次世代シーケンシングホットスポットパネルで初期評価された消化管間質腫瘍におけるKIT変異検出の向上におけるジデオキシシーケンシング

Gastrointestinal stromal tumors (GISTs) are predominantly characterized by mutations in KIT or PDGFRA. Mutation detection is important for optimal therapy. Next-generation sequencing (NGS) panels are useful in GIST assessment as they allow for simultaneous evaluation of multiple genes. However, inherent to use of NGS with short-read sequences on formalin-fixed specimens is the potential to miss larger insertion or deletion variants. Over 9 years, GIST testing was performed on specimens from 55 patients by amplicon-based, semiconductor NGS using the Ion AmpliSeq Cancer Hotspot Panel version 2, a Cancer Hotspot Panel version 2-based GIST panel, or the Oncomine Precision Assay. Negative cases were evaluated by dideoxy sequencing for detection of mutations in KIT and PDGFRA, as per the clinical protocol. Before reflexive sequencing, of 55 completed analyses, 47 (85%) were positive for KIT or PDGFRA mutations by NGS. Three cases were attributed to positivity for pathogenic variants in other genes. Of the five cases evaluated by dideoxy sequencing, all five (9% of all specimens) were positive for pathogenic or likely pathogenic KIT mutations. A total of 12% of KIT mutations required dideoxy sequencing for identification. Mutations were 12 to 39 nucleotide deletion or duplication variants. The results suggest that short-read, amplicon-based NGS assays may miss a significant number of clinically actionable KIT mutations and that follow-up of KIT and PDGFRA NGS-negative cases by alternative testing modalities should be considered.