AML-142: Implementation of Combined DNA and RNA Sequencing in Myeloid Neoplasms

Abstract

Context Identifying targetable and driver mutation and fusions in myeloid neoplasms through NGS platform. Objective The aim of this study was to optimize and validate Oncomine Myeloid Research Assay (comprising of 40 DNA target genes, 29 driver genes, and a broad fusion panel) for establishing assay sensitivity, specificity, and precision using Ion torrent PGM and implementation into clinics. Patients or other participants Archived DNA samples (n=17) with known mutations in (FLT3-ITD, CEBPA, NPM1, PTPN11, cMPL1, IDH1/2, TET, EZH2, U2AF1, SETBP1, ASLX1, TP53) and RNA samples (n=11) with FISH/RT-PCR-confirmed fusions (NUP 214, RUNX1, PML RARA BCR 1, PML RARA BCR 3, MLLT3, B14A3, B19A2) were retrieved as positive controls and negative controls for the validation. The sequences were analyzed using variant calling software (Ion Reporter Annotate variants version 5.4, 5.6, and 5.10) caller to identify variants relevant to the clinical indication. After thorough optimization and validation, this panel was used for prospective analysis in patients with suspected myeloid disorders. [AML (n=36), MDS (n=12), MPN (n=9)] AUL (n=1), Cytopenia/anaemia under evaluation (n=8) and CMML (n=3)]. Intervention FLT3-ITD by fragment analysis was performed in all cases after validation. Main outcome measures After implementation of NGS platform in myeloid neoplasms FLT3 and TET2 genes were the most common pathogenic variant in DNA sequencing and KMT2A rearrangement on RNA sequencing. Results In the four validation runs (318 chip - 4 DNA/RNA) comprising 28 known variants, all mutations/rearrangements were concordant except two cases (FLT3-ITD > 200bp and SETBP1, G870S 3.2%). For the prospective myeloid disorders (n=69), mutations were observed in 31 genes, maximum cases having FLT3 (n=8) and TET2 (n=8). The other frequently mutated genes were SRSF2 (n=7), NRAS (n=6), RUNX1 (n=6), ASXL1 (n=6), CEBPA (n=5), U2AF1 (n=4), SH2B3 (n=4), BCOR (n=4). All FLT3-ITD-positive and -negative cases were confirmed by Fragment Length Analysis. In eleven cases, fusions were detected; KMT2A (n=4), BCR-ABL (n=3), PML-RARA (bcr3) (n=1), and RUNX1T1 (n=2) and ETV6-ABL fusions Conclusions We demonstrate the validation and implementation of a combined targeted DNA and RNA sequencing for routine clinical use in myeloid malignancies.