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.

Mitoxantrone, a new anthracenedione, was administered to twenty-five evaluable patients with relapsed or refractory acute leukemia between January 1982 and September 1984. Two patients were not evaluable because of early death. There were 18 males and 7 females with a median age of 42 yrs (range 6-70 yrs). Four of these were less than 14 yrs and 6 more than 55 yrs. The initial dose employed was 3 mg/m2/day X 5 days. Eventually a starting dose of 10 mg/m2 X 5 days was used. Among 16 patients with acute nonlymphocytic leukemia, there was one complete and 3 partial remissions. One of 4 patients with acute lymphocytic leukemia achieved a complete remission. Also, a complete remission was obtained in a patient with T-cell lymphoma/leukemia. The overall remission rate was 24% with a complete remission rate of 12%. Remissions occurred at doses of more than 6 mg/m2/day X 5 days. Four of the 6 patients who had attained a remission received one of the anthracyclines. Bone marrow depression was the dose-limiting factor. Mucositis occurred in 6 patients to whom higher doses were administered. This mucositis was thought to be due to drug-related toxicity. The trials were too short to evaluate possible cardiac toxicity. These data indicate that mitoxantrone is a promising single drug for the treatment of relapsed or refractory acute leukemia.

During the past 18 and a half years, 285 patients with cancer of the pancreas have been treated in our hospital. By means of en-bloc resection of the lesion with the portal vein and/or hepatic artery, the resectable rate of cancer of the head of the pancreas was improved to 53% (1979-1983) from 22% (1966-1978). Postoperative deaths within 30 days after pancreatectomy were 9.8%. The five years survival rate in those with Stage I and II was 28.7%, but that with Stage III and IV was 15.2%. Intraoperative and/or external radiation therapy was adopted on 16 patients, in some of whom hyperthermia was further combined. As a result, remarkable effects on performance status were obtained. Intra-arterial infusion chemotherapy using a transfemorally implanted catheter was performed on ten patients with unresectable lesions, the median survival of whom was however, only four and a half months.

The effects of reduction surgery combined with chemotherapy used by MMC and ADM for DAB induced hepatoma were investigated. Results are as follows. Reduction surgery prolonged the survival time of rats with DAB hepatoma in which tumor was transplanted either in liver or subcutaneous tissue. Reduction surgery in combination with chemotherapy made survival time more prolonged in these rats with than reduction surgery alone. Reduction surgery combined with intraarterial infusion chemotherapy was carried out in three patients with advanced primary liver cancer. One survived for 9 months and another for 3 years after right trisegmentectomy and postoperative arterial infusion chemotherapy. The other is till alive without any recurrence 7 months after right trisegmentectomy with partial resection of a intrahepatically metastasized tumor in the lateral segment and arterial infusion chemotherapy used by ADM. In summary, it is suggested that reduction surgery in combination with chemotherapy is beneficial as a multidisciplinary treatment for advanced primary liver cancer.

Multimodality therapy for colorectal cancer is composed of surgery, chemotherapy and irradiation; and hyperthermia joins them recently. As patients with operable colorectal cancer are a good candidate for a chemotherapeutic approach, we began postoperative adjuvant chemotherapy since 1971. On the other hand, our treatment policy towards inoperable cases is various treatments combined with the four therapies described above. The most patients with hepatic metastasis are unamenable for surgery, and they have been mainly treated with intra-arterial chemotherapy. With conventional infusion treatment, however, the infusion drugs are eliminated rapidly from the drainage vein. Thus, we prepared biodegradable albumin microspheres containing MMC (mean diameter 45 +/- 8 micron); and in 6 patients with liver metastasis, we infused MMC microspheres into the proper hepatic artery, with marked tumor regression. Hyperthermia treatment has been performed with ThermaTech 2000 (International Institute for Medical Sciences, U.S.A.), which has a complete capacitive, 3-channel, "crossfire" heating system operating by RF at 13.56 MHz. Six patients with local recurrence and/or hepatic metastasis were treated by hyperthermia combined with chemotherapy or irradiation, with a fair success in tumor response and improvement of subjective symptoms.

Continuous hyperthermic peritoneal perfusion (CHPP) using the physiologic salt solution with the addition of Mitomycin C was performed for patients with peritoneal dissemination of gastric cancer. The result was favorable in some of them but unsatisfactory in survival. On the other hand, prophylactic CHPP for gastric cancer patients with serosal invasion revealed the favorable result in postoperative survival.

The effectiveness of Bestatin combined with Mitomycin-C, FT-207 for treating gastric cancer was investigated in a prospective randomized controlled study. All patients had undergone gastrectomy during the period from November 1980 to July 1983. Three-year survival rates revealed no difference between groups given or not given Bestatin. The effectiveness of Bestatin was confirmed by postoperative changes revealed by liver function test, especially GOT and GPT. No side effect due to Bestatin was detected.

Some of organ dysfunctions due to cancer therapy in children are common to those of adults, but others are specific for children. Even common toxicities may have aspects peculiar to children. For example, cisplatin nephrotoxicity easily causes hypomagnesemic, hypocalcemic tetany in children which is rare in adults. Occurrence of second primary malignancies is serious late effect of cancer therapy. Concerning this problem two factors are important in children. Firstly, children with heritable embryonal cancers are predisposed to develop additional malignancies related and unrelated to therapy. Secondly, proportion of different parts of body of children is different from one of adults and normal tissues distant from the primary radiation field may receive surprisingly large dose of irradiation. For example, a 8-year-old boy received 6 to 9% of dose of prophylactic skull irradiation for acute lymphocytic leukemia to his thyroid. Younger children who have smaller viscerocranium are expected to have larger dose. Children who received antileukemic therapy for 3 to 7 years showed significant delays of linear growth and bone age.