Sunder, Sham

Abstract Introduction/Objective Mutations in KIT and PDGFRA drive the majority of gastrointestinal stromal tumors (GIST) and inform targeted therapy selection. While single-driver mutations are typical, a subset of cases harbors multiple KIT mutations, complicating clinical interpretation. This study investigated the frequency, distribution, and inferred hierarchy of primary and secondary KIT mutations using DNA-based sequencing in a large GIST cohort. Methods/Case Report We retrospectively reviewed 579 GIST cases that underwent DNA-based next-generation sequencing using a custom panel targeting all coding exons of KIT and PDGFRA. Tumor-rich formalin-fixed paraffin-embedded (FFPE) tissue was required. In double-mutant cases, the mutation with higher variant allele frequency (VAF) was presumed to be primary. Results Mutations in KIT or PDGFRA were detected in 482/579 cases (83.2%). Among KIT-mutant cases, 28 had two mutations and one had three. Secondary mutations frequently occurred in exons 13, 14, and 17 and were significantly more common in non-gastric tumors (p < 0.0001). Primary mutations most often involved exon 11. Conclusion VAF analysis enabled classification of mutation hierarchy in cases with multiple KIT mutations. The distinct anatomical and exon-specific distribution of primary vs. secondary mutations highlights their potential therapeutic implications and warrants further study.