c-Met Mutations Research Articles

Abstract 4475: Targeting c-MET and MAPK pathway in metastatic castration-resistant prostate cancer

Abstract Identification of “agnostic” genetic drivers in cancer is foreseen as a major step forward in precision medicine. Thus, treatment selection could be based on the molecular background of the disease rather than on tumor histology in the near future. However clinical validation of most alterations is lacking and only preclinical models are available. We describe a prostate cancer patient refractory to standard therapies that was found to harbor both a c-Met activating mutation and a BRAF fusion that greatly responded to the MEK inhibitor (MEKi) trametinib. EXPERIMENTAL PROCEDURES The Next Generation Sequencing (NGS) platforms (Oncomine Assay and Archer Variant Plex) were used to characterize molecularly FFPE tissue from the primary tumor of a 73 years old man with metastatic castration-resistant prostate cancer (mCRPC) refractory to Docetaxel, Enzalutamide and Radium 223. Immunostaining was performed to evaluate the status of protumoral pathways activated by the identified genomic alterations. RESULTS Oncomine assay detected a pathogenic c-MET mutation that affected the splicing of exon 14 and the patient was included in a clinical trial with a c-MET inhibidor. He developed Grade 3 anemia, G3 bone pain which required major opioids, a rise of PSA over 900 ng/ml and progressive disease as best response. An extended screening using Archer platform detected a SND1/BRAF fusion. Immunohistochemistry revealed homogeneous tumor positive staining for c-MET, and a punctured staining on pERK in FFPE regions with perineural tumor invasion suggesting a critical role of the BRAF fusion in the spread of the disease. After a comprehensive review of the literature a similar model was identified, where the SND1/BAF function raised in a cell culture of gastric cancer as mechanism of resistance to MET inihibitors (Lee NV et al PLoS One 2012). In preclinical experiments the MEKi trametinib induced significant cell mortality. After given written consent, the patient was put on trametinib (2mg/day) under compassionate use. We observed a large decrease of PSA levels to 90 ng/ml after two weeks of treatment, an increase of hemoglobin levels and significant clinical benefit, with no need of analgesics. After two months on treatment the patient continues on tumor response by the time of communication of this abstract. CONCLUSIONS According to our knowledge, this is the first published study able to demonstrate that cancer patients harboring c-MET exon 14 splicing mutation and SND1-BRAF fusion genes could benefit from therapies with MEK inhibitors. Further experimental procedures are needed to validate this hypothesis. 1. Lee NV, Lira ME, Pavlicek A, Ye J, Buckman D, et al. (2012) A Novel SND1-BRAF Fusion Confers Resistance to c-Met Inhibitor PF-04217903 in GTL16 Cells though MAPK Activation. PLoS ONE 7(6): e39653. doi:10.1371/journal.pone.0039653. Citation Format: Sergio Ruiz, María Dolores Fenor de la Maza, Juan Francisco Rodríguez Moreno, Elena Sevillano, Paloma Navarro, Sandra Amarilla, Teresa García Donas, Eduardo Caleiras, Juan Carlos Torrego, Jesus García Donas. Targeting c-MET and MAPK pathway in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4475.

Discussion on several problems between tumor-associated venous thrombosis and driving genes status.

e13022 Background: The study aimed to explore clinical correlation between venous thrombosis and driving genes status in cancer patients (pts). Methods: The clinical data of cancer pts from 2014 to 2018 in our hospital were collected to calculate the overall thrombosis rate, driving gene mutation rate, and thrombosis rate in pts with mutated driving genes. Pts were divided into mutation group and non-mutation group based on the driving gene mutation status. Khorana model was used to divide each group into three subgroups and the thrombosis rate of each subgroup was calculated. The diagnostic value of the Khorana model was calculated in the whole patient set and each group. The correlation between driving gene mutation status and thrombus status was also analyzed. Results: A total of 2049 pts were enrolled, with 4.44% overall thrombosis rate and 72.13% driving gene mutation rate. The thrombosis rates in pts with EGFR, c-Met, ALK, HER-2, KRAS or ROS1 mutations were, in turn, 5.34%, 4.33%, 3.92%, 3.73%, 3.45% and 2.74%, however, there was no statistical difference in thrombosis rates between pts with or without the above gene mutations (EGFR, p = 0.90; c-Met, p = 0.23; ALK, p = 0.85; HER-2, p = 0.28; KRAS, p = 1.00; ROS1, p = 0.58). In the correlation analysis between the driving gene status and the thrombus status, the Spearman correlation coefficient was -0.024, and no statistically significant difference was observed (p = 0.69). Based on the Khorana model, the thrombosis rates of the low-, intermediate- and high-risk subgroups in the mutation group and non- mutation group were 0% vs 0%, 10.23 vs 8.33%, and 6.67% vs 12.50% respectively, which didn’t show statistically significant differences (low- risk, p = 1.00; intermediate-risk, p = 0.85; high-risk, p = 0.59). For the whole patient set, mutation group and non-mutation group, the Khorana model predicted venous thrombosis with the sensitivities of 13.33%, 10.00% and 20.00%, as well as the specificities of 92.02%, 92.55% and 90.67%; and the area under the ROC curve were 0.706 (p = 0.007), 0.719 (p = 0.02) and 0.681 (p = 0.18), respectively. Conclusions: The Khorana model has a moderate diagnostic value, and there is no correlation between the driving gene status of cancer pts and venous thrombosis.

Fishing wild-type sparing inhibitors of proto-oncogene c-met variants in renal cell carcinoma from a curated tyrosine kinase inhibitor pool using analog-sensitive kinase technology

A variety of missense mutations in proto-oncogene protein tyrosine kinase c-Met have been clinically observed in the patients and families of papillary renal cell carcinoma (pRCC), imparting that the kinase mutations can be exploited as a new and potential therapeutic target for pRCC. Here, a systematic inhibitor response-to-kinase mutation profile for ATP-competitive tyrosine kinase inhibitors (TKIs) against pRCC-related c-Met mutations is created using a rigorous thermodynamic cycle scheme, from which we are able to identify a number of representative inhibitor/mutation pairs with passivation and sensitization. It is revealed that passivation is commonly caused by steric hindrance between the mutated residue and inhibitor ligand, while sensitization usually results from the formation of favorable nonbonded interactions upon the mutation. The type II inhibitor Nintedanib possesses a high selectivity (7.2-fold) for c-MetY1248H variant over wild type. Structural and energetic analysis revealed that the Y1248H mutation is located in kinase's activation loop which can directly contact the extended moiety of type II inhibitor. The titratable variant residue His1248 is protonated with stabilization by its vicinal negatively charged residue Asp1246, which can form a geometrically satisfactory hydrogen bond and a weak cation-π interaction with the inhibitor ligand, thus conferring variant selectivity to the Nintedanib/Y1248H sensitization.

Correlation among genetic variations of c-MET in Chinese patients with non-small cell lung cancer.

BackgroundThe purpose of our research was to determine the correlation of amplification, protein expression and somatic mutation of c-MET in IIIb-IV stage NSCLC (Non-small cell lung cancer). We also explored correlation of c-MET variation with clinical outcome.Resultsc-MET expression was observed in 28.6% (56/196) cases, and among those 13.8% (27/196) were shown to be FISH positive. Only 2.67% patients in this study carried the c-MET mutation. Cases with c-MET FISH positive were all IHC positive ,but in IHC positive cases, only half were FISH positive. Among patients with IHC2+ staining, 35.5% was FISH positive, while cases with IHC3+ staining,64% was FISH positive. Both protein expression and copy number of c-MET did not significantly correlate with clinical prognosis in these patients treated with EGFR-TKIs.ConclusionsIHC could be used as a preliminary screening method for c-MET copy number amplification and should be confirmed by FISH only in IHC positive case which facilitate selection of ALK or MET inhibitor therapy.Methodsc-MET gene copy number, protein expression and somatic mutation for exon 14 were detected by fluorescent- In-Situ-Hybridization (FISH), Immunohistochemistry (IHC), and Denaturing-High-Performance-Liquid-Chromatography (DHPLC), respectively, in 196 NSCLC patients. The relationship between c-MET abnormalities and clinical outcome of targeted therapy was analyzed by McNemar's test.

Abstract 2773: A prospective observational study evaluating c-MET expression and EGFR gene mutation correlation with erlotinib response in second line treatment for advanced/metastatic non-small cell lung cancer patients

Abstract Background: The c-MET is associated with tumor progression and acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). The aim of this study was to evaluate the prevalence and predictive role of c-MET and EGFR mutation in non-small cell lung cancer (NSCLC). Methods: This was a multicenter prospective observational study for patients with stage IV or recurrent NSCLC who had progressive disease after 1st line chemotherapy and was followed by erlotinib for 2nd line treatment from September 2011 to September 2014. The primary outcome was to measure the rates of c-MET expression/amplification and EGFR mutation in tumor tissue. MET expression was evaluated by immunohistochemistry (IHC) and gene copy number was assessed by silver in situ hybridization (SISH). EGFR mutations were analyzed by Cobas™ version 1. Results: A total of 196 patients were enrolled and major histologic types were adenocarcinoma (67%). c-MET was overexpressed (MET IHC-positive) in 44.4% of the patients and dominant in female and non-squamous histology. c-MET amplification and high polysomy (MET SISH-positive) were observed in 1.5% and 8.7%, but there was no specific clinical characteristics. The detection rate of EGFR mutation was 10.2% (19 deletion: 11, L858R: 7, exon 20 insertion: 2 cases). The response rate of erlotinib was significantly higher in EGFR sensitive mutant group (55% vs. 4%, p<0.001) and the median progression-free survival (PFS) was significantly longer than wild type (173 vs 58 days, p<0.001). c-MET positivity (IHC- or SISH-positive) did not show significant correlation with response or PFS. Patients with SISH-positive for c-MET tended to have shorter overall survival (OS) than patients with SISH-negative (127 vs 303 days, p=0.051). Conclusion: In this observational study with 2nd line erlotinib treatment, we reconfirmed EGFR mutation is strong predictive marker, and EGFR-TKI use in EGFR wild-type patients should be considered carefully and may be harmful. c-MET overexpression was related to some clinical characteristics such as female and non-squamous histology. Although c-MET positivity was not associated with response or PFS, c-MET SISH may be predictive marker for OS. Citation Format: Cheol-Kyu Park, Yoo-Duk Choi, In-Jae Oh, Jung-Eun Lee, Shin Yup Lee, Tae-Won Jang, Jeong-Seon Ryu, Young-Chul Kim. A prospective observational study evaluating c-MET expression and EGFR gene mutation correlation with erlotinib response in second line treatment for advanced/metastatic non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2773. doi:10.1158/1538-7445.AM2017-2773

Abstract 1765: Molecular and immunological characterization of non-small cell lung cancer harboring cMET alterations

Abstract Purpose: To explore the correlation between the expression of immunological biomarkers in a subset of NSCLC tumors presenting cMET activation. Methods: We retrospectively reviewed 200 tumor samples from a cohort of patients diagnosed with NSCLC in 3 different hospitals, from Spain and France. cMET activation was defined by the presence of one or more of the following criteria: 1- cMET mutations inducing exon 14 skipping (METex14), 2- cMET amplification, assessed by FISH 3- cMET overexpression, assessed by immunohistochemistry (IHC). METex14 mutations were determined by genomic sequencing and RT-PCR. FISH was interpreted according to Capuzzo scoring system, considering 5 or more gene copies as the mean value for positive amplification. Concurrent genetic alterations in EGFR and ERBB2 tyrosine kinase receptors were also determined. PD-L1 expression and density of tumoral infiltrating CD8+ lymphocytes were assessed by IHC. Statistical correlation analysis was performed by Chi-square and Fisher’s exact test. Results: METex14, cMET amplification and cMET overexpression were detected in 2%, 7.8% and 31% of tumors, respectively. Histological subtypes included: 70% adenocarcinoma, 20% squamous cell carcinoma, 8% large cell, 2% pulmonary sarcomatoid. Curiously, one of the METex14 adenocarcinoma was concurrent with ERBB2 amplification. Those tumors harbouring METex14 or cMET amplification were correlated with positivity for PD-L1 expression (p = 0.02). However, this association was not observed in cMET overexpressed tumors. Conclusions: We observed a direct correlation between PD-L1 positivity and METex14 or cMET amplification. Functional analysis and a large cohort of tumors are required to better characterize the potential role of cMET pathway in the regulation of PD-L1 expression. Citation Format: Maria Saigi, Carolina Pereira, Eva Pros, Elisabeth Brambilla, Ernest Nadal, Montse Sánchez-Céspedes. Molecular and immunological characterization of non-small cell lung cancer harboring cMET alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1765. doi:10.1158/1538-7445.AM2017-1765

Correlation of c-MET Expression with PD-L1 Expression in Metastatic Clear Cell Renal Cell Carcinoma Treated by Sunitinib First-Line Therapy.

Clear cell renal cell carcinoma (ccRCC) is highly metastatic. Cabozantinib, an anti-angiogenic tyrosine kinase inhibitor that targets c-MET, provided interesting results in metastatic ccRCC treatment. To understand better the role of c-MET in ccRCC, we assessed its status in a population of patients with metastatic ccRCC. For this purpose, tumor samples were analyzed for c-MET expression by immunohistochemistry (IHC), for c-MET copy number alterations by fluorescence in situ hybridization (FISH), and for c-MET mutations by next generation sequencing (NGS) in a retrospective cohort of 90 primary ccRCC of patients with metastases treated by first-line sunitinib. The expression of c-MET was correlated with pathological, immunohistochemical (VEGFA, CAIX, PD-L1), clinical, and molecular criteria (VHL status) by univariate and multivariate analyses and to clinical outcome using Kaplan-Meier curves compared by log-rank test. Of ccRCC, 31.1% had low c-MET expression (absent to weak intensity by IHC) versus 68.9% with high expression (moderate to strong intensity). High expression of c-MET was associated with a gain in FISH analysis (p=0.0284) without amplification. No mutations were detected in NGS. Moreover, high c-MET expression was associated with lymph node metastases (p=0.004), sarcomatoid component (p=0.029), VEGFA (p=0.037), and PD-L1 (p=0.001) overexpression, the only factor that remained independently associated (p<0.001) after logistic regression. No difference was observed in clinical outcomes. This study is the first to analyse c-MET status in metastatic ccRCC. The high expression of c-MET in the majority of ccRCC and its independent association with PD-L1 expression, may suggest a potential benefit from combining c-MET inhibitors and targeted immunotherapy.

Multiregion sequencing of penile cancer to reveal distinct patterns of heterogeneous actionable mutations.

4518 Background: Penile cancer is a rare but mutilating disease. Due to its rarity few studies have investigated the molecular oncogenic changes. There is a huge need to improve the poor outcome of metastatic disease by advancing the standard practice of platinum based therapies. Treatment based on actionable mutations and the potential of immunotherapies may improve clinical outcomes. We present the first analysis of intra-tumour heterogeneity within penile cancer. Methods: Tissue samples, greater than 80% purity, were collected from four spatially distinct tumor regions together with matched normal and metastatic tissue. Deep whole exome sequencing was performed on the resulting 48 samples from eight patients. Mutations were called by using Mutect2 and annotated with Annovar. Copy number changes were estimated using Sequenza and phylogenetic trees created using pyclone. Results: A mean of 650 single nucleotide variants and insertion/deletion events were discovered per tumor sample. Extensive intra-tumor heterogeneity was found with a wide range of different predicted subclonal architectures. Two distinct patterns were noted: either the acquisition of mutations by the lymph node metastasis was an early event with a lower mutational load, or it was a later event with a high mutational load. In the two instances where patients were negative for human papillomavirus infection, the lymph node metastasis evolved at a later date. However in 5/6 cases of HPV infection, an early subclone developed which potentially led to the lymph node metastasis. Driver mutations in p53, cMET or FAT1 were found to be truncal early events in 80% of cases. Actionable mutations in PIK3CA and EGFR were found to be subclonal in two further samples. Conclusions: There is extensive intra- and inter-tumour heterogeneity within penile cancer. The main truncal drivers in penile cancer are cMET, FAT1 and p53. Samples infected with human papillomavirus appeared to have a distinct signature in terms of both copy number changes and clonal architecture. Actionable mutations PIK3CA and EGFR were found to be subclonal in origin. This may have profound effects on the clinical utility of targetable treatments such as tyrosine kinase inhibitors.

Hepatocyte Growth Factor/c-Met Signaling in Head and Neck Cancer and Implications for Treatment

Aberrant signaling of the hepatocyte growth factor (HGF)/c-Met pathway has been identified as a promoter of tumorigenesis in several tumor types including head and neck squamous cell carcinoma (HNSCC). Despite a relatively low c-Met mutation frequency, overexpression of HGF and its receptor c-Met has been observed in more than 80% of HNSCC tumors, with preclinical and clinical studies linking overexpression with cellular proliferation, invasion, migration, and poor prognosis. c-Met is activated by HGF through a paracrine mechanism to promote cellular morphogenesis enabling cells to acquire mesenchymal phenotypes in part through the epithelial-mesenchymal transition, contributing to metastasis. The HGF/c-Met pathway may also act as a resistance mechanism against epidermal growth factor receptor (EGFR) inhibition in advanced HNSCC. Furthermore, with the identification of a biologically distinct subset of HNSCC tumors acquired from human papillomavirus (HPV) infection that generally portends a good prognosis, high expression of HGF or c-Met in HPV-negative tumors has been associated with worse prognosis. Dysregulated HGF/c-Met signaling results in an aggressive HNSCC phenotype which has led to clinical investigations for targeted inhibition of this pathway. In this review, HGF/c-Met signaling, pathway alterations, associations with clinical outcomes, and preclinical and clinical therapeutic strategies for targeting HGF/c-Met signaling in HNSCC are discussed.

Understanding c-MET signalling in squamous cell carcinoma of the head & neck.

c-MET is a membrane spanning receptor tyrosine kinase for hepatocyte growth factor (HGF) also termed scatter factor. Transmitting signals from mesenchymal to epithelial cells, the HGF/c-MET axis mediates a range of biological processes that stimulate proliferation, motility, invasiveness, morphogenesis, apoptosis, and angiogenesis. Aberrant c-MET signal transduction favours tumorigenesis with the acquisition of invasive and metastatic phenotypes. Biological functions of c-MET may strongly vary according to microenvironmental changes, which occur at different stages of tumorigenesis and include also HGF/c-MET activation in stromal cells. In this review, we focused on abnormalities in non-nasopharyngeal squamous cell carcinoma of the head & neck. While the prevalence of c-MET mutations and amplifications ranges 0-25%, c-MET upregulation can be found in the majority of squamous head & neck carcinomas. Despite marked heterogeneity in published scoring methods, immunohistochemical overexpression of c-MET has been typically linked to advanced stages and associated with impaired survival and/or resistance to radiotherapy, chemoradiotherapy, and cetuximab. Experimental studies in cell lines and patient-derived xenografts using various c-MET antagonists (both as single-agents and in combination with cytotoxic and epidermal growth factor receptor [EGFR]-directed agents) yielded promising results, albeit benefit in clinical trials remains to be demonstrated. Consequently, selecting more active agents and integrating them effectively in studies, which incorporate predictive biomarkers such as c-MET gene mutations, amplifications, and overexpression, remains challenging. Further investigations should increase emphasis on disentangling the role of tumour-stromal interactions and analyse their potential as modifiers of drug response.

Correlation among Genetic Variations of c-MET in Chinese Patient with Non-Small Cell Lung Cancer

Aims: The aim of this study was to determine the relationship between amplification, protein expression and somatic mutation of c-MET in advanced Non-small cell lung cancer. The influence of c-MET abnormalities on clinical outcomes of patients undergoing Crizotinib therapy for treatment of Non-Small Cell Lung Cancer was also evaluated. Methods: c-MET protein expression, gene copy number (GCN) and somatic mutation for exon 14 were detected by Immunohistochemistry, fluorescent In Situ Hybridization and Denaturing High Performance Liquid Chromatography, respectively, in a large series of 196 NSCLC patients. The correlation of c-MET abnormalities and clinical outcome of targeted therapy was analyzed by McNemar’s test. Results: c-MET expression was observed in 28.6% (56/196) cases, and among those 13.8% (27/196) was shown to be FISH positive. Only 2.67% patients in this study carried the c-MET mutation. All cases that were c-MET FISH positive were also shown to express c-MET by IHC. However, only half of the cases that were positive for c- MET expression were found to be FISH positive. Among 31 patients with moderate c-MET IHC staining, 11 cases (35.5%) were FISH positive, while 16/25 cases with high IHC staining were also FISH positive. Six patients received Crizotinib as a first-line or second/third-line therapy. Among them, three cases showed ALK protein expression, two patients showed expression of the ROS1 fusion gene, and one was positive for c-MET expression by IHC. The response to Crizotinib in the three patients positive for ALK were all PR, while the c-MET positive patient showed SD and both cases with expression of the ROS1 gene showed PD. Conclusions: IHC could be a preliminary screening test to facilitate selection of patients with c-MET amplification for ALK inhibitor therapy.

P1.02-041 Characterization of MET-N375S as an Activating Mutation in Squamous Cell Carcinoma of the Lung

Over the years, significant progress has been made in the treatment of lung carcinoma. While targeting EGFR mutations in the tyrosine kinase domain and EML4-ALK rearrangements have yielded impressive therapeutic gains in lung adenocarcinoma; the rarity of genetic aberration in squamous cell carcinoma (SCC) has restricted the use of molecular-targeting agents. Next-Generation sequencing analysis has identified a high frequency of c-MET mutation in Asian lung SCC specimens, in particular the MET-N375S mutant. This study aims to characterize the functional significance and therapeutic implications of MET-N375S in lung SCC cells. MET (N375S) mutation in tumor tissues was verified with droplet digital PCR. c-MET mutant expressing clones were generated through site-directed mutagenesis and single cell clonal selection. The impact of MET-N375S in lung SCC cells was characterized by defining the downstream effectors (Proteome profiling), biological functions (anchorage-independent growth, invasion and migration assays), transciptomic regulation (RNAseq) and protein-protein interaction (SILAC). Novel binding partners of MET-N375S was verified using co-immunoprecipitation (co-IP) and proximity ligation assay (PLA). Sensitivity of c-MET mutant to various kinase inhibitors was determined with CellTiter-Glo assay. MET (N375S) mutation was confirmed in 9/45 lung SCC specimens (20%). Ectopic expression of MET-N375S in lung SCC cells significantly elevated the activation of downstream Src, p38 and ERK1/2 kinases, increased hsp27 expression, while enhanced migratory, invasive and anchorage-independent colony forming ability in vitro. Despite so, comparative transcriptomic analysis revealed that epithelial-mesenchymal signature genes were not induced in cells expressing mutant c-MET. On the contrary, SILAC and co-IP analyses on the MET-N375S interactome demonstrated an increase in binding affinity towards receptor tyrosine kinases (RTKs) as compared to wild type c-MET, which was confirmed with in situ PLA. Moreover, MET-N375S augmented in vitro sensitivity to selective MET inhibitors. These findings suggest the role of MET-N375S as an activating mutation that strongly enhance malignant transformation and metastatic potential in lung SCC cells. Mechanistically, the dysregulation of various oncogenic events could be associated with the formation of heterodimers with RTKs. Clinically, MET-N375S could be utilized as a potential predictive biomarker for patients with advanced SCC of the lung to be treated with selective MET inhibitors.

Analysis of a panel of druggable gene mutations and of ALK and PD-L1 expression in a series of thymic epithelial tumors (TETs)

IntroductionThymic epithelial tumors (TETs) are rare neoplasms with different prognosis lacking consistent molecular alterations possibly leading to targeted therapy. We collected a consecutive series of TETs aimed at investigating the mutational status of druggable genes (EGFR, c-KIT, KRAS, BRAF, PDGFR-alpha and −beta, HER2 and c-MET) and the expression of ALK and PD-L1. Patients and methodsOne hundred twelve consecutive cases of TETs and relative clinico-pathologic features were collected. Immunohistochemical expression of ALK (clone D5F3) and PD-L1 (clone E1L3N), molecular analysis of EGFR (exons 18–21), c-KIT (exons 9,11,13,14,17), KRAS (exon 2), BRAF (exon 15), PDGFR-alpha (exon 12) and -beta (exons 12, 14, 18), HER-2 (exons 19 and 20) and c-MET (exons 14, 17, 18, 19) mutations were performed. Immuno-molecular results were then statistically matched with clinico-pathologic characteristics. ResultsPatients were male in 54% of cases, with a median age of 61 years (range 19–83) and affected mainly by thymoma (78%) in stage II (45%). At molecular analysis, there were 4 c-KIT mutations (occurring in exon 11 V559A, L576P, Y553N and exon 17 D820E) in thymic carcinomas (typeC), but not in other tumor types (p=0.003). No mutations were detected in other genes and none case was ALK positive. Twenty-nine (26%) cases were PD-L1 positive (65% of thymic carcinomas and 18% of thymomas). High PD-L1 expression was statistically associated with WHO classification stage type C (p<0.001) and Masaoka stage III–IV disease (p=0.007). In univariate analysis, WHO classification type C, advanced Masaoka stage and absence of myasthenia, but not PD-L1 expressions were correlated with worse survival; at multivariate analysis, only WHO type C confirmed its negative prognostic role. ConclusionA subset of TETs as thymic carcinomas can harbor c-KIT mutations and elevated PD-L1 expression that could represent targets of potential therapeutic use.

Abstract 668: Induction of mammary carcinoma in tetracycline-inducible cMET(T1010I) transgenic mice

Abstract Gene amplification and mutations of cMET have been reported in a wide variety of tumors, including colorectal, lung, renal, liver, thyroid, and breast cancer. cMET(T1010I) mutation occurred in 5.2% out of 148 sibling pairs diagnosed with colorectal cancer in one study. This germline mutation occurs in about 1% of breast cancer patients, but its frequency increases to 2% in metastatic breast cancer. Furthermore, 16% of breast cancer cases with PI3KCA mutations have co-mutations of the cMET oncogene. To determine the functional consequences of this germline mutation and elucidate the molecular mechanisms of tumorigenesis induced by its oncogenic mutant protein, we developed a transgenic mouse model expressing human cMET(T1010I) under the control of a Tet-responsive promoter. MMTV promoter - directed expression of reverse tetracycline-controlled transactivator (rtTA) turns on the expression of the cMET(T1010I) transgene in murine mammary glands upon tetracycline administration. Thirty percent of these mice developed early onset mammary carcinoma within 5 months of transgene induction. This contrasts with previous reports of mammary tumor development in MMTV-cMET transgenic mice after more than a year. This strong phenotype is not the result of a random insertional effect of the transgene as no mammary tumors developed in the 29 transgene positive mice without mammary-specific expression of rtTA over a year in our study. Molecular characterization of these tumors is currently underway and the results may reveal mechanisms of tumorigenesis and novel therapeutic targets for patients harboring this mutation. In conclusion, we showed for the first time that the cMET(T1010I) mutation can trigger rapid breast tumorigenicity. By using organ-specific expression of rtTA to direct the expression of cMET(T1010I) in the organ of interest, our transgenic model may be used to understand the impact of cMET(T1010I) on many other cancer types such as colorectal, lung, renal, thyroid, and liver cancer. Citation Format: Chuanwei Yang, Ana M. Gonzalez, Naoto T. Ueno, Shuying Liu, Jinsong Zhang, Gordon B. Mills, John Mendelsohn, Prahlad Ram. Induction of mammary carcinoma in tetracycline-inducible cMET(T1010I) transgenic mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 668.

Abstract 4663: Combination of c-Met inhibitor tepotinib (MSC2156119J) and a third-generation EGFR inhibitor can overcome double resistance mediated by EGFR T790M mutation and c-Met amplification in non-small cell lung cancer models

Abstract In clinical practice, acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) causes EGFR TKI treatment failure. Several resistance mechanisms have been identified; EGFR T790M mutation and aberrant activation of c-Met through c-Met gene amplification and/or HGF upregulation are the two most common mechanisms. We have previously shown that combining the highly selective c-Met inhibitor tepotinib with 1st or 2nd-generation EGFR TKIs overcomes gefitinib resistance caused only by c-MET amplification. Third-generation EGFR TKIs such as rociletinib have been developed specifically to overcome T790M-mediated resistance to EGFR TKIs. We hypothesized that combining a 3rd-generation EGFR TKI with tepotinib could overcome resistance to a 1st or 2nd-generation EGFR TKI (e.g. gefitinib) regardless of the mechanism of resistance. We assessed the efficacy of tepotinib and a Merck-synthesized version of rociletinib (MSR) as monotherapy or in combination in a NSCLC cell line xenograft model (HCC827-GR-T790M) with an EGFR del 19 mutation, c-Met amplification, and exogenous T790M expression, and in a patient-derived xenograft model (DFCI081) with an EGFR del 19 mutation and c-Met amplification, but not a T790M mutation. Single-agent tepotinib, erlotinib, and afatinib did not show antitumor activity in HCC827-GR-T790M xenografts and tumors progressed on treatment (median tumor volume [TV] changes &amp;gt;73%). The combinations of tepotinib + erlotinib and tepotinib + afatinib delayed tumor regrowth more than single-agent therapy, but were no more effective than single-agent MSR. In contrast, the combination of tepotinib + MSR had strong antitumor activity in this model with double resistance due to c-Met amplification and exogenous T790M expression, resulting in complete tumor regression (median TV change -97%). In the patient-derived xenograft model DFCI081, tumors progressed under single-agent therapy with the EGFR TKIs erlotinib, afatinib, and MSR, as expected given that this cell line expresses c-Met. In contrast, tepotinib induced complete tumor regression as monotherapy and when combined with each of the EGFR TKIs (median TV change -100%). Complete regression was maintained until the end of the 60-day observation period after treatment termination. These findings are compatible with the hypothesis that combining tepotinib with a 3rd-generation EGFR TKI to overcome double resistance to 1st and 2nd-generation TKIs mediated by T790M and c-Met in NSCLC is more effective than using either agent alone. Furthermore, whereas EGFR TKIs were not effective in tumor models showing c-Met amplification and EGFR del 19 mutation but not T790M mutation, ie resistance is c-Met mediated, tepotinib monotherapy induced complete tumor regression. Citation Format: Manja Friese-Hamim, Giuseppe Locatelli, Friedhelm Bladt. Combination of c-Met inhibitor tepotinib (MSC2156119J) and a third-generation EGFR inhibitor can overcome double resistance mediated by EGFR T790M mutation and c-Met amplification in non-small cell lung cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4663.

Abstract 4043: Establishment of a mouse skin squamous cell carcinoma allograft model for in vivo pharmacological analysis of immunotherapy

Abstract The recent clinical success of novel therapeutics blocking immune checkpoints cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) and programmed cell death 1 (PD-1) has fueled an intense interest around immuno-oncology. However, the lack of relevant animal models is a major bottleneck for understanding the mechanism of action and evaluating the efficacy of such therapeutics. Syngeneic mouse tumor models, despite being widely used as experimental models for efficacy studies, are limited by the restricted number of models available and responsive to current checkpoint inhibitors. Genetically engineered mouse models (GEMMs) of human cancer are effective tools for mechanistic analysis, but they are often not suitable for efficacy studies due to usually un-synchronized tumor progression. Syngeneic allografts of spontaneous mouse tumors derived from GEMMs (MuPrime®) may be used as a new type of immuno-oncology models with the following advantages: 1) their primary nature of “stem cell diseases” and relevant tumor microenvironment mirror the patient derived xenograft (PDX) models; 2) the availability of diverse cancer types and oncogenic drivers deriving from a wide range of GEMMs. We set out to build a library of allografts of spontaneous mouse tumors, including those derived from GEMM, in an immunocompetent host to support pharmacological investigation, particularly of immuno-oncology agents. Recently, we observed the growth of spontaneous cutaneous tumors on the neck of the C57BL/6J-ApcMin/J GEMM, which is heterozygous for the Apc (APCMin/+). Histopathology suggests the tumors are well-differentiated skin squamous cell carcinoma. We subcutaneously transplanted these skin tumors into the syngeneic C57BL/6J mice. The allografts grow well, are serially transplantable and maintain the histopathology of the primary tumor. Transcriptome sequencing revealed that the allografts maintain the original ApcMin mutation, express high levels of HER2 (also confirmed by immunochemistry, or IHC), and present frameshift mutations in both c-met and EGFR. In vivo pharmacological assessment indicate that tumors respond to 5-FU, paclitaxel, gemcitabine, docetaxel, and cisplatin chemotherapy. They is also sensitive to anti-mouse CTLA-4 antibody, and the response is associated with an increased number of tumor infiltrating immune cells, including TILs. Alltogether, here we show that we have established an allograft model suitable for in vivo efficacy analysis of immunotherapy using surrogate anti-mouse antibodies. Citation Format: Gavin Jiagui Qu, Annie Xiaoyu An, Jinping Liu, Davy Xuesong Ouyang, Likun Zhang, Jie Cai, Jean Pierre Wery, Henry Q. X. Li. Establishment of a mouse skin squamous cell carcinoma allograft model for in vivo pharmacological analysis of immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4043.

Mechanism of c-MET in Non-small Cell Lung Cancer and Its Treatment and Testing

肝细胞生长因子/c-MET（hepatocyte growth factor/c-MET, HGF/c-MET）信号通路可通过多种机制如c-MET突变、扩增、过表达和HGF过表达被异常激活，并在非小细胞肺癌（non-small cell lung cancer, NSCLC）的发生发展以及表皮生长因子受体抑制剂（epidermal growth factor receptor tyrosine kinase inhibitor, EGFR-TKI）的耐药性方面发挥重要作用。因此，c-MET是继EGFR、ALK之后NSCLC的又一分子治疗靶点。目前c-MET抑制剂在一些临床试验中表现出了良好的的应用前景，但其在临床应用中的有效性和安全性的评估，以及对于c-MET检测方法的选择及判定标准还需进一步讨论。本文就c-MET在NSCLC中的作用机制、治疗前景和检测方法的选择作一综述。

Abstract 782: Modulation of receptor tyrosine kinase signaling by cMET inhibitor, INC280, in cMETmut/amp gastric carcinoma cell line Hs746.T

Abstract Aberrant Receptor Tyrosine Kinase (RTK) activity is one of the hallmarks of cancer. The genetic alternations of ABL and EGFR are classical examples of RTK-dependent oncogenesis. Recent data suggest that non mutated RTK signaling can form feedback pathways to serve as resistant mechanism against targeted therapies. Here, we examined the regulation of RTK signaling by a potent cMET inhibitor, INC280 (enzymatic EC50 against c-MET = 0.13 nM), in a cMET-dependent human gastric tumor model Hs746.T. Hs746.T is a heavily cMET dependent cell line with both cMET amplication (12.9 copies) and mutation (G-T mutation at slice donor site to cause juxtamembrane domain deletion). This model is exquisitely sensitive to cMET inhibitor, INC280 in vitro (GI50 = 9 nM) and in vivo. The treatment of INC280 lead to significant inhibition of pcMET. Furthermore, the treatment of INC280 resulted in the suppression of pEGFR, though INC280 does not actively inhibit EGFR (enzymatic EC50 against EGFR &amp;gt; 10 microM). This result supports prior reports that cMET and EGFR can form physical complex, with cMET modulating EGFR activation. Consequently, INC280 inhibited downstream signaling nodes of pAKT, pS6RP, pMEK and pERK in Hs746.T. To examine the RTK signaling underlying possible treatment relapse, we treated Hs746.T cells chronically in vitro under increasing concentrations of INC280. After 8 weeks of treatment, Hs746.T-R cells were generated that grew at 600 nM of INC280. RTK signaling was examined in Hs746.T-R cells under treatment of INC280. As expected, cMET signaling was significantly curtailed in Hs746.T-R cells, as was pEGFR. Interestingly, pPDGFRb was elevated in Hs746.T-R, compared to the parental cell line. We hypothesize that the increased signaling through PDGFRb may serve as resistance mechanism to INC280 in Hs746.T-R cells. In summary, we have examined RTK signaling in the cMET-dependent Hs746.T cell line. Our finding supported the notion of RTK cross-talk between cMET and EGFR in this setting. Additionally, our data reveal that additional RTK signaling (PDGFRb) can be induced under chronic treatment of INC280 to possibly mediate resistance to cMET inhibition. Citation Format: Maria C. Pinzon-Ortiz, Xianhui Rong, Jinsheng Liang, Hui Qin Wang, Alan Huang, Robert Schlegel, Z. Alexander Cao. Modulation of receptor tyrosine kinase signaling by cMET inhibitor, INC280, in cMETmut/amp gastric carcinoma cell line Hs746.T. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 782. doi:10.1158/1538-7445.AM2015-782

Retrospective Review of MET Gene Mutations.

C-MET proto-oncogene is a tyrosine kinase situated on chromosome 7. C-MET and its ligand hepatocyte growth factor/scatter factor (HGF/SF) play a role in proliferation, differentiation and organ development. C-MET genetic aberrations are found associated with driving tumorigenesis. In this retrospective study, we reviewed molecular analysis data gathered from a cancer institute during a two-year period (2010-2012). Upon detection of tumors harboring c-MET mutations, we determined the status of the other mutations tested and evaluated c-MET expression by fluorescent in-situ hybridization (FISH). Our search resulted in identification of 134 c-MET mutations, 44% of which had mutations of at least one of the other genes tested. No c-MET expression aberrancy was detected in this subset by FISH. Survival amongst the patients with surgically resected metastatic colorectal cancers (CRC) was slightly better in those with only a c-MET mutation compared to those with no mutation detected, although the difference was not statistically significant. When c-MET inhibition becomes an integrated part of chemotherapy practice, our observed frequency of co-mutations will be an argument for utilizing c-MET targeted treatment in combination with other targeted drugs and therapeutic strategies. Larger studies can aid to further parse out c-MET prognostic and therapeutic significance.

Multiplatform molecular analysis of biomarkers in renal cell carcinoma.

501 Background: Predictive biomarkers of response to targeted therapy are lacking in renal cell carcinoma (RCC). We evaluated a cohort of RCC patients referred for molecular profiling to identify potentially actionable recurrent molecular aberrations. Methods: 166 consecutive renal cases referred to Caris Life Sciences over 2 yrs were evaluated with central pathology review. Cases were subtyped into clear cell (ccRCC), n=100, papillary (PRCC), n=20, sarcomatoid, n=10, translocation, n=6, medullary, n=4 or unclassified, n=26. 63% of cases were metastatic. Testing included a combination of sequencing (Sanger or next generation sequencing [NGS]), protein expression (immunohistochemistry [IHC]), and/or gene amplification (CISH or FISH). Results: ccRCC had a 52% loss of PTEN, while PRCC had a 21% loss (p value=0.02). 100% of ccRCC with sarcomatoid features (4) showed aberrant expression of PD-L1 and were infiltrated with PD-1+ tumor infiltrating lymphocytes; 100% of 10 non-ccRCC sarcomatoids also had aberrant expression of PD-L1. Loss of PBRM1 expression was observed in 60% of ccRCC. Loss of histone 3 lysine 36 trimethylation (H3K36me3), which is associated with SETD2 mutations, was observed in 30% of ccRCC. TOP2A was overexpressed in ccRCC at 30% and in non-ccRCC at 50%. 100% of ccRCC and PRCC overexpressed EGFR; a single unclassified renal cell carcinoma did not overexpress EGFR. 50% of cases had cMET overexpression, amplification, or mutation. HER2 was not amplified or overexpressed in any RCC; a single rare HER2 mutation (R816H) was found in a collecting duct carcinoma. VHL mutations were identified in 50% of ccRCC tumors. We observed lower rates of TP53 (14%), ATM (6%), and PIK3CA(4% ccRCC, 7% PRCC) mutations compared to other cancers. Conclusions: Multiplatform molecular profiling of renal cell carcinoma identifies potential predictive biomarkers in ccRCC. The documented activity of everolimus in ccRCC (RECORD-1 trial) may be attributable to PIK3CA pathway alterations. RCC with sarcomatoid features may respond to PD1/PD-L1 targeted immunotherapies. The impact of molecular profiling in ccRCC to predict responses to currently available targeted therapy has important implications for trial design and patient selection.