39

Address correspondence to Richard B. Birrer, M.D., St Joseph’sRegional Medical Center, 703 Main Street, Patterson, NJ 07503,USA; tel 973 754 4366; fax 973 754 2044; e-mail birrerr@sjhmc.org.

Review: Hepatocellular Carcinoma and Hepatitis Virus

Richard B. Birrer,1 Danielle Birrer,1 and John V. Klavins 21 St. Joseph’s Regional Medical Center, Paterson, New Jersey, and2 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) andthe transcriptome profile are deregulated or altered in most HCC patients. The HBx protein, potentiallyoncogenic via multistep carcinogenesis, modifies apoptosis, inhibits nucleotide excision and repair of damagedcellular DNA, and modulates transcriptional activation of cellular growth regulating genes. Hepatocytetransformation may be indirectly influenced by HBV DNA integration, by the generation of mutagenicoxygen reactive species, or by acquisition of mutations in association with necroinflammatory disease. HBVreplication, which may occur in HCC, affects the long-term survival of patients. Prevention of HBV infectionis expected to decrease the incidence of endemic HCC. (received 24 April 2002, accepted 25 August 2002)

Keywords: hepatitis B virus, hepatocellular carcinoma

Introduction

About 80% of human hepatocellular carcinomas(HCC) are attributable to chronic hepatitis B virus(HBV) infection [1-3]. Chronic HBV carriers are100-400 times more likely to develop liver cancerthan noncarriers [4,5]. Approximately 20% of HCCarises de novo in otherwise healthy liver [6,7]. Suchpatients, compared to those with a background ofcirrhosis, are younger (mean 36 vs 57 yr), more likelyto be female (56 vs 98%), more frequently sympto-matic (nausea, vomiting, mass), and have longerduration of symptoms ( 9 vs 4 mo), less commonoccurrence of HBV markers ( 26 vs 72%), less likelyabnormalities of liver function tests, less frequentlyelevated serum alpha-fetoprotein (AFP, 46 vs 87%),higher resectability (10 vs 1%), increased respon-sivity to chemotherapy (30 vs 10%), and better one

yr survival rate (55 vs 8%). A vaccination programin infants and children can significantly reduce(>80%) the chronic carrier state and the long termpotential for HCC [8-10].

Epidemiology

Worldwide, 4% of all malignant tumors are HCC.HCC is the seventh most frequent cancer in malesand 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 HCCare HbsAg+. Antigenemia is unassociated with thepresence or absence of cirrhosis, but is more commonin younger patients [12].

An early study noted that HbsAg was seldomencountered in the Romanian area and that it wasnot involved in oncogenesis [13]. However, asubsequent case analysis found that 24% ofRomanian patients with HCC were HbsAg+ [14].In a 21 yr retrospective study in Greenland, HCCincidence was 5% of the HbsAg+ rate, indicating

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

Annals of Clinical & Laboratory Science, vol. 33, no. 1, 2003

40

that protective factors may be present or othercarcinogenic factors may be absent from theenvironment [15].

In nonendemic areas of the world, HBV has aprobable etiological role in HCC [16]. Of 32 HCCpatients of French origin, 3% were HbsAg+, whereas70% had elevated serum AFP [17]. In a study of333 Greek HCC patients, 58% were attributed toHBV, 12% to HCV, and 3% to dual infection [18].

In Asian and African countries, by contrast,hepatitis antigens have a statistically significantassociation with HCC [17]. The incidences of HCCand HBV infection rates were positively correlatedin a study of two nearby Chinese villages, one witha high rate (329/100,000 persons) and the otherwith a zero incidence rate of HCC [19]. Orientalpatients tend to be HbsAg+ compared to blacks andwhites (p <0.001) [20,21]. HCV and HbsAgprevalences are 33% and 68% in Chinese HCCpatients [22,23].

In a large prospective study, the HBV carrierstate in Taiwanese patients had >200-fold HCC risk,compared to non-carriers [24]. In 112 Koreanpatients with HCC, all gave positive tests for HBVinfection (97% positivity for HbsAg, 38% forHBeAg, 83% for AFP) [25]. Strong correlationbetween the prevalence of HbsAg carrier state (8-12%) 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 bloodtransfusion was noted in 40% of these patients [27].Higher incidence of HCC in Nagasaki vs Hiroshimais attributable to HBV infection, but other factors(ie, radiation-induced immunoincompetence)cannot be excluded [28]. After adjustment fortobacco usage, a strongly positive dose-responserelationship in HbsAg+ Japanese males was foundbetween drinking habits and HCC. Tobacco usagecorrelated with HCC occurrence, but no dose-response relationship was observed. Tobacco andethanol usage, therefore, may promote hepato-carcinogenesis in HbsAg+ patients [29].

The HbsAg carrier rate in HCC patients fromZaire is 57% [30]. HbsAg and HbcAg are morefrequent in non-neoplastic hepatocytes (53% and

23%, respectively) compared to HCC cells (13%and 3%) [31].

Risk factors for HCC include a history ofcirrhosis, transfusions, major surgery, HBV infection(Southeast Asia and sub-Saharan Africa), HCVinfection (Europe and Japan), long history of chronichepatitis, aflatoxin B1 exposure, excessive alcoholintake, prolonged HBV replication phase, andBudd-Chiari disease [32,33]. Chronic HBVinfection is associated with HCC in certain settings,depending on the sex of the carrier, immunologicstatus, size of innoculum, presence of wild-typeHBV, absence of HbeAb, severity of primaryinfection, age at primary infection, and geneticfactors [34,35]. The minimum relative risk for HCCamong HbsAg+ healthy blood donors is 12.7 [36].A genotype, Gt-1, is the most prevalent in HCCand may be an important independent risk factorfor the cancer [37]. There is 1-4% annual risk ofdeveloping HCC in cirrhotic patients with chronichepatitis C [38].

Pathology

In 165 HCC cases, 7-27% of liver tissue sampleswere HbsAg+. HbsAg positivity was related topreneoplastic hepatic changes. While the presenceof HbsAg in liver tissue was not uniform, in 75% ofcases dysplastic changes were observed in cellscontaining HbsAg [39]. In 223 autopsy cases ofHCC, there was good correlation between thesemiquantitative grade of dysplasia and the incidenceof HbsAg positivity [40]. Liver cell dysplasia isindirectly related to HbsAg, with no evidence ofpremalignancy [41].

HbsAg was frequently found in the cellularparenchyma (replacing and sinusoidal types) ofHCC cases. Hepatocytes were frequently retainedin the cancerous tissue, especially around the tumor-nontumor border. No HbsAg+ cells were seen inthe encapsulated type of HCC [42]. In a study of60 cases of HCC in Japanese patients, HbsAg wasfound mainly in the cytoplasm and HbcAg waspredominantly in the nuclei of normal-appearing,dysplastic, and tumor cells. Occasionally, bothHbsAg and HbcAg were present in normal andtumor cells [43].

Annals of Clinical & Laboratory Science

41

Etiopathogenesis

Viral hepatitis may progress to HCC through anintermediate postnecrotic cirrhotic stage [44]. HBVvirus may play an indirect role in hepatic oncogenesis[45]. The oncogenetic capacity of HBV is supportedby the finding of integrated HBV DNA in manyHCCs in which viral sequences are present, despitethe production of antibodies to HbsAg [46-48].Almost all cases of endemic HCC are associated withHBV. HBV DNA integration may occur long beforethe development of HCC and should be the targetof therapy [49]. Free, random and clonal integratedHBV DNA was detected in 83% of neoplastic and100% of nonneoplastic portions of the liver fromHCC cases. There was no HBV DNA in HCC casesnot associated with HBV markers [50].

HBV may synthesize reverse transcriptase fromc-pol fusion proteins in a manner similar to the wayretroviruses synthesize and process a precursor. Theaccumulation of intermediates of HBV reversetranscriptase in cancerous tissue and not other tissuesmay reflect the absence of viral core particles andpossibly lead to cellular transformation [51].

Liver cell injury during HBV infection may bea result of coinfection with a second cytopathic virus,namely, the delta agent. Persistent HBV infectionsin HCC patients are long-lasting, as suggested bylow serum HbsAg titers, rare occurrence of HBcAg,and absence of HbeAg in most HCC patients [52].Of male patients with chronic hepatitis B, 50% hadHCC with HbeAg detected in peripheral lympho-cytes, but they were seronegative for HbeAg. Hepaticoncogenesis may proceed from impaired immunemechanisms from HBV replication and productionin host lymphocytes [53]. The etiopathogenesis ofHCC may occur through death of susceptible livercells from HBV infection followed by integrationof HBV DNA in resistant hepatic cells [54].

The association rate of HCC is very high inHbsAg+ alcoholic (65%) and non-alcoholic (68%)patients. The rate is low (23%) in HbsAg- alcoholicpatients. Concomitant HBV infection in the settingof alcoholism has a major effect on the developmentof macronodular cirrhosis and HCC [55]. HBV-DNA integration is unrelated to the history ofalcohol intake. Furthermore, 100% of HbsAg-, non-

alcoholic HCC patients do not have HBV-DNAintegration detectable. HBV, therefore, does notappear to have a major role in the pathogenesis ofHCC in these patients [56].

There is growing evidence that HBV may actsynergistically with aflatoxin B1 (G to T transversionat codon 249) to induce HCC [57,58]. Of 41 treeshrews exposed to aflatoxin B1, 17 wereexperimentally infected with human HBV, and 53%developed primary liver cancer, compared to only 2of the controls (p <0.05) [59].

Markers

Hepatitis virus. An early study found that HbsAgwas present in 4.5% of HCC patients (n=22) [60].Subsequently, 14-64% of HCC cases (n=652) werenoted to be HbsAg+, but the positivity for HbcAband HbsAb was 4-89% [61-64]. The former wasoften associated with inactive cirrhosis, characterizedby minimal lymphocytic infiltration in the stroma.In HbsAg- HCC cases, active cirrhosis, as noted bysignificant lymphocytic infiltration, was observed.An altered immune mechanism was stronglysuggested [65-67].

A history of liver disease (cirrhosis, hepatitis)and positivity 90-93% for HBV markers wasassociated with both subclinical and clinical typesof HCC (n=138). The positive rate for HbsAb didnot correlate, being significantly lower in HCC andcirrhosis groups, compared to controls, suggestingan immune deficiency [68,69]. In antigen negativepatients, HBV and HCC were shown by C1q andconglutinin solid phase assays to be associated withthe identification of HbsAg- containing immunecomplexes [70].

The incidences of HbsAg, HbcAb and ‘e’antigen antibody were significantly increased inHCC (p <0.005) in patients with a history ofcirrhosis [71]. However, HCC in an asymptomatic,non-cirrhotic, 16 year-old male was reported whohad positive serum markers for HbsAg, HbeAg andHbcAb [72]. While the exposure rate to HBV forcirrhotic HCC patients was 90%, only 29% wereHbsAg+. HbsAb prevalence was 10% for the samegroup; 39% had serological evidence of continuousviral replication (isolated high titer HbcAb+ or

Hepatocellular carcinoma and hepatitis virus

42

HbsAg+), perhaps due to a defective clearancemechanism [73]. HbcAg positivity was morecommon in patients with cirrhosis (58%) than inHCC patients and it correlated with serologicalfeatures of high-level viral replication. There wereno gender differences in positivity for HCC orcirrhotic cases [74,75]. No specific intrahepaticdisplay pattern of HBV markers was identified inHCC. Tumor cells did not usually support thesynthesis of HbcAg or HbsAg. While 41% of livercirrhosis cases (n=144) were positive for HBsAg,HBsAb was present in 33%, HBeAg in 35%, HBeAbin 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 HBeAbwas 51% [76]. Of HCC patients (n=494),50-80%were positive for serum HBsAg, with positivity ratesfor uninvolved tissue and HCC tissue of 83% and35%, respectively (p <0.001). HBcAg was found in25% of liver tissue and 8% of HCC tissue (p <0.05).

The interval from HBV infection until clinicalrecognition of HCC may be as short as 9.5 yr, withseroconversion from HbeAg to HbeAb occurring aslong as 4 yr prior to clinical presentation [77]. Thereis a tendency of HBsAg to increase during the courseof HCC, often coincident with rapid tumor growth[78]. In a study of 391 blacks with HCC, there wassignificant association (p <0.001) between HCC andHBV infection and age, with 82% HBV positiveand 36% HBe positive in persons ≤30 yr old, vs30% and 11% respectively for those ≥50 yr old [79].The prevalence of HBeAg in patients with bothcirrhosis and HCC was significantly lower (p<0.0005) and did not correlate with age [80].

HbsAg, but not HbcAg or HBeAg, was foundin cytoplasm of some tumor cells of a well-differentiated human HCC cell-line transplanted inathymic (nude) mice [81]. Of these, 9% had HbcAgpresent in tumor cell cytoplasm [75]. HbsAgpositivity was observed predominantly in normalcells whereas HbcAg occurred in 7% of HCC cases,mostly in dysplastic cells.

Active replication of HBV in tumor cellsbecomes increasingly defective during the course ofmalignant degeneration, as evidenced by a lowincidence of HbsAg and complete absence of HbcAgpositive tumor cells [82]. HbsAg was present in 63-

93% of nontumor liver tissue and 14-50% of tumortissue samples (n=174). HbcAg was present in 46-53% of nontumor tissue samples, vs 0-9% of thetumor specimens, mostly in non-neoplastic hepato-cytes occurring in different intracellular distributionpatterns [83,84].

Of 29 HCC patients, 52% had microsatellitealterations (MSA) implying that MSA are unstablein genomic DNA of HBV-infected HCC [85]. MSAoccurred frequently with AFP elevation, but theywere not associated with the patients’ gender, age,tumor size, or differentiation. Of sera derived fromHBV positive hepatoma cell lines (PLC/PRF/5, Hep3B), 72% demonstrated voluminous intracyto-plasmic fluorescent inclusions. A 18kD protein wasidentified in this group, but it is unclear whether itwas a cellular protein or a product of the HBVgenome [86]. Only 4% of sera samples derived fromHCC patients were positive for serum HBV-DNA.An HBV-associated nuclear antigen, positive in 7-8% of HbsAg+ HCC cases, is expressed at an earlystage of in vitro culture (PLC/PRF/5), prior to thepeak of HbsAg expression [87].

Of 63 HCC tissue specimens, 5% were positiveonly in the surrounding cirrhotic liver; HbcAg anddelta were absent [88]. Of 95 HbsAg carriers withinflammatory liver disease and HCC, 6% werefound to be HDAg+ [89]. Of 155 histologicallyconfirmed HCC cases, 1.2% were HbsAg+ and 11%were HCV+ [90]. In 68 HCC patients, thepositivity rates for HCV-Ab and HbsAg were 84%and 69%, respectively. Both antibodies were presentin 34% of cases [91]. Of 42 non-B non-C HCCpatients, 47.6% contain HBV-DNA in non-neoplastic tissue [92-94]. The occult integrant maybe important in the development of HCC-HCVpatients that lack hepatic fibrosis [95]. The bindingis facilitated by an active enhancer-I, likelyfetoprotein transcription factor [96]. Within the coredomain of HBV enhancer 1 is a STAT-3 bindingsite (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 isenhanced function and viral gene expression.

The integration of HBV-DNA in HCC tissueof HbsAg carriers occurs into the host genome in

Annals of Clinical & Laboratory Science

43

every case in which it is present in the cell. IntegratedHBV-DNA is also found in tumors from patientswho are HbsAg-, but HbsAb+. Free viral DNA israrely found in HCC in addition to integrated HBV-DNA. Both free and integrated viral DNAs arefound in normal liver tissue adjacent to the tumor.

While integration of HBV-DNA into thehepatocyte precedes the development of HCC bymo or yr, this does not prove that HBV-DNA isoncogenic. However, integrated viral DNA inanimal cells often correlates with the ability of theviruses to transform the cells [98]. HBV-DNA wasfound to be stably integrated into 7 sites in DNA ofthe PLC/PRF/5 hepatoma cell line. The integrationappears to be in a head-to-tail tandem arrangementand the defective molecules may be involved in theprocess of neoplastic transformation by HBV [99].Two different hybridization techniques utilizing thePLC/PRF/5 cell line showed that the specificity ofthe DNA for HBV is represented in part bysequences coding for the surface and core antigens[100,101]. Shorter survival time is associated withhigh serum HBV-DNA concentrations [l02]. Novelgenes have been differentially expressed in HBVDNA associated-HCC [103]. Some (DNT10,HA6T4) are preferentially underexpressed in caseswith high AFP levels.

The FOCUS (Friendship of China and UnitedStates) carcinoma cell line, grown continuously for18 mo, contains integrated HBV sequences withinits genome [104]. The integration of HBV-DNA isassociated with a deletion of ≥13.5 kilobases ofcellular sequences in HCC. The integration anddeletion occurs on the short arm of chromosome11 at location 11p13-11p14, the significance ofwhich is unknown. However, Wilms’ tumor,hepatoblastoma, adrenal carcinoma, and rhabdo-myosarcoma have been associated with loss ofhetero-zygosity of DNA sequences in chromosome11p. The deleted cellular sequences are lost fromthe tumor cells, leaving a single copy of theremaining cellular allele. A second event may benecessary for oncogenesis to be initiated from thislocus [105]. Of 85 HCC patients, 27% wereHbsAg+ and 21% were HbsAb+. Of the HbsAg+cases, 83% had integrated HBV-DNA in tumorDNA whereas only 5% had integration in the HBV

antibody-positive patients. While there were noevident histological differences between HCCs withor without viral integration, the findings of highintegration rate in HCCs of carrier patients andextremely low rate of viral integration in HCCs ofnon-carrier patients contrasts strongly with previousresults from African and European cases[106].

HBV infection may manifest its oncogenicproperties after a shorter incubation period (2 yr)than is generally believed [107]. Neither the typeor differentiation of HCC nor the tumor local-ization of HBsAg or HBcAg correlate with HBV-DNA nuclear staining. Random cellular localizationof HBV-DNA sequences in HCC strengthens theepidemiologic association between HCC andinfection and suggests that HBV-DNA may beincorporated, or perhaps replicated, unevenly intumor cells [108].

HBV integration may be the cause or theconsequence of structural alterations in the hostgenome. There was deletion and/or mutation of an185 base-pair fragment of the core-polymeraseoverlap region of HBV in 8 cases of HCC [109].There is no significant association between theintegration of HBV and chromosomal losses oramong the various allelic losses in HCC tumorsinvolving 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 fromthe original biopsy material. The cell linedemonstrates multiple chromosome abnormalities(eg, pulverization, acentric fragments, breaks,minutes), some of which localize to the chromo-somal sites involved in abnormalities in the tumorcell line and some in the same area of the genome inwhich abnormalties were found in other HCCs. Inaddition, there is evidence of similar multiplechromosome instabilities in cells derived from non-neoplastic tissue (ie, peripheral blood cultures).Thus, the evidence does not support continueddisruptive recombination between integrated virionsin cultured cells [111].

HBV markers, especially HbcAg associated withviral replication, are rarely found in neoplastic cells,whereas they can be detected in nontumorous cellssurrounding the liver cancer. It is suggested that

Hepatocellular carcinoma and hepatitis virus

44

HBV replication becomes less active duringhepatocarcinogenesis and is consistent with the factthat HBV-RNA (mostly the pregenome-core genetranscript) is typically observed in noncancerous livertissue surrounding the cancer. In a study of 16 HCCpatients, HBV-RNA was observed only in highlyand moderately differentiated tissues that did notexpress alpha-fetoprotein or fetal insulin-like growthfactor II mRNA [112].

In woodchuck HCC, the degree of differen-tiation and the expression of IGF-II and woodchuckhepatitis virus transcripts are inversely related.Tumors with IGF-II mRNA and low viral RNAlevels are more anaplastic, while low IGF-II mRNAand high viral RNA levels are generally well differ-entiated [113].

Alpha-fetoprotein (AFP). In 269 HCC cases, 76-90% had AFP levels >4ng/ml [114-117]. AFPpositivity is associated with younger age, malegender, poorly differentiated tumors, and HbsAgpositivity. AFP elevation does not correlate withdisease stage, liver function tests, presence ofconcomitant cirrhosis, duration of symptoms priorto diagnosis, survival rate, or elevations of serumchorionic gonadotropin or serum prolinehydroxylase [118]. Also, 9% of metastatic livercancer and 28% of cholangiocarcinoma patientshave AFP levels >20ng/ml [119,120]. HBsantigenemia is related to AFP elevation in acutehepatitis, cirrhosis, and chronic active hepatitis, butnot in chronic persistent hepatitis or in healthyHbsAg carriers [121]. Simultaneous seropositivity(2-95%) for HbsAg and persistent or progressiveserum (>25 ng/ml) and tissue AFP levels suggestsHCC (2-72%) and, therefore, an etiologic relation-ship [122-125]. AFP production correlates withHbsAg in both serum and liver tissue, but does notcorrelate with HBV replication, the patient’s age,liver necrosis, or tumor histology [30,126,127]. Apositive predictive value of 95% was observed in 54HbsAg+ HCC patients with a serum AFP level of3200 ng/ml. However, in 93 patients, HbsAg titersdid 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 theincidence of HbsAg+ is greater in Chinese thanCaucasian HCC patients [130]. Of ChineseHbsAg+ HCC patients, 86.7% had AFP levels >20ng/ml; of HbsAg- HCC Chinese patients, 66.7%had levels >20 ng/ml. Although more than half ofChinese HCC patients have AFP levels >320 ng/ml(95% >20 ng/ml),not all HCC patients haveelevated AFP levels due to lower secretion.

Serum AFP elevations begin ≥ 2yr prior toclinically evident HCC [131]. In Alaskan nativeswith HbsAg+ HCC, 95% have elevated levels ofserum AFP. The increase of serum AFP can precedetumor development by 3 yr and has 90% sensitivity,92% specificity, 7% positive predictive value, and99% negative predictive value [140]. In patientswith chronic hepatitis, serum AFP >100 ng/mlpredicts the presence of HCC with a specificity of99%, but the sensitivity is only 67%.

The serum AFP levels correlate closely with thepresence of bridging hepatic necrosis, but not withage, gender, or HbeAg/Ab status. Elevated levels inassociation with HBV may be related to the virusitself (reactivation), or to individual differences invirus-induced hepatic injury, or to events subsequentto the injury (molecular interaction between HBVand AFP). Transient elevation of serum AFP suggestshepatic injury; increasing or persistent elevationindicates HCC [133]. In patients with sero-conversion from HBeAg to anti-Hbe, significantincreases of AFP levels occurred as long as 2 and 6yr before clinical onset of HCC [134,135]. SerumAFP levels also become markedly elevated inhepatitis virus-carrier woodchucks that developHCCs. The elevation may occur 3 to 11 mo beforeadvanced HCC [136].

HBV/HCV status should be evaluated whenserum AFP is measured as an independent test todiagnose HCC. in the early diagnosis of HCC,regular AFP determinations may be more useful inHbsAg- patients with chronic liver disease than inHbsAg+ patients [137]. The best use of AFPscreening may be to monitor HbsAg carriers afterconversion to HbeAb positivity [134]. Still, almost30% of HCC cases would be undetected at an earlystage [138]. AFP monitoring for all HbsAg carriersis therefore recommended, although the best yield

Annals of Clinical & Laboratory Science

45

is obtained from those positive for anti-Hbe [134].Rapid multisite high-affinity monoclonal antibodyradioimmunoassay (M-RIA) has high specificity forAFP-producing tumors, and little overlap withnonmalignant disorders, probably due in part to therecognition of epitopes unique to AFP. The M-RIAis technically convenient, rapid, and 4- to 10-foldmore sensitive than conventional polyvalent RIAs.Screening for early diagnosis of HCC and treatmentmonitoring in high-risk populations may beindicated with M-RIA [139].

The prevalences of elevated circulating immunecomplexes and elevated serum AFP were 89% and77%, respectively, in 93 HbsAg positive patientswith HCC. The levels of the former may be relatedto tumor mass and may be useful as markers fortherapeutic monitoring following transcatheterarterial embolization in patients with HbsAGg+HCC [140].

A well-differentiated human HCC cell-line thatwas transplanted in athymic (nude) mice producedAFP which increased exponentially, allowing quant-itative evaluation of tumor growth and responsivityto chemotherapeutic agents [141]. The develop-ment of HCC may be facilitated by the increasedserum AFP levels seen during hepatic parenchymalregeneration associated with viral hepatitis that maydepress or alter the host’s immune system [142].There is a rapid decrease in the synthesis andsecretion of specific plasma proteins (AFP) at thelevel of mRNA in HSV-2 infection of hepatomaMcA-RH7777 cells [143].

Serum AFP elevation significantly correlateswith HCV disease in Chinese patients with HCCdue to the presence of more advanced disease, olderage than HCV-negative patients, and less frequentscreening [144,145]. In Thailand elevations ofserum AFP are found in 76%, 89%, 79%, and 80%of patients with HbsAg, HCVAb, HBV DNA, andHCV RNA, respectively [146].

The presence of circulating HCC cells asdetected by a polymerase chain reaction-basedtechnique is suggested by the presence of AFPmRNA in the serum of HCC or hepatitis patients.Early hematogenous spread of the cancer issupported by the high incidence of AFP mRNA inthe blood of these patients, although its impact on

prognosis is unclear [147]. When the etiology ofHCC 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 serumAFP levels >200 ng/ml [148]. Sensitivity (66% vs46%) and diagnostic accuracy (78% vs 69%) werehigher in anti-HCV+ vs anti-HCV- patients,whereas the specificity (91%) was equivalent [149].The HCV core gene may modulate the expressionof the AFP gene independently from the albumingene. Intracellular localization of this viral proteindoes not appear to be a determining factor for thismodulation [150].

Serum AFP level >17.8 ng/ml strongly predictedthe presence of cirrhosis in a population of 299patients with chronic hepatitis C. The sensitivity,specificity and positive predictive value were 35%,98.6%, and 97.7% respectively. Of those whodeveloped HCC, 93% had an elevated serum AFP[151]. Elevated serum AFP levels occur in 15-58%of patients with chronic hepatitis and 11-47% ofpatients with cirrhosis [152]. There is no associationbetween increasing serum AFP values and degree ofhepatocyte proliferation, injury, immunochemicalstaining for AFP, or DNA synthesis. Alteredhepatocyte-hepatocyte interaction and loss of normalarchitectural relationships appear to be responsiblefor the elevated AFP values in serum, rather thanactive 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 withoutHCC [155]. The rise of AFP does not correlatewith serum aminotransferase activity, but correlateswith the severity of liver tissue damage (loss ofparenchymal gap junctions, fibrosis, and thepresence of bridging necrosis) with or withoutregenerative potential [156]. The lectin-reactivepatterns for serum AFP are similar in such cases andcompatible with benign liver disorders [165].

Other markers. The heterogeneity of HCC ischaracterized by down-regulated and altered geneexpression. G1/S transition, expression of cyclin D1and p27, and p53 signaling and apoptosis regulationare involved in HBV-associated HCC; a moreheterogeneous pattern with over-expression of

Hepatocellular carcinoma and hepatitis virus

46

distinct TGF-beta-induced gene typifies HCV-related HCC [158,159]. The development of HCCfrom dysplastic nodules (DN) could be related tothe presence of pRb in DN G1/S modulators.

The TONG/PHC HCC line secretes HbsAgand alpha-1-antitrypsin [160]. The modal chromo-some number is 70 and several HBV-DNA integ-ration 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% containa multifunctional polymorphic trans-activator Xgene of the HBV genome, the production of whichmay be related to HBV replication and developmentof HCC [162]. HBx occurs in 47% of HbsAg-HCCs [163]. HBx antigen was observed in 50-100% of tumorous and nontumorous liver tissue inpatients with HBV, HCV, and in HCC arising inpatients without evidence of either HBV or HCV[164]. Serum anti-X level increases with the lengthof chronic HBV infection and may suppress theexpression of the X protein in the liver [165]. HBx-positive cells are preferentially localized in theperiportal region (chronic hepatitis) or nodules(cirrhosis) where high necroinflammatory activityoccurs [94].

HBx-A31, a novel mutant of HBx prevalent inTaiwanese HCC patients, is less effective in sup-porting HBV replication, less potent in enhancingTNF-alpha induced increment of CPP32/caspase 3activities in Hep G2 cells, and less efficient intransactivating the HBV enhancer I-X promotercomplex [166]. The HBx-COOH terminus mayenhance the transforming ability of myc and ras,resulting in lost transcriptional activity, cellulartransformation, and proliferation [167].

X protein of HBV alone (decreased transcript-ional and co-transactivation of the NF-kappa B-driven luciferase reporter) or binding to p53, whichis a potent pro-apoptotic transactivator, mayinterfere with tumor suppression activity [168-171].P53 mutations are present in 25-45% of HCCs andare usually associated with a specific G to Ttransverse mutation in codon 249 with highexposure to aflatoxin B1 [57,58,172]. Of 77 HCCs,35% contained activated c-myc by DNA

amplification which occurs more frequently in HBVthan HCV infections [173]. A 3-16 fold amplifi-cation of cyclin D1 gene was present in 11% ofHCCs (n=45) associated with HBV or HCVinfection [174]. HCV, by comparison, is a noninteg-rating virus whose core protein has potential in vitrodirect carcinogenic effects, induces HCCs intransgenic mice, and appears to promote cell growththrough repression of the transcriptional activity ofp53 [175,176]. Up-regulation of p21 mRNA inHCV hepatitis may protect hepatocytes fromtumorgeneicity [177].

There is high frequency of HBV genomemutations at nucleotides 1762 (A to T) and 1764(G to A) in the core promoter region of HCCpatients [178]. Common amino acid substitutionsin HCC isolates were located in X, core, S, pre-S1,pre-S2 and polymerase proteins. Pre-S1 and pre-S2HBV proteins are expressed by some HCC [179].The expression of pre-S1 protein, an essentialcomponent of HBV, may be suppressed in HCC[180]. Integration and subsequent HCC develop-ment may be facilitated by precore/core HBVmutants (nt 1896) [181]. A proteosomal subunitmay disrupt the Rb pathway by Rb degradation andmethylation-dependent silencing of the p16/NK4Agene [182].

Sixty percent of HCCs have reduced expressionof E-cadherin due to methylation at CpG sitesadjacent to the promoter region and allelic deletionsof the E-cadherin gene itself [183]. Moreover, 19-40% of HCCs contain heterogeneous beta-catenin(BC) and axin gene mutations [184,185]. BC wasmutated in 41% of 22 HCCs associated with HCVinfection. BC is a submembrane component of thecadherin-mediated cell-cell adhesion system thatserves as a downstream transcriptional activator ofthe Wnt signaling pathway by forming complexeswith DNA-binding proteins (Tcf and Lef-1) [186].Gene mutation (exon 3 phosphorylation andubiquitination) is closely associated with nuclear(17% of HCCs) and cytoplasmic (62% of HCCs)accumulation. The latter are associated with largertumors, poorer differentiation, and shorter survival[185]. Seventy percent of the mutations were locatedat specific serine/threonine residues (codons33,37,41, and 45) of exon 3. These data suggest

Annals of Clinical & Laboratory Science

47

that the development of some HCCs may bepolyclonal. Intrahepatic metastases may occur and/or different mutations may be due to geneticheterogeniety within the tumor [187]. In 566 cases,BC mutation was associated with a favorableprognosis in low stage HCC [188].

In a study of 28 cases of fulminant hepatitis,improved survival rates were significantly associatedwith serum AFP levels >60 ng/ml (p <0.005) andnegative DNA polymerase activity (p <0.05). HbsAgand HbeAg status were not correlated with prognosis[189]. Hepatoma-specific alkaline phosphatase wassometimes elevated in Causcasian HCC patients(18%), but rarely in Chinese patients (2%) [190].

Higher levels of serum alpha-1-antitrypsin wereoriginally found in HbsAg- HCC cases [191], butlater work indicated that the levels are unrelated toHbsAg status [192]. Ferritin is detected in 75% ofnormal liver tissue and 40% of HCC tissue.Stainable iron is demonstrated in 65% of unaffectedlivers and 10% of HCC tissues, indicating that ironaccumulates in cells replicating HBV. Ferritin maybe produced by the tumor cells and is not due toincreased stainable iron [193,194]. Copper wasdetected in 60% of 20 human cirrhosis/HCC casesthat were HbsAg and HbcAg positive[195]. TheTat protein of HIV enhanced chemical hepatocarcin-ogenesis in tat-transgenic mice and may contributeto tumorigenesis in HIV infected patients [196].In summary, HBV is likely carcinogenic through anumber of cell cycle regulatory pathways. Theincidence of many HCCs could be decreased by theprevention of HBV infections.

References

1. World Health Organization Scientific Group onPrevention and Control of Hepatocellular Carcinoma.Prevention of primary liver cancer: Report on a meetingof a WHO scientific group. Lancet 1983;1:463-465.

2. Goncalves CS, Pereira FE, Zago M. Hepatocellularcarcinoma. Arq Gastroenterol 1988;25:207-217.

3. Brechot C, Gozuacik D, Murakami Y, Paterlini-BrechotP. Molecular basis for the development of hepatitis B virus(HBV)-related hepatocellular carcinoma (HCC). SeminCancer Biol 2000;10:211-231.

4. Beasley RP, Lin CC. Hepatoma risk among HbsAgcarriers. Am J Epid 1978;108:247.

5. Arbuthnot P, Kew M. Hepatitis B virus and hepatocellularcarcinoma. Int J Exp Pathol 2001;82:77-100.

6. Wilkinson ML, Melia W, Johnson PJ, Williams R.Hepatocellular carcinoma in the noncirrhotic liver: Adistinct clinical entity. Gut 1982;23:447.

7. Ohaki Y, Misugi K, Sasaki Y, Yonaguni E, Quasimoto H.Hepatitis B surface antigen positive hepatocellularcarcinoma in children: Report of a case and review of theliterature. Cancer. 1983;51:822-828.

8. Nishioka K. Hepatitis B virus and hepatocellularcarcinoma: Postulates for an etiological relationship. AdvViral Oncol 1985;5:173-199.

9. Goudeau A, Yvonnet B, Barin F, Denis F, Coursaget P,Chiron JP, Mar ID. Hepatitis B virus infection andhepatocellular carcinoma: perspectives for prevention. TheRole of Viruses in Human Cancer, vol II (Giraldo G, BethE, Eds), Elsevier, New York, 1984; pp 227-238.

10. Hann HW, Kim CY, London WT, Whitford P, BlumbergBS. Hepatitis B virus and primary hepatocellularcarcinoma: Family studies in Korea. Int J Cancer 1982;30:47-51.

11. Chlebowski RT, Tong M, Weissman J, Block JB,Ramming KP, Weiner JM, Bateman JR, Chlebowski JS.Hepatocellular carcinoma. Diagnostic and prognosticfeatures in North American patients. Cancer 1984;53:2701-2706.

12. Kew MC, Geddes E. Hepatocellular carcinoma in ruralsouthern African blacks. Med 1982;61:98-108.

13. Luca N, Luca R. Diagnostic significance of alpha-fetoprotein, bile antigen VIII and Australia antigen inprimary cancer of the liver. Rev Roum Med Interne1977;15:369-374.

14. Buligescu L, Visinoiu M, Buligescu S, Budanoff R.Epidemiological study of the risk factors and of theevolution of primary liver cancer (PLC) in Romania. SwedSoc Med Sci 1982;13:422-437.

15. Melbye M, Skinhj P, Nielsen NH, Vestergaard BF, EbbesenP, Hansen JP, Biggar RJ. Virus-associated cancers inGreenland: Frequent hepatitis B virus infection but lowprimary hepatocellular cancer incidence. J Nat CancerInst 1984;73:1267-1272.

16. Pirovino M, Heer M, Altorfer J, Akovbiantz A, Grob P,Schmid M. Hepatitis B virus infection and hepatocellularcarcinoma: Indication for a possible causal connectioneven in non-endemic Switzerland. Schweiz MedWochenschr 1983;113:824-826.

17. Bourgeaux C, Trepo C, Bordes M, Sizaret P, Martin F,Sananes R, Sepetjian M, Klepping C. Study of Australiaantigen and primary liver cancer: Relationship with alpha-fetoprotein. Biol Gastroenterol 1973;6:133-137.

18. Kuper HE, Tzonou A, Kaklamani E, Hadziyannis S,Tasopoulos N, Langiou P, Trichopoulos D, Stuver S.Hepatitis B and C viruses in the etiology of hepatocellularcarcinoma; a study in Greece using third-generationassays. Cancer Causes Control 2000;11:171-175.

19. Zhang F. The relationship between hepatitis B antigenand primary liver cancer. Zhonghua Liuxingbingxue Zazhi1982;3:49-52.

20. Chun H, Cheng E, Geller N, Silviotti I, Fortner J.Hepatocellular carcinoma (HC): Statistical analysis of 78

Hepatocellular carcinoma and hepatitis virus

48

consecutive patients. Proc Ann Mtg Am Soc Clin Oncol1984;3:6.

21. Kay P, Johnson PJ, Warnes TW, Prince R. Hepatoma-speciifc alkaline phosphatase in high and low incidenceareas of primary liver cancer. Swed Soc Med Sci 1982;27:438-450.

22. Tsai J-F, Chang WY, Jeng JE, Ho MS, Lin ZY, Tsai JH.Frequency of raised alpha-fetoprotein level among Chinesepatients with hepatocellular carcinoma related to hepatitisB and C. Br J Cancer 1994;69:1157-1159.

23. Lee HS, Chung YH, Kim CY. Specificities of serum alpha-fetoprotein in HbsAg+ and HbsAg- patients in thediagnosis of hepatocellular carcinoma. Hepatolog 1991;14:68-72.

24. Beasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellularcarcinoma and hepatitis B virus: A prospective study in22,707 men in Taiwan. Lancet 1981;2:1129-1132.

25. Chung WK, Sun HS, Park DH, Minuk GY, HoofnagleJH. Primary hepatocellular carcinoma and hepatitis Bvirus infection in Korea. J Med Virol 1983;11:99-104.

26. Pongpipat D, Suvatte V, Plengvanit U, Chinapak O,Bunyaphisit S, Assateerawatts A. Hepatitis B surfaceantigen and alpha-fetoprotein in hepatoma reports of 157cases. J Med Ass Thailand 1983;66:696-699.

27. Nishioka K, Watanabe J, Furuta S, Tanaka E, Iino S,Suzuki H, Tsuji T, Yano M, Kuo G, Choo QL, HoughtonM,Oda T. A high prevalence of antibody to the hepatitisC virus in patients with hepatocellular carcinoma in Japan.Cancer 1991;67:429-33.

28. Asano M, Kato H, Yoshimoto K, Seyama S, Itakura H,Hamada T, Iijima S. Primary liver carcinoma and livercirrhosis in atomic bomb survivors, Hiroshima andNagasaki, 1961-75, with special reference to hepatitis Bsurface antigen. J Nat Cancer Inst 1982;69:1221-1227.

29. Oshima A, Tsukuma H, Hiyama T, Fujimoto I, YamanoH, Tanaka M. Follow-up study of HBV Ag-positive blooddonors with special reference to effect of drinking andsmoking on development of liver cancer. Int J Cancer1984;30:775-779.

30. Kashala LO, Conne B, Kapanci Y, Frei PC, Lambert PH,Kalengayi MR, Essex M. Hepatitis B virus, alpha-feto-protein synthesis, and hepatocellular carcinoma in Zaire.Liver 1992;12:330-340.

31. Alward WL, McMahon BJ, Hall DB, Heyward WL,Francis DP, Bender TR. The long-term serological courseof asymptomatic hepatitis B virus carriers and thedevelopment of primary hepatocellular carcinoma. J InfectDis 1985;151:604-609.

32. Okuda K, Tsuchiya Y, Saisho H. Early stage cancerdiagnosis system for minute hepatocellular choledochaland pancreatic cancers: Current capabilities and problems.Nippon Rinsho 1982;40:146-150.

33. Tai DI, Changchien CS, Hung CS, Chen CJ. Replicationof hepatitis B virus in first-degree relatives of patients withhepatocellular carcinoma. Am J Trop Med Hyg 1999;61:716-719.

34. Ganem D. Persistent infection of humans with hepatitisB virus: Mechansism and consequences. Rev Infect Dis

1982;4:1026-1047.35. Kubo S, Hirohashi K, Tanaka H, Tsukamoto T, Shuto T,

Yamamoto T, Ikebe T, Wakasa K, Nishiguchi S, KinoshitaH. Effect of viral status on recurrence after liver resectionfor patients with hepatitis B virus-related hepatocellularcarcinoma. Cancer 2000;88:1016-1024.

36. Dodd RY. HbsAg as a risk factor for hepatocellularcarcinoma among American blood donors. In: ViralHepatitis and Liver Disease (Vyas GN, Dienbstag JL,Hoofnagle JH, Eds), Grune and Stratton, Florida, 1984;pp 638-651.

37. Findor JA, Tanno H, Villamil F, Finaski Z, Leopold D. IsHCV Genotype 1 a significant risk factor for hepato-cellular carcinoma? Hepatology 1998;28:338.

38. Fattovich GF, Giustina G, Degos F, Tremolada F,DiodatiG, Almasio P, Nevens F, Solinas A, Mura D,Brouwer JT, Thomas H, Njapoum C, Casarin C, Bonetti

P, Juschi P, Basho J, Tocco A, Bhalla A, Galassini R,Noventa F, Schalm SW, Realdi G. Morbidity andmortality in compensated cirrhosis type C: A retrospectivefollow-up study of 384 patients. Gastroenterol 1997;112:463-472.

39. Sarno EN, Vieira LM, Faulhaber DM. Hepatitis B surfaceantigen (HbsAg) and primary carcinoma of the liver: Aretrospective study. Rev Inst Med Trop Sao Paulo 1980;22:20-23.

40. Akagi G, Furuya K, Kanamura A, Chihara T, Otsuka H.Liver cell dysplasia and hepatitis B surface antigen in livercirrhosis and hepatocellular carcinoma. Cancer 1984;54:315-318.

41. Nakashima T, Okuda K, Kojiro M, Jimi A, YamaguchiR, Sakamoto K, Ikari T. Pathology of hepatocellularcarcinoma in Japan: 232 consecutive cases autopsied inten years. Cancer 1983;51:863-877.

42. Kawano Y. Localization of hepatitis B surface antigen inhepatocellular carcinoma. Acta Pathol Jpn 1983;33:1087-1093.

43. Hirohashi S, Shimosato Y, Ino Y, Kishi K. Distributionof hepatitis B surface and core antigens in human livercell carcinoma and surrounding nontumorous liver. J NatCancer Inst 1982;69:565-568.

44. Dammacco F, Miglietta A, Antonaci S, Bonomo L. Alpha-fetoprotein and Australia antigen in primary liver cancer.Digestion 1973;9:41-48.

45. Dai Y, Zheng B, Peng B, Dang Z, Fang UR. Analysis ofhepatitis B surface antigen and alpha-fetoprotein in livercarcinoma and its surrounding tissue by immunoperox-idase technics. Zhonghua Binglixue Zazhi 1982;1:104-108.

46. Shafritz DA. Hepatitis B virus DNA molecules in theliver of HBSAG carriers-mechanistic considerations in thepathogenesis of hepatocellular carcinoma. Hepatolog1982;2:35S-41S.

47. Zuckerman AJ. Primary hepatocellular carcinoma andhepatitis B virus. Trans R Soc Trop Med Hyg 1982;76:711-718.

48. Zuckerman AJ. Virological approach to the preventionof primary liver cancer. Hepatology 1982;2:67-71.

Annals of Clinical & Laboratory Science

49

49. Popper H, Gerber MA, Thung SN. The relation ofhepatocellular carcinoma to infection with hepatitis-B andrelated viruses in man and animals. Hepatol 1982;2:1-9.

50. Hino O, Kitagawa T, Koike K, Kobayashi M, Hara M,Mori W, Nakashima T, Hattori N, Sugano H. Detectionof hepatitis B virus DNA in hepatocellular carcinomas inJapan. Hepatol 1984;4:90-95.

51. Will H, Salfeld J, Pfaff E, Manso C, Theilmann L, SchalerH. Putative reverse transcriptase intermediates of humanhepatitis B virus in primary liver carcinomas. Science1986;231:594-596.

52. Robinson WS. Hepatitis B virus diseases. In: Conceptsin Viral Pathogenesis (Notkins AL, Oldstone MBA, Eds),Springer-Verlag, New York, 1984; pp 288-299.

53. Ding JL, Oon CJ. Detection of HbeAg in the lymphocytesof sero-HbeAg negative patients with chronic hepatitis Band primary hepatocellular carcinoma. Cytobios 1984;39:29-33.

54. Blumberg BS, London WT. Hepatitis B virus and hepato-cellular carcinoma. Cancer Treat Res 1984;18:71-91.

55. Nakanuma Y, Ohta G. Morphology of cirrhosis andoccurrence of hepatocellular carcinoma in alcoholics withand without HbsAg and in non-alcoholic HbsAg-positivepatients. A comparative autopsy study. Liver 1983;3:231-237.

56. Horiike N, Michitaka K, Onji M, Murota T, Ohta Y.HBV-DNA hybridization in hepatocellular carcinomaassociated with alcohol in Japan. J Med Virol 1989;28:189-192.

57. Cullen JM, Marion PL, Sherman GJ, Hong X, NewboldJE. Hepatic neoplasms in aflatoxin B1-treated, congenitalduck hepatitis B virus-infected, and virus-free Pekingducks. Cancer Res 1990;50:4072-4080.

58. Anthony PP. Hepatocellular carcinoma: an overview.Histopathology 2001;39:109-118.

59. Yan RQ. A study on primary liver cancer in tree shrewsinduced by human hepatitis B virus and aflatoxin B1.Chung-hua Ping Li Hsueh Tsa Chih 1989;18:19-22.

60. Dammacco F, Miglietta A, Antonaci S, Bonomo L. Alpha-fetoprotein and Australia antigen in primary liver cancer.Digestion 1973;9:41-48.

61. Craxi A, Pasqua P, Giannuoli G,Di Stefano R, SimonettiRG, Pagliaro L. Tissue markers of hepatitis B virusinfection in hepatocellular carcinoma and cirrhosis.Hepatogastroenterology 1984;31:55-59.

62. Ferrandez-Izquierdo,Llombart-Bosch A. Immunohisto-chemical characterization of 130 cases of primary hepaticcarcinomas. Pathol Res Pract 1987;182:783-791.

63. Dalgleish AG, Woods RL, Levi JA, Raghavan D,McCaughan GW, Tattersall MH. The role of hepatitis Bvirus in the etiology of hepatocellular carcinoma inAustralia. Aust NZ J Med 1983;13:605-607.

64. Phornphutkul K, Peerakpome S, Uthamachi C, Bosk-owica L, Lierstadiv G. Hepatitis B viral markers (HBV)and alpha fetoprotein(AFP) levels in hepatocellularcarcinoma (HCC) patients in northern Thailand. Cancer1987;12:38-47.

65. Sakurai M, Miyaji T. Alpha-fetoprotein and hepatitis Bantigen in hepatocarcinogenesis. Trans NY Acad Sci1975;36:822.

66. Stajic M, Milosavljevic J. Frequency of occurrence andsignificance of alpha-fetoprotein and Australia antigen inthe serum of 329 patients with chronic diseases andprimary malignant neoplasm of the liver. Acta Med Yugosl1978;32:243-251.

67. Chen DS, Sheu JC, Sung JL, Lai MY, Lee CS, SU CT,Tsang YM, How SW, Wang TH, Yu JY, Yang TH, WangCY, Hsu CY. Small hepatocellular carcinoma- A clinico-pathological study in thirteen patients. Gastroenterology1982;83:1109-1119.

68. Zumla A,Voller A. A serological study on primaryhepatocellular carcinoma in Zambia. Trans R Soc TropMed Hyg 1982;76:546-551.

69. Yang BH, Tang Zy. Hepatitis B virus and hepatocellularcarcinoma- clinical and serological aspects; In: SubclinicalHepatocellular Carcinoma (Tang ZY, Ed), Springer-VerlagNew York, 1984; pp 212-217.

70. Brown SE, Howard CR, Steward MW, Ajdukiewica AB,Whittle HC. Hepatitis B surface antigen conatiningimmune complexes occur in seronegative hepatocellularcarcinoma patients. Clin Exp Immunol 1984;55:355-359.

71. Bassendine MF, Della-Seta L, Salmeron J, Thomas HC,Sherlock S. Incidence of hepatitis B virus infection inalcoholic liver disease, HbsAg negative chronic active liverdisease and primary liver cell cancer in Britain. Liver1983;3:65-70.

72. Gasser RW, Judmaier G, Zur Nedden D, AufschnaiterM, Hofstaedter F. Primary hepatocellular carcinoma withhepatitis B virus infection in a 16-year-old noncirrhoticpatient. Am J Gastroeneterol 1983;78:305-308.

73. De Franchis R, Primignani M, Vecchi M, Antoniozzi F,Colombo M, Colucci G, Tommasini M. Case-controlstudy of hepatitis B virus infection in chronic liver diseaseand hepatocellular carcinoma. Ric Clin Lab 1984;14:81-88.

74. Lingao AL. The relationship of hepatocellular carcinomaand liver cirrhosis to hepatitis B virus infection in thePhilippines. Gastroenterol Jpn 1989;24:425-433.

75. Giuliani-Piccari G, Spongano P, Formica G, Funacelli T,Mozarella N, Ziti K. Hepatocellular carcinoma (HCC)and its relationship to HBV infection and chronic liverdisease in Italy. Swed Soc Med Sci 1982;13:321-335.

76. Sulaiman HA. Hepatitis B virus infection in liver cirrhosisand hepatocellular carcinoma in Jakarta Indonesia.Gastroenterol Jpn 1989;24:434-441.

77. Heyward W. Prospective serologic tests of hepatitis B virusantigens and antibodies in Alaskan Eskimos who subse-quently developed primary hepatocellular carcinoma. AmJ Epidemiol 1982;116:558.

78. Chen DS, Sung JL. Hepatitis B surface antigen and alpha-fetoprotein in patients with hepatocellular carcinoma.Gastroenterology 1978;74:163-165.

79. Kew MC, Macerollo P. Effect of age on the etiologic roleof the hepatitis B virus in hepatocellular carcinoma in

Hepatocellular carcinoma and hepatitis virus

50

blacks. Gastroenterology 1988;94:439-442.80. Liaw YF, Chu CM, Lin DY, Sheen IS, Yang CY, Huang

MJ. Age-specific prevalence and significance of hepatitisBe antigen and antibody in chronic hepatitis B virusinfection in Taiwan: A comparison among asymptomaticcarriers, chronic hepatitis, liver cirrhosis, and hepato-cellular carcinoma. J Med Virol 1984;13:385-391.

81. Bassendine MF, Arborgh AM, Shipton U, Monjardino J,Aranguibel F, Thomas HC, Sherlock S. Hepatitis B surfaceantigen and alpha-fetoprotein secreting human primaryliver cell cancer in athymic mice. Gastroenterology1980;79:528-532.

82. Busachi CA, Bianchi L, Gudat F, Ludavi H, Mondava W.Alpha-fetoprotein, alpha-antitrypsin and hepatitis B virusantigens in human hepatocellular carcinoma: Theirincidence and distribution. Eur Assoc Stud Liver 1982;23:45-53.

83. Yahalom J, Livni N, Fuks Z, Levij IS. Hepatocellularcarcinoma in Israel: Relationship to hepatitis B virusinfection. Israel J Med Sci 1985;21:1-5.

84. Zhang SP. Immunohistochemical study on HbsAg,HBCAg and AFP in 104 patients with small liver cancer.Chung Hua Chung Liu Tsa Chih 1988;10:353-356.

85. Yang EB, Zhao YN, Zhang K, Mack P. Microsatellitealterations in human hepatocellular carcinoma infectedwith hepatitis B virus: associated with the elevation ofserum alpha-fetoprotein. Int J Oncol 2001;19:513-518.

86. Roingeard P, Romet-Lemonne JL, Barin F, Stanzeri V.Hepatoma patients antibodies to a 18 kD cytoplasmicprotein in human hepatoma cell lines: PLC/PRF/5 andHep 3 B. Sym Comp Res Leuk Rel Dis 1985;22:121.

87. Wen YM. Hepatitis B virus and hepatocellular carcinoma-cellular and molecular aspects. In: Subclinical Hepato-cellular Carcinoma (Tang ZY, Ed), Springer-Verlag, NewYork, 1984; pp 218-231.

88. Chedid A, Chejfec G. Markers of hepatocellularcarcinoma: A histochemical and ultrastructural study. FedProc 1987;46:428.

89. Zhang YY, Hao LJ, Rizzetto M, Bonino F, Verme G.Immunohistochemical research on HDV infection inChinese patients with chronic liver disease. J Tongji MedUniv 1989;9:5-7.

90. Watanabe S, Kitade A, Deguchi A. Histopathologicalfindings of small hepatocellular carcinoma. Hepatology1998;28:759.

91. Khalifa A, Mady EA, Abadeer N, Kamal A. Differentialtumor markers and hepatitis markers profile in livertumors. Anticancer Res 1999;19:2495-2500.

92. Yotsuyanagi H, Shintani Y, Moriya K, Fujie H, TsutsumiT, Kato T, Nishioka K, Takayama T, Makuuchi M, IinoS, Kimura S, Koike K. Virologic analysis of non-B, non-C hepatocellular carcinoma in Japan: frequent involve-ment of hepatitis B virus. J Infect Dis 2000;18:1920-1928.

93. Kawai S, Yokosuka O, Imazeki F, Maru Y, Saisho H. Stateof HBV DNA in HbsAg-negative, anti-HCV-positivehepatocellular carcinoma: existence of HBV DNA

possibly as nonintegrated form with analysis by Alu-HBVDNA PCR and conventional HBV PCR. J Med Virol2001;64:410-418.

94. Jin YM, Yun C, Park C, Wang HJ, Cho H. Expression ofhepatitis B virus X protein is closely correlated with thehigh periportal inflammatory activity of liver diseases. JViral Hepat 2001;8:322-330.

95. Kubo S, Tamori A, Ohba K, Shuto T, Yamamoto T, TanakaH, Nishiguchi S, Wakasa K, Hirohashi K, Kinoshita H.Previous or occult hepatitis B virus infection in hepatitisC Virus-associated hepatocellular carcinoma withouthepatic fibrosis. Dig Dis Sci 2001;46:2408-2414.

96. Shamay M, Agami R, Shaul Y. HBV integrants of hepato-cellular carcinoma cell lines contain an active enhancer.Oncogene 2001;20:6811-6819.

97. Waris G, Siddiqui A. Interaction between STAT-3 andHNF-3 leads to the activation of liver-specific hepatitisB virus enhancer 1 function. J Virol 2002;76:2721-2729.

98. Brechot C, Hadchouel M, Scotto J, Degos F, Charnay P,Trepo C, Tiollais P. Detection of the hepatitis B virus DNAin liver and serum: A direct appraisal of the viral chroniccarrier state. Lancet 1981;2:765-768.

99. Monjardino JP, Fowler MJ, Thomas HC. Defectivehepatitis B virus DNA molecules detected in a stableintegration pattern in a hepatoma cell line, and in inducedtumours and derived cell lines. J Gen Virol 1983;64:2299-2303.

100. Marquardt O, Zaslavsky V, Hofschneider PH. Evidencefor non-chromosomal hepatitis B virus surface (HbsAg)and core antigen (HbcAg)-specific DNA sequences in ahepatoma cell line. J Gen Virol 1982;61:105-109.

101. Merten OW, Reiter S, Scheirer W, Katinger H. Purifi-cation of HbsAg produced by the human hepatoma cellline PLC/PRE/5 by affinity chromatography using mono-clonal antibodies and application for ELISA diagnostic.Dev Biol Stand 1983;55:121-127.

102. Kubo S, Hirohashi K, Tanaka H, Tsukamoto T, Shuto T,Higaki I, Takemura S, Yamamoto T, Nishiguchi S,Kinoshita H. Virologic and biochemical changes andprognosis after liver resection for hepatitis B virus-relatedhepatocellular carcinoma. Dig Surg 2001;18:26-33.

103. Kim MY, Park E, Park JH, Park DH, Moon WS, ChoBH, Shin HS, Kim DG. Expression profile of nine novelgenes differentially expressed in hepatitis B virus-associated hepatocellular carcinomas. Oncogene 2001;20:4568-4575.

104. He L, Isselbacher KJ, Wands JR, Goodman HM, Shih C,Quaroni A: Establishment and characterization of a newhuman hepatocellular carcinoma cell line. In Vitro 1984;20:493-504.

105. Rogler CE, Sherman M, Su CY, Shafritz DA, Summers J,Shows TB, Henderson A, Kew M. Deletion in chromo-some 11p associated with a hepatitis B integration site inhepatocellular carcinoma. Science 1985;230:319-322.

106. Hino O, Kitagawa T, Sugano H. Relationship betweenserum and histochemical markers for hepatitis B virusand rate of viral integration in hepatocellular carcinomas

Annals of Clinical & Laboratory Science

51

in Japan. Int J Cancer 1985;35:5-10.107. Tanaka T, Miyamoto H, Hino O, Kitagawa T, Koike M,

Iizuka T, Sakamoto H, Ooshima A. Primary hepatocellularcarcinoma with hepatitis B virus-DNA integration in a4-year-old boy. Hum Pathol 1986;17:202-204.

108. Brambilla C, Tackney C, Hirschman S, Tourneau F,Stanley V. Varying nuclear staining intensity of hepatitisB virus DNA in human hepatocellular carcinoma. LabInvest 1986;55:475-481.

109. Lo YM, Mehal WZ, Fleming KA. In vitro amplificationof hepatitis B virus sequences from liver tumour DNAand from paraffin wax embedded tissues using the poly-merase chain reaction. J Clin Pathol 1989;42:840-846.

110. Fujimori M, Tokino T, Hino O, Kitagawa T, Imamura T,Okamoto E, Mitsunobu M, Ishikawa T, Nakagama H,Harada H, Yagura M, Matsubara K, Nakamura Y.Allelotype study of primary hepatocellular carcinoma.Cancer Res 1991;51:89-93.

111. Simon D, Knowles BB. Hepatocellular carcinoma cell lineand peripheral blood lymphocytes from the same patientcontain common chromosomal alterations. Lab Invest1986;55:657-665.

112. Cariani E, Lasserre C, Kemeny F, Franco D, Brechot C.Expression of insulin-like growth factor II, alpha-feto-protein and hepatitis B virus transcripts in human primaryliver cancer. Hepatology 1991;13:644-649.

113. Fu XX, Su CY, Lee Y, Hintz R, Biempica L, Snyder R,Rogler CE. Insulin growth factor II expression and ovalcell proliferation associated with hepatocarcinogenesis inwoodchuck hepatitis virus carriers. J Virol 1988;62:3422-3430.

114. Emeljanchik EK. Immunodiffusion test for alpha-feto-protein, hepatocellular carcinoma, viral hepatitis and liver

cirrhosis. Oncol Biol Med 1981;2:196.115. Yamamoto B, Osaka A, Yokata D. Clinicopathological

analyses of high risk factors with hepatic cell carcinomain chronic liver diseases. Asian Pacif Assoc Stud Liver1982, pp 34-41.

116. Zumla A, Voller A. A serological study on primaryhepatocellular carcinoma in Zambia. Trans R Soc TropMed Hyg 1982;76:546-551.

117. Rajagopalan N, Sama SK, Krishnamurthy L, Gandhi G,Nayak NC. Hepatitis B antigen and alpha-fetoprotein inthe sera of patients with hepatocellular carcinoma. IndianJ Med Res 1977;65:95-100.

118. Vogel CL, Primack A, McIntire KR, Mercer V, Steussel J,Ford H. Serum alpha-fetoprotein in 184 Ugandan patientswith hepatocellular carcinoma: clinical, laboratory, andhistopathologic correlations. Cancer 1974;3:959-964.

119. Chang FY, Chen TC, Fong MS, Lin S, Yeh SH. Alphafeto-protein levels measured by radioimmunoassay in variousliver malignancies. Taiwan I Hsueh Hui Tsa Chih1982;81:224-231.

120. Deshpande AD, Patil SD, Talib SH. Comparative studyof needle biopsy and alphafetoprotein detection in thediagnosis of primary hepatocellular carcinoma. J AssocPhysicians India 1981;29:129-132.

121. Chen DS, Sung JL. Relationship of hepatitis B surfaceantigen to serum alpha-fetoprotein in nonmalignantdiseases of the liver. Cancer 1979;44:984-992.

122. Sakurai M, Miyaji T. Alpha-fetoprotein and hepatitis Bantigen in hepatocarcinogenesis. Ann NY Acad Sci1975;259:156-167.

123. Lok AS, Lai CL. Alpha-fetoprotein monitoring in Chinesepatients with chronic hepatitis B virus infection: Role inthe early detection of hepatocellular carcinoma. Hepat-ology 1989;9:110-115.

124. Ruoslahti E, Seppala M, Rasanen JA, Vuopio P, Helske T.Alphafetoprotein and hepatitis B antigen in acute hepatitisand primary liver cancer of the liver. Scand J Gastroenterol1973;8:197-202.

125. Heyward WL, Lanier AP, Carter ML, Stanford U, SmithLL. Early detection of primary hepatocellular carcinomain Alaskan natives through use of a hepatitis B registryand sereological screening for alphafetoprotein. In: ViralHepatitis and Liver Disease (Vyas GN, Dienstag JL,Hoofnagle JH, Eds), Grune and Stratton, Florida, 1984;pp 663-664.

126. Kashala LO, Kalengayi MMR, Essex M. Alphafetoproteinsynthesis in human hepatocellular carcinoma: Correlationwith hepatitis B surface antigen expression. Cancer Invest1992;10:513-522.

127. Zhou XD, DeTolla L, Custer RP, London WT. HepatitisB antigens and alphafetoprotein in hepatocellular carcin-oma. Gastroenterology 1987;92:1710.

128. Bowry TR, Shah MV. A study of hepatitis BS antigentitres and alphafetoprotein levels in primary hepatocellularcarcinoma in Kenya. East Afr Med J 1980;57:382-389.

129. Ohtoh K, Ohno H, Okuyama Y, Fukato Y, Fujii O.Clinico-pathological analyses of high risk factors withhepatic cell carcinoma in chronic liver diseases. Asian PacifAssoc Stud Liver 1982, pp 34-41.

130. Kay P, Johnson PJ, Warnes TW, Thomas R, Samsone N.Hepatoma-specific alkaline phosphatase in high and lowincidence areas of primary liver cancer. Swed Soc MedSci 1982;27:438-450.

131. Heyward W. Prospective serologic tests of hepatitis B virusantigens and antibodies in Alaskan Eskimos who subseq-uently developed primary hepatocellular carcinoma. AmJ Epidemiol 1982;116:558.

132. Heyward WL, Lanier AP, McMahon BJ, Fitzgerald MA,Kilkenny S, Paprocki TR. Early detection of primaryhepatocellular carcinoma. JAMA. 1985;254:3052-3054.

133. Liaw Y-F, Tai D-I, Chen T-J, Chu CM, Huang MJ. Alpha-fetoprotein changes in the course of chronic hepatitis:Relation to bridging hepatic necrosis and hepatocellularcarcinoma. Liver 1986;6:133-137.

134. Heyward WL, Bender TR, Lanier AP, Francis DP, McMahon BJ, Maynard JE. Serological markers of hepatitisB virus and alphafetoprotein levels preceding primaryhepatocellular carcinoma in Alaskan Eskimos. Lancet1982;2:889-891.

135. Ingle L, Kilkenny S, Kline K. Early detection of primaryhepatocellular carcinoma-Alaska. JAMA. 1984;251: 1134.

Hepatocellular carcinoma and hepatitis virus

52

136. Cote PJ, Pohl C, Boyd J, Tennant BC, Gerin JL. Alpha-fetoprotein in the woodchuck model of hepatitis virusinfection and disease: Immunochemical analysis of wood-chuck alpha-fetoprotein and measurement in serum byquantitative monoclonal radioimmunoassay. Hepatology1990;11:824-833.

137. Lee HY, Chung YH, Kim CY. Specificities of serumalphafetoproetin in HbsAg+ and HbsAg- patients in thediagnosis of hepatocellular carcinoma. Hepatology 1991;14:68-72.

138. Liaw YF. HBeAg/Anti-HBe and alphafetoprotein levelsin primary hepatocellular carcinoma. Lancet 1982;2:1458.

139. Bellet DH, Wands JR, Isselbacher KJ, Bohuon C. Serumalpha-fetoprotein levels in human disease: Perspectivefrom a highly specific monoclonal radioimmunoassay.Proc Natl Acad Sci USA 1984;81:3869-3873.

140. Tsai JF, Tsai JH, Chang WY, Ton TC. Elevation ofcirculating immune complexes and its relationship toalpha-fetoprotein levels in patients with hepatitis B surfaceantigen-positive hepatocellular carcinoma. Cancer Invest1991;9:137-143.

141. Bassendine MF, Arborgh AM, Shipton U, Monjardino J,Aranguibel F, Thomas HC, Sherlock S. Hepatitis B surfaceantigen and alpha-fetoprotein secreting human primaryliver cell cancer in athymic mice. Gastroenterology1980;79:528-532.

142. Panoyotopoulos S, Yiannacopoulos T, Margari-Papanic-olaou P, Christou A. The detection of alpha-fetoproteinin HbsAg and HbeAg carriers. Allergol Immunopathol1982;10:1-4.

143. Isom HC, Liao WS, Taylor JM, Willwerth GE, EadineTS. Rapid and selective shutoff of plasma protein prod-uction in herpes simplex virus type 2-infected hepatomacells. Virology 1983;126:548-562.

145. Lee HS, Chung YH, Kim CY. Specificities of serum alpha-fetoprotein in HbsAg+ and HbsAg- patients in thediagnosis of hepatocellular carcinoma. Hepatology1991;14:68-72.

146. Songsivilai S, Dharakul T, Sansnee S. Hepatitis B- andhepatitis C-associated hepatocellular carcinoma: Eval-uation of alphafetoprotein as a diagnostic marker. AsianPacif J Allergy Immunol 1995;13:167-171.

147. Jiang S-Y, Shyu R-Y, Huang M-F, Tang HS, Young TH,Roffler SR, Chiou YS, Yeh MY. Detection of alphafeto-protein-expressing cells in the blood of patients withhepatoma and hepatitis. Brit J Cancer 1997;75:928-933.

148. Findor JA, Tanno H, Villamil F, Mastromono T. Is HCVgenotype 1 a significant risk factor for hepatocellularcarcinoma? Hepatology 1998;28:338.

149. Trevisani F, Morselli-Labate AM, Calandra S et al.Influence of HbsAg and anti-HCV status on the diag-nostic accuracy of alpha-1-fetoprotein in patients withchronic liver disease. Hepatology 1997;26:172.

150. Verslype C, van Pelt JF, Wu CG, Eiderstein C, Plotz CP.HCV core protein expression increases the level of alpha-fetoprotein gene transcripts independently from the

albumin gene in a nontumorigenic immortalized humanhepatocyte line. Hepatology 1998;28:562.

151. Bayati N, Silverman AL, Gordon SC. Serum alphafeto-protein levels and liver histology in patients with chronichepatitis C. Am J Gastroenterol 1998;93:2452-2456.

152. Fiedler R, Baur A, Penner E. Elevation of alpha-1-feto-protein in patients with chronic hepatitis C. Hepatology1991;14:80.

153. Bayati N, Gordon SC, Silverman AL, Meserlongi V.Relationship of alpha-fetoprotein levels and hepatocyteproliferation and injury in hepatitis C. Hepatology 1998;28:281.

154. Goldstein NS, Blue DE, Hankin R, Hunter S, Bayati N,Silverman AL, Gordon SC. Serum alphafetoprotein levelsin patients with chronic hepatitis C. Am J Clin Pathol1999;111:811-816.

155. Bergmann JF, Lang F, Dechy H, Dorra M. Alpha-feto-protein in hepatitis superimposed on cirrhosis: A case ofconcomitant hepatitis B surface antigen and antibodyassociated with a major but transient increase in the alpha-fetoprotein level. Am J Gastroenterol 1985;80:810-812.

156. DiBisceglie AM, Hoofnagle JH. Elevations in serumalphafetoprotein levels in patients with chronic hepatitisB. Cancer 1989;64:2117-2120.

157. Koda M, Hori T, Maeda N, Kato S, Murawaki Y, HorieY, Kawasaki H, Hirayama C, Taketa K. Lectin-reactivepatterns of markedly elevated serum alpha-fetoportein inpartients with chronic active hepatitis. Am J Gastroenterol1991;86:861-865.

158. Delpuech O, Trabut JB, Carnot F, Feuillard J, BrechotC, Kremsdorf D. Identification, using cDNA macroarrayanalysis, of distinct gene expression profiles associated withpathological and virological features of hepatocellularcarcinoma. Oncogene 2002;21:2926-2937.

159. Choi YL, Jang JJ, Park CK. Expression of the G1-Smodulators in hepatitis B virus (HBV) relatedhepatocellular carcinoma (HCC) and dysplastic nodule(DN): Association of cyclin D1 and p27 with theprogression of HCC. Lab Invest 2000;80:181A.

160. Lin JH, Tong MJ, Stevenson D. A new human hepato-cellular carcinoma cell line secreting hepatitis B surfaceantigen and alpha-fetoprotein. In: Viral Hepatitis andLiver Disease (Vyas GN, Dienstag JL, Hoofnagle JH, Eds),Grune and Stratton, Florida, 1984; pp 673-685.

161. Moriarty AM, Alexander H, Lerner RA, Thornton GB.Antibodies to peptides detect new hepatitis B antigen:serological correlation with hepatocellular carcinoma.Sciience 1985;227:4290-4293.

162. Rabe C, Cheng B, Caselmann WH. Molecular mech-anisms of hepatitis B virus-associated liver cancer. DigDis 2001;19:279-287.

163. Shi DR. The HBX protein expression in liver cancer.Chung-hua Ping Li Hsueh Tsa Chih 1991;20: 85-87.

164. Garcia EJ, Cohen, Clayton MM, Thomasson M, MaloneyP. HBx immunohistochemical staining in hepatocellularcarcinoma: Relationship to hepatitis B virus and hepatitisC virus. Lab Invest 1999;79:162.

Annals of Clinical & Laboratory Science

53

165. Katayama K, Hayashi N, Sasaki Y, Kasahara A, Ueda K,Fusamoto H, Sato N, Chisaka O, Matsubara K, KamadaT. Detection of hepatitis B virus X gene protein and anti-body in type B chronic liver disease. Gastroenterology1989;97:990-998.

166. Yeh CT, Shen CH, Tai DI, Chu CM, Liaw YF.Identification and characterization of a prevalent hepatitisB virus X protein mutant in Taiwanese patients withhepatocellular carcinoma. Oncogene 2000;19:5213-5220.

167. Tu H, Bonura C, Giannini C, Mouly H, Soussan P, KewM, Paterlini-Brechot P, Brechot C, Kremsdorf D.Biological impact of natural COOH-terminal deletionsof hepatitis B virus X protein in hepatocellular carcinomatissues. Cancer Res 2001;61:7803-7810.

168. Wang XW, Forrester K, Yeh H, Feitelson MA, Gu JR,Harris CC. Hepatitis B virus X protein inhibits p53sequence-specific DNA binding, transcriptional activity,and association with transcription factor ERCC3. ProcNatl Acad Sci USA.1994;91:2230-2234.

169. Shiota G, Oyama K, Udagawa A, Tanaka K, Nomi T,Kitamura A, Tsutsumi A, Noguchi N, Takano Y, YashimaK, Kishimoto Y, Suou T, Kawasaki H. Occult hepatitis Bvirus infection in HBs antigen-negative hepatocellularcarcinoma in a Japanese population: involvement of HBxand p53. J Med Virol 2000;62:151-158.

170. Venard V, Corsaro D, Kajzer C, Bronowicki JP, LeFaouA. Hepatitis B virus X gene variability in French-bornpatients with chronic hepatitis and hepatocellularcarcinoma. J Med Virol 2000;62:177-84.

171. Huo TI, Wang XW, Forgues M, Wu CG, Spillare EA,Giannini C, Brechot C, Harris CC. Hepatitis B virus Xmutants derived from human hepatocellular carcinomaretain the ability to abrogate p53-induced apoptosis.Oncogene 2001;20:3620-3628.

172. Montesano R, Hainaut P, Wild CP. Hepatocellular carcin-oma: from gene to public health. J Natl Cancer Inst1997;89:1844-1851.

173. Peng SY, Lai PL, Hsu HC. Amplification of the c-mycgene in human hepatocellular carcinoma: biologicalsignificance. J Formos Med Assoc 1993;92:866-870.

174. Nishida N, Fukuda Y, Komeda T, Kita R, Sando T,Furukawa M, Amenomori M, Shibagi I, Nakao K, IkenagaM. Amplification and overexpression of the cyclin D1gene in aggressive human hepatocellular carcinoma.Cancer Res 1994;54:3107-3110.

175. Ray RB, Steele R, Meyer K, Ray R. Transcriptionalrepression of p53 promoter by hepatitis C virus coreprotein. J Biol Chem 1997;272:10983-10986.

176. Moriya K, Fujie H, Shintani Y, Yotsuyanagi H, TsutsumiT, Ishibashi K, Matsuura Y, Kimura S, Miyamura T, KoikeK. The core protein of hepatitis C virus induces hepato-cellular carcinoma in transgenic mice. Nature Med 1998;4:1065-1067.

177. Kobayashi S, Matsushita K, Saigo K, Urashima T, AsanoT, Hayashi H, Ochiai T. P21WAF1/CIP1 messengerRNA expression in hepatitis B, C virus-infected humanhepatocellular carcinoma tissues. Cancer 2001;91:2096-2103.

178. Lin X, Qian GS, Lu PX, Wu L, Wen YM. Full-lengthgenomic analysis of hepatitis B virus isolates in a patientprogressing from hepatitis to hepatocellular carcinoma. JMed Virol 2001;64:299-304.

179. Hu KQ. An immunohistochemical study on pre-Sproteins of hepatitis B virus. Chung-hua Ping Li HsuehTsa Chih 1989;18:27-29.

180. Hu KQ, Hao LJ, Zhang YY, Schaller H. A preliminarystudy on expression and significance of pre-S1 proteinin liver tissue of patients with HBV infection. J TongjiMed Univ 1989;9:8-12.

181. Zhong S, Chan JY, Yeo W, Tam JS, Johnson PJ. Frequentintegration of precore/core mutants of hepatitis B virusin human hepatocellular carcinoma tissues. J Viral Hepat2000;7:115-13.

182. Buendia MA. Genetics of hepatocellular carcinoma.Semin Cancer Biol 2000;10:185-200.

183. Hirohashi S. Inactivation of the E-cadherin-mediated celladhesion system in cancers. Am J Pathol 1998;153:333-339.

184. Miyoshi Y, Iwao K, Nagasawa Y, Aihara T, Sasaki Y,Imaoka S, Murata M, Shimano T, Nakamura Y. Activationof the beta-catenin gene in primary hepatocellular carcin-omas by somatic alterations involving exon 3. Cancer Res1998;58:2524-2527.

185. Wong CM, Fan ST, Ng IO. Beta-catenin mutation andoverexpression in hepatocellular carcinoma: clinico-pathologic and prognostic significance. Cancer 2001;92:136-145.

186. Xu XR, Huang J, Xu ZG, Qian BZ, Zhu ZD, Yan Q, CaiT, Zhang X, Xiao HS, Qu J, Liu F, Huang QH, ChengZH, Li NG, Du JJ, Hu W, Shen KT, Lu G, Fu G, ZhongM, Xu SH, Gu WY, Huang W, Zhao XT, Hu GX, GuJR, Chen Z, Han ZG. Insight into hepatocellular carcino-genesis at transcriptome level by comparing gene expres-sion profiles of hepatocellular carcinoma with those ofcorresponding noncancerous liver. Proc Natl Acad SciUSA 2001; 98:15089-15094.

187. Huang H, Fujii H, Sankila A, Mahler-Araujo BM,Matsuda M, Cathomas G, Ohgaka H. Beta-cateninmutations are frequent in human hepatocellular carcin-omas associated with hepatitis C virus infection. Am JPathol 1999;155:1795-1801.

188. Hsu HC, Jeng YM, Mao TL, Chu JS, Lau PL, Peng SY.Beta-catenin mutations are associated with a subset oflow-stage hepatocellular carcinoma negative for hepatitisB virus and with favorable prognosis. Am J Pathol 2000;157:763-770.

189. Pastore G, Dentico G, Angarano G, Zanetti AR, FerroniP, Frappampina V, Schiraldi O, Roggendorf M, FrosnerG. Hepatitis B virus markers, alpha-fetoprotein andsurvival in fulminant hepatitis. J Med Virol 1981;7:97-103.

190. Kay P, Johnson PJ, Warnes TW. Hepatoma-specificalkaline phosphatase in high and low incidence areas ofprimary liver cancer. Swed Soc Med Sci 1982;27:438-450.

Hepatocellular carcinoma and hepatitis virus

54

191. Sparos L, Tountas Y, Chapuis-Cellier C, TheodoropoulosG, Trichopoulos D. Alpha-1-antitrypsin levels and pheno-types and hepatitis B serology in liver cancer. Br J Cancer1984;49:567-570.

192. Hong WS, Hong SI. Clinical usefulness of alpha-1-anti-tryspsin in the diagnosis of hepatocellular carcinoma. JKorean Med Sci 1991;6:206-213.

193. Lustbader ED, Hann HL, Blumberg BS. Serum ferritinas a predictor of host response to hepatitis B virusinfection. Science 1983;220:423-425.

Annals of Clinical & Laboratory Science

194. Zhou X-D, DeTolla L, Custer P, London WT. Iron,ferritin, hepatitis B surface and core antigens in the liversof Chinese patients with hepatocellular carcinoma. Cancer1987;59:1430-1437.

295. Valyi-Nagy I, Schaff ZS, Lapis K: Distribution of hepatitisB surface and core antigens. Alpha-fetoprotein and copperin human liver cirrhosis and hepatocellular carcinoma.Path Res Pract 1985;180:320.

196. Altavilla G, Caputo A, Lanfredi M, Piola C, Barbanti-Brodano G, Corallini A. Enhancement of chemicalcarcinogenesis by the HIV-1 tat gene. Am J Path 2000;157:1081-1089.