Annals of Clinical & Laboratory Science, vol. 33, no.

1, 2003

39

Review: Hepatocellular Carcinoma and Hepatitis Virus

1 2

Richard B. Birrer,1 Danielle Birrer,1 and John V. Klavins 2 St. Josephs Regional Medical Center, Paterson, New Jersey, and Department of Pathology, Albert Einstein College of Medicine, New York City, New York

Abstract. Integrated hepatitis B virus (HBV) DNA is present in many hepatocellular carcinomas (HCC), suggesting that HBV has a direct oncogenic effect through interaction with transformation-associated genes. Many genes involved in cell cycle regulation (cyclins, kinases, negative regulators, Wnt-beta-catenin) and the transcriptome profile are deregulated or altered in most HCC patients. The HBx protein, potentially oncogenic via multistep carcinogenesis, modifies apoptosis, inhibits nucleotide excision and repair of damaged cellular DNA, and modulates transcriptional activation of cellular growth regulating genes. Hepatocyte transformation may be indirectly influenced by HBV DNA integration, by the generation of mutagenic oxygen reactive species, or by acquisition of mutations in association with necroinflammatory disease. HBV replication, which may occur in HCC, affects the long-term survival of patients. Prevention of HBV infection is expected to decrease the incidence of endemic HCC. (received 24 April 2002, accepted 25 August 2002) Keywords: hepatitis B virus, hepatocellular carcinoma yr survival rate (55 vs 8%). A vaccination program in infants and children can significantly reduce (>80%) the chronic carrier state and the long term potential for HCC [8-10]. Epidemiology Worldwide, 4% of all malignant tumors are HCC. HCC is the seventh most frequent cancer in males and the ninth in females. In North America, 52% of HCC patients have positive tests for HbsAg. Patients <45 yr old have a more favorable prognosis [11]. Of black males with HCC, 62% with HCC are HbsAg+. Antigenemia is unassociated with the presence or absence of cirrhosis, but is more common in younger patients [12]. An early study noted that HbsAg was seldom encountered in the Romanian area and that it was not involved in oncogenesis [13]. However, a subsequent case analysis found that 24% of Romanian patients with HCC were HbsAg+ [14]. In a 21 yr retrospective study in Greenland, HCC incidence was 5% of the HbsAg+ rate, indicating

Introduction About 80% of human hepatocellular carcinomas (HCC) are attributable to chronic hepatitis B virus (HBV) infection [1-3]. Chronic HBV carriers are 100-400 times more likely to develop liver cancer than noncarriers [4,5]. Approximately 20% of HCC arises de novo in otherwise healthy liver [6,7]. Such patients, compared to those with a background of cirrhosis, are younger (mean 36 vs 57 yr), more likely to be female (56 vs 98%), more frequently symptomatic (nausea, vomiting, mass), and have longer duration of symptoms ( 9 vs 4 mo), less common occurrence of HBV markers ( 26 vs 72%), less likely abnormalities of liver function tests, less frequently elevated serum alpha-fetoprotein (AFP, 46 vs 87%), higher resectability (10 vs 1%), increased responsivity to chemotherapy (30 vs 10%), and better one
Address correspondence to Richard B. Birrer, M.D., St Josephs Regional Medical Center, 703 Main Street, Patterson, NJ 07503, USA; tel 973 754 4366; fax 973 754 2044; e-mail birrerr@ sjhmc.org.

0091-7370/03/0100/0039; $4.00; 2003 by the Association of Clinical Scientists, Inc.

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that protective factors may be present or other carcinogenic factors may be absent from the environment [15]. In nonendemic areas of the world, HBV has a probable etiological role in HCC [16]. Of 32 HCC patients of French origin, 3% were HbsAg+, whereas 70% had elevated serum AFP [17]. In a study of 333 Greek HCC patients, 58% were attributed to HBV, 12% to HCV, and 3% to dual infection [18]. In Asian and African countries, by contrast, hepatitis antigens have a statistically significant association with HCC [17]. The incidences of HCC and HBV infection rates were positively correlated in a study of two nearby Chinese villages, one with a high rate (329/100,000 persons) and the other with a zero incidence rate of HCC [19]. Oriental patients tend to be HbsAg+ compared to blacks and whites (p <0.001) [20,21]. HCV and HbsAg prevalences are 33% and 68% in Chinese HCC patients [22,23]. In a large prospective study, the HBV carrier state in Taiwanese patients had >200-fold HCC risk, compared to non-carriers [24]. In 112 Korean patients with HCC, all gave positive tests for HBV infection (97% positivity for HbsAg, 38% for HBeAg, 83% for AFP) [25]. Strong correlation between the prevalence of HbsAg carrier state (812%) and HCC was found in Thailand, where 56% of HCC patients were HbsAg+; 6% of the HbsAg+ HCC cases had HbeAb but no HbeAg [26]. In Japan, only 25% of HCC cases were HbsAg+, whereas 76% were HCVAb+. History of blood transfusion was noted in 40% of these patients [27]. Higher incidence of HCC in Nagasaki vs Hiroshima is attributable to HBV infection, but other factors (ie, radiation-induced immunoincompetence) cannot be excluded [28]. After adjustment for tobacco usage, a strongly positive dose-response relationship in HbsAg+ Japanese males was found between drinking habits and HCC. Tobacco usage correlated with HCC occurrence, but no doseresponse relationship was observed. Tobacco and ethanol usage, therefore, may promote hepatocarcinogenesis in HbsAg+ patients [29]. The HbsAg carrier rate in HCC patients from Zaire is 57% [30]. HbsAg and HbcAg are more frequent in non-neoplastic hepatocytes (53% and

23%, respectively) compared to HCC cells (13% and 3%) [31]. Risk factors for HCC include a history of cirrhosis, transfusions, major surgery, HBV infection (Southeast Asia and sub-Saharan Africa), HCV infection (Europe and Japan), long history of chronic hepatitis, aflatoxin B1 exposure, excessive alcohol intake, prolonged HBV replication phase, and Budd-Chiari disease [32,33]. Chronic HBV infection is associated with HCC in certain settings, depending on the sex of the carrier, immunologic status, size of innoculum, presence of wild-type HBV, absence of HbeAb, severity of primary infection, age at primary infection, and genetic factors [34,35]. The minimum relative risk for HCC among HbsAg+ healthy blood donors is 12.7 [36]. A genotype, Gt-1, is the most prevalent in HCC and may be an important independent risk factor for the cancer [37]. There is 1-4% annual risk of developing HCC in cirrhotic patients with chronic hepatitis C [38]. Pathology In 165 HCC cases, 7-27% of liver tissue samples were HbsAg+. HbsAg positivity was related to preneoplastic hepatic changes. While the presence of HbsAg in liver tissue was not uniform, in 75% of cases dysplastic changes were observed in cells containing HbsAg [39]. In 223 autopsy cases of HCC, there was good correlation between the semiquantitative grade of dysplasia and the incidence of HbsAg positivity [40]. Liver cell dysplasia is indirectly related to HbsAg, with no evidence of premalignancy [41]. HbsAg was frequently found in the cellular parenchyma (replacing and sinusoidal types) of HCC cases. Hepatocytes were frequently retained in the cancerous tissue, especially around the tumornontumor border. No HbsAg+ cells were seen in the encapsulated type of HCC [42]. In a study of 60 cases of HCC in Japanese patients, HbsAg was found mainly in the cytoplasm and HbcAg was predominantly in the nuclei of normal-appearing, dysplastic, and tumor cells. Occasionally, both HbsAg and HbcAg were present in normal and tumor cells [43].

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41

Etiopathogenesis Viral hepatitis may progress to HCC through an intermediate postnecrotic cirrhotic stage [44]. HBV virus may play an indirect role in hepatic oncogenesis [45]. The oncogenetic capacity of HBV is supported by the finding of integrated HBV DNA in many HCCs in which viral sequences are present, despite the production of antibodies to HbsAg [46-48]. Almost all cases of endemic HCC are associated with HBV. HBV DNA integration may occur long before the development of HCC and should be the target of therapy [49]. Free, random and clonal integrated HBV DNA was detected in 83% of neoplastic and 100% of nonneoplastic portions of the liver from HCC cases. There was no HBV DNA in HCC cases not associated with HBV markers [50]. HBV may synthesize reverse transcriptase from c-pol fusion proteins in a manner similar to the way retroviruses synthesize and process a precursor. The accumulation of intermediates of HBV reverse transcriptase in cancerous tissue and not other tissues may reflect the absence of viral core particles and possibly lead to cellular transformation [51]. Liver cell injury during HBV infection may be a result of coinfection with a second cytopathic virus, namely, the delta agent. Persistent HBV infections in HCC patients are long-lasting, as suggested by low serum HbsAg titers, rare occurrence of HBcAg, and absence of HbeAg in most HCC patients [52]. Of male patients with chronic hepatitis B, 50% had HCC with HbeAg detected in peripheral lymphocytes, but they were seronegative for HbeAg. Hepatic oncogenesis may proceed from impaired immune mechanisms from HBV replication and production in host lymphocytes [53]. The etiopathogenesis of HCC may occur through death of susceptible liver cells from HBV infection followed by integration of HBV DNA in resistant hepatic cells [54]. The association rate of HCC is very high in HbsAg+ alcoholic (65%) and non-alcoholic (68%) patients. The rate is low (23%) in HbsAg- alcoholic patients. Concomitant HBV infection in the setting of alcoholism has a major effect on the development of macronodular cirrhosis and HCC [55]. HBVDNA integration is unrelated to the history of alcohol intake. Furthermore, 100% of HbsAg-, non-

alcoholic HCC patients do not have HBV-DNA integration detectable. HBV, therefore, does not appear to have a major role in the pathogenesis of HCC in these patients [56]. There is growing evidence that HBV may act synergistically with aflatoxin B1 (G to T transversion at codon 249) to induce HCC [57,58]. Of 41 tree shrews exposed to aflatoxin B1, 17 were experimentally infected with human HBV, and 53% developed primary liver cancer, compared to only 2 of the controls (p <0.05) [59]. Markers Hepatitis virus. An early study found that HbsAg was present in 4.5% of HCC patients (n=22) [60]. Subsequently, 14-64% of HCC cases (n=652) were noted to be HbsAg+, but the positivity for HbcAb and HbsAb was 4-89% [61-64]. The former was often associated with inactive cirrhosis, characterized by minimal lymphocytic infiltration in the stroma. In HbsAg- HCC cases, active cirrhosis, as noted by significant lymphocytic infiltration, was observed. An altered immune mechanism was strongly suggested [65-67]. A history of liver disease (cirrhosis, hepatitis) and positivity 90-93% for HBV markers was associated with both subclinical and clinical types of HCC (n=138). The positive rate for HbsAb did not correlate, being significantly lower in HCC and cirrhosis groups, compared to controls, suggesting an immune deficiency [68,69]. In antigen negative patients, HBV and HCC were shown by C1q and conglutinin solid phase assays to be associated with the identification of HbsAg- containing immune complexes [70]. The incidences of HbsAg, HbcAb and e antigen antibody were significantly increased in HCC (p <0.005) in patients with a history of cirrhosis [71]. However, HCC in an asymptomatic, non-cirrhotic, 16 year-old male was reported who had positive serum markers for HbsAg, HbeAg and HbcAb [72]. While the exposure rate to HBV for cirrhotic HCC patients was 90%, only 29% were HbsAg+. HbsAb prevalence was 10% for the same group; 39% had serological evidence of continuous viral replication (isolated high titer HbcAb+ or

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HbsAg+), perhaps due to a defective clearance mechanism [73]. HbcAg positivity was more common in patients with cirrhosis (58%) than in HCC patients and it correlated with serological features of high-level viral replication. There were no gender differences in positivity for HCC or cirrhotic cases [74,75]. No specific intrahepatic display pattern of HBV markers was identified in HCC. Tumor cells did not usually support the synthesis of HbcAg or HbsAg. While 41% of liver cirrhosis cases (n=144) were positive for HBsAg, HBsAb was present in 33%, HBeAg in 35%, HBeAb in 42%, and HBcAb in 16%. For HCC cases (n=82) positivity for HBsAg was 48%, HBsAb was 26%, HbcAg was 16%, HBeAg was 26%, and HBeAb was 51% [76]. Of HCC patients (n=494),50-80% were positive for serum HBsAg, with positivity rates for uninvolved tissue and HCC tissue of 83% and 35%, respectively (p <0.001). HBcAg was found in 25% of liver tissue and 8% of HCC tissue (p <0.05). The interval from HBV infection until clinical recognition of HCC may be as short as 9.5 yr, with seroconversion from HbeAg to HbeAb occurring as long as 4 yr prior to clinical presentation [77]. There is a tendency of HBsAg to increase during the course of HCC, often coincident with rapid tumor growth [78]. In a study of 391 blacks with HCC, there was significant association (p <0.001) between HCC and HBV infection and age, with 82% HBV positive and 36% HBe positive in persons 30 yr old, vs 30% and 11% respectively for those 50 yr old [79]. The prevalence of HBeAg in patients with both cirrhosis and HCC was significantly lower (p <0.0005) and did not correlate with age [80]. HbsAg, but not HbcAg or HBeAg, was found in cytoplasm of some tumor cells of a welldifferentiated human HCC cell-line transplanted in athymic (nude) mice [81]. Of these, 9% had HbcAg present in tumor cell cytoplasm [75]. HbsAg positivity was observed predominantly in normal cells whereas HbcAg occurred in 7% of HCC cases, mostly in dysplastic cells. Active replication of HBV in tumor cells becomes increasingly defective during the course of malignant degeneration, as evidenced by a low incidence of HbsAg and complete absence of HbcAg positive tumor cells [82]. HbsAg was present in 63-

93% of nontumor liver tissue and 14-50% of tumor tissue samples (n=174). HbcAg was present in 4653% of nontumor tissue samples, vs 0-9% of the tumor specimens, mostly in non-neoplastic hepatocytes occurring in different intracellular distribution patterns [83,84]. Of 29 HCC patients, 52% had microsatellite alterations (MSA) implying that MSA are unstable in genomic DNA of HBV-infected HCC [85]. MSA occurred frequently with AFP elevation, but they were not associated with the patients gender, age, tumor size, or differentiation. Of sera derived from HBV positive hepatoma cell lines (PLC/PRF/5, Hep 3B), 72% demonstrated voluminous intracytoplasmic fluorescent inclusions. A 18kD protein was identified in this group, but it is unclear whether it was a cellular protein or a product of the HBV genome [86]. Only 4% of sera samples derived from HCC patients were positive for serum HBV-DNA. An HBV-associated nuclear antigen, positive in 78% of HbsAg+ HCC cases, is expressed at an early stage of in vitro culture (PLC/PRF/5), prior to the peak of HbsAg expression [87]. Of 63 HCC tissue specimens, 5% were positive only in the surrounding cirrhotic liver; HbcAg and delta were absent [88]. Of 95 HbsAg carriers with inflammatory liver disease and HCC, 6% were found to be HDAg+ [89]. Of 155 histologically confirmed HCC cases, 1.2% were HbsAg+ and 11% were HCV+ [90]. In 68 HCC patients, the positivity rates for HCV-Ab and HbsAg were 84% and 69%, respectively. Both antibodies were present in 34% of cases [91]. Of 42 non-B non-C HCC patients, 47.6% contain HBV-DNA in nonneoplastic tissue [92-94]. The occult integrant may be important in the development of HCC-HCV patients that lack hepatic fibrosis [95]. The binding is facilitated by an active enhancer-I, likely fetoprotein transcription factor [96]. Within the core domain of HBV enhancer 1 is a STAT-3 binding site (activator of transcription and signal transducer), which interacts with hepatocyte nuclear factor (HNF-3) through epidermal growth factor (EGF) and interleukin-6 stimulation [97]. The result is enhanced function and viral gene expression. The integration of HBV-DNA in HCC tissue of HbsAg carriers occurs into the host genome in

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every case in which it is present in the cell. Integrated HBV-DNA is also found in tumors from patients who are HbsAg-, but HbsAb+. Free viral DNA is rarely found in HCC in addition to integrated HBVDNA. Both free and integrated viral DNAs are found in normal liver tissue adjacent to the tumor. While integration of HBV-DNA into the hepatocyte precedes the development of HCC by mo or yr, this does not prove that HBV-DNA is oncogenic. However, integrated viral DNA in animal cells often correlates with the ability of the viruses to transform the cells [98]. HBV-DNA was found to be stably integrated into 7 sites in DNA of the PLC/PRF/5 hepatoma cell line. The integration appears to be in a head-to-tail tandem arrangement and the defective molecules may be involved in the process of neoplastic transformation by HBV [99]. Two different hybridization techniques utilizing the PLC/PRF/5 cell line showed that the specificity of the DNA for HBV is represented in part by sequences coding for the surface and core antigens [100,101]. Shorter survival time is associated with high serum HBV-DNA concentrations [l02]. Novel genes have been differentially expressed in HBV DNA associated-HCC [103]. Some (DNT10, HA6T4) are preferentially underexpressed in cases with high AFP levels. The FOCUS (Friendship of China and United States) carcinoma cell line, grown continuously for 18 mo, contains integrated HBV sequences within its genome [104]. The integration of HBV-DNA is associated with a deletion of 13.5 kilobases of cellular sequences in HCC. The integration and deletion occurs on the short arm of chromosome 11 at location 11p13-11p14, the significance of which is unknown. However, Wilms tumor, hepatoblastoma, adrenal carcinoma, and rhabdomyosarcoma have been associated with loss of hetero-zygosity of DNA sequences in chromosome 11p. The deleted cellular sequences are lost from the tumor cells, leaving a single copy of the remaining cellular allele. A second event may be necessary for oncogenesis to be initiated from this locus [105]. Of 85 HCC patients, 27% were HbsAg+ and 21% were HbsAb+. Of the HbsAg+ cases, 83% had integrated HBV-DNA in tumor DNA whereas only 5% had integration in the HBV

antibody-positive patients. While there were no evident histological differences between HCCs with or without viral integration, the findings of high integration rate in HCCs of carrier patients and extremely low rate of viral integration in HCCs of non-carrier patients contrasts strongly with previous results from African and European cases[106]. HBV infection may manifest its oncogenic properties after a shorter incubation period (2 yr) than is generally believed [107]. Neither the type or differentiation of HCC nor the tumor localization of HBsAg or HBcAg correlate with HBVDNA nuclear staining. Random cellular localization of HBV-DNA sequences in HCC strengthens the epidemiologic association between HCC and infection and suggests that HBV-DNA may be incorporated, or perhaps replicated, unevenly in tumor cells [108]. HBV integration may be the cause or the consequence of structural alterations in the host genome. There was deletion and/or mutation of an 185 base-pair fragment of the core-polymerase overlap region of HBV in 8 cases of HCC [109]. There is no significant association between the integration of HBV and chromosomal losses or among the various allelic losses in HCC tumors involving chromosomes 5q, 10q, 11p, 16q and 17p [110]. Multiple sites of integration (eg, 1,7,14,17, 26) remain unchanged in a human cell line (Hep10) despite a long and selective derivation history from the original biopsy material. The cell line demonstrates multiple chromosome abnormalities (eg, pulverization, acentric fragments, breaks, minutes), some of which localize to the chromosomal sites involved in abnormalities in the tumor cell line and some in the same area of the genome in which abnormalties were found in other HCCs. In addition, there is evidence of similar multiple chromosome instabilities in cells derived from nonneoplastic tissue (ie, peripheral blood cultures). Thus, the evidence does not support continued disruptive recombination between integrated virions in cultured cells [111]. HBV markers, especially HbcAg associated with viral replication, are rarely found in neoplastic cells, whereas they can be detected in nontumorous cells surrounding the liver cancer. It is suggested that

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HBV replication becomes less active during hepatocarcinogenesis and is consistent with the fact that HBV-RNA (mostly the pregenome-core gene transcript) is typically observed in noncancerous liver tissue surrounding the cancer. In a study of 16 HCC patients, HBV-RNA was observed only in highly and moderately differentiated tissues that did not express alpha-fetoprotein or fetal insulin-like growth factor II mRNA [112]. In woodchuck HCC, the degree of differentiation and the expression of IGF-II and woodchuck hepatitis virus transcripts are inversely related. Tumors with IGF-II mRNA and low viral RNA levels are more anaplastic, while low IGF-II mRNA and high viral RNA levels are generally well differentiated [113]. Alpha-fetoprotein (AFP). In 269 HCC cases, 7690% had AFP levels >4ng/ml [114-117]. AFP positivity is associated with younger age, male gender, poorly differentiated tumors, and HbsAg positivity. AFP elevation does not correlate with disease stage, liver function tests, presence of concomitant cirrhosis, duration of symptoms prior to diagnosis, survival rate, or elevations of serum chorionic gonadotropin or serum proline hydroxylase [118]. Also, 9% of metastatic liver cancer and 28% of cholangiocarcinoma patients have AFP levels >20ng/ml [119,120]. HBs antigenemia is related to AFP elevation in acute hepatitis, cirrhosis, and chronic active hepatitis, but not in chronic persistent hepatitis or in healthy HbsAg carriers [121]. Simultaneous seropositivity (2-95%) for HbsAg and persistent or progressive serum (>25 ng/ml) and tissue AFP levels suggests HCC (2-72%) and, therefore, an etiologic relationship [122-125]. AFP production correlates with HbsAg in both serum and liver tissue, but does not correlate with HBV replication, the patients age, liver necrosis, or tumor histology [30,126,127]. A positive predictive value of 95% was observed in 54 HbsAg+ HCC patients with a serum AFP level of 3200 ng/ml. However, in 93 patients, HbsAg titers did not correlate with serum AFP levels [78,128]. Of Caucasians with HCC and HbsAg+ status, 80% have AFP levels >200 ng/ml. Very few (<2%) of 150 HCC patients without cirrhosis were AFP

and HbsAg+ [129]. The serum AFP level and the incidence of HbsAg+ is greater in Chinese than Caucasian HCC patients [130]. Of Chinese HbsAg+ HCC patients, 86.7% had AFP levels >20 ng/ml; of HbsAg- HCC Chinese patients, 66.7% had levels >20 ng/ml. Although more than half of Chinese HCC patients have AFP levels >320 ng/ml (95% >20 ng/ml),not all HCC patients have elevated AFP levels due to lower secretion. Serum AFP elevations begin 2yr prior to clinically evident HCC [131]. In Alaskan natives with HbsAg+ HCC, 95% have elevated levels of serum AFP. The increase of serum AFP can precede tumor development by 3 yr and has 90% sensitivity, 92% specificity, 7% positive predictive value, and 99% negative predictive value [140]. In patients with chronic hepatitis, serum AFP >100 ng/ml predicts the presence of HCC with a specificity of 99%, but the sensitivity is only 67%. The serum AFP levels correlate closely with the presence of bridging hepatic necrosis, but not with age, gender, or HbeAg/Ab status. Elevated levels in association with HBV may be related to the virus itself (reactivation), or to individual differences in virus-induced hepatic injury, or to events subsequent to the injury (molecular interaction between HBV and AFP). Transient elevation of serum AFP suggests hepatic injury; increasing or persistent elevation indicates HCC [133]. In patients with seroconversion from HBeAg to anti-Hbe, significant increases of AFP levels occurred as long as 2 and 6 yr before clinical onset of HCC [134,135]. Serum AFP levels also become markedly elevated in hepatitis virus-carrier woodchucks that develop HCCs. The elevation may occur 3 to 11 mo before advanced HCC [136]. HBV/HCV status should be evaluated when serum AFP is measured as an independent test to diagnose HCC. in the early diagnosis of HCC, regular AFP determinations may be more useful in HbsAg- patients with chronic liver disease than in HbsAg+ patients [137]. The best use of AFP screening may be to monitor HbsAg carriers after conversion to HbeAb positivity [134]. Still, almost 30% of HCC cases would be undetected at an early stage [138]. AFP monitoring for all HbsAg carriers is therefore recommended, although the best yield

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is obtained from those positive for anti-Hbe [134]. Rapid multisite high-affinity monoclonal antibody radioimmunoassay (M-RIA) has high specificity for AFP-producing tumors, and little overlap with nonmalignant disorders, probably due in part to the recognition of epitopes unique to AFP. The M-RIA is technically convenient, rapid, and 4- to 10-fold more sensitive than conventional polyvalent RIAs. Screening for early diagnosis of HCC and treatment monitoring in high-risk populations may be indicated with M-RIA [139]. The prevalences of elevated circulating immune complexes and elevated serum AFP were 89% and 77%, respectively, in 93 HbsAg positive patients with HCC. The levels of the former may be related to tumor mass and may be useful as markers for therapeutic monitoring following transcatheter arterial embolization in patients with HbsAGg+ HCC [140]. A well-differentiated human HCC cell-line that was transplanted in athymic (nude) mice produced AFP which increased exponentially, allowing quantitative evaluation of tumor growth and responsivity to chemotherapeutic agents [141]. The development of HCC may be facilitated by the increased serum AFP levels seen during hepatic parenchymal regeneration associated with viral hepatitis that may depress or alter the hosts immune system [142]. There is a rapid decrease in the synthesis and secretion of specific plasma proteins (AFP) at the level of mRNA in HSV-2 infection of hepatoma McA-RH7777 cells [143]. Serum AFP elevation significantly correlates with HCV disease in Chinese patients with HCC due to the presence of more advanced disease, older age than HCV-negative patients, and less frequent screening [144,145]. In Thailand elevations of serum AFP are found in 76%, 89%, 79%, and 80% of patients with HbsAg, HCVAb, HBV DNA, and HCV RNA, respectively [146]. The presence of circulating HCC cells as detected by a polymerase chain reaction-based technique is suggested by the presence of AFP mRNA in the serum of HCC or hepatitis patients. Early hematogenous spread of the cancer is supported by the high incidence of AFP mRNA in the blood of these patients, although its impact on

prognosis is unclear [147]. When the etiology of HCC is hepatitis C rather than hepatitis B infection, a raised serum AFP level is more common [144]. Of 48 HCV-positive HCC patients , 46% had serum AFP levels >200 ng/ml [148]. Sensitivity (66% vs 46%) and diagnostic accuracy (78% vs 69%) were higher in anti-HCV+ vs anti-HCV- patients, whereas the specificity (91%) was equivalent [149]. The HCV core gene may modulate the expression of the AFP gene independently from the albumin gene. Intracellular localization of this viral protein does not appear to be a determining factor for this modulation [150]. Serum AFP level >17.8 ng/ml strongly predicted the presence of cirrhosis in a population of 299 patients with chronic hepatitis C. The sensitivity, specificity and positive predictive value were 35%, 98.6%, and 97.7% respectively. Of those who developed HCC, 93% had an elevated serum AFP [151]. Elevated serum AFP levels occur in 15-58% of patients with chronic hepatitis and 11-47% of patients with cirrhosis [152]. There is no association between increasing serum AFP values and degree of hepatocyte proliferation, injury, immunochemical staining for AFP, or DNA synthesis. Altered hepatocyte-hepatocyte interaction and loss of normal architectural relationships appear to be responsible for the elevated AFP values in serum, rather than active regeneration or necrosis [153,154]. Transient but marked elevations of serum AFP (3000-13,500 ng/ml) may occur in cases of HbsAG+ and HbsAb+ cirrhosis/chronic hepatitis without HCC [155]. The rise of AFP does not correlate with serum aminotransferase activity, but correlates with the severity of liver tissue damage (loss of parenchymal gap junctions, fibrosis, and the presence of bridging necrosis) with or without regenerative potential [156]. The lectin-reactive patterns for serum AFP are similar in such cases and compatible with benign liver disorders [165]. Other markers. The heterogeneity of HCC is characterized by down-regulated and altered gene expression. G1/S transition, expression of cyclin D1 and p27, and p53 signaling and apoptosis regulation are involved in HBV-associated HCC; a more heterogeneous pattern with over-expression of

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distinct TGF-beta-induced gene typifies HCVrelated HCC [158,159]. The development of HCC from dysplastic nodules (DN) could be related to the presence of pRb in DN G1/S modulators. The TONG/PHC HCC line secretes HbsAg and alpha-1-antitrypsin [160]. The modal chromosome number is 70 and several HBV-DNA integration sites are present [160]. A 28-kD protein (p28) was detected in HCC tissue infected with HBV, suggesting an additional marker for infectivity status [161]. Of 83 HbsAg+HCC patients, 46-75% contain a multifunctional polymorphic trans-activator X gene of the HBV genome, the production of which may be related to HBV replication and development of HCC [162]. HBx occurs in 47% of HbsAgHCCs [163]. HBx antigen was observed in 50100% of tumorous and nontumorous liver tissue in patients with HBV, HCV, and in HCC arising in patients without evidence of either HBV or HCV [164]. Serum anti-X level increases with the length of chronic HBV infection and may suppress the expression of the X protein in the liver [165]. HBxpositive cells are preferentially localized in the periportal region (chronic hepatitis) or nodules (cirrhosis) where high necroinflammatory activity occurs [94]. HBx-A31, a novel mutant of HBx prevalent in Taiwanese HCC patients, is less effective in supporting HBV replication, less potent in enhancing TNF-alpha induced increment of CPP32/caspase 3 activities in Hep G2 cells, and less efficient in transactivating the HBV enhancer I-X promoter complex [166]. The HBx-COOH terminus may enhance the transforming ability of myc and ras, resulting in lost transcriptional activity, cellular transformation, and proliferation [167]. X protein of HBV alone (decreased transcriptional and co-transactivation of the NF-kappa Bdriven luciferase reporter) or binding to p53, which is a potent pro-apoptotic transactivator, may interfere with tumor suppression activity [168-171]. P53 mutations are present in 25-45% of HCCs and are usually associated with a specific G to T transverse mutation in codon 249 with high exposure to aflatoxin B1 [57,58,172]. Of 77 HCCs, 35% contained activated c-myc by DNA

amplification which occurs more frequently in HBV than HCV infections [173]. A 3-16 fold amplification of cyclin D1 gene was present in 11% of HCCs (n=45) associated with HBV or HCV infection [174]. HCV, by comparison, is a nonintegrating virus whose core protein has potential in vitro direct carcinogenic effects, induces HCCs in transgenic mice, and appears to promote cell growth through repression of the transcriptional activity of p53 [175,176]. Up-regulation of p21 mRNA in HCV hepatitis may protect hepatocytes from tumorgeneicity [177]. There is high frequency of HBV genome mutations at nucleotides 1762 (A to T) and 1764 (G to A) in the core promoter region of HCC patients [178]. Common amino acid substitutions in HCC isolates were located in X, core, S, pre-S1, pre-S2 and polymerase proteins. Pre-S1 and pre-S2 HBV proteins are expressed by some HCC [179]. The expression of pre-S1 protein, an essential component of HBV, may be suppressed in HCC [180]. Integration and subsequent HCC development may be facilitated by precore/core HBV mutants (nt 1896) [181]. A proteosomal subunit may disrupt the Rb pathway by Rb degradation and methylation-dependent silencing of the p16/NK4A gene [182]. Sixty percent of HCCs have reduced expression of E-cadherin due to methylation at CpG sites adjacent to the promoter region and allelic deletions of the E-cadherin gene itself [183]. Moreover, 1940% of HCCs contain heterogeneous beta-catenin (BC) and axin gene mutations [184,185]. BC was mutated in 41% of 22 HCCs associated with HCV infection. BC is a submembrane component of the cadherin-mediated cell-cell adhesion system that serves as a downstream transcriptional activator of the Wnt signaling pathway by forming complexes with DNA-binding proteins (Tcf and Lef-1) [186]. Gene mutation (exon 3 phosphorylation and ubiquitination) is closely associated with nuclear (17% of HCCs) and cytoplasmic (62% of HCCs) accumulation. The latter are associated with larger tumors, poorer differentiation, and shorter survival [185]. Seventy percent of the mutations were located at specific serine/threonine residues (codons 33,37,41, and 45) of exon 3. These data suggest

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that the development of some HCCs may be polyclonal. Intrahepatic metastases may occur and/ or different mutations may be due to genetic heterogeniety within the tumor [187]. In 566 cases, BC mutation was associated with a favorable prognosis in low stage HCC [188]. In a study of 28 cases of fulminant hepatitis, improved survival rates were significantly associated with serum AFP levels >60 ng/ml (p <0.005) and negative DNA polymerase activity (p <0.05). HbsAg and HbeAg status were not correlated with prognosis [189]. Hepatoma-specific alkaline phosphatase was sometimes elevated in Causcasian HCC patients (18%), but rarely in Chinese patients (2%) [190]. Higher levels of serum alpha-1-antitrypsin were originally found in HbsAg- HCC cases [191], but later work indicated that the levels are unrelated to HbsAg status [192]. Ferritin is detected in 75% of normal liver tissue and 40% of HCC tissue. Stainable iron is demonstrated in 65% of unaffected livers and 10% of HCC tissues, indicating that iron accumulates in cells replicating HBV. Ferritin may be produced by the tumor cells and is not due to increased stainable iron [193,194]. Copper was detected in 60% of 20 human cirrhosis/HCC cases that were HbsAg and HbcAg positive[195]. The Tat protein of HIV enhanced chemical hepatocarcinogenesis in tat-transgenic mice and may contribute to tumorigenesis in HIV infected patients [196]. In summary, HBV is likely carcinogenic through a number of cell cycle regulatory pathways. The incidence of many HCCs could be decreased by the prevention of HBV infections. References
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