Analytical validation of a 47-gene NGS panel for molecular profiling of myeloid neoplasms.

Abstract

e18526 Background: Molecular profiling can help diagnose, classify, and guide treatment of myeloid neoplasms. Next generation sequencing (NGS) is a powerful platform that can facilitate identification of actionable genetic alterations in multiple genes simultaneously. To serve clinical management needs, we developed and validated a NGS panel for 47 genes associated with diagnostic, prognostic, and/or therapeutic utilities for each of the subclasses of myeloid neoplasms: acute myeloid leukemia (AML, 42 genes), myelodysplastic syndrome (MDS, 36 genes), and myeloproliferative neoplasms (MPN, 26 genes). Methods: A total of 154 unique de-identified specimens were analyzed. Intra- and inter-assay precision studies were performed by standard laboratory practice. Single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 partial tandem duplications (PTD) were assessed for the entire coding regions of 23 genes and targeted exons of 24 genes. Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. Sequence reads obtained were analyzed using a bioinformatics pipeline that was developed in-house. At least 500 unique reads with base quality (Q) ≥20 (99% confidence) were required for coverage QC, except targets on X chromosome for male specimens, which required 250 unique reads. Results: On average, each sample generated 13.2 million reads, 100% (SD = 0.2%) mapped reads to the reference sequence, 61.7% (SD = 8.8%) on-target reads, and 1,952 (SD = 873) mean coverage. Data using ≥Q20 unique coverage of all target regions across all validation samples demonstrated that all target regions, even in the extreme end of GC spectrum (range: 12%-87%), met coverage requirements. Intra- and inter-assay precision studies, using 7 and 21 specimens, respectively, resulted in 100% concordance among replicates. An accuracy study of 131 unique specimens (a subset of the 154 specimens) yielded in 99.6% (755/758, 95% CI: 99.4%-100%) concordance for variants with allele frequencies ≥5% in comparison to other molecular methods including Sanger sequencing. No reportable variants were detected from any normal specimens (n = 17). Conclusions: We developed and validated a NGS panel for 47 genes with diagnostic, prognostic, and/or therapeutic utilities for each of the subclasses of myeloid neoplasms: AML (42 genes), MDS (36 genes), and MPN (26 genes). This panel will allow simultaneous analysis of the 47 genes with high sensitivity and specificity and may help manage clinical needs.