy in patients with transplant-eligible newly-diagnosed multiple myeloma (NDMM) by addition of the anti-CD38 monoclonal antibody isatuximab (Isa) to lenalidomide / bortezomib / dexamethasone (Isa-RVd), as compared to RVd alone (Goldschmidt H et al., 2021, ASH Annual Meeting). Aims: Here we present a subgroup analysis on patients with high-risk cytogenetics. Methods: Patients with transplant-eligible NDMM were equally randomized to receive three 42-day cycles of RVd (lenalidomide 25 mg/d p.o., d1–14 and d22-35; bortezomib 1.3 mg/m2 s.c. d1, 4, 8, 11, 22, 25, 29, 32; dexamethasone 20 mg/d d1-2, 4-5, 8-9, 11-12, 15, 22-23, 25-26, 29-30, 32-33) in both study arms. Isa was added to Isa-RVd as follows: 10 mg/kg i.v., cycle 1: d 1, 8, 15, 22, 29; cycles 2-3: d 1, 15, 29. Randomization for induction was stratified by Revised International Staging System. Primary endpoint of the trial was MRD negativity rate assessed by next-generation flow (NGF, cut-off 1x10-5) after induction therapy. Fluorescence in-situ hybridization (FISH) analysis was performed centrally on CD138-purified plasma cells. High-risk and ultra high-risk cytogenetics were defined as at least one or two of the following aberrations, respectively: del17p, t(4;14), t(14;16), gain1q21 (≥ 3 copies). Data cut-off for the present analysis was December 2021. Results: 660 patients (Isa-RVd: 331 and RVd: 329) were eligible for intention-to-treat analysis. The study met its primary endpoint, demonstrating superiority of NGF-MRD negativity rates with Isa-RVd compared to RVd (50.1% vs. 35.6%; odds ratio [OR]=1.82, 95% confidence interval [95% CI]: 1.33-2.48, p<0.001). High-risk cytogenetics were well balanced between the treatment arms. 264 of 584 (45.2%) and 82 of 580 (14.1%) evaluable patients had high-risk and ultra high-risk cytogenetics, respectively. Del17p, t(4;14), t(14;16) and gain 1q21 were present in 59 of 615 (9.6%), 67 of 613 (10.9%), 17 of 609 (2.8%) and 218 of 583 (37.4%) evaluable patients, respectively. Among patients with high-risk cytogenetics, MRD negativity rates were 50.4% (63/125) vs. 37.4% (52/139; OR=1.70, 95% CI: 1.04-2.79, p=0.03) with Isa-RVd vs. RVd. MRD negativity rates for ultra high-risk patients were 56.3% (27/48) vs. 44.1% (15/34; OR=1.63, 95% CI: 0.67-3.99, p=0.28) with Isa-RVd vs. RVd. Similar results were observed for Isa-RVd vs. RVd among the common major single high-risk cytogenetic features: del17p: 56.0% (14/25) vs. 35.3% (12/34), OR=2.33, 95% CI: 0.82-6.88; t(4;14): 57.6% (19/33) vs. 47.1% (16/34), OR=1.53, 95% CI: 0.58-4.06; t(14;16): 66.7% (6/9) vs. 50.0% (4/8), OR=2.00, 95% CI: 0.28-15.67; gain1q21: 48.2% (55/114) vs. 35.6% (37/104), OR=1.69, 95% CI: 0.98-2.92. Dividing evaluable patients in either standard risk (absence of any high-risk aberration; 320/578, 55.4%) vs. high-risk (exactly one high-risk aberration; 176/578, 30.4%) vs. ultra high-risk (≥2 high-risk aberrations; 82/578, 14.2%) yielded similar efficacy results. MRD negativity rates for Isa-RVd vs. RVd were 49.7% (86/173) vs. 36.7% (54/147; OR=1.70, 95% CI: 1.09-2.68) in standard risk patients, 46.7% (35/75) vs. 34.7% (35/101; OR=1.65, 95% CI: 0.90-3.05) in high-risk patients and 56.3% (27/48) vs. 44.1% (15/34; OR=1.63, 95% CI: 0.67-3.99) in ultra high-risk patients. Summary/Conclusion: Isa-RVd induction therapy is superior to RVd in patients with transplant-eligible NDMM and high-risk or ultra high-risk cytogenetics, consistent with the benefit observed in the overall trial population. P931: PROSPECTIVE OBSERVATIONAL MULTICENTER STUDY OF GAUCHER DISEASE PREVALENCE IN PATIENTS AFFECTED BY MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE G. Giuffrida1,*, U. Markovic1,2,3, C. Conticello1, D. Nicolosi1, V. Calafiore1, A. Romano1,4, A. Condorelli1, S. Grasso1, A. Duminuco1, M. Calagna1, A. Nardo1, C. Riccobene1, B. Esposito1, U. Consoli5, V. Di Giacomo6, S. Neri6, M. R. Cingari7, G. Iaria8, V. Innao5,9, M. Spina10, J. Gentile11, C. Zizzo12, G. Duro12, F. Di Raimondo1,4 1Division of Hematology, AOU “Policlinico G. Rodolico-San Marco”, Catania; 2Division of Oncohematology and BMT, Istituto Oncologico del Mediterraneo, Viagrande; 3Department of Biomedical, Dental, Morphological and Functional Imaging Sciences, University of Messina, Messina; 4Postgraduate School of Hematology, University of Catania; 5Unità Operativa Complessa (UOC) di Ematologia, Azienda Ospedaliera di Rilievo Nazionale e di Alta Specializzazione (ARNAS), Catania; 6UOC di Ematologia, Azienda Ospedaliera Papardo, Messina; 7Unitá Operativa Semplice Dipartimentale Ematologia, Ospedale San Vincenzo, Taormina; 8Hemato-Oncology and Radiotherapy Department, Azienda Ospedaliera “Bianchi Melacrino Morelli”, Reggio Calabria; 9Division of Hematology, Department of Human Pathology in Adulthood and Childhood, University of Messina, Messina; 10UO Medicina Generale, Ospedale Nuovo di Gragnano, Gragnano; 11Sanofi Genzyme, Milano; 12Institute for Biomedical Research and Innovation (IRIB-CNR), National Research Council of Italy, Palermo, Italy Background: Gaucher disease (GD) is a rare, autosomal recessive genetic disorder, caused by deficiency of glucocerebrosidase, leading to glucosylceramide accumulation in tissue macrophages of hematological, visceral, and skeletal organ systems. Type 1 GD accounts for more than 90% all patients. Monoclonal gammopathy of undetermined significance is the most common plasma cell disorder, occurring in 3% of the population older than 50 years and is typically detected as an incidental finding. Type 1 GD It is frequently associated with polyclonal and monoclonal gammopathy (MGUS) and is hypothetically caused by long-term immune activation due to accumulated lysolipids, particularly glucosylsphingosine, although the cause remains unknown. Aims: We evaluated the prevalence of GD in MGUS patients, particularly when other signs and symptoms are present, in order to determine its potential usefulness as screening tool and inclusion in diagnostic framework of GD. Methods: Between January 2018 and February 2022, dried blood spots (DBS) samples were collected and tested for the acid β-glucosidase (glucocerebrosidase) enzyme activity from MGUS patients, both adult and pediatric, followed in seven hematology units of Sicily, Calabria and Naples. The glucocerebrosidase activity was measured with multiplexed tandem mass spectrometry (MS/MS) using the NeoLSD® assay system, and pathological range was within 0.2 and 2.5 nmol/h/ml. In case of DBS positive result, a confirmatory test was carried over in order to confirm the diagnosis of GD. The study was approved by the local institutional review board and supported by Sanofi Genzyme. All patients provided informed consent for the prospective collection of their data. Results: A total of 600 patients with MGUS was enrolled at last study update. Around half of the study population was male, with median age of 65 years. Immunoglobulin G was most frequently isolated with serum immunofixation, median level of monoclonal protein was 0.66 g/dL, while median kappa and lambda values based on monoclonal serum free light chain type were 24.8 mg/L and 32.7 mg/L, respectively. Out of 161 patients with available complete blood count median hemoglobin value was 13.5 g/dL with 15 patients having less than 11 g/dL, median platelet count was 224.000/mmc with 10 patients having less than 100.000/mmc platelets and median neutrophil count of 4.280/mmc and seven patients with less than 2.000/mmc neutrophils. Median ferritin level was 77 ng/mL, with more than 400 ng/mL in 10 patients. The median glucocerebrosidase activity in the entire cohort was 6.9 nmol/h/ml (range 0.3-58.8), with thirteen patients having pathological enzyme activity (median value 1.8 nmol/h/ml, range 0.2-2.5). Sequence analysis of GBA gene evidenced compound heterozygous mutation of the GBA1 gene in three patients (c.1226A>G - N370S and c.1448T>C -L444P in two and c.1226A>G -N370S and c.355G>A -G119R in one respectively), and homozygous mutation (c.1226A>G -N370S) in one patient associated with glucocerebrosidase activity below normal limits. All three patients with compound heterozygous mutational status had signs of GD (hepatosplenomegaly and mild thrombocytopenia). As for the rest of the MGUS patients, in a total of 114 patients sequence analysis evidenced single heterozygous mutation in 16 patients. Summary/Conclusion: Although type 1 GD remains a rare lysosomal storage disorder, patients with the diagnosis of MGUS could be considered for GD screening with DBS, especially when other symptoms (thrombocytopenia, splenomegaly, hyperferritinemia) are present. P932: ON-DEMAND PLERIXAFOR WITH CYCLOPHOSPHAMIDE AND G-CSF FOR HEMATOPOIETIC STEM-CELL MOBILIZATION IN MULTIPLE MYELOMA PATIENTS: FINAL RESULTS OF THE MOZOBL06877 STUDY R. Mina1,*, F. Bonello1, F. Fazio1, R. Saccardi1, V. Bongarzoni1, F. Marchesi1, G. Bertuglia1, P. Curci1, R. M. Lemoli1, S. Ballanti1, T. Dentamaro1, G. Benevolo1, A. Capra1, R. Floris1, P. Tosi1, A. Olivieri1, D. Rota-Scalabrini1, C. Cangialosi1, M. Cavo1, P. Corradini1, G. Milone1, M. Boccadoro1, A. Larocca1 1European Myeloma Network, (EMN), Italy Background: High-dose melphalan and autologous stem-cell transplant (ASCT) are pillars for the upfront treatment of ASCT-eligible newly diagnosed multiple myeloma (ND)MM patients (pts) or as salvage strategy at relapse. Failure to collect an adequate number of hematopoietic stem cells (HSC) to receive ASCT (<2×106 CD34+ cells/kg) occurs in 5%-15% of MM pts undergoing HSC mobilization with granulocyte colony-stimulating factor (G-CSF) or G-CSF+cyclophosphamide (G-CSF/CY). Aims: We report the final results of the observational MOZOBL06877 study (NCT03406091; partially supported by Sanofi investigation funds) to prospectively assess the performance of HSC mobilization with G-CSF/CY plus on-demand CXCR4 inhibitor plerixafor (PLX) in NDMM pts treated with novel agents. Methods: NDMM pts undergoing HSC mobilization with CY (2-4 g/m2) and G-CSF (5-10 mcg/kg/day) were enrolled and observed up to 30 days after mobilization. According to its label, “on-demand” PLX was administered in pts with <20 CD34+ cells/ul after ≥4 days of G-CSF or in case <1×106 CD34+ cells/kg were collected on the first apheresis day. Pts gave written informed consent. The primary endpoint was the poor mobilizer rate, defined as the rate of pts collecting <2×106 CD34+ cells/kg or requiring PLX. Secondary endpoints were the identification of predictive factors for PLX use and safety during mobilization. Results: 301 NDMM pts were enrolled and analyzed; 72% received induction with bortezomib-thalidomide-dexamethasone, 9% a lenalidomide (Len)-based and 3% a daratumumab (Dara)-based regimen. Overall, 48 pts (16%) were poor mobilizers: 14 (5%) failed to yield ≥2×106/Kg CD34+, while 34 (11%) required rescue with PLX. Among pts yielding <2×106/Kg CD34+ cells, 4 received PLX, while 10 did not. Among pts who successfully collected ≥2×106/Kg CD34+ (n=287, 95%), 34 (12%) required PLX: 23 (68%) due to a CD34+/uL count ≤20 after ≥4 days of G-CSF and 11 (32%) to a collection <1×106/Kg after the first apheresis day. In pts mobilized with PLX, median number of CD34+×106/L cells increased from 17.5 (IQR 10.8-25.6) before PLX to 58.3 (IQR 34.2-100.2) after PLX. Median number of CD34+/Kg collected in pts who did not require PLX was 10.2×106 (IQR 8.3-13.2). In pts who required PLX, a median of 6.5×106 CD34+/Kg (IQR 4.6-9.6) was collected. An adequate HSC yield for ≥2 ASCTs (≥4×106/Kg) was obtained in 96% and 85% of pts not receiving or receiving PLX, respectively. Median number of apheresis days was 1 (IQR 1-2) in pts not requiring PLX and 2 (IQR 1-2) in pts requiring PLX. In a multivariate analysis (Table), factors predicting PLX use were advanced disease stage (R-ISS 3 vs 1-2, OR 5.5, P=0.01), bone marrow (BM) plasma cell (PC) infiltration at diagnosis (PC>60% vs ≤60%, OR 4.2, P<0.001), pre-mobilization absolute neutrophil count (ANC, < vs >2500/mmc, OR 2.8, P=0.01), and the use of Len-based (Yes vs No, OR 3.4, P=0.02) or Dara-based induction regimens (Yes vs No, OR 5.3; P=0.04). Grade 3 infections occurred in 3 pts (1%), with no grade 4-5 reported. Image: Summary/Conclusion: Among NDMM pts treated with novel-agent-based induction and undergoing HSC mobilization with CY/GCSF, “on-demand” PLX in case of low CD34+ cell count or insufficient HSC yield was a safe and effective rescue strategy, reducing mobilization failure from 16% to 5%. Predictive factors for PLX use were advanced disease stage, high BM plasmacytosis, low ANC values before mobilization and the use of Len/Dara during induction. Preemptive PLX in pts with ≥1 risk factors should be investigated to further reduce the risk of mobilization failure. P933: DARATUMUMAB (D) IN COMBINATION WITH VD OR D-RD IN RELAPSED OR REFRACTORY MULTIPLE MYELOMA: SUBGROUP ANALYSIS OF CASTOR AND POLLUX STUDIES IN PATIENTS WITH EARLY OR LATE RELAPSE AFTER INITIAL THERAPY A. Spencer1,*, P. Moreau2, M.-V. Mateos3, H. Goldschmidt4, K. Suzuki5, M.-D. Levin6, P. Sonneveld7, S.-S. Yoon8, S. Z. Usmani9, K. Weisel10, D. Reece11, T. Ahmadi12, H. Pei13, W. Garvin Mayo14, X. Gai15, J. Carey16, R. Carson16, M. A. Dimopoulos17 1Malignant Haematology and Stem Cell Transplantation Service, Alfred Health-Monash University, Melbourne, Australia; 2Hematology Department, University Hospital Hôtel-Dieu, Nantes, France; 3University Hospital of Salamanca/IBSAL/Cancer Research Center-IBMCC (USAL-CSIC), Salamanca, Spain; 4University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany; 5Department of Hematology, Japanese Red Cross Medical Center, Tokyo, Japan; 6Albert Schweitzer Hospital, Dordrecht; 7Erasmus MC Cancer Institute, Rotterdam, Netherlands; 8Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea; 9Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 10Department of Oncology, Hematology and Bone Marrow Transplantation With Section of Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 11Department of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Canada; 12Genmab US, Inc., Plainsboro, NJ; 13Janssen Research & Development, LLC, Titusville, NJ; 14Janssen Research & Development, LLC, Raritan, NJ, United States of America; 15Janssen Research & Development, LLC, Beijing, China; 16Janssen Research & Development, LLC, Spring House, PA, United States of America; 17National and Kapodistrian University of Athens, Athens, Greece Background: High-risk multiple myeloma (MM) is often defined based on cytogenetic abnormalities (ie, t[4;14], t[14;16], and/or del17p); however, patients who relapse early (12-18 months) after initial therapy are considered a functional high-risk group that is also associated with poor prognosis. Daratumumab (DARA), a human IgGk monoclonal antibody targeting CD38, is approved in combination with standard-of-care regimens for MM. In the phase 3 CASTOR and POLLUX studies, DARA in combination with bortezomib plus dexamethasone (D-Vd) and lenalidomide plus dexamethasone (D-Rd) significantly improved progression-free survival (PFS), regardless of cytogenetic risk, and achieved higher rates of complete response or better (≥CR) and minimal residual disease (MRD)–negativity vs Vd or Rd alone in patients with RRMM. Aims: In this post-hoc analyses of CASTOR and POLLUX we evaluated D-Vd vs Vd and D-Rd vs Rd in patient subgroups with 1 prior line of therapy based on timing of relapse (early or late) after initiation of the first line of therapy. Methods: In CASTOR and POLLUX, patients with RRMM and ≥1 prior line of therapy were randomized to D-Vd/Vd or D-Rd/Rd, respectively. The primary endpoint was PFS. In this analysis, the early relapse subgroup included patients with 1 prior line of therapy who relapsed <18 months after initiating their first line of therapy; patients with 1 prior line of therapy who relapsed ≥18 months after initiating their first line of therapy were included in the late relapse subgroup. Results: 49 and 186 patients from CASTOR and 99 and 196 patients from POLLUX were included in the early relapse and late relapse subgroups, respectively. Median follow-up was 72.6 months (CASTOR) and 79.7 months (POLLUX). PFS consistently favored the DARA-containing regimens across subgroups (Table). In CASTOR, ≥CR rates were higher with D-Vd vs Vd in the early relapse (21% vs 17%; P = 0.7360) and late relapse (51% vs 14%; P <0.0001) subgroups. In POLLUX, ≥CR rates were higher with D-Rd vs Rd in the early relapse (53% vs 12%; P <0.0001) and late relapse (62% vs 38%; P = 0.0012) subgroups. MRD-negativity rates (10–5) were higher with D-Vd/D-Rd vs Vd/Rd regardless of relapse timing (CASTOR: early, 13% vs 0%; P = 0.1476; late, 23% vs 3%; P <0.0001; POLLUX: early, 30% vs 4%; P = 0.0006; late, 34% vs 14%; P = 0.0009). Image: Summary/Conclusion: These post hoc analyses of CASTOR and POLLUX showed PFS and depth of response benefits of DARA-containing regimens in patients with 1 prior line of therapy, regardless of relapse timing (early or late). Our results support the use of D-Vd and D-Rd in RRMM, including in patients who are considered functional high risk. P934: DARATUMUMAB + LENALIDOMIDE, BORTEZOMIB, AND DEXAMETHASONE IN TRANSPLANT-ELIGIBLE NEWLY DIAGNOSED MULTIPLE MYELOMA: A POST HOC ANALYSIS OF SUSTAINED MINIMAL RESIDUAL DISEASE NEGATIVITY FROM GRIFFIN C. Rodriguez1,*, J. L. Kaufman2, J. Laubach3, D. W. Sborov4, B. Reeves5, A. Chari6, R. Silbermann7, L. J. Costa8, L. D. Anderson Jr9, N. Nathwani10, N. Shah11, N. Bumma12, A. Jakubowiak13, R. Z. Orlowski14, H. Pei15, A. Cortoos16, S. Patel16, T. S. Lin16, P. G. Richardson3, P. M. Voorhees17 1Wake Forest University School of Medicine, Winston-Salem; 2Winship Cancer Institute, Emory University, Atlanta; 3Dana-Farber Cancer Institute, Boston; 4Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City; 5University of North Carolina – Chapel Hill, Chapel Hill; 6Tisch Cancer Institute, Mount Sinai School of Medicine, New York; 7Knight Cancer Institute, Oregon Health & Science University, Portland; 8University of Alabama at Birmingham, Birmingham; 9Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas; 10Judy and Bernard Briskin Center for Multiple Myeloma Research, City of Hope Comprehensive Cancer Center, Duarte; 11Department of Medicine, University of California San Francisco, San Francisco; 12Division of Hematology, The Ohio State University Comprehensive Cancer Center, Columbus; 13University of Chicago Medical Center, Chicago; 14Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston; 15Janssen Research & Development, LLC, Titusville; 16Janssen Scientific Affairs, LLC, Horsham; 17Levine Cancer Institute, Atrium Health, Charlotte, United States of America Background: In the primary analysis of the phase 2 randomized GRIFFIN study, Daratumumab (DARA) + lenalidomide, bortezomib, and dexamethasone (D-RVd) improved the stringent complete response (sCR) rate by end of consolidation for transplant-eligible newly diagnosed multiple myeloma (NDMM) (42.4% vs 32.0%; 1-sided P = 0.068). With longer follow-up (median, 38.6 mo), D-RVd vs RVd improved minimal residual disease (MRD)-negativity (10–5) rates in clinically relevant subgroups (ISS stage III, 71% vs 36%; high cytogenetic risk, 44% vs 29% [del17p, t(4;14), or t(14;16)]; revised high cytogenetic risk, 55% vs 32% [del17p, t(4;14), t(14;16), t(14;20), or gain 1q]). Aims: In this post-hoc analysis we present results of sustained MRD negativity (median follow-up, 38.6 mo) in the same subgroups and in patients (pts) with ≥CR. Methods: Transplant-eligible NDMM pts were randomized 1:1 to 4 D-RVd/RVd induction cycles, ASCT, 2 D-RVd/RVd consolidation cycles, and 2 years of maintenance therapy with lenalidomide (R) ± DARA. For induction/consolidation (21-day cycles), pts received R (25 mg PO Days [D] 1-14), V (1.3 mg/m2 SC D1, 4, 8, 11), and d (40 mg PO weekly) ± DARA (16 mg/kg IV D1, 8, 15 of Cycles 1-4 and D1 of Cycles 5-6). In maintenance (28-day cycles), pts received R (10 mg PO D1-21; if tolerated, 15 mg in Cycles 10+) ± DARA (16 mg/kg IV Q8W/Q4W or 1800 mg SC per protocol amendments). The primary endpoint was sCR rate by end of consolidation. Results: The following features were balanced among randomized pts (D-RVd, n = 104; RVd, n = 103): high cytogenetic risk (16; 14), revised high cytogenetic risk (42; 37), gain 1q (34; 28), and ISS stage III (14; 14). Sustained MRD-negativity rates at 10–5 lasting ≥6 and ≥12 months were higher for D-RVd vs RVd among all high-risk subgroups (Table). D-RVd was superior to RVd for rates of sustained MRD negativity lasting ≥12 months for pts with ≥CR (53.7% vs 20.3%) and sCR (59.1% vs 17.4%; Table). Among all pts with sustained MRD negativity, only 1 D-RVd pt subsequently had disease progression, and 1 RVd pt died. Additional data on MRD at 10–6 and PFS will be presented. Image: Summary/Conclusion: MRD data in GRIFFIN show that the addition of DARA to RVd induction/consolidation and R maintenance may lead to durable MRD-negativity (10–5) rates in pts with transplant-eligible NDMM with high cytogenetic risk, ISS stage III, and those who achieve ≥CR or sCR, however larger studies are needed. P935: REAL WORLD COMPARATIVE ANALYSIS OF THE EFFICACY OF TECLISTAMAB VERSUS CURRENT TREATMENTS IN PATIENTS WITH TRIPLE-CLASS EXPOSED RELAPSED/REFRACTORY MULTIPLE MYELOMA FROM THE LOCOMOTION STUDY H. Einsele1,*, P. Moreau2, M. Delforge3, N. W. van de Donk4, F. Ghilotti5, J. Diels6, A. Elsada7, V. Strulev6, L. Pei8, R. Kobos8, J. Smit9, M. Slavcev10, K. Weisel11, M.-V. Mateos12 1Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Wuerzburg, Germany; 2Hematology Clinic, University Hospital Hôtel-Dieu, Nantes, France; 3University of Leuven, Leuven, Belgium; 4Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 5Janssen-Cilag SpA, Cologno Monzese, United States of America; 6Janssen Pharmaceutica NV, Beerse, Belgium; 7Janssen-Cilag, High Wycombe, Buckinghamshire, United Kingdom; 8Janssen Research & Development, Raritan; 9Janssen Research & Development, Spring House; 10Janssen Global Services, LLC, Raritan, United States of America; 11University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 12University Hospital of Salamanca/IBSAL, CIC, Salamanca, Spain Background: Patients with triple-class exposed relapsed/refractory multiple myeloma (TCE RRMM) have poor prognosis and limited treatment options after being treated with ≥3 lines of therapy (LOT). Teclistamab (tec), a B-cell maturation antigen × CD3 bispecific antibody, is currently being evaluated in the MajesTEC-1 (NCT04557098) study. MajesTEC-1 is a single-arm, phase 1/2 study in patients with TCE RRMM, previously exposed to an immunomodulatory drug, a proteasome inhibitor, and an anti-CD38 antibody and received ≥3 LOT. Aims: Since MajesTEC-1 lacks a control arm, we performed a real-world comparative analysis of the efficacy of tec vs currently used treatments in clinical practice by creating an external real-world control arm from LocoMMotion (NCT04035226), a prospective study of real-world clinical practice (RWCP) efficacy and safety outcomes in patients with TCE RRMM who received ≥3 LOT. Methods: Patients from LocoMMotion (248 patients, clinical cutoff May 21, 2021) who met MajesTEC-1 eligibility criteria were used to create the external control arm for MajesTEC-1. Individual patient-level data were included for 150 patients from MajesTEC-1 treated with tec (1.5 mg/kg weekly) at a clinical cutoff of Sep 7, 2021. Inverse probability of treatment weighting with average treatment effect on the treated was used to adjust for imbalances in prognostically significant baseline covariates: Eastern Cooperative Oncology Group performance status, gender, type of MM, prior transplant, refractory status, International Staging System stage, time to progression on prior LOT, extramedullary disease, number of prior LOT, time since diagnosis, average duration of prior LOT, age, hemoglobin, lactate dehydrogenase and creatinine clearance. Efficacy of tec vs RWCP was measured for overall response rate (ORR), very good partial response (VGPR) rate, complete response or better (≥CR) rate, duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Odds ratio, transformed into a response-rate ratio (RR) (along with 95% confidence interval [CI]), derived from weighted logistic regression was used for binary endpoints (ORR, VGPR rate, and ≥CR rate). Hazard ratios (HRs) and 95% CIs for time-to-event endpoints (DOR, PFS, and OS) were computed using a weighted Cox proportional hazards model. Results: After reweighting the external RWCP cohort, the 2 cohorts had well balanced baseline characteristics. Patients treated with tec had improved outcomes vs current treatments used in RWCP: ORR (RR: 2.31; 95% CI 1.75–2.87; P<0.0001), VGPR rate (RR: 5.54; 95% CI 3.38–7.70; P<0.0001), ≥CR rate (RR 91.50; 95% CI 12.66–661.43; P<0.0001), DOR (HR 0.17; 95% CI 0.08–0.36; P<0.0001), PFS (HR 0.47; 95% CI 0.34–0.67; P<0.0001), and OS (HR 0.69; 95% CI 0.46–1.05; P=0.08). Summary/Conclusion: In this analysis, significantly improved efficacy for almost all outcomes was observed with tec when compared with RWCP. This highlights the potential of tec as a highly effective treatment option for patients with TCE RRMM who have been exposed to ≥3 LOT. P936: TIME TO RESPONSE, DURATION OF RESPONSE, AND PATIENT-REPORTED OUTCOMES WITH DARATUMUMAB PLUS RD VS RD ALONE IN TRANSPLANT-INELIGIBLE PATIENTS WITH NDMM: SUBGROUP ANALYSIS OF THE PHASE 3 MAIA STUDY T. Facon1,*, S. K. Kumar2, T. Plesner3, P. Moreau4, N. Bahlis5, H. Goldschmidt6, M. O’Dwyer7, A. Perrot8, C. P. Venner9, K. Weisel10, J. R. Mace11, N. Raje12, M. Tiab13, M. Macro14, L. Frenzel15, X. Leleu16, H. Pei17, F. Borgsten18, S. Z. Usmani19 1University of Lille, CHU Lille, Service des Maladies du Sang, Lille, France; 2Department of Hematology, Mayo Clinic Rochester, Rochester, MN, United States of America; 3Vejle Hospital and University of Southern Denmark, Vejle, Denmark; 4Hematology Department, University Hospital Hôtel-Dieu, Nantes, France; 5Arnie Charbonneau Cancer Research Institute, University of Calgary, Calgary, AB, Canada; 6University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany; 7Department of Medicine/Haematology, NUI, Galway, Ireland; 8CHU de Toulouse, IUCT-O, Université de Toulouse, UPS, Service d’Hématologie, Toulouse, France; 9Cross Cancer Institute, University of Alberta, Edmonton, AB, Canada; 10Department of Oncology, Hematology and Bone Marrow Transplantation with Section of Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 11Florida Cancer Specialists, St. Petersburg, FL; 12Center for Multiple Myeloma, Massachusetts General Hospital Cancer Center, Boston, MA, United States of America; 13CHD Vendée, La Roche sur Yon; 14Centre Hospitalier Universitaire (CHU) de Caen, Caen; 15Department of Clinical Haematology, Hopital Necker-Enfants Malades, Paris; 16CHU Poitiers, Hôpital la Milétrie, Poitiers, France; 17Janssen Research & Development, LLC, Titusville, NJ, United States of America; 18Janssen-Cilag, Birkerød, Denmark; 19Memorial Sloan Kettering Cancer Center, New York, NY, United States of America Background: In the phase 3 MAIA study, adding daratumumab (DARA) to lenalidomide and dexamethasone (Rd) improved progression-free survival (primary endpoint), overall survival, duration of response, and patient-reported outcomes (PROs) in transplant-ineligible patients with newly diagnosed multiple myeloma (NDMM). Aims: Here we report a MAIA subgroup analysis of time to response, duration of response, and PROs. Methods: Transplant-ineligible patients with NDMM received 28-day cycles of Rd (R 25 mg PO on Days 1-21; d 40 mg PO QW) ± DARA (16 mg/kg IV QW in Cycles 1-2, Q2W in Cycles 3-6, and Q4W thereafter) until disease progression or unacceptable toxicity. Secondary endpoints included time to response and duration of response. PROs were measured using the EORTC QLQ-C30, with treatment effects assessed via mixed-effects model with repeated measures. Results: In total, 368 patients were assigned to the DARA plus lenalidomide and dexamethasone (D-Rd) group and 369 patients to the Rd group; 162 (44%) D-Rd patients and 142 (38%) Rd patients had renal impairment (defined as baseline CrCl ≤60 mL/min). At a 56.2-mo median follow-up, median times to very good partial response or better (≥VGPR) and complete response or better (≥CR) were shorter with D-Rd vs Rd in the overall study population and in the subgroups of patients with and without renal impairment (Table). Among patients who achieved ≥CR or partial response or better (≥PR), higher proportions of D-Rd vs Rd patients had not experienced disease progression at 48 mo (Table). Among patients with renal impairment, greater improvements from baseline in patient-reported pain, fatigue, and nausea and vomiting symptom scores were observed with D-Rd vs Rd across most timepoints; a notably greater meaningful reduction in pain symptom score was seen with D-Rd vs Rd as early as Cycle 6 Day 1 (least squares mean change from baseline, −14.9 vs −7.0; P=0.0241). Analyses for additional patient subgroups will be presented. Image: Summary/Conclusion: In transplant-ineligible patients with NDMM, D-Rd showed more rapid deep responses as well as more durable responses vs Rd, regardless of renal function. Improvements in patient-reported symptoms were generally greater with D-Rd vs Rd in patients with renal impairment. Our results support the use of D-Rd in transplant-ineligible patients with NDMM. P937: PRECLINICAL DISCOVERY AND EARLY FINDINGS FROM THE PHASE 1, DOSE-ESCALATION STUDY OF WVT078, A BCMA-CD3 BISPECIFIC ANTIBODY, IN PATIENTS WITH R/R MULTIPLE MYELOMA M. S. Raab1,*, Y. C. Cohen2,3, F. Schjesvold4,5, K. Aardalen6, A. Oka6, A. Spencer7, M. Wermke8, P. Hari9, J. L. Kaufman10, A. M. Cafro11, E. M. Ocio12, N. Doki13, K. Henson6, G. Trabucco6, P.-E. Juif14, R. Chawla14, J. Cotton6, A. Fessehatsion6, L. Fan6, J. Blankenship6, B. Granda6, H. Lu6, S. De Vita6 1Department of Internal Medicine, Heidelberg University Hospital, Heidelberg, Germany; 2Tel-Aviv Sourasky (Ichilov) Medical Center; 3Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 4Oslo Myeloma Center, Department of Hematology, Oslo University Hospital; 5KG Jebsen Center for B cell malignancies, University of Oslo, Oslo, Norway; 6Novartis Institutes for BioMedical Research, Cambridge, MA, United States of America; 7The Alfred Hospital, Melbourne, VIC, Australia; 8NCT/UCC Early Clinical Trial Unit, Universitätsklinikum Carl Gustav Carus an der Technische Universität, Dresden, Germany; 9Division of Hematology & Oncology, Medical College of Wisconsin, Milwaukee, WI; 10Winship Cancer Institute, Emory University, Atlanta, GA, United States of America; 11Department of Hematology, ASST Grande Ospedale Metropolitano, Niguarda, Milan, Italy; 12Hospital Universitario Marqués de Valdecilla (IDIVAL), Universidad de Cantabria, Santander, Spain; 13Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center, Tokyo, Japan; 14Novartis Institutes for BioMedical Research, Basel, Switzerland Background: B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily with selective expression on benign and malignant plasma cells. Signaling through BCMA mediates survival and proliferation of multiple myeloma (MM) cells. BCMA is a validated clinical target with multiple BCMA-directed therapies approved or in development for patients with relapsed and/or refractory (r/r) MM. Bispecific antibodies (BsAbs) offer a convenient alternative to chimeric antigen receptor–modified T-cell (CAR-T) therapies targeting BCMA and are demonstrating high response rates in patients with MM. Here we describe the discovery of WVT078, a novel BCMA-CD3 BsAb and report initial clinical data from the ongoing phase 1 study (NCT04123418) evaluating ascending doses of WVT078 in patients with r/r MM. Aims: To evaluate the safety and tolerability of the novel BCMA-CD3 BsAb WVT078 in patients with r/r MM. Methods: Multiple BCMA-CD3 BsAb candidates were characterized in vitro and in vivo for T cell activation and anti-MM activity. The activity and tolerability of prioritized candidates were further assessed in an integrated pharmacology study in cynomolgus monkeys. WVT078 was selected for the phase 1 clinical study and is being tested in patients with MM who are r/r to ≥2 standard of care regimens including an immunomodulatory drug, a proteasome inhibitor, and an anti-CD38 agent (if available). Patients who have received a prior BCMA CAR-T or BCMA-antibody-drug conjugates therapy are included, whereas those with prior treatment with BCMA-CD3 bispecifics are excluded. Results: WVT078, a BCMA-CD3 BsAb with high affinity binding to BCMA (with subnanomolar KD), was selected to effectively target MM cells with a low BCMA expression, an emerging mechanism of resistance to BCMA-targeted therapies. WVT078 demonstrated potent anti-MM activity and efficient T-cell activation in preclinical models, and was well tolerated in cynomolgus monkeys. As of December 08, 2021, a total of 33 patients with r/r MM were treated with intravenous WVT078 once weekly at the following dose levels: 3, 6, 12, 24, 48, 64, 96, 192, and 250 µg/kg of body weight. Twenty-five patients (75.8%) discontinued treatment due to progressive disease (n=22), physician decision (n=2) or adverse event (AE, n=1). The most frequent (≥20%) treatment-related AEs (TRAEs) of all grades across all doses included cytokine release syndrome (CRS, 60.6%), pyrexia (39.4%), increased alanine aminotransferase (ALT, 30.3%), increased aspartate aminotransferase (AST) and anemia (24.2% each), and the most common grade ≥3 TRAEs (≥10%) included increased AST, lymphopenia (18.2% each), increased ALT (15.2%), CRS and neutropenia (12.1% each). Clinical activity was observed starting at the dose of 48 µg/kg. At the active doses of 48-250 µg/kg (n=26), the overall response rate (partial response or better) was 34.6% (n=9; 90% CI, 19.4-52.6) and the complete response rate (stringent complete response+complete response) was 11.5% (n=3; 90% CI, 3.2-27.2). The maximum tolerated dose has not been reached. Pharmacokinetic data showed a trend towards dose-proportional relationship and no evidence of accumulation. Image: Summary/Conclusion: WVT078, a novel BCMA-CD3 BsAb, demonstrated potent anti-MM activity and favorable tolerability in preclinical models. In the phase 1 study, WVT078 exhibited an acceptable safety profile and preliminary evidence of clinical activity. Clinical evaluation of WVT078 as a single agent and in combination with a gamma secretase inhibitor is ongoing. P938: CARFILZOMIB-LENALIDOMIDE-DEXAMETHASONE CONSOLIDATION IN MYELOMA PATIENTS WITH A POSITIVE FDG PET/CT AFTER UPFRONT AUTOLOGOUS STEM CELL TRANSPLANTATION: A PHASE II STUDY (CONPET) J. N. Nørgaard1,2,3,*, N. Abildgaard4,5, A. Lysén1,3, G. Tsykunova6, A. J. Vangsted7, C. Joao8, N. Remen1, L. Osnes9, J. P. Connelly10, M.-E. R. Revheim2,10, F. Schjesvold1,3 1Oslo Myeloma Center, Department of Hematology, Oslo University Hospital; 2Institute of Clinical Medicine; 3KG Jebsen Center for B-cell Malignancies, University of Oslo, Oslo, Norway; 4Department of Hematology, Odense University Hospital; 5Department of Clinical Research, University of Southern Denmark, Odense, Denmark; 6Division of Hematology, Haukeland University Hostpital, Bergen, Norway; 7Departmen of Hematology, Rigshospitalet, Copenhagen, Denmark; 8Department of Hematology, Champalimaud Centre for the Unknown, Lisboa, Portugal; 9Department of Immunology; 10Division for Radiology and Nuclear Medicine, Oslo University Hospital, Oslo, Norway Background: [18F]-Fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) positivity after first line treatment with autologous stem cell transplantation (ASCT), is strongly correlated with reduced progression free survival and overall survival (Moreau et al., JCO, 2017). However, FDG PET/CT positive patients who obtain FDG PET/CT negativity after treatment seem to have comparable outcomes to patients who were FDG PET/CT negative at baseline (Davies et al., Haematologica 2018). Aims: Aiming for FDG PET/CT negativity may be an important goal in myeloma treatment. The use of FDG PET/CT positivity as an indication for consolidation therapy after ASCT has not been studied before. We here present the complete data of the primary endpoint, PET/CT negativity after KRd consolidation. Methods: Patients with multiple myeloma who had received standard first line treatment including ASCT and achieved very good partial response (VGPR) or better, were examined by FDG PET/CT. Patients who were FDG PET/CT positive defined by the Italian Myeloma criteria for PET USe (IMPETUS) (Nanni C et al., EJNMMI 2016 and 2018) were included in the treatment phase of the study and were assessed for minimal residual disease (MRD) by Euroflow (sensitivity: 10-5) before treatment. FDG PET/CT examinations were centrally read by two experienced nuclear medicine radiologists. The treatment consisted of four 28-day cycles of KRd (carfilzomib 36 mg/m2 day 1,2,8,9,15 and 16 (except 20 mg/m2 day 1 and 2 first cycle), lenalidomide 25 mg day 1-21 all cycles and dexamethasone 40 mg day 1,8,15 and 22 all cycles). After 4 cycles, FDG PET/CT and Euroflow for MRD were repeated for response evaluation. Both patients with FDG PET/CT negativity and patients with FDG PET/CT positivity at baseline are followed for progression free survival and overall survival. Results: 159 patients were screened with FDG PET/CT. Fifty-three patients of 159 (33%) had a positive FDG PET/CT result. Among FDG PET/CT positive patients, a higher proportion had ISS score III, high-risk cytogenetics and VCd induction was more common (Table 1). Forty-eight patients completed KRd treatment. Sixteen patients (33%) converted into FDG PET/CT negativity. More patients with previous VRd induction than VCd induction converted from FDG PET/CT positive to FDG PET/CT negative (38% vs 20%). No patients stopped treatment due to adverse events. Twenty-seven of 49 (55%) patients with a FDG PET/CT positive result were MRD negative before KRd consolidation. A higher proportion of MRD negative patients vs MRD positive patients before KRd treatment, converted into FDG PET/CT negativity, with 37% vs 28%, respectively. For the proportion of patients that after KRd treatment was both FDG PET/CT negative and MRD negative by EuroFlow, the difference was even higher (37% vs 5% in MRD negative vs MRD positive patients, respectively). In total, the MRD negativity rate increased from 55% to 76% Image: Summary/Conclusion: Before KRd consolidation, 33% of patients in VGPR or better after first line treatment including ASCT were FDG PET/CT positive. High-risk cytogenetics, ISS score III and VCd induction were more common in FDG PET/CT positive patients. After four cycles of KRd, 33% of patients converted from FDG PET/CT positivity to negativity. A higher proportion converted into FDG PET/CT negativity in patients who were MRD negative before KRd treatment, and in patients who had received VRd. KRd consolidation is feasible and converts a clinically significant proportion of FDG PET/CT positive patients to negativity. Patients are followed for PFS and OS. P939: SYNERGISTIC EFFECTS OF LOW DOSE BELANTAMAB MAFODOTIN IN COMBINATION WITH A GAMMA-SECRETASE INHIBITOR (NIROGACESTAT) IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM): DREAMM-5 STUDY A. Nooka1,*, S. Lonial1, S. Grosicki2, M. Hus3, K. Song4, T. Facon5, N. S. Callander6, V. Ribrag7, K. Uttervall8, H. Quach9, V. Vorobyev10, C.-K. Min11, S. Cheng12, L. M. Smith12, J. Yu13, T. Collingwood13, B. Holkova13, B. E. Kremer13, I. V. Gupta13, P. G. Richardson14, M. C. Minnema15 1Emory University, Winship Cancer Institute, Atlanta, GA, United States of America; 2Department of Hematology and Cancer Prevention, Medical University of Silesia, Katowice; 3Katedra i Klinika Hematoonkologii i Transplantacji Szpiku, Lublin, Poland; 4Vancouver General Hospital, Vancouver, BC, Canada; 5Department of Haematology, Lille University Hospital, Lille, France; 6University of Wisconsin, Carbone Cancer Center, Madison, WI, United States of America; 7Institut Gustave Roussy, Villejuif, France; 8Karolinska University Hospital, Stockholm, Sweden; 9University of Melbourne, St. Vincent’s Hospital Melbourne, Melbourne, VIC, Australia; 10S P Botkin City Clinical Hospital, Moscow, Russia; 11Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, South Korea; 12SpringWorks Therapeutics, Stamford, CT; 13GlaxoSmithKline, Upper Providence, PA; 14Dana-Farber Cancer Institute, Boston, MA, United States of America; 15University Medical Center Utrecht, Utrecht, Netherlands Background: Preclinical data demonstrate that nirogacestat, a gamma-secretase inhibitor, may increase cell-surface levels of a B-cell maturation antigen (BCMA) and reduce soluble BCMA levels, which could enhance anti-BCMA agent activity in multiple myeloma. In the DREAMM-5 (NCT04126200) Phase I/II platform trial belantamab mafodotin (belamaf; BLENREP), a BCMA-targeting antibody-drug conjugate, is being evaluated in combination with nirogacestat. Aims: The aim of this study is to determine if the combination can result in similar efficacy and an improved ocular safety profile compared to the currently approved belamaf schedule (single agent dose 2.5 mg/kg Q3W) in patients with RRMM which showed a 31% overall response rate (ORR) and 44.5% Gr3/4 keratopathy (BLENREP US prescribing information). Methods: This cohort within the DREAMM-5 nirogacestat combination sub-study has a sequential dose-exploration (DE) phase evaluating 0.95 mg/kg Q3W belamaf with 100 mg BID nirogacestat continuously, followed by a randomized cohort expansion (CE) comparing the combination to a belamaf 2.5 mg/kg Q3W arm. Results: Preliminary results from the 10 patients in the DE cohort with low-dose belamaf + nirogacestat, are presented in this abstract. Patients had a median (range) of 4.5 (3–10) prior lines of therapy. At time of data cut-off (Nov 15, 2021), patients received a median (range) of 7 cycles (1–26). The ORR was 60% (n=6/10) and 20% (n=2) achieved a very good partial response (Table). The key emergent adverse events (AEs) included ocular events (n=7 [70%]; ≥Grade [Gr] 3, n=2 [20%] of which Gr 3 keratopathy was reported in 1 patient [10%]), diarrhea (n=7 [70%]; Gr3, n=1 [10%]) and hypophosphatemia (n=7 [70%]; Gr3, n=1, [10%]). There were 2 Grade 5 AEs, neither of which were related to study treatment. No patient permanently discontinued study due to treatment related AEs. Image: Summary/Conclusion: Encouraging clinical activity and a manageable safety profile is observed with low dose belamaf (0.95 mg/kg Q3W) + nirogacestat (100 mg BID continuously) in patients with RRMM. This ongoing sub-study is actively recruiting patients and will continue to evaluate belamaf + nirogacestat efficacy and safety. Updated results will be reported at the congress. Funding Statement: GSK (208887); drug linker technology licensed from Seagen Inc.; mAb produced using POTELLIGENT Technology licensed from BioWa. This abstract was previously submitted to the American Society of Clinical Oncology (ASCO) Annual Meeting, June 3–7, 2022, and is submitted on behalf of the original authors with their permission. ©2022 American Society of Clinical Oncology, Inc. Reused with permission. All rights reserved. P940: SAFETY AND CLINICAL ACTIVITY OF BELANTAMAB MAFODOTIN WITH PEMBROLIZUMAB IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM): DREAMM-4 STUDY A. Suvannasankha1,*, N. Bahlis2, S. Trudel3, K. Weisel4, C. Koenecke5, A. Oriol6, P. M. Voorhees7, A. A. Alonso8, N. S. Callander9, M. V. Mateos Manteca10, N. Reddy11, S. Hakim12, N. Patel13, D. Williams12, R. C. Jewell13, X. Zhou12, I. Gupta14, A. K. Nooka15 1Indiana University Simon Cancer Center and Roudebush VAMC, Indianapolis, IN, United States of America; 2Arnie Charbonneau Cancer Research Institute, University of Calgary, Calgary, AB; 3Princess Margaret Cancer Centre, Toronto, ON, Canada; 4University Medical Center Hamburg-Eppendorf, Hamburg; 5Hannover Medical School, Clinic for Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany; 6Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain; 7Levine Cancer Institute, Atrium Health, Charlotte, NC, United States of America; 8Hospital Universitario Quirónsalud Madrid, Madrid, Spain; 9Carbone Cancer Center, University of Wisconsin, Madison, WI, United States of America; 10Instituto de Investigación Biomédica de Salamanca and Centro de Investigación del Cáncer, Hospital Universitario de Salamanca, Salamanca, Spain; 11Merck & Co., Inc., Kenilworth, NJ; 12GlaxoSmithKline, Upper Providence, PA; 13GlaxoSmithKline, Research Triangle Park, NC; 14GlaxoSmithKline, Philadelphia, PA; 15Winship Cancer Institute, Emory University Hospital, Atlanta, GA, United States of America Background: Belantamab mafodotin (belamaf; BLENREP), a B-cell maturation antigen (BCMA) targeted antibody–drug conjugate approved for adult patients with RRMM, has a multimodal mechanism that eliminates myeloma cells via direct cytotoxicity and a systemic anti-tumor immune response, which may be augmented by an immune checkpoint inhibitor. Aims: DREAMM-4 (NCT03848845) assessed safety and clinical activity of belamaf with pembrolizumab (pembro) in RRMM. Methods: This was a Phase I/II, single-arm, open-label study of adults with RRMM after ≥3 lines of therapy (LOT, including anti-CD38 monoclonal antibody, proteasome inhibitor, and immunomodulator). Part 1 established the dose of belamaf 2.5 mg/kg with pembro 200 mg, both IV Q3W up to 35 cycles, for the Part 2 expansion. Primary efficacy endpoint was investigator-assessed overall response rate (ORR, ≥partial response [PR] per IMWG criteria by investigator). Secondary endpoints included duration of response (DoR), progression-free survival (PFS), adverse events (AEs) per NCI-CTCAE v4.03, and pharmacokinetics (PK). Results: This primary analysis of all treated pts (as of Oct 18, 2021) included 34 pts (6 in Part 1 and 28 in Part 2). In both parts, median prior LOT was 5 (range 3–13); 10 pts (29%) had high-risk cytogenetics and 9 (26%) had extramedullary disease. ORR was 47%, with most responses (10/16 pts) ≥ very good PR (VGPR, Table). Median follow-up was 14.7 months (mo); median (95% CI) DoR was 8.0 (2.1–not reached) mo; median PFS was 3.4 (1.4–5.6) mo. Most pts had ≥1 AE (any grade [Gr]: 97%; Gr ≥3: 74%) and treatment-related AE (TRAE, any Gr: 97%; Gr ≥3: 65%). Most common (≥35%) AEs were keratopathy (any Gr: 76%; Gr ≥3: 38%), vision blurred (any Gr: 38%; Gr ≥3: 0%), and thrombocytopenia (any Gr: 35%; Gr ≥3: 29%). AEs led to dose delays (65%) and dose reductions (32%), but not discontinuation. Nine pts had a serious AE (SAE); 4 pts had ≥1 SAE related to study treatment. Two pts had immune-related AEs of Gr 1 (gout and autoimmune hypothyroidism). Preliminary PK and soluble BCMA data were consistent with single-agent belamaf therapy. Image: Summary/Conclusion: Belamaf + pembro demonstrated a favorable ORR compared with single-agent belamaf in heavily pre-treated RRMM. No new TRAEs were identified; AE frequency and severity were similar to single-agent belamaf. Correlative biomarker studies are ongoing. Funding Statement: GSK (205207) in collaboration with Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. Drug linker technology licensed from Seagen Inc. mAb produced using POTELLIGENT technology licensed from BioWa. This abstract was previously submitted to the American Society of Clinical Oncology (ASCO) Annual Meeting, June 3–7, 2022, and is submitted on behalf of the original authors with their permission. ©2022 American Society of Clinical Oncology, Inc. Reused with permission. All rights reserved. P941: SAFETY AND CLINICAL ACTIVITY OF BELANTAMAB MAFODOTIN WITH LENALIDOMIDE PLUS DEXAMETHASONE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM): DREAMM-6 ARM-A INTERIM ANALYSIS H. Quach1,*, M. Gironella2, C. Lee3, R. Popat4, P. Cannell5, R. S. Kasinathan6, B. Chopra7, R. Rogers6, G. Ferron-Brady6, S. Shafi-Harji8, N. Patel7, J. Opalinska6, I. Gupta6, B. Augustson9 1University of Melbourne, St. Vincent’s Hospital Melbourne, Melbourne, Australia; 2Department of Hematology, University Hospital Vall d’Hebron, Barcelona, Spain; 3Royal Adelaide Hospital, Adelaide, Australia; 4NIHR UCLH Clinical Research Facility, University College London Hospitals, NHS Foundation Trust, London, United Kingdom; 5Department of Medicine, Royal Perth Hospital, Perth, Australia; 6GlaxoSmithKline, Upper Providence, PA, United States of America; 7GlaxoSmithKline, London; 8GlaxoSmithKline, Stevenage, United Kingdom; 9Department of Haematology, Sir Charles Gairdner Hospital, Perth, Australia Background: Belantamab mafodotin (belamaf; BLENREP) is a B-cell maturation antigen-targeting antibody–drug conjugate approved for patients with RRMM as monotherapy at 2.5 mg/kg Q3W. Preclinical data demonstrate synergy between belamaf and lenalidomide (Len), suggesting added benefit when combined with standard of care such as Len + dexamethasone (Dex). Aims: DREAMM-6 (NCT03544281) Arm A is evaluating belamaf in combination with LenDex in patients with RRMM. Methods: This ongoing, two-part, two-arm, open-label study included patients with RMMM previously treated with ≥1 line of therapy (LOT). Patients received 4 belamaf doses/schedules (1.9 mg/kg Q8W or Q4W; 2.5 mg/kg Q4W or Q4W SPLIT dose [50% on Days (D)1, 8] IV) in combination with Len (20 mg PO D1–21) and Dex (20 mg PO/IV D1, 8, 15, 22). Primary objectives were safety (including treatment-related [TR]AEs related to the combination belamaf and LenDex), tolerability, and efficacy (including overall response rate [ORR] defined as ≥partial response). Results: As of this interim analysis (data cut: July 23, 2021), 45 patients received ≥1 dose (12 at 1.9 mg/kg Q8W; 4 at 1.9 mg/kg Q4W; 16 at 2.5 mg/kg Q4W; 13 at 2.5 mg/kg Q4W SPLIT). Across cohorts, the median age was 68 years (range: 36–80). Thirteen patients (29%) had high-risk cytogenetics and 6 (13%) had extramedullary disease. Median prior LOT was 3 (range: 1–11) and 26 (58%) had prior Len treatment. The median duration of follow-up and ORR ranged across cohorts (Table). The median duration of response was only reached in the 1.9 mg/kg Q4W cohort (11.1 mo [95% CI: 3.7–not reached [NR]). At the time of data cut, median progression-free survival was not reached in the 1.9 mg/kg Q8W or 2.5 mg/kg Q4W cohorts. Gr ≥3 TRAEs occurred in 42–85%. Gr ≥3 keratopathy occurred in 0 patients in 1.9 mg/kg Q8W, 1 patient (25%) in 1.9 mg/kg Q4W, 8 patients (50%) in 2.5 mg/kg Q4W, and 6 patients (46%) in 2.5 mg/kg Q4W SPLIT cohorts. Image: Summary/Conclusion: Belamaf + LenDex had a tolerable safety profile, with no new safety signals identified in patients with RRMM. AEs, including keratopathy, were common but manageable with dose modifications. Encouraging clinical activity is observed with this combination in patients with RRMM. Follow-up/correlative studies are ongoing. P942: DREAMM-9: PHASE I STUDY OF BELANTAMAB MAFODOTIN PLUS STANDARD OF CARE IN PATIENTS WITH TRANSPLANT-INELIGIBLE NEWLY DIAGNOSED MULTIPLE MYELOMA S. Z. Usmani1,*, M. Mielnik2, Y. Koh3, A. Alonso Alonso4, X. Leleu5, H. Quach6, C.-K. Min7, W. Janowski8, A.-O. Abdallah9, M. Garg10, I. Sandhu11, E. M. Ocio San Miguel12, A. Oriol13, P. Rodriquez-Otero14, K. Ramasamy15, K. Weisel16, B. Besemer17, M. Cavo18, X. L. Zhou19, M. C. Kaisermann20, C. M. Bego Marques21, D. Williams20, F. Carreno20, B. E. Kremer20, I. V. Gupta20, M. Hus2 1Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 2Department of Hematooncology and Bone Marrow Transplantation, Medical University of Lublin, Lublin, Poland; 3Seoul National University Hospital, Seoul, South Korea; 4University Hospital Quirón Madrid, Madrid, Spain; 5CHU de Poitiers, Poitiers, France; 6St.Vincent’s Hospital Melbourne, Melbourne, Australia; 7Catholic University of Korea Seoul St. Mary’s Hospital, Seoul, South Korea; 8Calvary Mater Newcastle, Newcastle, Australia; 9University of Kansas, Kansas City, KS, United States of America; 10Leicester Royal Infirmary, Leicester, United Kingdom; 11University of Alberta, Edmonton, Canada; 12Hospital Universitario Marqués de Valdecilla (IDIVAL), Universidad de Cantabria, Santander; 13Institut Català d’Oncologia and Institut Josep Carreras - Hospital Universitari Germans Trias i Pujol (HUGTP), Badalona; 14Department of Hematology, Clínica Universidad de Navarra, Pamplona, Spain; 15Churchill Hospital, Headington, Oxford, United Kingdom; 16University Medical Center Hamburg-Eppendorf, Hamburg; 17University of Tübingen, Tübingen, Germany; 18IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istitutodi Ematologia “Seràgnoli”, Universitàdegli Studi di Bologna, Bolonga, Italy; 19GlaxoSmithkline, Waltham, MA; 20GlaxoSmithKline, Upper Providence, PA, United States of America; 21GlaxoSmithKline, Barcelona, Spain Background: The bortezomib, lenalidomide, and dexamethasone (VRd) regimen is a standard of care for newly diagnosed multiple myeloma (NDMM). Belantamab mafodotin (belamaf) is a B-cell maturation antigen-binding antibody-drug conjugate that eliminates myeloma cells by a multimodal mechanism: direct cell kill and anti-myeloma tumor immune response. Belamaf has demonstrated deep and durable responses as a monotherapy in the DREAMM-2 study of patients (pts) with relapsed/refractory multiple myeloma (RRMM). Preclinical evidence of belamaf in combination with bortezomib or lenalidomide suggests enhanced anti-myeloma activity, providing rationale for this treatment combination. Aims: To evaluate the safety and tolerability of this combination in adult pts with transplant-ineligible (TI) NDMM and establish the recommended Phase III dose. Methods: DREAMM-9 (NCT04091126) is an ongoing Phase I, open-label, randomized study of belamaf + VRd. The belamaf dose cohorts currently being evaluated are Cohort 1 (1.9 mg/kg Q3/4W), Cohort 2 (1.4 mg/kg Q6/8W), Cohort 3 (1.9 mg/kg Q6/8W), Cohort 4 (1.0 mg/kg Q3/4W), and Cohort 5 (1.4 mg/kg Q3/4W). Belamaf is given with VRd Q3W until Cycle 8, and with Rd Q4W thereafter. After evaluation of safety data for Cohort 1, Cohorts 2–5 were opened in parallel and enrolled pts were randomized 1:1:1:1. Primary endpoint is safety. Secondary endpoints include efficacy, tolerability, and pharmacokinetics (PK). Results: As of data cutoff (07 Dec 2021), 64 pts were analyzed across all cohorts. Median age (range) was 73.0 (51–88) years, 55% were male, 80% were white, 8% had extramedullary disease, 59% were International Staging System stage II or III, 20% had amp1q, and 17% had high-risk cytogenetics (≥1 of: t(4;14), t(14;16), del17p). The median duration of follow-up varied: Cohort 1 (17.4 months [mo]), Cohort 2 (5.9 mo), Cohort 3 (6.1 mo), Cohort 4 (4.7 mo), Cohort 5 (5.8 mo). Median number of belamaf cycles were: Cohort 1 (6), Cohort 2 (3), Cohort 3 (3.5), Cohort 4 (4.5), and Cohort 5 (5). Most common adverse events (AEs) across cohorts included thrombocytopenia (49%), constipation (43%), diarrhea (32%), and peripheral sensory neuropathy (30%). AEs related to study treatment were experienced by 61 (97%) pts. Belamaf-related grade 3/4 AEs occurred in 24 (38%) pts. Belamaf dose reductions occurred in 11 (18%) pts, with dose delays in 10 (16%) pts. Three pts experienced a fatal severe AE (unrelated to study treatment); 2 due to COVID-19 infection, 1 due to pancreatic adenocarcinoma. Early deep responses were observed; 67–92% pts achieved ≥very good partial response (VGPR) (Table), with median time to VGPR of 2.1–2.9 months across cohorts. Of pts with ≥VGPR, 17 were minimal residual disease (MRD) negative, 10 in Cohort 1. As of data cutoff, 8–75% of pts achieved best response of complete response (CR) or stringent CR (sCR). Grade 3 corneal exam findings were reported in 25–58% of pts; grade 3 visual acuity changes were reported in 21–75% of pts. No grade 4 corneal exam findings or visual acuity changes were reported in pts receiving belamaf Q6/8W, compared with 0–17% and 0–8%, respectively, in the Q3/4W cohorts. Belamaf PK profile was similar to that in pts with RRMM, accounting for baseline characteristics. Image: Summary/Conclusion: Belamaf + VRd demonstrated high response rates in pts with TI NDMM, with a high rate of MRD negativity indicating deep responses. No new safety signals were observed relative to DREAMM-2. Study is ongoing to evaluate the safety and efficacy of variable dose intensities of belamaf in combination with VRd. P943: SURVIVAL OUTCOMES OF PATIENTS WITH MULTIPLE MYELOMA IN FRANCE: A COHORT STUDY USING THE FRENCH NATIONAL HEALTHCARE DATABASE (SNDS) X. Leleu1,*, B. Gorsh2, A. Bessou3, P. Paka2, J. De Nascimento3, X. Colin4, S. Landi5, P. F. Wang2 1Department of Hematology, Centre Hospitalier Universitaire, Université de Poitiers, Poitiers, France; 2GlaxoSmithKline, Upper Providence, PA, United States of America; 3IQVIA, Paris; 4GlaxoSmithKline, Rueil-Malmaison, France; 5GlaxoSmithKline, Research Triangle Park, NC, United States of America Background: While treatment options for multiple myeloma (MM) have expanded with the introduction of novel therapies, it remains unclear how this has translated into better patient outcomes. Although some patients can achieve long-term remission, MM remains incurable. In oncology, overall survival (OS) is the gold standard for measuring the clinical benefit of an agent. Real-world studies in several countries have shown increases in OS in recent years for patients with MM; up-to-date real-world data on MM survival in France are limited. Aims: To assess the OS of patients with MM in France from diagnosis and by line of therapy (LOT) and post triple-class exposure (TCE). Secondary aims were survival outcomes by different subpopulations based on standard of care (SoC) treatment regimens and transplant status. Methods: This longitudinal retrospective cohort study used data from the French National Healthcare database (Système National des Données de Santé – SNDS) from Jan 2013‒Dec 2019, which included records of outpatient claims, hospital discharges and death registry, demographics, and reimbursement data for hospital and outpatient services. Eligible patients (≥18 years) had ≥2 records of an MM diagnosis (ICD-10 codes C90/C90.0), and/or long-term disease during the study period; ≥1 dispensation/administration of MM drug treatment and date of death available. LOT was algorithmically defined based on drug regimen, time since administration, and gap between regimens. OS was analyzed from five time points: 1) from time of diagnosis; 2) from the start of line of LOT1 to LOT4; 3) from the start of first TCE (defined as prior exposure to a proteasome inhibitor, an immunomodulatory agent and an anti-CD38 monoclonal antibody); 4) from the subsequent treatment following TCE, and 5) from the subsequent treatment/no treatment following TCE and LOT5+ (indexed at the date of the last observed treatment). Patient OS was assessed by Kaplan–Meier method and death rates were calculated. Results: A total of 14,309 patients were included in this analysis. Median age was 71 years at diagnosis and 51% of patients were male. Patient survival outcomes deteriorated as their LOT advanced (Figure). Median OS from initial diagnosis was >5 years with a slight increase for patients diagnosed more recently (63.8 months for patients diagnosed in 2017 or later vs 60.8 months in those diagnosed in 2014 or later). From LOT1 to LOT4 OS decreased from 61 to 14.8 months or about 30% to 50% with each additional LOT. At the time of first TCE, daratumumab monotherapy/combination therapy were the most common regimens. Median OS following TCE was 3.8 months. Retreatment with previous agents were the SoC following TCE. Among TCE patients who had 4 prior LOTs, 43% of them received a subsequent LOT with a median OS estimate of 8.2 months. For those who discontinued treatment, median OS was only 1 month, indexing at the date of the last observed treatment. Image: Summary/Conclusion: Patients with MM experience worsening survival outcomes with increasing time from diagnosis and with subsequent LOT. Prognosis was especially poor among TCE patients who received subsequent therapy and worse for those who did not receive additional treatment. Despite therapeutic advances and slight increase in OS for patients diagnosed more recently, an unmet need for improved access to novel therapies remains, especially in those TCE patients. P944: A PHASE I STUDY OF C-CAR088, A NOVEL HUMANIZED ANTI-BCMA CAR T CELL THERAPY IN RELAPSED/REFRACTORY MULTIPLE MYELOMA X. Qu1,*, G. An2, W. Sui2, T. Wang2, X. Zhang3, J. Yang3, Y. Zhang4, L. Zhang4, D. Zhu5, J. Huang5, S. Zhu5, X. Yao5, J. Li5, C. Zheng5, S. Chen5, K. Zhu6, Y. Wei5, X. Lv5, L. Lan5, Y. Yao5, D. Zhou4, P. Lu3, L. Qiu2, J. Li1 1Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Nanjing; 2Department of Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin; 3Department of Hematology, Hebei Yanda Lu Daopei Hospital, Langfang; 4Department of Hematology, Peking Union Medical College Hospital, Beijing; 5Cellular Biomedicine Group Inc, Shanghai; 6University of Maryland School of Medicine, Maryland, China Background: Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor-T cell (CAR T) therapy shows remarkable efficacy in patients with relapsed or refractory multiple myeloma (RRMM). Aims: C-CAR088, a novel second-generation humanized anti-BCMA CAR T cell therapy, was developed. Methods: This phase I dose-escalation and expansion study assessed the safety and efficacy of three dosages of C-CAR088 in patients with RRMM. As of July 2, 2021, 31 patients had been infused with C-CAR088. Results: Any grade cytokine release syndrome (CRS) developed in 29 patients (93.5%), and grade 3 CRS occurred in three patients (9.7%). One patient from the high-dose group (4.5-6.0 × 106 CAR T cells/kg) developed grade 1 neurotoxicity. No dose-limiting toxicities were observed in any dose group, and all adverse events were reversible after proper management. The overall response, stringent complete response, complete response (CR), and very good partial response rates were 96.4%, 46.4%, 10.7%, and 32.1%, respectively. The CR rate in the medium-dose (3.0 × 106 CAR T cells/kg) and high-dose (4.5-6.0 × 106 CAR T cells/kg) groups was 54.5% and 71.4%, respectively. In the CR group, 15 (93.7%) patients achieved minimal residual disease (MRD) negativity (test sensitivity >1/10-5). All seven patients with double-hit or triple-hit multiple myeloma achieved MRD-negative CR. Summary/Conclusion: Our findings demonstrate the manageable safety profile and potential efficacy of C-CAR088. P945: SUBCUTANEOUS ISATUXIMAB ADMINISTRATION BY AN ON-BODY DELIVERY SYSTEM IN COMBINATION WITH POMALIDOMIDE-DEXAMETHASONE IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS: INTERIM PHASE 1B STUDY RESULTS H. Quach1,*, G. Parmar2, E. M. Ocio3, H. M. Prince4, A. Oriol5, N. Tsukada6, K. Sunami7, P. Bories8, C. Karanes9, S. Madan10, D. Semiond11, M. Inchauspe12, S. Macé13, P. B. Musholt14, F. Suzan13, P. Moreau15 1Clinical Haematology Service, St Vincent’s Hospital, University of Melbourne, Vic; 2Illawarra Cancer Care Centre, Wollongong, NSW, Australia; 3Hospital Universitario Marqués de Valdecilla (IDIVAL), Universidad de Cantabria, Santander, Spain; 4Molecular Oncology and Cancer Immunology, Epworth Healthcare and University of Melbourne, Melbourne, Vic, Australia; 5Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Barcelona, Spain; 6Department of Hematology, Japanese Red Cross Medical Center, Tokyo; 7National Hospital Organization Okayama Medical Center, Okayama, Japan; 8Early Phase Unit, Institut Claudius Regaud, Institut Universitaire du Cancer Toulouse, Toulouse, France; 9Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, CA; 10Banner MD Anderson Cancer Center, Gilbert, AZ; 11Sanofi, Cambridge, MA, United States of America; 12IT&M Stats for Sanofi, Neuilly sur-Seine; 13Sanofi Research & Development, Chilly-Mazarin, France; 14Sanofi Research & Development, Frankfurt, Germany; 15Department of Hematology, University Hospital of Nantes, Nantes, France Background: Intravenous (IV) isatuximab (Isa) + pomalidomide-dexamethasone (Pd) is approved for the treatment of relapsed/refractory multiple myeloma (RRMM) patients (pts). Subcutaneous (SC) delivery would optimize convenience of administration. Prior results showed that SC Isa administered by syringe pump has efficacy and safety profiles comparable to IV Isa; the recommended Phase 2 dose (RP2D) was 1400 mg (IMW21 P-207). Aims: This multicenter Phase 1b study evaluated safety, pharmacokinetics (PK), and efficacy of SC vs IV Isa + Pd in RRMM pts after ≥2 prior treatment lines. Methods: Pts were randomized 2:1 to SC1000 mg or IV 10 mg/kg and to SC1400 mg or IV. An expansion cohort was later implemented with SC Isa administered at the RP2D via on-body delivery system (OBDS), a wearable bolus injector applied to the abdomen by a healthcare professional. Primary endpoints (EPs) were safety, including injection site reactions (ISRs), and PK. Main secondary EPs were overall response rate (ORR) and progression-free survival (PFS). Results: Fifty-six pts were randomized and treated: 12 Isa IV, 12 Isa SC1000, 10 Isa SC1400, and 22 OBDS pts. At study entry, ISS stage II–III was 67% in IV, 33% in SC1000, 60% in SC1400, and 50% in OBDS pts. On Jan 20, 2022, 33% IV, 25% SC1000, 50% SC1400, and 86% OBDS pts remained on treatment. Due to sequential accrual, median follow-up (FU) was longer in IV (20.6 mo) and SC1000 (23.8 mo) than SC1400 (18.1 mo) and OBDS (6.5 mo) pts. Infusion reactions (IRs) were infrequent (≤10% in each cohort, all Grade [G] 2), only at first IV or SC infusion/injection, with no IRs in OBDS. Local tolerability of OBDS was very good, with 5 (22.7%) pts experiencing 7 ISR episodes, all G1, out of 305 administrations (2.3%): 5 injection site erythemas, 1 injection site hemorrhage, and 1 injection site induration. Median duration of OBDS injection was 10 min. Incidence of G4 neutropenia was lower in SC1000 (42%) vs the other cohorts (55–60%). The lower percentage of ≥G3 treatment-related adverse events in OBDS (77%) vs the other cohorts (≥80%) may be due to shorter FU. Best overall responses are shown in table; longer FU is needed for OBDS pts. Median PFS was 22 mo in IV, 12.5 mo in SC1000, and not reached in SC1400 and OBDS pts. PK and CD38 receptor occupancy results in OBDS pts are consistent with those observed in SC1400 with pump. Image: Summary/Conclusion: SC Isa administered by OBDS shows a safety profile consistent with IV administration with no IRs and excellent local tolerability. Efficacy in the SC cohorts was comparable to the Phase 3 ICARIA results. Isa SC administration by OBDS is well tolerated, requires a short duration of injection, and provides a convenient hands-free option. Clinical trial information: NCT04045795. Research Sponsor: Sanofi. P946: A PHASE I/II SINGLE ARM STUDY OF BELANTAMAB MAFODOTIN, CARFILZOMIB AND DEXAMETHASONE IN PATIENTS WITH RELAPSED MULTIPLE MYELOMA: AMARC 19-02 BELACARD STUDY. M. Lasica1,2,*, A. Spencer2,3,4, P. Campbell2,5,6, C. Wallington-Gates2,7,8, N. Wong Doo2,9,10, W. Janowski2,11, G. McCaughan2,12,13, A. Puliyayil2,14, F. Yuen2,3, K. Le2,3, J. Reynolds2,3,4, H. Quach1,2,15 1Department of Haematology, St Vincent’s Hospital Melbourne, Fitzroy; 2Australasian Myeloma Research Consortium; 3Department of Haematology, Alfred Hospital, Melbourne; 4Faculty of Medicine, Monash University, Clayton; 5Department of Haematology, Barwon Health, Geelong; 6Deakin University, Burwood; 7Flinders University; 8Department of Haematology, Flinders Medical Centre, Bedford Park; 9Department of Haematology, Concord Hospital, Concord; 10University of Sydney, Camperdown; 11Department of Haematology, Calvary Mater Newcastle, Newcastle; 12Department of Haematology, St Vincent’s Hospital Sydney; 13St Vincent’s Clinical School, University of New South Wales, Sydney; 14Department of Haematology, Border Cancer Hospital, Albury; 15University of Melbourne, Parkville, Australia Background: Belantamab Mafodotin (Belamaf), a first in class anti- B-cell maturation antigen (BCMA) antibody-drug conjugate is efficacious in patients with triple-class exposed/refractory multiple myeloma (RRMM). Combining Belamaf (B) with carfilzomib and dexamethasone (Kd) is potentially synergistic through direct myeloma-cell kill and immune response against myeloma. Aims: To characterize the safety, tolerability, and preliminary efficacy of BelaMaf in combination with carfilzomib and dexamethasone (Kd) in patients with early relapsed MM. Methods: BelaCarD is an ongoing, two-part, single-arm, multicentre phase I/II study evaluating an extended schedule of B every 8 weeks in combination with Kd in patients with RRMM after 1-3 prior lines of treatment. Prior refractoriness to proteasome inhibitors was allowed. Here we report a pre-planned analysis of the safety run-in phase of the first 10 patients who have completed at least 1 treatment-cycle. Belamaf (2.5mg/kg) was administered on day (D) 1 of every 2nd 28-day cycle, K 70mg/m2 iv D1 (20mg/m2 on C1D1), D8 and D15 of every cycle and dexamethasone 40mg weekly (20mg for patients >75 years). Treatment was continued until disease progression. Adverse events (AEs) were graded per CTCAEv4, except for corneal AEs which were graded by the pre-specified keratopathy and visual acuity (KVA) scale. Response was assessed by the International Myeloma Working Group (IMWG) criteria. Results: At cut-off (Feb 3rd 2022), 19 patients had received B-Kd. The median age of the 10 safety run-in patients was 65 years (range, 48-77); One, five and four patients had 3, 2 and 1 prior lines of therapy respectively including (exposed/refractory %) bortezomib (100/30%), carfilzomib (10/0%) lenalidomide (60/50%), pomalidomide (10/10%), ASCT (70/0%), anti-CD38 monoclonal Ab (mAb) (40/40%). The median number of treatment-cycles was 7 (5-11). The median number of cycles commenced was 9 (range, 2-13). The most frequent AE during cycle 1 was thrombocytopenia (all grade 30%, Gr 3/4 20%) and blurred vision (all grade 20%, Gr 3/4 0%) One patient experienced Gr 4 neutropenia. From cycle 2 onwards, the most frequent AEs included blurred vision (all grade 40%, Gr 3/4 20%), peripheral neuropathy (all grade 30%, Gr 3/4 10%), upper respiratory tract infection (all grade 30%, Gr 3/4 20%), dry eyes (all grade 20%, Gr 3/4 0%), neutropenia (all grade 20%, Gr 3/4 10%) and nausea (all grade 20%, Gr 3/4 0%). Six patients had an SAE, one was related to Belamaf (Gr 1 infusion reaction). Keratopathy occurred in 8 patients; grade 1, 20%; grade 2, 0%; grade 3, 60%. Decline in best corrected visual acuity (BCVA) by at least 2 lines occurred in 8 patients (Gr 3 n=6, Gr 2 n=2). One patient discontinued therapy due to corneal toxicity. Two patients died (progressive disease n=1, unrelated cause n=1). Of the 10 patients in the safety run-in, 9 achieved a PR or better (CR=3, VGPR=3, PR=3) and 2 are known to have subsequently progressed (Figure 1). At estimated median potential follow-up of 9.95 months, median PFS had not been reached (95%CI: 1.08 –NR). Image: Summary/Conclusion: B-Kd with an extended B schedule, has a safety profile that is in keeping with that expected for each individual drug. Deep responses were seen. Recruitment is ongoing in an expansion phase based on the preliminary safety and efficacy. P947: COMPARATIVE EFFECTIVENESS OF ORAL IXAZOMIB-LENALIDOMIDE-DEXAMETHASONE (IRD) AFTER INITIAL BORTEZOMIB (V)-BASED INDUCTION VS PARENTERAL V-BASED THERAPY IN NEWLY DIAGNOSED MULTIPLE MYELOMA (NDMM) R. M. Rifkin1,*, C. L. Costello2, R. E. Birhiray3, S. Kambhampati4, J. Richter5, R. Abonour6, H. C. Lee7, Y. J. Kim8, K. Ren9, D. M. Stull10, D. Cherepanov9, K. Bogard10, S. J. Noga9, S. Girnius11 1Rocky Mountain Cancer Centers/US Oncology Research, Denver; 2Moores Cancer Center, University of California San Diego, La Jolla; 3Hematology Oncology of Indiana/American Oncology Network, Indianapolis; 4Kansas City Veterans Affairs Medical Center, Kansas City; 5Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York; 6Indiana University School of Medicine, Indianapolis; 7MD Anderson Cancer Center, Houston, United States of America; 8Data Analytics, Evidera, St-Laurent, Canada; 9Takeda Development Center Americas, Inc. (TDCA); 10Takeda Pharmaceuticals U.S.A., Inc., Lexington; 11TriHealth Cancer Institute, Cincinnati, United States of America Background: Long-term proteasome inhibitor (PI)-based treatment can improve outcomes for patients (pts) with multiple myeloma (MM). However, prolonged parenteral PI therapy (e.g. with V) can be challenging to achieve in routine clinical practice, and outcomes for pts are often poorer in this setting compared with clinical trials. The phase IV, community-based, single-arm US MM-6 study (NCT03173092) is assessing in-class transition (iCT) from V-based induction to all-oral IRd in transplant ineligible NDMM pts treated in routine clinical practice, with the objective of increasing the duration of PI-based treatment while maintaining quality of life. INSIGHT MM is the largest global, prospective, observational study of MM pts (>4,200), and provided a subset of patients as the comparator cohort. This enabled assessment of iCT vs V-based therapy in NDMM pts in routine clinical practice in the US. Aims: To examine the comparative effectiveness of IRd following initial V-based induction (3 cycles; US MM-6 pts; ‘IRd’ cohort) vs continued V-based therapy (INSIGHT MM pts; ‘V-based’ cohort) in NDMM pts. Methods: A secondary analysis of non-transplant eligible US NDMM pts with ≥stable disease after 3 cycles of V-based induction and baseline Eastern Cooperative Oncology Group performance status (ECOG PS) of 0, 1 or 2 from the US MM-6 (Manda CLML 2020) and INSIGHT MM (Costello Future Onc 2019) studies was performed. Study outcomes included first-line duration of treatment (DOT), overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and reasons for treatment discontinuation. All analyses were weighted using the inverse probability of treatment weighting (IPTW) approach to reduce the imbalance of potential confounding factors between the two cohorts (adjusted analyses). Kaplan–Meier methods were used to examine DOT, PFS, OS, and associated 95% confidence intervals (CIs); the log-rank test was used to compare distribution of time to events. The Clopper-Pearson method was applied to estimate 95% CIs for ORR. Statistical significance was evaluated at alpha=0.05. Results: 100 pts from the IRd cohort (MM-6) and 111 pts from the V-based cohort (INSIGHT) were included. After IPTW, in the IRd vs V-based cohorts: median age was 75.0 vs 74.8 yrs; 56.7 vs 51.3% of pts were male; 37.4 vs 29.1% had an ECOG PS of ≥2; 48.8 vs 41.4% had International Staging System stage III at initial diagnosis, and 79.5/17.7/2.8 vs 77.3/19.5/3.1% pts had received VRd/ V-cyclophosphamide-d (VCd)/ VRCd as initial induction therapy. Adjusted ORRs in the IRd vs V-based cohorts were 74.1 (95% CI 66.0–82.2) vs 57.5% (95% CI 47.9–67.1; p<0.0001). After a median follow-up of 20.3 and 15.8 months in the IRd and V-based cohorts, respectively, DOT was 10.8 (95% CI 6.5–24.4) vs 5.3 months (95% CI 4.3–7.0; p<0.0001) (see Figure). Median PFS was not estimable (NE) in either cohort; 24-month PFS rates were 85.7 (95% CI 68.1–94.0; IRd cohort) vs 76.5% (95% CI 62.6–85.8; V-based cohort). Median OS was NE in either cohort; 24-month OS rates were 94.0 (95% CI 77.7–98.5; IRd cohort) vs 84.9% (95% CI 70.6–92.6; V-based cohort). In the IRd and V-based cohorts, 16.8 and 16.9% of pts discontinued IRd and V, respectively, due to an adverse event. Image: Summary/Conclusion: US MM-6 NDMM pts who transitioned to IRd after 3 initial cycles of V-based induction had a significantly higher ORR and longer DOT compared with pts who received continued V-based therapy in INSIGHT MM. The results suggest that iCT from continued V-based therapy to all-oral IRd may improve outcomes in pts treated at community oncology clinics. P948: INCIDENCE AND CLINICAL OUTCOME OF SARS-COV-2 INFECTION AFTER VACCINATION IN PATIENTS WITH MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE (MGUS) N. Sgherza1,*, P. Curci1, R. Rizzi1,2, D. Roccotelli2, M. Croce2, M. Avantaggiato2, L. Ruga2, A. Vitucci1, A. Palma1, D. Di Gennaro2, P. Musto1,2 1Hematology and Bone Marrow Transplantation Unit, AOUC Policlinico, Bari, Italy; 2Department of Emergency and Organ Transplantation, “Aldo Moro” University School of Medicine, Bari, Italy Background: Our group recently reported that MGUS patients investigated during the early waves of pandemic, when vaccines were still not available, neither have an increased risk of contracting SARS-CoV-2, infection, nor show poorer COVID-19 outcomes with respect to healthy controls (Sgherza N. et al. Haematologica. 2022). Aims: Aiming to specifically address the clinical effects of vaccines, we compared incidence and outcome of SARS-CoV-2 infection of 1,454 previously described, not vaccinated MGUS patients (91 of whom were SARS-CoV-2 positive), with those observed in a similar population during the national vaccination campaign. Methods: We obtained retrospective information from 86 individuals found to be SARS-CoV-2 positive among 1,265 MGUS patients analyzed after at least two doses of anti-SARS-CoV-2 vaccine received between April 2021, and January, 2022. Seventy-one subiects positive before vaccination or after only one dose were escluded from analysis. Results: The mean age of this group was 65.7 +/- 13.3 years (range 31-90); 32 patients were female (37%), and 54 were male (63%). About MGUS-subtypes, the most frequent one was IgG-kappa (n=35; 40.7%), followed by IgG-lambda (n=28; 32.5%), IgM-kappa (n=11; 12.8%) and others (n=12; 14%). Most of patients (81/46, 94.2%) were at low or low-intermediate risk, according to Mayo Clinic prognostic model. Thirty-seven (43%) patients developed SARS-CoV-2 infection after two doses (9, 26 and 2 patients receiving ChAdOx1-S, BNT162b2 mRNA and mRNA-1273 vaccines, respectively), fourty-nine (57%) after three doses (BNT162b2 mRNA or mRNA-1273 as “booster” dose, repectively). The mean number of days between last dose of vaccine and SARS-CoV-2 infection was 103.04 +/-80.28 (range 2-285). The two populations of SARS-CoV2 positive MGUS patients (before and after vaccination) were comparable for age, sex and presence of co-morbidities (data not shown). Overall, rates of symptoms (59.3% vs 26.7%), hospitalization (20.9% vs 2.3%), hospitalization in Intensive Care Unit (11% vs 1.2%) and rate of deaths (8.8% vs 1.2%) were significantly higher in still not vaccinated MGUS patients than in those who had received vaccines (Table 1). Image: Summary/Conclusion: Our data indicate that the incidence of SARS-CoV-2 infection is not significantly reduced in fully vaccinated MGUS patients (even after three doses), likely also because of the higher diffusion capacity of the recently recognized Omicron viral variants. However, as observed in normal population and in other hematological contexts, the clinical outcome of COVID-19 may be significantly improved after vaccination in these patients, with symptoms present in less of one third of subjects and no case of hospitalization or death in our series. These observations support extensive vaccination programs in patients with MGUS. P949: A PHASE 2 TRIAL OF ELRANATAMAB, A B-CELL MATURATION ANTIGEN (BCMA)-CD3 BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA: INITIAL SAFETY RESULTS FOR MAGNETISMM-3 A. M. Lesokhin1,*, B. Arnulf2, R. Niesvizky3, M. Mohty4, N. J. Bahlis5, M. H. Tomasson6, P. Rodrίguez-Otero7, H. Quach8, N. S. Raje9, S. Iida10, M.-S. Raab11, A. Czibere12, S. Sullivan12, E. Leip12, A. Viqueira13, X. Leleu14 1Division of Hematology and Oncology, Memorial Sloan Kettering Cancer Center/Weill Cornell Medical College, New York, United States of America; 2Hôpital Saint-Louis, Paris, France; 3Weill Cornell Medical College - New York Presbyterian Hospital, New York, United States of America; 4Sorbonne University, Hôpital Saint-Antoine, and INSERM UMRs938, Paris, France; 5Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada; 6Holden Comprehensive Cancer Center, University of Iowa, Iowa City, United States of America; 7Clinica Universidad de Navarra, Madrid, Spain; 8University of Melbourne, Melbourne, Australia; 9Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, United States of America; 10Department of Hematology & Oncology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 11Heidelberg Myeloma Center, Department of Hematology/Oncology, Heidelberg University Hospital, Heidelberg, Germany; 12Pfizer Inc, Cambridge, United States of America; 13Pfizer SLU, Madrid, Spain; 14Centre Hospitalier Universitaire de Poitiers, Poitiers, France Background: Elranatamab (PF-06863135) is a humanized bispecific antibody that targets both BCMA-expressing multiple myeloma (MM) cells and CD3-expressing T cells. MagnetisMM-3 (NCT04649359) is an open-label, multicenter, non-randomized, phase 2 study to evaluate the safety and efficacy of elranatamab monotherapy in patients with relapsed/refractory (R/R) MM. Aims: Initial safety results from the MagnetisMM-3 trial in patients with R/R MM and no prior BCMA-targeted treatment are presented. Methods: MagnetisMM-3 enrolled patients who are refractory to at least 1 proteasome inhibitor, 1 immunomodulatory drug, and 1 anti-CD38 antibody. Patients were assigned to 1 of 2 independent, parallel cohorts: those naïve to BCMA-directed therapies (Cohort A) and those with previous exposure to BCMA-directed antibody-drug conjugates or CAR-T cells (Cohort B). Patients received subcutaneous elranatamab 76 mg QW on a 28-day cycle with a 2-step-up priming dose regimen administered during the first week. Dose modifications were permitted for toxicity. Treatment-emergent adverse events (TEAEs) were graded by CTCAE (v5.0), and cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) by ASTCT criteria. Results: As of the data cutoff on Dec 31, 2021, 60 patients in Cohort A had received ≥1 dose of elranatamab; the last patient’s first dose was ~2 months prior to the cutoff. Median age was 69.0 years (range, 44−89), 48.3% were male, 63.3% were white, 18.3% were Asian and 11.7% were Black/African American. At baseline, 60.0% of patients had an ECOG performance status 1−2 and patients had received a median of 5 (range, 2−12) prior therapies. Median duration of elranatamab treatment was 9.57 weeks (range, 0.1−46.1); median relative dose intensity was 87.4% (range, 23.1−101.4). TEAEs were reported in 100% (Grade [G] 3/4, 75.0%) of patients. Most common (≥30%) hematologic TEAEs were neutropenia (36.7% [G3/4, 35.0%]), anemia (36.7% [G3/4, 30.0%]) and thrombocytopenia (30.0% [G3/4, 21.7%]). Among patients who received the 2-step-up priming regimen (n=56), CRS and ICANS, respectively, were reported in 58.9% (G3/4, 0%) and 3.6% (G3/4, 0%); of those patients, 57.6% (n=19/33) and 100% (n=2/2) received tocilizumab and/or steroids. Most common (≥30%) non-hematologic TEAE, other than CRS/ICANS, was fatigue (31.7% [G3/4, 3.3%]). Infections were reported in 46.7% (G3/4, 18.3%) of patients; most frequently reported were upper respiratory tract infections (11.7% [G3/4, 0%]). Discontinuations due to adverse events were reported in 5.0% of patients. No patients permanently discontinued treatment due to CRS or ICANS. There were 10 deaths; causes were MM progression (n=8), septic shock (n=1) and unknown (n=1). Data will be updated at the time of presentation to include ~90 patients. Summary/Conclusion: Preliminary results of MagnetisMM-3 in patients with R/R MM and no prior BCMA-targeted treatment suggest that 76 mg QW elranatamab with a 2-step-up priming regimen is well tolerated, with no G ≥3 CRS or ICANS observed. P950: COMBINED PROTEASOME AND AUTOPHAGY INHIBITION IN RELAPSED/REFRACTORY MULTIPLE MYELOMA – A PHASE I TRIAL OF HYDROXYCHLOROQUINE, CARFILZOMIB AND DEXAMETHASONE T. S. Slørdahl1,2,*, F. B. Askeland3,4, M. S. S. Hanssen5, S. M. Sundt-Hansen1, N. T. Vethe6, H. Hjorth-Hansen1,2, M. H. Fenstad7, A. Waage2, Ø. Hjertner1, F. Schjesvold3,4, A. Sundan2 1Department of Hematology, St. Olavs Hospital; 2Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim; 3Oslo Myeloma Center, Department of Hematology, Oslo University Hospital; 4KG Jebsen Center for B Cell Malignancies, University of Oslo, Oslo; 5Department of Ophthalmology, St. Olavs Hospital, Trondheim; 6Department of Pharmacology, Oslo University Hospital, Oslo; 7Department of Immunology and Transfusion Medicine, St. Olavs Hospital, Trondheim, Norway Background: Multiple myeloma (MM) is characterized by malignant cells which produce large amounts of monoclonal immunoglobulin. MM cells are highly sensitive to drugs that target protein degradation. There are two important intracellular pathways for protein degradation (Lamark, 2010). The proteasome is responsible for the degradation of poly-ubiquitinated damaged, modified, misfolded and redundant proteins (Jung, 2009). This pathway of degradation is the target of proteasome inhibitors. The other main pathway is that of autophagy and lysosomal degradation, eliminating protein aggregates and damaged organelles. Hydroxychloroquine (HCQ) is an inhibitor of autophagy. In vitro studies have shown that HCQ potentiates carfilzomib (K) toxicity towards myeloma cells and could therefore be an attractive treatment option in myeloma (Baranowska, 2016). Here we present safety and efficacy data from a phase 1 study on the combination of HCQ, K and dexamethasone (d). Aims: This study aimed to answer the question whether combined inhibition of the proteasome and the autophagosome is safe and tolerable. The primary endpoint was to establish a maximum tolerated dose (MTD) of the combination HCQ-Kd. Methods: This phase I, single arm, open label dose escalation study (3 + 3 design), included patients with relapsed and/or refractory MM with at least two prior lines of therapy including bortezomib and an immunomodulatory agent. All patients started a 14-day run-in of HCQ at their intended HCQ dose level (DL) before the first cycle of HCQ-Kd. The HCQ DL were 200 mg (DL1), 400 mg (DL2), 600 mg (DL3), and 800 mg (DL4&5) daily. Intravenous carfilzomib was given in a dose of 20 mg/m2 in cycle 1 day 1, and thereafter 56 mg/m2 (DL1-4) and 70 mg/m2 (DL5) on day 8, 15, and in all subsequent cycles and doses. Oral dexamethasone 40 mg (20 mg pts >75y) was administered on day 1, 8, 15 and 22. Patients were treated with HCQ-Kd for a maximum of six 28-day cycles and continued Kd if still responding thereafter. The dose limiting toxicity (DLT) observation period was 28 days. Adverse events (AE) grade (G) 1-5 were registered from day 1 of run-in cycle and until 90 days post last dose of HCQ. Results: The clinical trial is completed, and we here report the data for MTD, safety and efficacy. 19 patients were included. One patient withdrew consent prior to first treatment cycle and was therefore not included in MTD and efficacy analyses. Median previous lines of therapy were 4 (2-9), 50% had high-risk cytogenetics. Only one DLT occurred at DL2 (G3 pneumonia), and MTD was not reached. 104 AEs were recorded, of which G1 45%, G2 32%, G3 22% and G4 1%. No deaths occurred. Most common AEs were anemia (74%), neutropenia (42%), thrombocytopenia (19%), skeletal pain (32%), respiratory tract infections (26%), insomnia (26%) and vomiting/nausea (21%). A total of 13 serious adverse events (SAEs) occurred during the study of which 2 led to treatment discontinuation. Responses were seen in 8 of 18 (ORR 44 %) including 3 PR (18%), 3 VGPR (18%), 1 CR (6%), 1 sCR (6%). Clinical benefit rate was 78 % when including 6 MR (33%) (figure 1). One patient reached MRD negativity. 7 patients did not complete all 6 cycles of HCQ-Kd, 5 due to PD and 2 due to SAEs. Image: Summary/Conclusion: HCQ given up to 800 mg daily in combination with weekly carfilzomib and dexamethasone is well tolerated in patients with relapsed/refractory multiple myeloma. Adverse events were mostly grade 1 and 2. The study results indicate a meaningful clinical efficacy of the combination. P951: CLINICAL AND LABORATORY CHARACTERISTICS, COURSE AND OUTCOME OF COVID19 INFECTION IN PATIENTS WITH MULTIPLE MYELOMA A. Sretenovic1,*, M. Mitrovic1, T. Adzic Vukicevic2, Z. Bukumiric3, O. Markovic4, M. Zdravkovic4, D. Antic5, J. Bila1 1Myeloma department, Clinic of hematology University Clinical centre of Serbia; 2Pulmology, COVID Hospital “Batajnica”, University Clinical Center of Serbia; 3Medical Faculty, University of Belgrade; 4Hematology, Clinical-Hospital Center “Bezanijska Kosa”; 5Lymphoma department, Clinic of hematology University Clinical centre of Serbia, Belgrade, Serbia Background: During the Covid19 pandemic, patients with multiple myeloma (MM) are particularly vulnerable due to the characteristics of their disease. Aims: The aim of study was to analyze clinical and laboratory characteristics, course and outcome of Covid19 infection in patients with multiple myeloma. Methods: The study included 53 patients with MM and Covid 19 infection, diagnosed during period March 2020 - November 2021 (27 male; 26 female, mean age 62 yrs, range 37- 87 yrs). IgG MM was present in 28pts (53%), IgA in 8 (15%), IgM in 3pts (6%), and BJ in 14 (26%). According to the clinical stage (CS, Durie-Salmon), distribution was as follows: III 44pts (83%); II 4pts (8%); I CS 5pts (9%). Renal impairement existed in 18pts (34%). Regarding ISS score, the group included: ISS1 had 18pts (34%), ISS2 12pts (23%) and 23pts (43%) had ISS3. According to the Revised ISS (R-ISS) score, R-ISS1 was found in 9pts (17%), R-ISS2 in 20pts (58%) and R-ISS3 was present in 13pts (25%). All pts were treated according to National Protocol for the Treatment of Covid19 infection, including: antibiotics in 53pts (100%), corticosteroids in 50pts (94%), low molecular weight heparin in 52pts (98%), and intravenous immunoglobulins in 26pts (49%). During period January 2021 - November 2021, 9 of 24pts (37.5%) were vaccinated against Covid19. Variables of importance were analyzed using descriptive and analytical statistics. All calculations were made in SPSS program version 26.0. Results: At the moment of detected Covid19 infection, 37pts (70%) had active MM, and 16pts (30%) were in the state of follow-up during remission of disease. Immunoparesis was present in all of 37pts (74%) with active MM. Elevated interleukin-6 (IL-6) was found in 19pts (36%), of which 15pts (78.9%) had active MM. Similarly, elevated d-dimer was found in 46pts (87%), of which 35pts had active disease. During the course of Covid19 infection, pneumonia was registered in 52pts (98%), bleeding in 2pts (4%) and thrombosis in 4pts (8%). Bleeding and thrombosis were registered in patients with active MM. In the Intensive Care Units were treated 7pts (13%), with lethal outcome of 6pts. In the Semi-Intensive Care were treated 36pts (64%). A total of 36pts (64%) were cured and 17pts (36%) died. The average time to recovery of our pts was 14 days (max up to 45 days). After recovery 20pts (55%) had active myeloma, reinfection was found in 5pts (13.5%) and they were in active phase of disease. All of 5pts (10%), who were previously vaccinated against Covid19 during MM remission, completely recovered from Covid19 infection. Additionally, 4pts (8%) were vaccinated after recovery from Covid19 infection. Patients with active MM had signficantly worse outcome of Covid19 infection, (Chi-Square, p=0.003). Immunoparesis was highly frequent in pts with acitive disease (Fisher Exact Test, p=0.000), as well as elevated d-dimer (Fisher Exact Test, p=0.002). Increased IL-6 was observed in pts with active MM, but statistical significance was not established (Chi-Square p=0.088). The poor treatment outcome of pts with MM and Covid 19 was influenced by: Age (T-Test, p=0.022), Renal impairement (Chi-Square, p=0.032), and high R-ISS score 3 (Mann Whitney, p=0.042). Summary/Conclusion: A significantly worse outcome of Covid19 infection may be expected in patients with active MM and renal impairement, accompanied with findings of elevated d-dimer and IL-6, as well as immunoparesis and R-ISS score 3. All vaccinated patients recovered from Covid 19 infection. P952: THE ROLE OF AGE IN THE LONG-TERM TREATMENT OF MULTIPLE MYELOMA - A LONGITUDINAL ANALYSIS OF REGULAR CARE DATA H. T. Steinmetz1,*, M. Heinz1, U. Totzke2 1Onkologie Köln, Outpatient Clinic for Hematology and Oncology, Cologne, Cologne, Germany; 2TOTZKE & DREHER SCIENTIFIC SA, Basel, Switzerland Background: Recently, we demonstrated in a longitudinal approach that the percentage of multiple myeloma (MM) patients reaching consecutive treatment lines in regular care is much higher than presumed, i.e. 88%, 66%, 44%, and 30% in 2nd, 3rd, 4th, and 5th line, respectively (Oncol Res Treat 44, 662-71). Aims: As the annual risk of death generally increases with age, we investigated here the impact of age on the course of MM treatment. Methods: MM patients treated in our outpatient clinic in Germany over 8 years were assigned to three groups of equal size according to age at diagnosis. Resulting subgroups were analyzed regarding their outcome and the probabilities of reaching consecutive therapy lines using a linear statistical model, as previously described for the analysis of the whole cohort. Results: Age groups were <65, 65-74, and ≥75 years. As compared to the two remaining groups, the proportion of male patients and of those with ISS 1 was considerably higher in the youngest age group while the median observation time was on average 7 months shorter in the oldest age group (Tab.). Age <65 years Age 65-74 years Age ≥75 years Patientsb 50 (34.5%) 46 (31.7%) 49 (33.8%) womenb 18 (36.0%) 25 (54.3%) 23 (46.9%) Time observed [month]a 40.5 (0-198) 40 (2-152) 33 (0-197) Stage at diagnosisb ISS R-ISS ISS R-ISS ISS R-ISS I 19 (38%) 11 (22%) 6 (13%) 9 (20%) 10 (20%) 3 (6%) II 11 (22%) 13 (26%) 21 (46%) 17 (37%) 17 (35%) 16 (33%) III 11 (22%) 6 (12%) 13 (28%) 2 (4%) 16 (33%) 6 (12%) Unknown 9 (18%) 20 (40%) 6 (13%) 18 (39%) 6 (12%) 24 (49%) Deathb 12 (24.0%) 8 (17.4%) 27 (55.1%) Cause of deathb MM 3 (25.0%) 1 (12.5%) 10 (37.0%) Other 5 (41.7%) 6 (75.0%) 13 (48.1%) Unknown 4 (33.3%) 1 (12.5%) 4 (14.8%) a median (range); b N (%) The probability of reaching consecutive treatment lines decreased in all age groups, but most strikingly in that of the oldest patients (Fig.). Age does not appear to play a major role for reaching subsequent lines at ages up to 75 years though. Up to this age, patients were thus even more likely to reach subsequent lines than previously shown for the total cohort, increasing to about a 50% chance for the 5th line of therapy. The mean age at death was 83 (SD 4.8) years in the oldest age group, but in none of the age groups, the leading cause of death was MM. Image: Summary/Conclusion: Only age beyond 75 years at baseline significantly reduces the probability of reaching 2nd or higher lines of MM therapy. P953: PHASE I STUDY OF TQB3602 CAPSULE, AN ORAL PROTEASOME INHIBITOR, IN RELAPSED/REFRACTORY MULTIPLE MYELOMA W. Tang1, Y. Li1, X. Zhong1, Q. Liang1, Y. Liu2, Y. Zeng3, B. Fang2, L. Zheng4, T. Niu1,* 1Department of Hematology, West China Hospital, Sichuan University, Chengdu; 2Department of Hematology, Henan Cancer Hospital, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou; 3Department of Hematology, First Affiliated Hospital of Kunming Medical University, Hematology Research Center of Yunnan Province, Kunming; 4GCP Center/National Institute of Drug Clinical Trial, West China Hospital, Sichuan University, Chengdu, China Background: The proteasome inhibitor is an effective treatment strategy for multiple myeloma (MM). TQB3602 is a novel oral proteasome inhibitor derived from multiple rounds of optimization based on ixazomib. It has an addition of an acridine ring to the structure of ixazomib, which maintains high activity and selectivity, resulting in better metabolic stability. In preclinical models of MM, TQB3602 displayed potent kinase inhibiting activity for 20S proteasome enzyme with IC50 15.3 nM. It also inhibited cell proliferation in the MM.1S cell line with IC50 10.1 nM. In the MM.1S MM CB-17 CDX model, TQB3602 also showed antitumor efficacy. TQB3602 represents a promising clinical candidate for treating MM. Aims: This study presents the first-in-human trial designed to evaluate the safety, tolerance, pharmacokinetics, dose-limiting toxicity and maximum tolerated dose of TQB3602 in relapsed/refractory MM (RRMM). Methods: This study is a multicenter, open-label, 3 + 3 dose-escalation phase I trial (NCT04275583). Adult patients with RRMM who had received at least two kinds of therapy, including bortezomib, lenalidomide or thalidomide, are enrolled. TQB3602 is orally administered at a dose of 0.5mg, 1mg, 2mg, 3mg, 4mg, 5mg, 5.5mg, 6mg or 7mg on days 1, 8, 15 in 28-day cycle until limiting toxicity or disease progression. Results: Twenty-five patients with RRMM (16 males, 9 females, median age 65, range 37-73) were enrolled between May 2020 and October 2021, with all patients exposed to bortezomib and three patients exposed to ixazomib. Patients received a median of three cycles of TQB3602 (range 1-11). Grade ≥3 drug-related adverse events included anemia (28%), thrombocytopenia (8%), neutropenia (8%) and diarrhea (4%). No grade ≥3 drug-related peripheral neuropathy occurred. Two dose-limiting toxicities occurred at 7mg, including diarrhea and grade 2 peripheral neuropathy. The maximum tolerated dose was determined as 6mg. TQB3602 was rapidly absorbed, resulting in a time to plasma peak concentration from 0.8 to 1.5 hours. After multiple dosing, the terminal half-life was 23 to 152 hours. Following administration of 6 mg, the mean half-life was approximately 82 hours. TQB3602 slightly accumulated in the human body. At a dose of 6 mg, the AUClast and Cmax were approximately 1.9 times higher on day 15 than day 1. The mean plasma concentration-time profiles of day 1 and day 15 are shown in Figures A and B, and the geometric mean plasma Cmax and AUClast on day 1 and day 15 are shown in Figures C and D. Among 25 response-evaluable patients, 65% of patients achieved stable disease or better. Image: Summary/Conclusion: TQB3602 is well tolerated with no severe neuropathy adverse events and has shown preliminary efficacy in patients with RRMM. Based on the efficacy, safety, and pharmacokinetic data, 6mg was recommended as the anticipated therapeutic dose and adopted in an ongoing dose-expansion phase. P954: ESTABLISHING THE NATIONAL INSTITUTE FOR HEALTH RESEARCH (NIHR) HEALTH INFORMATICS COLLABORATIVE (HIC) MULTIPLE MYELOMA (MM) REGISTRY S. Tayabali1,*, S. Moore2, M. Jenner3, F. Djebbari2, L. Romao1,4, L. English1,4, R. Rodriguez1,4, B. Briot Ribeyre1,4, G. Roadknight2,5, K. Varnai2,5, S. Little2,5, J. Davies2,5, K. Woods2,5, C. Davis6, F. Borca6, J. Olza Meneses6, H. Phan7, W. K. Wong1,4, R. Popat1 1University College London Hospitals NHS Foundation Trust, London; 2Oxford University Hospitals NHS Foundation Trust, Oxford; 3University Hospital Southampton NHS Foundation Trust, Southampton; 4NIHR University College London Hospitals Biomedical Research Centre, London; 5NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford; 6NIHR Southampton Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust; 7NIHR Southampton Biomedical Research Centre, University of Southampton, Southampton, United Kingdom Background: Real world data provides unique insights into the natural history of MM, resource utilisation, adherence to standards and outcomes of treatments outside clinical trials. However, the quality of such initiatives can be poor due to the need for manual data entry, inability to integrate multiple data sources, lack of harmonisation of data across centres and limited generalisability due to centre bias. Exemplar registries collate and transform multidisciplinary routinely collected data from Electronic Health Records (EHRs) to a common data language allowing integration, analysis and expansion. Aims: To develop a secure national MM registry beginning with a three site pilot. Methods: Potential registry providers were evaluated against the following criteria: previous experience, infrastructure and technical capability, data governance, compliance with legal standards and cost effectiveness. The platform required ethical and data sharing approvals. A common data model was investigated and a myeloma focused multidisciplinary dataset was designed and mapped to source data. Data extraction, transformation and analysis is planned. Results: 8 providers were assessed for suitability. The NIHR HIC was chosen due to its established data sharing network across 30 UK sites and overarching governance framework. This platform permitted the establishment of harmonised data flow, comparable across sites and retaining clinical interpretability. A minimal myeloma dataset of routinely collected information was agreed by clinicians and data scientists to contain the following fields: baseline and disease characteristics including imaging findings eg: extramedullary/paramedullary disease, skeletal/spinal disease, comorbidities, mode of presentation, cytogenetics, treatments including chemotherapy, stem cell transplants/CAR-T cells, spinal interventions, anti-infectives, bisphosphonates and other supportive care. Each variable was mapped to identify the location of raw data in the EHR, its format and suitability for extraction. For unstructured data, eg: cytogenetics and imaging reports, natural language processing is being developed to extract key fields. To enable harmonisation of data from different systems, data is transformed into a common data model. The Observational Medical Outcomes Partnership common data model (OMOP CDM) was chosen as it provided a framework for systematic analysis of data from disparate data sources, typical of those held within health care information systems. This model allows for international benchmarking and collaboration for research. OMOP CDM maps commonly used coding systems and vocabularies, eg ICD-10, SNOMED-CT into standard concepts permitting raw data entering the registry to be standardised into a research ready format that is person-centric, disease agnostic and syntactically and semantically defined. Anonymised data from multiple sites will be electronically transferred to a ISO27001 certified, GDPR compliant UCL data safe haven for analysis. Oversight is provided by the Steering Committee of clinicians, data scientists, myeloma patients and Myeloma UK with patient involvement at every step. Data will be analysed to answer 3 initial themes (Image 1), and extended further to include control data for Health Technology Appraisals. To date we have successfully identified an initial cohort of over 4000 myeloma cases since 2015. Image: Summary/Conclusion: The NIHR HIC Myeloma registry represents a research ready platform to integrate and analyse UK wide real word data. Detailed data analysis and progress in answering the initial 3 themes is ongoing. Funding: Myeloma UK & NIHR P955: CARFILZOMIB IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS: THE REAL-LIFE EXPERIENCE OF EMMY C. Hulin1,*, M. Macro2, A. Perrot3, B. Royer4, D. Caillot5, K. Belhadj6, L. Frenzel7, R. Benramdane8, H. Demarquette9, B. Bareau10, S. Darre11, C. Calmettes12, R. Le Calloch13, M. Bouketouche14, K. Laribi15, N. Texier16, M. Willaime16, C. Deal17, P. Moreau18, O. Decaux19 1CHU Bordeaux, Bordeaux; 2CHU Caen, Caen; 3CHU de Toulouse, IUC T-O, Toulouse; [e-mail], paris; 5CHU Dijon, Dijon; 6Hôpital Henri Mondor, Creteil; 7Hôpital Necker, paris; 8CH Cergy Pontoise, Cergy Pontoise; 9CH. Dunkerque, Dunkerque; 10CH Cesson Sévigné, Cesson Sévigné; 11CH. Arras, Arras; 12CH. Périgueux, Périgueux; 13CH Cornouaille, Quimper; 14CH. St Quentin, St. Quentin; 15CH. Le Mans, Le Mans; 16kappa santé; 17IFM, paris; 18CHU Nantes, Nantes; 19CHU Rennes, Rennes, France Background: EMMY is a large-scale epidemiological study to assess the epidemiology and real-life management of multiple myeloma (MM). Proteasome inhibitors (PI), immunomodulators (IMID) and anti-CD38 (aCD38) provide broad solutions to treat patients. Following pivotal clinical trials, carfilzomib, a second-generation PI, is approved in relapsed/refractory (RR) MM. Its use in real-life setting can be assessed in EMMY. Aims: To assess the use of carfilzomib in RRMM patients in real-life conditions. Methods: EMMY is a descriptive, multicenter, national, non-interventional study conducted in 72 IFM (Intergroupe Francophone du Myélome, sponsor) sites in France. Any patient initiating treatment for MM over a 3-month annual observation period, is included, since 2017 This dynamic cohort has included approximately 900 patients each year (2765 patients included in 2019). Data are updated annually from hospital records. Patients receiving carfilzomib (K) for RRMM at inclusion or follow up were identified and classified by the regimens received. The median progression-free survival (mPFS), and median overall survival (mOS) were estimated per regimen and per treatment line. Results: 512 patients (18.5%) (median age 68.4 years (y), 20.5% ≥75y) received K for RRMM, 111 (21.7%) for a second (L2), 97 (18.9%) a third (L3) and 304 (59.4%) a fourth or more (L4+) line. K was used in a double regimen with dexamethasone (Kd) in 232 patients (45.3%) and in a triple regimen either with an IMID (K-IMID) in 198 (38.7%) patients or with other agents in 82 patients (16%) (K-aCD38 for 35 (6.8%), K-alkylant for 27 (5.3%) and K-venetoclax for 19 (3.7%) patients). 25.4% patients (62/244) were at high cytogenetic risk. K-IMID was used in patients of 66,8y, with an ECOG ≥ 2 for 20.8%, and comorbidities (≥ 1) for 24.3% including 3% of cardiovascular diseases. K-IMID was used in L2 (42.9%), L3 (21.7%) and L4+ (35.4%). 63.1% were priorly exposed to lenalidomide (len) of which 35,9% were refractory. K was combined with len in 144 (28,1%) and with pomalidomide (pom) in 54 (10,5%) patients. The use of K-pom increased over years: 14,4% in 2018, 28,2% in 2019, 73,9% in 2020. mPFS was overall 10.4 months (m) 95%CI [7.3; 14.7]; it was 23m 95%CI [14.7; -] in L2, 6.2m 95%CI [1.3; 12.2] in L3 and 7.1m 95%CI [4.6; 9.2] in L4+. mOS was not reached in L2, L3 or L4+. Kd was used in patients of 70.1y, with an ECOG ≥ 2 for 31.3% and comorbidities for 29.3%. 93.5% were priorly exposed to len of which 66.4% were refractory. Kd was used in L2(6%), L3 (17.2%) and L4+ (76.8%). mPFS and mOS were 4.4m [3.7; 5.8] and 19.5m [13.7; 22.3] in L4+. K-aCD38 was used in patients of 63.5y, with an ECOG ≥ 2 for 10%, and comorbidities (≥ 1) for 37.1%. 91.4% were priorly exposed to len including 45.7% len-refractory. K-aCD38 (33/35 with daratumumab) was used in L2 (31.4%), L3/L4 (35.7%) and L5+ (22.9%). mPFS and mOS (all lines included) were 6m [3.8; 15.1] and 21.3m [16.9; -]. K-alkylant (5/27 with bendamustine, 22/27 cyclophosphamide) and K-venetoclax were used in older patients (71.8y and 70.4y) and in L4 or L5 for >80% of them. More than 90% were pre-exposed to len including 65% of len-refractory patients. K discontinuation was recorded for 63.4% of patients for progression in 44% and adverse event in 11.7% of them. Summary/Conclusion: The EMMY study shows that carfilzomib is used in many combinations in extended real-life settings compared to pivotal clinical trials including older patients, largely-exposed to lenalidomide and at advanced disease. The use of lenalidomide in early stages has led to a shift in the management of RRMM patients with carfilzomib. P956: EMMY COHORT: MANAGEMENT OF NEWLY DIAGNOSED OR RELAPSED/ REFRACTORY MULTIPLE MYELOMA IN PATIENTS AGED OF 80 YEARS AND OVER M. Macro1,*, O. Decaux2, A. Perrot3, B. Royer4, M. Chretien5, K. Belhadj6, M. Mohty7, L. Frenzel8, X. Leleu9, M. Dib10, O. Allangba11, P. Zunic12, I. Botoc13, J. Malfuson14, P. Moreau15, R. Garlantazec16, N. Texier17, R. Germain17, C. Deal18, C. Hulin19 1CHU Caen, Caen; 2C.H.U de Rennes, Rennes; 3Institut Universitaire du Cancer Toulouse Oncopole, Toulouse; 4hôpital st louis, aphp, Paris; 5CHU Dijon, Dijon; 6Hôpital Henri Mondor, Creteil; 7Hôpital Saint-Antoine; 8Hôpital Necker, paris; 9CHU de Poitiers, Potiers; 10CHU Angers, Angers; 11C.H. Yves le Foll, ST Brieuc; 12CHU Réunion Sud, St Pierre; 13CH Saint-Malo, Saint Malo; 14Hôpital d’Instruction des Armées Percy, Clamart; 15CHU nantes, Nantes; 16CHU Rennes, Rennes; 17kappa santé; 18IFM, Paris; 19CHU Bordeaux, Bordeaux, France Background: Multiple myeloma (MM) patients aged 80 years and older are a population more prone to comorbidities, frailty, cognitive impairment or physical decline and require appropriate management of their myeloma. They are often underrepresented in pivotal clinical trials, with little data available on their management. The EMMY study is a large-scale epidemiological study to assess the epidemiology and real-life management of MM and can focus on the use and real-life efficacy of treatments for newly diagnosed or relapsed/ refractory elderly MM patients. Aims: To describe the management of MM in newly diagnosed or relapsed patients aged 80 years and over and to assess the real-life effectiveness of the treatments received. Methods: EMMY is a descriptive, multicenter, national, non-interventional study conducted in 72 IFM (Intergroupe Francophone du Myélome, sponsor) centers in France. Any patient initiating treatment for MM over a 3-month observation period, from October to December, is included, since 2017. It is a dynamic cohort with the inclusion of approximately 900 patients each year: 2765 patients included at the end of 2019 of which 561 were aged 80 and over (20.3%). Data are updated annually from hospital records up to 2020 at the time of the analysis. Four cohorts of patients aged 80 years and older were analysed according to the line of treatment initiated during their follow-up: L1, L2, L3 or L4+. Median time to next treatment (mTNT), median progression-free survival (mPFS) and median overall survival (mOS) were assessed in months(m). Results: Patients ≥80 years initiated 19.8% of L1s (237/1199) 22.7% (232/1022) of L2s, 22.2% (167/751) of L3s and 18.7% (288/1540) of L4+. They are 7.5% patients ≥85 years old to initiate L1, 8.7% L2, 7.4% L3 and 5.1% L4+. When the line is started, an ECOG ≥2 is reported for 39.4% of L1, 27.7% of L2, 35.1% of L3 and 38.5% of L4 patients and comorbidities recorded in 45.1% of L1, 39.6% of L2, 35.4% of L3 and 38.6% of L4+. In L1, the ISS is level I, II, III for 20.2%, 27.4% and 52.4% of patients. In L1, over the 2017-2020 study period, patients received treatment with PI (58.6%), IMID (32.1%), PI/IMID (4.2%), anti-CD38 (1.3%) or other molecules (3.8%). In L2, they received PI (22.8%), IMID (48.7%), PI/IMID (11.7%), anti-CD38 (14.2%) or other (2.6%), in L3 PI (12.6%), IMID (38, 9%), PI/IMID (9.6%), anti-CD38 (29.3%) or other (9.6%) and in L4+ a PI (23.3%), IMID (20.1%), PI/IMID (5.6%), anti-CD38 (29.2%) or other (21.9%). More than half received a double combination in L1 (50.2%), L2 (59.9%), L3 (54.4%) and L4+ (53.8%). Regarding efficacy, mTNT is 20.3m [15.2; 24.6] in L1, 14.7m [12.3; 20.4] in L2, 10.8m [9; 13.5] in L3 and 8.2m [6.5; 11.2] in L4+. The mPFS is 19.3m [15.1; 23.2] in L1, 12.6m [10.1; 17.5] in L2, 8.9m [10.1; 17.5] in L3 and 5.9m [4.5;7.4] in L4+. The mOS is not reached in L1 (rate at 30m: 76.7% [70; 83.3]) and L2 (rate at 30m: 58.1% [48.1; 68]; it is 25.7m [17.1; NE] in L3 and 18.3m [13.4; 22.4] in L4+. Summary/Conclusion: Patients ≥80 years of age account for more than 20% of de novo and relapsed myeloma patients treated in EMMY. Double combinations are used in half of the situations. Over the study period, PI-based treatments were preferred in L1, IMID-based in L2 and antiCD38 for 30% of patients in L3 and L4+. The benefits in terms of survival are real and this specific population is likely to increase significantly in the near future, requiring human and logistical resources. P957: CARFILZOMIB, LENALIDOMIDE, DEXAMETHASONE FOLLOWED BY A SECOND AUTO-HCT IS AN EFFECTIVE STRATEGY IN FIRST RELAPSE MULTIPLE MYELOMA: A STUDY OF THE CHRONIC MALIGNANCIES WORKING PARTY OF EBMT R. Tilmont1,*, I. Yakoub-Agha1,2, D.-J. Eikema3, N. Zinger4, M. Haenel5, N. Schaap6, C. Herrera Arroyo7, C. Schuermans8, W. Bethge9, M. Engelhardt10, J. Kuball11, M. Michieli12, N. Schub13, K. M. O. Wilson14, J. H. Bourhis15, M. V. Mateos16, N. Robin17, E. Jost18, N. Kröger19, J. M. Moraleda20, S. Sica21, P. J. Hayden22, M. Beksac23, S. Schönland24, S. Manier1,25 1Hematologie Clinique, CHU de Lille; 2CHU de Lille, Univ Lille, INSERM U1286, Infinite, Lille, France; 3EBMT Statistical Unit; 4EBMT Leiden Study Unit, Leiden, Netherlands; 5Klinikum Chemnitz gGmbH, Chemnitz, Germany; 6Nijmegen Medical Centre, Nijmegen, Netherlands; 7Hosp. Reina Sofia, Cordoba, Spain; 8GZA Hospitals, Antwerp, Belgium; 9Universitaet Tuebingen, Tuebingen; 10University of Freiburg, Freiburg, Germany; 11University Medical Centre, Utrecht, Netherlands; 12Centro di Riferimento Oncologico, Aviano, Italy; 13University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Germany; 14Department of Haematology, Cardiff, United Kingdom; 15Gustave Roussy Cancer Campus, Villejuif, France; 16Hospital Clínico, Salamanca, Spain; 17University College London Hospital, London, United Kingdom; 18University Hospital Aachen, Aachen; 19University Hospital Eppendorf, Hamburg, Germany; 20Hospital Universitario Virgen de la Arrixaca, Murcia, Spain; 21Universita Cattolica S. Cuore, Rome, Italy; 22Department of Haematology, Trinity College Dublin, St. James’s Hospital, Dublin, Ireland; 23Ankara University Faculty of Medicine, Ankara, Turkey; 24Medizinische Klinik u. Poliklinik V, University of Heidelberg, Heidelberg, Germany; 25Univ Lille, Canther, INSERM UMR-S1277 CNRS UMR9020, Lille, France Background: Multiple myeloma (MM) is an incurable hematologic malignancy despite recent therapeutic advances. In the context of lenalidomide-sensitive relapse, one of the preferred options from EHA/ESMO and IMWG recommendations is the combination of carfilzomib-lenalidomide-dexamethasone (KRd). This regimen has been approved based on the ASPIRE trial with a PFS of 26.3 months vs 17.6 months for Rd alone (HR = 0.69, p=0.0001). In younger patients, a second autologous hematopoietic cell transplantation (auto-HCT) remains an option in case of a prolonged remission after frontline auto-HCT. Only few data are available on the combination of KRd followed by a second auto-HCT in first relapse. Aims: Primary objective was to estimate progression-free survival (PFS) and overall survival (OS) in patients who received a second line of treatment with KRd followed by a second auto-HCT. Secondary objectives were to assess the response rates and identify statistically significant co-variables on PFS and OS in this population. Methods: This international retrospective study was performed in 22 centers affiliated to the European Society for Blood and Marrow Transplant Society (EBMT). Patients with MM were included if they received a second line of treatment with KRd induction followed by a second auto-HCT between January 2016 and December 2018. Results: A total of 51 patients were included, with a median age of 62 years (range 35 – 69). ISS at diagnosis was stage I for 18 patients (35.3%), stage II for 11 (21.6%),stage III for 14 (27.5%) and missing for 8 patients (15.7%). Twenty-seven patients (52.9%) were of standard cytogenetic risk and 11 patients (21.6%) of high cytogenetic risk according to IMWG criteria, data was missing for 13 patients (25.5%). The median time between the 1st to the 2nd transplant was 40.4 months (range 17.9–87.9). Regarding the number of cycles of KRd received in induction, 25 patients received 3 or 4 cycles (49.0%), 17 patients 5 or 6 cycles (33.3%) and 9 patients 7 to 12 cycles (17.7%). The conditioning chemotherapy was melphalan alone in 46 patients (90.2%), a combination of melphalan and another drug in 4 patients (7.8%) and cyclophosphamide alone in 1 patient (2.0%). The median follow-up was 36.7 months (range 5.3-58.0). The median PFS was 32.6 months (95%CI: 30–39.9) and the median OS was not reached, while 36- and 48-months OS rates were 87.5% (95%CI: 78.5–97.4) and 72.8% (95%CI: 57.0–92.8) respectively. In univariate analysis, 2 co-variates were found to be associated with a longer median PFS: an interval between the first and the 2nd auto-HCT of more than 4 years (mPFS of 36.1 vs. 30.6 months, p = 0.02) and the achievement of a very good partial response (VGPR) or better at the 2nd transplant (mPFS of 33.5 vs. 27.8 months, p = 0.01). These results were also observed in multivariate analysis, with HR of 0.41 (95%CI: 0.17-0.96, p = 0.04) and HR of 0.45 (95%CI: 0.21-0.98, p = 0.04), respectively. No statistical association was found between the duration of PFS and cytogenetic risk profile, with the limitation of a small number of patients with high-risk cytogenetics. Regarding OS, no significantly relevant co-variables were found in uni- or multivariate analysis. Image: Summary/Conclusion: A second auto-HCT after KRd induction is an effective treatment for patients with a first lenalidomide-sensitive relapse of MM. Our study suggests that patients with at least 4 years of remission after a frontline auto-HCT and who achieved at least a VGPR after KRd induction, benefit the most from this treatment strategy. P958: REAL-LIFE CURRENT STANDARD OF CARE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA: SUBGROUP ANALYSES FROM THE LOCOMMOTION STUDY H. Einsele1,*, P. Moreau2, V. De Stefano3, D. Dytfeld4, E. Angelucci5, R. Benjamin6, H. Goldschmidt7, N. W. van de Donk8, B. Besemer9, C. Scheid10, R. Vij11, E. I. ’. Groen-Damen12, M. Semerjian13, V. Strulev14, J. M. Schecter15, T. Roccia16, T. Nesheiwat17, R. Wapenaar12, K. Weisel18, M.-V. Mateos19 1Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Würzburg, Germany; 2University Hospital Hôtel-Dieu, Nantes, France; 3Section of Hematology, Catholic University, Fondazione Policlinico A Gemelli, IRCCS, Rome, Italy; 4Poznań University of Medical Sciences, Poznań, Poland; 5Hematology and Transplant Center, IRCCS Ospedale Policlinico San Martino, Genova, Italy; 6Department of Haematology, King’s College Hospital, London and School of Cancer and Pharmaceutical Sciences, King’s College, London, United Kingdom; 7University Hospital Heidelberg, Heidelberg, Germany; 8Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 9University Hospital Tubingen, Tubingen, Germany; 10University of Cologne, Cologne, Germany; 11Washington University School of Medicine, St. Louis, MO, United States of America; 12Janssen-Cilag, Breda, Netherlands; 13Janssen-Cilag, Issy-Les-Moulineaux, France; 14Janssen Pharmaceutica NV, Beerse, Belgium; 15Janssen R&D, Raritan, NJ, United States of America; 16Janssen R&D, High Wycombe, United Kingdom; 17Legend Biotech USA, Piscataway, NJ, United States of America; 18University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 19University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca, Spain Background: Patients (pts) with relapsed/refractory multiple myeloma (RRMM) who have had previous triple-class exposure to a proteasome inhibitor (PI), immunomodulatory drug (IMiD), and anti-CD38 monoclonal antibody have limited treatment options and represent an urgent and unmet clinical need. LocoMMotion (NCT04035226) is the first prospective multinational study to assess real-life standard of care (SOC) treatment in pts with triple-class exposed RRMM. Aims: To assess efficacy in subgroups of pts treated with SOC therapies in the LocoMMotion study. Methods: All pts provided informed consent. This noninterventional study was conducted across 76 sites (63 in Europe, 13 in the United States). Pts were included if they had received ≥3 prior lines of therapy (LOT) or were refractory to a PI and an IMiD (double-refractory); received a PI, an IMiD, and anti-CD38 monoclonal antibody and had documented progressive disease during/after their last LOT. Real-life SOC therapies were defined as those used in local clinical practice. Responses and disease progression were based on International Myeloma Working Group criteria and assessed by response review committee. Patient subgroups were categorized according to the following baseline characteristics: age, Eastern Cooperative Oncology Group performance status (ECOG PS), renal function, International Staging System stage, extramedullary plasmacytoma, lactate dehydrogenase, bone marrow plasma cells percentage, number of prior LOT, triple-class or penta-drug exposure, and refractoriness. Results: 248 pts were enrolled as of May 21, 2021 (median follow-up: 11.0 months). Pts received a median of 4.0 (range: 1-20) cycles of SOC treatment. Efficacy outcomes were generally worse in pts with refractoriness to 3 classes of antimyeloma therapy, presence of extramedullary plasmacytomas, high LDH, and ECOG PS ≥1 compared with pts who did not have these characteristics (Table). Across all subgroups, overall response rate ranged from 20.0% to 43.1%. Efficacy outcomes were not affected by age and number of prior LOT. Image: Summary/Conclusion: In this first prospective study of real-life SOC treatment in pts with triple-class exposed RRMM, subgroup analyses indicate that specific pt and disease characteristics were associated with poor outcomes. Outcomes were poor in triple-class refractory and non-triple-class refractory pts; however, the latter subgroup had longer median progression-free survival. These data may help inform bridging strategies for chimeric antigen receptor T-cell therapy. P959: CILTACABTAGENE AUTOLEUCEL IN LENALIDOMIDE-REFRACTORY PATIENTS WITH PROGRESSIVE MULTIPLE MYELOMA AFTER 1-3 PRIOR LINES OF THERAPY: CARTITUDE-2 BIOLOGICAL CORRELATIVE ANALYSES AND UPDATED CLINICAL DATA J. Hillengass1,*, A. D. Cohen2, M. Delforge3, H. Einsele4, H. Goldschmidt5, K. Weisel6, M.-S. Raab7, C. Scheid8, J. M. Schecter9, K. C. De Braganca9, H. Varsos9, T.-M. Yeh9, P. Mistry10, T. Roccia10, C. Corsale9, M. Akram11, L. Pacaud11, T. Nesheiwat11, M. Agha12, Y. C. Cohen13 1Roswell Park Comprehensive Cancer Center, Buffalo, NY; 2Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, United States of America; 3University Hospitals (UZ) Leuven, Leuven, Belgium; 4Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Würzburg; 5University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg; 6University Medical Center Hamburg-Eppendorf, Hamburg; 7University Hospital Heidelberg, and Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center, Heidelberg; 8University of Cologne, Cologne, Germany; 9Janssen R&D, Raritan, NJ, United States of America; 10Janssen R&D, High Wycombe, United Kingdom; 11Legend Biotech USA, Piscataway, NJ; 12UPMC Hillman Cancer Center, Pittsburgh, PA, United States of America; 13Tel-Aviv Sourasky (Ichilov) Medical Center and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Background: Cohort A of the multicohort phase 2 CARTITUDE-2 (NCT04133636) study is assessing ciltacabtagene autoleucel (cilta-cel), a B-cell maturation antigen (BCMA)-directed chimeric antigen receptor T-cell (CAR-T) therapy, in patients with multiple myeloma (MM) who received 1-3 prior lines of therapy (LOT) and were refractory to lenalidomide (len). This population is difficult to treat and has poor prognosis. Aims: To present updated results from CARTITUDE-2 Cohort A. Methods: All patients provided informed consent. Eligible patients had progressive MM after 1-3 prior LOT that included a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD). Patients were len-refractory and had no prior exposure to BCMA-targeting agents. Patients received a single cilta-cel infusion (target dose: 0.75×106 CAR+ viable T cells/kg) after lymphodepletion. Cilta-cel safety and efficacy were assessed. The primary endpoint was minimal residual disease (MRD) negativity at 10-5 by next generation sequencing. Patient management strategies were used to reduce the risk of movement and neurocognitive adverse events (MNTs). Other assessments included pharmacokinetic (PK) analyses (Cmax and Tmax of CAR+ T-cell transgene levels in blood), levels of cytokine release syndrome (CRS)-related cytokines (e.g., IL-6) over time, peak levels of cytokines by response and CRS, association of cytokine levels with immune effector cell-associated neurotoxicity syndrome (ICANS), and CAR+ T cell CD4/CD8 ratio by response, CRS, and ICANS. Results: As of January 2022 (median follow-up: 17.1 months [range: 3.3-23.1]), cilta-cel was administered to 20 patients (male: 65%; median age: 60 years [range: 38-75]). Median number of prior LOT was 2 (range: 1-3); median time since MM diagnosis was 3.5 years (range: 0.7-8.0). 95% of patients were refractory to their last LOT; 40% were triple-class refractory. Overall response rate was 95%, with 90% of patients achieving ≥complete response and 95% achieving ≥very good partial response. Median time to first response was 1.0 month (range: 0.7-3.3); median time to best response was 2.6 months (range: 0.9-13.6). All MRD-evaluable patients (n=16) achieved MRD negativity at 10-5. Median duration of response was not reached. The 12-month progression-free survival rate was 75% and the 12-month event-free rate was 79%. CRS occurred in 95% of patients (grade 3/4: 10%), with a median time to onset of 7 days (range: 5-9) and median duration of 3 days (range: 2-12). 30% of patients had neurotoxicity (5 grade 1/2 and 1 grade 3/4). ICANS occurred in 3 patients (15%; all grade 1/2); 1 patient had facial paralysis (grade 2). No MNTs were observed. 1 death due to COVID-19 occurred and was assessed as treatment-related by the investigator; 2 deaths due to progressive disease and 1 due to sepsis (not related to treatment) also occurred. Based on preliminary PK analyses of CAR transgene by qPCR, peak expansion of CAR-T cells occurred at day 10.5 (range: 8.7-42.9); median persistence was 153.5 days (range: 57.1-336.8). Summary/Conclusion: A single cilta-cel infusion led to deepening and durable responses at this longer follow-up (median 17.1 months) in patients with MM who had 1-3 prior LOT and were len-refractory. Follow-up is ongoing. We will present updated and detailed PK, cytokine, and CAR-T subset analyses as well as clinical correlation to provide novel insights into biological correlates of efficacy and safety in this difficult-to-treat patient population, which is being further evaluated in the CARTITUDE-4 study (NCT04181827; enrollment concluded). P960: HEALTH-RELATED QUALITY OF LIFE IN THE LOCOMMOTION STUDY OF REAL-LIFE CURRENT STANDARD OF CARE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA M. Delforge1,*, P. Moreau2, H. Einsele3, V. De Stefano4, J. Lindsey-Hill5, L. Vincent6, S. Mangiacavalli7, A. Perrot8, E. Ocio9, S. ten Seldam10, E. I. ’. Groen-Damen11, M. Semerjian12, V. Strulev13, J. M. Schecter14, T. Roccia15, K. S. Gries14, T. Nesheiwat16, R. Wapenaar11, M.-V. Mateos17, K. Weisel18 1University Hospitals (UZ) Leuven, Leuven, Belgium; 2University Hospital Hotel-Dieu, Nantes, France; 3Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Wuerzburg, Germany; 4Section of Hematology, Catholic University, Fondazione Policlinico A Gemelli, IRCCS, Rome, Italy; 5Nottinghamshire University Hospitals NHS Trust, Nottingham, United Kingdom; 6Département d’hématologie Clinique, Centre Hospitalier Universitaire de Montpellier, Montpellier, France; 7Fondazione IRCCS Policlinico San Matteo, University of Pavia, Pavia, Italy; 8Centre Hospitalier Universitaire de Toulouse, Service d’Hématologie, Toulouse, France; 9Hospital Universitario Marqués de Valdecilla (IDIVAL), Universidad de Cantabria, Santander, Spain; 10Myeloma Patients Europe, Brussels, Belgium; 11Janssen-Cilag, Breda, Netherlands; 12Janssen-Cilag, Issy-les-Moulineaux, France; 13Janssen Pharmaceutica NV, Beerse, Belgium; 14Janssen R&D, Raritan, NJ, United States of America; 15Janssen R&D, High Wycombe, United Kingdom; 16Legend Biotech USA Inc, Piscataway, NJ, United States of America; 17University Hospital of Salamanca/IBSAL/CIC, Salamanca, Spain; 18University Medical Center Hamburg-Eppendorf, Hamburg, Germany Background: Assessment of patient-reported outcomes (PRO) can inform how real-life standard of care (SOC) treatments affect health-related quality of life (HRQoL) for patients with relapsed/refractory multiple myeloma (RRMM). We present measures of symptoms, functioning, and overall HRQoL from LocoMMotion (NCT04035226), the first prospective, multinational study of real-life SOC in triple-class exposed patients with RRMM. Aims: To assess HRQoL in patients with RRMM receiving real-life current SOC in the LocoMMotion study. Methods: All patients provided informed consent. LocoMMotion is a noninterventional study across 76 sites (63 Europe, 13 United States) in patients who had received ≥3 prior lines of therapy (LOT) or were refractory to proteasome inhibitor (PI) and immunomodulatory drug (IMiD). Patients received PI, IMiD, and anti-CD38 monoclonal antibody and had disease progression during/after their last LOT. Real-life SOC treatments were defined as those used in local clinical practice. The following questionnaires were used: European Organisation for Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30), 4 single items from EORTC QLQ-myeloma-specific module (EORTC QLQ-MY20), and EuroQol 5-Dimension 5-Level (EQ 5D-5L). HRQoL data were collected at baseline (BL), day 1 of each treatment cycle, end of treatment visit, and during follow-up (every 4 weeks). Established thresholds were used to evaluate improvement compared with BL health status. Mixed models for repeated measures were used to assess within-group change. Results: The questionnaire completion rate was 75.6% during SOC treatment in the LocoMMotion study (N=248; male: 54.4%; median age: 68 years; median cycles of SOC: 4.0 [range: 1-20]). Most patients did not achieve meaningful improvement (defined by a literature-based minimally important difference of 10 points in mean score) in PRO scores. This was most pronounced in pain symptoms, with 62% of patients showing no meaningful improvement during the first 3 months of treatment and 55% showing no improvement during the full treatment duration. Least square (LS) mean changes from BL during SOC treatment and subsequent LOT for the overall population are described (Table). Patients with ≥very good partial response during SOC treatment showed greater improvement in PRO scores, including LS mean change for pain score (-14.9 [95% confidence interval: -22.9, -7.0]). Image: Summary/Conclusion: This first prospective study of real-life current SOC in triple-class exposed patients with RRMM reported limited gains in HRQoL, most notably in pain symptoms. There is an urgent and unmet need for therapies that lead to deep responses and delayed disease progression, as these are associated with improvements in HRQoL. P961: CILTACABTAGENE AUTOLEUCEL, A BCMA-DIRECTED CAR-T CELL THERAPY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA: 2-YEAR POST LPI RESULTS FROM THE PHASE 1B/2 CARTITUDE-1 STUDY Y. Lin1,*, T. Martin2, J. G. Berdeja3, A. Jakubowiak4, M. Agha5, A. D. Cohen6, A. Deol7, M. Htut8, A. Lesokhin9, N. C. Munshi10, E. O’Donnell11, C. C. Jackson12, T.-M. Yeh12, A. Banerjee13, E. Zudaire13, D. Madduri12, C. Zhou14, L. Pacaud14, S. Z. Usmani9, S. Jagannath15 1Mayo Clinic, Rochester, MN; 2UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA; 3Sarah Cannon Research Institute, Nashville, TN; 4University of Chicago, Chicago, IL; 5UPMC Hillman Cancer Center, Pittsburgh, PA; 6Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA; 7Karmanos Cancer Institute, Wayne State University, Detroit, MI; 8City of Hope Comprehensive Cancer Center, Duarte, CA; 9Memorial Sloan Kettering Cancer Center, New York, NY; 10Dana-Farber Cancer Institute, Harvard Medical School; 11Massachusetts General Hospital, Harvard Medical School, Boston, MA; 12Janssen R&D, Raritan, NJ; 13Janssen R&D, Spring House, PA; 14Legend Biotech USA, Piscataway, NJ; 15Mount Sinai Medical Center, New York, NY, United States of America Background: The phase 1b/2 CARTITUDE-1 study (NCT03548207) reported early, deep, and durable responses with ciltacabtagene autoleucel (cilta-cel), a chimeric antigen receptor T (CAR-T) cell therapy with 2 B-cell maturation antigen (BCMA)–targeting single-domain antibodies, in heavily pretreated patients with relapsed/refractory multiple myeloma (RRMM). At a median follow-up of ~1 year, the overall response rate (ORR) was 97%, with 67% of patients achieving stringent complete response (sCR). Progression-free survival (PFS) and overall survival (OS) rates at 1 year were 77% and 89%, respectively. Aims: To report updated 2-year post last patient in (LPI) results (total median follow-up of ~30 months). CARTITUDE-1 results at a median follow-up of 21.7 months are reported here. Methods: All patients provided informed consent. Patients with RRMM had received ≥3 prior lines of therapy (LOT) or were refractory to a proteasome inhibitor (PI) and immunomodulatory drug (IMiD). Patients had received a PI, IMiD, and anti-CD38 antibody. After apheresis, bridging therapy was allowed. A single cilta-cel infusion was administered at a target dose of 0.75×106 CAR+ viable T cells/kg 5-7 days after lymphodepletion. The primary objectives were to evaluate the safety and efficacy of cilta-cel. Response assessments were based on International Myeloma Working Group criteria by independent review committee. Minimal residual disease (MRD) negativity at 10-5 was assessed by next-generation sequencing. Results: 97 patients (male: 59%; median age: 61 years) received cilta-cel as of July 22, 2021. The median number of prior LOT was 6 (range: 3-18). 84% of patients were penta-drug exposed, 88% were triple-class refractory, 42% were penta-drug refractory, and 99% were refractory to their last LOT. The ORR was 97.9% (95% CI: 92.7-99.7), with 94.9% of patients achieving very good partial response and 82.5% achieving sCR. Median time to first response was 1.0 month, median time to best response was 2.6 months, and median time to ≥CR was 2.9 months. Median duration of response was not reached. Among 61 MRD-evaluable patients, 92% were MRD negative at 10-5, which was sustained in 44% of patients (27/61) for ≥6 months and in 18% of patients (11/61) for ≥12 months. Median PFS and OS were not reached. The 2-year PFS rate was 60.5% (95% CI: 48.5-70.4) overall. The 2-year PFS rate was 91% for patients with sustained MRD negativity ≥6 months and 100% for those with sustained MRD negativity ≥12 months. No new safety signals or new cases of CAR-T cell neurotoxicity, movement/neurocognitive treatment-emergent adverse events, or treatment-related deaths were reported since 1-year median follow-up. Over a median follow-up of ~2-years, 15 second primary malignancies occurred in 11 patients. Summary/Conclusion: A single cilta-cel infusion resulted in deepening and durable responses in heavily pretreated patients with RRMM at a median follow-up of ~2 years. The safety profile was manageable. Follow-up in CARTITUDE-1 is ongoing. We will present landmark 2-year post LPI data, with ~8 months of additional follow-up (~30 months total median follow-up). Cilta-cel is also being evaluated in earlier LOT and outpatient settings across the CARTITUDE program, including NCT04133636, NCT04181827, NCT04923893. P962: CHANGE IN SOLUBLE BCMA LEVEL MAY BE A SURROGATE MARKER OF EARLY RESPONSE TO THERAPY IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM): PRELIMINARY RESULTS FROM A PHASE I STUDY OF CEVOSTAMAB R. Nakamura1,*, R. Hendricks1, R. Dokhaei1, M. Susilo1, D. Wilson1, S. Trudel2, A. Cohen3, S. J. Harrison4, A. Krishnan5, R. Fonseca6, J. I. Adamkewicz1, M. Li1, C. Wong1, J. Cooper1, T. Sumiyoshi1 1Genentech, Inc., South San Francisco, CA, United States of America; 2Princess Margaret Cancer Centre and University of Toronto, Toronto, ON, Canada; 3Abramson Cancer Center and University of Pennsylvania, Philadelphia, PA, United States of America; 4Peter MacCallum Cancer Centre, Sir Peter MacCallum Department of Oncology, Melbourne University, and The Royal Melbourne Hospital, Melbourne, VIC, Australia; 5City of Hope, Duarte, CA; 6Mayo Clinic in Arizona, Phoenix, AZ, United States of America Background: Cevostamab is an FcRH5xCD3 bispecific antibody that facilitates T-cell directed killing of myeloma cells, and has shown promising activity and manageable safety in a Phase I study in patients (pts) with RRMM (NCT03275103; Trudel et al. ASH 2021). B-cell maturation antigen (BCMA) is a membrane-bound protein that is expressed preferentially by malignant plasma cells and has become an important therapeutic target in MM. Shedding of membrane-bound BCMA, mediated by γ-secretase, gives rise to the soluble form of BCMA (sBCMA) which is often present at elevated levels in pts with MM. Normalization of sBCMA may be a predictor of response to therapy, which may be independent of treatment and target. Aims: To evaluate sBCMA as a biomarker of early response in pts enrolled in the single step-up dosing cohorts of the cevostamab Phase I study. Methods: Plasma samples were collected at baseline and at Cycle (C) 1 Day (D) 1 (pre-infusion and end of infusion [EOI]), C1D2, C1D4, C1D8 (pre-infusion and EOI), C1D9, C1D11, and C2D1 (pre-infusion and EOI). sBCMA levels were quantified using hybrid immunoaffinity capture with LC-MS/MS. Pts were stratified by refractory status, prior therapy, prior transplant, cytogenetic risk and response (responder [≥PR] or non-responder [<PR]), and samples from each category were grouped for analysis. Associations between baseline sBCMA levels and baseline patient characteristics or response to treatment were evaluated using an unpaired 2-sample Wilcoxon test (significance level: p≤0.05). The relationship between sBCMA dynamics (percent change in sBCMA level from baseline to C2D1 EOI) and best response was evaluated using logistic linear regression. All pts provided informed consent. Results: At cut-off (January 7, 2022), 97/103 pts in the single step-up cohorts were biomarker evaluable. No clear differences in baseline sBCMA levels were observed in pts stratified by refractory status, prior transplant, or prior anti-CD38 or anti-BCMA therapy. Analysis of a subset of pts with cytogenetic data showed a trend towards lower baseline sBCMA levels in the standard-risk group (n=18) vs the high-risk group (n=42; p=0.032). In the active dose cohorts (3.6/20mg+; n=77), baseline sBCMA levels were comparable between responders and non-responders. However, baseline sBCMA levels were lower in pts achieving a VGPR or better vs those achieving a PR or no response (median: 47.7ng/mL vs 120.5ng/mL; p=0.037). At C2D1 EOI, most responders had a reduction in sBCMA relative to baseline, with the pts who achieved sCR having the greatest decrease (median: –92.10%). In comparison, an increase in sBCMA was observed in non-responders at C2D1 EOI, with the pts who had PD having the greatest increase (median: 93.14%). The extent of reduction in sBCMA from baseline to C2D1 EOI was significantly correlated with best overall response rate (PR or better) (p=1.8x10–5). Summary/Conclusion: In this study, baseline sBCMA was not associated with refractory status, prior transplant or treatment in pts with RRMM. Patients with standard-risk cytogenetics had lower baseline sBCMA levels vs those with high-risk cytogenetics. The kinetics of sBCMA change from baseline to C2D1 EOI corresponded with response to cevostamab, with greater reductions increasing the probability of best response. These early data suggest that sBCMA dynamics may be a potential tool for early disease monitoring and identification of response in RRMM. Updated data will be presented. P963: EVOLUTION OF TREATMENT PATTERNS AND OVERALL SURVIVAL IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA RECEIVING A SECOND LINE OF THERAPY BETWEEN 2012 AND 2020: ANALYSIS OF THE PREAMBLE COHORT D. Kuter1,*, H. Goldschmidt2, D. Cella3, M. T. Petrucci4, P. Moreau5, B. Durie6, A. Juarez-Garcia7, L. Trong7, J. Gu7, G. Hernandez Rivera8, S. Dhanasiri8, T. Marshall7, J. Wang7, L. Lacoin9, K. Ramasamy10, R. Vij11 1Hematology Division, Massachusetts General Hospital, Boston, MA, United States of America; 2University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany; 3Department of Medical Social Sciences and Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, United States of America; 4Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy; 5Service d’hématologie clinique, CHU Hôtel Dieu, Nantes, France; 6Cedars Sinai Cancer Center, Los Angeles, CA; 7Bristol Myers Squibb, Princeton, NJ, United States of America; 8Bristol-Myers Squibb, Boudry, Switzerland; 9Epi-Fit, Bordeaux, France; 10Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom; 11Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, United States of America Background: Treatment (Tx) options such as immunomodulatory agents (IMiDs), proteasome inhibitors (PIs), and more recently anti-CD38 monoclonal antibodies (mAbs) have improved clinical outcomes for patients (pts) with multiple myeloma (MM); yet many experience disease relapse or become refractory to Tx. PREAMBLE is a prospective, observational cohort study of pts with MM in North America and Europe. Aims: To assess Tx patterns and overall survival (OS) evolution over time in pts with relapsed/refractory MM (RRMM) who received a second-line therapy (2L) between 2012 and 2020 in the PREAMBLE study. Methods: This analysis focused on a subset of pts enrolled in PREAMBLE at the time of initiating 2L Tx for MM. The index date was defined as the enrollment date. Pts were followed until death, completion of 3 years of follow-up (FU), date of loss to FU, or date of last visit prior to database extraction date. Pt characteristics, previous Tx since diagnosis and 2L regimens received were described at index date. Pts were grouped by date of enrollment in 3 cohorts (2012-15, 2016-18, and 2019-20). Duration of 2L Tx and OS were analyzed using the Kaplan Meier method. Results: 611 pts were included with a median age of 71 years at the start of 2L Tx, 56.8% were male, and 72.7% and 27.3% were from Europe and North America, respectively. 352 pts received 2L in 2012-2015, 199 in 2016-2018 and 60 in 2019-2020. Median FU was 22.8, 32.3 and 18.4 months in the 3 cohorts. The median time from diagnosis to the start of 2L Tx was 33.3 months, increasing from 31.8 months in the 2012-15 cohort to 40.2 months in the 2019-20 cohort. Prior to the study entry, during first line (1L), 31.6% of pts received a stem cell transplant (SCT) (34.9%, 27.6% and 25.0% respectively for the 3 cohorts). Overall, the main 1L regimens received were PI-based regimens (48.0%), IMiD-based regimens (24.9%), and combinations of IMiD and PI (22.7%). Fewer than 5 pts received an anti-CD38 mAb in 1L. The proportion of pts who received a combination of IMiD and PI in 1L increased from 19.3% in 2012-15 to 25.1% in 2016-18 and 35.0% in 2019-20. Regarding 2L Tx received, single class regimens (IMiD only and PI only) use dropped from 88.0% in 2012-15 to 62.8% in 2016-18 and 18.4% in 2019-20. It was replaced by various combinations of Tx as shown in the figure. The combinations of Tx containing anti-CD38 mAb had increased from 9.0% to 60.1% since the first launch of anti-CD38 in 2016. 9.0% of pts received an SCT during 2L (10.5%, 8.0% and 3.3% for the 3 cohorts). The unadjusted median duration of 2L for the 3 cohorts was of 6.0, 8.1 and 9.9 months (log rank test, p=0.01). A significant improvement in unadjusted OS was observed (log rank test, p<0.001). One year-OS (95% confidence interval [CI]) was 78.4% (73.9-82.9), 84.8% (79.7-89.9) and 93.0% (86.3-99.7); 2 year-OS (95%CI) was 61.3% (55.8-68.5), 74.5% (68.2-80.6) and 77.7% (65.0-90.4) in 2015-16, 2016-18 and 2019-20 cohorts, respectively. Median OS was not reached. Image: Summary/Conclusion: Major changes in 2L Tx patterns were observed in pts with RRMM between 2012 and 2020 with the decreased use of single class regimens and the increased use of combination Tx. Pts were likely to be exposed to all 3 main classes of drugs (IMiD, PIs and anti-CD38) earlier in the Tx pathway. A trend towards improvement in OS from start of 2L was also observed over time, likely driven by the changes in Tx patterns. Future analyses of this on-going study will allow to evaluate the long-term impact of those new Tx and the remaining unmet needs. P964: IMMUNOPHENOTYPED-SUSPENSION-MULTIPLEX FISH BY IMAGING FLOW CYTOMETRY FOR THE SIMULTANEOUS DIAGNOSIS OF THREE PIVOTAL IGH TRANSLOCATIONS IN MULTIPLE MYELOMA T. Tsukamoto1,*, M. Kinoshita2, K. Yamada2, T. Yamaguchi3, Y. Chinen1, S. Mizutani1, T. Fujino1, Y. Shimura1, T. Kobayashi1, J. Inazawa4, J. Kuroda1 1Division of Hematology and Oncology, Kyoto Prefectural University of Medicine, Kyoto; 2Sysmex Corporation, Hyogo; 3General Laboratory, Bio Medical Laboratories, Inc.; 4Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Background: The cytogenetic abnormalities including t(4;14)(p16;q32), t(14;16)(q32;q23), and t(11;14)(q13;q32) are associated with the molecular profiles, clinical features, and treatment outcome in multiple myeloma (MM). Although the double-color interphase fluorescence in situ hybridization (DC-FISH) is well established to detect those translocations, the conventional method can analyze only one abnormality on each slide. Aims: To analyze multiple chromosomal abnormalities simultaneously with high sensitivity and specificity, we aimed to develop a new diagnostic modality of four-color FISH for multiple translocations in immunophenotyped cells in suspension using imaging flow cytometry (immunophenotyped-suspension-multiplex (ISM)-FISH). Methods: For the ISM-FISH, we first subjected Carnoy-fixed cells in suspension to immunostaining using a brilliant violet 421-conjugated anti-CD138 antibody. Then, we hybridized with four different FISH probes, i.e., Texas Red (TxRed)-conjugated probe for IGH, fluorescein isothiocyanate (FITC)-conjugated probe for FGFR3, Gold-conjugated probe for cyclin D1 (CCND1), and Cy5-conjugated probe for c-MAF, in suspension. Then, more than 2.5 × 104 nucleated cells for each sample were analyzed with imaging flow cytometer MI-1000 (Sysmex) by six channels: one for immunophenotyping, four for FISH and one for bright field image. In the process of imaging flowcytometric analysis, we sorted CD138-positive cell fraction, computed the distance between FISH probes using originally developed spot counting tool, and determined to be positive for translocation when the distance is under the threshold. Results: By this ISM-FISH system, we simultaneously examined three chromosomal translocations, including t(4;14), t(14;16), and t(11;14), in three MM cell lines, KMS-11, KMS-21-BM, KMS-26, and successfully detect the abnormalities. Using different proportions of transformation-positive MM cells spiked with transformation-negative leukemic cells, HL-60, ISM-FISH showed a sensitivity of less than 0.1%. Next, ISM-FISH and conventional DC-FISH were performed for bone marrow nucleated cells from 70 patients with MM or monoclonal gammopathy of undetermined significance (MGUS) and the experiments showed a promising qualitative diagnostic ability in detecting t(11;14), t(4;14), and t(14;16) of our ISM-FISH, which was more sensitive than standard DC-FISH, examining 200 interphase cells with its optimal sensitivity of up to 1.0%. Furthermore, the ISM-FISH showed a positive concordance of 83.3% and negative concordance of 97.4% using standard DC-FISH, examining 200 interphase cells. Furthermore, in three patients (4%), t(4;14) or t(11;14) were positive by ISM-FISH but negative by DC-FISH examining 200 cells. Extensive DC-FISH study examining additional 1,000 cells showed positive in these ISM-FISH-positive/DC-FISH (200cells)-negative samples, suggesting higher sensitivity of ISM-FISH than that of the standard DC-FISH. Image: Summary/Conclusion: This study developed the new diagnostic system of ISM-FISH, which enables the simultaneous diagnosis of three clinically pivotal chromosomal translocations, t(4;14), t(14;16), and t(11;14), in MGUS and MM. This system may facilitate rapid and reliable cytogenetic diagnosis and promote patient-oriented therapy according to the type of chromosomal translocation in the clinical practice setting. P965: FOLLOW-UP ANALYSIS OF THE RANDOMIZED PHASE II TRIAL OF BORTEZOMIB, LENALIDOMIDE, DEXAMTHASONE WITH/WITHOUT ELOTUZUMAB FOR NEWLY DIAGNOSED, HIGH RISK MULTIPLE MYELOMA (SWOG-1211) S. Usmani1,*, A. Hoering2, S. Ailawadhi3, R. Sexton2, B. Lipe4, J. Valent5, M. Rosenzweig6, J. Zonder7, M. Dhodapkar8, N. Callander9, T. Zimmerman10, P. Voorhees11, B. Durie12, S. V. Rajkumar13, P. Richardson14, R. Orlowski15 1Memorial Sloan Kettering Cancer Center, New York; 2Cancer Research And Biostatistics, Seattle; 3Mayo Clinic, Jacksonville; 4University of Rochester Medical Center, Rochester, NY; 5Cleveland Clinic, Cleveland; 6City of Hope, Los Angeles; 7Karmanos Cancer Institute, Detroit, MI; 8Emory Winship Cancer Institute, Atlanta GA; 9University of Wisconsin, Madison; 10Beigene, Chicao; 11Levine Cancer Institute, Charlotte; 12Cedar Sinai Medical Center, Los Angeles; 13Mayo Clinic, Rochester MN; 14Dana Farber Cancer Institute, Boston MA; 15MD Anderson Cancer Center, Houston TX, United States of America Background: The introduction of immunomodulatory agents, proteasome inhibitors, and autologous stem cell transplantation (ASCT) has improved outcomes for patients with multiple myeloma (MM), but those with high risk MM (HRMM) have a poor long-term prognosis. Herein we provide survival outcomes on the first randomized trial in newly diagnosed HRMM, S1211, to follow-up on the previously reported progression-free survival (PFS) (NCT01668719, Usmani SZ et al, Lancet Haem 2021). Aims: S1211 is a randomized phase II trial comparing 8 cycles of lenalidomide, bortezomib and dexamethasone (RVd) induction followed by dose-attenuated RVd maintenance until disease progression with or without elotuzumab (RVd-Elo). Stem cell collection was allowed, but ASCT was deferred until progression. Methods: HRMM was defined by one of the following: gene expression profiling high-risk (GEPhi), t(14;16), t(14;20), del(17p), amplification 1q21, primary plasma cell leukemia (pPCL), or elevated serum LDH (≥ 2X ULN). Median PFS was the primary endpoint, using a one-sided stratified log-rank test at a one-sided significance level of 0.1. Secondary endpoints included overall response rate (ORR), adverse events (AE), serious adverse events (SAE) and OS. Response was assessed using the IMWG 2009 criteria. Results: S1211 enrolled 103 evaluable patients, RVd n=54, RVd-Elo n=49. 74% had ISS II/III, 48% amp1q21, 38% del(17p), 11% t(14;16), 9% GEPhi, 7% pPCL, 5% t(14;20) and 4% elevated LDH (17% ≥1 feature). With median follow-up of 72 months (mos.), no difference in median PFS was observed [RVd-Elo=29 mos., RVd= 34 mos., HR = 1.11 (80% CI=0.82, 1.49, p=0.66]. No difference in OS was observed [RVd-Elo = median not reached (NR), RVd= 68 mos., HR = 0.85 (80% CI: 0.59, 1.23), p-value = 0.58]. 76% pts had ≥Grade 3 AEs, no differences in the safety profile were observed. Amongst patients with gain/amp 1q21, median PFS was [RVd-Elo=31 mos., RVd= 37 mos., HR = 1.48 (80% CI= 0.95, 2.31), p=0.25], median OS was [RVd-Elo = 61 mos., RVd= 68 mos., HR = 1.23 (80% CI: 0.72, 2.10), p-value = 0.63]. In patients with del(17p), median PFS was observed [RVd-Elo=41 mos., RVd= 30 mos., HR = 0.98 (80% CI= 0.60, 1.58), p=0.95], median OS was [RVd-Elo = NR, RVd= 72 mos., HR = 0.77 (80% CI: 0.40, 1.48), p-value = 0.61]. Summary/Conclusion: In the first randomized HRMM study reported to date, the addition of Elo to RVd induction and maintenance did not improve PFS and OS with a median follow-up of 6 years. Although the median PFS for Del17p subgroup on RVd-Elo arm is higher than RVd, it did not achieve statistical significance. The PFS and OS observed for gain/amp 1q21 and del17p in the RVd control arm may serve as important benchmarks for future enrichment design HRMM clinical trials. The PFS and OS in both arms of the study exceeded the original statistical assumptions and support the role for PI/IMiD combination induction/maintenance therapy for this population. P966: DARATUMUMAB, CARFILZOMIB, POMALIDOMIDE AND ELOTUZUMAB FOR THE TREATMENT OF POEMS SYNDROME- THE MAYO CLINIC EXPERIENCE I. Vaxman1,2,*, S. Kumar1, F. buadi1, M. Lacy1, D. Dingli1, A. Fonder1, M. Hobbs1, S. Hayman1, L. Y. hwa1, T. Kouralis1, R. Warsame1, E. Muchtar1, L. Nelson1, P. Kapoor1, M. Grogan1, R. Go1, W. Gonsalves1, M. Siddiqi1, K. Robert1, V. Rajkumar1, M. Gertz1, A. Dispenzieri1 1hematology, Mayo Clinic, Rochester, United States of America; 2hematology, Beilinson, Hod Hasharon, Israel Background: POEMS (Polyneuropathy, Organomegaly, Endocrinopathies, Monoclonal protein, Skin changes) syndrome is a rare paraneoplastic syndrome and therapies are directed against plasma cells that produce the proteins that cause this syndrome. Novel therapies are widely used in multiple myeloma aiming for plasma cell eradication. However, data on their use in POEMS syndrome are lacking. Aims: To provide the Mayo Clinic experience in treating 16 patients with relapsed POEMS syndrome with novel agents (daratumumab, carfilzomib, pomalidomide, and elotuzumab). Methods: We identified all POEMS patients seen at Mayo Clinic Rochester, Minnesota using a prospectively maintained database of patients seen at our center between June 1979 and May 2021. Of these patients, we identified all the patients that were treated with a “novel” agent, defined as daratumumab, carfilzomib, pomalidomide or elotuzumab. The primary endpoints were response to therapy (hematological, PET, VEGF and clinical, which will be referred to as responseH, P, V, C, respectively), and time to next therapy (TTNT), defined as time from institution of regimen of interest to next therapy. The secondary outcome was safety. Results: The median age at POEMS diagnosis was 57 years (range 39-79) and 15 patients (93%) were men. Among all patients at diagnosis, 10 (63%) had skin changes, 15 (94%) had signs of extravascular volume overload, and 15 (94%) patients had endocrine disorders. Ten and six patients had IgA and IgG isotypes, respectively, and all the patients had lambda light chain isotype. Radiological evidence for sclerotic lesions were found in 13 (81%) patients. The median time from diagnosis to novel agent’s first dose administration was 50 months (IQR 23-122) and the median lines of therapy prior to novel agent was 2 (range 1-4). Twelve patients (75%) underwent prior autologous stem cell transplantation (ASCT), and 5 patients had prior lenalidomide. The median age at novel agent administration was 63 years (IQR 51-70) and 4 patients (24%) were 70 years or older. The patients were treated with a a doublet including dexamethasone (N=5) (31%) or in various combinations with other agents: DRd (N=6), DC(V)d (N=3), KRd (N=3), KPd (N=1), DP(V)d (N=5), and EloRd (N=1). The outcomes with novel agent therapies were favorable (Table 1). Among patients treated with daratumumab based therapies (N=17), 9 patients achieved CR/VGPRH, 7 patients achieved CRV, and 5 patients achieved CRP. Among patients treated with carfilzomib-based therapies (N=6), 3 patients achieved CR/VGPRH and one achieved PRH. Only one patient treated with carfilzomib-based therapies achieved a clinical response. Neither patient who received pomalidomide and dexamethasone or elotuzumab with lenalidomide and dexamethasone responded. At a median follow-up of 38 months since starting of the novel agent (IQR 24-57), 15 of the patients (94%) are still alive, and the median TTNT was not reached. None of the patients discontinued therapy due to adverse events and no deaths occurred on therapy. Novel therapies were safe with 7 events of hospitalization due to pneumonia (4 in daratumumab-based therapies and 3 on carfilzomib-based therapies), and 4 patients were hospitalized due to volume overload (all received dexamethasone with therapy). Three patients experienced infusion-related reactions (IRR) to the first dose of IV daratumumab. Image: Summary/Conclusion: Response rate was high and the responses were deep. Novel agent therapies were safe, and no death case occurred on therapy. Future studies are needed to clarify the optimal sequence of novel agents and the best combination. P967: TUMOR BURDEN AS A CRITICAL PROGNOSTIC FACTOR OF PRIMARY EXTRAMEDULLARY DISEASE M. Vlachová1,*, M. Štork2, S. Ševčíková1, T. Jelínek3, J. Minařík4, J. Radocha5, P. Krhovská4, L. Pospíšilová6, I. Špička7, J. Straub7, P. Pavlíček8, A. Jungová9, V. Sandecká2, V. Maisnar5, R. Hájek3, L. Pour2 1Babak Myeloma Group, Department of Pathophysiology, Faculty of Medicine, Masaryk University; 2Clinic of Internal Medicine, Hematology and Oncology, University Hospital Brno, Brno; 3Department of Hematooncology, University Hospital Ostrava and Faculty of Medicine University of Ostrava, Ostrava; 4Department of Hemato-Oncology, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc; 54th Department of Internal Medicine – Hematology, Faculty Hospital and Charles University in Hradec Kralove, Hradec Kralove; 6Institute of Biostatistics and Analyses, Ltd., Brno; 71st Medical Department - Clinical Department of Haematology, the First Faculty of Medicine and General Teaching Hospital Charles University; 8Department of Internal Medicine and Hematology, University Hospital Kralovske Vinohrady, Prague; 9Hematology and Oncology Department, Charles University Hospital, Pilsen, Czechia Background: Multiple myeloma (MM) is the second most common hematological malignancy characterized by malignant plasma cell (PC) infiltration of the bone marrow (BM). In the case of extramedullary MM (EMD), plasma cells survive and proliferate outside of the bone marrow microenvironment. Primary EMD is found in newly diagnosed MM (NDMM) patients. Aims: The aim of this study was to describe clinical features and treatment outcomes of primary EMD patients and to define possible risk factors. Methods: In total, 724 primary EMD patients were diagnosed in the Czech Republic between 2004 and 2021 by using modern imaging methods. As a reference group, 2440 MM patients without any evidence of EMD were used. All patients enrolled into the analysis were treated by novel agents. Patients’ data were analyzed from the Registry of Monoclonal Gammopathies of the Czech Myeloma Group (RMG). Results: Primary EMD patients had less frequently advanced ISS (ISS 3) (OR 0.51 [95% CI: 0.42–0.63], p<0.001), high paraprotein levels (>20g/l) (OR 0.64 [95% CI: 0.54–0.76], p<0.001) and high PC infiltration of bone marrow (>10%) (OR 0.35 [95% CI: 0.30–0.42], p<0.001) when compared to reference MM patients. Median PFS in newly diagnosed primary EMD patients was comparable to reference MM patients (23.2 months [95% CI: 21.2–26.2] vs. 23.3 months [95% CI: 22.5–24.8], p=0.800). Median OS was comparable to reference patients (52.6 months [95% CI: 46.3–62.0] vs. 55.0 months [95% CI: 51.6–58.9], p=0.504). By multivariate analysis, adjusted for ISS, we found high levels of BM PCs (>10%) together with 3 and more EMD lesions as an independent risk factor for inferior survival in primary EMD patients (PFS: HR 1.92 [95 % CI: 1.28–2.87]; OS: HR 2.06 [95 % CI: 1.35–3.14], both p=0.001). Summary/Conclusion: We found primary EMD patients distinct from reference MM patients. Survival intervals of both groups of patients were comparable. We found that the intra- and extramedullary tumor burden is an independent key factor influencing primary EMD prognosis. This work was supported by grant AZV NU21-03-00076. P968: ADDITION OF IXAZOMIB TO POMALIDOMIDE AND DEXAMETHASONE IMPROVES PROGRESSION-FREE SURVIVAL FOR MULTIPLE MYELOMA PATIENTS PROGRESSING ON LENALIDOMIDE AS PART OF 1ST LINE THERAPY: ALLIANCE A061202 P. Voorhees1,*, V. Suman2, Y. Efebera3, N. Raje4, S. Tuchman5, C. Rodriguez6, K. Santo2, M. Bova-Solem7, D. Carlisle7, U. Saad8, P. McCarthy9, P. Richardson10 1Department of Hematologic Oncology and Blood Disorders, Levine Cancer Institute, Atrium Health, Charlotte; 2Alliance Statistics and Data Management Center, Mayo Clinic, Rochester; 3Department of Hematology and Oncology, Ohio Health, Columbus; 4Division of Hematology and Oncology, Massachusetts General Hospital, HArvard Medical School, Boston; 5Division of Hematology, Lineberger Comprehensive Cancer Center, the University of North Carolina, Chapel Hill; 6Tisch Cancer Institute, Mount Sinai School of Medicine, New York City; 7Protocol Operations Program, Alliance for Clinical Trials in Oncology, Chicago; 8Division of Hematologic Oncology, Memorial Sloan Kettering Cancer Center, New York City; 9Transplant and Cellular Therapy Program, Roswell Park Comprehensive Cancer Center, Buffalo; 10Department of Hematology and Oncology, Dana Farber Cancer Institute / Partners Cancer Care, Harvard Medical School, Boston, United States of America Background: Lenalidomide (LEN) maintenance and continuous LEN-based induction therapy until disease progression have become standard of care for frontline therapy of multiple myeloma (MM). As such, an increasing number of patients (pts) in need of 2nd line therapy have LEN-refractory disease. Optimal treatment in this setting has not been rigorously assessed in randomized studies. The phase I portion of Alliance A061202 demonstrated the safety of the ixazomib-pomalidomide-dexamethasone (IXA-POM-DEX) combination for the treatment of pts with LEN and proteasome inhibitor (PI)-refractory MM. Aims: In the randomized phase II portion, we evaluated the addition of IXA to POM-DEX for PI naïve / sensitive pts progressing on LEN as part of 1st line therapy. The primary endpoint was progression-free survival (PFS). Key secondary endpoints included overall response rate (ORR), depth of response, survival and safety. Methods: Pts were randomized 1:1 to IXA-POM-DEX or POM-DEX and stratified by prior bortezomib exposure, International Staging System stage (1 and 2 vs 3) and the presence of high-risk cytogenetics. POM was administered at 4 mg on days 1 – 21; IXA 4 mg on days 1, 8 and 15; and DEX 20 mg (>75 years (yrs)) or 40 mg (≤75 yrs) on days 1, 8, 15 and 22 of a 28-day cycle. Treatment was continued until disease progression, the emergence of unacceptable side effects or withdrawal of treatment consent. Results: 38 and 39 eligible pts were assigned to IXA-POM-DEX and POM-DEX, respectively. The median age was 66 yrs (range 41 – 83) and 64 yrs (range 52 – 85). A planned first interim analysis was conducted after 43 out of 57 required events had occurred. PFS favored the IXA-POM-DEX arm (one-sided log rank test value = 4.6345, p=0.03134 [< p-value boundary of 0.058]), yielding a hazard ratio of 0.528 (upper 90% bound = 0.777). A stratified log-rank test found that PFS was superior for the triplet after adjusting for stratification factors (one-sided stratified log rank test value = 5.8371; p=0.0157), adjusted hazard ratio 0.451 (upper 90% bound = 0.694). The ORR favored IXA-POM-DEX (63.2% vs 43.6%, p=0.0853), and the ≥very good partial response was 26.3% vs 5.1%, respectively (p=0.01). The clinical benefit rate (ORR + minimal response rate) was 73.7% and 56.4%. The most common grade 3/4 adverse events included lymphopenia, neutropenia, anemia, and fatigue in 40%, 37%, 16% and 16% of IXA-POM-DEX-treated pts and 26%, 21%, 13%, and 15% of POM-DEX-treated pts. Therapy was discontinued for disease progression in 47.4% of pts on IXA-POM-DEX and 76.9% of pts on POM-DEX and for adverse events in 7.9% and 7.7% of pts, respectively. Summary/Conclusion: The addition of IXA to the POM-DEX backbone improved the depth of response and PFS for pts relapsing on LEN as part of 1st line therapy. Hematologic toxicity was increased with the addition of IXA, but side effects were manageable. The ease of administration of this all-oral combination allowed for safer, uninterrupted treatment during the COVID pandemic. Our results should be confirmed in phase III trials but lend support for this regimen as part of 2nd line therapy for this patient population. P969: COMPARISON OF VENOUS THROMBOEMBOLISM INCIDENCE IN MULTIPLE MYELOMA PATIENTS RECEIVING LENALIDOMIDE-BASED REGIMENS WITH RIVAROXABAN OR ASPIRIN THROMBOPROPHYLAXIS Y. Wang1, X. Tang2, W. Zheng1, Z. Wang1, J. Xu1, X. Tan2, Y. Tian2, R. Xu1, S. Cui1,* 1Affiliated Hospital of Shandong University of Traditional Chinese Medicine; 2Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China Background: Thromboprophylaxis is routinely used with lenalidomide-based regimens in multiple myeloma because of a substantial risk of venous thromboembolism (VTE). Aspirin is one of the most common thromboprophylaxis agent but also increases bleeding complications.Rivaroxaban represents a selective direct inhibitor of activated coagulation factor X (FXa) having peroral bioavailability and prompt onset of action which can mitigate the risk of higher incidence of VTE without an observable increase in bleeding rates.However, little is known about the incidence of VTE in lenalidomide-based regimens with rivaroxaban thromboprophylaxis. Aims: The purpose of our study was to compare of venous thromboembolism incidence in multiple myeloma patients receiving lenalidomide-based regimen with rivaroxaban or aspirin thromboprophylaxis. Methods: We conducted a single-centre retrospective study designed to assess the rates of VTE, including pulmonary embolism (PE) and deep vein thrombosis (DVT), in MM patients receiving lenalidomide-based regimen with aspirin or rivaroxaban thromboprophylaxis.A total of 130 patients who were not anticoagulated for other clinical indications and received at least 1 cycle of lenalidomide-based regimen between January 2015 and December 2021 were included. Clinical bleeding events after treatment were also important indicators in our study. Results: The incidence of venous thromboembolism in the rivaroxaban group was significantly lower than that in the aspirin prevention group (P<0.05). The incidence of bleeding in the rivaroxaban group was also lower than that in the aspirin prevention group,but there was no statistical significance(P﹥0.05). Table II. Analysis of VTE and Bleeding. Regimen + prophylaxis R-based regimens+ASA（n=84） R-based regimens+XA（n=46） P-value VTE 13（15.5%） 1（2.2%） 0.041 Bleeding 6（7.1%） 0 0.081 ASA, aspirin; XA, rivaroxaban; VTE, venous thromboembolism. Image: Summary/Conclusion: In this pilot study, we found that rivaroxaban was safe and well-tolerated as the primary preventive measure for venous thromboembolism in patients with multiple myeloma receiving lenalidomide-based regimen. However, these findings warrant further investigation in a larger randomized study to be validated. P970: SAFETY AND EFFICACY OF BCMA-CAR-T IMMUNE CELLS DERIVED FROM CORD BLOOD IN THE TREATMENT OF RELAPSED AND REFRACTORY MULTIPLE MYELOMA Y. Wang1, X. Hu1, Q. Gao1, H. Wang1, Y. Gao1,*, W. Zhang1, X. Ru1, L. Hou1, W. Zhou1, H. Zhang1, Y. Zhang1, F. Wang2, X. Li3, F. Guan3 1Department of Hematology, Shaanxi Provincial People’s Hospital, Xi’An; 2School of Medicine, Shanghai Jiao Tong University, Shanghai; 3School of Medicine, Northwest University, Xi’An, China Background: Multiple myeloma (MM) is the second most prevalent hematologic malignancy. The prognosis of MM patients varies, with low-risk patients surviving more than 10 years with standard care and high-risk patients surviving less than 1 year. CAR-T therapy shows promising clinical results on relapsed or refractory MM (R/RMM) patients. However, generating clinically significant doses of CAR-T cells from heavily pre-treated patients for autologous CAR-T therapy is not always possible. Although allogeneic CAR-T cells therapy could potentially overcome such limitations, it poses a considerable risk of graft-versus-host disease (GVHD). Cord blood (CB) is a convenient source of T cells with clear advantages. T cells in CB have low immunogenicity and a relatively low risk of GVHD. Here we present a novel approach to CAR-T cells production from CB, which we hope could overcome the constraints mentioned above. Aims: The study aims to evaluate the safety and efficacy of the CB-generated BCMA-CAR-T infusion in R/RMM patients. Methods: This study is based on a clinical investigation during Jan 2021 and Dec 2021 in Shaanxi Provincial People’s Hospital. In this study, CD3+ T cells from the CB samples were selected, activated, and modified by lentivirus to produce anti-BCMA CAR-T cells. The cells were administrated intravenously to patients with R/RMM after expanding for 5-10 days in vitro. 2-3 days after being administered the lymphodepleting chemotherapy regimen of cyclophosphamide and fludarabine, patients received CAR-T cells infusion. We followed the occurrence of adverse events, the level of inflammatory cytokine concentration, CAR-T cells expansion in the peripheral blood, serum monoclonal protein levels, and the proportion of plasma cells in bone marrow smears after CAR-T cells infusion. Results: This study included 11 patients with R/RMM (7 males and 4 females). The median age was 58 years (ranging from 44 to 65). 18.2% (2/11) of the patients had received other CAR-T therapies and relapsed before the enrolment. 72.73% (8/11) of the patients had the extramedullary disease (EMD). 63.64% (7/11) of the patients had received autologous hematopoietic stem cell transplantation (Auto-HSCT). All patients received CAR-T infusion at the average dose of 7.11 (4.4-15) ×106/kg. After infusion, 18.2% (2/11) of the patients had a fever lasting for 48 hours, and 18.2% (2/11) of them had an increase in heart rate. Nausea and diarrhea occurred in 18.2% (2/11) of the patients. No patients had transient consciousness disorder. No patients received dexamethasone to relieve symptoms. CAR-T cells had expanded in all patients, with no increase in the levels of IL-6, IL-8 and IFN-r in peripheral blood in 2 weeks after infusion. Grade 1 cytokine release syndrome (CRS) was found in 2 patients. No patients had GVHD after infusion. The ORR was 54.5% (6/11), with 18.2% (2/11) of the patients achieving CR, and 36.4% (4/11) achieving PR. Except 1 female died from severe pulmonary infection, other 10 patients were followed up for more than 1 month. Unfortunately, 1 patient who reached CR relapsed during 3 months of follow-up. Those 4 patients who didn’t achieve PR received autologous CAR-T therapy afterwards and had responses better than PR (1 CR, 1 VGPR, 2 PR). Summary/Conclusion: BCMA-CAR-T cells manufactured from CB show a promising safety profile and certain clinical efficacy for R/RMM patients who are not eligible for autologous CAR-T cells therapy. We also found allogeneic CAR-T cells manufactured from CB could prepare patients for later subsequent autologous CAR-T therapy. P971: ADJUSTED COMPARISON OF PATIENT REPORTED OUTCOMES FROM CARTITUDE-1 AND LOCOMMOTION COMPARING CILTACABTAGENE AUTOLEUCEL VERSUS REAL WORLD CLINICAL PRACTICE IN TRIPLE-CLASS EXPOSED MULTIPLE MYELOMA K. Weisel1,*, M.-V. Mateos2, L. Vincent3, T. Martin4, J. G. Berdeja5, A. Jakubowiak6, S. Jagannath7, Y. Lin8, P. Thilakarathne9, F. Ghilotti10, J. Diels9, B. Haefliger11, C. Hague12, A. Gonzalez11, J. M. Schecter13, K. S. Gries14, V. Strulev15, T. Nesheiwat16, L. Pacaud16, H. Einsele17, P. Moreau18 1Oncology, Hematology and BMT, University Medical-Center Hamburg-Eppendorf, Hamburg, Germany; 2CIC, University Hospital of Salamanca/IBSAL, Salamanca, Spain; 3Clinical Haematology Department, Montpellier University Hospital, Montpellier, France; 4UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco; 5Sarah Cannon Research Institute, Nashville; 6University of Chicago, Chicago; 7Mount Sinai Medical Center, New York; 8Mayo Clinic, Rochester, United States of America; 9Health Economics, Market Access & Reimbursement, Janssen Pharmaceutica NV, Beerse, Belgium; 10Health Economics, Market Access & Reimbursement, Janssen-Cilag SpA, Cologno Monzese, Italy; 11Health Economics, Market Access & Reimbursement, Cilag GmbH International, Zug, Switzerland; 12Health Economics, Market Access & Reimbursement, Janssen, High Wycomb, United Kingdom; 13Research and Development, Janssen R&D, Raritan; 14Research and Development, Janssen R&D, Los Angeles, United States of America; 15EMEA Medical Affairs, Janssen Pharmaceutica NV, Beerse, Belgium; 16Legend Biotech USA, Piscataway, United States of America; 17Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany; 18Clinical Hematology, University Hospital Hotel-Dieu, Nantes, France Background: Adjusted comparisons of the single-arm CARTITUDE-1 clinical trial of ciltacabtagene autoleucel (cilta-cel) and the prospective LocoMMotion study of therapies used in real-world clinical practice (RWCP) have shown the clinical benefits of cilta-cel on response and survival outcomes in patients with triple-class exposed multiple myeloma (TCE-MM). Yet, patients with TCE-MM have a reduced health-related quality of life (HRQoL) compared with age- and sex-matched populations. Aims: To present adjusted comparisons of patient-reported outcomes (PROs) from TCE-MM patients who received cilta-cel in CARTITUDE-1 vs. RWCP in LocoMMotion. Methods: EQ-5D-5L, EORTC QLQ-C30 and EORTC QLQ-MY20 questionnaires were administered to all patients in the CARTITUDE-1 Phase 2 and LocoMMotion studies at baseline, day 7, day 28 and every 4 weeks up to 52 weeks. Mixed model repeated measures (MMRM) analyses were performed to analyze changes from baseline (CFB) for each patient cohort and the difference in CFB between cilta-cel and RWCP over time. MMRM models included the baseline PRO score and prognostic characteristics as covariates to balance patient cohorts and to adjust for confounding bias. A sensitivity analysis was implemented assigning worst PRO values to patients who dropped out of the analyses due to death. Results: A total of 61 patients in CARTITUDE-1 and 202 patients in LocoMMotion had PRO assessments at baseline and during follow-up. At day 7, PRO scores worsened versus baseline in both cohorts, and worsening was more pronounced for cilta-cel in physical, role and social functioning, fatigue and lack of appetite and constipation, coinciding with short term adverse events associated with the cilta-cel infusion and lymphodepleting chemotherapy. From the next assessment at week 4 onwards, PRO values significantly improved over time for cilta-cel patients versus baseline, while improvement from baseline was lower for RWCP for most domains and symptoms. The average improvement versus baseline from week 4 onwards, represented by the absolute difference in CFB between both patient cohorts, was significantly in favor of cilta-cel for Visual Analogue Scale (8.0), Global health status (8.5), pain (-11.4), dyspnea (-8.9), constipation (-8.3), future perspective (16.5) (all p<0.01), emotional functioning (7.4) and feeling restless or agitated (-7.2) (p<0.05) (Table). All other PROs were numerically in favor of cilta-cel across the follow-up period, except for nausea and vomiting and lack of appetite, for which small differences in improvement are numerically in favor of RWCP. The sensitivity analyses assigning worst PRO values to patients who dropped out of the main analyses due to death illustrate that these results are inherently biased against the more effective treatment on survival and underestimate the PRO benefit for cilta-cel. Image: Summary/Conclusion: Patients with TCE-MM treated with cilta-cel demonstrated significant improvements vs. RWCP increasing over time in multiple PRO endpoints. These findings indicate that cilta-cel can significantly improve patients’ HRQoL in addition to significant efficacy benefits on response, progression free and overall survival and can help address unmet patient needs. P972: INDIRECT COMPARISON OF TECLISTIMAB IN MAJESTEC-1 VERSUS PHYSICIAN’S CHOICE OF THERAPY IN LONG-TERM FOLLOW-UP OF TRIPLE-CLASS EXPOSED RELAPSED/REFRACTORY MULTIPLE MYELOMA IN DARATUMUMAB TRIALS K. Weisel1,*, A. Chari2, S. Z. Usmani3, H. Goldschmidt4, M.-V. Mateos5, K. Qi6, A. Londhe6, S. Nair7, X. Lin8, L. Pei9, E. Ammann10, R. Kobos9, J. Smit11, T. Parekh12, M. Slavcev10, P. Moreau13 1University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2Mount Sinai School of Medicine; 3Memorial Sloan Kettering Cancer Center, New York, United States of America; 4University Hospital Heidelberg and National Center of Tumor Diseases, Heidelberg, Germany; 5University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca, Spain; 6Janssen Research & Development, Titusville, United States of America; 7Janssen Pharmaceutica NV, Beerse, Belgium; 8Janssen Global Services, Horsham; 9Janssen Research & Development; 10Janssen Global Services, Raritan; 11Janssen Research & Development, Spring House; 12Janssen Research & Development, Bridgewater, United States of America; 13Hematology Clinic, University Hospital Hotel-Dieu, Nantes, France Background: Teclistamab (tec), a B-cell maturation antigen × CD3 bispecific antibody, is currently being investigated in MajesTEC-1, a phase 1/2, single-arm trial (NCT04557098) in patients with triple-class exposed relapsed/refractory multiple myeloma (TCE RRMM) who had received ≥3 prior lines of therapy (LOT) and previously exposed to an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 monoclonal antibody. Currently, there are no head-to-head trials comparing the efficacy of tec versus physician’s choice of therapy. Aims: To evaluate the comparative efficacy of tec versus physician’s choice of therapy. Methods: Patients in the long-term follow-up of 4 clinical trials of daratumumab (CASTOR, POLLUX, EQUULEUS, and APOLLO) who met the eligibility criteria for MajesTEC-1 were used to create an external control arm for MajesTEC-1. After discontinuation of trial treatments, patients (N=427) subsequently received physician’s choice of therapy, with disease progression and best treatment response assessed by the investigator. The MajesTEC-1 cohort included individual patient-level data from patients who received tec (1.5 mg/kg weekly) at a clinical cutoff of Sep 7, 2021. Inverse probability of treatment weighting with average treatment effect on the treated population was used to adjust for imbalances in baseline covariates of prognostic significance such as International Staging System stage, number of prior LOT, extramedullary plasmacytoma, time since diagnosis, age, hemoglobin, cytogenetic risk, refractory status and progression on last LOT. Efficacy assessments included overall response rate (ORR), rate of complete response or better (≥CR), rate of very good partial response or better (≥VGPR), progression-free survival (PFS), time to next treatment (TTNT), and overall survival (OS) were assessed. For binary endpoints (ORR, ≥CR rate, ≥VGPR rate), odds ratio (OR) along with 95% confidence interval (CI) derived from a weighted logistic regression analysis was used to estimate the relative effect of tec vs physician’s choice of therapy. Hazard ratios (HRs) and 95% CIs were computed using a weighted Cox proportional hazards model for time-to-event endpoints (PFS, OS, and TTNT). Multiple sensitivity analyses were also conducted. Results: Baseline characteristics were comparable between the 2 cohorts after applying inverse probability of treatment weighting. Patients treated with tec had improved outcomes vs physician’s choice of therapy: ORR (OR 4.58; 95% CI 2.83–7.53; P <0.0001); ≥CR rate (OR 12.62; 95% CI 5.20–38.55; P<0.0001); ≥VGPR rate (OR 11.64; 95% CI 6.49–21.98; P<0.0001); PFS (HR 0.62; 95%CI 0.45–0.84; P=0.0024); TTNT (HR 0.38; 95% CI 0.27–0.52; P<0.0001); and OS (HR 0.47; 95% CI 0.32–0.69; P=0.0001). All sensitivity analyses showed similar results. Summary/Conclusion: In the present analysis, improved efficacy in all clinical outcomes was observed with tec versus physician’s choice of therapy, highlighting the therapeutic potential of tec to address unmet needs in patients with TCE RRMM who received ≥3 prior LOT. P973: REAL-WORLD STUDY ON ADOPTION OF STANDARD OF CARE (SOC) FOR FRONTLINE TRANSPLANT-ELIGIBLE MULTIPLE MYELOMA (FLTEMM) PATIENTS BETWEEN 2017 AND 2020/2021 ACROSS FRANCE, GERMANY, SPAIN, AND ITALY K. Weisel1,*, A. Wadlund2, G. Gungor3, E. Dergarabetian4, C. Pacheco5, N. Masurkar5, P. Rodriguez-Otero6 1Oncology, Hematology and BMT, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2Janssen, Janssen-Cilag AB, Kolonnvägen 45, Solna, Sweden; 3Janssen, Kavacik, Keçeli Plaza, Ertürk Sk. No:13, 34810 Beykoz, Istanbul, Turkey; 4Janssen-Cilag Limited, 50-100 Holmers Farm Way, High Wycombe, Buckinghamshire, United Kingdom; 5Cerner Enviza, France SAS, 198 avenue de France, Paris, France; 6Department of Hematology, Clinica Universidad de Navarra, Pamplona, Spain Background: Newer therapies for frontline transplant-eligible multiple myeloma (FLTEMM) are underway, but an accurate and updated overview of standard MM treatment management is lacking. Repeated analyses and real-world data are warranted to describe current MM standard of care (SoC), treatment lines, and patient clinical outcomes in daily practice. Aims: This non-interventional, cross-sectional, retrospective observational database study described the current SoC for FLTEMM patients in France, Germany, Spain, and Italy, and recorded the evolution in regimen adoption in distinct elements of frontline treatment (induction, consolidation, and maintenance) during 2017-2020/2021. Methods: Clinical information on ongoing (population I) or previous (population II) FLTEMM patients was extracted from the Cancerology database. The primary objective was to describe the treatment regimen considered current SoC for FLTEMM management in all four countries using study population I, and to study the evolution in the use of different regimens in induction phase using study population II. The secondary objective was to describe clinical outcomes, including best tumor response and minimal residual disease (MRD) status, associated with each induction regimen in study population II. Results: In study populations I and II, the median (min, max) age of the patients at induction initiation was 59.0 (35.0, 86.0) and 60.0 (23.0, 80.0) years and 62.9% (227/361) and 64.8% (287/443) patients were male, respectively. In study population I, the most common induction regimens were bortezomib/lenalidomide/dexamethasone (VRd) in France (75.3%, 33/44) and Spain (44.1%, 53/120), bortezomib/thalidomide/dexamethasone (VTd) in Italy (65.2%, 76/116), and others in Germany (58.9%, 48/81) (including daratumumab-based [19.8%, 16/81] or not). Maintenance was ongoing or planned for 78.3% (34/44), 62.3% (51/81), 65.2% (78/120), and 61.4% (71/116) patients in France, Germany, Spain, and Italy, respectively. Among study population I patients on an ongoing maintenance regimen, lenalidomide was received by 99.6% (20/20) patients in France, 86.8% (34/40) in Germany, 80.2% (45/56) in Spain, and 90.7% (30/33) in Italy. In study population II, VRd use as 1L induction increased from 27.0% (10/36) in 2017 to 65.7% (12/18) in 2019 in France and was relatively low in Germany, Spain, and Italy. The most common 1L induction regimen in Germany was VCd, but its use decreased from 85.2% (31/37) in 2017 to 64.1% (24/38) in 2019. VTd was the most common 1L induction in Spain and Italy, but its use declined from 58.3% (16/27) and 72.4% (34/47) in 2017 to 17.3% (4/22) and 52.8% (19/36) in 2019, respectively. In total, 37.5% (166/443) study population II patients achieved at least a complete response, which was the highest for those on VRd (59.2% versus 34.3% for VTd, 33.4% for VCd, and 33.7% for other non-daratumumab-based regimens, respectively). In total, 42.5% (188/443) study population II patients were tested for MRD status and 18.2% (81/443) were found negative. MRD testing rate varied from 26.2% in France to 14.8% in Germany, 84.4% in Spain, and 47.7% in Italy. Summary/Conclusion: The use of bortezomib triplets in induction varied markedly over time and between selected countries. Despite not being approved by the European Medicines Agency specifically for FLTEMM patients, VRd use tended to increase with time in France and to a lesser extent in Spain and Italy in this population. P974: THE CLINICAL CHARACTERISTICS AND PROGNOSIS OF PATIENTS WITH PRIMARY PLASMA CELL LEUKEMIA (PPCL) UNDER THE NEW IWMG DEFINITION CRITERIA W. Yan1,*, J. Xu1, H. Fan1, J. Liu1, L. Li1, L. Qiu1, G. An1 1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China Background: Primary plasma cell leukemia has been described as a distinct entity of plasma cell disorders with special features and poor prognosis. In 2021, IMWG proposed that revised the diagnostic criteria of PCL as CPCs ≥ 5% by peripheral blood (PB) smear. As the new diagnostic standard has been established recently, it is of great significance that illustrate the clinical characteristics and outcomes of the new pPCL cohort. Aims: Given the limited data for the rare entity, we performed a large retrospective analysis about the clinical characteristics, survival outcomes, and risk factors for the new-defined pPCL patients treated in our hospital. Methods: We conducted a retrospective analysis of 158 pPCL patients diagnosed from 2000 to 2019 in our hospital. This pPCL cohort was redefined from NDMM patients who appear CPCs ≥ 5% by morphologic evaluation of their PB smear. And we compared them to a control group with 485 NDMM patients. Statistical analyses were performed using the R version 4.1.2. Results: The characteristics and comparisons between pPCL patients and NDMM group are depicted in Table 1.In general, bone marrow suppression and adverse prognostic biomarkers (ie, anemia, thrombocytopenia, elevated LDH, hypodiploidy, and high-risk cytogenetics) were more common in pPCL patients compared with NDMM patients (P<0.05). For 130 pPCL patients whose treatment and prognosis data were available, with a median follow-up time of 54.7 months, the median PFS and OS were 16.9 months and 30.0 months respectively, both significantly longer than the control NDMM patients (P<0.001). Interestingly, we find the cohort who attended after 2007 got obvious longer PFS than those who received treatment in 2000-2006(20.6 vs 10.0 months, P=0.017), which showed a similar result in OS (31.6 vs 16.0 months, P=0.034). A Cox-regression multivariate analysis was performed among the baseline variables. The presence of hypodiploidy and elevated serum LDH were found to be prognostic for worse PFS, whereas age＞60 and elevated LDH were the independent predictors for worse OS. As for the cytogenetic aberrations, did not play an important role in the outcome of pPCL patients other than the presence of del(17p) which could impact OS in the univariate analysis. There are 98 patients who had response data known, with 80(81.6%) patients achieving objective response in the first-line treatment, 56.4%≥VGPR, and 38.9% CR. When stratifying the cohort by the best response, the median PFS for patients achieved: NR, PR, and ≥VGPR were 2.4, 11.2, and 31.0 months, respectively (P＜0.001). The OS for patients who had ever achieved NR, PR, and≥VGPR were 2.4, 24.9, and 62.1 months, respectively (P＜0.001). Then, we divided the cohort who had achieved≥PR into early response group (≤2 courses), intermediate response group (3-4 courses), and late response group (＞4 courses). Whereas, there is no statistical difference in the prognosis of 3 groups with diverse remission speed rates (PFS: P=0.35; OS: P=0.77). We also found that achieving deep remission was the best independent favorable predictor for long survival in the multivariate analysis. Image: Summary/Conclusion: In conclusion, primary plasma cell leukemia (pPCL) defined by the new revised diagnostic criteria (CPCs≥5%), remains an aggressive disease characterized with poor prognosis despite the advancement of treatment regimens. And achieving deep remission (≥VGPR) in the first-line therapy predicts the best prognosis regardless of response rate. P975: INTERIM ANALYSIS OF PHASE II STUDY OF DARATUMUMAB IN COMBINATION WITH BORTEZOMIB AND DEXAMETHASONE IN PATIENTS WITH MULTIPLE MYELOMA WHO RECEIVED 1 PRIOR LINE OF THERAPY (KMM1906) K. H. Yoo1,*, G. W. Gang2, J. H. Yi2, M. K. Kim3, H. J. Kim4, S.-H. Kim5, J. S. Park6, J.-J. Lee7, C.-K. Min8, J. H. Lee1, D. Cho9, K. Kim10 1Division of Hematology, Department of Internal Medicine, Gachon University Gil Medical Center, Gachon University College of Medicine, Incheon; 2Division of Oncology and Hematology, Department of Internal Medicine, Korea University Medical Center, Seoul; 3Department of Internal Medicine, Yeungnam University College of Medicine, Daegu; 4Division of Hematology/Oncology, Department of Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang; 5Department of Internal Medicine, Dong-A University College of Medicine, Busan; 6Department of Hematology-Oncology, Ajou University School of Medicine, Suwon; 7Department of Hematology-Oncology, Chonnam University Hwasun Hospital, Hwasun; 8Department of Internal Medicine, Seoul St. Mary’s Hospital, The Catholic University of Korea; 9Department of Laboratory Medicine and Genetics; 10Division of Hematology and Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea Background: Daratumumab in combination with bortezomib and dexamethasone (DVd) demonstrated efficacy in heavily pretreated patients with relapsed or refractory multiple myeloma (MM). In a previous pivotal phase 3 study, DVd showed better efficacy in patients receiving only first-line treatment, while the limited administration of bortezomib and dexamethasone for 8 cycles was criticized. Aims: We conducted a phase 2 study with DVd regimen in patients with MM who received 1 prior line of therapy. Methods: We evaluated daratumumab (16 mg/kg IV), bortezomib (1.3 mg/m2 SQ), and dexamethasone (20 mg IV or PO) with maintaining all three drugs after 9 cycles until disease progression or unacceptable toxicity. The primary endpoint was objective response rate ≥ very good partial response (VGPR). Secondary objectives included progression-free survival (PFS), overall survival (OS), safety and tolerability, and minimal residual disease (MRD)-negativity. MRD was assessed in patients who achieved in stringent complete response (sCR) or CR, and who maintained VGPR over 6 months, with EuroFlow-based next-generation flow (NGF). Results: From June 2020 to June 2021, 26 MM patients who received 1 prior line of therapy and disease progressed were enrolled at 10 centers in Korea. The median age of all patients was 72 years (range, 47-85), and 8 patients were male. Five patients (19%) had high-risk cytogenetic abnormalities (t(4;14), t(14;16), or del17p), and 6 patients (23%) had extramedullary disease. All patients were treated with 1 prior line, including VTD (N = 11, 42%), VMP (N = 8, 31%), Rd (N = 6, 23%), and CMP (N = 1, 4%). Nineteen patients (73%) and 18 patients (69%) were exposed to bortezomib and immunomodulatory drugs, respectively, from previous treatment (Table 1). During the median follow-up period of 11.7 months, 15 patients (58%) maintained treatment, and 11 patients (42%) discontinued the study due to disease progression (N = 6), death from other causes (N = 2), or withdrawal of consent (N =3). 16 patients (62%) achieved ≥ VGPR (2 sCR, 7 CR, and 7 VGPR). The MRD-negativity (10-5) was 5/12 (42%) (Table 2). The treatment response and PFS of each patient were given in the swimmer plot (Figure 1). The most common adverse event (AEs) ≥ grade 3 was thrombocytopenia (N = 6, 23%). Serious AEs were reported in 8 patients (31%). Dose delay or dose reduction have occurred in 17 patients (65%). Table 2. Response and MRD-negativity rates Response category No. with response (%) CR or better 9 (35%) sCR 2 (8%) CR 7 (27%) VGPR or better 16 (62%) VGPR 7 (27%) PR 6 (23%) MR 2 (8%) SD 2 (8%) PD 0 MRD-negativity (10-5) N = 12 Negative 5 (42%) Image: Summary/Conclusion: Among patients with MM who received 1 prior line of therapy, DVd regimen with maintenance strategy showed an acceptable clinical response and MRD-negativity with manageable toxicity profile. P976: BENDAMUSTINE-POMALIDOMIDE-DEXAMETHASONE (BPD) FOR RELAPSED AND/OR REFRACTOR MULTIPLE MYELOMA WITH EXTRAMEDULLARY DISEASE Z. Yuping1,*, H. Y. Wu1, X. Chu2, X. Deng3, X. Feng4, C. Yuan5, X. Ran6, G. Liu7, C. Fan8, H. Hao5, X. Zhou1 1Qingdao Municipal Hospital, Qingdao City, Shandong Province; 2The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai City, Shandong Province; 3Weihai municipal hospital, Weihai City, Shandong Province; 4The Affiliated Hospital of Qingdao University (West Coast Hospital area), Dongying City, Shandong Province; 5QILU HOSPITAL OF SHANDONG UNIVERSITY(Qingdao), Qingdao City, Shandong Province; 6Weifang people’s Hospital, Weifang City, Shandong Province; 7Shengli Oilfield Central Hospital, Dongying City, Shandong Province; 8Department of Hematology, Qingdao Hospital of Traditional Chinese Medicine (Qingdao Hiser Hospital), Qingdao City, Shandong Province, China Background: Extramedullary disease (EMD) is a rare manifestation of multiple myeloma (MM). The incidence is about 6-20% in the relapsed MM patients. Although novel agents prolong the survival of patients with MM, patients with EMD are prone to relapse or progress again easily,EMD may be associated with decreased overall survival in MM. No standard therapy has been established for this high-unmet need population. Bendamustine is an old bi-functional alkylating agent which has proved to be effective in Multiple Myeloma (MM). Pomalidomide is a potent immunomodulatory drug agent with anti-angiogenic, anti-proliferative, and immunomodulatory activity against MM. Preclinical studies showed that bendamustine and pomalidomide had higher penetration rate of cerebrospinal fluid. In China, the price of two drugs is relatively economical. In a phase I/II study,the combination of BPd demonstrates deep Response(ORR:61%) and has a favorable tolerance profile in patients with RRMM. To summarize,BPD is an safe,effective, and economical treatment, and there are no studies of BPD in the regimen of RRMM with EMD,so it is worth exploring in domestic clinical trials. Aims: To evaluate the efficacy and safety of bendamustine, Pomalidomide, and dexamethasone (BPd) for relapsed / refractor multiple myeloma with EMD. Methods: This is an open-label, multicenter, prospective study（ChiCTR2100048829）.Eligible patients were aged≥18 years, and had a diagnosis of RRMM with EMD, and an Eastern Cooperative Oncology Group performance status of 0-2,Patients received bendamustine 75 mg/m2 on days 1 and 2(If there is no severe side effects in the first cycle, it can be increased to 80mg/m2), 6 cycles. Pomalidomide 4 mg on days 1-21,until progression or intolerable toxicity. Dexamethasone 20mg on days1-4, 15-18, until progression or unacceptable toxicity. Each cycle is 28days. The primary endpoint is overall response rates (ORR). Results: A total of 21 patients were enrolled in the study,12(57.14%) patients with first relapse multiple myeloma. The median age is 57.4years(47-72). According to IMWG criteria, all first relapse patients responded(ORR100%)and CR:3pts(25%),VGPR:7pts(58.33%),PR:2pts(16.67%).Patients with ≥2nd relapese, the ORR was44.44%,≥VGPR:0pts(0%), PR:4pts(44.44%),SD:3pts (33.33%),PD:2pts(22.22%).This results established that BPD has a deep Response in first relapse MM with EMD.The 1-year PFS was79.16%of all patients.The most common grade 3 or 4 hematologic toxicities were neutropenia7(33.33%),thrombocytopenia4(19.05%),anemia 2(9.52%). Overall,the severe toxicities are manageable. Table 1. Patient characteristics Age, years, median (range) 57.4(47-72) ECOG performance status(n,%) ISS disease stage(n,%) Number of prior therapies,median (range)(n,%) 0 4 19.05% I 2 9.52% 1 12 57.14% 1 10 47.62% II 3 14.29% ≥1 9 42.86% 2 7 33.33% III 16 76.19% Table 2. International Myeloma Working Group best response IMWG first relapse(n，%) ≥2nd relapese(n，%) ALL(n，%) ORR 12 100.00% 4 44.44% 16 76.19% SCR 0 0.00% 0 0.00% 0 0.00% CR 3 25.00% 0 0.00% 3 14.29% VGPR 7 58.33% 0 0.00% 7 33.33% PR 2 16.67% 4 44.44% 6 28.57% SD 0 0.00% 3 33.33% 3 14.29% PD 0 0.00% 2 22.22% 2 9.52% Image: Summary/Conclusion: Safety data from the study demonstrates that BPD regimen has a favorable tolerance profile in patients for RRMM with EMD. Early efficacy is encouraging, Longer-term results still require more patients have been enrolled and further follow-up. The combination of BPD will be an effective treatment strategy in RRMM with EMD. P977: BISPECIFIC CS1-BCMA CAR-T CELLS ARE CLINICALLY ACTIVE IN RELAPSED OR REFRACTORY MULTIPLE MYELOMA C. Li1,2, X. Wang1,2, Z. Wu1,2, W. Luo1,2, Y. Zhang1,2, M. Du1,2, C. Lu1,2, H. Kou1,2, Y. Kang1,2, J. Xu1,2, P. Huang1,2, W. Xiong3, J. Zheng1, J. Deng1, Y. Hu1,2, H. Mei1,2,* 1Institute of Hematology, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology; 2Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease; 3Wuhan Sian Medical Technology Co., Ltd, Wuhan, China Background: BCMA-targeted CAR-T cell therapy has showed high response rates in multiple myeloma (MM); however, remission is transient in most of patients (pts). Variable and even absent BCMA expression on MM cells have been documented after single BCMA-targeted CAR-T cell treatment. CS1 (CD319, SLAMF7) plays a vital role in myeloma pathogenesis, including promoting MM cells growth, survival and adhesion. CS1 is highly expressed on tumor cells in almost all MM pts, and is also retained at significant levels at relapse. Therefore, we propose to augment BCMA targeting with CS1. Bispecific CS1-BCMA CAR-T cells are effective in targeting MM cells in preclinical studies (Biomedicines 2021, 9, 1422). Aims: Here we report the outcomes of 13 pts with refractory or relapsed (RR) MM in our phase I clinical trial (NCT 04662099). Methods: CS1-BCMA bispecific CAR contained a murine anti-CS1 scFv (clone 7A8D5) and a murine anti-BCMA scFv (clone 4C8) in tandem, and a 4-1BB costimulatory domain (Figure 1A). The enrolled pts must have received at least 2 prior lines of therapy, and previous BCMA- or CS1-targeted immunotherapies were allowed. Pts were subjected to lymphodepleting regimens with cyclophosphamide (250 mg/m2, d-5 to d-3) and fludarabine (30 mg/m2, d-5 to d-3) daily prior to the CAR-T infusion (d0). Planned dose levels were 0.75, 1.5, and 3.0x106 CAR+T cells/kg, and repeated infusions were allowed. Primary objectives were incidence of adverse events. Cytokine release syndrome (CRS) and neurotoxicity were graded using the ASTCT criteria, and other AEs using CTCAE v5.0. Secondary objectives were overall response rate(ORR), overall survival(OS), duration of response (DOR), and progress-free survival (PFS). Response was assessed per the IMWG criteria (2016). Other objectives included in vivo kinetics of CAR-T cells. Results: As of February 10, 2022, 13 pts received the infusion of CS1-BCMA CAR-T cells and were included in the final analysis. Six pts (46%) carried cytogenetic abnormalities, and 3 pts had only extramedullary diseases (EMD) without detectable MM cells in bone marrow (BM). BCMA and CS1 were highly expressed on MM cells in BM (median BCMA+ 77.2% and CS1 + 96.5%) and EMD (Figure 1B). Eight pts (62%) were refractory, and median prior lines of treatment was 5.5 (range 2-10), and 7 pts (54%) received autologous stem cell transplantation. Four pts (31%) experienced CRS, and grade 3 CRS occurred once and lasted for 5 days. Neurotoxicity was not observed. The ORR is 76.9% (10/13), including 4 stringent complete, 2 very good partial, and 4 partial response (Figure 1C). Although CAR-T cells infiltrated into the tumor tissue, response was not observed in the 3 pts with only EMD (Figure 1D). Soluble BCMA decreased remarkably in peripheral blood (PB) and BM (Figure 1E). The median follow-up and DOR were 290 days. The median OS and PFS weren’t reached, and OS and PFS at 9-month were 77.1%. On day 14 after infusion, CAR-T cells peaked at 464842 copies/ug DNA in PB (n=13) and 282920 copies/ug DNA in BM (n=10) by droplet digital PCR; CAR-T cell expanded at 400 CAR+T cells/ul PB (n=13) and 271 CAR+T cells/ul BM (n=10) by flow cytometry. CAR was detected in vivo at a median of 5 months. Moreover, we systematically evaluated the immune characteristics of infused products (Figure 1F). Limited by small sample size, correlation analysis wasn’t done Image: Summary/Conclusion: Our study demonstrates bispecific CS1-BCMA CAR-T cells are clinical active with a good safety profile in heavily pretreated patients with MM. P978: IXAZOMIB VERSUS LENALIDOMIDE OR IXAZOMIB AND LENALIDOMIDE COMBINATION AS MAINTENANCE REGIMEN FOR PATIENTS WITH MULTIPLE MYELOMA: INTERIM ANALYSIS OF A MULTI-CENTER PROSPECTIVE STUDY IN CHINA Z. Zhuang1, Y. Tian2, H. Yu3, L. Shi4, W.-W. Tian5, Q. Liu6, D. Zou7, F. Dong8, Y. Ma9, R. Feng10, S. Liu1, H. Liu10, H. Jing8, W. Sun7, L.-M. Ma5, L. Bao4, R. Fu3, Y. Wu2, W. Chen2, J. Zhuang1,* 1Department of Hematology, Peking Union Medical College Hospital; 2Department of Hematology, Beijing Chao-Yang Hospital of Capital Medical University, Beijing; 3Department of Hematology, Tianjin Medical University General Hospital, Tianjin; 4Department of Hematology, Beijing JishuitanHospital, Beijing; 5Department of Hematology, Shanxi Bethune Hospital of Shanxi Medical University, Taiyuan; 6Department of Hematology, The First Affiliated Hospital of Anhui Medical University, Hefei; 7Department of Hematology, Xuanwu Hospital of Capital Medical University; 8Department of Hematology, Peking University Third Hospital, Beijing; 9Department of Hematology, The second Hospital of Shanxi Medical University, Taiyuan; 10Department of Hematology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Science, Beijing, China Background: Maintenance therapy (MT) deepens response and prolongs progression free survival (PFS) in patients with newly diagnosed multiple myeloma (NDMM) after frontline regimens. Ixazomib, a 2nd generation oral proteasome inhibitor (PI), has been approved for MT because of its convenience and tolerability. Aims: We conducted this prospective multi-center study to compare the efficacy and safety of Ixazomib (I-MT) or Ixazomib plus Lenalidomide (IL-MT) to Lenalidomide (L-MT) as maintenance regimen in NDMM patients. Methods: This study was approved by the Institutional Review Board of Peking Union Medical College Hospital and registered (NCT04217967). NDMM patients were enrolled from 10 centers of North China MM Registry since September 2019. After 4 cycles of front-line induction therapy, patients reached partial response (PR) would receive autologous stem cell transplantation (ASCT) if eligible, or keep up to 5 cycles of front regimens if ineligible, then start maintenance therapy. Patients did not reach PR would switch to 2nd-line induction for 2-5 cycles and start MT once PR was achieved. For MT, 4mg of Ixazomib was given on day 1,8,15, and 25mg of Lenalidomide every other day on days 1–21 of 28day cycles. Patients in dual drug group were administrated with both Ixazomib and Lenalidomide, dose as listed above. The primary endpoint was PFS from MT. Results: A total of 176 patients were enrolled, including 58 in I-MT, 72 in L-MT and 46 in IL-MT. The demographic and clinical characteristics, including gender ratio, age, paraprotein isotype, ISS, R-ISS, were comparable among different MT regimen groups either receiving ASCT or not at baseline (Table 1). The proportions of patients with high-risk cytogenetic abnormalities (HRCAs), defined as amplification 1q21, deletion 17p, t(4,14) and t(14,16), were lower in L-MT without ASCT and I-MT with ASCT. The median follow-up duration since MT was 10.6, 12.6 and 11.1 months in I-MT, L-MT and IL-MT groups in non-ASCT patients, while 9.3, 14.0 and 12.2 months in ASCT patients, respectively. In non-ASCT groups, the disease progression rates were 13.5%, 10.2% and 10.8%, whereas 0, 21.7% and 0 in ASCT groups. The median PFS and OS were not reached (NR) in all groups. There were 73.1%, 73.4% and 78.3% of the patients reached VGPR or better before MT in non-ASCT patients, while the rates improved to 80.8%, 81.6% and 86.5% during follow-up. The depth of response did not change in ASCT patients. In the whole cohort, the prevalence of peripheral neuropathy (PN) was 18.9% on I-MT, 8.3% on L-MT and 21.7% on IL-MT. Grade 2 PN occurred in 3, 0 and 2 patients, respectively. The incidence of gastrointestinal (GI) events was 12.1%, 1.4% and 15.2%, respectively. Grade 3 GI events occurred in 3, 0 and 1 patients. The incidence of grade 3-4 hematologic toxicities was 1.7%, 4.2%, and 2.2%. Infections developed in 10.3%, 2.8% and 4.3% patients. No drug withdrawal was related to adverse events. Image: Summary/Conclusion: Due to inadequate access to melphalan and low rate of ASCT, the PFS of NDMM in Chinese patients was relatively inferior. We design this multi-centered prospective study to evaluate if dual drug MT will further strengthen response and make up the gap. Though the primary endpoint--PFS has not been reached in all treatment groups, dual MT improves response most and is quite tolerable. P979: EXOSOMES IN POLYCYTHEMIA VERA: “MINI PLATELETS“ WITH THROMBOGENIC POTENTIAL A. Abulafia1,2,*, G. Spectre1,2, E. Ziv3, K. R. Geiger1, Z. Sarsor3, N. Ron2,3, E. Beery3, S. Revel-Vilk4,5, M. Naamad6, P. Raanani1,2,3, O. Uziel2,3, U. Rozovski1,2 1Institute of Hematology, Rabin Medical Center, Petah Tikva; 2Sackler School of Medicine, Tel Aviv University, Tel Aviv; 3Felsenstein Medical Research Center, Rabin Medical Center, Petah Tikva; 4Gaucher Unit, Pediatric Hematology/Oncology Unit, Shaare Zedek Medical Center; 5Faculty of Medicine, Hebrew University of Jerusalem; 6Flow Cytometry Unit, Shaare Zedek Medical Center, Jerusalem, Israel Background: Polycythemia vera (PV) is characterized by clonal proliferation of myeloid progenitor cells that is caused by activating mutation in the Janus Kinase (JAK)2 gene. Patients with PV tend to develop thrombotic complications across the vasculature tree. Yet, how PV-clonal cells induce pro-thrombotic environment is still under investigation. Secreted by all types of cells, exosomes are nano-scaled particles which carry a molecular cargo that reflects their cell of origin. Exosomes travel in blood, may reach distant sites and affect various physiological and pathological processes. These features led us to think that PV-derived exosomes (hereafter PV-exosomes) carry the mutated JAK2 oncogene and contribute to the thrombotic manifestations seen in PV. Aims: Determine whether PV-exosomes carry mutated JAK2 transcripts. Determine the prothrombotic potential of PV-exosomes and their effect on endothelium. Methods: Exosomes were isolated from eythroleukemia (HEL 92.1.7) cell line (positive for mutated JAK2) and from patients with PV by serial ultracentrifugation. To confirm the presence of exosomes, electron microscopy images were obtained and to quantify their concentration nanoscale tracking analysis was applied (Malvern). The exosomes were stained with the FM-4-11 lipophilic dye and their uptake by HUVECs and HaCaT cells was followed by flow cytometry. To determine the presence of JAK2 transcript, a 465 bp long DNA segment within exon14 of JAK2 was amplified by qRT-PCR and by Sanger sequencing JAK2 mutational status was determined. Thrombin generation assay (TGA) (Ceveron®, Alpha, Technoclone) was used to assess the thrombotic potential of PV-exosomes. Platelet activation status was assessed by the expression of PAC-1, CD-62 and CD-63 in platelets and Trans Epithelial Electric Resistance (TEER) (Electric cell substrate impedance sensing device, Applied Biophysics) was used to assay endothelial dysfunction in HUVECs. Results: We isolated exosomes from the sera of 24 healthy donors and 31 patients with PV. We found the presence of the mutated JAK2 mRNA in all samples confirming that the mutated JAK2 transcript is packaged into sera-exosomes. Yet, while healthy donors’ derived exosomes carry only the wild type JAK2 mRNA, we detected both the wild type and the mutated JAK2 isoforms in patients with PV. We showed that PV-exosomes induced thrombin generation. Then we compared PV-exosomes and healthy-individuals’ derived exosomes and found that thrombin generation potential of PV-exosomes is higher in all measured parameters (p< 0.05 in all parameters). Furthermore, using various platelet markers (CD62, CD63, PAC1) we confirmed that PV-exosomes induced platelet activation. Together, these findings suggest that PV-exosomes may promote thrombosis directly. Under physiological conditions the integrity of the vascular wall prevents uncontrolled thrombi. Therefore, we wondered whether PV-exosomes induce endothelial dysfunction, and in this way contribute indirectly to the pro-thrombotic phenotype. PV-exosomes were taken up by HUVEC endothelial cells in a dose- and time- dependent manner and TEER assay revealed that exosomal uptake by HUVECs induced endothelial dysfunction by breaking the tight junction between adjacent endothelial cells. Summary/Conclusion: Exosomes may act as “mini platelets” that promote thrombosis in patients with PV. PV-exosomes may promote thrombosis either directly by activating platelets and inducing thrombin generation or indirectly by inducing endothelial dysfunction. P980: JAK2V617F-DEPENDENT DOWN REGULATION OF SHP1 EXPRESSION PARTICIPATES IN THE SELECTION OF MPN CELLS IN THE PRESENCE OF TGFΒ C. Aoun1,*, N. Salomao1, N. Maslah1, S. Awan Toor1, G. Letort1, P. Gou1, J.-J. Kiladjian2, S. Giraudier1, B. Cassinat3 1INSERM U1131-hopital saint louis, Paris, France; 2Centre d’investigations cliniques; 3Laboratoire de biologie cellulaire, INSERM U1131-Hopital Saint Louis, Paris, France Background: Myeloproliferative neoplasms (MPNs) are characterized by an increased production of blood cells, due to acquisition in HSC of mutations in the JAK2, CALR or MPL genes. The JAK2V617F mutation, leading to a constitutively active form of JAK2, is the most frequent mutation. During disease evolution the clonal selection of mutated cells is dependent on cell-intrinsic and extrinsic factors present in the niche microenvironment such as cytokines. TGFβ, whose local secretion is increased in MPNs, and more particularly in myelofibrosis is known to negatively regulate normal hematopoietic stem cells (HSC) proliferation. However, its mode of action is not clearly established. We hypothesized that JAK2V617F mutant HSC are resistant to TGFβ antiproliferative effect explaining the pathological stem cell invasion in myelofibrosis. Using multiomics approaches to study the intracellular signaling pathways in MPNs we recently identified the down regulation of SHP-1 expression in JAK2V617F cells compared to wild type cells. Of note, the phosphatase SHP-1 has recently been shown to be required for the TGFβ-induced quiescence of HSCs. Aims: We hypothesized a relationship between SHP-1 down regulation and the resistance of JAK2V617F mutated cells to the antiproliferative effect of TGFβ. The work presented herein aimed at verifying this hypothesis. Methods: We used the HEL erythroleukemia cell line which carries several copies of the JAK2V617F mutated gene. We also used an isogenic model obtained by transducing either the JAK2 wild type (JAK2wt) of JAK2V617F allele in the UT-7 megakaryoblastic cell line. Proliferation was measured using the CCK8. Protein expression was measured using western blot while gene expression was measured using RT-qPCR. Results: The expression of SHP-1 was reduced by 30% at the RNA level and 50% at the protein level in UT-7 cells transduced with JAK2V617F compared to JAK2wt -transduced cells. In bone marrow cells taken from JAK2V617F knock-in mice the protein level of SHP1 was reduced by 70% compared to wild-type mice. EPO treatment of UT-7 cells (which express the EPO-R) showed a down regulation of SHP-1 expression which was abrogated in the presence of the JAK2 inhibitor ruxolitinib. These results suggest that SHP-1 expression is modulated by JAK2 signaling leading to its down regulation in JAK2V617F cells. Although TGFβ receptor and SMAD2/3 expression was similar in both cell type, JAK2V617F UT-7 cells were less responsive to TGFβ signaling compared to JAK2wt cells (reduced phosphorylation of SMAD2/3). The proliferation of JAK2wt UT-7 cells was significantly reduced in the presence of TGFβ 10ng/mL while JAK2V617F UT-7 cells were not affected. To confirm the involvement of SHP-1 we transduced HEL cells with the cDNA coding for SHP-1 in order to restore higher levels of the protein in this JAK2V617F mutated cell line and observed a significant reduction in the proliferation when treated with TGFβ compared to cells transduced with the empty vector. Finally, we performed an in vitro competitive assay where JAK2V617F UT-7 cells transduced by an m-cherry fluorescent vector were mixed 50/50 with JAK2wt UT-7 and cultured with TGFβ. After 14 days of culture with TGFβ 10ng/mL we observed a significant positive selection of the JAK2 mutant cells over JAK2wt cells. Summary/Conclusion: In conclusion, we found a JAK2-dependent down-regulation of SHP1 expression which is related to the resistance of JAK2V617F cells to the antiproliferative effect of TGFβ explaining clonal selection of mutant stem cells in myelofibrosis P981: NEXT GENERATION SEQUENCING (NGS): AN IMPORTANT TOOL TO CHARACTERIZE MYELOPROLIFERATIVE DISEASES IN CHILDREN S. Bianchi1,*, G. Palumbo1, V. Filipponi1, N. Monaco1, G. Pileggi1, R. Maglione1, M. Rousseau1, M. L. Moleti1, F. Giona1 1Translational and Precision Medicine, Sapienza, University of Rome, Rome, Italy Background: Mutations in the JAK2, MPL and CALR driver genes are reported in over 90% of adults with BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) and in 22-40% of children, where inherited forms, such as familial erythrocytosis (FE) and hereditary thrombocytosis (HT), are common. Next Generation Sequencing (NGS) is useful to identify clonal markers, other than those found using standardized methods. Aims: This study was carried out in order to: a) confirm the results obtained in our preliminary experience, using NGS in a larger number of children and adolescents with myeloproliferative diseases (MPDs); b) identify non-canonical driver and/or non-driver mutations in patients (pts) with MPDs; c) evaluate long-term outcome of disease in different subgroups of pts. Methods: 86 pts (46 male, 41 female; median age at diagnosis: 15 years) with a BM evaluation, molecular analysis for driver mutations for MPN (JAK2V617F, JAK2 exon 12, CALR, MPL), and genes involved in the FE (HIF2α) and HT (MPL), were studied in NGS. Results: Driver mutations were found in 52/87 pts (60%) (16 JAK2V617F, 10 CALR, 21 MPLS505N, 2 MPLV501A, and 3 HIF2α). A 44-NGS gene panel providing diagnostic information in myeloid malignancies and in rare inherited erythrocytosis/thrombocytosis were performed in 71/86 pts (82.5%), 28 of them (39.5%) without any mutations. The 44-NGS gene panel detected non canonical driver mutations in 5/28 (18%) triple negative pts, and additional non canonical driver mutations in 2 JAK2V617F and CALR type 2 pts. Overall, driver mutations were found in 61/86 (71%) of pts. The NGS panel revealed 28 additional functional non-driver mutations (12 mutations in 5 genes of the signaling group, 5 mutations in 2 genes of the mRNA splicing group, 6 mutations involving the ASXL1 gene of the histone modification group, and 5 mutations involving 2 genes of the DNA methylation group) in 29 pts, 16 of them with no driver mutations. Pts with acquired MPNs were retrospectively classified according to the WHO 2016 criteria; pts with HIF mutations and/or anamnestic criteria of familial erythrocytosis were considered as FE, pts with MPLS505N, or MPLV501A mutations were defined as HT (Table1). One or more non-driver mutations were found in 17/37 (46%) TE, 4/15 (26.5%) PV and mPV, 6/9 (66.6%) FE pts, and in 0/23 HT pts. During follow-up, 20 pts (12 ET, 5 HT, 2 PV and 1 pre-PMF) presented progressive splenomegaly, after a median of 120 months; in addition, 10 (6 ET, 3 HT, and 1 pre-PMF) of them developed a ≥2 BM fibrosis, after a median of 188 months. Considering driver mutations, 8/20 pts were JAK2V617Fmutated, 5 had CALR mutations, and 5 were MPLS505N mutated; 2 pts were wild type. One or more additional non-driver mutations were present in 3/5 CALR, and in 3/8 JAK2V617Fmutated pts. Overt MFI developed in 3 pts, 2 of them needed hematopoietic stem cell transplantation. Two pts developed a malignant neoplasia. Six (7%) pts, 3 MPLS505N, 2 JAK2V617F, and one wild type, without thrombophilic abnormalities, experienced a thrombotic event. All pts are alive after a median time of 15 years from initial diagnosis. Image: Summary/Conclusion: In our experience, the use of a 44-gene NGS panel enabled us to identify non canonical driver, and functional non-driver mutations, in addition to those detected by conventional methods, in about 90% of pediatric pts with acquired and familial MPDs. Additional clonal mutations have been identified in two thirds of FE pts, but none in HT pts. Interestingly, the development of BM fibrosis was observed in pts with HT, but not in FE group. P982: MYELOID-DERIVED SUPPRESSOR CELLS: CHARACTERIZATION IN PERIPHERAL BLOOD AND SPLEEN TISSUE OF PATIENTS WITH MYELOFIBROSIS R. Campanelli1,*, A. Bonometti2,3, M. Massa4, C. Abbà1, L. Villani1, A. Carolei1, P. Catarsi1, M. Paulli3, G. Barosi1, E. Boveri3, V. Rosti1 1Center for the Study of Myelofibrosis, General Medicine 2—Center for Systemic Amyloidosis and High-Complexity Diseases, IRCCS Policlinico San Matteo Foundation, Pavia; 2IRCCS Humanitas Research Hospital, Milan; 3Department of Diagnostic Medicine, Anatomic Pathology Unit; 4General Medicine 2—Center for Systemic Amyloidosis and High-Complexity Diseases, IRCCS Policlinico San Matteo Foundation, Pavia, Italy Background: Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that accumulate in patients with malignancies, sepsis or chronic inflammation. Features of MDSCs are: myeloid origin, immature state, strong ability to reduce cytotoxic functions of T/NK cells, potential to differentiate into endothelial cells favoring neoangiogenesis. MDSCs are conventionally divided into polymorphonuclear (PMN)- and monocytic (M)-MDSCs. PMN-MDSCs are characterized by the production of reactive oxygen species and arginase-1 (Arg-1), that directly inhibits T-cell proliferation and activation, while M-MDSCs predominantly express inducible nitric oxide synthase and produce nitric oxide. Myelofibrosis (MF) is characterized by clonal expansion of a hematopoietic stem cell, extramedullary hematopoiesis, bone marrow (BM) fibrosis, splenomegaly, BM and splenic neoangiogenesis and an acquired mutation of JAK2, CALR or MPL gene. Inflammation plays a relevant role in MF pathogenesis, as proven by high levels of inflammatory cytokines with prognostic significance and by a state of chronic oxidative stress. Aims: To characterize circulating MDSCs and assess their localization and quantification in spleen samples from MF patients. Methods: Peripheral blood mononuclear cells (PBMCs) from MF patients (n=15) and healthy subjects (HDs; n=10) were stained with surface antibodies (CD11b, CD14, HLA-DR, CD15, CD33) and intracellular Arg-1 and analysed by flow cytometry. Formalin-fixed paraffin-embedded samples of spleen specimens (MF n=41; HDs n=20) were immunostained with anti-Arg-1 antibody using the automated platform Dako Omnis Envision Flex with the EnVision FLEX, High pH, HRP Rabbit/Mouse High pH [GV800] revelation kit. Results: In PBMCs, the percentage of PMN-MDSCs (identified as CD11b+HLA-DRlow/-CD33dimCD14-CD15+) was higher (p=0) in patients (median 9.2, range 1-59) than HDs (median 1.7, range 0.1-4.5) and directly correlated with the allelic burden (R=0.86, p=0.006) in JAK2-mutated patients. Patients in pre-fibrotic phase (n=7) had levels of PMN-MDSCs (median 9.2, range 1-31.4) comparable to HDs; on the contrary, the percentage of PMN-MDSCs in patients with BM fibrosis (n=8; median 13.3, range 3.5-59) was higher (p=0) than in HDs. All PMN-MDSCs were Arg-1+, both in MF and in HDs. Gene expression studies are ongoing. The expression of the CXCR4 receptor on PMN-MDSCs was lower, although not statistically significant, in MF (median 1.1, range 0-9.3) than in HDs (median 2.5, range 0-11.9). PMN-MDSCs in MF inversely correlated (R=-0.89 p=0.007) with CXCR4 expressed on CD34+ cells. The percentage of M-MDSCs (identified as CD11b+HLA-DRlow/-CD33hiCD14+CD15-) was higher (p=0.015) in HDs (median 0.49, range 0.25-1.5) than in MF (median 0.29, range 0.02-0.76). Immunohistochemical staining of spleen sections showed a higher (p=0) percentage of Arg-1+ cells (calculated on total spleen cells) in MF (median 20, range 5-60) than in HDs (median 7, range 2-20); Arg-1+ cells were present in all patients and predominantly located in the red pulp in MF patients, while in HDs were detected in the white pulp. Summary/Conclusion: The identification of MDSCs and their relation with the mutational status suggest a new mechanism of MF pathogenesis, either related to the chronic inflammatory status of patients or as players of the neoangiogenesis that characterizes the disease. Moreover, the low expression of CXCR4 on PMN-MDSCs could be, on one hand, related to the low expression of this receptor on CD34+ cells or suggest a MF-specific recruitment mechanism, other than that observed in solid tumors. P983: SINGLE CELL ANALYSIS ALLOWS THE EARLY DETECTION OF LEUKEMIC CLONES IN MPN PATIENTS C. Carretta1,*, S. Parenti1, S. Mallia1, S. Rontauroli1, C. Chiereghin2, S. Castellano1, E. Bianchi1, E. Genovese1, S. Sartini1, L. Tavernari1, M. Mirabile1, M. G. Della Porta2,3, R. Manfredini1 1Center for Regenerative Medicine “S. Ferrari”, University of Modena and Reggio Emilia, Modena; 2Humanitas Clinical and Research Center -IRCCS, Rozzano; 3Department of Biomedical Sciences, Humanitas Unviersity, Milan, Italy Background: Myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders resulting in the overproduction of myeloid differentiated cells. Primary Myelofibrosis (PMF) is characterized by the worst prognosis and 15-20% of cases develop secondary Acute Myeloid Leukemia (AML). MPNs driver mutations affect JAK2, CALR or MPL genes. Moreover, mutations in epigenetic regulators can exacerbate the disease and alter response to treatment. Our group recently demonstrated through single cell analysis that MPNs progression is due to increased genetic heterogeneity, loss of heterozygosity and parallel AML evolution. Aims: In this work we sought to define the genomic architecture of MPN patients during disease evolution, and gain insight into the chromatin and transcriptional perturbations induced by epigenetic modifiers’ mutations. Methods: In order to describe MPNs clonal hierarchy at the single cell level, we employed Mission Bio Tapestri platform. We analyzed the CD34+ compartment of 3 patients during the chronic phase of the disease and after leukemic transformation through a custom panel comprising 29 genes frequently mutated in myeloid neoplasms. CNV assessment was performed on the same data through Mosaic algorithm. On one patient, single nucleus RNA (snRNA-seq) and ATAC-seq were then conducted through 10x genomics instrument on CD34+ cells from the chronic and blast phase. Data were analyzed through Seurat R package. Results: Genomic analysis was performed on 20.652 single cells coming from 7 samples. The analyzed patients suffered from Essential Thrombocythemia (n=2) or PMF (n=1); 2 patients harbored CALR type 1 driver mutation, while one patient carried JAK2V617F variant. In all patients, the first mutational hit occurred on epigenetic remodeler genes (i.e. TET2, ASXL1). Driver mutations’ allele frequency remained stable during disease progression and did not seem to drive leukemic transformation; in one case, JAK2V617F variant was lost in blast phase. In one patient, single cell analysis revealed the acquisition of 3 mutually exclusive pathogenic variants in RAS pathway (two NRAS mutations and a KRAS mutation). Notably, leukemic driver mutations, affecting genes such as IDH2, TP53 and KRAS, were already traceable at very low allele frequency in the chronic phase of the disease of all patients, despite being undetectable by bulk diagnostic NGS analysis. For all patients, CNV analysis highlighted a higher gene dosage imbalance in the leukemic clones when compared with those in the chronic phase. Preliminary analysis of single cell ATAC+RNAseq data showed that the leukemic sample is enriched in more primitive cell types (e.g. multipotent progenitors). Moreover, genes found to be upregulated and more accessible in blast phase erythroid progenitors and HSC clusters are associated with AML poor prognosis. Summary/Conclusion: Altogether this analysis suggests that MPNs’ first mutational hit frequently occurs in chromatin remodeler genes and affects a large fraction of neoplastic cells, confirming their impact on MPNs pathogenesis. Single cell multiomic analysis suggests that epigenetic alterations contribute to alter CD34+ cells differentiation state and activate the expression of pro-leukemic genes. Moreover, genomic analysis highlighted that clones carrying driver mutations remain stable during time and do not seem to drive leukemic transformation. On the other hand, genetic alterations, such as SNVs and CNVs, driving AML evolution are early identified by single cell analysis despite being undetectable by bulk sequencing. P984: MUTATIONAL PATTERNS IN PH-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS (MPN) I. C. Casetti1,*, D. Pietra2, V. V. Ferretti2, A. De Silvestri2, O. Borsani1, D. Vanni1, C. Trotti1, S. Catricalà2, C. Favaron1, M. Cazzola1, L. Arcaini1,2, E. Rumi1,2 1University of Pavia; 2Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Background: According to the 2016 WHO classification, Ph-negative MPN include essential thrombocythemia (ET), polycythemia vera (PV), prefibrotic myelofibrosis (prePMF) and overt fibrotic myelofibrosis (PMF). The molecular basis of MPN was clarified with the discovery of driver mutations in JAK2, MPL and CALR genes, but the biological landscape is more complex than initially assumed. Subclonal somatic mutations, also found in other myeloid neoplasms (MN), have been described and are associated with disease progression and poor prognosis. Those mutations target epigenetic regulators of DNA (DNMT3A, TET2, IDH1/IDH2), genes involved in the chromatin structure (ASXL1, EZH2), spliceosome genes (SF3B1, SRSF2), transcription factors and tumor suppressor genes (RUNX1, TP53). Aims: To correlate driver mutations, subclonal variants, clinical data and diagnosis in 509 MPN patients (236 ET, 91 prePMF, 182 PMF) and to define a diagnostic and prognostic role of these variants. Methods: We studied DNA variants with the Illumina Nextera Rapid Capture Custom Enrichment Kit and HiSeq2500 platform. We selected a panel of 81 genes based on prior implication in the pathogenesis of MN. Inclusion criteria were (i) a diagnosis of ET, prePMF or overt PMF according to the 2016 WHO criteria, (ii) a peripheral blood sampling at diagnosis or before therapy administration. Results: Overall, we detected 598 additional somatic variants. PMF showed a larger proportion of patients (pts) with at least one additional variant compared with prePMF and ET (86.3% vs 42.9% vs 34.8%, p<0.001) and a higher average number of variants per pts. These findings suggest that PMF is not an advanced stage of prePMF, but a different clinical entity with high tendency in variants accumulation. Considering the driver mutation, the number of pts with at least one additional variant was significantly different among the molecular subgroups (CALR 46.5%, JAK2 58.9%, MPL 82.1% TN 31.1%, p<0.001). Somatic mutations were grouped based on their role in the cell cycle. In ET the most commonly mutated genes belong to the DNA methylation regulation and the most frequently involved gene was TET2. In PMF, chromatin structure, RNA splicing and DNA methylation were the most recurrently involved pathway and ASXL1 and TET2 were the most frequently mutated genes. In prePMF most of the variants impaired DNA methylation and RNA splicing and TET2 and SF3B1 were the most frequently mutated genes. RNA splicing and DNA methylation are often involved in myelodisplastic syndromes (MDS). Thus, PMF and prePMF share molecular signatures with MDS, suggesting that MN are part of a continuum spectrum of diseases. There was no association between driver mutations and additional variants, except for the spliceosome genes. The rate of splicing factors alterations in CALR mutants was significantly lower compared with JAK2 or MPL mutants (3.9% vs 22.3% vs 35.7%, p<0.001). We finally correlated additional mutations and clinical data (blood count, splenomegaly, LDH, CD34+ circulating cells), overall survival (OS) and progression to blast phase (BP). We focused on the most commonly mutated genes, defined as mutations detected in at least 10 pts (>5%): ASXL1, DNMT3A, TET2, EZH2, SRSF2. ASXL1 was associated with anemia and splenomegaly, a higher risk of leukemic evolution (sHR=4.5, 95%CI:1.7-11.8, p=0.003) and shorter OS (HR=6.2 (4.3-9.0) p<0.001); SRSF2 had an impact on BP evolution and OS, while EZH2 showed an impact on OS. Image: Summary/Conclusion: We suggest that not only histological and clinical criteria, but also distinct mutational patterns might differentiate ET, prePMF and overt PMF. P985: THE ALLELIC RATIO OF DRIVER AND ASXL1 MUTATIONS IS PROGNOSTICALLY RELEVANT IN PMF G. Coltro1,2,*, G. Rotunno1,2, F. Mannelli1,2, G. G. Loscocco1,2, N. Bartalucci1,2, C. Mannarelli1,2, C. Maccari1,2, P. Guglielmelli1,2, A. M. Vannucchi1,2 1Department of Experimental and Clinical Medicine, University of Florence; 2CRIMM, Center for Research and Innovation of Myeloproliferative Neoplasms, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy Background: Primary myelofibrosis (PMF) is a clonal disorder driven by mutations in JAK2, CALR, and MPL. Somatic mutations in myeloid-associated genes were shown to impact prognosis of PMF patients (pts). Among these, ASXL1 mutations (ASXL1 mt) are by far the most frequent and prognostically relevant, in fact they are included in the category of “high molecular risk” (HMR), along with EZH2, IDH1/2, SRSF2, and U2AF1 Q157 mutations. Aims: To investigate the phenotypic and prognostic implications of ASXL1 mt and variant allele frequency (VAF) in PMF. Methods: After IRB approval, consecutive pts with WHO-defined PMF were included in the study. Mutational analysis by targeted NGS was performed by previously described methods (Guglielmelli P et al., JCO 2017). Results: The study enrolled a total of 384 pts, including 190 (49%) prefibrotic and 194 (51%) overt PMF. Median age was 60 (18-90) years, 236 (61%) were male. JAK2, CALR and MPL mutations were found in 255 (66%), 83 (22%), and 17 (4%) pts, respectively; 36 (9%) were triple negative (TN). Among HMR mutations, ASXL1 was mutated in 88 (23%) pts, SRSF2 in 35 (9%), EZH2 in 32 (8%), U2AF1 in 15 (5%), and IDH1/2 in 11 (3%). VAF distribution of driver and HMR mutations was as reported in Fig.1A. We did not find any significant correlation of ASXL1 mt VAF with other clinical and molecular characteristics, including PMF subtype, gender, age, hemoglobin, leukocyte, platelet and blast counts, splenomegaly, BM fibrosis grade, cytogenetics, mutational status for driver and other HMR genes and their VAFs. The only exception was the association of CALR-mutated PMF with lower ASXL1 VAF (median 28% vs 40%, p=.0332). Median overall survival (OS) was 120 (102-151) months. In univariate Cox analysis, ASXL1 mt VAF did not correlate with OS, nor did other single driver and HMR mutations. When considered as a whole (for the purpose of this study, TN pts were considered has having driver VAF equal to 0), the VAF of driver mutations inversely correlated with survival (HR 0.4 [0.2-0.9], p=.0219). We next investigated the allelic ratio between driver and ASXL1 mutations (d/Aratio) and its prognostic correlates. Median (range) of d/Aratio was 1.24 (0-13), and ROC analysis with death as an endpoint identified 1.31 as the optimal cut-off value. In univariate analysis, pts with a d/Aratio <1.31 had a significantly worse OS compared to those with a ratio ≥1.31 (median 45 vs 104 months, p=.0014; HR 2.4 [1.4-4.1]) (Fig.1B). When TN pts with ASXL1 mt were considered apart, having a d/Aratio <1.31 retained its inferior prognostic impact compared to the d/Aratio ≥1.31 group (median OS 57 vs 104 months, p<0.0453; HR 1.8 [1-3.2]), with TN/ASXL1 mt pts being associated with the worst outcome (median OS 24 months) (Fig.1C). In a multivariate Cox model including VAF-adjusted ASXL1 mutant status (ASXL1 wt vs ASXL1 mt with d/Aratio ≥1.3 vs ASXL1 mt with d/Aratio <1.3) and other HMR mutations, the former and mutated SRSF2 were confirmed to be independent predictors of inferior OS. Both d/Aratio <1.31 and ≥1.31 remained significant compared to ASXL1 wt with respective HRs of 3.4 (2.3-5.1; p<.0001) and 1.6 (1-2.6; p=.0342). Notably, d/Aratio <1.31 still retained its inferior prognostic impact compared to d/Aratio ≥1.31 (HR 2 [1.2-3.5]; p=.0095). Image: Summary/Conclusion: This study explores the implications of ASXL1 mt VAF and its interplay with driver mutant burden. The adverse prognosis of pts with a d/Aratio <1.31 suggests that this disease entity is predominantly driven by ASXL1 mt-clones characterized by a more aggressive biology. Further research is needed. P986: CYTOGENETIC DIAGNOSIS WITH NEXT-GENERATION CYTOGENETICS BY OPTICAL GENOME MAPPING IN PATIENTS WITH MYELOFIBROSIS. Á. Díaz González1,*, G. Avetisyán1, E. Mora1, M. Santiago2, A. Liquori1, S. Furió1, S. García1, A. Villalba1, Á. López1, J. V. Gil1, C. García1, E. González1, C. Martínez1, B. Fernández1, M. Guaita1, B. Martín1, L. Cordon1, E. Barragán1, J. de la Rubia1, J. Cervera2, E. Such1 1Hematology; 2Genetics Unit, Hospital Universitario y Politécnico La Fe, Valencia, Spain Background: The prognosis of myelofibrosis patients is highly variable. Due to this heterogeneity, it is important to have prognostic tools that correctly stratify the patients. The most current prognostic scales such as the MIPSS70+ v2.0 or the GIPSS require cytogenetic data. The main limitation in classifying these patients is the lack of information on cytogenetic alterations because of bone marrow fibrosis and absence of metaphases. Recently, a new strategy has emerged that characterize all cytogenetic alterations using Optical Genome Mapping considered Next Generation Cytogenetics which overcomes the limitations of conventional cytogenetics by using ultra high molecular weight DNA instead of metaphases. This DNA can be obtained from peripheral blood or bone marrow samples. Aims: The main objective of this project is to perform a complete genomic characterization of 10 patients with myelofibrosis by karyotype, FISH, Next Generation Sequencing (NGS) and Optical Genome Mapping (OGM). Methods: We have performed the karyotype staining the chromosomes by G-banding and the results were reported according to the International System for Human Cytogenetic Nomenclature (ISCN, 2020). FISH was performed to discard rearrangements of PDGFRA, PDGFRB, FGFR1 and JAK2. NGS libraries were prepared using a panel SOPHiA Myeloid Solution™ kit which includes SNV and CNV of 30 genes related to myeloproliferative neoplasms and leukemia. For study by OGM we obtained ultra-high molecular weight (UHMW) DNA from peripheral blood samples which are labeled with DLGreen fluorophores using Enzyme 1 (DLE-1) reactions following the manufacturer’s protocols (Bionano Genomics). Labeled DNA was loaded on a Saphyr chip and run on a Saphyr instrument (Bionano Genomics). The novo genome map assembly will be performed using BionanoSolve™ and structural variants will be called against the human reference hg38 assembly. Data was analyzed with Bionano Access™ (Bionano Genomics). Results: Preliminary results were obtained: - Patient 1 had unsuccessful karyotype and JAK2 (p.Val617Phe, VAF 13%) mutation by NGS. - Karyotype result of patient 2 suggest that is normal: 46,XY[6]. NGS detected pathogenic mutations in JAK2 (p.Val617Phe, VAF 41%), SRSF2 (p.Pro95His, VAF 42%) and ETV6 (p.Arg105*, VAF 41%) and likely pathogenic mutation in ASXL1 (p.?; c.1720-2A>G, VAF 45%). - Patient 3 had a normal karyotype: 46,XX[18] with CALR type 1 mutation (p.Leu367Thrfs*?, VAF 37%) by NGS. We have not detected rearrangements by FISH in none of the 3 patients. 1500 Gbp of DNA were collected to perform the OGM for each patient and we selected structural variants that were present in less than or equal to 0,5 % of the control samples with the same enzyme. Patients 1 and 2 who had unsuccessful karyotype could be characterized by OGM. We have found by OGM a median of 1 insertion (range 0-4), 8 deletions (4-10) and 2 duplications (0-3). No inversion or translocation were detected. No cytogenetic alterations classified as very high-risk karyotype in MIPSS70+ v2.0 scale were detected. After an exhaustive analysis of these structural alterations initially detected, all alterations were ruled out as they are present in general population and therefore correspond to a normal karyotype. Summary/Conclusion: Optical Genome Mapping suggest to be very promising diagnostic tool that can complete the study of patients with myelofibrosis, especially in those with non-assessable karyotype. The extension to a larger number of patients will confirm the results obtained and detect variables that remain cryptic to the karyotype. P987: A DIFFERENT BALANCE IN OXIDATIVE STRESS RESPONSE IN CALR AND JAK2 MUTATED MYELOFIBROSIS PATIENTS CORRELATES WITH CLINICAL OUTCOME E. Genovese1,*, M. Mirabile1, S. Rontauroli1, S. Sartini1, S. Fantini1, L. Tavernari1, M. Maccaferri2, P. Guglielmelli3, E. Bianchi1, S. Parenti1, C. Carretta1, S. Mallia1, S. Castellano4,5,6, C. Colasante5, M. Balliu3, N. Bartalucci3, R. Palmieri7, T. Ottone7,8, B. Mora9, L. Potenza5, F. Passamonti9, M. T. Voso7,8, M. Luppi5, A. M. Vannucchi3, E. Tagliafico4,5, R. Manfredini1 1Life Sciences Department, Centre for Regenerative Medicine, University of Modena and Reggio Emilia; 2Department of Laboratory Medicine and Pathology, Diagnostic Hematology and Clinical Genomics, AUSL/AOU Policlinico, Modena; 3Department of Experimental and Clinical Medicine, Center of Research and Innovation of Myeloproliferative Neoplasms (CRIMM), University of Florence, Careggi University Hospital, Florence; 4Center for Genome Research, University of Modena and Reggio Emilia; 5Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, AUSL/AOU Policlinico; 6PhD Program in Clinical and Experimental Medicine, University of Modena and Reggio Emilia, Modena; 7Department of Biomedicine and Prevention, University of Tor Vergata; 8Santa Lucia Foundation, I.R.C.C.S., Neuro-Oncohematology, Rome; 9Division of Hematology, Ospedale ASST Sette Laghi, University of Insubria, Varese, Italy Background: Myelofibrosis (MF) is the Philadelphia-negative myeloproliferative neoplasm characterized by the worst prognosis and poor response to conventional therapy. Driver mutations in JAK2 and CALR impact on JAK-STAT pathway activation but also on the production of reactive oxygen species (ROS). ROS play a pivotal role in inflammation-induced oxidative damage to cellular components including DNA, that leads to greater genomic instability and promotes cell transformation. Aims: In order to unveil the role of driver mutations in oxidative stress response, we evaluated oxidative stress markers levels in CD34+ hematopoietic stem/progenitor cells and plasma from MF patients. Methods: Firstly, we have measured the levels of ROS, 8-hydroxy-2-deoxy-guanosine (8-OHdG) and activity of superoxide dismutase (SOD) in CD34+ cells from 20 JAK2 and 14 CALR myelofibrosis (MF) patients compared with 17 healthy donors (HD). In particular, the redox-sensitive CM-H2DCFDA was used to measure the intracellular ROS. The levels of 8-OHdG were detected by enzyme-linked immunosorbent assay (ELISA). Moreover, SOD activity was measured by means of colorimetric assay. Subsequently, we evaluated whether a different oxidative status could be detected in 129 plasma samples of MF patients (n=86 JAK2, n=43 CALR), as the level of total antioxidant capacity (TAC), an analyte frequently used to assess the antioxidant status of biological samples. Finally, we evaluated the association between TAC plasma levels and overall survival (OS) by means of multivariate analysis. Results: Our results demonstrated that ROS production in CD34+ cells from CALR-mutated MF patients is strongly increased compared with patients harboring JAK2 mutation, leading to increased oxidative DNA damage. Moreover, CALR-mutated cells show less SOD antioxidant activity than JAK2-mutated ones. Subsequently, we found that high TAC plasma levels correlate with detrimental clinical features, such as high levels of lactate dehydrogenase (LDH) and circulating CD34+ cells. Moreover, high TAC plasma levels are also associated with a poor OS in JAK2-mutated patients. Multivariate analysis demonstrated that high plasmatic TAC is an independent prognostic factor for OS that allows the identification of patients with inferior OS in both DIPSS lowest and highest categories. Summary/Conclusion: Altogether, our results confirmed that CALR mutation has a higher impact on the oxidative stress status in MF cells compared to JAK2 variant. Furthermore, plasma samples from CALR mutated patients have significantly lower TAC levels leading to a lower responsiveness to oxidative injury. On the other hand, increased TAC levels correlate with the presence of JAK2 mutation and several detrimental clinical features. MF patients with high plasmatic TAC display inferior survival and multivariate analysis demonstrated that increased TAC activity might represent a novel prognostic biomarker independent from DIPSS classification. We speculated that the high increase in oxidative stress in CALRmutated patients could be involved in the activation of protective mechanisms which ultimately promote cell death. On the contrary, in JAK2 mutated patients, the slight increase in oxidative stress can determine the persistence of cells with damaged DNA where the accumulation of mutations promotes the disease progression. P988: LACTATE RESHAPES TUMOR MICROENVIRONMENT AND METABOLIC PROFILE IN MYELOFIBROSIS S. Giallongo1,*, D. Tibullo1, M. Spampinato1, C. Giallongo2, E. La Spina3, L. Longhitano1, A. Romano3, I. Dulcamare3, A. Barbato3, G. Scandura3, A. M. Amorini1, G. Lazzarino1, T. Zuppelli2, R. Caltabiano2, G. Li Volti1, F. Di Raimondo3, G. A. M. Palumbo2 1BIOMETEC; 2Department of Medical, Surgical Sciences and Advanced Technologies G.F. Ingrassia, A.O.U. “Policlinico-Vittorio Emanuele; 3Department of General Surgery and Medical-Surgical Specialties, Division of Hematology, A.O.U. “Policlinico-Vittorio Emanuele”, University of Catania, CATANIA, Italy Background: Primary myelofibrosis (PMF) is myeloproliferative neoplasm depicted by bone marrow (BM) fibrosis, ineffective clonal hematopoiesis, and splenomegaly. These outcomes are related with changes in bone marrow microenvironment, sustaining cancer glycolytic metabolism, fibrosis, immune evasion, and thrombotic events. Lactate, a glycolysis byproduct, may represent the driving-force towards the acquisition of these features. For this reason, we investigated PMF CD34+ cells lactate production as factor prompting the metabolic rewire of BM niche. Aims: In this work we aimed to target lactate metabolism as stricking factor improving PMF proliferative features. Methods: Primary mesenchymal stem cells (MSCs) and HS-5 proliferation assays were performed in vitro. Protein expression level was assessed using immunoblot and immunohistochemical assays. Moreover, Cytokines were also investigated ex vivo, using Multiplex immunobead assay technology. These results were also mirrored in vivo on TPOhigh Zebrafish (Danio rerio) model. Results: The monocarboxylate transporters (MCT-1 and -4), orchestrating lactate intake, were significatively upregulated in CD34+ cells and sera derived from MF-patients compared to healthy donor. MF-patients are characterized by increased percentage of circulating immunosuppressive cells. Incubation of peripheral blood mononucleated cells (PBMNCs) with MF-sera increased Treg and Myeloid-derived suppressor cells (M-MDSCs) expansion. This effect was disrupted by addition of MCT1 inhibitor AZD3965. The role of lactate on BM niche was evaluated by culturing healthy primary MSCs. Lactate (20 mM) and MF-sera exposition induced extracellular matrix (ECM) reorganization as supported by increased level of metalloproteases (MMP9, MMP2) together with reticulin and collagen deposition. Moreover, lactate induces calcium accumulation and soluble osteogenic molecular signals production (i.e. osteoprotegerin, osteonectin). On the epigenetic side, lactate treatment reshapes chromatin architecture eventually affecting histones lactylation, acetylation and methylation levels. These features were rescued upon AZD3965 treatment. Cancer-associated fibroblast (CAF) play an outstanding role in cancer development by ECM remodeling.Upon lactate exposure, expression of CAF markers α-SMA, FAP1 and TGFβ were upregulated in MSCs. The effect was reverted by AZD3965 treatment. The data achieved in vivo, and ex vivo were corroborated in a new pre-clinical TPOhigh Zebrafish model eventually showing increased Whole Kidney Marrow lactate. Summary/Conclusion: In this work we report MCT1 inhibition as a strategy to treat invasive and metastatic cancers. Our results corroborate the idea that targeting lactate metabolism may represent a promising path to counteract inflammation, osteosclerosis and fibrosis in PMF patients. P989: DYNAMIC CHANGES IN THE HISTONE H3 LYSINE 27 TRIMETHYLATION EPIGENETIC LANDSCAPE FOLLOWING RUXOLITINIB ADMINISTRATION G. Greenfield1,*, M. F. McMullin2, K. Mills3 1PGJCCR; 2CME, Queen’s University Belfast; 3PGJCCR, Queen’s Universtiy Belfast, Belfast, United Kingdom Background: Dysregulation of normal epigenetic processes is increasingly recognised as a feature of myeloid malignancy generally and implicated in the pathogenesis of MPN. Epigenetic changes may directly result from the mutation of key epigenetic regulators including TET2, ASXL1 and EZH2 or indirectly from the activation of intracellular signalling cascades. We have previously observed that ruxolitinib, a JAK1/2 inhibitor, may alter the epigenetic landscape of histone modifications in MPN. CUT&RUN is a novel experimental approach providing high resolution profiling of epigenetic modifications and transcription factor binding. Aims: To comprehensively profile the H3K27me3 epigenetic landscape of JAK2 V617F positive cells resulting from JAK inhibition and determine the dynamic changes occurring between short term and persistent ruxolitinib therapy to identify potential genetic contributors to pathogenesis and options for future therapeutics. Methods: JAK2 V617F positive UKE1 cells were cultured in duplicate in the presence of 1000nM ruxolitinib or DMSO 0.1% vehicle control continuously for approximately five weeks. Samples were harvested at 37 days for CUT&RUN analysis using H3K27me3, EZH2 and IgG (negative control) antibodies. CUT&RUN performed using Epicypher CUTANA Kit according to manufacturer instructions. Libraries prepared using NEBNext® Ultra™ II DNA Library Prep kit with 50 base pair, paired end sequencing undertaken on Illumina NovaSeq 6000. Resulting FASTQ files were trimmed using Trim Galore! and aligned to reference genome hg38 using Bowtie2. Duplicate reads were removed using SAMtools rmdup and peaks called with MACS2 callpeak on the usegalaxy.eu platform. Differential peak analysis undertaken with Diffbind package in R. Samples were normalised to library size prior to differential analysis with DESeq2 methodology. Bigwig files generated using deepTools bamcoverage for visualisation. Results: UKE1 cells remain sensitive to ruxolitinib after a period of treatment with high dose ruxolitinib lasting over 5 weeks. CUT&RUN libraries were sequenced to a minimum depth of 3.6 million unique reads per sample. Initial comparisons between Day 37 high dose treated cells and vehicle control revealed 1004 differentially enriched H3K27me3 peaks. Of these, 545 (54.2%) peaks were increased in the ruxolitinib treated cells and 459 (45.7%) peaks were increased in the control cells. Analysis of the difference between day 37 and day 1 high dose ruxolitinib treated cells revealed 290 peaks differentially enriched. Of these peaks, 279 (96.2%) were increased and 11 (3.8%) were decreased at day 37. Analysis of the overlap between these results identified 33 peaks increased at D37 in comparison to DMSO control and day 1 treatment samples. Analysis of the differential binding patterns and direct visualisation of the results in a genome browser revealed particular genetic locations of interest including the promoter region of SPRY1, a potential regulator of erythropoiesis and JAK signalling, UGCG and BTBD11. Figure 1 shows an example. Differential H3K27me3 was also noted at regions associated with PLXNA1 and NRP2 genes which act as co-receptors for class III semaphorin signalling. Image: Summary/Conclusion: The dynamic changes in the H3K27me3 epigenetic landscape of JAK2 V617F positive cells following persistent high dose ruxolitinib administration has potential therapeutic implications and may sensitise cells to epigenetic therapies. Specific gene loci identified demonstrating differential H3K27me3 may offer some insight into JAK2 V617F mediated pathology and require further investigation. P990: T CELL RESPONSE AND OMICRON VARIANT NEUTRALISATION FOLLOWING VACCINATION AGAINST SARS-COV-2 IN PATIENTS WITH CHRONIC MYELOID DISORDERS P. Harrington1,2,*, J. Saunders1, C. Saha1, A. Sheikh1, R. Dillon1, K. Raj1, L. Cadman-Davies1, T. Lechmere3, H. Khan3, C. Woodley1, S. Asirvatham1, N. Curto-Garcia1, J. O’Sullivan1, S. Kordasti1, D. Radia1, D. McLornan1, M. Malim3, K. Doores3, C. Harrison1, H. de Lavallade1,2 1Clinical Haematology, Guy’s & St Thomas’ NHS Foundation Trust; 2School of Cancer & Pharmaceutical Science; 3Infectious Diseases, King’s College London, London, United Kingdom Background: Concerns remain over the response to vaccination against SARS-CoV-2 in patients with haematological malignancy. Aims: Herein, we report a comprehensive immunological evaluation of the response to SARS-CoV-2 vaccination in patients with chronic myeloid neoplasms (MPN). Methods: Antibody response was assessed using anti-Spike (anti-S) IgG with anti-nucleocapsid (anti-N) IgG used to determine previous infection. Neutralising antibody analysis was performed assessing inhibitory effect of plasma on entry of HIV-1 particles expressing Wuhan and omicron variant spike proteins into cells expressing ACE-2 receptor. T cell response was assessed using flow cytometric evaluation of intracellular pro-inflammatory cytokines and surface exhaustion markers upon re-exposure to S peptides (Miltenyi) in T cell subsets. Subsequently, T cell response was evaluated using a fluorospot assay assessing IFNg/IL-2 secretion upon re-exposure to S peptides (Mabtech). Plates were analysed using the IRIS reader providing both spot-forming unit (SFU) frequency and relative spot volume (RSV). Results: Samples were collected in 102 patients with MPN or post allo-SCT. Testing was performed in 37 MPN patients after one dose, 61 after 2 doses and 30 after 3 doses to date. An anti-S IgG response was observed in 81.1% (30/37) of MPN patients after a first dose, increasing after two doses to 91.7% (55/60). Those with previous infection had higher anti-S IgG and neutralising antibody levels following two doses of vaccine (p=0.0002/p<0.0001). MPN patients receiving the BNT162b2 vaccine had higher anti-S IgG and neutralising antibody levels compared with those receiving ChAdOx-1 (p=0.05/p=0.04). The proportion of patients on ruxolitinib (rux) with a negative or borderline response after two doses was higher than observed in other patients (5/10 vs 5/47, p=0.01). Preliminary analysis in 20 patients showed reduced neutralisation of omicron compared with the Wuhan spike, with neutralising antibody ID50 of 93 vs 218 (p=0.06, Fig. 1a). Using fluorospot analysis after two doses, an overall T-cell response was observed in 88.3% (53/60) patients. Patients on rux had lower SFUs for IFN and IL-2 and amongst those on treatment, RSV of IFNg was again lowest in patients on rux, with mean RSV of 2942 vs 7990 in patients on hydroxycarbamide and 6687 in peg-IFNa (p=0.016/p=0.0008). T cells expressed increased exhaustion markers upon re-exposure to S peptides in patients with CML following vaccination. After a third dose, T cell response was assessed in 30 patients with a positive response in 90% (27/30). IFNg SFUs and RSV were significantly higher after 3 vs 2 doses at 242 and 9184 vs 72 and 5031 (p=0.0004/<0.0001, Fig. 1b,c). Patients on rux after 3 doses had lower SFU and RSV of IFNg than other patients at 79 and 7690 vs 302 and 9727 (p=0.035/0.04 Fig 1d,e). Patients on rux were more likely to have a negative T cell response than other patients (4/8 vs 20/22, p=0.0294). Patients with a diagnosis of MF had lower SFU and RSV for IFNg than patients with CML, PV and ET (SFU: 93 vs 294, 224 and 446, p=0.09/0.002/0.15, RSV: 7532 vs 9528, 10499 and 10630, p=0.08/0.001/0.01, Fig. 1f) Image: Summary/Conclusion: We show overall high frequency of seroconversion and memory T cell responses but demonstrate the need for alternative strategies in some groups, including patients taking ruxolitinib. The increase in T cell reactivity after 3 doses is of particular significance in view of the reduced neutralisation of the now dominant omicron variant, with studies showing T cell functionality is maintained against omicron. Updated results will be presented. P991: IMPAIRMENT OF TCR SIGNALLING IN PATIENTS WITH MYELOFIBROSIS TREATED WITH RUXOLITINIB P. Harrington1,2,*, R. Dillon1, D. Radia1, D. McLornan1, N. Curto-Garcia1, C. Woodley1, S. Asirvatham1, S. Kordasti1,2, C. Harrison1,2, H. de Lavallade1,2 1Clinical Haematology, Guy’s & St Thomas’ NHS Foundation Trust; 2School of Cancer & Pharmaceutical Science, King’s College London, London, United Kingdom Background: Ruxolitinib (rux) is a JAK1/JAK2 inhibitor with varied immune effects including on T cell, NK cell and dendritic cell function. Therapeutic effects are largely attributed to marked reduction in pro-inflammatory cytokines. Aims: We hypothesised that impairment of T cell receptor (TCR) signalling through off-target binding of Src kinases, including Lck, would be an additional mechanism of immune dysfunction. Methods: We performed phosphoflow cytometry in Tregs, T effectors and NK cells to assess the effect on signalling downstream from the TCR and activating NK cell receptors. 11-colour flow cytometry was performed after cells were activated with H2O2 for 15 minutes, due to its activity as a potent phosphatase inhibitor and cells were analysed for phosphorylation of ZAP70 as a surrogate of TCR signalling and STAT5. A gating strategy of CD4+/CD25+/FOXP3+/CD127lo cells was used for identification of Tregs. Results: Phosphoflow analysis was performed in 7 patients with a diagnosis of MF managed with rux and compared with 7 healthy controls (HC). 4 (57%) of patients were male and mean age was 61 (range 44-76). Patients were taking a median rux dosage of 30mg daily (10-50). Three patients had a DIPPS+ score of Int-2, 2 Int-1 and 2 low-risk. Results of phosphoflow analysis are expressed as relative fluorescence intensity (RFI) defined as MFI in H2O2 stimulated sample/MFI in unstimulated sample. As expected, patients on rux had significantly reduced phosphorylation of pSTAT5 when compared with HC in all cell subsets evaluated. The mean RFI of pSTAT5 in CD4+ and CD8+ cells in HC was 21.9 and 31.1 respectively, compared with 6 and 8.5 in rux patients (p=0.001/p=0.002). In CD56+ cells this was 46.2 vs 7.7 (p=0.005) and in Tregs this was 21.9 vs 6.1 (p=0.019). Of note, a difference in phosphorylation was also observed for pZAP70 indicative of inhibition of TCR proximal signalling. For pZAP70 mean RFI in CD4+ cells was 8.8 in controls compared with 2.2 in rux patients (p=0.05). In CD8+ cells mean RFI was 11.4 in controls and 2.4 in rux patients (p=0.04). In CD56+ cells mean RFI was 17.8 vs 2.4 (p=0.02), whilst in Tregs, significance was not reached with mean RFI of 6.3 vs 2.2 (p=0.13). Univariate analysis of rux patients showed no effect of age, comorbidities, DIPPS+ score or rux dosage on TCR or STAT5 signalling. Findings were compared with those observed in 4 patients with CML and deep molecular response on nilotinib, in view of the highly specific ABL1 binding properties of this TKI. As expected, patients on nilotinib had significantly higher RFI for pSTAT5 compared with rux patients, at 23, 32.8, 21.2 and 46.5 in CD4+, CD8+, Treg and NK cells respectively (p=0.002/p=0.002/p=0.006/p=0.003). Interestingly, patients on nilotinib also had significantly higher RFI for pZAP70 in CD4+, CD8+ and NK cells at 6.7, 7.4 and 8.1 compared with rux patients (p=0.035/p=0.025/p=0.037). Image: Summary/Conclusion: Studies have shown that rux inhibits T cell proliferation and reduces CD4+ cell cytokine secretion. Previous in vitro analysis of rux on CD4+ cells failed to identify an effect on TCR signalling after CD3/CD28 stimulation. In our ex-vivo analysis, using H2O2 stimulation, we observe TCR signalling inhibition in CD4+, CD8+ and NK cells, although reduced compared with effects on STAT5. It has been shown that rux inhibits Lck with an IC50 of 3.6uM. Whilst this is significantly less than the inhibitory effect on JAK kinases, there is ~34% sequency homology between Src and JAK family kinases in the kinase domain, suggesting off target kinase inhibition is implicated in the impairment of TCR signalling. P992: OUTCOMES OF GLOBAL COAGULATION ASSAYS IN PATIENTS WITH PHILADELPHIA-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS WITH RESPECT TO THEIR CLINICAL AND GENETIC DETERMINANTS OF CLONAL EVOLUTION A. Lucchesi1,*, R. Napolitano2, M. T. Bochicchio2, G. Simonetti2, G. Micucci1, M. Poggiaspalla1, V. Di Battista1, G. Musuraca1, F. Foca3, G. Giordano4, L. Catani5, M. Napolitano6 1Hematology Unit; 2Biosciences Laboratory; 3Unit of Biostatistics and Clinical Trials, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola; 4Internal Medicine Division, Hematology Service, Regional Hospital “A. Cardarelli”, Campobasso; 5IRCCS Azienda Ospedaliero-Universitaria di Bologna, Department of Experimental, Diagnostic and Specialty Medicine, School of Medicine, Institute of Hematology “Seràgnoli”, University of Bologna, Bologna; 6Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties and Haematology Unit, University Hospital “P. Giaccone”, Palermo, Italy Background: Classical Myeloproliferative Neoplasms (MPNs) are hematopoietic stem cell diseases characterized by inflammation, promotion of atherosclerosis, hypercoagulability, fibrosis, and clonal evolution. Our previous studies in cytofluorimetry have shown that the reduced expression of platelet fibrinogen receptor (PFR) - common to all MPNs - can be due to the hyperactivation of plasma-dependent mechanisms, such as tissue factor (TF) release, an unbalanced thrombin generation and the marked involvement of Protease Activated Receptors (PARs) and their signaling crosstalk with TGF-ß. Acetylsalicylic acid (ASA) seemed to be able to restore the expression of PFRs. Aims: The aim of the present study is to verify the existence of imbalances in hemostatic function in rotational thromboelastometry (ROTEM), with respect to the different classical forms of MPN, their cell counts, the presence of genetic mutations in Next Generation Sequencing (NGS), PFR expression, the use of ASA and the history of vascular events. Methods: We enrolled 53 patients affected by MPNs, naïve from any therapy, with the only exception of ASA (taken by 50% of patients). We used the samples to run assays of genetics (30-gene panel in NGS - SOPHiA Myeloid Solution™, SOPHiA Genetics, Switzerland), cytofluorimetric determination of PFRs, global coagulation by rotational tromboelastometry (ROTEM® Delta, Werfen, Spain) for INTEM, EXTEM, FIBTEM parameters. Results: ROTEM parameters appear to exhibit substantial variations, depending on the type of disease under investigation, cell counts, and selected mutations. Since most of the values are in the normal range, the results should be read in terms of “efficiency” or “preferential use” of one pathway over another. In particular, essential thrombocythemia (ET) and CALR mutation seem to be related to an increased efficiency of both classical coagulation pathways, with significantly more contracted clot formation times (CFT). In contrast, primary myelofibrosis (PMF) and polycythemia vera (PV) show greater imbalances in the hemostatic system. PV patients have longer CFT (EXTEM 114 sec, p= 0.008, INTEM 82 sec, p=0.027) and at the same time a selective contraction of the parameters in INTEM for higher platelet (Plt), white blood cells (WBC) and hemoglobin (Hb) levels. PMF - on the contrary - seems to exploit more the extrinsic pathway (EXTEM) - figure A-B. In multivariate analysis, the major contribution to the alterations is credited to Hb values for PV, while only Plt retains statistical significance for PMF and ET. Selected mutations are associated with changes in ROTEM parameters. Among the DTA mutations, the presence of DNMT3A shows a significant reduction in clotting time (CT) in EXTEM, while ASXL1 is associated with reduced maximum lysis (ML). EZH2 could be responsible for CFT elongations in the INTEM assay. In addition, increased expression of PFRs is associated with bleeding history and sustained CT time at FIBTEM under ASA prophylaxis. Image: Summary/Conclusion: In the most indolent MPNs (such as ET, especially if CALR mutated) the balance and efficiency of the classical pathways is preserved. In PMF, the hemostatic system is unbalanced towards the extrinsic pathway, confirming the connection between increasing fibrosis and plasma-dependent hypercoagulable states. In contrast, rheological characteristics and endothelial stress in PV seem to result in a greater involvement of the intrinsic pathway. ROTEM and PFR expression confirm the existence of dynamic states of hypercoagulability in MPNs and could be used to assess the efficacy of prophylaxis. P993: SETD2 IS A BONA FIDE TUMOR SUPPRESSOR IN SYSTEMIC MASTOCYTOSIS M. Mancini1,*, C. Monaldi2, S. De Santis2, C. Papayannidis1, M. Rondoni3, C. Sartor1, S. Bruno2, A. Curti1, R. Zanotti4, M. Bonifacio4, L. Scaffidi5, L. Pagano5, M. Criscuolo5, F. Ciceri6, C. Elena7, P. Tosi8, P. Valent9, M. Cavo10, S. Soverini2 1IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; 2Department of Experimental Diagnostic and Specialty Medicine - DIMES, Institute of Hematology “L. e A. Seràgnoli”, University of Bologna, Bologna; 3Azienda USL della Romagna, Ravenna; 4Azienda Ospedaliera di Verona, Verona; 5Fondazione Policlinico Universitario A. Gemelli, Roma; 6Servizio Immunoematologia Trasfusionale (SIMT), IRCCS Ospedale San Raffaele, Milano; 7Fondazione I.R.C.S.S. Policlinico San Matteo, Pavia; 8U. O. di Oncologia ed Oncoematologia-Osp. Infermi Azienda Unità Sanitaria-Locale di Rimini, Rimini, Italy; 9Department of Hematology, Internal Medicine I, Medical University of Vienna, Vienna, Austria; 10IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; Department of Experimental Diagnostic and Specialty Medicine - DIMES, Institute of Hematology “L. e A. Seràgnoli”, University of Bologna, Bologna, Italy Background: The trimethylation of histone H3 at lysine 36 (H3K36me3), catalyzed by the SETD2 methyltransferase, is a post-translational modification involved in the fidelity of transcription and splicing and in the recruitment of DNA repair machinery. Recent studies have emphasized the tumor-suppressive role of SETD2, especially in renal cancer where SETD2 is often deleted or mutated. A novel mechanism of SETD2 non genomic loss of function due to proteasome-mediated degradation has recently been reported in advanced systemic mastocytosis (advSM). Proteasome inhibition has been found to inhibit clonogenic growth and induce apoptosis, but whether this is due to SETD2 re-expression remains to be demonstrated. Aims: The aim of our study is to demonstrate unequivocally that forced expression of SETD2 in a cellular context in which it was lost, may restore proliferation control, clonogenic capacity and DNA damage repair mechanisms. Methods: The SETD2-deficient HMC-1.2 mast cell leukemia cell line was used as in vitro model. Forced SETD2 re-expression was obtained by nucleofection: 106 HMC-1.2 cells were resuspended in 85μl of cell line Nucleofector Solution V and 2μg of a SETD2 (GFP-tagged) construct were added. An empty vector coding for GFP was used as negative control. Cells were transfected by using the Lonza Nucleofector 2b and the Lonza Amaxa Cell Line Nucleofector Kit V according to manufacturer’s instructions. GFP expression was assessed using a Cytoflex flow cytometer 24 and 48 hours post transfection; 78% of fluorescence positivity was observed at 48 hours. Neomycin selection (1mg/ml) was performed to obtain stable SETD2 expression. One month after selection, SETD2 and H3K36me3 were assessed by Western Blotting (WB). Clonogenic capacity was tested by clonogenic assays. Immunofluorescence experiments were performed to assess if SETD2 forced expression was able to restore DNA damage repair by using p-H2AX (S139), RAD51, MSH6 and THEX1 antibodies. Results: We previously characterized the HMC-1.2 cell line as deficient for SETD2/H3K36me3, as virtually all patients with advSM. To investigate whether SETD2 may indeed play a tumor suppressor role in SM, we transfected HMC-1.2 cells with an ectopic SETD2 plasmid. After transfection, the morphology of cells dramatically changed; moreover, HMC-1.2tsSETD2 showed a 70% increase in doubling time. Co-immunoprecipitation demonstrated that tsSETD2 was able to interact with p53 and to restore its expression and activity. We thus observed an increase in p21 and p27 associated with an accumulation of cells at the G1/S checkpoint. Moreover, SETD2 stable transfection restored DNA damage responses, as demonstrated by an increase in H2AX phosphorylation and RAD51 (HR), THEX1 (DNA replication) and MSH6 (MMR) expression after UV exposure. Finally, clonogenic assays in control and HMC-1.2tsSETD2 cells showed that: 1) SETD2 re-expression restores cell proliferation control; 2) reduction of clonogenic growth observed after proteasome inhibition is indeed SETD2-dependent. Summary/Conclusion: In advSM, SETD2 is a bona fide tumor suppressor and its loss impairs proliferation control and DNA damage responses. Its overexpression restores cell proliferation control by stabilizing p53 activity, and DNA damage repair by rescuing the H3K36me3 mark. This validates the therapeutic potential of interfering with the mechanisms responsible for SETD2 loss. Supported by AIRC IG 2019 grant (23001). P994: HEDGEHOG PATHWAY INVOLVEMENT IN MASTOCYTOSIS DEVELOPMENT L. Polivka1,2, V. Parietti3, J. Bruneau4, E. Soucie5, P. Dubreuil5, C. Bodemer2, O. Hermine6, L. Maouche Chretien7,* 1IMAGINE Institute, Paris-Centre University, INSERM U1163; 2Department of Dermatology, Hôpital Necker-Enfants Malades; 3Department of animal experimentations, Institut Universitaire d’Hématologie, Université Paris Diderot, Sorbonne Paris Cité; 4Department of Pathology, Necker-Enfants Malades Hospital, Paris; 5Centre de Recherche en Cancérologie de Marseille, INSERM U1068, Marseille; 6Department of Hematology, Necker-Enfants Malades Hospital; 7Imagine Institute, Paris-Centre University, INSERM 1163, Paris, France Background: Mastocytosis is a rare and heterogenous disease characterized by abnormal accumulation and activation of mast cells in one or several organs. The clinical spectrum of the pathology varies from relatively mild forms with isolated skin lesions to very aggressive forms with wide systemic involvement, often fatal. The disease can affect both children and adults. While most pediatric forms resolve during or in late puberty, adult cases are persistent and may evolve into aggressive forms. A somatic KIT D816V mutation is detected in 85% of patients but all attempts to demonstrate its oncogenic effect have failed. There is strong evidence that the KIT D816V mutation alone cannot trigger the disease and that other genes may be involved. Aims: The French national reference center of mastocytosis (CEREMAST) has recorded more than 2000 patients with sporadic mastocytosis and 60 familial cases. Since KIT mutations do not fully explain the pathophysiology of the disease, we sought to identify other mutations that could act synergistically with KIT D816V to induce the disease. Methods: We focused on patients presenting with mastocytosis in particular syndromic contexts within the pediatric cohort followed at Necker hospital; we first investigated the molecular mechanisms underlying mastocytosis onset in patients with both congenital mastocytosis and an extremely rare polymalformative syndrome. Results: From 3 children presenting with both Greig cephalopolysyndactyly syndrome (GCPS) and congenital mastocytosis, we could demonstrate the implication of the hedgehog (Hh) pathway in mastocytosis. GCPS is a very rare syndrome resulting from haploinsufficiency of GLI3, the major repressor gene of the hedgehog (Hh) family. We showed that the Hh pathway is barely active in normal primary mast cells (MCs) and overactive in neoplastic MCs. Using a GCPS mouse model, we demonstrated that mutations in GLI3 and KIT have a synergistic, tumorigenic effect leading to mastocytosis onset. We also showed that Hh inhibitors suppress abnormal mast cell proliferation, in vitro, and extend the survival of mice with aggressive systemic mastocytosis (ASM). Summary/Conclusion: Thus, for the first time, we revealed the involvement of Hh signaling pathway in the pathophysiology of mastocytosis and demonstrated the cooperative effects of the KIT and Hh oncogenic pathways in ASM. Finally, we provided the first evidence that Hh inhibitors represent a promising new therapeutic target. P995: MYELOID NEOPLASMS-ASSOCIATED GENE VARIANTS IN 639 PATIENTS WITH POST-POLYCYTHEMIA VERA AND POST-ESSENTIAL THROMBOCYTHEMIA MYELOFIBROSIS: AN ANALYSIS OF THE MYSEC COHORT B. Mora1,2,*, P. Guglielmelli3, A. Kuykendall4, M. Maffioli1, G. Rotunno3, R. S. Komrokji4, F. Palandri5, J.-J. Kiladjian6, A. Iurlo7, G. Auteri5, D. Cattaneo7, V. De Stefano8, S. Salmoiraghi9,10, T. Devos11, F. Cervantes12, M. Merli1, A. Campagna13, G. Benevolo14, M. Brociner1, F. Albano15, J. Gotlib16, M. Caramella17, M. Ruggeri18, D. M. Ross19, F. Orsini20, C. Pessina20, I. Colugnat20, F. Pallotti2,20, T. Barbui10, L. Bertù2, M. G. Della Porta13, A. M. Vannucchi3, F. Passamonti1,2 1Hematology, Ospedale di Circolo, ASST Sette Laghi; 2Department of Medicine and Surgery, University of Insubria, Varese; 3Center for Research and Innovation of Myeloproliferative Neoplasms, University of Florence, Florence, Italy; 4Malignant Hematology Department, Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America; 5Institute of Hematology, Azienda Ospedaliero-Universitaria S.Orsola-Malpighi, Bologna, Italy; 6Centre d’Investigations Cliniques, Hôpital Saint-Louis, Paris, France; 7UOC Hematology, Foundation IRCCS Ca’Granda Ospedale Maggiore Policlinico, Milan; 8Hematology, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, Rome; 9Hematology and Bone Marrow Transplant Unit; 10FROM Research Foundation, ASST Papa Giovanni XXIII, Bergamo, Italy; 11Hematology, University Hospitals Leuven, Rega Institute, Leuven, Belgium; 12Hematology, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain; 13Cancer Center, IRCCS Humanitas Clinical and Research Center, Rozzano; 14Hematology, Azienda Universitario-Ospedaliera Città della Salute e della Scienza di Torino, Turin; 15Hematology - Dept. of Emergency and Organ Transplantation, University of Bari, Bari, Italy; 16Division of Hematology, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, California, United States of America; 17Hematology, ASST Grande Ospedale Metropolitano Niguarda, Milan; 18Hematology, San Bortolo Hospital, Vicenza, Italy; 19Haematology Directorate, SA Pathology, Royal Adelaide Hospital and Flinders Medical Centre, Adelaide, Australia; 20Laboratory of Cytogenetics and Molecular Biology, Ospedale di Circolo, ASST Sette Laghi, Varese, Italy Background: Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms evolving to post-PV (PPV-) and post-ET (PET-) myelofibrosis (MF), called secondary MF (SMF). In primary MF, knowledge on non-driver myeloid neoplasms-associated gene variants (M-GVs) influences clinical decision-making. In SMF, information on M-GVs is scant. Aims: The primary aim was to assess the pattern of distribution of M-GVs, their correlations with SMF subtype and with driver mutations, in a large cohort of SMF patients studied by next generation sequencing (NGS). Methods: The study involved SMF patients of the MYSEC (Myelofibrosis Secondary to PV and ET) project. In 97% of cases, NGS were performed within one-year pre/post-SMF diagnosis. Characteristics of study cohort were described by standard statistic. Associations were investigated by Chi-square or Fisher exact test. Results: The clinical features of the 639 NGS-annotated patients entering the analysis are reported in Table 1. Table 1. Characteristics of 639 NGS-annotated SMF. SMF (639) PPV-MF (290) PET-MF (349) p Age at SMF (years), mean (SD) 62.2 (11.8) 63.2 (10.7) 61.4 (12.5) 0.17 Male gender, n (%) 323 (51) 151 (52) 172 (49) 0.48 JAK2 mutated, n (%) 477 (75) 290 (100) 187 (53) CALR mutated, n (%) 125 (19) 125 (36) MPL mutated, n (%) 24 (4) 24 (7) Triple negative, n (%) 13 (2) 13 (4) Hb (g/dl), mean (SD) 11.3 (2.1) 12.0 (2.1) 10.7 (1.9) <0.0001 WBC (x10^9/l), mean (SD) 13.7 (12.7) 15.8 (13.9) 11.9 (11.3) <0.0001 PLT (x10^9/l), mean (SD) 368.5 (248.4) 310.6 (211.6) 417.8 (266.4) <0.0001 Blasts (%), mean (SD) 0.7 (1.9) 0.7 (1.6) 0.7 (2.1) 0.59 Constitut. symptoms, n (%) 259 (41) 134 (46) 125 (36) 0.01 A total of 198 (31%) patients did not harbor any M-GV, with no imbalance as for SMF subtype. Among the 441 (69%) with M-GVs, 223 (51%) had one, 137 (31%) two, 52 (12%) three, 23 (5%) four and 6 (1%) five or more. PPV-MF subjects reported more frequently one M-GV, while those with PET-MF at least three (p=0.02). Mean number of M-GVs was 1.4 per patient (range, 0-7). In detail, it was 1.2 (range, 0-4) and 1.5 (range, 0-7) per patient in PPV- and in PET-MF, respectively (p=0.01). The most frequent (≥5% of dataset) M-GVs involved: ASXL1 (n=181, 41%), TET2 (n=145, 33%), DNMT3A (n=49, 11%), TP53 (n=43, 10%), EZH2 (n=39, 9%), SF3B1 (n=31, 7%), U2AF1 (n=29, ~7%), ZRSR2 (n=27, 6%), CBL and RUNX1 (n=21 each, 5%). In PET-MF there was a significantly higher frequency of M-GVs in ASXL1 (47% vs 34%, p=0.01), SRSF2 (5% vs 1%, p=0.01), U2AF1 (9% vs 4%, p=0.04) and CBL (7% vs 2%, p=0.01) compared to PPV-MF. The latter was significantly associated with ETV6 alterations (5% vs 1% in PET-MF, p=0.04). As regards driver mutations, we found an association between “triple negative” status (TN) and M-GVs in SETBP1 (38%, p=0.002), IDH2 (25%, p=0.02), EZH2 (25%, p=0.05), and SRSF2 (25%, p=0.01). Figure 1 shows the frequency of M-GVs found in the MYSEC cohort, distinguished by SMF subtype (a) and driver mutations (b). Image: Summary/Conclusion: Among 639 NGS-annotated SMF cases, 69% presented at least one M-GV with a mean of 1.4 per patient. Overall, the most frequent (≥10%) M-GVs were in ASXL1, TET2, DNMT3A and TP53 genes. Different pathways of progression among PPV- and PET-MF have been disclosed. PPV-MF showed an increased rate of ETV6 alterations, opening the question of possible predisposing genetic factors. TN cases clustered with specific M-GVs, potentially targetable (IDH2). This is the first study exploring the mutational landscape of a wide cohort of SMF patients, paving the way for further investigations on the topic. P996: UTILITY OF KIT P.D816 IN MYELOID NEOPLASM WITHOUT DOCUMENTED SYSTEMIC MASTOCYTOSIS TO DETECT HIDDEN MAST CELLS IN BONE MARROW D. H. Kim1,*, F. Jia1, C. Y. Ok1 1Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: KIT p.D816 mutation is strongly associated with systemic mastocytosis (SM). Although it is not specific for SM, it is one of the diagnostic criteria for SM since its presence in the right morphologic context (i.e. presence of atypical mast cell clusters) is a strong support for neoplastic nature of mast cells. Next-generation sequencing (NGS) is now routinely performed in almost all bone marrow sample and KIT mutations are detected from patients who are not known or suspected to have SM. Aims: Therefore, we wanted to assess if KIT mutations in this patient population are associated with unsuspected SM. Methods: We searched NGS result in our institution between 1/1/2013 and 9/30/2021 with positive result for KIT mutation from patients with known/suspected myeloid neoplasms. Patients with previously documented history of systemic mastocytosis were excluded. Bone marrow biopsies from patients with KIT mutation were assessed with immunohistochemical stains for CD117 and mast cell tryptase (MST). Results: A total of 49 patients who had KIT mutation and had available formalin-fixed paraffin embedded blocks were identified. Thirty-eight (77.6%) patients has acute myeloid leukemia (AML) and 6 patients had chronic myelomonocytic leukemia (CMML, 12.2%). The remaining patients had myelodysplastic syndrome (n=1), myelodysplastic/myeloproliferative neoplasm, unclassifiable (n=1), myeloproliferative neoplasm (MPN, n=1), AML in remission (n=1) and therapy-related myeloid neoplasm (n=1). A total of 41 patients had a single KIT mutation and 8 patients had 2 or more KIT mutations. KIT p.D816V mutation was the most common mutation (n=38, 64.4%), followed by D816Y (n=8, 13.6%), D816H (n=5, 8.5%), Y418_D419insFF (n=2, 3.4%), R815_D816insVL (n=1, 1.7%), S197L (n=1, 1.7%), K558E (n=1, 1.7%), D419del (n=1, 1.7%), N822K (n=1, 1.7%) and R815_D816insIPP (n=1, 1.7%). Immunohistochemical stains for CD117 and MST were performed in all 49 patients. The MST stain was more suitable since 10 patients (20%) had increased blasts with CD117 expression which obscured morphologic review. A total of 4 patient (8.2%) showed mast cell nodules where spindled shaped mast cells were present, meeting the criteria for SM. All four patients had KIT p.D816V mutation and had high mutant allelic frequency (~50%) except one patient (1%). These patients had MPN (n=1), AML (n=1), AML in remission (n=1) and CMML (n=1), respectively. One (2%) patient has increased (>10%) mast cells but scattered throughout the bone marrow space without nodules and mast cells were round-to-ovoid shape. Eleven patients (22.4%) showed rare scattered mast cells and 33 patients (67.3%) did not show mast cells at all. Summary/Conclusion: We discovered approximately 8% of patients who had myeloid neoplasms with unexpected KIT mutations are met for systemic mastocytosis after additional immunohistochemical studies. Our data support that application of additional immunohistochemical studies are recommended to identify underrecognized SM when KIT mutations are unexpectedly found by molecular assays. P997: PROTEOMIC ANALYSIS ON PLATELETS OF ESSENTIAL THROMBOCYTHEMIA PATIENTS UNDERSCORES THE ROLE OF MITOCHONDRIA IN JAK2 V617F PLATELET REACTIVITY AND FUNCTION X. Guerrero-Carreño1, S. Smits2, A. Esteban Lasso3, J. A. Escudero García3, M. Samiotaki4, A. Alvarez-Larrán5, A. Angona6, A. J. Sáen Marín7, V. García Gutiérrez7, D. Dekkers8, J. Demmers8, C. M. Benavente Cuesta9, F. Ferrer-Marín10, J. C. Hernández-Boluda11, F. J. Iborra12, I. M. De Cuyper13, P. Vandenberghe14, P. Papadopoulos1,* 1Hematology, IdISSC, Madrid, Spain; 2Human Genetics, KULeuven, Leuven, Belgium; 3Sanidad Animal and VISAVET, Universidad Complutense de Madrid, Madrid, Spain; 4Institute for Bioinnovation, BSRC “Al. Fleming”, Vari, Greece; 5Hematology, Hospital Clínic; 6Hematology, Hospital del Mar, Barcelona; 7Hematology, Hospital Ramón y Cajal, IRICYS, Madrid, Spain; 8Proteomics Center, ErasmusMC, Rotterdam, Netherlands; 9Hematology, Hospital Clínico San Carlos, Madrid; 10Hematology, Hospital Morales Meseguer, UCAM, IMIB, Murcia; 11Hematology, Hospital Clínico Universitario, INCLIVA; 12Biological Noise and Cell Plasticity, CIPF, Centro de Investigación Príncipe Felipe, Valencia, Spain; 13Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Amsterdam, Netherlands; 14Hematology, UZLeuven, Leuven, Belgium Background: Essential thrombocythemia (ET) is a heterogeneous disease subdivided into five genetic groups according to WHO, based on the common MPN driver mutations (JAK2 V617F, MPL W515K/L, and CALR Type I&II). Current treatment strategies include anti-platelet (e.g. acetylsalicylic acid (ASA)) or cytoreductive agents (e.g. hydrea (HU)) and target mainly complications related to platelet dysfunction (i.e. hemorrhage/thrombosis). Platelet activation is an energy demanding process fueled mainly by a dynamic equilibrium between mitochondria oxidative phosphorylation and glycolysis1. Importantly, altering the platelet catabolic response to activation has been shown to prevent thrombus formation2. JAK2 V617F platelets have been reported to be more activated than CALR mutated platelets3, as well as JAK2 mutated patients have a greater thrombotic risk in comparison to other ET groups. However, the cause of this phenotype is not completely understood. Aims: In this study we aimed to analyze the proteome of ET platelets and to characterize their functional properties according to the mutational background and treatment regimens. Methods: 22 healthy donors (HD) and 67 ET patients have been included in the study. Most of the patients were treated with low dose aspirin (ASA), anagrelide (ANA) or hydrea (HU) or a combination of these. Specifically, 35 out of the 67 ET platelet samples and 8 out of 22 HD were subjected to label-free quantitation mass spectrometry (LFQ-MS). All HD and ET platelets were also analyzed for surface marker expression, degranulation and aggregation capacity by flow cytometry. Results: Hierarchical clustering of the mass spectrometry data revealed different proteomic profiles between HD and ET mutational groups and specifically between JAK2 V617F and CALR I and CALR Type II treated and/or untreated platelets. In general, HD platelets were more enriched for proteins related to platelet activation, and degranulation as compared to ET platelets and clustered next to JAK2 V617F ASA-treated platelet samples (Figure 1). In particular, the JAK2 V617F ASA-treated platelets presented significant enrichment in platelet activation metabolic and mitochondrial proteins in contrast to the CALR Type I and Type II ET platelets. Of note, no peptides corresponding to the mutant CALR were detected using label-free MS methods. Surface marker expression was variable among ET patients. However, CD49B and CD36 surface markers were the most affected among the JAK2V617F and CALR Type I and II platelets and their levels (mean fluorescence intensity-MFI-) were inversely correlated. With regards to platelet aggregation, JAK2 V617F platelets presented similar or lower levels when compared to HD platelets, in line with their proteomic profiles. Differences in aggregation and degranulation levels were also observed between CALR Type I and Type II platelets specifically in the untreated condition. Image: Summary/Conclusion: ET platelets are functional but they show different capacities to respond upon various stimuli and treatment regimens. JAK2 V61F platelets are more activated than any other ET group according to their proteome profile, which is not fully reflected by the functional assays. Mitochondrial activity arises as an important factor in the control of platelet reactivity and it could be critical in disease management and treatment strategies, specifically for the JAK2 V617F patients. References 1. Aibibula, M., et al. J Thromb Haemost. 2018; 16(11):2300-2314, 2. Nayak, M.K., et al. Blood Adv. 2018; 2(15):2029-2038, 3. Hauschner, H., et al. Am J Hematol, 2020. 95(4): p. 379-386. P998: REGULATION OF BCL-X SPLICING BY SRC KINASES INHIBITORS IN MYELOPROLIFERATIVE NEOPLASMS J. Petiti1,*, F. Itri1, C. Fava1, D. Cilloni1 1Dept. of Clinical and Biological Sciences, University of Turin, Orbassano, Italy Background: Myeloproliferative neoplasms (MPN) are a group of incurable diseases that include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Both ET and PV are associated with prolonged clinical courses, with symptoms that affect their quality of life, and risk of progression to MF and acute leukemia. MF is the MPN subtype with the most adverse prognosis and is associated with a high risk of leukemic transformation. The FDA approved the use of ruxolitinib, a JAK1/2 kinase inhibitor, that proved to be effective in reducing symptoms and spleen volume, but not in inducing complete molecular remission. Although hematopoietic stem cell transplantation involves a high risk of mortality, it is nowadays the only curative treatment for MF. Therefore, it is relevant to identify new therapeutic strategies to improve the clinical outcome of MPN. Recently, there has been a growing interest in Bcl‐xL, the long isoform encoded by alternative splicing of the Bcl‐x gene, that acts as an anti‐apoptotic regulator. Bcl-xL appears to be up-regulated in MPN patients and we previously showed that its modulation correlates with the clinical severity of MPN subgroups. We also demonstrated that ABT-737, a specific Bcl-xL inhibitor, has a synergistic effect in combination with Ruxolitinib in HEL cell lines and MPN patients’ cells, suggesting that Bcl-xL inhibition could increase the power of treatment (Petiti et al. J Cell Mol Med. 2020). Unfortunately, ABT-737 is characterized by poor bioavailability, so a new approach to target Bcl-xL must be investigated. Paronetto et al. found that tyrosine phosphorylation of Sam68 in cancer cells may protect them from apoptosis by altering the Bcl-xS/L ratio in favor of Bcl-xL. Intriguingly, c-Src is one of the tyrosine kinases that phosphorylate Sam68 and several papers indicated its involvement in the MPN pathogenesis, without explaining the molecular mechanism. Aims: We aimed to molecular investigate the mechanism linking c-Src kinase activity with the deregulation of Bcl-x splicing in MPN, intending to consider c-Src kinase inhibitors, widely bioavailable, as indirect modulators of Bcl-xL to improve MPNs outcome. Methods: HEL cell lines, JAK2 V617F-mutated in homozygosis, has been incubated with different concentrations of ruxolitinib (JAK1/2 inhibitor) and dasatinib (SRC kinases inhibitor) at different concentrations and time points. Then, proliferation, and apoptosis were evaluated. Subsequently, the c-Src pathway, Sam68, and Bcl-xL/S ratio were investigated at both RNA and protein levels. Results: Performing in vitro study on HEL cell lines, we identified that dasatinib induced apoptosis and inhibition of proliferation in HEL cell lines, with a synergistic effect in combination with ruxolitinib. We also showed that dasatinib alone inhibited c-Src and Sam68 phosphorylation and down-modulated Bclx-L protein expression. Combination treatment with ruxolitinib and dasatinib increased both pro-apoptotic Cleaved Caspase 3 and BAX protein expression. Furthermore, we noted that treatment with ruxolitinib alone decreases the mRNA levels of Bclx-L, but also of the Bcl-xS isoform. On the contrary, combined drug therapy modulated the Bcl-xL/S mRNA ratio in favor of the pro-apoptotic Bcl-xS isoform. Summary/Conclusion: Although further studies will be necessary to better understand the c-Src role in Bcl-x splicing regulation in MPN, our preliminary data suggest that indirect downmodulation of Bcl-xL through SRC kinases inhibitors in combination with standard therapy could be useful in the future for the treatment of MPN patients. P999: ERK1/2 INHIBITION REDUCES OSTEOPONTIN PLASMA LEVELS AND BONE MARROW FIBROSIS IN A MYELOFIBROSIS MOUSE MODEL S. Rontauroli1,*, E. Bianchi1, L. Tavernari1, M. Dall’Ora2, G. Grisendi3, M. Mirabile1, S. Sartini1, E. Genovese1, C. Carretta1, S. Mallia1, S. Parenti1, L. Fabbiani4, N. Bartalucci5, L. Losi6, M. Dominici3, A. M. Vannucchi5, R. Manfredini1 1Centre for Regenerative Medicine, Life Sciences Department, University of Modena and Reggio Emilia, Modena; 2Rigenerand S.R.L., Medolla (MO); 3Division of Oncology, Laboratory of Cellular Therapy, Department of Medical and Surgical Sciences of Children & Adults; 4Department of Medical and Surgical Sciences of Children & Adults, Pathology Unit, University of Modena and Reggio Emilia, Modena; 5Department of Experimental and Clinical Medicine, and Center Research and Innovation of Myeloproliferative Neoplasms (CRIMM), University of Florence, Careggi University Hospital, Florence; 6Department of Life Sciences, Pathology Unit, University of Modena and Reggio Emilia, Modena, Italy Background: Primary myelofibrosis (PMF) is a stem cell disorder belonging to Philadelphia-negative myeloproliferative neoplasms (MPNs). The disruption of bone marrow (BM) microenvironment due to the extensive deposition of extracellular matrix fibers is the distinctive trait of PMF and is accompanied by hematopoietic stem cells (HSCs) mobilization and extramedullary hematopoiesis. BM fibrosis is caused by the complex interaction between stromal and hematopoietic cells belonging to the neoplastic clone, in particular megakaryocytes and monocytes play a pivotal role through the production of pro-fibrotic cytokines. Supporting this hypothesis, we have previously demonstrated that osteopontin (OPN) contribute to the development of BM fibrosis. OPN plasma levels are increased in PMF patients and correlate with higher BM fibrosis grade, circulating CD34+ cells and inferior survival. Megakaryocytes and monocytes turned out as the main source for OPN production that can induce fibroblasts and mesenchymal stromal cells proliferation, as well as collagen upregulation. Aims: To determine whether OPN might represent a druggable target in PMF we assessed the inhibition of signaling pathways responsible for its production. In particular, we evaluated the effect of ERK1/2 inhibition over OPN expression and myelofibrosis development in a MF mouse model. Methods: The activity of inhibitors of signaling pathways affecting OPN expression was evaluated in vitro in human primary monocytes, given their key role in OPN secretion. The effects on cell viability were assessed by XTT assay at 72 hours of treatment, while OPN expression and production were evaluated by qRT-PCR and ELISA, respectively, at 72 and 96 hours of treatment. Inhibitors of OPN production selected from the in vitro screening were analyzed for their effect on OPN plasma levels in a MF mouse model induced by the treatment with a thrombopoietin receptor agonist Romiplostim (Rom). Hematological parameters and splenomegaly were monitored over time while BM and spleen fibrosis were evaluated at sacrifice. Results: Our in vitro analysis demonstrated that drug inhibitors of ERK1/2, MEK1/2, p38 and a Ca2+ channel antagonist were able to reduce OPN expression in primary monocytes, both at RNA and protein levels, without affecting cell viability. Next, we moved to a MF mouse model obtained through the stimulation of thrombopoietin receptor that develops bone marrow and spleen fibrosis, together with increased OPN expression, faster than JAK2V617F knock-in mice. ERK1/2 inhibition by Ulixertinib did not affect the development of thrombocytosis and splenomegaly but was able to significantly reduce OPN plasma levels. Interestingly, ERK1/2 inhibition also counteracted the development of bone marrow fibrosis in Rom-treated mice. The same results were observed in mice when Ulixertitnib was given together with the JAK inhibitor Ruxolitinib. Drug combination resulted in a reduction of both OPN plasmatic levels and bone marrow fibrosis development. Summary/Conclusion: Our results demonstrated that Ulixertinib treatment reduced OPN production both in vitro and in vivo. Moreover, in a MF mouse model, the concurrent inhibition of ERK1/2 and JAK2 signaling pathways displayed synergistic effects by diminishing OPN plasma levels and constraining BM fibrosis. These results provide a rational for the development of novel combination therapeutic approach for PMF patients. P1000: INCREASED PLASMA LEVELS OF LNCRNAS ARE POTENTIAL PROGNOSTIC BIOMARKERS IN MYELOFIBROSIS S. Sartini1,*, S. Fantini1, S. Rontauroli1, M. Mirabile1, E. Bianchi1, F. Badii1, M. Maccaferri2, P. Guglielmelli3, T. Ottone4,5, R. Palmieri4, E. Genovese1, C. Carretta1, S. Parenti1, S. Mallia1, L. Tavernari1, C. Salvadori3, F. Gesullo3, C. Maccari3, M. Zizza3, A. Grande6, S. Salmoiraghi7, B. Mora8, L. Potenza9, V. Rosti10, F. Passamonti8, A. Rambaldi7, M. T. Voso4,5, C. Mecucci11, E. Tagliafico9,12, M. Luppi9, A. M. Vannucchi3, R. Manfredini1 1Center for Regenerative Medicine, Life Sciences Department, University of Modena and Reggio Emilia; 2Department of Laboratory Medicine and Pathology, Diagnostic Hematology and Clinical Genomics, AUSL/AOU Policlinico, Modena; 3Department of Experimental and Clinical Medicine, and Center Research and Innovation of Myeloproliferative Neoplasms (CRIMM), University of Florence, Careggi University Hospital, Florence; 4Department of Biomedicine and Prevention, University of Tor Vergata; 5Neuro-Oncohematology, Santa Lucia Foundation, Istituto di Ricovero e Cura a Carattere Scientifico (I.R.C.C.S.), Rome; 6Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena; 7Hematology, ASST Papa Giovanni XXIII, Bergamo; 8Division of Hematology, Ospedale ASST Sette Laghi, University of Insubria, Varese; 9Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, AOU Policlinico, Modena; 10Center for the Study of Myelofibrosis, Foundation Policlinico San Matteo, Istituto di Ricovero e Cura a Carattere Scientifico (I.R.C.C.S.), Pavia; 11Department of Medicine and Surgery, Section of Hematology and Clinical Immunology, University of Perugia, Perugia; 12Center for Genome Research, University of Modena and Reggio Emilia, Modena, Italy Background: Myelofibrosis (MF) displays the worst prognosis among Philadelphia-negative myeloproliferative neoplasms and is characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and frequent transformation to acute myeloid leukemia. Different therapeutic approaches are being used depending on the severity and the specific clinical manifestations of the disease in each patient. Unfortunately, no curative therapy is currently available for MF, except for bone marrow transplantation, which however has a consistent percentage of failure. There is therefore a great urgency to identify biomarkers correlated with the different stages of the disease and to treat patients with a more tailored treatment. Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. Moreover, circulating lncRNAs have already been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis. Aims: The aim of this study was to identify circulating lncRNAs whose plasmatic concentration differs between MF patients and healthy donors (HDs). Subsequently, we evaluated their potential role as non-invasive disease biomarkers in MF. Methods: As a preliminary result we analyzed the expression of 38 lncRNAs in CD34+ cells collected from 83 MF patients and 26 HDs leading to the identification of 26 differentially expressed lncRNAs. To confirm these results and to move to a more accessible sample type we collected plasma from 143 MF patients and 65 HDs. RNA was extracted from these samples and the relative abundance of lncRNAs, including some of those deregulated in CD34+ cells or already described as involved in hematological malignancies and myeloid differentiation, was assessed using qRT-PCR. According to the plasmatic levels of each lncRNA patients were split into two groups (low- or high-) and this subdivision was used to unveil the potential correlation between the various lncRNAs and the clinical features of MF patients. Results: Our analysis identified 7 lncRNAs significantly upregulated in MF patients’ plasma compared to HDs. Among these, high levels of LINC01268, MALAT1 or GAS5 correlated with several detrimental clinical features of MF, such as high counts of leukocytes and CD34+ cells, a severe grade of bone marrow fibrosis and the presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (Log-rank p-value = 0.0018), GAS5 (Log-rank p-value = 0.0008) or MALAT1 (Log-rank p-value = 0.0348) were associated with a poor overall-survival (OS) while high levels of LINC01268 correlated also with a shorter leukemia-free-survival (LFS). Finally, multivariate analysis demonstrated that a high plasma concentration of LINC01268 was an independent prognostic variable for both OS (HR = 2.104; confidence interval (CI) = 1.08–4.12; p = 0.0297) and LFS (HR = 8.190; CI = 1.02–65.78; p = 0.0479). Summary/Conclusion: To our knowledge, this is the first study describing the expression profile of circulating lncRNAs in MF patients’ plasma and focusing on their putative role as biomarkers in clinical practice. In particular, our results demonstrated that increased levels of circulating LINC01268, GAS5 or MALAT1 are associated with disease detrimental features and correlate with an inferior OS in MF patients. Notably, multivariate analysis confirmed that LINC01268 plasma levels might improve the identification of patients with a poor prognosis. If the prognostic value of this lncRNA will be confirmed in independent patients’ cohorts it might be used to integrate contemporary prognostic models. P1001: DNMT3A/TET2/ASXL1 MUTATIONS DETERMINE THROMBOTIC RISK IN POLYCYTHAEMIA VERA A. Segura Diaz1, R. Stuckey1,*, Y. Florido Ortega1, M. Sobas2, A. Álvarez Larrán3, F. Ferrer-Marín4, M. Pérez Encinas5, G. Carreño6, M. Fox7, B. Tazón Vega7, B. Cuevas8, J. López Rodriguez1, N. Farías Sánchez1, C. Bilbao Sieyro1, M. Gómez Casares1 1H.U.Dr. Negrin de GC., Las Palmas, Spain; 2Wroclaw Medical University, Wrocław, Poland; 3H. Clínic de Barcelona, Barcelona; 4Hospital Morales Messeguer, IMIB, UCAM, Murcia; 5Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela; 6Hospital Universitario 12 de Octubre, Madrid; 7, Hospital Universitari Vall d’Hebron, Barcelona; 8Hospital Universitario de Burgos, Burgos, Spain Background: Polycythaemia vera (PV) is the myeloproliferative neoplasm with the highest incidence of thrombosis, with cardiovascular events being the main cause of death in these patients. Recent studies have identified clonal haematopoiesis (CHIP) in healthy individuals, which is associated with older age and an increased risk of developing both myeloid neoplasms and vascular events. Aims: In a previous study we saw an association between the presence of mutations in DTA genes (DNMT3A, TET2 and ASXL1, the most frequently mutated genes in CHIP), and vascular events in patients with PV. We aimed to confirm this association in a consecutive series of PV patients from our centre and in an age-matched case control study with patients from various European centers. Methods: All patients aged 18 years and above with a confirmed diagnosis of PV in our hospital were recruited consecutively between 2003 and 2018. NGS was performed on 200 ng genomic DNA extracted from peripheral blood at diagnosis. Sequencing was performed using a MiSeq (Illumina) with the 30-gene panel Myeloid Solution (SOPHiA Genetics). Only variants with an allelic frequency (VAF) ≥ 2% and annotated as pathogenic or probably pathogenic were considered. Additional mutations are defined as pathogenic mutations in any non-driver gene from the myeloid panel. The case-control study included PV patients with a thrombotic event in their patient history and the results of a myeloid NGS panel available from 9 Spanish and 1 Polish hospital. Cases were gender- and age-matched with control patients (with a diagnosis of PV but no thrombotic event) from the consecutive series. Results: A total of 79 patients with PV were analysed in our consecutive series. An association was observed between DTA mutation and thrombotic event (OR 4.5; p=0.002; χ2). An association was also observed between any non driver mutation from the myeloid panel and thrombotic event (OR 7.7; p=<0.0001; χ2). However, no association was seen between non-DTA mutation and thrombotic events (p=0.231; χ2). The association between DTA mutation and thrombotic event was confirmed in multivariate analysis (p=0.021, Table 1. Thrombotic event was associated with age (p=0.020) and marginally with leukocyte count at diagnosis (p=0.065) but not with JAK2 VAF. We saw that when the JAK2 mutation comes before DTA, there is a tendency for the risk of thrombosis to be higher than for DTA first. We also observed a positive association between cardiovascular risk factors (CVRF) and thrombotic events (Table 1), as expected. However, its significance was lost in the multivariate analysis. The DTA and CVRF variables are closely related, with a positive association between them (OR 6.77; p=0.009; χ2). Fifty eight of 79 patients (73.4%) were hypertensive; however, an association was still found between thrombotic event and DTA mutation in this group of hypertensive patients (p=0.033). The association between DTA and thrombotic events in the uni and multivariate analyses was confirmed by the case-control study of 47 cases and 47 gender- and age-matched controls (OR 2.7; p=0.036; χ2). Image: Summary/Conclusion: We were able to confirm the previously observed association in PV between the presence of DTA mutations and higher risk of developing a vascular event. Thus, detection of DTA mutation by NGS could help predict thrombotic risk in PV patients, including those with pre-existing CVRF. P1002: ANTI-FIBROTIC ACTIVITY OF BMP2 IN BONE MARROW-DERIVED MESENCHYMAL STROMAL CELLS OF MYELOPROLIFERATIVE NEOPLASMS T. Subotički1,*, E. Živković1, O. Mitrović Ajtić1, M. Vukotić1, D. Đikić1, T. Dragojević1, D. Šefer2, S. Bižić2, J. F. Santibanez1, M. Gotić3, V. P. Čokić1 1Department for Molecular Oncology, Institute for Medical Research, National Institute of Republic of Serbia; 2Clinic of Hematology, University Clinical Center of Serbia; 3Faculty of Medicine, University of Belgrade, Belgrade, Serbia Background: Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Bone marrow fibrosis (BMF) is a shared feature of all MPN, although it is most pronounced in PMF and represents a major diagnostic criteria. Studies suggest a correlation between the grade of BMF and prognosis of MPN, with more fibrosis associated with worse outcome. A large body of evidence has suggested that transforming growth factor beta (TGF-β) is among the most prominent inducers of fibrotic processes. Bone morphogenetic proteins (BMPs) are major regulators of cell fate in tissue homeostasis. In MPN, bone marrow-derived mesenchymal stromal cells (BM-MSC) are identified as a major cellular source of fibrosis, but exact molecular mechanism involved have not been identified so far. Aims: In addition to apoptosis and prolifration, we analyzed the effect of BMP2 and TGF- β / SMAD signaling pathway on the fibrotic phenotype of BM-MSC isolated from MPN patients and healthy donors. Methods: Bone marrow aspirates from 5 newly diagnosed MPN patients (3 PMF and 2 PV patients) and 3 healthy donors were analyzed by immunofluorescence expression of fibronectin and alpha smooth muscle Actin (αSMA), after treatment with TGF-β and / or BMP2. Using immunocytochemistry, we analyzed HEL 92.1.7 cells with a homozygous expression of JAK2V617F for proliferation (Ki67) and apoptosis (ssDNA) during exposure to BMP2 and selective BMP signaling inhibitor LDN-193189. Results: Our results showed that TGF-β significantly increased fibronectin expression, in contrast to BMP2, in BM-MSC of healthy donors. Also, the joint treatment of TGF-β and BMP2 reduced the level of fibronectin expression relative to TGF-β. In addition, TGF-β increased αSMA expression in BM-MSC from healthy donors. In contrast, BMP2 reduces the expression of αSMA and fibronectin in BM-MSC of healthy donors.BMP2 significantly reduced fibronectin expression in BM-MSC compared to untreated cells of patients with MPN. BMP2 dose dependently and significantly (p<0.01) increased the proliferation of HEL 92.1.7 cells, while the BMP signaling inhibitor LDN-193189 also demonstrated dose dependence in stimulation of proliferation (p<0.001). Apoptosis of HEL 92.1.7 cells was slightly affected by BMP2 and LDN-193189. Summary/Conclusion: Our results show that BMP2 has anti-fibrotic activity that is antagonistic to TGF-β. This indicate that BMP2 may modify the TGF-β signaling pathway. P1003: PERIPHERAL BLOOD CYTOTOXIC T CELLS SHOW EARLY EXHAUSTED FEATURES IN MYELOFIBROSIS PATIENTS L. Tavernari1,*, S. Rontauroli1, M. Maccaferri2, B. Mora3, E. Bianchi1, S. Parenti1, E. Genovese1, P. Guglielmelli4, C. Carretta1, S. Mallia1, M. Mirabile1, S. Sartini1, C. Colasante5, L. Potenza5, F. Passamonti3, E. Tagliafico5,6, M. Luppi5, A. M. Vannucchi4, R. Manfredini1 1Centre for Regenerative Medicine, Life Sciences Department, University of Modena and Reggio Emilia; 2Department of Laboratory Medicine and Pathology, Diagnostic Hematology and Clinical Genomics, AUSL/AOU Policlinico, Modena; 3Division of Hematology, Ospedale ASST Sette Laghi, University of Insubria, Varese; 4Department of Experimental and Clinical Medicine, and Center Research and Innovation of Myeloproliferative Neoplasms (CRIMM), University of Florence, Careggi University Hospital, Firenze; 5Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, AOU Policlinico; 6Center for Genome Research, University of Modena and Reggio Emilia, Modena, Italy Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by megakaryocyte hyperplasia, bone marrow fibrosis and extramedullary hematopoiesis. Compelling evidence are suggesting that persistent antigen stimulation in the tumor microenvironment can differentiate T effector cells into terminally exhausted T cells, a functional state characterized by decrease in proliferation, cytotoxicity, and cytokine production. Cutting-edge therapies are focused on the reversion of exhausted state through immune checkpoint inhibition to recover the immune response against the tumor. Aims: Despite MPNs are myeloid neoplasms, T cells are showing dysfunctional features. To assess if immune checkpoint inhibition could be a new effective therapy in combination with those currently approved, we investigated T cell exhaustion in MF. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 38 MF patients and 20 healthy donors (HD). The expression profile of the following inhibitory receptors (IRs): PD1, CD244, CD160, TIM3, LAG3, CTLA4 was characterized by flow cytometry in CD3+CD8+ T cells. Results were correlated with clinical features such as Dynamic International Prognostic Scoring System (DIPSS) classification, splenomegaly, hemoglobin level and other negative prognostic factors. For T cell activation assays we stimulated PBMCs overnight in the presence of coated anti-CD3, anti-CD28 and Brefeldin A, whilst cytokine production was then assessed by intracellular flow cytometry staining. Chi-squared and Mann Whitney tests were used for statistical analysis. Total cellular RNA was isolated from peripheral blood granulocytes of 141 primary and secondary MF patients and 28 HDs, and we performed Gene expression profiling (GEPs) by microarray platform. Results: We reported expansion of PD1+CD244+, PD1+CD160+, PD1+CD57- cytotoxic T cell populations from MF patients, together with increased expression of all the IRs assessed on both total CD3+CD8+ T cells and PD1+CD57- T cells fraction. Furthermore, we observed decreased secretion of IFNɣ and TNFɑ by CD3+CD8+ T cells of MF patients, while significant reduction of Granzyme B production was observed mainly in patients with expansion of PD1+CD57- T cell population. GEPs analysis highlighted increased expression of IR-ligands like CD274, CEACAM1, CD48 on granulocytes from MF patients compared to HDs. The correlation analysis of IRs expression with patients’ clinical features showed that higher levels of PD1, CD244, CD160, CTLA4, LAG3 or TIM3 associates with detrimental features like splenomegaly, low hemoglobin levels, HMR mutation number and more severe DIPSS classification. Summary/Conclusion: Our data evidenced in MF patients the presence of an impaired population of peripheral blood cytotoxic T cells expressing multiple IRs and with reduced cytokine production after in vitro activation. Moreover, granulocytes from MF patients display higher level of IR-ligands transcripts hinting an immunosuppressive interplay between the myeloid neoplastic clone and T cells. Lastly, clinical correlations suggest a more severe disease in patients with higher IRs expression on cytotoxic T cells. Taken together this data highlights an early exhausted state likely implicated in immune escape which will be investigated for immune checkpoint inhibition. P1004: EFFECT AND MOLECULAR MECHANISM OF TQ05105, A NOVEL SMALL MOLECULE INHIBITOR OF JAK2 IN MYELOPROLIFERATIVE NEOPLASM W. Zhang1,2,3,4,*, J. Liu1,2,3,4, Z. Shi4, L. Yang1,2,3,4, B. Li1,2,3,4, Z. Xiao1,2,3,4 1State Key Laboratory of Experimental Hematology; 2National Clinical Research Center for Blood Diseases; 3Institute of Hematology & Blood Diseases Hospital, Tianjin; 4Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China Background: The JAK2V617F gene mutation leads to upregulation of the JAK/STAT signaling pathway, which causes excessive bone marrow cell proliferation, elevated inflammatory cytokines, constitutional symptoms and shortened survival in myeloproliferative neoplasms (MPN) patients. The JAK1/2 kinase inhibitor ruxolitinib can dramatically improve disease-related symptoms, but side effect such as anemia and thrombocytopenia limit its use in a substantial proportion of patients. Several JAK2 kinase inhibitor III Clinical trials have yielded unsatisfactory outcomes recently. TQ05105 is a new small molecule inhibitor that targets JAK2 kinase. Aims: We aim to investigate the effect and molecular mechanism of TQ05105 on MPN cell lines and mouse models. Methods: We systematically measured the function of TQ05105 in MPN cell lines, including UKE1 and SET2 cells, by performing proliferation, apoptosis and colony-forming assays. RT-PCR and inflammatory factor multi-assay kit was used to detect the secretion of inflammatory factors such as TNF-α, IL-1β, MCP-1 in mRNA level and protein level respectively. CFU-E and BFU-E assays were performed on MPN cell lines and primary cells. In vivo, TQ05105 was evaluated in JAK2V617F Jak2V617F transplant mouse model and MPL515 retroviral mouse model. Mice were orally administrated with placebo, 25 mg/kg (bid) and 50 mg/kg (bid) every day for 4 weeks. Flow cytometry analysis was performed to determine the proportion of different hematopoietic progenitor cell populations. In addition, HE staining and Gomori staining was used to detect the changes in the spleen and bone marrow. Results: In UKE1 and SET2 cell lines, TQ05105 can inhibit the proliferation and induced cell apoptosis, notably reduce the expression of cytokines, including TNF-α, IL-1 and MCP-1. According to Western blot results, the phosphorylation of JAK/STAT signaling pathway associated proteins JAK2, STAT3 and STAT5 were significantly suppressed by TQ05105. Furthermore, compared to the control group, TQ05105 can strongly limit the ability of UKE1 cell lines, SET2 cell lines and primary cells from MPN patients (PMF, PV, ET) to form BFU-E and CFU-GM. TQ05105 can striking reduce mouse spleen volume, along with favorable impact on leukocyte and erythrocyte counts in Jak2V617F transplant mouse model. The proportion of stem and progenitor cells, especially CMPs and MEPs, was considerably reduced in mice after treatment with TQ05105. HE staining showed remarkably Spleen response such as recovering structure, reduced infiltration of erythrocytes and suppressed proliferation of megakaryocytes. MPLW515L retroviral mouse model recapitulated the feature of myelofibrosis, application of TQ05105 in this model resulted in reduction of the spleen volume, extramedullary hematopoiesis, leukocyte and platelets count, bone marrow fibrosis and decreased fraction of CMPs and MEPs. Summary/Conclusion: TQ05105, as a novel small molecule JAK2 inhibitor, can inhibit cell proliferation, induce cell apoptosis and decrease inflammatory cytokines by affecting the JAK/STAT signaling pathway. In vivo experiments further proved that TQ05105 can reduce spleen size and improve disease-related symptoms of MPN, indicating that it is a promising therapeutic drug for MPN. P1005: A PHASE 1, OPEN-LABEL, DOSE-ESCALATION STUDY OF SELINEXOR PLUS RUXOLITINIB IN PATIENTS WITH TREATMENT-NAÏVE MYELOFIBROSIS H. Ali1,*, A. Kishtagari2, K. Maher3, S. Mohan2, A. Mazumder4, K. Chamoun5, I. Karasik5, E. Sbar5, L. Dugom5, S. Tamir5, X. Wang5, J. Prchal6, S. Tantravahi6 1City of Hope, Duarte; 2Vanderbilt Ingram Cancer Center, Nashville; 3VCU Massey Cancer Center, Richmond; 4The Oncology Institute of Hope & Innovation, St. Petersburg; 5Karyopharm Therapeutics, Newton; 6Division of Hematology and Hematologic Malignancies, Huntsman Cancer Institute, University of Utah, Salt Lake City, United States of America Background: Myelofibrosis (MF) is a myeloproliferative neoplasm commonly associated with gene mutations in JAK2, CALR, or MPL caused by the unregulated proliferation of clonal myeloid precursors in the bone marrow. The JAK 1/2 inhibitor, ruxolitinib (RUX), has shown reductions in spleen volume and improvement in MF-related symptoms when used in the frontline. Despite the significant improvements of RUX, most patients (pts) eventually progress and lose response to treatment over time. Therefore, novel combinations are critical to improve responses and delay progression. Selinexor (SEL) is an oral selective inhibitor of nuclear export (SINE) compound, specifically inhibiting exportin-1 (XPO1), that has been approved for use in multiple myeloma and diffuse large B-cell lymphoma. Preclinical studies of the combination SEL and RUX have demonstrated significant activity. In clinical studies of MF refractory to JAK inhibitors, SEL monotherapy has exhibited robust clinical activity with a tolerable safety profile (NCT03627403). Aims: Here, we present the initial results of a phase 1 dose escalation study to determine the optimal dose and preliminary efficacy of once weekly (QW) SEL in combination with RUX in pts with treatment-naïve MF. Methods: In the ongoing multicenter, open-label, Phase 1/2 study (NCT04562389) using a 3 + 3 design, two dose levels of SEL were evaluated, 40 mg and 60 mg QW plus RUX twice daily (BID) as per label in 28-day cycles. For nausea prophylaxis, all pts received a 5-HT3 antagonist. Primary study objectives include safety, maximum tolerated dose (MTD), recommended Phase 2 dose (RP2D), and preliminary efficacy. Secondary objectives include spleen volume, symptom and anemia response, and overall survival (OS). Results: As of 24 Feb 2022, 10 pts have been dosed in 2 dose levels 40 mg (n=3), and 60 mg (n=7) SEL QW plus RUX. The starting dose of RUX was 20 mg in 8 pts, 15 mg in one patient and 10 mg in one patient. The median age was 64 (range 45-76) and 7 pts had primary MF and 3 had post-essential thrombocythemia (ET) MF. The Dynamic International Prognostic Scoring System (DIPSS) risk category was int-1 (n=4), int-2 (n=4) and high risk (n=2). There were no dose limiting toxicities reported for either SEL dose levels. Due to dizziness, one patient had a dose interruption and after 5 months of therapy discontinued treatment from new onset of atrial fibrillation and pulmonary hypertension (unrelated to SEL and RUX). Currently, all other pts remain on study. Hemoglobin levels were maintained without significant worsening in majority of patients. Low grade nausea (30%) was the most common treatment-emergent adverse event. All pts experienced an improvement in their white blood cell count. Of the 6 evaluable pts, 5 had a ≥35% spleen volume reduction at week 12. Summary/Conclusion: In pts with treatment-naïve MF, QW SEL in combination with RUX is well tolerated with a manageable side effect profile. Based on current data there have been no observed dose limiting toxicities in cohort 1 of QW oral SEL 40 and 60 mg with RUX. P1006: NEXT GENERATION SEQUENCING (NGS) IN PEDIATRIC MASTOCYTOSIS A. Angi1,*, S. Bianchi1, G. Palumbo1, V. Filipponi1, M. Rousseau1, M. L. Moleti1, F. Giona1 1EMATOLOGIA PEDIATRICA, Policlinico Umberto I Università Sapienza Roma, Roma, Italy Background: Mastocytosis is a clonal disorder characterized by the accumulation of mast cells in the skin and, less frequently, in other organs. The disease is limited to the skin in children and may occur in one of three cutaneous variants: maculopapular cutaneous mastocytosis (MPCM), diffuse cutaneous mastocytosis (DCM) and mastocytoma. Systemic mastocytosis (SM) typically occurs in adults with KIT D816V mutation (3). Additional genetic mutations (TET2, N-RAS, SF3B1, ASXL1, etc.) have been detected using NGS in adults with mastocytosis (4). Currently, there is no data concerning the use of NGS in pediatric mastocytosis. Aims: The aims of the monocentric study, named MAS_PED1, that was approved by Ethics Committee, are to: a) identify patients (pts) with different cutaneous forms at risk of developing aggressive systemic disease; b) detect the known KIT D816V mutation using RT-PCR; c) identify different KIT, and/or additional gene mutations using NGS; and d) evaluate potential role of different mutations in the outcome of pts with mastocytosis, aged < 18 years at lesion onset. Methods: Pts with pediatric onset of mastocytosis, followed at the study Center were included. Peripheral blood samples were collected to detect KITD816V mutation, using both RT-PCR and ddPCR techniques, and to investigate other molecular mutations using NGS panels for rare and myeloid genes. Results: All 36 pts included in this study, 19 male and 17 female, with median age of lesion onset ranged from birth to 17.81 years (median 4.74 months), had a cutaneous mastocytosis (CM). Twenty-one of the 36 pts (58%) underwent cutaneous biopsy after a median from lesion onset of 3.77 months (range: 2.49 months – 11.6 years). According to the type of cutaneous lesions at diagnosis, pts were classified as: 20 (55%) MPCM, 10 (28%) DCM and 6 (17%) mastocytoma. The median tryptase value at the onset was 5 ng/ml, higher in MPCM (range: 1.2 - 141 ng/ml), than in DCM (range: 2.71 - 19.4 ng/ml), and in mastocytoma (range: 3.8 - 7.3 ng/ml). Two of the MPCM pts developed indolent SM (ISM) after 10 and 20 years from the onset disease. RT-PCR and NGS assessment were performed in 17 pts (47%), 15 CM and 2 ISM. RT-PCR identified KIT D816V mutation in 4 pts (2 MPCM, 1 DCM, 1 ISM) while NGS revealed KIT D816V in 3 pts and other KIT mutations, KIT D816Y and KIT Y553C, in 2 pts. An additional 10 myeloid gene mutations were detected by NGS technique: 5 were already known (ASXL1 G1397S; JAK2 L393V; KITD816Y; LNK E208Q; TET2 Y867H) while the other 5 have not been previously described (ETV6A215P; KIT Y553C; NFE2 1291T; SH2B3 G382D; SH2B3 L438V). Mutations in myeloid genes were prevalent in the signalling functional group. A single mutation was found in 7 pts (3MPCM, 3 DCM, 1ISM), while two or more mutations were found in 3 DCM pts, 2 of them had a spontaneous regression. Two different mutations (KIT Y553C and SH2B3 L438V) were found in two sisters. Overall, 9/36 pts (5 DCM, 3 MPCM, 1 mastocytoma) presented spontaneous complete regression of cutaneous lesions after a median time of 25 months (range: 17 months - 25 years). Summary/Conclusion: KIT mutations resulted in 35% of the children tested. The RT-PCR technique resulted more sensitive in finding KIT D816V, while NGS in detecting other mutations. ASXL1, JAK2 and TET2 mutations, detected in our population, are different to those reported in adult pts with SM. The presence of multiple mutations in the DCM form, does not appear to influence the evolution to a systemic form, unlike in adults. P1007: PHASE 2, OPEN-LABEL, MULTICENTER, SINGLE-ARM STUDY INVESTIGATING THE EFFICACY AND SAFETY OF ROPEGINTERFERON ALFA-2B IN JAPANESE PATIENTS WITH POLYCYTHEMIA VERA K. Kirito1,*, Y. Edahiro2,3,4, K. Ohishi5, A. Gotoh6, K. Takenaka7, H. Shibayama8, T. Shimizu9, K. Usuki10, K. Shimoda11, M. Ito12, S. A. VanWart13, O. Zagrijtschuk14, A. Qin15, H. Kawase16, N. Miyachi16, T. Sato16, N. Komatsu2,3,4,16 1Department of Hematology and Oncology, University of Yamanashi, Chuo-shi, Yamanashi; 2Department of Hematology; 3Laboratory for the Development of Therapeutics Against MPN; 4Department of Advanced Hematology, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo; 5Department of Transfusion Medicine and Cell Therapy, Mie University Hospital, Tsu-shi, Mie; 6Department of Hematology, Tokyo Medical University, Shinjuku-ku, Tokyo; 7Department of Hematology, Clinical Immunology, and Infectious Diseases, Ehime University Graduate School of Medicine, Toon-shi, Ehime; 8Department of Hematology, National Hospital Organization Osaka National Hospital, Chuo-ku, Osaka; 9Division of Hematology, Department of Medicine, Keio University School of Medicine, Shinjuku-ku, Tokyo; 10Department of Hematology, NTT Medical Center Tokyo, Shinagawa-ku, Tokyo; 11Division of Hematology, Diabetes, and Endocrinology, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Kiyotake-cho, Miyazaki; 12Department of Pathology, Japanese Red Cross Aichi Medical Center Nagoya Daiichi Hospital, Nagoya-shi, Aichi, Japan; 13Enhanced Pharmacodynamics, LLC, NY; 14PharmaEssentia Corporation USA, MA, United States of America; 15PharmaEssentia Corporation, Taipei, Taiwan; 16PharmaEssentia Japan K.K., Minato-ku, Tokyo, Japan Background: Polycythemia vera (PV) is a BCR–ABL-negative myeloproliferative neoplasms with a JAK2 gene mutation, that can be complicated by thrombosis and hemorrhage. It may also evolve to secondary acute myeloid leukemia, which has a poor prognosis and is potentially fatal. Ropeginterferon alfa-2b, a peg-proline-interferon alfa-2b with improved pharmacokinetic properties, allowing for less frequent administration and better tolerability, has been shown in clinical studies (PEGINVERA, PROUD-PV, CONTINUATION-PV) to be efficacious and have a favorable safety profile. However, its efficacy and safety have not been evaluated in Japanese PV patients. Aims: The aim of this phase 2, single-arm study (NCT04182100) was to investigate the efficacy and safety of ropeginterferon alfa-2b in Japanese patients with PV to assess the clinical utility of this cytoreductive therapy as a treatment option. Methods: Japanese patients aged ≥20 years with a PV diagnosis according to World Health Organization 2008/2016 criteria and who were unsuitable candidates for current standard treatment were included, and patients being refractory to hydroxyurea (HU), symptomatic splenomegaly, or previous treatment with interferon alfa were excluded. Ropeginterferon alfa-2b was administered subcutaneously every 2 weeks for 12 months (M) at a starting dose of 100μg (50μg in patients receiving HU). This was increased by 50μg every 2 weeks until patients had achieved complete hematological response (CHR): hematocrit <45% without phlebotomy in the preceding 3M, platelets ≤400×109/L, leukocytes ≤10×109 /L, or reached the maximum recommended single dose (500μg). The primary endpoint was phlebotomy-free CHR at 9M and 12M (end of treatment [EOT]). Secondary endpoints were change in hematologic parameters, CHR in patients with or without prior HU use, JAK2V617F allele burden, molecular response, and frequency of phlebotomy. Safety endpoints were incidence of treatment-emergent adverse events (TEAEs) and AEs of special interest. Results: A total of 29 patients comprised the intent-to-treat population (median age, 54 years; mean hematocrit, 46.85%; mean platelets, 747.3×109/L; mean leukocytes, 17.07×109/L at baseline). Twenty-seven patients had a JAK2V617F mutation (mean allele burden, 72.19%). Two patients discontinued the study (one withdrew consent, the other due to an AE [silent thyroiditis]). The primary outcome of durable CHR without phlebotomy at 9M and EOT was achieved by 8/29 (27.6%) patients. Hematocrit, platelets, and leukocytes decreased over time (mean±SD change from baseline to EOT: –5.45±6.66%, –493.6±374.9×109/L, –11.71±8.38×109/L, respectively), and all parameters improved to their target value range. The proportion of patients with CHR at EOT was similar in patients with or without prior HU use (Table). JAK2V617F allele burden decreased over time (change from baseline to EOT: mean ±SD, –19.17±22.64; Median [min, max], -11.62 [-74.80,33.80]; n=26). Molecular response increased over time. Eight (27.6%) patients did not require phlebotomy. TEAEs occurred in all patients; however, no event of grade ≥ 3 occurred. The most common TEAEs were alopecia (55.2%), fatigue (27.6%) and influenza-like illness (27.6%). AEs of special interest, which could be both disease and treatment-related, occurred in 9 (31.0%) of the patients and were related to the study treatment in 5 (17.2%) of the patients. Image: Summary/Conclusion: Ropeginterferon alfa-2b is a safe and efficacious treatment option in Japanese patients with polycythemia vera. P1008: ASSOCIATION BETWEEN FRAILTY AND CLINICAL OUTCOMES IN MYELOPROLIFERATIVE NEOPLAMS: A POPULATION-BASED STUDY FROM ONTARIO, CANADA A. Bankar1,*, W. Chan2, N. Liu2, M. Cheung3, S. Alibhai4, V. Gupta1 1Medical Oncology and Hematology, Princess Margaret Cancer Center, University Health Network; 2ICES; 3Sunnybrook Health Sciences Centre; 4General Internal Medicine, Toronto General Hospital- University Health Network, Toronto, Canada Background: Frailty independently predicts adverse outcomes in several different cancers and community-dwelling older adults. Its impact on clinical outcomes in myeloproliferative neoplasms (MPN) is unknown. Aims: To measure the association between frailty and all-cause mortality and thrombosis in MPN patients. Methods: Method: A retrospective, population-based study using province-wide administrative databases of Ontario. Study population: We included MPN patients in the Ontario Cancer Registry from 2004 to 2019 ((Total n= 10,336: ET, n=5,108; PV, n=3,843; MF, n=1,385). Baseline frailty was measured during two years prior to date of MPN diagnosis using either (i) the Johns Hopkins Adjusted Clinical Groups® frailty indicator (ACG-F), categorized as fit or frail if any of the ten frailty-defining diagnoses were present or (ii) the McIsaac’s cumulative deficit frailty index (mFI), categorized as fit, prefrail, or frail if mFI <0.10, 0.10-0.19, > 0.19 respectively. Main outcome measures: Cox proportional hazard model was used to generate the Hazard Ratios (HRs) with 95% confidence interval (CI) for all-cause mortality comparing frail vs. prefrail vs. fit patients. Subdistribution hazard ratios (SHR) using the Fine-Gray models were used to evaluate the effect of frailty on development of thrombosis, taking death as the competing event. Analyses were adjusted for age, comorbidities, prior thrombosis, and level of marginalization. Results: Results: The mean duration of follow-up for ET, PV and MF was 3.8, 4.0 and 2.9 years, respectively. 16% MPN cases were categorized as mFI-frail and 51% as mFI-prefrail. Patient with MF were more likely to be mFI-frail or mFI-prefrail compared to ET and PV (34%, 23%, and 20% respectively, p<0.001). In all MPN subtypes, frailty was independently associated with increased risk of mortality after adjusting for age, sex, and comorbidities. Compared to fit patients, the HRs for all-cause mortality for prefrail and frail patients were: 1.6 (1.3-1.9), and 3.6 (2.9-4.4) in ET; 1.3 (1.1-1.5) and 2.7 (2.1-3.4) in PV, and 1.2 (1.0-1.5) and 2.0 (1.5-2.7) in MF. Other predictors associated with increased all-cause mortality were advanced age (compared to age <40 years, HRs for 40-65 years, 65-75 years, >75 years: ET: 3.3 (2.0-5.5), 5.34 (3.2-8.7), 12.9 (7.9-21.2); for PV: 3.2 (1.6-6.7), 7.36 (3.6-15.0), 17.3 (8.5-35.2), for MF: 1.8 (0.8-3.8), 3.0 (1.4-6.5), 4.8 (2.2-10.4)) and comorbidities (1 and ≥ 2 comorbidities vs no comorbidities: ET: 1.3 (1.1-1.5), 2.4 (2.0-2.8); for PV: 1.0 (0.8-1.2), 1.2 (1.0-1.5); for MF: 1.2 (1.0-1.5), 1.4 (1.1-1.8)). With ACG-F, frailty was noted in 11% in MPN cases and was associated with increased mortality after adjusting for comorbidities and advanced age in all MFN subtypes [HRs in ET 2.5 (2.2-2.8), PV: 2.1 (1.8-2.4), and MF: 1.8 (1.5-2.2)]. In multivariable Fine-Gray regression, neither mFI-prefrail nor mFI-frail patients had an increased risk of thrombosis in all MPN subtypes. Compared to fit patients, frail patients using ACG-F had lower SHRs of thrombosis (ET: 0.6 (0.4-0.8), PV: 0.6 (0.4-0.8), MF: 0.2 (0.05-0.8), but higher SHRs for the competing event of death (ET: 3.0 (2.6-3.5), PV: 2.3 (1.9-2.9), MF: 1.9 (1.5-2.4)). Image: Summary/Conclusion: Conclusions: Using large population-based databases, we found that a significant proportion of MPN patients are frail or prefrail at diagnosis despite younger age or less comorbidity burden. After adjusting for confounding from advanced age and increasing co-morbidity burden, frailty independently predicts increased risk of all-cause mortality in ET, PV, and MF. P1009: PREDICTORS OF HOSPITALIZATION AND SEVERE OUTCOMES IN PATIENTS WITH MPN AND COVID-19 T. Barbui1,*, A. Carobbio1, A. Masciulli1, A. Iurlo2, M. A. Sobas3, E. M. Elli4, E. Rumi5, V. De Stefano6, F. Lunghi7, M. Marchetti8, R. Daffini9, M. Gasior Kabat10, B. Cuevas11, M. L. Fox12, M. M. Andrade Campos13, F. Palandri14, P. Guglielmelli15, G. Benevolo16, C. Harrison17, M. A. Foncillas18, M. Bonifacio19, A. Alvarez-Larran20, J.-J. Kiladjian21, E. Bolanos Calderon22, A. Patriarca23, K. S. Quiroz Cervantes24, M. Griesshammer25, V. Garcia-Gutierrez26, A. Marin Sanchez27, E. Magro Mazo28, M. Ruggeri29, J. C. Hernandez-Boluda30, S. Osorio31, G. Carreno-Tarragona32, M. Sagues Serrano33, R. Kusec34, B. Navas Elorza35, A. Angona36, B. Xicoy Cirici37, E. Lopez Abadia38, S. Koschmieder39, E. Cichocka40, A. Kulikowska de Nałęcz41, M. Bellini42, D. Cattaneo2,43, C. Bucelli2, F. Cavalca4, O. Borsani5, S. Betti6, N. Curto-Garcia17, S. Carbonell20, L. Benajiba21, A. Rambaldi42,43, A. M. Vannucchi15 1FROM Research Foundation, Bergamo; 2Hematology division, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 3Department of Hematology, Blood neoplasms and Bone marrow transplantation, Wroclaw Medical University, Wroclaw, Poland; 4Hematology division and Bone marrow transplant unit, San Gerardo Hospital, ASST Monza, Monza; 5Department of Molecular medicine, University of Pavia, Pavia; 6Fondazione Policlinico “A. Gemelli” IRCCS, Rome; 7IRCCS Ospedale San Raffaele, Milan; 8AOU SS. Antonio e Biagio e C. Arrigo, Alessandria; 9ASST Spedali Civili, Brescia, Italy; 10Hospital Univeristario La Paz, Madrid; 11Hospital Universitario de Burgos, Burgos; 12Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus; 13Hospital del Mar, Barcelona, Spain; 14IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna; 15Center Research and Innovation of Myeloproliferative Neoplasms (CRIMM), Department of Experimental and Clinical Medicine, Azienda Ospedaliera Universitaria Careggi, University of Florence, Florence; 16AOU Città della Salute e della Scienza di Torino, Torino, Italy; 17Guy’s and St. Thomas’ NHS Foundation Trust, London, United Kingdom; 18Hospital Universitario Infanta Leonor, Madrid, Spain; 19Ospedale Policlinico “G.B. Rossi”, Borgo Roma, Verona, Italy; 20Hospital Clinic de Barcelona, Barcelona, Spain; 21Hospital Saint-Louis, Paris, France; 22Hospital Clinico San Carlos, Madrid, Spain; 23AOU Maggiore della Carità, Novara, Italy; 24Hospital Universitario de Mostoles, Madrid, Spain; 25University Clinic for Hematology, Oncology, Hemostaseology and Palliative Care, Johannes Wesling Medical Center, Minden, Germany; 26Hospital Ramon y Cajal, IRYCIS, Madrid; 27Hospital General Universitario de Albacete, Albacete; 28Hospital Universitario Principe de Asturias, Alcalà de Henares, Madrid, Spain; 29Ospedale San Bortolo, Vicenza, Italy; 30Hospital Clinico Universitario, INCLIVA, Valencia; 31Hospital Gregorio Maranon; 32Hospital Universitario 12 de Octubre, Madrid; 33ICO L’Hospitalet-Hospital Moises Broggi, Sant Joan Despì, Barcelona, Spain; 34Department of haematology, Clinic of internal medicine, University Hospital Dubrava-School of Medicine University of Zagreb, Zagreb, Croatia; 35Hospital Moncloa, Madrid; 36ICO Girona Hospital Josep Trueta, Girona; 37ICO Hospital Germans Trias i Pujol, Badalona (Barcelona); 38Hospital General de Elche, Elche (Alicante), Spain; 39Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen, Germany; 40Department of Hematology and Bone Marrow Transplantation, Nicolaus Copernicus Hospital, Torun; 41Department of Hematology and Internal Diseases, State Hospital, Opole, Poland; 42ASST Papa Giovanni XXIII, Bergamo; 43Università degli Studi di Milano, Milano, Italy Background: Risk factors for severe COVID-19 in myeloproliferative neoplasms (MPN) have been extensively explored. However, no information is available on risk factors to hospitalization at COVID-19 diagnosis. Aims: To provide an evidence-based triage and inform for a timely and antiviral therapy prescription in early phases of viral replication. Methods: The MPN-COVID study is still enrolling consecutive adult MPN patients with COVID-19 infection since February 15, 2020. Among 479 patients (ET n=175, PV n=158, MF n=91, and pre-PMF n=55) with COVID-19 from Feb 2020 to Jun 2021, 248 (52%) were managed at home and 231 (48%) were hospitalized. Results: Univariate analysis Compared to outpatients, those admitted to hospital were more likely to be men (58.9% vs. 45.2%, p=0.003), older than 70 years (61.3% vs. 29.0%, p<.001), with at least one comorbidity (79.7% vs. 55.5%, p<.001) and a history of thrombosis (26.5% vs. 16.6%, p=0.008). Overt myelofibrosis (MF) cases were more frequent in hospital (38.5% vs. 18.1%, p<.001) than PV, ET or pre-PMF. Ruxolitinib was more frequently used in patients who underwent to hospital than managed at home. (25.7% vs. 12.1%, p<.001). In comparison with outpatients, hospitalized cases had a significantly lower median values of hemoglobin (12.1 vs. 13.3 g/dL, p<.001), platelets (250 vs. 390 x109/L, p<.001), absolute lymphocytes (0.8 vs. 1.4 x109/L, p<.001) and higher neutrophil counts (5.1 vs. 4.5 x109/L, p=0.022), leading to a significant increase of neutrophil to lymphocyte ratio (NLR) (6.6 vs. 3.2, p<.001). Compared to lymphocytopenia, the best sensitivity and specificity was found for NLR, whose AUC was 77.28% by ROC analysis. Multivariate analysis By adjusting for sex, comorbidity, fever, systemic symptoms, O2 saturation, previous thrombosis, MPN type, ruxolitinib exposure, and first vs. subsequent waves and vaccination period, three factors emerged as independent predictors of hospitalization: age over 70 years, (OR=3.02, p=0.038), dyspnea (OR=7.23, p<.001) and NLR ≥4, (OR=6.94, p<.001). Interaction model of risk factors In a model fitted to test the interaction terms of the three significant variables, we evaluated the marginal effect of NLR and dyspnea across different age classes (Figure) and found that in younger patients (i.e., from 50 to 70 years) dyspnea was the stronger predictor than increased NLR; conversely, both dyspnea and NLR showed a high and comparable marginal effect in age >80 years. Remarkably, the probability of hospitalization consistently exceeded 90% for any age group when dyspnea and NLR were concomitantly present, and their combination was more prevalent in MF (42%) than in the other phenotypes (24%, 25% and 29% in pre-PMF, PV and ET patients, respectively). In addition to predict hospitalization, dyspnea and NLR≥4 were also associated with severity of COVID-19 illness in terms of respiratory support at hospitalization (OR=2.44, p=0.023). Of note, only age >70 years and NLR higher than 6 were predictors of survival in hospitalized patients (OR=3.24, p=0.007 and OR=5.41, p=0.041, respectively). Image: Summary/Conclusion: For triage purposes of MPN patients tested positive for COVID-19, dyspnea, age and NLR are powerful predictors of hospitalization and identify patients at higher probability of invasive or non-invasive respiratory support and survival, particularly in overt MF. This MPN subgroup at high risk for progression to severe COVID-19 disease should be prioritized for antiviral therapy, even in the ambulatory setting. P1010: RUXOLITINIB IN MYELODEPLETIVE MYELOFIBROSIS: RESPONSE, TOXICITY, AND OUTCOME F. Palandri1,*, D. Bartoletti1,2, M. Breccia3, G. Auteri1,2, E. M. Elli4, M. M. Trawinska5, N. Polverelli6, M. Tiribelli7, G. Benevolo8, A. Iurlo9, A. Tieghi10, F. H. Heidel11, G. Caocci12, E. Beggiato13, G. Binotto14, F. Cavazzini15, M. Miglino16,17, C. Bosi18, M. Crugnola19, M. Bocchia20, B. Martino21, N. Pugliese22, A. D. Romagnoli1,2, C. Mazzoni1,2, L. Scaffidi23, A. Isidori24, D. Cattaneo9, M. Krampera23, F. Pane22, D. Cilloni25,26, G. Semenzato14, R. M. Lemoli16,17, A. Cuneo15, E. Abruzzese5, N. Vianelli1, M. Cavo1,2, M. Bonifacio23, G. A. Palumbo27 1IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; 2Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna; 3A.O.U. Policlinico Umberto I, Università degli Studi di Roma “La Sapienza”, Rome; 4Ospedale San Gerardo, ASST Monza, Monza; 5Ospedale S. Eugenio, Università Tor Vergata, Rome; 6ASST Spedali Civili di Brescia, Brescia; 7A.O.U. Integrata di Udine, Udine; 8A.O.U. Città della Salute e della Scienza, Torino; 9Foundation IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano; 10Azienda USL - IRCCS di Reggio Emilia, Reggio Emilia, Italy; 11Innere Medicine C, Universitätsmedizin Greifswald, Greifswald, Germany; 12Polo oncologico “A. Businco”, Università degli studi di Cagliari, Cagliari; 13Dipartimento di Oncologia, Università di Torino, Torino; 14A.O.U. di Padova, Padova; 15A.O.U. Arcispedale S. Anna, Ferrara; 16IRCCS Policlinico San Martino; 17Dipartimento di Medicina interna e Specialità mediche, Università di Genova, Genova; 18AUSL di Piacenza, Piacenza; 19Azienda Ospedaliero-Universitaria di Parma, Parma; 20Policlinico S. Maria alle Scotte, AOU Senese, Siena; 21Division of Hematology, Azienda Ospedaliera ‘Bianchi Melacrino Morelli’, Reggio Calabria; 22Dipartimento di Medicina clinica e Chirurgia, Università degli Studi di Napoli Federico II, Napoli; 23A.O.U. Integrata Verona, Verona; 24A.O. Ospedali Riuniti Marche Nord (AORMN), A.O. San Salvatore, Pesaro; 25A.O. Ordine Mauriziano di Torino; 26AOU San Luigi Gonzaga, Torino; 27Dipartimento di Scienze Mediche, Chirurgiche e Tecnologie Avanzate “G.F. Ingrassia”, Università di Catania, Catania, Italy Background: Around 30% of Myelofibrosis (MF) either primary (PMF) or secondary to polycythemia vera/essential thrombocythemia (SMF) may present a myelodepletive phenotype (MyD) (ie, thrombocytopenia, leukopenia, anemia). Pts with MyD MF represent a challenging population, as prognosis is poorer compared to pts with myeloproliferative (MyP) MF and therapeutic options, including the JAK1/2 inhibitor ruxolitinib (RUX), are limited or must be given at reduced doses. Aims: In light of the upcoming new drugs that may be used in MyD MF, we explored prognostic correlates of MyD phenotype in RUX-treated MF pts. Methods: After IRB approval, the “RUX-MF” retrospective real-world study collected 801 chronic phase MF pts treated with RUX in 26 Hematology Centers. MyD was defined as: WBC <4×109/L and/or Hb <11/<10 g/dL (males/females) and/or PLT <100×109/L with no increase of other blood cells (WBC >15×109/L, Hb >16.5/>16 g/dL in males/females, Plt >450×109/L). 219 (27.3%) had a MyD MF, including 140 (17.5%) PMF and 79 (9.8%) SMF. Spleen and symptoms response (SR/SyR) were defined according to IWG-MRT criteria. NGS mutational analysis was available for 167 pts. Results: In multivariable analysis (MVA), PMF diagnosis (p=0.001) and unfavorable karyotype (p=0.01) confirmed their significant association with MyD. In PMF pts, MyD was due to leukopenia, anemia and thrombocytopenia in 7.1%, 52.2% and 9.3%, respectively; in SMF, corresponding figures were 5%, 51.9% and 11.4%. Two or more cytopenias were found in 31.4% and 31.7% of PMF and SMF patients, respectively. In MVA, lower peripheral blast count (p=0.03), higher TSS (p=0.04) and BM fibrosis grade ≥2 (p=0.03) confirmed their association with MyD in PMF pts. In univariate/MVA, MyD SMF patients were more likely to have higher peripheral blast count (p=0.003/p=0.04), a higher MYSEC-PM risk (p<0.001/p=0.001), and to be triple negative (p=0.005/0.03). RUX starting, median at 3 months, and median overall dose was more frequently ≤10 mg BID in MyD than in MyP pts (44.9% vs 67.5%, p<0.001; 39.5% vs 59.9%, p<0.001; 34.9% vs 57.3%, p<0.001, respectively). This was confirmed also in PMF and SMF separately. The rate of SR was comparable in MyD and MyP patients. However, SR at 3 and 6 months was lower in pts with PLT<100 x 109/l (p=0.02). In SMF, MyD pts had lower rates of SR (10.8% vs 28.0% at 3 mos, p=0.004; 20.0% vs 33.1% at 6 mos, p=0.05). SyR was significantly lower in MyD MF (51.9% vs 62.5% in MyP at 3 mos, p=0.01; 59.8% vs 71.0% at 6 mos, p=0.008). In particular, anemia and thrombocytopenia were significantly associated with lower SyR. After a median RUX exposure of 2.3 yrs (0.1-12.6), 364 (45.4%) pts stopped RUX, 110 (13.7%) had a blast phase and 366 (45.7%) died. After competing risk analysis, the cumulative incidence of RUX discontinuation was higher in MyD MF patients overall (p<0.001), only PMF (p=0.03) and only SMF (p<0.001) (Fig.1a). Incidence of RUX stop was significantly higher in MyD patients with ≥2 cytopenias (p=0.03). Leukemia-free survival was not influenced by MyD/MyP phenotype (Fig.1b). In Cox regression analysis adjusted for DIPSS score, OS was significantly shorter in MyD vs MyP MF (median, 4.5 vs 5.7 yrs; p=0.03) (Fig.1c). This was confirmed considering only SMF patients (p=0.02). Image: Summary/Conclusion: MyD phenotype is associated with baseline high-risk clinical and molecular features, with lower responses to RUX, particularly in case of low PLT count, and higher risk of drug discontinuation and death. Newer strategies are warranted in this setting. P1011: PREDICTORS OF COVID-19 DISEASE AND SURVIVAL TO COVID-19 IN MPN PATIENTS TREATED WITH RUXOLITINIB F. Palandri1,*, D. Bartoletti1,2, E. M. Elli3, G. Auteri1,2, M. Bonifacio4, G. Benevolo5, F. Heidel6, M. M. Trawinska7, E. Rossi8,9, C. Bosi10, A. Tieghi11, M. Tiribelli12, A. Iurlo13, N. Polverelli14, G. Caocci15, G. Binotto16, F. Cavazzini17, E. Beggiato18, D. Cilloni19, C. Tatarelli20, F. Mendicino21, M. Miglino22,23, M. Bocchia24, M. Crugnola25, C. Mazzoni1,2, A. D. Romagnoli1,2, G. Rindone3, S. Ceglie8, A. D’Addio26, E. Santoni4, D. Cattaneo13, R. M. Lemoli22,23, M. Krampera4, A. Cuneo17, G. Semenzato27, R. Latagliata28, E. Abruzzese7, N. Vianelli1, M. Cavo1,2, A. Andriani29, V. De Stefano8,9, G. Palumbo30, M. Breccia31 1IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; 2Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna; 3Ospedale San Gerardo, ASST Monza, Monza; 4A.O.U. Integrata Verona, Verona; 5A.O.U. Città della Salute e della Scienza, Torino, Italy; 6Innere Medicine C, Universitätsmedizin Greifswald, Greifswald, Germany; 7Ospedale S. Eugenio, Università Tor Vergata; 8Section of Hematology, Department of Radiological and Hematological Sciences, Catholic University School of Medicine; 9Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome; 10AUSL di Piacenza, Piacenza; 11Azienda USL - IRCCS di Reggio Emilia, Reggio Emilia; 12A.O.U. Integrata di Udine, Udine; 13Foundation IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano; 14ASST Spedali Civili di Brescia, Brescia; 15Polo oncologico “A. Businco”, Università degli studi di Cagliari, Cagliari; 16A.O.U. di Padova, Padova; 17A.O.U. Arcispedale S. Anna, Ferrara; 18Dipartimento di Oncologia, Università di Torino, Torino; 19A.O. Ordine Mauriziano di Torino, Torino; 20Azienda Ospedaliera Universitaria Sant’Andrea di Roma, Rome; 21Unit of Hematology, Hospital of Cosenza, Cosenza; 22IRCCS Policlinico San Martino; 23Dipartimento di Medicina interna e Specialità mediche, Università di Genova, Genova; 24Policlinico S. Maria alle Scotte, AOU Senese, Siena; 25Azienda Ospedaliero-Universitaria di Parma, Parma; 26Division of Hematology, Onco-hematologic Department, AUSL della Romagna, Ravenna; 27Unit of Hematology and Clinical Immunology, University of Padova, Padova; 28Hematology Unit, Ospedale Belcolle, Viterbo; 29Hematology, Fabrizio Spaziani Hospital, Frosinone; 30Department of Scienze Mediche, Chirurgiche e Tecnologie Avanzate “G.F. Ingrassia”, University of Catania, Catania; 31Division of Cellular Biotechnologies and Hematology, University Sapienza, Rome, Italy Background: Ruxolitinib (RUX) use and discontinuation are risk factors for severe COVID-19 and death in MPN patients (pts). In pts on RUX therapy, predictors for COVID-19 disease and survival (OS) to COVID-19 are unknown. Aims: The aims of this study were to distinguish RUX-treated pts at higher risk of COVID-19 and to assess prognostic factors for OS. Methods: We performed a sub-analysis of the RUX-MF and the PV-ARC observational studies that include consecutive adult pts with myelofibrosis (MF) and polycythemia vera (PV), respectively. Overall, 815 MF and 172 PV pts treated with RUX outside clinical trials have been registered. At pandemic start, 494 pts (359 MF and 135 PV) on RUX were included in this analysis. Results: Among 66 (13.6%) pts (PV n=11, MF n=55) with COVID-19 from Feb 2020 to Jan 2022, 1 (1.5%), 14 (21.2%), 9 (13.6%), 17 (25.8%), 4 (6.1%) and 21 (31.8%) pts had an asymptomatic, mild, moderate, severe, critical, and fatal infection, respectively; 42 (63.7%) were hospitalized. Overall, 14, 38 and 14 infections were observed during the 1st (Feb-Jun 2020), 2nd (Jul 2020-Jun2021) and 3rd (Jul 2021-Jan 2022) wave of the pandemic, with an overall incidence rate of 10.2 per 100 pt-yrs. Incidence rates in the 3 waves were 8, 10.2 and 7 per 100 pt-yrs respectively. Hospitalized cases were significantly less frequent during the 3rd wave (35.7% vs 64.3%/73.7% in the 1st/2nd wave, p=0.04). Overall, 283/390 evaluable pts (72.6%) received ≥1 dose of Comirnaty vaccine (19/66 COVID-19 pts; 5, 7 and 7 pts had received 1, 2 or 3 vaccine doses, respectively). At COVID-19 diagnosis, RUX was reduced in 10 (15.1%) pts and discontinued in 9 (13.6%) pts, comparably in MF and PV. In the total cohort, COVID-19 infection was more frequent in pts with MF (15.3% vs. 8.2% PV pts, p=0.04), with ≥1 comorbidity (15% vs. 8.7%, p=0.04). Also, COVID-19 infections after vaccine availability were more frequent in unvaccinated pts (37.4 vs. 6.3%, p<0.001). COVID-19 requiring hospitalization was more frequently observed in pts ≥70 yrs (12.2% vs. 6.8% in pts <70 yrs, p=0.04), and without COVID-19 vaccine (32.4% vs. 2.9%, p<0.001). No additional predictors for COVID-19 were noted analyzing MF and PV separately. In COVID-19 pts, hospitalized cases had a significantly lower median platelet count (275 vs. 168 x109/L, p=0.02), were receiving lower RUX doses (33.3% <10 mg BID vs. 8.3%, p=0.02) and more frequently presented comorbidities (40.5% vs. 13.6%, p=0.03) compared to outpatients. MF vs. PV, median hemoglobin levels, age≥70 yrs and sex were not associated with hospitalization. MF pts who were not in spleen response at COVID-19 infection had higher risk of hospitalization (73% vs. 44.4% in responders, p=0.04). After multivariable Cox analysis including previous anti-SARS-Cov-2 vaccine, need for hospitalization, age≥70 and male sex, OS to COVID-19 was significantly improved in pts who had previously received anti-SARS-Cov-2 vaccine (HR=0.10, p=0.02) (Fig.1), in pts with COVID-19 not requiring hospitalization (HR=0.19, p=0.03) and in patients <70 yrs (HR=0.38, p=0.03). The COVID-19 wave did not impact OS (p=0.53). Image: Summary/Conclusion: Among RUX-treated pts, lower RUX doses, comorbidities and no spleen response are significant predictors of hospitalization. Vaccine was the most protective factor against COVID-19 disease, hospitalization, and mortality. RUX-treated pts, regardless of MPN type, should be sensitized to adherence to the vaccine program and prioritized for antiviral therapy in case of infection. P1012: A NUMBER OF CONGENITAL ERYTHROCYTOSIS ARE MULTIGENIC A. Benetti1,*, G. Biagetti1, E. Cosi1, I. Bertozzi1, G. Ceolotto1, M. L. Randi1 1Department of Medicine, University of Padua, Padua, Italy Background: Imbalance in erythropoiesis and oxygen homeostasis could lead to the onset of Erythrocytosis, a clinical condition characterized by persistently raised hemoglobin (Hb) and hematocrit (Ht) levels. Nowadays, a high number of patients are classified as “Idiopathic Erythrocytosis” (IE) meaning that it is not possible to identify the cause of hematocrit increase. Recently, HFE mutations has been found to be often present in idiopathic erythrocytic patients. The HFE mutations could facilitate the increase in red cell mass, and this indicates the possible involvement of Iron Metabolism in causes of IE. Aims: To identify the possible mutations of IE patients using our gene panel for Next Generation Sequencing (NGS). Methods: In 118 patients with IE, regularly followed in our surgery, we used a gene panel for NGS composed by fifteen genes: JAK2, EGLN1 (PHD2), EPO-R, FTL, FTH, ASXL1, HFE, HFE2, TFR2, HAMP, SLC40A1, SLC11A2, VHL, BPMG, EPAS1 (HIF-2α). We analyzed all the exons parts of these genes. The approach used for the library preparation was a multiplexing PCR and data were analyzed by bioinformatics tools. In all patients, the mutations found were confirmed with Sanger Sequencing. Results: In 78 out of the 118 patients (66%) evaluated, we found one 1 mutated gene in 55 patients (7 EGLN1, 1 VHL, 34 HFE, 6 TFR2, 3 JAK2 and 4 EPO-R), 2 mutated genes in 18 patients (1 EGLN1/JAK2, 4 EGLN1/HFE, 2 EPAS1/HFE, 1 EPAS1/JAK2, 1 EPAS1/EGLN1, 2 EPO-R/HFE, 2 HFE/JAK2, 1 JAK2/TFR2, 2 with TFR2/EGLN1, 1 VHL/HFE, 1 TFR2/HFE) and 3 mutated genes in 5 patients (1 EGLN1/EPO-R/HFE, 1 VHL/HFE/EGLN1, 1 EPAS1/EPO-R/HFE, 1 EPAS1/HFE/EGLN1 and HFE/TFR2/JAK2). All the mutations found were germline. Interestingly, 21 out of the 27 (78%) patients carrying HFE mutation displayed 2 or more mutations. Summary/Conclusion: The use of NGS panels in various clinical conditions has frequently demonstrated that multiple mutations coexist in various patients and that the monogenic diseases are quite rare. This study is the first at our best knowledge that gives evidence of more than one mutated gene in erythrocytotic patients, and this is the results of the use of an appropriate NGS panel. The data here reported suggest that we can confirm that HFE mutations are common in IE patients, in some cases associated with other mutations, but the present data ask some questions: HFE mutations alone can induce erythrocytosis? or other genetic imbalances are needed? The presence of multiple mutations could lead to a greater severity of the disease? A significant number of IE patients displayed TFR2 mutations, giving more relevance to the hypothesis of the iron metabolism implication in IE genesis. Finally, the presence of 11 patients carrying germlines JAK2 mutations is surprising, and we suppose it will be necessary to strictly watch these patients in the suspicion of a future appearance of a polycythemia vera. The present study demonstrates the need to extend the biomolecular knowledge in patients with idiopathic erythrocytosis. P1013: OVERALL SURVIVAL IN PATIENTS WITH SYSTEMIC MASTOCYTOSIS WITH ASSOCIATED HEMATOLOGIC NEOPLASM TREATED WITH AVAPRITINIB VERSUS BEST AVAILABLE THERAPY A. Reiter1,*, J. Gotlib2, I. Álvarez-Twose3, D. H. Radia4, J. Luebke1, P. J. Bobbili5, A. Wang5, C. Norregaard6, S. Dimitrijević7, E. Sullivan6, M. Louie-Gao6, J. Schwaab1, I. A. Galinsky8, C. Perkins2, W. R. Sperr9, P. Sriskandarajah4, A. Chin5, S. R. Sendhil5, M. S. Duh5, P. Valent9, D. J. DeAngelo8 1Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany; 2Stanford Cancer Institute/Stanford University School of Medicine, Stanford, United States of America; 3Institute of Mastocytosis Studies of Castilla La Mancha (CLMast) ─ Spanish Reference Center (CSUR) for Mastocytosis and CIBERONC, Virgen del Valle Hospital, Toledo, Spain; 4Guy’s & St Thomas’ NHS Foundation Trust, Guy’s Hospital, London, United Kingdom; 5Analysis Group, Inc., Boston; 6Blueprint Medicines Corporation, Cambridge, United States of America; 7Blueprint Medicines Corporation, Zug, Switzerland; 8Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States of America; 9Department of Internal Medicine I, Division of Hematology and Hemostaseology, and Ludwig Boltzmann Institute for Hematology and Oncology, Medical University of Vienna, Vienna, Austria Background: Avapritinib, a selective inhibitor of KIT D816V, was approved in the United States (US) for treatment of adults with advanced systemic mastocytosis (AdvSM), a rare myeloid neoplasm with poor overall survival (OS), based on data from the Phase 1 EXPLORER (NCT02561988) and Phase 2 PATHFINDER (NCT03580655) single-arm trials. Systemic mastocytosis with an associated hematologic neoplasm (SM-AHN) is both the most common and most heterogenous subtype of AdvSM. No randomized control trial (RCT) has been conducted comparing efficacy of avapritinib with best available therapy (BAT) in SM-AHN. Aims: This study (NCT04695431) compared OS between SM-AHN patients treated with avapritinib in the EXPLORER and PATHFINDER trials versus SM-AHN patients treated with BAT in standard clinical practice. Methods: A multi-center, global, observational, retrospective chart review study was conducted at 6 study sites (4 European, 2 US) to identify and collect data from AdvSM patients who received BAT. SM-AHN patients were identified using inclusion/exclusion criteria similar to the EXPLORER and PATHFINDER trials. Patients receiving BAT could contribute data on multiple lines of therapy (LOTs) to the analysis; these data were compared with patient-level data from the EXPLORER and PATHFINDER trials. OS was defined as the time from avapritinib or BAT initiation to death from any cause; patients were censored at date of last follow-up if alive. Kaplan-Meier analysis was used to assess OS. Inverse probability of treatment weighting (IPTW) was used to adjust for differences in key variables between the treatment cohorts, including age, sex, region, anemia, thrombocytopenia, leukocyte count, skin involvement, number and type of prior treatments, performance status, serum tryptase level, and presence of SRSF2/ASXL1/RUNX1 mutations. An IPTW-weighted Cox proportional hazards model, adjusted for variables that remained unbalanced after weighting, was used to compare OS between treatment groups. Results: This analysis included 119 avapritinib patients and 83 BAT patients with SM-AHN (the latter contributed data on 121 LOTs). Median (range) age of the avapritinib and BAT cohorts was 70 (45−88) and 71 (38−88) years, and 61% vs. 76% of patients were male, respectively. Prior systemic therapy was received by 58% of avapritinib and 44% of BAT patients. In the avapritinib cohort, 76% of patients from both trials were dosed at ≤200mg and 24% of patients, all from EXPLORER, were dosed at ≥300mg. BAT patients were most frequently treated with tyrosine kinase inhibitors (73 out of 121 LOTs, 60%) or cytoreductive therapies (46 out of 121 LOTs, 38%). Mean durations of follow-up were 17.6 and 18.1 months, with 29 (24%) and 56 (68%) deaths in avapritinib and BAT cohorts, respectively. Median OS for avapritinib was 46.9 months (95% CI: 44.9, not estimable) and 18.0 months (95% CI: 13.0, 26.8) for BAT (Figure 1). IPTW-adjusted median OS was 46.9 months (95% CI: 21.4, 49.0) for avapritinib and 19.5 months (95% CI: 13.0, 32.2) for BAT. Weighted Cox analysis showed that OS was significantly improved for avapritinib versus BAT (hazard ratio [95% CI]: 0.42 [0.24, 0.74]; P<0.001), even with further adjustment for unbalanced variables. Image: Summary/Conclusion: The results of this analysis indicate that patients with SM-AHN treated with avapritinib in clinical trials had significantly longer OS compared to patients treated with BAT. With no RCTs, these data offer crucial insights into the survival benefit of SM-AHN patients treated with avapritinib compared to other treatments for AdvSM. P1014: OVERALL SURVIVAL IN PATIENTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS RECEIVING AVAPRITINIB VERSUS MIDOSTAURIN OR CLADRIBINE A. Reiter1,*, J. Gotlib2, I. Álvarez-Twose3, D. H. Radia4, J. Luebke1, P. J. Bobbili5, A. Wang5, C. Norregaard6, S. Dimitrijević7, E. Sullivan6, M. Louie-Gao6, J. Schwaab1, I. A. Galinsky8, C. Perkins2, W. R. Sperr9, P. Sriskandarajah4, A. Chin5, S. R. Sendhil5, M. S. Duh5, P. Valent9, D. J. DeAngelo8 1Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany; 2Stanford Cancer Institute/Stanford University School of Medicine, Stanford, United States of America; 3Institute of Mastocytosis Studies of Castilla La Mancha (CLMast) ─ Spanish Reference Center (CSUR) for Mastocytosis and CIBERONC, Virgen del Valle Hospital, Toledo, Spain; 4Guy’s & St Thomas’ NHS Foundation Trust, Guy’s Hospital, London, United Kingdom; 5Analysis Group, Inc., Boston; 6Blueprint Medicines Corporation, Cambridge, United States of America; 7Blueprint Medicines Corporation, Zug, Switzerland; 8Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States of America; 9Department of Internal Medicine I, Division of Hematology and Hemostaseology, and Ludwig Boltzmann Institute for Hematology and Oncology, Medical University of Vienna, Vienna, Austria Background: Avapritinib, a selective KIT D816V inhibitor, was approved for the treatment of adults with advanced systemic mastocytosis (AdvSM) in the United States (US), based on two single-arm trials: Phase 1 EXPLORER (NCT02561988) and Phase 2 PATHFINDER (NCT03580655). No randomized controlled trial (RCT) has been conducted comparing efficacy of avapritinib versus alternative therapies for AdvSM. Aims: This study (NCT04695431) compared overall survival (OS) between AdvSM patients treated with avapritinib in the EXPLORER and PATHFINDER trials and those treated with midostaurin or cladribine in real-world clinical practice. Methods: Pooled data from the EXPLORER and PATHFINDER trials were compared with data for patients with AdvSM obtained through a multi-center, global, observational, retrospective chart review study conducted at 6 study sites (4 European, 2 US). Patients treated with midostaurin or cladribine were identified using inclusion/exclusion criteria similar to the trials and could contribute multiple lines of therapy (LOTs) to the analysis. OS, assessed using Kaplan-Meier analysis, was defined as time from initiation of avapritinib, midostaurin, or cladribine to death from any cause; patients were censored at date of last follow-up if alive. Inverse probability of treatment weighting (IPTW) was used to adjust for differences in key variables between treatment cohorts, including age, sex, AdvSM subtype, anemia, thrombocytopenia, leukocyte count, skin involvement, number and type of prior treatments, performance status, serum tryptase level, and presence of SRSF2/ASXL1/RUNX1 mutations. IPTW-weighted Cox proportional hazards models, adjusted for variables that remained unbalanced after weighting, were used to compare OS between cohorts. Results: This analysis included 176 patients treated with avapritinib, 94 treated with midostaurin (LOT, n=99), and 44 treated with cladribine (LOT, n=49). Median (range) age was 68 (31−88) for avapritinib, 69 (26−87) for midostaurin, and 66 (45−88) years for cladribine patients. 103 (59%) avapritinib patients were male, versus 64 (68%) in the midostaurin and 27 (61%) in the cladribine cohort. In the avapritinib cohort, 136 (77%) patients from both trials were dosed at ≤200mg and 40 (23%) patients, all from EXPLORER, were dosed at ≥300mg. In unweighted analysis of avapritinib versus midostaurin, mean follow-up durations were 17.9 and 27.9 months, respectively, during which 34 (19%) avapritinib patients and 56 (60%) midostaurin patients died. Median OS was not reached (NR) (95% CI: 46.9, not estimable) in the avapritinib cohort and was 28.6 months (95% CI: 18.2, 44.6) in the midostaurin cohort (Figure 1). In weighted Cox analysis, OS was significantly improved in the avapritinib versus midostaurin cohort (hazard ratio [HR] [95% CI]: 0.59 [0.36, 0.97]; P<0.001). The mean-follow-up duration for the cladribine cohort was 24.2 months, during which 29 (66%) patients died. The median OS in the cladribine cohort was 23.4 months (95% CI: 14.8, 40.6). Weighted Cox analysis showed that OS was significantly improved in the avapritinib versus cladribine cohort (HR [95% CI]: 0.32 [0.15, 0.67]; P=0.003). Image: Summary/Conclusion: The results from this study indicate that AdvSM patients treated with avapritinib in clinical trials experienced significantly improved survival compared with patients treated with midostaurin or cladribine in the real world. Given the lack of RCTs, these data offer essential insights into the improved survival of patients treated with avapritinib compared to alternative therapies for AdvSM. P1015: DURATION OF TREATMENT AND REDUCTION IN SERUM TRYPTASE LEVELS IN PATIENTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS TREATED WITH AVAPRITINIB VERSUS BEST AVAILABLE THERAPY A. Reiter1,*, J. Gotlib2, I. Álvarez-Twose3, D. H. Radia4, J. Luebke1, P. J. Bobbili5, A. Wang5, C. Norregaard6, S. Dimitrijević7, E. Sullivan6, M. Louie-Gao6, J. Schwaab1, I. A. Galinsky8, C. Perkins2, W. R. Sperr9, P. Sriskandarajah4, A. Chin5, S. R. Sendhil5, M. S. Duh5, P. Valent9, D. J. DeAngelo8 1Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany; 2Stanford Cancer Institute/Stanford University School of Medicine, Stanford, United States of America; 3Institute of Mastocytosis Studies of Castilla La Mancha (CLMast) ─ Spanish Reference Center (CSUR) for Mastocytosis and CIBERONC, Virgen del Valle Hospital, Toledo, Spain; 4Guy’s & St Thomas’ NHS Foundation Trust, Guy’s Hospital, London, United Kingdom; 5Analysis Group, Inc., Boston; 6Blueprint Medicines Corporation, Cambridge, United States of America; 7Blueprint Medicines Corporation, Zug, Switzerland; 8Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States of America; 9Department of Internal Medicine I, Division of Hematology and Hemostaseology, and Ludwig Boltzmann Institute for Hematology and Oncology, Medical University of Vienna, Vienna, Austria Background: Avapritinib, a selective and highly potent KIT D816V inhibitor, was approved in the United States (US) for treatment of adults with advanced systemic mastocytosis (AdvSM) based on results from two single-arm trials: EXPLORER (Phase 1; NCT02561988) and PATHFINDER (Phase 2; NCT03580655). There is no comparative response data for avapritinib versus best available therapy (BAT) for AdvSM. Aims: This study (NCT04695431) compared duration of treatment (DOT) and maximum reduction in serum tryptase levels between patients treated with avapritinib at a starting dose ≤200mg versus BAT. Methods: A multi-center, global, observational, retrospective chart review study was conducted at 6 study sites (4 European, 2 US) to identify and collect data from AdvSM patients who received BAT. Patients were identified using inclusion/exclusion criteria similar to EXPLORER and PATHFINDER trials. AdvSM patients receiving BAT could contribute data on multiple lines of therapy (LOT); these data were compared with patient data from the trials. DOT was defined as time from initiation to discontinuation of each LOT. Maximum reduction in serum tryptase on treatment was defined during each LOT. DOT was compared between cohorts using a Cox proportional hazards model and reduction in serum tryptase levels was compared using a generalized estimating equation linear model. Both models were weighted by inverse probability of treatment weights (IPTW) to adjust for confounding by key variables: age, sex, region, AdvSM subtype, anemia, thrombocytopenia, leukocyte count, skin involvement, number and type of prior treatments, performance status, serum tryptase level, and presence of SRSF2/ASXL1/RUNX1 mutations. Both models further adjusted for key covariates that remained unbalanced after weighting. Subgroup analyses were conducted in patients who received ≥1 prior LOT (2L+), and a 200mg starting dose of avapritinib. Results: The DOT analysis included 136 avapritinib patients and 137 BAT patients. Median (range) age for avapritinib and BAT patients was 68 (31−88) and 69 (21−88) years, and 59% versus 66% of patients were male, respectively. In the avapritinib group, 67%, 17%, and 16% of patients were diagnosed with SM with an associated hematological neoplasm (SM-AHN), aggressive SM (ASM), and mast cell leukemia (MCL), respectively, while in the BAT group, 55%, 29%, and 16% were diagnosed with SM-AHN, ASM, and MCL. Among 188 BAT LOTs with agent-level data available, most common treatments were midostaurin (53%) and cladribine (24%). Median DOT was 32.1 months (95% CI: 23.8, not estimable) for avapritinib and 5.5 months (95% CI: 5.1, 7.0) for BAT. In IPTW-weighted Cox analysis, DOT was significantly longer for avapritinib versus BAT (hazard ratio [95% CI]: 0.35 [0.23, 0.51]; P<0.001). The analysis of reduction in serum tryptase included 135 avapritinib and 116 BAT patients. Unadjusted maximum percentage reduction in serum tryptase levels was -84.8% (standard deviation [SD]: 19.9%) for avapritinib versus -9.2% (SD: 161.4%) for BAT. The adjusted mean difference in maximum percentage reduction in serum tryptase comparing avapritinib to BAT was -69.8% (95% CI: -89.4%, -50.2%; P<0.001). Results were similar in subgroups (Table 1). Image: Summary/Conclusion: AdvSM patients treated with avapritinib experienced significantly longer DOT and greater reduction in serum tryptase levels compared to patients treated with BAT. In the absence of RCTs, these data offer important and meaningful insight into the improved effectiveness of avapritinib compared to alternative therapies for AdvSM. P1016: POLYCYTHEMIA VERA AND IMMUNE THROMBOGENESIS R. Cacciola1,*, V. Vecchio2, E. Gentilini Cacciola3, E. CACCIOLA4 1Experimental and Clinical Medicine, Haemostasis Unit - University of Catania; 2University of Catania, University of Catania School of Medicine, Catania; 3Public Health and Infectious Diseases, Sapienza University, Roma; 4Medical, Surgical Sciences and Advanced Technologies “G.F. Ingrassia”, Haemostasi Unit, Catania, Italy Background: Polycythemia Vera (PV) is a Ph1-negative myeloproliferative neoplasm characterized by vascular thrombosis and poor survival. The polycythemic thrombogenesis is associated with erythrocytosis and red cell adhesiveness angiopathy. Recent studies reported a JAK/STAT-mediated reduction of the regulatory T cells (Tregs) (CD4+, CD25high, CD127low, FoxP3+) and loss of self-tolerance. Aims: We investigated Tregs, anti-endothelial cell antibodies (AECA), and endothelial, platelet and coagulation activation, in Polycythemia Vera (PV) and thrombosis. Methods: We enrolled 60WHO-defined PV patients (30 men, 30 women; mean age 45±10 years) without cardiovascular risk factors, autoimmune disease or thrombotic history. Of PV patients, 40/60 had thromosis includingmyocardial infarction (MI) (10/60) according to the WHO critera, deep vein thrombosis (20/60) and pulmonary embolism (10/60) on lower-limb ultrasonography and computed tomography angiography, respectively. All patients were evaluated for JAK2V617F allele burden, Tregs, AECA,Endothelial Leukocyte Adhesion Molecule-1 (ELAM-1), Intercellular Adhesion Molecule-1 (ICAM-1),prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib) and D-dimer (DD). JAK2V617F allele burden were analyzed by Polymerase chain reaction, Tregs were measured by flow cytometry,AECA, ELAM-1 and ICAM-1 by ELISA, PT and APTT by coagulometric test, Fib using Clauss method, and DD using ELISA.Complete blood hemostasis was studied by PFA-100 on Collagen/ADP (CT-ADP) and Collagen/Epinephrine (CT-EPI) cartridges and Thromboelastometry method on Clotting Time (CT), Clotting Formation Time (CFT), Maximum Clot Firmness (MCF), and clot lysis at 30 minutes (LY-30). Results: The patients with thrombosis had JAK2V616F allele burden higher (> 50%) compared to patients without thrombosis (< 50Kroll%), lower Tregs (1,5±0,5% vs 3.5±1%),higher AECA (200±20% vs 110±10%), ELAM-1 (80±10 ng/ml vs 45±5 ng/ml), and ICAM (170 ng/mL±10 vs 110±10 ng/mL), longer PT (30±10 s vs 20±2 s) and PTT (60±10 s vs 38±5 s), lower Fib (90±20 mg/dl vs 120±20 mg/dl), higher DD (650±100 mg/l vs 300±50 mg/l) and shorter C/ADP and C/EPI (C/ADP, n.v. 68-121 s (40±10 s vs 55±20 s) and C/EPI n.v. 84-160 s (35±5 s vs 60±10 s). The patients with thrombosis had shorter CT (INTEM 35±20 s vs 70±20 s, EXTEM 20±10 s vs 30±5 s), shorter CFT (INTEM 15±10 s vs 25±5 s EXTEM 18±10 s vs 28±5 s), longer MCF (INTEM 130±10 mm vs 90±10 mm, EXTEM 120±10 mm vs 82±10 mm), and lower LY-30 (INTEM 0.9% vs 15%, EXTEM 0.8% vs 15%). A positive correlation there was between Tregs and AECA, ELAM-1 and ICAM-1 and thrombosis. Summary/Conclusion: These findings shed new light on thrombotic pathogenesis in patients with PV. P1017: HARBOR: A PHASE 2/3 STUDY OF BLU-263 IN PATIENTS WITH INDOLENT SYSTEMIC MASTOCYTOSIS AND MONOCLONAL MAST CELL ACTIVATION SYNDROME M. Castells1,*, R. Scherber2,3, V. Bhavsar2, K. He2, C. Akin4 1Mastocytosis Center, Brigham and Women’s Hospital and Harvard Medical School, Boston; 2Blueprint Medicines Corporation, Cambridge; 3UT Health San Antonio, MD Anderson Cancer Center, San Antonio; 4University of Michigan, Ann Arbor, United States of America Background: The KIT D816V mutation plays a key role in the aggregation and accumulation of aberrant mast cells which characterize systemic mastocytosis (SM), a rare, clonal mast cell neoplasm. The disease subtype of indolent SM (ISM) is characterized by the presence of mast cell aggregates in bone marrow and other organs which can lead to chronic, debilitating, and potentially life-threatening symptoms. Mast cell infiltration in the skin is common in ISM but is not always present. Similarly, monoclonal mast cell activation syndrome (mMCAS) is a clonal KIT D816V–positive mast cell disease without mast cell aggregates which can be accompanied by similar symptoms. Neither ISM nor mMCAS currently have approved molecularly targeted therapies, and significant symptom burden exists despite best supportive care (BSC) symptom-based therapy. BLU-263 is a novel, oral, next-generation tyrosine kinase inhibitor, exhibiting potent inhibition of KIT D816V and an evolved preclinical profile with limited central nervous system penetration. In our phase 1 study in normal healthy volunteers, BLU-263 was safe, with linear pharmacokinetics across all tested doses and a half-life allowing once-daily dosing, and thus supporting continued development for patients with SM. Aims: HARBOR (NCT04910685) is a randomized, double-blind, placebo-controlled, phase 2/3 study assessing efficacy and safety of orally administered BLU-263 in patients with ISM whose symptoms are not adequately controlled by standard therapies. Methods: Part 1 of the study includes 3 dose groups (25 mg, 50 mg, and 100 mg) and a placebo group combined with BSC therapies to determine the recommended dose (RD) of BLU-263 in approximately 40 patients with ISM (Figure). After determination of the RD and analysis of Part 1, Part 2 will open for enrollment of approximately 303 patients undergoing BSC therapies, randomized 2:1 to BLU-263 or placebo. The primary endpoint of Part 2 is the proportion of patients achieving a ≥30% reduction in total symptom score as assessed by the Indolent Systemic Mastocytosis Symptom Assessment Form (ISM-SAF). Patients completing Part 1 or Part 2 will roll over to Part 3 for open-label long-term evaluation of BLU-263 at the RD. The exploratory, open-label study Part M will explore BLU-263 at the RD in patients diagnosed with mMCAS. Other endpoints including but not limited to Mastocytosis Quality of Life scores, KIT D816V allele burden, and bone marrow mast cell involvement will also be assessed. Two pharmacokinetic (PK) groups will enroll patients with ISM prior to or concurrent with the Part 1 and Part 2 study cohorts to better characterize the PK and safety of BLU-263 in specific patient populations. Results: Topline results of Part 1 of the study are anticipated in the second half of 2022. Image: Summary/Conclusion: Overall, the goal of HARBOR is to investigate the safety and efficacy of the selective, targeted KIT inhibitor BLU-263 as a potential treatment option to reduce symptom burden for patients with ISM. P1018: TREND OF CIRCULATING CD34+ CELLS IN MYELOFIBROSIS PATIENTS TREATED WITH RUXOLITINIB D. Cattaneo1,*, N. Galli1, C. Bucelli2, S. Artuso3, A. Iurlo3 1Oncology and Hemato-Oncology, University of Milan; 2Hematology, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 3Hematology, Foundation IRCCS Ca’ Granda Policlinico, Milano, Italy Background: BCR-ABL1-negative myeloproliferative neoplasms (MPNs) are variably characterized by an increase in peripheral blood (PB) and bone marrow (BM) progenitor cells. More specifically, in myelofibrosis (MF) patients (pts) it has already been reported that the number of circulating hematopoietic precursors is consistently high, with a relative circulating CD34+ cells count of 15 ×106/L as the most frequently used criterion to discriminate between MF and other MPNs. Nowadays, ruxolitinib (RUX) is used for the treatment of splenomegaly and related symptoms in MF. Aims: To evaluate CD34+ cells levels in a series of primary (PMF) and secondary (SMF) MF pts at baseline and during treatment with RUX and identify any possible correlation with response to treatment. Methods: Between Oct 2014 and Dec 2021, CD34+ cells count from 49 consecutive pts with PMF or SMF (32 males and 17 females; median age at RUX start, 70.6 years) was retrospectively assessed at baseline and after 3, 6, 12 and 24 months (mts) from RUX start. All pts were treated with RUX according to current indications, requiring an IPSS risk of at least intermediate-1. At the time of CD34+ cells measurement, a complete blood cells count was available for all pts and their spleen measurement [expressed as the distance from the left costal margin (BCM) in cm] was taken. Results: 24 pts were classified as having PMF (10 in the pre-fibrotic and 14 in the overt fibrotic stage) and 25 as having SMF (15 had PPV- and 10 PET-MF). JAK2V617F mutation was detected in 38 (77.6%) cases, CALR mutations in 8 (16.3%), and MPL in 1 (2%). The remaining 2 pts (4.1%) were defined as “triple-negative”. The median absolute number of circulating CD34+ cells in the overall population at diagnosis was 83.5/mcL (range, 1-1528/mcL), with 31 (63.3%) pts showing >15 CD34+ cells/mcL. At RUX start (after a median time from MF diagnosis of 33.9 mts), median absolute number of CD34+ cells was 123/mcL (range, 2-1528/mcL), with 43 (87.7%) cases showing higher than normal levels. As expected, spleen measurements progressively decrease during the first mts of RUX (Fig. 1A): in particular, the spleen was palpable at a median of 10 cm BCM at RUX start, 5 and 3.5 cm after 3 and 6 mts of therapy, respectively. On the contrary, it increases up to 5.5 cm after 12 mts, without significant changes after 24 mts. Interestingly, with the exception of a transient increase after 3 mts of RUX therapy, a progressive reduction in the absolute number of PB CD34+ cells after 6 and 12 mts was documented in the whole cohort, with only a slight increase after 24 mts (Fig. 1B). However, considering PMF and SMF separately, a different behavior of CD34+ cells was detected: in detail, in PMF pts circulating hematopoietic precursors gradually decreased from 3 to 24 mts of RUX therapy in parallel with the persistence of a reduction in the spleen size (Fig. 1C). On the contrary, in SMF pts both CD34+ cells and spleen size still improve up to 6 mts of RUX, while they both regrow after 12 and 24 mts of treatment (Fig. 1D). Image: Summary/Conclusion: Preliminary results of our study confirm that PB CD34+ cells are increased in the majority of MF pts, both at diagnosis and during follow-up. To the best of our knowledge, we have first reported the changes in circulating CD34+ cells count during RUX, showing a parallel decrease in spleen diameter and circulating hematopoietic precursors. These findings suggest that this tool could facilitate the assessment of RUX responses in MF pts, although this needs to be confirmed by further studies. P1019: A PREDICTION RULE TO GUIDE JAK2 MUTATION TESTING IN PATIENTS WITH SUSPECTED POLYCYTHEMIA VERA: RESULTS FROM THE JAK2 PREDICTION COHORT (JAKPOT) STUDY B. Chin-Yee1,*, P. Bhai2, I. Cheong2, M. Matyashin1, C. Hsia1, E. Kawata3, J. Ho1, M. Levy2, A. Stuart2, H. Lin2, I. Chin-Yee2, M. Kadour2, B. Sadikovic2, A. Lazo-Langner1 1Hematology; 2Pathology and Laboratory Medicine, Western University, London, Canada; 3Hematology and Oncology, Kyoto Prefectural University of Medicine, Kyoto, Japan Background: The widespread availability of molecular testing for JAK2 mutations in patients referred for elevated hemoglobin has facilitated the diagnosis of polycythemia vera (PV) but also raises concerns of test overuse. A prediction rule could be useful to improve test utilization. Aims: In this study, we aimed to derive and validate of a simple rule using complete blood count and white blood cell differential (CBC) parameters to predict the likelihood of having a JAK2 mutation in patients referred for elevated hemoglobin. Methods: We examined all adult patients with elevated hemoglobin (≥160 g/L for women, or ≥165 g/L for men) who underwent JAK2 mutation testing using either quantitative polymerase chain reaction (qPCR), single nucleotide polymorphism (SNP) allelotyping or Next Generation Sequencing (NGS) panel between 2015 and 2021 at London Health Sciences Centre in Ontario, Canada. We extracted data on age, sex, and complete blood count and white blood cell differential (CBC) at the time of testing. All CBCs were performed on a Sysmex XN Analyzer. Patients were randomly divided into derivation and validation cohorts including all methods of JAK2 mutation testing. JAK2-positive and -negative groups were compared using Student’s t-tests or χ2 tests, as appropriate. Continuous variables were dichotomized at optimal cut-off points using receiving operating characteristic curves. Potentially significant predictors were evaluated using multiple variable stepwise logistic regression analysis with JAK2 positivity as the dependent variable. A score was derived and internally validated using non-parametric bootstrapping. The model was tested in the validation cohort and sub-analyses for each method were conducted in a similar fashion. Test accuracy for the score was evaluated in all cohorts. Results: The total study cohort included 901 patients (derivation n=616, validation n=285). Population characteristics are shown in Table 1A. The final model included 1-point for any of the following: erythrocytes >6.45 × 1012/L, platelets >350 × 109/L, and neutrophils >6.2 × 109/L. Patients with a score of 0 were considered low-risk; all others were classified as high-risk. The percentage of JAK2 positive patients in patients with a score of 1-3 was 24% versus 0.8% in patients with a score of 0. The model had a sensitivity of 94.7% and a negative predictive value of 98.8% in the derivation cohort; both sensitivity and negative predictive value were 100% in the validation cohort. The percent of false negatives was 0.6% and 0% in the derivation and validation cohorts, respectively (Table 1B). The results were consistent for each testing method. Image: Summary/Conclusion: We developed and validated a simple rule using CBC parameters to predict the likelihood of JAK2 mutation positivity in patients with elevated hemoglobin with demonstrated high sensitivity and negative predictive value across different JAK2 mutation testing platforms. In our cohort, the use of this rule to guide molecular testing would have resulted in over 50% fewer tests. Further studies to prospectively validate this prediction rule in different populations are planned. P1020: SCREENING FOR PORTAL HYPERTENSION IN PATIENTS WITH PHILADELPHIA NEGATIVE MYELOPROLIFERATIVE NEOPLASMS M. Davidson1,*, F. Wong1, M. Atri1, H. Sibai1, D. Maze1, V. Cheung1, T. Nye1, J. Callum2,3, E. Atenafu1, V. Gupta1 1Princess Margaret Hospital; 2University of Toronto, Toronto; 3Queen’s Unviversity, Kingston, Canada Background: Portal hypertension (PH) is a well-recognized complication of BCR-ABL negative myeloproliferative neoplasms (MPNs) and is associated with significant morbidity and mortality. However, the prevalence of PH and its associated risk factors have not been studied systematically in this patient population. Screening by esophagogastroduodenoscopy (EGD) can facilitate early diagnosis of PH and opportunity for intervention to prevent complications. Aims: To investigate the prevalence of PH and its patient and disease-related risk factors. Methods: From May 2013 to March 2019, MPN patients ≥ 18 years old with spleen >5 cm below the costal margin in myelofibrosis (MF) or any size in Polycythemia Vera (PV) or Essential Thrombocythemia (ET) were enrolled prospectively (NCT01816256). Patients with a known history of portal or hepatic vein thrombosis, liver cirrhosis, and esophageal varices were excluded. Screening for PH was done by EGD to evaluate for esophageal varices (EV) or gastric varices (GV), and portal hypertensive gastropathy (PHG). Abdominal Doppler ultrasonography (USG) was performed to evaluate for portal vein thrombosis and spleen size. PH severity was based on the size of varices or severity of PHG. For sample-size calculation, a precision approach was used, considering scenarios where PH prevalence ranged from 5 to 10% and an upper limit for sample size of 100. At a prevalence estimate of 8%, the 90% confidence interval (CI) was 4-14%, with an acceptable lower limit for moving forward with a screening program. Prevalence of PH was calculated as a proportion and CI are exact using Clopper-Pearson method. Results: Among 102 enrolled patients, EGD and USG were completed in 85 and 84, respectively. Primary and Post-PV/ET MF comprised the majority of the study population (93% [79/85]). Evidence of PH was found in 32 patients (38%, [90% CI 29-47]) of whom 12 (14% [90% CI 9-22]) had moderate to severe PH. EV, GV, and PHG were found in 81% (26/32), 13% (4/32), and 31% (10/32) of patients, respectively. The prevalence of PH among MF and PV/ET patients was 35% (28/79) and 67% (4/6), respectively. In MF patients, PH was moderate-to-severe in 13% (90% CI 8-20). No cases of portal vein thrombosis were identified on USG. Apart from a palpable liver observed more frequently in non-PH patients, there were no other significant differences in baseline characteristics among patients with and without PH. The frequency of JAK2, MPL and CALR mutations among PH patients was 78% (25/32),13% (4/32), and 6% (2/32). There was a trend towards more JAK2 mutations in PH patients (78% [25/32] vs 64% [34/53], p=0.23), but no difference in DIPSS risk or frequency of high molecular-risk mutations. Overall survival was similar in both groups. PH-directed interventions were initiated in 59% (19/32); 28% (9/32) were started on beta blocker (BB) prophylaxis, 16% (5/32) were treated with BB and variceal banding, and 16% (5/32) continued on surveillance EGDs alone. Three patients with moderate PH at baseline developed variceal bleeding (2) and ascites (1). All 3 patients were on BB prophylaxis, and 1 had prior prophylactic banding. There were no adverse events related to study procedures EGD or USG. Image: Summary/Conclusion: Given the study population, our results are generalizable to MF patients. Our study shows that clinically significant PH is common in MF patients, and led to additional clinical interventions in significant number of patients. These data support the inclusion of asymptomatic PH screening in the work up of MF patients. P1021: ERYTHROCYTOSIS, THROMBOCYTOSIS AND RATE OF RECURRENT THROMBOEMBOLIC EVENT - A POPULATION BASED COHORT STUDY A. Enblom-Larsson1,*, B. Andreasson2, H. Holmberg1, M. Liljeholm3, A. Själander1 1Department of Public Health and Clinical Medicine, Umeå University, Umeå; 2Department of Hematology, NU Hospital Group, Uddevalla; 3Department of Radiation Sciences, Umeå University, Umeå, Sweden Background: The diagnosis and management of myeloproliferative neoplasms (MPN), polycythemia vera (PV) and essential thrombocythemia (ET) are well established. Other conditions with elevated hemoglobin (Hb), hematocrit (HCT) or platelets (Plt) are not as studied and there are no international accepted guidelines of how to use phlebotomy and anticoagulants to reduce thromboembolic complications. Aims: To assess the frequency of elevated blood values in patients diagnosed with a thromboembolic event, defined as myocardial infarction, ischemic stroke, transient ischemic attack, peripheral arterial thromboembolism, pulmonary embolism, deep venous thrombosis or abdominal thrombosis, and how many of these patients should be examined further to find an underlying MPN, according to the 2016 revised WHO classification. The second aim is to investigate if individuals with elevated Hb, HCT or Plt have a higher rate of first recurrent thromboembolic event depending on underlying condition. Methods: All patients over the age of 18 years with a thromboembolic event as defined above during 2017 and 2018 in the county of Norrbotten (population n=250 230) were included (n=3931). From medical records data of elevated blood values according to the 2016 revised WHO criteria of PV and ET (Hb >16.5g/dL in men, Hb >16.0g/dL in women, HCT >0.49 in men, HCT >0.48 in women or Plt >450 x109) were retrieved, as well as underlying health conditions, recurrent thromboembolic events and death. Results: In total 29.6% (1165/3931) had elevated Hb/HCT or Plt. Elevated Hb/HCT was seen in 12.7% (501/3931), 4% (159/3931) had elevated Hb/HCT at the time of initial thromboembolic event. Underlying conditions causing secondary erythrocytosis was found in 25.5% (128/501). A MPN-diagnosis was found in 1.8% (9/501), and 72.7% (364/501) had an unexplained erythrocytosis. Elevated Plt were found in 17.3% (680/3931). Of these, 91.0% (619/680) had an obvious reactive thrombocytosis while 2.4% (16/680) were diagnosed with MPN. The remaining 6.6% (45/680) had unexplained thrombocytosis. Thrombocytosis at the time of initial thromboembolic event was found in 1.3% (51/3931). In total 35% (405/1165) of the patients with elevated Hb/HCT or Plt should be investigated further to rule out MPN according to current guidelines. Of these, 41 patients had thrombocytosis, 360 erythrocytosis and 4 patients had both. A recurrent thromboembolic event was found in 11% (434/3931), of these 9.7% (42/434) had elevated Hb or HCT and 18.7% (81/434) elevated Plt. The highest rate of recurrent events per 100 years was seen in the group with unexplained elevated platelets (8.11, 95% CI 2.37-13.84). Patients with secondary erythrocytosis and unexplained thrombocytosis had the most inferior event-free survival, 2.30 years (95% CI 1.95-2.64) and 2.51 (1.98-3.04). On the other hand, patients with unexplained erythrocytosis displayed an event free survival even surpassing that of patients without erythrocytosis or thrombocytosis 3.72 (3.55-3.89) vs 3.27 (3.2-3.3) years respectively. Image: Summary/Conclusion: Elevated Hb/HCT or Plt is common in patients with arterial or venous events, affecting almost one third of all patients (1165/3931), out of these one third (405/1165) should be investigated further to rule out MPN. Secondary erythrocytosis and unexplained thrombocytosis were associated with the worst event-free survival while patients with unexplained erythrocytosis were less prone to a recurrent thromboembolic event. These results highlights the importance of finding underlying cause of elevated blood values to reduce risk of recurrent thromboembolic event. P1022: OUTCOMES IN MYELOPROLIFERATIVE NEOPLASM PATIENTS WITH OR WITHOUT PRIOR HISTORY OF CANCER: A POPULATION-BASED STUDY FROM ONTARIO J. T. England1,*, A. Bankar1, W. C. Chan2, N. Liu2, M. C. Cheung3, V. Gupta1 1Medical Oncology/Hematology, Princess Margaret Cancer Centre; 2ICES; 3Hematology, Sunnybrook Health Sciences Centre, Toronto, Canada Background: Therapy-related myeloid neoplasms (tMNs) exist as a distinct diagnostic entity in the 2016 WHO classification, and include MDS, MDS/MPN overlap, and AML that develop following iatrogenic exposure to mutagenic agents and ionizing radiation. Patients with the classical myeloproliferative neoplasms (MPNs), essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF), are excluded from the tMN category. Prior studies have observed an increased risk of other cancers in patients with MPNs, possibly from shared risk factors including older age and chronic inflammation. The impact of prior history of cancer on outcomes in MPN patients is not well understood. A single centre study of MF (Masarova et al, Blood Advances, 2017) found no survival difference between patients exposed to chemotherapy/radiation and those managed with surgery/observation/hormone therapy. Aims: To evaluate the clinical outcomes of MPN patients with or without prior non-MPN cancer, and the impact of tMN-implicated therapy on survival following MPN diagnosis. Methods: We conducted a population-based, retrospective cohort study using the ICES provincial health databases. Included patients were residents of Ontario aged ≥18 years with an MPN diagnosis from Jan 1, 2004 to Dec 31, 2019. Diagnoses of MPN and non-MPN (other cancers excluding AML) cancers were based on International Classification of Diseases for Oncology, third edition (ICD-O-3) codes. Therapy for prior non-MPN cancer were grouped as: therapy implicated in tMN per 2016 WHO criteria (alkylating agents, antimetabolites, topoisomerase inhibitors, antitubulin agents, and ionizing radiation), non-implicated therapies (targeted inhibitors, hormone therapy, other), and surgery/observation alone. The primary outcome was overall survival (OS) from the MPN diagnosis, with multivariable Cox regression adjusted for age and comorbidities. In addition, to understand the patterns of non-MPN cancer type, therapy exposure, and latency period, we compared the MPN cohort with a control cohort with no MPN history matched (1:4) for age (±3 years), gender, geographic location, and neighbourhood income quintile. Results: A total of 10 336 MPN patients (5108 ET, 3843 PV, and 1385 MF) matched to 41 344 controls were identified in the study. MPN patients more frequently had a history of non-MPN cancer compared to the control cohort (n=1421[13.5%] vs. n=5509[11.9%], P<0.001). The most common non-MPN cancers were male genitourinary (24%), breast (18%), gastrointestinal (18%), urothelial (9%), and melanoma (5%). Comparing MPN patients to matched controls, there was no difference in the types of non-MPN cancer, treatment exposures, or latency time. After adjustment for age and comorbid conditions, the OS from PV/ET diagnosis was predicted by prior cancer managed with surgery/observation hazard ratio (HR)[95% confidence interval] 1.36 [1.22-1.52], tMN-implicated therapy HR1.83 [1.55-2.18] and non-implicated therapy HR1.88 [1.49-2.38] (Fig 1a). In MF patients the OS was predicted by tMN therapy, HR1.44 [1.04-1.98]; but not surgery/observation, HR1.17 [0.94-1.46], or non-implicated therapy, HR1.25 [0.83-1.87](Fig 1b). Lack of availability of cytogenetics and mutation profile in this population based dataset is the main limitation of this study. Image: Summary/Conclusion: Compared with matched controls, patients with MPNs are more likely to have had a history of prior cancer. MPN patients with prior cancer have worse OS; and those treated with systemic therapies have shorter OS than patients managed with surgery/observation alone. P1023: COHORT ANALYSIS OF MPN PATIENTS WITH CONCOMITANT MGUS DIAGNOSIS: A RETROSPECTIVE STUDY C. Fatigati1,*, F. Antonucci1, L. De Fazio1, R. Iannotta1, M. Lamagna1, A. Leone1, F. Muriano1, G. Scairati1, F. Pane1, N. Pugliese1 1University of Naples Federico II, University of Naples Federico II, Napoli, Italy Background: Patients with a Ph-negative myeloproliferative neoplasm (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), may harbor or develop plasma-cell dyscrasia such as monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM), however, the clinical and molecular determinants of this co-occurrence are still uncertain. Aims: The main aim of this study was to evaluate correlation between MGUS and MPN related features and clinical course. Methods: We retrospectively analyzed clinical data from 448 patients with a diagnosis of MPNs according to 2016 WHO criteria, performed from 1989 to 2020 at our Institution. Here, we described the clinical and genetic features of MPN patients with concomitant MGUS. Data collection included NGS panel, cytogenetic analysis, clinical history (including thrombotic events) and pharmacological therapy during the entire MPNs follow up. Results: Among the 448 consecutive patients analyzed, 33 (7.4%) displayed both MPN and MGUS. Twenty-three of them were females while 10 males. MPN diagnosis were ET (12/33, 36%), MF (11/33, 33%), PV (8/33, 24%), or pre-MF (2/33, 6%) at a median age of 61 years (28-84); 25 patients showed JAK2 V617F mutation (75.8 %), 3 patients type I CALR mutation (9.1%), 1 patient harbored W515K MPL mutation (3%), and 4 were triple negative (12.1%). Half of them incurred a MPN diagnosis antecedent (19/33, 57.6%) or synchronous (11/33, 33%) with MGUS diagnosis, while only 3 patients (9.1%) had MGUS diagnosed before MPN. Serum M protein consisted in a majority of IgG (84.8%, 14 cases ʎ e 14 ƙ light chain), 2 cases of IgA and 1 case of IgM protein with a median count of 1.04 plasma-cells in bone marrow aspirate at diagnosis. Eventually, after a median follow-up of 7 years (range 1-30 years), 5 patients (15.2%) originated a second hematological malignancy and 2 patients (6.1%) evolved in secondary myelofibrosis. In particular, 3 patients (10%) developed Non-Hodgkin lymphoma, 1 patient MM, and 1 patient developed acute myeloid leukemia (AML). NGS, cytogenetic, clinical history and MPN specific therapies don’t seem to influence clinical course of analyzed patients. Summary/Conclusion: In literature there are few publications that highlight the coexistence of MGUS in MPN patients. Currently little is known about the underlying molecular mechanisms and there are no guidelines clarifying whether a particular treatment or follow-up is necessary. The incidence of MGUS in our MPN cohort is higher than general population matched for sex and age. Although, this data can be influenced by an accurate hematological follow-up, we can’t exclude a real increased rate of MGUS in MPN patient. It is known MPN can predispose to an higher risk of developing second primary malignancies such as B cellular lymphoproliferative disorders. Interestingly, in our cohort the percentage of patients with coexisting MGUS and MPN who developed second hematological malignancies seemed slightly higher than literature data. Further studies with case-control cohort are necessary to better understand the possible prognostic impact and mutual influence between MPN and MGUS. P1024: IMPACT OF TP53 IN MYELOFIBROSIS UNDERGOING STEM CELL TRANSPLANTATION N. Gagelmann1,*, A. Badbaran1, V. Panagiota2, C. Wolschke1, F. Ayuk1, F. Thol2, M. Ditschkowski3, B. Cassinat4, M. Robin4, T. Schroeder3, M. Heuser2, H. C. Reinhardt3, N. Kröger1 1University Medical Center Hamburg-Eppendorf, Hamburg; 2Medical School Hannover, Hannover; 3University Hospital Essen, Essen, Germany; 4APHP, Paris, France Background: Most of myelofibrosis (MF) cases harbor somatic mutations in the driver genes JAK2, CALR, or MPL. Other somatic nondriver mutations have been increasingly detected with the use of high-throughput sequencing. The role of TP53 in prognosis of MF has long been unclear, but recent first studies suggested worse survival for patients harboring this mutation. Aims: While allogeneic stem cell transplantation still remains the only curative treatment option, outcome of patients with TP53 has not been evaluated yet. Methods: Here, we analysed in 417 patients with PMF or post ET/PV MF incidence and impact on otcome of TP53. We detected 46 patients with detectable TP53 mutation using next-generation sequencing, and compared characteristics and outcome after allograft with 371 non-TP53 patients with primary or secondary MF. Samples were collected at time of transplantation. Primary end points were overall survival (OS) and nonrelapse mortality. Secondary objectives were relapse, relapse-free survival, and graft-versus-host disease (GVHD). Results: Median follow-up from transplantation was 6.1 years for the TP53 group and 7.0 years for the non-TP53 group, respectively (P=0.50). Baseline characteristics were generally balanced, whereas more patients in the TP53 group had Karnofsky performance score <90 (57% versus 40%; P=0.04). Median OS was 2.1 years (0.1-5.4 years) for the TP53 group versus 15.4 years (11.5-19.3 years) for the non-TP53 group (P=0.002), and the 6-year OS was 40% (24-55%) for the TP53 group versus 65% (60-70%) for the non-TP53 group. 6-year nonrelapse mortality was 35% (21-49%) for the TP53 group versus 27% (22-32%) for the non-TP53 group (P=0.16) and 6-year incidence of relapse was 26% (13-39%) for the TP53 group versus 18% (14-21%) for the non-TP53 group (P=0.09). 6-year relapse-free survival was 35% (20-50%) versus 53% (48-58%; P=0.01). Acute GVHD occurred in 63% versus 57% (P=0.43). Of patients with relapse, 92% in the TP53 group versus 63% in the non-TP53 group died (P=0.05). Risk for death appeared to be associated with variant allele frequency (P=0.06). Summary/Conclusion: MF patients with TP53 appeared to have higher nonrelapse mortality and relapse incidence but significantly worse relapse-free and overall survival, mainly because of worse survival after relapse. P1025: RUXOLITINIB-ASSOCIATED CRYPTOCOCCOSIS: A SYSTEMATIC REVIEW AND META-ANALYSIS M.-A. Gaman1,2,* 1Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy; 2Hematology Department, Centre of Hematology and Bone Marrow Transplantation, Fundeni Clinical Institute, Bucharest, Romania Background: The use of the JAK 1/2 inhibitor ruxolitinib (RUX) in myeloproliferative neoplasms (MPNs), namely primary (PMF) or secondary myelofibrosis (MF) and polycythemia vera (PV), has been associated with the development of opportunistic infections due to the immunosuppressive actions of this drug on cellular immunity. However, there is still controversy regarding the association between ruxolitinib treatment and the occurrence of cryptococcosis, a fungal infection caused by the yeast Cryptococcus neoformans or Cryptococcus gattii. Aims: To report on cryptococcosis cases in RUX-treated MPNs and report on epidemiology, clinical manifestations, RUX associations, outcomes. Methods: Systematic search computed in PubMed/Medline, Web of Science and SCOPUS, from the inception of these databases until 28.02.2022, to identify relevant English-written articles. Eligibility criteria: 1. Confirmed MPNs diagnosis, 2. Confirmed diagnosis of cryptococcosis, 3. MPNs patients receiving RUX therapy. Results: The systematic search yielded 60 potentially relevant papers. After removal of duplicates (n=20) and screening of abstracts/titles (n=29), 11 manuscripts were selected for full-text review. After applying the eligibility criteria, 10 articles were entered into the qualitative and quantitative analysis. In total, 10 case reports on cryptococcosis occurring in RUX-treated MPNs patients were detected: 80.0% males, mean age 66.8±8.3 years, range 51-79 years. Most reports (70.0%) came from Asia (3 from Japan, 2 from Thailand, 1 from India and Taiwan each) and from the USA (30.0%) and were published between 2013 and 2021. In total, 70% were diagnosed with PMF, whereas cryptococcosis also occurred in PV, post-PV MF or post-ET MF (10.0%, 1 subject each). The average time from RUX initiation was 22.1±14.5 (6-48) months. HIV status was reported in 60.0% of cases and all subjects were apparently immunocompentent and had negative HIV screening. Cryptococcosis was caused by Cryptococcous neoformans only and occurred most commonly as disseminated infection (40.0%), followed by cryptococcal meningitis (30.0%) or pulmonary cryptococcosis (30.0%). Cryptococcosis co-occurred with tuberculosis (20.0%), disseminated histoplasmosis (10.0%) or Mycobacterium haemophilum cellulitis (10.0%). Most common presentations were fever (70.0%), neurological symptoms/signs (headache, consciousness/visual disturbances, 40.0%), shortness of breath (30.0%) or fatigue/lethargy (30.0%). Positive cryptococcosis diagnosis was established based on serum/cerebrospinal fluid (CSF) cryptococcal antigen positivity (80.0%) and/or positive CSF, blood or bronchoalveolar fluid cultures (70.0%). Specific treatment consisted in amphotericin B (50.0%) and/or fluconazole (70.0%). Amphotericin B was given in combination with flucytosine (20.0%) or fluconazole (20.0%) or in monotherapy (30.0%). Other combinations or drugs, i.e., isavuconazole, voriconazole, were used due to renal toxicity related to amphotericin B use. RUX was discontinued in 70.0% of cases and restarted in 20.0% of subjects. Cryptococcosis-related death occurred in 30.0% of cases. Image: Summary/Conclusion: Cryptococcosis can developed during ongoing RUX therapy in MPNs subjects, even if apparently immunocompetent, potentially due to the immunosuppressive actions of this drug on cellular immunity. Screening for Cryptococcus neoformans in selected individuals might reduce the risk of disseminated infection and death. RUX discontinuation during antifungal treatment is advised due to reported progression of lesions and drug-drug interactions with antifungal agents. P1026: COMPARATIVE GENOMIC PROFILING OF MYELOPROLIFERATIVE NEOPLASMS PRESENTING WITH AND WITHOUT SPLANCHNIC VEIN THROMBOSIS M. Garrote1,*, M. López-Guerra1, E. Arellano-Rodrigo2, M. Magaz3, A. Triguero4, S. Carbonell4, J. R. Alamo1, F. Turon3, V. Hernandez-Gea3, A. Baiges3, D. Colomer1, J. C. García-Pagán3, F. Cervantes4, A. Alvarez-Larrán4 1Pathology - Hematopathology Unit; 2Hemotherapy and Hemostasis; 3Hepatology; 4Hematology, Hospital Clínic de Barcelona, Barcelona, Spain Background: Splanchnic vein thrombosis (SVT) is the initial manifestation in 5% of myeloproliferative neoplasms (MPN). MPN patients presenting with SVT are usually younger, have less abnormal blood counts and lower JAK2V617F allele burden than other MPN patients. However, it is unknown whether they have a different genomic background. Aims: To describe the clinical and genomic profile of MPN presenting with SVT and to compare it with a matched group of MPN without SVT. Methods: Samples of 190 MPN patients were selected: 64 MPN presenting with SVT (SVT+) and 126 MPN without SVT (SVT-). SVT+ and SVT- were matched according to sex, age, MPN clinical subtype and driver mutation. Next Generation Sequencing was performed using Myeloid Solution by Sophia Genetics, which includes 30 genes recurrently mutated in MPN. Variants were selected using Sophia DDM software and were subsequently classified into 5 categories: 5 (definitely pathogenic), 4 (likely pathogenic), 3 (uncertain), 2 (likely benign), and 1 (benign). Only variants included into 4 and 5 categories were taken into account. Patients were classified according to the genomic classification proposed by Grinfeld and colleagues. Results: One hundred and three patients were women (54.2%) and 87 patients were men (45.8%), without significant differences between SVT+ and SVT- groups. Median age was 44 years (range: 15-85) and 50 years (range: 15-87) in SVT+ and SVT-, respectively (p not significant). Regarding MPN clinical subtype in SVT+ group, 39 (60.9%), 15 (23.4%) and 10 (15.6%) corresponded to Polycythemia Vera (PV), Essential Thrombocythemia (ET) and MPN unclassifiable, respectively. In the SVT- group, 75 (59.5%) and 51 (40.5%) corresponded to PV and ET, respectively. Genomic classification in the SVT+ and SVT- groups is shown in Table 1. The proportion of patients with high molecular risk (TP53 disruption/aneuploidy, chromatin/spliceosome mutation, or homozygous JAK2 mutation) was 14.1% and 26.2% for SVT+ and SVT-, respectively (p=0.06). Median number of pathogenic mutations was 1 (range: 1-4) in both SVT+ and SVT- groups. Pathogenic mutations in TET2, DNMT3A and ASXL1 were present in 10%, 3.7% and 3.7%, respectively, without significant differences between both groups. With a mean follow-up of 10.1 years for cases and 9.7 years for controls, 27 patients died (13 SVT+ and 14 SVT-). Median survival was 24 years and not reached for SVT+ and SVT-, respectively (p=0.1). On multivariate analysis, SVT+ group showed a higher risk of death (HR: 2.4, 95%CI: 1.1-5.5, p=0.03) after correction by age, sex and genomic classification. Transformation to myelofibrosis and acute leukemia was low and similar in both groups: 4.7% vs. 5.6% for myelofibrosis in SVT+ and SVT-, respectively (p not significant); and 0% vs. 2.4% for acute leukemia (p not significant). On multivariate analysis, patients with TP53 disruption/aneuploidy, chromatin/spliceosome mutation, or homozygous JAK2 showed a higher risk of disease progression to myelofibrosis or acute leukemia (HR: 14.5, 95%CI: 3.1-68.7, p=0.001) regardless of the presence of SVT at diagnosis, age and sex. Image: Summary/Conclusion: MPN presenting with SVT and MPN-matched controls showed a low molecular complexity. Genomic classification was helpful in identifying a small proportion of MPN patients presenting with SVT who are at high risk of disease progression. SVT at MPN diagnosis is associated with a higher risk of death independent of sex, age and genomic classification. P1027: RESPONSES TO AVAPRITINIB IN PATIENTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS: HISTOPATHOLOGIC ANALYSES FROM EXPLORER AND PATHFINDER CLINICAL STUDIES T. George1,*, K. H. Karner1, K. A. Moser1, A. Rets1, A. Reiter2, D. H. Radia3, M. Deininger4, H.-M. Lin5, S. Dimitrijević6, D. J. DeAngelo7 1Department of Pathology, ARUP Laboratories, University of Utah, School of Medicine, Salt Lake City, United States of America; 2Department of Hematology and Oncology, University Hospital Mannheim, Heidelberg University, Mannheim, Germany; 3Guy’s & St Thomas’ NHS Foundation Trust, London, United Kingdom; 4Versiti Blood Research Institute, Milwaukee; 5Blueprint Medicines Corporation, Cambridge, United States of America; 6Blueprint Medicines Corporation, Zug, Switzerland; 7Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States of America Background: Systemic mastocytosis (SM) is a mast cell (MC) neoplasm driven by the KIT D816V mutation in ~95% of cases. Diagnosis includes evaluation of MC aggregates in extracutaneous organs, atypical MC morphology, and MC immunophenotype. Avapritinib, a potent and selective KIT D816V inhibitor, has been approved in the US for patients with advanced SM (AdvSM) based on the phase 1 EXPLORER and phase 2 PATHFINDER studies. Aims: Here, we describe a comprehensive evaluation of the effect of avapritinib on bone marrow (BM) pathology in patients treated in these studies. Methods: Patients aged ≥18 years with diagnoses of AdvSM per local investigator were treated with once-daily avapritinib <200 mg (n=17), 200 mg (n=126), and ≥300 mg (n=50). Bone marrow biopsies (BMBs), aspirates (BMAs), and peripheral blood (PB) smears were obtained at screening, and after 8 and 24 weeks of treatment with avapritinib. Evaluations were performed using standard Wright-Giemsa and H&E staining, and IHC was performed on formalin-fixed EDTA-decalcified BMBs using standard techniques for CD25 and CD30. Changes in fibrosis were assessed by the European Consensus on grading of BM fibrosis (MF score). Results: As of April 2021, 193 patients enrolled in EXPLORER (n=86) and PATHFINDER (n=107) were included in analyses with centrally confirmed diagnoses of aggressive SM (n=29), SM with an associated hematologic neoplasm (n=119), mast cell leukemia (n=28), indolent SM (n=14), smoldering SM (n = 2) or CMML (n=1). Median (range) age was 67 years (31–88), 56% were male and 88% had a KIT D816V mutation. Following treatment with avapritinib, a decrease in the proportion of patients with multifocal dense aggregates of MCs in BMBs was observed from 93% at screening to 50% by Week 8 (Table). Avapritinib treatment reduced the proportion of aberrant CD25+ and CD30+ MCs in BMBs by Week 8 and Week 24 (Table). In BMAs, avapritinib reduced the mean MC burden from 11% of total nucleated cells at screening to 2% at Week 8 and by Week 24, with reduced proportions of immature and spindle-shaped MCs (Table). Of 9 patients with circulating MCs at screening and post-screening sample measurements (6 with SM with associated hematologic neoplasm diagnoses and 3 with MCL), 8 had no detectable MCs in PB by Week 8. The proportion of patients with fibrosis in BMBs decreased from 96% (178/186) at screening to 90% (142/158) at Week 8 to 84% (103/123) through week 24, and MF scores were reduced during avapritinib treatment in those patients presenting with increased fibrosis at screening (Table). Image: Summary/Conclusion: Patients treated with avapritinib were observed to have rapid (Week 8) and marked (Week 24) reductions in neoplastic BM MCs with a return to a normal morphologic appearance and immunophenotype. This was accompanied by an improvement of fibrosis and a decrease in circulating MCs. These improvements in disease histopathology provide further support that treatment with this highly selective and potent KIT D816V inhibitor is disease modifying in AdvSM. P1028: PTG-300 (RUSFERTIDE) TREATMENT INTERRUPTION REVERSES HEMATOLOGICAL GAINS AND RESTORES THERAPEUTIC BENEFIT ON REINITIATION IN SUBJECTS WITH POLYCYTHEMIA VERA A. Kuykendall1,*, M. Kremyanskaya2, Y. Ginsburg2, N. Pemmaraju3, E. Ritchie4, J. Gotlib5, F. Valone6, S. Khanna6, S. Gupta6, R. Hoffman2, S. Verstovsek3 1Hematology and Med Oncology, USF, Moffitt Cancer Center, Tampa; 2Hematology, Mt. sinai, New york; 3Dept of Leukemia, M.D. Anderson, Houston, Tx; 4Hematology, Weill Cornell medical College, New york; 5Hematology, school of Medicine, Stanford, Palo Alto, Ca; 6Clinical Research and Development, Protagonist Therapeutics, Newark, United States of America Background: Polycythemia vera (PV) patients are treated with periodic therapeutic phlebotomy (TP) and if needed, cytoreductive therapy to maintain hematocrit (HCT) <45% to reduce the incidence of thrombosis. Constitutive erythropoiesis and repeated TP result in iron deficiency which further stimulates iron absorption and availability for robust erythropoiesis. Iron deficiency contributes to fatigue, which is the most severe and prevalent symptom for patients with PV. We have previously shown that adding Rusfertide, a hepcidin mimetic, to existing PV treatment, significantly, and effectively reverses iron deficiency and essentially eliminates TP requirement for >1 year in PV patients [Hoffman ASH 2021]. The ongoing PTG-300-04 (NCT04057040) Phase 2 study of Rusfertide in TP-dependent PV patients experienced an FDA-mandated brief hold which resulted in dosing interruption of Rusfertide for all patients. Aims: To examine the impact of temporary rusfertide withdrawal on iron homeostasis and TP requirements after temporary suspension in the ongoing PTG-300-04 study. Methods: Eligibility criteria for PTG-300-04 study include PV diagnosis [by 2016 WHO criteria] and ≥3 phlebotomies with/without concurrent cytoreductive therapy in the 24 weeks before enrollment. Rusfertide 10-120 mg weekly SQ injection was added to existing cytoreductive treatment and adjusted to maintain HCT <45%. During the brief clinical hold patients were maintained on their cytoreductive regimens and had TP for HCT >45%. Most patients returned to Rusfertide add-on treatment 2-3 months after the dosing interruption and reinititation. Results: Pre-enrollment, PV patients were treated with TP alone (30) or with TP+cytoreductive therapy (33). TP requirements in the 24 weeks before enrollment ranged from 3 to 9. Rusfertide treatment enabled consistent HCT control <45%, essentially eliminated TPs in all sub-groups, decreasing RBC count and increased MCV and MCH values. Pre-enrollment, mean iron-related parameters were consistent with systemic iron deficiency. Rusfertide treatment resulted in progressive normalization of serum ferritin, suggesting a redistribution of systemic iron. During Rusfertide dosing interruption, all patients had significant (p<0.01) increase in TPs, HCT, and RBC count; and a simultaneous decrease in MCV and serum ferritin. Patients also reported increase in PV-related symptoms (i.e., increased fatigue and worsening concentration). Reinitiating Rusfertide resulted in rapid normalization of hematologic parameters, elimination of TP, and improvement in symptoms demonstrating potential effectiveness of this approach. Most adverse events were grade 1-2. The most frequent AE was transient injection site reaction reported in 59% of patients. Summary/Conclusion: The current results support Rusfertide as an effective treatment option in PV, reversing iron deficiency and essentially eliminating TP requirements in PV patients with/without cytoreductive agents. Reversal of hematological parameters during dosing interruption further confirms the effect of Rusfertide on both iron homeostasis and erythrocytosis. Taken together, Rusfertide is a very promising novel biologic agent in both low and high-risk PV patients. A global Phase 3 trial of rusfertide in PV, VERIFY, has been initiated. P1029: MATCHING-ADJUSTED INDIRECT COMPARISON (MAIC) OF PELABRESIB (CPI-0610) IN COMBINATION WITH RUXOLITINIB VS RUXOLITINIB OR FEDRATINIB MONOTHERAPY IN PATIENTS WITH INTERMEDIATE OR HIGH-RISK MYELOFIBROSIS V. Gupta1,*, J. Mascarenhas2, M. Kremyanskaya2, R. K. Rampal3, M. Talpaz4, J.-J. Kiladjian5, A. Vannucchi6, S. Verstovsek7, G. Colak8, D. Dey9, C. Harrison10 1Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada; 2Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai; 3Memorial Sloan Kettering Cancer Center, New York, NY; 4University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, United States of America; 5Hôpital Saint-Louis, Université de Paris, Paris, France; 6Azienda Ospedaliero-Universitaria Careggi, University of Florence, Florence, Italy; 7University of Texas MD Anderson Cancer Center, Houston, TX; 8Constellation Pharmaceuticals a MorphoSys Company, Boston, MA, United States of America; 9MorphoSys AG, Planegg, Germany; 10Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom Background: Pelabresib (CPI-0610), an investigational product, in combination with ruxolitinib (ruxo) has shown encouraging responses in terms of ≥35% reduction in spleen volume from baseline (SVR35) and ≥50% reduction in total symptoms score (TSS) from baseline (TSS50) in Janus kinase inhibitor (JAKi) treatment-naïve patients with intermediate or high-risk myelofibrosis (MF) in Arm 3 of the open-label Phase 2 MANIFEST study (NCT02158858). A cross-trial comparison with ruxo or fedratinib monotherapy has limitations in the presence of the potential imbalances in baseline characteristics. Aims: Here we report findings from matching-adjusted indirect comparisons (MAICs), conducted to correct for potential imbalances in baseline characteristics, comparing the primary endpoint SVR35 and secondary endpoint TSS50 at Week (Wk) 24 for the combination of pelabresib and ruxo (MANIFEST Arm 3) with Phase 3 ruxo monotherapy (COMFORT-I and II, SIMPLIFY-1) and fedratinib monotherapy (JAKARTA) data. Methods: Individual patient level data were available for MANIFEST Arm 3, while only published summary data were available for COMFORT-I and II, SIMPLIFY-1 and JAKARTA. An unanchored MAIC was conducted wherein patients in MANIFEST Arm 3 were reweighted to adjust for imbalances in the following key prognostic baseline characteristics: gender, MF subtype, International Prognostic Scoring System risk status, previous hydroxyurea use, platelet count, hemoglobin levels, spleen volume and JAK2V617F status. Weighted mean outcomes of the treatment effects were estimated together with their 95% confidence interval (CI) using robust sandwich estimators for variance. The weights were also used to calculate the Effective Sample Size (ESS). Treatment effect outcomes for SVR35 and TSS50 at Wk 24 are presented in terms of Response Rates (RR) and Response Rate Ratio (RRR, defined as RR in MANIFEST Arm 3/RR in comparator arm). An RRR >1 favors the pelabresib + ruxo arm in MANIFEST Arm 3 over the JAKi monotherapy comparator arm. A limitation of a MAIC is that bias due to potential unmeasured differences in patient baseline characteristics between trials cannot be excluded. Results: The MAIC analysis resulted in complete summary-level balance in the weighted distributions of the prognostic factors in MANIFEST Arm 3 versus comparators. The figure presents the weighted (or MAIC-adjusted) and unweighted (or unadjusted) treatment effect comparisons along with their 95% CI for SVR35 and corresponding ESS at Wk 24. Statistically significant MAIC-adjusted (weighted) RRRs of 1.57 (95% CI 1.10, 2.24; p-value: 0.012), 1.82 (95% CI: 1.17, 2.83; p-value: 0.008), 2.13 (95% CI: 1.51, 3.02; p-value: <0.001), 1.76 (95% CI: 1.16, 2.66; p-value: 0.008) and 1.55 (95% CI: 1.06, 2.27; p-value: 0.023) were observed for MANIFEST Arm 3 (pelabresib + ruxo) versus COMFORT-I and -II and SIMPLIFY-1 (ruxo only) and JAKARTA (fedratinib 400 mg and 500 mg), respectively. These weighted analyses are consistent with statistically significant results of unadjusted analyses. RRRs >1 were also observed for TSS50 at Wk 24 for all the comparisons. Image: Summary/Conclusion: The MAIC analysis, adjusting for eight key baseline prognostic factors, suggests an improvement in SVR35 and TSS50 at Wk 24 for JAKi treatment-naïve patients with MF treated with a combination of pelabresib and ruxo over ruxo or fedratinib monotherapy. MANIFEST-2 (NCT04603495), a Phase 3 double-blind, randomized study assessing the efficacy and safety of pelabresib or placebo in combination with ruxo in patients with MF, is currently open for enrollment. P1030: MANIFEST-2, A GLOBAL, PHASE 3, RANDOMIZED, DOUBLE-BLIND, ACTIVE-CONTROL STUDY OF PELABRESIB (CPI-0610) AND RUXOLITINIB VS PLACEBO AND RUXOLITINIB IN JAK INHIBITOR-NAÏVE MYELOFIBROSIS PATIENTS C. Harrison1,*, R. K. Rampal2, V. Gupta3, S. Verstovsek4, M. Talpaz5, J.-J. Kiladjian6, R. Mesa7, A. Kuykendall8, A. Vannucchi9, F. Palandri10, S. Grosicki11, T. Devos12, E. Jourdan13, M. J. Wondergem14, H. K. Al-Ali15, V. Buxhofer-Ausch16, A. Alvarez-Larrán17, S. Akhani18, R. Muñoz-Carerras19, Y. Sheykin19, G. Colak19, M. Harris19, J. Mascarenhas20 1Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom; 2Memorial Sloan-Kettering Cancer Center, New York, NY, United States of America; 3Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada; 4Leukemia Department, University of Texas MD Anderson Cancer Center, Houston, TX; 5University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, United States of America; 6Hôpital Saint-Louis, Université de Paris, Paris, France; 7Mays Cancer Center at UT Health San Antonio MD Anderson Cencer Center, San Antonio, TX; 8Mofitt Cancer Center, Tampa, FL, United States of America; 9Azienda Ospedaliero-Universitaria Careggi, University of Florence, Florence; 10IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy; 11Medical University of Silesia in Katowice, Katowice, Poland; 12University Hospitals Leuven and Laboratory of Molecular Immunology (Rega Institute), KU Leuven, Leuven, Belgium; 13C.H.U, Nîmes, France; 14Amsterdam University Medical Centers, Amsterdam, Netherlands; 15University Hospital Halle, Halle, Germany; 16KRANKENHAUS DER ELISABETHINEN Linz GmbH, Linz, Austria; 17Hospital Clínic Barcelona, Barcelona, Spain; 18MorphoSys AG, Planegg, Germany; 19Constellation Pharmaceuticals a MorphoSys Company, Boston, MA; 20Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America Background: Myelofibrosis (MF) is characterized by bone marrow fibrosis, anemia, splenomegaly and constitutional symptoms. Progressive bone marrow fibrosis results from aberrant megakaryopoeisis and expression of proinflammatory cytokines, both of which are heavily influenced by bromodomain and extraterminal domain (BET)-mediated gene regulation and lead to myeloproliferation and cytopenias. Pelabresib (CPI-0610) is an oral small-molecule investigational inhibitor of BET protein bromodomains currently being developed for the treatment of patients with MF. It is designed to downregulate BET target genes and modify nuclear factor kappa B (NF-κB) signaling. MANIFEST-2 was initiated based on data from Arm 3 of the ongoing Phase 2 MANIFEST study (NCT02158858), which is evaluating the combination of pelabresib and ruxolitinib in Janus kinase inhibitor (JAKi) treatment-naïve patients with MF. Primary endpoint analyses showed splenic and symptom responses in 68% and 56% of 84 enrolled patients, respectively. Aims: MANIFEST-2 (NCT04603495) is a global, Phase 3, randomized, double-blind, active-control study of pelabresib and ruxolitinib versus placebo and ruxolitinib in JAKi treatment-naïve patients with primary MF, post-polycythemia vera MF or post-essential thrombocythemia MF. The aim of this study is to evaluate the efficacy and safety of pelabresib in combination with ruxolitinib. Here we report updates from a recent protocol amendment. Methods: The MANIFEST-2 study schema is shown in Figure 1. Key eligibility criteria include a Dynamic International Prognostic Scoring System (DIPSS) score of Intermediate-1 or higher, platelet count ≥100 × 109/L, spleen volume ≥450 cc by computerized tomography or magnetic resonance imaging, ≥2 symptoms with an average score ≥3 or a Total Symptom Score (TSS) of ≥10 using the Myelofibrosis Symptom Assessment Form v4.0, peripheral blast count <5% and Eastern Cooperative Oncology Group performance status ≤2. Patient randomization will be stratified by DIPSS risk category (Intermediate-1 vs Intermediate-2 vs High), platelet count (>200 × 109/L vs 100–200 × 109/L) and spleen volume (≥1800 cm3 vs <1800 cm3). Double-blind treatment (pelabresib or matching placebo) will be administered once daily for 14 consecutive days, followed by a 7-day break, which is considered one cycle of treatment. Ruxolitinib will be administered twice daily for all 21 days of the cycle. The primary endpoint is SVR35 response (≥35% reduction in spleen volume from baseline) at Week 24, and the key secondary endpoint is TSS50 response (≥50% reduction in TSS from baseline) at Week 24. Other secondary endpoints include safety, pharmacokinetics, changes in bone marrow fibrosis, duration of SVR35 response, duration of TSS50 response, progression-free survival, overall survival, conversion from transfusion dependence to independence and rate of red blood cell transfusion for the first 24 weeks. Results: Study recruitment is ongoing; 400 patients (200 per arm) from North America, Europe, Asia and Australia will be enrolled. The study opened for enrollment in November 2020. Image: Summary/Conclusion: MANIFEST-2 was initiated based on data from the ongoing Phase 2 MANIFEST study with the aim of assessing the efficacy and safety of pelabresib and ruxolitinib in JAKi treatment-naïve patients with MF. MANIFEST-2 is currently open for enrollment. P1031: LOW-DOSE DOACS IN VERY HIGH-RISK MYELOPROLIFERATIVE NEOPLASMS: LESS BLEEDING BUT MORE ARTERIAL THROMBOTIC EVENTS L. Herbreteau1,*, L. Papageorgiou2,3, L. Le Clech4, G. Garcia5, C. James5, B. Pan-Petesch1,6, F. Couturaud7, E. Lippert8,9, G. Gerotziafas2,3, J.-C. Ianotto1,6,9 1Service d’Hématologie Clinique, Centre Hospitalier Universitaire de Brest, Brest; 2Service d’Hématologie Biologique, Hôpital Tenon, Hôpitaux Universitaires de l’Est Parisien, Assistance Publique Hôpitaux de Paris; 3Research Group “Cancer, Hémostase et Angiogenèse” INSERM UMR_S 938, Centre de Recherche Saint-Antoine Faculté de Médecine, Institut Universitaire de Cancérologie, Sorbonne Universités, Paris; 4Service de Médecine Interne, Maladies Infectieuses et du Sang, Centre Hospitalier de Cornouaille, Quimper; 5Laboratoire d’Hématologie, Centre Hospitalier Universitaire de Bordeaux, Bordeaux; 6GETBO, Groupe d’Etude des Thromboses en Bretagne Occidentale, EA3878; 7Département de Médecine Interne et de Pneumologie; 8Laboratoire d’Hématologie, Centre Hospitalier Universitaire de Brest, Brest; 9France Intergroupe des néoplasies Myéloprolifératives, Paris, France Background: Direct oral anticoagulants (DOACs) have recently proven their efficacy and safety, as primary and secondary prevention agents, for thrombosis in cancer patients. Thrombotic and haemorrhagic events are major causes of morbidity and mortality in Philadelphia-negative myeloproliferative neoplasm (MPN) patients. However, the best agent to prevent thrombotic events without increasing the bleeding incidence among these patients remains uncertain. Aims: In this study, we aimed to determine if DOACs might be an interesting choice to reduce the thrombotic risk in high-risk MPN patients. Methods: We analysed a large multicentric cohort of MPN patients treated with DOACs for atrial fibrillation (AF) or thrombotic events. We evaluated the overall thrombosis and haemorrhage rates related to DOACs, and also by the DOAC type (apixaban or rivaroxaban) as well as the daily dosage prescribed. Results: We included 135 MPN patients with a median follow-up of 23.8 months since DOAC initiation. Twenty patients (14.8%) developed 30 thrombotic events (28 arterial thromboses in 19 patients) for a global incidence of 6.5% patient-years. No difference was highlighted between apixaban and rivaroxaban in terms of thrombosis risk, but the incidence of arterial thrombosis was significantly higher on low-dose DOACs (11.9 vs. 4.5% patient-years, p=0.04). Bleeding events were more frequent in the full-dose group (15.2 vs. 41.2%, p=0.006). However, major and clinically relevant non major (CRNM) bleeding events occurred in 18 patients (13.3%), with no difference between the DOAC types or the DOAC doses. In multivariate analysis, age was the only identified thrombotic risk factor, whereas risk factors for major or CRNM bleeding were a full-dose treatment regimen and a combination of DOAC and low-dose aspirin. Image: Summary/Conclusion: DOACs seem effective in preventing venous thrombosis in MPN patients with AF or thrombotic events. For these high-risk patients, low-dose DOACs exposed patients to more arterial thrombosis but fewer bleeding events. Prospective studies are now needed to evaluate and compare DOACs to the currently recommended antithrombotic drugs for high-risk MPN patients. P1032: REAL-LIFE VALIDATION OF MIPSS70: A RETROSPECTIVE MULTICENTER AND NGS ANALYSIS FROM THE GEMFIN DATABASE A. Hernández-Sánchez1,2,*, Á. Villaverde-Ramiro2, A. Álvarez-Larrán3, E. Arellano-Rodrigo3, M. Pérez Encinas4, F. Ferrer Marín5,6,7,8, E. Such9, G. Carreño Gómez-Tarragona10, N. de las Heras Rodríguez11, M. A. Durán Pastor12, M. I. Mata Vázquez13, L. Fox14, J. M. Raya Sánchez15, E. Magro Mazo16, M. T. Gómez Casares17, M. J. Ramírez Sánchez18, B. Xicoy Cirici19, Á. Ramírez Páyer20, M. J. Fernández Llavador21, A. Á. Martín López1,2, A. Mosquera Orgueira4, J. Martínez Elicegui2, M. Garrote3, B. Bellosillo22, T. González1,2,23, J. Hernández-Rivas1,2,23, J. C. Hernández Boluda24 1Hospital Universitario de Salamanca; 2Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca; 3Hospital Clínic de Barcelona, Barcelona; 4Complexo Hospitalario Universitario de Santiago, Santiago de Compostela; 5Hospital Morales Meseguer; 6Universidad Católica San Antonio de Murcia (UCAM); 7Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER); 8Instituto Murciano de Investigación Biosanitaria, Murcia; 9Hospital Universitari I Politècnic La Fe, Valencia; 10Hospital Universitario 12 de Octubre, Madrid; 11Hospital Universitario de León, León; 12Hospital Universitari Son Espases, Palma de Mallorca; 13Complejo Hospital Costa Del Sol, Marbella; 14Hospital Universitari Vall D’Hebron, Barcelona; 15Hospital Universitario de Canarias, Santa Cruz de Tenerife; 16Hospital Universitario Príncipe de Asturias, Alcalá de Henares; 17Hospital Universitario de Gran Canaria Dr. Negrín, Las Palmas de Gran Canaria; 18Hospital Universitario de Jerez, Jerez de la Frontera; 19Institut Català d’Oncologia-Hospital Universitari Germans Trias I Pujol de Badalona, Badalona; 20Hospital Universitario Central de Asturias, Oviedo; 21Hospital Universitario Dr. Peset, Valencia; 22Hospital del Mar, Barcelona; 23Centro de Investigación del Cáncer (CIC), Salamanca; 24Hospital Clínico Universitario de Valencia, Valencia, Spain Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the presence of driver mutations (JAK2, CALR or MPL) in most of the patients. The development of next generation sequencing (NGS) has favored the incorporation of mutational information in novel prognostic scores. However, most of these risk stratification models also include cytogenetic information, which is only available for a fraction of patients with this disease, thus reducing their real-life applicability. Mutation-enhanced international prognostic scoring system for transplant-age patients (MIPSS70) is based on clinical and mutational risk factors and it is useful in the absence of cytogenetic information, but it has not been validated yet in a real-life clinical setting. Aims: To validate MIPSS70 risk stratification model for transplant-age PMF patients from a multicenter database. Methods: From the GEMFIN multicenter database, 668 myelofibrosis patients with enough information to calculate MIPSS70 were selected. As the score is intended for patients aged ≤70 years with PMF, the final analysis was carried out in 218 patients meeting these criteria. Patients who received allogeneic stem cell transplantation were censored on the date of transplantation. Results: Median age was 61 years (range 18-70), 69% of patients were male and median follow-up time was 4.65 years. Regarding the presence of MIPSS70 variables at disease diagnosis, 45% of patients had constitutional symptoms, 43% had hemoglobin <100g/L, 15% leukocytes >25 (x109/L) and 33% platelets <100 (x109/L). Circulating blasts were ≥2% in 30% and most patients presented BM fibrosis grade ≥2 (74%). CALR type 1 mutation was detected only in 12%, while any high-molecular risk (HMR) mutation was present in 31% (8% for ≥2 HMR mutations). In brief, in comparison to MIPSS70 training cohort, the patients in the present series were older and presented a higher proportion of adverse risk factors (Figure 1A). More than half of the patients were classified as high risk according to MIPSS70 (52%), while 35% were intermediate and 13% low risk. The 5-year overall survival (OS) was 44% (median 4.6 years), 77% (median 13 years) and 95% (median not reached) for high, intermediate and low risk patients respectively (p<0.001, Figure 1B). The 5-year leukemia-free survival (LFS) was 42%, 77% and 95% for high, intermediate and low risk groups (p<0.001). Univariate analysis of each MIPSS70 variable on OS was carried out, with statistically significant differences being confirmed for all variables except fibrosis grade and HMR mutations. ASXL1 was mutated in 27%, but it did not impact OS in our patients (HR 0.96, p=0.89). SRSF2 was the only MIPSS70 mutated gene (8%) associated with inferior OS (HR 2.5, p=0.04), while EZH2 and IDH1/2 mutations were only present in 2.5% of patients each. However, the analysis of the complete NGS panel allowed the identification of other genes associated with shorter OS in our series: CBL (HR 3.7, p=0.014), SETBP1 (HR 4.9, p=0.034) and KRAS (HR 4.6, p=0.041), with a tendency for U2AF1 (HR 2.3, p=0.061) and NRAS (HR 3.6, p=0.083). Image: Summary/Conclusion: This study constitutes the first real-life validation of MIPSS70 in a multicenter analysis. The score was able to stratify our cohort in three categories with significant differences in OS and LFS. Of note, mutation on ASXL1 gene was not associated with shorter OS in our patients. By contrast, several other genes with potential prognostic significance were identified. Further cooperative studies are needed in order to unravel PMF mutational landscape and its prognostic implications. P1033: A PHASE 2 STUDY OF THE LSD1 INHIBITOR IMG-7289 (BOMEDEMSTAT) FOR THE TREATMENT OF ESSENTIAL THROMBOCYTHEMIA (ET) F. Palandri1,*, D. M. Ross2, T. Cochrane3, C. Tate3, S. W. Lane4, S. R. Larsen5, A. T. Gerds6, A. B. Halpern7, J. Shortt8, J. M. Rossetti9, K. M. Pettit10, J. Liang11, A. Mead12, M. Marchetti13, A. Vannucchi14, A. Wilson15, J. R. Göthert16, M. Hanna17, A. Jones18, J. Peppe18, G. Natsoulis18, T. McClure18, W.-J. Hong18, W. S. Stevenson19, C. N. Harrison20, M. Talpaz10, N. Vianelli21, H. Y. Rienhoff Jr.18 1Institute of Hematology “L. & A. Seràgnoli”, Sant’Orsola-Malpighi University Hospital, Bologna, Italy; 2Department of Haematology, Royal Adelaide Hospital and SA Pathology, Adelaide; 3Department of Haematology, Gold Coast University Hospital, Southport; 4QIMR Berghofer Medical Research Institute, Brisbane; 5Institute of Haematology, Royal Prince Alfred Hospital, Sydney, Australia; 6Cleveland Clinic Taussig Cancer Institute, Cleveland; 7Department of Medicine, Division of Hematology, University of Washington, Seattle, United States of America; 8Monash Haematology, Monash Health, Clayton, Australia; 9UPMC Hillman Cancer Center, Pittsburgh; 10Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan, Ann Arbor, United States of America; 11Middlemore Clinical Trials, Auckland, New Zealand; 12MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; 13Azienda Ospedaliera Nazionale SS. Antonio e Biagio e Cesare Arrigo, Alessandria; 14University of Florence, AOU Careggi, CRIMM, Center for Research and Innovation of Myeloproliferative Neoplasms, Florence, Italy; 15University College Hospital, NHS Foundation Trust, London, United Kingdom; 16Department of Hematology, West German Cancer Center (WTZ), University Hospital Essen, Essen, Germany; 17North Shore Hospital. Waitemata District Health Board, Auckland, New Zealand; 18Imago BioSciences, Inc., South San Francisco, United States of America; 19Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, Australia; 20Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom; 21Institute of Hematology, “L. & A. Seragnoli”, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy Background: Lysine-specific demethylase-1 (LSD1) is critical for the self-renewal malignant stem cells and maturation of megakaryocytes, the cell central to ET pathogenesis. Bomedemstat is an oral LSD1 inhibitor active in mouse models of MPNs including improved survival (Kleppe et al. 2015; Jutzi et al. 2018). IMG-7289-CTP-201 is an ongoing, global, open-label, Phase 2b study of bomedemstat taken QD for 24+ weeks in patients with ET who are resistant/intolerant to at least one standard treatment (NCT04254978). Aims: Key objectives are safety and response, defined as platelet (plt) count ≤400x109/L without thromboses. Exploratory endpoints include durability of response (defined as plt ≤400x109/L for ≥12 wks) symptom improvement (TSS) and reduction of WBCs and mutation burden. Methods: Key eligibility criteria: cytoreduction required, platelet count >450x109/L, haemoglobin (Hb) ≥10 g/dL. The starting dose is 0.6 mg/kg/d and titrated, if needed, to a platelet count of 200-400x109/L. Results: At data cut-off (3Feb’22), 44 patients had enrolled. Median age was 68 (42-92) yrs; of the patients most recently treated with hydroxyurea, 88% met ELN criteria for resistance/ intolerance. The Day 1 mean plt, WBC and Hb values were 825x109/L (457-2220), 8.9x109/L (3.9-30.6), and 13.1 g/dL (9.4-16.5), respectively. Median TSS at baseline was 15 (0-74). Sequencing 261 genes (N=42) identified mutations in JAK2 (45%), CALR (43%), and MPL (5%). Other mutations (e.g., EZH2, ASXL1, SF3B1, TP53) were present in 29%. Median time on treatment is 29 weeks (0-68). For patients treated ≥12 weeks, 91% (31/34) achieved a plt count of ≤400x109/L in a median time of 8.1 weeks. 83% of patients (20/24) treated for >24 weeks achieved a durable response as defined above. Of the 9/30 (30%) patients Day 1 WBC count ≥10x109/L, 89% (8/9) had a WBC count <10x109/L; all maintained a stable hemoglobin (see figures). In patients with baseline TSS ≥10 (29/43), at Week 12 69% (11/16) showed reductions; 38% (6/16) had improvements >10-points. The median baseline TSS score for fatigue was 5 (N=41). At Week 24, the median score for evaluable patients fell to 3 and dropped to a median of 1 at Week 48. At Week 24 (N=14), JAK2 and CALR mutations were equally sensitive: 87% had a decrease in allele frequencies (mean: -29%; [S.D. 5%]; range -3 to -91). No new mutations were found. The most common AEs regardless of causality were dysgeusia (52%), fatigue (34%), constipation (32%), arthralgia (27%), thrombocytopenia (25%), and contusion (21%). Eight patients reported a total of 12 serious AEs (SAE), with two (thrombocytopenia and mouth haemorrhage reported in 1 patient) deemed related per the PI. Seven patients discontinued treatment, 6 due to AEs (fatigue, invasive ductal breast carcinoma, headache, respiratory failure, dysgeusia x 2) and one withdrew consent on Day 1. Similar to an ongoing MF study of bomedemstat (NCT03136185), there have been no safety signals or deaths related to drug. At the censor date, 84% (37/44) of patients remain on study. Image: Summary/Conclusion: To date, in this patient population, bomedemstat is generally well-tolerated, reduces platelets, improves symptoms, reduces the mutation burden, and moderates WBC counts without affecting haemoglobin. For those patients treated for at least 24 weeks, 79% achieved a durable reduction in platelet count to ≤400x109/L without thromboembolic events. A Phase 3 study of bomedemstat for the treatment of ET is being planned. P1034: HEALTHCARE UTILIZATION OF PATIENTS WITH HYPEREOSINOPHILIC SYNDROME IN EUROPE J. Hwee1,*, N. Kwon2, R. Alfonso-Cristancho3, L. Baylis4, G. Requena5, S. Du6, A. Khanal6, L. Huynh6, M. S. Duh6, R. W. Jakes5 1Value Evidence & Outcomes, GSK, Mississauga, ON, Canada; 2Respiratory Research & Development, GSK, Brentford, Middlesex, United Kingdom; 3Value Evidence & Outcomes, GSK, Collegeville, PA, United States of America; 4Global Medical Affairs, GSK, Durham, NC, United States of America; 5Epidemiology, GSK, London, United Kingdom; 6Analysis Group, Inc., Boston, MA, United States of America Background: Hypereosinophilic syndrome (HES) is a rare group of blood disorders characterized by prolonged eosinophilia that can lead to tissue and organ damage. HES was previously considered to be largely idiopathic, but myeloid and lymphocytic variants are also now recognized. The disease may manifest in multiple organ systems, most commonly the heart, nervous system, gastrointestinal tract, skin, and lungs, due to eosinophilic infiltration. Treatment aims to reduce eosinophil counts, and the current standard of care consists of high-dose glucocorticosteroids, despite the risks associated with high doses or long exposure, and antineoplastic agents for those with more severe disease or who do not respond to steroids. Healthcare resource utilization (HCRU) in patients with HES is not well characterized, and data from clinical trials may not be representative of the general disease population encountered in real-world settings. Aims: To describe HES-related HCRU among a real-world cohort of patients with a confirmed diagnosis of HES in Europe. Methods: This retrospective chart review study included patients ≥6 years old with a confirmed diagnosis of HES and ≥1 year of follow-up data from index (first physician encounter: Jan 2015–Dec 2019). Chart review was conducted by physicians in 5 European countries (France, Germany, Italy, Spain, and the United Kingdom [UK]). Data from a similar number of patients were reviewed and included across each country. HES-related hospitalizations and outpatient and emergency room (ER) visits were assessed and reported descriptively across all patients and separately by country. Results: Of the 280 patients included, the majority were male (65.0%) and had idiopathic HES (55.4%); patients had a mean (standard deviation [SD]) age at HES diagnosis of 42.4 (16.2) years. The most common comorbidity was asthma (45.0%). The mean disease duration was 4.0 (4.5) years, and the mean length of follow-up was 2.8 (1.4) years. Overall, 85 (30.4%) patients had ≥1 HES-related hospitalization. For patients with a HES-related hospitalization, the mean (SD) length of stay (LoS) was 11.0 (9.4) days. HES-related ER visits were reported for 72 (25.7%) patients, and HES-related outpatient visits were reported for 243 (86.8%) patients. Patients had a mean of 0.4 (1.2) hospitalizations, 0.3 (0.8) ER visits, and 4.3 (4.9) outpatient visits (of which 1.0 [3.3] were unscheduled outpatient visits) per year (Table). HCRU varied between countries. The proportion of patients requiring hospitalization was highest in the UK (43.5%) and lowest in Spain (11.5%). Mean (SD) days of LoS during a HES-related hospitalization was highest in Spain (15.3 [4.2]) and lowest in the UK (9.9 [11.0]) and France (9.9 [7.3]). The proportion of patients with outpatient visits was highest in France (93.4%) and lowest in Italy (78.8%). The proportion of patients with ER visits was highest in the UK (37.1%) and lowest in Italy (13.5%). Image: Summary/Conclusion: These results demonstrate that real-world patients with HES have substantial HCRU, including lengthy hospitalizations, ER visits, and outpatient visits, which is higher than that reported in previous clinical trials for control (placebo-treated) patients with eosinophilic diseases such as EGPA and severe eosinophilic asthma. These observations highlight the need for novel approaches to treatment to relieve the disease-related burden of HES for patients and to ameliorate the associated HCRU burden. P1035: COUNTRY DIFFERENCES IN THE BURDEN OF PATIENTS WITH HYPEREOSINOPHILIC SYNDROME IN EUROPE J. Hwee1,*, N. Kwon2, R. Alfonso-Cristancho3, L. Baylis4, G. Requena5, S. Du6, A. Khanal6, L. Huynh6, M. S. Duh6, R. W. Jakes5 1Value Evidence & Outcomes, GSK, Mississauga, ON, Canada; 2Respiratory Research & Development, GSK, Brentford, Middlesex, United Kingdom; 3Value Evidence & Outcomes, GSK, Collegeville, PA, United States of America; 4Global Medical Affairs, GSK, Durham, NC, United States of America; 5Epidemiology, GSK, London, United Kingdom; 6Analysis Group, Inc., Boston, MA, United States of America Background: Hypereosinophilic syndrome (HES) is a group of rare blood disorders characterized by peripheral eosinophilia that can lead to tissue and organ damage. Diagnosis and management of HES can be challenging owing to differences in patient characteristics, disease characteristics and clinical manifestations. At present, there is limited information available on the burden of HES in Europe. Aims: To describe real-world patient demographics, disease characteristics, clinical manifestations, treatment patterns, and clinical outcomes for patients with HES across Europe. Methods: This retrospective chart review study included patients ≥6 years old with a confirmed diagnosis of HES and ≥1 year of follow-up data from index (first physician encounter: Jan 2015–Dec 2019). Chart review was conducted by physicians in 5 European countries (France, Germany, Italy, Spain, and the United Kingdom [UK]). Data from a similar number of patients were reviewed and included across each country. Patients could be newly diagnosed during the patient identification window or could be diagnosed prior to their index date, allowing for the inclusion of patients with differing disease duration and organ involvement. Patient demographics, disease characteristics, clinical manifestations, treatment patterns, and clinical outcomes were summarized and described across the overall population and separately by country. Results: Overall, 280 patients were included in the study. Most patients were male (65.0%), with the highest proportion of male patients in Spain (73.1%) and France (75.4%). The majority of patients (55.4%) had idiopathic HES. The most common comorbidity overall was asthma (45.0%); however, in the UK, anxiety/depression was more common than asthma (51.6% vs 41.9%), and in France, both anxiety/depression and hypertension were more common than asthma (37.7% vs 23.0% and 36.1% vs 23.0%, respectively) (Table). Most patients (88.6%) were newly diagnosed with HES during the study time frame. Italy had the highest proportion of patients (17.3%) diagnosed prior to the study while Germany had the lowest (1.9%). The overall mean (standard deviation [SD]) number of distinct clinical manifestations was 3.7 (3.7), with the highest mean (SD) numbers of clinical manifestations observed in Italy (4.6 [4.7]) and Spain (4.7 [3.7]). Patients across all 5 countries were treated with 4 classes of therapy: oral corticosteroids (OCS; 89.3%), immunosuppressants or cytotoxic agents (63.6%), and biologics (43.9%), other therapies (10.7%). Spain had the highest mean (SD) maximum daily dose of OCS (42.4 [19.6] mg). In total, 22.9% of patients experienced a flare, and the mean (SD) number of flares in patients with ≥1 flare was 0.5 (0.3) flares/year. Patients in France, Germany, and the UK had the highest mean (SD) numbers of flares (0.7 [0.6], 0.6 [0.3], and 0.6 [0.3] flares/year, respectively). The overall mean (SD) duration of flares was 2.9 (3.2) months, with the longest flare duration observed in France (4.8 [6.3] months) and the shortest in the UK (1.6 [1.2]). Image: Summary/Conclusion: These results demonstrate that in patients with HES, there are differences in patient and disease characteristics as well as treatment strategies across Europe. These differences likely contribute to challenges in the diagnosis and management of HES and highlight the need for a common strategy to address these difficulties. P1036: VOLUMETRIC SPLENOMEGALY IN PATIENTS WITH POLYCYTHEMIA VERA M.-W. Lee1,*, S.-H. Yeon1, H. Ryu1, I.-C. Song1, H.-J. Lee1, H.-J. Yun1, S. Y. Kim2, J. E. Lee3, K. S. Shin3, D.-Y. Jo1 1Department of Internal Medicine; 2Department of Laboratory Medicine; 3Department of Radiology, Chungnam National University College of Medicine, Daejeon, South Korea Background: At present, palpable splenomegaly is relatively uncommon in patients with polycythemia vera (PV), possibly because of early diagnosis facilitated by a lowering of the diagnostic thresholds for hemoglobin and hematocrit levels as well as the wide application of driver gene mutation tests. Radiological volumetric analysis of spleen size is now readily available. However, non-palpable splenomegaly in patients with PV has seldom been addressed. Aims: In this retrospective study, we evaluated non-palpable, volumetric splenomegaly defined based on age- and body surface area (BSA)–matched criteria in patients with PV diagnosed according to the 2016 World Health Organization diagnostic criteria. Methods: Patients with PV who underwent abdominal computed tomography (CT) and who had palpable splenomegaly at diagnosis from January 1991 to December 2020 at Chungnam National University Hospital were enrolled in the study. The spleen volume of each patient was determined by volumetric analysis of abdominal CT and adjusted for the patient’s age and BSA. And then the degree of splenomegaly was classified as follows: no splenomegaly, spleen volume less than the mean plus 2 SD of the reference volume based on both age and BSA; overt volumetric splenomegaly, spleen volume greater than the mean plus 3 SD of the reference volume based on both age and BSA; borderline volumetric splenomegaly, a spleen volume between no splenomegaly and overt splenomegaly; or palpable splenomegaly, spleen palpable below the left costal margin. Results: Of the 87 PV patients enrolled in the study, 15 (17.2%) had no splenomegaly, whereas 17 (19.5%), 45 (51.7%), and 10 (11.5%) had borderline volumetric, overt volumetric, and palpable splenomegaly, respectively. Spleen volume was significantly positively correlated with lactate dehydrogenase level (r = 0.227, P = 0.042) and tended to be positively related to white blood cell count (r = 0.209, P = 0.055) and monocyte count (r = 0.210, P = 0.059) at diagnosis. Spleen volume was not correlated with hemoglobin level, platelet count, or JAK2V617F burden at diagnosis. The degree of splenomegaly did not affect the cumulative incidence of thrombotic vascular events (10-year incidence: 7.7%, 0%, 22.3%, and 50.7%, respectively, P = 0.414). By contrast, splenomegaly tended to adversely affect myelofibrotic transformation (10-year cumulative incidence: 0%, 0%, 7.1%, and 30.3%, respectively, P = 0.062). Moreover, the cumulative incidence of myelofibrotic transformation was significantly higher in patients with overt volumetric or palpable splenomegaly than those with no or borderline volumetric splenomegaly (10-year incidence: 0% vs. 10.3%, respectively; 15-year incidence: 0% vs. 26.3%, respectively, P = 0.020). Overall survival (OS) differed among patients with different degrees of splenomegaly (15-year OS: 100%, 78.6%, 71.7%, and 51.9%, respectively, P = 0.021). The degree of splenomegaly was independent risk factor for both myelofibrotic transformation (hazard ratio [HR]: 7.75; 95% confidence interval [CI]: 1.87-21.10; P = 0.005) and OS (HR: 6.70; 95% CI: 1.89-23.72; P = 0.003). Summary/Conclusion: The degree of splenomegaly, including volumetric splenomegaly, based on age- and BSA-matched reference spleen volumes at diagnosis reflects disease progression in PV patients. Therefore, volumetric splenomegaly should be evaluated at the time of PV diagnosis and taken into consideration when predicting the prognosis of patients with this disorder. P1037: DOES EARLY INTERVENTION IN MYELOFIBROSIS IMPACT OUTCOMES? A POOLED ANALYSIS OF THE COMFORT I AND II STUDIES C. Harrison1,*, J.-J. Kiladjian2, A. M. Vannucchi3, R. A. Mesa4, R. M. Scherber5, J. Hamer-Maansson5, S. Verstovsek6 1Guy’s and St. Thomas’ NHS Foundation Trust, Guy’s Hospital, London, United Kingdom; 2Hôpital Saint-Louis, Assitance Publique – Hôpitaux de Paris, Université de Paris, Inserm, Paris, France; 3CRIMM, Center for Research and Innovation of Myeloproliferative Neoplasms, AOU Careggi, University of Florence, Florence, Italy; 4Mays Cancer Institute at UT Health San Antonio MD Anderson, San Antonio; 5Incyte Corporation, Wilmington; 6The University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Myelofibrosis (MF) is characterized by cytopenias, splenomegaly, burdensome symptoms, and poor overall survival (OS). Few studies have investigated if earlier intervention with targeted MF therapies affects response and OS. In a pooled analysis of the COMFORT I and II trials of ruxolitinib (RUX), patients (pts) who received RUX at randomization or after crossover from placebo (PBO) or best available therapy (BAT) had improved OS. Aims: To assess the association of MF disease duration before treatment with disease outcomes using pooled COMFORT data Methods: COMFORT I (NCT00952289) and COMFORT II (NCT00934544) were phase 3 trials of RUX vs PBO or BAT, respectively, in pts with intermediate-2 or high-risk MF who provided written informed consent. In this post hoc analysis, data from RUX-treated pts in both studies were combined (RUX group), and data from the PBO/BAT arms were pooled (control group). Pt subgroups were defined based on disease duration before treatment initiation (≤12 mo or >12 mo from diagnosis). Assessments included frequency of thrombocytopenia (platelets [PLT] <100 Gi/L or PLT transfusion) and anemia (hemoglobin <100 g/L or red blood cell transfusion) events, spleen volume response (SVR; spleen volume reduction ≥35% from baseline [SVR35]), symptom response (MF-Symptom Assessment Form total symptom score [TSS] reduction ≥50% from baseline [TSS50]; available in COMFORT I only), and OS. OS was assessed using the Kaplan-Meier method; pts randomized to PBO/BAT were included in the PBO/BAT group regardless of crossover. A multivariable analysis (MVA) using a logistic regression model was used to examine factors associated with SVR. Results: There were 525 pts in the analysis (RUX: ≤12 mo, n=84; >12 mo, n=216; PBO/BAT: ≤12 mo, n=66; >12 mo, n=159). Median age across groups ranged from 65 to 70 y. Baseline clinical characteristics were generally similar across subgroups, but pts with shorter vs longer disease duration were slightly younger with higher blood counts. Among pts who received RUX, fewer thrombocytopenia events were observed among those treated earlier (≤12 mo vs >12 mo), with differences seen as early as Wk 4–8 on treatment (18% vs 33%) and sustained over time; a similar trend was observed for anemia events (Wk 4–8, 59% vs 72%). The proportion of pts with SVR35 was greater among those who initiated RUX earlier (≤12 mo vs >12 mo) at Wk 24 (48% vs 33%; P=0.0610) and 48 (44% vs 27%; P=0.0149; Table). A numerically greater proportion of pts who initiated RUX at ≤12 mo vs >12 mo achieved TSS50 at Wk 24 (56% vs 40%; P=0.0829; Table). OS at Wk 240 was improved among pts who initiated RUX at ≤12 mo vs >12 mo (63% [95% CI, 51%–73%] vs 57% [95% CI, 49%–64%]; P=0.0430; Table). Comparatively, OS was longer with RUX vs PBO/BAT regardless of disease duration. A sensitivity analysis using a 24-mo cutoff was also conducted but yielded weaker associations between disease duration and SVR, TSS, and OS. In the MVA, a significantly greater binary SVR was seen among pts with shorter (≤12 mo) vs longer (>12 mo) MF disease duration (odds ratio, 2.1; P=0.022). Image: Summary/Conclusion: These findings suggest that earlier RUX initiation in MF may improve clinical outcomes, including cytopenias, SVR, symptom burden, and OS. While “watch and wait” remains a common treatment approach for newly diagnosed patients, these data suggest that pts with MF may benefit from earlier intervention. Additional studies to further evaluate the impact of early intervention are warranted. P1038: CLINICOPATHOLOGIC AND MOLECULAR CORRELATES OF ORGAN DAMAGE ACROSS THE SPECTRUM OF ADVANCED SYSTEMIC MASTOCYTOSIS E. Liang1,*, C. Perkins2, R. Lu3, H. Shi4, S. Dimitrijevic5, G. Hoehn4, J. Abuel2, C. Langford2, P. Abidi2, L. Fechter2, W. Shomali2, J. Gotlib2 1Department of Medicine, Stanford University School of Medicine; 2Division of Hematology, Stanford Cancer Institute/Stanford University School of Medicine; 3Quantitative Sciences Unit, Stanford University School of Medicine, Stanford, CA; 4Blueprint Medicines Corporation, Cambridge, MA, United States of America; 5Blueprint Medicines Corporation, Zug, Switzerland Background: Advanced SM (AdvSM) comprises 3 subtypes: aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN), and mast cell leukemia (MCL). The patterns of organ damage across the spectrum of AdvSM have not been well characterized. The definition of hematologic and non-hematologic organ damage has evolved from World Health Organization (WHO) C-findings, to organ damage criteria specified by the International Working Group-Myeloproliferative Neoplasms Research and Treatment and European Competence Network on Mastocytosis (IWG). More recently, modified IWG (mIWG) criteria have been used to define eligible organ damage in trials of AdvSM. Aims: 1) To describe the distribution of organ damage in AdvSM patients (pts) at initial presentation or at time of trial enrollment using WHO, IWG, and mIWG criteria; 2) to evaluate clinicopathologic and molecular correlates of organ damage. Methods: Our cohort included 244 AdvSM pts: 68 pts from our Stanford IRB-approved MPN registry and 176 pts from the phase I EXPLORER (NCT02561988) and phase II PATHFINDER (NCT03580655) studies of avapritinib in AdvSM. Comparisons between continuous and categorial variables were performed using the Wilcoxon rank sum test and the Fisher’s exact test, respectively. The Benjamini and Hochberg method was used to control the false discovery rate in multiple comparisons. Results: Median age at diagnosis was 68 years (range, 24-88) and 146 pts (60%) were male. Forty-one pts (17%) had ASM, 165 (68%) had SM-AHN, and 38 (16%) had MCL. The median number of prior anti-neoplastic therapies was 1 (range, 0-6), with 87 pts (36%) previously treated with midostaurin. Comparison of AdvSM Subtypes: Pts with MCL had the highest transfusion requirement, with 13% and 8% of pts requiring RBC and platelet transfusions, respectively. SM-AHN pts had the highest median alkaline phosphatase (ALP) (214 U/L), while pts with MCL had the largest median liver (2860 cm3) and spleen (1120 cm3) volumes by imaging. Among the two largest SM-AHN subtypes, pts with SM-CMML (n = 72) had a lower ANC (p = 0.02) and tryptase levels (p = 0.04) than pts with MDS/MPN-U (n = 36). Pts with SM-AHN fulfilled the WHO thrombocytopenia criterion (<100 x 109/L) more frequently than MCL and ASM pts, and more WHO hematologic (p = 0.02) and IWG non-hematologic (p = 0.008) criteria than ASM pts. Prior Therapy: Pts who received ≥1 prior anti-neoplastic therapy (compared to none) had greater tryptase levels (p = 0.02) and bone marrow (BM) MC burden (p = 0.03), lower ALP (p = 0.04), and higher likelihood of meeting WHO malabsorption criteria (hypoalbuminemia and/or weight loss) (p = 0.03). Compared to midostaurin-naïve pts, those who had received midostaurin had a greater BM MC burden (p = 0.045), but less frequently met mIWG splenomegaly criteria (p = 0.04) and met fewer WHO non-hematologic criteria (p < 0.001). Mutation Profiles: Compared to pts with KIT D816V only, pts who also had ≥1 SRSF2, ASXL1, or RUNX1 mutations had a lower median hemoglobin (p = 0.02), platelet count (p = 0.02), and greater direct bilirubin (p = 0.005), ALP (p = 0.04), and spleen volume (p = 0.06). They were more likely to fulfill the WHO hypersplenism criterion (p = 0.03) and exhibited more WHO-defined cytopenias (p = 0.006). Image: Summary/Conclusion: In AdvSM, patterns of organ damage reflect disease subtype, prior therapy, and mutation profile beyond KIT D816V (Figure). These data can help inform the clinical heterogeneity of AdvSM patients who are being evaluated for clinical trials which currently require 1 or more organ damage findings defined by the WHO or (m)IWG criteria. P1039: PALPABLE SPLEEN SIZE IS PROGNOSTIC IN PRIMARY BUT NOT SECONDARY MYELOFIBROSIS M. Lucijanic1,*, I. Krecak2, E. Soric1, D. Galusic3, H. Holik4, V. Perisa5, M. Moric Peric6, I. Zekanovic6, R. Kusec1 1Hematology Department, University hospital Dubrava, Zagreb; 2Internal medicine department, General Hospital Sibenik-Knin county, Sibenik; 3Internal medicine department, University hospital center Split, Split; 4Internal medicine department, Slavnoski Brod, General hospital Slavonski Brod; 5Hematology Department, University hospital center Osijek, Osijek; 6Internal medicine department, General hospital Zadar, Zadar, Croatia Background: Splenomegaly is a clinical hallmark of primary (PMF) and secondary myelofibrosis (SMF). Although not a part of disease specific risk scores, spleen size represents disease burden and correlates with unfavorable disease features. Spleen size reduction is a function of baseline spleen size and was recently shown to be independent prognostic factor in composite PMF and SMF cohort of ruxolitinib treated patients. Aims: To estimate prognostic properties of palpable splenomegaly and potential differences between PMF and SMF patients. Methods: We analyzed a multicentric cohort of 191 patients from 6 Croatian centers with either PMF or post polycythemia vera (PV) and essential thrombocythemia (ET) SMF diagnosed in period from 2004-2021 who had available data on palpable spleen size. Diagnoses were established according to 2016 and 2008 criteria. Risk was stratified according to the DIPSS in PMF and according to the MySEC-PM in SMF patients. Palpable spleen size was assessed at the time of diagnosis or referral and stratified in three categories: non-palpable, <10 cm and ≥10 cm. Results: A total of 191 patients were analyzed, among them 144 PMF (63 early, 81 overt fibrotic) and 47 SMF (27 post PV; 20 post ET). Median age was 67 years, there were 64% males. Intermediate-2 or high risk disease was present in 45% PMF and 49% SMF patients. Non-palpable spleen, <10 cm and ≥10 cm splenomegaly were present in 31%, 43% and 26% PMF patients, respectively, and 23%, 49% and 28% SMF patients, respectively. Palpable spleen size was similar between patients with PMF and SMF (median 3 vs 4 cm, P=0.325). Patients with palpable spleen ≥10 cm were significantly grouped among patients with higher risk disease in PMF (P=0.026) but not SMF (P=0.654). Degree of bone marrow fibrosis was significantly associated with higher spleen size in PMF (P=0.008) but not SMF (0.809). On the contrary, JAK2 mutational status had no significant association with spleen size in PMF (P=0.847) but was significantly associated with higher spleen size in SMF (P=0.035). Regarding overall survival, larger spleen size was significantly associated with shorter overall survival in PMF (P<0.001) but not SMF (P=0.825) as shown in Figure. Time to thrombosis had no significant association with larger spleen size in neither PMF nor SMF (P>0.05). Time to bleeding was significantly shorter in PMF patients with both <10 cm and ≥10 cm in comparison to non-palpable spleen (P<0.05) whereas palpable spleen size had no significant association with in SMF patients (P>0.05). Image: Summary/Conclusion: Various clinical features are differently associated with palpable spleen size in PMF and SMF patients. Larger spleen size is prognostic of worse survival and higher bleeding risk in PMF but not SMF patients. This might have repercussions on prognostic scores based on palpable spleen size that might differently perform in PMF and SMF patients. P1040: HIGHER ESTIMATED PLASMA VOLUME STATUS IS ASSOCIATED WITH INCREASED THROMBOTIC RISK AND IMPAIRED SURVIVAL IN PATIENTS WITH PRIMARY MYELOFIBROSIS M. Lucijanic1,*, I. Krecak2, E. Soric1, D. Galusic3, H. Holik4, V. Perisa5, M. Moric Peric6, I. Zekanovic6, R. Kusec1 1Hematology Department, University hospital Dubrava, Zagreb; 2Internal medicine Department, General hospital Sibenik-Knin county, Sibenik; 3Hematology Department, University hospital center Split, Split; 4Internal medicine Department, General hospital Slavonski Brod, Slavonski Brod; 5Hematology Department, University hospital center Osijek, Osijek; 6Internal medicine Department, General hospital Zadar, Zadar, Croatia Background: Blood plasma experiences substantial changes in both volume and composition in patients with chronic myeloproliferative neoplasms (MPN) and represents large reservoir of cytokines and other mediators of inflammation. Higher estimated plasma volume status (ePVS) has been shown to correlate with increased thrombotic risk in polycythemia vera patients, but its clinical and prognostic associations are unknown in patients with other MPN subsets. Aims: To estimate clinical and prognostic associations of ePVS in patients with primary myelofibrosis (PMF). Methods: We retrospectively analyzed a cohort of 167 patients with myelofibrosis treated in 6 Croatian hematology centers in period from 2004-2021. Diagnosis was established according to the WHO 2016 criteria. ePVS was calculated using the Strauss derived Duarte formula: 100-hematocrit (%)/hemoglobin (g/dL) and expressed as dl/g. ePVS associations with baseline clinical parameters and its prognostic associations with overall survival (OS), time to thrombosis (TTT) and time to bleeding (TTB) were analyzed. Results: There were 167 patients with PMF analyzed (72 pre-fibrotic and 95 overt fibrotic). Median age was 68 years, there were 65% males, 67% were JAK2 mutated. Intermediate-2 or high DIPSS risk was present in 48% of patients. Median ePVS was 5.62 dl/g. Higher ePVS was significantly associated with higher degree of bone marrow fibrosis, absence of JAK2 mutation, presence of constitutional symptoms, presence of circulating blasts, transfusion dependency, larger palpable spleen size, higher Charlson comorbidity index, history of bleeding, lower WBC, lower platelet count, lower hemoglobin, higher RDW, higher LDH, higher CRP, higher ferritin, lower albumin, higher fibrinogen, higher risk DIPSS status (P<0.05 for all analyses). Median follow-up of our cohort was 57 months. During follow-up period a total of 79 patients died, 18 experienced thrombotic event (14 arterial and 5 venous thromboses) and 14 experienced bleeding event. Higher ePVS stratified at the ROC curve analysis defined cut-off points was significantly associated with worse OS (ePVS>6.2 dl/g, HR 1.74, P=0.017) and shorter TTT (ePVS>6.6 dl/g, HR 2.69, P=0.047) as shown in Figure. There was no significant association with TTB. In a multivariate Cox regression analysis model association of higher ePVS with increased thrombotic risk (HR 3.22, P=0.019) was present independently of WBC (HR 1.02, P=0.003), whereas JAK2 mutational status (HR 2.74, P=0.080) and cardiovascular risk factors (HR 7.12, P=0.060) retained marginal significance. Association with OS diminished in multivariate analysis after adjustment for DIPSS. Image: Summary/Conclusion: PMF patients with more advanced disease features (more pronounced cytopenias and splenomegaly) have higher ePVS indicative of expanded plasma volume. Higher ePVS is associated with higher thrombotic risk and impaired survival. P1041: IMPACT OF FEDRATINIB ON SPLEEN VOLUME AND MYELOFIBROSIS SYMPTOMS IN PATIENTS WITH SUBSTANTIAL SPLENOMEGALY: POST HOC ANALYSES FROM THE JAKARTA AND JAKARTA2 TRIALS J.-J. Kiladjian1,*, A. Tefferi2, F. Passamonti3, A. Vannucchi4, M. Talpaz5, F. Cervantes6, C. N. Harrison7, R. A. Mesa8, J. Mascarenhas9, N. Schaap10, S. Verstovsek11, T. Devos12, S. Rose13, J. Zhang13, O. Sy13, A. Pardanani2 1Hôpital Saint-Louis; Université de Paris, INSERM, Paris, France; 2Mayo Clinic of Rochester, Rochester, United States of America; 3University of Insubria, Varese; 4CRIMM, University of Florence, AOU Careggi, Florence, Italy; 5University of Michigan Comprehensive Cancer Center, Ann Arbor, United States of America; 6Hospital Clinic Provincial de Barcelona, Barcelona, Spain; 7Guy’s and St Thomas’ Hospital, London, United Kingdom; 8Mays Cancer Center at UT Health San Antonio MD Anderson Cancer Center, San Antonio; 9Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, United States of America; 10Radboud University Medical Centre, Nijmegen, Netherlands; 11The University of Texas MD Anderson Cancer Center, Houston, United States of America; 12University Hospitals Leuven and Laboratory of Molecular Immunology (Rega Institute), KU Leuven, Leuven, Belgium; 13Bristol Myers Squibb, Princeton, United States of America Background: In myelofibrosis (MF), splenomegaly is a marker of disease progression, and larger spleen size (SS) is correlated with poorer survival and debilitating symptoms. Fedratinib (FEDR) is an oral, selective Janus kinase 2 (JAK2) inhibitor approved as first-line (1L) treatment (Tx) of patients (pts) with MF and in pts previously treated with ruxolitinib (RUX). In the phase 3, placebo-controlled JAKARTA trial (NCT01437787) and the phase 2, single-arm JAKARTA2 trial (NCT01523171), pts experienced substantial improvements in spleen volume and MF symptoms after receiving 6 FEDR Tx cycles. Given the prognostic impact of splenomegaly, these analyses explore whether these clinical benefits were influenced by pre-Tx SS. Aims: Investigate the efficacy of FEDR 400 mg/d in JAKARTA and JAKARTA2, in pt subgroups defined by baseline (BL) SS. Methods: The JAKARTA trial assessed FEDR 400 mg/d, FEDR 500 mg/d, and placebo in pts with JAK inhibitor-naïve MF, and the JAKARTA2 trial evaluated FEDR 400 mg/d (starting dose) in pts resistant/intolerant to prior RUX. Both studies enrolled pts with intermediate- or high-risk MF, SS >5 cm from the left costal margin by palpation, platelets ≥50×109/L, and ECOG PS ≤2. These post hoc analyses included pts from both studies who were allocated to receive FEDR 400 mg/d (the approved dosage) in continuous 28-d Tx cycles. Pt subgroups were divided according to BL median SS (mSS) in each trial. Changes from BL in spleen volume and Myelofibrosis Symptom Assessment Form (MFSAF) total symptom score (TSS) were measured at the end of cycle 6 (EOC6). Spleen volume was assessed by MRI/CT scan; MFSAF TSS was the sum of 6 MF symptom scores: night sweats, early satiety, pruritus, pain under ribs-left side, abdominal discomfort, and bone/muscle pain. Eligible pts must have had spleen volume and/or TSS data at BL and EOC6. Results: Overall, 96 pts in JAKARTA and 97 pts in JAKARTA2 received FEDR 400 mg/d; mSS (range) at BL was 16 (5–40) cm and 18 (5–36) cm, respectively. In the JAKARTA <mSS and ≥mSS cohorts, median BL spleen volume was 1771 (316–3396) mL and 3329 (983–6430) mL, respectively, and median TSS was 13.0 (0.0–54.9) and 17.2 (0.4–57.0). In JAKARTA2, median BL spleen volume was 1937 (737–4305) mL and 4002 (1821–7815) mL for pts with <mSS and ≥mSS, respectively; BL TSS was 17.2 (0.7–48.0) and 23.6 (1.0–44.0). Overall, 75 (78%) pts in JAKARTA and 51 (53%) pts in JAKARTA2 had spleen volume data available at BL and EOC6; the <mSS and ≥mSS cohorts, comprised 37 and 38 pts, respectively, in JAKARTA, and 31 and 20 pts in JAKARTA2. Median spleen volume reduction (SVR) from BL was similar between the <mSS and ≥mSS cohorts in each trial, and 97% of pts with ≥mSS in both trials achieved some degree of SVR. In JAKARTA, median SVR at EOC6 was −38% (95% CI, −43 to −31) in the <mSS and −40% (95% CI, −40 to −28) in the ≥mSS cohort; in JAKARTA2, median SVR was −37 (95% CI, −43 to −30) in the <mSS and −38.5% (95% CI, −49 to −14) in the ≥mSS cohort. TSS data were available at BL and EOC6 for 71 (74%) pts in JAKARTA and 51 (53%) pts in JAKARTA2. Changes from BL in TSS at EOC6 were similar in the JAKARTA <mSS and ≥mSS cohorts, with median TSS reductions of −49% and −51%, respectively. Median TSS changes at EOC6 in JAKARTA2 were greater for pts with larger spleens, −36% and −49% in the <mSS and ≥mSS cohorts, respectively. Image: Summary/Conclusion: Pts who completed 6 Tx cycles with FEDR 400 mg/d, whether used as 1L MF Tx or after RUX Tx, experienced substantial improvements in spleen volume and MF symptom severity regardless of the extent of splenomegaly at BL. P1042: SAFETY AND TOLERABILITY RESULTS FROM THE PHASE 3B FREEDOM TRIAL OF FEDRATINIB (FEDR), AN ORAL, SELECTIVE JAK2 INHIBITOR, IN PATIENTS WITH MYELOFIBROSIS (MF) PREVIOUSLY TREATED WITH RUXOLITINIB (RUX) V. Gupta1,*, A. Yacoub2, S. Verstovsek3, R. A. Mesa4, C. N. Harrison5, G. Barosi6, J.-J. Kiladjian7, H. J. Deeg8, S. Fazal9, L. Foltz10, R. J. Mattison11, C. B. Miller12, V. Parameswaran13, C. Hernandez14, J. Zhang14, M. Talpaz15 1Princess Margaret Cancer Centre, Toronto, Canada; 2University of Kansas Medical Center, Kansas City; 3The University of Texas MD Anderson Cancer Center, Houston; 4Mays Cancer Center at UT Health San Antonio MD Anderson Cancer Center, San Antonio, United States of America; 5Guy’s and St Thomas’ Hospital, London, United Kingdom; 6Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 7Hôpital Saint-Louis, Université de Paris, INSERM, Paris, France; 8Fred Hutchinson Cancer Center, Seattle; 9Allegheny Health Network Cancer Institute, Pittsburgh, United States of America; 10St. Paul’s Hospital, University of British Columbia, Vancouver, Canada; 11University of Wisconsin Carbone Comprehensive Cancer Center, Madison; 12Ascension Saint Agnes Hospital, Baltimore; 13Avera Cancer Institute, Sioux Falls; 14Bristol Myers Squibb, Princeton; 15University of Michigan Comprehensive Cancer Center, Ann Arbor, United States of America Background: FEDR is an oral, selective Janus kinase 2 (JAK2) inhibitor approved for treatment (Tx) of patients (pts) with MF, including those previously treated with RUX. In the single-arm, phase 2 JAKARTA2 trial in pts with MF resistant/intolerant to prior RUX, the most common adverse events (AEs) during FEDR Tx were gastrointestinal (GI) events. JAKARTA2 ended in 2013, when a temporary clinical hold was placed on FEDR due to suspected cases of Wernicke’s encephalopathy (WE). The safety and efficacy of FEDR in pts with MF previously treated with RUX are being further evaluated in the ongoing, single-arm, phase 3b FREEDOM trial (NCT03755518), which includes prospective strategies for preventing or mitigating GI AEs, thiamine decreases, and potential WE. Aims: To assess the safety of FEDR and the effectiveness of AE mitigation strategies in the FREEDOM trial. Methods: Eligible pts had intermediate- or high-risk MF, platelets ≥50×109/L, and spleen volume ≥450cm3 by MRI/CT or palpable spleen ≥5 cm below the left costal margin. Pts must have received prior RUX for ≥3 mo, or for ≥28 d with development of red blood cell transfusion requirement (≥2 units/mo for 2 mo) or grade ≥3 thrombocytopenia, anemia, hematoma, or hemorrhage. All pts received FEDR 400 mg/d in continuous 28-d cycles. AE mitigation strategies include prophylactic and symptomatic use of anti-nausea/vomiting and anti-diarrheal Tx, thiamine supplementation, FEDR dosing modifications, and administration of FEDR with food. Results: In all, 34 pts were enrolled and 16 pts continued to receive FEDR at data cutoff (April 9, 2021). Reasons for FEDR discontinuation (D/C) in >1 pt were lack of efficacy (n=5), AEs (n=4), disease progression (n=2), pt decision (n=2), and to undergo transplant (n=2). Median FEDR Tx duration at cutoff was 28.3 (range, 1.6–101.3) wk; 14 (41%) pts had completed >12 cycles. At baseline (BL), median (range) age was 68.5 (49–82) y, time from MF diagnosis was 3.4 (0.1–17.4) y, and spleen size was 15 (3–31) cm. Most pts (62%) had primary MF, and all pts had received ≥3 mo of prior RUX; the most common reason for RUX D/C was loss of response/Tx failure (41%). During FEDR Tx, 22 (65%) pts received ondansetron and 11 (32%) pts received loperamide. GI AEs in >10% of pts were constipation (47%), diarrhea (35%), nausea (26%), abdominal pain (24%), and vomiting (18%). Nausea, vomiting, and diarrhea decreased as Tx continued. Most GI AEs were grade (G) 1/2 (including nausea, vomiting, and diarrhea) and there were no Tx-related G3/4 GI AEs. No pt required FEDR reduction, interruption, or D/C due to a Tx-related GI AE. Tx-related G3/4 AEs were reported in 11 (32%) pts, including anemia in 7 (21%) pts, and neutropenia, thrombocytopenia, and hyperkalemia in 2 (6%) pts each. At BL, 1 pt had thiamine below the 70 nmol/L lower limit of normal (LLN), which normalized before the pt received FEDR. During FEDR Tx, thiamine levels dropped below the LLN for 4 pts between cycles 2 and 3 (and for 1 pt at end of Tx); for these pts, levels returned to normal with thiamine supplementation and no FEDR interruption or reduction was required (Figure). Five other pts received prophylactic thiamine supplementation. There were no cases of WE. Image: Summary/Conclusion: FEDR was generally well tolerated in pts with MF previously treated with RUX. The frequency and severity of GI AEs were substantially lower in FREEDOM than in previous FEDR clinical trials, likely due to early implementation of GI prophylaxis. Thiamine decreases were uncommon and easily managed with routine monitoring and oral supplementation as needed. P1043: OUTCOME OF PATIENTS WITH PREFIBROTIC MYELOFIBROSIS FROM A LARGE ACADEMIC CENTER L. Masarova1,*, P. Bose1, N. Pemmaraju1, H. Chifotides1, L. Zhou1, Z. Estrov1, H. Kantarjian1, S. Verstovsek1 1Leukemia, MD Anderson Cancer Center, Houston, United States of America Background: The 2016 WHO classification includes prefibrotic primary myelofibrosis (pre-PMF; fibrosis grading 0-1) and overt PMF (fibrosis grading 2–3). Pre-PMF has favorable survival when compared to overt PMF, however pre-PMF patients with intermediate -2 or high risks IPSS still have poor outcome (Guglielmelli et al. Blood. 2017;129(24):3227–3236). Aims: We aimed to characterize pre-PMF patients referred to our institution between the years of 2000-2020. We specifically investigated the role of known IPSS prognostic factors separated into i) clinical factors (CF): presence of either hemoglobin [hgb] < 10 g/dL / white cells [WBC] > 25 x109/L] or peripheral blasts [bl] ≥1%]; ii) age > 65 years and iii) systemic symptoms (IPSS defined; Cervantes, et al. Blood. 2009;113(13):2895–901). Methods: This retrospective study included 130 patients with newly diagnosed pre-PMF (median 1.5 months from diagnosis to presentation) and available bone marrow fibrosis grading (Thiele, J. et al. Haematologica. 2005. 90, 1128–1132). Demographics were expressed by descriptive statistics. Overall survival was estimated from the time of presentation using Kaplan Meier method with log rank test. Results: Clinical characteristics are detailed in Table 1, PART I. Fibrosis degree included grade 0 in 9, grade 0/1 in 7 and grade 1 in 114 patients, respectively. IPSS risks were as follows: low (N 22), intermediate 1 (N 50), intermediate 2 (N 30) and high (N 28). Thirty-five patients had no i-iii) factors (27%), symptoms only were in 49 patients (38%), one CF without and with symptoms was noticed in 9 (7%) and 22 (17%) patients, respectively, and 15 (11%) patients had 2 CF +/- symptoms. Median OS of the entire cohort was 68 months (95% CI 42-94) with 54% of patients being alive at 5 years. Overall survival (95% CI) per IPSS scores: low-intermediate 1 and 2 - high were not reached, 179 months (65-not expressed), 65 months (32-98) and 27 months (5-49), respectively. Median OS (95% CI) of patients without i-iii) factors (age < 65 years, no symptoms and no CF) and patients with only symptoms (all ages included) was not reached; median OS of patients with one CF with and without symptoms was of 48 months (33-63) and 47 months (37-57), respectively and median OS of patients with 2 CF +/- symptoms was of 16 months (8-24). Detailed separation of survivals per age categories and IPSS subgroups is provided in Table 1, PART II. Over the median follow-up of 43 months (95% CI 28-59), 7 patients progressed to acute leukemia with time to progression of 40 months (95% CI 9-178). Forty-one percent of patients (n = 53) required MF therapy during their follow-up, including 35 patients with JAK inhibitors and 8 with stem cell transplantation, respectively. Patients without the need for therapy or those who received JAK inhibitor had median OS of 81 (95% CI 65-not estimated) and 100 months (95% CI 17-183), respectively. This was superior to treated patients without exposure to JAK inhibitors (median OS of 45 months, 95% CI 16-47). Image: Summary/Conclusion: Presence of more adverse clinical factors is the strongest predictor of unfavorable outcome of patients with pre-PMF. Use of JAK inhibitors might have significantly improved the outcome of patients with constitutional symptoms. P1044: REAL-WORLD RUXOLITINIB TREATMENT PATTERN IN MYELOFIBROSIS PATIENTS WITH THROMBOCYTOPENIA J. Mei*1,, Y. Wang*2, S. Kabir1, N. Ichikawa3, S. Iino3, C. Lebedinsky1 1Clinical Development, Sumitomo Dainippon Pharma Oncology, Inc, Cambridge; 2Computational Research, Sumitovant Biopharma, New York, United States of America; 3Oncology Clinical Development Unit, Sumitomo Dainippon Pharma Co., Ltd, Osaka, Japan Background: Ruxolitinib (RUX) is the standard treatment for patients (pts) with myelofibrosis (MF) to reduce splenomegaly and improve MF-associated symptoms. RUX treatment is titrated based on platelet counts and is associated with high rates of cytopenia effect. The incidence of moderate thrombocytopenia (platelet count <100 x 109/L) is about 25% in newly diagnosed MF pts; however, the prevalence is about 67% in all MF pts. Low platelet count has been identified as an important surrogate marker of survival in MF pts with associated poor clinical features. Thrombocytopenia is also an independent predictor in the Dynamic International Prognostic Scoring System plus (DIPPS plus), with an actual 1.4-fold increased risk of death. Thus, treatment of MF pts with thrombocytopenia confers a unique challenge and unmet medical need. Real world studies will provide a perspective on the current management of MF pts with thrombocytopenia. Aims: To characterize the disease and RUX treatment pattern in MF pts with thrombocytopenia using de-identified insurance claims data in the US. Methods: The IBM MarketScan® Medicare Supplemental Database data was retrospectively analyzed to identify pts aged ≥18 years with ≥1 claim for RUX and ≥2 non-diagnostic medical claims for MF (ICD-10 D47.4, D7581) from 2017–2019. The first RUX claim on or after the first MF claim defined the index date. Pts who continuously enrolled in a health plan for 3 months before the index date and 6 months after the index date were included. Clinical characteristics and RUX treatments were compared in MF pts with or without a history of thrombocytopenia prior to RUX initiation. Results: The database contained 136 MF pts who had received treatment with RUX. Median age was 61 years, 61% were male, 62% of pts had primary MF, and 38% had post-polycythemia vera or essential thrombocythemia MF. Prior to initial RUX treatment, 57% of pts had a diagnosis of splenomegaly, 56% had anemia, 25% had thrombocytopenia, 19% had received RBC transfusion, and 7% had neutropenia. During RUX treatment, 25% of pts had a new diagnosis of thrombocytopenia, 22% had neutropenia, and 19% had anemia. Pts with a history of thrombocytopenia also had more anemia (79% vs 48%, p=0.001) and more RBC transfusions (44% vs 11%, p<0.001) than those without a history of thrombocytopenia. History of thrombocytopenia was associated with a lower starting dose of RUX (average starting daily dose 20 mg vs 27 mg, p=0.005), whereas history of anemia, leukocytosis, neutropenia, MF type, and splenomegaly were found to have no difference in the RUX starting dose. In addition, MF pts with history of thrombocytopenia continued RUX treatment at lower doses compared with those without a history of thrombocytopenia. During RUX treatment, 57% of pts with history of thrombocytopenia experienced anemia compared with 42% in pts without history of thrombocytopenia (OR 1.88, p=0.43). Summary/Conclusion: In this real-world data analysis, history of thrombocytopenia was a major factor influencing the RUX dose for treatment of MF pts, consistent with prescribing data that RUX dose is adjusted based on platelet counts. These data show that pts with thrombocytopenia were treated with lower doses of RUX in clinical practice. Additionally, history of thrombocytopenia was associated with history of anemia; thus, these MF pts may be more prone to develop anemia during RUX treatment compared with pts without a history of thrombocytopenia. Our findings underscore the need for more treatment options to manage MF pts with thrombocytopenia. *Equal contribution. P1045: IS JAK2V617F BUT NOT CALR AS DRIVER MUTATION ENOUGH BY ITSELF IN THE PATHOGENESIS OF UNUSUAL TYPE VENOUS THROMBOSIS IN MPN PATIENTS? E. Morsia1,*, E. Torre1, G. Tarantino2, G. Svegliati Baroni2, G. Goteri3, S. Mancini1, A. Tassoni1, F. Alessandro1, A. Olivieri1, S. Rupoli1 1Hematology; 2Gastroenterology and hepatology; 3Pathological Anatomy and Histopathology, Ospedali Riuniti di Ancona, Ancona, Italy Background: Myeloproliferative neoplasms (MPN) are a heterogenous group of hematopoietic stem cell disorders and clinically they constitute the most frequent underlying cause of venous thrombosis (VTE) in unusual site, including splanchnic vein thrombosis (SVT) and cerebral vein thrombosis (CVT). This subgroup of patients has been showed to have distinct characteristics compared to MPN patients with VTE in usual site, including deep vein thrombosis and pulmonary embolism. Aims: The aim of this study is analyzed in a retrospective cohort of patients with MPN clinical characteristics, molecular features and outcome data in patients who have experienced an unusual site thrombosis. Methods: In our study, we retrospectively analyzed a cohort of 577 consecutive patients with MPN according to WHO 2016 criteria who referred to our institute between 2009 and 2020. In all, 113 patients (19.58%) had a vascular event during a median follow up of 94.3 months (range, 2.4-416.0). Usual and unusual site venous thrombosis occurred in 40.7% and 33.6% in MPN patients with thrombosis, respectively. Results: From the 38 MPN patients with unusual site VTE, 19 (50%) were male. Twenty-seven patients had an SVT and 11 a CVT. The driven mutation was JAK2V617F in 79% with a median allelic burden of 20% using NGS. MPN patients with SVT and CVT are younger (ORR 0.87 (0.82-0.93), pValue < 0.0001), with higher PLT count at diagnosis (ORR 0.06 (0.01-0.38), pValue=0.0085), and higher splenomegaly rate (ORR 0.97 (0.90-0.99), pValue=0.0003) compared to MPN patients with usual site VTE. Globally survival data in our population reported an overall survival in MPN with thrombosis of 238 months (95% I.C. 177-336), poorer in patients with arteriosus events compared to VTE (usual and unusual sites; pValue=0.017). At all, only few patients experienced recurrence thrombosis during the follow up and the recurrence thrombosis free survival rate at 5 years in MPN with thrombosis was 85%. Moreover, older age and level of hemoglobin at time of thrombosis significantly influence survival in MPN patients with unusual site VTE. Using NGS on 20 MPN patients with unusual site VTE, molecular analysis identified the mutation in the hotspot exon 14 region of JAK2 in 13 patients, among them the 92% as isolated alteration. The most frequent additional mutations were found in CALR mutated patients, including high risk mutation as ASXL1 and U2AF1 (Figure 1). Image: Summary/Conclusion: This finding underlies the central role of JAK2V617F mutation in the pathogenesis of unusual type VTE in MPN, which occurs as sole molecular alteration in more than 90% of cases. However, in our cohort most patients who carried CALR as driver mutation seem to need one or more additional mutations. Our interpretation on these findings is that JAK2V617F is per se a strong risk factor for unusual venous thrombosis while CALR mutation must be enriched for additional non-driver alterations, needing a second hit to drive to thrombosis. P1046: SEVERE COMPLICATIONS IN JAK2 V617F POSITIVE PEDIATRIC PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS W. Novak1,*, C. Annamária1, R. Crazzolara2, J. Neil3, K. Pirolt4, M. Dworzak1,5, L. Kager1,5 1St. Anna Children’s Hospital, Department of Pediatrics, Medical University Vienna, Vienna; 2Department of Pediatrics, Medical University of Innsbruck, Innsbruck; 3Department of Pediatrics, Paracelsus Medical University, Salzburg; 4Department of Pediatrics, Klinikum Klagenfurt, Klagenfurt; 5St. Anna Children’s Cancer Research Institute (CCRI), Vienna, Austria Background: Myeloproliferative neoplasms (MPNs) are very rare diseases in children and adolescents and neither detailed risk stratification nor treatment guidelines are available. Aims: The aim was to evaluate disease characteristics, complications, and treatment response of pediatric patients diagnosed with MPNs. Methods: We retrospectively analyzed clinical data from a cohort of 14 pediatric patients diagnosed and treated in Austria over the past 12 years. Results: Of these 14 patients (female, N=9), 10 patients were diagnosed with essential thrombocythemia (ET), three patients with polycythemia vera (PV) and one patient with primary myelofibrosis (PMF). One of the three main driver mutations known to cause MPN (JAK2, MPL or CALR mutation) was found in nine patients, eight of whom carrying a JAK2 V617F mutation (ET, N=5; PV, N=3; female, N=5; male, N= 3) and the patient with PMF harbored a CALR type 1 mutation. Thrombotic complications occurred in 3/5 female (0/3 male) JAK2 V617F positive patients; one patient with ET developed recurrent sinus vein thrombosis (SVT) and two patients with PV developed a Budd-Chiari syndrome (BCS). One patient with BCS died. A transformation into a pre-PMF was observed in the other patient with BCS. In addition, another JAK2 V617F positive patient showed transformation from ET to PV seven years after diagnosis. Although 7/11 tested patients developed an acquired von Willebrand syndrome, there were no major bleeding complications. Two symptomatic JAK2 V617F positive female ET patients were treated off-label with ropeginterferon alfa-2b. The indications for treatment were microvascular symptoms (headache) and recurrent SVT. To our knowledge, this new long-acting interferon has not previously been used in children and adolescents. The treatment was well tolerated, no complications occurred and both patients quickly resolved their clinical symptoms. Summary/Conclusion: Our data support results from studies in adult MPN patient populations, which showed that JAK2 V617F positivity is a risk factor for thrombotic complications and female gender is an additional risk factor for atypical thrombosis (BCS and SVT). P1047: REAL-WORLD SAFETY OF RUXOLITINIB IN PATIENTS WITH INTERMEDIATE OR HIGH RISK OF PRIMARY MYELOFIBROSIS, POST-POLYCYTHEMIA VERA MYELOFIBROSIS OR POST-ESSENTIAL THROMBOCYTHEMIA MYELOFIBROSIS IN CHINA Z. Xu1, M. Duan2, Q. Jiang3, Q. Leng4, N. Xu5, Y. Zhang6, C. Zhao7, W. Wu8, Q. Zhang9, J. Fu10, J. Zhang11, R. Fu12, Z. Yan13, J. Zhang14, C. Lin15, G. Ouyang16, Z. Wang17, L. Ma18, H. Hao19, X. Li20, S. Ran21, Y. Chen22, T. Li23, Z. Xiao1,* 1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin; 2Peking Union Medical College Hospital; 3Peking University People’s Hospital, Beijing; 4Anshan Central Hospital, Liaoning; 5Nanfang Hospital, Southern Medical University, Guangdong; 6Henan Provincial People’s Hospital, Henan; 7Affiliated Hospital of Qingdao University, Shandong; 8Ruijin Hospital, Shanghai Jiao Tong University, Shanghai; 9Gansu Provincial People’s Hospital, Gansu; 10Shaoxing People’s Hospital, Zhejiang; 11The Second Hospital of Hebei Medical University, Hebei; 12Tianjin Medical University General Hospital, Tianjin; 13North China University of Technology Affiliated Hospital, Hebei; 14Nanchang University Affiliated Ganzhou Hospital, Jiangxi; 15Zhangzhou Affiliated Hospital of Fujian Medical University, Fujian; 16Ningbo First Hospital, Zhejiang; 17Wuxi Second People’s Hospital, Jiangsu; 18Sun Yat Sen Memorial Hospital of Sun Yat Sen University, Guangdong; 19Hebei General Hospital, Hebei; 20Affiliated Hospital of Southwest Medical University, Sichuan; 21Beijing Novartis Pharma Co., Ltd, Beijing; 22China Novartis Institutes For Biomedical Research Co., Ltd; 23China Novartis Institutes For Biomedical Research Co., Ltd., Shanghai, China Background: Ruxolitinib, a potent and selective inhibitor of Janus kinase 1 (JAK1)/JAK2 tyrosine kinases, was approved for treatment of intermediate- and high-risk patients with myelofibrosis (MF) based on the findings from the pivotal studies, COMFORT I and II. Although an Asia regional study was conducted, the enrolled Chinese population was limited (N=63). Aims: We aimed to evaluate the safety profile and the treatment of ruxolitinib in Chinese patients with MF under routine clinical practice. Methods: This was a retrospective, non-interventional, multicenter, post-marketing surveillance study collecting data up to 48 weeks after ruxolitinib initiation. Patients aged ≥18 years with a confirmed diagnosis of intermediate or high-risk primary MF (PMF), post-polycythemia vera MF (PPV-MF) or post-essential thrombocythemia MF (PET-MF) who had received or were currently receiving ruxolitinib treatment per clinical judgment according to China approved label were included. Results: In total, 480 patients from 20 sites were enrolled and 428 patients completed the study treatment. The most reported reasons for treatment discontinuation were death (17.3%), adverse events (AE, 7.7%) and abnormal laboratory value (5.8%). Gender distribution were balance (male 51.6% vs female 48.4%) with median age of 61.0 years and median BMI of 22.5 kg/m2. Majority of the patients were diagnosed with PMF (69.8%), followed by PPV-MF (15.7%), and PET-MF (14.5%). The information on dosing and safety is detailed in Table 1. Ruxolitinib dosing was according to the local approved label (median initial daily dose 30.0 mg). The median time from first diagnosis to medication was 1.5 months. Overall, patients received ruxolitinib for a median of 11.1 months with median dose intensity of 28.7 mg/day. The most prescribed dose was 15 mg BID across varying durations of exposure to ruxolitinib (up to 48 weeks). A total of 196 (40.8%) patients had dose modification. On-treatment AE and serious AE (SAE) were reported in 361 (75.2%) and 57 (11.9%) patients, respectively. Of those, 60.2% and 6.0%, were related to study drug, respectively. The severity of majority of the AE were grade 2 and 3 (26.7 & 25.2%, respectively) while SAE were grade 3 & 4 (3.5% & 4.4%, respectively). A total of 4.0% and 2.3% of patients reported AEs and SAEs leading to discontinuation. The most commonly reported AEs were anemia (47.3%), decreased platelet count (22.1%), and abnormal liver function (12.7%). The most commonly reported SAEs were anemia (3.8%), platelet count decreased (1.0%), infectious pneumonia and pulmonary inflammation (both 0.8%). On-treatment AESI was reported by 18 (3.8%) patients, consisted of bleeding events (2.3%), serious or opportunistic infections (0.8%), and second primary malignancies (SPM, 0.6%). The SPM were pulmonary malignant tumors (n=2) and liver cancer (n=1). A total of 18 on-treatment deaths (MF progression=9, AE=4, and others=5) were reported. During the study period, we found that the number of patients with efficacy data were decreasing with time (baseline=183&116, Week 16=126 & 96, Week 32=90 & 62, Week 48=38 & 19 for spleen size measurement and total symptom score, respectively). This reflects the need for improve compliance in disease management. Image: Summary/Conclusion: This was the largest ruxolitinib real-world study in China. The safety profile of ruxolitinib in patients with intermediate- or high-risk PMF, PPV-MF, or PET-MF was generally consistent with previous global and Chinese studies of MF. The incidence of SPM was lower than that reported previously4. No new unexpected safety signals have been identified. P1048: MYF3001: A RANDOMIZED OPEN LABEL, PHASE 3 STUDY TO EVALUATE IMETELSTAT VERSUS BEST AVAILABLE THERAPY IN PATIENTS WITH INTERMEDIATE-2 OR HIGH-RISK MYELOFIBROSIS REFRACTORY TO JANUS KINASE INHIBITOR J. Mascarenhas1,*, C. N. Harrison2, J.-J. Kiladjian3, R. S. Komrokji4, S. Koschmieder5, A. M. Vannucchi6, T. Berry7, D. Redding7, L. Sherman7, S. Dougherty7, L. Peng7, L. Sun7, F. Huang7, Y. Wan7, F. M. Feller7, A. Rizo7, S. Verstovsek8 1Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America; 2Guy’s and St Thomas’ Hospital, London, United Kingdom; 3Hôpital Saint-Louis, Université Paris, Paris, France; 4H Lee Moffitt Cancer Center, Tampa, FL, United States of America; 5RWTH Aachen University, Aachen, Germany; 6AOU Careggi, University of Florence, Florence, Italy; 7Geron Corporation, Parsippany, NJ; 8The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America Background: Myelofibrosis (MF) is a life-threatening myeloproliferative neoplasm. The Janus Kinase inhibitors (JAKi) ruxolitinib and fedratinib are the only FDA approved treatment options for MF. Despite benefits reported with ruxolitinib in the front-line setting, a high proportion of patients discontinue treatment (Abdelrahman 2015), and the median overall survival (OS) is 11-16 months (Kuykendall 2018; Newberry 2017; Schain 2019; Palandri 2019; Mascarenhas 2020), highlighting a great unmet need for pts non-responsive to a JAKi treatment. Imetelstat, a first-in-class telomerase inhibitor, has shown meaningful clinical improvement in IMbark, a Phase 2 study in patients with intermediate-2 or high-risk MF who have relapsed after or are refractory to JAKi (Mascarenhas JCO 2021). Treatment with 9.4 mg/kg imetelstat resulted in 32.2% symptom response (total symptom score [TSS] reduction ≥50%) at Week 24 and median overall survival (OS) of 29.9 months with overall study follow up of 27.4 months. Dose-dependent inhibition of telomerase with imetelstat resulted in on-target activity that correlated with clinical benefits; dose-dependent reduction in variant allele frequency of MF driver mutations indicated targeting of the underlying malignant clone. Aims: The Phase 2 results support continued study of imetelstat 9.4 mg/kg in a Phase 3 randomized controlled study, for which the study design is described here. Methods: Study MYF3001 (IMpactMF; NCT04576156) is an open label, randomized (2:1), multicenter, Phase 3 study of imetelstat compared with best available therapy (BAT) in ~320 patients with intermediate 2 or high-risk MF refractory to JAKi treatment. Patients will be randomized to receive imetelstat 9.4 mg/kg IV every 21 days or investigator selected BAT including hydroxyurea, thalidomide, interferon, danazol, hypomethylating agents, chemotherapy, or other non-JAKi containing therapy as appropriate). Eligible patients will be stratified based on a) Intermediate 2 or high-risk per Dynamic International Prognostic Scoring System; b) platelet count at entry (platelets ≥ 75 and < 150 x 109/L vs ≥ 150 x 109/L). Patients who meet progressive disease criteria and discontinue BAT may be eligible to crossover to imetelstat. Results: The primary endpoint is OS and one interim analysis is planned when approximately >71% of death events have occurred. Secondary endpoints include symptom and spleen response rates at week 24, progression-free survival, clinical response assessment per modified 2013 International Working Group - Myeloproliferative Neoplasms Research and Treatment criteria, time to and duration of response, reduction in degree of bone marrow fibrosis, safety, pharmacokinetics and patient-reported outcomes. Biomarkers and mutation analyses will be performed to evaluate the impact of imetelstat on reduction/depletion of malignant clones. (Figure 1) Image: Summary/Conclusion: Approximately 180 sites are planned in North and South America, Europe, Middle East, Australia and Asia. The study is open for enrollment. P1049: A PHASE 2 STUDY OF BEZUCLASTINIB (CGT9486), A NOVEL, HIGHLY SELECTIVE, POTENT KIT D816V INHIBITOR, IN ADULTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS (APEX): METHODS, BASELINE DATA, AND EARLY INSIGHTS D. J. DeAngelo1,*, V. Pullarkat2, M. Piris-Villaespesa3, T. I. George4,5, J. L. Patel4,5, C. Ustun6, P. Bose7, M. L. Heaney8, A. Pilla9, M. Massaro9, B. Exter9, H. A. Jolin9, Z. Mikhak9, T. Tashi5 1Dana-Farber Cancer Institute, Boston; 2City of Hope, Duarte, United States of America; 3Hospital Universitario Ramón y Cajal, Madrid, Spain; 4ARUP Laboratories; 5Huntsman Cancer Institute, University of Utah, Salt Lake City; 6Rush University Cancer Center, Chicago; 7The University of Texas MD Anderson Cancer Center, Houston; 8Columbia University Medical Center, New York; 9Cogent Biosciences, Cambridge, United States of America Background: Systemic mastocytosis (SM) is a rare disease characterized by accumulation of pathogenic mast cells in bone marrow and extracutaneous tissues with or without skin involvement. In approximately 95% of adult patients, SM pathogenesis is driven by a gain-of-function mutation (D816V) in exon 17 of the KIT gene. Advanced systemic mastocytosis (AdvSM) is an aggressive, life-threatening form of SM with three variants: aggressive systemic mastocytosis (ASM), SM with an associated hematologic neoplasm (SM-AHN), and mast cell leukemia (MCL). Overall survival ranges from <6 months to 3-4 years. Bezuclastinib (CGT9486) is an orally administered, highly selective and potent tyrosine kinase inhibitor (TKI) that targets KIT D816V, avoiding other closely related kinases with clinical liabilities such as PDGFRα, PDGFRβ, and CSF1R. Bezuclastinib has demonstrated low brain penetration in preclinical tissue distribution studies, potentially addressing challenges associated with other targeted agents such as CNS effects and intracranial hemorrhage. Bezuclastinib has been administered to >100 participants in clinical trials, including 51 patients with advanced solid tumors (e.g., GIST) in a Phase 1b/2a study. In that study, bezuclastinib led to a reduction in KIT exon 17 mutational burden, temporally correlated with a reduction in tumor burden, and an acceptable safety profile. Aims: To describe the Apex study methods and present baseline data and preliminary findings from Part 1 of this Phase 2 clinical trial evaluating bezuclastinib in patients with AdvSM (NCT04996875). Methods: This open-label, 2-part, multicenter study aims to enroll approximately 140 adult patients with AdvSM per WHO criteria assessed with SM-related organ damage and baseline serum tryptase of ≥20 ng/mL. Part 1 patients are randomized 1:1:1:1 to 50, 100, or 200 mg of bezuclastinib twice daily, or 400 mg once daily. Data from Part 1 will help determine the optimal dose of bezuclastinib, which will be utilized to further assess safety, efficacy, PK, and PD in Part 2. The primary efficacy endpoint is overall response rate (% patients classified as confirmed responders; CR, CRh, PR and CI) according to mIWG-MRT-ECNM response criteria, assessed by a Central Response Review Committee. Results: Patients diagnosed with ASM, SM-AHN, and MCL have been enrolled in Part 1 (dose optimization) at centers in Europe and the United States. Baseline values include median (min, max) serum tryptase of 170 ng/mL (95.7-303), KIT D816V variant allele fraction 8.7% (7.04-13.48) by ddPCR, and bone marrow mast cells of 70% (7-80) by IHC. Dosing continues in these patients and the study is actively enrolling. Preliminary safety, PK, and PD data from Part 1 will be presented. Safety data will include AEs/SAEs/AECIs and dose modification frequencies while PD biomarker data, as early evidence of target engagement and clinical activity, will include changes in mast cell and KIT D816V mutational burden. Summary/Conclusion: There is a significant unmet need for novel, safe, and effective treatment options for patients with AdvSM. Bezuclastinib, an orally administered and highly selective TKI with potent activity against KIT D816V, may be a treatment option in AdvSM. Part 1 of the Apex study aims to determine the optimal dose to further evaluate in Part 2. Early insights into the safety and tolerability profile and biomarker data will further inform clinical development of bezuclastinib in AdvSM. P1050: THROMBOCYTOPENIC MYELOFIBROSIS (MF) PATIENTS PREVIOUSLY TREATED WITH A JAK INHIBITOR IN A PHASE 3 RANDOMIZED STUDY OF MOMELOTINIB (MMB) VERSUS DANAZOL (DAN) [MOMENTUM] A. Vannucchi1,*, R. Mesa2, A. Gerds3, H. K. Al-Ali4, D. Lavie5, A. Kuykendall6, S. Grosicki7, A. Iurlo8, Y. T. Goh9, M. Lazaroiu10, M. Egyed11, M. L. Fox12, D. McLornan13, A. Perkins14, S.-S. Yoon15, V. Gupta16, J.-J. Kiladjian17, R. Donahue18, J. Kawashima18, S. Verstovsek19 1Center Research and Innovation of Myeloproliferative Neoplasms, AOU Careggi, University of Florence, Florence, Italy; 2UT Health San Antonio Cancer Center, San Antonio, TX; 3Cleveland Clinic Department of Hematology and Medical Oncology, Avon, OH, United States of America; 4University Hospital of Halle, Halle, Hungary; 5Hadassah Hebrew University Medical Center, Jerusalem, Israel; 6Moffitt Cancer Center, Tampa, FL, United States of America; 7Medical University of Silesia, Katowice, Poland; 8Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 9Singapore General Hospital, Singapore, Singapore; 10Policlinica de Diagnostic Rapid Brasov, Brasov, Romania; 11Somogy County Mór Kaposi General Hospital, Kaposvár, Hungary; 12Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain; 13Guy’s and Saint Thomas’ NHS Foundation Trust, London, United Kingdom; 14Monash University, Melbourne, Australia; 15Seoul National University Hospital, Seoul, South Korea; 16Princess Margaret Cancer Centre, Toronto, ON, Canada; 17Saint-Louis Hospital (AP-HP), Paris, France; 18Sierra Oncology, Inc., San Mateo, CA; 19The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America Background: MMB, a novel oral ACVR1/ALK2 and JAK1/2 inhibitor, showed clinical activity on MF symptoms, red blood cell (RBC) transfusion requirements (anemia), and spleen volume in the SIMPLIFY trials, including in MF patients (pts) with thrombocytopenia. Aims: MOMENTUM is a pivotal phase 3 study of symptomatic and anemic MF pts previously treated with a JAK inhibitor (JAKi) testing MMB vs DAN. This analysis evaluated MOMENTUM pts with baseline (BL) platelet counts (PLT) ≤150 x 109/L on key symptom, anemia, and spleen volume endpoints at 24 weeks (wks). Methods: Eligibility: Primary or post-ET/PV MF; DIPSS high risk, Int-2, or Int-1; MF Symptom Assessment Form Total Symptom Score (MFSAF TSS) ≥10; hemoglobin (Hgb) <10 g/dL; prior JAKi for ≥90 days, or ≥28 days if RBC transfusions ≥4 units in 8 wks or Gr 3/4 thrombocytopenia, anemia, or hematoma; palpable spleen ≥5 cm; PLT ≥25 x 109/L. JAKi taper and washout was ≥21 days. Randomization: 2:1 to MMB 200 mg QD plus DAN placebo or DAN 600 mg QD plus MMB placebo for 24 wks. Primary endpoint: TSS response (≥50% reduction from BL) rate at wk 24. Key secondary endpoints, assessed sequentially at wk 24: RBC transfusion independence (TI) rate, splenic response rate (SRR; ≥25% reduction in volume from BL), change from BL in TSS, SRR (≥35% reduction from BL) and rate of zero transfusions since BL. Informed consent was obtained from all participants. Results: 64% of the MOMENTUM pts had BL PLT of ≤150 x 109/L. Of this subset, 60 (74%) of 81 MMB pts and 25 (58%) of 43 DAN pts completed the 24-week randomized treatment (RT) phase. In this subset, median BL TSS were 29 (MMB) and 24 (DAN), Hgb were 7.9 (MMB) and 8.0 (DAN) g/dL, and PLT were 67 x 109/L (MMB) and 64 x 109/L (DAN). BL mean spleen volume was 2504 (MMB) and 2282 (DAN) cm3. Prior JAKi was ruxolitinib in 124 pts (100%) and fedratinib in 6 pts (5%); mean duration of prior JAKi was 136 weeks. Efficacy results are in Table. These results are consistent with the overall intent-to-treat (ITT) analysis set (N=195). Most common Gr ≥3 treatment-emergent adverse events (TEAEs) in the RT phase were thrombocytopenia (MMB, 31%; DAN, 16%) and anemia (MMB, 7%; DAN, 14%); Gr ≥3 bleeding events occurred in 9% of MMB and 5% of DAN pts. TEAEs led to study drug discontinuation in 15% of MMB and 19% of DAN pts, and serious TEAEs were reported in 36% of MMB and 40% of DAN pts, in RT phase. A trend toward improved OS up to wk 24 was seen with MMB vs DAN [HR (95% CI)=0.490 (0.195, 1.235)]. Additional analyses of pts with BL PLT <100 x 109/L (N=100) and BL PLT <50 x 109/L (N=31) show similar treatment effects of MMB vs DAN. Image: Summary/Conclusion: In thrombocytopenic MF pts who were symptomatic and anemic, MMB was superior to DAN for symptom responses, transfusion requirements, and spleen responses and showed comparable safety and favorable survival. MMB may address a critical unmet need in thrombocytopenic MF pts. NCT04173494. P1051: A PHASE 2 STUDY OF IMG-7289 (BOMEDEMSTAT) IN PATIENTS WITH ADVANCED MYELOFIBROSIS H. Gill1,*, A. Yacoub2, K. Pettit3, T. Bradley4, A. Gerds5, M. Tatarczuch6, J. Shortt7, N. Curtin8, J. Rossetti9, K. Burbury10, A. Mead11, J. Göthert12, S. Koschmieder13, A. Jones14, J. Peppe14, J. Dias15, G. Natsoulis16, T. McClure17, M. Kleppe15, W.-J. Hong14, W. Stevenson18, J. Ewing19, J. Chacko20, E. Rumi21, A. Halpern22, F. Palandri23, N. Vianelli23, F. Passamonti24, R. Mesa25, M. Marchetti26, C. Harrison27, A. Vannucchi28, J. Watts29, D. Ross30, M. Talpaz31, H. Rienhoff32 1Medicine, University of Hong Kong, Hong Kong, China; 2Internal Medicine, The University of Kansas Cancer Center, Leawood; 3Internal Medicine, University of Michigan, Ann Arbor; 4Hematology and Medical Oncology, University of Miami, Sylvester Comprehensive Cancer Center, Miami; 5Hematology and Medical Oncology, Cleveland Clinic, Cleveland, United States of America; 6Haematology and Oncology, Monash Health; 7Clinical Sciences, Peter MacCallum; 8Monash Health, Monash University, Melbourne, Australia; 9Medicine, UPMC Hillman Cancer Center, Pittsburgh, United States of America; 10Oncology, Peter MacCallum Cancer Centre, Melbourne, Australia; 11MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; 12Hematology, University Hospital Essen, Essen; 13Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Aachen University, Aachen, Germany; 14Hematology; 15Basic Sciences; 16Genetics; 17Biometrics, Imago BioSciences, South San Francisco, United States of America; 18Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, Australia; 19Haematology, University Hospitals Birmingham, Birmingham; 20Haematology, The Royal Bournemouth & Christchurch Hospitals, Bournemouth, United Kingdom; 21Haematology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 22Medicine, University of Washington, Seattle, United States of America; 23Azienda Ospedaliero, Universitaria di Bologna Policlinico S.Orsola-Malpighi, Bologna; 24Haematology, University of Insubria, Varese, Italy; 25UT Health San Antonio Cancer Center, University of Texas Health Science Center, San Antonio, United States of America; 26Haematology, Azienda Ospedaliera SS Antonio e Biagio e Cesare Arrigo, Alessandria, Italy; 27Haematology, Guy’s and St Thomas’, London, United Kingdom; 28Center for Research and Innovation of Myeloproliferative Neoplasms, University of Florence, Florence, Italy; 29Medicine, University of Miami, Sylvester Comprehensive Cancer Center, Miami, United States of America; 30Haematology, Flinders Medical Centre and University, Adelaide, Australia; 31Medicine, University of Michigan, Ann Arbor; 32Hematology, Imago BioSciences, San Carlos, United States of America Background: Lysine-specific demethylase-1 (LSD1) is an activity critical for the self-renewal of malignant myeloid cells and maturation of megakaryocytes, cells central to the pathogenesis of MF. Bomedemstat is an orally active LSD1 inhibitor that in mouse models ameliorated the hallmarks of MPNs and improved survival (Kleppe et al. 2015; Jutzi et al. 2018). Aims: IMG-7289-CTP-102 is an ongoing, global, open-label Phase 2 study evaluating bomedemstat dosed QD in MF patients (NCT03136185). Key eligibility criteria include patients intolerant, refractory, resistant, or inadequately controlled by approved therapy, and platelet count ≥100 x 109/L. Key objectives are safety and reduction of spleen volume (SVR) by MRI/CT and total symptoms scores (TSS) using the MPN-SAF instrument. Methods: Serial bone marrow (BM) biopsies and imaging studies are read centrally. 261 genes are serially sequenced to quantify changes in the allelic frequencies of mutations (MAF) and identify new mutations. The starting dose is 0.6 mg/kg/d titrating, as needed, to a platelet count of 50-75x109/L. Results: At 89 patients, the study is now fully enrolled: 46% primary MF, 33% post-essential thrombocythaemia-MF, 21% post-polycythaemia vera-MF. Median age is 68 (35-88) with 52% males. Prior treatment with ruxolitinib was reported in 83% (74/89); 46% had also received at least 1 additional treatment. 30% of patients (27/89) had received ≥1 RBC transfusion prior to dosing. By IPSS, 53% were high-risk, 40% int.-2, and 7% int.-1. At screening (N=103), sequencing to a mean depth of >1000 bp, JAK2 was mutated in 69%, CALR in 21%, MPL in 6%; 59% had ≥2 mutations of which 71% were high-molecular risk mutations in ASXL1, IDH1/2, EZH2, U2AF1, TP53 and/or SRSF2. At data cutoff (3 Feb 2022), the median duration of treatment is 28 weeks (2-131). Of patients for whom TSS data is available at 24 weeks in those with baseline values ≥20, 72% (18/25) had a reduction in TSS; 24% (6/25) reported a ≥50% reduction. Of patients evaluable for SVR (N=50), 64% had a reduction in spleen volume from baseline; in 28%, SVR was ≥20%. Of evaluable patients (N=41), 90% had stable (∆ <±1.0 g/dL) or improved (≥1.0 g/dL) hemoglobin. Of patients with BM fibrosis scoring post-baseline (N=52), 31% improved by 1 grade and 50% were stable. CCL5 and S100A8/A9 cytokines were elevated at baseline in 50% (16/32) and 78%, respectively; at Day 84, 81% and 68%, respectively, showed reductions of at least 10% and of those, 100% (CCL5) and 47% (S100A8/A9) normalized. In follow-up sequencing at around Week 24, of 60 mutant alleles in 32 patients, the mean MAF fell by 39% in 48%. JAK2 MAFs fell by 31% [SD 5%] in 46% (N=24); ASXL1 MAFs fell 40% in 71%. Clones with JAK2 and/or ASXL1 mutations were most affected. No new mutations have been identified and no patient has transformed to AML. The most common non-hematologic AEs reported by patients was dysgeusia in 36% (32/90) and diarrhoea in 34% (31/90). All dysgeusia events were grade 1/2 and 1 led to treatment discontinuation. Of 14 related SAEs, 4 were Grade 2, 9 Grade 3 and 1 Grade 4 (thrombocytopenia). Twenty-nine patients remain on bomedemstat. Early terminations due to AEs occurred in 18 (20%) patients (9 related to bomedemstat), and 13 discontinued for other reasons. There have been no safety signals, DLTs, or deaths related to drug. Summary/Conclusion: In patients with advanced MF, bomedemstat alone had an acceptable tolerability profile, relieved symptoms, reduced spleen volume and mutation burden while improving fibrosis and anemia without safety signals. P1052: IMPACT OF SF3B1 MUTATION IN MYELOFIBROSIS J. Senapati1,*, S. Verstovsek1, L. Masarova1, N. Pemmaraju1, G. Montalban Bravo1, S. Pierce1, L. Zhou1, G. Garcia-Manero1, H. Kantarjian1, P. Bose1 1Leukemia, MD Anderson Cancer Center, Houston, United States of America Background: Splicing factor 3B subunit 1 (SF3B1) mutations have been shown to confer a unique phenotype in MDS and MDS/MPN overlap syndromes, with ring sideroblasts, thrombocytosis and a relatively favorable prognosis. In myelofibrosis (MF), the frequency of SF3B1 mutation is <10% and it may play a less important role in disease outcomes (Lasho et. al, Leukemia, 2011). Aims: We aimed to characterize the phenotypic and genotypic associations of SF3B1 mutations in pts with MF and compare them to SF3B1 wild type MF. Methods: We retrospectively analyzed all pts with WHO-defined MF (primary, including pre-fibrotic MF, or progressed from PV or ET) seen at our center from 1/2017 through 7/2021. All pts had a BM biopsy with karyotyping and most of the pts had an 81-gene myeloid mutation panel performed by next-generation sequencing (including JAK2, CALR, MPL and SF3B1) at presentation or at progression to MF from PV or ET. We compared the disease phenotypes, MPN driver and co-occurring mutations, cytogenetics, DIPSS, transfusion requirements, treatment characteristics and outcomes between pts with SF3B1 -mutated (SF3B1 +) and -wild type (SF3B1 -) MF. Results: 381 pts with MF were identified, 29 (8%) of whom were SF3B1 +. There were similar frequencies of JAK2, CALR and MPL driver mutations and “triple negative” MF in the SF3B1 + and SF3B1 - groups (Table 1). The median number of SF3B1 mutations was 1 (range, 1-2); K666N being the most common (52%), followed by K700E (14%). 10% SF3B1 + pts were DIPSS high risk, compared to 19% SF3B1 - pts (p=0.3). There were no significant differences between the 2 groups in terms of baseline hemoglobin level, white blood cell count, platelets, co-occurring mutations, and symptom burden but more SF3B1 + pts were red blood cell (RBC)-transfusion dependent at presentation than SF3B1 - pts (38% vs. 15%, p=0.003). A total of 152 (43%) SF3B1 - pts and 14 (48%) SF3B1 + pts received ruxolitinib-based therapy (p=0.7). At a median follow up (mFU) of 17.2 months (mos) for the entire cohort (range, 0.1- 52.2 mos; mFU 22.6 mos for SF3B1 + and 16.8 mos for SF3B1 -), 41 pts had died [4 (14%) SF3B1 + and 37(10%) SF3B1 -] and 7 pts (1 SF3B1 + and 6 SF3B1 -) had experienced leukemic transformation. The median estimated OS was 230 mos for SF3B1 - vs. not reached for SF3B1 + pts (p = 0.57, Fig. 1). Table 1: Patient chracteristics, treatment and outcomes Characteristics SF3B1+ (N= 29) SF3B1- (N= 352) p value N/Median (%) [range] Age, years 72 [54-91] 69 [21-89] Gender Males 24 (83) 202 (57) Diagnosis PMF 21 (72) 226 (64) Post ET/PV MF 8 (28) 126 (36) Baseline CBC Hb (g/dl) 9.7 [6.9-18.4] 10.5 [5.5-17.6] 0.4 WBC (109/L) 8.3 [1.3-73.3] 8.5 [1.1-212.1] 0.9 Platelet (109/L) 230 [19-1188] 216 [1-1271] 0.5 Driver mutation JAK2 18 (62) 214 (60) 0.9 CALR 5 (17) 82 (23) 0.6 MPL 3 (10) 29 (8) 0.7 Triple negative 3 (10) 32 (9) 0.7 JAK2 allele burden (n=28 SF3B1+,343 SF3B1-) 45 [1-81]% 47 [1-99]% 0.5 Co-mutations (n=20 SF3B1+, 205 SF3B1-) Splicing 3 (15) 59 (29) 0.3 Epigenetic 15 (75) 157 (76) 0.9 TP53 2 (10) 10 (5) 0.3 CTG (n=25 SF3B1+, 264 SF3B1-) Non diploid karyotype 12 (48) 102 (39) 0.4 BM fib (n=29 SF3B1+, 274 SF3B1-) Grade 3 3 (24) 113 (41) 0.001 DIPSS High 3 (10) 67 (19%) 0.3 Transfusion dependency RBC 11 (38) 52 (15) 0.003 Platelet 0 (0) 4 (1) 0.99 Events Leukemic transformation 1 (3) 6 (2) 0.4 Death 4 (14) 37 (10) Image: Summary/Conclusion: SF3B1 mutation is an uncommon event in MF and does not substantially affect the disease phenotype and outcomes. SF3B1 mutated MF pts appear to be more be RBC transfusion dependent given the underlying biology of SF3B1 mutation. P1053: ALBUMIN AND C-REACTIVE PROTEIN PROVIDE PROGNOSTIC INFORMATION INDEPENDENTLY FROM MIPSS70 IN OVERT MYELOFIBROSIS. A RETROSPECTIVE STUDY. N.-M. Messerich1, T. Volken2, S. Cogliatti3, T. Lehmann4, A. Holbro5, I. Demmer3, R. Benz6, W. Jochum3, T. Silzle4,* 1Department of Intensive Care, Cantonal Hospital St. Gallen, St. Gallen; 2Institute of Public Health, ZHAW School of Health Sciences, Winterthur; 3Institute of Pathology; 4Clinic for Medical Oncology and Hematology, Cantonal Hospital St. Gallen, St. Gallen; 5Division of Hematology, University Hospital Basel, Basel; 6Clinic for Hematology and Oncology, Spital Thurgau AG, Münsterlingen, Switzerland Background: Accurate prognostication is essential for patients with myelofibrosis (MF). The Dynamic Prognostic Scoring System (DIPSS) relies on age and several clinical and laboratory parameters and can be refined by information about chromosomal and/or molecular aberrations. C-reactive protein (CRP) and albumin have been reported to add prognostic information to both DIPSS and DIPSS-plus, which incorporates certain chromosomal aberrations and to scoring systems including both molecular and cytogenetic data (e.g. Molecular International Prognostic Scoring System 70 (MIPSS70)-plus V2). The impact of CRP and albumin in the context of the MIPSS70, which is useful, if metaphase cytogenetics are not obtainable, but next generation sequencing (NGS)-data are on hand, has not yet been reported. Aims: To define the prognostic impact of albumin and CRP in the context of the MIPSS70. Methods: We performed a retrospective chart review of all patients diagnosed at our institution with MF between 2000 and 2020 and collected information from hospital records as documented at diagnosis. For cases lacking NGS during diagnostic-workup, NGS was performed retrospectively, if DNA was available from diagnostic samples (Oncomine Myeloid Research Assay, Thermo Fisher Scientific, Waltham, MA, USA). Results: 79 patients (median age 72 years, range 28-87; primary MF n=54, MF following ET or PV: n=22) were evaluable. Thirty-eight (48.1%) died during follow-up (median 34 months; range 0-184). CRP-values were available for 71/79 (89.9%), albumin-values in 62/79 (78.5%) and both parameters in 57/79 (72.2%) patients. A CRP-elevation (>8mg/l, n=24) was associated with a shorter overall survival (OS) compared to a CRP <8mg/l (n=47, median 44 vs. 89 months, p<0.001, see fig A), as was an albumin below the median of the population (</≥ 40 g/l, n=31 each, median 50 vs. 101 months, p=0.018; see fig B). In univariate analyses, both CRP >8mg/l and albumin <40 g/l were associated with a higher risk of death (HR 3.85, 95% CI 1.85-8.0, p<0.001 and HR 2.49, 95% CI 1.13-5.49, p=0.024). A logistic regression model showed a continuously rising probability of death with lower albumin levels even in the range of normal (at our institution: 34-45 g/l; OR=0.85, 95% CI 0.73-0.99; p=0.043) The MIPSS70 was available for 60/79 (76%) patients. Due to the low number of low-risk-patients (n=2) we split the cohort into a “MIPSS70dichlow/intermediate” risk group (MIPSS70-low and MIPSS70-intermediate, n=45) and a “MIPSSdichhigh” risk group, comprising the 15 MIPSS70 high-risk patients, for further analyses. CRP >8 mg/l and albumin <40 g/l retained their prognostic value, if included each into a bivariate model together with the MIPSS70dich (HR 2.498, 95% CI 1.13-5.52, p=0.0236 and HR 5.486, 95% CI 1.89-15.96, p=0.0018). After inclusion of CRP >8 mg/l and albumin< 40 g/l together with MIPSS70dich into a multivariate model, only albumin <40 g/l added prognostic information (HR 3.8, 95% CI 1.27-11.48, p=0.0173). Image: Summary/Conclusion: Albumin and CRP deserve further evaluation as prognostic factors in myelofibrosis, since they add prognostic information irrespective of the molecular risk profile. In a common view, CRP and hypalbuminaemia reflect the extent of MF-associated inflammation. However, already albumin-levels in the lower range of normal are associated with an increased mortality. Given its role as main antioxidant in the extracellular space and its potential anti-inflammatory/anti-thrombotic properties, higher-albumin levels could be associated with an increased capability to counteract MF-associated inflammation. P1054: MYLOX-1: AN OPEN-LABEL, PHASE IIA STUDY OF THE SAFETY, TOLERABILITY, PHARMACOKINETICS AND PHARMACODYNAMICS OF ORAL LOXL2 INHIBITOR, GB2064, IN MYELOFIBROSIS C. Harrison1, J. Mascarenhas2, R. Rampal3, D. Cilloni4, B. Lindmark5,*, B. Singh5, B. Jacoby5, S. Verstovsek6 1Guy’s and St Thomas Hospital, London, United Kingdom; 2Icahn School of Medicine at Mount Sinai; 3Memorial Sloan Kettering, New York, United States of America; 4University Hospital San Luigi Gonzaga, Orbassano, Italy; 5Galecto Inc, Copenhagen, Denmark; 6MD Anderson Cancer Center, Houston, United States of America Background: GB2064 is a high-affinity, selective, pseudo-irreversible, small-molecule inhibitor of LOXL2, a secreted glycoprotein that crosslinks extracellular matrix collagens and elastin which contributes to stiffness and loss of function of fibrotic organs. GB2064 is being developed as an oral treatment for myelofibrosis (MF), a rare myeloproliferative disease with high morbidity and mortality. Janus kinase (JAK) inhibitor therapy has brought significant advancements in the treatment of MF, but a significant proportion of patients would eventually discontinue treatment, predominantly due to the development of cytopenia (Kyukendall et al Ann, Hematol 2018). Thus, there remains a substantial unmet need for developing well-tolerated disease-modifying treatments that reduce bone marrow fibrosis to improve haematologic parameters, splenomegaly, symptom burden and quality of life. Aims: To assess the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and clinical effects of oral GB2064 (1000 mg twice daily [BID]) dosed for 9 months to participants with primary or secondary myelofibrosis (PMF/SMF). Methods: Open-label study in 16 adult participants diagnosed with PMF or SMF in accordance with World Health Organization diagnostic criteria (Barbui et al, Blood Cancer 2018; Cruz et al, Expert Rev Hematol 2020), who are not taking a JAK inhibitor and therefore likely to be refractory, intolerant or ineligible for such inhibitors, with Eastern Cooperative Oncology Group performance status 0-2 and clinical laboratory parameters within appropriate limits per protocol. Primary endpoint is safety and tolerability. Safety and tolerability, PK, PD and appropriate MF-specific assessments will take place at all visits, except Day 7, Day 15 and Month 4 when only safety and tolerability will be assessed (Fig A). Bone marrow biopsies, magnetic resonance imaging (MRI) of spleen and quality of life measures, MPN-10 and EQ-5D-5L, are performed at prespecified timepoints within the protocol. Exploratory endpoints include LOXL2 binding assay in the circulation, relationships between PK plasma exposures, PD markers, and markers of clinical activity, fibrosis and inflammation biomarkers (YKL-40, PAI-1, PDGF, CCN2, collagen formation and degradation neoepitopes). The study is not formally powered. Participants who derive benefit may continue therapy for an additional 3 years (Fig B). Results: More than half of the intended participants have been enrolled and are on treatment with GB2064 as of February 2022.There have been no significant safety concerns observed at a dose of 1000 mg BID to date. Image: Summary/Conclusion: MYLOX-1 is designed to explore the safety and clinical effects of GB2064, a novel small-molecule LOXL-2 inhibitor, addressing bone marrow fibrosis as a main element of myelofibrosis, with the aim of decreasing extramedullary haematopoesis and improving haematological parameters, symptom burden and the quality of life for patients with MF. P1055: CLINICAL AND GENETIC RESULTS OF THE PHASE IB/II TRIAL MPNSG-0212: RUXOLITINIB PLUS POMALIDOMIDE IN MYELOFIBROSIS WITH ANEMIA F. Stegelmann1,*, E. Jahn1, S. Koschmieder2, F. Heidel3, A. Hochhaus4, H. Hebart5, S. Isfort2, A. Reiter6, M. Bangerter7, C. F. Waller8, D. Wolleschak9, C. Scheid10, J. Göthert11, P. Schafhausen12, T. Kindler13, M. P. Radsak13, N. Gattermann14, R. Möhle15, N. von Bubnoff16, A. Schrade1, T. Brümmendorf2, H. Döhner1, M. Griesshammer17, K. Döhner1 1Internal Medicine III, University Hospital of Ulm, Ulm; 2Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, RWTH Aachen University, Aachen; 3Klinik und Poliklinik für Innere Medizin C, Universitätsmedizin Greifswald, Greifswald; 4Klinik für Innere Medizin 2, Universitätsklinikum Jena, Jena; 5Internal Medicine, Stauferklinikum, Mutlangen; 6III. Medical Department, University Medical Centre Mannheim, Mannheim; 7Hematology and Oncology, Practice, Augsburg; 8Department of Internal Medicine I, University Hospital of Freiburg, Freiburg; 9Medical Center, Otto-von-Guericke University, Magdeburg; 10Department I of Internal Medicine, University of Cologne, Cologne; 11Department of Hematology, University Hospital of Essen, Essen; 122nd Medical Clinic, University Hospital of Hamburg-Eppendorf, Hamburg; 13Internal Medicine III, Johannes Gutenberg University, Mainz; 14Hematology, Oncology and Clinical Immunology, Heinrich Heine University, Düsseldorf; 15Hematology and Oncology, University Tübingen, Tübingen; 16Hematology and Oncology, University Hospital of Schleswig-Holstein, Lübeck; 17Hematology/Oncology, Universitätsklinik Minden, Minden, Germany Background: Ruxolitinib (RUX) alleviates disease-associated symptoms including splenomegaly in patients (pts) with myelofibrosis (MF). However, management of cytopenia remains challenging. Aims: As single-agent pomalidomide (POM) improved cytopenia in 14-29% of MF pts in our previous MPNSG-0109 trial, we sought to investigate the combination of RUX plus POM in MF pts with anemia (Hb <10 g/dL and/or RBC transfusion dependency [RBC-TD]). Methods: MPNSG-0212 is a multicenter, open-label, phase-Ib/II trial (NCT01644110) comprising 39 pts in cohort 1 (co1, recruited 2013-2017) and 52 pts in co2 (2017-2021). Co1 pts received RUX 10 mg BID plus POM 0.5 mg QD, while POM was intended to be increased in co2 to 1 and 2 mg QD after 3 and 6 28-day-cycles, respectively. Primary endpoint was response according to IWG-MRT and RBC-TI criteria at end of cycle 12 (EOC12). In addition, genomic landscape was characterized in all pts using targeted NGS of 269 candidate genes (Illumina NextSeq550). Results: Co1 and co2 pts had similar characteristics: median age was 71 years (range 49-86), median Hb level 8.6 g/dL (5.4-11.7), and median spleen size 17.5 cm (11.4-36); 30% were RBC-TD, 66% intermediate-2, and 25% high-risk according to DIPSS; mutations (muts) in JAK2, CALR, or MPL were identified in 57%, 23%, and 20%, respectively; 55% had ≥1 high-molecular risk (HMR) mut, with ASXL1 being the most common (41%), followed by SRSF2 (24%), EZH2 (10%) and IDH2 (9%). Of note, pts in co2 were more frequently pre-treated with RUX compared to co1 (44% vs 15%; p=.006). Median treatment time at data cut-off was 12 cycles in co1 (2-98) and co2 (3-46); 3 pts of co2 have not yet reached EOC12. In co1, 8/39 pts (21%) achieved response at EOC12: partial remission (PR, n=1), clinical improvement (CI, n=6), or RBC-TI (n=1); 18/39 pts (46%) were treated for >12 cycles due to response (n=8), clinical benefit (CB, n=5) defined as Hb increase ≥1 g/dL or >50% improvement of ≥1 quality of life (QoL) symptom according to MPN-SAF, or stable disease (SD, n=5). Median Hb level at EOC12 was 9.3 g/dL; 15/39 pts (38%) were on treatment for >30 cycles, and 3 pts remained on long-term treatment (cycle 76, 97, and 98). In 73% and 40% of pts in co2, POM dose was increased to 1 mg and 2 mg QD after cycle 3 and 6, respectively. 5/49 pts (10%) showed response at EOC12 (CI, n=3 and RBC-TI, n=2), while 14 additional pts (29%) had CB; median Hb level at EOC12 was 8.6 g/dL; 22/49 pts (45%) were treated for >12 cycles; 3 pts were still on treatment (cycle 21, 36, and 46). Targeted NGS did not reveal a distinct mutational pattern associated with treatment response. Median overall survival (OS) of co1 and co2 was 3.6 and 2.6 years, respectively (p=.66). Among all, CALR, but not JAK2 or MPL muts were associated with better OS (p=.04), whereas HMR muts were in trend prognostically adverse (p=.057). Pts with more than 3 muts had a worse median OS (1.2 vs 4 years, p=.002). Combination therapy was well tolerated in both cohorts. Most common grade 1/2 adverse events (AE) were dyspnea (28%) and fatigue (23%), while the most frequent grade 3 AE was worsening of anemia (32%) in the first weeks of combination treatment not limiting therapy. Summary/Conclusion: Treatment with RUX and POM was safe and feasible in our study of advanced MF with an adverse genetic profile. Almost half of co1 and co2 pts were treated >12 cycles due to clinical benefit of the combination therapy with a subset of 38% of pts in co1 receiving treatment for >30 cycles. Dose increase of POM in co2 did not result into better anemia response or improvement of OS. Authors FS and EJ contributed equally. P1056: SCREENING OF JAK2 EXON 12 SOMATIC MUTATIONS BY HIGH-RESOLUTION MELTING CURVE ANALYSIS D. Kurochkin1,*, I. Maslyukova1, T. Subbotina1,2, A. Khazieva3, E. Dunaeva4, K. Mironov4 1Siberian Federal University; 2The Federal State-Financed Institution «Federal Siberian Research Clinical Centre under the Federal Medical Biological Agency»; 3Regional Clinical Hospital, Krasnoyarsk; 4Central Research Institute of Epidemiology of The Federal Service on Customers’ Rights Protection and Human Well-being Surveillance, Moscow, Russia Background: JAK2 exon 12 mutations are seen in about 2-5% of JAK2V617F-negative cases of polycythemia vera (PV). Nowadays about 40 different JAK2 exon 12 mutations associated with PV have been identified and classified. To identify all possible variants, it is necessary to use sequencing. However, due to the high cost of sequencing, developing a two-stage algorithm for detect mutations in JAK2 exon 12 using inexpensive screening is of immediate practically necessity. We have previously proposed a two-stage algorithm for detect mutations in JAK2 exon 12 using inexpensive screening test by heteroduplex analysis (Subbotina T et al, Haematologica 2017). Aims: The aim of this study was to demonstrate the feasibility of HRM analysis using the CFX96 thermocycler and the Precision Melt Analysis software (Bio-Rad, USA) as the preliminary screening test for detection of JAK2 exon 12 mutations. Methods: DNA samples of 5 JAK2 exon 12 mutation positive PV patients were included in this study. The identification of the JAK2 exon 12 mutation types and allele burden measurement was carried out by pyrosequencing (Subbotina T et al, Haematologica 2014). All 5 patients have different mutation variant in the 12 exon of the JAK2 and different levels of allelic burden: c.1624_1629delAATGAA – 67%; с.1619_1627TCAgAAATg>AAA – 14%; c.1623_1628delAAATGA – 15%; c.1622_1627delGAAATG – 33%; c.1612_1616CACAA>TT – 21%. HRM analysis was performed using a Precision Melt Supermix reagent kit in the presence of Eva Green dye (Bio-Rad, USA). PCR with an additional high-resolution melting step was carried out on a CFX96 thermocycler (Bio-Rad, USA) according to the following program: denaturation at 95°C for 2 minutes, then 40 cycles at 95°C for 10 seconds, 58°C in for 30 seconds. The high resolution melting program consisted of denaturation at 95°C for 30 seconds, renaturation at 60°C for 1 minute, and melting at 65°C to 95°C with a 0.2°C gradient in 10 seconds. Each DNA sample was analyzed in duplicate. For the two out five JAK2 exon 12 mutations a threshold determination of the mutant allele presence was analyzed. To analyze the threshold for determining the proportion of the mutant allele, dilution of cloned wild-type and mutated samples was performed to obtain samples with different levels of allelic burden: 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%. Results: Figure 1 shows the differential melting plots of the DNA fragments. The analyzed samples were divided into five clusters: first cluster – melting curves of wild type DNA; second cluster – melting curves of DNA from samples with deletion type mutations: c.1624_1629delAATGAA and c.1622_1627delGAAATG; the third cluster – the DNA melting curves from the sample with the combined mutation: c.1612_161616CACAA>TT; the fourth cluster – DNA melting curves from the sample with deletion type mutation: c.1623 _1628delAAATGA; fifth cluster – DNA melting curves from a sample with the combined mutation: c.1619_1627TCAGAAATG>AAA. The detection thresholds in case of the c.1624_1629delAATGAA and с.1619_1627TCAgAAATg>AAA mutation analysis are 6.25% of the presence of the mutant allele in the samples (data not shown). Image: Summary/Conclusion: Therefore, the HRM analysis that was conducted on the CFX96 allows to screen highly specific for the PV diagnosis mutations in exon 12 of the JAK2 gene. The inclusion of this screening research in the laboratory testing algorithm improves the efficiency and accessibility of molecular genetic technologies in the diagnosis of PV. P1057: REAL-WORLD UTILIZATION OF FEDRATINIB FOR MYELOFIBROSIS FOLLOWING RUXOLITINIB FAILURE C. Harrison1,*, J. Mascarenhas2, P. Abraham3, J. A. Nadal3, A. Balanean4, A. McBride3, J. K. Kish4, D. Liassou4, B. A. Feinberg4, A. T. Gerds5 1Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom; 2The Tisch Cancer Intitute, Icahn School of Medicine at Mount Sinai, New York; 3Bristol Myers Squibb, Princeton; 4Cardinal Health, Dublin; 5Cleveland Clinic Taussig Cancer Institute, Cleveland, United States of America Background: In August 2019, fedratinib (FEDR) became the second treatment, after ruxolitinib (RUX), to be approved for intermediate (Int)- or high-risk (HR) primary or secondary myelofibrosis (MF). Aims: To characterize real-world patient characteristics and treatment patterns for patients with MF receiving FEDR after RUX therapy in US clinical practices. Methods: Adults with Int- or HR MF initiating FEDR on or after August 16, 2019 (FEDR approval date) after discontinuing RUX were identified from community oncology practices. Eligible patients had ≥90 days of follow-up from FEDR initiation, completed ≥1 FEDR cycle, and had a spleen size assessment at FEDR initiation. Treating physicians completed electronic case report forms for the selected eligible patients, capturing patient characteristics at time of MF diagnosis and during RUX and FEDR therapy, including bone marrow fibrosis grade, biomarkers, risk score, performance status score, spleen size (cm above the costal margin), symptoms (per Modified Myelofibrosis Symptom Assessment Form version 4.0), and complete blood count data. Providers reported treatment dosing and modifications and dates/rationale for RUX treatment failure (defined a priori, including refractory or suboptimal response, disease progression [PD], or intolerance). Results: Among 150 eligible patients, the median age at diagnosis was 68 years; 55% were male, 68% were non-Hispanic White, 85% had primary MF, 55% were JAK2 V617F-positive, and 9% were JAK2/MPL/CALR-negative. RUX initiation dosage was ≥20 mg twice/daily (bid) for approximately 50% of patients. At RUX initiation, 65% had ECOG scores of 0/1, and 90% had palpable spleen (Table). A total of 19 patients (13%) had RUX dosage reductions due to neutropenia (7), thrombocytopenia (6), other adverse event (AE)/toxicity (3), patient request (2), and anemia (1); 4 patients (3%) had treatment interruptions due to thrombocytopenia (2), neutropenia (1), other AE/toxicity (1), and patient request (1); and 18 patients (12%) had dosage increases due to titration (13) and persistent MF symptoms (5). Primary reasons for RUX discontinuation were PD (70%), refractory or suboptimal response (25%), or intolerance (5%). RUX discontinuation due to PD was most often attributed to MF symptom return (65%), increased spleen size to >50% vs best achieved response (23%), and other reasons (12%). Median RUX duration was 4.0 months (IQR, 3.0–6.1) for refractory or intolerant patients and 11.7 months (IQR, 6.5–17.1) for those with PD. FEDR was initiated at 400 mg daily for 74% of patients. At FEDR initiation, 43% had International Prognostic Scoring System (IPSS)/Dynamic IPSS HR, 37% Int-2 risk, and 88% had palpable spleen; mean spleen size was 16.0 cm, and median platelet count was 98.0×109/L (Table). Symptoms reported at FEDR initiation included fatigue (72%), abdominal discomfort (51%), night sweats (44%), early satiety (25%), bone pain (18%), itching (13%), and pain under the left rib (13%). At the end of follow-up, 44 patients (29.3%) were deceased, and mean time to death among deceased patients was 6.5 (standard deviation, 2.7) months from initiation of FEDR. Image: Summary/Conclusion: This study showed a short duration of RUX therapy for real-world patients with MF, and poorer clinical status at FEDR initiation than at RUX initiation. Patients at risk of early failure of RUX should be considered for earlier FEDR interventional treatment. P1058: REAL-WORLD OUTCOMES WITH FEDRATINIB THERAPY IN PATIENTS WITH PRIMARY MYELOFIBROSIS POST-RUXOLITINIB DISCONTINUATION F. Passamonti1,*, Y. Lou2, M. Chevli3, P. Abraham4 1University of Insubria and ASST Sette Laghi, Ospedale di Circolo, Varese, Italy; 2Bristol Myers Squibb, Lawrenceville, United States of America; 3Bristol Myers Squibb, Uxbridge, United Kingdom; 4Bristol Myers Squibb, Princeton, United States of America Background: Dysregulation of the Janus kinase 2 (JAK2) hematopoiesis-signaling pathway in myelofibrosis (MF) disrupts bone marrow production of blood cells, causing anemia, fatigue, and splenomegaly. Dual JAK1/JAK2 inhibitor ruxolitinib (RUX) was the first drug approved in the USA for intermediate/high-risk MF, but many patients only have a partial response or develop cytopenia, ultimately discontinuing treatment. Fedratinib (FEDR), approved in August 2019, is active against wild-type and mutationally activated JAK2 and receptor tyrosine kinase FLT3, but its real-world effectiveness in patients discontinuing RUX has not been evaluated. Aims: To compare baseline characteristics and survival outcomes for patients with MF receiving FEDR vs those not receiving FEDR after discontinuing RUX in routine US clinical practice. Methods: Patients receiving RUX for primary MF were identified using Flatiron Health’s nationwide electronic health record-derived database. Eligible patients were those with ≥2 recorded visits in the database between Jan 1, 2011 and Oct 31, 2020, ≥18 years of age at the index date (start of FEDR therapy for patients receiving FEDR and last date of RUX therapy for those not receiving FEDR), with data ≥1 month before and after index, and no record of receiving unclassified clinical study drugs prior to index. Patients were stratified by treatment with FEDR post-RUX (FEDR and non-FEDR group); the non-FEDR group was further stratified by time of RUX discontinuation (before [subgroup A] and after [subgroup B] US approval of FEDR) to enable a contemporaneous comparison with the FEDR group. Demographics and clinical characteristics were assessed at index. Overall survival (OS) was defined as time from index until death or censoring and assessed by Kaplan–Meier analysis; landmark survival was defined as the proportion of patients who survived at a given point. Associations of baseline variables and survival were assessed by Cox proportional hazards model. Results: A total of 229 patients were evaluated (FEDR group: n=70; non-FEDR group: n=159). Median age at index was 71.0 for the FEDR group and 70.0 years for the non-FEDR group. Baseline demographic characteristics were broadly similar for the 2 groups. Median follow-up from index in the FEDR and non-FEDR group was 7.0 and 6.0 months, respectively. At index, 90.2% (46/51) and 74.3% (84/113) of patients had an ECOG performance status (PS) score of 0–1, respectively. Median duration of FEDR therapy in the FEDR group was 3.7 (range, 0–12.2) months, and 47.1% (33/70) received 400 mg FEDR once/day. Median OS was not reached in the FEDR group (vs 17 months in the non-FEDR group). Landmark survival in the FEDR and non-FEDR groups was 71.6% and 53.5% at 12 months, respectively. In the non-FEDR subgroup B, the corresponding survival rate at 12 months was 47.9%. The Cox proportional hazards model suggested that being male, of ‘other’ race (non-White, non-Black/African–American, non-Asian, non-missing), and having Charlson comorbidity index ≥1, ECOG PS 2–4, and body mass index <18.5 kg/m2 were significantly associated with poorer survival. Based on the risk-adjusted analysis, we witnessed an apparent trend toward increased OS with FEDR, but the observed differences did not achieve statistical significance (HR [95% CI]: 0.6 [0.3–1.2]; P=0.1466). Image: Summary/Conclusion: FEDR treatment of real-world patients with primary MF post-RUX may offer improved likelihood of survival rate up to 1 year after index compared with non-FEDR therapy. These differences in survival rate were sustained when the FEDR group and the non-FEDR B subgroup were compared. P1059: COMPARATIVE EFFICACY OF FEDRATINIB AND PACRITINIB FOR THE TREATMENT OF MYELOFIBROSIS IN PATIENTS WITH LOW PLATELET COUNTS: A SIMULATED TREATMENT COMPARISON STUDY G. Tremblay1,*, P. Daniele1, P. Abraham2, S. Rose2, A. McBride2 1Purple Squirrel Economics, Montreal, Canada; 2Bristol Myers Squibbs, Princeton, United States of America Background: Myelofibrosis (MF) is a Philadelphia chromosome-negative myeloproliferative neoplasm with an estimated prevalence of 4–6/100,000 persons in the United States (US). Among intermediate- to high-risk patients, Janus kinase 2 (JAK2) inhibitors are the primary treatment for MF. Fedratinib was approved by the US Food and Drug Administration (FDA) based on the results of the JAKARTA trials (NCT01437787; NCT01523171), and a new drug application for pacritinib was submitted to the FDA for the treatment of MF in patients with severe thrombocytopenia. Aims: To assess the comparative efficacy of fedratinib and pacritinib in patients with MF and thrombocytopenia (platelets < 100 × 109/L), in the absence of head-to-head clinical trials, an indirect treatment comparison (ITC) was conducted. Methods: According to the results of a systematic literature review, JAKARTA, JAKARTA2, and PERSIST-2 (NCT01773187) trials formed the basis of the ITC. A pooled analysis data set was developed using individual patient-level data from the fedratinib 400-mg arms of JAKARTA and JAKARTA2 including patients with platelets < 100 × 109/L. Published summary data from the pacritinib 200-mg arm of PERSIST-2 served as the comparator. Simulated treatment comparisons (STCs) were used to compare spleen volume reduction (SVR) ≥ 35% while adjusting for mutually reported baseline patient characteristics such as age, sex, Dynamic International Prognostic Scoring System status, Eastern Cooperative Oncology Group (ECOG) performance status (PS), JAK2 V617F mutation status, prior ruxolitinib exposure, and laboratory tests. Final adjustment models were selected based on fit statistics and the literature review. Indirect relative risk (RR) was estimated with 95% confidence intervals (CIs) using unanchored naive ITC (unadjusted) and STC (adjusted) methodologies. A naive ITC was also conducted for the subgroup of patients who received ruxolitinib in prior lines of therapy. In addition, a sensitivity analysis was conducted to evaluate the impact of differential median baseline platelet counts, which PERSIST-2 did not report. Using the full pooled population of the JAKARTA trials, outcomes were simulated at 3 median baseline platelet counts (25, 50, and 75 × 109/L) and compared using similar methodology to the main analysis. Results: The main analysis suggests that fedratinib is associated with a greater proportion of SVR ≥ 35% than pacritinib. According to the naive ITC, fedratinib was numerically favored over pacritinib in terms of SVR ≥ 35% (RR, 1.67 [95% CI, 0.94–2.97]). After the STC adjustment, fedratinib was statistically favored relative to pacritinib for SVR ≥ 35% (RR, 1.76 [95% CI, 1.00–3.10]). The naive ITC results were similar for patients who had received ruxolitinib in prior lines of therapy (RR, 2.82 [95% CI, 1.02–7.82]). Results of the platelet-count-adjusted sensitivity analysis showed a significant difference in favor of fedratinib with respect to SVR ≥ 35%. Differential baseline platelet count had minimal impact on the ITC results, with similar RRs regardless of simulated median baseline platelet count. Image: Summary/Conclusion: This analysis used a population-adjusted ITC to assess the comparative efficacy of pacritinib and fedratinib in patients with MF and thrombocytopenia. Following population adjustment, fedratinib was associated with a greater proportion of patients achieving SVR ≥ 35% than pacritinib. Further real-world evidence studies should be conducted to assess the effectiveness of these treatments in clinical settings. P1060: REAL-WORLD CLINICAL OUTCOMES AFTER 3 AND 6 MONTHS OF TREATMENT WITH FEDRATINIB FOLLOWING RUXOLITINIB FAILURE J. Mascarenhas1,*, A. T. Gerds2, J. K. Kish3, P. Abraham4, A. Balanean3, J. A. Nadal4, D. Liassou3, B. A. Feinberg3, A. McBride4, C. Harrison5 1The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York; 2Cleveland Clinic Taussig Cancer Institute, Cleveland; 3Cardinal Health, Dublin; 4Bristol Myers Squibb, Princeton, United States of America; 5Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom Background: Relief of symptoms and splenomegaly is an important treatment (Tx) goal for patients with myelofibrosis (MF). Fedratinib (FEDR) and ruxolitinib (RUX) are approved in the USA for Intermediate (Int)- or High-risk (HR) primary or secondary MF. Aims: To describe spleen size, and hematologic and MF-specific symptoms, during the first 6 months of FEDR therapy after prior Tx with RUX in US clinical practices. Methods: Adults with Int- or HR MF who initiated FEDR on or after August 16, 2019 (FEDR approval date) and with ≥90 days of follow-up were identified from community oncology practices. Eligible patients were those treated with RUX and then FEDR, who had spleen palpation at FEDR initiation, and who had completed ≥1 cycle of FEDR. Treating physicians completed electronic case report forms for the selected eligible patients, extracting patient characteristics, Tx pattern data, and clinical outcomes such as spleen size, MF-related symptoms per the Myelofibrosis Symptom Assessment Form version 4.0 (MFSAF v4.0), hemoglobin (Hb), and platelet and white blood cell (WBC) counts, at each visit in the first 6 months of FEDR Tx. Data were collected from February to March 2021. Mean spleen size, symptom count, Hb, and platelet and WBC counts at FEDR initiation vs at 3 and 6 months post-FEDR were compared via paired 2-sided t-test. Percentage reduction in spleen size and proportion of transfusion-dependent patients between FEDR initiation and 3 and 6 months post-FEDR were compared via 1-sided chi-square test. Only patients assessed at FEDR initiation (with palpable spleen or complete blood count available) and with a corresponding assessment at 3 or 6 months were included in the comparative analysis. Results: Among 150 eligible patients, the mean age at FEDR initiation was 68 years (range, 36–85). Median duration of RUX Tx prior to FEDR was 7.6 months (range, 0.7–65.5). At FEDR initiation, MF risk was categorized as International Prognostic Scoring System (IPSS)/Dynamic IPSS HR in 43% of patients, Int-2 in 37%, Int-1 in 9%, and unknown in 10%. A total of 74% of patients started FEDR at the recommended dosage of 400 mg daily. Median duration of post-FEDR follow-up was 5.1 months (IQR, 3.0–17.3) for patients initiated at FEDR 400 mg daily and 4.4 months (IQR, 3.0–14.3) for those initiating at <400 mg daily. At data cutoff, 55% of patients were still on FEDR. The primary reason for FEDR discontinuation was disease progression (43%). Mean duration of FEDR Tx was 4.7 months. Of 67 patients who discontinued FEDR, 44 were deceased at data cutoff. At FEDR initiation, 88% of patients had palpable spleen; mean spleen size was 16.0 cm (standard deviation [SD] 5.8) above the costal margin, which decreased to 13.2 cm (SD 7.9) at 3 months (P=0.0001) and 7.2 cm (SD 7.4) at 6 months (P=0.012). Mean number of MF-related symptoms declined significantly at 3 and 6 months; mean platelet count increased significantly at 6 months, from 160×109/L to 186×109/L (Table). Overall, 65.2% of patients experienced spleen size reduction, 18.8% had no change, and 16.0% had an increase. Mean best percentage change in spleen size was −29.2% (median −9.8%); complete resolution of palpable spleen occurred in 21% of patients. Image: Summary/Conclusion: This study illustrates the real-world effectiveness of FEDR after RUX failure in patients with Int- or HR MF. FEDR provided significant reductions in spleen size and reported number of MF-related symptoms after 3 months of Tx, including complete spleen responses. Greater benefits were observed with longer Tx duration. P1061: INDIRECT TREATMENT COMPARISONS OF FEDRATINIB VERSUS NAVITOCLAX PLUS RUXOLITINIB: EFFECT ON SPLEEN VOLUME AND SYMPTOMS IN RUXOLITINIB-EXPOSED MYELOFIBROSIS PATIENTS P. Abraham1,*, X. Liao2, M. Chevli3, S. Smith2 1Bristol Myers Squibb, Princeton, United States of America; 2BresMed Health Solutions Ltd, Sheffield; 3Celgene, a Bristol-Myers Squibb Company, Uxbridge, United Kingdom Background: Myelofibrosis (MF) is a life-threatening myeloproliferative neoplasm characterized by stem cell-derived clonal myeloproliferation, bone marrow fibrosis, anemia, and splenomegaly. The Janus kinase (JAK) pathway is the critical pathway in its pathogenesis. Ruxolitinib (RUX), a JAK1/2 inhibitor, was the first therapy for intermediate- and high-risk MF approved in the United States; however, a high unmet need remains for alternative treatment options for patients who discontinue or are no longer responding to RUX. The efficacy and safety of fedratinib (FEDR), a JAK2 inhibitor approved by the US Food and Drug Administration in 2019, was investigated post RUX in the single-arm JAKARTA2 trial (NCT01523171). Clinical data from a similar population treated with navitoclax plus RUX (NAV+RUX) have been reported. The efficacy of FEDR relative to NAV+RUX in patients with MF previously treated with RUX has not been evaluated. Aims: To explore the comparative efficacy of FEDR versus NAV+RUX in patients with MF previously treated with RUX for the binary endpoints of ≥35% spleen volume reduction (SVR) from baseline to the end of cycle 6 (EOC6; 24 weeks) and ≥50% reduction in total symptom score (TSS) from baseline to the EOC6. Methods: Evidence for FEDR was informed by JAKARTA2 patient-level data, and evidence for NAV+RUX was informed by known reported evidence from the REFINE study (NCT03222609). The suitability of these studies for indirect treatment comparison (ITC) was assessed by considering the comparability of study design, population, intervention, and outcomes. Given the lack of a common comparator in the identified studies, unanchored ITCs were performed for SVR using matching-adjusted indirect comparison (MAIC) and simulated treatment comparison (STC) methods. MAICs used effective sample size (ESS) of the FEDR cohort as an indicator of the amount of overlap. Univariable and multivariable regression models were used to identify potential prognostic factors to adjust for in the ITCs. Additionally, all Dynamic International Prognostic Scoring System (DIPSS)-Plus criteria reported were considered. Results: A subgroup of 58 JAKARTA2 patients with an Eastern Cooperative Oncology Group performance status (ECOG PS) score of 0 or 1 and intermediate-2 or high-risk disease most closely aligned with the REFINE population (N=34) was used in the analyses. Baseline mean platelet count was similar between subgroups. Across all analyses, results suggested FEDR consistently increased the odds/risk of a spleen response compared with NAV+RUX. The MAIC, matching on ECOG PS score, suggested that the odds of having an SVR for patients in the FEDR group was 2.19 times that of the NAV+RUX group (95% confidence interval [CI], 1.26 to 3.66), and the risk of having an SVR for patients in the FEDR group was 17.6% higher (95% CI, −2.1 to 37.0) (Figure). The results from the MAIC that additionally matched on all possible DIPSS-Plus criteria (age, hemoglobin, and platelet count) were consistent. Results from the 2 methods (MAIC and STC) were also consistent. For TSS reduction, the sample size (N=20) in REFINE was considered too small to perform a meaningful ITC; however, the absolute response rates for TSS reduction were similar across the 2 groups (29% [16/56] in the FEDR group and 30% [6/20] in the NAV+RUX group). Image: Summary/Conclusion: In patients with MF previously treated with RUX, these analyses suggest treatment with FEDR was associated with a greater proportion of patients achieving a spleen response compared with NAV+RUX. Limited data were available for comparison of TSS. P1062: A REAL-WORLD EVALUATION OF THE ASSOCIATION BETWEEN ELEVATED BLOOD COUNTS AND THROMBOTIC EVENTS IN POLYCYTHEMIA VERA: AN ANALYSIS OF DATA FROM THE REVEAL STUDY A. T. Gerds1,*, R. Mesa2, J. M. Burke3, M. R. Grunwald4, R. Scherber5, J. Yu5, J. Hamer-Maansson5, S. T. Oh6 1Cleveland Clinic Taussig Cancer Institute, Cleveland; 2UT Health San Antonio MD Anderson Cancer Center, San Antonio; 3Rocky Mountain Cancer Centers, Aurora; 4Levine Cancer Institute, Atrium Health, Charlotte; 5Incyte Corporation, Wilmington; 6Washington University School of Medicine, St. Louis, United States of America Background: Polycythemia vera (PV) is characterized by clonal hematopoiesis leading to elevated peripheral blood counts and an increased risk of thrombotic events (TEs). Advanced age and TE history form the conventional risk model used to determine TE risk/treatment strategy. Associations between TEs and elevated hematocrit (HCT) levels exist, but associations with white blood cell (WBC) or platelet (PLT) counts have not been assessed consistently. The large, real-world, p r ospective Obs e rvational Study of Pts with Polycythemia Ve ra in US Clinic al Practices (REVEAL; NCT02252159) followed pts with PV treated in community or academic centers. Aims: This analysis evaluated associations between elevated blood counts and TEs in pts with PV using data from REVEAL. Methods: Eligible pts had ≥3 lab values (blood counts) post-enrollment; pts with a post-enrollment TE but no lab value <6 mo before that TE were excluded. The association between blood counts and TEs was assessed using a time-dependent covariate Cox proportional hazards model. Time to first post-enrollment TE was modeled with time censored at last known visit for pts with no TE. Each lab parameter was modeled with sex, age, disease duration, and TE history at enrollment as baseline covariates and treatment as a time-dependent covariate. Blood counts were included as binary time–dependent covariates using the following thresholds: HCT >45%, WBC >11×109/L, PLT >400×109/L. Linear interpolation was used to determine lab values between observed lab values. Alternative thresholds for WBC (<7, ≥7 to <8.5; ≥8.5 to <11, and ≥11×109/L, and >12×109/L with HCT controlled at ≤45%) and PLT counts (>600×109/L) were evaluated. Statistical significance was considered at P<0.05. Results: 2271/2510 pts were eligible (median age, 66 y [range, 22–95]; male, 54.1%). Median disease duration was 4.1 y (range, 0–56.3), 456 (20.1%) had TE history; 52.6% of pts received hydroxyurea. Of 106 pts who had TEs, 30 had arterial TEs (most commonly, transient ischemic attack [n=15]) and 76 had venous TEs (most commonly, deep vein thrombosis [n=37]). Elevated HCT levels (>45%, hazard ratio [HR]=1.84 [95% CI, 1.234–2.749], P=0.0028), WBC (>11×109/L, HR=2.35 [1.598–3.465], P<0.0001), and PLT counts (>400×109/L, HR=1.60 [1.088–2.359], P=0.0170) were each associated with increased TE risk (Table 1). WBC count ≥11×109/L is associated with the highest TE risk compared with WBC count <7×109/L (HR=2.61 [95% CI, 1.594–4.262], P<0.0001). Elevated WBC >12×109/L was significantly associated with increased risk of TE with HCT controlled at ≤45%. PLT count (>600×109/L) increased TE risk (HR=1.37 [95% CI, 0.763–2.468]) compared with PLT count ≤600×109/L, but this was not statistically significant (P>0.05). In all models, advanced age, female sex, and TE history were associated with increased TE risk. Image: Summary/Conclusion: This analysis of REVEAL, the largest real-world cohort of PV pts to date, demonstrated that elevated HCT levels (>45%), WBC (>11×109/L), and PLT counts (>400×109/L) were associated with increased TE risk. An association of elevated WBC >12×109/L with increased risk of TE was also observed when HCT was controlled, indicating that TE risk may be reduced by controlling WBC as well as HCT. These data support the need to incorporate blood count into risk stratification and treatment strategies for pts with PV in clinical practice and to move beyond the conventional risk model. Further studies to understand the causal relationship between elevated blood counts and TEs are warranted. P1063: EFFICACY AND SAFETY OF PARSACLISIB-RUXOLITINIB COMBINATION THERAPY IN MYELOFIBROSIS PATIENTS WITH LOW VS HIGHER BASELINE PLATELET COUNT: A SUBGROUP ANALYSIS OF DATA FROM A PHASE 2 STUDY A. Yacoub1,*, U. Borate2, R. Rampal3, H. Ali4, E. Wang5, A. Gerds6, G. Hobbs7, M. Kremyanskaya8, E. Winton9, C. O’Connell10, S. Goel11, S. Oh12, G. Schiller13, A. Assad14, S. Erickson-Viitanen14, F. Zhou14, N. Daver15 1University of Kansas Cancer Center, Westwood; 2Oregon Health & Science University, Portland; 3Memorial Sloan Kettering Cancer Center, New York; 4City of Hope National Medical Center, Duarte; 5Roswell Park Comprehensive Cancer Center, Buffalo; 6Cleveland Clinic, Cleveland; 7Massachusetts General Hospital, Boston; 8Icahn School of Medicine at Mount Sinai, New York; 9Emory University, Atlanta; 10University of Southern California, Los Angeles; 11Montefiore Medical Center, Bronx; 12Washington University School of Medicine, St. Louis; 13David Geffen School of Medicine, University of California Los Angeles, Los Angeles; 14Incyte Corporation, Wilmington; 15University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Despite the demonstrated efficacy of ruxolitinib (a potent and selective JAK1 and JAK2 inhibitor), patients with myelofibrosis (MF) often exhibit inadequate or loss of response to chronic ruxolitinib therapy, possibly due to persistent PI3K pathway activation. Parsaclisib (INCB50465) is a potent and highly selective next generation PI3Kδ inhibitor. In the ongoing phase 2 INCB 50465-201 trial (NCT02718300), add on parsaclisib has shown preliminary efficacy in patients with MF who experienced a suboptimal response to ruxolitinib. JAK inhibitors, including ruxolitinib, are associated with thrombocytopenia; therefore, patients with low platelet count (PC) are commonly more difficult to treat. Aims: Here we present a subgroup analysis of efficacy and safety data from the ongoing INCB 50465-201 study by baseline PC. Methods: Eligible adults had primary or secondary MF with suboptimal response (palpable spleen >10 cm below left subcostal margin [LSM]; or palpable spleen 5–10 cm below LSM and active symptoms) after ≥6 months of ruxolitinib monotherapy (5–25 mg BID; stable dose ≥8 weeks). Patients remained on their last stable ruxolitinib dose and received add on parsaclisib (10 or 20 mg QD for 8 weeks; same dose QW thereafter) or parsaclisib (5 or 20 mg QD for 8 weeks; 5 mg QD thereafter). For this analysis, spleen volume (SV), total symptom score (TSS) assessed by Myelofibrosis-Symptoms Assessment Form (MFSAF) v3.0 daily diary, and safety, were evaluated by baseline PC (low PC, 50 to <100×109/L; higher PC, ≥100×109/L). Results: At data cutoff (August 27, 2020), 67 patients were enrolled (low PC, n=21; higher PC, n=46; median age 68 years). Median prior duration of ruxolitinib use was 34.7 months for low PC compared with 14.9 months for higher PC; baseline symptoms were worse for patients with low PC than higher PC (median [range] MFSAF-TSS, 21.4 [0.6–47] vs 10 [0–43]). Table 1 summarizes the responder analysis for SV reduction (SVR) based on baseline PC. At week 12, slightly more patients with low PC achieved ≥10% SVR compared with patients with higher PC (50.0% vs 39.4%), whereas, at week 24, responses were similar between the 2 groups (35.2% vs 37.1%). Of patients with ≥10% SVR at week 24, 4/6 with low PC and 9/13 with higher PC were on all daily dosing regimens. Median (range) percentage change in MFSAF-TSS was −20.5 (−56.6 to +17.1) for patients with lower PC compared with −22.2 (−100 to +500) for patients with higher PC at week 12; and −26.1 (−54.7 to +2.4) compared to −23.1 (−91.3 to +222.5) at week 24, respectively. In both subgroups, nonhematologic treatment-emergent adverse events (TEAEs) were mostly grade 1 or 2. Most common (≥20%) TEAEs were dyspnea (33%), falls (33%), peripheral edema (29%), and nasal congestion (24%) for low PC; diarrhea (28%), nausea (24%), abdominal pain (24%), cough (20%), and fatigue (20%) for higher PC. For patients with low PC compared to higher PC, 9/21 (43%) compared to 3/46 (7%) patients had parsaclisib dose interruption due to thrombocytopenia. One patient with low PC had ruxolitinib interruption due to thrombocytopenia. Image: Summary/Conclusion: Add-on parsaclisib showed efficacy in patients from both low and higher baseline PC groups. Given the acceptable safety profile and efficacy of add-on parsaclisib, MF patients with both low and higher PC may be able to benefit from parsaclisib-ruxolitinib combination therapy. Phase 3 trials in ruxolitinib-treated and ruxolitinib-naive patients are underway to further assess the combination of JAK and PI3K inhibitors. P1064: CIRCULATING CYTOKINE LEVELS IN PATIENTS WITH PHILADELPHIA-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS – PROFILE AND CLINICAL CORRELATIONS – A SINGLE CENTER STUDY G. Tsvetkova1,*, S. Mihailova2, N. Ivanov2, M. Ivanova2, E. Bekirova1, G. Balatzenko3, A. Vlahova4, E. Hadjiev1 1Medical University - Sofia, Faculty of Medicine, Department of Internal diseases, Clinic of clinical hematology; 2Medical University - Sofia, Faculty of Medicine, Department of Clinical Immunology, University Hospital “Alexandrovska”-Sofia; 3Laboratory of Cytogenetics and Molecular Biology, National Specialized Hospital for Active Treatment of Hematological Diseases; 4Medical University - Sofia, Faculty of Medicine, Department of General and Clinical Pathology - Sofia, University Hospital “Alexandrovska”-Sofia, Sofia, Bulgaria Background: Myeloproliferative neoplasms(MPNs) are a group of disorders with heterogeneous features and often progressive clinical course. Over the past few years a remarkable advance has been made in the knowledge on the pathogenesis and therapeutic management of the Philadelphia(Ph)-negative MPNs. Despite the wide variety of studies in this field, there are still unsolved issues regarding their biology, disease course, prognosis and treatment. The improved recognition of molecular markers is not sufficient for a comprehensive explanation of the variable clinical symptoms and complications among these patients. The role of the chronic inflammation has been widely considered as a co-factor, involved in the pathogenesis and progression of MPNs. Aims: To analyze the serum levels of a panel of cytokines, associated with the mechanisms of chronic inflammation, among patients with Ph-negative MPNs and healthy controls and the possible correlations of these cytokines with the disease’s features. Methods: One hundred and fifty adults were included in the study, distributed in comparable groups, as follows: 36 patients with Essential thrombocytemia (ET), 49 patients with Polycythemia vera (PV), 30 patients with Myelofibrosis (MF) and 35 age-matched healthy controls. The patients with MPNs were diagnosed according to the latest WHO criteria. The cytokines’ levels were tested in serum samples with a specific kit Cytokine Human Panel for Luminex™. Assessments of the symptoms burden were performed with MPN-SAF- questionnaire. Results: Among the 11 tested cytokines we detected significantly higher serum levels of Interleukin-2, Interleukin-5, Interleukin-6, Interleukin-8, Interleukin-18 and GM-CSF among the MF patients (p<0.05), in comparison with the healthy controls, as well as significantly higher levels of Interleukin-6 in the MF-group, compared to the PV-patients. Elevated serum Interleukin-6, Interleukin-8, Interleukin-18 and GM-CSF were observed in the ET-group versus the control group levels (p<0.05). The cytokine profile analysis also revealed higher serum levels of Interleukin-2, Interleukin-5, Interleukin-6 and Interleukin-18 in the patients with PV, than in the healthy subjects. In terms of estimation of the clinical impact of the circulating inflammatory cytokines among the analyzed MPN cohort - for the ET-patients a positive correlation was present between GM-CSF and the fatigue severity and between Interleukin-6 levels and the weight loss severity index, both assessed according to the MPN-SAF score. A positive correlation was detected between Interleukin-10 levels and the bone pain burden and the degree of weight loss for the patients with MF. Interleukin-18 values demonstrated a positive correlation with the weight loss among the patients with PV. Summary/Conclusion: Our single center study results can give a ground to assume the potential role of the chronic inflammation and the pathogenetic impact of the circulating inflammatory cytokines on the MPNs clinical features and course. The cytokine profile of the different subgroups of MPNs is a topic of interest and can be monitored as a potential predictor of disease progression. It can motivate future research attempts, aiming to alter the clinical evolution of MPNs by affecting the pathogenetic mechanisms of the inflammatory process. It can create prerequisites for future studies regarding more effective therapeutic targets, related to both – the molecular pathways of the mutations in MPNs and the molecular mechanisms of the chronic inflammation. P1065: CONGENITAL ERYTHROCYTOSIS DUE TO HETEROZYGOUS VARIANTS IN THE BISPHOSPHOGLYCERATE MUTASE GENE M. J. van Dijk1,*, B. A. van Oirschot1, M. C. Stam-Slob1, B. van der Zwaag1, E. J. van Beers1, J. J. Jans1, P. van der Linden2, J. M. Torregrosa Diaz3, B. Gardie4,5,6, F. Girodon6,7,8, R. Schots9, N. Thielen10, R. van Wijk1 1University Medical Center Utrecht, Utrecht; 2Spaarne Gasthuis, Haarlem, Netherlands; 3University Hospital of Poitiers, Poitiers; 4L’institut du Thorax, Inserm, CNRS, University of Nantes, Nantes; 5Ecole Pratique des Hautes Etudes (EPHE), Université Paris Sciences et Lettres; 6Laboratory of Excellence GR-Ex, Paris; 7Service d’Hématologie Biologique, Pôle Biologie, Centre Hospitalier Universitaire (CHU) de Dijon; 8INSERM U1231, Université de Bourgogne, Dijon, France; 9Universitair Ziekenhuis Brussel, Brussels, Belgium; 10Diakonessenhuis, Utrecht, Netherlands Background: Congenital erythrocytosis is an inherited condition characterized by increased red blood cell mass, reflected by persistently elevated hemoglobin (Hb) and hematocrit levels. Erythrocytosis can be primary due to an intrinsic defect of erythropoiesis, or secondary, including germline variants in genes involved in the oxygen-sensing pathway and Hb variants with altered oxygen affinity. A very rare cause involves variants in bisphosphoglyerate mutase (BPGM), which regulates the red blood cell concentration of 2,3-bisphosphoglycerate (2,3-BPG), an important modifier of Hb-oxygen affinity. Aims: We report three European families with erythrocytosis and partial BPGM deficiency due to heterozygosity for variants in the BPGM gene, including one novel variant. Methods: The study was performed on adults of three families with JAK2-negative erythrocytosis. DNA analysis concerned sequencing of relevant coding exons, including flanking splice-site consensus sequences, of the eight major genes involved in secondary congenital erythrocytosis (EPOR, HBB, HBA1, HBA2, VHL, EPAS1, ELGN1, and BPGM). Identified variants were evaluated using Ensembl, gnomAD, PolyPhen-2, and SIFT. BPGM enzyme activity was determined as described by Beutler. Quantitative analysis of 2,3-BPG was performed using liquid chromatography tandem mass spectrometry. Hb-oxygen affinity, reflected by p50 levels (the oxygen pressure when Hb is 50% saturated with oxygen), was analyzed using the Hemox Analyzer (TCS Scientific). Results: In the first family (Table 1), a novel missense variant (c.535C>T p.(Arg179Cys) was identified in BPGM. Both the 65-year-old mother with complaints of headaches, and her 39-year-old son with a medical history of retinal vein occlusion were heterozygous for this variant. Allele frequency in gnomAD was 3.98-5. The predicted effect ranged from either deleterious (SIFT) to benign (HumVar model of Polyphen-2). Subsequent functional analysis showed low-normal BPGM activity, whereas 2,3-BPG and p50 levels were decreased (Table 1). In the second and third families, three male individuals were heterozygous for the c.269G>A p.(Arg90His) missense variant in BPGM. This variant was only once previously reported, and has an allele frequency of 1.06-5. All affected subjects had mild erythrocytosis without any additional clinical features, and showed decreased BPGM activity, decreased 2,3-BPG levels and decreased p50 (Table 1). Image: Summary/Conclusion: We describe five individuals from three families in which heterozygosity for missense variants in BPGM is associated with congenital erythrocytosis. Our findings further strengthen the hypothesis of an autosomal dominant inheritance pattern of BPGM variants. Our data also demonstrate the added value of functional analyses to validate gene panel and in silico analysis results for their clinical relevance. Future cases may help to interpret the impact of BPGM variants on hematological and clinical phenotype. P1066: OUTCOMES OF COVID-19 IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS - AN OBSERVATIONAL COHORT STUDY FROM A LARGE ACADEMIC CANCER CENTER IN THE UNITED STATES S. Venugopal1,*, H. Song2, C. Young2, M. A. Cutherell2, J. A. Baganz2, S. Zatorsky2, S. E. Woodman3, N. Pemmaraju1, P. Bose1, L. Masarova1, L. Zhou1, S. Pierce1, H. Kantarjian1, S. Verstovsek1 1Leukemia; 2Data Engineering-Analytics; 3Genomic Medicine, The University of Texas, MD Anderson Cancer Center, Houston, United States of America Background: Infections are one of the main causes of morbidity and mortality in patients with Myeloproliferative Neoplasms (MPN). Several studies from all over the world has reported on outcomes of the SARS-CoV-2 infection (COVID-19) pandemic in MPN pts but there is no robust data on characterizing the clinical outcomes of patients with MPN and COVID-19 in the United States (US). Aims: Therefore, we aimed to investigate the effect of COVID-19 on MPN patients’ clinical outcomes including the effect of MPN-subtypes, and SARS-CoV-2 vaccination on COVID-19 infection. Methods: We conducted a single center, retrospective, observational cohort study. Between April 2020 – December 2021, data was obtained from 388 patients flagged in the electronic health records system with MPN and COVID-19, of which 53 MPN patients with positive SARS-CoV-2 test were identified. COVID-19 infection was ascertained by a positive real-time reverse transcriptase polymerase chain reaction from nasal swab. Presenting clinical information was abstracted from review of unstructured notes as well as structured data from the foundry. Demographic and baseline parameters were summarized. The Kaplan-Meier method was utilized to compare the median time from date of diagnosis to date of last follow up or death. Results: Among 53 patients with MPN and COVID-19 infection, the median age was 58 yrs (range,21-83) with equal distribution between men and women of which 46% had a body mass index ≥30. Most common comorbidities included hypertension (60%), and diabetes (19%). 17 (33%) patients required hospitalization of which all (33%) of them required oxygen supplementation. 12 pts (23%) had radiological evidence of pneumonia of which 4 pts (8%) required high flow oxygen and 3 pts (6%) required mechanical ventilation. 2 pts had new thrombosis of portal vein and coronary artery each. 10 pts (19%) received remdesivir and 4 (8%) received tocilizumab, and 9 pts (17%) received steroids. At a median follow up of 11.8 months, 7 pts died at a median of 24 days after COVID-19 diagnosis and the case fatality rate was 13%. Except for 1 pt,all the other 6 pts who died had BMI≥30. In the US, COVID-19 predominantly occurred in pts with myelofibrosis (56%), polycythemia vera (29%), and essential thrombocythemia (15%). 54% received MPN directed therapy of which 17 (33%) pts received ruxolitinib, and 11 pts (21%) received hydroxyurea. At the time of infection, median WBC was 6.2 x 109/µL (range,0.3-16.3),Hb was 11.6 gm/dL (range,6.8-15.8),platelet count was 178 x 109/µL (range,2-766), and the median neutrophil to lymphocyte ratio was 4.1. In this cohort, 22 pts (42%) were infected prior to the availability of vaccination. 42% of the entire cohort has received vaccination of which 45% pts were infected after first dose of vaccination. Among the 7 deaths, 6 patients had underlying myelofibrosis, and one pt had polycythemia vera, and all deaths occurred in unvaccinated population except for one death that occurred post vaccination. Hospitalization rate was higher in the unvaccinated (33%) pts than those who were vaccinated (22%). Summary/Conclusion: To date this is the largest cohort of MPN patients with COVID-19 infection in the US accounting for 14% of the MPN patients with COVID-19 in our center. BMI≥30 appear to be an important risk factor for COVID-19 infection and mortality in MPN pts. SARSCOV2-vaccination appear to decrease the risk of mortality and hospitalization. Patterns of inflammatory markers, and long term follow up of recovered MPN pts with COVID-19 will be presented in the meeting. P1067: PHASE 2 STUDY OF ORAL THALIDOMIDE-CYCLOPHOSPHAMIDE-DEXAMETHASONE FOR RECURRENT/REFRACTORY ADULT LANGERHANS CELL HISTIOCYTOSIS J.-N. Wang1,*, T. Liu1, A.-L. Zhao1, B.-J. Pan2, J. Sun2, J. Li1, D.-B. Zhou1, M.-H. Duan1, X.-X. Cao1 1Department of Hematology; 2Department of Pathology, Peking Union Medical College Hospital, Beijing, China Background: Langerhans cell histiocytosis (LCH) is a clonal histiocytic neoplasm with various clinical manifestations and heterogeneous prognoses. No standard therapy is currently available for adults with recurrent/refractory LCH. Aims: To determine the efficacy and safety of oral thalidomide combined with cyclophosphamide and dexamethasone (TCD) regimens in the treatment of recurrent/refractory LCH among adult patients. Methods: This single-center, single-arm, phase 2 study enrolled 32 recurrent/refractory LCH patients between October 2019 and August 2021. The final follow-up date was October 31st, 2021.The TCD regimen (thalidomide 100 mg daily, cyclophosphamide 300 mg/m2 Day 1, 8, 15, and dexamethasone 40 mg Day 1, 8, 15, 22 every 4 weeks) was administered for 12 cycles and then thalidomide alone as maintenance for 12 months. The primary endpoint was event-free survival (EFS). Events were defined as progression during or after TCD therapy or death from any cause. Results: The median number of prior lines of systemic treatment was 1 (range 1-4), and all patients received a cytarabine-based regimen as first-line or second-line therapy. After a median follow-up of 22 months (range 5-24 months), no patient died of all causes. The overall response rate was 87.5%, including 18 patients (56.3%) achieving complete remission and 10 patients (31.3%) as partial remission. The estimated 24-month EFS was 64.0%. Fourteen patients (43.7%) had risk organ involvement. Patients with risk organ involvement had a similar EFS compared to patients without risk organ involvement (P=0.38). The common toxicities of the TCD regimen include grade 1-2 constipation (12.5%), grade 1-2 tiredness (9.4%) and grade 2 peripheral neuropathy (12.5%). Summary/Conclusion: Oral thalidomide, cyclophosphamide and dexamethasone are effective and safe regimen for recurrent/refractory LCH patients, particularly for patients with risk organ involvement. P1068: RISK-ADJUSTED SAFETY ANALYSIS OF PACRITINIB IN PATIENTS WITH MYELOFIBROSIS A. Vannucchi1, N. Pemmaraju2,*, B. Scott3, M. Savona4, S. Oh5, F. Palandri6, H. K. Al-Ali7, M. Sobas8, M. F. McMullin9, V. Gupta10, A. Yacoub11, R. Mesa12, S. Buckley13, K. Roman-Torres13, S. Verstovsek2, C. Harrison14 1Azienda Ospedaliera Universitaria Careggi, University of Florence, Florence, Italy; 2The University of Texas MD Anderson Cancer Center, Houston, TX; 3Fred Hutchinson Cancer Research Center, Seattle, WA; 4Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN; 5Washington University School of Medicine, St. Louis, MO, United States of America; 6IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy; 7Krukenberg Cancer Center, University Hospital Halle, Halle, Germany; 8Wroclaw Medical University, Wroclaw, Poland; 9Queen’s University Belfast, Belfast, United Kingdom; 10Princess Margaret Cancer Centre, Toronto, ON, Canada; 11The University of Kansas Cancer Center, Kansas City, KS; 12UT Health San Antonio Cancer Center, San Antonio, TX; 13CTI BioPharma, Seattle, WA, United States of America; 14Guy’s and St Thomas’ NHS Trust, London, United Kingdom Background: Pacritinib is a novel JAK2/IRAK1 inhibitor that has shown clinically significant activity in patients with myelofibrosis, including those with platelet counts <50 x 109/L. Recently, JAK inhibitors have come under increased scrutiny due to specific, emerging toxicities with drugs in this class. Aims: This safety analysis focuses on these toxicities of interest for patients treated with pacritinib 200 mg twice daily (BID) and best available therapy (BAT), including ruxolitinib, on the phase 3 PERSIST-2 and phase 2 PAC203 studies. Data are presented as risk-adjusted incidences to account for differential time at risk for adverse events (AEs) between arms due to cross-over. Methods: Patients treated with pacritinib 200 mg BID on PERSIST-2 and PAC203, and those treated with BAT, including ruxolitinib on PERSIST-2, were included. Risk-adjusted AEs, representing event rate per 100 patient-years, were calculated for the following: overall and fatal AEs, bleeding AEs (determined by Standardized Medical Dictionary for Regulatory Activities Query [SMQ]), cardiac AEs (by SMQ), major cardiac events (determined by major adverse cardiovascular events [MACE] classification), infections, thromboses, and secondary malignancies. Results: A total of 160 patients were analyzed as the pooled pacritinib 200 mg BID group (n=106 in PERSIST-2; n=54 in PAC203) and 98 patients in the BAT group (44 on ruxolitinib). At baseline, the median platelet count was 57 x 109/L for the pooled pacritinib group and BAT group; hemoglobin was 9.2 vs 9.7 g/dL and 66% vs 53% had prior JAK2 inhibitor therapy respectively. The rate of AEs was higher on pacritinib versus BAT, while the rate of fatal AEs was lower (Table 1). Both bleeding and cardiac events occurred at slightly lower rates on pacritinib compared to BAT. There were no MACE for pacritinib, whereas there were for BAT, including in patients treated with ruxolitinib. Malignant neoplasms (excluding worsening myelofibrosis and leukemia) occurred at similar rates on pacritinib and BAT, though rate of non-melanoma skin cancers was lower in the pooled pacritinib group (3/100 patient-years) versus BAT (7/100 patient-years), including ruxolitinib (11/100 patient-years). Infection occurred more frequently on pacritinib versus BAT, though fungal and viral infections occurred less frequently, as did Herpes Zoster reactivation (pooled pacritinib: 0/100 patient-years vs BAT: 2.4/100 patient-years, including ruxolitinib: 5.5/100 patient-years). Thrombosis occurred at similar rates on pacritinib and BAT. Image: Summary/Conclusion: Risk-adjusted analysis demonstrates that the safety profile of pacritinib 200 mg BID is comparable or superior to BAT, including ruxolitinib. Pacritinib 200 mg BID may represent a full-dose therapeutic option for patients with myelofibrosis, including those with thrombocytopenia. P1069: RETROSPECTIVE COMPARISON OF PATIENT OUTCOMES ON PACRITINIB VERSUS RUXOLITINIB IN PATIENTS WITH MYELOFIBROSIS AND THROMBOCYTOPENIA C. Harrison1,*, P. Bose2, R. Mesa3, A. Gerds4, S. Oh5, J.-J. Kiladjian6, V. García-Gutierrez7, A. Vannucchi8, C. Scheid9, M. Sobas10, S. Verstovsek2, S. Buckley11, K. Roman-Torres11, J. Mascarenhas12 1Guy’s and St Thomas’ NHS Trust, London, United Kingdom; 2The University of Texas MD Anderson Cancer Center, Houston, TX; 3UT Health San Antonio Cancer Center, San Antonio, TX; 4Cleveland Clinic Taussig Cancer Institute, Cleveland, OH; 5Washington University School of Medicine, St. Louis, MO, United States of America; 6Hôpital Saint-Louis, Université de Paris, Paris, France; 7Hospital Universitario Ramón y Cajal, Madrid, Spain; 8Azienda Ospedaliera Universitaria Careggi, University of Florence, Florence, Italy; 9University of Cologne, Cologne, Germany; 10Wroclaw Medical University, Wroclaw, Poland; 11CTI BioPharma, Seattle, WA; 12Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America Background: Pacritinib is a JAK2/IRAK1 inhibitor for patients with myelofibrosis and thrombocytopenia. Unlike the JAK1/2 inhibitor ruxolitinib, which is used at lower doses in patients with thrombocytopenia, pacritinib has been studied at full dose regardless of platelet count. Aims: We retrospectively analyzed the phase 3 PERSIST-2 study, comparing pacritinib with ruxolitinib, with a focus on disease symptomatology. Methods: PERSIST-2 enrolled patients with platelet counts ≤100 x 109/L and randomized them 1:1:1 to pacritinib 200 mg twice daily (BID), pacritinib 400 mg once daily (QD), or best available therapy (BAT). This analysis focused on the 200-mg BID dose and patients who received ruxolitinib prior to week 24. Endpoints included the percentage of patients with ≥35% spleen volume reduction (SVR), ≥50% modified total symptom score (mTSS) reduction, and improvement in disease symptoms measured by Patient Global Impression of Change (PGIC). Safety analyses were based on all treated patients; efficacy analyses were based on the intention-to-treat population randomized at least 22 weeks prior to study end. The Fisher Exact test was used to describe differences in response. Logistic regression was used to adjust for differences in baseline characteristics. Results: Safety analysis included 106 patients on pacritinib and 44 on ruxolitinib; efficacy analysis included 74 on pacritinib and 32 on ruxolitinib (median daily dose of 10 mg). Baseline characteristics were similar between the groups, including median platelet count (55 vs 61 x 109/L) and percentage receiving RBC transfusion (46% vs 43%). The following differences in baseline characteristics were accounted for in multivariable modeling: percentage of patients with grade 3 fibrosis (58% vs 36%), primary myelofibrosis (77% vs 50%), ≥1% peripheral blasts (45% vs 61%), and prior JAK2 inhibitor (48% vs 73%). Patients treated with pacritinib vs ruxolitinib achieved higher rates of SVR (22% vs 3%, p=0.02) and numerically higher rates of mTSS response (35% vs 19%; p=0.11) at week 24 (Figure 1A). A greater percentage of patients on pacritinib reported “much” or “very much” improved symptoms (35% vs 16%, p=0.06; Figure 1A). Among ruxolitinib-treated patients with an available PGIC measure at week 24, 50% reported either no improvement or worsening symptoms, while 76% of pacritinib-treated patients reported improvement (Figure 1B). After adjusting for imbalances in baseline characteristics, there was no diminution of treatment effect on SVR or mTSS, and the hazard ratio for survival on pacritinib vs ruxolitinib was 0.46 [95% CI: 0.15-1.43]. Fatal adverse events occurred at slightly lower rates on pacritinib vs ruxolitinib (8% vs 11%). Bleeding occurred at similar rates on pacritinib and ruxolitinib (43% vs 41%). There were low rates of herpes zoster reactivation (n=0 vs 1), fungal skin infection (n=0 vs 1), pulmonary aspergillosis (n=1 vs 0), deep venous thrombosis (n=0 vs 1), and pulmonary embolism (n=1 vs 0) on pacritinib and ruxolitinib, respectively. Image: Summary/Conclusion: Pacritinib yielded higher response rates and a similar safety profile to lower-dose ruxolitinib in patients with myelofibrosis who have moderate or severe thrombocytopenia. P1070: NAVITOCLAX MONOTHERAPY IN PATIENTS WITH MYELOFIBROSIS PREVIOUSLY TREATED WITH JAK-2 INHIBITORS: SAFETY AND TOLERABILITY V. Pullarkat1, A. Cruz-Chacon2, S. Gangatharan3, A. Melnyk4, G. A. Palumbo5, M. Bellini6, S. K. Tantravahi7, Q. Qin8, J. Potluri8, P. Vachhani9,* 1Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, CA, United States of America; 2Hospital Centro Comprensivo de Cancer Universidad de Puerto Rico, San Juan, Puerto Rico; 3Department of Haematology, Fiona Stanley Hospital, University of Western Australia, Perth, Western Australia, Australia; 4Texas Oncology-Abilene, Abilene, TX, United States of America; 5Dipartimento di Science Mediche Chirurgiche e Tecnologie Avanzate “GF Ingrassia”, Università degli Studi di Catania, Catania; 6ASST Papa Giovanni XXIII, Bergamo, Italy; 7University of Utah and Huntsman Cancer Institute, Salt Lake City, UT; 8AbbVie Inc., North Chicago, IL; 9O’Neal Comprehensive Cancer Center at UAB, Birmingham, AL, United States of America Background: Navitoclax is an oral, small-molecule inhibitor of anti-apoptotic B-cell lymphoma 2 family proteins (BCL-XL, BCL-2, BCL-W) that is being evaluated in the ongoing, multicenter, multi-cohort, phase 2 trial (REFINE; NCT03222609). Previous results from REFINE Cohort 1a indicated that addition of navitoclax to ruxolitinib resulted in clinically meaningful outcomes with acceptable safety profile in patients with myelofibrosis (MF) with progression or suboptimal response to ruxolitinib monotherapy (Pemmaraju et al. J Clin Oncol. 2022). Aims: To report preliminary safety results of navitoclax monotherapy (Cohort 2) in patients with suboptimal response to prior Janus kinase inhibitors (JAKis). Methods: This phase 2, open-label trial in patients with MF enrolled patients into 4 cohorts according to JAKi experience; all patients provided informed consent. Patients in Cohort 2 had discontinued prior JAKi therapy and received navitoclax monotherapy orally at the starting dose of 100 mg/day (QD) or 200 mg QD if baseline platelet count was 75–150 × 109/L or >150 × 109/L, respectively. Eligible patients had previously received JAKi for ≥12 weeks, or ≥28 days with red blood cell transfusion dependence (≥2 units/month for 2 months) or with ≥ grade 3 adverse event (AE) of thrombocytopenia or anemia, while on JAKi; all patients had splenomegaly. The primary endpoint was spleen volume reduction of ≥35% (SVR35) from baseline (BL) assessed centrally at Week 24. Secondary endpoints included change in grade of bone marrow fibrosis according to the European consensus grading system and anemia response as per International Working Group criteria. Exploratory endpoints included duration of response of SVR35 and overall survival. Safety and AEs were monitored throughout the study. Results: As of Oct 4, 2021, 30 patients were enrolled and received navitoclax. A majority of patients were male (63%), median age was 68 years (range 55–84), with a median prior ruxolitinib exposure of 100 weeks (range 11–496). Fifteen patients (50%) had secondary MF, of which 8 patients (27%) had post–polycythemia vera MF and 7 patients (23%) had post-essential thrombocytopenia MF (Table). Fourteen patients (47%) started navitoclax at a dose of 100 mg QD and 16 patients (53%) started at 200 mg QD. The median follow-up time was 4.2 months (range 0.2–15.5). Median duration of navitoclax exposure was 9.4 weeks (range 0.1–67.1). Twenty-seven patients (90%) experienced ≥1 AE, the most common being: thrombocytopenia (n=16; 53%), diarrhoea (n=9; 30%), and nausea (n=8; 27%). Grade ≥3 AEs were experienced by 63% of patients, with thrombocytopenia (n=11; 37%) and anemia (n=7; 23%) being the most common. Three patients had serious AEs of dyspnea, hypoxia, and pulmonary hypertension. Navitoclax dose reductions and interruptions were experienced by 15 patients (50%) and 16 patients (53%), respectively. Three patients experienced AEs leading to navitoclax discontinuation (pulmonary hypertension, increased bilirubin, and low platelets; n=1 each). Eight patients discontinued navitoclax due to: AE (n=3), consent withdrawal (n=1), physician decision (n=1), disease relapse (n=1), and progressive disease (n=2). Two patients died >30 days after the last dose of navitoclax. Details will be included in the presentation. Image: Summary/Conclusion: Navitoclax monotherapy in patients with MF after prior JAKi had a similar safety profile as previously reported in Cohort 1a. Efficacy analyses are underway to evaluate the activity of navitoclax monotherapy in patients with MF and will be available for the presentation. P1071: IMPACT OF ACETYLSALICYLIC ACID DOSAGE ON INCIDENCE OF THROMBOSIS AND MORTALITY IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS. A. Wild1,2,*, S. Wimmerová3 1Haematology department, Teaching F.D.Roosevelt Hospital, Banská Bystrica; 2Medical faculty; 3Public Health Faculty, Slovak Medical University, Bratislava, Slovakia Background: Bcr/abl – negative myeloproliferative neoplasms (MPN) are associated with a higher risk of thrombosis and higher vascular mortality. Acetylsalicylic acid (ASA) used in prevention decreases risk of vascular events in patients with MPN. There is some evidence that ASA in dose 100 mg once daily results in incomplete inhibition of a platelet function over the whole 24-hour interval in most patients with essential thrombocythemia (ET). High on-treatment reactivity (HOTR) was corrected with dosing ASA twice daily. Evidence of clinical consequences of these findings has been missing. Aims: Authors compared thrombosis incidence, mortality and safety among cohorts of MPN patients with adequate on-treatment reactivity (AOTR) on ASA dosed once daily, twice daily and patients with HOTR. Methods: Response to ASA in patients with MPN was evaluated by a platelet light transmission aggregometry induced by arachidonic acid. AOTR was defined as maximal amplitude up to 20% just before the next dose of ASA. In patients with HOTR, ASA dosing was changed to twice daily with a dose from 50 mg step by step up to 150 mg till AOTR was reached. A few patients didn´t change their doses at their own decision despite HOTR. The patients were regularly followed-up. Incidence of thrombosis, adverse events and mortality were compared among the cohorts. Results: The platelet aggregation was tested in 69 patients with MPN on ASA. Patients with ET, polycythemia vera (PV), primary myelofibrosis, post-PV plus post-ET myelofibrosis and MPN not otherwise specified presented 16, 42, 36, 5 and 1%, respectively. The adequate responses (AOTR) were found in 57 (83%), cohort 1. The insufficient responses were found in 12 persons (17%), in 30% of patients on an enterosolvent form of ASA. In these 12 patients the dose of ASA was changed to 2x50mg in 4 and to 2x100mg in 1 patient (cohort 2) and remained unchanged in seven (cohort 3). There were no differences in frequency of risk factors besides higher frequency of diabetes mellitus in group 3 compared to group 1 (p=0,021). Non-adherence to regular administration of ASA was found in 10% of patients. The follow-up was 36,8 months. All-cause mortality was higher in the cohort 3 compared to the cohort 1 (p=0,009), see fig. 1, but thrombotic episodes incidence not. The cohort 2 had not different incidence of both thrombotic episodes and deaths compared to the cohort 1. No proximal gastrointestinal bleeding and other gastropathies occurred. There was no bleeding episode on ASA in dose of 2x50mg daily. A non-severe bleeding dependant on any ASA dose were found. Repeated measurement of platelet aggregation was done in 29 patients in median 49,5 months after previous measurements. There was no statistically significant difference of adequate/high platelet reactivity on ASA between the first and the last measurement. Image: Summary/Conclusion: The frequency of MPN patients with higher platelet reactivity on ASA was lower than in the most data in literature. Correction of an ASA dosage to twice daily reaching AOTR resulted in lower mortality. Other studies are required to confirm these data. Initial dose 2x50mg seems to be effective and safer than higher dosage. The long-term reproducibility of platelet aggregation testing results was preserved. P1072: NATURAL HISTORY OF JUVENILE MYELOMONOCYTIC LEUKEMIA W. Yang1,*, X. Zhu1 1Pediatric department, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical Colleg, Tianjin, China Background: Juvenile Myelomonocytic leukemia (JMML) is a rare clonal disease that occurs in infants and young children, and most patients respond poorly to chemotherapy. Hematopoietic stem cell transplantation (HSCT) is the only way to cure this disease. The clinical heterogeneity of JMML is large, and the disease progresses rapidly in most patients, whereas a few patients progress slowly and survive for long periods without treatment. Aims: This study retrospectively analyzed clinical data of untreated JMML patients from our department. Explore their natural course of disease and prognosis factors and provide clinical research data for screening the prognostic factors for natural history of JMML. Methods: Patients who were diagnosed JMML from January 2008 to December 2021 in our center were included in this study. The clinical characteristics, bone marrow morphology, cytogenetics, mutated genes, hypersensitivity of GM-CSF, abdominal ultrasound and other laboratory test results were retrospectively analyzed. Results: In this study，88 patients were diagnosed JMML from 2008 till now in our single institute, 44 cases (50%) without treatment (including chemotherapy and HSCT) in the whole course. The median age of onset was 13 months (range 2-48), and the incidence was mostly male, with the male to female ratio =32: 12, median time between onset and diagnosis was 2 (0.3-36.0) months. Among them, 15 cases (34.1%) had simple abnormal blood image as chief complaint, and 17 cases (38.6%) had non-specific rash on physical examination. Lymphadenopathy was found in 23 cases (52.3%) with a median subcostal value of 3.9 (0-11) cm in liver and 5.4 (1-16) cm in spleen. The median WBC count at initial diagnosis was 28.2 (range 9.1-128.6) ×109/L, and the hemoglobin value was 88.8 (range 52.0-153.0) g/L. Platelet count(PLT) was 44.0 (range 1.0-389.0) ×109/L, absolute value of monocyte was 4.5 (1.3-30.5) ×109/L, absolute value of median lymphocyte was 8.2 (2.4-48.7) ×109/L, and median fetal hemoglobin was 10 (0-88) %. Thirty-four patients underwent cytogenetic examination, of which 31 (91.2%) had normal karyotype and 3 patients had chromatid 7. Thirty patients underwent gM-CSF hypersensitive examination, with a median CFU-GM-3 (0-73) BMMNC. JMML mutations were detected in 36 children, including 6 patients with second-generation sequencing, and 30 patients with first-generation sequencing JMML hotspot mutations, including 13 (36.1%) PTPN11 mutations, 4 (11.1%) KRAS mutations, 9 (25.0%) NRAS mutations, and 1 (2.8%) NF1 mutations. One (2.8%) had mutations of NRAS and PTPN11, and 8 (22.2%) had no known JMML mutants. The study was followed up to 2022.1, with a median follow-up time of 20.6 (range 1-132) months. Among the 44 patients, 8 cases (18.2%) were lost to follow-up, 28 cases (63.6%) died, and 8 cases (18.2%) survived. To explore the influence of clinical characteristics and laboratory test results on patients’ OS, the study found that PLT < 40×109/L, spleen subcostal > 5 cm and PTPN11 were important adverse factors affecting OS. The 5-year overall survival rate of 44 children was 40%, and the 5-year overall survival rate of those with PTPN11 mutation was significantly lower than that of other mutation gene types. Summary/Conclusion: The natural history of JMML was significantly different among different mutated gene types, among which PLT, spleen size and PTPN11 mutated gene type were independent prognostic factors affecting the natural course of JMML. P1073: MUTATIONAL LANDSCAPE AND CLINICAL OUTCOMES OF PATIENTS WITH MYELOID AND LYMPHOID NEOPLASMS WITH EOSINOPHILIA (MLN-EOS) AND ABNORMALITIES OF PDGFRA, PDGFRB, FGFR1, FLT3 AND JAK REARRANGEMENT Y. Zhang1,*, L. Nguyen2, C. Lu3, E. Wang4, M. Lauw4, S. Ball1, N. Dong1, L. Moscinski5, O. Chan6, S. Yun6, D. Sallman6, L. Sokol6, B. Shah6, J. Lancet6, R. Komrokji6, A. Kuykendall6, E. Padron6, L. Zhang6 1Moffitt Cancer Center; 2Department of Pathology, James A Haley Veterans’ Hospital, Tampa; 3Department of Pathology, Duke University School of Medicine, Durham; 4Department of Pathology, University of California San Francisco, San Francisco; 5Department of Pathology; 6Department of Hematologic Malignancies, Moffitt Cancer Center, Tampa, United States of America Background: To recognize the growing list of recurrent genetically defined eosinophilia driven by constitutively active tyrosine kinase fusion genes, WHO created a provisional entity of myeloid/lymphoid neoplasms with eosinophilia (MLN-Eo) with rearrangement of PDGFRA, PDGFRB, FGFR1, or PCM1- JAK2. Aims: We aim to describe the mutational landscape and outcome with upfront targeted tyrosine kinase inhibitor (TKI) therapy in patients (pts) with MLN-Eo. Methods: We retrospectively reviewed clinical and molecular data on ptss with MLN-Eos with PDGFRA, PDGFRB, FGFR1, JAK2 or FLT3 rearrangement at Moffitt Cancer Center, Duke Cancer Center, and UCSF Comprehensive Cancer Center. The next generation sequencing of the bone marrow or peripheral blood was either done using our in-house targeted 54 gene myeloid panel or the commercially available panel, FoundationOneTM Heme Genomic profile. Pts were divided into two categories based on morphology at diagnosis: chronic and blastic phase disease. Median time to blastic phase and overall survival (mOS) were calculated using Kaplan Meier method and compared with log-rank test. Hazard ratio was calculated using the Cox proportional hazards (PH) model. Results: A total of 41 pts were included in the analysis. Thirteen patients had available comprehensive NGS results. 23 pts had PDGFRA fusion (21) or activating mutations (2); 4 had PDGFRB; 8 had FGFR1; 2 had JAK2, PCM-JAK2, and BCR-JAK2; 4 had FLT3, ETV6-FLT3 (3) and FLT3-TRIPP (1). Twelve (92%) patients had at least one other coexisting mutation. The mutations in at least two patients included TET2, RUNX1, TP53, and PTPN11 (Fig 1). Pathogenic PTPN11 mutation was present in all activating PDGFRA mutation but absent in other cases. Among 23 patients in chronic-phase disease 14 patients (61%) received the corresponding TKI to their fusion in the frontline setting. None transformed into blastic-phase disease. Six patients in the de novo blastic phase received the upfront TKI alone or combined with chemotherapy. All achieved complete remission (CR) and remained alive at a median follow-up of 44 months. In the OS analysis, upfront TKI use was associated with longer survival (HR 0.065; 95%CI, 0.008-0.50, p=0.008) in univariate analysis. The improved OS emained significant (HR 0.043, 95% CI 0.003-0.63, p=0.021) in multivariate analysis when adjusted for sex, age, hypereosinophilia in the blood, PDGFRA/B fusion status, complex cytogenetics at diagnosis, the blastic-phase at diagnosis, and alloHSCT. Image: Summary/Conclusion: Our study showed the high frequency of secondary somatic mutations in MLN-eos and described the mutational landscape similar to other myeloid malignancies. Targeted TKI therapies in the upfront setting are associated with excellent outcomes in patients with MLN-Eos. The elucidation of potential mechanisms needs further exploration. P1074: PRIMARY REFRACTORY HODGKIN’S LYMPHOMA: A MOROCCAN MONOCENTRIC RETROSPECTIVE STUDY M. Ababou1,*, A. Hammani1, A. Siham1, W. Elfaria1, E. M. Mahtat1, S. Jennane1, H. EL Maaroufi1, K. Doghmi1 1Clinical Hematology, Military hospital Mohammed v, Rabat, Morocco, Rabat, Morocco Background: Primary refractory Hodgkin’s lymphoma (PRHL)is defined as disease progression during initial first-line therapy or early relapse of less than 3 months [1], this entity is a therapeutic challenge for hematologists, as the response rate to second-line therapy remains lower (51% vs. 83%, P < 0.0001)[2] Aims: Compare different parameters between patients with PRHL and patients with non-PRHL to reveal factors that could predict the primary refractory character of our patients. Evaluate the evolution in terms of therapeutic response and survival of our patients with PRHL compared to non-PRHL Methods: This is a monocentric retrospective descriptive study that collected all the files of patients followed for Hodgkin Lymphoma at the clinical hematology department of the Military Hospital of Instruction Mohammed V in Rabat between January 2010 and December 2020. Their epidemiological, clinical and prognostic characteristics were recorded, and the efficacy and tolerance of the therapies used were evaluated. We used the t-student, Mann-Whitney, χ2 tests (or Fisher’s exact test) to compare the differences between PRHL and non-PRHL patients. To assess the difference in survival between the two groups, we used the Log-rank test on Kaplan-Meier survival curves.Treatment outcomes were measured in terms of response rate, overall survival (OS) and progression-free survival (PFS). Statistical analyses were performed using JAMOVI software (version 1.6) Results: Classical Hodgkin lymphoma was diagnosed in 122 patients. 27 patients were primary refractory (22.1%). Our comparison between the group of PRHL and non-PRHL patients in terms of age, sex, time to consultation, performance status (PS) score, clinical signs at admission, histological subtype, presence of Bulky mass on chest X-ray, the Ann Arbor stage and the prognostic risk group did not show any significant difference, which makes the identification of primary refractory patients in pre-therapy a difficult challenge. In our serie 83(68%) patients were re-evaluated at 2 courses by PET-Scan (Positron Emission Tomography, 45(54.3%) patients were in Complete Remission (CR), 34(41%) in Partial Remission (PR). Comparing PRHL and non-PRHL patients according to the response at 2 courses, a highly significant difference was observed (p=0.001). Using a univariate logistic regression model, we found that a PR at 2 courses was more likely to be PRHL than a complete response (OR= 4.95; CI95% [1.55-15.78]; p=0.007). At the end of first-line treatment, 77.1% of all patients achieved complete remission, 11.5% achieved partial response, 1 patient had a stable response and 10.6% were in progression. The overall response at the end of treatment for PRHL patients was 44.4% vs 93.6% for the non-PRHL group (p<0.001). 10.7% of patients relapsed within a median of 23 months. With a median follow-up of 65.5 months, the OS and PFS of all patients at 5 years were 91.2% and 90.6% respectively. Comparing primary refractory and non-refractory patients, the OS at 5 years was 73% [56.2-94.8] vs 96.5% [92.7-100] (p<0.0001) (Figure 1). Image: Summary/Conclusion: Primary refractory Hodgkin’s lymphoma remains a form with a poor prognosis, the overall 5-year survival of this form in our series is 73% compared to 96.5% for the non-PRHL patients. PET-2 may be a predictive step for this form, studies are needed to identify the place of immunotherapy in the management of these patients P1075: EVALUATION OF CYTOKIN GENE POLYMOPHISMS AND GENE EXPRESSIONS IN PATIENTS WITH HODGKIN LYMPHOMA H. Haydaroglu Sahin1,*, D. D. Demirbas1, D. M. Akkurd1, V. Okan1 1Haematology Department, Gaziantep University Sahinbey Research and Practice Hospital, Gaziantep, Turkey Background: In Hodgkin Lymphoma (HL), Reed-Sternberg (RS) cells play a role in the etiopathogenesis and clinical course of the disease and that is thought to be the result of expression of cytokines. Aims: In this study; it was aimed to evaluate the role of gene polymorphisms and gene expression of five cytokines (tumour necrosis factor alfa (TNF-α), transforming growth factor beta-1 (TGF-β1), interferon gamma (IFN-γ), interleukin-6 (IL-6), and interleukin-10 (IL-10)) in the etiopathogenesis of the disease, clinical parameters and prognosis in HL patients. Methods: A total of 70 patients and 70 control group who were diagnosed, treated and followed up for HL in the Hematology clinic of Gaziantep University Faculty of Medicine were included in the study. Cytokine gene polymorphisms and expression levels were analyzed by DNA isolation from peripheral blood of the patient and control groups and by using of Sequence Specific Primary polymerase chain reaction (PCR-SSP) method. Results: TNF-α (-308) gene variant GA genotype (high expression), IFN-γ (+874) gene variant TT genotype (high expression), IL6 (-174) gene variant GG genotype (high expression), TGF-β1 (T10/C10) gene variant TT genotype, TGF-β1 (C25/G25) gene variant GC genotype, IL10 (-1082) gene variantGG genotype, IL10 (-819) gene variantCC genotype and IL10 (-592) gene variantCC genotype was significantly higher in HL patients compared to healthy controls (respectively; OR= 0.3636, p= 0.0259; OR= 0.3164, p= 0.0349, OR= 0.4189, p = 0.0299, OR= 0.4329, p= 0.0257, OR= 0.4889, p<0.001, OR= 0.4189, p= 0.0299, OR= 0.3951, p= 0.0193, OR= 0.3951, p= 0.0193). While IL6 (-174) G allele (p = 0.0302), TGF-β1 (T10 / C10) T allele (p = 0.0224), IL10 (-1082) G allele (p = 0.0232), IL10 (-819) C allele (p = 0.0156), IL10 (-592) C allele (p = 00156) was higher in HL patients compared to the control group, expression of IL6 (-174) C allele (p = 0.0302), TGF-β1 (T10 / C10) C allele (p = 0.0224), IL10 (-1082) A allele (p = 0.0232), IL10 (-819) T allele, IL10 (-592) A allele (p = 0.0156) was found to be low. In haplotype analysis; TGF-β1 (T / C10, C / G25) gene “TCGC, CCGG, TTGC” (moderately increased plasma TGF-β1 level) and IL10 (-1082, -819, -592) gene “GCC GCC” (high IL10 plasma level) haplotype variants was significantly higher in HL patients than in the control group (respectively; OR= 0.3406, p= 0.0161; OR= 0.4149, p= 0.0299).In multivariate analysis; Bone marrow involvement (p <0.001) and carrier of IL6 (-174) gene polymorphism CC variant genotype (p= 0.02) were detected as an independent risk factor associated with poor prognosis on 5-year PFS. Summary/Conclusion: The results of the study suggests that TNF-α (-308) GA genotype, IFN-γ (+874) TT genotype, IL6 (-174) GG genotype, TGF-β1 (T10 / C10) TT genotype, TGF-β1 (C25 / G25) GC genotype, IL10 (-1082) GG genotype, IL10 (-819) CC genotype, IL10 (-592) CC genotype, TGF-β1 (T / C10, C / G25) gene “TCGC, CCGG, TTGC” and IL10 (-1082, -819, -592) gene “GCC GCC” haplotype may be a risk factor for HL development. P1076: A RETROSPECTIVE ANALYSIS ON OLD AND EMERGING PROGNOSTIC FACTORS IN CLASSICAL HODGKIN’S LYMPHOMA IN THE PET-GUIDED ERA A. Cellini1,*, C. A. Cavarretta1, S. Pravato1, M. Pizzi2, M. Gregianin3, F. Crimì4, F. Piazza1, L. Trentin1, A. Visentin1 1Hematology and Clinical Immunology Unit, Department of Medicine; 2Surgical Pathology and Cytopathology Unit, Department of Medicine, University of Padua, Padua; 3Nuclear Medicine Unit, Veneto Institute of Oncology IOV-IRCCS, Castelfranco; 4Institute of Radiology, Department of Medicine, University of Padua, Padua, Italy Background:: Hodgkin’s Lymphoma (HL) has been commonly recognized as a chemo-sensitive and potentially curable neoplastic disease. However, some patients are refractory or relapse after frontline therapy, and their prognosis is severely impacted by such events. Baseline prognostic factors that can reliably identify patients with high-risk disease are currently lacking, as classical prognostic factors have lost their meaning in the modern era, where a PET-guided approach and novel therapies are being incorporated in the treatment of HL. Such therapeutic options, however, need to be directed to the right subset of patients to avoid overtreatment, unwanted toxic effects, or financial burden. In recent years lymphocyte to monocyte ratio (LMR) and neutrophil to lymphocyte ratio (NLR) have been evaluated as potential outcome predictors in patients affected by HL. These factors, however, need to be further evaluated to be included in prognostic scores that reliably predict outcomes in HL patients treated in the current era. Aims: This study’s aim was to identify potential predictors of worse overall survival (OS) and progression free survival (PFS) in patients affected by HL treated with a PET-adapted chemotherapeutic approach. Methods: We included HL patients diagnosed at the University Hospital of Padova between 2004 and 2020. All patients were treated with ABVD, were staged with PET-TC at baseline, and evaluated after 2 cycles (interim, iPET) and at the end of therapy. iPET positive patients (Deauville score 4-5) were intensified with escalated BEACOPP. The number of cycles and the addition of radiation therapy as mandated by stage and local policy. Low LMR was considered as ≤2.1 and high NLR as ≥6. Survival curves were compared with Log-rank test, Cox proportional hazards test was used for multivariate analysis. 95% Confidence intervals (CI) were also reported. Results: Of the 261 patients analyzed, 101 (39%) had early-stage and 160 (61%) had advanced-stage (IIB or higher) HL. Median age was 32 years, 127 (49%) of patients were male, 134 (51%) presented with B-symptoms and 91 (35%) had bulky disease. After a median follow up of 59 months the 5-year OS and PFS for the whole population were 93.4% (CI 88.7–96.1) and 73.6% (CI 67.3–78.9), respectively. In univariate analysis stage ≥III, age ≥65 years, B symptoms, hemoglobin <105 g/L, low LMR, high NLR were associated with a shorter PFS (p<0.05), while stage ≥III, advanced age, low LMR and high NLR were associated with worse OS (p<0.05). In multivariate analysis, only stage ≥III significantly predicted worse PFS (HR 2.04; 1.13–3.69), while age ≥65 years (HR 5.38; 1.49–19.40) and high NLR (HR 4.14; 1.15–14.87) were associated with decreased OS. Focusing on the 160 advanced stage patients’ subgroup, stage ≥III and low LMR predicted a decreased PFS both in univariate and multivariate analysis with a HR of 2.14 (CI 1.30–4.08) and 2.14 (CI 1.10-4.31), respectively. Age ≥65 years and high NLR were associated with a worse OS both in univariate and multivariate analysis, with a HR of 7.48 (CI 2.14–26.17) and of 6.57 (CI 1.70–25.48), respectively. Summary/Conclusion: In this study, we provide evidence that current practice, based on a PET-guided therapy intensification, is unsatisfactory, since the survival of stage III-IV is worse than the one reached by earlier stage patients. We confirmed the reliability prognostic efficacy of NLR and LMR. Whether advanced stage patients with low LNR and high NLR might benefit from a frontline brentuximab or nivolumab-based therapies is unknown, and deserve further investigation. P1077: HODGKIN LYMPHOMA IN PATIENTS WITH HIV: NATIONAL RETROSPECTIVE MULTICENTER STUDY A. Chekalov1,*, M. Popova1, I. Tsygankov1, Y. Rogacheva1, N. Volkov1, A. Beynarovich1, K. Lepik1, M. Demchenkova2, T. Schneider3, Y. Kopeikina3, V. Potapenko4, I. Zyuzgin5, M. Kolesnikova6, T. Pospelova6, E. Zinina7, A. Myasnikov8, K. Kaplanov9, T. Ksenzova10, E. Pavlyuchenko11, N. Mikhaylova12, V. Baykov13, A. Kulagin13 1Department of Oncology, Hematology and Transplantation for teenagers and adults, Raisa Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, Saint Petersburg; 2Irkutsk Regional Cancer Center, Irkutsk; 3Leningrad Regional Clinical Hospital, Saint Petersburg; 4Municipal educational hospital №31, Saint-Petersburg; 5National Medicine Research Center of oncology named after N.N. Petrov, Saint Petersburg; 6Municipal Clinical Hospital No. 2 of Novosibirsk Region, Center of Hematology, Novosibirsk; 7Surgut’s Clinical Hospital, Surgut; 8Republican hospital named under V.A. Baranov, Petrozavodsk; 9Volgograd Regional Clinical Oncology Dispensary, Volgograd; 10Regional Clinical Hospital, Tumen; 11II Mechnikov North-Western State Medical University; 12Department of Oncology, Hematology and Transplantation for teenagers and adults, Raisa Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation; 13Raisa Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, Saint Petersburg, Russia Background: Patients with HIV infection have a significantly higher risk of developing cancer than the general population. In the era of antiretroviral therapy (ART) the cancer risk and mortality have been decreased, however, the risk of developing Hodgkin lymphoma (HL) increased. The ART allows treating HIV-infected patients with lymphomas with protocols for patients in the general population. Withal the data on the results of the treatment HL in patients with HIV controversy. Aims: To study epidemiology and evaluate the results of the treatment of HL in patients with HIV in national multicenter study. Methods: The study included 45 patients with HL in patients with HIV who received treatment in 9 Russian centers from 2007 to 2021. The median follow-up was 9 months (1-129). Patients and treatment characteristics were analyzed. Overall survival (OS) and progression-free survival (PFS) were calculated within two years from the diagnosis using the Kaplan-Meier method. Results: The median age was 39 years (25-66), men - 25 (55.6%), women - 20 (44.4%). Histological variants of HL in most cases were represented by nodular sclerosis (56%) and mixed-cell variant (41%). The advanced stage of the disease (3-4 Ann Arbor) was observed in 72.7% of patients, B-symptoms at the onset of the disease - 68.2%. The majority of patients (97.7%) received ART at the diagnosis of HL. The median number of CD4+ cells/µl at the onset of HL was 352.8 (50-692) cells/μl. General somatic status at the start of chemotherapy ECOG 0-1 - 34 (82.9%), ECOG≥2 - 7 (17.1%). As the first line of therapy, patients with localized stages of HL received ABVD (75%) and BEACOPP (25%), with advanced stages - ABVD (61.3%) and BEACOPP (38.7%). The median courses of first-line therapy were 4 (1-10). Radiation therapy in first-line therapy was performed in 4 patients (8.9%). The structure of response to first-line therapy were complete response - 51.4%, partial response - 25.7%, disease stabilization - 2.9%, disease progression - 20%. Fourteen and 8 patients received second and third-line therapy, respectively. Autologous hematopoietic stem cell transplantation was performed in 6 patients - the only 42.8% of patients with relapsed / refractory HL. OS in the study group was 81%, PFS - 38% (median PFS - 23 months). The level of CD4+ cells at the onset of HL less than 250/µl was associated with a statistically significant worsening of OS during 1 year (50% vs 100%, p=0.014). Factors such as gender, age, stage of the disease, ECOG status, the presence of B-symptoms at the onset of the disease, and the treatment regimen did not statistically significantly affect the two years OS and PFS from the diagnosis of HL in patients with HIV. Summary/Conclusion: The national multicenter study allowed characterization of HL in patients with HIV and evaluation of the efficacy of first-line therapy, which was found to be lower than in the general population. The level of CD4+ cells was the only factor that affect 2-year OS. The obtained data can form the basis for further prospective studies aimed at improving the results of HL treatment in HIV-infected patients. P1078: PATIENTS WITH HODGKIN LYMPHOMA DEVELOP ADEQUATE HUMORAL SEROLOGICAL RESPONSE TO VACCINATION WITH TWO DOSES OF BNT162B2 AND THEIR IGG ANTIBODY LEVELS MARKEDLY INCREASE AFTER A THIRD VACCINE DOSE E. Dann1,2,*, T. Inbar1, T. Mashiach1, J. Eisa1, S. Ringelstein-Harlev1, N. Horowitz1 1Rambam Health Care Campus; 2The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Background: In patients (pts) with hematological malignancies, COVID-19 is considered to be associated with a high risk of severe morbidity and mortality. While anti-COVID-19 vaccination of such pts has become the standard of care, pts undergoing lymphodepleting therapy fail to generate protective serological response due to either the nature of their underlying disease or exposure to therapy. Aims: This study aimed to assess serological response to vaccination with BNT162b2 (Pfizer) as well as COVID-19-related morbidity and mortality in Hodgkin lymphoma (HL) pts. Methods: The above vaccine was available in Israel from January 2021 and all pts with hematological malignancies were recommended to undergo vaccination with 2 doses of this vaccine, injected 21 days apart. Six months later a 3rd dose was recommended and in another 3 months a 4th dose was available for pts at risk. Serology tests were performed at least 2 weeks after the 2nd vaccination. The SARS-CoV-2 IgG II Quant (Abbott©) assay was used to measure levels of IgG antibodies (Abs) against the SARS-CoV-2 spike protein. A result was considered positive if the IgG level was ≥150 AU/ml, which was defined as an adequate serological response. Results: The current non-interventional single-center study evaluated the outcome of 55 HL pts (median age 46 years, 53% females); 51% of pts had advanced HL. Study participants received 1-9 lines of therapy (median 1 line). Six pts had COVID-19 prior to vaccination, 49 were vaccinated: 9 with 2 doses, 36 with 3 doses and 4 with 4 doses of BNT162b2. Following initial 2 vaccine doses and after a 3rd dose Ab levels >150 AU/ml were developed in 85% and 89.5% of pts, respectively. At a median of 95 days post-2nd vaccination, Ab levels were 2024 (1-29400) and 4 (0-7539) in 48 patients with no background disease versus 7 pts treated with lymphodepleting drugs or having a background disease, respectively. During the follow-up, 5 vaccinated pts were diagnosed with COVID-19 when the Delta variant was prevalent and 9 - during the Omicron wave. Notably, similar Ab levels were observed in those infected with Omicron and in non-infected pts, reflecting the genetic drift of this variant. A further analysis was performed to compare findings in a subgroup of 7 pts who had an additional background disease along with HL, such as chronic lymphocytic leukemia, s/p kidney transplantation, solid tumor, or those who were heavily pretreated, including therapy with bendamustine, versus the values observed in the rest 48 pts. The median age in the former subgroup was 58 (31-80) years, which was significantly older than in the remaining pts [median 45 (18-78) years] and median Ab levels were 4 (0-7539) AU/ml and 2024 (1-2940) AU/ml, respectively. Notably, after the 3rd vaccination, the median Ab level in both groups was 7000 AU/ml. Summary/Conclusion: The results of the current study show that at least 85% of HL pts develop a high titer of anti-spike antibodies after vaccination with 2 BNT162b2 doses. These titers substantially increased post the 3rd vaccine dose. Only a minority of HL pts who had additional background diseases or were heavily pretreated, failed to develop an adequate serological response; however, some of them had high Ab titers post-3rd and 4th vaccinations. In this study, morbidity and mortality rates of HL pts infected with COVID-19 were lower than those reported in pts with other lymphoma types. P1079: COMPARISON OF NOVEL SALVAGE REGIMENS AND TRADITIONAL SALVAGE CHEMOTHERAPY IN RELAPSED AND REFRACTORY CLASSIC HODGKIN LYMPHOMA D. Ermann1,*, V. Vardell2, E. Zacholski3, A. Fegley3, D. Modi4, K. Fedak4, S. Sundaram5, S. Rajeeve5, G. Goyal6, H. Hatic7, P. Torka8, S. Ba Aqeel8, A. Borogovac9, K. MacDougall9, S. Kothari10, A. Kress11, E. Travers12, N. Chilakamarri13, E. Brem14, L. Fitzgerald15, C. Wagner16, B. Hu15, D. Stephens15, H. Shah15 1Hematology/Oncology, Huntsman Cancer Institute, University of Utah; 2Internal Medicine, University of Utah, Salt Lake City; 3Clinical Pharmacology, Virginia Commonwealth University, Richmond; 4Hematology/Oncology, Karmanos Cancer Institute, Detroit; 5Hematology/Oncology, Tisch Cancer Institute, Mount Sinai, New York; 6Hematology; 7Hematology/Oncology, University of Alabama, Birmingham; 8Hematology/Oncology, Roswell Park Comprehensive Cancer Center, Buffalo; 9Hematology/Oncology, University of Oklahoma, Oklahoma City; 10Hematology; 11Hematology/Oncology, Yale Comprehensive Cancer Center, New Haven; 12Hematology, University of Kentucky, Lexington; 13Internal Medicine, Pomona Valley Medical Center, Pomona; 14Hematology/Oncology, University of California, Irvine; 15Hematology; 16Clinical Pharmacology, Huntsman Cancer Institute, University of Utah, Salt Lake City, United States of America Background: Traditional salvage chemotherapy followed by autologous stem cell transplant (ASCT) is the standard second line approach for treatment of relapsed/refractory (r/r) Classical Hodgkin Lymphoma (cHL) and can cure approximately 50-60% of patients (pts). Novel agents such as brentuximab vedotin (BV) and check-point inhibitors (CPIs) have high response rates in the r/r setting and have recently been used as salvage regimens to induce remissions before ASCT. Limited data exists comparing efficacy of these two approaches. Aims: Our aim was to compare outcomes of pts receiving salvage chemotherapy (CT) as opposed to novel treatments (NT) for their first salvage regimen for r/r cHL. Methods: Adult pts with r/r cHL who received their first salvage regimen (SR1) between January 2018 and June 2020 were retrospectively identified across 9 academic centers in the United States. Baseline characteristics were compared across CT and NT cohorts based on SR1. Endpoints were to compare complete response (CR), overall response rate (ORR), and event free survival (EFS) between the CT and NT cohorts. ORR was defined as CR+ partial response (PR). Events were defined as stable disease (SD) or progressive disease (PD) after SR1, less than a complete response (CR) at 90 days post-ASCT, PD after ASCT, or death. Results: In total, 120 pts were identified with a median age of 33 years (range 18-85). 68% had advanced disease and 13% were early stage unfavorable (NCCN) at diagnosis. 88% received ABVD based front-line regimen. Many pts had poor prognostic characteristics including 43% with primary refractory disease, 52% with relapse <12 months from diagnosis, 38% with extranodal disease, and 26% with B-symptoms at time of relapse (Table 1). 65% of pts (n=78) received CT and 35% (n=42) received NT for SR1. 90% of CT pts received Ifosfamide, Carboplatin, and Etoposide (ICE) as SR1. Regimens used for NT treated pts included BV + Bendamustine (43%), BV alone (36%), BV + CPI (12%), BV + CT (5%), and other CPI (4%). No significant difference was found in the ORR and CR rates according to pts treated with CT vs. NT of 67% vs. 69% and 47% vs. 55%, respectively. Of the 104 patients who received ASCT, 88% received CT and 83% received NT for SR1. At a median follow-up of 32 months, 1-year EFS was 57% vs 69% and 2-year EFS was 56% vs 66% between CT and NT cohorts (p=0.25) (Figure 1). All pts who progressed after CT for SR1 (n=31) received NT for SR2; whereas 73% (n=11) of those progressing after NT for SR1 received CT for SR2. ORR for CT in SR2 was 91% vs 70% for NT (p=.48). The most common SR2 CT regimen was ICE (82%), and NT regiment for SR2 was BV alone (40%), BV + Bendamustine (31%), BV + CPI (23%), and other CPI (6%). 79% vs 89% remained progression-free post-ASCT at their last follow-up after receiving CT vs. NT at last salvage respectively (p=0.15). Of the 16 pts who did not proceed to ASCT, 7 died within an average of 19.5 months from the receipt of SR1. Interestingly, pts who did not proceed to ASCT had a significantly improved 2-year EFS if they received NT (n=7) at SR1 (71% vs. 13%) when compared to CT at SR1 (n=9) (p=0.03). Image: Summary/Conclusion: There was a numerical trend towards better CR and EFS for novel therapy compared to traditional chemotherapy for first salvage, however the outcomes were not statistically significant. This demonstrates that CT is still a useful salvage therapy that can effectively get patients to an ASCT for curative intent. Prospective studies comparing novel therapy to traditional chemotherapy for salvage are warranted. P1080: COMPARISON OF NIVOLUMAB 40 MG EFFICACY VERSUS 3 MG/KG IN PATIENTS WITH RELAPSED AND REFRACTORY CLASSIC HODGKIN LYMPHOMA L. Fedorova1,*, K. Lepik1, N. Mikhailova1, E. Kondakova1, Y. Komarova1, M. Popova1, P. Kotselyabina1, E. Borzenkova1, V. Baykov1, I. Moiseev1, A. Kulagin1 1RM Gorbacheva Research Institute, Pavlov University, St. Petersburg, Russia Background: Nivolumab 3 mg/kg was shown to be effective in patients with relapsed and refractory classic Hodgkin lymphoma (r/r cHL). However, previously obtained data on pharmacokinetics, as well as financial toxicity of therapy, raised the issue of studying necessity of efficacy of reduced doses of PD-1 inhibitors. Efficacy of nivolumab 40 mg has previously been demonstrated in patients with r/r cHL in a prospective phase 2 study (Lepik KV et al., 2020). Continued accumulation of experience in the use of low-dose PD-1 inhibitors in patients with r/r cHL is required to support previous data. Aims: To compare the efficacy of nivolumab (Nivo) 40 mg therapy with 3 mg/kg for patients with r/r cHL. Methods: The Nivo40 trial (NCT03343665) expanded prospective cohort of patients (group 1, n=50) treated with Nivo 40 mg was compared with the retrospective group 2 (n=116) of patients treated with Nivo 3 mg/kg. Patients characteristics are demonstrated in the Table 1. The response was evaluated every 3 months by PET-CT using LYRIC criteria. Adverse events (AE) were analyzed by NCI CTCAE 4.0.3. Overall response rate, progression-free survival (PFS) and overall survival (OS) were compared between group 1 and 2. During the survival analysis the PFS was censored by the time of additional therapy initiation. Variable Nivo 40 mgN=50 Nivo 3 mg/kgN=116 p Median age, years (range) 36 (20-54) 38 (14-65) 0.129 Male/female, n (%) 17/33 (34/66) 56/60 (48/52) 0.089 Primary chemoresistance, n (%) 38 (76) 74 (64) 0.124 Early relapse, n (%) 7 (14) 14 (12) 0.731 Prior autologous stem cell transplantation, n (%) 17 (34) 44 (38) 0.630 Prior brentuximab vedotin, n (%) 18 (36) 62 (53) 0.039 Therapy lines before Nivo therapy, n (range) 4 (1-8) 5 (2-10) 0.011 B symptoms at Nivo therapy initiation, n (%) 26 (52) 71 (61) 0.269 Disease stage at Nivo therapy initiation, n (%) 2 8 (16) 11 (9) 0.294 3 5 (10) 7 (6) 4 37 (74) 97 (84) Progression at Nivo therapy initiation, n (%) 46 (92) 92 (79) 0.272 ECOG status at Nivo therapy initiation, n (%) 0-1 31 (62) 70 (60) 0.758 2 14 (28) 29 (25) 3 4 (8) 14 (12) 4 1 (2) 2 (2) Results: Median follow up was 44 (11-55) months in group 1 and 60 (6-70) months in group 2. Median Nivo cycles was 19 (2-49) and 20 (1-32) respectively. The best response to Nivo therapy was detected at 6 (2-24) and 6 (1-27) cycles respectively. Overall response rate was 66% in group 1 and 67% in group 2. The structure of response in group 1 was: complete response (CR) in 38% of patients, partial response (PR) in 28%, stable disease (SD) in 6%, indeterminate response (IR) in 22% and progressive disease (PD) in 6%; in group 2: CR in 34%, PR in 33%, SD in 5%, IR in 20% and PD in 8%. Median OS was not achieved in both groups, 3-year OS was 97,8% and 96,5% respectively (p=0.356). Median PFS was 21,9 months (95%CI: 16,75-27,05) in group 1 and 18,8 months (95%CI: 13,44-24,16) in group 2, 3-year PFS was 25,6% and 27% respectively (p=0.356). Additional therapy after Nivo monotherapy was started in 78% of patients after Nivo 40 mg and in 84% after Nivo 3 mg/kg. Allogeneic stem cell transplantation after Nivo therapy was performed in 5 (10%) patients in group 1 and in 26 (22%) patients in group 2. Any grade adverse events were detected in 66% of patients in group 1 and in 79% in group 2 (p=0.068), 3-4 grade AE were detected in 10% and 19% respectively (p=0.151). Summary/Conclusion: Nivolumab 40 mg therapy is comparable to the standard dose of 3 mg/kg in terms of overall response rate as well as survival in patients with r/r cHL. However, a direct comparison of different doses of nivolumab in a prospective study is required. P1081: PET-ADAPTED THERAPY AFTER THREE CYCLES OF ABVD FOR ALL STAGES OF HODGKIN LYMPHOMA: LONG TERM FOLLOW UP OF THE GATLA LH-05 TRIAL A. Pavlovsky1,*, N. L. Fiad1, M. V. Prates1, I. Fernandez1, N. Kurgansky1, A. Cerutti1, F. Sackmann1, F. Negri Aranguren1, P. Negri Aranguren1, G. Remaggi1, L. Ferrari1, R. Mariano1, L. Guanchiale1, J. Maradei1, F. Giuliani1, E. Roveri1, A. Enrico1, S. Zabaljauregui1, M. D. R. Cabrejo1, C. Gumpel1, A. I. Varela1, M. Riddick1, S. Pavlovsky1 1GATLA, Buenos Aires, Argentina Background: PET-CT adapted treatment for first line Hodgkin Lymphoma has been widely studied in the last decades. Long-term follow-up is important to judge both effcacy and safety of this approach. Aims: Evaluate the efficacy of the PET3 adapted treatment in patients with classical Hodgkin’s lymphoma (HL) in Ann Arbor stages I-IV. Methods: We analyzed updated follow-up data on all patients (pts) treated within the LH-05 GATLA trial. Newly diagnosed pts with HL Stages I-IV were included. All patients received 3 ABVD and were evaluated with a PET-CT-3 (PET3). Pts with a negative PET3 (DS 1 and 2) were consideredin metabolic CR and received no further therapy. Pts with DS 3 and 4 completed 6 ABVD and IFRT on PET-CT positive areas. Pts with progressive disease (DS 5) after 3 ABVD received salvage chemotherapy. We present the updated results of 490 pts with a median follow-up of 10 years. With a median age of 35 yrs., 300 presented with localized stage and 190 with advanced stage. Results: In LH-05, of all pts, 338 (69%) achieved CR with negative PET3, 152 (31 %) were PET3 positive. With a median follow up of 120 months the PFS and OS for all pts at 5 years is 79.2%and 94.3% respectively. Pts with negative PET3 had an PFS of 89% and 80% for localizedand advanced stage, compared to 63% for all pts with positive PET3 (p <0.0001). We performed a multivariate analysis for PFS which included age, stage, IPS, bulky disease, extranodalareas and the result of the PET3. This last parameter, together with age, were the only ones with statistical significance (p=0.001 and 0.046 respectively). Stage at diagnosis was not significant. With long term follow up the OS at 5 years is 97.3% and 87.3% for all PET3 negative vs PET3 positive pts. When comparing the results LH-05 with our previous clinical trial (LH-96) there is no difference in PFS and OS at 5 years but in LH-05 only 31% received more than 3 cycles of ABVD and IFRT compared to 61% and 100% in LH-96. This PET adapted approach reduces exposure to chemo andradiotherapy with no negative effect on long term outcome. Image: Summary/Conclusion: This long term follow up data support the PET-CT adapted approach for all stages ofHL after a short course of ABVD. In the Cox regression model, PET-CT at completion of treatment was the most significant factor associated to PFS. Treatment with 3 cycles of ABVD can be adequate for pts with negative PET3 regardlesstheir stage at diagnosis. Nevertheless, this long term follow up demonstrated that there is still a room for improvement trying to identify PET3 negative pts that will relapse and escalating treatment in PET3 positive pts to improve outcome. GATLA is designing a trial with the aim to improve these two different risk groups. P1082: SAFETY AND EFFICACY ANALYSIS IN OLDER PATIENTS TREATED WITHIN THE GATLA LH-05 PROTOCOL: PET-ADAPTED THERAPY AFTER 3 CYCLES OF ABVD FOR ALL STAGES OF HODGKIN LYMPHOMA N. L. Fiad1,*, M. V. Prates1, I. Fernandez1, N. Kurgansky1, A. Cerutti1, F. Negri Aranguren1, L. Guanchiale1, F. Sackmann1, J. Maradei1, A. Enrico1, P. Negri Aranguren1, A. Pavlovsky1 1GATLA, Buenos Aires, Argentina Background: Treatment for classic Hodgkin lymphoma (cHL) in older age population continues to be a challenge. Comorbidity prevalence, increase toxicity to the standard treatments and the lack of inclusion in clinical trials all contribute to this phenomenon. The obtained results are inferior in comparison to young adults and there is shortage of evidence regarding effective therapeutic strategies. Recently, the GATLA LH-05 protocol has published the long-term follow-up of the results of the PET/CT adapted strategy after 3 cycles of ABVD, regardless of the presentation stage and no upper age limit. Aims: To assess the effectiveness and safety of the interim PET/CT adapted treatment in cHL patients older than 60 years old. Methods: A retrospective analysis of the LH-05 database was performed. Patients were included ≥ aged 60 with recent diagnosis of cHL stage I-IV and HIV negative. All patients received 3 cycles of ABVD and were evaluated with PET-TC (PET3). Those patients with negative PET (Deauville score 1 and 2) were considered to be in complete remission (CR) and they finalized the treatment. Patients with DS 3 and 4 completed 6 ABVD cycles and involved-field radiotherapy in hypermetabolic areas in interim PET3. Patients with DS 5 were considered to have progressive disease. Progression-free survival (PFS) and overall survival (OS) were evaluated. Kaplan-Meier method and Log-rank test were used for survival analysis. Results: Of a total of 490 patients included in the GATLA LH-05 protocol, 59 met the inclusion criteria. The mean age was of 66 years (range: 60-89), 90% presented a PS<2. The most frequent histological subtype was Nodular Sclerosis (54%) and 75% presented in localized stage (I-II). All patients received initial treatment with 3 cycles of ABVD and were evaluated by PET: 81% presented negative PET (DS 1-2) and 19% were positive. No GIII-IV toxicity or treatment-related deaths were recorded. With a median follow-up of 10 years, median PFS and OS were not reached. At 36 and 60 months, PFS was 86% and 78,9%, and OS was 90% and 85.5%, respectively. Negative PET3 patients had a PFS of 85.4%, while positive PET3 patients had a PFS of 43% (p=0,0001). In the multivariable analysis that included age (>60 vs. <60), stage (localized vs. advanced), IPS (<2 vs. >2), extranodal areas, bulky disease and PET3 result, only age and PET3 result had significant impact in PFS (p=0.046 and p=0,001 respectively). PFS of PET/TC- positive patients was significantly lower than in the cohort of patients younger than 60. OS at 36 and 60 months in PET negative patients was 97.5% and 94.5% respectively, versus 63.6% and 53% in PET/TC positive patients. Image: Summary/Conclusion: With the PET/TC adapted treatment after 3 cycles of ABVD in 59 patients > 60 years old, 81% of patients achieved negative PET and therefore received no further treatment. These patients had an excellent result with a PFS of 85.4% at 3 years, similar to the younger patient population. However, a significant reduction in PFS was observed in PET3 positive patients > 60 years old compared to younger ones. Implementation of this PET/TC guided strategy, regardless of stage at diagnosis, resulted in reduced exposure to chemotherapy and radiotherapy, contributing to the absence of severe treatment-related morbidity and mortality. P1083: BRENTUXIMAB VEDOTIN, ETOPOSIDE, SOLUMEDROL, HIGH DOSE ARA-C & PLATINUM FOLLOWED BY HDT & APBSCT FOR REFRACTORY/RELAPSED HODGKIN LYMPHOMA PATIENTS: LONG-TERM RESULTS OF THE GELTAMO GROUP BRESHAP STUDY R. Garcia-Sanz1,*, C. Martínez2, F. De la Cruz3, A. P. González4, A. Rodríguez5, B. Sánchez-González6, E. Domingo-Domenech7, M. Moreno8, J. López9, J. L. Piñana10, M. Bastos11, M. Canales12, A. Gutiérrez13, M. J. Rodríguez-Salazar14, A. Navarro1, A. Sureda7 1hematology, Hospital Universitario, Salamanca; 2hematology, Hospital Clinic, Barcelona; 3hematology, Hospital Virgen del Rocío, Sevilla; 4hematology, Hospital Universitario Central de Asturias, Oviedo; 5hematology, Hospital 12 de Octubre, Madrid; 6hematology, Hospital del Mar; 7hematology, Hospital Duran i Reynals, Barcelona; 8hematology, Hospital Germans Trias i Pujol, Badalona; 9hematology, Hospital Ramón y Cajal, Madrid; 10hematology, Hospital Clínico, Valencia; 11hematology, Hospital Gregorio Marañón; 12hematology, Hospital La Paz, Madrid; 13hematology, Hospital Son Espases, Palma de Mallorca; 14hematology, Hospital Universitario de Canarias, Tenerife, Spain Background: For Refractory/Relapsed Hodgkin Lymphoma (RRHL) patients, the combination of BRentuximab vedotin, Etoposide, Solumedrol, High dose Ara-C and Platinum (BRESHAP) as induction therapy prior to high-dose therapy and Autologous Stem Cell Transplantation (APBSCT) induces an overall (OR) and complete remission (CR) rate of 93% and 71% of patients with an initial 75% Time to Tumor Progression at three years (Garcia-Sanz et al, Ann Oncol 2019). In these patients, the primary efficacy endpoint is usually the CR pre-APBSCT, but long-term results including time to tumor progression (TTP) or overall survival are challenges that should be evaluated in these recently adopted strategies. Aims: In this work, we evaluate long-term results of the phase II trial with the combination of BV and ESHAP [BRESHAP] as 2nd line therapy for RRHL prior to APBSCT (ClinicalTrials.gov #NCT02243436). Methods: Data update with an additonal follow-up period of four years compared with the initial publication in the EHA-2018 congress Results: The trial included 66 patients with relapsed or refractory classical HL (cHL) who had failed to one prior line of therapy were eligible. There were 35 females & 31 males, with a median age of 36 years (18-66). Forty patients were primary refractory, 16 early relapses (CR ≤1 year) and 10 late relapses (CR >1 year). During the follow-up, excluding progressions, there were 39 Severe Adverse Events (SAEs) reported in 22 patients (hospitalizations and AEs around transplant were not considered SAEs), which means that no new SAEs were reported after the initial publication of the results. Transplant was done after BRESHAP in 61 patients with no problems reported about the engraftment in the long-term evaluation. As reported, pre-transplant OR was 93% (including a 71% CR). At a mean follow-up of 75 months, 16 patients have progressed and 3 died without progression, providing 7-year time to treatment failure (TTP) and progression free survival (PFS) of 74% and 70%, respectively. Eight patients have died: 5 due to progression, and the 3 due to complications in the absence of relapse or refractory disease: pneumonia, sepsis and pulmonary embolism. The overall survival (OS) at seven year was 88% (figure 2). There were three variables related to a lower PFS and TTP: CR after BRESHAP, bone marrow infiltration at relapse/refractoriness and B symptoms at inclusion in the trial. In the multivariate analysis, only response to BRESHAP maintained the independent predictive capacity. Respect to OS, the only prognostic factor was CR to 2nd line therapy Image: Summary/Conclusion: BRESHAP followed by high dose therapy and autologous peripheral blood stem transplant is a safe and effective strategy for patient with refractory or relapsed Classical Hodgkin lymphoma with excellent long-term results. P1084: CHECK-POINT INHIBITORS IN PATIENTS WITH RELAPSE/REFRACTORY HODGKIN’S LYMPHOMA: A RETROSPECTIVE ANALYSYS BY THE RETE EMATOLOGICA PUGLIESE (REP). F. Gaudio1,*, G. Loseto2, V. Bozzoli3, P. R. Scalzulli4, A. M. Mazzone5, L. Tonialini6, V. Fesce7, G. Quintana8, G. De santis9, P. Masciopinto10, E. Arcuti10, F. Clemente2, S. Scardino3, G. Tarantini9, D. Pastore8, L. Melillo7, V. Pavone6, P. Mazza5, A. M. Carella4, N. Cascavilla4, N. Di Renzo3, A. Guarini2, P. Musto1,10 1Unit of Hematology and Stem Cell Transplantation, AOUC Policlinico; 2Hematology Unit, Giovanni Paolo II IRCCS Cancer Institute Oncology Hospital, Bari; 3Hematology and Stem Cell Transplant Unit, “Vito Fazzi” Hospital, Lecce; 4Hematology Unit, IRCCS “Casa Sollievo della Sofferenza”, S. Giovanni Rotondo (FG); 5Hematology Unit, Department of Hematology-Oncology, Moscati Hospital, Taranto; 6Hematology and Transplant Unit, Cardinal Panico Hospital, Tricase (LE); 7Hematology Unit, Azienda Ospedaliero Universitaria-Ospedali Riuniti, Foggia; 8Hematology Unit, A. Perrino Hospital, Brindisi; 9Hematology Unit, Dimiccoli Hospital, Barletta; 10Department of Emergency and Organ Transplantation, “Aldo Moro” University, Bari, Italy Background: The majority of patients with either limited stage or advanced-stage classical Hodgkin lymphoma (cHL) can be cured with chemotherapy; however, 5–10% will have refractory disease to frontline therapy and approximately 10–30% will relapse. In recent years, checkpoint/PD-1 inhibitors have significantly changed the prognosis of patients with relapsing refractory cHL, demonstrating exceptional results in heavily pretreated patients. There is limited data on the real-world experience with PD-1 inhibitors in cHL and it is unknown whether fewer selected patients treated with these agents receive benefits similar to those observed in published trials. Aims: We performed a retrospective multicentre analysis of the Rete Ematologica Pugliese (REP) of 66 patients with relapsing refractory cHL who received PD-1 inhibitors in the non-trial setting. Methods: Forty-three patients (65%) were treated with nivolumab and 23 (35%) with pembrolizumab. Median age at diagnosis was 36 years (range, 18–81) and median age at initiation of PD-1 inhibitor therapy was 43 years (range 19–86). Forty-four (67%) and 23 (35%) patients underwent autologous stem cell transplantation (SCT) and allogeneic SCT, respectively, prior to checkpoint inhibitor therapy. The median lines of treatment attempted prior to PD-1 inhibitor therapy was 4 (range, 3 to 7). All patients were treated with brentuximab vedotin prior to receiving checkpoint inhibitor therapy. Results: The best overall response rate to PD-1 inhibitor therapy was 70%: 47% complete remission (CR) and 23% partial remission (PR). Five patients (8%) achieved stable disease (SD), while 14 patients (21%) showed progressive disease (PD). In patients treated with nivolumab the response was: CR in 23 (54%), PR in 9 (21%), SD in 4 (9%), PD in 7 (16%) patients, respectively; in those treated with pembrolizumab CR occurred 8 (35%), PR in 6 (26%), SD in 1 (4%), PD in 8 (35%) patients, respectively. Twenty-four immune-related adverse events were documented (4 gastrointestinal, 4 hepatic, 6 fever, 4 hematological, 3 dermatological, 3 allergic rhinitis). Toxicity resolved in all patients and there were no deaths attributed to checkpoint inhibitor therapy. After a median follow-up of 26 months (range 3-72 months), 54 patients (82%) are alive and 12 (18%) died. The cause of death was attributed to disease progression in 9 patients, and sepsis in 3 patients. After PD-1 inhibitor therapy, 22 patients (33%) relapsed or progressed. The overall survival and progression free survival at five years were 65% and 54%, respectively. Summary/Conclusion: We demonstrate similar response rates, clinical outcome and toxicity profiles compared to clinical trials. These results support the effectiveness and tolerability of PD-1 inhibitors therapy in relapsing refractory cHL in a real-world setting. P1085: EVALUATION OF GONADAL FUNCTION IN YOUNG WOMEN DIAGNOSED WITH HODGKIN AND NON-HODGKIN LYMPHOMA A. N. Georgopoulou1,*, A. Giannakou1, T. P. Vassilakopoulos1, M. Siakantaris1, N. A. Georgopoulos2, S. Kalantaridou3, E. Lalou1, K. Keramaris1, E. Loukari1, M. Arapaki1, I. Konstantinou1, A. Machairas1, A. Kopsaftopoulou1, I. Mammali2, I. Vassilopoulos1, D. Galopoulos1, M. K. Angelopoulou1 1Department of Hematology and BMT of the National and Kapodistrian University of Athens, Laikon, laikon general hospital, Athens; 2Department of Endocrinology of the University Hospital of Patras, University Hospital of Patras, Patras; 3B Obstetrics and Gynecology Department of the National and Kapodistrian University of Athens, Areteion Hospital, Athens, Greece Background: ABVD chemotherapy (CT) is the standard treatment approach for Hodgkin lymphoma (HL), with a negative interim PET. R-DA-EPOCH and R-CHOP are the most frequently used regimens used in primary mediastinal/high grade B-NHL and DLBCL, respectively. Young women are often affected by these types of lymphoma and may be cured in >80% of the cases. Thus, treatment-related complications are being increasingly recognized, among which, gonadal insufficiency with its major psychological consequences. This is more prevalent in female patients, in whom collection and cryopreservation of oocytes/ovarian tissue are not applied in everyday clinical practice. Published data are scarce on this subject. Moreover, little is known about the kinetics of gonadal function and sex hormones during chemotherapy to safely guide contraceptive measures. Aims: The aim of this study is the prospective evaluation of gonadal function in young women with malignant lymphoma who are receiving CT. We here present our preliminary results on 50 patients with HL and B-NHL. Methods: This is a prospective study of gonadal function in female patients≤40 years. Hormonal measurements were performed at pre-specified time points: before treatment (t0), during CT(t1), at the end of CT(t2) and every six months(t6,t12) thereafter. The following hormones were measured: follicle-stimulating hormone (FSH), lutenizing hormone (LH), progesterone (PG), estradiol (Ε2), anti-Mullerian hormone (ΑΜΗ). The study included:32 HL [median age: 29 years, 32 ABVD] and 18 B-NHL patients [median age:27 years, 9 RDAEPOCH, 7 RCHOP, 2 other]. FSH reflects gonadal function in women (increased levels indicate gonadal dysfunction). AMH is considered to be the most sensitive biomarker for gonadal reserve (decreasing values correlate with ovarian insufficiency). E2 and progesterone are the major sex hormones. Results: HL-ABVD: FSH constantly increased from the beginning, peaking in the middle (fsh0-1 p<0.0001), remaining high until the end (fsh0-2 p<0.0001), without reaching normal levels at 6 months (fsh0-6 p=0.002), finally normalizing at 12 months after the end of treatment. Consistently with FSH, AMH sharply decreased during treatment: [median values: 2,85IU/mL(t0), 0,45IU/mL(t1), 0,62IU/mL(t2), p<0.0001 for both]. A rebound increase was observed at 6 months and slowly decreased again to normal values at 12 months after the end of CT. Estradiol and progesterone were not affected throughout the treatment. B-NHL: Gonadal damage, reflected by the increase in FSH, was evident, though with slightly different kinetics compared to HL: [median values: 4,1IU/mL(t0), 8,3IU/mL(t2), fsh0-2 p=0,035]. FSH gradually increased during treatment, reaching a peak towards the end. FSH slowly reached normal values after six months from the end of treatment. In parallel to HL, AMH values sharply decreased during CT: [median values: 7,3IU/mL(t0), 0,17IU/mL(t1), 1,57IU/mL(t2), amh0-1 p=0.002, amh0-2 p=0,043). However, in contrast to HL, no clear rebound was seen and AMH values remained extremely low, even at 18 months after the end of treatment (median values: 0,23IU/mL), highlighting the gonadotoxic effect of CT. Estradiol and progesterone did not change significantly. Image: Summary/Conclusion: Gonadal function in female patients with malignant lymphomas is affected during CT in both HL and B-NHL, though with different kinetics. In HL patients treated with ABVD, gonadal function normalized at 6 months, whereas, in B-NHL gonadal dysfunction remained even at 18 months after the end of CT, possibly indicating a chemotherapy-dependent genotoxic effect. P1086: FAVEZELIMAB (ANTI–LAG-3) AND PEMBROLIZUMAB CO-BLOCKADE IN ANTI–PD-1–NAIVE PATIENTS WITH RELAPSED OR REFRACTORY CLASSICAL HODGKIN LYMPHOMA: AN OPEN-LABEL PHASE 1/2 STUDY G. Gregory1,*, J. Timmerman2, D. Lavie3, P. Borchmann4, A. F. Herrera5, L. Minuk6, V. Vucinic7, P. Armand8, A. Avigdor9, R. Gasiorowski10, Y. Herishanu11, C. Keane12, J. Kuruvilla13, J. Palcza14, P. Pillai14, P. Marinello14, N. A. Johnson15 1School of Clinical Sciences at Monash Health, Monash University, Melbourne, Australia; 2UCLA Medical Center, Los Angeles, United States of America; 3Hadassah Medical Center, Jerusalem, Israel; 4University Hospital of Cologne, Cologne, Germany; 5City of Hope, Duarte, United States of America; 6CancerCare Manitoba, Winnipeg, Canada; 7University of Leipzig Medical Center, Leipzig, Germany; 8Dana-Farber Cancer Institute, Boston, United States of America; 9Sheba Medical Center, Ramat Gan, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 10Concord Hospital, University of Sydney, Concord, Australia; 11Tel Aviv Sourasky Medical Center, Tel Aviv-Yafo, Israel; 12Princess Alexandra Hospital, Brisbane, Australia; 13Princess Margaret Cancer Centre, Toronto, Canada; 14Merck & Co., Inc., Kenilworth, United States of America; 15Jewish General Hospital, Montreal, Canada Background: Programmed cell death 1 (PD-1) inhibitors are a standard of care in patients with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL), but clinical interest persists for new approaches to deepen and lengthen responses. Dual blockade of PD-1 and lymphocyte-activation gene 3 (LAG-3) has demonstrated antitumor activity in preclinical models. Aims: The multicohort phase 1/2 MK-4280-003 study (NCT03598608) evaluated the safety and efficacy of favezelimab (MK-4280), a humanized Immunoglobulin G4 LAG-3 inhibitor, plus pembrolizumab (a PD-1 inhibitor) in patients with R/R hematologic malignancies. This analysis focused on anti–PD-1–naïve patients with R/R cHL (cohort 1). Methods: This study included a safety lead-in phase (part 1) to determine the recommended phase 2 dose (RP2D) followed by a dose-expansion phase (part 2). Eligible patients in cohort 1 must have had R/R cHL after autologous stem cell transplantation (ASCT) or been ineligible for ASCT and have had no prior anti–PD-1 therapy. In part 1, patients from all cohorts received intravenous (IV) pembrolizumab 200 mg every 3 weeks (Q3W) and favezelimab IV 200 mg or 800 mg Q3W. Dose-finding based on occurrence of dose-limiting toxicities (DLTs) was determined using a modified toxicity probability interval design. In part 2, patients received pembrolizumab + favezelimab at the established RP2D for up to 2 years (35 cycles), or until documented disease progression, adverse events (AEs), or withdrawal from the study. Primary end point was safety, including DLTs and AEs. Secondary end point was objective response rate (ORR). Duration of response (DOR), progression-free survival (PFS), and overall survival (OS) were exploratory end points. Results: Only 1 DLT (autoimmune hepatitis [grade 4]) was identified among the first 6 patients from all cohorts included in part 1 at the favezelimab 200 mg dose; thus, the dose was escalated to 800 mg. No DLTs were observed in the 15 additional patients treated at the 800 mg dose. The RP2D for the combination was defined as 800 mg Q3W + pembrolizumab 200 mg Q3W. In cohort 1, 30 patients were enrolled; median age was 40 years, 53% had ECOG PS 0, and 80% had no more than 3 prior lines of therapy. As of the database cutoff (Nov 1, 2021), 9 of 30 (30%) patients had discontinued treatment either due to adverse events (n=3) or disease progression (n=6). After a median follow-up of 13.5 months, ORR for cohort 1 was 73% (95% CI, 54-88; complete response, 7 patients [23%]; partial response, 15 patients [50%]). 28 of 30 patients (93%) had a reduction from baseline in target lesions. Median DOR was not reached ([NR]; 95% CI, 0+ to 23+ months) and 6 patients (51%) had response ≥12 months. The median PFS was 19 months (95% CI, 8-NR) and the 12-month PFS rate was 57%. The median OS was NR (95% CI, NR-NR) and the 12-month OS rate was 94%. Treatment-related AEs (TRAE) occurred in 26 patients (87%); most common (≥10%) were hypothyroidism (27%), fatigue (20%), infusion-related reactions (20%), headache (17%), and arthralgia, hyperthyroidism, myalgia, and nausea (10% each). Grade 3 or 4 TRAEs occurred in 6 patients (20%) and 10% of patients discontinued due to TRAEs. No treatment-related deaths occurred. Summary/Conclusion: Favezelimab 800 mg + pembrolizumab 200 mg Q3W demonstrated an acceptable safety profile and effective antitumor activity in anti–PD-1–naïve patients with R/R cHL. Further studies are merited to compare the activity of this combination to that of pembrolizumab alone. P1087: FAVEZELIMAB (ANTI–LAG-3) AND PEMBROLIZUMAB CO-BLOCKADE IN PATIENTS WITH RELAPSED OR REFRACTORY CLASSICAL HODGKIN LYMPHOMA WHO PROGRESSED AFTER ANTI–PD-1 THERAPY: AN OPEN-LABEL PHASE 1/2 STUDY A. F. Herrera1,*, D. Lavie2, N. A. Johnson3, A. Avigdor4, P. Borchmann5, C. Andreadis6, A. Bazargan7, G. Gregory8, C. Keane9, T. Inna10, V. Vucinic11, P. L. Zinzani12, H. Zhang13, P. Pillai13, P. Marinello13, J. Timmerman14 1City of Hope, Duarte, United States of America; 2Hadassah Medical Center, Jerusalem, Israel; 3Jewish General Hospital, Montreal, Canada; 4Sheba Medical Center, Ramat Gan, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 5University Hospital of Cologne, Cologne, Germany; 6UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, United States of America; 7University of Melbourne, Melbourne, and St Vincent’s Hospital, Fitzroy; 8School of Clinical Sciences at Monash Health, Monash University, Melbourne; 9Princess Alexandra Hospital, Brisbane, Australia; 10Rambam Health Care Campus, Haifa, Israel; 11University of Leipzig Medical Center, Leipzig, Germany; 12IRCCS Azienda Ospedaliero-Universitaria di Bologna Istituto di Ematologia “Seràgnoli” and Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy; 13Merck & Co., Inc., Kenilworth; 14UCLA Medical Center, Los Angeles, United States of America Background: PD-1 inhibitors are a standard of care for relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL), but optimal therapy is yet to be defined for patients whose disease progresses after anti–PD-1 treatment. Dual blockade of PD-1 and lymphocyte-activation gene 3 (LAG-3) has demonstrated antitumor activity in preclinical models. Aims: This multicohort phase 1/2 study (NCT03598608) evaluated safety and efficacy of favezelimab (MK-4280), a humanized IgG4 LAG-3 inhibitor, plus the PD-1 inhibitor pembrolizumab in patients with R/R hematologic malignancies. Cohort 2 focused on patients with R/R cHL refractory to anti–PD-1 therapy. Methods: This study included a safety lead-in phase (part 1) to determine recommended phase 2 dose (RP2D) followed by a dose-expansion phase (part 2). Eligible patients in cohort 2 had R/R cHL, relapsed after or were ineligible for autologous stem cell transplantation (ASCT), and progressed after ≥2 doses of anti–PD-1 therapy (within 12 weeks of last dose). In part 1, patients from all cohorts received intravenous (IV) pembrolizumab 200 mg every 3 weeks (Q3W) and favezelimab IV 200 mg or 800 mg Q3W. Dose-finding based on occurrence of dose-limiting toxicities (DLT) was determined using a modified toxicity probability interval design. In part 2, patients received pembrolizumab + favezelimab at the established RP2D for up to 2 years (35 cycles), or until documented disease progression, adverse events (AEs), or withdrawal from study. Primary end point was safety, including DLTs and AEs. Secondary end point was objective response rate (ORR). Duration of response (DOR), progression-free survival (PFS), and overall survival (OS) were exploratory end points. Results: Only 1 DLT (autoimmune hepatitis [grade 4]) was observed among the first 6 patients from all cohorts included in part 1 at the favezelimab 200 mg dose; thus, the dose was escalated to 800 mg. No DLTs were observed in the 15 additional patients at the 800 mg dose. Favezelimab RP2D was defined as 800 mg Q3W + pembrolizumab 200 mg Q3W. In cohort 2, 33 patients were enrolled; median age was 37 years, 64% had ECOG PS 0, and 94% had at least 4 prior lines of therapy. At database cutoff (Nov 1, 2021), 20 patients had discontinued treatment due to adverse events (n=7); disease progression or clinical progression (n=11); or withdrawal/physician decision (n=2). After a median follow-up of 16.5 months, ORR for patients receiving favezelimab 800 mg (n=29) was 31% (95% CI, 15-51; complete response, 2 [7%]; partial response, 7 [24%]). 19 of 29 responders (66%) had an anti–PD-1–based regimen as their most recent line of therapy at study entry. 23 of 29 patients (79%) had a reduction from baseline in target lesions. Median DOR for patients who received favezelimab 800 mg was not reached ([NR]; 95% CI, 0+ to 14+ months). For all patients in cohort 2, the median PFS was 9 months (95% CI, 5-15); 12-month PFS rate was 39%. Median OS was 26 months (95% CI, 26-NR); 12-month OS rate was 91%. Treatment-related AEs (TRAE) occurred in 28 patients (85%); most common (>10%) were hypothyroidism (18%), nausea and fatigue (15% each), and arthralgia and diarrhea (12% each). Grade 3 or 4 TRAEs occurred in 6 patients (18%) and 18% of patients discontinued treatment due to TRAEs. No treatment-related deaths occurred. Summary/Conclusion: Favezelimab 800 mg + pembrolizumab 200 mg Q3W showed a tolerable safety profile and effective antitumor activity in heavily pretreated patients with R/R cHL whose disease had progressed after anti–PD-1 therapy, suggesting that the combination may reinduce a response in these patients. P1088: UPDATED DATA FROM A PHASE 1/2 STUDY OF BRENTUXIMAB VEDOTIN COMBINED WITH CHEMOTHERAPY IN PEDIATRIC PATIENTS WITH ADVANCED STAGE CLASSICAL HODGKIN LYMPHOMA F. Locatelli1,*, F. Luisi2, M. Pianovski3, M. A. Salvino4, F. Fagioli5, S. Epelman6, L. Britto De Abreu Lima7, R. Norris8, V. Odone Filho9, M. Zecca10, C. Favre11, R. Kobayashi12, Y. Koga13, Y. Sidi14, X. Zhou14, X. Bai14, F. Campana14, E. J. Leonard14, A. R. Franklin15 1Department of Pediatric Haematology and Oncology and Cell and Gene Therapy, IRCCS Ospedale Pediatrico Bambino Gesù, Sapienza, University of Rome, Rome, Italy; 2Pediatria, GRAACC/UNIFESP, São Paulo; 3Department of Pediatric Oncology, Hospital Erastinho, Liga Paranaense de Combate ao Câncer, Jardim das Américas, Curitiba, Parana; 4Hematology, IDOR, Salvador, Hospital São Rafael, Salvador, Brazil; 5Department of Sciences of Public Health and Pediatrics, Regina Margherita Children’s Hospital, Turin, Italy; 6Pediatric Oncology Department, Hospital Santa Marcelina, São Paulo; 7Hematology, INCA - Instituto Nacional de Câncer, Rio de Janeiro, Brazil; 8Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cancer and Blood Diseases Institute, Cincinnati, OH, United States of America; 9ITACI – Instituto De Tratamento Do Câncer Infantil – Departament De Pediatria – Faculdade De Medicina Da Universidade De São Paulo, São Paulo, Brazil; 10Oncoematologia Pediatrica, Fondazione IRCCS Policlinico San Matteo, Pavia; 11Dipartimento di Oncoematologia, Azienda Ospedaliero Universitaria Ospedale Pediatrico Meyer, Firenze, Italy; 12Department of Hematology/Oncology for Children and Adolescents, Sapporo Hokuyu Hospital, Sapporo; 13Department of Pediatrics, Kyushu University Hospital, Fukuoka-shi, Fukuoka-Ken, Japan; 14Takeda Development Center Americas, Inc. (TDCA), Lexington, MA; 15Center for Cancer and Blood Disorders, Children’s Hospital Colorado, Aurora, CO, United States of America Background: Brentuximab vedotin is a CD30-directed monoclonal antibody-drug conjugate approved for use in the first-line adult classical Hodgkin lymphoma (cHL) setting in combination with doxorubicin, vinblastine, and dacarbazine (A+AVD). The safety, tolerability, recommended dosing, immunogenicity, and antitumor activity of A+AVD have not previously been evaluated as a first-line therapy in treatment-naïve pediatric patients with cHL. Aims: To provide updated safety and efficacy data from an open-label phase 1/2 study of A+AVD in previously untreated pediatric patients with stage III or IV cHL (NCT02979522). Methods: Patients with newly diagnosed stage III or IV cHL who were aged 5 to <18 years were treated with A+AVD on days 1 and 15 of 28-day cycles, for a maximum of 6 cycles. In phase 1, patients (n = 8) received brentuximab vedotin 48 mg/m2 + AVD; dose-limiting toxicity (DLT) was assessed from day 1 of cycle 1 to day 56. In phase 2, 51 additional patients received the same treatment regimen. Progression-free survival (PFS) was defined as the time between first dose and time of disease progression, and event-free survival (EFS) was defined as the time between first dose and time of treatment failure; EFS events were disease progression, treatment withdrawal, and death. All patients had been on study for at least 2 years at the data cutoff (September 24, 2021). The data presented refer to phases 1 and 2 combined. Results: All patients (n = 59) in phases 1 and 2 completed 6 cycles of A+AVD. Among 6 DLT-evaluable patients in phase 1, no DLTs were observed. The maximum tolerated dose of brentuximab vedotin was not reached and the recommended dose was determined to be 48 mg/m2. Treatment-emergent adverse events (TEAEs) occurred in all patients; the most common TEAEs were vomiting (85%), nausea (75%), and neutropenia (58%). Fourteen patients (24%) experienced treatment-emergent peripheral neuropathy (PN); of these, 10 patients had PN resolved by end of treatment (EOT), 13 patients had PN resolved by last contact, and one patient had ongoing grade 1 paraesthesia at last contact. No on-study deaths occurred. At the end of cycle 2, the PET-negative rate (Deauville score 1, 2, or 3) was 90%. Objective response rate (complete response [CR] + partial response) according to independent review facility was 88%, with 76% of patients achieving a CR. Median duration of response and duration of CR were not estimable. Median PFS, EFS, and overall survival were not reached, suggesting promising efficacy of A+AVD in pediatric patients. Overall PFS, EFS and overall survival data will be presented at the meeting. Summary/Conclusion: Brentuximab vedotin 48 mg/m2 plus AVD every two weeks was generally well tolerated with an acceptable safety profile, and demonstrated an efficacy benefit in CD30+ pediatric patients with previously untreated advanced cHL. These data support A+AVD as a suitable frontline therapy option in this patient population. P1089: BRENTUXIMAB VEDOTIN, NIVOLUMAB, DOXORUBICIN, AND DACARBAZINE (AN+AD) FOR ADVANCED STAGE CLASSIC HODGKIN LYMPHOMA: PRELIMINARY SAFETY AND EFFICACY RESULTS FROM THE PHASE 2 STUDY (SGN35 027 PART B) H. Lee1, I. W. Flinn2, J. Melear3, R. Ramchandren4, J. Friedman5, J. M. Burke3, Y. Linhares6, M. Raval3, R. Chintapatla7, T. A. Feldman8, H. Yimer3, M. Islas-Ohlmayer3, A. Dean3, V. Rana9, M. D. Gandhi3, J. Renshaw3, A. L. Gillespie-Twardy3, L. Ho10,*, M. Puhlmann10, W. Guo10, C. A. Yasenchak11 1MD Anderson, Houston; 2Sarah Cannon Research Institute and Tennessee Oncology, Nashville; 3US Oncology Research, The Woodlands; 4University of Tennessee Medical Center, Nashville; 5University Hospitals Seidman Cancer Center, Cleveland; 6US Oncology Research, Woodlands; 7Kadlec Clinic, Kennewick; 8Lymphoma Division, John Theurer Cancer Center at Hackensack Meridian Health, Hackensack; 9University of Colorado Health Hematology and Oncology, Colorado Springs; 10Seagen, Bothell; 11Willamette Valley Cancer Institute and Research Center, Eugene, United States of America Background: Brentuximab vedotin (BV) and nivolumab are both active and well-tolerated in patients (pts) with classical Hodgkin lymphoma (cHL) and were previously studied in first salvage (overall response rate [ORR] 85%; complete response [CR] 67%) (Advani 2021) and as firstline (1L) therapy in older adults (ORR 95%; CR 79%) (Yasenchak 2019). The combination of BV plus nivolumab demonstrated promising activity as a frontline treatment option for pts over 60 yrs of age with cHL (Friedberg 2018). Additionally, in pts with non-bulky Stage I or II cHL, treatment with BV plus doxorubicin and dacarbazine (AD) resulted in a CR rate of 97% at end of treatment (EOT), as well as a promising 4-year progression-free survival (PFS) estimate of 91%. There were no cases of ≥Grade 3 peripheral neuropathy and only 9% were Grade 2 (Abramson 2021). Aims: This abstract will present the preliminary safety and efficacy results from Part B of the SGN35-027 study, which enrolled pts with Ann Arbor Stage II cHL with bulky mediastinal disease (defined as a single node or nodal mass ≥10 cm as determined by CT imaging), or Stage III or IV cHL. Methods: SGN35-027 (NCT03646123) is an open-label, multiple part, multicenter, phase 2 clinical trial. Pts received up to 6 cycles of AN+AD (consisting of BV 1.2 mg/kg, nivolumab 240 mg, doxorubicin 25 mg/m2, and dacarbazine 375 mg/m2). All study drugs were administered by IV infusion on Days 1 and 15 of each 28-day cycle. The primary endpoint was CR rate at EOT. Key secondary endpoints included safety, tolerability, ORR, and PFS. Disease response and progression was assessed by investigators using the Lugano Classification Revised Staging System for nodal non-Hodgkin and Hodgkin Lymphomas (Cheson 2014) and Lymphoma Response to Immunomodulatory Therapy Criteria (LYRIC) (Cheson 2016) at Cycle 2 and EOT. Results: In Part B, 58 pts were enrolled and 57 pts received at least 1 dose of study treatment. Of these 57 pts, 30 (53%) were male, 27 (47%) were female, 50 (88%) were white, 46 (81%) were not of Hispanic or Latino/a or Spanish origins, and 54 (95%) were <65 years old. Median age was 35 yrs (range: 19-78 yrs); 30% had Stage II cHL with bulky mediastinal disease, while 18% and 51% had Stage III and IV cHL, respectively. Of 58 pts enrolled, 52 (90%) completed treatment and 4 (7%) discontinued due to AEs. The most frequently reported TEAEs were nausea, fatigue, and diarrhea (70%, 51%, and 46% of pts, respectively). Grade 3 or higher TEAEs were reported in 30 pts (53%), with neutropenia and increased ALT in 5 pts (9%) being the most frequent. Treatment-related AEs occurred in 98% of pts; the most frequent were nausea, fatigue, and peripheral sensory neuropathy (65%, 46%, and 39% of pts, respectively). Treatment-related SAEs occurred in 8 pts (14%), of which pneumonitis was the most common (3 pts [5%]). Immune-mediated AEs were observed in 18 pts (32%), and hypothyroidism (4 pts [7%]) was the most frequently reported. No febrile neutropenia was observed, and there were no Grade 5 AEs. Preliminary results show at EOT, 52 pts had a response to treatment (ORR 93% [95% CI:82.7-98.0]), with 49 pts having a complete response (CR rate: 88% [95% CI: 75.9-94.8]). Summary/Conclusion: Preliminary results demonstrate that AN+AD has promising clinical activity and is well-tolerated, with no new safety signals observed. The omission of bleomycin and vinblastine may have contributed to the absence of certain AEs, such as febrile neutropenia. AN+AD may provide another active treatment option for patients with 1L advanced cHL. P1090: CAMRELIZUMAB (CAM) COMBINED WITH GEMOX CHEMOTHERAPY RESULTS IN HIGH COMPLETE METABOLIC RESPONSE RATES IN RELAPSED/REFRACTORY CLASSIC HODGKIN LYMPHOMA (CHL): A PHASE II TRIAL Y. Xie1,*, Y. Tang1, W. Zheng1, L. Ping1, N. Lin1, M. Tu1, C. Zhang1, Z. Ying1, W. Liu1, L. Deng1, M. Wu1, L. Mi1, T. Du1, X. Wang1, J. Zhu1, Y. Song1 1Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Lymphoma, Peking University Cancer Hospital & Institute, Beijing, China Background: Approximately 30-35% of patients with classic Hodgkin Lymphoma will prove refractory to frontline therapy or relapse subsequently. Traditional second-line chemotherapy regimens result in complete response rates about 20-40%. Achievement of complete metabolic response (CMR) assessed by PET/CT imaging prior to hematopoietic stem cell transplant (HSCT) predicts favorable progression free survival (PFS) and overall survival (OS). PD‐1 antibody has shown remarkable efficacy in relapsed or refractory hodgkin lymphoma (R/R HL). Aims: we designed a clinical trial to evaluate the efficacy and safety of PD‐1 antibody camrelizumab combined with GEMOX chemotherapy in R/R HL patients. The preliminary results had been obtained. Methods: This is an open‐label, single‐center, non‐randomized, phase 2 study (NCT04239170). The main inclusion criteria included age ≥ 18, ECOG < 2, histopathology confirmed classical HL, no more than 3 lines of previous chemotherapy treatment, having measurable lesion(s) assessed by Lugano 2014 criteria and preparing to receive autologous stem cell transplantation (ASCT). Patients were treated with 2 cycles of camrelizumab (CAM, 200 mg IV, q2w) followed by 2 cycles (28-day cycle) of CAM (IV, day 1 and day 15) combined with standard GEMOX (gemcitabine 1000 mg/m2 and oxaliplatin 100mg/m2, IV, day 1 and day 15). Patients achieved CR were transferred to stem cell mobilization/collection stage; patients achieved PR/SD could have another cycle of CAM plus GEMOX, patients who achieved PR/CR underwent stem cell mobilization/collection. For patients waiting more than 4 weeks for ASCT, 1-2 cycles of CAM monotherapy were allowed. The primary endpoint is the proportion of patients who achieved PET‐CT‐confirmed complete response (CR) according to Lugano 2014 criteria. Results: From March 2020 to December 2021, 30 patients were enrolled. One patient withdrew informed consent. The median age of remaining 29 patients was 34 years old (ranged from 22 to 63), with a male‐to‐female ratio of 16:13. At the first tumor response evaluation, 69% (20/29) patients achieved CR and 17% (5/29) patients achieved partial response (PR), resulting an overall response rate (ORR) of 86%. Only one patient had progression disease (PD). The patients who achieved PR or stable disease (SD) received an additional cycle of CAM plus GEMOX treatment, and one patient who had PR in the first evaluation was found to achieve CR. Up to now, collection of the autologous hematopoietic stem cells was completed in thirteen patients. With a cutoff date of February 10, 2022, the 1-year (progression-free survival) PFS rate was 74% and the 1-year (overall survival) OS rate was 100%, median PFS and OS were not reached with too few events (Figure). Most adverse events (AEs) were of grade 1 to 2. Common AEs included reactive capillary endothelial proliferation (RCEP), alanine aminotransferase (ALT)/ aspartate aminotransferase (AST) elevation, vomiting, nausea, hyperuricemia (all > 30%). Hematologic AEs included neutropenia (> 34%). No grade 4 or above AEs occurred. Grade 3 AE occurred in 6 patients, including 1 pulmonary infection, 1 nausea, 1 fever, 1 hypertriglyceridemia, 1 ALT elevation, and 1 neutropenia. Serious adverse events included 1 case of interstitial pneumonia (grade 2) and 1 case of fever (grade 3) resulting in hospitalization. Image: Summary/Conclusion: Camrelizumab combined with GEMOX chemotherapy showed encouraging clinical efficacy and tolerable toxicity in R/R HL patients and merited further study. P1091: FEASIBILITY OF ASCT AFTER ANTI-PD-1 THERAPY FOR R/R CLASSICAL HODGKIN LYMPHOMA N. Mochkin1,*, V. Sarzhevskiy1, Y. Protopopova2, E. Demina1, V. Melnichenko1, V. Bogatyrev1, A. Samoylova1, A. Mamedova1, A. Rukavitsyn1, A. Bannikova1, E. Smirnova1, N. Shorokhov1 1FSBI National Medical Surgical Center n.a. N.I. Pirogov; 2I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia Background: Immunotherapy with checkpoint inhibitors (anti-PD-1) is highly effective in relapsed/refractory (R/R) classical Hodgkin Lymphoma (cHL) and leads to more than 70% overall response rate. However, long-term progression-free survival rate is not sufficient. Consolidation with ASCT after anti-PD-1 could be a very promising option even among previously chemorefractory patients. Nevertheless, there is not enough experience about the safety and feasibility of ASCT after anti-PD-1 in the context of possible immune-related adverse events (AE). Aims: to assess the safety and feasibility of ASCT after anti-PD-1 therapy in patients with R/R cHL. Methods: We retrospectively analysed patients with cHL who previously underwent ASCT from November 2018 until December 2021 after treatment with anti-PD-1. A median patient age was 34 years (range, 20-55), 22 patients were female, 21 - male. Patients received anti-PD-1 as monotherapy (n=17), combination of anti-PD-1 and chemotherapy (n=4), anti-PD-1 monotherapy followed by combination of anti-PD-1 and chemotherapy (n=14), anti-PD-1 monotherapy followed by salvage therapy (n=3) and other (n=2). A median number of anti-PD-1 cycles was 6 (range, 1-45). The median time from the last dose of anti-PD1 was 69 days (range, 14-365). Conditioning regimen for ASCT consisted of BeEAM (n=12), BeAC (n=30) and other (n=1). Safety assessment was performed by Common Terminology Criteria for Adverse Events (CTCAE) (v5.0). Results: Forty-three eligible patients were enrolled in this analysis. A median time to engraftment was 10.5 days (range, 9-26). A median time to platelet count > 20x109/L was 13.5 days (range, 7-43). A median use of G-CSF after high-dose chemotherapy followed by ASCT was 9 days (range, 0-26). PEG-G-CSF (empegfilgrastim 7.5 mg/ml once) was used in 10 patients (23.3%). Plerixafor was used in 6 patients (14%). Neutropenic fever was observed in 32 patients (74.4%). Documented infections were reported in 19 patients (44.2%) among which were 5 cases of clostridial colitis (11.6%). A median intravenous administration of antibiotics was 8 days (range, 0-23). Grade 3-4 mucositis and enteropathy was reported in 2 patients (4.6%) and 6 (14%), respectively. Other non-hematological toxicity was observed in 10 patients (23.4%): 8 patients (18.6%) developed engraftment syndrome (that was successfully treated with steroids), grade 4 autoimmune toxic myocarditis was reported in 1 patient (2.3%). One death due to grade 5 autoimmune myocarditis in combination with grade 5 autoimmune pneumonitis was reported (2.3%). A median duration of hospitalization was 25 days (range, 14-72). Transplant-related mortality (TRM) was reported in 2 patients (4.6%). Image: Summary/Conclusion: The obtained data on safety of ASCT after anti-PD-1 treatment generally correspond to the data of international studies. Toxicity profile tends to be very similar to the published data about ASCT without anti-PD-1 previous treatment, especially in terms of TRM. However, we observe a high incidence of engraftment syndrome that can lead to fulminant immune-related AE (myocarditis and pneumonitis) probably. Attention should be paid to the defining of optimal “wash-out” period after last dose of anti-PD-1 and early defining of possible life-threatening immune related AE (especially myocarditis and pneumonitis). P1092: NODULAR LYMPHOCYTE PREDOMINANT HODGKIN LYMPHOMA (NLPHL): HISTOLOGYCAL AND CLINICAL REVIEW IN A SINGLE CENTRE. E. Rámila1,*, N. Papaleo2, A. Arrieta2, C. Blazquez2, M. Solorzano2, M. Vidal2, X. Vilaseca1, J. Piedra1, J. I. Martinez1 1Department of Hematology; 2Department of Pathology, Hospital Universitari Parc Taulí Sabadell (Barcelona), Sabadell, Spain Background: NLPHL constitutes a subtype of Hodgkin lymphoma with distinct clinical characteristics and evolution. Aims: To review the histopatologycal diagnosis and clinical data in patients with NLPHL in our institution between 1999-2021. Methods: We retrospectively reviewed the histology of lymph node biopsy and clinical data, treatment and outcome of 21 adult patients consecutively diagnosed of a NLPHL between 1999 and 2021. Results: Twenty-one adult patients with diagnosis of NLPHL (3 of which presented transformation areas to diffuse large B-cell lymphoma (t-DLBCL) in the biopsy), were identified. After the histological review of the biopsies 1 patient was reclassified as diffuse large B cell lymphoma (DLBCL) and another patient as NLPLH with t-DLBCL. Six (30%) patients showed a typical histological pattern (A-B) in the biopsy whereas 14 (70%) presented histopathological variants (pattern C-F) including the 4 patients with transformation areas to DLBCL. Thus, the clinical data of a total number of 20 patients with NLPLH (4 of them with transformation areas) were reviewed. Twelve (60%) patients were males and 8 (40%) female with a median age of 46 years (range 17-79). Regarding the clinical stage at diagnosis, 9 (45%), 2 (10%), 5 (25%) and 4 (20%) were in favourable IA stage, favourable IIA stage, unfavourable I-IIA stage (according the German Study Hodgkin Group and European Organisation for Research and Treatment of Cancer criteria) and advanced III-IV stage respectively. Three of the 4 patients in advance stage presented variant histological patterns. Among the 9 patients in favourable clinical IA stage the treatment administered was the following: 2 of them didn’t receive any further treatment (in one by rejection and other patient was in surveillance after lymph node resection), 5 received Radiotherapy and 2 received combined treatment (chemotherapy with ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) and radiotherapy). The 2 patients in favourable IIA stage received combined treatment. Among the 5 patients in unfavourable I-II clinical stage, 3 received combined treatment (ABVD and radiotherapy), 1 patient was treated with ABVD and 1 with R-CHOP (rituximab, ciclophosphamide, doxorubicin, vincristine and prednisone) due to areas of t-DLBCL in the biopsy. Patients (4) in advanced stage were treated with chemotherapy: 2 with ABVD, 1 with CVP (ciclophosphamide, vincristine and prednisone) and 1 with rituximab and platinum based regimen (transformation areas to DLBCL in the biopsy and previous episode of DLBCL). Regarding to responses, 15 (83.3%) of the 18 treated patients achieved a complete remission and 3 (16.7%) were refractory (2 of them received second line therapy incuding rituximab with a complete remission). One patient relapsed; he was refractory to ABVD and achieved a complete remission with R-CHOP. With a median follow-up of 5.9 years (range 0.8-18) the overall survival is 90%; 2 patients have died (1 of them, a 79 years old patient with comorbidities, due to lymphoma). Summary/Conclusion: We have seen a 95% of concordance with the initial diagnosis after a histological review in our patients with NLPLH. 20% of the biopsies showed transformation areas to DLBCL. Most of the patients (80%) presented in a localized stage. Refractory patients responded well to antiCD20 treatment. The prognosis is good with an overall survival of 90%. P1093: A NEW PROGNOSTIC MODEL FOR INDIVIDUAL OUTCOME PREDICTION IN ADVANCED-STAGE HODGKIN LYMPHOMA. R. Rask Kragh Jørgensen1,2,*, S. Eloranta3, M. Tang Severinsen1,2, A. Kiesbye Øvlisen1, K. Bjøro Smeland4, C. Kiserud4, J. Haaber Christensen5, M. Hutchings6, R. Bo-Dahl Sørensen7, P. Kamper8, I. Glimelius3,9, K. E. Smedby3,10, F. Bergström3,11, T. C. El-Galaly1,2, L. Hjort Jakobsen1,2,12 1Department of Hematology, Clinical Cancer Research Centre, Aalborg University Hospital; 2Department of Clinical Medicine, Aalborg University, Aalborg, Denmark; 3Department of Medicine Solna, Division of Clinical Epidemiology, Karolinska Institutet, Stockholm, Sweden; 4Department of Oncology, Oslo University Hospital, Oslo, Norway; 5Department of Hematology, Odense Universitets Hospital, Odense; 6Department of Hematology, Copenhagen University Hospital, Copenhagen; 7Department of Hematology, Zealand University Hospital, Roskilde; 8Department of Hematology, Aarhus University Hospital, Aarhus, Denmark; 9Department of Immunology, Genetics and Pathology, Unit of Oncology, Uppsala University, Uppsala; 10Department of Hematology, Karolinska University Hospital; 11Department of Mathematics, Stockholm University, Stockholm, Sweden; 12Department of Mathematical Sciences, Aalborg University, Aalborg, Denmark Background: Patients diagnosed with advanced-stage Hodgkin lymphoma (aHL) are commonly risk stratified based on prognostic scores like the International Prognostic Score (IPS). These scores are often based on dichotomized clinical predictors, leading to loss of information and thus inaccurate prognoses. Aims: This study investigated if a machine learning approach utilizing real world data from aHL patients can outcompete the IPS in predicting overall survival (OS). Methods: This study included patients with classical aHL registered in the Danish Lymphoma Registry (LYFO). We developed a new prognostic model for predicting OS using stacking, which combined several predictive survival models into a single model. The models used in the stacking, were Cox porportional hazard (CPH), Flexible parametric survival (FPS), Random Survival forest and IPS models, with different combinations of covariates included depending on the model. The time-varying area under the curve (AUC), integrated Brier score (IBS), and concordance index (C-Index) were used to measure performance of the new model as well as the IPS and a simplified version containing three risk-factors (IPS-3). The performance measures were computed using 10-fold cross-validation. Results: A total of 1,110 aHL patients were included and the 5-year OS was 81% (95% CI, 79-84%). The IBS for the stacking, IPS, and IPS-3 model was 0.078, 0.099, and 0.096, respectively, while the C-Index was 0.86, 0.73, and 0.71. The time-varying AUC was consistently higher for the stacking-model compared to IPS-models within the first five years after diagnosis (Figure). According to the IPS-model, 143 (12.9%) patients were high-risk (IPS 5-7). The 5-year OS for these patients was 62% (95% CI, 54-71%). We defined an alternative high-risk group by including the 143 patients with the lowest predicted 5-year OS based on the stacking-model. This high-risk group had a 5-year OS of 40% (95% CI, 32-50%). Image: Summary/Conclusion: The new prognostic model for aHL based on machine-learning technique (stacked model) demonstrated a substantial improvement in predictive performance compared to the IPS-models. The model also detected a high-risk group with worse OS than the IPS high-risk group. The model is planned for international validation. P1094: CHARACTERISTICS, MANAGEMENT, AND OUTCOMES OF PATIENTS WITH T-CELL LARGE GRANULAR LYMPHOCYTIC LEUKEMIA AT A LARGE TERTIARY CARE CENTER H. Audil1,*, M. Shah1, K. Rabe1, W. Ding1, P. Hampel1, N. N. Bennani1, T. Call1, C. Hanson1, Y. Wang1, A. Koehler1, S. Schwager1, J. Leis2, S. Slager1, S. Kenderian1, A. Al-Kali1, G. Nowakowski1, M. Shi1, S. Parikh1 1Mayo Clinic, Rochester; 2Mayo Clinic, Phoenix, United States of America Background: Large granular lymphocytic leukemia (LGLL) comprises 2-6% of chronic lymphoproliferative disorders. T-LGLL accounts for approximately 85% of cases, while NK-LGLL represents 15% of cases. There is a significant knowledge gap regarding diagnosis, management, and outcomes of LGLL. Aims: Our aims were to describe the clinical characteristics, time to first therapy (TTFT), type of first line therapy, response to treatment, and overall survival (OS) in patients with LGLL seen at our institution. Methods: We identified LGLL patients from the Mayo Clinic Chronic Lymphoproliferative Disorders Database seen between 1/1995-7/2021. At least 3 of the following 4 criteria were used to diagnose LGLL (T-LGLL or NK-LGLL): (1) a distinct T-cell or NK-cell population by flow cytometry; (2) a clonal T-cell or NK-cell population; (3) intrasinusoidal cytotoxic T-cell or NK-cell infiltrates in bone marrow, spleen, or liver; and (4) persistence of the abnormal T-cell or NK-cell population or unexplained cytopenia for more than 6 months (Salama, BCJ, 2022). Baseline clinical characteristics, TTFT, first line therapies, and OS were analyzed for all patients. The Mayo Clinic IRB approved this study. Results: We identified 217 patients who met inclusion criteria for LGLL diagnosis; 196 (90.3%) patients had T-LGLL and 21 (9.7%) patients had NK-LGLL. The median age at diagnosis was 65 years [range 21-86], and 120 (55.3%) were male. 159 (73.3%) patients had evidence of cytopenia at the time of diagnosis. Frequently associated clinical characteristics included dependence on red cell or platelet transfusions (44.3%), colony-stimulating factors (20.0%), or both (21.7%); splenomegaly (32.9%); history of autoimmune disease (24.9%); history of hematologic disorder (18.4%) or malignancy (5.6%); and constitutional symptoms (11.1%). The median follow-up was 8.4 years; 133 patients died, and 111 patients were treated for LGLL during this period. The median TTFT after diagnosis was 47.0 months (42.8 for T-LGLL and 68.0 for NK-LGLL) with a median of 1 (range 0-11) line of treatment. First-line therapy consisted of methotrexate in 33 (29.7%) patients, cyclophosphamide in 25 (22.5%), cyclosporine in 6 (5.4%), chlorambucil in 6 (5.4%), rituximab in 5 (4.5%), steroids alone in 17 (15.3%), and splenectomy in 7 (6.3%); 5 (4.5%) patients received combination therapy and 2 (1.8%) received IVIG. Alemtuzumab, cladribine, fludarabine, lenalidomide, and vincristine were first-line therapy in 1 (0.9%) patient each. Response to first-line therapy (Lamy, Blood, 2011) consisted of complete response (CR) in 31%, partial response (PR) in 33%, and treatment failure or progressive disease in 36% patients. The median OS was 11.0 years: 11.0 years for T-LGLL and 8.1 years for NK-LGLL (p = 0.90; Figure 1). Image: Summary/Conclusion: In this retrospective study of LGLL, we utilized stringent pathology inclusion criteria to allow for analyses of a homogenous cohort of patients seen at our institution. One in 4 patients with LGLL had a concomitant autoimmune condition, and 25% had a concomitant hematologic condition or hematologic malignancy at the time of initial diagnosis. Approximately a third of all patients who needed LGLL treatment did not achieve a response to frontline therapy, suggesting that newer treatments are needed to improve outcomes of this rare disease. P1095: TREATMENT ATTRIBUTES FOR 3RD LINE FOLLICULAR LYMPHOMA TREATMENT DECISION-MAKING: PHYSICIAN PERSPECTIVES FROM A SURVEY ACROSS WESTERN EUROPE AND THE UNITED STATES P. C. Johnson1,*, A. Bailey2, N. Milloy2, E. Clayton2, R. G. Quek3, Q. Ma3 1Department of Medicine, Division of Hematology & Oncology, Massachusetts General Hospital Cancer Center & Harvard Medical School, Boston, United States of America; 2Oncology, Adelphi Real World, Bollington, United Kingdom; 3Health Economics & Outcomes Research, Regeneron Pharmaceuticals Inc., Sleepy Hollow, United States of America Background: Follicular lymphoma (FL) is the second most common subtype of non-Hodgkin lymphoma (NHL), and is the most common of the clinically indolent NHLs. Treatment (tx) selection must integrate a myriad of clinical and patient factors, including disease resistance, cumulative toxicities and treatment efficacy, as well as patient quality of life (QoL) and the costs of therapies. However, real-world data regarding physician perspectives on tx selection is lacking, especially at later lines of therapy. Aims: This study aims to assess physician perspectives regarding key tx selection attributes including those beyond efficacy and safety, as well as physician perception of patient’s tx preference, when selecting a 3rd line (3L) FL tx. Methods: Descriptive data were drawn from the Adelphi FL Disease-Specific Programme™, a point-in-time study that was fielded June 2021-Jan 2022. Haematologists, haem-oncologists and medical oncologists (med-onc) in EUR [France, Germany, Italy, Spain, United Kingdom (UK)] and the US completed online surveys regarding self-reported demographics and the top 7 tx attributes contributing to their rationale for 3L FL tx selection from a comprehensive prespecified list of 28 attributes derived from expert opinions. Physicians also provided their perception on patient’s preference regarding tx attributes for choosing 3L tx. Results: Of 251 physicians surveyed in EUR (France: n=46; Germany: n=40; Italy: n=43; Spain: n=41; UK: n=31) and the US (n=50), 49% were haem-oncologists, 41% haematologists and 10% med-oncs. 52% primarily worked at academic hospitals. Progression free survival (74.1%), overall survival (70.1%), duration of response (59.8%), evidence-based efficacy overall / overall response rate (57.4%) and ability to achieve complete response (46.2%) were most frequently physician reported top 7 attributes for 3L tx selection. Notably, long-term safety and impact on patients’ QoL were reported by 38.6% and 34.3% of physicians, respectively. Fewer physicians reported cost (4.8%), patient acceptability of lag time between leukapheresis and CAR-T infusion (7.2%), patient acceptability of frequency of administration (8.0%), less monitoring needed (8.8%) and fewer outpatient / inpatient consultations needed (5.6% and 7.6% respectively) as their top 7 3L tx attributes considered. Frequency of some 3L tx selection attributes reported differed substantially between EUR and the US including long-term safety (35.8% vs 50.0%), impact on patients’ QoL (37.8% vs 20.0%), and suitability for patients aged over 60 years (10.9% vs 22.0%). Summary/Conclusion: Treatment efficacy and improved survival were the most important attributes for physicians’ 3L FL tx selection whilst safety, impact on patient QoL, frequency of inpatient / outpatient consultations, monitoring requirements, and cost were amongst those attributes considered less important by physicians. Despite literature indicating that patient QoL and cost of tx should both be key considerations for physicians when choosing FL tx, our study highlights these are less commonly reported attributes for treatment selection. Future study is warranted to further explore reasons for physician treatment selection. P1096: TREATMENT PATTERNS AND OUTCOMES IN RELAPSED/REFRACTORY MANTLE CELL LYMPHOMA A. Bock1,*, K. Poonsombudlert2, M. Larson3, J. Gile1, R. Tawfiq1, S. Maliske2, M. Maurer3, J. Cerhan4, X. Andrade-gonzalez1, A. Saliba1, J. Paludo1, D. Inwards1, S. Ansell1, T. Witzig1, T. Habermann1, B. Link2, S. Ayyappan2, G. Nowakowski1, U. Farooq2, Y. Wang1 1Division of Hematology, Mayo Clinic, Rochester; 2Division of Hematology, University of Iowa, Iowa City; 3Division of Biostatistics; 4Division of Epidemiology, Department of Quantitative Health Sciences, Mayo Clinic, Rochester, United States of America Background: Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma with heterogeneous clinical presentation and no established standard of care. In the last two decades, the pattern of frontline therapy for MCL has evolved, and multiple new agents became available for relapsed/refractory (R/R) MCL. The pattern of treatment, its evolution with time, and the association with treatment outcomes in R/R MCL are not well understood. Aims: To characterize second line (2L) treatment patterns and outcomes in patients with R/R MCL initially diagnosed in the rituximab era. Methods: Patients with newly diagnosed MCL between August 2002 and April 2015 and followed through 2021 were identified from the Mayo Clinic/University of Iowa Molecular Epidemiology Resource (MER) prospective cohort study. Patients who initiated therapy for R/R MCL after relapse or progression to first line (1L) therapy were included in this analysis. Clinical characteristics and 1L and 2L therapies were abstracted from MER and by chart review; treatment outcomes were analyzed. Results: Among a total of 343 MCL patients with a median follow-up of 11.8 years, 188 had documented progression/relapse to 1L therapy, of which 170 had 2L treatment information available for this analysis. At the time of 2L therapy, 123 (72%) patients had an age >60, 136 (80%) were male, 132 (78%) had stage III/IV, and simplified MIPI was 0-3 (low) in 37 (35%), 4-5 (intermediate) in 40 (37%), and 6-11 (high) in 30 (28%; simplified MIPI missing in 63 patients). The median time from diagnosis to 2L was 27 months (range 0.5-141). The patterns of 1L and 2L treatment are shown in Figure 1. 1L treatment included HiDAC-based (n=31, 13 had autologous stem cell transplant [ASCT]), R-CHOP-like (n=79, 45 had ASCT), R-Bendamustine (BR, n=22, 2 had ASCT), other systemic (n=29), and non-systemic (n=9). 15 patients (9%) received rituximab maintenance after induction or ASCT. 2L treatment included salvage therapy followed by ASCT (n=13; salvage with HiDAC-based n=2, R-CHOP-like n=5, BR n=3, platinum-based n=2, other n=1) or Allogeneic SCT (AlloSCT, n=5), HiDAC-based n=9, R-CHOP-like n=6, BR n=33, BTK inhibitor (BTKi, n=21), other targeted therapy (lenalidomide, bortezomib, mTOR inhibitors, n=26), other systemic (n=47), and non-systemic (n=10). The median follow-up from 2L therapy was 8.2 years. The objective response rate to 2L therapy was 71% (45% complete response). The median progression-free survival (PFS) and overall survival (OS) were 1.1 and 4.0 years, respectively. The 2-year PFS rate and 5-year OS rate were 55% (95% CI 36-84) and 71% (95% CI 49-100), respectively for patients treated with BTKi at 2L, and 42% (95% CI 28-63) and 46% (95% CI 32-68), respectively, for those treated with BR. In patients intended to undergo salvage therapy followed by ASCT (n=18, n=13 transplanted) or AlloSCT (n=5, all transplanted), the 2-year PFS rate and 5-year OS rate were 80% (95% CI 52-100) and 60% (95% CI 41-88), and 80% (95% CI 52-100) and 61% (95% CI 42-88), respectively. Patients treated with HiDAC-based, R-CHOP-like, other targeted therapy, or other systemic therapy had poorer outcomes (2-year PFS 0-27%, 5-year OS 0-35%). Image: Summary/Conclusion: Second line treatment for MCL was very heterogenous in terms of treatment and outcomes and was likely impacted by 1L treatment and other clinical factors. The evolution of 1L and R/R MCL treatment patterns and the associated patient characteristics and outcomes requires further exploration in larger datasets, given extensive heterogeneity in practice patterns. P1097: MEK-INHIBITORS IN TREATMENT OF LANGERHANS CELL HISTIOCYTOSIS E. Burtsev1,*, G. Bronin1 1HSCT and BMT, Morozov Children’s Hospital, Moscow, Russia Background: There are increasing data of targeted therapy efficacy of different types of Langerhans cell histiocytosis (LCH) with inhibitors of BRAF-specific serin-threonine kinase (BRAF-inhibitors) in cases with BRAFV600e mutation published last years. At the same time there are lack of published data of MAPK/ERK pathway inhibitors (MEK-inhibitors) use in pediatric patients with BRAF-negative forms of LCH. Aims: Aim of the study is to evaluate efficacy and safety of MEK-inhibitor (cobimetinib) in eight pediatric BRAFV600e-negative refractory LCH patients. Methods: The study included 8 children with various forms of LCH. BRAFV600e mutation was not found in any case included in the trial. All patients received therapy according to the LCH IV protocol and were diagnosed with progression of LCH during or after termination of the treatment. Monotherapy with cobimetinib was used as a second line therapy in all cases. The response to the targeted therapy was assessed in accordance with the international scale Response Evaluation Criteria in Solid Tumors (RECIST v.1.1). The assessment of the toxicity was performed in accordance with the international scale of Common Terminology Criteria for Adverse Events (CTCAE v.5.0). Results: Complete response to cobimetinib was not achieved in any patient. Partial response was established in 62,5% cases. One patient was diagnosed with disease progression in three months after termination of the therapy. The incidence of adverse events was high (75%). The most common side effects were diarrhea (75%), rush (50%) and electrolyte disturbances (37,5%). Summary/Conclusion: Cobimetinib monotherapy is effective in BRAF V600e- negative refractory pediatric LCH patients. The response to the treatment can be delayed. All cases of the toxicity were dose dependant and successfully resolved after dose correction. Further research is needed to define duration of treatment and optimal dosage regimens. P1098: OUTCOME OF FOLLICULAR LYMPHOMA PATIENTS IN MAINTENANCE TREATMENT WITH ANTICD20 MONOCLONAL ANTIBODIES IN SARS-COV2 ERA: RESULTS FROM A MULTICENTER, RETROSPECTIVE- PROSPECTIVE ITALIAN STUDY. A. Castellino1,*, C. Castellino1, C. Boccomini2, M. Clerico3, P. Nicoli4, A. Vanazzi5, F. Fanelli6, T. Perrone7, F. Marchesi8, F. Cocito9, M. Merli10, S. Bigliardi11, B. Mecacci12, V. Bozzoli13, G. Margiotta-Casaluci14, E. Meli15, A. Anastasia16, L. Farina17, O. Annibali18, D. Gottardi19, M. Zanni20, A. Conconi21, C. Ciochetto22, N. Cenfra23, F. Rotondo24, S. Ratotti25, A. Cuneo26, C. Selleri27, S. Galimberti28, M. Massaia1 1Hematology Unit, AO Santa Croce e Carle di Cuneo, Cuneo; 2Hematology Unit, Citta’ della Salute e della Scienza; 3Hematology Unit, Citta della Salute e della Scienza, Torino; 4Hematology Unit, San Luigi Gonzaga Hospital and University, Orbassano; 5Hematology Unit, Istituto Europeo di Oncologia, Milano; 6Hematology Unit, San Camillo Hospital, Roma; 7Hematology Unit, Policlinico Hospital, Bari; 8Hematology Unit, IRCCS Regina Elena National Cancer Institute, Roma; 9Hematology Unit, San Gerardo Hospital, Monza; 10Hematology Unit, Ospedale di Circolo ASST Sette Laghi, Varese; 11Hematology Unit, Ospedale Civile, Sassuolo; 12Hematology Unit, University of Siena, Siena; 13Hematology Unit, Vito Fazzi Hospital, Lecce; 14Hematology Unit, Department of Translation Medicine, AOU Maggiore della Carità, Novara; 15Hematology Unit, ASST Grande Ospedale Metropolitano Niguarda, Milano; 16Hematology Unit, Ospedali Civili, Brescia; 17Hematology Unit, Istituto Nazionale Tumori, Milano; 18Hematology Unit, Università Campus Bio-Medico, Roma; 19Hematology Unit, Ospedale Mauriziano, Torino; 20Hematology Unit, SS Arrigo e Biagio Hospital, Alessandria; 21Hematology Unit, Ospedale degli Infermi, Biella; 22Hematology Unit, ASL TO4, Ivrea; 23Hematology Unit, Ospedale Santa Maria Goretti, Latina; 24Hematology Unit, Ospedale Papardo, Messina; 25Hematology Unit, IRCCS Fondazione San Matteo, Pavia; 26Hematology Unit, Sant’Anna Hospital, Ferrara; 27Hematology Unit, AOU S. Giovanni di Dio e Ruggi D’Aragona Salerno, Salerno; 28Hematology Unit, Santa Chiara Hospital and University, Pisa, Italy Background: Maintenance in FL patients (pts) improves progression free survival (PFS). SARS-Cov2 pandemic posed unique challenges for immunocompromised pts. Aims: The aim is to evaluate the outcome of FL pts in maintenance with antiCD20-MoAb during SARS-Cov2 pandemic and how suspension of therapy affected lymphoma outcome and the risk of SARS-Cov2 infection and its morbidity and mortality. Methods: This is an observational, multicenter, retrospective and prospective study. Results: A total of 420 from 18 Italian Hematological Centers were included in the analysis. Median age was 62 years old (range 27-91 years), 216 pts (51%) were male. Main clinical characteristics of the population were: histological grade 1-2 vs 3A in 288 (69%) vs 109 (26%), while not valuable in 23 (5%) pts; limited I-II vs advanced III-IV stage in 57 (14%) vs 361 (86%) pts, not reported in 2 cases. FLIPI score was low vs intermediate vs high in 71 (17%) vs 151 (36%) vs 192 (46%) patients, respectively, not valuable in 6 cases. All 420 patients included were in maintenance treatment with antiCD20 MoAb at the time of the onset of SARS-Cov2 pandemic (March 2020): 333 (79%) pts were receiving maintenance after a first line, while 87 (21%) after a second line. 342 (81%) pts were receiving Rituximab, while 75 (18%) Obinutuzumab, 3 patients did not start the planned maintenance because of pandemic spread. Status of disease after induction was complete remission (CR) in 374 (89%), partial response (PR) in 41 (10%), progressive disease (PD) in 1, not evaluated in 4 pts, respectively. At the end of maintenance was CR in 265 (63%), PR in 19 (4%), stable disease (SD) in one and PD in 14 (3%) patients, respectively, maintenance is stiil ongoing in 121 (29%) pts. Because of SARS-Cov2 pandemic from March 2020 consequences on maintenance treatment were: temporary suspension in 122 (29%), definitively interruption in123 (29%), no modification in 175 (42%) of pts, respectively. Median number of maintenance treatment administered at the time of SARS-Cov2 pandemic onset was 2 (range 1-12), median number of courses administered at the time of analysis was 8 (range 0-12), in patients who modified treatment because of pandemic median number of performed courses was 7 (range 0-11) and median number of lost cycles were 2 (range 1-12). Pts were divided into two groups according to type of approach to maintenance during pandemic: pts who interrupted maintenance (temporary or definitively): groups A (245 (58%) pts) vs pts who did not modified maintenance: group B (175 (42%) pts). No differences in clinical characteristics, type of therapy and response were observed between the two groups. 29(7%) relapses were observed: 16 (7%) vs 13 (7%) in group A vs B, respectively. 70 (17%) pts experienced SARS-Cov2 positivity: 47 (19%) vs 23 (13%) in group A vs B, respectively. 53 (76%) pts had symptomatic COVID syndrome and 43 (61%) were hospitalized, with no differences between the two groups. Anti-SARS-Cov2 vaccine was administered in 349 patients, serology assessment was done in 46% of cases, showing 21 (13%) reactive vs 138 (87%) not reactive pts, with no differences between the two groups. 21 (30%) pts died because of COVID: 9 (19%) vs 12 (52%) in groups A vs B, respectively. Summary/Conclusion: Suspension of maintenance treatment during SARS-Cov2 pandemic did not show a protection in terms of SARS-Cov2 positivity and morbidity. A trend in lower mortality is suggested. No differences in terms of relapse rate were observed, but longer follow up is needed. P1099: THE SPECTRUM OF SECOND PRIMARY MALIGNANCIES AND CAUSE-SPECIFIC MORTALITY AMONG PATIENTS WITH FOLLICULAR LYMPHOMA IN THE UNITED STATES: A POPULATION-BASED STUDY. K. Chamarti1,*, V. Rudraraju2, S. Kiani1 1Department of Internal Medicine, Texas Tech University Health Sciences Center at Permian Basin, Odessa, United States of America; 2Department of Internal Medicine, Jawaharlal Nehru Medical College, Belgaum, India Background: Follicular lymphoma (FL) is the second most common type of non-Hodgkin lymphoma (NHL). FL accounts for 35 percent of NHLs and has an estimated incidence of 3.18 cases per 1,00,000 people in the United States. The clinical course of FL is variable, and it has median overall survival of 10 to 12 years. In the era of targeted therapies with anti-CD20 antibodies, there is data lacking on the spectrum of second primary malignancies (SPMs) and cause-specific mortality in patients with follicular lymphoma. Aims: In the current study, we aim to identify the risk of SPM and cause specific mortality in FL patients in the era of targeted therapies with anti CD-20 antibodies using a national registry from the United States. Methods: Using the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER)-18, we conducted a retrospective study with patients diagnosed with FL (ICD-0-3 code 9690/3) between 2012 and 2018. We calculated the risk of SPMs developing ≥ 2 months using standardized incidence ratios (SIRs) and cause-specific mortality by standardized mortality ratios (SMRs) and absolute excess risk (AER)/10,000 population. Results: A total of 6,215 patients with follicular lymphoma (FL) were included in this study. The median age at diagnosis was 66 years (range 3 to 85 years) and the median follow-up duration was 31 months (range 0 to 81 months). A total of 355 patients (5.7%) developed 376 SPMs, of which 56% were solid organ malignancies, and 44% were hematologic neoplasms. The latency period for SPM development was 20 months (range 2 to 80 months). The SPM risk for the cohort was significantly elevated (SIR= 1.99, 95% confidence interval [CI] = 1.79-2.2, P<0.05) when compared with the general population. The SIR was higher in females (SIR= 2.16, 95% CI=1.85-2.51) when compared to males (SIR= 1.86, 95% CI= 1.61- 2.13). The SIR for age groups were as follows: age < 65 years (SIR= 2.84, 95% CI= 2.43-3.31), age > 65 years (SIR= 1.59, 95% CI= 1.38-1.82). The risk for hematopoietic SPMs was higher (SIR= 8.66, 95% CI= 7.33-10.15) than solid organ tumors (SIR= 1.30, 95% CI= 1.13-1.49). Specific SPMs with the highest risk included NHL (SIR= 16.24, 95% CI= 13.62-19.22), Hodgkin lymphoma (SIR= 14.31, 95% CI= 5.25-31.15), followed by thyroid cancer (SIR= 4.63, 95% CI= 2.53-7.76), kidney and renal pelvis cancer (SIR= 1.43, 95% CI= 1.55-4.14), and lung cancer (SIR= 2.16, 95% CI= 1.63-2.79). The risk of developing Hodgkin lymphoma, thyroid, and kidney cancers was high in the first year of FL diagnosis. While, the risk for NHL was elevated throughout the follow-up period. At last follow-up, 724 (11.5%) patients had died, of which 65% were due to malignant neoplasms. The risk of mortality due to all malignant cancers combined was statistically significant (SMR= 6.26, 95% CI= 2.00-2.31), largely due to mortality from NHL (SMR= 139.5, 95% CI= 126-154, AER= 286.8), leukemia (SMR= 5.23, 95% CI= 2.99-8.49, AER= 9.63), and infectious diseases (SMR= 2.58, 95% CI= 1.04-5.31, AER=3.19). Summary/Conclusion: Our study reveals that despite therapeutic advances in FL, hematologic neoplasms (including NHL and leukemia) remain the major cause of death in our cohort. Patients with FL are at higher risk of SPM than the general population, most notably for NHL, Hodgkin lymphoma, followed by thyroid, kidney, and lung cancer. These patients may benefit from cancer-specific screening during follow-up. P1100: BRUIN MCL-321: A PHASE 3 OPEN-LABEL, RANDOMIZED STUDY OF PIRTOBRUTINIB VS INVESTIGATOR CHOICE OF BTK INHIBITOR IN PATIENTS WITH PREVIOUSLY TREATED, BTK INHIBITOR NAÏVE MCL (TRIAL IN PROGRESS) T. A. Eyre1, N. N. Shah2, S. Le Gouill3, M. Dreyling4,*, E. Vandenberghe5, W. Jurczak6, Y. Wang7, C. Y. Cheah8, M. Gandhi9, C. Chay10, J. P. Sharman11, D. J. Andorsky12, M. Yin13, M. Balbas13, J. Kherani13, M. L. Wang14 1Oxford University Hospitals NHS Foundation Trust, Churchill Cancer Center, Oxford, United Kingdom; 2Medical College of Wisconsin, Milwaukee, United States of America; 3Service d’hématologie clinique du CHU de Nantes, INSERM CRCINA Nantes-Angers, NeXT Université de Nantes, Nantes, France; 4Medicine III, LMU University Hospital, Munich, Germany; 5Hope Directorate St. James Hospital, Dublin, Ireland; 6Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland; 7Division of Hematology, Mayo Clinic, Rochester, United States of America; 8Linear Clinical Research and Sir Charles Gairdner Hospital, Perth, Australia; 9Virginia Cancer Specialists, Fairfax; 10Messino Cancer Centers, Asheville; 11Willamette Valley Cancer Institute and Research Center, US Oncology Research; 12Rocky Mountain Cancer Centers, US Oncology Research, Eugene; 13Loxo Oncology at Lilly, Stamford; 14University of Texas MD Anderson Cancer Center, Houston, United States of America Background: Covalent Bruton’s Tyrosine Kinase (BTK) inhibitors (BTKi) have transformed the management of relapsed mantle cell lymphoma (MCL), but these treatments are not curative and the majority of patients will require additional treatment. Covalent BTKi share pharmacologic liabilities (e.g. low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage especially in rapidly proliferating tumors with high BTK protein turnover such as MCL. To address these limitations, pirtobrutinib, a highly selective, non-covalent BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency was developed. In the phase 1/2 BRUIN study, pirtobrutinib achieved pharmacokinetic exposures that exceeded its BTK IC96 at trough, was well tolerated, and demonstrated promising efficacy in heavily pretreated, poor-prognosis MCL patients, most of whom had prior treatment with a covalent BTKi. Aims: The purpose of this randomized study is to determine whether pirtobrutinib is superior to investigator’s choice of covalent BTKi in patients with previously treated MCL. Methods: BRUIN MCL-321 is a randomized, open-label, global phase 3 study comparing pirtobrutinib monotherapy versus investigator’s choice of covalent BTKi monotherapy (ibrutinib, acalabrutinib, or zanubrutinib) in patients with previously treated, BTKi naïve MCL. Approximately 500 patients will be randomized 1:1. Randomization will be stratified by sMIPI risk (low/intermediate vs high), comparator BTKi (ibrutinib vs acalabrutinib/ zanubrutinib), and number of prior lines of therapy (1 vs ≥ 2). Eligible patients are adults aged ≥18 years with a confirmed diagnosis of MCL (cyclin D1 overexpression, and ≥ 1 B-cell marker) who have received ≥ 1 prior line of systemic therapy for MCL that did not include a prior BTKi. Patients must have measurable disease per Lugano criteria and must have progressed on or relapsed following the most recent line of therapy prior to study enrollment. Key exclusion criteria include a history of current or prior central nervous system (CNS) involvement, significant cardiovascular disease, stroke, or intracranial hemorrhage within 6 months of randomization, and allogeneic stem cell transplant (SCT), autologous SCT or chimeric antigen receptor (CAR) T-cell therapy within 60 days of randomization. The primary endpoint is progression-free survival (PFS) per Lugano criteria assessed by an independent review committee (IRC), with the goal of demonstrating superiority of pirtobrutinib over investigator’s choice of covalent BTKi. Secondary endpoints include overall response rate (ORR), duration of response (DoR), investigator-assessed PFS per Lugano criteria, overall survival, event-free survival, time to treatment failure, time to next treatment, PFS2 (time from randomization to disease progression on next line of treatment or death from any cause), safety and tolerability, and patient reported outcomes. This global study is currently enrolling patients (NCT04662255). Results: This study is a Trial in Progress. The results will be presented at a later date. Summary/Conclusion: This study is a Trial in Progress. The conclusions will be presented at a later date. P1101: PIRTOBRUTINIB, A HIGHLY SELECTIVE, NON-COVALENT (REVERSIBLE) BTK INHIBITOR IN PREVIOUSLY TREATED MANTLE CELL LYMPHOMA: UPDATED RESULTS FROM THE PHASE 1/2 BRUIN STUDY T. A. Eyre1,*, M. L. Wang2, N. N. Shah3, A. J. Alencar4, J. N. Gerson5, M. R. Patel6, B. Fakhri7, E. Vandenberghe8, W. Jurczak9, X. N. Tan10, K. L. Lewis10, T. Fenske3, Y. Wang11, C. C. Coombs12, I. Flinn13, D. Lewis14, S. Le Gouill15, M. Gandhi16, C. Chay17, M. L. Palomba18, J. A. Woyach19, J. M. Pagel20, N. Lamanna21, J. P. Sharman22, D. J. Andorsky23, J. B. Cohen24, M. A. Barve25, P. Ghia26,27, M. Yin20, P. L. Zinzani28, C. Ujjani29, Y. Koh30, K. Izutsu31, E. Lech-Maranda32, J. Kherani20, C. Tam33, S. Sundaram34, B. Nair20, D. E. Tsai20, M. Balbas20, A. R. Mato18, C. Y. Cheah10 1Oxford University Hospitals NHS Foundation Trust, Churchill Cancer Center, Oxford, United Kingdom; 2MD Anderson Cancer Center, Houston; 3Medical College of Wisconsin, Milwaukee; 4Sylvester Comprehensive Cancer Center, Miami; 5University of Pennsylvania, Philadelphia; 6Florida Cancer Specialists/Sarah Cannon Research Institute, Sarasota; 7University of California San Francisco, San Francisco, United States of America; 8Hope Directorate St. James Hospital, Dublin, Ireland; 9Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland; 10Linear Clinical Research and Sir Charles Gairdner Hospital, Perth, Australia; 11Division of Hematology, Mayo Clinic, Rochester; 12University of North Carolina at Chapel Hill, Chapel Hill; 13Sarah Cannon Research Institute, Nashville, United States of America; 14University Hospitals Plymouth NHS, Plymouth, United Kingdom; 15Service d’hématologie clinique du CHU de Nantes, INSERM CRCINA Nantes-Angers, NeXT Université de Nantes, Nantes, France; 16Virginia Cancer Specialists, Fairfax; 17Messino Cancer Centers, Asheville; 18Memorial Sloan Kettering Cancer Center, New York; 19The Ohio State University Comprehensive Cancer Center, Columbus; 20Loxo Oncology at Lilly, Stamford; 21Herbert Irving Comprehensive Cancer Center, Columbia University, New York; 22Willamette Valley Cancer Institute and Research Center, US Oncology Research; 23Rocky Mountain Cancer Centers, US Oncology Research, Eugene; 24Winship Cancer Institute, Emory University, Atlanta; 25Mary Crowley Cancer Research, Dallas, United States of America; 26Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy; 27Medicine III, LMU University Hospital, Munich, Germany; 28Institute of Hematology “Seràgnoli” University of Bologna, Bologna, Italy; 29Fred Hutchinson Cancer Research Center, Seattle, United States of America; 30Internal Medicine, Seoul National University Hospital, Seoul, South Korea; 31National Cancer Center Hospital, Tokyo, Japan; 32Institute of Hematology and Transfusion Medicine, Warsaw, Poland; 33Peter MacCallum Cancer Center, Royal Melbourne Hospital, and University of Melbourne, Melbourne, Australia; 34Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, United States of America Background: Covalent BTK inhibitors (BTKi) have transformed the management of mantle cell lymphoma (MCL), but these treatments are not curative and the majority of patients (pts) will require additional treatment. Covalent BTKi share pharmacologic liabilities (e.g. low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage, for example in rapidly proliferating tumors with high BTK protein turnover such as MCL. To address these limitations, pirtobrutinib, a highly selective, non-covalent (reversible) BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency was developed. Aims: BRUIN is a multicenter phase 1/2 study (NCT03740529) of oral pirtobrutinib monotherapy in pts with advanced B-cell malignancies who have received ≥2 prior therapies. Methods: Pirtobrutinib was dose escalated in a standard 3 + 3 design in 28-day cycles. The primary objective for phase 1 was to determine the recommended phase 2 dose (RP2D) and the primary objective of phase 2 was overall response rate (ORR); secondary objectives included duration of response (DoR), progression-free survival (PFS), overall survival (OS), safety and tolerability, and pharmacokinetics. Efficacy evaluable pts included all dosed pts who underwent their first response evaluation or discontinued therapy. Response was assessed every 8 weeks from cycle 3, and every 12 weeks from cycle 13 and was measured according to Lugano Classification. Safety was assessed in all pts (CLL/SLL and NHL). Results: As of 27 September 2020, 323 pts (170 CLL/SLL, 61 MCL, 26 WM, 26 DLBCL, 13 MZL, 12 FL, 9 RT, and 6 other NHL [other transformation, B-PLL and hairy cell leukemia]) were treated on 7 dose levels (25-300mg QD). Median age was 69 (range 50-87) years for MCL pts. Among the 61 MCL pts, median number of prior lines of therapy was 3 (range, 1-8) and a majority of them had received a prior BTKi (93%), an anti-CD20 antibody (98%) or chemotherapy (92%). No DLTs were reported and MTD was not reached (n=323). 200mg QD was selected as the RP2D. Fatigue (20%), diarrhea (17%) and contusion (13%) were the most frequent treatment-emergent adverse events regardless of attribution or grade seen in ≥10% of pts (n=323). The most common adverse event of grade ≥3 was neutropenia (10%). Treatment-related hemorrhage and hypertension occurred in 5 (2%) and 4 (1%) pts, respectively. Five (1%) pts discontinued due to treatment-related adverse events. At the efficacy cutoff date, 52 prior BTKi treated MCL pts were efficacy evaluable with an ORR of 52% (95% confidence interval 38-66; 13 complete response (CR) [25%], 14 partial response (PR) [27%], 9 stable disease (SD) [17%]), 11 progressive disease (PD) [21%] and 5 [10%] discontinued prior to first response assessment). Median follow up was 6 months (range 0.7-18.3+). Responses were observed in 9/14 pts (64%) with prior autologous or allogeneic stem cell transplant, and 2 of 2 with prior CAR-T cell therapy. Summary/Conclusion: Pirtobrutinib demonstrated promising efficacy in heavily pretreated, poor-prognosis MCL following multiple prior lines of therapy, including a covalent BTKi. Pirtobrutinib was well tolerated and exhibited a wide therapeutic index. Updated data, including approximately 60 new pts with MCL and an additional 10 months since the prior data cut will be presented. P1102: A PHASE 1 STUDY OF PARSACLISIB IN COMBINATION WITH RITUXIMAB, BENDAMUSTINE + RITUXIMAB, OR IBRUTINIB IN PATIENTS WITH PREVIOUSLY TREATED B-CELL LYMPHOMA (CITADEL-112): PRELIMINARY SAFETY RESULTS J.-M. Sancho1,*, A. Lopez-Guillermo2, P. Abrisqueta3, A. Kumar4, R. Cordoba5, M. Tani6, W. Zhao7, E. Rappold7, P. Langmuir7, M. Mims8 1Clinical Hematology Department, Hospital Germans Trias i Pujol, Institut Català d’Oncologia; 2Clinic Barcelona; 3Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), Hospital Universitari Vall d’Hebron, Barcelona, Spain; 4University of Arizona, Tucson, United States of America; 5Lymphoma Unit, Department of Hematology, Fundacion Jimenez Diaz University Hospital, Health Research Institute IIS-FJD, Madrid, Spain; 6Haematology Unit, Santa Maria delle Croci Hospital, Ravenna, Italy; 7Incyte Corporation, Wilmington; 8Department of Medicine, Hematology/Oncology, Baylor College of Medicine, Houston, United States of America Background: Rituximab-based chemoimmunotherapy regimens are backbone treatment (Tmt) for both indolent (follicular [FL], marginal zone [MZL]) and aggressive (diffuse large B-cell [DLBCL], mantle cell [MCL]) B-cell lymphomas. Standard of care (SoC) for relapsed or refractory (R/R) disease includes anti-CD20 in combination with chemotherapy and targeted therapies, such as Bruton’s tyrosine kinase inhibitors (eg, ibrutinib) and phosphoinositide 3-kinase (PI3K) inhibitors. Parsaclisib is a potent and highly selective next generation PI3Kδ inhibitor that is currently being investigated in hematological malignancies. Aims: CITADEL-112 (NCT03424122) is an open-label phase 1 study evaluating the safety and tolerability of adding parsaclisib to investigator choice SoC Tmt rituximab (RIT), RIT + bendamustine (BEN), or ibrutinib (IBR) in patients (pts) with R/R B-cell lymphoma. Methods: Enrolled pts were ≥18 years and had histologically confirmed DLBCL, FL, MCL, or MZL, ECOG PS 0–2, were R/R to ≥1 (≥2 for FL) prior systemic therapy, and ineligible for stem cell transplant. Pts received parsaclisib 20 mg orally once daily (QD) for 8 weeks then 20 mg once weekly (QW) in combination with either: RIT 375 mg/m2 IV QW for 4 doses in cycle 1 (± cycle 2) (Tmt A); RIT 375 mg/m2 IV on day 1 + BEN 90 mg/m2 on day 1 and day 2 of each 28-day cycle for ≤6 cycles (Tmt B); or IBR 560 mg QD (Tmt C). Pts received treatment until disease progression, unacceptable toxicity, or withdrawal. Results: At data cutoff (May 14, 2021), 50 pts were treated (16 pts each in Tmt A and C, 18 pts in Tmt B) and 13 pts were ongoing treatment (3 pts in Tmt A, 8 pts in Tmt B, 2 pts in Tmt C). Most pts had received ≥2 prior systemic treatments (81.3%, 61.1%, and 68.8% in Tmt A [range 1–4], B [range 1–4], and C [range 1–7], respectively). The most common reasons for discontinuation were progressive disease (56.3%, 38.9%, and 50.0%) and adverse events (AEs) (12.5%, 11.1%, and 6.3% in Tmt A, B, and C, respectively). One pt in Tmt B experienced a dose-limiting toxicity of grade 4 neutropenia for >14 days. All pts experienced at least 1 treatment-emergent AE (TEAE); in Tmt A, 75.0% had grade ≥3 and 37.5% had serious TEAEs; Tmt B, 83.3% had grade ≥3 and 27.8% had serious TEAEs; and Tmt C, 62.5% had grade ≥3 and 43.8% had serious TEAEs. Common any-grade TEAEs (≥30%) included neutropenia (62.5%), diarrhea (37.5%), and anemia (31.3%) in Tmt A; neutropenia (50.0%), abdominal pain, asthenia, diarrhea, and nausea (each 33.3%) in Tmt B; neutropenia (50.0%) and increased ALT and increased AST (each 37.5%) in Tmt C. Most common grade ≥3 TEAEs (≥15%) were neutropenia (50.0%) and diarrhea (18.8%) in Tmt A, and neutropenia (38.9% and 25.0%) in Tmt B and Tmt C, respectively. Serious TEAEs occurring in >1 pt were COVID-19, diarrhea, and pneumonia (n = 2 each) in Tmt A, and atrial fibrillation (n = 2) in Tmt C. TEAEs with fatal outcome were reported in 2 pts in Tmt A (COVID-19 and COVID-19 pneumonia [n = 1], interstitial lung disease [n = 1]) and 1 pt in Tmt C (COVID-19, acute kidney injury). Parsaclisib dose interruption or dose reduction due to TEAEs occurred in 75.0% and 18.8% of pts, respectively, in Tmt A; 66.7% and 27.8% of pts, respectively, in Tmt B; and 56.3% and 18.8% of pts, respectively, in Tmt C. Summary/Conclusion: Parsaclisib 20 mg QD for 8 weeks followed by 20 mg QW can be safely combined with RIT, RIT + BEN, or IBR in pts with R/R B-cell lymphomas. The tolerability profile of the combination regimens was manageable, with no unexpected safety concerns. P1103: INMIND: A PHASE 3 STUDY OF TAFASITAMAB PLUS LENALIDOMIDE AND RITUXIMAB VERSUS PLACEBO PLUS LENALIDOMIDE AND RITUXIMAB FOR RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA (FL) OR MARGINAL ZONE LYMPHOMA (MZL) L. H. Sehn1,*, K. Hübel2, S. Luminari3, A. Salar4, B. E. Wahlin5, A. K. Gopal6, C. Bonnet7, S. Paneesha8, M. Trneny9, H. Mashegu10, C. Lihou10, D. Li10, C. W. Scholz11 1BC Cancer Centre for Lymphoid Cancer and The University of British Columbia, Vancouver, Canada; 2Department of Internal Medicine I Oncology and Hematology, University Hospital Cologne, Cologne, Germany; 3Hematology Unit, Azienda USL-IRCCS di Reggio Emilia, Reggio Emilia, Italy; 4Department of Haematology, Hospital del Mar-IMIM, Barcelona, Spain; 5Department of Medicine, Unit of Hematology, Karolinska Institute, Stockholm, Sweden; 6Division of Medical Oncology, University of Washington Medicine, Seattle, United States of America; 7Clinical Hematology, Centre Hospitalier Universitaire, University of Liège, Liège, Belgium; 8University Hospitals Birmingham NHS Foundation Trust, Birmingham, United Kingdom; 9First Department of Medicine, First Faculty of Medicine, Charles University, General Hospital, Prague, Czechia; 10Incyte Corporation, Wilmington, United States of America; 11Department of Hematology and Oncology, Vivantes Klinikum Am Urban, Berlin, Germany Background: Patients (pts) with indolent non-Hodgkin lymphoma (NHL) subtypes FL or MZL respond to first-line treatment but relapse is common; there is no standard treatment for pts with relapsed/refractory (R/R) FL or MZL. Tafasitamab (TAFA) is an Fc-engineered humanized monoclonal antibody (mAb) against CD19, which is broadly expressed in FL and MZL and regulates proliferation via B-cell receptor signaling. In preclinical studies, TAFA has shown activity against NHL cell lines in combination with rituximab (anti-CD20 mAb) and lenalidomide (LEN). TAFA monotherapy demonstrated promising clinical activity in a phase 2a study in pts with R/R NHL (NCT01685008), with an ORR of 29% (n/N=10/34) in pts with FL and 33% (n/N=3/9) in pts with MZL. In an ongoing phase 2, single-arm study (L-MIND, NCT02399085), TAFA + LEN followed by TAFA alone demonstrated an ORR of 57.5% (n/N=46/80) with CR of 40% (n/N=32/80) and median DOR of 34.6 mo (95% CI: 26.1–NR), in pts with R/R diffuse large B-cell lymphoma (FDA approved indication). These observations suggest a potential clinical benefit of TAFA + LEN and rituximab for pts with R/R FL or MZL. Aims: Phase 3 double-blind, placebo-controlled, randomized study designed to investigate whether TAFA + LEN and rituximab provides improved clinical benefit compared with LEN and rituximab in pts with R/R FL or MZL. Methods: Pts will be randomized 1:1 to receive TAFA (12 mg/kg IV on days 1, 8, 15, and 22 of 28-day cycle [cycles 1–3], then days 1 and 15 [cycles 4–12]) + LEN (20 mg PO QD [or starting dose 10 mg PO QD if creatinine clearance ≥30 to <60 mL/min], days 1–21/cycle for 12 cycles) and rituximab (375 mg/m2 IV on days 1, 8, 15, and 22 of cycle 1, then day 1 of cycles 2–5), or placebo (0.9% saline solution IV) + LEN and rituximab. Stratification will include progression of disease within 24 mo (FL; yes vs no), refractoriness to prior anti-CD20 mAb therapy (FL; yes vs no), and number of prior lines of therapy (FL or MZL; <2 vs ≥2). Radiological (PET) assessment will be performed at baseline, every 12 (± 2) weeks in year 1, 16 (± 2) weeks in years 2–3, and 24 (± 3) weeks in years 4–6; additional assessment performed within 4 (± 2) weeks if progressive disease confirmed before end-of-treatment (EOT) visit. The primary study endpoint is PFS (investigator assessed [INV] by Lugano 2014 criteria) for pts with FL. Key secondary endpoints are PFS (INV) in the overall population (FL and MZL), PET-CR rate (INV) at EOT (90 days after last treatment) and OS in pts with FL. Other secondary endpoints include PET-CR rate (INV) at EOT and OS in the overall population, and ORR (INV), DOR (INV), health related quality of life, safety, and minimum residual disease-negativity rate at EOT in FL and the overall population. Inclusion criteria include age ≥18 y, histologically confirmed FL (grade 1, 2, or 3a) or MZL (nodal, splenic, or extranodal), documented R/R disease, ≥1 prior systemic anti-CD20 therapy (including anti-CD20 refractory disease), ECOG PS ≤2, adequate systemic organ function, and high tumor burden (per GELF criteria). Exclusion criteria include prior rituximab + LEN treatment, history of radiotherapy for other diseases (≥25% of bone marrow), nonhematologic malignancy, congestive heart failure (LVEF <50%), active systemic infection, known CNS lymphoma, or severe immunocompromised state. inMIND (NCT04680052, EudraCT2020-004407-13) is currently enrolling pts; planned enrollment is 528 pts with R/R FL and 60–90 pts with R/R MZL across North America, Europe, and Asia Pacific. Results: Not applicable. Summary/Conclusion: Not applicable. P1104: A PHASE 1 STUDY EVALUATING SAFETY AND EFFICACY OF PARSACLISIB IN COMBINATION WITH BENDAMUSTINE + OBINUTUZUMAB IN PATIENTS WITH RELAPSED OR REFRACTORY FOLLICULAR LYMPHOMA (CITADEL-102) M. Hamadani1,*, M. Coleman2, R. Boccia3, J. Duras4, M. Hutchings5, P. L. Zinzani6, R. Cordoba7, M. B. Oreiro8, V. Williams9, M. Stouffs9, P. Langmuir9, J.-M. Sancho10 1Division of Hematology and Oncology, Medical College of Wisconsin, Milwaukee; 2Clinical Research Alliance Inc, Westbury; 3Center for Cancer and Blood Disorders, Bethesda, United States of America; 4Department of Haematooncology, University Hospital Ostrava and Faculty of Medicine, University of Ostrava, Ostrava, Czechia; 5Department of Haematology and Phase 1 Unit, Rigshospitalet, Copenhagen, Denmark; 6Institute of Hematology “Seràgnoli”, University of Bologna, Bologna, Italy; 7Lymphoma Unit, Department of Hematology, Fundación Jimenez Diaz University Hospital; 8Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; 9Incyte Corporation, Wilmington, United States of America; 10Clinical Hematology Department, Institut Català d’Oncologia-Hospital Germans Trias i Pujol, Barcelona, Spain Background: Patients (pts) with follicular lymphoma (FL) generally respond well to first-line CD20-targeted therapies, such as obinutuzumab or rituximab-based regimens. However, many pts relapse and studies suggest that each subsequent relapse is associated with shorter durations of response to the next treatment. Parsaclisib is a potent and highly selective next generation PI3Kδ inhibitor. The combination of bendamustine + obinutuzumab is approved for pts with relapsed/refractory (R/R) FL. We hypothesized that adding parsaclisib may improve clinical benefit with a manageable safety profile in this pt population. Aims: CITADEL-102 (NCT03039114) is an open-label, phase 1, dose-finding study that investigated safety and efficacy of parsaclisib in combination with bendamustine + obinutuzumab in pts with R/R FL following rituximab-containing regimens. Methods: Pts enrolled were ≥18 years with histologically confirmed CD20-positive FL, R/R to any prior rituximab-containing regimen, ECOG PS 0–2, ≥1 measurable lesion, and ≤4 prior therapies. Pts received parsaclisib 20 mg orally once daily (QD) for 8 weeks then 20 mg once weekly (QW); bendamustine 90 mg/m2 infusion on days 1 and 2 of cycles 1–6; and obinutuzumab 1000 mg infusion on days 1, 8, and 15 of cycle 1, and day 1 of cycles 2–6, and on every second cycle of cycles 8–30 in pts having complete response/complete metabolic response (CR/CMR), partial response/partial metabolic response (PR/PMR), or stable disease/no metabolic response. Part 1 (safety run-in) used a 3 + 3 design with dose de-escalation to identify the maximum tolerated dose (MTD) of parsaclisib in combination with bendamustine + obinutuzumab. In Part 2 (dose expansion), the safety and efficacy of this combination were further evaluated. The primary study endpoint was safety and tolerability; secondary endpoints included efficacy outcomes (ORR, DOR, PFS, and OS). Results: A total of 26 pts were enrolled and treated; median (range) age was 65.0 (44–80) years, 25 (96.2%) had ECOG PS ≤1, 11 (42.3%) had ≥2 prior systemic therapies, and 6 (23.1%) had received prior bendamustine. Median (range) parsaclisib exposure was 10.6 (0.4–32.8) months. Main reasons for treatment discontinuation included adverse events (AEs) (8 pts, 30.8%) and progressive disease (6 pts, 23.1%). All pts experienced treatment-emergent AEs (TEAEs); most common any-grade TEAEs (≥10 pts) were pyrexia (53.8%), neutropenia (50%), diarrhea (46.2%), thrombocytopenia, and nausea (each 38.5%). Grade ≥3 TEAEs were experienced by 88.5% of pts; most common grade ≥3 TEAEs (≥2 pts) were neutropenia (34.6%), febrile neutropenia (23.1%), thrombocytopenia (19.2%), ALT and AST increase (each 11.5%), and diarrhea, neutrophil count decreased, and rash maculopapular (each 7.7%). One of 6 evaluable pts in Part 1 had a DLT of grade 4 QTc elongation. The MTD was not reached, and parsaclisib 20 mg QD for 8 weeks then 20 mg QW was the selected dosage for dose expansion in Part 2. Treatment discontinuation due to TEAEs was 30.8%, 7.7%, and 15.4% for parsaclisib, bendamustine, and obinutuzumab, respectively. One fatal TEAE (COVID-19 pneumonia) occurred. ORR (95% CI) as reported by the investigator was 76.9% (56.4–91.0), with 17 pts (65.4%) achieving CR/CMR and 3 pts (11.5%) achieving PR/PMR as the best overall response. Median DOR, PFS, and OS were not reached. Summary/Conclusion: Parsaclisib in combination with bendamustine + obinutuzumab appears to have a manageable safety profile and demonstrated promising efficacy in pts with R/R FL. P1105: FINAL RESULTS OF THE PHASE I/II HOVON124/ECWM-R2 STUDY INCLUDING 2-YEAR RITUXIMAB MAINTENANCE AFTER INDUCTION WITH IXAZOMIB, RITUXIMAB AND DEXAMETHASONE IN RELAPSED WALDENSTRÖM’S MACROGLOBULINEMIA M.-A. Dimopoulos1,*, K. Amaador2,3, M. C. Minnema4, K. Nasserinejad5, M. Kap5, E. Kastritis1, M. Gavriatopoulou1, W. Kraan3,6, M. E. D. Chamuleau7, D. Deeren8, L. Tick9, J. K. Doorduijn10, F. Offner11, L. H. Böhmer12, R. D. Liu2, S. T. Pals3,6, J. M. Vos2,3, M. J. Kersten2,3 1Department of Clinical Therapeutics, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 2Department Of Hematology, Amsterdam UMC, University of Amsterdam, Cancer Center Amsterdam; 3LYMMCARE (Lymphoma and Myeloma Center Amsterdam), Amsterdam; 4Department of Hematology, University Medical Center Utrecht, University Utrecht, Utrecht; 5HOVON Data Center, Department of Hematology, Erasmus MC Cancer Institute, Rotterdam; 6Department of Pathology, Amsterdam UMC, University of Amsterdam, Cancer Center Amsterdam; 7Department of Hematology, Amsterdam UMC, VU University, Amsterdam, Netherlands; 8Department of Hematology, AZ Delta, Roeselare, Belgium; 9Department of Hematology, Maxima Medical Center, Eindhoven; 10Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, Netherlands; 11Department of Hematology, University Hospital Gent, Gent, Belgium; 12Department of Hematology, Haga Teaching Hospital, The Hague, Netherlands Background: In the phase I/II HOVON124/ECWM-R2 trial, induction treatment with the combination of ixazomib, subcutaneous (s.c.) rituximab and dexamethasone (IRd) showed promising efficacy with manageable toxicity in patients with relapsed Waldenström’s Macroglobulinemia (WM). Aims: Here we report the final analysis of the trial after two years of rituximab maintenance and with a median follow-up (FU) on study of 45.6 months (range, 12.4-72.2). Methods: In total, 59 patients were enrolled (median age, 69 years; range, 46-91 years) of which 48 patients completed at least six cycles of induction with IRd; 41 patients (median age 66 years, 66% male) with at least a minimal response (MR) continued to 2 years of rituximab maintenance (1400 mg s.c., q 3 months) starting 3 months after the last induction cycle. The primary endpoint of the study was overall response rate (ORR, ≥ MR)) after 8 induction cycles and was 71% (Kersten/Amaador et al, JCO, 2022). Secondary endpoints included progression-free survival (PFS), overall survival (OS) and ORR after 2 years of rituximab maintenance and improvement of response after maintenance. Results: In total, 22 (54%) out of 41 patients completed 2 years of rituximab maintenance. The median number of cycles was 8 (range, 1-8). Nineteen patients did not complete maintenance treatment due to progression (n=16), excessive toxicity (n=1), non-compliance (n=1), and other unknown reason (n=1). The best ORR after maintenance was 85% (2% complete response [CR], 24% very good partial response [VGPR], 39% partial response [PR], and 20% MR). Improvement of response after maintenance occurred in 9 (22%) patients; VGPR to CR in one (2%), PR to VGPR in 6 (15%), and MR to PR in 2 (5%). A further decrease in IgM levels was seen after 2 years of maintenance (IgM 3.62 g/dl at baseline; 1.3 g/dl after induction and 0.42 g/dl after end of maintenance, p-value<0.001; Figure 1A), accompanied by a further increase in Hb levels (Hb 10.5 to 14.3 g/dl, p-value<0.001; Figure 1B). The median progression-free survival (PFS) was 23.6 months (95% CI, 13.4 to 43.2; Figure 1C), and median overall survival (OS) was not reached; at 45 months, 85% of patients were alive (95% CI, 0.72 to 0.92; Figure 1D). The median time to best response was 7 months, the median duration of response (DOR) was 34.8 months, and the median time to next treatment (TTNT) was 36 months[MM(1]. After a median FU of 45.6 months, 29 patients had received subsequent therapy and the median time to progression after subsequent therapy was 53.7 months[MM(2]. During maintenance, rituximab was administered i.v. instead of s.c. in 7 (17%) patients. None of the patients treated with s.c. rituximab developed systemic hypersensitivity reactions but 2 patients had an injection site reaction (1 grade 1, 1 grade 2). Grade 3/4 toxicity was seen in 10% and 5% of patients, respectively (grade 3/4 neutropenia (n=1 and n=2), grade 3 chronic kidney disease (n=1), and grade 3 elevations of transaminases (n=2)). In the 41 patients who started maintenance, 8 SAEs were reported in 6 patients, of which 4 occurred during maintenance (mostly infections) and 4 (mostly secondary malignancies, considered unrelated to study drug) during FU. Three patients died during maintenance therapy due to progressive disease, intracranial bleeding, and acute myeloid leukemia, respectively. Image: Summary/Conclusion: Rituximab maintenance after IRd induction is feasible and well tolerated. The ORR improved in 22% of patients, confirming the efficacy of this chemo-free regimen in combination with rituximab maintenance in relapsed/refractory WM. P1106: PRELIMINARY RESULTS OF A PHASE 1 STUDY OF NOVEL BCL-2 INHIBITOR LISAFTOCLAX (APG-2575) IN CHINESE PATIENTS (PTS) WITH RELAPSED OR REFRACTORY (R/R) NON-HODGKIN LYMPHOMAS (NHLS) M. Sun1,*, J. Qi1, Y. Song2, K. Zhou2, H. Liu3, A. Shen3, L. Geng3, F. Zhou4, J. Huang4, Z. Chen5, H. Zhang5, M. Lu6, M. Ahmad6, L. Men5, D. Yang7, Y. Zhai6, J. Wang1 1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin; 2Department of Hematology, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou; 3First Affiliated Hospital of University of Science and Technology of China (USTC) Anhui Provincial Hospital, Hefei; 4Zhongnan Hospital, Wuhan University, Wuhan; 5Ascentage Pharma (Suzhou) Co., Ltd., Suzhou, China; 6Ascentage Pharma Group Inc., Rockville, United States of America; 7State Key Laboratory of Oncology in South China Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China Background: Many B-cell malignancies evade apoptosis by overexpressing BCL-2 proteins. Although indicated for the management of certain hematological malignances (HMs), BCL-2 inhibitor (BCL-2i) venetoclax requires a slow dose ramp-up to reduce the risk of tumor lysis syndrome (TLS) and is associated with severe neutropenia. Investigational lisaftoclax is a novel, potent, selective BCL-2i, which was previously reported to have activity against HMs in US and Australian pts. Lisaftoclax also offers a daily (rather than weekly) ramp-up to the target dose. Aims: The objective of this study is to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of lisaftoclax in Chinese pts with R/R NHLs. Methods: In this study, pts were dosed with lisaftoclax orally once daily in 28-day cycles. A daily ramp-up schedule was used in pts with chronic lymphocyte leukemia (CLL) or NHL with medium/high-risk TLS. A regular schedule was used in pts without CLL and those with NHL with low-risk TLS. Results: As of January 1, 2022, 40 pts (median age 55.5 [range 30-79] years; 72.5% male) had been treated with lisaftoclax (dose range 20-800 mg), including 20 who remain in the trial. The median number of prior therapies was 4 (range 0-14). NHL subtypes included: 12 CLL, 9 follicular, 7 mantle cell (MCL), 4 marginal zone (MZL), 4 T-cell, 2 diffuse large B-cell, and 2 Waldenström macroglobulinemia. DLT, MTD, and laboratory/clinical TLS were not observed, and the RP2D was 600 mg. Treatment-emergent adverse events (TEAEs) were reported in 36 pts (90%), of which most were grade 1-2 (67.5%). Common TEAEs (≥ 15% all grades) were hyperuricemia (38.6%), hypertriglyceridemia and anemia (each 36.4%), thrombocytopenia (29.5%), hyperphosphatemia (27.3%), neutropenia (25%), proteinuria (22.7%), leukopenia (18.2%), and diarrhea (15.9%). Common grade 3-4 TEAEs (> 5%) were neutropenia (13.6%) and thrombocytopenia (9.1%). A total of 4 (10%) pts experienced serious AEs, including 1 small intestinal obstruction, 1 pneumonia, 1 wound hemorrhage, and 1 case each of anemia and thrombocytopenia. One pt had dose reduction because of grade 2 colitis, and 1 pt receiving 100 mg discontinued because of grade 4 thrombocytopenia. With a medium treatment duration of 4 (range 4-20) cycles, 12 of 32 (37.5%) evaluable pts reached a complete response (CR; n = 4) or partial response (PR; n = 8), including 7 with CLL, 2 MCL, 2 MZL, and 1 T-cell NHL. In pts with CLL, the overall response rate (ORR) was 63.6%, CR 27.3%, and PR rate 36.4%. At doses ≥ 200 mg, the ORR was 87.5%. A total of 8 of 11 pts had at least 1 adverse prognostic factor (TP53 mutation/17p-/IGHV unmutated/complex chromosomal abnormalities). Of 11 pts, 5 had disease that previously failed BTKi or CD20 monoclonal antibody therapy. Systemic exposure increased across lisaftoclax doses from 20 to 800 mg, with an average half-life of 4 to 6 hours. There was no significant accumulation following the once-daily dosing. Summary/Conclusion: Here, we report for the first time clinical data for lisaftoclax in Chinese pts with R/R NHLs. Lisaftoclax was well tolerated at doses of up to 800 mg/day, with no TLS observed (despite the daily ramp-up), and was active against CLL/SLL, MZL, MCL, and T-cell NHL subtypes. Lisaftoclax may offer a more convenient treatment alternative, with a daily ramp-up schedule that may be more pt friendly. ClinicalTrials.gov: NCT03913949. P1107: CLINICAL OUTCOMES OF SOLID ORGAN TRANSPLANT PATIENTS WITH EBV+ PTLD WHO FAIL RITUXIMAB PLUS CHEMOTHERAPY: A MULTINATIONAL, RETROSPECTIVE CHART REVIEW STUDY V. Dharnidharka1, D. Thirumalai2, U. Jaeger3,*, W. Zhao2, D. Dierickx4, P. Xun2, P. Minga5, A. Sawas6, N. Sadetsky7, P. Chauvet8, E. Sundaram9, A. Barlev7, H. Zimmerman10, R. U. Trappe10 1Washington University School of Medicine & St. Louis Children’s Hospital, St Louis; 2Atara Biotherapeutics, Thousand Oaks, United States of America; 3Medical University of Vienna, Vienna, Austria; 4Universitair Ziekenhuis Leuven, Leuven, Belgium; 5Niguarda Ca’ Granda Hospital, Milan, Italy; 6Herbert Irving Comprehensive Cancer Center, New York; 7Atara Biotherapeutics, South San Francisco, United States of America; 8CHU Lille, Lille, France; 9Nashville Bioscences, Nashville, United States of America; 10DIAKO Bremen, Bremen, Germany Background: Post-transplant lymphoproliferative disease (PTLD) is a rare and often aggressive disease i n the setting of immunosuppression following solid organ transplant (SOT). Epstein-Barr virus (EBV) infection of B cells is responsible for about 50% of cases, either due to reactivation of the virus after transplantation or primary EBV infection. Although there is no approved therapy for patients (pts) with PTLD, guidelines include reduction of immunosuppression (RIS) as a part of initial treatment and may be sufficient for pts with early lesions. Rituximab, either as monotherapy or in combination with chemotherapy (CT), is used in addition to RIS as initial treatment. Pts with EBV+ PTLD following SOT who fail rituximab plus CT have poor outcomes with limited treatment options. Published data on clinical outcomes of these pts remain limited and not well documented. Aims: To characterize the outcomes for pts diagnosed with EBV+ PTLD following SOT who fail initial rituximab plus CT in a multi- national real-world setting. Methods: We conducted a large multinational, multicenter, retrospective chart review study of pts with EBV+ PTLD following allogeneic hematopoietic cell transplantation (HCT) or SOT who received rituximab or rituximab plus CT between January 2000‒December 2018 and were refractory (failed to achieve complete response [CR] or partial response [PR]) or relapsed at any point after such therapy. Data were collected from 29 centers across North America (United States and Canada) and the European Union. This analysis includes pts with EBV+ PTLD following SOT who were refractory/relapsed to rituximab plus CT. The Kaplan-Meier method was utilized to estimate the overall survival (OS). Results:: A total of 86 pts with EBV+ PTLD following SOT who failed rituximab plus CT were included in the analysis; 65 (75.6%) pts were refractory while 21 (24.4%) relapsed after initial response of CR or PR. Median age at PTLD diagnosis was 43 years (range 1‒78) and median time to PTLD onset from transplant was 1.7 years (range 0.1‒27.9). Median follow up time was 12.9 months from the date of PTLD diagnosis. PTLD histological subtypes were 66 (76.7%) monomorphic, 18 (20.9%) polymorphic, and 2 (2.3%) early lesions. The most common PTLD subtype was diffuse large B-cell lymphoma (DLBCL) (58, 67.4%). Of the 86 pts, 49 (57%) received CT following rituximab monotherapy while 37 (43%) pts received CT concurrently with rituximab. Overall, 63 (73.3%) pts died. PTLD-specific mortality was observed in 41 (65.1%) pts, treatment-related mortality in 10 (15.9%) pts, mortality due to organ rejection/failure in 2 (3.2%) pts, mortality due to other causes in 7 (11.1%) pts, and mortality due to unknown causes in 3 (4.8%) pts. Median OS was 15.5 months (95% confidence interval [CI]: 8.3‒22.9) from PTLD diagnosis, and was 4.1 months (95%CI: 1.9‒8.5) from the earliest date when pts became refractory or relapsed following rituximab plus CT. Image: Summary/Conclusion: The prognosis for pts with EBV+ PTLD following SOT who fail rituximab plus CT remains poor, with an estimated median OS of about 4 months and a majority of pts dying from PTLD and related treatment. In this specific population, there remains a significant unmet medical need for effective and well-tolerated therapies. P1108: HIGH COMPLETE RESPONSE RATE FOLLOWING POINT-OF-CARE ANTI CD19 CAR T-CELL THERAPY IN PATIENTS WITH RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA S. Fried1,2,*, M. J. Besser3,4, E. Shkury1,2, R. Yerushalmi1,2, N. Shem-Tov1,2, I. Danylesko1,2, E. Jacoby2,5, O. Itzhaki4, R. Shouval1,2,6, M. Kedmi1,2,7, A. Shimoni1,2, A. Nagler1,2, A. Avigdor1,2 1Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel Hashomer; 2Sackler School of Medicine; 3Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel-Aviv; 4Ella Lemelbaum Institute for Immuno Oncology; 5Department of Pediatric Hematology-Oncology, Safra Children’s Hospital, Chaim Sheba Medical Center, Tel Hashomer, Israel; 6Adult BMT Service, Memorial Sloan Kettering Cancer Center, New York, United States of America; 7The Mina and Everard Goodman faculty of life sciences, Bar Ilan University, Ramat Gan, Israel Background: Patients with relapsed/refractory follicular lymphoma (R/R FL) often experience multiple relapses and require various lines of therapy. The ELARA and ZUMA-5 trials demonstrated high response rates along with acceptable safety profiles. We perform a phase 1b/2 single-center clinical trial of autologous point-of-care (POC) academic anti-CD19 chimeric antigen receptor (CAR) T-cells for patients with R/R FL treated with at least 2 lines of systemic therapy (NCT02772198). Aims: To report outcomes of POC CAR T-cell therapy in patients with R/R FL. Methods: Adults with R/R FL underwent a single leukapheresis procedure. Fresh peripheral blood mononuclear cells were isolated, activated, and transduced with a gammaretrovirus encoding for a CD19 CAR (based on an FMC63-derived ScFv, a CD28 costimulatory domain, and a CD3-ζ signaling domain). Lymphodepletion included fludarabine 25 mg/m2 over 3 days (days −4 to −2) and cyclophosphamide 900 mg/m2 once (day −2), followed by infusion of 1×106/kg CAR T-cells in the inpatient setting. Primary endpoints were response (by PET-CT, per Lugano criteria) at day 28, best response, and safety. Secondary endpoints included overall survival, progression-free survival (PFS), and production feasibility. Last follow-up was as of 02/2022. Results: All 19 patients enrolled received CAR T-cell infusion in a median of 11 days (IQR 10-11) after leukapheresis. The median age was 61 years (IQR 52-66). Five (26%) patients had Karnofsky performance status < 90%. Disease stage at enrollment was III-IV in 16 (84%) patients. Two (11%) patients had bulky disease; 8 (42%) had LDH > upper limit of normal; and 16 (84%) had Follicular Lymphoma International Prognostic Index ≥ 3. Disease status at enrollment was progressive disease (n=14, 74%), stable disease (n=3, 16%), or partial response (PR; n=2, 11%). Twelve patients (64%) were refractory to last treatment. Disease grade at most recent lymph node biopsy was 1 (n=3, 16%), 2 (n=11, 58%), or 3a (n=5, 26%). The median time from FL diagnosis was 3.9 years (IQR 2.5-4.6). Sixteen (84%) patients had progression of disease within 24 months of initial therapy. The number of prior therapies was ≥ 4 in 6 (32%) patients; and 5 (26%) patients underwent prior autologous transplantation. Grade III-IV cytokine release and immune effector cell-associated neurotoxicity syndromes occurred in 1 (5%) and 4 (21%) patients, respectively. One patient was infected with COVID-19 on the 5th day following cell infusion and was admitted to the intensive care unit. One patient had grade 3 atrial fibrillation. Severe neutropenia (absolute neutrophil count <500/µL), thrombocytopenia (platelets <50K/µL) and anemia (hemoglobin <10g/dl) occurred in 15 (79%), 5 (26%), and 7 (37%) patients, respectively. No bleeding events or death were recorded following cell infusion. Response was evaluated in all patients. Overall response rate on day 28 was 84% (79% complete response [CR]). One patient with PR on day 28 achieved a CR after a year of follow-up. Three patients (16%) continued to progress following CAR infusion. All patients were alive at the last follow-up (median follow-up, 11.5 months [IQR 4-21]). One-year PFS was 74% (95% CI, 53-100). The median duration of response (DOR) was not reached (95% CI, 12.5-not reached). Estimated DOR at 1-year was 89% (95% CI, 71-100). Image: Summary/Conclusion: Point-of-Care anti-CD19 CAR T-cell therapy, performed following a very short production time, induced high CR rate with an acceptable safety profile in a cohort of patients with high-risk R/R FL. P1109: TRIAL IN PROGRESS: PHASE 1B/2 TRIAL OF TAZEMETOSTAT IN COMBINATION WITH VARIOUS TREATMENTS IN PATIENTS WITH RELAPSED OR REFRACTORY HEMATOLOGIC MALIGNANCIES A. Bessudo1,*, D. Greenwald2, M. H. Kazemi3, A. Mahindra4, L. Shunyakov5, M. Sondhi6, H. O’Connor6, Y. Chen6, J. Yang6, G. Salles7 1cCare, Encinitas; 2UCLA, Santa Barbara; 3Astera Cancer Care, East Brunswick; 4Scripps MD Anderson Cancer Center, La Jolla; 5Central Care Cancer Center, Bolivar; 6Epizyme, Inc., Cambridge; 7Memorial Sloan Kettering Cancer Center, New York, United States of America Background: Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator, suppresses key cellular checkpoints and differentiation pathways to maintain the oncogenic characteristics of germinal center B cells. In addition to activity in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), EZH2 inhibitors induced cell cycle arrest and apoptosis in preclinical studies of multiple myeloma (MM). Tazemetostat (TAZ), an oral selective EZH2 inhibitor, is approved for treatment of relapsed or refractory (R/R) follicular lymphoma. Preclinical studies demonstrated antiproliferative and synergistic effects of TAZ + lenalidomide (LEN), a Bruton tyrosine kinase inhibitor (BTKi), and daratumumab, pomalidomide, and dexamethasone (mAbPD) in DBLCL, MCL, and MM cell lines, respectively. LEN is effective for DLBCL and is approved in combination with tafasitamab-cxix, a CD19-directed cytolytic antibody (CD19 antibody [Ab]), for R/R DLBCL. Acalabrutinib, a BTKi, is approved in MCL, and mAbPD showed clinical activity in R/R MM. Aims: The aim of this study is to examine the efficacy and safety of TAZ + CD19 Ab, LEN, BTKi, or mAbPD in patients (pts) with R/R hematologic malignancies. Methods: This trial is a 2-part, multicenter, open-label, safety, efficacy, signal-finding, multi-arm phase 1b/2 study of TAZ plus various treatment regimens for hematologic malignancies (NCT05205252). Phase 1b is a dose escalation safety run-in; phase 2 is a dose expansion study. Currently, there are 4 treatment arms (Table). Written informed consent will be obtained upon enrollment. Eligible pts are adults (aged ≥18 y) with an ECOG performance status 0–1 (phase 1b) or 0–2 (phase 2) and measurable disease by Lugano classification for arms 1–3 or IMWG 2016 criteria for arm 4. Pts with severe concurrent disease are excluded from this study. During the phase 1b safety run-in portion of the study, pts will be enrolled in 3 dose escalation cohorts of TAZ 400 mg, 600 mg, and 800 mg. Pts in all novel treatment combinations (arms 1, 3, 4) will start at the lowest TAZ dose of 400 mg, and treatment combinations previously studied (arm 2) will start at TAZ 800 mg. All pts will receive TAZ orally twice daily in continuous cycles. All combination partners will be administered per their respective package insert or investigator brochure. The phase 1b portion endpoint summarizes treatment-emergent dose-limiting toxicities and adverse events to inform the recommended phase 2 dose for each arm. The primary endpoint for all arms in phase 2 is objective response rate; key secondary endpoints include progression-free survival, duration of response, overall survival, and safety. Results: As of February 9, 2022, no pts are enrolled at 1 active site; additional sites are in startup. Image: Summary/Conclusion: This phase 1b/2 study will provide insights into the efficacy and safety of TAZ + CD19 Ab, LEN, BTKi, or mAbPD in pts with R/R hematologic malignancies. P1110: EFFICACY AND SAFETY OF IMMUNOCHEMOTHERAPY IN TREATMENT OF FOLLICULAR NON-HODGKIN’S LYMPHOMA DURING COVID-19 PANDEMIC: A STUDY OF KROHEM, THE CROATIAN COOPERATIVE GROUP FOR HEMATOLOGIC DISEASES D. Galusic1,2, S. Basic - Kinda3, A. Pijuk1, V. Milunovic4, B. Dreta3,*, N. Franjic5, B. Coha6, J. Sincic – Petricevic7, P. Gacina8, V. Pejsa9, M. Lucijanic9, I. Aurer3,10 1Division of Hematology, Department of Internal Medicine, University Hospital of Split; 2School of Medicine, University of Split, Split; 3Division of Hematology, Department of Internal Medicine, University Hospital Centre Zagreb; 4Division of Hematology, Department of Internal Medicine, University Hospital Merkur, Zagreb; 5Division of Hematology, Department of Internal Medicine, University Hospital Centre Rijeka, Rijeka; 6Department of Internal Medicine, Dr. “Josip Bencevic” General Hospital, Slavonski Brod; 7Division of Hematology, Department of Internal Medicine, University Hospital Centre Osijek, Osijek; 8Division of Hematology, Department of Internal Medicine, Sestre Milosrdnice University Hospital Center; 9Division of Hematology, Department of Internal Medicine, University Hospital Dubrava; 10School of Medicine, University of Zagreb, Zagreb, Croatia Background: Follicular lymphoma (FL) is a systemic neoplasm of the lymphoid tissue arising from B cell proliferation. The novel monoclonal anti-CD20 antibody obinutuzumab in combination with chemotherapy has been widely accepted as the first choice in front line treatment of FL. Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), responsible for coronavirus disease 2019 (COVID-19) is causing increased mortality among patients with lymphoproliferative disorders compared with the general population. Furthermore, there are some concerns in terms of morbidity and mortality for patients with FL because of their immunocompromised status induced by recent exposure to cytotoxic chemotherapy, especially bendamustine and anti-CD20. Aims: To investigate efficacy and safety of immunochemotherapy protocols for patients with newly diagnosed FL during COVID-19 pandemic. Methods: We retrospectively investigated medical data of all patients with newly diagnosed FL grade 1, 2 or 3A from Croatian hematologic registry in period from April 2019 to March 2021. Only patients which required systemic treatment were included in the analysis. All patients received obinutuzumab (G) in combination with either CHOP, bendamustine (B) or CVP chemotherapy protocol. Treatment response was evaluated using international lymphoma response criteria. Results: We analyzed a total of 114 FL patients treated with G-chemotherapy. Mean age was 62.4 ±10.5 years. Majority of patients were female (71/114 (62.3%)). FL grade I was present in 45/114 (39.5%), grade II in 28/114 (24.6%), grade III in 27/114 (23.7%) and not specified (but not IIIB) in 14/114 (12.3%) patients. A total of 61/114 (53.5%) patients were treated with G-B, 49/114 (43%) with G-CHOP and 4/114 (3.5%) with G-CVP immunochemotherapy. Similar rates of adverse events were observed in patients treated with G-CHOP and G-B Median follow up was 17 months. Overall response rate was 94%, complete remission (CR) in 68% and partial remission (PR) in 25% of patients. Median overall survival (OS) and progression free survival (PFS) were not reached with 12-months rates of 94% and 92%, respectively. Patients treated with G-CHOP had statistically significantly superior OS and PFS compared to patients treated with G-B (P=0.002 and P=0.006, respectively, Fig. 1). More favorable survival course associated with G-CHOP in comparison to G-B persisted in multivariate analysis (P=0,026, HR=15,12) after adjustment for age, sex, FLIPI grade and SARS-CoV-2 infection. Total of 12 patients died during the follow up and COVID-19 was cause of death in 5 patients. During the follow-up SARS-CoV-2 infection was diagnosed in 20/114 (17,5%) patients with overall mortality rate of 25%. All of the 7 patients treated with G-CHOP recovered from SARS-CoV-2 infection and mortality rate in infected group of patients treated with G-B was 33% (4/12 patients). Image: Summary/Conclusion: Increased COVID-19 mortality in patients with lymphoproliferative disorders was observed in this study. Our group of patients had reduced OS and PFS compared to the GALLIUM trial and SARS‐CoV‐2 infection was the most pronounced risk factor for death. Even though in some studies bendamustine has shown to be less toxic and more effective than CHOP in FL, there are some important pandemic aspects that must be considered. Bendamustine exposure seems to be associated with worse outcome in case of the infection with SARS-CoV-2. These intriguing differences could play important role in treatment approach in COVID-19 pandemic. Future studies investigating hematological malignancies in COVID-19 pandemic are warranted. P1111: COMPARATIVE EFFICACY AND SAFETY OF TISAGENLECLEUCEL AND AXICABTAGENE CILOLEUCEL IN RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA M. Dickinson1,*, J. Martinez-Lopez2, E. Jousseaume3, C. Anjos4, H. Yang5, X. Chai5, T. Wang5, R. Ramos4, C. Lobetti-Bodoni3, S. Schuster6, N. Fowler7 1Peter MacCallum Cancer Centre, Royal Melbourne Hospital and the University of Melbourne, Melbourne, VIC, Australia; 2Hospital Universitario 12 de Octubre, Complutense University, CNIO, Centro de Investigación Biomédica en Red Cáncer (CIBERONC), Madrid, Spain; 3Novartis Pharmaceuticals AG, Basel, Switzerland; 4Novartis Pharmaceuticals Corporation, East Hanover, NJ; 5Analysis Group Inc., Boston, MA; 6Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA; 7The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America Background: The chimeric antigen receptor T-cell (CAR-T) therapies, tisagenlecleucel (tisa-cel) and axicabtagene ciloleucel (axi-cel) have demonstrated clinical benefits in treating relapsed/refractory follicular lymphoma (r/r FL). Aims: To compare the efficacy and safety outcomes of tisa-cel and axi-cel in r/r FL using matching-adjusted indirect comparison (MAIC). Methods: Individual patient-level data (IPD) in ELARA (tisa-cel; NCT03568461; 03/2021 data-cut; N=97 [infused set]; N=90 [efficacy-evaluable set]) were weighted to match the patient population in ZUMA-5 (axi-cel; NCT03105336; 03/2020 data-cut; N=124 [infused set]; N=84 [efficacy-evaluable set]). Because patients from ZUMA-5 did not use bridging chemotherapy, the primary analysis compared a patient subgroup not exposed to bridging chemotherapy in ELARA (N=53 [infused set]; N=50 [efficacy-evaluable set]) with patients in ZUMA-5. Baseline characteristics available in both trials were adjusted in MAIC including age, sex, ECOG performance status, Ann Arbor stage, FLIPI, tumor burden per GELF criteria, number of prior therapy lines, refractory status to the most recent regimen, progression of disease within 24 months from first anti-CD20 monoclonal antibody-containing therapy, and prior autologous stem cell transplant. Efficacy outcomes, including overall response rate (ORR), complete response (CR) rate, and progression-free survival (PFS), were compared among the efficacy-evaluable set, defined as patients with ≥12 months of follow-up in both trials. Selected safety outcomes, including cytokine release syndrome (CRS), neurological events (NE), and tocilizumab and corticosteroid use for CRS, were compared between the infused populations. A sensitivity analysis was conducted to compare patients with and without bridging chemotherapy in ELARA with ZUMA-5. Results: After adjusting for differences in baseline characteristics, ORR, CR rate, and PFS were comparable between tisa-cel and axi-cel efficacy-evaluable patients (Figure 1A). In the primary analysis, the hazard ratio for PFS post-weighting was 0.90 (95% confidence interval: 0.39, 2.06; p=0.81) (Figure 1B). The adverse event rates for CRS and NE of any grade were 32.70% (p<0.01) and 47.70% (p<0.001) lower, respectively, in patients infused with tisa-cel compared to those infused with axi-cel. The proportion of patients who used tocilizumab and corticosteroids for CRS were 35.57% (p<0.001) and 12.69% (p<0.01) lower, respectively, in patients infused with tisa-cel (Figure 1C). Similar results were observed in the sensitivity analysis among patients with and without bridging chemotherapy in ELARA (Figure 1A-C). Image: Summary/Conclusion: The MAIC results indicated that tisa-cel and axi-cel were comparable in response rates and PFS, while tisa-cel was associated with better safety outcomes than axi-cel. These results confirm and reinforce the relevance of CAR-T therapies for r/r FL. Future analyses using IPD from both trials and real-world data are warranted. P1112: TREATING BING-NEEL SYNDROME USING A TAILORED APPROACH: EXPERIENCE OF A SPECIALIST NEUROHAEMATOLOGY CLINIC J. Khwaja1,*, D. Smyth2, A. Rismani1, C. Hoskote3, C. Kyriakou1, M. P. Lunn2, S. D’Sa1 1Department of Haematology, University College London Hospital; 2Centre for Neuromuscular Disease; 3Department of Neuroradiology, National Hospital for Neurology and Neurosurgery, London, United Kingdom Background: Bing-Neel syndrome (BNS) is a rare complication of lymphoplasmacytic lymphoma (LPL) comprising LPL infiltration in the central nervous system (CNS). Clinical and radiological features are diverse; the diagnosis is confirmed by cerebrospinal fluid (CSF) analysis using immunological and molecular techniques. Rarely, a tissue biopsy is required. The pattern of presentation including systemic involvement and CSF features inform treatment strategies, which include CNS-penetrating therapies. Aims: To evaluate the diagnostic characteristics of patients with BNS and their influence on therapy. Methods: Data from patients referred between 2011-2021 for management of BNS to our academic neurohaematology centre were retrospectively reviewed. Those with imaging features alone or where it was not possible to distinguish from high-grade transformation were excluded. Results: Thirty-five patients (22 male, 13 female) were identified. Median age at diagnosis of BNS was 65 years (range 48-85). All patients were symptomatic. In 12 patients (34%) BNS was the de novo presentation of the IgM-related disorder, of which 3 (25%) had no detectable bone marrow (BM) infiltration of LPL at diagnosis. Approximately half (17; 49%) had previously received therapy for LPL; median time to BNS diagnosis in these was 49 months (range 3-125). At BNS diagnosis, BM involvement with LPL ranged from 0-95%. More than half (14/26; 54%) had ≤10% infiltrate and almost a fifth (4/26) >60%. All patients had leptomeningeal involvement and 8 (23%) additionally had parenchymal CNS disease. The majority had kappa light-chain predominance: IgMκ (n=26), non-IgMκ (n=5), IgMλ (n=3), one unknown. The BNS diagnosis was made on CSF analysis (n=28; 80%), leptomeningeal tissue biopsy (n=3; 9%) where CSF was non-informative, or by expert opinion based on supportive clinical, radiological and non-definitive CSF features (n=4; 11%). Of those with a diagnosis based on CSF studies, B-cell clonality was confirmed by flow cytometry (27/28; 96%), MYD88L265P mutation (18/28; 64%) and immunoglobulin gene rearrangement (12/28; 43%). In 22 samples with a full dataset, median CSF white cell count was 25/ul (1-233), CSF protein 1.69g/l (0.35-6), CSF IgM 9.49mg/l (1.07-61.5). The majority were treated with intensive regimens (rituximab, methotrexate (MTX), cytarabine (ARA-C) ± thiotepa/idarubicin; n=30) due to the presence of CNS disease bulk and clinical need, and less commonly ibrutinib (n=3), bendamustine-rituximab (BR, n=1); one patient had intrathecal therapy (MTX, ARA-C) at the height of the COVID pandemic. Of those who received 2 cycles of intensive chemotherapy, 3 had ≥4 cycles followed by BCNU/thiotepa autologous stem cell transplant; 10 proceeded to ‘consolidation’ (indefinite) ibrutinib to limit intensive chemotherapy or tackle systemic disease. At a median follow up of 26 months (range 1-121), median survival was not reached; 2-year overall survival was 91% (95% CI 74-97). Three patients died during treatment (1 invasive fungal infection post COVID-19 during ibrutinib consolidation post MTX/ARA-C based therapy) and 2 during MTX-ARA-C based therapy; 7 patients relapsed or progressed and were treated with ibrutinib: 1 relapsed after ibrutinib use, 1 patient was intolerant of ibrutinib and switched to BR. Image: Summary/Conclusion: Our cohort confirms that BNS may present with leptomeningeal disease and/or parenchymal disease, de novo and without systemic disease. Overall outcomes are excellent with intensive regimens, consolidated with or followed by ibrutinib; however, there are treatment-related toxicities emphasising the need for a tailored approach. P1113: PROGNOSTIC ROLE OF GENETIC ABNORMALITIES IN MANTLE CELL LYMPHOMA E. Kleina1,*, S. Voloshin1, J. Vokueva1, O. Petukhova1, L. Martynenko1, M. Bakai1, J. Ruzhenkova1, S. Linnikov1, E. Kariagina2, O. Uspenskaia3, I. Ziuzgin4, S. Bessmelcev1, S. Sidorkevich1, I. Martynkevich1 1Russian Research Institute of Hematology and Transfusiology, Federal Medical and Biological Agency; 2St. Petersburg GBUZ “City Hospital No. 15; 3GBUZ Leningrad Regional Clinical Hospital; 4”The N.N. Petrov National Medicine Research Institute of oncology” Ministry of Health of Russia, Saint-Petersburg, Russia Background: Mantle cell lymphoma (MCL) – B-cell lymphoma with a high frequency of genome instability associated with a recurrent clinical course, short non-progressive and overall survival. Special attention is paid to the “double-hit” MCL (simultaneous presence of translocations of CCND1 and C-MYC genes), characterized mainly by a pleomorphic/blastoid morphological variant, refractory clinical course and unfavorable prognosis. Aims: To identify the spectrum and frequency of occurrence of genetic anomalies and their effect on the course of MCL. Methods: The results of a standard cytogenetic study (G-banding) in 51 and FISH studies in 75 patients with MCL are presented. Results: In a standard cytogenetic study, a pathological karyotype was found in 25/51 (49,0%) patients. Translocation t(11;14)(q13;q32) was detected in 23/51 (45,1%) patients. As part of the complex karyotype, t(11;14) was determined in 16/51 (31,4%) cases. The FISH study made it possible to additionally identify genetic aberrations in patients with MCL. In patients with normal karyotype t(11;14) was found in 18/51 (35,3%) cases. C-MYC gene rearrangements were detected in 16/75 (21,3%) cases. In this group, genetic abnormalities were found in 7/11 (63,6%) patients (in 5 patients, mitoses were not obtained). In all patients t(11;14) was detected either as a single genetic aberration (in 1/7 (14,3%)) or as part of a complex karyotype (in 6/7 (85,7%)) in combination with genetic abnormalities 7 and 17 chromosomes. In 8/16 (50,0%) patients, C-MYC gene rearrangements were combined with a deletion of the TP53 gene. In 59/75 (78,7%) cases, C-MYC gene rearrangements were not detected. Chromosomal aberrations were found in 18/39 (46,2%) patients, including complex karyotype changes in 10/18 (55,6%) patients. Translocation t(11;14) - in 16/18 (88,9%) cases. TP53 gene deletion was detected in 10/59 (16,9%) patients. Data analysis showed a significant adverse effect of the complex karyotype, TP53 deletion, C-MYC rearrangement on the clinical course of MCL compared with the standard risk group. The median progression-free survival in high molecular risk patients was 12,5 months; the median progression-free survival was not achieved in the standard risk group. Summary/Conclusion: An integrated approach to the genetic diagnosis of MCL using standard cytogenetic and FISH studies makes it possible to identify a high-risk group: “double-hit” and with complex changes in the karyotype. It was also shown that in the group of patients with changes in the C-MYC gene, additional genetic anomalies were more often detected, which are an unfavorable prognostic factor: a complex karyotype (the presence of three or more aberrations) – 37,5% and 16,9%, and aberrations involving the gene TP53 – 50,0% and 16,9%. Changes in these genes are associated with shorter progression-free survival rates. P1114: ZANDELISIB ON INTERMITTENT DOSING AS A SINGLE AGENT OR IN COMBINATION WITH RITUXIMAB OR ZANUBRUTINIB IN RELAPSED/REFRACTORY (R/R) FOLLICULAR LYMPHOMA (FL): RESULTS FROM A MULTI-ARM PHASE 1B STUDY J. D. Soumerai1,*, D. Jagadeesh2, H. Salman3, F. Samaniego4, K. Patel5, A. Stathis6, N. Reddy7, V. P. Kenkre8, A. Asch9, C. Diefenbach10, I. S. Lossos11, D. Persky12, F. Awan13, W. Huang14, K. Vandever14, A. D. Zelenetz15 1Massachusetts General Hospital, Boston; 2Cleveland Clinic, Cleveland; 3Stony Brook University, Stony Brook; 4The University of Texas MD Anderson Cancer Center, Houston; 5Swedish Cancer Institute, Seattle, United States of America; 6Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 7Vanderbilt University Cancer Center, Nashville; 8University of Wisconsin Carbone Cancer Center, Madison; 9Oklahoma University Health Sciences Center, Oklahoma City; 10Perlmutter Cancer Center at NYU Langone Health, New York; 11Sylvester Comprehensive Cancer Center, Miami; 12University of Arizona, Tucson; 13University of Texas Southwestern Medical Center, Dallas; 14MEI Pharma, Inc., San Diego; 15Memorial Sloan Kettering Cancer Center, New York, United States of America Background: PI3Kδ inhibitors administered daily in patients (pts) with FL were limited by immune-related adverse events (AEs) due to regulatory T cell (Treg) suppression following continuous on-target inhibition. Zandelisib is a structurally differentiated oral PI3Kδ inhibitor with high specificity and target-binding affinity. We hypothesized that zandelisib administered on an intermittent dosing (ID) schedule (one capsule daily on days 1-7 of a 28-day cycle) would enable Treg repopulation during treatment breaks and improve safety. Aims: We conducted this analysis to assess the safety and efficacy of zandelisib administered by ID as a single agent or in combination therapy in pts with R/R FL enrolled in a 3-arm phase 1b study in various B-cell malignancies. Methods: Eligible pts ≥18 years with FL Grade I-IIIA, ECOG PS 0-2, progressive disease after ≥1 prior therapy and no prior PI3Kδ inhibitor, provided consent prior to enrollment (NCT02914938). A dose escalation stage assessed single-agent zandelisib daily continuously and is not reported here (Soumerai et al, J Clin Oncol 2018;36:#7519). Subsequent cohorts evaluated zandelisib 60 mg daily for two 28-day cycles then on ID as a single agent (Group 1) or in combination with rituximab 375 mg/m2 weekly x4 in cycle 1 and on Day 1 of cycles 3-6 (Group 2). Group 3 evaluated zandelisib 60 mg on ID from cycle 1 and zanubrutinib 80 mg twice daily. Treatment was continued until disease progression or intolerance. Imaging scans were obtained after 2 and 6 cycles 6 and then every 6 months. Response was assessed by investigators using the Lugano Criteria. Results: 69 FL pts were treated: 18 in Group 1, 19 in Group 2, and 32 in Group 3. Enrollment began in September 2017 for Group 1 and 2 and June 2019 for Group 3, and is completed in all 3 groups. Median age was 66 years (range 38-87), median prior therapies was 2 (range 1-5), 40 pts (58%) received ≥2 prior therapies, 45 pts (65%) were POD24, 21 pts (30.4%) were refractory to last therapy, and 28 pts (41%) had tumors ≥5 cm. With a median follow-up of 10.8 months (range 1.8-49.5), 6 pts (8.7%) have discontinued therapy due to an AE. Grade 3 AEs of special interest (AESI) were rash in 6 pts (8.7%), ALT increased in 6 (8.7%), diarrhea in 3 (4.3%), colitis in 2 (2.9%), and AST increased in 3 (4.3%). No grade 4-5 AESI were reported. 1 pt in Group 3 had reversible grade 4 drug rash with eosinophilia and systemic symptoms (DRESSS) syndrome. Grade 3-4 neutropenia was observed in 11 pts (16%). The overall response rate (ORR) in 65 evaluable pts was 84.6% (55/65), 95% CI 73.5-92.4, and the CR rate was 24.6% (16/65), 95% CI 14.8-36.9. The ORR was 77.8% (14/18) in Group 1, 94.7% (18/19) in Group 2, and 82.1% (23/28) in Group 3. The median duration of response (DOR) was 31.1 months in Group 1, 25.8 months in Group 2, and not yet mature in Group 3, with median drug exposure of 17.1, 20.5, and 7.1 months, respectively (Figure). Image: Summary/Conclusion: Zandelisib was generally well tolerated when administered by ID as a single agent or in combination with rituximab or zanubrutinib, with a low rate (8.7%) of grade 3 AESI and discontinuations due to AEs. The high ORR and prolonged DOR are encouraging. This profile supports further evaluation of zandelisib on ID as a single agent and in combination in various B-cell malignancies, both in R/R disease and in earlier lines of therapy. P1115: A MULTICENTER PHASE 1/2 TRIAL OF EO2463, A MICROBIAL-DERIVED PEPTIDE THERAPEUTIC VACCINE, AS MONOTHERAPY AND IN COMBINATION WITH LENALIDOMIDE AND RITUXIMAB, FOR TREATMENT OF PATIENTS WITH INDOLENT NHL P. L. Zinzani1,*, S. M. Ansell2, F. Bosch3, J. W. Friedberg4, J. P. Marolleau5, L. Arcaini6, R. Garcia-Sanz7, A. K. Gopal8, C. Grande9, R. Merryman10, A. Pinto11, S. D. Smith12, J. C. Villasboas2, D. Wallace13, J. Fagerberg14, J. G. Magalhaes14, P. Armand10 1Institute of Hematology “L. e A. Seràgnoli”, University of Bologna, Bologna, Italy; 2Division of Hematology, Mayo Clinic, Rochester, United States of America; 3Department of Hematology, Vall d’Hebron Institute of Oncology, Barcelona, Spain; 4University of Rochester Medical Center, Rochester, United States of America; 5CHU Amiens, Amiens, France; 6Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 7Hospital Universitario de Salamanca, Salamanca, Spain; 8Division of Medical Oncology, University of Washington, Seattle, United States of America; 9Universidad de Navarra, Madrid and Pamplona, Spain; 10Dana-Farber Cancer Institute, Boston, United States of America; 11Istituto Nazionale Tumori “Fondazione G.Pascale”- IRCCS, Naples, Italy; 12University of Washington/Fred Hutchinson Cancer Research Center, Seattle; 13Department of Medicine, University of Rochester, Rochester, United States of America; 14Enterome, Paris, France Background: EO2463 is a therapeutic vaccine designed to activate existing commensal bacteria-specific memory T cells that cross-react with B cell markers in order to drive anti-tumor immune activity against B-cell malignancies. EO2463 contains four microbial-derived, synthetically produced peptides (OMP72, OMP64, OMP65, and OMP66), which correspond to cytotoxic CD8 T cell HLA-A2 restricted epitopes, and exhibit molecular mimicry with the B cell markers CD20, CD22, CD37, and CD268 (BAFF-receptor), respectively. In pre-clinical models, these peptides can generate strong immune responses and specifically stimulate cross-reactive cytotoxic CD8 T cells to recognize the chosen B cell targets. EO2463 also contains a CD4 helper peptide referred to as universal cancer peptide 2, derived from the human telomerase reverse transcriptase catalytic subunit. The present study is a first-in-human clinical trial of this microbiome-derived peptide therapeutic cancer vaccine approach in patients with follicular lymphoma (FL) and marginal zone lymphoma (MZL). Aims: The primary objectives of the phase 1 part of the trial are to define the recommended phase 2 dose (RP2D) for EO2463 monotherapy, and to confirm the safety of EO2463 at the monotherapy RP2D in combination with lenalidomide (EL), rituximab (ER), and lenalidomide/rituximab (ER2). The primary objective of the phase 2 part of the trial is to estimate the objective response rate (ORR) according to the Lugano Classification 2014 during EO2463 monotherapy Methods: This four-cohort phase 1/2 trial will investigate EO2463 monotherapy, and combinations of EO2463/lenalidomide (EL), EO2463/rituximab (ER), and EO2463/lenalidomide/rituximab (ER2), for treatment of patients with FL and MZL. Cohort 1 is a safety lead-in dose-finding in patients with relapsed/refractory (RR) disease, with a 3-by-3 design to establish the recommended phase 2 dose (RP2D) for EO2463 monotherapy and to confirm the safety of the RP2D for combination schedules of EL, and ER2. Maximal eighteen patients will be included in cohort 1. After the recommended EO2463 monotherapy dose is established, cohorts 2, 3, and 4 will open to accrual. Fifteen patient will be included in each of these cohorts. Cohort 2 will investigate EO2463 monotherapy in patients with newly diagnosed FL/MZL who are not in need of treatment; cohort 3 will investigate EO2463 monotherapy, followed by ER in patients with limited tumor burden who need treatment, and cohort 4 will further investigate EL, followed by ER2 in the RR setting. EO2463 will be administered subcutaneous 4 times at 2-week intervals, followed by continued booster administrations every 4 weeks for 9 (Cohorts 2 and 3) or 12 (Cohorts 1 and 4) months. Inclusion/exclusion criteria, and the design and schedule of the intense immune and safety monitoring will be presented. Results: The safety lead-in dose-finding is currently ongoing, and no safety concerns have been observed thus far. Summary/Conclusion: This is a first in human clinical trial to evaluate safety, determine the recommended phase 2 dose and estimate efficacy of a novel microbial-derived peptide therapeutic vaccine, EO2463, in patients with indolent non-hodgkin lymphoma as monotherapy and in combination with lenalidomide and rituximab. P1116: SAFETY, PHARMACOKINETIC (PK), PHARMACODYNAMIC (PD) AND ACTIVITY OF THE HIGHLY SELECTIVE PHOSPHOINOSITIDE 3-KINASE INHIBITOR DELTA (PI3KΔ) INHIBITOR IOA-244 IN PATIENTS WITH FOLLICULAR LYMPHOMA (FL) C. Carlostella1,*, M. Lahn2, T. Hammett2, L. van der Veen2, Z. Johnson2, C. Pickering2, A. Santoro1 1IRCSS Humanitas Research Hospital, Milan, Italy; 2iOnctura, Geneva, Switzerland Background: IOA-244 is a highly selective inhibitor of PI3Kδ, and has a favourable safety and ADME profile in patients (pts) with solid malignancies. In pts with uveal melanoma, IOA-244 also reduced circulating Tregs counts at the biologically effective dose (BED) range. Aims: As part of the First-in-human (FiH) dose escalation study, pts with FL received IOA-244 at the BED range established in patients with solid malignancies to confirm its favourable safety and PK profile in pts with haematologic malignancies. Methods: IOA-244 was investigated in a two-part FIH study. Part A explored the continuous daily dosing of IOA-244 at 10, 20, 40 and 80 mg in pts with solid malignancies and at 20 mg and 80 mg in pts with FL. Part B consists of expansion cohorts of specific tumour indications, including pts with lymphoma. Primary objective: safety of the anticipated BED, or the recommended phase 2 dose (RP2D). Secondary objectives: PK; PD (e.g., inhibition of CD63 expression on basophils, changes in immune cell subsets in peripheral blood); Lugano-based responses; PFS and OS Results: While Part A for solid malignancies have previously been reported (Di Giacomo et al 2021), we here report for the first time the safety, PK, PD and activity of pts with FL. This part of the study is still ongoing in patients with FL and is anticipated to be completed in 2022. Pts at the first cohort of 20 mg QD daily (4/4; 2 female and 2 males) had no DLT and no dose interruptions. Transient platelet reduction (G3) and AST/ALT elevation (G2) were observed in 1/4 pts, which improved while pt was on therapy. The PK and ADME profile was consistent with the ones observed in pts with solid malignancies. One patient had FL and DLBCL as prior co-primary malignancy with 7 prior lines of therapy. The other 3 pts had 1-3 prior lines of therapy. The best response was SD (1/3 pts) and all pts progressed after completing 2 cycles. LDH was initially elevated in 2/4 pts which subsequently decreased on therapy. 3/4 pts had low Treg counts at baseline. Additional pts are being recruited at the RP2D for solid malignancies, at 80 mg QD. Reference: Di Giacomo et al. Annals of Oncology (2021) 32 (suppl_7): S1428-S1457. 10.1016/annonc/annonc787 Summary/Conclusion: The PK and ADME profile of IOA-244 in pts with FL appears to match that in pts with solid malignancies. Additional pts at the 80 mg QD dose are expected to match the safety observed in pts with solid malignancies. In contrast to other PI3Kd inhibitors, IOA-244 is highly selective and may therefore target Tregs in pts with FL without off-target effects induced by inhibition of other PI3K isoenzymes. P1117: THREE-YEAR FOLLOW-UP OF OUTCOMES WITH KTE-X19 IN PATIENTS WITH RELAPSED/REFRACTORY MANTLE CELL LYMPHOMA IN ZUMA-2 M. L. Wang1,*, J. Munoz2, A. Goy3, F. L. Locke4, C. A. Jacobson5, B. T. Hill6, J. M. Timmerman7, H. Holmes8, I. W. Flinn9, D. B. Miklos10, J. M. Pagel11, M. J. Kersten12, R. Houot13, A. Beitinjaneh14, W. Peng15, X. Fang15, R. R. Shen15, R. Siddiqi15, I. Kloos15, P. M. Reagan16 1The University of Texas MD Anderson Cancer Center, Houston, TX; 2Banner MD Anderson Cancer Center, Gilbert, AZ; 3John Theurer Cancer Center, Hackensack University, Hackensack, NJ; 4Moffitt Cancer Center, Tampa, FL; 5Dana-Farber Cancer Institute, Boston, MA; 6Cleveland Clinic Foundation, Cleveland, OH; 7David Geffen School of Medicine at UCLA, Los Angeles, CA; 8Texas Oncology, Dallas, TX; 9Sarah Cannon Research Institute and Tennessee Oncology, Nashville, TN; 10Stanford University School of Medicine, Stanford, CA; 11Swedish Cancer Institute, Seattle, WA, United States of America; 12Amsterdam UMC, University of Amsterdam, Amsterdam, Cancer Center Amsterdam, The Netherlands, on behalf of HOVON/LLPC, Amsterdam, Netherlands; 13CHU Rennes, Université Rennes, INSERM & EFS, Renne, France; 14University of Miami, Miami, FL; 15Kite, a Gilead Company, Santa Monica, CA; 16University of Rochester Medical Center, Rochester, NY, United States of America Background: Brexucabtagene autoleucel (KTE-X19) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of patients (pts) with relapsed/refractory (R/R) mantle cell lymphoma (MCL). In ZUMA-2, a 93% objective response rate (ORR; 67% complete response [CR] rate) was reported with KTE-X19 in pts with R/R MCL (median follow-up: 12.3 mo; 60 efficacy-evaluable pts; Wang et al. N Engl J Med. 2020). Aims: To present updated outcomes from ZUMA-2 with 2 years of additional follow-up. Methods: Adult pts (≥18 years) with R/R MCL underwent leukapheresis and conditioning chemotherapy followed by a single infusion of KTE-X19. Minimal residual disease (MRD) was an exploratory endpoint (sensitivity 10-5) evaluated in peripheral blood using next-generation sequencing. Updated results are reported for all 68 treated pts. Results: After 35.6 mo median follow-up, the ORR (CR + partial response) was 91% (95% CI, 81.8-96.7), with a 68% CR rate (95% CI, 55.2-78.5). The median duration of response (DOR) was 28.2 mo (95% CI, 13.5-47.1), with 25 of 68 treated pts (37%) still in ongoing response (all CR) at data cutoff. Late relapse >24 mo post-infusion was infrequent (n=3). The medians for progression-free survival (PFS) and overall survival (OS) were 25.8 mo (95% CI, 9.6-47.6) and 46.6 mo (95% CI, 24.9-not estimable), respectively. MRD was analyzed in 29 pts total; 24 of 29 were MRD-negative at mo 1, and 15 of 19 with available data were MRD-negative at mo 6. At data cutoff, the medians for DOR, PFS, and OS in the 15 MRD-negative pts were all not reached, vs 6.1, 7.1, and 27.0 mo, in the 4 MRD-positive pts, respectively. MRD-negative status at mo 1, 3, and 6 was associated with durable response, with 55%, 71%, and 69% of MRD-negative pts at those timepoints remaining in ongoing CR at data cutoff. Circulating tumor DNA analysis of MRD at mo 3 and 6 was predictive of relapse (AUC 0.80 and 0.75, respectively). No new safety signals were observed. Only 3% of treatment-emergent adverse events (AEs) of interest occurred since the primary report. The most frequent Grade ≥3 AE was neutropenia (1 [1%] Grade 3; 7 [10%] Grade 4). Two pts had KTE-X19-related Grade 3 serious infections: pneumonia and upper respiratory tract infection (n=1); influenza (n=1). There were no new cytokine release syndrome AEs and 1 new serious neurologic AE of Grade 3 encephalopathy (13.0 mo post-infusion) that was considered not related to study treatment. Three new Grade 5 AEs occurred, none of which were considered related to study treatment: Salmonella bacteremia (24.9 mo post-infusion), myelodysplastic syndrome (25.2 mo post-infusion), and acute myeloid leukemia (37.5 mo post-infusion). Summary/Conclusion: These data represent the longest follow-up of CAR T-cell therapy in pts with MCL to date and suggest that KTE-X19 induces durable long-term responses with manageable safety and low late relapse potential in R/R MCL. P1118: PROGNOSTIC SIGNIFICANCE OF ABSOLUTE MONOCYTE COUNT AND LYMPHOCYTE TO MONOCYTE RATIO IN MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT) LYMPHOMA Y. Li1,*, C. Shang1, Y. Ren1, L. Wang1, J. Li1, W. Xu1 1Hematology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China Background: Extranodal marginal zone B-cell lymphoma of the MALT lymphoma is a unique type of indolent lymphoma. It usually presents with local disease and exhibit an indolent clinical course, but some patients have disseminated systemic symptoms and require systemic treatment. Monocytes, as part of the important immune cells in tumor microenvironment, are closely linked to the pathogenesis and progression of malignancies. In recent studies, the absolute monocyte count (AMC) and lymphocyte to monocyte ratio (LMR) are shown to reflect the host systemic immunity states and have prognostic significance in different kinds of lymphoma. Aims: Our study aims to explore the prognostic significance of AMC and LMR in MALT lymphoma, and to propose a more accurate prognostic index based on MALT-IPI. Methods: Baseline clinical characteristics collected from the medical records at diagnosis. PFS and OS were analyzed with the Kaplan-Meier method and compared using the log-rank test. X-tile analysis was performed to identify cut-off value. Chi-square test was performed for comparisons between groups. Cox proportional-hazards regression models were used for univariate and multivariate analysis. Statistical analyses were performed with MedCalc 19.5.6, SPSS software 26.0, R software version 4.0.4 and Graphpad Prism 9.0. Results: 316 MALT lymphoma patients were enrolled in this study. A statistically evident dominance showed that LMR was related to age, LDH level, β2-MG level, B symptom, ECOG PS and systemic therapy. With a median follow-up time of 39.1 months (1–237 months), median PFS was 146.4 months and median OS was not reached, estimated PFS rate at 3 years and 5 years were 84.1% and 79.6%, estimated OS rate at 3 years and 5 years were 94.9% and 92.4%, respectively. According to Figure 1, high AMC group (>0.6×109/L) and low LMR group (<1.8) were associated with poor outcomes. We performed Cox proportional-hazards regression for PFS and OS, MALT-IPI, ECOG PS and LMR were identified to have independent prognostic significance for PFS while MALT-IPI, β2-MG and LMR were independently associated with poor OS. Figure 2A showed that low level of LMR in subgroup was related to a poor PFS. The prognostic value of low level of LMR for PFS was more significant in age (p value for interaction =0.020). Across a subgroup analysis of OS (Figure 2B), the prognostic value of low level of LMR was consistent. Since we have confirmed AMC showed prognostic significance in MALT lymphoma survival. We combined MALT-IPI and AMC to generate a new prognostic index named ‘MALT-IPI-M’, which included four parameters: age≥70 years, Ann Arbor stage III or IV, serum LDH level >UNL, and LMR <1.8. MALT-IPI-M classified patients into low-risk group (the MALT-IPI-M=0), intermediate-risk group (the MALT-IPI-M=1) and high-risk group (the MALT-IPI-M≥2), the proportions of different stratifications in our cohort are 52.5%, 32.9% and 14.6%, respectively. As shown in Figure 3, we generated Receiver-operator characteristic (ROC) curves to compare the prognostic prediction capability of MALT-IPI and MALT-IPI-M. The area under the curves (AUCs) for MALT-IPI-M were 0.682 for PFS and 0.804 for OS, which was larger than AUCs for MALT-IPI, 0.654 for PFS and 0.788 for OS. This illustrated that MALT-IPI-M has a better capability of distinguishing MALT patients with different risk. Image: Summary/Conclusion: In conclusion, our findings suggest that low level LMR at diagnosis might be associated with inferior PFS and OS in patients with MALT lymphoma. Incorporating LMR into MALT-IPI may permit a more accurate risk assessment of disease progression. P1119: THE ORAL PI3KΔ INHIBITOR LINPERLISIB FOR THE TREATMENT OF RELAPSED OR REFRACTORY FOLLICULAR LYMPHOMA: A SINGLE-ARM MULTICENTER PHASE 2 CLINICAL TRIAL T. wang1, X. sun2, L. qiu3, H. su4, J. cao5, Z. li6, Y. song7, L. zhang8, S. yi9, L. qiu9,*, J. zhou10, H. wu11, W. zhang12, J. li13, K. zhou14, H. zhou15, Y. yang16, Z. li17, H. cen18, Z. cai19, Z. zhang20, W. fu21, J. jin19, F. li22, W. wu23, X. gu24, W. zhu25, L. liu26, Z. li27 1Department of Lymphoma and Myeloma, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin; 2department of medical oncology, the second hospital of dalian medical university, dalian; 3Department of Lymphoma, Tianjin Medical University Cancer Institute and Hospital, Tianjin; 4Department of Lymphoma, The Fifth Medical Center of PLA General Hospital, Beijing; 5Fudan University Shanghai Cancer Center, Shanghai; 6Sun Yat-sen University Cancer Center, guangzhou; 7Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing; 8West China Hospital, Sichuan University, chengdu; 9Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin; 10Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology; 11Department of Medical Oncology, Hubei Cancer Hospital, Wuhan; 12Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing; 13Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai; 14Department of Hematology, The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Zhengzhou; 15Department of Lymphoma & Hematology, Hunan Cancer Hospital, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha; 16Department of Lymphoma and Head and Neck Cancer, Fujian Provincial Cancer Hospital, The Affiliated Tumor Hospital of Fujian Medical University, Fuzhou; 17Department of Hematology, The First Affiliated Hospital of Xiamen University and Institute of Hematology, School of Medicine, Xiamen University, Xiamen; 18Department of Haematology/Oncology and Paediatric Oncology, Guangxi Medical University Affiliated Cancer Hospital, Nanning, China, Nanning; 19Department of Hematology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou; 20Department of Medical Oncology, Sichuan Cancer Hospital and Institute, Chengdu; 21Department of Hematology, The Myeloma & Lymphoma Center, Shanghai Changzheng Hospital, The Second Military Medical University, Shanghai; 22Department of Hematology, The First Affiliated Hospital of Nanchang University, Nanchang; 23Department of Internal Medicine and Oncology, Zhongshan Hospital, Xiamen University, Xiamen; 24Department of Hematology, The First Affiliated Hospital of Guangzhou University of Chinese Medicine; 25Department of Oncology, Zhujiang Hospital of Southern Medical University, Guangzhou; 26Department of Hematology, The Fourth Hospital of Hebei Medical Universit, Shijiazhuang; 27Hematological Disorders, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China Background: The orally active phosphatidylinositol 3-kinase delta (PI3Kδ) inhibitor linperlisib which conveyed acceptable safety and notable efficacy for B-cell lymphoma medications in a previous phase 1 dose escalation study was investigated in this phase 2 study for the treatment of relapsed and/or refractory follicular lymphoma (FL) patients who had received at least two prior systemic treatments Aims: To explore the satefy and efficacy of linperlisib in FL Methods: Linperlisib was administered at 80 mg qd, po in a 28-day cycle until disease progression or intolerable toxicity occurred. The primary outcome was the objective response rate (ORR), while secondary outcomes included the duration of response (DOR), progression-free survival (PFS), overall survival (OS), disease control rate (DCR) and the drug safety profile. Results: For 84 FL patients in the full analysis set (FAS) the ORR was 79.8% (95% CI: 69.6-87.8, 67 patients), with 13 patients achieving a complete response and 54 a partial response. The median DOR was 12.3 months (range, 9.3-15.9) with 6-month and 12-month DORs of 80.0% (95% CI: 67.4-88.1) and 55.3% (95% CI: 40.6-67.8). The median PFS was 13.4 months (95% CI: 11.1-16.7), with 6-month and 12-month PFS rates of 78.7% (95% CI: 67.5-86.3) and 53.1% (95% CI: 40.3-64.3). The median OS was not reached, while 6-month and 12-month OS rates were 97.6% (95% CI: 90.6-99.4) and 91.4% (95% CI: 82.7-95.8), respectively. The most frequent any-grade treatment related AEs (TRAE) were neutropenia (47%), hypertriglyceridemia (25%), while the most frequent TRAEs ≥ grade 3 were neutropenia (15%), infectious pneumonia (19.4%) and interstitial lung disease (6.5%). AEs that were potentially immune-mediated, such as diarrhea, colitis, pneumonitis, and transaminitis, were observed at significantly lower incidences of occurrence. Summary/Conclusion: Linperlisib demonstrated compelling clinical efficacy and a promising safety profile, which has the potential to surpass the approved PI3K inhibitors for the treatment of relapsed or refractory FL patients after two prior therapies. P1120: THE EFFICACY AND SAFETY OF ZANUBRUTINIB AND DEXAMETHASONE IN SYMPTOMATIC WALDENSTROM MACROGLOBULINNEMIA A. Liu1, J. Yin1, J. Lu2, Y. Ma3, D. Gao4, L. Hua5, Y. Tian1, Y. Jian1, W. Chen1,* 1Beijing Chaoyang Hospital, affiliated to Capital Medical University; 2Peking University People’s Hospital, Beijing; 3Second hospital of Shanxi Medical University, Taiyuan; 4The Affiliated Hospital of Inner Mongolia Medical University, Huhehaote; 5Affiliated Hospital of Hebei University, Shijiazhuang, China Background: Bruton tyrosine kinase (BTK) inhibition is an effective treatment approach for patients with Waldenström macroglobulinemia (WM)，but the efficacy of single drug is limited. Aims: This single arm study (ChiCTR2000038140) evaluated the efficacy and safety of the combination of zanubrutinib, a novel, highly selective BTK inhibitor, and dexamethasone in patients with WM. Methods: Symptomatic patients with WM were enrolled to the regimen of zanubrutinib and dexamethasone (ZD)*. The primary endpoint was objective response rate (ORR), progression-free survival (PFS). Key secondary endpoints included the proportion of patients achieving a complete or very good partial response (CR or VGPR), duration of response (DOR), Time to response (TOR), disease burden, and safety. The control group** were matched patients treated by chemotherapy or immunechemotherapy previously in Beijing Chaoyang Hospital. Results: A total of 22 Patients with WM were enrolled in this study, median age 67(36-89); 68.2% males, 12 patients were untreated, others were treated patients. IPSS assessment grade1 23.8%; grade2 19.0%; grade3 57.2%. 90.9% (20/22) patients with MYD88L265P mutation, 27.3% (3/11) patients with CXCR4 mutation. 21 patients received ≥1 dose of study treatment. Median follow-up of 8.2 months, median DOR and PFS were not reached; 95% of patients were progression-free at 6 months. ORR was 95% in those (17/18) received ZD regimen more than 2 months. No patient achieved a CR. 33.3% of patients in ZD group achieved a VGPR, time to VGPR within 3 months in 57.1% of patients，a statistically significant difference with control group (0%, P = 0.001). Time to PR in ZD group was 2 months, much faster than control group (11 months) (P = 0.023) by K-M analysis. The study-safety profile was consistent with previous BTK inhibitor clinical trial data. 45% of patients had any grade AEs. In which, the most frequent grade <=2 AEs were hemorrhage(18.2% all grade 1), rash(9.1%), hyperglycemia (13.6%), infection(9.1%), nausea and vomiting (9.1%), hypogammaglobulinemia(4.5%), neutropenia(9.1%). Grade 3/4 AEs were atrial fibrillation(4.5%), leading to treatment discontinuation. Other cause of treatment discontinuation is hyperglycemia, bowel obstruction by disease. Comments *The regimen of ZD: Zanubrutinib 240mg d1-28, dexamethasone 20mg D1-4,15-18. Patients more than 75 years old, Zanubrutinib 160mg d1-28, dexamethasone 10mg D1-4,15-18. After 8 cycle, Zanubrutinib use as maintenance. ** The control group: 22 treated patients with WM, median age 69.5(39-84); 68.2% males. IPSS Grade1 21.4%; Grade2 32.1%; Grade3 42.9%; Unknown 3.6%. 75%(9/12) patients with MYD88L265P mutation, 33%(1/3) patients with CXCR4 mutation. Treatment included chemotherapy(containing nitrogen mustard phenylbutyrate, fludarabine, cyclophosphamide), proteasome inhibitor regimens, rituximab regimens and immunomodulator regimens. Summary/Conclusion: These results demonstrate that zanubrutinib and dexamethasone are quickly effective in the treatment of WM, with more deeper response and less toxicity. P1121: TAKEAIM LYMPHOMA- AN OPEN-LABEL, DOSE ESCALATION AND EXPANSION TRIAL OF EMAVUSERTIB (CA-4948) IN COMBINATION WITH IBRUTINIB IN PATIENTS WITH RELAPSED OR REFRACTORY HEMATOLOGIC MALIGNANCIES E. Joffe1,*, G. Nowakowski2, H. Tun3, A. Rosenthal4, M. Lunning5, R. Ramchandren6, C.-C. Li7, L. Zhou7, E. Martinez7, R. von Roemeling7, R. Earhart7, M. McMahon7, I. Isufi8, L. Leslie9 1MSKCC, NY; 2Mayo Clinic-Minnesota, Rochester, Rochester; 3Mayo Clinic-Florida, Jacksonville; 4Department of Hematology, Mayo Clinic- Arizona, Phoenix; 5University of Nebraska, Omaha; 6University of Tennessee Medical Center, Knoxville; 7Curis, Lexington; 8Yale New Haven Hospital, New Haven; 9John Theurer Cancer Center, Hackensack, United States of America Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4), which is essential for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in B cell proliferation. IRAK4 forms a Myddosome complex with MYD88 adaptor protein and drives overactivation of nuclear factor-kappa B (NF-κB), causing inflammation and tumor growth. Emavusertib has been reported to be well tolerated and active as monotherapy in heavily pretreated patients with relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL). Preclinical studies demonstrated that tumor resistance and survival via IRAK4 activation could be delayed or reversed. Emavusertib crossed the blood-brain barrier in a murine PDX model of pCNS lymphoma, resulting in tumor response and prolonged survival. In combination with Bruton tyrosine kinase (BTK) inhibitors, emavusertib showed in vivo synergy in B-cell NHL. Here we present an update on the preliminary efficacy data of emavusertib + ibrutinib in R/R hematologic malignancies. Aims: Assessment of safety and clinical activity of emavusertib in combination with ibrutinib at full prescribed dose. Methods: This is an ongoing open-label trial (NCT03328078) of emavusertib as monotherapy and in combination with ibrutinib. Part A1 (completed) dose escalation of emavusertib as monotherapy; the recommended phase 2 dose (RP2D) is 300 mg BID with continuous oral dosing. Part A2 (dose escalation in combination with ibrutinib), and Part B (a basket design of 4 expansion cohorts of emavusertib and ibrutinib: BTK-naïve MZL, DLBCL, or PCNSL and NHL with adaptive resistance to ibrutinib). The primary endpoints of Parts A1 and A2 include safety, tolerability, and RP2D. The primary endpoints of Part B include CR or ORR, with key secondary endpoints of DOR, DCR, PFS and OS following treatment of emavusertib at dose levels of 200 (DL1) or 300 mg BID (DL2) with ibrutinib at full prescribed dose. Results: As of December 7th, 2021, 35 heavily pretreated NHL patients have received emavusertib monotherapy (median age 66 years, range 50-87), of which six patients have been on emavusertib for approximately 1 year or longer, suggesting emavusertib has a long-term acceptable safety and tolerability profile at RP2D (dose level of 300 mg BID). In Part A2 , 10 patients are treated with emavusertib + ibrutinib (median age 65 years, range 56-82). Median number of prior lines of anti-cancer therapies is 3 (range 1-8). No DLTs were observed at 200 or 300 mg dose levels to date. The preliminary efficacy data of seven evaluable patients with combination therapy showed 1 CR (MCL), 2 PR (MCL and MZL), 3 SD, and 1 PD, 3 of whom had failed prior ibrutinib. The preliminary data indicate the combination therapy may overcome ibrutinib resistance. Summary/Conclusion: Emavusertib as a monotherapy and in combination with ibrutinib is well tolerated with an acceptable long term safety profile and promising efficacy. Part A2 is transitioning to Part B basket cohorts of MZL, ABC-DLBCL, PCNSL and NHL with adaptive resistance to ibrutinib. P1122: EFFICACY OF ALPELISIB IN PI3K-DRIVEN LANGERHANS CELL HISTIOCYTOSIS R. Mazor1,*, B. Durham2, O. Abdel-Wahab2, E. Diamond3, G. Itchaki4, M. Ben-Sasson5,6,7, O. Hershkovitz-Rokah1,8,9, O. Shpilberg1,9,10 1Clinic of Histiocytic Neoplasms, Institute of Hematology, Assuta Medical Center, Tel Aviv, Israel; 2Human Oncology and Pathogenesis Program; 3Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, United States of America; 4Department of Hematology, Rabin Medical Center, Petah Tikva; 5The Institute for Pain Medicine, Rambam Medical Center; 6The Rappaport school of medicine, Technion, Haifa; 7Meuhedet Health Maintenance Organization, Zikhron Ya’akov; 8Department of Molecular Biology, Faculty of Natural Sciences, Ariel University, Ariel; 9Translational Research Lab, Assuta Medical Center, Tel Aviv; 10The Adelson School of Medicine, Ariel University, Ariel, Israel Background: Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm characterized by the accumulation of clonal dendritic cells expressing CD1a and CD207 (Langerin). LCH affects both children and adults and typically ranges from an indolent unifocal form to a progressive multisystemic disease. In the past decade, in depth molecular profiling of LCH as well as other histiocytic neoplasms demonstrated that these diseases are fundamentally dependent on somatic MAPK activating mutations, with the canonical V600E BRAF mutation emerging in more than 50% of LCH patients. Nonetheless, MAPK independent driver mutations were previously reported and anecdotal evidence of successful targeted treatment of various histiocytoses harboring non-canonical drivers such as RET, ALK and CSF1R were previously described in the literature. Aims: To report a case of PI3K driven LCH treated with an isoform specific PIK3CA inhibitor. Methods: Case summary: a 46 year-old mother of 3 presented with complaints of progressive headaches, polydipsia and polyuria. Following a pathological water deprivation test and identification of pituitary stalk thickening on MRI, a diagnosis of central diabetes insipidus (CDI) was established and desmopressin treatment was initiated. However, 10 months following her CDI diagnosis, the patient began to complain of sub febrile episodes of fever and worsening dry cough. Imaging studies including PET/CT disclosed extensive bilateral reticulo-nodular infiltration, nodules with cystic transformation and ground glass opacity, suspicious of pulmonary LCH. Histological examination of a wedge biopsy specimen obtained from the lungs confirmed the diagnosis of LCH and consequent molecular studies identified the M1043V PIK3CA mutation. During her medical investigation, the patient’s conditioned worsened, with new disease foci emerging – including cervical lymphadenopathy and a lytic vertebral lesion involving D11. The patient then received palliative radiotherapy directed at the D11 lytic lesion followed by escalating systemic therapy with single agent Alpelisib, via the Managed Access Program at Novartis. Response to treatment was assessed clinically and using PET/CT. At the molecular level, the effect of Alpelisib was assessed by measuring PTEN expression levels and cell cycle regulating non-coding RNA molecules by using quantitative real-time polymerase chain reaction (qRT-PCR). Results: Treatment with Alpelisib resulted in rapid amelioration of night sweats within 6 days, decrease in back pain within 3 weeks and normalization of all abnormally FDG avid disease foci within 3 months of treatment. The complete metabolic response observed persists currently more than a year following treatment initiation. Treatment with Alpelisib is well tolerated except for a mild increase in HbA1C levels to 6.7%. Moreover, PTEN, the main regulator of the PI3K pathway was found to be upregulated following 3 and 9 months of treatment, in parallel to downregulation of small non-coding RNAs molecules that regulates its expression. Image: Summary/Conclusion: This is the first study demonstrating that the alpha catalytic subunit of PI3K is a targetable non-canonical driver of LCH. P1123: TEMPO: A PHASE 2, RANDOMIZED, OPEN-LABEL, 2-ARM STUDY COMPARING TWO INTERMITTENT DOSING SCHEDULES OF DUVELISIB IN SUBJECTS WITH INDOLENT NON-HODGKIN LYMPHOMA (INHL) V. Vorobyev1,*, D. H. Yoon2, M. Kaźmierczak3, S. Grosicki4, C. Tarella5, A. Genua6, J. S. Kim7, D. Cohan8, M. Daugherty8, I. W. Flinn9, P. L. Zinzani10, L. I. Gordon11 1S. P. Botkin City Clinical Hospital, Moscow, Russia; 2University of Ulsan College of Medicine, Seoul, South Korea; 3Poznań University of Medical Sciences, Poznań; 4Silesian Medical University, Katowice, Poland; 5IEO Istituto Europeo di Oncologia IRCCS, Milano; 6Università degli studi di Perugia, Terni, Italy; 7Yonsei University College of Medicine, Severance Hospital, Seoul, South Korea; 8Secura Bio, Las Vegas; 9Sarah Cannon Research Institute-Tennessee Oncology, Nashville, United States of America; 10University of Bologna, Istituto Di Ematologia, Bologna, Italy; 11Northwestern University Feinberg School of Medicine and the Robert H. Lurie Comprehensive Cancer Center, Chicago, United States of America Background: Dosing of duvelisib at 25 mg orally BID has been shown to be active in iNHL, as the pivotal phase 2 DYNAMO study (NCT01882803) in iNHL met its primary endpoint of overall response rate (ORR) assessed by an IRC. Aims: The TEMPO study (NCT04038359; supported by Secura Bio) examined the effects of prespecified 2-week dose holidays on tumor responses and safety/tolerability in patients (pts) with iNHL based on data from a phase 3 trial (DUO) in CLL/SLL that demonstrated no negative impacts of (unscheduled) dose interruptions on response (Flinn I et al. J Clin Oncol. 2019;37(15):7523). Methods: In TEMPO, patients were randomized to receive duvelisib 25 mg BID for one 10-week cycle followed by 25 mg BID on Weeks 3 and 4 of each subsequent 4-week cycle (Arm 1) or duvelisib 25 mg BID on Weeks 1, 2, 5, 6, 9, and 10 of one 10-week cycle, and then on Weeks 3 and 4 of each subsequent 4-week cycle (Arm 2). Pts continued treatment until progressive disease (PD), unacceptable toxicity, or study withdrawal. ORR by investigator assessment, based on 2007 revised International Working Group (IWG) criteria was the primary endpoint. Results: As of data cutoff on 5 May 2021, 65 treated pts had at least 4 months of follow-up on or after study drug treatment. Across both arms, the median age was 63 years (range 34-85), and 47 (72.3%) of patients remained on duvelisib at the time of this analysis (see Table). The patient population was heavily pretreated (total prior lines: 36.9% = 1; 26.2% = 2; 10.8% = 3; and 26.2% ≥ 4); 93.8% of pts received prior rituximab and 43.1% prior bendamustine. As of data cutoff, 27.7% of pts have discontinued duvelisib, 19.7% due to PD (21.2% in Arm 1; 18.8% in Arm 2). Four patients, all in Arm 1, had a TEAE that led to treatment discontinuation. Across both arms, the ORR was 60% (39 of 65 pts), and the complete response (CR) rate was 12.3% (8 of 65 pts). Results were similar in pts who received prior rituximab (59.0%) and those who received prior bendamustine (57.1%) and were relatively consistent regardless of the number of prior treatments received (62.5%, 64.7%, 54.2% for pts with 1, 2, or ≥ 3 prior lines of therapy, respectively). Overall, the incidence of TEAEs was higher in Arm 1 vs Arm 2, including grade ≥ 3 AEs (ALT elevation 30.3% vs 3.1%; AST elevation 27.3% vs 3.1%; GI disorders 9.1% vs 0; infections 9.1% vs 3.1%; rash 6.1% vs 3.1%; and drug reaction with eosinophilia 3.0% vs 0); the incidence of grade ≥ 3 neutropenia was higher in Arm 2 (9.1% vs 18.8%). TEAEs leading to discontinuation of study drug occurred only in Arm 1 (15.2%; 5 of 33 pts) and included ALT/AST elevations (2 of 33 pts; 6.1%), skin and subcutaneous tissue disorders (2 of 33 pts; 6.1%), and pneumonia (1 of 33 pts; 3.0%). There was 1 death on study in Arm 1 due to disease progression. No TEAE-related deaths occurred in either arm. Image: Summary/Conclusion: Based on this interim analysis, continuous followed by intermittent dosing (Arm 1) and intermittent dosing (Arm 2) are both safe and effective in patients with iNHL. Efficacy was seen irrespective of prior lines of therapy, including those heavily pretreated and/or treated with prior rituximab or bendamustine. The ORR and CR are consistent with those seen in DYNAMO. Because these are early data, the differences between arms in certain types of AEs or discontinuations due to AEs are difficult to interpret. The study confirms the overall therapeutic profile of duvelisib and demonstrates that intermittent dosing may decrease the incidence of AEs without compromising efficacy. P1124: MOSUNETUZUMAB RETREATMENT IS EFFECTIVE AND WELL-TOLERATED IN PATIENTS WITH RELAPSED OR REFRACTORY B-CELL NON-HODGKIN LYMPHOMA C. Y. Cheah1,*, N. L. Bartlett2, S. Assouline3, S. J. Schuster4, W. Seog Kim5, M. Shadman6, I. Isufi7, S. Yin8, M. Y. Doral8, J. Sit8, V. Chen8, H. Huang9, M. Zhou10, M. C. Wei8, L. E. Budde11 1Linear Clinical Research, Sir Charles Gairdner Hospital and The University of Western Australia, Perth, Australia; 2Washington University School of Medicine, Siteman Cancer Center, St Louis, MO, United States of America; 3Jewish General Hospital, Montreal, Canada; 4University of Pennsylvania, Philadelphia, PA, United States of America; 5Samsung Medical Centre, Seoul, South Korea; 6Fred Hutchinson Cancer Research Center, Seattle, WA, Seattle, WA; 7Yale School of Medicine, New Haven, CT; 8Genentech, Inc., South San Francisco, CA, United States of America; 9Hoffmann-La Roche Ltd, Mississauga, Canada; 10F. Hoffmann-La Roche, Shanghai, China; 11City of Hope, Duarte, CA, United States of America Background: The prognosis for heavily pretreated patients (pts) with relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL) is poor and there is a need for effective treatment options (Batlevi, et al. 2020; Crump, et al. 2017). Mosunetuzumab (M) is a T-cell engaging CD20xCD3 bispecific monoclonal antibody that redirects T cells to eliminate malignant B cells (Sun, et al. 2015; Budde, et al. 2022). Results from a single-arm, Phase I/II study (NCT02500407) of M in pts with R/R aggressive and indolent NHL showed durable complete responses (CR) and a manageable safety profile (Budde, et al. 2022). Specifically, in pts with R/R follicular lymphoma (FL) after ≥2 prior therapies, M demonstrated a 60% CR rate and an 80% objective response rate with a median duration of response (DoR) of 22.8 months (Budde, et al. ASH 2021). Initial M treatment had a fixed duration (8 cycles for pts who achieved CR; 17 cycles for pts with partial response or stable disease after 8 cycles); however, for pts who achieved a CR but had progressive disease (PD) after initial treatment completion, M retreatment could be provided. We report additional data from this ongoing study to describe the M retreatment experience of pts with R/R NHL. Aims: To evaluate the efficacy and safety of M monotherapy (mono) retreatment in pts with R/R NHL from an ongoing Phase I/II study (NCT02500407). Methods: Pts with R/R NHL who achieved CR after initial treatment with intravenous (IV) M mono but subsequently developed PD after treatment completion were eligible for retreatment. Pts were retreated with IV M mono at a previously tested dose and schedule, which included step-up dosing for cytokine release syndrome (CRS) mitigation. All pts provided informed consent. Results: At the clinical cut-off date, March 15, 2021, 12 pts received M retreatment; 6 pts completed retreatment and 6 pts discontinued retreatment (PD, n=5; start of another anti-lymphoma therapy, n=1). M retreatment step-up dose schedules were as follows (Cycle [C] 1, Day [D] 1/C1D8/C1D15/C2D1/C3+D1): 1/2/13.5mg (n=1), 1/2/20mg (n=1), 1/2/27mg (n=1), 1/2/40.5mg (n=1) and 1/2/60/60/30mg (n=8). Median number of M retreatment cycles received was 8.0 (range, 2–13). Overall, 8 pts with R/R FL, 2 pts with R/R diffuse large B-cell lymphoma (DLBCL), 1 pt with R/R transformed FL (trFL) and 1 pt with R/R mantle cell lymphoma (MCL) were included in the analysis. During retreatment, 9 (75.0%) pts had an objective response (6/8 pts with R/R FL; 2/2 pts with R/R DLBCL; 1/1 pt with trFL; and 0/1 pt with MCL; Table). Six/12 pts achieved a CR (4/8 pts with R/R FL, 1/2 pts with R/R DLBCL; 1/1 pt with R/R trFL; and 0/1 pt with MCL). All pts who received M retreatment were included in the safety population and had ≥1 adverse event (AE). The most common any-grade AE and Grade 3–4 AE was hypophosphatemia (n=7, 58.3% [each]; none were serious and all events resolved with or without treatment). Four pts experienced ≥1 serious AE. No Grade 5 events were reported and no pts had an AE that led to treatment discontinuation. Three pts (25.0%) had low-grade CRS (by Lee, 2014; Grade 1, n=2; Grade 2, n=1) and there were no Grade 3–4 CRS events. Image: Summary/Conclusion: Our results show that IV M mono retreatment was efficacious in heavily pretreated pts with R/R NHL who initially achieved a CR with M treatment, but subsequently developed PD. M retreatment had a manageable safety profile, consistent with the initial treatment. P1125: CELESTIMO: A PHASE III TRIAL EVALUATING THE EFFICACY AND SAFETY OF MOSUNETUZUMAB PLUS LENALIDOMIDE VERSUS RITUXIMAB PLUS LENALIDOMIDE IN PATIENTS WITH RELAPSED OR REFRACTORY FOLLICULAR LYMPHOMA L. Nastoupil1,*, F. Morschhauser2, C. W. Scholz3, M. Bishton4, S.-S. Yoon5, P. Giri6, M. C. Wei7, A. Knapp8, C.-C. Li7, A. Bottos8, H. Li9, E. Purev7, N. L. Bartlett10 1Emory University, Atlanta, GA, United States of America; 2University of Lille, CHU Lille, Lille, France; 3Vivantes Klinikum am Urban, Berlin, Germany; 4Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; 5Seoul National University Hospital, Seoul, South Korea; 6Royal Adelaide Hospital, Adelaide, Australia; 7Genentech, Inc., South San Francisco, CA, United States of America; 8F. Hoffmann-La Roche Ltd, Basel, Switzerland; 9Hoffmann-La Roche Ltd, Mississauga, ON, Canada; 10Washington University School of Medicine, St. Louis, United States of America Background: Despite significant progress with first-line immunochemotherapy, most patients with follicular lymphoma (FL) will eventually relapse with increasing refractoriness and decreasing duration of response to subsequent therapy lines (Rivas-Delgado et al. 2019). Mosunetuzumab (M) is a CD20xCD3 bispecific antibody that engages and redirects T-cells to eliminate malignant B cells (Sun et al. 2015). In an ongoing, pivotal Phase I/II trial of M monotherapy, patients with relapsed/refractory (R/R) FL who have received ≥2 prior treatment lines achieve deep and durable responses (NCT02500407; Budde et al. ASH 2021). Preliminary data from a Phase Ib study have suggested favorable safety and promising activity of M in combination with lenalidomide (Len), a potent immunomodulatory agent that has shown additive/synergistic activity with an anti-CD20 antibody in R/R indolent lymphoma (Leonard et al. 2019), in patients with R/R FL who have received ≥1 prior therapy (NCT4246086; Morschhauser et al. ASH 2021). The chemotherapy-free M-Len combination may represent a promising outpatient therapy option for future management of patients with R/R FL. The randomized, multicenter Phase III study has been initiated. Aims: CELESTIMO (NCT04712097) is a randomized, multicenter, open-label Phase III study evaluating the efficacy and safety of M-Len versus rituximab plus Len (R-Len) in patients with previously treated R/R FL. Methods: Patients must have histologically documented CD20+ FL (Grades 1–3a) requiring systemic therapy and have received ≥1 prior line of systemic therapy. Patients are randomized (1:1) to receive M-Len (M intravenously [IV] on Days [D] 1, 8 and 15 of Cycle [C] 1 [21-day cycle] and D1 of C2–12 [28-day cycles], plus Len orally [PO] on D1–21 of C2–12) or R-Len (R IV on D1, 8, 15 and 22 of C1 then on D1 of C3, 5, 7, 9, and 11, plus Len PO on D1–21 of C1–12 [all 28-day cycles]), and stratified by disease progression within 24 months of initial treatment (yes/no), number of prior lines of therapy (1 versus ≥2), and refractoriness to anti-CD20 therapy (refractory/non-refractory). All patients must provide informed consent. Results: The primary endpoint is progression-free survival (PFS) assessed by independent review committee; secondary endpoints include investigator-assessed PFS, complete and objective response, overall survival, and safety. Biomarkers predictive of response to M-Len and R-Len will also be investigated as exploratory endpoints. Summary/Conclusion: The study started recruitment in 2021 and plans to enroll ~400 patients from approximately 16 countries and 150 sites globally. Further study details will be presented. P1126: MOSUNETUZUMAB IS EFFICACIOUS AND WELL TOLERATED IN PATIENTS AGED <65 AND ≥65 YEARS WITH RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA AND ≥2 PRIOR THERAPIES: SUBGROUP ANALYSIS OF A PIVOTAL PHASE II STUDY M. Matasar1,*, N. L. Bartlett2, L. H. Sehn3, S. J. Schuster4, S. Assouline5, P. Giri6, J. Kuruvilla7, M. Canales8, S. Dietrich9, K. Fay10, M. Ku11, L. J. Nastoupil12, M. C. Wei13, S. Yin13, I. To13, D. Turner13, H. Huang14, J. Min15, E. Penuel13, L. E. Budde16 1Memorial Sloan Kettering Cancer Center, New York, NY; 2Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, United States of America; 3BC Cancer Centre for Lymphoid Cancer and University of British Columbia, Vancouver, BC, Canada; 4Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, United States of America; 5Jewish General Hospital, Montreal, QC, Canada; 6Royal Adelaide Hospital, Adelaide, Australia; 7Princess Margaret Cancer Centre, Toronto, ON, Canada; 8Hospital Universitario La Paz, Madrid, Spain; 9Universitat Heidelberg, Heidelberg, Germany; 10St Vincent’s Hospital and Royal North Shore Hospital, Sydney; 11St Vincent’s Hospital, University of Melbourne, Melbourne, Australia; 12MD Anderson Cancer Center, Houston, TX; 13Genentech, Inc., South San Francisco, CA, United States of America; 14Hoffmann-La Roche Ltd, Mississauga, ON, Canada; 15Roche Products Ltd, Welwyn Garden City, United Kingdom; 16City of Hope, Duarte, CA, United States of America Background: Advancing age is associated with a decline in immune function and an increase in the incidence of comorbidity (Castellino et al. 2017). Vulnerability to treatment-related toxicities may also increase with age. Mosunetuzumab is a T-cell engaging CD20xCD3 bispecific monoclonal antibody (Ab) that redirects T cells to eliminate malignant B cells. In a pivotal Phase II study (NCT02500407), mosunetuzumab induced deep and durable remissions and had a favorable safety profile in patients (pts) with relapsed/refractory (R/R) follicular lymphoma (FL) and ≥2 prior therapies (Budde et al. ASH 2021). Aims: In the current study, we evaluated the efficacy and safety of mosunetuzumab in pts aged <65 and ≥65 years (age stratification per ESMO Clinical Practice Guidelines for management of newly diagnosed and R/R FL; Dreyling et al. 2021) in the pivotal Phase II study. Methods: All pts had Grade (Gr) 1–3a FL and ECOG performance status (PS) 0–1, and were R/R to ≥2 prior therapies, including an anti-CD20 Ab and an alkylating agent. Intravenous mosunetuzumab was given in 21-day cycles with Cycle (C) 1 step-up dosing: C1 Day (D) 1: 1mg; C1D8 2mg; C1D15 and C2D1: 60mg; D1 of C3+: 30mg. Pts with a complete response (CR) by C8 completed therapy; pts with a partial response or stable disease continued treatment for up to 17 cycles, unless disease progression or unacceptable toxicity occurred. The primary endpoint was CR (as best response) rate by PET/CT assessed by Independent Review Committee using standard response criteria (Cheson et al. 2007). Cytokine release syndrome (CRS) was graded using ASTCT criteria (Lee et al. 2019). All pts provided informed consent. Results: As of August 27, 2021, 90 pts had received mosunetuzumab; 67% were aged <65 years and 33% were aged ≥65 years. Of 30 pts aged ≥65 years, median age was 71 years (range: 65–90) and 43% had ECOG PS 1 at entry, 53% had FLIPI 3–5, and 70% had Ann Arbor stage III/IV disease. Median number of prior therapies in the older age group was 3 (range: 2–8); 77% were refractory to a prior anti-CD20 Ab and 43% were double-refractory to a prior anti-CD20 Ab and an alkylating agent. Compared with younger pts, those aged ≥65 years had a numerically higher objective response rate (87% [95% CI: 69–96] vs 77% [95% CI: 64–87]) and CR rate (70% [95% CI: 51–85] vs 55% [95% CI: 42–68]). The 18-month event-free rate for duration of response was 54% (95% CI: 31–76) in pts aged ≥65 years and 59% (95% CI: 43–74) in those aged <65 years. The rate of Gr 3–4 adverse events (AEs) was comparable in the older and younger age groups (73% vs 68%, respectively). A lower rate of serious AEs of any Gr was observed in pts aged ≥65 years (37% vs 52%). One patient in each age group discontinued treatment due to mosunetuzumab-related AEs. CRS occurred less frequently in pts aged ≥65 years than in those aged <65 years (30% vs 52%), while CRS events were predominantly low-Gr in both groups (age ≥65 vs <65 years: Gr 1, 20% vs 28%; Gr 2, 7% vs 22%); high-Gr CRS was uncommon (one patient in each group). All CRS events resolved. No cases of aphasia, seizures, encephalopathy or cerebral edema occurred. Rates of serious AEs of infection (any Gr) were comparable in pts aged ≥65 and <65 years (17% vs 22%, respectively). The PK disposition of mosunetuzumab was also comparable in pts aged ≥65 and <65 years. Summary/Conclusion: Mosunetuzumab is efficacious and well tolerated in older and younger pts with R/R FL and ≥2 prior therapies. In older pts (aged ≥65 years), numerically lower rates of CRS and serious AEs were observed. P1127: WATCHFUL WAITING IS AN ACCEPTABLE TREATMENT OPTION FOR PRIMARY OCULAR ADNEXAL MUCOSA-ASSOCIATED LYMPHOID TISSUE LYMPHOMA: A RETROSPECTIVE STUDY K. Mizuhara1,*, T. Kobayashi1, M. Nakao2, R. Takahashi3, H. Kaneko4, K. Shimura4, K. Hirakawa5, N. Uoshima6, Y. Kamitsuji6, K. Wada7, E. Kawata7, R. Isa1, T. Fujino1, Y. Matsumura-Kimoto1, T. Tsukamoto1, S. Mizutani1, Y. Shimura1, M. Taniwaki8, J. Kuroda1 1Division of Hematology and Oncology, Department of Medicine, Kyoto Prefectural University of Medicine, Kyoto; 2Department of Hematology, Otsu City Hospital, Otsu; 3Department of Hematology, Omihachiman Community Medical Center, Omihachiman; 4Department of Hematology, Aiseikai Yamashina Hospital, Kyoto; 5Department of Hematology, Fukuchiyama City Hospital, Fukuchiyama; 6Department of Hematology, Japanise Red Cross Kyoto Daini Hospital, Kyoto; 7Department of Hematology, Matsushita Memorial Hospital, Moriguchi; 8Center for Molecular and Therapeutics, Kyoto Prefectural University of Medicine, Kyoto, Japan Background: Primary ocular adnexal mucosa-associated lymphoid tissue (MALT) lymphoma (POAML) is the most frequent subtype of ocular adnexal lymphomas. Radiotherapy (RT) is the standard of care for localized POAML, despite several complications, such as cataracts, retinitis, dry eye, and optic neuropathy. Watchful waiting (WW) is expected to be a therapeutic option for POAML, however, its efficacy for POAML remains to be verified. Aims: The aim of the study was to clarify the validity of WW for POAML. Methods: We retrospectively analyzed the background, disease status, treatments, and clinical outcomes of patients histologically diagnosed with POAML between 2008 and 2019 at seven institutes belonging to the Kyoto Clinical Hematology Study Group. The primary ocular adnexal disease sites were defined as involvement of the conjunctiva, orbit, eyelid, or lacrimal gland. Bilateral ocular adnexal lymphoma without other lymphomatous involvement was defined as stage IE disease. Results: One hundred and three patients were diagnosed with POAML, of which 11 were excluded from analysis because of lost follow-up, 11 due to missing data, and 6 due to short follow-up within 1 year. As a result, 75 patients were analyzed in this study. The median age was 68 years, and 41 patients (54.7%) were females. The most common involved site was the conjunctiva (42.7%), followed by the orbit (36.0%), lacrimal gland (12.0%), and eyelid (8.0%). Sixty-nine patients (92.0%) presented with stage IE disease, and 61 patients (81.3%) presented with a unilateral ocular region. According to the International Prognostic Index, most patients (92.0%) were classified as low risk. The tumor was completely resected at diagnosis in 17 patients (22.7%), while a residual tumor existed after biopsy in 58 patients (77.0%). WW was the most frequent first-line modality, selected for 29 patients (38.7%), including 14 patients with complete tumor resection at diagnosis. Twenty-four patients (32.0%) were treated with RT and 19 (25.3%) with rituximab monotherapy (R). In the RT group, most patients had a residual tumor after the biopsy, and the orbit was the most frequent site of lymphoma, which caused treatment-emergent symptoms that potentially impair visual function. Complete response rates were 79.2% and 42.1% in the RT and R groups, respectively. With a median follow-up of 48.8 months, there were no significant differences in the time to start of new treatment between WW and RT groups (median: both not reached [NR], p = 0.187) and between WW and R groups (median: NR vs. 69.0 months, p = 0.554). At 60 months follow-up, the estimated proportions of POAML patients not requiring new treatment were 69.4%, 85.2%, and 53.8% in the WW, RT, and R groups, respectively. All patients enrolled in this study were alive at the time of analysis. In the WW group, 9 patients (31.0%) showed disease progression, seven of whom received treatments with a median time to next treatment of 15.6 months, but the remaining two patients continued WW even after disease progression because of no subjective symptoms. In the RT group, 16 patients (66.7%) developed adverse events, most of which were mild, while grade 3 cataracts occurred in five patients. We compared the clinical characteristics to identify predictive factors for disease progression in the WW group, however, all factors analyzed were not significantly associated with disease progression. Summary/Conclusion: WW may be an acceptable treatment option for POAML, especially in asymptomatic patients. P1128: CLINICAL FEATURES AND OUTCOME OF PATIENTS WITH CASTLEMAN DISEASE: A SPANISH MULTICENTRIC STUDY OF 134 PATIENTS FROM GELTAMO J. T. Navarro1,2,3,*, C. Celades2,3, O. García1,2,4,5, E. González-Barca6,7,8, F. Climent7,9, A. Feu9, A. Jiménez10, A. Gutiérrez de la Peña10, M. Bastos-Oreiro11,12, T. Aldamiz-Echevarria13, A. Gutiérrez14,15, L. Bento14,15, P. Abrisqueta16,17, C. M. Alonso18, C. Tejada Chavez18, E. M. Ocio19,20, N. Fernández Escalada19,20, M. B. Navarro Matilla21, J. M. Mateos Pérez21, A. López-García22, C. Castillo-Girón23, S. F. Pinzón24, E. Pérez Ceballos25, J. Á. Hernández Rivas26,27, R. del Campo García15,28, E. Pardal de la Mano29, R. García-Sanz30,31,32,33, J. Rovira34, J. M. Sancho1,2,3, G. Tapia35,36 1Hematology, Institut Català d’Oncologia-Germans Trias i Pujol Hospital; 2Lymphoid Neoplasms, Josep Carreras Leukaemia Research Institute; 3Medicine, Universitat Autònoma de Barcelona; 4Institut Germans Trias i Pujol; 5Universitat Autònoma de Barcelona, Badalona; 6Hematology, Institut Català d’Oncologia, Hospitalet de Llobregat; 7IDIBELL; 8Universitat de Barcelona, Hospitalet; 9Pathology, Hospital Universitari de Bellvitge, Hospitalet de Llobregat; 10Hematology, Hospital 12 de Octubre; 11Hematology, Hospital Gregorio Marañón; 12Instituto de Investigación Sanitaria Gregorio Marañón; 13Infectious Diseases-Clinical Microbiology, Hospital Gregorio Marañón, Madrid; 14Hematology, Son Espases University Hospital; 15IdISBa, Palma de Mallorca; 16Hematology, Hospital Universitari Vall d’Hebron; 17Vall d’Hebron Institute of Oncology (VHIO), Barcelona; 18Hematology and Hemotherapy, Hospital Arnau de Vilanova, Valencia; 19Hematology, Hospital Universitario Marqués de Valdecilla (IDIVAL); 20Universidad de Cantabria, Santander; 21Hematology, Hospital Universitario Puerta de Hierro Majadahonda; 22Hematology, Hospital Universitario Fundación Jiménez Diaz, Madrid; 23Amyloidosis and Myeloma, Hospital Clinic; 24Hematology, Hospital del Mar, Barcelona; 25Hematology, Hospital Universitario Morales Meseguer, Murcia; 26Hematology and Hemotherapy, Hospital Universitario Infanta Leonor; 27Universidad Complutense de Madrid, Madrid; 28Hematology, Hospital Universitari Son Llàtzer, Palma de Mallorca; 29Hematology and Hemotherapy, Hospital Virgen del Puerto, Plasencia; 30Hematology, University Hospital of Salamanca & Cancer Research Center (CiC-IBMCC, CSIC/USAL); 31Center for Biomedical Research in Network of Cancer (CIBERONC); 32Institute of Biomedical Research of Salamanca (IBSAL); 33Universidad de Salamanca, Salamanca; 34Hematology, Hospital Joan XXIII-ICO Tarragona, Tarragona; 35Pathology, Hospital Germans Trias i Pujol, Badalona; 36Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain Background: Castleman disease (CD) is a heterogeneous rare disorder, characterized by lymph node enlargement with a common histopathological spectrum. This disease comprises the subtypes unicentric (UCD), HHV-8 multicentric CD (HHV-8 MCD), POEMS-asociated MCD (POEMS MCD) and idiopathic multicentric CD (iMCD).2 Whereas UCD is a localized reversible disease, the other three are systemic diseases with multiple lymphadenopathies. The TAFRO (thrombocytopenia, anasarca, fever, reticulin myelofybrosis, organomegaly) syndrome is an aggressive form of iMCD. Each CD subtype has different clinical features, prognosis and treatment. Aims: This study aims to study a large series of patients diagnosed with CD to describe the clinical and biological characteristics, as well as the outcome, of the different CD subtypes. Methods: Multicentric retrospective study which includes patients diagnosed with different subtypes of CD in 19 Spanish hospitals from 2006 to 2020. Clinical and biological data were retrieved from the clinical records. The project has been approved by the Ethical Committee of Germans Trias I Pujol Hospital (PI-20-103). Statistical analyses were performed using SPSS v24.0 (IBM, Somer, NY). Results: One hundred and thirty-four patients with available data were included; 47 with UCD and 87 with any of the 3 subtypes of MCD. The median follow-up was 3.4 years. The main clinical and biological characteristics of the 4 CD subtypes are shown in Table 1 and differences in OS in figure 1. Most patients with HHV-8 MCD were HIV-infected (73%); median CD4 lymphocyte count 0.229x109/L (range: 0.016, 1.2) and 46% of them had concomitant or prior Kaposi’s sarcoma. Three iMCD patients had TAFRO. All patients with UCD were treated with surgery. Most patients with HHV-8 MCD were treated at front-line with rituximab (69%) or polychemotherapy plus rituximab (21%). Patients with iMCD were treated with anti-IL-6 (25%), polychemotherapy (25%), rituximab (25%) and steroids (25%). Treatment of POEMS MCD was also diverse; 3 patients received lenalidomide plus dexamethasone, 2 polychemotherapy, 1 anti-IL-6 and 1 auto SCT (3 patients unrecorded). Eleven patients (8.2%) had concomitant or evolved to lymphoma (2 UCD, 2 iMCD and 7 HHV-8 MCD). Thirty-two patients are dead (20 HHV8-MCD, 7 iMCD, 2 UCD, 3 POEMS), 6 of them from CD (4 HHV8-MCD, 2 iMCD). Table 1. Main clinical and biological characteristics of the CD subtypes Variable UCD (n=47) iMCD (n=24) HHV8-MCD (n=52) POEMS (n=11) TOTAL (n=134) p value Male gender, % 36 46 90 36 59 <0.001 Age, median (range) 40 (5, 84) 47 (10, 82) 47 (25, 88) 64 (30, 78) 47 (5, 88) 0.154 ECOG<2, n (%) 36/38 (95) 13/19 (68) 24/39 (62) 5/8 (63) 78/104 (75) 0.005 Bulky mass, n (%) 3/41 (7) 0/19 1/41 (2) 0/9 4/110 (4) 0.422 B symptoms, n (%) 4/41 (10) 6/17 (35) 31/41 (76) 3/9 (33) 44/108 (41) <0.001 Hemoglobin g/L, median (range) 135 (74, 990) 132 (40, 355) 106 (43, 332) 144 (83, 172) 126 (40, 990) <0.001 Leukocytes x10 9 /L, median (range) 7.1 (3.8, 21.1) 7.2 (2, 13.2) 6.3 (0.5, 23.7) 7.9 (2.4, 23) 6.7 (0.5, 23.7) 0.075 Platelets x10 9 /L, median (range) 247(67, 483) 241 (13.5, 501) 190.5 (3.5, 461) 467 (1.4, 560) 232 (1.4, 560) 0.05 High LDH, n (%) 2/35 (6) 7/16 (44) 6/41 (15) 0/9 15/101 (15) 0.002 High Ferritin, n (%) 1/24 (4) 2/9 (22) 19/29 (66) 0/6 22/68 (32) <0.001 High Beta-2 microglobulin, n (%) 1/15 (7) 4/11 (36) 20/26 (77) 4/5 (80) 29/57 (51) <0.001 Low albumin, n (%) 3/35 (9) 6/14 (43) 19/39 (49) 2/8 (25) 30/96 (31) 0.002 Image: Summary/Conclusion: Castleman Disease subtypes have different characteristics and outcomes. First-line treatment is heterogeneous in MCD subtypes pointing a need for national guidelines. P1129: ACALABRUTINIB IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) MARGINAL ZONE LYMPHOMA (MZL): RESULTS OF A PHASE 2, MULTICENTER, OPEN-LABEL TRIAL P. Strati1,*, M. Coleman2, D. Stevens3, S. Ma4, C. Patti5, M. Levy6, I. S. Lossos7, P. Ramakrishnan Geethakumari8, S. Lam9, R. Calvo10, K. Higgins11, L. Budde12 1The University of Texas MD Anderson Cancer Center, Houston, TX; 2Clinical Research Alliance/Weill Cornell Medicine, New York City, NY; 3Norton Cancer Institute, Louisville, KY; 4Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, United States of America; 5A.O.O.R. Villa Sofia-Cervello, U. O. Ematologia I, Palermo, Italy; 6Baylor University Medical Center, Dallas, TX; 7Sylvester Comprehensive Cancer Center, University of Miami−Miller School of Medicine, Miami, FL; 8Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Cente, Dallas, TX, United States of America; 9London Health Sciences Centre, London, Ontario, Canada; 10AstraZeneca, Gaithersburg, MD; 11AstraZeneca, South San Francisco, CA; 12City Of Hope National Medical Center, Duarte, CA, United States of America Background: MZL is a rare indolent B-cell malignancy considered incurable at recurrent stage. Bruton tyrosine kinase (BTK) inhibitors have produced durable responses in patients (pts) with R/R MZL. Acalabrutinib (acala) is a potent next-generation BTK inhibitor with high selectivity for BTK. Aims: We report data for acala monotherapy from the R/R MZL cohort (phase 2) of a phase 1b/2 clinical trial (NCT02180711). Methods: Pts with histologically confirmed MZL, ECOG performance status ≤2, and ≥1 prior therapy (including ≥1 CD20-directed regimen) received oral acala 100 mg twice daily until disease progression or unacceptable toxicity ± R. The primary objective was overall response rate (ORR; Lugano criteria as assessed by the investigator). Secondary objectives were duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Data were analyzed descriptively (no formal hypothesis testing). Results: Forty-two pts received acala (median age 69 y [range 42–84]; median 2 prior systemic regimens [range 1–4]). MZL subtypes were extranodal (43%), nodal (31%), and splenic (26%). At data cutoff (Oct 15, 2021), median follow-up duration was 10.7 mo (range 0.4–42.8). Sixteen (38%) pts discontinued acala, most commonly due to disease progression (26%). Among pts evaluable for response (n=37; 3 pts had not reached the first assessment timepoint and 2 pts exited the study without response assessment), ORR was 54% (95% CI 37%–71%) with 6 complete (16%) and 14 partial (38%) responses; 17 (46%) pts had stable disease. ORRs in extranodal, nodal, and splenic subtypes were 65%, 44%, and 45%, respectively. Median time to initial response was 3.0 mo; median DOR was 19.3 mo (95% CI 8.4–not estimable). Median PFS was 27.4 mo with a 12-mo PFS rate of 66%. Four pts died (disease progression, n=2; transformation to diffuse large B-cell lymphoma after stopping treatment, n=1; adverse event [AE], n=1 [septic shock unrelated to treatment]); median OS was not reached. Treatment was well tolerated with most AEs being grade 1 or 2. Sixteen pts (38%) had grade ≥3 AEs, most commonly (in ≥2 pts) anemia, dyspnea, neutrophil count decrease (n=3 each), fatigue, thrombocytopenia, and neutropenia (n=2 each). AEs led to treatment discontinuation in 2 pts (grade 3 hypotension and grade 1 myalgia). Among AEs of clinical interest, hypertension was reported in 2 pts (both grade 2); no cases of atrial fibrillation/flutter or major hemorrhage were reported. Summary/Conclusion: These early results indicate that acala is efficacious and well tolerated in pts with R/R MZL. The AE profile is consistent with the known safety profile of acala. P1130: ACALABRUTINIB IN TREATMENT-NAIVE OR RELAPSED/REFRACTORY WALDENSTRÖM MACROGLOBULINEMIA: 5-YEAR FOLLOW-UP OF A PHASE 2, SINGLE-ARM STUDY R. Owen1,*, H. McCarthy2, S. Rule3,4, S. D’Sa5, S. Thomas6, O. Tournilhac7, F. Forconi7, M. José Kersten8, P. Luigi Zinzani9, S. Iyengar10, J. Kothari11, M. Minnema12, E. Kastritis13, B. Cheson14, H. Walter15, D. Greewald16, R. Calvo17, C.-C. Wun18, R. Furman19 1St. James’s University Hospital, Leeds; 2Royal Bournemouth Hospital, Bournemouth; 3Plymouth University Medical School, Plymouth, United Kingdom; 4AstraZeneca, Mississauga, Ontario, Canada; 5University College London Hospitals NHS Trust, London, United Kingdom; 6MD Anderson Cancer Center, Houston, TX, United States of America; 7University of Southampton Hospital Trust, Southampton, United Kingdom; 8Amsterdam University Medical Centers, University of Amsterdam, Cancer Center Amsterdam, Amsterdam, Netherlands; 9Institute of Hematology University of Bologna, Bologna, Italy; 10Royal Marsden Hospital, London; 11Churchill Hospital, Oxford, United Kingdom; 12University Medical Center Utrecht, University Utrecht, Utrecht, The Netherlands; on behalf of the Lunenburg Lymphoma Phase I/II Consortium – HOVON/LLPC, Utrecht, Netherlands; 13National and Kapodistrian University of Athens, Athens, Greece; 14Georgetown University Hospital, Lombardi Comprehensive Cancer Center, Washington, DC, United States of America; 15Ernest and Helen Scott Haematological Research Institute and Leicester Cancer Research Centre, University of Leicester, Leicester, United Kingdom; 16Cancer Center of Santa Barbara, Santa Barbara; 17AstraZeneca, Gaithersburg, MD; 18AstraZeneca, South San Francisco; 19Weill Cornell Medical College, New York Presbyterian Hospital, New York, NY, United States of America Background: Bruton tyrosine kinase (BTK) inhibitors are effective in the treatment of Waldenström macroglobulinemia (WM). Acalabrutinib is a highly selective, potent, covalent BTK inhibitor that demonstrated less toxicity and improved tolerability in a head-to-head trial versus ibrutinib in chronic lymphocytic leukemia. Aims: The aim of this trial was to determine the efficacy and safety of acalabrutinib in patients with treatment-naive (TN) or relapsed/refractory (R/R) WM. Interim results at 25 months (mo) of median follow-up have been previously reported. At a 5-year median follow-up, we report the final results of this trial. Methods: In this single-arm, multicenter, phase 2 trial, after informed consent was obtained, patients with TN or R/R WM received 100 mg oral acalabrutinib twice daily (or 200 mg acalabrutinib once daily, later switched to 100 mg twice daily). The primary endpoint was investigator-assessed overall response rate (ORR). Key secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Efficacy parameters were based on the modified 3rd International Workshop on Waldenström Macroglobulinemia (IWWM) criteria (Kimby 2006). Mutation data were available for a minority of patients and therefore are not reported. Results: Of 106 patients treated with acalabrutinib, 14 were TN and 92 were R/R; median age was 69 years (range 39–90); 94% had ECOG PS ≤1; median serum IgM was 3615 mg/dL (range 291–9740). Patients with R/R disease had a median of 2 prior therapies (range 1–7). ORR was 93% (95% CI: 66, 100) in the TN cohort and 95% (88, 98) in the R/R cohort; major response rates (≥partial response) were 79% (49, 95) and 82% (72, 89), respectively. Median DOR was not estimable (NE) (12, NE) in the TN cohort and 65 mo (55, NE) in the R/R cohort; the estimated 66-mo DOR was 90% (TN; 47, 99) and 45% (R/R; 27, 61). At a 63.7-mo median follow-up, median PFS rate was NE (TN; 19, NE) and 68 mo (R/R; 53, NE); estimated 66-mo PFS rate was 84% (TN; 48, 96) and 52% (R/R; 39, 63). Median OS rate was NE for the TN and R/R cohorts; estimated 66-mo OS rate was 91% (TN; 51, 99) and 71% (R/R; 60, 80). Of the patients who remained on treatment, 7 (50%) were in the TN cohort and 43 (47%) were in the R/R cohort. The main reasons for discontinuing acalabrutinib were adverse events (AEs) (4 [29%] patients in the TN cohort, 15 [16%] patients in the R/R cohort) and progressive disease (PD) (1 [7%] patient in the TN cohort, 20 [22%] patients in the R/R cohort). The most common AEs and selected events of clinical interest are listed in the Table. There were 12 grade 5 AEs (11 and 1 patients in the R/R and TN cohorts, respectively); 1 (intracranial hematoma [R/R]) was considered treatment-related. Image: Summary/Conclusion: Acalabrutinib is a highly effective treatment that achieved durable responses with a favorable safety profile in patients with TN or R/R WM. P1131: POST HOC ANALYSIS OF PATIENTS WITH HIGHLY PROLIFERATIVE VARIANTS OF MANTLE CELL LYMPHOMA TREATED WITH ACALABRUTINIB S. Le Gouill1,*, M. Długosz-Danecka2, S. Rule3,4, P. Luigi Zinzani5, A. Goy6, S. D. Smith7, J. K. Doorduijn8, C. Panizo9, B. Shah10, A. Davies11, R. Eek12, E. Jacobsen13, A. P. Kater14, T. Robak15, P. Jain16, R. Calvo17, H. Yang18, M. Wang16 1Institut Curie, Paris, France; 2Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland; 3Plymouth University Medical School, Plymouth, United Kingdom; 4AstraZeneca, Mississauga, Ontario, Canada; 5IRCCS Azienda Ospedaliero-Universitaria di Bologna Istituto di Ematologia “Seràgnoli,” Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale Università di Bologna, Bologna, Italy; 6John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ; 7University of Washington, Fred Hutchinson Cancer Research Center, Seattle, WA, United States of America; 8Erasmus MC Cancer Institute, HOVON Lunenburg Lymphoma Phase I/II Consortium, Rotterdam, Netherlands; 9Clínica Universidad de Navarra, Pamplona, Spain; 10Moffitt Cancer Center, Tampa, FL, United States of America; 11Cancer Research UK Experimental Cancer Medicines Centre, University of Southampton Faculty of Medicine, Southampton, United Kingdom; 12Border Medical Oncology, Albury, NSW, Australia; 13Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, United States of America; 14Amsterdam University Medical Center, Amsterdam, on behalf of HOVON, Amsterdam, Netherlands; 15Copernicus Memorial Hospital, Medical University of Lodz, Lodz, Poland; 16MD Anderson Cancer Center, University of Texas, Houston, TX; 17AstraZeneca, Gaithersburg, MD; 18AstraZeneca, South San Francisco, CA, United States of America Background: Blastoid and pleomorphic variants of mantle cell lymphoma (MCL) are considered high risk and have poor prognoses. Patients with these variants rarely achieve durable remission or prolonged clinical outcomes with currently available chemotherapies and targeted therapies. Final results from ACE-LY-004, an open-label, multicenter, single-arm phase 2 study of the Bruton tyrosine kinase (BTK) inhibitor acalabrutinib in adult patients with relapsed/refractory (R/R) MCL confirmed acalabrutinib was highly active in patients with R/R MCL, with a median overall survival of 59.2 months, a median progression-free survival (PFS) of 22.0 months, and a median duration of response (DOR) of 28.6 months after a median 38.1 months of follow-up. In a subgroup analysis, we found that patients with 1 prior line of therapy had unexpectedly shorter DOR than those who had ≥2 prior lines of therapy. Aims: To investigate a possible reason for shorter DOR in patients with R/R MCL treated with acalabrutinib who received only 1 prior line of therapy, we conducted a post hoc analysis on a subgroup of patients with blastoid/pleomorphic disease. Methods: ACE-LY-004 enrolled adult patients with confirmed R/R MCL and an Eastern Cooperative Oncology Group performance status (ECOG PS) of ≤2. Informed consent was obtained. Acalabrutinib 100 mg was administered orally twice daily until occurrence of progressive disease or unacceptable toxicity. Results: A total of 124 patients were enrolled; median age was 68 years and 80% of patients were male. As of December 4, 2020, median follow-up was 38.1 months and median treatment exposure was 17.5 months (range 0.1–65.3). Fifty-nine of the 124 patients (48%) had 1 prior therapy. The remaining 65 patients (52%) had 2 prior therapies (n=37) or ≥3 prior therapies (n=28). Median DOR was longer in patients with ≥2 prior lines of therapy versus those with 1 prior therapy (33.8 and 25.6 months, respectively). Similarly, median PFS was longer in patients who received acalabrutinib in later lines of therapy than in those who received acalabrutinib as second-line therapy (24.8 and 19.2 months, respectively). Among the 59 patients with 1 prior therapy, 16 (27%) had blastoid or pleomorphic disease and 43 (73%) did not, compared with 10 patients (15%) with blastoid/pleomorphic disease and 55 (85%) who did not among the 65 patients with ≥2 prior therapies. Overall response rate (ORR) in those with 1 prior therapy was 88% (n=14; 95% CI 61.7–98.4) in those with blastoid/pleomorphic disease (complete response [CR] in 50%, n=8) versus 81% (n=35; 95% CI 66.6–91.6) in those without blastoid/pleomorphic disease (CR in 47%, n=20). Median DOR for these patients was 20.3 (95% CI 3.5–46.9) and 31.8 (95% CI 14.9–not estimable) months, respectively. Summary/Conclusion: In this post hoc analysis of the ACE-LY-004 study of acalabrutinib in adult patients with confirmed R/R MCL, a shorter DOR was observed among patients who received acalabrutinib after 1 prior therapy compared with those who received acalabrutinib after ≥2 prior therapies, likely explained by a high proportion of patients with blastoid/pleomorphic variants in the group of patients receiving acalabrutinib after 1 prior therapy. While patients with blastoid/pleomorphic variant had comparable ORR, DOR was shorter but still substantial at 20.3 months, indicating that some patients with highly proliferative disease and poorer prognosis also achieve clinical benefit from acalabrutinib. P1132: PET INTERIM RESULTS PREDICT PROGRESSION-FREE-SURVIVAL IN FOLLICULAR LYMPHOMA PATIENTS WITH ADVANCED STAGE M. Poza1,*, I. Zamanillo1, R. Íñiguez1, P. Sarandeses2, A. Rodriguez1, T. Baumann1, S. Barrio3, C. Grande1, A. Martin3, L. Rufian1, J. Martinez-Lopez1, A. Jiménez-Ubieto1 1Hematology; 2Department of Nuclear Medicine, Hospital 12 Octubre; 3Altum Sequencing Co,., Madrid, Spain Background: Follicular lymphoma (FL) is the second most common NHL. It is generally associated with an indolent course, but a subgroup of patients has worse outcome, lower progression-free-survival (PFS) and higher risk of histological transformation (HT). Since Lugano classification, the Deauville criteria using the 5-point scale (D5PS) is validated as the interpretation method for response assessment PET scans in HL and NHL. Nevertheless, the prognostic value of D5PS in the interim evaluation in FL (PETINT) is not well stablished. Additionally, the quantitative value of SUVmax parameter in the response assessment is almost not studied. Aims: The aim of this study is to evaluate the prognostic value of PETINT evaluation and quantitative mid-induction SUVmax parameter in a cohort of FL patients followed and treated in a single, academic centre with chemo-immunotherapy. Methods: 179 consecutive FL patients were newly diagnosed between 2011 and 2020. 40 patients that did not require systemic treatment and 68 that did not have a PETINT evaluation were excluded. As well, patients with HT (9) and FL grade 3B (4) were excluded. From the 58 remaining patients, clinical, laboratory and PET examinations data was recorded. All patients were Ann-Arbor stage III-IV and were treated with chemo-immunotherapy. PET/CT examinations were acquired at baseline, mid-induction (after 4 cycles of treatment) and end of treatment (EOT). PET positive was considered when Deauville score (DS) was 4 or 5. Statistical analysis was performed with SPSS® using Cox regression, Kaplan-Meier survival curves and log-rank test. Results: The median age at diagnosis was 64 years (26-95) and 29 (50%) patients were female. 33/58 (57%) patients had a high risk FLIPI and 18/55 (33%) had a high risk PRIMA-PI. 37 (64%) patients were treated with R-CHOP and 21 with R-bendamustine. 44 (76%) patients received maintenance with rituximab. After a median follow-up of 43 months, 20 (34%) patients had progression. On PETINT, 19 (32%) patients were PET(+), and 10 of them remained PETEOT(+). Only one patient with PETINT(-), became PETEOT(+). PETINT(+) patients had a median PFS of 31 months compared with a median PFS of 88 months in PETINT(-) (p < 0.001), Figure 1. 38.2% PETINT(+) presented progression of disease in less than 24 months (POD24) vs 5.2% on those who were in PETINT(-), with a p<0.001. PETINT(-) presented a negative predictive value of 95%. No differences in OS was observed. 3/19 (16%) patients with PETINT(+) presented HT vs 1/39 (2%) with PETINT(-). Transformation-free survival was lower in patients with PETINT(+), with a trend towards significance (p= 0.057). Similar PFS results were obtained independently of prognostic index, treatment or maintenance received. Analysing the prognostic value of quantitative SUVmax, patients with SUVmax< 3,8 (28/58; 48%) presented better outcomes, with larger PFS (p < 0.001) and less HT (p= 0.004). Figure 2. Additionally, PET/CT findings at EOT were predictive of relapse, with a median PFS of 30 months in PETEOF (+) vs 88 months in PETEOT(-) (p = 0.005). PETEOT (+) presented a higher risk of HT (p= 0.004). Figure 3. Image: Summary/Conclusion: This study suggests that response assessment by PET at mid-induction is predictive of relapse in advanced stage FL patients treated with 1st line chemo-immunotherapy. With previous conflicting results, further studies are needed to confirm this preliminary data. Probably, new laboratory tests such as MRD monitored by liquid biopsy could improve the detection of this subgroup of high risk patients and help to develop a response-adapted precision therapy. P1133: SUB-CUTANEOUS RITUXIMAB INDUCTION FOLLOWED BY SHORT RITUXIMAB MAINTENANCE IMPROVES PFS IN PATIENTS WITH LOW-TUMOR BURDEN FOLLICULAR LYMPHOMA. FINAL RESULTS OF FLIRT PHASE III TRIAL, A LYSA STUDY. G. Cartron, MD, PhD, E. Bachy, MD, PhD, H. Tilly, MD3, N. Daguindau, MD4, G.-M. Pica, MD5, F. Bijou, MD6, C. Mounier, MD7, A. M. Clavert, MD8, G. L. Damaj, MD, PhD, B. Slama, MD10, O. Casasnovas, MD11, R. Houot, MD, PhD, K. Bouabdallah, MD13, D. Sibon, MD, PhD, O. Fitoussi, MD15, N. Morineau, MD16, C. Herbaux, MD, PhD, T. Gastinne, MD18, L.-M. Fornecker, MD19, C. Haioun, MD, PhD, V. Launay, MD21, C. Araujo, MD22, O. Benbrahim, MD23, L. Sanhes, MD24, R. Gressin, MD25, H. Gonzalez, MD26, F. Morschhauser, MD, PhD, L. Xerri, MD, PhD, K. Tarte, PhD12, D. Pranger, MD29 1Haematology, CHU de Montpellier, MONTPELLIER Cedex 5; 2CHU Lyon Sud, Pierre Benite; 3Centre Henri Becquerel, Rouen; 4Centre Hospitalier Annecy-Genevois - Site d’Annecy, Pringy Cedex; 5CH de Chambéry, Chambéry; 6Institut Bergonié, Bordeaux; 7Institut de Cancérologie Lucien Neuwirth, St-Priest-en-Jarez; 8CHU Angers, Angers; 9CHU Côte de Nacre, Caen; 10CH d’Avignon - Hôpital Henri Duffaut, Avignon; 11CHU Dijon - Hôpital d’Enfants, Dijon; 12CHU Rennes, Rennes; 13CHU Bordeaux, Bordeaux; 14Hôpital Necker, Paris; 15Polyclinique Bordeaux Nord Aquitaine, Bordeaux; 16CH Départemental Vendée, La Roche sur Yon; 17CHU Montpellier, Montpellier; 18CHU de Nantes - Hôtel Dieu, Nantes; 19Hôpitaux Universitaires de Strasbourg - Hôpital de Hautepierre, Strasbourg; 20Hôpital Henri Mondor, Créteil; 21CH de Saint-Brieuc - Hôpital Yves Le Foll, Saint- Brieuc; 22Centre Hospitalier de la Côte Basque - Hôpital de Bayonne, Bayonne; 23CHR de la Source, Orléans; 24Hôpital Saint Jean, Perpignan; 25CHU de Grenoble - Hôpital Albert Michallon, Grenoble; 26CH René Dubos, Pontoise; 27CHRU de Lille - Hôpital Claude Hurriez, Lille; 28Departement de bio-pathologie, Institut Paoli Calmette, Marseille, France; 29GHdC, Charleroi, Belgium Background: In low–tumor burden follicular lymphoma (FL), maintenance rituximab (MR) has been shown to improve progression-free survival (PFS) when compared with observation. RESORT study (Kahl et al. 2014) clearly showed that Rituximab (R) retreatment strategy provided similar time to treatment failure that maintenance strategy with less rituximab use. Aims: It is not known whether short MR using sub-cutaneous (sc) route could improve PFS while reducing R infusions. Methods: Patients with the diagnosis of low-tumor burden FL (GELF criteria) within the last 4 months before signing informed consent were randomly assigned to either Iv-Arm: 4-weekly iv infusions of R 375 mg/m2 or Sc-Arm: one iv infusion of R (D1, 375 mg/m2) followed by 3 sc infusions of R (1400 mg, on days 8, 15 and 22) followed by Rsc maintenance on months (M) 3, 5, 7 and 9. The primary endpoint was PFS and secondary endpoints included safety, response rates (M3, M12), duration of response (DOR), time to next anti-lymphoma treatment (TTNTL) and overall survival (OS). Results: A total of 202 patients were included, 102 in Iv-Arm and 100 in Sc-Arm and constitute the intent to treat population. The median uses of R were 4 infusions (range: 1-4, Iv-Arm) and 8 infusions (range: 2-8, Sc-Arm). With a median follow-up of 50.2 months (95% CI: 48.3-54.5), 4‐year PFS was 41.2% (95% CI: 30.6%; 51.6%) in Iv-Arm and 58.1% (95% CI: 47.5%; 67.4%) in Sc-Arm. Median PFS was then 36.1 months (95% IC: 23.9-52.6) in Iv-Arm and 73.8 months (95% CI: 39.4-NA) in Sc-Arm (Fig 1.) (HR: 0.58; 95% CI: 0.39-0.87; P = 0.0076). Patients with at least one AE grade ≥ 3 were 8 (7.8%) and 12 (12.4%) in Iv-Arm and Sc-Arm, respectively. According to Cheson criteria, ORR at M3 were: 83% and 80% including 38% and 29% of CR/CRu, in Iv-Arm and Sc-Arm, respectively. According to Lugano criteria, 36.3% (Iv-Arm; 95% CI: 27.0%. 46.4%) and 59.0% (Sc-Arm; 95% CI: 48.7%; 68.7%) were in CMR at M12. The median DOR was 32.7 months (95% IC: 20.6-49.7) and 70.8 months (36.4-NR) (HR: 0.56; 95% IC: 0.37-0.84) in Iv-Arm and Sc-Arm, respectively. 4-year TTNLT was 54% (95% CI: 42.9%; 63.8%) in Iv-Arm and 61.8% (95% CI: 50.8.6%; 71.0%) in Sc-Arm (HR: 0.81, 95% IC: 0.53-1.24). 4-year TTNLT chemotherapy was 60.8 % (95% CI: 49.6%; 70.3%) in Iv-Arm and 71.4% (95% CI: 60.7%; 79.8%) in Sc-arm (HR: 0.69, 95% IC: 0.42-1.12) (Fig 2.). OS was not different according to treatment arm, 4-year OS was 95.0% (95% CI: 88.5%; 97.9%) in Iv-Arm and 96.7% (95% CI: 89.9%; 98.9%) in Sc-Arm. Summary/Conclusion: This phase III study met its primary endpoint and demonstrated that Rsc induction followed by a short MRsc improves PFS of patients with low-tumor burden. MRsc did not however improved TTNLT. R in low-tumor burden FL allowed to avoid cytotoxic use in most patients 6 years after treatment initiation. P1134: OUTCOMES FOR PATIENTS WITH MANTLE CELL LYMPHOMA POST-CBTK INHIBITOR THERAPY IN THE UNITED STATES AND JAPAN: A STUDY OF TWO REAL-WORLD DATABASES S. Rai1,*, L. Hess2, Y. Chen2, P. Abada2, H. Konig2, R. Walgren2, Y. Tanizawa3, Z. Cai3, M. Tajimi3 1Kindai University Faculty of Medicine, Osakasayama, Japan; 2Eli Lilly and Company, Indianapolis, United States of America; 3Eli Lilly Japan K.K, Kobe, Japan Background: Patients with mantle cell lymphoma (MCL) have limited treatment options following covalent Bruton’s tyrosine kinase inhibitor (cBTKi) therapy, with no standard regimens defined. Aims: The aim of this study was to investigate real-world treatment patterns and the outcomes for patients with MCL following cBTKi treatment utilizing two separate datasets—one from the US and one from Japan. Methods: De-identified patient-level electronic medical record data from two real-world databases (ConcertAI in the US and Medical Data Vision in Japan) were utilized for this study. Eligible patients were ≥18 years old and were diagnosed with MCL between January 2011 and October 2020 in the US or between December 2010 and July 2020 in Japan. Data were available through 2020 to allow a minimum of 1 year of follow-up for all patients. Patients were required to have completed treatment with at least one cBTKi during the first through third lines of therapy. Time-to-event analyses utilized the Kaplan-Meier method. Results: 946 US patients (median age 72 years, male 75.1%) and 196 Japanese patients (median age 78 years, male 73.5%) met the eligibility criteria. 352 (37.2%) patients in the US and 103 (52.6%) in Japan received subsequent post-cBTKi therapy. The remaining 387 (40.9%) patients in the US and 93 (47.5%) patients in Japan did not receive further treatment. In the US, immediate post-cBTKi regimens included rituximab (with or without other chemotherapy agents, n=152, 43.2%) or additional cBTKi-based 146 (41.5%) or B-cell lymphoma 2 inhibitor–based (n=30, 8.5%) therapy. In Japan, immediate post-cBTKi regimens were nearly exclusively chemotherapy based (n=92, 89.3%), and only 11 (10.7%) patients were retreated with cBTKi-containing regimens. Median time from the end of cBTKi therapy to discontinuation of the immediate post-cBTKi therapy or death was 3.9 months (95% confidence interval [CI]: 3.3-4.6) in the US and 2.4 months (n=196, 95% CI: 1.7-3.0) in Japan. Among those who received post-cBTKi therapy, the duration of therapy was 2.6 months (95% CI: 2.1-3.3) in the US and 1.8 months (n=103, 95% CI: 1.1-2.3) in Japan. Median overall survival (OS) from discontinuation of cBTKi therapy was 10.3 months (95% CI: 8.0-13.0 in US patients and 7.1 months (n=196, 95% CI: 4.6-12.7) in Japanese patients. Summary/Conclusion: Patients with MCL previously treated with cBTKi experience very poor outcomes, as measured by their duration of subsequent therapy and OS. Data were generally consistent between the US and Japan. The development of new safe and effective therapies after cBTKi is needed to address the growing unmet medical need in this treatment setting. P1135: SUBCUTANEOUS EPCORITAMAB IN COMBINATION WITH RITXUIMAB + LENALIDOMIDE IN RELAPSED OR REFRACTORY FOLLICULAR LYMPHOMA: PHASE 1/2 TRIAL UPDATE D. Belada1,*, S. Leppä2, B. E. Wahlin3, M. Nijland4, J. H. Christensen5, S. de Vos6, H. Holte7, K. M. Linton8, A. Abbas9, L. Wang9, M. Dinh10, B. Elliott9, L. Falchi11 14th Department of Internal Medicine – Hematology, University Hospital and Faculty of Medicine, Hradec Králové, Czechia; 2Helsinki University Hospital Comprehensive Cancer Center and University of Helsinki, Helsinki, Finland; 3Karolinska Institutet, Stockholm, Sweden; 4University Medical Center Groningen and University of Groningen, Groningen, Netherlands; 5Odense University Hospital, Odense, Denmark; 6Ronald Reagan University of California Los Angeles Medical Center, Los Angeles, United States of America; 7Oslo University Hospital and KG Jebsen Center for B-cell Malignancies, Oslo, Norway; 8The Christie NHS Foundation Trust and Manchester Cancer Research Centre, Manchester, United Kingdom; 9Genmab, Princeton; 10AbbVie, North Chicago; 11Lymphoma Service, Memorial Sloan Kettering Cancer Center, New York, United States of America Background: Patients (pts) with relapsed or refractory (R/R) follicular lymphoma (FL) experience less frequent and shorter responses with each line of treatment. Although the rituximab + lenalidomide (R2) regimen is effective and has an acceptable safety profile in R/R FL, FL remains incurable; therefore, better treatment options are needed to help pts achieve durable responses. Epcoritamab is a subcutaneously administered bispecific antibody that binds to CD3 on T cells and CD20 on malignant B cells. In the dose-escalation portion of the first-in-human phase 1/2 trial (EPCORE NHL-1), epcoritamab monotherapy (0.76–48 mg) resulted in an overall response rate (ORR) of 90% and a complete response rate of 50% in pts with R/R FL. Because epcoritamab and R2 have distinct mechanisms of action and generally non-overlapping toxicity profiles, combining these treatments may safely enhance antitumor response. Aims: To present updated results for epcoritamab with R2 in pts with R/R FL (EPCORE NHL-2 arm 2; NCT04663347). Methods: Adult pts with R/R CD20+ FL received subcutaneous epcoritamab + R2 for 12 cycles (28 d/cycle). In the dose-escalation part of the trial, epcoritamab was administered at 24 or 48 mg; in the expansion part, the dose was 48 mg. Pts received their assigned epcoritamab dose as follows: every wk, cycles 1–3; every 2 wk, cycles 4–9; and every 4 wk, cycles ≥10 up to 2 y. Corticosteroid prophylaxis and step-up epcoritamab dosing were required during cycle 1 to mitigate CRS. PET-CT was used to assess response. All pts provided informed consent before enrollment. Results: As of the data cutoff date of December 1, 2021, 30 pts had received epcoritamab (24 mg, n=3; 48 mg, n=27) + R2. Median age was 68 y. Overall, 70% of pts had stage IV disease, 67% had FLIPI scores 3–5, 30% had primary refractory disease, and 40% had disease progression within 24 mo after starting first-line treatment (20% within 24 mo after starting immunochemotherapy). The median number of prior lines of therapy was 1 (range, 1–5). With a median follow-up of 5.1 mo (range, 0.8–12.3), 25 pts (83%) remained on study treatment; 5 pts had discontinued due to disease progression (n=2), AEs (n=2), or withdrawal of consent (n=1). Frequent treatment-emergent AEs (TEAEs; any grade [G]) included infections (57%), injection-site reactions (50%), constipation (37%), fatigue (37%), and neutropenia (37%). CRS was reported in 15 pts (50%; G1 30%, G2 13%, G3 7%). Most CRS events occurred in cycle 1, and all CRS events resolved with standard management (median time to resolution, 3 d; range, 1–8). Tocilizumab was used in 3 pts with CRS, and 1 pt discontinued study treatment due to CRS. ICANS (G2) was reported in 1 pt and resolved. There were no fatal TEAEs. Response profiles for individual pts are shown in Figure 1. Among the efficacy-evaluable pts, the ORR was 100% (27/27), with 93% (25/27) achieving a complete metabolic response (CMR) and 7% (2/27) achieving a partial metabolic response. As of the data cutoff date, the longest duration of response was 7.0+ mo and ongoing. Image: Summary/Conclusion: Subcutaneous epcoritamab + R2 demonstrated promising efficacy in pts with R/R FL, with a high CMR rate. The safety profile remained consistent with prior data. CRS events occurred mostly in cycle 1 and were generally low grade. Updated data with 30 additional pts will be presented at the meeting. P1136: INTERIM IMAGING DOES NOT PREDICT CLINICAL EVOLUTION IN FOLLICULAR LYMPHOMA IN A REAL-LIFE CLINICAL SETTING V. Ramos De Ascanio1,*, L. Sánchez Paz1, M. S. Infante1, J. Churruca Sarasqueta1, M. Á. Foncillas García1, E. Landete Hernández1, K. D. C. Marín Mori1, C. Muñoz Novas1, J. Á. Hernández Rivas1, I. González Gascón y Marín1 1Hematology, Infanta Leonor University Hospital, Madrid, Spain Background: Interim scan evaluation is a common practice during the treatment of some types of lymphomas with (immuno)-chemotherapy (ICT). In follicular lymphoma (FL) its role remains controversial and few studies have looked at this topic specifically. However, it is performed in clinical practice, clinical trials and recommended by some international guidelines (ESMO). Aims: In this study, we analyzed the clinical and prognostic value of interim imaging (iPET/iCT) during the first line-treatment of patients diagnosed with FL. Methods: In order to execute this, we carried out a retrospective analysis of all FL patients that received a first line treatment (ICT) at our institution and underwent an iPET/iCT between 2008 and 2021. Patients with Grade 3b or histologic transformation were excluded from the analysis. All data was extracted from electronical medical records. The Lugano response criteria were used to assess disease response. Statistical analysis was performed using SPSS version 25.0 and survival was estimated by Kaplan-Meier curves and log rank tests. Results: Baseline characteristics of patients included in this study are described in Table 1. Median follow-up was 71 months (6-153). All patients underwent iPET/iCT (iCT=52 (81.3%); iPET=12 (18.8%)). Most iPET/iCT showed a partial response (PR) (n=48, 75%), and a minority a complete response (CR) (n=14, 21.9%). Two patients (3.1%) did not respond to induction. The 14 patients with CR maintained the response at the end of induction; 34 of 48 PR patients (70.8%), achieved a CR on final imaging; whereas 14 patients (29.2%) maintained a PR. Out of the 64 patients, 17 (26.6%) had disease progression during the observation period. In 6 patients (9.4%) interim imaging conditioned a treatment modification (premature discontinuation of therapy, increase/decrease in the number of ICT cycles or ICT dose attenuation). No significant differences were found in progression-free survival (PFS) (p=0.3) between the 14 patients with CR and the 48 patients who showed PR or less in the interim imaging. Nevertheless, patients in PR who converted to CR at the end of therapy had a significantly higher PFS than patients who did not achieve CR at the end of induction (p<0.01). Consequently, patients who reached a CR at the end of therapy achieved a significantly higher PFS compared to those who did not (p<0.01). Image: Summary/Conclusion: Obtaining an early CR on interim imaging did not predict PFS in FL patients treated with IQT. In addition, clinical decisions based on iPET/iCT were only made in a minority of the patients, with the detection of early refractoriness being residual. Conversely, end of treatment imaging assessment was a useful tool capable of predicting PFS. P1137: ACCURACY AND PROGNOSTIC IMPACT OF FDG PET/CT AND BIOPSY IN BONE MARROW ASSESSMENT OF FOLLICULAR LYMPHOMA AT DIAGNOSIS: A NATION-WIDE STUDY. I. Ródenas Quiñonero1,*, T. Chen-Liang1, T. Martín-Santos2, A. Salar3, M. Fernández-González2, C. Celades4, J. T. Navarro5, A. B. Martinez6, R. Andreu7, A. Balaguer7, A. Martín8, M. Baile8, J. López-Jiménez9, J. Marquet9, A. I. Teruel10, M. J. Terol10, C. Benet11, L. Frutos12, J. L. Navarro12, J. Uña13, M. Suarez14, M. Cortes15, J. Contreras16, C. Ruiz17, P. Tamayo18, J. Mucientes19, P. Sopena20, L. Reguilón-Gallego1, J. J. Sánchez-Blanco1, E. Pérez-Ceballos1, A. Jerez1, F. J. Ortuño1 1Hematology, H. Morales Meseguer, Murcia; 2Hematology, H. Universitario de Canarias, Tenerife; 3Hematology, H. del Mar, Barcelona; 4Hematology, Josep Carreras Leukaemia Research Institute (IJC); 5Hematology, ICO-H. Germans Trias i Pujol, Badalona; 6Hematology, H. Santa Lucía, Cartagena; 7Hematology, H. La Fe, Valencia; 8Hematology, H. Clínico Universitario, Salamanca, Salamanca; 9Hematology, H. Ramon y Cajal, Madrid; 10Hematology, H. Clínico, Valencia; 11Hematology, H. Arnau de ViIlanova, Valencia; 12Nuclear Medicine, H. Virgen de la Arrixaca, Murcia; 13Nuclear Medicine, H. Universitario N.S. de la Candelaria, Tenerife; 14Nuclear Medicine, H. del Mar; 15Nuclear Medicine, H. Universitari de Bellvitge-IDIBELL, Barcelona; 16Nuclear Medicine, H. Santa Lucía, Cartagena; 17Nuclear Medicine, H. La Fe, Valencia; 18Nuclear Medicine, H. Clínico Universitario de Salamanca/IBSAL, Salamanca; 19Nuclear Medicine, H. Puerta de Hierro, Madrid; 20Nuclear Medicine, H. 9 de Octubre, Valencia, Spain Background: In the workout of Follicular Lymphoma (FL), bone marrow biopsy (BMB) assessment is a key component of FLIPI and FLIPI2, the most widely used outcome scores. During the previous decade, several studies explored the role of FDG-PET/CT for detecting nodal and extranodal disease, with only one large study comparing both techniques. Aims: The aim of this study was to evaluate the diagnostic accuracy and the prognostic impact of both procedures in a retrospective cohort of 299 FL patients. Methods: Two hundred and ninety-nine, ≥18 y.o. patients diagnosed with FL between June 2005 and December 2018 were included. We used the Kaplan-Meier and the Cox method to analyze overall survival (OS) and progression free survival (PFS). To avoid collinearity in the multivariate regressions, we deconstructed the FLIPI2 composite in its foundational factors, considering bone marrow involvement (BMI) by mean of different measures in four models: BMB+, PET/CT+, “BMB+ and PET/CT+” and “BMB+ or PET/CT+”. In Cox regressions, examination of log (-log) survival plots ant partial residuals was performed to assess that the underlying assumption of proportional hazards was met. Results: The median follow-up was 57.3 months (range 3.6-185.8 months). The PET/CT was positive in 58 patients and negative in 238. Among those positive, 37 also had a BMB+, whereas 151 patients had a BMB+ with a PET/CT-. The BMB was positive in 124 patients and negative in 172. Among those negative, 21 had a PET/CT+. Considering PET/CT, the sensitivity was 63.7% (95% confidence interval(CI);50.5-77.0), specificity was 63.4% (95%CI;57.1-69.7), negative predictive value (NPV) was 87.7% (95%CI;82.6-92.9), accuracy 63.5% (95%CI:57.8-69.1). Focusing BMB, the sensitivity was 29.8% (95%CI;21.3-38.3), specificity was 87.7% (95%CI;82.6-92.9), NPV was 63.4% (95%CI;57.1-63.7) and accuracy was 63.5% (95%CI:57.8-69.1). Either beta2-microglobulin (B2M) over the upper normal limit (ULN), a diameter of the largest involved node (LoDLIN) over 6cm and a hemoglobin lower than 120g/L, were significantly associated with a shorter PFS in the univariate analysis. Also in univariate, a positive BMB and the combined “PET/CT or BMB positive”, were significantly associated with a shorter PFS. Neither BMI positive or the combined “PET/CT or BMB positive”, could add an independent prognostic value to the two factors than remained significant in the multivariate model: B2M higher than ULN and a LoDLIN over 6cm. Regarding OS, an elevated B2M, a LoDLIN over 6cm, a hemoglobin lower than 120g/L and an age older than 60y.o., were significantly associated with a shorter OS in the univariate analysis. A positive result by BMB, a positive PET/CT and the combined “PET/CT or BMB positive” result, were significantly associated with a shorter OS in univariate regression. The combined “PET/CT or BMB positive” and an age older than 60y.o. remained significant for a shorter OS in the multivariate model (p=0.030, HR 2.318, 95%CI 1.083-4.961 and p= 0.020, HR 2.473, 95%CI 1.151-5.314, respectively). Image: Summary/Conclusion: In our FL series, the combined “PET/CT or BMB positive” was the only BMI variable that showed an independent prognostic value in FL. Therefore, our data support that both procedures should be carried out in the upfront work of all suitable FL patients. P1138: COPANLISIB + RITUXIMAB VS RITUXIMAB + PLACEBO IN PATIENTS WITH RELAPSED INDOLENT NON-HODGKIN LYMPHOMA (NHL): UPDATED SAFETY AND EFFICACY FROM THE PHASE III CHRONOS-3 TRIAL P. L. Zinzani1,2,*, M. Özcan3, K. Sapunarova4, W. Jurczak5, A. Hamed6, K. Bouabdallah7, G. Saydam8, K. Geissler9, Á. Szomor10, M. Lazaroiu11, A. Salar12, A. Tempescul13, M. Nalcaci14, L. Gercheva15, M. Egyed16, P. Panayiotidis17, L. Mongay Soler18, A. Cao18, C. Phelps18, B. H Childs18, M. J Matasar19 1IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seragnoli”; 2Dipartimento di Medicina Specialistica, Diagnostica e Sperintale, Universita di Bologna, Bologna, Italy; 3Ankara University School of Medicine, Ankara, Turkey; 4Medical University, Plovdiv, Bulgaria; 5Maria Skłodowska Curie National Research Institute of Oncology, Krakow, Poland; 6Petz Aladár Megyei Oktató Kórház, Gyor, Hungary; 7Hematology and Cellular Therapy Department, University Hospital of Bordeaux, Bordeaux, France; 8Ege Üniversitesi Tip Fakültesi, Izmir, Turkey; 9Sigmund Freud University, Vienna, Austria; 10Pécsi Tudományegyetem Klinikai Központ, Pécs, Hungary; 11S.C. Policlinica de Diagnostic Rapid S.A., Brasov, Romania; 12Hospital del Mar, Barcelona, Spain; 13Hôpital Morvan - Brest, Brest, France; 14Istanbul Universitesi İstanbul Tip Fakultesi, Istanbul, Turkey; 15MHAT Sveta Marina EAD, Varna, Bulgaria; 16Somogy Megyei Kaposi Mor Oktato Korhaz, Kaposvar, Hungary; 17First Department of Propaedeutic Internal Medicine, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 18Bayer HealthCare Pharmaceuticals, Inc., Whippany, NJ; 19Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America Background: The Phase III CHRONOS-3 trial of copanlisib plus rituximab (C+R) vs placebo plus rituximab (P+R) in patients (pts) with indolent NHL (iNHL) showed a significant improvement in progression-free survival (PFS; hazard ratio [HR] 0.52; Matasar et al. Lancet Oncol 2021). At the time of data cut-off (August 31, 2020), 23% of pts were ongoing on treatment in the C+R arm and 43% of pts had discontinued treatment and entered active follow-up; corresponding numbers in the P+R arm were 19% and 17%. Aims: Here we report an updated 1-year follow-up analysis of efficacy and safety for all pts from CHRONOS-3 based on a data cut-off of August 6, 2021. Methods: Eligible pts with iNHL who relapsed after the last R-containing regimen and were progression-free and treatment-free for ≥12 months (mo) after the last R-containing regimen, or for >6 mo if unwilling/unfit to receive chemotherapy, were randomized 2:1 to receive C+R or P+R. All pts provided informed consent. Treatment continued until progression or unacceptable toxicity and was administered i.v. on a 28-day cycle, with C 60 mg/P given on days 1, 8, and 15 and R 375 mg/m2 given on days 1, 8, 15, and 22 of cycle 1 and on day 1 of cycles 3, 5, 7, and 9. Primary endpoint was centrally assessed PFS and secondary efficacy endpoints included overall survival (OS), objective response rate (ORR), duration of response (DoR), complete response rate (CRR), and treatment-emergent adverse events (TEAEs). Results: 307 pts were randomized to C+R and 151 to P+R. As of the August 2021 cut-off, 46 pts (15.0%) receiving C+R and 16 (10.6%) receiving P+R were ongoing on treatment. The most common reason for discontinuation was adverse events (AEs) unrelated to clinical progression (35.8%) for C+R and radiologic progression (49.7%) for P+R. Similar to the initial disclosure, C+R reduced the risk of progression or death vs P+R by 47%, with median PFS of 22.3 mo (95% confidence interval [CI] 19.4, 30.7) for C+R and 13.8 mo (10.8, 17.5) for P+R (HR 0.53 [95% CI 0.41, 0.70]; p=0.000001). PFS benefit was preserved across all cancer subtypes (HR [95% CI]): follicular lymphoma (n=275; 0.57 [0.41, 0.80]); marginal zone lymphoma (n=95; 0.53 [0.28, 0.99]); small lymphocytic lymphoma (n=50; 0.22 [0.10, 0.49]); lymphoplasmacytic/Waldenström macroglobulinemia (n=38; 0.42 [0.15, 1.16]). Updated ORRs were 80.8% vs 49.7% and CRRs were 34.9% vs 14.6% for C+R vs P+R, respectively. Median OS was not estimable and no significant benefit was seen for C+R over P+R, although the risk of death was reduced (HR 0.86 [95% CI 0.55, 1.32]) vs the August 2020 disclosure (HR 1.07 [0.63, 1.82]). Median DoR was 25.7 mo (17.6, 31.5) for C+R vs 17.3 mo (12.6, 25.3) for P+R; for pts with complete responses, median DoR was 42.3 mo (29.2, not estimable) vs 24.7 mo (10.2, 29.7) (HR 0.46 [0.24, 0.87]). Safety profiles for C+R and P+R were mostly unchanged from the initial disclosure, and the most common treatment-related AEs for pts receiving C+R were hyperglycemia (69.4%), hypertension (49.5%), and diarrhea (33.9%). For C+R, 6 additional pts (2.0%) discontinued C due to an AE, bringing the total to 33.2%. The incidence of grade 3/4 treatment-related TEAEs was generally unchanged with the longer follow-up (2 new grade 4 for C+R; 1 new grade 4 for P+R), with no new grade 5 events. Summary/Conclusion: 1 year after the primary disclosure of CHRONOS-3, C+R continued to demonstrate acceptable safety and superior efficacy with durable complete responses vs P+R in pts with relapsed iNHL. These data support the long-term use of C+R in pts with relapsed iNHL. P1139: A POPULATION-BASED STUDY OF ISOLATED ADULT PULMONARY LANGERHANS CELL HISTIOCYTOSIS IN THE UNITED STATES: 2010-2017 G. Ruan1,*, J. Abeykoon1, A. Hazim2, A. Ravindran3, K. Rech3, J. Young4, C. Cox4, J. Ryu5, M. Koster6, O. Tobin7, M. Shah1, N. Bennani1, R. Vassallo5, G. Goyal1,8, R. Go1 1Hematology; 2Internal Medicine; 3Laboratory Medicine and Pathology; 4Radiology; 5Pulmonology; 6Rheumatology; 7Neurology, Mayo Clinic Rochester, Rochester; 8Hematology, University of Alabama, Birmingham, United States of America Background: Isolated adult pulmonary Langerhans cell histiocytosis (pLCH) is a rare histiocytic neoplasm that involves the lungs as single system disease. Retrospective studies have suggested that patients with isolated adult pLCH have better overall survival compared to multisystem LCH. While population-based studies have been done for LCH with multisystem disease, there has been no epidemiologic studies done to date on isolated adult pLCH. Aims: To characterize the incidence and outcomes of isolated adult pLCH in the United States. Methods: We queried the Surveillance, Epidemiology, and End Results Program (SEER) 18 Registries (2010-2017) using ICD-0-3 code 9751/3 with the primary site of involvement being the lung. Patients were included if they were ≥ 18 years and had localized pulmonary involvement. Patients with extrapulmonary disease or unknown staging were excluded. The primary outcome was overall survival (OS), which was analyzed using the Kaplan-Meier method. Data on incidence rates (IR) and relative survival (RS) were calculated using the SEER*Stat software. IR (cases/1,000,000) was age-adjusted to the U.S. 2000 standard population. RS was defined as the ratio of the proportion of observed survivors in a cohort of isolated adult pLCH to the proportion of expected survivors in a comparable set of individuals that do not have isolated adult pLCH adjusting for the general survival of the US population for race, sex, age, and time when the diagnosis was established. Results: In SEER, there were a total 147 patients with isolated adult pLCH. Between the years 2010-2017, the median IR of isolated adult pLCH was 0.3 (range 0.1-0.5) and did not change significantly. The median IR from 2010-2013 was 0.2 (range 0.1-0.2) and 2014-2017 was 0.5 (range 0.3-0.5). The median age at diagnosis was 53 years (range 23-79) and 54 (37%) were male. 115 (78%) were white, 21 (14%) were black, and 11 were labeled as other. 116 (79%) were diagnosed on histopathologic evaluation while the remainder were diagnosed without microscopic confirmation. No patients were documented to have secondary malignancies. With a median follow up of 35 months, the median OS was not reached (95% CI: not reached-not reached) and the 3-year OS was 89%. The 3-year OS between males and females was 93% and 87% (p=0.16) respectively. The 3-year OS between white and non-white was 90% and 87% (p=0.98) respectively. In the general U.S. population, the expected survival for 1, 2, 3 years were 99.2%, 98.5%, 97.8% respectively. In contrast, the RS for patients with isolated adult pLCH at the same time points were 98.3%, 94.8%, 90.9% (Figure). A total of 14 patients died. 3 patients died from isolated adult pLCH while 11 patients died of other causes, including chronic obstructive pulmonary disease (3), cardiac disease (3), chronic liver disease (1), and unknown (4). Image: Summary/Conclusion: The incidence of isolated adult pLCH has not changed over the years from 2010-2017. Patients with isolated adult pLCH have lower relative survival compared to the general population. Among patients who died, the majority died from cause other than pLCH. P1140: TIME TO TUMOR, SYMPTOMATIC AND LABORATORY RESPONSES FOLLOWING SILTUXIMAB TREATMENT IN IDIOPATHIC MULTICENTRIC CASTLEMAN DISEASE F. van Rhee1,*, A. Rosenthal2, K. Kanhai3, R. Martin3, K. Nishimura2, A. Hoering2, D. Fajgenbaum4 1University of Arkansas for Medical Sciences, Little Rock; 2Cancer Research And Biostatistics, Seattle, United States of America; 3EUSA Pharma, Hemel Hempstead, United Kingdom; 4University of Pennsylvania, Philadelphia, United States of America Background: Idiopathic multicentric Castleman disease (iMCD) is a rare, heterogeneous disorder involving multicentric lymphadenopathy, systemic inflammation, and cytokine-driven organ dysfunction. Siltuximab—a monoclonal antibody against interleukin-6—is the only FDA- and EMA-approved treatment for iMCD. A phase 2, randomized, double-blind, placebo-controlled trial of siltuximab for the treatment of iMCD (NCT01024036) showed that 18/53 patients (34%) responded to treatment, compared with 0% in the placebo group, but it is not known how long patients should be maintained on siltuximab before deciding whether the treatment is beneficial. Aims: To determine the time to and sequence of normalization of laboratory, clinical, and lymph node responses in patients who responded to siltuximab. Methods: The phase 2 trial enrolled adults with symptomatic iMCD who were human immunodeficiency virus seronegative and human herpesvirus 8 negative. In this post hoc analysis, we aimed to determine the sequence of and time to normalization of laboratory, clinical, and lymph node responses in patients who responded to siltuximab. The Kaplan–Meier and cumulative incidence with competing risks methods with corresponding log-rank tests were produced to estimate and compare the progression-free survival (PFS), overall survival, time to treatment failure, time to lymph node response, time to durable symptomatic response, and time to normalization of laboratory values. Results: Seventy-nine patients were enrolled in the trial (n=53 siltuximab, n=26 placebo; all patients received best supportive care). The median duration of follow-up was 422 days (range 55‒1051). Siltuximab treatment improved PFS compared with placebo (p=0.0001). The median PFS was 14.5 months (95% CI 13.6–upper bound not reached) for patients on placebo, whereas it was not reached for patients on siltuximab. The 2-year estimates for overall survival were 93% (95% CI 85–100%) for the siltuximab arm and 77% (95% CI 55–98%) for the placebo arm (p=0.11). Time to treatment failure in the placebo arm was 4.8 months and was not reached with siltuximab (p=0.005). In the 18 siltuximab-treated patients who achieved the primary endpoint of durable tumor (radiologic) and symptomatic response, most first achieved rapid normalization of various laboratory parameters and symptomology (median of 0.8 months to normalization based on a 34-point MCD symptom score) (Table 1). Lymph node responses occurred later (median of 4.1 months to normalization). Image: Summary/Conclusion: Significant improvements in biochemical parameters such as platelet count, albumin, and symptomatic responses occur within 3 months in the majority of responders, showing them as early indicators of response. Lymph nodes were slower to show an improvement in response to treatment, and so involution of these should not be used to drive early therapeutic decisions in iMCD patients following siltuximab. The present study shows the recovery sequence of different parameters over time following initiation of treatment with siltuximab. P1141: IBRUTINIB AND RITUXIMAB AS FIRST LINE THERAPY FOR MANTLE CELL LYMPHOMA: A MULTICENTRE, REAL-WORLD UK STUDY A. Tivey1,*, R. Shotton1, T. A. Eyre2, D. Lewis3, N. Crosbie3, E. Nga4, R. Guerrero Camacho5, W. Swe6, H. Marr7, C. Rees8, S. Moule8, T. Sutton9, D. Wrench10, N. Thomas11, M. Wilson11, J. Bailey12, M. Prahladan13, A. Hodson13, M. Koppana13, S. Smith14, S. Jones14, F. Miall15, J. Norman16, E. Davies16, C. Hildyard17, L. Lowry18, S. Paneesha19, I. Qureshi19, A. Beech20, C. Bedford21, A. Everden21, D. Tucker21, J. Wright22, J. Goddard22, T. Nicholson23, J. Wilson24, A. Lord25, B. Jackson25, M. Flont26, A. Gibb27, K. Linton27 1Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester; 2Oxford University Hospitals NHS Foundation Trust, Oxford; 3University Hospitals Plymouth NHS Foundation Trust, Plymouth; 4Airedale NHS Foundation Trust, Airedale; 5Blackpool Teaching Hospitals NHS Foundation Trust, Blackpool; 6Calderdale and Huddersfield NHS Foundation Trust, Huddersfield; 7The Freeman Hospital, Newcastle; 8Frimley Health NHS Foundation Trust, Frimley; 9Gateshead Health NHS Foundation Trust, Gateshead; 10Guy’s and St Thomas’ NHS Foundation Trust, London, London; 11Harrogate and District NHS Foundation Trus, Harrogate; 12Hull University Teaching Hospitals NHS Trust, Hull; 13East Suffolk and North Essex NHS Foundation Trust, Ipswich; 14Sherwood Forest Hospitals NHS Foundation Trust, Sutton in Ashfield; 15University Hospitals of Leicester NHS Foundation Trust, Leicester; 16Manchester University NHS Foundation Trust, Manchester; 17Milton Keynes University Hospital, Milton Keynes; 18Somerset NHS Foundation Trust, Taunton; 19University Hospitals Birmingham NHS Foundation Trust, Birmingham; 20Nottingam University Hospitals, Nottingham; 21Royal Cornwall Hospitals NHS Trust, Truro; 22Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield; 23St Helens & Knowsley Teaching Hospitals NHS Trust, Whiston; 24University Hospitals Sussex NHS Foundation Trust, Chichester; 25Torbay and South Devon NHS Foundation Trust, Torbay; 26York and Scarborough Teaching Hospitals NHS Foundation Trust, York; 27Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, Manchester, United Kingdom Background: Ibrutinib (IBR) is an oral covalent Bruton tyrosine kinase inhibitor (BTKi), licensed for treatment of relapsed or refractory mantle cell lymphoma (MCL). Under NHS interim Covid-19 agreements in England, IBR with or without rituximab (R) was approved for the frontline treatment for MCL patients (pts) as a safer alternative to conventional immunochemotherapy. Although recent phase 2 studies have reported high response rates in low-risk patients for this combination in the frontline setting, randomised phase 3 and real-world data are currently lacking. Aims: To describe the real-world response rates (overall response rate (ORR), complete response (CR) rate) and toxicity profile of IBR +/- R in adult patients with previously untreated MCL. Methods: Following institutional approval, adults commencing IBR +/- R for untreated MCL under interim Covid-19 arrangements were prospectively identified by contributing centres. Hospital records were interrogated for demographic, pathology, response, toxicity and survival data. ORR/CR were assessed per local investigator according to the Lugano criteria using CT and/or PET-CT. Results: Data were available for 66 pts (72.7% male, median age 71 years, range 41-89). Baseline demographic and clinical features are summarised in Table 1. 23/66 pts (34.8%) had high-risk disease (defined as presence of TP53 mutation/deletion, blastoid or pleomorphic variant MCL, or Ki67%/MiB-1 ≥30%). IBR starting dose was 560mg in 56/62 pts (90%) and was given with R in 22/64 pts (34%). At a median follow up of 8.7 months (m) (range 0-18.6), pts had received a median of 7 cycles of IBR. 19/60 pts (32%) required a dose reduction or delay in IBR treatment. New atrial fibrillation and grade ≥3 any-cause toxicity occurred in 3/59 pts (5.8%) and 8/57 (14.0%) respectively. For the whole population and high-risk pts only, ORR was 74.4% and 64.7% respectively (p=0.2379), with a median time to response of 3.8m, coinciding with the first response assessment scan. Seven pts (16.7%), of whom 2 had high-risk disease, attained CR at a median of 6.0m. ORR for pts receiving vs not receiving R were 84.2% and 66.7% respectively (p=0.1904). IBR was discontinued in 20/61 pts (32.8%) at a median time to discontinuation of 4.1m, due to progressive disease (PD, 19.7%), toxicity (4.9%), death (3.3%; 1 pt each of Covid-19 and E. coli infection), pt choice (3.3%) and other unspecified reasons (1.6%). 15/66 pts (22.7%) overall and 7/23 (30.4%) with high-risk disease progressed on IBR at a median time to PD of 4.0m. No pts underwent autologous stem cell transplantation consolidation during the study period. 12/57 pts (21.1%) received second line treatment (R-chemotherapy n=7, Nordic MCL protocol n=2, VR-CAP n=2, pirtobrutinib n=1). Response to second line treatment was CR in 4/11 pts, PD in 7/11. Of the 2 Nordic-treated patients, 1 had CR after cycle 2 and 1 PD. Fourteen pts (21.2%) died during the follow up period, due to MCL (n=11), Covid-19 (n=2) and congestive cardiac failure (n=1). Overall survival was lower for patients with high-risk disease (HR 0.55, p=0.038). Image: Summary/Conclusion: In this real-world UK cohort of pts receiving first-line IBR +/-R for MCL, including older and high-risk pts, we report high ORR rates in a similar range to the phase II Geltamo IMCL-2015 study of combination IBR-R in an exclusively low-risk population. Documented CR rates were lower, possibly reflecting a low usage of rituximab in the Covid-19 pandemic as well as CT assessment of response. Treatment was generally well tolerated, with low rates of toxicity-related treatment discontinuation. The study is ongoing. P1144: RADIOIMMUNOTHERAPY (RIT) VERSUS AUTOLOGOUS HEMATOPOIETIC STEM-CELL TRANSPLANTATION (ASCT) IN RELAPSED/REFRACTORY (R/R) FOLLICULAR LYMPHOMA: A FONDAZIONE ITALIANA LINFOMI (FIL) PHASE III TRIAL. M. Ladetto1,*, R. Tavarozzi1, A. Evangelista2, M. Zanni3, A. Tucci4, A. Anastasia4, B. Botto5, C. Boccomini6, S. Bolis7, S. Volpetti8, V. R. Zilioli9, B. Puccini10, A. Arcari11, V. Pavone12, G. Gaidano1, P. Corradini13, M. Tani14, S. Ferrero15, F. Cavallo15, G. Milone16, C. Ghiggi17, A. Pinto18, D. Pastore19, A. J. Ferreri20, G. Latte21, C. Patti22, F. Re23, L. Arcaini24, F. Benedetti25, S. V. Usai26, S. Luminari27, D. Mannina28, A. Pulsoni29, C. Stelitano30, E. Pennese31, G. Pietrantuono32, F. Gherlinzoni33, F. Pomponi34, A. Olivieri35, T. Perrone36, D. Rota Scalabrini37, C. Califano38, B. Falini39, G. Ciccone2, U. Vitolo37 1Department of Translational Medicine, University of Eastern Piedmont, Novara; 2Unit of Clinical Epidemiology, A.O.U. Città della Salute e della Scienza, Torino; 3SCDU of Hematology, AO SS Antonio e Biagio e Cesare Arrigo, Alessandria; 4Hematology, ASST Spedali Civili, Brescia; 5Hematology, A.O.U. Città della Salute e della Scienza; 6Hematology, A. O. U. Città della Salute e della Scienza, Torino; 7Hematology Department, ASST San Gerardo University Hospital, Monza; 8Institute of Hematology, University and Hospital of Udine, Udine; 9ASST Grande, Ospedale Metropolitano Niguarda, Milano; 10Hematology Department, AOU Careggi, Firenze; 11Hematology, Ospedale Guglielmo da Saliceto, Piacenza; 12Hematology, Card. Panico Hospital S.Pio X, Tricase; 13Division of Hematology and Stem Cell Transplantation, University of Milano Istituto Nazionale Tumori, Milano; 14Hematology, Santa Maria delle Croci Hospital, Ravenna; 15Department of Molecular Biotechnologies and Health Sciences, Division of Hematology, University of Turin, Torino; 16Hematology and BMT Unit, Azienda Policlinico Vittorio Emanuele, Catania; 17Clinic of Hematology, IRCCS Ospedale San Martino, Genova; 18Hematology-Oncology and Stem-Cell Transplantation Unit, National Cancer Institute, Fondazione ‘G. Pascale, Napoli; 19Ospedale Antonio Perrino, Brindisi; 20Onco-Hematology Department, Fondazione Centro San Raffaele, Milano; 21Unità di Ematologia e Trapianto di Midollo Osseo, San Francesco hospital, Nuoro; 22Divisione di Oncoematologia, Azienda Villa Sofia-Cervello, Palermo; 23Hematology, A.O.U. di Parma, Parma; 24Department of Onco-Hematology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Pavia; 25Department of Medicine, Section of Hematology and Bone Marrow Transplant Unit, University of Verona, Verona; 26Ospedale Businco, Cagliari; 27Hematology, IRCCS Reggio Emilia, Reggio Emilia; 28Hematology, Azienda Ospedaliera Papardo, Messina; 29Hematology, Department of Translational and Precision Medicine, Sapienza University of Rome, Roma; 30Hematology, Azienda Ospedaliera Bianchi-Melacrino-Morelli, Reggio Calabria; 31Lymphoma Unit, Department of Hematology, Ospedale Spirito Santo, Pescara; 32IRCCS Centro Oncologico Della Basilicata, Rionero In Vulture; 33Division of Hematology, Ca’ Foncello Hospital, Treviso; 34ULSS8 Berica, Vicenza; 35Hematology, AOU Ospedali Riuniti, Ancona; 36Unit of Hematology with Transplantation, Dept. of Emergency and Organ Transplantation, University of Bari, Bari; 37Candiolo Cancer Institute, FPO-IRCCS, Candiolo; 38U.O.C. Ematologia, P.O. “Andrea Tortora”, Pagani; 39Department of Medicine, Division of Hematology and Clinical Immunology, University of Perugia, Perugia, Italy Background: Optimal consolidation for young R/R FL in the rituximab age remains uncertain and the benefit of ASCT is not clearly established. Aims: The FIL FLAZ12 trial (NCT01827605) is a prospective, multicenter, randomized, phase 3 trial, comparing RIT versus ASCT, as consolidation after chemoimmunotherapy, both followed by R maintenance in R/R FL. Methods: Pts aged 18-65 yrs, with R/R FL after 1 or 2 lines of chemoimmunotherapy, without significant comorbidities were enrolled. Patients received 3 courses of therapy chosen by the investigator among RCHOP, R-DHAP, R-FM, R-ICE, R-IEV or R-B. Pts achieving at least PR (according to Cheson et al. 2007) were randomized 1:1 to either RIT or ASCT before CD34+ collection. Conditioning for ASCT was BEAM or TEAM. RIT was given as previously described (Morschhauser et al., 2008). After consolidation, pts received R maintenance every 3 months for eight courses. Primary endpoint was PFS. Considering ASCT toxicity, it was hypothesized to be a superior choice, if capable of increasing 3-years PFS from 40% to 60% (two-side log-rank test with alpha of 5% and a power of 85%). Clinical secondary endpoints were ORR, CRR, OS, EFS and TTF. Results: Between Aug 2012, and Sep 2019, 164 pts were screened and 159 enrolled by 38 FIL Centers (enrolled population). Unfortunately, the study was prematurely closed due to low accrual. The data were analyzed on an ITT basis on May 2, 2021 with a median follow-up (mFU) from enrollment of 43 months and 75 PFS events. The two arms were clinically well balanced, with median age of 57 yrs (IQR 49- 62), 55% male, 57% stage IV, 20% bulky disease. Tumor re-biopsy was performed in 79% pts. POD-24, retrospectively assessed was observed in 32% of pts. Two pts (1%) did not start treatment (non-confirmed histology and withdrawal). Sixteen (10%) pts discontinued before randomization (7 SD, 3 PD, 3 AE, 1 withdrawal, 2 poor compliance) and 141 (89%) were randomized to either RIT (71) or ASCT (70) (randomized population). Of these 19 (13%) (RIT 8, ASCT 11) did not receive the planned consolidation due to 7 PD, 4 AE, 1 medical decision, 2 poor mobilization, 2 withdrawals, 1 poor compliance, 2 protocol breaches, while 63 (89%) received RIT and 59 (84%) ASCT. After RIT, 61% of pts achieved CR and 23% PR, while after ASCT these were 70% and 9%. Estimated PFS at 3 yrs was 60% (95% CI: 46%-71%) in the RIT arm vs. 59% (95% CI:45%-70%) in the ASCT arm, p = 0.8613 (HR 0.96, 95%CI: 0.57,1.59). (Figure 1) 3yrs-OS was again superimposable in the two arms: 83% (95%CI: 69%-91%) in the RIT vs 85% (95% CI: 72%-91%) in the ASCT, p = 0.8310 (HR 1.10, 95%CI: 0.45,2.72). Grade ≥ 3 hematological toxicity was 46% in the RIT vs 94% in the ASCT arm (p < 0.001). For ASCT vs RIT grade ≥3 neutropenia occurred in 94% vs 41% of pts (p < 0.001). During follow-up, 4 pts died in remission: 1 AML (RIT), 2 SARS- COV2 infections (RIT) and 1 pneumonia (ASCT). Second cancers occurred in 3 pts after RIT and 7 after ASCT (p = 0.480). Multivariable analysis for PFS indicated POD-24, male sex, LDH and refractory disease as adverse parameters. Subgroup analysis for PFS including gender, age, LDH, POD-24 and extranodal disease show no subgroup favoring RIT nor ASCT. Image: Summary/Conclusion: Even if prematurely interrupted, our study demonstrated no meaningful difference in efficacy between ASCT and RIT, but ASCT was more toxic and more demanding for pts and health service. Both strategies induced a similar and favorable long-term outcome suggesting that consolidation programs milder than ASCT require further investigation in R/R FL. P1145: OUTCOMES AND TREATMENT PATTERNS AFTER FIRST RELAPSE IN PATIENTS WITH WALDENSTRÖM MACROGLOBULINEMIA R. Tawfiq1,*, J. Abeykoon2, S. Zanwar2, J. Paludo2, P. Kapoor2 1Department of Medicine; 2Division of Hematology, Mayo Clinic, Rochester, United States of America Background: Waldenström macroglobulinemia (WM) is a rare, B cell lymphoma, with a relapsing-remitting course. Despite expansion of therapeutic options, WM remains incurable. Data on outcomes of patients (pts) after first relapse or primary refractory disease are sparse. Frequently used salvage therapies include chemoimmunotherapy (CIT), proteasome inhibitor (PI) based regimens and Bruton tyrosine kinase inhibitors (BTKi). Aims: Analyze the outcomes of pts with WM after initial relapse and/or refractory (RR) disease and the impact of the type of therapy used in the second line setting. Methods: ecords of pts with WM seen at Mayo Clinic between 2000 and 2021 were reviewed. Pts with RRWM requiring second line therapy were included in the primary analysis. Response rates were assessed per the modified IWWM-6 criteria. All time to event analyses, barring progression free survival 2 (PFS2), were calculated from the start of the second line treatment, using the Kaplan Meier method. Pts were grouped based on sequence of therapy and response to frontline treatment for additional subgroup analysis. Results: Records of 220 pts with WM were reviewed; 92 had RRWM and were treated with a second line therapy. For the entire cohort, frontline regimens included dexamethasone, rituximab, cyclophosphamide (DRC) (n=93, 42%), bendamustine rituximab (BR) (n=71, 32%), bortezomib, dexamethasone, rituximab (BDR) (n=33, 15%), BTKi (n=20, 9%), and other (n=3, 1%). Median follow-up of RRWM cohort from second line therapy was 5.1 years (95% CI: 4.5-6.3). Pts with RRWM received the following therapies at first relapse: BR (n=24, 26%), BTKi (n=24, 26%), PI (n=17, 19%), DRC (n=6, 7%), autologous transplant (n=4, 4%), and other (n=17, 18%). Following second-line treatment, the overall response rate and major response rate (MRR) were 72% and 67% (CR=5%, VGPR=15%, and PR=47%), respectively. The median PFS was 2.7 years (CI: 1.7-4). 2-year overall survival (OS) rate was 87%. The median PFS2 from frontline therapy was 7.03 years (CI: 5.5-9.8). Baseline characteristics were similar for subgroups in all the following subgroup analyses. With first-line therapy, 54% of pts (n=50) achieved MRR vs. 46% (n=42) who did not. The former group had similar progression-free survival (PFS) rates as well as overall survival (OS) rates and response rates (MRR and ORR) to the second-line treatment compared to their counterparts who did not achieve MRR with the frontline regimen (Table). Thiry one pts received BTKi as either first or second line and 61 pts received no BTKi in either line. As expected, the BTKi group was enriched for pts with MYD88 L265P mutation (n=29, 94%) vs 49% (n=30) in the no BTKi group, p<0.001. The outcomes between the 2 groups were comparable (Table). The group that received frontline BTKi followed by CIT/PI based Rx as second line (n=7) was compared to that receiving frontline CIT/PI based Rx followed by BTKi (n=21). The outcomes were similar (Table). Image: Summary/Conclusion: Currently available second line therapies are effective in RRWM. Achieving MRR with frontline regimen does not significantly affect overall outcomes with the second-line therapy. Although follow up is short, using a BTKi as the first- or second-line therapy appears to show comparable outcomes after second line treatment as compared to not treating with a BTKi in the upfront setting or in second-line setting. Similarly, using frontline BTKi followed by CIT as second line shares similar outcomes to treating with CIT initially followed by a BTKi. These findings require confirmation in prospective studies. P1147: SERUM ALBUMIN AND NEUTROPHIL-TO LYMPHOCYTE RATIO TWO INDEPENDENT FACTOR PREDICTING SURVIVAL IN FOLLICULAR LYMPHOMA. MULTI-INSTITUTIONAL 763 COHORT LA M. A. Torres Viera1,* 1LYMPHOMAS, Hematologia Oncologia 360 Clinica Sta Sofia, Caracas, Venezuela Background: Introduction: The neutrophil-lymphocyte ratio (NLR) is a measure of systemic inflammation that appears prognostic in different cancers. Although the exact mechanism remains to be elucidated, reduced lymphocyte intratumoral infiltration coupled with the formation of neutrophil extracellular traps (or NETosis) have been postulated as endogenous mechanisms for tissue damage and inflammation. Along this line, serum albumin has also been studied as a biomarker of inflammation and has been associated to prognosis in certain cancers. We have previously reported on the prognostic value of the NLR and serum albumin in diffuse large B-cell lymphoma (Villela, ASH meeting, 2019; Castro, ASH meeting, 2019) and peripheral T-cell lymphoma, not otherwise specified (Idrobo, ASH meeting, 2019), but nothing on follicular lymphoma (FL) yet. Aims: Therefore, we aim to investigate the role of different biomarkers on the prognosis of patients with FL diagnosed and managed in Latin America Methods: We analyzed patients with FL diagnosed between 2010 and 2020 from 30 centers in 10 Latin American countries. The study outcomes were overall survival (OS) and progression-free survival (PFS) in relation to different biomarkers. Kaplan-Meier and log-rank test were used for survival analysis. Univariate and multivariate Cox regression analysis were used to estimate hazard ratios (HR) with a 95% confidence interval (CI) and adjusted to the Follicular Lymphoma International Prognostic Index (FLIPI) score. Outcomes with a p-value <0.05 were considered statistically significant Results: We identified 939 FL patients; 741 were included for the final analysis (median age 58 y, female 52%). There was no significant correlation between the NLR and other clinical factors such as: age, clinical stage, histological FL grading, and chemotherapy regimen used. A cutoff of 2.15 for NLR was defined as the maximum point for sensitivity and specificity based on ROC analysis. Table 1 and 2 summarizes the results from the univariate and multivariate analysis for 2 years OS and PFS, respectively. Both, serum albumin <3.5 g/dL and a NLR >2.15 were independently associated with worse OS (adjusted, aHR 2.48 [1.26-4.91], p=0.009; and 2.55 [1.21-5.37], p=0.014) and PFS (aHR 1.62 [1.03-2.55], p=0.038; 2.22 [1.45-3.40], p<0.001), respectively. The lymphocyte:monocyte ratio (LMR) was not found to be prognostic for OS or PFS, although with a trend for worse PFS with a LMR ≤2.5. With a median follow of 43 months, (95% CI: 40-47), the survival rates in patients with FL and albumin <3.5 were OS of 83% (vs. 95%) and PFS of 70% (vs. 83%); whereas in patients with NLR >2.15 the survival rates were OS of 91% (vs. 96%) and PFS of 75% (vs. 88%) (Figures 1 and 2; Table 3). Summary/Conclusion: In this study, serum albumin and NLR emerge as reliable predictors for survival for FL patients in Latin America. Although these markers have been associated to an increased inflammatory state in cancer patients; other factors such as poor nutritional status, and advanced disease stage due to delayed access to specialized cancer care in our region may have contributed to the observed outcome. Further studies are needed to better understand the role of these biomarkers on lymphoma care and to validate our findings. Lastly, we are currently working on evaluating these biomarkers on existing prognostic models and to improve prognostication for FL patients in Latin America P1148: REAL WORLD DATA (RWD) AMONG FOLLICULAR LYMPHOMA (FL) PATIENTS IN GERMANY WITH AT LEAST TWO PRIOR LINES OF SYSTEMIC THERAPY AND COMPARISON WITH CLINICAL DATA FOR MOSUNETUZUMAB N. Marschner1,*, J. Hanselmann2, S. McGough3, S. Braun4, S. Pfizler-Dempfle5, R. Sandner6, J. Mohm7, M. Jaber8, D. Hamm9, N. Hamm10, M. Jaenicke11, A. Shewade3 1Outpatient Centre for Interdisciplinary Oncology and Haematology; 2Biostatistics, iOMEDICO, Freiburg, Germany; 3Roche Global Product Development Data Sciences, Genentech, Inc., South San Francisco, CA, United States of America; 4Biometrics and Epidemiology. Roche Pharma AG, Grenzach-Wyhlen; 5Oncology and Haematology Outpatient Centre, Kaiserslautern; 6MVZ for Haematology and Oncology, Passau; 7Oncology and Haematology Outpatient Centre, Dresden, Germany; 8Roche Global Access, F. Hoffmann-La Roche Ltd, Basel, Switzerland; 9Medical Affairs Excellence, Roche Pharma AG, Grenzach-Wyhlen; 10Medical Department, iOMEDICO; 11Clinical Epidemiology, iOMEDICO, Freiburg, Germany Background: FL is characterized by its indolent nature, with patients (pts) experiencing multiple relapses and successively poorer outcomes with each relapse (Link et al. 2019). Treatment with immunochemotherapy regimens has significantly improved outcomes in pts with newly diagnosed FL, but limited treatment options are available in Europe, with no standard of care for pts with multiple relapses, such as those who have received at least two prior lines of systemic therapy (3L+ FL). Mosunetuzumab (M) is a T-cell engaging CD20xCD3 bispecific antibody that has shown high rates of complete response and favorable safety in pts with 3L+ FL in a single-arm Phase II trial (NCT02500407; Budde et al. ASH 2021). RWD can provide relevant context to interpret data from the single-arm trial (SAT) of M as a treatment option in 3L+ FL. Aims: This study summarizes RWD in pts with 3L+ FL utilizing the Tumor Registry Lymphatic Neoplasms extension (TLNext) and plans to perform a matching-adjusted indirect comparison (MAIC) between the M SAT and TLNext. Methods: TLN was a multicenter, longitudinal, observational, prospective cohort study collecting data from medical charts of pts with lymphoid B-cell neoplasms receiving care at office-based hematology practices in Germany (NCT00889798; Knauf et al. 2019). Patients were enrolled between 2009–14 and followed for up to 5 years. TLNext collected additional data up to November 2021 from consented pts originally enrolled in TLN as well as additional newly-consented pts with 3L+ FL. Baseline characteristics, treatment patterns, and outcomes were summarized for pts with 3L+ FL in TLNext. A MAIC is planned between the M SAT and a TLNext cohort selected by applying key eligibility criteria from the M SAT. An index line of therapy (LoT) will be selected for all pts with more than one recorded LoT. Imbalance adjustment will be performed by re-weighting the TLNext cohort using weights determined from distributions of prognostic clinical factors observed in M SAT. Results: TLNext included 69 pts who had received 112 documented LoTs in 3L+ FL. Median follow-up was 40 months from the start of the 3rd LoT. 65 pts had received an anti-CD20 therapy and an alkylator therapy prior to index therapy and had a known relapse or refractory status (Table). Latest recorded LoT was selected as the index therapy for every patient. Compared to M SAT, the TLNext cohort was slightly older and had more female pts. A higher proportion of pts in TLNext had received only 2 prior LoT. Overall, M SAT had a higher proportion of pts with factors known to be associated with poorer outcomes in 3L+ FL, e.g. refractory to prior anti-CD20 therapy, double-refractory to prior anti-CD20 therapy and alkylator therapy. The most common regimens in the TLNext cohort were bendamustine-like regimens (34%), PI3K-like regimens (15%), and anti-CD20 monotherapy (14%). Comparative MAIC analyses are ongoing. Image: Summary/Conclusion: TLNext represents a real-world 3L+ FL cohort from Germany that provides relevant context for the M SAT. TLNext outcomes data will be re-weighted using the MAIC approach to generate a descriptive benchmark for outcomes observed in M SAT. While comparisons between clinical trials and RWD carry several limitations pertaining to measurement, reporting of data, and choice of analytical approach to conduct the comparison, RWD from TLNext may be useful to describe a benchmark for clinical practice in 3L+ FL in Germany and globally prior to the availability of randomized evidence on newer treatment options like mosunetuzumab. P1149: THE EVOLVING STATE OF PLAY IN 1000 PATIENTS WITH WALDENSTRÖM’S MACROGLOBULINAEMIA IN THE UNITED KINGDOM (UK): A REAL-WORLD DATA ANALYSIS FROM THE WMUK RORY MORRISON REGISTRY PROJECT E. Uppal1,*, A. Otamas1, J. Khwaja1, H. McCarthy2, J. Kothari3, A. Rismani1, D. El-Sharkawi4, C. Kyriakou1, S. D’Sa1 1Department of Haematology, University College London Hospitals NHS Foundation Trust, London; 2Department of Haematology, Royal Bournemouth Hospital, Bournemouth; 3Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford; 4Department of Haematology, The Royal Marsden Hospital NHS Foundation Trust, London, United Kingdom Background: Waldenström’s Macroglobulinaemia (WM) is a rare B-NHL, with an incidence of 0.55 per 100,000 per year in the UK (1). As novel therapies move from the bench to the clinic, real world data (RWD) is important to understand trends and supplement data from clinical trials. The Rory Morrison Registry (RMR) collects sequential data on diagnosis, treatment landscape and outlook for patients from 25 UK centres. Aims: To characterise therapy trends in the UK by an analysis of disease and treatment characteristics, and survival status. Methods: The RMR was searched for all patients with a diagnosis of WM. Information regarding demographics, disease characteristics, bone marrow (BM) results, treatment and survival status were collected retrospectively; the earliest diagnosis was in 1978 and the data lock was on 25/2/22. Results: 1007 patients with a diagnosis of WM using consensus criteria (2) at 25 UK Centres (94.5% academic, 5.5% district centres) were identified. The following data pertain to diagnosis; median age was 64 years (21-93); M:F ratio 1.6:1, 90% of white ethnicity. Diagnostic sub-entities included peripheral neuropathy (116), cryoglobulinaemia (68), amyloidosis (22), Bing-Neel syndrome (18) and Schnitzler’s syndrome (9); 53% (501/945) were symptomatic at diagnosis. The median IPSSWM for 496 patients was 2. MYD88 L265P by RT-PCR was found in 87.1% (304/349) and CXCR4 mutations in 28.9% (26/90). Of the total, 690 (68.5%) patients received treatment with a median time from diagnosis to first line (L1) treatment of 3 months (0-312). Indications for L1 were recorded as: 274/690 lymphoma related, 248/690 paraprotein-related, 65/690 B-Symptoms, 101/690 other indications. Regarding later lines, 402 (39.9%) patients received a 2nd treatment (L2); median time from end L1 to beginning of L2 was 15.1 months (0-163). A 3rd line (L3) was administered in 226 (22.4%) patients; median time from end of L2 to start of L3 was 9 months (0-158); 125 (12.4%) patients received ≥4 treatments. Between 1984 and 2021 the three most popular treatments received at L1 were: Dexamethasone-Rituximab-Cyclophosphamide (DRC) 20.1% (144/690), R-Bendamustine (BR) 15.2% (105/690), Fludarabine based (±Cyclophosphamide, Rituximab) 9.0% (62/690) [Figure 1]. The three most popular treatments at L2 were: Ibrutinib 24.9% (100/402), BR 9.5% (38/402), DRC 5.0% (20/402). Ibrutinib is the leading 2nd, 3rd, 4th line treatment option from 2018 across all centres, due to its availability on the national Cancer Drugs Fund from Nov 2017. The most received treatments from 2020 at: Ibrutinib 31.1% (60/193), R-Bendamustine 12.4% (24/193), DRC 10.4% (20/193). 38 patients underwent autologous and 7 allogeneic stem cell transplants between 2002 to 2020 at a median of L3 (range 1-7). At the time of data lock, 184 patients were labelled deceased. Cause of death was available for 78 patients: 15 due to WM, 7 patients high grade transformation, 3 amyloid, 3 during ASCT, 2 AML/MDS. Image: Summary/Conclusion: This RWD analysis of >1000 UK patients shows that inter/national consensus guidelines are resulting in more streamlined treatment approaches in WM. As targeted therapies have become available, there is a paradigm shift towards their use; however there is still much to learn about the optimal selection and sequencing of therapies in WM patients whose diseases are heterogenous by nature. Patient-related outcome studies are underway to complement ongoing follow-up of clinically meaningful remission duration and tolerance. P1150: REAL WORLD DATA ON BORTEZOMIB-BASED THERAPY IN WALDENSTRÖM’S MACROGLOBULINAEMIA: EFFECTIVE EVEN IN MULTIPLY TREATED PATIENTS INCLUDING PRIOR BTK-INHIBITORS E. Uppal1,*, J. Khwaja1, A. Rismani1, C. Kyriakou1, S. D’Sa1 1Department of Haematology, University College London Hospitals NHS Foundation Trust, London, United Kingdom Background: Waldenström macroglobulinemia (WM) is an indolent lymphoma with a prolonged disease course which typically follows a remitting and relapsing trajectory, eventually leading to treatment resistance. Several treatment options exist including Bruton tyrosine kinase inhibitors (BTKi), rituximab-containing regimens, and bortezomib-containing regimens. Treatment selection is based on patient performance status, disease characteristics, drug tolerability and availability. Aims: To assess the effectiveness and tolerability of bortezomib-based regimens in WM. Methods: Data for patients who had Bortezomib-containing regimens between 2010 and 2021 from 6 centres in the United Kingdom were retrospectively reviewed. Data was acquired from the WMUK Rory Morrison Registry. Research ethics approval was obtained. Results: Thirty-four patients were identified: 32/34 had Bortezomib-containing regimens once and 2/34 had >1 Bortezomib-containing regimens on separate occasions, giving a total of 38 subcutaneous Bortezomib-containing regimens administered at bi-weekly and weekly schedules. Median age was 62 years (37-87), with a median of 2 prior lines of therapy (0-7), at a median duration of 49.6 months from date of WM diagnosis (0.7-422). 5 patients received a prior BTKi, with the Bortezomib regimen prescribed following a median of 3 prior lines of therapy (2-5) in this group. Patients who were treated at first line had elected for non-chemotherapy regimens. Median performance status was 1 (0-2) in 23 evaluable patients. The median M-protein at initiation was 34.5g/l (8-60) with bone marrow infiltration 70%, and haemoglobin 94g/l (88-107). A median of 5 cycles (1-8) were delivered and 65% (13/20) received a dose of 1.6mg/m2 and 35% (7/20) received 1.3mg/m2. Grade (G) 1 to 2 neuropathy occurred in 19% (5/26) of evaluable patients but did not result in treatment cessation in any case. Six of 25 (24%) needed a dose reduction, the majority due to G1-2 neuropathy (67%; 4/6). Gastrointestinal disturbance occurred in 12% (3/26) patients, 1 required admission with G4 diarrhoea and remaining cases were G1. Of 34 evaluable cases, major response rate (≥ PR) was 74% (5 CR, 6 VGPR, 14 PR). 62% (8/13) of patients receiving 1.6mg/m2 achieved a major response and 86% (6/7) of those who received 1.3mg/m2. Three of 5 patients who had prior BTKi achieved PR, 1 MR, 1 SD. Two patients had treatment discontinued due refractory disease. The overall median time to best response was 81 days from end of treatment. Six of 19 (26%) evaluable patients achieved best response during therapy. Two patients died during treatment due to infection (COVID; respiratory sepsis), not attributable to disease relapse. Eighteen patients (60%; 18/30) had treatment after bortezomib regimens at a median of 5.3 months (0-75) and are alive. Median follow up was 30 months (1-111). Twenty-one evaluable patients (72%; 21/29) were alive at the end of follow up. Image: Summary/Conclusion: This retrospective real-world analysis shows that bortezomib-containing regimens have utility in WM with effective major response rates even in those with multiple prior lines of therapy and heavy marrow infiltration including BTKi failures. Lower bortezomib doses are effective, and GI and neurotoxicity are manageable with dose reductions but no treatment discontinuations in this real-world cohort indicating an acceptable safety profile. P1151: METABOLIC TUMOR VOLUME IMPROVES OUTCOME PREDICTION IN UNTREATED MANTLE CELL LYMPHOMA V. K. Vergote1,*, G. Verhoef1, A. Janssens1, F. S. Woei-a-jin2, A. Laenen3, T. Tousseyn4, D. Dierickx1, C. M. Deroose5 1Hematology; 2General Medical Oncology, UZLeuven; 3Biostatistics and Statistical Bioinformatics Center, KULeuven; 4Pathology; 5Nuclear Medicine, UZLeuven, 3000, Leuven, Belgium Background: Mantle cell lymphoma (MCL) is a rare, incurable subtype of B-cell non-Hodgkin’s lymphoma, with heterogenous clinical behavior ranging from indolent to very aggressive disease course with rapid relapses and short survival. Aims: The aim of this retrospective study is to analyze the prognostic value of baseline [18F]FDG-PET/CT metabolic parameters in untreated MCL. Methods: We retrospectively analyzed baseline [18F]FDG-PET/CT of patients in our institution (University Hospitals Leuven, Belgium) with histopathological confirmed newly diagnosed MCL between January 1st 2004 and 31st December 2020. Maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), peak standardized uptake value (SUVpeak), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) were analyzed. Lesion dissemination (Dmax) and lesion dissemination standardized to body surface area (SDmax) were calculated. Univariate and multivariate analysis were performed using Cox proportional hazard models. Kaplan Meier survival curves were constructed. Results: We included 83 patients with a median age of 66 years. The median MTV was 141.5 ml and the median Dmax was 0.6 m. A median follow-up of 47 months was achieved. In total 42 patients died. Higher MTV was an adverse factor for overall survival (OS) (p=0.0137; hazard ratio (HR): 1.48; 95% confidence interval (CI): 1.08-2.03), progression-free survival (PFS) (p=0.0106; HR: 1.41; 95% CI: 1.08-1.84) and disease-specific survival (DSS) (p=0.0366; HR: 1.56; 95% CI: 1.03-2.36). Furthermore, higher age at diagnosis, MCL International Prognostic Index-score (MIPI) and Eastern Cooperative Oncology Group Performance status (ECOG PS) were adverse factors for OS, PFS and DSS. We found no significant correlation between Dmax, SDmax and any of the outcomes considered. In multivariate analysis accounting for clinical characteristics, MTV was significantly associated with DSS (p=0.0072; HR: 1.53; 95% CI: 1.12-2.08), but not with OS and PFS. OS in the patients with MTV above the median (high, ≥141 ml) was significantly worse compared to the group with low MTV (<141 ml) (median OS 6.1 years versus 7.6 years; p=0.0077). Furthermore, there was a significant difference in PFS (median 2.9 versus 5.0 years; p=0.0055) and DSS (median 7.3 years versus not reached; p=0.0219) between the groups with high and low MTV. MTVspleen was an adverse factor for OS (p=0.0103; HR: 1.36; 95% CI: 1.08-1.72) and PFS (p=0.0019; HR: 1.40; 95% CI: 1.13-1.72) in multivariate analysis. Ann Arbor stage was not withheld as a significant factor for outcome prediction in univariate nor multivariate analysis. Summary/Conclusion: MTV is an important prognostic tool and can further improve patient risk stratification at staging of untreated MCL. We found no correlation between lesion dissemination (Dmax, SDmax) on [18F]FDG-PET/CT and outcome. P1152: RESULTS FROM A PHASE I PHARMACOKINETIC (PK) AND SAFETY STUDY OF TRPH-222, A NOVEL CD22-TARGETING ANTIBODY-DRUG CONJUGATE, IN PATIENTS WITH RELAPSED/REFRACTORY B-CELL NON-HODGKIN LYMPHOMA (R/R NHL) F. J. Hernandez-Ilizaliturri1, J. Kuruvilla2, B. A. Christian3, I. W. Flinn4, S. E. Assouline5, M. L. Ulrickson6, D. J. Landsburg7, M. Stuart8, H. Lowman8, N. Levin8, D. Maetzel9, N. N. Viller9, A. MacLaren8,* 1Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY, United States of America; 2The Princess Margaret Hospital, Toronto, ON, Canada; 3The Ohio State University, Columbus, OH; 4Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, United States of America; 5Division of Hematology, Sir Mortimer B. Davis-Jewish General Hospital, Department of Oncology, McGill University, Montreal, QC, Canada; 6Banner MD Anderson Cancer Center, Gilbert, AZ; 7Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA; 8Triphase Accelerator, La Jolla, CA, United States of America; 9Triphase Accelerator, Toronto, ON, Canada Background: TRPH-222 is a novel antibody-drug conjugate (ADC) comprised of a humanized anti-CD22 monoclonal antibody and maytansine, a potent microtubule inhibitor, joined by a novel third-generation linker-conjugation technology (SMARTag®). This approach enables site-specific conjugation of the maytansine payload while tightly controlling drug:antibody ratio (DAR), resulting in a highly stable anti-CD22 ADC with a non-cleavable linker designed to widen the therapeutic window. Aims: The primary objectives of this first in human study are to determine the safety, tolerability and pharmacokinetics (PK) of TRPH-222 monotherapy in patients with R/R NHL. Methods: TRPH-222-100 is an open-label, multicenter study comprised of dose-escalation and dose-expansion stages. TRPH-222 was administered IV once every 3 weeks. 22 patients were enrolled in dose-escalating cohorts of TRPH-222 (0.6 mg/kg to 10 mg/kg) from DLBCL, FL, TFL, MCL and MZL histologies, and 10 patients in a dose-expansion cohort (7.5 mg/kg) focusing on DLBCL and FL histologies. Results: As of January 7, 2022, 32 NHL patients have been enrolled: 15 indolent (14 FL and 1 MZL) and 17 aggressive histologies (15 DLBCL, 1 TFL and 1 MCL). Patients had a median age of 64.5 years, a median of 4 prior lines of therapy including 7 patients receiving prior CAR-T treatment. Three DLTs occurred in 2 patients during the study and comprised Grade 3 and 4 transaminase elevations; one each at 4.2 and 10 mg/kg and one Grade 3 thrombocytopenia at 4.2 mg/kg. Treatment-related serious adverse events (AEs) occurred in 2 patients (6.3%), caused by thrombocytopenia and pyrexia (both at 7.5 mg/kg TRPH-222). AEs were more prevalent at doses ≥7.5 mg/kg and less prevalent at lower doses. There was a trend to higher grade AEs in patients with aggressive histologies, compared to indolent ones. The most frequent (≥ 5%) treatment-emergent related AEs (Grade ≥3) included thrombocytopenia (34%), neutropenia (22%), ALT/AST elevation (6%), dry eye (6%) and blurred vision (6%). Cytopenias were non-febrile, infrequent, asymptomatic and resolved without significant intervention. Ocular findings were consistent with known epithelial keratopathy of ADCs and were generally low grade and resolved to ≤ Grade 1 with dose interruptions and/or reductions. Overall, TRPH-222 demonstrated a favourable safety profile with most AEs being predominantly low grade, tolerable, easily managed and reversible. Preliminary efficacy results suggest evidence of anti-tumor activity, most notably in patients with R/R FL. Of the 13 response-evaluable FL patients, 4 complete responses (CR) and 2 partial responses (PR) were observed, with an overall response rate (ORR) of 46% and a complete response rate (CRR) of 31%. Four patients with metabolic CRs maintained these CRs for long periods off treatment; 3 patients remain in CR with responses maintained for up to 25 months. Responses were generally early, durable and CRs were maintained off-therapy. Beyond FL, CRs were also observed in 1 DLBCL patient and in 1 MCL patient. Summary/Conclusion: TRPH-222 was found to be well tolerated at higher dose levels than evaluated for other ADCs. TRPH-222 monotherapy resulted in robust and durable CRs in FL across dose levels where patients were able to discontinue TRPH-222 while remaining in remissions. Collectively, these characteristics of TRPH-222 are favorable for further development in the indolent lymphoma setting either as monotherapy or in combination with other anti-tumor agents in B-cell lymphoma patients. P1153: LONG-TERM SURVIVAL OUTCOMES OF PATIENTS WITH PRIMARY OCULAR ADNEXAL MALT LYMPHOMA: A LARGE SINGLE-CENTER COHORT STUDY Y. Liang1, R.-Y. Fu1, X. Li1, Y.-S. Piao2, J.-M. Ma3, L. Wang1,* 1Hematology; 2Pathology; 3Eye center, Beijing Tongren Hospital, Capital Medical University, Beijing, China Background: Primary ocular adnexal extranodal marginal zone mucosa-associated lymphoid tissue lymphoma (OAML) is a rare subtype of non-Hodgkin’s lymphoma, and no consensus has been defined concerning the optimal treatment strategies. Aims: This study aims to investigate the associations of disease characteristics and different treatments with long-term outcomes of patients with localized OAML. Methods: A large retrospective cohort study was conducted in a single-center of China, and 166 patients with newly diagnosed primary localized OAML between April 2008 and August 2021 were enrolled in our analysis. Detailed data of disease characteristics at diagnosis and treatments were collected for all patients, and treatment response was evaluated at one month after therapy and every six months thereafter. We compared treatment response and progression-free survival (PFS) among patients with different characteristics and treatments. Results: Of the 166 patients, 52 received complete resection of neoplasm, whereas 114 had residual lesion after surgery. Among the 114 patients, 61 underwent watchful waiting and 53 received further treatment including localized radiotherapy, chemotherapy (including immunochemotherapy), or combined radiotherapy and chemotherapy. Median follow-up of these patients was 49 months (range: 2-156). A total of 31 patients had disease progression or relapse during follow-up, including four patients with such event more than five years after initial treatment. The 5-year PFS was 73.9%, 70.6% and 85.9%, whereas the 10-year PFS was 69.3%, 59.2% and 79.3%, among patients with complete resection of neoplasm, patients in the watchful waiting group and patients with further treatment, respectively. Patients with further treatment had longer PFS, compared with patients in the watchful waiting group (P=0.011), while no difference in PFS was noted between patients with further treatment and patients with complete resection of neoplasm (P=0.127). Bilateral involvement at diagnosis was associated with significantly inferior PFS (P=0.029), whereas age, IPI score, or TNM staging was not associated with PFS. No serious adverse reaction was reported among patients with further treatment. Image: Summary/Conclusion: Bilateral involvement was associated with poor prognosis. Among patients with residual lesions after surgery, further treatment was associated with improved survival. Patients with OAML might experience disease progression or relapse more than five years after initial treatment. P1154: STUDY ZILO-301: A PHASE 3, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED, MULTICENTER STUDY OF ZILOVERTAMAB PLUS IBRUTINIB VS IBRUTINIB IN PATIENTS WITH RELAPSED OR REFRACTORY MANTLE CELL LYMPHOMA S. Yazji1,*, S. Hamburger1, Y. Wang1, S. Yavrom1, A. Pietrofeso1, R. Bliss2, J. B. Breitmeyer1, M. Dreyling3, M. Wang4 1Oncternal Therapeutics, Inc., San Diego, CA; 2Veristat, LLC, Southborough, MA, United States of America; 3University of Munich, Munich, Bavaria, Germany; 4Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America Background: Zilovertamab (ZILO) is a humanized monoclonal antibody that inhibits the tumor promoting activity of ROR1 and has demonstrated additive/synergistic activity with anti-cancer agents including ibrutinib (Ibr). In the Phase 1/2 Study CIRM-0001, ZILO in combination with Ibr was well tolerated and demonstrated promising efficacy in heavily pretreated, relapsed and refractory (R/R) patients (pts) with mantle cell lymphoma (MCL). As of 1 Oct 2021, 26 evaluable pts were enrolled, the objective response rate (ORR) was 80.8 % of which 34.6% achieved complete response (CR), with a median progression free survival (mPFS) of 35.9 months (mos). The rationale for the novel enrichment design (Figure 1) of Study ZILO-301 is (1) there is an unmet medical need for pts with R/R MCL that only achieve a partial response (PR) or have stable disease (SD) on Ibr, (2) there is an early opportunity for marketing authorization based upon an accepted surrogate endpoint (namely ORR), and (3) a confirmatory study for regular approval is built into the study design based on PFS. The timeframe from initial accelerated marketing authorization to regular approval is shorter than historical approaches for this patient population. Additionally, in Study ZILO-302, the safety and efficacy of ZILO rescue therapy can be evaluated in pts that progress early on Ibr in Study ZILO-301. Aims: To evaluate the efficacy and safety of ZILO plus Ibr vs Ibr in R/R MCL pts. Methods: Study ZILO-301 is a Phase 3 randomized, double-blind, placebo-controlled, multicenter study under which pts with R/R MCL will initially receive single agent Ibr (560 mg daily). After 4 mos, pts who only have PR or SD will be randomized (1:1) to receive an infusion of ZILO or matching placebo (Pbo) and continue to receive Ibr. Study ZILO-301 key inclusion criteria: Aged ≥18 years, confirmed diagnosis of MCL, received ≥ 1 prior treatment for MCL (other than Bruton’s tyrosine kinase inhibitor), have measurable disease, and signed informed consent. Key exclusion criteria: History or current CNS involvement, significant cardiovascular disease, prior stem cell transplant requiring immunosuppressive therapy, or graft versus host disease. Efficacy assessments will be evaluated by a Blinded Independent Central Review Committee utilizing Lugano Classification (Cheson 2014). Results: Three analysis timepoints are planned. Analysis 1 is a futility assessment focused on ORR. Analysis 2 is an analysis for potential early marketing authorization (using accelerated assessment pathways by FDA and EMA), based upon ORR, duration of response (DOR), and safety. This analysis is expected to occur approximately 2 years after the first pt is enrolled in Study ZILO-301. Regardless of the Analysis 2 result, the study will proceed without modification. Analysis 3 is the end of study analysis for the primary endpoint of PFS and secondary endpoints of CR rate, duration of CR, time to CR on therapy, OS, proportion of pts experiencing Grade ≥ 3 neutrophil or platelet decrease, and overall safety. Regular marketing approvals would be sought based on Analysis 3. Figure 1 provides a schematic and subject flow for Study ZILO-301. Study ZILO-302 data may also support additional labeling for the combination of ZILO plus Ibr. Image: Summary/Conclusion: Study ZILO-301, a phase 3, multinational, double-blind, placebo-controlled study is open for enrollment in June 2022. P1155: BREXUCABTAGENE AUTOLEUCEL FOR RELAPSED/REFRACTORY MANTLE CELL LYMPHOMA IN ROUTINE PRACTICE: UPDATED REPORT FROM THE US LYMPHOMA CAR T CONSORTIUM Y. Wang1,*, P. Jain2, F. Locke3, M. Maurer1, M. Frank4, J. Munoz5, S. Dahiya6, A. Beitinjaneh7, M. Jacobs8, J. Mcguirk9, J. Vose10, A. Goy11, C. Andreadis12, B. Hill13, K. Dorritie14, O. Oluwole15, A. Deol16, B. Shah3, J. Paludo1, T. Wang7, R. Banerjee12, S. Neelapu2, D. Miklos4, A. Rapoport6, L. Lekakis7, A. Ghobadi8, Y. Lin1, M. Wang2, M. Jain3 1Mayo Clinic, Rochester; 2The University of Texas MD Anderson Cancer Center, Houston; 3Moffitt Cancer Center, Tampa; 4Stanford University Medical Center, Stanford; 5Mayo Clinic, Phoenix; 6University of Maryland School of Medicine, Greenebaum Comprehensive Cancer Center, Baltimore; 7University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami; 8Washington University School of Medicine, Siteman Cancer Center, St Louis; 9University of Kansas Medical Center, Kansas City; 10University of Nebraska Medical Center, Buffett Cancer Center, Omaha; 11John Theurer Cancer Center, Hackensack Meridian Health, Hackensack; 12University of California San Francisco, San Francisco; 13Cleveland Clinic, Cleveland; 14UPMC Hillman Cancer Center, Pittsburg; 15Vanderbilt-Ingram Cancer Center, Nashville; 16Wayne State University, Karmanos Cancer Institute, Detroit, United States of America Background: The ZUMA-2 trial and real-world studies showed high efficacy of brexucabtagene autoleucel (brexu-cel) in relapsed/refractory mantle cell lymphoma (MCL). We previously reported an objective response rate (ORR) of 89% in 95 patients receiving standard-of-care brexu-cel in the US Lymphoma CAR T Consortium (Wang et al, ASH 2021). Aims: To update the safety and efficacy results of the Consortium study with more patients and longer follow-up. Methods: Patients who underwent leukapheresis for standard-of-care brexu-cel manufacture at one of the 16 Consortium centers between 8/1/2020 and 12/31/2021 were included. Baseline characteristics, bridging therapy, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and treatment outcome data were collected. Eligibility for ZUMA-2 was retrospectively determined based on characteristics at leukapheresis. Time-to-event data were analyzed using the Kaplan-Meier method. Results: At the data cut-off date of 1/31/2022, 189 patients underwent leukapheresis; 167 (88%) completed infusion and 22 (12%) did not (manufacture failure n=7, death n=6, disease progression n=5, organ dysfunction n=2, complete response [CR] to bridging therapy n=1, patient decline n=1). The median age of the 167 infused patients was 67 years (range 34-89) and 76% were male. 16% had high risk simplified MIPI, 57% had Ki-67 ≥50%, 41% had blastoid or pleomorphic variant, 49% had TP53 alteration, 31% had complex karyotype, 14% had bulky disease (≥10 cm), and 10% had CNS involvement. The median prior lines of therapy was 3 (range 1-10), and 86% were BTKi-exposed (89% of which were refractory). 130 (78%) patients would not have met ZUMA-2 eligibility criteria, and the most common reasons were prior therapies (eg, anthracycline- or bendamustine-naïve [16%], BTKi-naïve [14%], >5 lines of prior therapy [11%]), renal dysfunction (21%), cardiac comorbidities (14%), cytopenias (11%), ECOG PS ≥2 (11%), and CNS involvement (10%). 113 (68%) patients received bridging therapy, which included immunochemotherapy (n=45), BTKi (n=45), venetoclax (n=24), radiotherapy (n=23), corticosteroid (n=18), and lenalidomide (n=6). The median time from leukapheresis to lymphodepletion chemotherapy was 28 days (range 17-140). The rate of CRS was 90% (8% grade ≥3, 1 grade 5), and the rate of ICANS was 61% (32% grade ≥3, 0 grade 5). Medications used to manage CRS and/or ICANS included tocilizumab (76%), corticosteroid (68%), anakinra (16%), and siltuximab (3%). 20% of patients required ICU admission, with a median stay of 3 days (range 1-12); 18% required vasopressors, 3% required mechanical ventilation, and 3% required dialysis. Among 159 evaluable patients, the day 30 ORR was 89%, with 70% CR and 19% partial response (PR). With continued follow-up, the best ORR was 89%, with 80% CR and 9% PR. The best ORR/CR rate were 89%/77% for high Ki-67% (≥50%), 88%/79% for blastoid or pleomorphic variant, 88%/73% for complex karyotype, 90%/72% for TP53 altered, 81%/75% for CNS involved, 89%/79% for BTKi-exposed, 91%/83% for BTKi-naïve, and 89%/78% for ZUMA-2 ineligible. With a median follow-up of 5.6 months (range 0.2-15.3), the 6-month estimates for duration of response, progression-free survival, and overall survival were 67% (95% CI 57-75), 63% (95% CI 54-71) and 85% (95% CI 77-90), respectively. Summary/Conclusion: This updated analysis demonstrated favorable safety and efficacy results of brexu-cel in R/R MCL that are consistent with the initial report. Despite 78% of patients being ineligible for ZUMA-2, the responses, CRS, ICANS and survival outcomes were comparable to ZUMA-2 data. P1156: MAGNIFY PHASE 3B STUDY OF LENALIDOMIDE + RITUXIMAB (R2) FOLLOWED BY MAINTENANCE IN RELAPSED/REFRACTORY INDOLENT NON-HODGKIN LYMPHOMA: COMPLETE INDUCTION PHASE ANALYSIS F. Lansigan1,*, D. J. Andorsky2, M. Coleman3, A. Yacoub4, J. M. Melear5, S. R. Fanning6, K. S. Kolibaba7, C. Reynolds8, G. S. Nowakowski9, M. Gharibo10, J. R. Ahn10, J. Li10, M. J. Rummel11, J. P. Sharman12 1Dartmouth–Hitchcock Medical Center, Lebanon, NH; 2Rocky Mountain Cancer Centers, US Oncology Research, Boulder, CO; 3Clinical Research Alliance Inc, Weill Cornell Medicine, New York, NY; 4University of Kansas Cancer Center, Westwood, KS; 5Texas Oncology — Austin, US Oncology Research, Austin, TX; 6Prisma Health, US Oncology Research, Greenville, SC; 7US Oncology Research, Vancouver, WA; 8IHA Hematology Oncology Consultants — Ann Arbor, Ypsilanti, MI; 9Mayo Clinic, Rochester, MN; 10Bristol Myers Squibb, Princeton, NJ, United States of America; 11Justus-Liebig-Universität, Giessen, Germany; 12Willamette Valley Cancer Institute and Research Center, US Oncology Research, Eugene, OR, United States of America Background: Patients with relapsed indolent NHL (iNHL) have limited standard treatment options. Lenalidomide combined with rituximab (R2) has shown complimentary clinical activity and is a tolerable regimen in both untreated and relapsed or refractory (R/R) patients with iNHL (RELEVANCE: N Engl J Med 2018;379:934 and AUGMENT: J Clin Oncol. 2019;37:1188). Aims: These analyses examine the MAGNIFY interim primary endpoint of overall response rate (ORR; 1999 IWG) for induction R2 in efficacy-evaluable patients receiving ≥ 1 treatment and who have available baseline and post-baseline assessments. Methods: MAGNIFY is a multicenter, phase 3b trial in patients with R/R follicular lymphoma (FL) grades 1–3b, transformed FL (tFL), marginal zone lymphoma (MZL), or mantle cell lymphoma (MCL; NCT01996865) exploring optimal lenalidomide duration. In the induction phase, lenalidomide 20 mg PO on days 1–21 of a 28-day cycle + rituximab IV at 375 mg/m2/week cycle 1 and then every 8 weeks starting with cycle 3 (R2) are administered for 12 cycles. Patients with stable disease, partial response, or complete response/complete response unconfirmed (CR/CRu) were randomized 1:1 to R2 vs rituximab maintenance for 18 months. Data presented here are the complete analysis from the induction phase in efficacy-evaluable patients with FL grades 1–3a or MZL (FL grade 3b, tFL, and MCL not included). Results: As of March 5, 2021, 394 patients (318 [81%] FL gr1–3a; 76 [19%] MZL) were enrolled. The median follow-up was 40.6 mo (range, 0.6–79.6). Median age was 66 y (range, 35–91), 328 (83%) had stage III/IV disease, with a median of 2 prior therapies (94% prior rituximab-containing). ORR was 71% (n = 279) with 42% (n = 164) CR/CRu (Table). All patients have completed R2 induction (n = 232, 59%) or discontinued study treatment (n = 162, 41%). 141 patients (36%) prematurely discontinued both lenalidomide and rituximab, primarily due to adverse events (AEs) (n = 54, 14%) or progressive disease (n = 42, 11%). The majority of patients who have completed induction have been randomized and entered maintenance (n = 217). Median duration of response in the induction period was not reached (95% CI, 43.9 mo–NR), and median progression-free survival in the induction safety population (n = 393) was 50.5 mo (95% CI, 39.5–NR). Efficacy results are reported in the table by histology subgroups (FL vs MZL), and rituximab-refractory, double-refractory, and early relapse statuses. Most common all-grade treatment emergent AEs (TEAEs) were 47% fatigue, 43% neutropenia, 37% diarrhea, 30% nausea, and 30% constipation. Grade 3/4 AEs occurring in ≥ 5% of patients included 37% neutropenia (10 patients [3%] had febrile neutropenia), 8% leukopenia, 6% thrombocytopenia, 5% anemia, and 5% fatigue. TEAEs led to discontinuation of lenalidomide in 19% of patients and rituximab in 12% of patients; reduction or interruption of lenalidomide in 64% of patients; and to interruption of rituximab in 30% of patients (dose reduction for rituximab was not allowed). Neutropenia was the most common TEAE leading to lenalidomide discontinuation in 6% and reduction/interruption in 32%, and rituximab discontinuation in 3%. Infusion-related reaction was the most common TEAE leading to rituximab interruption in 8%. Image: Summary/Conclusion: These data represent complete analysis of all patients in the induction phase of MAGNIFY which continue to support that R2 is active with a tolerable safety profile in patients with R/R FL grade 1–3a and MZL, including rituximab-refractory, double-refractory, and early relapse patients. P1157: REAL-WORLD TREATMENT PATTERNS AND COMPARATIVE EFFECTIVENESS OF BRUTON TYROSINE KINASE INHIBITORS IN PATIENTS WITH MANTLE CELL LYMPHOMA B. Shah1,*, K. Yang2, A. Klink3, T. Liu2, T. Zimmerman2, A. Gajra3, B. Tang2 1H. Lee Moffitt Cancer Center & Research Institute, Tampa; 2Beigene USA, San Mateo; 3Cardinal Health, Dublin, United States of America Background: Bruton tyrosine kinase inhibitor (BTKi) therapies, approved for relapsed or refractory (R/R) mantle cell lymphoma (MCL), have not been comprehensively evaluated in real world populations. Aims: This study aimed to assess patient characteristics, treatment patterns and associated outcomes in real world BTKi-treated MCL patients. Methods: The retrospective multicenter chart review was conducted in the Cardinal Health Oncology Provider Extended Network. EMR data were extracted for eligible patients diagnosed with MCL who initiated any of the approved BTKi (ibrutinib, acalabrutinib, zanubrutinib) from 2018 to 2021; patients enrolled in trials were excluded. Index date was defined as the use of any of the BTKis. Patients were required to have 12-month pre-index for medical history, and from index to last follow-up or death. Descriptive analyses were conducted to assess demographic/clinical characteristics, MCL baseline features, BTKi treatment patterns, adverse events (AE), and response rates by BTKi. Multivariable logistic regression was performed to assess factors associated with response and AE. Results: The study cohort consisted of 300 MCL patients (59% male; 69% white); most (64%) patients were covered by Medicare, 34% had commercial insurance. BTKis were given mainly as monotherapy (93%) and in R/R setting (86%). Patients in zanubrutinib group were significantly older (n = 100, median age = 71, range = 50-91) than patients in ibrutinib (n = 100, median age = 69, range = 39-87) and acalabrutinib (n = 100, median age = 70, range = 51-86) groups. Significantly fewer patients in the zanubrutinib group had baseline Ann Arbor stage I-II (4%) than ibrutinib (10%) or acalabrutinib (13%), while more zanubrutinib patients had presence of B symptoms (67%) than ibrutinib (44%) or acalabrutinib (57%). Patients in the zanubrutinib group also had significantly less with ECOG of 3+ (4%) compared to ibrutinib (8%) or acalabrutinib (6%). At BTKi initiation, significantly more patients in zanubrutinib group (18%) had history of atrial fibrillation than ibrutinib (1%) or acalabrutinib (5%). BTKis were given mainly as second-line (86%) and as monotherapy (93%). Most patients were started at an on-label BTKi dose. In zanubrutinib patients, the dose of 160 mg BID was more commonly administered (64%) than 320 mg QD (31%). Multivariable regression reported a significant association of age, gender, extranodal/splenic involvement, and timing of BTKi initiation with response and AE (Table). Image: Summary/Conclusion: This study provides the first real world evidence on comparative effectiveness of ibrutinib, acalabrutinib, and zanubrutinib in MCL patients. While patients treated with zanubrutinib were older and had more complex MCL baseline features at initiation, multivariable regression suggested a trend favoring zanubrutinib over ibrutinib or acalabrutinib for both response and AE. Frontline initiation of BTKi therapy was also associated with improved tolerability. Future real world studies are needed to discern long-term outcomes. P1158: REAL-WORLD TREATMENT PATTERNS AND ECONOMIC BURDEN OF PATIENTS WITH MARGINAL ZONE LYMPHOMA K. Yang1, T. Liu1, B. Tang1, B. Shah2,* 1Beigene USA, San Mateo; 2H. Lee Moffitt Cancer Center & Research Institute, Tampa, United States of America Background: Marginal zone lymphoma (MZL) is an indolent non-Hodgkin lymphoma that is treatable, yet incurable with a remitting and relapsing disease course. Given its disease rarity and underlying heterogeneity, MZL remains understudied with limited real-world evidence on how current treatment pattern conform to clinical guidelines, and the economic outcomes associated with current treatments. Aims: This study aimed to assess real-world treatment patterns, costs, and healthcare resource utilization (HRU) in US MZL patients. Methods: A retrospective, observational study was conducted using the IBM MarketScan® commercial and Medicare supplemental claims dataset (2017-2020). Newly diagnosed adult MZL patients (≥18 years) continuously enrolled 6 month pre- and 3-month post-index date, defined as the first diagnosis date, were included. Treatment regimens were identified by line of therapy and mutually exclusively categorized as rituximab monotherapy (R-mono), bendamustine + rituximab (BR), rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), ibrutinib, or other. Descriptive analyses were conducted to assess patient sociodemographic and clinical characteristics, and treatment utilization patterns including the frequency, duration, and discontinuation of each treatment regimen. Costs and HRU assessed included inpatient, outpatient, and pharmacy visits per-patient-per-month (PPPM). Treatment regimens, costs, and hospitalizations were examined overall, and by line of therapy. Multivariable logistic regression was conducted to examine predictors of costs and HRU. Results: Among the 2491 newly-diagnosed MZL patients (median age = 63 years; 52% male), 59% were commercially insured (median age = 57 years) and 41% in Medicare (median age = 76 years). The most common comorbidities were hypertension (44%), diabetes (17%), atrial fibrillation (AF; 16%), and gastroesophageal reflux disease (15%). Mean time from diagnosis to treatment initiation was 223 days. A total of 1,781 (72%) patients received first-line (1L), 518 (29%) patients received second-line (2L) and 239 (13%) patients received third-line (3L) therapies. R-mono was the most common regimen across both commercial and Medicare patients and all treatment lines (Table). R-CHOP and BR were the second most used regimen in 1L with decreased use in 2L+. Ibrutinib was used more in 2L+ setting but had the lowest 1L PPPM cost (median $2958.9) than other regimens. Overall MZL patients had PPPM 4.6 outpatient visits, 0.5 hospitalization, and mean length of stay of 2.6 days. Total PPPM healthcare cost was $19,895.8. Multivariable regression showed that baseline comorbidities (AF, renal disease, neutropenia) and treatment discontinuation were significant predictors of higher costs and HRU. Image: Summary/Conclusion: This real-world data suggested that the overall US MZL real-world treatment pattern across lines of therapy follows the regimen recommendations by the National Comprehensive Cancer Network clinical practice guidelines and that MZL patients incur high economic burden. Future studies are needed to evaluate long-term outcomes and the impact of heterogenous MZL subtypes. P1159: A MULTICENTER, INTERNATIONAL COLLABORATIVE STUDY EVALUATING FRONTLINE THERAPY WITH BENDAMUSTINE RITUXIMAB FOR WALDENSTRÖM MACROGLOBULINEMIA S. Zanwar1,*, J. Abeykoon1, J. Castillo2, E. Durot3, E. Kastritis4, E. Uppal5, P. Morel6, R. Tawfiq7, L. Montes8, J. Paludo1, S. Sarosiek2, S. Kumar1, O. Ogunbiyi9, P. Cornillet-Lefebvre10, R. Kyle1, A. Delmer3, M. Gertz1, M. Dimopoulos11, S. Ansell1, S. Treon2, S. D’Sa12, P. Kapoor1 1Hematology, Mayo Clinic, Rochester, Rochester; 2Hematology, Dana Farber Cancer Institute, Boston, United States of America; 3Hematology, University Hospital of Reims and UFR Médecine, Reims, France; 4Department of Clinical Therapeutics, National and Kapodistrian University of Athens, Athens, Greece; 5Hematology, University College London Hospitals (UCLH) NHS Foundation Trust, London, United Kingdom; 6Hematology, Hôpital Schaffner, Lens, France; 7Internal Medicine, Mayo Clinic, Rochester, Rochester, United States of America; 8Service d’Hématologie Biologique, CHU Amiens, Amiens, France; 9Hematology, Imperial College of London, London, United Kingdom; 10Laboratory of Hematology, University Hospital of Reims and UFR Médecine, Reims, France; 11Department of Clinical Therapeutics, National and Kapodistrian University of Athens, Athens, Greece; 12Hematology, University College London Hospitals, London, United Kingdom Background: Bendamustine rituximab (BR) is a frequently used chemoimmunotherapy for indolent lymphomas, including Waldenström macroglobulinemia (WM), a rare B-cell malignancy. The promising results of a small subset analysis (n= 41) of the Study group indolent Lymphomas (StiL) trial, demonstrating a median progression-free survival (PFS) of 69.5 months in the frontline setting served as the basis for widespread adoption of BR. Whether the more recently identified somatic mutations within the MYD88 and CXCR4 genes impact the outcomes of patients (pts) treated with BR remains less clear. Aims: To study a large cohort of BR-treated pts with active WM through an international, multicenter collaborative effort. Methods: Records of pts with newly diagnosed active WM who received BR between January 2012 and July 2021, in the US and Europe, were reviewed. The MYD88 L265P and CXCR4 mutation status were captured, if available. All time-to-event analyses were performed from the frontline therapy initiation, using the Kaplan-Meier method. Results: Among 248 pts who were treated with BR, 208 pts received BR induction without rituximab maintenance, and were included in the primary analysis. The median age at treatment initiation was 65 (range 40-86) years; 64 % were males. The baseline characteristics are outlined in Table 1. The median follow-up was 4 (95% CI: 3.6-4.6) years. The estimated median PFS was 5.9 years [95% CI: 5.3-not reached (NR)]. The estimated 5-year overall survival (OS) rate was 90%. Among 174 pts evaluable for response, the overall response rate (ORR), major response rate (MRR) and very good partial response (VGPR) rates were 95%, 93% and 31%, respectively, per the modified IWWM-6 criteria based on serum IgM level alone. Pts with progression of disease (POD) within 24 months of BR therapy (11%) had an inferior subsequent survival compared to those without POD within 24 months, the reference group [5-year subsequent survival rate, 71 % versus (vs) 86%, p=0.02]. Among 131 (63%) pts with a known MYD88 L265P status, 88% (n=116) had MYD88 L265P genotype. The 4-year PFS was 71% for both pts with MYD88 L265P and MYD88 WT genotypes (p=0.44), Figure 1A. The VGPR rates were also comparable between the two groups (41% for MYD88 L265P and 50% for MYD88 WT genotypes, p=0.55). Among 42 (20%) pts with a known CXCR4 mutation status, 28% harbored a CXCR4 mutation. The ≥VGPR rate for pts with CXCR4 MUT genotype was numerically lower; 33% vs 57% for those with CXCR4 WT genotype, p=0.3. A trend towards shorter PFS among pts with CXCR4 MUT genotype [estimated median PFS for 3.9 years (95% CI: 0.8-NR) vs 5.5 years (95% CI 5.3-NR) for pts with CXCR4 WT genotype, p=0.05, Figure 1B] was observed. The PFS rates for pts without a known MYD88 or CXCR4 mutational status was comparable to their counterparts with known genotypes, respectively. Among 40 (16%) pts who had received rituximab maintenance following BR, the median age at initiation of therapy was 67 years (range 44-88). After 1:1 matching for age, the 4-year PFS for the rituximab maintenance group was 89% vs 73% for pts who did not receive rituximab maintenance (p=0.09); OS was comparable (5-year OS rate, 85% for both groups, p=0.99). Image: Summary/Conclusion: Fixed-duration BR is a highly effective regimen for pts with previously untreated symptomatic WM, irrespective of the MYD88 L265P mutation status, although early POD, within 2 years of initiation of BR, is associated with inferior subsequent survival. Our preliminary analysis, suggesting that the presence of CXCR4 mutation confers resistance to BR, warrants confirmation in prospective studies. P1160: RESULTS OF A PHASE 2 EXPANDED ACCESS STUDY OF ZANUBRUTINIB IN PATIENTS WITH WALDENSTRÖM MACROGLOBULINEMIA J. J. Castillo1,*, E. C. Kingsley2, M. Narang3, H. A. Yimer4, C. A. Dasanu5, J. M. Melear6, M. Coleman7, C. M. Farber8, M. Gupta9, J. Shulman10, E. H. Mantovani11, X. Zhang11, A. Cohen11, J. Huang11 1Dana-Farber Cancer Institute, Boston, MA; 2Comprehensive Cancer Centers of Nevada, Las Vegas, NV; 3US Oncology Research, Maryland Hematology Oncology, Columbia, MD; 4Texas Oncology, US Oncology Research, Tyler, TX; 5Lucy Curci Cancer Center, Eisenhower Health, Rancho Mirage, CA; 6US Oncology Research, Texas Oncology, Austin Midtown, Austin, TX; 7Clinical Research Alliance, New York, NY; 8Atlantic Hematology Oncology, Morristown Medical Center, Morristown, NJ; 9Ridley-Tree Cancer Center at Sansum Clinic, Santa Barbara, CA; 10Icahn School of Medicine at Mount Sinai, New York, NY; 11BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA, United States of America Background: Bruton tyrosine kinase (BTK) inhibition is an emerging standard of care for Waldenström macroglobulinemia (WM). Zanubrutinib (ZANU; BGB-3111) is a next-generation BTK inhibitor designed to maximize BTK occupancy and minimize off-target inhibition of TEC- and EGFR-family kinases. ZANU was recently approved by the United States (US) Food and Drug Administration, Health Canada, and the European Union at a dose of 320 mg once daily (QD) or 160 mg twice daily (BID) for the treatment of adult patients (pts) with WM. BGB-3111-216 (NCT04052854) is a single-arm, expanded access study of ZANU for treatment-naïve (TN) pts who are unsuitable for standard chemoimmunotherapy or pts with relapsed refractory (R/R) WM. Aims: To provide real-world experience with ZANU in pts with WM. Methods: Eligible pts with TN or R/R WM were assigned to receive ZANU at a dose of 320 mg QD or 160 mg BID. The primary endpoint was the number of pts enrolled/treated and the number of enrolling sites. Secondary endpoints of safety and efficacy included treatment emergent adverse events (TEAEs) of special interest, disease response (overall response rate [ORR] and very good partial response or better [VGPR+]), progression-free survival (PFS), and overall survival (OS). Response was evaluated by investigator-assessment according to the 6th International Workshop on WM (Br J Haematol. 2013;160(2):171-6) every 6 months at minimum. The study was closed by the sponsor in July 2021 and active pts were transitioned to commercial ZANU via a patient assistance program. Results: Fifty pts with WM (17 TN; 33 R/R), were enrolled from December 2019 to June 2021 across 10 academic and community medical centers in the US. At study entry, median age was 72 years, 54% had intermediate-risk disease, 40% had high-risk disease, and median number of prior therapies for R/R pts was 2. Median treatment exposure was 9.2 months (range 1.4 to 20.0). Thirty-eight (76%) pts had ≥1 TEAE, and 36 (72%) experienced ≥1 TEAE of special interest (Table). Grade ≥3 TEAEs of special interest included hypertension (8%), infection (8%), atrial fibrillation or flutter (2%), neutropenia (2%), and second primary malignancy (2%). No new safety signals were observed. In the 41 pts with ≥1 response evaluation, 39.0% achieved a best overall response (BOR) of VGPR (16; 95% CI, 24.2-55.5). ORR was 85.4% (35; 95% CI, 70.8-94.4), and major response rate was 73.2% (30; 95% CI, 57.1-85.8). Of the 4 pts who achieved a BOR of progressive disease, 3 had IgM values that met partial response criteria before the first 6 months response assessment. PFS and OS were immature due to short follow-up, and the median was not met. Image: Summary/Conclusion: The results of this real-world expanded access study were consistent with the established ZANU profile in WM and other B-cell malignancies when administered as monotherapy at a daily dose of 320 mg orally (either as 160 mg BID or 320 mg QD) in pts with intermediate or high-risk R/R or TN WM. P1161: ASPEN: LONG-TERM FOLLOW-UP RESULTS OF A PHASE 3 RANDOMIZED TRIAL OF ZANUBRUTINIB (ZANU) VS IBRUTINIB (IBR) IN PATIENTS (PTS) WITH WALDENSTRÖM MACROGLOBULINEMIA (WM) M. Dimopoulos1,*, S. Opat2, S. D’Sa3, W. Jurczak4, H.-P. Lee5, G. Cull6, R. G. Owen7, P. Marlton8, B. E. Wahlin9, R. Garcia-Sanz10, H. McCarthy11, S. Mulligan12, A. Tedeschi13, J. J. Castillo14, J. Czyz15, C. Fernandez De Larrea Rodriguez16, D. Belada17, E. Libby18, J. Matous19, M. Motta20, T. Siddiqi21, M. Tani22, M. Trneny23, M. Minnema24, C. Buske25, V. Leblond26, S. P. Treon14, J. Trotman27, W. Y. Chan28, J. Schneider28, H. Allewelt28, A. Cohen28, J. Huang28, C. S. Tam29 1National and Kapodistrian University of Athens, Athens, Greece; 2Monash Health and Monash University, Clayton, Victoria, Australia; 3Centre for Waldenström’s Macroglobulinemia and Associated Disorders, University College London Hospital Foundation Trust, London, United Kingdom; 4Maria Sklodowska-Curie National Institute of Oncology, Krakow, Poland; 5Flinders Medical Centre, Adelaide, SA; 6Sir Charles Gairdner Hospital, University of Western Australia, Perth, WA, Australia; 7St. James University Hospital, Leeds, United Kingdom; 8Princess Alexandra Hospital, University of Queensland, Brisbane, Queensland, Australia; 9Karolinska Universitetssjukhuset and Karolinska Institutet, Stockholm, Sweden; 10Hospital Universitario de Salamanca, Salamanca, Spain; 11Royal Bournemouth and Christchurch Hospital, Bournemouth, United Kingdom; 12Royal North Shore Hospital, Sydney, New South Wales, Australia; 13ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 14Dana-Farber Cancer Institute, Boston, MA, United States of America; 15Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, Poland; 16Hospital Clinic de Barcelona, Barcelona, Spain; 17FN Hradec Kralove, Hradec Králové, Czechia; 18University of Washington/Seattle Cancer Care Alliance - Clinical Research, Seattle, WA; 19Colorado Blood Cancer Institute, Denver, CA, United States of America; 20AO Spedali Civili di Brescia, Lombardia, Italy; 21City of Hope National Medical Center, Duarte, CA, United States of America; 22Ospedale Civile S.Maria delle Croci, AUSL Ravenna, Italy; 23Vseobecna fakultni nemocnice v Praze, Prague, Czechia; 24University Medical Center Utrecht, Utrecht, Netherlands; 25CCC Ulm - Universitätsklinikum Ulm, Ulm, Baden-Württemberg, Germany; 26Sorbonne University, Pitié Salpêtrière Hospital, Paris, France; 27Concord Repatriation General Hospital, Sydney, New South Wales, Australia; 28BeiGene USA, Inc., San Mateo, CA, United States of America; 29Royal Melbourne Hospital, Parkville, Victoria, Australia Background: ZANU is a potent and selective next-generation Bruton tyrosine kinase inhibitor (BTKi) designed to have greater affinity to BTK while minimizing off-target inhibition of TEC-and EGFR-family kinases. ASPEN (NCT03053440) is a randomized, open-label, phase 3 study comparing ZANU with the first-generation BTKi IBR in pts with WM. We present data with a median follow-up of 43 months. Aims: To compare the efficacy and safety of ZANU vs IBR in pts with MYD88 mutant (MYD88 mut) WM and ZANU in pts with wild-type MYD88 (MYD88 wt) WM. Methods: Pts with MYD88 mut WM were assigned to cohort 1 and randomized 1:1 to receive ZANU 160 mg twice daily or IBR 420 mg once daily. Pts with MYD88 wt were assigned to cohort 2 and received ZANU 160 mg twice daily until disease progression. Randomization was stratified by CXCR4 mutational status by Sanger sequencing and lines of prior therapy (0, 1-3, or >3). All pts gave informed consent. The primary endpoint was proportion of pts achieving very good partial response or better (VGPR + complete response [CR]). Primary analysis occurred at 19 months median follow-up, and final analysis is planned to occur ~4 years after the first pt enrolled. Results: A total of 201 pts (102 ZANU; 99 IBR) were enrolled in cohort 1 and 28 pts in cohort 2. Baseline characteristics in cohort 1 differed between pts treated with ZANU vs IBR in CXCR4 mutations by next-generation sequencing (32% vs 20%, or 33 of 98 vs 20 of 92 available samples, respectively) and pts aged >75 years (33% vs 22%, respectively). Median duration of treatment was 42 months (ZANU) and 41 months (IBR), with 67% and 58% remaining on treatment, respectively. The VGPR+CR rate by investigator was 36% with ZANU vs 22% with IBR (descriptive p = 0.02) in cohort 1, and 31% in cohort 2. One pt in cohort 2 obtained a CR. In pts with wild-type (65 ZANU; 72 IBR) or mutant CXCR4 (33 ZANU; 20 IBR) from cohort 1, VGPR+CR rates with ZANU vs IBR were 45% vs 28% (p = 0.04) and 21% vs 5% (p = 0.15), respectively. Median progression-free survival and overall survival were not reached. Consistent with less off-target inhibition, rates of atrial fibrillation, diarrhea, hypertension, localized infection, hemorrhage, muscle spasms, pneumonia, and adverse events (AEs) leading to discontinuation or death were lower with ZANU vs IBR (Table); neutropenia (including grade ≥3) was the only AE of interest that was higher with ZANU (33.7%) vs IBR (19.4%). Rate of grade ≥3 infection was lower with ZANU (20.8%) vs IBR (27.6%). AE incidence with ZANU was similar across cohorts 1 and 2. Annual prevalence analysis of cohort 1 AEs showed reduced prevalence of hemorrhage over time and lower prevalence with ZANU vs IBR at all intervals. In pts treated with ZANU, neutropenia and infection prevalence decreased over time. Prevalence of infection was lower in pts treated with ZANU vs IBR, and neutropenia was similar between arms (8.8% vs 9.7%, respectively) at >24-36 months of treatment. Prevalence of atrial fibrillation remained ≤5% and hypertension remained stable with ZANU, each with a lower prevalence at all intervals vs an increasing trend seen with IBR. Consistently, exposure-adjusted incidence rates of atrial fibrillation/flutter and hypertension were lower with ZANU vs IBR (0.2 vs 0.8 and 0.5 vs 1.0 persons per 100 person-months, respectively; p < 0.05). Image: Summary/Conclusion: ASPEN is the largest phase 3 trial with head-to-head BTKi comparison in WM. At a median follow-up of 43 months, ZANU was associated with a higher VGPR+CR rate and demonstrated clinically meaningful advantages in long-term safety and tolerability vs IBR. P1162: ZANUBRUTINIB IN OLDER PATIENTS (PTS) WITH RELAPSED/REFRACTORY (R/R) MARGINAL ZONE LYMPHOMA (MZL): SUBGROUP ANALYSIS OF THE MAGNOLIA STUDY S. Opat1,*, B. Hu2, A. Tedeschi3, K. M. Linton4, P. McKay5, H. Chan6, J. Jin7, M. Sun8, M. Sobieraj-Teague9, P. L. Zinzani10, M. Coleman11, P. Browett12, X. Ke13, C. A. Portell14, C. Thieblemont15, K. Ardeshna16, F. Bijou17, P. Walker18, E. A. Hawkes19, S.-J. Ho20, K.-S. Zhou21, M. Co22, J. Xu22, Z. Liang22, J. Anderson22, C. Tankersley22, J. Huang22, J. Trotman23 1Monash Health and Monash University, Clayton, Victoria, Australia; 2Levine Cancer Institute/Atrium Health, Charlotte, NC, United States of America; 3ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 4The Christie Hospital NHS Foundation Trust, Manchester; 5Beatson West of Scotland Cancer Centre, Glasgow, United Kingdom; 6North Shore Hospital, Auckland, New Zealand; 7The First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang; 8Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China; 9Flinders Medical Centre, Bedford Park, South Australia, Australia; 10Institute of Hematology “Seràgnoli” University of Bologna, Bologna, Italy; 11Clinical Research Alliance/Weill Cornell Medicine, Lake Success, NY, United States of America; 12Auckland City Hospital, Grafton, New Zealand; 13Peking University Third Hospital, Beijing, China; 14University of Virginia Health System, Charlottesville, VA, United States of America; 15APHP, Hôpital Saint-Louis, Hemato-oncology, Paris University Diderot, Paris, France; 16University College London Hospitals, London, United Kingdom; 17Institut Bergonié, Bordeaux, France; 18Peninsula Private Hospital, Frankston, Victoria; 19Box Hill Hospital, Box Hill, Victoria; 20St. George Hospital, Kogarah, New South Wells, Australia; 21Henan Cancer Hospital, Zhengzhou, Henan, China; 22BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA, United States of America; 23Concord Repatriation General Hospital, University of Sydney, Concord, New South Wales, Australia Background: MZL is the second most common lymphoma in older pts. Choosing an optimal treatment can be challenging because of patient- or disease-related risk factors and treatment-related toxicities (Curr Opin Oncol. 2019;31(5):386-393). Zanubrutinib is a potent, irreversible next-generation Bruton tyrosine kinase (BTK) inhibitor designed to maximize BTK occupancy and minimize off-target kinase inhibition, which may improve efficacy outcomes and minimize toxicities, such as cardiac arrythmias and bleeding events. Zanubrutinib received accelerated approval from the United States FDA for the treatment of pts with R/R MZL (Haematologica. 2022;107(1):35-43). Aims: We aim to present a subgroup analysis of efficacy and safety of zanubrutinib in pts aged ≥65 years with R/R MZL enrolled in MAGNOLIA (BGB-3111-214; NCT03846427). Methods: MAGNOLIA is a phase 2, multicenter, single-arm study of adults with R/R MZL who had received ≥1 line of therapy including ≥1 CD20-directed regimen. All were treated with zanubrutinib 160 mg twice daily until disease progression or unacceptable toxicity. Use of long-term antiplatelet and anticoagulation agents was permitted. The primary endpoint was overall response rate (ORR; complete response [CR] and partial response [PR]) determined by an independent review committee (IRC) in accordance with the Lugano classification. Secondary endpoints include ORR by investigator assessment (INV), duration of response (DOR), progression-free survival (PFS), and safety. All pts gave informed consent. Results: As of 18 January 2021, a total of 68 pts were enrolled (Table). Forty (61%) pts were ≥65 years old with a median age of 73 (range, 65-85); 18 pts were ≥75 years old. Median number of prior therapies was 2 (range, 1-6) and 10 (25%) pts were refractory to last therapy. Most pts received prior rituximab + cyclophosphamide + vincristine + prednisone (48%) or bendamustine + rituximab (30%), while 5 (13%) pts received rituximab monotherapy. MZL subtypes included extranodal (n=17, 43%), nodal (n=14, 35%), and splenic (n=8, 20%). Median duration of treatment was 14.4 months (mo; range, 0.9-19.6). At a median follow-up of 15.8 mo (range, 2.8-21.8), ORR by IRC was 75% (CR 25%, PR 50%; Table). Responses were observed in all subtypes, with an ORR of 71%, 86%, and 75% in extranodal, nodal, and splenic subtypes, respectively (CR 41%, 21%, and 0%, respectively). Median DOR and PFS were not reached; 15-month PFS was 87% and 12-month DOR was 93%. Most (63%) pts are continuing zanubrutinib. Treatment discontinuation due to disease progression was 28% by INV. Most common treatment-emergent adverse events (AEs) observed in ≥20% of pts include contusion (28%), diarrhea (25%), and constipation (20%). Grade ≥3 neutropenia occurred in 5% of pts. The most common infection was upper respiratory tract infection (10%). Two (5%) pts discontinued zanubrutinib due to unrelated fatal AEs (COVID-19 pneumonia and myocardial infarction in a patient with pre-existing coronary artery disease). Atrial fibrillation/flutter and hypertension occurred in 2 (5%) pts each and did not lead to treatment discontinuation. No pts required dose reductions, or experienced major or serious hemorrhage. Image: Summary/Conclusion: The safety profile of zanubrutinib observed in older pts was consistent with previously published results (Clin Cancer Res. 2021;27(23):6323-6332). Zanubrutinib was well tolerated and effective, as demonstrated by a high response rate and durable disease control in older pts with R/R MZL. P1163: OUTCOMES OF HIGH-RISK LARGE B-CELL LYMPHOMA TREATED WITH STANDARD OF CARE CHEMOIMMUNOTHERAPY: A NON-RANDOMIZED COMPARISON TO FRONTLINE CAR-T CELL THERAPY O. Albanyan1,*, O. Castaneda-Puglianini1, J. Chavez2 1Blood and Marrow Transplant and Cellular Immunotherapy; 2Department of Malignant Hematology, Moffitt Cancer Center, Tampa, United States of America Background: High-risk LBCL is associated with poor prognosis after standard of care (SOC) first-line anti-CD20 mAb-containing chemoimmunotherapy (CIT), therefore here is a need for novel approaches. ZUMA-12 reported high overall response (ORR) and complete response (CR) rates in high risk LBCL defined as IPI score of ≥3 and/or double or triple-hit status [MYC and BCL2 and/or BCL6 translocations (double-hit/triple-hit lymphoma – DHL/THL)] and positive interim PET scan (iPET+) Aims: Here, we report the outcomes of these high risk LBCL patients (pts) who did not qualify due to a negative (-) iPET or declined or were ineligible to be treated in the ZUMA-12 clinical trial. Methods: Eligible patients were older than 18 and had high-risk LBCL, defined as DHL/THL status and/or an IPI score ≥3. Patients could have had an available iPET (or CT scan). Patients did not pursue ZUMA-12 due to trial ineligibility (i.e., iPET-) or did not want to pursue the trial. Patients continued SOC CIT per discretion of treating physician. The primary endpoint was end of treatment ORR and CR. Secondary endpoints included progression-free survival (PFS), overall survival (OS). Results: During the time period ZUMA-12 was open, 61 pts were approached for the trial, 8 patients were excluded from the final analysis due to incomplete data. The remaining 53 pts did not qualify to ZUMA-12 due to iPET- (n=31, 58.4%), declined (n=10, 18.8%)), ineligible (n= 8, 15.1%) and other (n= 4). Median age was 67 (26 – 78), 11 (20.1 %) had DHL/THL, 46 (86.8 %) had IPI score 3-5. Patients were treated with DA-EPOCH-R (n=11, 21%) and R-CHOP (n=41, 77%). The ORR and CR rates was 83 and 79.2%, respectively in this cohort. Four patients did not complete therapy due to PD (n=3) and death for other causes (n=1). The ORR and CR rates for the ZUMA-12 study were reported at 90 and 80%, respectively. In our study, the median PFS and OS was 27.4 months (95% CI: 23.7 – 31.1) and 30.8 months (95% CI: 27.7 – 33.8), and the 30-months PFS and OS were 60 and 71%, respectively. In comparison, the ZUMA-12 reported a PFS and OS that was not reached. Summary/Conclusion: In this nonrandomized study of high risk LBCL, the efficacy of axi-cel in the ZUMA-12 was numerically higher than SOC CIT. The PFS and OS in our cohort was lower despite majority of patients achieving early CR per iPET. This analysis supports a potential trial of frontline CAR-T versus SOC CIT for high risk LBCL. P1164: IMAGE-BASED DETECTION OF HIGH-GRADE B CELL LYMPHOMAS (DH-L) I. Avivi1,2,*, C. Perry1,2, O. Ahron2,3, N. Hershkovitz4, D. Hershkovitz2,5, A. Avinoam6, I. Gazy6, N. Paz-Yaacov6 1Hematology department, Tel Aviv Sourasky Medical Center; 2Sackler Faculty of Medicine., Tel Aviv University; 3Pathology department; 4Hematology department, Tel Aviv Medical Center; 5Pathology department, Tel Aviv Sourasky Medical Center; 6Imagene AI, Tel Aviv, Israel Background: Aggressive B cell Non-Hodgkin Lymphomas (B-NHL) are divided into two main categories: diffuse large B cell lymphoma (DLBCL) accounting for 90% of cases, and high-grade B-cell lymphoma (HGBL). Diagnosis of high-grade lymphoma with MYC and BCL2 and/or BCL6 rearrangements (double-hit lymphoma-DHL) is confirmed by fluorescence in situ hybridization (FISH) analysis, demonstrating c-MYC-rearrangement in combination with BCL-2 / BCL-6 rearrangement in lymphoma cells. Accurate and rapid diagnosis of DHL is obligatory, when considering more aggressive treatment regimens (other than R-CHOP), suggested in these patients. Aims: To establish a novel tool for diagnosing DLBCL and DHL, directly on the scanned Hematoxylin and Eosin (H&E) biopsy slides, by applying digital imaging technologies supported by machine learning algorithms. Methods: H&E whole slide images, prepared from biopsies obtained mainly from lymph nodes, but also from extra-nodal organs of patients with aggressive B cell lymphoma histology, were collected from the pathology department at Tel-Aviv Medical center (TASMC). Cases were randomly divided into a training (n=43) and a validation (n=35) set. On-the-fly augmentation was applied to images, together with advanced Convolutional Neural Network (CNN) analysis, to generate the aggressive B-NHL classifier (powered by Imagene-AI). Proprietary multiple instance learning (MIL) algorithms and training scheme, based on cases ranking, rather than absolute values, were applied. The classifier was validated through a testing set in a blinded study scheme, and results were compared to the FISH results for c-MYC and BCL2/6 rearrangements (Figure 1A). Results: The classifier training set was composed of 36 DLBCL NOS and 7 DHL cases. The aggressive B-NHL classifier was validated on a cohort of 35 cases, including 14 DHL cases (diagnosed by FISH) and 21 DLBCL cases. The model demonstrated 100% sensitivity and 90.48% specificity, with an accuracy rate of 94.29% and Area Under the Curve (AUC) of 0.99 (Figure 1B). Two cases were concluded as false positive, including one case that demonstrated rearrangements in substantial percentages of cells, just below the required positivity threshold. Image: Summary/Conclusion: Herein, we demonstrate for the first time a machine learning-based genomic testing solution for the detection and classification of aggressive B-NHL, based on H&E stained slide images. Implementation of this solution in clinical practice can support the diagnosis of DHL, which currently has both technical and financial challenges. Applying the proposed accessible and standardized AI-based method, can improve and facilitate the diagnosis of DHL, thereby directly improving patient care. P1165: BETALUTIN® IN PATIENTS WITH RELAPSED/ REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL) NOT ELIGIBLE FOR AUTOLOGOUS STEM CELL TRANSPLANT T. Illidge1,*, M. Beasley2, J. G. Codina3, F. Cavallo4, V. Pascal5, V. Wills6 1Haematology/Oncoloy, The Christie NHS Foundation Trust, Withington; 2Haematology/Oncoloy, Bristol Haematology and Oncology Centre, Bristol, United Kingdom; 3Hospital Universitari i Politècnic La Fe, Hospital Universitari i Politècnic La Fe, Valencia, Valencia, Spain; 4Division of Hematology, Department of Molecular Biotechnology and Health Sciences, University of Torino, University of Torino, A.O.U. Città della Salute e della Scienza di Torino, Turin, Italy; 5Bioanalytics; 6Clinical Sciences, Nordic Nanovector ASA, Oslo, Norway Background: There remains a significant unmet medical need for the treatment of patients with relapsed/refractory DLBCL. Betalutin® (lilotomab labelled with the beta-emitter 177Lu), targets the CD37 antigen which permits delivery of a therapeutic dose of radiation directly to the DNA of tumour cells. Betalutin® is under development for the treatment of relapsed/refractory Non-Hodgkin’s Lymphoma (NHL). Aims: This was an open label Phase I, multi-centre, single ascending dose study of four different treatment regimens of Betalutin® administered after rituximab pre-treatment and lilotomab pre-dosing. The purpose of this study was to define the maximum tolerated dose (MTD) of Betalutin®. Methods: Eligible participants received 375 mg/m2 rituximab on Day -14 and escalating doses of both lilotomab (60 and 100 mg/m2) and Betalutin® (10, 15 and 20 MBq/kg) on Day 0. Participants were followed-up for dose limiting toxicities (DLTs) for up to 12 weeks. Participants were followed-up until disease progression, the start of further anticancer treatment or until withdrawal from the study for any reason. Tumour response was determined using Cheson et al, 2014. Clinical benefit was assessed using the ECOG performance and quality of life (QoL) assessments using the Functional Assessment of Cancer Therapy-Lymphoma (FACT-Lym) scale. Results: A total of 16 participants (8 males and 8 females) with median age 75 years (range: 45 to 94 years) received lilotomab and Betalutin® after pre-treatment with rituximab. Overall ECOG scores at screening was 0 in 5 (31.3%), 1 in 7 (43.8%) and 2 in 4 (25.0%) participants. DLBCL stage at screening was Stage III or IV in 13 (81.3%). Median time since last relapse was 1.3 months, and 11 (68.8%) participants were refractory to the last anti-lymphoma therapy. Cell surface expression of CD37 on tumour cells was demonstrated for all patients before treatment. A single case of DLT (grade 4 neutropenia) was reported in an 83-year-old female at the dose of 100 mg/m2 lilotomab and 20 MBq/kg Betalutin® on day 43; there was no fever or infections associated with neutropenia; the event was treated with filgastrim and resolved after 2 weeks on day 57. A total of 14 (87.5%) participants had at least one treatment emergent adverse event (TEAE). Most events occurred between Days 1-92 and the most commonly reported events (in 4 [25.0%] participants each) were diarrhoea, decrease in lymphocyte count, and decrease in neutrophil count. Grade ≥3 TEAEs were reported in 10 (62.5%) participants, of which only four (25.0%) participants had treatment related events (decrease in lymphocyte count - 2 participants, decrease in neutrophil count - 1 participant and decrease in white blood cell count -1 participant). No SAEs assessed were deemed to be related to lilotomab and Betalutin®, by the investigators. Nine (56.3%) deaths were reported, all were determined to be related to disease progression and none was considered related to the study drugs. Two participants (one each from the 2 highest dose cohorts) had complete responses that lasted 2.8 and 3.0 months, respectively. Summary/Conclusion: Betalutin® is well tolerated in relapsed/ refractory DLBCL and a MTD was not reached in this study. The initial efficacy and safety findings from this study warrant further investigation of Betalutin® as a single agent or in combination with a standard of care treatment in relapsed/refractory DLBCL. References: Cheson et al, J Clin Oncol 2014:32: 3059-67 P1166: REPEAT BIOPSY IN RELAPSED OR REFRACTORY DIFFUSE LARGE B CELL LYMPHOMA: A NATIONWIDE SURVEY AND RETROSPECTIVE STUDY T. Berger1,2,*, K. R. Geiger1,2, M. Yeshurun1,2, A. Gafter-Gvili1,2,3, T. Shochat4, R. Gurion1,2, P. Raanani1,2, O. Pasvolsky1,2 1Hematology division, Davidoff cancer center, Beilinson hospital, Rabin medical center, Petah Tikva; 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; 3Department of Medicine A; 4Bio-Statistical Unit, Rabin Medical Center, Beilinson Campus, Petah-Tikva, Israel Background: Almost half of patients with diffuse large B-cell lymphoma (DLBCL) will have relapsed/refractory (R/R) disease after frontline immunochemotherapy. Although guidelines recommend histological confirmation of R/R disease, repeat biopsies are not always performed. Aims: To better appreciate the extent and reasoning for not performing repeat biopsies in R/R DLBCL. Methods: We conducted a two-part study: (1) using a nationwide electronic questionnaire consisting of 8 clinical vignettes, we evaluated the views of practicing hematologists in Israel regarding the need for repeat biopsies in suspected R/R DLBCL; (2) a single center retrospective study was utilized to describe real-life clinical practice patterns of performing procedures aimed at achieving tissue diagnosis for relapsed/refractory aggressive NHL (aNHL) patients: we included all consecutive adult (≥18 years of age) patients diagnosed with aNHL treated at a tertiary center in Israel, between 1/1/2013 and 1/1/2020, we identified patients who were diagnosed with R/R disease and analyzed data pertaining to utilization of histological confirmation via repeat biopsy. Each timepoint of management decision regarding biopsy performance was considered as a separate “episode of R/R lymphoma”. We extracted data regarding reasons for not performing the biopsies from the computerized system, according to the treating physician. Results: In the survey part, all 64 participating physicians opted not to perform a repeat biopsy in at least one clinical case scenario. Physicians’ age, gender, tenure or clinical experience with DLBCL patients did not correlate with the decision to perform a biopsy, whereas more physicians chose to perform biopsies in relapsed compared to refractory disease cases. In the retrospective part, 116 episodes of R/R aNHL among 61 patients were identified. In 72% of these episodes a repeat biopsy was not performed, mostly due to low likelihood of an alternative diagnosis or problematic location for biopsy. Patients with relapsed disease were more likely to undergo biopsy, as compared to those with refractory disease (47% vs 19%, p=0.002). Focusing on the group of patients with DLBCL (excluding primary CNS lymphoma, primary mediastinal B cell lymphoma and Burkitt lymphoma), there were 43 patients with refractory (n=26) or relapsed (n=17) DLBCL. In 61.4% out of the 83 episodes of R/R disease in these 43 patients, a biopsy was not performed: 74.5% of refractory DLBCL episodes versus 35.7% of relapsed DLBCL episodes (p=0.001). Stratifying biopsy utilization for each patient according to the timing of event, i.e first, second or third event of R/R disease, revealed that less biopsies were performed at more advanced relapse episodes. The most common reason for not performing a repeat biopsy was having stable radiological findings and other diagnoses deemed unlikely, as depicted in Figure 1. Image: Summary/Conclusion: Our study suggests that contrary to guideline recommendations, in clinical practice many patients do not undergo repeat biopsy in R/R DLBCL, especially in refractory cases. Since this is a common dilemma faced in clinical practice, future studies and recommendations should address the necessity of repeat biopsy, according to patient and disease related characteristics. P1168: CHARACTERISTICS PREDICTIVE OF PROGRESSION DURING FRONTLINE TREATMENT IN AGGRESSIVE B-CELL LYMPHOMAS A. Bock1,*, R. Mwangi2, M. Maurer2, J. Cerhan3, W. L. Ng1, R. King4, N. N. Bennani1, S. Ansell1, T. Habermann1, T. Witzig1, Y. Wang1, G. Nowakowski1 1Division of Hematology; 2Division of Biostatistics; 3Division of Epidemiology, Department of Quantitative Health Sciences; 4Department of Pathology, Mayo Clinic, Rochester, United States of America Background: Primary refractory diffuse large B-cell lymphoma (DLBCL) represents 10-15% of DLBCL cases. The subgroup of refractory patients who have no response or progressive disease (PD) by the end of frontline treatment (EOT) is a high-risk group with particularly poor outcomes. It is challenging to identify such patients at diagnosis. Aims: To identify clinical, pathological, and cytogenetic characteristics of patients with progression during frontline treatment. Methods: Adult patients with newly diagnosed DLBCL, including patients with high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (DHL/THL), between 2002 and 2019 and seen at Mayo Clinic Rochester were identified from the prospective Molecular Epidemiology Resource (MER) cohort study of the University of Iowa/Mayo Clinic Lymphoma SPORE. We analyzed patients who had no response or PD during treatment or by EOT (early PD group) and compared to non-early PD (non-ePD) patients (including relapsed patients). We compared demographics, clinical characteristics, cell of origin (COO), and MYC rearrangements using Chi-square analysis. Results: Out of 1,505 patients with DLBCL, there were 66 patients (4.3%) with early PD (N=20 PD/stable disease at interim PET-CT, N=46 PD by EOT). At baseline, 61% were male and the median age was 60. Early PD patients had more advanced stage III-IV (72.7% vs 53.3%), elevated serum lactate dehydrogenase (LDH) (64.0% vs 47.3%), >1 extranodal site involved (36.4% vs 23.3%), and International Prognostic Index (IPI) ≥3 (52.0% vs 33.6%)(all p <0.05 compared to non-ePD). COO distribution was similar between early PD and non-ePD patients. Among patients with complete pathological and cytogenetic data available (early PD, n=47; non-ePD, n=813), DHL or THL was observed in 23.4% of early PD patients compared to 9.2% in non-ePD patients (p=0.001). Of the DHL patients in the early PD group (n=8), MYC/BCL2 rearrangement was present in 87.5%, MYC/BCL6 in 12.5%. Dual Myc/Bcl2 expressor (DEL) was present in 42.2% of early PD patients (n=45) compared to 25.5% in non-ePD patients (n=736)(p=0.013). First-line treatment regimens in the early PD group included R-CHOP (74.2%), R-EPOCH (7.6%), and other immunochemotherapy (IC)(18.2%), which was similar to non-ePD patients. Summary/Conclusion: High-risk characteristics including DHL/THL and DEL are significantly associated with progressive disease during frontline treatment, suggesting that MYC-signaling may mediate chemoresistance. Known high-risk clinical features including advanced stage, elevated LDH, >1 extranodal site, and IPI ≥3 are also associated with early PD. Early identification of patients at high risk for early PD through clinical, pathological, and cytogenetic features is of utmost importance given the availability of clinical trials incorporating novel therapies in both frontline and consolidative approaches. P1169: ELEVATED CRP AT INFUSION OF AXICABTAGENE CILOLEUCEL PREDICTS HIGH-GRADE CAR-T TOXICITY AND OUTPERFORMS MORE COMPLEX SCORES S. Boyle1,*, A. Kuhnl1, M. Correia de Farias1, P. E. Patten1,2, D. Yallop1, R. Benjamin1, V. Potter1, R. Sanderson1 1Dept of Haematology, King’s College Hospital; 2King’s College London, London, United Kingdom Background: Predicting CRS and ICANS prior to CAR-T infusion is desirable as it may help to plan management. Two groups1,2 have recently shown that a modification of the Endothelial Activation and Stress Index (EASIX) score, developed to predict toxicity in bone marrow transplant recipients3, can predict CRS and ICANS severity in CAR-T patients, however these have not been externally validated or are difficult to apply in practice. Aims: We aimed to determine whether the EASIX or related scores could predict CAR-T toxicity in our patients and if not, then to identify other potential predictors from the available clinical information. Methods: We reviewed the medical records of adult patients who received axicabtagene ciloleucel (axi-cel) as standard of care at our centre over a 2yr period, collecting data on timing and severity of CRS and ICANS as well as baseline demographic and biochemical factors. We calculated an EASIX score for all patients and an ‘EASIX-F’ score based on the values provided by Greenbaum et al (EASIX-F1) and our own interquartile values (EASIX-F2). We then performed a linear regression of each of: the EASIX score; EASIX-F1; EASIX-F2; and other variables of interest with maximum grade of CRS, with a plan to perform a multivariate analysis with any significant variables (p<0.05). Results: 67 patients were eligible for analysis. Baseline demographics were similar to those described on the licensing trial4. On univariate analysis (UVA), neither EASIX score (p=0.10), nor either iteration of the EASIX-F score (p= 0.08 and 0.15 respectively), was associated with severity of CRS for our patients. Pre-infusion C-Reactive Protein (D0 CRP) however appeared strongly associated with CRS severity, both on UVA (p=0.001) and by multiple linear regression, where it was the only variable identified that showed any association with CRS severity (p=0.003). Given the strength of the D0 CRP and CRS association, we evaluated the association of D0 CRP with ICANS severity which was significant (p=0.012) and D0 CRP with a combined outcome of ‘high grade CAR-T toxicity’ (HGT), defined as development of either grade 3+ CRS or grade 3+ ICANS, where it was again strongly associated (p<0.001). We then dichotomised D0 CRP to determine whether we could identify a practically useful cut-off to predict HGT. We looked at D0 CRP values of ≥50, ≥100 and ≥150 individually and all cut-offs performed well with increasing likelihood of HGT with higher CRP values. With D0 CRP ≥50, probability of developing HGT was 42% vs 13% (OR 5.09, p=0.007); with CRP ≥100 it was 55% vs 14% (OR 7.2, p=0.006); and with CRP ≥150 it was 100% vs 13% (OR undefined, p<0.001). Finally, given these unexpected findings we performed a prospective review of the 19 axi-cel patients treated from our initial data-cut off to 1 Feb 2022 and found that for these patients, a D0 CRP ≥100 or ≥150 predicted HGT well. HGT incidence was 75% vs 7% (OR 42, Fisher’s p=0.016) for both cut-offs because 3 of the 4 patients with HGT had a CRP of >150 (Table 1). The single patient with HGT and D0 CRP <100 interestingly was a non-responder. For the full 86-patient cohort, D0 CRP ≥150 was the best cut-off, predicting a 90% incidence of HGT vs 12% if CRP <150 (OR 42, p<0.001). Image: Summary/Conclusion: On retrospective analysis of 67 axi-cel patients, D0 CRP was more strongly associated with CRS severity than more complex prognostic scores. D0 CRP level, especially if ≥150, showed strong association with high grade CAR-T toxicity and remained predictive in a 19-patient validation cohort. This has potential implications for management of axi-cel patients. P1170: INCIDENCE, TREATMENT AND OUTCOME OF PATIENTS WITH RICHTER’S SYNDROME: A POPULATION-BASED COHORT STUDY IN THE NETHERLANDS F. Huisman1, D. Al-sarayfi1, M. Bellido1, R. Mous2, J. S. Vermaat3, A. P. Kater4, M. Nijland5, M. Brink6,* 1Department of Hematology, University Medical Center Groningen, University of Groningen, Groningen; 2Department of Hematology, UMC Utrecht Cancer Center, Utrecht; 3Department of Hematology, Leiden University Medical Center, Leiden; 4Department of Hematology, Academic Medical Centre, Amsterdam; 5Department of Hematology, University Medical Center Groningen, Groningen; 6Department of Research and Development, Netherlands Comprehensive Cancer Organisation, Utrecht, Netherlands Background: Richter’s syndrome is a rare aggressive transformation of chronic lymphocytic leukaemia (CLL) into a diffuse large B-cell lymphoma (RS-DLBCL) or Hodgkin lymphoma. Patients with RS-DLBCL have an average overall survival (OS) of 6-12 months. Survival of RS-DLBCL patients previously treated for their CLL is inferior as compared to patients with a previously untreated CLL. In recent years, management of CLL has changed with the introduction of novel agents. Aims: This study evaluates the clinical characteristics, treatment and survival of RS-DLBCL patients in a contemporary era of CLL management in the Netherlands. Methods: DLBCL patients ≥18 years diagnosed in 2014-2020 were identified in the Netherlands Cancer Registry (NCR), with survival follow-up through February 1st, 2021. A prior CLL, diagnosed in 2014-2019, was identified through cross-linkage with the NCR. For these patients, detailed information on CLL and RS-DLBCL treatment was available. Patients with CLL and DLBCL diagnosis within a time interval of 3 months were defined as concurrent RS-DLBCL (concurrent RS). The primary endpoint was overall survival (OS). OS was defined as the time between diagnosis and death from any cause and presented for three subgroups of RS-DLBCL patients, 1) concurrent RS, 2) RS-DLBCL following treated CLL (treated RS), and 3) RS-DLBCL following untreated CLL (untreated RS). Results: A total of 110 patients with RS-DLBCL (median age 72 years, range 43-89 years; 61% males) were selected, including 22 (20%) concurrent RS, 38 (35%) treated RS, and 50 (45%) untreated RS. The majority of patients were diagnosed with stage III/IV RS-DLBCL, i.e., 55% of the concurrent RS were diagnosed with advanced disease as compared to 70% and 79% of the untreated and treated RS, respectively. Median time from CLL to RS diagnosis was for untreated RS 23.2 months (range, 3.4-74.4 months) and for treated RS 17.4 months (range, 4.7-67.5 months). Of the 38 treated RS patients, 30 patients had received rituximab in combination with either chlorambucil (n=11), purine analogues (n=8) or chemotherapeutic regimens such as cyclophosphamide-vincristine-prednisolonie (n=11). Seven patients received treatment including ibrutinib or venetoclax, and 3 patients received treatment not including one or more of the abovementioned regimens. Regarding primary treatment of all RS-DLBCL patients, 19 (86%) out of 22 concurrent RS patients received R-CHO(E)P, 2 patients received another R-based chemotherapy, and 1 patient did not receive therapy. Of the 88 (un)treated RS patients, 59 (67%) received R-CHO(E)P of whom 6 patients were consolidated with autologous stem cell transplantation (SCT) and 2 patients with allogenic SCT. Of the 29 remaining patients, 17 (19%) were treated with a R-based chemotherapy regimen including R-PECC and R-bendamustine, and 12 (14%) patients did not receive therapy. The overall response rate (≥partial remission) of concurrent RS patients was superior as compared to untreated and treated RS patients (86% vs. 70% vs. 55%, respectively; p<0.01). With a median follow-up of 9.5 months (range 0.3-78.3), 3-years OS was 64% for patients with concurrent RS, 53% for untreated RS patients and 5% for treated RS patients (Figure; p<0.01). Image: Summary/Conclusion: In this contemporary population-based study, concurrent RS patients have superior OS, while survival among treated RS patients is very poor. Our data underline the importance of development of new strategies, specifically aiming at treated RS patients. P1171: REFLECT: PROSPECTIVE NON-INTERVENTIONAL STUDY ON THE EFFECTIVENESS AND SAFETY OF SANDOZ RITUXIMAB (SDZ-RTX) WITH CHOP FOR PATIENTS WITH PREVIOUSLY UNTREATED CD20-POSITIVE DIFFUSE LARGE B-CELL LYMPHOMA M. Welslau1,*, B. Kubuschok2, J. Topaly3, B. Otremba4, T. Wolff5, G. Bryn6 1Department of Oncology, Klinikum Aschaffenburg, Aschaffenburg; 2Department of Hematology/Oncology, Augsburg University Medical Center, Augsburg; 3Klinik für Hämatologie und Onkologie, CaritasKlinikum Saarbrücken, Saarbrücken; 4Onkologische Praxis Oldenburg, Oldenburg; 5OncoResearch Lerchenfeld, Hamburg; 6Sandoz, Holzkirchen, Germany Background: Sandoz biosimilar rituximab (SDZ-RTX) was approved in the EU for all the indications of reference rituximab. REFLECT is a real-world, multicenter, open-label, single-arm, non-interventional trial, and is the first prospective, post-approval study of SDZ-RTX in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) as a curative therapy for patients with CD20-positive diffuse large B-cell lymphoma (DLBCL). Aims: The aim of the REFLECT study was to evaluate the 2-year effectiveness and safety of SDZ-RTX as first-line treatment for DLBCL. Methods: Treatment-naïve, CD20-positive adult patients (aged ≥18 years) with DLBCL eligible for therapy with R-CHOP were treated with SDZ-RTX-CHOP every 2 or 3 weeks for 6–8 cycles according to the product label, as per routine clinical practice. Data was collected at enrollment, at every patient contact during the 12-month treatment period, and at one time point ≥30 days after the last dose of R-CHOP. Complete response was defined as the best result, as assessed by the treating physician using the Response Evaluation Criteria in Lymphoma 2017 (RECIL criteria). Kaplan–Meier estimates of progression free survival (PFS) were assessed at 24 months. No imputation for missing data was used; endpoints were summarized descriptively. All patients provided written informed consent prior to study entry. The cut-off for data collection was March 31, 2021. Results: A total of 169 patients (52.1% female, mean [standard deviation] age 67.3 [13.4] years) with DLBCL were included in the full analysis set. At baseline, 19.5% and 24.3% of patients had Ann Arbor disease stage III or IV, respectively, and most patients (80.5%) had Eastern Cooperative Oncology Group performance score of 0 or 1. In terms of International Prognostic Index, 92 (54.5%) patients had a score of ≥2 at baseline. A total of 100 (59.2%) patients completed the 24-month observation period. In total, 110 (65.1%; 95% confidence interval [CI] 57.4–72.3) patients achieved complete response as the best response and 50 (29.6%; 95% CI 22.8–37.1) patients achieved partial response. The overall response rate was 94.7% (95% CI 90.1–97.5). One-year PFS was 84.9% (95% CI 78.2–89.6), while 2-year PFS was 78.5% (95% CI 70.9–84.4) (Figure 1); median PFS was not reached within the observational period. A total of 143 (84.6%) patients experienced at least one adverse event (AE), 53 (31.4%) of which were suspected to be related to the study drug. The most common AEs were anemia (24.3%), fatigue (20.7%), polyneuropathy (17.2%), and nausea (12.4%). Serious AEs were reported in 63 (37.3%) patients, with those suspected to be related to the study drug reported in 11 (6.5%) patients. AEs requiring dose interruption and/or dose change were reported in 24 (14.2%) patients. There were 8 (4.7%) deaths recorded during the study, of which 3 (1.8%) occurred during the on-treatment period. Image: Summary/Conclusion: This real-world, post-approval study reconfirms that first-line treatment of CD20-positive DLBCL with R-CHOP using SDZ-RTX is effective over a 24-month observation period, with no unexpected safety signals. These data may help to broaden patient access to rituximab-based chemotherapy and support the sustainability of cancer care. P1172: DUVELISIB IN PATIENTS WITH RELAPSED/REFRACTORY PERIPHERAL T-CELL LYMPHOMA FROM THE PHASE 2 PRIMO TRIAL: UPDATED EXPANSION PHASE ANALYSIS P. L. Zinzani, MD, PhD, J. Zain, MD2, M. Mead, BS, MD, C. Casulo, MD4, E. D. Jacobsen, MD5, G. Gritti, MD, PhD6, L. Pinter-Brown, MD7, K. Isutzu, MD8, D. Cohan, MD9, M. Daugherty, PharmD10, J. E. Brammer, MD11, N. Mehta-Shah, MD12, B. Pro, MD13, S. M. Horwitz, MD14 1Institute of Hematology “Seràgnoli”, University of Bologna, Bologna, Italy; 2Department of Hematology and Hematopoietic Cell Transplantation, City of Hope Comprehensive Cancer Center, Duarte; 3Department of Medicine, Division of Hematology/Oncology, UCLA Medical Center, Santa Monica; 4University of Rochester Medical Center, James P. Wilmot Cancer Center, Rochester; 5Dana-Farber Cancer Institute, Boston, United States of America; 6Hematology and Bone Marrow Transplant Unit, Ospedale Papa Giovanni XXIII, Bergamo, Italy; 7University of California-Irvine, Irvine, United States of America; 8National Cancer Center Hospital, Tokyo, Japan; 9Secura Bio, Inc.; 10SecuraBio, Inc, Las Vegas; 11Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus; 12Washington University School of Medicine in St. Louis, Saint Louis; 13Columbia University Herbert Irving Comprehensive Cancer Center; 14Memorial Sloan Kettering Cancer Center, New York, United States of America Background: Peripheral T-cell lymphoma (PTCL) is a family of aggressive lymphomas, with a short median overall survival when relapsed or refractory (R/R). Current single-agent therapies for R/R PTCL have modest overall response rates (ORR) of <30%. Use of duvelisib (DUV), an oral dual inhibitor of phosphatidylinositol 3-kinase (PI3K)-δ and PI3K-γ isoforms, in PTCL is as an investigational agent only. Both Phase 1 data and prior interim analyses of this Phase 2, open-label, multi-center, parallel cohort PRIMO Trial (NCT03372057; supported by Secura Bio) demonstrated promising efficacy (ORR ~ 50%) of DUV in patients (pts) with R/R PTCL. Based on interim PRIMO data, DUV was added to the National Comprehensive Cancer Network® T-cell Lymphoma Guidelines® (Version 1.2022, 12/22/21) as a Category 2A other recommended regimen for pts with R/R PTCL of all subtypes. Here we present the latest PRIMO Expansion Phase (EP) data. Aims: The primary objective of the PRIMO EP is to determine the efficacy of DUV given at a recommended dose in pts with R/R PTCL; secondary objectives include additional efficacy measures, safety, and pharmacokinetics. Methods: EP eligibility criteria includes adults with pathologically confirmed PTCL, defined per WHO criteria, after ≥ 2 cycles of 1 prior standard regimen, and a CD4 lymphocyte count of ≥ 50/mm3. Based on the dose optimization results, the dose in the EP is DUV at 75 mg BID for 2 cycles, to maximize disease control, followed by 25 mg BID, to mitigate late toxicities, until progressive disease (PD) or unacceptable toxicity. Pneumocystis jirovecii prophylaxis is required; herpes simplex and varicella zoster virus prophylaxis are strongly recommended. The primary endpoint is ORR by IRC assessment using the Lugano 2014 criteria; efficacy is assessed in all pts that received at least 1 dose of DUV. Results: This updated analysis of the EP included 101 pts (data cutoff Oct 1, 2021); median 10.4 months follow-up from first dose. Pts had a median age of 67.0 years (range, 21-92) with a median of 3 prior lines of therapy (range 1-9). Fourteen pts remain on treatment with 87 pts withdrawn from treatment for: PD (44), clinical deterioration due to PD (4), adverse events (AE; 20), death (5), other (13), and withdrawal of consent (1). The ORR by IRC was 49% with a CR rate of 34%; responses were seen across multiple subtypes (Table 1 Median PFS was 3.6 months, though not yet fully mature. Five pts discontinued therapy to undergo stem cell transplantation. There were 3 treatment-related AEs associated with death (1 each): pneumonitis, Epstein-Barr associated lymphoproliferative disorder, and sepsis. AEs of special interest ≥ Grade 3 (all causality, number of events) were transaminase elevation increased (23), neutropenia (19), infections (10), cutaneous reactions (9), diarrhea (7), pneumonia (2), and pneumonitis (1). There was 1 event of colitis (Grade 1). ALT and/or AST elevations were the most common AEs leading to treatment discontinuations (15). Image: Summary/Conclusion: This updated analysis of 101 patients in the PRIMO EP demonstrated an ORR of 49% and a CR rate of 34%, which compares favorably to currently available single-agent options. Duvelisib was generally manageable with per-protocol dose modifications in this population, showing an AE profile consistent with what has been observed previously and no unexpected or novel toxicities. These data confirm duvelisib to be a promising agent for a disease set with high unmet needs, poor prognoses, and limited effective treatment options. P1173: CLINICAL APPLICABILITY OF TARGETED NEXT-GENERATION SEQUENCING FOR PRECISION MEDICINE IN DIFFUSE LARGE B CELL LYMPHOMA D. Huang1,*, J. Li1, T. Yang1, Y. Nan1, L. Zhang1, Y. Xiang1, Q. Li1, Y. Liu1 1Department of Hematology-Oncology, Chongqing University Cancer Hospital, Chongqing, China Background: The cure rate of diffuse large B-cell lymphoma is only 60-70%, and new treatment methods need to be found. Aims: Comprehensive genomic analysis based on next-generation sequencing (NGS) allows a better molecular characterization of diffuse large B-cell lymphoma, offering a roadmap toward precision medicine. Methods: We analyzed somatic alterations of 37 DLBCL tissue or blood samples by a hybridization capture-based NGS panel targeting 188 lymphoma-related genes. Results: Totally, 279 single-nucleotide variants and small insertions or deletions affecting 103 genes were identified, with at least 1 mutation detected in 100% (37/37) of cases. The most frequently mutated genes were those involved in epigenetic modifiers (DNMT3A, KMT2B and KMT2D), activated signaling (TYK2, MYD88, NOTCH2 and BCR) and transcription factors/transcriptional regulator (RUNX1, SPEN, IRF4, KLF2 and ASXL1). Significant co-occurrences were shown between RUNX1 mutations with NOTCH2, IRF4, ARID1A and BCR (P<0.05, respectively), BCR and IRF4 (P<0.01), etc. Additionally, we analyzed the association of genomic aberrations with clinicopathological features in DLBCL. Patients aged >60 years harbored less LRP1B mutations than those aged ≤60 years (5.3% vs. 33.3%, P< 0.05). The mutation frequency of TP53 was significantly higher in LDH-high (>244 U/L) group than that in LDH-low (≤244 U/L) group (40% vs. 6.25%, P<0.05). Compared with germinal center B-cell (GCB), the mutation frequency of TYK was significantly lower in non-GCB (5% vs. 36.4%, P<0.05). New potential therapeutic targets (CREBBP, EP300, EZH2, SMARCB1, CD79A/B, BTK, CARD11, MYD88, THFAIP3, PIK3CA, PTEN and JAK) were identified in 55% (11/20) of newly diagnosed patients and in 41.2% (7/17) of relapsed/refractory patients. Ibrutinib, as a single agent, has demonstrated limited activity in DLBCL with mutant MYD88 and wildtype CD79. In this study, we identified 3 patients with mutant MYD88 and wildtype CD79A/B (8.1%), among whom 2 harbored MYD88 L265P variants and 1 had MYD88 S219C mutation. Summary/Conclusion: The utility of mutational profiling may facilitate the identification of potential drug targets, which in turns may represent new therapeutic possibilities. P1174: THE COMBINATION OF QUALITATIVE AND QUANTITATIVE METABOLIC PARAMETERS PREDICTS PROGRESSION-FREE SURVIVAL IN PATIENTS WITH LARGE B-CELL LYMPHOMAS (LBCL) TREATED WITH ANTI-CD19 CAR-T CELLS. A. Dodero1,*, A. Guidetti1, A. Lorenzoni2, S. Pizzamiglio3, G. Argiroffi2, P. Verderio3, A. Chiappella1, F. Bagnoli4, C. Carniti1, M. Pennisi1, A. Alessi2, P. Corradini1 1Hematology; 2Nuclear Medicine; 3Unit of Bioinformatics and Biostatistics, Fondazione IRCCS Istituto Nazionale dei Tumori; 4Hematology, Policlinico di Milano, Ospedale Maggiore Ca’ Granda, Milano, Italy Background: Autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is an effective therapy for 40% of patients (pts) with relapsed/refractory (R/R) LBCL. Qualitative and quantitative metabolic parameters from the 18-FDG PET-CT scan are tools able to predict outcomes in lymphomas, but their value in the CAR-T setting is still under investigation. Aims: Prognostic role of PET-CT performed before lymphodepletion and 1 month after CAR-T cell infusion, analyzed by qualitative and quantitative parameters Methods: We conducted an observational a prospective study on pts affected by R/R aggressive lymphomas receiving axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel). PET-CT was evaluated before lymphodepletion, at 1 month (M1) and at 3 months (M3) post-infusion using visual assessment with a 5-point Deauville score (DS). Quantitative PET parameters were evaluated with PET-VCAR software (GE Healthcare). The contour of pathological lesions was delineated using semiautomatic contouring systems including a gradient-based algorithm. SUVmax, SUVmean, Metabolic tumor volume (MTV), and Total lesions glycolysis (TLG) were automatically recorded. Total MTV (TMTV) and total TLG (TTLG) were calculated as the sum of all individual lesions. The percentage change in FDG uptake of each parameter between baseline and M1 was defined as Δ=(baseline-M1)/baseline x 100%. Logistic and Cox regression models were applied to evaluate the association between PET parameters at baseline, at M1 and in terms of Δ with respect to clinical response and progression free survival (PFS), respectively. Results: 47 pts were analyzed: median age was 55 years (range, 22-70); histologies included diffuse-large B-cell lymphomas (n=33) and primary mediastinal B-cell lymphomas (n=14); pts received treatment with axi-cel (n=31) or tisa-cel (n=16). Before infusion, 18 (38%) had high LDH, 14 (30%) had extranodal disease and 16 (34%) had bulky disease. Majority of pts [n=41 (84%)] received bridging therapy. At a median follow-up of 12 months (range, 3-29 months), 38 patients are alive and 9 died of disease progression with an estimated 1-year PFS and OS of 46% (95%CI; 30%-60%) and 83% (95%CI; 66%-92%), respectively. Baseline values of SUVmax, but not SUVmean, TTLG, TMTV correlated with poor clinical response [HR 1,49; (95%CI; 1,01-2,2), p=0,04]. By univariate logistic model, a higher ΔSUVmean between baseline and M1 was associated with better response at M3 [odds ratio 3,49, (95%CI; 1,25-9,77), p=0.02]. Univariable Cox analyses showed that a higher ΔSUVmean and ΔTMTV between baseline and M1 were associated with a better PFS [HR 0,63, (95%CI; 0,41-0,96), p=0,03; HR 0,67 (95%CI; 0,48-0,94), p=0,02], respectively. In terms of visual assessment, pts with DS 4-5 at M1 had a significantly higher risk of failure as compared to those with DS 1-3 [HR 2,92, (95%CI; 1,12-7,64), p=0,02]. A combination of M1 DS score and ΔSUV mean showed a better prognosis for patients with DS1-3 (PFS 60%), an intermediate prognosis for those with DS4/5 and high ΔSUVmean (PFS 49%) and a poor prognosis for those with DS4/5 and low ΔSUVmean (PFS 22%), [p=0,009]. In a multivariable analyses, evaluating clinical prognostic factors and PET parameters, a higher risk of failure was observed in pts with high LDH [HR 3,2 (95%CI; 1,30-8,14), p=0,01] whereas a higher ΔSUVmean was associated to a better PFS [HR 0,61 (95%CI, 0,41-0,92), p=0,02]. Summary/Conclusion: Assessment of DS combined with variation of SUVmean at M1 provides informations about early risk of CAR-T failure. A validation group are needed to extend and confirm our observations. P1175: REAL LIFE OUTCOMES OF RELAPSED/REFRACTORY DIFFUSE LARGE B CELL LYMPHOMA: A SINGLE CENTRE EXPERIENCE I. Dogliotti1,*, M. Clerico1, F. Vassallo2, S. Ragaini1, V. Peri1, B. Botto2, C. Consoli1, L. Orsucci2, R. Freilone2, S. Ferrero1, A. Lonardo1, G. De Luca1, D. Ferrero1, F. Cavallo1 1Division of Hematology 1U, Department of Biotechnology and Health Sciences, University of Torino; 2Division of Hematology 2, University Hospital AOU Città della Salute e della Scienza, Torino, Italy Background: Nearly 40% diffuse large B cell lymphoma (DLBCL) patients relapse after first line treatment (Crump, Blood 2017) and around 15% are chemorefractory (Feugier, JCO 2005). Despite modern treatments availability, outcome of these patients remains suboptimal (Crump, Blood 2017). Aims: With the observational retrospective study STRIDER, we aimed at determining real-life outcome of relapsed/refractory (R/R) DLBCL patients. Methods: Patients were selected based on local diagnosis of DLBCL between Jan 2010 and Dec 2019 within Hematology Units at the University Hospital Città della Salute e della Scienza, Torino, Italy. R/R disease was documented by biopsy, imaging, or clinical evaluation; refractoriness was defined as reappearance or progression of DLBCL within 12 months from initial diagnosis. Demographics, laboratory, imaging, pathological and clinical data were collected retrospectively from hospital health records. Results: Overall, 413 DLBCL patients aged >18 years old were included. 151 (36%) patients had an event of relapse (n=51 patients, 12%) or refractory disease (n=100, 24%). Median age was 67 (range 21-89) years for non R/R patients (NRR) vs 72 (19-89) for R/R group (p <0.001). Advanced stage (stage 3-4: 68% vs 87%, p <0.001), baseline B symptoms (28 vs 43%, p <0.001), higher LDH level (p <0.001) and >1 extranodal site involvement (39 vs 53%, p =0.007) were linked to R/R disease, and consequently, IPI score highly affected R/R disease distribution. Kidney or adrenal involvement was less frequent in the NRR group (6% vs 11%, p =0.042). Central nervous system (CNS) involvement was present at diagnosis in only 8/413 (2%) patients, while median CNS-IPI score was 2 vs 3 for the 2 groups (p <0.001). Expression of immunohistochemical markers was not significantly different between groups. Treatment intent was curative (R-CHOP/COMP, R-Magrath, R-DA-EPOCH, MATRIX) for 391/413 (95%) patients; R/R group included reduced dose or palliative intent treatments (11 vs 2%, p <0.001). For CNS prophylaxis, intrathecal methotrexate was used in 78/413 (19%) patients, while intravenous in 22 (5%) patients. After first line completion, 99/413 (24%) patients received consolidative radiotherapy on previous bulky areas, while 4 (1%) received upfront autologous stem cell transplantation (ASCT). Interim response was not different, but end of treatment response was inferior for R/R group (p <0.001). After a median follow up of 50 months, median overall survival (OS) from initial diagnosis was NR for NRR patients, 38 months (IQR 25-64) for relapsed, 11 (5-20) for refractory patients (Figure 1). Median progression free survival (PFS) was 21 (IQR 16-30) months for relapsed vs 6 (4-9) for refractory patients. Treatments performed in II line were platinum-based (rituximab + OxDHA/DHAP/ICE/GemOx) for 50 patients (22 relapsed, 28 refractory), lenalidomide for 17 patients (5 vs 12); 14 patients were enrolled in clinical trials. Palliative intent treatments (oral chemotherapy +/- rituximab) were administered to 17 patients. II line consolidation with RT was performed in 11, ASCT consolidation in 6 patients. After II line, best response was CR in 29%, PR in 17% (ORR 45%); 45 patients received a third line, 10 more than three. Image: Summary/Conclusion: Our retrospective series confirmed good survival for NRR patients, but 36% patients were R/R after first line, with refractory patients showing a dismal outcome despite intensive treatments. Survival analyses after II line and multivariate analysis are ongoing and will be presented at the meeting. P1176: RITUXIMAB IN PRIMARY CNS LYMPHOMA – LONG TERM FOLLOW-UP OF THE PHASE III HOVON 105/ALLG NHL 24 STUDY J. Doorduijn1,*, S. Issa2, B. van der Holt3, M. Minnema4, T. Seute5, M. Durian6, G. Cull7, M. van der Poel8, W. Stevens9, J. Zijlstra10, M. Nijland11, K. Mason12, A. Beeker13, D. Brandsma14, M. van den Bent15, M. Gonzalez16, D. de Jong17, J. Bromberg15 1Hematology, Erasmus MC Cancer Institute Rotterdam, Rotterdam, Netherlands; 2Hematology, Middlemore Hospital, Auckland, New Zealand; 3HOVON Data Center, Erasmus MC, Rotterdam; 4Hematology; 5neurology, University Medical Center Utrecht, Utrecht; 6Hematology, ETZ Hospital, Tilburg, Netherlands; 7Hematology, Sir Charles Gairdner Hospital and PathWest Laboratory Medicine, Nedlands, Australia; 8Hematology, University Medical Center, Maastricht; 9Hematology, Radboud University Medical Center, Nijmegen; 10Hematology, Amsterdam UMC, Amsterdam; 11Hematology, University Medical Center Groningen, Groningen, Netherlands; 12Hematology, Royal Melbourne Hospital, Melbourne, Australia; 13Hematology, Spaarne Gasthuis, Haarlem; 14neurology, Netherlands Cancer Institute, Amsterdam; 15neurology, Erasmus MC Cancer Institute Rotterdam, Rotterdam, Netherlands; 16pathology, Royal Melbourne Hospital, Melbourne, Australia; 17pathology, Amsterdam UMC, Amsterdam, Netherlands Background: The efficacy of rituximab in Primary CNS Lymphoma (PCNSL) is still under debate. We performed an international randomized phase III study to investigate the efficacy of rituximab when added to methotrexate, BCNU, teniposide and prednisolone (MBVP) in PCNSL. The primary endpoint, event-free survival (EFS) at one year, was similar in both treatment groups and was previously reported (Bromberg et al, Lancet Oncology 2019; 20: 216-228). Aims: Here we present long-term follow up results with a median follow-up of 82 months. Methods: Between August 2010 and May 2016 200 newly-diagnosed, non-immunocompromised patients with PCNSL aged 18-70 years and WHO performance status 0-3 were randomized between treatment with MBVP chemotherapy with (arm B) or without (arm A) rituximab. The rituximab was given weekly in the first MBVP cycle, fortnightly in the second (in total 6 rituximab administrations). Responsive patients received consolidation with high-dose cytarabine, and patients aged ≤ 60 were subsequently treated with low-dose WBRT if in CR/CRu; in case of PR with an additional boost on the tumor. Patients > 60 were not irradiated. All patients gave written informed consent. Results: The modified intention-to-treat (m-ITT) population consisted of 199 eligible patients, 55% were men. The median age was 61 yrs (range 26-70), the median WHO performance status 1 (range 0-3). The primary endpoint EFS at one year was 49% (95% CI 39-58)(MBVP) vs 55% (95% CI 44-64) (R-MBVP). The EFS at 5 years was 25% (17-34) vs 36% (27-46) respectively, hazard ratio (HR) 0.85, 95% CI 0.61-1.18, p=0.33 (adjusted for age and WHO performance status). The progression-free survival (PFS) at one and 5 years were 58% (47-67) and 29% (21-39) (MBVP) and 65% (54-73) and 43% (33-53) (R-MBVP)) (HR 0.73, 95% CI 0.52-1.02, p=0.07). 80 patients were still alive. Overall survival (OS) at 5 years for MBVP and R-MBVP was 49% (39-59) and 53% (43-63) respectively. The median OS was 57 months (95% CI 38-75) in the MBVP arm and 85 months (95% CI 43–104) in the R-MBVP arm (HR 0.87, 95% CI = 0.61-1.26, p=0.47). A total of 111 patients had progression or relapse, 63 after MBVP and 48 after R-MBVP. 79% of these patients received further treatment. The median OS after progression/relapse was 9.7 months (5.9-19.9) in the MBVP arm, and 6.1 months (2.4-13.1) in the R-MBVP arm (HR 1.25, 95% CI 0.83-1.87, p=0.29). 119 patients died, 64 in the MBVP arm and 55 in the R-MBVP arm. Causes of death were PCNSL in 69% of the patients (both arms), complication of treatment (6% vs 5%), secondary malignancy (5% vs 2%) and other or unknown causes (20% vs 24%). Age was the strongest prognostic factor for EFS, PFS and OS in multivariate analysis. An unplanned subgroup analysis by age (≦60 vs > 60 yrs) showed a significant different EFS at one year for younger patients with R-MBVP, not found in patients > 60 yrs (Bromberg et al, 2019). With longer follow up the 5-year EFS in the younger group was 28% (16-41)(MBVP) vs 55% (40-68) (R-MBVP), HR 0.48, 95% CI 0.28-0.81, p=0.006. 5-year PFS was 31% (18-44) versus 57% (42-70), HR 0.47, 95% CI 0.27-0.81, p=0.007. The OS at 5 years was 54% (39-68) vs 70% (55-81) respectively (HR 0.65, 95% CI 0.36-1.19, p=0.16). Image: Summary/Conclusion: In the modified-ITT population we found no statistically significant benefit of the addition of rituximab to MBVP on EFS, PFS and OS in patients with PCNSL, even after a long follow-up of median 82 months. Therefore, the results of this study do not support the use of rituximab with MBVP in the treatment of primary CNS lymphoma. P1177: PROGNOSTIC VALUE OF TP53 VARIANTS IN CHILDREN WITH MATURE B-CELL LYMPHOMA TREATED ACCORDING TO REDUCED-INTENSITY B-NHL-2010M PROTOCOL V. Egor1,*, A. Yuliya1, K. Brenning1, A. Itov1, D. Abramov2, A. Sharlai2, M. Senchenko2, D. Konovalov2, G. Novichkova1, N. Miakova1 1Oncohematology; 2Pathology, Dmitry Rogachev National Medical Research Center Of Pediatric Hematology, Oncology and Immunology, Moscow, Russia Background: Reduced-intensity rituximab-based therapy leads to long-term remission in more than 80-85% of pediatric patients with B-cell non-Hodgkin lymphoma (B-NHL), but in case of relapse or refractoriness the prognosis in poor with less then 20% survival rate. Therefore, the identification of adverse prognostic factors is critically important. Recent data show that the status of the TP53 gene may be of prognostic value in children with B-NHL. We evaluated the clinical significance of TP53 abnormalities in children with B-NHL enrolled in the Russian-Belorussian B-NHL-2010mab multicenter clinical trial. Aims: To investigate the prognostic value of TP53 gene status in pediatric B-NHL receiving reduced-intensity therapy Methods: Due to reference status of pathology department in Dmitry Rogachev National Medical Research Center for Pediatric Hematology, Oncology, and Immunology, samples from 79 pediatric B-NHL patients (77 with Burkitt lymphoma and 2 with diffuse large B-cell lymphoma) treated according to B-NHL-2010mab protocol in 2012-2021 were included in this study. The median age was 9 years (range 2-17 years), m:f ratio was 6:1. 19 patients (24%) had limited stage and 60 patients (76%) had advanced-stage of disease. DNA was extracted from FFPE tissue, TP53 variants were detected using Sanger sequencing of exons 5 to 8 according to the International Agency for Research on Cancer (IARC) protocol. B-NHL samples were obtained following informed consent from participants or their parents/guardians. Results: Median follow up was 16 months (range 5-117 months). The 5-year event free and overall survival for all patients was 86,3% (95%CI=78, 3-94%) and 86,5% (95%CI=78,3-94,8%) respectively. DNA suitable for sequencing was obtained from 66 samples. TP53 pathogenic variants were present in 26% of cases and were associated with a significantly inferior event free and overall survival compared to those without a TP53 abnormality: 45,3% (95%CI = 21-69,6%) vs 97,9% (95%CI=93,9-100%) (p < 0.001) and 47,1% (95%CI=21,8-72,4%) vs 97,9% (95%CI= 93,9-100%) respectively (p = 0.001). 13 patients with unknown TP53 status are alive without relapsed of disease. Summary/Conclusion: Our findings show the independed prognostic value of TP53 pathogenic variants in pediatric B-NHL patients treated with rituximab-based reduced-intensity therapy. Therefore, given the poor survival rate of patients with TP53 mutations, this marker can be used to define high risk groups within pediatric B-NHL, those who need new therapeutic approaches to achieve better results. It is necessary to study this prognostic factor in large groups of patients. P1178: BRENTUXIMAB VEDOTIN IN COMBINATION WITH LENALIDOMIDE AND RITUXIMAB IN PATIENTS WITH RELAPSED/REFRACTORY DLBCL: SAFETY AND EFFICACY RESULTS FROM THE SAFETY RUN-IN PERIOD OF THE PHASE 3 ECHELON-3 STUDY N. L. Bartlett1,*, C. A. Yasenchak2, K. K. Ashraf3, W. N. Harwin4, J. Orcutt5, P. Kuriakose6, P. L. Zinzani7,8, A. Mamidipalli9, K. Fenton9, C. Glenn9, G. Nowakowski10 1Washington University School of Medicine, St. Louis, MO; 2Willamette Valley Cancer Institute and Research Center/US Oncology Research, Eugene, OR; 3Hem-Onc Assocs, Birmingham, AL; 4Florida Cancer Specialists and Research Institute, Fort Myers, FL; 5Charleston Hematology Oncology Associates, Charleston, SC; 6Henry Ford Health System, Detroit, MI, United States of America; 7Institute of Hematology “L. e A. Seràgnoli”, University of Bologna; 8Department of Specialized Medicine, Diagnostic and Experimental, University of Bologna, Bologna, Italy; 9Seagen Inc., Bothell, WA; 10Division of Hematology, Mayo Clinic, Rochester, MN, United States of America Background: Patients (pts) with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) who relapse after or are ineligible for hematopoietic stem cell transplant (HSCT) or chimeric antigen receptor T cell (CAR-T) therapy generally have poor outcomes. Preclinical data provide the rationale for combining brentuximab vedotin (BV), lenalidomide (len), and rituximab for the treatment of R/R DLBCL. BV+len showed promising clinical activity in a Phase 1 trial with a 57% overall response rate (ORR), 10.2-month median progression-free survival, and 14.3-month overall survival (Ward 2021). Aims: We report the results of the open-label safety run-in that was conducted prior to the randomized portion of the study. Methods: ECHELON-3 (NCT04404283) is a randomized, double-blind, placebo-controlled, active-comparator, multicenter Phase 3 study. Prior to randomization, an open-label safety run-in was conducted; pts received BV (1.2 mg/kg) and rituximab (375 mg/m2) both q3w, and len 20 mg qd. Pts must have received ≥2 prior lines of therapy and be previously treated with or were ineligible for HSCT or CAR-T, ECOG score ≤2, and PET-avid, bidimensional measurable disease (>1.5 cm by CT). Pts with negligible CD30 expression (<1%) were eligible. Response was assessed by the investigator according to the Lugano Classification Revised Staging System (Cheson 2014). Results: 10 pts with R/R DLBCL were enrolled. Median age was 70.5 years, 7 pts were male, and all had an ECOG ≤1. Median prior lines of therapy was 3 (range, 2 to 6); no pts received prior HSCT and 6 pts received prior CAR-T. At a median follow-up of 6.9 months (range, 2.3 to 14.1), the most common treatment emergent adverse events (TEAE) were fatigue (n=5), anemia (n=4) and constipation (n=4). Grade ≥3 events were experienced by 8 pts, most commonly anemia and pneumonia (n=3 each), and neutropenia and thrombocytopenia (n=2 each). Serious adverse events were observed in 7 pts. Seven pts each had BV and rituximab dose modifications, and 6 pts had a len dose modification. The most common reasons for any dose modification were anemia (n=3), neutropenia, peripheral neuropathy, or pneumonia (n=2 each). 4 pts discontinued treatment due to an AE, 2 of which were treatment related (n=1 each of Grade 2 fatigue; Grade 3 anemia). There was 1 death due to a TEAE that was not treatment related. The ORR (best response) was 70%, including 5 pts with a complete metabolic response and 2 pts with a partial metabolic response; 3 pts had progressive disease. Responses were seen in both CD30 (+) and (-) pts, as well as in 4 pts who received prior CAR-T. Summary/Conclusion: This novel triplet regimen appears active in R/R DLBCL with an acceptable safety profile. The randomized portion of the study is currently enrolling. P1179: EFFECTIVENESS OF CENTRAL NERVOUS SYSTEM PROPHYLAXIS STRATEGIES IN DIFFUSE LARGE B-CELL LYMPHOMA F. Z. Eraslan1, T. Toptas2,*, T. Tuglular2, I. Atagunduz2 1Internal Medicine; 2Hematology, Marmara University School of Medicine Hospital, İSTANBUL, Turkey Background: Central nervous system recurrence is seen in 5% of patients diagnosed with DLBCL. CNS relapse is associated with survival limited to months. Therefore, determining the patients who are at high risk of CNS relapse and selecting the most efficient prophylactic approach is one of the mainstays of treatment. IV-MTX and IT-MTX are widely used in CNS prophylaxis. Nevertheless, the efficiency of IV-MTX and IT-MTX in preventing CNS relapse was not proven in large study groups or in prospective studies. Aims: Our study aims to verify the efficiency of current CNS prophylaxis in preventing CNS relapse in DLBCL patients. Additionally, association of CNS relapse with survival and the correlation of CNS-IPI score, age, albumin, origin of cell, and DHL/DEL status with cumulative CNS relapse incidence. Methods: The data from 190 patients were analyzed retrospectively. Among the 89 patients who had been classified as high-risk group for CNS relapse, seven were administered IT-MTX, 17 received IV-MTX, and remaining 65 patients received no prophylaxis. Results: Median age was 57(18-88) and male to female ratio was almost equal (male: 48.8%). At the time of diagnosis, 66.8% of the patients were defined to have advanced stage disease. All of the cohort included 89 patients (46.8%) who were at high risk for CNS relapse. Cumulative CNS relapse risk at one-year was estimated as 12.2%, 9.5%, 4.5% for patients received IV-MTX, IT-MTX and no prophylactic treatment, respectively. Two-year cumulative CNS relapse risk was 24.5%, 12.2%, 6.0% for patients who received IV-MTX, IT-MTX and no prophylaxis, respectively (p=0.008). The frequency of DEL or DHL were higher in patients who received IV-MTX (41.1%). CNS relapse rate was 25% and 50% for patients who did not receive prophylaxis and who did, respectively. In patients with DEL, CNS relapse rate (33.3%) did not differ for those who received prophylaxis and those who did not. In our study, we found overall CNS relapse incidence 6.8% in DLBCL. Median time to CNS relapse was 12 months in all cohort. Median overall survival after occurrence of CNS relapse was two (95% CI: 0-4.9) months. We found that the patients who had high CNS-IPI score were at six times greater risk of CNS relapse than those with low and intermediate CNS-IPI score (9.0% and 1.5% after 12 months, 11.6% and 2% after 24 months) (p=0.03). Despite the univariate analyses revealed a statistically significant association, multivariate analysis has shown that albumin level is not an independent predictor for CNS relapse. Additionally, we have shown that CNS-IPI score, DHL/DEL status are strong predictors of CNS relapse in DLBCL patients. Image: Summary/Conclusion: Independent predictors of CNS relapse in DLBCL are DHL/DEL status and CNS-IPI score. No predictive relationship was proven to exist between CNS relapse and gender, albumin levels, cell of origin classification by Hans-algorithm. Patients with CNS relapse had poor prognosis (median survival two months). IV-MTX prophylaxis has been shown to have no preventive effect on CNS relapse in high risk DLBCL population. Due to this data, significant changes in prophylactic approach against CNS relapse in high risk DLBCL patients is required. P1180: ARE EXTRANODAL LIMITED-STAGE DIFFUSE LARGE B-CELL LYMPHOMA MORE AGRESSIVE THAN NODAL FORMS? C. Fernandes1,*, S. Alves1, F. Moita1, I. Dlouhy1, S. Esteves2, M. Gomes da Silva1 1Hematology; 2Clinical Research Unit, Instituto Português de Oncologia de Lisboa Francisco Gentil, Lisboa, Portugal Background: Limited-stage diffuse large B-cell lymphoma (DLBCL) is associated with favourable outcomes with overall survival of 70-80% at 10 years. Stage-modified IPI (sm-IPI) predicts the risk of relapse and death based on clinical factors that do not include extranodal (EN) involvement, although in some studies it has been associated with worse outcomes. Aims: To analyse the characteristics and outcomes of limited-stage DLBCL in a single center series and evaluate the prognostic impact of EN involvement. Methods: We conducted a retrospective analysis of consecutive patients with newly diagnosed stage I-II DLBCL treated with curative intent at a single centre from 2015 to 2019. Primary central nervous system (CNS) lymphoma, primary testicular lymphoma, primary mediastinal B-cell lymphoma and transformed lymphoma were excluded. Response to therapy was assessed according to Lugano criteria. Overall (OS) and progression free survival (PFS) were calculated by Kaplan-Meier method and comparisons done by the log rank test. Cumulative incidence of relapse was calculated using competing risk models and Gray test for group comparisons. Cox regression was used to identify prognostic factors for PFS. Results: Of 152 patients identified, 53 (35%) had EN disease and 99 (65%) were nodal (N). The most common EN sites were stomach (30%), sinus/nasal cavity (13%), skin and soft tissue (9%) and breast (9%). Seventy-two (47%) had low risk sm-IPI (45 N; 27 EN). Clinical characteristics were similar between both groups, except for stage (stage I in 50.9% of EN vs 32.3% of N cases) and bulky disease (EN 15.1% vs N 37.4%). All patients received R-CHOP (median 6 cycles; range 1-8). Eight patients, all with EN involvement (3 sinus/nasal cavity, 2 breast, 2 bone and 1 orbit), received CNS prophylaxis. Seventeen out of 152 patients received additional radiotherapy (RT), 71% of whom had nodal involvement; 16/17 were irradiated due to bulky disease and/or treatment with abbreviated immunochemotherapy. Globally, 89% patients had a complete response (CR) to treatment: 85% in the N and 96% in the EN group (p=0.212). Nine patients relapsed at a median of 18 (7-52) months after obtaining CR (6 from the N and 3 from the EN group). The 4-year cumulative incidence of relapse was similar: 11.2% (95%CI 9.0-13.9%) in EN and 6.7% (95%CI 5.9-7.5%) in N group (p=0.778). No gastric lymphomas relapsed. The most common sites of relapse were lymph nodes (3), soft tissue (3) and CNS (2). Nineteen (14 N; 5 EN) patients died at a median of 12 (0-47) months after starting treatment, mostly (52.6%) due to lymphoma progression. With a median follow-up of 43 months, 4-year OS and PFS were 87% and 82%, respectively. No significant survival differences were observed in patients with N or EN presentation with a 4-year PFS of 81.8% and 81.3% (p=0.428), respectively. On multivariable analysis adjusting for RT, sm-IPI and bulky disease, EN involvement was not associated with a decreased PFS compared with N involvement (HR 0.77; 95%CI 0.31-1.90; p=0.570). Image: Summary/Conclusion: Our study corroborates the very favourable outcomes of localized DLBCL. We could not find differences in PFS according to N and EN presentations when controlling for other prognostic factors. These results do not support different treatment strategies for EN disease; however, due to limited sample size the role of RT and the prognostic impact of specific locations cannot be fully ascertained. P1181: REAL-WORLD CHARACTERISTICS AND CLINICAL OUTCOMES IN RELAPSE/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA POST CAR-T FAILURE C. Flores Avile1,*, L. Liao2, L. Wilson1, A. Lau2, L. Chen2 1Genesis Research, Hoboken; 2ADC Therapeutics, New Providence, United States of America Background: Progressive disease following chimeric antigen receptor T-cell (CAR-T) therapy for diffuse large B-cell lymphomas (DLBCL) is a common scenario. There are limited treatment options after CAR-T failure with a poor prognosis for patients at this stage in their disease. The effectiveness of existing treatment options following CAR-T failure is still being investigated in the real-world setting. Aims: To further understand the clinical outcomes of CAR-T failure in relapse/refractory (RR) DLBCL patients in the real-world setting. Methods: This retrospective analysis identified adult patients diagnosed with RR-DLBCL [01/01/2014 – 03/31/2021] who received CAR-T therapy and experienced a subsequent disease progression or death. COTA’s Real World Evidence (RWE) database is comprised of longitudinal, HIPAA-compliant data abstracted from electronic health records (EHR) from over 200 sites of care in US (60% academic, 40% community). The first post CAR-T therapy was categorized as checkpoint inhibitor +/- other therapies (CPI), investigational therapies, tafasitamab +/- lenalidomide, polatuzumab-containing regimen (pola-containing), lenalidomide +/- anti-CD20, BTK inhibitors (BTKi), chemotherapy/chemoimmunotherapy (CT/CIT), allogenic stem-cell transplant (allo-SCT), or anti-CD20 monoclonal antibody. Overall response rate (ORR), complete response (CR), and overall survival (OS) were reported for treatment groups with at least 5 patients. Results: Of the 97 CAR-T patients identified, 57 (59%) patients failed CAR-T therapy due to receiving subsequent line of therapy, having documented progression event, or death. Patients who failed CAR-T were 67% male and on average 59 years old. Within a median follow-up of 12.4mo, 44 (77%) initiated further therapy whereby 7 (16%) initiated investigational therapies, 9 (20%) CPI, 7 (16%) tafasitamab +/- lenalidomide, 6 (14%) pola-containing, 5 (11%) lenalidomide +/- anti-CD20, 3 (7%) CT/CIT, 3 (7%) anti-CD20 monoclonal antibody, 2 (5%) BTKi, and 2 (5%) allo-SCT as their first post CAR-T therapy. Of these patients, 48% received more than two lines of therapies after CAR-T. Response rates of select first therapies received after CAR-T are detailed in Table 1. Outside clinical trials, ORR and CRs were highest for lenalidomide +/- anti CD20 (60% & 20%), followed by CPI (33% & 0%), pola-containing (33% & 0%) and tafasitamab +/- lenalidomide (14% & 0%). OS of first post CAR-T therapy was highest for lenalidomide+/- anti CD20 (12.5 mo), then CPI (11 mo), pola-containing therapy (6.37 mo), and tafasitamab +/- lenalidomide (2.7 mo). Image: Summary/Conclusion: There is no existing standard of care after patients fail CAR-T therapy. Although further research is warranted in a larger sample population, poor clinical outcomes in treatment response and longevity were observed with existing treatment options. There is still a high unmet need for more effective therapies after CAR T-cell therapy has failed. P1182: REAL-WORLD CHARACTERISTICS AND CLINICAL OUTCOMES IN RELAPSE/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA PATIENTS WHO RECEIVED CAR-T THERAPY L. Liao1, L. Nastoupil2, L. Wilson3, C. Flores Avile3,*, T. Kilavuz1, L. Chen1 1ADC Therapeutics, New Providence; 2Department of Lymphoma–Myeloma, Division of Cancer Medicine, University of Texas, Houston; 3Genesis Research, Hoboken, United States of America Background: A significant proportion of patients diagnosed with diffuse large B-cell lymphoma (DLBCL) experience refractory or relapse (RR) disease. Approval of chimeric antigen receptor T-cell (CAR-T) therapies has resulted in a novel therapeutic option for eligible patients with RR-DLBCL. However, progressive disease post CAR-T remains a common scenario as patient identification, timing, and effectiveness of CAR-T in the real-world setting is still evolving. Aims: To further understand clinical outcomes of standard of care CAR-T in RR-DLBCL in clinical practice. Methods: This retrospective analysis identified adult patients diagnosed with RR-DLBCL [01/01/2014 – 03/31/2021] who received CAR-T therapy. COTA’s Real World Evidence (RWE) database is comprised of longitudinal, HIPAA-compliant data abstracted from the electronic health records (EHR) of healthcare provider sites, representing diverse treatment U.S settings from over 200 sites of care; roughly 60% of patients are seen at academic sites and 40% are seen at community sites. Patients were categorized as having received CAR-T therapy in 2L, 3L, 4L, or 5L. Baseline characteristics was reported for CAR-T patients. Best response rate, treatment failure, and overall survival (OS) were reported by line of therapy. Disease characteristics were derived from the EHR, including the presence of high-grade lymphoma (positive rearrangement in C-MYC and BCL-2 or BCL-6 biomarkers) and primary refractory disease (2L started within 6 months not due to patient preference, drug shortage, insurance reasons, toxicity, or pandemic reasons). CAR-T treatment failure was defined as the earliest of death, initiation of subsequent line of therapy, or documented progression event after CAR-T. Results: A total of 97 CAR-T patients were identified whereby 17 received CAR-T therapy in a clinical trial setting and were excluded from this real-world evidence study. Of the 80 patients that remained, 10 (13%) received CAR-T in 2L, 31 (39%) in 3L, 24 (30%) in 4L, and 15 (19%) in 5L+. CAR-T patients had a mean age of 57 years, most were male (58%), 16% were diagnosed with high-grade lymphoma, and 60% were primary refractory. Median time from diagnosis to initiation of CAR-T was 15 months. Overall, 38% of patients achieved a complete response with a decrease in response in later lines (2L: 70%, 3L: 58%, 4L: 29%, 5L+: 33%). Within a median follow up of 13.5 months (2L: 13.5 mo, 3L: 15.2 mo, 4L: 12.8 mo, 5L+: 9.5 mo), treatment failure occurred in 45% of patients, with an increase in later lines (2L: 20%, 3L: 48%, 4L: 63%, 5L+: 80%). Median OS was 31.2 months (Not reached (NR); 2L: NR, 3L: NR, 4L: 26.3 mo, 5L+: 13.5 mo) with unequal survival probabilities across lines of therapy (Log-rank test: p=0.004) (Figure 1). Image: Summary/Conclusion: CAR T-cell therapies are considered a major advance in DLBCL, yet over half of those patients eventually fail. Outcomes are inferior in later lines with a decrease in complete response rates and shorter survival by line of therapy, thus, highlighting the need to provide CAR T-cell therapies in earlier settings. P1183: CAR-T MORPHOLOGICAL CHARACTERISTICS AND DYNAMICS IN PERIPHERAL BLOOD, EVALUATED BY A NOVEL MICROSCOPY PLATFORM G. Fridberg1,*, B.-Z. Katz1, D. Benisty1, C. Perry1, R. Ram1, I. Avivi1 1Hematology Division, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Background: Chimeric Antigen Receptor T-cells (CAR-T) therapy becomes a preferable therapeutic approach in patients with relapsed and refractory (R/R) non-Hodgkin’s large B cell lymphomas (LBCL) who failed ≥2 therapeutic regimens. However, there are no commercial assays for routine CAR-T measurements and clinical studies often employ PCR or FACS, both expensive and relatively complicated. Detection of CAR-T in peripheral blood smear (PBS) is challenging, mostly due to the lack of data regarding their morphology prior to transfusion and the low sensitivity of currently available technologies. As oppose to the currently available digital morphology, Scopio Labs Full Field Morphology (FFM) is a novel digital microscopy platform that provides high-resolution images combined with a full field analysis, incorporating artificial intelligence capabilities, thereby enabling the detection and classification of rare cells in PBS, including CAR-T cells. Aims: The dynamics of CAR-T quantity and subtypes in PBS were evaluated, and correlated with responsiveness to treatment and treatment-related toxicities. Methods: Morphological library of CAR-T cells was established from cells obtained directly from the CAR-T transfusion-bag (Fig. 1A). FFM analysis of consecutive PBS samples obtained from 26 R/R DLBCL patients, treated either with tisagenlecleucel (tisa-cel) or axicabtagene ciloleucel (axi-cel) at the Tel Aviv Sourasky Medical Center between October 2019-October 2020 was performed. Results: We identified in the CAR-T cell transfusion bags 5 distinct CAR-T morphological sub groups prior to transfusion: regular, activated, apoptotic, multinucleated and cells in mitosis (Fig. 1A). First, 79 smears, obtained from 18 patients, were examined with the FFM platform vs the CellaVision DM1200 system. Overall, 24,247 WBC were detected by the FFM platform (average 303 cells per PBS), compared with 4,335 detected by CellaVision DM1200 system (average 54.4 cells per PBS). Detection of CAR-T was superior with the FFM platform (median difference per PBS - 2, IQR 0-6, p<0.001). Then, PBS from 26 patients, 21 (80.7%) treated with tisa-cel, and 5 (19.2%) treated with axi-cel, were analyzed by the FFM platform. The absolute number of CAR-T cells in the PBS post infusion was higher in patients treated with axi-cel (across days 1 to 15). The average numbers of total, regular, and activated CAR-T cells, measured on day 5 post CAR-T-transfusion, were all higher in patients that obtained CR vs those who did not (Fig. 1B). Longer duration of cytokine release storm (CRS) was associated with higher number of CAR-T cells with activated morphology, and a lower presence of apoptotic cells on day 14. Higher number of apoptotic cells on day 14 were associated with a deferred CRS onset, while lower number of activated morphology cells on day 14 was associated with earlier onset of CRS. There was a positive correlation between the number of cells, measured on day 7 by FACS, and time to peak of regular morphological CAR-T cells (r=0.67, p=0.034, n=10). Image: Summary/Conclusion: In this study, we show that CAR-T cells has heterogeneous morphology identified in PBS. Specific morphological features in specific days were associated with outcome and therapy related toxicities, emphasizing that CAR-T monitoring require quantitative, qualitative and repeated measurements. Thus, morphological CAR-T surveillance using FFM might serve as an available, simple and inexpensive method to provide clinically relevant insights. P1184: PHASE I TRIAL OF MB-CART2019.1 IN PATIENTES WITH RELAPSED OR REFRATORY B-CELL NON-HODGKIN LYMPHOMA: 2 YEAR FOLLOW-UP REPORT P. Borchmann1,*, A. Lohneis1, P. Gödel1, H. Balke-Want1, C. Schmid2, F. Ayuk3, S. Holtkamp4, L. Hanssens4, G. Zadoyan4, B. Friedrichs4, M. Assenmacher5, I. Bürger5, D. Schneider6, T. Overstijns4, C. Scheid7, U. Holtick7, S. Miltenyi5, M. Hallek1 1Department of Internal Medicine I, University Hospital of Cologne, Cologne; 2II. Med. Klinik Hämatologie/Onkologie, Universitätsklinikum Augsburg, Augsburg; 3Department of Stem Cell Transplantation, Universitätsklinikum Hamburg-Eppendorf, Hamburg; 4Miltenyi Biomedicine GmbH; 5Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany; 6Lentigen Technology Inc., Gaithersburg, United States of America; 7Leukapheresis and Stem Cell Transplant Unit, Department of Internal Medicine I, University Hospital of Cologne, Cologne, Germany Background: A tandem chimeric antigen receptor (CAR) targeting CD20 and CD19 (pLTG1497) has shown superior efficacy as compared to each single-CAR in pre-clinical models. MB-CART2019.1 consists of autologous CD4 and CD8 enriched T-cells, which are transduced with a lentiviral vector that encodes the CAR construct for both CD20 and CD19 incorporating a 4-1BB co-stimulatory domain. Aims: This first-in-human, Phase I study had the objective to assess feasibility, safety and toxicity of ex vivo generated MB-CART2019.1 in mainly elderly patients (pts) with relapsed or refractory (r/r) CD20 and CD19 positive aggressive B-cell Non-Hodgkin lymphoma (B-NHL) without curative treatment option (NCT03870945). Methods: The trial included two predefined dose levels (DL1 1.0x106 and DL2 2.5x106 CAR T-cells/kg body weight (BW), respectively). In DL1 pts with multiple prior treatment lines were included while in DL2 the patient population was limited to pts with r/r DLBCL and 1 prior line of treatment who were transplant-ineligible. Fludarabine and cyclophosphamide were used for lymphodepletion. Infusion of fresh MB-CART2019.1 was scheduled 14 days after leukapheresis. The primary endpoint was to evaluate the maximum tolerated dose of MB-CART2019.1 as determined by dose limiting toxicities (DLT). Secondary endpoints included adverse events, overall response rate (ORR), maximum concentration (Cmax), area under the curve (AUCd0-d28), and persistence of CAR T-cells, measured by flow-cytometry. Results: A total of 12 pts, 6 per dose level, have been treated in the trial after obtaining informed consent. Median age was 72 y (range 20, 78 y), with 8 pts >70 y. Histologies included aggressive B-NHL (9), transformed follicular lymphoma (2), and mantle cell lymphoma (1). No grade ≥3 cytokine release syndrome (CRS) or neurotoxicity were observed. Haematotoxicity was very limited with no anemia or thrombocytopenia ≥grade 3 beyond day 28 and intermittent neutropenia ≥grade 3 in only two pts beyond week 8 after treatment. No DLT were observed. The ORR was 75% (3/6 pts in DL1 and 6/6 pts in DL2) with 5/12 pts (3/6 pts in DL1 and 2/6 pts in DL2) achieving PET-CT negative complete remission (CR) (CR-group). Among those 5 pts, all had ongoing CR as radiologically documented by PET-CT or CT at 12 months and all completed a 2-year follow-up visit without evidence of relapse as per investigator assessment. Patients with only partial response or stable disease as best overall response ultimately progressed. All pts with CR had a Cmax ≥450 cells/µL with mean Cmax 1,092.5 cells/µL (range 460.1, 3,147.0) while pts with no CR had lower values with mean Cmax of 111.0 cells/µL (3.9, 458.0). Mean AUCd0-d28 was 7,901 d*cells/µL (2,399.2; 19,574.7) in the CR-group as opposed to mean AUCd0-d28 of 942.0 d*cells/µL (39.3; 3,471.62) in the non-CR group. Mean time of CAR T-cell persistence in the CR-group was 491 days (274, 736 d) and all 5 CR pts had detectable CAR T-cells beyond month 6. Summary/Conclusion: As reported previously, recommended dose of MB-CART2019.1 is 2.5x106 CAR T-cells/kg BW. Importantly, all 5 pts with CR were still in CR 12 months after treatment and have now completed the 2-year follow-up visit without evidence of relapse. MB-CART2019.1 is currently challenging conventional immune-chemotherapy in a randomized Phase II trial for elderly, non-transplant eligible pts with first progression or relapse of aggressive B-NHL (NCT04844866). P1185: ANTITUMOR ACTIVITY AND SAFETY OF ZANUBRUTINIB COMBINED WITH R-CHOP REGIMEN IN THE TREATMENT OF NEWLY DIAGNOSED NON-GCB DLBCL WITH EXTRANODAL INVOLVEMENT: A PROSPECTIVE PHASE II STUDY. C. Li1,2,*, H. Geng1, X. Zong1,2, J. Zhou1,2, Y. Zhang1, J. Li1, S. Jia1, D. Wu1,2 1National Clinical Research Center for Hematologic Diseases, Jiangsu Institute of Hematology, The First Affliated Hospital of Soochow University; 2Institute of Blood and Marrow Transplantation, Collaborative Innovation Center of Hematology, Soochow University, suzhou, China Background: Approximately one-third of Diffuse large B-cell lymphoma(DLBCL) arises primarily from extranodal sites,less than 70% showed extranodal involvement in at least 1 site.Patients with multiple extranodal involvement indicates adverse clinical outcomes,and non-GCB subtype is another poor prognostic factor.Study shows that the rates of complete remission(CR),five-year OS,five-year PFS were significantly lower for the non-GCB subtype(77.5% vs 52.6%,78% vs 54%,76% vs 48%).Patients who failed in initial treatment had poor prognosis and short overall survival.In the genetic taxonomy, with non-GCB tumors enriched for MCD and BN2 subtype are suitable for the treatment of BTK inhibitor.Zanubrutinib has shown very promising future in newly diagnosed and relapsed/refractory B-cell lymphoma. Aims: We conducted a single-arm,phase II trial for newly diagnosed non-GCB DLBCL with extranodal involvement to evaluate the efficacy and safety of the zanubrutinib combined with R-CHOP(number NCT 04835870). Methods: 20 patients enrolled in this prospective single-center study with newly diagnosed non-GCB DLBCL from October 2020 to February 2022.Patients received therapies:R-CHOP(intravenously rituximab(375mg/m2 on Day0), cyclophosphamide(750mg/m2on Day1),doxorubicin(50mg/m2 on Day1),vincristine(1.4mg/m2 on Day1),and oral prednisone(50mg/day Day1-5) with Zanubrutinib(160mg bis in die orally) regimen. Particularly,patients with a weak constitution or older than 70 years old were administered ZR-miniCHOP. Results: 20 patients received ZR-CHOP regimen treatments, Patients had a median age of 60 years(31~83years) and 4 patients were over 70 years old,11 patients (55%) had IPI-PS ≥3 score,16 patients (80%) had stage III/IV disease,13 patients (65%) had ≥2 extranodal organs involved,2 patients (10%) had bulky disease and 9 patients (45%) were double-expression lymphoma confirmed by immunohistochemistry and FISH (Table 1). The median follow-up time of all patients was 8.7 months(0.4~16.1months) and there are 12 patients received 6 ~ 8 cycles of treatment who can evaluate the efficacy. The objective response rate(ORR) of 12 patients was 91.67%(11/12),11 patients(91.67%) reached complete remission(CR),of which 9 achieved CR at the interim evaluation, and 1 patient(8.3%) reached progression disease(PD).The median OS and PFS of the 12 patients who finished treatment were 10.7months(5.6~16.1months) and 10.2months(5.6~16.1months).One patient relapsed(Figure 1).13(65%) patients reduced the dose during treatment, the most common AEs were hematological toxicity, fatigue and apositia. all-grade and grade 3 or higher hematological toxicity were 100% and 65%,11 patient observed grade 3 or higher neutropenia and 3 patients had severe pneumonia during treatment. Hemorrhage were observed in 3 patients,2 patients developed atrial fibrillation after the first medication.No patient had fatal adverse events(Table2). Image: Summary/Conclusion: Our results showed that Zanubrutinib plus R-CHOP(ZR-CHOP) is a safe and effective scheme for this part of non-GCB DLBCL patients with extranodal involvement.It indicate that the addition of zanubrutinib to the standard first-line treatment can enable these patients to achieve remission in the early stage of treatment and without significantly increasing toxicity. P1186: A LARGE FRENCH REAL WORLD MULTICENTRIC PROSPECTIVE COHORT OF PATIENTS WITH LYMPHOMA (REALYSA STUDY): DESCRIPTION OF THE DIFFUSE LARGE B CELL LYMPHOMA PATIENTS IN REAL WORLD IN FRANCE H. Ghesquieres1,*, F. Cherblanc2, A. Belot2, V. Camus3, K. Thokagevistk4, K.-K. Bouabdallah5, C. Esnault4, L.-M. Fornecker6, S. Micon4, F. Bijou7, C. Haioun8, N. Morineau9, L. Ysebaert10, G. Damaj11, S. Le Gouill12, S. Guidez13, F. Morschhauser14, C. Thiéblemont15, A. Chauchet16, R. Gressin17, F. Jardin3, C. Fruchart18, G. Labouré19, L. Fouillet20, P. Lionne-Huyghe21, A. Bonnet22, L. Lebras23, S. Amorim24, C. Leyronnas25, G. Olivier26, R. Guieze27, T. Lamy28, V. Launay29, B. Drenou30, O. Fitoussi31, L. Detourmignies32, J. Abraham33, C. Soussain34, F. Lachenal35, P. Fogarty2, P. Cony-Makhoul2, A. Bernier2, S. Le Guyader-Peyrou36, A. Monnereau36, F. Boissard37, C. Rossi38 1Hopital Lyon Sud, Pierre Benite; 2LYSARC, Pierre-Benite; 3Centre Henri Becquerel, Rouen; 4Roche, Boulogne-Billancourt; 5CHU Haut Lévêque, Pessac; 6ICANS, Strasbourg; 7Institut Bergonié, Bordeaux; 8Hôpital Henri Mondor, Créteil; 9CHD Vendée, La Roche sur Yon; 10IUCT Oncopôle, Toulouse; 11Institut d’Hématologie de Basse Normandie, Caen; 12Centre Hospitalier Universitaire de Nantes, Nantes; 13Centre Hospitalier Universitaire de Poitiers, Poitiers; 14CHRU de Lille, Lille; 15Hôpital Saint-Louis, Paris; 16CHU Jean Minjoz, Besancon; 17Centre Hospitalier Universitaire Michallon, La Tronche; 18Centre Hospitalier de Dunkerque, Dunkerque; 19Centre Hospitalier de Libourne, Libourne; 20Institut de Cancérologie Lucien Neuwirth, Saint-Priest-en-Jarez; 21Centre Hospitalier d’Arras, Arras; 22Centre Hospitalier Bretagne Atlantique, Vannes; 23Centre Léon Bérard, Lyon; 24Hôpital Saint-Vincent-de-Paul, Lille; 25Groupe Hospitalier Mutualiste de Grenoble, Grenoble; 26Centre Hospitalier de Niort, Niort; 27CHU de Clermont-Ferrand, Clermont-Ferrand; 28CHU Pontchaillou, Rennes; 29Centre Hospitalier Yves Le Foll, Saint-Brieuc; 30Groupe Hospitalier Mulhouse et Sud Alsace, Mulhouse; 31Polyclinique Bordeaux Nord Aquitaine, Bordeaux; 32Centre Hospitalier de Roubaix, Roubaix; 33Centre Hospitalier Universitaire de Limoges, Limoges; 34Institut Curie, Saint-Cloud; 35Centre Hospitalier Pierre Oudot, Bourgoin-Jallieu; 36Université de Bordeaux, Bordeaux, France; 37Roche - F.Hoffman-La Roche Ltd, Basel, Switzerland; 38Centre Hospitalier Universitaire Francois Mitterand, Dijon, France Background: In France, there were an estimated 18.000 incident lymphoma cases in 2018. Currently, most of the knowledge comes from clinical trials (CT), with stringent inclusion criteria and poor representativeness. Real world data (RWD) is essential to complement CT data, but major challenges such as feasibility and data quality remain. In this context, the REal world dAta in LYmphoma and Survival in Adults (REALYSA) multicentric prospective cohort started in 2018 (NCT03869619), with a recruitment objective of 6000 patients and a 9-year follow-up (FU). Aims: The aims of this study are (i) to evaluate the capacity of a real-world program to provide high-quality data that translate into meaningful clinical endpoints, and (ii) to describe the demographic and clinical characteristics as well as therapeutic management and treatment effectiveness of 1st line (1L) DLBCL patients. Methods: Patients diagnosed with 7 histological lymphoma subtypes are included in REALYSA at diagnosis after signing informed consent. Patients receive standard routine care. Data collection includes demographic, clinical, quality of life and epidemiological data at inclusion and every 6 to 12 months. A biobank is also constituted. A strong data validation system inspired from clinical research standards, including care pathway visualisation tools, is regularly running. Meaningful real-life endpoints were derived, such as end of treatment (EoT) evaluation and event-free survival (EFS). We present here the results of a proof-of-concept analysis for REALYSA patients with a DLBCL diagnosis and who received a 1L treatment. Results: Thirty-five hospitals/clinics are currently participating to REALYSA across France. As of Jan 31st 2022, 1217 patients with DLBCL were included in REALYSA with a mean number of 60 patients per month; 645 epidemiological questionnaires were collected; and there were more than 1500 samples in the biobank. The analysis was conducted on 645 DLBCL patients included in REALYSA before March 31st 2021. Median age was 66.3 years [54-75]. Most patients were male (344; 53.3%), with advanced-stage disease (Ann-Arbor stage III/IV for 472 patients (73.3%)), extra-nodal locations (499; 77.4%) and elevated LDH level (402; 64.0%). The international prognostic index was 2 to 5 for 486 patients (76.2%). Treatments received at 1L were R-CHOP (482; 74.7%), R-miniCHOP (86; 13.3%), high dose anthracycline-based regimen (62; 9.6%) and non-anthracycline based regimen (15; 2.3%). Among patients with EoT evaluation done (603), 483 (80.1%) had a complete response, 51 (8.5%) a partial response, 7 (1.2%) a stable disease and 62 (10.3%) a progressive disease. Of note, EoT evaluation was performed by PET/scan for 92% of patients. Median FU was 9.9 months [4.5-17.2]. Results for effectiveness at 12 months were: (i) EFS of 77.9% [95% CI 73.8-81.4]; (ii) PFS of 79.7% [75.6-83.1]; and (iii) overall survival of 90.0% [86.5-92.5]. With the limitation of the short FU, and using backward stepwise regression model, we confirmed that EFS was associated with age, ECOG PS, LDH level and bulk disease. Summary/Conclusion: This analysis showed that REALYSA, the first nationwide real-life cohort on lymphoma, is able to provide high-quality data, with results for 1L DLBCL patients consistent with the literature, including recent phase 3 trials. These data will be useful for numerous purposes, including a better characterization of lymphoma population in France (clinic, epidemiology, biology), as well as innovative study designs (e.g. new outcome endpoints, synthetic control arm). P1187: END OF TREATMENT 18F FDG-PET/CT RESPONSE IS PROGNOSTIC FOR OVERALL AND PROGRESSION-FREE SURVIVAL IN PERIPHERAL T-CELL LYMPHOMA: RESULTS OF THE UK NCRI PHASE 2 RANDOMISED CHEMO-T TRIAL PET/CT SUBSTUDY M. Gleeson1,2,*, C. Arias2, D. Cunningham2, C. Peckitt2, Y. Du2, N. Hujairi2, Y. M. To2, H.-C. Chen2, S. Patel2, I. Chau2, P. Johnson3, K. M. Ardeshna4, A. Wotherspoon2, A. Attygalle2, E. A. Hawkes5,6, M. P. Macheta7, G. P. Collins8, J. Radford9, A. Forbes10, A. Hart11, S. Montoto12, P. McKay13, K. Benstead14, N. Morley15, N. Kalakonda16, Y. Hasan17, D. Turner18, S. Chua2 1Guy’s and St. Thomas’ NHS Foundation Trust, London; 2The Royal Marsden NHS Foundation Trust, London and Surrey; 3Cancer Research UK Centre, University of Southampton, Southampton; 4University College London Hospitals, London, United Kingdom; 5Olivia Newton John Cancer Research Centre, Austin Health; 6Monash University, Melbourne, Australia; 7Blackpool Victoria Hospital, Blackpool; 8Oxford Cancer and Haematology Centre Churchill Hospital, Oxford; 9University of Manchester and the Christie NHS Foundation Trust, Manchester; 10Royal Cornwall Hospital, Truro; 11New Victoria Hospital, Glasgow; 12St Bartholomew’s Hospital, London; 13Beatson West of Scotland Cancer Centre, Glasgow; 14Gloucestershire Hospitals NHS Foundation Trust, Cheltenham; [e-mail], Sheffield; 16The Clatterbridge Cancer Centre NHS Foundation Trust, Liverpool; 17Sandwell and West Birmingham Hospitals NHS Trust, Birmingham; 18Torbay Hospital, Torquay, United Kingdom Background: The role of 18F FDG PET (PET) in peripheral T-cell lymphoma (PTCL) remains under evaluation. The UK NCRI phase 2 randomised CHEMO-T trial compared cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) with gemcitabine, cisplatin and methylprednisolone (GEM-P) in previously untreated PTCL patients. The primary endpoint, to demonstrate a superior complete response (CR) rate for GEM-P by contrast-enhanced CT (CECT) at end of treatment (EOT) was not met. Aims: To investigate the role of PET in determining baseline bone marrow (BM) involvement in PTCL and as a prognostic tool at EOT. Methods: Patients required a baseline PET (acquired as per protocol) within 28 days of randomisation and a PET at EOT at least 28 days post chemotherapy. Site accreditation, data collation, and quality control for PET imaging was performed by the UK PET Core Lab. BM biopsy was required within 6 weeks of randomisation. PET scans were centrally reviewed at The Royal Marsden by 2 expert nuclear medicine physicians (YD and NH). Baseline BM involvement by PTCL on PET was evaluated. PET response was assessed according to the revised response criteria for malignant lymphoma and by the Deauville score (DS) 5-point scale. Where discordance existed between reviewers, a third reviewer (SC) adjudicated. EOT PET response and PET status (DS 1-3=PET-negative, DS 4-5=PET-positive) were correlated with PFS and OS. Results: Baseline PET data was available for 82/84 patients, 80/82 (98%) had FDG-avid disease (n=2 with non-avid disease (n=1 AITL, n=1 EATL) were excluded). Clinical characteristics were similar to the main trial cohort: median age=63 yrs, male=73% and high-risk IPI=23%. PTCL subtypes were AITL n=33 (41%), PTCL-NOS n=32 (40%), ALK- ALCL n=14 (18%) and EATL n=1 (1%). For 20/79 (25%) patients with biopsy-proven BM infiltration 9/19 (47%) had BM uptake on baseline PET. Of 20/80 patients (25%) with BM uptake on baseline PET, 9/20 (45%) had biopsy-confirmed infiltration. PET had a sensitivity of 47% and a specificity 81% for determination of BM involvement. EOT PET was available for 69/80, n=4 were excluded as data was acquired following a later line (n=3) or complete imaging was not retrievable (n=1); therefore 65/80 had evaluable EOT PET data. The median number of days from EOT to PET was 41. At EOT, PET response was CR n=41 (63%), partial response (PR) n=15 (23%), stable disease n=0 and progressive disease (PD) n=9 (14%); 41 (63%) were PET-negative and 24 (37%) were PET-positive. At a median follow-up of 27 months, PET-negative vs. PET-positive patients at EOT had a 2-yr OS of 72% vs. 52% [HR 0.58; 95% CI 0.24–1.41), p=0.231] and 2-yr PFS of 55% and 29% [HR 0.45 (95% CI 0.23–0.88), p=0.021] respectively. EOT PET response correlation with 2-yr OS was CR=72%, PR=67%, PD=29%; and with 2-yr PFS was CR=55%, PR=32%, PD=25%. A CR by PET (vs. PD) was associated with improved OS [HR 0.245 (95% CI 0.083–0.730), p=0.012] (Figure 1) and PFS [HR 0.265 (95% CI 0.102–0.687), p=0.006]. Adjusting for stratification parameters at randomisation (IPI risk group, PTCL subtype) PET response remained prognostic for both OS and PFS while EOT PET status (positive vs. negative) did not. CECT response remained prognostic for PFS only. Image: Summary/Conclusion: Our findings indicate that PET has insufficient sensitivity for detection of BM involvement for baseline staging. Furthermore, while both EOT CECT and PET response were prognostic for PFS and OS, only PET response remained prognostic for OS following adjustment for stratification factors suggesting it is a superior prognostic tool in PTCL. P1188: PERSONALIZED TREATMENT ACCORDING TO BIOLOGICAL PROFILE IMPROVES OUTCOME OF NEWLY DIAGNOSED PATIENTS AFFECTED BY DIFFUSE LARGE B-CELL LYMPHOMA: A REAL-LIFE EXPERIENCE A. Guidetti1,2,*, R. Pasquale3, A. Dodero1, A. Chiappella1, L. Devizzi1, L. Farina1, C. Rusconi1, M. Pennisi1, P. Corradini1,2 1Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori; 2Pathophysiology and Transplantation, University of Milano; 3Hematology, Ospedale Maggiore Policlinico, Milano, Italy Background: Diffuse Large B-Cell Lymphoma (DLBCL) is a heterogeneous disease characterized by biological and clinical aspects conditioning different prognosis. Chemo-immunotherapy treatment with R-CHOP proved to be less effective in patients with double hit (DH)/triple hit (TH) or double expressor (DE) DLBCL who require more intensive regimens Aims: To investigate survival outcomes of DLBCL patients treated according to biological profile in the real-life clinical practice at Istituto Nazionale dei Tumori in Milano. Primary objective was to verify whether personalized treatment could overcome well known differences in Progression Free Survival (PFS) observed between DE, DH/TH and non DE/DH/TH patients when they are uniformly treated with R-CHOP Methods: We retrospectively collected clinical and survival data of all newly diagnosed DLBCL patients referred at our Hematology Division from January 2013 to August 2021. All DLBCL patients are evaluated for BCL2, BCL6 and MYC expression in immunohistochemistry and gene rearrangements by fluorescent in situ hybridization, and they are assigned to treatment based on the biologic profile: non DE/DH/TH patients receive 6 cycles R-CHOP-21 + 2R, DE patients receive 6 cycles R-DA-EPOCH and DH/TH receive R-CODOX-M/R-IVAC. Central Nervous System (CNS) prophylaxis with lumbar puncture and/or intravenous high dose methotrexate is reserved for DE or DH patients and for those with CNS-IPI 3-5 Results: Since 2013, 243 patients affected by DLBCL at diagnosis were consecutively treated according to biological risk: 139 non DE/DH/TH patients received R-CHOP, 80 DE patients received R-DA-EPOCH and 24 high risk patients received R-CODOX-M/R-IVAC (n= 13 DH/TH patients and n=11 DE patients with CNS IPI 3-5). Median age was 62 years (range, 21-86), 178 patients (73%) presented in stage 3-4 and 97 patients (40%) had an IPI score of 3-5. The three groups were not significantly different in terms of clinical characteristics, except for median age, which was higher in the R-CHOP group (66 years vs. 58 and 57 years in R-DA-EPOCH and R-CODOX-M/R-IVAC groups, respectively). Despite the toxicity expected with an intensified therapy, 95% of patients completed the treatment and no toxic deaths were registered. Febrile neutropenia was observed in 3%, 15% and 29% of patients in RCHOP, RDAEPOCH and RCODOX-M/R-IVAC, respectively. At a median follow-up of 42 months (range, 2-106 months), the estimated 3 years PFS and OS for the entire population were 80% and 89%, respectively. Patients with low IPI, limited stage and germinal center B cell of origin had significant better PFS as compared to the rest. Three years PFS and OS according to biological entity and relative treatment group were not significantly different, being 84%, 77% and 66%, (p=ns) and 93%, 85% and 74%, (p=ns) in non DE/DH/TH, DE and high risk patients, respectively. Similar PFS between non DE/DH/TH, DE and high risk patients were observed also in the subgroup of patients with IPI3-5 with PFS of 77%, 65% and 57%, respectively (p=ns). CNS prophylaxis was administered to 177 patients (73%) and 9 cases of CNS recurrence (4%) were observed Summary/Conclusion: In this study, we describe the feasibility of a personalized treatment according to biological risk in a real-life setting. This approach seems to overcome the poor outcome of DLBCL patients at high risk of relapse and ameliorate the prognosis of the overall DLBCL population. Biological characteristics should be carefully considered at diagnosis in order to give patients a personalized program and improve their outcome P1189: INITIAL SAFETY RUN-IN RESULTS OF THE PHASE III POLARGO TRIAL: POLATUZUMAB VEDOTIN PLUS RITUXIMAB, GEMCITABINE, AND OXALIPLATIN IN PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA A. McMillan1,*, C. Haioun2, J.-M. Sancho3, A. Viardot4, A. Rodriguez Izquierdo5, E. M. Donato Martin6, A. M. García-Sancho7, J. Sandoval-Sus8, H. Tilly9, E. Vandenberghe10, J. Hirata11, P. Choudhry11, Y. M. Chang12, L. Musick11, M. Matasar13 1Centre for Clinical Haematology, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; 2Lymphoid Malignancies Unit, Groupe Hospitalier Henri Mondor, Créteil Cedex, France; 3ICO-HU Germans Trias I Pujol, Barcelona, Spain; 4University Hospital of Ulm, Ulm, Germany; 5Hospital Universitario 12 de Octubre Servicio de Hematología, Madrid; 6Hospital Universitario Dr Peset Servicio de Hematología, Valencia; 7Hospital Clínico Universitario de Salamanca Servicio de Hematología, Salamanca, Spain; 8Moffitt Cancer Center at Memorial Healthcare Institute, Pembroke Pines, United States of America; 9Centre Henri Becquerel and University of Rouen, Rouen, France; 10St James Hospital Cancer Clinical Trials Office, Dublin, Ireland; 11Genentech, Inc., South San Francisco, United States of America; 12Hoffmann-La Roche Ltd, Mississauga, Canada; 13Memorial Sloan Kettering Cancer Center, New York, United States of America Background: Transplant-ineligible patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) have a poor prognosis (Gisselbrecht C, et al. Br J Haematol 2018). Several treatment options are available, including platinum-based chemotherapies such as oxaliplatin plus rituximab and gemcitabine (R-GemOx). Adding polatuzumab vedotin to R-GemOx (Pola-R-GemOx) may improve outcomes for patients with a continued unmet medical need. Pola-R-GemOx is an investigational combination. The safety of polatuzumab vedotin and platinum-based therapy combinations must be considered as both are associated with neuropathy. POLARGO (NCT04182204; MO40598) is a Phase III, multicenter, open-label, randomized trial evaluating the safety and efficacy of Pola-R-GemOx vs R-GemOx in patients with R/R DLBCL. Aims: To present the results from the safety run-in stage of POLARGO. Methods: The primary endpoint is the safety and tolerability of polatuzumab vedotin (1.8 mg/kg) + R-GemOx (R, 375 mg/m2; Gem, 1000 mg/m2; Ox, 100 mg/m2) given every 21 days for up to 8 cycles. Safety was assessed by the incidence, nature, and severity of adverse events (AEs; NCI CTCAE v5.0), with a focus on peripheral neuropathy (PN). Dose interruptions and reductions were used to assess tolerability. Granulocyte-colony stimulating factor was given as primary prophylaxis with each cycle (C) of therapy; anti-infective prophylaxis for pneumocystis and herpes virus was mandatory. All patients provided informed consent. Results: As of October 26, 2021, 15 patients were enrolled, and 11 (73%) received ≥4 cycles of Pola-R-GemOx. Median age was 76 (range 47–87) years, 10 (