MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non–Small-Cell Lung Cancer

Abstract

Genetic alterations in MET that lead to exclusion of exon 14 from the transcript are now targetable in non–small-cell lung cancer (NSCLC) after recent drug approvals for this indication. However, this class of alteration is much more diverse than activating mutations in other targetable oncogenes, which presents challenges for clinical detection by molecular diagnostic assays. Therefore, MET exon 14 (METex14) skipping was highlighted in the Association for Molecular Pathology Emerging and Evolving Biomarkers webinar series (https://www.amp.org/education/emerging-and-evolving-biomarkers, last accessed April 26, 2022). Eukaryotic DNA contains noncoding sequences called introns that are interspersed between coding sequences called exons. The removal of introns is important for gene regulation and occurs by RNA splicing. This is a carefully orchestrated molecular process specifically reliant upon key genomic sequences: namely 5′ and 3′ splice sites, a branch site, and a polypyrimidine tract. A broad range of diverse mutations, inclusive of single nucleotide variants and insertion/deletion mutations of various sizes, have been shown to disrupt these conserved genomic elements (or delete exon 14 altogether), leading to aberrant splicing and METex14 skipping. Underlying all laboratory and clinical considerations is the molecular pathogenesis of METex14 skipping that results from significant diversity in both mutation type, size, and position. The MET gene is a proto-oncogene that encodes for a receptor tyrosine kinase protein. The protein product is commonly referred to by a variety of names including MET, c-Met (cellular mesenchymal-epithelial transition factor), or HGFR (hepatocyte growth factor receptor). Normally, in the presence of ligand, MET dimerization results in phosphorylation of multiple intracellular tyrosine kinase residues, activating downstream pro-growth and survival cell signaling cascades. Ligand binding also results in receptor internalization into endosomes, after which the MET receptor can either recycle to the cell surface or undergo lysosomal degradation. MET, similar to other receptor tyrosine kinases, is marked for degradation by the E3 ubiquitin ligase, c-CBL. The MET juxtamembrane domain, encoded in part by exon 14, contains a direct binding site for c-CBL [tyrosine 1003 (Y1003)]. Genomic variants that result in exon 14 skipping generate a mutant form of the MET receptor lacking the c-CBL binding site in the juxtamembrane domain. Impaired receptor degradation is a leading hypothesis for oncogenicity of METex14 skipping mutations. NSCLC specimens typically are small, yet requirements for molecular biomarker analysis are ever-expanding. Consequently, iterative single biomarker testing generally is considered impractical. In addition, immunohistochemistry, although applicable for some biomarker analyses, has been shown to be of limited clinical utility in NSCLC for the detection of METex14 skipping mutations, and therefore not useful for predicting response to targeted therapy. Increasingly, laboratory analysis of METex14 skipping events is occurring via next-generation sequencing. The use of this technology is clinically relevant given its potential to broadly interrogate the diverse genomic variation underpinning METex14 skipping. However, properly leveraging this potential requires designing a next-generation sequencing–based assay that is informed by the underlying molecular pathogenesis alongside genomic laboratory considerations including choice of analyte (DNA and/or RNA), library preparation approach, and bioinformatic methodology. DNA sequencing permits indirect assessment for METex14 skipping because DNA variants potentially underlying skipping of exon 14 at the mature RNA level are assessed. Unless a DNA variant of concern has been documented previously to definitively result in METex14 skipping, confirmatory RNA-based testing to officially determine the outcome of the suspect DNA mutation is worth consideration. RNA sequencing, on the other hand, permits direct detection of altered splicing (observed as the fusion of exons 13 and 15). Ultimately, it is the latter, the abnormal fusion product, that is the clinically relevant outcome regardless of the underlying DNA mutation. A point worth considering in the context of RNA sequencing, however, is low basal rates of alternative spicing (nonpathogenic), which, if not accounted for, risk false-positive results. Testing performance implications also stem from the library preparation method. A primary disadvantage of DNA-based amplicon methodology is allele dropout with resultant false-negative results. This is owing to primer mismatch or primer failure secondary to mutations impacting primer binding sites. Alternatively, it generally is accepted that hybrid capture methodology is preferred for optimizing DNA-based detection of METex14 skipping variants because it circumvents allelic dropout. However, issues that have the potential to negatively impact assay sensitivity with hybrid capture include relatively poor intronic coverage of some assays, failure to use algorithms to detect relevant large deletions, and the potential for off-target sequencing, thereby reducing the sequencing coverage in the regions of interest. Ultimately, RNA-based assays for detection of METex14 skipping mutations have been shown to have superior performance when compared with some DNA-based assays (particularly those based on amplicon methodology). Yet, the success of RNA-based sequencing is reliant upon RNA integrity, and related testing protocols are best served with pertinent RNA quality control measures. Lastly, if a laboratory performs iterative testing, meaning DNA sequencing potentially followed by RNA sequencing, then there is a risk of running out of specimen unless extraction protocols are optimized for total nucleic acid in advance. METex14 skipping mutations occur in approximately 3% to 4% of NSCLC cases across all histologic subtypes (incidence varies by histology: between approximately 1% squamous cell carcinoma and approximately 13% pulmonary sarcomatoid carcinoma). Although there are clinical characteristics associated with METex14 skipping (nonsmoking status, female sex, older age compared with patients with other actionable driver mutations), these should not be used to determine which tumors are analyzed. METex14 skipping mutations now are incorporated into professional guidelines for the clinical management of NSCLC. This category of mutations is considered clinically actionable in NSCLC because clinical trial data showed the association of METex14 alterations with therapeutic responsiveness to oral MET tyrosine kinase inhibitors. These data further resulted in recent Food and Drug Administration approvals of capmatinib and tepotinib, both oral tyrosine kinase inhibitors that selectively inhibit MET, for patients with metastatic NSCLC who are positive for METex14 skipping mutations. Capmatinib was the first oral tyrosine kinase inhibitor selective for MET approved by the Food and Drug Administration for the treatment of metastatic disease positive for METex14 skipping. This approval was based on impressive data from the GEOMETRY trial, which assessed capmatinib as a monotherapy and also showed therapeutic efficacy for intracranial tumor burden. Subsequently, the Food and Drug Administration granted accelerated approval to tepotinib for adult patients with metastatic NSCLC positive for METex14 skipping mutations. The latter approval was granted after the VISION trial showed a favorable overall response rate and response duration. Before the approval of these two selective agents, crizotinib, a multikinase oral tyrosine kinase inhibitor approved for ALK- or ROS1-rearranged advanced NSCLC, was shown in the PROFILE 1001 trial to have therapeutic benefit in the treatment of advanced NSCLC that harbors METex14 skipping mutations. Although National Comprehensive Cancer Network guidelines acknowledge crizotinib as a treatment option for METex14 skipping mutation–positive metastatic NSCLC, capmatinib and tepotinib are recommended as preferred therapies in the first-line setting. Data will continue to emerge with ongoing assessment of the most recently approved targeted agents. Moreover, additional investigational selective agents for METex14 skipping mutations are in clinical trials. Lastly, it has been shown and acknowledged in professional treatment guidelines that immunotherapy, regardless of programmed death-ligand 1 (PD-L1) levels, is not efficacious for certain classes of oncogene-driven NSCLC (eg, EGFR+, ALK+). Currently, there are conflicting data regarding the impact of METex14 skipping mutations on immunotherapy and this consideration is under active investigation. The complex molecular pathogenesis of this biomarker, which often is simplified as METex14 skipping, is important because it results from a broad range of mutations that can only undergo comprehensive molecular interrogation with assays properly designed in accordance with the molecular biology. METex14 skipping mutations are clinically relevant because they are predictive for response to Food and Drug Administration–approved targeted therapies and are deemed a part of routine biomarker analysis in professional guidelines for the clinical and laboratory management of NSCLC. Broad molecular profiling is strongly advised in NSCLC given the other oncogenic biomarkers recommended for routine analysis in this tumor type using tissue biopsy specimens that often are composed of limited tissue. Although DNA-based assays may be used for successful detection of METex14 skipping mutations, the underlying molecular biology requires use of hybrid capture–based target enrichment coupled with bioinformatic analyses enabled for large indel detection. RNA-based assays, by directly detecting omission of exon 14 from the transcript, overcome many of the limitations of DNA-based analyses. However, RNA-based testing may be hampered by a higher rate of poor-quality RNA in clinical tumor samples. For more information and an in-depth presentation on this content, please see the Emerging and Evolving Biomarkers webinar (https://educate.amp.org/local/catalog/view/product.php?productid=218, last accessed February 10, 2022). Socinski MA, Pennell NA, Davies KD: MET exon 14 skipping mutations in non-small-cell lung cancer: an overview of biology, clinical outcomes, and testing considerations. JCO Precis Oncol 2021, 5:653-663 Awad MM: Impaired c-Met receptor degradation mediated by MET exon 14 mutations in non-small-cell lung cancer. J Clin Oncol 2016, 34:879-881 Frampton GM, Ali SM, Rosenzweig M, Chmielecki J, Lu X, Bauer TM, et al: Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors. 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