Method to predict tumor response to therapy

A method is disclosed for predicting response of a tumor patient to therapy, and selecting appropriate therapy for malignant neoplasms such as breast, ovarian and gastrointestinal cancer. A sample of tumor cells is cultured in the presence of 10 .mu.g/ml estradiol, and inhibition of cell growth in the culture paradoxically indicates an estrogen dependent tumor that will respond to antiestrogenic therapy. Another sample of tumor cells is cultured in the presence of a chemotherapeutic agent which predicts response of the tumor to in vivo administration of a cytotoxic agent. The chemotherapeutic agent is preferably a cytotoxic drug, or a drug having both cytotoxic and antiestrogenic mechanisms of action. A particularly suitable substance having both mechanisms of action is: ##STR1## wherein R.sup.1 H, OH, OOC(CH.sub.2).sub.2 CO.sub.2 H or CH.sub.3 COO; R.sup.2 is C.sub.6 H.sub.4 OH, C.sub.6 H.sub.4 OCOCH.sub.3, C.sub.6 H.sub.4 OOC(CH.sub.2)CO.sub.2 H, or C.sub.6 H.sub.5 ; and X is C.sub.6 H.sub.3 -2,4(NO.sub.2).sub.2, C.sub.6 H.sub.5, C.sub.6 H.sub.4 -4(NO.sub.2), C.sub.6 H.sub.4 -3(NO.sub.2), or C.sub.6 H.sub.3 -2,4(NO.sub.2).sub.2 ; Predictive power of the assay is increased by combining information from the estradiol and chemotherapeutic assays. These assays are preferably performed in culture flasks having removable bases that function as conventional microscope slides.

BACKGROUND OF THE INVENTION 
Field of the Invention 
This invention concerns the testing of tumor cells for their sensitivity to 
estrogens and hydrazones. More specifically, it concerns a method and test 
kit useful in selecting an appropriate treatment for cancer, and 
predicting the probable response of a patient to therapy. 
Discussion of the Background of the Invention 
Approximately 55% of all human breast cancers and 20% of ovarian cancers 
are considered to be estrogen dependent. These tumors require an estrogen 
for growth, and contain cells that bind estrogens through an absorption 
and transport mechanism that facilitates the entry of estrogens into the 
cell. The absorption and transport mechanism is believed to be an estrogen 
receptor (ER), which is a protein that originates in the cytoplasm of the 
cell and initially interacts with extracellular estrogen in the cell 
membrane. After interacting with the estrogen, the ER folds over the 
estrogen to complete the tertiary structure of the receptor complex. This 
structural change stimulates translocation of the receptor-estrogen 
complex through the pores of the nuclear membrane. Once inside the 
nucleus, the estrogen binds to DNA and helps direct the activity of the 
cell. 
Estrogen receptors are believed to play an important biological role in 
several types of hormone sensitive cancers, such as some breast and 
ovarian tumors, in which estrogens are essential for continued cell 
growth. Some therapeutic modalities take advantage of this hormone 
dependence by surgically removing a patient's ovaries or adrenal glands to 
reduce endogenous production of estrogen. Some anti-cancer drugs also take 
advantage of the hormone dependence of these cells. Tamoxifen and certain 
nitrophenylhydrazones, for example, have an antiestrogenic activity that 
interferes with the interaction between the ER and endogenous estrogen. 
This interference prevents estrogen incorporation into the cell, and can 
inhibit growth of estrogen dependent tumor cells. 
The prognostic importance of a patient's estrogen receptor status is 
illustrated by the significantly improved treatment outcome seen in 
patients who are ER+. It has been found, for example, that approximately 
50-70% of ER+ patients respond to endocrine manipulations or 
antiestrogenic therapy, while ER- patients have a response rate between 0% 
and 10%. Patients with ER+ tumors also tend to have a better prognosis 
with lower recurrence rates and a longer disease-free interval. 
In addition to ER status, it has been found that the presence or absence of 
a progesterone receptor (PgR) in the tumor cells helps select those ER+ 
patients who are most likely to respond to endocrine treatment. The 
presence of PgR and ER together appears to be a better indicator of a 
favorable treatment outcome than ER values alone. PgR is detected in 
approximately two-thirds of ER+ tumors, and is only occasionally found in 
ER- tumors. Its presence has been shown to improve the prediction of an 
objective response to endocrine therapy. 
The use of quantitive methods to detect and quantitate estrogen and 
progesterone receptors has provided one basis for prescribing 
antiestrogens and other hormone manipulation therapies to treat patients 
with hormone dependent malignancies. Ligand binding assays, monoclonal 
antibody assays, and enzyme immunochemical analyses have been used to 
detect and quantitate these receptors, as disclosed in ACTA Oncologica, 
27:1-19 (1988). Using such techniques, it has been determined that breast 
cancer tissue containing ER concentrations greater than 5 fentomoles/ng 
and PgR concentrations greater than 3 fentomoles/ng is considered 
sensitive to antiestrogen therapy (J. Clinical Oncology, 1:227-241 
(1985)). The higher the ER/PgR concentrations, the more sensitive the 
tissue should be to the antiestrogen therapy. 
Unfortunately, a tumor's ER and PgR status is not always useful in 
predicting a response to endocrine therapies. The insensitivity of 
receptor screening is even more pronounced with respect to predicting 
response to non-hormonal chemotherapeutic agents, such as doxorubicin, 
methotrexate or 5-fluorouracil. In the ER-/PgR- case, for example, one can 
conclude that the tumor will probably not respond to hormonal therapy, but 
these results give no guidance about the likelihood of response to 
non-hormonal chemotherapy. When the tumor is ER+/PgR-, some response to 
antiestrogens is expected, but only about 30% of the tumors will respond 
to such therapy. Finally, even patients who are ER+/PgR+ may fail to 
respond to antiestrogenic therapy, especially if the quantitative level 
and cell distribution of receptors is low. These problems illustrate that 
hormone receptor status alone is often inadequate in selecting appropriate 
treatment or predicting outcome of chemotherapy in patients with breast 
cancer. 
Efforts have been made to develop in vitro tests of cell sensitivity that 
would avoid such problems by predicting in vivo responses of tumors to 
chemotherapeutic drugs. Vescio et al., for example, described a three 
dimensional, gel-supported culture system for growing human tumors in the 
presence of a variety of different cytotoxic drugs to predict differential 
drug sensitivities of multiple cell types within individual cultured 
tumors. Proc. Natl. Acad. Sci., 84:5029-5033 (1987). In vitro tests for 
predicting response to hormonal agents, however, have been found to be 
particularly unhelpful. Wantanabe et al., Japanese Journal of Cancer 
Research, 81:536-43 (1990). In addition, such in vitro assays have 
typically monitored cell division and growth to determine cell viability 
with time. These assays are laborious procedures designed to select the 
most effective drugs to stop the cells from replicating and growing. As 
many as twenty drugs are often required in such assays to provide a 
productive profile of drug sensitivities for any one cancer tissue. 
Hence, a need still exists for a relatively rapid, simple and efficient in 
vitro method of predicting a patient's response to chemotherapy and other 
modalities of treatment. 
Therefore, it is one object of the present invention to provide an 
efficient test system to determine drug sensitivities of living cancer 
cells, and predict responses of cancer patients to hormonal therapies. 
It is yet another object of the present invention to provide such an 
improved test system to determine sensitivities of cancer cells to 
non-hormonal chemotherapy, and predict patient response to a variety of 
non-hormonal chemotherapeutic agents. 
Yet another object of the invention is to provide an efficient, 
cost-effective in vitro prognosticative assay for predicting probable 
response of patients to medical and surgical intervention. 
These and other objects of the invention will be understood more clearly by 
reference to the following detailed description and drawing. 
SUMMARY OF THE INVENTION 
The foregoing objects are achieved by providing a method of predicting in 
vivo response of tumor cells to therapy by culturing a sample of the cells 
in the presence of a cancer chemotherapeutic drug and quantitating the 
degree of inhibition of cell growth in the cultured sample. The drug is 
preferably a nitrophenylhydrazone such as: 
##STR2## 
wherein R.sup.1 is hydrogen, hydroxy, acetate, succinate or another water 
soluble group; R.sup.2 is C.sub.6 H.sub.4 OH, C.sub.6 H.sub.4 OCOCH.sub.3, 
C.sub.6 H.sub.4 OCO(CH.sub.2)CO.sub.2 H or C.sub.6 H.sub.5 ; and X is 
C.sub.6 H.sub.3 -2,4(NO.sub.2).sub.2, C.sub.6 H.sub.5, C.sub.6 H.sub.4 
-4(NO.sub.2), C.sub.6 H.sub.4 -3(NO.sub.2), or C.sub.6 H.sub.3 
-2,4(NO.sub.2).sub.2. In especially preferred embodiments, R.sup.1 is OH, 
R.sup.2 is C.sub.6 H.sub.4 OH and X is C.sub.6 H.sub.3 
-2,4(NO.sub.2).sub.2. These hydrazones have been found to have both 
cytotoxic and antiestrogenic mechanisms of action. 
When assaying cells that may be hormone dependent, such as breast or 
ovarian tumor cells, a first sample of the cells may be cultured in the 
presence of a sufficient amount of an estrogen to inhibit growth of 
estrogen sensitive cells in the culture. A second sample of the cells is 
cultured in the presence of a chemotherapeutic agent that, when cultured 
with a sample of the tumor cells, predicts a patient's probable response 
to therapeutic interventions. The degree of inhibition of cell growth in 
each cultured sample is determined, which allows selection of a treatment 
modality based on the degree of inhibition of cell growth. Specifically, 
an antiestrogen therapy is selected if the degree of inhibition of cell 
growth in the presence of estrogen is significant. A non-hormonal therapy 
is indicated if cell growth in the estrogen-containing first sample is 
stimulated or not inhibited. In the latter case, if an IC.sub.50 is noted 
at lower concentrations (.ltoreq.10 .mu.g/ml) of the hydrazone, 
sensitivity to a cancer chemotherapies is suggested. If the cell growth is 
inhibited only at higher concentrations of the hydrazone in the second 
culture, the patient will probably not improve when given either hormonal 
or non-hormonal therapy. 
The invention also includes a test kit designed to help select treatment 
for cancer patients or predict probable response of a patient to a 
proposed therapy. The kit includes a control culture container, and a 
culture container for a chemotherapeutic agent that predicts a patient's 
probable therapeutic response to therapeutic interventions when cultured 
with a sample of the tumor cells. In preferred embodiments, the 
chemotherapeutic agent is a nitrophenylhydrazone, particularly 
4,4'-dihydroxydiphenyl-methylene-2,4-dinitrophenylhydrazone (A-007), which 
has been found to predict response to chemotherapy. Tumor cells are 
cultured in the presence of the agent, and cell growth is compared to 
control culture growth to determine the relative degree of inhibition of 
tumor cell growth in the presence of the agent. 
The kit preferably includes a plurality of culture flasks that are provided 
with a supply of different amounts of the agent to assess the 
concentration of drug at which an IC.sub.50 is observed. In the disclosed 
embodiment, three culture flasks respectively contain 10, 15 and 20 
.mu.g/ml of A-007. Tumor cells are cultured in each of these flasks, and 
cell growth is compared to growth in the control culture medium flask. A 
significant advantage may be obtained by using A-007, because a 
particularly strong correlation has been observed between the IC.sub.50 in 
the presence of A-007 and the response of a patient to both hormonal and 
non-hormonal cancer therapies. 
Alternatively, the kit can include a culture flask that contains an 
estrogen, such as 10 .mu.g/ml of estradiol. Tumor cells are cultured in 
the presence of estradiol, and cell growth is compared to growth in a 
control culture medium flask. Paradoxically, estrogen dependent tumors 
(such as those having high levels of estrogen receptors) will exhibit 
growth inhibition in the presence of this supraphysiological amount of 
estradiol. Equally unexpectedly, growth of non-estrogen dependent tumor 
cells is unaffected or stimulated. 
A particularly powerful degree of prognostication is achieved by combining 
the results of the separate tumor cell culture growths in the presence of 
estradiol and in the presence of the chemotherapeutic agent.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
The in vitro assay of the present invention provides a relatively simple 
test that increases the effectiveness with which appropriate therapy can 
be selected for a cancer patient. The assay also provides prognostic 
information that is not presently available from quantitative assays for 
estrogen and progesterone receptors alone. In its simplest embodiments, 
the assay exposes tumor cells in culture to a substance that, when 
cultured with a sample of the tumor cells, predicts a patient's probable 
response to proposed therapies. The substance is preferably an 
antiestrogenic hydrazone such as: 
##STR3## 
wherein R.sup.1 is H, OH, CH.sub.3 COO, or HO.sub.2 C(CH.sub.2).sub.2 COO; 
R.sup.2 is C.sub.6 H.sub.4 OCOCH.sub.3, C.sub.6 H.sub.4 
OOC(CH.sub.2).sub.2 CO.sub.2 H or C.sub.6 H.sub.5 ; and X is C.sub.6 
H.sub.3 -2,4(NO.sub.2).sub.2, C.sub.6 H.sub.5, C.sub.6 H.sub.4 
-4(NO.sub.2), C.sub.6 H.sub.4 -3(NO.sub.2), or C.sub.6 H.sub.3 
-2,4(NO.sub.2).sub.2. In especially preferred embodiments, R.sup.1 is OH, 
R.sup.2 is C.sub.6 H.sub.4 OH and X is C.sub.6 H.sub.3 
-2,4(NO.sub.2).sub.2. 
Several examples of these hydrazones are set forth below: 
TABLE I 
__________________________________________________________________________ 
Antiestrogenic Hydrzaones 
Compound 
R.sup.1 R.sup.2 X 
__________________________________________________________________________ 
A-007 HO C.sub.6 H.sub.4 OH 
C.sub.6 H.sub.3 -2,4(NO.sub.2).sub.2 
A-070 HO C.sub.6 H.sub.4 OH 
C.sub.6 H.sub.5 
A-100 HO C.sub.6 H.sub.4 OH 
C.sub.6 H.sub.4 -4(NO.sub.2) 
A-034 HO C.sub.6 H.sub.4 OH 
C.sub.6 H.sub.4 -3(NO.sub.2) 
A-032 CH.sub.3 COO 
C.sub.6 H.sub.4 OCOCH.sub.3 
C.sub.6 H.sub.3 -2,4(NO.sub.2).sub.2 
A-033 H C.sub.6 H.sub.5 
C.sub.6 H.sub.3 -2,4(NO.sub.2).sub.2 
A-106 HO.sub.2 C(CH.sub.2).sub.2 COO 
C.sub.6 H.sub.4 -OOC(CH.sub.2).sub.2 CO.sub.2 H 
C.sub.6 H.sub.3 -2,4(NO.sub.2).sub.2 
__________________________________________________________________________ 
The structures and methods of synthesis for these compounds have already 
been described in U.S. Pat. No. 4,732,904, for A-007 
(4,4'-dihydroxydiphenyl-methylene-2,4-dinitrophenylhydrazone); A-100 
(4,4'-dihydroxydiphenyl-methylene-4-nitrophenylhydrazone); and A-033 
(diphenyl-methylene-2,4-dinitrophenylhydrazone). U.S. Pat. No. 4,732,904 
is incorporated herein by reference. 
The synthesis of A-032 (4,4'-dihydroxybenzophenone phenylhydrazone) was 
performed by dissolving 0.389 (0.0072 mole) ammonium chloride, 4.28 g 
(0.02 mole) 4,4'-dihydroxybenzophenone and 6.48 g (0.06 mole) 
phenylhydrazone in 50 ml water. The mixture was refluxed for 48 hours. The 
reaction mixture was cooled and the tan solid product filtered. Light tan 
crystals (1.38 g) were recrystallized from 50% ethanol, m.p. 
183-185.degree.. 
Analytical calculation for C.sub.19 H.sub.16 N.sub.2 O.sub.2 was C, 74.98; 
H,5.30. 
Found: C, 75.07; H,5.29. 
The use of the invention is illustrated in the following Examples: 
EXAMPLE I 
Samples (1 cm) of sterile fresh human cancer tissue are obtained from a 
surgical biopsy or resection. The tissue can be stored in 20-50 cc 
RPMI-1640 tissue culture medium (Gibco Laboratories) at 
5.degree.-10.degree. C. for up to five days. The specimen(s) are 
transferred under sterile conditions to a laboratory where, under sterile 
conditions, the tissue is removed from the holding solution and is minced 
by scalpel and scissors. A cellular suspension is made by cutting the 
specimen into 0.1 mm fragments in petri dishes of 60 ml RPMI-1640 (Gibco 
Laboratories) containing 10% fetal bovine serum (FBS) (Gibco 
Laboratories), 100 .mu.g/ml streptomycin (Sigma Chemical Co.) and 100 
units/ml penicillin (Sigma Chemical Co.). 
One (1) ml of the cellular suspension is added to each of ten (10) NUNC 
SlideFlasks which are available from Nunc, Inc. (Catalog No. 170920 or 
Flask Style 177453). These flasks are described more fully in U.S. Pat. 
No. 3,726,764 which is incorporated herein by reference. Such a flask 10 
is shown in accompanying FIG. 1 to include a baseplate 12 and a housing 14 
having a 9 cm.sup.2 rectangular horizontal cross section and a square 
vertical cross section about 2 cm in height. Housing 14 covers a 9 
cm.sup.2 /culture area 16 of baseplate 12, leaving a portion 18 of the 
baseplate protruding beyond the area covered by the housing. The bottom 
edge of housing 14 is sealed to baseplate 12 by a thermoplastic or 
adhesive seal which is substantially fluid impervious. 
A flange 17 projects outwardly from the sidewalls of housing 14 spaced from 
and parallel to baseplate 12. The seal between the bottom edge of the 
housing and the baseplate can be selectively broken by prying baseplate 12 
away from housing 14, for example, by inserting a lever such as opener 19 
below flange 17 and prying the housing away from the base by exerting 
pressure on opener 19 in the direction shown by arrow 20. Once the 
baseplate 12 is removed from the housing, the baseplate acts as a 
conventional microscope slide that can be handled by portion 18 and 
examined under a microscope. 
Near the center of a square vertical sidewall of housing 14 is an opening 
surrounded by a cylindrical, externally threaded neck. The threads on the 
neck are configured to engage internal threads of a cap 22 such that 
access can be selectively obtained to the interior of housing 14 by 
unscrewing cap 22 from the neck. Culture media and additives are 
introduced through the open neck, and the flask is then re-sealed by 
threading cap 22 back on the threaded neck. 
The SlideFlask is used as an ordinary small-size culture flask with a 
culture area of 9 cm.sup.2. After culturing, the bottom of the flask (the 
slide) may be removed at any step in the preparation procedure (fixation, 
staining, etc.), by using the SlideFlask opener a shown in FIG. 1. The 
bottom of the flask has standard slide microscopy dimensions and is 
handled exactly as a standard microscopy slide. Employing light 
microscopy, the cell growth on each slide is counted. 
EXAMPLE II 
Using A-007 as the test hydrazone, the assay consists of two flasks each 
labelled: 
A. Control 
B. Estradiol (E.sub.2) (10 .mu.g/ml) 
C. A-007 (10 .mu.g/ml) 
D. A-007 (15 .mu.g/ml) 
E. A-007 (20 .mu.g/ml) 
The cellular suspension of tumor in each flask is diluted as described 
below. 
Flasks A are diluted to 6 ml each with RPMI-1640 containing 10% FBS, 100 
.mu.g/ml streptomycin and 100 units/ml penicillin. 
Flasks B are diluted to 6 ml with the same culture medium as in Flasks A, 
but which additionally contain estradiol (Sigma Chemical Co.) in a 
concentration of 10 .mu.g/ml in the culture medium. 
Flasks C are similarly diluted to 6 ml with RPMI-1640 plus FBS, penicillin 
and streptomycin as in flasks A, but the culture medium also contains 
A-007 (10 .mu.g/ml). 
Flasks D are diluted to 6 ml with RPMI-1640 plus FBS, penicillin and 
streptomycin as in flasks A, but the culture medium also contains A-007 
(15 .mu.g/ml). 
Flasks E are diluted to 6 ml with RPMI-1640 plus FBS, penicillin and 
streptomycin as in flasks A, but the culture medium also contains A-007 
(20 .mu.g/ml). 
The flasks are incubated for seven (7) to twenty-one (21) days in a 5% 
CO.sub.2 incubator at 37.degree. C. The cells may need to be cultured for 
a longer time to obtain sufficient cell numbers to count. Sufficient cell 
growth is arbitrarily said to be present in this example when at least 10 
cells/HPF or ten clumps of cell/HPF are present in the control culture. It 
is desireable that at least 5 cells or clumps of cells/HPF be present to 
ensure adequate control culture growth. The importance of establishing a 
standard control growth is to provide a basis for determining relative 
cell growth inhibition or stimulation in the presence of estrogen or the 
hydrazone. 
After sufficient cell growth appears in the flasks, the medium and contents 
are poured out and the slide bottom peeled off using the SlideFlask opener 
as shown in FIG. 1. The slides, which have previously been labelled A-E, 
are air dried for a few minutes and sprayed with a cytofixative 
(Pro-Fixx.TM., Lerner Labs) then examined under a conventional light 
microscope as any cytological or histological slides would ordinarily be 
examined by one skilled in the art. If the laboratory has an inverted 
field microscope, the above slide preparation can be eliminated and the 
flasks containing cells and liquid content can be counted directly, 
without preparing and staining the slides. High power fields are scanned, 
counted and compared in all five tissue culture systems (Flasks A-E). Ten 
(10) cells/HPF arbitrarily is considered 100% growth, because sufficient 
cell growth in the control culture was set at 10 cells/HPF. Results from 
each of the two corresponding flasks are averaged. 
One object of the assay is to find the lethal dose of hydrazones that 
inhibit 50% of the cells from growing. This dose is referred to as the 
IC.sub.50, and is present when there are 5 or fewer cells/HPF (compared to 
the 10 cells/HPF control). Another object is to determine the cell growth 
response to 10 .mu.g/ml estradiol in the culture medium. These results, 
both separately and together, provide important information about the 
receptor status of the cells and the likely response of the patient to 
hormonal or non-hormonal therapy. 
The results of the assay are interpreted as follows: 
1. Patients whose cancer cells in flask B (in the presence of 10 .mu.g/ml 
estradiol) that have .ltoreq.50% growth (IC.sub.50) as compared to 
control, are considered sensitive to estrogens and are candidates for 
antiestrogen therapy. 
2. If estradiol (E.sub.2) in flask B either has no influence or stimulates 
cell growth, the tumor cells are considered not to be estrogen sensitive. 
This is paradoxical because growth of the estrogen sensitive cells would 
be expected to be stimulated by estradiol. Instead, growth stimulation 
here signals probable unresponsiveness to therapeutic estrogen 
deprivation. This paradoxical result is believed to be strictly an in 
vitro phenomenon. The high estrogen content of the flask (.mu.g/ml) as 
compared to (ng/ml) physiological concentrations of estrogen in the body 
apparently is responsible for this inhibition. 
3. If the cell growth is inhibited by exposure to estrogens in flask B, 
then an IC.sub.50 should also be observed in flasks C and D with A-007. If 
the cells are 100% sensitive to estradiol (no cell growth in flask B) and 
completely inhibited by low concentrations of A-007 (no growth in flask 
C), the patient should be treated by an antiestrogen therapy. Examples of 
such therapy include tamoxifen, A-007, glutethimide, or surgical removal 
of the ovaries or adrenal glands. 
4. If an IC.sub.50 is noted in flask B as well as in flasks C (A-007, 10 
.mu.g/ml) or D (A-007, 15 .mu.g/ml), the cells should be considered 
sensitive to non-hormonal cancer chemotherapy, as well as antiestrogen 
therapies. Examples of non-hormonal cancer chemotherapy include the CMF 
regimen (cyclophosphamide, methotrexate and 5-fluorouracil) as well as 
A-007 at cytotoxic doses. 
5. If the cells only show IC.sub.50 inhibition in flask C (A-007, 10 
.mu.g/ml) or D (A-007, 15 .mu.g/ml), but not in flask B (with estradiol), 
the cells should be considered sensitive only to non-hormonal 
chemotherapy. 
6. If the cells only show inhibition in flask E (A-007, 20 .mu.g/ml) and 
are not inhibited by estradiol (flask A), the cells are considered poorly 
sensitive to both antiestrogens and other types of chemotherapy. 
7. For cells such as colon and other cancers that are not dependent on 
estrogens for growth, flask B may be eliminated from use in the test 
system. 
Antiestrogenic/Cytotoxic Activities 
The use of estrogen and progesterone receptors to predict sensitivities of 
breast cancer to antiestrogen therapy has been a major step forward in 
cancer management, as recounted by McGuire, et al., Cancer Res. 37:637-9 
(1977); Powell, et al., Cancer Res. 39:1678-82 (1979); Clark, et al., 
Semin Oncol; 125:Suppl 1:20-5 (1988). Unfortunately, information regarding 
chemotherapy sensitivities and prognosis cannot be obtained from these 
studies. 
In accordance with the present invention, cancer cells are grown under 
physiological conditions to document viability and growth patterns. In the 
case of breast and ovarian cancers, which contain ER and PgR in up to 55% 
and 20% of cases, the influence of .mu.g/ml of estradiol are assayed. 
Natural estradiol and other estrogens in ng/ml quantities or less can 
stimulate the growth and replication of ER positive breast cancer 
resulting in well-defined stellate cells. Paradoxically .mu.g/ml 
concentrations of estradiol inhibit breast cancer growth. Breast cancer 
cells that are estrogen sensitive are stellate types of cells. The 
estrogen independent cells are small undifferentiated cells. The 
sensitivities of all the cultured tissues to A-007 or one of the described 
hydrazones are evaluated. 
The in vitro data for breast cancer, ovarian cancer and colon cancer are 
provided in Tables II, III and IV. This data illustrates that even though 
agents other than the hydrazones are not routinely included in the culture 
assays, the use of A-007 predicts patient response to agents such as 
doxorubicin and 5-fluorouracil (5-FU). 
The data regarding results from breast cancer testing are: 
TABLE II 
__________________________________________________________________________ 
Breast Cancer Cases 
Specimen 
(+&gt;19)ER* 
(+&gt;10)PgR* 
(Flask A)Control** 
(10 .mu.g/ml)E.sub.2 *** 
##STR4## 
__________________________________________________________________________ 
1 &lt;4 &lt;3 100% 100% 5 
2 54 43 50% 10% 1 
3 82 346 0% 30% 3 
4 59 378 75% 10% 3 
5 121 179 100% 20% 3 
6 82 289 100% 100% &lt;5 
7 796 3418 10% 0% &lt;1 
8 440 998 100% 10% 5 
9 62 67 100% 10% 3 
10 35 80 85% 10% 4 
11 289 256 75% 65% 3 
12 &gt;450 89 10% 90% 3 
13 171 497 50% 5% 3 
14 214 201 50% 90% 2 
15 9 14 35% 100% &lt;1 
16 &lt;5 &lt; 3 100% 100% 10 
17 30 &lt;3 100% 100% 10 
18 &lt;5 &lt;3 100% 90% 12 
19 &lt;4 &lt;3 100% 100% 15 
20 &lt;5 &lt;3 90% 87% 15 
21 &lt;5 &lt;3 100% 100% 15 
22 73 57 100% 100% 12 
23 19 7 100% 95% 15 
24 &lt;3 &lt;5 100% 100% &gt;10 
25 
26 17 45 100% 100% 20 
27 &lt;5 &lt;3 100% 100% 17 
28 16 106 50% 65% 18 
29 &lt;5 &lt;3 100% 80% &gt;20 
__________________________________________________________________________ 
*ER and PgR: Receptor values in fmol/.mu.g protein; ER &gt; 5 and PgR &gt; 3 ar 
considered positive for estrogen sensitivity by most laboratories. 
**Control: 10 cells or clumps/HPF = 100% growth 
***Estradiol: 10 cells or clumps/HPF = 100% growth 
****IC.sub.50 : Conc. of A007 or another hydrazone required to inhibit 50 
of cancer cell growth. 
Data regarding ovarian cancer cells are: 
TABLE III 
______________________________________ 
Ovarian Cancer Cells 
Specimen 
Growth(Flask A)Control* 
Growth(10 .mu.g/ml)E.sub.2 ** 
##STR5## 
______________________________________ 
30 100% 100% 17 
31 100% 95% 10 
32 60% 55% 8 
33 50% 50% 7 
34 30% 25% 18 
35 100% 90% 15 
36 100% 100% 15 
37 0% 100% 15 
38 100% 100% 17 
39 100% 100% 12 
40 100% 100% 7 
41 100% 100% 6 
______________________________________ 
*Control: 10 cells or clumps/HPF = 100% growth 
**Estradiol: 10 cells or clumps/HPF = 100% growth 
***IC.sub.50 : Conc. of A007 or another hydrazone required to inhibit 50% 
of cancer cell growth. 
Data regarding inhibition of colon cancer cells in the test system is 
presented in TABLE IV: 
TABLE IV 
______________________________________ 
Colon Cancer Cells 
Control* A-007 (.mu./ml)** 
Specimen (Flask A) IC.sub.50 
______________________________________ 
42 100% 6 
43 100% 7 
44 100% 6 
45 100% 15 
46 100% &gt;20 
47 100% &gt;20 
48 50% 17 
49 100% &gt;20 
50 100% &gt;20 
51 100% 17 
52 100% 15 
______________________________________ 
*Control: 10 cells or clumps/HPF = 100% growth 
**IC.sub.50 : Conc. of A007 or another hydrazone required to inhibit 50% 
of cell growth. 
METHOD OF USE OF THE TEST SYSTEM 
Breast Cancer 
Case 1 (Specimen 22): 
A 73 year old white female with advanced breast cancer that was already 
metastatic to bone had ER/PgR values of 73/57. These values would 
conventionally be taken to indicate that the tumor is estrogen dependent, 
hence the patient was treated with the antiestrogenic drug tamoxifen. Her 
tumor enlarged, however, while on tamoxifen therapy, and she required 
chemotherapy (doxorubicin, 5-fluorouracil and cyclophosphamide). On 
chemotherapy, the patient's condition improved. Her tumor tissue's 
IC.sub.50 =12 with A-007 and growth in estradiol was 100%. Hence these 
values would have predicted that her tumor would be insensitive to 
treatment with an antiestrogen (such as tamoxifen) but would respond to 
cytotoxic agents such as doxorubicin, 5-fluorouracil and/or 
cyclophosphamide. It is notable that inhibition of cell growth in the 
presence of A-007 does not merely predict tumor sensitivity to A-007, but 
instead is a marker for sensitivity to cytotoxic agents. 
Case 2 (Specimen 26): 
A 76 year old black female with advanced breast cancer had an ER/PgR of 
17/45 and tumor spread to the chest wall. In view of her "positive" 
receptor status, she was treated with tamoxifen and did not respond. 
Patient 26 was then treated with 5-fluorouracil, methotrexate and 
cyclophosphamide. She did not respond and expired. Her tissue results 
indicated no response to estrogens or to chemotherapy. This outcome would 
have been predicted (in spite of her "positive" receptor status) by an 
IC.sub.50 =20 in A-007 and no growth suppression by estradiol. Hence the 
method of the present invention could have predicted a poor prognosis, in 
advance of chemotherapy, that may have averted an unfruitful course of 
drug treatment with its attendant side effects. This contrasts with the 
prior art approach of treating all "receptor positive" patients with 
hormone and/or chemotherapy. 
Case 3 (Specimen 16): 
A 68 year old white female had severe breast cancer spread to her chest 
wall and lung and a "negative" receptor status (ER/PgR &lt;5/3). Her tissue 
demonstrated no growth inhibition to estradiol and an IC.sub.50 of 10 
.mu.g/ml in A-007. The absence of growth inhibition in estradiol confirms 
the negative receptor status, but an IC.sub.50 .mu.g/ml in A-007 predicted 
absence of multi-drug resistance to cytotoxic agents. This patient was 
treated with doxorubicin and had a complete response that has lasted for 
six months. 
Case 4 (Specimen 27): 
A 66 year old white female with breast cancer spread to axillary lymph 
nodes was not considered a candidate for tamoxifen (ER/PgR=&lt;5/&lt;3). She was 
treated with cyclophosphamide, methotrexate and 5-fluorouracil post 
surgery. She failed with aggressive recurrence in ten months and expired. 
Her tissue demonstrated an IC.sub.50 =17 for A-007 and no growth 
inhibition in the presence of estradiol. The test assays would have 
predicted multi-drug resistance to cytotoxic agents, as was observed in 
fact. 
Ovarian Cancer 
Case 5 (Specimen 31): 
A 65 year old female with advanced ovarian cancer spread to the abdomen was 
treated with doxorubicin. She had an impressive response after one 
treatment. Her tissue had an IC.sub.50 =10 in A-007, but only 5% growth 
inhibition in the presence of E.sub.2. Hence a trial of antiestrogen 
therapy would have probably been unsuccessful, while the low IC.sub.50 
predicted the response to doxorubicin and other agents. 
Case 6 (Specimen 33): 
A 65 year old black female with advanced unresectable ovarian cancer was 
treated with paraplatin (Carboplatin). She has undergone a complete 
remission and is doing well. Her cancer tissue had an IC.sub.50 of 7 in 
A-007, but no growth inhibition was observed with E.sub.2 (the 50% growth 
with E.sub.2 was the same as the 50% growth in control flask A). The low 
IC.sub.50 with A-007 predicted response to non-hormonal chemotherapy. 
Case 7 (Specimen 30): 
A 68 year old white female with advanced ovarian cancer was treated 
aggressively with doxorubicin and paraplatin (Carboplatin) with no 
response and she expired. Her tissue had an IC.sub.50 =17 in A-007 and was 
not inhibited by estradiol. These tissue findings support the treatment 
outcome, which was characterized by multi-drug resistance. 
Colon Cancer 
Case 8 (Specimen 48): 
A 50 year old white male with advanced colon cancer was treated with 
5-fluorouracil. His tumor did not demonstrate any response and required 
exploration and surgery. His tissue demonstrated an IC.sub.50 =17 in 
A-007. This relatively high IC.sub.50 is consistent with multi-drug 
resistance to cytotoxic agents. 
Case 9 (Specimen 43): 
A 75 year old white female with advanced colon cancer was treated with 
5-fluorouracil and has done well. Her cancer tissue demonstrated an 
IC.sub.50 =7 in A-007, which is consistent with her observed response to 
the cytotoxic agent. Hence the IC.sub.50 of A-007 predicts tumor 
susceptibility to agents other than A-007. 
A-007 and the other disclosed nitrophenylhydrazones are useful in 
confirming sensitivity to antiestrogenic agents that is separately 
suggested by culture inhibition in the presence of .mu.g/ml concentrations 
of estradiol. The nitrophenylhydrazones are also helpful in predicting 
tumor response to non-hormonal cytotoxic agents. This dual predictive 
capacity is believed to be due to the hydrazones' bipotential effect on 
tumor cells. These hydrazones demonstrate both an antiestrogenic mechanism 
of action at lower doses and a direct cytotoxic action at higher doses. 
The direct cytotoxic action begins to predominate at concentrations of 10 
.mu.g/ml for A-007. 
Definitions 
In summary, the foregoing examples and data illustrate a method of 
predicting response of a tumor patient to therapy. As used herein, the 
term "tumor patient" refers to a person having a neoplasm, particularly a 
malignant neoplasm and, in most especially preferred embodiments, a 
hormone dependent neoplasm, such as ovarian or breast cancer. In one 
embodiment, the method includes culturing a first sample of the cells in 
the presence of a sufficient amount of an estrogen to inhibit growth of 
estrogen sensitive cells in the culture. The term "culturing" implies that 
the first sample includes a culture medium suitable for allowing growth of 
the tumor cells, such as RPMI-1640 containing 10% FBS 100 .mu.g/ml 
streptomycin and 100 units/ml penicillin. The term "sufficient amount of 
an estrogen to inhibit growth of estrogen sensitive cells" refers to an 
amount of estrogen which decreases the number of cells per high power 
field to below the number of cells/HPF observed in a control culture. 
A second sample of the cells is cultured in the presence of a sufficient 
amount of a cancer chemotherapeutic agent to inhibit growth of cells 
sensitive to the agent. As used herein, a cancer chemotherapeutic agent 
includes any drug useful in treating malignant neoplasms, and includes 
such substances as methotrexate, vincristine, 5-fluorouracil, 
cis-diamminedichloroplatinum, doxorubicin, cyclophosphamide and others. 
Inhibition of cell growth, in this context, is determined by a reduction 
in the number of cells compared to a control. In one specific embodiment 
disclosed herein, wherein the control contains 10 cells/HPF, inhibition is 
indicated by a reduction of cell number to less than 10 cells/HPF, most 
preferably less than 5 cells/HPF. 
The degree of inhibition of cell growth in each cultured sample is 
quantitated in accordance with this method. Such quantitation can take the 
form of counting the number of cells per high power field in each cultured 
sample. A particularly useful form of quantitation is to determine the 
IC.sub.50 of each sample, or the dose needed to inhibit the growth of 
.gtoreq.50% of the cells. 
The term "estrogen" refers to a substance that tends to promote estrus and 
stimulate the development of female secondary sexual characteristics. 
Included in the scope of this term are estradiol, estrone, estriol, and 
other substances. In particular disclosed embodiments, cells are exposed 
to supraphysiologic amounts of estradiol in culture to determine tumor 
cell sensitivity to antiestrogenic drugs. Supraphysiologic amounts are 
those above the basal level seen in a normal human female. An example of a 
supraphysiologic amount is a concentration in the (.mu.g/ml) range, or 
greater. Supraphysiologic amounts of estrone, estriol or other estrogens 
are easily determined, and their inhibitory concentrations in culture with 
estrogen dependent tumor cells are easily determined without undue 
experimentation. 
The assay of the present invention preferably employs a chemotherapeutic 
agent that has antiestrogenic properties, such as tamoxifen. More 
preferably, the antiestrogenic agent also has cytotoxic properties, as 
with the nitrophenylhydrazones of some embodiments of the present 
invention. Antiestrogenic properties refer to an ability to interfere with 
the interaction between estrogen and estrogen receptors in cells. 
Cytotoxic properties refer to a specific toxic action upon a cell, and not 
merely interference with interaction between an estrogen receptor and its 
ligand. 
The present method further includes selecting a treatment modality based on 
the degree of inhibition of cell growth in culture. The term "selecting a 
treatment" can refer to a broad variety of activities, including 
recommending a course of treatment or actually treating a patient with a 
particular therapeutic modality. The term "treatment modality" includes 
such things as drug therapy, surgical therapy (for example, 
adrenalectomies and oophorectomies) or even refraining from treatment in 
cases of poor treatment prognosis. The term "antiestrogen therapy" refers 
to using antiestrogenic drugs (such as tamoxifen or nitrophenylhydrazones, 
or adrenal cortical suppressors such as glutethimide), or surgical 
interventions such as oophorectomies or adrenalectomies. 
Antiestrogen therapies are selected, in accordance with some embodiments of 
this invention, when the degree of inhibition of cell growth in the 
estrogen exposed sample is significant. Significant inhibition, in this 
context, refers to an inhibition of at least about 50%, or 5 or less 
cells/HPF compared to a 10 cell/HPF control. If cell growth in the 
estrogen exposed sample is stimulated or not inhibited, something other 
than an antiestrogen therapy is selected. Examples of such other therapies 
include doxorubicin, 5-FU, methotrexate, cyclophosphamide and other drugs 
which are not known solely to interfere with estrogen receptors as a 
principal mechanism of action. Antiestrogen therapy also includes 
treatment with drugs, such as A-007 and other nitrophenylhydrazones, that 
possess both antiestrogenic properties and direct cytotoxic activity. 
Differential exhibition of antiestrogenic and cytotoxic properties may 
occur at different dosage ranges, for example, a cytotoxic mechanism of 
action may predominate at higher doses while an antiestrogenic mechanism 
may be most evident at lower doses. 
In other embodiments, an antiestrogen treatment modality is selected if the 
degree of inhibition of cell growth in both the estrogen exposed and drug 
exposed culture is substantially complete. A substantially complete degree 
of inhibition is, for example, at least a 50% inhibition, most preferably 
90-100% inhibition. 
In other embodiments of the invention, either an antiestrogenic or 
non-hormonal therapy is provided for the patient if cell growth in the 
first and second sample is partially inhibited. The term "partially 
inhibited" refers to an inhibition below the level of growth seen in the 
control culture. Such inhibition is at least about 10%, preferably at 
least about 50%, or between 10% and 50%. 
A disclosed embodiment of the invention uses A-007 in culture 
concentrations of 10 .mu.g/ml, 15 .mu.g/ml and 20 .mu.g/ml. These culture 
concentrations are "calibrated" such that an IC.sub.50 at 10 .mu.g/ml 
indicates a high degree of tumor susceptibility to a broad variety of 
chemotherapeutic agents. An IC.sub.50 at 15 .mu.g/ml indicates an 
intermediate likelihood of response to chemotherapy, while an IC.sub.50 
above 15 .mu.g/ml suggests multi-drug resistance and a poor prognosis, 
even with chemotherapy. It is a particular advantage of A-007 that it is 
so finely calibrated to predict therapeutic outcome. These precise 
calibrations, if they exist, have not yet been found for some of the other 
hydrazones or other chemotherapeutic agents, such as doxorubicin or 
methotrexate. Grosser indications of culture growth inhibitions are used 
with these agents, for example any inhibition of growth compared to 
controls. The method is still useful with these other drugs, but A-007 is 
a particularly preferred embodiment because of its high degree of 
calibration. 
The antiestrogenic hydrazones (of which A-007 is a paradigm) contain 
substituents R.sup.1, R.sup.2 and X. R.sup.1 is preferably a water soluble 
group, and can include hydrogen, hydroxy, succinate, esters or other 
hydrophilic groups or hydrogen bonding groups. Substituent R.sup.2 is a 
phenyl or substituted phenyl, preferably a hydroxy or ester substituted 
phenyl, wherein the ester substitution is preferably acetate or succinate. 
Substituent X is phenyl or substituted phenyl, wherein X is preferably 
nitro or dinitro substituted. 
Having illustrated and described the principles of the invention in a 
preferred embodiment, it should be apparent to those skilled in the art 
that the invention can be modified in arrangement in detail without 
departing from such principles. I claim all modifications coming within 
the spirit and scope of the following claims.