Clinical Validation of a Myeloid Next-Generation Sequencing Panel for Single-Nucleotide Variants, Insertions/Deletions, and Fusion Genes

Abstract

Myeloid neoplasms are a heterogeneous group of neoplasms including acute myeloid leukemia (AML), myeloproliferative neoplasms, myelodysplastic syndrome, and myeloproliferative neoplasms/myelodysplastic syndrome. Genetic abnormalities are used as diagnostic, prognostic, and predictive biomarkers in patients with these diseases. Herein, we describe the clinical validation of the Oncomine Myeloid Research (OMR) next-generation sequencing panel that interrogates for 40 genes and 29 fusion genes commonly seen in myeloid neoplasms. Our validation set of 77 DNA samples included acute and chronic myeloid neoplasms, with 91 single-nucleotide variants and small insertions/deletions. The 71 RNA samples from patients with AML included most of the AML-defining translocations. The OMR on the Ion Torrent S5 platform shows good performance in terms of depth of coverage, on-target reads, and uniformity. The panel achieved 91.3% and 100% concordance with reference DNA and RNA samples, respectively, with a clinical sensitivity and specificity of 96.7% and 100% for DNA and 99.8% and 100% for RNA, respectively. Precision and reproducibility were 100%, and the lower limit of detection was generally 5% variant allele fraction for DNA and 2-log reduction from initial value for RNA fusion genes. In conclusion, the OMR panel is a highly accurate and reproducible next-generation sequencing panel for the detection of common genetic alterations in myeloid neoplasms.

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

“…Among many others, amplicon-based commercial panels, e.g. Illumina TruSight Myeloid panel, Thermo Fisher Oncomine Myeloid research panel, and Archer VariantPlex Core Myeloid panel are most commonly used in clinical laboratories [12,21,22]. However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS, especially with amplicon-based or RNA bait capture-based panels.…”

“…However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS, especially with amplicon-based or RNA bait capture-based panels. For example, technical difficulties hinder the ability to capture targets with high GC content, such as CEBPA, which is associated with poor prognosis of AML, or repetitive genomic regions, such as FLT3-ITD, which is associated with poor prognosis of AML in the absence of NPM1 mutation [10,12,[21][22][23][24][25][26][27]. Aguilera-Diaz and colleagues evaluate the performance of four different targeted NGS gene panels, three commercial panels and one in-house developed panel, and noted that CEBPA, CALR and FLT3 genes remain challenging the use of NGS for diagnosis of myeloid neoplasms in compliance with current guidelines [21].…”

“…Aguilera-Diaz and colleagues evaluate the performance of four different targeted NGS gene panels, three commercial panels and one in-house developed panel, and noted that CEBPA, CALR and FLT3 genes remain challenging the use of NGS for diagnosis of myeloid neoplasms in compliance with current guidelines [21]. Similarly, Lzevbaye and colleagues evaluated an amplicon based commercial panel and noted that the panel is unable to detect specific variants in ASXL1 and CEBPA in long homopolymer regions [22]. The application of NGS in clinical settings has additional challenges.…”

Broad‐spectrum next‐generation sequencing‐based diagnosis of a case of Nager syndrome

“…The similar phenotypes, along with the small number of reported cases, make it difficult to diagnose this rare disease. Next‐generation sequencing (NGS) is widely used to diagnose genetic diseases 12 . Here, we report a case which was initially diagnosed as TCS.…”

“…Next-generation sequencing (NGS) is widely used to diagnose genetic diseases. 12 Here, we report a case which was initially diagnosed as TCS. However, gene detection by next-generation sequencing helped us to arrive at a final diagnosis of Nager syndrome.…”