1. Exposure Data

1.1. Structure and biology of hepatitis B virus (HBV)

1.1.1. Structure of the virus

The structure of hepatitis B virus (HBV) has been characterized in great detail (Tiollais & Buendia, 1991). HBV belongs to a group of hepatotropic DNA viruses (hepadnaviruses) that includes the hepatitis viruses of the woodchuck (Summers et al., 1978), ground squirrel (Marion et al., 1980), Pekin duck (Mason et al., 1980) and heron (Sprengel et al., 1988).

HBV is a small virus, about 42 nm in diameter (‘Dane particle’), composed of a lipid-bilayer envelope containing hepatitis B surface antigen (HBsAg) and an internal nucleocapsid structure (core). The nucleocapsid consists of the core protein and the viral DNA genome, which is about 3200 base pairs (about 2100 kDa) in length, with an associated DNA polymerase/reverse transcriptase (Tiollais et al, 1985; Blum et al, 1989a).

1.1.2. Structure of HBV genome and gene products

The viral genome is a partially double-stranded, circular DNA molecule. The genome has four open reading frames, three of which encode for viral proteins whose structures and functions have been well characterized (Fig. 1) (Blum et al., 1989a).

Fig. 1.

The hepatitis B viral DNA genome consists of a complete minus strand and an incomplete plus strand, with a cohesive overlap of their 5′ regions. In the cohesive region, there are two direct repeat sequences (DR1 and DR2), which are important in (more...)

(a) HBsAg

Hepatitis B surface antigen, formerly termed ‘Australia antigen’ (Le Bouvier, 1971), serum hepatitis antigen, hepatitis antigen and hepatitis associated antigen, is encoded by the pre-surface and surface genes; with lipid, it makes up the envelope of the virus. Excess HBsAg occurs abundantly in serum as small (22 nm) spherical or filamentous, non-infectious particles. In natural infection, the ratio of non-infectious HBsAg particles to virions is about 1000 to 1. Three different HBsAgs are synthesized: the large HBsAg (encoded by pre-S1, pre-S2 and S genes), the middle HBsAg (encoded by pre-S2 and S genes) and the major HBsAg (encoded by the S gene) (Tiollais et al., 1985; Blum et al, 1989a). The large HBsAg presumably mediates binding of the virus to the cell (Neurath et al., 1986, 1990). The function of the middle HBsAg is unknown. The major HBsAg represents the predominant structural protein of the viral envelope (Tiollais et al, 1985) (Fig. 2).

Fig. 2

Structure of hepatitis B virus, showing surface (HBsAgs) and core (HBcAg) antigens

HBsAg carries a group-specific determinant, a, common to all subtypes of this antigen, and two additional subtypic determinants, d or y and w or r. As a result, four major subtypes of HBsAg exist: adw, adr, ayw and ayr (Le Bouvier, 1971; Bancroft et al., 1972). They have distinct distributions worldwide and may therefore be useful for tracing the source of infection, e.g. w is commoner than r in the USA but r is commonest in Thailand (Bancroft et al., 1972).

The group-specific determinant a is encoded by the S genic region, encompassing roughly codons 124 to 147. This epitope is highly immunogenic, resulting in an anti-HBs response after natural infection (Carman et al, 1993) or vaccination (Halliday et al., 1992).

(b) HBsAg and HBeAg

The hepatitis B core antigen (HBcAg) and its antigenically distinct processed product, hepatitis B envelope antigen (HBeAg), are encoded by the pre-core/core (HBe-C/C) gene. The core (C) gene is transcribed into a core protein which packages the pre-genomic RNA to yield ‘core particles’. The pre-C/C gene is transcribed into a pre-C/C fusion protein (Blum et al, 1989a). The core antigen represents the major structural component of the nuclear capsid (Fig. 2). After truncation at its amino and carboxy termini, this protein is detectable in serum as HBeAg, usually indicating a high level of viral replication in the liver.

(c) Viral DNA polymerase/reverse transcriptase

The hepadnaviral polymerase gene encodes for a protein with a calculated molecular weight of 93.2 kDa. Genetic analysis has demonstrated that the enzyme consists of several functional domains arranged in order from the amino to the carboxy terminus: (i) terminal protein, which presumably serves as a protein primer for reverse transcription of the RNA pre-genome into minus-strand DNA (ii) a spacer region, which can be deleted without loss of enzyme activity; (iii) DNA polymerase/reverse transcriptase activity; and (iv) RNase H activity. HBV and duck hepatitis B virus polymerases have been expressed in vitro (Bartenschlager et al., 1992; Howe et al., 1992; Wang & Seeger, 1992).

(d) HBxAg

The fourth open reading frame, which is conserved in all mammalian but not avian hepadnaviruses, encodes for a small protein, X. The biological functions of this protein for the viral life cycle and for the pathobiology of HBV have not been firmly established. The X gene product (HBxAg) has been shown to activate transcription of HBV, other viral sequences and a variety of cellular genes (Kekulé et al., 1993). HBxAg does not seem to be required for HBV replication or gene expression in vitro (Blum et al., 1992). Data in the woodchuck model suggest, however, that a woodchuck hepatitis virus X-minus mutant is not infectious in vivo (Chen et al., 1993).

1.1.3. Replication of HBV

The hepadnaviral genomes are of similar size and structure and replicate asymmetrically via reverse transcription of an RNA intermediate (Summers & Mason, 1982). The replication strategy of HBV has been analysed in great detail both biochemically and genetically (Seeger et al., 1986; Will et al., 1987). Although hepadnaviruses are similar to retroviruses, their mode of replication is unique (Miller & Robinson, 1986), with homologies only to the cauliflower mosaic DNA virus (Toh et al., 1983).

1.1.4. HBV and related animal viruses

Similarities and differences between HBV and the related mammalian viruses of the woodchuck (WHV) and ground squirrel (GSHV) and the related avian viruses of Pekin duck (DHBV) and heron (HHBV) have been reviewed (Wain-Hobson, 1984; Mason & Taylor, 1989; Schödel et al., 1989, 1991). All hepadnaviruses have a similar sized, partially double-stranded genome of about 3000 base pairs, which replicates asymmetrically by reverse transcription of an intermediate RNA template, the pre-genome (Ganem & Varmus, 1987; Feitelson, 1992). Further, the genetic organization of these genomes is identical, except that the avian viruses (DHBV and HHBV) lack the X open reading frame. While hepadnaviruses have a high species specificity of infection, the DNA sequences of HBV and WHV are highly homologous, which results in cross-reactivity between HBsAg and WHsAg (Wain-Hobson, 1984). Further, GSHV has been shown to infect woodchucks (Seeger et al., 1991). Avian hepadnaviruses are more divergent in genomic structure and sequence. Calculations show DNA homologies of about 82% between GSHV and WHV and about 55% between GSHV and HBV, while only scattered homologies were apparent between DHBV and the other hepadnaviruses (reviewed by Sherker & Marion, 1991).

1.1.5. HBV mutants

The existence of HBV mutants was suspected for many years on the basis of the finding of HBV DNA in liver and serum from HBsAg-seronegative patients with or without antibodies to HBV. Conventional cloning techniques or polymerase chain reaction (PCR) amplification were used to clone and sequence viral DNA from sera and liver biopsy specimens, and naturally occurring mutations were identified in all viral genes. PCR allows amplification and detection of viral DNA at a sensitivity equal to that of tests of transmission in chimpanzees in vivo (Ulrich et al., 1989). The PCR product can be sequenced directly or after cloning into an appropriate vector.

In the woodchuck model, the mutation rate has been estimated to be less than or equal to 2 × 10⁻⁴ base substitutions per genome and year of replication (Girones & Miller, 1989). The hepadnaviral genome therefore appears to be relatively stable during replication in its natural host. The mutation rate of hepadnaviruses is 100–1000 times lower than that of RNA viruses but about 100 times higher than that of other DNA viruses. Since HBV infection frequently persists in humans for many years or decades, base changes can accumulate over time and may eventually result in a significant number of mutations. In addition, defective viral genomes containing major deletions occur frequently in individuals chronically infected with HBV (Takeda et al., 1990). These defective viruses probably arise during active viral replication, but their contribution to the pathogenesis of HBV-related liver disease remains unclear.

(a) Pre-S and S gene mutants (Carman et al., 1993)

While various naturally occurring mutations in the pre-S and S genes have been described, including deletions and point mutations leading to subtypic changes (Le Bouvier, 1971; Okamoto et al., 1987; Lai et al., 1991), a potentially important naturally occurring mutation affects the group-specific determinant a. A child from southern Italy, for example, who was infected by HBV despite passive-active immunization at birth and development of an anti-HBs response was found to carry a virus with a mutation in codon 145 of the S gene, resulting in a glycine to arginine substitution. This substitution results in loss of the a determinant against which the vaccine-induced anti-HBs response is mainly directed (Carman et al., 1990; Harrison et al., 1991; Waters et al., 1992). Similar findings have been reported from Japan (Fujii et al., 1992; Okamoto et al., 1992), where not only the mutation in codon 145 but a further mutation in codon 126 of the S gene was detected (an asparagine to threonine or isoleucine substitution), which also resulted in loss of the a determinant (Okamoto et al., 1992). In liver transplant recipients with chronic hepatitis B treated with a human monoclonal anti-HBs antibody, mutations similar to those described above have been identified in the S gene (McMahon, G. et al., 1992). The transmission efficiency of these mutants has not been established.

Other mutations in pre-S and S genes may affect the sensitivity of antigen-antibody tests routinely used to detect HBsAg in serum. This may be especially relevant for assays based on antigen capture or detection using monoclonal anti-HBs antibodies that may not bind to the mutant HBsAg.

(b) Pre-C and C gene mutants

Attention has recently focused on mutations identified in the pre-core (pre-C) or core (C) gene region of the viral genome in patients who seroconverted from HBeAg to anti-HBe without loss of viral replication. The mutations in the pre-C gene identified to date most frequently induce a stop codon at the end of the pre-C region (Carman et al., 1989; Akahane et al., 1990; Brunetto et al., 1990; Santantonio et al, 1991a), resulting in an inability to produce HBeAg. While a pre-C stop codon mutation does not interfere with viral replication (Tong et al., 1990), these mutations were found in clinically asymptomatic individuals, in patients with severe and active liver disease ( Carman et al., 1989; Akahane et al., 1990; Brunetto et al., 1990; Tong et al., 1990;Naoumov et al., 1992) and in patients with a fulminant course of HBV infection (Terazawa et al., 1990; Carman et al., 1991a,b; Hasegawa et al., 1991; Kojima et al., 1991; Kosaka et al., 1991; Liang et al., 1991a). Given the different clinical presentations of patients with pre-C stop codon mutants and the fact that in any patient with chronic HBV infection many mutants may coexist (‘quasispecies’) and multiple mutations may be found in a single viral genome, the causal relationship between the pre-C stop codon mutation and a particular course of the disease is unclear. A study in the woodchuck model suggests, however, that the pre-C stop codon may prevent persistence of WHV infection without affecting acute pathogenicity (Chen, H.-S. et al., 1992).

Relatively few mutations have so far been identified in the C gene. In a patient seropositive for human immunodeficiency virus, cytomegalovirus, HBsAg and HBeAg and seronegative for anti-HBc, Bhat et al. (1990) identified a viral mutant with two point mutations in the pre-C and C genes as well as an in-frame 36-base pair insertion in the pre-C region. In contrast, in a study of children with HBV infection seronegative for anti-HBc who were undergoing chemotherapy for malignancies, no pre-C or C mutation was found (Melegari et al., 1991). Recent evidence suggests, however, that mutations in the core gene (codons 84–101) are correlated with the severity of liver disease, possibly by altering recognition of infected cells by cytotoxic T cells (Ehata et al., 1992).

(c) X gene mutants

Three naturally occurring mutations in the X gene have been described: a replication-competent HBV genome with a pre-X open reading frame (Loncarevic et al., 1990); an HBV variant with an 8-base pair deletion at the 3′ end of the X gene (Repp et al., 1992); and a replication-competent HBV variant with a fused X–C reading frame, resulting from a single nucleotide insertion in the X–C overlapping region (Kim et al., 1992). The functional significance of X gene mutations is unclear.

(d) Polymerase gene mutants

In a patient serologically immune to HBV infection, a viral genome was identified with a point mutation in the protein region of the polymerase gene which terminated HBV replication through loss of RNA encapsidation function; this defect could be transcomplemented by a normal polymerase (Blum et al., 1991). A further naturally occurring polymerase-defective variant was detected in the DHBV system, as a point mutation in the region of the gene that encodes for RNase H activity (Chen, Y. et al., 1992). The functional significance of this mutation is unclear.

1.1.6. Host range and target cells of HBV infection

The host range of HBV and the related viruses in woodchuck, ground squirrel, Pekin duck and heron is very narrow. HBV, for example, infects only humans and chimpanzees. This narrow host range is believed to reflect the specificity of the liver-cell receptor for HBV, which interacts with an epitope in the pre-S1 region, 21–47, and which is also found on cells of extrahepatic origin (Neurath et al., 1986, 1990).

In permissive hosts, viral antigens and nucleic acids are found primarily in liver cells. By use of molecular techniques, hepadnaviruses have also been detected in cells other than hepatocytes (Blum et al., 1989b), e.g. bile-duct epithelial cells, endothelial cells in liver (Blum et al., 1983), pancreas, adrenal cortex, kidney, skin, spleen and bone-marrow cells (Halpern et al., 1983, 1984; Tagawa et al., 1985; Tiollais et al., 1985; Freiman et al., 1988; Yoffe et al., 1990; Mason et al., 1992) and various peripheral white blood cells. The biological significance of HBV in cells other than hepatocytes remains largely undefined (Ornata, 1990). Activation of HBV has been observed in peripheral blood mononuclear cells (Bouffard et al., 1992), and persistent HBV infection of mononuclear blood cells without concomitant liver infection has been demonstrated (Feray et al., 1990).

1.2. Methods of detection

Infection is detected on the basis of assays for viral antigens, antibodies and nucleic acids.

1.2.1. In serum and plasma

(a) HBsAg and anti-HBs

Tests for HBsAg developed in 1965 (Sherker & Marion, 1991) have since been improved significantly with regard to sensitivity and specificity. The early, less sensitive methods identified only patients with high titres of surface antigen. More recent methods, such as reverse passive haemagglutination and enzyme immunoassay/radioimmunoassay (EIA/RIA), are highly sensitive and specific and allow detection of HbsAg at 100–200 pg/ml serum, that is, about 3 × 10⁷ HBsAg particles/ml (Dusheiko et al., 1992), and these are the assays used most commonly for HBsAg in serum. In reverse passive haemagglutination, fixed erythrocytes coated with anti-HBs are added to test samples, and haemagglutination patterns are read. In EIA/RIA a sandwich method, with anti-HBs as both absorbed reagent and label or conjugate, has been employed. Anti-HBs is measured by passive haemagglutination of fixed erythrocytes coated with HBsAg or by an EIA/RIA sandwich method with HBsAg as the adsorbed reagent and label or conjugate.

(b) Pre-S antigens and antibodies

Pre-S antigens and antibodies are measured by research procedures (Itoh et al., 1986; Coursaget et al., 1990). The significance of these markers in natural infection or protection is not known.

(c) HBcAg and anti-HBc

HBcAg is not routinely detected in serum; in contrast, anti-HBc is a useful serological marker for current or past HBV infection. Total anti-HBc is measured by the haemagglutination inhibition method (Iizuka et al., 1992) or by competitive binding EIA/RIA. Anti-HBc tests have limited specificity, especially at low titres. Commercial tests for both immunoglobulin (Ig) M and total anti-HBc are available; high titres of IgM class anti-HBc are typically present in acute HBV infection. As IgG class anti-HBc appears and is predominant in the course of chronic infection, IgM-anti-HBc may be a useful marker to differentiate between acute and chronic infection.

(d) HBeAg and anti-HBe

HBeAg can be measured by sandwich EIA/RIA using anti-HBe as the capture antibody. Early tests for HBeAg, such as gel diffusion, had little sensitivity, and the results of studies based on such tests must be interpreted with caution. Anti-HBe can be measured by competitive binding.

(e) HBxAg and anti-HBx

HBxAg and anti-HBx are determined by an enzyme-linked immunoabsorbent assay (ELISA) (Horiike et al., 1991), which is not available commercially.

(f) HBV DNA

HBV DNA in serum can be detected by hybridization analysis (filter hybridization or liquid-phase hybridization) or PCR amplification followed by hybridization.

Hybridization assays: In filter hybridization, a test sample is denatured by the addition of sodium hydroxide and filtered through a nitrocellulose membrane to bind DNA. The membrane is then incubated with cloned labelled HBV DNA. If the test sample contains HBV DNA the labelled probe is annealed to the membrane-bound viral DNA and can be detected by autoradiography. The sensitivity of this assay is 0.1–1 pg HBV DNA or about 10³–10⁵ virions. In liquid-phase hybridization, HBV DNA exposed by virion lysis is mixed with a labelled HBV probe. This test system is available commercially, is better standardized than filter hybridization and has the same sensitivity; it is, however, costly and time-consuming (Dusheiko et al., 1992).

Polymerase chain reaction (PCR): The amplification of HBV DNA by PCR is an extremely sensitive test: theoretically, one genome equivalent per sample, at least 10 000 times more sensitive than dot-blot hybridization or RIA of HBsAg; it also facilitates analysis of the sequence of the amplified genomes. Contamination remains the major difficulty of this method, and extreme care must be taken at each step to avoid it. Negative and positive control samples, including reaction mixtures without DNA should be analysed in each test (Dusheiko et al., 1992; Seelig et aL, 1992). PCR followed by sequencing has also been used for subtyping HBV and for characterizing and identifying HBV mutants.

Table 1 gives information on the relative sensitivities and specificities of the tests for HBV markers.

Table 1.

Relative sensitivities and specificities of tests for hepatitis B viral markers in serum.

1.2.2. In liver tissues

(a) HBsAg and HBcAg

Both HBsAg and HBcAg can be detected by a direct immunofluorescence method in formalin-fixed, paraffin-embedded liver specimens (Yoshizawa et al., 1977). [The sensitivity is limited, however, as shown by the fact that 35–40% of individuals seropositive for HBV markers have no detectable level of antigen in tissues.] HBsAg can also be detected in infected liver cells by histochemical staining, such as with orcein and other reagents for staining elastic fibres (Shikata et al., 1974). These methods were used to locate HBsAg and HBcAg in liver cells.

(b) HBV DNA

HBV DNA can be detected in liver tissue by Southern blot hybridization of extracted DNA or by in-situ hybridization. The major contribution of Southern blot analysis is physical characterization of HBV DNA and especially the distinction between extrachromosomal viral replication and integration of viral sequences into the cellular genome (Tiollais et al., 1985). HBV-specific antigen and DNA can be detected simultaneously in paraffin-embedded liver tissue by immunohistochemistry and in-situ hybridization using a digoxigenin-label probe, without significant reduction in the sensitivity of either assay (Han et al., 1992). HBV DNA can also be detected at high sensitivity in formalin-fixed, paraffin-embedded liver tissue by PCR, at a level correlated with serological and immunohistochemical markers (Lampertico et al., 1990; Diamantis et al., 1992).

1.2.3. Interpretation of serological markers of HBV infection

Typical patterns of serological markers in HBV infection are summarized in Table 2. Further information and correlations with the clinical course of disease are given in section 1.4.

Table 2.

Typical serological patterns in HBV infection.

1.3. Epidemiology of infection

1.3.1. Transmission

Hepatitis B virus is transmitted from a person who has circulating virus and is HBsAg seropositive. The person may have an acute infection or be a carrier, a carrier being defined as a person who is seropositive for HBsAg on at least two occasions six months apart. Individuals who are HBeAg seropositive are particularly infectious, since the presence of this antigen is correlated with the level of serum HBV DNA.

The mode of transmission of virus to a susceptible individual varies with age. Transmission occurs at three important times of life: at birth (Mitsuda et al., 1989), in early childhood and in adult life. Neonates born to HBeAg-seropositive carriers have an approximately 85% chance of becoming infected, whereas children of HBeAg-seronegative carriers have only a 31% probability of infection (Beasley et al., 1977). The precise mode of perinatal transmission is unclear.

Infection in childhood is associated with living in households in which there is one or more infected sibling; the risk of infection increases with their number (Whittle et al., 1990). The mode of transmission in childhood is unclear. Traditional practices, such as scarification, ear piercing, circumcision and tatooing, have been proposed (Struve, 1992), but controlled studies have failed to confirm them as risk factors (Fox et al., 1988). HBV transmission through the use of contaminated needles, syringes and acupuncture equipment has been well documented (Kent et al., 1988). Skin lesions, in particular tropical ulcers, have been proposed as a source of infection (Foster et al., 1984), but, again, the evidence is not strong. Arthropods have been suggested as a means of transmission on the basis of studies of mosquitoes (Prince et al., 1972), bedbugs (Wills et al., 1977) and tampans (Joubert et al., 1985). One study found a significant association between infection of HBeAg-seropositive people and infestation of beds with bugs (Vail Mayans et al., 1990). Actual arthropod transmission has not been confirmed.

In adult life, parenteral and sexual transmission are the most important routes. The use of contaminated needles by intravenous drug users¹ is a very well documented form of transmission. For example, in a study of drug users in Sweden, 74% of men and 80% of women had markers of past infection, whereas only 1% and 5%, respectively, in the general population did so (Struve, 1992). In surveillance programmes, it was estimated that 27% of patients with acute hepatitis B in the USA in 1988 (Alter et al., 1990) and 24% in the United Kingdom in 1985–88 (Polakoff, 1990) were intravenous drug users. Blood transfusion and administration of blood products for bleeding disorders were important sources of parenteral exposure, but the risk has now been virtually eliminated by screening blood sources and by treatment of blood products.

Szmuness et al. (1975a) indicated the importance of sexual intercourse in transmission of HBV. They compared the prevalence of past infection in spouses of 280 people with and 238 without persistent infection and the cumulative prevalence in women, homosexual men and attendees at sexually transmitted disease clinics. The results clearly showed that the virus could be transmitted sexually. The prevalence of past infection was 10% in spouses of non-carriers and 27% in spouses of carriers. Homosexual men had a prevalence of 48%, but no increase was seen in homosexual women. Subsequent studies of sexual activity showed that the number of sexual partners, duration of sexual activity and a history of sexually transmitted disease were all risk factors for HBV infection (Alter et al., 1989; Rosenblum et al., 1992; Osmond et al., 1993). In surveillance studies of patients with acute hepatitis B, a history of multiple sexual partners was also found to be an important risk factor; 7% of all such patients in the USA and the United Kingdom had a history of homosexuality (Alter et al., 1990; Kingsley et al., 1990; Polakoff, 1990), and 26% in the USA were heterosexual (Alter et al., 1990). The largest proportion of patients in these surveillance studies reported no known risk factor. Some infection may be intra-familial (Szmuness et al., 1975b), from a household carrier. This form of transmission was associated with skin lesions in one case-control study (Bernier et al., 1982). In both developed (Szmuness et al., 1978a) and developing (Toukan, 1987) countries, infection is associated with low socio-economic status.

1.3.2. Determinants of chronic infection

Age at infection is the major determinant of whether a person becomes a carrier. Perinatal transmission confers the highest probability of becoming a carrier, with 80 to about 100% of infected children becoming carriers (Beasley et al., 1977; Wong et al, 1984). In children aged 1–10, the risk is 20–40% and appears to decline across this interval (McMahon et al., 1985; Coursaget et al., 1987). In adolescence and adult life, the probability of chronic infection following infection is in the range 0–10% (Nielsen et al, 1971; McMahon et al., 1985). The relationship has been reviewed recently (Edmunds et al, 1993), and there is no evidence of geographical heterogeneity.

This profile of risk for chronic infection contrasts with the risk for acute clinical hepatitis. Symptomatic infection is unusual in childhood but affects 30–60% of individuals in adolescence and adult life (McMahon et al., 1985).

The risk of becoming a carrier after infection is greater in males than in females in a ratio of about 1.6:1 (London, 1979). The sex ratio of prevalent carriers in the population increases with age because of longer chronic infection in males (Coursaget et al, 1987).

People whose immune system is suppressed, for example by cytotoxic drugs or the human immunodeficiency virus, appear to have a higher risk of chronic infection. They may also convert from apparent immunity to active infection.

1.3.3. Global patterns of chronic infection

WHO classifies hepatitis B endemicity by the proportion of the adult population who are hepatitis B carriers. Populations with 0–2% carriers are regarded as having low endemicity, 2–7% as intermediate and 8% or greater as high endemicity (Fig. 3).

Fig. 3.

Geographic pattern of the prevalence of hepatitis B

The prevalence of infection is low in North America, western Europe, Australia and South America, with the exception of the Amazon basin. In these populations, a steady increase in prevalence of viral markers is seen with age. In the USA, blacks have a higher prevalence of infection than whites, particularly at older ages (McQuillan et al., 1989). Intermediate levels of prevalence are found in eastern and southern Europe, in the Middle East, Japan and South Asia; high prevalences are found in China, Southeast Asia and sub-Saharan Africa (Prince, 1970; Szmuness, 1975).

High endemicity is associated with infection in childhood, as shown by the results of population-based surveys of hepatitis B markers before vaccination became widespread (Table 3). In Asia, perinatal transmission plays an important role in childhood infection: 30–40% of carriers are infected around the time of birth; in contrast, only 5–10% of carriers in Africa had perinatal infection. The proportion of mothers who are carriers is similar in Asia and sub-Saharan Africa (15–20%); however, 50% of carrier mothers in Asia are highly infectious (HBeAg seropositive), compared with only 10% in Africa. The reasons for this difference are not known.

Table 3.

Hepatitis B seroprevalence in children and adults in selected countries.

Childhood infection plays an important role in countries of both high and intermediate endemicity, whereas adult transmission is predominant in areas of low endemicity (Table 4). Perinatal transmission does occur in countries of low endemicity and is particularly important among migrants from highly endemic regions and their descendants. Adoption of carrier children from highly endemic countries can lead to intra-familial transmission (Christenson, 1986; Friede et al., 1988).

Table 4.

Endemicity of chronic infection with HBV by area of the world and predominant mode of transmission.

Variation may be seen within countries, as in Nigeria, with high prevalences in the north of the country but intermediate levels in the south (Fakunle et al., 1980; Nasidi et al., 1986); in Italy, with a low prevalence of infection in the north but an intermediate prevalence in the south (D’Amelio et al., 1992); and in China, with a markedly higher prevalence of persistent infection in adults in the southeast of the country than in the northern inland areas (Beasley et al., 1982). Variation in infection is also seen at the village level in Africa (Whittle et al., 1983, 1990) and the Middle East (Toukan et al, 1990): adults in adjacent villages have significantly different prevalences of persistent infection which are associated with the age at infection of children. Urban-rural differences in infection vary, some countries having higher urban rates and some higher rural rates (Soběslavský, 1980). In some countries, minority groups have significantly different risks of infection from the general population. For example, Maoris in New Zealand have higher rates than Caucasians (Milne et al., 1985), and Aborigines in Australia have intermediate to high rates of infection in comparison with the low rate in non-aboriginal Australians (Holman et al., 1987).

1.4. Clinical diseases (other than cancer)

The natural history and clinical manifestations of HBV infection are highly variable. In industrialized societies, about 45% of all HBV infections result in acute disease, and 1% have a fatal outcome. Chronic infections develop in 5% of infected people, and the remaining 50% of cases follow an asymptomatic course. There are multiple subtypes of the virus, but there is no known difference between the subtypes with respect to pathogenesis. In contrast, recently described HBV mutants may play a role in the clinical manifestations and natural history of HBV infection.

Whereas HBV replication and gene expression in infected individuals do not appear to be directly cytopathic, hepatic injury appears to be immune-mediated. Cytotoxic T cells are directed against HBcAg (Mondelli et al., 1982; Ferrari et al., 1987; Milich et al., 1989) and HBeAg (Ferrari et al., 1992; Tsai et al., 1992) but not against HBsAg (Mondelli et al., 1982), and the exact nature of the target antigens for cytotoxic immune reactions is unknown (Ferrari et al., 1987; Vento & Eddleston, 1987; Ferrari et al, 1992). Conversely, immune suppression by co-infection with human immune deficiency virus or by treatment for organ transplantation reduces inflammatory reactions in the liver and frequently results in normalization of biochemical parameters, with no resolution of liver disease (Davis, 1989; Todo et al., 1991; Martin et al., 1992; McNair et al., 1992).

1.4.1. Acute infection

The lag between exposure to HBV and onset of hepatitis B is 2–26 weeks, and the clinical expression of this infection is heterogeneous. Subclinical episodes of acute hepatitis are common, as indicated by the large number of chronically infected patients with no history of acute hepatitis B (Redeker, 1975). The usual clinical attack of hepatitis caused by HBV in adults is more severe than that in either children or patients with hepatitis caused by hepatitis A or C virus. The self-limited bout of icteric hepatitis usually lasts less than three months and only occasionally has a prolonged or cholestatic course. A minority of patients become jaundiced and have symptoms and signs of hepatitis, such as fever, fatigue, hepatosplenomegaly and dark urine. Fulminant hepatic failure is an occasional result of acute hepatitis (Junge & Deinhardt, 1985; Krogsgaard et al., 1985).

In uncomplicated hepatitis, HBV DNA is the first serum marker to appear, during the first four weeks of exposure, followed by HBsAg (Fig. 4a) (Krogsgaard et al., 1985). In acute self-limited hepatitis, HBsAg persists for several weeks. After a variable time (window period), anti-HBs appears. Detection of IgM antibodies to anti-HBc, another early marker of HBV infection, is useful in differentiating acute hepatitis B from other forms of acute liver damage which could also be present in healthy carriers of HBsAg (Chau et al., 1983; Perrillo et al., 1983). HBeAg appears concurrently with HBsAg as a fourth marker in the serum (Krugman et al., 1979). Recovery from acute hepatitis is heralded by clearance of serum HBV DNA HBsAg and HBeAg and sequential appearance of anti-HBe and anti-HBs (Fig. 4).

Fig. 4.

Serological patterns of acute infection (a) and chronic infection (b) with HBV

1.4.2. Chronic infection

Chronic HBV infection may follow acute symptomatic or asymptomatic infection but is more frequent after asymptomatic infection. It occurs more frequently in men than in women, in children than in adults and in immunocompromised patients than in immunocompetent patients (Taylor et al., 1988) (see section 1.3.2). The risk of chronicity declines from 80–90% following perinatal infection to 0–10% in older children and immunocompetent adults (McMahon et al., 1985; Edmunds et al., 1993).

Persistence of HBV infection is associated with variable degrees of hepatic inflammation; seroconversion to anti-HBe is paralleled by exacerbation of hepatitis caused by immune-mediated liver-cell necrosis and progressive clearance of infected hepatocytes and serum HBV DNA (Fig. 4). After seroconversion to anti-HBe, many patients show long-term, non-replicating, latent HBV infection. An important molecular process in HBsAg carriers is integration of HBV DNA into the liver-cell genome, and the majority of such carriers show no evidence of replication (De Franchis et al., 1993). Patients with replicating HBV display various degrees of liver damage—from no histological change to benign forms of chronic lobular hepatitis to more severe forms of active hepatitis and cirrhosis. Chronic active hepatitis is the result of host immune attacks on infected liver cells.

The annual rate of HBeAg/anti-HBe seroconversion and disease remission was 10–15% in adult Italian patients (Fattovich et al., 1986), but the rate varies by geographical location and age at infection. Children do not show reactivation of replication of HBV after anti-HBe seroconversion (Bortolotti et al., 1990). Although HBeAg to anti-HBe seroconversion in adults was accompanied by clinical, biochemical and histological remission of disease, in a study of 88 patients followed-up for a mean of five years, 10 (22%) had transient spontaneous reactivation of HBV infection and exacerbation of disease (Fattovich et al., 1990). Thus, seroconversion from HBeAg to anti-HBe during adulthood is not always stable, in contrast to infantile HBV infections (Lok et al., 1987). HBV reactivation may lead to deterioration of the underlying liver disease, from quiescent chronic hepatitis to active cirrhosis (Fattovich et al., 1990). Reactivation of a latent HBV infection occurs more frequently in immunocompromised patients infected with human immunodeficiency virus (Vento et al., 1989) and in patients receiving cytotoxic therapy (Lok et al., 1991).

HBV replication is instrumental in progression of the disease to cirrhosis. In 105 Italian patients with chronic hepatitis B (Fattovich et al., 1991) who were followed prospectively for a mean of 5.5 years, cirrhosis was documented in 34% of patients with persistent serum HBV DNA but in only 15% of those without serum HBV DNA. In these patients, bridging hepatic necrosis was another important predictor of cirrhosis. In 43 Dutch patients with compensated cirrhosis, five-year survival was 72%, but the risk of death was decreased by a factor of 2.2 when HBeAg seroconversion occurred during follow-up (De Jongh et al., 1992).

1.4.3. Extrahepatic manifestations

Extrahepatic clinical disease is infrequent during hepatitis B and has often been associated with circulating immune complexes containing viral antigens. Some patients in the prodromal phase of acute hepatitis have symptoms indicating immune complex disease, such as serum sickness-like syndrome, fever, urticarial skin lesions and symmetrical arthropathy. Systemic necrotizing vasculitis (polyarteritis) affecting the gastrointestinal tract (Shusterman & London, 1984), peripheral or central nervous system has been reported (Tabor, 1987). The presence of circulating complexes correlated well with disease activity. Membranous or membrano-proliferative glomerulonephritis due to HBeAg immunocomplexes has been found either alone or as part of a generalized vasculitis (Shusterman & London, 1984). The Guillain-Barré syndrome was reported, with HBsAg-containing immunocomplexes in serum and cerebrospinal fluid (Penner et al., 1982). Aplastic anaemia complicating hepatitis B is extremely uncommon and shows typical features of refractory marrow failure or hepatitis-dependent pancytopenia (McSweeney et al., 1988).

2. Studies of Cancer in Humans

Reports of epidemiological studies of liver cancer relevant to these monographs have employed a variety of terms to describe this disease, for example, liver cancer, primary liver cancer, primary hepatocellular carcinoma and hepatocellular carcinoma. We chose to use only the term hepatocellular carcinoma (HCC) in describing these studies. This choice was made because most of the studies have specified primary cancer of the liver or HCC, and the vast majority of primary liver cancers in most areas of the world are HCC (Colombo, 1992). A large number of case-control studies and fewer cohort studies have been conducted on the association between HBV and HCC. The many case reports, case series and descriptive studies are therefore not described in detail.

HBsAg is the marker of infection with HBV most often measured in epidemiological studies of HBV infection and HCC. While it is not often measured twice, six months apart (as required by the strict definition of carrier status), its presence in adults without acute hepatitis has generally been taken to indicate chronic infection with the virus.

The presence of other markers of HBV infection has also been documented in many studies, but most studies in which these markers were measured were not analysed with the intention of estimating their relationship with HCC. In addition, in some countries, the large majority of the population may have been exposed to HBV, and most have some marker of infection. Thus, an appropriate—uninfected—reference population of sufficient size may not be available for comparison with individuals with anti-HBc or anti-HBs alone or in combination. Lastly, in populations in which a suitable reference group may exist, it has usually not been reported separately.

2.3. Cohort studies

Cohort studies of HBV and HCC can be divided into three broad groups: studies of general population groups, studies of blood donors and studies of populations with pre-existing disease.

2.3.1. Prospective studies of general population groups (Table 5, p. 73)

Beasley and colleagues recruited 22 707 male Government employees in Taiwan, China, to evaluate their risk for HCC (Beasley & Lin, 1978; Beasley et al., 1981; Beasley & Hwang, 1991). Subjects were recruited at the time of routine physical examinations offered by the Government between March 1976 and June 1978. Study subjects were Chinese men aged from their twenties to over 70; 82% were 40–59 years of age. Follow-up for cancer incidence and mortality from all causes was conducted through medical and life insurance records and also by annual physical examinations of both HBV carriers and a subset of non-carriers. At the time of enrolment into the study, 3454 of the 22 707 men were HBsAg seropositive, 15 570 were anti-HBs seropositive and anti-HBc seropositive, 2411 were only anti-HBc positive and 1272 had no HBV marker. As at 30 June 1989, none of the HBsAg-seronegative men who were re-tested had become HBsAg seropositive. All HBsAg-seropositive men were re-tested annually; about 1% per year became HBsAg seronegative. At 30 June 1989, 194 cases of HCC had occurred, 184 (95%) in the HBsAg seropositive men, conferring a relative risk (RR) of 103 (95% CI, 57–205) as compared with HBsAg-seronegative subjects. The remaining 10 cases of HCC all occurred among the 17 981 anti-HBc-seropositive men. Among HBsAg-seronegative men, the difference in risk between those who were anti-HBc seropositive and those with no marker of HBV infection was not significant. The occurrence of HCC in the entire population was related to age, evidence of cirrhosis, HBeAg seropositivity and IgM anti-HBe seropositivity. The only causes of marked excess mortality found in relation to HBV markers were HCC and cirrhosis.

Table 5.

Prospective studies of HBV surface antigen (HBsAg)-seropositive people for the development of hepatocellular carcinoma (HCC).

Tu et al. (1985) studied all men over the age of 40 from four communes on Chongming Island, China, for development of HCC; 98% (12 222 men) of those eligible participated and were followed for three years. Of the 12 222 men, 1971 (16.1%) were classified as HBV carriers (defined as being either HBsAg seropositive or anti-HBs seronegative and anti-HBc seropositive at the time of the survey). Thirty-seven of the 70 deaths from HCC occurred among the HBV carriers and 33 occurred among the non-carriers, giving an RR of 6.7 [95% CI, 4.2–10.7]. The authors also studied the effects of water source, cigarette smoking, maize consumption (as a measure of exposure to aflatoxin B₁) and alcohol consumption on the risk for HCC. A significantly higher rate of HCC was seen among carriers who smoked 20 cigarettes or more per day. None of the other factors had a significant effect.

Yeh et al. (1989) conducted a prospective study of the effects of HBV and aflatoxins on risk for HCC in the southern Guangxi Autonomous Region, China. The entire cohort consisted of 7917 men who were between the ages of 25 and 64 at enrolment, lived in one of five communities and did not have HCC at the initiation of the study. The cohort was assembled between July 1982 and June 1983, at which time demographic information was collected and a blood sample was drawn; the sera were banked. In 1987, the sera of 2072 men were tested for HBsAg. The 2072 men included 149 men who had died as of 31 July 1986, of whom 76 had died of HCC, and a 25% random sample of the men (1923) still alive. Four live controls were matched to each death from HCC. Of the 76 cases, 69 had occurred in HBsAg-seropositive men (90.7%), whereas 68 of the 304 matched controls were HBsAg seropositive (22.4%) (RR, 39; 95% CI, 16–117). In a geographical analysis of these data, mortality from HCC was not correlated with the mean prevalence of HBsAg seropositivity by commune, but the range of HBsAg seropositivity was narrow (19.5–24.8%). A positive correlation was found between the estimated mean level of anatoxin B₁ consumed in food and the rate of HCC by commune, and the range of aflatoxin B₁ levels was broad (0.3–51.8 mg/person per year).

Ding et al. (1988) carried out a prospective investigation of residents of the Guangxi Autonomous Region, China. A total of 22 830 people [sex distribution unspecified] over the age of 20 years were stratified on the basis of HBsAg status, hepatic enlargement, alanine aminotransferase levels and residence in an area with a high or low rate of HCC. The average period of follow-up was 6.8 years. The highest RR for HCC occurred in HBsAg-seropositive people with evidence of both liver enlargement and abnormal liver function, regardless of place of residence. In the group from the high-rate area, 30.6% who were HBsAg seropositive and 2.7% who were HBsAg seronegative developed HCC. In the group from the low-rate area, 10% of the HBsAg-seropositive and 2.4% of the HBsAg-seronegative people developed HCC. In the total population, the RR for HCC, adjusted for age, was 8.2 [95% CI, 4.5–15] in HBsAg-seropositive as compared with HBsAg-seronegative subjects.

In a study in Shanghai, China, an association between HBsAg seropositivity and HCC was reported among men participating in a prospective study of diet and cancer (Ross et al., 1992). Between January 1986 and September 1989, 18 244 male subjects aged 45–64 were enrolled in the cohort; 22 cases of HCC had occurred by 1 March 1990. A nested case-control study was conducted with 140 controls matched by age (within one year), sample date (within one month) and residence, who had no history of liver cancer when the case was diagnosed. Five cases have been confirmed by biopsy. Conditional logistic regression, controlling for education, urinary aflatoxins, smoking and alcohol use, yielded an RR of 8.5 (95% CI, 2.8–26) for HBsAg seropositivity as compared with HBsAg seronegativity. The RR associated with detectable urinary aflatoxins was 3.8 (1.2–12), adjusted for educational level, HBsAg seropositivity, smoking and alcohol use; the RR associated with both HBsAg seropositivity and detectable urinary aflatoxins was 60 (95% CI, 6.4–562).

In the Japan-Hawaii Cancer Study of 7498 men of Japanese ancestry born between 1900 and 1919 and living in Hawaii, sera were collected in 1967–70 and stored. From 1967 through 1980, 18 histologically confirmed incident cases of HCC were identified; serum was available for 16. In a case-control analysis (Nomura et al., 1982), each of the 16 cases was matched to three controls from the study cohort by age and serum collection date. Ten of the cases were seropositive for HBsAg as compared with none of the controls (p < 0.0001). Another indicator of persistent infection, anti-HBc without anti-HBs, was detected in 7 of the 16 patients and in two of the 48 controls.

Iijima et al. (1984) and Sakuma et al. (1988) studied prospectively two overlapping cohorts of male Japanese employees of Japan National Railways. The first cohort was identified in 1973 and 1978. A total of 6918 men, 126 of whom were found to be HBsAg seropositive at the time of the study (1.8%) were followed to March 1985. Average follow-up was 8.5 years (range, 6.5–11.5 years) and was conducted using annual health examinations, self-reported disease (mandatory for any illness lasting more than six days), follow-up of all men who failed to report for an annual examination, and death certificates. Four HCCs developed in the HBsAg-seropositive group and six in the HBsAg-seronegative group, giving an RR of 30 [95% CI, 8.1–77]. The second cohort consisted of 25 547 men who were identified in 1977–79; the purpose of this study was to determine whether HBeAg status among HBsAg-seropositive men (n = 513) was associated with the risk for developing HCC. Average follow-up to 1985 was 7.3 years. RRs were calculated in relation to the risks for HBsAg-seronegative men. HCC was observed in 21 HBsAg-seronegative and 9 HBsAg-seropositive individuals. The RR was 50 [95% CI, 0.66–280] for the 30 HBsAg-seropositive, HBeAg-seropositive men (one HCC), 9.5 [1.1–34] for the 238 HBsAg-seropositive, anti-HBe-seropositive carriers (two HCCs) and 29 [11–63] for the 245 HBsAg-seropositive, HBeAg- and anti-HBe-seronegative carriers (six HCCs).

A population-based study of risk for HCC was conducted among Alaskan natives (Alward et al., 1985; Heyward et al., 1985; McMahon et al., 1990a,b). About two-thirds of the Alaskan native population was classified according to HBV infection status in a statewide screening programme begun in 1983 with the establishment of a HBV carrier registry. The authors identified 1400 HBV carriers (824 men, 576 women) and followed them up until 1 July 1987 (when 1292 were left) for a total of 7815 person-years. The study cohort comprised people of all three Alaskan native groups (85% Inuit, 7.5% Indian, 7.5% Aleut). Review of HBV sequelae from January 1975 to July 1987 showed that 20 cases of HCC had occurred, 19 of which were histologically confirmed. The annual incidence among men was 387/100 000, and that among women was 63/100 000. The incidence of HCC in HBsAg-seronegative people was estimated from the prevalence of HBsAg seronegativity in the Alaskan native population (97%) and the seven HCCs diagnosed in HBsAg-seronegative Alaskan natives between 1975 and 1987, to give an RR of 148 [95% CI, 59–305] for carriers versus non-carriers (McMahon et al., 1990a).

2.3.2. Prospective studies of blood donors (Table 5)

Oshima et al. (1984) followed a cohort of 8646 HBsAg-seropositive male Japanese blood donors in the Osaka Red Cross Blood Center, who were found to be HBsAg seropositive in 1972–75. Follow-up was conducted by examining data in the Osaka Cancer Registry and the death certificate files of Osaka Prefecture through the end of 1980. The mean length of follow-up was 6.2 years (range, 5–8.5 years). The expected number of cases was based on the age-specific incidence rates among the general population of Osaka. The study cohort developed 20 HCCs during the follow-up period, with 3.0 expected, giving a significant RR of 6.6 [95% CI, 4.0–10]. In a nested case-control analysis, alcohol drinking was significantly related to the risk of developing HCC, with RRs of 5.5 (95% CI, 1.2–26) for moderate drinkers and 8.0 (95% CI, 1.3–50) for heavy drinkers in comparison with non-drinkers.

Fukao (1985) identified 1000 HBsAg-seropositive and 10 000 HBsAg-seronegative blood donors from blood donation records in Miyagi Prefecture, Japan. All cohort members were men over the age of 30 years who had donated blood between 1971 and 1977. The HBsAg-seronegative group was matched 10:1 to the HBsAg-seropositive group by age, district of residence and month of blood donation. Cancer incidence was determined from the records of the Migayi Prefectural Cancer Registry for the years 1971–80. Three HCCs were detected in the HBsAg-seropositive group and one in the HBsAg-seronegative group, giving an RR of 30 [95% CI, 6.0–88] in carriers as compared with non-carriers.

Tokudome et al. (1987, 1988) estimated the risk for HCC among HBV carriers in Japan. A cohort of 3769 HBsAg-seropositive women was identified from among blood donors at the Fukuoka Red Cross Blood Center by examining Red Cross records of donations between the years 1977 and 1982. Mortality follow-up was completed until 1985; vital status was determined by checking against each donor’s home residence card. Death certificates were obtained for each donor known to be dead. The 220 women (5.8%) who were lost to follow-up were assumed in the analysis to be alive at the end of the study. The average period of follow-up was five years. Seventeen deaths occurred during the study period, four of which were due to HCC. In comparison with the age-adjusted mortality rates among all women in Fukuoka Prefecture in 1980, the RR for HCC was 5.6 [95% CI, 1.5–14] (Tokudome et al., 1987). In a cohort of 2595 HBsAg-seropositive male blood donors identified during 1977–79 and followed up to 1983 (9.2% lost to follow-up; average length of follow-up, six years), 15 HCCs developed as compared with 2.1 expected on the basis of age-specific mortality rates for Fukuoka Prefecture in 1980. The RR in HBsAg-seropositive men in relation to the general male population was 7.3 [95% CI, 4.1–12] (Tokudome et al, 1988).

Prince and Alcabes (1982) identified a cohort of HBsAg-seropositive men from four sources in the USA: the health departments of New Jersey, New York State and New York City and blood donors in the Greater New York Blood Program. The three health departments maintain lists of HBsAg-seropositive people, most of whom are blood donors who were found to be HBsAg seropositive during routine testing. Men identified as being HBsAg seropositive between 1971 and 1979 and who were residents of either New York City (n = 5353) or New York State (n = 1497) were included. Causes of death were determined by record matching with the New York State and New York City health departments. Four deaths from HCC occurred (three in New York City), as compared with 0.40 expected on the basis of mortality rates for the general populations of New York City and New York State [RR, 10; 95% CI, 2.7–25].

Dodd and Nath (1987) conducted a mortality study of people who had given blood during 1971–80 in the national Red Cross Donor Deferral Registry and identified 15 166 HBsAg-seropositive and 18 144 HBsAg-seronegative people. Vital status was determined by matching records with those of the Social Security Administration, and cause of death was determined from death certificates. The mean length of follow-up for the HBsAg-seropositive group was 3.7 years and that for the HBsAg-seronegative group, 3.3 years. Men comprised 70% of the HBsAg-seropositive group and 63% of the HBsAg-seronegative group. Six HCCs occurred in the HBsAg-seropositive group and none in the HBsAg-seronegative group. The authors give a standardized mortality ratio (SMR) for HCC of 27 [95% CI, 10–39] on the basis of mortality rates in the general population.

A prospective mortality study of HBsAg-seropositive people who donated blood in England and Wales between 1971 and 1981 (Hall et al., 1985) comprised 2880 men and 1054 women, of whom more than 92% could be followed for death to the end of 1983. Seventy-seven cohort members died during the period. Five deaths from HCC were reported among men in contrast to the 0.1 expected, giving a RR of 42 (95% CI, 13–98). No death from HCC occurred among the female cohort members (0.02 expected).

2.3.3. Prospective studies of populations with pre-existing disease

The incidence of HCC in HBsAg-seropositive and HBsAg-seronegative subjects has been studied among patients with pre-existing liver disease, as indicated variously by abnormal liver function, ‘chronic hepatitis’ and cirrhosis. The RRs for HCC associated with HBsAg seropositivity in these studies were generally small, e.g. [RR, 2.1 (95% CI, 0.3–17)] (Liaw et al., 1986). Their interpretation is complicated by the fact that causes of liver disease other than HBV may also be associated with an increased risk for HCC (Liaw et al., 1986; Dodd & Nath, 1987; Colombo et al., 1991; Johnson, 1991; Kato et al., 1992).

2.4. Case-control studies

2.4.1. Hepatocellular carcinoma

Many case-control studies have been published on the relationship between HCC and HBV infection. The prevalences of seropositivity are summarized in Table 6 (p. 90). In all studies, tests for HBV markers were performed on one occasion only, and ‘carriers’ were taken to be subjects in whom HBsAg was detected at that time. Unless otherwise noted in the Table, testing for HBV markers was done by radioimmunoassay. RRs, as measured by the odds ratio (OR) and 95% Cornfield confidence intervals (CIs), were calculated by the Working Group wherever the data reported in the original papers allowed it. In general, only ORs and p values are reported in the text, while ORs and CIs are reported in Table 6. Studies of clinical series (typically, patients with liver disease) in which cases of HCC were a subgroup but in which there was no specifically defined control group were not included.

Table 6.

Summary of results of case-control studies of hepatocellular carcinoma and presence versus absence of hepatitis B surface antigen (HBsAg).

(a) Africa

Prince et al. (1970) reported a comparison of patients with HCC and other chronic liver diseases with various control groups in relation to the prevalence of HBsAg seropositivity. They tested sera from subjects from Senegal, Uganda and the USA using two serological assays—agar-gel diffusion and high-voltage immunoelectroosmophoresis; the latter was 10 times more sensitive than the former. The prevalences of HBsAg seropositivity among the subjects examined were: in Senegal, 42% of 210 HCC patients, 9% of 201 adult males and 12.7% of 959 army personnel; in Uganda, 12% of 34 HCC patients, 23% of 26 with cirrhosis and 2% of 311 healthy subjects [OR for HCC, 6.8; 1.8–25]; in the USA, 4% of 55 HCC patients, 29% of 42 with chronic active hepatitis, 8% of 124 with cirrhosis and 0.1% of 55 956 blood donors. The authors remarked that, although the controls were not matched to the cases by age, sex or place of residence, hepatitis virus appeared to play an important role in at least a proportion of the cases of chronic liver disease.

Vogel et al. (1972) reported the results of a study conducted among in-patients at Mulago Hospital, Kampala, Uganda, or the Solid Tumour Centre in Uganda between October 1969 and May 1970 (Vogel et al., 1970) and January 1970 to May 1971. The 90 HCC cases (in 73 men and 17 women) had a significantly higher frequency of HBsAg in their sera (40%) than the 224 (149 non-neoplastic and 75 neoplastic) controls combined (151 men, 73 women) (3%; p < 0.001). Control patients with cirrhosis or hepatitis were excluded; histological confirmation was available for 71 of the HCC cases. There was no difference between cases and controls with respect to anti-HBs seropositivity (28–37%). A nonsignificant association was noted between HBsAg seropositivity in HCC patients and the presence of cirrhosis (44% in comparison with 27% without cirrhosis). Serum was tested by complement fixation, counter immunoelectrophoresis and passive haemagglutination.

Kew et al. (1974) investigated 75 male Bantu miners in South Africa with HCC confirmed at necropsy. The control group of 18 377 healthy miners was comparable with respect to age and tribal distribution. Testing of sera for HBsAg by complement fixation and counter Immunoelectrophoresis revealed a difference in the prevalence of persistent HBV infection: 40% versus 7% [p < 0.05]. No significant relationship was noted between HBsAg seropositivity and the presence of cirrhosis (46% in comparison with 31% without cirrhosis) among cases. [No information was provided as to how and when cases and controls were identified.]

A study conducted at Le Dantec Hospital, Dakar, Senegal, between October 1972 and July 1974 involved 165 cases of HCC (in 127 men and 38 women), 154 controls with other cancers (102 men, 52 women) and 328 non-cancer controls (226 men, 102 women) (Michon et al., 1975; Prince et al., 1975). A diagnosis of HCC was histologically confirmed for 80 cases. Controls were matched to cases on sex, age (within 10 years) and admission date (within 30 days) and were of similar ethnicity and religion; non-cancer controls were free of liver disease. A higher frequency of HBsAg seropositivity was reported among the cases than in each control group: 61.2% versus 11.8 and 11.3% in the two control groups, respectively [p < 0.05]. The prevalence of anti-HBs seropositivity (tested by passive haemagglutination) alone was lower among cases (18.2%) than controls (45.4 and 42.1%, respectively) (Michon et al., 1975). In 94 documented cases of HCC, the presence of cirrhosis was not related to HBsAg seropositivity but was related to anti-HBs seropositivity (6% versus 20%; p < 0.05) (Prince et al., 1975).

Larouzé et al. (1976) matched 28 cases of HCC (in 22 men and 6 women) from Le Dantec Hospital, Dakar, Senegal, to healthy individuals from the same urban neighbourhood or rural village, of the same sex, age and ethnic group. Blood was also collected from parents and siblings. Histological confirmation was obtained in 24 cases. Although no significant association was observed between the presence of HBsAg (by radioimmunoassay) and HCC (79% in cases, 57% in controls; p = 0.15 [OR, 2.8]), the prevalence of HBsAg among the controls was very high. Cases were markedly less likely to be seropositive for anti-HBs (by passive haemagglutination) (25% versus 64%; p = 0.006) and more likely to be seropositive for anti-HBc (by counter immunoelectrophoresis and immunodiffusion) (89% versus 64%; p = 0.05). [Neither the time frame nor the case selection method was described.]

In a study in Addis Ababa, Ethiopia, HBsAg seroprevalence was compared in 46 HCC cases (in 31 men and 15 women) and 90 healthy hospital employees without a history of liver disease, blood transfusion, leprosy, leukaemia or Down’s syndrome (Tsega et al., 1976; Tsega, 1977). The cases from whom serum was collected were a subset of 100 consecutive HCC patients admitted to St Paul’s or Haile Selassie I hospitals between June 1972 and November 1974. The diagnosis of HCC was confirmed by biopsy in 25 cases. HBsAg seropositivity was significantly associated with the occurrence of HCC: 50% among cases, 7% among controls [OR, 14, p < 0.001].

Tabor et al. (1977) analysed serum samples from 47 cases of HCC confirmed by biopsy in Uganda (previously studied by Vogel et al., 1972), 19 in Zambia and 27 in the USA the controls were 50 in-patients with melanoma or Kaposi’s sarcoma in Uganda, 40 healthy Zambian villagers (from the same geographic region as the cases) and three US blood donor groups (6726 total), respectively. Evidence of active HBV infection (HBsAg seropositivity with or without anti-HBs seropositivity or anti-HBc seropositivity with anti-HBs seronegativity) was significantly associated with HCC in each comparison: 72% of cases in Uganda, 68% in Zambia and 41% in the USA. Anti-HBs was tested by radioimmunoassay and passive haemagglutination and anti-HBc by complement fixation and counter immunoelectrophoresis.

In a study involving blood donor controls, 32 histologically confirmed cases of HCC in 19 men and 13 women in Mozambique were studied (Reys et al., 1977). Their 60% HBsAg seropositivity was significantly higher than the 3–15% [9% overall] observed in 231 male African blood donors from the Hôpital Central de Maputo by subgroup (p < 0.01). Anti-HBs was measured by radioimmunoprecipitation (12% compared with 33–49% had anti-HBs). [Neither the methods of subject selection nor the time period were further specified in either study.]

Van Den Heever et al. (1978) conducted a study at H.F. Verwoerd Hospital in Pretoria, South Africa, during 1973–76 of 92 histologically confirmed cases of HCC from the Pretoria area (in 75 men and 17 women) matched to 92 orthopaedic out-patients from the same area, of the same age and sex with no history of liver disease; all subjects were black. The association between seropositivity for HBsAg and HCC status was significant (34% of cases, 9% of controls; p < 0.01) [RR, 5.3].

In another case-control study in South Africa (Kew et al., 1979), a 62% seroprevalence of HBsAg was found among 289 blacks (280 men, nine women) with histologically confirmed HCC over a four-year period; the prevalence in the 213 healthy controls (gold miners matched by age, sex and ethnic group) was significantly lower, 11% (p < 0.001). The cases had been referred consecutively to the South African Primary Liver Cancer Unit from mine hospitals or admitted to two teaching hospitals; most were miners, and the majority were not from South Africa. A significant inverse association was observed for anti-HBs reactivity and HCC (17% in cases, 42% in controls; p < 0.001) [OR, 0.28]. In a subset of 74 cases and 104 controls tested for anti-HBc, a significant difference was found (89% versus 38%; p < 0.001). A detailed serological analysis of 131 HBsAg-seropositive (98 cases, 33 controls) and 222 HBsAg-seronegative (50 cases, 172 controls) male miners was performed (Kew et al., 1981), using the more sensitive radioimmunoassay to detect HBeAg, anti-HBe and anti-HBc. Significant positive correlations with HCC were observed for anti-HBc (p < 0.01) and anti-HBe (p < 0.05) among the HBsAg-seronegative subjects, and significant inverse relationships for anti-HBe (p < 0.02) among HBsAg-seropositive individuals and for anti-HBs (p < 0.05) among HBsAg-seronegative subjects. No relationship was observed between HBsAg seropositivity and the presence of cirrhosis among HCC cases (Kew et al., 1979). [The case identification period was not further specified. The later study (Kew et al., 1981) may have included additional subjects not in the earlier one.]

Seventy-six cases of HCC (in 60 men and 16 women) and 33 controls matched for age, sex and tribe were studied by Bowry and Shah (1980) in Nairobi, Kenya. The cases attended Kenyatta National Hospital between January 1976 and April 1979; histological or cytological confirmation was obtained for 56. Of the controls, 28 were relatives of hospital patients and five were hospital patients. HBsAg was detected (by passive haemagglutination) in 51% of cases and 6% of controls [p < 0.05]. A report published a year later by the same group (Bowry et al., 1981) probably involved a subset of 60 of these HCC cases [subject selection was not described]. Seropositivity only for anti-HBc was found in eight [13 %] of the 60 HCC cases, 15% of 20 matched hospital controls and 4% of 104 volunteer blood donors; the prevalence was higher among the 13 HCC patients with known cirrhosis (31%). [The blood donors were younger than the cases.]

Coursaget et al. (1981) conducted a study in Senegal of 134 cases of HCC (in 114 men and 20 women) diagnosed at Le Dantec Hospital and 100 blood donor controls from the National Blood Center of Dakar, in which anti-HBc was measured by counter immunoelectrophoresis. A significant association was found between HCC and active HBV infection [OR, 14] (67% versus 13%; p < 10⁻⁶). A significantly lower frequency of past infection (HBsAg seronegativity and anti-HBs seropositivity or anti-HBc seropositivity by radioimmunoassay) was noted for cases (33%) in comparison with controls (74%; p < 10⁻⁶). HBsAg was detected in 63% of the HCC patients and in 12% of the 100 blood donors [OR, 12], 14% of 833 rural country dwellers (72 men, 761 women) [OR, 11] and 25% of 560 leprosy patients from the Pavilion de Malte (410 men, 150 women) [OR, 50]. Two earlier reports of this study (Maupas et al., 1977; Coursaget et al., 1980) showed consistent findings. [Little detail could be found as to subject selection.]

Gombe (1984) found a significantly higher prevalence of HBsAg seropositivity among 65 cases of HCC (in 47 men and 18 women) (74%) than among 120 blood donors (115 men, five women) (9% [p < 0.05]) or 71 other cancer controls (13 men, 58 women) (3%; p < 0.05) in the Congo. The blood donors were tested in two centres by radioimmunoassay and passive haemagglutination, and the HCC cases and cancer controls from the Brazzaville General Hospital by passive haemagglutination. [The process for selecting subjects was not clear.]

Sebti (1984) in Rabat, Morocco, reported a significant but weaker association between HCC and HBsAg seropositivity (17% in 46 cases and 5% in 379 controls) [OR, 4.2]. The HCC patients were a subset of 63 cases hospitalized at Avicenne Hospital between 1976 and 1983; the controls were healthy subjects and people with non-hepatic disease. [The process for selecting subjects was not clear.]

Another study by Kew et al. (1986a) focused on southern African blacks living in an urban environment. Markers of HBV infection were assayed in 62 urban-born patients with histologically confirmed HCC (41 men, 21 women) and in pair-matched urban-born hospital controls, matched according to race, sex, age, tribe (when possible), hospital and ward. Subjects were identified from two large general hospitals, Baragwanath in Soweto and Hillbrow in Johannesburg. HBsAg was detected significantly more frequently among the cases (40%) than the controls (3%) (p < 0.001). No difference was observed with regard to past infection (HBsAg seronegativity and anti-HBs or anti-HBc seropositivity). [Information about the period of subject selection was not provided.]

Otu (1987) studied 200 consecutive, histologically confirmed cases of HCC (in 180 men and 20 women) at the University of Calabar Teaching Hospital, Nigeria, between January 1978 and December 1982. Two symptomless controls matched for sex and age (within five years) were selected per case from the general out-patient department. HBsAg was detected in 49% of the cases and 8% of the controls (p < 0.01). [Little detail was provided about selection of controls.]

Gashau and Mohammed (1991) compared the prevalence of HBV markers in 65 HCC patients (57 men, eight women) and 69 sex- and age-matched healthy controls in Nigeria. The cases of HCC were examined consecutively at the University of Maiduguri Teaching Hospital between 1986 and 1987; needle biopsy was used for diagnosis in 21. The controls were examined at the hospital during the same period; most were blood donors. The cases had a significantly higher rate of HBsAg seropositivity (65%), measured by ELISA and reverse passive haemagglutination, than the controls (35%) [OR, 3.2]. Seropositivity for anti-HBc, measured by ELISA, was about the same in those tested in the two groups; HBeAg seropositivity was higher and that of anti-HBe lower in the cases. [Few details were provided about the controls.]

Mohamed et al. (1992) examined the prevalence of current (HBsAg seropositivity) and past (seronegative for HBsAg and seropositive for anti-HBs or anti-HBc) infection among 101 black South Africans with HCC, matched for ethnic origin, sex, age (within two years) with patients from the wards of Baragwanath Hospital, Johannesburg (77 men, 24 women). Controls were excluded if they had a disease caused by alcohol abuse or were unable to answer questions. Histological confirmation was obtained for 85 cases of HCC. Among men, 35% of cases and 5% of controls were currently HBsAg seropositive; the OR, adjusted for alcohol and smoking, was 7.5 (95% CI, 2.2–25). Among women, HBsAg seropositivity was 25% for cases and 4% for controls, with an adjusted OR of 12 (95% CI, 1.0–154). [The time frame for subject selection was not given.]

At the Parirenyatwa Teaching Hospital in Zimbabwe, Tswana and Moyo (1992) studied 182 HCC cases (in 128 men and 54 women) and 100 non-liver disease patient controls (50 men, 50 women). Pregnant women, cigarette smokers and alcohol consumers were excluded from the study. The diagnosis of HCC was made clinically and confirmed by a-fetoprotein level. Controls were selected randomly and were comparable to the cases with respect to age and sex; subjects were 20–65 years old. A significant correlation (p < 0.0001) was reported between HBsAg seroprevalence and HCC [OR, 10] (56% in cases versus 11% in controls); anti-HBc was detected in 54% of cases and 4% of controls [OR, 29; p < 0.05]. Among HBsAg-seropositive subjects, the seroprevalence of HBeAg was 20% among cases and 9% among controls. HBV markers were determined by ELISA. [The time frame for the study was not given.]

Ryder et al. (1992) studied HCC and HBV infection in the Gambia between 1 December 1981 and 30 November 1982. In a community-based surveillance system, they identified 70 cases (in 61 men and nine women); 44 were confirmed histologically. Patients were interviewed within one month of diagnosis in their village, at which time the person living closest to the case, of the same sex and age (within five years) was identified as a control. All potential subjects agreed to participate, and the two groups were not significantly different with respect to length of residence, alcohol consumption, smoking habits or family size. After adjustment for age, the following associations were reported: HBsAg (64 cases and 67 controls tested), OR, 6.9 (p < 0.01); anti-HBs (63 cases and 68 controls tested), OR, 0.31 (not significant); HBeAg (63 cases and 68 controls tested), OR, undefined (p < 0.001); and anti-HBe (62 cases and 66 controls tested), OR, 2.3 (not significant). The prevalence of HBsAg seropositivity was 63% among cases and 21% among controls; that of HBeAg was 17% among cases and 0 among controls. In all instances, the relationships were strongest among people under 50 years of age.

Serological HBV markers were investigated by the immunoperoxidase procedure in 40 cases of HCC selected in 1985 among 223 cases collected at the Department of Pathology of the University Hospital and Medical School of Kinshasa, Zaire, and in 68 age- and sex-matched controls selected from among blood donors (Kashala et al., 1992). The proportion of seropositive individuals among cases of HCC was significantly higher for HBsAg (57.6% vs 7.35%) and for anti-HBeAg (27.5% vs 16.2%) but was significantly lower for anti-HBs (25% vs 63%), and no significant difference was observed for anti-HBc, HBe or HBV DNA.

(b) Americas

Yarrish et al. (1980) analysed sera collected from patients attending hospitals in Philadelphia, USA, between 1968 and 1977. The sera from 34 HCC cases (in 28 men and six women) were then matched to those of 38 patients (30 men, eight women) with colon cancer, 45 (36 men, nine women) with lung cancer and 56 blood donors (48 men, eight women) matched for age (within five years) and sex. All but one HCC case was histologically confirmed. Blood donor samples were collected prior to routine screening for HBsAg and were stored on average 39 months longer than the sera from HCC cases; the sera of the colon cancer controls were stored for four months less and those of the lung cancer controls for 17 months less than those of the HCC cases. Five HCC cases (15%) were seropositive for HBsAg (assayed by radioimmunoassay), which was significantly higher than in any control group (p < 0.05). Seropositivity for anti-HBs, as assayed by passive haemagglutination, was not significantly different between cases and controls. [The details of subject selection were not provided.]

In the Japan-Hawaii Cancer Study, described in detail on p. 69, Nomura et al. (1982) found a significant excess of HBsAg in HCC patients in Hawaii (63%, with none in controls).

Austin et al. (1986) performed a multicentre study of 67 HCC patients (45 men, 22 women), aged 18–84, from 12 US hospitals, for whom HBsAg status was known (49 assayed by radioimmunoassay, 18 from medical records). The 18% (all in men) HBsAg prevalence among these cases was significantly higher (p = 0.0002) than the 0 prevalence for the 63 controls, who had no liver disease or a condition related to tobacco use and were matched by sex, year of birth (within five years), race and current residence (59 assayed by radioimmunoassay, four from records). Of the people seronegative for HBsAg who were tested, 7/40 (18%) of cases and 5/58 (9%) of controls were seropositive for anti-HBs [not significant]. [Details were not given about the time and method of case selection.]

Yu et al. (1990) described a study of black and white residents of Los Angeles County, USA 18–74 years of age. Histologically confirmed incident cases of HCC were identified between January 1984 and August 1989; 392 cases were eligible, but only 51 (12 blacks, 39 white; 35 men, 16 women) were analysed, either because of death (290), refusal (29), inability to locate (9), incorrect diagnosis (7) or serum sample depletion (6). Controls were selected from among 404 community control subjects used in a case-control study of lymphoma from 1978 to 1982; 128 of 404 controls were randomly selected to be frequency matched by sex and age (10-year intervals) to the cases (1 black, 127 white; 81 men, 47 women). All interviews were performed in the subjects’ homes. The age- and sex-adjusted ORs for the various HBV markers, tested by radioimmunoassay with no markers as the reference level, were: HBsAg, infinity (p = 0.002); anti-HBc, 7.3 (p < 0.0005); anti-HBs, 5.2 (p < 0.0005). Neither adjustment for level of education nor a separate analysis of US-born or white subjects affected the results.

In a study conducted in Baltimore, USA, 99 consecutive histologically confirmed HCC patients at the Johns Hopkins Oncology Center were compared between January 1987 and May 1988 with 98 consecutive patients with other malignancies seen at the same centre between November 1987 and January 1988 (Di Bisceglie et al., 1991). The cases were from the eastern half of the USA and were referred for inclusion in therapeutic radiation trials. No significant difference was reported between the two groups with regard to age, sex or race. HBsAg and IgM anti-HBc were detected only in the cases (7% and 8%, respectively), at significantly higher prevalences than in the controls (p = 0.009 and 0.004). Cases and controls were similar with regard to the presence of the other HBV markers measured. Anti-HBc was determined by enzyme immunoassay. [The fact that the patients had advanced disease might have affected HBsAg levels.]

(c) Asia

In Taiwan, China, Tong et al. (1971) examined the prevalence of detectable antigen in 55 cases of HCC (in 52 men and three women) and 943 male personnel at the Tsoying Naval Base. The cases were from the Chinese Veterans’ Hospital, and 25 diagnoses were confirmed by needle biopsy or autopsy. No subject had Down’s syndrome, leprosy, leukaemia or a recent transfusion (within 12 months); the controls had no past or present liver disease. A significant difference in HBsAg seropositivity was found by a modified immunodiffusion technique: 80% among cases and 15% among controls (p < 0.001) [OR, 23]. The cases were older than the controls (mean, 48 versus 30 years). [No details were provided concerning the timing or selection of subjects.]

Simons et al. (1972) studied HBsAg seroprevalence among 156 Chinese HCC patients in Singapore; 114 (87 men, 27 women) had been reported previously (Simons et al., 1971). The control groups consisted of 1516 male blood donors, 260 women attending antenatal clinics and 207 patients investigated for suspected nasopharyngeal carcinoma at one of the hospitals; all controls were Chinese. HBsAg seropositivity was markedly higher among the HCC cases (35%) than in any of the control groups (8%, 2% and 6%, respectively). [The associations calculated by the Working Group were all statistically significant; the blood donors and women attending antenatal clinics are combined in Table 6 as ‘normal controls’. No details were found concerning selection of controls.]

Lee (1975) compared the prevalence of HBsAg seropositivity in 100 cases of HCC (in 85 men and 15 women) and 120 patient controls (98 men, 22 women) in Hong Kong with no history of liver disease or blood transfusion; the diagnosis of HCC was confirmed by biopsy in 81 cases. Testing by immunoelectroosmophoresis and complement fixation showed a significant difference (49% versus 9%; p < 0.001). [No data were provided on the timing or process of subject selection or on the gender distribution of the cases.]

Chainuvati et al. (1975) reported a higher frequency of HBsAg seroprevalence, measured by crossover immunoelectrophoresis, among 49 HCC patients with cirrhosis (16%) than among 87 hospitalized controls (74 men, 13 women) without liver disease (2%) in Thailand (p < 0.005). No HBsAg was found in eight HCC patients without cirrhosis. The cases in this study had been identified between August 1972 and April 1973 and were histologically confirmed.

Kubo et al. (1977) studied 124 cases of HCC (in 107 men and 17 women) seen at Kurume University and Chiba University Schools of Medicine, Japan; the diagnosis was histologically confirmed in 108 cases. Healthy employees of the Japan National Railways (290 men, nine women) seen at regular physical check-up were used as controls. The difference in sero-prevalence of HBsAg between cases and controls was significant (46% versus 4%; p < 0.01) [OR, 20], as were smaller differences in anti-HBc seropositivity (73% versus 30%; p < 0.01) [OR, 6.2] and the presence of any marker (81% versus 34%; p < 0.01) [OR, 8.1]. No difference between cases and controls was observed for anti-HBs, as tested by passive haemagglutination. Immune adherence haemagglutination was used to detect anti-HBc. [No details were given as to when subjects were diagnosed.]

In Taiwan, China, 127 HCC cases admitted to the National Taiwan University Hospital between May 1974 and December 1976 were compared with 729 healthy controls (Chen & Sung, 1978). The controls comprised 241 40–67-year-old adults from a cancer education programme and 488 18–22-year-old university students receiving a regular check-up in 1975. HCC was histologically confirmed in 63 cases. HBsAg (assayed by reverse passive haemagglutination) was found in 83% of cases and 15% of controls [OR, 28; p < 0.05], and anti-HBs (detected by passive haemagglutination) in 14% and 45%, respectively [OR, 0.21; p < 0.05]. An analysis of 68 HCC cases with and without cirrhosis revealed no association with HBsAg seropositivity (81% in each group). [The gender breakdown for the subjects was not given.]

Chien et al. (1981) conducted a study among Chinese HCC patients seen at the Taiwan Veterans General Hospital (Tong et al., 1971). The 102 cases (in 97 men and five women) were matched by age and sex to 100 healthy controls from the out-patient clinic; histological confirmation was obtained for 36 cases. A larger proportion of cases than of controls were seropositive for HBsAg (71% versus 12%) [OR, 18] or anti-HBc (98% versus 84%) [OR, 4.7], and a lower proportion for anti-HBs (27% versus 54%). HBsAg-seropositive patients had higher levels of HBeAg and lower levels of anti-HBe than controls. All differences except those for HBeAg and anti-HBe were significant [p < 0.05].

Lam et al. (1982) performed a study in Hong Kong which included 107 Chinese cases (in 95 men and 12 women) of HCC at the Queen Mary Hospital, who were matched by sex and age (within five years) to 107 control (94 men, 13 women) trauma patients in the orthopaedic ward of the same hospital; 106 cases were histologically confirmed. Between March 1977 and September 1980, 149 Chinese HCC patients were admitted to the hospital, 72% of whom were interviewed; controls were interviewed within one month of the index case. After adjustment for age and sex, the OR for HCC associated with the presence of HBsAg was 21 (95% CI, 10–46).

In the Republic of Korea, Sjøgren et al. (1984) reported an HBsAg seroprevalence of 82% among 110 histologically confirmed HCC cases (in 90 men and 20 women) and 14% among 63 controls with other cancers matched for sex and age (p < 0.001). Subjects were identified between 1973 and 1981 at St Mary’s Hospital in Seoul; the control patients had no evidence of liver disease, although five had metastatic liver cancer (Chung et al., 1983). IgM anti-HBc was present in 74 cases (67%) and one control (2%) (p < 0.001) [OR, 127]; the association between HCC and presence of IgM anti-HBc was also seen among the HBsAg-seropositive subjects (81% versus 11%; p < 0.005). [No details of subject selection methods were given.]

A report from Yamanashi Prefecture, Japan (Inaba et al., 1984), described a matched analysis of 62 cases (in 49 men and 13 women) of HCC from seven hospitals between April 1977 and August 1979. Patient controls with no hepatic disease were selected by sex, age (within five years) and hospital. Confirmation of the diagnosis in liver biopsies was obtained for 36 of the HCC cases. There was a significant, 10-fold increase in risk for liver cancer associated with HBsAg seropositivity, assayed by reverse passive haemagglutination (36% in cases versus 3% in controls) (p < 0.01), and a weaker association with anti-HBs (27% versus 18%) (p < 0.05) [crude OR, 1.7].

A matched analysis of cases of HCC in Guangxi Autonomous Region, China, revealed a high OR (17; 95% CI, 4.3–99) for the relationship between HBsAg seropositivity and HCC (Yeh et al., 1985a,b), based on 50 cases (in 47 men and three women) and 49 controls without liver disease matched by sex, age (within five years) and ward/clinic; 86% of the cases were seropositive for HBsAg versus 22.45% of the controls. Case identification began on 1 July 1982 and continued until 50 cases were obtained at the College Hospital; only four diagnoses were based on histological examination. In the HBsAg-seropositive subjects assayed, HBeAg reactivity was not significantly greater in HCC patients (31%) than in controls (18%); the seroprevalence of anti-HBe was lower among cases than controls (50% versus 64%) (Luo et al., 1988).

In Riyadh, Saudi Arabia, a significant difference (p < 0.001) was found between cases of HCC and local population controls for HBsAg seropositivity [60% versus 12%] (Arya et al., 1988). The 30 histologically confirmed cases (in 25 men and five women) were a subset of cases in 75 local HCC patients hospitalized at King Fahad Central Hospital from September 1984 to October 1985, from whom serum was available. The control group comprised 326 patients aged 20– > 40 treated for minor ailments in the area. Among the HBsAg-seropositive subjects, a significant inverse association was reported between HCC and anti-HBe seroprevalence [OR, 0.15] (24% in cases versus 67% of controls; p < 0.01), but no significant association was seen for HBeAg [OR, 2.7]. ELISA was used to assay all HBV markers. [Little detail was available about control selection.]

A matched analysis by Lu, C.Q. et al. (1988) of 30 HCC patients and 60 matched controls with other tumours or anorectal diseases in the same hospital in Tianjin, China, showed significant associations with reactivity to HBsAg (p < 0.001) (OR, 5) and anti-HBc (p < 0.05) (OR, 39). The prevalences among the cases were 57% and 83%, respectively, and those among controls were [17%] and [20%]. HBV markers were assayed by passive haemagglutination and ELISA.

Lingao (1989) conducted a study in the Philippines of 340 HCC cases (in 288 men and 52 women) individually matched by age and sex to asymptomatic population-based controls from five rural areas. About 90% of the diagnoses of HCC were confirmed histologically. The presence of HBsAg was evaluated by reverse passive haemagglutinin, radioimmunoassay and ELISA only in patients with HBV infection and was significantly higher for HCC patients than for the controls [75% versus 14%; p < 0.0001; OR, 19 (12–29)]. Among the HBsAg-reactive subjects, a significant association was found between HCC and anti-HBe seropositivity (73% versus 52%; p < 0.01) [RR, 2.6] but not with HBeAg; these markers were determined by gel diffusion followed by radioimmunoassay and ELISA. Among 99 cirrhosis patients studied, the HBsAg seroprevalence was 58%. In a preliminary study of 104 histopathologically confirmed HCC cases (in 88 men and 16 women), which probably represented a subset of the larger case group (Lingao et al., 1981), the control group consisted of 84 asymptomatic controls (42 men, 42 women). [No details of subject selection were provided.]

In the study of Yeh et al. (1990) in southern Guangxi Autonomous Region, China (see p. 68), 91% of 76 HCC cases were HBsAg seropositive compared with 22% of 304 controls (OR, 39; 95% CI, 16–117).

In a study by Tsukuma et al. (1990) in Japan, of 229 (192 men, 37 women) newly diagnosed HCC patients admitted to the Center for Adult Diseases in Osaka between November 1983 and June 1987, 221 (96.5%) were interviewed; 87 cases were histologically confirmed. One control per male case and two per female case (266 in all) were selected from among patients admitted for gastroenterology, people admitted for health check-ups and those admitted for gastroenterological endoscopy. People with liver disease, malignancy, smoking- and alcohol-related disease, and lack of HBsAg testing were excluded. All subjects were interviewed at admission or at the time of endoscopic examination. In the analysis, confounding by sex, age, history of blood transfusion, heavy drinking, cigarette index and family history of liver cancer was controlled by unconditional logistic regression; the OR for HBsAg seropositivity was 14 (95% CI, 5.7–36; p < 0.0001). HBsAg was determined by reverse passive haemagglutination; the results were abstracted from medical records.

Lin et al. (1991) studied cases of HCC and hospital controls at the Chang-Gung Memorial and Kaohsiung Medical College Hospitals in Taiwan, China, and interviewed them between 20 February 1985 and 20 December 1986. Preliminary results were reported previously (Lu, S.N. et al., 1988). The subjects were 243 hospitalized or out-patient cases of HCC (in 218 men and 25 women) and 302 orthopaedic and ophthalmic in-patient controls (260 men, 42 women). Two controls were matched to each case by age (within three years) and sex, but some subsequently refused to have blood drawn; the authors stated that no significant difference was found between cases and controls with regard to age or sex. Adjustment for age, sex and hepatitis markers yielded significantly increased risks in association with seropositivity for HBsAg (OR, 10; 77% in cases versus 19% in controls) and HBeAg (OR, 3.2; 18% versus 2%) and significantly decreased risks in association with seropositivity for anti-HBs (OR, 0.1; 28% versus 78%) and anti-HBc (OR, 0.1; 97% versus 100%). [No information was given about how many subjects refused to have blood drawn or how many cases were histologically confirmed.]

Chen et al. (1991) examined 200 male HCC patients from the same two hospitals in Taiwan who were recruited consecutively between September 1985 and July 1987. Healthy community controls were matched individually to cases by age (within three years), ethnic group and residence, using a roster from household registration offices. Seventeen female pairs had also been identified but were excluded owing to their small number. Only two cases and three controls were seronegative for markers of HBV infection; thus, the authors focused on detection of HBsAg and HBeAg. In comparison with those seronegative for both antigens, those only seropositive for HBsAg had a 17-fold higher risk for HCC (95% CI, 7.4–38) and those seropositive for both had an OR of 58 (95% CI, 27–124). [Refusal rates were not provided. The cases of HCC overlapped with those analysed by Lin et al. (1991); the number of histologically confirmed diagnoses was not stated.]

Srivatanakul et al. (1991) studied subjects from three hospitals in several areas of northeast Thailand as part of a larger study of liver cancer (Parkin et al., 1991). Sixty-five cases (in 47 men and 18 women) were compared with 65 controls matched by sex, age (within five years), residence and hospital. Controls were either in-patients or clinic patients with nonmalignant, nonhepatic diseases and diseases unrelated to tobacco or alcohol. All subjects were under 75 years, were recruited during 1987–88 and were interviewed in hospital; histological confirmation was obtained for 20 HCC cases. Conditional multivariate analysis, controlling for consumption of alcohol, shrimp paste, powdered peanuts and fresh vegetables and for betel-nut chewing gave a significant OR of 15 (95% CI, 2.3–103) for the relationship between HCC and seropositivity for HBsAg (p < 0.001); 42% of the cases were HBV carriers versus 8% of the controls. HBV markers were determined by ELISA

A study in Japan involved 204 patients with HCC (31 were not studied ‘for logistic reasons’) admitted to Kyushu University Hospital between December 1985 and June 1989 (Tanaka, K. et al., 1988, 1992). The cases were diagnosed within one year of identification, were aged 40–69 and were residents of Fukuoka or Saga Prefecture (168 men, 36 women). The diagnosis of HCC was confirmed by histology in 82 cases. The 410 controls selected were residents of Fukuoka City, had undergone a health examination between January 1986 and July 1989 at a nearby public health centre, did not have chronic liver disease and had had a blood specimen taken; they were frequency-matched by sex and age to the cases (291 men, 119 women). Cases and controls were similar with respect to education and occupation and were interviewed in the hospital wards or at the health centre. The OR for HBsAg seropositivity as a risk factor for HCC was 14 (95% CI, 5.9–33) after adjustment for sex, age, history of blood transfusion, family history of liver disease, alcohol consumption and smoking (19% prevalence in cases versus 2% in controls). Case sera were tested by radioimmunoassay or reverse passive haemagglutination and control sera by the latter method.

In Kaohsiung Medical College Hospital, Taiwan, China, Chuang et al. (1992) studied 128 histologically or cytologically confirmed cases of HCC (in 112 men and 16 women) and 384 community controls (336 men, 48 women) matched for age (within five years) and sex; no significant difference was found in age and sex distributions. HBsAg was detected in 77% of the cases, which was significantly higher than in controls (28%) (p < 0.001). Using these data, Leandro and Duca (1993) calculated an OR of 14 for HBsAg seropositivity (95% CI, 7.8–25).

A case-control study of HCC was carried out in Hanoi, Viet Nam, between 1989 and 1992 (Cordier et al., 1993). A total of 152 male cases were recruited from two hospitals and frequency-matched on sex, age, hospital and residence to 241 controls admitted to the abdominal surgical departments of the same hospitals. HBsAg status was investigated using a second-generation ELISA test. One hundred and thirty-eight (93%) cases and 44 (18%) controls were seropositive for HBsAg (OR, 62; 95% CI, 30–128). The effect of alcohol consumption was significant only among HBsAg-seronegative individuals.

(d) Europe

Trichopoulos et al. (1978) reported the findings of a study of 80 HCC patients (69 men, 11 women; 47 histologically confirmed cases) admitted to one of eight large hospitals in Athens, Greece, between April 1976 and June 1977. Two control patients were matched by sex and age (within five years) to each case, who had diagnoses exclusive of neoplasm and liver disease. After Mantel-Haenszel adjustment for age and sex, the OR for an association between HCC and active HBV infection (HBsAg seropositivity or anti-HBc seropositivity and anti-HBs seronegativity) was 10 (p < 0.001) by comparison with people with no evidence of active infection. No relationship with any antigen was observed in 40 metastatic liver cancer patients (OR, 1.2). In addition, the presence only of anti-HBs did not confer a greater risk for HCC than the absence of HBV markers (OR, 0.8; 95% CI, 0.3–2.1). Among the HCC cases, the prevalence of active HBV infection was significantly higher in the 45 with cirrhosis (67%) than in the 35 without cirrhosis (26%) (p < 0.001).

The prevalence of active HBV infection (HBsAg seropositivity or anti-HBc seropositivity and anti-HBs seronegativity) was determined in 34 cases of HCC (22 with cirrhosis) and 100 healthy general population controls of similar age and sex in Barcelona, Spain (Pedreira et al., 1980). Eighteen cases were verified by biopsy. A strong association was observed, with 52% of cases and 5% of controls having this HBV status [OR, 21; p < 0.05]. The prevalence was somewhat higher for HCC cases without cirrhotic liver (58%) than for those with (50%); 38% of 139 cirrhotic patients (47 alcoholics, 92 not) had active HBV infection. HBsAg was determined by reverse passive haemagglutination. [No details of subject selection methods were given.]

Goudeau et al. (1981) reported the prevalence of HBV markers in 46 histologically confirmed cases of HCC (in 39 men and seven women) and in 10 000 blood donors in Tours, France. All subjects were Caucasian. Significant differences were found for HBsAg seroprevalence (4% versus 0.5%; p < 0.01) and the seroprevalence of anti-HBc alone (15% versus 2%; p < 10⁻⁶). [No information was given on the timing and method of subject selection.]

De Franchis et al. (1982) studied 42 subjects with HCC (33 men, nine women) and two groups of controls matched for age (within five years) and sex, comprising 42 patients with chronic liver disease and 84 patients with diagnoses other than neoplasm or liver disease. Subjects were identified from 1974 at the University Hospital in Milan, Italy, and histological examination was used to diagnose HCC. The cases were significantly different from the other hospital controls with respect to all HBV markers analysed, the greatest differences (p < 0.0005) being for the presence of HBsAg (36% versus 2%) [OR, 23], of anti-HBc (95% versus 51%) [OR, 19], of HBeAg (19% versus 0%) and of active infection (seropositivity for HBsAg or a high titre of anti-HBc alone; 44% versus 2%) [OR, 32]. The only significant associations (p < 0.05) for HCC cases in the comparison with chronic liver disease controls were for HBsAg seropositivity (12% of controls) [OR, 4.1], anti-HBs seropositivity (43%) [OR, 0.14] and active infection (17%) [OR, 3.9]. The direction of the associations between HCC and markers of HBV infection was the same in comparison with both control groups. [No information was given about the timing of subject selection.]

In a multicentre case-control study in Italy (Pagliaro et al., 1982), a significant OR of 14 (1.4–∞) was reported for HBsAg seropositivity in relation to HCC (80% confirmed histologically) in a matched analysis of 50 case-control pairs (37 men, 13 women). Consecutive prevalent cases were collected from 23 hospitals and university medical departments from December 1974 through December 1976; controls were diagnosed with non-surgical diseases other than liver disease. Matching was by sex, age (within five years), hospital and admission date (within six months). HBsAg seropositivity was assayed by the same method for each case-control pair, by radioimmunoassay, counter electroimmunophoresis or reverse passive haemagglutination.

A study from 17 centres in Italy of patients newly diagnosed with HCC during 1979–80 (Pagliaro et al., 1983) comprised 286 HCC cases with cirrhosis (in 250 men and 36 women), who were compared with 3629 patients with cirrhosis (2340 men, 1289 women), and 64 HCC cases without cirrhosis (in 52 men and 12 women), who were compared with 1545 patients with chronic, non-hepatic disease (1038 men, 507 women). The latter control group represented a random sample of non-surgical disease. Among the cirrhotic subjects, cases were significantly different from controls for seropositivity for all HBV markers except HBeAg and anti-HBe: HBsAg, 30% versus 17% (p < 0.0005); anti-HBs, 26% versus 37% (p < 0.005); anti-HBc alone, 26% versus 16% (p < 0.005). Among those without liver cirrhosis, a significant difference was reported for seropositivity for HBsAg (33% versus 2%; p < 0.0005). HBsAg was detected by radioimmunoassay or ELISA anti-HBs and anti-HBc were assayed by radioimmunoassay in a subset of the sample that was similar to the whole group with respect to sex, age and HBsAg reactivity. [The data were incompletely reported.]

In London, United Kingdom, 27 consecutive HCC patients (20 men, seven women) who had undergone liver biopsy between 1979 and 1981 were compared with 112 hospital in-patient and staff controls (60 men, 52 women) (Bassendine et al., 1983); the subjects were Caucasian. The cases had significantly higher frequencies than the controls of HBsAg (15%; controls, 0.9%; p < 0.005), anti-HBc (48%; controls, 11%; p < 0.005) and anti-HBe (26%; controls, 7%; p < 0.001). [Details about the control selection process were not available.]

An association with HBsAg seropositivity was reported by Pirovino et al. (1983) in Switzerland: 31% in 65 HCC cases and 17% in 115 liver cirrhosis controls (p < 0.05) [RR, 2.3]. The cases were a subset of 75 histologically confirmed HCC cases diagnosed between 1975 and 1982 at the City Hospital Waid in Zurich on whom HBV testing was done. [Although all of the controls had cirrhosis, it is not known what proportion of cases did.]

Filippazzo et al. (1985), in Palermo, Italy, enrolled 120 consecutive in-patients with HCC (99 men, 21 women) between December 1980 and December 1983 and three controls from the same hospital matched for sex and age (within five years), who had either cirrhosis, solid tumour or chronic non-neoplastic disease. Biopsy or laparoscopy was used to verify the diagnosis in 62 cases of HCC. The difference in HBsAg prevalence between cases (17%) and controls was greater for the two non-cirrhotic control groups (2–3%) than for the cirrhotic controls (15%). [The prevalences reported in the paper did not correspond exactly to those calculable from the data given. No information was provided on how HBsAg status was determined.]

Colloredo Mels et al. (1986) conducted a study in Bergamo, Italy, which included 72 histologically confirmed HCC cases (in 60 men and 12 women), 57 of whom also had cirrhosis. Cases were identified between January 1980 and December 1984, as were two control groups from the same hospital: 199 without liver disease (159 men, 40 women) and 156 with liver cirrhosis (114 men, 42 women). The OR for the relationship between seropositivity for HBsAg and HCC was 11.5 among the non-cirrhotic patients (p < 0.001) and 1.0 among the cirrhotic patients: 47% of the HCC cases without cirrhosis, 7% of their controls and 28% of those with cirrhosis and 26% of their controls were seropositive for HBsAg. The authors calculated an overall OR of 4.6 on the basis of an HBsAg seroprevalence of 9.1% in the general population. Among the subset of HBsAg-seronegative subjects assayed, no significant difference was found for the prevalence of past HBV infection in either comparison. [The sample sizes were small, and the means of subject selection could not be determined.]

A case-control study in Greece (Trichopoulos et al., 1987) involved 194 cases of HCC (in 81 cirrhotic subjects out of 173 men and 21 women) and 456 in-patient controls (400 men, 56 women). Cases admitted to eight hospitals in Athens between April 1976 and October 1984 were interviewed in hospital; 113 were confirmed by histology. Controls with diagnoses other than neoplasm or liver disease were selected from the same hospitals (as well as the hospital for accidents and orthopaedic disorders) and were also interviewed in hospital. All subjects were Caucasians of Greek nationality and were comparable with respect to education and birthplace. Data on about one-third of these subjects were analysed previously (see above: Trichopoulos et al., 1978; 1980a), but all assays were repeated for this study. Multiple logistic regression was used to control for age, sex and anti-HCV status (Kaklamani et al., 1991). A significant, 11-fold association was observed between HBsAg seropositivity(46% in cases; 7% in controls) and HCC on the basis of 185 cases (in 166 men and 19 women; 108 confirmed by histology) and 432 controls (381 men, 51 women). This estimate was slightly lower than the earlier one, in which HCV infection was not controlled for (OR, 14; 95% CI, 8.0–24); the association with HBsAg seropositivity was stronger in comparison with subjects with no HBV marker (OR, 19; 95% CI, 10–38) (Trichopoulos et al., 1987). An additional analysis of these cases according to the presence of cirrhosis (Tzonou et al., 1991) revealed a stronger association with HBsAg seropositivity among 78 cases with cirrhosis (65% positive; OR, 33) than those 107 without (32% positive; OR, 6.7), after control for age, sex, anti-HCV seropositivity and smoking.

Vall Mayans et al. (1990) investigated 96 cases of HCC (in 67 men and 29 women; 83 cases with cirrhosis) admitted to the University Hospital in Barcelona, Spain, between October 1986 and March 1988; 74 cases were confirmed histologically or cytologically. Two controls were chosen for each case from the same hospital and matched for sex and age (within five years), within one month after identification of the case; controls with diagnoses related to the risk factors of interest (HBV infection, alcohol consumption, smoking and oral contraceptive use) were not eligible, leaving 199 controls for analysis. Cases and controls were Caucasian and had comparable histories of occupation and blood transfusion. All interviewing took place in hospital. A significant, nearly five-fold association was observed for HBsAg seropositivity after adjustment for age and sex (OR, 4.9, exact 95% CI, 1.3–22); the seroprevalence of HBsAg was low (9% in cases, 2% in controls). The OR for anti-HBc seropositivity (2.3; 95% CI, 1.3–3.9) was also significant, 50% of cases and 31% of controls being seropositive. The authors reported that adjustment for alcohol drinking did not change the association with HBV infection.

Leandro et al. (1990) studied 457 patients with liver cirrhosis in Italy: 140 (117 men, 23 women) had confirmed cases of HCC, and 317 without HCC (209 men, 108 women) were used as controls. HCC patients were diagnosed in 1980–88 at a hospital in either Bari or Bergamo; the controls were admitted to the same centres between 1 January 1984 and 3 December 1985. After control for age and sex by logistic regression, the association with HBsAg seropositivity was significant (p < 0.05), with an OR of 2.3 [similar to the crude estimated OR of 1.8 (95% CI, 1.1–3.1)]. [The ORs are related to the probability of developing HCC given the presence of pre-existing cirrhosis.]

A study was carried out in four cities in Italy (Stroffolini et al., 1992) to investigate HBsAg seropositivity in 65 incident cases of HCC with underlying cirrhosis (in 47 men and 18 women) admitted to four teaching hospitals during 1990. Patients with chronic nonhepatic disease, matched for age (within five years) and sex and admitted consecutively to the same hospitals in the same year were selected as controls (75 men, 23 women). Multiple logistic regression methods were used to control for age, sex, anti-HCV status and HBV markers. A significant OR of 12 (95% CI, 3.1–41) was found for the association between HBsAg reactivity and HCC; 25% of cases and 6% of controls were seropositive for the antigen. HBV markers were determined by ELISA. [Not all cases were confirmed histologically.]

(e) International collaborative studies

Not included in Table 6 are the results of two large collaborative studies. In one, patients with liver disease (including HCC) were compared with healthy subjects in Burma, China, Hong Kong, India, Indonesia, Japan, Kenya, Papua New Guinea, the Philippines and Thailand (Nishioka et al., 1975). HBsAg was determined by immune adherence haemagglutination and anti-HBs by passive haemagglutination. The seroprevalence of HBsAg ranged from 33 to 80% among HCC patients and from 3 to 18% among healthy controls; that for anti-HBs was 0–26% among HCC patients and 12–43% among controls. In each country, HBsAg seropositivity was always higher and anti-HBs lower among HCC cases than among controls.

In the other collaborative study, data on blacks in Senegal, Burundi and Mali were combined (Coursaget et al., 1984, 1985) to give a total of 453 HCC patients, 221 cirrhotic patients and 7051 adult controls. HBsAg seropositivity was 58–65% among HCC cases, 4–17% among controls and 63% among cirrhotic patients. HBeAg was detected in 25% of cases and 13–19% of controls, and anti-HBe was detected in 60% of cases and 75% of controls. An analysis of HBsAg-seropositive Senegalese subjects (Coursaget et al., 1986a) revealed an OR of 6.2 (95% CI, 4.1–9.6) for HCC; HBsAg/IgM complexes were detected in 14% of controls, 40% of cirrhotics and 50% of HCC cases. [No details were provided about subject selection.]

Some studies have addressed immunohistochemical identification of HBV antigens in liver tissues and detection of HBV DNA in serum or liver tissue. Their results are important in elucidating pathogenetic mechanisms but cannot provide directly interpretable estimates of effect parameters like the OR, since it is inherently difficult to assess the suitability of the comparison groups. These studies provide strong support for the hypothesis that HBV is an important factor in the etiology of HCC (Nayak et al., 1977; Tan et al., 1977; Turbitt et al., 1977; Omata et al., 1979; Bréchot et al., 1981a, 1985; Röckelein & Hecken-Emmel, 1988; Sjøgren et al., 1988; Guan et al., 1989).

(f) Factors that modify the risk for HCC associated with HBV

Male HBsAg carriers are more likely to develop HCC than female carriers (Anthony, 1984; Coursaget et al., 1987), and there is some evidence that establishment of the carrier state prenatally or early in life is associated with a higher OR for HCC than establishment of a similar state in adulthood (Larouzé et al., 1976; London, 1981; Hsieh et al., 1992).

Several factors other than HBV have been evaluated as causally associated with HCC. In particular, aflatoxins, drinking of alcoholic beverages and oral contraceptives have been determined to be human carcinogens (IARC, 1987, 1988, 1993). Whether these factors modify the effect of HBV in the causation of HCC is inconclusive. The modifying effect of concurrent infection with HCV on the action of HBV is discussed in the monograph on HCV (p. 165).

2.4.2. Cholangiocarcinoma

Two case-control studies found no association between HBsAg seropositivity and the occurrence of cholangiocarcinoma. Parkin et al. (1991) conducted a case-control study in north-east Thailand involving 103 cases and 103 hospital controls matched for sex, age and hospital; patients with tobacco- and alcohol-related disease and other liver disease were excluded. No association was found with HBsAg seropositivity (OR, 1.0; 95% CI, 0.4–2.7). In Taiwan, China, Chen and Sung (1978) reported that of seven cases one (14%) was HBsAg seropositive, giving a similar rate to that seen among 729 controls (15%).

2.4.3. Other cancers

The relationship between HBsAg seroprevalence (as measured by reverse passive haemagglutination) and the occurrence of oral and uterine cervical cancer was examined in one study (Vijayakumar et al., 1984). The subjects analysed were 350 oral cancer patients (232 men, 118 women), 150 cervical carcinoma patients and 100 healthy controls (50 men, 50 women); all were 40–60 years old and had no history of jaundice. Significant differences (p < 0.001) were found for all sex-specific comparisons between cases and controls: the seroprevalence of HBsAg was 11% in male and 12% in female oral cancer cases, 13% in cervical cancer cases and 4% in both male and female controls. [No data were provided on social class, nor was it clear whether the carrier state preceded treatment of the disease.]

3. Studies of Cancer in Experimental Animals

3.2. Transgenic mice

3.2.1. With no concomitant administration of chemical carcinogens

The expression of various HBV gene sequences in livers of transgenic mice has been examined in several studies (for reviews, see Chisari, 1991; Slagle et al., 1992). Only those transgenic models in which hepatic neoplasia was the end-point are included in this section (for a discussion of gene expression and mechanisms of tumour induction, see also section 4.3.2).

Male mice of three transgenic lineages producing the HBV large surface antigen were back-crossed with normal C57B1/6J females (see Table 7). Mice from the first generation (36 animals from lineage 50–4, 12 from lineage 45–2, eight from lineage 45–3 and four non-transgenic controls) were observed for two years for the development of liver injury (as indicated by increased levels of serum glutamic and pyruvic transaminases) and neoplasms (determined by abdominal palpation and serum levels of α-fetoprotein). Variable expression of large surface antigen (as measured by western blot of total liver protein) was correlated with lineage, being highest in lineage 50–4, medium in lineage 45–2 and lowest in lineage 45–3. Lineages that expressed the highest levels of large surface antigen and filamentous protein in hepatocytes had the highest level of liver injury, and liver tumours developed in animals that had hepatocellular injury. Tumours were first detectable in lineages 50–4 and 45–2 at 9–12 months after the onset of injury, and virtually all mice of the 50–4 lineage with pre-existing chronic liver-cell injury developed HCC by 18–21 months of age. One or two large tumours (1.0–2.0 cm) usually predominated, and numerous smaller tumours were scattered throughout the livers. Thirty-eight animals (25 males and 13 females) [lineage unspecified] with palpable abdominal masses were examined histologically at necropsy. Males developed more palpable liver tumours and displayed more HCCs (18/25) than did females (4/13). Adenomas predominated (5/7) in younger males (10–15 months) and carcinomas (13/18) in older males (16-21 months). All tumours occurred concurrently with hepatocellular injury, characterized by ground-glass hepatocytes, necrosis and inflammation. Neither metastases nor fibrosis or cirrhosis were observed (Chisari et al., 1989). [The Working Group noted that the terms ‘hepatoma’ and ‘hepatocellular carcinoma’ appear to have been used interchangeably.]

Table 7.

Transgenic mice that express hepatitis B surface antigen as the major product.

A group of 59 male and female transgenic mice (lineage 50-4; see Table 7) were examined for abdominal masses every four months for 24 months. Selected animals were killed at monthly intervals from 1 to 23 months, and nine control nontransgenic animals were killed at 3, 11, 18 and 24 months [exact number of animals killed at each time point not specified]. Liver sections were examined histologically for the presence of hepatocellular adenomas and carcinomas. Livers of nontransgenic mice were normal histologically at all time points. Starting at two months, mice progressively developed liver injury and inflammation, including hepatocellular necrosis, Kupffer-cell hyperplasia and mono-nuclear-cell infiltration, with concurrent preneoplastic lesions which appeared by seven months of age. Preneoplastic lesions consisted of hepatocellular dysplasia and foci of altered hepatocytes, which progressively developed into larger compressive nodular masses. Seventy-five adenomas, characterized by masses of neoplastic hepatocytes which compressed adjacent parenchyma, occurred in 18 mice from eight months and peaked in incidence around the 17th month of the study. HCC (29 in all) had occurred in all surviving transgenic mice by 20 months of age (Dunsford et al., 1990).

Transgenic mice containing the entire coding region of the HBx gene, including the X promoter, the principal RNA start sites, transcriptional enhancer and polyadenylation site, were created by microinjecting embryos from outbred CD₁ mice (see Table 7). Six transgenic mice, each with at least one intact, stably expressed copy of the X gene, were identified by Southern blot analysis, and three animals with a high level of expression were bred into permanent lines (lineages C11, H9 and E1) [strain and sex of crosses and total numbers of transgenic and nontransgenic offspring from all three lineages unspecified]. Livers were examined histologically at various times. At four months, preneoplastic lesions consisting of multifocal areas of altered hepatocytes were observed in progeny from all three lines of transgenic mice but not in nontransgenic littermates. Neoplastic nodules [sizes unspecified], which occurred by 8-10 months of age, compressed surrounding hepatocytes and accumulated high levels of HBx protein. The authors reported that fewer than 10% of control male CD₁ mice develop hepatic neoplasms during an average lifespan of 24 months [no data shown]. HCCs were observed in 19/21 males and 12/20 females of line Cll, in 8/10 males and 4/6 females of line H9 and in the E₁ line [details not given]. Most males of the Cll line died with HCC between 11 and 15 months of age, and most females between 17 and 21 months of age. There was no difference in the incidence of liver tumours in male and female mice. Liver damage, determined by concentration of serum alanine aminotransferase, was not observed, and the levels were consistently within normal range (Kim et al., 1991). [Detailed data on liver lesions in nontransgenic littermates were not provided.]

In another study of a transgenic lineage expressing the X gene driven by the α-1-anti-trypsin promoter, mice did not exhibit liver disease or tumour development. This lineage exhibited an early but transient expression of the HBx protein (Lee et al., 1990).

3.2.2. With concomitant administration of known chemical carcinogens

The transgenic mouse strain E36 was derived from founder (C57B1/6 × SJL/J)F₁ mice containing all of the HBV genome except for the core gene, allowing expression of HBsAg under control of the HBV promoter and enhancer sequences (see Table 7). Two hundred and four transgenic and nontransgenic mice (F₁ hybrids resulting from crosses of males of the transgenic strain E36 with C3H/He females) were allocated to three treatment groups. Animals of group 1 (23 HBV-seropositive males, 21 HBV-seropositive females, 19 HBV-seronegative males and 22 HBV-seronegative females still alive at 30 weeks) were treated at seven days of age by oral gavage with a single dose of 10 mg/kg bw NDEA in 0.9% saline solution. Animals of group 2 (12 HBV-seropositive and 22 HBV-seronegative males still alive at 30 weeks) were treated with a single dose of 150 mg/kg bw para-dimethyl-aminoazobenzene (DAB) in corn oil by gavage at seven days of age. Group 3 consisted of 52 untreated controls (15 HBV-seropositive males, 11 HBV-seropositive females, 14 HBV-seronegative males and 12 HBV-seronegative females still alive at 30 weeks). Survivors at 30 weeks were 92% of those treated with NDEA and 98% of those given DAB. Animals were killed at 30 weeks of age and examined both grossly and microscopically for the presence of liver tumours. No tumour was observed in the 52 control animals. Liver nodules > 220 µm in diameter were counted, and those 5 mm in diameter were classified as either adenomas or carcinomas by histological criteria. In NDEA-treated male groups, the total number of nodules per cubic centimetre of liver was about the same for HBV-seropositive and HBV-seronegative animals, but larger nodules (> 330 µm diameter) occurred at about twice the frequency in HBV-seropositive mice as compared with HBV-seronegative mice (p < 0.05, Wilcoxon test). The frequency of nodules in the DAB-treated group was much lower than that in NDEA-treated animals. The frequency of nodules per cubic centimetre of liver was 1.5-2 times higher in transgenic than in nontransgenic animals, but the increase was significant only for nodules < 110 µm. The incidence of hepatocellular adenomas and carcinomas was higher in HBV-seropositive (18/56) than in HBV-seronegative (14/63) animals treated with either NDEA or DAB; this difference was not significant (Dragani et al., 1990).

Six groups of 10 female transgenic mice that produce the HBV large surface antigen (lineage 50-4; see Table 7) and of 10 nontransgenic littermates were treated as follows. Group 1 served as untreated controls; group 2 received five monthly intraperitoneal injections of 0.25 μg/g bw aflatoxin B₁ as a suspension in tricaprylin beginning at three or four months of age; group 3 received a single intraperitoneal injection of 0.25 μg/g bw aflatoxin B₁ suspended in tricaprylin at three or four months of age; group 4 received three weekly intraperitoneal injections of 2.0 μg/g bw aflatoxin B₁ suspended in tricaprylin at four months of age; group 5 received a single intraperitoneal injection of 50 μg/g bw NDEA dissolved in sterile saline at four or five months of age; and group 6 received 0.1% phenobarbital in powdered diet beginning at six months of age for one year. The study was terminated when the animals were 15 months of age. Survival rates were approximately 90%, except for group 6 in which survival was about 50%. Liver nodules and tumour masses were observed grossly post mortem and by histological examination. Nodules were classified by size into three categories: 0.1–1.9 mm, 2–4.9 mm and > 4.9 mm in diameter. Adenomas and HCCs were distinguished histologically. No gross or histological lesions were seen in the livers of nontransgenic control mice, whereas the livers of control transgenic mice contained multiple nodules of different sizes. Livers of transgenic mice treated with aflatoxin B₁ had 15–23 nodules (0.1–1.9 mm in diameter) per liver, as compared with 0.1–0.2 nodules of the same size per liver in nontransgenic aflatoxin B₁-treated mice and five nodules per liver in transgenic control mice not treated with aflatoxin B₁. Similar results were obtained for the incidence of larger nodules. Aflatoxin B₁-treated transgenic mice had 6.2–8.8 nodules (2.0–4.9 mm in diameter) per liver, whereas untreated transgenic mice had an average of 3.7 nodules per liver and aflatoxin B₁-treated nontransgenic mice had 0–0.1. Adenomas and HCCs were seen only in transgenic mice treated with aflatoxin B₁ or NDEA. In the three aflatoxin-treated groups (2, 3 and 4), a total of 20 adenomas and two HCCs were observed in 26 transgenic mice and none in 27 nontransgenic mice. In the NDEA-treated group (5), nine adenomas and two HCCs were seen in eight transgenic mice and none in nine nontransgenic mice examined. Livers of transgenic mice fed phenobarbital showed increased nodularity but no adenoma or HCC; however, survival was poor (Sell et al., 1991).

3.4. Ground squirrels, ducks and other species

3.4.1. Beechey ground squirrels

GSHV infecting Beechey ground squirrels was discovered in 1979 in northern California, USA, as a result of a search for a virus similar to HBV in animals related to woodchucks (Marion et al., 1980). The biology, genetic structure, gene products and viral replication of GSHV have been reviewed recently (Marion, 1991). The virus was originally detected in sera taken from apparently healthy animals. To date, the only known location of this virus is on the San Francisco Peninsula, although the virus that putatively infects

Richardson ground squirrels (Spermophilus richardsonii) (see section 3.4.2) (and viruses that possibly infect other ground squirrel species) may be a variant of the Beechey squirrel virus.

In an experiment described in a series of reports (Marion et al., 1983, 1986, 1987), Beechey ground squirrels, estimated to be one to two years of age, were trapped live at various locations on the San Francisco Peninsula between 1980 and 1984. Animals were held individually in quarantine for one month, during which time their serum was tested for (i) surface antigen (GSHsAg), by a commercial solid-phase radioimmunoassay for cross-reacting HBsAg; (ii) anti-GSHs, by a virus-specific solid-phase radioimmunoassay; and (iii) virion-associated DNA polymerase activity, as a measure of virus load (Marion et al., 1983). Animals with serum GSHsAg and DNA polymerase activity were housed in a room separate from GSHsAg-seronegative animals (Marion et al., 1986). Marion et al. (1987) reported that 24/103 ground squirrels examined at necropsy had tumours at various sites; all tumour-bearing squirrels were 4.5–8 years of age and had been in captivity for a minimum of 2.4 years. Among animals under 4.5 years, no tumour of any kind was observed in 19 persistent carriers of GSHV, 22 seropositive for anti-GSHs or 19 with no serological marker of GSHV. Of the tumours observed in older squirrels, 11 were found in 17 GSHV carriers, eight in 11 squirrels seropositive for anti-GSHs and five in 15 GSHV marker-free squirrels. The predominant type of tumour observed in squirrels over 4.5 years of age was HCC, which was detected in 10/17 persistent GSHV carriers and in 3/11 squirrels seropositive for anti-GSHs, but in none of 15 GSHV marker-free squirrels in the same age range, resulting in a highly significant association between HCC and the GSHV carrier state (p = 0.0005, Fisher’s exact test) and a weaker association with seropositivity for anti-GSHs. Development of HCC in carrier squirrels may be related either to age or to the length of the carrier state, as all animals appeared to have become carriers before 1.5 years of age. HCC was seen at necropsy in six of nine carrier squirrels (67%) over six years of age but in only three of nine carriers aged four to six years and none of 17 carrier squirrels less than four years of age. All HCCs except one were of the same histological type: a trabecular, highly differentiated liver carcinoma; the only non-trabecular HCC was seen in one of the three squirrels seropositive for anti-GSHs and was of the medullary type and less differentiated. The diameters of the major tumours were generally larger in squirrels that were older when the HCC was detected. Single nodules of HCC were commoner in the younger squirrels, while older squirrels usually had more than one nodule. Four of the five oldest squirrels with HCC also had metastases to or adhesions of the tumour in the spleen. While viral DNA was integrated into the host DNA of some of the HCCs examined, the majority of those from squirrels with GSHV markers did not have detectable integrated viral DNA. Chronic active hepatitis and cirrhosis were not seen (Marion et al., 1986, 1987).

In a further assessment of the development of HCC in the squirrel colonies after nine years of observation (Marion & Cullen, 1992), 18 cases (45% of all neoplasms) were observed in the study population of 24 GSHV-infected, 20 anti-GSHs-seropositive and 26 GSHV marker-free ground squirrels over four years of age. Eleven of the liver tumours were seen in carrier animals, five in anti-GSHs-seropositive squirrels and two in GSHV marker-free animals. The association of HCC with the GSHV carrier state was significant (p = 0.0016). As in WHV-infected woodchucks, the incidence of HCC in animals that had recovered from infection was relatively high (20%). Anti-GSHs-seropositive squirrels that developed tumours experienced only a brief period of viraemia. No sex difference was noted. The average age of carrier squirrels at the time of detection of HCC was 6.5 years.

3.4.2. Richardson ground squirrels

HCC has been observed in Richardson ground squirrels from the southern half of the Canadian province of Alberta. The hepadnavirus thought to be associated with these tumours has not been characterized genetically or biologically, nor has it been transmitted experimentally to other animals. In a study by Minuk et al. (1986), animals were trapped and kept in captivity for less than one month. Two of 25 adult squirrels but none of 15 juveniles had HCC at necropsy (Table 8). Anti-GSHs was found in 7 of the 25 adult animals, and the serum of one animal reacted positively when tested with a commercial radioimmunoassay for HBsAg known to detect GSHsAg. Serum was not tested for the presence of virions. Anti-GSHs seropositivity was assayed with a commercial radioimmunoassay for anti-HBs. Of the animals in which HCC was found, one had GSHsAg reactivity in the serum, while the other was seropositive for anti-GSHs. No viral DNA was detectable in the HCC of the seropositive animal or in the DNA of adjacent liver tissue. [The assay to detect anti-GSHs was unspecific and insensitive.]

Table 8.

Studies of hepatocellular carcinoma (HCC) in Richardson ground squirrels trapped or born in captivity in Alberta, Canada, according to age at necropsy

In a study by Tennant et al. (1991), several groups of Richardson ground squirrels were examined for the presence of masses in the liver at necropsy. The majority, collected at Picture Butte, Alberta, Canada, and not maintained in captivity, were not tested for hepadnavirus markers or examined histologically. None of 618 squirrels ranging in age from three to four months to three years or more had evidence of liver cancer (Table 8). Squirrels held in captivity for 14 months or longer for various experiments were also examined for HCC at necropsy; nodules or histological evidence of HCC were detected in some animals (Table 8). HCCs were found in squirrels trapped at Picture Butte only after the animals had been maintained in captivity. Hepadnavirus markers were assayed in the sera of the five squirrels trapped near Edmonton and the seven from Picture Butte. None cross-reacted with HBsAg, but most had evidence of anti-GSHs. Viral DNA was detected in two of four HCCs; no anti-GSHc was detectable in any sample using an assay which readily detects this antibody. Non-neoplastic lesions in the livers of animals kept for three years in captivity included mild to moderate portal inflammation, with somewhat more severe inflammation adjacent to tumours. In the livers of six of seven animals with moderate portal inflammation, focal hepatocellular necrosis and inflammation were seen. [Limited data are available to support hepadnavirus infection per se.]

3.4.3. Ducks

Observations of liver tumours in domestic ducks (Anas domesticus) were first described in China by Wang et al. (1980), which led to the discovery of duck hepatitis B virus (DHBV) (Mason et al., 1980). The biology, genetic structure, gene products and viral replication of DHBV have been reviewed (Schödel et al., 1991).

Studies of the oncogenic potential of DHBV are of three types: (i) assessment of liver tumours and markers of DHBV in ducks collected on farms or free-ranging in communities; (ii) prospective studies of the development of HCC in ducks of known DHBV status and history; and (iii) experimental studies of the joint effects of DHBV infection and aflatoxin B₁ exposure in the development of HCC in ducks.

(a) Liver tumours and markers of duck hepatitis B virus in ducks collected on farms and in free-ranging flocks

After the initial discovery of DHBV in ducks with hepatitis and liver tumours in the Chinese Province of Qidong, several studies were carried out to determine whether the presence of HCC and hepatitis in domestic ducks was linked to current or past replication of DHBV in the same animals (see Table 9).

Table 9.

Presence of hepatocellular carcinoma (HCC) and duck hepatitis B virus (DHBV) infection in populations of ducks on farms and free-ranging.

DHBV was found in 70/195 ducks from three of five locations in China but in none of 17 ducks from Chiba, Japan (Table 9); HCC was found only in four ducks from Qidong, and evidence of present or past DHBV infection was seen in three of them. Moderate to severe hepatitis was observed in both DHBV-seropositive and -seronegative ducks from Qidong, where there are known to be relatively high levels of aflatoxin B₁ a known cause of liver disease in ducks (IARC, 1993). Moderate hepatitis consisted of mild portal inflammation, with rare necrosis of hepatocytes. Severe hepatitis was associated with dense chronic inflammation of portal tracts, which extended into adjacent parenchyma and was accompanied by focal necrosis of hepatocytes. Severe hepatitis was sometimes accompanied by septal fibrosis, focal areas of parenchymal collapse and regenerative nodules; cirrhosis was seen in one duck with HCC (Marion et al., 1984). Three of the four HCCs were observed in Chinese ducks and none in white Pekin ducks. Overall, while there was concomitant presence of DHBV and HCC in some ducks from Qidong, the two have not been firmly linked, nor has the simultaneous presence of DHBV replication and liver inflammation been associated in these ducks.

(b) Prospective studies of hepatocellular carcinoma in ducks of known duck hepatitis B viral status

Ducks infected either congenitally or by injection with DHBV as hatchlings were monitored for development of HCC in four studies (Table 10). HCC was seen in only 1/37 experimentally infected ducks aged 0.3–1.8 years and in none of eight congenitally infected and none of 26 uninfected ducks of similar ages. The single HCC observed was in a white Pekin duck similar to those used in all of the studies (Cullen et al., 1991).

Table 10.

Presence of hepatocellular carcinoma (HCC) in ducks of known duck hepatitis B virus (DHBV) status.

Inflammation of the liver was much less severe in two breeds of domestic ducks from California inoculated with known amounts of DHBV than in the free-range Chinese ducks (above). The majority of domestic birds (25/25 nonviraemic and 17/28 viraemic) showed only insignificant or mild inflammation; seven viraemic birds exhibited moderate inflammation (Marion et al., 1984).

[The relative absence of both inflammation and cancer in experimentally infected ducks is noteworthy. Further, the prospective experimental studies have been of limited duration relative to the lifespan of ducks.]

(c) Synergy between infection with duck hepatitis B virus and treatment with aflatoxin B₁ in inducing hepatocellular carcinoma in ducks

Ducks have been reported to be sensitive to the effects of aflatoxin B₁ and to the development of HCC as a consequence of treatment with this mycotoxin (IARC, 1993). Studies of the combined effect of DHBV infection and exposure to aflatoxin B₁ in the development of liver cancer all involved white Pekin ducks and a variety of dosing schedules (Table 11). Aflatoxin B₁ was highly toxic, increasing the mortality rate in treated over that in untreated ducks. The rate of appearance of HCC was not significantly different in DHBV-infected aflatoxin B₁-treated ducks from that in DHBV marker-free aflatoxin B₁ treated ducks, suggesting a lack of synergy between current viral infection and exposure to aflatoxin B₁. Integrated viral DNA was found in three of the eight DHBV-associated HCCs examined.

Table 11.

Development of hepatocellular carcinoma (HCC) in ducks with and without duck hepatitis B virus (DHBV) infection treated with aflatoxin B₁ (AFB₁).

[The existence of species-specific hepadnaviruses closely related to HBV, which produce HCC in two species (woodchuck and Beechey ground squirrels), strengthens the plausibility of the conclusion that HBV is carcinogenic]

3.4.4. Other species

The DNA of a hepadnavirus that infects herons (HHBV) has been cloned and characterized genetically, but it has not been characterized biologically nor has infection with the virus been associated with the development of liver cancer (Sprengel et al., 1988).

Evidence that a hepadnavirus infects tree squirrels (Sciurus carolinensis pennsylvanicus) was reported from studies of their livers, but viraemia has never been described in tree squirrels, and the virus remains uncharacterized both genetically and biologically (Feitelson et al., 1986a,b). Liver cancer has not been observed in tree squirrels with evidence of hepadnaviral infection.

4. Other Relevant Data

4.2. Molecular biology

The molecular biology of HBV in relation to HCC has been reviewed (Rogler, 1991; Buendia, 1992; Feitelson, 1992; Slagle et al., 1992). The virus was first associated with HCC in epidemiological studies. During the last 10 years, the role of HBV in liver-cell transformation has been investigated by different approaches, either directly in human HCC or in experimental models, including human HCC cells in culture, transgenic mice carrying part or all of the viral genome (see section 3.2) and naturally occurring animal models of hepadnavirus infection (see sections 3.3 and 3.4). The studies have focused on three main subjects:

1. integration of HBV: the integrated state of HBV DNA in human HCCs, integration of other hepadnaviruses, structure of viral inserts, cellular target sites for viral integration and insertional mutagenesis;
2. expression of HBV genes in human HCCs and their role in the tumorigenic process (mainly surface proteins and X transactivator); and
3. genetic alterations in human HCCs, activation of cellular oncogenes and inactivation of tumour suppressor genes.

4.2.1. Integration of HBV DNA

Viral integration has been detected only in hepatocytes, despite the presence of viral DNA in extrahepatic tissues (Bréchot, 1987).

(a) Integrated state of HBV DNA in chronic hepatitis and hepatocellular carcinomas from hepatitis B surface antigen-seropositive patients

Initial studies using Southern blot analysis showed the presence of integrated HBV sequences in cellular DNA of established HCC cell lines and in human HCCs from HBsAg-seropositive patients (Bréchot et al., 1980; Chakraborty et al., 1980; Edman et al., 1980).

It is at present uncertain whether or not integration occurs in the early stages of natural acute hepatitis. In contrast, multiple integrations have been observed in tissues from patients with chronic hepatitis (Bréchot et al., 1981b; Shafritz et al., 1981; Boender et al., 1985; Tanaka, Y. et al., 1988), indicating that viral integration takes place prior to tumour development. Analysis of 83 cirrhotic nodules from the livers of 11 HBV carriers with cirrhosis revealed discrete bands at a higher molecular weight region in 26 of 83 nodules (31.3%), indicating clonal outgrowth of altered hepatocytes with viral integration (Yasui et al., 1992). Single-site HBV insertions are common in childhood HCC but are less common later in life (Chang et al., 1991), suggesting that multiple-site integration occurring during the course of long-term HBV infections might accumulate within single cells, as indicated by sequence divergence among HBV inserts in the same tumour (Imai et al., 1987).

Integrated HBV DNA has been identified in HCC specimens from chronic HBV carriers in numerous other studies (Bréchot et al., 1981a; Shafritz et al., 1981; Hino et al., 1984, 1985; Horiike et al., 1989; Sakamoto et al., 1989), indicating that HBV DNA integration is present in more than 80% of HCCs developing in HBsAg-seropositive patients.

(b) Presence of HBV DNA in hepatocellular carcinomas from hepatitis B surface antigen-seronegative patients

The presence of serum anti-HBs and anti-HBc in HBsAg-seronegative HCC subjects has been reported (Kew et al., 1986b; Bréchot, 1987; Blum et al., 1990). In spite of the fact that these antibodies generally reflect past, resolved infection, HBV DNA sequences have nonetheless been detected in some tumours in this group (Bréchot et al., 1982, 1985; Hino et al., 1985; Bréchot, 1987; Tabor, 1989; Lai et al., 1990). Improvements in the sensitivity of assays for HBsAg and HBV DNA have made it possible to identify cases of chronic HBV infection with low viral replication (Bréchot et al., 1985; Liang et al., 1989; Bréchot et al., 1991; Kremsdorf et al., 1991; Liang et al., 1991b). The finding of HBV DNA in HCCs in HBsAg-seronegative patients (Pontisso et al., 1987; Paterlini et al., 1993) has been questioned because the estimated copy number of HBV DNA sequences per cell is only 0.001–0.1. The significance of these findings is unclear.

(c) Integrated hepadnavirus DNA in animal models

Integrated WHV sequences have been detected in chronically infected woodchuck liver and in a majority ( > 90%) of woodchuck HCCs in chronic carriers (Ogston et al., 1982; Rogler & Summers, 1984; Hsu et al., 1990), although Korba et al. (1989) reported a lower frequency of integration. Viral integration appears to be less frequent in tumours associated with GSHV and even less in those associated with DHBV (Yokosuka et al., 1985; Marion et al., 1986; Imazeki et al., 1988; Transy et al., 1992).

(d) Structure and expression of viral inserts

Studies of the organization of cloned HBV inserts in liver tissues and HCCs show that HBV sequences are fragmented and rearranged and that integration and recombination sites are dispersed over the viral genome. These data indicate that HBV integration does not occur through a unique mechanism, as is the case for other retroelements and retroviruses. Virtually all HBV inserts consist either of linear subgenomic fragments or of rearranged fragments in different orientations, in the absence of a complete HBV genome, showing that these integrated sequences cannot serve as a template for viral replication. Integrated HBV sequences may be rearranged both during the integration process and after formation of viral inserts (Mizusawa et al., 1985; Nagaya et al., 1987; Tokino et al., 1987). Integrated forms, made up of a continuous genome or subgenomic fragment, which are frequent in tissues from children with HCC and chronic hepatitis (Yaginuma et al., 1987), are believed to represent primary products of integration.

Highly preferred integration sites have been mapped in the HBV genome within the ‘cohesive end’ region that lies between two 11-base pair direct repeats (DR1 and DR2) which are highly conserved in hepadnaviruses (Koshy et al., 1983; Dejean et al., 1984; Nagaya et al., 1987). A narrow region encompassing DR1 has been shown to be particularly prone to recombination (Yaginuma et al., 1987; Hino et al., 1989; Buendia, 1992; Quade et al., 1992). This region coincides with a short terminal redundancy of the minus-strand DNA, which confers a triple-stranded structure to the circular viral genome (Shih et al., 1987). Integration sites are tightly clustered at both the 5’ and 3’ ends of minus-strand DNA, suggesting that replication intermediates and specially relaxed circular DNA might be preferential preintegration substrates (Nagaya et al., 1987; Shih et al., 1987). Invasion of cellular DNA by single-stranded HBV DNA using mainly free 3′ ends, might take place through a mechanism of illegitimate recombination, also suggested by frequent patch homology between HBV and cellular sequences at the recombination breakpoints (Matsubara & Tokino, 1990). Different minor changes in flanking cellular DNA have been associated with viral integration, including microdeletions and short duplications (Yaginuma et al., 1985; Dejean et al., 1986; Berger & Shaul, 1987; Nakamura et al., 1988; Hino et al., 1989). The hypothesis that topoisomerase I might promote illegitimate recombination of hepadnavirus DNA/n vivo has been proposed (Wang & Rogler, 1991). The mechanisms underlying HBV DNA integration have still not been fully identified. Analysis of a limited number of WHV and DHBV insertions suggests that a similar mechanism of integration occurs in these hepadnaviruses (Ogston et al., 1982; Rogler & Summers, 1984; Hsu et al., 1988; Imazeki et al., 1988; Fourel et al., 1990). No similar information is available for GSHV.

As a consequence of the viral integration process, sequences of the S and X genes and of the enhancer I element are present almost systematically in HBV inserts, whereas those of the C gene are less frequently represented (Buendia, 1992). It has been shown that the pre-S2/S promoter is transcriptionally active in its integrated form in human HCC (Freytag von Loringhoven et al., 1985; Caselmann et al., 1990) and that HBsAg might be produced from viral inserts (Zhou et al., 1987). Highly rearranged HBV inserts show virus-virus junctions scattered throughout the viral genome (Nagaya et al., 1987), and recombination breakpoints have been mapped in the S coding region of some of them (Buendia, 1992). Truncation of the S gene between residues 77 and 221 (Buendia, 1992) confers transcriptional activation activity on the mutated pre-S2/S products (Caselmann et al., 1990; Kekulé et al., 1990). Other studies have shown that a large percentage of virus-host junctions are located in the carboxy terminal of the viral X gene, predicting a fusion of the X open reading frame with flanking cellular sequences in a way that might preserve the functional capacity of the X transactivator. Transcripts have been demonstrated from integrated X sequences in tumours and chronically infected livers (Miyaki et al., 1986; Wollersheim et al., 1988; Takada & Koike, 1990; Hilger et al., 1991).

(e) Cellular targets for viral integration in human hepatocellular carcinomas

Studies of different viral insertions in many human HCCs have revealed that integration can take place at multiple sites on various chromosomes; insertion sites have been mapped on many different human chromosomes, with higher than average rates on chromosomes 11 and 17 (Tokino & Matsubara, 1991; Slagle et al., 1991; Quade et al., 1992). These studies did not demonstrate the presence of known dominant oncogenes or tumour suppressor genes in the immediate vicinity of any integration site. Alu-type repeats and minisatellite-like, satellite III and variable-number terminal repeat sequences have frequently been identified near HBV insertion sites (Shaul et al., 1986; Berger & Shaul, 1987; Nagaya et al., 1987; Buendia, 1992). A small cellular DNA compartment (H3), characterized by a base composition similar to that of HBV DNA and a high concentration of Alu repeats, has been designated as a major target for stable HBV integration (Zerial et al., 1986; Buendia, 1992).

In many tumours, large inverted duplications, deletions, amplifications and chromosomal translocations have been associated with HBV insertions, suggesting that HBV DNA integration may enhance chromosomal instability (Koch et al., 1984; Mizusawa et al., 1985; Rogler et al., 1985; Yaginuma et al., 1985; Hino et al., 1986; Tokino et al., 1987; Hatada et al., 1988). It has also been shown that HBV DNA promotes homologous recombination at a distance from the insertion site (Hino et al., 1991). Roles for most of these chromosomal abnormalities have not been assigned as yet, however, although amplification of hst-1 loci has been associated with HBV integration in the same chromosomal region (Hatada et al., 1988).

(f) Insertional mutagenesis by HBV DNA

Evidence for a direct cis-acting promoter insertion mechanism in HCC has been provided. In two cases, viral integration disrupted the structure of the gene (Dejean et al., 1986; de-The et al., 1987; Dejean & de-Thé, 1990; Wang et al., 1990) in early tumours that developed in non-cirrhotic livers from clonal proliferation of a cell containing a single, specific viral integration. In one case, the HBV insertion occurred in an exon of the retinoic acid receptor β gene (RARβ) and fused the amino terminal domain of the viral pre-S1 gene to the DNA and hormone binding domains of the gene (Dejean et al., 1986; de-Thé et al., 1987; Buendia, 1992). The predicted chimeric HBV/RARβ protein might have altered transcriptional capacity and thus participate in the tumorigenic process (Buendia, 1992). In the second case, HBV sequences were found to be integrated in an intron of the human cyclin A gene, resulting in production of spliced HBV/cyclin A fusion mRNAs initiated at the pre-S2/S promoter (Wang et al., 1990, 1992). In the deduced polypeptide, the amino terminal domain of cyclin A (a target for proteolytic degradation of cyclin A at the end of the M phase) was replaced by an amino acid sequence from the terminus of pre-S1. Cyclins are important in the control of cell division, and disruption of the cyclin A gene by viral insertion probably contributed to oncogenesis (Buendia, 1992).

In a third human HCC, integration of HBV DNA into a cellular gene related to the epidermal growth factor receptor (c-erbB) has been described (Zhang et al., 1992).

(g) Insertional activation of mycfamily genes in woodchuck hepatocellular carcinoma

The search for transcriptional activation of already known proto-oncogenes and for viral insertion sites in woodchuck HCCs has revealed that WHV acts as an insertional mutagen which activates myc family genes (c-myc and N-myc) in more than half of the tumours examined (Möröy et al., 1986; Hsu et al., 1988; Fourel et al., 1990). Analysis of the mutated c-myc alleles in two tumours showed integration of WHV sequences in the vicinity of the c-myc coding domain, either 5′ of the first exon or in the 3′ untranslated region (Hsu et al., 1988). Deregulated expression of the oncogene driven by its normal promoters resulted from deletion or displacement of c-myc regulatory regions known to exert a negative effect on c-myc expression and their replacement by viral sequences encompassing the enhancer I element (Buendia, 1992).

Insertional activation of N-myc genes was observed more frequently. In particular, the woodchuck N-myc2 gene (a functional processed pseudogene or ‘retroposon’) represents by far the most frequent target for WHV DNA integration. In about 40% of tumours, viral insertions were detected either upstream of the gene or in a short sequence of the 3′ untranslated region (Fourel et al., 1990; Wei et al., 1992). The N-myc2 gene is also present in ground squirrels, although insertional mutagenesis has not been demonstrated in this animal model (Transy et al., 1992). Furthermore, woodchucks infected with GSHV do not show insertional mutagenesis (Hansen et al., 1993). Therefore, a direct role of WHV DNA integration in myc gene activation might account for the higher incidence and more rapid onset of HCC in the woodchuck model. Finally, there is no evidence that HBV integrates into myc family genes in human HCC (Buendia, 1992).

4.2.2. Expression and potential oncogenic properties of HBV gene products

The HBV genome encodes seven proteins from four open reading frames. Experimental evidence has been presented for an oncogenic role of two of the viral proteins, the large surface (HBs) protein and the transcriptional transactivator X.

(a) Surface proteins

In natural HBV infection, the production of infectious virions and HBsAg particles depends on tight regulation of the relative levels of the three envelope glycoproteins. Neither liver lesions nor HCCs have been observed in any of the published transgenic lineages that carry and replicate complete HBV genomes or produce the middle and major surface (HBs) proteins from HBV-derived regulatory sequences (Babinet et al., 1985; Chisari et al., 1985; Burk et al., 1988; Farza et al., 1988; Araki et al., 1989). The appearance and rate of development of preneoplasic nodules and liver tumours following administration of carcinogens are, however, slightly increased in HBsAg-seropositive transgenic mice over that in seronegative littermates, suggesting that HBsAg expression might enhance the effects of hepatocarcinogens (Dragani et al., 1990).

In contrast, when the endogenous pre-S1 promoter is replaced by an exogenous promoter (the metallothionein or albumin promoter), the production of roughly equimolar ratios of large HBs protein with respect to middle and major HBs proteins leads to intracellular accumulation of nonsecretable filamentous envelope particles within the endoplasmic reticulum of transgenic mouse hepatocytes (Chisari et al., 1986, 1987). This leads to histological and ultrastructural features similar to those of ‘ground-glass’ hepatocytes, which have been described in chronic hepatitis B in humans. Cell death follows, accompanied by mild persistent hepatitis, which is followed by the development of regenerative nodules and eventually HCC by 12 months of age (Chisari et al., 1989). The preneoplastic nodules and tumours display a marked reduction in transgene expression, suggesting that hepatocytes that express low levels of the large HBs polypeptide would have a selective survival advantage. Chemical carcinogens are not required for tumour induction in this model, but exposure of adult transgenic mice to hepatocarcinogens produced more rapid and extensive development of preneoplastic lesions and HCCs under conditions that do not alter the liver morphology of nontransgenic controls (Sell et al., 1991). These data show that inappropriate expression of the large HBs protein can be directly cytotoxic to hepatocytes and may initiate a cascade of events that ultimately progress to malignant transformation (Buendia, 1992).

Studies of integrated HBV sequences in human HCC suggest a possible role for abnormal expression of rearranged viral S genes in the development of HCC. Deletion of the carboxy terminal region of the S gene generates a novel transcriptional trans-activation activity (Caselmann et al., 1990; Kekulé et al., 1990; Lauer et al., 1992). Both integrated HBV sequences from a human tumour and from a hepatoma-derived cell line and different constructs bearing similarly truncated pre-S2/S sequences can stimulate the SV40 promoter in transient transfection assays; transactivation occurs at the transcriptional level and is dependent on the SV40 enhancer. The c-myc P2 promoter is also activated in trans. These findings support the hypothesis that accidental 3′ truncation of integrated pre-S2/S genes could be a causative factor in HBV-associated oncogenesis (Buendia, 1992).

(b) HBx: a transcriptional transactivator

Evidence for expression of the HBV Xgene was obtained by Moriarty et al. (1985) and by Kay et al. (1985), who reported that the sera of HBV-related HCC patients recognize synthetic peptides made on X sequences. Expression of the X reading frame in prokaryotic and eukaryotic cells, using various vectors, allowed identification of a 17-kDa polypeptide that reacted with serum samples from a number of HBV-infected individuals (Elfassi et al., 1986; Meyers et al., 1986; Schek et al., 1991). Anti-HBx has been detected in a minor proportion of acutely infected patients about three to four weeks after the onset of clinical signs, and more frequently in chronic HBsAg carriers who have markers of active viral replication. Very few patients are seropositive for anti-HBx after seroconversion to anti-HBs or at the time an HCC is discovered (Levrero et al., 1991). Conflicting results have been obtained regarding the association between anti-HBx and other viral markers and with HCC. The problem may be related to the weak antigenicity of HBx protein or to its sequestration into cellular compartments that render it inaccessible to the host immune system. HBxAg has been detected in the livers of HBsAg carriers and has been correlated with current viral replication and chronic liver disease (Levrero et al., 1990; Haruna et al., 1991; Wang et al., 1991). The HBxprotein is located mainly in the cytoplasm of cells infected in vivo, at or near the plasma membrane and at the nuclear periphery (Vitvitski et al., 1988; Levrero et al., 1990; Wang et al., 1991). The HBx protein has been detected in the nuclear compartment only in transfected cell lines (Höhne et al., 1990; Seifer et al., 1990).

The recent finding that the X gene product can trans-activate transcription from a number of HBV and heterologous promoters is of considerable importance in defining its role in the pathogenesis of HCC (for review, see Rossner, 1992). More clues to the possible role of HBx protein in HBV-associated pathogenesis were provided by three lines of evidence: in studies in vitro and in vivo and by direct analysis of biopsy samples of human liver and of HCC (Buendia, 1992).

High levels of expression of the X gene may induce malignant transformation of certain cultured cells, such as the mouse fibroblast NIH3T3 cell line (Shirakata et al., 1989) and immortalized hepatocytes that express the SV40 large tumour antigen (Höhne et al., 1990; Seifer et al., 1990). It has been shown that the c-myc, c-jun and c-fos proto-oncogene promoters can be trans-activated by the X gene product (Balsano et al., 1991; Seifer et al., 1991; Twu et al., 1993). Activation of protein kinase C (Kekulé et al., 1993) and formation of a p53-HBx protein complex have been reported (Feitelson et al., 1993).

Studies of transgenic mice carrying the X reading frame controlled by its natural HBV enhancer and promoter sequences or by heterologous liver-specific promoters have given rise to conflicting results. In three lines of mice (C11, H9 and E1) derived from the outbred CD1 strain (see Table 7) carrying a 1.15-kilobase HBV fragment (spanning the enhancer, the complete X coding region and the polyadenylation signal), preneoplastic lesions were observed in the liver, which were followed by carcinomas at 8–10 months of age (Kim et al., 1991). In contrast, a transgene in which the X coding domain was placed under the control of the α-1-antitrypsin regulatory region failed to induce tumours in ICR × B6C3F1 transgenic mice, although X mRNAs were detected in liver tissues (Lee et al., 1990).

Analysis of integrated viral sequences in tumour DNA has shed new light on one of the mechanisms leading to overexpression of the X gene in chronically infected livers and in HCCs. HBV sequences are frequently interrupted between or around the viral direct repeats DR1 and DR2 upon integration into host cell DNA (Buendia, 1992), and overproduction of hybrid viral and host transcripts may result from HBV DNA integration in a hepatoma cell line (Freytag von Loringhoven et al., 1985; Ou & Rutter, 1985). The presence of viral and host transcripts containing a 3′ truncated version of the HBx coding region fused with flanking cellular sequences and retaining trans-activating capacity was first described in a human HCC (Wollersheim et al., 1988). Moreover, enhanced trans-activating capacity of the integrated X gene product has been related to substitution of viral carboxy terminal residues by cellular amino acids (Koshy & Wells, 1991). trans- Activating ability of similarly truncated X gene products made from fusion of integrated HBV sequences with adjacent cell DNAhas also been shown in many tissues from patients with chronic hepatitis (Takada & Koike, 1990).

This suggests that the integrated X gene might be essential for maintaining the tumour phenotype that develops at the early stages of carcinogenesis. Consistent with this model is the finding that viral and host junctions can be mapped in the carboxy terminal region of X in most human HCCs (Nagaya et al., 1987; Buendia, 1992).

4.2.3. Genetic alterations in hepatocellular carcinoma

Genetic alterations that cannot be associated clearly with a direct effect of viral infection have been observed in human HCCs. Such somatic changes include allele losses on several chromosomal regions, mutation and activation of cellular genes that show oncogenic potential and deletion or mutation of tumour suppressor genes.

(a) Chromosomal losses and tumour suppressor genes

Several groups have reported loss of heterozygosity in a large number of HCCs (for a review, see Buendia, 1992). Loss of heterozygosity on the distal 1p region and on chromosomes 4q, 11p, 13q and 16q has frequently been detected in human HCCs by restriction fragment length polymorphism (Pasquinelli et al., 1988; Wang & Rogler, 1988; Buetow et al., 1989; Tsuda et al., 1990; Simon et al., 1991). These studies showed no relation to tumour histology or grade, presence of HBV, cirrhosis or ethnic origin of the patient. Thus, it has been suggested that these parts of the human genome might contain genes, the functional loss of which might be involved in hepatocellular carcinogenesis. Allele loss of the short arm of chromosome 17p, which includes the p53 gene, has also been observed frequently in human HCC (Fujimori et al., 1991; Slagle et al., 1991). Viral insertions at chromosome 17p in some HCCs are not physically linked to the p53 gene (Hino et al., 1986; Tokino et al., 1987; Zhou et al., 1988). Accumulation of allelic loss on different chromosomes (e.g. 13q, 16q) has been associated with advanced stages of HCC (Nishida et al., 1992). Whether HBV DNA integration, which has been shown to promote genetic instability, contributes to these events has not been elucidated.

Tumour suppressor genes are identified on the basis of their loss or inactivation in tumour cells, as the product of such genes are negative regulators of cell growth (Marshall, 1991). Mutational inactivation of p53 is the commonest known genetic alteration in human cancer (Hollstein et al., 1991). p53 and retinoblastoma genes are the only genes known to be involved in HCC (Bressac et al., 1990, 1991; Hsu, I.C. et al., 1991; Murakami et al., 1991). The frequency of p53 mutations in HCC was reported to be high (≥ 45%) in Qidong, China (Hsu, I.C. et al., 1991; Scorsone et al., 1992; Li et al., 1993) and Mozambique (Bressac et al., 1991; Ozturk et al., 1991); intermediate (15–35%) in Japan (Murakami et al., 1991; Oda et al., 1992; Nishida et al., 1993) and in Shanghai (Buetow et al., 1992; Li et al., 1993), Xian (Buetow et al., 1992) and Taiwan, China (Hosono et al., 1991; Sheu et al., 1992); and low (0–15%) in Germany (Kress et al., 1992), the United Kingdom (Challen et al., 1992), Thailand (Hollstein et al., 1993) and Alaska, USA (Buetow et al., 1992). As shown in Table 12, the relationship between chronic infection with HBV (as determined by serum HBsAg) and the frequency of p53 mutation in HCC was addressed directly in three studies, and p53 mutations were shown to occur at similar frequencies in HbsAg-seropositive and HBsAg-seronegative patients.

Table 12.

Frequency of p53 mutations in patients seropositive and seronegative for hepatitis B surface antigen (HBsAg).

A ‘hot spot’ mutation (Buendia, 1992) at the last guanine residue of codon 249 (AGG to AGT) was identified in more than 45% of HCCs from Mozambique and Qidong, China (Bressac et al., 1991; Hsu, I.C. et al., 1991). The codon 249 mutation (AGG→AGT) of p53 is not a mutational hot spot in non-HCC tumours (Hollstein et al., 1991). The presence of codon 249 mutations in HCC was reported in both HBsAg-seropositive and HBsAg-sero-negative patients (Ozturk et al. ,1991; Buetow et al., 1992; Oda et al., 1992; Sheu et al., 1992; Hollstein et al., 1993; Yap et al., 1993) as well as in the presence and absence of tumour HBV DNA (Scorsone et al., 1992; Li et al., 1993). An association between HBxAg and p53 both in vitro and in vivo has been described (Feitelson et al., 1993).

(b) Activation of cellular oncogenes

The search for activated oncogenes in DNA from HCCs using the NIH3T3 cell transformation assay has not been successful in most cases. A transforming DNA, lca (liver cancer), was characterized in a very small number of tumours (2/4) (Ochiya et al., 1986). This new oncogene is expressed at a proliferative stage in fetal liver; its expression in liver cancer has not been associated with gross rearrangement of the gene (Shiozawa et al., 1988). Another transforming gene, hst-1, was identified by this method in a tumour from an HBsAg-sero-negative patient (Yuasa & Sudo, 1987); and co-amplification of integrated HBV sequences and hst-l was reported in another case (Hatada et al., 1988). Conflicting results have been obtained concerning the ras genes in the NIH3T3 cell transformation assay. Direct sequencing of the c-Ha-ras, c-Ki-ras and N-ras genes has shown a very low incidence of point mutations in liver tumours (Tsuda et al., 1989; Tada et al., 1990; Ogata et al., 1991).

Increased expression of c-myc protooncogene has been described in a majority of human HCCs but also occurs in cirrhosis (Gu et al., 1986; Himeno et al., 1988). In rare cases, it was associated with genetic amplification of the c-myc locus (Trowbridge et al., 1988).

5. Summary of Data Reported and Evaluation

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