MDS-227: Digital PCR for Sensitive Detection and Accurate Quantification of KIT D816V Allele Burden in Patients with Suspected Systemic Mastocytosis

Context: Detection of the KIT D816V mutation in the bone marrow (BM) is one of the minor criteria for the diagnosis of systemic mastocytosis (SM). Because in most SM patients, the number of neoplastic mast cells (MCs) in the BM is low, the use of molecular techniques, such as ASO-qPCR, with elevated sensitivity is recommended. Droplet digital PCR (ddPCR) is a promising alternative to ASO-qPCR for D816V testing. Objective: We evaluated a commercial ddPCR assay for the detection of KIT D816V in SM. ddPCR was performed on a BioRad QX200 instrument using the ddPCR™ Mutation Assay: KIT p.D816V, Human (Bio-Rad). Patients: PB samples from 13 healthy donors (HDs) were used as negative controls; the D816V-positive HMC-1.2 cell line was used as positive control. Thirty-five BM samples from patients with suspected SM, either with neoplastic mast cell infiltration Results: In HMC 1.2 cells, ddPCR measured an allele burden (AB) of 50%, consistent with the heterozygous status. No D816V-positive events were detected in the HDs. Comparison between ddPCR and ASO-qPCR revealed high concordance in mutation detection and quantitation, even at low AB levels. ddPCR identified D816V in 70/151 patients prospectively evaluated for diagnosis of SM. According to WHO criteria, 49 were later diagnosed with ISM, 13 with aggressive SM, 4 with smouldering SM, 2 with SM with an associated clonal hematopoietic non-MC disease, and 2 with MC leukemia. The median AB in all positive SM patients was 0.34%. Patients with advanced SM showed a higher D816V AB (median, 15.23%) compared with patients with ISM and smouldering SM (median, 0.27%). All patients positive for D816V in BM also tested positive in the PB. Conclusion: ddPCR proved accurate and precise and was able to detect D816V in PB when the BM was positive. We propose ddPCR as a valid alternative to ASO-qPCR for straightforward and sensitive detection of D816V in SM.