MET Exon 14 Variants in Non-Small Cell Lung Carcinoma: Prevalence, Clinicopathologic and Molecular Features

: Somatic MET exon 14 skipping mutations (MET ex14) are targetable driver mutations for non-small cell lung cancer (NSCLC), responsive to MET inhibitors. Objective: This study seeks to further characterize the clinicopathologic features and mutational proﬁle of MET ex14 variant NSCLC. Design: Retrospective review of all MET ex14 tested NSCLC. Testing for selected BRAF, EGFR, HER2, KRAS, and MET mutations was performed using a clinically validated NGS assay, followed by MiSeq sequencing. Variants were classiﬁed as signiﬁcant (Tier1/2) or variants of uncertain signiﬁcance (VUS) per 2017 AMP/ASCO/CAP Joint Consensus Guidelines. PD-L1 expression was assessed by immunohistochemistry. Results: Of 2296 NSCLCs tested between 2017-7/2019, MET ex14 variants were present in 44 (1.9%). A total of 32 of 44 variants were MET exon 14 skipping, while the other 12 mutations were signiﬁcant missense (3) or VUS (9). Of nine VUS, ﬁve were adjacent to the canonical splice site and likely to impact splicing. Four cases had concomitant mutations. Of 35 cases with known clinical staging, stage 1–2 = 20 (57%), stage 3 = 3 (9%), and stage 4 = 12 (34%). Of 19 resected NSCLSs, histological types and growth pattern included 7 lepidic pattern-predominant. A high percentage of tumors with MET ex14 mutations are positive for PD-L1, and the percentage of cases with PD-L1 expression >50% trends higher in more advanced disease. Conclusions: Most MET variants identiﬁed in our cohort (73%) are MET ex14 skipping. The prevalence of MET ex14 variants is 1.9%, and a large percentage of tumors has lower clinical stage and less aggressive pathologic features.


Introduction
The mesenchymal-to-epithelial transition (MET) proto-oncogene encodes a tyrosine kinase receptor that controls cell growth and migration and plays an important role in tumor invasion and metastasis [1]. MET exon 14 encodes the juxta membrane region of the receptor which is known to modulate receptor downregulation via Cbl binding and ubiquitin-mediated degradation; mutations to MET exon 14 exert their effect through altered splicing that results in exon 14 skipping [2]. MET exon 14 skipping leads to loss of a phosphorylation site required for Cbl binding, reducing ubiquitin-mediated degradation and promoting oncogenesis [3]. Splice site mutations in exon 14 of MET are reported to occur in 3% of lung adenocarcinomas and 7.7-32% of sarcomatoid carcinomas [4][5][6]. MET amplification can also lead to MET activation in tumors and has been reported to occur up to 5.6% of non-small cell lung cancer (NSCLC) [6,7]. Similar to other driver oncogenes such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), that can be targeted with small molecule tyrosine kinase inhibitors (TKIs); driver alterations in the MET gene have also been identified as a target in NSCLC. Currently, FDA has approved Capmatinib and Tepotinib for use in patients with NSCLC harboring MET exon 14 skipping mutations [8].
With the advent of improved sequencing technologies, routine detection of MET exon 14 mutations has become more feasible. This study seeks to further characterize the clinicopathologic features and mutational profile of cases with MET ex14 variants in patients with NSCLC. As the development of acquired resistance to targeted therapy is very common in NSCLC patients with actionable driver alterations, immunotherapy also plays an important role in the management of these patients. We thus further analyzed the percentage of tumors with MET ex14 mutations that are positive for program death ligand 1 (PD-L1), and the percentage of cases with PD-L1 expression >50% in more advanced disease.

Subjects and Methods
A retrospective review of all NSCLC cases at the Cleveland Clinic tested for MET ex14 skipping mutations was conducted. For MET exon 14 testing, genomic DNA was extracted from FFPE or FNA tumor specimens and tested for selected BRAF, EGFR, HER2, KRAS, and MET mutations using a clinically validated targeted next generation sequencing (NGS) assay. Definitions of variant categories are as follows: (1) "splice": involve consensus splice site (−1, −2, +1, +2) or has been previously reported in the literature as exon 14 skipping; (2) "VUS, possible splice": occurs near the exon/intron junction (in the region of other known exon 14 skipping variants) but does not involve consensus splice site (−1, −2, +1, +2) and has not been previously reported in the literature as MET exon 14 skipping; (3) "missense, significant": has been previously reported as a significant somatic mutation in lung carcinoma but does not involve MET exon 14 splicing; (4) "VUS, missense": not expected to involve MET exon 14 splicing and has not been previously reported as a significant somatic mutation in NSCLC. PD-L1 expression was assessed by immunohistochemistry (IHC) using a mouse monoclonal PD-L1 antibody (22-c3, Dako, CA, USA). Clinicopathologic features for MET ex14 positive cases were evaluated by chart review.
The study was performed in accordance with the Institutional review board (IRB) of the Cleveland Clinic (IRB# 19-1245).

Results
Of the 2296 cases of NSCLC analyzed by DNA-based NGS between 2017-7/2019, MET ex14 variants were present in 44 cases (1.9%), of which 26 (59%) were males and 18 (41%) were females. The median age of positive cases was 76 years (±9.6, 59% men; 41% women). Specimen type was FNA in 46.7%. A total of 32 of 44 variants were MET exon 14 skipping mutations (previously reported and/or involve the canonical recognition site), while the other 12 mutations were significant missense (3) or VUS (9). Of nine VUS, five were adjacent to the canonical splice site and likely to impact splicing, while four were missense variants ( Figure 1A).

Discussion
Lung cancer continues to be the leading cause of cancer-related mortality among men and women worldwide [9], driving the need to discover new targetable genomic mutations. MET exon 14 skipping is a primary oncogenic driver sensitive to MET inhibition. In addition to existing FDA approved therapeutic options for MET exon 14 skipping positive tumors such as capmatinib, crizotinib, and tepotinib [1], several selective MET inhibitors are being investigated, including emibetuzumab and savolitinib, and are promising therapies with maximized efficacy and reduced off-target toxicity [10].
Previous studies have reported the prevalence of MET ex14 to be approximately 3.0% in NSCLC with highest prevalence in the pulmonary sarcomatoid subtype [3]. While Awad et al. [4] and Schrock et al. [5] reported a prevalence of 3% and 2.7% of MET ex14 variants, in studies conducted by Zheng et al. [11] and Liu et al. [12], the prevalence was observed to be 1.3% and 1%, respectively, possibly reflecting a lower incidence in Asians. In our cohort, MET ex14 variants were observed in 1.9% cases (44/2296). Our lower observed prevalence may be due in part to universal testing of NSCLC at our institution rather than testing of only advanced disease. Additionally, although our MET test is expected to detect most relevant exon 14 skipping variants, our lower reported prevalence may be partially attributable to variants lying outside the test's covered regions and/or alterations not efficiently detected by next generation sequencing methodology such as large deletions.
Recent studies by Poirot et al. and Davies et al. have demonstrated the superiority of RNA-based assays in detecting MET exon 14 skipping mutations compared with DNAbased assays [13,14]. In both these studies, DNA-based assays were able to identify only approximately 60% of the mutations as any variant outside the amplified area or preventing binding due to mutation of the primer would not be detected. In contrast, in RNA-based assays, any variant causing skipping of exon 14 in vivo is identified as fusion on exon 13 to exon 15. In general, RNA-based targeted approach analyses and quantifies directly fusion transcripts and is more accurate than DNA panels on tumor tissue, but it can be limited by RNA quality and quantity. However, amplicon-based RNA NGS panels can detect gene fusions in poor quality RNA samples, such as those obtained from FFPE tissue samples (PMID: 32726941). This is a major limitation of our study, as adding RNA-based NGS assays or using combined DNA-RNA testing might have allowed us to better classify the samples with non-canonical DNA mutations, including missense and VUS variants in this cohort.
In contrast with other targetable mutations seen in NSCLC such as ALK and ROS1 rearrangements and KRAS, EGFR, and BRAF mutations, MET ex14 mutations have been identified in older patients. In a review of 933 patients with NSCLC, Awad et al. identified MET exon 14 skipping mutations in 28 patients with a median age of 72.5 years [4]. Whereas 68% and 60.4% patients with MET ex14 variants were females in the study conducted by Awad et al. [4] and Shrock et al. [5], respectively, in a study conducted by Wang et al., 66% patients were males [15]. In our cohort, the mean age of the patients was 76 years (53 to 91 years) which is consistent with previous studies. However, there is male preference is our study, with 59% (26) males and 41% (18) females.
MET ex14 mutations have been observed across various histological subtypes of nonsmall cell lung carcinoma, with a decreasing order of prevalence seen in sarcomatoid carcinoma, adenosquamous carcinoma, adenocarcinoma, and squamous cell carcinoma [4,16]. In our study, of the 19 patients who underwent resection, 16 patients had adenocarcinoma (including five tumors with lepidic-predominant pattern), 2 adenosquamous carcinoma, and 1 sarcomatoid carcinoma. As primary lung tumors, adenosquamous carcinoma and sarcomatoid carcinoma are relatively rare, comprising 0.3-1.3% and 0.4-4% of all lung carcinomas, respectively [17,18]. Our data are consistent with the previous studies showing that MET ex14 appears enriched in these two aggressive histologic subtypes [17,18]. However, a large percentage of tumors also had lower clinical stage and less aggressive pathologic features, both possibly reflecting sampling differences attributed to universal testing of NSCLC at our institution rather than testing of only advanced diseases was common in early studies due to the emphasis on targeted therapies for advanced disease. This seemingly paradoxical observation is consistent with studies from other centers where MET ex14 variants testing is performed routinely for all cases of NSCLC [4].
The spectrum of known MET exon 14 skipping mutations must be considered when designing diagnostic tests for their detection. In literature, hundreds of distinct genetic alterations leading to MET exon 14 skipping have been reported, including base substitutions and insertions or deletions at the splice acceptor site, at the splice donor site, and in intronic non-coding regions immediately adjacent to the splice acceptor site, as well as whole exon deletions [19]. In our cohort, 32 of the 44 (73%) MET ex14 mutations were previously reported and/or canonical recognition sites skipping variants. Nine patients had VUS, five of which were adjacent to a canonical splice site and were likely to impact splicing and four were missense variants. Three patients had significant missense mutations. To detect these potential alterations, it is important to sequence both exon 14 and its surrounding regions, although a plurality of MET exon 14 skipping events occur at the donor site [20].
MET exon 14 skipping mutations are usually mutually exclusive of other characteristic driver mutations in NSCLC. Whereas the cancer genome atlas research network [21] and Awad et al. [4] identified no concomitant mutation with MET ex14 skipping mutation, in the study conducted by Shrock et al., concurrent KRAS mutation and EGFR amplification was observed in 3% and 6.4% cases, respectively [5]. In our cohort, four cases (9%) had concomitant mutations with three harboring KRAS mutations and one with EGFR mutation. However, none of the co-mutations occurred with a known functional pathogenic MET ex14 skipping mutation. The EGFR mutation was co-mutated with a VUS possible splice of MET ex14. The KRAS mutations were co-mutated with a significant missense mutation of METe x14, a VUS missense of MET ex14, and a VUS missense mutation together with a possible splice site mutation of MET exon 16, respectively. Further investigations of these cases would be required to confidently establish true functionally co-mutated status. Moreover, the testing methodology used does not distinguish whether the mutations are part of the same clone (co-mutational status as a subclone) or reflect tumoral heterogeneity.
The development of monoclonal antibodies against PD-1 receptor and its ligand, PD-L1 has provided a major breakthrough in the management of patients with NSCLC in the last decade. In a review of 147 patients with MET exon 14 skipping mutations, PD-L1 expression of > 1% was seen in 57 0f 111 patients, although the overall survival did not improve in patients who were administered PD-L1 immunotherapy [22]. In a similar study conducted by Xu et al., 205 of 401 patients with NSCLC and MET exon14 skipping had PD-L1 expression of > 1% [23]. In our cohort, 27 patients had tissue available for evaluation of PD-L1 expression. PD-L1 expression of 0%, 1-49%, and >50% was observed in 19% (5/27), 41% (11/27), and 41% (11/27) of patients, respectively, which is higher compared with previous studies (approximately 80% versus 50%). Patients with stage 4 tumors showed a significantly higher percentage of PD-L1 expression of >50% (67%, 4/6) compared with stage 1-3 tumors (33%, 6/18). In contrast, although four of five lepidic-predominant tumors were positive for PD-L1, they were all at the level of 1-49%.
Engagement of programmed cell death-1 (PD-1) receptor by its ligands PD-L1 is an important adaptor immune mechanism by tumor cells. This leads to downregulation of T-cells in the tumor microenvironment which can be reversed by PD1 and PD-L1 blocking antibodies such as pembrolizumab, nivolumab, atezolizumab, and durvalumab [24]. These have shown promise in the treatment of patients with non-small cell lung carcinoma with response rates of over 20% in treatment-naïve patients [25]. Testing for PD-L1 expression remains current standard in patients with NSCLC who are likely to respond to immunotherapy. It has been seen that in patients with higher PD-L1 expression have better response to immunotherapy than patients with lower PD-L1 expression [26,27]. Presently, IHC remains the gold standard for quantifying PD-L1 expression in tumor samples [28]. Although these findings have potential therapeutic implications, NCCN recognizes that "Patients with MET ex14 skipping mutations have a modest response (16%, single-agent ICIs) to immunotherapy, even those with high PD-L1 levels." (NCCN NSCLC version 1.2023, . Additionally, the guidelines support that any patient with a targetable oncogenic driver should receive targeted therapy before immunotherapy. Larger studies are required to clarify the PD-L1 status in patients with MET exon 14 skipping mutations and the potential contribution of immunotherapies in these patients, especially in patients with high-stage diseases. In conclusion, roughly three quarters of MET variants identified in our cohort are MET ex14 skipping, another one tenth likely result in exon 14 skipping, while the other 16% are missense variants presumably unrelated to splicing. Our prevalence of MET ex14 variants is 1.9%, and a large percentage of tumors has lower clinical stage and less aggressive pathologic features. Notably, a high percentage of tumors with MET ex14 mutations are positive for PD-L1, and the percentage of cases with PD-L1 expression >50% trends higher in more advanced stage disease. It is unclear whether certain MET exon 14 mutations are more responsive to c-Met inhibition than others and more prospective clinical trials will be necessary to determine if immunotherapy or combination strategies (i.e., immunotherapy and chemotherapy) in addition to targeted therapy in this population will provide survival advantage. The presence of existing and potential targeted therapies for MET inhibition highlight the importance of including testing for MET in the molecular evaluation of NSCLC.
Author Contributions: L.Y. and D.B. contributed to study conception, data acquisition, data analysis and interpretation, and manuscript writing and editing. H.M. contributed to manuscript writing and editing. X.H. contributed to data acquisition. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement:
The study was performed in accordance with the Institutional review board (IRB) of the Cleveland Clinic (IRB# 19-1245).

Informed Consent Statement: No patient involved.
Data Availability Statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.