RNA-based testing uncovers great diversity in lung cancer mutations

New research has shown that RNA-based testing can improve the detection and interpretation of clinically actionable genomic alterations leading to MET exon 14 skipping in lung cancer patients — important biomarkers that may be missed by DNA-only approaches.

The findings from the study in The Journal of Molecular Diagnostics, AMP's official journal, are poised to lead to improved diagnostic accuracy and help determine patients' eligibility for MET-targeted therapies.

The mutations leading to MET exon 14 skipping occur in 3% to 4% of non–small cell lung cancer (NSCLC) patients. They are exceptionally heterogeneous and occur across a broad splice-site region, creating significant analytical challenges for both DNA- and RNA-based next-generation sequencing (NGS) assays.

This study aimed to improve assay design, variant interpretation, and diagnostic workflows in molecular pathology laboratories. In a large real-world cohort of NSCLC patients, the researchers systematically characterized the spectrum of MET exon 14-associated alterations encountered in routine clinical practice, assessed their functional consequences at the RNA level, identified potential diagnostic pitfalls, and evaluated laboratory performance through multinational external quality assessment (EQA) schemes.

Key findings

The research uncovered the extraordinary diversity of genomic alterations capable of causing MET exon 14 skipping; a total of 171 distinct mutations affecting the MET exon 14 splice region were identified.

"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," explained lead investigator Carina Heydt, Ph.D., of the University of Cologne Faculty of Medicine and University Hospital Cologne Institute of Pathology.

RNA analysis confirmed exon 14 skipping for more than 100 different alteration types and demonstrated that certain clinically relevant events, including large genomic deletions and a synonymous variant affecting splicing, may be missed or incorrectly interpreted by DNA-based testing alone.

The EQA schemes, using both tissue and liquid biopsy samples, demonstrated excellent laboratory performance for tissue-based testing, with an overall success rate of 98%, indicating that reliable detection of MET exon 14 alterations can be achieved across laboratories using appropriately validated methodologies.

In contrast, liquid biopsy testing proved considerably more challenging. While performance improved substantially between the first EQA round in 2022 (37.5% success rate) and the second in 2024 (63% success rate), this shows that detection of MET exon 14 alterations in circulating tumor DNA remains technically demanding.

Clinical and therapeutic implications

These findings demonstrate that certain clinically relevant MET exon 14 skipping events may be missed by DNA-only approaches due to assay design limitations, bioinformatic filtering, or variant interpretation challenges. "To improve diagnostic accuracy, incorporating RNA-based confirmation, where feasible, can improve sensitivity and specificity for identifying true exon-skipping events," noted Heydt.

From a therapeutic perspective, accurate detection of MET exon 14 skipping is essential because it determines eligibility for MET-targeted therapies.

"This research has important implications for routine clinical molecular pathology," concluded Heydt. "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."

About The Journal of Molecular Diagnostics

The Journal of Molecular Diagnostics, the official publication of the Association for Molecular Pathology, co-owned by the American Society for Investigative Pathology, and published by Elsevier, seeks to publish high-quality original papers on scientific advances in the translation and validation of molecular discoveries in medicine into the clinical diagnostic setting, and the description and application of technological advances in the field of molecular diagnostic medicine.

The editors welcome review articles that contain: novel discoveries or clinicopathologic correlations, including studies in oncology, infectious diseases, inherited diseases, predisposition to disease, or the description of polymorphisms linked to disease states or normal variations; the application of diagnostic methodologies in clinical trials; or the development of new or improved molecular methods for diagnosis or monitoring of disease or disease predisposition.