Title: Pathogenesis of hepatitis B virus—related hepatocellular carcinoma: Old and new paradigms
Abstract: Chronic infection with the hepatitis B virus (HBV) is a major risk factor for development of hepatocellular carcinoma (HCC). The pathogenesis of cancer in HBV infection has been extensively analyzed, and multiple factors appear to play a role. A major factor is chronic inflammation and the effects of cytokines in the development of fibrosis and liver cell proliferation. Also important is the role of integration of HBV DNA into host cellular DNA, which, in some situations, acts to disrupt or promote expression of cellular genes that are important in cell growth and differentiation. In addition, expression of HBV proteins may have a direct effect on cellular functions, and some of these gene products can favor malignant transformation. Several HBV genes have been found in infected tissues more frequently than others, including truncated pre-S2/S, hepatitis B X gene, and a novel spliced transcript of HBV, referred to as the hepatitis B spliced protein. The proteins expressed from these integrated genes have been shown to have intracellular activities that may account for their association with HCC, including effects on cellular growth and apoptosis. Finally, some patients with HCC have no detectable hepatitis B surface antigen in serum but do have low levels of HBV DNA in serum and integrated molecules of HBV DNA in tissue. Occult HBV infection may account for a proportion of cases of HCC that occur in patients without serologic markers for hepatitis B and C and may be a cofactor in HCC in patients with chronic hepatitis C who have coexistent occult HBV infection. Chronic infection with the hepatitis B virus (HBV) is a major risk factor for development of hepatocellular carcinoma (HCC). The pathogenesis of cancer in HBV infection has been extensively analyzed, and multiple factors appear to play a role. A major factor is chronic inflammation and the effects of cytokines in the development of fibrosis and liver cell proliferation. Also important is the role of integration of HBV DNA into host cellular DNA, which, in some situations, acts to disrupt or promote expression of cellular genes that are important in cell growth and differentiation. In addition, expression of HBV proteins may have a direct effect on cellular functions, and some of these gene products can favor malignant transformation. Several HBV genes have been found in infected tissues more frequently than others, including truncated pre-S2/S, hepatitis B X gene, and a novel spliced transcript of HBV, referred to as the hepatitis B spliced protein. The proteins expressed from these integrated genes have been shown to have intracellular activities that may account for their association with HCC, including effects on cellular growth and apoptosis. Finally, some patients with HCC have no detectable hepatitis B surface antigen in serum but do have low levels of HBV DNA in serum and integrated molecules of HBV DNA in tissue. Occult HBV infection may account for a proportion of cases of HCC that occur in patients without serologic markers for hepatitis B and C and may be a cofactor in HCC in patients with chronic hepatitis C who have coexistent occult HBV infection. Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). This review will summarize the current knowledge on the mechanisms involved in HBV-related liver carcinogenesis. It will also demonstrate how viruses can be viewed as tools to discover and dissect new cellular pathways involved in cancer development. Cirrhosis is the long-term histologic consequence of chronic inflammation and fibrosis and is found to be present in most patients who present with HCC. A number of in vivo studies, particularly those using transgenic mice, have now allowed for the dissection of the relative roles of chronic inflammation and cytokines in development of fibrosis and liver cell proliferation and malignant transformation expansion.1Chisari F.V. Viruses, immunity and cancer lessons from hepatitis B.Am J Pathol. 2000; 156: 1117-1132Abstract Full Text Full Text PDF PubMed Google Scholar In addition, there is now solid evidence for the synergistic effects of HBV with chronic inflammation in liver carcinogenesis. Interestingly, consistent with this view, the pattern of genetic alterations has been found to be markedly different in HBV- vs. hepatitis C virus (HCV)-related HCC. These observations further substantiate the hypothesis of a direct involvement of these etiologic agents in liver cell transformation. Along the same line, studies on gene expression using microarrays show distinctively different patterns between HBV- and HCV-related HCC.2Delpuech O. Buffello-Le Guillou D.B. Rubinstein E. Feray C. Petit M.A. The hepatitis C virus (HCV) induces a long-term increase in interleukin-10 production by human CD4+ T cells (H9).Eur Cytokine Netw. 2001; 12: 69-77PubMed Google Scholar, 3Ye Q.H. Qin L.X. Forgues M. He P. Kim J.W. Peng A.C. Simon R. Li Y. Robles A.I. Chen Y. Ma Z.C. Wu Z.Q. Ye S.L. Liu Y.K. Tang Z.Y. Wang X.W. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning.Nat Med. 2003; 9: 416-423Crossref PubMed Scopus (728) Google Scholar HBV DNA integration into cellular DNA is not necessary for viral replication4Brechot C. Thiers V. Kremsdorf D. Nalpas B. Pol S. Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen clinically significant or purely “occult”?.Hepatology. 2001; 34: 194-203Crossref PubMed Scopus (496) Google Scholar but allows for persistence of the viral genome. HBV DNA sequences have been shown to be integrated into cellular DNA in samples of HCC and can also be identified in nontumorous tissue from patients with chronic hepatitis.4Brechot C. Thiers V. Kremsdorf D. Nalpas B. Pol S. Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen clinically significant or purely “occult”?.Hepatology. 2001; 34: 194-203Crossref PubMed Scopus (496) Google Scholar Moreover, previous observations based on Southern blotting methods have been confirmed recently using polymerase chain reaction (PCR)-based approaches and show that, at least in some cases, integration of the viral DNA occurs during the acute or early stages of infection.5Lugassy C. Bernuau J. Thiers V. Krosgaard K. Degott C. Wantzin P. Schalm S.W. Rueff B. Benhamou J.P. Tiollais P. Sequences of hepatitis B virus DNA in the serum and liver of patients with acute benign and fulminant hepatitis.J Infect Dis. 1987; 155: 64-71Crossref PubMed Scopus (47) Google Scholar, 6Murakami Y. Minami M. Daimon Y. Okanoue T. Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen.J Med Virol. 2004; 72: 203-214Crossref PubMed Scopus (99) Google Scholar Integration, therefore, precedes development of HCC, and comparative analyses of the various restriction profiles at different times during the course of HBV infection suggest that there is a progressive clonal expansion of certain infected cells. The functional consequences of HBV DNA integration must be viewed as a dynamic process (Figure 1). Long-term chronic inflammation, associated with increased liver cell proliferation, induces rearrangements of the integrated viral sequences. Deletion of part of the viral genome as well as more complex rearrangements is frequently observed. These changes may be related to oxidative DNA damage during chronic inflammation.7Dandri M. Burda M.R. Burkle A. Zuckerman D.M. Will H. Rogler C.E. Greten H. Petersen J. Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly(ADP-ribosyl)ation.Hepatology. 2002; 35: 217-223Crossref PubMed Scopus (88) Google Scholar HBV insertion can induce chromosomal deletions at the HBV integration sites. Moreover, transpositions from one chromosome to another of the viral sequences, together with the flanking cellular sequences, have been recently confirmed.7Dandri M. Burda M.R. Burkle A. Zuckerman D.M. Will H. Rogler C.E. Greten H. Petersen J. Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly(ADP-ribosyl)ation.Hepatology. 2002; 35: 217-223Crossref PubMed Scopus (88) Google Scholar, 8Wang H.P. Zhang L. Dandri M. Rogler C.E. Antisense down-regulation of N-myc1 in woodchuck hepatoma cells reverses the malignant phenotype.J Virol. 1998; 72: 2192-2198PubMed Google Scholar Thus, integrated HBV can generate chromosomal instability, and viral DNA sequences encompassing the encapsidation signal of HBV may exhibit intrinsic recombinogenic activity via binding to a putative “recombinogenic” cellular protein.9Aoki H. Kajino K. Arakawa Y. Hino O. Molecular cloning of a rat chromosome putative recombinogenic sequence homologous to the hepatitis B virus encapsidation signal.Proc Natl Acad Sci U S A. 1996; 93: 7300-7304Crossref PubMed Scopus (35) Google Scholar The insertion of viral DNA into a cellular gene as well as modification of its expression (ie, cis-activation) is another potential consequence of HBV DNA integration. The impact of cis-activation in hepatic carcinogenesis has been controversial. Results of studying the animal model of woodchuck hepatitis virus (WHV) have been different from those of human HBV infection and HCC. In WHV-related HCC, the insertion of WHV DNA into the c-myc or, predominantly, N-my 2c oncogenes, has been found frequently.10Jacob J.R. Sterczer A. Toshkov I.A. Yeager A.E. Korba B.E. Cote P.J. Buendia M.A. Gerin J.L. Tennant B.C. Integration of woodchuck hepatitis and N-myc rearrangement determine size and histologic grade of hepatic tumors.Hepatology. 2004; 39: 1008-1016Crossref PubMed Scopus (46) Google Scholar In contrast, in HBV-related HCCs, integration of the viral genome into known oncogenes has been found to be a rare event. We recently revisited this issue using a PCR-based approach, allowing a much faster analysis of the flanking cellular sequences and thus the investigation of a much larger number of HBV insertion sites.11Paterlini-Brechot P. Saigo K. Murakami Y. Chami M. Gozuacik D. Mugnier C. Lagorce D. Brechot C. Hepatitis B virus–related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene.Oncogene. 2003; 22: 3911-3916Crossref PubMed Scopus (267) Google Scholar With this approach, we demonstrated that HBV insertion into cellular genes is, in fact, frequent (around 70%) and that HBV integration can occur in genes encoding for proteins that are important in the control of cell signaling, proliferation, and viability (Figure 2). Importantly, our work, combined with recent investigations from other groups, has shown that the telomerase gene is targeted for integration in different HBV-related HCCs, thus suggesting common pathways in HBV-related carcinogenesis (Figure 3, Figure 4). These recent observations point out that this might also hold true for genes regulating calcium-homeostasis and MAP kinase-dependent signalling, genes not previously known to be involved in malignant transformation (Figure 3).11Paterlini-Brechot P. Saigo K. Murakami Y. Chami M. Gozuacik D. Mugnier C. Lagorce D. Brechot C. Hepatitis B virus–related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene.Oncogene. 2003; 22: 3911-3916Crossref PubMed Scopus (267) Google Scholar, 12Horikawa I. Barrett J.C. cis-Activation of the human telomerase gene (hTERT) by the hepatitis B virus genome.J Natl Cancer Inst. 2001; 93: 1171-1173Crossref PubMed Scopus (91) Google Scholar, 13Ferber M.J. Thorland E.C. Brink A.A. Rapp A.K. Phillips L.A. McGovern R. Gostout B.S. Cheung T.H. Chung T.K. Fu W.Y. Smith D.I. Preferential integration of human papillomavirus type 18 near the c-myc locus in cervical carcinoma.Oncogene. 2003; 22: 7233-7242Crossref PubMed Scopus (142) Google Scholar Thus, HBV DNA might be viewed as a “proviral tag” capable of identifying new cellular genes or unraveling new mechanisms controlling known gene expression.14Li M. Xie Y.H. Kong Y.Y. Wu X. Zhu L. Wang Y. Cloning and characterization of a novel human hepatocyte transcription factor, hB1F, which binds and activates enchancer II of hepatitis B virus.J Biol Chem. 1998; 273: 29022-29031Crossref PubMed Scopus (101) Google Scholar, 15Sanchez-Prieto R. de Alava E. Palomino T. Guinea J. Fernandez V. Cebrian S. Leonart M.L. Cabella P. Martin P. San Roman C. Bornstein R. Pardo J. Martinez A. Diaz-Espada F. Barrios Y. Ramon y Cajal S. An assocation between viral genes and human oncogenic alterations the adenovirus E1A induces the Ewing tumor fusion transcript EWS-FLI1.Nat Med. 1999; 5: 1076-1079Crossref PubMed Scopus (32) Google ScholarFigure 3The telomerase-encoding gene (hTERT) is targeted by HBV DNA integration in several independent HCCs and in HCC-derived cell lines (see references in the text).View Large Image Figure ViewerDownload (PPT)Figure 4Schematic representation of the HBx biologic actions.View Large Image Figure ViewerDownload (PPT) Examination of the viral DNA sequences present in HCC has shown that sequences encoding for the hepatitis B X gene (HBx) and/or truncated envelope PreS2/S viral proteins are retained in a large proportion of tumor cells. Moreover, a novel viral hepatitis B spliced protein (HBSP) has been identified in HBV-infected patients, which is encoded by spliced HBV RNA and is also expressed during the course of chronic HBV infection. This review will focus on HBx and HBSP. In most integrated subviral DNA, the open reading frame for HBx is maintained and transcribed. Several studies have now demonstrated both HBx RNA and protein expression in human HCC tumor cells. The HBx gene is highly conserved among all mammalian hepadnaviruses and encodes a small polypeptide, which is expressed at low levels during acute and chronic hepatitis.16Murakami Y. Minami M. Daimon Y. Okanoue T. Hepatitis B virus DNA in liver, serum and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen.J Med Virol. 2004; 72: 203-214Crossref PubMed Scopus (50) Google Scholar, 17Klein N.P. Bouchard M.J. Wang L.H. Kobarg C. Schneider R.J. Src kinases involved in hepatitis B virus replication.EMBO J. 1999; 18: 5019-5027Crossref PubMed Scopus (104) Google Scholar In addition, antibodies to HBx (anti-HBx) can be detected in the serum of HBV-infected patients. In some studies, anti-HBx was more frequent among patients with HCC than those with chronic hepatitis B without cancer.18Moriarty A.M. Alexander H. Lerner R.A. Thornton G.B. Antibodies to peptides detect new hepatitis B antigen serological correlation with hepatocellular carcinoma.Science. 1985; 227: 429-433Crossref PubMed Scopus (123) Google Scholar, 19Hwang G.Y. Lin C.Y. Huang L.M. Wang Y.H. Wang J.C. Hsu C.T. Yang S.S. Wu C.C. Detection of the hepatitis B virus X protein (HBx) antigen and anti-HBx antibodies in cases of human hepatocellular carcinoma.J Clin Microbiol. 2003; 41: 5598-5603Crossref PubMed Scopus (73) Google Scholar HBx appears to function as a major regulatory protein for viral replication and is required for efficient infectivity of the WHV. In addition, studies of in vitro expression of HBx in different cell types and studies of in vivo expression of HBx protein in transgenic mice have suggested an important role for HBx in the control of cell proliferation and viability.4Brechot C. Thiers V. Kremsdorf D. Nalpas B. Pol S. Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen clinically significant or purely “occult”?.Hepatology. 2001; 34: 194-203Crossref PubMed Scopus (496) Google Scholar, 17Klein N.P. Bouchard M.J. Wang L.H. Kobarg C. Schneider R.J. Src kinases involved in hepatitis B virus replication.EMBO J. 1999; 18: 5019-5027Crossref PubMed Scopus (104) Google Scholar, 20Yen T.S. Hepadnaviral X. Protein review of recent progress.J Biomed Sci. 1996; 3: 20-30Crossref PubMed Scopus (145) Google Scholar Overall, the available in vivo data suggest that HBx expression in transgenic mice likely induces HCC by sensitization of the animals to chemical carcinogens or by altering cellular oncogenes such as c-myc. The biologic actions of HBx have been studied in detail. The results of studies have varied, depending on the amount of HBx expressed in cells, the cell type utilized, and their status of differentiation. The findings of these studies can be summarized as follows (Figure 4): (1) HBx transactivates a number of cellular promoters, acting on cis-acting regulatory elements.20Yen T.S. Hepadnaviral X. Protein review of recent progress.J Biomed Sci. 1996; 3: 20-30Crossref PubMed Scopus (145) Google Scholar The HBx protein is localized largely in the cytoplasm and does not bind directly to DNA but rather acts on cellular promoters by protein-protein interactions. Several of these interactions lead to activating cellular signalling pathways, which regulate target gene expression and modulate apoptosis, cell proliferation, and response to DNA damage. (2) HBx regulates proteasome function and, thus, controls degradation of cellular and viral proteins. (3) HBx has an effect on mitochondria function, a possible, important target of HBx action.21Rahmani Z. Huh K.W. Lasher R. Siddiqui A. Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential.J Virol. 2000; 74: 2840-2846Crossref PubMed Scopus (272) Google Scholar (4) HBx protein may also modulate calcium homeostasis, which provides novel clues for its biologic actions.22Chami M. Ferrari D. Nicotera P. Paterlini-Brechot P. Rizzuto R. Caspase-dependent alterations of Ca2+ signaling in the induction of apoptosis by hepatitis B virus X protein.J Biol Chem. 2003; 278: 31745-31755Crossref PubMed Scopus (101) Google Scholar, 23Bouchard M.J. Wang L.H. Schneider R.J. Calcium signaling by HBx protein in hepatitis B virus DNA replication.Science. 2001; 294 (2376–238)Crossref PubMed Scopus (345) Google Scholar These various biologic effects have overlapping effects on cell proliferation and viability. HBx also appears to act as a paracrine factor.24Tralhao J.G. Roudier J. Morosan S. Giannini C. Tu H. Goulenok C. Carnot F. Zavala F. Joulin V. Kremsdorf D. Brechot C. Paracrine in vivo inhibitory effects of hepatitis B virus X protein (HBx) on liver cell proliferation an alternative mechanism of HBx-related pathogenesis.Proc Natl Acad Sci U S A. 2002; 99: 6991-6996Crossref PubMed Scopus (68) Google Scholar Natural mutants of HBx have been described in liver and serum of patients with different clinical conditions. We refined these observations by using laser-based microdissection of individual cells and subsequent cloning of the HBx gene. HBx sequences were identified in HCC tumor cells that had deletions in the C-terminal portion of the gene.25Iavarone M. Trabut J.B. Delpuech O. Carnot F. Colombo M. Kremsdorf D. Brechot C. Thiers V. Characterisation of hepatitis B virus X protein mutants in tumour and non-tumour liver cells using laser capture microdissection.J Hepatol. 2003; 39: 253-261Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar These findings can be used to address the problem of the biologic actions of HBx genetically, comparing the effects of HBx and its mutants from human HCC on cell growth and viability.8Wang H.P. Zhang L. Dandri M. Rogler C.E. Antisense down-regulation of N-myc1 in woodchuck hepatoma cells reverses the malignant phenotype.J Virol. 1998; 72: 2192-2198PubMed Google Scholar, 26Tu H. Bonura C. Giannini C. Mouly H. Soussan P. Kew M. Paterlini-Brechot P. Brechot C. Kremsdorf D. Biological impact of natural COOH-terminal deletions of hepatitis B virus X protein in hepatocellular carcinoma tissues.Cancer Res. 2001; 61: 7803-7810PubMed Google Scholar Thus, deletion of the C-terminal region of HBx, observed in several HBx mutants isolated from HCC tissue, leads to abrogation of HBx-dependent transactivation, cell-cycle arrest, and apoptosis inhibition; the results also suggest that such deletions may enhance HBx-transforming capacity (Figure 5).26Tu H. Bonura C. Giannini C. Mouly H. Soussan P. Kew M. Paterlini-Brechot P. Brechot C. Kremsdorf D. Biological impact of natural COOH-terminal deletions of hepatitis B virus X protein in hepatocellular carcinoma tissues.Cancer Res. 2001; 61: 7803-7810PubMed Google Scholar Overall, these results are consistent with selection of HBx sequences in clonally expanding liver cells that favor cell proliferation and transformation. Other HBx mutations have been described in patients with or without HCC. Mutations in amino acids 130 and 131 have been frequently reported and may be associated with the severity of chronic hepatitis27Yotsuyanagi H. Hino K. Tomita E. Toyoda J. Yasuda K. Iino S. Precore and core promoter mutations, hepatitis B virus DNA levels and progressive liver injury in chronic hepatitis B.J Hepatol. 2002; 37: 355-363Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar; HBx with these mutations have also been detected in HCC,26Tu H. Bonura C. Giannini C. Mouly H. Soussan P. Kew M. Paterlini-Brechot P. Brechot C. Kremsdorf D. Biological impact of natural COOH-terminal deletions of hepatitis B virus X protein in hepatocellular carcinoma tissues.Cancer Res. 2001; 61: 7803-7810PubMed Google Scholar, 28Hsia C.C. Yuwen H. Tabor E. Hot-spot mutations in hepatitis B virus X gene in hepatocellular carcinoma.Lancet. 1996; 348: 625-626Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar and a recent study of serum samples indicated that these mutations arise before the development of HCC.29Kuang S.Y. Jackson P.E. Wang J.B. Lu P.X. Munoz A. Qian G.S. Kensler T.W. Groopman J.D. Specific mutations of hepatitis B virus in plasma predict liver cancer development.Proc Natl Acad Sci U S A. 2004; 101: 3575-3580Crossref PubMed Scopus (149) Google Scholar However, the functional consequences of these mutated HBx proteins are still unclear. We have demonstrated the presence of a spliced HBV transcript, which can be reverse transcribed and encapsidated in defective HBV particles or expressed as a new HBV protein, referred to as HBV spliced protein (HBSP).30Soussan P. Garreua F. Zylberberg H. Ferray C. Brechot C. Kremsdorf D. In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA.J Clin Invest. 2000; 105: 55-60Crossref PubMed Scopus (125) Google Scholar In vitro, HBSP expression induces apoptosis without cell-cycle block. Antibodies to HBSP (anti-HBSP) can be detected in serum of approximately 45% of patients with chronic hepatitis.31Soussan P. Tuveri R. Nalpas B. Garreau F. Zavala F. Masson A. Pol S. Brechot C. Kremsdorf D. The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis.J Hepatol. 2003; 38: 343-348Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Interestingly, there appears to be an independent correlation between the presence of anti-HBSP, viral replication, and liver fibrosis. In vitro findings suggest that HBSP can induce apoptosis and may modulate transforming growth factor (TGF)-β-dependent signaling. Thus, these results offer a novel mechanism for the role of HBV in inducing liver fibrosis. Collectively, these studies show that several HBV proteins can trigger immune responses and may participate directly in different steps of liver carcinogenesis. These observations further substantiate the importance to treat patients with cirrhosis and decrease viral multiplication. The issue of persisting HBV infections in patients without detectable hepatitis B surface antigen (HBsAg) detection in the serum has been a matter of debate for many years.4Brechot C. Thiers V. Kremsdorf D. Nalpas B. Pol S. Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen clinically significant or purely “occult”?.Hepatology. 2001; 34: 194-203Crossref PubMed Scopus (496) Google Scholar, 32Brechot C. Gozuacik D. Murakami Y. Paterlini-Brechot P. Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC).Semin Cancer Biol. 2000; 10: 211-231Crossref PubMed Scopus (256) Google Scholar, 33Torbenson M. Thomas D.L. Occult hepatitis B.Lancet Infect Dis. 2002; 2: 479-486Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar Despite these controversies, most findings indicate that occult HBV infections occur and that their prevalence varies widely in different geographic areas. Highest rates of occult HBV infection have been reported in patients with HCC. In particular, there is now more direct evidence for an impact of occult HBV infections in the occurrence of HCC in patients with chronic HCV infection,34Pollicino T. Squadrito G. Cerenzia G. Cacciola I. Raffa G. Crax A. Farinati F. Missale G. Smedile A. Tiribelli C. Villa E. Raimondo G. Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection.Gastroenterology. 2004; 126: 102-110Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar making concomitant infection by HBV and HCV a major emerging issue. Occult HBV infections have also been identified in resolved acute infections. Thus, there is likely to be a “spectrum” of liver disease associated with occult infection as there is with HBsAg-positive hepatitis, the severity of which depends on other environmental as well as genetic factors. There are several mechanisms that could account for absence of detectable HBsAg in serum in these patients, including progressive decline in HBV replication, genetic mutations in the HBsAg gene, and environmental factors. In patients with HCC and occult HBV infection, HBx protein expression and cellular gene cis-activation can be identified in tumor cells; the mechanisms described above for HBsAg-positive HCCs are likely to apply to HBsAg-negative subjects as well. Thus, persistence of HBV DNA has been shown to be a risk factor for development of HCC, a finding consistent with those reported in woodchucks with WHsAg-negative persistent WHV infections. The role of occult HBV infection is a major issue for epidemiologic studies on HCC, which should include screening for HBV DNA in serum and tumor tissue using sensitive, PCR-based, assays.