A comparison of sequential polymerase chain reaction–based cascade testing vs next-generation sequencing in molecular profiling of myeloproliferative neoplasms: improving testing strategies in light of evolving molecular landscapes

Introduction: Somatic mutations in the JAK2, CALR, and MPL genes are traditionally tested using a cascading reflex algorithm in BCR::ABL1–negative myeloproliferative neoplasms (MPNs). However, next-generation sequencing (NGS) has revealed that these variants may coexist, exposing limitations in current testing practices. Methods: This pilot study analyzed 3 JAK2 p.V617F–positive MPN cases and 3 cases negative for classical driver mutations using the Oncomine Myeloid Assay GX v2 assay (Thermo Fisher Scientific) on the Genexus Integrated Sequencer (Thermo Fisher Scientific). Results: JAK2 p.V617F status was 100% concordant between polymerase chain reaction (PCR) and NGS. Next-generation sequencing detected a concurrent driver MPL mutation in 1 case, an SF3B1 variant in 1 case, and an IDH2 variant in a JAK2-positive case that have established prognostic and therapeutic significance. In cases negative for conventional targets, NGS detected DNMT3A and TET2 variants, which are associated with clonal hematopoiesis and MPN initiation. One case had a JAK2 p.V617F alteration at a variant allele frequency of 0.9%, below the NGS-reportable range but detectable by PCR, adding another caveat to profiling of MPNs. Discussion: Next-generation sequencing provides comprehensive molecular profiling in patients with MPNs, identifying additional prognostic and therapeutic markers. However, PCR remains superior for detecting low–variant allele frequency variants. We propose an updated MPN testing strategy that integrates PCR and NGS within a reflex algorithm to optimize diagnostics and therapeutic guidance.