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The landscape of gene mutations in cirrhosis and hepatocellular carcinoma

  • Miryam Müller
    Affiliations
    Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
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  • Thomas G. Bird
    Correspondence
    Corresponding authors. Addresses: Cancer Research UK, Beatson Institute, Switchback Road, Glasgow, G61 1BD, UK; Tel.: +44 (0)14133019222, fax: +44 (0)1419426521
    Affiliations
    Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK

    MRC Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh, EH164TJ, UK

    Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Glasgow, G61 1QH, UK
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  • Jean-Charles Nault
    Correspondence
    Service d'hépatologie, hôpital Jean Verdier, Avenue du 14 juillet, 93140 Bondy; Tel.: +33 6 10 67 94 61, fax: +33 1 53 72 51 92
    Affiliations
    Service d'Hépatologie, Hôpital Jean Verdier, Hôpitaux universitaires Paris-Seine-Saint-Denis, Assistance publique Hôpitaux de Paris, Bondy, France

    Unité mixte de Recherche 1162, Génomique fonctionnelle des Tumeurs solides, Institut national de la Santé et de la Recherche médicale, Paris, France

    Unité de Formation et de Recherche Santé Médecine et Biologie humaine, Université Paris 13, Communauté d'Universités et Etablissements Sorbonne Paris Cité, Paris, France
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Published:February 07, 2020DOI:https://doi.org/10.1016/j.jhep.2020.01.019

      Summary

      Chronic liver disease and primary liver cancer are a massive global problem, with a future increase in incidences predicted. The most prevalent form of primary liver cancer, hepatocellular carcinoma, occurs after years of chronic liver disease. Mutations in the genome are a causative and defining feature of all cancers. Chronic liver disease, mostly at the cirrhotic stage, causes the accumulation of progressive mutations which can drive cancer development. Within the liver, a Darwinian process selects out dominant clones with selected driver mutations but also leaves a trail of passenger mutations which can be used to track the evolution of a tumour. Understanding what causes specific mutations and how they combine with one another to form cancer is a question at the heart of understanding, preventing and tackling liver cancer. Herein, we review the landscape of gene mutations in cirrhosis, especially those paving the way toward hepatocellular carcinoma development, that have been characterised by recent studies capitalising on technological advances in genomic sequencing. With these insights, we are beginning to understand how cancers form in the liver, particularly on the background of chronic liver disease. This knowledge may soon lead to breakthroughs in the way we detect, diagnose and treat this devastating disease.

      Keywords

      Introduction

      Liver disease and liver cancer, specifically hepatocellular carcinoma (HCC), are an increasing global pandemic. Chronic liver disease is responsible for approximately 2 million deaths annually worldwide with liver cancer responsible for nearly 800,000 deaths.
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      The trends in incidence of primary liver cancer caused by specific etiologies: results from the Global Burden of Disease Study 2016 and implications for liver cancer prevention.
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      Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      HCC mostly forms after years of chronic liver disease, against a background of severe liver scarring and typically cirrhosis. Even HCC related to non-alcoholic fatty liver disease (NAFLD) in individuals without liver cirrhosis is a process that occurs over many years and can be established as early as childhood.
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      • Holst C.
      • Sorensen T.I.
      • Baker J.L.
      Body mass index in childhood and adult risk of primary liver cancer.
      Mutations are ubiquitous in cancer, and the link between somatic mutations and cancer has been clearly appreciated for many years.
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      The clonal evolution of tumor cell populations.
      ,
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      Somatic mutation in cancer and normal cells.
      Whilst mutations are necessary they are not always sufficient to drive cancer progression.
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      The cancer genome.
      Instead, we are now appreciating that combinations of mutations are required, the amount dependent on the type of cancer.
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      Universal patterns of selection in cancer and somatic tissues.
      In the last decade sequencing of cancer genomes and related non-cancerous tissue has revolutionised our understanding of how cancers form in many tissues. Herein, we will summarise how sequencing studies in the liver have shed light on the genetic progression from a healthy liver to HCC through the key ‘breeding ground’ of chronic liver disease. The hepatocyte is the principle cell of origin of HCC. However, it is now appreciated that there is plasticity between biliary epithelial cells and hepatocytes.
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      Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice.
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      Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.
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      Hepatic progenitor cells of biliary origin with liver repopulation capacity.
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      Cholangiocytes act as facultative liver stem cells during impaired hepatocyte regeneration.
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      De novo formation of the biliary system by TGFβ-mediated hepatocyte transdifferentiation.
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      Chronic liver injury induces conversion of biliary epithelial cells into hepatocytes.
      An intermediate population may represent a form of adult stem cell in the liver and is associated with the severity of liver disease.
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      Oval cell numbers in human chronic liver diseases are directly related to disease severity.
      Nonetheless, little is known about mutations within a putative liver stem cell compartment and their role in subsequent HCC development. For these reasons we will focus on mutations affecting the hepatocellular liver parenchyma and their role in the early steps of HCC development.

      General concepts surrounding the mutational events leading to HCC development

      The development of HCC is almost never a sporadic event. It can be viewed as a Darwinian progression through a spectrum, ranging from an entirely healthy liver to a clonal aggregation of cells able to escape both intrinsic programmes regulating and enforcing normal cell behaviour and the exogenous restraints on cell proliferation imposed by the environment (Fig. 1). Once these processes controlling equitable hepatocellular proliferation are evaded, a dominant hepatocyte clone may become established. Additional mutations may then cause further escape of restriction, with subclones becoming capable of unlimited and unregulated proliferation. However, at each stage of this process, further mutation and selection continue. In response to liver damage all hepatocytes are capable of regeneration (reviewed in
      • Michalopoulos G.K.
      Advances in liver regeneration.
      ), but are kept under a tight control in a healthy liver. Yet it only takes a small replicative advantage to circumvent the restrictions and outgrow the surrounding cells.
      Mutations are integral to the formation of hepatocellular carcinoma.
      Figure thumbnail gr1
      Fig. 1Early genomic events in cirrhotic hepatocytes and their role in malignant transformation.
      Schematic of the main somatic genetic alterations responsible for the malignant transformation of premalignant lesions developed from chronic liver disease, with examples given of transition through low–grade dysplastic and high–grade dysplastic nodules. This stepwise transition is typical but not exclusive. The establishment of cirrhosis with fibrotic scar separating nodules is associated with mutations and establishment of mutant clones. Coloured hepatocytes depict those of a distinct lineage. Over time and with progression to HCC the overall mutational rate and burden increases. Specific driver mutations are associated with progression to HCC. Some are associated with premalignant stages of the disease e.g. TERT whilst others are associated with HCC; particularly TP53 in late stage disease. HCC, hepatocellular carcinoma.
      Although cirrhosis may be regarded as a premalignant stage, the development of HCC in cirrhosis requires additional and progressive pre-neoplastic stages as additional stepping stones towards HCC.
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      • et al.
      Real impact of liver cirrhosis on the development of hepatocellular carcinoma in various liver diseases-meta-analytic assessment.
      This spectrum of development can be crudely broken down into distinct stepwise progression events from injured, often cirrhotic, liver to focal patches of low-grade dysplastic nodules, then through high-grade dysplastic nodules onto HCC
      • Park Y.N.
      Update on precursor and early lesions of hepatocellular carcinomas.
      (Fig. 1). The risk of malignant transformation increases incrementally at each stage. Whilst these lesions may progress to frank HCC, each can be seen as a stage along a continuum from normal to cancer. At each stage, competition occurs with successful clones outcompeting their neighbours and increasing in number. This deregulated growth results in microscopic nodules which may progress to nodules visible either macroscopically or radiologically. If the overall nodule enlarges significantly within a fibrotic lobule then it compresses the surrounding tissue. Though clonal nodules are not necessarily visible by clinical imaging methods they have become identifiable thanks to our ability to visualise them by their genetic profile.
      The human genome, spread across 23 chromosome pairs, comprises approximately 3 billion base pairs in a diploid cell. Hepatocytes can be both multinucleate and polyploid,
      • Duncan A.W.
      Aneuploidy, polyploidy and ploidy reversal in the liver.
      meaning any one usually has up to 12 billion base pairs. The presence of multiple gene copies in hepatocytes impacts the probability of genetic mutations affecting all copies of a specific gene required for a loss of function phenotype. Therefore, gain-of-function mutations as drivers of disease are more prominent in the liver. Thus, polyploidy in the liver likely acts to protect against loss of tumour suppressing genes in the same way that losing function is more difficult when a cell has multiple copies of a gene.
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      Hepatostat: liver regeneration and normal liver tissue maintenance.
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      Potential mechanisms for cancer resistance in elephants and comparative cellular response to DNA damage in humans.
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      • et al.
      TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants.
      In contrast to the protective effect of polyploidy, aneuploidy (an imbalance in the copy number of the 23 chromosome pairs) is associated with a higher risk of cancer development including HCC.
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      • et al.
      Induction of aneuploidy by increasing chromosomal instability during dedifferentiation of hepatocellular carcinoma.
      Aneuploidy is also associated with chronic liver disease and aging
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      • et al.
      DNA ploidy pattern in human chronic liver diseases and hepatic nodular lesions. Flow cytometric analysis on echo-guided needle liver biopsy.
      as well as shortened telomeres.
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      • Kreipe H.
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      • et al.
      Telomere shortening correlates with increasing aneuploidy of chromosome 8 in human hepatocellular carcinoma.
      As aneuploidy results from mistakes in chromosomal separation during cell division this is consistent with the concept that repeated division, both in healthy aging or through forced regeneration during chronic liver disease, promotes both aneuploidy in hepatocytes and subsequent HCC.
      Aside from changes in chromosome number, the genetic code of chromosomes may be altered by mutations. Most mutations affecting the genome do not confer a selection advantage and can be termed passenger mutations. However, they can be utilised by mankind, both as potential therapeutic targets
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      • et al.
      Passenger deletions generate therapeutic vulnerabilities in cancer.
      and as markers of cellular heritage. Conversely, driver mutations do instil a selective advantage and thus are selected over time. This may be, but is not necessarily, linked to malignant transformation. To understand how cancers form we must understand not only how the mutated genes make cells cancerous what but causes these mutations.

      Constitutional predisposition to cirrhosis and HCC

      Mutations are either inherited or arise within an individual cell. Inherited mutations, or those occurring in gametes, are known as germline, whilst those which cannot be inherited are somatic. Somatic mutations are then passed to descendants of the original cell in which the mutation formed. They can have multi-organ effects if they occur during development. However, if they occur after early development, when cellular differentiation and geography are more restricted, their descendants are more local. This is particularly the case in the liver. We are now appreciating that somatic mutations are restricted, particularly during chronic liver disease states, within individual lobules of the liver. On the other hand, germline mutations will, by definition, be present throughout the liver, having been disseminated during the organ's development.
      Several germline mutations predispose individuals to chronic liver disease. This is mainly due to iron (haemochromatosis due to C282Y/C282Y mutations of HFE) or copper overload (Wilson disease due to ATP7B germline mutations), protein deficiency (alpha-1 antitrypsin deficiency due to SERPINA1 mutations) or metabolic disorders (tyrosinemia due to FAH mutations).
      • Zucman-Rossi J.
      • Villanueva A.
      • Nault J.C.
      • Llovet J.M.
      Genetic landscape and biomarkers of hepatocellular carcinoma.
      With the exception of germline HNF1A mutations or glycogenosis type 1A (due to G6PC inactivating mutations) which both predispose to benign liver tumours with potential malignant transformation in a non-fibrotic liver, all these inherited mutations foster HCC development on a background of cirrhosis.
      • Bluteau O.
      • Jeannot E.
      • Bioulac-Sage P.
      • Marques J.M.
      • Blanc J.F.
      • Bui H.
      • et al.
      Bi-allelic inactivation of TCF1 in hepatic adenomas.
      ,
      • Labrune P.
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      • Duvaltier I.
      • Chevalier P.
      • Odievre M.
      Hepatocellular adenomas in glycogen storage disease type I and III: a series of 43 patients and review of the literature.
      Single-nucleotide polymorphisms (SNPs) are frequent constitutional variants in the general population (generally at a frequency of more than 5%) and some of them modulate the risk of developing various human diseases.
      • Nahon P.
      • Zucman-Rossi J.
      Single nucleotide polymorphisms and risk of hepatocellular carcinoma in cirrhosis.
      Several SNPs, belonging to different pathways such as inflammation, DNA repair, cell cycle regulation, oxidative stress, iron metabolism and growth factors, have been involved in HCC development but few of them have been robustly validated in the literature
      • Nahon P.
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      Single nucleotide polymorphisms and risk of hepatocellular carcinoma in cirrhosis.
      (Table 1). PNPLA3 and TM6SF2 genes encode for proteins involved in lipid metabolism and in the composition of lipid droplets.
      • Trepo E.
      • Romeo S.
      • Zucman-Rossi J.
      • Nahon P.
      PNPLA3 gene in liver diseases.
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • Pertsemlidis A.
      • Cox D.
      • Pennacchio L.A.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      • Singal A.G.
      • Manjunath H.
      • Yopp A.C.
      • Beg M.S.
      • Marrero J.A.
      • Gopal P.
      • et al.
      The effect of PNPLA3 on fibrosis progression and development of hepatocellular carcinoma: a meta-analysis.
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • Nordestgaard B.G.
      • Zhou H.H.
      • Tybjaerg-Hansen A.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      • Stickel F.
      • Buch S.
      • Nischalke H.D.
      • Weiss K.H.
      • Gotthardt D.
      • Fischer J.
      • et al.
      Genetic variants in PNPLA3 and TM6SF2 predispose to the development of hepatocellular carcinoma in individuals with alcohol-related cirrhosis.
      • Yang J.
      • Trepo E.
      • Nahon P.
      • Cao Q.
      • Moreno C.
      • Letouze E.
      • et al.
      PNPLA3 and TM6SF2 variants as risk factors of hepatocellular carcinoma across various etiologies and severity of underlying liver diseases.
      • BasuRay S.
      • Wang Y.
      • Smagris E.
      • Cohen J.C.
      • Hobbs H.H.
      Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis.
      Moreover, recent studies have identified an SNP of rs72613567 HSD17B13 leading to a truncated protein and loss of function associated with a reduced risk of cirrhosis occurrence in alcohol-related liver disease (ALD) and NAFLD and a decreased HCC occurrence in ALD.
      • Abul-Husn N.S.
      • Cheng X.
      • Li A.H.
      • Xin Y.
      • Schurmann C.
      • Stevis P.
      • et al.
      A protein-truncating HSD17B13 variant and protection from chronic liver disease.
      ,
      • Yang J.
      • Trepo E.
      • Nahon P.
      • Cao Q.
      • Moreno C.
      • Letouze E.
      • et al.
      A 17-beta-hydroxysteroid dehydrogenase 13 variant protects from hepatocellular carcinoma development in alcoholic liver disease.
      The exact function of this gene and the consequence of the loss of function in the liver remains to be explored.
      Liver damage and aging promote the establishment of mutant clones.
      Table 1Driver mutations associated with HCC. Summary of the most frequent driver mutations associated with HCC.
      Mutations associated with HCCStageReferences in text
      Constitutional mutations/SNPs
      ATP7BWilson disease: cirrhosis/HCC predisposition
      • Zucman-Rossi J.
      • Villanueva A.
      • Nault J.C.
      • Llovet J.M.
      Genetic landscape and biomarkers of hepatocellular carcinoma.
      FAHTyrosinemia: cirrhosis/HCC predisposition
      • Zucman-Rossi J.
      • Villanueva A.
      • Nault J.C.
      • Llovet J.M.
      Genetic landscape and biomarkers of hepatocellular carcinoma.
      G6PCGlycogenosis 1a: HCA-HCC predisposition
      • Calderaro J.
      • Labrune P.
      • Morcrette G.
      • Rebouissou S.
      • Franco D.
      • Prevot S.
      • et al.
      Molecular characterization of hepatocellular adenomas developed in patients with glycogen storage disease type I.
      HFEHaemochromatosis: cirrhosis/HCC predisposition
      • Zucman-Rossi J.
      • Villanueva A.
      • Nault J.C.
      • Llovet J.M.
      Genetic landscape and biomarkers of hepatocellular carcinoma.
      HNF1AMODY 3 diabetes and HCA predisposition
      • Bluteau O.
      • Jeannot E.
      • Bioulac-Sage P.
      • Marques J.M.
      • Blanc J.F.
      • Bui H.
      • et al.
      Bi-allelic inactivation of TCF1 in hepatic adenomas.
      HSD17B13 rs72613567Cirrhosis/HCC predisposition (SNP)
      • Abul-Husn N.S.
      • Cheng X.
      • Li A.H.
      • Xin Y.
      • Schurmann C.
      • Stevis P.
      • et al.
      A protein-truncating HSD17B13 variant and protection from chronic liver disease.
      ,
      • Yang J.
      • Trepo E.
      • Nahon P.
      • Cao Q.
      • Moreno C.
      • Letouze E.
      • et al.
      A 17-beta-hydroxysteroid dehydrogenase 13 variant protects from hepatocellular carcinoma development in alcoholic liver disease.
      PNPLA3 rs738409Cirrhosis/HCC predisposition (SNP)
      • Trepo E.
      • Romeo S.
      • Zucman-Rossi J.
      • Nahon P.
      PNPLA3 gene in liver diseases.
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • Pertsemlidis A.
      • Cox D.
      • Pennacchio L.A.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      • Singal A.G.
      • Manjunath H.
      • Yopp A.C.
      • Beg M.S.
      • Marrero J.A.
      • Gopal P.
      • et al.
      The effect of PNPLA3 on fibrosis progression and development of hepatocellular carcinoma: a meta-analysis.
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • Nordestgaard B.G.
      • Zhou H.H.
      • Tybjaerg-Hansen A.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      • Stickel F.
      • Buch S.
      • Nischalke H.D.
      • Weiss K.H.
      • Gotthardt D.
      • Fischer J.
      • et al.
      Genetic variants in PNPLA3 and TM6SF2 predispose to the development of hepatocellular carcinoma in individuals with alcohol-related cirrhosis.
      • Yang J.
      • Trepo E.
      • Nahon P.
      • Cao Q.
      • Moreno C.
      • Letouze E.
      • et al.
      PNPLA3 and TM6SF2 variants as risk factors of hepatocellular carcinoma across various etiologies and severity of underlying liver diseases.
      • BasuRay S.
      • Wang Y.
      • Smagris E.
      • Cohen J.C.
      • Hobbs H.H.
      Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis.
      SERPINA1Alpha-1 antitrypsin deficiency: cirrhosis/HCC predisposition
      • Zucman-Rossi J.
      • Villanueva A.
      • Nault J.C.
      • Llovet J.M.
      Genetic landscape and biomarkers of hepatocellular carcinoma.
      TM6SF2 rs58542926Cirrhosis/HCC predisposition (SNP)
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • Nordestgaard B.G.
      • Zhou H.H.
      • Tybjaerg-Hansen A.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      • Stickel F.
      • Buch S.
      • Nischalke H.D.
      • Weiss K.H.
      • Gotthardt D.
      • Fischer J.
      • et al.
      Genetic variants in PNPLA3 and TM6SF2 predispose to the development of hepatocellular carcinoma in individuals with alcohol-related cirrhosis.
      • Yang J.
      • Trepo E.
      • Nahon P.
      • Cao Q.
      • Moreno C.
      • Letouze E.
      • et al.
      PNPLA3 and TM6SF2 variants as risk factors of hepatocellular carcinoma across various etiologies and severity of underlying liver diseases.
      Somatic mutations
      TERT promoterTumour (early) (40–60%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • 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.
      • Paterlini-Brechot P.
      • Saigo K.
      • Murakami Y.
      • Chami M.
      • Gozuacik D.
      • Mugnier C.
      • et al.
      Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene.
      • Nault J.C.
      • Datta S.
      • Imbeaud S.
      • Franconi A.
      • Mallet M.
      • Couchy G.
      • et al.
      Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.
      • La Bella T.
      • Imbeaud S.
      • Peneau C.
      • Mami I.
      • Datta S.
      • Bayard Q.
      • et al.
      Adeno-associated virus in the liver: natural history and consequences in tumour development.
      • Tatsuno K.
      • Midorikawa Y.
      • Takayama T.
      • Yamamoto S.
      • Nagae G.
      • Moriyama M.
      • et al.
      Impact of AAV2 and hepatitis B virus integration into genome on development of hepatocellular carcinoma in patients with prior hepatitis B virus infection.
      ,
      • 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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      ACVR2ATumour (5%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ARID1ATumour (5–15%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • 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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      ARID2Tumour (3–15%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • 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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      AXIN1Tumour (5–15%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • 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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      CTNNB1Tumour (15–35%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • de La Coste A.
      • Romagnolo B.
      • Billuart P.
      • Renard C.A.
      • Buendia M.A.
      • Soubrane O.
      • et al.
      Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas.
      ,
      • 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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      FGF19Tumour (4–6%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • 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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      KEAP1Tumour (2–8%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • 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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      KRASTumour (1%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Delire B.
      • Starkel P.
      The Ras/MAPK pathway and hepatocarcinoma: pathogenesis and therapeutic implications.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      MLL4Tumour (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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      NFE2L2Tumour (3–6%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • 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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      RB1Tumour (3–8%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      RPS6KA3Tumour (2–9%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • 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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      SF3B1Tumour (3%)
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      TP53Tumour (15–45%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • 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.
      ,
      • 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.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      VEGFATumour (3–5%)
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      ,
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; MODY, maturity onset diabetes of the young; SNP, single-nucleotide polymorphism.
      In contrast to germline mutations that cause chronic liver diseases, some SNPs are not pathogenic per se, but require an additional cause of chronic liver disease. These SNPs are associated with only a slight increase in cirrhosis and HCC risks, with odds ratios below 2 in most of the studies.
      • Trepo E.
      • Nahon P.
      • Bontempi G.
      • Valenti L.
      • Falleti E.
      • Nischalke H.D.
      • et al.
      Association between the PNPLA3 (rs738409 C>G) variant and hepatocellular carcinoma: evidence from a meta-analysis of individual participant data.
      Consequently, these SNPs only marginally increase our ability to predict HCC compared to the combination of classical clinical features and are not currently used in clinical practice.
      • Guyot E.
      • Sutton A.
      • Rufat P.
      • Laguillier C.
      • Mansouri A.
      • Moreau R.
      • et al.
      PNPLA3 rs738409, hepatocellular carcinoma occurrence and risk model prediction in patients with cirrhosis.
      Interestingly, some constitutional variants predispose individuals to a specific molecular subtype of liver tumours. For example, germline HNF1A mutations predispose individuals to liver adenomatosis composed of HNF1A-inactivated hepatocellular adenoma (HCA). Conversely HCA developed after glycogenosis are never HNF1A inactivated. This is likely due to the similar metabolic defects observed with HNF1A inactivation and G6PC deficiency.
      • Bacq Y.
      • Jacquemin E.
      • Balabaud C.
      • Jeannot E.
      • Scotto B.
      • Branchereau S.
      • et al.
      Familial liver adenomatosis associated with hepatocyte nuclear factor 1alpha inactivation.
      • Nault J.C.
      • Couchy G.
      • Balabaud C.
      • Morcrette G.
      • Caruso S.
      • Blanc J.F.
      • et al.
      Molecular classification of hepatocellular adenoma associates with risk factors, bleeding, and malignant transformation.
      • Calderaro J.
      • Labrune P.
      • Morcrette G.
      • Rebouissou S.
      • Franco D.
      • Prevot S.
      • et al.
      Molecular characterization of hepatocellular adenomas developed in patients with glycogen storage disease type I.
      In contrast, PNPLA3, TM6SF2 or HSD17B13 SNPs were not associated with development of a specific molecular subgroup of HCC.

      Clonal and subclonal evolution of cirrhotic hepatocytes

      Recently somatic mutations and clonal evolution have come into focus as we aim to better understand disease development in the majority of patients with HCC. Early studies examining clonal selection and dominance needed to overcome the limitations of polyclonality in tissue. Bulk-sequencing approaches at low read depth are insensitive and unable to capture clonal and subclonal dynamics. Instead investigators made use of genetic markers of clones detected through immunohistochemistry in patients with cirrhosis. These showed clonal spatial restriction within cirrhotic nodules sequestered by the extracellular matrix. Two different approaches were used to identify these clones, using either X-Chromosome inactivation or mitochondrial DNA mutations as markers.
      • Aihara T.
      • Noguchi S.
      • Sasaki Y.
      • Nakano H.
      • Imaoka S.
      Clonal analysis of regenerative nodules in hepatitis C virus-induced liver cirrhosis.
      • Paradis V.
      • Laurendeau I.
      • Vidaud M.
      • Bedossa P.
      Clonal analysis of macronodules in cirrhosis.
      • Lin W.R.
      • Lim S.N.
      • McDonald S.A.
      • Graham T.
      • Wright V.L.
      • Peplow C.L.
      • et al.
      The histogenesis of regenerative nodules in human liver cirrhosis.
      These early reports have been reinforced by more recent microbiopsy sequencing studies,
      • Zhu M.
      • Lu T.
      • Jia Y.
      • Luo X.
      • Gopal P.
      • Li L.
      • et al.
      Somatic mutations increase hepatic clonal fitness and regeneration in chronic liver disease.
      ,
      • Kim S.K.
      • Takeda H.
      • Takai A.
      • Matsumoto T.
      • Kakiuchi N.
      • Yokoyama A.
      • et al.
      Comprehensive analysis of genetic aberrations linked to tumorigenesis in regenerative nodules of liver cirrhosis.
      including a landmark study from Peter Campbell's group, where Brunner et al. used mapped microbiopsies within cirrhotic nodules.
      • Brunner S.F.
      • Roberts N.D.
      • Wylie L.A.
      • Moore L.
      • Aitken S.J.
      • Davies S.E.
      • et al.
      Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.
      All approaches showed that the majority of regenerative nodules are monoclonal in nature. Some nodules are oligoclonal, but all contain genetically mutant hepatocytes. In every case, clonality is restricted to each individual nodule (Fig. 2). This finding that many cirrhotic nodules are monoclonal or oligoclonal suggests a non-neutral drift towards mutations conferring a selective advantage. Whilst oligoclonality may be an intermediate stage of one clone displacing another we are, as yet, unable to predict whether, or indeed which, clone will become dominant. Driver mutations are not necessarily stepping stones to cancer, however, and in some instances they may actually protect nodules from the liver injury driving chronic disease, as well as promoting regeneration of these clones.
      • Zhu M.
      • Lu T.
      • Jia Y.
      • Luo X.
      • Gopal P.
      • Li L.
      • et al.
      Somatic mutations increase hepatic clonal fitness and regeneration in chronic liver disease.
      Overall, in cirrhosis, as a nodule develops over time it is formed from a single clone possibly with a selective advantage over its original cohabitants within the lobule.
      Figure thumbnail gr2
      Fig. 2Clonality and subclonality within cirrhotic nodules.
      Within cirrhotic nodules, each separated by fibrosis, clones of hepatocytes form and are selected for by a process of natural selection. Over time, clones (represented by colours – brown with passenger mutations; yellow, pink and green for driver mutations) become selected. Within nodule 1, a driver mutation (yellow) grows to replace the other hepatocytes in the nodules. Expansion between nodules is limited by the fibrotic boundaries. A neoplastic subclone (red) then forms within this area. Most nodules however do not progress to malignancy e.g. nodule 2 where a stable clone expands to repopulate the nodule. Other nodules are oligoclonal e.g. nodule 3.
      While some data also suggest clonal patches in the healthy liver,
      • Fellous T.G.
      • Islam S.
      • Tadrous P.J.
      • Elia G.
      • Kocher H.M.
      • Bhattacharya S.
      • et al.
      Locating the stem cell niche and tracing hepatocyte lineages in human liver.
      this is harder to confirm. The lack of physical restriction enforced by cirrhotic fibrous bands makes microdissection of related patches challenging. Additionally, without high cell turnover in healthy liver compared to chronic disease the forces driving clonal selection may be lessened. Additional studies utilising either microdissection or spatially registered single cell analyses are needed to confirm whether or not clonal expansions are typical in the healthy liver. Assuming that clonal expansions become more prevalent in chronic disease an interesting concept arises; the potential role of fibrous bridging acting to restrict the spread of clonal populations (Fig. 2). This may have important implications for reducing the risk of future cancer by preventing the spread of clones with harmful mutations to more distant sites in the liver and by limiting clonal size.
      Healthy livers steadily accumulate mutations over time, with approximately 33–40 per year per diploid genome, with only moderate variation between individuals.
      • Blokzijl F.
      • de Ligt J.
      • Jager M.
      • Sasselli V.
      • Roerink S.
      • Sasaki N.
      • et al.
      Tissue-specific mutation accumulation in human adult stem cells during life.
      Insertion/Deletions (InDels) and substitutions are heterogeneous between and within individuals, but structural variants and copy number alterations are more often found in patients with chronic liver disease. Also chromothripsis, a localised massive chromosomal rearrangement probably due to a catastrophic event during cell proliferation, is more often found in patients with chronic liver disease.
      • Brunner S.F.
      • Roberts N.D.
      • Wylie L.A.
      • Moore L.
      • Aitken S.J.
      • Davies S.E.
      • et al.
      Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.
      Mutational rates and patterns are influenced by the causative factors of chronic liver disease as well as the change in microenvironment, such as inflammation.
      • Letouze E.
      • Shinde J.
      • Renault V.
      • Couchy G.
      • Blanc J.F.
      • Tubacher E.
      • et al.
      Mutational signatures reveal the dynamic interplay of risk factors and cellular processes during liver tumorigenesis.
      • Barash H.
      • Gross E.R.
      • Edrei Y.
      • Ella E.
      • Israel A.
      • Cohen I.
      • et al.
      Accelerated carcinogenesis following liver regeneration is associated with chronic inflammation-induced double-strand DNA breaks.
      • Matsumoto T.
      • Shimizu T.
      • Nishijima N.
      • Ikeda A.
      • Eso Y.
      • Matsumoto Y.
      • et al.
      Hepatic inflammation facilitates transcription-associated mutagenesis via AID activity and enhances liver tumorigenesis.
      Overall, the mutational burden correlates with both fibrosis stage and tissue damage and increases during malignant transformation.
      It is plausible that the increased risk of progressing to HCC on a background of chronic liver disease is driven more by the constant evolution of countless numbers of clones that can independently acquire sufficient driver mutations than by the presence of one specific driver mutation. Consistent with this, it may be more informative to evaluate gene-expression in non-tumoural adjacent tissue with regards to the risk of de novo HCC recurrence.
      • Hoshida Y.
      • Villanueva A.
      • Kobayashi M.
      • Peix J.
      • Chiang D.Y.
      • Camargo A.
      • et al.
      Gene expression in fixed tissues and outcome in hepatocellular carcinoma.
      Mutations in the TERT promoter, while not found in healthy or cirrhotic tissue, are one of the first indicators of malignant transformation, since they already arise in dysplastic nodules.
      • Kim S.K.
      • Takeda H.
      • Takai A.
      • Matsumoto T.
      • Kakiuchi N.
      • Yokoyama A.
      • et al.
      Comprehensive analysis of genetic aberrations linked to tumorigenesis in regenerative nodules of liver cirrhosis.
      ,
      • Nault J.C.
      • Calderaro J.
      • Di Tommaso L.
      • Balabaud C.
      • Zafrani E.S.
      • Bioulac-Sage P.
      • et al.
      Telomerase reverse transcriptase promoter mutation is an early somatic genetic alteration in the transformation of premalignant nodules in hepatocellular carcinoma on cirrhosis.
      Usually only 2 to 6 driver mutations are found in HCC, but they are not restricted in the order in which they appear.
      • Martincorena I.
      • Raine K.M.
      • Gerstung M.
      • Dawson K.J.
      • Haase K.
      • Van Loo P.
      • et al.
      Universal patterns of selection in cancer and somatic tissues.
      ,
      • Letouze E.
      • Shinde J.
      • Renault V.
      • Couchy G.
      • Blanc J.F.
      • Tubacher E.
      • et al.
      Mutational signatures reveal the dynamic interplay of risk factors and cellular processes during liver tumorigenesis.
      Interestingly, it has been suggested that not all mutations found in cirrhotic tissue are necessarily linked to malignant transformation and some might be beneficial for regeneration without carrying the risk of progression to cancer.
      • Zhu M.
      • Lu T.
      • Jia Y.
      • Luo X.
      • Gopal P.
      • Li L.
      • et al.
      Somatic mutations increase hepatic clonal fitness and regeneration in chronic liver disease.
      Conversely some mutations, well described as cancer drivers, are found in non-cancerous cirrhotic nodules and even non-cirrhotic liver. These appear to be present only in a very small minority (<5%) of nodules or hepatocytes respectively.
      • Zhu M.
      • Lu T.
      • Jia Y.
      • Luo X.
      • Gopal P.
      • Li L.
      • et al.
      Somatic mutations increase hepatic clonal fitness and regeneration in chronic liver disease.
      ,
      • Brunner S.F.
      • Roberts N.D.
      • Wylie L.A.
      • Moore L.
      • Aitken S.J.
      • Davies S.E.
      • et al.
      Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.

      Mutational signatures and viral insertional mutagenesis in HCC development

      Mutational signatures are the consequences of endogenous and/or exogenous processes on the genome.
      • Nik-Zainal S.
      • Alexandrov L.B.
      • Wedge D.C.
      • Van Loo P.
      • Greenman C.D.
      • Raine K.
      • et al.
      Mutational processes molding the genomes of 21 breast cancers.
      ,
      • Helleday T.
      • Eshtad S.
      • Nik-Zainal S.
      Mechanisms underlying mutational signatures in human cancers.
      As a fingerprint of early and late events occurring in the cell, the analysis of mutational signatures could be used to understand the mechanisms of transformation of normal cells into malignant cells and to identify new risk factors for tumour development.
      • Nik-Zainal S.
      • Kucab J.E.
      • Morganella S.
      • Glodzik D.
      • Alexandrov L.B.
      • Arlt V.M.
      • et al.
      The genome as a record of environmental exposure.
      In each tumour, the mutational spectrum observed is due to a combination of several mutational processes operating at different times during the life of a cell. The different mutational signatures in one HCC can be derived from mathematical analysis of the type of substitution, taking into account the trinucleotide context (single base substitution signature) as well as the type of larger rearrangement in the genome (rearrangement signature).
      • Alexandrov L.B.
      • Nik-Zainal S.
      • Wedge D.C.
      • Aparicio S.A.
      • Behjati S.
      • Biankin A.V.
      • et al.
      Signatures of mutational processes in human cancer.
      In HCC a number of common and unique signatures have been observed (Fig. 3 and Table 2). Ubiquitous mutational signatures related to age (signature 1 and 5), as well as others considered as liver specific (signature 12 and 16), have been identified in most HCC genomes.
      • Letouze E.
      • Shinde J.
      • Renault V.
      • Couchy G.
      • Blanc J.F.
      • Tubacher E.
      • et al.
      Mutational signatures reveal the dynamic interplay of risk factors and cellular processes during liver tumorigenesis.
      ,
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      In contrast, sporadic signatures were identified with a high intra- and inter-patient variability and were related to mismatch repair deficiency (signature 6), exposure to aflatoxin B1 (signature 24), tobacco (signature 4) or aristolochic acid (signature 22).
      • Letouze E.
      • Shinde J.
      • Renault V.
      • Couchy G.
      • Blanc J.F.
      • Tubacher E.
      • et al.
      Mutational signatures reveal the dynamic interplay of risk factors and cellular processes during liver tumorigenesis.
      ,
      • Hoang M.L.
      • Chen C.H.
      • Sidorenko V.S.
      • He J.
      • Dickman K.G.
      • Yun B.H.
      • et al.
      Mutational signature of aristolochic acid exposure as revealed by whole-exome sequencing.
      ,
      • Poon S.L.
      • Pang S.T.
      • McPherson J.R.
      • Yu W.
      • Huang K.K.
      • Guan P.
      • et al.
      Genome-wide mutational signatures of aristolochic acid and its application as a screening tool.
      Importantly, the mechanisms explaining several sporadic signatures (such as signature 23) remain to be identified. These studies also suggest that some mutational signatures are dependent on the aetiology of the underlying liver diseases and exposure to carcinogens.
      Cirrhotic nodules are formed from clonal subpopulations of hepatocytes.
      Figure thumbnail gr3
      Fig. 3Mutational processes in liver carcinogenesis.
      Summary of the main mutational processes operating in hepatocellular carcinoma and chronic liver disease and their relationship to specific risk factors and carcinogens. In one tumour, several mutational processes may operate synchronously or at different times. Some mutational signatures are ubiquitous whereas other mutational signatures are identified only in a subset of tumours and are considered as sporadic. Some have been associated with specific environmental pathogens, including smoking and alcohol.
      Table 2Mutational signatures associated with HCC.
      Mutational signatureStageOccurrence in HCCAetiologyMutational features
      Signature ACirrhosis/HCClowUbiquitousEndogenous mutational processT>C substitutions
      Viral insertionCirrhosis/HCCSporadicVirus infection (AAV2, HBV)Insertional mutagenesis
      Signature 1Cirrhosislow/HCCUbiquitousEndogenous mutational process (Age)Deamination of 5–methylcytosine
      Signature 4HCCSporadicExposure to tobacco mutagensC>A mutations
      Signature 5Cirrhosis/HCCUbiquitousEndogenous mutational process (Age)T>C substitutions in ATN trinucleotides
      Signature 6HCCSporadicDefective DNA mismatch repairC>T substitutions, small insertions and deletions
      Signature 12Cirrhosislow/HCCUbiquitousUnknown, hallmark of liver cancerT>C substitutions
      Signature 16Cirrhosislow/HCCUbiquitousExposure to alcohol, hallmark of liver cancerT>C mutations in ATN trinucleotides
      Signature 22HCCSporadicExposure to aristolochic acidT>A mutations in CTG trinucleotides
      Signature 24HCCSporadicExposure to aflatoxinC>A mutations in GCC trinucleotides
      Summary of key mutational signatures described in chronic liver disease and HCC. A comprehensive list of signatures can be found at https://cancer.sanger.ac.uk/cosmic/signatures. AAV, adeno-associated virus; HCC, hepatocellular carcinoma.
      Moreover, the link between mutational signatures with specific mutational processes and carcinogens could explain the association observed between mutations in driver genes and a specific aetiology. For example, the increased likelihood of TP53 mutations in aflatoxin B1-related HCC is mainly due to R249S TP53 mutations, a result of C to A substitutions in the GCC trinucleotide context (signature 24) induced by aflatoxin B1.
      • Hsia C.C.
      • Kleiner Jr., D.E.
      • Axiotis C.A.
      • Di Bisceglie A.
      • Nomura A.M.
      • Stemmermann G.N.
      • et al.
      Mutations of p53 gene in hepatocellular carcinoma: roles of hepatitis B virus and aflatoxin contamination in the diet.
      ,
      • Bressac B.
      • Kew M.
      • Wands J.
      • Ozturk M.
      Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa.
      We also recently showed that signature 16, linked with alcohol consumption, is the most important contributor to mutations in CTNNB1, explaining the fact that CTNNB1 mutations were more frequent in alcohol-related HCC.
      • Letouze E.
      • Shinde J.
      • Renault V.
      • Couchy G.
      • Blanc J.F.
      • Tubacher E.
      • et al.
      Mutational signatures reveal the dynamic interplay of risk factors and cellular processes during liver tumorigenesis.
      The mutational signatures in cirrhosis are subtly different to those found in HCC. Based on the analysis of different adult human stem cells, a study showed that adult stem-like liver cells harboured a predominant signature with T:A to C:G transitions and a transcriptional bias close to the features of signature 5 (age-related) observed in HCC.
      • Bluteau O.
      • Jeannot E.
      • Bioulac-Sage P.
      • Marques J.M.
      • Blanc J.F.
      • Bui H.
      • et al.
      Bi-allelic inactivation of TCF1 in hepatic adenomas.
      However, the significance of the enrichment of this mutational signature in adult liver stem cells remains unknown. In the adult liver, a recent study has described the mutational signatures operative at the subclonal and clonal level in cirrhotic hepatocytes.
      • Brunner S.F.
      • Roberts N.D.
      • Wylie L.A.
      • Moore L.
      • Aitken S.J.
      • Davies S.E.
      • et al.
      Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.
      They reported that signature 5, related to aging, and signature A, described in haematopoietic stem cells,
      • Lee-Six H.
      • Obro N.F.
      • Shepherd M.S.
      • Grossmann S.
      • Dawson K.
      • Belmonte M.
      • et al.
      Population dynamics of normal human blood inferred from somatic mutations.
      account for most of the mutations observed in cirrhotic nodules. Interestingly, although both signatures were conserved in HCC, signature A was present in HCC at a lower proportion than other mutational signatures, suggesting that mutations related to signature A are probably surpassed by other mutational processes during progression from cirrhosis to HCC. Signature 1, related to age, and signatures 12 and 16, specific to liver tumours, were identified in cirrhosis but account for a lower rate of the mutations in cirrhotic hepatocytes compared to the higher frequencies observed in HCC. This observation suggests an active role for the agents causing these mutational signatures during malignant transformation. Brunner et al. also identified mutational signatures related to exposure to aflatoxin B1 and aristolochic acid in cirrhotic liver with regional variability in terms of mutagen exposure.
      • Brunner S.F.
      • Roberts N.D.
      • Wylie L.A.
      • Moore L.
      • Aitken S.J.
      • Davies S.E.
      • et al.
      Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.
      Importantly, this study was able to predict the clinical risk factors from the mutational spectrum. For example, all cases of smoking signatures were found in known smokers and rarer signatures, resulting from environmental factors, were found in patients with known risk of exposure to these factors. Therefore, mutational signatures in cirrhosis are not only indicative of the drivers of mutational processes and may reflect the underlying disease but are also subtly different to those in HCC.
      Another imprint in the liver and tumour genome is due to viral insertional mutagenesis.
      • Levrero M.
      • Zucman-Rossi J.
      Mechanisms of HBV-induced hepatocellular carcinoma.
      HBV is one of the most frequent causes of HCC worldwide. A subset of HCCs, particularly in some sub-Saharan African and Asian populations,
      • Kew M.C.
      Epidemiology of hepatocellular carcinoma in sub-Saharan Africa.
      develop in minimally fibrotic livers of young patients infected by HBV, suggesting a direct oncogenic effect of the virus. HBV can insert in the host genome in the liver and deep sequencing data identified non-clonal HBV insertion that occurs randomly in all chromosomes in a subset of hepatocytes. Yet, most of the detected HBV breakpoints are located in the X gene and the viral enhancer.
      • Ding D.
      • Lou X.
      • Hua D.
      • Yu W.
      • Li L.
      • Wang J.
      • et al.
      Recurrent targeted genes of hepatitis B virus in the liver cancer genomes identified by a next-generation sequencing-based approach.
      In some cases, the insertion of the HBV genome near a cancer gene modifies its expression and function, promoting malignant transformation of hepatocytes.
      • Brechot C.
      • Pourcel C.
      • Louise A.
      • Rain B.
      • Tiollais P.
      Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma.
      ,
      • Wang J.
      • Chenivesse X.
      • Henglein B.
      • Brechot C.
      Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma.
      In HCC a subset of HBV-related tumours harbour clonal insertion of HBV in major driver genes such as TERT, MLL4, CCNE1 or CCNA2 whereas insertions in non-tumour liver are subclonal and inserted in various locations in the genome.
      • 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.
      ,
      • Paterlini-Brechot P.
      • Saigo K.
      • Murakami Y.
      • Chami M.
      • Gozuacik D.
      • Mugnier C.
      • et al.
      Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene.
      A similar mechanism is observed in a subset of HCC developed in normal liver that harboured a clonal insertion of adeno-associated virus type 2 (AAV2).
      • Nault J.C.
      • Datta S.
      • Imbeaud S.
      • Franconi A.
      • Mallet M.
      • Couchy G.
      • et al.
      Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.
      AAV2 and AAV2/AAV13 strand insertions have been observed in the hepatocyte genomes of approximately 20% of patients.
      • La Bella T.
      • Imbeaud S.
      • Peneau C.
      • Mami I.
      • Datta S.
      • Bayard Q.
      • et al.
      Adeno-associated virus in the liver: natural history and consequences in tumour development.
      Most of these insertions were subclonal and randomly inserted into the genome of normal hepatocytes. However, a subset of HCC harboured clonal insertion of the 3' inverse tandem repeat region of AAV2 in driver genes such as TERT, CCNA2, CCNE1, TNFSF10 and GLI1 underlining a role of AAV2-insertional mutagenesis in liver carcinogenesis on the background of a healthy liver.
      • Nault J.C.
      • Datta S.
      • Imbeaud S.
      • Franconi A.
      • Mallet M.
      • Couchy G.
      • et al.
      Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.
      • La Bella T.
      • Imbeaud S.
      • Peneau C.
      • Mami I.
      • Datta S.
      • Bayard Q.
      • et al.
      Adeno-associated virus in the liver: natural history and consequences in tumour development.
      • Tatsuno K.
      • Midorikawa Y.
      • Takayama T.
      • Yamamoto S.
      • Nagae G.
      • Moriyama M.
      • et al.
      Impact of AAV2 and hepatitis B virus integration into genome on development of hepatocellular carcinoma in patients with prior hepatitis B virus infection.
      The reasons for the rare occurrence of AAV2-related HCC in the context of the high incidence of AAV2 strand insertion in the general population remain to be better understood. In contrast, the direct oncogenic role of HCV outside the background of cirrhosis remains controversial.
      • Hoshida Y.
      • Fuchs B.C.
      • Bardeesy N.
      • Baumert T.F.
      • Chung R.T.
      Pathogenesis and prevention of hepatitis C virus-induced hepatocellular carcinoma.
      Mutations in specific driver genes promote malignant transformation of hepatocytes.

      Driver mutations involved in tumour initiation and malignant transformation on cirrhosis

      Next-generation sequencing (RNA sequencing, whole-exome sequencing and whole-genome sequencing) has enabled the genetic landscape of HCC to be mapped (Table 1). The most frequent alterations in driver genes were mutations in the TERT promoter (40–60%), TP53 (15–40%), CTNNB1 (10–35%), ARID1A (5–17%), ARID2 (3–18%), AXIN1 (5–15%), RPS6KA3 (2–9%), NFE2L2 (3–6%), KEAP1 (2–8%), RB1 (3–8%) and VEGFA (5%) and FGF19 (5–10%) amplifications.
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • de La Coste A.
      • Romagnolo B.
      • Billuart P.
      • Renard C.A.
      • Buendia M.A.
      • Soubrane O.
      • et al.
      Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas.
      • 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.
      • 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.
      Beyond the most frequent somatic mutations there is also a long list of rarer mutations in other cancer driver genes. The transforming growth factor β (TGFβ) pathway is also involved in the pathogenesis of HCC with a dual role in liver carcinogenesis: aberrant activation of this pathway has been observed in some HCCs whereas other HCCs harboured inactivating mutations of genes belonging to the pathway, such as ACVR2A (5%).
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Chen J.
      • Zaidi S.
      • Rao S.
      • Chen J.S.
      • Phan L.
      • Farci P.
      • et al.
      Analysis of genomes and transcriptomes of hepatocellular carcinomas identifies mutations and gene expression changes in the transforming growth factor-beta pathway.
      ,
      • Coulouarn C.
      • Factor V.M.
      • Thorgeirsson S.S.
      Transforming growth factor-beta gene expression signature in mouse hepatocytes predicts clinical outcome in human cancer.
      Moreover, frequent activation of the Ras/Raf/Map-kinase pathway at the protein level has been described in human HCC
      • Calvisi D.F.
      • Ladu S.
      • Gorden A.
      • Farina M.
      • Conner E.A.
      • Lee J.S.
      • et al.
      Ubiquitous activation of Ras and Jak/Stat pathways in human HCC.
      as well as rare somatic mutations leading to the constitutive activation of the pathway, such as RPS6KA3 mutations (2-9%) and Kras mutations (1%).
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Delire B.
      • Starkel P.
      The Ras/MAPK pathway and hepatocarcinoma: pathogenesis and therapeutic implications.
      Interestingly, this pathway could be targeted using MEK inhibitors such as trametinib or refametinib.
      • Caruso S.
      • Calatayud A.L.
      • Pilet J.
      • La Bella T.
      • Rekik S.
      • Imbeaud S.
      • et al.
      Analysis of liver cancer cell lines identifies agents with likely efficacy against hepatocellular carcinoma and markers of response.
      Altogether, each HCC is a unique combination of somatic alterations, with 40 to 60 non-synonymous mutations in the coding sequence per tumour and around 2 to 6 mutations in driver genes.
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      ,
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      A recent study has highlighted an enrichment of mutations in TP53, RB1 and SF3B1 in advanced HCC and in patients with poor prognosis, supporting an important role of mutations in genes controlling the cell cycle (TP53, RB1) and the spliceosome machinery (SF3B1) for tumour progression.
      • Nault J.C.
      • Martin Y.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Calderaro J.
      • et al.
      Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma.
      Even though the genetic landscape of HCC is relatively well characterised, the early mechanisms of transformation of hepatocytes into cancer cells remain only partially understood, particularly the key steps from cirrhosis to HCC. Studying progressive mutations during dysplastic nodule formation has given us some insights into this process, however. Senescence, a state of permanent cell cycle arrest, together with short telomeres in hepatocytes are hallmarks of cirrhosis.
      • Hoare M.
      • Das T.
      • Alexander G.
      Ageing, telomeres, senescence, and liver injury.
      Telomeres are shortened in chronic liver injury due to a combination of lack of expression of telomerase in the adult liver and long-standing regenerative proliferation.
      • Nault J.C.
      • Ningarhari M.
      • Rebouissou S.
      • Zucman-Rossi J.
      The role of telomeres and telomerase in cirrhosis and liver cancer.
      One of the key mechanisms involved in malignant transformation of cirrhotic hepatocytes is reactivation of telomerase, which is observed in most HCCs and occurs progressively from low to high-grade dysplasia
      • Kolquist K.A.
      • Ellisen L.W.
      • Counter C.M.
      • Meyerson M.
      • Tan L.K.
      • Weinberg R.A.
      • et al.
      Expression of TERT in early premalignant lesions and a subset of cells in normal tissues.
      (Fig. 1). TERT promoter mutations are observed in around to 10 to 20% of low-grade and high-grade dysplastic nodules and in up to 60% of early HCC.
      • Nault J.C.
      • Calderaro J.
      • Di Tommaso L.
      • Balabaud C.
      • Zafrani E.S.
      • Bioulac-Sage P.
      • et al.
      Telomerase reverse transcriptase promoter mutation is an early somatic genetic alteration in the transformation of premalignant nodules in hepatocellular carcinoma on cirrhosis.
      ,
      • 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.
      No other recurrent genetic alterations in other driver genes have been observed in premalignant nodules in cirrhosis to date. Whilst TERT promoter mutations are considered a key event in HCC occurrence, currently no study has found subclonal TERT promoter mutations in cirrhosis. These data suggest that the TERT promoter is a gatekeeper involved in tumour initiation and malignant transformation of hepatocytes through reactivation of telomerase.
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      Other studies highlighted that transcriptomic dysregulation of signalling pathways, such as TGFβ, WNT, and NOTCH, as well as extensive epigenetic modifications occurred after malignant transformation, mainly in progressed HCC, confirming that genomic and epigenomic diversity occurred in the late stages of liver carcinogenesis.
      • Nam S.W.
      • Park J.Y.
      • Ramasamy A.
      • Shevade S.
      • Islam A.
      • Long P.M.
      • et al.
      Molecular changes from dysplastic nodule to hepatocellular carcinoma through gene expression profiling.
      • Marquardt J.U.
      • Seo D.
      • Andersen J.B.
      • Gillen M.C.
      • Kim M.S.
      • Conner E.A.
      • et al.
      Sequential transcriptome analysis of human liver cancer indicates late stage acquisition of malignant traits.
      • Jee B.A.
      • Choi J.H.
      • Rhee H.
      • Yoon S.
      • Kwon S.M.
      • Nahm J.H.
      • et al.
      Dynamics of genomic, epigenomic, and transcriptomic aberrations during stepwise hepatocarcinogenesis.
      Although recently our understanding of mutations and mutational signatures in human patients at different stages of liver disease has improved greatly, there remain key questions of how a particular cirrhotic nodule is most likely to become a cancer depending on its current genetic profile. One question is whether every nodule holds the potential for malignant transformation. If not, are dysplastic nodules required as an intermediary for HCC? If HCC may arise independently of a dysplastic nodule precursor this would be a non-linear model for HCC development. This model has been proposed,
      • Joung J.G.
      • Ha S.Y.
      • Bae J.S.
      • Nam J.Y.
      • Gwak G.Y.
      • Lee H.O.
      • et al.
      Nonlinear tumor evolution from dysplastic nodules to hepatocellular carcinoma.
      however, it is difficult to trace clonal evolution longitudinally in patients. The lack of inter-relationships between dysplastic nodules and separate HCCs in this study would be predicted given that each cirrhotic nodule is distinct by high resolution whole-genome sequencing.
      • Zhu M.
      • Lu T.
      • Jia Y.
      • Luo X.
      • Gopal P.
      • Li L.
      • et al.
      Somatic mutations increase hepatic clonal fitness and regeneration in chronic liver disease.
      ,
      • Brunner S.F.
      • Roberts N.D.
      • Wylie L.A.
      • Moore L.
      • Aitken S.J.
      • Davies S.E.
      • et al.
      Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.
      An important study in human tissue will be to examine dysplastic nodules containing early forms of HCC. Even though dysplastic nodules and HCC may be different at the time of the study it is impossible to determine what status they had in the past or how they will progress in the future. It would therefore be ideal to investigate these questions in various model systems in addition to humans.

      How to model early genomic events of liver carcinogenesis?

      Characterising the role of driver genes and distinct mutational progression from health, through fibrosis, to HCC may be possible using large-scale human tissue sequencing, however, to mechanistically unravel these roles we will require manipulation using preclinical model systems. In vitro systems exist for human hepatocyte culture both in 2D and as 3D organoids
      • Blokzijl F.
      • de Ligt J.
      • Jager M.
      • Sasselli V.
      • Roerink S.
      • Sasaki N.
      • et al.
      Tissue-specific mutation accumulation in human adult stem cells during life.
      ,
      • Huch M.
      • Gehart H.
      • van Boxtel R.
      • Hamer K.
      • Blokzijl F.
      • Verstegen M.M.
      • et al.
      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      • Hu H.
      • Gehart H.
      • Artegiani B.
      • LO-I C.
      • Dekkers F.
      • Basak O.
      • et al.
      Long-term expansion of functional mouse and human hepatocytes as 3D organoids.
      • Peng W.C.
      • Logan C.Y.
      • Fish M.
      • Anbarchian T.
      • Aguisanda F.
      • Alvarez-Varela A.
      • et al.
      Inflammatory cytokine TNFalpha promotes the long-term expansion of primary hepatocytes in 3D culture.
      • Broutier L.
      • Mastrogiovanni G.
      • Verstegen M.M.
      • Francies H.E.
      • Gavarro L.M.
      • Bradshaw C.R.
      • et al.
      Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.
      • Westra I.M.
      • Mutsaers H.A.
      • Luangmonkong T.
      • Hadi M.
      • Oosterhuis D.
      • de Jong K.P.
      • et al.
      Human precision-cut liver slices as a model to test antifibrotic drugs in the early onset of liver fibrosis.
      • Paish H.L.
      • Reed L.H.
      • Brown H.
      • Bryan M.C.
      • Govaere O.
      • Leslie J.
      • et al.
      A bioreactor technology for modeling fibrosis in human and rodent precision-cut liver slices.
      • Collin de l'Hortet A.
      • Takeishi K.
      • Guzman-Lepe J.
      • Morita K.
      • Achreja A.
      • Popovic B.
      • et al.
      Generation of human fatty livers using custom-engineered induced pluripotent stem cells with modifiable SIRT1 metabolism.
      with the potential for overlaying chronic liver disease conditions. However, modelling of HCC formation over prolonged time, especially given the role of both disease and aging in the mutational landscape, within the complex multicellular environment will likely require complex in vivo modelling systems. Preclinical in vivo models offer an opportunity to monitor and manipulate hepatocytes as they progress from a native state to cancer, including studying the complex interactions within the multicellular liver environment. Mouse models have proven to be widely used particularly in view of their flexibility for genetic targeting, immunomodulation (including immunodeficiency) and combinations with transplantation or xenotransplantation.
      • Brown Z.J.
      • Heinrich B.
      • Greten T.F.
      Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research.
      Nonetheless, all the models have limitations when it comes to recapitulation of the progression to HCC in man. Murine models vary greatly in the rapidity of tumour formation, with rapid modelling being attractive from a practical standpoint. Yet, a balance needs to be found between quick models, that are still able to capture the mutational processes developed over decades in human disease, and models that accurately represent human disease. Unfortunately, there is still no consensus model that reproduces human cirrhosis closely and often damage is induced by chemicals that are very different to the exogenous toxins that cause liver disease in humans. This leads to the dilemma that tumours induced by chemical agents in mice differ greatly from human HCC, whereas long-term spontaneous tumourigenesis in mice more closely resembles mutational patterns found in human HCC.
      • Connor F.
      • Rayner T.F.
      • Aitken S.J.
      • Feig C.
      • Lukk M.
      • Santoyo-Lopez J.
      • et al.
      Mutational landscape of a chemically-induced mouse model of liver cancer.
      Another study analysing 4 different mouse models of HCC at the molecular level confirmed that chemically induced mouse models are differ considerable from human HCC at a molecular level, whereas genetic models mimic them more closely.
      • Dow M.
      • Pyke R.M.
      • Tsui B.Y.
      • Alexandrov L.B.
      • Nakagawa H.
      • Taniguchi K.
      • et al.
      Integrative genomic analysis of mouse and human hepatocellular carcinoma.
      Therefore, it will be crucial to investigate a broader array of genetic models of liver disease in detail, particularly those modelling stages from genetic predispositions to chronic liver disease, through to early carcinogenesis. This may enable dissection of the natural history of HCCs as they form in these model systems. The development of mouse models where chronic liver disease and subsequent HCC development is driven by exogenous factors relevant to human disease, like ALD or NAFLD, will be crucial. Modelling of disease states including fibrosis
      • Kim Y.O.
      • Popov Y.
      • Schuppan D.
      Optimized mouse models for liver fibrosis.
      and NAFLD
      • Tsuchida T.
      • Lee Y.A.
      • Fujiwara N.
      • Ybanez M.
      • Allen B.
      • Martins S.
      • et al.
      A simple diet- and chemical-induced murine NASH model with rapid progression of steatohepatitis, fibrosis and liver cancer.
      is possible in the mouse and could be combined with genetic modelling of HCC.
      Understanding the mutational patterns of individual HCCs may aid diagnosis and treatment.
      More recently, specific mutations that drive HCC-like tumour expansions have been modelled in the mouse liver. An early observation from these targeted genetically engineered mouse models is that therapeutic responses may be dependent upon the genetic makeup of a tumour.
      • Wang C.
      • Vegna S.
      • Jin H.
      • Benedict B.
      • Lieftink C.
      • Ramirez C.
      • et al.
      Inducing and exploiting vulnerabilities for the treatment of liver cancer.
      • Ruiz de Galarreta M.
      • Bresnahan E.
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      p53 represses the mevalonate pathway to mediate tumor suppression.
      Whether or not these will be applicable to human HCC subtypes based on the mutational drivers remains to be seen. However, it is promising that the lack of responsiveness to immunotherapy observed in murine tumours models driven by β-catenin mutations
      • Ruiz de Galarreta M.
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      beta-catenin activation promotes immune escape and resistance to anti-PD-1 therapy in hepatocellular carcinoma.
      is also reported in some relatively early observational studies in man.
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      Prospective genotyping of hepatocellular carcinoma: clinical implications of next-generation sequencing for matching patients to targeted and immune therapies.
      Model systems therefore offer the opportunity to understand and develop treatments for these early stages of disease which could then be applied to the clinic with implications for the way that tumours are detected, monitored, diagnosed and treated.

      Implications for disease and therapy

      Currently there are many shortcomings in our clinical approach to patients at risk of and with HCC. For disease detection we generally apply a ‘one size fits all’ surveillance approach. Early detection of HCC through surveillance is recommended but notoriously inaccurate and cost inefficient.
      EASL Clinical Practice Guidelines: Management of hepatocellular carcinoma.
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      Diagnosis, staging, and management of hepatocellular carcinoma: 2018 practice guidance by the American Association for the Study of Liver Diseases.
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      Asia-Pacific clinical practice guidelines on the management of hepatocellular carcinoma: a 2017 update.
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      Surveillance imaging and alpha fetoprotein for early detection of hepatocellular carcinoma in patients with cirrhosis: a meta-analysis.
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      Disease prevention is centred upon avoiding or treating the underlying aetiology. Diagnosis of HCC disease is typically radiological and gives little information about tumour biology, patient prognosis or the likely response to therapy. Each of these areas could theoretically be improved by appreciating the mutational steps leading to HCC development (Fig. 4). Profiling the mutational landscape of the liver could be achieved either by liver biopsy (targeted and/or untargeted) or using a liquid biopsy using either cells or DNA from the liver present in the blood steam. The source and fidelity of this material will be critical for clinical decision making.
      Figure thumbnail gr4
      Fig. 4Implications for diseases and therapies.
      Pathways to translate improved understanding of the mutational landscape of cirrhosis and HCC to impact clinical practice. Profiling can be achieved from blood as a liquid biopsy (sequencing either cell–free DNA or circulating tumour cells). Alternatively, a liver biopsy sample can be sequenced from either an untargeted area of the liver or directly from nodules targeted based on imaging characteristics. At the population level, primary prevention requires identification of risk factors which may be identified by their mutational signatures. For individual patients at risk of HCC their genetic profile could inform both surveillance and targeted prevention. For individuals with HCC, genetic tumour profiling may aid precision medicine for the tumour itself and guide tertiary prevention and surveillance for recurrence after tumour treatment.
      At the population level, identifying exogenous mutational signatures can link specific risk factors to HCC development. The causative agents of several current mutational signatures remain unidentified. Should further causative agents become apparent then this would have major public health implications, like the initial linkage between aflatoxin and HCC.
      At the patient level, characterising mutational signatures could be used clinically. As the mutational signature is relatively conserved across the liver,
      • Brunner S.F.
      • Roberts N.D.
      • Wylie L.A.
      • Moore L.
      • Aitken S.J.
      • Davies S.E.
      • et al.
      Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.
      an untargeted biopsy can still give information on particular exogenous risk factors that drive mutagenesis. This could then be used to focus prevention measures specific to the individual. However, the absence of driver mutations in one nodule does not predict absence in other nodules. Therefore, cautious interpretation will be required for untargeted biopsies. A potential use for overall mutational rate might be to stratify HCC risk. Mutational load could be used to target HCC surveillance. Similarly, this could be used to predict risk of recurrent disease, as has already been suggested for epigenetic signatures.
      • Hoshida Y.
      • Villanueva A.
      • Kobayashi M.
      • Peix J.
      • Chiang D.Y.
      • Camargo A.
      • et al.
      Gene expression in fixed tissues and outcome in hepatocellular carcinoma.
      Targeted biopsies can provide information on specific mutations within a nodule. For example, the presence of TERT promoter mutation in low or high-grade dysplastic nodules could be useful to identify the premalignant lesions at high risk of malignant transformation.
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      • Zafrani E.S.
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      Telomerase reverse transcriptase promoter mutation is an early somatic genetic alteration in the transformation of premalignant nodules in hepatocellular carcinoma on cirrhosis.
      This could be used in a similar fashion to our current risk stratification of hepatic adenoma based on high risk β-catenin mutations.
      EASL Clinical Practice Guidelines on the management of benign liver tumours.
      Additionally, treatment decisions for other conditions could be tailored to the mutational load of the liver. It is becoming apparent that systemic chemotherapy results in the accumulation of higher mutational load in other organs
      • Lee-Six H.
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      The landscape of somatic mutation in normal colorectal epithelial cells.
      and the same is likely to be true in the liver also. Therefore, the use of non-selective chemotherapy, or even repeated irradiation may prove to be disadvantageous for patients with a high mutational burden in a cirrhotic liver
      Studying specific mutations in preclinical models may highlight subtype specific treatment vulnerabilities.
      As a characterised handful of mutations drive the majority of HCC, detecting these may aid HCC surveillance. Through analysis of circulating tumour cells (CTCs) or tumour cell-free DNA (cfDNA) it is possible that tumours could be detected and even phenotyped using liquid biopsy from a blood sample.
      • Ye Q.
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      Liquid biopsy in hepatocellular carcinoma: circulating tumor cells and circulating tumor DNA.
      ,
      • Su Y.H.
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      • Jain S.
      Liquid biopsies for hepatocellular carcinoma.
      However, there are well described limitations to analysis of CTCs and cfDNA. These include the difficulty of differentiating between potential tumours within a mixed liquid biopsy due to the low allelic frequency using either ctDNA or bulk sequencing from CTCs.
      • Heitzer E.
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      The potential of liquid biopsies for the early detection of cancer.
      The co-occurrence of relevant driver mutations together might increase this yield but would require single cell analysis and be outside the availability and budget of most healthcare providers. CTCs are associated with larger tumours and hence this approach may not be applicable to early stage disease. Crucially, genetic drivers of HCC are also more rarely found in the myriad of non-tumoural cirrhotic nodules (approximately half a million in the average liver),
      • Zhu M.
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      Somatic mutations increase hepatic clonal fitness and regeneration in chronic liver disease.
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      Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.
      as well as other non-malignant tissues outside the liver.
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      Tissue-specific mutation accumulation in human adult stem cells during life.
      ,
      • Lee-Six H.
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      • Ellis P.
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      • Moore L.
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      The landscape of somatic mutation in normal colorectal epithelial cells.
      ,
      • Yokoyama A.
      • Kakiuchi N.
      • Yoshizato T.
      • Nannya Y.
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      • Takeuchi Y.
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      Age-related remodelling of oesophageal epithelia by mutated cancer drivers.
      • Martincorena I.
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      • Wabik A.
      • Lawson A.R.J.
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      Somatic mutant clones colonize the human esophagus with age.
      • Martincorena I.
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      Tumor evolution. High burden and pervasive positive selection of somatic mutations in normal human skin.
      Thus, the detection of HCC cancer drivers does not equate to the presence of HCC.
      The most obvious role for genetic analysis in HCC is for treatment stratification. Although identifying driver mutations may deliver effective therapies, the present potential for druggable targets in HCC based on current knowledge and pharmacotherapy remains poor.
      • Zehir A.
      • Benayed R.
      • Shah R.H.
      • Syed A.
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      • Kim H.R.
      • et al.
      Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.
      Nonetheless, as we begin to examine the role for precision medicine in HCC, treatment decisions will become increasingly dependent upon genetic stratification. It is becoming apparent that tumour biology
      • Sia D.
      • Jiao Y.
      • Martinez-Quetglas I.
      • Kuchuk O.
      • Villacorta-Martin C.
      • Castro de Moura M.
      • et al.
      Identification of an immune-specific class of hepatocellular carcinoma, based on molecular features.
      and treatment responses, even for immunotherapy,
      • Harding J.J.
      • Nandakumar S.
      • Armenia J.
      • Khalil D.N.
      • Albano M.
      • Ly M.
      • et al.
      Prospective genotyping of hepatocellular carcinoma: clinical implications of next-generation sequencing for matching patients to targeted and immune therapies.
      may be predictable based on mutational subtypes. However, inter and intratumour genomic and epigenomic heterogeneity have been described in HCC, which will potentially impact on clinical practice. A better understanding of genetic heterogeneity will be helpful to dissect the mechanisms of primary and secondary resistance to systemic treatments.
      • Ding X.
      • He M.
      • Chan A.W.H.
      • Song Q.X.
      • Sze S.C.
      • Chen H.
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      Genomic and epigenomic features of primary and recurrent hepatocellular carcinomas.
      Another important application could be using our knowledge about mutations which predict extrahepatic spread. This could, for example, have profound implications upon the decision to proceed to liver transplantation.

      Unmet needs and conclusion

      Understanding how normal hepatocytes and cirrhotic hepatocytes develop into cancer is central to tackling the HCC pandemic and managing patients with chronic liver disease. Whilst we already know a lot about the genomic defects that shape HCC, the early events occurring in the genome of normal and cirrhotic livers need to be better described. It is not clear how the subclonal or clonal events observed in cirrhosis are involved in malignant transformation, if they provide plasticity and survival advantages in a context of chronic liver disease or could be a dead-end for the cell. More data are required to confirm the robustness and reproducibility of the observation of subclonal and clonal mutations in cirrhosis using a larger number of samples. More data are also warranted to understand the relationship between the different aetiologies of chronic liver diseases, exposure to carcinogens and the early modifications of the hepatocyte genome, as well as the effect of the treatment of underlying liver disease on genomic dysregulation observed in cirrhosis. New model systems to help understand the effects of underlying aetiology and subclonal mutations on liver carcinogenesis and that better mimic human diseases are essential. Finally, a better understanding of early changes in cirrhotic hepatocytes, including both driver genes and overall mutational signature and burden, will be helpful to translate this knowledge, develop preventive strategies and adapt treatment to patients with chronic liver disease and early HCC.

      Abbreviations

      AAV, adeno-associated virus; ALD, alcohol-related liver disease; cfDNA, cell-free DNA; CTC, circulating tumour cell; HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; InDel, insertion/deletion; NAFLD, non-alcoholic fatty liver disease; SNP, single-nucleotide polymorphism; TGF, transforming growth factor.

      Financial support

      MM and TGB are supported by the CRUK Beatson Institute Core funding ( A171196 ), CRUK / AECC / AIRC Accelerator Award ( C9380/A26813 ) and the Wellcome Trust ( WT107492Z ). JCN is supported by Inserm with the « Cancer et Environnement » (plan Cancer), MUTHEC and TELOTHEP projects (INCa).

      Authors' contributions

      Drafting of the manuscript, revision of the manuscript and approval of the final version of the manuscript: MM, TGB, and JCN.

      Conflicts of interest

      The authors declare no conflicts of interest that pertain to this work.Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

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