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

<i>MET</i> exon 14 (<i>MET</i>ex14) skipping mutations differ from other non-small cell lung cancer (NSCLC) genomic biomarkers as they result in aberrantly spliced <i>MET</i> transcripts and increased MET-signaling. However, the most accurate method for their detection remains debated. We conducted a retrospective study of previously identified <i>MET</i>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 <i>MET</i>ex14 and supplemented by targeted RNA sequencing for selected cases. Using this approach, we identified <0.2% patients with <i>MET</i>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 <i>MET</i>ex14-positive patients (3.5%) and allowed us to identify <i>MET</i>ex14 skipping transcripts. Collectively, data from our cohort (n = 34) demonstrated that optimal diagnostics of <i>MET</i>ex14 variants require a complementary DNA-NGS performed with targeted panels covering both <i>MET</i>ex14 splice sites, and RNA-NGS. Consequently, we propose a new workflow for interpretation of concordant and discordant findings in <i>MET</i>ex14 detection. Finally, the potential of DNA-identified <i>MET</i>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 <i>MET</i>ex14, which also produced the <i>METex14</i> 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.