At the cellular level, loss of exon 14 disrupts the juxtamembrane domain of the MET receptor, impairing CBL-mediated ubiquitination and leading to sustained MET signaling through downstream pathways including MAPK and PI3K-AKT that contributes to aggressive tumor behaviour [4].
Clinically, this molecular phenotype shows limited responsiveness to conventional chemotherapy and immunotherapy, while showing marked sensitivity to MET-selective tyrosine kinase inhibitors (TKIs) such as tepotinib and capmatinib [5,6]. Hence, it emphasizes the crucial need for their accurate molecular identification.
Genomically, METex14 can arise from a heterogeneous spectrum of alterations affecting splice regulatory regions flanking exon 14, such as changes within intronic splice acceptor site or donor site, polypyrimidine tract, branch points, or adjacent regulatory motifs [7,8]. Despite advances in combined DNA-RNA based next-generation sequencing approaches, non-canonical intronic variants are frequently classified as variants of uncertain significance (VUS) due to limited functional evidence [9,10].
Here, we report two cases of advanced NSCLC harboring novel MET splice-site deletions within the exon 14 acceptor region, initially annotated as VUS. Transcript-level analysis using reverse transcription PCR confirmed METex14 skipping in both cases, enabling variant reclassification and directly informing targeted therapy. These findings support the routine incorporation of transcript-level assays for the evaluation of MET splice-site variants and highlight the need for systematic integration of functionally validated variants into clinical databases to improve molecular interpretation and therapeutic decision making.
Case 1
A 71-year-old woman presented with metastatic lung adenocarcinoma involving the left upper lobe with mediastinal and osseous metastasis. Tumor cells were positive for thyroid transcription factor-1 (TTF-1), with wild-type EGFR status (PCR), ALK (IHC) negativity, and PD-L1 tumor proportion score (TPS) of 90%. The patient received one cycle of gemcitabine-carboplatin chemotherapy and remained symptomatic but clinically stable.
Comprehensive molecular profiling using a customized NGS lung cancer panel performed on formalin-fixed paraffin-embedded (FFPE) tumor tissue identified a novel intronic MET splice-site deletion (NM_001127500.3: c.2942-18_2942-4delCTTTCTCTCTGTTTT;VAF 93.78%) within the exon 14 acceptor region, initially classified as a VUS. Matched RNA sequencing demonstrated exon 13–15 junction reads (total mapped reads: 37,066; 132372 reads per million), consistent with METex14 skipping (Figure 1A). No additional actionable genomic alterations were detected. Functional validation by reverse transcription PCR (RT-PCR) performed on cDNA synthesized from freshly extracted RNA using primers flanking exons 13-15, yielded a shorter 140-bp amplicon (Figure 1B), confirming METex14 skipping alteration leading to reclassification of the variant as a Tier II variant of potential clinical significance according to AMP/ASCO/CAP guidelines (Table I).
In accordance with current guidelines and evidence from the VISION trial, the patient was initiated on oral tepotinib (225 mg twice daily). At early follow-up, the patient was clinically stable with mild gastrointestinal toxicity and continued tepotinib with supportive care.
Case 2
A 70-year-old man with metastatic pulmonary sarcomatoid carcinoma presented with a left upper lobe mass and skeletal metastases. Tumor profiling showed PDL1 TPS of 100%, with negative EGFR (PCR) and ALK (IHC) testing. The patient was started on weekly paclitaxel-carboplatin with symptomatic improvement.
Subsequent DNA–RNA NGS using the same customized panel identified a novel MET splice-site deletion (NM_001127500.3: c.2942-19_2942-10delTCTTTCTCTC; VAF 20.87%) in the exon 14 acceptor region, along with a truncating TP53 frameshift mutation (p. Cys229Tyrfs*10; VAF 24.72%). Similar to case 1, matched RNA sequencing demonstrated exon 13–15 junction reads (total mapped reads: 50,179; 92,125.5 reads per million), consistent with METex14 skipping, which was orthogonally confirmed by RT-PCR. The variant was reclassified as a clinically actionable METex14 alteration (Table I), prompting transition to oral tepotinib (225 mg twice daily) with symptomatic improvement at early follow-up.
To summarise, both cases with noncanonical intronic MET splice-site variants initially classified as VUS on DNA-based analysis were functionally validated by RNA sequencing and RT-PCR, enabling reclassification and directly guiding targeted therapy decisions.
Biological and Molecular Context
METex14 skipping results in loss of juxtamembrane domain of the MET receptor, impairing CBL-mediated ubiquitination, prolonging receptor stability, and leading to sustained downstream signaling through pathways such as RAS-RAF-MEK-ERK and PI3K-AKT [4]. Genomically, these alterations arise from a heterogeneous spectrum of variants, including substitutions, small insertions/deletions, and complex rearrangements affecting splice regulatory regions flanking exon 14 [7,8].
Large sequencing datasets have shown enrichment of such variants within the intron 13 splice acceptor region [7], consistent with deletions identified in this study, which were not previously catalogued in curated databases and therefore initially classified as VUS.
Role of RNA-Based Confirmation: NGS and RT-PCR
A key diagnostic challenge highlighted by these cases is the limitations of DNA-only NGS approaches in interpreting noncanonical splice-site variants. Such alterations are frequently intronic, variably covered, and not reliably interpreted by standard bioinformatic pipelines, leading to under-recognition of clinically actionable events [9,11]. In contrast, RNA-based assays overcome this limitation by directly detecting exon 14-skipping transcripts, providing definitive functional evidence [10,12]. In both cases, RNA sequencing demonstrated high-confidence exon 13-15 junction reads, confirming the functional consequence of the identified variants.
Importantly, orthogonal validation using RT-PCR provided an additional rapid and cost-effective method to confirm exon 14 exclusion. This is particularly relevant in settings where RNA NGS is unavailable or inconclusive and supports the incorporation of RT-PCR as a practical adjunct in molecular diagnostic workflow [13]. Together, these findings support the use of transcript-level evidence as strong functional data (PS3) for variant interpretation and reclassification.
Figure 2: Proposed diagnostic workflow for evaluation of MET splice-site variants.
Clinical Implications and Therapeutic Relevance
From a clinical perspective, accurate identification of METex14 alterations has direct therapeutic implications. METex14-positive NSCLC is associated with inferior outcomes when treated with chemotherapy or immunotherapy, even in the context of high PD-L1 expression [6,14]. In contrast, MET-targeted tyrosine kinase inhibitors have demonstrated robust and durable activity. Prospective trials have reported meaningful response rates, prolonged progression-free survival, and manageable toxicity with agents such as tepotinib and capmatinib, supporting prioritization of MET-targeted therapy once METex14 is confirmed [5,6]. In the present cases, integrated RNA-based enabled reclassification of ambiguous splice-site VUS as likely pathogenic Tier II METex14 alterations, prompting a timely transition from chemotherapy to tepotinib. Early clinical stability in both patients is consistent with outcomes reported in prospective trials and reinforces the clinical utility of transcript-level confirmation [5,11,12].
Proposed Diagnostic Workflow for MET Splice-Site Variants (Figure 2)
Based on our findings and existing literature, we propose a practical diagnostic framework for the evaluation of MET splice-site variants identified on DNA-based NGS:
1. Variant Localization: Intronic variants located within the exon 14 flanking regions (particularly intron 13 splice acceptor and intron 14 splice donor sites) should be flagged for potential splicing impact, even when noncanonical.
2. Initial Classification: Variants should not be dismissed as VUS solely based on lack of database annotation if they occur within known splice regulatory regions.
3. Splice Impact Assessment: In silico splice prediction tools may be used as supportive evidence but should not be solely relied upon for classification.
4. RNA-Based Validation: RNA sequencing should be performed to directly assess exon 14 skipping by identifying exon 13–15 junction transcripts.
5. Orthogonal Confirmation: In resource-limited settings or where RNA NGS is inconclusive, RT-PCR using primers flanking exons 13–15 provides a cost-effective and reliable alternative for detecting exon 14 exclusion.
6. Variant Reclassification: Detection of METex14 skipping at the transcript level should be considered strong functional evidence (PS3) supporting reclassification under AMP/ASCO/CAP or ACMG frameworks.
7. Clinical Reporting: Reports should integrate DNA and RNA findings, explicitly stating when transcript-level evidence supports classification of a previously reported VUS as a clinically actionable METex14 alteration.
8. Therapeutic Implication: Confirmed METex14 alterations should prompt consideration of MET-targeted therapies in accordance with current clinical guidelines.
Limitations of this study include short follow-up duration, precluding assessment of long-term outcomes. However, the primary objective was to demonstrate a reproducible diagnostic and interpretative framework for evaluation of noncanonical MET splice-site variants. The concordance between RNA sequencing and RT-PCR strengthens the validity of the proposed approach with RT-PCR serving as an additional low-cost orthogonal validation, strengthening confidence in accurate variant classification and reporting.
Systematic curation of such functionally validated alterations in genomic databases such as ClinVar and COSMIC is essential to improve future variant interpretation, reduce VUS burden, and facilitate timely precision oncology care in NSCLC.
1) Paik PK, Drilon A, Fan PD, Yu H, Rekhtman N, Ginsberg MS, Borsu L, Schultz N, Berger MF, Rudin CM, Ladanyi M. Response to MET inhibitors in patients with stage IV lung adenocarcinomas harboring MET mutations causing exon 14 skipping. Cancer Discov. 2015;5(8):842-9. doi:10.1158/2159-8290.CD-14-1467
2) Schrock AB, Frampton GM, Suh J, Chalmers ZR, Rosenzweig M, Erlich RL, Halmos B, Goldman J, Forde P, Leuenberger K, Peled N, Kalemkerian GP, Ross JS, Stephens PJ, Miller VA, Ali SM, Ou SH. Characterization of 298 patients with lung cancer harboring MET exon 14 skipping alterations. J Thorac Oncol. 2016;11(9):1493-502. doi:10.1016/j.jtho.2016.06.004
3) Liu SY, Gou LY, Li AN, Lou NN, Gao HF, Su J, Yang JJ, Zhang XC, Shao Y, Dong ZY, Zhou Q, Zhong WZ, Wu YL. The unique characteristics of MET exon 14 mutation in chinese patients with NSCLC. J Thorac Oncol. 2016;11(9):1503-10. doi:10.1016/j. jtho.2016.05.016
4) Kong-Beltran M, Seshagiri S, Zha J, Zhu W, Bhawe K, Mendoza N, Holcomb T, Pujara K, Stinson J, Fu L, Severin C, Rangell L, Schwall R, Amler L, Wickramasinghe D, Yauch R. Somatic mutations lead to an oncogenic deletion of met in lung cancer. Cancer Res. 2006;66(1):283-9. doi:10.1158/0008-5472.CAN-05-2749
5) Paik PK, Felip E, Veillon R, Sakai H, Cortot AB, Garassino MC, Mazieres J, Viteri S, Senellart H, Van Meerbeeck J, Raskin J, Reinmuth N, Conte P, Kowalski D, Cho BC, Patel JD, Horn L, Griesinger F, Han JY, Kim YC, Chang GC, Tsai CL, Yang JC, Chen YM, Smit EF, van der Wekken AJ, Kato T, Juraeva D, Stroh C, Bruns R, Straub J, Johne A, Scheele J, Heymach JV, Le X. Tepotinib in non-small-cell lung cancer with MET exon 14 skipping mutations. N Engl J Med. 2020;383(10):931-43. doi:10.1056/NEJMoa2004407
6) Wolf J, Seto T, Han JY, Reguart N, Garon EB, Groen HJM, Tan DSW, Hida T, de Jonge M, Orlov SV, Smit EF, Souquet PJ, Vansteenkiste J, Hochmair M, Felip E, Nishio M, Thomas M, Ohashi K, Toyozawa R, Overbeck TR, de Marinis F, Kim TM, Laack E, Robeva A, Le Mouhaer S, Waldron-Lynch M, Sankaran B, Balbin OA, Cui X, Giovannini M, Akimov M, Heist RS; GE-OMETRY mono-1 Investigators. Capmatinib in MET exon 14-mutated or MET-amplified non-small-cell lung cancer. N Engl J Med. 2020;383(10):944-57. doi:10.1056/NEJMoa2002787
7) Frampton GM, Ali SM, Rosenzweig M, Chmielecki J, Lu X, Bauer TM, Akimov M, Bufill JA, Lee C, Jentz D, Hoover R, Ou SH, Salgia R, Brennan T, Chalmers ZR, Jaeger S, Huang A, Elvin JA, Erlich R, Fichtenholtz A, Gowen KA, Greenbowe J, Johnson A, Khaira D, McMahon C, Sanford EM, Roels S, White J, Greshock J, Schlegel R, Lipson D, Yelensky R, Morosini D, Ross JS, Collisson E, Peters M, Stephens PJ, Miller VA. Activation of MET via diverse exon 14 Splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors. Cancer Discovery. 2015;5(8):850-9. doi:10.1158/2159-8290.CD-15-0285
8) Liao J, Wu S, Min Y, Li Y, Nie Y, Chen Q, Mao Z, Zong Q, Gao N, Zhang D, Liang W. The landscape of MET exon 14 skipping mutations in patients with lung cancer identified by next-generation sequencing. JTO Clinical and Research Reports. 2025;6(7):100826. doi:10.1016/j.jtocrr.2025.100826
9) Owen D, Ben-Shachar R, Feliciano J, Gai L, Beauchamp KA, Rivers Z, Hockenberry AJ, Harrison G, Guittar J, Catela C, Parsons J, Cohen E, Sasser K, Nimeiri H, Guinney J, Patel J, Morgensztern D. Actionable structural variant detection via RNA-NGS and DNA-NGS in patients with advanced non–small cell lung cancer. JAMA Netw Open. 2024;7(11):e2442970. doi:10.1001/jamanetworkopen.2024.42970
10) Davies KD, Lomboy A, Lawrence CA, Yourshaw M, Bocsi GT, Camidge DR, Aisner DL. DNA-based versus RNA-based detection of MET exon 14 skipping events in lung cancer. J Thorac Oncol. 2019;14(4):737-41. doi:10.1016/j.jtho.2018.12.020
11) Benayed R, Offin M, Mullaney K, Sukhadia P, Rios K, Desmeules P, Ptashkin R, Won H, Chang J, Halpenny D, Schram AM, Rudin CM, Hyman DM, Arcila ME, Berger MF, Zehir A, Kris MG, Drilon A, Ladanyi M. High yield of RNA sequencing for targetable kinase fusions in lung adenocarcinomas with no mitogenic driver alteration detected by DNA sequencing and low tumor mutation burden. Clin Cancer Res. 2019;25(15):4712-22. doi:10.1158/1078-0432.CCR-19-0225
12) Cohen D, Hondelink LM, Solleveld-Westerink N, Uljee SM, Ruano D, Cleton-Jansen AM, von der Thüsen JH, Ramai SRS, Postmus PE, Graadt van Roggen JF, Hoppe BPC, Clahsen PC, Maas KW, Ahsmann EJM, Ten Heuvel A, Smedts F, van Rossem RN, van Wezel T. Optimizing mutation and fusion detection in NSCLC by sequential DNA and RNA sequencing. J Thorac Oncol. 2020;15(6):1000-14. doi:10.1016/j.jtho.2020.01.019
){kind=link}
){kind=link}
){kind=link}