In vitro chemosensitivity was evaluated by SDI test in various human tumors including 1 lymph node metastasis of esophageal cancer, 10 gastric cancers, 4 colo-rectal cancers, 1 hepatoma, 2 lung cancers, 2 breast cancers and 1 gallbladder cancer. Tumor fragments cut with scissors were exposed to twelve kinds of antitumor drugs at five to ten times peak plasma concentration. After 3 days at 37 degrees C, each tumor fragment suspension was washed with phosphate-buffered saline and assayed for succinate dehydrogenase (SD) activity using 3-(4,5- dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) as a hydrogen acceptor. When the SD activity of the drug-treated cells was reduced to below 50% that of control cells, the chemosensitivity to the antitumor drug was considered positive. The chemosensitivity of each tumor varied individually. Mitomycin C or 5-fluorouracil are regularly used to treat gastric cancer patients, but, some specimens of gastric cancer in this study showed a resistance to these drugs and an unexpected sensitivity to other drugs. Our results show that the SDI test is a convenient method for clinical use and gives significant information about drug sensitivity.

The effects of the anticancer drugs Nimustine (ACNU), Aclacinomycin A (ACR), Adriamycin (ADM), Bleomycin (BLM), Cisplatin (CDDP), and 5-Fluorouracil (5-FU) on the multicellular spheroid of a chemically-induced 9L rat glioma was studied. The multicellular spheroid in which cells grow in vitro as three-dimensional aggregates represents a biological model, which is intermediate between monolayer cells in vitro and solid tumors. Spheroids were initiated in bacteriological grade petri dishes seeded with 10(6) 9L rat glioma cells, cultured for four days and thereafter transferred and further developed in a spinner flask. Spheroids of 200-400 micron diameter were sorted and exposed for 24 hours to 5-FU and one hour for other drugs. After treatment both cytotoxic effect and growth delay were analyzed. Following disaggregation using collagenase, pronase and DNAase, cytotoxic effect on multicellular spheroids was measured by colony forming assay and were compared with those effects on 9L monolayer culture cells in the exponential growth. For growth delay assay, multicellular spheroids were individually transferred to 16 mm well containing 0.4 ml agarose base and 2 ml culture medium. Spheroid size was measured twice a week and growth curves were drawn. The growth delay was determined as the treated group vs. control differences in time required to a size four times that of the initial volume. For cells both in the monolayer culture and the multicellular spheroid, the dose response curve for ADM, BLM and 5-FU was "biphasic" and that for ACNU, ACR and CDDP "shoulder-threshold" type.(ABSTRACT TRUNCATED AT 250 WORDS)

A phase II study of oral 5'-deoxy-5-fluorouridine (5'-DFUR) was conducted on a multi-institutional basis. Six hundred and ninety-two cases were entered into this study and evaluated by the extramural review committee. In 437 evaluable cases the response rate was 16.0%, 13 CR and 57 PR. The response rate of gastric cancer was 14.3%, 1 CR and 19 PR out of 140 evaluable cases, that of colorectal cancer was 9.2%, 1 CR and 6 PR out of 76 evaluable cases, and that of breast cancer was 35.9%, 11 CR and 26 PR out of 103 evaluable cases. The response rate was high with a long-lasting efficacy in breast cancer. Although the number of cases was small, PR was observed in 3 cases of head & neck, and in 1 case each of esophagus, gall-bladder and thyroid cancer. The optimal daily dosage was considered to be 800-1,200 mg/body/day. The tumor began to decrease in size within 8 weeks of treatment in most of the responded cases. Side effects were observed in 44.4% of 513 cases. Diarrhea occurred with the highest rate (26.3%), but this was controllable.

A phase I study of a new fluorinated pyrimidine compound, 5'-deoxy-5-fluorouridine (5'-DFUR), was performed in 37 patients with various malignant cancers. Starting dose was 600 mg/m2/day (900 mg/body/day) and escalated up to 3900 mg/body/day. The dose given was divided into 3 administrations a day for 5 consecutive days. Subjective symptoms were observed in cases given a dose of over 2,100 mg/body/day. Gastro-intestinal disturbances such as nausea, vomiting and anorexia were the major side effects. In the hematological and urinary examinations, no severe abnormal signs were observed. The maximum tolerated dose was considered to be 2.100 mg/body/day, and the dose-limiting factor was gastro-intestinal disturbance. 5-FU levels were determined in the serum and tumor tissues. 5'-DFUR was well absorbed. The 5-FU level in tumor tissue was very high at 2 to 3 hours post-dose and then rapidly decreased, being 0.05 microgram/g 12 hours after administration. The optimal dosage for a phase II study was suggested to be less than 2,100 mg/body/day.

We have attempted to grow several kinds of malignant tumors using human tumor stem cell assay. Formation of colonies in vitro was seen in 65 of 132 primary tumors (49%), including 25 of 41 (61%) uroepithelial cancers, 12 of 19 (63%) renal cancers, five of 12 (42%) testicular cancers, five of 21 (24%) gastrointestinal malignancies, five of 12 (42%) lung cancers, five of 11 (45%) hematopoietic malignancies and five of 16 (31%) other malignancies. Growth sufficient for in vitro chemosensitivity tests of CDDP developed from seven cases of uroepithelial cancer, three of them (43%) were sensitive to 2.5 micrograms X hour/ml of CDDP. The specimens from a metastatic testicular tumors that received several courses of PVB chemotherapy resulted in the resistance of the in vitro chemosensitivity test at a higher dosage of CDDP. Nine cases of renal cancer had sufficient growth for an in vitro chemosesitivity test of interferon. One of them was sensitive for alpha 2 type interferon. Three of seven cases were sensitive for alpha type of interferon. To predict clinical correlation, 19 patients were tested with the same drugs used in the in vitro chemosensitivity test. The predictability resulted in more than 60% of true positive, 91% of true negative and 86% of overall predictability.

Acquired multidrug resistance as well as innate drug resistance are directly related to ineffectiveness and failure of the cancer chemotherapy. The mechanisms of such resistance, especially those of innate resistance, have not been fully elucidated. Drug resistant tumor cells, however, usually bear biochemical changes which are related to resistance mechanisms. New modalities with high selectivity against resistant cells could, therefore, be possible if we could target these biochemical changes. Vincristine (VCR)-and adriamycin (ADM)-resistant tumor cells (pleiotropic drug resistant cells) usually show an enhanced outward transport of these antitumor agents, and they express unique glycoproteins in the plasma membrane. By targeting for these biochemical changes characteristic to the resistant tumor cells, we establish new modality which shows high selectivity against drug resistant tumor cells. In this review, I will describe genetic origin of drug resistance, biochemical mechanisms of drug resistance and reversal of drug resistance in tumor cells. The modality to utilize calcium channel blockers which inhibit the enhanced outward transport of VCR and ADM from resistant tumor cells will be reviewed.

Over the past year, we have attempted to grow 132 different human tumor samples (78 from urological and 54 from non urological tumors) using a soft agar colony formation assay similar to that originally described by Salmon and colleagues. Formation of colonies in vitro occurred in 44 of 78 primary urological tumors (56%), including 63% (12/19) of renal cancers, 61% (25/41) of uroepithelial cancers and 42% (5/12) of testicular cancers. The effects of in vitro chemosensitivity were analysed using the inhibition of colony growth (more than 70%) at two different cut-off doses which were equilibrated with the achievable AUC doses for the higher cut-off point and to one-tenth of AUC for the lower cut-off point. Five of 13 (38%) drugs showed an effective rate between 10% and 38% for the lower cut-off point in vitro. On the other hand, eleven of 13 (85%) drugs. Showed an effective rate between 20% and 67% for the higher cut-off point in vitro. In the tested uroepithelial cancers, none of seven for the lower cut-off and three of seven for the higher cut-off were demonstrated to be sensitive cases from the dose corresponding colony inhibition curve for cisplatinum. Nine of the renal cancers were demonstrated for the dose corresponding colony inhibition curve for Interferon. To predict clinical correlation, 16 patients (20 drugs) were treated with identical drugs which were estimated from in vitro chemosensitivity testing. The predictability results were 75%-100% true positive and 85%-100% true negative, with 87% overall predictability. This assay can therefore be used to study differences of biological character including drug sensitivity.

The antitumor activity of 27 anticancer drugs against a human breast cancer tumor (MX-1) has been studied using the subrenal capsule assay developed by A.E. Bogden et al. in 1978. Without immunosuppression with cyclophosphamide, BDF1 mice transplanted with MX-1 were treated with various drugs. The antitumor activity was evaluated by the tumor growth inhibition rate on day 6 after treatment. Among the 27 anticancer drugs tested, 10 compounds (37%) which showed more than 75% tumor growth inhibition rate were considered to be active. On the other hand, 8 compounds out of 27 drugs (30%) which showed less than 50% tumor growth inhibition rate were considered to be inactive. When the antitumor activity between the subrenal capsule assay in BDF1 mice and the subcutaneous transplantation assay in nude mice were compared, both assays were well correlated (r = 0.787, p less than 0.001). These results suggest that the antitumor activity of drugs can be evaluated faster, cheaper and easier by the subrenal capsule assay compared with the subcutaneous transplantation assay in nude mice.

As a tumor factor possibly responsible for chemosensitivity of human cancer xenografts in nude mice, the vascular architecture of tumors growing in mice was investigated in 15 kinds of cancer lines. These consisted of 7 gastric, 3 colorectal, 3 breast and 2 pancreatic cancers. Whole body angiograms of tumor-bearing mice were obtained by perfusing a radiopaque silicone rubber compound (Microfil) through the left ventricle of each mouse. Each cancer retained a characteristic vascular architecture comparable to its histopathological finding. According to the vascularity of the viable part of the tumor, the 15 lines of cancer were classified into 5 groups. Compared with colorectal cancers, stomach cancers had a tendency to decline to a more hypervascular group. There was no apparent relation between vascularity and growth rate, or histological differentiation of the tumors. Among 14 anticancer agents studied, statistically significant correlation between chemosensitivity and vascularity of the 15 cancer lines was observed in 2 drugs, 5'-DFUR and adriamycin. The vascular architecture of the tumors would have some influence in their chemosensitivity through drug accessibility to cancer cells.

The author reviewed his experience to date with chemosensitivity testing of 162 gastric and 144 colorectal cancers. All human tumor clonogenic assays were performed using a double-layer soft agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Significant growth was defined as greater than or equal to 30 colonies/control plate. Clinical responses were determined according to standard criteria. Forty-six per cent of gastric cancer specimens and 67% of colorectal cancer specimens plated produced significant growth in vitro. When greater than or equal to 50% inhibition of colony formation was used as the criterion for differentiating sensitivity from resistance, the assay was 67% (8/12) reliable for predicting in vivo sensitivity, and 91% (10/11) reliable for in vivo resistance.

It would be helpful for successful chemotherapy in cancer patients if a drug-sensitivity test in vitro could predict the exact response of an individual patient's tumor. We have investigated a drug-sensitivity test using human tumor clonogenic assay since 1980. In this paper, results obtained in lung cancer patients are discussed. Specimens for testing were obtained from primary tumor, metastatic mass, malignant pleural and pericardial effusion, and affected bone marrow. Drugs tested in this study were adriamycin, aclarubicin , THP-adriamycin, mitoxantrone, mitomycin C, cis-platinum, 40497 S (an active compound derived from ifosfamide), and methotrexate. Out of 88 specimens tested, 41 (47%) successfully yielded more than 30 colonies per control dish, and were able to evaluate drug-sensitivity. Of those, 32 instances were valid for examination in an in vitro-in vivo association. As a result, 3 were in vitro sensitive-in vivo sensitive, 2 were in vitro sensitive-in vivo resistant, and 27 were in vitro resistant-in vivo resistant. Accordingly, the true positive rate was 60%, and the true negative rate was 100%. In summary, the human tumor clonogenic assay appeared to be an excellent method for testing drug-sensitivity for an individual patient with lung cancer.

The human tumor clonogenic assay (HTCA) developed by Hamburger and Salmon was evaluated in 135 fresh samples of breast cancer. Successful tumor colony growth (greater than or equal to 5 colonies/plate) was obtained in 100 (74%) of the 135 samples, and adequate growth for drug testing (greater than or equal to 30 colonies/plate) in 75 (56%). With regard to the success rates of growing colonies categorized by specimen source, tumor sites, histology, prior chemotherapy and estrogen receptor (ER), specimens from solid tumors and primary tumors showed higher success rates than those from pleural effusions and metastatic tumors. The effects of prior chemotherapy, histology type and ER status on the success rate of colony formation were not significant. The overall median plating efficiency was 0.012%. Higher plating efficiencies were found in pleural effusion, metastatic tumors and samples from patients with prior chemotherapy. These findings appeared to indicate that aggressiveness of disease might be related to plating efficiency. Defining a greater than or equal to 50% inhibition of colony formation (ICF) as in vitro drug sensitivity, the in vitro response rates to anticancer drugs tested were as follows: adriamycin 33%, mitomycin C 39%, 5-fluorouracil 32%, methotrexate 42%, L-PAM 31%, cisplatin 38%, vincristine 32%, vinblastine 54%. The group of patients without prior chemotherapy showed higher sensitivity in vitro compared with the group of patients who had prior chemotherapy (38% vs. 27%). Correlation between in vitro drug sensitivity (greater than or equal to 70% ICF) and clinical response in 12 patients treated with the same drugs were analyzed retrospectively. The predictive accuracy was 0% (0/1) for true positive and 91% (10/11) for resistance. Thus, overall predictive accuracy was 83%. Based on these results, HTCA appeared to be useful chemosensitivity test for evaluation of antitumor drugs for human cancers in vitro and prediction of the chemotherapeutic effect in clinical use.

For use in routine clinical studies, modifications to Salmon and Hamburger's human tumor stem cell assay were made. A multiplate with 24 wells made the handling of a large number of samples feasible. The addition of anticancer drugs to the bottom layer of agar facilitated avoidance of exposure to drugs before cell plating and evaluation of the effect of long-acting drugs such as 5-fluorouracil. Storage of test plates including anticancer drugs in a freezer produced no loss of colony-forming activity. Specimens from 32 patients with advanced malignancies of the GI tract were tested for sensitivity to anticancer drugs. Forty-seven percent formed enough colonies for the performance of drug testing. Two patients showed sensitivity to drugs from both in vitro and in vivo results; the ascites in one disappeared while the other showed more than 50 percent regression of hepatic metastatic foci after treatment with suitable drugs. Nine patients showed resistance to drugs from both in vitro and in vivo results. Eight showed resistance to all tested drugs.

The subrenal capsule assay (SRCA) has been developed by Bogden. This simple method using fresh human tumor has much more approximation with clinical procedure than other sensitivity tests. Even from our as yet limited small experiences, its high evaluability and high specificity are promising. Problems inherent to the actual assay and its limitations were reviewed and discussed, including our trial with bone marrow puncture specimens of leukemia, our evaluation criteria and assay-clinical correlations. This method will be widely utilized in several fields of cancer treatment in the near future.

The human tumor clonogenic assay (HTCA) is a double-layer agar technique, which provides an in vitro prediction of the response of an individual patient's tumor to an antitumor agent. This paper briefly provides an outline of HTCA and its potential use in chemotherapy on patients with lung cancer. In our experience with culturing 123 carcinomas of the lung, 105 specimens (85%) could be subject to more than 5 chemosensitivity tests each by modifying the preparation method of single cell suspension in this system. Growth rate was improved in all types of primary human lung cancer with reasonable consistency. Further, metastatic tumors were capable of being successfully grown in a high percentage of cases, which was comparable to the results obtained for other kinds of tumors. There was no correlation of growth or cloning efficiency with histology, source, or previous chemotherapy. Using 50% or more inhibition on to colony formation as the criterion for chemosensitivity, response rates to vindesine or mitomycin C were 19% or 16%, respectively. The in vitro response rates of these or almost all other antitumor drugs seemed to be comparable to the clinical responses reported by various investigators. Correlation between in vitro chemosensitivity in HTCA and clinical response has been evaluated by various investigators, and the pooled data have demonstrated a good association between in vitro drug sensitivity and clinical response or lack of response. In lung cancer, HTCA had a 57% true positive rate and an 85% true negative rate for the prediction of drug sensitivity and resistance, respectively, of cancer patients to specific chemotherapeutic drugs. Although the system still has to undergo modification to resolve a number of theoretical and practical problems, it has potential uses in lung cancer chemotherapy.

The in vitro evaluation of anticancer drug efficacy was performed using the human tumor clonogenic assay developed by Hamburger and Salmon, and correlation between the in vitro and clinical efficacy was analyzed retrospectively. The in vitro colony assay method used in this study was a minor modification of the above method. Thirty-two out of forty-eight samples from patients with ovarian cancer formed more than five colonies per plate on in vitro colony assay. The median plating efficiency was 0.06% (range 0.02-1.3%) and the median colony count per plate was 279 (range: 8-4,000). With regard to colony formation of ovarian cancer according to the source of the specimen, the colony-forming rate of solid tumor was high (72%) as compared with 63% for ascites and 43% for pleural effusion. In vitro chemosensitivity was defined as more than a 50% decrease in colony formation and the rates for standard drugs on ovarian cancer were as follows: adriamycin (0.04 micrograms/ml) 29%, bleomycin (0.1 micrograms/ml) 24% cisplatin (0.2 micrograms/ml) 31%, 5-FU (1.0 micrograms/ml) 22%, hexamethylmelamine (1.0 micrograms/ml) 19%, L-PAM (0.4 micrograms/ml) 44%, mitomycin C (0.1 micrograms/ml) 38% and THP-adriamycin (0.5 micrograms/ml) 36%. A group of patients who had not been exposed to any anticancer drug showed higher sensitivity in vitro as compared with a group of patients who had received prior chemotherapy (35% vs 22%, p less than 0.05). Correlation between in vitro drug sensitivity and clinical responses in patients treated with the same drugs were analysed retrospectively. In all twenty cases, two were true positive cases (29%), while in ten cases, the results were true negative (77%), The overall predictive accuracy was 60%. In conclusion, ovarian cancer cells can form colonies well when the soft agar method is used and this assay method is suitable for the evaluation of various anticancer drugs in vitro.