RNA-Based and DNA-Based Next-Generation Sequencing of KIT and PDGFRA Mutations in Gastrointestinal Stromal Tumors: Analytical Performance and Epidemiologic Insights.

Mutations in the KIT and PDGFRA proto-oncogenes are key drivers in gastrointestinal stromal tumors (GIST) and guide targeted therapy. While DNA-based next-generation sequencing (NGS) is standard for mutation detection, RNA sequencing (RNA-Seq) may offer complementary advantages. This study evaluates RNA-Seq performance and presents molecular epidemiologic data from a large DNA-sequenced GIST cohort. RNA-Seq was performed on 24 GIST cases previously analyzed by DNA-based NGS (16 KIT mutants, 5 PDGFRA mutants, 3 wild-type) using the Agilent SureSelectXT RNA Direct Library Prep Kit and an in-house analysis pipeline. RNA-Seq was successful in 21 cases, identifying 13 of 14 KIT mutations and all PDGFRA mutations, including single nucleotide variants (SNV), insertions, and in-frame deletions (3-27 bp). One complex 45 bp deletion-insertion was not detected, though low-level evidence (<10% of reads) was present. Separately, DNA-based NGS results from 579 GIST cases were reviewed to assess mutation prevalence and clinicopathologic associations. Mutations were found in 83.2% of cases (403 KIT, 79 PDGFRA), with secondary KIT mutations in 6.0%. Mutation site correlated with tumor location and patient age; secondary mutations were more frequent in non-gastric tumors. RNA-Seq demonstrates high accuracy for detecting clinically relevant KIT and PDGFRA mutations and may complement DNA-based profiling. The DNA cohort provides broader context for mutation prevalence and patterns in clinical practice.