Targeted ultradeep next‐generation sequencing as a method for KIT D 816 V mutation analysis in mastocytosis

Next‐generation sequencing ( NGS ) is becoming increasingly used for diagnostic mutation analysis in myeloid neoplasms and may also represent a feasible technique in mastocytosis. However, detection of the KIT D 816 V mutation requires a highly sensitive method in most patients due to the typically low mutation levels. In this study, we established an NGS ‐based KIT mutation analysis and analyzed the sensitivity of D 816 V detection using the I on T orrent platform. Eighty‐two individual NGS analyses were included in the study. All samples were also analyzed using highly sensitive KIT D 816 V mutation‐specific qPCR . Measurements of the background level in D 816 V ‐negative samples supported a cutoff for positivity of 0.2% in three different NGS panels. Clinical samples from patients with SM that tested positive using qPCR with a D 816 V allele burden >0.2% also tested positive using NGS . Samples that tested positive using qPCR with an allele burden <0.2% tested negative using NGS . We thereby demonstrate that caution should be taken when using the potentially very sensitive NGS technique for KIT D 816 V mutation analysis in mastocytosis, as many patients with SM have D 816 V mutation levels below the detection limit of NGS . A dedicated and highly sensitive KIT D 816 V mutation analysis therefore remains important in mastocytosis diagnostics.