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Supplementary Figure 1. Sensitivity to selective MET inhibitors in liver cancer cell lines is related to MET gene amplification. Supplementary Figure 2. Tivantinib sensitivity is associated with high expression of genes involved in cell cycle regulation. Supplementary Table 1. Liver cancer cell lines. Supplementary Table 2. Clinical features of HCC series. Supplementary Table 3. List of the 188 genes analyzed by quantitative RT-PCR. Supplementary Table 4. Mutations and copy number variations identified in 13 genes among the 35 liver cancer cell lines (GRCh37). Supplementary Table 5. Association between tivantinib sensitivity and mutation status of the 12 most frequently mutated genes in HCC tumors and MET across the panel of 35 liver cancer cell lines.

Purpose: Tivantinib was initially reported as a selective MET inhibitor and is under phase III evaluation in "MET-high" hepatocellular carcinoma (HCC) patients. However, it has been also proposed as an antimitotic agent. We aimed to evaluate the antitumor effect of tivantinib in HCC cells by combining pharmacologic and molecular profiling.Experimental Design: Sensitivity to tivantinib, JNJ-38877605, PHA-665752, vinblastine, and paclitaxel was tested in a panel of 35 liver cancer cell lines analyzed with exome sequencing, mRNA expression of 188 genes, and protein expression. Drug effect was investigated by Western blot analysis and mitotic index quantification. Expression of candidate biomarkers predicting drug response was analyzed in 310 HCCs.Results: Tivantinib sensitivity profiles in the 35 cell lines were similar to those obtained with antimitotic drugs. It induced blockage of cell mitosis, and high cell proliferation was associated with sensitivity to tivantinib, vinblastine, and paclitaxel. In contrast, tivantinib did not suppress MET signaling, and selective MET inhibitors demonstrated an antiproliferative effect only in MHCC97H, the unique cell line displaying MET gene amplification. HCC tumors with high expression of cell proliferation genes defined a group of patients with poor survival. Interestingly, highly proliferative tumors also demonstrated high MET expression, likely explaining better therapeutic response of MET-high HCC patients to tivantinib.Conclusions: Tivantinib acts as an antimitotic compound, and cell proliferation markers are the best predictors of its antitumor efficacy in cell lines. Ki67 expression should be tested in clinical trials to predict tivantinib response. Clin Cancer Res; 23(15); 4364–75. ©2017 AACR.

International audience; BACKGROUND AND AIMS: Hepatocellular carcinomas (HCCs) are heterogeneous aggressive tumors with low rates of response to treatment at advanced stages. We screened a large panel of liver cancer cell lines (LCCLs) to identify agents that might be effective against HCC and markers of therapeutic response.METHODS: We performed whole-exome RNA and microRNA sequencing and quantification of 126 proteins in 34 LCCLs. We screened 31 anticancer agents for their ability to decrease cell viability. We compared genetic, RNA, and protein profiles of LCCLs with those of primary HCC samples and searched for markers of response.RESULTS: The protein, RNA and mutational signatures of the LCCLs were similar to those of the proliferation class of HCC, which is the most aggressive tumor type. Cell lines with alterations in genes encoding members of the Ras-MAPK signaling pathway and that required fibroblast growth factor (FGF)19 signaling via FGF receptor 4 for survival were more sensitive to trametinib than to FGF receptor 4 inhibitors. Amplification of FGF19 resulted in increased activity of FGF19 only in tumor cells that kept a gene expression pattern of hepatocyte differentiation. We identified single agents and combinations of agents that reduced viability of cells with features of the progenitor subclass of HCC. LCCLs with inactivating mutations in TSC1 and TSC2 were sensitive to the mammalian target of rapamycin inhibitor rapamycin, and cells with inactivating mutations in TP53 were sensitive to the Aurora kinase A inhibitor alisertib. Amplification of MET was associated with hypersensitivity to cabozantinib and the combination of sorafenib and inhibitors of MAP kinase 1 and MAP kinase2 had a synergistic antiproliferative effect.CONCLUSION: LCCLs can be screened for drugs and agents that might be effective for treatment of HCC. We identified genetic alterations and gene expression patterns associated with response to these agents. This information might be used to select patients for clinical trials.

Purpose: Tivantinib was initially reported as a selective MET inhibitor and is under phase III evaluation in "MET-high" hepatocellular carcinoma (HCC) patients. However, it has been also proposed as an antimitotic agent. We aimed to evaluate the antitumor effect of tivantinib in HCC cells by combining pharmacologic and molecular profiling. Experimental Design: Sensitivity to tivantinib, JNJ-38877605, PHA-665752, vinblastine, and paclitaxel was tested in a panel of 35 liver cancer cell lines analyzed with exome sequencing, mRNA expression of 188 genes, and protein expression. Drug effect was investigated by Western blot analysis and mitotic index quantification. Expression of candidate biomarkers predicting drug response was analyzed in 310 HCCs. Results: Tivantinib sensitivity profiles in the 35 cell lines were similar to those obtained with antimitotic drugs. It induced blockage of cell mitosis, and high cell proliferation was associated with sensitivity to tivantinib, vinblastine, and paclitaxel. In contrast, tivantinib did not suppress MET signaling, and selective MET inhibitors demonstrated an antiproliferative effect only in MHCC97H, the unique cell line displaying MET gene amplification. HCC tumors with high expression of cell proliferation genes defined a group of patients with poor survival. Interestingly, highly proliferative tumors also demonstrated high MET expression, likely explaining better therapeutic response of MET-high HCC patients to tivantinib. Conclusions: Tivantinib acts as an antimitotic compound, and cell proliferation markers are the best predictors of its antitumor efficacy in cell lines. Ki67 expression should be tested in clinical trials to predict tivantinib response. Clin Cancer Res; 23(15); 4364–75. ©2017 AACR.

Genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. Analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereas FGF3, FGF4, FGF19 or CCND1 amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)-approved drugs. In conclusion, we identified risk factor-specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.

Background and Aims: Hepatocellular carcinoma (HCC) is a heterogeneous disease mainly developing in chronic liver disease and caused be a variety of etiologies. Hepatocarcinogenesis is a multi-step process in which pre-cancerous lesions can ultimately transform into advanced liver cancer. Genomic analyses promise to unravel the mutagenic processes in order to identify molecular profiles and to optimize personalized patient care. Methods: We performed exome sequences of 243 liver tumors from European patients (France, Italy and Spain), associated with cirrhosis (F4, n = 118), fibrosis (F2+F3, n = 46), or non-fibrotic livers (F0+F1, n = 79). Cirrhosis-associated tumors presented different stages along HCC progression: 7 dysplastic macronodules, 7 early, 17 small and progressed, 58 classic, and 29 poor-prognosis HCC. Tumors were categorized by varying risk factors: Significant alcohol intake (41%), HCV (26%), non-alcoholic steatohepatitis (18%), HBV (14%), hemochromatosis (7%), no known etiology (11%). Results: We identified 8 mutational signatures of which 6 (Signatures 1A, 1B, 4, 5, 6, and 16) were previously validated in a pan-cancer analysis, while 2 (Signatures 23 and 24) are novel. Hierarchical clustering, based on mutational signatures, revealed 6 groups (MSig1 to 6) and 4 singletons which were significantly associated with specific risk factors, mainly combined alcohol/tobacco consumption, and aflatoxin B1. Next, we identified 161 putative driver genes associated with 11 recurrent pathways. Associations of mutations defined 3 groups of genes centered on CTNNB1 (alcohol), TP53 (HBV), and AXIN1. Analyses according to tumor stage progression revealed TERT promoter mutation as an early event whereas FGF/CCND1 amplification, TP53 and CDKN2A alterations, appeared at more advanced stages in aggressive tumors. Altogether, 28% of patients harbored at least one damaging alteration potentially targetable by an FDA-approved drug and 86% by a drug which has been studied in phase I to phase III clinical trials. Conclusions: In conclusion, our study identified relationships between environmental exposures and mutational patterns in HCC as well as the landscape of driver genes and pathways altered in different clinical stages and etiological backgrounds. For patient care, genomic alterations identified in targetable genes will be useful to determine HCC patients that could potentially benefit from targeted treatment in future clinical trials. Citation Format: Kornelius Schulze, Sandrine Imbeaud, Eric Letouzé, Ludmil B. Alexandrov, Julien Calderaro, Sandra Rebouissou, Gabrielle Couchy, Clément Meiller, Jayendra Shinde, Shalini Datta, Frederic Soysouvanh, Anna-Line Calatayud, Roser Pinyol, Laura Pelletier, Charles Balabaud, Alexis Laurent, Jean-Frederic Blanc, Viccenzo Mazzaferro, Fabien Calvo, Augusto Villanueva, Jean-Charles Nault, Paulette Bioulac-Sage, Michael R. Stratton, Josep M. Llovet, Jessica Zucman-Rossi. Exome sequencing of 243 liver tumors identifies new mutational signatures and potential therapeutic targets. [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 2973. doi:10.1158/1538-7445.AM2015-2973