Improved diagnostic screening and classification of clonal mast cell diseases by ultrasensitive <i>KIT</i> p.D816V detection

Abstract

Abstract Detection of KIT p.D816V is a cornerstone in the diagnosis and classification of mast cell activation syndromes (MCAS) and mastocytosis. However, KIT p.D816V may be undetected due to a low mutated-cell burden in the blood and bone marrow (BM) of many patients, particularly those without skin lesions. These findings underscore the need for ultrasensitive molecular techniques for the detection of KIT p.D816V in these clinical settings. Here, we evaluated, to our knowledge, for the first time, the sensitivity and specificity of a novel Flow-SuperRCA KIT p.D816V assay, compared with conventional allele-specific oligonucleotide quantitative polymerase chain reaction (ASOqPCR), through the analysis of 548 blood and BM samples from 337 adult patients with MCAS and mastocytosis. Our results demonstrated greater sensitivity of the new technique vs ASOqPCR (limit of detection: 0.001% vs 0.01% variant allele frequency), with higher rates of positivity for KIT p.D816V in whole blood and/or BM of 64% of patients with monoclonal MCAS and 55% of patients with BM mastocytosis (P&amp;lt; .0001). Notably, the sensitivity of the new assay went beyond that of ASOqPCR in purified BM mast cells (P&amp;lt; .0001). Additionally, clonality was newly identified in 18% of patients previously diagnosed with nonclonal MCAS, who presented with a unique cutaneous MCA-related symptomatic profile. These results confirm the high specificity and ultrasensitivity of the Flow-SuperRCA assay for the detection of KIT p.D816V, emerging as a well-suited whole-blood and whole-BM test for diagnostic screening and classification of patients with MCAS and mastocytosis. These findings further highlight the clonal nature of an unprecedently high fraction of patients who presented with anaphylaxis and MCAS, with important pathogenic, diagnostic, and clinical implications.