Journal of Gastroenterology and Hepatology (1992) 7, 622-638
REVIEW ARTICLE
The role of hepatitis B virus in the development of primary hepatocellular carcinoma: Part I W I L L I A M S . ROBINSON
Stanford University School of M.dicine, Stanford, California, USA Abstract Chronic infections with hepatitis B virus (HBV) of humans and animal hepadnavirus infections in their natural hosts are strongly associated with primary hepatocellular carcinoma (HCC). Although viral integrations are found in cells of many HCC, no general viral-specific hepatocarcinogenic mechanism for hepadnaviruses has been identified. In approximately one half of HCC in woodchuck hepatitis virus (WHV) infected woodchucks, viral integrations near the c-myc or N-myc genes have been reported which result in enhanced expression of the respective gene. Such host gene-specific insertional mutagenesis has not been found in HCC of other hepadnavirus infected hosts. Thus in humans, ground squirrels and ducks hepadnaviral integrations appear to be at different host chromosomal DNA sites in each HCC and few integrations have been found within or near any cellular gene. Other possible hepadnavirus-specific carcinogenic mechanisms that are being investigated include transactivation of cellular gene expression by an hepadnavirus gene product (e.g. the X-gene), and mutation of host genes by unknown hepadnavirusspecific mechanisms. It should be noted, however, that chronic hepadnavirus infection is associated with chronic necroinflammatory liver disease with hepatocellular necrosis and regeneration (sometimes leading to cirrhosis in humans), a pathological process that is common to numerous other risk factors for HCC. This suggests the possibility that this pathological process is hepatocarcinogenic irrespective of the inciting agent and the role of hepadnavirus infection is no different from that of other risk factors in causing chronic necroinflammatory liver disease.
Key words: hepadnaviruses, hepatitis B virus, hepatocellular carcinoma, hepatoma, insertional mutagenesis, oncogenes, primary liver cancer, viral DNA, viral integration, virus replication.
INTRODUCTION Primary hepatocellular carcinoma (HCC) is one of the most common cancers occurring in humans and there is strong epidemiological evidence suggesting that persistent hepatitis B virus (HBV) infection is the most important risk factor for its development. Hepatitis B virus infections commonly occur at an early age and become persistent in populations in which this virus is highly endemic such as in eastern Asia and sub-Saharan Africa.' Such persistent HBV infections may be associated with chronic hepatitis. Macronodular cirrhosis develops in a significant fraction of persistently infected individuals, usually after many years.',' Long standing persistent HBV infection is associated with a greatly increased risk of HCC compared with the risk in uninfected population^,'^^-^ and the presence of macronodular cirrhosis in HBV infected individuals appears to further increase the risk of HCC by at least l O - f ~ l dThe . ~ strong association of HCC with persistent HBV infection in humans3-' and with other natural hepadnavirus infec-
tions in woodch~cks,6.~ ground squirrels' and duck^^-'^ suggests an important role for infection with members of this virus family in the development of HCC. T h e finding of a clonal pattern of hepadnavirus DNA integration in cellular DNA of many HCC in and in other hosts naturally infected with hepadnaviruses indicates that these tumours arise from a single cell with integrated v i r ~ s . ' - ' ~ ~ 'This ~ ~ ' finding has stimulated interest in whether viral integration plays a role in HCC. Although the most important risk factor for HCC in humans appears to be chronic or persistent HBV infection, several other specific factors appear to increase the risk of developing liver cancer and almost all HCC cases appear to be associated with HBV or one of the other recognized risk factors. It has been estimated that 80% of all HCC in the world occur in HBV infected individuals" and careful prospective studies have indicated that 40% or more of middle aged Chinese males with chronic HBV infection die of HCC.3 Since chronic infection can occur in 10% or more of the population in high prevalence area^,"^ the number of liver cancers in those populations
Correspondence: W. S. Robinson, Stanford University School of Medicine, Stanford, California, USA. Accepted for publication 11 May 1992. Editor's note: This is the first of a two part article. Part I1 will appear in the next issue (vol. 8, no. 1) of the Journal.
HBV in the development of carcinoma is very great. Among all cancers of humans associated with recognized environmental risk factors, only the number of cigarette smoking-associated lung cancers appears to exceed HBV-associated HCC. Important risk factors associated with the remaining 20% of HCC cases are certain factors that cause chronic necroinflammatory liver disease and cirrhosis including chronic alcohol-induced liver disease,19720 chronic hepatitis C,21-28 h a e m o c h r ~ m a t o s i s , ~ ~a*1-antitrypsin ~~ defi~iency,~'cryptogenic cirrhosis,32 primary biliary cirrhosis,33 hereditary tyrosinaemia,20and other causes of cirrhosis; and exposure to certain chemical carcinogens such as dietary aflatoxin (toxins produced by fungal organisms which infest improperly stored grain, peanuts and other food),34-36androgenic-anabolic steroids37 and oral contraceptive s t e r o i d ~ , ~ ~undefined -~' substances (possibly pe~ticides)~' in polluted ground water used for drinking in parts of China4'-43 and possibly other chemical carcinogens (Table 1). There is an uneven geographic distribution of HCC in the world because the most important risk factors for HCC are unevenly distributed.' The highest prevalence is in Eastern Asia (China and adjacent regions) and sub-Saharan Africa and these are areas of the world with the highest prevalence of chronic HBV infection.' The prevalence of HCC is much lower in the United States and Western Europe where HBV infections are much less common and more important risk factors for HCC are alcoholic liver disease and probably chronic hepatitis C. Recent research has provided much information about the molecular and genetic structure of HBV, its molecular mechanism of replication, its phylogenetic relationship Table 1 Established and possible risk factors for HCC in humans Associated with chronic necroinflammatory liver disease and/or cirrhosis. HBV (and other hepadnaviruses in their natural hosts) I .3.4,6-Y964
HC-21-28
Alcohol 19.20.3 19.33 1.332 Cryptogenic c i r r h o ~ i s ? ~ ~ . ~ ~ ~ Primary biliary cirrhosis?33 Autoimmune CAH?334q335 Membranous obstruction of inferior vena cava?336-337 a-1-antitrypsin d e f i c i e n ~ y ? ~ ~ ? ~ ~ * - ~ ~ ~ Hemochromatosis?2Y~30 Wilson's disease?3447345 Glycogen storage d i ~ e a s e ? ~ ~ ~ . ~ ~ ' Hereditary t y r ~ s i n a n e m i a ? ~ ~ . ~ ~ ~ ~ ~ ~ ' Parasitic infestations of ~ i v e r ? ~ ~ ~ - ~ ~ ~ Methotrexate the rap^?^^^,'^^ Other causes of cirrhosis? Without necroinflammatory liver disease Aflatoxin B1?34-36.358 Polluted drinking water in China?4'-43 Contraceptive and anabolic Cigarette s m o k i x ~ g ? ~ ~ ' - ~ ~ ~ Other chemical carcinogens? ~
? indicates a clinical association with HCC without definitive epidemiologic evidence for an independent causal role.
623 with other viruses and its molecular state in cells of HCC. This information suggests possible mechanisms by which HBV infections can lead to HCC. This review will consider the role of HBV in the development of HCC by reviewing important features of the virus, describing evidence that HBV infection plays an important role in HCC, and considering possible carcinogenic mechanisms for the virus.
Hepatitis B and related viruses (hepadnaviruses) Hepatitis B surface antigen (HBsAg) was discovered serendipitously in human serum in the mid 1 9 6 0 It~ ~ ~ was later associated with acute serum he pa ti ti^^^-^' and eventually was identified as a viral (HBV) antigen. This led to rapid advances in understanding the epidemiology of the virus, the course of infection and recognition of associated diseases, and the physical nature of the virus. Early during this period HBV infection was recognized to be associated with HCC.48
Biological and epidemiological features of HBV Many primary HBV infections were shown to be asymptomatic, infections could persist for many years, the virus was found to be hepatotropic (i.e. to infect primarily liver cells) although less frequent and less permissive infection sometimes occurs in bone marrow, circulating leucocytes, B cells and T cells, and p a n c r e a ~ . ~ Virtually ~-~' all active infections were accompanied by high concentrations of HBsAg and often infectious virus in the blood. Persistent infections were often associated with minimal liver disease or with chronic hepatitis that sometimes led to cirrhosis.2 The risk of HCC was shown to be more than 100 times higher in some 'HBsAg carrier' populations than in similar uninfected population^.^
Hepadnavirus family The discovery of several closely related viruses with similar epidemiological features and associated with similar disease syndromes including HCC occurring naturally in certain rodent and avian species indicates that there may be many members of this virus family which has been named Hepadnaviridae for hepatotropic DNA v i r ~ s e s . ~ The ' - ~ ~hepadnavirus family includes hepatitis B virus of humans, woodchuck hepatitis virus (WHV) of Marmata rn0nax,6~ ground squirrel hepatitis virus (GSHV) of Spermophilus bee~heyi,6~ duck hepatitis B virus (DHBV) found in several varieties of domestic ducks (Summers, London, Sun, Blumberg et al. unpubl. data)49@' and similar viruses in tree ~quirrels,6~ and herons6' Less well documented findings in other rodents, marsupials and cats suggest that other hepadnaviruses may exist. A characteristic feature of all hepadnaviruses is a moderately narrow host range. For example HBV infects only humans, chimpanzees and possibly other great apes but not monkeys or other species. All hepadnaviruses exhibit strong relative tropism for h e p a t o c y t e ~ ~and ~,~~*~~ production by infected hepatocytes of large amounts of
W.S. Robinson
624
non-infectious viral envelope particles (as well as infectious virus) that can be detected in high concentrations (up to 500 pg/mL) in the blood,"9,64,65,69-71and the common occurrence of persistent infections with viral forms in liver and blood continuously for years and often for the lifetime of the host. Infections with hepadnaviruses may be associated with acute and chronic hepatitis characterized by hepatocyte necrosis, inflammatory reaction, lymphocytic infiltration and liver regeneration;'* immune com lex (viral surface antigen-antibody) mediated d i ~ e a s e ;74 ~ and HCC.',3-'0364 T he mechanism of liver injury in acute and chronic hepatitis associated with hepadnavirus infection is not completely defined but a cellular immune mechanism is considered to be likely. Some evidence indicates that cytoxic T cells directed at the viral core antigen (HBcAg) displayed on the surface of hepatocytes can lead to cell Other studies have suggested that HBcAg expression in infected cells may have a direct cytopathic effect.77 Another Eonimmune mechanism involving accumulation of viral envelope polypeptides in liver cells and resulting liver cell necrosis has been demonstrated in transgenic mice78s7yand, in theory, could play some role in in wiwo hepadnavirus infections. Hepadnaviruses share unique features of virion (virus particle) size and ultrastructure with an envelope surrounding an electron dense spherical nucleocapsid or characteristic polypeptide and antigenic composition;80 common virion DNA size, structure,81i82and genetic 0rganization;8~the presence of DNA polymerase activity in the virion;84i85and an unusual mechanism of viral DNA replication which includes reverse transcription of a greater than genome length viral RNA transcript.86-88
Y,
.
Viral genome structure The virion DNA of these viruses is among the smallest of all known animal viruses consisting of an approximately 3200 base pair (bp) circular molecule that contains a single stranded region of different length in different molecules81782 (Fig. 1) reflecting the fact that DNA molecules are packaged into virions before viral DNA replication (i.e. synthesis of the DNA plus strand) is complete. The short DNA strand of variable length is the second synthesized plus strand and the full length strand is the first synthesized minus strand. Neither viral DNA strand is a covalently closed circle and single breaks occur in the two strands at unique positions in the nucleotide sequence approximately 225 nucleotide pairs apart in mammalian h e p a d n a v i r u ~ e s(Fig. ~ ~ ~1). ~ ~The full length DNA minus strand is longer than the plus strand due to a nine nucleotide terminal redundancy. The circular conformation of the DNA is maintained by non-covalent base pairing of the cohesive overlapping 5' ends of the two DNA strands. 'The virion contains DNA polymerase a c t i ~ i t y ~that ~ . ~catalyses ' the repair of the single stranded region to make fully double stranded relaxed circular DNA molecules81~82 and this enzyme appears to be the reverse transcriptase involved in viral DNA replication in infected liver The complete nucleotide sequences of the cloned ~ - ~ ' DHBV isoDNA of 13 HBV i ~ o l a t e s , 8 ~ ' ~two
2458
833
Figure 1 Circular map of the HBV (3dw2) genome. The inner circles represent the virion DNA strands and the broken (dashed) line in the short ( + ) DNA strand represents the region within which the 3' end of the + strand may occur in different molecules, and the corresponding region of the long strand is that which .3ay be single-stranded in different molecules. A line of dots represents the oligoribonucleotide primer covalently attached to the 5' end of the +DNA strand and a single dot represents the protein primer covalently attached to the 5' end of the - DNA strand. The large arrows represent the recognized functional open reading frames (ORF) with the direction of transcription from the minus DNA strand indicated. The small arrows indicate the 5'-ends of the three major size classes of transcripts of 3.4, 2.4 and 2.1 Kb, all of which are identically terminated near the poly A addition signal (TATAAA). The nucleotide sequence locations of the initiation and termination codons of each ORF are given with reference to map position 1 at the single EcoRI cleavage site in this DNA. The map position of the first nucleotide of the glucocorticoid responsive element (GRE), DR2, DRI, the US-like sequence and the poly A addition sequence (TATAAA) are indicated, as is the general regions exhibiting enhancer 1 (ENH 1) and enhancer 2 (ENH 2) activity.
lates,lOO~lO1 and two of WHV,'02*'03one each of GSHVIo4 and the heron virus68 have been reported, The genomes of the three mammalian hepadnaviruses have four long open reading frames in the complete or long (minus) virion DNA strand and these have similar locations in each virus with respect to the cohesive ends of the DNA (Fig. 1). T h e genes for the major virion polypeptides have been identified. All of the genome appears to be translated and overlapping genes are translated in different reading frames. Evidence suggests that none of these genes is interrupted by intervening sequences. T h e C gene specifies the major viral core or nucleocapsid polypeptide of 21 000 daltons and a truncated 16 000 dalton form of this polypeptide with HBeAg specificity. This open reading frame includes a short preC (precore) sequence delineated by an in-frame initiation codon. The
HBV in the development of carcinoma
S gene, including preS,, preS,, and S regions delineated by three in-frame initiation codons, specifies the viral surface antigen reactive polypeptides in the virion envelope and in incomplete viral forms (surface antigen particles) found in serum and liver of infected individuals. These consist of the glycosylated and non-glycosylated forms of three polypeptides of 24 000, 33 000 and 39 000 daltons coded respectively by S alone, by preS, and S , and by preSl, preS, and S regions of the S open reading frame. The P gene which covers three-fourths of the genome and overlaps a carboxyl-terminal portion of the C-gene, the entire S-gene and an amino-terminal portion of the X-gene can specify a basic polypeptide of approximately 90 000 daltons and e v i d e n ~ e ~ ~ suggests * ’ ~ ~ - that ’~~ this contains the virion associated DNA polymerase (or reverse transcriptase) activity, ribonuclease H activity and a protein primer for minus DNA strand ~ynthesis.”~ The small X-gene appears to specify a polypeptide of 154 amino acids or more in some viruses. It was first shown to activate transcription regulated by regulatory elements of the P-interferon gene by Twu and S c h l ~ e m e r and ’ ~ ~ it is now known to activate transcription of HBV”’*”’ as well as several heterologous viral”’-”5 and cellular genes.109,1 16-118 T h e codon preference of the X-gene is more like that of typical cellular genes than viral genes suggesting it may be of cellular origin.lo6 The C, P, preS, and S open reading frames are approximately the same relative size and occupy the same relative genomic positions in all mammalian hepadnaviruses. The preS, region varies in size in different viruses suggesting that the polypeptide sequence specified by this DNA sequence is less functionally critical than that specified by the preS, and S regions. The preC sequence similarly varies in size in different sequenced HBV genomes and the C gene of the avian hepadnaviruses is significantly larger than in the mammalian viruses. The size of the X-gene varies by up to 50% among nine different HBV isolates (largest in HBV adr subtype genomes) and the X open reading frame is not present in avian hepadnavirus genomes. Thus avian hepadnaviruses contain only three open reading frames (S, C and P). Other functionally important elements in hepadnavirus genomes include 11 bp direct repeat sequences designated DRl and DR2 (Fig. 1) which are approximately 225 bp apart in the mammalian viruses. They appear to play a role in viral DNA replication.87q88 This sequence is not completely conserved but exhibits significant variation in different viruses.”’ T h e 5’ end of the minus DNA strand occurs within DR1 and the 5’ end of the plus DNA strand at the 3’ boundary of DR2.87,i20 No consensus TATAA elements have been detected within nucleotide distances of the known hepadnavirus RNA initiation sites expected for usual eukaryotic promoters. However, studies of transcription and RNA mapping in infected cells in culture, in liver tissue and in cell free systems, and use of isolated hepadnavirus DNA fragments have provided preliminary evidence for the existence and general locations of four promoter elements. One (called the preS, promoter) upstream from the start of the preS, sequence (Fig. 1)”’ may initiate synthesis of a 2.4 kb messenger RNA (mRNA) for a protein containing preS,, preS, and S sequences. Another
625 (called the preS, promoter) within the preS, appears to direct synthesis of a 2.1 kb RNA transcript that probably functions as messenger for preS, and S gene specified polypeptides. Both are within the coding sequence for the P gene and the action of neither appears to be cell or tissue specific. A sequence upstream from the start of the C-gene and within the coding region of the X-gene appears to direct synthesis of a greater than genome length (3.5 kb) RNA transcript which may function as messenger for the synthesis of C and P-gene specified polypeptide^.'^^-'^^ This RNA also appears to serve as a template for synthesis of the virus minus DNA strand by reverse transcription. This promoter (called the C-promoter) appears to exhibit some specificity for liver cells.’27 A fourth promoter element has been described upstream from the X-gene and within the P open reading frame’” and this promoter (called the X-promoter) appears to direct synthesis of minor transcripts which function as X-gene messengers. A transcriptional enhancer element (enhancer 1) has been localized to a region approximately 450 bp upstream from the C-gene promoter (Fig. It is either immediately upstream of the start of the X-gene in mammalian viruses with a short (typical) X-gene or within the 5’ end of that open reading frame in viruses with a long X-gene (e.g. in HBV subtype adr) and within the P open reading frame in all viruses. Functional analysis has suggested liver cell specificity for this enhancer.’” The C - p r ~ m o t e r ’ ~ ~ .and ’ ~ ’ other HBV promoter^'^' under control of the HBV enhancer 1 (and not other enhancers) have been shown to be much more active in differentiated liver than other cell types. In nuclear extracts of human liver cells, several proteins have been shown (by DNA footprint analysis) to bind specific sequences within HBV-DNA fragments that contain functional enhancer activity. These include the genomic region of approximately 300 bp which contains overlapping binding sites for multiple cellular transcription factors, including NF-1,’33*’34 C/EBP,135-’36 AP-l,i37 CREB’37 and ATF.138’139 A second cis-acting transcription regulatory element has been described within an approximately 100 bp sequence located in the downstream half of the X open reading frame. This is within or just upstream of the core promoter (Fig. 1); it also has properties of a strong liver cell specific enhancer (enhancer 2).’40-’43 Another hepadnavirus genomic region (within the S coding region) is necessary for a five-fold enhancement of gene expression in the presence of glucocorticoids.’44 This glucocorticoid responsive enhancer-like element appears to be independent of orientation in the genome and without apparent species or cell type specificity. An 18 nucleotide sequence in HBV similar to known glucocorticoid responsive DNA elements was identified in that region (approximately 2097-21 12 on the map in Fig. 1). Glucocorticoids increase levels of HBsAg expression in HBV infected patients in v i ’ u ~ , ’in~ ~HCC cell lines expressing HBsAg in culture’46 and in transgenic mice.I4’ This effect might in part explain some of the differences in the behaviour of HBV in males and females. A polyadenylation signal that appears to be used by all major transcripts lies within the beginning of the C-gene,
626 approximately 20 bp upstream from the 3’ end of the minus DNA strand and just downstream of a TATAAA (AATAAA in DHBV) Sequences downstream from the TATAAA sequence also appear to play a role in correct polyadenylation of HBV transcript^.'^^ Finally, a highly conserved 60-70 bp sequence with a high degree of homology with the U5 sequence of the LTR of certain retroviruses is present just downstream from DR1 and within the preC region of the genome.Io6 T h e function of this sequence is unknown. The hepadnavirus genome is unusually compact and efficiently organized in that much of the genome is used for multiple functions including overlapping genes so that the same nucleotide sequence encodes more than one protein and all cis-acting regulatory sequences (e.g. transcriptional enhancer and promoter elements) are contained in genomic sequences also encoding protein (Fig. 1).
Viral genome replication
W. S. Robinson General features of hepadnavirus integrations have been identified by comparing the structure of viral and flanking cellular DNA of many published examples (almost all being HBV integrants in human HCC).’51,152,157 H epatocellular carcinomas containing integrated virus sometimes have a single clonal integrant but more often there are multiple clonal integrants each at a different cellular DNA site (most often three to four T hese would appear to but rarely 10 or arise from separate viral integration events. The apparent cellular DNA sites of integration have been different in every human HCC studied. Most HCC with integrated virus do not contain replicating viral DNA forms, which suggests that cells of such HCC are non-permissive for virus replication by an unknown mechanism. Some integrants consist of simple contiguous linear sequences of HBV-DNA without rearrangement and others are complex and may have arisen by integration of multiple viral genomes at one site or rearrangement after integration by recombination between viral sequences of different i n t e g r a n t ~ . ’The ~ ~ complex integrants contain one or virus-virus junctions. Complete viral genOmeShave not been found in any integrants and deletion of viral sequence has been found in all that have been sequenced whether they arose from single or mul54,157,160 The long terminally tiple genome redundant HBV transcript that as a template for viral genomic DNA synthesis be synthesized from such viral integrations and virtually all integrants are defective for virus replication. Thus hepadnavirus integrants are not involved in virus replication as is the integrated DNA provirus of retroviruses. be preferred sites for integration There appear within the viral genome. More than 50y0 of viral integrants in H C C ~ 5 0 - ~ 5 2 . ~ 5 7 , ~ 6 1 and non-HCC liver
The first step in viral genome replication involves conversion of infecting virion DNA molecules into covalently closed circular molecules, this takes place in liver cell It is by formation Of a greater than genome length RNA transcript with a terminally repeated sequence as well as shorter transcripts which function as messenger RNA. The long transcript, made transcriptase and protein primer are packaged in ‘Ore particles that are found in hepatocyte ‘ytoplasm. Synthesis of new viral DNA molecules is accomplished by reverse transcription of the long RNA transcript using a viral encoded protein primer to form the first synthesized viral DNA (minus) strand and a capped oligoribonucleotide derived from the 5’ end of the long RNA template as primer for synthesis ofthe second t i s s U e ~ ~ 4 , 1 5 5 appear to have one viral DNA end which DNA s y n t h e s i ~ ~ ~ ~ joins ~:~ DNA (Plus) ’Wand (Fig’ 2)’ cell DNA (or viral DNA in the case of virus-virus exclusively within cytoplasmic viral core particles DNA junctions) near or between the cohesive 5’ ends A nine nucleotide terminal redundancy in the minus which are within the direct repeat (DR) sequences of viral strand is thought to be involved in template switching for DNA (Fig. l). Those with ends near the 5 , end of the synthesis of the second (plus) DNA strand.87 Further minus DNA strand (and DRI) exceed those near the 5‘ details of hepadnavirus replication have been described end of the plus DNA strand (and DR2) by approximately el~ewhere.~~.’~ 3 : 1 (Table 2; Fig. 3). T h e most frequent site for a viral end is near the left end (first nucleotide) of DR1. No Hepadnavirus integration integrant with an intact cohesive end region has been Hepadnavirus integration in cellular DNA has been found. In simple linear contiguous viral sequence intefound in infected liver as well as HCC. In hepadnavirus grants, when one end of the viral DNA joining cellular infected hosts many HCC contain viral integrations with DNA is within or near a DR sequence, the other DR a clonal pattern. It follows that these tumours must have sequence is not present. arisen by clonal expansion of an original cell with that T h e other end of viral DNA which joins cell DNA (or viral integration. The possible role of such integrations in viral DNA in the case of virus-virus junctions) in integrants joining cell DNA near a DR sequence at the development of HCC has been intensively investigated. Much of the evidence of the fine structure of one end, occurs at variable positions in the viral hepadnavirus integrations comes from investigation of g e n ~ m e . ’ ~ ’ > This ’ ~ ~ *suggests ’ ~ ~ random recombination those in h ~ m a n ’ ~ ~and - ’ ~woodchuck * HCC;’53there has with cell DNA, although there may be a slight preference been much less investigation of viral integrations in for the preS region as a viral site of integrati~n.’~’ non-tumorous l i ~ e r . ” ~ -It’ ~is~ not known whether These findings suggest that the cohesive end region of viral integration occurs in every hepadnavirus infected viral DNA is involved in the mechanism of viral DNA cell in viva or only in Some cells. T o date, however, no integration into cellular DNA. This implies that replicaapparent difference in the structure of viral integrations of tion intermediates (probably linear molecules) with single HCC and non-tumorous liver have been identified stranded DNA at the cohesive end region rather than closed circular viral DNA are substrates for integration (Table 2).
627
HBV iv the development of carcinoma Transcription
Form I DNA free in cell nucleus
DR 1
-
R DNARNA 51.. +
J.
.I.. ................/!.3'
*
DNA synthesis , in immature cores in cell cytoplasm DR2
DR1
I
I
r
r
Extracellular virions
'
.... RNA
-
DR2
DNA Protein primer for -DNA strand synthesis DR-l,DR-2 = 12 nucleotide direct repeat sequences R = 200 nucleotide terminal redundancy in long RNA transcript r = Terminal redundancy in -DNA strand Figure 2 Scheme of proposed mechanism of hepadnavirus DNA replication. DK-1 and DR-2 represent 11 nucleotide pair direct repeat sequences. R represents the approximately 200 nucleotide terminal redundancy in the long KNA transcript and r represents the short terminal redundancy of the -DNA strand. Solid lines represent DNA strands, dotted lines KNA and the stippled area the protein primer for DNA strand synthesis.
(Fig. 2). It also suggests that viral integrations occur at a stage of infection when virus replication is proceeding and appropriate replication intermediates are present. The common structural features of viral integrants suggest that the substrates for integration are replication
intermediates with DNA strands of variable length, all of which would have the same 5' end (near a DR sequence) and 3' ends at variable positions along the viral genome (Figs 2, 3). Of the many integrants that contain viral ends near D R l , most contain viral sequences to the left ofDR1
628
W.S. Robinson
Table 2 Hepadnavirus integration
Configuration of viral integrants
Tissue source of integrant (# positives/# integrants
examined)
One end of viral DNA insert which joins cell DNA DNA is near or between viral DNA cohesive ends (i.e. near a DR sequence) Shih integration patterns (150) I (DR1) I1 (DRl) 111
(DR2)
IV
(DR2)
Neither end of viral DNA insert which joins cell DNA is near or between viralcohesive ends Viral insert contains complete or near complete X-gene: Viral insert contains no X gene sequence
One end of viral DNA at virus-virus DNA junction is near or between viral DNA cohesive ends Viral insert contains truncated preS,/S sequence
HCC
Non-HCC liver
30145 8/45 14/45 5/45 3/45
4/4* 014 314 1/4* 014
17/45
014
6/17 7/17
-
11/45
114
5/45
114
Percentage of 30 HCC integrants with one end near a DR sequence
26.6 46.7 16.6 10.1
Fraction and end of X-gene contained in integrants
< 10% of < 90% of z 50% of z 50% of
3’ 5’ 5’ 3‘
end end end
end
-
*One WHV integration in WHV infected woodchuck liver (153) has integration pattern 111; 3 HBV integrations in HBV infected human liver have integration pattern 11. Integration of HCC reported in ref. 149,150,152 and 153; and integrants of non-HCC liver in ref. 152-1 54.
(integration pattern 11). This is consistent with minus strand DNA invasion (Table 2, Figs 2, 3). Fewer integrants contain viral ends near DR2 and most of those have viral sequences to the right of DR2 (integration pattern III), consistent with plus strand DNA invasion. An alternative viral DNA substrate for integration has been suggested r e ~ e n t l y . ’ ~ *Preferred .’~~ topoisomerase I cleavage sites were identified in WHV-DNA near and between DR1 and DR2 (including the 9 bp terminally redundant region of the viral minus-strand DNA). It was proposed that cleavage of these sites by topoisomerase I could lead to integration of the resulting linear viral DNA molecules by illegitimate recombination. 162,163 Small regions of DNA homology near crossover points appear to be involved in partially homologous recomb i n a t i ~ n . ‘Moderate ~~ sequence homology between the ends of HBV integrants and flanking cellular DNA have been noted.’52,’61,’65-167 T he degree of homology of these regions is not as great, however, and crossover points do not coincide as precisely as do the homologous regions found in systems of homologous recombination. 164 Almost all data on viral sites that join cell DNA in integrations come from integrations in HCC. It is possible, therefore, that the cohesive region represents a biased selection of sites that are special for HCC. However, the small number of integrants that have been examined from infected non-HCC liver appear to confirm that a common viral site that joins cellular DNA in
integrants of non-HCC liver is also in the cohesive end region (Table 2). Several models of hepadnavirus integration mechanism based on known features of viral integrants have been proposed. 150-152.1 57,162,163,165,168 While there are preferred sites for integration within the hepadnavirus genome (e.g. the cohesive end region), viral DNA appears to be integrated at many different sites of cellular DNA. Whether there may be somewhat preferred cellular sites of HBV integration as appears to be the case for some retroviruses is not yet ~ 1 e a r .There I~~ may be some preference for HBV integration within repeating sequences such as Alu DNA,’” satellite 111 DNA,’51,’70 a-satellite DNA,’ 55 and minisatellite or VNTR DNA.’52,’55,’67,’68,’70 One half of WHV integrations in woodchuck HCC appear to be near either the c-myc or the N-myc gene. These cellular sites have been found only for this virus and HCC of this host; this site has not been identified for integration of other hepadnaviruses in HCC of their natural hosts, or in non-HCC liver. Whether myc genes represent true preferential cellular sites for WHV integration in woodchuck liver cells is uncertain. An alternative and more likely explanation is that liver cells with WHV integration near myc genes have been selected because such integrations impart oncogenic attributes to the cells, thereby resulting in their clonal expansion and HCC formation. Hepatitis B virus integrations have been found on at least 12 different chromosomes in HCC.’57 Five integrations assigned to chromosome 11 represent a higher
HBV in the development of carcinoma
,
1300
Virion DNA
-strand +strand
629
15r
18[6
2400
----
5’
-----
4
- - - - - - - -b
~
*# - -
p*
Integration pattern I
I I Z I 26.6%
I1 I I I I I11
IV
20,OO
-- -- -- --
IIII
DR2
46.6% 16.6% 10.0%
DR1
Figure 3 Hepadnavirus integration patterns described for integrants in references 149,150,152-1 55. T h e upper horizontal scale indicates sequence positions in the HBV genome as shown in Fig. 1, the large closed circle represents the protein bound to the 5’ end of the virion DNA minus strand, the dotted line indicates the RNA attached to the 5’ end of the virion DNA plus strand, and DKl and DR2 represent the 11 bp direct sequence repeats in the viral genome. The horizontal double lines (part solid and part dashed (indicating that one end can be at various sequence positions)) opposite the indications of integrations patterns I, 11, 111 and IV represent DNA strands of idealized viral integrants for each integration pattern. The numbers to the right of each integration pattern are the percentages of each pattern found among 30 integrants with one viral DNA end near or within a DR sequence.
frequency than expected for a random d i ~ t r i b u t i o n . ’ ~ ’ * ~ integration^.'^^ ~~ Such translocations appear to be the Because of the small number of integrations studied for result of a virus-dependent mechanism by which chromosomal aberrations can be generated. However, no comchromosomal location and virtually all such data are from HCC, it is not possible to determine whether chromomon chromosomal translocation associated with or without a viral integration and activating a protosome 11 is a preferred chromosome for integratim of oncogene as found in many human cancers has been these viruses or whether integrations in chromosome 11 found in HCC.176 are found more often in HCC because they play a (4) Inverted duplication of integrated virus and functional role in oncogenesis. It has not been excluded flanking cellular DNA is a common rearrangement that integration can occur on any or all of the chromoof host chromosomal DNA found in human somes not yet found to contain a viral integration. HCC. 151.1 55,165,166,168,174.1 77,l 78 These consist of inverted Structural alterations in host chromosomal DNA at the identical sequences of integrated viral DNA and flanking site of viral integrations are common as follows: cellular DNA. Such structures suggest that an initial (1) All HBV integrations are associated with microsimple integration had been amplified and then two deletions (e.g. 10 bp) in cellular DNA at the site of copies underwent head-to-tail recombination. Head-tointegrati~n.’~’This suggests that such microdeletions tail repeats of integrated viral DNA have also been arise as an essential part of the integration mechanism as described.157In all such integrated viral DNA rearrangeis the case for integration of some other DNA viruses by ments, the viral cohesive end region is commonly found illegitimate r e ~ 0 m b i n a t i o n . l ~ ~ at virus-cell and virus-virus junctions. (2) In sporadic HCC, larger deletions of chromosomal ( 5 ) Amplification of a region of chromosomal DNA at DNA have been described at sites of viral DNA the site of integrated HBV-DNA has been described.”’ i n t e g r a t i ~ n ; ’ ~ they ~ . ’ ~appear ~ to be formed by a different (6) Alterations in host chromosomal DNA have also mechanism than the microdeletions. been detected in HCC without virus integration or at host (3) Translocations involving cellular DNA from two DNA sites a great distance from a viral integration. These different chromosomes joined to the respective ends of a include allelic d e l e t i ~ n s and ~ ~ point ~ - ~m~ u~ t a t i ~ n s . ~ ~ ~ - ~ viral DNA sequence have been described; no two have Since these alterations are not topographically near viral involved the same chromosomal DNA region. In one integrations, it seems unlikely that hepadnavirus intehuman HCC, a translocation was noted between chromogration could play a direct role. However, since viral somes 17 and 18.174There was deletion of 1.3 kb of DNA integrations can undergo rearrangement including deleof chromosome 18 at the translocation site and occurring tions involving both viral and cellular sequences after the at the site of an HBV integration with a large inverted primary integration, it has been s u g g e ~ t e dthat ~~~~~~ repeat of viral DNA.174 These findings suggest that integrated virus could be completely deleted by such postintegration rearrangement of integrated HBV and mechanisms in some HCC. This would leave an altered cellular DNA resulted in the translocation. Other transor mutated cellular sequence without residual integrated locations involving chromosomes 5 and 9, and X and 17 viral DNA. Whether this ever occurs in infected liver (or respectively have also been found at sites of viral
W. S.Robinson
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HCC) is unknown but such a mechanism seems unlikely to account for the frequent allelic deletions and point mutations described for HCC. There is no precedent for a mechanism by which viral integration could cause point mutations. Thus HBV can act as a non-host DNA sequencespecific insertional mutagen and cause secondary rearrangements of host chromosomal DNA. However, HCC appear to contain frequent chromosomal DNA alterations, for example, point mutations and allelic deletions, without apparent relationship to hepadnavirus integrations.
Hepadnavirus infection and HCC
Hepadnaviruses are related to retroviruses
Epidemiological evidence indicates that there is a geographic correspondence of relative rates of HBV and HCC. Although HCC is rare in most parts of the world, it occurs commonly in sub-Saharan Africa, Southeast and Eastern Asia, native populations in the Arctic, Oceania, Greece and Italy. Geographic areas with the highest incidence of HCC are also areas where HBV infection is common and persistent HBV infections occur at the highest known frequencies. Apparent unusual exceptions to this close correspondence in high prevalence regions suggest the possibility of important additional factors such as dietary aflatoxin in these p o p ~ 1 a t i o n s . lIn ~ ~ certain areas of Asia and Africa, where the prevalence of chronic HBV infection is the highest, HCC is the most common cancer in males.
Hepadnaviridae appear to be phylogenetically related to members of two other virus families: cauliflower mosaic virus and Retroviridae and related transposable elements. Similarity in gene number, function and order; shared genome nucleotide sequence homology in genomic regions of similar f u n c t i ~ n ; ’ ~and ~ ”utilization ~~ by all of a reverse transcriptase step in genome replication indicate these virus families are related.86,’x6-’88Reverse transcription is an unusual mechanism of genome replication for DNA viruses (known at this time only for hepadnaviruses and cauliflower mosaic virus). Undoubtedly it is a mechanism used by these viruses because of their evolution from a common ancestor shared with retroviruses and different from the evolutionary pathway of other DNA viruses. Notable differences in the viruses of these families include differences in the form of the genome packaged in virions (linear RNA in the case of retroviruses and nicked or gaped double stranded circular DNA in hepadnaviruses and cauliflower mosaic virus), retroviruses package two copies of the viral genome in each virion resulting in high rates of homologous recombination and hepadnaviruses one genome copy.’89 Details of the replication mechanisms are also different. Another important difference is in the mechanism of viral integration and the requirement for DNA genome integration in cellular DNA for virus replication. The orderly integration of retroviral DNA ‘provirus’ mediated by a virus encoded integrase with preservation of the genome sequence and including a terminally repeated sequence (the long terminal repeat or LTR) containing cis-acting transcriptional and other regulatory elements, and expression of viral genes and synthesis of new RNA genomes from the integrated provirus is an integral feature of retrovirus replication.Ix6 This feature is not shared by hepadnaviruses whose replication appears to involve only episomal (unintegrated) viral nucleic acid form^.^^,'^ Hepadnaviral integration appears to be a less well-controlled event. The mechanism includes illegitimate recombination without involvement of a viral integrase and without preservation of the genome sequence, thereby making it impossible for the viral integrant to function as a template for virus replication. The relationship of hepadnaviruses and retroviruses is of particular interest because many retrovirus infections in animals are associated with neoplasia or malignant tumour formation and intense investigation of these viruses has led to understanding certain viral oncogenic mechanisms.
The best evidence that chronic hepadnavirus infection plays an important role in the development of HCC is the epidemiological association in all of the well-studied hepadnavirus-host systems. This association was first suggested for chronic HBV infection and HCC in humans,48and the recognition of HCC in woodchuck^^^ and d ~ ~ kprompted s ~ ~ the , search ~ ~ for, and discovery of, hepadnaviruses in these hosts.
Geographic correspondence of HBV infections and HCC
Increased incidence of H C C in hepadnavirus-infected populations T h e incidence of HCC has been shown to be much higher in hepadnavirus-infected than uninfected w o o d c h ~ c k s , 6 .ground ~ ~ ~ ~ squirrels,’ and in some duck populations (Table 3).9 In humans, this appears to be true for HBV in both high and low incidence HCC populations.’ A prospective study of more than 22 000 male government workers in Taiwan has shown the incidence of HCC to be more than 100-fold higher in HBsAg positive than HBsAg negative individuals. 3 ~911 Prospective studies in China’93 and Alaskan natives’94 have indicated 30- to 100-fold higher risks of HCC among HBsAg carriers. The few cases of HCC in HBsAg negative patients in the large prospective study3,I8’ had serum anti-HBc indicating past HBV infection. These studies document that HBV infection precedes the development of HCC and they quantitate the risk. The incidence of HCC in HBsAg carriers in these populations rises steeply after age 40 years, while cirrhosis in HBsAg carriers increases the risk of HCC by more than 10-fold compared with noncirrhotic HBsAg carrier^.^.^.^^^,^^^-^^^ T h e risk of HCC is significantly higher in male than in female carriers,’*191 and this sex difference is greatest in the presence of cirrhosis. Some evidence suggests that most HBV infections in HCC patients occurred early in life and had continued for many years when HCC developed. The high incidence of persistent HBV infection in mothers of HCC patients, in contrast to that in fathers,”’ suggests that transmission from mothers to newborn or infant children is a frequent
HBV in the development of carcinoma
63 1
these models appears to be the same for males and females. In both the woodchuck and ground squirrel colonies, HCC developed in a few HBsAg negative, anti-HBc/anti-HBs positive animals (evidence of past Approximate infection) but in no animals without any serum viral Infection incidence of HCC marker. These studies suggest that chronic hepadnavirus Host status (cases per 100 000 x year) infection alone, without a recognizable cofactor, can result in HCC. This process appears to be the most Humans3 All HBsAg' 1158 efficient in the case of WHV-infected woodchucks. In Age 30-33, HBsAg' 251 individuals ( h ~ m a n swoodchucks"' ,~ or ground squirrels 40-49, HBsAg' 283 (P. L. Marion & W. S. Robinson unpubl. data) with 50-59, HBsAg' 2 149 evidence of past hepadnavirus infection the risk of devel60-69, HBsAg' 3 398 oping HCC is significantly higher than in individuals Cirrhosis, HBsAg' 12 500 with no viral markers but is much lower than in those HBsAg- , anti-HBc' ? with chronic infection (serum HBsAg positive). ? All markerThe association of DHBV infection and HCC in ducks is less clear. Duck HBV was first found in small brown 10 000 Ground squirrel All GSHsAg' domestic ducks in a region of China where HCC is (8,200-a) GSHsAg- , anti-GSHc' 5 000 common in these animals and in humans.203In addition, All marker -0 there appears to be a high content of aflatoxin in many human and animal foods in that region, a factor which All WHsAg' Woodchuck > 20 000 complicates the assessment of the role of hepadnaviruses WHsAg- , anti-WHc' ? (199,200) All markerin HCC. Although the DHBV was first discovered in ZO these ducks, no correlation of virus infection with HCC has been reported in this location. Infected brown ducks Duck (200-C) DHBsAg' All marker from this region of China (and not uninfected animals) followed prospectively in Japan were noted to develop HCC.204On the other hand, white Peking ducks infected with DHBV in the USA have not been observed to develop HCC. It is not clear whether this apparent mode and time of HBV infection in HCC patients. If difference is related to a critical difference in the viruHBV infection does occur frequently at very early ages in lence of Chinese and USA DHBV strains, susceptibility HCC patients in high-HCC-incidence areas, the age of different hosts (e.g. Chinese brown v s Peking ducks) or distribution of patients with clinically recognized HCC to other non-viral factors. would suggest that these tumours usually appear after The apparent differences in incidence and time of continuous HBV infection of 30 or more years. Few cases onset of HCC in the different hepadnavirus-host systems of HCC occur in ~ h i l d r e n . " ~ Up to 90% of HCC patients have co-existing c i r r h o s i ~ . ~ ~Projections ' ~ ' ~ ' ~ ~made ~ ~ ~ ~ (Table 4) could be due to variability in viral virulence and/or in host susceptibility factors that determine the from the large prospective study in Taiwan have sugdevelopment of HCC. The ability to infect woodchucks gested that over 40?h of middle aged male HBsAg carriers with GSHV, and different duck varieties with different in Taiwan will die of HCC.3 DHBV strains should permit direct comparison of differAlthough the argument for a role of HBV in human ent viruses in the same host. In this regard, a recent study HCC is persuasive, the association with two other mamshowed that woodchucks chronically infected with malian hepadnaviruses is even stronger. One hundred per GSHV developed HCC an average of 18 months later cent of both wild-caught WHV-infected and colony-born than woodchucks chronically infected with WHV."' experimentally-infected woodchucks died of HCC within This indicates that there are important differences in the 3 years, while no HCC developed in animals that had oncogenic effects of the two viruses in that host. never been infected.20'*202T h e animals with HCC have active hepatitis with significant components of liver cell necrosis, regeneration and inflammation but cirrhosis has Table 4 Estimated time of HCC occurrence in different not been obser~ed.~?' In a colony of captive ground hepadnavirus infected hosts squirrels infected with GSHV in the wild, no HCC were seen before 4.5 years but by 7 years more than two-thirds of the infected animals had died of HCC; no HCC were Estimated Estimated fraction of Host life span observed in animals without serologic evidence of current life span when 40% of hepadnavirus carriers (years) or past infection (P. L. Marion & W. S. Robinson unpubl. are projected to have data).' As with woodchucks, infected ground squirrels developed HCC ( O h ) have significant hepatitis but not cirrhosis. The absence of cirrhosis in these animals may be related to an intrinsic 70 100 Humans3 difference in the liver of rodents and primates. Cirrhosis Ground squirrel (8,200-a) 10 60 and fibrosis are common responses to chronic liver injury 10-15 Woodchuck (6,7,199,200) 10 in primates and not in rodents. In contrast to humans, the Duck (200-C) ? > prevalence of the carrier state and incidence of HCC in Table 3 Approximate incidence of HCC in different hepadnavirus infected hosts
'
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632
Development of HCC in cirrhotic liver and the presence of virus in tumours Hepadnaviruses have been found in the cells of many, although not all, HCC of hepadnavirus infected hosts. Hepadnavirus DNA has been detected in approximately 85% of HCC but not in the remaining 15% of surface antigen carrier humans,'3'15~206-208woodchuck^^^^ and ground squirrels (P. L. Marion & W. S. Robinson unpubl. data). Thus persistence of viral DNA in tumour cells, as detected by Southern blot analysis, does not appear to be essential for the development of HCC in hepadnavirus infected liver. Episomal viral DNA forms have been observed in only a small fraction of HCC of HBV infected patients and integrated viral DNA can be detected in most (75-85%) although not all HCC.'33'5,206-208 In humans, 60-90% of patients with HCC have underlying cirrhosi~.',~,'~','~~-~~~ In such cirrhotic liver, HCC arise in adenomatous foci that form within regenerative nodules of liver cells. 196,209 Examination of individual cirrhotic nodules of HBV infected cirrhotic livers has revealed that the DNA of most nodules of most cirrhotic Other nodules livers does not contain integrated contain a clonal pattern of one or more viral DNA integrations (i.e. viral integrations in the same cellular DNA sites in many cells of the nodule). Such nodules are undoubtedly formed by proliferation and clonal expansion of an original hepatocyte that contained one or more viral integrations. This suggests that the integration event(s) may have imparted a growth advantage to the original cell containing the integrant(s) and this resulted in clonal expansion of that cell. Other nodules contain a non-clonal pattern of viral integration (many integrations at different cellular DNA sites).'58 Thus the state of HBV-DNA is not the same in all regenerative nodules of HBV-infected cirrhotic liver. Of the approximately 85% of HCC in HBV-infected livers that have integrated HBV-DNA in a clonal integration pattern, most have multiple viral integrants and only a few have single integrants.l 1-15,157,158,206-208 I n most HBV-infected cirrhotic livers, only a small minority of cirrhotic nodules contain multiple clonal integrant~.'~'This suggests that HCC arise preferentially (but not exclusively) from cells of cirrhotic nodules with multiple HBV integrations. It follows, therefore, that the viral integrations could play a role in the development of HCC. As described in Part 11, the role played by viral integrations in the development of HCC remains unclear in most cases. T h e fact that each integration studied has been found at a different cellular DNA site and on any of several different chromosomes in different human tumours would appear to rule out a viral integration-site-specific mechanism for most human HCC. Multiple tumours in the same liver usually have the same clonal integration pattern in each tumour nodule indicating that they are metastases arising from the same original tumour. Occasionally, however, integration sites can be different in different HCC tumours within the same liver.'58 This demonstrates that HCC can be multicentric in origin in the cirrhotic liver. Immunofluorescent and immunoperoxidase staining of HCC tissue has demonstrated that in patients with
HBsAg in the blood and in whom non-tumorous liver cells are positive for HBsAg and/or HBcAg, tumour cells appear most often to be negative. Conversely, other studies have reported small numbers of HBsAg-positive cells in turn our^.'^' HBcAg has been detected even more rarely. Thus few tumour cells appear to express either viral gene product in amounts that can be detected by immunofluorescent staining, while cells within the same tumour do not uniformly express these antigens. These findings, as well as the failure to detect episomal forms of viral DNA, support the concept that HCC cells are non-permissive for expression of these viral structural genes or for virus replication.
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199. STEINER P. E. Cancer of the liver and cirrhosis in primed RNA templates. Nucleic Acids Res. 1985; 13: trans-Saharan Africa and the b nited States of America. 4557-76. Cancer 1960; 13: 1085. 189. Hu W-S. & TEMIN H. M. Retroviral recombination and 200. KEWM. C. & POPPERH. Relationship between hepatocelreverse transcription. Science 1990; 250: 1227-33. M a r carcinoma and cirrhosis. Sem. Liver Dis. 1985; 4: 190. DELONG S. The relationship of hepatitis B virus infection 130. to hepatoma: pros and cons. In: Szmuness W., Alter 201. GERIN J. L. Experimental WHV infection of woodchucks: H. T., Maynard J. E., eds, Viral Hepatitis. Franklin an animal model of hepadnavirus-induced liver cancer. Institute Press, Philadelphia, 1981; 253-9. Gastroenterol. Jpn. 1990; 25 (Suppl.): 38-42. 191. BEASLEY R. P. Hepatitis B virus, the major etiology of 202. KORBA B. E., WELLSF. V., BALDWIN B. ef al. Hepatocelluhepatocellular carcinoma. Cancer 1988; 61: 1942-56. lar carcinoma in woodchuck hepatitis virus-infected 192. IJIMA T., SAITOH N., NOROTUMO K., NAMRU M. & SAKUMA woodchucks: presence of viral DNA in tumor tissue from chronic carriers and animals serologically recovered from K. A prospective cohort study of hepatitis B surface acute infections. Hepatology 1989; 9: 461-70. antigen carriers in a working population. Gann. 1984; 75: 203. ZHOUY. Z. A virus possibly associated with hepatitis and 571-3. hepatoma in ducks. Shanghai Med. J. 1980; 3: 641-4. 193. YEH F., Yu M. C., Mo C-C., Luo S., TONG M. J. & 204. OMATAM. Liver diseases associated with hepadnavirus HENDERSON B. E. Hepatitis B virus aflatoxins, and hepatoinfection. A study in duck model. Cancer Defect. Prev. cellular carcinoma in southern Guangxi China. Cancer 1989; 14: 231-3. Res. 1989, 49: 2506-9. 205. SEEGER C., BALDWIN B., HORNBCCKI.F. W. E. et al. Wood194. MCMAHONB. J., LANIER A., WAINWRIGHT R. B. & KILchuck hepatitis virus is a more efficient oncogenic agent K E N N Y J. J. Hepatocellular carcinoma in Alaska Eskimos: than ground squirrel hepatitis virus in a common host. J . epidemiology, clinical features and early detection. Prog. Virol. 1991; 65: 1673-8. Liver Dis. 1990; 9: 643-55. C., HADCHOUEI. M., SCOTTOJ. et al. State of 206. BRECHOT D., VIOI.AKIM., SPARROS L. & XIROUCHAKI 195. TRICHOPOLT.OS hepatitis B virus DNA in hepatocytes of patients with E. Epidemiology of hepatitis B and primary hepatic hepatitis B surface antigen-positive and -negative liver disease. Proc. Natl Acad. Sci. USA 1981; 78: 3906-10. carcinoma. Lancer 1975 ii: 1038. 207. BRECHOT, C., POURCEL. C., LOUISE A,, RAINB. & TIOI.I.AIS 196. PETERSR. L. Pathology of hepatocellular carcinoma. In: P. Detection of hepatitis B virus DNA sequences in Okuda K., Peters R. L., eds, Hepatocellular carcinoma. human hepatocellular carcinoma in an integrated form. John Wiley & Sons, New York, 1975; 107-68. Prog. Med Virol. 1981; 27: 99-102. 197. KEWM. D. 1978. Hepatoma and HBV. In: Vyas G. M., 208. SHAFRITZ D. A. & KEW M. Identification of integrated Cohen S. N., Schmid R., eds, Viral Hepatitis: A Contemhepatitis B virus DNA sequences in human hepatocelluporary Assessment of Etiology, Epidemiology, Pathogenesis lac carcinoma. Hepatology 1981; 1: 1-8. and Prevention. Franklin Institute Press, Philadelphia, M., KAGEM., SUCIHARA S. et al. Emergence of 209. ARAKAWA 1979; 439. malignant lesions within an adenomatous hyperplastic 198. LAROLZE B., LONDONW. T., SAIMOTC. et al. Host nodule in a cirrhotic liver. Observations in five cases. responses to hepatitis-B infection in patients with primary Gastroenterology 1986; 91: 198-208. hepatic carcinoma and their families: A case-control References 210-365 will appear in Part I1 of this article which study in Senegal, West Africa. Lancet 1976; ii: 534. will be published in vol. 8 no. 1 of the Journal.
REVIEW ARTICLE
The role of hepatitis B virus in the development of primary hepatocellular carcinoma: Part I W I L L I A M S . ROBINSON
Stanford University School of M.dicine, Stanford, California, USA Abstract Chronic infections with hepatitis B virus (HBV) of humans and animal hepadnavirus infections in their natural hosts are strongly associated with primary hepatocellular carcinoma (HCC). Although viral integrations are found in cells of many HCC, no general viral-specific hepatocarcinogenic mechanism for hepadnaviruses has been identified. In approximately one half of HCC in woodchuck hepatitis virus (WHV) infected woodchucks, viral integrations near the c-myc or N-myc genes have been reported which result in enhanced expression of the respective gene. Such host gene-specific insertional mutagenesis has not been found in HCC of other hepadnavirus infected hosts. Thus in humans, ground squirrels and ducks hepadnaviral integrations appear to be at different host chromosomal DNA sites in each HCC and few integrations have been found within or near any cellular gene. Other possible hepadnavirus-specific carcinogenic mechanisms that are being investigated include transactivation of cellular gene expression by an hepadnavirus gene product (e.g. the X-gene), and mutation of host genes by unknown hepadnavirusspecific mechanisms. It should be noted, however, that chronic hepadnavirus infection is associated with chronic necroinflammatory liver disease with hepatocellular necrosis and regeneration (sometimes leading to cirrhosis in humans), a pathological process that is common to numerous other risk factors for HCC. This suggests the possibility that this pathological process is hepatocarcinogenic irrespective of the inciting agent and the role of hepadnavirus infection is no different from that of other risk factors in causing chronic necroinflammatory liver disease.
Key words: hepadnaviruses, hepatitis B virus, hepatocellular carcinoma, hepatoma, insertional mutagenesis, oncogenes, primary liver cancer, viral DNA, viral integration, virus replication.
INTRODUCTION Primary hepatocellular carcinoma (HCC) is one of the most common cancers occurring in humans and there is strong epidemiological evidence suggesting that persistent hepatitis B virus (HBV) infection is the most important risk factor for its development. Hepatitis B virus infections commonly occur at an early age and become persistent in populations in which this virus is highly endemic such as in eastern Asia and sub-Saharan Africa.' Such persistent HBV infections may be associated with chronic hepatitis. Macronodular cirrhosis develops in a significant fraction of persistently infected individuals, usually after many years.',' Long standing persistent HBV infection is associated with a greatly increased risk of HCC compared with the risk in uninfected population^,'^^-^ and the presence of macronodular cirrhosis in HBV infected individuals appears to further increase the risk of HCC by at least l O - f ~ l dThe . ~ strong association of HCC with persistent HBV infection in humans3-' and with other natural hepadnavirus infec-
tions in woodch~cks,6.~ ground squirrels' and duck^^-'^ suggests an important role for infection with members of this virus family in the development of HCC. T h e finding of a clonal pattern of hepadnavirus DNA integration in cellular DNA of many HCC in and in other hosts naturally infected with hepadnaviruses indicates that these tumours arise from a single cell with integrated v i r ~ s . ' - ' ~ ~ 'This ~ ~ ' finding has stimulated interest in whether viral integration plays a role in HCC. Although the most important risk factor for HCC in humans appears to be chronic or persistent HBV infection, several other specific factors appear to increase the risk of developing liver cancer and almost all HCC cases appear to be associated with HBV or one of the other recognized risk factors. It has been estimated that 80% of all HCC in the world occur in HBV infected individuals" and careful prospective studies have indicated that 40% or more of middle aged Chinese males with chronic HBV infection die of HCC.3 Since chronic infection can occur in 10% or more of the population in high prevalence area^,"^ the number of liver cancers in those populations
Correspondence: W. S. Robinson, Stanford University School of Medicine, Stanford, California, USA. Accepted for publication 11 May 1992. Editor's note: This is the first of a two part article. Part I1 will appear in the next issue (vol. 8, no. 1) of the Journal.
HBV in the development of carcinoma is very great. Among all cancers of humans associated with recognized environmental risk factors, only the number of cigarette smoking-associated lung cancers appears to exceed HBV-associated HCC. Important risk factors associated with the remaining 20% of HCC cases are certain factors that cause chronic necroinflammatory liver disease and cirrhosis including chronic alcohol-induced liver disease,19720 chronic hepatitis C,21-28 h a e m o c h r ~ m a t o s i s , ~ ~a*1-antitrypsin ~~ defi~iency,~'cryptogenic cirrhosis,32 primary biliary cirrhosis,33 hereditary tyrosinaemia,20and other causes of cirrhosis; and exposure to certain chemical carcinogens such as dietary aflatoxin (toxins produced by fungal organisms which infest improperly stored grain, peanuts and other food),34-36androgenic-anabolic steroids37 and oral contraceptive s t e r o i d ~ , ~ ~undefined -~' substances (possibly pe~ticides)~' in polluted ground water used for drinking in parts of China4'-43 and possibly other chemical carcinogens (Table 1). There is an uneven geographic distribution of HCC in the world because the most important risk factors for HCC are unevenly distributed.' The highest prevalence is in Eastern Asia (China and adjacent regions) and sub-Saharan Africa and these are areas of the world with the highest prevalence of chronic HBV infection.' The prevalence of HCC is much lower in the United States and Western Europe where HBV infections are much less common and more important risk factors for HCC are alcoholic liver disease and probably chronic hepatitis C. Recent research has provided much information about the molecular and genetic structure of HBV, its molecular mechanism of replication, its phylogenetic relationship Table 1 Established and possible risk factors for HCC in humans Associated with chronic necroinflammatory liver disease and/or cirrhosis. HBV (and other hepadnaviruses in their natural hosts) I .3.4,6-Y964
HC-21-28
Alcohol 19.20.3 19.33 1.332 Cryptogenic c i r r h o ~ i s ? ~ ~ . ~ ~ ~ Primary biliary cirrhosis?33 Autoimmune CAH?334q335 Membranous obstruction of inferior vena cava?336-337 a-1-antitrypsin d e f i c i e n ~ y ? ~ ~ ? ~ ~ * - ~ ~ ~ Hemochromatosis?2Y~30 Wilson's disease?3447345 Glycogen storage d i ~ e a s e ? ~ ~ ~ . ~ ~ ' Hereditary t y r ~ s i n a n e m i a ? ~ ~ . ~ ~ ~ ~ ~ ~ ' Parasitic infestations of ~ i v e r ? ~ ~ ~ - ~ ~ ~ Methotrexate the rap^?^^^,'^^ Other causes of cirrhosis? Without necroinflammatory liver disease Aflatoxin B1?34-36.358 Polluted drinking water in China?4'-43 Contraceptive and anabolic Cigarette s m o k i x ~ g ? ~ ~ ' - ~ ~ ~ Other chemical carcinogens? ~
? indicates a clinical association with HCC without definitive epidemiologic evidence for an independent causal role.
623 with other viruses and its molecular state in cells of HCC. This information suggests possible mechanisms by which HBV infections can lead to HCC. This review will consider the role of HBV in the development of HCC by reviewing important features of the virus, describing evidence that HBV infection plays an important role in HCC, and considering possible carcinogenic mechanisms for the virus.
Hepatitis B and related viruses (hepadnaviruses) Hepatitis B surface antigen (HBsAg) was discovered serendipitously in human serum in the mid 1 9 6 0 It~ ~ ~ was later associated with acute serum he pa ti ti^^^-^' and eventually was identified as a viral (HBV) antigen. This led to rapid advances in understanding the epidemiology of the virus, the course of infection and recognition of associated diseases, and the physical nature of the virus. Early during this period HBV infection was recognized to be associated with HCC.48
Biological and epidemiological features of HBV Many primary HBV infections were shown to be asymptomatic, infections could persist for many years, the virus was found to be hepatotropic (i.e. to infect primarily liver cells) although less frequent and less permissive infection sometimes occurs in bone marrow, circulating leucocytes, B cells and T cells, and p a n c r e a ~ . ~ Virtually ~-~' all active infections were accompanied by high concentrations of HBsAg and often infectious virus in the blood. Persistent infections were often associated with minimal liver disease or with chronic hepatitis that sometimes led to cirrhosis.2 The risk of HCC was shown to be more than 100 times higher in some 'HBsAg carrier' populations than in similar uninfected population^.^
Hepadnavirus family The discovery of several closely related viruses with similar epidemiological features and associated with similar disease syndromes including HCC occurring naturally in certain rodent and avian species indicates that there may be many members of this virus family which has been named Hepadnaviridae for hepatotropic DNA v i r ~ s e s . ~ The ' - ~ ~hepadnavirus family includes hepatitis B virus of humans, woodchuck hepatitis virus (WHV) of Marmata rn0nax,6~ ground squirrel hepatitis virus (GSHV) of Spermophilus bee~heyi,6~ duck hepatitis B virus (DHBV) found in several varieties of domestic ducks (Summers, London, Sun, Blumberg et al. unpubl. data)49@' and similar viruses in tree ~quirrels,6~ and herons6' Less well documented findings in other rodents, marsupials and cats suggest that other hepadnaviruses may exist. A characteristic feature of all hepadnaviruses is a moderately narrow host range. For example HBV infects only humans, chimpanzees and possibly other great apes but not monkeys or other species. All hepadnaviruses exhibit strong relative tropism for h e p a t o c y t e ~ ~and ~,~~*~~ production by infected hepatocytes of large amounts of
W.S. Robinson
624
non-infectious viral envelope particles (as well as infectious virus) that can be detected in high concentrations (up to 500 pg/mL) in the blood,"9,64,65,69-71and the common occurrence of persistent infections with viral forms in liver and blood continuously for years and often for the lifetime of the host. Infections with hepadnaviruses may be associated with acute and chronic hepatitis characterized by hepatocyte necrosis, inflammatory reaction, lymphocytic infiltration and liver regeneration;'* immune com lex (viral surface antigen-antibody) mediated d i ~ e a s e ;74 ~ and HCC.',3-'0364 T he mechanism of liver injury in acute and chronic hepatitis associated with hepadnavirus infection is not completely defined but a cellular immune mechanism is considered to be likely. Some evidence indicates that cytoxic T cells directed at the viral core antigen (HBcAg) displayed on the surface of hepatocytes can lead to cell Other studies have suggested that HBcAg expression in infected cells may have a direct cytopathic effect.77 Another Eonimmune mechanism involving accumulation of viral envelope polypeptides in liver cells and resulting liver cell necrosis has been demonstrated in transgenic mice78s7yand, in theory, could play some role in in wiwo hepadnavirus infections. Hepadnaviruses share unique features of virion (virus particle) size and ultrastructure with an envelope surrounding an electron dense spherical nucleocapsid or characteristic polypeptide and antigenic composition;80 common virion DNA size, structure,81i82and genetic 0rganization;8~the presence of DNA polymerase activity in the virion;84i85and an unusual mechanism of viral DNA replication which includes reverse transcription of a greater than genome length viral RNA transcript.86-88
Y,
.
Viral genome structure The virion DNA of these viruses is among the smallest of all known animal viruses consisting of an approximately 3200 base pair (bp) circular molecule that contains a single stranded region of different length in different molecules81782 (Fig. 1) reflecting the fact that DNA molecules are packaged into virions before viral DNA replication (i.e. synthesis of the DNA plus strand) is complete. The short DNA strand of variable length is the second synthesized plus strand and the full length strand is the first synthesized minus strand. Neither viral DNA strand is a covalently closed circle and single breaks occur in the two strands at unique positions in the nucleotide sequence approximately 225 nucleotide pairs apart in mammalian h e p a d n a v i r u ~ e s(Fig. ~ ~ ~1). ~ ~The full length DNA minus strand is longer than the plus strand due to a nine nucleotide terminal redundancy. The circular conformation of the DNA is maintained by non-covalent base pairing of the cohesive overlapping 5' ends of the two DNA strands. 'The virion contains DNA polymerase a c t i ~ i t y ~that ~ . ~catalyses ' the repair of the single stranded region to make fully double stranded relaxed circular DNA molecules81~82 and this enzyme appears to be the reverse transcriptase involved in viral DNA replication in infected liver The complete nucleotide sequences of the cloned ~ - ~ ' DHBV isoDNA of 13 HBV i ~ o l a t e s , 8 ~ ' ~two
2458
833
Figure 1 Circular map of the HBV (3dw2) genome. The inner circles represent the virion DNA strands and the broken (dashed) line in the short ( + ) DNA strand represents the region within which the 3' end of the + strand may occur in different molecules, and the corresponding region of the long strand is that which .3ay be single-stranded in different molecules. A line of dots represents the oligoribonucleotide primer covalently attached to the 5' end of the +DNA strand and a single dot represents the protein primer covalently attached to the 5' end of the - DNA strand. The large arrows represent the recognized functional open reading frames (ORF) with the direction of transcription from the minus DNA strand indicated. The small arrows indicate the 5'-ends of the three major size classes of transcripts of 3.4, 2.4 and 2.1 Kb, all of which are identically terminated near the poly A addition signal (TATAAA). The nucleotide sequence locations of the initiation and termination codons of each ORF are given with reference to map position 1 at the single EcoRI cleavage site in this DNA. The map position of the first nucleotide of the glucocorticoid responsive element (GRE), DR2, DRI, the US-like sequence and the poly A addition sequence (TATAAA) are indicated, as is the general regions exhibiting enhancer 1 (ENH 1) and enhancer 2 (ENH 2) activity.
lates,lOO~lO1 and two of WHV,'02*'03one each of GSHVIo4 and the heron virus68 have been reported, The genomes of the three mammalian hepadnaviruses have four long open reading frames in the complete or long (minus) virion DNA strand and these have similar locations in each virus with respect to the cohesive ends of the DNA (Fig. 1). T h e genes for the major virion polypeptides have been identified. All of the genome appears to be translated and overlapping genes are translated in different reading frames. Evidence suggests that none of these genes is interrupted by intervening sequences. T h e C gene specifies the major viral core or nucleocapsid polypeptide of 21 000 daltons and a truncated 16 000 dalton form of this polypeptide with HBeAg specificity. This open reading frame includes a short preC (precore) sequence delineated by an in-frame initiation codon. The
HBV in the development of carcinoma
S gene, including preS,, preS,, and S regions delineated by three in-frame initiation codons, specifies the viral surface antigen reactive polypeptides in the virion envelope and in incomplete viral forms (surface antigen particles) found in serum and liver of infected individuals. These consist of the glycosylated and non-glycosylated forms of three polypeptides of 24 000, 33 000 and 39 000 daltons coded respectively by S alone, by preS, and S , and by preSl, preS, and S regions of the S open reading frame. The P gene which covers three-fourths of the genome and overlaps a carboxyl-terminal portion of the C-gene, the entire S-gene and an amino-terminal portion of the X-gene can specify a basic polypeptide of approximately 90 000 daltons and e v i d e n ~ e ~ ~ suggests * ’ ~ ~ - that ’~~ this contains the virion associated DNA polymerase (or reverse transcriptase) activity, ribonuclease H activity and a protein primer for minus DNA strand ~ynthesis.”~ The small X-gene appears to specify a polypeptide of 154 amino acids or more in some viruses. It was first shown to activate transcription regulated by regulatory elements of the P-interferon gene by Twu and S c h l ~ e m e r and ’ ~ ~ it is now known to activate transcription of HBV”’*”’ as well as several heterologous viral”’-”5 and cellular genes.109,1 16-118 T h e codon preference of the X-gene is more like that of typical cellular genes than viral genes suggesting it may be of cellular origin.lo6 The C, P, preS, and S open reading frames are approximately the same relative size and occupy the same relative genomic positions in all mammalian hepadnaviruses. The preS, region varies in size in different viruses suggesting that the polypeptide sequence specified by this DNA sequence is less functionally critical than that specified by the preS, and S regions. The preC sequence similarly varies in size in different sequenced HBV genomes and the C gene of the avian hepadnaviruses is significantly larger than in the mammalian viruses. The size of the X-gene varies by up to 50% among nine different HBV isolates (largest in HBV adr subtype genomes) and the X open reading frame is not present in avian hepadnavirus genomes. Thus avian hepadnaviruses contain only three open reading frames (S, C and P). Other functionally important elements in hepadnavirus genomes include 11 bp direct repeat sequences designated DRl and DR2 (Fig. 1) which are approximately 225 bp apart in the mammalian viruses. They appear to play a role in viral DNA replication.87q88 This sequence is not completely conserved but exhibits significant variation in different viruses.”’ T h e 5’ end of the minus DNA strand occurs within DR1 and the 5’ end of the plus DNA strand at the 3’ boundary of DR2.87,i20 No consensus TATAA elements have been detected within nucleotide distances of the known hepadnavirus RNA initiation sites expected for usual eukaryotic promoters. However, studies of transcription and RNA mapping in infected cells in culture, in liver tissue and in cell free systems, and use of isolated hepadnavirus DNA fragments have provided preliminary evidence for the existence and general locations of four promoter elements. One (called the preS, promoter) upstream from the start of the preS, sequence (Fig. 1)”’ may initiate synthesis of a 2.4 kb messenger RNA (mRNA) for a protein containing preS,, preS, and S sequences. Another
625 (called the preS, promoter) within the preS, appears to direct synthesis of a 2.1 kb RNA transcript that probably functions as messenger for preS, and S gene specified polypeptides. Both are within the coding sequence for the P gene and the action of neither appears to be cell or tissue specific. A sequence upstream from the start of the C-gene and within the coding region of the X-gene appears to direct synthesis of a greater than genome length (3.5 kb) RNA transcript which may function as messenger for the synthesis of C and P-gene specified polypeptide^.'^^-'^^ This RNA also appears to serve as a template for synthesis of the virus minus DNA strand by reverse transcription. This promoter (called the C-promoter) appears to exhibit some specificity for liver cells.’27 A fourth promoter element has been described upstream from the X-gene and within the P open reading frame’” and this promoter (called the X-promoter) appears to direct synthesis of minor transcripts which function as X-gene messengers. A transcriptional enhancer element (enhancer 1) has been localized to a region approximately 450 bp upstream from the C-gene promoter (Fig. It is either immediately upstream of the start of the X-gene in mammalian viruses with a short (typical) X-gene or within the 5’ end of that open reading frame in viruses with a long X-gene (e.g. in HBV subtype adr) and within the P open reading frame in all viruses. Functional analysis has suggested liver cell specificity for this enhancer.’” The C - p r ~ m o t e r ’ ~ ~ .and ’ ~ ’ other HBV promoter^'^' under control of the HBV enhancer 1 (and not other enhancers) have been shown to be much more active in differentiated liver than other cell types. In nuclear extracts of human liver cells, several proteins have been shown (by DNA footprint analysis) to bind specific sequences within HBV-DNA fragments that contain functional enhancer activity. These include the genomic region of approximately 300 bp which contains overlapping binding sites for multiple cellular transcription factors, including NF-1,’33*’34 C/EBP,135-’36 AP-l,i37 CREB’37 and ATF.138’139 A second cis-acting transcription regulatory element has been described within an approximately 100 bp sequence located in the downstream half of the X open reading frame. This is within or just upstream of the core promoter (Fig. 1); it also has properties of a strong liver cell specific enhancer (enhancer 2).’40-’43 Another hepadnavirus genomic region (within the S coding region) is necessary for a five-fold enhancement of gene expression in the presence of glucocorticoids.’44 This glucocorticoid responsive enhancer-like element appears to be independent of orientation in the genome and without apparent species or cell type specificity. An 18 nucleotide sequence in HBV similar to known glucocorticoid responsive DNA elements was identified in that region (approximately 2097-21 12 on the map in Fig. 1). Glucocorticoids increase levels of HBsAg expression in HBV infected patients in v i ’ u ~ , ’in~ ~HCC cell lines expressing HBsAg in culture’46 and in transgenic mice.I4’ This effect might in part explain some of the differences in the behaviour of HBV in males and females. A polyadenylation signal that appears to be used by all major transcripts lies within the beginning of the C-gene,
626 approximately 20 bp upstream from the 3’ end of the minus DNA strand and just downstream of a TATAAA (AATAAA in DHBV) Sequences downstream from the TATAAA sequence also appear to play a role in correct polyadenylation of HBV transcript^.'^^ Finally, a highly conserved 60-70 bp sequence with a high degree of homology with the U5 sequence of the LTR of certain retroviruses is present just downstream from DR1 and within the preC region of the genome.Io6 T h e function of this sequence is unknown. The hepadnavirus genome is unusually compact and efficiently organized in that much of the genome is used for multiple functions including overlapping genes so that the same nucleotide sequence encodes more than one protein and all cis-acting regulatory sequences (e.g. transcriptional enhancer and promoter elements) are contained in genomic sequences also encoding protein (Fig. 1).
Viral genome replication
W. S. Robinson General features of hepadnavirus integrations have been identified by comparing the structure of viral and flanking cellular DNA of many published examples (almost all being HBV integrants in human HCC).’51,152,157 H epatocellular carcinomas containing integrated virus sometimes have a single clonal integrant but more often there are multiple clonal integrants each at a different cellular DNA site (most often three to four T hese would appear to but rarely 10 or arise from separate viral integration events. The apparent cellular DNA sites of integration have been different in every human HCC studied. Most HCC with integrated virus do not contain replicating viral DNA forms, which suggests that cells of such HCC are non-permissive for virus replication by an unknown mechanism. Some integrants consist of simple contiguous linear sequences of HBV-DNA without rearrangement and others are complex and may have arisen by integration of multiple viral genomes at one site or rearrangement after integration by recombination between viral sequences of different i n t e g r a n t ~ . ’The ~ ~ complex integrants contain one or virus-virus junctions. Complete viral genOmeShave not been found in any integrants and deletion of viral sequence has been found in all that have been sequenced whether they arose from single or mul54,157,160 The long terminally tiple genome redundant HBV transcript that as a template for viral genomic DNA synthesis be synthesized from such viral integrations and virtually all integrants are defective for virus replication. Thus hepadnavirus integrants are not involved in virus replication as is the integrated DNA provirus of retroviruses. be preferred sites for integration There appear within the viral genome. More than 50y0 of viral integrants in H C C ~ 5 0 - ~ 5 2 . ~ 5 7 , ~ 6 1 and non-HCC liver
The first step in viral genome replication involves conversion of infecting virion DNA molecules into covalently closed circular molecules, this takes place in liver cell It is by formation Of a greater than genome length RNA transcript with a terminally repeated sequence as well as shorter transcripts which function as messenger RNA. The long transcript, made transcriptase and protein primer are packaged in ‘Ore particles that are found in hepatocyte ‘ytoplasm. Synthesis of new viral DNA molecules is accomplished by reverse transcription of the long RNA transcript using a viral encoded protein primer to form the first synthesized viral DNA (minus) strand and a capped oligoribonucleotide derived from the 5’ end of the long RNA template as primer for synthesis ofthe second t i s s U e ~ ~ 4 , 1 5 5 appear to have one viral DNA end which DNA s y n t h e s i ~ ~ ~ ~ joins ~:~ DNA (Plus) ’Wand (Fig’ 2)’ cell DNA (or viral DNA in the case of virus-virus exclusively within cytoplasmic viral core particles DNA junctions) near or between the cohesive 5’ ends A nine nucleotide terminal redundancy in the minus which are within the direct repeat (DR) sequences of viral strand is thought to be involved in template switching for DNA (Fig. l). Those with ends near the 5 , end of the synthesis of the second (plus) DNA strand.87 Further minus DNA strand (and DRI) exceed those near the 5‘ details of hepadnavirus replication have been described end of the plus DNA strand (and DR2) by approximately el~ewhere.~~.’~ 3 : 1 (Table 2; Fig. 3). T h e most frequent site for a viral end is near the left end (first nucleotide) of DR1. No Hepadnavirus integration integrant with an intact cohesive end region has been Hepadnavirus integration in cellular DNA has been found. In simple linear contiguous viral sequence intefound in infected liver as well as HCC. In hepadnavirus grants, when one end of the viral DNA joining cellular infected hosts many HCC contain viral integrations with DNA is within or near a DR sequence, the other DR a clonal pattern. It follows that these tumours must have sequence is not present. arisen by clonal expansion of an original cell with that T h e other end of viral DNA which joins cell DNA (or viral integration. The possible role of such integrations in viral DNA in the case of virus-virus junctions) in integrants joining cell DNA near a DR sequence at the development of HCC has been intensively investigated. Much of the evidence of the fine structure of one end, occurs at variable positions in the viral hepadnavirus integrations comes from investigation of g e n ~ m e . ’ ~ ’ > This ’ ~ ~ *suggests ’ ~ ~ random recombination those in h ~ m a n ’ ~ ~and - ’ ~woodchuck * HCC;’53there has with cell DNA, although there may be a slight preference been much less investigation of viral integrations in for the preS region as a viral site of integrati~n.’~’ non-tumorous l i ~ e r . ” ~ -It’ ~is~ not known whether These findings suggest that the cohesive end region of viral integration occurs in every hepadnavirus infected viral DNA is involved in the mechanism of viral DNA cell in viva or only in Some cells. T o date, however, no integration into cellular DNA. This implies that replicaapparent difference in the structure of viral integrations of tion intermediates (probably linear molecules) with single HCC and non-tumorous liver have been identified stranded DNA at the cohesive end region rather than closed circular viral DNA are substrates for integration (Table 2).
627
HBV iv the development of carcinoma Transcription
Form I DNA free in cell nucleus
DR 1
-
R DNARNA 51.. +
J.
.I.. ................/!.3'
*
DNA synthesis , in immature cores in cell cytoplasm DR2
DR1
I
I
r
r
Extracellular virions
'
.... RNA
-
DR2
DNA Protein primer for -DNA strand synthesis DR-l,DR-2 = 12 nucleotide direct repeat sequences R = 200 nucleotide terminal redundancy in long RNA transcript r = Terminal redundancy in -DNA strand Figure 2 Scheme of proposed mechanism of hepadnavirus DNA replication. DK-1 and DR-2 represent 11 nucleotide pair direct repeat sequences. R represents the approximately 200 nucleotide terminal redundancy in the long KNA transcript and r represents the short terminal redundancy of the -DNA strand. Solid lines represent DNA strands, dotted lines KNA and the stippled area the protein primer for DNA strand synthesis.
(Fig. 2). It also suggests that viral integrations occur at a stage of infection when virus replication is proceeding and appropriate replication intermediates are present. The common structural features of viral integrants suggest that the substrates for integration are replication
intermediates with DNA strands of variable length, all of which would have the same 5' end (near a DR sequence) and 3' ends at variable positions along the viral genome (Figs 2, 3). Of the many integrants that contain viral ends near D R l , most contain viral sequences to the left ofDR1
628
W.S. Robinson
Table 2 Hepadnavirus integration
Configuration of viral integrants
Tissue source of integrant (# positives/# integrants
examined)
One end of viral DNA insert which joins cell DNA DNA is near or between viral DNA cohesive ends (i.e. near a DR sequence) Shih integration patterns (150) I (DR1) I1 (DRl) 111
(DR2)
IV
(DR2)
Neither end of viral DNA insert which joins cell DNA is near or between viralcohesive ends Viral insert contains complete or near complete X-gene: Viral insert contains no X gene sequence
One end of viral DNA at virus-virus DNA junction is near or between viral DNA cohesive ends Viral insert contains truncated preS,/S sequence
HCC
Non-HCC liver
30145 8/45 14/45 5/45 3/45
4/4* 014 314 1/4* 014
17/45
014
6/17 7/17
-
11/45
114
5/45
114
Percentage of 30 HCC integrants with one end near a DR sequence
26.6 46.7 16.6 10.1
Fraction and end of X-gene contained in integrants
< 10% of < 90% of z 50% of z 50% of
3’ 5’ 5’ 3‘
end end end
end
-
*One WHV integration in WHV infected woodchuck liver (153) has integration pattern 111; 3 HBV integrations in HBV infected human liver have integration pattern 11. Integration of HCC reported in ref. 149,150,152 and 153; and integrants of non-HCC liver in ref. 152-1 54.
(integration pattern 11). This is consistent with minus strand DNA invasion (Table 2, Figs 2, 3). Fewer integrants contain viral ends near DR2 and most of those have viral sequences to the right of DR2 (integration pattern III), consistent with plus strand DNA invasion. An alternative viral DNA substrate for integration has been suggested r e ~ e n t l y . ’ ~ *Preferred .’~~ topoisomerase I cleavage sites were identified in WHV-DNA near and between DR1 and DR2 (including the 9 bp terminally redundant region of the viral minus-strand DNA). It was proposed that cleavage of these sites by topoisomerase I could lead to integration of the resulting linear viral DNA molecules by illegitimate recombination. 162,163 Small regions of DNA homology near crossover points appear to be involved in partially homologous recomb i n a t i ~ n . ‘Moderate ~~ sequence homology between the ends of HBV integrants and flanking cellular DNA have been noted.’52,’61,’65-167 T he degree of homology of these regions is not as great, however, and crossover points do not coincide as precisely as do the homologous regions found in systems of homologous recombination. 164 Almost all data on viral sites that join cell DNA in integrations come from integrations in HCC. It is possible, therefore, that the cohesive region represents a biased selection of sites that are special for HCC. However, the small number of integrants that have been examined from infected non-HCC liver appear to confirm that a common viral site that joins cellular DNA in
integrants of non-HCC liver is also in the cohesive end region (Table 2). Several models of hepadnavirus integration mechanism based on known features of viral integrants have been proposed. 150-152.1 57,162,163,165,168 While there are preferred sites for integration within the hepadnavirus genome (e.g. the cohesive end region), viral DNA appears to be integrated at many different sites of cellular DNA. Whether there may be somewhat preferred cellular sites of HBV integration as appears to be the case for some retroviruses is not yet ~ 1 e a r .There I~~ may be some preference for HBV integration within repeating sequences such as Alu DNA,’” satellite 111 DNA,’51,’70 a-satellite DNA,’ 55 and minisatellite or VNTR DNA.’52,’55,’67,’68,’70 One half of WHV integrations in woodchuck HCC appear to be near either the c-myc or the N-myc gene. These cellular sites have been found only for this virus and HCC of this host; this site has not been identified for integration of other hepadnaviruses in HCC of their natural hosts, or in non-HCC liver. Whether myc genes represent true preferential cellular sites for WHV integration in woodchuck liver cells is uncertain. An alternative and more likely explanation is that liver cells with WHV integration near myc genes have been selected because such integrations impart oncogenic attributes to the cells, thereby resulting in their clonal expansion and HCC formation. Hepatitis B virus integrations have been found on at least 12 different chromosomes in HCC.’57 Five integrations assigned to chromosome 11 represent a higher
HBV in the development of carcinoma
,
1300
Virion DNA
-strand +strand
629
15r
18[6
2400
----
5’
-----
4
- - - - - - - -b
~
*# - -
p*
Integration pattern I
I I Z I 26.6%
I1 I I I I I11
IV
20,OO
-- -- -- --
IIII
DR2
46.6% 16.6% 10.0%
DR1
Figure 3 Hepadnavirus integration patterns described for integrants in references 149,150,152-1 55. T h e upper horizontal scale indicates sequence positions in the HBV genome as shown in Fig. 1, the large closed circle represents the protein bound to the 5’ end of the virion DNA minus strand, the dotted line indicates the RNA attached to the 5’ end of the virion DNA plus strand, and DKl and DR2 represent the 11 bp direct sequence repeats in the viral genome. The horizontal double lines (part solid and part dashed (indicating that one end can be at various sequence positions)) opposite the indications of integrations patterns I, 11, 111 and IV represent DNA strands of idealized viral integrants for each integration pattern. The numbers to the right of each integration pattern are the percentages of each pattern found among 30 integrants with one viral DNA end near or within a DR sequence.
frequency than expected for a random d i ~ t r i b u t i o n . ’ ~ ’ * ~ integration^.'^^ ~~ Such translocations appear to be the Because of the small number of integrations studied for result of a virus-dependent mechanism by which chromosomal aberrations can be generated. However, no comchromosomal location and virtually all such data are from HCC, it is not possible to determine whether chromomon chromosomal translocation associated with or without a viral integration and activating a protosome 11 is a preferred chromosome for integratim of oncogene as found in many human cancers has been these viruses or whether integrations in chromosome 11 found in HCC.176 are found more often in HCC because they play a (4) Inverted duplication of integrated virus and functional role in oncogenesis. It has not been excluded flanking cellular DNA is a common rearrangement that integration can occur on any or all of the chromoof host chromosomal DNA found in human somes not yet found to contain a viral integration. HCC. 151.1 55,165,166,168,174.1 77,l 78 These consist of inverted Structural alterations in host chromosomal DNA at the identical sequences of integrated viral DNA and flanking site of viral integrations are common as follows: cellular DNA. Such structures suggest that an initial (1) All HBV integrations are associated with microsimple integration had been amplified and then two deletions (e.g. 10 bp) in cellular DNA at the site of copies underwent head-to-tail recombination. Head-tointegrati~n.’~’This suggests that such microdeletions tail repeats of integrated viral DNA have also been arise as an essential part of the integration mechanism as described.157In all such integrated viral DNA rearrangeis the case for integration of some other DNA viruses by ments, the viral cohesive end region is commonly found illegitimate r e ~ 0 m b i n a t i o n . l ~ ~ at virus-cell and virus-virus junctions. (2) In sporadic HCC, larger deletions of chromosomal ( 5 ) Amplification of a region of chromosomal DNA at DNA have been described at sites of viral DNA the site of integrated HBV-DNA has been described.”’ i n t e g r a t i ~ n ; ’ ~ they ~ . ’ ~appear ~ to be formed by a different (6) Alterations in host chromosomal DNA have also mechanism than the microdeletions. been detected in HCC without virus integration or at host (3) Translocations involving cellular DNA from two DNA sites a great distance from a viral integration. These different chromosomes joined to the respective ends of a include allelic d e l e t i ~ n s and ~ ~ point ~ - ~m~ u~ t a t i ~ n s . ~ ~ ~ - ~ viral DNA sequence have been described; no two have Since these alterations are not topographically near viral involved the same chromosomal DNA region. In one integrations, it seems unlikely that hepadnavirus intehuman HCC, a translocation was noted between chromogration could play a direct role. However, since viral somes 17 and 18.174There was deletion of 1.3 kb of DNA integrations can undergo rearrangement including deleof chromosome 18 at the translocation site and occurring tions involving both viral and cellular sequences after the at the site of an HBV integration with a large inverted primary integration, it has been s u g g e ~ t e dthat ~~~~~~ repeat of viral DNA.174 These findings suggest that integrated virus could be completely deleted by such postintegration rearrangement of integrated HBV and mechanisms in some HCC. This would leave an altered cellular DNA resulted in the translocation. Other transor mutated cellular sequence without residual integrated locations involving chromosomes 5 and 9, and X and 17 viral DNA. Whether this ever occurs in infected liver (or respectively have also been found at sites of viral
W. S.Robinson
630
HCC) is unknown but such a mechanism seems unlikely to account for the frequent allelic deletions and point mutations described for HCC. There is no precedent for a mechanism by which viral integration could cause point mutations. Thus HBV can act as a non-host DNA sequencespecific insertional mutagen and cause secondary rearrangements of host chromosomal DNA. However, HCC appear to contain frequent chromosomal DNA alterations, for example, point mutations and allelic deletions, without apparent relationship to hepadnavirus integrations.
Hepadnavirus infection and HCC
Hepadnaviruses are related to retroviruses
Epidemiological evidence indicates that there is a geographic correspondence of relative rates of HBV and HCC. Although HCC is rare in most parts of the world, it occurs commonly in sub-Saharan Africa, Southeast and Eastern Asia, native populations in the Arctic, Oceania, Greece and Italy. Geographic areas with the highest incidence of HCC are also areas where HBV infection is common and persistent HBV infections occur at the highest known frequencies. Apparent unusual exceptions to this close correspondence in high prevalence regions suggest the possibility of important additional factors such as dietary aflatoxin in these p o p ~ 1 a t i o n s . lIn ~ ~ certain areas of Asia and Africa, where the prevalence of chronic HBV infection is the highest, HCC is the most common cancer in males.
Hepadnaviridae appear to be phylogenetically related to members of two other virus families: cauliflower mosaic virus and Retroviridae and related transposable elements. Similarity in gene number, function and order; shared genome nucleotide sequence homology in genomic regions of similar f u n c t i ~ n ; ’ ~and ~ ”utilization ~~ by all of a reverse transcriptase step in genome replication indicate these virus families are related.86,’x6-’88Reverse transcription is an unusual mechanism of genome replication for DNA viruses (known at this time only for hepadnaviruses and cauliflower mosaic virus). Undoubtedly it is a mechanism used by these viruses because of their evolution from a common ancestor shared with retroviruses and different from the evolutionary pathway of other DNA viruses. Notable differences in the viruses of these families include differences in the form of the genome packaged in virions (linear RNA in the case of retroviruses and nicked or gaped double stranded circular DNA in hepadnaviruses and cauliflower mosaic virus), retroviruses package two copies of the viral genome in each virion resulting in high rates of homologous recombination and hepadnaviruses one genome copy.’89 Details of the replication mechanisms are also different. Another important difference is in the mechanism of viral integration and the requirement for DNA genome integration in cellular DNA for virus replication. The orderly integration of retroviral DNA ‘provirus’ mediated by a virus encoded integrase with preservation of the genome sequence and including a terminally repeated sequence (the long terminal repeat or LTR) containing cis-acting transcriptional and other regulatory elements, and expression of viral genes and synthesis of new RNA genomes from the integrated provirus is an integral feature of retrovirus replication.Ix6 This feature is not shared by hepadnaviruses whose replication appears to involve only episomal (unintegrated) viral nucleic acid form^.^^,'^ Hepadnaviral integration appears to be a less well-controlled event. The mechanism includes illegitimate recombination without involvement of a viral integrase and without preservation of the genome sequence, thereby making it impossible for the viral integrant to function as a template for virus replication. The relationship of hepadnaviruses and retroviruses is of particular interest because many retrovirus infections in animals are associated with neoplasia or malignant tumour formation and intense investigation of these viruses has led to understanding certain viral oncogenic mechanisms.
The best evidence that chronic hepadnavirus infection plays an important role in the development of HCC is the epidemiological association in all of the well-studied hepadnavirus-host systems. This association was first suggested for chronic HBV infection and HCC in humans,48and the recognition of HCC in woodchuck^^^ and d ~ ~ kprompted s ~ ~ the , search ~ ~ for, and discovery of, hepadnaviruses in these hosts.
Geographic correspondence of HBV infections and HCC
Increased incidence of H C C in hepadnavirus-infected populations T h e incidence of HCC has been shown to be much higher in hepadnavirus-infected than uninfected w o o d c h ~ c k s , 6 .ground ~ ~ ~ ~ squirrels,’ and in some duck populations (Table 3).9 In humans, this appears to be true for HBV in both high and low incidence HCC populations.’ A prospective study of more than 22 000 male government workers in Taiwan has shown the incidence of HCC to be more than 100-fold higher in HBsAg positive than HBsAg negative individuals. 3 ~911 Prospective studies in China’93 and Alaskan natives’94 have indicated 30- to 100-fold higher risks of HCC among HBsAg carriers. The few cases of HCC in HBsAg negative patients in the large prospective study3,I8’ had serum anti-HBc indicating past HBV infection. These studies document that HBV infection precedes the development of HCC and they quantitate the risk. The incidence of HCC in HBsAg carriers in these populations rises steeply after age 40 years, while cirrhosis in HBsAg carriers increases the risk of HCC by more than 10-fold compared with noncirrhotic HBsAg carrier^.^.^.^^^,^^^-^^^ T h e risk of HCC is significantly higher in male than in female carriers,’*191 and this sex difference is greatest in the presence of cirrhosis. Some evidence suggests that most HBV infections in HCC patients occurred early in life and had continued for many years when HCC developed. The high incidence of persistent HBV infection in mothers of HCC patients, in contrast to that in fathers,”’ suggests that transmission from mothers to newborn or infant children is a frequent
HBV in the development of carcinoma
63 1
these models appears to be the same for males and females. In both the woodchuck and ground squirrel colonies, HCC developed in a few HBsAg negative, anti-HBc/anti-HBs positive animals (evidence of past Approximate infection) but in no animals without any serum viral Infection incidence of HCC marker. These studies suggest that chronic hepadnavirus Host status (cases per 100 000 x year) infection alone, without a recognizable cofactor, can result in HCC. This process appears to be the most Humans3 All HBsAg' 1158 efficient in the case of WHV-infected woodchucks. In Age 30-33, HBsAg' 251 individuals ( h ~ m a n swoodchucks"' ,~ or ground squirrels 40-49, HBsAg' 283 (P. L. Marion & W. S. Robinson unpubl. data) with 50-59, HBsAg' 2 149 evidence of past hepadnavirus infection the risk of devel60-69, HBsAg' 3 398 oping HCC is significantly higher than in individuals Cirrhosis, HBsAg' 12 500 with no viral markers but is much lower than in those HBsAg- , anti-HBc' ? with chronic infection (serum HBsAg positive). ? All markerThe association of DHBV infection and HCC in ducks is less clear. Duck HBV was first found in small brown 10 000 Ground squirrel All GSHsAg' domestic ducks in a region of China where HCC is (8,200-a) GSHsAg- , anti-GSHc' 5 000 common in these animals and in humans.203In addition, All marker -0 there appears to be a high content of aflatoxin in many human and animal foods in that region, a factor which All WHsAg' Woodchuck > 20 000 complicates the assessment of the role of hepadnaviruses WHsAg- , anti-WHc' ? (199,200) All markerin HCC. Although the DHBV was first discovered in ZO these ducks, no correlation of virus infection with HCC has been reported in this location. Infected brown ducks Duck (200-C) DHBsAg' All marker from this region of China (and not uninfected animals) followed prospectively in Japan were noted to develop HCC.204On the other hand, white Peking ducks infected with DHBV in the USA have not been observed to develop HCC. It is not clear whether this apparent mode and time of HBV infection in HCC patients. If difference is related to a critical difference in the viruHBV infection does occur frequently at very early ages in lence of Chinese and USA DHBV strains, susceptibility HCC patients in high-HCC-incidence areas, the age of different hosts (e.g. Chinese brown v s Peking ducks) or distribution of patients with clinically recognized HCC to other non-viral factors. would suggest that these tumours usually appear after The apparent differences in incidence and time of continuous HBV infection of 30 or more years. Few cases onset of HCC in the different hepadnavirus-host systems of HCC occur in ~ h i l d r e n . " ~ Up to 90% of HCC patients have co-existing c i r r h o s i ~ . ~ ~Projections ' ~ ' ~ ' ~ ~made ~ ~ ~ ~ (Table 4) could be due to variability in viral virulence and/or in host susceptibility factors that determine the from the large prospective study in Taiwan have sugdevelopment of HCC. The ability to infect woodchucks gested that over 40?h of middle aged male HBsAg carriers with GSHV, and different duck varieties with different in Taiwan will die of HCC.3 DHBV strains should permit direct comparison of differAlthough the argument for a role of HBV in human ent viruses in the same host. In this regard, a recent study HCC is persuasive, the association with two other mamshowed that woodchucks chronically infected with malian hepadnaviruses is even stronger. One hundred per GSHV developed HCC an average of 18 months later cent of both wild-caught WHV-infected and colony-born than woodchucks chronically infected with WHV."' experimentally-infected woodchucks died of HCC within This indicates that there are important differences in the 3 years, while no HCC developed in animals that had oncogenic effects of the two viruses in that host. never been infected.20'*202T h e animals with HCC have active hepatitis with significant components of liver cell necrosis, regeneration and inflammation but cirrhosis has Table 4 Estimated time of HCC occurrence in different not been obser~ed.~?' In a colony of captive ground hepadnavirus infected hosts squirrels infected with GSHV in the wild, no HCC were seen before 4.5 years but by 7 years more than two-thirds of the infected animals had died of HCC; no HCC were Estimated Estimated fraction of Host life span observed in animals without serologic evidence of current life span when 40% of hepadnavirus carriers (years) or past infection (P. L. Marion & W. S. Robinson unpubl. are projected to have data).' As with woodchucks, infected ground squirrels developed HCC ( O h ) have significant hepatitis but not cirrhosis. The absence of cirrhosis in these animals may be related to an intrinsic 70 100 Humans3 difference in the liver of rodents and primates. Cirrhosis Ground squirrel (8,200-a) 10 60 and fibrosis are common responses to chronic liver injury 10-15 Woodchuck (6,7,199,200) 10 in primates and not in rodents. In contrast to humans, the Duck (200-C) ? > prevalence of the carrier state and incidence of HCC in Table 3 Approximate incidence of HCC in different hepadnavirus infected hosts
'
W. S. Robinson
632
Development of HCC in cirrhotic liver and the presence of virus in tumours Hepadnaviruses have been found in the cells of many, although not all, HCC of hepadnavirus infected hosts. Hepadnavirus DNA has been detected in approximately 85% of HCC but not in the remaining 15% of surface antigen carrier humans,'3'15~206-208woodchuck^^^^ and ground squirrels (P. L. Marion & W. S. Robinson unpubl. data). Thus persistence of viral DNA in tumour cells, as detected by Southern blot analysis, does not appear to be essential for the development of HCC in hepadnavirus infected liver. Episomal viral DNA forms have been observed in only a small fraction of HCC of HBV infected patients and integrated viral DNA can be detected in most (75-85%) although not all HCC.'33'5,206-208 In humans, 60-90% of patients with HCC have underlying cirrhosi~.',~,'~','~~-~~~ In such cirrhotic liver, HCC arise in adenomatous foci that form within regenerative nodules of liver cells. 196,209 Examination of individual cirrhotic nodules of HBV infected cirrhotic livers has revealed that the DNA of most nodules of most cirrhotic Other nodules livers does not contain integrated contain a clonal pattern of one or more viral DNA integrations (i.e. viral integrations in the same cellular DNA sites in many cells of the nodule). Such nodules are undoubtedly formed by proliferation and clonal expansion of an original hepatocyte that contained one or more viral integrations. This suggests that the integration event(s) may have imparted a growth advantage to the original cell containing the integrant(s) and this resulted in clonal expansion of that cell. Other nodules contain a non-clonal pattern of viral integration (many integrations at different cellular DNA sites).'58 Thus the state of HBV-DNA is not the same in all regenerative nodules of HBV-infected cirrhotic liver. Of the approximately 85% of HCC in HBV-infected livers that have integrated HBV-DNA in a clonal integration pattern, most have multiple viral integrants and only a few have single integrants.l 1-15,157,158,206-208 I n most HBV-infected cirrhotic livers, only a small minority of cirrhotic nodules contain multiple clonal integrant~.'~'This suggests that HCC arise preferentially (but not exclusively) from cells of cirrhotic nodules with multiple HBV integrations. It follows, therefore, that the viral integrations could play a role in the development of HCC. As described in Part 11, the role played by viral integrations in the development of HCC remains unclear in most cases. T h e fact that each integration studied has been found at a different cellular DNA site and on any of several different chromosomes in different human tumours would appear to rule out a viral integration-site-specific mechanism for most human HCC. Multiple tumours in the same liver usually have the same clonal integration pattern in each tumour nodule indicating that they are metastases arising from the same original tumour. Occasionally, however, integration sites can be different in different HCC tumours within the same liver.'58 This demonstrates that HCC can be multicentric in origin in the cirrhotic liver. Immunofluorescent and immunoperoxidase staining of HCC tissue has demonstrated that in patients with
HBsAg in the blood and in whom non-tumorous liver cells are positive for HBsAg and/or HBcAg, tumour cells appear most often to be negative. Conversely, other studies have reported small numbers of HBsAg-positive cells in turn our^.'^' HBcAg has been detected even more rarely. Thus few tumour cells appear to express either viral gene product in amounts that can be detected by immunofluorescent staining, while cells within the same tumour do not uniformly express these antigens. These findings, as well as the failure to detect episomal forms of viral DNA, support the concept that HCC cells are non-permissive for expression of these viral structural genes or for virus replication.
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