High-resolution melting assay for rapid, simultaneous detection ofJAK2,MPLandCALRvariants

Abstract

AimsIdentification of recurrent genetic alterations inJAK2,MPLandCALRremains crucial in the diagnosis of Philadelphia-negative myeloproliferative neoplasms (MPNs). Current laboratory testing algorithms may entail batching and/or sequential testing, involving multiple testing modalities and sometimes send-out testing that increase the technical and economic demands on laboratories while delaying patient diagnoses. To address this gap, an assay based on PCR and high-resolution melting (HRM) analysis was developed for simultaneous evaluation ofJAK2exons 12–14,MPLexon 10 andCALRexon 9, embodied in the HemeScreen® (hereafter ‘HemeScreen’) MPN assay.MethodsThe HemeScreen MPN assay was validated with blood and bone marrow samples from 982 patients with clinical suspicion for MPN. The HRM assay and Sanger sequencing were performed in independent Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories with Sanger sequencing (supported by droplet digital PCR) serving as the gold standard.ResultsHRM and Sanger sequencing had an overall concordance of 99.4% with HRM detecting 133/139 (96%) variants confirmed by sequencing (9/10 MPL, 25/25 CALR, 99/104 JAK2), including 114 single nucleotide variants and 25 indels (3–52 bp). Variants consisted of disease-associated (DA) variants (89%), variants of unclear significance (2%) and non-DA variants (9%) with a positive predictive value of 92.3% and negative predictive value of 99.5%.ConclusionsThese studies demonstrate the exquisite accuracy, sensitivity and specificity of the HRM-based HemeScreen MPN assay, which serves as a powerful, clinically applicable platform for rapid, simultaneous detection of clinically relevant, somatic disease variants.