P094 | HIGH-SENSITIVITY DETECTION OF LOW ALLELE FREQUENCY V617F VARIANT IN THE <i>JAK2</i> GENE USING DIGITAL PCR (DPCR): A NOVEL TOOL FOR DIAGNOSIS OF MYELOPROLIFERATIVE NEOPLASMS

Accurate quantification of the V617F variant in the JAK2 gene using highly sensitive technologies is crucial for the diagnosis, therapeutic monitoring and prognostic assessment of patients with myeloproliferative neoplasms (MPNs). Although the clinical relevance of variant allele frequency (VAF) in predicting disease phenotype and progression risk remains uncertain, the 2022 International Consensus Classification (ICC) strongly recommends the detection of variants with VAF below 1%. The integration of a sensitive and standardized dPCR assay into routine diagnostics became essential. In this study, we developed a dPCR assay based on the QuantStudio Absolute Q Digital PCR System (Applied Biosystems), which employs Microfluidic Array Plate (MAP) technology to enable absolute quantification across 20,480 fixed-array microchambers, without the need for a calibration curve. In 2024, the assay was validated using well-characterized diagnostic samples and WHO international reference material, with comparative analyses conducted as a part of the GIMEMA-JakNet external quality assurance program. A TaqMan-based chemistry with allele-specific FAM and VIC labeled probes was optimized for V617F detection. PCR conditions were refined to ensure optimal MAP coverage (≥20,000 microchambers) and a limit of detection of 0.01% (1 in 10,000 copies/μL). The assay showed high concordance with expected values, including the WHO sample with a VAF of 0.03% (Standard deviation ± 0.01%). Between January 2022 and March 2025, over 1,000 patients with suspected MPNs were screened using quantitative real-time PCR on DNA extracted from peripheral blood. Among these, 190 patients tested positive for the V617F variant. Notably, 28 cases exhibited a VAF below 1%, confirmed by repeat testing. In 22 of these low-VAF cases, a definitive diagnosis of essential thrombocythemia or polycythemia vera was established. Since 2025, the dPCR assay has been incorporated into our routine diagnostic workflow as an alternative method for low-level V617F detection, with 6 patients analyzed to date. In conclusion, dPCR represents a robust, high-sensitivity approach for detecting low-frequency V617F variants. Nonetheless, clinical interpretation remains challenging in distinguishing pathogenic driver mutations from variants associated with clonal hematopoiesis of indeterminate potential (CHIP).