title: Targeted ultradeep next-generation sequencing as a method for KIT D816V mutation analysis in mastocytosis.
abstract: 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 D816V 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 D816V detection using the Ion Torrent platform. Eighty-two individual NGS analyses were included in the study. All samples were also analyzed using highly sensitive KIT D816V mutation-specific qPCR. Measurements of the background level in D816V-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 D816V 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 D816V mutation analysis in mastocytosis, as many patients with SM have D816V mutation levels below the detection limit of NGS. A dedicated and highly sensitive KIT D816V mutation analysis therefore remains important in mastocytosis diagnostics.
authors.affiliation: Department of Pathology, Odense University Hospital, Odense, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kristensen
authors.affiliation: Department of Dermatology and Allergy Centre, Odense University Hospital, Odense, Denmark.
authors.forename: Sigurd
authors.initials: S
authors.lastname: Broesby-Olsen
authors.affiliation: Department of Hematology, Odense University Hospital, Odense, Denmark.
authors.forename: Hanne
authors.initials: H
authors.lastname: Vestergaard
authors.affiliation: Department of Dermatology and Allergy Centre, Odense University Hospital, Odense, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Bindslev-Jensen
authors.affiliation: Department of Pathology, Odense University Hospital, Odense, Denmark.
authors.forename: Michael Boe
authors.initials: MB
authors.lastname: Møller
mesh_terms: D000328:Adult; D000483:Alleles; D016022:Case-Control Studies; D004252:DNA Mutational Analysis; D005260:Female; D015870:Gene Expression; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D034721:Mastocytosis, Systemic / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D019009:Proto-Oncogene Proteins c-kit
keywords: KIT D816V mutation; gene panels; next-generation sequencing; systemic mastocytosis