Tumours of the Liver M. C. KEW Dept. of Medicine, Witwatersrand University Medical School and
Johannesburg and Baragwanath Hospitals, Johannesburg, South Africa
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Auckland on 11/11/14 For personal use only.
Kew MC. Tumours of the liver. Scand J Gastroenterol 1992;27 Suppl 192:3942. One of the major debates in hepatocellular carcinogenesis at present is whether the hepatitis-B and -C viruses are directly carcinogenic or exert their effect indirectly by causing chronic necro-inflammatory hepatic disease, which in turn is responsible for malignant transformation of hepatocytes. This debate has been fueled by the observation that hepatitis Cvirus is a single-stranded RNA virus with no precedent for inducing cancer but with a marked propensity to cause chronic necro-inflammatory hepatic disease and by the findings in Chisari's transgenic mouse model, which suggest that severe and prolonged hepatocellular injury per se induces a proliferative response that progresses to tumour formation. Recent reports of a guanine to thymine mutation of the third base of codon 249 of the tumour suppressor gene, p53, in 50% of patients with hepatocellular carcinoma in regions of high aflatoxin exposure, and mutagenic experiments showing that aflatoxin B 1 binds particularly to guanine residues in G-C-rich domains and that codon 249 is a preferred target have suggested a mechanism whereby aflatoxin might induce malignant transformation. Key words: Aflatoxin; hepatitis-B virus; hepatitis-B X gene; hepatitis-c virus; hepatocellular carcinogenesis; p53; transgenic mice Michael C. Kew, M. D . , Dept. of Medicine, Witwatersrand University Medical School and Johannesburg and Baragwanath Hospitals. Johannesburg, South Africa
The symptoms and physical signs produced by hepatic tumours are by now well known, the sensitivity and specificity of the imaging modalities and serological tests used in their diagnosis have been established, and there is little to enthuse over when considering the results of treatment. In recent years most interest in the field of hepatic tumours has focused on the aetiology and pathogenesis of hepatocellular carcinoma, and it is here that the major advances in knowledge are occurring. These advances are not concerned as much with the recognition of new aetiologic associations of the tumour as with the way in which known risk factors may be causing hepatic carcinogenesis. Another aspect that is attracting attention is the detection and treatment of presymptomatic and hence potentially resectable hepatocellular carcinomas, and whether programs designed t o achieve this goal are cost-effective. CHRONIC NECROINFLAMMATORY HEPATIC DISEASE One of the major subjects of debate at present concerning the aetiology and pathogenesis of hepatocellular carcinoma is whether the hepatitis-B virus is directly carcinogenic or plays only an indirect role by inducing chronic necroinflammatory hepatic disease, which in turn is responsible for the malignant transformation of hepatocytes. The recent epidemiologic evidence of an association between hepatocellular carcinoma and the hepatitis-c virus, a singlestranded R N A virus with no precedent for causing tumours but with a marked propensity to induce chronic hepatitis
and cirrhosis, has fueled the argument and lends credence to the belief that the role of the hepatitis viruses in the pathogenesis of hepatocellular carcinoma is a n indirect one. A similar conclusion may be drawn from the experiments in which hepatitis-B virus D N A encoding the entire open reading frame for surface polypeptides has been placed directly into the germ-line of mice (1). The transgenic mice overproduce large envelope polypeptide, which accumulates within the endoplasmic reticulum of hepatocytes, producing severe and prolonged hepatocellular injury that initiates a response characterized by inflammation, regenerative hyperplasia, transcriptional deregulation, and aneuploidy, with ultimate progression to neoplasia. These findings suggest that severe and prolonged cellular injury induces a proliferative response that fosters secondary genetic events that program the cell for unrestrained growth. This effect acts synergistically with the chemical carcinogens aflatoxin B1 and diethylnitrosamine in experimental models (2). AFLATOXIN Another subject of ongoing debate is the role of aflatoxin in the aetiology and pathogenesis of hepatocellular carcinoma. There is long-standing epidemiologic evidence of a positive correlation between ingestion of aflatoxin and the occurrence of hepatocellular carcinoma. Reports published during the past year have suggested a possible mechanism whereby the mycotoxin might be involved in hepatic carcinogenesis. Mutation o r loss of the putative tumour-suppressor gene, p53, has been demonstrated in several human tumours,
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M. C. Kew
including some of intestinal origin (3-5). Loss of heterozygosity has been described in hepatocellular carcinomas, which suggested a possible pathogenetic role for tumour-suppressor genes, and in 1990 Bressac et al. (6) detected abnormalities of p53 in six of seven human hepatocellular carcinoma-derived cell lines (6). The latter consisted either of mutations of the gene, loss of the transcript, or changes in the concentration or half-life of p53 protein. The belief that p53 may be involved in the transition of hepatocytes to a malignant phenotype received substantial support during 1991, when mutations of the p53 gene were demonstrated in 50% of hepatocellular carcinomas from Chinese patients living in the Quidong province of China (7) and from southern African blacks (8). Furthermore, in nearly every tumour the mutation was a G to T transversion at the third base of codon 249. In some of the patients the wild-type allele appeared to be lost as a result of deletion. Non-tumour tissue contained the normal p53 gene. In other tumours p53 mutations have involved more than 30 different codons spread over 4 of the highly conserved domains of the p53 gene in chromosome 17 (3-5). The finding of a mutational hotspot in a particular type of tumour implies both that there is a selective advantage for the mutation and that it could be a marker for a specific carcinogen. The latter possibility is supported by the observation that to date mutations at codon 249 of the p53 gene have been reported only in tumours and tumour-derived cell lines from patients living in regions of high aflatoxin exposure (9). Moreover, aflatoxin B1 has been shown in mutagenic experiments to bind particularly to G residues in G-C-rich regions (lo), and codon 249 is a preferred target (9). If this specific association is proved, this would provide the first clue to how an environmental toxin may contribute to tumour development. Aflatoxin and the hepatitis-B virus could, of course, act as cofactors in the pathogenesis of hepatocellular carcinoma; for example, a virally encoded protein could interact with p53 protein to confer a growth advantage on cells harbouring the mutation (4). HEPATITIS-B VIRUS Although the hepatitis-B virus has for some years been suspected of being the cause of approximately 80% of human hepatocellular carcinoma, the mechanism or mechanisms by which it might be directly carcinogenic, if indeed it is, remain to be elucidated. There is no evidence that the virus contains an oncogene. The finding in a few tumours of viral integrants within or near proto-oncogenes or near genes critical in cell cycling raises the possibility of insertional mutagenesis in these patients (11, 12). However, in the vast majority of hepatocellular carcinomas associated with hepatitis-B virus infection viral integrations are in different sites in cellular DNA in different tumours, and they are not in domains of known proto-oncogenes. Moreover, integrated viral DNA cannot be detected in 10% to 15% of tumours (13). The
situation appears to be quite different in woodchucks with tumours induced by the woodchuck hepatitis virus. In 50% of these tumours viral integration in or near C-myc or Nmyc has been demonstrated, indicating the potential importance of insertional mutagenesis in this model (14; P. Tiollais, personal communication, 1991). Disparity between different members of the hepadnaviridae family in this regard is confirmed by the failure of the same laboratory to find insertion of ground sqirrel virus DNA in or near protooncogenes in tumours in ground squirrels (P. Tiollais, personal communication, 1991). Another possible mechanism of hepatitis-B virus-related hepatic carcinogenesis is the expression of factors with transactivational properties from integrated intact or truncated viral genes. Demonstration that the hepatitis-B virus X-gene protein acts as a transcriptional trans-activator of viral genes, at least in tissue culture (15, 16), led to speculation that this might alter host gene expression in such a manner as to lead ultimately to malignant transformation of hepatocytes. Evidence in support of this putative mechanism has recently been published. When the X gene under its own regulatory elements was placed directly into the germ-line of mice, the mice developed, in turn, multifocal areas of altered hepatocytes, benign hepatic adenomas, and, finally, malignant carcinomas (17). When the hepatitis-B virus X gene had earlier been introduced under the transcriptional control of the human alpha-1-antitrypsin promoter, no liver tumours developed (18). Expression of the X-gene protein in these mice was shut off within 4 weeks of birth, and progression to neoplastic change would not be expected. The X gene is often interrupted or missing from integrated hepatitis-B virus DNA because it spans the cohesive end region of hepadnaviral DNA, which is the preferred site for viral integration. Moreover, the virus could not influence tumorigenesis without being integrated. This suggests that X-gene expression in hepatocellular carcinoma cannot be a common event. Nevertheless, this model supports the belief that the hepatitis-B virus may play a direct role in hepatic carcinogenesis and that diffuse chronic necro-inflammatory hepatic disease may not be a prerequisite for neoplastic transformation. PreS/S is the only hepatitis-B virus gene to be integrated in almost every hepatitis-B virus-related hepatocellular carcinoma. The notion that integrated DNA can contribute to carcinogenesis by activation of cellular genes in t r a m is supported by a recent report that 3' truncated preS/S sequences in integrated hepatitis-B virus DNA in hepatocellular carcinomas encode a transcriptional trans-activator (19). The fact that integrated sequences of viral DNA cannot be demonstrated in 10% to 15% of hepatitis-B virus-related hepatocellular carcinomas implies that integration is not essential for carcinogenesis. It is, however, possible that integration is invariable in these tumours, but that it is not detectable, for one of several reasons, at the time of analysis.
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Tumours of the Liver
There is no preferred site for integration within chromosomal DNA, although there are preferred sites within the viral DNA. In most integrations the site at which the viral genome joins the cellular DNA is very near the 5‘ end of one of the virion DNA strands and within or near the D R l or DR2 sequence. This suggests that the ends of the DNA strands (particularly the minus strand) may be involved in the integration process. Integrated viral DNA almost invariably has one or more deletions or is rearranged in other ways (13). These changes have been thought to occur during or after integration, but an alternative explanation is that the mutations preceded integration. Mutant forms of woodchuck hepatitis virus have recently been detected in the serum of woodchucks with hepatocellular carcinoma (Kew MC, Miller RH, Purcell RH. Unpublished observations). These mutations cluster near DR1 and DR2 at the preferred sites of action of topo-isomerase I, an enzyme thought to play an important role in the process of integration of viral DNA (20). These observations raise the possibility that mutant forms of hepadnaviruses may become integrated into cellular DNA and hence may play a role in hepatic carcinogenesis. HEPATITIS-C VIRUS Epidemiologic data supporting a link between chronic infection with the hepatitis-c virus and the development of hepatocellular carcinoma continue to accumulate. However, almost all of the published prevalences are still based on first-generation enzyme-linked immunoabsorbent assays for antibodies to the hepatitis-C virus (anti-HCV), and the question of how many of the positive results are falsely positive remains to be resolved. Second-generation enzymelinked immunoabsorbent assays making use of a greater number and wider distribution of hepatitis-C virus epitopes, currently under trial, and recombinant immunoblot assays will reduce the number of false-positive results. However, the prevalence of hepatitis-c virus RNA (after amplification by the polymerase chain reaction) in the serum of patients with hepatocellular carcinoma will need to be ascertained before the closeness of the association between the virus and the tumour in different populations can be accurately gauged. Three patterns of correlation between hepatocellular carcinoma and hepatitis-B and -C virus infections have emerged. In Chinese and black African patients hepatitis-B virus is the major risk factor, and the hepatitis-C virus plays a relatively minor role (21,22). In Japan, Italy, and Spain the opposite pattern is found, with hepatitis-c virus infection being present in half or more of the patients and hepatitisB virus in only about a quarter (23-25); in other countries, usually with lower incidences of the tumour, the two virsuses play a minor but equal part (26,27). The interaction, if any, between these viruses in the pathogenesis of hepatocellular carcinoma remains uncertain. Studies in Chinese, Japanese, Italian, and African patients have shown that anti-HCV is
41
positive appreciably more often in hepatitis-B surface antigen (HBsAg)-negative than in HBsAg-positive patients (2123,25). This phenomenon has not been seen in ‘Western’ patients, and in two recent analyses of Greek (26) and North American patients (28), respectively, the relative risk of hepatocellular carcinoma in HBsAg-positive patients was significantly greater than that in those negative for this marker, suggesting that the two viruses may interact in the pathogenesis of hepatocellular carcinoma. With the present state of knowledge, the most plausible explanation of the link between hepatitis-C virus and hepatocellular carcinoma is that the virus causes chronic necroinflammatory hepatic disease, and this, rather than the virus, is responsible for neoplastic transformation. EARLY DIAGNOSIS Because symptomatic hepatocellular carcinoma is rarely amenable to surgical cure and responds poorly to chemotherapy and irradiation, there is a pressing need either to prevent this tumour or to diagnose it at a presymptomatic stage. when resection is possible. Screening of whole populations can be contemplated only in countries having the highest incidences of hepatocellular carcinoma, but even then the enormity of the task is daunting. Moreover, the very nature of this type of program demands that serum alpha-foetoprotein is the sole screening tool. Approximately 45% of small subclinical hepatocellular carcinomas are now known not to produce a diagnostically raised serum alphafoetoprotein concentration, and these tumours will inevitably be missed in mass population surveys: the less sensitive (and hence the cheaper) the method for detecting alphafoetoprotein, the more tumours will be overlooked (29). Long-term surveillance of individuals considered to be at high risk of developing hepatocellular carcinoma involves periodic testing for alpha-foetoprotein and hepatic imaging. Again there is the problem that only 45% of presymptomatic tumours produce a diagnostically raised serum alphafoetoprotein level. In addition, difficulty is experienced in interpreting levels raised to less than 400 ng/ml because various forms of benign hepatic disease (chronic hepatitis and cirrhosis) may produce such concentrations. More sensitive serum markers of small hepatocellular carcinomas have not emerged. Measurement of the differential reactivity of alpha-foetoprotein to certain lectins may be useful in this respect, but the methods used are of such a nature that they are unlikely to be available in small peripheral hospitals in those countries where hepatocellular carcinoma occurs most commonly and screening and surveillance programs are most needed. Ultrasonography remains the first choice for initial screening, with the more expensive, less readily available, or more hazardous imaging modalities being used in doubtful cases or in special circumstances. With the use of lipiodol in conjunction with computerized tomography it has become
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M. C. Kew
apparent that even small tumours may b e accompanied by satellite nodules. Failure to detect these tiny nodules largely accounts for the unexpectedly high early recurrence rate after resection of small hepatocellular carcinomas (30). T h e cost-effectiveness of screening and surveillance programs remains the subject of debate. Recent reports in the literature have n o t helped t o resolve this issue because they contain data that support both sides of the argument.
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REFERENCES 1 . Chisari FV, Klopchin K, Moriyama T, et al. Molecular pathogenesis of hepatocellular carcinoma in hepatitisB virus transgenic mice. Cell 1989;5:1145-56. 2. Sell S, Hunt JM, Dunsord HA, Chisari FV.Synergism between hepatitis-B virus expression and chemical hepatocarcinogens in transgenic mice. Cancer Res 1991;51:1278-85. 3. Stanbridge EJ. Human tumour-suppressor genes. Annu Rev Genet 1990;24:615-57. 4. Harris AL. Telling changes of base. Nature 1991;350:377-8. 5. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49-53. 6. Bressac B, Galvin KM, Liang TJ, Isselbacher KJ, Wands JR, Ozturk M. Abnormal structure and expression of p53 gene in human hepatocellular carcinoma. Proc Natl Acad Sci USA 1990;87:1973-7. 7. Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC. Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature 1991 ;350:427-8. 8. Bressac B, Kew MC, Wands JR, Ozturk M. Selective G to T mutations of p53 in hepatocellular carcinoma from southern Africa. Nature 1991;350:429-31. 9. Ozturk M, and collaborators. p53 in hepatocellular carcinoma after aflatoxin exposure. Lancet 1991 ;338:13569. 10. Muench KF, Misra RP, Humayun MZ. Sequence specificity in aflatoxin Bl-DNA interactions. Proc Natl Acad Sci USA 1983;80:610. 11. DeJean A, Bougueleret L, Grzeschik KH, Tiollais P. HepatitisB virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma. Nature 1986;322:70-2. 12. Wang J, Chenivesse X, Henglein B, Brechot C. Hepatitis-B virus integration in a cyclin A gene in a hepatocellular carcinoma. Nature 1990;343:555-7. 13. Robinson WS, Klote L, Aoki N. Hepadnaviruses in cirrhotic liver and hepatocellular carcinoma. J Med Virol 1990;31:1%32.
14. Hsu T, Moroy T, Etiemble J, et al. Activation of C-myc by woodchuck hepatitis virus insertion in hepatocellular carcinoma. Cell 1988;55:627-35. 15. Twu JS, Schloemer RH. Transcriptional trans-activating function of hepatitis-B virus. J Virol 1987;61:3448-53. 16. Spandau DF, Lee CH. Trans-activation of viral enhancers by the hepatitis-B virus X gene protein. J Virol 1988;62:427-34. 17. Kim C-M, Koike K, Saito 1, Miyamura T, Jay G. HBx gene of hepatitis-B virus induces liver cancer in transgenic mice. Nature 1991;351:317-20. 18. Lee T-H, Finegold M-J, Shen RF, DeMoyo JL, Woo SLC, Butel JS. Hepatitis-B virus trans-activator X protein is not tumorigenic in transgenic mice. J Virol 1990;64:5939-47. 19. Kekule A, Lauer U, Meyer M, Caselmann W,Hofschneider PA, Koshy K. The preS/S region of integrated hepatitis-B virus encodes a transcriptional trans-activator. Nature 1990;343:457-
60. 20. Wang HP, Rogler CE. Topoisomerase I-mediated integration of hepadnavirus DNA in vitro. J Virol 1991;65:2381-92. 21. Jeng J-E, Tsai J-F. Hepatitis-C virus antibody in hepatocellular carcinoma in Taiwan. J Med Virol 1991;34:747. 22. Kew MC, Houghton M, Choo Q-L, Kuo G. Hepatitis-C antibodies in southern African blacks with hepatocellular carcinoma. Lancet 1990;335:873-4. 23. Nishioka K, Watanabe J, Furuta S. et al. A high prevalence of antibody to the hepatitis-c virus in patients with hepatocellular carcinoma in Japan. Cancer 1991 ;67:429-33. 24. Bruix J , Barrera JM, Calvet X, et al. Prevalence of antibodies to hepatitis-c virus in Spanish patients with hepatocellular carcinoma and hepatic cirrhosis. Lancet 1989;334:1004-6. 25. Columbo M, Kuo G , Choo Q-L, et al. Prevalence of antibodies to hepatitis-c virus in Italian patients with hepatocellular carcinoma. Lancet 1989;334:10068. 26. Kaklamani E, Trichopoulos D, Tzonou A , et al. Hepatitis B and C viruses and their interaction in the origin of hepatocellular carcinoma. JAMA 1991;265:1974-6. 27. DiBisceglie AM, Order SE, Klein JL, et al. The role of chronic viral hepatitis in hepatocellular carcinoma in the United Stgues. Am J Gastroenterol 1991;86:335-8. 28. Yu MC, Tong MJ, Coursaget P, Ross RK, Govindarajan S, Henderson BE. Prevalence of hepatitis B and C viral markers in black and white patients in the United States. J Natl Cancer Inst 1990;82:1038-41. 29. Kew MC. Detection and treatment of small hepatocellular carcinomas. In: Hollinger EB, Lemon SM, Margolis HS, editors. Viral hepatitis and liver disease. Baltimore; Williams & Wilkins, 1991 535-40. 30. Editorial. Lipiodol computed tomography in the diagnosis of small hepatocellular carcinomas. Lancet 1991;337:333-1.
Johannesburg and Baragwanath Hospitals, Johannesburg, South Africa
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Auckland on 11/11/14 For personal use only.
Kew MC. Tumours of the liver. Scand J Gastroenterol 1992;27 Suppl 192:3942. One of the major debates in hepatocellular carcinogenesis at present is whether the hepatitis-B and -C viruses are directly carcinogenic or exert their effect indirectly by causing chronic necro-inflammatory hepatic disease, which in turn is responsible for malignant transformation of hepatocytes. This debate has been fueled by the observation that hepatitis Cvirus is a single-stranded RNA virus with no precedent for inducing cancer but with a marked propensity to cause chronic necro-inflammatory hepatic disease and by the findings in Chisari's transgenic mouse model, which suggest that severe and prolonged hepatocellular injury per se induces a proliferative response that progresses to tumour formation. Recent reports of a guanine to thymine mutation of the third base of codon 249 of the tumour suppressor gene, p53, in 50% of patients with hepatocellular carcinoma in regions of high aflatoxin exposure, and mutagenic experiments showing that aflatoxin B 1 binds particularly to guanine residues in G-C-rich domains and that codon 249 is a preferred target have suggested a mechanism whereby aflatoxin might induce malignant transformation. Key words: Aflatoxin; hepatitis-B virus; hepatitis-B X gene; hepatitis-c virus; hepatocellular carcinogenesis; p53; transgenic mice Michael C. Kew, M. D . , Dept. of Medicine, Witwatersrand University Medical School and Johannesburg and Baragwanath Hospitals. Johannesburg, South Africa
The symptoms and physical signs produced by hepatic tumours are by now well known, the sensitivity and specificity of the imaging modalities and serological tests used in their diagnosis have been established, and there is little to enthuse over when considering the results of treatment. In recent years most interest in the field of hepatic tumours has focused on the aetiology and pathogenesis of hepatocellular carcinoma, and it is here that the major advances in knowledge are occurring. These advances are not concerned as much with the recognition of new aetiologic associations of the tumour as with the way in which known risk factors may be causing hepatic carcinogenesis. Another aspect that is attracting attention is the detection and treatment of presymptomatic and hence potentially resectable hepatocellular carcinomas, and whether programs designed t o achieve this goal are cost-effective. CHRONIC NECROINFLAMMATORY HEPATIC DISEASE One of the major subjects of debate at present concerning the aetiology and pathogenesis of hepatocellular carcinoma is whether the hepatitis-B virus is directly carcinogenic or plays only an indirect role by inducing chronic necroinflammatory hepatic disease, which in turn is responsible for the malignant transformation of hepatocytes. The recent epidemiologic evidence of an association between hepatocellular carcinoma and the hepatitis-c virus, a singlestranded R N A virus with no precedent for causing tumours but with a marked propensity to induce chronic hepatitis
and cirrhosis, has fueled the argument and lends credence to the belief that the role of the hepatitis viruses in the pathogenesis of hepatocellular carcinoma is a n indirect one. A similar conclusion may be drawn from the experiments in which hepatitis-B virus D N A encoding the entire open reading frame for surface polypeptides has been placed directly into the germ-line of mice (1). The transgenic mice overproduce large envelope polypeptide, which accumulates within the endoplasmic reticulum of hepatocytes, producing severe and prolonged hepatocellular injury that initiates a response characterized by inflammation, regenerative hyperplasia, transcriptional deregulation, and aneuploidy, with ultimate progression to neoplasia. These findings suggest that severe and prolonged cellular injury induces a proliferative response that fosters secondary genetic events that program the cell for unrestrained growth. This effect acts synergistically with the chemical carcinogens aflatoxin B1 and diethylnitrosamine in experimental models (2). AFLATOXIN Another subject of ongoing debate is the role of aflatoxin in the aetiology and pathogenesis of hepatocellular carcinoma. There is long-standing epidemiologic evidence of a positive correlation between ingestion of aflatoxin and the occurrence of hepatocellular carcinoma. Reports published during the past year have suggested a possible mechanism whereby the mycotoxin might be involved in hepatic carcinogenesis. Mutation o r loss of the putative tumour-suppressor gene, p53, has been demonstrated in several human tumours,
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Auckland on 11/11/14 For personal use only.
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M. C. Kew
including some of intestinal origin (3-5). Loss of heterozygosity has been described in hepatocellular carcinomas, which suggested a possible pathogenetic role for tumour-suppressor genes, and in 1990 Bressac et al. (6) detected abnormalities of p53 in six of seven human hepatocellular carcinoma-derived cell lines (6). The latter consisted either of mutations of the gene, loss of the transcript, or changes in the concentration or half-life of p53 protein. The belief that p53 may be involved in the transition of hepatocytes to a malignant phenotype received substantial support during 1991, when mutations of the p53 gene were demonstrated in 50% of hepatocellular carcinomas from Chinese patients living in the Quidong province of China (7) and from southern African blacks (8). Furthermore, in nearly every tumour the mutation was a G to T transversion at the third base of codon 249. In some of the patients the wild-type allele appeared to be lost as a result of deletion. Non-tumour tissue contained the normal p53 gene. In other tumours p53 mutations have involved more than 30 different codons spread over 4 of the highly conserved domains of the p53 gene in chromosome 17 (3-5). The finding of a mutational hotspot in a particular type of tumour implies both that there is a selective advantage for the mutation and that it could be a marker for a specific carcinogen. The latter possibility is supported by the observation that to date mutations at codon 249 of the p53 gene have been reported only in tumours and tumour-derived cell lines from patients living in regions of high aflatoxin exposure (9). Moreover, aflatoxin B1 has been shown in mutagenic experiments to bind particularly to G residues in G-C-rich regions (lo), and codon 249 is a preferred target (9). If this specific association is proved, this would provide the first clue to how an environmental toxin may contribute to tumour development. Aflatoxin and the hepatitis-B virus could, of course, act as cofactors in the pathogenesis of hepatocellular carcinoma; for example, a virally encoded protein could interact with p53 protein to confer a growth advantage on cells harbouring the mutation (4). HEPATITIS-B VIRUS Although the hepatitis-B virus has for some years been suspected of being the cause of approximately 80% of human hepatocellular carcinoma, the mechanism or mechanisms by which it might be directly carcinogenic, if indeed it is, remain to be elucidated. There is no evidence that the virus contains an oncogene. The finding in a few tumours of viral integrants within or near proto-oncogenes or near genes critical in cell cycling raises the possibility of insertional mutagenesis in these patients (11, 12). However, in the vast majority of hepatocellular carcinomas associated with hepatitis-B virus infection viral integrations are in different sites in cellular DNA in different tumours, and they are not in domains of known proto-oncogenes. Moreover, integrated viral DNA cannot be detected in 10% to 15% of tumours (13). The
situation appears to be quite different in woodchucks with tumours induced by the woodchuck hepatitis virus. In 50% of these tumours viral integration in or near C-myc or Nmyc has been demonstrated, indicating the potential importance of insertional mutagenesis in this model (14; P. Tiollais, personal communication, 1991). Disparity between different members of the hepadnaviridae family in this regard is confirmed by the failure of the same laboratory to find insertion of ground sqirrel virus DNA in or near protooncogenes in tumours in ground squirrels (P. Tiollais, personal communication, 1991). Another possible mechanism of hepatitis-B virus-related hepatic carcinogenesis is the expression of factors with transactivational properties from integrated intact or truncated viral genes. Demonstration that the hepatitis-B virus X-gene protein acts as a transcriptional trans-activator of viral genes, at least in tissue culture (15, 16), led to speculation that this might alter host gene expression in such a manner as to lead ultimately to malignant transformation of hepatocytes. Evidence in support of this putative mechanism has recently been published. When the X gene under its own regulatory elements was placed directly into the germ-line of mice, the mice developed, in turn, multifocal areas of altered hepatocytes, benign hepatic adenomas, and, finally, malignant carcinomas (17). When the hepatitis-B virus X gene had earlier been introduced under the transcriptional control of the human alpha-1-antitrypsin promoter, no liver tumours developed (18). Expression of the X-gene protein in these mice was shut off within 4 weeks of birth, and progression to neoplastic change would not be expected. The X gene is often interrupted or missing from integrated hepatitis-B virus DNA because it spans the cohesive end region of hepadnaviral DNA, which is the preferred site for viral integration. Moreover, the virus could not influence tumorigenesis without being integrated. This suggests that X-gene expression in hepatocellular carcinoma cannot be a common event. Nevertheless, this model supports the belief that the hepatitis-B virus may play a direct role in hepatic carcinogenesis and that diffuse chronic necro-inflammatory hepatic disease may not be a prerequisite for neoplastic transformation. PreS/S is the only hepatitis-B virus gene to be integrated in almost every hepatitis-B virus-related hepatocellular carcinoma. The notion that integrated DNA can contribute to carcinogenesis by activation of cellular genes in t r a m is supported by a recent report that 3' truncated preS/S sequences in integrated hepatitis-B virus DNA in hepatocellular carcinomas encode a transcriptional trans-activator (19). The fact that integrated sequences of viral DNA cannot be demonstrated in 10% to 15% of hepatitis-B virus-related hepatocellular carcinomas implies that integration is not essential for carcinogenesis. It is, however, possible that integration is invariable in these tumours, but that it is not detectable, for one of several reasons, at the time of analysis.
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Tumours of the Liver
There is no preferred site for integration within chromosomal DNA, although there are preferred sites within the viral DNA. In most integrations the site at which the viral genome joins the cellular DNA is very near the 5‘ end of one of the virion DNA strands and within or near the D R l or DR2 sequence. This suggests that the ends of the DNA strands (particularly the minus strand) may be involved in the integration process. Integrated viral DNA almost invariably has one or more deletions or is rearranged in other ways (13). These changes have been thought to occur during or after integration, but an alternative explanation is that the mutations preceded integration. Mutant forms of woodchuck hepatitis virus have recently been detected in the serum of woodchucks with hepatocellular carcinoma (Kew MC, Miller RH, Purcell RH. Unpublished observations). These mutations cluster near DR1 and DR2 at the preferred sites of action of topo-isomerase I, an enzyme thought to play an important role in the process of integration of viral DNA (20). These observations raise the possibility that mutant forms of hepadnaviruses may become integrated into cellular DNA and hence may play a role in hepatic carcinogenesis. HEPATITIS-C VIRUS Epidemiologic data supporting a link between chronic infection with the hepatitis-c virus and the development of hepatocellular carcinoma continue to accumulate. However, almost all of the published prevalences are still based on first-generation enzyme-linked immunoabsorbent assays for antibodies to the hepatitis-C virus (anti-HCV), and the question of how many of the positive results are falsely positive remains to be resolved. Second-generation enzymelinked immunoabsorbent assays making use of a greater number and wider distribution of hepatitis-C virus epitopes, currently under trial, and recombinant immunoblot assays will reduce the number of false-positive results. However, the prevalence of hepatitis-c virus RNA (after amplification by the polymerase chain reaction) in the serum of patients with hepatocellular carcinoma will need to be ascertained before the closeness of the association between the virus and the tumour in different populations can be accurately gauged. Three patterns of correlation between hepatocellular carcinoma and hepatitis-B and -C virus infections have emerged. In Chinese and black African patients hepatitis-B virus is the major risk factor, and the hepatitis-C virus plays a relatively minor role (21,22). In Japan, Italy, and Spain the opposite pattern is found, with hepatitis-c virus infection being present in half or more of the patients and hepatitisB virus in only about a quarter (23-25); in other countries, usually with lower incidences of the tumour, the two virsuses play a minor but equal part (26,27). The interaction, if any, between these viruses in the pathogenesis of hepatocellular carcinoma remains uncertain. Studies in Chinese, Japanese, Italian, and African patients have shown that anti-HCV is
41
positive appreciably more often in hepatitis-B surface antigen (HBsAg)-negative than in HBsAg-positive patients (2123,25). This phenomenon has not been seen in ‘Western’ patients, and in two recent analyses of Greek (26) and North American patients (28), respectively, the relative risk of hepatocellular carcinoma in HBsAg-positive patients was significantly greater than that in those negative for this marker, suggesting that the two viruses may interact in the pathogenesis of hepatocellular carcinoma. With the present state of knowledge, the most plausible explanation of the link between hepatitis-C virus and hepatocellular carcinoma is that the virus causes chronic necroinflammatory hepatic disease, and this, rather than the virus, is responsible for neoplastic transformation. EARLY DIAGNOSIS Because symptomatic hepatocellular carcinoma is rarely amenable to surgical cure and responds poorly to chemotherapy and irradiation, there is a pressing need either to prevent this tumour or to diagnose it at a presymptomatic stage. when resection is possible. Screening of whole populations can be contemplated only in countries having the highest incidences of hepatocellular carcinoma, but even then the enormity of the task is daunting. Moreover, the very nature of this type of program demands that serum alpha-foetoprotein is the sole screening tool. Approximately 45% of small subclinical hepatocellular carcinomas are now known not to produce a diagnostically raised serum alphafoetoprotein concentration, and these tumours will inevitably be missed in mass population surveys: the less sensitive (and hence the cheaper) the method for detecting alphafoetoprotein, the more tumours will be overlooked (29). Long-term surveillance of individuals considered to be at high risk of developing hepatocellular carcinoma involves periodic testing for alpha-foetoprotein and hepatic imaging. Again there is the problem that only 45% of presymptomatic tumours produce a diagnostically raised serum alphafoetoprotein level. In addition, difficulty is experienced in interpreting levels raised to less than 400 ng/ml because various forms of benign hepatic disease (chronic hepatitis and cirrhosis) may produce such concentrations. More sensitive serum markers of small hepatocellular carcinomas have not emerged. Measurement of the differential reactivity of alpha-foetoprotein to certain lectins may be useful in this respect, but the methods used are of such a nature that they are unlikely to be available in small peripheral hospitals in those countries where hepatocellular carcinoma occurs most commonly and screening and surveillance programs are most needed. Ultrasonography remains the first choice for initial screening, with the more expensive, less readily available, or more hazardous imaging modalities being used in doubtful cases or in special circumstances. With the use of lipiodol in conjunction with computerized tomography it has become
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M. C. Kew
apparent that even small tumours may b e accompanied by satellite nodules. Failure to detect these tiny nodules largely accounts for the unexpectedly high early recurrence rate after resection of small hepatocellular carcinomas (30). T h e cost-effectiveness of screening and surveillance programs remains the subject of debate. Recent reports in the literature have n o t helped t o resolve this issue because they contain data that support both sides of the argument.
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Auckland on 11/11/14 For personal use only.
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