HTSCA was used to study the chemotherapy sensitivity of gynecological malignant tumors. 34 specimens were obtained from 32 patients. 11 of the 34 specimens (32%) yielded a sufficient number of colonies for testing. On these, 51 separate drug assays were done, each at the 1/10 peak plasma level. 20 drugs were identified as active among 51 drugs tested. Clinical correlations of sensitivity (S) and resistance (R) were studied in 6 patients treated with combination therapies, including drugs selected on the basis of in vitro sensitivity. (formula; see text) We conclude that HTSCA can aid in identifying active drugs for use in designing treatment protocols. However, because of difficulty in preparing a single cell suspension, and the small proportion of tumors suitable for testing, HTSCA will not be used in routine drug sensitivity testing until these drawbacks are eliminated.

One of the major causes of failure of cancer chemotherapy is the proliferation of specific drug-resistant tumor cells during treatment. Drug-resistant tumor cells, however, usually bear biochemical changes which are related to the resistance mechanisms. New modalities against resistant cells could be possible if we were able to characterize these biochemical changes. Vincristine (VCR)- and adriamycin (ADM)-resistant tumor sublines show cross-resistance (pleiotropic drug resistance) to other unrelated drugs. VCR- and ADM-resistant sublines possess an enhanced outward transport of antitumor agents, which results in a low accumulation of antitumor agents in the cells. The cells express unique glycoproteins in the plasma membrane, and possess a higher calcium content in the cells. They also have double-minute chromosomes and homogeneously staining regions in chromosomes. By targeting for these biochemical changes, we have established new modalities against drug-resistant tumor cells. Calcium channel blockers inhibited the enhanced outward transport of VCR and ADM from resistant tumor cells, and thus overcame resistance to these agents. This approach showed potential usefulness in clinical trials. Another possible approach against drug-resistant tumor cells could be the utilization of monoclonal antibodies against unique glycoproteins in the plasma membrane of resistant tumor cells. We have developed monoclonal antibodies against adriamycin-resistant human myelogenous leukemia K562. Our recent progress with work on calcium channel blockers and monoclonal antibodies are discussed in this paper.

We have attempted to investigate the usefulness of the subrenal capsule assay (SRCA). Initially, we compared the results of antitumor activities of 27 anticancer drugs obtained using the SRCA in BDF1 mice and the subcutaneous transplantation assay in nude mice, and it became clear that these results correlated well. It was thus apparent that the antitumor activity of drugs could be evaluated faster, more cheaply and more easily using the SRCA. In the SRCA, the overall success rate for growing tumors (with a tumor growth rate of more than 1.0) from patients was 79% (106/134) and the success rates for each tumor type were as follows: malignant lymphoma 90%, ovarian cancer 87%, sarcoma 77%, breast cancer 76%, colorectal cancer 73% and renal cancer 58%. In a retrospective analysis of correlation between the antitumor activities assayed by the SRCA and the respective clinical responses, 11 out of 25 trials were true positive (79%) and 7 trials were true negative (64%). The sensitivity and specificity levels of this test were 73% and 70%, respectively. The tumor growth inhibition rates were correlated well with the clinical responses. The advantages of the SRCA are: it has a high success rate; it allows rapid evaluation; it can be applied for masked compounds; and it has a good clinical correlation. These advantages indicate that the SRCA is of potential value as a predictive test for selection of anticancer drugs on an individual patient basis.

Human tumor clonogenic assay(HTCA) not only offers potential advantages for prediction of the sensitivity of individual patients, to anticancer drugs, but also for the development and preclinical testing of prospective new antineoplastic agents. However, HTCA has several theoretical and technical problems, such as a low success rate, the requirement of large numbers of tumor cells and the long time period necessary for evaluation. In order to solve these problems, the MINI-hybrid assay has been developed. We reviewed our experiences to date with chemosensitivity testing by MINI-hybrid assay. Twenty-two of 23 tumors gave evaluable chemosensitivity results (95.6%), and the range of thymidine incorporation for evaluable assays was 7 X 10(2)-1.2 X 10(5)cpm. In addition, the results of drug sensitivity testing of cultured cell lines by MINI-hybrid assay were well correlated with those obtained by HTCA. With its high evaluability rates, the need for fewer cells, the short duration (5 days) required and ease of quantitation, the MINI-hybrid assay is widely applicable to the chemosensitivity testing of human tumors.

CPT-11 is a new derivative of camptothecin, a plant alkaloid developed in Japan. We investigated the antitumor activity of CPT-11 on the subrenal capsule assay by using 39 tumor samples from patients with various tumors. The overall chemosensitivity rate of CPT-11 was 44% and the mean tumor growth inhibition rate was 44% in all the tumors. In 18 ovarian cancer cases, the chemosensitivity rate was 56% and the mean tumor growth inhibition rate was 46%. The antitumor activity obtained by CPT-11 is comparable to that of adriamycin, cyclophosphamide and 5-fluorouracil in the same assay system. Based upon these results, CPT-11 is considered to be a good candidate for clinical trials.

Recently, it has been shown clearly that various tumor cells can be induced by differentiation inducers including biological response modifiers, synthetic chemicals and conventional anticancer drugs to differentiate terminally both in vitro and in vivo into cells with normal characteristics. On differentiation, the cells cease to proliferate and lose their transplantability in either nude mice or syngeneic animals. Furthermore, prolongation by the differentiation inducers of survival times of animals inoculated with various tumors was confirmed. These findings suggest that induction of terminal cell differentiation by differentiation inducers is another approach to tumor therapy, that is "differentiation therapy" of tumors. In this review, recent results of basic and clinical studies on the differentiation therapy of tumors are described. The problems and perspectives of differentiation therapy are also discussed.

A phase II study of the oral agent methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-glucopyranoside (MCNU tablet) for myeloproliferative disorders was performed. Fifty-two patients were treated with MCNU tablets and 43 patients were evaluated for clinical effects and 45 for adverse effects. The standard regimen was as follows; oral administration of 50mg (one tablet)/body/day every 4-6 days was considered as one course, and this was repeated at 6-8-week intervals if possible, with certain modifications according to dosage, period of administration and dose interval wherever necessary. Of 16 patients with chronic myelogenous leukemia (CML) in the chronic phase, 13 achieved complete remission (CR), and 3 achieved partial remission (PR). The overall response ratio was 100%. Rapid reduction of leucocytes was detected within two weeks. One patient with CML in blast crisis achieved PR (100%). Of 15 patients with polycythemia vera, 13 showed an excellent effect (87%), and 1 a moderate effect (6.7%), the overall response ratio being 93%. In essential thrombocythemia, an excellent effect (70%) was obtained in 7 of 10 patients. One patient with myelofibrosis showed an excellent effect (100%). Nausea & vomiting (33%) and anorexia (13%) were major adverse effects, but these symptoms were observed only transiently. Liver dysfunction was also seen in 8.9% of patients, but no patient showed severe manifestations. Our study supports the contention that MCNU tablet is a useful agent against myeloproliferative disorders.

Patients with specific types of cancer have achieved substantial prolongation of survival after successful cancer chemotherapy. Furthermore, cancer chemotherapeutic agents are being widely used as immunosuppressive drugs in patients with benign conditions. Both of these patient populations are at risk for development of late complications of treatment. Though the late effects of chemotherapy are less well defined, they are insidious in onset and not manifested clinically until damage have become overt and irreversible. Many reports on the late effects of chemotherapy demonstrate the importance of assessing not only the therapeutic results of various treatment program but also of evaluating the long-term complications of therapy in order to maximize the benefit of the treatment. Thus, skillful and judicious application of chemotherapy produces a minimum of delayed consequences.

The subrenal capsule (SRC) assay for cancer chemotherapy was tested according to Bogden's methodology. Of 37 patients providing tumor tissue for assay, 29 cases were considered suitable for evaluable assays. Fourteen patients had clinically evaluable diseases and 10 cases were evaluable for SRC assays. Correspondence between sensitive assay and clinical sensitivity was seen in 2 cases, and that between resistant assay and clinical resistance was seen in 4 cases. Discordance between sensitive assay and clinical resistance was seen in 4 cases. In histological studies, cancer tissues implanted in the subrenal space in immunocompetent mice did not show marked proliferation and were replaced by prominent leukocyte infiltration and fibrosis on day 6 after inoculation. The degree of leukocyte infiltration in the xenografts in the mice administered some anti-cancer drugs was slight in comparison with that in untreated control mice, which showed a remarkable trend in xenografts treated with 5-fluorouracil and cyclophosphamide, respectively. Our study suggests that there are many problems involved in the SRC assay methodology of Bogden, and that careful examination of this aspect will be required.

Although the human tumor clonogenic assay (HTCA) is extremely reliable in determining clinical correlations, it is a complicated process requiring considerable time in order to obtain results. Thus, an experimental study on cytopathologic observation (cytologic assay) and comparative evaluation between it and HTCA were performed in order to establish a more rapid and accurate drug sensitivity test. Materials included Colon 26, a cell line established in our department, malignant effusion and surgical specimens. In carrying out HTCA according to the Hamburger-Salmon method, the cell suspension samples following exposure to anti-tumor agents (MMC, L-PAM, ADM, CDDP) were cultivated in test tubes for 3-8 hours and stained by the Papanicolaou and Giemsa methods. According to Tokita's criteria, when cellular changes showed as nuclear pyknosis and nuclear destruction were found to have increased significantly in comparison with a control group, the cells were judged to be sensitive. Very similar and parallel results were obtained between HTCA and cytologic assay in this study, with a significant correlation. Cytologic assay was proved to be an easy, rapid and accurate method for testing drug sensitivity and its clinical application can be expected in the future.

Subrenal Capsule Assay (SRCA) as a chemosensitivity test was performed on 14 esophageal squamous cell carcinomas in order to select a more effective form of chemotherapy. Of the 14 assays, 12 were evaluable. Mice were treated with anticancer agents (e.g. Cisplatin, Bleomycin, Methotrexate, Vindesine) on days 1 and 3 after transplantation, and on day 6, the sensitivities were determined. Fresh esophageal cancers yielded an evaluable assay rate of 74%. The implant grew progressively for six days in the remaining group of control mice. Histologically, host cell infiltration at the border of the implant was observed from day 3 after transplantation, and cells had degenerated or had been partially replaced by scar tissue by day 6. The results of chemosensitivity tests differed according to the anticancer agent used or from case to case. Clinically, correspondence between the assay results and clinical results was obtained in 5 out of 7 cases. SRCA is a new promising chemosensitivity test which is clinically useful, and the present results indicated the feasibility of its use in developing an effective chemotherapy for esophageal cancer.

With the purpose of obtaining more potent and less toxic camptothecin (CPT) analogs, we prepared many derivatives of CPT. Among them, 7-ethyl-CPT (SN 22) and 7-ethyl-10-hydroxy-CPT (SN 38) showed strong antitumor activity with less toxicity. They were, however, insoluble and when they were made soluble, their activity was markedly diminished, as a result of cleavage of the delta-lactone ring. We therefore attempted to make soluble derivatives without breaking the delta-lactone ring and obtained 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxy-CPT (CPT-11), which showed very strong antitumor activity by i.p., i.v. or p.o. administration against the ascites type of L1210 leukemia, P388 leukemia, sarcoma 180, Meth A fibrosarcoma, B16 melanoma, Ehrlich carcinoma and MH134 hepatoma and the solid type of sarcoma 180, Meth A fibrosarcoma, Lewis lung carcinoma, C3H/HeN mammary carcinoma, Ehrlich carcinoma and MH134 hepatoma. The antileukemic activity of CPT-11 against L1210 was much higher than that of adriamycin. The acute toxicity of CPT-11 was extremely low, particularly in the case of oral administration, the LD50 being 765.3 mg/kg, 22 times greater than that of CPT-Na.

Hitherto, the selection of anticancer drug has been based on clinical experience. But to succeed for chemotherapy to succeed, effective drugs must be selected for individual patients with cancer. We have developed the following new in vitro micromethod. Method. In brief, tumor cells obtained by surgery were incubated with anticancer drugs for 48 hrs at 37 degrees C in 5% CO2 in a microplate. After incubation, the surviving cells were fixed with methanol and stained with crystal violet. Then the stained cells were solubilized with 1% lauryl sulfate and the absorbance of each well was measured at 540 nm with a multiscan spectrophotometer. In this method, cytotoxicity was quantitated by the absorbance. The method is simple, rapid (48 hrs) and reproducible, and it requires only a small amount of cells (5 X 10(3) approximately 1 X 10(4]. This method correlates well with sensitivity tests using isotopes. We have examined the chemosensitivity of 22 specimens from gynecologic malignancies by this method. The success rate is 77% and higher than with any other in vitro method. This method will be widely utilized in several fields of cancer treatment in the near future.

A calmodulin inhibitor, trifluoperazine, was found to enhance the cytotoxic action of ACNU in C6, especially in ACNU-resistant (C6/ACNU) glioma cells in vitro. In order to clarify the efficacy of trifluoperazine in vivo, 1 X 10(7) C6 or C6/ACNU cells were percutaneously implanted into the cisterna magna of Wistar rats to produce meningeal gliomatosis (MG) models as a chemosensitivity assay system. MG rats were treated with ACNU and trifluoperazine according to a variety of schedules. Trifluoperazine in doses of 250 to 500 micrograms/kg intrathecally (it) administered with 1 mg/kg ACNU 1 day after the tumor inoculation significantly increased the life span of the C6/ACNU bearing (C6/ACNU MG) rats. At doses of 250 and 500 micrograms/kg of trifluoperazine in the C6/ACNU MG rats, values of increased life span of 22 and 30% were obtained with a 1 mg/kg dose of ACNU, respectively. These values were statistically significant compared with that obtained in the C6/ACNU MG rats treated with ACNU alone at 1 mg/kg. It might be concluded that the combination chemotherapy with ACNU and such a calmodulin inhibitor as trifluoperazine could overcome ACNU resistance in malignant brain tumors.

Sister chromatid exchanges (SCEs) induced by four anticancer drugs, 3-(4-amino-2-methyl-5-pyrimidyl) methyl-1-(2-chloroethyl)-1-nitrosourea (ACNU), 1-3-bis (2-chloroethyl)-1-nitrosourea (BCNU), nitrogen mustard (HN2), cis-diamminedichloroplatinum (II) (cis-Pt) were examined on five cell lines derived from human malignant glioma biopsy specimens, and compared to results obtained with colony-forming efficiency (CFE) assay. Treatment of the five cell lines with these four drugs produced concentration-dependent increases in SCEs. Treatment with ACNU induced the most SCEs in SF-126 cells decreasing in SF-268 cells followed by SF-210 cells, SF-295 cells, and the least SCEs in SF-188 cells. The results of the SCE assay with BCNU in these cell lines were similar with ones with ACNU. In contrast to results obtained with nitrosoureas, the most SCEs were induced in SF-188 cells and the least were induced in SF-126 cells by the treatment of HN2. The frequency of SCEs induced with cis-Pt was almost similar in the five cell lines. The number of SCEs induced by the treatment of ACNU, BCNU, HN2 and cis-Pt in five cells lines showed a good correlation with cytotoxicity measured by CFE assays, and induction of SCEs occurred at much lower concentrations of these anticancer drugs than those required to induced cell kill. These results suggest that measurement of induced SCEs in human brain tumor cells treated with some anticancer drugs provide a more sensitive indicator of drug action than CFE assay and that SCE assays may be a useful method of the in vitro sensitivity test to some anticancer drugs.

Kazusamycins A and B and leptomycin B have a structure characteristic of an unsaturated, branched-chain fatty acid with a terminal delta-lactone ring, and show antibacterial activity on some kinds of fungi. Kazusamycin A (KZM-A) showed cytotoxic activity on mammalian cells at very low concentrations (ng/ml) in vitro. The antibiotic inhibited not only the growth of transplantable murine tumors and their metastases to the lung but also human mammary tumors inoculated into nude mice. KZM-A became immediately distributed to the main organs of mice, and a certain quantity of the antibiotic was inactivated by binding to high-molecular-weight substances such as albumin. A large quantity of KZM-A was carried to the liver and excreted into the bile, but was then reabsorbed by the small intestine. The growth of tumor metastases (L5178Y cells) in the liver was suppressed by KZM-A. The antibiotic induced severe diarrhea by causing necrosis and/or lysis of the mucous membrane of the small intestine. In contrast to this, the degree of myelotoxicity was relatively slight. The active site of the fatty acid of KZM-A appeared to consist of conjugated double bonds, carboxylic acid and hydroxyl moieties.