Challenges in Amplicon-Based DNA NGS Identification of MET Exon 14 Skipping Events in Non-Small Cell Lung Cancers

Introduction: MET Exon 14 skipping alterations are drivers of non-small cell lung carcinoma (NSCLC) with responses to tyrosine kinase inhibitors. Amplicon-based DNA NGS assays (DNA NGSs) for the detection of METex14 skipping can yield false-negative results. We examined the efficacy of METex14 skipping with a DNA NGS and reflex RNA-based NGS (RNA NGS) strategy. Materials and Methods: Clinical cases with definitive or suspected lung adenocarcinoma (LungCa), lacking driver mutations with targeted DNA NGS, underwent the RNA NGS to identify oncogenic drivers. Samples with METex14 skipping identified on reflex RNA NGSs were confirmed with Sanger sequencing. Results:METex14 skipping events were detected in 22/762 (2.9%) samples by DNA NGS. RNA NGS identified 10 additional samples, for an overall frequency of 32/762 (4.1%). All 22 METex14 DNA variants affected the donor splice site. Sanger sequencing revealed that missed METex14 variants were largely deletions spanning the ~30 bp intronic region upstream of the splice acceptor site. The failure of DNA NGS to detect all METex14 mutants was due to a lack of coverage of the 3′ splice acceptor site of intron 13, branch point, and polypyrimidine tract. Conclusions: Modification of amplicon-based DNA hotspot assays, with primers that cover both donor and acceptor splice sites, can identify a larger number of METex14 skipping events. Clinical data show that patients with advanced NSCLC and METex14 variants responded to targeted therapy.