Abstrict
The present invention involves a method for early detection of
developing tamoxifen resistance in breast cancer. Tamoxifen is the
drug of choice for hormonal therapy of a first recurrence of breast
cancer, but its use is always associated eventually with emergence
of resistant tumors. Whereas initial treatment is usually followed
by tumor regression, resistant tumors may actually resume growth
under continued tamoxifen treatment. Because such growth may actually
be augmented by the tamoxifen, it is essential to identify the onset
of resistance as early as possible so alternative therapy may be
promptly instituted.
Claims
What is claimed is:
1. A method for detecting in vivo development of tamoxifen-resistant
breast tumors comprising:
obtaining ratios of cis-4-hydroxy-tamosifen concentration to trans-4-hydroxy-tamoxifen
concentration in tissue samples from a breast cancer patient being
subjected to a course of tamoxifen treatment; and
following said ratios during the course of tamoxifen treatment
to determine onset of treatment resistance as characterized by an
increase in the ratio of cis-4-hydroxy-tamoxifen concentration to
trans-4-hydroxy-tamoxifen concentration.
2. The method according to claim 1 wherein said tissue is breast
tumor.
3. The method according to claim 1 wherein said tissue is blood.
4. A method for detecting in vivo development of tamoxifen-resistant
breast tumors comprising:
obtaining breast tumor tamoxifen concentration in a patient during
a course of tamoxifen treatment; and
following said concentration during the course of tamoxifen treatment
to determine onset of treatment resistance as characterized by a
decrease in said tamoxifen concentration.
5. A method for detecting in vivo development of tamoxifen-resistant
breast tumors comprising:
obtaining breast tumor tamoxifen concentrations and tissue ratios
of cis-4-hydroxy-tamoxifen concentration to trans-4-hydroxy-tamoxifen
concentration in a patient being subjected to a course of tamoxifen
treatment; and
following said tamoxifen concentrations and ratios during the course
of tamoxifen treatment to determine onset of treatment resistance
as characterized by a decrease in tamoxifen concentration and an
increase in the ratio of cis-4-hydroxy-tamoxifen concentration to
trans-4-hydroxy-tamoxifen concentration.
6. The method according to claim 5 wherein said tissue is breast
tumor.
7. The method according to claim 5 wherein said tissue is blood.
8. A method for detecting in vivo development of resistance in
breast tumors to treatment by a triphenylethylene estrogen comprising:
obtaining concentration ratios tissue of cis to trans forms of
triphenylethylene antiestrogen in a patient being subjected to a
course of treatment with a triphenylethylene antiestrogen; and
following said ratios during the course of treatment to determine
onset of treatment resistance as characterized by an increase in
the ratio of concentration of cis to trans forms of said triphenylethylene
antiestrogen.
9. The method according to claim 8 wherein said tissue is breast
tumor.
10. The method according to claim 8 wherein said tissue is blood.
11. The method according to claim 1, 5 or 8 wherein said obtaining
step comprises photoactivation of a tissue sample extract.
12. The method of claim 11 wherein the photoactivation involves
irradiation with ultraviolet light.
13. The method of claim 1, 5 or 8 wherein the obtaining step comprises
high performance liquid chromatography.
14. A method for detecting in vivo development of tamoxifen-resistant
breast tumors in a breast cancer patient being subjected to a course
of tamoxifen treatment, the method comprising:
obtaining tissue ratios of cis-4-hydroxy-tamoxifen concentration
to trans-4-hydroxy-tamoxifen concentration in said patient; and
following said ratios during the course of tamoxifen treatment
to determine onset of treatment resistance as characterized by an
increase in the ratio of cis-4-hydroxy-tamoxifen concentration to
trans-4-hydroxy-tamoxifen concentration.
Description BACKGROUND OF THE INVENTION
The present invention relates to methods for monitoring the effectiveness
of tamoxifen (2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine)
therapy in the treatment of breast cancer, and more particularly
to methods of detecting the emergence of tamoxifen-resistant tumors
and the resulting treatment resistance by means of an analytical
assay for antiestrogenic and other forms of tamoxifen and its metabolites.
Breast cancer is the most common form of malignant disease among
women of the Western world, and it is the most common cause of death
among those between 40 and 45 years of age. This disease will develop
in about six to seven percent of women in the United States, and
at the present time about one half of this group can be cured. The
treatment of breast cancer involves surgery, radiation, chemotherapeutics
and hormonal therapy, the last category including consideration
of antiestrogens for treatment of endocrine-responsive tumors.
Tumors sensitive to estrogen stimulation may regress following
competitive inhibition of estrogen receptors by tamoxifen (an antiestrogen),
and response is currently predicted based on the stage of disease
and on the basis of assays for estrogen receptors (ER) and progesterone
receptors (PR) in the tissue. Many breast cancers and all normal
estrogen-responsive tissues contain these labile cytoplasmic proteins
which bind estrogen and progesterone. Patients with positive assays
for these proteins have an objective response to hormone therapy
of about 65%, while those with negative assays have an objective
response rate of <10%. For postmenopausal women having a first
recurrence of breast cancer with an ER+ or PR+ assay, tamoxifen
therapy is the treatment of choice.
Notwithstanding strong interest in the use of antiestrogens in
breast cancer treatment, however, incomplete knowledge of their
basic pharmacology persists. Substituted triphenylethylenes (including
tamoxifen) have antiestrogen effects which appear to be dependent
on geometric isomerism. For example, trans-tamoxifen (the isomer
used in tamoxifen therapy) is an antiestrogen, whereas the cis isomer
is a weak estrogen. The present invention makes use of newly acquired
knowledge about in vivo interconversion of geometric isomers to
improve treatment of breast cancer.
Although tamoxifen is the most widely used antiestrogen for treating
breast cancer, development of tamoxifen resistance and subsequent
tumor progression during tamoxifen therapy represents a major reason
for treatment failures. The mechanism of tamoxifen resistance has
been unknown, but an estrogenic metabolite of tamoxifen which would
promote growth in ER+ tamoxifen-resistant tumors has been identified.
SUMMARY OF THE INVENTION
The present invention relates to the use of tamoxifen metabolites
as early indicators of tamoxifen resistance (prior to clinical treatment
failure). Tamoxifen is representative of a group of compounds called
triphenylethylene antiestrogens, whose effect is to slow or stop
the growth of estrogen-dependent tumors. While it is the most commonly
used drug for treatment of breast cancer today, tamoxifen is associated
with the development of drug resistance in virtually all patients
who take it. With the onset of resistance, tumor growth resumes
or accelerates and tamoxifen therapy should be discontinued. Renewed
tumor growth will eventually become obvious, of course, but an objective
of the present invention is to provide early warning of tamoxifen
resistance so that therapy can be changed promptly as needed.
The mechanism of tamoxifen resistance is unknown, but it is known
that tamoxifen exists as two geometric isomers, the trans form (an
antiestrogen) and the cis form (a weak estrogen). The trans form,
of course, is the therapeutic drug for breast cancer. Further, however,
it has been shown that there can be interconversion from one isomer
to the other in tissue culture cells and also, presumably, in patients
(though this has not been demonstrated).
The model with which the present invention was developed consists
of human breast cancer cells growing subcutaneously in athymic nude
mice. Treatment of the mice with tamoxifen results in tumor growth
inhibition for four to six months, followed by the onset of tumor
resistance and regrowth (much as in humans). Further, tumor regrowth
is actually stimulated by tamoxifen. Even when transplanted into
different mice, the resistant tumors will not grow unless stimulated
by tamoxifen or estrogen. Tamoxifen, in these cases, appears to
mimic the action of estrogen.
One of the possible mechanisms for development of tamoxifen resistance
is hypothesized to be conversion over time of both tamoxifen and
its metabolites from trans (antiestrogen) forms to cis forms which
have estrogen-like activity. Assays for the presence of both trans
and cis forms can be accomplished with a high performance liquid
chromatography (HPLC) system. In three separate blinded experiments
using such a system, it has been shown that:
1) tumors from tamoxifen-resistant mice have significantly lower
concentrations of tamoxifen than sensitive tumors (whose growth
is still arrested); and
2) there is an increase in the cis-4-hydroxytamoxifen/trans-4-hydroxytamoxifen
(C/T-OH-TAM) ratio in resistant tumors.
Actual values of the C/T-OH-TAM ratio are about 0.4-0.5 in sensitive
tumors and 0.8-0.9 in resistant tumors. While it can not be confirmed
that the increased estrogen effect resulting from an elevated C/T-OH-TAM
ratio is the reason for development of resistance, the association
appears to be consistent.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows HPLC tracings of 400 ng/ml stock solutions of trans
and cis 4-hydroxytamoxifen (4-OH-TAM), respectively.
FIG. 2 shows HPLC chromatographs indicating that the cis/trans
ratio of 4-OH-TAM is lower in sensitive than in resistant tumors
isolated from athymic nude mice.
FIG. 3 shows the approximately linear inverse relationship which
exists between the logarithm of total tumor tamoxifen concentration
and the cis/trans 4-OH-TAM ratio (N=32,R=0.83, linear regression
analysis).
FIG. 4 shows a scatter plot of tamoxifen concentrations in sensitive
and resistant MCF-7 tumors (N=32).
FIG. 5 shows a scatter plot of 4-OH-TAM ratios (cis/trans) in sensitive
and resistant MCF-7 tumors (N=32).
FIG. 6 shows a scatter plot of tamoxifen versus cis/trans 4-OH-TAM
ratios in resistant (open symbols) and sensitive (solid symbols)
tumors.
FIG. 7 shows HPLC chromatographs of a resistant tumor analyzed
for total tumor tamoxifen, cytosol tamoxifen, and tamoxifen found
in nuclear pellets extracted with KC1 (from top to bottom, respectively).
FIG. 8 shows example HPLC chromatographs demonstrating cis >trans
concentrations of 4-OH-TAM extracted from tumor nuclear pellets
representing patients with clinically-evident tumor resistance.
These findings must be contrasted with analogous measurements for
tamoxifen-sensitive tumors in the mouse model (showing trans >cis
4-OH-TAM) because sensitive tumors in human patients are not biopsied
and the information is not available.
FIG. 9 shows example HPLC chromatographs demonstrating detection
of tamoxifen, des-tamoxifen, and cis and trans 4-OHTAM following
treatment with 20 mg tamoxifen per day. These measurements demonstrate
the capability to measure tamoxifen and its metabolites in the serum.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Those skilled in the art will recognize that changes analogous
to those detected in the C/T-OH-TAM ratio might occur in cis-tamoxifen
or other (monophenol and bisphenol) tamoxifen metabolites or in
any of the triphenylethylene antiestrogens related to tamoxifen.
Nevertheless, the best mode of implementing the present invention
now appears to be in conjunction with treatment of estrogen-responsive
breast cancer with tamoxifen and detection of the onset of tamoxifen
resistance.
Those skilled in the art will also recognize that if geometric
isomerization or metabolism to other estrogenic metabolites proves
to be the mechanism of antiestrogen resistance, synthesis of antiestrogens
which preclude isomerization may be a fruitful path to improved
treatment. At the present time, however, tamoxifen is the drug of
choice for hormonal treatment of recurrent breast cancer, and its
invariable association with the onset of tumor resistance and tamoxifen-enhanced
tumor growth makes the present invention a valuable aid to therapy.
Claims are directed to the two main indicators which have been
discovered to be closely associated with the onset of tumor tamoxifen
resistance. Elevation of the C/T ratio for 4-OH-TAM in tumor or
blood, and depression of the tamoxifen concentration in tumor are
both shown by experimental data to be useful predictors of recrudescence
of tumor growth despite (and perhaps in-part because of) tamoxifen
treatment. Either indicator can be used alone, or they may be used
together.
For each indicator, small changes (about a few percent) in the
direction indicated by this specification and claims are believed
to indicate the early stages of tumor resistance. Larger indicator
changes (up to about 80 to 100 percent) can be expected when tumor
resistance is well established. Examples of in vitro and in vivo
tests are provided to illustrate the specific utility of the claimed
invention and to support both the approximate magnitude of indicator
changes and the extrapolations suggested in this specification.
These examples are presented to describe preferred embodiments
and utilities of the present invention and are not meant to limit
the present invention unless otherwise stated in the claims appended
hereto. Taken together, the examples illustrate the best mode of
implementing the invention as it is currently understood. |