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Single nucleotide polymorphisms and risk of hepatocellular carcinoma in cirrhosis

  • Pierre Nahon
    Correspondence
    Corresponding author. Address: Service d’Hépatologie, Hôpital Jean Verdier, 93140 Bondy, France. Tel.: +33 1 48 02 62 80; fax: +33 1 48 02 62 02.
    Affiliations
    Service d’Hépatologie, Hôpital Jean Verdier, AP-HP, Bondy, and Université Paris 13/UFR SMBH, Bobigny, France

    Inserm U773, CRB3, Université Paris 7, Paris, France
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  • Jessica Zucman-Rossi
    Affiliations
    Inserm, UMR-674, Génomique fonctionnelle des tumeurs solides, IUH, Paris F-75010, France

    Université Paris Descartes, Labex Immuno-oncology, Sorbonne Paris Cité, Faculté de Médecine, Paris, France

    Service d’Oncologie, HEGP, AP-HP, Paris, France
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Open AccessPublished:May 18, 2012DOI:https://doi.org/10.1016/j.jhep.2012.02.035

      Summary

      Liver carcinogenesis is a complex and multi-factorial process, in which both environmental and genetic features interfere and contribute to malignant transformation. Patients with cirrhosis are particularly exposed and justify periodical screenings in order to detect the early development of hepatocellular carcinoma (HCC). The risk of HCC is, however, not identical from one patient to another. The identification of host factors that may also play an important role in HCC development may improve our understanding of the implications of the various biological pathways involved in liver carcinogenesis; such progress may as well help refine the selection of patients who could benefit from specific preventative measures or could be given adapted screening policies. Numerous candidate-gene studies have reported associations between single nucleotide polymorphisms (SNPs) and the presence of HCC. Some of these publications unfortunately suffer from major methodological drawbacks because of their case–control, retrospective and monocentric aspect. Prospective cohort studies conducted in large homogeneous populations and comprising a sufficient number of events during follow-up may overcome these pitfalls, but require a long time to be conducted and are still scarce. More recently, the first Genome Wide Association studies (GWAs) have enabled the identification of unsuspected loci that may be involved in various steps implicated in liver tumourigenesis. Taken together, these studies highlight variants that modulate oxidative stress, iron metabolism, inflammatory and immune responses, DNA repair mechanisms or systems involved in cell-cycle regulation as genetic traits susceptible to modify the natural history of cirrhotic patients and partly explain the observed differences in the risk of HCC occurrence. However, large genetic epidemiology studies in the field of cancer diseases have suggested the limited ability of polymorphic traits, alone, to refine individual prognosis. The integration of various panels of genes into clinical scores may in the near future define a “genomic risk prediction” specific to liver cancer developed in cirrhotic patients.

      Abbreviations:

      AFB1 (aflatoxin B1), AFP (α-fetoprotein), FPRP (false-positive report probability), GWAs (Genome-Wide Association study), HBV (hepatitis B virus), HCC (hepatocellular carcinoma), HCV (hepatitis C virus), HR (hazard ratio), OR (odds ratio), SNP (single-nucleotide polymorphisms)

      Keywords

      Introduction

      Cirrhosis is a precancerous condition that predisposes to the development of hepatocellular carcinoma (HCC) [
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      Hepatocellular carcinoma.
      ]. European cohort studies have reported that HCC-related mortality accounts for 54–70% of deaths in patients with compensated cirrhosis [
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      ], with an annual incidence ranging from 2% to 6% [
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      ]. This trend is increasing in both Europe and North America, and is due to a decline in mortality due to other liver-related causes in such patients [
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      ]. This latter finding reflects the improvement in the management of liver-related complications and suggests that the decrease of competing risks occurring during the outcome of cirrhosis may explain, at least in part, the increased incidence of HCC in these patients.
      The risk of HCC development is, however, not identical from one patient to another. Several risk factors for the occurrence of HCC have been identified and extensively studied in large prospective cohorts of patients with cirrhosis. The identified features so far encompass the cause of cirrhosis, older age over 55–60 years and male gender [
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      ]. The additional contribution of yet undefined biological factors obtained through analyses of serum or tissue samples from cirrhotic patients, represents a new exciting challenge in this field.

      Methodological issues for translational genetic research in the field of hepatocarcinogenesis

      The identification of genetic factors influencing the risk of developing HCC in patients with cirrhosis is crucial. This will improve our understanding of the implications of the various biological pathways involved in liver carcinogenesis and may, in the near future, help identify subgroups of patients at high risk, which could benefit from specific preventative measures or could be given adapted screening policies. The search for easily assessable genetic markers in these patients has been greatly facilitated by advances in molecular biology, in parallel with the constitution of large cohorts of patients encompassing exhaustive databases and referenced biobanks in clinical research units.
      These advances have resulted in the publication of numerous studies. However, many have been inconclusive and sometimes contradictory. Several facts may explain these disappointing results. Firstly, the majority of these studies have been conducted on small case–control cohorts and have compared genotypic distributions between HCC patients and individuals with or without cirrhosis. This approach may be considered as an approximation in the assessment of a given genetic risk factor and is subjected to major selection bias, particularly in a population exposed to fatal liver or extra-hepatic complications not related to HCC, introducing major competitive risks. Thus, the selection of appropriate control patients represents a major drawback in these types of studies; if cases include non-cirrhotic patients, it is impossible to conclude that a given single nucleotide polymorphism (SNP) is actually implicated in the development of cirrhosis or that it exerts a direct role in hepatocarcinogenesis. Conversely, if cirrhotic patients are included in the control group, selection bias may artificially impair genotype comparisons between cases and controls. Indeed, the genotypic distribution of ‘survivors’, when including compensated outpatients or, conversely, patients with a worse prognosis who are selected in the context of liver transplantation or end-stage liver disease, may be affected. Secondly, accumulating data suggest that the mechanisms of liver carcinogenesis strongly depend upon the origin of the underlying liver disease. Thus, the study of risk factors for the occurrence of HCC in heterogeneous cohorts of patients may also introduce confounding factors: the development of cirrhosis and subsequent tumour development may indeed be considered as common steps in diseases with different pathophysiologies. It therefore becomes obvious that the study of such genetic factors should now be assessed according to a rigorous clinical methodology in order to meet quality criteria regarding the constitution of large cohorts of well-defined patients. In addition, these studies should be conducted in prospectively followed-up patients with cirrhosis who are regularly screened for HCC according to international guidelines in order to take into account all confounding factors that could influence hepatocarcinogenesis.

      Genetic predisposition and HCC

      Several lines of evidence indicate that development of HCC is a multistep process affected by both inherited and acquired factors leading to the transformation of normal hepatocytes into malignant clones. SNPs correspond to a modification of a DNA sequence due to the change of a single nucleotide; they account for >90% of allelic disparities scattered throughout the human genome. Although the vast majority of these modifications are situated in non-coding regions, some can modify gene-product expression and function, which may affect biological pathways. If the genes are involved in liver carcinogenesis, these modifications may partly explain the genetic heritability thought to influence individual susceptibility to HCC.
      In addition to the above mentioned epidemiological and environmental risk factors, many studies have reported numerous associations between various SNPs and the presence of HCC. Most of them test the hypothesis that a known variant, with an identified functional involvement in a biological pathway implicated in hepatocarcinogenesis, may be over-represented in HCC patients included in case–control studies (Fig. 1A), or may be associated with a higher risk of liver cancer occurrence in cohorts of prospectively followed-up cirrhotic patients (Fig. 2). Conversely, Genome-Wide Association studies (GWAs) propose to compare the genotypic distributions of several hundreds of thousands of SNPs in HCC patients and ‘controls’ without a prior hypothesis, in order to reveal previously unsuspected variants associated with HCC (Fig. 1B). This approach, by providing public availability of data, also offers the opportunity to validate associations that have been previously reported by candidate-gene studies. Unfortunately, many of these publications suffer from major methodological biases as they have been conducted in heterogeneous populations. As a consequence, the lack of reproducibility of some data is a major drawback in genetic epidemiology. Many studies, that have attempted to explore previously published statistically significant associations, have failed to reproduce these findings, suggesting the existence of some false-positive results [
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      ]. As a whole, when considering false-positive report probability (FPRP) in the field of genetic epidemiology of cancer diseases, it has been shown that less than one-third of reported gene-variant tumour associations are actually statistically significant [
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      ]. This trend towards false-positive reports is further increased when conducting specific association studies such as meta-analysis.
      Figure thumbnail gr1
      Fig. 1Examples of case–control studies. (A) Meta-analyses. The publication of numerous candidate gene case–control studies now allows large meta-analyses to be conducted that encompass several thousand patients. The main limitation of such analyses is the heterogeneity of the included studies in terms of their patients’ ethnicities, control-group variabilities and/or aetiologies of the underlying liver diseases, Wang et al.
      [
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      ]
      . (B) Genome-Wide Association studies. In contrast to gene-candidate approaches, in which the SNPs are a priori chosen on the basis of the known functional consequences of a pathway involved in liver carcinogenesis, GWAs identify unsuspected variants, sometimes of unknown function, associated with the presence of HCC in large case–control studies. Adapted by permission from Macmillan Publishers Ltd.: Nature Genetics, Kumar et al.
      [
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      ]
      , copyright 2011.
      Figure thumbnail gr2
      Fig. 2Examples of prospective studies. (A) Combined influence of various SNPs on the risk of HCC. Populations under study may be stratified according to genotypic associations that define different subgroups with various risks of HCC occurring during follow-up. Nahon et al.
      [
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      Table 1 refers to the most robust and reliable published data regarding associations between some genetic variants and the risk of HCC. The criteria for selection of main studies reporting reliable associations in this table have been defined according to their methodological qualities. Thus, only studies referenced in PubMed, published in English and meeting the following criteria were considered:
      • (1)
        Large-scale case–control studies that included at least several hundred patients,
      • (2)
        meta-analyses,
      • (3)
        prospective studies, and
      • (4)
        GWAs conducted in HCC patients or data obtained from GWAs that studied liver fibrosis or anti-viral treatments.
      Table 1Published SNPs associated with the risk of HCC.
      OR or HR take into account the highest reported value according to allelic or genotypic expression of results and, when available, after multivariate analysis.
      Case–control studies conducted in cirrhotic patients with or without HCC.
      #OR for genotypic associations in the same publication.
      Table 1 clarifies (1) the ethnicity of patients and the causes of the underlying liver disease, (2) the main biological pathways identified as involved in the mechanisms of liver carcinogenesis and subjected to a possible genetic heterogeneity, and (3) the confidence in the drawn conclusions that can be granted according to strict methodological criteria (number of included patients, selection of cases, characteristics of the study, reported odds or hazards ratio). This selection of publications [
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      ] highlights both the tremendous efforts conducted by various teams to report the possible influence of genetic traits on hepatocarcinogenesis, as well as the complex integration of such data in both biological and clinical settings. The present review will summarize the influence of this inherited background on the risk of HCC with the constant will to illustrate both the limitations and the progress made in this field. For a more exhaustive overview, free online databases providing comprehensively and systematically compiled genetic associations have been recently established [
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      ].

      Candidate-gene approach: limitations of case–control studies, strengths of prospective cohorts

      With this approach, candidate genes are a priori chosen on the basis of biological plausibility, assuming that some genetic variations would render their carriers more prone to developing HCC. This approach is based on the hypothesis that a given variant in a specific gene, involved in a pathway that influences HCC development, could sufficiently alter either protein function or expression, and result in the modulation of cancer risk. Thus, case–control studies have been widely conducted to compare allelic or genotypic distributions of SNPs that are thought to modulate liver carcinogenesis pathways in patients with or without HCC. Because the number of publications has grown over the last ten years, some of the results have been reproduced by numerous teams, but have also been challenged by others. Despite their limitations, the ability to perform large meta-analyses that encompass several thousands of patients is now possible. In parallel, the constitution of prospective cohorts of cirrhotic patients with a long follow-up and a large number of events now enables to perform multivariate analyses taking into account, along with genetic traits, other clinical and environmental factors known to influence HCC occurrence. Thus, variants that modulate the capacity to detoxify free radicals and carcinogens, iron metabolism, inflammatory and immune responses, DNA repair mechanisms or systems involved in cell-cycle regulation may now be validated in this setting.

      Oxidative stress and detoxifying systems

      Damage to cellular macromolecules mediated by reactive oxygen species (ROS) has been shown to accumulate with older age and leads to deleterious effects associated with hepatocarcinogenesis [
      • Sarsour E.H.
      • Kumar M.G.
      • Chaudhuri L.
      • Kalen A.L.
      • Goswami P.C.
      Redox control of the cell cycle in health and disease.
      ]. Various SNPs modulate the activity of several anti- or pro-oxidant enzymes, and have been associated with the emergence of numerous cancers [
      • Choi J.Y.
      • Neuhouser M.L.
      • Barnett M.J.
      • Hong C.C.
      • Kristal A.R.
      • Thornquist M.D.
      • et al.
      Iron intake, oxidative stress-related genes (MnSOD and MPO) and prostate cancer risk in CARET cohort.
      ,
      • Hu Y.J.
      • Diamond A.M.
      Role of glutathione peroxidase 1 in breast cancer: loss of heterozygosity and allelic differences in the response to selenium.
      ,
      • Reynolds W.F.
      • Chang E.
      • Douer D.
      • Ball E.D.
      • Kanda V.
      An allelic association implicates myeloperoxidase in the etiology of acute promyelocytic leukemia.
      ,
      • Sutton A.
      • Nahon P.
      • Pessayre D.
      • Rufat P.
      • Poire A.
      • Ziol M.
      • et al.
      Genetic polymorphisms in antioxidant enzymes modulate hepatic iron accumulation and hepatocellular carcinoma development in patients with alcohol-induced cirrhosis.
      ], including HCC. Among these systems, glutathione S-transferases (GSTs) are a large family of detoxifying enzymes protecting against oxidative DNA damage. The influence of genetic traits involved in GSTM1 and GSTT1 activities has been extensively studied in various cancers, with controversial results obtained in small-sample case–control studies for HCC. Although a first meta-analysis only reported a moderate effect of these variants [
      • White D.L.
      • Li D.
      • Nurgalieva Z.
      • El-Serag H.B.
      Genetic variants of glutathione S-transferase as possible risk factors for hepatocellular carcinoma: a HuGE systematic review and meta-analysis.
      ], the meta-analysis by Wang et al. [
      • Wang B.
      • Huang G.
      • Wang D.
      • Li A.
      • Xu Z.
      • Dong R.
      • et al.
      Null genotypes of GSTM1 and GSTT1 contribute to hepatocellular carcinoma risk: evidence from an updated meta-analysis.
      ], by including a large number of studies (n = 24, cases: 3349, controls: 5609) and by assessing the between-study heterogeneity, was able to report the association between null genotypes of GSTM1 and GSTT1 (alone or combined), conferring low enzymatic activity, and HCC, mostly in HBV-infected Asian cohorts (Fig. 1A).
      Other pro- or antioxidant systems are modulated by genetic heterogeneity and act at different subcellular levels. Myeloperoxidase (MPO) is expressed in neutrophils and Kupffer cells [
      • Brown K.E.
      • Brunt E.M.
      • Heinecke J.W.
      Immunohistochemical detection of myeloperoxidase and its oxidation products in Kupffer cells of human liver.
      ], and leads to the formation of highly reactive hypochlorous acid (HOCl) and anion (OCl) [
      • Klebanoff S.J.
      Myeloperoxidase: friend and foe.
      ]. Manganese superoxide dismutase (SOD2) generates H2O2 in mitochondria [
      • Wallace D.C.
      Mitochondrial diseases in man and mouse.
      ]. Unless detoxified into water at the mitochondrial level by GPx1 or in the cytoplasm by peroxisomal catalase (CAT), hydrogen peroxide can form hypochlorous acid in the presence of MPO, or the hydroxyl radical in the presence of iron. In large prospective cohorts of cirrhotic patients, the implication of variants modulating the activity of these enzymes has been underlined, and suggested both similarities and differences in HCV- and alcohol-induced hepatocarcinogenesis. Indeed, alcoholic cirrhotic patients bearing the high-activity-conferring Ala-SOD2 allele and the low activity-associated Leu-GPx1 allele(s), had a higher incidence of HCC during follow-up [
      • Sutton A.
      • Nahon P.
      • Pessayre D.
      • Rufat P.
      • Poire A.
      • Ziol M.
      • et al.
      Genetic polymorphisms in antioxidant enzymes modulate hepatic iron accumulation and hepatocellular carcinoma development in patients with alcohol-induced cirrhosis.
      ], suggesting that these genotypic associations may represent surrogate markers for high mitochondrial hydrogen-peroxide accumulation (Fig. 2A). Conversely, this genetic heterogeneity does not impact on HCV-related hepatocarcinogenesis in which cytoplasmic ROS accumulation seems to play a critical role [
      • Nahon P.
      • Sutton A.
      • Rufat P.
      • Charnaux N.
      • Mansouri A.
      • Moreau R.
      • et al.
      A variant in myeloperoxidase promoter hastens the emergence of hepatocellular carcinoma in patients with HCV-related cirrhosis.
      ]. Finally, the implication of variants modulating the activity of extra-hepatic MPO, in both alcohol and HCV-induced emergence of HCC, highlights the possible intervention of inflammatory and Kupffer cells in this complex balance [
      • Nahon P.
      • Sutton A.
      • Rufat P.
      • Ziol M.
      • Akouche H.
      • Laguillier C.
      • et al.
      Myeloperoxidase and superoxide dismutase 2 polymorphisms comodulate the risk of hepatocellular carcinoma and death in alcoholic cirrhosis.
      ,
      • Nahon P.
      • Sutton A.
      • Rufat P.
      • Charnaux N.
      • Mansouri A.
      • Moreau R.
      • et al.
      A variant in myeloperoxidase promoter hastens the emergence of hepatocellular carcinoma in patients with HCV-related cirrhosis.
      ]. As the benefit of using antioxidant treatments to prevent cancer remains controversial [
      • Bjelakovic G.
      • Nikolova D.
      • Gluud L.L.
      • Simonetti R.G.
      • Gluud C.
      Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis.
      ], future strategies that incorporate this therapeutic axis should not only take into account the cause of liver disease but also the genetic-host factors, which seem to influence the individual pro- or antioxidant status.

      Iron metabolism

      Iron overload is considered to be a co-factor in the onset and progression of almost all liver diseases, including the development of HCC [
      • Deugnier Y.
      • Turlin B.
      Pathology of hepatic iron overload.
      ]. The risk of HCC in patients with genetic haemochromatosis is well established, particularly in patients with overt cirrhosis [
      • Elmberg M.
      • Hultcrantz R.
      • Ekbom A.
      • Brandt L.
      • Olsson S.
      • Olsson R.
      • et al.
      Cancer risk in patients with hereditary hemochromatosis and in their first-degree relatives.
      ]. Several teams have reported that the prevalence of HFE C282Y mutations was increased in patients with HCC [
      • Fargion S.
      • Stazi M.A.
      • Fracanzani A.L.
      • Mattioli M.
      • Sampietro M.
      • Tavazzi D.
      • et al.
      Mutations in the HFE gene and their interaction with exogenous risk factors in hepatocellular carcinoma.
      ,
      • Hellerbrand C.
      • Poppl A.
      • Hartmann A.
      • Scholmerich J.
      • Lock G.
      HFE C282Y heterozygosity in hepatocellular carcinoma: evidence for an increased prevalence.
      ]. However, other studies have failed to confirm these associations [
      • Boige V.
      • Castera L.
      • de Roux N.
      • Ganne-Carrie N.
      • Ducot B.
      • Pelletier G.
      • et al.
      Lack of association between HFE gene mutations and hepatocellular carcinoma in patients with cirrhosis.
      ,
      • Cauza E.
      • Peck-Radosavljevic M.
      • Ulrich-Pur H.
      • Datz C.
      • Gschwantler M.
      • Schoniger-Hekele M.
      • et al.
      Mutations of the HFE gene in patients with hepatocellular carcinoma.
      ,
      • Lauret E.
      • Rodriguez M.
      • Gonzalez S.
      • Linares A.
      • Lopez-Vazquez A.
      • Martinez-Borra J.
      • et al.
      HFE gene mutations in alcoholic and virus-related cirrhotic patients with hepatocellular carcinoma.
      ]. A recent prospective study enabled a link to be made between hepatic iron overload, HFE variations and development of HCC in alcoholic cirrhosis [
      • Nahon P.
      • Sutton A.
      • Rufat P.
      • Ziol M.
      • Thabut G.
      • Schischmanoff P.O.
      • et al.
      Liver iron, HFE gene mutations, and hepatocellular carcinoma occurrence in patients with cirrhosis.
      ] while neither of these two factors affected the risk of liver cancer in HCV-infected cirrhotic patients. This finding has been partially confirmed in HCV-infected patients included in the HALT-C trial [
      • Lambrecht R.W.
      • Sterling R.K.
      • Naishadham D.
      • Stoddard A.M.
      • Rogers T.
      • Morishima C.
      • et al.
      Iron levels in hepatocytes and portal tract cells predict progression and outcomes of patients with advanced chronic hepatitis C.
      ], in whom HFE gene mutations did not correlate with the development of HCC. In addition to the impact of the underlying liver disease, iron overload also depends on a complex set of polygenic and epidemiological factors that are still poorly understood. In clinical practice, liver iron content and/or HFE gene mutations could be assessed to improve identification of cirrhotic patients with a higher risk of HCC and who may benefit from iron depletion [
      • Kato J.
      • Miyanishi K.
      • Kobune M.
      • Nakamura T.
      • Takada K.
      • Takimoto R.
      • et al.
      Long-term phlebotomy with low-iron diet therapy lowers risk of development of hepatocellular carcinoma from chronic hepatitis C.
      ].

      Inflammation, cytokine, and chemokine systems

      HCC is a tumour that slowly develops in a background of chronic inflammation, a common denominator in the main causes of liver disease. At the origin of this cancer lies the perpetuation of a wound-healing response triggered by parenchymal cell death and inflammation [
      • El-Serag H.B.
      • Rudolph K.L.
      Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.
      ]. The influence of external factors (alcohol, viral infections, oxidative stress etc.) induces the activation of macrophages and liver Kupffer cells, in particular through the interleukin 1 pathway (IL-1), which leads to secretion of proinflammatory cytokines such as interleukin 6 (IL-6) and TNF. These cytokines are involved in the regulation of proliferation and cell differentiation as well as neo-angiogenesis [
      • Berasain C.
      • Castillo J.
      • Perugorria M.J.
      • Latasa M.U.
      • Prieto J.
      • Avila M.A.
      Inflammation and liver cancer: new molecular links.
      ]. Polymorphisms that affect the production of these pro-inflammatory molecules may constitute the most studied genetic background in the HCC setting, mostly in HCV- or HBV-infected Asian populations. One of the first case–control studies in this field reported that the high IL-1 production-conferring alleles in two SNPs, in the promoter region of the IL-1β gene (namely IL-1β C-31T and IL-1β C-511T polymorphisms), were associated with the presence of HCV-related HCC after adjustment for confounding factors, which included the presence of cirrhosis [
      • Wang Y.
      • Kato N.
      • Hoshida Y.
      • Yoshida H.
      • Taniguchi H.
      • Goto T.
      • et al.
      Interleukin-1beta gene polymorphisms associated with hepatocellular carcinoma in hepatitis C virus infection.
      ]. As the number of reports has accumulated, two recent meta-analyses have reported that SNPs affecting TNFα production were candidate genes that increased the risk of virally-induced HCC, particularly TNFα G-308A SNP, with a higher risk for individuals bearing at least one high-activity A-308 allele [
      • Wei Y.
      • Liu F.
      • Li B.
      • Chen X.
      • Ma Y.
      • Yan L.
      Polymorphisms of tumor necrosis factor-alpha and hepatocellular carcinoma risk: a huge systematic review and meta-analysis.
      ,
      • Yang Y.
      • Luo C.
      • Feng R.
      • Bi S.
      The TNF-alpha, IL-1B and IL-10 polymorphisms and risk for hepatocellular carcinoma: a meta-analysis.
      ]. Numerous other variants affecting the production/activity of other pro- or anti-inflammatory molecules warrant further validation and may also participate in this complex regulation [
      • Migita K.
      • Miyazoe S.
      • Maeda Y.
      • Daikoku M.
      • Abiru S.
      • Ueki T.
      • et al.
      Cytokine gene polymorphisms in Japanese patients with hepatitis B virus infection–association between TGF-beta1 polymorphisms and hepatocellular carcinoma.
      ,
      • Wei Y.G.
      • Liu F.
      • Li B.
      • Chen X.
      • Ma Y.
      • Yan L.N.
      • et al.
      Interleukin-10 gene polymorphisms and hepatocellular carcinoma susceptibility: a meta-analysis.
      ]. Other mediators of inflammation such as chemokines and their receptors [
      • Boisvert J.
      • Kunkel E.J.
      • Campbell J.J.
      • Keeffe E.B.
      • Butcher E.C.
      • Greenberg H.B.
      Liver-infiltrating lymphocytes in end-stage hepatitis C virus: subsets, activation status, and chemokine receptor phenotypes.
      ] are implicated in the promotion of tumour growth and the invasion in the liver [
      • Yoong K.F.
      • Afford S.C.
      • Jones R.
      • Aujla P.
      • Qin S.
      • Price K.
      • et al.
      Expression and function of CXC and CC chemokines in human malignant liver tumors: a role for human monokine induced by gamma-interferon in lymphocyte recruitment to hepatocellular carcinoma.
      ]. Genetic variations commonly occur in their regulatory regions, affecting chemokines gene transcription [
      • Promrat K.
      • McDermott D.H.
      • Gonzalez C.M.
      • Kleiner D.E.
      • Koziol D.E.
      • Lessie M.
      • et al.
      Associations of chemokine system polymorphisms with clinical outcomes and treatment responses of chronic hepatitis C.
      ]. Although no clear associations with the development of HCC were initially reported [
      • Nahon P.
      • Sutton A.
      • Rufat P.
      • Faisant C.
      • Simon C.
      • Barget N.
      • et al.
      Lack of association of some chemokine system polymorphisms with the risks of death and hepatocellular carcinoma occurrence in patients with alcoholic cirrhosis: a prospective study.
      ,
      • Nahon P.
      • Sutton A.
      • Rufat P.
      • Simon C.
      • Trinchet J.C.
      • Gattegno L.
      • et al.
      Chemokine system polymorphisms, survival and hepatocellular carcinoma occurrence in patients with hepatitis C virus-related cirrhosis.
      ], a recent prospective study suggested that chemokine RANTES G-403A dimorphism influenced the occurrence of HCC in patients with alcoholic cirrhosis [
      • Charni F.
      • Sutton A.
      • Rufat P.
      • Laguillier C.
      • Mansouri A.
      • Moreau R.
      • et al.
      Chemokine RANTES promoter dimorphisms and hepatocellular carcinoma occurrence in patients with alcoholic or hepatitis C virus-related cirrhosis.
      ].

      DNA synthesis and repair mechanisms

      SNPs in the methylene tetrahydrofolate reductase (MTHFR) gene, which lead to alterations in folate metabolism, an essential component of DNA synthesis and methylation, seem to be associated with HCC development [
      • Yuan J.M.
      • Lu S.C.
      • Van Den Berg D.
      • Govindarajan S.
      • Zhang Z.Q.
      • Mato J.M.
      • et al.
      Genetic polymorphisms in the methylenetetrahydrofolate reductase and thymidylate synthase genes and risk of hepatocellular carcinoma.
      ]. In this large case–control study, which included HCV- and HBV-infected patients from two cohorts of distinct ethnicities (Asians and non-Asians), the authors were able to observe a joint protective effect of genotypic associations in two variants conferring a low MTHFR activity (namely rs1801133 and rs1801131). This finding was further strengthened by the combined effect of variants modulating thymidylate synthase (TYMS) activity, an enzyme also implicated in DNA protection; with patients bearing an increasing number of mutant alleles of these 3 SNPs having the lowest risk of HCC. This approach, by combining several genotypic risk factors for HCC development, points to the complex polygenic aspect of hepatocarcinogenesis, which is further influenced by environmental factors. Indeed, large studies that have included several thousands of Chinese individuals have elegantly demonstrated a synergic effect between genetic variants in DNA repair genes, such as XRCC3 or XPC, with environmental exposure to aflatoxin (AFB1): the risk of HCC was indeed higher in patients cumulating both the at-risk genotypes and the longest AFB1 exposure [
      • Long X.D.
      • Ma Y.
      • Qu de Y.
      • Liu Y.G.
      • Huang Z.Q.
      • Huang Y.Z.
      • et al.
      The polymorphism of XRCC3 codon 241 and AFB1-related hepatocellular carcinoma in Guangxi population, China.
      ,
      • Long X.D.
      • Ma Y.
      • Zhou Y.F.
      • Ma A.M.
      • Fu G.H.
      Polymorphism in xeroderma pigmentosum complementation group C codon 939 and aflatoxin B1-related hepatocellular carcinoma in the Guangxi population.
      ].

      Other pathways involved in hepatocarcinogenesis

      The loss of p53 function, enabling damaged cells to escape the cell-cycle checkpoint control and become carcinogenic, plays a critical role in carcinogenesis, and places variants of the p53 gene as fair candidates in the modulation of HCC risk [
      • Staib F.
      • Hussain S.P.
      • Hofseth L.J.
      • Wang X.W.
      • Harris C.C.
      TP53 and liver carcinogenesis.
      ]. MDM2 is an important regulator of p53 that represents a negative auto-regulatory feedback loop with p53 protein [
      • Bond G.L.
      • Hu W.
      • Bond E.E.
      • Robins H.
      • Lutzker S.G.
      • Arva N.C.
      • et al.
      A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans.
      ]. Some other polymorphic traits affecting the promoter region of MDM2 may be associated with HCV-related HCC [
      • Dharel N.
      • Kato N.
      • Muroyama R.
      • Moriyama M.
      • Shao R.X.
      • Kawabe T.
      • et al.
      MDM2 promoter SNP309 is associated with the risk of hepatocellular carcinoma in patients with chronic hepatitis C.
      ]. This latter finding has been confirmed in a large case–control study that included 583 HBV-infected Asian patients in whom the same MDM2 G-309 allele, conferring higher protein expression, was more prevalent in patients with liver cancer [
      • Yoon Y.J.
      • Chang H.Y.
      • Ahn S.H.
      • Kim J.K.
      • Park Y.K.
      • Kang D.R.
      • et al.
      MDM2 and p53 polymorphisms are associated with the development of hepatocellular carcinoma in patients with chronic hepatitis B virus infection.
      ]. In addition, a recent meta-analysis of various SNPs reported that the MDM2 G-309T polymorphism was one of the most reliable candidate genes to be linked with the risk of HCC [
      • Jin F.
      • Xiong W.J.
      • Jing J.C.
      • Feng Z.
      • Qu L.S.
      • Shen X.Z.
      Evaluation of the association studies of single nucleotide polymorphisms and hepatocellular carcinoma: a systematic review.
      ]. This conclusion was reached after the authors applied FPRP analysis and Venice guidelines [
      • Ioannidis J.P.
      • Boffetta P.
      • Little J.
      • O’Brien T.R.
      • Uitterlinden A.G.
      • Vineis P.
      • et al.
      Assessment of cumulative evidence on genetic associations: interim guidelines.
      ,
      • Wacholder S.
      • Chanock S.
      • Garcia-Closas M.
      • El Ghormli L.
      • Rothman N.
      Assessing the probability that a positive report is false: an approach for molecular epidemiology studies.
      ] which have become recent recommendations evaluating the reliability of the associations reported in genetic epidemiological studies.
      The epidermal growth factor (EGF) is implicated in malignant transformation and tumour progression, [
      • Blanc P.
      • Etienne H.
      • Daujat M.
      • Fabre I.
      • Zindy F.
      • Domergue J.
      • et al.
      Mitotic responsiveness of cultured adult human hepatocytes to epidermal growth factor, transforming growth factor alpha, and human serum.
      ] and seems to actively participate in liver carcinogenesis in animal models [
      • Schiffer E.
      • Housset C.
      • Cacheux W.
      • Wendum D.
      • Desbois-Mouthon C.
      • Rey C.
      • et al.
      Gefitinib, an EGFR inhibitor, prevents hepatocellular carcinoma development in the rat liver with cirrhosis.
      ]. Furthermore, high levels of serum EGF have been associated with an increased incidence of diverse malignancies in epidemiological studies [
      • Lanuti M.
      • Liu G.
      • Goodwin J.M.
      • Zhai R.
      • Fuchs B.C.
      • Asomaning K.
      • et al.
      A functional epidermal growth factor (EGF) polymorphism, EGF serum levels, and esophageal adenocarcinoma risk and outcome.
      ]. A functional polymorphism involves an A–G transition at position 61 in the 5′ untranslated region of the EGF gene, leading to higher EGF levels and the emergence of various cancers in individuals who are homozygous for the G allele [
      • Shahbazi M.
      • Pravica V.
      • Nasreen N.
      • Fakhoury H.
      • Fryer A.A.
      • Strange R.C.
      • et al.
      Association between functional polymorphism in EGF gene and malignant melanoma.
      ]. Tanabe et al. [
      • Tanabe K.K.
      • Lemoine A.
      • Finkelstein D.M.
      • Kawasaki H.
      • Fujii T.
      • Chung R.T.
      • et al.
      Epidermal growth factor gene functional polymorphism and the risk of hepatocellular carcinoma in patients with cirrhosis.
      ] extensively studied the consequences of this polymorphic trait on the progression of hepatic injury in two distinct cohorts of cirrhotic Caucasian patients, either with HCV-related (Massachusetts cohort: 59/207 HCC cases) or alcoholic (French cohort: 44/141 HCC cases) liver disease. They were able to report that patients bearing two copies of this variant not only had higher EGF-serum levels but also displayed increased hepatic expression of this factor. Furthermore, this same homozygous G/G genotype was over-represented in HCC patients in both cohorts and was independently associated with the presence of a liver tumour after adjustment for confounding factors. This finding, also observed in two case–control cohorts with different causes of liver disease, was recently confirmed in patients included in the HALT-C trial [
      • Abu Dayyeh B.K.
      • Yang M.
      • Fuchs B.C.
      • Karl D.L.
      • Yamada S.
      • Sninsky J.J.
      • et al.
      A functional polymorphism in the epidermal growth factor gene is associated with risk for hepatocellular carcinoma.
      ]. With the advantage of a long prospective follow-up, Abu Dayyeh et al. were able to observe an increased risk of subsequent HCC occurrence in G/G homozygotes, who also exhibited higher EGF serum levels. When incorporating these genetic data for the creation of a predictive score along with other clinical features associated with HCC, the authors stratified their study population according to three levels of liver-cancer risk, ranging from 2.3% to 26% at 6 years (Fig. 2B).
      MicroRNAs (miRNAs) are a class of short non-coding RNAs with post-transcriptional regulatory functions [
      • Pillai R.S.
      MicroRNA function: multiple mechanisms for a tiny RNA?.
      ]. The binding of miRNA to mRNA is critical in the regulation of mRNA level and protein expression [
      • Wiemer E.A.
      The role of microRNAs in cancer: no small matter.
      ]. Accumulating evidence has linked the dysregulated expression of miRNAs with HCC [
      • Huang S.
      • He X.
      The role of microRNAs in liver cancer progression.
      ]. Functional SNPs in miRNA genes can lead to changes in expression of mature miRNAs [
      • Chen K.
      • Song F.
      • Calin G.A.
      • Wei Q.
      • Hao X.
      • Zhang W.
      Polymorphisms in microRNA targets: a gold mine for molecular epidemiology.
      ]. A G>C polymorphism in the miR-146a gene leads to an increased production of the mature product for the G allele and promotes cell proliferation [
      • Jazdzewski K.
      • Murray E.L.
      • Franssila K.
      • Jarzab B.
      • Schoenberg D.R.
      • de la Chapelle A.
      Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma.
      ]. Xu et al. [
      • Xu T.
      • Zhu Y.
      • Wei Q.K.
      • Yuan Y.
      • Zhou F.
      • Ge Y.Y.
      • et al.
      A functional polymorphism in the miR-146a gene is associated with the risk for hepatocellular carcinoma.
      ] reported, in a large case–control study involving almost 1000 HBV- or HCV-infected Chinese patients, an increased risk of liver cancer in patients homozygous for the G allele. Other polymorphic traits can affect the miRNA target site, which can modify the binding sites to the mRNA and thus influence its expression [
      • Chen K.
      • Rajewsky N.
      Natural selection on human microRNA binding sites inferred from SNP data.
      ]. This is the case for rs3783553, an insertion/deletion polymorphism located within the IL-1α 3′ untranslated region, which significantly affects the binding of miR-122 and miR-378 and influences the regulation of IL-1α expression [
      • Landi D.
      • Gemignani F.
      • Barale R.
      • Landi S.
      A catalog of polymorphisms falling in microRNA-binding regions of cancer genes.
      ]. More than 3000 Chinese patients (including an independent validation set), of whom half with HCC, were genotyped for this SNP [
      • Gao Y.
      • He Y.
      • Ding J.
      • Wu K.
      • Hu B.
      • Liu Y.
      • et al.
      An insertion/deletion polymorphism at miRNA-122-binding site in the interleukin-1alpha 3’ untranslated region confers risk for hepatocellular carcinoma.
      ]. The authors observed that patients homozygous for the deletion allele were more frequent in HCC groups with a fair genotype–phenotype correlation.

      Genome-Wide Association studies (GWAs): identification of unsuspected genetic traits associated with the risk of HCC

      Genome-wide association studies have recently changed our approach to study genetic susceptibility to liver disease [
      • Karlsen T.H.
      • Melum E.
      • Franke A.
      The utility of genome-wide association studies in hepatology.
      ]. The recent development of high-throughput genomics technology enables several hundred-thousand SNPs to be assessed, and compares differences in the allelic or genotypic distribution of these SNPs in large case–control studies, which usually encompass thousands of patients [
      • Stadler Z.K.
      • Gallagher D.J.
      • Thom P.
      • Offit K.
      Genome-wide association studies of cancer: principles and potential utility.
      ] in order to limit the probability of false positive report. This new tool allows investigators to scan the genome using surrogate markers for specific susceptibility loci that harbour one or more variant(s) that may account for the genetic differences between cases and controls. Compared to candidate-gene studies, GWAs have the advantage of being able to identify genes without any previously suspected role in the pathophysiology of a given disease. In this setting, convergent data obtained by GWAs have enabled the identification of SNPs associated with indirect factors that may modulate the risk of HCC, such as IL-28B polymorphisms that influence HCV clearance [
      • Balagopal A.
      • Thomas D.L.
      • Thio C.L.
      IL28B and the control of hepatitis C virus infection.
      ] or PNPLA3 variants involved in steatosis and fibrosis progression in alcoholic or non-alcoholic fatty liver disease [
      • 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.
      ,
      • Tian C.
      • Stokowski R.P.
      • Kershenobich D.
      • Ballinger D.G.
      • Hinds D.A.
      Variant in PNPLA3 is associated with alcoholic liver disease.
      ,
      • Yuan X.
      • Waterworth D.
      • Perry J.R.
      • Lim N.
      • Song K.
      • Chambers J.C.
      • et al.
      Population-based genome-wide association studies reveal six loci influencing plasma levels of liver enzymes.
      ]. Following theses publications, the influence of these variants on the subsequent development of HCC has been hypothesized and has been so far reported in case–control studies [
      • Fabris C.
      • Falleti E.
      • Cussigh A.
      • Bitetto D.
      • Fontanini E.
      • Bignulin S.
      • et al.
      IL-28B rs12979860 C/T allele distribution in patients with liver cirrhosis: role in the course of chronic viral hepatitis and the development of HCC.
      ,
      • Valenti L.
      • Rumi M.
      • Galmozzi E.
      • Aghemo A.
      • Del Menico B.
      • De Nicola S.
      • et al.
      Patatin-like phospholipase domain-containing 3 I148M polymorphism, steatosis, and liver damage in chronic hepatitis C.
      ,
      • Trepo E.
      • Guyot E.
      • Ganne-Carrie N.
      • Degre D.
      • Gustot T.
      • Franchimont D.
      • et al.
      PNPLA3 (rs738409 C>G) is a common risk variant associated with hepatocellular carcinoma in alcoholic cirrhosis.
      ,
      • Corradini S.G.
      • Burza M.A.
      • Molinaro A.
      • Romeo S.
      Patatin-like phospholipase domain containing 3 sequence variant and hepatocellular carcinoma.
      ,
      • Nischalke H.D.
      • Berger C.
      • Luda C.
      • Berg T.
      • Muller T.
      • Grunhage F.
      • et al.
      The PNPLA3 rs738409 148M/M genotype is a risk factor for liver cancer in alcoholic cirrhosis but shows no or weak association in hepatitis C cirrhosis.
      ]. These data now warrant further validation in larger prospective studies.
      This non-hypothesis-driven approach has recently revealed novel genetic-risk loci associated with HCC. The first GWAs that directly focused on HCC explored 440,794 SNPs in a homogeneous population of HBV-infected Chinese patients, half of them with liver cancer [
      • Zhang H.
      • Zhai Y.
      • Hu Z.
      • Wu C.
      • Qian J.
      • Jia W.
      • et al.
      Genome-wide association study identifies 1p36.22 as a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers.
      ]. Based on this first screening process, the progressive selection of the most associated SNPs was subsequently assessed in five additional independent cohorts, consisting of 1962 HCC patients and 1430 ‘controls’ (and 159 family trios). This stepwise procedure enabled the identification of a susceptibility locus mapping to chromosome 1p36.22, a region that has been reported to be commonly affected by chromosomal losses or gains in numerous cancers, including HCC [
      • Bagchi A.
      • Mills A.A.
      The quest for the 1p36 tumor suppressor.
      ]. Further experiments conducted in tumoral and non-tumoral tissues have assessed the intra-hepatic expression of the corresponding susceptibility locus genes (KIF1B, PDG, and UBE4B) and suggested their possible influence on liver carcinogenesis through the tumour-suppressing pathway.
      Figure thumbnail fx3

      Conclusions: integration of genetic information in large prospective cohorts of cirrhotic patients

      Progress in genomics technology has enabled identification of numerous polymorphic traits that may, in part, account for the inheritable aspect of this multifactorial disease [
      • Ioannidis J.P.
      • Castaldi P.
      • Evangelou E.
      A compendium of genome-wide associations for cancer: critical synopsis and reappraisal.
      ]. The replication of published data, particularly for yet poorly investigated Caucasians and patients with alcoholic-related HCC, may contribute to a better understanding of the genetic heterogeneity that modulates the various biological pathways involved in liver carcinogenesis. Such progress will also benefit from the coordinated work of international research consortia and in the establishment of very large cohorts of well-defined patients, as suggested in Fig. 3, which will allow complex multivariate analyses of all confounding factors.
      Figure thumbnail gr3
      Fig. 3Options for clinical methodology. (A) The optimal exploratory case–control study. Case–control studies aimed at performing candidate-gene studies or GWAs should be conducted in large homogeneous cohorts of patients of the same ethnicity and affected by the same cause and severity of liver disease. To avoid any misinterpretation of the observed associations, it is mandatory to take into account other host or environmental factors that influence liver carcinogenesis. As a consequence, the presence of cirrhosis is a prerequisite for the selection of both cases and controls, which should only differ according to the absence or presence of HCC. Other external features, such as viral clearance, alcohol consumption or the presence of features of a metabolic syndrome, must also be incorporated into the multivariate analysis. (B) The ideal validation prospective study. The influence of SNPs reported as associated with the risk of liver cancer should be validated in large prospective cohorts of homogeneous patients undergoing periodical surveillance for HCC. Their predictive value should be assessed according to multivariate analyses taking into account non-genetic baseline features or events that occur during follow-up, such as competitive risks of death (liver-related or not) or control of the cause of liver disease. The incorporation of numerous variants should enable to highlight gene–gene interactions and to assess polygenic predictive scores. Their additional performance in existing risk-assessment models to predict HCC occurrence may allow implementation of genetic-based screening strategies.
      The adaptation of preventive measures or therapeutic procedures based on inter-individual susceptibilities forms the basis and ultimate goal of personalized medicine. Unfortunately, the SNPs identified so far only partly explain a small proportion of the overall variability in susceptibility to HCC and do not permit good prediction at the individual and population levels. The additional integration of genetic information, which may improve the performance of pre-existing risk-assessment models for HCC, now needs to be tested in large cohorts of prospective follow-up patients with cirrhosis (Fig. 2). The addition of genetic variants into risk-assessment models has been recently performed for various diseases, such as breast cancer, to give, as yet, modest improvements in risk prediction [
      • Wacholder S.
      • Hartge P.
      • Prentice R.
      • Garcia-Closas M.
      • Feigelson H.S.
      • Diver W.R.
      • et al.
      Performance of common genetic variants in breast-cancer risk models.
      ]. Genetic-based screening strategies or therapeutic management of cirrhotic patients may become feasible by incorporating various panels of SNPs into complex models of ‘genomic risk prediction’, which take into account both host and environmental factors that can influence liver carcinogenesis.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Financial support

      This work was supported by the Ligue Nationale Contre le Cancer (“Cartes d’identité des tumeurs” program), the PAIR-CHC 2009 project (funded by INCa and Association pour la recherche contre le Cancer, ARC), the Association Nationale pour la Recherche sur le Sida et les Hépatites Virales (ANRS), and l’Association Française pour l’Etude du Foie (AFEF).

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