Evaluation of a 42-Gene Next-Generation Sequencing Panel for Detection of Acute Myeloid Leukemia Mutations for Clinical Use

Objectives Identification of genetic alterations by molecular and cytogenetic testing has an important role in the classification, risk stratification, and management of acute myeloid leukemia (AML). We evaluated a next-generation sequencing (NGS) panel that assesses IDH2, FLT3, NPM1, and TP3 and 38 other genes associated with diagnostic, prognostic, and/or therapeutic utility in AML, with a turnaround time of 8 to 14 days, using a DNA input of 50 ng. Methods In total, 110 patients, 65 males and 45 females, at a median age of 58 years (range: 2-68 years) with clinical diagnosis of AML were included in this study. Targeted regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. Sequence reads were analyzed using a bioinformatics pipeline that was developed in house. Analytical sensitivity of the assay is ~5% mutated alleles with 100% specificity. Results A total of 296 pathogenic mutations in 38 genes were detected in 95 of 110 samples (86%). On average, three mutations (range 1-10) were detected per positive sample. Variant frequency ranged from 3.0% to 96.1%. Most frequently mutated genes were TET2 (18.2%), FLT3 (18.2%), TP53 (17.3%), RUNX1 (15.5%), NPM1 (14.5%), ASXL1 (12.7%), NRAS (11.8%), and CEBPA (10.9%). In total, 166 of 296 (56%) mutations from 13 genes had prognostic significance, 42 of 296 (14%) mutations from three genes (CEBPA, NPM1, RUNX1) had diagnostic significance, and 40 of 296 (14%) mutations from three genes (IDH1, IDH2, FLT3) had therapeutic significance. Conclusion This focused 42-gene targeted panel identifies mutations of clinical significance in over 85% of patients with a clinical diagnosis of AML.