Therapeutic treatment of breast cancer based on c-MAF status

Abstract

To provide a therapeutic treatment of breast cancer based on c-MAF status.SOLUTION: The present invention relates to the design of a customized therapy for a subject with breast cancer based on the c-MAF expression level and the menopausal status of the subject. In some embodiments, the customized therapy comprises an agent for avoiding or preventing bone degradation. In some embodiments, the agent for avoiding or preventing bone degradation is zoledronic acid. In one aspect, provided is an in vitro method for designing a customized therapy for a subject having breast cancer. The method comprises: (i) quantifying the c-MAF gene expression level, copy number, amplification, or gain in a sample of the subject; and (ii) comparing the expression level, copy number, amplification, or gain obtained in (i) with a reference value.SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing Reference The contents of the electronically submitted Sequence Listing submitted with this application ("3190_015PC02_SeqListing.txt", 58,739 bytes, created May 18, 2017) are incorporated herein by reference in their entirety.

[0002] 2. Background of the Invention FIELD OF THEINVENTION The present invention relates to the design of a customized therapy for a subject with breast cancer, the customized therapy being selected based on the subject's c-MAF expression level, copy number, amplification, gain or translocation and menopausal status. In some embodiments, the customized therapy comprises an agent for avoiding or preventing bone remodeling. In some embodiments, the agent for avoiding or preventing bone remodeling is zoledronic acid. [Background technology]

[0003] Breast cancer is the second most common type of cancer (10.4%; after lung cancer) and the fifth most common cause of cancer deaths worldwide (after lung, stomach, liver and colon cancer). Among women, breast cancer is the most common cause of cancer deaths. In 2005, breast cancer caused 502,000 deaths worldwide (7% of cancer deaths; almost 1% of all deaths). The number of cases worldwide has increased significantly since the 1970s, a phenomenon that is partly attributable to modern lifestyles in the Western world.

[0004] Breast cancer is classified into stages according to the TNM system (see American Joint Committee on Cancer. AJCC Cancer Staging Manual. 6th ed. New York, NY: Springer, 2002, which is incorporated herein by reference in its entirety). Prognosis is closely related to the outcome of staging, and staging is also used to assign patients to treatments in both clinical trials and medical practice. The information for staging is as follows:

[0005] TX: Primary tumor cannot be assessed. T0: No evidence of tumor. Tis: Carcinoma in situ, non-invasive. T1: Tumor is 2 cm or less. T2: Tumor is more than 2 cm but less than 5 cm. T3: Tumor is more than 5 cm. T4: Tumor of any size growing in the chest wall or skin, or inflammatory breast cancer.

[0006] NX: Adjacent lymph nodes cannot be evaluated. N0: Cancer has not spread to regional lymph nodes. N1: Cancer has spread to 1-3 axillary lymph nodes or 1 internal mammary lymph node. N2: Cancer has spread to 4-9 axillary lymph nodes or multiple internal mammary lymph nodes. N3: One of the following:

[0007] Cancer has spread to 10 or more axillary lymph nodes, or cancer has spread to the subclavian lymph nodes, or cancer has spread to the supraclavicular lymph nodes, or cancer has affected the axillary lymph nodes and has spread to the internal mammary lymph nodes, or cancer has affected 4 or more axillary lymph nodes and a minimal amount of cancer is in an internal mammary lymph node or sentinel lymph node biopsy.

[0008] MX: the presence of distant spread (metastasis) cannot be assessed. M0: there is no distant spread. M1: spread to distant organs has occurred, not including the supraclavicular lymph nodes.

[0009] The fact that most patients with solid tumor cancer die after metastasis means that it is important to understand the molecular and cellular mechanisms that allow tumor metastasis. Recent publications have demonstrated how metastasis is triggered by complex mechanisms that are still largely unknown, and how different metastatic cell types have tropism for specific organs. These tissue-specific metastatic cells have a set of acquired features that allow them to colonize specific organs.

[0010] All cells have receptors on their surface, in their cytoplasm and in the cell nucleus. Certain chemical messengers, such as hormones, bind to the receptors, which cause changes in the cell. There are three important receptors that can affect breast cancer cells: estrogen receptor (ER), progesterone receptor (PR) and HER2 / neu. To name cells that have any of these receptors, a plus sign is added to it if the receptor is present, and a minus sign is added to it if it is absent: ER positive (ER+), ER negative (ER-), PR positive (PR+), PR negative (PR-), HER2 positive (HER2+) and HER2 negative (HER2-). Receptor status has become an important assessment for all breast cancers, as it determines the appropriateness of using certain treatments, such as tamoxifen or trastuzumab.

[0011] Unsupervised gene expression array profiling has provided biological evidence for breast cancer heterogeneity through the identification of intrinsic subtypes, such as luminal A, luminal B, HER2+ / ER- and basal-like subtypes.

[0012] Triple-negative cancers are defined as tumors that do not express the genes for estrogen receptor (ER), progesterone receptor (PR) and HER2. This subgroup accounts for 15% of all types of breast cancer and a higher proportion of breast cancers occurring in premenopausal African and African-American women. Triple-negative breast cancers have a very different relapse pattern than estrogen receptor-positive breast cancer: the risk of relapse is significantly higher during the first 3-5 years, but then declines sharply and is substantially lower than that of estrogen receptor-positive breast cancer.

[0013] The basal-like subtype is characterized by low expression of genes in both the ER and HER2 clusters and is therefore typically ER-negative, PR-negative and HER2-negative in clinical trials; for this reason it is often referred to as "triple-negative" breast cancer (Breast Cancer Research 2007,9(Suppl 1):S13). Basal-like carcinomas express genes normally found in "basal" / myoepithelial cells of the normal breast, including high molecular weight cytokeratins (5 / 6, 14 and 17), P-cadherin, caveolin 1 and 2, nestin, αB crystallin and epidermal growth factor receptor (Reis-Fiho J et al., http: / / www.uscap.org / site~ / 98th / pdf / companion03h03.pdf).

[0014] Given that there is no internationally accepted definition of basal-like breast cancer, it is not surprising that there is great confusion as to whether triple-negative and basal-like breast cancers are synonymous. Although some groups use these terms interchangeably, it should be noted that not all basal-like cancers lack ER, PR, and HER2, and not all triple-negative cancers exhibit a basal-like phenotype. The majority of triple-negative cancers are of basal-like phenotype. Similarly, the majority of tumors expressing "basal" markers are triple-negative. However, it should be noted that there is a significant number of triple-negative cancers that do not express basal markers, and a small but still significant subgroup of basal-like cancers that express either hormone receptors or HER2. Bertucci et al. (Int J Cancer. 2008 Jul 1;123(1):236-40) addressed this issue directly and found that not all triple-negative tumors were classified as basal-like when analyzed by gene expression profiling (i.e., only 71% were of basal-like phenotype), and not all basal-like breast carcinomas classified by expression arrays displayed a triple-negative phenotype (i.e., 77%).

[0015] The main focus of breast cancer treatment is surgery with possible adjuvant hormonal therapy (with tamoxifen or aromatase inhibitors), chemotherapy and / or radiation therapy if the tumor is localized. Currently, the postoperative treatment proposal (adjuvant therapy) follows a pattern. This pattern is subject to change, as a world conference is held every two years in St. Gallen, Switzerland, to discuss the actual results of global multicenter studies. Similarly, the pattern is also reviewed according to the consensus criteria of the National Institutes of Health (NIH). Based on these criteria, more than 85-90% of patients without metastases in lymph nodes would be candidates for adjuvant systemic therapy.

[0016] Currently, PCR assays, such as Oncotype DX, or microarray assays, such as MammaPrint, can predict the risk of breast cancer recurrence based on the expression of certain genes. In February 2007, the MammaPrint assay became the first breast cancer indicator to receive official approval from the Food and Drug Administration.

[0017] European Patent Application Publication No. 1961825 describes a method for predicting the occurrence of breast cancer metastasis to bone, lung, liver or brain, comprising determining the expression level of one or more markers (including c-MAF) in tumor tissue samples relative to the corresponding expression level in control samples.However, this document requires the simultaneous determination of several genes to enable the determination of breast cancer patient survival, and the correlation between the ability of gene signatures to predict the probability of survival without bone metastasis is not statistically significant.

[0018] US Patent Application Publication No. 2011 / 0150979 describes a method for predicting the prognosis of basal-like breast cancer, comprising detecting levels of FOXC1.

[0019] US Patent Application Publication No. 2010 / 0210738 relates to a method for predicting cancer prognosis in a subject with triple-negative breast cancer, comprising detecting the expression levels of a set of genes that are randomly up- or down-regulated in a sample. US Patent Application Publication No. 2011 / 0130296 relates to the identification of marker genes useful in the diagnosis and prognosis of triple-negative breast cancer. There is a need to identify subsets of breast cancer patients who will benefit from a particular treatment and, conversely, subsets of breast cancer patients who will not benefit from or may be harmed by a particular treatment. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] European Patent Application Publication No. 1961825 [Patent Document 2] US Patent Application Publication No. 2011 / 0150979 [Patent Document 3] US Patent Application Publication No. 2010 / 0210738 [Patent Document 4] US Patent Application Publication No. 2011 / 0130296 [Non-patent literature]

[0021] [Non-Patent Document 1] American Joint Committee on Cancer.AJCC Cancer Staging Manual.6th ed.New York,NY:Springer,2002 [Non-Patent Document 2] Breast Cancer Research 2007,9(Suppl 1):S13 [Non-Patent Document 3] Bertucci et al., Int J Cancer.2008 Jul 1;123(1):236-40 Summary of the Invention [Means for solving the problem]

[0022] In one embodiment, the present invention relates to an in vitro method for designing a customized therapy for a subject with breast cancer, comprising: i) quantifying the c-MAF gene expression level, copy number, amplification or gain in a sample of said subject; and ii) comparing said expression level, copy number, amplification or gain obtained in i) with a reference value, wherein if said expression level, copy number, amplification or gain is not increased with respect to said reference value, said subject is admitted to a therapy aimed at preventing and / or treating bone remodeling and improving disease-free or overall survival.

[0023] In some embodiments, the subject is non-postmenopausal. In other embodiments, the subject is postmenopausal.

[0024] In one embodiment, the present invention relates to an in vitro method for designing a customized therapy for a non-postmenopausal subject with breast cancer, comprising: i) quantifying the c-MAF gene expression level, copy number, amplification or gain in a sample from said subject; and ii) comparing said expression level, copy number, amplification or gain obtained in i) with a reference value, If said expression level, copy number, amplification or gain is increased relative to said reference value, then said subject is not permitted to receive a treatment aimed at preventing and / or treating bone remodeling and improving disease-free or overall survival.

[0025] In one embodiment, the present invention provides an in vitro method for designing a customized therapy for a postmenopausal subject with breast cancer, comprising: i) quantifying the c-MAF gene expression level, copy number, amplification or gain in a sample from said subject; and ii) comparing the expression level, copy number, amplification or gain obtained in i) with a reference value. Including, If said expression level, copy number, amplification or gain is increased relative to said reference value, then said subject is permitted to receive a treatment aimed at preventing and / or treating bone remodeling and improving disease-free or overall survival.

[0026] In some embodiments, the subject is administered a therapy aimed at preventing and / or treating bone remodeling and improving disease-free or overall survival, while in other embodiments, the subject is not administered a therapy aimed at preventing and / or treating bone remodeling and improving disease-free or overall survival.

[0027] In certain embodiments, the treatment aimed at preventing and / or treating bone remodeling or improving disease-free or overall survival is an agent intended to prevent or inhibit bone degradation and improve disease-free or overall survival, selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranelate, DKK-1 inhibitors, dual MET and VEGFR2 inhibitors, estrogen receptor modulators, calcitonin, radium-223, CCR5 antagonists, Src kinase inhibitors, COX-2 inhibitors, mTor inhibitors and cathepsin K inhibitors. In some embodiments, the RANKL inhibitor is selected from the group consisting of RANKL-specific antibodies, RANKL-specific nanobodies and osteoprotegerin. In certain embodiments, the RANKL-specific antibody is denosumab. In some embodiments, the bisphosphonate is zoledronic acid. In other embodiments, the RANKL-specific nanobody is ALX-0141. In certain embodiments, the dual MET and VEGFR2 inhibitor is Cabozantinib.

[0028] In some embodiments, quantifying the c-MAF gene expression level comprises quantifying the messenger RNA (mRNA) of said gene or a fragment of said mRNA, the complementary DNA (cDNA) of said gene or a fragment of said cDNA, or quantifying the level of the protein encoded by said gene. In certain embodiments, the expression level, copy number, amplification or gain is quantified by quantitative polymerase chain reaction (PCR) or DNA or RNA array, or nucleotide hybridization techniques. In embodiments, the protein level is quantified by Western blot, ELISA, immunohistochemistry or protein array. In certain embodiments, the protein level is quantified using an antibody comprising a heavy chain CDR1 of SEQ ID NO: 21 and / or a heavy chain CDR2 of SEQ ID NO: 22 and / or a heavy chain CDR3 of SEQ ID NO: 23; and / or a light chain CDR1 of SEQ ID NO: 18 and / or a light chain CDR2 of SEQ ID NO: 19 and / or a light chain CDR3 of SEQ ID NO: 20. In some embodiments, the amplification or gain of the c-MAF gene is determined by using a c-MAF gene specific probe. In a particular embodiment, the c-MAF gene specific probe is a Vysis LSI / IGH MAF dual color dual fusion probe.In other embodiments, amplification or gain is determined by in situ hybridization or PCR.

[0029] In certain embodiments, the reference value is from a breast cancer tumor tissue sample from a subject not suffering from metastasis.

[0030] In one embodiment, the present invention relates to a method for treating bone metastasis in a subject having breast cancer, wherein the c-MAF expression level in the metastatic tumor sample is not increased relative to a control sample, comprising administering an agent capable of preventing or inhibiting bone remodeling or improving disease-free or overall survival, wherein the agent capable of preventing or inhibiting bone remodeling or improving disease-free or overall survival is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranelate, DKK-1 inhibitors, dual MET and VEGFR2 inhibitors, estrogen receptor modulators, EGFR inhibitors, calcitonin, radium-223, CCR5 antagonists, Src kinase inhibitors, COX-2 inhibitors, mTor inhibitors and cathepsin K inhibitors.

[0031] In certain embodiments, the subject is non-postmenopausal. In other embodiments, the subject is postmenopausal.

[0032] In one embodiment, the present invention relates to a method for treating bone metastasis in a postmenopausal subject with breast cancer, the subject having an increased level of c-MAF expression in a metastatic tumor sample relative to a control sample, comprising administering an agent capable of preventing or inhibiting bone remodeling or improving disease-free or overall survival, wherein the agent capable of avoiding or preventing bone remodeling is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranelate, DKK-1 inhibitors, dual MET and VEGFR2 inhibitors, estrogen receptor modulators, EGFR inhibitors, calcitonin, radium-223, CCR5 antagonists, Src kinase inhibitors, COX-2 inhibitors, mTor inhibitors and cathepsin K inhibitors.

[0033] In certain embodiments, the RANKL inhibitor is selected from the group consisting of a RANKL-specific antibody, a RANKL-specific nanobody, and osteoprotegerin. In further embodiments, the RANKL-specific antibody is denosumab. In other embodiments, the bisphosphonate is zoledronic acid. In yet other embodiments, the RANKL-specific nanobody is ALX-9141. In certain embodiments, the dual MET and VEGFR2 inhibitor is cabozantinib.

[0034] In one embodiment, the present invention relates to a method for classifying subjects suffering from breast cancer into cohorts, comprising: a) determining the expression level, copy number, amplification or gain of c-MAF in a breast tumor sample of the subject; b) comparing the expression level, copy number, amplification or gain of c-MAF in the sample with a predefined reference level of c-MAF expression; and c) classifying the subject into a cohort based on the expression level, copy number, amplification or gain of c-MAF in the sample and the subject's status as postmenopausal or non-postmenopausal.

[0035] In certain embodiments, subjects are administered different treatments based on c-MAF expression levels and / or postmenopausal or non-postmenopausal status.

[0036] In some embodiments, quantifying c-MAF expression level comprises quantifying messenger RNA (mRNA) of said gene or a fragment of said mRNA, complementary DNA (cDNA) of said gene or a fragment of said cDNA, or quantifying the level of protein encoded by said gene. In a particular embodiment, an antibody comprising heavy chain CDR1 of SEQ ID NO: 21 and / or heavy chain CDR2 of SEQ ID NO: 22 and / or heavy chain CDR3 of SEQ ID NO: 23; and / or light chain CDR1 of SEQ ID NO: 18 and / or light chain CDR2 of SEQ ID NO: 19 and / or light chain CDR3 of SEQ ID NO: 20 is used to quantify the level of protein. In a particular embodiment, amplification is determined by in situ hybridization or PCR. In a further embodiment, in situ hybridization is fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or silver in situ hybridization (SISH). In still further embodiments, the in situ hybridization is fluorescent in situ hybridization (FISH).

[0037] In some embodiments, the copy number of c-MAF as measured using FISH is ≧2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0. In certain embodiments, the copy number of c-MAF as measured using FISH is ≧2.2. In further embodiments, the copy number of c-MAF as measured using FISH is ≧2.3. In still further embodiments, the copy number of c-MAF as measured using FISH is ≧2.4. In certain embodiments, the copy number of c-MAF as measured using FISH is ≧2.5. In other embodiments, the copy number of c-MAF as measured using FISH is <2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0.

[0038] In one embodiment, the present invention relates to an in vitro method for predicting IDFS in a patient with breast cancer, comprising: i) quantifying the expression level, copy number, amplification or gain of the c-MAF gene in a sample of said subject; and ii) comparing said expression level, copy number, amplification or gain obtained in step i) with a reference value, wherein an increase in the expression level, copy number, amplification or gain of said gene relative to said reference value is indicative of poor IDFS.

[0039] In one embodiment, the present invention relates to an in vitro method for predicting IDFS in a patient with breast cancer, comprising determining a c-MAF gene expression level, copy number, amplification or gain in a sample of said subject compared to a reference, wherein an increase in said c-MAF gene expression level, copy number, amplification or gain relative to said reference is indicative of poor IDFS.

[0040] In one embodiment, the present invention relates to an in vitro method for predicting IDFS (excluding bone recurrence) in a patient with breast cancer, comprising determining a c-MAF gene expression level, copy number, amplification or gain in a sample of said subject compared to a reference, wherein an increase in said c-MAF gene expression level, copy number, amplification or gain relative to said reference is indicative of poor IDFS (excluding bone recurrence).

[0041] In some embodiments, the agent capable of preventing or inhibiting bone remodeling is an agent capable of preventing or inhibiting bone degradation.

[0042] In some embodiments, quantifying c-MAF expression level comprises quantifying messenger RNA (mRNA) of said gene or a fragment of said mRNA, complementary DNA (cDNA) of said gene or a fragment of said cDNA, or quantifying the level of protein encoded by said gene. In a particular embodiment, an antibody comprising heavy chain CDR1 of SEQ ID NO: 21 and / or heavy chain CDR2 of SEQ ID NO: 22 and / or heavy chain CDR3 of SEQ ID NO: 23; and / or light chain CDR1 of SEQ ID NO: 18 and / or light chain CDR2 of SEQ ID NO: 19 and / or light chain CDR3 of SEQ ID NO: 20 is used to quantify the level of protein. In other embodiments, amplification is determined by in situ hybridization or PCR. In further embodiments, in situ hybridization is fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or silver in situ hybridization (SISH). In still further embodiments, the in situ hybridization is fluorescent in situ hybridization (FISH).

[0043] In some embodiments, the copy number of c-MAF as measured using FISH is ≧2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0. In certain embodiments, the copy number of c-MAF as measured using FISH is ≧2.2. In other embodiments, the copy number of c-MAF as measured using FISH is ≧2.3. In further embodiments, the copy number of c-MAF as measured using FISH is ≧2.4. In yet further embodiments, the copy number of c-MAF as measured using FISH is ≧2.5. In some embodiments, the copy number is determined as the average copy number per cell.

[0044] In some embodiments, the breast cancer is ER+ breast cancer. In certain embodiments, the breast cancer is ER- breast cancer. In other embodiments, the breast cancer is triple-negative breast cancer. In different embodiments, the breast cancer is a basal-like subtype. In some embodiments, the breast cancer is HER2+ breast cancer.

[0045] In some embodiments, the expression level, copy number, amplification or gain of the c-MAF gene is determined by determining the expression level, copy number, amplification or gain of the 16q23 or 16q22-q24 locus.

[0046] In some embodiments, the treatment is an mTOR inhibitor or a CDK4 / 6 inhibitor, hi other embodiments, the treatment is hormonal therapy extended beyond standard treatment.

[0047] In some embodiments, the present invention relates to a method for treating a subject with breast cancer, in which the c-MAF expression level, copy number, amplification or gain in the metastatic tumor sample is increased relative to a control sample, comprising administering an mTOR inhibitor or a CDK4 / 6 inhibitor. In some embodiments, the present invention relates to a method for treating a subject with breast cancer, in which the c-MAF expression level, copy number, amplification or gain in the metastatic tumor sample is increased relative to a control sample, comprising administering an extended hormonal therapy beyond the standard of care. In some embodiments, the present invention relates to a method for treating a subject with breast cancer, in which the c-MAF expression level, copy number, amplification or gain in the metastatic tumor sample is not increased relative to a control sample, comprising not administering an mTOR inhibitor or a CDK4 / 6 inhibitor. In some embodiments, the present invention relates to a method for treating a subject having breast cancer, in which the c-MAF expression level, copy number, amplification or gain in a metastatic tumor sample is not increased relative to a control sample, comprising not administering extended hormonal therapy beyond standard of care.

[0048] In one embodiment, the invention relates to a method for predicting disease-free survival status of a patient, comprising measuring c-MAF gene expression level, copy number, amplification or gain relative to a reference sample level, and using the c-MAF gene expression level, copy number, amplification or gain to predict overall survival of the patient. In some embodiments, an increase in c-MAF gene expression level, copy number, amplification or gain relative to a reference sample level predicts a shorter disease-free survival than patients in whom the c-MAF gene expression level, copy number, amplification or gain is not increased relative to the reference sample level.

[0049] In one embodiment, the invention relates to a method for predicting the overall survival status of a patient, comprising measuring c-MAF gene expression level, copy number, amplification or gain relative to a reference sample level, and using said c-MAF gene expression level, copy number, amplification or gain to predict the overall survival of said patient. In another embodiment, an increase in c-MAF gene expression level, copy number, amplification or gain relative to a reference sample level predicts a shorter overall survival than patients in whom the c-MAF gene expression level, copy number, amplification or gain is not increased relative to the reference sample level.

[0050] In some embodiments, the menopausal status of the patient is also used to predict the survival status of the patient. In some embodiments, the subject is non-postmenopausal. In certain embodiments, the subject is premenopausal. In certain embodiments, the subject is postmenopausal. In certain embodiments, for example, the following items are provided: (Item 1) 1. An in vitro method for designing a customized therapy for a subject with breast cancer, comprising: i) quantifying the c-MAF gene expression level, copy number, amplification or gain in a sample from said subject; and ii) comparing the expression level, copy number, amplification or gain obtained in i) with a reference value. Including, An in vitro method wherein if the expression level, copy number, amplification or gain is not increased relative to the reference value, the subject is permitted to receive a treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival. (Item 2) 2. The method of claim 1, wherein the subject is non-postmenopausal. (Item 3) 2. The method of claim 1, wherein the subject is postmenopausal. (Item 4) 1. An in vitro method for designing a customized therapy for a non-postmenopausal subject with breast cancer, comprising: i) quantifying the c-MAF gene expression level, copy number, amplification or gain in a sample from said subject; and ii) comparing the expression level, copy number, amplification or gain obtained in i) with a reference value. Including, An in vitro method, wherein if the expression level, copy number, amplification or gain is increased relative to the reference value, the subject is not permitted to receive a therapy aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival. (Item 5) 1. An in vitro method for designing a customized therapy for a postmenopausal subject with breast cancer, comprising: i) quantifying the c-MAF gene expression level, copy number, amplification or gain in a sample from said subject; and ii) comparing the expression level, copy number, amplification or gain obtained in i) with a reference value. Including, An in vitro method, wherein if the expression level, copy number, amplification or gain is increased relative to the reference value, the subject is permitted to receive a treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival. (Item 6) The method according to any one of items 1 to 3 or 5, wherein the subject is administered a treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival. (Item 7) 5. The method according to item 4, wherein the subject is not administered a therapy aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival. (Item 8) 8. The method according to any one of items 1 to 7, wherein said treatment aimed at preventing and / or treating bone remodeling (including but not limited to degradation) or improving disease-free survival or overall survival is an agent intended to prevent or inhibit bone remodeling or improve disease-free survival or overall survival, selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogues, strontium ranelate, DKK-1 inhibitors, dual MET and VEGFR2 inhibitors, estrogen receptor modulators, calcitonin, radium-223, CCR5 antagonists, Src kinase inhibitors, COX-2 inhibitors, mTor inhibitors and cathepsin K inhibitors. (Item 9) 9. The method of claim 8, wherein the RANKL inhibitor is selected from the group consisting of a RANKL-specific antibody, a RANKL-specific nanobody and osteoprotegerin. (Item 10) 10. The method of claim 9, wherein the RANKL-specific antibody is denosumab. (Item 11) 9. The method of claim 8, wherein the bisphosphonate is zoledronic acid. (Item 12) The method of claim 9, wherein the RANKL-specific nanobody is ALX-0141. (Item 13) 9. The method of claim 8, wherein the dual MET and VEGFR2 inhibitor is Cabozantinib. (Item 14) 14. The method according to any one of items 1 to 13, wherein quantifying the c-MAF gene expression level comprises quantifying the messenger RNA (mRNA) of the gene or a fragment of the mRNA, the complementary DNA (cDNA) of the gene or a fragment of the cDNA, or quantifying the level of a protein encoded by the gene. (Item 15) 15. The method according to any one of items 1 to 14, wherein the expression level, copy number, amplification or gain is quantified by quantitative polymerase chain reaction (PCR) or DNA or RNA array, FISH or nucleotide hybridization techniques. (Item 16) 15. The method according to any one of items 1 to 14, wherein the level of the protein is quantified by Western blot, ELISA, immunohistochemistry or protein array. (Item 17) 15. The method according to any one of items 1 to 14, wherein the level of the protein is quantified using an antibody comprising a heavy chain CDR1 of SEQ ID NO: 21 and / or a heavy chain CDR2 of SEQ ID NO: 22 and / or a heavy chain CDR3 of SEQ ID NO: 23; and / or a light chain CDR1 of SEQ ID NO: 18 and / or a light chain CDR2 of SEQ ID NO: 19 and / or a light chain CDR3 of SEQ ID NO: 20. (Item 18) 15. The method according to any one of items 1 to 14, wherein said amplification or gain of said c-MAF gene is determined by using a c-MAF gene specific probe. (Item 19) 19. The method of claim 18, wherein the c-MAF gene specific probe is a Vysis LSI / IGH MAF dual-color dual fusion probe. (Item 20) 20. The method according to any one of items 1 to 19, wherein the reference value is from a breast cancer tumor tissue sample from a subject not suffering from metastasis. (Item 21) 16. The method according to any one of items 1 to 15, wherein the amplification or gain is determined by in situ hybridization or PCR. (Item 22) 1. A method for treating bone metastasis in a subject having breast cancer, the subject having an increased or non-increased c-MAF expression level in a metastatic tumor sample relative to a control sample, comprising administering an agent capable of preventing or inhibiting bone remodeling or improving disease-free or overall survival, wherein the agent capable of avoiding or preventing bone degradation or improving disease-free or overall survival is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranelate, DKK-1 inhibitors, dual MET and VEGFR2 inhibitors, estrogen receptor modulators, EGFR inhibitors, calcitonin, radium-223, CCR5 antagonists, Src kinase inhibitors, COX-2 inhibitors, mTor inhibitors and cathepsin K inhibitors. (Item 23) 23. The method of claim 22, wherein the subject is non-postmenopausal. (Item 24) 23. The method of claim 22, wherein the subject is postmenopausal. (Item 25) 1. A method for treating bone metastasis in a postmenopausal subject having breast cancer, the subject having an increased level of c-MAF expression in a metastatic tumor sample relative to a control sample, comprising administering an agent capable of preventing or inhibiting bone remodeling or improving disease-free or overall survival, the agent being selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranelate, DKK-1 inhibitors, dual MET and VEGFR2 inhibitors, estrogen receptor modulators, EGFR inhibitors, calcitonin, radium-223, CCR5 antagonists, Src kinase inhibitors, COX-2 inhibitors, mTor inhibitors and cathepsin K inhibitors. (Item 26) 26. The method according to any one of items 22 to 25, wherein the RANKL inhibitor is selected from the group consisting of a RANKL-specific antibody, a RANKL-specific nanobody and osteoprotegerin. (Item 27) 27. The method of claim 26, wherein the RANKL-specific antibody is denosumab. (Item 28) 26. The method according to any one of items 22 to 25, wherein the bisphosphonate is zoledronic acid. (Item 29) The use according to item 26, wherein the RANKL-specific nanobody is ALX-9141. 26. The use according to any one of items 22 to 25, wherein the dual MET and VEGFR2 inhibitor is Cabozantinib. (Item 31) 1. A method for classifying a subject suffering from breast cancer into a cohort, comprising: a) determining the expression level, copy number, amplification or gain of c-MAF in a breast tumor sample of the subject; b) comparing the expression level, copy number, amplification or gain of c-MAF in the sample with a predetermined reference level of c-MAF expression; and c) classifying the subject into a cohort based on the expression level, copy number, amplification or gain of c-MAF in the sample and the subject's status as postmenopausal or non-postmenopausal. (Item 32) 32. The method of claim 31, wherein the subject is administered a different treatment based on the c-MAF expression level and / or postmenopausal or non-postmenopausal status. (Item 33) The method according to any one of items 22 to 32, wherein the quantification of the c-MAF expression level comprises quantifying the messenger RNA (mRNA) of the gene or a fragment of the mRNA, the complementary DNA (cDNA) of the gene or a fragment of the cDNA, or quantifying the level of a protein encoded by the gene. The method according to any one of items 22 to 33, wherein the level of the protein is quantified using an antibody comprising a heavy chain CDR1 of SEQ ID NO: 21 and / or a heavy chain CDR2 of SEQ ID NO: 22 and / or a heavy chain CDR3 of SEQ ID NO: 23; and / or a light chain CDR1 of SEQ ID NO: 18 and / or a light chain CDR2 of SEQ ID NO: 19 and / or a light chain CDR3 of SEQ ID NO: 20. (Item 35) 35. The method according to items 1 to 34, wherein the amplification is determined by in situ hybridization or PCR. (Item 36) 36. The method of claim 35, wherein the in situ hybridization is fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or silver in situ hybridization (SISH). (Item 37) 37. The method of claim 36, wherein the in situ hybridization is fluorescent in situ hybridization (FISH). (Item 38) 38. The method according to items 1 to 37, wherein the copy number of c-MAF as measured using FISH is ≧2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0. (Item 39) 39. The method according to items 1 to 38, wherein the copy number of c-MAF is ≧2.2 as measured using FISH. (Item 40) 40. The method according to items 1 to 39, wherein the copy number of c-MAF as measured using FISH is ≧2.3. (Item 41) 41. The method according to items 1 to 40, wherein the copy number of c-MAF is ≧2.4 as measured using FISH. (Item 42) 42. The method according to items 1 to 41, wherein the copy number of c-MAF is ≧2.5 as measured using FISH. (Item 43) 38. The method according to items 1 to 37, wherein the copy number of c-MAF as measured using FISH is <2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0. (Item 44) 38. The method according to any one of items 1 to 37, wherein the copy number is determined as an average copy number per cell. (Item 45) 1. An in vitro method for predicting IDFS in a patient with breast cancer, comprising: i) quantifying the expression level, copy number, amplification or gain of the c-MAF gene in a sample from said subject; and ii) comparing the expression level, copy number, amplification or gain obtained in step i) with a reference value. Including, The in vitro method, wherein an increase in expression level, copy number, amplification or gain of said gene relative to said reference value is indicative of poor IDFS. (Item 46) An in vitro method for predicting IDFS in a patient with breast cancer, comprising determining a c-MAF gene expression level, copy number, amplification or gain in a sample from the subject compared to a reference, wherein an increase in the c-MAF gene expression level, copy number, amplification or gain relative to the reference indicates poor IDFS. (Item 47) An in vitro method for predicting IDFS excluding bone recurrence in a patient with breast cancer, comprising determining a c-MAF gene expression level, copy number, amplification or gain in a sample from the subject compared to a reference, wherein an increase in the c-MAF gene expression level, copy number, amplification or gain relative to the reference indicates poor IDFS excluding bone recurrence. (Item 48) The method according to any one of Items 45 to 47, wherein the quantification of the c-MAF expression level comprises quantifying the messenger RNA (mRNA) of the gene or a fragment of the mRNA, the complementary DNA (cDNA) of the gene or a fragment of the cDNA, or quantifying the level of a protein encoded by the gene. 49. The method according to any one of items 45 to 48, wherein the level of the protein is quantified using an antibody comprising a heavy chain CDR1 of SEQ ID NO: 21 and / or a heavy chain CDR2 of SEQ ID NO: 22 and / or a heavy chain CDR3 of SEQ ID NO: 23; and / or a light chain CDR1 of SEQ ID NO: 18 and / or a light chain CDR2 of SEQ ID NO: 19 and / or a light chain CDR3 of SEQ ID NO: 20. (Item 50) 49. The method according to any one of items 45 to 48, wherein the amplification is determined by in situ hybridization or PCR. (Item 51) 51. The method of claim 50, wherein the in situ hybridization is fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or silver in situ hybridization (SISH). (Item 52) 52. The method of claim 51, wherein the in situ hybridization is fluorescent in situ hybridization (FISH). (Item 53) 53. The method according to items 45 to 52, wherein the copy number of c-MAF as measured using FISH is ≧2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0. (Item 54) 54. The method according to items 45 to 53, wherein the copy number of c-MAF is ≧2.2 as measured using FISH. (Item 55) 55. The method according to items 45 to 54, wherein the copy number of c-MAF as measured using FISH is ≧2.3. (Item 56) 56. The method according to items 45 to 55, wherein the copy number of c-MAF is ≧2.4 as measured using FISH. (Item 57) 57. The method according to items 45 to 56, wherein the copy number of c-MAF as measured using FISH is ≧2.5. (Item 58) 58. The method according to any one of items 45 to 57, wherein the copy number is determined as an average copy number per cell. (Item 59) 59. The method of any one of items 1 to 58, wherein the breast cancer is ER+ breast cancer. (Item 60) 59. The method of any one of items 1 to 58, wherein the breast cancer is ER- breast cancer. (Item 61) 59. The method of any one of items 1 to 58, wherein the breast cancer is triple-negative breast cancer. (Item 62) 59. The method of any one of items 1 to 58, wherein the breast cancer is of the basal-like subtype. (Item 63) 59. The method of any one of items 1 to 58, wherein the breast cancer is HER2+ breast cancer. (Item 64) 31. The method according to any one of items 1 to 30, wherein the agent capable of preventing or inhibiting bone remodeling is an agent capable of preventing or inhibiting bone degradation. (Item 65) 65. The method according to any one of items 1 to 64, wherein the expression level, copy number, amplification or gain of the c-MAF gene is determined by determining the expression level, copy number, amplification or gain of locus 16q23 or 16q22-q24. (Item 66) 31. The method of any one of items 1 to 30, wherein the treatment that improves disease-free survival or overall survival is an mTOR inhibitor or a CDK4 / 6 inhibitor. (Item 67) 31. The method according to any one of items 1 to 30, wherein said treatment that improves disease-free survival or overall survival is hormonal therapy extended beyond standard treatment. (Item 68) A method for treating a subject having breast cancer in which c-MAF expression levels, copy number, amplification or gain in a metastatic tumor sample is increased relative to a control sample, the method comprising administering an mTOR inhibitor or a CDK4 / 6 inhibitor. (Item 69) A method for treating a subject having breast cancer in which c-MAF expression levels, copy number, amplification or gain in a metastatic tumor sample is increased relative to a control sample, comprising administering extended hormonal therapy beyond standard treatment. (Item 70) A method for treating a subject having breast cancer, in which c-MAF expression levels, copy number, amplification or gain in a metastatic tumor sample is not increased relative to a control sample, comprising not administering an mTOR inhibitor or a CDK4 / 6 inhibitor. (Item 71) A method for treating a subject having breast cancer in which c-MAF expression levels, copy number, amplification or gain in a metastatic tumor sample is not increased relative to a control sample, comprising not administering extended hormonal therapy beyond standard treatment. (Item 72) A method for predicting disease-free survival status of a patient, comprising measuring c-MAF gene expression level, copy number, amplification or gain relative to a reference, and using the c-MAF gene expression level, copy number, amplification or gain to predict overall survival of the patient. (Item 73) 73. The method of item 72, wherein an increase in the c-MAF gene expression level, copy number, amplification or gain relative to the reference predicts a shorter disease-free survival than patients whose c-MAF gene expression level, copy number, amplification or gain is not increased relative to the reference. (Item 74) A method for predicting a patient's overall survival status, comprising measuring c-MAF gene expression level, copy number, amplification or gain relative to a reference, and using the c-MAF gene expression level, copy number, amplification or gain to predict the patient's overall survival. (Item 75) 75. The method of item 74, wherein an increase in the c-MAF gene expression level, copy number, amplification or gain relative to the reference predicts shorter overall survival than patients whose c-MAF gene expression level, copy number, amplification or gain is not increased relative to the reference. (Item 76) 74. The method of any one of items 68 to 73, wherein the subject is non-postmenopausal. (Item 77) 74. The method of any one of items 68 to 73, wherein the subject is premenopausal. (Item 78) 74. The method of any one of items 68 to 73, wherein the subject is postmenopausal. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 is a summary of the assay parameters.

[0052] [Diagram 2] Figure 2 shows the AZURE study design.

[0053] [Diagram 3] Figure 3 is an H&E analysis of AZURE samples. Evaluable and non-evaluable samples are shown.

[0054] [Figure 4A] 4A and 4B show the MAF positivity rate. [Figure 4B] 4A and 4B show the MAF positivity rate.

[0055] [Diagram 5]Figure 5 shows the MAF cutoff optimized FISH data. The sharp increase on the cut point graph indicates that the MAF FISH value is truly a threshold event. In addition, the defined cutoff is close to the optimized cutoff.

[0056] [Figure 6] FIG. 6 shows the risk of bone recurrence based on MAF FISH values.

[0057] [Figure 7] FIG. 7. Time to bone recurrence by MAF FISH value using a bone optimized cutoff of 2.3.

[0058] [Figure 8] Figure 8: %IDFS by FISH. An optimal cutoff of 2.2 was used.

[0059] [Figure 9] Figure 9: Overall survival by FISH. An optimal cutoff of 2.2 was used.

[0060] [Figure 10] Figure 10: Time to bone recurrence by FISH in AZURE control patients only. A bone optimization cutoff of 2.3 was used.

[0061] [Figure 11] Figure 11 is IDFS by FISH in AZURE control patients only. An optimized cutoff of 2.2 was used.

[0062] [Figure 12] Figure 12: Time to IDFS (excluding bone recurrence) by FISH in AZURE control patients only. An optimized cutoff of 2.2 was used.

[0063] [Figure 13]Figures 13A and B: Time to bone metastasis in patients in the control arm and in the zoledronic acid-treated arm. (A) Cumulative incidence of bone metastasis as a first event and (B) at any time during follow-up. Analysis was by intention to treat. HR - hazard ratio.

[0064] [Figure 14] Figure 14: Evaluation of time to bone metastasis as first event in AZURE control patients and zoledronic acid treated patients. A bone optimized cutoff of 2.3 was used.

[0065] [Figure 15] Figure 15A and B: Disease-free survival (DFS) and invasive disease-free survival (IDFS) between the control arm and zoledronic acid-treated patients. Kaplan-Meier curves for (A) disease-free survival and (B) invasive disease-free survival. Analysis was by intention to treat. HR = hazard ratio.

[0066] [Figure 16] FIG. 16 is the time to distant recurrence between the control arm and zoledronic acid treated patients.

[0067] [Figure 17] Figure 17: Time to bone metastasis events (any time point) according to treatment. In the time to bone metastasis (any time point), death is used as a competing event.

[0068] [Figure 18] FIG. 18: Time to bone metastasis event (any time point) according to MAF copy number (according to a pre-specified MAF cutoff of 2.5).

[0069] [Figure 19] Figure 19A and B, IDFS by menopausal status in the AZURE trial. Kaplan-Meier curves of invasive disease-free survival by menopausal status. (A) Premenopausal, perimenopausal and unknown menopausal status and (B) >5 years postmenopausal. Heterogeneity test by menopausal status χ2 14.71; p=0.03.

[0070] [Figure 20] FIG. 20: Time to bone metastasis event (any time point) according to MAF copy number in postmenopausal patients (data according to a prespecified cutoff of 2.5).

[0071] [Figure 21] FIG. 21: Time to bone metastasis event (any time point) according to MAF copy number in non-postmenopausal patients (data according to a pre-specified cutoff of 2.5).

[0072] [Figure 22] FIG. 22 is IDFS (excluding bone metastases in postmenopausal women) for the zoledronic acid treated arm and the control arm.

[0073] [Diagram 23] FIG. 23 is IDFS (excluding bone metastases in non-postmenopausal women) in the zoledronic acid treated arm and the control arm.

[0074] [Figure 24] Figure 24: Overall survival (OS) by treatment arm. Treatment of MAF FISH positive patients with zoledronic acid had a significant impact on OS.

[0075] [Diagram 25] FIG. 25 is the prognostic value of MAF FISH for disease-free survival (DFS) in the Azure control arm.

[0076] [Figure 26] FIG. 26 is the prognostic value of MAF FISH for overall survival (OS) in the Azure control arm.

[0077] [Figure 27] FIG. 27 is the predictive value of MAF FISH for the effect of zoledronic acid treatment on disease-free survival (DFS) outcomes.

[0078] [Figure 28] FIG. 28 is the predictive value of MAF FISH for the effect of zoledronic acid treatment on disease-free survival (DFS) outcomes in postmenopausal patients.

[0079] [Figure 29] FIG. 29 is the predictive value of MAF FISH for the effect of zoledronic acid treatment on disease-free survival (DFS) outcomes in non-postmenopausal patients.

[0080] [Diagram 30] FIG. 30 is the predictive value of MAF FISH for the effect of zoledronic acid treatment on OS outcome.

[0081] [Diagram 31] FIG. 31 is the predictive value of MAF FISH for the effect of zoledronic acid treatment on OS outcomes in postmenopausal patients.

[0082] [Diagram 32] FIG. 32 is the predictive value of MAF FISH for the effect of zoledronic acid treatment on OS outcomes in non-postmenopausal patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] Detailed Description of the Invention Definitions of common terms and expressions As used herein, "and / or" should be considered as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein.

[0084] The c-MAF gene (v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian) also known as MAF or MGC71685) is a transcription factor containing a leucine zipper that acts as a homodimer or heterodimer. Depending on the DNA binding site, the encoded protein can be a transcription activator or a transcription repressor. The DNA sequence encoding c-MAF is listed in the NCBI database under the accession number NG_016440 (SEQ ID NO: 1) (code). The genomic sequence of c-MAF is shown in SEQ ID NO: 13. The method of the present invention can utilize either the coding sequence or the genomic DNA sequence. Two messenger RNAs are transcribed from the DNA sequence, each of which gives rise to one of the two c-MAF protein isoforms (α and β isoforms). The complementary DNA sequences of each of the isoforms are listed in the NCBI database under the accession numbers NM_005360.4 (SEQ ID NO: 2) and NM_001031804.2 (SEQ ID NO: 3), respectively. The use of c-MAF gene to predict the prognosis of ER+ breast cancer can be found in US Patent Application No. 13 / 878,114, which is incorporated herein by reference in its entirety. The use of c-MAF gene to predict the prognosis of triple negative and ER+ breast cancer is described in US Patent Application No. 14 / 391,085, which is incorporated herein by reference in its entirety. The use of c-MAF gene to predict the prognosis of thyroid cancer is described in US Provisional Application No. 61 / 801,769, which is incorporated herein by reference in its entirety. The use of c-MAF gene to predict the prognosis of renal cell carcinoma is described in US Provisional Application No. 14 / 776,390, which is incorporated herein by reference in its entirety. The use of genes of interest (including c-MAF and the c-MAF locus, as well as probes to said locus) to determine the prognosis of individuals with breast cancer is described in U.S. Patent Application Serial No. 14 / 776,412, which is incorporated herein by reference in its entirety.The use of c-MAF gene to predict the prognosis of lung cancer can be found in US Patent Application No. 14 / 405,724, which is incorporated herein by reference in its entirety. The use of c-MAF gene to predict the prognosis of prostate cancer can be found in US Patent Application No. 14 / 050,262 and US Patent Application No. 14 / 435,128, which are incorporated herein by reference in their entirety. The use of c-MAF gene to predict the prognosis of HER2+ cancer can be found in US Patent Application No. 15 / 027,946, which is incorporated herein by reference in its entirety. The use of c-MAF downstream genes to predict the prognosis of cancer can be found in US Patent Application No. 15 / 014,916 and US Patent Application No. 14 / 776,453, which are incorporated herein by reference in their entirety.

[0085] As used herein, the terms "basal-like", "basal-like subtype", "basal-like subtype breast cancer", etc., refer to a specific subtype of breast cancer characterized by two negative receptors ER and HER2, and at least one positive receptor from the group consisting of CK5 / 6, CK14, CK17 and EGFR. Thus, all texts in this application that refer to triple-negative breast cancer (ER, HER-2, PgR) may also refer to basal-like breast cancer that is negative for ER and HER2, and positive for at least one of CK5 / 6, CK14, CK17 and EGFR. Alternatively, "basal-like" also refers to breast cancers characterized by a gene expression profile based on upregulation and / or downregulation of the following 10 genes: (1) forkhead box CI (FOXC1); (2) melanoma inhibitory activity (MIA); (3) NDC80 homolog, kinetochore complex component (KNTC2); (4) centrosomal protein 55 kDa (CEP55); (5) anillin, actin-binding protein (ANLN); (6) maternal embryonic leucine zipper kinase (MELK); (7) G protein-coupled receptor 160 (GPR160); (8) transmembrane protein 45B (TMEM45B); (9) estrogen receptor 1 (ESR1); and (10) forkhead box Al (FOXA1). Because the gene expression profile used to classify breast cancer tumors as a basal-like subtype does not include estrogen receptor, progesterone receptor, or Her2, both triple-negative and non-triple-negative breast cancers can be classified as basal-like subtypes.

[0086] As used herein, "triple-negative breast cancer" refers to breast cancer characterized by the absence of detectable expression of both ER and PR (preferably when the measurement of ER and PR expression is performed by the method disclosed in M. Elizabeth H et al., Journal of Clinical Oncology, 28(16):2784-2795, 2010), and the tumor cells do not have amplified epidermal growth factor receptor type 2 (HER2 or ErbB2), the receptor normally located on the cell surface. Tumor cells are considered negative for ER and PR expression if less than 5% of tumor cell nuclei stain for ER and PR expression using standard immunohistochemical techniques. As used herein, tumor cells are considered negative for HER2 overexpression if they result in a test result score of 0 or 1+ or 2+ when tested using the HercepTest™ Kit (Code K5204, Dako North America, Inc., Carpinteria, Calif.), a semi-quantitative immunohistochemistry assay using a polyclonal anti-HER2 primary antibody, or if they are HER2 FISH negative.

[0087] As used herein, "ER+ breast cancer" refers to breast cancer whose tumor cells express estrogen receptor (ER). This makes the tumor sensitive to estrogen, which means that estrogen causes cancerous breast tumors to grow. In contrast, "ER- breast cancer" refers to breast cancer whose tumor cells do not express estrogen receptor (ER). ER+ breast cancer includes luminal A and B subtypes.

[0088] As used herein, "HER2+" refers to breast cancer characterized by tumor cells with detectable expression of epidermal growth factor receptor type 2 (HER2 or ErbB2) and / or amplification of HER2 gene, a receptor that is usually located on the cell surface.As used herein, tumor cells are considered to be negative for HER2 overexpression if they produce a test result score of 0 or 1+ or 2+ when tested using HercepTest™ Kit (Code K5204, Dako North America, Inc., Carpinteria, CA), a semi-quantitative immunohistochemistry assay using polyclonal anti-HER2 primary antibody, or if they are HER2 FISH negative.

[0089] In the context of the present invention, a "postmenopausal" subject is understood to be a woman who has undergone menopause and has experienced 60 consecutive months of absence of menstruation. See Coleman et al., Lancet Oncol 2014;15:997-1006. In certain embodiments, women may confirm their postmenopausal status through measurement of follicle stimulating hormone (FSH).

[0090] In the context of the present invention, a "non-postmenopausal" subject is any subject who has not undergone menopause and has experienced 60 consecutive months of absence of menstruation. "Non-postmenopausal" subjects include premenopausal, perimenopausal and women of unknown menopausal status.

[0091] In the context of the present invention, "metastasis" is understood as the spread of cancer from the organ where it started to a different organ. It generally occurs through the blood or lymphatic system. When cancer cells spread to form new tumors, the latter are called secondary or metastatic tumors. The cancer cells that form secondary tumors are similar to those of the original tumor. For example, when breast cancer spreads (metastasizes) to bone, the secondary tumors are formed from malignant breast cancer cells. The disease in bone is metastatic breast cancer, not bone cancer. In a particular embodiment of the method of the present invention, the metastasis is breast cancer that spreads (metastasizes) to bone.

[0092] In the context of the present invention, "recurrence" refers to the return of breast cancer after a period of time when the cancer was not detected. Breast cancer may reoccur locally in the breast or in tissues surrounding the breast. Breast cancer may also reoccur in lymph nodes near or in non-surrounding areas. When breast cancer reoccurs by spreading to other tissues or travels through the bloodstream and reoccurs in bone or other organs, it is also referred to as metastasis. As used herein, recurrence also includes the risk of recurrence.

[0093] In the context of the present invention, "relapse" refers to a situation where cancer returns when symptoms have been reduced but the subject is cancer-free. Breast cancer can relapse locally in the breast or in tissues surrounding the breast. Breast cancer can also relapse in lymph nodes near or in non-surrounding areas. When breast cancer relapses by spreading to other tissues or travels through the bloodstream and recurs in bone or other organs, it is also referred to as metastasis. As used herein, relapse also includes the risk of relapse.

[0094] As used herein, the term "disease-free survival" refers to the length of time after the primary treatment for cancer is completed that a patient survives without any signs or symptoms of the cancer. In some embodiments, disease-free survival is referred to as DFS, relapse-free survival or RFS.

[0095] As used herein, the term "overall survival" or "OS" refers to the length of time from either the date of cancer diagnosis or the date of initiation of cancer treatment that a patient diagnosed with the disease is still alive.

[0096] As used herein, the term "subject" or "patient" refers to all animals classified as mammals, including, but not limited to, domestic and farm animals, primates and humans, such as humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats or rodents. Preferably, the subject is a human male or female of any age or race.

[0097] The term "poor" or "good", when used herein to refer to a clinical outcome, means that the subject exhibits a favorable or unfavorable outcome. As will be understood by those skilled in the art, such an assessment of probability is preferably accurate for 100% of the subjects diagnosed, but need not be accurate. However, the term requires that a statistically significant portion of the subjects can be identified as having a predisposition for the given outcome. Those skilled in the art can easily determine whether a portion is statistically significant using various well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-tests, Mann-Whitney tests, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%. The p-value is preferably 0.05, 0.01, 0.005 or 0.0001 or less. More preferably, at least about 60 percent, at least about 70 percent, at least about 80 percent or at least about 90 percent of the subjects in the population can be suitably identified by the methods of the invention.

[0098] In the present invention, "tumor sample" is understood as a sample (e.g., tumor tissue, circulating tumor cells, circulating tumor DNA) derived from primary breast cancer tumor. Said sample can be obtained by conventional methods, for example, biopsy, using methods well known to those skilled in the art of related medical technology. Methods for obtaining biopsy samples include dividing tumor into large pieces, or microdissection, or other cell separation methods known in the art. In addition, tumor cells can be obtained by cytology by aspiration using a small gauge needle. For easy storage and handling of samples, samples can be fixed with formalin and immersed in paraffin, or first frozen, and then immersed in tissue freezing medium such as OCT compound by immersion in a cryogenic medium that allows rapid freezing.

[0099] In the context of the present invention, a "functionally equivalent variant of the c-MAF protein" is understood to be (i) a variant of the c-MAF protein (SEQ ID NO: 4 or SEQ ID NO: 5) in which one or more of the amino acid residues have been replaced by a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), such a substituted amino acid residue may or may not be encoded by the genetic code, or (ii) a variant comprising an insertion or deletion of one or more amino acids, which variant has the same function as the c-MAF protein (i.e. acts as a DNA-binding transcription factor). Variants of the c-MAF protein can be identified using methods based on the ability of c-MAF to promote in vitro cell proliferation, as shown in International Patent Application WO2005 / 046731, which is incorporated herein by reference in its entirety, methods based on the ability of so-called inhibitors to block the transcriptional ability of a reporter gene under the control of the cyclin D2 promoter or a promoter containing a c-MAF response region (MARE or c-MAF response element) in cells expressing c-MAF, as described in International Publication No. WO2008098351, which is incorporated herein by reference in its entirety, or methods based on the ability of so-called inhibitors to block the expression of a reporter gene under the control of the IL-4 promoter in response to stimulation with PMA / ionomycin in cells expressing NFATc2 and c-MAF, as described in US2009048117, which is incorporated herein by reference in its entirety.

[0100] The variants of the present invention preferably have at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence similarity with the amino acid sequence of any of the c-MAF protein isoforms (SEQ ID NO: 4 or SEQ ID NO: 5). The degree of similarity between a variant and a specific c-MAF protein sequence as defined above is determined using algorithms and computer processes that are widely known to those skilled in the art. The similarity between two amino acid sequences is preferably determined using the BLASTP algorithm [BLAST Manual, Altschul, S et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S et al., J. Mol. Biol. 215: 403-410 (1990)].

[0101] As used herein, "agents for preventing or avoiding bone remodeling" refers to any molecule that can prevent, inhibit, treat, reduce or stop bone degradation by stimulating osteoblast proliferation or inhibiting osteoclast proliferation or fixing bone structure. Agents for preventing or avoiding bone remodeling include agents for preventing or avoiding bone degradation, including agents for preventing or avoiding bone synthesis.

[0102] As used herein, "c-MAF inhibitor" refers to any molecule that can completely or partially inhibit c-MAF gene expression by preventing the production of the expression product of the c-MAF gene (interfering with c-MAF gene transcription and / or blocking translation of mRNA resulting from c-MAF gene expression) and by directly inhibiting c-MAF protein activity. C-MAF gene expression inhibitors can be identified using methods based on the ability of the so-called inhibitors to block the ability of c-MAF to promote in vitro cell proliferation, as shown for example in International Patent Application WO2005 / 046731, the entire contents of which are incorporated herein by reference, methods based on the ability of the so-called inhibitors to block the transcriptional ability of a reporter gene under the control of the cyclin D2 promoter or a promoter containing a c-MAF response region (MARE or c-MAF response element) in cells expressing c-MAF, as described for example in WO2008098351, the entire contents of which are incorporated herein by reference, or methods based on the ability of the so-called inhibitors to block the expression of a reporter gene under the control of the IL-4 promoter in response to stimulation with PMA / ionomycin in cells expressing NFATc2 and c-MAF, as described for example in US2009048117, the entire contents of which are incorporated herein by reference.

[0103] As used herein, mammalian target of rapamycin (mTOR) or "mTor" refers to a protein corresponding to EC 2.7.11.1. The mTor enzyme is a serine / threonine protein kinase that regulates cell proliferation, cell motility, cell growth, cell survival and transcription.

[0104] As used herein, "mTor inhibitor" refers to any molecule that can fully or partially inhibit mTor gene expression by preventing the production of the expression product of the mTor gene (interfering with mTor gene transcription and / or blocking translation of the mRNA resulting from mTor gene expression) and by directly inhibiting mTor protein activity. Includes inhibitors with dual or more targets, particularly mTor protein activity.

[0105] As used herein, "Src" refers to a protein corresponding to EC 2.7.10.2. Src is a non-receptor tyrosine kinase and proto-oncogene. Src may play a role in cell growth and embryonic development.

[0106] As used herein, "Src inhibitor" refers to any molecule that can completely or partially inhibit Src gene expression by preventing the production of the expression product of the Src gene (interfering with Src gene transcription and / or blocking translation of mRNA resulting from Src gene expression) and by directly inhibiting Src protein activity.

[0107] As used herein, "prostaglandin-endoperoxide synthase 2," "cyclooxygenase-2," or "COX-2" refers to a protein corresponding to EC 1.14.99.1. COX-2 is involved in the conversion of arachidonic acid to prostaglandin endoperoxide H2.

[0108] As used herein, "COX-2 inhibitor" refers to any molecule that can fully or partially inhibit COX-2 gene expression by preventing the production of the expression product of the COX-2 gene (interfering with COX-2 gene transcription and / or blocking translation of mRNA resulting from COX-2 gene expression) and by directly inhibiting COX-2 protein activity.

[0109] As used herein, "outcome" or "clinical outcome" refers to the resulting course of disease and / or progression of disease, and may be characterized, for example, by recurrence, time to recurrence, relapse, metastasis, time to metastasis, number of metastases, number of metastatic sites, and / or death from disease. For example, good clinical outcomes include cure, prevention of recurrence, prevention of metastasis, and / or survival (without recurrence) within a certain period of time, and bad clinical outcomes include disease progression, metastasis, and / or death within a certain period of time.

[0110] As used herein, "invasive disease-free survival" or "IDFS" refers to the length of time after the completion of primary cancer treatment that a patient survives without any signs or symptoms of the cancer invading the same breast parenchyma or other tissue as the original primary tumor. In some embodiments, IDFS includes death from any cause, including ipsilateral invasive breast tumor recurrence, local or regional invasive breast cancer recurrence, metastasis or distant recurrence, breast cancer, contralateral invasive breast cancer, and second primary invasive cancer (non-breast cancer, but excluding basal cell carcinoma or squamous cell carcinoma). See Coleman et al., Lancet Oncol 2014;15:997-1006.

[0111] In the present invention, "diagnosis of metastasis in a subject with breast cancer" is understood as identifying the disease (metastasis) by studying its symptoms, i.e. in the context of the present invention, by an increase in the expression level of the c-MAF gene (i.e. overexpression) in breast cancer tumor tissue relative to a control sample.

[0112] In the present invention, "prognosis of the tendency to develop metastasis in a subject with breast cancer" is understood to mean knowing whether the breast cancer that said subject has will metastasize in the future based on a symptom. In the context of the present invention, the symptom is c-MAF gene overexpression in tumor tissue.

[0113] In the context of the present invention, it is understood that "said subject has a positive diagnosis of metastasis" if the breast cancer suffered by the subject has metastasized to other organs of the body, in a particular embodiment to the bone. The term is used similarly for recurrence and relapse.

[0114] Those skilled in the art will understand that prediction of the tendency of a primary tumor to metastasize, relapse or recur is not intended to be accurate for all subjects to be identified (i.e., 100% of subjects). Nevertheless, the term requires that it allows for the identification of a statistically significant portion of subjects (e.g., a cohort in a cohort study). Those skilled in the art can easily determine whether a portion is statistically significant using various well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. Details are provided in Dowdy and Wearden, Statistics for Research, John Wiley and Sons, New York 1983. Preferred confidence intervals are at least about 90%, at least about 95%, at least about 97%, at least 98% or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005 or 0.0001. More preferably, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the subjects in the population can be suitably identified by the methods of the present invention.

[0115] As used herein, "poor prognosis" indicates that a subject is expected (e.g., predicted) not to survive and / or to have recurrence, relapse or distant metastasis within a given time period, or is at high risk of having them. The term "high" is a relative term, and in the context of this application, refers to the risk of the "high" expression group in terms of clinical outcome (recurrence, distant metastasis, etc.). A "high" risk may be considered to be a risk higher than the average risk of a heterogeneous cancer patient population. In the study of Paik et al. (2004), an overall "high" risk of recurrence was considered to be higher than 15 percent. The risk will also vary depending on the time period. The time period may be, for example, 5 years, 10 years, 15 years, or even 20 years after the initial diagnosis of cancer or after the implementation of the prognosis.

[0116] "Reference value" as used herein refers to a test value used as a standard for the values / data obtained by clinical testing of a patient or a sample collected from a patient. Reference value or reference level can be an absolute value; a relative value; a value with upper and / or lower limits; a range of values; an average value; a median value, a mean value, or a value that is compared to a specific control value or baseline value. Reference value can be based on individual sample values, for example, values ​​obtained from samples from the subject being tested, but at an earlier time point. Reference value can be based on multiple samples from a population of subjects, for example, a chronological age-matched group, or based on a pool of samples that includes or excludes the sample to be tested.

[0117] The term "treatment" as used herein refers to any type of therapy aimed at terminating, preventing, ameliorating, or reducing susceptibility to a clinical condition as described herein. In an embodiment, the term treatment relates to prophylactic treatment of a disorder or condition defined herein (i.e., treatment to reduce susceptibility to a clinical condition). Thus, "treatment", "treat" and their equivalent terms refer to obtaining a desired pharmacological or physiological effect, covering any treatment of a pathological condition or disorder in a mammal, including a human. The effect may be preventive in terms of complete or partial prevention of the disorder or its symptoms, and / or therapeutic in terms of partial or complete cure of the disorder and / or adverse effects caused by the disorder. That is, "treatment" includes (1) preventing the occurrence or recurrence of the disorder in a subject; (2) inhibiting the disorder, e.g., halting its development; (3) halting or terminating the disorder or at least a symptom associated therewith, e.g., causing regression of the disorder or a symptom thereof, such that the host is no longer afflicted by the disorder or a symptom thereof, e.g., by restoring or repairing a loss, deficiency or defect in function or stimulating an inefficient process; or (4) alleviating, alleviating or ameliorating the disorder or a symptom associated therewith (amelioration is used in a broad sense to refer to at least a decrease in the magnitude of a parameter, e.g., inflammation, pain, or immune deficiency).

[0118] As used herein, "sample" or "biological sample" refers to a biological material isolated from a subject. A biological sample may contain any biological material suitable for determining the expression level of the c-MAF gene. A sample may be isolated from any suitable biological tissue or fluid, such as tumor tissue, blood, plasma, serum, urine or cerebrospinal fluid (CSF).

[0119] As used herein, the term "expression level" of a gene refers to the measurable amount of a gene product produced by a gene in a sample of a subject, where the gene product can be a transcription product or a translation product. Thus, the expression level can relate to a nucleic acid gene product, such as an mRNA or cDNA, or a polypeptide gene product. The expression level can be derived from a sample of a subject and / or a reference sample, and can be detected, for example, de novo, or correspond to a previous determination. The expression level can be determined or measured, for example, using microarray methods, PCR methods (e.g., qPCR), and / or antibody-based methods, as known to those skilled in the art.

[0120] "Increased expression level" is understood as an expression level when referring to the c-MAF gene level in a reference sample or a control sample. These increased levels may be caused by amplification, copy gain or translocation of the gene or 16q23 or 16q22-24 chromosomal locus, without excluding other mechanisms. In particular, a sample may be considered to have a high c-MAF expression level if the expression level in the sample isolated from the patient is at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more than that of the reference or control. In an embodiment, "increased expression level" is a "high" expression level. A "not increased" or "non increased" expression level is any value not included in the definition of an "increased" expression level, including a value equal to a reference or control level, or an expression level that is decreased compared to a reference or control level.

[0121] "Decreased expression level" is understood as an expression level referring to a c-MAF gene level that is lower than that in a reference sample or control sample. This decreased level may be caused by, but not excluding, a deletion of the gene or chromosomal locus 16q23 or 16q22-24. In particular, if the expression level in the sample isolated from the patient is at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even lower than that in the reference or control, the sample may be considered to have a decreased c-MAF expression level. In an embodiment, the "decreased expression level" is a "low" expression level.

[0122] As used herein, the term "gene copy number" refers to the copy number of a nucleic acid molecule in a cell. Gene copy number includes the gene copy number in the genomic (chromosomal) DNA of a cell. In normal cells (non-tumor cells), gene copy number is usually 2 copies (one copy in each member of a chromosome pair). Gene copy number may also include half the gene copy number taken from a sample of a cell population.

[0123] In the present invention, "increased gene copy number" is understood as the case where the c-MAF gene copy number exceeds the copy number that the reference sample or control sample has.These increased gene copy numbers can be caused by amplification, copy gain or translocation of the gene or 16q23 or 16q22-24 chromosomal locus, without excluding other mechanisms.In particular, when the copy number is more than 2 copies, for example 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9 or 10 copies, or even more than 10 copies of the c-MAF gene, the sample can be considered to have increased c-MAF copy number.In an embodiment, "increased gene copy number" is determined based on the average copy number per counted cell. In an embodiment, a sample may be considered to have an increased c-MAF copy number if the average number of copies per cell counted is more than 2 copies, such as 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9 or 10 copies, or even more than 10 copies, of the c-MAF gene.

[0124] In the present invention, "reduced gene copy number" is understood as the case where the c-MAF gene copy number is less than the copy number that reference sample or control sample has.These reduced gene copy numbers can be caused by the deletion of gene or 16q23 or 16q22-24 chromosomal locus, without excluding other mechanisms.In particular, when the copy number of the c-MAF gene is less than 2 copies, the sample can be considered to have reduced c-MAF copy number.

[0125] In the present invention, "non-increased gene copy number" is understood as the case where c-MAF gene copy number or average c-MAF gene copy number is less than the copy number that reference sample or increase positive sample has.Non-increased gene copy number can be caused by non-increased gene or 16q23 or 16q22-24 chromosomal locus amplification, copy gain or translocation, without excluding other mechanisms.In particular, when the copy number of c-MAF gene is less than 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 copies, the sample can be considered to have non-increased c-MAF copy number or c-MAF average copy number.

[0126] As understood herein, the term "gene amplification" refers to the process in which various copies of a gene or gene fragment are formed in an individual cell or cell line. The copies of a gene are not necessarily located on the same chromosome. The overlapping region is often referred to as an "amplicon". Usually, the amount of mRNA produced, i.e., the gene expression level, also increases in proportion to the copy number of a particular gene.

[0127] The term "gain" refers to any increase in chromosomal copy number from the norm (i.e., in a diploid organism, 3 copies of a gene in a cell is a gain). In some embodiments, "gain" includes the term "copy gain", which is used synonymously with "copy number".

[0128] "Probe" as used herein refers to an oligonucleotide sequence that is complementary to a particular nucleic acid sequence of interest. In some embodiments, the probe may be specific to a region of a chromosome known to undergo translocation. In some embodiments, the probe has a specific label or tag. In some embodiments, the tag is a fluorophore. In some embodiments, the probe is a DNA in situ hybridization probe whose labeling is based on stable coordinate binding of platinum to nucleic acids and proteins ... The results are described in “Hybridization probes” Nucleic Acids Research 40(3):e20(2012).

[0129] "Tag" or "label" as used herein refers to any physical molecule that is directly or indirectly associated with a probe and that allows for visualization, marking or other capture of the probe or its location.

[0130] "Translocation" as used herein refers to the exchange of unequal or equal amounts of chromosomal material between chromosomes. In some cases, the translocation is on the same chromosome. In some cases, the translocation is between different chromosomes. Translocations occur frequently in many types of cancer, including breast cancer and leukemia. Translocations can be primary reciprocal translocations or more complex secondary translocations. There are several primary translocations involving the immunoglobulin heavy chain (IgH) locus that are thought to constitute the initiating event in many cancers (Eychene, A., Rocques, N., and Puoponnot, C., A new MAFia in cancer. 2008. Nature Reviews: Cancer. 8: 683-693.).

[0131] "Polyploid" or "polyploidy" as used herein refers to a cell containing more than two copies of a gene of interest. In some cases, the gene of interest is MAF. In some embodiments, polyploidy is associated with the accumulation of expression of the gene of interest. In some embodiments, polyploidy is associated with genomic instability. In some embodiments, genomic instability can lead to chromosomal translocations.

[0132] "Whole genome sequencing," as used herein, is the process of sequencing an organism's entire genome in one go. See, e.g., Ng., PC and Kirkness, EF, Whole Genome Sequencing. 2010. Methods in Molecular Biology. 628:215-226.

[0133] "Exome sequencing" as used herein is the process of sequencing the entire coding region of an organism's DNA. In exome sequencing, mRNA is sequenced. In exome sequencing, the untranslated regions of the genome are not included. See, for example, Choi, M et al., Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. 2009. PNAS. 106(45):19096-19101.

[0134] As used herein, "binding member" refers to one member of a pair of molecules that bind to each other. The members of a binding pair can be naturally occurring or wholly or partially synthetically produced. One member of a pair of molecules has a surface area or cavity that binds to the other member of the pair of molecules and thus is complementary to the specific spatial and polar configuration of the other member. Examples of types of binding pairs are antigen-antibody, receptor-ligand and enzyme-substrate. In some embodiments, the binding member is an antibody. In some embodiments, the binding member is an antibody that binds to the c-MAF antigen.

[0135] As used herein, "CDR region" or "CDR" is intended to refer to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department of Health and Human Services, Public Service, NIH, Washington. Antibodies typically contain three heavy chain CDRs, designated HCDR1, HCDR2, and HCDR3, and three light chain CDRs, designated LCDR1, LCDR2, and LCDR3. The term CDR(s) is used herein to refer to one of these regions that contains most of the amino acid residues involved in binding, or several or even the entirety of these regions, depending on the affinity of the antibody for the antigen or epitope it recognizes. Among the six CDR sequences, the third CDR of the heavy chain (HCDR3) has the greatest degree of variability (i.e., greater diversity), essentially due to a mechanism known in the art as the V(D)J rearrangement of the V, D, and J gene segments of the germline immunoglobulin heavy chain locus. HCDR3 can be as short as 2 amino acids or as long as 26 amino acids, or have any length between these two extremes. The length of the CDR can also vary according to the length that can be accommodated by a particular base framework.Functionally, HCDR3 may play an important role in determining antibody specificity (Segal et al., (1974) Proc Natl Acad Sci USA. 71(11):4298-302; Amit et al., (1986) Science 233(4765):747-53; Chothia et al., (1987) J. Mol. Biol. 196(4):901-17; Chothia et al., (1989) Nature 342(6252):877-83; Caton et al., (1990) J. Immunol. 144(5):1965-8; Sharon (1990a) PNAS USA.87(12):4814-7, Sharon (1990b) J. Immunol.144:4863-4869, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department of Health and Human Services, Public Service, NIH, Washington).

[0136] As used herein, "antibody", "antibody molecule" or "antibodies" refers to immunoglobulins, whether naturally produced or partially or wholly synthetically produced. The term also encompasses any polypeptide or protein that contains an antibody antigen-binding site. It should be understood herein that the present invention does not relate to antibodies in their natural form (in other words, they are not in their natural environment, but they may be isolated or obtained by purification from a natural source, or obtained by genetic recombination or by chemical synthesis), and therefore they may contain non-naturally occurring amino acids. Antibody fragments that contain the antibody antigen-binding site include Fab, Fab', F(ab') and / or Fab'-binding fragments. 2These include, but are not limited to, molecules such as Fab'-SH, scFv, Fv, dAb, and Fd. A variety of other antibody molecules have been engineered that contain one or more antigen-binding sites of an antibody, including, for example, Fab2, Fab3, diabody, triabody, tetrabody, camelbody, nanobody, and minibody. Antibody molecules and methods for their construction and use are described in Hollinger & Hudson (2005) Nature Biot. 23(9):1126-1136.

[0137] As used herein, "antibody molecule" should be construed to include any binding member or substance having an antibody antigen-binding site with the required specificity and / or binding to the antigen. Thus, the term includes functional antibody fragments and derivatives, including any polypeptide comprising an antibody antigen-binding site, whether natural or wholly or partially synthetic. Thus, chimeric molecules or equivalents comprising an antibody antigen-binding site fused to another polypeptide (e.g., from another species or belonging to another antibody class or subclass) are included. Cloning and expression of chimeric antibodies are described, for example, in European Patent Application Publication No. 0120694A (Boss et al.) and European Patent Application Publication No. 0125023A (Cabilly et al.), which are incorporated herein by reference in their entireties.

[0138] As used herein, for example, a "functional fragment or variant" of a binding member of the invention means a fragment or variant of the binding member that retains at least some function of the intact binding member (e.g. the ability to specifically bind an antigen such as Maf).

[0139] "Tumor tissue sample" is understood to mean a tissue sample derived from a breast cancer tumor, including but not limited to circulating tumor cells and circulating tumor DNA. Said sample can be obtained by conventional methods, for example biopsy, using methods well known to those skilled in the relevant medical technology.

[0140] "Osteolytic bone metastasis" refers to a type of metastasis in which bone resorption (progressive loss of bone density) occurs in the vicinity of the metastasis due to stimulation of osteoclast activity by tumor cells, and is characterized by severe pain, pathological fractures, hypercalcemia, spinal cord compression, and other syndromes due to nerve compression.

[0141] Methods for designing customized treatments of the present invention in patients with breast tumors The present invention is directed to identifying subjects suffering from breast cancer who would benefit from treatment with certain drugs and / or therapies. In some embodiments, the present invention is directed to identifying subjects suffering from breast cancer who would not benefit from treatment with certain drugs and therapies. In some embodiments, the subject has high expression levels, copy numbers, amplifications, gains, and / or translocations of c-MAF. In certain embodiments, the subject has low expression levels, copy numbers, amplifications, gains, and / or translocations of c-MAF. In certain embodiments, the cancer is triple-negative breast cancer. In other embodiments, the cancer is ER+ breast cancer. In further embodiments, the cancer is ER- breast cancer. In still further embodiments, the cancer is HER2+ breast cancer. In some embodiments, the cancer is basal-like breast cancer. In one embodiment, the subject is postmenopausal. In an embodiment, the subject is non-postmenopausal. As described in U.S. Patent Application No. 14 / 391,085, U.S. Provisional Application No. 61 / 801,769, U.S. Provisional Application No. 14 / 776,390, U.S. Patent Application No. 14 / 776,412, U.S. Patent Application No. 14 / 405,724, U.S. Patent Application No. 14 / 050,262, U.S. Patent Application No. 14 / 435,128, U.S. Patent Application No. 15 / 027,946, U.S. Patent Application No. 15 / 014,916 and U.S. Patent Application No. 14 / 776,453 (each of which is incorporated herein by reference in its entirety), levels of c-MAF can be used to diagnose metastasis, relapse or recurrence or to predict the propensity of a tumor to undergo metastasis, relapse or recurrence. Therefore, considering that c-MAF gene overexpression in breast cancer cells is associated with the presence of metastasis, relapse or recurrence as described in the present invention, the c-MAF gene expression level allows for the decision on the most appropriate treatment for the subject suffering from said cancer.In an embodiment, the present invention comprises quantifying only c-MAF gene expression level as a single marker (i.e., the method does not involve determining the expression level of any additional marker).

[0142] Therefore, in one embodiment, the present invention provides an in vitro method for designing customized therapy for a subject with breast cancer, comprising a) quantifying the expression level, copy number, amplification or gain of c-MAF gene in a tumor sample of the subject, and b) comparing the expression level, copy number, amplification or gain obtained with the expression level, copy number, amplification or gain of the gene in a control sample, and determining therapy based on the expression level, copy number, amplification or gain of c-MAF gene in the subject. In some embodiments, the subject has a high expression level of c-MAF gene. In other embodiments, the subject has a low expression level of c-MAF gene. In certain embodiments, the subject is administered an agent that prevents and / or prevents bone remodeling (including an agent that prevents or prevents bone degradation). In an embodiment, the subject is administered an agent that treats cancer. In a further embodiment, the subject is administered a c-MAF inhibitor. In certain embodiments, the agent that avoids and / or prevents bone remodeling or the c-MAF inhibitor is any agent disclosed in U.S. Patent Application Publication Nos. 2014 / 0057796 and 2015 / 0293100 and U.S. Patent Application No. 15 / 027,946, which are incorporated by reference in their entireties.

[0143] In one embodiment, the present invention provides an in vitro method for designing a customized treatment for a subject with breast cancer, comprising: i) quantifying the expression level, copy number, amplification or gain of the c-MAF gene in a sample of said subject; and ii) comparing said expression level, copy number, amplification or gain obtained in i) with a reference value, and if said expression level, copy number, amplification or gain is not increased relative to said reference value, said subject is allowed to receive a treatment aimed at preventing and / or treating bone remodeling or improving disease-free or overall survival. In some embodiments, the subject is non-postmenopausal. In other embodiments, the subject is postmenopausal. In an embodiment, a drug aimed at preventing and / or treating bone remodeling is administered to the subject. In an embodiment, a drug that improves disease-free or overall survival is administered to the subject. In a further embodiment, a c-MAF inhibitor is administered to the subject.

[0144] In another embodiment, the present invention relates to an in vitro method for designing a customized treatment for a non-postmenopausal subject with breast cancer, comprising: i) quantifying the expression level, copy number, amplification or gain of the c-MAF gene in a sample of said subject; and ii) comparing said expression level, copy number, amplification or gain obtained in i) with a reference value, wherein if said expression level, copy number, amplification or gain is increased with respect to said reference value, said subject is not allowed to receive a treatment aimed at preventing and / or treating bone remodeling and / or improving disease-free or overall survival. In some embodiments, the subject is not administered a drug aimed at preventing and / or treating bone remodeling and / or improving disease-free or overall survival.

[0145] In another embodiment, the present invention relates to an in vitro method for designing a customized treatment for a postmenopausal subject with breast cancer, comprising: i) quantifying the expression level, copy number, amplification or gain of the c-MAF gene in a sample of said subject; and ii) comparing said expression level, copy number, amplification or gain obtained in i) with a reference value, wherein if said expression level, copy number, amplification or gain is increased with respect to said reference value, said subject is allowed to receive a treatment aimed at preventing and / or treating bone remodeling and / or improving disease-free or overall survival. In some embodiments, a drug aimed at preventing and / or treating bone remodeling and / or a treatment for improving disease-free or overall survival is administered to the subject.

[0146] In another embodiment, the present invention relates to an in vitro method for designing a customized treatment for a postmenopausal subject with breast cancer, comprising: i) quantifying the expression level, copy number, amplification or gain of the c-MAF gene in a sample of said subject; and ii) comparing said expression level, copy number, amplification or gain obtained in i) with a reference value, wherein if said expression level, copy number, amplification or gain is not increased with respect to said reference value, said subject is not allowed to receive a treatment aimed at preventing and / or treating bone remodeling and / or improving disease-free or overall survival. In some embodiments, the subject is not administered a drug aimed at preventing and / or treating bone remodeling and / or improving disease-free or overall survival.

[0147] In one embodiment, the present invention relates to a method for treating bone metastasis in a subject with breast cancer, the subject having a decreased c-MAF level in metastatic tumor samples relative to control samples, comprising administering an agent capable of preventing or inhibiting bone remodeling and / or improving disease-free or overall survival, wherein the agent capable of preventing or inhibiting bone remodeling or improving disease-free or overall survival is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranelate, DKK-1 inhibitors, dual MET and VEGFR2 inhibitors, estrogen receptor modulators, EGFR inhibitors, calcitonin, radium-223, CCR5 antagonists, Src kinase inhibitors, COX-2 inhibitors, mTor inhibitors and cathepsin K inhibitors. In some embodiments, the subject is non-postmenopausal. In other embodiments, the subject is post-menopausal.

[0148] In another embodiment, the present invention relates to a method for treating bone metastasis in a postmenopausal subject with breast cancer, wherein the subject has increased c-MAF levels in metastatic tumor samples versus control samples, comprising administering an agent capable of preventing or inhibiting bone remodeling and / or improving disease-free or overall survival, wherein the agent capable of avoiding or preventing bone remodeling and / or improving disease-free or overall survival is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranelate, DKK-1 inhibitors, dual MET and VEGFR2 inhibitors, estrogen receptor modulators, EGFR inhibitors, calcitonin, radium-223, CCR5 antagonists, Src kinase inhibitors, COX-2 inhibitors, mTor inhibitors and cathepsin K inhibitors.

[0149] In certain embodiments, an agent that avoids and / or prevents bone remodeling (including an agent that avoids or prevents bone degradation) is administered to the subject. In embodiments, an agent that avoids or prevents bone degradation is administered to the subject.

[0150] When the c-MAF gene expression level, copy number, amplification or gain is measured in a sample and compared to a control sample, the expression level, copy number, amplification or gain of said gene in combination with the menopausal status of the subject indicates whether the subject is permissible to receive a treatment aimed at preventing (if the subject has not yet undergone metastasis) and / or treating (if the subject has already experienced metastasis) metastasis, relapse or recurrence, and / or a treatment or drug intended to avoid or prevent bone remodeling.

[0151] In some embodiments, a copy number of MAF per cell or an average copy number of MAF > 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0 as measured using FISH is considered to be a high value. In an embodiment, the MAF FISH value is > 2.2. In a particular embodiment, the MAF FISH value is > 2.3. In another embodiment, the MAF FISH value is > 2.4. In a further embodiment, the MAF FISH value is > 2.5. In another embodiment, the copy number of c-MAF as measured using FISH is < 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 copies of the c-MAF gene.

[0152] In certain embodiments, the subject has a metastasis or is prognosticated to experience a metastasis. In some embodiments, the metastasis is a bone metastasis. In further embodiments, the bone metastasis is an osteolytic metastasis.

[0153] In some embodiments, the method comprises in a first step quantifying the c-MAF gene expression level, copy number, gain or amplification in a tumor sample of a subject suffering from breast cancer.

[0154] In some embodiments, the sample is a primary tumor tissue sample of the subject. In the second step, the obtained c-MAF gene expression level, copy number, amplification or gain in the tumor sample of the subject is compared with the expression level, copy number, amplification or gain of said gene in a control sample. The determination of the c-MAF gene expression level, copy number, amplification or gain must be related to the value of a control sample or a reference sample. Depending on the type of tumor to be analyzed, the exact nature of the control sample may vary. Thus, in some embodiments, the reference sample is a tumor tissue sample of a subject with breast cancer that does not have metastasis, relapse or recurrence, or a tumor tissue sample of a subject with breast cancer that corresponds to the median value of the c-MAF gene expression level, copy number, amplification or gain measured in a tumor tissue collection in a biopsy sample of a subject with breast cancer that does not have metastasis, relapse or recurrence.

[0155] In one embodiment, the method of the invention comprises in a second step comparing the obtained c-MAF gene expression level, copy number, amplification or gain in a tumor sample (including but not limited to a primary tumor biopsy, circulating tumor cells and circulating tumor DNA) from the subject with the expression level of said gene in a control sample.

[0156] The determination of c-MAF gene expression level, copy number, amplification or gain must be correlated with the value of control sample or reference sample.Depending on the type of tumor to be analyzed, the exact nature of the control sample may vary.Therefore, in the case of evaluating diagnosis, the reference sample is the tumor tissue sample from the subject with breast cancer that does not metastasize, or the tumor tissue sample from the subject with breast cancer that corresponds to the median value of the c-MAF gene expression level measured in the tumor tissue collection in the biopsy sample from the subject with breast cancer that does not metastasize.

[0157] Said reference sample is typically obtained by combining equal amounts of samples from a subject population. In general, a typical reference sample will be obtained from a subject who is clinically well documented and well characterized for the absence of metastasis. In such a sample, the normal concentration (reference concentration) of the biomarker (c-MAF gene) can be determined, for example, by providing the average concentration of the reference population. When determining the reference concentration of the marker, various considerations are taken into account. Such considerations are the age, weight, sex, general physical condition, etc. of the patient. For example, the reference group is a group of equal amounts of at least about 2, at least about 10, at least about 20, at least about 25, at least about 50, at least about 75, at least about 100, at least about 250, at least about 500 to more than about 1000 subjects classified according to said considerations, for example, according to various age categories. The sample collection from which the reference level is derived will preferably be formed by subjects suffering from the same type of cancer (e.g., breast cancer) as the patient subjects of the study. Similarly, a reference value within a cohort of patients can be established by using a receiver operating curve (ROC) and measuring the area under the curve for all de sensitivity and specificity pairs to determine which pair provides the best value and what the corresponding reference value is. ROC is a standard statistical concept. A description is given in Stuart G. Baker “The Central Role of Receiver Operating Characteristic (ROC) curves in "Evaluating Tests for the Early Detection of Cancer" Journal of The National Cancer Institute (2003) Vol 95, No. 7, 511-515.

[0158] Once this median or reference value is established, the levels of this marker expressed in tumor tissue from patients with this median value can be compared and assigned, for example, to an "increased" expression level. Due to subject-to-subject variability (e.g., aspects related to age, race, etc.), it is very difficult (if not virtually impossible) to establish an absolute reference value for c-MAF expression. Thus, in certain embodiments, the reference value for "increased" or "decreased" expression of c-MAF expression is determined by calculating percentiles by conventional means, which involves performing an assay on one or more samples isolated from subjects whose disease has been well documented for c-MAF expression levels by any of the above methods. Preferably, a "decreased" c-MAF level can then be assigned to samples in which the c-MAF expression level is equal to or less than the 50th percentile of the normal population (including, for example, expression levels equal to or less than the 60th percentile of the normal population, expression levels equal to or less than the 70th percentile of the normal population, expression levels equal to or less than the 80th percentile of the normal population, expression levels equal to or less than the 90th percentile of the normal population, and expression levels equal to or less than the 95th percentile of the normal population). An "increased" c-MAF gene expression level can then preferably be assigned to samples in which the c-MAF gene expression level is equal to or exceeds the 50th percentile of the normal population (including, for example, an expression level equal to or exceeds the 60th percentile of the normal population, an expression level equal to or exceeds the 70th percentile of the normal population, an expression level equal to or exceeds the 80th percentile of the normal population, an expression level equal to or exceeds the 90th percentile of the normal population, and an expression level equal to or exceeds the 95th percentile of the normal population).

[0159] In a particular embodiment, the degree of amplification or gain of the c-MAF gene can be determined by determining the amplification or gain of the chromosomal region containing said gene.Preferably, the chromosomal region whose amplification or gain indicates the presence of the amplification or gain of the c-MAF gene is the locus 16q22-q24, which contains the c-MAF gene. The locus 16q22-q24 is located on chromosome 16, on the long arm of said chromosome, and in the range between bands 22 and 24. This region corresponds to contigs NT_010498.15 and NT_010542.15 in the NCBI database.In another preferred embodiment, the degree of amplification or gain of the c-MAF gene can be determined using a probe specific for said gene.

[0160] In some embodiments, the amplification or gain is in the region of the 16q23 locus. In some embodiments, the amplification or gain is in any part of the chromosomal region of chromosome 16 from 79,392,959bp to 79,663,806bp (from centromere to telomere). In some embodiments, the amplification or gain is in the genomic region of chromosome 16 from 79,392,959bp to 79,663,806bp (excluding DNA repeat elements). In some embodiments, the amplification or gain is measured using a probe specific to the region.

[0161] In an embodiment, the c-MAF gene is amplified relative to the reference gene copy number when the c-MAF gene copy number is greater than the copy number of a reference or control sample. In one example, the c-MAF gene is said to be "amplified" when the genomic copy number or average genomic copy number of the c-MAF gene is increased by at least about 2- (i.e., 6 copies), 3- (i.e., 8 copies), 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, or 50-fold in the test sample compared to the control sample. In another example, a c-MAF gene is said to be "amplified" if the genomic copy number or average genomic copy number of the c-MAF gene per cell is at least about 2.1, 2.2, 2.3, 2.4, 25., 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.

[0162] In some embodiments, when copy number is measured, the control sample refers to a tumor sample of a subject with breast cancer who does not suffer from metastasis, or a tumor sample of a subject with breast cancer who corresponds to the median c-MAF gene copy number measured in a tumor tissue collection in a biopsy sample of a subject with breast cancer who does not suffer from metastasis. The reference sample is typically obtained by combining equal amounts of samples from a subject population. If the c-MAF gene copy number is increased relative to the copy number of the gene in the control sample, the subject has a positive diagnosis of metastasis or has a higher tendency to develop metastasis. In another embodiment, the reference gene copy number is the gene copy number in a breast cancer sample from a subject who does not suffer from bone metastasis.

[0163] In another embodiment, the amplification or gain is determined by in situ hybridization or PCR.

[0164] In another embodiment, and as described in the present invention, given that chr16q22-24, including the c-MAF gene, is amplified in breast cancer cells and is associated with the presence of metastasis, relapse or recurrence, amplification or gain of chr16q22-24, including the c-MAF gene, allows for decisions regarding the most appropriate treatment for a subject suffering from said cancer.

[0165] The determination of the amplification of the c-MAF gene requires correlation with a control or reference sample value corresponding to the level of amplification of the c-MAF gene measured in tumor tissue samples from subjects with breast cancer and not suffering from metastases, or the median value of amplification of the c-MAF gene measured in a collection of tumor tissues in biopsy samples from subjects with breast cancer and not suffering from metastases, said reference sample being typically obtained by combining equal amounts of samples from a population of subjects.

[0166] In general, a typical reference sample will be obtained from a subject who is clinically well documented and well characterized for the absence of metastasis.The sample collection from which the reference level is derived will preferably be composed of subjects suffering from the same type of cancer as the patient subjects of the study.Once this median value is established, the level of amplification of c-MAF in the patient's tumor tissue can be compared with this median value, and if there is amplification, the subject has a positive diagnosis of metastasis or has a higher tendency to develop metastasis.

[0167] In another aspect, the present invention relates to an in vitro method for designing a customized therapy for a patient suffering from breast cancer, comprising determining whether the c-MAF gene is translocated in a sample from said subject.

[0168] In some embodiments, the translocation gene is derived from the region of the 16q23 locus. In some embodiments, the translocation gene is derived from any part of the chromosomal region of chromosome 16 from 79,392,959 bp to 79,663,806 bp (from centromere to telomere). In some embodiments, the translocation gene is derived from the genomic region of chromosome 16 from 79,392,959 bp to 79,663,806 bp (excluding DNA repeat elements). In some embodiments, the translocation is measured using a probe specific to the region.

[0169] In a particular embodiment, the translocation of the c-MAF gene can be determined by determining the translocation of the chromosomal region containing said gene. In one embodiment, the translocation is a t(14,16) translocation. In another embodiment, the translocated chromosomal region originates from the locus 16q22-q24. The locus 16q22-q24 is located on chromosome 16, on the long arm of said chromosome, and in the range between bands 22 and 24. This region corresponds to contigs NT_010498.15 and NT_010542.15 in the NCBI database. The c-MAF gene is translocated to the locus 14q32 on chromosome 14, resulting in the translocation t(14,16)(q32,q23). This translocation places the MAF gene next to a strong enhancer in the IgH locus, which in some cases leads to overexpression of MAF (Eychene, A., Rocques, N., and Puoponnot, C., A new MAFia in cancer. 2008. Nature Reviews:Cancer.8:683-693.).

[0170] In an embodiment, the translocation of the c-MAF gene may be determined by using a probe specific for said translocation.

[0171] One embodiment of the present invention comprises a method which in a first step determines whether the c-MAF gene is translocated in a sample from a subject. In an embodiment, the sample is a tumor tissue sample.

[0172] In a particular embodiment, the method of the present invention for prognosis of the tendency to develop bone metastasis in a subject with breast cancer comprises determining the c-MAF gene copy number in a sample from said subject in which the c-MAF gene is translocated, and comparing said copy number with the copy number of a control or reference sample, wherein if the c-MAF copy number is higher relative to the c-MAF copy number of the control sample, the subject has a greater tendency to develop bone metastasis.

[0173] In some embodiments, the amplification, gain and copy number of c-MAF gene are determined after the translocation of c-MAF gene is determined. In some embodiments, a probe is used to determine whether a cell is polyploid for c-MAF gene. In some embodiments, the determination of ploidy is performed by determining whether there are more than two signals from the gene of interest. In some embodiments, the ploidy is determined by measuring the signal from the probe specific to the gene of interest and comparing it with a centromere probe or other probe.

[0174] How c-MAF can be used to predict survival, including IDFS The present invention is directed to predicting IDFS of subjects suffering from breast cancer. In certain embodiments, the subject has high expression level, copy number, amplification or gain of c-MAF. In other embodiments, the subject has low expression level, copy number, amplification or gain of c-MAF. In some embodiments, the cancer is triple negative breast cancer. In other embodiments, the cancer is ER+ breast cancer. In further embodiments, the cancer is ER- breast cancer. In certain embodiments, the cancer is basal-like breast cancer. In still further embodiments, the cancer is HER2+ breast cancer. In some embodiments, the subject is postmenopausal. In other embodiments, the subject is non-postmenopausal.

[0175] In some embodiments, the present invention is directed to an in vitro method for predicting IDFS in a patient with breast cancer, comprising: i) quantifying the expression level, copy number, amplification or gain of the c-MAF gene in a sample of said subject; and ii) comparing said expression level, copy number, amplification or gain obtained in step i) with a reference value, wherein an increase in the expression level, copy number, amplification or gain of said gene relative to said reference value is indicative of a poor IDFS prognosis.

[0176] In one embodiment, the present invention is directed to an in vitro method for predicting IDFS in a patient with breast cancer, comprising determining a c-MAF gene expression level, copy number, amplification or gain in a sample from said subject compared to a reference, wherein an increase in the c-MAF gene expression level, copy number, amplification or gain relative to said reference is indicative of a poor IDFS prognosis.

[0177] In a further embodiment, the present invention is directed to an in vitro method for predicting IDFS (excluding bone recurrence) in a patient having breast cancer, comprising determining a c-MAF gene expression level, copy number, amplification or gain in a sample from said subject compared to a reference, wherein an increase in the c-MAF gene expression level, copy number, amplification or gain relative to said reference is indicative of a poor IDFS prognosis (excluding bone recurrence).

[0178] In some embodiments, a copy number of MAF per cell or an average copy number of MAF > 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0 as measured by FISH is considered to be a high value. In certain embodiments, the MAF FISH value is > 2.2. In other embodiments, the MAF FISH value is > 2.3. In further embodiments, the MAF FISH value is > 2.4. In yet further embodiments, the MAF FISH value is > 2.5.

[0179] In some embodiments, the c-MAF status of a subject predicts the duration of the subject's overall survival or disease-free survival.In certain embodiments, the c-MAF status in any of the embodiments herein comprises amplification, copy gain or translocation or lack thereof at 16q23 or 16q22-24 chromosomal locus, or deletion at 16q23 or 16q22-24 chromosomal locus.In certain embodiments, a subject with an increase in c-MAF gene expression level, copy number, amplification or gain relative to a reference has a shorter disease-free survival than a subject without an increase in c-MAF gene expression level, copy number, amplification or gain relative to a reference. In an embodiment, the disease-free survival of a subject with an increase in c-MAF gene expression level, copy number, amplification or gain relative to the reference is at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 18 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years shorter than the disease-free survival of a subject without an increase in c-MAF gene expression level, copy number, amplification or gain relative to the reference. In a particular embodiment, a subject with an increase in c-MAF gene expression level, copy number, amplification or gain relative to the reference has a shorter overall survival than a subject without an increase in c-MAF gene expression level, copy number, amplification or gain relative to the reference. In an embodiment, the overall survival of the subject with an increase in c-MAF gene expression level, copy number, amplification or gain relative to the reference is at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 18 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years shorter than the disease-free survival of the subject with no increase in c-MAF gene expression level, copy number, amplification or gain relative to the reference.In an embodiment, the subject is postmenopausal.In another embodiment, the subject is non-postmenopausal.In some embodiments, the subject is premenopausal.

[0180] In an embodiment, the disease-free survival of a subject who does not have an increase in c-MAF gene expression level, copy number, amplification or gain relative to the baseline is longer than the disease-free survival of a subject who has an increase in c-MAF gene expression level, copy number, amplification or gain relative to the baseline after treatment with a bone-modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid). In embodiments, the disease-free survival of a subject who does not have an increase in c-MAF gene expression level, copy number, amplification or gain relative to the baseline is at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 18 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more longer after treatment with zoledronic acid than the disease-free survival of a subject who has an increase in c-MAF gene expression level, copy number, amplification or gain relative to the baseline after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid). In an embodiment, the overall survival of subjects who do not have an increase in c-MAF gene expression level, copy number, amplification or gain relative to the baseline is longer than the overall survival of subjects who have an increase in c-MAF gene expression level, copy number, amplification or gain relative to the baseline after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid). In an embodiment, the overall survival of subjects who do not have an increase in c-MAF gene expression level, copy number, amplification or gain relative to the baseline is at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 18 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more after treatment with zoledronic acid than the overall survival of subjects who have an increase in c-MAF gene expression level, copy number, amplification or gain relative to the baseline after treatment with zoledronic acid. In an embodiment, the subject is postmenopausal. In another embodiment, the subject is non-postmenopausal. In some embodiments, the subject is premenopausal.

[0181] In an embodiment, the disease-free survival of a subject having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline is shorter than the disease-free survival of a subject not having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid). In embodiments, the disease-free survival of a subject having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline is at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 18 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years shorter after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid) than the disease-free survival of a subject not having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline. In an embodiment, the overall survival of a subject having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline is shorter than the overall survival of a subject not having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid).In an embodiment, the overall survival of a subject having an increase in c-MAF gene expression level, copy number, amplification or gain relative to the baseline is at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 18 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid) than the overall survival of a subject not having an increase in c-MAF gene expression level, copy number, amplification or gain relative to the baseline after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid). In an embodiment, the subject is postmenopausal. In other embodiments, the subject is non-postmenopausal. In some embodiments, the subject is premenopausal.

[0182] In an embodiment, the disease-free survival of a non-postmenopausal subject having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline is shorter than the disease-free survival of a subject not having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline after treatment with a bone-modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid). In embodiments, the disease-free survival of a non-postmenopausal subject having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline is at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 18 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years shorter after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid) than the disease-free survival of a subject not having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline.

[0183] In an embodiment, the overall survival of a subject having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline is shorter than the disease-free survival of a subject not having an increased c-MAF gene expression level, copy number, amplification or gain relative to the baseline after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid). In an embodiment, the overall survival of a subject having an increase in c-MAF gene expression level, copy number, amplification or gain relative to baseline after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid) is at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 18 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more shorter after treatment with a bone modifying agent and / or an agent that avoids or prevents bone degradation (i.e., zoledronic acid) than the overall survival of a subject having no increase in c-MAF gene expression level, copy number, amplification or gain relative to baseline. In an embodiment, the subject is postmenopausal. In another embodiment, the subject is non-postmenopausal. In some embodiments, the subject is premenopausal.

[0184] In some embodiments, MAF predicts OS or DFS of subject based on the menopausal status of the subject.In some embodiments, MAF is predictive in postmenopausal, unknown and peri-menopausal subjects who are at risk of shorter DFS or worst OS.In other embodiments, in premenopausal subjects, MAF positive subjects are lower risk subjects and are more likely to have longer DFS and better OS.

[0185] In an embodiment, the MAF status of a subject predicts the treatment that the subject should receive. In an embodiment, the c-MAF status in any of the embodiments herein includes amplification, copy gain or translocation or lack thereof at 16q23 or 16q22-24 chromosomal locus, or deletion at 16q23 or 16q22-24 chromosomal locus. In an embodiment, postmenopausal patients with increased c-MAF gene expression levels, copy number, amplification or gain relative to baseline (and therefore at high risk of poor DFS or OS outcome) can be administered any treatment disclosed herein. In some embodiments, postmenopausal patients with increased c-MAF gene expression levels, copy number, amplification or gain relative to baseline (and therefore at high risk of poor DFS or OS outcome) can be treated by extending their hormone treatment beyond the 5 years prescribed by the use of hormone treatment as standard treatment. In a particular embodiment, the hormone treatment is tamoxifen and / or aromatase inhibitor. Patients who do not have an increase in c-MAF gene expression levels, copy number, amplification or gain relative to the baseline should not be administered the treatments disclosed herein.

[0186] In certain embodiments, the subject has a metastasis or is prognosticated to experience a metastasis. In some embodiments, the metastasis is a bone metastasis. In further embodiments, the bone metastasis is an osteolytic metastasis.

[0187] In some embodiments, the sample is a primary tumor tissue sample of the subject. In the second step, the obtained c-MAF gene expression level, copy number, amplification or gain in the tumor sample of the subject is compared with the expression level, copy number, amplification or gain of said gene in a control sample. The determination of the c-MAF gene expression level, copy number, amplification or gain must be related to the value of a control sample or a reference sample. Depending on the type of tumor to be analyzed, the exact nature of the control sample may vary. Thus, in some embodiments, the reference sample is a tumor tissue sample of a subject with breast cancer that does not have metastasis, relapse or recurrence, or a tumor tissue sample of a subject with breast cancer that corresponds to the median value of the c-MAF gene expression level, copy number, amplification or gain measured in a tumor tissue collection in a biopsy sample of a subject with breast cancer that does not have metastasis, relapse or recurrence.

[0188] In one embodiment, the method of the invention comprises in a second step comparing the obtained c-MAF gene expression level, copy number, amplification or gain in a tumor sample (including but not limited to a primary tumor biopsy, circulating tumor cells and circulating tumor DNA) from the subject with the expression level of said gene in a control sample.

[0189] After measuring the c-MAF gene expression level, copy number, amplification or gain in a tumor tissue sample, circulating tumor cells or circulating tumor DNA from a subject with breast cancer and comparing it with a control sample, if the expression level of said gene is increased relative to its expression level in the control sample, it can be concluded that said subject has a positive diagnosis of metastasis or has a greater propensity to develop metastasis.

[0190] The determination of c-MAF gene expression level, copy number, amplification or gain must be correlated with the value of control sample or reference sample.Depending on the type of tumor to be analyzed, the exact nature of the control sample may vary.Therefore, in the case of evaluating diagnosis, the reference sample is the tumor tissue sample from the subject with breast cancer that does not metastasize, or the tumor tissue sample from the subject with breast cancer that corresponds to the median value of c-MAF gene expression level measured in the tumor tissue collection in the biopsy sample from the subject with breast cancer that does not metastasize.

[0191] Said reference sample is typically obtained by combining equal amounts of samples from a subject population. In general, a typical reference sample will be obtained from a subject who is clinically well documented and well characterized for the absence of metastasis. In such a sample, the normal concentration (reference concentration) of the biomarker (c-MAF gene) can be determined, for example, by providing the average concentration of the reference population. When determining the reference concentration of the marker, various considerations are taken into account. Such considerations are the age, weight, sex, general physical condition, etc. of the patient. For example, the reference group is a group of equal amounts of at least about 2, at least about 10, at least about 20, at least about 25, at least about 50, at least about 75, at least about 100, at least about 250, at least about 500 to more than about 1000 subjects classified according to said considerations, for example, according to various age categories. The sample collection from which the reference level is derived will preferably be formed by subjects suffering from the same type of cancer (e.g., breast cancer) as the patient subjects of the study. Similarly, a reference value within a cohort of patients can be established by using a receiver operating curve (ROC) and measuring the area under the curve for all de sensitivity and specificity pairs to determine which pair provides the best value and what the corresponding reference value is. ROC is a standard statistical concept. A description is given in Stuart G. Baker “The Central Role of Receiver Operating Characteristic (ROC) curves in "Evaluating Tests for the Early Detection of Cancer" Journal of The National Cancer Institute (2003) Vol 95, No. 7, 511-515.

[0192] Once this median or reference value is established, the levels of this marker expressed in tumor tissue from patients with this median value can be compared and assigned, for example, to an "increased" expression level. Due to subject-to-subject variability (e.g., aspects related to age, race, etc.), it is very difficult (if not virtually impossible) to establish an absolute reference value for c-MAF expression. Thus, in certain embodiments, the reference value for "increased" or "decreased" expression of c-MAF expression is determined by calculating percentiles by conventional means, which involves performing an assay on one or more samples isolated from subjects whose disease has been well documented for c-MAF expression levels by any of the above methods. Preferably, a "decreased" c-MAF level can then be assigned to samples in which the c-MAF expression level is equal to or less than the 50th percentile of the normal population (including, for example, expression levels equal to or less than the 60th percentile of the normal population, expression levels equal to or less than the 70th percentile of the normal population, expression levels equal to or less than the 80th percentile of the normal population, expression levels equal to or less than the 90th percentile of the normal population, and expression levels equal to or less than the 95th percentile of the normal population). An "increased" c-MAF gene expression level can then preferably be assigned to samples in which the c-MAF gene expression level is equal to or exceeds the 50th percentile of the normal population (including, for example, an expression level equal to or exceeds the 60th percentile of the normal population, an expression level equal to or exceeds the 70th percentile of the normal population, an expression level equal to or exceeds the 80th percentile of the normal population, an expression level equal to or exceeds the 90th percentile of the normal population, and an expression level equal to or exceeds the 95th percentile of the normal population).

[0193] In a particular embodiment, the degree of amplification or gain of the c-MAF gene can be determined by determining the amplification or gain of the chromosomal region containing said gene.Preferably, the chromosomal region whose amplification or gain indicates the presence of the amplification or gain of the c-MAF gene is the locus 16q22-q24, which contains the c-MAF gene. The locus 16q22-q24 is located on chromosome 16, on the long arm of said chromosome, and in the range between bands 22 and 24. This region corresponds to contigs NT_010498.15 and NT_010542.15 in the NCBI database.In an embodiment, the degree of amplification or gain of the c-MAF gene can be determined using a probe specific to said gene.

[0194] When measuring copy number, the control sample refers to a tumor sample of a subject with breast cancer who does not suffer from metastasis, or a tumor sample of a subject with breast cancer who does not suffer from metastasis, which corresponds to the median value of the c-MAF gene copy number measured in a tumor tissue collection in a biopsy sample of a subject with breast cancer who does not suffer from metastasis.The reference sample is typically obtained by combining equal amounts of samples from a subject population.If the c-MAF gene copy number is increased relative to the copy number of the gene in the control sample, the subject has a positive diagnosis of metastasis or has a stronger tendency to develop metastasis.In an embodiment, the copy number is determined as the average copy number per cell.

[0195] In some embodiments, the amplification or gain is in the region of the 16q23 locus. In some embodiments, the amplification or gain is in any part of the chromosomal region of chromosome 16 from 79,392,959bp to 79,663,806bp (from centromere to telomere). In some embodiments, the amplification or gain is in the genomic region of chromosome 16 from 79,392,959bp to 79,663,806bp (excluding DNA repeat elements). In some embodiments, the amplification or gain is measured using a probe specific to the region.

[0196] In an embodiment, the c-MAF gene is amplified relative to the reference gene copy number when the c-MAF gene copy number is greater than the copy number possessed by a reference or control sample. In one example, the c-MAF gene is said to be "amplified" when the genomic copy number or average genomic copy number of the c-MAF gene is increased by at least about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45- or 50-fold in the test sample compared to the control sample. In another example, a c-MAF gene is said to be "amplified" if the genomic copy number or average genomic copy number of the c-MAF gene per cell is at least about 2.1, 2.2, 2.3, 2.4, 25., 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.

[0197] In another embodiment, the reference gene copy number is the gene copy number in a breast cancer sample from a subject not suffering from bone metastasis.

[0198] In another embodiment, the amplification or gain is determined by in situ hybridization or PCR.

[0199] In another embodiment, and as described in the present invention, given that chr16q22-24, including the c-MAF gene, is amplified in breast cancer cells and is associated with the presence of metastasis, relapse or recurrence, amplification or gain of chr16q22-24, including the c-MAF gene, allows for decisions regarding the most appropriate treatment for a subject suffering from said cancer.

[0200] The determination of the amplification of the c-MAF gene requires correlation with a control or reference sample value corresponding to the level of amplification of the c-MAF gene measured in tumor tissue samples from subjects with breast cancer and not suffering from metastases, or the median value of amplification of the c-MAF gene measured in a collection of tumor tissues in biopsy samples from subjects with breast cancer and not suffering from metastases, said reference sample being typically obtained by combining equal amounts of samples from a population of subjects.

[0201] In general, a typical reference sample will be obtained from a subject who is clinically well documented and well characterized for the absence of metastasis.The sample collection from which the reference level is derived will preferably be composed of subjects suffering from the same type of cancer as the patient subjects of the study.Once this median value is established, the level of amplification of c-MAF in the patient's tumor tissue can be compared with this median value, and if there is amplification, the subject has a positive diagnosis of metastasis or has a higher tendency to develop metastasis.

[0202] In another embodiment, the invention relates to determining whether the c-MAF gene is translocated in a sample from said subject.

[0203] In some embodiments, the translocation gene is derived from the region of the 16q23 locus. In some embodiments, the translocation gene is derived from any part of the chromosomal region of chromosome 16 from 79,392,959 bp to 79,663,806 bp (from centromere to telomere). In some embodiments, the translocation gene is derived from the genomic region of chromosome 16 from 79,392,959 bp to 79,663,806 bp (excluding DNA repeat elements). In some embodiments, the translocation is measured using a probe specific to the region.

[0204] In certain embodiments, the translocation of the c-MAF gene can be determined by determining the translocation of the chromosomal region containing said gene. In one embodiment, the translocation is a t(14,16) translocation. In another embodiment, the translocated chromosomal region originates from locus 16q22-q24. Locus 16q22-q24 is located on chromosome 16, on the long arm of said chromosome, and in the range between bands 22 and 24. This region corresponds to contigs NT_010498.15 and NT_010542.15 in the NCBI database. In some embodiments, the c-MAF gene is translocated to locus 14q32 on chromosome 14, resulting in the translocation t(14,16)(q32,q23). This translocation places the MAF gene next to a strong enhancer in the IgH locus, which in some cases leads to overexpression of MAF (Eychene, A., Rocques, N., and Puoponnot, C., A new MAFia in cancer. 2008. Nature Reviews:Cancer.8:683-693.).

[0205] In an embodiment, the translocation of the c-MAF gene may be determined by using a probe specific for said translocation.

[0206] One embodiment of the present invention comprises a method which in a first step determines whether the c-MAF gene is translocated in a sample from a subject. In an embodiment, the sample is a tumor tissue sample.

[0207] In a particular embodiment, the method of the present invention for prognosis of the tendency to develop bone metastasis in a subject with breast cancer comprises determining the c-MAF gene copy number in a sample from said subject in which the c-MAF gene is translocated, and comparing said copy number with the copy number of a control or reference sample, wherein if the c-MAF copy number is higher relative to the c-MAF copy number of the control sample, the subject has a greater tendency to develop bone metastasis.

[0208] Methods for determining whether the c-MAF gene or chromosomal region 16q22-q24 is translocated are widely known in the state of the art, including those previously described for the amplification of c-MAF. Said methods include, but are not limited to, in situ hybridization (ISH) (e.g., fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or silver in situ hybridization (SISH)), genomic comparative hybridization or polymerase chain reaction (e.g., real-time quantitative PCR). For any ISH method, amplification, gain, copy number or translocation can be determined by counting the number of fluorescent, colored or silver-bearing dots in chromosomes or nuclei. In other embodiments, detection of copy number changes and translocations can be detected by using whole genome sequencing, exome sequencing, or by using any PCR-derived technology. For example, PCR can be performed on a sample of genomic DNA to detect translocations. In one embodiment, quantitative PCR is used. In one embodiment, PCR is performed using a primer specific for the c-MAF gene and a primer specific for the IGH promoter region; if a product is produced, the translocation has occurred.

[0209] In some embodiments, the amplification, gain and copy number of c-MAF gene are determined after the translocation of c-MAF gene is determined. In some embodiments, a probe is used to determine whether a cell is polyploid for c-MAF gene. In some embodiments, the determination of ploidy is performed by determining whether there are more than two signals from the gene of interest. In some embodiments, the ploidy is determined by measuring the signal from the probe specific to the gene of interest and comparing it with a centromere probe or other probe.

[0210] Methods for measuring c-MAF expression, copy number, amplification, gain and translocation In some embodiments, c-MAF gene expression levels, copy number, amplification, gain or translocation is measured using any method known in the art or described herein.

[0211] The c-MAF protein expression level can be quantified by any conventional method that allows the detection and quantification of said protein in a sample from a subject.As a non-limiting example, said protein level can be quantified, for example, by using an antibody (or its fragment containing an antigenic determinant) with c-MAF binding capacity, followed by quantifying the complex formed.The antibodies used in these assays can be labeled or unlabeled.Examples of markers that can be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, etc. There is a wide range of known assays that can be used in the present invention, using unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies); these techniques include Western blot or Western transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme-linked immunosorbent assay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of protein microarrays or biochips containing specific antibodies, or assays based on colloidal precipitation in formats such as dipsticks. Other methods for detecting and quantifying the c-MAF protein include affinity chromatography techniques, ligand binding assays, etc. When immunological methods are used, any antibody or reagent known to bind to c-MAF protein with high affinity can be used to detect its amount. This includes, but is not limited to, the use of antibodies, such as polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, scFv, humanized diabodies, triabodies, tetrabodies, antibodies, nanobodies, alphabodies, stapled peptides and cyclopeptides.There are commercially available anti-c-MAF protein antibodies that can be used in the context of the present invention, such as antibodies ab427, ab55502, ab55502, ab72584, ab76817, ab77071 (Abcam plc, 330 Science Park, Cambridge CB4 0FL, United Kingdom), O75444 monoclonal antibody from AbD Serotec (Mouse Anti-Human MAF Azide free Monoclonal antibody, Unconjugated, Clone 6b8), etc. There are many commercial companies that provide anti-c-MAF antibodies, such as Abnova Corporation, Bethyl Laboratories, Bioworld Technology, GeneTex, etc.

[0212] In some embodiments, c-MAF protein levels are detected by an antigen binding member or fragment thereof. In some embodiments, the binding member is an antigen binding molecule or fragment thereof that binds to human c-MAF, and the antibody binding molecule or fragment thereof has a heavy chain CDR1 at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical to the amino acid sequence of SEQ ID NO:21, and / or a heavy chain CDR2 at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical to the amino acid sequence of SEQ ID NO:22, and / or a heavy chain CDR2 at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical to the amino acid sequence of SEQ ID NO:23. and / or a light chain CDR1 at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical to the amino acid sequence of SEQ ID NO:18, and / or a light chain CDR2 at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical to the amino acid sequence of SEQ ID NO:19, and / or a light chain CDR3 at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical to the amino acid sequence of SEQ ID NO:20.

[0213] In some embodiments, the antibody or fragment thereof has a V sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 99%, or at least about 100% identical to the amino acid sequence of SEQ ID NO:15. H Includes the domain.

[0214] In some embodiments, the antigen-binding molecule or fragment thereof has a V sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 99%, or at least about 100% identical to the amino acid sequence of SEQ ID NO: 17. L Includes the domain.

[0215] In some embodiments, the antibody or fragment thereof comprises a heavy chain sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 99%, or at least about 100% identical to the amino acid sequence of SEQ ID NO:14.

[0216] In some embodiments, the antibody or fragment thereof comprises a light chain sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 99%, or at least about 100% identical to the amino acid sequence of SEQ ID NO:16.

[0217] In some embodiments, the antigen-binding molecule or fragment thereof is an antibody. In some embodiments, the antibody is a rabbit antibody, a mouse antibody, a chimeric antibody or a humanized antibody. In one aspect, the present invention is directed to a binding member, a functional fragment, an antibody or a variant thereof that specifically binds to the epitope encoded by SEQ ID NO: 24. In some embodiments, the antibody is any antibody described in International Patent Application No. PCT / IB2015 / 059562, which is incorporated herein by reference in its entirety.

[0218] In certain embodiments, c-MAF protein levels are quantified by Western blot, immunohistochemistry, ELISA or protein array.

[0219] As will be understood by those skilled in the art, gene expression levels can be quantified by measuring the messenger RNA levels of the gene or of the protein encoded by the gene. In some embodiments, gene expression levels can be quantified by any means known in the art.

[0220] For this purpose, biological samples can be treated to physically or mechanically disrupt tissue or cellular structures, releasing intracellular components into aqueous or organic solutions for nucleic acid preparation. Nucleic acids are extracted by commercially available methods known to those skilled in the art (Sambroock, J et al., "Molecular cloning: a Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1-3.).

[0221] Thus, the c-MAF gene expression level can be quantified from the RNA (messenger RNA or mRNA) resulting from transcription of said gene, or alternatively from the complementary DNA (cDNA) of said gene. Thus, in a particular embodiment of the present invention, quantification of the c-MAF gene expression level comprises quantification of the messenger RNA of the c-MAF gene or a fragment of said mRNA, a complementary DNA of the c-MAF gene or a fragment of said cDNA, or a mixture thereof.

[0222] To detect and quantify the mRNA level of the c-MAF gene or its corresponding cDNA, virtually any conventional method can be used within the scope of the present invention. As a non-limiting example, the mRNA level of the gene can be quantified using conventional methods, such as methods including mRNA amplification and quantification of the mRNA amplification product, such as electrophoresis and staining, or alternatively by Southern blot and the use of a suitable probe, Northern blot and the use of a specific probe of the mRNA of the gene of interest (c-MAF) or its corresponding cDNA, mapping with S1 nuclease, RT-PCR, hybridization, microarray, etc., preferably by real-time quantitative PCR using a suitable marker. Similarly, the cDNA level corresponding to the mRNA encoded by the c-MAF gene can also be quantified by using conventional techniques; in this case, the method of the present invention includes a step for synthesizing the corresponding cDNA by reverse transcription (RT) of the corresponding mRNA, followed by amplification and quantifying the cDNA amplification product. Conventional methods for quantifying expression levels can be found, for example, in Sambrook et al., 2001 (supra). These methods are known in the art, and those skilled in the art are familiar with the normalization required for each technique.For example, the expression measurements produced using multiplex PCR should be normalized by comparing the expression of the measured gene with so-called "housekeeping" genes (whose expression is constant in all samples, and therefore should provide the baseline expression for comparison) or other control genes whose expression is known to be modulated by cancer.

[0223] In a particular embodiment, the c-MAF gene expression level is quantified by quantitative polymerase chain reaction (PCR) or DNA, RNA array or nucleotide hybridization techniques.

[0224] In addition, the c-MAF gene expression level can also be quantified by quantifying the expression level of the protein encoded by said gene (i.e., c-MAF protein (c-MAF) [NCBI, accession number O75444]) or any functionally equivalent variant of the c-MAF protein. There are two c-MAF protein isoforms (α isoform (NCBI, NP_005351.2) (SEQ ID NO: 4) composed of 403 amino acids and β isoform (NP_001026974.1) (SEQ ID NO: 5) composed of 373 amino acids). The c-MAF gene expression level can be quantified by quantifying the expression level of any of the c-MAF protein isoforms. Thus, in certain embodiments, quantifying the level of the protein encoded by the c-MAF gene includes quantifying the c-MAF protein.

[0225] The method for determining whether c-MAF gene or chromosome region 16q22-q24 is amplified is widely known in the state of the art.The method includes, but is not limited to, in situ hybridization (ISH) (e.g., fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or silver in situ hybridization (SISH)), genomic comparative hybridization or polymerase chain reaction (e.g., real-time quantitative PCR).For any ISH method, amplification, gain or copy number can be determined by counting the number of fluorescent, colored or silver spots in chromosome or nucleus.

[0226] Fluorescence in situ hybridization (FISH) is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences in chromosomes. FISH uses fluorescent probes that bind only to parts of chromosomes with which they show a high degree of sequence similarity. In a typical FISH procedure, the DNA probe is typically fluor-dU The probe is labeled with a fluorescent molecule or hapten in the form of dUTP, digoxigenin-dUTP, biotin-dUTP or hapten-dUTP (which is incorporated into DNA using an enzymatic reaction such as nick translation or PCR). The sample containing the genetic material (chromosomes) is placed on a glass slide and denatured by formamide treatment. The labeled probe is then hybridized with the sample containing the genetic material under appropriate conditions, as determined by the skilled artisan. After hybridization, the sample is observed directly (in the case of a fluorine-labeled probe) or indirectly (using a fluorescently labeled antibody to detect the hapten).

[0227] For CISH, probes are labeled with digoxigenin, biotin or fluorescein and hybridized under appropriate conditions to a sample containing the genetic material.

[0228] Methods for determining whether the c-MAF gene or chromosomal region 16q22-q24 is translocated are widely known in the state of the art, including those previously described for the amplification of c-MAF. Said methods include, but are not limited to, in situ hybridization (ISH) (e.g., fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or silver in situ hybridization (SISH)), genomic comparative hybridization or polymerase chain reaction (e.g., real-time quantitative PCR). For any ISH method, amplification, gain, copy number or translocation can be determined by counting the number of fluorescent, colored or silver-bearing dots in chromosomes or nuclei. In other embodiments, detection of copy number changes and translocations can be detected by using whole genome sequencing, exome sequencing, or by using any PCR-derived technology. For example, PCR can be performed on a sample of genomic DNA to detect translocations. In one embodiment, quantitative PCR is used. In one embodiment, PCR is performed using a primer specific for the c-MAF gene and a primer specific for the IGH promoter region; if a product is produced, the translocation has occurred.

[0229] Any marking or labeling molecule that can be bound to DNA can be used to label the probe used in the method of the present invention, allowing detection of nucleic acid molecules.Examples of labels for labeling include, but are not limited to, radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes and their combinations.Guidelines for selecting methods for labeling and suitable labels for different purposes can be found, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1998).

[0230] In some embodiments, the probes of the invention are dual color probes. In some embodiments, the probes of the invention are dual fusion probes. In some embodiments, the probes of the invention are dual color dual fusion probes. In some embodiments, two separate probes are used.

[0231] In another embodiment, one of the following probes is used to measure the c-MAF gene (including translation of the c-MAF gene): Vysis LSI IGH / MAF dual color dual fusion probe (http: / / www.abbottmolecular.com / us / products / analyte-specific-reagent / fish / vysis-lsi-igh-maf-dual-color-dual-fusion-probe.html; last accessed 11 / 5 / 2012) (which includes probes for 14q32 and 16q23); Kreatech diagnostics MAF / IGH gt(14;16) fusion probe (https: / / www.leicabiosystems.com / fileadmin / img_uploads / kreatech / ifu / PI-KI-10610_D2.1.pdf; last accessed 05 / 18 / 2017), Abnova MAF FISH probe (http: / / www.abnova.com / products / products_detail.asp?Catalog_id=FA0375; last accessed 11 / 5 / 2012), Cancer Genetics Italia IGH / MAF two-color two-fusion translocation probe (http: / / www.cancergeneticsitalia.com / dna-fish-probe / ighmaf / ; last accessed 11 / 5 / 2012), Creative Bioarray IGH / MAF-t(14;16)(q32;q23) FISH probe (http: / / www.creative-bioarray.com / products.asp?cid=35&page=10; last accessed 11 / 5 / 2012), and Arup by FISH. Laboratories multiple myeloma panel (http: / / www.aruplab.com / files / technical-bulletins / Multiple%20Myeloma%20%28MM%29%20by%20FISH.pdf; last accessed 11 / 5 / 2012), Agilent probes specific for 16q23 or 14q32 (http: / / www.genomics.agilent.com / ProductSearch.aspx?chr=16&start=79483700&end=79754340;last accessed 11 / 5 / 2012;http: / / www.genomics.agilent.com / ProductSearch.aspx?Pageid=3000&ProductID=637;last accessed 11 / 5 / 2012), Dako probes specific for 16q23 or 14q32 (http: / / www.dako.com / us / ar42 / psg42806000 / baseproducts_surefish.htm?setCountry=true&purl=ar42 / psg42806000 / baseproducts_surefish.htm?undefined&submit=Accept%20country;last accessed 11 / 5 / 2012), Cytocell IGH / MAF translocation, double fusion probe (http: / / www.zentech.be / uploads / docs / products_info / prenatalogy / cytocell%202012-2013%20catalogue%5B3%5D.pdf; last accessed 11 / 5 / 2012), Metasystems XL IGH / MAF translocation-double fusion probe (http: / / www.metasystems-international.com / index.php?option=com_joodb&view=article&joobase=5&id=12%3Ad-5029-100-og&Itemid=272; last accessed 11 / 5 / 2012), Zeiss FISH probe XL, 100 μl, IGH / MAFB (https: / / www.micro-shop.zeiss.com / ?s=440675675dedc6&l=en&p=uk&f=r&i=5000&o=&h=25&n=1&sd=000000-0528-231-uk; last accessed 11 / 5 / 2012) or Genycell Biotech IGH / MAF double fusion probe (http: / / www.google.com / url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCQQFjAA&url=http%3A%2F%2Fwww.genycell.es%2Fimages%2Fproductos%2Fbrochures%2Flphmie6__86.ppt&ei=MhGYUOi3GKWH0QGlt4DoDw&usg=AFQjCNEqQMbT8vQGjJbi9riEf3lVgoFTFQ&sig2=V5IS8juEMVHB18Mv2Xx_Ww;last accessed 11 / 5 / 2012).

[0232] In some embodiments, the label on the probe is a fluorophore. In some embodiments, the fluorophore on the probe is orange. In some embodiments, the fluorophore on the probe is green. In some embodiments, the fluorophore on the probe is red. In some cases, the fluorophore on the probe is yellow. In some embodiments, one probe is labeled with a red fluorophore and one is labeled with a green fluorophore. In some embodiments, one probe is labeled with a green fluorophore and one is labeled with an orange fluorophore. In some cases, the fluorophore on the probe is yellow. For example, if the MAF-specific probe is labeled with a red fluorophore and the IGH-specific probe is labeled with a green fluorophore, if white is seen, it indicates that the signals are overlapping and translocation is occurring.

[0233] In some embodiments, the fluorophore is SpectrumOrange. In some embodiments, the fluorophore is SpectrumGreen. In some embodiments, the fluorophore is DAPI. In some embodiments, the fluorophore is PlatinumBright405. In some embodiments, the fluorophore is PlatinumBright415. In some embodiments, the fluorophore is PlatinumBright495. In some embodiments, the fluorophore is PlatinumBright505. In some embodiments, the fluorophore is PlatinumBright550. In some embodiments, the fluorophore is PlatinumBright547. In some embodiments, the fluorophore is PlatinumBright570. In some embodiments, the fluorophore is PlatinumBright590. In some embodiments, the fluorophore is PlatinumBright647. In some embodiments, the fluorophore is PlatinumBright495 / 550. In some embodiments, the fluorophore is PlatinumBright415 / 495 / 550. In some embodiments, the fluorophore is DAPI / PlatinumBright495 / 550. In some embodiments, the fluorophore is FITC. In some embodiments, the fluorophore is Texas Red. In some embodiments, the fluorophore is DEAC. In some embodiments, the fluorophore is R6G. In some embodiments, the fluorophore is Cy5. In some embodiments, the fluorophore is FITC, Texas Red and DAPI. In some embodiments, DAPI counterstaining is used to visualize translocations, amplifications, gains or copy number changes.

[0234] Agents and Therapies for Use in Methods for Treating or Preventing Breast Cancer - Patent application In some embodiments, the method of the present invention herein comprises treating a subject with an agent for preventing or avoiding bone remodeling.As used herein, "agent for preventing or avoiding bone remodeling" refers to any molecule (including agent for preventing or avoiding bone decomposition) that can treat or stop bone decomposition by stimulating osteoblast proliferation or inhibiting osteoclast proliferation.In an embodiment, the agent for preventing or avoiding bone remodeling is a bone modifying agent and / or an agent for preventing or avoiding bone decomposition.Illustrative examples of agents used to prevent and / or prevent bone decomposition include, but are not limited to: -Parathyroid hormone (PTH) inhibitors and parathyroid-like hormone (PTHLH) inhibitors (including blocking antibodies) or their recombinant forms (teriparatide, corresponding to amino acids 7-34 of PTH). This hormone acts by stimulating osteoclasts and increasing their activity. - Strontium ranelate is an alternative oral treatment and forms part of a group of drugs called "dual action bone agents" (DABAs) as they stimulate the proliferation of osteoblasts and inhibit the proliferation of osteoclasts. "Estrogen receptor modulator" (SERM) refers to a compound that interferes with or inhibits the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, among others, estrogen progestagens, estradiol, droloxifene, raloxifene, lasofoxifene, TSE-424, tamoxifen, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate 4,4'dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646. -Calcitonin directly inhibits osteoclast activity via calcitonin receptors, which have been identified on the surface of osteoclasts. -Bisphosphonates are a group of medicines used for the prevention and treatment of diseases involving bone resorption and resorption, such as osteoporosis and cancers involving bone metastasis (the latter being related to breast cancer, with or without hypercalcemia).Examples of bisphosphonates that can be used in the treatment designed by the fifth method of the present invention include, but are not limited to, nitrogen-containing bisphosphonates (e.g., pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, incadronate, zoledronate or zoledronic acid, etc.) and nitrogen-free bisphosphonates (e.g., etidronate, clodronate, tiludronate, etc.). "Cathepsin K inhibitor" refers to a compound that interferes with the activity of Cathepsin K cysteine ​​protease. Non-limiting examples of Cathepsin K inhibitors include 4-amino-pyrimidine-2-carbonitrile derivatives (described in International Patent Application WO03 / 020278 in the name of Novartis Pharma GMBH), pyrrolo-pyrimidines described in publications WO03 / 020721 (Novartis Pharma GMBH) and WO04 / 000843 (ASTRAZENECA AB), and inhibitors described in publications WO00 / 55126 of Axys Pharmaceuticals, WO01 / 49288 of Merck Frosst Canada&Co. and Axys Pharmaceuticals. -As used herein, "DKK-1 (Dickkopf-1) inhibitor" refers to any compound capable of decreasing DKK-1 activity. DKK-1 is a soluble Wnt pathway antagonist that is primarily expressed in adult bone and is upregulated in myeloma patients with osteolytic lesions. Agents that target DKK-1 may play a role in preventing osteolytic bone disease in multiple myeloma patients. BHQ880 from Novartis is a first-in-class fully human anti-DKK-1 neutralizing antibody. Preclinical studies support the hypothesis that BHQ880 promotes bone formation, thereby inhibiting tumor-induced osteolytic disease (Ettenberg S et al., American Association for Cancer Research Annual Meeting. April 12-16, 2008; San Diego, Calif. Abstract). - As used herein, "dual MET and VEGFR2 inhibitor" refers to any compound that is a potent dual inhibitor of the MET and VEGF pathways, designed to block MET-driven tumor escape. MET is expressed not only in tumor cells and endothelial cells, but also in osteoblasts (cells that form bone) and osteoclasts (cells that remove bone). HGF binds to MET in all of these cell types, giving the MET pathway a key role in multiple autocrine and paracrine loops. Activation of MET in tumor cells appears to be important in the establishment of metastatic bone lesions. At the same time, activation of the MET pathway in osteoblasts and osteoclasts can lead to pathological features of bone metastasis, including abnormal bone growth (i.e., blastic lesions) or destruction (i.e., lytic lesions). Thus, targeting the MET pathway may be a viable strategy in preventing the establishment and progression of metastatic bone lesions. Cabozantinib (Exelixis, Inc), previously known as XL184 (CAS849217-68-1), is a potent dual inhibitor of the MET and VEGF pathways designed to block MET-driven tumor escape. In multiple preclinical studies, cabozantinib has been shown to kill tumor cells, reduce metastasis, and inhibit angiogenesis (the formation of new blood vessels necessary to support tumor growth). Another suitable dual inhibitor is E7050 (N-[2-fluoro-4-({2-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonylaminopyridin-4-yl}oxy)phenyl]-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (2R,3R)-tartrate) (CAS928037-13-2) or foretinib (also known as GSK1363089, XL880, CAS849217-64-7). -As used herein, "RANKL inhibitor" refers to any compound that can reduce RANK activity. RANKL is found on the surface of the stroma and osteoblast membrane of T-lymphocyte cells, which are the only cells that have been shown to secrete it. Its main function is the activation of osteoclasts, the cells involved in bone resorption. RANKL inhibitors can act by blocking the binding of RANKL to its receptor (RANK), blocking RANK-mediated signal transduction, or reducing the expression of RANKL by blocking the transcription or translation of RANKL. RANKL antagonists or inhibitors suitable for use in the present invention include, but are not limited to: A suitable RANK protein capable of binding to RANKL and comprising the whole or a fragment of the extracellular domain of the RANK protein. Soluble RANK may comprise the signal peptide and the extracellular domain of mouse or human RANK polypeptide, or alternatively the mature form of the protein in which the signal peptide has been removed may be used. o Osteoprotegerin or its variants having RANKL binding ability. ○RANKL-specific antisense molecules. A ribozyme capable of processing the RANKL transcript. Specific anti-RANKL antibodies. By "anti-RANKL antibodies or antibodies against RANKL" is herein understood all antibodies capable of specifically binding to the ligand of the receptor for activator of nuclear factor kappa B (RANKL) which inhibit one or more functions of RANKL. Antibodies can be prepared using any method known to those skilled in the art. Thus, polyclonal antibodies are prepared by immunizing animals with the protein to be inhibited. Monoclonal antibodies are prepared using the method described in Kohler, Milstein et al. (Nature, 1975, 256:495). Suitable antibodies in the context of the present invention include intact antibodies, fragments "Fab", "F(ab')2" and "Fab'", Fv, scFv, diabodies and bispecific antibodies, which contain variable antigen binding regions and constant regions. Specific anti-RANKL nanobodies. Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. Nanobody technology was originally developed following the discovery that camelids (camels and llamas) possess fully functional antibodies that lack light chains. The general structure of a nanobody is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, ○FR1-FR4 are framework regions 1-4, and CDR1-CDR3 are complementarity determining regions 1-3. These heavy chain antibodies contain one variable domain (VHH) and two constant domains (CH2 and CH3). Importantly, the cloned and isolated VHH domains are fully stable polypeptides with the full antigen-binding capacity of the original heavy chain antibody. These newly discovered VHH domains with unique structural and functional properties form the basis of a new generation of therapeutic antibodies that Ablynx has named nanobodies.

[0235] In one embodiment, the RANKL inhibitor is selected from the group consisting of RANKL-specific antibodies, RANKL-specific nanobodies and osteoprotegerin. In a specific embodiment, the anti-RANKL antibody is a monoclonal antibody. In an even more specific embodiment, the anti-RANKL antibody is denosumab (Pageau, Steven C. (2009). mAbs 1(3):210-215, CAS number 615258-40-7), the entire contents of which are incorporated herein by reference. Denosumab is a fully human monoclonal antibody that binds to RANKL and prevents its activation (it does not bind to the RANK receptor). Various aspects of denosumab are covered by U.S. Patent No. 6,740,522; U.S. Patent No. 7,411,050; U.S. Patent No. 7,097,834; U.S. Patent No. 7,364,736, the entire contents of which are each incorporated herein by reference. In another embodiment, the RANKL inhibitor is an antibody, antibody fragment or fusion construct that binds to the same epitope as denosumab.

[0236] In an embodiment, the anti-RANKL nanobody is any of the nanobodies described in WO2008142164, the contents of which are incorporated herein by reference. In an embodiment, the anti-RANKL antibody is ALX-0141 (Ablynx). ALX-0141 is designed to inhibit bone loss associated with postmenopausal osteoporosis, rheumatoid arthritis, cancer and certain drug applications, and to restore balance to healthy bone metabolism.

[0237] In an embodiment, the agent that prevents bone degradation is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH and PTHLH inhibitors or PRG analogs, strontium ranelate, DKK-1 inhibitors, dual MET and VEGFR2 inhibitors, estrogen receptor modulators, radium-223, calcitonin and cathepsin K inhibitors.In an embodiment, the agent that prevents bone degradation is a bisphosphonate.In an embodiment, the bisphosphonate is zoledronic acid.

[0238] In one embodiment, the CCR5 antagonist is administered to prevent or inhibit metastasis or relapse or recurrence of primary breast cancer tumors to bone. In one embodiment, the CCR5 antagonist is a large molecule. In another embodiment, the CCR5 antagonist is a small molecule. In some embodiments, the CCR5 antagonist is Maraviroc. In some embodiments, the CCR5 antagonist is Vicriviroc. In some embodiments, the CCR5 antagonist is Aplaviroc. In some embodiments, the CCR5 antagonist is a spiropiperidine CCR5 antagonist (Rotstein DM et al., 2009. Spiropiperidine CCR5 antagonists. Bioorganic&Medicinal Chemistry Letters. 19(18):5401-5406). In some embodiments, the CCR5 antagonist is INCB009471 (Kuritzkes, DR 2009. HIV-1 entry inhibitors: an overview. Curr. Opin. HIV AIDS. 4(2):82-7).

[0239] In embodiments, the dual MET and VEGFR2 inhibitor is selected from the group consisting of Cabozantinib, Foretinib, and E7050.

[0240] In an embodiment, the MAF status predicts the treatment that the subject should receive. In an embodiment, the c-MAF status in any of the embodiments herein includes amplification, copy gain or translocation or lack thereof at 16q23 or 16q22-24 chromosomal locus, or deletion at 16q23 or 16q22-24 chromosomal locus. In an embodiment, postmenopausal patients with increased c-MAF gene expression levels, copy number, amplification or gain relative to baseline (and therefore at high risk of poor DFS or OS outcome) can be administered any treatment disclosed herein. In some embodiments, postmenopausal patients with increased c-MAF gene expression levels, copy number, amplification or gain relative to baseline (and therefore at high risk of poor DFS or OS outcome) can be treated by extending their hormone treatment beyond the 5 years prescribed by the use of hormone treatment as standard treatment. In a particular embodiment, the hormone treatment is tamoxifen and / or aromatase inhibitor. Patients who do not have an increase in c-MAF gene expression levels, copy number, amplification or gain relative to the baseline should not be administered the treatments disclosed herein.

[0241] In another embodiment, the treatment is an mTor inhibitor. In some embodiments, the mTor inhibitor is a dual mTor / PI3 kinase inhibitor. In some embodiments, the mTor inhibitor is used to prevent or inhibit metastasis, relapse or recurrence. In some embodiments, the mTor inhibitor is: ABI009 (sirolimus), Rapamycin (sirolimus), Abraxane (paclitaxel), Absorb (everolimus), Afinitor (everolimus), Afinitor in combination with Gleevec, AS703026 (pimasertib), Axxess (umirolimus), AZD2014, BEZ235, Biofreedom (umirolimus), BioMatrix (umirolimus), BioMatrix flex (umirolimus), CC115, CC223, Combo Bio-engineered Sirolimus Eluting Stent ORBUSNEICH (sirolimus), Curaxin CBLC102 (mepacrine), DE109 (sirolimus), DS3078, Endeavor DES (zotarolimus), Endeavor Resolute (zotarolimus), Femara (letrozole), Hocena (antroquinonol), INK128, Inspiron (sirolimus), IPI504 (retaspimycin hydrochloride), KRN951 (tivozanib), ME344, MGA031 (teplizumab), MiStent SES (sirolimus), MKC1, Nobori (umirolimus), OSI027, OVI123 (cordycepin), Palomid 529, PF04691502, Promus Element (everolimus), PWT33597, Rapamune (sirolimus), Resolute DES (zotarolimus), RG7422, SAR245409, SF1126, SGN75 (vorsetuzumab mafodotin), Synergy (everolimus), Taltorvic (ridaforolimus), Tarceva (erlotinib), Torisel (temsirolimus), Xience Prime (everolimus), Xience V (everolimus), Zomaxx (zotarolimus), Zortress (everolimus), Zotarolimus-Eluting Peripheral Stent Stent)MEDTRONIC (zotarolimus), AP23841, AP24170, ARmTOR26, BN107, BN108, Canstatin GENZYME (canstatin), CU906, EC0371, EC0565, KI1004, LOR220, NV128, Rapamycin ONCOIMMUNE (sirolimus), SB2602, Sirolimus PNP SAMYANG BIOPHARMACEUTICALS (sirolimus), TOP216, VLI27, VS5584, WYE125132, XL388, Advacan (everolimus), AZD8055, Cypher Select Plus Sirolimus-Eluting Coronary Stent (sirolimus), Cypher Sirolimus-Eluting Coronary Stent (Sirolimus), Drug Coated Balloon (Sirolimus), E-Magic Plus (Sirolimus), Emtor (Sirolimus), Esprit (Everolimus), Evertor (Everolimus), HBF0079, LCP-Siro (Sirolimus), Limus CLARIS (Sirolimus), mTOR Inhibitor CELLZOME, Nevo Sirolimus-Eluting Coronary Stent (Sirolimus), nPT-mTOR, Rapacan (Sirolimus), Renacept (Sirolimus), ReZolve (Sirolimus), Rocas (Sirolimus), SF1126, Sirolimus, Sirolimus NORTH CHINA (Sirolimus), Sirolimus RANBAXY (Sirolimus), SirolimusThe mTor inhibitor is selected from the group consisting of WATSON (sirolimus), Siropan (sirolimus), Sirova (sirolimus), Supralimus (sirolimus), Supralimus-Core (sirolimus), Tacrolimus WATSON (tacrolimus), TAFA93, Temsirolimus ACCORD (temsirolimus), Temsirolimus SANDOZ (temsirolimus), TOP216, Xience Prime (everolimus), Xience V (everolimus). In a specific embodiment, the mTor inhibitor is Afinitor (everolimus) (http: / / www.afinitor.com / index.jsp?usertrack.filter_applied=true&NovaId=4029462064338207963; last accessed November 28, 2012). In another embodiment, mTor inhibitors can be identified by methods known in the art. (See, e.g., Zhou, H. et al., Updates of mTor inhibitors. 2010. Anticancer Agents Med.Chem. 10(7):571-81, which is incorporated herein by reference). In another embodiment, mTor inhibitors can be identified by methods known in the art. (See, e.g., Zhou, H. et al., Updates of mTor inhibitors. 2010. Anticancer Agents Med.Chem. 10(7):571-81, which is incorporated herein by reference). In some embodiments, mTor inhibitors are used to treat or prevent or inhibit metastasis in patients positive for hormone receptors. (See, e.g., Baselga, J., el al., Everolimus in Postmenopausal Hormone-Receptor Positive Advanced Breast Cancer. 2012.(See N. Engl. J. Med. 366(6):520-529). In some embodiments, mTor inhibitors are used to treat or prevent or inhibit metastasis in patients with advanced breast cancer. In some embodiments, mTor inhibitors are used in combination with a second treatment. In some embodiments, the second treatment is any treatment described herein.

[0242] In another embodiment, the treatment is a Src kinase inhibitor. In some embodiments, the Src inhibitor is used to prevent or inhibit metastasis, relapse or recurrence. In some embodiments, the Src kinase inhibitor is selected from the following groups: AZD0530 (saracatinib), Bosulif (bosutinib), ENMD981693, KD020, KX01, Sprycel (dasatinib), Yervoy (ipilimumab), AP23464, AP23485, AP23588, AZD0424, c-Src kinase inhibitor KISSEI, CU201, KX2361, SKS927, SRN004, SUNK706, TG100435, TG100948, AP23451, Dasatinib HETERO (dasatinib), Dasatinib Selected from VALEANT (dasatinib), Fontrax (dasatinib), Src kinase inhibitor KINEX, VX680 (tozasertib lactate), XL228 and SUNK706. In some embodiments, the Src kinase inhibitor is dasatinib. In another embodiment, the Src kinase inhibitor can be identified by methods known in the art (see, for example, Sen, B. and Johnson, FM Regulation of Src Family Kinases in Human Cancers. 2011. J. Signal Transduction. 2011:14 pages, which is incorporated herein by reference). In some embodiments, the Src kinase inhibitor is used to treat or prevent or inhibit metastasis, relapse or recurrence in patients positive for SRC responsive signature (SRS). In some embodiments, the patient is SRS+ and ER- (see, e.g., Zhang, C.H.-F et al., Latent Bone Metastasis in Breast Cancer Tied to Src-Dependent survival signals. 2009. Cancer Cell. 16:67-78, which is incorporated herein by reference). In some embodiments, Src kinase inhibitors are used to treat or prevent or inhibit metastasis in patients with advanced breast cancer. In some embodiments, Src kinase inhibitors are used in combination with a second treatment. In some embodiments, the second treatment is any treatment described herein.

[0243] In another embodiment, the treatment is a COX-2 inhibitor. In some embodiments, the COX-2 inhibitor is used to prevent or inhibit metastasis, relapse or recurrence. In some embodiments, the COX-2 inhibitor is selected from the following groups: ABT963, Acetaminophen ER JOHNSON (acetaminophen), Acular X (ketorolac tromethamine), BAY1019036 (aspirin), BAY987111 (diphenhydramine, naproxen sodium), BAY11902 (piroxicam), BCIBUCH001 (ibuprofen), Capoxigem (apricoxib), CS502, CS670 (pelubiprofen), Diclofenac HPBCD (diclofenac), Diractin (ketoprofen), GW406381, HCT1026 (nitroflurbiprofen), Hyanalgese-D (diclofenac), HydrocoDex (acetaminophen, dextromethorphan, hydrocodone), Ibuprofen Sodium PFIZER, Ibuprofen with Acetaminophen PFIZER, Impracor (ketoprofen), IP880 (diclofenac), IP940 (indomethacin), ISV205 (diclofenac sodium), JNS013 (acetaminophen, tramadol hydrochloride), Ketoprofen TDS (ketoprofen), LTNS001 (naproxen etemesil), Mesalamine SALIX (mesalamine), Mesalamine SOFAR (mesalamine), Mesalazine (mesalamine), ML3000 (licofelone), MRX7EAT (etodolac), Naproxen IROKO (naproxen), NCX4016 (nitroaspirin), NCX701 (nitroacetaminophen), NuprinSCOLR (ibuprofen), OMS103HP (amitriptyline hydrochloride, ketoprofen, oxymetazoline hydrochloride), Oralease (diclofenac), OxycoDex (dextromethorphan, oxycodone), P54, PercoDex (acetaminophen, dextromethorphan, oxycodone), PL3100 (naproxen, phosphatidylcholine), PSD508, R-Ketoprofen (ketoprofen), Remura (bromfenac sodium), ROX828 (ketorolac tromethamine), RP19583 (ketoprofen lysine), RQ00317076 , SDX101 (R-etodolac), TDS943 (diclofenac sodium), TDT070 (ketoprofen), TPR100, TQ1011 (ketoprofen), TT063 (S-flurbiprofen), UR8880 (cimicoxib), V0498TA01A (ibuprofen), VT122 (etodolac, propranolol), XP20B (acetaminophen, dextropropoxyphene), XP21B (diclofenac potassium), XP21L (diclofenac potassium), Zoenasa (acetylcysteine, mesalamine), Acephen, Actifed Plus, Actifed-P, Acular, Acular LS, Acular PF, Acular IBRAHIM, Aleve-D, Alka-Seltzer, Alka-Seltzer BAYER, Alka-Seltzer Extra Strength, Alka-Seltzer Lemon-Lime, Alka-Seltzer Original, Alka-Seltzer Plus, Alka-Seltzer plus Cold and Cough, Alka-Seltzer plus Cold and CoughFormula、Alka-Seltzer Plus Day and Night Cold Formula、Alka-Seltzer Plus Day Non-Drowsy Cold Formula、Alka-Seltzer Plus Flu Formula、Alka-Seltzer Plus Night Cold Formula、Alka-Seltzer Plus Sinus Formula、Alka-Seltzer Plus Sparkling Original Cold Formula、Alka-Seltzer PM、Alka-Seltzer Wake-Up Call、Anacin、Anaprox、Anaprox MINERVA、Ansaid、Apitoxin、Apranax、Apranax abdi、Arcoxia、Arthritis Formula Bengay、Arthrotec、Asacol、Asacol HD、Asacol MEDUNA ARZNEIMITTEL、Asacol ORIFARM、Aspirin BAYER、Aspirin Complex、Aspirin Migran、AZD3582、Azulfidine、Baralgan M、BAY1019036、BAY987111、BAYl1902、BCIBUCH001、Benadryl Allergy、Benadryl Day and Night、Benylin 4 Flu、Benylin Cold and Flu、Benylin Cold and Flu Day and Night、Benylin Cold and Sinus Day and Night、Benylin Cold and Sinus Plus、Benylin Day and Night Cold and Flu Relief、Benylin1 All-In-One、Brexin、Brexin ANGELINI、Bromday、Bufferin、Buscopan Plus、Caldolor、Calmatel、Cambia、Canasa、Capoxigem、Cataflam、Celebrex、Celebrex ORIFARM、Children’s Advil Allergy Sinus、Children’sTylenol、Children’s Tylenol Cough and Runny Nose、Children’s Tylenol plus cold、Children’s Tylenol plus Cold and Cough、Children’s Tylenol plus cold and stuffy nose、Children’s Tylenol plus Flu、Children’s Tylenol plus cold & allergy、Children’s Tylenol plus Cough & Runny Nose、Children’s Tylenol plus Cough & Sore Throat、Children’s Tylenol plus multi symptom cold、Clinoril、Codral Cold and Flu、Codral Day and Night Day Tablets、Codral Day and Night Night Tablets、Codral Nightime、Colazal、Combunox、Contac Cold plus Flu、Contac Cold plus Flu Non-Drowsy、Coricidin D、Coricidin HBP Cold and Flu、Coricidin HBP Day and Night Multi-Symptom Cold、Coricidin HBP Maximum Strength Flu、Coricidin HBP Nighttime Multi-Symptom Cold、Coricidin II Extra Strength Cold and Flu、CS502、CS670、Daypro、Daypro Alta、DDS06C、Demazin Cold and Flu、Demazin Cough、Cold and Flu、Demazin day / night Cold and Flu、Demazin PE Cold and Flu、Demazin PE day / night Cold and Flu、Diclofenac HPBCD、Dimetapp Day Relief、Dimetapp Multi-Symptom Cold and Flu、Dimetapp Night Relief、Dimetapp Pain and Fever Relief、Dimetapp PE Sinus Pain、Dimetapp PE Sinus Pain plus Allergy、Dipentum、Diractin、Disprin Cold ‘n’ Fever、Disprin Extra、Disprin Forte.Disprin Plus、Dristan Cold、Dristan Junior、Drixoral Plus、Duexis、Dynastat、Efferalgan、Efferalgan Plus Vitamin C、Efferalgan Vitamin C、Elixsure IB、Excedrin Back and Body、Excedrin Migraine、Excedrin PM、Excedrin Sinus Headache、Excedrin Tension Headache、Falcol、Fansamac、Feldene、FeverAll、Fiorinal、Fiorinal with Codeine、Flanax、Flector Patch、Flucam、Fortagesic、Gerbin、Giazo、Gladio、Goody’s Back and Body Pain、Goody’s Cool Orange、Goody’s Extra Strength、Goody’s PM、Greaseless Bengay、GW406381、HCT1026、He Xing Yi、Hyanalgese-D、HydrocoDex、Ibuprofen Sodium PFIZER、Ibuprofen with、Acetaminophen PFIZER、Icy Hot SANOFI AVENTIS、Impracor、Indocin、Indomethacin APP PHARMA、Indomethacin MYLAN、Infants’ Tylenol、IP880、IP940、Iremod、ISV205、JNS013、Jr.Tylenol、Junifen、Junior Strength Advil、Junior Strength Motrin、Ketoprofen TDS、Lemsip Max、Lemsip Max All. in One、Lemsip Max All Night、Lemsip Max Cold and Flu、Lialda、Listerine Mouth Wash、Lloyds Cream、Lodine、Lorfit P、Loxonin、LTNS001、Mersyndol、Mesalamine SALIX、Mesalamine SOFAR、Mesalazine、Mesasal GLAXO、Mesasal SANOFI、Mesulid、Metsal Heat Rub、Midol C omplete、Midol Extended Relief、Midol Liquid Gels、Midol PM、Midol Teen Formula、Migranin COATED TABLETS、ML3000、Mobic、Mohrus、Motrin、Motrin Cold and Sinus Pain、Motrin PM、Movalis ASPEN、MRX7EAT、Nalfon、Nalfon PEDINOL、Naprelan、Naprosyn、Naprosyn RPG LIFE SCIENCE、Naproxen IROKO、NCX4016、NCX701、NeoProfen LUNDBECK、Nevanac、Nexcede、Niflan、Norgesic MEDICIS、Novalgin、Nuprin SCOLR、Nurofen、Nurofen Cold and Flu、Nurofen Max Strength Migraine、Nurofen Plus、Nuromol、NyQuil with Vitamin C、Ocufen、OMS103HP、Oralease、Orudis ABBOTT JAPAN、Oruvail、Osteluc、OxycoDex、P54、Panadol、Panadol Actifast、Paradine、Paramax、Parfenac、Pedea、Pennsaid、Pentasa、Pentasa ORIFARM、Peon、Percodan、Percodan-Demi、PercoDex、Percogesic、Perfalgan、PL2200、PL3100、Ponstel、Prexige、Prolensa、PSD508、R-Ketoprofen、Rantudil、Relafen、Remura、Robaxisal、Rotec、Rowasa、ROX828、RP19583、RQ00317076、Rubor、Salofalk、Salonpas、Saridon、SDX101、Seltouch、sfRowasa、Shinbaro、Sinumax、Sinutab、Sinutab、sinus、Spalt、Sprix、Strefen、Sudafed Cold and Cough、Sudafed Head Cold and Sinus、Sudafed PE Cold plus Cough、Sudafed PE Pressure plus Pain、Sudafed PE、Severe Cold、Sudafed PE Sinus Day plus Night Relief Day Tablets、Sudafed PE Sinus Day plus Night Relief NightTablets、Sudafed PE Sinus plus Anti-inflammatory Pain Relief、Sudafed Sinus Advance、Surgam、Synalgos-DC、Synflex、Tavist allergy / sinus / headache、TDS943、TDT070、Theraflu Cold and Sore Throat、Theraflu Daytime Severe Cold and Cough、Theraflu Daytime Warming Relief,Theraflu Warming Relief Caplets Daytime Multi-Symptom Cold、Theraflu Warming Relief Cold and Chest Congestion、Thomapyrin、Thomapyrin C、Thomapyrin Effervescent、Thomapyrin Medium、Tilcotil、Tispol、Tolectin、Toradol、TPR100、TQ1011、Trauma-Salbe、Trauma-Salbe Kwizda、Treo、Treximet、Trovex、TT063、Tylenol、Tylenol Allergy Multi-Symptom、Tylenol Back Pain、Tylenol Cold & Cough Daytime、Tylenol Cold & Cough Nighttime、Tylenol Cold and Sinus Daytime、Tylenol Cold and Sinus Nighttime、Tylenol Cold Head Congestion Severe、Tylenol Cold Multi Symptom Daytime、Tylenol Cold Multi Symptom Nighttime Liquid、Tylenol Cold Multi Symptom Severe、Tylenol Cold Non-Drowsiness Formula、Tylenol Cold Severe Congestion Daytime、Tylenol Complete Cold,Cough and Flu Night time、Tylenol Flu Nighttime、Tylenol Menstrual、Tylenol PM、Tylenol Sinus Congestion & Pain Daytime、Tylenol Sinus Congestion & Pain Nighttime、Tylenol Sinus Congestion & Pain Severe、Tylenol Sinus Severe Congestion Daytime、Tylenol Ultra Relief、Tylenol with Caffeine and Codeine phosphate、Tylenol with Codeine phosphate、Ultra Strength Bengay Cream、Ultracet、UR8880、V0498TA01A、Vicks NyQuil Cold and Flu Relief、Vicoprofen、Vimovo、Voltaren Emulgel、Voltaren GEL、Voltaren NOVARTIS CONSUMER HEALTH GMBH、Voltaren The COX-2 inhibitor is selected from XR, VT122, Xefo, Xefo Rapid, Xefocam, Xibrom, XL3, Xodol, XP20B, XP21B, XP21L, Zipsor and Zoenasa. In another embodiment, the COX-2 inhibitor can be identified by methods known in the art (see, for example, Dannhardt, G. and Kiefer, W. Cyclooxygenase inhibitors-current status and future prospects. 2001. Eur. J. Med. Chem. 36:109-126, which is incorporated herein by reference). In some embodiments, the COX-2 inhibitor is used to treat or prevent or inhibit metastasis in patients with advanced breast cancer. In some embodiments, the COX-2 inhibitor is used in combination with a second treatment. In some embodiments, the second treatment is any treatment described herein. In some embodiments, the COX-2 inhibitor is used in combination with a second treatment selected from the group consisting of Denosumab, Zometa (http: / / www.us.zometa.com / index.jsp?usertrack.filter_applied=true&NovaId=2935376934467633633; last accessed December 2, 2012), Carbozantinib or Cabozantinib, antibodies or peptides that block PTHLH (parathyroid hormone-like hormone) or PTHrP (parathyroid hormone-related protein).

[0244] In one embodiment, the treatment is Radium-223. In an embodiment, the Radium-223 treatment is Alpharadin (aka Xofigo) (Radium-223 dichloride). Alpharadin uses alpha radiation from Radium-223 decay to kill cancer cells. Radium-223 naturally self-targets to bone metastases due to its properties as a calcimimetic. Alpha radiation has a very short range of 2-10 cells (compared to current radiation therapy based on beta or gamma radiation), so it causes less damage to surrounding healthy tissues (especially bone marrow). Due to its similar properties to calcium, Radium-223 is attracted to locations in the body where calcium is used to build bone, including sites of faster abnormal bone growth. After injection, Radium-223 is carried by the bloodstream to the site of abnormal bone growth. The location in the body where cancer begins is known as the primary tumor. Some of these cells may break off and be carried by the bloodstream to other parts of the body. Those cancer cells can then colonize that location in the body and form new tumors. When this happens, it is called a secondary cancer or metastasis. The purpose of using Radium-223 is to selectively target this secondary cancer. Any Radium-223 that is not taken up by the bone is quickly sent to the intestine to be excreted.

[0245] In some embodiments, the treatment is a CDK4 / 6 inhibitor. In certain embodiments, the CDK4 / 6 inhibitor is selected from any known CDK4 / 6 inhibitor. In yet further embodiments, the CDK4 / 6 inhibitor is palbociclib (PD-0332991), ribociclib (LEE011) or abemaciclib (LY2835219). The use of CDK4 / 6 inhibitors is described in Finn et al., Breast Cancer Research 18:17 (2016).

[0246] Alternatively, combination treatments may be performed combining one or more of the above agents to treat and / or prevent metastasis, relapse or recurrence, or the agents may be combined with other supplements, such as calcium or vitamin D, or with hormonal treatments.

[0247] In an embodiment, MAF-positive postmenopausal patients with high risk of DFS or bad OS outcome are treated with any therapy in adjuvant setting to improve patient outcome.These therapies include any therapy disclosed herein, including drugs for avoiding or preventing bone remodeling, drugs for improving disease-free survival or overall survival, c-MAF inhibitors, chemotherapy, hormone therapy, m-Tor inhibitors, CDK4 / 6 inhibitors, radium-223, CCR5 antagonists, Src kinase inhibitors or COX-2 inhibitors and combinations thereof.Patients who are not MAF-positive should not be administered such drugs or therapies.

[0248] If cancer has metastasized, systemic treatments are used, including but not limited to chemotherapy, hormonal treatments, immunotherapy or combinations thereof.In addition, radiation therapy and / or surgery may be used.The choice of treatment usually depends on the type of primary cancer, its size, the location of metastasis, the age of the patient, general health, and the type of treatment used previously.

[0249] Systemic treatments are those that reach the entire body: -Chemotherapy is the use of medicines to destroy cancer cells. Medicines are generally administered by oral or intravenous routes. In other embodiments, the treatment is chemotherapy. In some embodiments, the chemotherapy is any chemotherapy known in the art. In certain embodiments, the chemotherapy is adjuvant chemotherapy. In certain embodiments, the chemotherapy is a taxane. In further embodiments, the taxane is paclitaxel (taxol), docetaxel (taxotere) or cabazitaxel. Medicines are generally administered by oral or intravenous routes. Chemotherapy may be used together with radiation treatment. Hormonal therapy is based on the fact that some hormones promote cancer growth. For example, in women, estrogen produced by the ovaries can promote breast cancer growth. There are several ways to stop the production of these hormones. One way is to remove the organs that produce them (ovaries in women, testes in men). More frequently, medicines to prevent these organs from producing hormones or to prevent hormones from acting on cancer cells may be used. In an embodiment, the treatment is hormonal therapy. In a particular embodiment, the hormonal therapy is tamoxifen and / or aromatase inhibitor. -Immunotherapy is a treatment that supports the patient's immune system itself to fight cancer. There are several types of immunotherapy that are used to treat metastatic patients. These include, but are not limited to, cytokines, monoclonal antibodies, and antitumor vaccines.

[0250] Agents for avoiding or preventing bone remodeling are typically administered in combination with a pharma- ceutically acceptable carrier.

[0251] The term "carrier" refers to a diluent or excipient with which an active ingredient is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, such as those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or saline solutions, particularly for injection solutions, as well as aqueous dextrose and glycerol solutions, are preferably used as carriers. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin, 1995. Preferably, the carriers of the present invention are those approved by state or federal regulatory agencies or listed in the United States Pharmacopoeia or other pharmacopoeias generally recognized for their use in animals, more particularly in humans.

[0252] The carriers and auxiliary substances necessary to prepare the desired pharmaceutical dosage form of the pharmaceutical composition of the present invention depend inter alia on the pharmaceutical dosage form selected. Said pharmaceutical dosage form of the pharmaceutical composition will be prepared according to conventional methods known to those skilled in the art. A review of the various methods for administering active ingredients, the excipients to be used and the processes for producing them can be found in "Tratado de Farmacia Galenica", C. Fauli i Trillo, Luzan 5, SA 1993 Edition. Examples of pharmaceutical compositions include any solid composition (tablets, pills, capsules, granules, etc.) or liquid composition (solution, suspension or emulsion) for oral, topical or parenteral administration. In addition, the pharmaceutical composition may contain stabilizers, suspending agents, preservatives, surfactants, etc., if deemed necessary.

[0253] In medicine, bone remodeling agent can be found in the form of prodrug, salt, solvate or clathrate, either alone or in combination with additional active agent, and can be formulated with pharmaceutically acceptable excipients.Preferred excipients for its use in the present invention include sugar, starch, cellulose, gum and protein.In certain embodiments, the pharmaceutical composition of the present invention will be formulated as solid pharmaceutical dosage form (e.g., tablet, capsule, pill, granule, suppository, sterile crystalline or amorphous solid that can be reconstituted to obtain liquid form, etc.), liquid pharmaceutical dosage form (e.g., solution, suspension, emulsion, elixir, lotion, ointment, etc.) or semi-solid pharmaceutical dosage form (e.g., gel, ointment, cream, etc.). The pharmaceutical compositions of the present invention may be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. A review of the various methods for administering active ingredients, the excipients to be used and their manufacturing processes can be found in Tratado de Farmacia Galenica, C. Fauli i Trillo, Luzan 5, SA, 1993 Edition and Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 20 th edition, Williams & Wilkins PA, USA (2000). Examples of pharma- ceutically acceptable carriers are known in the state of the art and include phosphate buffered saline solutions, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, etc. Compositions containing said carriers can be formulated by conventional processes known in the state of the art.

[0254] The agent for preventing and preventing bone remodeling or the pharmaceutical composition containing them can be administered at a dose of less than 10 mg per kilogram of body weight, preferably at least about 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or less than 0.00001 mg per kg of body weight. The unit dose can be administered by injection, inhalation or topical administration. In certain embodiments, the agent is administered at its approved dose.

[0255] The dose depends on the severity and response of the condition being treated and may vary from a few days to a few months or until the condition subsides. The optimal dose may be determined by periodically measuring the concentration of the drug in the patient's body. The optimal dose may be determined from an EC50 value obtained by prior in vitro or in vivo assays in animal models. The unit dose may be administered once a day or less than once a day, preferably less than once every 2, 4, 8 or 30 days. Alternatively, after the initial dose, one or several maintenance doses (usually less than the initial dose) may be administered. The maintenance regimen may involve treating the patient with a dose range of 0.01 μg to 1.4 mg / kg body weight / day, for example, 10, 1, 0.1, 0.01, 0.001 or 0.00001 mg / kg body weight / day. The maintenance dose is preferably administered at most once every 5, 10 or 30 days. Treatment must be continued for a period of time that varies according to the type of disorder the patient suffers from, its severity and the condition of the patient. After treatment, the patient's progress must be monitored to determine whether the dosage should be increased if the disease does not respond to treatment, or whether the dosage should be decreased if improvement of the disease is observed or if undesirable side effects are observed.

[0256] Kits of the Invention In another aspect, the present invention relates to a kit for determining a treatment for a subject suffering from breast cancer, comprising: a) means for quantifying the expression level, copy number, amplification, gain or translocation of c-MAF in a sample from the subject; b) means for comparing the quantified expression level, copy number, amplification, gain or translocation of c-MAF in the sample with a reference c-MAF expression level; and c) means for determining a treatment or excluding a treatment from consideration for the subject based on a comparison of the quantified expression level with the reference expression level.

[0257] The means for quantifying the expression level of c-MAF in the sample of the subject has been described in detail previously, including amplification, gain and / or translocation of 16q23 and 16q22-24 loci. In some embodiments, the means for quantifying c-MAF expression is any antibody, antigen-binding molecule or fragment described herein. In some embodiments, the antibody is any antibody described in International Patent Application No. PCT / IB2015 / 059562, which is incorporated herein by reference in its entirety.

[0258] In a preferred embodiment, the means for quantifying expression comprises a set of probes and / or primers that specifically bind to and / or amplify the c-MAF gene.

[0259] All of the particular embodiments of the methods of the invention are applicable to the kits of the invention and their uses.

[0260] A method for classifying a subject suffering from breast cancer. In another embodiment, the present invention relates to a method for classifying subjects suffering from breast cancer into cohorts, comprising: a) determining the expression level, copy number, amplification, gain or translocation of c-MAF in a sample of said subject; b) comparing said expression level, copy number, amplification, gain or translocation of c-MAF in said sample with a predefined reference level of c-MAF expression; and c) classifying said subjects into cohorts based on said expression level, copy number, amplification, gain or translocation of c-MAF in said sample.

[0261] In some embodiments, the c-MAF gene expression level is used to stratify patients into treatment groups based on c-MAF expression level. In embodiments, patients with high c-MAF expression levels receive a different treatment than patients with low c-MAF expression levels. In embodiments, patients are further stratified based on menopausal status. In embodiments, patients are stratified based on being postmenopausal or non-postmenopausal. In certain embodiments, subjects are administered different treatments based on c-MAF expression level and / or postmenopausal or non-postmenopausal status. In some embodiments, the stratified patients are administered an agent that prevents or prevents bone remodeling. In embodiments, the agent that prevents or prevents bone remodeling is an agent that prevents or prevents bone degradation. In further embodiments, the agent that prevents or prevents bone degradation is zoledronic acid. In other embodiments, the c-MAF gene expression level is used to select patients for treatment. In some embodiments, patients are stratified into groups for clinical trials.

[0262] Means for quantifying the expression level of c-MAF in a sample from said subject, including amplifications, gains and / or translocations of the 16q23 and 16q22-24 loci, have been described in detail previously.

[0263] In another preferred embodiment, the cohort is for conducting a clinical trial.

[0264] In a preferred embodiment, the sample is a tumor tissue sample.

[0265] The following examples illustrate the invention without limiting its scope. EXAMPLES

[0266] Example 1: Validation of c-MAF as a metastasis marker. The IHC and FISH assays used to test c-MAF in AZURE samples were analytically validated. An overview of the assay validation parameters can be seen in Figure 1.

[0267] The MAF FISH assay was developed by Kreatech for Inbiomotion based on KREATECH's proprietary FISH technology. The probe set contains two probes (MAF 16q23 probe + D16Z3 probe) as a control for the centromeric region of chromosome 16. The assay was validated using the 16q23 / D16Z3 probe (Inbiomotion) and Kreatech's Poseidon tissue digestion kit. In the fact sheet, the scoring criteria were defined as follows: there were two evaluable FISH results per patient, and the highest was selected. The scoring algorithm was as follows: for target and centromere amplification, 20 cells were counted, and if the gene count was >2 and ≦3, 50 cells were counted.

[0268] The MAF IHC assay was based on recombinant monoclonal antibodies (described in International Patent Application No. PCT / IB2015 / 059562, which is incorporated herein by reference in its entirety). Antibodies were selected based on IHC. DAKO The assay was validated using the MAF RecMab (Inbiomotion) by AS LINK platform and the control specimen protocol provided by Inbiomotion. Scoring criteria were predefined in the fact sheet, with a single IHC H-score per patient. The scoring algorithm was the H-score.

[0269] Patients were included in the AZURE clinical trial used to validate MAF (Coleman et al., N Eng J Med 2011;365:1396-1405 and AZURE Current Controlled Trials number, ISRCTN79831382 and ClinicalTrials.gov identifier NCT00072020) An overview of the study design is provided in Figure 2. MAF was validated in a retrospective analysis of the AZURE study using patient tumor samples collected in a prospective study under regulatory compliant conditions. Of the 3360 patients recruited, 1769 donated tumor tissue (52.4%). There were 13 TMAs (tissue microarrays) (150 patient samples each) (1769 patients). There were 4 replicas of each TMA using different tissue cores (6326 (4 × patients)). One TMA had only 1 replica and 2 TMAs had 3 replicas. Based on H&E analysis (hematoxylin and eosin) ( Fig. 3 ) (6,326), 3978 cores were evaluable (63%) and 2348 were non-evaluable.

[0270] For FISH assays, there were 2,067 evaluable FISH cores (56%). 865 patients (49%) had two evaluable FISH cores (26% of AZURE patients) and 1,202 patients had a single FISH score (68%). 567 patients were not evaluable by FISH in any of the four replicas (32%).

[0271] IHC assays were evaluated by pathologist and VisoPharm computer-assisted evaluation. For pathologist evaluation, 2,232 cores were evaluated (59% evaluable for H-score). 1390 patients had an IHC H-score (74%) (representing 39% of all AZURE patients). 460 patients were not evaluable by IHC in any of the four replicas. For VisioPharm computer-assisted IHC staining evaluation, 1299 IHC patients were evaluated out of 1309 pathologist-scored for H-score and mean stain / nucleus. MAF positivity rates can be seen in Figures 4A and 4B.

[0272] Cutoff optimized FISH data can be seen in Figure 5 and were calculated as described in Vipery et al., JCO 2014 DOI:10.1200 / JCO.2013.53.3604.

[0273] For molecular variables of FISH analysis: MAF copy number: numerical and categorical (+ / - cutoff >=2.5) variable; % of nuclear MAF amplified (MAFCN>2): numerical + categorical (cutoff TBD). For IHC analysis: IHC H-score: numerical + categorical (cutoff =>200), IHC OD: numerical + categorical (cutoff tBD). The following clinical variables were analyzed: disease-free survival (DFS), invasive disease-free survival (IDFS), overall survival (OS), first recurrence in bone, bone recurrence at any time point, time to first DFS event in bone, time to first DFS event outside bone, response to zoledronic acid treatment.

[0274] For the analysis of MAF FISH prognostic value, patients in the control and treatment arms were pooled for the initial analysis. Optimized cutoffs for each variable to be analyzed were used where indicated. Death was used as a competing event in time to bone metastasis (any time point). The following clinical variables were analyzed: time to bone metastasis (any time point), time to bone metastasis (as first event), IDFS (including death due to any cause including ipsilateral invasive breast tumor recurrence, locally invasive breast cancer recurrence, metastatic disease-breast cancer, breast cancer, contralateral invasive breast cancer, second primary non-breast invasive cancer), and overall survival.

[0275] Figure 6 shows that, when analyzing all patients, MAF FISH-positive patients had a 40% higher risk of bone metastasis (>=2.3) (p=0.007) (time to bone metastasis (any time point) using death as a competing event). Figure 7 shows that MAF FISH-positive patients had a 42% higher risk of bone as the first metastatic site (>=2.3) (p=0.02, multivariate analysis). As can be seen in Figure 8, MAF FISH-positive patients had a 38% higher risk of IDFS (>=2.2) (p=0.0002, multivariate analysis) (very early separation by 2 years, after which the curves become parallel). Figure 9 shows that MAF FISH-positive patients had a 33% lower overall survival (>=2.2) (p=0.02, multivariate analysis) (early separation by 3 years for overall survival).

[0276] As can be seen in Figure 10, MAF FISH-positive patients in the control arm had a shorter time to bone as first recurrence (>=2.3), a difference that was significant in multivariate analysis (HR=0.47, p=0.013).

[0277] As shown in Figure 11, untreated MAF positive patients tended to have a shorter time to recurrence (>=2.2) compared to untreated MAF non-positive patients (HR=0.72, p=0.08, multivariate analysis). Figure 12 shows time to IDFS (excluding bone recurrence) by FISH in AZURE control patients only. An optimized cutoff of 2.2 was used.

[0278] In summary, the predefined cut-offs for stratifying patients according to MAF FISH levels were very close to the computer-based defined optimized cut-offs. The threshold effect allows clear group delineation for appropriate (relevant) treatment (or avoidance of treatment). Based on the prognosis of MAF FISH-positive patients in the control arm, we found that the time to bone as first metastasis was shorter (HR=0.53, multivariate p=0.03) and the time to recurrence (invasive disease) tended to be shorter (HR=0.72, p=0.08).

[0279] Example 2: Evaluation of zoledronic acid treatment effect by MAF FISH stratification. The control and zoledronic acid treatment arms of the AZURE study, described in Example 1, were evaluated to determine the effect of zoledronic acid treatment on MAF stratified patients.

[0280] Figure 13 (Coleman et al., Lancet Oncol 2014;15:997-1006, Figure 3) shows the time to bone metastasis in patients in the control arm and in the zoledronic acid-treated arm of the Azure trial. Figure 14 shows the evaluation of the time to bone metastasis as first event. As can be seen in Figure 14, MAF FISH-positive (>=2.3) patients in the control arm had a shorter time to bone as first recurrence, a difference that was significant in multivariate analysis (HR=0.47, p=0.013, multivariate analysis). Zoledronic acid treatment reduced the difference in the incidence of bone as the site of first recurrence between MAF-positive and non-positive patients, and MAF-positive patients treated with zoledronic acid had no significant difference in the risk of bone metastasis at any time point compared to MAF-non-positive patients.

[0281] Figure 15 (Coleman et al., Lancet Oncol 2014;15:997-1006, Fig. 2) shows an analysis of disease-free survival (DFS) and invasive disease-free survival (IDFS) between control arm and zoledronic acid-treated patients in the AZURE trial.

[0282] Figure 16 shows the time to distant recurrence between control arm and zoledronic acid-treated patients. Untreated MAF-positive patients tended to have a shorter time to distant recurrence (>=2.2) (HR=0.72, p=0.08, multivariate analysis). MAF-positive patients in the zoledronic acid-treated arm had a significantly shorter time to recurrence (invasive disease) (HR=0.52, p<0.001, multivariate analysis). Treatment with zoledronic acid worsened IDFS compared to untreated MAF-positive patients.

[0283] Figure 17 shows the time to bone metastasis events (any time point) according to treatment. In the time to bone metastasis (any time point), death is used as a competing event. In the control arm, MAF FISH positive patients (>=2.3), the increased risk of bone metastasis was not significant (HR=0.72, p=0.18). In MAF FISH non-positive patients, zoledronic acid treatment significantly reduced the risk of bone metastasis at any time point (<2.3) compared to MAF FISH positive patients (HR=0.52, p=0.01).

[0284] Figure 18 shows the time to bone metastasis event (any time point) according to MAF copy number (according to a pre-specified MAF cut-off of 2.5). In the time to bone metastasis (any time point), death is used as a competing event. In MAF FISH non-positive patients, zoledronic acid treatment significantly reduced the risk of bone metastasis (HR=0.65, p=0.03). In MAF-positive patients, zoledronic acid treatment showed a trend towards an increased risk of bone metastasis. The difference was not significant (HR=1.54, p=0.22).

[0285] FIG. 19 shows IDFS by menopausal status in the AZURE trial when patients are not stratified according to MAF (Coleman et al., Lancet Oncol 2014;15:997-1006, FIG. 5).

[0286] Figure 20 shows the time to bone metastasis events (any time point) according to MAF copy number in postmenopausal patients (data according to a prespecified cutoff of 2.5) (using death as a competing event). Treatment outcomes in MAF-positive postmenopausal patients (>2.5) showed a trend towards a reduced number of bone metastasis events (HR=0.46, p=0.26, limited number of events). Treatment outcomes in MAF-non-positive postmenopausal patients treated with zoledronic acid were less effective than in MAF-positive postmenopausal patients (HR=0.63 vs. HR=0.46, limited number of events), suggesting a clear benefit of zoledronic treatment to prevent bone metastases in MAF-positive postmenopausal patients.

[0287] Figure 21 shows the time to bone metastasis events (any time point) according to MAF copy number in non-postmenopausal patients (data according to a prespecified cutoff of 2.5). MAF-positive non-postmenopausal patients had significantly worse zoledronic acid treatment outcomes, leading to an increase in bone metastasis events (HR=2.44, p=0.045). MAF-non-positive non-postmenopausal patients had a trend toward better zoledronic acid treatment outcomes (HR=0.66, p=0.08).

[0288] Figure 22 shows the IDFS (excluding bone metastases in postmenopausal women) of the zoledronic acid treatment arm and the control arm. As can be seen in Figure 22, treatment of postmenopausal patients with zoledronic acid significantly improved IDFS (excluding bone) in MAF FISH-positive patients (>=2.2), reducing the number of invasive disease events, with no difference in IDFS (excluding bone) in MAF-non-positive patients.

[0289] Figure 23 shows the IDFS (excluding bone metastases in non-postmenopausal women) of the zoledronic acid treatment arm and the control arm. As can be seen in Figure 23, treatment of non-postmenopausal women with zoledronic acid significantly worsened the IDFS (excluding bone) of MAF FISH positive patients (>=2.2), and no difference was observed in the IDFS (excluding bone) of MAF FISH non-positive patients.

[0290] Figure 24 shows overall survival (OS) by treatment arm. Treatment of MAF FISH positive patients with zoledronic acid significantly impacted OS.

[0291] Figure 25 shows the prognosis of disease-free survival (DFS) in the AZURE control arm. As can be seen in Figure 25, disease-free survival is significantly lower in untreated MAF-positive postmenopausal patients. Regarding disease-free survival: (in control patients) Significance of FISH status and menopausal status interaction covariates in multivariate analysis; χ 2 = 6.23, p-value = 0.013. Figure 26 shows the prognosis of overall survival in AZURE control arm patients. There is a trend towards shorter OS in untreated MAF-positive postmenopausal patients. Regarding OS: Significance of FISH status and menopausal status interaction covariates in multivariate analysis (in control patients); χ 2 = 3.62, p-value = 0.057. Figure 27 shows the effect of zoledronic acid treatment on DFS according to MAF FISH value. As can be seen in Figure 27, zoledronic acid treatment resulted in differential DFS outcomes between MAF FISH positive and negative patients, and these differences occur in postmenopausal and non-postmenopausal women.

[0292] FIG. 28 shows the impact of zoledronic acid treatment on DFS according to the MAF FISH value in postmenopausal patients. As can be seen in FIG. 28, in MAF-negative postmenopausal patients, zoledronic acid treatment leads to a better DFS outcome (HR=0.56, (95%CI., 0.33-0.95). FIG. 29 shows the impact of zoledronic acid treatment on DFS in non-postmenopausal women. As can be seen in FIG. 29, in MAF-positive non-postmenopausal patients, zoledronic acid treatment leads to the worst DFS outcome. FIG. 30 shows the impact of zoledronic acid treatment on overall survival according to MAF FISH value. As can be seen in FIG. 30, in MAF-positive patients, zoledronic acid treatment leads to a significantly shorter overall survival. These differences occur in postmenopausal and non-postmenopausal women. FIG. 31 shows the impact of MAF FISH value in postmenopausal patients. Figure 31 shows the effect of zoledronic acid treatment on overall survival according to FISH level. As can be seen in Figure 31, in MAF-negative postmenopausal patients, zoledronic acid treatment shows a trend towards better overall survival outcomes (HR=0.56, (95%CI., 0.31-1.01)), but in zoledronic FISH-positive patients, the effect is greater. Figure 32 shows the effect of zoledronic acid treatment on overall survival in non-postmenopausal women according to MAF FISH value. As can be seen in Figure 32, in MAF-positive non-postmenopausal patients, zoledronic acid treatment leads to the worst overall survival outcomes.

[0293] A summary of the predictive value of gene of interest (GOI) MAF on patient risk for DFS and OS divided according to menopausal status can be found in Table 1 . [Table 1]

[0294] As can be seen, MAF is predictive for postmenopausal, unknown and peri-menopausal patients who are at risk for shorter DFS or worst OS. However, in premenopausal women, MAF-positive patients are at lower risk and more likely to have longer DFS and better OS.

[0295] In summary, MAF FISH-positive patients in the control arm have a significantly increased risk of bone metastases as a first site of recurrence compared to non-positive patients (HR=0.47, p=0.013, cutoff=2.3), and treatment with zoledronic acid reduces this difference. In addition, in MAF FISH-non-positive patients, zoledronic acid treatment significantly reduced the risk of bone metastases at any time point (HR=0.65, p=0.03, cutoff=2.5). In MAF-positive patients, zoledronic acid treatment shows an increased risk of bone metastases at any time point. The difference is not significant (HR=1.54, p=0.22, cutoff=2.5). This effect is driven by menopausal status, with the greatest effect in the non-postmenopausal group. Zoledronic acid significantly improves the outcome of MAF FISH-positive postmenopausal patients. However, zoledronic acid worsens the outcome of MAF FISH-positive non-postmenopausal patients. This effect was dependent on an increase in invasive disease (reduction in IDFS) upon treatment with zoledronic acid, suggesting that in non-postmenopausal patients, prevention of bone metastases may promote metastases elsewhere, ultimately leading to bone metastases as a secondary event.

[0296] MAF FISH-positive patients not treated with zoledronic acid have a higher risk of bone metastasis and invasive disease (reduced IDFS regardless of inclusion and exclusion of bone events). In patients treated with zoledronic acid, MAF-positive patients have a worse outcome compared to untreated patients in terms of bone metastasis, IDFS (regardless of inclusion and exclusion of bone events) and overall survival at any time point. In terms of bone metastasis at any time point, MAF-negative patients treated with zoledronic acid have a better outcome compared to untreated patients. In postmenopausal women, MAF-positive patients treated with zoledronic acid have a better outcome in terms of IDFS (excluding bone). In non-postmenopausal women, MAF-positive patients treated with zoledronic acid have a worse outcome in terms of IDFS (excluding bone).

[0297] All publications, patents, patent applications, internet sites and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site or accession number / database sequence was specifically and individually indicated to be incorporated by reference.

Claims

1. An in vitro method of using c-MAF gene expression level, copy number, amplification, or gain in a sample from a subject having breast cancer as an indicator of whether said subject is amenable to receiving customized therapy, comprising: i) quantifying the c-MAF gene expression level, copy number, amplification, or gain in a sample from said subject; and ii) comparing the expression level, copy number, amplification or gain obtained in i) with a reference value. Including, If the expression level, copy number, amplification, or gain is not increased relative to the baseline value, the subject is permitted to receive zoledronic acid treatment at a dose of less than about 0.5, about 0.1, about 0.05, about 0.01, about 0.005, about 0.001, about 0.0005, about 0.0001, about 0.00005, or about 0.00001 mg / kg of body weight.

2. The method of claim 1, wherein the subject is non-postmenopausal.

3. The method of claim 1, wherein the subject is postmenopausal.

4. An in vitro method of using c-MAF gene expression level, copy number, amplification, or gain in a sample from a subject having breast cancer, and the menopausal status of the subject, as indicators of the subject's sensitivity to zoledronic acid, comprising determining the c-MAF gene expression level, copy number, amplification, or gain in the sample from the subject relative to a reference value, and determining the menopausal status of the subject, wherein the subject is postmenopausal and having an increase in c-MAF gene expression level, copy number, amplification, or gain relative to the reference value indicates that the subject is tolerant to receiving zoledronic acid at a dose of less than about 0.5, about 0.1, about 0.05, about 0.01, about 0.005, about 0.001, about 0.0005, about 0.0001, about 0.00005 or about 0.00001 mg per kg of body weight.

5. A method according to any one of claims 1 to 4, wherein the zoledronic acid is for administration at a dose of less than about 0.05 mg per kg of body weight or less than about 0.1 mg per kg of body weight.

6. The method of claim 1, wherein quantifying the c-MAF gene expression level comprises quantifying messenger RNA (mRNA) of the gene or a fragment of the mRNA, complementary DNA (cDNA) of the gene or a fragment of the cDNA, or quantifying the level of a protein encoded by the gene.

7. A method according to any one of claims 1 to 5, wherein the expression level, copy number, amplification or gain is quantified by quantitative polymerase chain reaction (PCR) or DNA or RNA array, FISH or nucleotide hybridization techniques.

8. The method of claim 6, wherein the level of the protein is quantified by Western blot, ELISA, immunohistochemistry or protein array.

9. A method according to any one of claims 1 to 5, wherein the amplification or gain of the c-MAF gene is determined by using a c-MAF gene specific probe.

10. The method of claim 4, wherein the copy number of c-MAF as measured using FISH is ≧2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.

0.

11. The method of any one of claims 1 to 3, wherein the copy number of c-MAF as measured using FISH is <2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.

0.

12. The method of claim 1, wherein the copy number is determined as an average copy number per cell.

13. The method of any one of claims 1 to 12, wherein the breast cancer is ER+ breast cancer, ER- breast cancer, triple-negative breast cancer, basal-like subtype, or HER2+ breast cancer.

14. A method according to any one of claims 1 to 5, wherein the expression level, copy number, amplification or gain of the c-MAF gene is determined by determining the expression level, copy number, amplification or gain of locus 16q23 or 16q22-q24.

15. A method according to any one of claims 1 to 3, wherein the treatment aims to prevent and / or treat bone remodeling and improve disease-free survival or overall survival.

16. The method of claim 6 or 8, wherein the level of the protein is quantified using an antibody that specifically binds to c-MAF, the antibody comprising a heavy chain CDR1 of SEQ ID NO: 21, a heavy chain CDR2 of SEQ ID NO: 22, and a heavy chain CDR3 of SEQ ID NO: 23; and a light chain CDR1 of SEQ ID NO: 18, a light chain CDR2 of SEQ ID NO: 19, and a light chain CDR3 of SEQ ID NO:

20.

17. The method of any one of claims 1 to 5, 9 and 14, wherein the amplification is determined by in situ hybridization or PCR.

18. The method of claim 17, wherein the in situ hybridization is fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or silver in situ hybridization (SISH).

19. The method of any one of claims 1 to 18, wherein the zoledronic acid is for intravenous administration.

20. The method of any one of claims 1 to 19, wherein the zoledronic acid is for administration once every 30 days.

21. The method of any one of claims 1 to 20, wherein the method further comprises calcium and / or vitamin D for administration.

22. A pharmaceutical composition for use in a method of treating a subject having breast cancer, comprising zoledronic acid, wherein the subject has been identified as having no increased c-MAF gene expression levels, copy number, amplification, or gain in a tumor sample relative to a baseline value, the baseline value being a breast cancer tumor tissue sample from a subject not suffering from metastasis, and the pharmaceutical composition is for administration of zoledronic acid at a dose of less than about 0.5, about 0.1, about 0.05, about 0.01, about 0.005, about 0.001, about 0.0005, about 0.0001, about 0.00005 or about 0.00001 mg per kg of body weight of the subject.

23. A pharmaceutical composition for use in a method of treating a subject having non-metastatic breast cancer comprising zoledronic acid, wherein the subject has been identified as having no increased c-MAF gene expression levels, copy number, amplification, or gain in a tumor sample relative to a baseline value, the baseline value being a breast cancer tumor tissue sample from a subject not suffering from metastasis, and the pharmaceutical composition is for administration of zoledronic acid at a dose of less than about 0.5, about 0.1, about 0.05, about 0.01, about 0.005, about 0.001, about 0.0005, about 0.0001, about 0.00005 or about 0.00001 mg per kg of body weight of the subject.

24. A pharmaceutical composition for use in a method for treating a subject having breast cancer, said pharmaceutical composition comprising zoledronic acid, said method comprising: i) quantifying the c-MAF gene expression level, copy number, amplification, or gain in a sample from said subject; and ii) comparing the expression level, copy number, amplification or gain obtained in i) with a reference value, wherein the reference value is from a breast cancer tumor tissue sample from a subject not suffering from metastasis. Including, If the expression level, copy number, amplification, or gain is not increased relative to the baseline value, the subject is administered the pharmaceutical composition, wherein the pharmaceutical composition is for administration of zoledronic acid at a dose of less than about 0.5, about 0.1, about 0.05, about 0.01, about 0.005, about 0.001, about 0.0005, about 0.0001, about 0.00005 or about 0.00001 mg / kg of body weight of the subject.

25. A pharmaceutical composition for use in a method for treating a subject having non-metastatic breast cancer, said pharmaceutical composition comprising zoledronic acid, said method comprising: i) quantifying the c-MAF gene expression level, copy number, amplification, or gain in a sample from said subject; and ii) comparing the expression level, copy number, amplification or gain obtained in i) with a reference value, wherein the reference value is from a breast cancer tumor tissue sample from a subject not suffering from metastasis. Including, If the expression level, copy number, amplification, or gain is not increased relative to the baseline value, the subject is administered the pharmaceutical composition, wherein the pharmaceutical composition is for administration of zoledronic acid at a dose of less than about 0.5, about 0.1, about 0.05, about 0.01, about 0.005, about 0.001, about 0.0005, about 0.0001, about 0.00005 or about 0.00001 mg / kg of body weight of the subject.

26. A pharmaceutical composition for use in a method for treating bone metastases in a subject having breast cancer, said pharmaceutical composition comprising zoledronic acid, said subject having been identified as having no increased c-MAF gene expression levels, copy number, amplification, or gain in a tumor sample relative to a baseline value, said pharmaceutical composition for administration of zoledronic acid at a dose of less than about 0.5, about 0.1, about 0.05, about 0.01, about 0.005, about 0.001, about 0.0005, about 0.0001, about 0.00005 or about 0.00001 mg per kg of body weight of the subject.

27. ​​The pharmaceutical composition described in any one of claims 22 to 26, wherein the subject is non-postmenopausal.

28. The pharmaceutical composition described in any one of claims 22 to 26, wherein the subject is premenopausal.

29. The pharmaceutical composition described in any one of claims 22 to 26, wherein the subject is postmenopausal.

30. The pharmaceutical composition of any one of claims 22 to 29, wherein the breast cancer is ER+ breast cancer, ER- breast cancer, triple-negative breast cancer, basal-like subtype, or HER2+ breast cancer.

31. The pharmaceutical composition of any one of claims 22 to 30, wherein quantifying the c-MAF gene expression level, amplification, gain or copy number comprises quantifying messenger RNA (mRNA) of the gene or a fragment of the mRNA, complementary DNA (cDNA) of the gene or a fragment of the cDNA, or quantifying the level of a protein encoded by the gene.

32. The pharmaceutical composition of any one of claims 22 to 30, wherein the amplification is determined by in situ hybridization or PCR.

33. The pharmaceutical composition of claim 32, wherein the in situ hybridization is fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or silver in situ hybridization (SISH).

34. The pharmaceutical composition of claim 31, wherein the level of the protein is quantified using an antibody that specifically binds to c-MAF, the antibody comprising a heavy chain CDR1 of SEQ ID NO: 21, a heavy chain CDR2 of SEQ ID NO: 22, a heavy chain CDR3 of SEQ ID NO: 23, a light chain CDR1 of SEQ ID NO: 18, a light chain CDR2 of SEQ ID NO: 19 and a light chain CDR3 of SEQ ID NO:

20.

35. The pharmaceutical composition of any one of claims 22 to 34, wherein the pharmaceutical composition comprises less than about 0.05 mg of zoledronic acid per kg of the subject's body weight.

36. The pharmaceutical composition of any one of claims 22 to 35, wherein the zoledronic acid is formulated for intravenous administration.

37. The pharmaceutical composition of any one of claims 22 to 36, wherein the zoledronic acid is formulated for administration once every 30 days.

38. The pharmaceutical composition described in any one of claims 22 to 37, further comprising calcium and / or vitamin D.

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