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Twenty-eight patients with choriocarcinoma have received the three kinds of combination chemotherapy since 1983 at our department, i.e., MOA consisting of moderate dose methotrexate (MTX), actinomycin-D (Act-D) and vincristine, MEA (moderate dose MTX, Act-D and etoposide) and FA (high dose 5-Fluorouracil and Act-D). The clinical and laboratory data obtained in the 28 patients were summarized as follows; 1. The MOA regimen was administered to 4 patients primarily and to 2 secondarily. All of the 6 patients attained remission, but finally two (33.3%) developed relapse. 2. The MEA regimen was administered to 12 patients primarily and to 12 secondarily. Of the 24 patients, five (20.8%) were found to be resistant to the MEA regimen. Nineteen patients (79.2%) attained remission, but 2 (10.5%) developed recurrence. 3. The FA regimen was attempted in one patient primarily and in 6 secondarily. Although one patient died, the remaining 6 achieved remission and one relapse has been observed in the 6 cases. 4. By applying the above mentioned 3 combination chemotherapy regimens, the overall survival rate was pushed up from 64% to 90% in choriocarcinoma patients. 5. Three patients finally died of the disease but not from the side effects of the combination chemotherapies. The major adverse effects were alopecia, nausea, vomiting and myelosuppression. In particular, serious myelosuppression was caused by the MEA or FA regimen in 5-7% of all chemotherapy courses.

The sensitivity to methotrexate (MTX) and mechanisms of the drug resistance of seven kinds of human choriocarcinoma cell lines were studied in vitro. The results were as follows. Each cell line of HCCM-5 , BeWo, IMa, and NUC-1 secreted more than 200ng/10(6) cells/48 hours of human chorionic gonadotropin (hCG), whereas GCH-1, ENAMI-1, and SCH secreted less than 20ng/10(6) cells/48 hours. HCCM-5 cell line was the most sensitive to MTX judging from the inhibition of both 3H-thymidine uptake and cell growth in vitro. Each cell line of BeWo, IMa, and NUC-1 revealed sensitivity to 2 X 10(-8)M MTX, but such cell lines as GCH-1, ENAMI-1, and SCH showed resistance to MTX. The cell lines which revealed sensitivity to MTX incorporated more 3H-MTX and showed less intracellular dihydrofolate reductase activity than resistant cell lines.

Cis-platinum (CDDP) was investigated in vitro and in vivo for its ability to inhibit the growth of Methotrexate (MTX)-resistant choriocarcinoma cell line (BeWo) and was compared with the effect of MTX and Actinomycin D (ACD). Each drug was added into medium (RPMI 1640 containing 10% FBS) for 1 hr. at the concentration of peak plasma level in clinical use (CDDP 7 X 10(-6)M, MTX 10(-6)M, and ACD 8 X 10(-8)M), or for 48 hrs. at one-tenth of the level. CDDP inhibited the cell growth to 40% of control at 7 X 10(-6)M for 1 hr., and 25% at 7 X 10(-7) M for 48 hrs. And ACD suppressed the cell growth to 10% at both 8 X 10(-8)M for 1 hr. and 8 X 10(-9)M for 48 hrs. But MTX did not inhibit the cell growth at 10(-6)M for 1 hr., and inhibited to 60% of control at 10(-7)M for 48 hrs. On the other hand, BeWo transplanted to athymic nude mice (CD-1(ICR) nu/nu) was treated with intraperitoneal injections of CDDP (1.4 mg/kg/day for 4 consecutive days in a week and repeated for 2 and 4 weeks), MTX (2mg/kg/day for 4 consecutive days in a week and repeated for 2 weeks), and ACD (90 micrograms/kg/day for 4 consecutive days in a week and repeated for 2 and 4 weeks). The suppression of the tumor growth was seen in CDDP-treated group (TRW/CRW = 6.5%) and ACD-treated group (TRW/CRW = 29%) after 2 courses of treatment, but no apparent suppression was shown in the MTX-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)

To establish a new method for treating choriocarcinoma, basic and clinical studies were made on Etoposide, a newly developed anticancer drug. I. Basic study 1) The crystal violet uptake sensitivity tests for anticancer drugs were examined with 5 human choriocarcinoma cell lines, BeWo, SCH, HCCM-5, JEG and SMT-CCl. 2) A drug concentration which could reduce the survival rate of SMT-CCl cells to 50% was regarded as the standard concentration. 3) Etoposide showed the same strong anticancer activity on all the choriocarcinoma cell lines as Act-D and CPA. II. Clinical study 1) In the treatment of trophoblastic diseases (choriocarcinoma 15 cases and invasive mole 14 cases), Etoposide therapy manifested a much higher effective rate (93.3%) than conventional Act-D therapy (50.0%) and MTX therapy (21.0%). But side effects of Etoposide therapy were milder than those of the other drugs. 2) Especially in the high risk choriocarcinoma group, the remission rate was 50% (PR 3, NC 2 and PD 1) for the conventional MAC protocol and 100% (CR 3 and PR 2) for the MECA protocol. In conclusion, it was demonstrated basically and clinically that Etoposide was very effective in the treatment of choriocarcinoma.

The cytotoxicity of methotrexate (MTX) and actinomycin-D (Act-D), and the influence of Citrovorum factor on MTX-treated cells were analyzed in vitro, using two choriocarcinoma cell lines (BeWo and SCH). HCG and beta-HCG in medium were measured with a radioimmunoassay kit, and 3H-MTX,-thymidine, and-uridine with a liquid scintillation counter. The following results were obtained. The MTX resistant cell line of BeWo, named BeWo, was selected by continuous exposure to increasing concentration of MTX in culture medium. The resistant cell was 50 to 100 times as resistant to MTX as the parent BeWo to the inhibition of cell growth and the development of resistance was accompanied by two-third decrease in 3H-MTX transport. The cytotoxicity of MTX and Act-D were found to increase linearly with the drug dose for concentrations between 10(-9) and 10(-4)M, 10(-10) and 10(-7)M, respectively. Act-D caused lethal damage in three choriocarcinoma cells (BeWo, SCH and BeWo) at one-thousandths the concentration of MTX. Citrovorum factor given within twelve hours following the start of methotrexate administration decreased in 3H-MTX transport and inhibited MTX-induced growth.

The sensitivity to methotrexate (MTX) of ten kinds of human choriocarcinoma cell lines was studied in vitro. The cell line most sensitive to MTX was HCCM-5 and it was fed with a medium containing increasing concentrations of MTX. A resistant subline (HCCM-5R) which could proliferate in the medium containing 5 X 10(-7)M MTX was obtained after about 80 weeks of feeding. The properties of HCCM-5R cells were compared with those of the parent HCCM-5 cells. i) There were no apparent differences in morphology between the HCCM-5 and HCCM-5R lines. The population doubling time was almost the same for both cell lines. ii) The hCG level in the culture fluid of the HCCM-5R cells was about twice as high as that of the HCCM-5 cells. iii) The concentration of MTX which inhibited 50% of DNA synthesis in HCCM-5R cells was 10,000 times higher than that for HCCM-5 cells. iv) Each X and Y chromosome was identified in 90% of the HCCM-5 cells, while no Y chromosome was detected in the HCCM-5R cells. v) The DNA distribution pattern for HCCM-5R cells consisted of a large fraction of each cell with DNA G1 phase content and distributed in the tetraploid range, whereas that of HCCM-5 showed no particular cell cycle pattern. vi) The incorporation of 3H-MTX in the HCCM-5R cells was one tenth of that in the HCCM-5. vii) The intracellular DHFR activity of the HCCM-5R cells was about 5 times higher than that of HCCM-5. These results suggest that MTX-resistant cells among the HCCM-5 cells were selected in long-term contact with MTX, and sensitivity to MTX was concerned with both the MTX transport and intracellular DHFR levels.

Serial transplantation in nude mice of human choriocarcinoma showed the drug resistance in clinic was succeeded and experimental chemotherapy has been studied to improve the prognosis of the patients with choriocarcinoma. The experimental tumors were two lines. CC-I-JCK (JCK) and CC-HM-I- (HM). In this paper, single and/or combination chemotherapy with methotrexate (MTX), actinomycin D (ACD), cyclophosphamide (CMP), and vincristine (VCR) were discussed. 1) Effects of the single agent: In JCK, the suppressive effects against tumor growth under administration of CPM or VCR, and some responses in histological finding were obtained. While, in HM, few suppressive effects against tumor growth and some responses in histological findings were obtained. 2) Effects of the combined agents: In JCK, the suppressive effects against tumor growth were as follows; nontreatment group less than MTX + ACD less than MTX + CPM less than MTX + ACD + CPM less than ACD + CPM less than ACD + CPM + VCR. The complete disappearance of tumors were gained under administration of the combination therapy with ACD and CPM. While in in HM, the effects were as follows; nontreatment group less than MTX + ACD less than ACD + CPM less than MTX + ACD + CPM less than MTX + CPM less than MTX + CPM + VRC. But no complete disappearance of tumor was obtained. 3) The good relationship between the results of experimental chemotherapy and the response of clinical patients who provided the tissues could be seen. It has been defined that the experimental chemotherapy with the tumors transplanted to nude mice in useful models for the establishment of rational chemotherapy for drug resistant choriocarcinoma.

Experimental chemotherapy has been studying with choriocarcinoma in nude mice transplanted from drug resistant patients. The following results: 1) the combined therapy with MTX and ACD was not always effective and in place of one of them, the combination of CPM was effective 2) addition of VCR was effective; were obtained. These results were applied to two patients in clinic. 1) The first patient failed to obtain complete remission by the course of ACD, MTX + ACD, and MTX + ACD + CPM (MAC) therapy. Then the combination therapy with ACD + CPM made her HCG titer regressed to negative immediately. She is free from disease now. 2) The HCG titer and pulmonary metastasis of the second patient showed resistance to MAC therapy, CHAMOMA therapy, and the combined therapy with MTX. The combination chemotherapy with ACD + CPM + VCR made her HCG titer become to regressed and the pulmonary metastasis reduce to 1/4 in size. The clinical application of the results from experimental chemotherapy was defined to be possible. The experimental model will contribute to establish the schedule of the rational chemotherapy for drug-resistant choriocarcinoma.

A new anticancer drug, VP16-213 was studied in vitro to determine its effect upon 3H-thymidine uptake and the survival rate of choriocarcinoma cells in comparison with the effect of MTX and Act-D. Three human choriocarcinoma cell lines, BeWo, HCCM-5 and SCH were used. The results were as follows; Serum VP16-213 levels were 10.3 +/- 1.19 micrograms/ml and 1.35 +/- 0.38 micrograms/ml at one hour and 12 hours after the administration of 100mg of VP16-213, respectively. In those three cell lines, VP16-213 suppressed cellular 3H-thymidine uptake by 70% as compared with the control. On the sixth day after exposure to VP16-213, the survival rate of choriocarcinoma cells was less than 10% in each of the three cell lines. One of the most prominent pharmacodynamic characteristics of VP16-213 was a rapid influx and efflux mechanism as seen in MTX. From the above, the anticancer effect of VP16-213 upon those three cell lines was supposed to be equal to or more than those of MTX and Act-D.

Since the introduction of MAC therapy in 1973, choriocarcinoma has become one of the most curable gynecologic malignancies. But in MAC resistant cases, the therapeutic results have been unsatisfactory. To establish the proper therapy for MAC resistant choriocarcinoma, fundamental experiments with MTX resistant HM cell lines were carried out with the aim of their clinical application. The effective combination in the treatment of choriocarcinoma appeared to be 10 to the minus 5th power mol of MTX and 10 to the minus 8th power mol of Act-D, considering its effect in cell proliferation inhibition, deoxyuridine uptake inhibition and dihydrofolate reductase activity suppression. Taking the effects of Oncovin confirmed by the experiments with nude mice into consideration, a combined moderate dose of MTX, Oncovin and Act-D, namely MOA therapy was used. The protocol of MOA therapy is as follows: The first day, MTX 150mg bolus, 300mg drip infusion for 4 hours, Oncovin 2mg bolus and Act-D 0.5mg bolus. From the second to the fifth day, Act-D bolus. MAC was effective in only one of the eight recurrent cases, but on the other hand, MOA was effective in five cases of choriocarcinoma including two MAC resistant cases. Therefore, MOA seemed to be a more effective therapy than MAC.

The remission rates of 373 patients with trophoblastic diseases were analysed according to the change in therapeutic methods. The following results were obtained. 1) In patients in the "low risk" and nonmetastatic "high risk" group, the remission rate was 100%. On the other hand, in those in the metastatic "high risk" group, the remission rate was only 71.4%, even if triple chemotherapy with methotrexate (MTX), actinomycin D (Act-D) and cyclophosphamide (CPM) was given primarily or secondarily. 2) Thirty-two of the patients in the metastatic "high risk" group underwent primary triple chemotherapy. Eleven additional patients in the same group who had never responded to initial chemotherapy of a different regimen had triple chemotherapy. The overall remission rates were 65.6% (21/32) and 45.5% (5/11), respectively. 3) The remission rate in those with recurrence who underwent triple chemotherapy was 44.4% (4/9). But two of these patients had a re-relapse. 4) Six of the patients in the metastatic "high risk" group had MEA protocol with a moderate dose of MTX, Act-D and etoposide. Five of them achieved remission. 5) The side effects of MEA protocol were not so serious, although alopecia and severe anemia were found in 100% and 75.8% of the patients, respectively.

In order to establish the value of ultrasonography in the diagnosis and management of trophoblastic tumor, we have performed ultrasonic examination on 32 patients. In 24 out of 32 cases, the focal echo could be detected within the uterus. The ultrasonically estimated volume of the focus was correlated with the level of urinary hCG. Concerning 17 hysterectomized cases, the ultrasound imaging of focuses was quite consistent with the post-operative findings. The echo patterns of these focuses could be classified into 4 types, that is, Type I, massive echogenic; Type II, multiple echo free; Type III, solitary echo free; and Type IV, subserous cystic. Type I echoes were macroscopically recognized to be clot, molar tissue and cancer tissue. In Types II, III and IV, multifarious hemorrhages were proven to be echo sources. The histopathological results showed that it is difficult to discriminate choriocarcinoma from invasive mole clearly by this echo pattern analysis. A notable decrease in focal size and change in echo pattern could be observed after chemotherapy, but these echographic changes were much slower than the rapid response in the urinary hCG value.

Hematoporphyrin derivative (HpD) is known to have an affinity for tumor cells. Photoradiation therapy (PRT), in which HpD is activated by an argon dye laser, is not always effective in treating all gynecological malignancies. Fundamental studies were performed to elucidate the mechanism acting on two cell lines and to evaluate efficiency when applied to them. Two cell lines established from endometrial adenocarcinoma and uterine choriocarcinoma were used in this study. There were apparent differences in 3H-thymidine incorporation on two cell lines treated by single HpD. In both cell lines, single HpD has some effects on neutral red uptake depending on the HpD concentration and length of treatment time. In PRT, a proportional increase in effects is obtained by extending the irradiation time. More than 90% effectiveness is obtained by pretreatment with HpD 40 micrograms/ml and irradiation for 3 minutes or more. On the DNA patterns of the cells, a shift in the histogram peak to diploid and a remarkable decrease in the number of high ploidy cells above tetraploidy occurred but were not so noteworthy in these two cell lines as in SKG-1 cells.

16 cases with gestational choriocarcinoma treated at our clinic from 1978 to 1987 were reviewed in connection with the surgical treatment for metastases in the brain and lung. 9 patients were classified as high risk, 5 patients as medium risk and 2 patients as low risk according to Bagshawe's prognostic score. An overall remission rate of 87.5% was achieved. 10 patients underwent surgical treatment with chemotherapy in 14 of 16 patients in whom metastases were identified, followed by complete remission. Pulmonary metastases were noted in 14 of 16 patients and lobectomy was performed in 10 of 14 patients, resulting in complete remission. Brain metastasis also was noted in 3 of 16 patients and the tumorectomy for the site of metastasis with irradiation was performed in 2 of the 3 patients, resulting in complete remission. One of 3 patients treated with chemotherapy alone, died. Our satisfactory results suggest the aggressive surgical therapy for the site of metastases in selected patients as indicated is apparently effective with vigorous systemic chemotherapy and radiotherapy.

The study dealt with anti-tumor and adverse effects of etoposide and cisplatin on human choriocarcinoma cell line (GCH-1) transplantable in nude mice in relation to the rate of their uptake into tumor tissue. Nude mice were divided into 3 groups (etoposide, cisplatin and MTX-group), 7 to 9 per group and received drug treatment which started when their tumor grew to 100 approximately 300 mm3 in volume. All drugs were intraperitoneally administrated to nude mice (each drug: 1/4 mouse LD50 dose X3, weekly). The inhibiting action on the tumor was observed to be etoposide greater than cisplatin greater than MTX. The body weight loss of nude mice was observed to at its maximum in the cisplatin-treated group. The serum beta-HCG level did not rise in the drug-treated groups but rose in the non-treated control group. No histopathological changes characteristic of each drug were found. A concentration of etoposide and cisplatin in various tissues was serially measured after one shot intraperitoneal injection of etoposide 25 mg/kg or cisplatin 5 mg/kg. Etoposide reached its peak concentration after 30 minutes in tumor, liver and kidney and, after 4 hours, was left with 37.1%, 10.4% and 2.3% concentrations after 30 minutes in tumor, liver and kidney, respectively. Cisplatin showed similar kinetics, but was found to remain at a comparatively high concentration in both liver and kidney. Thus, it was demonstrated that etoposide and cisplatin were active against the human choriocarcinoma cell line. Furthermore, the less adverse effect of etoposide was felt to be partially due to its lower residue in liver and kidney.