WO2011130381A1

WO2011130381A1 - Diagnostic assays for breast cancer treatment and progression

Info

Publication number: WO2011130381A1
Application number: PCT/US2011/032277
Authority: WO
Inventors: David Allen Ralph; Gary Dean; Sohaib Khan
Original assignee: Estrocept Diagnostics, Inc.
Priority date: 2010-04-13
Filing date: 2011-04-13
Publication date: 2011-10-20

Abstract

The present invention provides for assays that identify women with estrogen receptor alpha expressing breast tumors (ER+ tumors) that are best treated with aromatase inhibitors instead of specific estrogen receptor modulators (SERMs) such as tamoxifen (TAM). In one embodiment, the invention provides for the use of an H222 ELISA to identify those postmenopausal women with ER+ breast tumors that must be treated with aromatase inhibitors because treatment with tamoxifen or other similarly acting SERM will be ineffective.

Description

DIAGNOSTIC ASSAYS FOR BREAST CANCER TREATMENT AND

PROGRESSION

Cross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application

Serial No. 61/323,517, filed April 13, 2011 , which application is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] The human estrogen receptor alpha (ERoc or just ER) was affinity purified from the human breast cancer cell line MCF7 and used to immunize Lewis rats at the University of Chicago in the laboratory of Elwood Jensen. This work was performed in the early 1980's as part of a collaborative effort involving Abbott Laboratories. Geoffrey Green, working in the Jensen laboratory, fused splenic lymphocytes from these immunized rats with cells from mouse myeloma cells to create a library of thirteen hybridoma cell lines that produced monoclonal antibodies recognizing epitopes on the ER protein (1).

[0003] One of these hybridomas was named H222. Another was named D547.

Hereafter, H222 and D547 may be used to refer to either the cell lines or the monoclonal antibodies that they secrete. Abbott Laboratories used H222 and D547 to create two immunoassays that were commercially distributed until 2000. One of these was an immunohistochemical (IHC) stain that determined what portion of cells in a patient's breast cancer tumor that (over) expressed the estrogen receptor. Breast tumors containing cells that over express ER are said to be ER positive (ER+). The other immunoassay was an ELISA that determined the amount of estrogen receptor present in a lysate made from a fragment of a patient's tumor. This ELISA used D547 as the capture antibody and then used a peroxidase labeled H222 antibody to detect the captured ER. In this disclosure this assay will be called the H222 ELISA. Prior to 2000, both Abbott's IHC and H222 ELISA tests were in common usage to determine if women's breast cancer over expressed ER with those women with ER+ tumors frequently being offered adjuvant hormone therapy for their cancers. Soon after the H222 ELISA assay was developed, an unexpected and highly interesting behavior of H222 in the ELISA assay was discovered (2, 3). This discovery was that in the presence of added estrogens, tamoxifen (Tarn) and other selective estrogen receptor modulators (SERMs) the amount of H222 that became bound to D547 captured ER increased dramatically. This enhanced H222 binding occurred to differing extents in tumor lysates derived from different patients. For some patients' tumor lysates, the addition of Tarn increased H222 binding by two or three fold. For other patients' tumor lysates, addition of Tam resulted in little or no increase in the binding of H222. Approximately one quarter of ER+ breast tumors fall into this second class in which Tam has minimal effect on H222 binding. These observations are interesting because SERMs and especially tamoxifen are frequently used as adjuvant therapies for ER+ breast cancers. SERMs interact with ER and block its ability to signal in breast cancer cells and thus prevent ER from promoting cancer cell growth in ER+ tumors. It has long been known that adjuvant tamoxifen therapy of ER+ breast cancers reduces the rate of disease recurrence. In addition in 2000, Naundorf et al (4) published findings from a small clinical study of women with ER+ tumors treated with adjuvant tamoxifen. This study showed that those women with tumors

demonstrating tamoxifen mediated increases in H222 binding were less likely to suffer a disease recurrence than those women without increased tamoxifen mediated H222 binding. Specifically, 20% of women with tumors demonstrating increased Tam mediated H222 binding experienced a disease recurrence while 50% of women with tumors not showing a Tam mediated increase in H222 binding recurred. In 2000 when this paper was published, women with ER+ tumors were frequently offered adjuvant tamoxifen therapy. This finding provided preliminary evidence that by screening tumors for increased tamoxifen mediated H222 binding, it would be possible to determine in advance of treatment which women would most likely benefit from tamoxifen therapy by comparing the results of the Abbott H222 ELISA test performed with and without tamoxifen. These results were not followed up with a larger clinical study possibly due to the withdrawal from the market of the ELISA test by Abbott Laboratories in 2000. Another event that occurred in the 1990's was the introduction on the pharmaceutical market of aromatase inhibitors (AI) for use as an alternative to tamoxifen for hormonal adjuvant therapy of ER+ breast cancers. Aromatase is an enzyme that converts androstenedione to estrone and testosterone to estradiol. Postmenopausal women produce modest amounts of estrogens from androgens via this aromatase pathway. In ER+ postmenopausal breast cancers, this modest amount of estrogens enables sufficient signaling through the ER to promote cancer cell growth and replication. By blocking this estrogen synthesis, aromatase inhibitors will starve ER for estrogens in postmenopausal women and prevent ER from promoting cancer growth. Several large studies (reviewed in 5) have shown that the aromatase inhibitor, anastrozole, when administered as an adjuvant hormone therapy in postmenopausal women with ER+ breast cancers, is more effective than tomoxifen in preventing disease recurrence. The hazard ratio for disease recurrence for postmenopausal ER+ women treated with anastrozole compared to tamoxifen is about 0.77 but might be lower under certain circumstances. As a result, there has been a recent shift away from tamoxifen and other SERMs and towards the use of anastrozole and other AI for the adjuvant treatment of postmenopausal ER+ breast cancer. The interactions between tamoxifen and other SERMs with the estrogen receptor are more complex than the previous discussion may have indicated. Tamoxifen and several other SERMs can act both as estrogen receptor agonists or antagonists in the same patient depending upon which tissue is being examined. In some cell types, as for example most ER+ breast cancers, tamoxifen acts as an estrogen receptor antagonist preventing estrogen mediated cell growth and division. In other tissues, as for example the endometrium and bone, tamoxifen acts as an agonist promoting signaling through the estrogen receptor. This creates an interesting set of side effects associated with long term use of tamoxifen and some other SERMs, which include a modest elevation in the risks for endometrial cancer and thromboembolic disease. Conversely, tamoxifen and some other SERMs can slow and possibly reverse the progression of osteoporosis, a leading cause of morbidity and mortality in

postmenopausal women. In fact, one SERM, raloxifene, has been approved by the FDA for the treatment and prevention of osteoporosis. Aromatase inhibitors are not associated with either endometrial cancer or thromboembolic disease. On the other hand, aromatase inhibitors have their own set of adverse side effects, which are consequences of the extreme estrogen deprivation they induce in postmenopausal women. Among these side effects is the loss of bone mineral density, as in osteoporosis, and a concurrent modest but significant increase in fractures. Women on aromatase inhibitors have a higher incidence of bone or joint pain, which in some patients can be severe. Aromatase inhibitors are also associated with increases in serum cholesterol, but a possible derivative association with cardiovascular disease has not yet been demonstrated. The adverse side effect profiles of tamoxifen and aromatase inhibitors have been extensively reviewed (for example see 6).

[0007] Currently, aromatase inhibitors are displacing tamoxifen as the preferred adjuvant hormone therapy for postmenopausal women with ER+ breast tumors. This shift is largely being driven by the improved suppression of disease recurrence in this patient population afforded by aromatase inhibitors. It is unclear that aromatase inhibitors would be the preferred treatment in all cases if tamoxifen were equally effective as aromatase inhibitors in suppressing disease recurrence. This may be especially true for women who have osteopenia, osteoporosis, have high levels of serum cholesterol, have had a hysterectomy or are experiencing severe joint or bone pain as a result of taking aromatase inhibitors. The optimal treatment for women with ER+ breast cancers may depend on determining if their cancers are best prevented from recurring with an aromatase inhibitor or if their adjuvant hormone therapy would be just as effective with tamoxifen (or similarly acting SERM).

[0008] The key to directing women with ER+ breast cancer towards their most effective adjuvant hormonal therapies with the least undesirable side effects may be facilitated through a reinterpretation of the results presented by Naundorf et al. (4). While their results were preliminary, Naundorf and colleagues differentiated the subset of ER+ breast cancer patients that benefit from adjuvant therapy with tamoxifen from those that do not. They examined both pre- and post-menopausal patients and did not anticipate the impact of aromatase inhibitors on the development of adjuvant breast cancer therapy. Reevaluation of their results within the context of what is now known about the relative efficacies of SERMs and AI for the prevention of recurrent breast cancer in postmenopausal patients with ER+ disease indicate that the H222 ELISA test evaluated by Naudorf et al has an application or utility not realized by these authors. It is evident that this H222 ELISA test performed with and without tamoxifen can be used to identify postmenopausal women that must be treated with aromatase inhibitors and should not be treated with tamoxifen or similarly acting SERM. From previous studies (7), it is known that the five year recurrence free survival of lymph node negative ER+ tumors treated surgically but not given adjuvant therapy was approximately 70%. If this same population of patients were given tamoxifen, then the five year disease free survival is approximately 83%. Stated differently, the recurrence rate of an untreated population is approximately 30%, which can be reduced to 17%o with adjuvant tamoxifen hormonal therapy. More recent trials that have compared the efficacy of tamoxifen with aromatase inhibitors alone or in combination (4, 8) but do not include an untreated control group; however, these studies show that the recurrence rate of ER+ postmenopausal breast cancer patients treated with aromatase inhibitors is about 77% that of similar patients treated with tamoxifen. This difference in efficacy can be explained by the difference in the results of the H222 ELISA assay if those patients with ER+ tumors whose ER does not increase binding to the antibody in the presence of tamoxifen are responding to aromatase inhibitors. Thus, the H222 ELISA assay of the present invention is defining two classes of estrogen receptors: those that will respond equally well to either tamoxifen or aromatase (H222 binding increased by tamoxifen) and those that will only respond to the estrogen starvation brought on by aromatase inhibitors in postmenopusal women (H222 binding not increased by tamoxifen). The application the IT222 ELISA to identify postmenopausal breast cancer patients who must be treated with aromatase inhibitors is one of the inventions described herein.

[0010] BRIEF SUMMARY OF THE INVENTION

[0011] The present invention relates generally to the use of an assay that identifies subjects with estrogen receptor alpha expressing breast tumors (ER+ tumors) that are best treated with aromatase inhibitors instead of specific estrogen receptor modulators (SERMs) such as tamoxifen (TAM).

[0012] In another embodiment, the invention provides for a method for selecting a breast cancer patient for therapy comprising (a) determining whether the cancer comprises a tumor with estrogen receptor alpha expressing breast tumors (ER+ tumors) and (b) selecting the patient for therapy with an with aromatase inhibitors instead of specific estrogen receptor modulators (SERMs) only if the cancer is determined to comprise an ER+ tumor.

[0013] In another embodiment, the determination of presence of an ER+ tumor is made in a tissue sample of the patient by obtaining a positive result in an assay. In another embodiment, the assay is selected from the group consisting of ligand binding assays, immunohistochemical assays and combinations thereof. In another embodiment, the assay is an immunohistochemical assay using the monoclonal antibody, H222. In another embodiment, the method further comprises determining, for a tumor found to be ER+, whether the tumor is resistant or responsive to selective estrogen receptor modulator (SERM) treatment.

[0014] In another embodiment, the invention provides for a method for treating breast cancer in a patient, comprising (a) determining whether the cancer comprises a tumor with estrogen receptor alpha expressing breast tumors (ER+ tumors) and (b) selecting the patient for therapy with an with aromatase inhibitors instead of specific estrogen receptor modulators (SERMs) only if the cancer is determined to comprise an ER+ tumor; and (c) administering to the selected patient.

[0015] In another embodiment, the estrogen receptor alpha expressing breast tumors (ER+ tumors) are treated with an aromatase inhibitor. In another embodiment, the non-estrogen receptor alpha expressing breast tumors (ER- tumors) are treated with a specific estrogen receptor modulator. In another embodiment, the aromatase inhibitor comprising at least one compound selected from the group consisting of aminoglutethimide, anastrozole, exemestane, fadrozole, formestane, letrozole, vorozole, and pharmaceutically acceptable salts, prodrugs and active metabolites thereof. In another embodiment, the SERM comprising at least one compound selected from the group consisting of acolbifene, arzoxifene, bazedoxifene, droloxifene, HMR-3339, idoxifene, lasofoxifene, levormeloxifene, ospemifene, raloxifene, tamoxifen, toremifene, and pharmaceutically acceptable salts, prodrugs and active metabolites thereof.

[0016] In certain embodiments, the patient is female. In other embodiments, the patient is male. There are still further provided kits comprising therapeutic combinations as described above.

[0017] In another embodiment, the invention provides for the use of the monoclonal antibody, H222, in immunoassays that identify women with estrogen receptor alpha expressing breast tumors (ER+ tumors) that are best treated with aromatase inhibitors instead of specific estrogen receptor modulators (SERMs) such as tamoxifen (TAM).

[0018] In another embodiment, the invention provides for the use of an H222

ELISA to identify those postmenopausal women with ER+ breast tumors that must be treated with aromatase inhibitors because treatment with tamoxifen or other similarly acting SERM will be ineffective. [0019] In another embodiment, the invention provides for the use of an H222

ELISA to identify women with ER+ tumors that are more likely to fail hormonal adjuvant therapy in general regardless of whether they are treated with aromatase inhibitors or tamoxifen. These patients for whom hormonal therapy is likely to be ineffective can be directed towards other therapies that may decrease their chances of experiencing a disease recurrence. Conversely, postmenopausal patients with increased tamoxifen mediated binding of H222 would be given aromatase inhibitors. The H222 ELISA test would be used to better identify those women who would benefit from adjuvant therapy with aromatase inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates generally to the use of an assay that identifies women with estrogen receptor alpha expressing breast tumors (ER+ tumors) that are best treated with aromatase inhibitors instead of specific estrogen receptor modulators (SERMs) such as tamoxifen (TAM).

[0021 ] In another embodiment, the invention provides for a method for selecting a breast cancer patient for therapy comprising (a) determining whether the cancer comprises a tumor with estrogen receptor alpha expressing breast tumors (ER+ tumors) and (b) selecting the patient for therapy with an with aromatase inhibitors instead of specific estrogen receptor modulators (SERMs) only if the cancer is determined to comprise an ER+ tumor.

[0022] In another embodiment, the determination of presence of an ER+ tumor is made in a tissue sample of the patient by obtaining a positive result in an assay. In another embodiment, the assay is selected from the group consisting of ligand binding assays, immunohistochemical assays and combinations thereof. In another embodiment, the assay is an immunohistochemical assay using the monoclonal antibody, ESR1 Rat anti-Human Monoclonal (H222) Antibody (which is commercially available). H222 antibody reacts specifically with the ESR1 antigen (also known as ESR1, DKFZp686N23123, ER, er alpha, ER-alpha, Era, Eralpha, ESR, ESRA, Estrogen Receptor, Estrogen receptor alpha, NR3A1) In another embodiment, the method further comprises determining, for a tumor found to be ER+, whether the tumor is resistant or responsive to selective estrogen receptor modulator (SERM) treatment.

[0023] In another embodiment, the invention provides for a method for treating breast cancer in a patient, comprising (a) determining whether the cancer comprises a tumor with estrogen receptor alpha expressing breast tumors (ER+ tumors) and (b) selecting the patient for therapy with an with aromatase inhibitors instead of specific estrogen receptor modulators (SERMs) only if the cancer is determined to comprise an ER+ tumor; and (c) administering to the selected patient.

[0024] In another embodiment, the estrogen receptor alpha expressing breast tumors (ER+ tumors) are treated with an aromatase inhibitor. In another embodiment, the non-estrogen receptor alpha expressing breast tumors (ER- tumors) are treated with a specific estrogen receptor modulator. In another embodiment, the aromatase inhibitor comprising at least one compound selected from the group consisting of aminoglutethimide, anastrozole, exemestane, fadrozole, formestane, letrozole, vorozole, and pharmaceutically acceptable salts, prodrugs and active metabolites thereof. In another embodiment, the SERM comprising at least one compound selected from the group consisting of acolbifene, arzoxifene, bazedoxifene, droloxifene, HMR-3339, idoxifene, lasofoxifene, levormeloxifene, ospemifene, raloxifene, tamoxifen, toremifene, and pharmaceutically acceptable salts, prodrugs and active metabolites thereof. [0025] In certain embodiments, the patient is female. In other embodiments, the patient is male. There are still further provided kits comprising therapeutic combinations as described above.

[0026] In another embodiment, the invention provides for the use of the monoclonal antibody, H222, in immunoassays that identify women with estrogen receptor alpha expressing breast tumors (E + tumors) that are best treated with aromatase inhibitors instead of specific estrogen receptor modulators (SERMs) such as tamoxifen (TAM).

[0027] In another embodiment, the invention provides for the use of an H222

ELISA to identify those postmenopausal women with ER+ breast tumors that must be treated with aromatase inhibitors because treatment with tamoxifen or other similarly acting SERM will be ineffective.

[0028] In another embodiment, the invention provides for the use of an H222

[0029] In another embodiment, the H222 ELISA is used to identify ER+

postmenopausal women that would receive specific benefit from adjuvant hormonal therapy with aromatase inhibitors.

[0030] In another embodiment, the present invention is used to determine the same information that is obtained with H222 ELISA with a cytology based non-ELISA test format that provides the same information that is obtained with the IHC test format to determine which breast tumors are ER+.

[0031] The term "treat," "treating" or "treatment" herein includes preventive or prophylactic use of therapeutic agents in a subject at risk of, or having a prognosis including, breast cancer, as well as use of such an agent in a subject already experiencing breast cancer, as a therapy to alleviate, relieve, reduce intensity of or eliminate one or more symptoms of the disease or an underlying cause thereof. Thus treatment includes (a) preventing a condition or disease from occurring in a subject that may be predisposed to the condition or disease but in whom the condition or disease has not yet been diagnosed; (b) inhibiting the condition or disease, including retarding or arresting its development; and/or (c) relieving, alleviating or ameliorating the condition or disease, or primary or secondary signs and symptoms thereof, including promoting, inducing or maintaining remission of the disease.

[0032] The H222 ELISA assay described herein is used to analyze a sample of fresh or frozen breast tumor tissue by first creating a lysate and then binding the estrogen receptors with a capturing antibody. In existing protocols, the capture antibody has been D547 that has been attached to polystyrene beads, however; other capture antibodies could be substituted for D547 and the capture antibodies could be secured to any one of a number of alternative substrates. The captured estrogen receptor/capture antibody/polystyrene bead complex (ECP complex) is then washed with a buffer solution to remove unbound material. After washing, the detection antibody - H222 conjugated to some observable label - is added to the ECP complex. In standard protocols, the observable label is a peroxidase enzyme, but many alternatives such as but not limited to other enzymes or florescent molecules could be substituted for peroxidase. The H222 with its conjugated label will then bind to any captured estrogen receptor creating an immune complex consisting of the label, H222, an estrogen receptor molecule and the capture antibody (i.e. D547) all of which is attached to a support (i.e. polystyrene bead). The immune complex is then washed to remove any unbound H222 and label. The label is detected by adding the appropriate enzyme substrate or by exciting a florescent label. The amount of label detected is proportional to the amount of H222 that is bound to estrogen receptors. This ELISA assay is run in duplicate. One run is performed in presence of tamoxifen or other SERM while the other is run without any added tamoxifen or other SERM. In previously used protocols, tamoxifen was added to a concentration of 1.0 microMolar, however; other concentrations could be used. In Martin et al (2), the tamoxifen mediated increase in H222 binding was observed with either 50 nM tamoxifen or with 50 nM MHT (4-monohydroxytamoxifen). MHT is a more potent metabolic derivative of tamoxifen. This assay has two outputs. It can determine the extent of tamoxifen (or other SERM) mediated increase in H222 binding to estrogen receptors, and, if appropriately calibrated, can determine the amount or concentration of estrogen receptor protein present in the sample.

In another embodiment, the assay is performed using the H222 immunoassay with and without tamoxifen (or other SERM) on a cytology specimen rather than as an ELISA performed on tissue lysates. The cytology specimen is obtained from a fine needle aspirate, other biopsy technique or a "touch prep" or scraping of a resected tumor. The cells in this cytology specimen are attached to a microscope slide perhaps with the aid of a "Cytospin" or other common laboratory instrument. In another embodiment, the cells can be fixed and permeablized with either alcohol or methanol and/or picric acid using standard protocols. In another embodiment, the estrogen receptor is secured within the matrix of the cell. In another embodiment, the H222 antibody plus its conjugated label is the only antibody used. In another embodiment, the H222 antibody plus its conjugated label is used in combination with one or more additional types of antibodies. In one embodiment, the fixed cells in the cytology specimen are exposed or "stained" with the labeled H222 antibody. In one embodiment, the unbound antibody and label are washed away; then the label is detected by addition of the appropriate substrate or florescent excitation. In one embodiment, the assay is performed in duplicate: once with and once without the presence of tamoxifen (or other similarly acting SERM). In one embodiment, the average amount of H222 antibody bound to cells in each replicate as indicated by the amount of detected conjugated label is determined through image analysis. In one embodiment, the difference in the average amount of bound H222 in the tamoxifen treated and untreated replicates is a measure of the amount of tamoxifen mediated increase in H222 binding.

Those skilled in the art will realize that there may be some technical challenges related to uniformity of staining (H222 antibody binding) encountered when attempting to perform the simple version of the test as described in the previous paragraph. These may be overcome by performing the assay by adding a second antibody conjugated to a different label in the same reaction. This second antibody could recognize any one of many proteins or epitopes and serves the purpose of normalizing the amount of H222 detected to a uniform standard with the cells, thus mediating any issues related to uniformity of staining. For example, the second antibody could recognize a constitutively expressed protein (a protein expressed at a constant level regardless of physiologic state) such as perhaps GAPDH or tubulin. In this variation, the antibody recognizing the constitutively expressed protein and H222 would probably best be labeled with fiorescent tags that when excited emit light of different wavelengths permitting the binding of one antibody to be discriminated from the other. Again this assay would be performed in duplicate with and without tamoxifen (on similarly acting SERM). The amount of florescence associated with the binding of the two antibodies would be measured for numerous cells in both replicates (with and without tamoxifen). Numerous statistics could be derived from this data. In one embodiment, the proportion of cells expressing the estrogen receptor could be calculated. So to, the amount of estrogen receptor being expressed in the cells could be determined. These two statistics - the portion of cells expressing ER and their relative level of expression - are the outputs for the IHC assay previously offered by Abbott and currently used IHC assays, such as the one offered by DAKO (9). In fact, the proposed immunoassay could provide a precise quantitative determination of the amount of estrogen present in a cell while traditional IHC can provide only a qualitative estimate of estrogen receptor amounts. In one embodiment, the assay will be able to determine in which cellular compartment the estrogen receptor resides. This is also information provided by other IHC assays for the estrogen receptor. The added information provided by the newly described assay is that the tamoxifen mediated increase in H222 binding is also determined. Thus, this variation in the immunocytochemical assay provides all the information that was previously provided by both the IHC and ELISA assay performed with and without tamoxifen.

Another variation on the proposed immunocytochemical assay would be to use D547 or other anti-estrogen receptor antibody whose epitope is not affected by tamoxifen as a second antibody in this assay. In one embodiment, the D547 (or equivalent antibody) and H222 are labeled with florescent tags that when excited emit light of different wavelengths. This variation of the test would not be run in duplicate but instead would only be performed in the presence of tamoxifen (or other similarly acting SERM). Since both antibodies detect the estrogen receptor but only H222 binding is affected by tamoxifen, this assay could be calibrated such that the ratio of the H222 signal to the D547 (or other anti-estrogen antibody) signal would indicate the degree that tamoxifen has increased the binding of H222. In one embodiment, the assay - like the IHC assay - would also determine the proportion of cells that express the estrogen receptor and possibly also the amount estrogen receptor expressed in the cells, however; this later quantitative measurement might be challenging to implement with precision due to routinely experienced variation in the efficiency of immunostains.

[0036] Given the features of the two variations to the H222 immunoassay

involving two antibodies as described in the previous paragraphs, it is now possible to describe an immunoassay with three antibodies that combines the best attributes of both previously described assays. In one embodiment, the three antibodies used in this assay would be H222, D547 (or equivalent) and an antibody recognizing a constitutively expressed protein. Each would be labeled with a fluorescent tag that when excited would emit light at a different wavelength. Other means of labeling the antibodies can be imagined.

[0037] Like the second variation described previously, this assay would not be performed in duplicate and would be preformed in the presence of tamoxifen. In one embodiment, the cytology specimen would be incubated with a mixture of the three labeled antibodies. After washing away unbound antibodies, the amount of each antibody bound to a cell would be determined by quantitative fluorescent microscopy and image analysis at the three specific wavelengths emitted by the respective antibodies fluorescent tags. Numerous cells would be examined. In one embodiment, the proportion of cells to which either H222 and/or D547 bound would determine the portion of cells expressing the estrogen receptor. A precise quantization of the amount of estrogen receptor in each cell or the average amount of estrogen receptor in cells could be determine by examining the ratio of the amount of D547 to the amount of anti-constitutive protein antibody bound to a cell or cells. In one embodiment, the amount of tamoxifen mediated increase in H222 binding can be determined by examining the ratio of H222 to D547 binding. This assay has all the functionality of both the IHC and H222 ELISA assays except that all the measurements are quantitative, whereas some IHC determinations are qualitative, and the entire assay can be performed in a single procedure, whereas the ELISA has to be performed in duplicate. In addition, pathologists are accustom to performing slide based

immunoassays, and while its format is different from the IHC assays performed on formalin-fixed tissue with which they are most accustom, the proposed cytology based immunoassay is also a slide based assay and procedurally similar to IHC assays and likely to be preferred by pathologists over the H222 ELISA assay.

While the present invention makes use of the H222 and D547 antibodies, it is recognized that other antibodies and/or other binding molecules - such as aptimers or small selected peptides from a phage display library - if they recognized the same or similar epitopes as H222 and D547 could be substituted for H222 and/or D547 in these inventions. Furthermore, it is recognized that the labels or tags on H222, D547 or their functional equivalents do not have to be covalently conjugated to H222, D547 or their functional equivalents to perform adequately in these assays. Those skilled in the art will realize that the labels or tags could be conjugated to other antibodies or other high affinity molecules that themselves recognize and bind specifically to H222, D547 or their functional equivalents and that such a system of secondary antibodies or other high affinity molecules could substitute for the direct conjugation of the labels or tags to H222, D547 or their functional equivalents. Also, the previous descriptions used conjugated enzymes and florescent molecules as examples of labels and tags for the primary antibodies. Those skilled in the art will realize that other labels or tags are possible. For example, radiolabeled molecules or chemoluminescent detection is also possible

[0039] Certain compounds useful according to the present invention have acid and/or base moieties that, under suitable conditions, can form salts with suitable acids. Internal salts can also be formed. In one embodiment, the compound can be used in its free acid/base form or in the form of an internal salt, an acid addition salt or a salt with a base.

[0040] Acid addition salts can illustratively be formed with inorganic acids such as mineral acids, for example sulfuric acid, phosphoric acids or hydrohalic (e.g., hydrochloric or hydrobromic) acids; with organic carboxylic acids such as (a) C|.₄ alkanecarboxylic acids which may be unsubstituted or substituted (e.g., halo-substituted), for example acetic acid, (b) saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or terephthalic acids, (c) hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acids, (d) amino acids, for example aspartic or glutamic acids, or (e) benzoic acid; or with organic sulfonic acids such as C alkanesulfonic acids or arylsulfonic acids which may be unsubstituted (e.g., halo-substituted), for example methanesulfonic acid or p-toluenesulfonic acid. [0041] Salts with bases include metal salts such as alkali metal or alkaline earth metal salts, for example sodium, potassium or magnesium salts; or salts with ammonia or an organic amine such as morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkyl amine, for example ethylamine, tert-butylamine, diethylamine, diisopropylamine, triethyl amine, tributylamine or dimethylpropylamine, or a mono-, di- or tri-(hydroxy lower alkyl) amine, for example monoethanolamine, diethanolamine, or triethanolamine.

[0042] Alternatively, a prodrug of the compound or a salt of such prodrug can be used. A prodrug is a compound, typically itself having weak or no pharmaceutical activity, that is cleaved, metabolized or otherwise converted in the body of a subject to an active compound. In one embodiment, the prodrug is cleaved, metabolized or otherwise converted in the body of a subject to an active drug. In another embodiment, the prodrug is cleaved, metabolized or otherwise converted in the body of a subject to a renin inhibitor. Examples of prodrugs are esters, particularly alkanoyl esters and more particularly C i-₆ alkanoyl esters. Other examples include carbamates, carbonates, ketals, acetals, phosphates, phosphonates, sulfates, and sulfonates.

[0043] The drug, prodrug, or a salt of such drug or prodrug should be

administered according to a treatment regimen effective to reduce growth, invasiveness and/or metastasis of the tumor. One of skill in the art, having the benefit of the present disclosure, will readily and without undue experimentation select a suitable regimen, adjusting it as necessary or desirable in the course of treatment based on clinical response and occurrence of adverse side effects, if any. The term "regimen" in the present context includes dosage amount and frequency, duration of treatment, route of administration and other factors that may be prescribed by the clinician.

[0044] Dosages stated herein on a daily or per diem basis should not be

interpreted as necessarily being administered on a once daily frequency. Indeed the drug, prodrug, or a salt of such drug or prodrug can be administered at any suitable frequency, for example as determined conventionally by a physician taking into account a number of factors including number, size and invasiveness of tumors, but typically about four times a day, three times a day, twice a day, once a day, every second day, twice a week, once a week, twice a month or once a month. The drug, prodrug, or a salt of such drug or prodrug can alternatively be administered more or less continuously, for example by parenteral infusion in a hospital setting. In some situations a single dose may be administered, but more typically administration is according to a regimen involving repeated dosage over a treatment period. In such a regimen the daily dosage and/or frequency of administration can, if desired, be varied over the course of the treatment period, for example introducing the subject to the compound at a relatively low dose and then increasing the dose in one or more steps until a full dose is reached.

[0045] Suitable daily dosage amounts depend on the particular drug, prodrug, or a salt of such drug or prodrug used, as these vary in properties such as receptor affinity, bioavailability, metabolic half-life, etc., and on the route and method of administration. In general, a daily dosage amount should be sufficient to deliver to the target site, i.e., in the present case a breast tumor, a sustained concentration of at least about 30 nM, for example at least about 100 nM, at least about 300 nM or at least about 1 μΜ, and at most about 1 mM, for example at most about 300 μΜ, at most about 100 μΜ or at most about 30 μΜ, of the administered drug and/or active metabolite(s) thereof. Daily dosage amounts capable of delivering such concentrations when administered systemically will typically be about 0.01 to about 100 mg/kg, more typically about 0.02 to about 50 mg/kg, for example about 0.05 to about 25 mg/kg or about 0.1 to about 20 mg/kg. Illustratively, a daily systemic (e.g., oral or parenteral) dose for an adult woman with breast cancer can be about 1 to about 3000 mg, for example about 3 to about 1500 mg or about 5 to about 1000 mg.

[0046] The aromatase inhibitor may comprising at least one compound selected from the group consisting of aminoglutethimide, anastrozole, exemestane, fadrozole, formestane, letrozole, vorozole, pharmaceutically acceptable salts, prodrugs and active metabolites thereof.

[0047] Suitable dosages, routes of administration and other aspects of the

treatment regimen for the anti-hormone drug will typically be within the normal therapeutic range for the drug when used in monotherapy.

However, in some instances it may be possible, when the drug is used in combination therapy with a specific estrogen receptor modulator (SERM).

[0048] A still further embodiment of the invention comprises a therapeutic

combination comprising a specific estrogen receptor modulator (SERM) and an aromatase inhibitor in amounts effective in combination to reduce growth, invasiveness, and/or metastasis of a breast tumor. Suitable absolute and relative amounts of the specific estrogen receptor modulator (SERM) and the aromatase inhibitor will be based on therapeutically effective dosage amounts of each, but in some instances it will be found possible to reduce the dosage amount of one or other component of the therapeutic combination without loss of efficacy.

[0049] The components of the therapeutic combination of the present

embodiment can be present in separate pharmaceutical compositions or in a single pharmaceutical composition. Such a single pharmaceutical composition, comprising a specific estrogen receptor modulator (SERM), an aromatase inhibitor, and at least one pharmaceutically acceptable excipient, is a further embodiment of the present invention.

[0050] A method for treating a breast tumor in a patient, comprising

administering to the patient a therapeutic combination comprising a specific estrogen receptor modulator (SERM) and an aromatase inhibitor, is a still further embodiment of the invention.

[0051 ] Such a tumor can be ER- or ER+; if ER+ it can be SERM-responsive or

SERM-resistant. The tumor can be a ductal or lobular carcinoma; in a particular embodiment, the tumor is primary infiltrating ductal carcinoma. The combination can be administered separately or together; if together, the components of the combination can be administered in separate pharmaceutical compositions or in a single pharmaceutical composition.

[0052] Other delivery systems providing extended release of a drug are also

available and adaptable for use in the present invention. Such systems include, for example, nanoparticulate systems that can provide sustained and targeted delivery of a drug within or in close proximity to a tumor. In one embodiment, such drug delivery systems deliver an aromatase inhibitor. In another embodiment, such drug delivery systems deliver a specific estrogen receptor modulator (SERM).

[0053] The present methods are directed to selection, screening, and/or treatment of patients. Patients herein are generally human patients, but it will be understood that the methods are adaptable to other species, including animal models for human disease and to animals requiring veterinary care. References

1. Greene GL, Sobel NB, King WJ, Jensen EV. Immunochemical studies of estrogen receptors. J Steroid Biochem. 1984 Jan;20(l):51-6.

2. Martin PM, Berthois Y, Jensen EV. Binding of antiestrogens exposes an occult antigenic determinant in the human estrogen receptor. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2533-7.

3. Giambiagi N, Pasqualini JR. Immunological differences between the estradiol-, tamoxifen- and 4-hydroxy-tamoxifen-estrogen receptor complexes detected by two monoclonal antibodies. J Steroid Biochem. 1988;30(l-6):213-7

4. Naundorf H, Jost-Reuhl B, Becker M, Reuhl T, Neumann C, Fichtner I. Differences in immunoreactivity of estrogen receptor (ER) in tamoxifen-sensitive and - resistant breast carcinomas: preclinical and first clinical investigations. Breast Cancer Res Treat. 2000 Mar;60(l):81-92.

5. Ay diner A, Tas F. Meta-analysis of trials comparing anastrozole and tamoxifen for adjuvant treatment of postmenopausal women with early breast cancer. Trials. 2008 Jul 29;9:47.

6. E. A. Perez. Safety profiles of tamoxifen and the aromatase inhibitors in adjuvant therapy of hormone-responsive early breast cancer. Annals of Oncology. 2007. 18 (Supplement 8): viii26-viii35.

7. Bernard Fisher, Jong-Hyeon Jeong, James Dignam, Stewart Anderson, Eleftherios Mamounas, D. Lawrence Wickerham, Norman Wolmark. Findings From Recent National Surgical Adjuvant Breast and Bowel Project Adjuvant Studies in Stage I Breast Cancer. JNCI Monographs 2001 2001 (30): 62-66.

8. Baum M, Buzdar A, Cuzick J, Forbes J, Houghton J, Howell A, Sahmoud T; The ATAC (Arimidex, Tamoxifen Alone or in Combination) Trialists' Group.

Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early-stage breast cancer: results of the ATAC (Arimidex, Tamoxifen Alone or in Combination) trial efficacy and safety update analyses. Cancer. 2003 Nov 1 ;98(9): 1802-10.

9. http://www.dakousa.com/index/piOd_search/piOd_pi ducts.htm?pi ductareaid= 39&baseprodidver=A235371003

Claims

Claims:

1. A method for selecting a breast cancer patient for therapy comprising (a) determining whether the cancer comprises a tumor with estrogen receptor alpha expressing breast tumors (ER+ tumors) and (b) selecting the patient for therapy with aromatase inhibitors instead of specific estrogen receptor modulators (SERMs) only if the cancer is determined to comprise an ER+ tumor.

2. The method of claim 1, wherein determination of presence of an ER+ tumor is made in a tissue sample of the patient by obtaining a positive result in an assay for estrogen receptor alpha.

3. The method of claim 2, wherein the assay is selected from the group

consisting of ligand binding assays, immunohistochemical assays and combinations thereof.

4. The method of claim 2, wherein the assay is an immunohistochemical assay using the monoclonal antibody H222.

5. The method of claim 1, further comprising determining, for a tumor found to be ER+, whether the tumor is resistant or responsive to selective estrogen receptor modulator (SERM) treatment.

6. A method for treating breast cancer in a patient, comprising (a)

determining whether the cancer comprises a tumor with estrogen receptor alpha expressing breast tumors (ER+ tumors) and (b) selecting the patient for therapy with an aromatase inhibitor instead of specific estrogen receptor modulators (SERMs) only if the cancer is determined to comprise an ER+ tumor; and (c) administering to the selected patient an aromatase inhibitor.

7. The method of claim 6, wherein the patient determined to have estrogen receptor alpha expressing breast tumors (ER+ tumors) is treated with an aromatase inhibitor.

8. The method of claim 6, wherein the patients determined to have non- estrogen receptor alpha expressing breast tumors (ER- tumors) is treated with a specific estrogen receptor modulator.

9. The method of claim 7, wherein the aromatase inhibitor comprises at least one compound selected from the group consisting of aminoglutethimide, anastrozole, exemestane, fadrozole, formestane, letrozole, vorozole, and pharmaceutically acceptable salts, prodrugs and active metabolites thereof.

10. The method of claim 8, wherein the SERM comprises at least one

compound selected from the group consisting of acolbifene, arzoxifene, bazedoxifene, droloxifene, HMR-3339, idoxifene, lasofoxifene, levormeloxifene, ospemifene, raloxifene, tamoxifen, toremifene, and pharmaceutically acceptable salts, prodrugs and active metabolites thereof.