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

PLoS One. 2021 Apr 28;16(4):e0243683. doi: 10.1371/journal.pone.0243683. eCollection 2021.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Hematologic Neoplasms* / genetics
  • Hematologic Neoplasms* / metabolism
  • High-Throughput Nucleotide Sequencing*
  • Humans
  • Leukemia, Myeloid, Acute* / genetics
  • Leukemia, Myeloid, Acute* / metabolism
  • Mutation*
  • Myelodysplastic Syndromes* / genetics
  • Myelodysplastic Syndromes* / metabolism
  • Neoplasm Proteins* / genetics
  • Neoplasm Proteins* / metabolism

Substances

  • Neoplasm Proteins

Grants and funding

This research was supported by Quest Diagnostics. The specific roles of these authors are articulated in the ‘author contributions’ section. The authors received no specific funding for this work. Quest Diagnostics provided support in the form of salaries for all authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.