SUMMARY OF ACTIVITIES

From April I to March 31, 1981, the major activities of the Cancer Biology, Immunology, and Diagnosis Program have consisted of seminars and scientist exchanges. This area of the U.S.-Japan Cooperative Cancer Research Program is divided into three components: Cellular and Molecular Biology Area; Immunology Area (Biology, Genetics, and Preclinical Immunotherapy); and Diagnosis Area (Automated Cytology and Development of New Techniques). Each of these components held one seminar during the past year. A total of 35 scientists from the United States and 33 scientists from Japan participated in these seminars. Agendas and lists of participants for each meeting appear later in this report.
The 1981 Cellular and Molecular Biology Seminar on "Genetic and Epigenetic Aspects of Cancer" was held at the Hotel Napualani in Honolulu, Hawaii, January 19 to January 21, 1981. Seven Japanese and eight United States participants attended. There were 15 formal presentations of 30 to 45 minutes, with each followed by at least 15 minutes of discussion. Discussion and exchange of ideas was extensive, and the meeting was judged a success by all participants.
Two general approaches to understanding the biology of cancer were taken. The first involved the use of animal models to study the relative role of genetic and epigenetic factors in the development of cancer. The second approach involved the use of tissue culture systems to study the molecular basis of transformation by viral agents.
The 1980 Immunology Seminar on "Analysis of Mechanisms for Inducing Tumor-Specific Immunity and Experimental Approaches for Immunotherapy" was held October 20 to October 26, 1980, in Osaka, Japan. This Second Joint Seminar of the United States-Japan Immunology Area exchange was jointly organized by Professors Yuichi Yamamura and Toshiyuki Hamaoka and Dr. Richard Hodes. The program consisted of three major segments: (1) antigenic nature of tumor cells, (2) fundamental immunologic processes in host responses to tumors, and (3) new approaches and preclinical trials for tumor immunotherapy and prevention.
The detection of tumor-associated antigens was discussed in presentations by Drs. Ronald Levy and Yoshifumi Ishii. Dr. Levy discussed the identification of idiotypic cell surface markers on lymphoblastoid cell lines derived from normal B cells or from acute lynphocytic leukemias. The ability to utilize these idiotypic markers in immunodiagnosis, as well as their potential for the targeting of immunotherapy, were discussed. Dr. Ishii presented data which demonstrate the identification of a melanoma antigen which may be detectable in the serum of tumor-bearing patients.
An extensive series of presentations discussed the fundamental immunologic processes in the host-tumor interaction. Effector mechanisms, which may play a significant role in host defense to tumor, were evaluated. Dr. C. Henney discussed the basis for target susceptibility to natural killer (NK) activity, with emphasis upon those procedures capable of enhancing NK activity, as well as determining the characteristics of certain tumor cells which appear resistant to such activity. Dr. Katsutoshi Komuro presented data which described the role of antibody dependent cell-mediated cytotoxicity (ADCC) in resistance to tumors in rats, and related this activity to circulating complement levels in tumor-bearing animals. The role played by immune regulatory mechanisms in the host immune response was evaluated in several experimental systems. Dr. Frances Noonan and Dr. Mark Greene presented evidence for the function of tumor-specific suppressor cells in the susceptibility of mice to ultraviolet (UV) irradiation induced sarcomas. The ability of UV light to induce defects in accessory cell function, and to concomitantly predispose to the generation of suppressor T cells, was demonstrated and discussed as a model for tumor-specific immunosuppression. Dr. Toshiyuki Hamaoka described a system in which a killer cell helper factor augmented the induction of cytotoxic T lynphocytes, a mechanism with potential relevance to tumor-specific cytotoxic T cell responses. Dr. Yoshiyuki Hashimoto described the production of a lymphokine with functional activity in the establishment of cytotoxic killer T cell lines specific for either alloantigens or tumor-associated antigens. The host immune response to tumors in humans was discussed by Dr. Kokichi Kikuchi who described the infiltration of human tumors by T cells and discussed the relevance of the level of T cell infiltration to clinical disease status. Dr. Kazumasa Yamada presented the result of attempts to in vitro sensitize human lymphocytes to leukemia cell surface antigens.
The final area of presentations emphasized the application of basic immunologic experimental findings on the development of new approaches to immunoprophylaxis and immunotherapy of malignant tumors. Dr. T. Tachibana reported tumor regression in tumor-bearing mice following the inoculation of these animals with an imnunogenic somatic hybrid cell population in combination with cyclophosphamide chemotherapy. Dr. David Terman described the use of Staphylococcal protein A columns as extracorporeal immunoadsorbants for the treatment of neoplastic disease, with successful regression of metastatic tumor in a dog model and with preliminary results in humans. Dr. Michael Berendt described the use of endotoxin and endotoxin-induced mediators in the regression of established animal tumors. Nonspecific immunotherapies were described by Dr. Kosei Yasumoto, who has employed regional ilnmunotherapy of autochthonous murine tumors, and Dr. Fumio Hirao, who has employed BCG and Nocardia rubra cell wall skeleton preparations and has succeeded in modifying the induction of lung cancer by chemical carcinogens in animal model systems. Drs. Takeshi Ogura and Yuichi Yamamura further described their analysis of the immunotherapeutic effects of Nocardia rubra cell wall skeleton in both preclinical and clinical immunotherapy.
The final session of this second exchange meeting was a forum discussion of the potential applications of basic immunologic findings to the generation of models for applied preclinical and clinical immunotherapy. Specific therapeutic concepts which were discussed include the use of monoclonal antibodies directed at tumor-specific antigens either for their direct antitumor effect or through the targeting of chemotherapeutic agents. The feasibility of generating cloned cytotoxic T cell or natural killer populations, which would be functional in adoptive immunotherapy, was discussed at length. The controversial role of NK cells in host defense to tumors was discussed, and the potential means of amplifying NK activity in the tumor-bearing host was enumerated. The experimental and clinical evidence for interferon-related antitumor effects was actively debated. This open discussion constituted an important part of this meeting and provided a productive and rare opportunity for exchange of infor-mation and ideas among scientists working both in basic immunology and in the clinical and preclinical application of basic science to the treatment of cancer.
A particularly gratifying aspect of this meeting was the high quality of interaction between the areas of basic tumor immunology and preclinical/ clinical immunotherapy. It has been and continues to be a unique character of these meetings that they permit the exchange of information and ideas between investigators in areas of basic immunology and investigators whose orientation is toward applied immunotherapy. Throughout the meeting, and in particular during an extensive discussion session at the end of the meeting, ideas were generated for the application of basic immunology to the development of new forms of experimental immunotherapy.
The 1980 Diagnosis Area Seminar on "Oncodevelopmental Proteins: Basic Biological and Clinical Aspects" was held December 4 and 5, 1980, in San Diego, California. Co-organizers of the Conference were Dr. William H. Fishman, President of La Jolla Cancer Research Foundation (the host institution), and Professor Hidematsu Hirai of Hokkaido University School of Medicine, Sapporo, Japan.
Three topics were discussed on the first day of the meeting: modification of oncodevelopmental gene expression with anti-AFP antibodies, hepatoma diagnosis, and radioimmunodetection of cancer.
In approaching the first topic, a Japanese team led by Dr. Hirai detailed their experiences with horse antibodies to human or rat alphafetoprotein (AFP) which exhibited apparent cytotoxic effects on cultured hepatoma cells and on transplanted rat hepatoma. Only slight effects were seen on human hepatoma or testicular tumors transplanted in nude mice. Additional work reported the use of daunomycin-linked antibodies to AFP in cytotoxicity studies in vitro and in rats bearing transplanted hepatoma.
Dr. Haruo Kaneda (Nihon University, Tokyo) reported that elevation of Serum AFP levels and development of hepatoma in rats were greatly inhibited by injection of horse antiserum to AFP. In the subsequent immunohistochemical study of such livers, fatty degeneration was seen. Somewhat disconcerting was the observation that normal horse IgG is also taken up by both hepatocytes and hepatoma cells.
In examining the mechanism of AFP gene expression, Dr. Shinzo Nishi (Yamanashi Medical School, Yamanashi), after evaluating mRNA and AFP production in a variety of biological situations, concluded that AFP synthesis is controlled at transcription with no indication so far of rearrangement of the AFP gene during ontogenesis and oncogenesis.
The hepatoma diagnosis section centered on human hepatoma. Dr. Barbara Knowles (The Wistar Institute, Philadelphia) described two human hepatoma-derived cell lines which between them secrete 30 different plasma proteins. One possesses the hepatitis B virus (HBV) genome and the other does not. Both can activate polycyclic hydrocarbons to proximate carcinogens. These cell lines thus provide significant opportunities for basic studies of chemical carcinogens and viral oncogens.
Dr. Yasuo Endo (University of Tokyo) described the complexities of the dynamics of AFP in patients with hepatitis, liver cirrhosis, and hepatoma. In hepatoma, 64 percent showed highly elevated AFP values (>10,000 ng/ml) whereas in chronic hepatitis the levels are usually normal; cirrhosis (33%) exhibited modest elevations of serum AFP. Two other observations were presented: (1) administration of pyridoxine and ATP decreased AFP levels in cirrhotics but not in hepatoma, and (2) the Con-A nonreactive fraction of AFP in hepatoma is less than 5 percent in contrast to 40 to 50 percent of patients with metastatic liver cancer.
Dr. Irwin Arias (Albert Einstein College of Medicine, New York City) stressed several aspects in viewing primary hepatocellular carcinoma: (1) geographic and sociologic differences relate to HBV status and the natural history of human hepatoma. Screening of susceptibility for hepatoma can now be considered by probing for integrated HBV DNA in patients' liver cells. (2) Serum AFP is most useful in cancer detection when the lesion is not clinically obvious. (3) High serum ligandin (GSH transferase) levels appear to correlate with well-differentiated liver cancer whereas high AFP correlates with less well-differentiated types.
The last session, which was on the radioimmunodetection of cancer, dealt with the use of heterologous radioiodinated antibodies to oncodevelopmental antigenic proteins in localizing cancer in animals and humans through exterior radioscanning. Dr. David Goldenberg (University of Kentucky, Lexington), a pioneer in such studies, reviewed his experiences with over 300 cancer patients with radioactive antibodies to CEA, AFP, and hCG. On the whole, these studies were impressive; sometimes more occult tumors 2cm in diameter were detected only by this procedure, and with each marker study there was a high correlation with true positives. Dr. Toshihiko Koji (Nagasaki University, Nagasaki) utilized petrified radiolabeled horse antibodies to human and rat AFP while Dr. Goldenberg prepared his antibodies in the goat. The results on rats bearing transplanted hepatoma and on patients with hepatoma were qualitatively similar to Goldenberg's.
In the final hour of discussion led by Dr. Erkki Ruoslahti (La Jolla Research Foundation) emphasis was placed on the need to continually test for the specificity and mechanism of the tumor-modifying effects of heterologous antibodies administered to rats or humans. In other words, are the results due entirely to the antibody to the marker protein, to heterologous IgG, or to both? A comparison was suggested of administering labeled antibodies to AFP and albumin in nude mice bearing two xenografts: one an AFP-producing and the other a nonAFP- but albumin-producing tumor. One would expect only the former tumor to be a target for radiolabeled AFP antibody.
On the second day, four topics were presented: differentiation and neoplastic transformation of hepatocytes, testicular and yolk sac tumors, oncotrophoblast proteins, and conceptual perspectives.
Dr. Janice Chou (National Institutes of Health, Bethesda, Maryland) reported on the transformation of rat fetal liver cells by the temperature-sensitive A (ts A) mutants of simian virus 40 (SV40). AFP, albumin, and transferrin were secreted by such cells at the restrictive temperature in good amount, much less so at the permissive temperature. However, in the latter state, cortisol addition to the medium enhanced intracellular cyclic AMP accumulation by glucagon. Dr. Michio Mori and Dr. Kimimaro Dempo (Sapporo Medical College, Sapporo) found two types of hyperplastic nodules during the early states of 3'-Me-DAB hepatocarcinogenesis: one rich in esterase and the other poor in this enzyme activity. The latter was typed as L-1 isozyme which appears only in the neonatal period of ontogeny and is associated with a subsequent high frequency of liver cancer development.
Events during the early stages of carcinogenesis with 2-fluorenylacetamide (AAF) were also examined by Dr. Stewart Sell (University of California, San Diego) using autoradiographic analysis. Oval cells arise from a small number of portal cells, not hepatocytes, and spread rapidly across the entire liver lobule. A technique was described for physically separating "oval" cells from other hepatocytes; one which will, when perfected, undoubtedly bring more valuable new information to bear on the origin of the hepatoma cell.
A team, led by Dr. Hyam Leffert (University of California, San Diego), has concentrated its attention on developmental programs and procarcinogen AAF processing in primary adult hepatocyte cultures during a two-week period. Ten hepatocyte-specific traits and four nonhepatocyte-specific characteristics typify these hepatocytes. Hepatocyte-specific functions are inversely related to growth in a number of variables. Much information has been obtained which is relevant to procarcinogen processing.
Testicular and yolk sac tumors were discussed separately. Dr. Robert McIntire (National Institutes of Health) discussed the clinical value of serum and biopsy tissue levels of hCG, AFP, and newer tumor markers with reference to tumor classification, selection of therapy, localization of metastases, and monitoring of treatment. He concluded that tumor markers will continue to be invaluable in the management of these tumors. Yukada Tsukada (Hokkaido University, Sapporo) reported the interesting finding that dibutyryl-cyclic AMP induces in cloned yolk sac tumor cell lines myotube and neuron-like cells which elaborated the respective tissue-specific enzymes, creating phosphokinase and acetylcholinesterase. A basic protein purified from the tumor is under study.
Two research reports on oncotrophoblast proteins were presented. In the first, Dr. Eva Engvall (La Jolla Cancer Research Foundation) described advances in the purification and assay of SP1, an oncoplacental protein. The new assay principle of the two-site sandwich ELISA utilizing monoclonal antibodies was introduced, resulting in greater specificity and speed of measurement than has hitherto been possible. Dr. Shinichi Tokumitsu and Dr. William Fishman (La Jolla Cancer Research Foundation) succeeded in demonstrating for the first time, the presence of term placental alkaline phosphatase in cytoplasmic organelles such as Golgi apparatus and endoplasmic reticulum in HeLa TCRC-l subline. The dynamics of the synthesis and processing of the enzyme will now be approachable experimentally.
The final session of this conference dealt with three conceptual perspectives in oncodevelopmental biology. Dr. Erkki Ruoslahti (La Jolla Cancer Research Foundation) proposed that extracellular matrix and basement membrane specificities, such as the presence or absence of fibronectin and/or laminin, by influencing cellular adhesion, may play an important role in normal development and biological behavior of malignant cells. The work of his laboratory indicates that multiple interactions between the different matrix components could be required for the formation of insoluble extracellular matrix and that proteoglycans capable of interacting with collagen, fibronectin, and laminin could play a central role in such a process. Dr. Van R. Potter (University of Wisconsin, Madison), in evaluating the present status of the "blocked ontogeny" hypothesis of neoplasia, pointed to the genetic basis of the hemoglobinopathies as a model on which to develop more direct tests of the hypothesis. In addition, recent findings in the literature on epidermal growth factor and sarcoma growth factor suggest that they are normal embryonic products which are counteracted in the fully differentiated cell by inhibitors of growth. Finally, Dr. Davor Solter (The Wistar Institute, Philadelphia, Pennsylvania) illustrated the interrelationship between embryology and oncology with current research on teratocarcinoma. Focusing attention on cell surface antigens and utilizing monoclonal antibodies, several antigenic determinants prove to be branched glycosphingolipids similar to human blood group antigens. These and others serve as useful and reliable markers of differentiation of embryonal carcinoma cells.