MPN-182 Droplet Digital PCR for Non-Invasive Detection of the KIT D816V Mutation in the Peripheral Blood of Patients With Suspected Systemic Mastocytosis.

Abstract

Systemic mastocytosis (SM) is a rare and underdiagnosed hematologic neoplasm. Detection of the D816V KIT mutation in the bone marrow (BM) is one of the minor criteria for the diagnosis of SM and requires sensitive methods like ASO-qPCR. In patients with suspected SM, non-invasive pre-screening of peripheral blood (PB) by ASO-qPCR has been shown to detect the D816V mutation with a concordance with BM results of 92% in indolent SM (ISM) patients with skin lesions and of 66% in those without skin lesions. However, ASO-qPCR is not standardized, and commercial kits are not available. Droplet digital PCR (ddPCR) might be a valuable alternative to ASO-qPCR. To validate a commercially available ddPCR assay for the detection and quantitation of the KIT D816V mutation and to evaluate its potential as a non-invasive screening tool in patients with suspected SM. To assess specificity and calculate the limit of blank, PB samples from 30 healthy donors (HDs) were used. The limit of detection was determined by mixing KIT D816V-mutated (HMC-1.2) and unmutated (HMC-1.1) cell lines in order to mimic different allele burdens, from 50% down to 0.01%. Accuracy was investigated by analyzing, in parallel, 35 samples from patients with confirmed or suspected ISM by ASO-qPCR and ddPCR. Concordance between ddPCR results in BM and PB was assessed in 60 matched PB/BM samples. ddPCR was performed using 50 ng/µL of genomic DNA with the KIT p.D816V human mutation assay on a QX200 instrument (Bio-Rad). No KIT D816V-positive events were detected in any of the HD samples. In HMC 1.2 cell dilutions, KIT D816V mutation could be detected down to 0.01% allele burden. Comparison of ddPCR and ASO-qPCR revealed very high concordance in mutation detection and quantitation. All patients positive for KIT D816V in the BM also tested positive in the PB by ddPCR. Moreover, ddPCR revealed high concordance in mutation quantitation between BM and PB, even at very low variant allele frequencies. ddPCR is an attractive alternative to ASO-qPCR for KIT D816V mutation detection and quantification in SM and may reliably detect the D816V mutation non-invasively in PB in patients with suspected SM.