Superior detection of low-allele burden Janus kinase 2 V617F mutation and monitoring clonal evolution in myeloproliferative neoplasms using chip-based digital PCR

Abstract

The JAK2 V617F is a prevalent driver mutation in Philadelphia chromosome-negative myeloproliferative neoplasms (Ph−MPNs), significantly affecting disease progression, immunophenotype, and patient outcomes. The World Health Organization (WHO) guidelines highlight the JAK2 V617F mutation as one of the key diagnostic criterions for Ph−MPNs. In this study, we analyzed 283 MPN samples with the JAK2 V617F mutation to assess the effectiveness of three detection technologies: chip-based digital PCR (cdPCR), real-time quantitative PCR (qPCR), and next-generation sequencing (NGS). Additionally, we investigated the relationship between JAK2 V617F mutant allele burden (% JAK2 V617F) and various laboratory characteristics to elucidate potential implications in MPN diagnosis. Our findings demonstrated high conformance of cdPCR with qPCR/NGS for detecting % JAK2 V617F, but the mutant allele burdens detected by qPCR/NGS were lower than those detected by cdPCR. Moreover, the cdPCR exhibited high sensitivity with a limit of detection (LoD) of 0.08% and a limit of quantification (LoQ) of 0.2% for detecting % JAK2 V617F in MPNs. Clinical implications were explored by correlating % JAK2 V617F with various laboratory characteristics in MPN patients, revealing significant associations with white blood cell counts, lactate dehydrogenase levels, and particularly β2-microglobulin (β2-MG) levels. Finally, a case report illustrated the application of cdPCR in detecting low-allele burdens in a de novo chronic myeloid leukemia (CML) patient with a hidden JAK2 V617F subclone, which expanded during tyrosine kinase inhibitor (TKI) treatment. Our findings underscore the superior sensitivity and accuracy of cdPCR, making it a valuable tool for early diagnosis and monitoring clonal evolution.