A Systematic Review of Mesenchymal Epithelial Transition Factor (MET) and Its Impact in the Development and Treatment of Non-Small-Cell Lung Cancer

Simple Summary Lung cancer is the type of cancer that kills the most people in the world each year. It is difficult to diagnose lung cancer in the early stages and there are only few treatment options available once the cancer has spread. The mesenchymal epithelial transition factor (MET) gene is of importance in lung cancer development, and mutations in this gene are related to poor prognosis. Consequently, it is important to develop new treatment options that specifically target the MET protein. In this systematic review, we aimed to summarize the existing knowledge on the impact of MET on lung cancer development and the effect of currently available medications. Our hope is that the findings of this systematic review will deepen the understanding of other researchers, possibly providing a guiding hand as to what may be most interesting to focus on in future research projects on this subject. Abstract Lung cancer represents the leading cause of annual cancer-related deaths worldwide, accounting for 12.9%. The available treatment options for patients who experience disease progression remain limited. Targeted therapeutic approaches are promising but further understanding of the role of genetic alterations in tumorigenesis is imperative. The MET gene has garnered great interest in this regard. The aim of this systematic review was to analyze the findings from multiple studies to provide a comprehensive and unbiased summary of the evidence. A systematic search was conducted in the reputable scientific databases Embase and PubMed, leading to the inclusion of twenty-two articles, following the PRISMA guidelines, elucidating the biological role of MET in lung cancer and targeted therapies. The systematic review was registered in PROSPERO with registration ID: CRD42023437714. MET mutations were detected in 7.6–11.0% of cases while MET gene amplification was observed in 3.9–22.0%. Six studies showed favorable treatment outcomes utilizing MET inhibitors compared to standard treatment or placebo, with increases in PFS and OS ranging from 0.9 to 12.4 and 7.2 to 24.2 months, respectively, and one study reporting an increase in ORR by 17.3%. Furthermore, patients with a higher mutational burden may derive greater benefit from treatment with MET tyrosine kinase inhibitors (TKIs) than those with a lower mutational burden. Conversely, two studies reported no beneficial effect from adjunctive treatment with a MET targeted therapy. Given these findings, there is an urgent need to identify effective therapeutic strategies specifically targeting the MET gene in lung cancer patients.


Introduction
Lung cancer is one of the most common malignancies, causing 12.9% of cancer-related deaths worldwide, resulting in 1.3 million deaths annually [1][2][3][4]. Between 80.0 and 85.0% of lung cancer cases are non-small cell lung cancer (NSCLC) [3,5,6]. Late diagnosis is a major problem, contributing to the short median survival of approximately 18 months and the overall 5-year survival rate for lung cancer of 15-21%, depending on gender [7,8]. Up to 75.0% of patients with newly diagnosed NSCLC have locally advanced or metastasized disease at diagnosis, with a 5-year survival rate below 5.0% [1]. To this day, locally advanced or metastasized NSCLC is commonly treated with platinum-based chemotherapy, offering modest efficacy, with response rates of 20.0-30.0% and a plethora of side effects, or immunotherapy [3,5,9]. Targeted therapies for several types of cancer, including NSCLC, have emerged as a beneficial option for subsets of patients. Current treatment guidelines for advanced NSCLC call for broad molecular profiling to identify and guide the choice of potential targeted therapy options [10]. The proportion of patients with NSCLC receiving next generation sequencing (NGS) is low, which is in part due to insufficient availability of tumor tissue at the time of diagnosis. Approximately 60.0-65.0% of patients undergo testing for mutations in the epidermal growth factor receptor gene (EGFR) and less than 25.0% of patients are tested for alterations in the mesenchymal epithelial transition factor gene (MET) [11].
EGFR is a transmembrane receptor that is involved in several signaling pathways; it promotes cell proliferation and is anti-apoptotic. Overexpression of the EGFR gene is a wellknown pathological mechanism in NSCLC, present in 43.0-89.0% of NSCLC cases, which can lead to poorer outcomes [12]. Several EGFR TKIs exist; however acquired resistance to these therapies is common and alterations in the MET gene have been proven to be a contributing factor [13]. The protein MET is a transmembrane receptor tyrosine kinase (RTK) with a central role in cell motility, morphogenesis, proliferation, survival, and invasion ( Figure 1A) [5,[14][15][16]. Alterations in the MET gene, such as gene copy number (GCN) gain, mutation, or overexpression of the protein, have been reported in NSCLC [17]. The only known ligand to MET is hepatocyte growth factor (HGF) [6]. HGF is found in healthy lung tissue but is often overexpressed in NSCLC. Aberrant signaling through the HGF/MET pathway has clinically been linked to oncogenic potential and poor outcomes in NSCLC, with shortened overall survival (OS) and progression-free survival (PFS). However, studies showing the opposite also exist, introducing a contradiction in this area of research. One proven cause of unfavorable outcomes in MET altered lung cancer is acquired resistance to EGFR TKIs, underlining the need for more efficient targeted therapies [1,5,6,14,15,[17][18][19][20]. The frequency of any form of dysregulation of MET in NSCLC ranges from 3.0 to 7.0%. Sporadic GCN gain of MET is detected in 1.0-4.0% of wild type EGFR NSCLC cases. MET exon 14 skipping mutations occur in approximately 3.0% of NSCLC cases [2,3,18,20]. Amplification of the MET gene is the most common type of dysregulated signaling in NSCLC with acquired resistance to EGFR TKIs, with reported frequencies between 5.0 and 26.0% [20,21]. Alterations in MET have been shown to upregulate the expression of EGFR ligands, which in turn increases EGFR signaling, promoting cell proliferation, angiogenesis, and apoptosis [15]. Increased expression of HGF can also promote resistance to EGFR TKIs by supporting clonal selection of a subpopulation of cells with MET amplification [1]. Despite the fact that acquired resistance to EGFR TKIs is very common, they remain the preferred first-line treatment for locally advanced or metastatic EGFR mutation-positive NSCLC [15,22,23]. For patients with acquired resistance to EGFR TKIs caused by upregulation or amplification of MET, it may be beneficial to treat with a combination of inhibitors of both MET and EGFR, as this has been shown to have a synergistic inhibitory effect on the proliferation of cancer cells [2,21,22]. A combination of EGFR and MET TKIs has been shown to possibly delay the occurrence of resistance to EGFR TKIs [24]. For an overview of the EGFR and MET TKIs discussed in this article, see Table 1 ( Figure 1B).  leading to enhanced morphogenesis, cell survival, motility, proliferation, and invasion. MET is activated by its ligand, HGF, and the MET receptor can interact in various ways with the EGFR receptor. (B) The MET targeted therapies presented according to specific targets, either extracellular or intracellular. The pipe-line drugs are highlighted in cursive. MET: mesenchymal epithelial transition factor, HGF: hepatocyte growth factor, EGFR: epidermal growth factor receptor, SRC: v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog, PI3K: phosphatidylinositol 3-kinase, GRB2: growth factor receptor-bound protein 2, GAB1: GRB2-associated binding protein 1, SHC: src homology 2 domain-containing, SOS: son of sevenless, RAS: rat sarcoma, RAF: rapidly accelerated fibrosarcoma, MEK: MAPK effector kinase, ERK: extracellular signal-regulated kinase, RAC1: ras-related C3 botulinum toxin substrate 1, JNK: janus kinase 1, AKT: ak strain transforming, NF-kB: nuclear factor kappa B, mTOR: mammalian target of rapamycin, FAK: focal adhesion kinase, STAT3: signal transducer and activator of transcription 3, ATP: adenosine triphosphate. Created in biorender.com. Table 1. Overview of included EGFR and MET TKIs. EGFR and MET targeted therapies included in this systematic review. EGFR: epidermal growth factor receptor, MET: mesenchymal epithelial transition factor, TKI: tyrosine kinase receptor, VEGFR2: vascular endothelial growth factor receptor 2, RET: ret proto-oncogene, ROS1: ROS proto-oncogene 1, KIT: CD117, TIE-2: tyrosine kinase with immunoglobin and EGF homology domains 2, AXL: AXL receptor tyrosine kinase, RTK: receptor tyrosine kinase, NSCLC: non-small cell lung cancer, HGF: hepatocyte growth factor, ATP: adenosine triphosphate, CYP2C19: cytochrome P450 2C19. Tivantinib A selective, non-ATP-competitive MET inhibitor metabolized by CYP2C19. Yoshioka et al. [5] An issue with these types of targeted therapies remains the inherent genetic heterogeneity of NSCLC. Several mutations may co-exist in the same patient and complicate the interpretation of specific drug effects [3,25]. An additional aspect to take into consideration is the variability in methodologies employed to quantify aberrant MET expression. Some studies use immunohistochemistry (IHC) with the definition of MET positivity ranging from 1+ to 3+ and MET negativity varying from 0 to 1+ [2,17,18,20]. Other studies apply gene copy number (GCN) ≥ 5 or MET/centromere 7 ratio ≥ 2.0 as the lower limit for defined MET positivity [20].

Drug Name Effect References
Genetic profiling carried out by NGS is a rapidly progressing area of research within oncology. The methods used are becoming increasingly cost and time efficient, allowing for improved individual genetic testing and tailored therapy options [26]. The aim of this systematic review was to provide a comprehensive and unbiased summary of the evidence investigating the role of MET and aberrant expression of the protein in lung cancer development and treatment thereof.

Search Strategy
The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines were used in conducting this systematic review; for the full PRISMA 2020 Checklist, see Appendix B [27] (Tables A1 and A2)

Search Strategy
The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines were used in conducting this systematic review; for the full PRISMA 2020 Checklist, see Appendix B [27] (Tables A1 and A2)

Exclusion and Inclusion Criteria
After the initial search in Embase and PubMed, all duplicates were removed. All remaining articles were screened independently by two of the authors, E.B. and F.S., for relevance based on the title, abstract, and full article. Articles were included if one or both parties deemed it relevant to the subject. Articles that were published in 2012 or earlier were excluded as well as supplemental materials and conference abstracts. A full text screening of all eligible articles was performed independently by the two parties, excluding irrelevant and ongoing trials. A flow diagram of exclusion steps is presented in Figure  3.

Exclusion and Inclusion Criteria
After the initial search in Embase and PubMed, all duplicates were removed. All remaining articles were screened independently by two of the authors, E.B. and F.S., for relevance based on the title, abstract, and full article. Articles were included if one or both parties deemed it relevant to the subject. Articles that were published in 2012 or earlier were excluded as well as supplemental materials and conference abstracts. A full text screening of all eligible articles was performed independently by the two parties, excluding irrelevant and ongoing trials. A flow diagram of exclusion steps is presented in Figure 3.

Data Extraction and Processing
The included papers were grouped by investigated drug; the main characteristics are described in Tables 2 and 3. The main outcome measures presented and analyzed in this article are PFS and OS. Data on prevalence of MET mutations and amplifications in lung cancer were also analyzed and included in this article.

Data Extraction and Processing
The included papers were grouped by investigated drug; the main characteristics are described in Tables 2 and 3. The main outcome measures presented and analyzed in this article are PFS and OS. Data on prevalence of MET mutations and amplifications in lung cancer were also analyzed and included in this article. Table 2. Characteristics of included articles reporting the prevalence and impact of MET and HGF expression. Summary of characteristics of the six included articles reporting numbers regarding the prevalence of aberrant MET expression and results regarding the impact of the protein HGF on outcome. NSCLC: non-small cell lung cancer, EGFR: epidermal growth factor receptor, WT: wild type, NS: mutational status not specified, TKI: tyrosine kinase inhibitor, HGF: hepatocyte growth factor, ORR: objective response rate, PFS: progression-free survival, OS: overall survival, MET: mesenchymal epithelial transition factor, GCN: gene copy number, METex14: MET exon 14 skipping mutations.   In a subcohort with METex14 mutations receiving second or third-line (2/3L) treatment with capmatinib, overall response rate was 36.4%. In a second cohort with MET GCN ≥ 10, overall response rate to 2/3L capmatinib was 45.5%.

Bias
Publication bias is a factor inevitably impacting systematic reviews due to positive selection bias in the publication of research articles. This may lead to overestimation of the therapeutic effect of the investigated drugs. In this systematic review, several articles reported no effect of the evaluated drugs on PFS and OS among patients with lung cancer. By simultaneous screening of all articles by separate parties, the risk of bias in the process of selecting articles to be included in this systematic review was minimized. Furthermore, the Cochrane Risk of Bias Tool "robvis" was adapted to create risk of bias plots for all RCT's and clinical trials included in this systematic review, see Appendix C ( Figures A1 and A2) [33]. None of the authors of this systematic review declare any conflicts of interest related to the article.

Results
This systematic review identified a total of 786 eligible articles by the two search queries (Appendix A) employed following the PRISMA guidelines. Exclusion of 256 duplicates resulted in 530 remaining articles. After the assessment of article titles, 314 articles remained followed by further exclusion based on the abstract, resulting in 114 remaining articles. Six articles were excluded based on publication year and 71 studies were excluded due to being conference abstracts or supplements. Consequently, 37 full articles remained for the final full text screening. A total of 22 articles were judged to be relevant to this systematic review ( Figure 3). Six of the included articles present data regarding the prevalence and significance of aberrant signaling through the HGF/MET pathway in lung cancer. The remaining 16 articles present data on the effect of specific targeted therapies on mortality and morbidity of lung cancer patients.

Epidemiology
The reported patient characteristics varied greatly between the included studies, see Appendix C (Table A3) [18,29]. In regard to the included subjects' gender, Matsumoto et al. included a vast overweight of women compared to men in their clinical trial (80.3% women), while Okamoto et al. reported the highest percentage of men included (77.0%) [3,19]. Similar to age and gender, smoking history also varied between the studies. The highest reported prevalence of never smokers was seen in the clinical trial conducted by Matsumoto et al., with 89.1% of the patients being never smokers in an EGFR WT population [19]. Interestingly, both of the MET-negative cohorts in a RCT by Spigel et al. held smaller fractions of patients with no smoking history (7.0% in the treated subgroup and 3.0% in the placebo subgroup) compared to the MET+ cohorts in the same study (20.0% in the treated subgroup and 19.0% in the placebo subgroup) [6]. In the study by Moro-Sibilot et al., the authors reported double the amount of patients with a no smoking history in the MET mutated cohort compared to the MET amplified cohort (MET mutated: 48.0%, MET amplified: 24.0%) (Table A3) [29].

Prevalence of Aberrant MET Expression
The reported prevalence of the different MET gene alterations varies greatly between the articles included in this systematic review. In a prospective clinical trial by Palmero et al., genetic testing of treatment naïve NSCLC patients was carried out to identify genetic alterations. Next generation sequencing (NGS) of circulating cell free tumor DNA in blood was compared to standard-of-care tissue-based biopsy testing in order to determine the patients' mutational burden. The results showed no difference between the methods' ability to identify mutations in NSCLC. Out of 186 tested patients, 11.0% harbored MET exon 14 skipping mutations (METex14) and 22.0% harbored amplification of the MET gene [11].
In a retrospective clinical study by Okamoto et al., 295 patients were tested for MET mutations and 229 patients were tested for MET amplification. The results showed a prevalence of 7.6% for mutations in the MET gene and 3.9% for MET amplification, with a median GCN of 8.8. The median OS was found to be non-significantly longer among patients without MET amplification [3].
Sacher et al. carried out a single arm, single center clinical trial, testing the EGFR TKI erlotinib on 22 NSCLC patients with varying EGFR mutational statuses. All included patients were naïve to any systemic anti-cancer treatment. MET amplification was determined by fluorescence in situ hybridization (FISH) and MET positivity was defined as GCN ≥ 4. Protein expression was assessed through IHC and MET positivity was defined as an H-score ≥ 100. The prevalence of any aberrant MET expression in this study was 45.0%, while MET amplification specifically was seen in 9.0% of the patients [28].
Helman et al. conducted a retrospective study that performed NGS on plasma samples from patients suffering from NSCLC harboring EGFR mutations. NGS was carried out at study enrolment before receiving treatment and after disease progression to evaluate the effect of the EGFR tyrosine kinase inhibitor (TKI) rociletinib. Prior to treatment with rociletinib, 15.0% of the NGS screened patients were positive for genetic alterations in the MET gene. After disease progression on treatment with rociletinib, another 7.6% of the patients with acquired resistance to the drug had acquired amplification of the MET gene. This amplification was found to be caused by focal amplification in 4.5% of the patients and by aneuploidy in the remaining 3.1% [25].
In a phase II study by Arrieta et al., the levels of the HGF protein were analyzed in 66 patients with either EGFR mutated or EGFR wild type lung adenocarcinoma. All patients received treatment with the EGFR TKI afatinib after disease progression on first-line platinum-based chemotherapy. Patients with reduction of HGF levels after treatment with afatinib had a significantly longer PFS, OS, and objective response rate (ORR) compared to patients with higher levels of HGF after treatment with afatinib. These differences were most prominent among patients with EGFR mutated disease. The authors of the study suggest that HGF has a direct role in acquired resistance to EGFR TKIs, making HGF an interesting target in this field of research [1].
In a different phase II study by Matsumoto et al., HGF levels were measured and MET mutational status was determined in 47 patients with EGFR wild type NSCLC treated with erlotinib. Tumors expressing HGF had a poor response to erlotinib, and the patients had a shorter median PFS compared to the HGF negative study population. MET mutational status did not impact PFS or the response to erlotinib in this clinical trial [19].

Targeted Therapies
Sixteen of the papers included in this systematic review are studies investigating targeted therapies for the MET gene. The different MET TKIs evaluated in the clinical trials are presented in Table 3. The most common MET TKIs were onartuzumab and crizotinib, occurring in five and four of the included articles each.
In a phase II study conducted by Hirsch et al., treatment with the combination of onartuzumab, paclitaxel, and carboplatin/cisplatin was compared to treatment with only paclitaxel and carboplatin/cisplatin in 106 NSCLC patients. The included patients were all EGFR wild type, with or without mutations in the MET gene. The results showed no significant benefit of added treatment with onartuzumab, regardless of MET mutational status, with similar PFS (5 months in the onartuzumab group and 5.2 in the placebo group) and OS (10.8 months vs. 7.9 months) in the two groups. In this study, MET mutational status was evaluated by immunohistochemistry, with IHC3+ and IHC2+ considered MET positive, and IHC1+ and IHC0 considered MET negative [17].
Similar results were found in a phase II study conducted by Wakelee et al., including 259 NSCLC patients in two cohorts. Cohort 1 tested the addition of onartuzumab vs. placebo to treatment with bevacizumab, carboplatin/cisplatin, and paclitaxel. In cohort 2, patients received either onartuzumab or placebo in addition to carboplatin/cisplatin and pemetrexed. The subjects had varying mutational statuses regarding both EGFR and MET. MET positivity was defined as IHC3+ or IHC2+. In cohort 1, the overall median PFS was 5.0 months in the onartuzumab group compared to 6.8 months in the placebo group.
In a MET+ subgroup, median PFS was 4.8 months and median OS was 9.9 months in the onartuzumab arm compared to 6.9 months and 16.5 months in the placebo arm. Cohort 2 revealed similar results, with a median PFS of 5.1 months in the placebo group compared to 4.9 months in the onartuzumab group. Median OS was 13.7 months in the placebo treated group and 8.5 months in the onartuzumab treated group. In the MET+ subgroup of cohort 2, median PFS was 5.0 in for both onartuzumab and placebo while median OS was marginally longer in the onartuzumab arm (8.0 months) compared to the placebo arm (7.6 months) [32].
In another phase III study evaluating onartuzumab, 636 NSCLC patients with varying EGFR and MET mutational statuses were included and treated with either erlotinib and onartuzumab or erlotinib and placebo. In this study, high doses of onartuzumab were associated with a longer median PFS compared to lower doses of erlotinib or placebo (high dose PFS = 4.37; low dose PFS = 2.5 months; placebo PFS = 2.5 months). No significant differences were found in OS regardless of MET mutational status [14].
In contrast to this, a phase II study by Spigel et al. performed on NSCLC patients found that both median PFS and OS were significantly longer in a MET positive subgroup (defined as MET IHC3+ or IHC2+) treated with onartuzumab and erlotinib compared to a subgroup treated with erlotinib and placebo (dual treatment PFS = 2.9 months, OS = 12.6 months; single treatment PFS = 1.5 months, OS = 3.8 months). All 136 included participants were EGFR wild type but had varying MET mutational statuses. Interestingly, the patients without any mutations in the MET gene had earlier progression when treated with the combination of onartuzumab and erlotinib compared to erlotinib and placebo [6].
A third phase II study, titled the global METLung study (OAM4971g), presented results on treatment with onartuzumab and erlotinib without comparing the effects to a control group. This study included 61 patients with MET and EGFR mutation positive NSCLC but was terminated early due to lack of efficacy of onartuzumab. Patients with MET IHC3+ or IHC2+ as well as a total number of MET genes in 20 cancer cells ≥ 90 as determined by a gene amplification assay were considered MET positive. The preliminary results showed a median PFS of 8.5 months, a median OS of 15.6 months, and an overall response rate of 68.9% in the patients treated with onartuzumab and erlotinib [15].
In a phase II clinical trial, Landi et al. evaluated treatment with the targeted MET inhibitor crizotinib in an EGFR wild type NSCLC population of 26 patients with either MET amplification or METex14 mutation. The median PFS was 4.4 months, median OS was 5.4 months, and the ORR was 27.0%. MET amplification was defined as a MET/centromere 7 (CEP7) ratio > 2.2 [18]. Jänne et al. treated 67 NSCLC patients with mixed EGFR mutational statuses with crizotinib and dacomitinib in a phase I clinical trial. The included patients had experienced progression on first-line treatment with either chemotherapy or another targeted therapy and were included in either an escalation phase cohort or in an expansion phase cohort. The median PFS was 3.0 months in the escalation cohort, with 61.0% of these patients having stable disease during the treatment. In the expansion cohort, the median PFS was 2.1 months and 32.0% of the patients had stable disease during treatment. No association was seen between overexpression of MET and PFS. MET positivity was defined as IHC3+, IHC2+, or MET GCN ≥ 2.1 [21].
In another phase II clinical trial conducted by Moro-Sibilot et al., 53 NSCLC patients with varying EGFR mutational statuses were treated with crizotinib. The study included two cohorts with aberrant MET expression, one with MET GCN ≥ 6 (n = 25) and one with positivity for MET exon skipping mutations in exon 14 or 16-19 (n = 28). In the MET amplified cohort (GCN ≥ 6), the ORR was 16.0%, the median PFS was 3.2 months, and the median OS was 7.7 months. In the MET mutated cohort, the OS was longer while the PFS and ORR were inferior (ORR = 10.7%, median PFS = 2.4 months, median OS = 8.1 months) [29].
In a phase I study by Ou et al., crizotinib was evaluated in combination with erlotinib on 26 NSCLC patients with previous progression of disease on one or two prior treatments with chemotherapy. The patients' EGFR and MET mutational statuses were not reported in this article. Only 20 of the 26 included patients were evaluated for response to treatment. Of these, two had partial response, eight had stable disease, and ten had progressive disease as defined by the RECIST version 1.1 guidelines [24].
One study evaluated the MET targeted drug savolitinib in combination with osimertinib in a phase Ib clinical trial. A total of 180 patients with EGFR mutation positive and MET amplified NSCLC received treatment with savolitinib and osimertinib after previous treatment with one, two, or three different EGFR TKIs. In this study, MET amplification was defined as MET GCN ≥ 5, MET/CEP7 ratio ≥ 2, MET IHC3+, or MET expression in ≥20.0% of tumor cells as determined by NGS. Patients were stratified into one of two cohorts according to type of EGFR mutation and number of prior EGFR TKI treatments. Only 161 patients were eligible for final evaluation of treatment effect. Of these, 89 patients had a partial response according to the RECIST version 1.1 guidelines. One of the two cohorts included 138 patients and had a median PFS of 7.6 months, while the other cohort, including 23 patients, had a median PFS of 9.1 months [22].
In a phase Ib/II trial by Wu et al., standard platinum doublet chemotherapy was compared to treatment with the targeted drugs tepotinib and gefitinib. In this clinical trial, 55 patients were included, all with NSCLC, positive for EGFR mutation, and MET overexpression or amplification. MET overexpression was defined as IHC2+ or IHC3+, and amplification was defined as GCN ≥ 5. All patients included in the phase II part of the trial had acquired resistance to other first or second-generation EGFR TKIs. There were no significant differences in median PFS or OS when comparing chemotherapy to targeted therapy. Subgroup analyses were carried out on groups of patients harboring either MET amplification or high MET overexpression defined as IHC3+. In the MET amplified group, both PFS and OS were significantly longer in the targeted therapy treated group compared to the chemotherapy treated group (targeted therapy median PFS = 16.6, chemotherapy median PFS = 4.2; targeted therapy OS = 37.7, chemotherapy OS = 13.1). In the MET IHC3+ group, the median PFS was 8.3 months, and the OS was 37.3 months compared to a PFS of 4.4 months and an OS of 17.9 in the chemotherapy group [23].
A phase I study evaluating capmatinib in NSCLC patients with MET amplification or MET overexpression showed that patients with a high MET GCN or METex14 mutations may benefit from treatment with MET inhibitors. Of 55 enrolled patients treated with capmatinib, 26 were included in a dose expansion group with varying EGFR mutational statuses and either MET overexpression or amplification. The remaining 29 patients were all EGFR wild type and had high overexpression of MET, defined as IHC3+. Complete response was observed in one of the 55 patients and partial response was seen in ten patients. The median PFS was 3.7 months for the entire cohort, whereas the MET IHC3+ group had a median PFS of 5.1 months. An even greater PFS was seen among patients with MET GCN ≥ 6 (median PFS of 9.3 months). Furthermore, in four patients with METex14 mutations, a reduction in the tumor burden between 14.0 and 83.0% could be seen. This trial was terminated early due to disease progression and frequent adverse events [20].
A phase II trial by Seto et al. tested the effect of capmatinib on 45 EGFR wild type NSCLC patients divided into several cohorts depending on MET mutational burden and prior systemic anti-cancer treatment. Aberrant MET expression was defined as the presence of the METex14 mutation or amplification of the MET gene. The results showed that treatment with capmatinib as a second or third-line option in METex14 positive subjects (n = 11) yielded an overall response rate of 36.4%. The overall response rate in a cohort of MET amplified patients with GCN ≥ 10 (n = 11) was found to be 45.5%. In contrast to this, in a cohort with MET GCN ≥ 4 but <6 (n = 10), the overall response rate was only 10.0%. The remainder of the patients were further subdivided into considerably smaller cohorts with inconclusive results [31].
Yoshioka et al. conducted a phase III clinical trial comparing treatment with tivantinib and erlotinib to treatment with erlotinib and placebo in 303 EGFR wild type NSCLC patients. All included patients had received one or two prior treatments, one of them being platinumbased chemotherapy. The patients had varying MET mutational statuses, including high and low expression of MET as well as elevated and normal MET GCN. The authors found no significant difference in the median OS when comparing the dual treatment group to the erlotinib plus placebo group. In the patients treated with both tivantinib and erlotinib, a significantly longer median PFS could be seen compared to the placebo group (dual treatment PFS = 2.9 months, placebo PFS = 2.0 months). Furthermore, high expression of HGF, defined as an H-score ≥ 200 as measured by IHC, was associated with a significant benefit in OS in the tivantinib plus erlotinib group compared to the placebo group. This clinical trial was terminated early due to increased incidence of interstitial lung disease, with 14 cases in the dual treatment group and 6 cases in the placebo group [5]. This is coherent with the results of a phase III study conducted by Scagliotti et al., which also compared erlotinib plus tivantinib to erlotinib plus placebo in 109 EGFR mutated NSCLC patients with varying MET mutational statuses. Here the median PFS was 13.0 months and the median OS was 25.5 months in the dual treatment group compared to a median PFS of 7.5 months and a median OS of 20.3 months in the erlotinib plus placebo group [30].
In a phase II clinical trial, Neal et al. assessed treatment with cabozantinib in EGFR wild type NSCLC patients with varying MET mutational statuses. This study comprised three treatment arms, one with both erlotinib and cabozantinib, one with only cabozantinib, and one with only erlotinib. All patients had previously been treated with one or two other therapeutic agents prior to enrolment in this trial. The median PFS was significantly longer in the erlotinib plus cabozantinib arm (median PFS = 4.7 months) and in the cabozantinib arm (median PFS = 4.3 months) compared to the erlotinib arm (median PFS = 1.8 months). The median OS was also longer in the erlotinib plus cabozantinib arm (median OS = 13.3 months) compared to the erlotinib arm (median OS = 5.1 months). The median OS in the cabozantinib arm was 9.2 months and was not significantly longer compared to the erlotinib arm. In this clinical trial, no association was found between MET IHC status and median PFS in the patient groups receiving treatment with cabozantinib or erlotinib plus cabozantinib [2].
In summary, many of the targeted drugs mentioned in this review show promising results that encourage further research. Onartuzumab, as an added treatment to erlotinib, has been shown in several trials to lead to a longer PFS and OS in NSCLC patients, compared to treatment with placebo and erlotinib. One clinical trial showed a reversed effect of onartuzumab as an added treatment to chemotherapy, as compared to placebo, with a shortened PFS and OS among the patients treated with onartuzumab. Crizotinib alone has been proven to greater increase OS among NSCLC patients harboring MET mutations compared to patients with MET amplified cancer genotypes. Crizotinib, as an addition to the EGFR TKIs dacomitinib or erlotinib, has resulted in a greater fraction of patients with stable disease. Treatment with savolitinib in combination with osimertinib has led to a fraction of patients with partial response of more than 55% in cohorts of NSCLC patients with previous progression of disease on EGFR TKIs. Tepotinib and gefitinib, as a treatment for NSCLC patients harboring high overexpression or amplification of MET, has been shown to prolong both the PFS and OS compared to treatment with chemotherapy. Similarly, in NSCLC patients with a high MET GCN or high levels of overexpression of MET, treatment with capmatinib has prolonged PFS and led to a reduced tumor burden and higher overall response rates compared to cohorts with lower grades of overexpression or amplification. The addition of tivatinib to treatment with erlotinib has been proven to be effective and has led to increases in PFS and OS compared to placebo. Lastly, use of cabozantinib alone or in combination with erlotinib has led to significantly longer PFS compared to treatment with erlotinib alone. For an overview of the results, see Figure A3 (Appendix C).

Discussion
Lung cancer continues to be the leading cause of global annual cancer-related mortality, and the need for more efficient therapies is evident. Targeted therapies are a promising area of research, offering new possibilities for inhibiting genetic alterations that are involved in driving tumorigenesis, but to this day, there are no known clinically useful biomarkers [34][35][36][37]. Lung cancer patients harboring alterations in the MET gene detected in circulating DNA in blood or tumor tissue have been shown to have poorer outcomes.
In a study by Andreasson et al., the authors were able to show that the MET protein could be found in exhaled breath as well as in blood plasma, and that the expression of the protein diminished after surgical removal of the lung cancer, underlining the proteins role in lung cancer tumorigenesis [34].
There is a pressing need for novel and more efficient therapies that can target the MET signaling pathway, but for this to be possible, the understanding of how abundant certain genetic alterations are, and how they affect and drive lung cancer development, needs to be further deepened. Therefore, this systematic review aimed to summarize and enlighten the current status of genetic mapping and prevalence of different alterations in the MET gene in NSCLC patients, as well as existing and pipe-line targeted therapies towards this protein.
The articles reviewed in this paper reported that MET mutations were found in 7.6-11.0% of lung cancer patients and that amplification was found in 3.9-22.0% of cases [3,11]. One study reported that genetic alterations in the MET gene were present in 15.0% of the lung cancer cases analyzed [25]. These numbers are in line with previously published data [38][39][40]. One study presented results on prevalence, which differed significantly from the rest of the included papers, with a reported frequency of aberrant MET expression of 45.0% [28]. The reported prevalence of aberrant MET expression varies in different studies. This may, in part, be due to the fact that different studies analyze samples from different genotypes of lung cancer, with differing characteristics, using different quantification methods and definitions. Some of the articles included in this systematic review analyzed the prevalence of aberrant MET signaling in EGFR mutated patients, while other studies do not specify EGFR mutational status [3,11,25]. It has previously been shown that alterations in the MET gene are a known mechanism of resistance to EGFR TKIs; therefore, considering the impact of previous treatments on genetic analyses results may offer valuable insights for included patients [22].
The varying patient characteristics in the included studies make it difficult to predict any specific groups of the population that are at increased risk of developing NSCLC with MET mutated genotypes. Some trials do not specify MET mutational status or present their data on patient characteristics in cohorts of patients with varying MET mutational statuses [6,21,24]. However, some trends can be seen among the studies presenting results on patient characteristics in cohorts with specified MET mutations [6,15,18,20,22,23,29,31]. The median age among patients harboring MET mutations ranges from 56.0 to 72.0 years. MET mutations appear equally among both genders, with the greatest difference being reported by a single study being 32% men and 68% women [29]. Cohorts with MET positive patients reported smoking history as "never smokers" ranging from 19 to 72%, compared to cohorts consisting of MET negative patients, with the lowest reported percentage of never smokers being 3.0% in the study by Spigel et al. [6]. This could be indicative of a trend with higher fractions of MET positive NSCLC patients being never smokers. However, more research that is more directly aimed at this research question needs to be conducted to verify these results.
Five of the included studies evaluated the MET TKI onartuzumab. Spigel et al. found a significantly longer OS and PFS in MET positive patients receiving treatment with both onartuzumab and erlotinib compared to patients receiving erlotinib alone. Interestingly, they also found that the MET negative patients suffered earlier progression of disease and increased mortality when treatment with onartuzumab was added compared to treatment with placebo alone [6]. This is in line with the findings of Han et al., who showed a significantly longer PFS among patients treated with high doses of onartuzumab together with erlotinib compared to single treatment with erlotinib or erlotinib plus lower doses of onartuzumab [14]. Kishi [17]. MET has previously been suggested to act as both a suppressor and an oncogene, which is in line with the presented results [6]. These findings underline the importance of genetic testing upon lung cancer diagnosis in order to customize targeted therapies and postpone or prevent progression caused by treatment with ill-suited therapies.
Two separate trials investigated the effect of dual treatment with tivantinib and erlotinib compared to single treatment with erlotinib. They both found a prolongation in the median PFS in the dual treatment group compared to treatment with erlotinib alone, although to very differing extents. No difference was seen in the median OS [5,30]. One study included only EGFR wild type subjects while the other had only EGFR mutated subjects. The variation in EGFR mutational status is likely a contributing factor to the varying results of the studies included in this review. Some clinical trials included only EGFR wild type lung cancers, others included only EGFR positive cases or a combination of wild type and positive cases. Two studies evaluated the effect of additional treatment with onartuzumab to erlotinib in patients with varying EGFR mutational statuses [6,14]. As erlotinib is an EGFR TKI, it seems likely that the patients with an EGFR positive cancer genotype would benefit more from treatment with erlotinib than the patients with an EGFR wild type genotype, making it difficult to evaluate the actual effect of the added onartuzumab or capmatinib treatment. Therefore, in these studies, the effect of added treatment with onartuzumab or capmatinib should be interpreted carefully and further research in larger clinical trials is needed to determine the effect.
Multiple studies performed on different MET TKIs have presented varying results on PFS, ranging from 2.1 to 9.1 months, without comparisons to control groups [18,[20][21][22]29]. Interestingly, two of these articles showed that treatment with the targeted drug crizotinib had a greater effect in a ROS1 translocated NSCLC cohort compared to MET altered cohorts [18,29]. Other studies evaluating crizotinib did not report ROS1 mutational status, rendering the results more difficult to interpret [21,24]. Neal et al. reported that treatment with cabozantinib and erlotinib and single treatment with cabozantinib was superior to treatment with erlotinib alone in lung cancer patients with EGFR wild type lung cancer. These results support the statement that patients with EGFR wild type lung cancer may not respond to treatment with EGFR TKIs alone. In the clinical trial conducted by Neal et al., the authors did not find any association between MET IHC+ status and prolonged PFS in the treatment groups receiving cabozantinib. It is not possible to draw any conclusions regarding the association between prolonged time to progression and death, and inhibition of the MET receptor specifically, as cabozantinib has multiple gene targets [2,22]. Several clinical trials present similar results, indicating that patients with a high mutational burden in the MET gene (defined as high overexpression, high GCN gain, and high IHC+ status) benefit more from treatment with MET TKIs compared to patients with a lower mutational burden [5,20,23,31]. This further emphasizes the importance of thorough genetic mapping of cancer patients prior to treatment.
An important aspect in trials testing novel drugs is the fact that the included cohorts differ greatly in cancer genotype [6,14]. This puts the reliability and comparability of the results into question. Another aspect is the fact that the drugs presented in these trials typically show a gain in PFS and OS months at best [5,6,14,17]. This needs to be put in relation with the potential side effects and adverse events that the patients experience during treatment [5,20]. MET TKIs are still considered experimental, and treatment with targeted MET inhibitors is currently a second, third, or even fourth-line treatment option. It is known that cancers generally harbor fewer driver mutations in the beginning of tumorigenesis compared to the later stages of disease and so it is possible that earlier implementation of MET inhibitors could provide a greater effect on PFS and OS [41,42]. However, the timing of treatment will need to be investigated in further clinical trials.
The different cancer genotypes also contribute to potential bias in reporting more or less promising pipe-line drugs. However, there are several interesting ongoing clinical trials evaluating different pipe-line drugs on more targeted patient groups harboring different MET alterations (Table 4) [43]. Two of these ongoing trials have already presented results from their studies. The NCT02544633 trial tested the MET inhibitor MGCD265 on patients with NSCLC and MET activating mutations or MET amplifications. The patients were divided into four study arms, one with MET activating mutations in tumor tissue, one with MET activating mutations in circulating tumor DNA in the bloodstream, one with MET amplification in tumor tissue, and one with MET amplification in circulating tumor DNA. Interestingly, in the case of MET amplification, the patients with MET amplification identified in circulating tumor DNA had a poorer PFS (2.76 months) and OS (4.08 months) compared to the patients with MET amplification identified in tumor tissue (PFS 4.85 months, OS 7.04 months). The patients with MET activating mutations in tumor tissue had a longer OS of 16.32 months compared to all other study arms, but not PFS (3.95 months). The last treatment group with MET activating mutations in circulating tumor DNA had a similar PFS to the other three study arms (3.39 months). No results were available regarding OS in this fourth study arm [44]. This data is similar to results from earlier studies on MET TKIs presented in this systematic review. A possible explanation to why the patients with MET amplification detected in circulating tumor DNA had a poorer outcome is the greater tumor burden associated with circulating tumor cells compared to that of a localized tumor.
One other study had available preliminary results at the time of writing this systematic review (trial ID: NCT02648724). In this phase I/II study, the monoclonal anti MET antibody mixture entitled Sym015 was investigated. The study included 57 patients divided into three different treatment arms, one with KRAS wild type patients with MET amplifications, one with MET amplified NSCLC, and one where the patients harbored METex14 deletions. The outcome measure presented was the ORR and the results showed that the KRAS wild type cohort had 0.0% ORR while the MET amplified and the METex14 deletion cohort both had an ORR of 25.0% [45]. A vast difference between the already existing and published clinical trials and the ongoing ones is the greatly improved consistence in genotypes included in the newer trials. The majority of the current trials on pipe-line targeted therapies consist of cohorts of patients that are all genetically altered in the MET gene rather than cohorts of mixed cancer genotypes. This will make future results more easily interpreted and clinically useful.

Strengths and Limitations
The most apparent limitation of the articles included in this review is the lack of control groups in most of the phase I trials, leading to difficulties in interpreting the results. Furthermore, some articles have a low number of included patients and overall, the number of participants varies greatly. Since the included studies have a high grade of variation in patient characteristics, such as age, gender, and smoking history, the comparison between the studies' results need to be interpreted with some caution. As the targeted therapies are tested on different patient categories, with varying cancer genotypes, it is difficult to compare the presented results and draw conclusions on the efficacy of the investigated drugs. On the same note, the definition of the criteria for what is considered aberrant MET expression varies between the included trials, with some research groups applying IHC, some performing NGS, and some using different methods. A strength of this review is the variation in location of the included trials, leading to more generalizable and widely applicable conclusions. The exclusion criterion of a publication year prior to 2013 might be considered a limitation; however, this also ensured that only current results were included in this systematic review.

Conclusions
It remains difficult to compare different targeted therapies and to draw conclusions regarding their potential place in the future treatment panorama. The inter and intra study variation in cohort composition and included cancer genotypes is large. Onartuzumab has shown prolonged PFS and OS among MET positive NSCLC patients but no convincing results in cohorts with mixed MET mutational statuses. The studies on crizotinib lacked control groups for comparison of the outcome measures. Many ongoing trials on pipe-line targeted therapies exist, which are investigating anti-MET agents on clearly defined cohorts of patients with aberrant MET expression. This systematic review summarized the current status of publications on the MET gene's implications in lung cancer development and the status of existing and up and coming targeted therapy options. More research is needed and should be encouraged to fully understand how, when, and to whom these drugs should be recommended in order to improve patient outcomes. Selection process 8 Specify the methods used to decide whether a study met the inclusion criteria of the review, including how many reviewers screened each record and each report retrieved, whether they worked independently, and if applicable, details of automation tools used in the process.

Page 5, paragraph 2
Data collection process 9 Specify the methods used to collect data from reports, including how many reviewers collected data from each report, whether they worked independently, any processes for obtaining or confirming data from study investigators, and if applicable, details of automation tools used in the process. Describe the processes used to decide which studies were eligible for each synthesis (e.g., tabulating the study intervention characteristics and comparing against the planned groups for each synthesis (item #5)). Page 8, Table 3 13b Describe any methods required to prepare the data for presentation or synthesis, such as handling of missing summary statistics, or data conversions. N/A 13c Describe any methods used to tabulate or visually display results of individual studies and syntheses. Page 8, Table 3 13d Describe any methods used to synthesize results and provide a rationale for the choice(s). If meta-analysis was performed, describe the model(s), method(s) to identify the presence and extent of statistical heterogeneity, and software package(s) used.
N/A 13e Describe any methods used to explore possible causes of heterogeneity among study results (e.g., subgroup analysis, meta-regression). N/A 13f Describe any sensitivity analyses conducted to assess robustness of the synthesized results. N/A Reporting bias assessment 14 Describe any methods used to assess risk of bias due to missing results in a synthesis (arising from reporting biases). Page 11, paragraph 1 Certainty assessment 15 Describe any methods used to assess certainty (or confidence) in the body of evidence for an outcome. N/A

Study selection 16a
Describe the results of the search and selection process, from the number of records identified in the search to the number of studies included in the review, ideally using a flow diagram. Page 6, Figure 3 16b Cite studies that might appear to meet the inclusion criteria, but which were excluded, and explain why they were excluded. N/A Report which of the following are publicly available and where they can be found: template data collection forms; data extracted from included studies; data used for all analyses; analytic code; any other materials used in the review.

Included studies 7
Give the total number of included studies and participants and summarise relevant characteristics of studies. Yes

Synthesis of results 8
Present results for main outcomes, preferably indicating the number of included studies and participants for each. If meta-analysis was done, report the summary estimate and confidence/credible interval. If comparing groups, indicate the direction of the effect (i.e., which group is favoured).

Limitations of evidence 9
Provide a brief summary of the limitations of the evidence included in the review (e.g., study risk of bias, inconsistency and imprecision).    Figure A3. Forest plots presenting the median PFS and median OS with 95% confidence intervals (CI) from the included articles in this systematic review presenting results on treatment with a MET TKI. Results that are specific to a MET+ subgroup are presented separately from the overall cohort results [2,5,6,14,15,17,18,[20][21][22][23]29,30,32]. (A) Median PFS with 95% CIs. (B) Median OS with 95% CIs.