REPORTS ON SEMINARS

(1) Seminar on "Cellular and Molecular Biology"
The conference on "Cell Interactions and Cancer" was organized by Drs. Ikawa and Takeichi in Japan and Drs. Pastan and Barondes in the United States. There were 15 formal presentations followed by informal discussions. Discussion and exchange of ideas was extensive and the meeting was judged a success. The meeting focused on cell-cell interactions and ranged from considerations of primitive cells, such as hydra and slime molds, to mammalian cancer cells. Special emphasis was placed on new information concerning adhesive and recognition factors.
Studies of cellular associations in lower organisms were discussed in the first session. The organisms studied, cellular slime and hydra both have very favorable experimental properties for studies of cell-cell adhesion and morphogenetic pattern formation. Analysis of the underlying mechanisms may facilitate similar work in mammals.
Dr. I. Takeuchi discussed differentiation of the cellular slime mold, D. discoideum, into two cell types, prestalk and prespore cells and the segregation of these cell types. He presented evidence that differentiation precedes sorting and is not, therefore, a consequence of sorting. Instead it appears that segregation of the two cell types is a consequence of their differentiation.
Using studies with several species of cellular slime molds, Dr. S. Barondes described evidence that lectins, a class of carbohydrate binding proteins, play a role in cell adhesion. These lectins are synthesized with cell differentiation and become associated with the cell surface. They appear to influence the direct association of the cells in aggregates and may also organize extracellular matrix late in differentiation. Similar developmentally regulated lectins recently described in vertebrate tissues may play similar roles.
Dr. T. Sugiyama discussed the factors that determine the proportions of the six cell types found in hydra. For some cells, such as epithelial cells, the number that are in a state of proliferation is regulated. For interstitial cells there is a continuous high rate of proliferation but there is regulation of the proportion that are then converted to another cell type, the nematocytes. Attempts are being made to determine the cell surface and humoral factors that control these proliferative and differentiative events.
The second session was devoted to studies of cell interactions, the cell surface and development. Dr. Luis Glaser discussed his recent work on control of cell growth and cell differentiation by cell-to-cell contact. He has been studying Schwann cells which grow in mixed culture with neuronal cells. The Schwann cells require the neuronal cells present to grow. Imbedded in the surface of the neuronal cell is a growth factor or hormone-like factor. This factor is able to stimulate Schwann cell growth and development. Thus an immobilized substance can act like a soluble hormone or growth factor to control cell differentiation and growth. This novel system will no doubt serve as a model for other types of cellular interactions.
Dr. M. Takeichi described his studies on cellular reassociation. He has identified both a calcium-dependent and a calcium-independent aggregation process. These two processes are mediated by different types of molecules. For calcium-dependent site reassociation of teratocarcinoma cells a cell surface component with a molecular weight of about 140,000 seems to be involved and a molecule about 150,000 daltons in fibroblastic cells.
Dr. Fujisawa described his studies on the central connections of retinal fibers in amphibians. He has developed a novel method to trace the pathway of retinal fiber connections with the tectum. His findings indicate that some forms of recognition between ingrowing retinal fibers and tectal neurons are involved in the selective maintenance of appropriate axonal branches during regeneration and development of the retinotectal projection system in amphibians.
Dr. Tochinai next described his studies on the homing of thymic lymphoid stem cells in Xenopus larvae. In these studies he grafted diploid thymus rudiments from different ages into artificially induced histocompatible, triploid 40-day tadpoles. He finds that the lymphoid precursor cells of the thymus are of extrinsic origin and actively migrate into rudiments 3 to 4 days after fertilization and then differentiate these into immunocompetent lymphocytes.
Finally Dr. Irv Weissmann, Chairman of the session, discussed his studies on lymphocyte receptors for cellular interactions. He spent much of his time discussing the possibility that lymphocyte receptors on the surface of B cells and T cells may play an important role in the development of B cell and T cell leukemias.
Session 3 was devoted to microenvironments in cancer. The first presentation was by Dr. Kitamura who has been studying the differentiation of tissue mast cells. He has used mice which have giant granules so that he can readily identify the origin and fate of mast cells. One phase of his studies concerned the effects of carcinogenic substances on the differentiation of mast cells. Both dimethylbenzanthracene and a phorbol ester (TPA) increase the concentration of mast cells in the skin, but the underlying mechanism by which these substances acted was different.
Dr. Suzuki presented his studies on proteoheparan sulfate on cell surfaces and whether or not it is involved in the control of differentiation of Friend erthroleukemic cells. One novel feature of these studies was to synthesize various exloside derivatives of chrondroitins.
Dr. Kenneth Yamada closed the session with a discussion of the structure and function of fibronectin. Fibronectin is a multifunctional adhesive glycoprotein that is often decreased in transformed and malignant cells. Dr. Yamada and his co-workers have found that the fibronectin molecule can be dissected into functional domains including domains that interact with collagen, heparin and the cell surface.
Session 4 was devoted to studies on cell adhesion and cancer. Dr. Ira Pastan reported on studies done in collaboration with Dr. Benoit de Crombrugghe on the structure and function of the collagen gene. This gene is the first gene for an adhesive protein to be cloned and whose structure has been established. It has a very unusual structure with more than 45 exons. Using this gene they have shown that the levels of collagen messenger RNA are specifically reduced in some types of malignant transformation. Such a change in differentiation probably accounts in part for the abnormal adhesiveness of transformed cells.
Next Dr. Urushihara described her studies on a calcium independent adhesion mechanism that exists in BHK cells. Some abnormalities in the system have been detected in transformed BHK cells.
Dr. Hideo Hayashi described the properties of a 70,000 molecular weight cell surface adhesive glycoprotein isolated from hepatoma cells. This appears to be a different factor than other adhesive proteins previously isolated. The activity of this protein is inhibited by sugars suggesting it is some type of lectin.
The final paper of this session was by Dr. Fujita who has been studying cell adhesion and cell migration in incipient gastric carcinoma, a common type of carcinoma of the stomach. The progress of this tumor has been examined in detailed morphological studies.
In addition to the regular planned program Dr. Hiroshi Hiai was able to attend and present some of his recent exciting data on the role of thymic microenvironments in leukemogenesis. He has been able to develop a method to isolate complexes of leukemia cells and thymic epithelial cells from leukemic thymuses. In such a system there is a complex interaction between these cell types. They have been able to trace the natural history of one type of murine leukemogenesis and they propose that the thymus provides a permissive microenvironment for developing leukemias to progress to autonomously growing neoplastic cells.
The public symposium, which followed after the closing of the 'formal' meetings, attracted an audience of 100, some from as far away as Nagoya and Tokyo, made up of professionals and interested students. This was titled simply "Cell Interaction" and four speakers from the United States presented their views and work during the three-hour session. This type of sharing of knowledge is perhaps too rare among the different generations of scientists and the fact that it was so well attended would appear to testify to the fact that more such presentations would be welcomed.

(2) Seminar on "Antigenic Nature of Tumor Cells"
The conference on the "Antigenic Nature of Tumor Cells" was organized by Dr. Hamaoka in Japan and Dr. Hodes in the United States. A total of 24 presentations was made and these were followed by lively discussions. The meeting focused on the definition, identification and regulation of expression of various types of tumor antigens and the fundamental immunologic processes in host responses to these tumors.
The identification of melanoma tumor antigens was described in the presentations by Drs. Kikuchi and Shinohara. Dr. Shinohara described the use of multiple monoclonal hybridoma antibodies generated against the B16 melanoma tumor line and resulting in the identification of one group of antibodies with apparent specificity for the B16 tumor alone, and a second group of antibodies reacting with melanoma cells of both human and murine origin. The studies presented by Dr. Kikuchi involved the use of radio-labeled antibody which was demonstrated to persist selectively in areas of in vivo tumor involvement, and the use of anti-melanoma antibodies conjugated to the chemo therapeutic agent neocarcinostatin in preliminary trials of immunotherapy. Dr. Hashimoto presented the results of the studies employing monoclonal antibodies to both rat and human bladder carcinomas, and characterized both unique tumor-associated antigens and antigens common to both bladder cancers and normal epithelial cells.
Tumor antigens associated with cells of lymphoid origin were discussed in presentations by Drs. Nadler, Vitetta, and Kishimoto. Dr. Nadler's studies described the expression on human lymphomas of the antigens B1 and B2, both of which are also expressed as differentiation antigens on normal B cell subpopulations. A relationship between the expression of these antigens and the occurrence of carcinogenic transformation in normal lymphoid cells of a given differentiation stage was discussed. Dr. Kishimoto described a human CLL of B cell origin which was inducable to differentiation and immunoglobulin synthesis under the influence of T cell factors. Dr. Vitetta presented analogous studies in the induction of differentiation in the murine BCL₁ model.
The molecular biology of tumor antigens and the relationship of tumor antigens to oncogenic transformation were described in studies of Dr. Marc Greene. The technique of gene transfection was employed in studies of both virus induced tumors and methylcholanthrene-induced tumors. In these important studies, both the expression of tumor associated antigens and the oncogenic transformation event were induced in recipient cells through transfection with DNA.
Fundamental Immunologic Processes in Host Response to Tumors.
T cell responses as effector mechanisms of tumor specific immunity were discussed by Dr. Hashimoto, Dr. Mazumder, Dr. Hamaoka, and Dr. Fujimoto. Dr. Hashimoto described the use of a mixed lymphocyte-tumor reaction for detection of tumor associated transplantation antigens. Dr. Mazumder described the generation of cytotoxic alloreactive and tumor specific T cell lines, and the ability of such cytotoxic T cells to function in experimental models of tumor immunotherapy. Dr. Fujimoto analyzed cytotoxic T cell lines with tumor specificity, and emphasized the function of accessory populations in the effector mechanism of the cytotoxic cloned T cells. The function of at least three soluble lymphokines in the generation of cytotoxic T lymphocytes was demonstrated by Dr. Hamaoka, employing the generation of cytotoxic T lymphocytes to TNP modified syngeneic cells as a model system.
Drs. Koren and Tokunaga presented experimental data concerning the role of activated macrophages as an anti-tumor effector mechanism. The behavior of activated macrophages was studied at the single cell level in work by Dr. Koren, who characterized both the cationic and metabolic requirements for tumor cell kill by monocyte effectors. Dr. Tokunaga employed a murine tumor line which could be induced to differentiate into a macrophage-like cell with efficient killing activity against tumor cells and employed this model to study the importance of asialo GM1 in the function of cytotoxic monocyte effector cells.
Natural killer cells as an anti tumor effector mechanism were discussed by Drs. Henney, Kumagai, and Koren. NK cells were demonstrated to be responsive to both poly I-C and IL-2 in the studies presented by Dr. Henney, in which both NK cell survival and natural killer function were enhanced by exposure to these agents. Dr. Kumagai was able to clone murine NK cells and to study the functional potency of these cells in anti tumor assays, characterizing both the cell surface phenotypes of these cells and the effects of lymphokines on their functional activity. Dr. Koren similarly described the effects of modulating agents on the functional activity of human NK cells, and in addition presented the results of phase I clinical studies of the administration of lymphoblastoid interferon to patients with colon carcinoma.
Regulatory effects of anti tumor responses were discussed in presentations by Dr. Hodes and Dr. Greene. Dr. Hodes presented the results of studies characterizing the cytotoxic T cell response to the products of mutant major histocompatibility genes, and demonstrated the effects of T cell maturation environment upon the cytotoxic T cell repertoire. Dr. Greene described the function of suppressor cell network regulation in responses to both conventional and tumor associated antigens.
Preclinical Applications of Tumor Immunology.
A model system for the evaluation of NK cell effects in limiting the growth of lymphoma in vivo was presented by Dr. Henney. Tumor cell clones which were specifically resistant or susceptible to NK effects in vitro were examined for susceptibility to NK-mediated immunotherapy in vivo, and the results of such studies suggested strongly that NK-Susceptibility can in fact influence the in vivo growth of tumor lines.
The administration of in vitro PHA-activated autologous peripheral blood lymphocytes as an immuno therapeutic modality was described by Dr. Mazumder. In vitro studies demonstrated the cytotoxic T cell activity in such preparations of human cells. It was further demonstrated that patients who had received in vitro activated autologous lymphocytes had detectable levels of circulating T cells which were cytotoxic to autologous fresh tumor. A preclinical murine model for enhanced T cell mediated anti tumor activity was presented by Dr. Hamaoka. In PPD pre-sensitized mice, therapy with tuberculin-coupled tumor cells was able to increase survival in animals bearing the homologous (unmodified) tumor.
Sero therapy with ricin-conjugated tumor specific antibody was presented as a tumor immunotherapy model by Dr. Vitetta. Using the murine BCL₁ model, it was shown that in vitro treatment of bone marrow preparations from tumor bearing mice was able to deplete these populations of malignant cells without compromising their stem cell activity. The additional use of ricin conjugated anti BCL₁ antibody in vivo was described in combination with a cytoreductive radiation therapy or chemotherapy regimen. Dr. Nadler described the administration of monoclonal antibodies specific for human lymphoma-associated antigens in preliminary clinical studies. Difficulties associated with circulating free antigen as well as modulation of cell surface antigen were described in these studies. Dr. Nadler further described the use of autologous bone marrow transplantation in association with in vitro treatment of bone marrow cells to eliminate antigen-positive malignant cells.
Dr. Kishimoto reported the use of both T cell lines and hybridomas of human T cells. Using such lines, it was demonstrated that distinct lymphokines could be isolated with inter-leukin-2 activity, with killer helper activity, with T cell replacing activity, or with interferon activity.
A general discussion was conducted emphasizing new perspectives on the application of preclinical modalities to the generation of approaches for clinical immunoprophylaxis and immunotherapy. All formal meeting participants participated in these discussions, with the additional contributions of several guests from the Harvard Medical School community and the Sidney Farber Cancer Center.
The importance of demonstrating antigens with adequate tumor specificity was discussed. In particular, the limitation in current ability to demonstrate tumor specificity with presently available reagents and techniques was emphasized. The practical issues of undesirable toxicities toward normal tissues, as well as the escape mechanism of tumors from antigen specific effector mechanisms were discussed.
The present status of evidence supporting the physiologic significance of T cell effectors, NK effectors, and activated macrophages was presented and discussed. Significant experimental evidence exists supporting the relevance of each of these effector mechanisms in certain model systems. It would appear from available evidence that individual tumor cell populations may be differentially susceptable to one or another of these mediating effector mechanisms. The results of preclinical studies in recent years have demonstrated the potential relevance of highly specific monoclonal antibody preparations, cloned effector cell populations, and highly purified lymphokine preparations. It was stressed that the application of these forms of immunotherapy to clinical studies should occur only after optimal preclinical evaluation, and should be accompanied by intensive efforts to study the effects of therapy on the host immune system, as well as the final anti tumor effects of such therapies.
(3) Application of Cytology Automation in Cancer Cytology and Cell Biology Purpose
The conference on "Application of Cytology Automation in Cancer Cytology and Cell Biology" was organized by Dr. Tenjin in Japan and Dr. Melamed in the United States. A total of 31 papers were presented at this conference during the 8 sessions that occurred during a two day period. The conference covered topics that included Instrumentation, Imaging and Data Analysis; the Effects of Chemotherapy; Characterization of Normal and Reactive Lymphocytes and Leukemic Cells; Sample Preparation, Enrichment Techniques and Cell Staining; DNA and RNA Measurements and Cell Psychoanalysis; Clinical Diagnostic Trials on Gynecologic and Urinary Tract Specimens; and Studies of Solid Tumors.
This seminar, which follows the previous one held in January 1980 in Los Angeles, is the second of the U.S.-Japan Cooperative Research Group's meeting. The meeting concentrated on the diagnosis of cancer cells. This was not pathomorphologic diagnosis, but automated diagnosis and automated analysis of cancer cells. The participants discussed how these techniques can be applied in clinical situations. In automated cytology, both flow system and image analysis have been used, although these techniques have not yet been perfected. The progress of both soft and hard techniques in flow cytometry (FCM) and computer systems has been marked. For this reason the advance of automated cell analysis has been rapid, giving us information which was until now not available.
Since there are slight differences between the research carried out in the U.S. and that carried out in Japan, this type of seminar is necessary for the exchange of ideas and discussion of the results of these studies. This seminar opened new paths of research. One of the goals that is hoped to be achieved is the perfection of instrumentation for the automated diagnosis and automated analysis of cancer cells.
Results
Four major topics were taken up at this seminar. They are 1) Instrumentation and Data Analysis, 2) Application in Cell Biology, 3) New Technological Approach for Sample Preparation and 4) Application in Cancer Detection. The reports by individuals are discussed below.

I. Instrumentation and Data Analysis.
New research on automated cytology and its results were discussed, centering around image analysis.
Drs. Sakai and Kawamura reported on the characteristics of malignant cells and their attempts at the diagnosis of early cancer of the uterine cervix by the HITAC M-180 computer system. Dr. Wheeless has developed a multidimentional slit-scan flow system which was shown to be more advanced and precise than the three-dimensional slit-scan system. This combines both the fiow system and the image analysis system. Cell clusters are a difficult problem in image analysis. Dr. Onoe took advantage of these clusters to differentiate adenocarcinoma and normal cells. He has achieved a rather high rate of success. Using a system which is based on multispectral images provided as three scanned monochromic images illuminated with lights with wavelengths of 610 nm, 535 nm and 470 nm, Dr. Noguchi was able to detect a malignant cell in a clump of cells. Dr. Masayoshi Takahashi introduced a new system of cyto-image analysis using a line-type change coupled device (CCD). It consists of 2048 sensor elements. This system makes possible the representation by histogram of the amount of DNA per cell of cells in a Feulgen reaction. An interactive system of color graphic display programs, developed by Dr. Wied during the course of the TICAS project, enables the performance of quantitative cytometry and assessment of the main cyto-morphologic descriptors in an objective and rapid manner. Dr. Manabu Takahashi reported on a new method of analysis of DNA histograms (DHA8202). The interpretation of G₁ and G₂ M peaks in the DNA histograms has been difficult. This system is able to reveal more accurately the distribution of DNA in cells in the cell cycle and to identify abnormal cells. This is important as this system may be applicable to image analysis system.
II. Application in Cell Biology
Dr. Andreeff reported that although acute leukemia is classified into acute lymphoblastic (ALL) and acute non-lymphoblastic leukemia (ANLL), flow cytometric investigation has revealed a biophenotypic letikemia. This challenges the "dogma" of non-clonal origin of leukemia. Monitoring of aneuploid cells permits the identification of minimal residual disease and allows the detection of impending relapse. Dr. Okumura reported on the applications of flow cytometry and cell sorting for the analysis of lymphocyte subpopulations. The surface phenotypes of two cells line identified by Dr. Okumura, which have distinct suppressor activity in antiformation characterized by FACS as well as their detailed function, was discussed. Dr. Takamoto has carried out research on monoclonal antibodies which are produced against leukemia. Analysis by flow cytometry provided precise quantitative data and histogram patterns of positive cells. Dr. Colvin reported on the application of flow cytometry to monitor patients treated with OKT3 monoclonal antibody for acute renal allograft rejection. This will be useful in clinical situations where monoclonal antibodies are employed for therapy. Dr. Nomura reported on the application of flow cytometric analysis to brain tumor chemotherapy. Dr. Sugishita evaluated FCM data on the post-chemotherapeutic sensitivity to CIS-platin of cancer cells of ascites in cases of ovarian cancer. He discovered a point of differentiation between effective and ineffective cases in the DNA histograms. Dr. Takakura reported on the chemotherapeutic schedule of treating malignant brain tumors based on flow cytometric studies.
III. New Technological Approach for Sample Preparation
Dr. Wied reported on preparations suitable for automatic assessment. These steps involve the depositing of the cell sample in a collection fluid, the adjustment of cell concentration to about 30,000 cells per milliliter, dispersion of the sample by syringing, and the deposition of the cellular content onto microscopic slides by centrifugation. Dr. Kishigami reported on his work on the removal of leukocytes by the spontaneous sedimentation technique using single layer Ficoll gradient and the coating of a glass slide with poly-L-lysin to prevent cell exfoliation from the slide's glass surface. Drs. Melamed and Adreeff introduced a new technique which combines blood cell separation by density fractionation with subsequent FC analysis for the detection of aneuploid cells when neither morphology nor routine FC of mononuclear blood cells provide evidence of leukemic cells. To eliminate the weakpoints of flow cytometry, Dr. Tenjin developed a method of dispersing the tissue block into cells. He applied this method in cases of dysplasia, CIS and invasive cancer of the uterine cervix and compared the dispersed tissue block cells with exfoliated cells and their DNA histograms. Dr. Colvin discussed studies, using monoclonal antibodies to T cell subsets, in which he monitored lymphocyte cell population in renal allograft recipients treated with either conventional immunosuppression or monoclonal antibody. The use of monoclonal antibodies and automated flow cytometry allows the precise determination of changes in T cell subsets. Dr. Tanaka developed an automated sample pretreatment system for preparation of cells to be analyzed by CYBEST. Dr. Nishiya discussed the results of his study which evaluated the influences of steroid hormones to human endometrial adenocarcinoma cells in vitro using flow cytometry. Dr. Melamed reported on the use of acridine orange as a DNA/RNA stain. There were many points concerning single strand RNA that are still unclear, but he gave an explanation on a theory of the emission of red and green.
IV. Application in Cancer Detection
Dr. Wied reported on cytomophometric markers for uterine cancer in intermediate and dysplastic cells. He suggested that automated prescreening might center on the assessment of a limited number of readily available intermediate cells and to the diagnostic evaluation of dysplastic cells. Dr. Wheeless presented the results of his multidimensional slit-scan flow system used to analyze 438 clinical specimens. Applying flow cytometric analysis, using acridine orange stain to 300 patients with or without urinary bladder cancer for the detection and assessment of urinary bladder cancer, Dr. Melamed was able to obtain results which compared favorably with those of conventional cytologic diagnosis by light microscopy. Dr. Andreeff reported that the clinical response of patients with biphenotypic leukemia was considerably poorer than those with ALL and ANLL. Dr. Manabu Takahashi reported on the complexity of DNA staining with propidium iodide. Dr. Uei reported that the number of abnormal cells decreased by approximately 33% following their special preparation of cells for flow cytology study. Dr. Tanaka reported his field test data using the CYBEST model-4.
Summary
1) The discussion of the automated diagnosis and automated analysis of cancer cells progressed a step beyond the previous seminar.
2) The rise of the level of the diagnostic ability of borderline lesions by image analysis is anticipated with the improvement of both hard and soft techniques. There are also new methods of diagnosis for adenocarcinoma. This research will be useful in future studies.
3) Research on FCM is progressing rapidly. There are great expectations not only for the ability of the system to analyze cells and its speed, but also expect it to be valuable in the selection of therapeutic method and for the determination of the reaction to chemotherapy. Both the U.S. and Japanese sides agree on this point. There is still much basic research being done, but it is expected to be applied in clinical situations.
4) Collection and preparation of samples are very important. At this seminar both sides reported on new techniques of cell collection. This should raise the level of the reliability of the instrumentation.
5) Reports were given on the clinical applications and results of both image analysis and FCM. Cancers of the uterine cervix, ovaries and urinary, and leukemia were discussed. This included not only diagnosis, but also prediction of recurrence and selection of therapeutic methods.
6) The slight differences in the research carried out by the two sides is extremely valuable in reaching the common goal, and much has already been gained.



SEMINAR AGENDA AND PARTICIPANTS

(1) U.S.-Japan Seminar on "Cell Interaction and Cancer"
Kyoto International Conference Hall-Room F
November 5-7, 1981

AGENDA