Next generation sequencing of HCC from European and Asian HCC cohorts. Back to p53 and Wnt/β-catenin
Authors: Andreas Teufel, Jens U. Marquardt, Peter R. Galle
Journal: Journal of Hepatology (2012)

COMMENTARY ON: Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma Guichard C, Amaddeo G, Imbeaud S, Ladeiro Y, Pelletier L, Maad IB, Calderaro J, Bioulac-Sage P, Letexier M, Degos F, Clément B, Balabaud C, Chevet E, Laurent A, Couchy G, Letouzé E, Calvo F, Zucman-Rossi J. Nat Genet 2012 May 6;44(6):694–8. Copyright (2012). Abstract reprinted by permission of McMillan Publishers Ltd. http://www.ncbi.nlm.nih.gov/pubmed/22561517 Abstract. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Here, we performed high-resolution copy-number analysis on 125 HCC tumors and whole-exome sequencing on 24 of these tumors. We identified 135 homozygous deletions and 994 somatic mutations of genes with predicted functional consequences. We found new recurrent alterations in four genes (ARID1A, RPS6KA3, NFE2L2 and IRF2) not previously described in HCC. Functional analyses showed tumor suppressor properties for IRF2, whose inactivation, exclusively found in hepatitis B virus (HBV)-related tumors, led to impaired TP53 function. In contrast, inactivation of chromatin remodelers was frequent and predominant in alcohol-related tumors. Moreover, association of mutations in specific genes (RPS6KA3-AXIN1 and NFE2L2-CTNNB1) suggested that Wnt/β-catenin signaling might cooperate in liver carcinogenesis with both oxidative stress metabolism and Ras/mitogen-activated protein kinase (MAPK) pathways. This study provides insight into the somatic mutational landscape in HCC and identifies interactions between mutations in oncogene and tumor suppressor gene mutations related to specific risk factors. AND Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators Fujimoto A, Totoki Y, Abe T, Boroevich KA, Hosoda F, Nguyen HH, Aoki M, Hosono N, Kubo M, Miya F, Arai Y, Takahashi H, Shirakihara T, Nagasaki M, Shibuya T, Nakano K, Watanabe-Makino K, Tanaka H, Nakamura H, Kusuda J, Ojima H, Shimada K, Okusaka T, Ueno M, Shigekawa Y, Kawakami Y, Arihiro K, Ohdan H, Gotoh K, Ishikawa O, Ariizumi S, Yamamoto M, Yamada T, Chayama K, Kosuge T, Yamaue H, Kamatani N, Miyano S, Nakagama H, Nakamura Y, Tsunoda T, Shibata T, Nakagawa H. Nat Genet 2012 May 27;44(7):760–4. Copyright (2012). Abstract reprinted by permission of McMillan Publishers Ltd. http://www.ncbi.nlm.nih.gov/pubmed/22634756 Abstract. Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. We sequenced and analyzed the whole genomes of 27 HCCs, 25 of which were associated with hepatitis B or C virus infections, including two sets of multicentric tumors. Although no common somatic mutations were identified in the multicentric tumor pairs, their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns. Statistical and functional analyses yielded a list of recurrently mutated genes. Multiple chromatin regulators, including ARID1A, ARID1B, ARID2, MLL and MLL3, were mutated in ∼50% of the tumors. Hepatitis B virus genome integration in the TERT locus was frequently observed in a high clonal proportion. Our whole-genome sequencing analysis of HCCs identified the influence of etiological background on somatic mutation patterns and subsequent carcinogenesis, as well as recurrent mutations in chromatin regulators in HCCs. Hepatocellular carcinoma (HCC) ranks among the most deadly cancers worldwide and is characterized by phenotypic and molecular heterogeneity. Besides surgical resection or liver transplantation, chemotherapeutic or even targeted therapeutic options are limited, with sorafenib currently being the only approved systemic treatment. Thus, the need for a more detailed understanding of underlying molecular changes resulting in liver cancer is imperative for effective therapeutic targeting [[1]Teufel A. Staib F. Kanzler S. Weinmann A. Schulze-Bergkamen H. Galle P.R. Genetics of hepatocellular carcinoma.World J Gastroenterol. 2007; 13: 2271-2282Crossref PubMed Scopus (109) Google Scholar]. Triggered by the Human Genome Project, major progress was achieved in unravelling the genetic basis of HCC. Following the first studies from the Thorgeirsson and other laboratories, it became increasingly clear that gene expression profiles of tumors are clearly linked to prognosis, suggesting a high therapeutic potential for specific targeting of identified genetic changes [[2]Lee J.S. Chu I.S. Heo J. Calvisi D.F. Sun Z. Roskams T. et al.Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling.Hepatology. 2004; 40: 667-676Crossref PubMed Scopus (688) Google Scholar]. However, since these first studies were performed almost a decade ago, many genetic signatures have been proposed, but only very few have been validated independently [[3]Marquardt J.U. Galle P.R. Teufel A. Molecular diagnosis and therapy of hepatocellular carcinoma (HCC): an emerging field for advanced technologies.J Hepatol. 2012; 56: 267-275Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar]. Thus, it became apparent that the molecular pathogenesis of HCC mirrors the observed phenotypic heterogeneity and that a more detailed understanding of the complexity of genetic changes involved in HCC development is needed in order to achieve efficient therapeutic targeting. Over the past several years the revolution in technological progress promoted the availability of high throughput ("next generation", NGS) sequencing technologies. As a result, it is now possible to sequence whole genomes within days. As for many other diseases, great promise rests on the expectation that these NGS technologies will provide a significantly higher resolution of the genetic landscape of HCC as well. So far, the number of studies utilizing NGS to investigate molecular alterations in HCC is very limited [[3]Marquardt J.U. Galle P.R. Teufel A. Molecular diagnosis and therapy of hepatocellular carcinoma (HCC): an emerging field for advanced technologies.J Hepatol. 2012; 56: 267-275Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar]. In recent volumes of Nature Genetics, Guichard et al. and Fujimoto et al. reported on the first representative cohorts of HCC patients in which tumor samples and surrounding tissues were sequenced by NGS [4Guichard 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 (992) Google Scholar, 5Fujimoto 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 (657) Google Scholar]. These data obtain valuable insights into HCC biology in many ways: (1) for an analysis of the mutational spectrum in HCC; (2) for a detailed genetics comparison between cohorts of different ethnical or etiological backgrounds, an issue that was previously repeatedly adressed with respect to treatment response to sorafenib in comparison to the SHARP [[6]Llovet J.M. Ricci S. Mazzaferro V. Hilgard P. Gane E. Blanc J.F. et al.Sorafenib in advanced hepatocellular carcinoma.N Engl J Med. 2008; 359: 378-390Crossref PubMed Scopus (8534) Google Scholar] and Asia Pacific trials [[7]Cheng A.L. Kang Y.K. Chen Z. Tsao C.J. Qin S. Kim J.S. et al.Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial.Lancet Oncol. 2009; 10: 25-34Abstract Full Text Full Text PDF PubMed Scopus (4222) Google Scholar]; (3) for a detailed comparative analysis of the mutational spectrum with other cancers thereby identifying both key driver genes as well as distinct entity specific carcinogenic alterations; (4) for a detailed comparison of molecular changes obtained by different high-throughput analyses such as gene expression profiling and NGS on a genome wide level; (5) for multi-level integrative analyses of transcriptomic, genomic and epigenomic changes to further pinpoint critical molecular changes in hepatocarcinogenesis [[8]Teufel A. Marquardt J.U. Staib F. Galle P.R. Snapshot liver transcriptome in hepatocellular carcinoma.J Hepatol. 2012; 56: 990-992Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. The landscape of molecular alterations in HCC observed in both studies was relatively broad with various numbers of mutations found in the investigated HCCs. Comparable to other tumor entities, in the European cohort this number ranged from 5 to 121 mutations per tumor [[4]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 (992) Google Scholar]. This high and heterogeneous number of genetic variations in HCC was confirmed in the Asian study [[5]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 (657) Google Scholar]. Thus, in contrast to previous hypotheses on HCC genetics, a rather complex interaction of multiple mutations in one tumor finally resulting in HCC development must now be assumed. These data are in accordance with other cancers, such as colon or breast cancer, where an average 60–80 mutations per tumor have been described [[9]Wood L.D. Parsons D.W. Jones S. Lin J. Sjoblom T. Leary R.J. et al.The genomic landscapes of human breast and colorectal cancers.Science. 2007; 318: 1108-1113Crossref PubMed Scopus (2528) Google Scholar]. One of the biggest challenges of the upcoming decade will be the development of molecular and bioinformatics strategies to distinguish true genetic driver changes critical to HCC development from by-stander changes that are not relevant to cancer biology, in order to filter for the most promising druggable targets for HCC treatment. An interesting aspect is the assessment of genetic data from diverse backgrounds of ethnicity and etiology of the underlying liver disease, leading to distinct carcinogenic mechanisms. Thus, an integration of the coincidental results from the European cohort with large numbers of alcoholic liver disease and the Asian cohort with a large number of hepatitis B induced HCC may lead to genetically robust insights into hepatocarcinogenesis [4Guichard 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 (992) Google Scholar, 5Fujimoto 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 (657) Google Scholar]. Both groups reported a high number of mutations in p53 and Wnt signaling genes. By number and significance level, both pathways are clearly dominant among other genetic alterations. However, these results are to a large extent in agreement with previously published results from transcriptomic and proteomic investigations. Thus, the here presented in-depth analyses strengthen existing evidence from multiple biological layers that p53 and Wnt/β-catenin signaling is among the most common molecular changes involved in HCC development. β-Catenin is a well known oncogene described in many types of cancer, including liver cancer. Furthermore, disruption of this pathway is supposed to occur frequently (around 30%) and early during hepatocarcinogenesis and has been linked to prognosis. Similarly, p53 mutations were commonly detected before. However, the incidence of these mutations varied significantly between African or Asian (10–60%) and Western countries (10–20%). This was mainly attributed to environmental factors such as aflatoxin. Another interesting result of both studies was the genetic alteration of genes involved in chromatin remodeling [[10]Nault J.C. Zucman-Rossi J. Genetics of hepatobiliary carcinogenesis.Semin Liver Dis. 2011; 31: 173-187Crossref PubMed Scopus (125) Google Scholar], an alteration predominatly found in alcohol-related tumors. Overall, around 16–24% of HCCs showed genetic alterations in this pathway, thereby suggesting a causative association with hepatocyte transformation and highlighting recent evidence for the key role of epigenetics in hepatocarcinogenesis [[11]Andersen J.B. Factor V.M. Marquardt J.U. Raggi C. Lee Y.H. Seo D. et al.An integrated genomic and epigenomic approach predicts therapeutic response to zebularine in human liver cancer.Sci Transl Med. 2010; 2: 54ra77Crossref PubMed Scopus (95) Google Scholar]. Both studies extensively analyzed single nucleotide variations, in particular nucleotide transversions, and demonstrated an over-representation of G>T transversions. This profile of nucleotide transversion distiguishes HCC development from other studies where in similar experiments a predominant C:G>T:A transition was reported. Intriguingly, in the European study, these G>T transversions were even more significantly enriched in the non-transcribed DNA strand, less efficiently repaired genotoxic injury [[4]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 (992) Google Scholar]. Furthermore, these changes were significantly more frequent in HCCs that developed in non-cirrhotic livers as well as in well-differentiated tumors, suggesting that exposure to genotoxic agents could contribute to hepatocarcinogenesis as a mechanism independent of liver cirrhosis. Although these data were established on a small cohort and definitely require independent validation, this aspect may shed light on ongoing discussions of HCC development in non-cirrhotic livers [[12]Hashimoto E. Tokushige K. Hepatocellular carcinoma in non-alcoholic steatohepatitis: Growing evidence of an epidemic?.Hepatol Res. 2012; 42: 1-14Crossref PubMed Scopus (91) Google Scholar]. This will be of particular significance given the increasing importance of NASH-derived HCCs [[12]Hashimoto E. Tokushige K. Hepatocellular carcinoma in non-alcoholic steatohepatitis: Growing evidence of an epidemic?.Hepatol Res. 2012; 42: 1-14Crossref PubMed Scopus (91) Google Scholar]. Linking this novel genetic information to the effectiveness of systemic treatment strategies for HCC in particular, will be challenging, but may subsequently lead to major progress in the development of more effective, stratified treatment options. In other cancer entities, such as melanoma or colorectal cancer, such genetics-based stratification of treatment resulted in some major breakthroughs for systemic treatment. For example, one of the most commonly found mutation in melanoma, BRAFV600E, leads to the activation of ERK and, subsequently, to an increase in proliferation and survival advantage, and defines a subgroup of patients sensitive to treatment with BRAF or MEK inhibitors. [13Romano E. Schwartz G.K. Chapman P.B. Wolchock J.D. Carvajal R.D. Treatment implications of the emerging molecular classification system for melanoma.Lancet Oncol. 2011; 12: 913-922Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 14Tsao H. Chin L. Garraway L.A. Fisher D.E. Melanoma: from mutations to medicine.Genes Dev. 2012; 26: 1131-1155Crossref PubMed Scopus (356) Google Scholar]. Similarly, in colorectal cancer, activating mutations within KRAS predict resistance to anti-EGFR monoclonal antibody treatment [[15]Normanno N. Tejpar S. Morgillo F. De Luca A. Van Cutsem E. Ciardiello F. Implications for KRAS status and EGFR-targeted therapies in metastatic CRC.Nat Rev Clin Oncol. 2009; 6: 519-527Crossref PubMed Scopus (347) Google Scholar], warranting genetic stratification prior to treatment. With respect to HCC, it became clear from both publications, that p53 and Wnt signaling pathway mutations (in particular CTNNB1) are rarely coincidental but mostly exclusive to HCC. Thus, increasing stratification of HCCs using p53 or CTNNB1 mutations as molecular classifiers and subsequent evaluation of specific treatment options in the respective subgroups may be valuable for the development of successful targeting strategies. In summary, the two recently published NGS analyses of HCC offer valuable novel insights into the genetics of liver cancer on a unprecedented resolution compared to previous genetics data available. They further validated an important role for p53 and Wnt/β-catenin signaling in populations of distinct ethnical and etiological backgrounds. Finally, these data may be a major aid for entering a novel era in treatment and development of more stratified treatment algorithms for systemic therapies in HCC (Fig. 1). The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.