Introduction: Systemic mastocytosis (SM) is a rare, clonal, hematopoietic neoplasm characterized by excessive proliferation and hyperactivation of abnormal mast cells (MCs). Approximately 95% of patients with SM harbor the KIT D816V mutation within the KIT receptor tyrosine kinase, which results in constitutive activation of the KIT receptor. Abnormal activation of KIT leads to increased proliferation, survival, and hyperactivation of MCs, which can cause debilitating constitutional symptoms such as pruritus, flushing, headaches, bone pain, nausea, vomiting, diarrhea, and life-threatening anaphylaxis, across all SM subtypes.Polymerase chain reaction (PCR)-based detectionof the KIT D816V mutation in bone marrow (BM) aspirates or other extracutaneous tissues is an important tool to confirm clinical and pathological diagnosis. In addition, genomic determination of the KIT D816V variant allelic fraction (VAF) in peripheral blood (PB) or BM can serve as a potential prognostic tool and may also be used to monitor treatment efficacy over time. However, low numbers of neoplastic MCs in BM or PB samples, especially from patients with non-advanced forms of the disease (ISM and smoldering SM) can result in misdiagnosis and underestimation of disease burden. Here, we report on the performance of an adapted ddPCR assay compared with NGS for measuring the KIT D816V VAF in PB samples from patients with moderate to severe ISM and symptoms inadequately controlled with best supportive care enrolled in part 1 (randomized dose finding part to determine recommended dose) of the ongoing, randomized, double-blind, placebo-controlled phase 2 PIONEER clinical study (NCT03731260) of avapritinib, a potent and selective inhibitor of KIT D816V mutant kinases.

Methods: Part 1 of PIONEER included 39 patients (3 cohorts with 10 patients at 25, 50 and 100 mg of avapritinib, and a placebo cohort of 9 patients) with a confirmed diagnosis of ISM based on World Health Organization criteria and moderate-to-severe symptoms based on Total Symptom Score as measured on the ISM-System Assessment Form (ISM-SAF). Samples of PB were collected at screening for all enrolled patients in part 1 and were evaluated by local quantitative and qualitative KIT testing, central ddPCR assay for detection of KIT D816V, and central NGS (TruSight™ Myeloid Panel) for KIT D816V and other co-mutations (central assays performed by MolecularMD, Portland, OR). Results were expressed as the percentage of patient PB samples testing positive for KIT D816V mutation (all genomic assays) and the log percent of VAF as measured by both central assay methods. Correlations of KIT D816V VAF to objective measure of MC burden, including serum tryptase levels and BM MC numbers, were performed. KIT D816V VAF was also evaluated serially in each patient over the treatment period.

Results: The central ddPCR assay method detected the KIT D816V mutation in 37/39 (95%) of PB samples compared with 11/39 (28%) assayed by NGS and 30/39 (80%) of PB samples assayed locally (Table). ddPCR also demonstrated ~30-fold greater sensitivity over NGS for measuring VAF: median percent VAF (range) by ddPCR was 0.36 (0.02-30) and by NGS was 11 (1.9-31) (Figure). The lower limit of detection of VAF by ddPCR on PB samples was 95-fold lower than that of NGS (0.02% and 1.9%, respectively). In addition, ddPCR provided greater diagnostic sensitivity for ISM compared with serum tryptase >20 ng/mL (30/39, 77%) and presence of BM MC aggregates (35/39, 90%).

Conclusions: The high-sensitivity ddPCR assay method detected the KIT D816V mutation in 95% of PB samples from patients with previously confirmed ISM, which provided greater sensitivity for detection of KIT D816V mutation than either NGS or local assessments, greater sensitivity over NGS for KIT D816V VAF in PB samples, and higher diagnostic sensitivity than clinical assessments of serum tryptase and BM MC aggregates. These results have implications for assay sensitivity in the diagnosis of SM and suggest that higher sensitivity testing for KIT D816V mutation using a minimally invasive PB ddPCR assay could be used as a screening tool to facilitate identification of ISM patients, including those with variable disease characteristics or low MC burden.


George:Blueprint Medicines Corporation: Consultancy, Other: I have received no funding for this research. ARUP Laboratories, owned by the University of Utah, has received funding; Allakos: Consultancy; Celgene: Consultancy; Deciphera: Other: consultancy, but has received no financial compensation for the past 12 months. Hoehn:Blueprint Medicines Corporation: Current Employment, Current equity holder in publicly-traded company. Lin:Blueprint Medicines Corporation: Current Employment, Current equity holder in publicly-traded company. Miller:Blueprint Medicines Corporation: Current Employment, Current equity holder in publicly-traded company. Akin:Novartis: Consultancy; Blueprint Medicines Corporation: Consultancy, Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.