Genetics of hepatocellular carcinoma: The next generation
Authors: Jean-Charles Nault, Jessica Zucman-Rossi
Journal: Journal of Hepatology (2013)

COMMENTARY ON: Identification of driver genes in hepatocellular carcinoma by exome sequencing. Cleary SP, Jeck WR, Zhao X, Kuichen, Selitsky SR, Savich GL, Tan TX, Wu MC, Getz G, Lawrence MS, Parker JS, Li J, Powers S, Kim H, Fischer S, Giundi M, Ghanekar A, Chiang DY. Hepatology. 2013 May 31. doi: 10.1002/hep.26540. Copyright © 2013. Abstract reprinted by permission from the American Association for the Study of Liver Diseases. http://www.ncbi.nlm.nih.gov/pubmed/23728943 Abstract: Genetic alterations in specific driver genes lead to disruption of cellular pathways and are critical events in the instigation and progression of hepatocellular carcinoma. As a prerequisite for individualized cancer treatment, we sought to characterize the landscape of recurrent somatic mutations in hepatocellular carcinoma. We performed whole exome sequencing on 87 hepatocellular carcinomas and matched normal adjacent tissues to an average coverage of 59×. The overall mutation rate was roughly 2 mutations per Mb, with a median of 45 non-synonymous mutations that altered the amino acid sequence (range 2–381). We found recurrent mutations in several genes with high transcript levels: TP53 (18%), CTNNB1 (10%), KEAP1 (8%), C16orf62 (8%), MLL4 (7%) and RAC2 (5%). Significantly affected gene families include the nucleotide-binding domain and leucine rich repeat containing family, calcium channel subunits, and histone methyltransferases. In particular, the MLL family of methyltransferases for histone H3 lysine 4 were mutated in 20% of tumors. Conclusion: The NFE2L2-KEAP1 and MLL pathways are recurrently mutated in multiple cohorts of hepatocellular carcinoma. AND Whole genome sequencing identifies recurrent mutations in hepatocellular carcinoma. Kan Z, Zheng H, Liu X, Li S, Barber T, Gong Z, Gao H, Hao K, Willard MD, Xu J, Hauptschein R, Rejto PA, Fernandez J, Wang G, Zhang Q, Wang B, Chen R, Wang J, Lee NP, Zhou W, Lin Z, Peng Z, Yi K, Chen S, Li L, Fan X, Yang J, Ye R, Ju J, Wang K, Estrella H, Deng S, Wei P, Qiu M, Wulur IH, Liu J, Ehsani ME, Zhang C, Loboda A, Sung WK, Aggarwal A, Poon RT, Fan ST, Hardwick J, Wang J, Reinhard C, Dai H, Li Y, Luk JM, Mao M. Genome Res. 2013 Jun 20, doi: 10.1101/gr.154492.113. Copyright © 2013, Published by Cold Spring Harbor Laboratory Press. http://www.ncbi.nlm.nih.gov/pubmed/23788652 Abstract: Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease. Like other cancers, hepatocellular carcinoma (HCC) could be considered as an acquired genetic disorder defined by an accumulation of somatic genetic alterations in tumor hepatocytes [[1]Vogelstein B. Papadopoulos N. Velculescu V.E. Zhou S. Diaz Jr., L.A. Kinzler K.W. Cancer genome landscapes.Science. 2013; 339: 1546-1558Crossref PubMed Scopus (5026) Google Scholar]. Recently, several technological breakthroughs were performed and now we can decode the whole sequence of an individual or of tumor genomes in a few days exploring more than 20,000 coding genes. Following this technological revolution, several pioneering studies have refined our knowledge of the mutational landscape and the related signaling pathway involved in liver carcinogenesis (Fig. 1) [2Guichard C. Amaddeo G. Imbeaud S. Ladeiro Y. Pelletier L. Maad I.B. et al.Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.Nat Genet. 2012; 44: 694-698Crossref PubMed Scopus (1001) Google Scholar, 3Fujimoto A. Totoki Y. Abe T. Boroevich K.A. Hosoda F. Nguyen H.H. et al.Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators.Nat Genet. 2012; 44: 760-764Crossref PubMed Scopus (665) Google Scholar, 4Huang J. Deng Q. Wang Q. Li K.Y. Dai J.H. Li N. et al.Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma.Nat Genet. 2012; 44: 1117-1121Crossref PubMed Scopus (309) Google Scholar, 5Sung W.K. Zheng H. Li S. Chen R. Liu X. Li Y. et al.Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma.Nat Genet. 2012; 44: 765-769Crossref PubMed Scopus (618) Google Scholar, 6Li M. Zhao H. Zhang X. Wood L.D. Anders R.A. Choti M.A. et al.Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma.Nat Genet. 2011; 43: 828-829Crossref PubMed Scopus (317) Google Scholar]. First, they have confirmed that activation of the Wnt/β-catenin pathway was the main oncogenic pathway in HCC with recurrent mutations of CTNNB1 (coding for β-catenin, 11–32%) and AXIN1 (15%) [2Guichard C. Amaddeo G. Imbeaud S. Ladeiro Y. Pelletier L. Maad I.B. et al.Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.Nat Genet. 2012; 44: 694-698Crossref PubMed Scopus (1001) Google Scholar, 3Fujimoto A. Totoki Y. Abe T. Boroevich K.A. Hosoda F. Nguyen H.H. et al.Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators.Nat Genet. 2012; 44: 760-764Crossref PubMed Scopus (665) Google Scholar, 4Huang J. Deng Q. Wang Q. Li K.Y. Dai J.H. Li N. et al.Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma.Nat Genet. 2012; 44: 1117-1121Crossref PubMed Scopus (309) Google Scholar, 7Nault J.C. Zucman-Rossi J. Genetics of hepatobiliary carcinogenesis.Semin Liver Dis. 2011; 31: 173-187Crossref PubMed Scopus (126) Google Scholar]. Cell cycle regulatory genes were also frequently altered. In addition to the well-known alterations in TP53 and CDKN2A [[7]Nault J.C. Zucman-Rossi J. Genetics of hepatobiliary carcinogenesis.Semin Liver Dis. 2011; 31: 173-187Crossref PubMed Scopus (126) Google Scholar], next generation studies have identified new recurrent inactivating mutations of IRF2 (5%) [[2]Guichard C. Amaddeo G. Imbeaud S. Ladeiro Y. Pelletier L. Maad I.B. et al.Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.Nat Genet. 2012; 44: 694-698Crossref PubMed Scopus (1001) Google Scholar], leading to a functional inactivation of TP53, and of ATM (5%) [[3]Fujimoto A. Totoki Y. Abe T. Boroevich K.A. Hosoda F. Nguyen H.H. et al.Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators.Nat Genet. 2012; 44: 760-764Crossref PubMed Scopus (665) Google Scholar], a protein involved in cell cycle regulation and DNA damage repair. Moreover, sequencing analyses revealed frequent alterations in genes coding for proteins involved in chromatin structure and maintenance. In particular, recurrent somatic mutations inactivating ARID1A and ARID2 were identified in around 10% of HCC [2Guichard C. Amaddeo G. Imbeaud S. Ladeiro Y. Pelletier L. Maad I.B. et al.Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.Nat Genet. 2012; 44: 694-698Crossref PubMed Scopus (1001) Google Scholar, 3Fujimoto A. Totoki Y. Abe T. Boroevich K.A. Hosoda F. Nguyen H.H. et al.Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators.Nat Genet. 2012; 44: 760-764Crossref PubMed Scopus (665) Google Scholar, 4Huang J. Deng Q. Wang Q. Li K.Y. Dai J.H. Li N. et al.Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma.Nat Genet. 2012; 44: 1117-1121Crossref PubMed Scopus (309) Google Scholar, 6Li M. Zhao H. Zhang X. Wood L.D. Anders R.A. Choti M.A. et al.Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma.Nat Genet. 2011; 43: 828-829Crossref PubMed Scopus (317) Google Scholar]. ARID1A and ARID2 belong to the chromatin remodeling gene family, encoding subunits of SWI/SNF complexes and are considered tumor suppressor genes although they are altered in several other cancer types. Another pivotal signaling pathway identified by next generation sequencing was the activation of the NRF2/KEAP1 pathway. NRF2 (coded by NFE2L2) is a transcription factor that is physiologically degraded by the proteasome in a complex with KEAP1 and CUL3. When activated by increase of reactive oxygen species or by mutations identified in 6% of HCC [[2]Guichard C. Amaddeo G. Imbeaud S. Ladeiro Y. Pelletier L. Maad I.B. et al.Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.Nat Genet. 2012; 44: 694-698Crossref PubMed Scopus (1001) Google Scholar], NRF2 dissociates from KEAP1, translocates to the nucleus and activates the transcription of antioxidant genes (Fig. 1). The antioxidant response gives proliferative and survival advantages to tumor cells. Finally, activating mutations of PIK3CA, FGF19 amplification and inactivating mutations of RPS6KA3 are also recurrent genetic alterations in HCC, leading to a constitutive activation of PI3K/Akt/mTOR and Ras/Raf/MAP kinase pathways [2Guichard C. Amaddeo G. Imbeaud S. Ladeiro Y. Pelletier L. Maad I.B. et al.Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.Nat Genet. 2012; 44: 694-698Crossref PubMed Scopus (1001) Google Scholar, 3Fujimoto A. Totoki Y. Abe T. Boroevich K.A. Hosoda F. Nguyen H.H. et al.Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators.Nat Genet. 2012; 44: 760-764Crossref PubMed Scopus (665) Google Scholar]. However, these seminal next generation studies were limited by the small number of samples analyzed (from 10 to 27). In two recent publications, large series of HCC cases were analyzed [8Cleary S.P. Jeck W.R. Zhao X. Kuichen Selitsky S.R. Savich G.L. et al.Identification of driver genes in hepatocellular carcinoma by exome sequencing.Hepatology. 2013; (in press)PubMed Google Scholar, 9Kan Z. Zheng H. Liu X. Li S. Barber T. Gong Z. et al.Whole genome sequencing identifies recurrent mutations in hepatocellular carcinoma.Genome Res. 2013; 23: 1422-1433Crossref PubMed Scopus (360) Google Scholar]. In the first publication, Clearly et al. used whole exome sequencing (exploring the entire coding sequence of the genome) of 80 HCC and they found a mean number of 66 mutations per tumor [[8]Cleary S.P. Jeck W.R. Zhao X. Kuichen Selitsky S.R. Savich G.L. et al.Identification of driver genes in hepatocellular carcinoma by exome sequencing.Hepatology. 2013; (in press)PubMed Google Scholar]. This value is in the middle range of mutation solid tumors that vary from 4 mutations per samples for pediatric rhabdoid tumors to 200 in melanoma and lung cancer [[1]Vogelstein B. Papadopoulos N. Velculescu V.E. Zhou S. Diaz Jr., L.A. Kinzler K.W. Cancer genome landscapes.Science. 2013; 339: 1546-1558Crossref PubMed Scopus (5026) Google Scholar]. It indicates that accumulation of several somatic genetic alterations is required during liver carcinogenesis. Among them, functional mutations in driver genes (directly involved in carcinogenesis) should be distinguished from the stochastic background of mutations in passenger genes. As pivotal driver genes, Clearly et al. confirmed frequent alterations of TP53 (18% of the cases) and CTNNB1 (10% of the cases) in hepatocarcinogenesis [[8]Cleary S.P. Jeck W.R. Zhao X. Kuichen Selitsky S.R. Savich G.L. et al.Identification of driver genes in hepatocellular carcinoma by exome sequencing.Hepatology. 2013; (in press)PubMed Google Scholar]. They also identified recurrent mutations in MLL (Myeloid/Lymphoid or Mixed-Lineage Leukemia, 2% of mutations), MLL2 (4.5%), MLL3 (1%) and MLL4 (7%) [[8]Cleary S.P. Jeck W.R. Zhao X. Kuichen Selitsky S.R. Savich G.L. et al.Identification of driver genes in hepatocellular carcinoma by exome sequencing.Hepatology. 2013; (in press)PubMed Google Scholar]. These genes belong to the histone methylation writer gene family and encode H3K4 methyltransferases that are involved in methylation, acetylation and remodeling of nucleosomes (Fig. 1). Interestingly, recurrent hepatitis B virus integration targeting MLL4 has also been recently described [[5]Sung W.K. Zheng H. Li S. Chen R. Liu X. Li Y. et al.Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma.Nat Genet. 2012; 44: 765-769Crossref PubMed Scopus (618) Google Scholar]. Together with ARID1A and ARID2 mutations, these data reinforce the link between cancer genome defects and epigenetic alterations in liver tumorigenesis. Finally, Clearly et al. identified inactivating mutations of KEAP1 in 8% of the cases that are mutually exclusive of NFE2L2 mutations and allow the activation of the antioxidant gene response [[8]Cleary S.P. Jeck W.R. Zhao X. Kuichen Selitsky S.R. Savich G.L. et al.Identification of driver genes in hepatocellular carcinoma by exome sequencing.Hepatology. 2013; (in press)PubMed Google Scholar]. In the second publication, Kan et al. analyzed 88 HCC, mainly related to HBV infection, using whole genome sequencing [[9]Kan Z. Zheng H. Liu X. Li S. Barber T. Gong Z. et al.Whole genome sequencing identifies recurrent mutations in hepatocellular carcinoma.Genome Res. 2013; 23: 1422-1433Crossref PubMed Scopus (360) Google Scholar]. In this study, the authors confirmed most of the previous results but they also enlightened frequent activations of the JAK-STAT pathway with activating mutations of JAK1 (9.1%), a JAK1/2 inhibitor [[9]Kan Z. Zheng H. Liu X. Li S. Barber T. Gong Z. et al.Whole genome sequencing identifies recurrent mutations in hepatocellular carcinoma.Genome Res. 2013; 23: 1422-1433Crossref PubMed Scopus (360) Google Scholar]. Whereas these results warrant validation in HCC of other etiologies, they are promising for patient care because JAK1 activations are targetable by ruxolitinib. In conclusion, both studies have refined our understanding of the HCC genome and they revealed new recurrent somatic mutations in putative driver genes. But what are the next steps in the field of HCC genomics? First, new insights have emerged from non-coding tumor sequences: we recently described somatic mutations in the promoter of the telomerase reverse transcriptase (TERT) gene [[10]Nault J.C. Mallet M. Pilati C. Calderaro J. Bioulac Sage P. Laurent C. et al.High frequency of telomerase reverse-transcriptase promoter somatic mutations in hepatocellular carcinoma and preneoplastic lesions.Nat Commun. 2013; 4: 2218Crossref PubMed Scopus (414) Google Scholar]. Now, these mutations represent the most frequent genetic alterations in HCC (59%) and the first recurrent genetic alterations in cirrhotic preneoplastic lesions (25%). TERT promoter mutations increase the promoter activity and lead to an increased transcription of telomerase. We can assume that other driver genetic alterations will be discovered in the dark matter of the genome. Next, among the huge number of genes mutated in the cancer genome, functional analysis will be warranted to understand the consequences for tumorigenesis and for targeted treatment. Moreover, data generated by massive parallel sequencing have to be integrated with transcriptomic, methylome analysis, proteomic, and metabolomic in order to capture the full complexity of liver cancer. Finally, next generation sequencing will be available very soon routinely at hospitals and a huge effort has to be made to translate this knowledge into a new paradigm of personalized clinical care for HCC patients. This work was supported by the INCa with the ICGC project, the PAIR-CHC project NoFLIC (funded by INCa and Association pour la Recherche sur le Cancer, ARC), the Réseau national CRB Foie, HEPTROMIC (FP7) and ARC (grant n°3194). J.-C.N. is supported by a fellowship from the INCa. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.