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Tumor heterogeneity and in vitro chemosensitivity testing in ovarian cancer
Bernd-Uwe Sevin, MD, PhD, and James P. Perras, PhD
Miami, Florida
OBJECTIVE: Our purpose was to study the heterogeneity of drug response in fresh human ovarian tumors to chemotherapeutic agents in an in vitro chemosensitivity assay. STUDY DESIGN: This assay evaluates total tumor cell kill by measuring the intracellular adenosine triphosphate levels of untreated controls and drug-exposed cells at various doses after culture for 6 days. The surviving fraction is calculated by dividing the treated values with the control values. One hundred tumors were tested against four single drugs (cisplatin, the active metabolite of cytoxan, 4-hydroxy- peroxy-cyclophosphamide, Taxol, and carboplatin) and two drug combinations (cisplatin plus 4-hydroxy- peroxy-cyclophosphamide; cisplatin plus Taxol). RESULTS: There is great variation in the degree of cell death for single drugs and drug combinations among the 1 O0 tumors tested. CONCLUSION: More effective clinical response to chemotherapy may be achieved in patients with ovarian cancer by selecting the most active drugs for chemotherapy, on the basis of in vitro chemosensitivity test results for individual patients. (Am J Obstet Gynecol 1997;176:759-68.)
Key words: Human ovarian cancer, chemosensitivity assay, heterogeneity in drug response
Patients with ovarian cancer are primarily treated with surgel)7 and chemotherapy.~ Surgery is the first step of
therapy, during which staging and tumor resection are roudnely perfbrmed. All patients, with the exception of
those with early-stage disease, receive postoperative che-
motherapy that combines potent cytotoxic drugs such as
cisplatin or carboplatin with cyclophosphamide. Re-
cently, the combination of cisplatin and Taxol has been
shown to be superior to the standard combination of
cisplatin and cyclophosphamide in advanced ovarian carcinoma. 2 Clinical response rates to these regimens
center around 70% of cases. Patients without evidence of
disease at the end of six to eight courses of therapy may be offered surgical reexploration (so-called "second-
look" procedure) to assess the presence or absence of disease. Patients with no or only microscopic disease at second-lo0k laparotomy have a 5=year survival expectancy
of >50%, :~ whereas those with macroscopic disease and those who do not have a response to first-line chemo-
therapy generally have a life expectancy of <2 years.
Persistent and recurrent ovarian carcinoma is treated
either by second-line chemotherapy or by second surgi- cal tumor resection followed by chemotherapy; however, response rates of only 20% to 40% are observed, e
t,)om /he Divisio~ oJ C3,necologic Ontology, Univ~:s'ily of MiamL Presenled al the lqfleenlh Annual Meeting of the American ())'nea)log- it al and Obsletfical Sociely, Asheville, North Carolina, September 5 7, 1996. R@rinl requesls: Bernd-Uwe Sevin, MJ), Phi), Moyo Clinic Jackson- ville, 4500 San Pablo Road, Jacksonville, lff~ 32224. Copyright © 1997 by Mos@-Year Book, h~e. 0002-9378/97 $5.00 + 0 6/6/80058
It is currently accepted that not all neoplasms behave in the same manner. Ovarian epithelial neoplasms, in
particular, occur in a great variety of histologic cell types
and grades of differentiation. 1' :~ Histologic evaluation does not provide information in regard to chemotherapy
response. In general, adenocarcinomas respond differ-
ently compared with squamous cell carcinomas, germ
cell tumors, and sarcomas, but histologie type does not
offer any further information. Hormone receptor analy-
sis serves as a relative predictor fbr response to progestin
therapy, but no reliable method predicts response to
chemotherapy. Recent studies such as ploidy, prolifera- tive cell fraction, and oneogenes so far have shown no
correlation with chemotherapy response. :~ The clinician empirically selects chemotherapy for an individual pa-
tient on the basis of results of clinical trials in which large numbers of patients have been treated with a predefined
treatment regimen. These clinical trials, however, ignore differences among individual patients by averaging treat-
ment responses of many patients. In contrast, every
practicing oncologist has experienced significant varia-
tions in drug response among individual patients who, by all available clinical parameters, appear to have compa-
rable disease. Tumor heterogeneity among tumors of identical morphologic type has been ignored in our daily clinical practice. One of the reasons why chemosensitivity testing has not been incorporated into standard onco- logic therapy has been the lack of a good drug sensitivity assay. In vitro chemosensitivity testing of solid tumors has been diificult because of the low growth rate of tumors in vitro, the low viability of tumor cells after disaggregation
759
760 Sevin and Perras April 1997 Am J Obstct Gynecol
required for plating cells in culture, and other reasons. 4
The human tumor stem cell assay, pioneered in 1977 by
Hamburger and Salmon ~ and reported on by others, ~-s
initially created a high level of enthusiasm. However, the
assay failed to perform clinically, because of low evalu-
ability rates of <50% and because it mainly predicted
drug resistance. The initial great expectations were un-
fullfilled, resulting in a philosophic rejection of the
entire concept of in vitro testing to predict chemother- apy response. Recently, a revival of in vitro drug testing
has occurred.~q. 2 The adenosine triphosphate (ATP)-cell viability assay,
which was developed as a clinically useful chemosensitiv-
ity test, is designed to evaluate both sensitivity and resistance to chemotherapy in solid tumors.~l' 12 Data on
clinical response to chemotherapy in 65 patients with
ovarian cancer (41 primary and 24 recurrent), whose
tumor tissues were evaluated with the ATP-cell viability
assay, demonstrated a sensitivity of 90% and a specificity
of 80%. is The rate of true-sensitive results was 94% and
the rate of true-resistant results was 71%. Preliminary
results in patients with metastatic breast cancer showed
almost identical results, with a sensitivity of 90% and a specificity of 86%. 14 Within the limitations of nonran-
domized studies these are encouraging results, indicat-
ing that the ATP-cell viability assay is a promising method to predict in vivo patient response to chemother-
apy. Individualized therapy, which is based on maximal
knowledge of tumor biologic characteristics and disease
extent, seems to offer the best hope for long-term
survival. In ovarian cancer surgery always precedes che- motherapy and thus provides tissue for the study of
tumor-specific prognostic characteristics that may aid in
the treatment selection for an individual patient. This publication will not address the issue of predictive value
of in vitro chemosensitivity testing but will present some of our data on variability in drug-response patterns of primary ovarian carcinoma to different single drugs, as
well as to drug combinations.
Material and methods
In the gynecologic oncology research laboratory at the
University of Miami we developed a new in vitro chemo- sensitivity assay that uses decrease of total tumor cell viability as a measure of in vitro drug response. 1 i, 12 This
assay incorporates the agar underlayer technique of the clonogenic assay to retard growth of nonneoplastic tissue components 15 and analyzes total cell viability by measur-
ing intracellular ATP in treated cells compared with untreated cells. Cell death results in a rapid loss of cellular ATP because of the ubiquitous presence of ATPases. ATP is measured with a luminometer after in
situ extraction of the ATP from each well with 4% trichloroacetic acid. An aliquot of the trichloroacetic
acid extraction is neutralized with Tris buffer, and a 50 txl aliquot of the neutralized sample is placed in a polysty-
rene tube. The tube with the sample is placed in the
luminometer. The luminometer injects 50 Ixl of lucif-
erase-luciferin reagent into the tube. The light generated is proportional to the amount of ATP present in the
sample. The assay uses the differential in ATP values between
drug-treated wells and untreated controls on day 6 of in
vitro culture as the measure of drug response. The
effectiveness of agar underlayer or agarose coating to suppress cell growth of nonmalignant cell elements has been demonstrated in our test system. 15 In addition,
untreated control samples of each assay are evaluated by
a cytologist who determines the fraction of nonmalig-
nant cells on day 6, which is usually <5%. 11 In vitro drug
concentrations are standardized in reference to pub-
lished peak plasma concentrations for each drug, except
for cyclophosphamide. The active metabolite of cytoxan,
4-hydroxy-peroxy-cyclophosphamide (4-HC), is used with
an estimated peak plasma concentration equivalent of 20% of the in vivo cytoxan peak plasma concentration.
Quality control is provided for each assay in the
following two ways: (1) measuring the variability of individual ATP values, which is done in sextuplicate on
day 6 for untreated controls and in triplicate for each
drug-treated sample; (2) testing each drug or drug
combination at four to five drug concentrations, ranging
from 0.1 to 5.0 × Peak plasma concentration, which
provides another indirect quality control through the
generation of a dose-response curve. For this analysis we chose 100 consecutive assays from
our database of approximately 800 tumor assays. Results were entered into a Microsoft Excel spreadsheet on a
Macintosh computer, and the data were analyzed with Data Desk and Systat (Systat, Evanston, Ill.) statistical
programs.
Results
Untreated controls. We analyzed the variability in
measurements of ATP from the individual wells for the
control triplicates of day 0 and day 3 and the sextupli- cates of day 6. The means -+ SDs of the coefficients of variation for these 100 untreated controls of primary ovarian tumors were 0.138 -+ 0.074 for day 0, 0.132 _+ 0.083 for day 3, and 0.127 _+ 0.067 for day 6. The mean relative luminescence data and error bars for individual values are illustrated in Fig. 1. These results from the untreated tumor cells demonstrate that heterogeneity of drug response is not the result of variability of control in vitro ATP analyses.
Response patterns after single-drug exposure. The coefficients of variation for treated specimens varied
more than those of untreated controls and generally increased with increasing degree of cytotoxicity, because
Volume t76, Number 4 Sev in and Perras 761 Am.] Obstet Gynccol
o c- 6) o ¢0 r-
E
..I
._>
N
35000
25000
15000
5000 i I I
Day 0 Day 3 Dav 6
Mean
Fig. 1. Mean luminescence values of controls for 100 tumor assays. Error bars represent SEM.
1.2
1.0
g 0.8
LL 0.6
"'~ 0.4 g
0.2
0.0 • • • . • • = , l
.01 ,1
• n o - . - DDP
8 4-HC
Taxol
-- Carbo
1 10
Dose
Fig. 2. Mean survival fraction tk)r each dose of each single chemotherapeutic drug tested. £'r'~r bars represent SD. DDP, Cisplatin; Ca~'bo, carboplatin.
of differenlL degrees of cell death. All cases taken together
show a direct correlat ion between drug concent ra t ion
and cytotoxicity. Fig. 2 shows the average dose responses
of the 100 tumors for the drugs cisplatin, carboplatin,
Taxol, and 4-HC used as single agents. Except for Taxol,
almost l inear dose-response curves were observed. Cyclo-
phosphamide appears to be the most active drug,
whereas Taxol showed a bimodal dose response with sur-
xdval fractions at 0.1 × Peak plasma concentration being
<0.2 and <0.5 × Peak plasma concentra t ion values (Fig.
2). Individual drug-response patterns, however, varied
widely. Some tumors showed almost comple te cell kill at
the lowest drug concent ra t ion (0.l x Peak plasma con-
centrat ion) whereas others showed only l imited response
at 1.0 × Peak plasma concentrat ion. Fig. 3 shows repre-
sentative dose-response curves from l0 randomly se-
lected tumors, demonst ra t ing the range of response
patterns for cisplatin, carboplatin, 4-HC, and Taxol.
Of particular interest to us was the degree of correla-
tion in drug response patterns among the four drugs
tested, cisplatin, carboplatin, 4-HC, and Taxol. As ex-
pected, comparisons between cisplatin and 4-HC, which
are quite different chemical compounds with different
mechanisms of action, showed significant differences in
response with values between r = 0.226 and r = 0.477. In
addition, 4-HC demons t ra ted a h igher degree of cytotox-
icity for each concentrat ion, compared with cisplatin and
carboplat in (Fig. 2). However, resistance to 4-HC was
obselwed in 7 patients with tumors that were sensitive to
cisplatin. When the survival fraction at 0.2 × Peak plasma
762 Sevin and Perras April 1997 Am.] Obstet Gynecol
t- O 0 LI. ._~
O)
1.2,
1.011
0.8=
0.6=
0.4-
0.2-
0.0 .01
A 1.2- B
1.0
0.8
06
0.4
0.2
. . . . . . . . m . . . . . . . . , . . . . . . . . , 0 . 0 , . . . . . . . . ,
.1 1 10 .01 .1 I 10
1.2 t C 1.2" D
1 .o • 1.0
.o 01 U.
.>-- - - ,:> (0 0"61 ~ 0 6
0.4 0.4 O0
0.2 0.2
0 .0 . . . . . . . . , . . . . . 0 0
.01 .1 1 10 .01 .1 1 10
D o s e D o s e
Fig. 3. Representative dose-response cmwes from 10 randomly selected tumors. A, Cisptatin; B, 4-HC; C, Taxol; D, carboplatin.
concentra t ion was compared for cisplatin and 4-HC in 10 cases, the survival fractions were the same. In the remain-
ing 83 cases the survival fraction was less for 4-HC. In
contrast, cisplatin and carboplatin are chemically closely related analogs, which are frequently used by clinicians
as equal substitutes. One would expect a very close
correlation of drug-response patterns between these two
drugs. A graphic comparison of the mean values suggests that these two drugs are indeed very similar (Fig. 9).
However, a correlation analysis of survival fractions be-
tween the two drugs for each tmnor, at each concentra- tion, demonstra ted an enormous range of variability
(Fig. 4). Except for the highest drug concentrat ion, resulting in almost complete cell kill from both drugs,
significant differences were observed with values between r = 0.344 and r = 0.462 for all o ther concentrat ions.
Response patterns after exposure to drug combina- tions. We observed overall increased cytotoxicity with drug combinations, compared with single drugs, which led us to test drug combinat ions in lower concentra t ions ranging from 0.1 m 1.0 × Peak plasma concentra t ion only. We used fixed pairs of concentrat ions to permit the evaluation of synergistic and antagonistic drug interac- tions with the median effect analysis method described in detail by Chou and Talalay/a Fig. 5 shows the mean values of 100 assays for cisplatin-Taxol and cisplatin-4-
HC, whereby for each dose cisplat in-4-HC appears more cytotoxic than cisplatin-Taxol.
Drug-response patterns were quite variable for com- b ined drug exposures as well. The combina t ion was no t
always more active than each drug alone. Four patterns of drug interaction were observed; most commonly tire two
drugs showed additive (Fig. 6, A) or subadditive (Fig. 6, B)
responses, and less commonly there were antagonistic (Fig. 6, C) or synergistic (Fig. 6, D) drug interactions.
A correlat ion analysis between cisplatin-Taxol and
cisplat in-4-HC for each tumor, at each concentrat ion,
demonstrates significant variability (Fig. 7). A closer correlation was observed with drug combinat ions as compared with single drugs; values were between r =
0.419 and r = 0.677 for the four concentrat ions. Again the cisplatin-zbHC combinat ion appears more cytotoxic than cisplatin-Taxol.
C o m m e n t
These findings in 100 flesh tumor specimens from patients with primary ovarian carcinoma are similar to the ones observed in cell lines, conf i rming the existence
of heterogeneity of the in vitro drug response in mor- phologically similar neoplasms. ~ Neither the response to a single drug nor that to a drug combina t ion could be predicted for a particular tumor specimen other than by
V o l u m e 1 7 6 , N u m b e r 4 Sevin and Perras 763 A[ll. J Obste/Gynecol
8
S t . X -~ o p
,?,
1.10
0.85
0.65
0.44
0.22
R = 0.442 1.10
,•
I I I I I
0.00 0.22 0.44 0.65 0.85 1.10
0 . 2 X C a r b o p l a t l n
Survival F r a c t i o n
1.10 R = 0.344 1.10
0.00
0.85
_8 ~, ~ 065 x o ~ ,~$ 044
0.85
_8
x
,s-~ 044 g
0.22
0.00
0.85
_8 ~ 065 ,., ..,-
x o ~ ,~ ~o.44
,?,
0.22 , " • " ; •
0.00 i i i I , i 0.00
0.00 0 .22 0 . 4 4 0.6.5 0.85 1.10
1.0 X C a r b o p l a t l n
Survlval Frac t ion
0.22
R = 0,462
I I I I I
0.00 0.22 0.44 0.65 0.85 1.10
0.5 X C a r b o p l a t l n
Survival F r a C t i o n
R = 0.526
• . i i 4 t
0.00 0.22 0,44 0.65 0.85 1.10
5 . 0 X C a r b o p l a t l n
Survival F r a c t i o n
Fig. 4. Correlation scatmrplots of survival fractions bev,~,een cisplatin and carboplatin for fnur different drug co*~centrations: 0.2, 0.5, 1.0, and 5.0 × Peak plasma concentration. Regression analysis with 95% confidence intmwals and individual coetficients of correlation, expressed as r values, are listed in each distribution pattern.
t - O . B
O
I&
- I
1.2 =
1.0
0.8
0.6"
0.4"
0.2"
0.0 0.0
i ~ DDP/4-HC ¢ DDP/raxol
i
|
0.2 0.4 0.6 0.8 1.0 1.2
Dose Fig. 5. Mean survival fraction for each dose of two drug combinations tested, cisplatin plus 4-HC and cisplatin plus Taxol. Error bars represent SD. DDP, Cisplatin.
d i rec t testing. This u nde r s co r e s the n e e d to charac te r ize
chemosens idvi ty profi les tb r individual pa t ien ts be fore
d rug t r ea tmen t .
T u m o r he t e rogene i ty r ema ins a controvers ia l issue.
The old c o n c e p t of cance r o r ig ina t ing f rom one cancer-
ous ceil appears to be too simplistic. In a r ecen t review
article Nicho lson ]7 discussed the in te rp lay of oncogenes ,
suppressor genes, a n d the complex in te rac t ions be tween
764 Sev in and Pe r ras April 1997
Am J Obste t Gynecol
1- .9
U.
.> -,i
1.2-
1.0,
08.
0.6.
0.4-
0.2.
0.0 0.0
A DDP
DDP/4-HC
0.2 0.4 0.6 0.8 1.0 1.2 Dose
1 '2~B
¢- 1 . 0 ~ . ~ " ~ t DDP .9 4-HC
elk 0.4 .>
i f} 0.2
0.0 . , . • 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Dose
12.
g 1.0.
'~ 0.6'
U. 0.6'
.~ 0.4'
"~ 0.2. u)
0.0 00
C -'--O--- DDP ; Taxol
~ ODPf1"axol g
n
- m • | - m • | • | - I
0.2 0.4 0.6 03 1.0 1.2 Dose
1.2-
1.0t
08-
0.6-
0.4.
0.2-
0.0 0.0
D DDP • Taxol
• ---0--.. DDP/Taxol
• | . | • i • i |
0.2 0.4 0.6 0.6 1.0 1.2 Dose
Fig, 6. Representative dose-response curves of individual tumors tested againSt single drugs and drug combinations. DDP, Cisplatin.
tumor and host microenvi ronment , which together ex-
plain the evolution of he te rogeneous tumor phenotypes
with different biologic characteristics such as protein
expression and deoxyribonucleic acid content. These
tumors also respond differently to chemotherapy, which
contributes to t rea tment failure and tumor recurrence.
To be able to study tumor heterogenei ty in vitro, the test
system itself has to be reproducible , without much vari-
ability if the same tissue specimen is repeatedly tested.
We have demonst ra ted the reproducibi l i ty of the assay
itself. As an example, almost identical dose-response
patterns were observed in three different generat ions of
a human ovarian cancer growing as nude mouse xeno-
grafts./3 However, we also observed different degrees of
chemosensitivity of fresh tumor specimens harvested
f rom various sites in the same pat ient (unpubl ished
data). Even though some heterogenei ty was observed, in
the majority of cases no significant differences in dose-
response patterns were observed in tumor tissues t rom
different sites in the same patient. The issue of hetero-
geneity needs to be studied further. Because the A T P -
cell viability assay requires relatively few cells per assay,
tumor tissue f rom primary and metastatic sites can easily
be tested in parallel to study heterogenei ty in drug
response, as well as differences between primary and recurrent disease in individual patients.
Clinical practice requires an in vitro m e t h o d that has a
high success rate in providing useful results. The evalu-
ability rate was 91% for primal T ovarian carcinoma.
Reasons for lower evaluability rates relate to selection of
poorly vascularized rumor specimens by the surgeon,
length of transit t ime from surgical resection to process-
ing in the laboratory, and handl ing of the tissue dur ing
preparat ion for plating of tumor samples in culture.
For the clinician the most interest ing aspect of chemo-
sensitivity testing is the selection of active drugs, the
exclusion of inactive drugs, or both. The concept of drug
response and drug resistance has received considerable
at tention in recent years. For the oncologist drug resis-
tance means the failure of a pat ient to have a clinical
response to treatment. For tumor biologists drug resis-
tance is a cellular p h e n o m e n o n and can be def ined as
failure to obtain cytotoxicity at physiologically achievable
drug concentrat ions. Cellular mechanisms of drug resis-
tance can be subclassified into those that are located in
the cell membrane , cytoplasm, or nucleus. The ones in
the cell m e m b r a n e relate to t ransmembrane drug trans-
port; the most popular is the p170 glycoprotein that
affects drug efflux of a variety of drugs and is control led
by the multi-drug-resistance (mdr-1) gene. ~ Resistance
mechanisms in the cytoplasm, such as elevated levels of
glutathione, result in increased resistance to alkylating
agents and rad ia t ion] 9 Nuclear mechanisms of drug
resistance relate to deoxyribonucleic acid repair mecha-
V o l u m e I 7 6 , N u m b e r 4 Sevin and Perras 765 -kin .J O b s t e t G y n e c o
1.10
0.85
4 : x o ~ "6 0.65
÷
8~ 044 × g
(5 0.22
0.00
1.10
0 0.85 -r 4 c x o
,':. ~ 0.65
e, p 0.44
g 0.22
R = 0.677
0.00 0.22 0.44 0,65 0.85 1.10
0.1 X DDP + 0.1 X Taxol
Survival Fraction
R = 0 . 4 1 9
1.10
0.85 O
x ~ e~ ~ 0.65 o u_ , "~
x
(5 0.22
0.00
1.10
O 0.85 =, = '¢ _o
o x ~0.~ ,.:~. +
x ~ o.
0.22
0.00 0.00
0.00 0.22 0.44 0.65 0,85 1.10
0 . 5 X DDP ÷ 0.5 X Taxol
Survival F r a c t i o n
0.00
R = 0 . 5 ~
• . • ° •
0.22 0.44 0,65 0.85 1.10
0 . 2 X DDP + 0,2 X Taxol Sun/Ival F r a c t i o n
R = 0.477
0.00 0.22 0.44 0,65 0,85 1.10
1.0 X DDP + 1 .O X Taxol
Survival F r a c t i o n
Fig. 7. Correlation scatterplots ofsutwival fractions between cisplatin-4-HC and cisplatin-Taxol for four different drug concentrations: 0.1, 0.2, 0.5, and 1.0 × Peak plasma concentration. Regression analysis with 95% confidence intervals and individual coefficients of correlation, expressed as rvalues, are listed in each distribution pattern.
nisms, involving complex enzyme systems such as de-
oxyribonucleic acid polymerases or the production of
deoxyribonucleic acid precursors (e.g., increased dihy-
drofolate reductase activity results in methotrexate resis- tance). 19 Most of these mechanisms have been investi-
gated in highly selected tumor models (cell lines) and are difficult to apply to fresh tumor specimens in clinical practice. Most previously described chemosensitivity as-
says are quite good at predicting a lack of response with
reported negative predictive values greater than the 90% rate. m Prediction of resistance is more likely than pre-
diction of response to be successful with in vitro testing,
because, if the end organ does not respond, the other
biologic and pharmacologic variables have a lesser fin- pact on overall response. Using an unphysiologically high concentration will further increase the probability
of predicting in vitro resistance and with that the lack of clinical response. However, exclusion of ineffective drugs is less attractive to clinicians than is selection of active
drugs. In clinical practice the variables that influence the
overall chemotherapy response in patients include host factors (e.g., age, nutritional status, immune compe- tence) and treatment variables (dose, frequency, route of drug application, plasma concentration, tissue levels), as
well as tumor variables (size, localization, degree of
vascularization). These variables between the "end or-
gan" of drug response at the cellular level and the overall
clinical response to chemotherapy in the patient define
the limitations of information that can be obtained even with an optimal method of chemosensitivity testing.
It is obvious that the in vitro growth environment
cannot reproduce the in vivo conditions of solid tumors. One specific limitation relates to drug concentration and
length of drug exposure. Neither drug tissue levels nor
duration data are available. In the design phase of our
assay we selected published peak plasma concentration
values as reference values to standardize the assay condi- tions and allow comparison of results. Drugs that need
metabolic conversion in vivo pose additional difficulties in choosing appropriate in vitro drug concentrations. From our results it appears that the dose we chose for 4-HC, the active metabolite of cyclophosphamide, may have been too high, relative to the other three drugs, assuming that the average dose-response curves should be similar. If this assumption is correct, it may explain
the disproportionately higher cytotoxicity for 4-HC com- pared with the other single-agent results (Fig. 2) and the skewing of data toward the cisplatin-g-HC combination (Figs. 5 and 7). The other interesting observation is the
766 Sevin and Perras April 1997 Am J Obstet Gynecol
b imoda l response to Taxol (Figs. 2 a n d 3, C). Taxol
inhibi ts cell p ro l i fe ra t ion by in te r fe r ing with the sp indle
appara tus d u r i n g cell division. At this p o i n t we have no
good exp lana t ion for this observed p h e n o m e n o n .
It shou ld be obvious tha t the best one can expec t f rom
an in vitro chemosensi t iv i ty test is an increase in p roba-
bility of clinical response (or lack of response) for a
par t icu lar d rug or d r u g combina t ion . T he long-s tand ing
discussion of the super ior i ty of c o m b i n a t i o n t r e a t m e n t
over s ingle-agent c h e m o t h e r a p y may also be resolved
with chemosensi t iv i ty testing. Unless significantly h i g h e r
cytotoxicity is observed in vitro with the d rug combina-
t ion (Fig. 6, A and D), it may b e c o m e unaccep t ab l e to
t rea t a pa t i en t with c o m b i n a t i o n c h e m o t h e r a p y tha t
general ly p roduces m o r e serious side effects, if one d rug
d o m i n a t e s the overall cytotoxicity.
In summary, i n f o r m a t i on a b o u t d rug sensitivity or
resis tance at the e n d o rgan level shou ld increase the
overall probabi l i ty of clinical r e sponse if the mos t active
drug(s) is se lected for t r ea tmen t . Indiv idual ized the rapy
select ion tha t is based o n in vitro chemosensi t iv i ty studies
promises to b r ing s ignif icant i m p r o v e m e n t to cance r
t r ea tmen t .
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Discussion
DR. STEPHEN L. CURRY, Har t ford , Connect icu t . Dr. Sevin a n d his co l labora tors have b e e n working on this chemosensi t iv i ty test for > 1 0 years. They have pub l i shed extensively. Every gynecologic oncologis t u n d e r s t a n d s tha t the ability to use agents tha t have b e e n shown to be effective against a pa t i en t ' s specific ovarian cance r would
be a ma jo r advance. This p r e s e n t a t i o n of the au tho r s ' work in this area a t tempts to verify, the p remise tha t like t umors r e s p o n d differently to the same chemothe rapy . They reviewed 100 consecut ive sets of ovar ian cance r data f rom the i r labora tory a n d showed signif icant vari- ability in chemosensi t iv i ty f rom case to case.
Previous chemosensi t iv i ty tests fai led because of tech- nical diflfculties a n d because the test m e a s u r e d the ability of cells to multiply. Jus t because cells w o n ' t mult iply does no t m e a n they w o n ' t persist. This test evaluates cell kill by a single c h e m o t h e r a p y agen t or c o m b i n a t i o n of agents. It is based o n the well-proved fact tha t ATP rapidly disap- pears f rom cells w h e n they die, a n d the p re sence or absence of ATP is easily measured .
Over the years Dr. Sevin has car r ied ou t his work in a very careful stepwise fashion to ensu re no t only tha t this test is accura te in p red ic t ing c h e m o t h e r a p y response in vivo bu t also tha t it is technical ly feasible in the pa t i en t care setting. He first es tab l i shed r ep roduc ib l e dose- response a n d t ime-response curves in an ovar ian cance r cell line. He subsequen t ly showed tha t the test could be d o n e with as few as 50 cells a n d tha t the test is evaluable in > 9 5 % of clinical cases. T h e g r o u p has p u b l i s h e d clinical r e sponse data in b reas t and u t e r i ne cancer .
In this p r e sen t a t i on we have seen tha t ovar ian cancers show a very di f ferent response to known active agents a n d tha t of ten agents tha t are active individual ly are no t necessari ly synergistic or even addit ive in comb ina t i on . So they p o r t e n d to have p roved the i r p o i n t a n d conc lude tha t fu tu re protocols shou ld r a n d o m l y evaluate individ- ual ized the rapy o n the basis of this test. But I submi t tha t to show t rue variability they n e e d to do regress ion
V,:flumc 176, Number 4 Sevin and Perras 767 Mn.J Obstet Gynccol
analysis, inc luding histologic typing and differentiation, or, bet ter yet, only include data on a specific subset such as grade 2 papillary serous carcinoma. For years we have known that response is d e p e n d e n t on mult iple factors, including grade and histologic type. Is a series of 100 consecutive patients enough to evaluate three different grades and five different histologic types? I presume the authors conf ined this presentat ion to epithelial ovarian cancer. They failed to discuss these two variables in their presentation.
Obviously, ongoing randomized protocols will defini- tively prove this point, and I congratulate the authors and their colleagues for persisting in this endeavor because I for one gave up on chemosensitivity testing long ago, and 1 was wrong.
In closing, I have two questions for Dr. Sevin: (1) Why not look at a specific grade and histologic type to make your point even stronger? (2) Do you have any experi- ence with stromal tumors or border l ine tumors, for which chemotherapy m a n a g e m e n t is even more difficult?
DR. HOWARD W. JONFS III, Nashville, Tennessee. That 's a very interest ing discussion of work that you've done over the years.
In addt ion to the heterogenei ty you have observed among individuals with differert types of tumors, is there no t heterogenei ty within a single individual 's tumor? Also, is it no t possible that over time a patient 's resistance pattern might change as she is exposed to one chemo- therapy or another?
Another problem that we've had clinically is drug delivery to the tumor. You might find that in vitro you have excellent sensitivity, and yet you ' re not able to deliver the drug to the tumor. So, in vivo the tumor never receives that chemotherapy, and as a result we may no t get a clinical tumor response.
These are all problems that I think have caused most clinicians to be very discouraged with various types of in vitro chemosensitivity testing that have been tried over the last 10 or 15 years. Do you know of any plans for randomized trials looking at this or other chemosensitiv- ity tests in the clinical setting, because thus far no one has shown that the promise of in vitro testing translates to clinical value for the patient?
DR. ARTHUR L. HERBST, Chicago, Illinois. I have a question. From your in vitro data for the ovarian tumors, do you have sufficient prel iminary clinical informat ion to tell us whether this is going to be different from or similar to the o ther types of chemosensi tMty assays, which I think, at least in most of our experience, have been more effective in showing resistance to chemother- apy than in showing sensitivity. I wonder whether you have any data you could share with us even prel iminari ly on that.
DR. SEWN (Closing). Dr. Curry, thank you very much for the discussion. I have tried, as you said, for the last 10 years to br ing the issue of chemosensitivity testing a little more to the at tent ion of oncologists. The biggest obsta- cles have been our colleagues, the medical oncologists, who seem to have a part icular problem with accepting
the idea of drug selection that is based on a laboratory test rather than their clinical j udgment .
To answer Dr. Curry's last questions first, regarding tumor grade, stromal and border l ine tumors, we do have some in vitro data on these issues. The border l ine tumors show very little response in vitro. They are no t fast growing, have a low proliferation rate, and therefore do not demonstra te a large differential response between treated and unt rea ted cells in vitro dur ing the 6 days of culture. Preliminary data suggest that tumors with high rates of proliferation show stronger responses to chemo- therapeutic agents. This impression is supported by in vitro data on cell lines. Cells treated in log-phase growth have steeper dose-response curves than cells in plateau phase. This may be one reason why most cell l ine work is done with cells in log-phase growth, which increases the probability of obta in ing positive results. However, in vivo, especially in solid tumors, we are no t deal ing with log-phase growth but with cells in plateau phase, which is one of the biologic problems we are facing in treating ovarian carcinoma with chemotherapy.
To limit our in vitro analysis to specific grades and histologic types would certainly s t rengthen our a rgumen t regarding heterogenei ty in drug response. However, the purpose of this study was to demonstra te heterogeneity in ovarian cancers as we receive them from our patients. 1 believe we have done that. All tumors presented were grade 2 or 3 adenocarc inomas of the ovat T. As clinicians we do not select drugs on the basis of grade or cell type in patients with advanced adenocarc inoma either. Cur- rently we treat all these patients with the same chemo- therapy regimen (e.g., Taxol and cisplatin) and in the clinical setting ignore the heterogenei ty we have ob- served in vitro. More detailed and statistically elaborate studies on tumor heterogeneity within a better-defined histologic subset are desirable, and we may do those in the future.
Stromal tumors cannot be tested with our system. Because the assay is designed to suppress growth of normal stromal cells and blood components , with the aid of the agar underlayer, to reduce "contaminat ion noise" of the assay, sarcomas and stromal tumors have not been evaluated.
I thank Dr. Jones for his questions; they are, as always, excellent, because they give me the oppor tuni ty to discuss some other impor tan t issues. In addi t ion to in te r tumor heterogeneity to drug response between tu- mors from different patients, there may also be signifi- cant differences within the tumor of an individual pa- tient, especially between the primary tumor and metastatic sites, such as lymph node metastases. We are trying to answer this question but do not have sufficient data yet. We did, however, observe in many cases in which we analyzed multiple tissue specimens from the same pat ient a fairly high degree of consistency in drug-response patterns, but also at times there were impressive differences between metastatic sites. This issue certainly needs further study.
Changes in drug sensitivity between the unt rea ted
768 Sevin and Perras April 1997 Am J Obstet Gynecol
primary and the treated recurrent disease in the same patient have been observed many times, generally resis- tance to the drugs the patients had been exposed t o before, but again our experience is limited.
The other issues relate to differences in pharmacology and drug delivery to the tumor in vivo compared with the in vitro enviromnent. We are very aware of the multiple problems associated with in vitro drug testing in solid tumors. There is no question that in vitro assays cannot predict in vivo response completely. With the in vitro assay we are only looking at the end organ response to chemotherapy in the tumor cell, and we do so in an artificial growth environment. We know that chemother- apy response is influenced by many factors, including interactions of oncogenes, cellular drug resistance mech- anisms, the microenvironment of the tumor and the host, and tumor vascularization and drug tissue levels. At this point we know very little, and we certainly cannot assess these variables or their impact in an individual patient. As oncologic surgeons we try through tumor debulking to create a favorable starting point for chemotherapy. The best we can hope to achieve with in vitro drug testing is to increase the probability of end organ drug response. If we could thus increase overall drug response by 20% or 30%, we could potentially improve patient treatment outcome substantially in patients with advanced ovarian carcinoma, compared to our current results.
The last question by Dr. Jones addresses the issue of chemosensitivity testing in randomized clinical trials. Most published data in the past have been correlative studies comparing in vivo and in vitro drug responses. Most studies on treatment selection that are based on in vitro test results have used the clonogenic assay, and I refer to a review byAapro (see reference 13 of article) for details. These studies suggest an increase in clinical response when treatment is based on in vitrO test results; however, the numbers were small and most studies were not randomized. Von Hoff et al. (see reference 10 of article) published the Southwest Oncology Group expe- rience with the modified human tumor cloning assay in ovarian cancer, which did not show a treatment advan- tage. Last year I submitted a concept proposal to the Gynecologic Oncology Group (GOG) to randomize che- motherapy selection in patients with advanced primary ovarian cancer that was based on in vitro ATP-cell viability assay results versus the most active "standard
treatment regimen" (e.g., Taxol-cisplatin). A similar mul- ticenter study is currently underway in Switzerland. The principal investigator is Dr. Ossi Koechli, who studied in Miami and then established the ATP-cell viability assay at the University of Zfirich. In this study the most active drug combination on the basis of the ATP-cell viability assay is randomly compared to "the physician's choice" chemotherapy selected by the treating oncologist. I be- lieve only such prospective studies will be able to answer the question regarding the value of in vitro chemosensi- tivity testing for patient care.
To answer Dr. Herbst's question would take a little more time. There are basically three different, clinically applicable in vitro test systems that are based on the biologic end point to define drug response. I refer to our recent monograph on chemosensitivity testing for details (see reference 13 of article). One system relies on inhibition of cell proliferation as exemplified by the modified clonogenic assay measuring short-term inhibi- tion of deoxyribonucleic acid precusor incorporation. This method exposes cells to high drug concentrations and therefore primarily measures drug resistance. An- other method measures the effect of drugs on cell membrane integrity. Most notable is the Fluorescent Cytoprint Assay developed by Rotman et al. (see refer- ence 13 of article). The third method measures cell viability, the ATP-cell viability assay and the MMT (m,- 0t-methyl-m-tyrosine). Test results cannot be easily com- pared because of the different methods used and their different end points. We believe that measuring changes in cell viability, or cell death, is the most logical end point and the one clinicians are most interested in. The ATP-cell viability assay assesses cell viability by measur- ing intracellular ATP with a high degree of sensitivity. We believe that comparing cell viability of drug-treated tu- mor cells at four to five drug concentrations, in compar- ison with untreated controls, offers the best in vitro measure of end organ cytotoxicity. With this method we obtain dose-response patterns that provide information in regard to both drug resistance and drug sensitivity. We believe that in the future more oncologists will be convinced that this method offers the potential of im- proving treatment outcome. This technology needs to be tested in the clinical setting, preferably in a prospec- tive clinical trial in patients with advanced ovarian carcinoma.
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