WO2014152640A1 - Assay for predictive biomarkers of anti-androgen efficacy - Google Patents

Abstract

Biomarkers associated with anti-androgen sensitivity in cancers, methods for detecting and quantitating the biomarkers, and methods for treating cancer patients that exhibit the biomarkers are provided. The biomarkers are activated androgen receptor foci (AAF) found in the nuclei of certain tumor cells. The methods provide new information to guide the intention to treat patients with anti-androgens, allowing selection of individual patients and patient populations that are likely to respond to treatment.

Description

ASSAY FOR PREDICTIVE BIOMARKERS OF ANTI- ANDROGEN EFFICACY TECHNICAL FIELD

[0001] The present invention relates to biomarkers associated with anti-androgen sensitivity in cancers, methods for detecting and quantitating the biomarkers, and methods for treating cancer patients that exhibit the biomarkers.

BACKGROUND

[0002] Prognostic and predictive biomarkers are routinely used in clinical management of patients with prostate cancer. One such biomarker is the androgen receptor (AR), which is a nuclear transcription factor activated by androgens, testosterone (T) or its metabolite dihydrotestosterone (DHT) to regulate growth and differentiation of normal prostate epithelial cells. Androgens also stimulate growth of tumor cells which express AR, and AR expression in tumors is a good prognostic factor for prostate cancer patients. As AR is the target receptor of T and DHT, it is highly predictive of tumor sensitivity to therapeutic intervention with anti- androgens. AR is present to some degree in nearly all prostate cancers (Ruizeveld de Winter JA, Janssen PJ, Sleddens HM, Verleun-Mooijman MC, Trapman J, Brinkmann AO, et al. Androgen receptor status in localized and locally progressive hormone refractory human prostate cancer. Am J Pathol 1994; 144:735-46. Chodak GW, Kranc DM, Puy LA, Takeda H, Johnson K, Chang C. Nuclear localization of androgen receptor in heterogeneous samples of normal, hyperplastic and neoplastic human prostate. J Urol 1992; 147:798-803. Sadi MV, Walsh PC, Barrack ER. Immunohistochemical study of androgen receptors in metastatic prostate cancer. Comparison of receptor content and response to hormonal therapy. Cancer 1991; 67:3057-64.).

[0003] The standard medical treatment is to lower androgen exposure, also called androgen-deprivation. After androgen deprivation treatment, 12-18 months later, the disease progresses in an androgen independent state in its "truly" malignant phase, as even though AR are still present, prostate cancer progresses independently from androgen exposure and standard anti-androgen treatments are not active. However, AR remains a critical factor for prostate cancer progression. AR is activated via other mechanisms than ligand binding. Anti- androgens include direct receptor antagonists (AR Antagonists), CYP17 inhibitors, and LH-RH agonists. After treatment failure, alternate anti-androgen treatments frequently fail despite the presence of AR and treatments such as chemotherapy may be used. [0004] The simple determination of AR presence in a tumor is not therefore a guarantee of activity or indication of anti-androgens. The recent physiopathological reviews do explain possible mechanisms by which tumors might escape hormonal control, but do not provide any method that could determine which patients would benefit from anti-androgen treatment. (Nelson, N Engl J Med 2003;349:366-81., Heinlein Endoc Rev 2004).

[0005] Standard AR expression in tumors is evaluated in immunohistochemical assays using peroxidase labeled antibodies targeting the receptor. Typically, formalin-fixed paraffin- embedded tumor specimens are evaluated under direct microscopic visualization, and the number of stained cells is quantitated as a percentage of total tumor cells. There is significant variation in positivity by this method, and AR-expressing tumors may contain from almost 0 to nearly 100% positive cells. Although there is a correlation between the likelihood of clinical response to hormonal therapies and the level of AR expression, even tumors expressing very low levels (e.g., 1-10% positive cells) may show a significant response, whereas AR-negative tumors are essentially completely unresponsive.

[0006] The immunohistochemical assays described above provide only a quantitation of the androgen receptor itself. That is, they do not take into account whether or not it is bound to its ligand and/or to DNA, the Androgen Receptor Element (ARE). Current immunohistochemical assays therefore do not provide any information on whether or not the androgen receptor that is detected is also biologically or transcriptionally activated.

[0007] On the other hand, experimentally, the transcriptional activity of AR can be analyzed in engineered cells and in vitro. Fluorescent- AR are engineered and their movements can be visualized. To this end, Green Fluorescent Protein (GFP) tagged AR is transfected in cell systems. In vitro, in the absence of ligand binding to AR and DNA, the androgen receptor is evenly distributed and seen as a diffuse nuclear staining in fluorescence assays. Academic studies in experimental biology have reported that when nuclear receptors such as androgen receptor are biologically active, i.e. they transactivate gene expression, they form nuclear aggregates or foci in the presence of ligand, and that these structure are visible by fluorescence microscopy. These subnuclear structures may also be referred to as speckles and the nuclei containing them as hyperspecked nuclei. Upon ligand binding the receptor moves into the subnuclear aggregate structure to activate transcription of a wide variety of genes. . (Van Royen, The Journal of Cell Biology, Vol. 177, No. 1, April 9, 2007 63-72). [0008] As mentioned previously, the most commonly used methods in studies visualizing the formation of androgen receptor foci have employed cell lines transiently transfected with green fluorescent protein (GFP)-tagged AR. One such commercially available assay is the AR Redistribution® Assay from Thermo Scientific (Biolmage Products, Lafayette, CO). This assay is designed to assay compounds for their ability to modulate accumulation of AR in nuclear foci using GFP to monitor translocation. Nuclear foci are detected and analyzed by an image analysis algorithm, revealing ligand-regulated movement of the transfected androgen receptors into subnuclear foci. While these types of assays are useful for elucidating possible biologic mechanisms of androgen receptor foci formation, they do not provide any information concerning the process, how it occurs, or its relevance to disease in naturally- occurring cancers. As these systems are experimental, they are not applicable in the clinic. The current AR determination by histology does not provide information on the transcriptional activity or any method helping to determine adequate treatment (Asian J Androl. 2008 Nov;10(6):855-63). Resistance to anti-androgen is classified according to current genetic interpretation common to all cancers disease (Shifting the Paradigm of Testosterone and Prostate Cancer: The Saturation Model and the Limits of Androgen-Dependent Growth Abraham Morgentaler a,*, Abdulmaged M. Traish in European Urology 55 ( 2 0 0 9 ) 310- 321). Recent drug such as Enzalutamide prevent the binding of AR to DNA but there is no method that allows predicting which patient will benefit from these drugs.

[0009] Better targeting assists in avoiding unnecessary treatment and in developing more relevant treatment strategies. For example, if anti-androgens were known in advance to be ineffective in a particular tumor (particularly in a tumor that is AR positive by conventional methods), unnecessary treatment would be avoided and other treatment options could be initiated more quickly with better expected outcomes.

[0010] The present invention satisfies these needs. In contrast to the assays of the prior art, such as the Thermo Scientific Androgen Receptor alpha Redistribution^® Assay, the present invention provides analysis of AR foci in primary tumor tissue, irrespective of the presence of an AR ligand or a drug. In one aspect, the exemplary methods described herein relate to the presence of AR foci in the nuclei of cells in naturally-occurring tumors indicating an anomaly that can be used to predict the efficacy in that patient of an anti-androgen that has AR antagonist properties. In another aspect, the characterization of constitutively activated AR in the clinic has now been found to be a new and useful indicator of tumors and cancers that are susceptible to treatment with anti-androgens. If AR expression is a characteristic of prostate cancer, and a natural target, other biological pathways have been described to drive the growth of these cancers. Thus, the determination of whether or not AR still is a player in driving the cancer growth is critical to guide the choice, the combination or the sequence of use of available active drugs.

SUMMARY

[0011] We have determined that there are two different types of tumors with respect to

AR expression. Subsets of prostate cancers express AR, but the nuclei morphology indicates that the receptor is not biologically active. Even though there is sufficient androgen plasma level to activate AR, these prostate cancer cells which have AR with this pattern do not seem to be particularly involved in cancer progression. On the other hand, there are cancers where the nuclei morphology is in agreement with a transcriptional activation and therefore AR is a driving factor and a suitable therapeutic target. Supplemental pathways are activated and progressively AR becomes less and less dependent on the actual androgen exposure. As AR seem to be biologically active though, they are a relevant target and their activation could be interrupted by classic androgen antagonism or any drug which has the capacity to prevent the binding of AR to DNA. In this sense these drugs might not per se be considered anti-androgens in the physiological sense, i.e. simply opposing the ligand-receptor binding but rather preventing the ligand-receptor complex to bind to DNA, and the ligand in cancer may not necessarily be the natural and normal ligand. It has been shown in cancer that activated genes are different in cancer cell lines than in their normal counterparts. For example, a genetic anomaly found in prostate cancer, TMPRSS2-ERG fusion, produces aberrantly regulated oncogenic transcription factor ERG. ERG binds to nearly 50% of the promoters in the human genome in prostate cancer cells. However, a huge number of these genes are neither expressed in these cells, nor in patient samples. This illustrates the complexity of nature and development, as different cancers seem to find their own ways to aberrantly regulate the leaders of cell's transcriptional machinery, and strongly underlines the context dependency (Itkonen, Mol and Cel Endoc , pp45-51, 2012). Thus, the activated AR may have specific properties only pertinent to the malignant cells and not found in normal cells, and this test differentiates tumors that are driven by AR from those that aren't, based on histological patterns which indicate the involvement of AR even in the absence of androgens. The activated AR as described here is a pattern integrating the multiple underlying biological anomalies that vary from case to case in a context dependent manner.

[0012] Applicants have determined that the foregoing needs in the art for improved androgen receptor (including AR) assays employing primary prostate cancer tissue samples can be met by immunohistochemical or cytological assays that distinguish cancers in which the androgen receptor is biologically activated from those in which androgen receptor is present but biologically inactive. These assays detect the presence of AR foci or aggregates in the nuclei of cancer cells (biologically, transcriptionally active AR) as an indication of AR- positive status of the tumor, as opposed to AR-negative status indicated by diffuse nuclear staining (biologically inactive AR) and/or no nuclear staining. The assay establishes for the first time that the patient population with AR expressing tumors can be separated in two subgroups, one with AR which would not play an active role in the growth process, and a different population where there are indications that AR is active biologically and where therefore anti-androgen treatment could be beneficial. The presence of biologically active AR provides an indication that the androgen receptor that is present is engaged in the biology of the tumor and is therefore an appropriate target for anti-androgen therapy. In contrast, diffuse nuclear staining of androgen receptors indicates that, although the tumor would be considered AR-positive by conventional methods, it is unlikely to be sensitive to anti-androgen therapy due to the biologically inactive status of the receptor.

[0013] The present invention therefore provides a method for identification of a subset of AR-positive tumors most likely to benefit from treatment with anti-androgens such as aberatirone, nilutamide, flutamide, bicalutamide, Enzalutamide, or ARN-509. Androgen receptor foci have been studied in homogeneous, artificial experimental models. However, each naturally-occurring tumor is different and each naturally-occurring tumor is heterogeneous. The clinical significance of distinguishing tumor cells expressing androgen receptor in biologically activated form (i.e., bound to ligand in foci or aggregates) from tumor cells expressing androgen receptor in biologically inactive form (not bound to ligand and in a diffuse nuclear distribution) was not previously recognized. Expression of biologically active (i.e., transcriptionally active) androgen receptors, visualized as foci within the nuclei of cancer cells, provides a potential target for intervention by anti-androgens and disruption of androgen- stimulated pathways of tumor cell growth. That is, in such tumor cells anti-androgens can potentially inactivate ligand activated AR. In contrast, expression of transcriptionally inactive AR, visualized as diffuse nuclear staining on immunohistochemical or cytological evaluation, would indicate that in spite of the presence of androgen receptor in tumor cells it is unlikely to be an appropriate target for anti-androgen intervention.

[0014] AR foci in tumor cells can therefore serve as a biomarker for selection of an appropriate treatment, including the decision whether or not to employ anti-androgens as treatment. Recognition of this biomarker allows development of an assay which can serve as an indicator of the likelihood that a prostate cancer patient will benefit from therapy with anti- androgens.

[0015] In a first embodiment, the diagnostic assay is a method for identifying patients having tumors which express activated AR (i.e., activated AR foci or "AAF"). These patients are more likely to benefit from treatment with an anti-androgen than patients that do not express activated AR (typically seen as diffuse nuclear staining). Inactivation of the AAF by an anti-androgen may occur by any of a variety of mechanism, including dissociation of the foci and inhibition of activation of the foci without substantially altering their structure. Patients that do not express activated AR foci (AAF) may include those that are androgen receptor negative by the conventional assay, or those that are androgen receptor positive by the conventional assay. Any tumor which exhibits AAF is believed to be a candidate for treatment with such anti-androgens, including prostate cancer. In particular breast cancer cells might also contain AAF.

[0016] In a second embodiment, the invention relates to an in vitro method for identifying a tumor treatable with an AAF-active anti-androgen, comprising:

a) exposing cancer cells or a primary tissue specimen containing cancer cells obtained from a patient to an anti-androgen receptor antibody under conditions appropriate for binding of the antibody to androgen receptors in nuclei of the cancer cells; and

b) detecting presence or absence of focal binding of the antibody to androgen receptors in the nuclei;

wherein the presence of focal binding indicates sensitivity of the tumor to treatment with the AAF-active anti-androgen and the absence of focal binding indicates lack of sensitivity of the tumor to treatment with the AAF-active anti-androgen.

[0017] In yet another embodiment, the above method further comprises treating a patient positive for focal binding of the antibody with an androgen receptor antagonist. [0018] In yet another embodiment, focal binding of the antibody is detected in the absence of diffuse binding of the antibody in the nuclei.

[0019] In yet another embodiment, focal binding of the antibody is detected in addition to diffuse binding of the antibody in the nuclei.

[0020] In yet another embodiment, the cancer cells are prostate cancer cells or the primary tumor tissue specimen is a prostate cancer specimen.

[0021] In yet another embodiment, presence or absence of focal binding is detected by fluorescence.

[0022] In yet another embodiment, presence or absence of focal binding is detected in a colorimetric reaction, such as an enzymatic reaction.

[0023] In yet another embodiment, the method further comprises detecting presence or absence of focal binding in a quantitative or semi- quantitative manner.

[0024] In a third embodiment, the invention relates to a method for treating a tumor in a patient, comprising:

a) exposing cancer cells or a primary tissue specimen containing cancer cells obtained from a patient to an anti-androgen receptor antibody under conditions appropriate for binding of the antibody to androgen receptors in nuclei of the cancer cells;

c) detecting presence or absence of focal binding of the antibody to androgen receptors in the nuclei; and

d) treating the patient with an AAF-active anti-androgen if focal binding is present.

[0025] In yet another embodiment, the invention relates to use of an AAF-active anti- androgen for treating a tumor, following identification of the presence of focal binding of anti- androgen receptor antibody to androgen receptors in cancer cell nuclei.

[0026] In any of the foregoing embodiments, presence of focal binding may mean 1- 100%, 5-100%, 25-100%, 50-100% , 80-100%, 80-90%, 50-80% or 30-40% of cancer cell nuclei exhibit focal binding.

[0027] In any of the foregoing embodiments, when focal binding is present, the intensity or density of such focal binding may be quantitated.

[0028] In any of the foregoing embodiments the AAF-active anti-androgen may be a receptor antagonist, or the tumor of interest may be prostate cancer. [0029] In yet another embodiment, the invention provides an in vitro method for screening an antitumor drug or antitumor drug candidate for AAF inactivating activity which comprises:

a) providing cancer cells or a primary tumor tissue specimen containing cancer cells, wherein the cancer cells express AAF and the AAF are detectably stained with an anti- androgen receptor antibody;

b) exposing the cancer cells or the primary tumor tissue specimen to the antitumor drug; and

c) detecting a decrease in detectable staining of the AAF as an indication of AAF inactivating activity of the antitumor drug, or detecting no substantial decrease in detectable staining of the AAF as an indication of lack of AAF inactivating activity of the antitumor drug.

[0030] In any of the foregoing embodiments, the androgen receptor detected may be

AR and the anti-androgen receptor antibody used may be anti-AR.

DESCRIPTION OF THE DRAWINGS

[0031] Fig. 1A - Fig. 1J show the chemical structures of various anti-androgens.

[0032] Fig. 2 shows prostate tumor cells of the "D" phenotype, wherein the nuclei are diffusely and homogeneously stained and do not contain AAF.

[0033] Fig. 3 shows prostate tumor cells, wherein the nuclei contain AAF which are seen as clumps or aggregates.

[0034] Fig. 4A and Fig. 4B show a close-up comparison of the "D" phenotype nuclei of

Fig. 2 and the AAF nuclear phenotype of Fig. 3, respectively.

DETAILED DESCRIPTION

[0035] Before describing several exemplary embodiments of the invention, it is to be understood that the invention is not limited to the details of construction or process steps set forth in the following description. The invention is capable of other embodiments and of being practiced or being carried out in various ways.

[0036] Reference throughout this specification to "one embodiment," "certain embodiments," "one or more embodiments" or "an embodiment" means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases such as "in one or more embodiments," "in certain embodiments," "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the invention. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0037] As used herein, the terms "cancer cells" and "tumor cells" are generally interchangeable and refer to malignant cells which may be present in a solid tumor or which may be circulating in the blood. The solid tumor may be a primary tumor or a metastatic tumor, and any circulating cancer cells may be derived from either solid tumor type. Analysis of solid tumors for purposes of the invention (i.e., histological analysis) may be performed using a primary biopsy specimen. Alternatively, cancer or tumor cells for analysis according to the invention (i.e., cytological analysis) may be obtained by fine needle aspiration of a solid tumor as well as by separation from blood.

[0038] As used herein, the phrases "treating a tumor," "treatment of a tumor" and the like mean to inhibit the replication of tumor cells, inhibit the spread of the tumor, decrease tumor size, lessen or reduce the number of tumor cells in the body, or ameliorate or alleviate the symptoms of the disease caused by the tumor. Tumors include cancers. The treatment is considered therapeutic if there is a decrease in mortality and/or morbidity, or there is a decrease in disease burden as may be manifested by reduced numbers of tumor cells in the body or decreased tumor size.

[0039] As used herein, the term "androgen receptor" or "AR" means the nuclear receptor that is activated by binding of an androgenic hormone (e.g., testosterone (T) or dihydrotestosterone (DHT)) in the cytoplasm of a cell followed by translocation into the nucleus. AR may be referred to as NR3C4 (nuclear receptor subfamily 3, group C, member 4).

[0040] As used herein, the term "AAF-active anti-androgen" and its equivalents refer to an anti-androgen drug which exhibits an ability to inactivate, dissolve or dissociate activated AR foci (AAF) in the nuclei of cells, indicating that its mechanism of action is via the AR activation pathway of the cell.

[0041] The terms "AAF-positive", "AR foci positive", "activated AR", "ARs in a functional state" and the like refer to the presence of androgen receptor aggregates in the nuclei of cells. [0042] The term "degree of focal distribution" refers to the relative number or intensity of AR foci present in the nuclei of AR-positive cells, or to the relative number of cells that exhibit AR foci (AR focal binding). The degree of focal distribution can be determined quantitatively or qualitatively. For example, the use of a colorimetric, enzymatic, or radiolabeled ligand such as anti-androgen receptor antibody, can be used to bind to androgen receptors in cell nuclei. The degree of focal distribution can be determined quantitatively, for example, by measuring color intensity, fluorescence, or the level of radioactivity emitted by the labeled antibody in foci in the nucleus. The degree of focal distribution can be determined qualitatively by comparing the intensity of focal binding, relative number of labeled foci, or relative number of cells exhibiting focal binding between a control sample and a labeled sample using a light microscope at an appropriate magnification or techniques including, but not limited to, radiolabeling, or other surrogates for measuring AR foci.

[0043] The term "diffuse pattern" refers to a finely granular pattern which is indicative of the absence of focal distribution and lack of AR activation.

[0044] The term "focal binding," "foci" and the like refers to the appearance of aggregates in the nuclei of cells that are larger than the even, diffuse staining pattern indicative of AR that are not activated. The large aggregates or fine aggregates of focal binding may be observed in a background of a diffuse staining pattern in the nuclei.

[0045] The term "androgen" refers to a natural or synthetic androgenic substance that mimics some or all of the actions of T and/or DHT, also referred to as androgen receptor modulators (ARM) or selective androgen receptor modulators (SARM).

[0046] The term "anti-androgen" refers to a substance that inhibits the formation, transport, or action of, or which inactivates androgenic agents, nilutamide (AR antagonist,) flutamide (AR antagonist), bicalutamide (AR antagonist), enzalutamide (AR antagonist), ARN-509 (AR antagonist), orteronel (CYP17 inhibitor), galeterone (CYP17 inhibitor + anti AR), goserelin (LH-RH and GnRH agonist), leuprolide (LH-RH and GnRH agonist), and triptorelin (GnRH agonist) are examples of anti-androgens.

Nilutamide (AR antagonist) 5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl) phenyl] imidazolidine- 2,4-dione is shown in Fig. 1A. Flutamide (AR antagonist) 2-methyl-N-[4-nitro-3- (trifluoromethyl)phenyl]propanamide is shown in Fig. IB. Bicalutamide (AR antagonist) N-[4- cyano-3-(trifluoromethyl)phenyl]-3-(4-fluorophenyl) sulfonyl-2-hydroxy-2- methylpropanamide is shown in Fig. 1C. Enzalutamide (AR antagonist) 4-[3-[4-cyano-3- (trifluoromethyl)phenyl]-5,5-dim

methylbenzamide is shown in Fig. ID. ARN-509 (AR antagonist) 4-[7-[6-cyano-5- (trifluoromethyl)pyridin-3-yl]-8-oxo-6-sulfanylidene-5,7-diazaspiro[3.4]octan-5-yl]-2-fluoro- N-methylbenzamide is shown in Fig. IE. Orteronel (CYP17 inhibitor) 6-[(7S)-7-hydroxy-5,6- dihydropyrrolo[l,2-c]imidazol-7-yl]-N-methylnaphthalene-2-carboxamide is shown in Fig. IF. Galeterone (CYP17 inhibitor + anti AR) (3S,8R,9S,10R,13S,14S)-17-(benzimidazol-l-yl)- 10,13-dimethyl-2,3,4,7,8,9,l l,12,14,15-decahydro-lH-cyclopenta[a]phenanthren-3-ol is shown in Fig. 1G.

[0047] LH-RH Agonists: Goserelin (LH-RH and GnRH agonist) is shown in Fig. 1H. Leuprolide (LH-RH and GnRH agonist) is shown in Fig. II. Triptorelin (GnRH agonist) is shown in Fig. 1J.

[0048] An "AAF-active anti-androgen" or "AAF-active drug" is a drug that dissociates

AAF or otherwise reduces the number, size or staining intensity of AAF in cell nuclei. In one example, an AAF-active drug causes the conversion of an A or AD staining pattern into a D staining pattern in the cell. AAF-active drugs include anti-androgens and androgen receptor antagonists as well as drugs that exhibit activity against AAF but do no operate by an anti- hormonal mechanism.

[0049] The term "antibody" or "antibodies" refers to a protein which is capable of specifically binding to an antigen and includes any substance, or group of substances, which has a specific binding affinity for the antigen to which it is directed, with little or no binding affinity for other substances. Generally, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, antibodies derived from humans or animals, humanized antibodies (e.g., non-binding portions derived from a human, binding portions derived from an animal) and fragments thereof.

[0050] The term "anti-AR antibody" refers to antibodies directed the androgen receptor. "Anti-AR antibody" refers generically to an antibody capable of binding AR. Specific antibodies suitable for use in accordance with aspects herein include, but are not limited to anti-AR monoclonal antibodies available from Leica Biosystems (Buffalo Grove, IL).

[0051] In one aspect, the invention provides a method of inhibiting the growth of a tumor susceptible to growth inhibition by anti-androgens by determining the degree of focal binding of anti-AR antibody in nuclei of cells in a cell sample or tissue obtained from a patient and suspected of containing tumor cells. If the degree of focal distribution is greater than about 5% of cells (i.e., greater than about 5% of cells contain AAF foci), for example from about 5% to 100%, 25-100% or 50-100% of cells, an anti-androgen is administered to the patient to inhibit growth of the tumor. Potentially useful anti-androgens include nilutamide (5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl)phenyl]-2,4-imidazolidinedione), flutamide (2- methyl-N-[4-nitro-3 (trifluoromethyl) phenyl] propanamide.), bicalutamide (N [4 cyano-3- (trifluoromethyl)phenyl]-3-[(4-fluorophenyl)sulfonyl]-2-hydroxy-2-methyl-, (+-)),

enzalutamide (4-{3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5dimethyl-4-oxo-2- sulfanylideneimidazolidin-l-yl}-2-fluoro-N-methylbenzamide), or ARN-509 (4-(7-(6-cyano-5- (trifluoromethyl )pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N- methylbenzamide) .

[0052] In one aspect, the invention relates to a method for identifying a tumor treatable with an AAF-active anti-androgen, comprising:

a) exposing cancer cells or a primary tumor tissue specimen containing cancer cells obtained from a patient to an anti-androgen receptor antibody under conditions appropriate for binding of the antibody to androgen receptors in nuclei of the cancer cells; and b) detecting presence or absence of focal binding of the antibody to androgen receptors in the nuclei;

wherein the presence of focal binding indicates sensitivity of the tumor to treatment with the AAF-active anti-androgen and the absence of focal binding indicates lack of sensitivity of the tumor to treatment with the AAF-active anti-androgen.

[0053] The foregoing assay for focal AR binding provides a more sensitive and predictive test than currently-used conventional AR assays, and focal AR binding can be identified in patients classified in conventional AR assays as AR-negative as well as those that are conventionally AR-positive. Patients classified in conventional AR assays as AR-negative as well as those that are conventionally AR-positive may test positive for focal AR nuclear binding and therefore be considered candidates for treatment with anti-androgens. The absence of AR foci in patients conventionally tested as AR-positive would explain the apparently anomalous result that anti-androgens are ineffective in some of these patients. The assay method of the invention therefore makes anti-androgen treatment an efficacious choice for a greater number of cancer patients. [0054] The cancer cells analyzed in any of the assays for focal binding according to the invention may be contained in a specimen of tumor tissue taken directly from a patient. These specimens are typically referred to as primary biopsies, and may be derived from either primary or metastatic solid tumors (a histological analysis). Alternatively, the cancer cells analyzed in any of the assays for focal binding according to the invention may be individual cancer cells or small clusters of cancer cells obtained, for example, by needle aspiration of a tumor or by separation of cancer cells from blood (a cytological analysis). Cytological analysis has several advantages, including being less invasive for the patient and it providing an analysis of the relevant cellular compartment without interference from surrounding tissue architecture.

[0055] As AR is a commonly used biomarker for prostate cancer, antibodies which specifically bind to AR may be most appropriate for the methods of the invention.

[0056] Certain cytological methods suitable for use in the invention include immunocytochemical methods applied to circulating tumor cells (CTC). CTC can be analyzed by a variety of methods, for example, Miyamoto et al, Cancer Discovery 2012, analyzed prostate cancer CTC and used immunofluorescence to build a model and predict response to abiraterone. However, these authors do not rely on analysis of specific histological pattern and subnuclear structure. Kirby PLOSone, 2012 ( Kirby BJ, Jodari M, Loftus MS, Gakhar G, Pratt ED, et al. (2012) Functional Characterization of Circulating Tumor Cells with a Prostate- Cancer-Specific Microfluidic Device. PLoS ONE 7(4): e35976. doi: 10.1371/journal.pone.0035976) analyze CTC to determine specific molecular signature. This is different from a specific cellular or nuclear pattern as described in histopathology. So far, the diagnosis of cancer is made solely on histology or cytology patterns and not any biochemical or molecular pattern. It may be however that the pattern would be analyzed through specific software for image analyses. Our method includes the differentiation of tumor cell nuclei with activated AR vs. non-activated AR as identified by a specific pattern immunohistochemistry, i.e., focal binding in the nucleus. Fixed slides may be exposed to a primary antibody specific for the AR, preferably a monoclonal antibody, which binds to AR. Binding of the antibody may be detected using any one of the methods known in the art for detection of antibody binding.

[0057] One appropriate method for detection of binding of an antibody to its target is a colorimetric assay, typically an enzymatic colorimetric assay. One such method employs peroxidase to produce a colored stain visible under the light microscope. Endogenous peroxidase in the tissue specimen is blocked using hydrogen peroxide and endogenous biotin is blocked using a biotin-blocking reagent prior to incubation with the antibody or antibodies. In one example, binding of primary antibody is followed by biotinylated secondary antibody targeting the primary antibody. Binding of the secondary antibody is then detected using avidin or streptavidin conjugated to peroxidase, typically horse radish peroxidase (HRP). The conjugate is added to bind the enzyme to the antibody- target complex. AR is visualized by enzymatic conversion of the chromogenic substrate 3,3'-Diaminobenzidine (DAB) to a brown stain at the site of AR localization. If the primary antibody is a mouse antibody, it is subsequently bound to a biotinylated anti-mouse immunoglobulin. The cytological or tissue specimen may be counterstained with fast green to increase visibility of the peroxidase stain.

[0058] Alternatively, a fluorescence method may be used to detect antibody binding to

AR. In this case, a fluorescently-labeled primary antibody may be bound to the AR target and detected directly under a fluorescence microscope. However, a method employing binding of an unlabeled primary antibody to the AR followed by binding a fluorescently-labeled secondary (e.g., anti-mouse immunoglobulin) antibody to the primary antibody may reduce non-specific fluorescence. Any fluorescent label known for use in immunocytological or immunohistochemical assays may be used in the methods of the invention, for example FITC.

[0059] Both monoclonal and polyclonal antibodies may be useful in the present methods. A non-exhaustive list of suitable monoclonal antibodies is available commercially from Santa Cruz Biotechnology, Inc., (http://www.scbt.com/table-androgen_receptor.html).

[0060] Binding of the antibody to AR is typically detected by observation of the stained slide under a light microscope or fluorescence microscope as appropriate. Magnification is typically about 50X; however, to improve sensitivity for detection of AAF it may be desirable to evaluate the slides at 100X-400X to facilitate study of subnuclear structures. To more clearly identify the various AR phenotypes described herein, it is even more desirable to use particularly high magnification, such as 400X-900X, e.g., 800X.

[0061] Samples that are apparently negative by microscopy may be evaluated by flow cytometry to detect positivity that is below the threshold of light or fluorescence microscopy. If flow cytometry indicates rare positive cells, high magnification microscopy (100X-400X, or 400X-900X, e.g., 800X) may be used to study subnuclear structures and identify AAF. However, if the positive cells detected by flow cytometry are too rare to be reliably detected by microscopy for analysis of AAF, a fluorescence-activated cell sorter (FACS) can be used to separate positive cells from the cells in suspension based on their fluorescence. As positive cells are concentrated but not damaged by this process, the reliability and probability of successfully visualizing AAF on subsequent microscopic evaluation is substantially increased.

[0062] The presence or absence of AAF in individual tumor cell nuclei may be detected visually under a light or fluorescence microscope, or by any other appropriate means, such as fluorescence or colorimetric measurements. Typically, visual means for detection will be used. The results of staining may be quantitated by simply noting presence or absence of AAF, or by counting the number or percentage of positive cells. Alternatively, specific characteristics of the staining may be quantitated. For example, detection may include notation of whether or not focal binding in the form of AAF is accompanied by diffuse nuclear staining, quantitation of positive cells by number or percentage, and/or quantitation of intensity or number/density of AAF. Relative size of AAF may also be used to characterize, classify and quantitate. Quantitation of AAF density may be determined as the average number of foci/cell, or using an arbitrary scale (e.g., "few", "moderate" or "many"). Intensity may similarly be determined using an arbitrary scale, e.g., low/medium/high or a numerical scale such as 1-5. Typically, the results of the analysis of the patient's tumor tissue will be compared to positive and/or negative controls.

[0063] A cytological or tumor tissue specimen is judged as AAF-positive when 1- 100%, 5-100%, 25-100%, 50-100%, 80-100%, 80-90%, 50-80% or 30-40% of cells in the specimen exhibit AAF. As therapeutic efficacy of an AAF-active anti-androgen may also be correlated with the intensity of AAF staining or with the number, size or density of AAF, these parameters may also be used to determine the sensitivity of the tumor to treatment with the AAF-active anti-androgen. In general, it is anticipated that sensitivity of the tumor to treatment with AAF-active anti-androgen will increase with increasing number or percentage of positive cells, increasing intensity of AAF and/or increasing number or size of AAF in the cells of the cytological or tumor tissue specimen

[0064] The above methods for determining the sensitivity of a tumor to AAF-active anti-androgens may be either manual (e.g., visual detection using a fluorescence microscope) or they may be automated or semi- automated using methods for rapid scanning, detection and quantitation of colorimetrically- or fluorescently-labeled cytological or tissue specimens. For example, a fully automated scanning and analysis system may be developed and used in the present invention. Alternatively, a system similar to the InScape® ER/PR quantitative immunohistochemistry (IHC) system, which requires manual selection of specific regions to be analyzed may be used. However, the preferred system for scanning and analysis of AAF in cell nuclei will be designed to provide automated whole- specimen scanning and analysis of the antigen-specific immunohistochemistry stained specimen. Image recognition will create a digital image of the entire stained tissue section. An antigen- specific computer algorithm will then analyze the results of the digital image representing the whole specimen. For use in the methods of the invention, the software will distinguish foci from diffuse background staining in the nucleus, and measure fluorescence intensity and size of foci on a cell-by-cell or cluster- by-cluster basis, repeating the process for each cell or cluster over the entire specimen. These automated methods will result in improved accuracy by performing a function that is not possible manually, with reduced cost. Full automation will also make the test accessible to non-expert medical centers.

[0065] The present methods for determining the sensitivity of a tumor to AAF-active anti-androgens allow medical practitioners to identify a subset of tumor patients that are more likely to benefit from such treatment. That is, by first classifying the tumor as AAF-positive or AAF-negative in the diagnostic method, the medical practitioner is able to select the most appropriate treatment for the patient. Accordingly, in another aspect the invention also provides a method for treating a tumor in a patient, comprising:

a) exposing cancer cells or a primary tissue specimen containing cancer cells obtained from a patient to an anti-androgen receptor antibody under conditions appropriate for binding of the antibody to androgen receptors in nuclei of the cancer cells;

b) detecting presence or absence of focal binding of the antibody to androgen receptors in the nuclei; and

c) treating the patient with an AAF-active anti-androgen if focal binding is present.

[0066] The details of the detection of presence or absence of focal binding of the antibody to androgen receptors in the nuclei of cells are discussed above. The decision whether to treat the patient based on the results of the diagnostic assay will be based on the number/percentage, intensity and/or density of AAF when they are present. A decision to treat the patient with anti-androgen may be made when 1-100%, 5-100%, 25-100%, 50-100%, 80- 100%, 80-90%, 50-80% or 30-40% of cancer cell nuclei exhibit focal binding in the diagnostic assay. Generally, it is anticipated that the efficacy of treatment with an AAF-active anti- androgen will increase with increasing number or percentage of positive cells, increasing intensity of AAF and/or increasing number or size of AAF in the cells of the tumor tissue specimen or cancer cell sample. Based on these parameters the medical practitioner may also determine the dosing, timing and length of treatment. Accordingly, in another embodiment the invention relates to use of an AAF-active anti-androgen for treating an AAF-positive tumor.

[0067] The tumor to be identified or treated according to the above methods may include any cancerous or non-cancerous tumor in which AAF occur, and in which the presence of AAF can be determined. In specific examples, the tumor tissue or cancer cells for analysis or treatment may be selected from the group consisting of AR-positive tissue or cells, or prostate or breast tissue or cells. About 20% of triple negative breast cancers are positive for the androgen receptor. Such cancers particularly include prostate cancer.

[0068] The AAF-active anti-androgen of the foregoing treatment methods may be any anti-androgen drug having the ability to inactivate AAF (for example by dissolving or dissociating the aggregates). Such drugs include AR antagonists and/or CYP17 inhibitors, LH- RH agonists and GnRH agonists, but others with the ability to inactivate AAF as the mechanism of action are also included for use in the present invention. In certain aspects, the anti-androgen is selected from the group consisting of nilutamide, flutamide, bicalutamide, enzalutamide, or ARN-509.

[0069] In yet another aspect, the anti-androgen is administered to a patient having an AAF positive tumor in an amount from 10 to about 200 mg per day depending upon the potency, bioavailability, and safety profile of the selected anti-androgen or combination of anti-androgens. Without being bound by theory, it is believed that by identifying patients with tumors that are more susceptible to treatment with anti-androgens, a lower dose of the anti- androgen may be used, resulting in a lower risk of toxic side effects. Thus, a lower dosage within the range of about 10 to about 200 mg may be useful for patients exhibiting greater than about 5% focal binding of AAF. In one aspect, highly AAF positive classification of AR in a tumor could result in a different dosing regimen than classification as both AAF positive and diffusely staining cells, due to a higher degree of activation in the highly AAF positive tumor. In contrast, a predominantly diffuse staining pattern (indicating unactivated AR) would indicate that treatment with an anti-androgen treatment is not warranted.

[0070] In yet another aspect, the invention provides methods for screening antitumor drugs and antitumor drug candidates for the ability to inactivate AAF. These methods are useful to identify additional AAF-active anti-androgens which may be candidates for use in treating of AAF-positive tumors according to the methods of the invention. Accordingly, the method for screening an antitumor drug or antitumor drug candidate for AAF inactivating activity comprises:

a) providing cancer cells or a primary tumor tissue specimen wherein cancer cells in the tissue specimen express AAF and the AAF are detectably stained with an anti-androgen receptor antibody;

b) exposing the cells or tumor tissue specimen to the antitumor drug or antitumor drug candidate; and

c) detecting a decrease in detectable staining of the AAF compared to staining of the AAF prior to exposure to the antitumor drug or antitumor drug candidate as an indication of AAF inactivating activity of the antitumor drug or detecting no substantial decrease in detectable staining of the AAF compared to staining of the AAF prior to exposure to the antitumor drug or antitumor drug candidate as an indication of lack of AAF inactivating activity of the antitumor drug.

[0071] In an alternative embodiment of the foregoing screening method, two AAF expressing tumor tissue specimens or two samples of AAF-expressing cancer cells from the same tumor can be used for comparison. In this embodiment, both of the tumor tissue specimens, or both of the cancer cell samples, are detectably stained for AAF with an anti- androgen receptor antibody, then one of the specimens or samples is exposed to the antitumor drug or antitumor drug candidate and the other is not. If the staining of the AAF in the treated specimen or sample is decreased compared to the untreated specimen or sample, the antitumor drug or antitumor drug candidate, the antitumor drug or antitumor drug candidate has AAF inactivating activity. Conversely, if the staining of the AAF in the treated specimen or sample is not decreased compared to the untreated specimen or sample, the antitumor drug or antitumor drug candidate, the antitumor drug or antitumor drug candidate does not have AAF inactivating activity. The foregoing screening method will provide further understanding of the mechanism of action of known anti-androgens and anti-androgens yet to be discovered. Those which are AAF-active (i.e., have AAF inactivating activity) will likely be useful in treating tumors in patient populations identified as having AAF-positive tumors using the methods of the invention. Examples of anti-androgens that may be screened as AAF-active include any of the anti-androgens approved for use by regulatory authorities and any of the unapproved anti-androgens in development.

[0072] If an antitumor drug is negative for AAF activity in the screening method described above, the lack of ability to inactivate AAF can be interpreted as an indication that the antitumor drug may be effective in combination therapy with a an AAF-active anti- androgen due to complementarity of the different mechanisms of action. For example, an AAF-active anti-androgen may be used in combination with additional treatment that does not act by an AAF inactivation mechanism (e.g., antibody therapeutics such as bevacizumab) to achieve improved therapeutic efficacy as compared to either agent alone. Alternatively, an AAF-active anti-androgen may be used in combination with one or more conventional chemotherapeutic agents which are negative for AAF activity in the screening assay to achieve improved therapeutic efficacy as compared to either agent alone (e.g., mitozantrone, doxorubicin, , paclitaxel, docetaxel, or carboplatin).

[0073] In yet another aspect, detecting the presence of focal distribution of the antibody to AAF in the nuclei may be used as an indication that the tumor of a patient previously treated with an antitumor drug, which has become resistant to that drug, is still sensitive to an AAF- inactivating anti-androgen. In one aspect, the method can be adapted to determine whether chemoresistance of a tumor resulting from previous chemotherapy can be reversed by treatment with an AAF-inactivating anti-androgen. Reversal of such chemoresistance may be based on the different mechanisms of action of the previous chemotherapy and the AAF- inactivating anti-androgen.

Example 1

[0074] Ten tumor tissue samples from patients diagnosed with prostate cancer

(adenocarcinomas) and having a tumor content >20 were obtained from Indivumed GmbH (Hamburg, DE). The tissues had been fixed in 4% formalin fixative and embedded in paraffin (FFPE). Basic clinical/medical data for each sample including age, gender, height and weight of the patient (BMI), surgical diagnosis, grading, TNM classification, UICC stage, pathological/histological diagnosis, exact ischemia time and Gleason Score were provided by Indivumed.

[0075] Immunohistochemistry (IHC) was performed on 3-4 μιη sections of the prostate tumor tissues. The sections were deparaffinized, hydrated and washed in working buffer (0.05 mol/L Tris/HCl, 0.15 mol/L NaCl, 0.05% Tween 20, pH 7.6, Dako, Denmark, code S3006). Antigen retrieval was carried out with the Dako Target Retrieval Solution pH 9 (Tris/EDTA buffer, pH 9, Dako, Denmark, code S2367) in a water bath at 98°C for 20 min. Then, the sections were covered with the Dako Peroxydase Block solution at room temperature (RT) for 5 min in order to block endogenous peroxides (Dako En Vision® +/HRP Rabbit (DAB+) Kit, Dako, Denmark, code K4011), washed and incubated with the primary antibodies at the optimal dilutions at RT for 60 min in a humidified chamber (Table 1). Following a 5-min. wash with working buffer, the appropriate Dako Labelled Polymer (Dako En Vision® +/HRP Rabbit (DAB+) Kit, Dako, Denmark, code K4011 or Dako En Vision® +/HRP Mouse (DAB+) Kit, Dako, Denmark, code K4007) was used for the detection of the primary antibody binding at RT for 30 min. Chromogen (DAB) was then carried out with Substrate-Batch at RT for 5-10 min and the sections were lightly counterstained with Gill's hematoxylin n°2.

Table 1. Antibodies used for immunohistochemistry

Antibody Clone Dilutions Host / Isotype Supplier Code against

A AR27 1:50 (0,9 μg/m\) Mouse IgGl Novocastra AR-318-CE

PR A 16 1:100 (3.6 μg/m\) Mouse IgGl Novocastra PGR-312-L-CE

PR B SAN27 1:100 (0.4 μ§/ΓηΙ) Mouse IgGl k Novocastra PGR-B-CE

ER SP1 1:200 (5 μ§/ΓπΙ) Rabbit IgG ThermoScientific MA1-39540

[0076] Negative controls were obtained by substitution of the primary antibodies with isotype controls (Table 2) or with antibody diluent alone (wash buffer negative control) in the immunohistochemichal staining procedure.

Table 2. Isotype controls used for immunohistochemistry

Antibody against Clone Dilutions Host / Isotype Supplier Code

Negative control DAK-GOl 1:125 (0.8 μg/ml) Mouse IgGl Dako X0931

Mouse

Negative control - 1:3000 (5 μg/ml) Rabbit IgG Dako X0936

Rabbit

[0077] Immunohistochemistry analysis was performed using a Zeiss Axioscope microscope, equipped with an Imaging Model ROHS digital camera. Immunoreactive signals were classified as unequivocal brown labeling of tumor cell nuclei. The intensity of labeling was defined as 0 for negative, + for weak, ++ for moderate and +++ for strong. On each labeled sample, positivity rate was defined as the percentage of labeled tumor cells in the entire tumor tissue, excluding necrotic areas. [0078] The results are presented in the Table 3 below. The % tumor cells is the percentage of prostate cancer cells in the specimen, the remainder being normal prostate cells, as usually found in early stage routine biopsies. This result essentially shows that the diagnosis was made on a relatively small fraction of malignant cells. % positivity refers to the percentage of tumor cells expressing AR. As shown, usually about 90 to 100% of the cancer cells were AR positive as expected. Intensity is measured as a score from 1 = weak to 3 = strong staining. As is typical, prostate cancer cells in these cases were strongly expressing AR, which is expected early in the disease history. Nuclei Pattern is the information gathered from the invention: 7 cases had a D pattern which means that even though AR is present, it doesn't participate in proliferation. This is a in agreement with recent development where it is observed that AR is in fact over-exposed to androgen hormones, raising doubt about its actual implication as a driving factor in early disease (Shifting the Paradigm of Testosterone and Prostate Cancer: The Saturation Model and the Limits of Androgen-Dependent Growth Abraham Morgentaler a,*, Abdulmaged M. Traish in European Urology 55 ( 2 0 0 9 ) 310- 321). 3 samples are described as AD i.e. a mix of D cells and A cells suggesting that these cancer samples include a subset of cells that have activated AR and thus targetable. In this small patient sample, only 4 cases would benefit from treatment at this stage.

Table 3

% tumor

cells % Positivity Intensity Nuclei Pattern

20 90 3 AD

30 80 2 D

20 50 2 D

40 90 3 D

30 100 3 D

25 90 3 AD

55 90 3 D

25 90 3 AD

30 90 3 D

20 80 2 D

[0079] Two subnuclear patterns were observed on IHC: 1) a homogeneous diffuse pattern with even staining of the nuclei, and 2) a granular patter of aggregates (AAF foci) on a background of diffuse staining. The cells with a homogenous and diffuse staining were characterized as "D" (Diffuse), cells with a granular pattern and diffuse staining were characterized as "AD" (Aggregates and Diffuse cell type mix). Thus any combination of D cells with A cells in the cancer tissue indicates that the AR is transcriptionally active in the tissue and anti-androgen treatment would be effective. However, the absence of aggregates in the cells of the tissue indicates that the anti-androgen treatment would be ineffective.

Example 2

[0080] The ten prostate cancer samples were processed for immunohistofluorescence (IHF) analysis.

[0081] Immunohistofluorescence was performed using a Zeiss fluorescent microscope equipped with a CCD camera and Smart Capture software, specific for capture of fluorescent images. IHF was performed on 3-4 μιη sections of the archival prostate tumor tissues. The sections were deparaffinized, hydrated and washed in working buffer (0.05 mol/L Tris/HCl, 0.15 mol/L NaCl, 0.05% Tween 20, pH 7.6, Dako, Denmark, code S3006). Antigen retrieval was carried out with the Dako Target Retrieval Solution (modified citrate buffer, pH 6.1, Dako, Denmark, code S1699) in a water bath at 98°C for 20 min. Then, the sections were incubated with the primary anti-AR antibodies at the appropriate optimal dilutions at RT for 60 min in a black humidified chamber. Following a 5 -minute wash with working buffer, appropriate secondary antibody conjugated to Alexa Fluor 488 was used for the detection of the primary antibody binding at RT for 30 min (Anti-mouse IgG (H+L), F(ab')2, Cell Signaling, USA, code 4408S, dilution 1: 1000 ; Anti-rabbit IgG (H+L), F(ab')2, Cell Signaling, USA, code 4412S, dilution 1: 1000). All slides were then washed and coverslipped using Vectashield® HardSet Mounting Medium (Vector Labs, USA, code H-1400) and stored refrigerate in the dark until analysis, to preserve fluorescence. Negative controls were obtained by substitution of the primary antibodies with isotype control mouse IgGl or rabbit serum (see IHC table) or with antibody diluent alone (wash buffer negative control) in the immunohistofluorescence staining procedure.

[0082] For each labeled tumor sample, positive focal distribution was defined as the percentage of labeled tumor cells in the entire tumor tissue, excluding necrotic areas. Various subnuclear patterns were observed: 1) a diffuse pattern with even staining of the nuclei, 2) a finely granular pattern with aggregates measuring about 0.1 micron, and 3) large aggregates measuring about 1 micron. At the tissue level, heterogeneity in the staining of cells was observed with respect to their nuclei pattern. Cells with a homogenous and diffuse staining are characterized as "D" (Diffuse), cells with a fine granular pattern are characterized as "FA" (Fine Aggregates), and cells including large aggregates are characterized as "LA" (Large Aggregates). Thus any combination of D cells with FA or LA cells in the cancer tissue indicates that the AR is transcriptionally active in the tissue and anti-androgen treatment would be effective. However, the absence of aggregates in the cells of the tissue indicates that the anti-androgen treatment would be ineffective. The coexistence of FA or LA with D describes the "AD" phenotype (for Aggregates and Diffuse type cell mix), as opposed to "D" phenotype where only D cells are present. If there is a majority of FA or LA cells, the phenotype is referred to as "A".

[0083] IHF showed that seven samples had a D phenotype and three had an AD phenotype. In each sample, normal tissue was used as an internal control and was always of the D phenotype. ER, PRA and PRB nuclear receptors were tested and all were negative in these samples.

[0084] Conclusions: Thus, two basic patterns were found: a diffuse AR nuclear staining

"D," indicating an absence of activated ARs, as shown in Fig. 2, and heterogeneous staining "A" where aggregates, indicating AAF, can be recognized within the nucleus of the cells, as shown in Fig. 3. A section of Fig. 2 is enlarged in Fig. 4A and a section of Fig. 3 is enlarged in Fig. 4B to show the distinct difference in staining patterns. The "D" nuclei appear evenly stained in Fig. 4A, but Fig. 4B clearly shows a pattern of unstained areas and darkly stained areas in the "A" nuclei. AR foci are visibly larger than elements of a diffuse D pattern. These basic staining patterns were observed upon staining of tissue samples with anti-AR antibodies using IHC and in samples processed using IHF. The diffuse D pattern was similar to the results obtained in gene-engineered cells that express a fluorescent receptor when no steroid or steroid-agonist is present.

[0085] The active A pattern observed in formalin fixed, paraffin embedded tumor tissue may differ from images obtained in fresh cells. This is expected because formalin- fixation and paraffin embedding tissue will result in changes to the cellular contents, thereby resulting in a different pattern of AR. Another difference relative to the research publications which utilized IHF is related to the method. In the research setting, a confocal microscope (i.e. using two laser beams) provides high resolution and 3D images. The IHC pattern results from a chemical reaction that modifies the cellular content. In contrast to IHC, a traditional wide- field microscope is used for reading the standard tumor slices (e.g., 4 microns). The IHC technique described results in some loss of resolution.

[0086] The IHF technique is less chemically aggressive for tumor tissues, in that it does not alter the microscopic cellular architecture. IHF requires specialized, equipment, a pathologist experienced with the technique, and is much more time-consuming. IHF cannot be easily coupled with other pathology analyses such as standard histology that requires formalin- fixed paraffin embedded tissues. Thus, in one aspect, IHC may be used as a routine pathological laboratory procedure. In the developed IHC technique used herein, 4 micrometer tissue sections (a commonly used thickness for routine clinical analysis) were used for all analyses.

Example 3 - Activated androgen receptor in androgen-independent prostate cancer

[0087] After androgen- suppressive treatment with drugs such as Casodex or flutamide, prostate cancer inevitably progresses again. Nevertheless, androgen antagonists may still work. Recently, more potent drugs (abiraterone, enzalutamide) have been found to work in about half of the cases but there is no way to predict which patients will benefit.

[0088] A study of 51 androgen-resistant tumor samples was conducted to determine the presence and the frequency of the activated androgen receptor pattern. Tissues samples were obtained from patients with prostate cancer progressing on standard first-line androgen- suppression therapy. The samples were obtained from Hospital Foch, Suresnes, France. The immunohistochemistry was performed as described previously. The results were: one case had no analyzable tumor tissue, 5 cases did not express AR at all, and 45 cases expressed an average of 48% AR positive tumor cells (range 1 - 90%). In 28/45 (62%) AR positive cases a Diffuse (D) pattern was observed. The AD pattern was observed in 17/45 (38%) of AR positive cases. Cases with the AD pattern, i.e. the mix of D and A cells, are expected to benefit from treatment, while D cases which consist of cells with no constitutively activated AR are not expected to benefit from treatment.

Example 4 - Activated androgen receptor in breast cancer

[0089] It is generally reported that about two-thirds of breast cancers have cells expressing the AR. Anti-androgen treatments with recently developed potent anti-androgens have been tested (5 studies with abiraterone and 5 studies with enzalutamide), demonstrating the potential for efficacy of anti-androgens (source clinicaltrial.gov). In this study, 490 cases were obtained from Oscar-Lambret Cancer Cencer, Lille, France and analyzed. Mean Age was 57 (17-89). Histology was ductal 82%, lobular 15%, and other 3%; Disease Stage was I 44%, II 48%, III 8%. Tumor histology Grade was I 25%, II 50%, III 25%. Breast cancer tumors were positive for detection of the AR (AR-pos) in 51% of the cases. Activated AR status was diffuse (D-AR) in 66% and aggregated (activated, A-AR) in 34% of the AR-pos biopsies. AR-pos and D-AR were associated with lower tumor histology grade (p <0. 04, p = 0. 1), ERa (p <0. 000, = 0. 03), and PR (p <0. 000, 0. 1), but not HER2, Ki67, or Staging. Conclusions: A-AR is associated with grade and other steroid receptors indicating biological relevance. Thus, in breast cancer the Activated pattern of AR would indicate patients which may benefit from anti- androgen treatment by indicating which cancer cases have constitutively active AR which could be antagonized

Example 5

[0090] Prostate tumor cell lines are grown in monolayers, with a culture media adapted for each line, after amplification for 3 days. Each cell line is cultured in either Normal FBS or charcoal-stripped FBS. Each serum condition is exposed to growth factors or hormones: either Vehicle, RPMI, testosterone, DHT, EGF or FGF. Each experimental setting is supplemented with Vehicle (DMSO), and an anti-androgen test substance such as enzalutamide. Cell viability is assessed with trypan blue exclusion.

[0091] For cytoblocks, WB and cytotoxic assays, cells are incubated in 75 cm² flasks,

25 cm flasks and 6 well plates, respectively, and treated with hormones and anti-androgen test substance at T=0 hours and retreated similarly at T=4 days. For cytoblocks and WB, the cells are collected at T=6 hours, T=4 days and T=7 days. For cytotoxicity assays, the cells are observed at T=7 days. The experiment is performed twice with duplicate conditions within each experiment.

[0092] The in vitro cytotoxic activity of the test substance is revealed by an MTS assay. For each condition of treatment, the ratio between growth factor or hormone treated only and control (RPMI) conditions is calculated to show the effect of hormone on growth. The ratio of anti-androgen test substance + growth factor over growth factor (or control) is calculated to estimate anti-androgen effect of the reference condition.

[0093] Cytoblocks of formalin-fixed paraffin embedded cells are produced at time=6 hours, 4 days and 7 days. Test blocks are evaluated with Hematin-Eosine Saffran and IHC for AR receptors. If the coloration is too intense for nuclear analysis, separated HES slides and AR immunochemistry w/o background may be performed.

[0094] IHC for AR is performed on 3-4 μιη sections. The sections are deparaffinized, hydrated and washed in working buffer. Antigen retrieval is carried out. Then, the sections are covered with the Dako Peroxydase Block solution to block endogenous peroxides at room temperature (RT) for 5 min., washed and incubated with the primary antibodies at the appropriate optimal dilutions at RT for 60 min. in a humidified chamber. Following a 5-min. wash with working buffer, the Dako Labelled Polymer is used for the detection of the primary antibody binding at RT for 30 min. Chromogen (DAB) is then carried outwith Substrate-Batch at RT for 5-10 min. and the sections are lightly counterstained with Gill's hematoxylin no. 2. Negative controls are obtained by substitution of the primary antibodies with isotype control mouse IgGl or with antibody diluent alone (wash buffer negative control) in the immunohistochemical staining procedure. Immunohistochemical analysis is performed using a Zeiss Axioscope microscope, equipped with a digital camera or scanner.

[0095] Immunoreactive signals are analyzed at x40 magnification and classified as unequivocal brown labeling of tumor cell nuclei. The intensity of labeling is defined as 0 for negative, + for weak, ++ for moderate and +++ for strong. The percentage of tumor positive cells is calculated. Subsequently, subnuclear morphology is analyzed at xlOO magnification using a microscope and photos are scanned at high resolution with a Zeiss microscope. As described previously, subnuclear morphology is described as D when a diffuse staining is observed or FA/LA when a mottled focal binding pattern is observed. The sample is characterized as AD when cells of D and FA or LA are mixed.

[0096] It is found that when cells express the D staining pattern there is no sensitivity to the anti-androgen test substance, even if the cells are AR-positive by conventional methods. All cells that are sensitive to the anti-androgen test substance are either FA or LA staining pattern. It is also observed that cells convert from FA or LA pattern to D pattern even when they are growing with various stimuli other than hormones. This indicates a biological effect that does not affect viability when the stimulus is of a different nature. In controls, there is no change in AAF present at baseline.

[0097] It is demonstrated that AR itself is not predictive of a drug effect, but baseline expression of AAF is predictive of a drug effect. Further, the conversion of AAF into an inactive state signals a biological event relevant to the receptor.

[0098] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the method and apparatus of the present invention without departing from the spirit and scope of the invention. Thus, it is intended that the present invention include modifications and variations that are within the scope of the appended claims and their equivalents.

Claims

What is claimed is:

1. An in vitro method for identifying a tumor treatable with an AAF-active anti-androgen, comprising:

a) exposing cancer cells or a primary tumor tissue specimen containing cancer cells obtained from a patient to an anti-androgen receptor antibody under conditions appropriate for binding of the antibody to androgen receptors in nuclei of the cancer cells; and

b) detecting presence or absence of focal binding of the antibody to androgen receptors in the nuclei;

wherein the presence of focal binding indicates sensitivity of the tumor to treatment with the AAF-active anti-androgen and the absence of focal binding indicates lack of sensitivity of the tumor to treatment with the AAF-active anti-androgen.

2. The method of claim 1, further comprising detecting presence or absence of diffuse binding of the antibody to androgen receptors in the nuclei.

3. The method of claim 1 or 2, wherein presence or absence of focal binding is detected by fluorescence.

4. The method of claim 1 or 2, wherein presence or absence of focal binding is detected colorimetrically.

5. The method of claim 4, wherein presence or absence of focal binding is detected using peroxidase.

6. The method of any of claims 1-5, wherein presence or absence of focal binding is quantitated.

7. The method of claim 6, wherein presence or absence of focal binding is expressed as a percentage of positive cells.

8. The method of claim 6, wherein presence or absence of focal binding is expressed as a relative intensity or density of activated androgen receptor foci.

9. The method of any of claims 1-8, further comprising treating the patient with an AAF- active anti-androgen if focal binding is present.

10. The method of any of claims 1-9, wherein focal binding is detected in 1-100%, 5- 100%, 25-100%, 50-100%, 80-100%, 80-90%, 50-80% or 30-40% of cancer cell nuclei. A method for treating a tumor in a patient, comprising:

a) exposing cancer cells or a primary tumor tissue specimen containing tumor cells obtained from a patient to an anti-androgen receptor antibody under conditions appropriate for binding of the antibody to androgen receptors in nuclei of the cancer cells;

b) detecting presence or absence of focal binding of the antibody to androgen receptors in the nuclei; and

c) treating the patient with an AAF-active anti-androgen if focal binding is present. The method of claim 11, wherein the patient is treated with an AR antagonist.

The method of claim 11, wherein the patient is treated with an aromatase inhibitor. The method of claim 11, wherein the patient is treated with a non- AAF-active antitumor drug if focal binding is absent.

Use of an AAF-active anti-androgen for treatment of a tumor previously identified as AAF-positive.

The use of claim 15, wherein AAF-positive status of the tumor is determined in a method comprising;

a) exposing cancer cells or a primary tumor tissue specimen containing cells obtained from a patient to an anti-androgen receptor antibody under conditions appropriate for binding of the antibody to androgen receptors in nuclei of the cancer cells; and b) detecting presence or absence of focal binding of the antibody to androgen receptors in the nuclei;

wherein the presence of focal binding indicates sensitivity of the tumor to treatment with the AAF-active anti-androgen and the absence of focal binding indicates lack of sensitivity of the tumor to treatment with the AAF-active anti-androgen.

The use of claim 15 or 16, wherein the tumor is treated with an AR- antagonist.

The use of claim 17, wherein the tumor is treated with CYP17 inhibitor, an LH-RH agonist and/or an GnRh agonist.

A method for screening an antitumor drug or antitumor drug candidate for AAF inactivating activity which comprises:

a) providing cancer cells or a primary tumor tissue specimen containing cancer cells, wherein the cancer cells express AAF and the AAF are detectably stained with an anti-androgen receptor antibody; b) exposing the cells or tissue specimen to the antitumor drug or antitumor drug candidate; and

c) detecting a decrease in detectable staining of the AAF as an indication of AAF inactivating activity of the antitumor drug or antitumor drug candidate or detecting no substantial decrease in detectable staining of the AAF as an indication of lack of AAF inactivating activity of the antitumor drug or antitumor drug candidate.

The method or use of any of claims 1, 11, 15 or 19, wherein the anti-androgen receptor antibody is a monoclonal antibody.