WO2019195889A1

WO2019195889A1 - Methods of assessing risk of cancer recurrence and response to treatment

Info

Publication number: WO2019195889A1
Application number: PCT/AU2019/050323
Authority: WO
Inventors: Cameron SNELL
Original assignee: The University Of Queensland
Priority date: 2018-04-12
Filing date: 2019-04-11
Publication date: 2019-10-17

Abstract

The present invention relates generally to a method of identifying a subject at risk of recurrence of breast cancer following ablation of a breast cancer tumour, the method comprising: (i) obtaining a sample of breast cancer cells from a subject with breast cancer; (ii) contacting the sample with a first binding agent that binds to an estrogen receptor (ER) in the sample and a second binding agent that binds to a progesterone receptor (PR) in the sample; and (iii) detecting the first and second binding agents when bound to an ER-PR complex in the sample; wherein the level of expression of ER-PR complexes detected in the sample in step (iii) relative to a level of expression of ER-PR complexes detected in a control sample is indicative of the risk of recurrence of breast cancer in the subject.

Description

METHODS OF ASSESSING RISK OF CANCER RECURRENCE AND

RESPONSE TO TREATMENT

TECHNICAL FIELD

[0001] The invention relates generally to a method of determining the likelihood of cancer recurrence and response to cancer treatment in a subject, more particularly to a method of determining the likelihood of breast cancer recurrence and response to breast cancer treatment in a subject in need thereof.

BACKGROUND

[0002] According to Cancer Council Australia, breast cancer is the most common cancer in women in Australia and the second most common cancer to cause death in women, after lung cancer. Breast cancer is typically defined as the abnormal growth of the cells lining the breast lobules or ducts. These cells become malignant and have the capacity to metastasize to other areas of the body. Both men and women can develop breast cancer, although it is generally uncommon in men. Cancer Council Australia reports that in 2013, 15,902 women and 142 men were diagnosed with breast cancer in Australia and in 2014, 2814 women and 30 men died of breast cancer.

[0003] The risk of being diagnosed with breast cancer by age 85 is 1 in 8 for women and 1 in 631 for men. In Australia, the overall five year survival rate for breast cancer in females is 90%. If the cancer is limited to the breast, 96% of patients will be alive five years after diagnosis, noting that this figure excludes those who die from other diseases. If the cancer has spread to the regional lymph nodes, five year relative survival drops to 80%.

[0004] Surgery (also referred to as ablation) is still considered the primary treatment for breast cancer, the aim of which is the complete resection of the primary tumor, with negative margins to reduce the risk of local recurrences. This is typically followed by pathologic staging of the tumor and axillary lymph nodes to provide necessary prognostic information. Several different types of surgical procedures are available, including partial or segmental mastectomy and mastectomy. Partial or segmental mastectomy (also referred to as breast conserving surgery, lumpectomy or wide local excision), is typically defined as the complete surgical resection of a primary tumour within the breast with the goal of achieving widely negative margins ( e.g ., 1 cm). It may be performed with palpation guidance or with image guidance and is applicable in most patients with stage I or II invasive carcinomas. Whereas partial or segmental mastectomy involves the removal of the part of the breast affected by the cancer, mastectomy typically involves the total removal of all breast tissue to the clavicle superiorly, the sternum medially, the inframammary crease inferiorly, and the anterior axillary line laterally, with en bloc resection of the pectoralis major fascia. Variants to this approach include modified radical mastectomy (total mastectomy with axillary lymph node dissection (ALND)), radical mastectomy (total mastectomy with en bloc resection of the pectoralis major and ALND), extended radical mastectomy (radical mastectomy with resection of the internal mammary lymph nodes), skin-sparing total mastectomy (SSM) and nipple-sparing total mastectomy (NSM). In most cases, breast cancer surgery also involves the removal of one or more axillary lymph nodes. Patients may also receive adjunct therapy such as chemotherapy, radiotherapy, ovarian ablation and/or hormone therapy prior to surgery with a view to reducing the size of the tumour before surgery.

[0005] Breast conserving surgery followed by radiotherapy has been shown to be as effective as mastectomy for most women with early breast cancer, such that the chance of the breast cancer metastasizing to other parts of the body is, for most women, the same after either breast conserving surgery or mastectomy. However, breast conserving surgery followed by radiotherapy is typically associated with a higher incidence of recurrence (also referred to as relapse); that is, where the tumour reappears in the breast area. Regular clinical follow-up after treatment for breast cancer, such as regular physical examinations and breast imaging (mammogram and/or ultrasound), is therefore strongly recommended so as to ensure that if the breast cancer returns, or if a new breast cancer tumour develops, it can be treated promptly.

[0006] Recurrence can occur as: 1) local recurrence when the relapsing lesion is in the same area of the breast (ipsilateral) or in the mastectomy scar; 2) regional recurrence (lymph nodes in the ipsilateral axilla); and/or 3) distant recurrence (more frequently mediastinal lymph nodes, bones, lungs, liver and brain.). Studies have shown that around two-thirds of breast recurrences are local and occur more frequently within 5 years of the initial diagnosis. Age is also a relevant prognostic factor, with a greater incidence of recurrence in younger patients.

[0007] Current strategies for detecting breast cancer recurrence include breast examination, ultrasonography and magnetic resonance imaging (MRI). Digital tomosynthesis has also been used to evaluate radiographically dense breasts. However, these strategies have their limitations. For instance, as reported by Piva et al. (2017, Breast Cancer (Dove Med Press ).; 9:461-471), the sensitivity of clinical breast examination and mammography in the detection of local recurrences ranges from 55% to 68%, being influenced by an insufficient morphological distinction between therapy-induced edema and lymphangiosis carcinomatosa or between radially striated scar tissue and tumor recurrence. Moreover, the treated breast may be more difficult to position because of surgical deformities or may be relatively noncompressible because of pain or structural changes, such as the presence of hematoma, seroma, fat tissue necrosis, scar tissue development and dystrophic calcifications. Radiotherapy-induced alterations can include vascular dilatation, capillary damage, microcirculatory changes and consequent edema. The summation of these changes complicates the interpretation of clinical breast examination and mammography due to focal thickening, decreased compressibility and increased density at the surgical site.

[0008] Early detection of regional breast cancer recurrence in asymptomatic patients will typically have a positive influence on patient survival, although this can be difficult for several reasons. First, there is currently no reliable screening or diagnostic tool to evaluate the axillary and supra-clavicular lymph node areas, which are useful predictors of concurrent or subsequent distant metastasis. Moreover, the false-negative rate of the axilla at physical examination is relatively high, reaching up to 39%. Physical examination of regional lymph nodes is also hindered by factors such as anatomic location (beneath the clavicle or deep in subcutaneous fat tissue), scar tissue due to previous axillary lymph node dissection or radiation, obesity and small size of the recurrence. Since the false-negative rate of physical examination of the axilla is so high and the field of view of mammography does not include this entire area, ultrasonography after breast cancer surgery is often recommended, also in asymptomatic patients, as this will assist in detecting lymph node region recurrence. However, ultrasonography sensitivity is also limited, as it is user-dependent and may be diminished in the presence of small or noninvasive lesions, especially in fatty breasts.

[0009] MRI is typically superior to traditional imaging in the diagnosis of recurrence in the presence of scar tissue when performed at least 12-18 months after breast conserving surgery and at greater intervals from radiotherapy. It is to be noted, however, that MRI sensitivity in the diagnosis of suspected breast cancer recurrence may range from about 75% to 100% while specificity ranges from about 66% to 100%. Sensitivity and specificity are generally higher for MRI as compared with mammography or ultrasonography alone. MRI has also shown an extremely high negative predictive value (98.8%) in the detection of breast cancer recurrence, including lesions not related to the surgical scar, thus allowing avoidance of the need for further biopsy in the presence of negative MRI. However, given its significantly higher cost and lower availability, MRI is not used for routine surveillance of breast cancer recurrence and is typically only used in cases of suspected recurrence when clinical, mammographic and/or sonographic findings are inconclusive.

[0010] Despite recent advances in monitoring patients after breast cancer surgery, there is still a significant risk of recurrence, in particular following partial or segmental mastectomy. Hence, there remains a critical need to develop improved methods of determining whether a patient is at risk of recurrence following breast cancer surgery so that these higher risk individuals can be stratified to suitable post-surgical treatment regimens. The methods disclosed herein solve, or at least partly alleviate, these problems by allowing a patient’s risk of recurrence to be determined following breast cancer surgery. The methods disclosed herein can also be used to determine a patient’s response to breast cancer treatment.

SUMMARY OF THE INVENTION

[0011] In an aspect disclosed herein, there is provided a method of identifying a subject at risk of recurrence of breast cancer following ablation of a breast cancer tumour, the method comprising:

(i) obtaining a sample of breast cancer cells from a subject with breast cancer;

(ii) contacting the sample with a first binding agent that binds to an estrogen receptor (ER) in the sample and a second binding agent that binds to a progesterone receptor (PR) in the sample; and

(iii) detecting the first and second binding agents when bound to an ER-PR complex in the sample;

wherein the level of expression of ER-PR complexes detected in the sample in step (iii) relative to a level of expression of ER-PR complexes detected in a control sample is indicative of the risk of recurrence of breast cancer in the subject.

[0012] In another aspect disclosed herein, there is provided a method of predicting a subject’s response to a treatment regimen for treating breast cancer, the method comprising:

wherein the level of expression of ER-PR complexes detected in the sample in step (iii) relative to a level of expression of ER-PR complexes detected in a control sample is predictive of the subject’s response to the treatment regimen.

[0013] In another aspect disclosed herein, there is provided a kit comprising (i) a first binding agent that binds to an estrogen receptor (ER) in breast cancer cells, (ii) a second binding agent that binds to a progesterone receptor (PR) in breast cancer cells; and (iii) a third agent, wherein the third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex. [0014] In another aspect disclosed herein, there is provided a kit for use in the methods described herein, wherein the kit comprises (i) a first binding agent that binds to an estrogen receptor (ER) in breast cancer cells, (ii) a second binding agent that binds to a progesterone receptor (PR) in breast cancer cells; and (iii) a third agent, wherein the third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex.

[0015] In an aspect disclosed herein, there is provided a method of stratifying a subject to a treatment regimen for breast cancer, the method comprising:

(ii) contacting the sample with a first binding agent that binds to an estrogen receptor (ER) in the sample and a second binding agent that binds to a progesterone receptor (PR) in the sample;

(iii) detecting the first and second binding agents when bound to an ER-PR complex in the sample; wherein the level of expression of ER-PR complexes detected in the sample relative to a level of expression of ER-PR complexes detected in a control sample is indicative of the risk of recurrence of breast cancer in the subject; and

(iv) exposing the subject identified in step (iii) as being at risk of recurrence to a treatment regimen for treating breast cancer.

[0016] In another aspect disclosed herein, there is provided a complex formed by (i) a first binding agent that binds to an estrogen receptor (ER) of an ER-PR complex in the cell, wherein the ER-PR complex comprises an estrogen receptor (ER) and a progesterone receptor (PR); (ii) a second binding agent that binds to the PR of the ER-PR complex; and (iii) a third agent, wherein third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to the ER-PR complex in the cell.

[0017] In another aspect disclosed herein, there is provided a complex comprising (i) a first binding agent bound to an estrogen receptor (ER) of an ER-PR complex in a breast cancer cell, wherein the ER-PR complex comprises an estrogen receptor (ER) and a progesterone receptor (PR); (ii) a second binding agent bound to the PR of the ER-PR complex; and (iii) a third agent, wherein third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex in the cell.

[0018] In another aspect disclosed herein, there is provided a complex comprising a conjugate of an estrogen receptor (ER) and a progesterone receptor (PR), a first binding agent bound selectively to the ER, a second binding agent bound selectively to the PR and a third agent bound selectively to the first and second binding agents.

BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1 shows the development of a proximity ligation assay (PLA) to detect interactions between the estrogen and progesterone receptors in patient-derived breast cancer tumour explants and T47D cells. A. Breast cancer explants were treated with either Vehicle, estradiol (E₂), R5020 or the combination of E₂ and R5020. Explants were immunohistochemically stained for the estrogen receptor (ER), progesterone receptor (PR) and the ER-PR proximity ligation assay (PLA). Scale bar (bottom right, applies to all images) is 1 Omih B. Bar graph indicates mean number of ER-PR interactions and standard deviation per cell for three explants with each condition. C. T47D cells were treated with either Vehicle, E₂, progesterone (P) or the combination of E₂ and P. Cells were immunohistochemically stained for ER, PR and stained using the ER-PR PLA.

[0020] Figure 2 shows the association between PR expression with the number of ER- PR complexes by PLA and example photomicrographs. 227 patients with ER+, HER2- breast cancer with paired evaluable expression of PR by immunohistochemistry (Allred score) and ER-PR interactions by proximity ligation assay (signals/cell) are represented. Line indicates median with interquartile range. P value is the result of the Mann-Whitney U test. Example immunostains for ER, PR and PLA images for selected patients (red signals) are shown at each comer. Scale bar (bottom right, applies to all images) is 10pm.

[0021] Figure 3 shows Kaplan-Meier curves by PR expression and the number of ER- PR complexes (A) and the number of ER-PR complexes stratified by type of adjuvant endocrine agent taken (B). P values quoted are the result of the log-rank test. [0022] Figure 4 shows the receiver operating characteristic (ROC) curves of the number of ER-PR complexes detected per cell in tissue samples obtained from Tamoxifen- (A) and AI-treated (B) patients with relapse (recurrence).

[0023] Figure 5 shows the receiver operating characteristic (ROC) curves of ER expression in tissue samples obtained from Tamoxifen- (A) and AI-treated (B) patients with relapse (recurrence).

DETAILED DESCRIPTION

[0024] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0025] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0026] It is to be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. For example, reference to "an ER-PR complex" includes a single ER-PR complex, as well as two or more ER-PR complexes. Similarly, reference to "a tissue sample" includes a single tumour sample, as well as two or more tumour samples.

[0027] Nucleotide and amino acid sequences are referred to by sequence identifier numbers (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>l, <400>2, etc. A summary of sequence identifiers is provided herein.

[0028] The present invention is predicated, at least in part, on the inventors’ surprising finding that the level of expression of ER-PR interactions in breast cancer cells from a patient with breast cancer, as reflected in the number of ER-PR complexes detected in the cells, is detenninative of the risk of recurrence (or relapse) of breast cancer in the patient. Thus, in an aspect disclosed herein, there is provided a method of identifying a subject at risk of recurrence of breast cancer following ablation of a breast cancer tumour, the method comprising:

[0029] As used herein, the terms "ablation”, "ablate", "ablating" and the like refer to the substantial alteration of biological tissue, specifically, cancerous tissue or cells or tumours. The term also applies to the alteration of any hyperplastic growth. In embodiments disclosed herein, the terms "ablation”, "ablate", "ablating" and the like refer to the physical destruction or removal of target cells ( e.g ., cancer cells). Suitable methods for ablating tissue will be familiar to persons skilled in the art, illustrative examples of which include surgical ablation (e.g., surgical removal of the tumour or a portion thereof), cryoablation, thermal ablation, photoacoustic ablation, radiotherapy, chemotherapy, radiofrequency ablation, electroporation, alcohol ablation, high-intensity focused ultrasound, photodynamic therapy and the use of monoclonal antibodies and/or immunotoxins. Whilst it is generally desirable that ablation of a breast cancer tumour results in the destruction or removal of the entire tumour, in some embodiments ablation will result in the destruction or removal of only a portion of the breast cancer tumour (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the breast cancer tumour) from the subject. For example, access to the entire breast cancer tumour may be prohibited or otherwise obstructed by the presence of adjacent healthy tissue structures. [0030] The terms "subject", "patient", "individual" and the like are used interchangeably herein to refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, that is susceptible to developing breast cancer. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates ( e.g ., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes), rodents, lagomorphs, bovines, ovines, caprines, porcines, equines, canines, felines and avians. In an embodiment, the subject is a nonhuman primate. In an embodiment, the subject is a human. In an embodiment, the subject is a female. In an embodiment, the subject is a pre-menopausal female.

[0031] In an embodiment disclosed herein, the subject is on aromatase inhibitor (AI) therapy. A subject is considered to be“on aromatase inhibitor (AI) therapy” if they have been exposed to or otherwise administered an AI as part of a treatment regimen, preferably as part of a treatment regimen for breast cancer. The subject may have been exposed to or otherwise administered an AI before, during or subsequent to initial surgical intervention (e.g., partial, segmental or complete mastectomy). Suitable aromatase inhibitors will be familiar to persons skilled in the art, illustrative examples of which include anastrozole, exemestane and letrozole.

[0032] Suitable methods of obtaining a sample of breast cancer cells from a subject with breast cancer will be familiar to persons skilled in the art, whereby the cells are extracted, excised or otherwise removed from the subject. Illustrative examples of suitable methods include resection, biopsy and/or fine needle aspiration. As used herein, the phrase“sample of breast cancer cells” refers to any sample obtained from the subject that is reasonably expected to comprise one or more breast cancer cells, whether the breast cancer cells are derived from a primary breast cancer tumour or from a secondary (e.g, metastatic) carcinoma. For example, a sample may comprise one or more breast cancer cells that have been shed from the primary tumour or the secondary carcinoma and collected from biological fluids and tissue such as lymph fluid and/or lymph nodes ( e.g ., axillary lymph nodes). Illustrative examples of samples that are reasonably expected to comprise one or more breast cancer cells include breast cancer tissue, blood, serum, plasma, saliva, urine, peritoneal fluid, ascitic fluid, breast fluid, breast milk, lymph fluid and lymph nodes.

[0033] In an embodiment disclosed herein, the sample is selected from the group consisting of a primary breast cancer tumour, a secondary ( i.e ., metastatic) breast cancer carcinoma, blood, serum, plasma, saliva, urine, peritoneal fluid, ascitic fluid, breast fluid, breast milk, lymph fluid and lymph node. In another embodiment, the sample comprises lymph node tissue, an illustrative example of which includes axillary lymph node tissue. In another embodiment disclosed herein, the sample comprises a tissue biopsy of a primary or metastatic breast cancer tumour. In another embodiment, the sample comprises a resected primary or secondary breast cancer tumour.

[0034] In an embodiment disclosed herein, the sample comprises one or more breast cancer cells derived from a primary or secondary breast cancer tumour (e.g., a tissue biopsy, as herein before described).

[0035] In some embodiments disclosed herein, the sample comprises one or more breast cancer cells derived from a primary breast cancer tumor. Suitable samples can be obtained by any means known to persons skilled in the art, illustrative examples of which include tissue biopsy and/or surgical ablation. In some embodiments disclosed herein, the tissue sample is obtained from the primary breast cancer tumour that has been removed by surgical ablation (e.g., by partial, segmental or complete mastectomy).

[0036] The sample may be processed and analyzed in accordance with the methods disclosed herein almost immediately following collection (e.g, as a fresh sample), or it may be stored for subsequent processing and/or analysis. If storage of the sample is necessary or desirable, it would be understood by persons skilled in the art that it should ideally be stored under conditions that preserve the integrity of ER-PR complexes within the sample. In some embodiments disclosed herein, the sample is a fresh frozen tissue sample of a primary breast cancer tumour. In other embodiments, the sample is a formalin-fixed paraffin embedded (FFPE) tissue sample of a primary breast cancer tumour, such as those prepared by pathologists for immunohistochemical and/or histological analyses.

[0037] The term“carcinoma” would be understood by persons skilled in the art as a tumour comprising cells derived from putative epithelial cells that have become malignant. The term also encompasses a carcinoma in situ , which is often used to describe a carcinoma in its pre-malignant stage; that is, having the cytological appearance of a malignant carcinoma but showing no signs of invasion through the epithelial basement membrane.

[0038] As used herein, the term“binding agent” refers to an interactive molecule that is capable of binding specifically to its target antigen. Thus, in an embodiment disclosed herein, the first binding agent is capable of specifically binding to an estrogen receptor (ER) in the sample. In another embodiment disclosed herein, the second binding agent is capable of specifically binding to a progesterone receptor (PR) in the sample. In another embodiment, the first and second binding agents are capable of specifically binding to their respective target receptors in the sample. It is to be understood, however, that the first and/or second binding agents, as herein described, may possess at least some cross-reactivity with other proteins in the sample without negatively impacting the ability of the first and second binding agents to identify ER-PR complexes in the sample.

[0039] Suitable binding agents will be familiar to persons skilled in the art, illustrative examples of which include antibodies or antigen-binding fragments thereof. In an embodiment disclosed herein, the first binding agent is an antibody or an ER-binding fragment thereof. In another embodiment disclosed herein, the second binding agent is an antibody or a PR-binding fragment thereof. Antibodies or antigen-binding fragments suitable for use in accordance with the methods disclosed herein would be familiar to persons skilled in the art, illustrative examples of which include polyclonal antibodies, monoclonal antibodies, mono-specific antibodies, poly-specific antibodies (including bi-specific antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies and CDR-grafted antibodies. Illustrative examples of suitable antigen-binding fragments include Fab, F(ab)₂, Fv and scFv. Various techniques for producing antibodies or antigen-binding fragments thereof will be familiar to persons skilled in the art. Antibodies or antigen-binding fragments thereof may be derived from any species, illustrative examples of which include rat, mouse, goat, guinea pig, donkey, rabbit, horse, lama, camel, or any avian species ( e.g ., chicken, duck). Antibodies or antigen-binding fragments thereof may be of any suitable isotype, such as IgG, IgM, IgA, IgD, IgE or any subclass thereof.

[0040] In an embodiment, the first binding agent is a monoclonal antibody or an ER- binding fragment thereof. In an embodiment, the second binding agent is a monoclonal antibody or a PR-binding fragment thereof. The monoclonal antibodies can be a humanised or deimmunised form of a non-human antibody. In another embodiment, the monoclonal antibody is a human antibody.

[0041] In an embodiment disclosed herein, the first and/or second binding agents are selected for their ability to specifically bind to the target ER and PR antigens within an ER- PR complex, with minimal or negligible binding to non-complexed ER and PR. This may be achieved, for example, by selecting first and second binding agents that specifically bind to antigenic regions of the ER and the PR that are only exposed when the ER interacts with the PR (i.e., within an ER-PR complex). Alternatively, the first and/or second binding agents will specifically bind to the target ER and PR in the sample, whether the ER and PR are within an ER-PR complex or not.

[0042] The terms "bind", "binding" and the like, as used herein, mean an event in which one substance, such as a binding agent, directly or indirectly interacts with a target molecule in such a way that the interaction with the target may be detected. In some examples, a binding agent may react with a target, or directly bind to a target, or indirectly react with or bind to a target by directly binding to another substance that in turn directly binds to or reacts with a target. The terms "specific for", "specifically" and the like, as used herein in the context of describing binding between two or more entities, mean that the binding is through a specific interaction between complementary binding partners, rather than through non specific aggregation.

[0043] Estrogen receptors are typically classified into two groups: ER-alpha (ERa, ESR1) and ER-beta (ERp, ESR2). ERa is encoded by genes located on chromosome 6, whereas the ERP is encoded by genes located on chromosome 14. ERa is predominantly expressed in mammary glands, pituitary, hypothalamus, ovarian theca cells and the reproductive tract. In contrast, ERP is primarily expressed in ovarian granulosa cells, lung and prostate cells.

[0044] In an embodiment, the first binding agent is capable of binding specifically to ERa. In an embodiment, the ER is a human ERa comprising the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 70% sequence identity thereto:

[0045] SEQ ID NO: 1 - Human ERa (GenBank accession no. : AAA52399.1)

MTMTLHTKAS GMALLHQIQG NELEPLNRPQ LKIPLERPLG EVYLDSSKPA VYNYPEGAAY EFNAAAAANA QVYGQTGLPY GPGSEAAAFG SNGLGGFPPL NSVSPSPLML LHPPPQLSPF LQPHGQQVPY YLENEPSGYT VREAGPPAFY

RPNSDNRRQG GRERLASTND KGSMAMESAK ETRYCAVCND YASGYHYGVW

SCEGCKAFFK RSIQGHNDYM CPATNQCTID KNRRKSCQAC RLRKCYEVGM

MKGGIRKDRR GGRMLKHKRQ RDDGEGRGEV GSAGDMRAAN LWPSPLMIKR SKKNSLALSL TADQMVSALL DAEPPILYSE YDPTRPFSEA SMMGLLTNLA DRELVHMINW AKRVPGFVDL TLHDQVHLLE CAWLEILMIG LVWRSMEHPV KLLFAPNLLL DRNQGKCVEG MVEIFDMLLA T S SRFRMMNL QGEEFVCLKS IILLNSGVYT FLSSTLKSLE EKDHIHRVLD KITDTLIHLM AKAGLTLQQQ

HQRLAQLLLI LSHIRHMSNK GMEHLYSMKC KNVVPLYDLL LEMLDAHRLH

APTSRGGASV EETDQSHLAT AGSTSSHSLQ K Y YIT GE AEG FPATV

[0046] In an embodiment, the ER comprises an amino acid sequence that has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or preferably at least 99% sequence identity to SEQ ID NO: 1.

[0047] Human PR is typically encoded by a single gene located on chromosome 11 (Hq22-q23). Expression of PR isoforms is controlled by two promoters to produce two major mRNA transcripts that encode two proteins. The full-length 116 kDa PR-B is controlled by the distal PR-B promoter region and the 94 kDa PR-A is controlled by the proximal PR-A promoter region. [0048] In an embodiment, the second binding agent is capable of binding specifically to PR. In an embodiment, the PR is a human PR comprising the amino acid sequence of SEQ ID NO:2 or a sequence having at least 70% sequence identity thereto:

[0049] SEQ ID NO:2 - Human PR (GenBank accession no.: AAA60081.1)

MTELKAKGPR APHVAGGPPS PEVGSPLLCR PAAGPFPGSQ TSDTLPEVSA

IPISLDGLLF PRPCQGQDPS DEKTQDQQSL SDVEGAYSRA EATRGAGGSS SSPPEKDSGL LDSVLDTLLA PSGPGQSQPS PPACEVTSSW CLFGPELPED

PPAAPATQRV LSPLMSRSGC KVGDSSGTAA AHKVLPRGLS PARQLLLPAS

ESPHW SGAPV KPSPQAAAVE VEEEDGSESE ESAGPLLKGK PRALGGAAAG GGAAAVPPGA AAGGVALVPK EDSRFSAPRV ALVEQDAPMA PGRSPLATTV MDFIHVPILP LNHALLAART RQLLEDESYD GGAGAAS AF A PPRSSPCASS TPVAVGDFPD CAYPPDAEPK DDAYPLYSDF QPPALKIKEE EEGAEASARS

PRSYLVAGAN PAAFPDFPLG PPPPLPPRAT PSRPGEAAVT AAPASASVSS

ASSSGSTLEC ILYKAEGAPP QQGPFAPPPC KAPGASGCLL PRDGLPSTSA

SAAAAGAAPA LYPALGLNGL PQLGYQAAVL KEGLPQVYPP YLNYLRPDSE

ASQSPQYSFE SLPQKICLIC GDEASGCHY G VLTCGSCKVF FKRAMEGQHN YLCAGRNDCI VDKIRRKNCP ACRLRKCCQA GMVLGGRKFK KFNKVRVVRA LDAVALPQPV GVPNESQALS QRFTFSPGQD IQLIPPLINL LMSIEPDVIY AGHDNTKPDT SSSLLTSLNQ LGERQLLSVV KWSKSLPGFR NLHIDDQITL

IQYSWMSLMV FGLGWRSYKH VSGQMLYFAP DLILNEQRMK ESSFYSLCLT

MWQIPQEFVK LQVSQEEFLC MKVLLLLNTI PLEGLRSQTQ FEEMRSSYIR

ELIKAIGLRQ KGVVSSSQRF YQLTKLLDNL HDLVKQLHLY CLNTFIQSRA LSVEFPEMMS EVIAAQLPKI LAGMVKPLLF HKK

[0050] In an embodiment, the PR comprises an amino acid sequence that has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or preferably at least 99% sequence identity to SEQ ID NO:2.

[0051] As noted elsewhere herein, the present invention is predicated, at least in part, on the inventors’ surprising finding that the level of expression of ER-PR interactions in breast cancer cells from a patient with breast cancer, as reflected in the level of expression of ER-PR complexes detected in breast cancer cells (e.g., the number of ER-PR complexes detected in breast cancer cells), is determinative of the risk of recurrence (or relapse) of breast cancer in the patient.

[0052] In an embodiment, ER-PR complexes are detected by selecting, as noted elsewhere herein, first and second binding agents that bind specifically to the target ER and PR antigens within an ER-PR complex, with minimal or negligible binding to non- complexed ER and PR. This may be achieved, for example, by selecting first and second binding agents that specifically bind to antigenic regions of the ER and the PR that are only exposed when the ER interacts with the PR (i.e., within an ER-PR complex). Hence, the detection of the first and second binding agents in the sample is indicative of the presence of an ER-PR complex in the sample. The first and second binding agents (bound to an ER- PR complex in the sample) can be detected by any means known to persons skilled in the art. In an embodiment, the first and second binding agents comprise a detectable moiety.

[0053] Suitable detectable moieties will be familiar to persons skilled in the art, illustrative examples of which include any molecule that may be detected directly or indirectly so as to reveal the presence of a target (e.g., ER and PR) in the sample. Examples of detectable moieties that may be used in accordance with the present invention include fluorophores, radioactive isotopes, chromophores, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, beads or other solid surfaces, gold or other metal particles or heavy atoms, spin labels, haptens, myc, nitrotyrosine, biotin and avidin. Others include phosphor particles, doped particles, nanocrystals or quantum dots.

[0054] In an embodiment disclosed herein, a direct detectable moiety is used. Direct detectable moieties may be detected per se without the need for additional molecules. In another embodiment, an indirect detectable moiety is used, which requires the employment of one or more additional molecules so as to a form detectable molecular complex. Suitable indirect detectable moieties will be familiar to persons skilled in the art, illustrative examples of which include biotin, avidin, peroxidase, and short nucleic acid sequences (oligonucleotides). [0055] In an embodiment, the first and second binding agents comprise the same detectable moiety. In another embodiment, each of the first and second binding agents comprise a different detectable moiety. In an illustrative example, the first binding agent may comprise a red fluorophore ( e.g ., Red 613, Texas Red) and the second binding agent may comprise a yellow fluorophore (e.g., Cy3, Alexa fluor 546/555), whereby the detection of orange fluorescence is indicative of the presence of the first and second binding agents bound to an ER-PR complex in the sample.

[0056] In an embodiment of the methods disclosed herein, step (iii) comprises contacting the sample with a third agent, wherein the third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex in the sample.

[0057] The third agent can be used to increase the sensitivity of detection of the methods described herein. Alternatively, or in addition, the third agent may comprise the detectable moiety for detecting the first and second binding agents in accordance with the methods described herein. In some embodiments, the third agent comprises a binding agent. As used herein, the term "third binding agent" means any substance that is capable of binding to or otherwise recognizing the first and/or second binding agents, as herein described. Suitable third binding agents would be familiar to persons skilled in the art, illustrative examples of which include antibodies, or antigen-binding fragments thereof, such as polyclonal, monoclonal, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated and CDR-grafted antibodies, as described elsewhere herein. In an embodiment, the third binding agent is an antibody or an antigen-binding fragment thereof that binds specifically to the first and/or second binding agents. In an embodiment, the third binding agent comprises a detectable moiety, as described elsewhere herein. The detectable moiety of the third binding agent may be the same as the detectable moiety of the first and/or second binding agents (where present) or it may be different to the detectable moiety of the first and/or second binding agents (where present).

[0058] In another embodiment disclosed herein, the ER-PR complex (to which the first and second binding agents are bound) is detected by a method selected from the group consisting of a proximity ligation assay (PLA), biomolecular fluorescence complementation, chemical cross-linking followed by high mass MALDI mass spectrometry and fluorescence resonance energy transfer (FRET). In an embodiment, the ER-PR complex is detected by PLA.

[0059] A PLA is a sensitive technique that utilises first and second binding agents to detect the interaction of two or more target proteins with high specificity and sensitivity. Targets proteins can typically be detected and localized with single molecule resolution and objectively quantified in unmodified cells and tissues. Each of the two binding agents (the first and second binding agents; also referred to as the primary binding agents) will bind specifically to the two or more proteins of interest ( e.g ., ER and PR). In an embodiment, each of the first and second (primary) binding agents will typically comprise a detectable moiety such as a short nucleic acid molecule (e.g., an oligonucleotide).

[0060] In an embodiment, each of the first and second binding agents comprises a detectable moiety. Suitable detectable moieties will be familiar to persons skilled in the art, an illustrative example of which includes a unique short nucleic acid molecule (e.g., a short oligonucleotide strand). In an embodiment, the detectable moiety comprises, consists, or consists essentially of a short oligonucleotide strand. Thus, when the first and second binding agents are in close proximity (i.e., where the two target proteins to which the first and second binding agents are bound are in close proximity, such as part of an ER-PR protein complex), the short oligonucleotides of the first and second binding agents can be detected by performing nucleic acid amplification with appropriate substrates and enzymes. Suitable methods of nucleic acid amplification will be familiar to persons skilled in the art, an illustrative example of which includes rolling circle DNA amplification. Once the nucleic acid has been amplified, a complementary nucleic acid molecule can be used to detect the amplified nucleic acid. For example, fluorescent-labeled complementary oligonucleotide probes can be added, which will bind to the amplified nucleic acid. The resulting high concentration of fluorescence can be visualized under fluorescence.

[0061] In an embodiment, the ER-PR complex is detected in a cell nucleus.

[0062] As used herein, the term "risk" denotes a subject's likelihood, based on the level of expression of ER-PR complexes in a sample of breast cancer cells from the subject, of the recurrence of breast cancer after breast cancer ablation. Accordingly, the terms "risk" and "likelihood" are used interchangeably herein, unless otherwise stated.

[0063] It would be apparent to persons skilled in the art that the risk of recurrence of breast cancer will vary, for example, from being at low or decreased risk of recurrence to being at high or increased risk of recurrence. By "low or decreased risk" is meant that recurrence of breast cancer is less likely in that subject as compared to a subject determined to be a "high or increased risk" subject. Conversely, a "high or increased risk" subject is a subject in whom recurrence is more likely as compared to a subject who is not at risk or a "low risk" subject. For example, a healthy subject may be regarded as being at low or no risk of recurrence.

[0064] Likelihood is suitably based on mathematical modeling. An increased likelihood, for example, may be relative or absolute and may be expressed qualitatively or quantitatively. For instance, an increased risk may be expressed as simply determining the subject's level of expression of an ER-PR complex and placing the test subject in an "increased risk" category, based upon a corresponding reference level of ER-PR expression as determined, for example, from previous population studies. Alternatively, a numerical expression of the test subject's increased risk may be determined based upon the level of expression of ER-PR complexes in the sample from that subject.

[0065] As used herein, the term "probability" refers to the probability of class membership for a sample as determined by a given mathematical model and is construed to be equivalent likelihood in this context.

[0066] In some embodiments, likelihood is assessed by comparing the level of expression of an ER-PR complex to one or more preselected levels of expression of an ER- PR complex in a control sample, also referred to herein as a threshold or reference level. Threshold or reference levels may be selected that provide an acceptable ability to predict risk, treatment success, etc. In illustrative examples, receiver operating characteristic (ROC) curves are calculated by plotting the value of a variable versus its relative frequency in two populations in which a first population is considered at risk of recurrence of breast cancer after ablation and a second population that is not considered to be at risk, or have a low risk, of recurrence of breast cancer after ablation (called arbitrarily, for example, "healthy controls").

[0067] As noted elsewhere herein, the present inventors have shown that patients with a lower level of expression of ER-PR interactions (complexes) in breast cancer cells are at greater risk of recurrence (or relapse) of breast cancer as compared to patients with a higher level of expression of ER-PR interactions in their breast cancer cells. Thus, in an embodiment disclosed herein, the subject is identified as being at high risk of recurrence of breast cancer where the level of expression of ER-PR complexes in the sample is lower than a level of expression of ER-PR complexes in a control sample.

[0068] Methods of measuring the level of expression of ER-PR complexes in a sample will depend on the method by which the ER-PR complex in the sample is detected in accordance with the methods described herein and, in particular, the type of detectable moiety or moieties that are employed, as described elsewhere herein. For example, where the detectable moiety comprises one or more fluorophores, the level of expression of ER-PR complexes in a sample may be determined by measuring the intensity of fluorescence ( e.g ., via fluorescence microscopy or fluorescence-activated cell sorting). In another illustrative example, the level of expression of ER-PR complexes in a sample may be determined by counting the number of ER-PR complexes in the sample (e.g., by counting the number of detectable moieties). Thus, in an embodiment, the subject is identified as being at high risk of recurrence of breast cancer where the number of ER-PR complexes per cell in the sample is lower than the number of ER-PR complexes per cell in a control sample. Illustrative examples of methods of identifying and counting the number of ER-PR complexes in a sample of breast cancer cells are described elsewhere herein.

[0069] It is to be understood that the level of expression of ER-PR complexes for subjects who are at risk or not at risk of recurrence of breast cancer may overlap kinder such conditions, a test may not absolutely distinguish a subject who is at risk of recurrence from a subject who is not at risk of recurrence with absolute (i.e., with 100%) accuracy/certainty, and the area of overlap indicates where the test cannot distinguish the two subjects. A threshold can be selected above or below which the assessment is considered to be "positive" and above or below which the test is considered to be "negative". The area under the ROC curve (AUC) can provide a C-statistic, which is a measure of the probability that the perceived measurement will allow correct identification of a condition (see, e.g., Hanley el al., Radiology 143 : 29-36 (1982)).

[0070] Alternatively, or in addition, thresholds may be established by obtaining the level of expression of ER-PR complexes from the same patient, to which later results may be compared. In these embodiments, the individual in effect acts as their own "control sample". Hence, a decrease in the level of expression of ER-PR complexes in a sample from the same patient over time can indicate an increased risk of recurrence in that patient.

[0071] In some embodiments, a positive likelihood ratio, negative likelihood ratio, odds ratio, and/or AETC or receiver operating characteristic (ROC) values are used as a measure of a method’s ability to predict risk of recurrence. As used herein, the term "likelihood ratio" is the probability that a given test result would be observed in a subject with a likelihood of such risk, divided by the probability that that same result would be observed in a subject without a likelihood of such risk. Thus, a positive likelihood ratio is the probability of a positive result observed in subjects with the specified risk divided by the probability of a positive results in subjects without the specified risk. A negative likelihood ratio is the probability of a negative result in subjects without the specified risk divided by the probability of a negative result in subjects with specified risk. The term "odds ratio," as used herein, refers to the ratio of the odds of an event occurring in one group (e.g., a healthy control group) to the odds of it occurring in another group (e.g., a group of patients with recurrent breast cancer), or to a data-based estimate of that ratio. The term "area under the curve" or "AUC" refers to the area under the curve of a receiver operating characteristic (ROC) curve, both of which are well known in the art. AUC measures are useful for comparing the accuracy of a classifier across the complete data range. Classifiers with a greater AUC have a greater capacity to classify unknowns correctly between two groups of interest (e.g., a healthy control group and a breast cancer recurrence group). ROC curves are useful for plotting the performance of a particular feature (e.g, level of expression of ER- PR complexes, as described herein, and/or any additional biomedical information) in distinguishing or discriminating between two populations (e.g., cases having a condition and controls without the condition). Typically, the feature data across the entire population (e.g., the cases and controls) are sorted in ascending order based on the value of a single feature. Then, for each value for that feature, the true positive and false positive rates for the data are calculated. The sensitivity is determined by counting the number of cases above the value for that feature and then dividing by the total number of cases. The specificity is determined by counting the number of controls below the value for that feature and then dividing by the total number of controls. Although this definition refers to scenarios in which a feature is elevated in cases compared to controls, this definition also applies to scenarios in which a feature is lower in cases compared to the controls (in such a scenario, samples below the value for that feature would be counted). ROC curves can be generated for a single feature as well as for other single outputs, for example, a combination of two or more features can be mathematically combined ( e.g ., added, subtracted, multiplied, etc.) to produce a single value, and this single value can be plotted in a ROC curve. Additionally, any combination of multiple features, in which the combination derives a single output value, can be plotted in a ROC curve. These combinations of features may comprise a test. The ROC curve is the plot of the sensitivity of a test against the specificity of the test, where sensitivity is traditionally presented on the vertical axis and specificity is traditionally presented on the horizontal axis. Thus, "AUC ROC values" are equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. An AUC ROC value may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.

[0072] In the case of a positive likelihood ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "recurrence risk" and "healthy control" groups; a value greater than 1 indicates that a positive result is more likely in the recurrence risk group; and a value less than 1 indicates that a positive result is more likely in the healthy control group. In this context, "recurrence risk group" refers to a population of reference individuals considered to be at risk of breast cancer recurrence and a "control group" refers to a group of subjects considered not to be at risk of recurrence (e.g., healthy controls). In the case of a negative likelihood ratio, a value of 1 indicates that a negative result is equally likely among subjects in both the "recurrence risk" and "control" groups; a value greater than 1 indicates that a negative result is more likely in the "recurrence risk" group; and a value less than 1 indicates that a negative result is more likely in the "control" group. In the case of an odds ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "recurrence risk" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the "recurrence risk" group; and a value less than 1 indicates that a positive result is more likely in the "control" group. In the case of an AUC ROC value, this is computed by numerical integration of the ROC curve. The range of this value can be 0.5 to 1.0. A value of 0.5 indicates that the classifier (the level of expression of ER-PR complexes) is no better than a 50% chance to classify unknowns correctly between two groups of interest, while 1.0 indicates the relatively best diagnostic accuracy.

[0073] In an embodiment, the level of expression of ER-PR complexes is selected to exhibit a positive or negative likelihood ratio of at least about 1.5 or more or about 0.67 or less, at least about 2 or more or about 0.5 or less, at least about 5 or more or about 0.2 or less, at least about 10 or more or about 0.1 or less, or at least about 20 or more or about 0.05 or less.

[0074] In some embodiments, the level of expression of ER-PR complexes is selected to exhibit an odds ratio of at least about 2 or more or about 0.5 or less, at least about 3 or more or about 0.33 or less, at least about 4 or more or about 0.25 or less, at least about 5 or more or about 0.2 or less, or at least about 10 or more or about 0.1 or less.

[0075] In some embodiments, the level of expression of ER-PR complexes is selected to exhibit an AETC ROC value of greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95.

[0076] In some embodiments, an optimal cut-off of less than or equal to 5 ER-PR complexes per cell is selected as indicative of a high risk of recurrence. This, in an embodiment disclosed herein, the subject is determined to be at risk of recurrence where the number of ER-PR complexes per cell in the tissue sample is less than or equal to 5.

[0077] In another embodiment, an optimal cut-off of less than or equal to 1 ER-PR complex per cell is selected as indicative of a high risk of recurrence. This, in another embodiment disclosed herein, the subject is determined to be at risk of recurrence where the number of ER-PR complexes per cell in the tissue sample is less than or equal to 1.

[0078] In some embodiments, multiple thresholds may be determined in so-called "tertile," "quartile," or "quintile" analyses. In these methods, the "recurrence risk" and "control" groups are considered together as a single population, and are divided into 3, 4, or 5 (or more) "bins" having equal numbers of individuals. The boundary between two of these "bins" may be considered "thresholds." The degree of risk can then be assigned based on which "bin" a test subject falls into.

[0079] In other embodiments, particular thresholds for the level of expression of ER- PR complexes measured are not relied upon to determine if the corresponding level of expression obtained from a subject is correlated to risk of recurrence. For example, a temporal change in the level of expression of ER-PR complexes can be used to rule in or out such risk. For example, an increase, decrease, or other change ( e.g ., slope over time) in the level of expression of ER-PR complexes in the sample may be sufficient to indicate the degree of risk of recurrence in a subject.

[0080] The phrases "assessing the likelihood" and "determining the likelihood," as used herein, refer to methods by which persons skilled in the art can predict a subject's risk of breast cancer recurrence. It is to be understood that these phrases include within their scope an increased probability that the subject will develop breast cancer recurrence; that is, such risk is more likely to be present or absent in a subject. For example, the probability that an individual identified as being at risk of recurrence may be expressed as a "positive predictive value" or "PPV." Positive predictive value can be calculated as the number of true positives divided by the sum of the true positives and false positives. PPV is typically determined by the characteristics of the predictive methods of the present invention, as well as the prevalence of the condition in the population analysed. The statistical algorithms can be selected such that the positive predictive value in a population considered to be at risk of recurrence is in the range of 70% to 99% and can be, for example, at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. [0081] In other examples, the probability that a subject is identified as not being at risk of breast cancer recurrence may be expressed as a "negative predictive value" or "NPV." Negative predictive value can be calculated as the number of true negatives divided by the sum of the true negatives and false negatives. Negative predictive value is determined by the characteristics of the diagnostic or prognostic method, system, or code as well as the prevalence of risk in the population analysed. The statistical methods and models can be selected such that the negative predictive value in a population considered at risk of recurrence is in the range of about 70% to about 99% and can be, for example, at least about 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0082] In some embodiments, a subject is determined as being at significant risk of breast cancer recurrence. By "significant risk" is meant that the subject has a reasonable probability ( e.g ., 0.6, 0.7, 0.8, 0.9 or more) of breast cancer recurrence.

[0083] The methods of the present invention, as broadly described herein, also permit the generation of high-density data sets that can be evaluated using informatics approaches. High data density informatics analytical methods are known and software is available to those in the art, e.g., cluster analysis (Pirouette, Informetrix), class prediction (SIMCA-P, Umetrics), principal components analysis of a computationally modeled dataset (SIMCA-P, Umetrics), 2D cluster analysis (GeneLinker Platinum, Improved Outcomes Software), and metabolic pathway analysis (biotech.icmb.utexas.edu). The choice of software packages offers specific tools for questions of interest (Kennedy et al., Solving Data Mining Problems Through Pattern Recognition. Indianapolis: Prentice Hall PTR, 1997; Golub et al., (2999) Science 286:531-7; Eriksson et al., Multi and Megavariate Analysis Principles and Applications: Umetrics, Umea, 2001). In general, any suitable mathematic analyses can be used to evaluate the level of expression of ER-PR complexes with respect to determining the likelihood that the subject is at risk of breast cancer recurrence. For example, methods such as multivariate analysis of variance, multivariate regression, and/or multiple regression can be used to determine relationships between dependent variables (e.g, clinical measures) and independent variables (e.g, levels of biomarkers). Clustering, including both hierarchical and non-hierarchical methods, as well as nonmetric Dimensional Scaling can be used to detennine associations or relationships among variables and among changes in those variables.

[0084] In some embodiments, the level of expression of ER-PR complexes is used to assign a risk score which describes a mathematical equation for evaluation or prediction of risk. The evaluation of risk may also take into account genotype, and other clinical risk factors of breast cancer recurrence into account. Other clinical risk factors of breast cancer recurrence will be familiar to persons skilled in the art.

[0085] In addition, principal component analysis is a common way of reducing the dimension of studies, and can be used to interpret the variance-covariance structure of a data set. Principal components may be used in such applications as multiple regression and cluster analysis. Factor analysis is used to describe the covariance by constructing "hidden" variables from the observed variables. Factor analysis may be considered an extension of principal component analysis, where principal component analysis is used as parameter estimation along with the maximum likelihood method. Furthermore, simple hypothesis such as equality of two vectors of means can be tested using Hotelling’s T squared statistic.

[0086] Persons identified as being at risk of recurrence by the methods disclosed herein may suitably be stratified to a treatment regimen for minimising the likelihood of recurrence. Thus, in an embodiment disclosed herein, the methods disclosed herein further comprises exposing the subject identified as being at risk of recurrence following ablation of a breast cancer tumour to a treatment regimen for treating the breast cancer.

[0087] Suitable treatment regimens will be familiar to persons skilled in the art, illustrative examples of which include administering or otherwise exposing the subject to a selective CDK4/6 inhibitor, a histone deacetylase (HD AC) inhibitor, an mTOR inhibitor, a phophoinositide-3 -kinase (PI3K) inhibitor, a MAPK inhibitor, a selective estrogen receptor degrader or a combination of any two or more of the foregoing. Thus, in an embodiment disclosed herein, the treatment regimen comprises administering or otherwise exposing the subject in need thereof to an agent selected from the group consisting of a selective CDK4/6 inhibitor, a histone deacetylase (HD AC) inhibitor, an mTOR inhibitor, a phophoinositide-3- kinase (PI3K) inhibitor, a MAPK inhibitor, a selective estrogen receptor degrader and a combination of any two or more of the foregoing.

[0088] In an embodiment, the selective CDK4/6 inhibitor is selected from the group consisting of Palbociclib, Ribociclib and Abemaciclib. In another embodiment, the histone deacetylase (HDAC) inhibitor is selected from the group consisting of Panobinostat, Vorinostat and Entinostat. In another embodiment, the mTOR inhibitor is selected from the group consisting of Everolimus and Temsirolimus. In another embodiment, the phophoinositide-3 -kinase (PI3K) inhibitor is selected from the group consisting of Buparlisib, Tasekisib, Alpelisib and Pictilisib. In another embodiment, the MAPK inhibitor is Selumetinib. In another embodiment, the selective estrogen receptor degrader is Fulvestrant.

[0089] As noted elsewhere herein, the inventors have also surprisingly found that the level of expression of ER-PR interactions in breast cancer cells from a patient with breast cancer, as reflected in the number of ER-PR complexes detected in the cells, is determinative of the patient’s response to therapy. Thus, in another aspect disclosed herein, there is provided a method of predicting a subject’s response to a treatment regimen for treating breast cancer, the method comprising:

[0090] In an embodiment disclosed herein, step (iii) further comprises contacting the sample with a third agent, such as those described elsewhere herein, wherein the third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex in the sample. Suitable detectable moieties will be familiar to persons skilled in the art, illustrative examples of which are described elsewhere herein.

[0091] In an embodiment disclosed herein, the ER-PR complex is detected by a method selected from the group consisting of a proximity ligation assay (PLA), biomolecular fluorescence complementation, chemical cross-linking followed by high mass MALDI mass spectrometry and fluorescence resonance energy transfer (FRET)

[0092] In an embodiment disclosed herein, the ER-PR complex is detected by PLA.

[0093] In an embodiment disclosed herein, the treatment regimen comprises administering or otherwise exposing the subject to an aromatase inhibitor (AI). Suitable aromatase inhibitors will be familiar to persons skilled in the art, illustrative examples of which are described elsewhere herein. In another embodiment disclosed herein, the treatment regimen comprises administering or otherwise exposing the subject to an estrogen receptor inhibitor. Suitable estrogen receptor inhibitors will be familiar to persons skilled in the art, an illustrative example of which is tamoxifen.

[0094] Predicting a subject’s response to a treatment regimen, as used herein, suitably means making a determination, based on the level of expression of ER-PR complexes in a sample of breast cancer cells from the subject, the likelihood that the subject will respond to the treatment regimen in the manner to which the regimen is intended; for example, a showing of at least some improvement in one or more clinical indications relevant to the condition being treated. It is to be understood that the one or more clinical indications, as referred to herein, will depend on the therapeutic regimen in question. As an illustrative example, a subject with breast cancer is said to be responsive to treatment with a therapeutic agent ( e.g ., an aromatase inhibitor or an estrogen receptor inhibitor) where a breast cancer tumour in the subject either stops growing in size or grows at a much slower rate as compared to the growth rate of the tumour prior to treatment. Conversely, a subject with breast cancer is said to be non-responsive to treatment with a therapeutic agent (e.g., an aromatase inhibitor or an estrogen receptor inhibitor) where a breast cancer tumour in the subject continues to grow in size at a similar or faster rate as compared to the growth rate of the tumour prior to treatment. As a further illustrative example, a subject with breast cancer is said to be responsive to treatment with a therapeutic agent ( e.g ., an aromatase inhibitor or an estrogen receptor inhibitor) where there is an absence, or a fewer number, of detectable metastatic tumours in the subject for a period of at least 1 year (e.g., for 1 year, preferably for 2 years, preferably for 3 years, preferably for 4 years, preferably for 5 years) following treatment, as compared to a population of breast cancer patients who are either non-responders to the therapeutic agent or have not received treatment with the therapeutic agent. Conversely, a subject with breast cancer is said to be non-responsive to treatment with a therapeutic agent (e.g., an aromatase inhibitor or an estrogen receptor inhibitor) where there is no significant difference in the number of detectable metastatic tumours in the subject following treatment, as compared to a population of breast cancer patients who are either non-responders to the therapeutic agent or have not received treatment with the therapeutic agent.

[0095] As noted elsewhere herein, likelihood is suitably based on mathematical modeling. An increased likelihood, for example, may be relative or absolute and may be expressed qualitatively or quantitatively. For instance, an increased likelihood that the subject will respond to a treatment regimen may be expressed as simply determining the subject's level of expression of an ER-PR complex and placing the test subject in a "responder" category, based upon a corresponding reference level of ER-PR expression as determined, for example, from previous population studies. Alternatively, a numerical expression of the test subject's likelihood of responding to a treatment regimen may be determined based upon the level of expression of ER-PR complexes in the sample from that subject.

[0096] In some embodiments, likelihood is assessed by comparing the level of expression of an ER-PR complex to one or more preselected levels of expression of an ER- PR complex in a control sample, as described elsewhere herein.

[0097] As noted elsewhere herein, the present inventors have shown that patients with a lower level of expression of ER-PR interactions (complexes) in breast cancer cells are less responsive to treatment as compared to patients with a higher level of expression of ER-PR interactions in their breast cancer cells. Thus, in an embodiment disclosed herein, the subject is identified as a responder to a therapeutic regimen where the level of expression of ER-PR complexes in the sample is higher than a level of expression of ER-PR complexes in a control sample.

[0098] Methods of measuring the level of expression of ER-PR complexes in a sample will depend on the method by which the ER-PR complex in the sample is detected in accordance with the methods described herein and, in particular, the type of detectable moiety or moieties that are employed, as described elsewhere herein.

[0099] It is to be understood that the level of expression of ER-PR complexes for subjects who are considered responders or non-responders may overlap. ETnder such conditions, a test may not absolutely distinguish a subject who is a responder from a subject who is a non-responder with absolute ( i.e ., with 100%) accuracy/certainty, and the area of overlap indicates where the test cannot distinguish the two subjects. A threshold can be selected above or below which the assessment is considered to be "positive" and above or below which the test is considered to be "negative". The area under the ROC curve (AETC) can provide a C-statistic, which is a measure of the probability that the perceived measurement will allow correct identification of a condition, as described elsewhere herein.

[0100] In some embodiments, a positive likelihood ratio, negative likelihood ratio, odds ratio, and/or AUC or receiver operating characteristic (ROC) values are used as a measure of a method’s ability to predict a subject’s response to a therapeutic regimen, as described elsewhere herein.

[0101] In some embodiments, an optimal cut-off of less than or equal to 5 ER-PR complexes per cell is selected as predictive that a subject will be a poor respond to a treatment regimen for treating breast cancer. Thus, in an embodiment disclosed herein, the subject is determined to be a poor responder to the treatment regimen where the number of ER-PR complexes per cell in the tissue sample is less than or equal to 5.

[0102] In other embodiments, an optimal cut-off of less than or equal to 1 ER-PR complexes per cell is selected as predictive that a subject will be a poor responder to a treatment regimen for treating breast cancer. Thus, in an embodiment disclosed herein, the subject is determined to be a poor responder to the treatment regimen where the number of ER-PR complexes per cell in the tissue sample is less than or equal to 1. [0103] In some embodiments, multiple thresholds may be determined in so-called "tertile", "quartile" or "quintile" analyses, as described elsewhere herein. In other embodiments, particular thresholds for the level of expression of ER-PR complexes measured are not relied upon to determine if the corresponding level of expression obtained from a subject is correlated to a response to a treatment regimen. For example, as described elsewhere herein, a temporal change in the level of expression of ER-PR complexes can be used to rule in or out a subject’s likelihood of responding to a treatment regimen. For example, an increase, decrease, or other change ( e.g ., slope over time) in the level of expression of ER-PR complexes in the sample may be sufficient to indicate the subject’s likelihood of responding to a treatment regimen.

[0104] The phrases "assessing the likelihood" and "determining the likelihood," as used herein, refer to methods by which persons skilled in the art can predict whether or not a subject will respond to a treatment regimen. It is to be understood that these phrases include within their scope an increased probability that the subject will respond to a treatment regimen; that is, it is more likely that not the subject will respond to the treatment regimen. For example, the probability that an individual will respond to a treatment regimen may be expressed as a "positive predictive value" or "PPV", as described elsewhere herein. In other examples, the probability that an individual will not respond to a treatment regimen may be expressed as a "negative predictive value" or "NPV", as described elsewhere herein.

[0105] In some embodiments, a subject is determined as being very likely to respond to a treatment regimen. By "very likely" is meant that the subject has a reasonable probability (e.g., 0.6, 0.7, 0.8, 0.9 or more) of responding to the treatment regimen. In some embodiments, a subject is determined as being very unlikely to respond to a treatment regimen. By "very unlikely" is meant that the subject has a reasonable probability (e.g, 0.6, 0.7, 0.8, 0.9 or more) of not responding to the treatment regimen.

[0106] In some embodiments, the level of expression of ER-PR complexes is used to assign a risk score which describes a mathematical equation for evaluation or prediction of the likelihood of responding to a treatment regimen, as described elsewhere herein. [0107] In an embodiment disclosed herein, the method further comprises exposing the subject identified as likely to be a poor responder to the treatment regimen, to an alternative treatment regimen for treating breast cancer.

[0108] In another embodiment disclosed herein, the subject is determined to be a poor responder to the therapeutic agent where the number of ER-PR complexes per cell in the tissue sample is less than the number of ER-PR complexes per cell in a control sample.

[0109] In another embodiment disclosed herein, the subject is determined to be a poor responder to the treatment regimen where the number of ER-PR complexes per cell in the sample is less than or equal to 5. In yet another embodiment disclosed herein, the subject is determined to be a poor responder to the treatment regimen where the number of ER-PR complexes per cell in the sample is less than or equal to 1.

[0110] In another aspect, there is provided a kit comprising (i) a first binding agent that binds to an estrogen receptor (ER) in a breast cancer tissue sample, (ii) a second binding agent that binds to a progesterone receptor (PR) in a breast cancer tissue sample; and (iii) a third binding agent that selectively binds to the first and second binding agents; wherein the third binding agent comprises at least one detectable label that is detectable when the each of the first and second binding agents are bound to an ER-PR complex in the tissue sample.

[0111] In another aspect, there is provided a kit for use in the methods described herein, wherein the kit comprises (i) a first binding agent that binds to an estrogen receptor (ER) in a breast cancer tissue sample, (ii) a second binding agent that binds to a progesterone receptor (PR) in a breast cancer tissue sample; and (iii) a third binding agent that selectively binds to the first and second binding agents; wherein the third binding agent comprises at least one detectable label that is detectable when the each of the first and second binding agents are bound to an ER-PR complex in the tissue sample.

[0112] In some embodiments, the kits may comprise suitable controls to ensure the method is working reliably. Suitable controls may include one or more breast cancer cells with a known level of expression of ER-PR complexes. This acts as a positive control to help to ensure that false negative results are not obtained. Suitable negative controls may also be incorporated into the kits, such as mammary cells from normal subjects (e.g., subjects with no history of breast cancer). This provides a negative control to ensure against false positive results.

[0113] In another aspect there is provided a kit comprising one or more reagents and/or devices for use in performing the method of the present invention, as herein described. In some embodiments, kits for carrying out the methods of the present invention will include, in suitable container means, (i) a first binding agent that binds to an estrogen receptor (ER) in a breast cancer tissue sample, (ii) a second binding agent that binds to a progesterone receptor (PR) in a breast cancer tissue sample; and (iii) a third binding agent that selectively binds to the first and second binding agents; wherein the third binding agent comprises at least one detectable label that is detectable when the each of the first and second binding agents are bound to an ER-PR complex in the tissue sample; and (iv) instructions for how to perform the methods. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and/or other container into which the reagents may be placed and/or suitably aliquoted. Alternatively, a container may contain a mixture of more than one reagent to be used in accordance with the present invention. The kits of the present invention may also include means for containing the reagents in close confinement for commercial sale. Such containers may include injection and/or blow-molded plastic containers into which the desired vials are retained.

[0114] As noted elsewhere herein, the kits may further comprise positive and negative controls, including a reference samples, as well as instructions for the use of kit components contained therein, in accordance with the methods of the present invention.

[0115] In another aspect, there is provided a method of stratifying a subject to a treatment for breast cancer, the method comprising:

(i) obtaining a sample of breast cancer cells from a subject with breast cancer; and

[0116] In an embodiment disclosed herein, step (iii) comprises contacting the sample with a third agent, wherein the third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex in the sample.

[0117] In an embodiment disclosed herein, the ER-PR complex is detected by a method selected from the group consisting of a proximity ligation assay (PLA), biomolecular fluorescence complementation, chemical cross-linking followed by high mass MALDI mass spectrometry and fluorescence resonance energy transfer (FRET).

[0118] In an embodiment disclosed herein, the ER-PR complex is detected by PLA.

[0119] In an embodiment disclosed herein, the subject is on, or will be on, endocrine therapy. In an embodiment disclosed herein, the subject is on aromatase inhibitor (AI) therapy. In other embodiments, the subject will not be on AI therapy ( e.g ., at step (i)), but will go on to receive AI therapy, for example, as part of the treatment regimen.

[0120] In an embodiment disclosed herein, the subject is identified as being at high risk of recurrence of breast cancer where the level of expression of ER-PR complexes in the sample is lower than a level of expression of ER-PR complexes in a control sample.

[0121] In an embodiment disclosed herein, the subject is identified as being at high risk of recurrence of breast cancer where the number of ER-PR complexes per cell in the sample is lower than the number of ER-PR complexes per cell in a control sample.

[0122] In an embodiment disclosed herein, the treatment regimen of step (iv) comprises administering to the subject in need thereof an agent selected from the group consisting of a selective CDK4/6 inhibitor, a histone deacetylase (HD AC) inhibitor, an mTOR inhibitor, a phophoinositide-3 -kinase (PI3K) inhibitor, an MAPK inhibitor, an aromatase inhibitor, a selective estrogen receptor degrader and a combination of two or more of the foregoing.

[0123] In an embodiment disclosed herein, the selective CDK4/6 inhibitor is selected from the group consisting of Palbociclib, Ribociclib and Abemaciclib.

[0124] In an embodiment disclosed herein, the histone deacetylase (HD AC) inhibitor is selected from the group consisting of Panobinostat, Vorinostat and Entinostat.

[0125] In an embodiment disclosed herein, the mTOR inhibitor is selected from the group consisting of Everolimus and Temsirolimus.

[0126] In an embodiment disclosed herein, the phophoinositide-3 -kinase (PI3K) inhibitor is selected from the group consisting of Buparlisib, Tasekisib, Alpelisib and Pictilisib.

[0127] In an embodiment disclosed herein, the MAPK inhibitor is Selumetinib. In another embodiment disclosed herein, the treatment regimen of step (iv) comprises administering to the subject in need thereof (i) an aromatase inhibitor and (ii) an agent selected from the group consisting of a selective CDK4/6 inhibitor, a histone deacetylase (HDAC) inhibitor, an mTOR inhibitor, a phophoinositide-3 -kinase (PI3K) inhibitor, an MAPK inhibitor, a selective estrogen receptor degrader and a combination of any of the foregoing, as described herein. In another embodiment, the subject is on AI therapy and the treatment regimen of step (iv) comprises administering to the subject in need thereof an agent selected from the group consisting of a selective CDK4/6 inhibitor, a histone deacetylase (HDAC) inhibitor, an mTOR inhibitor, a phophoinositide-3 -kinase (PI3K) inhibitor, an MAPK inhibitor, a selective estrogen receptor degrader and a combination of any of the foregoing, as described herein. In another embodiment disclosed herein, the aromatase inhibitor is selected from the group consisting of anastrozole, letrozole and exemestane.

[0128] In an embodiment disclosed herein, the selective estrogen receptor degrader is Fulvestrant. [0129] In another aspect disclosed herein, there is provided a complex formed by (i) a first binding agent that binds to an estrogen receptor (ER) of an ER-PR complex in the cell, wherein the ER-PR complex comprises an estrogen receptor (ER) and a progesterone receptor (PR); (ii) a second binding agent that binds to the PR of the ER-PR complex; and (iii) a third agent, wherein third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to the ER-PR complex in the cell.

[0130] In another aspect disclosed herein, there is provided a complex comprising (i) a first binding agent bound to an estrogen receptor (ER) of an ER-PR complex in a breast cancer cell, wherein the ER-PR complex comprises an estrogen receptor (ER) and a progesterone receptor (PR); (ii) a second binding agent bound to the PR of the ER-PR complex; and (iii) a third agent, wherein third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex in the cell.

[0131] In another aspect disclosed herein, there is provided a complex comprising a conjugate of an estrogen receptor (ER) and a progesterone receptor (PR), a first binding agent bound selectively to the ER, a second binding agent bound selectively to the PR and a third agent bound selectively to the first and second binding agents.

[0132] As used herein, the term "treatment regimen" typically refers to the administration to, or exposure of, the subject of a therapeutic agent for treating breast cancer. The term "treatment regimen" encompasses natural substances and pharmaceutical agents (i.e., "drugs") as well as any other treatment regimen including, but not limited to, dietary treatments, physical therapy or exercise regimens, surgical interventions, and combinations thereof.

[0133] The term "treating" as used herein, unless otherwise indicated, means alleviating, inhibiting the progress of, or preventing, either partially or completely, the recurrence and/or progression of breast cancer, including breast cancer metastases and/or one or more symptoms of any of the foregoing. The term "treatment" as used herein, unless otherwise indicated, refers to the act of treating. [0134] Following diagnosis/prognosis of risk of recurrence or response to treatment, as herein described, a treatment regimen to be adopted or prescribed may depend on several factors, including the age, weight and general health of the subject. Another determinative factor may be the degree of risk of recurrence or response to a treatment regimen, as determined in accordance with the present invention, as described herein. For instance, where the subject is determined to be at high risk of recurrence, a more aggressive treatment regimen may be prescribed as compared to a subject who is determined to be at low risk of recurrence. The treatment regimen may also depend on existing clinical parameters relevant to breast cancer, such as whether or not the subject is pre-menopausal.

[0135] As used herein, the term“treatment regimen” refers to prophylactic and/or therapeutic ( i.e ., after onset of a specified condition) treatments, unless the context specifically indicates otherwise. The term “treatment regimen” encompasses natural substances and pharmaceutical agents {i.e.,“drugs”) as well as any other treatment regimen including but not limited to dietary treatments, physical therapy, exercise regimens, surgical interventions, radiation therapy and combinations thereof.

[0136] Treatment may suitable be decided according to the type or grade of breast cancer, its anatomical location in the subject and its size {i.e., its stage). The“stage” of a breast carcinoma {e.g., primary tumour) is a typically used descriptor (usually numbers I to IV) of how much the breast carcinoma has spread. The stage often takes into account the size of a primary and/or secondary tumour, how deep it has penetrated, whether it has invaded adjacent tissue, if and how many lymph nodes it has metastasized to, and whether it has spread to distant organs. Staging of a carcinoma is important because the stage at diagnosis is generally a predictor of survival, and treatments are often changed based on the stage.

[0137] In some embodiments, a subject at risk of recurrence is prescribed a chemotherapeutic agents selected from any one or more of the following categories:

[0138] (i) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan and nitrosoureas); antimetabolites (for example antifolates such as fluoropyridines like 5- fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea; anti-tumor antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like paclitaxel and docetaxel; and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin);

[0139] (ii) cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), UH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5a-reductase such as finasteride;

[0140] (iii) agents which inhibit cancer cell invasion (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function);

[0141] (iv) inhibitors of growth factor function, for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti-erbb2 antibody trastuzumab [Herceptin™] and the anti-erbbl antibody cetuximab [C225]), farnesyl transferase inhibitors, MEK inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example other inhibitors of the epidermal growth factor family (for example other EGFR family tyrosine kinase inhibitors such as N-(3- chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4- -amine

(gefitinib, AZD1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3- morpholinopropoxy)quinazoli- n-4-amine (Cl 1033)), for example, inhibitors of the platelet- derived growth factor family and for example inhibitors of the hepatocyte growth factor family; [0142] (v) anti-angiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [Avastin™], compounds such as those disclosed in International Patent Applications WO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354) and compounds that work by other mechanisms (for example linomide, inhibitors of integrin anb3 function and angiostatin);

[0143] (vi) vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/02166, WO00/40529, WO 00/41669, WOOl/92224, W002/04434 and W002/08213;

[0144] (vii) antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense; and

[0145] (viii) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy.

[0146] Examples of other suitable treatment regimen include phototherapy, cryotherapy, toxin therapy or pro-apoptosis therapy. One of skill in the art would know that this list is not exhaustive of the types of treatment modalities available for breast cancer.

[0147] The invention can also be practiced to evaluate whether a subject is responding ( i.e ., a positive response) or not responding (z.e., a negative response) to a treatment regimen. This aspect of the invention provides methods of correlating a level of expression of a ER- PR complexes in a sample of breast cancer cells from the subject with a positive and/or negative response to a treatment regimen. These methods generally comprise: (a) obtaining a sample of breast cancer cells from a subject with breast cancer, as described elsewhere herein; and (b) correlating the level of expression of a ER-PR complexes in the sample with a positive and/or negative response to the treatment regimen. [0148] This aspect of the invention can be practiced to identify responders or non responders relatively early in the treatment process, z.e., before clinical manifestations of efficacy. In this way, the treatment regimen can optionally be discontinued, a different treatment protocol can be implemented and/or supplemental therapy can be administered.

[0149] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non limiting examples.

EXAMPLES

Example 1 - Patients, Materials and Methods

A. Cell culture

[0150] T47D breast cancer cell line was acquired from the ATCC and cultured in DMEM supplemented with 10% FBS. The cells were regularly tested for mycoplasma infection. To stimulate ER and PR interactions, the cells were cultured in phenol red-free DMEM supplemented with charcoal stripped FBS (Gibco, no. 12676011) for 48 hours. The media was then supplemented with vehicle (ethanol), estradiol (E ₂; Sigma, no. E2758; 250mg), progesterone (Sigma, no. P6149; lmg) or the combination of E₂ and progesterone at a final concentration of 10hM for 24 hours. Cells were mechanically lifted, fixed in 10% neutral buffer formalin for 24 hours and then resuspended in 2% molten agarose dissolved in 10% formalin. The cells and agarose suspension were centrifuged to form a pellet. Cell pellets were processed in tissue cassettes at Mater Pathology (Queensland, Australia) as per clinical specimens.

B. Patient-derived tumour explants

[0151] Tumour samples were obtained following informed consent from women undergoing surgery for breast cancer at the Burnside War Memorial Hospital, Adelaide. This study was approved by the ETniversity of Adelaide Human Research Ethics Committee (approval numbers: H-065-2005; H-169-2011). Excised tissue samples were cultured ex vivo as previously described (10,17). Briefly, tumour explants were cultured on gelatine sponges for 36 hours then treated with the following conditions: vehicle (ethanol), E₂ (10hM), a synthetic progestin R5020 (lOnM) or the combination of E₂ and R5020 (both at lOnM) with treatment for 48 hours. Explants were fixed in 10% neutral buffered formalin overnight and processed as per clinical specimens.

C. Proximity-ligation assay (PLA)

[0152] Formalin-fixed, paraffin-embedded (FFPE) tissue was sectioned at 6pm. The tissue was deparaffmised and antigen retrieved in citrate buffer at pH 6 using a Decloaking Chamber (Biocare Medical). Sections were blocked and primary antibodies were diluted in antibody diluent (Roche, no. 251-018) and incubated overnight at 4°C. Monoclonal rabbit Anti-ER (Thermo Scientific, clone SP1) and monoclonal mouse anti -PR (Sigma, clone 3E11; raised against an immunogen from the N-terminal specific to the PR-B isoform) were both used at 1 : 100 dilution. Secondary antibodies used were Duolink in Situ PLA Probe Anti -Mouse PLETS (Sigma, no. DU092001-100RXN) and Anti -Rabbit MINUS (Sigma, no. DU092005-100RXN) as per manufacturer’s instructions. Detection was performed as per manufacturer’s recommendation using Duolink in Situ Detection reagents brightfield (Sigma, no. DU092012-100RXN) and counterstained with haematoxylin. Staining was independently scored by two breast histopathologists by counting the number of signals per nucleus in 20 cells in the areas of tumour with greatest numbers of signals, a similar method to that used to score HER2 detected by in situ hybridisation assays. Scores were averaged to determine a final score.

D. Immunohistochemistry

[0153] ER and PR immunohistochemistry was performed with an anti-ER antibody (Ventana, clone SP1) and an anti-PR antibody (Ventana, clone 1E2) using the Ventana BenchMark ULTRA automated slide Stainer (Roche). ER and PR immunohistochemistry was scored by two breast histopathologists using the‘Allred score’ on a scale of 0-8 (18). Differences were resolved by consensus. A cutoff of >2 was considered positive (weak positive staining in >1% of tumour cell nuclei) (19).

E. Patients

[0154] The cohort for these studies was comprised of a consecutive series of 229 patients with ER+, HER2- negative breast cancer with lymph node metastatic deposits of at least 2.0mm in size resected with curative intent (at least Nl, all patients were stage II and III (20)). Patients with node-positive disease were selected to amplify the effect of adjuvant therapies. Patients had their tumours resected at the Mater Hospital (Brisbane, Queensland, Australia) between January 2005 and December 2014. No patients had endocrine therapy prior to surgery. HER2 negativity was defined by negative immunohistochemistry and lacking amplification by in situ hybridisation of the ERBB2 gene. All patients were recommended adjuvant endocrine therapy post-surgery, and patients were considered not to have taken adjuvant endocrine therapy if they took a total of <2 months treatment. The use of clinical information and tumour blocks was approved by the Mater Health Services Human Research Ethics Committee (approval number: HREC/15/MHS/123). Treatment of all patients was discussed at a breast cancer multidisciplinary meeting and adjuvant therapy was recommended according to international guidelines. Cores of the primary tumour from each patient were assembled into tissue microarrays (four cores per patient, each measuring l .Omm in diameter) (21) using a semiautomated arrayer (Beecher Instruments). The study was designed to meet the REMARK guidelines for reporting tumour marker prognostic studies (22).

F. Study Population

[0155] A total of 229 patients had surgery with curative intent for ER+, HER2- node- positive breast cancer (Table 1). After surgery, recommendations for adjuvant treatment were made at the breast multidisciplinary meeting and treatment decisions were made by patients in conjunction with their treating specialists. The median age of patients was 54 years at resection (range 27 - 88 years) and 71.2% were postmenopausal. 86.0% of patients had adjuvant chemotherapy, 81.7% adjuvant radiation and 94.8% adjuvant endocrine therapy. Of those that had endocrine therapy, 69.6% were treated with an aromatase inhibitor and 29.0% with tamoxifen. The median follow up time was 5.1 years (range 0.9 - 11.3 years). Relapse occurred in 48 patients (21%) and the mean estimated relapse-free survival time was 8.8 years (SD 3.7 years). Table 1. Clinical and pathological variables of 229 included patients.

G. Statistical design and analysis

[0156] Statistical analysis was performed using SPSS V.22.0 (IBM) and GraphPad Prism V.7.03 (GraphPad Software, Inc). Correlations between ER-PR interactions and clinical and pathological factors were determined using the 2-tailed Spearman’s rank correlation coefficient as ER-PR interactions were not normally distributed. The Mann- Whitney U test was used to compare test whether number of ER-PR interactions differed between PR- and PR+ groups. Receiver operating characteristic (ROC) curves were used to determine the optimum cut-off of signals per cell with respect to relapse. Relapse-free survival analyses were carried out using Kaplan-Meier curves and significance determined by log-rank test. ETnivariate and multivariate Cox regression analyses were used to determine significant dependent and independent variables. Factors significant in the univariate analysis were included in the multivariate analysis. Associations for 2 x 2 tables were carried out using a Fisher’s exact test, due to small numbers in some subgroups. The 3 x 2 table for association of tumour histologic type was tested for significance using a Fisher’s exact test. All other 3 x 2 tables and 4 x 2 tables were tested for significance using the Cochran- Armitage test for trend, due to the variables being ordinal.

Example 2 - Development of an assay to detect interactions between ER and PR in FFPE tissue

[0157] ER-PR PLA was applied to three ER+, PR+ patient breast cancer explants which were treated with vehicle, E₂, the synthetic progestin R5020 or with the combination of E₂ and R5020. In the absence of both E₂ and R5020, ER-PR complexes (interactions) were not seen in tumour explants (Figure 1A and 1B). Moreover, there was no detectable difference in levels of ER and PR by immunohistochemistry.

[0158] T47D cells were treated for 24 hours with vehicle, E₂, progesterone or with the combination of E₂ and progesterone under steroid-depleted conditions. The proximity ligation assay (PLA) to detect ER-PR complexes (interactions) was applied to FFPE cell pellets and ER-PR complexes were only detected in cells treated with the combination of estrogen and progesterone (Figure 1C). Again, there was no detectable change in ER and PR by immunohistochemistry. Example 3 - Association between ER-PR interactions and PR immunohistochemistry in breast cancer

[0159] The ER-PR PLA was applied to primary tumours and scored by counting the number of signals {i.e., ER-PR complexes) per tumour cell nucleus. Tumours were also scored for ER and PR expression using the Allred score. There was a significant positive correlation between ER-PR interactions (i.e., the number of ER-PR complexes detected by the PLA) and PR expression (p = 0.003) (Table 2).

Table 2: Correlations between ER-PR interactions (dots/cell) and clinicopathological variables. The p value quoted is the result of a 2-tailed Spearman’s correlation. N is the number of patients with pairwise non-missing values. Bold indicates significant p values.

[0160] In 44 tumours that were negative for PR by immunohistochemistry (Allred score 0-2; representing weak positive staining in less than 1% of tumour cells), the median number of ER-PR complexes was 2.28 (inter-quartile range 1.8 - 10.3), significantly lower than the number of ER-PR complexes in tumour samples with a PR Allred score of 3 or more (median 6.45 signals per cell, inter-quartile range 0.26 - 7.99,/? = 0.001) (Figure 2). Many cases with positive PR expression showed very few detectable ER-PR interactions (example in bottom right panel, Figure 2) and conversely there were cases which showed significant number of ER-PR complexes despite the absence of detectable PR expression (example in top left panel, Figure 2). The inventors have shown that PLA is more sensitive at detecting PR than immunohistochemistry (see Snell et al., 2018; Br J Cancer, 119(11): 1316-1325).

[0161] All cases had detectable ER by immunohistochemistry. There was a significant positive correlation between the level of ER expression and number of ER-PR interactions (p = 0.001) (Table 2).

Example 4 - Correlations between ER-PR interactions and clinical and pathological variables

[0162] The average number of ER-PR complexes detected per tumour cell was correlated with clinical and pathological variables. There was a positive correlation between the number of ER-PR complexes and age (p = 0.001), and a higher number of ER-PR complexes in post-menopausal women (Table 2). Higher number of ER-PR complexes were correlated with lower tumour grade (p = 0.030) and lower mitotic score (p = 0.003). There was no significant correlation between the number of ER-PR complexes and T-stage, N- stage, the presence of multiple tumours, or type of surgical or adjuvant treatment.

Example 5 - Association of PR immunohistochemistry and ER-PR interactions with relapse-free survival

[0163] In the cohort as a whole, absent PR immunohistochemistry was associated with poorer relapse free survival (log-rank p = 0.021) (Figure 3A). ROC curve analysis was used to determine an optimal cut-off of number of ER-PR complexes per cell in patients that had taken adjuvant tamoxifen or an aromatase inhibitor (AI) with respect to relapse (Figure 4). In AI-treated patients, the area under the curve was 0.701 (p = 0.0013) and at a cut-off of 5 signals per cell, sensitivity for detecting relapse was 76.9% and specificity was 63.4%. Similar ROC curve analysis for ER expression (Allred Score) showed no significant association with relapse (Figure 5). When the entire cohort of patients was dichotomised into low (<5) and high (>5) ER-PR complexes per cell, those with low ER-PR complexed per cell had significantly poorer relapse free survival (log-rank p = 0.003) (Figure 3 A).

Example 6 - Univariate and multivariate Cox regression analyses of variables potentially affecting recurrence

[0164] In an analysis of the whole cohort of patients, higher pathological T-stage and N-stage were significantly associated with recurrence (relapse) in univariate analysis (Table 3). Patients that underwent chemotherapy and took prescribed endocrine therapy had significantly reduced risk of relapse. Absent PR expression was significantly associated with relapse (HR 2.028, Cl 1.100 - 3.731, p = 0.024) and a low number of ER-PR complexes was associated with a higher risk of relapse (HR 2.463, Cl 1.333 - 4.545, p = 0.004). There was no significant prognostic effect of age, grade, mitotic score, histologic type, multiple tumours, type of surgery, adjuvant radiotherapy or class of endocrine agent taken.

Table 3: ETnivariate and multivariate Cox regression analysis of clinicopathological factors influencing relapse-free survival in ER+, HER2-, node positive breast cancer patients. Factors significant in the univariate analysis are included in the multivariate model. Bold indicates significant p values.

[0165] In multivariate analysis, only lower numbers of ER-PR complexes (HR 2.475, Cl 1.297 - 4.717, p = 0.006), higher T-stage (HR 3.031, Cl 1.063 - 8.649 , p = 0.038) and taking endocrine therapy (HR 0.335, Cl 0.121 - 0.926, p = 0.035) were independent prognostic factors associated with recurrence (Table 3). Absent progesterone receptor expression, N-stage and having adjuvant chemotherapy were not significant independent prognostic factors for recurrence.

Example 7 - Prognostic effect of ER-PR interactions stratified by type of adjuvant endocrine agent taken

[0166] In an exploratory analysis of ER-PR interactions stratified by type of endocrine agent taken, low-ER-PR interactions was associated with relapse in patients taking AIs as adjuvant therapy (log-rank p = 0.0002), but not in those taking tamoxifen (log-rank p = 0.939) (Figure 3B). This equated to a hazard ratio of 4.831 (Cl 1.942 - 12.048, p = 0.001) for patients with low ER-PR interactions taking an AI (Table 4). A test for interaction was significant {p = 0.031).

Table 4: Cox regression analysis of ER:PR interactions influencing relapse-free survival stratified by adjuvant endocrine agent class taken. Percentages refer to the number of patients with high or low ER/PR interactions that had relapsed on endocrine treatment. Bold indicates significant p values.

[0167] Patients taking adjuvant endocrine therapy had significant clinical and pathological differences depending on the type of agent they were taking (Table 5). Patients taking tamoxifen were younger, more were pre-menopausal and had lower T-stage. They also had a larger proportion of patients with tumours that were of no special type. In patients taking adjuvant tamoxifen, there was also a trend towards a greater proportion of tumours with low levels of ER-PR interactions (p = 0.051).

Table 5: Associations between Tamoxifen- and Aromatase inhibitor (Al)-treated patients and clinicopathological variables. Abbreviations: PR-, progesterone receptor negative; PR+, progesterone receptor positive; pT, pathologic tumour; pN, pathologic node; A.I., aromatase inhibitor p values are the result of the two-sided Fisher’s exact test for 2 x 2 tables. For 2 x 3 and 2 x 4 tables, p values are the result of the Cochran- Armitage test for trend, except for Histology, which is a two-sided Fisher’s exact test. Bold indicates significant p values.

Table 6: Cox regression analysis of PR expression influencing relapse-free survival stratified by adjuvant endocrine agent class taken. Percentages refer to the number of patients with negative or positive PR expression that had relapsed on endocrine treatment.

Discussion

[0168] This work describes the development and application of an ER-PR interaction assay using PLA which can be used in FFPE tissue sections. In a breast cancer cell line and in patient-derived breast cancer explants, the interactions between ER and PR were dependent on the presence of both E₂ and a progestogen as no interactions were detected in the presence of only a single agonist ligand.

[0169] Whilst ER-PR interactions and PR immunohistochemistry were found to be positively correlated, that correlation was weak. Tumours negative for PR by immunohistochemistry can still have detectable ER-PR interactions by PLA, which is likely a function of the superior sensitivity of PLA over immunohistochemistry. Conversely, many tumours with detectable ER and PR showed no evidence of interaction, consistent with a lack of ligand activation, adding to the confidence of the specificity of the assay.

[0170] ETnexpectedly, ER-PR interactions were increased with age and were higher in post-menopausal women. Whilst post-menopausal levels of estrogen in breast tissues are high enough to promote the development of ER+ breast cancer, and forms the basis of clinical benefit from AIs, progesterone levels have been previously thought to be too low to play any significant role in signalling. The results described here provide first indirect evidence that a progesterone receptor ligand may be present at a level high enough to induce ER-PR interactions in post-menopausal women in breast cancer cells.

[0171] Lower levels of ER-PR interactions were associated with higher tumour grade and increased numbers of mitotic figures, intrinsic biological features of aggressive behaviour. Interactions were not associated with factors associated with stage (T-stage, N- stage).

[0172] It was also found that patients with tumours that were PR- also had poorer prognosis but the level of ER-PR interactions was more prognostic for relapse. The levels of ER, however, were not associated with relapse. The outcomes of subjects in the cohort of ER+, HER2-, node-positive breast cancer patients were highly dependent on the efficacy of adjuvant treatments, as evidenced by the observation that patients who did not have adjuvant chemotherapy or adjuvant endocrine therapy were more likely to relapse. Patients who did not take endocrine therapy were predominantly those that were non-compliant with adjuvant therapy recommendations.

[0173] In multivariate analysis, it was surprisingly found that PR expression was not an independent prognostic factor for relapse whilst low levels of ER-PR interactions were. This is an unexpected and significant finding, as it indicates that absent PR expression confers adverse prognosis on account of this selecting for a subgroup of patients with low ER-PR interactions. This result suggests that ER-PR interactions are the major determinant of the prognostic value of PR expression.

[0174] When stratified by type of endocrine agent taken, the prognostic effect of ER- PR interactions was limited to patients on AIs, predominantly post-menopausal women. A significant test for interaction raises the possibility that ER-PR interactions may be predictive of AI efficacy, although significant differences in the clinical and pathological factors of the subgroups precludes us from making this conclusion. Specifically, patients on tamoxifen were younger and more were pre-menopausal, on account of the lack of requirement to suppress ovarian function with this drug. Pre-menopausal women also formed only 28.8% of the cohort. [0175] Differences in ER-PR interactions between tumours may be due to either intrinsic factors within the tumour, or the availability of endogenous ligand, likely progesterone. The current findings of lower overall levels of ER-PR interactions in pre- menopausal women, who have higher progesterone levels, suggests tumour factors may be responsible for the differences in ER-PR interaction levels. Indeed, tumours that develop in pre-menopausal women would be expected to be able to survive and proliferate despite physiologic surges of endogenous progesterone and have developed mechanisms to resist the anti-proliferative and tumour suppressive effect of activated PR-mediated reprogramming of estrogen signalling.

[0176] Measuring the level of expression of ER-PR interactions may predict benefit from progestogen treatment. On the basis of the results presented here, adjuvant progestogen treatment may be an effective treatment strategy in post-menopausal women, who have higher levels of ER-PR interactions.

[0177] In conclusion, ER-PR interactions, which occur at a higher number in post menopausal women, are associated with relapse in patients on AIs. ER-PR interactions are therefore be predictive of AI efficacy and may also have utility in predicting response to progestogen therapy.

[0178] The data show that PR binds to ER in the nucleus in clinical samples of breast cancer and high levels of this interaction are a marker of good prognosis. This interaction is dependent on the presence of both an ER and PR ligand in vitro and ex vivo indicates that endogenous progesterone may have an effect on reprogramming ERa signalling in breast cancer even among post-menopausal women. Higher ER-PR interactions were associated with increasing age, indicating that PR ligand levels are high enough to elicit interaction between ER and PR in post-menopausal women. The data also show that low-levels or absent ER-PR interactions are specifically associated with relapse in women taking an adjuvant AI and ER-PR interactions may be used as a marker of AI response. Without being bound by theory or by a particular mode of action, these findings also raise the possibility that part of the benefit from AIs may arise from not only removing the trophic effects of estrogen, but by allowing endogenous progesterone to reprogram ER more effectively. ER- PR interactions may also be a potential biomarker of response to exogenous progestogen treatment.

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Claims

CLAIMS:

1. A method of identifying a subject at risk of recurrence of breast cancer following ablation of a breast cancer tumour, the method comprising:

2. The method of claim 1, wherein step (iii) comprises contacting the sample with a third agent, wherein the third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex in the sample.

3. The method of claim 1 or claim 2, wherein the ER-PR complex is detected by a method selected from the group consisting of a proximity ligation assay (PLA), biomolecular fluorescence complementation, chemical cross-linking followed by high mass MALDI mass spectrometry and fluorescence resonance energy transfer (FRET)

4. The method of claim 3, wherein the ER-PR complex is detected by PLA.

5. The method of claim 4, wherein the ER-PR complex is detected in a cell nucleus.

6. The method of any one of claims 1 to 5, wherein the subject is on aromatase inhibitor (AI) therapy.

7. The method of any one of claims 1 to 6, further comprising exposing the subject identified as being at risk of recurrence following ablation of a breast cancer tumour to a treatment regimen for treating the breast cancer.

8. The method of claim 7, wherein the treatment regimen comprises administering to the subject in need thereof an agent selected from the group consisting of a selective CDK4/6 inhibitor, a histone deacetylase (HD AC) inhibitor, an mTOR inhibitor, a phophoinositide-3- kinase (PI3K) inhibitor, a MAPK inhibitor, an aromatase inhibitor, a selective estrogen receptor degrader and a combination of any of the foregoing.

9. The method of claim 8, wherein the selective CDK4/6 inhibitor is selected from the group consisting of Palbociclib, Ribociclib and Abemaciclib.

10. The method of claim 8, wherein the histone deacetylase (HD AC) inhibitor is selected from the group consisting of Panobinostat, Vorinostat and Entinostat.

11. The method of claim 8, wherein the mTOR inhibitor is selected from the group consisting of Everolimus and Temsirolimus.

12. The method of claim 8, wherein the phophoinositide-3 -kinase (PI3K) inhibitor is selected from the group consisting of Buparlisib, Tasekisib, Alpelisib and Pictilisib.

13. The method of claim 8, wherein the MAPK inhibitor is Selumetinib.

14. The method of claim 8, wherein the aromatase inhibitor is selected from the group consisting of anastrozole, letrozole and exemestane.

15. The method of claim 8, wherein the selective estrogen receptor degrader is Fulvestrant.

16. The method of any one of claims 1 to 15, wherein the subject is identified as being at high risk of recurrence of breast cancer where the level of expression of ER-PR complexes in the sample is lower than a level of expression of ER-PR complexes in a control sample.

17. The method of claim 16, wherein the subject is identified as being at high risk of recurrence of breast cancer where the number of ER-PR complexes per cell in the sample is lower than the number of ER-PR complexes per cell in a control sample.

18. A method of predicting a subj ec s response to a treatment regimen for treating breast cancer, the method comprising:

19. The method of claim 18, wherein step (iii) comprises contacting the sample with a third agent, wherein the third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex in the sample.

20. The method of claim 18 or claim 19, wherein the ER-PR complex is detected by a method selected from the group consisting of a proximity ligation assay (PLA), biomolecular fluorescence complementation, chemical cross-linking followed by high mass MALDI mass spectrometry and fluorescence resonance energy transfer (FRET)

21. The method of claim 20, wherein the ER-PR complex is detected by PLA.

22. The method of any one of claims 18 to 21, wherein the treatment regimen comprises administration of an aromatase inhibitor (AI).

23. The method of any one of claims 18 to 22, further comprising exposing the subject, predicted to be a poor responder to the treatment regimen, to an alternative treatment regimen for treating breast cancer.

24. The method of any one of claims 18 to 23, wherein the subject is predicted to be a poor responder to the treatment regimen where the level of expression of ER-PR complexes in the sample is less than the level of expression of ER-PR complexes in a control sample.

25. The method of any one of claims 18 to 23, wherein the subject is predicted to be a poor responder to the therapeutic regimen if the number of ER-PR complexes per cell in the sample is less than the number of ER-PR complexes in a control sample.

26. A kit comprising (i) a first binding agent that binds to an estrogen receptor (ER) in breast cancer cells, (ii) a second binding agent that binds to a progesterone receptor (PR) in breast cancer cells; and (iii) a third agent, wherein the third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex.

27. A kit for use in the method of any one of claims 1 to 25, wherein the kit comprises

(i) a first binding agent that binds to an estrogen receptor (ER) in breast cancer cells, (ii) a second binding agent that binds to a progesterone receptor (PR) in breast cancer cells; and (iii) a third agent, wherein the third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex.

28. A method of stratifying a subj ect to a treatment regimen for breast cancer, the method comprising:

(iii)detecting the first and second binding agents when bound to an ER-PR complex in the sample; wherein the level of expression of ER-PR complexes detected in the sample relative to a level of expression of ER-PR complexes detected in a control sample is indicative of the risk of recurrence of breast cancer in the subject; and

(iv)exposing the subject identified in step (iii) as being at risk of recurrence to a treatment regimen for treating breast cancer.

29. The method of claim 28, wherein step (iii) comprises contacting the sample with a third agent, wherein the third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex in the sample.

30. The method of claim 28 or claim 29, wherein the ER-PR complex is detected by a method selected from the group consisting of a proximity ligation assay (PLA), biomolecular fluorescence complementation, chemical cross-linking followed by high mass MALDI mass spectrometry and fluorescence resonance energy transfer (FRET)

31. The method of claim 30, wherein the ER-PR complex is detected by PLA.

32. The method of any one of claims 28 to 31, wherein the subject is on aromatase inhibitor (AI) therapy.

33. The method of any one of claims 28 to 32, wherein the subject is identified as being at high risk of recurrence of breast cancer where the level of expression of ER-PR complexes in the sample is lower than a level of expression of ER-PR complexes in a control sample.

34. The method of claim 33, wherein the subject is identified as being at high risk of recurrence of breast cancer where the number of ER-PR complexes per cell in the sample is lower than the number of ER-PR complexes per cell in a control sample.

35. The method of any one of claims 28 to 34, wherein the treatment regimen of step (iv) comprises administering to the subject in need thereof an agent selected from the group consisting of a selective CDK4/6 inhibitor, a histone deacetylase (HDAC) inhibitor, an mTOR inhibitor, a phophoinositide-3 -kinase (PI3K) inhibitor, a MAPK inhibitor, an aromatase inhibitor, a selective estrogen receptor degrader and a combination of any of the foregoing.

36. The method of claim 35, wherein the selective CDK4/6 inhibitor is selected from the group consisting of Palbociclib, Ribociclib and Abemaciclib.

37. The method of claim 35, wherein the histone deacetyl ase (HD AC) inhibitor is selected from the group consisting of Panobinostat, Vorinostat and Entinostat.

38. The method of claim 35, wherein the mTOR inhibitor is selected from the group consisting of Everolimus and Temsirolimus.

39. The method of claim 35, wherein the phophoinositide-3 -kinase (PI3K) inhibitor is selected from the group consisting of Buparlisib, Tasekisib, Alpelisib and Pictilisib.

40. The method of claim 35, wherein the MAPK inhibitor is Selumetinib.

41. The method of claim 35, wherein the aromatase inhibitor is selected from the group consisting of anastrozole, letrozole and exemestane.

42. The method of claim 35, wherein the selective estrogen receptor degrader is Fulvestrant.

43. A complex formed by exposing a breast cancer cell to (i) a first binding agent that binds to an estrogen receptor (ER) of an ER-PR complex in the cell; wherein the ER-PR complex comprises an estrogen receptor (ER) and a progesterone receptor (PR); (ii) a second binding agent that binds to the PR of the ER-PR complex; and (iii) a third agent, wherein third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to the ER-PR complex in the cell.

44. A complex comprising (i) a first binding agent bound to an estrogen receptor (ER) of an ER-PR complex in a breast cancer cell, wherein the ER-PR complex comprises an estrogen receptor (ER) and a progesterone receptor (PR); (ii) a second binding agent bound to the PR of the ER-PR complex; and (iii) a third agent, wherein third agent comprises a detectable moiety that is detectable when each of the first and second binding agents is bound to an ER-PR complex in the cell.

45. A complex comprising a conjugate of an estrogen receptor (ER) and a progesterone receptor (PR), a first binding agent bound selectively to the ER, a second binding agent bound selectively to the PR and a third agent bound selectively to the first and second binding agent.

46. The complex of any one of claims 43 to 45, wherein the complex is located in a tumour cell.

47. The complex of claim 46, wherein the complex is located in the nucleus of the tumour cell.