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      Enriched Bone Marrow Derived Disseminated Neuroblastoma Cells Can Be a Reliable Source for Gene Expression Studies—A Validation Study

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          Abstract

          Background

          Metastases in the bone marrow (BM) in form of disseminated tumor cells (DTCs) are frequent events at diagnosis and also at relapse in high-risk neuroblastoma patients. The frequently highly diluted occurrence of DTCs requires adequate enrichment strategies to enable their detailed characterization. However, to avoid methodical artifacts we tested whether pre-analytical processing steps—including transport duration, temperature and, importantly, tumor cell enrichment techniques—are confounding factors for gene expression analysis in DTCs.

          Methods

          LAN-1 neuroblastoma cells were spiked into tumor free BM and/or peripheral blood and: i) kept at room temperature or at 4°C for 24, 48 and 72 hours; ii) frozen down at -80°C and thawed; iii) enriched via magnetic beads. The effect on the gene expression signature of LAN-1 cells was analyzed by qPCR arrays and gene expression microarrays.

          Results

          Neither storage at –80°C in DMSO and subsequent thawing nor enrichment of spiked-in neuroblastoma cells changed the expression of the analyzed genes significantly. Whereas storage at 4°C altered the expression of analyzed genes (14.3%) only at the 72h-timepoint in comparison to the 0h-timepoint, storage at room temperature had a much more profound effect on gene expression by affecting 20% at 24h, 26% at 48h and 43% at 72h of the analyzed genes.

          Conclusion

          Using neuroblastoma as a model, we show that tumor cell enrichment by magnetic bead separation has virtually no effect on gene expression in DTCs. However, transport time and temperature can influence the expression profile remarkably. Thus, the expression profile of routinely collected BM samples can be analyzed without concern as long as the transport conditions are monitored.

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          Most cited references45

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          Recent advances in neuroblastoma.

          John Maris (2010)
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            MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia.

            Acute lymphoblastic leukemias carrying a chromosomal translocation involving the mixed-lineage leukemia gene (MLL, ALL1, HRX) have a particularly poor prognosis. Here we show that they have a characteristic, highly distinct gene expression profile that is consistent with an early hematopoietic progenitor expressing select multilineage markers and individual HOX genes. Clustering algorithms reveal that lymphoblastic leukemias with MLL translocations can clearly be separated from conventional acute lymphoblastic and acute myelogenous leukemias. We propose that they constitute a distinct disease, denoted here as MLL, and show that the differences in gene expression are robust enough to classify leukemias correctly as MLL, acute lymphoblastic leukemia or acute myelogenous leukemia. Establishing that MLL is a unique entity is critical, as it mandates the examination of selectively expressed genes for urgently needed molecular targets.
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              Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling.

              Treatment of pediatric acute lymphoblastic leukemia (ALL) is based on the concept of tailoring the intensity of therapy to a patient's risk of relapse. To determine whether gene expression profiling could enhance risk assignment, we used oligonucleotide microarrays to analyze the pattern of genes expressed in leukemic blasts from 360 pediatric ALL patients. Distinct expression profiles identified each of the prognostically important leukemia subtypes, including T-ALL, E2A-PBX1, BCR-ABL, TEL-AML1, MLL rearrangement, and hyperdiploid >50 chromosomes. In addition, another ALL subgroup was identified based on its unique expression profile. Examination of the genes comprising the expression signatures provided important insights into the biology of these leukemia subgroups. Further, within some genetic subgroups, expression profiles identified those patients that would eventually fail therapy. Thus, the single platform of expression profiling should enhance the accurate risk stratification of pediatric ALL patients.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 September 2015
                2015
                : 10
                : 9
                : e0137995
                Affiliations
                [1 ]CCRI, Children’s Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria
                [2 ]Molecular Diagnostics, Health & Environment Department, AIT Austrian Institute of Technology GmbH, Vienna, Austria
                [3 ]Children's University Hospital, University of Tuebingen, Tuebingen, Germany
                [4 ]Department of Pediatrics, Medical University of Vienna, Vienna, Austria
                University of Navarra, SPAIN
                Author notes

                Competing Interests: The authors declare that they have no competing interests.

                Conceived and designed the experiments: PFA STM FR. Performed the experiments: FR MRA AW. Analyzed the data: MK FR. Contributed reagents/materials/analysis tools: AW RH PFA. Wrote the paper: FR PFA.

                Article
                PONE-D-15-28360
                10.1371/journal.pone.0137995
                4567134
                26360775
                036e302c-3054-4880-b2aa-0cdce4e93bf5
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 30 June 2015
                : 25 August 2015
                Page count
                Figures: 5, Tables: 0, Pages: 15
                Funding
                The research leading to these results has received funding from: St. Anna Kinderkrebsforschung (Vienna, Austria) and the European Union's Seventh Framework Program (FP7/2007-2013) under the project ENCCA, grant agreement HEALTH-F2-2011-261474.
                Categories
                Research Article
                Custom metadata
                The microarray raw data are available at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63294.

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