H. A. Neumann

Effects of cytostatic drugs and 40.5°C hyperthermia on human clonogenic tumor cells

Abstract A tumor colony-forming assay was used to investigate the effect of 40.5° C hyperthermia and drugs on the colony-forming ability of human clonogenic tumor cells. In order to be able to perform repeated incubations with identical tumor material, specimens were used that were augmented in the nude mouse system. Five tumor samples (two malignant melanomas, two squamous cell carcinomas of the lung and one small cell carcinoma of the lung) were incubated with seven drugs: doxorubicin, actinomycin-D, bleomycin, melphalan, vincristine, vinblastin and cisplatinum. One additional tumor specimen (chondrosarcoma) was incubated with doxorubicin immediately after resection. The incubation with drugs at 37° C revealed dose response curves typical for each tumor. Incubation at 40.5° C for 2 hr showed enhanced drug effects in five out of the six tumors tested. The drugs, with an enhanced effect and the pattern of enhancement were different in each individual tumor.

Clonogenicity of normal and malignant hematopoietic progenitor cells after exposure to synthetic alkyl-lymphospholipids

SummaryAlkyl-lysophospholipids (ALP) are synthetic analogues of natural lysophosphatidylcholine and represent a new class of anti-tumor agents. They are cytotoxic in vitro with a high selectivity for neoplastic cells which, in contrast to normal cells, lack an alkyl-cleavage enzyme to degrade the adsorbed ALP molecules. As ALP accumulates, it interferes with normal membrane phospholipid turnover and eventually causes disruption of membrane integrity. To evaluate the potential value of ALP in eliminating leukemic cells from remission marrows prior to autologous transplantation, we tested the effect of various ALPs on the clongenicity of normal human marrow cells and on promyelocytic leukemia HL-60. A remarkable difference in the dose response to ALP of normal marrow cells an HL-60 was observed. After an incubation period of 24 h, the same inhibition of clonogenicity in HL-60 occurred at ALP concentrations 4 times lower than in normal marrow cells. Reducing the exposure time to 6 h enhanced the selectivity further: whereas HL-60 colonies were nearly completely inhibited at 16 μg ALP/ml, more than 50% of normal CFU-c and BFU-E were recovered after incubation with 48 μg/ml. No further increase in selectivity was achieved by changing the incubation temperature. Both thioether- and alkyl-analogues were active and no difference was observed between methoxy- and acylamino-substituted ALPs. We conclude that this selective cytotoxicity makes ALP compounds worth further study as purging agents in autologous bone marrow transplantation programs.

Phase II trial of recombinant leukocyte A interferon in advanced malignant melanoma

SummaryA total of 21 patients with advanced metastatic malignant melanoma were treated in this efficacy study of recombinant leukocyte A interferon (interferon alfa-2a). Patients received 18x106 units interferon alfa-2a by i.m. injection daily for the first 10 weeks and then three times weekly for a further 4 months. The symptoms of toxicity observed in this study resembled those previously reported for alfa interferons and included fever, chills, fatigue, anorexia, myalgia, headache, occasional nausea and vomiting, dose-dependent reversible leukopenia, and hepatic transaminase elevations. Of the 21 patients, 12 had evidence of tumor progression, 6 had stable disease for at least 2 months, and complete remission was seen in 3 patients with stage III melanoma. We conclude that interferon alfa-2a appears to have some antiproliferative effect in metastatic malignant melanoma. While its use in stage IV patients with big tumor masses is doubtful, there seems to be therapeutic benefit in earlier stages.

Cytostatic drug effects on human clonogenic tumor cells and human bone marrow progenitor cells (CFU-C) in vitro

SummaryHuman tumor cells from a squamous cell carcinoma of the lung and from a malignant melanoma and human bone marrow progenitor cells were cultured in a methylcellulose monolayer system. To obtain tumor material for repeated experiments with human tumor specimens, human xenografts grown in the nude mouse system were used. The cultures were incubated continuously with adriamycin, actinomycin-D, bleomycin, cisplatinum, and melphalan. Dose response curves for bone marrow and tumor cell colonies were established. A comparison of bone marrow and tumor cell colony dose response curves might be useful for the assessment of the drug sensitivity of tumor cells independent of in vivo pharmacologic data and duration of drug exposure.

Tumor cell colonies in bone marrow cultures from patients with small cell carcinoma of the lung

SummaryBone marrow specimens from 27 patients with small cell carcinoma of the lung (17 with limited and 10 with extensive disease) were plated in a culture system that supports the growth of multilineage haemopoietic progenitors CFU-GEMM. In five patients (three with extensive and two limited disease) atypical colonies could be observed that were not identifiable as haemopoietic colonies. Cytological staining and cytochemical examination as well as electronic micrographs suggest that these colonies are derived from metastatic carcinoma cells. The histological examination of marrow cells from three out of these five patients revealed no bone marrow involvement. Additional studies might provide further evidence whether bone marrow cultures are a useful probe in order to monitor bone marrow involvement in patients with small cell carcinoma of the lung.

Comparison of bleomycin and peplomycin toxicity on clonogenic tumor cells from various human tumors.

SummaryThe cytotoxic effect of bleomycin and peplomycin was compared using a methylcellulose monolayer assay for the cultivation of human tumor cells. In 3 out of 4 samples from human malignant melanomas peplomycin proved to be more cytotoxic than bleomycin. Peplomycin was more cytotoxic than bleomycin in 1 of 5 myosarcoma samples, whereas 2 samples from squamous cell carcinomas of the lung showed identical dose response curves. In 1 carcinoma of the gall bladder peplomycin was more toxic than bleomycin.

Effects of the synthetic alkyl-lysophospholipid BM 41 440 on clonogeneity of human tumor cells from various histological types

Some human tumor cell lines are able to prevent chloroethylnitrosourea-ind~ced DNA-interStrand crosslinks by removing O -chloroethyl DNA monoadducts before cross,inks can form. The same cell lines can ~epair 0 -methyl-guanine adducts by guanine-O -~ethyl-transferase an~ were designated as Mer (methylation repair ). Methylating agents like MNNG or MNU are able to inactivate this repair system followed by an increase in chloroethylnitrosourea-induced DNA intersSrand crosslinks and in vitro cytotoxicity in Mer tumor cell lines. With regard to the potential benefit of such combinations for the treatment of human tumors knowledge of whole animal and organ toxicity is indispensible. We determined the combination toxicity index of MNU plus BCNU according to a method proposed by Skipper. The sums of the decimal fractions of the LDInS of both agents were opposed to the observed 9 ortality. The LDl0(sum) was calculated by probit analysis. Altogether 14 different combinations of both agents were investigated. The results revealed a combination toxicity index of 0.32, the lowest value reported so far for the combination of 2 cytostatic drugs. The main targets of toxicity were the intestinal ~ract and the bone marrow. The results suggest that a clinical application of such combinations should be performed with the respective precautions. Institut fGr Toxikologie und Chemotherapie, Deutsches Erebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg S 23

Cytotoxicity of recombinant leukocyte a interferon on human clonogenic tumor cells and human bone marrow progenitor cells in vitro

PHASE I I STUDY OF A COMBINATION THERAPY WITH HUIFN-ALPHA, VBL, TAMOXIFEN IN THE ADVANCED RENAL CELL CARCINOMA (RCC) H. Diedr ich, H.-J. Schmoll, P. vq Wuss~w, J~ Anagnou In t roduct ion: In the chemotherapy of the advanced RCC VBL and HulFN-alpha are the most act ive agents f o r which a response rate (PR,CR) o f about 20% is claimed. (R.A. Kempf, Proc Ann Meet Soc Cl in 0ncoI,3:59,1984) Several studies with tamoxifen repor t a remission rate of about 5% to 10% (V.J. Lanter i , Ur01.,9:623o625, 1982). An add i t i o nal e f f ec t o f VBL and HulFN-alpha in v i t r o was described (M.S. Aapro, Exp Chemotherapy, AACR Abstracts, 725,1982). Treatment plan: VBL 6mg/m2i.v. on day 1,8 and 15, HulFNalpha 5 Mio IU/m2s.c. three times a week f o r 4 weeks, tamoxifen 15mg/m2p.o.daily f o r 28 days. On day 29 the cycle was r e s t a r t e t . Re-evaluation a f t e r a minimum of 2 cycles. In cases of no change the therapy was con t i nued un t i l the tumor progressed. C r i t e r i as fo r pat ients ent ry : h i s t o l o g i c a l l y confirmed RCC, documented progressive disease. Exclusion c r i t e r i a : brain metastases, previous chemotherapy, p r i o r i r r a d i a t i o n . From 9/84 to 9/85 24 pts entered the study. 20 pts are e l i g i b l e , 16 males, 4 females, median age o f 54 years, median Karnofsky PS 85%. In 4/20 pts the primary tumor was unresected. Results: No CR, no PR were achieved, s table disease occurred in 12 pts (60%), 8/12 with metastases only o f the lung. 8/20 showed cont ineously progressive desease. 5/20 died (3 with progressive disease, 2 with no change a f t e r two cyc les) . The median surv iva l o f the group with stable disease was 25+ weeks. The median surv iva l o f the group with progressive disease was 17+ weeks. Tox ic i t y was acceptable: 6 pts showed leukopenia 2000. 1 pt achieved a severe per ipheral neu ro tox i c i t y , grade I I I WHO and VBL had to be stopped in the second cycle. Despite some an t i tumor a c t i v i t y has been observed i t can be concluded that the combination of VBL/HulFN-alpha/tamoxifen in these doses and th is regime is i ne f f ec t i ve in pts with metastat i c RCC and documented progression before treatment. Abt. Haematologie-Onkologie der Medizinischen Hochschule Hannover, Konstanty-Gutschow-Str. 8, D-3000 Hannover 61

Cytotoxicity of bleomycin and peplomycin on human tumor cells in vitro

CYTOTOX!CITY OF BLEOMYCIN AND PEPLOMYCIN ON HUNANTUMOR CELLS IN VITRO. H.A. Neumann, D.J.B. Herrmaun, M.H. Runge, H.H. Fiebig~ R. Engelhardt Human clonogenic tumor cells from various human tumors were cultured in a methylcellulose monolayer assay. Single cells were seeded in Iscoves modified Dulbeccos medium, fetal calf serum and methylcellulose. Bleomycin and Peplo~ mycin were added at concentrations between 1~and ~0 ug/ml. Incubation was performed at 37 C in 7,5 % C0~. After 8-10 days of incubation, colonies could be scored under an inverted microscope. Each tumor showed individual dose response curves. In two out of four malignant melanoma probes, Peplomycin showed a considerable stronger cytotoxicity than Bleomycin; in one case the difference was less pronounced and in one case the dose response curves were identic. In two out of four samples from myosarcomas, Peplomycin proved to be more cytotoxic than Bleomycin, in two probes the dose response curves showed identic effects. In two samples from colon carcinomas Bleomycin showed at reduction of colony formation ~0 ug/ml to 40-5~% and 60-65% of control, whereas the incubation with Peplomycin at the same concentration resulted in a reduction of 5 % and 20 % of controls. The dose response curves of two probes from squamous cell carcinomas of the lung were almost identic. Insofar our experiments coroborate the findings with the HeLa cells and Ehrlich cells that Peplomycin is more cytotoxic than Bleomycin. However, individual drug sensitivity of human tumors must be considered. Medizinische Klinik und Frauenklinik der Albert-Ludwigs-Universit~t Freiburg, Hugstetterstr. 55, 7800 Freiburg. VBR 28