Improvement of Diagnostics in NSCLC Patients with Exon 14 Mutations Using Complementary DNA/RNA-NGS and Identification of Two Novel Exonic Splicing Mutations.

exon 14 (ex14) skipping mutations differ from other non-small cell lung cancer (NSCLC) genomic biomarkers as they result in aberrantly spliced transcripts and increased MET-signaling. However, the most accurate method for their detection remains debated. We conducted a retrospective study of previously identified ex14 skipping NSCLC samples by using different, commercially available, diagnostic targeted DNA- /RNA-Next-Generation Sequencing (NGS) panels. We primarily used small DNA-NGS panels covering the 5' splice site of ex14 and supplemented by targeted RNA sequencing for selected cases. Using this approach, we identified <0.2% patients with ex14 mutations. Due to this low frequency, we validated and introduced complementary NGS testing using combined DNA/RNA-panels. This resulted in an increased number of ex14-positive patients (3.5%) and allowed us to identify ex14 skipping transcripts. Collectively, data from our cohort (n = 34) demonstrated that optimal diagnostics of ex14 variants require a complementary DNA-NGS performed with targeted panels covering both ex14 splice sites, and RNA-NGS. Consequently, we propose a new workflow for interpretation of concordant and discordant findings in ex14 detection. Finally, the potential of DNA-identified ex14 variants to cause aberrant splicing was in silico assessed by the MaxEntScan tool, providing a quantitative approach to splicing disruption. Interestingly, we also identified two novel variants located inside ex14, which also produced the skipping transcript despite being located outside the canonical splice sites. The altered binding site resulting from these exonic mutations was in silico determined by SpliceTransformer.