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      Accelerated Instability Testing Reveals Quantitative Mass Spectrometry Overcomes Specimen Storage Limitations Associated with PD-L1 Immunohistochemistry

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          Abstract

          Immunohistochemistry (IHC) using formalin-fixed, paraffin embedded (FFPE) tissue is limited by epitope masking, post-translational modification and immunoreactivity loss that occurs in stored tissue by poorly characterized mechanisms. Conformational epitopes recognized by many programmed death-ligand 1 (PD-L1) IHC assays are particularly susceptible to degradation and provide an ideal model for understanding signal loss in stored FFPE tissue. Here we assessed 1,206 tissue sections to evaluate environmental factors impacting immunoreactivity loss. PD-L1 IHC using 4 antibodies (22C3, 28-8, E1L3N, SP142), raised against intracellular and extracellular epitopes, was assessed in stored FFPE tissue alongside quantitative mass spectrometry (MS). Global proteome analyses were used to assess proteome-wide oxidation across an inventory of 3,041 protein groups (24,737 distinct peptides). PD-L1 quantitation correlated well with IHC expression on unaged sections (R 2=0.744; P<0.001), with MS demonstrating no loss of PD-L1 protein, even in sections with significant signal loss by IHC impacting diagnostic category. Clones 22C3 and 28-8 were most susceptible to signal loss, with E1L3N demonstrating the most robust signal (56%, 58% and 33% reduction respectively; p<0.05). Increased humidity and temperature resulted in significant acceleration of immunoreactivity loss, which was mitigated by storage with desiccant. MS demonstrated only modest oxidation of 274 methionine-containing peptides and aligned with IHC results suggesting peptide oxidation is not a major factor. These data imply immunoreactivity loss driven by humidity and temperature results in structural distortion of epitopes rendering them unsuitable for antibody binding following epitope retrieval. Limitations of IHC biomarker analysis from stored tissue sections may be mitigated by cost-effective use of desiccant when appropriate. In some scenarios, complementary MS is a preferred approach for retrospective analyses of archival FFPE tissue collections.

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          Most cited references 41

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          Programmed Death-Ligand 1 Immunohistochemistry Testing: A Review of Analytical Assays and Clinical Implementation in Non–Small-Cell Lung Cancer

          Purpose Three programmed death-1/programmed death-ligand 1 (PD-L1) inhibitors are currently approved for treatment of non-small-cell lung cancer (NSCLC). Treatment with pembrolizumab in NSCLC requires PD-L1 immunohistochemistry (IHC) testing. Nivolumab and atezolizumab are approved without PD-L1 testing, though US Food and Drug Administration-cleared complementary PD-L1 tests are available for both. PD-L1 IHC assays used to assess PD-L1 expression in patients treated with programmed death-1/PD-L1 inhibitors in clinical trials include PD-L1 IHC 28-8 pharmDx (28-8), PD-L1 IHC 22C3 pharmDx (22C3), Ventana PD-L1 SP142 (SP142), and Ventana PD-L1 SP263 (SP263). Differences in antibodies and IHC platforms have raised questions about comparability among these assays and their diagnostic use. This review provides practical information to help physicians and pathologists understand analytical features and comparability of various PD-L1 IHC assays and their diagnostic use. Methods We reviewed and summarized published or otherwise reported studies (January 2016 to January 2017) on clinical trial and laboratory-developed PD-L1 IHC assays (LDAs). Studies assessing the effect of diagnostic methods on PD-L1 expression levels were analyzed to address practical issues related to tissue samples used for testing. Results High concordance and interobserver reproducibility were observed with the 28-8, 22C3, and SP263 clinical trial assays for PD-L1 expression on tumor cell membranes, whereas lower PD-L1 expression was detected with SP142. Immune-cell PD-L1 expression was variable and interobserver concordance was poor. Inter- and intratumoral heterogeneity had variable effects on PD-L1 expression. Concordance among LDAs was variable. Conclusion High concordance among 28-8, 22C3, and SP263 when assessing PD-L1 expression on tumor cell membranes suggests possible interchangeability of their clinical use for NSCLC but not for assessment of PD-L1 expression on immune cells. Development of LDAs requires stringent standardization before their recommendation for routine clinical use.
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            Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation.

            Gene regulation is a complex process. Numerous factors appear to be required for the accurate temporal and spatial regulation of each gene. Often these factors are assembled into multiprotein complexes, contributing to specific gene regulation events. Understanding how all these factors are organized in the chromosome and how their function is regulated in vivo is a challenging task. One of the most useful techniques for studying this level of gene regulation is the in vivo fixation by formaldehyde crosslinking of proteins to proteins and proteins to DNA, followed by immunoprecipitation of the fixed material.
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              Tissue handling and specimen preparation in surgical pathology: issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue.

              Expression profiling by microarrays and real-time polymerase chain reaction-based assays is a powerful tool for classification and prognostication of disease; however, it remains a research tool, largely reliant on frozen tissue. Limiting the utility of expression profiling is the isolation of quality nucleic acids from formalin-fixed, paraffin-embedded tissue. The collection, handling, and processing of tissue directly impacts the biomolecules that can be recovered from it. High-quality nucleic acids can be obtained from formalin-fixed, paraffin-embedded tissue, but greater attention to all steps in the process of tissue handling and preparation is required. To summarize the current state-of-the-art of preanalytic factors in tissue handling and processing as they impact the quality of RNA obtainable from formalin-fixed, paraffin-embedded tissue. The goals are to provide recommendations that will improve RNA quality for expression profiling from formalin-fixed, paraffin-embedded tissue and highlight areas for additional research. Tissue is an analyte and it must be handled in a standardized fashion to provide consistent results. The literature was reviewed. Consultation with industry and academic leaders in the use of RNA for expression profiling was obtained to identify areas for additional research. Development of RNA-based assays from formalin-fixed, paraffin-embedded tissue is feasible. Greater attention to tissue handling and processing is essential to improve the quality of biospecimens for the development of robust RNA-based assays. Standardization of procedures and vigorous testing of alternative protocols are required to ensure that these assays function as designed.
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                Author and article information

                Journal
                0376617
                Lab Invest
                Lab. Invest.
                Laboratory investigation; a journal of technical methods and pathology
                0023-6837
                1530-0307
                10 December 2019
                02 January 2020
                June 2020
                10 June 2020
                : 100
                : 6
                : 874-886
                Affiliations
                [1 ]Institute of Translational Medicine, University of Liverpool, Liverpool, UK
                [2 ]Protypia, LLC, Nashville, TN, USA
                [3 ]Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA
                [4 ]H. Lee Moffitt Cancer Center, Tampa, FL, USA
                Author notes
                [* ]Corresponding author: Aaron M. Gruver, M.D., Ph.D., Diagnostic and Experimental Pathology, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana USA, gruver_aaron_m@ 123456lilly.com , 317.433.8641 (phone), 317.651.6346 (fax)
                Article
                EMS85151
                10.1038/s41374-019-0366-y
                7280177
                31896815

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                Pathology

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