Identification of Novel MET Exon 14 Skipping Variants in Non-Small Cell Lung Cancer Patients: A Prototype Workflow Involving in Silico Prediction and RT-PCR

HIGHLIGHTS: MET exon 14 skipping is an oncogenic targetable driver mutation in lung cancer. Two novel non-canonical splice site variants identified in MET genome. Predicted splicing strength using in silico splicing prediction tools. Tested routine cytological smear slides for RNA-based molecular diagnostics. RT-PCR and Sanger sequencing analysis confirmed MET exon 14 skipping. SIMPLE SUMMARY: Non-small Cell Lung cancer (NSCLC) contributes to 85% of total lung cancer diagnoses in the United States. With the discovery of various targetable genetic markers and FDA approval of drugs against these markers, genetic testing has become a routine part of the diagnosis and staging process of NSCLC. MET gain of function mutations have been of particular interest as FDA has recently approved two MET inhibitors for the treatment of NSCLC patients with MET exon 14 skipping (METex14) mutations. However, an effective workflow for the classification of various METex14 mutations in the clinical testing laboratory has not been explored. In this report, we reveal two novel METex14 variants and propose a cost-effective and robust workflow for molecular diagnosis of MET variants contributing to exon 14 skipping with the use of readily available specimen sources. ABSTRACT: Background and aims: The MET exon 14 skipping (METex14) is an oncogenic driver mutation that provides a therapeutic opportunity in non-small cell lung cancer (NSCLCs) patients. This event often results from sequence changes at the MET canonical splicing sites. We characterize two novel non-canonical splicing site variants of MET that produce METex14. Materials and Methods: Two variants were identified in three advanced-stage NSCLC patients in a next-generation sequencing panel. The potential impact on splicing was predicted using in silico tools. METex14 mutation was confirmed using reverse transcription (RT)-PCR and a Sanger sequencing analysis on RNA extracted from stained cytology smears. Results: The interrogated MET (RefSeq ID NM_000245.3) variants include a single nucleotide substitution, c.3028+3A>T, in intron 14 and a deletion mutation, c.3012_3028del, in exon 14. The in silico prediction analysis exhibited reduced splicing strength in both variants compared with the MET normal transcript. The RT-PCR and subsequent Sanger sequencing analyses confirmed METex14 skipping in all three patients carrying these variants. Conclusion: This study reveals two non-canonical MET splice variants that cause exon 14 skipping, concurrently also proposes a clinical workflow for the classification of such non-canonical splicing site variants detected by routine DNA-based NGS test. It shows the usefulness of in silico prediction to identify potential METex14 driver mutation and exemplifies the opportunity of routine cytology slides for RNA-based testing.