Download Inhibition of Human Ovarian Cancer Colony

[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.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/40/11/4109
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1980 American Association for Cancer Research.