Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms

Abstract

Identification of genomic mutations by molecular testing plays an important role in diagnosis, prognosis, and treatment of myeloid neoplasms. Next-generation sequencing (NGS) is an efficient method for simultaneous detection of clinically significant genomic mutations with high sensitivity. Various NGS based in-house developed and commercial myeloid neoplasm panels have been integrated into routine clinical practice. However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS panels (e.g., CEBPA, CARL, and FLT3). We report development and validation of a 48-gene NGS panel that includes genes that are technically challenging for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. A bioinformatics pipeline that was developed in-house was used to detect single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). An analytical validation study was performed on 184 unique specimens for variants with allele frequencies ≥5%. Variants identified by the 48-gene panel were compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 unique specimens. The developed assay was applied to a large cohort (n = 2,053) of patients with suspected myeloid neoplasms. Analytical validation yielded 99.6% sensitivity (95% CI: 98.9–99.9%) and 100% specificity (95% CI: 100%). Concordance of variants detected by the 2 tested panels was 100%. Among patients with suspected myeloid neoplasms (n = 2,053), 54.5% patients harbored at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN. Together, these findings demonstrate that the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms even in technically challenging genes.

Gene mutation analysis using next‐generation sequencing and its clinical significance in patients with myeloid neoplasm: A multi‐center study from China

“…The gene mutation structure was differed slightly from a recent study; the study demonstrated ASXL1, SRSF2 , and TET2 mutations were more common 17 . NPM1, DNMT3A, FLT3 , and NRAS had a high mutation frequency in AML 6,18 . In this study, epigenetic abnormalities were the most prominent in ML, where TET2, ASXL1, GATA2 , and FLT3 had the highest mutation frequency (≥20%), followed by NPM1, NRAS , and DNMT3A (10%–20%); the results were slightly different from those of previous studies; however, these genes are common in AML.…”

“… 17 NPM1, DNMT3A, FLT3 , and NRAS had a high mutation frequency in AML. 6 , 18 In this study, epigenetic abnormalities were the most prominent in ML, where TET2, ASXL1, GATA2 , and FLT3 had the highest mutation frequency (≥20%), followed by NPM1, NRAS , and DNMT3A (10%–20%); the results were slightly different from those of previous studies; however, these genes are common in AML.…”

Genomic Alterations, Gene Expression Profiles and Functional Enrichment of Normal-Karyotype Acute Myeloid Leukaemia Based on Targeted Next-Generation Sequencing

“…Although they performed WES sequencing with greater genome coverage, the number of somatic variants was relatively low, especially in the myeloid-related genes included in our targeted NGS panel. The number of somatic mutations detected in our cohort was lower than in other studies with similar targeted myeloid panel designs [16,51].…”

Precision Diagnostics in Myeloid Malignancies: Development and Validation of a National Capture‐Based Gene Panel

“…As a joint effort to keep up with state-of-the-art genomic characterization of myeloid malignancies, Genomic Medicine Sweden (GMS), a national initiative in precision medicine, in collaboration with the SciLifeLab Clinical Genomics platform, is currently developing and harmonizing the use of larger, capture-based gene panels for improved cancer diagnostics [18]. Most gene panels currently used in diagnostics of hematologic malignancies are PCR amplicon-based and rely on a small, fixed gene content (e.g., 40-70 genes) [19][20][21][22][23][24][25][26]. Probe-based capture enrichment, however, has several advantages compared with PCR amplicon-based, as it is easier to update, displays a much more even target-coverage, allows larger content to similar sequencing capacity, and performs especially well across complex, repetitive regions, and GCrich areas.…”