RNA Testing Confirms MET Exon 14 Skipping Missed by DNA-Only Approaches

These alterations occur in 3% to 4% of patients and can be missed by DNA-only next-generation sequencing (NGS). Interpretation difficulties in routine molecular pathology can therefore jeopardize patient selection for treatment. New findings demonstrate that RNA-based testing can clarify this biomarker and identify variants that drive exon 14 skipping.

Researchers at the University of Cologne evaluated RNA-based testing for MET exon 14 alterations in a large real-world cohort of patients with NSCLC. The analysis, detailed in The Journal of Molecular Diagnostics, focused on assay design and variant interpretation issues observed in routine practice. The work also examined laboratory performance through multinational external quality assessment programs. The approach centered on integrating RNA analysis with DNA-based NGS to resolve splicing events.

RNA analysis directly confirms exon 14 skipping at the transcript level, capturing events that may be overlooked or misclassified by DNA-only testing. Mutations that lead to skipping are exceptionally heterogeneous and extend across the splice-site region, creating known analytical pitfalls for both DNA- and RNA-based NGS assays. The study specifically identified challenges related to assay design limitations, bioinformatic filtering, and variant interpretation, and assessed how RNA confirmation can help address them.

In this large real-world cohort, investigators systematically characterized the full spectrum of MET exon 14-associated alterations encountered in routine clinical practice. They assessed functional consequences at the RNA level and cataloged potential diagnostic pitfalls. Laboratory performance was also evaluated through multinational external quality assessment (EQA) schemes using both tissue and liquid biopsy samples.

The research identified 171 distinct mutations affecting the MET exon 14 splice region. RNA analysis confirmed exon 14 skipping for more than 100 different alteration types and showed that large genomic deletions and a synonymous variant affecting splicing may be missed or misinterpreted by DNA-based testing alone. EQA results showed excellent performance for tissue-based testing, with an overall success rate of 98%, while liquid biopsy detection remained more challenging, improving from 37.5% in 2022 to 63% in 2024.

The authors note that certain clinically relevant MET exon 14 events may be missed by DNA-only approaches, underscoring the value of incorporating RNA-based confirmation where feasible to improve sensitivity and specificity. Because detection determines eligibility for MET-targeted therapies, reliable identification across laboratories remains a priority. The study was published online on June 4, 2026, in The Journal of Molecular Diagnostics.

“Although MET exon 14 is a well-established predictive biomarker, our findings highlight a level of molecular complexity that is often underappreciated in routine diagnostic practice,” said lead investigator Carina Heydt, Ph.D., University of Cologne, Faculty of Medicine, and University Hospital Cologne, Institute of Pathology.

"This research has important implications for routine clinical molecular pathology. Our study provides evidence supporting the complementary role of RNA-based testing in the assessment of MET exon 14 alterations and highlights the value of EQA programs in monitoring assay performance, identifying analytical limitations, promoting harmonization of testing strategies, and ensuring accurate and reproducible results for patient selection for MET-targeted therapies," said Heydt.

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University of Cologne, Faculty of Medicine
University Hospital Cologne