Histology	Japan	Hong Kong
Squamous cell	18%	27%
Adenocarcinoma	60	38
Small cell	9	21
Large cell	5	6
Other	4	8
Total	100	100
Number of cases	479	497

In 1984 cigarette smoking was reported by 65.5% males in Japan vs 32.8% in Hong Kong. The rates for women were 14.0% and 4.1 % respectively.
In another study 300 pairs of school children and their mothers were questioned about indoor air pollution, passive smoking and respiratory symptoms. Personal exposure to nitrous oxide was measured by a badge, and the urine was examined for the hydroxy-proline/creatinine ratio and cotinine. The air pollution in the two locales was similar. Stemmermann said that the same differences in cell types were noted in Chinese vs Japanese women in Honolulu, and the environment was not the cause.
Teratomas, Moles and Fragile Sites
In speaking on ovarian teratomas, Hecht (Arizona) said that studies of enzyme poly-morphisms showed that tumor tissue is frequently homozygous at loci for which the host is heterozygous. The same pattern is seen in centromeric chromosome markers. These findings suggested that ovarian teratomas arise from a single germ cell after the first meiotic division; ie, by parthenogenesis. More recent studies indicate three likely mechanisms for the origin of ovarian carcinomas: from a germ cell that has failed to undergo meiosis I; a failure subsequently but before meiosis II; or from a haploid egg that undergoes endoreduplication to give a normal 46, XX karyotype (Parington, JM et al: Med Genet 21: 4-12, 1984).
He went on to describe the three mechanisms that give rise to hydatidiform moles. In 90% of complete moles (molar tissue only), a sperm enters an empty ovum and duplicates itself. In 5% two sperm enter an empty ovum and fuse. Rarely the sperm and ovum produce a mole instead of an embryo.
With respect to fragile sites on chromosomes Hecht said that their relation to cancer is as yet unknown, but in some cancers there are structural chromosomal rearrangements with highly specific break-points. The relationship to fragile sites is not yet clear. McCaw-Hecht (Arizona) stressed that some fragile sites are almost always seen in properly prepared karyotypes. If they are missing, the quality of the preparation and interpretation of other cytogenetic findings should be questioned.
The uneven geographic distribution of chromosomal abnormalities reflects the un equal distribution of cancer internationally, she said. A series of workshops on cytogenetics, histology, immunology and pathology of CML and ALL revealed that earlier reported geographic variation in karyotypes for specific cancers had disappeared. The differences had been due to dissimilarities in cytogenetic techniques. Miller asked about the rate in Japan of Wilms' tumor and of the 11p- syndrome associated with it, both half of what they are in Western countries. This might reflect resistance of the short arm of chromosome 11 in the Japanese to the deletion that gives rise to the syndrome and neoplasm.
Summary
There are many opportunities for research into cancer etiology in Southeast Asia because of differences in cancer occurrence and high-risk groups in Japan as compared with other countries in the area. It appears to be best to move from clinical observations to epidemiologic and laboratory research rather than try to formulate hypotheses solely on epidemiologic observations.

(2) Seminar on "Recent Advance in Bladder Cancer Research"
The joint seminar in the Interdisciplinary area was held on the subject "Recent Advance in Bladder Cancer Research" at Nagoya Castle Hotel in Nagoya, Japan on March 24-25, 1986. The meeting was convened by Dr. Samuel M. Coen Department of Pathology and Microbiology, University of Nebraska Medical Center and Dr. Nobuyuki Ito, Department of Pathology, Nagoya City University Medical School.
Epidemiology:
Dr. Yoshiyuki Ohno presented an extensive overview of bladder cancer in Japan and also included some data regarding the epidemiology of renal pelvic cancer, a relatively uncommon tumor in Japan. Parameters evaluated by Dr. Ohno and presented at the meeting included the proportional rates of bladder cancer amongst all cancers on an international basis, as well as by age and diagnosis, and also the age-adjusted incidence rate. The range for the age-adjusted incidence rate was as low as 2.4 and as high as 30.2 per 100,000. Japan continues to be amongst the lower incidence rates and the United States is in the approximately middle to high end. A male predominance was usually observed, and there was usually an urban predominance over rural. He then focused on comparisons between the United States and Japan and showed that the rates have changed over the last thirty years. He also emphasized that migration studies tend to show that the Japanese in the United States, either Hawaii or California, have an increased rate of bladder cancer com-pared to the Japanese in Japan. He also indicated that the bladder cancer incidence in Japan is rising, and he felt that this could only be partially attributed to the increase in cigarette smoking occurring in Japan.
Dr. Alan Morrison then presented information concerning specific etiologic agents with respect to bladder cancer. Much of his data included results from an extensive international study which he and Dr. Ohno participated in comparing Boston, Massachusetts, Nagoya, Japan, and Manchester, England. Cigarette smoking has been shown to be a major risk factor for the development of bladder cancer in these three cities and in Western civilization in general. Attributable risk to cigarette smoking was approximately 50% in Boston, 30% in Nagoya, and 46% for males and 14% for females in Manchester, England. In general, there is a dose-response relationship with respect to cigarette smoking in the incidence of bladder cancer. Cigar smoking has not been found to be related to bladder cancer, but for pipe smoking there is a questionable risk. He then discussed the role of occupation as an etiologic factor in bladder cancer and stated that earlier studies were predominantly retrospective, whereas more recent studies have utilized the case-control method. Using this method, the details concerning occupation often are not well documented and specific tasks related to exposure are even less well documented. Nevertheless, specific occupations have been identified as having an appreciable risk of developing bladder cancer, including leather workers, truck drivers, tool and dye makers, and auto and truck mechanics.
Dr. Morrison then presented extensive data concerning the role of coffee as an etiologic factor in bladder cancer' and indicated that cigarette smoking is a major confounding factor with trying to determine the role of coffee. At this time, there is generally a positive relative risk found, but it is very low (1.0-1.6) and this may be attributable to the confounding factor of cigarette smoking. This remains a controversial area. In contrast, the numerous, extensive studies with artificial sweeteners indicate no etiologic relationship to the development of bladder cancer. Analgesic abuse has been linked to the development renal pelvic carcinoma, but, also, more recently, to bladder carcinoma. Dietary factors have received little close attention for an etiologic role in bladder cancer, but Bracken fern has been evaluated and shows no evidence of being a human bladder carcinogen. Vitamin A has been studied to some extent, but there is no clear relationship to the development of bladder cancer.
Dr. Robert Miller presented some graphic material illustrating that the death rate from bladder cancer in the United States has not been as high as one would have anticipated based on age-adjusted incidence rates from the 1950s. This appears to be primarily due to changes in individuals under the age of 65. It is not clear what the reason for this less than expected incidence rate is, but better responses to therapy might be one explanation.
Metabolism of Carcinogens:
Dr. Masashi Okada presented a summary of his extensive studies concerning the organ specificity of various nitrosamines, particularly as they relate to BBN. BCPN is the primary urinary metabolite of BBN. Dr. Okada and his colleagues have carried out extensive metabolite and carcinogenicity studies relating not only carcinogenic potency, but target organ specificity. Several of the related nitrosamines were shown to induce liver and esophagus cancer, but not bladder carcinoma. It has been assumed that alpha-hydroxylation is a critical step in the metabolic activation of nitrosamines. The lactone formed from the alpha-hydroxylated BCPN has been shown to be relatively stable, but it induces lung carcinomas in rats when given subcutaneously rather than bladder cancers. Also, this lactone has not been identified in the urine of rats given BBN. In response to questions, Dr. Okada state that it is not yet clear what the structure specificity of the nitrosamines are as related to bladder carcinogenicity. DNA adducts have not yet specifically been identified.
Dr. Terry Zenser then presented the results of extensive studies comparing the P450 metabolic activation of various aromatic amines as compared to the metabolic activation via the hydroperoxidase portion of the prostaglandin H-synthase enzyme complex. A brief summary of prostaglandin metabolism was presented, and then detailed studies of various aromatic amines, particularly benzidine. Benzidine is poorly activated by the liver, but is extensively metabolized by bladder, considerably more so than the inner medulla which has greater activity than the outer medulla of the kidney. The acetylated analog of benzidine is metabolized by P450 but benzidine itself is not metabolized by P450. Specific DNA adducts have been identified following peroxidase activation of benzidine, which occurs either via a free radical or via a diimine intermediate. Also of considerable interest was the recent finding by Dr. Zenser and his colleagues that 2-naphthylamine is metabolized not only to the N-hydroxylated naphthylamine, but to 1-hydroxy-2-naphthylamine. P450 activation leads only to the N-hydroxyl compound, whereas DNA adducts related to 2-amino-1-naphthol have been identified following peroxidase activation and in the target organ, the dog bladder. Lastly, the in vivo and in vitro studies with the nitrofuran, FANFT, and its urinary metabolic product, ANFT, were presented. ANFT is not metabolized by P450, but is readily activated by PHS. Also, FANFT bladder carcinogenesis is inhibited partially by co-administration with aspirin.
Pathology:
Dr. Nobuyuki Ito reported results from the Japanese National Autopsy Registry, and compared the results from 1964 through 1968 to the cases from 1982 through 1983. Metastases appear to be more frequent in the more recent cases, and this may be related to the longer survival of the more recent cases. He then presented the results of 311 bladders evaluated at autopsy less than 5 hrs after death. These were all normal-appearing bladders in patients without bladder cancer. There were 190 males and 121 females and were of an age of greater than 20 years old or older. Brunn's nests occurred in 80-100% of cases at all ages and occurred in all areas of the bladder. Squamous metaplasia, however, was predominantly in females with increasing incidence with age, and occurred predominantly in the trigone area. Hyperplasia was relatively infrequent and increased with age. Nevertheless, it was relatively uncommon even at older ages, with an incidence of less than 20%. Dysplasia was very uncommon and increased with age, appearing after age 50. Only one case of carcinoma in situ was detected. A histogenetic pathway was postulated.
Dr. Eigoro Okajima presented the results of his extensive studies of carcinogenesis in dogs. He has utilized as carcinogens, BBN, BCPN, and EHBN. Papillary carcinomas occurred predominantly with BBN, but non-papillary, invasive carcinoma has occurred more commonly with the other two nitrosamines, although the number of dogs is relatively small. These studies required 2–4 years for development of tumors. In a more recent study, the combination of methyl-nitrosourea instilled intravesically followed by BBN was tried to accelerate the process and also to evaluate the effect on tumor grade. It was observed that within one year with MNU followed by BBN, there was extensive evidence of dysplasia and carcinoma in situ in the bladder epithelium. It was usually multifocal as well. Dr. Pauli suggested that this model might be very useful in finally resolving the many questions still concerning the biological potential or carcinoma in situ in the bladder.
Dr. Bendicht Pauli presented several studies relating to possible mechanisms by which bladder cancer becomes invasive. Three possible mechanisms were discussed. First was invasion occurring entirely due to an increased pressure, with tumor cells gaining access to the stroma by increased pressure through the weakened areas between the collagen and protoglycan bundles. This was not considered to be of great importance in bladder carcinoma. The second mechanism was an activation of proteolytic enzymes or an inactivation of various inhibitors of these enzymes. It was indicated that the enzyme, collagenase, was probably important in remodeling, but not in tumor invasion. A "crosslinkase" of collagen followed by the action of other proteinases was considered to be more likely important. It was noted that proteolytic activity was much greater in tumor tissue and in low passage cell lines rather than high passage cell lines. A more recently developed model by Dr. Pauli and his colleagues was also presented concerning hyaluronic acid. It appears that invasive tumors are able to stimulate fibroblasts to greatly increase the synthesis of hyaluronic acid, and possibly various proteinases. This makes the stroma more soluble and easier for tumor cells to permeate.
Oncogenes:
Dr. Hung reviewed several aspects of transfection and cloning methodology involved with the original discovery of human oncogenes in the T24 bladder cancer cell line. This was the discovery of the Ha-ras gene with activation resulting from a single point mutation from the normal ras gene. In general, examination of extensive numbers of human cells has indicated that between 10 and 30% of the bladder tumors have activated Ha-ras gene. Dr. Hung then described more recent results relating to the neu gene. The gene product is a 185 KD phosphoprotein which is serologically related to epidermal growth factor receptor protein and the v-erb B gene. It is detectable by the transfection technique and is located on human chromosome 17 q 21. EGF receptor gene is on chromosome 7. It would appear that the transforming form of this gene compared to the normal cellular gene is an abnormality in the transmembrane portion of the protein. Neu-gene has been found to be altered in 5 of 11 bladder tumor cases, 3 of 8 cell lines, and 2 of 3 primary cancers. Dr. Oyasu made the suggestion that this may be related to one of the urinary factors which he has been studying, and collaborative efforts between Dr. Hung and Dr. Oyasu were discussed. If such a relationship is found, it would bring together two very separate active avenues of research in bladder carcinogenesis. Dr. Terada reviewed the various classes of oncogenes and indicated that numerous changes.occur in these genes which might result in cancer, including translocation, amplification, and mutation. He reviewed the evidence for the various ras genes being activated in bladder cancer, and indicated that it is relatively infrequent. This is true in humans as well as in F344 rat bladder tumors induced by EHBN in collaboration with Dr. Ito's laboratory. In the rat, only 2 of 9 samples so far examined have had transforming activity. Dr. Terada also briefly described recent studies in his laboratory regarding the hst gene as related to a human stomach cancer.
Modification of Bladder Carcinogenesis:
Dr. Cohen briefly reviewed the model of initiation and promotion as originally described in the mouse skin model and as it applies to bladder carcinogenesis in animals. He then described studies in his laboratory relating to sodium saccharin as a promoting substance, and indicated the importance of cell proliferation in the process of promotion. The importance of other factors beside the promoting substance itself was clearly illustrated by the finding that sodium saccharin had considerably more activity in short-term assays than potassium saccharin, and calcium saccharin and acid saccharin were without activity. This occurred despite essentially equal concentrations of saccharin anion in the urine of the animals administered the different forms of saccharin. However, there were marked differences in urinary pH, sodium, potassium, and calcium, as well as osmolality and urinary volume. Which of these factors, if any, are directly related to the promoting activity of substances like saccharin is yet unclear. However, it was indicated that one could tie the many areas of bladder carcinogenesis research, including studies of promoting substances as well as growth factors, oncogenes, and electrolytes, by their relationship to regulation of cell growth. Intracytoplasmic pH, sodium, and calcium are well known to be important factors in the control of cell proliferation, as are a variety of growth factors and oncogenes in the bladder.
Dr. Fukushima then presented similar studies in rats regarding sodium ascorbate as a promoter, whereas ascorbic acid was without promoting activity. He also indicated that sodium bicarbonate (NaHCO₃) as 3% of the diet also had promoting activity. When sodium ascorbate was fed with ammonium chloride, resulting in an acidified urine, promoting activity was lost. He also presented results of experiments with other sodium salts and various forms of carbonate, including potassium, calcium, and magnesium carbonate, which strongly suggested that sodium or potassium levels are important in the promoting activity of these substances, but by themselves are not promoting. For example, sodium hippurate and sodium chloride were without promoting activity. He also illustrated that there were at least 3 classes of tumor promoters for the bladder. One of these is the sodium or potassium salts of a variety of substances, such as saccharin or ascorbate. A second class includes the antioxidants, such as BHA, BHT, and ethoxyquin, which do not have effects on urinary ions. Another class of bladder tumor promoters are compounds such as diphenyl and uracil which act by the induction of calculi which act as stimulants of cell proliferation.
Dr. Oyasu presented an update of the results of his studies concerning urinary factors which act as cell promoters. He originally utilized an ornithine decarboxylase (ODC) in-ducibility assay for screening for the urinary components, but has more recently found that ³H-thymidine uptake provides somewhat different results. Extensive fractionation studies have been performed in his laboratory and indicate that the factor resulting in ODC inducibility might be different from that acting as a mitogen. Electrophoresis studies still indicate that the substance which he is purifying is not yet pure. The importance of this type of substance in the urine was actively discussed by several participants, and the potential relationship to the neu gene product presented by Dr. Hung was again discussed.
Diagnosis:
Dr. Hashimoto briefly described potential uses of monoclonal antibodies as related to diagnosis with therapy. He then discussed in detail three aspects of his research involving monoclonal antibodies. The first area concerned the study of anti-rat cytochrome P450 isozymes. In particular, he has used the P-448 L and P-448 H isozymes as antigens. He found that one monoclonal antibody which was specific for the bladder isozyme, P448 H, which he designated APH-8, did not react with normal rat bladder, but reacted with blad-der epithelium beginning four weeks after BBN administration and continuing through the development of carcinoma. The usefulness of this antibody in carcinogenesis studies were briefly summarized. The next area of research that Dr. Hashimoto presented was the development of various monoclonal antibodies with specificity against human bladder cancer. These monoclonal antibodies were developed against human bladder cancer cell lines and could be separated into several categories. The first group of these antibodies were specific for the various red blood cell isoantigen markers which are present on bladder epithelial cells. The second group appeared to be related to squamous epithelium, and might be related to keratin. The third group of antibodies appeared to be specific toward epithelia and the last group were active against tumors. He has utilized several of these monoclonal antibodies to purify the actual target proteins and characterize some of these. At this point, the specificity of the monoclonal antibodies does not allow for their use in diagnosis, but extensive research is continuing. The potential usefulness of multiple monoclonal antibodies used as a battery for diagnosis was briefly presented.
Dr. Hakala then presented an overview of the various types of monoclonal antibodies against the urinary bladder available in several laboratories. The first class of these antibodies were used to distinguish urothelial cancer compared to normal urothelium. Some of these appear to have specificity toward cytokeratins. Many of the antibodies are known to react with other tumor types as well, but this would not be a problem in evaluation of patients with bladder cancer. The potential usefulness of these antibodies was again discussed, and the usefulness of administering them by the intravesical route was emphasized since this would avoid many of the pharmacodynamic problems of systemic administration. The need for the use of multiple antibodies was also emphasized. Many antigens apparently are targets of these different antibodies, and thus far, for those evaluated, there would appear to be a broad range of molecular weights for these antigens. Another group of monoclonal antibodies appears to detect antigens which are present in tumors and in normal urothelia, but detect the antigen with quantitative differences between normal and tumor. In response to questions, Dr. Hakala discussed problems with respect to the specificity of these monoclonal antibodies and their potential usefulness for clinical studies.
Therapy:
Dr. Alan Yagoda presented a summary of the various chemotherapy clinical trials and described the importance of patient selection, response criteria, and site of response in these studies. In general, it has been found that single agents can be found which induce a partial remission, but do not usually induce a complete remission. Cis platin has induced an overall response of 25-35%, and similar levels of response have been found with methotrexate. Multi agent regimens have been evaluated, and in addition to a higher level of partial remissions than with the single agents alone, have induced some cases of complete remission. The best results appear to involve the combination of cis platin and adriamycin, although methotrexate has also been useful. Hexamethylmelamine has been useful against bilharzial squamous cell carcinoma, but it has not been efficaceous against transitional cell carcinoma, the more common type seen in Western countries. He then summarized in detail the current results with his multi agent regimen entitled, "M-VAC". This includes methotrexate, vinblastine, adriamycin, and cis platin. The overall response rate has been 74%, with occasional complete remissions. In general, relapses have involved disseminated disease and 8 have had brain recurrences as the only site. Prophylactic brain radiation therapy does not appear to be indicated for this disease yet, since the dose necessary for the prevention of recurrent bladder cancer would be prohibitively toxic. Dr. Yagoda concluded with a brief summary of the combination of chemotherapy and radiation therapy, and suggested that in preliminary trials so far, that there may be a synergistic effect. The studies presented by Dr. Yagoda were certainly encouraging, first of all for the increased overall response rate, but also the suggestion that some cures may actually occur. In response to questioning, Dr. Yagoda indicated that bladder cancer has been one of the better studied solid tumors with response to chemotherapy and is actually one of the more responsive solid tumors to therapy.
Dr. Kakizoe then presented experimental and clinical studies indicating that the presence of tumor promoters in the urine may be extremely important in the management of patients with papillary carcinoma of the bladder, particularly as related to the high recurrence rate of these tumors. Utilizing a concanavalin-A agglutination assay, he has found a strong correlation between a positive result in his assay and tumor promoting activity. In particular, he identified L-isoleucine, L-leucine, and L-valine as being positive in his screening assay and he was able to demonstrate that L-isoleucine and L-leucine acted as bladder tumor promoters in male F344 rats when administered following BBN initiation. The role of these amino acids and other possible substances in the recurrence of bladder cancer in humans was discussed.
Dr. Yoshida then presented a brief description of a double soft agar assay which he has developed for evaluating chemotherapy agents, or for the evaluation of carcinogenesis. He presented data showing that three weeks of BBN followed by nine weeks of the test chemical could be utilized in this model for detection of tumor promoters. Sodium saccharin, D-tryptophan, D,L-leucine, and D,L-isoleucine were also positive in his assay. L-tryptophan was negative.
Summary:
Dr. Cohen summarized the presentations by suggesting that progress in the clinical management of human bladder cancer required a multi-disciplinary approach as evidenced by the various presentations at this meeting. In particular, new developments with monoclonal antibodies, urinary modifying factors, carcinogen exposure, and oncogenes, particularly the interaction of multiple factors, would be important in the diagnosis and treatment of the disease. Considerably more research is necessary in all of these areas, and it was suggested that a multi-disciplinary and collaborative approach to the problems of clinical bladder cancer would be most productive.



SEMlNAR AGENDA AND PARTICIPANTS

SEMINAR ON CANCER EPIDEMIOLOGY IN SOUTHEAST ASIA
Honolulu, Hawaii, December 12-13, 1985

AGENDA