High-dose cytosine arobinoside (HDAra-C) was originally introduced in clinical hematology for the treatment of refractory or relapsed acute leukemia. Recently, however, this treatment has been tried for remission induction and/or consolidation in early leukemia cases, and resulted in a fairly percentage of long-term survival cases. HDAra-C in combination with anthracyclines is now considered to be a treatment which may afford some hope of a cure in a certain percentage of cases of adult acute non-lymphocytic leukemia. This treatment also has the advantage of being an effective treatment for meningeal leukemia, since a high concentration of Ara-C effective enough to kill leukemic cells is maintained in the cerebrospinal fluid after intravenous infusion of high-dose Ara-C. In this paper, the HDAra-C treatment of acute leukemia is reviewed and the results of our group study on the treatment of 30 cases of refractory acute leukemia with HDAra-C are discussed.

On twenty-three specimens from patients with osteosarcoma (biopsied tissue, resected specimens from primary and metastatic lesions), the human tumor clonogenic assay (HTCA: standard assay) as well as cultivated HTCA involving short-term cultivation of a single-cell suspension, was carried out. The percentage colony-forming ability, which was very small for standard HTCA, increased significantly when cultivated HTCA was performed. With cultivated HTCA, sensitivity tests were possible in 87% of the specimens, in contrast to only 28.6% with standard HTCA. Retrospective evaluation of the results obtained by cultivated HTCA with the clinical efficacy of anticancer drugs showed a true positive rate, 66.7%, and a true negative rate, 88.9%. This analysis revealed that drugs shown to be ineffective and that tumor cells are refractory to those drugs, so that clinical use of such drugs should be avoided.

In the multi-disciplinary treatment of head and neck tumor, anti-cancer chemotherapy was evaluated on the basis of survival rate, response rate, and preservation of function and shape. Chemotherapy significantly improved the survival rates in maxillary cancer, cervical esophago-hypopharyngeal cancer and malignant lymphoma in stage II. A better than partial response was obtained in 42% of the patients by a combination of Peplomycin, Vincristine, Endoxan, Mitomycin C, Predonine, 5-FU and Adriamycin. Functional and cosmetic preservation with combined chemotherapy was observed only in maxillary cancer. Chemosensitivities for head and neck malignant tumors were measured by both clonogenic assay and rapid scintillation assay in order to evaluate the sensitivity test. Successful growth rate of head and neck tumors in the clonogenic assay was 58%, while that in the scintillation assay was 54%. Squamous cell carcinoma in the head and neck region was sensitive to Peplomycin, Bleomycin, 5-FU, Mitomycin C, Adriamycin and Cisplatin in that order. Although no concrete merit of these tests has yet been achieved, inoperable recurrent cancer or maxillary carcinoma could be treated by more effective drugs selected by these methods.

Second malignancies were observed in 181 cases after treatment of antecedent breast cancer, among 5,302 primary breast cancer cases. The accumulated incidence of double cancer was as follows: 2.8% for 5 years, 5.2% for 10 years, 7.6% for 15 years, and 10.0% for 20 years. The observed incidence of second malignancy for all sites was 1.58 times as frequent as in the normal group. Statistically significant increased risks were observed for opposite breast cancer (O/E ratio 5.92), ovarian cancer (O/E ratio 4.47), corpus uterine cancer (O/E ratio 5.97) and thyroid cancer (O/E ratio 5.07). Among 5,302 cases, 2,431 (45.9%) underwent adjuvant chemotherapy. In chemotherapy groups, significantly increased risk of stomach cancer, thyroid cancer, leukemia and hepatoma was observed, but there were no remarkable differences between the MMC group and the CPA group. However, in the MMC + CPA combination treatment group, the risk of stomach cancer and leukemia was higher than in the single drug treatment groups. When multiple drugs were administered in large doses as long-term adjuvants, the risk of second malignancy seemed to become greater.

There is little difference in heat sensitivity in vitro between normal and cancer cells. However, rather selective tumor destruction is induced by in vivo heating without affecting normal tissues. This selective effect may be attributed to heat-induced vascular damage causing reduction in the supply of oxygen and nutrients, and a decrease in the pH of tumors. These cellular environmental changes greatly enhance hyperthermic cytotoxicity. Besides this, antitumor immunity of host animals is also involved in the selective effect of hyperthermia. Hyperthermia potentiated the actions of various antitumor agents including nitrosoureas, cisplatin and bleomycin. The effect of heating on adriamycin cytotoxicity depends on the cell lines used; in some cell lines the cytotoxic effect is enhanced by 42-43 degrees hyperthermia, but in some other cells it is enhanced only at 40-41 degrees and drug-resistance is induced at 43 degrees. Benzaldehyde is nontoxic at 37 degrees but has greatly enhanced cytotoxicity by combination with hyperthermia.

A rapid method using nude mice has been established as an in vivo model for assessing the chemosensitivity of individual human tumors, in which the final evaluation is made with 3H-thymidine (3H-TdR) incorporation into the treated tumor. In 234 of 289 cancers, the chemosensitivity of anticancer agents was evaluated by this method. This assay proved to be feasible in a sufficiently high percentage of human primary tumors (81.0%). The rate of positive sensitivity against all tumors was 23.8% for MMC, 12.3% for 5-FU, 29.1% for CPM and 23.5% for ADM, respectively. The sensitivity of anticancer agents varied according to the type of cancer. Correlation between the sensitivity test and the end results after chemotherapy in cases of inoperable gastrointestinal cancers was investigated, prospectively. Out of 19 cases, the 50% survival time of 11 patients treated with sensitive agents was longer than that of 8 patients treated with insensitive agents. From a prospective-correlative study carried out on 25 patients, this assay appeared to be correlated with clinical response (overall agreement, 76.0%) with specific agreements of sensitivity and resistance of 37.5% and 94.1%, respectively. From these results, it seems reasonable to conclude that this sensitivity test using a human/nude mouse system is a useful screening assay for revealing appropriate agents for the treatment of patients with cancer.

To improve the plating efficiency (PE) of cells used in the standard human tumor clonogenic assay (standard HTCA), an HTCA was developed using single-cell suspensions obtained from short-term cultures of surgical specimens of malignant bone and soft tissue tumors of the extremities. Consequently, the percentage possibility of application for the chemosensitivity test for anticancer drugs as well as the PE improved from 29.7% to 65.7% for bone tumors (37 specimens) and from 30.0% to 72.4% for soft tissue tumors (31 specimens). In bone tumors the correlation between these drug sensitivity tests and the clinical effectiveness of the drugs was retrospectively evaluated in 17 specimens from 13 cases mainly of osteosarcoma; they were well correlated with the true positive rate (71.0%) and the true negative rate (90%). In soft tissue sarcomas, retrospective evaluations were possible only in 9 cases and their true positive and negative rated were 75.0% and 100% respectively. These results indicate that cultivated HTCA seemed to be a clinically useful chemosensitivity test: the anticancer drugs judged useless by this test were likely to be clinically useless. Drugs such as resistance-acquired or non-sensitive drugs should be excluded from multidrug combination chemotherapy in the treatment of bone and soft tissue sarcoma.

The response to cancer chemotherapy varies from patient to patient with the same histologic type of tumor. However, anticancer drugs are considerably toxic to the patient, and it is therefore very important to have an accurate knowledge of the sensitivity of anticancer drugs against an individual patient's tumor in establishing successful chemotherapy. We have investigated the sensitivity testing of anticancer drugs using the human tumor clonogenic assay since 1980. Specimens were obtained by aspiration of ascites, pleural effusions and bone marrow and by biopsy of primary and metastatic tumors. Drugs tested in the present study were adriamycin, aclarubicin, THP-adriamycin, mitoxantrone, 40497s (an active compound derived from ifosfamide), mitomycin C, cisplatinum and methotrexate. One hundred and fifty-two specimens were obtained from cancer patients and tested for their drug sensitivity using the assay. Of the 152 specimens, 63 (41%) formed more than 30 colonies in control plates and could be used to evaluate drug sensitivity. Of these, 45 instances were evaluable for examination in an in vitro-in vivo association, and 42 (93%) showed a correlation between in vitro sensitivity and clinical response. In summary, the results indicated that the human tumor clonogenic assay was an excellent technique for testing the in vitro sensitivity of anticancer drugs. However, technical developments yielding higher colony efficiency would be required to facilitate practical use of the assay.

Several problems have been pointed out in colony formation assay (CFA) developed by Hamburger and Salmon. We studied those using 87 fresh renal carcinoma, comprising of 72 renal cell carcinoma and 15 transitional cell carcinoma of renal pelvis. Results are as follows: Pure single cell suspension was difficult to be yielded from these carcinoma. Our result suggests that cell suspension contains various sized growth unit as well as greater than or equal to 60 microns growth unit. Presence of substantial number of greater than or equal to 60 microns growth unit in plating cell suspension was revealed to influence on the cell growth, ie, the increase of newly formed greater than 60 microns growth unit. This indicates that initial growth unit count is mandatory for "quality control" in CFA. The current study in extent of growth for renal carcinoma revealed three different patterns, one of which did not seem suitable for in vitro chemosensitivity test with CFA. This finding brings for us the fact to prepare "proliferation control" which can check periodically the growth extent of renal carcinoma.

Through our retrospective analysis of HTCA and clinical effects as well as fundamental studies, the following results were obtained: In this retrospective study, HTCA for lung cancer showed a predictive accuracy of 71%, a true positive rate of 50% and a true negative rate of 77%. To obtain good predictive accuracy, HTCA should be modified to provide conditions comparable to those in vivo with regard to drug concentration and drug exposure time. More precise analysis of the pharmacokinetics of anticancer agents might yield methodological improvement. A decrease in the chemosensitivity spectrum in vitro was observed after chemotherapy. This might be related to evidence that patients with prior chemotherapy exhibited a poor response rate to chemotherapy. There were no active anticancer agents against specimens with aplating efficiency of more than 0.04%. More extensive prospective trials will be necessary to determine the clinical value of HTCA. HTCA could be a superior assay for detecting the anti-tumor activity of new agents and a useful method for in vitro phase II study.

The authors reviewed their experiences to date with chemosensitivity testing of 629 tumors by human tumor clonogenic assay (HTCA) and of 199 tumors by scintillation assay (SA). HTCA and SA were both performed using a double-layer-soft-agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Overall, 60% of specimens in HTCA and 58% in SA produced significant growth in vitro. HTCA was 52% (13/25) reliable for predicting in vivo sensitivity, and 95% (36/38) reliable for in vivo resistance, whereas SA was 40% (8/20) reliable for in vivo sensitivity and 88% (21/24) for in vivo resistance. The current results indicate that there is no particular difference in the success rate and in the predictive accuracy for in vivo sensitivity and resistance between HTCA and SA. Therefore, it is doubtful whether only clonogenic tumor cells among whole tumor cell populations should be selected in assessing the chemosensitivities of human tumors. In vitro success rates were variable, depending on the tumor histology. In vitro growth of gastric cancer specimens was characteristically lower than that of colon cancer specimens (48% and 60% in HTCA, and 46% and 68% in SA, respectively). (p less than 0.005). Optimal in vitro-in vivo drug concentrations and culture conditions are still being defined. Correlation studies of in vitro-in vivo responses of gastrointestinal cancers suggested that in vitro concentrations of 5-fluorouracil and mitomycin C used in this study were considerably higher than their optimal doses. Tumor cell heterogeneity poses significant problems in the clinical use of chemosensitivity assays. The question arises as to whether a single biopsy specimen is representative of a patient's disease. In this last study, we sought evidence of tumor heterogeneity by comparing chemosensitivity responses between: 1) different portions of a single tumor, 2) a primary and a metastatic biopsy taken from a patient on the same day, and 3) different metastases from a patient taken on the same day. The results demonstrated the presence of considerable heterogeneity of response to chemotherapy among different tumors from the same patient, and even within the same tumor. The reported discrepancies of in vitro and in vivo sensitivity may be due to such therapeutic heterogeneity among tumors.

The antitumor activity and mode of action of antitumor agents were assayed by a new method which takes a relatively short time for evaluation. The method was clinically applied to patients with malignant lymphoma or acute leukemia for estimating drug sensitivity. Fresh malignant cells were obtained from patients' bone marrow, lymph nodes, or peripheral blood using Lymphoprep methods in 26 patients with these malignant disorders. Each sample (5 X 10(5) cells/ml) was cultured in the medium with RPMI 1640 supplemented with 10% of FCS and 20% of patients' own sera containing every antitumor agent at predetermined concentrations for 30 minutes. Each sample was again cultured for another 72 hours at 37 degrees C in a 5% CO2 humidified atmosphere after rinsing. Microscopic observation was periodically carried out using a specially devised observation chamber with an inverted immersion microscope for 72 hours. Morphological changes of 200 to 300 cells of each sample were described according to morphological criteria. Of these, irreversible changes in number were considered to be indicative of antitumor activity of the agent tested. More than 50% cellular damage was categorized as effective and more than to 50% reduction of leukemia cells or decrease in size of lymph nodes the same degree in malignant lymphoma were considered to be clinically effective. Forty-four assay runs in 26 patients were done, and on 31 occasions were shown to be coincident. We conclude that this in vitro method is fully applicable for predicting the effectiveness of an agent to be clinically administered regardless of the various controversial problems to be solved.

In vitro drug testing using (3H) incorporation was utilized for specimens from 60 patients with advanced malignancies, mainly gastrointestinal cancers. Forty-six specimens had sufficient growth for drug testing. Forty-seven percent of stomach cancer specimens and 67% of colorectal cancer specimens demonstrated in vitro chemosensitivity. Among 9 chemotherapeutic agents tested, ara-C, 5-fluorouracil, and mitomycin C had higher percentages of positive sensitivity for stomach cancer, whereas actinomycin D, carboquone, and ACNU had a more pronounced effect on colorectal cancer. A comparison was made between the results of chemosensitivity tests and clinical response. In 46 drug assays, in vitro/in vivo drug responses were correlated in 78% with a true-positive predictive rate of 47% and a true-negative rate of 94%. This test appears to be a reliable indicator for clinical response to the same chemotherapeutic agents in human malignant disease.

Transitional thermotolerance develops during (at below circa 43 degrees C) or after (at above circa 43 degrees C) hyperthermic treatment, increases through the first 6 h, is maintained for circa 24 h and disappears at circa 72 h after the hyperthermia. Therefore, a once every three days modality of hyperthermic treatment is preferred among the clinical institutions where therapeutic hyperthermia is provided. In the present communication, the inhibition of thermotolerance development by combined treatment of anticancer drug or benzylidene glucopyranose with hyperthermia and its related problems are reported.

A 77-year-old male was admitted to the hospital because of left hemiparesis secondary to multifocal cerebral metastases from adenocarcinoma of the stomach. He was treated with combination of radiotherapy and chemotherapy consisting of ACNU, Tegafur and PSK. He was in good condition, but abruptly developed severe dyspnea 40 days after administration of Tegafur and 28 days after that of ACNU. Chest X-ray at that time revealed diffuse opacity involving entire lung fields associated with marked hypoxia. The patient expired 9 days after this episode. The autopsy revealed acute interstitial pneumonitis associated with hyaline membrane formation consistent with adult respiratory distress syndrome involving entire lobes of both lungs without metastases. As to the etiology of the ARDS in this case, we concluded that the administration of Tegafur was the most likely as to the cause, although the possibility of betamethasone was not ruled out. The remaining factors were not likely as to the cause of the ARDS in this case.

Monocyte-derived macrophages (M phi) from cancer patients injected with low doses of Neocarzinostatin (NCS, 500 units/day, three times a week) 12 times produced significantly more superoxide (O2-) than controls. Lymphocyte functions, such as PHA response, surface marker and serum IAP, before and after NCS injections were the same. M phi from normal persons cultured with NCS (0.4 microgram/ml) for three days produced more O2- than controls, but those cultured with rINF gamma did not. These results suggest that the increased O2- production of M phi from patients taking low doses of NCS may be due to the direct action of NCS on the M phi.

The antitumor activity and enzymology of 2'-deoxy-3', 5'-bis-O-(4-methoxyphenoxycarbonyl)-5-fluoro-3-(4-n-Propoxybenz oyl uridine (FF-707) were examined. It was found that stability in small intestine homogenate of FF-707 was higher than that of FF-705 [2'-deoxy-3', 5'-O-diacetyl-5-fluoro-3-(3 methylbenzoyl) uridine]. Reduction of the tumor weight was greater in mice bearing sarcoma-180 and rats bearing Yoshida sarcoma treated with the oral administration of FF-707 than that FF-705. The relationship between the antitumor activity and the inhibition of thymidylate synthase after the oral administration of FF-707 was examined. The extent of the inhibition of thymidylate synthase seemed to be parallel to that of the inhibition of the tumor growth.

The authors examined the sensitivities of esophageal cancer to Bleomycin (BLM), Peplomycin (PEP), Cisplatin (CDDP) and 5-FU by the INAS method using 3H-thymidine or 14C-formate as labeled precursors, and determined the concentrations of anticancer agents in cancer lesions by the Band Culture method. On the other hand, the authors investigated the superiority or inferiority of various methods of BLM administration by observing the prevention effect of BLM on the development of experimental esophageal cancer in rats. Forty-three cases out of 76, 57%, showed a sensitivity to BLM, 60% to PEP, 38% to CDDP and 56% to 5-FU. As to the types of roentgenological findings, the superficial and tumorous types showed a high sensitivity rate. As to the types of macroscopical findings, the protruded and superficial types showed a high sensitivity rate. As to the types of histological findings, well differentiated squamous cell carcinoma showed a high sensitivity rate. Sensitivity was higher in metastatic lymph nodes than in main cancer lesions. Tumor tissues which had undergone previous hyperthermic management (at 42 degrees C) showed a higher sensitivity than those which had not. PEP at a half dose brought about the same grade of anticancer effect as BLM. The sensitivities of esophageal cancer to various anticancer agents showed individual differences among clinical cases. Therefore, combination chemotherapy for esophageal cancer was thought to be an effective administration method. The divided administration of small doses of BLM was thought to be more superior than the one-shot administration of a large dose for esophageal cancer. The results of the INAS sensitivity test were perfectly coincident with the effects of chemotherapy in clinical cases of esophageal cancer.