[1582] Comparison of High-Throughput Molecular Profiling Platforms for Rapid Mutational Analysis of Myeloid Neoplasms

Shalini Verma, Wesley O Greaves, Bedia Barkoh, Keyur P Patel, Jawad H Manekia, Raj Patel, L Jeffrey Medeiros, Rajyalakshmi Luthra. The University of Texas M.D. Anderson Cancer Center, Houston, TX

Background: Recurrent mutations in cancer genes are drivers of oncogenesis and tumor progression in myeloid neoplasms. Currently available platforms can assess for gene mutations (e.g. Sanger sequencing or pyrosequencing), but cannot be used in a multiplex strategy to simultaneously detect all gene mutations. Next-generation sequencing (NGS) is an obvious approach as one can sequence the entire genome, but is expensive and bioinformatics aspects are not yet standardized. Two multiplex hotspot mutation screening approaches with potential application in clinical laboratories include primer extension followed by mass spectrometry (MS)-based (Sequenom® MassARRAY; Sequenom, San Diego, CA) or fluorescent capillary electrophoresis (FCE)-based detection (SNaPshot® multiplex system: Life Technologies, Carlsbad, CA). The aim of this study is to develop and assess the performance characteristics of the MS and FCE-based platforms using cell lines and patient samples and to evaluate the clinical utility of these platforms for myeloid neoplasms.
Design: Genomic DNA was extracted from 13 cell lines which harbor common myeloid neoplasm mutations and 10 patient samples (7 acute myeloid leukemia, 2 essential thrombocythemia, 1 primary myelofibrosis). An 8-well panel was developed to assess 58 hotspot regions in KIT, FLT3, GATA2, IDH1, IDH2, JAK2, KRAS, MDM2, MPL, NRAS, TP53 and WT1. 10 ng of DNA was PCR amplified. Separate aliquots were simultaneously subjected to single-base-primer extension (SBE) as per manufacturer protocols, followed by MALDI-TOF mass spectrometry and capillary electrophoresis, respectively.
Results: Twenty two sequence variations (KRAS=2; NRAS=3; IDH1=5; JAK2=1; MDM2=6; and TP53=5), documented previously by Sanger sequencing or pyrosequencing, were also detected by both platforms in cell lines. In patient samples, 2 patients demonstrated SNPs in TP53 and 1 patient demonstrated MPL mutation. There was a 100% concordance in the genotyping results from both platforms.
Conclusions: Both MS and FCE are reliable high-throughput platforms that can be used in the clinical laboratory to detect multiple mutations simultaneously using limited DNA. In our experience, FCE data are easier to interpret, especially in samples with low level mutation.
Category: Hematopathology

Tuesday, March 20, 2012 1:45 PM

Platform Session: Section G, Tuesday Afternoon


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