Abstract 01: Development of a targeted panel for acute myeloid leukemia (AML) by using next-generation sequencing (NGS)

Abstract

Abstract Background: AML is a clinically and genetically heterogeneous clonal disorder. Approximately 50% are characterized by recurrent clonal chromosome aberrations which have contributed to the classification of disease and are recognized as important prognostic factors. AML patients who lack these recurrent structural abnormalities have been grouped as “intermediate cytogenetic risk” and are being further subcategorized by the sequence alterations that are being identified. A more complete understanding of the genetic changes that are relevant to the pathogenesis of AML will improve the classification of risk and ultimately better selection of therapy. Our hypothesis is that mutational profiling and analysis of patient outcomes will help better define the risk subgroups of patients and predict prognosis in patients with AML. Methods: Archived DNA from 37 patients with various AML diagnoses were obtained with IRB approval (IRB#201502763). A panel of 30 commonly mutated genes in AML were designed using the Ion Ampliseq Designer (Ion Torrent) and targeted for “hotspot” or complete coding sequence (CDS). Genomic libraries were prepared using Ion AmpliSeq™2.0 (ThermoFisher) technology. Library clonal amplification was performed by emulsion PCR using Ion PGM™ Template OT2 Reagents Kit200. Simultaneous sequencing of clonally amplified DNA targets was performed by Ion-Torrent Personalized Genome Machine (IT-PGM) using 318v2 chip. Genomic variants were identified by variant caller3.4 and further filtered and analysed through a custom-made clinically validated pipeline. Novel mutations were confirmed by either single nucleotide primer extension or Sanger sequencing. Results: The assay successfully identified previously identified point mutations in FLT-3 and CEBPA genes. Additional variants were identified in 81% (30/37) of specimens; no mutations were found in 19% (7/37) specimens. The genes most frequently mutated were DNMT3A (23%), TET2 (17%), IDH2 (14%) and TP53 (14%). U2AF1 (11%) and RUNX1 (8%) were exclusively mutated in specimens with non-recurrent cytogenetic abnormalities. The identified variants will be used to risk stratify patients based on clinical outcome. Conclusion: This targeted multi-gene panel successfully identified several variants along with previously known mutations in archived specimens. Citation Format: Anup Tilak, Ramakrishna Sompallae, Aaron Stence, Natalya Guseva, Aaron Bossler. Development of a targeted panel for acute myeloid leukemia (AML) by using next-generation sequencing (NGS). [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 01.