Hepatitis B Virus Infection and Primary Hepatocellular Carcinoma

Transcription

1 CLINICAL MICROBIOLOGY REVIEWS, July 1992, p Vol. 5, No /92/ $02.00/0 Copyright 1992, American Society for Microbiology Hepatitis B Virus Infection and Primary Hepatocellular Carcinoma MARK FEITELSON Department of Pathology and Cell Biology, Room 219 Alumni Hall, Jefferson Medical School, Thomas Jefferson University, 1020 Locust Street, Philadelphia, Pennsylvania INTRODUCTION Genome and Gene Products of HBV HBV Replication and Natural History of Infection HBV-Like Viruses and PHC EPIDEMIOLOGICAL LINK BETWEEN HBV INFECTION AND PHC Significance of In Utero or Perinatal Transmission of HBV to PHC Factors Contributing to the High Relative Risk of PHC among HBV-Infected Patients Chemicals and other infectious agents Age, gender, and sex hormones Chronic liver disease PUTATIVE CELLULAR AND MOLECULAR MODELS FOR PHC ASSOCIATED WITH CHRONIC HBV INFECTION Integration Sites of HBV DNA in PHC Cells cis-acting Mechanisms of PHC Fusion proteins Promotion-insertion Putative role of other oncogenes Genetic Instability and Recessive Carcinogenesis Chromosomal rearrangements and loss of antioncogenes p53, AFBj, and hepatocarcinogenesis HBV Gene Expression: Putative Roles in PHC Genetic contribution of hepadnaviruses to cancer Immunological targeting of virus antigens: relevance to pathogenesis of PHC Evidence for trans-activating mechanisms in PHC: putative roles for pre-s and HBxAg gene products CONCLUSIONS: MODEL FOR HBV-INDUCED CARCINOGENESIS REFERENCES INTRODUCTION Chronic infection with hepatitis B virus (HBV) is associated with an increased risk for the development of primary hepatocellular carcinoma (PHC). Intensive efforts to describe how HBV causes PHC on the cellular and molecular levels have drawn attention to frequent characteristics that may be central to the pathogenesis of this tumor type. However, to discuss these characteristics in detail, it is important to present the salient features of the virus genome, its replication cycle, and the characteristics of the gene products which may contribute to the pathogenesis of PHC. Genome and Gene Products of HBV The genome of HBV has been obtained from infectious serum samples and characterized by both cloning and sequencing (111, 114, 267, 272, 383). In virus particles, the genome exists as a circular, partially double-stranded molecule with a nick at a unique site in the long strand of DNA (Fig. 1). The 5' end of the short strand of DNA is at a fixed position, but its 3' end is at different nucleotide positions in different virus particles. The genome can be made fully double stranded in vitro by further synthesis on the short strand by an endogenous DNA polymerase activity (291). The fully double-stranded genome is approximately 3.2 kb long and has four open reading frames (ORFs) on the long strand, which are ultimately translated into the virus gene 275 products. The first ORF encodes a family of hepatitis B surface antigen (HBsAg)-related polypeptides that make up the envelope of the virus. HBsAg polypeptides are also present in numerous small, spherical, and variably long filamentous particles in serum (92). The major HBsAg polypeptide is encoded by the S gene (114, 272). Several minor envelope polypeptides are made from the combination of the pre-s and S gene regions (100, 137, 401) and may encode the virus receptors for infection (Fig. 1) (2, 252). The second HBV ORF encodes the major hepatitis B core antigen (HBcAg) polypeptide, which is the major component of the virus nucleocapsid (4, 272). When a small precore region located contiguous to and just upstream from the core ORF is included in the protein product, such a polypeptide becomes proteolytically cleaved at the cell membrane and is then secreted into serum as the hepatitis B e antigen (HBeAg) (32, 223, 269, 382). The HBeAg in serum is associated with continued virus replication and high infectivity (207, 208). The third HBV ORF encodes a polypeptide known as the hepatitis B x antigen (HBxAg) (Fig. 1). A polypeptide of the size expected from the translation of the X ORF has been found in the serum of some HBV-infected patients with high levels of virus replication (96, 98). HBxAg has also been found associated with virus core particles, which is the site where virus replication actually occurs, again suggesting that this antigen is important to HBV replication (93). The trans-activating function of HBxAg (7,

2 276 FEITELSON CLIN. MICROBIOL. REV. extracellular virion infection DNA templates for transcription (p.',' -v~ FIG. 1. Schematic representation of the HBV genome as it appears in virus particles. The numbers, sizes, and relative positions of the ORFs are indicated. The positions and sizes of the major transcripts made during infection are also shown. The origin of minus (or long)-strand synthesis is at direct repeat 1 (DR1), while the origin of plus (or short)-strand synthesis is at direct repeat 2 (DR2). The dashed region of the short strand could be filled in and the genome made fully double stranded by the endogenous DNA polymerase activity associated with virus particles. Abbreviation: aa, amino acid. Reprinted with permission from Nature (London) (362). 56, 314, 331, 376, 378) may be important in stimulating virus or host cell gene expression during infection. HBcAg particles also include the products of the fourth HBV ORF, the HBV polymerase. The HBV-encoded polymerase is responsible for the endogenous DNA polymerase activity observed in virus particles from serum as well as for other activities which are necessary for viral replication (13, 14, 219, 336). HBV Replication and Natural History of Infection Upon infection of susceptible hepatocytes, HBV DNA becomes fully double stranded, and covalently closed circular (supercoiled) viral DNA then appears in the nuclei (233, 234, 374). It is believed that this supercoiled species encodes a greater-than-genome-length RNA (Fig. 2) that is then transported to the cytoplasm, where it becomes encapsidated in HBcAg particles (336). The long, or minus, strand of DNA is then synthesized from this template by reverse transcription, and during this process the RNA is degraded. The short, or plus, strand is then partially synthesized, and the HBcAg particles acquire envelopes and are secreted into the circulation as mature virions (Fig. 2). At 6 weeks to 6 months after a patient is exposed to HBV, HBsAg becomes detectable in the serum. The appearance of HBsAg is often accompanied by the appearance in serum of HBxAg and/or HBeAg, which are closely associated with viral replication (Fig. 3). Most people lose all of these antigens and seroconvert to corresponding viral antibodies within a few months, which usually indicates recovery from virus gene products immature core with pre-genome, DNA polymerase, and protein primer transitional core with full length minus strand core with packaging signal extracellular virion. reverse transcrption to DNA appearance of surface _0 antigen and virus in blood FIG. 2. Replication scheme of HBV and related viruses. Viral replication occurs in the cytoplasm of infected hepatocytes within immature core or nucleocapsid particles by reverse transcription of an encapsidated RNA pregenome. Modified and reprinted with permission from Cell (336). acute infection. Patients with persistent HBsAg in blood for more than 6 months usually become chronic carriers. Among carriers with detectable HBeAg, many lose HBeAg and spontaneously seroconvert to anti-hbe sometime during the course of chronic infection. This seroconversion is usually accompanied by an exacerbation of hepatitis in the liver prior to seroconversion and is followed by a decrease or disappearance of virus from the serum when anti-hbe appears. The HBsAg(+) chronic carrier state also spontaneously terminates after many years in some long-term infections, and the outcome may include seroconversion to anti-hbs. During the intervening period, however, patients may suffer multiple episodes of chronic hepatitis (95). Such patients are at a high risk for the development of cirrhosis and PHC. Hence, PHC develops most often among HBsAg carriers with progressive chronic liver disease.

3 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 277 A HBsAg AFP,,..ANTI-HBe anti-hbx B RR I TIME -I Infection > Chronic Carriers with Hepatitis -Cirrhosis/Dysplasia -Tumor - Death 0 0 Y WFECTION gr es 1. PERI-NATAL, CHILDHOOD INFECTION (FIRST EVENT) S CELLS: REPLICATION R CELLS: NO REPLICATK)N HOST NMUJNE RESPONSE 2. EARLY ADULT S CELLS: INFECTION, REPLICATION DEATH, CONTINUED INFECTION, REGENERATION AND SCARRING OF LIVER R CELLS: PRESERVATION, DIVISION, INTEGRATION 1.R S -*'R 3. MIDDLE AGE - INTEGRATION - SELECTION OF FAVORED CLONE - RAPID MULTIPUCATION OF R CELLS (SECOND EVENT) a~~~~~~~~~~a NOn"'' - 0s0...*00 4. FINAL EVENTS - TUMOR GROWTH.. METASTASIS FIG. 3. (A) Serological profile that often characterizes the HBV chronic carrier. Such carriers often have detectable HBsAg in serum for years or decades after the initial infection. Many patients with lengthy periods of virus replication also have detectable levels of HBeAg and/or HBxAg in blood. During chronic infection, many individuals lose HBxAg and seroconvert to anti-hbx while clearing virus from serum. This seroconversion is often followed by loss of HBeAg and the development of anti-hbe. Some carriers experience one or more episodes of chronic hepatitis, which can develop into cirrhosis and dysplasia. These patients are at high risk for the development of hepatocellular carcinoma, which is often accompanied by rises in serum alpha-fetoprotein (AFP). (B) Putative cellular model for PHC associated with chronic HBV infection. In this model (24, 25), a susceptible liver is rich in hepatocytes that support HBV replication (S cells). During chronic infection accompanied by bouts of hepatitis, S cells are recognized and lysed by immunological mechanisms, and the liver becomes increasingly rich in R cells, which do not support virus replication. Such R cells have integrated HBV DNA and in time may be selected for expansion. Eventually, some of these cells evolve into preneoplastic and neoplastic nodules. Modified and reprinted with permission from Cancer Detection and Prevention (25a). HBV-Like Viruses and PHC HBV is the prototype of a growing family of agents called hepadnaviruses (293). The best-characterized of these agents include HBV-like viruses that naturally infect woodchucks (woodchuck hepatitis virus [WHV]) (338), ground squirrels (ground squirrel hepatitis virus [GSHV]) (214), and some strains of ducks (duck hepatitis B virus [DHBV]) (220). Chronic infection of these hosts with their respective viruses results in the appearance of chronic hepatitis and PHC (215, 266, 276, 329, 330). This is especially true in WHV-infected woodchucks, of which nearly 100% develop PHC within 3 years (115, 275). These naturally occurring models further demonstrate a close relationship between hepadnavirus infection and liver cancer and have been very useful in teaching us more about the mechanisms responsible for PHC. EPIDEMIOLOGICAL LINK BETWEEN HBV INFECTION AND PHC There is much evidence suggesting a close association between HBV infection and the development of PHC. The importance of this relationship to public health is highlighted by the fact that PHC is among the most common cancers in the world (250,000 to 1 million new cases annually) (24, 94), that there are more than 300 million HBV carriers worldwide, and that chronic infection with HBV is closely associated with the development of liver cancer (25, 344, 387). The last point is supported by epidemiological data that show a close correlation between markers of HBV infection and PHC (16, 254, 256, 344, 409). In regions of the world where HBV infection is endemic (characterized by a high prevalence of carriers), there is a high incidence of liver cancer, whereas in most parts of the world with a low

4 278 FEITELSON prevalence of HBV carriers, there is a low incidence of liver cancer (203). About 80% of patients with PHC have evidence of HBV infection, as demonstrated by the presence of HBsAg and/or anti-hbc in blood, whether or not they reside in geographical regions where HBV is endemic, again suggesting a close correlation between markers of virus infection and PHC (175, 189, 348). Further, the fact that markers of HBV infection appear in the blood of infected patients years before the appearance of PHC argues for a cause-andeffect relationship (355). Among HBV carriers, liver cancer often appears after episodes of chronic active hepatitis (CAH) and cirrhosis (328). The close association of chronic WHV (115, 275) and GSHV (215) infections and PHC, as mentioned above, also underscores this linkage. Significance of In Utero or Perinatal Transmission of HBV to PHC In utero and perinatal transmission of HBV has provided additional evidence for a close relationship between HBV infection and PHC. In regions of the world where HBV is endemic, there are family clusters of HBV carriers and PHC patients (366). This clustering may reflect transmission of HBV from carrier mothers to children in the perinatal period or during the first few months of life (190, 257, 264, 335, 365). This type of transmission occurs most often from HBeAg(+) mothers (who are actively replicating virus) and accounts for almost half of the childhood carriers in populations with a high prevalence of HBV carriers (16). Further observations have shown that family members with PHC are more likely than age-matched controls to have mothers who are HBV carriers (16, 190). Hence, the incidence of the chronic carrier state after HBV infection is inversely related to the age at infection (16), with perinatal exposure often resulting in the development of the chronic carrier state and exposure later in childhood (or adulthood) often resulting in acute infection which resolves, instead of the chronic carrier state. The underlying cause for such a high carrier rate among infants infected with HBV may be an immature immune system. A number of recent results from a variety of systems provide support for this idea. First, T cells from HBeAg-expressing transgenic mice were unresponsive (or tolerant) to both HBeAg and HBcAg (230), suggesting that transplacental transmission of the soluble HBeAg from the blood of the carrier mother to the fetus could make the latter tolerant and allow ready establishment of the chronic carrier state. Second, T-cell-deficient nude mice injected intrahepatically with cloned HBV DNA developed many characteristics in common with human carriers, while normal littermates with mature T-cell functions did not become carriers (99), suggesting that mature T-cell functions are required to prevent establishment of the chronic carrier state. Third, treatment of adult woodchucks with cyclosporin A (which suppresses T-cell function) in the early phases of experimental WHV infection alters the outcome of such infections. Usually, experimental infection of adult woodchucks with WHV results in transient viremia or the appearance of viral antibodies in the absence of detectable virus, but when cyclo- administered during the early phases of experi- sporin A is mental infection, most animals develop the chronic carrier state (59). Hence, the development of the chronic carrier state and, later, PHC is heavily influenced by the mode and timing of infection. CLIN. MICROBIOL. REV. Factors Contributing to the High Relative Risk of PHC among HBV-Infected Patients Chemicals and other infectious agents. The importance of the chronic carrier state to the development of PHC is underscored by work showing that the relative risk that HBV carriers will develop PHC is more than 200 times that for noncarriers, which is one of the highest relative risks of an environmental agent known for a human cancer (16-18). In some populations, the lifetime risk that chronic carriers will develop PHC is as high as 40% in males, and almost half of all deaths in carriers 40 years or older have been attributed to PHC (16, 294). There is some evidence suggesting that the relative risk of PHC among chronic carriers is the same in regions of the world with a low incidence of HBV carriers as in regions with a high incidence of carriers (280), although the general applicability of these observations remains to be confirmed. Since other environmental carcinogens or promoting agents (for example, hepatitis C virus, aflatoxin B1 [AFB1], nitrosamines, alcohol, tobacco, and oral contraceptives) implicated in PHC (16, 171, 203, 347) are unequally distributed among regions with high and low incidences of chronic HBV carriers, the close correlation between chronic HBV infection and the development of PHC is consistent with the conclusion that HBV is able to cause PHC in the absence of other environmental agents. The latter point has been convincingly demonstrated by recent evidence showing that WHV can act as a complete carcinogen (275). If this is also true for HBV, other agents may either contribute to the incidence of PHC independently from HBV infection or act synergistically with HBV during chronic infection. Recent work, for example, has shown that HBV and AFB1 do not appear to act synergistically, since the ages of PHC patients with both HBV infection (i.e., chronic carriers) and AFB1 exposure (measured by determining the levels of aflatoxin- DNA adducts in liver) were not significantly different from the ages of PHC patients with evidence of HBV infection or AFB1 exposure alone (420). This is in apparent contrast to results of earlier work, which suggested that exposure to AFB1 in HBV carriers increases the risk of developing PHC (133, 205). However, exposure of WHV-carrier woodchucks to AFB1 had no effect on the appearance of PHC (357), which is compatible with a lack of synergy between WHV and AFB1. Exposure of DHBV-infected ducklings (which are highly susceptible to the effects of AFB1 [370]) to this carcinogen resulted in liver abnormalities and PHC independent of DHBV infection (60), again suggesting that DHBV and AFB1 do not act synergistically. However, the idea that chemical carcinogens contribute to the etiology of PHC in HBV-infected hosts has been supported by recent evidence in HBsAg-producing transgenic mice (73). Transgenic mice containing a single copy of HBV DNA without the core region were mated with another strain of mice that developed spontaneous hepatocellular carcinomas. The offspring (50% with HBV DNA and the other 50% without HBV DNA) were treated with a single dose of a known chemical hepatocarcinogen (diethylnitrosamine or p-dimethylaminoazobenzene). The numbers and sizes of proliferative nodules were significantly greater in carcinogen-treated HBVpositive mice than in carcinogen-treated HBV-negative mice. Further, HBV-positive mice treated with diethylnitrosamine developed more hepatocellular adenomas and carcinomas than did HBV-negative mice treated likewise, suggesting that transgenic mice with integrated HBV DNA sequences are more susceptible to the action of chemical hepatocarcinogens than are nontransgenic littermates (73).

5 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 279 The relevance of the observations in transgenic mice to chronic human infection, however, remains to be determined. Regardless of the contribution of other environmental agents to PHC, the finding of a higher incidence of PHC among HBV carriers than among noncarriers implies (i) that HBV infection may be all that is needed for PHC and (ii) that the continued expression of one or more viral antigens during chronic infection is important to the pathogenesis of liver cancer. Evidence in support of the first point comes from observations showing a very high incidence of PHC among experimentally infected neonatal woodchucks compared with uninfected controls (115, 275), suggesting that WHV acted as a complete carcinogen under controlled experimental conditions. Further observations have shown that HBsAg carrier children from an institution for the mentally retarded, which is free of most known chemical hepatocarcinogens, had a relative risk of developing PHC of more than 200 (200). Evidence in support of the second point comes from observations that HBxAg and HBsAg are common markers in the livers of chronically infected patients who are at high risk for the development of PHC (283, 393, 394, 418) and that each of these antigens may be expressed, in the absence of HBV replication, from viral nucleic acid templates integrated into one or more host chromosomes (see below). The finding that HBV gene products made from integrated templates stimulated the expression of many cellular genes (7, 42, 56, 169, 331, 378) is consistent with the possibility that such trans-activating properties result in alterations in cellular gene expression important to changing the patterns of cellular behavior from normal to neoplastic. In this way, HBV and like viruses may be complete carcinogens under experimentally controlled conditions. Under natural conditions, however, HBV-infected human populations are probably exposed to at least one other environmental agent, resulting in the very high incidence of PHC observed. The contribution of factors other than chronic HBV infection to PHC arises from observations that point to a discordance between the frequency of the HBV carrier state and the incidence of PHC in several parts of the world (133, 171). For example, some studies show a stronger correlation between PHC incidence and the estimated dietary intake of AFB1 than between PHC incidence and the geographic distribution of HBV infection (395, 412). A preliminary study has shown that in a population with a high HBV carrier rate and a low consumption of AFB1, the incidence of PHC was considerably lower than expected (226). Although the mechanism of AFB1-mediated liver carcinogenesis probably involves the formation of AFB1-DNA adducts in liver cells (62), suppression of cellular immunity by AFB1 may have contributed to the establishment or persistence of the HBV carrier state (205). Additional studies have shown that alcohol consumption, contaminated water, low selenium levels in the diet, tobacco smoking, certain occupations, and androgen therapy may also be important to the etiology of PHC and may contribute to the observed discordance between the frequency of PHC and of the HBV carrier state (133, 171). These observations suggest that the etiology of PHC could vary in different parts of the world. For example, PHC develops on a background of cirrhosis in both highincidence regions (Africa and eastern Asia) and low-incidence regions (Europe and North America) of the world (173). However, in high-incidence regions, CAH and cirrhosis develop as a consequence of the HBV chronic carrier state, whereas in low incidence areas, cirrhosis often develops as a consequence of prolonged high levels of alcohol intake (132, 415). Ultimately, the relative contributions of HBV and other factors to the etiology of PHC may become more apparent when children who live in areas of the world where HBV is endemic and who are undergoing HBV vaccination (345, 346) mature. If chronic infection with HBV is a leading cause of PHC worldwide, the incidence of this cancer should decrease, especially in endemic areas, in the coming decades. These observations would unequivocally show that the HBV vaccine prevents the development of one of the major cancers in the world. Age, gender, and sex hormones. Among HBV carriers, age, sex, chronic hepatitis, cirrhosis, and an elevated serum immunoglobulin M anti-hbc level have been identified as additional risk factors for PHC (16). On a worldwide basis, the incidence of PHC increases linearly with age in both male and female carriers (16). In areas of the world with a high prevalence of HBV carriers, PHC is roughly two- to fourfold more common in males than in females (243, 263). This difference in PHC incidence is thought to be due to hormonal differences between the sexes. Anabolic steroid therapy for bone marrow anaplasia (164, 225) and the use of oral contraceptives by women (12, 77, 211, 228) are both associated with increased incidence of benign and malignant liver tumors in the apparent absence of HBV infection. Estrogen receptors on human liver cells (74, 277) may mediate the association between oral contraceptives and PHC, but this remains to be shown. The finding of higher concentrations of functionally active androgen receptors on PHC tumor cells compared with normal hepatocytes (160, 241, 242), suggests that PHC growth may be stimulated by androgens (86, 138), which would contribute to the high incidence of PHC in males. Additional support comes from findings in transgenic mice that produce HBsAg. Pubescent male mice produced 5 to 10 times as much HBsAg as did age-matched female mice. Castration of the male mice resulted in concentrations of HBsAg reduced to the levels observed in female mice; replacement hormone therapy reestablished the high levels of HBsAg expression (88, 279). Additional support for a hormonal role in the appearance of benign and malignant liver tumors comes from work with PHC-prone transgenic mice with integrated HBV DNA (73). When these mice were treated with a single dose of a chemical hepatocarcinogen, male mice developed many more liver nodules and tumors than did female littermates similarly exposed, suggesting that the hormonal environment is an important determinant of tumor development. Further work showing that corticosteroids stimulate HBV replication in cell culture demonstrates that the hormonal environment could have a major impact on the regulation of virus gene expression and replication, which may be relevant to the establishment and maintenance of the chronic carrier state (373). The underlying basis for these observations is the presence of a glucocorticoid-responsive element in the genome of HBV and related viruses (372). By analogy with the way glucocorticoids change the patterns of gene expression among responsive cells, it is possible that selected glucocorticoids bind to appropriate receptors at the surface of infected cells and then migrate as a complex to the nucleus. Specific recognition and binding of this complex to the glucocorticoid responsive element within HBV DNA result in the stimulation in the expression of one or more HBV gene products. Sustained stimulation of HBV polypeptide synthesis and virus multiplication may promote the maintenance of the chronic liver state. Since the chronic carrier state is an important risk factor for the development

6 280 FEITELSON of chronic liver disease and PHC, continuing stimulation of the glucocorticoid responsive element of HBV may ultimately contribute to the high incidence of PHC in males. Chronic liver disease. Epidemiological evidence has also shown that PHC most often appears in HBV carriers with cirrhosis and/or CAH, although the risk for carriers without liver disease is also elevated, but less so (16, 149). PHC is common among HBV carriers with macronodular cirrhosis, whereas the risk of PHC in patients with micronodular cirrhosis is much lower (16). Further, the risk of developing PHC is much greater among symptomatic patients with cirrhosis than among asymptomatic patients with cirrhosis. It has been pointed out that PHC could arise from cells among regenerating cirrhotic nodules regardless of the cause of the cirrhosis (101, 165, 417). If so, the close association between cirrhosis and PHC among HBV carriers suggests that the chronic carrier state is an important cause of cirrhosis in endemic regions and that with the appearance of cirrhosis, the liver is ripe for the development of PHC (173). HBV may contribute to cirrhosis by stimulating the turnover of hepatocytes in response to removal of HBV-infected cells by immune responses during recurring bouts of CAH. This process of cellular turnover, which is an essential component of progressive liver disease associated with chronic HBV infection, is also fundamental to carcinogenesis (55, 61). Prolonged periods of rapid cellular turnover increases the probability of genetic mutations that are not repaired and that ultimately become fixed in rapidly dividing cells. The environment that stimulates this cellular proliferation will select cells which, by virtue of genetic alterations, have a selective growth advantage (24, 25). Many chemicals or other agents that increase cell proliferation (and, in so doing, act as promoters) during the course of chronic HBV infection may contribute to hepatocarcinogenesis, even though many are not genotoxic (55). For example, in areas characterized by a low incidence of HBV infection, PHC may arise in patients with micronodular cirrhosis associated with chronic alcohol consumption (173), although the increased risk is small (16). It is not clear whether alcohol (16, 262) (or other agents such as vinyl chloride, hormones, or cottonseed oil) acts as a tumor promoter or, in some cases, as a cocarcinogen (94) in conjunction with the HBV carrier state. In addition, any agent that stimulates cellular proliferation reduces the dose at which genotoxic chemical exposure results in cancer (55). Therefore, the contribution of various chemicals or other infectious agents to PHC may be partially dependent on whether progressive chronic liver disease is present. The importance of cellular proliferation to PHC has been convincingly demonstrated in transgenic mice making HBsAg polypeptides in the liver (50, 51). These polypeptides accumulated in the liver over time, and ultimately reached toxic levels, resulting in extensive hepatocellular necrosis and massive regeneration. Finally, nodules of PHC appeared. Similarly, PHC resulted from stimulation of hepatocellular growth by insertion of the simian virus 40 T (tumor) antigen and human growth hormone genes into the mouse genome (229, 271). Independent observations that PHC in children is associated with the rapid development of CAH and cirrhosis (153) also illustrate the central roles of severe liver injury and extensive regeneration in the pathogenesis of this tumor type. Recent work has shown that elevated levels of transformation growth factor alpha (which stimulates hepatocellular regeneration) and transformation growth factor beta (which is closely associated with enhanced procollagen synthesis and fibrosis) are likely to be important parts CLIN. MICROBIOL. REV. of the host response to hepatocellular necrosis, which accompanies the development of cirrhosis (43, 89, 126, 320). These results suggest that continued hepatocellular proliferation and regeneration during the course of HBV-associated chronic liver disease may promote the development of PHC after initiation by HBV, other infectious agents (e.g., hepatitis C virus [48, 57, 176, 261]), and/or hepatocarcinogenic chemicals. Even though progressive chronic liver disease was identified as a risk factor for PHC (16), PHC develops most often in cirrhotic patients who are also HBV carriers but not in cirrhotic patients who are not carriers (16, 256). These observations underscore the importance of the chronic carrier state to the pathogenesis of PHC. The facts that PHC is associated with a high mortality rate and that few effective treatments are available (108, 268, 305) emphasize that an understanding of the mechanism(s) responsible for the pathogenesis of PHC is crucial for determining and evaluating intervention strategies aimed at reducing the incidence of this tumor type. PUTATIVE CELLULAR AND MOLECULAR MODELS FOR PHC ASSOCIATED WITH CHRONIC HBV INFECTION Multiple lines of evidence on both the cellular and molecular levels demonstrate that HBV is a major etiologic agent of PHC. Hepadnavirus DNA has been found integrated into host DNA in both tumor and nontumor tissues. The sites of integration appear to be random with respect to cellular DNA. Integration is often accompanied by rearrangements of both virus and cellular DNA, resulting in translocation, amplification, deletion, and other alterations in host genetic material. Viral RNA and proteins have also been demonstrated in the cells of such primary tumors, although the functions of the viral proteins important to the pathogenesis of PHC are not known. Tissue culture cell lines derived from such primary tumors have provided additional evidence for a close association between HBV and PHC on the cellular and molecular levels. In all, the available literature suggests a number of mechanisms by which HBV and related viruses cause PHC. Integration Sites of HBV DNA in PHC Cells HBV DNA is integrated into host chromosomal DNA in both tumor and nontumor tissues (316, 404) in 80 to 90% of the published cases (292). Integration of hepadnavirus DNA in PHC cells from chronically infected woodchucks (329, 330, 338), ground squirrels (212, 215), and ducks (159, 413) has also been documented, demonstrating the generality of this property among these viruses. Integration of HBV at multiple sites within the host genome occurs early after infection (19, 29, 47, 153, 172, 355, 404). These observations imply that one or more integrated species may carry out functions important to the initiation and/or maintenance of PHC. This rationale has prompted a number of laboratories to carry out the cloning and partial sequencing of integrated viral DNA species (69, 143, 184, 406, 421). A salient feature of integration is that one virus-host junction often occurs in or around one of two 11-bp direct repeat sequences in viral DNA (69, 143) (Fig. 1). This finding has generated speculation that the viral templates for integration derive from replicative intermediates of HBV DNA, since HBV DNA replication initiates within these direct repeat sequences

7 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 281 (362) (Fig. 2). This suggestion is supported by additional evidence demonstrating that the pool of nuclear supercoiled viral DNA (234, 299) is maintained by the transport of DNA replication intermediates from the cytoplasm (234, 403). In the scheme of virus replication (Fig. 2), supercoiled viral DNA acts as a template for pregenomic RNA synthesis. The pregenomic RNA is then transported into the cytoplasm and becomes encapsidated in immature core particles. Reverse transcription of minus-strand DNA and synthesis of plusstrand DNA usually result in the production of mature virus particles. However, some of the core particles with these growing viral DNA strands (i.e., replication intermediates) are transported back into the nucleus, where both strands become full length and then ligated, so that they enter the pool of supercoiled molecules. With regard to integration, the finding of direct repeat sequences at one virus-host junction contrasts sharply with viral sequences found at the other virus-host junction, which vary with respect to the nucleotide position in viral DNA at the junction (321, 404). The facts that integration probably occurs by nonhomologous recombination (i.e., at sites with little or no homology with host DNA) and that it often occurs at the direct repeat sequences within viral DNA are consistent with the suggestion that one or more host enzymes carry out virus integration. Independent evidence has shown that the cellular enzyme topoisomerase I mediates integration of simian virus 40 DNA into host chromatin (34, 35), and recent experiments have shown that this may also be the case for HBV (390). Hence, the persistence of HBV in infected hepatocytes, some of which ultimately develop into PHC, occurs by integration of HBV DNA into one or more sites within the host DNA and is probably mediated by the action of one or more host enzymes, which include topoisomerase I. cis-acting Mechanisms of PHC Fusion proteins. Integration of virus DNA into chromosomal DNA may result in small deletions and/or duplications of virus and/or host sequences adjacent to the point of integration (143, 244, 349, 406, 421). Although the significance of these observations is uncertain, it is clear that many such integration events yield a structure in which the translation stop codon for the X gene is deleted. Expression of such integrated species can result in a unique polypeptide(s) consisting of sequences from adjacent virus and host ORFs (244, 270, 300, 349, 421). In some retroviral systems, such hybrid proteins are very important for transformation (287, 391, 399), and the same may be true for HBV. Fusion proteins consisting of HBV gene product(s) and one or more cell-encoded proteins, however, have not been reported. If fusion proteins are important to hepatocarcinogenesis mediated by HBV, then HBV integration should occur at a single site or at a very few sites within the host DNA so that the correct fusion proteins could be made. Instead, HBV integration has been reported to occur in chromosomes 2, 3, 4, 5, 6, 7, 9, 11, 15, 17, and 18 and in the X chromosome (22, 27, 66, 144, 222, 244, 292, 296, 421). It appears, then, that there is little specificity for the sites of HBV DNA integration into the host DNA during chronic infection and that integration occurs at many sites by illegitimate recombination. These considerations do not rule out the possibility that fusion proteins are made and play important roles in hepatocarcinogenesis, because it could be argued that it is the rare integration event at the appropriate gene that is important. In this context, there is evidence for integration of HBV DNA into sites of cellular DNA with limited homology to viral DNA (185, 349), implying that integration may not be completely random. In addition, there are short repetitive sequences within the human genome, referred to as variable number of tandem repeats, which have limited homology with DNA sequences within the X ORF (245). It is possible that these repetitive sequences are preferred sites of integration, although this has not been demonstrated. However, the fact that HBV DNA integrates at different host chromosomal sites in different patients (315, 316) argues against the hypothesis that integration at one or more common sites is crucial for the production of fusion proteins that are important to the development of PHC. In addition, the long latency period between HBV infection and the development of PHC in most cases (30 to >50 years) argues against the importance of the fusion protein(s) in transformation, since retroviruses which cause cancer by this mechanism are acutely transforming. The long incubation period also precludes the existence of a dominant oncogene encoded by the viral genome. Promotion-insertion. If there are sites within the host genome at which HBV integration commonly occurs, such integration events could result in enhanced or inappropriate expression of one or more neighboring genes. In this so called promotion-insertion model (136, 177, 303), viral integration could result in the altered expression of cellular oncogenes or proteins important to the regulation of the cell cycle. For example, several studies have shown that WHV DNA could integrate near the cellular c-myc locus, resulting in the apparent activation of this cellular oncogene (79, 156, 239). In this context, "activation" is defined as the introduction of a point mutation(s) into a cellular oncogene, the rearrangement of cellular oncogene sequences so that expression is enhanced under strong enhancer and/or promoter sequences, and/or the creation of many copies of the oncogene by the transformed cells. Similarities exist between this mechanism and that of c-myc activation in bursal lymphomas, which is mediated by integration of some avian leukosis viruses near the c-myc locus (274). It may be relevant that one of these avian leukosis viruses, MC29, causes PHC and has sequences related to c-myc within the region of the virus genome responsible for transformation (15, 319). In a study of 30 WVHV-infected woodchucks with PHC, 6 had insertions of WHV DNA next to c-myc. Eighteen of the 30 animals had elevated levels of c-myc RNA; some of the latter also had WHV DNA integrated near c-myc. Elevated c-myc expression in the absence of nearby integrated WHV DNA sequences may be due to transactivation of c-myc by one or more virus gene products (see below) and/or rearrangements in the c-myc locus. However, there are no reports demonstrating that HBV is integrated near c-myc in human tumor or nontumor tissues. In addition, the finding that c-myc expression is rarely elevated in HBV-associated PHC cells (91, 191, 384) suggests that promotion-insertion at a c-myc locus is not common and may not contribute significantly to the pathogenesis of PHC. However, amplification of the c-myc gene in tumor tissue compared with nontumor tissue has been observed in some cases (368), although the significance of this observation is not clear. The facts that PHC nodules consist of proliferating cells and that c-myc expression is elevated during liver regeneration (90, 91) suggest that the elevations of expression of c-myc (and other oncogene products) in tumors may be the consequence, rather than the cause, of cellular proliferation in tumors. Hence, the relationship of hepadnavirus DNA integration to c-myc activation and the importance of c-myc activation to PHC remain to be clarified.

8 282 FEITELSON The activation of c-myc in a fraction of woodchuck PHC cases is accompanied by changes in the number and size of c-myc-related transcripts (239). Additional characterization has shown that the c-myc locus in these tumors is rearranged and that in some cases, chromosomal rearrangements bring about a fusion between c-myc sequences and other cellular genes (79, 238). Such rearrangements may result in the production of chimeric gene products from two or more cellular genes, and these new products may contribute to hepatocarcinogenesis. In this regard, it is clear that hepadnavirus integration events are often associated with rearrangements in both viral and cellular DNA sequences (52, 144, 221, 259, 273, 297, 364) that may result in the production of chimeric cellular proteins. If such rearrangements are essential steps in the pathogenesis of PHC, a function of hepadnavirus integration may be to bring about insertional mutagenesis (67). The mechanisms by which viral DNA integration gives rise to mutations within cellular genes may be varied. For c-myc, direct insertion near the oncogene locus or rearrangement at such a locus alters the quantity and characteristics of the oncogene products made. In addition to using c-myc as a target for insertional mutation, one integrant of HBV DNA occurred near the gene that encodes the retinoic acid receptor (66). Further analysis showed that this integration results in the creation of a new gene consisting of the 5' end of the surface antigen gene fused with the gene encoding the retinoic acid receptor. The normal receptor binds vitamin A-related retinoic acid, which is important for the regulation of cell growth and differentiation (28, 67) and is related to a family of DNA-binding hormone receptor genes that are also involved in the same cellular processes (80). Integration of HBV DNA near the cyclin A gene has also been reported (392). The cyclin A gene encodes a protein important to the regulation of the cell cycle (90). The important roles of the products of these genes in the regulation of cell growth and differentiation are consistent with the conclusion that their altered expression may be a factor in the etiology of some cases of PHC, but such integration events seem to occur in only a tiny fraction of human PHC patients examined. In this context, it is important to point out that the patterns of HBV expression and replication are dependent on the state of differentiation of an infected cell (5, 45, 148, 246, 282, 369). Integration of HBV near genes encoding proteins important to the cell cycle may stimulate growth and thus contribute to the appearance of PHC. Changes in the patterns of cell growth in the liver are likely to be accompanied by changes in the state of cellular differentiation, so that the proliferating hepatocytes may be less capable of supporting HBV replication than are fully differentiated hepatocytes at rest. The observation that virus replication decreases as the duration of chronic HBV infection increases (316) is consistent with this hypothesis. A cellular model of chronic HBV infection (24, 25) (Fig. 3) proposes that early during infection (but after infancy), most of the hepatocytes in the liver are fully differentiated nondividing cells susceptible to HBV infection and capable of supporting virus replication. With the removal of such cells by various immune responses during chronic infection, and with increasing integration of HBV DNA that results in insertional mutagenesis, more and more of the cells in the liver become less differentiated proliferating cells which are less capable of being infected by HBV and of supporting HBV replication. Additional evidence in support of the idea that there is a relationship between hepadnavirus replication and the state of cellular differentiation comes from studies showing that CLIN. MICROBIOL. REV. DHBV replicates in primary duck hepatocytes that have been freshly plated but does not replicate in cultures that dedifferentiate in culture over several weeks (374, 375). Further, the susceptibility of primary duck hepatocytes to infection decreases as the time after plating increases, implying that changes in host cell differentiation have profound effects on the host-virus relationship. Moreover, it is known that the surface promoter (45, 81, 282, 361), pre-sl promoter (246), core promoter (148, 204, 361, 411), and enhancer of HBV (5, 148, 318, 361, 369) are most active in differentiated cell lines of hepatocellular origin, less active in undifferentiated hepatocellular lines, and often inactive in other cell lines. Probably as a result of these activities, HBV replicates in differentiated but not in undifferentiated hepatoma cell lines. Hence, infection of susceptible hepatocytes with HBV results in HBV integration that is likely to have a major impact on the host-virus relationship that evolves. Putative role of other oncogenes. In addition to the activation of c-myc, activation of other known or newly described oncogenes in PHC patients has been reported (110, 135, 324, 419). The associations of point mutations with chemical hepatocarcinogens and of genetic instability (see below) with chronic HBV infection are consistent with the hypothesis that activation of one or more cellular oncogenes may accompany hepatocarcinogenesis. For example, one report demonstrates coamplification of integrated HBV DNA sequences with a newly characterized transforming gene, hst-i (135). Other studies suggests that the oncofetal gene Ica may be reactivated in PHC cells (258, 324). AFB1 is capable of activating ras oncogenes associated with PHC in rats (224). C3B6F1, a mouse strain prone to the development of spontaneous PHC (105, 288), is also highly susceptible to oncogene activation in response to chemical hepatocarcinogens (289, 398). Again, one or more of the ras oncogenes are the major targets for these chemical carcinogens. Activation of the oncogene N-ras has been reported in PHC (350), but on close examination the gene was found to be activated only in a small fraction of tumor cells, suggesting that such activation may be secondary to the generation of PHC and, instead, may reflect tumor heterogeneity. An activated c-raf oncogene has also been documented in chemically induced rat PHC cells (161), again suggesting a close correlation between oncogene activation and hepatocarcinogenesis. It is not clear, however, whether activation of oncogenes actually contributes to the cause or is the consequence of transformation, since increased oncogene expression is characteristic of growth accompanying fetal liver development and liver regeneration (90, 91). The central importance of activated oncogenes to the genesis of various tumors (37, 58), including PHC (188), has prompted additional studies addressing a possible relationship between HBV DNA integration and alterations in oncogenes and/or oncogene expression. Investigations of integrated HBV DNA and flanking host sequences have failed to consistently show viral integration near one or more known oncogenes (52, 181). Other observations have shown that dominant oncogenes are not rearranged in PHC cells as a result of chromosomal alterations (384). However, several laboratories have shown the existence of activated oncogenes in PHC cells, in that DNA isolated from primary tumors or PHC-derived cell lines were capable of transforming NIH 3T3 cells from a nontumorigenic to a tumorigenic cell line (40, 119, 121, 161, 255, 258, 292, 408). The DNA sequences isolated from most of the transformants did not contain HBV DNA, implying that HBV was not required for maintenance of the transformed state. The combined data

9 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 283 suggest that although activation of cellular oncogenes could result in liver tumors under experimental conditions, these events are not common in HBV-associated PHC. Many changes in the patterns of cellular gene expression accompany hepatocarcinogenesis. These changes could come about as a result of hepatocyte dedifferentiation (381) and/or proliferation of immature stem cells in the liver (236, 278). Work has shown that the earliest cellular response to chemical carcinogen exposure in the liver involves the proliferation of small nonparenchymal cells referred to as oval cells (83, 311). These cells, which are morphologically and biochemically distinguishable from mature hepatocytes, proliferate under defined circumstances and differentiate into mature hepatocytes (202, 310). Oval cells are also the precursors of bile duct epithelium and pancreatic cells (310, 359). The fact that hepadnaviruses infect bile duct epithelium (23, 60, 379, 393) and subpopulations of cells within the pancreas (68, 127, 128, 163, 323) in addition to hepatocytes raises the question of whether infection of oval cells provides an important source of rapidly proliferating infected cells that give rise to tumors at these sites. The findings of hepadnavirus antigens in bile duct cells and tumor cells from patients with cholangiocarcinoma (393) and of HBsAg within nontumor cells from some patients with pancreatic carcinoma (146) are consistent with these ideas. The finding that oval cells proliferate only under conditions in which mature hepatocytes do not (e.g., because of the toxic effects of carcinogens on hepatocytes) (70, 83, 311) suggests that when mature hepatocytes supporting HBV replication are eliminated during the course of chronic liver disease, some regenerative nodules are derived from these dividing oval cells. If oval cells are infected, the genetic instability associated with hepadnavirus infections (see below) may provide some proliferating oval cells with the growth advantage needed to take the next step toward neoplasia. With rapid cell division, the expression of cellular oncogenes and other growth factors would increase significantly (109). In this way, oncogene expression would be tightly linked to the regenerative responses that occur during episodes of chronic liver disease and would be associated with an increased risk of developing PHC. In rat liver, there is sustained elevation of cellular oncogene expression in oval cells proliferating in response to chemical carcinogens (410). Thus, both chronic HBV infection and chemical carcinogen exposure are associated with increased oncogene expression (or oncogene activation) and may be important in better understanding the pathogenesis of PHC (55, 61). Genetic Instability and Recessive Carcinogenesis Chromosomal rearrangements and loss of antioncogenes. The persistence of hepadnavirus DNA in liver and tumor nodules has been central in implicating these viruses as causative agents of PHC (221, 292, 316). However, roughly 15% of HBsAg carrier patients with PHC do not have evidence of HBV DNA integrated into tumor tissue (104, 232, 317, 337). The absence of integrated hepadnavirus DNA has also been observed among infected woodchucks (275, 276), ducks (213), and ground squirrels (292). Among some populations of ducks, PHC is more closely associated with AFB1 exposure than with DHBV infection (60, 370, 379). Among chronic carrier woodchucks and ground squirrels, however, the absence of integrated virus DNA implies that hepadnavirus infection may be required for the initiation but not maintenance of PHC. The absence of integrated virus DNA in PHC from infected hosts may be the consequence of the genetic rearrangements and deletions that accumulate during the course of infection. In WHV infection, for example, the structures of integrated WHV DNAs from the livers of chronically infected animals were colinear with those found in cloned isolates of the virus genome (297), demonstrating that integration events occur without rearrangement of virus sequences. In liver tumors from WHV-infected animals, the structure of the integrated WHV DNA was highly rearranged (259), suggesting that rearrangements occur during the course of chronic infection. As presented above, genetic rearrangements occurred in both viral and cellular sequences, suggesting that integration may be associated with the generation of chromosomal aberrations. To test this hypothesis, the ability of an HBV DNA integrant from a human PHC to give rise to chromosomal aberrations in transgenic mice was assayed ( ). Transgenic mice were made by injection of a fragment of PHC DNA (containing an inverted repeat of HBV DNA at the origin of virus replication) that had previously been cloned from a PHC patient who had an HBV-associated chromosomal translocation between chromosomes 17 and 18 (144) as a result of gene rearrangements in vivo. Deletions in and rearrangements of the HBV DNA-containing clone were observed in descendants of some transgenic lineages ( ), indicating that the transgene was unstable during transmission. The results also demonstrate that these types of genetic changes occur in somatic cells undergoing mitosis (and not in gametes undergoing meiosis), which is a situation analogous to that of hepatocytes undergoing regeneration during chronic liver disease. The significance of such findings is underscored by previous work showing that agents which cause chromosomal aberrations are associated with an increased risk of cancer (120, 171, 177, 310, 315). Hence, integration of HBV DNA during chronic infection may create genetic instability, which is an important step in the pathogenesis of PHC. If a major effect of HBV DNA integration into host chromatin is to induce chromosomal mutations and instability (140) and if such alterations in the host DNA are not repaired, some of these changes will become permanent and confer growth advantages on colonies of cells beyond that necessary for regeneration. In this context, the regenerative stimulus provided by progressive liver disease promotes the development of foci that eventually develop into nodules of PHC. These relationships may explain the much higher risk of PHC among HBV carriers with progressive chronic liver disease than among HBV carriers with little or no liver disease. There is considerable evidence that cancer may be caused by the mutation or loss of genes encoding proteins responsible for maintaining cellular differentiation and for suppressing uncontrolled cellular proliferation (301). These tumor suppressor genes (178, 301), or antioncogenes (180), are capable of suppressing the tumorigenic phenotype of neoplastic cells in culture and in animals (178, 301, 332, 333). Given that HBV DNA is associated with chromosomal instability during the course of chronic liver disease, it is possible that the consequence of such genetic instability is somatic mutation that results in the inactivation and/or loss of one or more tumor suppressor genes. In this model of carcinogenesis, one of the two alleles from a putative tumor suppressor gene becomes nonfunctional through mutation at one or more points or through deletion (Fig. 4). When the gene is lost through deletion, the individual becomes hemizygous for the wild-type gene. This situation leaves an individual at high risk for the development of PHC. When

10 284 FEITELSON CLIN. MICROBIOL. REV. PHC in children PHC in Adults Heritable Sporadic 9? 1,2 2::2 *.+ +6'+ 1,2 2::2 1 '2 * 6+ 1,.2 Constitutional 1 2 2,, ,', ::2 Q. 1 : Downloaded from ( 9 1 '2 Predisposed cell * 1 :2 on September 2, 2018 by guest tumor 1 1 * U U I j 1 El ii 0 1 '2 *. 1 : 2 I

11 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 285 FIG. 4. Chromosomal mechanisms by which an initial predisposing recessive germinal or somatic mutation could be revealed. In a hereditary case (left) the child inherits from his or her parent a chromosome that carries a recessive defect at a tumor suppressor gene locus (-), so that the child is genotypically E/+ in all of his or her cells. A tumor would occur after the elimination of the dominant wild-type allele at the tumor suppressor gene locus by one of the mechanisms shown at the bottom of the diagram. In a sporadic case (right), a recessive mutation occurs in a single predisposed cell and a tumor occurs when the recessive mutation is unmasked by one of the mechanisms shown at the bottom. Modified and reprinted with permission from Cancer Research (130). the function of the second allele is lost through additional somatic mutations (point mutations or deletions), the loss in heterozygosity often correlates with the appearance of a particular tumor type (130). Cell proliferation during chronic liver disease promotes the loss of heterozygosity through nondisjunction with or without reduplication, recombination, or other genetic events (Fig. 4). Under these circumstances, genetic differences between tumor and surrounding nontumor tissue from the same patient can be detected by restriction fragment length polymorphism mapping. This multistep process, which results in the loss of function of both tumor suppressor alleles, often takes years or decades to occur and illustrates the recessive character of such tumors. Loss of heterozygosity on chromosomes 4 (33, 388, 419), 5 (110), 8 (327), 10 (110), 11 (296, 388, 389), 13 (388, 389), 16 (110, 327, 419), and/or 17 (110, 144, 327, 388) has been found in patients with PHC. These results imply the existence of one or more tumor suppressor genes within regions where loss of heterozygosity was detected. The importance of tumor suppressor genes to PHC will have to be demonstrated by using somatic cell hybridization and gene replacement experiments (11, 158, 367, 396). Regardless of the results, genomic instability associated with chronic HBV infection is consistent with the long latency period prior to the appearance of PHC, with observations that different fragments of HBV DNA are present in different tumors, and with the fact that some established tumors from infected patients have no detectable HBV DNA (110, 292). The last point implies a "hit-and run" mechanism for HBV-associated PHC and is consistent with the hypothesis that integrated HBV DNA could promote genetic aberrations resulting in the loss of tumor suppressor genes. Such genetic instability could also result in the amplification of oncogene products and/or the activation of growth regulatory molecules. Known tumor suppressor genes (44, 102, 193, 217) have not been found in association with most of the chromosomes with documented loss of heterozygosity. Loss of heterozygosity at chromosome llp results in the loss of the H-RAS1 oncogene (389) and the WAGR locus, both of which are also lost in Wilms' tumor (an embryonal carcinoma) (107, 286, 290). It has been proposed that the loss of one or more tumor suppressor genes from chromosome llp results in the activation of insulinlike growth factor II, which may stimulate tumor growth (109) analogous to that observed with Wilms' tumor (285, 304). Enhanced insulinlike growth factor II expression has been found in several cell lines derived from human PHC and in primary tumors from both HBV-infected humans and WHV-infected woodchucks (38, 39, 109, 295). The loss of heterozygosity at chromosome llp in PHC cells includes loss of the region that is responsible for the appearance of certain embryonal tumors such as rhabdomyosarcoma, Wilms' tumor, and hepatoblastoma (187). Chromosome 13q14 is the location of the retinoblastoma locus (RB-I), which encodes one or more tumor suppressor products, whose loss results in sporadic and hereditary retinoblastoma (44, 193). Aberrations in the RB-I gene have recently been found in other tumors such as breast carcinoma (356) and small cell lung cancer (131), although preliminary analysis suggest that the RB-I gene is structurally intact in Wilms' tumor and PHC (356). Most recently, however, loss of heterozygosity at 13q, which includes the RB-I gene, has been observed in several HBV-negative PHC patients (388). The finding that the loss of heterozygosity occurs at some chromosomes in several patients with PHC and at other chromosomes in other patients suggests that these deletions are not random but that the pathogenesis resulting in PHC may be heterogeneous on the molecular level (389). Further evidence consistent with this interpretation comes from a study that directly characterized the karyotype of a PHC (326). The results showed abnormalities in chromosomes 1, 5, 6, 9, 13, 16, and 22 that appeared to occur at fragile sites and/or near selected cellular oncogenes. Various rearrangements among most of these chromosomes were observed, and chromosomes 13 and 16 were deleted. Combined with the lack of HBV DNA associated with this tumor, the results again illustrate that PHC arises from cells that have undergone somatic mutations in the form of chromosomal rearrangements and deletions and that such aberrations appear to be central to the pathogenesis of PHC independent of HBV status. p53, AFBj, and hepatocarcinogenesis. There is some evidence that the p53 tumor suppressor (194) may also play an important role in the pathogenesis of PHC. The p53 gene, located on chromosome 17p, encodes a protein that negatively regulates cell proliferation (9, 253). One or more point mutations within the p53 gene result in a protein that promotes cellular proliferation instead. In the former case, wild-type p53 acts as a tumor suppressor, whereas in the latter case, mutant p53 acts as an oncogene (194). HBV DNA integration has been described near the p53 gene in a single PHC patient as an inverted repeat structure containing virus and cellular sequences (421). Further analysis showed that the genetic instability associated with this type of inverted repeat structure resulted in the loss of distinct but overlapping chromosome 17 fragments containing the p53 gene in more than half of the 19 PHC patients examined (327). This observation supports the hypothesis that the mutation or loss of p53 may be an important step in the pathogenesis of PHC. Significantly, the loss of the p53 locus has been detected in a number of other solid tissue tumors, including osteosarcoma (240, 363), astrocytoma (112, 162), colorectal carcinomas (9), neurofibrosarcoma (227), breast cancers (206), and lung cancers (352, 414). The importance of mutation and deletion in p53 alleles to carcinogenesis (194) was further explored by using liver cell lines derived from PHC patients. p53 gene mutations were observed in some (30, 150) but not all PHC cell lines tested (150). Further, the levels of p53-specific RNA in a human PHCderived cell line were unchanged relative to those in the same cell line containing replicating HBV (150), suggesting that HBV does not affect p53 transcription. In contrast, cloning and DNA sequence analysis of the p53 exon sequences from most but not all PHC tissues (from Taiwan)

12 286 FEITELSON showed no evidence of mutations within regions of the gene prone to mutagenesis in other tumors (150). PHC DNA from other populations of patients (from Africa and China), however, had a high frequency of p53 mutation at a single site within the p53 gene compared with nontumor DNA from the same patients (31, 155). Among 16 Chinese patients, half had a point mutation within codon 249 of exon 7 from the p53 gene (155). Among 10 PHC patients from southern Africa, 3 had this same point mutation and 2 others had different mutations within the same p53 exon (31). Three of the African patients with p53 point mutations also showed loss of heterozygosity at chromosome 17p. Given that AFB1 exposure is high in both of these geographical regions and that the point mutation at codon 249 is consistent with those caused by AFB1 in mutagenesis experiments (103, 224, 302), the study suggested that the mutations in p53 were likely to be due to AFB1. Hence, in regions of the world characterized by high AFB1 exposure, which are similar to regions of high HBV carrier prevalence, the very high rates of PHC may result from chromosomal rearrangement (at chromosome 17p and elsewhere) associated with chronic HBV infection and point mutations in p53 associated with longterm AFB1 exposure. In addition, AFB1 requires metabolic activation to exert its carcinogenic effects (62, 118). Recent experiments have shown that liver homogenates from HBVand WHV-infected hosts have enhanced abilities to activate AFB1 and several other chemical hepatocarcinogens compared with liver homogenates from uninfected hosts (64, 65), implying that viral and chemical hepatocarcinogens may act synergistically on the molecular level to produce PHC. A characteristic central to the pathogenesis of PHC in most patients is the fact that 30 to 50 years often pass from the time of HBV infection to the appearance of liver cancer. This long period may reflect the time needed for multiple, rare events to occur for tumor development. This multistep nature of carcinogenesis has been described in well-developed models of chemical hepatocarcinogenesis (84, 85, 310), as well as in sporadic carcinogenesis of other solid tumors (217). In this regard, there have been several reports in the literature documenting the presence of PHC in HBV-infected children (46, 153, 172, 260, 355, 404, 405). The relatively short incubation time for PHC in children is consistent with the hypothesis that these children have a genetic predisposition for the development of such tumors. Under these circumstances, tumor susceptibility would be inherited in the form of a critical mutation(s) in the germ line, and the somatic mutations accumulating as a result of HBV infection would significantly shorten the period between infection and tumor appearance. Documented examples of hereditary tumors (123, 180, 187) point out the importance of germ line mutations in one or more tumor suppressor genes as the major predisposing event. In this model, sporadic cases of PHC would require mutation or loss of both alleles of a tumor suppressor gene, but when a mutation in one of the alleles is inherited, mutation or loss of the remaining allele of a tumor suppressor gene is all that is needed for liver cancer to develop (Fig. 4). The age at which PHC appears would also depend on other factors, such as exposure to aflatoxins, HBV, and/or hepatitis C virus. Clearly, germ line mutations in the RB-I or p53 antioncogenes are associated with several hereditary cancers (20, 44, 179, 210), and it is possible that familial studies will reveal mutations in one or more tumor suppressor genes which contribute to the appearance of PHC in children. CLIN. MICROBIOL. REV. HBV Gene Expression: Putative Roles in PHC Genetic contribution of hepadnaviruses to cancer. In light of all these studies, the question still arises whether hepadnaviruses make a genetic contribution to PHC (384). The recent observation that GSHV could infect woodchucks may provide an important clue (308). It is clear from the natural history of GSHV and WHV infections that PHC developed in about 30% of GSHV-infected carrier ground squirrels 4 to 5 years of age (212, 215) compared with nearly 100% of WHV-infected carrier woodchucks 2 to 3 years of age (275, 329, 330). When woodchucks were infected with GSHV, however, only about 40% developed PHC at 4 to 5 years of age (306). The observed differences in the behavior of the two viruses (GSHV and WHV) in a single host (the woodchuck) suggest that the differences in tumorigenicity are in the viruses themselves. Since the genetic organizations of GSHV and WHV are very similar (113, 307), it is possible that differences in virus gene products acting in trans on the host cell are crucial for the observed variation in the tumorigenicity of these two viruses. Immunological targeting of virus antigens: relevance to pathogenesis of PHC. Immunohistochemical studies have demonstrated the presence of HBsAg (116, 145, 154, 248, 250, 283, 340), HBxAg (134, 170, 341, 393, 418), and, to a lesser extent, HBcAg (145, 154, 250, 340) and pre-s (71, 72, 106, 125, 139, 358, 360) in tumor and/or nontumor liver cells of patients with PHC. These results imply that the expression of one or more HBV antigens may be significant in the pathogenesis of PHC. HBsAg, for example, is associated with the membranes of hepatocytes in patients who are actively replicating HBV (283); this suggests that HBsAg determinants may be important targets for immune elimination during CAH. In transgenic mice making HBsAg, spleen cells from nontransgenic syngeneic mice previously immunized with purified HBsAg particles were capable of recognizing and destroying host HBsAg-positive hepatocytes (237). However, the facts that there is no correlation between HBsAg expression and liver abnormalities (3, 152, 283) and that HBsAg-specific T cells have not been detected in patients with CAH (75, 76, 385) suggest that HBsAg is not a target for cellular immune responses under most circumstances during infection. Alternatively, the finding that prolonged overproduction of HBsAg by transgenic mice results in massive hepatic necrosis, extensive cellular inflammatory responses, widespread hepatocellular regeneration, and, finally, PHC (50, 51) implies that high levels of antigen production could be directly cytotoxic and that the accompanying regeneration could be important to the creation and propagation of preneoplastic nodules. The facts that HBV replication in tissue culture cells is not accompanied by cytopathic effects (298, 312, 322, 339, 371) and that many HBsAg carriers do not have clinical or biochemical evidence of liver damage (i.e., they are asymptomatic carriers) (149) show that HBV is not directly cytopathic. Hence, HBsAg is probably not a target for immune elimination of hepatocytes during HBV infection. There is considerable evidence that HBcAg is a target for immune elimination (95). For example, there appears to be a close relationship between HBcAg and hepatocyte cell membranes in patients with high levels of virus replication (122, 247, 249, 407). The widespread distribution of HBcAg in the livers of immunosuppressed patients is also consistent with the conclusion that an immune response against core antigen may be important in the elimination of cells that are replicating virus (21). The inverse relationship between HBcAg

13 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 287 expression and the severity of liver disease further implicates HBcAg as a target of the immune system (21, 26, 36, 54). In addition, the close association between immunoglobulin M anti-hbc and virus replication during chronic infection (10, 182, 281) and between virus replication and chronic liver disease (CAH and cirrhosis) may explain why both immunoglobulin M anti-hbc and chronic liver disease are risk factors for PHC (16). In addition to HBcAg, the related HBeAg has been proposed as a target related to the pathogenesis of chronic liver disease (95), especially since its biosynthesis involves one or more membrane-associated steps (32). However, the finding of severe CAH in HBsAg carriers who are HBeAg(-) and anti-hbe(+) (53, 124, 201, 284) and the fact that such carriers often have circulating virus with one or more mutations in the precore region (which effectively prevent the synthesis of HBeAg) (1, 41, 380) suggest that another virus antigen target must be responsible for CAH under these circumstances. The finding of virus(es) containing precore mutants associated with cases of fulminant hepatitis (197, 265) also indicates that HBeAg is not involved in the pathogenesis of severe liver disease in such individuals. Hence, HBcAg and HBeAg may be important immunologically recognized targets at some but not other times during the course of chronic infection. There is some evidence that HBxAg is a target for immunological recognition and removal of infected cells. For example, HBxAg is present in the liver in both HBeAG(+) and anti-hbe(+) carriers (134, 167, 341). Forty to 50% of carrier patients with chronic hepatitis B have evidence of membranous HBxAg (394). The persistence of HBxAg may provide an immunological target throughout chronic HBV infection and may contribute significantly to the progression of chronic liver disease and the development of PHC. The detection of anti-hbx in blood (49, 78, 97, 168, 196, 334, 386) and HBxAg-reactive T lymphocytes in the peripheral circulation (166) is also consistent with the recognition and targeting of X determinants during HBV infection. It is likely, however, that host immune responses to several virus antigens are important for the recognition and removal of infected cells. Further work will undoubtedly focus on which HBV products are targets and when during infection targeting occurs. Evidence for trans-activating mechanisms in PHC: putative roles for pre-s and HBxAg gene products. Much work has focused on the possible cis-activating mechanisms that may contribute to hepatocarcinogenesis. However, increasing evidence suggests that one or more virus gene products may contribute to PHC by way of altering the expression of host cellular proteins during infection. This mechanism, referred to as trans-activation, proposes that integrated HBV DNA serves as a template for the production of one or more virus gene products that participate in altering the behavior of the host cell in ways relevant to the development of PHC. Recent reports have identified regions of integrated HBV DNA from PHCs that were capable of stimulating the expression of heterologous genes. One of the cloned regions contained the pre-s2 and a 3'-truncated portion of the S gene, which stimulated both the expression of reporter genes (e.g., chloramphenicol acetyltransferase or luciferase) in nucleic acid constructs and the expression of c-myc (42, 169). The importance of the latter point has been demonstrated by a 5- to 50-fold activation of c-myc expression in several PHC cases associated with chronic WHV infection (239). The lack of integrated WHV sequences within 5 kb of the c-myc locus implies that c-myc activation is not due to the proximity of a virus promoter but instead may be due to the trans-activating properties of a truncated pre-s2/s product. Interestingly, full-length pre-s2/s polypeptide (made from episomal or free virus DNA during viral replication) has no trans-activating properties. It is possible, then, that the deletions and rearrangements of viral DNA that accompany hepadnavirus DNA integration generate new protein species capable of distinctly different biological roles. The possible significance of a truncated pre-s2/s polypeptide to the pathogenesis of PHC remains to be explored. A similar set of observations for the X-gene region of HBV DNA have been reported. Many laboratories have shown that HBxAg is capable of trans-activating a growing list of virus and cellular genes. These include the HBV enhancer, HBV X and core promoters, herpes simplex virus tk promoter, simian virus 40 enhancer and early promoter, and long terminal repeat sequences of human immunodeficiency virus types 1 and 2 among virus sequences (56, 195, 314, 331, 377, 417) and beta interferon, AP1, AP2, RNA polymerase II and III promoters, c-myc, c-fos, and class I major histocompatibility complex among cellular sequences (7, 56, 157, 313, 325, 378, 421). Integrated HBV DNA sequences cloned from PHC tissue are capable of synthesizing HBxAg, which is, in turn, capable of trans-activation (351, 400, 416). Analogous to pre-s2, HBxAg may stimulate c-myc expression in at least some PHC cells by virtue of its trans-activating properties. Additional work has shown that HBxAg is not a DNAbinding protein and that it probably mediates trans-activation by binding to one or more intracellular protein factors (e.g., CREB and ATF-2) that regulate transcription (209, 313). It is thought that the binding of HBxAg to such regulatory proteins alters their DNA-binding characteristics and, as a result, the patterns of virus and host gene expression. These observations suggest that the ability of HBxAg to mediate trans-activation is dependent on the availability of such regulatory proteins, which varies according to the cell cycle (218) and the state of hepatocyte differentiation. The implication of these observations is that the transactivation function(s) of HBxAg may differ widely in asymptomatic carriers without liver disease (in whom hepatocytes are resting and fully differentiated) compared with carriers with CAH or cirrhosis (in whom hepatocytes are undergoing division and may be less differentiated). The correlation between trans-activation and the ability of HBxAg to stimulate HBV gene expression in one human hepatoma cell line, but not in another (183), also suggests that HBxAg function is sensitive to the state of cellular differentiation, which differs in these two cell lines. The mechanism(s) by which HBV mediates trans-activation may be multiple because HBxAg has been found in different regions of infected cells. For example, most of the HBxAg staining observed in liver cells from chronic carriers occurred in the cytoplasm (134, 167, 170, 341, 386, 393, 394, 418). It has been suggested that cytoplasmic HBxAg may interfere with the signal transduction pathway mediated by protein kinase C (186). However, the ability of HBxAg to act as a transcriptional trans-activator is consistent with its being found in the nucleus of hepatocytes. Nuclear localization of HBxAg has recently been observed by immunohistochemical staining in liver sections from more than 70% of HBV carriers with cirrhosis and dysplasia but fewer than 10% of patients with chronic hepatitis B (394). This staining pattern seems to parallel the finding that PHC develops most often on a background of cirrhosis and dysplasia and much less often on a background of chronic hepatitis. The results support the hypothesis that HBxAg made from integrated HBV DNA may play a role in the initiation of PHC by (i)

14 288 FEITELSON CLIN. MICROBIOL. REV. HBV -_ Infection sumiicient Immune responses anti-hbag, CMI HBeAg, HBxAg in 1. elimination of hepatocytes expressing membranous 0- HBAgs _ little or no HBV antigens or pathology in liver low risk -- for PHC serum HBeAg and/or HBxAg persists in serum, aborted or insufficient immune responses 2. persistence of HBAg+ hepatocyte virus replicative phase - CAH -- cirrhosis -- infection removal of new of HBAg + cells cells 3. * 4. transactivation of cellular --. genes and/or phosphorylation of cellular proteins by HBxAg nonreplicative phase FIG. 5. Schematic representation of the postulated role(s) for HBV antigens (HBAgs) and corresponding immune responses in the pathogenesis of PHC. HBV-infected hepatocytes release HBxAg into the serum along with HBV, HBsAg, and HBeAg. Some infected hepatocytes have cytoplasmic and/or nuclear HBAgs. A subset of infected cells also have plasma membrane-associated HBAgs. (1) If membrane-associated HBxAg, HBcAg, and/or HBeAg serves as the target for an antibody and/or cell-mediated immune (CMI) attack, an appropriate immune response will result in the removal of antigen-positive hepatocytes and recovery from HBV infection (top row). Such individuals would be at reduced risk for the development of PHC. (2) If an aborted or insufficient immune response occurs during infection, HBAgs will persist in liver and serum. Under these conditions, individuals would not seroconvert to viral antibody, and high levels of virus replication would persist. (3) Infection of new hepatocytes with HBV, combined with the ongoing expression of HBAgs in hepatocytes that are not removed, continues to stimulate partially effective immune responses that serve to perpetuate chronic hepatitis, which slowly gives way to cirrhosis (bottom row). (4) The destruction of cells with membranous HBAgs and those producing virus results in proliferation of hepatocytes that do not have membranous viral antigens. The latter group would not support virus replication because they represent a state of differentiation different from that among hepatocytes which do support replication. These nonpermissive hepatocytes may escape immune recognition and removal and are characterized by the nuclear accumulation of HBxAg. HBxAg in the nucleus may trans-activate one or more host genes, phosphorylate one or more host growth regulatory molecules, or both, which may contribute to the establishment of PHC. localizing in the nuclei of hepatocytes in patients with cirrhosis and dysplasia and (ii) trans-activating the expression of cellular genes responsible for the appearance of the transformed phenotype (Fig. 5). This may involve binding directly to transcription factors and altering their activity, which may result in increased expression of growth factors (possibly insulinlike growth factor II) or cellular oncogenes. HBxAg may also bind inhibitors of transcription factors, as do products from other DNA viruses (8). In addition, it is possible that HBxAg acts through binding to tumor suppressor gene products, which would result in the release of growth controls among selected hepatocytes during infection. The latter mechanism is used by a number of DNA viruses that cause cancer (47, 63, 117, 151, 194, 397). Several studies suggest that mutations or deletions in the p53 locus may be particularly important to the pathogenesis of PHC and that HBxAg binding to wild-type or mutant p53 could be an important step in hepatocarcinogenesis. The targets of HBxAg binding and/or of HBxAg trans-activation will undoubtedly be the focus of many future investigations. The X gene of hepadnaviruses has a number of other characteristics that suggest that it may be centrally involved high risk for PHC with hepatocarcinogenesis. For example, it is located at the extreme 3' end of the hepadnavirus genome, which is analogous to the position of the transforming gene in oncogenic retroviruses (231, 235). The codon usage in the X gene is more similar to that of cellular genes, compared with the rest of the virus genome, implying that the X region may have been acquired from cellular sequences (235). This is consistent with findings that the transforming protein(s) associated with other DNA tumor viruses may also have been acquired from host cell DNA. In such cases, the transduced cellular gene, now a viral oncogene, may be a mutant tumor suppressor gene, which may confer a growth advantage upon infected cells by binding to one or more native tumor suppressor gene products and inactivating them. The result of tumor suppressor gene product inactivation is the alteration in the patterns of cellular growth. Interestingly, there are parallels here with a number of transforming retroviruses that have acquired their transforming gene from cellular oncogene sequences (216, 342, 354). In the retroviruses, however, the transduced genetic material is from a cellular oncogene instead of a tumor suppressor gene. Some parallels may exist here as well, -_

15 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 289 because the trans-activating properties of HBxAg are similar to those of the human immunodeficiency virus type 1 tax and tat proteins. The tat protein is capable of producing tumors in transgenic mice (251), as is HBxAg (174). Perhaps the features that distinguish acutely transforming viruses from HBV is that the X region of HBV is really only part of a cellular gene capable of transformation and the other functional domains which would have been required to make HBxAg an acutely transforming protein were never acquired or were lost in the course of host-virus evolution. This would be consistent with a central role for HBxAg in hepatocarcinogenesis but would also take into account the fact that PHC usually occurs only after long-term infection. Additional characteristics of HBxAg are also compatible with its role in hepatocarcinogenesis. For example, integration of HBV DNA into host chromatin often results in the deletion of sequences at the extreme 3' end of the X gene and the trans-activating capability of such an integrated structure is often greater than that of the wild-type X gene (186). These observations suggest that if X trans-activation is important to PHC, integration would provide a mechanism of enhancing this activity. The recent findings that HBxAg has sequences potentially capable of binding divalent metal cations (199) and that HBxAg readily dimerizes (198) (perhaps as a result of metal ion binding) imply that the ability of HBxAg to form a dimer with itself (homodimer) may be important to its function. Dimerization, in turn, may be altered by a posttranslational modification such as phosphorylation. In this context, recent evidence has shown that HBxAg can become phosphorylated at serine and threonine residues (402). The evidence is also compatible with the hypothesis that HBxAg is a protein kinase (402). It has been proposed that the endogenous protein kinase activity associated with virus core particles is carried out by the HBxAg polypeptide, although HBxAg lacks homology with other known protein kinases (129). It is possible, though, that X affects the expression of protein kinases through its trans-activating properties and that such kinases affect the ability of X to mediate trans-activation by altering the state of X phosphorylation. Alternatively, or in addition, altered patterns of protein kinase expression resulting from HBxAg transactivation may affect the phosphorylation state of regulatory proteins of the cell cycle. Hence, several putative functions associated with HBxAg are also compatible with its role in carcinogenesis. A number of studies have further implicated a central role for HBxAg in the onset of PHC. HBV DNA successfully "transformed" NIH 3T3 fibroblasts in culture from a nontumorigenic cell line into one which formed tumors when injected into nude mice (325). In addition, a mouse hepatocyte cell line was transformed by HBV DNA so that the resulting transformants formed colonies instead of monolayers in soft agar and yielded tumors when injected into nude mice (147). The same mouse hepatocyte cell line could also be transformed by a fragment of HBV DNA containing only the virus enhancer and the X gene (309). It may be relevant that HBxAg trans-activates the promoters of the cellular oncogenes c-myc, c-fos, and c-jun (186). The enhanced expression of these genes early in the cell cycle (186) may explain how X stimulates hepatocellular growth in the liver. Several laboratories have constructed mice with the entire HBV genome or with the X region as the transgene, and although most attempts have not resulted in liver tumors in the transgenic progeny (6, 87, 192), one group has recently reported the development of tumors (174). One of the reasons for the discordant outcome of these studies may be that HBxAg production rapidly decreased with age or was undetectable among transgenic mice that did not develop tumors, whereas it remained high in those that eventually developed PHC. Therefore, continued high levels of HBxAg expression may be important for tumorigenesis. Interestingly, HBxAg(+) transgenic mice with PHC showed no histological or biochemical evidence of liver damage (168), suggesting that chronic hepatitis and cirrhosis are not required steps in hepatocarcinogenesis. In this context, it is of interest that asymptomatic chronic human carriers, who do not have clinical or biochemical evidence of liver disease, are at elevated risk for the development of PHC, although much less so than carriers with chronic liver disease (343). Together, these results strongly suggest that the persistent expression of HBxAg is an important step in multistep hepatocarcinogenesis, although the mechanism(s) used remain to be fully elucidated. Since HBxAg may mediate trans-activation of many cellular genes through protein-protein interactions, it may significantly affect the regulation of cellular growth and differentiation. If the function of HBxAg depends on the formation of homodimers or heterodimers, the quantity of HBxAg made and the cellular localization of HBxAg during infection may be crucial determinants of the outcome of the host-virus relationship on the molecular level. For example, trans-activation occurs most readily at low concentrations of HBxAg (82). On the other hand, high concentrations of HBxAg may be necessary to form the appropriate heterodimers with tumor suppressor gene products or similar growth regulatory molecules. Further, the presence of HBxAg in the nuclei of hepatocytes in most patients with cirrhosis and dysplasia, but in only a few patients with chronic hepatitis, means that HBxAg would have access to different cellular substrates according to the type of chronic liver disease. The availability of appropriate factors during chronic infection is also related to the state of hepatocellular differentiation. Hence, the environment in which HBxAg is expressed may determine the function of this HBV gene product. CONCLUSIONS: MODEL FOR HBV-INDUCED CARCINOGENESIS The multiple factors contributing to the appearance of PHC discussed above reflect the multistep nature of hepatocarcinogenesis (Fig. 6). This stepwise progression has been documented by the observation of discrete histological stages accompanying the changes from normal liver to PHC during chemical carcinogenesis in rats (84, 310), in transgenic mice with high levels of persistent HBxAg expression (174), and in humans with PHC (353). The important contributions can be summarized as follows. (i) HBV probably makes a genetic contribution to PHC by integration and expression of HBxAg (and/or pre-s), and the expression of such virus gene products from integrated templates is probably important for tumor initiation but not progression. This is consistent with the finding of persistent HBxAg in most carriers with progressive chronic liver disease and is also compatible with PHC cases lacking detectable HBxAg or integrated HBV DNA. (ii) The events surrounding tumor initiation are also likely to involve genetic aberrations resulting from integration of HBV DNA into multiple sites within the host genome. It may be less important where in the host genome the HBV integrates and more important that such integration events bring about genetic instability. The inability of hepatocytes to repair such anomalies yields cells

16 290 FEITELSON CLIN. MICROBIOL. REV. asymptomatic chronic carrer -- HBV+ mother resolution resolution FIG other HBV sources infection M w with HBV /~~~~~ persistent infection acute infection reso ilution viral replicative phase chemical carcinogens or HCV chronicactive cirrhosis * dysplasia _ PHC hepatitis integration of oncogene activation; HBV DNA; loss of suppressor growth genetic gene(s); advantage * instability persistent expression of HBV pre SorX increased susceptibility to chemical carcinoaens or promoters; increase rate of mutations infection nonreplicative phase ilnutam or early in childhood years 6. Summary of the factors that contribute to PHC amount HBV chronic carriers. Modified with permission from references 120 and whose states of differentiation and phenotypes are permanently altered. Such initiated cells will probably not be further selected for HBxAg expression or retention of integrated HBV DNA. (iii) Immunological responses to specific patterns of virus gene expression in infected hepatocytes probably result in the recognition and death of such cells, which may provide a regenerative stimulus to neighboring hepatocytes and/or oval (stem) cells. This regenerative stimulus favors cell proliferation over DNA repair and provides an environment which promotes the outgrowth of initiated cells into clones. The stronger the proliferative stimulus, the more likely it is that genetic mutations will accumulate and be propagated. Promotion in the context of progressive chronic liver disease may be attributed not only to the immunological responses against virus-infected cells but also to exposure to chemical promoters (Fig. 6). In selected cases of liver cancer, integration of hepadnavirus DNA near specific regulatory genes of cell growth may be central to the pathogenesis of PHC. Similarly, amplification of specific growth-regulatory genes, specific rearrangement or activation of cellular oncogenes, or loss of heterozygosity at one or more chromosomes may turn out to be a central element in understanding the pathogenesis of PHC in specific cases. It is also clear from such studies that the pathogenesis of PHC involves more than a single mechanism, and the contribution of each step to PHC may vary among HBV infected human populations. If there are many mechanisms by which multistep hepatocarcinogenesis could arise in association with HBV infection, it is important to find the steps common to these mechanisms. Identification of these common steps would be important to the scientist and clinician in attempting to understand the pathogenesis of PHC from the viewpoint of prognosis and in the construction of rational therapeutic approaches. REFERENCES 1. Akahane, Y., T. Yamanaka, H. Suzuki, Y. Sugai, F. Tsuda, S. Yotsumoto, S. Omi, H. Okamoto, Y. Miyakawa, and M. Mayumi Chronic active hepatitis with hepatitis B virus DNA and antibody against e antigen in the serum. Gastroenterology 99: Alberti, A., P. Pontisso, L. Chemello, E. Schiavon, and G. Realdi Virus receptors for polymerized human serum albumin and antireceptor antibody in hepatitis B virus infection, p In G. Verme, F. Bonino, and M. Rizzetto (ed.), Viral hepatitis and delta infection. Alan R. Liss, Inc., New York. 3. Alberti, A., G. Realdi, F. Tremolada, and G. P. Spina Liver cell surface localization of hepatitis B antigen and of immunoglobulins in acute and chronic hepatitis and in liver cirrhosis. Clin. Exp. Immunol. 25: Almeida, J. D., D. Rubinstein, and J. E. Stott New antigen-antibody system in Australia-antigen-positive hepatitis. Lancet ii: Antonucci, T. K., and W. J. Rutter Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner. J. Virol. 63: Araki, K., J.-I. Miyazaki, 0. Hino, N. Tomita, 0. Chisaka, K. Matsubara, and K.-I. Yamamura Expression and replication of hepatitis B virus genome in transgenic mice. Proc. Natl. Acad. Sci. USA 86: Aufiero, B., and R. J. Schneider The hepatitis B X-gene product trans-activates both RNA polymerase II and III promoters. EMBO J. 9: Bagchi, S., P. Raychaudhari, and J. R. Nevins Adenovirus ElA proteins can dissociate heteromeric complexes involving the E2F transcription factor: a novel mechanism for ElA trans-activation. Cell 62: Baker, S. J., E. R. Fearon, J. M. Nigro, S. R. Hamilton, A. C. Preisinger, J. M. Jessup, P. van Tuinen, D. H. Ledbetter, D. F. Barker, Y. Nakamura, R. White, and B. Vogelstein Chromosome 17 deletions and p53 gene mutations in colorectal

17 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 291 carcinoma. Science 244: Banninger, P., J. Altorfer, G. G. Frosner, M. Pirovino, F. Gudat, L. Bianchi, P. J. Grob, and M. Schmid Prevalence and significance of anti-hbc IgM (RIA) in acute and chronic hepatitis B and in blood donors. Hepatology 3: Barrett, J. C Cellular and molecular mechanisms for suppression and reversion of tumorigenicity-a chemical pathology study section workshop. Cancer Res. 47: Baum, J. K., J. J. Bookstein, F. Holtz, and E. W. Klein Possible association between benign hepatomas and oral contraceptives. Lancet ii: Bavand, M., M. A. Feitelson, and 0. Laub The hepatitis B virus-associated reverse transcriptase is encoded by the viral pol gene. J. Virol. 63: Bavand, M. R., and 0. Laub Two proteins with reverse transcriptase activities associated with hepatitis B virus-like particles. J. Virol. 62: Beard, J. W., E. A. Hillman, D. Beard, K. Lapis, and U. Heine Neoplastic response of the avian liver to host infection with strain MC-29 leukosis virus. Cancer Res. 35: Beasley, R. P., and L.-Y. Hwang Epidemiology of hepatocellular carcinoma, p In G. N. Vyas, J. L. Dienstag, and J. H. Hoofnagle (ed.), Viral hepatitis and liver disease. Grune and Stratton, Inc., Orlando, Fla. 17. Beasley, R. P., L.-Y. Hwang, C. C. Lin, and C. S. Chien Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22,707 men in Taiwan. Lancet ii: Beasley, R. P., and C. C. Lin Hepatoma risk among HBsAg carriers. Am. J. Epidemiol. 108: Beasley, R. P., I. S. Shaio, T. C. Wu, and L.-Y. Hwang Hepatoma in an HBsAg carrier-7 years after perinatal infection. J. Pediatr. 101: Benedict, W. F., A. L. Murphree, A. Banerjee, C. A. Spina, M. C. Sparkes, and R. S. Sparkes Patient with a 13 chromosome deletion: evidence that the retinoblastoma gene is a recessive cancer gene. Science 219: Bianchi, L., H. P. Spichtin, and F. Gudat Chronic hepatitis, p In R. N. M. MacSween, P. P. Antony, and P. J. Scheuer (ed.), Pathology of liver disease. Churchill Livingstone, New York. 22. Blanquet, V., F. Garreau, X. Chenivesse, C. Brechot, and C. Turleau Regional mapping to 4q32.1 by in situ hybridization of a DNA domain rearranged in human liver cancer. Hum. Genet. 80: Blum, H. E., L. Stowring, A. Figus, C. K. Montgomery, A. T. Haase, and G. N. Vyas Detection of hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization. Proc. Natl. Acad. Sci. USA 80: Blumberg, B. S., and W. T. London Hepatitis B virus: pathogenesis and prevention of primary cancer of the liver. Cancer 50: Blumberg, B. S., and W. T. London Hepatitis B virus and the prevention of primary cancer of the liver. JNCI 74: a.Blumberg, B. S., I. Milman, P. S. Venkateswaran, and S. P. Thyagarajan Hepatitis B virus and hepatocellular carcinoma-treatment of HBV carriers with Phyllanthus amarus. Cancer Detect. Prev. 14: Bortolotti, F., A. Alberti, P. Cadrobbi, M. Rugge, M. Armigliato, and G. Realdi Prognostic value of hepatitis B core antigen (HBcAg) expression in the liver of children with chronic hepatitis type B. Liver 5: Bowcock, A. M., M. R. Pinto, E. Bey, J. M. Kuyl, G. M. Dusheiko, and R. Bernstein The PLC/PRF/5 human hepatoma cell line. II. Chromosomal assignment of hepatitis B virus integration sites. Cancer Genet. Cytogenet. 18: Brand, N., M. Petkovitch, A. Krust, P. Chambon, H. de The, A. Marchio, P. Tiollais, and A. Dejean Identification of a second human retinoic acid-receptor. Nature (London) 332: Brechot, C., M. Hadchouel, J. Scotto, M. Fonck, F. Potet, G. N. Vyas, and P. Tiollais State of hepatitis B virus DNA in hepatocytes of patients with hepatitis B surface antigen-positive and -negative liver diseases. Proc. Natl. Acad. Sci. USA 78: Bressac, B., K. M. Galvin, T. J. Liang, K. J. Isselbacher, J. R. Wands, and M. Ozturk Abnormal structure and expression of p53 gene in human hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 87: Bressac, B., M. Kew, J. Wands, and M. Ozturk Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature (London) 350: Bruss, V., and W. Gerlich Formation of transmembraneous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein. Virology 163: Buetow, K. H., J. C. Murray, J. L. Israel, W. T. London, M. Smith, M. Kew, V. Blanquet, C. Brechot, A. Redeker, and S. Govindarajah Loss of heterozygosity suggests tumor suppressor gene responsible for primary hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 86: Bullock, P. J., J. J. Champoux, and M. Botchan Association of crossover points with topoisomerase I cleavage sites: a model for nonhomologous recombination. Science 230: Bullock, W., M. Forrester, and M. Botchan DNA sequence studies of simian virus 40 chromosomal excision and integration in rat cells. J. Mol. Biol. 174: Burrell, C. J., E. J. Gowans, R. Rowland, P. Hall, A. R. Jilbert, and B. P. Marmion Correlation between liver histology and markers of hepatitis B virus replication in infected patients: a study by in situ hybridization. Hepatology 4: Cairns, J The origin of human cancers. Nature (London) 289: Cariani, E., C. Lasserre, D. Seurin, B. Hamelin, F. Kemeny, D. Franco, M. P. Czech, A. Ullrich, and C. Brechot Differential expression of insulin-like growth factor II mrna in human primary liver cancers, benign tumors, and liver cirrhosis. Cancer Res. 48: Cariani, E., D. Seurin, C. Lasserre, D. Franco, M. Binoux, and C. Brechot Expression of insulin-like growth factor II (IGF-II) in human primary liver cancer: mrna and protein analysis. J. Hepatol. 11: Carloni, G., B. Champ, A. M. Szekely, R. Cassingena, and F. Galibert Transfection of oncogenes activated in human hepatocellular carcinomas, p In A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York. 41. Carman, W. F., S. Hadziyannis, M. J. McGarvey, M. R. Jacyna, P. Karayiannis, A. Makris, and H. C. Thomas Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet ii: Caselmann, W. H., M. Meyer, A. S. Kekule, U. Lauer, P. H. Hofschneider, and R. Koshy A trans-activator function is generated by integration of hepatitis B virus pres/s sequences in human hepatocellular carcinoma DNA. Proc. Natl. Acad. Sci. USA 87: Castilla, A., J. Prieto, and N. Fausto Transforming growth factors beta and alpha in chronic liver disease. Effects of interferon alfa therapy. N. Engl. J. Med. 324: Cavenee, W. K., T. P. Dryja, R. A. Phillips, W. F. Benedict, R. Godbout, B. L. Gallie, A. L. Murphree, L. C. Strong, and R. L. White Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature (London) 305: Chang, H.-Y., and L.-P. Ting The surface gene promoter of the human hepatitis B virus displays a preference for differentiated hepatocytes. Virology 170: Chang, M.-H., D.-S. Chen, H.-C. Hsu, M.-Z. Wu, H.-Y. Hsu, and C.-Y. Lee Childhood hepatocellular carcinoma in Taiwan: the prominent role of perinatally transmitted hepatitis B virus infection, p In A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York. 47. Chang, M.-H., H. C. Hsu, C. Y. Lee, D.-S. Chen, C.-H. Lee, and K.-S. Lin Fraternal hepatocellular carcinoma in young children in two families. Cancer 53:

18 292 FEITELSON 48. Chen, D.-S., G. C. Kuo, J.-L. Sung, M.-Y. Lai, J.-C. Sheu, P.-J. Chen, P.-M. Yang, H.-M. Hsu, M.-H. Chang, C.-J. Chen, L.-C. Hahn, Q.-L. Choo, T.-H. Wang, and M. Houghton Hepatitis C virus infection in an area hyperendemic for hepatitis B and chronic liver disease: the Taiwan experience. J. Infect. Dis. 162: Chen, M.-L., Y.-H. W. Lee, and S. J. Lo High level production of hepatitis B viral X protein in Escherichia coli using gene II promoter of bacteriophage M13. Gene 62: Chisari, F. V., P. Filippi, J. Buras, A. McLachlan, H. Popper, C. A. Pinkert, R. D. Palmiter, and R. L. Brinster Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice. Proc. Natl. Acad. Sci. USA 84: Chisari, F. V., K. Klopchin, T. Moriyama, C. Pasquinelli, H. A. Dunsford, S. Sell, C. A. Pinkert, R. L. Brinster, and R. D. Palmiter Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 59: Choo, K.-B., M.-S. Liu, P.-C. Chang, S.-M. Wu, M.-W. Su, C.-C. Pan, and S.-H. Han Analysis of six distinct integrated hepatitis B virus sequences cloned from the cellular DNA of a human hepatocellular carcinoma. Virology 154: Chu, C.-M., P. Karayiannis, M. J. F. Fowler, J. Monjardino, Y.-F. Liaw, and H. C. Thomas Natural history of chronic hepatitis B virus infection in Taiwan: studies of hepatitis B virus DNA in serum. Hepatology 5: Chu, C.-M., and Y.-F. Liaw Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis. Gastroenterology 92: Cohen, S. M., and L. B. Ellwein Cell proliferation in carcinogenesis. Science 249: Colgrove, R., G. Simon, and D. Ganem Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication. J. Virol. 63: Colombo, M., Q. L. Choo, E. Del Ninno, N. Dioguardi, G. Kuo, M. F. Donato, M. A. Tommasini, and M. Houghton Prevalence of antibodies to hepatitis C virus in Italian patients with hepatocellular carcinoma. Lancet ii: Cooper, G. M Cellular transforming genes. Science 218: Cote, P. J., B. E. Korba, H. Steinberg, C. Ramirez-Mejia, B. Baldwin, W. E. Hornbuckle, B. C. Tennant, and J. L. Gerin Cyclosporin A modulates the course of woodchuck hepatitis virus infection and induces chronicity. J. Immunol. 146: Cova, L., C. P. Wild, R. Mehrotra, V. Turusov, T. Shirai, V. Lanbert, C. Jacquet, L. Tomatis, C. Trepo, and R. Montesano Contribution of aflatoxin B1 and hepatitis B virus infection in the induction of liver tumors in ducks. Cancer Res. 50: Craddock, V. M Cell proliferation and induction of liver cancer, p In H. Remmer, H. M. Bolt, P. Bannasch, and H. Popper (ed.), Primary liver tumors. University Park Press, Baltimore. 62. D'Andrea, A. D., and W. A. Haseltine Modification of DNA by aflatoxin B1 creates alkali-labile lesions in DNA at positions of guanine and adenine. Proc. Natl. Acad. Sci. USA 75: DeCaprio, J. A., J. W. Ludlow, J. Figge, J.-Y. Shew, C.-M. Huang, W.-H. Lee, E. Marsilio, E. Paucha, and D. M. Livingston SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 54: De Flora, S., E. Hietanen, H. Bartsch, A. Camoirano, A. Izzotti, M. Bagnasco, and I. Millman Enhanced metabolic activation of chemical hepatocarcinogens in woodchucks infected with hepatitis B virus. Carcinogenesis 10: De Flora, S., M. Romano, C. Basso, D. Serra, M. Astengo, and CLIN. MICROBIOL. REV. A. Picciotto Metabolic activation of hepatocarcinogens in chronic hepatitis B. Mutat. Res. 144: Dejean, A., L. Bougueleret, K.-H. Grzeschik, and P. Tiollais Hepatitis B virus DNA integration in a sequence homologous to v-erb-a and steroid receptor genes in a hepatocellular carcinoma. Nature (London) 322: Dejean, A., and H. de The Hepatitis B virus as an insertional mutagene in a human hepatocellular carcinoma. Mol. Biol. Med. 7: Dejean, A., C. Lugassy, S. Zafrani, P. Tiollais, and C. Brechot Detection of hepatitis B virus DNA in pancreas, kidney and skin of two human carriers of the virus. J. Gen. Virol. 65: Dejean, A., P. Sonigo, S. Wain-Hobson, and P. Tiollais Specific hepatitis B virus integration in hepatocellular carcinoma DNA through a viral 11-base-pair direct repeat. Proc. Natl. Acad. Sci. USA 81: Dempo, K., N. Chisaka, Y. Yoshida, A. Keneko, and T. Onoe Immunofluorescent study on a-fetoprotein-producing cells in the early stage of 3'-methyl-4-dimethylaminoazobenzene carcinogenesis. Cancer Res. 35: Dienes, H. P., L. Bianchi, W. Gerlich, and G. Hess Different patterns of HBsAg and pre-s antigens in liver biopsies from healthy carriers, p In A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York. 72. Dienes, H. P., W. H. Gerlich, M. Worsdorfer, G. Gerken, L. Bianchi, G. Hess, and K. H. Meyer zum Buschenfelde Hepatic expression patterns of the large and middle hepatitis B virus surface proteins in viremic and nonviremic chronic hepatitis B. Gastroenterology 98: Dragani, T. A., G. Manenti, H. Farza, G. D. Porta, P. Tiollais, and C. Pourcel Transgenic mice containing hepatitis B virus sequences are more susceptible to carcinogen-induced hepatocarcinogenesis. Carcinogenesis 11: Duffy, M. J., and G. J. Duffy Estradiol receptors in human liver. J. Steroid Biochem. 9: Dusheiko, G. M., J. H. Hoofnagle, W. G. Cooksley, S. P. James, and E. A. Jones Synthesis of antibodies to hepatitis B virus by cultured lymphocytes from chronic hepatitis B surface antigen carriers. J. Clin. Invest. 71: Eddleston, A. L. W. F., and R. Williams Inadequate antibody response to HBsAg or suppressor T-cell defect in development of active chronic hepatitis. Lancet ii: Edmondson, H. A., B. Henderson, and B. Benton Liver cell adenomas associated with the use of oral contraceptives. N. Engl. J. Med. 294: Elfassi, E., W. A. Haseltine, and J. L. Dienstag Detection of hepatitis B virus X product using an open reading frame Escherichia coli expression vector. Proc. Natl. Acad. Sci. USA 83: Etiemble, J., T. Moroy, E. Jacquemin, P. Tiollais, and M.-A. Buendia Fused transcripts of c-myc and a new cellular locus, hcr, in a primary liver tumor. Oncogene 4: Evans, R. M The steroid and thyroid hormone receptor superfamily. Science 240: Faktor, O., T. de-medina, and Y. Shaul Regulation of hepatitis B virus S gene promoter in transfected cell lines. Virology 162: Faktor, O., and Y. Shaul The identification of hepatitis B virus X gene responsive elements reveals functional similarity of X and HTLV-1 tax. Oncogene 5: Farber, E Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylaminofluorene, and 3'-methyl-4-demethylaminoazobenzene. Cancer Res. 16: Farber, E Cellular biochemistry of the stepwise development of cancer with chemicals: G. H. A. Clowes Memorial Lecture. Cancer Res. 44: Farber, E., and D. S. R. Sarma Biology of disease. Hepatocarcinogenesis: a dynamic cellular perspective. Lab. Invest. 56: Farrell, G. C., D. E. Joshua, R. F. Uren, P. J. Baird, K. W.

19 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 293 Perkins, and H. Kronenberg Androgen-induced hepatoma. Lancet i: Farza, H., M. Hadchouel, J. Scotto, P. Tiollais, C. Babinet, and C. Pourcel Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome. J. Virol. 62: Farza, H., A. M. Salmon, M. Hadchouel, J. L. Moreau, C. Babinet, P. Tiollais, and C. Pourcel Hepatitis B surface antigen gene expression is regulated by sex steroids and glucocorticoids in transgenic mice. Proc. Natl. Acad. Sci. USA 84: Fausto, N., and J. E. Meade Regulation of liver growth: proto-oncogenes and transforming growth factors. Lab. Invest. 60: Fausto, N., and P. R. Shank Oncogene expression in liver regeneration and hepatocarcinogenesis. Hepatology 3: Fausto, N., and P. R. Shank Analysis of proto-oncogene expression during liver regeneration and hepatocarcinogenesis, p In K. Okuda, and K. G. Ishak (ed.), Neoplasms of the liver. Springer-Verlag, New York. 92. Feitelson, M. A Molecular components of hepatitis B virus, p In Y. Becker and J. Hadar (ed.), Developments in molecular virology. Martinus Nijhoff Publishers, Hingham, Mass. 93. Feitelson, M. A Products of the "X" gene in hepatitis B and related viruses. Hepatology 6: Feitelson, M. A Hepatitis B virus and cancer, p In A. L. Notkins and B. A. Oldstone (ed.), Concepts in viral pathogenesis, vol. 2. Springer-Verlag, New York. 95. Feitelson, M. A Hepatitis B virus gene products as immunological targets in chronic infection. Mol. Biol. Med. 6: Feitelson, M. A., and M. M. Clayton X antigen polypeptides in the sera of hepatitis B virus-infected patients. Virology 177: Feitelson, M. A., and M. M. Clayton X antigen/antibody markers in hepadnavirus infections. Antibodies to the X gene product(s). Gestroenterology 99: Feitelson, M. A., M. M. Clayton, and B. S. Blumberg The X antigen/antibody markers in hepadnavirus infections. Presence and significance of the hepadnavirus X gene products in serum. Gastroenterology 98: Feitelson, M. A., L. J. Detolla, and X.-D. Zhou A chronic carrierlike state is established in nude mice injected with cloned hepatitis B virus DNA. J. Virol. 62: Feitelson, M. A., P. L. Marion, and W. S. Robinson The nature of polypeptides larger in size than the major surface antigen components of hepatitis B and like viruses in ground squirrels, woodchucks, and ducks. Virology 130: Ferenc, P., B. Dragosics, L. Marosi, and F. Kiss Relative incidence of primary liver cancer in cirrhosis in Austria. Aetiological considerations. Liver 4: Finlay, C. A., P. W. Hinds, and A. J. Levine The p53 proto-oncogene can act as a suppressor of transformation. Cell 57: Foster, P. L., E. Eisenstadt, and J. H. Miller Base substitution mutations induced by metabolically activated aflatoxin B1. Proc. Natl. Acad. Sci. USA 80: Fowler, M. J. F., C. Greenfield, C.-M. Chu, P. Karayiannis, A. Dunk, A. S. F. Lok, C. L. Lai, E. K. Yeoh, J. P. Monjardino, B. M. Wankya, and H. C. Thomas Integration of HBV-DNA may not be a prerequisite for the maintenance of the state of malignant transformation. J. Hepatol. 2: Fox, T. R., and P. G. Watanabe Detection of a cellular oncogene in spontaneous liver tumors of B6C3F1 mice. Science 228: Fraise, A., P. Pontisso, D. Cavaletto, G. Fattovich, and A. Alberti Expression of pre-sl in the liver of chronic hepatitis B virus carriers. J. Hepatol. 7: Francke, U., L. B. Holmes, L. Atkins, and V. M. Riccardi Aniridia-Wilms' tumor association: evidence for specific deletion of 11pl3. Cytogenet. Cell Genet. 24: Friedman, M. A Chemotherapy for patients with hepatocellular carcinoma: prospects and possibilities, p In E. Tabor, A. M. DiBisceglie, and R. H. Purcell (ed.), Etiology, pathology, and treatment of hepatocellular carcinoma in North America. Gulf Publishing Co., Houston Fu, X.-X., C. Y. Su, Y. Lee, R. Hintz, L. Biempica, R. Snyder, and C. E. Rogler Insulinlike growth factor II expression and oval cell proliferation associated with hepatocarcinogenesis in woodchuck hepatitis virus carriers. J. Virol. 62: Fujimori, M., T. Tokino, 0. Hino, T. Kitagawa, T. Imamura, E. Okamoto, M. Mitsunobu, T. Ishikawa, H. Nakagama, H. Harada, M. Yagura, K. Matsubara, and Y. Nakamura Allelotype of primary hepatocellular carcinoma. Cancer Res. 51: Fujiyama, A., A. Miyanohara, C. Nozaki, T. Yoneyama, N. Ohtomo, and K. Matsubara Cloning and structural analysis of hepatitis B virus DNAs, subtype adr. Nucleic Acids Res. 11: Fults, D., R. H. Tippets, G. A. Thomas, Y. Nakamura, and R. White Loss of heterozygosity for loci on chromosome 17p in human malignant astrocytoma. Cancer Res. 49: Galibert, F., T. N. Chen, and E. Mandart Nucleotide sequence of a cloned woodchuck hepatitis virus genome: comparison with the hepatitis B virus sequence. J. Virol. 41: Galibert, F., E. Mandart, F. Fitoussi, P. Tiollais, and P. Charmay Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature (London) 281: Gerin, J. L., B. C. Tennant, H. Popper, F. J. Tyeryar, and R. H. Purcell The woodchuck model of hepadna virus infection and disease, p In F. Brown, F. Chanock, and R. Lemer (ed.), Vaccines '86: new approaches to immunization. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y Goudeau, A., P. Maupas, P. Coursaget, J. Drucker, J.-P. Chiron, F. Denis, I. D. Mar, and P. D. N'Diaye Detection of hepatitis B virus antigens in hepatocellular carcinoma tissues. Prog. Med. Virol. 27: Green, M. R When the products of oncogenes and anti-oncogenes meet. Cell 56: Groopman, J. D., W. F. Busby, and P. R. Donahue Aflatoxins as risk factors for liver cancer: an application of monoclonal antibodies to monitor human exposure, p In C. C. Harris (ed.), Biochemical and molecular epidemiology of cancer. Alan R. Liss, Inc., New York Gu, J.-R Oncogenes in human and duck primary hepatic cancer, p In W. Robinson, K. Koike, and H. Will (ed.), Hepadna viruses. Alan R. Liss, Inc., New York Gu, J.-R Molecular aspects of human hepatic carcinogenesis. Carcinogenesis 9: Gu, J.-R., T. Peikun, W. Dafang, W. Xiang, L. Hongnian, P. Zhimei, H. Lehong, L. Xiughen, and J. Huiqui Identification of human N-ras as the common oncogene in NIH3T3 cells transformed with DNAs from human primary hepatic cancer and hepatoma 7402 line. Sci. Sin. Ser. B 29: Gudat, F., and L. Bianchi Evidence for phasic sequences in nuclear HBcAg formation and cell membrane directed flow of core particles in chronic hepatitis B. Gastroenterology 73: Haber, D. A., and D. E. Housman Rate-limiting steps: the genetics of pediatric cancers. Cell 64: Hadziyannis, S. J., H. M. Lieberman, G. G. Karvountzis, and D. A. Shafritz Analysis of liver disease, nuclear HBeAg, viral replication, and hepatitis B virus DNA in liver and serum of HBeAg vs. anti-hbe positive carriers of hepatitis B virus. Hepatology 3: Hadziyannis, S. J., G. Raimondo, C. Papaioannou, C. Anastassakos, D. Wong, J. Sninsky, and D. Shafritz Expression of pre-s gene encoded proteins in liver and serum during chronic hepatitis B virus infection in comparison with other

20 294 FEITELSON CLIN. MICROBIOL. REV. markers of active virus replication, p In A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York Hahn, E. G., and D. Schuppan Pathogenic mechanisms: fibrosis, fibrogenesis and fibrolysis, p In N. Tygstrup and F. Orlandi (ed.), Cirrhosis of the liver: methods and fields of research. Elsevier Biomedical Press, Amsterdam Halpern, M. S., J. Egan, S. B. McMahon, and D. L. Ewert Duck hepatitis B virus is tropic for exocrine cells of the pancreas. Virology 146: Halpern, M. S., J. M. England, D. T. Deery, D. J. Petcu, W. S. Mason, and K. L. Molnar-Kimber Viral nucleic acid synthesis and antigen accumulation in pancreas and kidney of Pekin ducks infected with duck hepatitis B virus. Proc. Natl. Acad. Sci. USA 80: Hanks, S. K., A. M. Quinn, and T. Hunter The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241: Hansen, M. F., and W. K. Cavenee Genetics of cancer predisposition. Cancer Res. 47: Harbour, J. W., S.-L. Lai, J. Whang-Peng, A. F. Gazdar, J. D. Minna, and F. J. Kaye Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science 241: Hardell, L., N. 0. Bengtsson, U. Jonsson, S. Eriksson, and L. G. Larsson Aetiological aspects of primary liver cancer with special regard to alcohol, organic solvents and acute intermittent porphyria-an epidemiological investigation. Br. J. Cancer 50: Harris, C. C., and S. Tsung-Tang Interactive effects of chemical carcinogens and hepatitis B virus in the pathogenesis of hepatocellular carcinoma. Cancer Surv. 5: Haruna, Y., N. Hayashi, K. Katayama, N. Yuki, A. Kasahara, Y. Sasaki, H. Fusamoto, and T. Kamada Expression of X protein and hepatitis B virus replication in chronic hepatitis. Hepatology 13: Hatada, I., T. Tokino, T. Ochiya, and K. Matsubara Co-amplification of integrated hepatitis B virus DNA and transforming gene hst-1 in a hepatocellular carcinoma. Oncogene 3: Hayward, W. S., B. G. Neel, and S. M. Astrin Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis. Nature (London) 290: Heermann, K. H., U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich Large surface proteins of hepatitis B virus containing the pre-s sequence. J. Virol. 52: Henderson, J. T., J. Richmond, and M. D. Sumerling Androgenic-anabolic steroid therapy and hepatocellular carcinoma. Lancet i: Hess, G., W. H. Gerlich, G. Gerken, M. Manns, and K. H. Meyer zum Buschenfelde Relationship of pre-s encoded antigens in liver and clinical manifestations of chronic hepatitis B infection. Liver 7: Hino, O., K. Nomura, K. Ohtake, T. Kawaguchi, H. Sugano, and T. Kitagawa Instability of integrated hepatitis B virus DNA with inverted repeat structure in a transgenic mouse. Cancer Genet. Cytogenet. 37: Hino, O., K. Nomura, K. Ohtake, T. Kitagawa, H. Sugano, S. Kimura, M. Yokoyamo, and M. Katsuki Rearrangement of integrated HBV DNA in descendants of transgenic mice. Proc. Jpn. Acad. Ser. B 62: Hino, O., K. Nomura, K. Ohtake, H. Sugano, T. Kitagawa, S. Kimura, M. Yokoyama, and M. Katsuki Rearrangements of integrated hepatitis B virus DNA in transgenic mice, p In A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York Hino, O., K. Ohtake, and C. E. Rogler Features of two hepatitis B virus (HBV) DNA integrations suggest mechanisms of HBV integration. J. Virol. 63: Hino, O., T. B. Shows, and C. E. Rogler Hepatitis B virus integration site in hepatocellular carcinoma at chromosome 17;18 translocation. Proc. Natl. Acad. Sci. USA 83: Hirohashi, S., Y. Shimosato, Y. Ino, and K. Kishi Distribution of hepatitis B surface and core antigens in human liver cell carcinoma and surrounding nontumorous liver. JNCI 69: Hohenberger, P Detection of HBsAg in the pancreas in cases of pancreatic carcinoma. Hepato-gastroenterology 31: Hohne, M., S. Schaefer, M. Seifer, M. A. Feitelson, D. Paul, and W. H. Gerlich Malignant transformation of immortalized transgenic hepatocytes after transfection with hepatitis B virus DNA. EMBO J. 9: Honigwachs, J., 0. Faktor, R. Dikstein, Y. Shaul, and 0. Laub Liver-specific expression of hepatitis B virus is determined by the combined action of the core gene promoter and the enhancer. J. Virol. 63: Hoofnagle, J. H., D. A. Shafritz, and H. Popper Chronic type B hepatitis and the "healthy" HBsAg carrier state. Hepatology 7: Hosono, S., C.-S. Lee, M.-J. Chou, C.-S. Yang, and C. Shih Molecular analysis of the p53 alleles in primary hepatocellular carcinomas and cell lines. Oncogene 6: Howley, P. M Tumor suppressor genes and tumors associated with virus infection: interaction of HBV 16 and 18 with tumor suppressor gene products, p In E. Tabor, A. M. DiBisceglie, and R. H. Purcell (ed.), Etiology, pathology, and treatment of hepatocellular carcinoma in North America. Gulf Publishing Co., Houston Hsu, H.-C., M.-Y. Lai, I.-J. Su, D.-S. Chen, M.-H. Chang, P.-M. Yang, C.-Y. Wu, and H.-C. Hsieh Correlation of hepatocyte HBsAg expression with virus replication and liver pathology. Hepatology 8: Hsu, H.-C., M. Z. Wu, M. H. Chang, I. J. Su, and D. S. Chen Childhood hepatocellular carcinoma develops exclusively in hepatitis B surface antigen carriers in three decades in Taiwan. Repart of 51 cases strongly associated with rapid development of liver cirrhosis. J. Hepatol. 5: Hsu, H.-C., T.-T. Wu, J.-C. Sheu, C.-Y. Wu, T.-J. Chiou, C.-S. Lee, and D.-S. Chen Biologic significance of the detection of HBsAg and HBcAg in liver and tumor from 204 HBsAg-positive patients with primary hepatocellular carcinoma. Hepatology 9: Hsu, I. C., R. A. Metcalf, T. Sun, J. A. Welsh, N. J. Wang, and C. C. Harris Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature (London) 350: Hsu, T.-Y., T. Moroy, J. Etiemble, A. Louise, C. Trepo. P. Tiollais, and M.-A. Buendia Activation of c-myc by woodchuck hepatitis virus insertion in hepatocellular carcinoma. Cell 55: Hu, K.-Q., J. M. Vierling, and A. Siddiqui Transactivation of HLA-DR gene by hepatitis B virus X gene product. Proc. Natl. Acad. Sci. USA 87: Huang, H.-J. S., J.-K. Yee, J.-Y. Shew, P.-L. Chen, R. Bookstein, T. Friedmann, E. Y.-H. P. Lee, and W.-H. Lee Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science 242: Imazeki, F., K. Yaginuma, M. Omata, K. Okuda, M. Kobayashi, and K. Koike Integrated structures of duck hepatitis B virus DNA in hepatocellular carcinoma. J. Virol. 62: Iqbal, M. J., M. L. Wilkinson, P. J. Johnson, and R. Williams Sex steroid proteins in foetal, adult and malignant human liver tissue. Br. J. Cancer 48: Ishikawa, F., F. Takaku, K. Hayashi, M. Nagao, and T. Sugimura Activation of rat c-raf during transfection of hepatocellular carcinoma DNA. Proc. Natl. Acad. Sci. USA 83: James, C. D., E. Carlbom, M. Nordenskjold, V. P. Collins, and W. K. Cavenee Mitotic recombination of chromosome 17 in astrocytomas. Proc. Natl. Acad. Sci. USA 86: Jilbert, A. R., J. S. Freiman, E. J. Gowans, M. Holmes, Y. E.

21 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 295 Cossart, and C. J. Burrell Duck hepatitis B virus DNA in liver, spleen, and pancreas: analysis by in situ and Southern blot hybridization. Virology 158: Johnson, F. L Androgenic-anabolic steroids and hepatocellular carcinoma, p In K. Okuda and R. L. Peters (ed.), Hepatocellular carcinoma. John Wiley & Sons, Inc., New York Johnson, P. J., N. Krasner, B. Portmann, A. L. W. F. Eddieston, and R. Williams Hepatocellular carcinoma in Great Britain: influence of age, sex, HBsAg status and aetiology of underlying cirrhosis. Gut 19: Jung, M.-C., M. Stemler, T. Weimer, U. Spengler, J. Dohrmann, R. Hoffman, D. Eichenlaub, J. Eisenburg, G. Paumgartner, G. Riethmuller, H. Will, and G. R. Pape Immune response of peripheral blood mononuclear cells to HBx-antigen of hepatitis B virus. Hepatology 13: Katayama, K., N. Hayashi, Y. Sasaki, A. Kasahara, K. Ueda, H. Fusamoto, N. Sato, 0. Chisaka, K. Matsubara, and T. Kamada Detection of hepatitis B virus X gene protein and antibody in type B chronic liver disease. Gastroenterology 97: Kay, A., E. Mandart, C. Trepo, and F. Galibert The HBV HBx gene expressed in E. coli is recognized by sera from hepatitis patients. EMBO J. 4: Kekule, A. S., U. Lauer, M. Meyer, W. H. Caselmann, P. H. Hofschneider, and R. Koshy The pres2/s region of integrated hepatitis B virus DNA encodes a transcriptional trans-activator. Nature (London) 343: Ke-qin, H., H. Lian-jie, and H. Will A preliminary study on localization of HBxAg in liver tissue of patients with chronic liver disease and its significance. Chin. Med. J. 101: Kew, M. C The development of hepatocellular cancer in humans. Cancer Surv. 5: Kew, M. C., J. Hodkinson, A. C. Paterson, and E. Song Hepatitis-B virus infection in Black children with hepatocellular carcinoma. J. Med. Virol. 9: Kew, M. C., and H. Popper Relationship between hepatocellular carcinoma and cirrhosis. Semin. Liver Dis. 4: Kim, C.-M., K. Koike, I. Saito, T. Miyumura, and G. Jay HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature (London) 351: Kim, C.-Y., S.-K. Bae, H.-W. L. Hann, W. T. London, and B. S. Blumberg Prevalence of HBeAg and anti-hbe in chronic active hepatitis, cirrhosis and primary hepatocellular carcinoma in Korea. Hepatology 5: Kiyosawa, K., E. Tanaka, T. Sodeyama, K. Furuta, S. Usuda, M. Yousuf, and S. Furuta Transition of antibody to hepatitis C virus from chronic hepatitis to hepatocellular carcinoma. Jpn. J. Cancer Res. 81: Klein, G Evolution of tumors and the impact of molecular oncology. Nature (London) 315: Klein, G The approaching era of the tumor suppressor genes. Science 238: Knudson, A. G., Jr Model hereditary cancers of man. Prog. Nucleic Acid Res. Mol. Biol. 29: Knudson, A. G., Jr Hereditary cancer, oncogenes and antioncogenes. Cancer Res. 45: Koch, S., A. F. von Loringhoven, P. H. Hofschneider, and R. Koshy Amplification and rearrangement in hepatoma cell DNA associated with integrated hepatitis B virus DNA. EMBO J. 3: Koike, K., S. Iino, K. Kurai, K. Mitamura, Y. Endo, and H. Oka IgM anti-hbc in anti-hbe positive chronic type B hepatitis with acute exacerbations. Hepatology 7: Koike, K., Y. Shirakata, K. Yaginuma, M. Arii, S. Takada, I. Nakamura, Y. Hayashi, M. Kawada, and M. Kobayashi Oncogenic potential of hepatitis B virus. Mol. Biol. Med. 6: Koshy, R., S. Koch, A. Freytag von Loringhoven, R. Kahmann, K. Murray, and P. H. Hofschneider Integration of hepatitis B virus DNA: evidence for integration in the single stranded gap. Cell 34: Koshy, R., P. Maupas, R. Muller, and P. H. Hofschneider Detection of hepatitis B virus-specific DNA in the genomes of human hepatocellular carcinoma and liver cirrhosis tissues. J. Gen. Virol. 57: Koshy, R., and J. Wells Deregulation of cellular gene expression by HBV trans-activators in hepatocarcinogenesis, p In E. Tabor, A. M. DiBisceglie, and R. H. Purcell (eds.), Etiology, pathology, and treatment of hepatocellular carcinoma in North America. Gulf Publishing Co., Houston Koufos, A., M. F. Hansen, N. G. Copeland, N. A. Jenkins, B. C. Lampkin, and W. K. Cavenee Loss of heterozygosity in three embryonal tumours suggests a common pathogenetic mechanism. Nature (London) 316: Land, H., L. F. Parada, and R. A. Weinberg Cellular oncogenes and multistep carcinogenesis. Science 222: Larouze, B., B. S. Blumberg, W. T. London, E. D. Lustbader, M. Sankale, and M. Payet Forcasting the development of primary hepatocellular carcinoma by the use of risk factors: studies in West Africa. J. Natl. Cancer Inst. 58: Larouze, B., W. T. London, G. Saimot, B. G. Werner, E. D. Lustbader, M. Payet, and B. S. Blumberg Host responses to hepatitis B infection in patients with primary hepatic carcinoma and their families. A case control study in Senegal, West Africa. Lancet ii: Lee, H.-S., M. S. Rajagopalan, and G. N. Vyas A lack of direct role of hepatitis B virus in the activation of ras and c-myc oncogenes in human hepatocellular carcinogenesis. Hepatology 8: Lee, T.-H., M. J. Finegold, R.-F. Shen, J. L. DeMayo, S. L. C. Woo, and J. S. Butel Hepatitis B virus transactivator X protein is not tumorigenic in transgenic mice. J. Virol. 64: Lee, W.-H., R. Bookstein, F. Hong, L.-J. Young, J.-Y. Shew, and E. Y.-H. P. Lee Human retinoblastoma susceptibility gene: cloning, identification, and sequence. Science 235: Levine, A. J The p53 protein and its interactions with the oncogene products of the small DNA tumor viruses. Virology 177: Levrero, M., C. Balsano, G. Natoli, M. L. Avantaggiati, and E. Elfassi Hepatitis B virus X protein transactivates the long terminal repeats of human immunodeficiency virus types 1 and 2. J. Virol. 64: Levrero, M., 0. Jean-Jean, C. Balsano, H. Will, and M. Perricaudet Hepatitis B virus (HBV) X gene expression in human cells and anti-hbx antibodies detection in chronic HBV infection. Virology 174: Liang, T. J., K. Hasegawa, N. Rimon, J. R. Wands, and E. Ben-Porath A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. N. Engl. J. Med. 324: Lin, M.-H., and S. J. Lo Dimerization of hepatitis B viral X protein synthesized in a cell free system. Biochem. Biophys. Res. Commun. 164: Lo, S. J., M.-L. Chien, and Y.-H. W. Lee Characteristics of the X gene of hepatitis B virus. Virology 167: Lohiya, G., H. Pirkle, J. Hoefs, A. Lohiya, and V. T. Ngo Hepatocellular carcinoma in young mentally retarded HBsAg carriers without cirrhosis. Hepatology 5: Lok, A. S. F., S. J. Hadziyannis, I. V. D. Weller, M. G. Karvountzis, J. Moijardino, P. Karayiannis, L. Montano, and H. C. Thomas Contribution of low level HBV replication to continuing inflammatory activity in patients with anti-hbe positive chronic hepatitis B virus infection. Gut 25: Lombardi, B On the nature, properties and significance of oval cells, p In P. Pani, F. Feo, and A. Columbano (ed.), Recent trends in chemical carcinogenesis. ESA, Cagliari, Italy London, W. T Primary hepatocellular carcinoma-etiology, pathogenesis, and prevention. Hum. Pathol. 12: Lopez-Cabrera, M., J. Letovsky, K.-Q. Hu, and A. Siddiqui Multiple liver-specific factors bind to the hepatitis B

22 296 FEITELSON virus core/pregenomic promoter: trans-activation and repression by CCAAT/enhancer binding protein. Proc. Natl. Acad. Sci. USA 87: Lutwick, L. I Relation between aflatoxin, hepatitis B virus, and hepatocellular carcinoma. Lancet i: Mackay, J., C. M. Steel, P. A. Elder, A. P. M. Forrest, and H. J. Evans Allele loss on short arm of chromosome 17 in breast cancers. Lancet ii: Magnius, L Characterization of a new antigenantibody system associated with hepatitis B. Clin. Exp. Immunol. 20: Magnius, L. O., and J. A. Espmark New specificities in Australia antigen positive sera distinct from LeBouvier determinants. J. Immunol. 109: Maguire, H. F., J. P. Hoeffler, and A. Siddiqui HBV X protein alters the DNA binding specificity of CREB and ATF-2 by protein-protein interactions. Science 252: Malkin, D., F. P. Li, L. C. Strong, J. F. Fraumeni, Jr., C. E. Nelson, D. H. Kim, J. Kassel, M. A. Gryka, F. Z. Bischoff, M. A. Tainsky, and S. H. Friend Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250: Malt, R. A., J. J. Galdabini, and B. W. Jeppson Abnormal sex-steroid milieu in young adults with hepatocellular carcinoma. World J. Surg. 7: Marion, P. L., M. J. V. Davelaar, S. S. Knight, F. Salazar, G. Garcia, H. Popper, and W. S. Robinson Hepatocellular carcinoma in ground squirrels persistently infected with ground squirrel hepatitis virus. Proc. Natl. Acad. Sci. USA 83: Marion, P. L., S. S. Knight, B.-K. Ho, Y. Y.-Guo, W. S. Robinson, and H. Popper Liver disease associated with duck hepatitis B virus infection of domestic ducks. Proc. Natl. Acad. Sci. USA 81: Marion, P. L., L. S. Oshiro, D. C. Regnery, G. H. Scullard, and W. S. Robinson A virus in Beechey ground squirrels that is related to hepatitis B virus of humans. Proc. Natl. Acad. Sci. USA 77: Marion, P. L., H. Popper, R. R. Azcarraga, C. Steevens, M. J. V. Davelaar, G. Garcia, and W. S. Robinson Ground squirrel hepatitis and hepatocellular carcinoma, p In W. Robinson, K. Koike, and H. Will (ed.), Hepadna-viruses. Alan R. Liss, Inc., New York Marx, J. L The case of the misplaced gene. Science 218: Marx, J. L Many gene changes found in cancer. Science 246: Marx, J. L The cell cycle: spinning farther afield. Science 252: Mason, W. S., C. Aldrich, J. Summers, and J. M. Taylor Asymmetric replication of duck hepatitis B virus DNA in liver cells: free minus-strand DNA. Proc. Natl. Acad. Sci. USA 79: Mason, W. S., G. Seal, and J. Summers Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus. J. Virol. 36: Matsubara, K., and T. Tokino Integration of hepatitis B virus DNA and its implications for hepatocarcinogenesis. Mol. Biol. Med. 7: Mattei, M. G., H. de Thie, J. F. Mattei, A. Marchio, P. Tiollais, and A. Dejean Assignment of the human hap retinoic acid receptor RAR beta gene to the p24 band of chromosome 3. Hum. Genet. 80: McLachlan, A., D. R. Milich, A. K. Raney, M. G. Riggs, J. L. Hughes, J. Sorge, and F. V. Chisari Expression of hepatitis B virus surface and core antigens: influence of pre-s and pre-core sequences. J. Virol. 61: McMahon, G., E. F. Davis, J. Huber, Y. Kim, and G. N. Wogan Characterization of c-ki-ras and N-ras oncogenes in aflatoxin B,-induced rat liver tumors. Proc. Natl. Acad. Sci. USA 87: Meadows, A. T., J. L. Naiman, and M. Valdes-Dapena CLIN. MICROBIOL. REV. Hepatoma associated with androgen therapy for aplastic anemia. J. Pediatr. 84: Melbye, M., P. Skinhoj, N. H. Nielsen, B. F. Vestergaad, P. Ebbesen, P. H. Hansen, and R. J. Biggar Virus associated cancers in Greenland: frequent hepatitis B virus infection but low primary hepatocellular carcinoma incidence. JNCI 73: Menon, A. G., K. M. Anderson, V. M. Riccardi, R. Y. Chung, J. M. Whaley, D. W. Yandell, G. E. Farmer, R. N. Freiman, J. K. Lee, F. P. Li, D. F. Barker, D. H. Ledbetter, A. Kleider, R. L. Martuza, J. F. Gusella, and B. R. Seizinger Chromosome 17p deletions and p53 gene mutations associated with the formation of malignant neurofibrosarcomas in von Recklinghausen neurofibromatosis. Proc. Natl. Acad. Sci. USA 87: Menzies-Gow, N Hepatocellular carcinoma associated with oral contraceptives. Br. J. Surg. 65: Messing, A., H. Y. Chen, R. D. Palmiter, and R. L. Brinster Peripheral neuropathies, hepatocellular carcinomas and islet cell adenomas in transgenic mice. Nature (London) 316: Milich, D. R., J. E. Jones, J. L. Hughes, J. Price, A. K. Raney, and A. McLachlan Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero? Proc. Natl. Acad. Sci. USA 87: Miller, R. H., S. Kaneko, C. T. Chung, R. Girones, and R. H. Purcell Compact organization of the hepatitis B virus genome. Hepatology 9: Miller, R. H., S.-C. Lee, Y.-F. Liaw, and W. S. Robinson Hepatitis B viral DNA in infected human liver and in hepatocellular carcinoma. J. Infect. Dis. 151: Miller, R. H., P. L. Marion, and W. S. Robinson Hepatitis B viral DNA-RNA hybrid molecules in particles from infected liver are converted to viral DNA molecules during an endogenous DNA polymerase reaction. Virology 139: Miller, R. H., and W. S. Robinson Hepatitis B virus DNA forms in nuclear and cytoplasmic fractions of infected human liver. Virology 137: Miller, R. H., and W. S. Robinson Common evolutionary origin of hepatitis B virus and retroviruses. Proc. Natl. Acad. Sci. USA 83: Mintz, B., and R. A. Fleishman Teratocarcinoma and other neoplasms as developmental defects in gene expression. Adv. Cancer Res. 34: Moriyama, T., S. Guilhot, K. Klopchin, B. Moss, C. A. Pinkert, R. D. Palmiter, R. L. Brinster, 0. Kanagawa, and F. V. Chisari Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science 248: Moroy, T., J. Etiemble, L. Bougueleret, M. Hadchouel, P. Tiollais, and M.-A. Buendia Structure and expression of hcr, a locus rearranged with c-myc in a woodchuck hepatocellular carcinoma. Oncogene 4: Moroy, T., A. Marchio, J. Etiemble, C. Trepo, P. Tiollais, and M.-A. Buendia Rearrangement and enhanced expression of c-myc in hepatocellular carcinoma of hepatitis virus infected woodchucks. Nature (London) 324: Mulligan, L. M., G. J. Matlashewski, H. J. Scrable, and W. K. Cavenee Mechanisms of p53 loss in human sarcomas. Proc. Natl. Acad. Sci. USA 87: Nagasue, N., A. Ito, H. Yukaya, and Y. Ogawa Androgen receptors in hepatocellular carcinoma and surrounding parenchyma. Gastroenterology 89: Nagasue, N., H. Yukaya, Y.-C. Chang, Y. Ogawa, H. Kohno, and A. Ito Active uptake of testosterone by androgen receptors of hepatocellular carcinoma in humans. Cancer 57: Nagasue, N., H. Yukaya, T. Hamada, S. Hirose, R. Kanashima, and K. Inokuchi The natural history of hepatocellular carcinoma: a study of 100 untreated cases. Cancer 54: Nagaya, T., T. Nakamura, T. Tokino, T. Tsurimoto, M. Imai, T. Mayumi, K. Kamino, K. Yamamura, and K. Matsubara The mode of hepatitis B virus DNA integration in

23 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 297 chromosomes of human hepatocellular carcinoma. Genes Dev. 1: Nakamura, Y., M. Leppert, P. O'Connell, R. Wolff, T. Holm, M. Culver, C. Martin, E. Fujimoto, M. Hoff, E. Kumlin, and R. White Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235: Nakao, K., Y. Miyao, Y. Ohe, and T. Tamaoki Involvement of an AP1-binding site in cell-specific transcription of the pre-s1 region of the human hepatitis B virus surface antigen gene. Nucleic Acids Res. 17: Naumov, N. V., M. Mondelli, G. J. M. Alexander, R. S. Tedder, A. L. W. F. Eddleston, and R. Williams Relationship between expression of hepatitis B virus antigens in isolated hepatocytes and autologous lymphocyte cytotoxicity in patients with chronic hepatitis B virus infection. Hepatology 4: Nayak, N. C., A. Dhar, R. Sachdeva, A. Mittal, N. H. Seth, D. Sudarsonam, B. Reddy, U. L. Wagholikar, and C. R. R. Reedy Association of human hepatocellular carcinoma and cirrhosis with hepatitis B virus surface and core antigens in the liver. Int. J. Cancer 20: Nazarewicz-de Mezer, T., K. Krawczynski, T. Michalak, and A. Nowoslawski Intracellular localization of HB antigens in liver tissue, p In L. Bianchi, W. Gerok, K. Sickinger, and G. A. Stalder (ed.), Virus and the liver. MTP Press, Ltd., Lancaster, England Nazarewicz-de, Mezer, T., J. Slusarczyk, K. Krawczynski, and A. Nowoslawski Localization of hepatitis B virus antigens in hepatocellular carcinoma. Prog. Med. Virol. 27: Nerenberg, M., S. H. Hinrichs, R. K. Reynolds, G. Khoury, and G. Jay The tat gene of human T-lymphotropic virus type 1 induces mesenchymal tumors in transgenic mice. Science 237: Neurath, A. R., S. B. H. Kent, N. Strick, and K. Parker Biological role of pre-s sequences of the hepatitis B virus envelope protein, p In W. Robinson, K. Koike, and H. Will (ed.), Hepadna viruses. Alan R. Liss, Inc., New York Nigro, J. M., S. J. Baker, A. C. Preisinger, J. M. Jessup, R. Hostetter, K. Cleary, S. H. Bigner, N. Davidson, S. Baylin, P. Devilee, T. Glover, F. S. Collins, A. Weston, R. Modali, C. C. Harris, and B. Vogelstein Mutations in the p53 gene occur in diverse human tumor types. Nature (London) 342: Nishioka, K., A. G. Levin, and M. J. Simons Hepatitis B antigen, antigen subtypes and hepatitis B antibody in normal subjects and patients with liver disease. Bull. W.H.O. 52: Notario, V., S. Sukumar, E. Santos, and M. Barbacid A common mechanism for animal tumors, p In G. F. Van de Wonde, A. J. Levine, W. C. Topp, and J. D. Watson (ed.), Cancer cells, oncogenes, and viral genes. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y Obata, H., N. Hayashi, Y. Motoike, T. Hisamitsu, H. Okuda, S. Kobayashi, and K. Nishioka A prospective study on the development of hepatocellular carcinoma from liver cirrhosis with persistent hepatitis B infection. Int. J. Cancer 25: Obayashi, A Genetic and familial aspects of liver cirrhosis and hepatocellular carcinoma, p In K. Okuda, and R. L. Peters (ed.), Hepatocellular carcinoma. John Wiley & Sons, Inc., New York Ochiya, T., A. Fugiyama, S. Fukushige, I. Hatada, and K. Matsubara Molecular cloning of an oncogene from a human hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 83: Ogston, C. W., J. R. Jonak, C. E. Rogler, S. M. Astrin, and J. Summers Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks. Cell 29: Ohbayashi, A., K. Okochi, and M. Mayumi Familial clustering of asymptomatic carriers of Australia antigen and patients with chronic liver disease or primary liver cancer. Gastroenterology 62: Ohkoshi, S., H. Kojima, H. Tawaraya, T. Miyajima, T. Kamimura, H. Asakura, A. Satoh, S. Hirose, M. Hiikata, N. Kato, and K. Shimotohno Prevalence of antibody against non-a, non-b hepatitis virus in Japanese patients with hepatocellular carcinoma. Jpn. J. Cancer Res. 81: Ohnishi, K., S. Iida, S. Iwama, N. Goto, F. Nomura, M. Takashi, A. Mishima, K. Kono, K. Kimura, H. Musha, K. Kotota, and K. Okuda The effect of chronic habitual alcohol intake on the development of liver cirrhosis and hepatocellular carcinoma. Cancer 49: Okuda, K., and R. P. Beasley Epidemiology of hepatocellular carcinoma. UICC Tech. Rep. Ser. 17: Okuda, K., I. Kamiyama, M. Inomata, M. Imai, Y. Miakama, and M. Mayumi e antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. N. Engl. J. Med. 294: Omata, M., T. Ehata, 0. Yokosuka, K. Hosoda, and M. Ohto Mutations in the precore regions of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N. Engl. J. Med. 324: Omata, M., K. Uchiumi, Y. Ito, 0. Yokosuka, J. Mori, K. Terao, Y. Wei-Fa, A. P. O'Connell, W. T. London, and K. Okuda Duck hepatitis B virus and liver diseases. Gastroenterology 85: Ono, Y., H. Onda, R. Sasada, K. Igarashi, Y. Sugino, and K. Nishioka The complete nucleotide sequences of the cloned hepatitis B virus DNA: subtype adr and adw. Nucleic Acids Res. 11: Order, S. E., J. L. Klein, P. K. Leichner, and G. B. Stillwagon New therapeutic approaches for the management of hepatocellular carcinoma in the United States, p In E. Tabor, A. M. DiBiseglie, and R. H. Purcell (ed.), Etiology, pathology, and treatment of hepatocellular carcinoma in North America. Gulf Publishing Co., Houston Ou, J.-H., 0. Laub, and W. J. Rutter Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proc. Natl. Acad. Sci. USA 83: Ou, J.-H., and W. J. Rutter Hybrid hepatitis B virushost transcripts in a human hepatoma cell. Proc. Natl. Acad. Sci. USA 82: Palmiter, R. D., H. Y. Chen, A. Messing, and R. L. Brinster SV40 enhancer and large-t antigen are instrumental in development of choroid plexus tumors in transgenic mice. Nature (London) 316: Pasek, M., T. Goto, W. Gilbert, B. Zink, H. Schaller, P. MacKay, G. Leadbetter, and K. Murray Hepatitis B virus genes and their expression in E. coli. Nature (London) 282: Pasquinelli, C., F. Garreau, L. Bougueleret, E. Cariani, K. H. Grzeschik, V. Thiers, 0. Croissant, M. Hadchouel, P. Tiollais, and C. Brechot Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors. J. Virol. 62: Payne, G. S., J. M. Bishop, and H. E. Varmus Multiple arrangements of viral DNA and an activated host oncogene in bursal lymphomas. Nature (London) 295: Popper, H., L. Roth, R. H. Purcell, B. C. Tennant, and J. L. Gerin Hepatocarcinogenicity of the woodchuck hepatitis virus. Proc. Natl. Acad. Sci. USA 84: Popper, H., J. W.-K. Shih, J. L. Gerin, D. C. Wong, B. H. Hoyer, W. T. London, D. L. Sly, and R. H. Purcell Woodchuck hepatitis and hepatocellular carcinoma: correlation of histologic with virologic observations. Hepatology 1: Porter, L. E., M. S. Elm, D. H. van Thiel, M. C. Dugas, and P. K. Eagon Characterization and quantitation of human hepatic estrogen receptor. Gastroenterology 84: Potter, V. R Phenotypic diversity in experimental hepatomas: the concept of partially blocked ontogeny. Br. J. Cancer 38: Pourcel, C., M. Hadchouel, A.-M. Salmon, T. Moroy, P. Tiollais, C. Babinet, and H. Farza The use of transgenic

24 298 FEITELSON mice containing hepatitis B virus DNA as a model for HBV infection: developmental and hormonal regulation of viral gene expression, p In A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York Prince, A. M Round table discussion: prophylaxis/ vaccination, p In L. Bianchi, W. Gerok, K. Sickinger, and G. A. Stalder (ed.), Virus and the liver. MTP Press, Ltd., Lancaster, England Raimondo, G., G. Rodino, V. Smedile, S. Brancatelli, D. Villari, G. Longo, and G. Squadrito Hepatitis B virus (HBV) markers and HBV-DNA in serum and liver tissue of patients with acute exacerbation of chronic type B hepatitis. J. Hepatol. 10: Raney, A. K., D. R. Milich, A. J. Easton, and A. McLachlan Differentiation-specific transcriptional regulation of the hepatitis B virus large surface antigen gene in human hepatoma cell lines. J. Virol. 64: Ray, M. B Hepatitis B virus antigens in tissues. University Park Press, Baltimore Realdi, G., A. Alberti, M. Rugge, F. Bortolotti, A. M. Rigoli, F. Tremolada, and A. Ruol Seroconversion from hepatitis B e antigen to anti-hbe in chronic hepatitis B virus infection. Gastroenterology 79: Reeve, A. A., M. R. Eccles, R. J. Wilkins, G. I. Bell, and L. J. Millow Expression of insulin-like growth factor II transcripts in Wilms' tumor. Nature (London) 317: Reeve, A. E., P. J. Housiaux, and R. J. M. Gardner Loss of Harvey ras allele in sporadic Wilms' tumor. Nature (London) 309: Reynolds, F. H., Jr., T. L. Sacks, D. N. Deobaghar, and J. R. Stephenson Cells nonproductively transformed by Abelson murine leukemia virus expresses a high molecular weight polyprotein containing structural and nonstructural components. Proc. Natl. Acad. Sci. USA 75: Reynolds, S. H., S. J. Stowers, R. R. Maronpot, M. W. Anderson, and S. A. Aaronson Detection and identification of activated oncogenes in spontaneously occurring benign and malignant hepatocellular tumors of the B6C3F1 mouse. Proc. Natl. Acad. Sci. USA 83: Reynolds, S. H., S. J. Stowers, R. M. Patterson, R. R. Maronpot, S. A. Aaronson, and M. W. Anderson Activated oncogenes in B6C3F1 mouse liver tumors: implications for risk assessment. Science 237: Riccardi, V. M., H. M. Hittner, U. Francke, J. J. Yunis, D. Ledbetter, and W. Borges The aniridia-wilms' tumor association: the critical role of chromosome band 11pl3. Cancer Genet. Cytogenet. 2: Robinson, W. S The genome of hepatitis B virus. Annu. Rev. Microbiol. 31: Robinson, W. S., L. Klote, and N. Aoki Hepadnaviruses in cirrhotic liver and hepatocellular carcinoma. J. Med. Virol. 31: Robinson, W. S., P. L. Marion, M. A. Feitelson, and A. A. Siddiqui The hepadna virus group: hepatitis B and related viruses, p In W. Szmuness, H. J. Alter, and J. E. Maynard (ed.), Proceeding of the International Symposium on Viral Hepatitis. Franklin Institute Press, Philadelphia Robinson, W. S., R. H. Miller, and P. L. Marion Hepatitis B virus as an environmental carcinogen, p In H. A. Milman, and S. Sell (ed.), Application of biological markers to carcinogen testing. Plenum Publishing Corp., New York Rogler, C. E., 0. Hino, and D. A. Shafritz Mechanisms of hepatic oncogenesis during persistent hepadna virus infection, p In A. L. Notkins and M. B. A. Oldstone (ed.), Concepts in Viral Pathogenesis III. Springer-Verlag, New York Rogler, C. E., M. Sherman, C. Y. Su, D. A. Shafritz, J. Summers, T. B. Shows, A. Henderson, and M. Kew Deletion in chromosome llp associated with a hepatitis B integration site in hepatocellular carcinoma. Science 230: Rogler, C. E., and J. Summers Cloning and structural CLIN. MICROBIOL. REV. analysis of integrated woodchuck hepatitis virus sequences from a chronically infected liver. J. Virol. 50: Roingeard, P., S. Lu, C. Sureau, M. Freschlin, B. Arbeille, M. Essex, and J.-L. Romet-Lemonne Immunocytochemical and electron microscopic study of hepatitis B virus antigen and complete particle production in hepatitis B virus DNA transfected Hep G2 cells. Hepatology 11: Ruiz-Opazo, N., P. R. Chakarborty, and D. A. Shafritz Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum Dane particles: possible implications in persistent HBV infection. Cell 29: Rutter, W. J., M. Ziemer, J. Ou, Y. Shaul, 0. Laub, P. Garcia, and D. N. Standring Transcription units of hepatitis B virus genes and structure and expression of integrated viral sequences, p In G. N. Vyas, J. L. Dienstag, and J. H. Hoofnagle (ed.), Viral hepatitis and liver disease. Grune and Stratton, Inc., New York Sager, R Tumor suppressor genes: the puzzle and the promise. Science 246: Sambamurti, K., J. Callahan, X. Luo, C. P. Perkins, J. S. Jacobson, and M. Z. Humayun Mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position. Genetics 120: Schubach, W., and M. Groudine Alterations of c-myc chromatin structure by avian leukosis virus integration. Nature (London) 307: Scott, J., J. Cowell, M. E. Robertson, L. M. Priestly, R. Wadley, B. Hopkins, J. Pritchard, G. I. Bell, L. B. Rail, and C. F. Graham IGF-II gene expression in Wilms' tumor and embryonic tissues. Nature (London) 317: Scudamore, C. H., A. Hemming, and Y. Chow Resection of hepatocellular carcinoma in the cirrhotic patient, p In E. Tabor, A. M. DiBisceglie, and R. H. Purcell (eds.), Etiology, pathology, and treatment of hepatocellular carcinoma in North America. Gulf Publishing Co., Houston Seeger, C., B. Baldwin, W. E. Hornbuckle, A. E. Yeager, B. C. Tennant, P. Cote, L. Ferrell, D. Ganem, and H. E. Varmus Woodchuck hepatitis virus is a more efficient oncogenic agent than ground squirrel hepatitis virus in a common host. J. Virol. 65: Seeger, C., D. Ganem, and H. E. Varmus The nucleotide sequence of an infectious, molecularly cloned genome of the ground squirrel hepatitis virus. J. Virol. 51: Seeger, C., P. L. Marion, D. Ganem, and H. E. Varmus In vitro recombinants of ground squirrel and woodchuck hepatitis viral DNA produce infectious virus in squirrels. J. Virol. 61: Seifer, M., M. Hohne, S. Schaefer, and W. H. Gerlich Malignant transformation of immortalized cells by hepatitis B virus DNA, abstr. 619, p Int. Symp. Viral Hepatitis Liver Dis., Houston, Tex Sell, S., and H. L. Leffert An evaluation of cellular lineages in the pathogenesis of experimental hepatocellular carcinoma. Hepatology 2: Sell, S., and J. Salmon Light and electron-microscopic autoradiographic analysis of proliferating cells during the early stages of chemical hepatocarcinogenesis in the rat induced by feeding N-2-fluorenylacetamide in a choline-deficient diet. Am. J. Pathol. 114: Sells, M. A., M.-L. Chen, and G. Acs Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci. USA 84: Seto, E., P. J. Mitchell, and T. S. B. Yen Transactivation by the hepatitis B virus X protein depends on AP-2 and other transcription factors. Nature (London) 344: Seto, E., T. S. B. Yen, B. M. Peterlin, and J.-H. Ou Trans-activation of the human immunodeficiency virus long terminal repeat by the hepatitis B virus X protein. Proc. Natl. Acad. Sci. USA 85: Shafritz, D. A., G. Raimondo, H. M. Lieberman, R. D. Burk, S. J. Hadziyannis, H. Will, M. C. Kew, and G. M. Dusheiko Molecular biology of hepatocellular carcinoma: HBV

25 VOL. 5, 1992 HBV INFECTION AND PRIMARY HEPATOCELLULAR CARCINOMA 299 DNA molecular forms and viral gene products in human liver tissue, p In A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York Shafritz, D. A., and C. E. Rogler Molecular characterization of viral forms observed in persistent hepatitis infections, chronic liver disease and hepatocellular carcinoma in woodchucks and humans, p In G. N. Vyas, J. L. Dienstag, and J. H. Hoofnagle (ed.), Viral hepatitis and liver disease. Grune and Stratton, Inc., New York Shafritz, D. A., D. Shouval, H. I. Sherman, S. J. Hadziyannis, and M. C. Kew Integration of hepatitis B virus DNA into the genome of liver cells in chronic liver disease and hepatocellular carcinoma. N. Engl. J. Med. 305: Shaul, Y., W. J. Rutter, and 0. Laub A human hepatitis B viral enhancer element. EMBO J. 4: Sheiness, D. K., and J. M. Bishop DNA and RNA from uninfected vertebrate cells contain nucleotide sequences related to the putative transforming gene of avian myelocytomatosis virus. J. Virol. 31: Sherlock, S Hepatic cirrhosis, p In S. Sherlock (ed.), Diseases of the liver and biliary system. Blackwell Scientific Publications Ltd., Oxford Shih, C., K. Burke, M.-J. Chou, J. B. Zeldis, C.-S. Yang, C.-S. Lee, K. J. Isselbacher, J. R. Wands, and H. M. Goodman Tight clustering of human hepatitis B virus integration sites in hepatomas near a triple-stranded region. J. Virol. 61: Shih, C., M.-Y. W. Yu, L.-S. Li, and J. W.-K. Shih Hepatitis B virus propagated in a rat hepatoma cell line is infectious in a primate model. Virology 179: Shimoda, T., T. Shikata, T. Tsutomu, S. Tsukagoshi, M. Yoshimura, and I. Sakurai Light microscopic localization of hepatitis B virus antigens in the human pancreas. Possibility of multiplication of hepatitis B virus in the human pancreas. Gastroenterology 81: Shiozawa, M., T. Ochiya, I. Hatada, T. Imamura, Y. Okudaira, H. Hiraoka, and K. Matsubara The Ica as an onco-fetal gene: its expression in human fetal liver. Oncogene 2: Shirakata, Y., M. Kawada, Y. Fujiki, H. Sano, M. Oda, K. Yaginuma, M. Kobayashi, and K. Koike The X gene of hepatitis B virus induced growth stimulation and tumorigenic transformation of mouse NIH3T3 cells. Jpn. J. Cancer Res. 80: Simon, D., S. J. Munoz, W. C. Maddrey, and B. B. Knowles Chromosomal rearrangements in a primary hepatocellular carcinoma. Cancer Genet. Cytogenet. 45: Slagle, B. L., Y.-Z. Zhou, and J. S. Butel Hepatitis B virus integration event in human chromosome 17p near the p53 gene identifies the region of the chromosome commonly deleted in virus-positive hepatocellular carcinomas. Cancer Res. 51: Smuckler, E. A., L. Ferrell, and G. A. Clawson Proliferative hepatocellular lesions, benign and malignant, p In G. N. Vyas, J. L. Dienstag, and J. H. Hoofnagle (ed.), Viral hepatitis and liver disease. Grune and Stratton, Inc., New York Snyder, R. L., and J. Summers Woodchuck hepatitis virus and hepatocellular carcinoma, p In M. Essex, E. Todaro, and H. zur Hausen (ed.), Viruses in naturally occurring tumors. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y Snyder, R. L., G. Tyler, and J. Summers Chronic hepatitis and hepatocellular carcinoma. Am. J. Pathol. 107: Spandau, D. F., and C.-H. Lee trans-activation of viral enhancers by the hepatitis B virus X protein. J. Virol. 62: Stanbridge, E. J Base for genetic regulatory elements that control tumorigenic expression in human hybrid cells, p In J. C. Barrett, and R. W. Tennant (ed.), Carcinogenesis. Raven Press, New York Stanbridge, E. J., B. Fagg, and C. J. Der Differentiation and the control of tumorigenicity in human cell hybrids, p In C. C. Harris, and H. N. Autrup (ed.), Human carcinogenesis. Academic Press, Inc., San Diego, Calif Stemler, M., T. Weimer, Z.-X. Tu, D.-F. Wan, M. Levrero, C. Jung, G. R. Pape, and H. Will Mapping of B-cell epitopes of the human hepatitis B virus X protein. J. Virol. 64: Stevens, C. E., R. P. Beasley, J. Tsui, and W. Lee Vertical transmission of hepatitis B antigen in Taiwan. N. Engl. J. Med. 292: Summers, J., and W. S. Mason Replication of the genome of a hepatitis B like virus by reverse transcription of an RNA intermediate. Cell 29: Summers, J., A. O'Connell, P. Maupas, A. Goudeau, P. Coursaget, and J. Drucker Hepatitis B virus DNA in primary hepatocellular carcinoma tissue. J. Med. Virol. 2: Summers, J., J. M. Smolec, and R. Snyder A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc. Natl. Acad. Sci. USA 75: Sureau, C., J.-L. Romet-Lemonne, J. I. Mullins, and M. Essex Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Cell 47: Suzuki, K., T. Uchida, R. Horiuchi, and T. Shikata Localization of hepatitis B virus surface and core antigens in human hepatocellular carcinoma by immunoperoxidase methods. Cancer 56: Suzuki, K., T. Uchida, T. Shikata, M. Moriyama, Y. Arakawa, M. Mizokami, and F. Mima Expression of pre-sl, pre-s2, S and X peptides in relation to viral replication in livers with chronic hepatitis B. Liver 10: Swanstrom, R., R. C. Parker, H. E. Varmus, and J. M. Bishop Transduction of a cellular oncogene: the genesis of Rous sarcoma virus. Proc. Natl. Acad. Sci. USA 80: Sylvan, S. P. E., and U. B. Hellstrom Immunological mechanisms in asymptomatic carriers of HBsAg, p In A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York Szmuness, W Hepatocellular carcinoma and hepatitis B virus: evidence for a causal association. Prog. Med. Virol. 24: Szmuness, W., C. E. Stevens, E. J. Harley, E. A. Zang, W. R. Oleszko, D. C. William, R. Sadovsky, J. M. Morrison, and A. Kellner Hepatitis B vaccine. Demonstration of efficacy in a controlled clinical trial in a high-risk population in the United States. N. Engl. J. Med. 303: Szmuness, W., C. E. Stevens, E. A. Zang, E. J. Harley, and A. Kellner A controlled clinical trial of the efficacy of the hepatitis B vaccine (Heptavax B): a final report. Hepatology 1: Tabor, E Hepatocellular carcinoma: possible etiologies in patients without serologic evidence of hepatitis B virus infection. J. Med. Virol. 27: Tabor, E., R. J. Gerety, C. L. Vogel, A. C. Bayley, P. P. Anthony, C. H. Chan, and L. F. Barker Hepatitis B virus infection and primary hepatocellular carcinoma. J. Natl. Cancer Inst. 58: Takada, S., Y. Gotoh, S. Hayashi, M. Yoshida, and K. Koike Structural rearrangement of integrated hepatitis B virus DNA as well as cellular flanking DNA is present in chronically infected hepatic tissues. J. Virol. 64: Takada, S., and K. Koike Activated N-ras gene was found in human hepatoma tissue but only in a small fraction of the tumor cells. Oncogene 4: Takada, S., and K. Koike Trans-activation function of a 3' truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues. Proc. Natl. Acad. Sci. USA 87: Takahashi, T., M. Nau, I. Chiba, M. J. Birrer, R. K. Rosenberg, M. Vinocour, M. Levitt, H. Pass, A. F. Gazdar, and J. D. Minna p53: a frequent target for genetic abnormalities in lung cancer. Science 246: Takayama, T., M. Makuuchi, S. Hirohashi, M. Sakamoto, N.

26 300 FEITELSON Okazaki, K. Takayasu, T. Kosuge, Y. Motoo, S. Yamazaki, and H. Hasegawa Malignant transformation of adenomatous hyperplasia to hepatocellular carcinoma. Lancet ii: Takeya, T., and H. Hanafusa Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus. Cell 32: Tanaka, T., H. Miyamoto, 0. Hino, T. Kitagawa, M. Koike, T. lizuka, H. Sakamoto, and A. Ooshima Primary hepatocellular carcinoma with hepatitis B virus-dna integration in a 4-year-old boy. Hum. Pathol. 17: T'Ang, A., J. M. Varley, S. Chakraborty, A. L. Murphree, and Y.-K. T. Fung Structural rearrangement of the retinoblastoma gene in human breast carcinoma. Science 242: Tennant, B. C., W. E. Hormbuckle, A. E. Yeager, B. H. Baldwin, W. K. Sherman, W. I. Anderson, H. Steinberg, P. J. Cote, and J. L. Gerin Effects of aflatoxin B1 (AFB1) on experimental woodchuck hepatitis virus (WHV) infection and hepatocellular carcinoma (HCC), abstr. 579, p Int. Symp. Viral Hepatitis Liver Dis., Houston, Tex Theilmann, L., M.-Q. Klinkert, K. Gmelin, J. Salfeld, H. Schaller, and E. Pfaff Detection of pre-sl proteins in serum and liver of HBsAg-positive patients: a new marker for hepatitis B virus infection. Hepatology 6: Thorgeirsson, S. S., and R. P. Evarts Experimental hepatocarcinogenesis: relationship between oval cells and hepatocytes in rat liver, p In E. Tabor, A. M. DiBisceglie, and R. H. Purcell (ed.), Etiology, pathology, and treatment of hepatocellular carcinoma in North America. Gulf Publishing Co., Houston Thung, S. N., M. A. Gerber, E. J. Kasambalides, B. K. Gija, W. Keh, and W. H. Gerlich Demonstration of pre-s polypeptides of hepatitis B virus in infected livers. Hepatology 6: Ting, L.-P., H.-K. Chang, C. Chang, and C.-K. Chou Expression of HBV genes in well- and poorly-differentiated human hepatoma c Ul lines, p In W. Robinson, K. Koike, and H. Will (ed.), Hepadna viruses. Alan R. Liss, Inc., New York Tiollais, P., C. Pourcel, and A. Dejean The hepatitis B virus. Nature (London) 317: Toguchida, J., K. Ishizaki, Y. Nakamura, M. S. Sasaki, M. Ikenaga, M. Kato, M. Sugimoto, Y. Kotoura, and T. Yamamuro Assignment of common allele loss in osteosarcoma to the subregion 17p13. Cancer Res. 49: Tokino, T., S. Fukushige, T. Nakamura, T. Nagaya, T. Murotsu, K. Shiga, N. Aoki, and K. Matsubara Chromosomal translocation and inverted duplication associated with integrated hepatitis B virus in hepatocellular carcinomas. J. Virol. 61: Tong, M. J., M. W. Thursby, J.-H. Lin, J. Y. Weissman, and C. M. McPeak Studies on the maternal-infant transmission of the hepatitis B virus and HBV infection within families. Prog. Med. Virol. 27: Tong, M. J., J. M. Weiner, M. W. Ashcavai, and G. N. Vyas Evidence for clustering of hepatitis B virus infection in families of patients with primary hepatocellular carcinoma. Cancer 44: Trent, J. M., E. J. Stanbridge, H. L. McBride, E. V. Meese, G. Casey, D. E. Araujo, C. M. Witkowski, and R. B. Nagle Tumorigenicity in human melanoma cell lines controlled by induction of human chromosome 6. Science 247: Trowbridge, R., E. A. Fagan, F. Davison, A. L. W. F. Eddleston, R. Williams, M. H. K. Linskens, and F. Farzaneh Amplification of the c-myc gene locus in a human hepatic tumor containing integrated hepatitis B virus DNA, p In A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York Trujillo, M. A., J. Letovsky, H. F. Maguire, M. Lopez-Cabrera, and A. Siddiqui Functional analysis of a liver-specific enhancer of the hepatitis B virus. Proc. Natl. Acad. Sci. USA 88: CLIN. MICROBIOL. REV Tsung-tang, S., and W. Neng-jin Studies on human liver carcinogenesis, p In C. C. Harris and H. N. Autrup (ed.), Human carcinogenesis. Academic Press, Inc., San Diego, Calif Tsurimoto, T., A. Fujiyama, and K. Matsubara Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA. Proc. Natl. Acad. Sci. USA 84: Tur-Kaspa, R., R. D. Burk, Y. Shaul, and D. A. Shafritz Hepatitis B virus DNA contains a glucocorticoid-responsive element. Proc. Natl. Acad. Sci. USA 83: Tur-Kaspa, R., and 0. Laub Corticosteroids stimulate hepatitis B virus DNA, mrna and protein production in a stable expression system. J. Hepatol. 11: Tuttleman, J. S., C. Pourcel, and J. Summers Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47: Tuttleman, J. S., J. C. Pugh, and J. Summers In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus. J. Virol. 58: Twu, J.-S., and W. S. Robinson Hepatitis B virus X gene can transactivate heterologous viral sequences. Proc. Natl. Acad. Sci. USA 86: Twu, J.-S., C. A. Rosen, W. A. Haseltine, and W. S. Robinson Identification of a region within the human immunodeficiency virus type 1 long terminal repeat that is essential for transactivation by the hepatitis B virus gene X. J. Virol. 63: Twu, J.-S., and R. H. Schloemer Transcriptional transactivating function of hepatitis B virus. J. Virol. 61: Uchida, T., K. Suzuki, M. Esumi, A. Arii, and T. Shikata Influence of aflatoxin B1 intoxication on duck livers with duck hepatitis B virus infection. Cancer Res. 48: Ulrich, P. P., R. A. Bhat, I. Kelly, M. R. Brunetto, F. Bonino, and G. N. Vyas A precore-defective mutant of hepatitis B virus associated with e antigen-negative chronic liver disease. J. Med. Virol. 32: Uriel, J Cancer, retrodifferentiation, and the myth of Faust. Cancer Res. 36: Uy, A., V. Bruss, W. H. Gerlich, H. G. Kochel, and R. Thomssen Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein. Virology 155: Valenzuela, P., M. Quironga, J. Zaldivar, P. Gray, and W. J. Rutter The nucleic acid sequence of the hepatitis B viral genome and the identification of the major viral genes, p In V. Fields, R. Jaenisch, and C. F. Fox (ed.), Animal virus genetics. Academic Press, Inc., New York Varmus, H. E Do hepatitis B viruses make a genetic contribution to primary hepatocellular carcinoma? p In G. N. Vyas, J. L. Dienstag, and J. H. Hoofnagle (ed.), Viral hepatitis and liver disease. Grune and Stratton, Inc., New York Vento, S., J. E. Hegarty, A. Alberti, C. J. O'Brien, G. J. M. Alexander, A. L. W. F. Eddleston, and R. Williams T lymphocyte sensitization to HBcAg and T cell-mediated unresponsiveness to HBsAg in hepatitis B virus-related chronic liver disease. Hepatology 5: Vitvitski-Trepo, L., A. Kay, C. Pichoud, P. Chevallier, S. D. Dinechin, B.-M. Shamoon, E. Mandart, C. Trepo, and F. Galibert Early and frequent detection of HBxAg and/or anti-hbx in hepatitis B virus infection. Hepatology 12: Vyas, G. N., and H. E. Blum Hepatitis B virus infection-current concepts of chronicity and immunity. West. J. Med. 140: Walker, G. J., N. K. Hayward, S. Falvey, and W. G. E. Cooksley Loss of somatic heterozygosity in hepatocellular carcinoma. Cancer Res. 51: Wang, H. P., and C. E. Rogler Deletions in human chromosome arms lip and 13q in primary hepatocellular carcinomas. Cytogenet. Cell Genet. 48: Wang, H.-P., and C. E. Rogler Topoisomerase 1-medi-

27 VOL. 5, 1992,HBV INFECITION AND PRIMARY HEPATOCELLULAR CARCINOMA 301 ated integration of hepadnavirus DNA in vitro. J. Virol. 65: Wang, J From c-abl to v-abl. Nature (London) 304: Wang, J., X. Chenivesse, B. Henglein, and C. Brechot HBV integrates in a human cyclin A gene in an early hepatocellular carcinoma, abstr. 611, p Int. Symp. Viral Hepatitis Liver Dis., Houston, Tex Wang, W., W. T. London, and M. A. Feitelson Hepatitis B x antigen in hepatitis B carrier patients with liver cancer. Cancer Res. 51: Wang, W., W. T. London, L. Lega, and M. A. Feitelson HBxAg in the liver from carrier patients with chronic hepatitis and cirrhosis. Hepatology 14: Wang, Y., P. Yeh, W. Li, and Y. Liu Correlation between geographical distribution of liver cancer and aflatoxin B1 climate conditions. Sci. Sin. Ser. B 26: Weissman, B. E., P. J. Saxon, S. R. Pasquale, G. R. Jones, A. G. Geiser, and E. J. Stanbridge Introduction of a normal human chromosome 11 into a Wilms' tumor cell line controls its tumorigenic expression. Science 236: Werness, B. A., A. J. Levine, and P. M. Howley Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248: Wiseman, R. W., S. J. Stowers, E. C. Miller, M. W. Anderson, and J. A. Miller Activating mutations of the c-ha-ras proto-oncogene in chemically induced hepatomas of the male B6C3F1 mouse. Proc. Natl. Acad. Sci. USA 83: Witte, 0. N., N. Rosenberg, M. Paskind, A. Shields, and D. Baltimore Identification of an Abelson murine leukemia virus encoded protein present in transformed fibroblasts and lymphoid cells. Proc. Natl. Acad. Sci. USA 75: Wollersheim, M., U. Debelka, and P. H. Hofschneider A trans-activating function encoded in the hepatitis B virus X gene is conserved in the integrated state. Oncogene 3: Wong, D. T., N. Nath, and J. J. Sninsky Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame. J. Virol. 55: Wu, J. Y., Z.-Y. Zhou, A. Judd, C. A. Cartwright, and W. S. Robinson The hepatitis B virus-encoded transcriptional trans-activator HBx appears to be a novel protein serine/ threonine kinase. Cell 63: Wu, T.-T., L. Coates, C. E. Aldrich, J. Summers, and W. S. Mason In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway. Virology 175: Yaginuma, K., H. Kobayashi, M. Kobayashi, T. Morishima, K. Matsuyama, and K. Koike Multiple integration site of hepatitis B virus DNA in hepatocellular carcinoma and chronic active hepatitis tissues from children. J. Virol. 61: Yaginuma, K., H. Kobayashi, E. Yoshida, M. Kobayashi, and K. Koike Direct evidence for the expression of integrated hepatitis B virus DNA in human hepatoma tissue. Gann 75: Yaginuma, K., M. Kobayashi, E. Yoshida, and K. Koike Hepatitis B virus integration in hepatocellular carcinoma DNA: duplication of cellular flanking sequences at the integration site. Proc. Natl. Acad. Sci. USA 82: Yamada, G., Y. Sakamoto, M. Mizuno, T. Nishihara, T. Kobayashi, T. Takahashi, and H. Nagashima Electron and immunoelectron microscopic study of Dane particle formation in chronic hepatitis B virus infection. Gastroenterology 83: Yang, S. S., R. Modali, J.-B. Parks, and J. V. Taub Transforming DNA sequences of human hepatocellular carcinomas, their distribution and relationship with hepatitis B virus sequence in human hepatomas. Leukemia 2:102S-113S Yarrish, R. L., B. J. Werner, and B. S. Blumberg Association of hepatitis B virus infection with hepatocellular carcinoma in American patients. Int. J. Cancer 26: Yaswen, P., M. Goyette, P. R. Shank, and N. Fausto Expression of c-ki-ras, c-ha-ras, and c-myc in specific cell types during hepatocarcinogenesis. Mol. Cell. Biol. 5: Yee, J.-K A liver-specific enhancer in the core promoter region of human hepatitis B virus. Science 246: Yeh, F.-S., M. C. Yu, C.-C. Mo, S. Luo, M. J. Tong, and B. E. Henderson Hepatitis B virus, aflatoxins, and hepatocellular carcinoma in southern Guangxi, China. Cancer Res. 49: Yokosuka, O., M. Omata, Y.-Z. Zhou, F. Imazeki, and K. Okuda Duck hepatitis B virus DNA in liver and serum of Chinese ducks: integration of viral DNA in a hepatocellular carcinoma. Proc. NatI. Acad. Sci. USA 82: Yokota, J., M. Wada, Y. Shimosato, M. Terada, and T. Sugimura Loss of heterozygosity on chromosomes 3, 13, and 17 in small cell carcinoma and on chromosome 3 in adenocarcinoma of the lung. Proc. Natl. Acad. Sci. USA 84: Yu, M. C., T. Mack, R. Hamisch, R. L. Peters, B. E. Henderson, and M. C. Pike Hepatitis, alcohol consumption, cigarette smoking, and hepatocellular carcinoma in Los Angeles. Cancer Res. 43: Zahm, P., P. H. Hofschneider, and R. Koshy The HBV X-ORF encodes a transactivator: a potential factor in viral hepatocarcinogenesis. Oncogene 3: Zaman, S. N., R. D. Johnson, P. J. Johnson, W. M. Melia, B. C. Portmann, and R. Williams Risk factors in development of hepatocellular carcinoma in cirrhosis: prospective study of 613 patients. Lancet i: Zentgraf, H., G. Herrmann, R. Klein, P. Schranz, I. Loncarevic, D. HeLmnann, K. Hubner, and C. H. Schroder Mouse monoclonal antibody directed against hepatitis B virus X protein synthesized in Escherichia coli: detection of reactive antigen in liver cell carcinoma and chronic hepatitis. Oncology 47: Zhang, W., S. Hirohashi, H. Tsuda, Y. Shimosato, J. Yokota, M. Terada, and T. Sugimura Frequent loss of heterozygosity on chromosomes 16 and 4 in human hepatocellular carcinoma. Jpn. J. Cancer Res. 81: Zhang, Y.-J., C.-J. Chen, C.-S. Lee, B. Haghighi, G.-Y. Yang, L.-W. Wang, M. Feitelson, and R. Santella Aflatoxin B1-DNA adducts and hepatitis B virus antigens in hepatocellular carcinoma and nontumorous liver tissue. Carcinogenesis 12: Zhou, Y.-Z., B. L. Slagle, L. A. Donehower, P. van Tuinen, D. H. Ledbetter, and J. S. Butel Structural analysis of a hepatitis B virus genome integrated into chromosome 17p of a human hepatocellular carcinoma. J. Virol. 62: