Therapeutic treatment of breast cancer based on the status of c-MAF

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] Reference to Sequence Listing The content of the electronically submitted sequence listing submitted with this application ("3190_015PC02_SeqListing.txt", 58,739 bytes, created on May 18, 2017) is incorporated herein by reference in its entirety.

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

Background Art

[0003] Breast cancer is the second most common type of cancer in the world (10.4%; second to lung cancer) and the fifth most common cause of cancer death (after lung cancer, gastric cancer, liver cancer and colon cancer). Among women, breast cancer is the most common cause of cancer death. 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, and this phenomenon is partly due to the modern lifestyle in the Western world.

[0004] Breast cancer is staging 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 staging results, and staging is also used to assign patients to treatment in both clinical trials and medical practice. The information for staging is as follows:

[0005] TX: Primary tumor cannot be evaluated. T0: No evidence of tumor. Tis: Carcinoma in situ without invasiveness. T1: Tumor is 2 cm or less. T2: Tumor is greater than 2 cm but less than 5 cm. T3: Tumor is greater 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 applies:

[0007] The cancer has spread to 10 or more axillary lymph nodes, or to the subclavian lymph nodes, or to the supraclavian lymph nodes, or to the axillary lymph nodes and has spread to the internal mammary lymph nodes, or to 4 or more axillary lymph nodes with a minimal amount of cancer present in the internal mammary lymph nodes or sentinel lymph node biopsy.

[0008] MX: The presence of distant diffusion (metastasis) cannot be assessed. M0: No distant diffusion. M1: Diffusion to distant organs, excluding the supraclavicular lymph nodes, has occurred.

[0009] The fact that most patients with solid tumor cancer die after metastasis means that understanding the molecular and cellular mechanisms that enable tumor metastasis is crucial. Recent publications have demonstrated how metastasis is triggered by complex mechanisms that are still largely unknown, and how different metastatic cell types exhibit specific organ-specific targeting. These tissue-specific metastatic cells possess a set of acquired functions that enable them to colonize specific organs.

[0010] All cells have receptors on their surface, in their cytoplasm, and in their nucleus. Certain chemical messengers, such as hormones, bind to these receptors, causing 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 if the receptor is present, and a minus sign 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 is an important assessment for all breast cancers as it determines the appropriateness of using certain treatments, such as tamoxifen or trastuzumab.

[0011] Unregulated gene expression array profiling has provided biological evidence regarding heterogeneity in breast cancer by identifying endogenous subtypes, such as luminal A, luminal B, HER2+ / ER-, and basal-like subtypes.

[0012] Triple-negative breast cancer is defined as a tumor that does not express genes related to the estrogen receptor (ER), progesterone receptor (PR), and HER2. This subgroup accounts for 15% of all types of breast cancer and is more prevalent in premenopausal African women and African American women. Triple-negative breast cancer has a very different recurrence pattern than estrogen receptor-positive breast cancer: the risk of recurrence is considerably higher in the first 3-5 years, but then drops sharply, becoming 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 carcinoma expresses genes normally found in “basal” / myoepithelial cells of 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 the lack of an internationally recognized definition of basal-like breast cancer, it is not surprising that there is considerable confusion regarding whether triple-negative breast cancer and basal-like breast cancer are synonymous. While 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 exhibit a 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) directly addressed this issue and confirmed that not all triple-negative tumors are classified as basal-like carcinomas when analyzed by gene expression profiling (i.e., only 71% had a basal-like phenotype), and not all basal-like breast carcinomas classified by expression arrays exhibit a triple-negative phenotype (i.e., 77%).

[0015] The key considerations regarding the treatment of breast cancer are, when the tumor is localized, surgery with possible adjuvant hormone therapy (with tamoxifen or aromatase inhibitors), chemotherapy, and / or radiotherapy. Currently, the proposed postoperative treatment (adjuvant therapy) follows a pattern. This pattern is modified as a world conference is held every two years in St. Gallen, Switzerland, to discuss the actual results of global multicenter studies. Similarly, the aforementioned 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 lymph node metastasis would be candidates for systemic adjuvant 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 specific genes. In February 2007, the MammaPrint assay became the first breast cancer indicator to receive public approval from the Food and Drug Administration.

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

[0018] U.S. Patent Application Publication 2011 / 0150979 describes a method for predicting the prognosis of basal-like breast cancer, comprising detecting the level of FOXC1.

[0019] U.S. Patent Application Publication 2010 / 0210738 relates to a method for predicting the prognosis of cancer in a subject with triple-negative breast cancer, comprising detecting the expression levels of a set of genes that are randomly upregulated or downregulated in a sample. U.S. Patent Application Publication No. 2011 / 0130296 relates to the identification of marker genes useful in the diagnosis and prognosis of triple-negative breast cancer. It is necessary to identify a subset of breast cancer patients who would benefit from a particular treatment, and conversely, a subset of breast cancer patients who would not benefit from or might be harmed by such a 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 Document]

[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 Problems]

[0022] In one embodiment, the present invention is an in vitro method for designing a customized treatment for a subject having breast cancer, comprising: 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, wherein when the expression level, copy number, amplification or gain is not increased relative to the reference value, the subject is allowed to receive a treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival. The present invention relates to an in vitro method.

[0023] In some embodiments, the subjects are postmenopausal. In other embodiments, the subjects are postmenopausal.

[0024] In one embodiment, the present invention provides an in vitro method for designing a customized treatment for a postmenopausal subject with breast cancer, comprising: 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. The present invention relates to an in vitro method in which, if the expression level, copy number, amplification, or gain is elevated above the baseline, the subject is not permitted to receive treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival.

[0025] In one embodiment, the present invention is an in vitro method for designing a customized treatment for a postmenopausal subject with breast cancer, i) To quantify the c-MAF gene expression level, copy number, amplification, or gain in the sample of the subject, and ii) Compare the expression level, copy number, amplification, or gain obtained in i) with a reference value. Includes, The present invention relates to an in vitro method in which, if the expression level, copy number, amplification, or gain is increased compared to the baseline, the subject is permitted to receive treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival.

[0026] In some embodiments, subjects are given treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival. In other embodiments, subjects are not given treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival.

[0027] In certain embodiments, a treatment aimed at preventing and / or treating bone remodeling or improving disease-free survival or overall survival is an agent intended to prevent or inhibit osteolysis and 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 analogs, strontium ranerate, 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, quantification of c-MAF gene expression levels includes quantifying the messenger RNA (mRNA) or fragments of the mRNA of the gene, the complementary DNA (cDNA) or fragments of the cDNA of the gene, or the level of the protein encoded by the gene. In certain embodiments, expression levels, copy numbers, amplification, or gain are quantified by quantitative polymerase chain reaction (PCR), DNA or RNA arrays, or nucleotide hybridization techniques. In embodiments, protein levels are quantified by Western blotting, ELISA, immunohistochemistry, or protein arrays. In certain embodiments, protein levels are quantified using antibodies containing 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. In some embodiments, amplification or gain of the c-MAF gene is determined by using a c-MAF gene-specific probe. In certain embodiments, the c-MAF gene-specific probe is a Vysis LSI / IGH MAF bichromatic double 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 tumor tissue sample of breast cancer derived from a subject without metastasis.

[0030] In one embodiment, the present invention relates to a method for treating bone metastases in a subject having breast cancer in which the c-MAF expression level in a metastatic tumor sample is not elevated compared to a control sample, comprising administering an agent that can prevent or inhibit bone remodeling or improve disease-free survival or overall survival, wherein the agent that can avoid or prevent bone remodeling or improve disease-free survival or overall survival is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranerate, 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 subjects are postmenopausal. In other embodiments, the subjects are postmenopausal.

[0032] In one embodiment, the present invention relates to a method for treating bone metastases in a postmenopausal subject with breast cancer in which the c-MAF expression level in a metastatic tumor sample is elevated compared to a control sample, comprising administering an agent that can prevent or inhibit bone remodeling or improve disease-free survival or overall survival, wherein the agent that can avoid or prevent bone remodeling is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranerate, 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 another embodiment, 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 with 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 postmenopausal.

[0035] In certain embodiments, different treatments are administered to subjects based on their c-MAF expression level and / or whether they are postmenopausal or non-menopausal.

[0036] In some embodiments, quantification of c-MAF expression levels includes quantifying the messenger RNA (mRNA) or fragments of the mRNA of the gene, the complementary DNA (cDNA) or fragments of the cDNA of the gene, or the level of the protein encoded by the gene. In certain embodiments, the level of the protein is quantified using an antibody containing 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. In certain embodiments, amplification is determined by in-situ hybridization or PCR. In further embodiments, in-situ hybridization is fluorescence in-situ hybridization (FISH), chromogenic in-situ hybridization (CISH), or silver in-situ hybridization (SISH). In a further embodiment, in-situ hybridization is fluorescence in-situ hybridization (FISH).

[0037] In some embodiments, the number of c-MAF copies 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 a particular embodiment, the number of c-MAF copies measured using FISH is ≥2.2. In a further embodiment, the number of c-MAF copies measured using FISH is ≥2.3. In yet another embodiment, the number of c-MAF copies measured using FISH is ≥2.4. In a particular embodiment, the number of c-MAF copies measured using FISH is ≥2.5. In other embodiments, the number of c-MAF copies 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 the subject; and ii) comparing the expression level, copy number, amplification, or gain l obtained in step i) with a reference value, wherein an increase in the expression level, copy number, amplification, or gain of the gene relative to the reference value indicates 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 the c-MAF gene expression level, copy number, amplification, or gain in a sample of the subject compared to a baseline, wherein an increase in the c-MAF gene expression level, copy number, amplification, or gain relative to the baseline indicates 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 the c-MAF gene expression level, copy number, amplification, or gain in a sample of the subject compared to a baseline, wherein an increase in the c-MAF gene expression level, copy number, amplification, or gain compared to the baseline indicates poor IDFS (excluding bone recurrence).

[0041] In some embodiments, an agent that can prevent or inhibit bone remodeling is an agent that can prevent or inhibit osteolysis.

[0042] In some embodiments, quantification of c-MAF expression levels includes quantifying the messenger RNA (mRNA) or fragments of the mRNA of the gene, the complementary DNA (cDNA) or fragments of the cDNA of the gene, or the level of the protein encoded by the gene. In certain embodiments, the protein level is quantified using an antibody containing 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. In other embodiments, amplification is determined by in-situ hybridization or PCR. In further embodiments, in-situ hybridization is fluorescence in-situ hybridization (FISH), chromogenic in-situ hybridization (CISH), or silver in-situ hybridization (SISH). In a further embodiment, in-situ hybridization is fluorescence in-situ hybridization (FISH).

[0043] In some embodiments, the copy number of c-MAF 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 a particular embodiment, the copy number of c-MAF measured using FISH is ≥2.2. In another embodiment, the copy number of c-MAF measured using FISH is ≥2.3. In a further embodiment, the copy number of c-MAF measured using FISH is ≥2.4. In yet another embodiment, the copy number of c-MAF 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, breast cancer is ER+ breast cancer. In certain embodiments, breast cancer is ER- breast cancer. In other embodiments, breast cancer is triple-negative breast cancer. In different embodiments, breast cancer is a basal-like subtype. In some embodiments, 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 loci 16q23 or 16q22-q24.

[0046] In some embodiments, the treatment is an mTOR inhibitor or a CDK4 / 6 inhibitor. In other embodiments, the treatment is hormone 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 a metastatic tumor sample is increased compared 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 a metastatic tumor sample is increased compared to a control sample, comprising administering hormone therapy extended beyond standard treatment. 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 a metastatic tumor sample is not increased compared 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 has not increased compared to a control sample, and which includes not administering hormone therapy extended beyond standard treatment.

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

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

[0050] In some embodiments, the patient's menopausal status is also used to predict the patient's survival status. In some embodiments, the subjects are postmenopausal. In certain embodiments, the subjects are premenopausal. In certain embodiments, the subjects are postmenopausal. In certain embodiments, for example, the following items are provided: (Item 1) An in vitro method for designing customized treatment for subjects with breast cancer, i) To quantify the c-MAF gene expression level, copy number, amplification, or gain in the sample of the subject, and ii) Compare the expression level, copy number, amplification, or gain obtained in i) with a reference value. Includes, An in vitro method in which, if the expression level, copy number, amplification, or gain is not increased relative to the reference value, the subject is permitted to receive treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival. (Item 2) The method according to item 1, wherein the subject is postmenopausal. (Item 3) The method according to item 1, wherein the subject is postmenopausal. (Item 4) An in vitro method for designing customized treatment for postmenopausal subjects with breast cancer, i) To quantify the c-MAF gene expression level, copy number, amplification, or gain in the sample of the subject, and ii) Compare the expression level, copy number, amplification, or gain obtained in i) with a reference value. Includes, An in vitro method in which, if the expression level, copy number, amplification, or gain is increased relative to the reference value, the subject is not permitted to receive treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival. (Item 5) An in vitro method for designing customized treatment for postmenopausal subjects with breast cancer, i) To quantify the c-MAF gene expression level, copy number, amplification, or gain in the sample of the subject, and ii) Compare the expression level, copy number, amplification, or gain obtained in i) with a reference value. Includes, An in vitro method in which, if the expression level, copy number, amplification, or gain is increased relative to the reference value, the subject is permitted to receive 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 1-3 or 5, wherein the subject is subjected to treatment aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival. (Item 7) The method according to item 4, wherein the subject is not subjected to any treatment aimed at preventing and / or treating bone remodeling or improving disease-free survival or overall survival. (Item 8) The method according to any one of items 1 to 7, wherein the 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, and is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranerate, 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) The method according to item 8, wherein the RANKL inhibitor is selected from the group consisting of a RANKL-specific antibody, a RANKL-specific nanobody, and osteoprotegerin. (Item 10) The method according to item 9, wherein the RANKL-specific antibody is denosumab. (Item 11) The method according to item 8, wherein the bisphosphonate is zoledronic acid. (Item 12) The method according to item 9, wherein the RANKL-specific nanobody is ALX-0141. (Item 13) The method according to item 8, wherein the dual MET and VEGFR2 inhibitor is Cabozantinib. (Item 14) The method according to any one of items 1 to 13, wherein the quantification of the c-MAF gene expression level includes quantifying the messenger RNA (mRNA) or a fragment of the mRNA of the gene, the complementary DNA (cDNA) or a fragment of the cDNA of the gene, or the level of the protein encoded by the gene. (Item 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), DNA or RNA array, FISH, or nucleotide hybridization technique. (Item 16) The method according to any one of items 1 to 14, wherein the level of the protein is quantified by Western blotting, ELISA, immunohistochemistry, or protein array. (Item 17) The method according to any one of items 1 to 14, wherein the level of the protein is quantified using 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. (Item 18) The method according to any one of items 1 to 14, wherein the amplification or gain of the c-MAF gene is determined by using a c-MAF gene-specific probe. (Item 19) The method according to item 18, wherein the c-MAF gene-specific probe is a Vysis LSI / IGH MAF bichromatic double fusion probe. (Item 20) The method according to any one of items 1 to 19, wherein the aforementioned reference value is from a tumor tissue sample of breast cancer derived from a subject that has not suffered metastasis. (Item 21) 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) A method for treating bone metastases in subjects with breast cancer in which the c-MAF expression level in a metastatic tumor sample is increased or not increased compared to a control sample, comprising administering an agent that can prevent or inhibit bone remodeling or improve disease-free survival or overall survival, wherein the agent that can avoid or prevent osteolysis or improve disease-free survival or overall survival is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranerate, 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) The method according to item 22, wherein the subject is postmenopausal. (Item 24) The method according to item 22, wherein the subject is postmenopausal. (Item 25) A method for treating bone metastases in postmenopausal subjects with breast cancer in which the c-MAF expression level in metastatic tumor samples is elevated compared to a control sample, comprising administering an agent that can prevent or inhibit bone remodeling or improve disease-free survival or overall survival, wherein the agent that can avoid or prevent bone remodeling or improve disease-free survival or overall survival is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranerate, 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) 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) The method according to item 26, wherein the RANKL-specific antibody is denosumab. (Item 28) The method according to any one of items 22 to 25, wherein the bisphosphonate is zoledronic acid. (Item 29) The use described in item 26, wherein the RANKL-specific nanobody is ALX-9141. (Item 30) The use described in any one of items 22-25, wherein the dual MET and VEGFR2 inhibitor is Cabozantinib. (Item 31) A method for classifying subjects with 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 to 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 postmenopausal. (Item 32) The method according to item 31, wherein different treatments are administered to the subject based on the c-MAF expression level and / or whether the subject is postmenopausal or non-menopausal. (Item 33) The method according to any one of items 22 to 32, wherein the quantification of the c-MAF expression level includes quantifying the messenger RNA (mRNA) or a fragment of the mRNA of the gene, the complementary DNA (cDNA) or a fragment of the cDNA of the gene, or the level of the protein encoded by the gene. (Item 34) The method according to items 22-33, wherein the level of the protein is quantified using 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. (Item 35) The method according to items 1 to 34, wherein the amplification is determined by in situ hybridization or PCR. (Item 36) The method according to item 35, wherein the in-situe hybridization is fluorescent in-situe hybridization (FISH), chromogenic in-situe hybridization (CISH), or silver in-situe hybridization (SISH). (Item 37) The method according to item 36, wherein the in-situ hybridization is fluorescence in-situ hybridization (FISH). (Item 38) The method according to items 1 to 37, wherein the copy number of the c-MAF 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) The method according to items 1 to 38, wherein the number of copies of the c-MAF measured using FISH is ≥ 2.2. (Item 40) The method according to items 1 to 39, wherein the number of copies of the c-MAF measured using FISH is ≥ 2.3. (Item 41) The method according to items 1 to 40, wherein the number of copies of the c-MAF measured using FISH is ≥ 2.4. (Item 42) The method according to items 1 to 41, wherein the number of copies of the c-MAF measured using FISH is ≥ 2.5. (Item 43) The method according to items 1 to 37, wherein the copy number of the c-MAF 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) The method according to any one of items 1 to 37, for determining the copy number as the average copy number per cell. (Item 45) An in vitro method for predicting IDFS in patients with breast cancer, i) To quantify the expression level, copy number, amplification, or gain of the c-MAF gene in the sample of the subject, and ii) Comparing the expression level, copy number, amplification, or gain l obtained in step i) with a reference value. Includes, An in vitro method in which an increase in the expression level, copy number, amplification, or gain of the gene relative to the aforementioned reference value indicates poor IDFS. (Item 46) An in vitro method for predicting IDFS in a patient with breast cancer, comprising determining the c-MAF gene expression level, copy number, amplification, or gain in a sample of the subject compared to a reference, wherein an increase in the c-MAF gene expression level, copy number, amplification, or gain compared to the reference indicates poor IDFS. (Item 47) An in vitro method for predicting IDFS excluding bone recurrence in patients with breast cancer, comprising determining the c-MAF gene expression level, copy number, amplification, or gain in a sample of the subject compared to a standard, wherein an increase in the c-MAF gene expression level, copy number, amplification, or gain compared to the standard 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 includes quantifying the messenger RNA (mRNA) or a fragment of the mRNA of the gene, the complementary DNA (cDNA) or a fragment of the cDNA of the gene, or the level of the protein encoded by the gene. (Item 49) The method according to any one of items 45 to 48, wherein the level of the protein is quantified using 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. (Item 50) The method according to any one of items 45 to 48, wherein the amplification is determined by in situ hybridization or PCR. (Item 51) The method according to item 50, wherein the in-situe hybridization is fluorescence in-situe hybridization (FISH), colorimetric in-situe hybridization (CISH), or silver in-situe hybridization (SISH). (Item 52) The method according to item 51, wherein the in-situ hybridization is fluorescence in-situ hybridization (FISH). (Item 53) The method according to items 45-52, wherein the copy number of the c-MAF 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) The method according to items 45-53, wherein the number of copies of the c-MAF measured using FISH is ≥ 2.2. (Item 55) The method according to items 45-54, wherein the number of copies of the c-MAF measured using FISH is ≥ 2.3. (Item 56) The method according to items 45-55, wherein the number of copies of the c-MAF measured using FISH is ≥ 2.4. (Item 57) The method according to items 45-56, wherein the number of copies of the c-MAF measured using FISH is ≥ 2.5. (Item 58) The method according to any one of items 45 to 57, for determining the copy number as the average copy number per cell. (Item 59) The method described in any one of items 1 to 58, wherein the breast cancer is ER+ breast cancer. (Item 60) The method according to any one of items 1 to 58, wherein the breast cancer is ER-breast cancer. (Item 61) The method according to any one of items 1 to 58, wherein the breast cancer is triple-negative breast cancer. (Item 62) The method according to any one of items 1 to 58, wherein the breast cancer is of the basal-like subtype. (Item 63) The method according to any one of items 1 to 58, wherein the breast cancer is HER2-positive breast cancer. (Item 64) 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 osteolysis. (Item 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 the gene locus 16q23 or 16q22-q24. (Item 66) The method according to 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) The method according to any one of items 1 to 30, wherein the treatment that improves disease-free survival or overall survival is hormone therapy extended beyond standard treatment. (Item 68) A method for treating a subject with breast cancer in which the c-MAF expression level, copy number, amplification, or gain in a metastatic tumor sample is increased compared to a control sample, comprising administering an mTOR inhibitor or a CDK4 / 6 inhibitor. (Item 69) A method for treating a subject with breast cancer in which the c-MAF expression level, copy number, amplification, or gain in a metastatic tumor sample is increased compared to a control sample, comprising administering hormone therapy extended beyond standard treatment. (Item 70) A method for treating a subject with breast cancer in which the c-MAF expression level, copy number, amplification, or gain in a metastatic tumor sample is not increased compared to a control sample, the method comprising not administering an mTOR inhibitor or a CDK4 / 6 inhibitor. (Item 71) A method for treating a subject with breast cancer in which the c-MAF expression level, copy number, amplification, or gain in a metastatic tumor sample has not increased compared to a control sample, the method comprising not administering hormone therapy extended beyond standard treatment. (Item 72) A method for predicting disease-free survival in a patient, comprising measuring the c-MAF gene expression level, copy number, amplification, or gain relative to a criterion, and using the c-MAF gene expression level, copy number, amplification, or gain to predict the patient's overall survival. (Item 73) The method according to item 72, wherein an increase in the c-MAF gene expression level, copy number, amplification, or gain relative to a baseline predicts shorter disease-free survival than in patients in whom the c-MAF gene expression level, copy number, amplification, or gain has not increased relative to a baseline. (Item 74) A method for predicting the overall survival of a patient, comprising measuring the c-MAF gene expression level, copy number, amplification, or gain relative to a criterion, and predicting the patient's overall survival using the c-MAF gene expression level, copy number, amplification, or gain. (Item 75) The method according to item 74, wherein an increase in the c-MAF gene expression level, copy number, amplification, or gain relative to the baseline predicts shorter overall survival than in patients in whom the c-MAF gene expression level, copy number, amplification, or gain has not increased relative to the baseline. (Item 76) The method according to any one of items 68 to 73, wherein the subject is postmenopausal. (Item 77) The method according to any one of items 68 to 73, wherein the subject is premenopausal. (Item 78) The method according to any one of items 68 to 73, wherein the subject is postmenopausal. [Brief explanation of the drawing]

[0051] [Figure 1] Figure 1 shows an overview of the assay parameters.

[0052] [Figure 2] Figure 2 shows the AZURE research design.

[0053] [Figure 3] Figure 3 shows the H&E analysis of AZURE samples. Evaluable and unevaluable samples are shown.

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

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

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

[0057] [Figure 7] Figure 7 shows the time to bone recurrence based on MAF FISH values ​​using the bone optimization cutoff from section 2.3.

[0058] [Figure 8] Figure 8 shows the %IDFS obtained by FISH. The optimal cutoff from section 2.2 was used.

[0059] [Figure 9] Figure 9 shows overall survival by FISH. The optimal cutoff of 2.2 was used.

[0060] [Figure 10] Figure 10 shows the 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 shows the IDFS by FISH in AZURE control patients only. The optimized cutoff from section 2.2 was used.

[0062] [Figure 12] Figure 12 shows the time to IDFS (excluding bone recurrence) by FISH in AZURE control patients only. The optimized cutoff from section 2.2 was used.

[0063] [Figure 13]Figures 13A and 13B show the time to bone metastasis in patients in the control arm and the zoledronic acid-treated arm. (A) Cumulative incidence of bone metastasis as the first event, and (B) Cumulative incidence of bone metastasis at any point in time during follow-up. Analysis was based on treatment intent. HR-hazard ratio.

[0064] [Figure 14] Figure 14 shows the evaluation of the time to bone metastasis as the first event in AZURE control patients and zoledronic acid-treated patients. A bone optimization cutoff of 2.3 was used.

[0065] [Figure 15] Figures 15A and 15B show disease-free survival (DFS) and disease-free survival (IDFS) between the control arm and zoledronic acid-treated patients. (A) Kaplan-Meier curves for disease-free survival and (B) disease-free survival. The analysis was based on treatment intent. HR = Hazard Ratio.

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

[0067] [Figure 17] Figure 17 shows the time to bone metastasis event (at any given time) depending on the treatment. Death is used as a competing event in the time to bone metastasis (at any given time).

[0068] [Figure 18] Figure 18 shows the time to a bone metastasis event (at any given time point) depending on the MAF copy number (corresponding to a pre-specified MAF cutoff in 2.5).

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

[0070] [Figure 20] Figure 20 shows the time to bone metastasis event (at any given time) in postmenopausal patients, corresponding to the MAF copy number (data based on the pre-specified cutoff in 2.5).

[0071] [Figure 21] Figure 21 shows the time to bone metastasis events (at any given time) in non-menopausal patients, corresponding to the MAF copy number (data based on the pre-specified cutoff in 2.5).

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

[0073] [Figure 23] Figure 23 shows IDFS (excluding bone metastases in non-menopausal women) for the zoledronic acid-treated arm and the control arm.

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

[0075] [Figure 25] Figure 25 shows the prognostic values ​​of MAF FISH for disease-free survival (DFS) in the Azure control arm.

[0076] [Figure 26] Figure 26 shows the MAF FISH prognostic values ​​for overall survival (OS) in the Azure control arm.

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

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

[0079] [Figure 29] Figure 29 shows the MAF FISH predictive values ​​for the effect of zoledronic acid treatment on disease-free survival (DFS) outcomes in non-menopausal patients.

[0080] [Figure 30] Figure 30 shows the MAF FISH predictions regarding the effect of zoledronic acid treatment on overall survival (OS) outcomes.

[0081] [Figure 31] Figure 31 shows the MAF FISH predictive values ​​for the effect of zoledronic acid treatment on overall survival (OS) outcomes in postmenopausal patients.

[0082] [Figure 32] Figure 32 shows the MAF FISH predictive values ​​for the effect of zoledronic acid treatment on overall survival (OS) outcomes in non-menopausal patients. [Modes for carrying out the invention]

[0083] Detailed description of the invention Definitions of common terms and expressions When used herein, “and / or” should be considered as specific disclosures of each of the two designating features or components, with or without the other. For example, “A and / or B” should be considered as specific disclosures of (i) A, (ii) B, and (iii) A and B, as if each were shown separately herein.

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

[0085] As used herein, terms such as “basal-like,” “basal-like subtype,” and “basal-like subtype breast cancer” refer, as used herein, 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. Accordingly, all sentences in this application that cite and refer to triple-negative breast cancer (ER, HER-2, PgR) may also cite and 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 cancer characterized by a gene expression profile based on the 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) centrosome protein 55kDa (CEP55); (5) aniline, 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); (10) forkhead box Al (FOXA1). The gene expression profiles used to classify breast cancer tumors as basal-like subtypes do not include estrogen receptors, progesterone receptors, or Her2; therefore, 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 ER and PR expression is measured by the method disclosed in M. Elizabeth H et al., Journal of Clinical Oncology, 28(16):2784-2795, 2010), where tumor cells are not amplified by epidermal growth factor receptor type 2 (HER2 or ErbB2), a receptor normally located on the cell surface. Tumor cells are considered negative for ER and PR expression if, using standard immunohistochemical techniques, fewer than 5% of tumor cell nuclei are stained for ER and PR expression. When used herein, tumor cells are considered negative for HER2 overexpression if they yield a test result score of 0, 1+, or 2+ when tested with HercepTest® Kit (Code K5204, Dako North America, Inc., Carpinteria, CA), a semi-quantitative immunohistochemical assay using a polyclonal anti-HER2 primary antibody, or if they are negative for HER2 FISH.

[0087] As used herein, “ER+ breast cancer” is understood to mean breast cancer in which the tumor cells express the estrogen receptor (ER). This makes the tumor sensitive to estrogen, meaning that estrogen promotes the growth of the cancerous breast tumor. In contrast, “ER- breast cancer” is understood to mean breast cancer in which the tumor cells do not express the 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 having detectable expression of epidermal growth factor receptor type 2 (HER2 or ErbB2) and / or amplification of the HER2 gene, a receptor typically located on the cell surface. As used herein, tumor cells are considered negative for HER2 overexpression if they yield a test result score of 0, 1+, or 2+ when tested with HercepTest® Kit (Code K5204, Dako North America, Inc., Carpinteria, CA) (a semi-quantitative immunohistochemical assay using a polyclonal anti-HER2 primary antibody), or if they are HER2 FISH negative.

[0089] In the context of this invention, a “postmenopausal” subject is understood to be a woman who has gone through menopause and has experienced 60 consecutive months without menstruation. See Coleman et al., Lancet Oncol 2014;15:997-1006. In certain embodiments, a woman may confirm her postmenopausal status through the measurement of follicle-stimulating hormone (FSH).

[0090] In the context of this invention, a "non-menopausal" subject is any subject who has not gone through menopause and has experienced 60 consecutive months without menstruation. A "non-menopausal" subject includes women who are pre-menopausal, perimenopausal, or of unknown menopausal status.

[0091] In the context of this invention, “metastasis” is understood as the propagation of cancer from one organ to another. It generally occurs via the blood or lymphatic system. When cancer cells spread and form a new tumor, the latter is referred to as a secondary or metastatic tumor. The cancer cells that form a secondary tumor are similar to those of the original tumor. For example, when breast cancer spreads (metastasizes) to the bone, the secondary tumor is formed from malignant breast cancer cells. The disease in the bone is metastatic breast cancer, not bone cancer. In certain embodiments of the method of this invention, metastasis is breast cancer that has spread (metastasized) to the bone.

[0092] In the context of this invention, “recurrence” refers to the return of breast cancer after a period in which cancer was not detected. Breast cancer may recur locally in the breast or surrounding tissues. Breast cancer may also recur in lymph nodes near or outside the surrounding area. When breast cancer recurs by spreading to other tissues or by traveling through the bloodstream to recur in bone or other organs, this is also referred to as metastasis. As used herein, recurrence also encompasses the risk of recurrence.

[0093] In the context of this invention, “relapse” refers to a situation in which cancer recurs when symptoms have subsided but the subject no longer has cancer. Breast cancer can relapse locally in the breast or surrounding tissues. Breast cancer can also relapse in lymph nodes near or outside the surrounding area. When breast cancer relapses by spreading to other tissues or by traveling through the bloodstream to recur 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 completion of primary treatment for cancer in which a patient is alive without any signs or symptoms of that 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, the length of time a patient diagnosed with the disease is still alive.

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

[0097] When the terms “poor” or “good” are used herein to refer to clinical outcomes, they mean that a subject exhibits a favorable or unfavorable outcome. As will be understood by those skilled in the art, such an assessment of probabilities is preferably, but not necessarily, accurate for 100% of the subjects being diagnosed. However, the term requires that a statistically significant portion of the subjects can be identified as having a predisposition to a given outcome. Those skilled in the art can easily determine whether a portion is statistically significant using various well-known statistical assessment tools, such as determining confidence intervals, p-values, Student's t-tests, and Mann-Whitney tests. For further details, see 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%, and 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 adequately identified by the method of the present invention.

[0098] In this invention, “tumor sample” is understood to mean a sample derived from a primary breast cancer tumor (e.g., tumor tissue, circulating tumor cells, circulating tumor DNA). The sample may be obtained by conventional methods, such as biopsy, using methods well known to those skilled in the art of the relevant medical technology. Methods for obtaining a biopsy sample include dividing the tumor into larger pieces, or microdissection, or other cell separation methods known in the art. In addition, tumor cells may be obtained by cytology by aspiration using a small-gauge needle. To facilitate the storage and handling of the sample, the sample may be fixed in formalin and immersed in paraffin, or it may be frozen first and then immersed in a tissue freezing medium such as an OCT compound by immersion in a cryogenic medium that allows for rapid freezing.

[0099] In the context of the present invention, "functionally equivalent variants of the c-MAF protein" is understood to mean (i) a variant of the c-MAF protein (SEQ ID NO: 4 or SEQ ID NO: 5) in which one or more amino acid residues are substituted by conserved or non-conserved amino acid residues (preferably conserved amino acid residues) (such substituted amino acid residues may or may not be encoded by the genetic code), or (ii) a variant comprising one or more amino acid insertions or deletions that 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 reporter genes under the control of a promoter containing a cyclin D2 promoter or a c-MAF response region (MARE or c-MAF response element) in cells expressing c-MAF, as described in international publication 2008098351 (which is incorporated herein by reference in its entirety); or methods based on the ability of so-called inhibitors to block the expression of reporter genes under the control of an IL-4 promoter in response to PMA / ionomycin stimulation 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 sequence similarity of 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% to the amino acid sequence of either of the c-MAF protein isoforms (SEQ ID NO: 4 or SEQ ID NO: 5). The degree of similarity between the variant and the previously defined specific c-MAF protein sequence is determined using algorithms and computer processes widely known to those skilled in the art. The similarity between the 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 avoiding or preventing bone remodeling” refers to any molecule that can prevent, inhibit, treat, mitigate, or halt osteolysis by stimulating osteoblast proliferation, inhibiting osteoclast proliferation, or fixing bone structures. Examples of agents for avoiding or preventing bone remodeling include agents for avoiding or preventing osteolysis and agents for avoiding or preventing 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 c-MAF gene expression products (by interfering with c-MAF gene transcription and / or blocking the 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 so-called inhibitors to block the ability of c-MAF to promote in vitro cell proliferation, as shown in, for example, International Patent Application WO2005 / 046731 (the entire contents of which are incorporated herein by reference); methods based on the ability of so-called inhibitors to block the transcriptional ability of reporter genes under the control of a promoter containing a cyclin D2 promoter or a c-MAF response region (MARE or c-MAF response element) in cells expressing c-MAF, as described in, for example, International Publication No. 2008098351 (the entire contents of which are incorporated herein by reference); or methods based on the ability of so-called inhibitors to block the expression of reporter genes under the control of an IL-4 promoter in response to PMA / ionomycin stimulation in cells expressing NFATc2 and c-MAF, as described in, for example, US2009048117 (the entire contents of which are incorporated herein by reference).

[0103] As used herein, the mammalian target of rapamycin (mTOR) or "mTor" refers to the protein corresponding to EC2.7.11.1. 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 completely or partially inhibit mTor gene expression by preventing the production of mTor gene expression products (by interfering with mTor gene transcription and / or blocking the translation of mRNA resulting from mTor gene expression), and by directly inhibiting mTor protein activity. This includes inhibitors with dual or more targets, particularly those with mTor protein activity.

[0105] As used herein, "Src" refers to the protein corresponding to EC2.7.10.2. 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 Src gene expression products (by interfering with Src gene transcription and / or blocking the 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 the protein corresponding to EC1.14.99.1. 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 completely or partially inhibit COX-2 gene expression by preventing the production of COX-2 gene expression products (by interfering with COX-2 gene transcription and / or blocking the 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 the disease and / or progression of the disease, which may be characterized, for example, by relapse, time to relapse, exacerbation, metastasis, time to metastasis, number of metastases, number of metastatic sites, and / or death from the disease. For example, a good clinical outcome may be cure, prevention of relapse, prevention of metastasis, and / or survival within a certain period (no relapse), while a poor clinical outcome may be disease progression, metastasis, and / or death within a certain period.

[0110] As used herein, “Invasive Disease Survival” or “IDFS” means, in cancer, the length of time after the completion of primary treatment for the cancer in which the patient is alive 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 due to any cause, including ipsilateral invasive breast tumor recurrence, local or locally invasive breast cancer recurrence, metastasis or distant recurrence, breast cancer, contralateral invasive breast cancer, and a second primary invasive cancer (non-breast cancer, except 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 subjects with breast cancer" is understood to mean identifying the disease (metastasis) by studying its signs, that is, in the context of the present invention, by increasing the level of c-MAF gene expression in breast cancer tumor tissue compared to a control sample (i.e., overexpression).

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

[0113] In the context of this invention, if the breast cancer in a subject has metastasized to other organs of the body, in certain embodiments to the bones, then "the subject has a positive diagnosis of metastasis." This term is used similarly for recurrence and relapse.

[0114] Those skilled in the art will understand that predictions of the tendency for primary tumors to metastasize, relapse, or recur are not intended to be accurate for all subjects to be identified (i.e., 100% of the subjects). Nevertheless, this term requires the identification of a statistically significant portion of the 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, p-values, Student's t-tests, and Mann-Whitney tests. 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%. P-values ​​are 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 adequately identified by the method of the present invention.

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

[0116] When used herein, “reference value” refers to a test value used as a baseline for values / data obtained by clinical testing of a patient or a sample collected from a patient. A reference value or reference level may be an absolute value; a relative value; a value with an upper and / or lower limit; a value within a range; an average value; a median, mean, or a value compared to a specific control or baseline value. A reference value may be based on individual sample values, for example, values ​​obtained from a sample derived from the subject being tested, but based on values ​​obtained at an earlier point in time. A reference value may be based, for example, on a large number of samples from a population of subjects in a chronologically age-matched group, or on a pool of samples that include or exclude the sample to be tested.

[0117] When used herein, the term “treatment” refers to any type of therapy aimed at ending, preventing, improving, or reducing susceptibility to a clinical condition as described herein. In embodiments, the term “treatment” refers to a preventive treatment (i.e., therapy to reduce susceptibility to a clinical condition) of a disorder or condition as defined herein. Thus, “treatment,” “to treat,” and their equivalent terms refer to obtaining a desired pharmacological or physiological effect encompassing any treatment of a pathological condition or disorder in mammals, including humans. The effect may be preventive in the sense of complete or partial prevention of the disorder or its symptoms, and / or therapeutic in the sense of partial or complete cure of the disorder and / or adverse effects resulting therefrom. In other words, “treatment” includes (1) preventing the onset or recurrence of a disorder in the subject, (2) inhibiting the disorder, e.g., stopping its onset, (3) stopping or terminating the disorder or at least its associated symptoms, e.g., causing regression of the disorder or its symptoms, by restoring or repairing a loss, deficiency or defect of function, or by stimulating an inefficient process, so that the host no longer suffers the disorder or its symptoms, or (4) reducing, alleviating or improving the disorder or its associated symptoms (improvement is used in a broad sense to mean a reduction in the magnitude of at least a parameter, e.g., inflammation, pain or immunodeficiency).

[0118] As used herein, “sample” or “biological sample” means 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 means, as used herein, a measurable amount of gene product produced by a gene in a sample of a subject, and the gene product may be a transcript or a translation product. Thus, the expression level may relate to nucleic acid gene products, such as mRNA or cDNA, or polypeptide gene products. The expression level may originate from a sample of a subject and / or a reference sample and may be detected, for example, de novo, or correspond to a previous determination. The expression level may be determined or measured, for example, using microarrays, PCR (e.g., qPCR), and / or antibody-based methods, as are known to those skilled in the art.

[0120] "Increased expression levels" are understood to refer to expression levels that exceed those in reference or control samples of the c-MAF gene. These increased levels may be caused by amplification, copy gain, or translocation of the gene or the 16q23 or 16q22-24 chromosomal locus, although other mechanisms are not ruled out. In particular, if the expression levels in a sample isolated from a patient are at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more compared to reference or control, the sample may be considered to have high c-MAF expression levels. In embodiments, "increased expression levels" is a "high" expression level. An expression level that is "not increased" or "non-increased" is any value that is not included in the definition of an "increased" expression level (including values ​​equal to the baseline or control level, or expression levels that are decreased compared to the baseline or control level).

[0121] "Reduced expression level" is understood to refer to an expression level that is lower than that in a reference or control sample of the c-MAF gene. This reduced level may be caused by a deletion of the gene or the 16q23 or 16q22-24 chromosomal locus, although this does not rule out other mechanisms. In particular, a sample may be considered to have a reduced c-MAF expression level if the expression level in a sample isolated from a patient is at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or less than that compared to reference or control. In embodiments, "reduced expression level" is a "low" expression level.

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

[0123] In this invention, “increased gene copy number” is understood to mean that the c-MAF gene copy number exceeds the copy number present in the reference or control sample. These increased gene copy numbers may be caused by amplification, copy gain, or translocation of the gene or the 16q23 or 16q22-24 chromosomal locus, although this does not rule out other mechanisms. In particular, if the c-MAF gene has a copy number greater 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, the sample may be considered to have an increased c-MAF copy number. In embodiments, the “increased gene copy number” is determined based on the average copy number per counted cell. In one embodiment, if the average number of copies per counted cell 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 of the c-MAF gene, or even more than 10 copies, the sample may be considered to have an increased c-MAF copy number.

[0124] In this invention, "reduced gene copy number" is understood to mean a case where the c-MAF gene copy number is less than the copy number present in the reference or control sample. These reduced gene copy numbers may be caused by deletions of the gene or the 16q23 or 16q22-24 chromosomal locus, although this does not rule out other mechanisms. In particular, if the c-MAF gene has fewer than two copies, the sample may be considered to have a reduced c-MAF copy number.

[0125] In this invention, "unincreased gene copy number" is understood to mean that the c-MAF gene copy number or mean c-MAF gene copy number is less than the copy number present in the reference sample or the increased-positive sample. An unincreased gene copy number can be caused by amplification of the gene or the 16q23 or 16q22-24 chromosomal locus, copy gain, or non-increase of a translocation, although this does not rule out other mechanisms. In particular, if the c-MAF gene has a copy number less than 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, or 2.9 copies, the sample may be considered to have an unincreased c-MAF copy number or mean c-MAF copy number.

[0126] As understood herein, the term “gene amplification” refers to the process by which multiple copies of a gene or gene fragment are formed in an individual cell or cell line. These gene copies are not necessarily located on the same chromosome. The duplicated regions are often referred to as “amplicons.” Typically, the amount of mRNA produced, i.e., the gene expression level, also increases proportionally to the number of copies of a particular gene.

[0127] The term "gain" refers to any increase in chromosome copy number from the standard (i.e., in diploid organisms, three copies of a gene in a cell is the gain). In some embodiments, "gain" includes the term "copy gain" and is used synonymously with "copy number."

[0128] When used herein, “probe” refers to an oligonucleotide sequence complementary to a specific 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 a stable coordination bond of platinum to the nucleic acid and protein. In some embodiments, the probe is described in U.S. Patent No. 9,127,302 and U.S. Patent No. 9,134,237 (which are incorporated by reference in their entirety) or in Swennenhuis et al., “Construction of repeat-free fluorescence in situ This is described in "hybridization probes" in Nucleic Acids Research 40(3):e20(2012).

[0129] When used herein, “tag” or “label” means any physical molecule that directly or indirectly associates with a probe, enabling the visualization, marking, or other means of capturing the probe or the probe’s location.

[0130] As used herein, "translocation" refers to the exchange of unequal or equal amounts of chromosomal material between chromosomes. In some cases, translocations occur on the same chromosome. In other cases, translocations occur 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 immunoglobulin heavy chain (IgH) loci that are thought to constitute the initiation event in many cancers (Eychene, A., Rocques, N., and Puoponnot, C., A new MAFia in cancer. 2008. Nature Reviews: Cancer. 8: 683-693.).

[0131] When used herein, "ploidy" or "ploidy" refers to a cell containing more than two copies of the gene of interest. In some cases, the gene of interest is a multicellular affinity factor (MAF). In some embodiments, ploidy is associated with the accumulation of expression of the gene of interest. In some embodiments, ploidy is associated with genomic instability. In some embodiments, genomic instability can lead to chromosomal translocations.

[0132] When used herein, "whole genome sequencing" refers to the process of sequencing the entire genome of an organism in a single step. See, for example, Ng., PC and Kirkness, EF, Whole Genome Sequencing. 2010. Methods in Molecular Biology. 628:215-226.

[0133] When used herein, "exome sequencing" refers to the process of sequencing the entire coding region of an organism's DNA. Exome sequencing sequences mRNA. Exome sequencing does not include the untranslated regions of the genome. 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. Members of a binding pair may be of natural origin or may be produced entirely or partially by synthesis. One member of a pair of molecules binds to the other member of the pair and therefore has a surface region or cavity that is complementary to the particular 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, as well as three light chain CDRs designated LCDR1, LCDR2, and LCDR3. The term CDR(s) is used herein to refer to one, some, or all of these regions containing most of the amino acid residues involved in binding, depending on the antibody’s affinity for the antigen or epitope it recognizes. Of the six CDR sequences, the third heavy chain CDR (HCDR3) exhibits the greatest variability (i.e., greater diversity), essentially attributable to a mechanism known in the art as a 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 two amino acids, as long as 26 amino acids, or have any length between these two extremes. The length of the CDR can also vary according to lengths that can be adapted by a particular underlying framework.Functionally, HCDR3 can 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 occurring or partially or entirely synthesized. The term also encompasses any polypeptide or protein containing 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 present in their natural environment, but can be isolated or obtained from natural sources by purification, or by genetic engineering or chemical synthesis), and therefore they may contain amino acids that are not present in nature. Examples of antibody fragments containing an antibody antigen-binding site include, but are not limited to, molecules such as Fab, Fab', F(ab')2, Fab'-SH, scFv, Fv, dAb, and Fd. For example, various other antibody molecules containing one or more antibody antigen-binding sites, including Fab2, Fab3, diabody, triabody, tetrabody, camelbody, nanobody, and minibody, have been engineered. 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 interpreted to include any binding member or substance having an antigen-binding site of an antibody that possesses the necessary specificity and / or binding ability to an antigen. Therefore, this term includes functional antibody fragments and derivatives comprising any polypeptide containing an antigen-binding site of an antibody, whether natural or entirely or partially synthetic. Thus, it includes chimeric molecules or equivalents containing an antigen-binding site of an antibody, fused to another polypeptide (e.g., derived from another species or belonging to another antibody class or subclass). 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.) (both of which are incorporated herein by reference in their entirety).

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

[0139] "Tumor tissue sample" is understood to be a tissue sample derived from a breast cancer tumor (including, but not limited to, circulating tumor cells and circulating tumor DNA). The sample may be obtained by conventional methods, such as biopsy, using methods well known to those skilled in the art of the relevant medical technology.

[0140] "Osteolytic bone metastasis" refers to a type of metastasis in which bone resorption (progressive loss of bone density) occurs near 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 resulting from nerve compression.

[0141] Method for designing customized treatment of the present invention in patients with breast tumors The present invention aims to identify subjects with breast cancer who would benefit from treatment with specific drugs and / or therapies. In some embodiments, the present invention aims to identify subjects with breast cancer who would not benefit from treatment with specific drugs and / or therapies. In some embodiments, the subjects have high levels of c-MAF expression, copy number, amplification, gain, and / or translocation. In certain embodiments, the subjects have low levels of c-MAF expression, copy number, amplification, gain, and / or translocation. 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 even further embodiments, the cancer is HER2+ breast cancer. In some embodiments, the cancer is basal-like breast cancer. In one embodiment, the subjects are postmenopausal. In some embodiments, the subjects are non-menopausal. 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 may be used to diagnose metastasis, relapse or recurrence, or to predict the tendency of a tumor to undergo metastasis, relapse or recurrence. Therefore, given 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 enables a determination of the most appropriate treatment for a subject suffering from said cancer. In embodiments, the present invention includes quantifying only the c-MAF gene expression level as a single marker (i.e., the method does not involve determining the expression levels of any further markers).

[0142] Accordingly, in one embodiment, the present invention relates to an in vitro method for designing a customized treatment for a subject having breast cancer, comprising: a) quantifying the c-MAF gene expression level, copy number, amplification or gain in a tumor sample of the subject; and b) comparing the obtained expression level, copy number, amplification or gain with the expression level, copy number, amplification or gain of the gene in a control sample, and determining the treatment based on the c-MAF gene expression level, copy number, amplification or gain in the subject. In some embodiments, the subject has a high c-MAF gene expression level. In other embodiments, the subject has a low c-MAF gene expression level. In certain embodiments, the subject is administered an agent that avoids and / or prevents bone remodeling (including an agent that avoids or prevents osteolysis). In embodiments, the subject is administered an agent that treats cancer. In further embodiments, the subject is administered a c-MAF inhibitor. In certain embodiments, agents or c-MAF inhibitors that avoid and / or prevent bone remodeling are any agents disclosed in U.S. Patent Applications Publication No. 2014 / 0057796, No. 2015 / 0293100, and U.S. Patent Application No. 15 / 027,946 (these are incorporated herein by reference in their entirety).

[0143] In one embodiment, the present invention relates to an in vitro method for designing a customized treatment for a subject with breast cancer, comprising: 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, wherein if the expression level, copy number, amplification, or gain is not increased relative to the reference value, the subject is permitted to receive treatment aimed at preventing and / or treating bone remodeling or improving disease-free survival or overall survival. In some embodiments, the subject is postmenopausal. In other embodiments, the subject is postmenopausal. In embodiments, the subject is administered a drug aimed at preventing and / or treating bone remodeling. In embodiments, the subject is administered a drug aimed at improving disease-free survival or overall survival. In further embodiments, the subject is administered a c-MAF inhibitor.

[0144] In another embodiment, the present invention relates to an in vitro method for designing a customized treatment for a postmenopausal subject having breast cancer, comprising: 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) to a reference value, wherein if the expression level, copy number, amplification, or gain is increased relative to the reference value, the subject is not permitted to receive treatment aimed at preventing and / or treating bone remodeling and / or improving disease-free survival or overall survival. In some embodiments, the subject is not administered any agent aimed at preventing and / or treating bone remodeling and / or improving disease-free survival 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 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) to a reference value, 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 / or improving disease-free survival or overall survival. In some embodiments, the subject is administered a drug aimed at preventing and / or treating bone remodeling and / or a treatment aimed at improving disease-free survival or overall survival.

[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 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) to a reference value, wherein if the expression level, copy number, amplification, or gain is not increased relative to the reference value, the subject is not permitted to receive treatment aimed at preventing and / or treating bone remodeling and / or improving disease-free survival or overall survival. In some embodiments, the subject is not administered any agent aimed at preventing and / or treating bone remodeling and / or improving disease-free survival or overall survival.

[0147] In one embodiment, the present invention relates to a method for treating bone metastases in a subject having breast cancer in which c-MAF levels in a metastatic tumor sample are reduced compared to a control sample, comprising administering an agent that can prevent or inhibit bone remodeling and / or improve disease-free survival or overall survival, wherein the agent that can avoid or prevent bone remodeling or improve disease-free survival or overall survival is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranerate, 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 postmenopausal. In other embodiments, the subjects are postmenopausal.

[0148] In another embodiment, the present invention relates to a method for treating bone metastases in a postmenopausal subject with breast cancer in which c-MAF levels in a metastatic tumor sample are elevated compared to a control sample, comprising administering an agent capable of preventing or inhibiting bone remodeling and / or improving disease-free survival or overall survival, wherein the agent capable of avoiding or preventing bone remodeling and / or improving disease-free survival or overall survival is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH, PTHLH inhibitors (including neutralizing antibodies and peptides), PRG analogs, strontium ranerate, 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, a drug that avoids and / or prevents bone remodeling (including a drug that avoids or prevents osteolysis) is administered to the subject.

[0150] By measuring the c-MAF gene expression level, copy number, amplification, or gain in a sample and comparing it to a control sample, the expression level, copy number, amplification, or gain of the gene, combined with the subject's menopausal status, indicates whether the subject is tolerable to receive treatment aimed at preventing (if the subject has not yet experienced metastasis) and / or treating (if the subject has already experienced metastasis), as well as treatment or medication intended to avoid or prevent bone remodeling.

[0151] In some embodiments, a copy number of MAF per cell or mean 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 when measured using FISH is considered a high value. In one embodiment, the MAF FISH value is ≥ 2.2. In a specific 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 when measured using FISH is < 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, or 2.9 copies of the c-MAF gene.

[0152] In certain embodiments, the subject has metastases or is prognosed to experience metastases. In some embodiments, the metastases are bone metastases. In further embodiments, the bone metastases are osteolytic metastases.

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

[0154] In some embodiments, the sample is a primary tumor tissue sample from the subject. In a second step, the obtained c-MAF gene expression level, copy number, amplification, or gain in the subject's tumor sample is compared to the expression level, copy number, amplification, or gain of the gene in a control sample. The determination of the c-MAF gene expression level, copy number, amplification, or gain must be related to the values ​​in the control or reference sample. Depending on the type of tumor to be analyzed, the exact nature of the control sample may vary. Therefore, in some embodiments, the reference sample is a tumor tissue sample from a subject with breast cancer that has not metastasized, relapsed, or recurred, or a tumor tissue sample from a subject with breast cancer that corresponds to the median c-MAF gene expression level, copy number, amplification, or gain measured in a tumor tissue collection from a biopsy sample of a subject with breast cancer that has not metastasized, relapsed, or recurred.

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

[0156] The determination of c-MAF gene expression levels, copy number, amplification, or gain must be correlated with values ​​from a control or reference sample. Depending on the type of tumor to be analyzed, the exact nature of the control sample may vary. Therefore, when evaluating a diagnosis, the reference sample should be a tumor tissue sample from a subject with non-metastatic breast cancer, or a tumor tissue sample from a subject with breast cancer corresponding to the median c-MAF gene expression level measured in a tumor tissue collection from a biopsy sample from a subject with non-metastatic breast cancer.

[0157] The reference sample is typically obtained by combining equal volumes of samples from the subject population. Generally, a typical reference sample would be obtained from subjects that are clinically well-documented and whose absence of metastasis is well-characterized. In such samples, the normal concentration (reference concentration) of the biomarker (c-MAF gene) can be determined, for example, by providing the mean concentration of the reference population. When determining the reference concentration of the marker, various considerations are taken into account. Such considerations include the patient's age, weight, sex, and overall physical condition. For example, according to these considerations, a reference group of subjects ranging from 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, and at least about 500 to over 1000, classified according to various age categories, for example, would be used. The sample collection from which the reference level is derived would preferably be formed from subjects suffering from the same type of cancer as the patient population in the study (e.g., breast cancer). Similarly, reference values ​​within a patient cohort can be established by using receiver operating curves (ROCs) and measuring the area under the curve for all 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. For an explanation, see Stuart G. Baker, “The Central Role of Receiver Operating Characteristic (ROC) curves in This can be found 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 level of this marker expressed in tumor tissue derived from patients with this median can be compared and assigned, for example, to an "increased" expression level. Due to intersubjective variability (e.g., aspects relating to age, race, etc.), establishing an absolute reference value for c-MAF expression is extremely difficult (though not virtually impossible). Therefore, in certain embodiments, the reference value for "increased" or "decreased" c-MAF expression is determined by calculating percentiles by conventional means, which involve performing assays on one or more samples isolated from subjects whose disease has been well documented for c-MAF expression levels by one of the methods described above. Preferably, the “reduced” c-MAF level can then be assigned to samples whose c-MAF expression level is equal to or below the 50th percentile of the normal population (for example, including expression levels equal to or below the 60th percentile of the normal population, expression levels equal to or below the 70th percentile of the normal population, expression levels equal to or below the 80th percentile of the normal population, expression levels equal to or below the 90th percentile of the normal population, and expression levels equal to or below the 95th percentile of the normal population). Preferably, the “increased” c-MAF gene expression level can then be assigned to samples where the c-MAF gene expression level is equal to or above the 50th percentile of the normal population (including, for example, expression levels equal to or above the 60th percentile of the normal population, expression levels equal to or above the 70th percentile of the normal population, expression levels equal to or above the 80th percentile of the normal population, expression levels equal to or above the 90th percentile of the normal population, and expression levels equal to or above the 95th percentile of the normal population).

[0159] In certain embodiments, 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 the gene. Preferably, the chromosomal region in which the amplification or gain indicates the presence of amplification or gain of the c-MAF gene is locus 16q22-q24 containing the c-MAF gene. Locus 16q22-q24 is located on chromosome 16, on the long arm of the 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 to the gene.

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

[0161] In embodiments, if the c-MAF gene copy number is greater than that of the reference or control sample, the c-MAF gene is amplified relative to the reference gene copy number. For example, if the genomic copy number or mean genomic copy number of the c-MAF gene in the test sample is increased by at least approximately 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 compared to the control sample, the c-MAF gene is said to be "amplified." In another example, the 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 approximately 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 measuring copy numbers, the control sample refers to a tumor sample from a subject with breast cancer who has not developed metastases, or a tumor sample from a subject with breast cancer that corresponds to the median c-MAF gene copy number measured in a tumor tissue collection from a biopsy sample of a subject with breast cancer who has not developed metastases. The reference sample is typically obtained by combining equal volumes of samples from the subject population. If the c-MAF gene copy number is increased compared to the copy number of the gene in the control sample, the subject has a positive diagnosis of metastasis or a stronger tendency to develop metastases. In another embodiment, the reference gene copy number is the gene copy number in a breast cancer sample from a subject without bone metastases.

[0163] In another embodiment, 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 containing the c-MAF gene is amplified in breast cancer cells and associated with the presence of metastasis, relapse, or recurrence, the amplification or gain of chr16q22-24 containing the c-MAF gene allows for a determination of the most appropriate treatment for a subject suffering from said cancer.

[0165] Determining c-MAF gene amplification requires correlation with a control or reference sample corresponding to the level of c-MAF gene amplification measured in tumor tissue samples from subjects with breast cancer who have not metastasized, or with a control or reference sample corresponding to the median level of c-MAF gene amplification measured in tumor tissue collections from biopsy samples of subjects with breast cancer who have not metastasized. The reference sample is typically obtained by combining equal volumes of samples from the subject population.

[0166] Generally, a typical reference sample would be obtained from subjects that are clinically well-documented and whose absence of metastasis is well-characterized. The sample collection from which the reference level is derived would preferably consist of subjects with the same type of cancer as the patient subjects in the study. Once this median is established, the level of c-MAF amplification in the patient's tumor tissue can be compared to this median; if amplification is present, the subject has a positive diagnosis of metastasis or a stronger tendency to develop metastasis.

[0167] In another embodiment, the present invention relates to an in vitro method for designing a customized treatment for a patient with breast cancer, comprising determining whether the c-MAF gene is translocated in a sample of the subject.

[0168] In some embodiments, the translocation gene originates from a region of the 16q23 locus. In some embodiments, the translocation gene originates from any portion of the chromosomal region of chromosome 16, from 79,392,959 bp to 79,663,806 bp (centromere to telomere). In some embodiments, the translocation gene originates 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 that region.

[0169] In certain embodiments, the translocation of the c-MAF gene can be determined by determining the translocation of the chromosomal region containing the 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 the 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 translocates to locus 14q32 on chromosome 14, resulting in the translocation t(14,16)(q32,q23). This translocation places the MAF gene next to a potent enhancer at the IgH locus, which in some cases leads to MAF overexpression (Eychene, A., Rocques, N., and Puoponnot, C., A new MAFia in cancer. 2008). Nature Reviews:Cancer.8:683-693.).

[0170] In one embodiment, the translocation of the c-MAF gene can be determined by using a probe specific to the translocation.

[0171] One embodiment of the present invention includes a method for determining whether the c-MAF gene is translocated in a sample of a subject, in a first step. In this embodiment, the sample is a tumor tissue sample.

[0172] In a particular embodiment, the present invention's method for prognosis of the tendency to develop bone metastases in subjects with breast cancer comprises determining the c-MAF gene copy number in a sample of the subject in which the c-MAF gene is translocated, and comparing the copy number to the copy number of a control or reference sample, wherein if the c-MAF copy number is higher than that of the control sample, the subject has a stronger tendency to develop bone metastases.

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

[0174] How to predict survival, including IDFS, using c-MAF This invention relates to predicting IDFS in subjects with breast cancer. In certain embodiments, the subject has a high expression level, copy number, amplification, or gain of c-MAF. In other embodiments, the subject has a 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 further embodiments, the cancer is HER2+ breast cancer. In some embodiments, the subject is postmenopausal. In other embodiments, the subject is non-menopausal.

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

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

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

[0178] In some embodiments, a copy number of MAF per cell or mean 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 when measured using FISH is considered 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 even further embodiments, the MAF FISH value is ≥ 2.5.

[0179] In some embodiments, the subject's c-MAF status 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 includes amplification, copy gain, or translocation or absence thereof of the 16q23 or 16q22-24 chromosomal locus, or deletion thereof. In certain embodiments, subjects with increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline have shorter disease-free survival than subjects without increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline. In embodiments, disease-free survival of subjects having increased c-MAF gene expression levels, copy numbers, amplification, or gain relative to a baseline is shorter by 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 than disease-free survival of subjects without increased c-MAF gene expression levels, copy numbers, amplification, or gain relative to a baseline. In certain embodiments, subjects having increased c-MAF gene expression levels, copy numbers, amplification, or gain relative to a baseline have shorter overall survival than subjects without increased c-MAF gene expression levels, copy numbers, amplification, or gain relative to a baseline. In embodiments, overall survival of subjects with increased c-MAF gene expression levels, copy numbers, 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 than disease-free survival of subjects without increased c-MAF gene expression levels, copy numbers, amplification, or gain relative to the baseline. In embodiments, subjects are postmenopausal. In other embodiments, subjects are non-postmenopausal. In some embodiments, subjects are premenopausal.

[0180] In embodiments, disease-free survival of subjects without an increase in c-MAF gene expression level, copy number, amplification, or gain relative to the baseline is longer than that of subjects with 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 osteolysis (i.e., zoledronic acid). In embodiments, disease-free survival of subjects without an increase in c-MAF gene expression level, copy number, amplification, or gain relative to a 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 longer after treatment with zoledronic acid than disease-free survival of subjects with an increase in c-MAF gene expression level, copy number, amplification, or gain relative to a baseline. In embodiments, the overall survival of subjects without an increase in c-MAF gene expression level, copy number, amplification, or gain relative to the baseline is longer than that of subjects with 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 osteolysis (i.e., zoledronic acid). In embodiments, the overall survival of subjects without 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 longer after treatment with zoledronic acid than the overall survival of subjects with an increase in c-MAF gene expression level, copy number, amplification, or gain relative to the baseline. In embodiments, the subjects are postmenopausal. In other embodiments, the subjects are non-postmenopausal. In some embodiments, the subjects are premenopausal.

[0181] In embodiments, disease-free survival of subjects with increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline is shorter than that of subjects without increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline, after treatment with bone-modifying agents and / or agents that avoid or prevent osteolysis (i.e., zoledronic acid). In embodiments, disease-free survival of subjects having an increased c-MAF gene expression level, copy number, amplification, or gain relative to a baseline is shorter by 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 osteolysis (i.e., zoledronic acid) than disease-free survival of subjects without an increased c-MAF gene expression level, copy number, amplification, or gain relative to a baseline. In embodiments, the overall survival of subjects with increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline is shorter than that of subjects without increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline, after treatment with bone-modifying agents and / or agents that avoid or prevent osteolysis (i.e., zoledronic acid).In embodiments, the overall survival of subjects with increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline is shorter by 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 osteolysis (i.e., zoledronic acid) than the overall survival of subjects without increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline. In embodiments, the subjects are postmenopausal. In other embodiments, the subjects are non-menopausal. In some embodiments, the subjects are premenopausal.

[0182] In embodiments, disease-free survival in non-menopausal subjects with increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline is shorter than disease-free survival in subjects without increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline, after treatment with bone-modifying agents and / or agents that avoid or prevent osteolysis (i.e., zoledronic acid). In embodiments, disease-free survival of non-menopausal subjects having increased c-MAF gene expression levels, copy number, amplification, or gain relative to a baseline is shorter by 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 osteolysis (i.e., zoledronic acid) than disease-free survival of subjects without increased c-MAF gene expression levels, copy number, amplification, or gain relative to a baseline.

[0183] In embodiments, overall survival of subjects with increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline is shorter than disease-free survival of subjects without increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline, after treatment with bone-modifying agents and / or agents that avoid or prevent osteolysis (i.e., zoledronic acid). In embodiments, the overall survival of subjects with increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline is shorter by 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 a bone-modifying agent and / or an agent that avoids or prevents osteolysis (i.e., zoledronic acid) than the overall survival of subjects without increased c-MAF gene expression levels, copy number, amplification, or gain relative to the baseline.

[0184] In some embodiments, the predictive power of MAF regarding a subject's OS or DFS is based on the subject's menopausal status. In some embodiments, MAF is predictive in postmenopausal, unknown, and perimenopause subjects who are at risk of shorter DFS or the 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 embodiments, the subject's MAF status predicts the treatment the subject should receive. In embodiments, the c-MAF status in any of the embodiments herein includes amplification, copy gain, or translocation or absence thereof of the 16q23 or 16q22-24 chromosomal locus, or deletion thereof. In embodiments, postmenopausal patients having increased c-MAF gene expression levels, copy number, amplification, or gain relative to a baseline (and therefore at high risk of poor DFS or OS outcomes) may be subjected to any of the treatments disclosed herein. In some embodiments, postmenopausal patients having increased c-MAF gene expression levels, copy number, amplification, or gain relative to a baseline (and therefore at high risk of poor DFS or OS outcomes) may be treated by extending their hormonal treatment beyond the five years prescribed by the use of hormonal treatment as standard treatment. In certain embodiments, the hormonal treatment is tamoxifen and / or an aromatase inhibitor. Patients who do not have an increase in c-MAF gene expression level, copy number, amplification, or gain relative to the criteria should not be subjected to the treatments disclosed herein.

[0186] In certain embodiments, the subject has metastases or is prognosed to experience metastases. In some embodiments, the metastases are bone metastases. In further embodiments, the bone metastases are osteolytic metastases.

[0187] In some embodiments, the sample is a primary tumor tissue sample from the subject. In a second step, the obtained c-MAF gene expression level, copy number, amplification, or gain in the subject's tumor sample is compared to the expression level, copy number, amplification, or gain of the gene in a control sample. The determination of the c-MAF gene expression level, copy number, amplification, or gain must be related to the values ​​in the control or reference sample. Depending on the type of tumor to be analyzed, the exact nature of the control sample may vary. Therefore, in some embodiments, the reference sample is a tumor tissue sample from a subject with breast cancer that has not metastasized, relapsed, or recurred, or a tumor tissue sample from a subject with breast cancer that corresponds to the median c-MAF gene expression level, copy number, amplification, or gain measured in a tumor tissue collection from a biopsy sample of a subject with breast cancer that has not metastasized, relapsed, or recurred.

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

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

[0190] The determination of c-MAF gene expression levels, copy number, amplification, or gain must be correlated to values ​​in a control or reference sample. The exact nature of the control sample may vary depending on the type of tumor being analyzed. Therefore, when evaluating a diagnosis, the reference sample should be a tumor tissue sample from a subject with non-metastatic breast cancer, or a tumor tissue sample from a subject with breast cancer corresponding to the median c-MAF gene expression level measured in a tumor tissue collection from a biopsy sample from a subject with non-metastatic breast cancer.

[0191] The reference sample is typically obtained by combining equal volumes of samples from the subject population. Generally, a typical reference sample would be obtained from subjects that are clinically well-documented and whose absence of metastasis is well-characterized. In such samples, the normal concentration (reference concentration) of the biomarker (c-MAF gene) can be determined, for example, by providing the mean concentration of the reference population. When determining the reference concentration of the marker, various considerations are taken into account. Such considerations include the patient's age, weight, sex, and overall physical condition. For example, according to these considerations, a reference group of subjects ranging from 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, and at least about 500 to over 1000, classified according to various age categories, for example, would be used. The sample collection from which the reference level is derived would preferably be formed from subjects suffering from the same type of cancer as the patient population in the study (e.g., breast cancer). Similarly, reference values ​​within a patient cohort can be established by using receiver operating curves (ROCs) and measuring the area under the curve for all 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. For an explanation, see Stuart G. Baker, “The Central Role of Receiver Operating Characteristic (ROC) curves in This can be found 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 level of this marker expressed in tumor tissue derived from patients with this median can be compared and assigned, for example, to an "increased" expression level. Due to intersubjective variability (e.g., aspects relating to age, race, etc.), establishing an absolute reference value for c-MAF expression is extremely difficult (though not virtually impossible). Therefore, in certain embodiments, the reference value for "increased" or "decreased" c-MAF expression is determined by calculating percentiles by conventional means, which involve performing assays on one or more samples isolated from subjects whose disease has been well documented for c-MAF expression levels by one of the methods described above. Preferably, the “reduced” c-MAF level can then be assigned to samples whose c-MAF expression level is equal to or below the 50th percentile of the normal population (for example, including expression levels equal to or below the 60th percentile of the normal population, expression levels equal to or below the 70th percentile of the normal population, expression levels equal to or below the 80th percentile of the normal population, expression levels equal to or below the 90th percentile of the normal population, and expression levels equal to or below the 95th percentile of the normal population). Preferably, the “increased” c-MAF gene expression level can then be assigned to samples where the c-MAF gene expression level is equal to or above the 50th percentile of the normal population (including, for example, expression levels equal to or above the 60th percentile of the normal population, expression levels equal to or above the 70th percentile of the normal population, expression levels equal to or above the 80th percentile of the normal population, expression levels equal to or above the 90th percentile of the normal population, and expression levels equal to or above the 95th percentile of the normal population).

[0193] In certain embodiments, 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 the gene. Preferably, the chromosomal region in which the amplification or gain indicates the presence of amplification or gain of the c-MAF gene is locus 16q22-q24 containing the c-MAF gene. Locus 16q22-q24 is located on chromosome 16, on the long arm of the 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 embodiments, the degree of amplification or gain of the c-MAF gene can be determined using a probe specific to the gene.

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

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

[0196] In an embodiment, if the c-MAF gene copy number is greater than the copy number present in the reference or control sample, the c-MAF gene is amplified relative to the reference gene copy number. For example, if the genomic copy number or mean genomic copy number of the c-MAF gene in the test sample is increased by at least approximately 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, or 50-fold compared to the control sample, the c-MAF gene is said to be "amplified." In another example, the 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 approximately 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 derived from a subject without bone metastases.

[0198] In another embodiment, 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 containing the c-MAF gene is amplified in breast cancer cells and associated with the presence of metastasis, relapse, or recurrence, the amplification or gain of chr16q22-24 containing the c-MAF gene allows for a determination of the most appropriate treatment for a subject suffering from said cancer.

[0200] Determination of c-MAF gene amplification involves correlating the level of c-MAF gene amplification measured in a tumor tissue sample from a subject with breast cancer who has not suffered from metastasis, with the value of a control sample or reference sample corresponding thereto, or correlating with the value of a control sample or reference sample corresponding to the median of the c-MAF gene amplification measured in a tumor tissue collection in a biopsy sample from a subject with breast cancer who has not suffered from metastasis. The reference sample is typically obtained by pooling equal amounts of samples from a subject population.

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

[0202] In another aspect, the present invention relates to determining whether the c-MAF gene is translocated in a sample of the subject.

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

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

[0205] In embodiments, the translocation of the c-MAF gene can be determined by using a probe specific to said translocation.

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

[0207] In certain embodiments, the method of the present invention for prognosis of the tendency to develop bone metastasis in a subject having breast cancer comprises determining the c-MAF gene copy number in a sample of 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 stronger tendency to develop bone metastasis.

[0208] Methods for determining whether the c-MAF gene or the chromosomal region 16q22-q24 is translocated are widely known in the art, including those previously described for the amplification of c-MAF. These 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)), genome comparison 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 the chromosome or nucleus. In other embodiments, the detection of copy number variations and translocations can be detected by whole-genome sequencing, exome sequencing, or any PCR-derived technique. For example, PCR may 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 primers specific to the c-MAF gene and primers specific to the IGH promoter region; if a product is produced, a translocation has occurred.

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

[0210] Methods for measuring c-MAF expression, copy number, amplification, gain, and translocation. In some embodiments, the c-MAF gene expression level, 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 enables the detection and quantification of the protein in a sample derived from a subject. As a non-limiting example, the protein level may be quantified, for example, by using an antibody (or a fragment thereof containing an antigenic determinant) having c-MAF binding ability, followed by quantification of the formed complex. The antibodies used in these assays may or may not be labeled. Examples of markers that can be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, and the like. A wide range of known assays that can be used in the present invention include assays using unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies); these techniques include Western blotting or Western transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), 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 the form of dipsticks, etc. Other methods for detecting and quantifying the c-MAF protein include affinity chromatography techniques and ligand-binding assays. When immunological methods are used, any antibody or reagent known to bind to the c-MAF protein with high affinity may be used to detect its amount. This includes, but is not limited to, the use of antibodies, such as polyclonal serum, hybridoma supernatant or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, scFv, humanized diabody, triabody, tetrabody, antibodies, nanobody, alphabody, stapled peptide and cyclopeptide.Commercially available anti-c-MAF protein antibodies that can be used in the context of the present invention include, for example, antibodies ab427, ab55502, ab72584, ab76817, ab77071 (Abcam plc, 330 Science Park, Cambridge CB4 0FL, United Kingdom), and AbD Serotec's O75444 monoclonal antibody (Mouse Anti-Human MAF Azide free Monoclonal antibody, Unconjugated, Clone 6b8). Many commercial companies provide anti-c-MAF antibodies, such as Abnova Corporation, Bethyl Laboratories, Bioworld Technology, and GeneTex.

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

[0213] In some embodiments, the antibody or fragment has a 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: 15. H Includes the domain.

[0214] In some embodiments, the antigen-binding molecule or fragment has a 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 contains 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 contains 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 embodiment, the present invention relates to a binding member, a functional fragment, an antibody or variant thereof that specifically binds to an 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 blotting, immunohistochemistry, ELISA, or protein arrays.

[0219] As is understood by those skilled in the art, gene expression levels can be quantified by measuring the messenger RNA levels of the gene or 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 may 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] Therefore, the c-MAF gene expression level can be quantified from RNA (messenger RNA or mRNA) resulting from the transcription of the gene, or from the complementary DNA (cDNA) of the gene. Accordingly, in certain embodiments of the present invention, the quantification of the c-MAF gene expression level includes the quantification of messenger RNA or fragments of the mRNA of the c-MAF gene, complementary DNA or fragments of the cDNA of the c-MAF gene, or mixtures thereof.

[0222] Substantially any conventional method may be used within the scope of the present invention to detect and quantify the mRNA level encoded by the c-MAF gene or the mRNA level of its corresponding cDNA. Non-limiting examples include the mRNA level encoded by the gene, which may be quantified by conventional methods, such as methods including mRNA amplification and quantification of the mRNA amplification product, e.g., using electrophoresis and staining, or by Southern blotting and the use of appropriate probes, Northern blotting and the use of specific probes for the mRNA of the gene of interest (c-MAF) or its corresponding cDNA, mapping using S1 nucleases, RT-PCR, hybridization, microarrays, etc., preferably by real-time quantitative PCR using appropriate markers. Similarly, the cDNA level corresponding to the mRNA encoded by the c-MAF gene may also be quantified by conventional techniques; in this case, the method of the present invention includes the steps of synthesizing the corresponding cDNA by reverse transcription (RT) of the corresponding mRNA, subsequently amplifying it, and quantifying the cDNA amplification product. Conventional methods for quantifying expression levels can be found, for example, in Sambrook et al., 2001 (cited above). These methods are publicly known in the art, and those skilled in the art are familiar with the normalization required for each technique. For example, expression measurements generated using multiplex PCR should be normalized by comparing the expression of the measured gene to a so-called "housekeeping" gene (whose expression is constant across all samples and should therefore provide a baseline expression for comparison) or to other control genes whose expression is known to be modulated by cancer.

[0223] In certain embodiments, 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 consisting of 403 amino acids (NCBI, NP_005351.2) (SEQ ID NO: 4) and β isoform consisting of 373 amino acids (NP_001026974.1) (SEQ ID NO: 5)). 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, quantification of the level of the protein encoded by the c-MAF gene includes quantification of the c-MAF protein.

[0225] Methods for determining whether the c-MAF gene or chromosomal region 16q22-q24 is amplified are well known in the art. Such methods include, but are not limited to, in situ hybridization (ISH) (e.g., fluorescence 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-bearing dots in the chromosome or nucleus.

[0226] Fluorescence in situ hybridization (FISH) is a cytogenetic technique used to detect and localize the presence or absence of a specific DNA sequence in a chromosome. FISH uses fluorescent probes that bind only to a portion of the chromosome showing a high degree of sequence similarity. In a typical FISH method, the DNA probe is typically fluor-dU The samples are labeled with fluorescent molecules or haptens in the form of TP, digoxigenin-dUTP, biotin-dUTP, or hapten-dUTP (which is incorporated into DNA using enzymatic reactions such as nick translation or PCR). The sample containing genetic material (chromosomes) is placed on a glass slide and denatured by formamide treatment. The labeled probe is then hybridized with the sample containing genetic material under appropriate conditions determined by those skilled in the art. After hybridization, the sample is observed directly (in the case of fluorine-labeled probes) or indirectly (using fluorescently labeled antibodies to detect haptens).

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

[0228] Methods for determining whether the c-MAF gene or the chromosomal region 16q22-q24 is translocated are widely known in the art, including those previously described for the amplification of c-MAF. These 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)), genome comparison 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 the chromosome or nucleus. In other embodiments, the detection of copy number variations and translocations can be detected by whole-genome sequencing, exome sequencing, or any PCR-derived technique. For example, PCR may 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 primers specific to the c-MAF gene and primers specific to the IGH promoter region; if a product is produced, a translocation has occurred.

[0229] To label the probe used in the method of the present invention, any marking or labeling molecule capable of binding to DNA may be used to enable the 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 combinations thereof. Guidelines for methods of labeling and for selecting appropriate 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 probe of the present invention is a two-color probe. In some embodiments, the probe of the present invention is a double-fusion probe. In some embodiments, the probe of the present invention is a two-color double-fusion probe. 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) (this 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 dichromatic difusion 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), 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 to 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 to 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, dual 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-dual 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, 100μl, 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 dual 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 a MAF-specific probe is labeled with a red fluorophore and an IGH-specific probe is labeled with a green fluorophore, the presence of white 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, amplification, gain, or copy number changes.

[0234] Drugs and treatments for use in the treatment or prevention of breast cancer In some embodiments, the methods of the present invention as described herein involve treating a subject with an agent for avoiding or preventing bone remodeling. As used herein, “agent for avoiding or preventing bone remodeling” refers to any molecule (including agents for avoiding or preventing osteodegradation) that can treat or halt osteodegradation by stimulating osteoblast proliferation or inhibiting osteoclast proliferation. In embodiments, the agent for avoiding or preventing bone remodeling is a bone modifier and / or an agent for avoiding or preventing osteodegradation. Examples of agents used to avoid and / or prevent osteodegradation include, but are not limited to, the following: - Parathyroid hormone (PTH) inhibitors and parathyroid-like hormone (PTHLH) inhibitors (including blocking antibodies) or recombinants thereof (teriparatide, corresponding to amino acids 7-34 of PTH). This hormone acts by stimulating osteoclasts and increasing their activity. Strontium ranerate is an alternative oral treatment that stimulates osteoblast proliferation and inhibits osteoclast proliferation, thus forming part of a group of drugs known as "dual-acting bone agents" (DABAs). - An "estrogen receptor modulator" (SERM) refers to a compound that interferes with or inhibits the binding of estrogen to a receptor, regardless of the mechanism. Examples of estrogen receptor modulators include, among others, estrogen progestagens, estradiol, droloxifene, raloxifene, rasofoxifene, TSE-424, tamoxifene, doxifene, 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. Calcitonin receptors have been identified on the surface of osteoclasts. -Bisphosphonates are a group of pharmaceuticals used for the prevention and treatment of diseases involving bone resorption and resorption, such as osteoporosis and cancers with bone metastases (the latter being associated with breast cancer, with or without hypercalcemia). Examples of bisphosphonates that may be used in the treatments 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) and nitrogen-free bisphosphonates (e.g., etidronate, clodronate, tildronate, etc.). - "Cathepsin K inhibitor" refers to a compound that interferes with cathepsin K cysteine ​​protease activity. Non-limiting examples of cathepsin K inhibitors include 4-amino-pyrimidine-2-carbonitrile derivatives (described in international patent application WO03 / 020278 under the name of Novartis Pharma GMBH), pyrrolo-pyrimidines described in international publication 03 / 020721 (Novartis Pharma GMBH) and international publication 04 / 000843 (ASTRAZENECA AB), and inhibitors described in international publication 00 / 55126 of Axys Pharmaceuticals and international publication 01 / 49288 of Merck Frosst Canada & Co. and Axys Pharmaceuticals. -As used herein, “DKK-1 (Dickkopf-1) inhibitor” refers to any compound capable of reducing DKK-1 activity. DKK-1 is a soluble Wnt pathway antagonist primarily expressed in adult bone and upregulated in myeloma patients with osteolytic lesions. Drugs targeting DKK-1 may play a role in preventing osteolytic bone disease in multiple myeloma patients. Novartis' BHQ880 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 and is designed to block MET-driven tumor escapes. 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 and imparts a crucial role to the MET pathway in multiple autocrine and paracrine loops. Activation of MET in tumor cells appears to be important in the establishment of metastatic bone lesions. Simultaneously, activation of the MET pathway in osteoblasts and osteoclasts can lead to pathological features of bone metastases, including abnormal bone growth (i.e., blastic lesions) or destruction (i.e., lytic lesions). Therefore, targeting the MET pathway may be a viable strategy in preventing the establishment and progression of metastatic bone lesions. Cabozantinib (Exelixis, Inc.), formerly known as XL184 (CAS849217-68-1), is a potent dual inhibitor of the MET and VEGF pathways, designed to block MET-driven tumor escapes. Multiple preclinical studies have shown that cabozantinib kills tumor cells, reduces metastasis, and inhibits angiogenesis (the formation of new blood vessels necessary to support tumor growth). Another suitable double inhibitor is E7050(N-[2-fluoro-4-({2-[4-(4-methylpiperazine-1-yl)piperidine-1-yl]carbonylaminopyridine-4-yl}oxy)phenyl]-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide(2R,3R)-taltorate) (CAS 928037-13-2) or foretinib (also known as GSK 1363089, XL 880, CAS 849217-64-7). -As used herein, “RANKL inhibitor” refers to any compound capable of reducing RANK activity. RANKL is found on the surface of osteoblast membranes of bronchoscopy and T-lymphocyte cells, and these T-lymphocyte cells are the only cells that have been shown to be capable of secreting it. Its primary function is the activation of osteoclasts, cells involved in bone resorption. RANKL inhibitors may act by reducing RANKL expression by blocking the binding of RANKL to its receptor (RANK), blocking RANK-mediated signaling, or blocking the transcription or translation of RANKL. Suitable RANKL antagonists or inhibitors for use in the present invention include, but are not limited to, the following: ○A suitable RANK protein that can bind to RANKL and contains the entire or a fragment of the extracellular domain of the RANK protein. Soluble RANK may contain the signal peptide and extracellular domain of a mouse or human RANK polypeptide, or a mature form of the protein from which the signal peptide has been removed may be used. ○Osteoprotegerin or its variants that have RANKL binding ability. ○RANKL-specific antisense molecule ○ A ribozyme capable of processing RANKL transcripts. ○Specific anti-RANKL antibodies. “Anti-RANKL antibody or antibody against RANKL” is understood herein to be any antibody capable of specifically binding to the ligand (RANKL) of the activating receptor for nuclear factor κB, which inhibits the function of one or more RANKL. Antibodies can be prepared using any method known to those skilled in the art. 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 containing a variable antigen-binding region and a constant region, fragments “Fab”, “F(ab')2” and “Fab'”, Fv, scFv, diabody, and bispecific antibodies. ○Specific anti-RANKL nanobodies. Nanobodies are antibody-derived therapeutic proteins that possess the unique structural and functional properties of naturally occurring heavy-chain antibodies. Originally, nanobody technology was developed following the discovery that camelids (camels and llamas) possess fully functional antibodies lacking light chains. The general structure of nanobodies 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 domain is a fully stable polypeptide with the full antigen-binding capacity of the original heavy chain antibody. These newly discovered VHH domains, with their unique structural and functional properties, form the basis for 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 a RANKL-specific antibody, a RANKL-specific nanobody, and osteoprotegerin. In a specific embodiment, the anti-RANKL antibody is a monoclonal antibody. In a 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 embodiments of denosumab are covered by U.S. Patents 6,740,522; 7,411,050; 7,097,834; and 7,364,736 (the entire contents of which are incorporated herein by reference, respectively). In another embodiment, the RANKL inhibitor is an antibody, antibody fragment, or fusion construct that binds to the same epitope as denosumab.

[0236] In embodiments, the anti-RANKL nanobody is one of the nanobody described in International Publication No. 2008142164 (the contents of which are incorporated herein by reference). In embodiments, 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, as well as to restore a healthy balance of bone metabolism.

[0237] In embodiments, the agent for preventing osteolysis is selected from the group consisting of bisphosphonates, RANKL inhibitors, PTH and PTHLH inhibitors or PRG analogs, strontium ranerate, DKK-1 inhibitors, dual MET and VEGFR2 inhibitors, estrogen receptor modulators, radium-223, calcitonin, and cathepsin K inhibitors. In embodiments, the agent for preventing osteolysis is a bisphosphonate. In embodiments, the bisphosphonate is zoledronic acid.

[0238] In one embodiment, the CCR5 antagonist is administered to prevent or inhibit metastasis, relapse, or recurrence of primary breast cancer tumors to the 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 are selected from the group consisting of Cabozantinib, foretinib, and E7050.

[0240] In embodiments, the MAF status predicts the treatment the subject should receive. In embodiments, the c-MAF status in any of the embodiments herein includes amplification, copy gain, or translocation or absence thereof of the 16q23 or 16q22-24 chromosomal locus, or deletion thereof. In embodiments, postmenopausal patients having increased c-MAF gene expression levels, copy number, amplification, or gain relative to a baseline (and therefore at high risk of poor DFS or OS outcomes) may be subjected to any of the treatments disclosed herein. In some embodiments, postmenopausal patients having increased c-MAF gene expression levels, copy number, amplification, or gain relative to a baseline (and therefore at high risk of poor DFS or OS outcomes) may be treated by extending those hormonal treatments beyond the five years prescribed by the use of hormonal treatments as standard treatment. In certain embodiments, the hormonal treatment is tamoxifen and / or an aromatase inhibitor. Patients who do not have an increase in c-MAF gene expression level, copy number, amplification, or gain relative to the criteria should not be subjected to 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, mTor inhibitors include: 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 (Mepaclin), 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 (Umilolimus), 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 (Peripheral) 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 (everolilimus), AZD8055, Cypher Select Plus sirolimus-eluting coronary stent (sirolimus), Cypher Sirolimus-eluting coronary stents (sirolimus), drug-coated balloons (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 stents (sirolimus), nPT-mTOR, Rapacan (sirolimus), Renacept (sirolimus), ReZolve (sirolimus), Rocas (sirolimus), SF1126, Sirolim (sirolimus), Sirolimus NORTH CHINA (sirolimus), Sirolimus RANBAXY (sirolimus), SirolimusThe group selected consists 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), and 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 may be identified by methods known in the art. (See, for example, 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 may be identified by methods known in the art. (See, for example, 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, prevent, or inhibit metastasis in patients positive for hormone receptors. (See, for example, 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, 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, Src inhibitors are used to prevent or inhibit metastasis, relapse, or recurrence. In some embodiments, Src kinase inhibitors belong to 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 The Src kinase inhibitors are selected from VALEANT (dasatinib), Fontrax (dasatinib), Src kinase inhibitors KINEX, VX680 (tozasertib lactate), XL228, and SUNK706. In some embodiments, the Src kinase inhibitor is dasatinib. In other embodiments, the Src kinase inhibitor may 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, prevent, or inhibit metastasis, relapse, or recurrence in patients positive for the SRC-responsive signature (SRS). In some embodiments, the patient is SRS+ and ER- (see, for example, Zhang, CH.-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, the Src kinase inhibitor is used to treat, prevent, or inhibit metastasis in patients with advanced breast cancer. In some embodiments, the Src kinase inhibitor is 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, COX-2 inhibitors are used to prevent or inhibit metastasis, relapse, or recurrence. In some embodiments, COX-2 inhibitors belong to the following groups: ABT963, Acetaminophen ER JOHNSON (acetaminophen), Acular X (ketrolactromethamine), BAY1019036 (aspirin), BAY987111 (diphenhydramine, naproxen sodium), BAY1902 (piroxicam), BCIBUCH001 (ibuprofen), Capoxigem (apricoxib), CS502, CS670 (pelubiprofen), Diclofenac HPBCD (diclofenac), Diractin (ketoprofen), GW406381, HCT1026 (nitroflurbiprofen), Hyanarugese-D (diclofenac), HydrocoDex (acetaminophen, dextromethorphan, hydrocodone), Ibuprofen Sodium PFIZER (ibuprofen sodium), Ibuprofen with Acetaminophen PFIZER (acetaminophen, ibuprofen), 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 (ketrolactromethamine), RP19583 (ketoprofenlysine), RQ00317076 SDX101 (R-Etodolac), TDS943 (Diclofenac Sodium), TDT070 (Ketoprofen), TPR100, TQ1011 (Ketoprofen), TT063 (S-Flurbiprofen), UR8880 (Cimicoxib), V0498TA01A (Ibuprofen), VT122 (Etodolac, Propranolol), XP20B (Acetaminophen, Dextropropoxifen), 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 may 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, 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, COX-2 inhibitors are 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, and antibodies or peptides that inhibit PTHLH (parathyroid hormone-like hormone) or PTHrP (parathyroid hormone-related protein).

[0244] In one embodiment, the treatment is radium-223. In this embodiment, the radium-223 treatment is Alpharadin (also known as Zofigo) (radium-223 chloride). Alpharadin uses alpha rays from the decay of radium-223 to kill cancer cells. Due to its properties as a calcium mimetic, radium-223 spontaneously self-targets bone metastases. Because alpha rays have a very short range of 2 to 10 cells (compared to current radiotherapy based on beta or gamma rays), they cause less damage to surrounding healthy tissue (especially bone marrow). Due to its calcium-like properties, 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 site where cancer begins in the body is known as the primary tumor. Some of these cells can detach and be carried by the bloodstream to other parts of the body. Subsequently, these cancer cells can settle in that location in the body and form new tumors. When this happens, it is called secondary cancer or metastasis. The purpose of using radium-223 is to selectively target this secondary cancer. Any radium-223 that is not incorporated into the bone is rapidly transported to the intestines and 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 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 therapy may be performed using one or more of the above-mentioned drugs to treat and / or prevent metastasis, relapse, or recurrence, or the drugs may be combined with other supplements, such as calcium or vitamin D, or with hormonal treatments.

[0247] In embodiments, MAF-positive postmenopausal patients at high risk of DFS or poor OS outcomes are treated with any therapy in an adjuvant setting to improve patient outcomes. These therapys include any therapy disclosed herein, including agents to avoid or prevent bone remodeling, agents to improve 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 agents or therapy.

[0248] If the cancer has metastasized, systemic treatments including but not limited to chemotherapy, hormone therapy, immunotherapy, or a combination thereof are used. In addition, radiation therapy and / or surgery may be used. The choice of treatment usually depends on the type and size of the primary cancer, the location of metastases, the patient's age, overall health, and the type of treatment previously used.

[0249] Systemic treatment involves treatment of the entire body: - Chemotherapy is the use of pharmaceuticals to destroy cancer cells. Pharmaceuticals are generally administered orally or intravenously. In other embodiments, the treatment is chemotherapy. In some embodiments, chemotherapy is any chemotherapy known in the art. In certain embodiments, chemotherapy is adjuvant chemotherapy. In certain embodiments, chemotherapy is taxane. In further embodiments, taxane is paclitaxel (Taxol), docetaxel (Taxotere), or cabazitaxel. Pharmaceuticals are generally administered orally or intravenously. Chemotherapy may be used in conjunction with radiation therapy. Hormone therapy is based on the fact that certain 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, pharmaceuticals may be used to prevent these organs from producing hormones, or to prevent the hormones from acting on cancer cells. In some embodiments, the treatment is hormone therapy. In certain embodiments, the hormone therapy is tamoxifen and / or aromatase inhibitors. Immunotherapy is a treatment that fights cancer by supporting the patient's immune system itself. There are several types of immunotherapies used to treat patients with metastatic cancer. These include, but are not limited to, cytokines, monoclonal antibodies, and antitumor vaccines.

[0250] Drugs used to avoid or prevent bone remodeling are typically administered in combination with a pharmaceutically acceptable carrier.

[0251] The term "carrier" refers to a diluent or excipient through which the active ingredient is administered. Such pharmaceutical carriers may be sterile liquids, such as water, and oils, such as those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water or saline solutions 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 approved by a state or federal regulatory authority, or are listed in the United States Pharmacopeia, or in other pharmacopoeias generally recognized for their use in animals, and more specifically in humans.

[0252] The carriers and auxiliary substances required to produce the desired pharmaceutical dosage form of the pharmaceutical composition of the present invention depend, in particular, on the selected pharmaceutical dosage form. The pharmaceutical dosage form of the pharmaceutical composition will be produced according to conventional methods known to those skilled in the art. An overview of various methods for administering the active ingredient, 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 (solutions, suspensions, or emulsions) for oral, topical, or parenteral administration. Furthermore, the pharmaceutical composition may contain stabilizers, suspending agents, preservatives, surfactants, etc., if deemed necessary.

[0253] In pharmaceutical use, bone remodeling agents may be found alone or in combination with further activators, in the form of prodrugs, salts, solvates, or inclusion complexes, and may be formulated with pharmaceutically acceptable excipients. Preferred excipients for use in the present invention include sugars, starches, cellulose, rubber, and proteins. In certain embodiments, the pharmaceutical compositions of the present invention may be formulated as solid pharmaceutical dosage forms (e.g., tablets, capsules, pills, granules, suppositories, sterile crystals or amorphous solids which can be reconstituted to obtain liquid forms), liquid pharmaceutical dosage forms (e.g., solutions, suspensions, emulsions, elixirs, lotions, ointments, etc.), or semi-solid pharmaceutical dosage forms (gels, ointments, creams, 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, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal routes. An overview of various methods for administering the active ingredient, the excipients to be used, and their manufacturing processes is provided in Tratado de Farmacia Galenica, C. Fauli i Trillo, Luzan 5, SA, 1993 Edition and Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 20 th This can be found in edition, Williams & Wilkins PA, USA (2000). Examples of pharmaceutically acceptable carriers are known in the art and include phosphate buffered salt solutions, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, and sterile solutions. Compositions containing such carriers can be formulated by conventional processes known in the art.

[0254] Agents for avoiding and preventing bone remodeling, or pharmaceutical compositions containing them, may be administered in doses of less than 10 mg per kilogram of body weight, preferably less than 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 0.00001 mg per kg of body weight. The unit dose may be administered by injection, inhalation, or topical administration. In certain embodiments, the agent may be administered at its approved dose.

[0255] The dose may vary depending on the severity of the condition being treated and the response, for several days to several months or until the condition is sedated. The optimal dose may be determined by regularly measuring the concentration of the drug in the patient's body. The optimal dose may be determined from the EC50 value obtained by prior in vitro or in vivo assays in animal models. The unit dose may be administered once daily or less than once daily, preferably less than once every 2, 4, 8, or 30 days. Alternatively, one or more maintenance doses (usually less than the initial dose) may be administered after the initial dose. Maintenance regimens may involve treating the patient with a dose range of 0.01 μg to 1.4 mg / kg body weight / day, e.g., 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. The treatment must be continued for a duration that varies depending on the type and severity of the disorder the patient is suffering from and the patient's condition. After the treatment, the patient's progress should be monitored to determine whether to increase the dose if the disease does not respond to the treatment, or to decrease the dose if improvement in the disease is observed or if undesirable side effects are observed.

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

[0257] Means for quantifying the expression level of c-MAF in a sample of the subject have been previously described in detail, including amplification, gain, and / or translocation of the 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, means for quantifying expression include a set of probes and / or primers that specifically bind to and / or amplify the c-MAF gene.

[0259] All specific embodiments of the methods of the present invention are applicable to the kits of the present invention and their use.

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

[0261] In some embodiments, c-MAF gene expression levels are used to stratify patients into treatment groups based on their c-MAF expression levels. In embodiments, patients with high c-MAF expression levels receive different treatments than patients with low c-MAF expression levels. In embodiments, patients are further stratified based on their menopausal status. In embodiments, patients are stratified based on whether they are postmenopausal or non-menopausal. In certain embodiments, subjects receive different treatments based on their c-MAF expression levels and / or postmenopausal or non-menopausal status. In some embodiments, stratified patients are administered drugs that prevent or avoid bone remodeling. In embodiments, the drugs that prevent or avoid bone remodeling are drugs that prevent or avoid osteolysis. In further embodiments, the drugs that prevent or avoid osteolysis are zoledronic acid. In other embodiments, c-MAF gene expression levels are 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 the subject sample have been previously described in detail, including amplification, gain, and / or translocation of the 16q23 and 16q22-24 loci.

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

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

[0265] The following embodiments illustrate the present invention and do not limit its scope. [Examples]

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

[0267] Based on KREATECH's proprietary FISH technology, a MAF FISH assay was developed by Kreatech for Inbiomotion. The probe set contains two probes (MAF 16q23 probe + D16Z3 probe) as controls for the centromere 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 value was selected. The scoring algorithm was as follows: for targeted amplification 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 a recombinant monoclonal antibody (as described in International Patent Application No. PCT / IB2015 / 059562, which is incorporated herein by reference in its entirety). The antibody was selected based on IHC. DAKO The assay was validated using MAF RecMab (Inbiomotion) via the AS LINK platform and the control sample protocol provided by Inbiomotion. Scoring criteria were predefined in the fact sheet, with one single IHC H score per patient. The scoring algorithm was the H-score.

[0269] The AZURE clinical trial used patients to validate MAF (Coleman et al., N Eng J Med 2011;365:1396-1405 and AZURE Current A summary of the study design (Controlled Trials number, ISRCTN79831382 and ClinicalTrials.gov identifier NCT00072020) is provided in Figure 2. MAF was validated in a retrospective analysis of the AZURE trial using patient tumor samples collected in a prospective study under regulatory compliance conditions. Of the 3,360 recruited patients, 1,769 provided tumor tissue (52.4%). There were 13 tissue microarrays (TMAs) (150 patient samples each) (1,769 patients). There were 4 replicas of each TMA using different tissue cores (6,326 (4 × patient)). Each TMA had only 1 replica, and two TMAs had 3 replicas. Based on H&E analysis (hematoxylin and eosin) (Figure 3) (6,326 samples), 3,978 cores were evaluable (63%), and 2,348 were unevaluable.

[0270] The FISH assay yielded 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%). In any of the four replicas, 567 patients were unevaluable by FISH (32%).

[0271] In the IHC assay, pathologist evaluation and VisioPharm computer-aided evaluation were performed. In pathologist evaluation, 2,232 cores were evaluated (59% were evaluable by H-score). 1,390 patients had an IHC H-score (74%) (equivalent to 39% of all AZURE patients). In any of the four replicas, 460 patients were unevaluable by IHC. In the VisioPharm computer-aided IHC staining evaluation, 1,299 IHC patients were evaluated out of 1,309 patients whose H-score and mean stain / nucleus were scored by pathologists. The MAF positivity rate can be seen in Figures 4A and 4B.

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

[0273] Regarding molecular variables in FISH analysis: MAF copy number: numerical and categorical (+ / - cutoff >= 2.5) variable; % of amplified nuclear MAF (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), disease-free survival (IDFS), overall survival (OS), first relapse in bone, bone relapse at any point in time, time to first DFS event in bone, time to first DFS event outside of bone, and response to zoledronic acid treatment.

[0274] In the analysis of MAF FISH prognostic values, patients from the control and treatment arms were pooled for initial analysis. Optimized cutoffs were used for each variable to be analyzed, where indicated. Death was used as a competing event for 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 the 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, and a second primary non-invasive breast 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) (death was used as a competing event in the time to bone metastasis (at any given time point)). Figure 7 shows that MAF FISH-positive patients had a 42% higher risk of bone as the initial metastasis site (>=2.3) (p=0.02, multivariate analysis). As seen in Figure 8, MAF FISH-positive patients had a 38% higher risk of IDFS (>=2.2) (p=0.0002, multivariate analysis) (the curves become parallel after very early separation by 2 years). Figure 9 shows that MAF FISH-positive patients had a 33% lower overall survival (>=2.2) (p=0.02, multivariate analysis) (for overall survival, early separation was performed by 3 years).

[0276] As shown in Figure 10, MAF FISH-positive patients in the control arm had a shorter time to bone as the first recurrence (>=2.3), and this difference was statistically 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 with untreated MAF-non-positive patients (HR=0.72, p=0.08, multivariate analysis). Figure 12 shows the 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 cutoffs for stratifying patients according to MAF FISH levels were very close to the computer-based, predefined, optimized cutoffs. The threshold effect allows for clear group depiction regarding appropriate (relevant) treatment (or avoidance of treatment). Based on the prognosis of MAF FISH-positive patients in the control arm, we found that there was a tendency for shorter time to bone as the first metastasis (HR=0.53, multivariate p=0.03) and shorter time to recurrence (infiltrative disease) (HR=0.72, p=0.08).

[0279] Example 2: Evaluation of the effect of zoledronic acid treatment by MAF FISH stratification. The control arm and zoledronic acid-treated arm 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 zoledronic acid-treated arm of the Azure trial. Figure 14 shows the evaluation of the time to bone metastasis as the 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 the first recurrence, and this difference 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 first recurrence site between MAF-positive and non-positive patients, and there was no significant difference in the risk of bone metastasis at any time point in MAF-positive patients treated with zoledronic acid compared with MAF-non-positive patients.

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

[0282] Figure 16 shows the time to distant recurrence between the 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). Zoledronic acid treatment worsened IDFS compared to untreated MAF-positive patients.

[0283] Figure 17 shows the time to bone metastasis event (at any given time) for each treatment. Death is used as a competing event in the time to bone metastasis (at any given time). In the control arm, MAF FISH-positive patients (>=2.3) did not show a significant increase in the risk of bone metastasis (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 compared to MAF FISH-positive patients (<2.3) (HR=0.52, p=0.01).

[0284] Figure 18 shows the time to bone metastasis event (at any given time) in relation to MAF copy number (corresponding to a pre-specified MAF cutoff of 2.5). Death is used as a competing event in the time to bone metastasis (at any given time). In patients without MAF FISH positivity, zoledronic acid treatment significantly reduced the risk of bone metastasis (HR=0.65, p=0.03). In patients with MAF positivity, zoledronic acid treatment showed a trend toward an increased risk of bone metastasis. The difference was not statistically significant (HR=1.54, p=0.22).

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

[0286] Figure 20 shows the time to bone metastasis events (at any given time) in postmenopausal patients, corresponding to MAF copy number (data based on a pre-specified cutoff of 2.5) (death used as a competing event). Treatment outcomes in MAF-positive postmenopausal patients (>2.5) tended to reduce the 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 not as effective as in MAF-positive postmenopausal patients (HR=0.63 vs. HR=0.46, limited number of events), but this suggests a clear benefit of zoledronic acid treatment for preventing bone metastasis in MAF-positive postmenopausal patients.

[0287] Figure 21 shows the time to bone metastasis events (at any given time) in non-postmenopausal patients, corresponding to MAF copy number (data based on a pre-specified cutoff in 2.5). In MAF-positive non-postmenopausal patients, zoledronic acid treatment outcomes were significantly worse and led to an increase in bone metastasis events (HR=2.44, p=0.045). In non-MAF-non-positive non-postmenopausal patients, there was a tendency towards better zoledronic acid treatment outcomes (HR=0.66, p=0.08).

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

[0289] Figure 23 shows IDFS (excluding bone metastases) in the zoledronic acid-treated arm and the control arm. As seen in Figure 23, zoledronic acid treatment in non-menopausal women significantly worsened IDFS (excluding bone) in MAF FISH-positive patients (>=2.2), while there was no difference in IDFS (excluding bone) in MAF FISH-non-positive patients.

[0290] Figure 24 shows overall survival (OS) for the treated arm. Treatment of MAF FISH-positive patients with zoledronic acid had a significant impact on 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: Significance of FISH status and menopausal status interaction covariates in multivariate analysis (in control patients); χ² 2 =6.23, p-value=0.013. Figure 26 shows the overall survival prognosis in patients in the AZURE control arm. Untreated MAF-positive postmenopausal patients tend to have shorter OS. Regarding OS: Significance of FISH status and menopausal status interaction covariates in multivariate analysis (in control patients); χ² 2 =3.62, p-value = 0.057. Figure 27 shows the effect of zoledronic acid treatment on DFS according to MAF FISH values. 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 occurred in postmenopausal and non-postmenopausal women.

[0292] Figure 28 shows the effect of zoledronic acid treatment on DFS according to MAF FISH values ​​in postmenopausal patients. As can be seen in Figure 28, in MAF-negative postmenopausal patients, zoledronic acid treatment results in a better DFS outcome (HR=0.56, (95% CI, 0.33-0.95)). Figure 29 shows the effect of zoledronic acid treatment on DFS in non-postmenopausal women. As can be seen in Figure 29, in MAF-positive non-postmenopausal patients, zoledronic acid treatment results in the worst DFS outcome. Figure 30 shows the effect of zoledronic acid treatment on overall survival according to MAF FISH values. As can be seen in Figure 30, in MAF-positive patients, zoledronic acid treatment results in significantly shorter overall survival. These differences occur in postmenopausal and non-postmenopausal women. Figure 31 shows MAF in postmenopausal patients. The effects of zoledronic acid treatment on overall survival, according to FISH level, are shown. As can be seen in Figure 31, zoledronic acid treatment tends to lead to a better overall survival outcome in MAF-negative postmenopausal patients (HR=0.56, (95% CI, 0.31-1.01)), but the effect is greater in zoledronic acid FISH-positive patients. Figure 32 shows the effects of zoledronic acid treatment on overall survival in non-menopausal women, according to MAF FISH level. As can be seen in Figure 32, zoledronic acid treatment results in the worst overall survival outcome in MAF-positive non-menopausal patients.

[0293] A summary of the predictive values ​​of target gene (GOI)MAF for patient risk of DFS and OS, segmented according to menopausal status, can be seen in Table 1. [Table 1]

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

[0295] In summary, in the control arm, MAF FISH-positive patients had a significantly increased risk of bone metastasis as the first recurrence site compared to non-positive patients (HR=0.47, p=0.013, cutoff=2.3), and this difference decreased with zoledronic acid treatment. In addition, in MAF FISH-non-positive patients, zoledronic acid treatment significantly reduced the risk of bone metastasis at all time points (HR=0.65, p=0.03, cutoff=2.5). In MAF-positive patients, zoledronic acid treatment showed an increased risk of bone metastasis at all time points. The difference was not statistically significant (HR=1.54, p=0.22, cutoff=2.5). This effect was driven by menopausal status, showing the maximum effect in the non-postmenopausal group. Zoledronic acid significantly improved outcomes in MAF FISH-positive postmenopausal patients. However, zoledronic acid worsened outcomes in MAF FISH-positive non-postmenopausal patients. This effect depends on the increase in invasive disease (decrease in IDFS) when treated with zoledronic acid (this suggests that in non-menopausal patients, prevention of bone metastases may promote metastases elsewhere, ultimately leading to bone metastases as a secondary event).

[0296] MAF FISH-positive patients who have not been treated with zoledronic acid have a higher risk of bone metastases and invasive disease (reduced IDFS, regardless of inclusion or exclusion of bone events). In patients treated with zoledronic acid, MAF-positive patients have worse outcomes compared to untreated patients with respect to bone metastases, IDFS (regardless of inclusion or exclusion of bone events), and overall survival at any given time. Regarding bone metastases at any given time, MAF-negative patients treated with zoledronic acid have better outcomes compared to untreated patients. In postmenopausal women, MAF-positive patients treated with zoledronic acid have better outcomes regarding IDFS (excluding bone). In non-postmenopausal women, MAF-positive patients treated with zoledronic acid have worse outcomes regarding 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 invoked by reference in whole herein to the same extent as each individual publication, patent, patent application, internet site, or accession number / database sequence is specifically and individually indicated as being invoked by reference.

Claims

1. An in vitro method for using the c-MAF gene expression level, copy number, amplification, or gain in a sample of a subject with breast cancer as an indicator of whether the subject is permitted to receive customized treatment, i) To quantify the c-MAF gene expression level, copy number, amplification, or gain in the sample of the subject, and ii) Compare the expression level, copy number, amplification, or gain obtained in i) with a reference value. Includes, An in vitro method in which, if the expression level, copy number, amplification, or gain has not increased relative to the reference value, the subject is permitted to receive treatment with zoledronic acid at a dose of less than 0.5 mg per kg of body weight.

2. The method according to claim 1, wherein the subject is post-menopausal.

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

4. An in vitro method for using the c-MAF gene expression level, copy number, amplification, or gain in a sample of a subject with 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 a sample of the subject, and determining the menopausal status of the subject, wherein the subject is postmenopausal and has an increase in the c-MAF gene expression level, copy number, amplification, or gain compared to the reference value, indicating that the subject is tolerable to receive zoledronic acid at a dose of less than 0.5 mg per kg of body weight.

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

6. The method according to any one of claims 1 to 5, wherein the quantification of the c-MAF gene expression level includes quantifying the messenger RNA (mRNA) or a fragment of the mRNA of the gene, the complementary DNA (cDNA) or a fragment of the cDNA of the gene, or quantifying the level of the protein encoded by the gene.

7. The 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 technology.

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

9. The 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 according to claim 4, wherein the copy number of the c-MAF measured using FISH is ≥ 2.

1.

11. The method according to any one of claims 1 to 3, wherein the copy number of the c-MAF measured using FISH is < 3.

0.

12. The method according to any one of claims 1 to 5, wherein the copy number is determined as the average copy number per cell.

13. The method according to 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 breast cancer, or HER2+ breast cancer.

14. The 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 the gene locus 16q23 or 16q22-q24.

15. The method according to any one of claims 1 to 3, wherein the treatment is aimed at preventing and / or treating bone remodeling and improving disease-free survival or overall survival.

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

20.

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

18. The method according to claim 17, wherein the in-situe hybridization is fluorescent in-situe hybridization (FISH), chromogenic in-situe hybridization (CISH), or silver in-situe hybridization (SISH).

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

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

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

22. The method according to claim 1, wherein the subject is permitted to receive zoledronic acid treatment at a dose of less than 0.05 mg per kg of body weight.

23. The method according to claim 4, wherein the subject is permitted to receive zoledronic acid at a dose of less than 0.05 mg per kg of body weight.

24. The method according to claim 5, wherein the zoledronic acid is for administration in a dose of less than 0.01 mg per kg of body weight.

25. The method according to claim 10, wherein the copy number of c-MAF measured using FISH is ≥ 3.

0.

26. The method according to claim 11, wherein the copy number of the c-MAF measured using FISH is < 2.1.

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