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[CANCER RESEARCH 40, 4109-4112, November 1980] 0008-5472/80/0040-0000$02.00 Inhibition of Human Ovarian Cancer Colony Formation by Adriamycin and Its Major Metabolites Robert F. Ozols,1 James K. V. Willson, Martin D. Weltz, Karen R. Grotzinger, Charles E. Myers, and Robert C. Young Medicine Branch ¡R.F O., J. K. V. W.. K. R. G.. R. C. Y.] and Clinical Pharmacology Institute, Bethesda. Maryland 20205 ABSTRACT We have examined the in vitro sensitivity to Adriamycin of human ovarian cancer colonies cloned in soft agar. In the 26 patients tested, 3 different patterns of sensitivity to Adriamycin were observed: (a) in 75% of the previously untreated patients, there was greater than 70% reduction in colony-forming cells after exposure to Adriamycin (1.0 jug/ml), a level which ap proximates the peak plasma level after i.v. therapy; (fa) in all the patients who had progressive disease while on a chemo therapy regimen without Adriamycin, a greater than 70% re duction in colony-forming cells was observed only at a concen tration of 10 ^g/ml, a level not achievable by i.v. administration; (c) in 80% of patients with progressive disease after treatment with Adriamycin as part of the primary chemotherapy regimen, a 70% reduction in tumor colony-forming cells could not be achieved even at 10 jug/ml. These in vitro results are in agree ment with clinical observations regarding the effectiveness of Adriamycin in previously untreated patients (42% response rate) with ovarian cancer as well as its ineffectiveness (0 to 6% response rate) as a second-line therapy in relapsed patients. The results also have provided a rationale for an ongoing Phase I trial of i.p. Adriamycin in patients with ovarian cancer from Group b above since cytotoxic levels can be produced i.p. using large-volume dialysis via a Tenckhoff dialysis catheter. The relative cytotoxicity of Adriamycin to its two major me tabolites, adriamycinol and adriamycin aglycone, was also determined in the clonogenic assay. Both derivatives produced suppression of ovarian cancer colony formation; however, Ad riamycin was more cytotoxic than was either metabolite. INTRODUCTION Adriamycin is an active agent in the treatment of patients with ovarian cancer. The response rate of 42% in previously untreated patients (4) has led to its use in combination with other active agents in patients with advanced ovarian cancer (5, 19). However, as a second-line agent, i.e., in patients who have failed previous chemotherapy, Adriamycin does not have significant activity (9, 17). Although the activity of Adriamycin against many tumors has been established, the metabolism of Adriamycin and the mech anisms for its cytotoxicity remain areas of active investigation (11). In particular, the nature of the metabolites of Adriamycin and their relative cytotoxicity compared to the parent com pound is unclear (1, 2). This has been due in part to difficulties in the extraction of Adriamycin and its metabolites from plasma 1To whom requests for reprints should be addressed, and tissue. It is possible that techniques which are harsh enough to quantitatively extract Adriamycin from tissues may also result in the artifactual production of aglycones via chem ical cleavage of the glycosidic bond. It does appear that there are at least 2 major metabolites which can be detected in the plasma using nondestructive extraction techniques. However, the cytotoxicity of these 2 derivatives, AA2 and AOL, compared to Adriamycin in human tumors, has not been established. Hamburger and Salmon (6-8) have recently described a tissue culture system which allows for selective growth of human ovarian cancer colonies in soft agar. We have used this technique to investigate the sensitivity to Adriamycin of human ovarian cancer cells obtained from a variety of patients, as well as to determine the relative tumor cytotoxicity of Adriamycin and its 2 major metabolites. The results have provided, in part, a rationale for a novel therapeutic approach to selected patients with ovarian cancer consisting of the i.p. administration of Adriamycin in large volumes using the Tenckhoff dialysis cath eter, a device designed for chronic outpatient peritoneal di alysis. MATERIALS AND METHODS Drugs. Adriamycin (Adria Laboratories, Inc., Wilmington, Del.) was obtained from the Investigational Drug Branch, Divi sion of Cancer Treatment, National Cancer Institute. AA was synthesized by mild acid hydrolysis of Adriamycin (2 N acetic acid at 100°for 30 min). AOL was donated by Dr. John Driscoll (NIH, Bethesda, Md.) and Dr. Mervyn Israel (Sidney Farber Cancer Center, Boston, Mass.). The purity of the 3 compounds was established by Chromatographie methods including highpressure liquid chromatography in which Adriamycin and its metabolites can be identified on the basis of characteristic retention times (10). Specimens. Malignant effusions were obtained from patients with epithelial ovarian cancer. Peritoneal washings, as de scribed previously (14), were also used as a source of clono genic ovarian cancer cells. All specimens were collected aseptically with preservative-free heparin, 10 units/ml. Soft Agar Culture System. After centrifugation at 150 x g for 10 min, the cells were washed twice with Hanks' balanced salt solution containing 10% heat-inactivated fetal calf serum (Grand Island Biological Co., Grand Island, N. Y.). Trypan blue dye exclusion was used as a measure of cell viability. Prior to culture, smears were made of the single cell suspension and stained by Wright-Giemsa or Papanicolaou methods (American Histolabs, Silver Spring, Md.). The ovarian cancer cell suspensions were incubated with at Medicine Branch, Bldg. 10, Room 12N226, National Cancer Institute, Bethesda, Md. 20205. Received March 5. 1980; accepted August 1, 1980. NOVEMBER Branch ¡M.D. W.. C. E. M.I, Division of Cancer Treatment. National Cancer • The abbreviations used are: AA, adriamycin aglycone: AOL, adriamycinol. 1980 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1980 American Association for Cancer Research. 4109 R. F. Ozols et al. Adriamycin, AA, and AOL for 1 hr at 37° with each of the compounds tested at final concentrations of 10.0, 1.0, and 0.1 /¿g/ml. The concentrations were chosen to include the peak plasma level, 0.5 to 1.0 /¿g/mlobserved after an i.v. bolus of 60 mg/sq m body surface area (2), as well as a higher con centration, 10 /¿g/ml, which could be achieved by i.p. drug administration (15). This latter concentration was selected to determine whether the clinically apparent resistance to Adria mycin in previously treated patients (9, 18) was dose depend ent and could be overcome by increasing the concentration of Adriamycin. After incubation, the cells were washed free of drug and resuspended in enriched CMRL 1066 (Grand Island Biological Co.) media containing 15% heat-inactivated horse serum (Grand Island Biological Co.), other nutrients, and 0.3% agar (6-8). The cells (final concentration of either 0.5 or 1.0 x 106 cells in a 1.0 ml volume) were plated in triplicate in 35-mm Petri dishes, onto a feeder layer of 1.0 ml enriched McCoy's media (Grand Island Biological Co.) with 0.5% agar. The plates were incubated at 37°in a 7.0% CO2 humidified atmosphere. The number of ovarian cancer colonies in each plate was determined after 14 or 21 days of growth using an inverted microscope. The criteria for colony size were the same as established by Hamburger ef al. (8). Only colonies containing greater than 30 cells were counted. The number of colonies in the drug-treated plates was compared to control plates. Results were graphed as mean percentage of survival (± S.E.) of ovarian colonies versus concentration of drug. In Vitro Sensitivity to Adriamycin. Adriamycin was tested in vitro in 3 groups of ovarian cancer patients: those who had not received any chemotherapy (6 patients) or who were respond ing to a non-Adriamycin-containing regimen (2 patients); those who had progression of disease after treatment with a nonAdriamycin-containing combination (9 patients); and those who had progression of disease after being treated with an Adriamycin-containing combination (9 patients). greatest sensitivity to Adriamycin was observed in the group of patients whose cells were tested prior to any chemotherapy or at a time when they were still responding to a non-Adriamycincontaining combination (Chart 1). Seven of 9 patients in this group had less than 30% survival of colony-forming cells at a dose of 1 jug/ml. Two of the patients who were sensitive in vitro were treated with an Adriamycin-containing combination, and both patients had an objective response to therapy. The one patient who had no reduction in tumor colony-forming cells at either 1 or 10 /ig/ml was also resistant in vitro to melphalan and 5-fluorouracil. She was treated with a non-Adriamycincontaining combination and had rapid progression of disease. In the group of patients who had relapsed while undergoing therapy with non-Adriamycin-containing regimens (Chart 2), there was a considerable variation in the observed dose-re sponse curves. However, in 8 of 9 patients, more than 30% of the colony-forming cells survived a concentration of 1 jug/ml. All 8 of these patients, with demonstrable in vitro resistance to Adriamycin at doses comparable to peak plasma levels ob served after i.v. administration, were sensitive (greater than 70% reduction in colony-forming cells) at 10 /ig/ml. At this concentration of Adriamycin, which cannot be attained by conventional therapy but can be achieved using i.p. drug 0.2 RESULTS Characterization of Colonies. Under the conditions of growth, ovarian cancer colonies had a morphology similar to that described by Hamburger ef al. (8). In addition, colonies plucked from the plates or fixed in agar as described by Salmon and Buick (16), and subsequently stained with the Papanicolaou method, had cytological characteristics of human ovarian cancer cells. Previously, Hamburger ef al. (8) had used cytogenetic and cytochemical analyses to help establish the human ovarian cancer cell origin of the colonies grown in soft agar by these techniques. Effects of Adriamycin on Human Ovarian Colony Forma tion. There were marked differences in the observed doseresponse curves to Adriamycin in the 26 patients studied. However, there appear to be 3 patterns of sensitivity which correlate with the clinical status of the patients. In this assay, in vitro sensitivity has operationally been defined as at least a 70% reduction in tumor-colony-forming cells after exposure to a drug at clinically achievable plasma levels. Using these criteria, as well as area under the curve calculations, Salmon ef a/. (1 7) and Von Hoff (20) have demonstrated that this assay has a 90 to 95% accuracy in predicting clinical resistance and a 60 to 70% accuracy in predicting for a clinical response. The 4110 0.4 0.6 0.8 1.0 10.0 ADRIAMYCIN (Mg/mll Chart 1. Ovarian cancer cells from patients previously untreated or while they were responding to a non-Adriamycin-containing chemotherapy regimen were exposed for 1 hr to different concentrations of Adriamycin. The results are expressed for each patient as percentage of colonies in untreated controls. Bars. S.E. The mean number of colonies in untreated controls was 82 colonies/plate. 100 O (fi on 5_j BO D üj 1-0 a« |2 60 11 ujO 0-1 irO tuo "• 20 0.1 0.2 0.4 0.6 ADRIAMYCIN 0.8 1.0 10.0 (Mg/ml) Chart 2. Details are the same as for Chart 1, except that cells were obtained from ovarian cancer patients who had progressive disease while on a nonAdriamycin-containing chemotherapy regimen. The mean number of colonies in untreated controls was 113 colonies/plate. CANCER RESEARCH VOL. 40 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1980 American Association for Cancer Research. Inhibition of Human Ovarian Cancer Colony administration, the mean percentage of survival of colony-form ing cells was 10%. Three of these patients were treated in a Phase I trial of i.p. Adriamycin (15) administered in large volume (2 liters) via a Tenckhoff dialysis catheter. At a concentration of 10 /¿g/mlAdriamycin dialysate (20 mg Adriamycin in 2 liters Inpersol) (Abbott Laboratories, North Chicago, III.), 2 patients had a reduction in ascites formation. The 9 patients whose cells were cultured after progression of disease while on therapy with i.v. Adriamycin demonstrated the greatest overall degree of resistance to Adriamycin in vitro (Chart 3). Five of the patients in this group had i.v. Adriamycin as part of their initial chemotherapy, and 4 of the patients were treated with i.v. Adriamycin after having failed a non-Adriamycin-containing primary region. Those patients who had failed initial therapy with i.v. Adriamycin had a greater degree of resistance in vitro than did those patients who had been treated with Adriamycin as a second-line agent. This latter subset of patients (none of whom had a response in vivo to i.v. Adria mycin) has an in vitro pattern of sensitivity similar to those patients who had progressive disease on a non-Adriamycincontaining regimen and who were not treated with i.v. Adria mycin as a second-line agent, Chart 2; i.e., there was resist ance at clinically achievable plasma concentrations, but sen sitivity was observed at levels achievable i.p. In contrast, 4 of 5 patients who had failed i.v. Adriamycin as part of their induction therapy demonstrated a markedly greater degree of resistance in vitro. Even at a concentration of 10 tig/ml, the mean percentage of survival of tumor-colony-forming cells was 87%. Thus, some of the patients in this clinical subset may not be expected to benefit from i.p. Adriamycin since the resistance to Adriamycin in 80% of these patients was not dose dependent over the concentration range studied, 0.1 to 10 /ig/ml. Relative Cytotoxicity of Adriamycin, AA, and AOL. The effect of Adriamycin, AA, and AOL on the survival of human ovarian cancer colony-forming cells were examined only in 3 patients (Table 1), due to limited quantities of AA and AOL. In Patient A, Adriamycin produced more suppression of colony formation than did either of the metabolites at all 3 concentra140 - IV ADRIAMYCIN - IV ADRIAMYCIN AS INITIAL CHEMOTHERAPY AS A SECOND LINE AGENT 120 0.1 0.2 0.6 0.8 1.0 10.0 ADRIAMYCIN (Mg/ml) Chart 3. Details are the same as for Chart 1, except that cells were from ovarian cancer patients with progressive disease while on treatment with an Adriamycin-containing chemotherapy regimen. The mean number of colonies in untreated controls was 143 colonies/plate. Table 1 Cytotoxicity of Adriamycin and metabolites Single-cell suspensions of human ovarian cancer cells were exposed Adriamycin, AA. and AOL at 3 different concentrations. The percentage survival of ovarian cancer colonies was calculated at each concentration. to of % of Survival ovarian cancer colonies at following drug concentrations 0.1 jig/ml 1.0 fig/ml 10.0(jg/ml AAdriamycinAAAOLPatient Patient ±69 ±32 ±71 ±27 ±31 ±19 ±27 ±47 ±4 BAdriamycinAAAOLPatient ±98 ±42 ±46 ±79 ±31 ±48 ±54 ±19 ±19 CAdriamycinAAAOL24 ±1361103752" Mean ±S.E. ±48 ±26 ±62 ±67 ±27 ±3a63664711117 ±1351163535 tions studied. AOL was more cytotoxic than AA at 0.1 /ig/ml, both compounds were equitoxic at 1.0 jug/ml, whereas at 10 /¿g/ml,AA produced a greater suppression of colony formation than AOL. In Patient B, Adriamycin was more cytotoxic than were AA and AOL at 1.0 and 10.0 /¿g/mlwith AOL producing greater Cytotoxicity than did AA at all 3 concentrations. In Patient C, although Adriamycin was again the most active compound, AA produced more suppression than did AOL at the lower concentrations with both metabolites having the same effect at 10 jiig/ml (26 and 27% colony survival). Although considerable variation in activity of the 3 compounds was evident in the patients studied, the results suggest that Adria mycin is more active than either AA or AOL. DISCUSSION The studies reported here, utilizing a method for the cloning of human ovarian cancer, provide experimental support for the clinical observations regarding the efficacy of Adriamycin in the treatment of patients with ovarian cancer. The results have also provided, in part, a rationale for a Phase I trial of i.p. Adriamycin in certain patients with ovarian cancer. Further more, the potential of the clonogenic assay to serve as a model system in which to study pharmacological and pharmacokinetic questions in human solid tumor therapy has been demon strated. Three distinct patterns of Adriamycin sensitivity in vitro were observed which correlate with clinical observations regarding the efficacy of Adriamycin in patients with ovarian cancer. Adriamycin is an active agent (response rate, 40%) in previ ously untreated patients with ovarian cancer (4), and in this group of patients Adriamyciri demonstrated the greatest in vitro sensitivity (Chart 1). Those patients who had failed primary therapy with Adriamycin had a markedly different pattern of sensitivity than that observed in the untreated patients. In 80% of the patients Adriamycin did not produce a 70% reduction in colony-forming cells even at a concentration of 10 jug/ml (Chart 3). In contrast to the "sensitive" dose-response curves ob served in previously untreated patients and "resistant" doseresponse curves in patients who had failed therapy with Adria mycin, was the pattern of Adriamycin sensitivity observed in patients who had progressed on non-Adriamycin-containing NOVEMBER 1980 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1980 American Association for Cancer Research. 4111 R. F. Ozols et al. chemotherapy regimens (Chart 2). The relative resistance ob served at concentrations of 0.1 and 1.0 jug/ml could be over come by increasing the concentration of Adriamycin to 10 jug/ ml, which resulted in a greater than 70% reduction in colonyforming cells in all the patients tested. This concentration is not achievable after conventional intravenous therapy with Adriamycin, but can be attained using i.p. administration (15). This relative resistance correlates with the clinical observation that i.v. Adriamycin is of very limited benefit in patients who have failed on non-Adriamycin chemotherapy regimens (9,18). Two patients who demonstrated in vitro sensitivity to Adria mycin at 0.1 /¿g/ml(Chart 1), were treated with Adriamycin plus cyclophosphamide and had an objective response to therapy. Salmon ef al. (18) and Von Hoff (20) have previously demonstrated the potential of this assay to aid in the clinical selection of active antitumor agents. The results presented here, the comparison between in vitro sensitivity to Adriamycin, and the previously reported activity spectrum of this drug in patients with ovarian cancer provide further evidence that the stem cell assay may be useful in the individualization of chemotherapy. The observation that the resistance to Adriamycin was dose dependent in patients who had progressive disease while on a non-Adriamycin-containing regimen has added to the rationale for the i.p. use of Adriamycin in certain patients with ovarian cancer. Dedrick ef al. (3) have demonstrated previously with pharmacokinetic modeling, that the i.p. administration of cer tain antitumor agents could, on the basis of a differential between the peritoneal and plasma clearances, result in an increase in the intraabdominal drug levels and thus be of potential benefit in the treatment of i.p. tumors. However, if an increase in drug levels is not correlated with an increase in cytotoxicity, then this form of therapy would not likely be of clinical benefit. The demonstration that, for certain patients with ovarian cancer, Adriamycin levels which produce in vitro cytotoxicity cannot be achieved by i.v. administration but are attainable by i.p. therapy with a Tenckhoff catheter provides primary experimental support for this modality of treatment. Further support for this form of therapy has been provided by our previous studies with a murine ovarian tumor model (12, 13). In this model, which has a metastatic pattern similar to patients with ovarian cancer, i.p. Adriamycin was curative in 70% of mice inoculated with 106 tumor cells i.p. 2 days prior to chemotherapy, whereas an equitoxic i.v. dose was without benefit. Preliminary results from a Phase I trial of i.p. Adria mycin in patients with ovarian cancer suggest that up to 60 mg of Adriamycin, in 2 liters of dialysate, can be administered via a Tenckhoff dialysis catheter with apparently acceptable local toxicity (15). The effect of Adriamycin and its major metabolites on human ovarian cancer colony formation demonstrates that Adriamycin is more cytotoxic than is either AA or AOL (Table 1). These results are consistent with the known effects of Adriamycin and AOL on the inhibition of thymidine incorporation in cultured L1210 cells where it has been demonstrated that at 5 fiM Adriamycin produced a 52% inhibition of thymidine incorpo ration into nucleic acids and AOL produced a 32% inhibition (1). In addition, in P388 leukemia, AOL has significant activity (increasing survival by more than 150% of untreated controls), but it is less active than Adriamycin.3 Although the complete 3 M. K. Wolpert, personal communication, 4112 1980. data have not yet been published, the aglycone metabolites have been reported to have little biochemical activity in cell culture systems (1). In summary, the sensitivity patterns of human ovarian cancer colonies to Adriamycin correlate with the clinical observations regarding the efficacy of Adriamycin in patients with ovarian cancer. The results further support the validity of this system to predict the clinical efficacy of chemotherapy and also have provided a rationale for the i.p. use of Adriamycin in certain patients with ovarian cancer. ACKNOWLEDGMENTS We wish to thank Dr. John Driscoll and Dr. Mervyn Israel for providing us with adriamycinol and Dr. Mary Wolpert for the information regarding the cytotoxicity of Adriamycin metabolites. REFERENCES 1. Bachur, N. R. Adriamycin (NSC-123127) pharmacology. Cancer Chemother. Rep., 6 (Part 3). 153-158, 1975. 2. Benjamin, R. S., Riggs, C. E., Jr., and Bachur, N. R. Plasma pharmacokinetics of Adriamycin and its metabolites in humans with normal hepatic and renal function. Cancer Res., 37: 1416-1420, 1977. 3. Dedrick, R. L., Myers, C. E., Bungay, P. M., and DeVita, V. T., Jr. Pharma cokinetic rationale for peritoneal drug administration in the treatment of ovarian cancer. Cancer Treat. Rep., 62: 1-12, 1978. 4. DePaulo, G. M., DeLena, M.. DiRe, F., Luciani, L., Valagussa, P., and Bonadonna, G. Melphalan versus Adriamycin in advanced ovarian carci noma. Surg. Gynecol. Obstet., 141: 899-902, 1975. 5. Ehrlich, C. E., Einhorn, L., Williams, S. D., and Morgan, J. Chemotherapy for Stage III-IV epithelial ovarian cancer with cis-dichlorodiammine platinum (II), Adriamycin and cyclophosphamide: a preliminary report. Cancer Treat. Rep., 63. 281-288, 1979. 6. Hamburger, A. W., and Salmon. S. E. Primary bioassay of human tumor stem cells. Science (Wash. D. C.), 197: 461-463, 1977. 7. Hamburger, A. W., and Salmon, S. E. Primary bioassay of human myeloma stem cells. J. Clin. Invest., 60: 846-854, 1977. 8. Hamburger, A. W., Salmon, S. E., Kim, M. B., Trent, J. M., Soehnlen, B. J., Alberts, D. S., and Smith, H. J. Direct cloning in human ovarian carcinoma cells in agar. Cancer Res., 38: 3438-3444, 1978. 9. Hubbard, S. M., Barkes, P., and Young, R. C. Adriamycin therapy for advanced ovarian carcinoma recurrent after chemotherapy. Cancer Treat. Rep., 62: 1375-1377, 1978. 10. Israel, M., Pegg, W. J., Wilkinson, P. M., and Garnick, M. B. Liquid Chro matographie analysis of Adriamycin and metabolites in biological fluids. J. Liquid Chromatog., J. 795-809, 1978. 11. Myers, C. E. Antitumor antibiotics I: anthracyclines. In: H. M. Pinedo (ed.) Cancer Chemotherapy 1979, pp. 56-74. Amsterdam: Excerpta Medica, 1979. 12. Ozols, R. F., Grotzinger, K. R.. Fisher, R. I., Myers, C. E., and Young, R. C. Kinetic characterization and response to chemotherapy in a transplantable murine cancer. Cancer Res., 39: 3202-3208, 1979. 13. Ozols, R. F., Locker. G. Y., Doroshow, J. H., Grotzinger, K. R., Myers, C. E., and Young, R. C. Pharmacokinetics of Adriamycin and tissue penetration in murine ovarian cancer. Cancer Res., 39: 3209-3214, 1979. 14. Ozols, R. F., Willson, J. K. V., Grotzinger, K. R. and Young, R. C. Cloning of human ovarian cancer cells in soft agar from malignant effusions and peritoneal washings. Cancer Res., 40: 2743-2747, 1980. 15. Ozols, R. F., Young, R. C., Speyer, J. L., Weltz. M., Collins, J. M., Dedrick, R. L., and Myers, C. E. Intraperitoneal (IP) Adriamycin (Adr) in ovarian carcinoma (OC). Proc. Am. Assoc. Cancer Res., 21: 425, 1980. 16. Salmon. S. E., and Buick, R. N. Preparation of permanent slides of intact soft-agar colony cultures of hematopoietic and tumor stem cells. Cancer Res., 39: 1133-1136, 1979. 17. Salmon, S. E., Hamburger, A. W., Soehnlen, B., Durie, B. G. M., Alberts, D. S., and Moon, T. E. Quantitation of differential sensitivity of human tumor stem cells to anticancer drugs. N. Engl. J. Med., 298: 1321-1327, 1978. 18. Stanhope, R. C., Smith, J. P., and Rutledge, F. Second trial drugs in ovarian cancer. Gynecol. Oncol., 5: 52-58, 1977. 19. Vogl, S. W., Berenzweig, M., Kaplan, B. H., Moukhtan, M., and Bulkin, W. The CHAD and HAD regimens in advanced ovarian cancer: combination chemotherapy including cyclophosphamide, hexamethylmelamine, Adria mycin, and cis-dichlorodiammineplatinum (II). Cancer Treat. Rep., 63: 311317, 1979. 20. Von Hoff, D. D. Clinical correlations of drug sensitivity in tumor stem ce!! assay. Proc, Arn. Assoc. Cancer Res., 21: 134, 1980. CANCER RESEARCH VOL. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1980 American Association for Cancer Research. 40 Inhibition of Human Ovarian Cancer Colony Formation by Adriamycin and Its Major Metabolites Robert F. Ozols, James K. V. Willson, Martin D. Weltz, et al. Cancer Res 1980;40:4109-4112. 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