(1) Seminar on “US-Japan Differences in Cancer Experience”
A workshop on U.S.-Japan Differences in Cancer Experience, sponsored by the Interdisciplinary Area (Dr. Haruo Sugano and Dr. Robert W. Miller, co-organizers), was held in Maui, Hawaii, on March 12-13, 1992. There were seven speakers from each country.
Binational differences in cancer occurrence have been the subject of two workshops annually since 1979. The current workshop, for the most part, supplements especially interesting information presented in this series.
Overview from the U.S.: Ernst L. Wynder (American Health Foundation) spoke on differences in cancer rates which he attributed to cigarette smoking and diet He spoke of the need to use applied epidemiology to reduce cancer rates. He noted that cancer rates in the U.S. and Japan would be diminished by 70-80 percent if each type-specific cancer rate was reduced to the lowest level for it in the world. He called for cancer prevention centers where biology and epidemiology can be brought together, and stressed his view that among dietary fats, linoleic acid may be a ubiquitous cancer enhancer, especially of breast cancer, melanoma and metastatic spread. He lamented that physicians do not learn about nutrition, because they need to correct this and other risk factors early in life.
Overview from Japan: Kunio Aoki (Aichi Cancer Center) stated that these binational workshops have greatly stimulated cancer epidemiologists in Japan. He then described a new cohort study of 110,000 inhabitants of 34 cities and towns in Japan and 25,000 industrial workers, in which 200,000 serum samples from about 40,000 persons are being stored for future studies of biomarkers of among those who develop cancer. In addition, a cancer registration network, which covers about 40 percent of the population, is now undergoing improvements in the quality of the data based on a U.S.-Japan workshop on this subject held in Atlanta in 1990.
In addition to general cancer registries, Japanese clinicians have formed a number of special cancer registries, as for childhood cancers, bone tumors, and MEN type 1. There is no list of these -- of interest to scientists outside Japan. Dr. Greenwald spoke of his interest in considering registries to improve understanding of cause and prevention; e.g., registries for precancer, such as dysplasia and adenoma, or patients with certain biomarkers, such as p53. It may be better to think not of cancer but of carcinogenesis; that is, a disorder that develops earlier over time, well before clinical onset. The only such registry in Japan, Dr. Nanba said, was the tumor tissue registries (50,000 malignant, 50,000 premalignant) in Hiroshima and Nagasaki.
Melanoma: Margaret A. Tucker (NCI), a participant in a workshop on melanoma in 1987 (Fitzpatrick TB et al. J. Invest Dermatol 1989; 92 [Suppl]), spoke of the markedly higher rate of cutaneous malignant melanoma (CMM) in U.S. whites than in Japanese. The difference, thought to be due to protective pigmentation of Japanese skin, may in fact be due to a lack of susceptibility. Dysplastic nevi, found in 50 percent of CMM in whites, are apparently rare in Japanese. After the 1987 workshop Dr. Tucker consulted at the Radiation Effects Research Foundation (RERF) in Hiroshima on plans for a dermatologic survey, and urged that clinic patients be screened for dysplastic nevi. The survey has since been completed, but it concerned tumor pathology and not a clinical surveillance of skin. A screening survey for dysplastic nevi should be made now either in Japan or among Japanese-Americans. Another suggestion was that the high rate of meningeal melanomas in Japanese under 30 years of age -- 23 percent of all melanomas in this age-group, should be evaluated through a case-control study. The cases could come from the source of this observation: the Annual of Pathological Autopsy Cases in Japan, 1958-82.
Lymphoma: Koji Nanba (Hiroshima University) told of two workshops held in the early 1980s, which concerned binational differences in the frequencies of lymphocyic diseases (Miller RW et al. JNCI 1981; 67:789-740; Kadin M et al. Human Pathol 1983; 14:745-72). Certain lymphoproliferative disease ale more common in the U.S. than in Japan, whereas autoimmune diseases are less common -- as if lymphocytes that protect against lymphoma predispose to autoimmune disease (systemic lupus erythematosus, Takayasu’s aortitis and Hashimoto’s thyroiditis).
Important information not previously available was the rate of lymphoma in Japanese after immunosuppression for organ-transplantation. Dr. Nanba reported that among 7740 renal transplant patients in Japan, only 5 developed lymphoma -- all extranodal, as compared with 34 of 3823 U.S. patients. If differences in the dosage or type of immunosuppressive drugs are not the explanation, the finding would be dramatic evidence that the Japanese, even when given this tremendous stimulus to develop lymphoma seldom do so.
Dr. Nanba had high praise for the Tumor Tissue Registry at RERF, which he had used to determine that the rate for lymphomas in Japan was 5.1 per 100,000 as compared with 12.1 in U.S. whites. He confirmed the low rates of follicular NHL and Hodgkin’s disease among children and young adults in Japan. In Honolulu he later found intermediate rates of follicular NHL among Japanese-Americans there. He said that other Japanese pathologists would benefit by using the Tumor Tissue Registry.
In 1966 MacMahon published graphs of the age-distribution of Hodgkin’s disease in about a dozen countries (Cancer Res 1966; 26: 1189-1200). In all countries studied there, were two peaks, except in Japan where the early peak was absent Dr. Nanba stated that there is a small peak at 25-29 years of age in Japan, previously obscured by misclassification of another disease (HTLV-1). An updated graph should be published, possibly based on rates from Cancer Incidence in Five Continents.
Biostatistics: Although Japan has a 2000-year history of mathematics, biostatistics has not been given much attention. William J. Blot (NCI) noted that in 1978 a workshop in Hiroshima first brought American and Japanese biostatsticians together to discuss statistical and methodologicai issues in cancer research. Additional workshops in 1983 and 1988 stimulated further interchange of methodological approaches in cancer research, especially in epidemiologic studies. Proceedings from the first and third workshops were published in Environmental Health Perspectives in 1979 (vol 32) and 1990 (vol 87), and from the second workshop in a volume issued by the Radiation Effects Research Foundation (Blot WJ et al: Statistical Methods in Cancer Research, 1984).
As an example of a fractional factorial epidemiological design that reduces cost and effort, Dr. Blot presented the plan for a clinical trial involving various dietary influences and vitamin supplements in the prevention of esophageal cancer in un County, Chin2~ where the mortality rates from this neoplasm alone are equal to the total mortality from other cancers. The study has shown no relationship to eating an Ames-positive fermented pickle mix suspected of causing esophageal cancer in people of Lin County and gullet cancer in their chickens. The observation by Dr. Blot that cancer of the gastric cardia was very high in mainland Chinese led to the observation that it was also frequent in Taiwan (804 cases among 1381 with subsite specified [Cancer Registry Annual Report, 1 986, Veterans General Hospital, Taipei, and Taichung Hospital, p 22]).
Takashi Yanagawa (Kyushu University), the Japanese co-organizer of three workshops, stated that they were extremely helpful in introducing to Japan revolutionary statistical methods in cancer epidemiology, such as proportional hazard models, relative risk regression models and cancer maps. He thought the workshops played a role in raising the frequency of case-control studies reported at the annual meeting of the Japan Epidemiological Association from zero to 20 percent of all presentations. He then discussed and exemplified the need for research into measurement errors and their impact on cancer risks, and the need to develop methods to adjust for measurement errors.
Childhood Cancer: In 1965, before there were regular U.S.-Japan workshops on cancer, Hideo Nishimura and Robert W. Miller co-organized a workshop on a) childhood cancer and b) birth defects. It familiarized scientists of both countries with one another, to the great advantage of teratology and of childhood cancer registries in Japan (Miller RW, Nishimura H. Science 1966; 151:357-8). A registry had been established in Tokyo by Tadao Takatsu and Noboru Kobayashi, his successor at Tokyo University. Dr. Kobayashi, in reporting some observations from the 26,000 cases in the Childhood Cancer Registries in Six Metropolitan Areas of Japan since 1969, stated that among children with NF-1, 12 had leukemia and 8 had rhabdomyosarcoma, confirming previous reports by NCI. The leukemias have been FAB-classified for the first time, and the rhabdomyosarcomas raise the question, why should this neoplasm arise in a neural crest disorder? Lenkemia, whose relationship to NF-1 was equally puzzling in the past, is now thought to be due to mutation of a small gene (EVI2) within the large NF1 gene (Cawthon RM et al: Cell 1990; 62:193-201; Wallace MR et al: Science 1990; 249:181-6).
The registry data also show that Japanese children now exhibit a peak in the incidence of acute lymphocyic leukemia at 4 years of age. This peak started to form in the 1960s (Miller RW: Tohoku J Exper Med 1967; 91:103-7), 20 years after it arose in the U.S. and 40 years after it arose in Great Britain. Registry data are needed to depict it because incident cases must be graphed by single year of age to see the peak.
The rate of Wilms’ tumor in Japan, noted to be half as common there as in western countries, has since been found to be similar to that in other Asian countries (Stiller CA, Parkin DM: Br J Cancer 1990; 62:1026-30). Study was suggested of DNA adjacent to the WT-1 locus in Japanese as compared with the U.S., for differences which may influence the mutation rate.
It was brought out that dysgerminomas of the pineal gland are substantially more common in Japan than in the U.S. (Koide O et al: Cancer 1980; 45:2119-30). Germ cell tumors also occur excessively in Klinefelter’s syndrome (KS); 5 have been in the brain, 2 of them in Japanese (reviewed by Miller RW: Princess Takamatsu Symposium 1 988; 18:3-12). Question: is the excess of germ cell tumors of the brain in Japan related to KS? The answer can be determined by obtaining buccal smears from patients with pineal tumors and examining them for the presence of Barr bodies.
Cancer at 15-29 years of Age: Robert W. Miller (NCI) reported on a workshop held in 1985 on cancer under 30 years of age. At 1 5-29 years of age cancers were substantially less frequent in Japan than in the U.S., notably Ewing’s sarcoma, melanoma, NHL, Hodgkin’s disease, and cancers of the breast, uterine cervix, testis, brain, and thyroid. Only stomach cancer was more common in the Japanese. Equal frequencies were observed for osteosarcoma, ovarian cancer and colon carcinoma (At cancer centers in the U.S. a substantial number of young adults is obvious in the waiting rooms. The scene must be quite different in Japan.) New data should be reviewed at intervals to see if an increase is occurring in Japan which may be due to the environment:
Subtypes and Subsites: Workshops were held in 1984 and 1990 on geographic pathology and the importance of subtypes and subsites in cancer epidemiology. Haruo Sugano (Cancer Institute, Tokyo) stated that in Japan the rate of pancreatic cancer of a nonendocrine type has increased in Japan from 1.8 to 5.2 per 100,000 from 1960 through 1989, possibly due to better diagnosis. Subtypes of pancreatic tumors have been newly recognized by his group though advances in diagnostic techniques, including ultrasonography, CT scan and endoscopic retrograde cholangiopancreatography. Among these tumors are ductectatic-type mucinous cystadenomas and cystadenocarcinomas. They were analyzed for K-ras activation because pancreatic carcinomas of exocrine origin frequently have a point mutation at codon 12. Such activation, found again in these new entities, may be a feature in common in pancreatic exocrine carcinogenesis. In Japan the commonest point mutation in pancreatic cancers resulted in a glycine to aspartic acid transition, whereas in western countries other amino acid substitutions were found. Whether this geographic difference is inherent or environmentally induced remains to be determined.
Cancer Syndromes with Small Bowel Adenocarcinoma: Familial cancers and the genetics of human cancer have been recurrent topics for workshops over the years. Grant N. Stemmermann (Kuakini Hospital) described a study of adenocarcinoma of the small bowel in Hawaii. Four of the cases, all Japanese-American males, had multiple gastrointestinal cancers. Three of the men had several family members with gastrointestinal cancers; the family history of the fourth man was unknown. Family members in Japan might be sought with help from RERF experts in such tracing. Files of cancer centers in Japan could be searched for similar cases, keying on adenocarcinoma of the small bowel. It was suggested that paraffin blocks be used for DNA studies, and they have since been sent to Dr. Warren Nichols at Merck, Sharp and Dohme. Dr. Stemmermann also mentioned a family in which 6 members had gastric carcinoma, another adult had adrenal cortical carcinoma (ACC, and another had leukemia ACC and leukemia suggest a link to the Li-Fraumeni syndrome, which should be explored by molecular studies.
Discussion of this family led to the suggestion that a search be made for stomach cancer in the families of the 47 children with ACC studied by Tsunematsu et al: Japan J Cancer Rcs 1991; 82:893). If found, look for a germline p53 mutation. Also seek this mutation in any gastlic cancer family in which one or more members has a cancer other than gastric, preferably of types in the Li-Fraumeni syndrome.
Li-Fraumeni Syndrome: In 1988 a workshop sponsored by the U.S.-Japan Program (Miner RW et al. Japan J Cancer Res 1988; 79:1155-8)) kindled interest among the Japanese in the Li-Fraumeni syndrome (LFS) of diverse familial cancers (osteosarcoma, soft tissue sarcomas, breast cancer, brain tumor, leukemia and adrenocortical carcinoma). LFS had been reported in the U.S. and England, but not Japan. Japanese participants brought data to the workshop concerning familial cancers of the type seen in LFS, and several typical pedigrees were identified. In pedigrees presented by Dr. Yukiko Tsunematsu of the National Children’s Medical Research Center in Tokyo the index cases had adrenocortical carcinoma She and others subsequently published a series of these families drawn from the All-Japan Children’s Cancer Registry (Tsunematsu Y et al. Japan J Cancer Res 1991; 82:893-900). Following the workshop she spent a month studying the syndrome in the U.S., primarily with Dr. Louise Strong at the M.D. Anderson Cancer Center. In 1990 several families with the syndrome in the U.S. were found to have germline mutations of the p53 (tumor suppressor) gene (Malkin D et al. Science 1990; 250:1233-8).
At the current workshop David W. Yandell of the Massachusetts Eye and Ear Infirmary in Boston reported that he had collaborated with his Japanese co-worker, Dr. Junya Toguchida, and others in studying the prevalence and spectrum of germline mutations of the p53 gene among 196 patients with sarcoma, 167 of whom were Japanese. There were 100 control patients from each country who did not have cancer or a known family history of cancer. DNA from peripheral lymphocytes revealed germline p53 mutations in 5 patients with an unusual personal or family history of cancer, 3 with sarcoma but no known family history of cancer, and none among the 200 controls. Four mutations caused amino acid substitutions and 4 caused stop codons. Three mutations were novel in that they occurred outside the conserved domains of the p53 gene. The study revealed that p53 germline mutations are rare in the general population, and were found in this series only among a minority of patients with a personal or family history of multiple cancers, some of which differed from the 6 types previously reported as constituents of LFS. The report has since been published (Toguchida J et al. N Engl J Med 1992; 326: 1301-8).
Takao Sekiya (National Cancer Center Research Institute) described his Single-Strand Conformation Polymorphism (SSCP) analysis of DNA fragments, which can reveal not only single-nucleotide substitutions but also deletions or insertions of one to several nucleotides. A DNA chain length of less than 300 base-pairs is amplified and labeled by the polymemse chain reaction. This method was applied to genomic DNA from fresh tissue samples and from paraffin-embedded tumor samples from one typical and one atypical Li-Fraumeni families in which the index cases had adrenocorticai carcinoma The proband in one family was found to have a single-base deletion at the first nucleotide of codon 307 in exon 8 resulting in a premature stop codon. In the other family, tumor tissue from the proband and the father’s astrocytoma both had an A to C transversion at codon 286 in exon 8 (Sameshima Y et al. JNCI In press).
Colorectal Cancer Gene at 5q21: Yusuke Nakamura (Cancer Institute, Tokyo) stated that familial adenomatous polyposis (FAP) is about three times more common in Americans than in Japanese. His group identified the gene, known as the APC gene, at chromosome 5q21. In a study of 100 patients with FAP the gene was divided into 31 segments. Ninety-one germline mutations were found in unrelated patients. The mutations by type included 27 point 23 nonsense, 4 missense and 23 frameshift (particularly from codon 1-14). Ten percent of nucleotide changes were found in codon 1309 in Japanese, U.S. blacks and U.S. whites. A change from TTCA to TTAA easily identifies people destined to develop FAP, allegedly with 100 percent accuracy. If the test is negative, there is no need for a colonoscopy (Miyoshi Y et al. Proc Natl Acad Sci USA, in press).
Breast Cancer Genes: Dr. Nakamura presented his work on a newly recognized 17q21 breast cancer gene, the importance of which was not brought out at the meeting. When scientists from previously uncrossed disciplines participate in a meeting like this, new ideas can be generated - or, as here, important information may be underestimated. Dr. Yandell pointed out such a failure in a letter after the meeting. Ideally, the thoughts in the letter should have been in the discussion at the meeting: “I would like to reiterate how important Yusuke Nakamura’s finding of a 17q21 breast cancer gene is ..... He told me a number of things in private ..... First, his finding of a point mutation on one allele and loss of the second allele in at least one breast tumor is strong evidence that this is another tumor suppressor gene. Second, he is a great wizard at linkage, and if he says that this gene is tightly linked to the markers previously seen to segregate with the disease in breast cancer families, this is very compelling. Third, he found mutations of one sort or another in more than 50% of breast tumors, and particularly in intraductal tumors. In my experience, this is very significant because breast tumors are so contaminated with stromal material that a significant fraction of the samples .... will test as false negatives (thus 50% is most likely an underestimate). Finally the homology with the NF1 gene is very significant. In short; I think it is likely that this gene could be responsible for a significant fraction of familial breast cancers (and also, by analogy to RB, perhaps a significant fraction of simplex disease with pre- or peri-menopausal onset).
“The social implications of this finding (assuming that my speculations are even partly correct) are almost greater than the scientific implications ..... [T]his gene is small and hence diagnostic testing will be comparatively easy (about 1/3 to 1/2 the work of testing p53, which is about l/3 the work of testing RB, which is about 1/3 the work of testing the colon cancer gene APC). This means that many labs will be able to do family testing and, unfortunately, that many will also do it wrong (with disastrous consequences). The intrinsic difficulty in testing RB and APC has [limited the use of these procedures] so far; the complex process that we've seen begin in the last year for p53 testing, because this is near the limit of feasibility in a fairly large number of research labs, is hence just the tip of the iceberg of what we can expect from a familial breast cancer gene.”
Dr. Nakamura said that he was handicapped by a lack of specimens for study, because familial breast cancer is rare in Japan. Dr. Tucker subsequently searched the NCI Family Studies file for appropriate material, and has written to Dr. Nakamura as to what is available.
Distinguishing Chemical from Spontaneous Tumorigenesis: At a workshop in 1990 on high-risk subsites for aerointestinal-tract cancer, Warren W. Nichols (Merck, Sharp and Dohme, West Point, PA) described two methods his group uses to distinguish chemically induced from spontaneous liver tumors in CD-1 mice. In one method, PCR-amplified tumor DNA was sequenced in regions encompassing codons 12, 13 and 6 1 of threo genes (Ha-ras. Ki-ras and N-ras). Four chemical carcinogens were used: DMBA, DEN, AAB and MF. There was a low frequency of ras mutations in spontaneous CD-1 liver tumors, but among the three groups with tumors induced by chemicals, a higher frequency of ras activation was observed. Other studies revealed mutations of only a few tumor cells indicating a late event, of possible importance in tumor progression rather than initiation. The other method uses DNA fingerprinting (Ledwith BJ et al. Cancer Res 1990; 50:5245-9). Each tumor was assayed for genomic rearrangements by comparing its DNA fingerprint with the DNA fingerprint of its normal tissue counterpart. Changes in the bands of minisatellites were sought and more frequently found in chemically induced than in spontaneous CD-1 liver tumors.
He described the programs made since 1 990. In point mutation analysis of the ras genes in mice, based on only 1 g of DNA, multiplex PCR can be used for amplification and direct simultaneous sequencing of homologous sequences (Manam S, Nichols WW. Anal Biochem 1991; 199:106-11). CD-1 mouse lung tumors, whether spontaneous or DNBA-induced, had similar high frequencies of Ki-ras but not of Ha- and N-ras gene activation (7 of the mutations in the DNBA group differed qualitatively from those found when the tumor occurred spontaneously). DEN apparently induces these tumors by a different mechanism as evidenced by a substantially lower frequency of Ki-ras mutations (Manum S et al. Mol Carcinog, in press). In CD-1 mouse liver tumors, 4 genotoxic carcinogens each caused increased frequency and different profiles of ras mutations than that found in spontaneous tumors. The findings were consistent with their role in initiation. Additional findings indicate that mouse hepatocarcinogenesis involves multiple steps, one of which is ras gene activation that can occur in a differential time-sequence in different tumors. This approach may prove of value in screening animals for carcinogenesis of new drugs, and in the human for etiologic studies of cancer clusters, or in linking an individual cancer to a particular exposure.
Overview: In this updating of the best of past workshops, the crossing of disciplines was particularly effective. Participants primarily interested in environmental carcinogenesis, could appreciate the important role of inherent susceptibility, brought out by ethnic differences in cancer rates. Laboratory scientists could find in these differences ideas for their research, as could epidemiologists, who in addition heard about the most recent molecular findings in studies stimulated by their clinical and epidemiological studies.
There was a call for cross-cultural studies, possibly with special funding.
With regard to high rates of adenocarcinoma of the lung among Polynesians, it was suggested the metabolic phenotypes be studied, perhaps in comparison with Caucasians and Japanese-Americans in Hawaii. Current research at NCI should soon reveal if debrisoquine metabolic phenotype is a risk factor for lung cancer, and information from a cross-cultural study would be informative. Another suggestion, by Dr. Sugano, was that the time may have come for preparing a monograph on differences in cancer occurrence in Japan and the U.S. The workshop generated 18 suggestions or recommendations for research, which were sent to the participants and others who may implement them.
(2) Workshop on “Genetic Analysis of Hepatocarcinogenesis”
Introduction
The US-Japan Joint Workshop on Genetic Analysis of Hepatocarcinogenesis was held at the East-West Conference Center in Honolulu, Hawaii, on February 8-9, 1992. The conference was organized by Drs. Tomoyuki Kitagawa (Cancer Institute, Tokyo, Japan) and Norman Drinkwater (University of Wisconsin, Madison, Wisconsin, USA), and included seven investigators from Japan and six from the United States. The primary objective of the meeting was to bring together researchers working on the genetic regulation of both viral and chemically-induced hepatocarcinogenesis in both the human and experimental models in a format that maximized scientific interactions and discussion. The topics discussed at the meeting included mechanisms for the induction of liver cancer by hepatitis B virus, the roles of cellular oncogenes and tumor suppressor genes in the pathogenesis of liver cancer, genetic modulation of susceptibility to hepatocarcinogenesis, and alterations in gene regulation and cell proliferation in preneoplastic and neoplastic hepatic lesions.
Meeting Summary
Although hepatitis B virus (HBV) is causally associated with the development of a high proportion of hepatocellular carcinomas in humans, the mechanisms by which this virus contributes to tumor induction are unclear. Dr. F. Chisari (Scripps Research Institute, LaJolla) described a murine model for HBV-induced disease in which transgenic mice carrying the HBV surface antigen gene linked to the albumin promoter expressed high levels of the HBV protein in hepatocytes. These animals developed severe liver damage by 4 months of age and all of the mice developed hepatocellular carcinomas between 12 and 21 months of age. The onset of liver tumors was influenced by the genetic background of the animals, with an increase in latency observed when the transgene was carried on a C57BL/6J strain relative to the original lines derived from a hybrid between SJL and C57Bu6J mice. Perinatal treatment of transgenic animals with chemical carcinogens accelerated the development of liver tumors. Expression of the transgene resulted in a significant increase in both cell proliferation and the production of reactive oxygen species in the liver relative to that observed in control animals. The combined effects of enhanced cell replication and DNA damage induced by reactive oxygen species may play a causal role in the development of liver tumors in this model system. Dr. O. Hino (Cancer Institute, Tokyo) discussed an alternative mechanism for the induction of genetic alterations in cells infected by HBV in which integration of the viral genome could increase the rate of recombination or gene rearrangement at sites distant from the site of integration. In his studies, incubation of plasmid DNA molecules containing sequences derived from the HBV genome with extracts obtained from dividing mammalian cells resulted in an increased rate of recombination between the plasmid targets when compared with control plasmids not containing HBV sequences. Cell extracts derived from non-dividing tissues were not active in promoting plasmid recombination. The binding of proteins derived from active cell extracts to specific HBV DNA sequences was demonstrated by a filter-binding assay. Dr. C. Seeger (Institute for Cancer Research, Philadelphia) described an approach to identifying the viral determinants of HBV carcinogenesis. The abilities of various HBV species to induce liver tumors in their natural hosts varies considerably. Hepatocellular carcinoma occurs in approximately 5% of humans chronically infected with HBV with a latency period measured in decades; in contrast, 90% of woodchucks infected with Studies of liver tumor induction in woodchucks infected with recombinant viruses containing sequences derived from WHBV and GSHV will allow the identification of specific viral sequences that are responsible for the difference in pathogenicity.
Studies of hepatocarcinogenesis in rodent models have demonstrated that the role of mutations in cellular proto-oncogenes in the development of the tumors depends on the species and strain of animal as well as on the nature of carcinogenic treatment Dr. M. Anderson (NIEHS, Research Triangle Park) observed that a high proportion (60-90%) of spontaneous and chemically-induced liver tumors in C3H or B6C3F1 mice carried mutant H-ras genes. The dependence of the sequence of the activating mutation in the H-ras gene on the chemical used to induce the tumor is consistent with a model in which these mutations result in initiation of carcinogenesis. However, a significantly lower frequency (10-25%) of H-ras activation was observed in spontaneous or induced liver tumors in C57BU6 mice, indicating that an alternative pathway for initiation results in hepatocarcinogenesis in this inbred mouse strain. In tumors induced in C57BL/6 mice by vinyl carbamate, the frequency of H-ras activation decreased with increasing doses of the carcinogen. Dr. T. Kitagawa (Cancer Institute, Tokyo) reported that H-ras activation may also contribute to the progression of hepatic tumors in mice. Transgenic mice that express simian virus 40 (SV40) large T-antigen from the albumin promoter consistently developed hepatocellular carcinomas between 5 and 7 months of age. Analysis of tumors obtained at 6 months of age by PCR and oligonucleotide hybridization revealed that approximately 40% of the tumors also contained activating mutations in the H-ras gene. Analysis of cell lines established from tumors also demonstrated that the acquisition of H-ras mutations was associated with progression. Allelotype analysis of mouse liver tumors indicated that more than 80% of the tumors examined lost heterozygosity for a region of chromosome 2. Dr. K. Ogawa (Asahikawa Medical College, Nishikagura) described studies directed toward identifying genetic alterations occurdng during hepatocarcinogencsis In the rat In contrast to studies in the mouse, H-ras mutations were not observed in rat liver tumors. In a series of more than 100 liver tumors induced by treatment of rats with N-methyl-N-nitrosourea, approximately 5% of the tumors carried mutations in the K-ras gene. Mutations in the p53 gene were observed in a small number of tumors. Critical genetic alterations were not identified in the majority of rat liver tumors.
Mutations in the p53 tumor suppressor gene have been observed in significant proportions of a wide variety of human malignancies. Dr. C. Hanis (NIH, Bethesda) discussed the relationship between HBV inflection and atlatoxin exposure in causing hepatocellular carcinomas in some regions of China Combined exposure to these agents resulted in a 60-fold increase in the risk for the development of liver tumors. Analysis of the p53 gene in tumors from these patients demonstrated that approximately 50% of the tumors contained a specific G to T transversion mutation in codon 249 of the p53 gene. In areas of China with low levels of exposure to atlatoxin, 50-80% of the tumors carried mutant p53 genes but only 6% of the mutations were in codon 249, indicating that the aflatoxin exposure played a causal role in the induction of the transversion mutation at this site. Dr. Y. Murakami (National Cancer Center, Tokyo) reported on studies of the p53 gene in liver tumors from a series of patients in Japan. In this population, the prevalence of p53 mutations depended on the stage of progression of the disease. Among 21 early hepatocellular carcinomas, none of the tumors demonstrated mutations in the p53 gene, while 8 of 22 advanced tumors contained p53 alleles with mutations at various sites. In the tumors with p53 gene mutations, a high proportion also had genetic alterations in the Rb (retinoblastoma) gene. Dr. Y. Nakamura (Cancer Institute, Tokyo) provided evidence for the involvement of novel tumor supressor genes in the development of hepatocellular carcinoma in humans. Frequent loss of heterozygosity was observed in liver tumors for chromosomal regions 5q, 11p, 16q, and 17p. Genetic alterations in the latter two regions were also observed in a significant number of breast tumors. As model for the molecular analysis of tumor suppressor genes, he also described detailed molecular studies of the Apc gene, for which inherited mutations are the cause of familial adenomatous polyposis.
Studies of hepatocarcinogenesis in experimental animals have demonstrated that the genetic background of the host plays an important role in determining the risk for the development of liver cancer. Dr. N. Drinkwater (University of Wisconsin, Madison) discussed genetic studies of hepatocarcinogenesis in three highly susceptible inbred mouse strains. The C3H mouse is up to 100-fold more susceptible to spontaneous and chemically-induced liver tumors relative to C57BU6 mice as a consequence of the action of a single locus the Hcs gene, which regulates the proliferation of both preneoplastic and normal hepatocytes. The DBA/2 mouse has a low spontaneous incidence of liver tumors but is highly susceptible to perinatal induction of hepatomas by treatment with chemical carcinogens A single locus unlinked to the Hcs gene, is responsible for this phenotype. Evidence for a third gene that controls susceptibility to hepatocarcinogenesis was pro vided by studies of the C57BR mouse, in which the sensitivity to liver tumor induction is expressed primarily in female mice. Dr. N. Takeichi (Hokkaido University School of Medicine, Sapporo) described the development of liver disease in LEC rats. As a result of an autosomal recessive mutation, hts, these rats develop an acute hepatitis by 4 months of age, and surviving animals suffer from chronic hepatitis with the subsequent development of hepatocellular carcinoma Hepatic disease in these animals was linked to the accumulation of abnormally high levels of copper in the liver. The high levels of free copper can promote oxidative damage to DNA. Hepatic DNA isolated from mutant LEC rats contained significantly higher levels of 8-hydroxy-guanine relative to DNA isolated from the related non-mutant strain of LEA rats. This oxidative damage to DNA may contribute to both the development of hepatic necrosis and liver tumors in LEC rats.
Although alterations in gene expression are essential features in the development of preneoplastic hepatic lesions, the key regulatory factors that mediate these changes in transcription have not been identified. Dr. M. Muramatsu (University of Tokyo, Tokyo) discussed the regulation of the glutathione transferase P (GST-P) gene. Increased expression of the GST-P gene is the earliest and most consistent alteration in gene expression observed in preneoplastic lesions in rat liver. The promoter region of this gene contains two enhancer elements and a silencer region within the 3 kilobases upstream of the transcription start site. Specific protein factors that bind to these sites have been purified and one of the factors that binds to the silencer site was shown to be identical to a previously cloned transcription factor, NF-I16. The 3 kb promoter region is sufficient to confer the altered regulation observed for the GST-P gene in liver tumors. Transgenic rats carrying a chimeric gene consisting of the GST-P promoter and the chloramphenicol acetyltransferase coding sequence were constructed and treated with hepatocarcinogens. All of the induced hepatocellular carcinomas were found to express the transgene at high levels, while no expression was detected in surrounding normal liver. Dr. R. Jirtle (Duke University, Durham) discussed the possible role of TGF-!
!!in the promotion of hepatocarcinogenesis by phenobarbital. In normal hepatocytes, treatment with phenobarbital results in an increased uptake of TGF-!!!as a consequence of increased expression of the mannose6-phosphate/IGF2 receptor, while expression of this receptor is diminished or absent in neoplastic cells. The increase in intracellular levels of TFG-!!!may result in the inhibition of replication of normal hepatocytes, thus selecting for the growth of preneoplastic cells. Conclusions
This joint US-Japan workshop was highly successful in meeting the objectives stated above. The keys to this success were the small number of participants and the breadth of their research interests related to hepatocarcinogenesis. Discussion during and following each presentation was lively and extensive, involving in each case a large proportion of the participants. In particular, the strengths of various genetic, molecular, and biological approaches to understanding hepatocarcinogenesis, and the parallels and distinctions in human and experimental liver cancer were addressed. Equally important, this workshop provided a rare opportunity for investigators with similar research interests from Japan and the US to share research results and ideas through direct interaction. Although information regarding new collaborations that resulted from the meeting is not available, many of the participants subsequently remarked to the organizers that the workshop provided them with fresh perspectives regarding their research and that they found the meeting to be highly worthwhile.
SEMINAR AGENDA AND PARTICIPANTS
(1) SEMINAR ON “U.S.-JAPAN DIFFERENCES IN CANCER EXPERIENCE”*
March 12-13, 1992
Maui Marriott, 100 Nohea Kai Drive
Lahaina, Maui, HI 96761-1989
* Based largely on some of the best reports, updated, from past workshops of the Interdisciplinary Area, US-Japan Cooperative Cancer Research Program
AGENDA