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.
Highlights
Myeloid neoplasms are a heterogeneous group of malignancies of the hematopoietic stem/progenitor cells
Substantial clinical and genomic overlap exists among different subclasses of myeloid neoplasms that are currently classified by the World Health Organization (WHO): acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), and overlap myelodysplastic/myeloproliferative neoplasms (MDS/MPN) [1]
For MPN, certain mutations such as JAK2 V617F or exon 12 mutation satisfy diagnostic criteria to help establish a diagnosis of MPN [5]; mutations in ASXL1, SRSF2, EZH2, IDH1, and IDH2 categorize patients to high molecular risk in Primary Myelofibrosis (PMF) [6]; and mutations in IDH2, U2AF1, EZH2, TP53, SH2B3, and SF3B1 indicate adverse prognostic value in Essential Thrombocythemia (ET) and Polycythemia Vera (PV) [7, 8]
Summary
Myeloid neoplasms are a heterogeneous group of malignancies of the hematopoietic stem/progenitor cells. Morphologic, and immunophenotypic abnormalities, identification of genetic alterations by molecular testing has an important role in the classification, risk stratification, and management of myeloid neoplasms [2]. For patients with MDS, identification of mutations in genes including TET2, SF3B1, ASXL1 and TP53 is useful to help establish clonal hematopoiesis to make a definitive diagnosis of MDS [4]. For MPN, certain mutations such as JAK2 V617F or exon 12 mutation satisfy diagnostic criteria to help establish a diagnosis of MPN [5]; mutations in ASXL1, SRSF2, EZH2, IDH1, and IDH2 categorize patients to high molecular risk in Primary Myelofibrosis (PMF) [6]; and mutations in IDH2, U2AF1, EZH2, TP53, SH2B3, and SF3B1 indicate adverse prognostic value in Essential Thrombocythemia (ET) and Polycythemia Vera (PV) [7, 8]
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