Reclassification of Noncanonical MET Splice-Site Variants Using RNA-Based Validation: A Practical Diagnostic Approach in Non-Small Cell Lung Cancer.

Abstract

MET exon 14 (METex14) skipping alterations represent actionable oncogenic drivers in non-small cell lung cancer (NSCLC). However, noncanonical intronic splice-site variants are frequently classified as variants of uncertain significance (VUS) on DNA-based next-generation sequencing (NGS), limiting timely therapeutic decision making. We describe two patients with advanced NSCLC harboring novel intronic deletions affecting the MET exon 14 splice acceptor region, initially classified as VUS. Integrated transcript level analysis using RNA sequencing and orthogonal validation by reverse transcription PCR (RT-PCR) was performed to assess functional impact. RNA-based analysis in both cases demonstrated METex14 skipping through identification of exon 13-15 junction reads, which was further confirmed by RT-PCR. Based on functional evidence, both variants were reclassified as clinically actionable alterations (AMP/ASCO/CAP Tier II), directly influencing therapeutic management with MET-targeted tyrosine kinase inhibitors. Early clinical stabilization was observed in both patients. These findings support the routine incorporation of RNA-based validation strategies to improve accurate interpretation of noncanonical MET splice-site variants. We propose a practical diagnostic workflow for evaluation and reclassification of MET splice-site VUS, emphasizing the role of transcript-level evidence in enabling accurate molecular reporting and guiding targeted therapy.