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

## TL;DR

This study improves the detection of MET exon 14 mutations in lung cancer patients using combined DNA and RNA sequencing, identifying two new mutations that cause abnormal splicing.

## Contribution

The study introduces a new workflow for METex14 diagnostics and identifies two novel exonic splicing mutations previously unrecognized.

## Key findings

- Combined DNA/RNA NGS increased detection of METex14 mutations from 0.2% to 3.5%.
- Two novel METex14 mutations were found to cause skipping transcripts despite being outside canonical splice sites.
- A new workflow for METex14 detection was proposed based on complementary DNA and RNA sequencing.

## Abstract

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