Diagnostic and therapeutic methods for the treatment of breast cancer

Abstract

This document provides, in particular, predictive diagnostic, pharmacodynamic, and therapeutic methods for treating breast cancer. In the embodiments, these methods and compositions are based, at least in part, on the finding that estradiol (E2) induction scores or estrogen receptor (ER) pathway activity scores measured from samples of an individual (e.g., tissue samples, such as tumor tissue samples, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples) can be used to determine whether an individual with breast cancer is likely to respond to treatment, including endocrine therapy, in order to select therapy for the individual with breast cancer; to treat the individual with breast cancer; and to monitor the efficacy of endocrine therapy, and related kits.

Description

Technical Field

[0001] This article specifically provides diagnostic and treatment methods for breast cancer. For example, it provides methods for predicting treatment responsiveness, monitoring treatment response, selecting treatment methods, treatment approaches, and diagnostic kits. Prior Technology

[0002] Cancer remains one of the deadliest threats to human health. In the United States, cancer affects nearly 1.3 million new patients each year and is the second leading cause of death after heart disease, accounting for about one-quarter of all deaths. Solid tumors are the cause of the majority of these deaths. Although significant progress has been made in the medical treatment of some cancers, the overall 5-year survival rate for all cancers has only increased by about 10% over the past 20 years.

[0003] The estrogen receptor ("ER") is a ligand-activated transcriptional regulatory protein that mediates the induction of various biological effects through its interaction with endogenous estrogen. Estrogen and its receptor are involved in cancers such as breast cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, endometrial cancer, uterine cancer, and other diseases or conditions. However, estrogen receptor degradation measured by immunohistochemistry (IHC) is insufficient as a predictor of ER pathway activity or as a pharmacodynamic (PD) biomarker for endocrine therapy; ER protein levels are not frequently correlated with ER pathway status. For example, activators of ER signaling (such as the ER ligand estradiol (E2)) promote ER degradation. Furthermore, the progesterone receptor (PR) (a well-established ER target gene) is often measured as a readout of ER pathway activity; however, PR is not frequently present in ER+ breast tumors, and even when present, PR inhibition does not fully capture ER pathway status.

[0004] Therefore, there is an unmet need for biomarkers (e.g., transcriptional signatures) that comprehensively reflect ER pathway activity and can be used in both diagnosis and treatment. Summary of the Invention

[0005] This article provides, in particular, diagnostic methods, treatment methods, a suite of methods for notifying individuals with breast cancer about treatment, and a suite of methods for predicting an individual's responsiveness to breast cancer treatment.

[0006] In one state, a method is provided for identifying an individual with breast cancer who may benefit from treatment including endocrine therapy, the method comprising measuring an estrogen receptor (ER) pathway activity score from a sample of the individual, wherein when the ER pathway activity score from the sample is equal to or higher than a reference ER pathway activity score, the individual can be identified as a recipient of treatment including endocrine therapy.

[0007] In one state, a method is provided for selecting a therapy for an individual with breast cancer, the method comprising measuring an ER pathway activity score from a sample of the individual, wherein when the ER pathway activity score from the sample is equal to or higher than a reference ER pathway activity score, the individual can be identified as a beneficiary of treatment including endocrine therapy.

[0008] In one instance, a method for treating an individual with breast cancer is provided, the method comprising administering an effective dose of endocrine therapy to the individual, wherein the individual has been determined to be more likely to benefit from treatment including endocrine therapy as described herein.

[0009] In one instance, a method is provided for treating an individual with breast cancer who is determined to have an ER pathway activity score equal to or higher than a reference ER pathway activity score, the method comprising administering an effective dose of endocrine therapy to the individual.

[0010] In one state, a method for treating an individual with breast cancer is provided, the method comprising: (a) determining an ER pathway activity score from a sample of the individual, wherein the ER pathway activity score from the sample is determined to be equal to or higher than a reference ER pathway activity score; and (b) administering an effective amount of endocrine therapy to the individual.

[0011] In one state, a method is provided for monitoring the response of an individual with breast cancer to endocrine therapy, the method comprising: (a) measuring a first ER pathway activity score of a sample from the individual at a first time point; (b) after step (a), measuring a second ER pathway activity score of a sample from the individual at a second time point after administration of endocrine therapy; and (c) comparing the first ER pathway activity score with the second ER pathway activity score, wherein a reduction in the second ER pathway activity score relative to the first ER pathway activity score predicts an individual's response to endocrine therapy.

[0012] In one state, a method is provided for identifying an individual with breast cancer who may benefit from treatment including endocrine therapy, the method comprising measuring an estradiol (E2) induction score from a sample of the individual, wherein when the E2 induction score from the sample is equal to or higher than a reference E2 induction score, the individual can be identified as a recipient of treatment including endocrine therapy.

[0013] In one state, a method is provided for selecting a therapy for an individual with breast cancer, the method comprising measuring an E2 induction score from a sample of the individual, wherein when the E2 induction score from the sample is equal to or higher than a reference E2 induction score, the individual can be identified as a beneficiary of treatment including endocrine therapy.

[0014] In one instance, a method for treating an individual with breast cancer is provided, the method comprising administering an effective dose of endocrine therapy to the individual, wherein the individual has been determined to be more likely to benefit from treatment including endocrine therapy as described herein.

[0015] In one state, a method for treating an individual with breast cancer, the individual being determined to have an E2 induction score equal to or higher than a reference E2 induction score, the method comprising administering an effective dose of endocrine therapy to the individual.

[0016] In one state, a method for treating an individual with breast cancer is provided, the method comprising: (a) determining an E2-inducible score from a sample of the individual, wherein the determined E2-inducible score from the sample is equal to or higher than a reference E2-inducible score; and (b) administering an effective amount of endocrine therapy to the individual.

[0017] In one state, a method is provided for monitoring the response of an individual with breast cancer to endocrine therapy, the method comprising: (a) measuring a first E2-induced score of a sample from the individual at a first time point; (b) measuring a second E2-induced score of a sample from the individual at a second time point after administration of endocrine therapy following step (a); and (c) comparing the first E2-induced score with the second E2-induced score, wherein a reduction in the second E2-induced score relative to the first E2-induced score predicts an individual's response to endocrine therapy.

[0018] In one embodiment, a method is provided for detecting estrogen receptor (ER) pathway activity in a subject with breast cancer, the method comprising detecting the following expression levels: at least five genes described in Table 1 and at least five genes described in Table 4; at least five genes described in Table 2 and at least five genes described in Table 5; or at least five genes described in Table 3 and at least five genes described in Table 6.

[0019] In one embodiment, a method is provided comprising: detecting, by means of one or more processors, a first expression level of at least five genes described in Table 1, at least five genes described in Table 2, or at least five genes described in Table 3; detecting, by means of one or more processors, a second expression level of at least five genes described in Table 4, at least five genes described in Table 5, or at least five genes described in Table 6; and detecting, at least based on the first expression level and / or the second expression level, estrogen receptor (ER) pathway activity in a subject with cancer.

[0020] In one state, a kit is provided comprising a plurality of nucleic acids, wherein the plurality of nucleic acids are at least 5 nucleotides in length and are at least 95% identical to, or 95% identical to, a sequence complementary to, the 5 nucleotides in the at least five genes described in Table 1 and Table 4; the at least five genes described in Table 2 and Table 4; or the at least five genes described in Table 3 and Table 6. Simple Explanation of the Diagram

[0021] This application file contains at least one drawing executed in color. A copy of this patent or patent application with a color drawing will be provided by the office upon request and payment of the necessary fees.

[0022] [picture] [1A] Overview of estradiol (E2)-inducing genes shown in red (rows) in seven breast cancer cell lines (columns). E2 induction was defined as a ≥2-fold change in expression after E2 treatment compared to DMSO treatment (p-value ≤0.05).

[0023] [picture] [1B] Overview of E2 repressor genes shown in blue (rows) in 7 breast cancer cell lines (columns). E2 repression was defined as a ≤1 / 2-fold change in expression after E2 treatment compared to DMSO treatment (p-value ≤0.05).

[0024] [picture] [1C] A heatmap showing the z-score performance of 23 E2-inducing genes and 18 E2-repressing genes from 989 breast tumors from The Cancer Genome Atlas (TCGA), with annotations based on estrogen receptor (ER) immunohistochemical (IHC) status and PAM50 subtype. []

[0025] [picture] [1D] A plot showing the superimposed reference density curves of E2 induction scores (defined as the average z-score performance of the 23 E2-inducing genes shown in Figure 1C) for 726 ER IHC+ and 213 ER IHC- breast tumors from TCGA RNA-seq data.

[0026] [picture] [1E] A graph showing the superimposed reference density curves of E2 inhibition scores (defined as the average z-score performance of the 18 E2 inhibition genes shown in Figure 1C) for 726 ER IHC+ and 213 ER IHC- breast tumors from TCGA RNA-seq data.

[0027] [picture] [1F] A plot showing the superimposed reference density curves of ER pathway activity scores (defined as the difference between E2 induction scores and E2 inhibition scores) for 726 ER IHC+ and 213 ER IHC- breast tumors from TCGA RNA-seq data.

[0028] [picture] [2A] A graph showing the in vivo efficacy of treatment with compound A (selective estrogen receptor degrader (SERD)) in a mouse model of HCI-013 patient-derived xenograft (PDX) with hormone receptor (HR)-positive breast cancer, as assessed by tumor volume over time at specified compound A concentrations.

[0029] [picture] [2B] A heatmap showing the relative changes in z-score performance of 20 designated E2-inducing genes and 14 E2-repressing genes in HCI-013 tumors after treatment with compound A. Treatment regimens are noted above.

[0030] [picture] [2C] A set of bar charts showing the E2 induction score, E2 inhibition score, and ER pathway activity score as a mean log10⁻ fold change in HCI-013 tumors after exposure to compound A relative to mediated animals. The bar charts show the mean relative scores and standard errors, where n=4. One-sided t-test: *, p<0.05; **, p<0.01; ***, p<0.001; black for 1 mg / kg vs. 0.1 mg / kg; red for 10 mg / kg vs. 1 mg / kg.

[0031] [picture] [2D] A graph showing the in vivo efficacy of treatment with the mordant, or compound B (SERD), compound C (SERD / SERM mixture) or compound F (SERD) in an HCI-013 PDX mouse model of HR-positive breast cancer, as assessed by tumor volume over time at specified concentrations.

[0032] [picture] [2E] A heatmap showing the relative changes in z-scores of 20 designated E2-inducing genes and 14 E2-repressing genes in HCI-013 tumors after treatment with the specified therapies. Treatment regimens are described in the comments above.

[0033] [picture] [2F] A set of bar plots representing the mean log10⁻⁶ change in E2 induction score, E2 inhibition score, and ER pathway activity score of HCI-013 tumors relative to those treated with the catalyst after exposure to the specified compounds. The bar plots show the mean relative scores and standard errors, where n=4. One-sided t-tests: *, p<0.1; **, p<0.05; ***, p<0.01; ****, p<0.001; comparisons of compound E to catalyst, compound C to compound E, compound D to compound C, and compound B to compound D.

[0034] [picture] [2G] A graph showing the in vivo efficacy of treatment with the mordant, compound B, compound C or compound F in an HCI-011 PDX mouse model of HR-positive breast cancer, as assessed by tumor volume over time at specified endocrine therapy concentrations.

[0035] [picture] [2H] A heatmap showing the relative changes in z-scores of 20 designated E2-inducing genes and 14 E2-repressing genes in HCI-011 tumors after treatment with the specified therapies. Treatment regimens are described in the comments above.

[0036] [picture] [2I] The HCI-011 tumors, as represented by the mean log10-fold change in E2 induction score, E2 inhibition score, and ER pathway activity score relative to the mediated animals after exposure to the specified compounds, are presented in a set of bar plots. The bar plots show the mean relative scores and standard errors, where n=4. One-sided t-tests: *, p<0.1; **, p<0.05; ***, p<0.01; ****, p<0.001; comparisons of compound E to mediated; compound C to compound E, compound B to compound C, and compound A to compound B.

[0037] [picture] [3A] A heatmap showing the relative changes in z-score performance of 21 designated E2-inducing genes and 17 designated E2-repressing genes in a collection of 139 hormone receptor-positive / human epidermal growth factor receptor 2-negative (HR+ / HER2-) breast tumors.

[0038] [picture] [3B] [to] [3D] A series of reference density curves for E2 induction score (Figure 3B), E2 inhibition score (Figure 3C), and ER pathway activity score (Figure 3D) of a collection of 139 HR+ / HER2- breast tumors. Data from six patients before and after treatment with compound B are superimposed: pre-treatment scores are indicated by diamonds; post-treatment scores are indicated by circles. Arrows indicate the magnitude and direction of change in ER pathway activity score / patient.

[0039] [picture] [3E] is a scatter plot of the pre-treatment ER pathway activity score versus the pre-treatment ESR1 performance in six patients treated with compound B.

[0040] [picture] [3F] is a scatter plot of the difference in ER pathway activity before and after treatment in six patients treated with compound B against the level of ER pathway activity before treatment.

[0041] [picture] [3G] is a scatter plot of the difference in E2-induced scores before and after treatment in six patients treated with compound B against the pre-treatment E2-induced scores.

[0042] [picture] [3H] [to] [3J] A series of reference density curves for E2 induction score (Fig. 3H), E2 inhibition score (Fig. 3I), and ER pathway activity score (Fig. 3J) of a collection of 139 HR+ / HER2- breast tumors. Data from seven patients before and after treatment with compound A were superimposed: pre-treatment scores are indicated by diamonds; post-treatment scores are indicated by circles. Arrows indicate the magnitude and direction of change in ER pathway activity score / patient.

[0043] [picture] [3K] is a scatter plot of the pre-treatment ER pathway activity score versus the pre-treatment ESR1 performance of seven patients treated with compound A.

[0044] [picture] [3L] is a scatter plot of the difference in ER pathway activity before and after treatment in seven patients treated with compound A against the level of ER pathway activity before treatment.

[0045] [picture] [3M] is a scatter plot of the difference in E2 induction scores before and after treatment in seven patients treated with compound A against the pre-treatment E2 induction scores.

[0046] [picture] [4A] [to] [4C] A series of bar graphs representing the E2 induction score, E2 inhibition score, and ER pathway inhibition based on the mean log10-fold change of HCI-013 PDX breast tumors relative to mediated animals after exposure to compound A, using complete 41-gene signature (Fig. 4A), 19-gene signature (Fig. 4B), or 14-gene signature (Fig. 4C). The bar graphs show the mean relative scores and standard errors, where n=4. One-sided t-test: *, p<0.05; **, p<0.01; ***, p<0.001; black for 1 mg / kg vs. 0.1 mg / kg; red for 10 mg / kg vs. 1 mg / kg.

[0047] [picture] [4D] [to] [4F] A series of reference density curves for ER pathway activity scores based on 41-gene signatures (Fig. 4D), 19-gene signatures (Fig. 4E), or 14-gene signatures (Fig. 4F) for a collection of 139 HR+ / HER2- breast tumors. Pre- and post-treatment data for six patients are overlaid: pre-treatment scores are indicated by diamonds; post-treatment scores are indicated by circles. Arrows indicate the magnitude and direction of change in ER pathway activity scores / patients.

[0048] [picture] [4G] [to] [4I] A series of reference density curves for E2-induced scores based on 41-gene signatures (Fig. 4G), 19-gene signatures (Fig. 4H), or 14-gene signatures (Fig. 4I) for a collection of 139 HR+ / HER2- breast tumors. Pre- and post-treatment data for six patients are overlaid: pre-treatment scores are indicated by diamonds; post-treatment scores are indicated by circles. Arrows indicate the magnitude and direction of change in E2-induced scores / patients.

[0049] [picture] [4J] A table showing the characteristics of 41-genes (23 E2-inducing genes and 18 E2-repressing genes).

[0050] [picture] [4K] is a table showing the characteristics of the 19-genes (11 E2-inducing genes and 8 E2-repressing genes).

[0051] [picture] [4L] is a table showing the characteristics of 14 genes (8 E2-inducing genes and 6 E2-repressing genes).

[0052] [picture] [5] Scatter plots of ER pathway activity scores for 60 tissue samples, where performance data were prepared using RIBO-ZERO TRUSEQ® or RNA ACCESS®. Formalin-fixed and paraffin-embedded (FFPE) samples are shown as triangles; fresh frozen samples are shown as circles. Breast tumors are shown in pink; other tissues are shown in black.

[0053] [picture] [6] A graph showing the E2 induction score of ER+ / HER2- breast cancer cell lines.

[0054] [picture] [7] A graph showing the effect of E2 induction score on fulvestrant on cell growth rate. Implementation

[0055] [Cross-reference to related applications] []

[0056] This application claims priority to U.S. Provisional Application No. 62 / 719,545, filed August 17, 2018, the entire disclosure of which is incorporated herein by reference and for all purposes. [Reference to "Sequence List"] []

[0057] The full text of the sequence list (machine format IBM-PC, MS-Windows operating system) created on August 13, 2019, written in file 048893-517001WO_SEQUENCE_LISTING_ST25.txt, 560,154 bytes, is incorporated herein by reference for all purposes. [I.] [General Technology] []

[0058] The techniques and procedures described or referenced in this article are generally well-known and employed by those skilled in the art, such as, for example, the following widely used methodologies: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (edited by FM Ausubel et al., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (edited by MJ MacPherson, BD Hames, and GR Taylor, (1995)), edited by Harlow and Lane, (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (edited by RI Freshney, (1987)); Oligonucleotide Synthesis (edited by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis (edited, 1998) Academic Press; Animal Cell Culture (edited by RI Freshney, 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, JB Griffiths and DG Newell, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Edited by Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos (eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al. (eds., 1994)); Current Protocols in Immunology (JE Coligan et al. (eds., 1991)); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty. (ed., IRL Press, 1988-1989)); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean (eds., Oxford University Press, 2000)); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and JD Capra (eds., Harwood Academic Press)). Publishers, 1995); and Cancer: Principles and Practice of Oncology (edited by VT DeVita et al., JB Lippincott Company, 1993). [II.] [definition] []

[0059] It should be understood that the states and embodiments described herein include “comprising” states and embodiments, consisting of states and embodiments, and substantially consisting of states and embodiments. As used herein, unless otherwise specified, the singular forms “a / an” and “the” include a plurality of indicators.

[0060] As used herein, the term "approximately" refers to the typical range of error for each value that is readily known to those skilled in the art. The "approximately" values ​​or parameters mentioned herein include... [] (and the description) refers to an embodiment of the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0061] As used herein, “administer” means a method of delivering a dose of a compound (e.g., an endocrine therapy or composition as described herein, e.g., a pharmaceutical composition, such as a pharmaceutical composition comprising an endocrine therapy (e.g., a selective estrogen receptor modulator (SERM) (e.g., a selective estrogen receptor degrader (SERD)), a gonadotropin-releasing hormone (GnRH) agonist, and / or an aromatase inhibitor (AI))) to a subject. The compounds and / or compositions used in the methods described herein may be administered, for example, orally, intramuscularly, intravenously (e.g., by intravenous infusion), subcutaneously, percutaneously, percutaneously, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, or intravaginally. Administration methods include intraperitoneal, transrectal, transtumoral, transperitoneal, transconjunctival, transcystic, transmucosal, transpericardial, transumbilical, transocular, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by direct local perfusion of target cells, by catheter, by irrigation, and in the form of creams or lipid compositions. The method of administration may vary depending on various factors, such as the type of compound or composition being administered and the severity of the condition, disease, or symptom being treated.

[0062] The term "anticancer therapy" refers to any therapy that can be used to treat cancer (e.g., breast cancer, e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)) and / or metastatic or locally advanced breast cancer). Examples of anticancer therapeutic agents include (but are not limited to) endocrine therapies as described herein, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, apoptotic agents, anti-microtubule agents, and other agents for treating cancer, such as anti-CD20 antibodies, platelet-derived growth factor inhibitors (e.g., GLEEVEC™ (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies), other bioactive agents, and organic chemical agents and the like. Combinations thereof are also included herein. The anticancer therapy used in this article may also be referred to as "non-endocrine therapy," which in turn refers to any anticancer therapy that excludes endocrine therapy as defined in this article.

[0063] The term "endocrine therapy" refers to a treatment or therapy that can be used to regulate (e.g., modulate, reduce, block, or inhibit) the effects of one or more hormones found to cause or otherwise contribute to the progression of breast cancer as described herein. Endocrine therapies as described herein include non-hormonal and hormonal therapies, such as, for example, selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), gonadotropin-releasing hormone (GnRH) agonists, selective estrogen receptor covalent antagonists (SERCAs), selective human estrogen receptor partial agonists (ShERPA), aromatase inhibitors (AIs), or combinations thereof. In one embodiment, endocrine therapy includes one or more compounds derived from Section IV-A of this document.

[0064] Additional exemplary endocrine therapies used in the methods described herein include (but are not limited to): anti-estrogenic agents, such as tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, FARESTON® (toremifene citrate), nafoxidine, clomifene, anordrin, bazedoxifene, broparestrol, cyclofenil, lasofoxifene, ormeloxifene, acolbifene, and elacestrant. (RAD1901), clomifenoxide, etacstil, ospemifene, fulvestrant (FASLODEX®), EM800, brilanestrant (GDC-0810), LX-039, AZ9496, GDC-0927 (SRN-0927); GDC-9545, G1T48 (G1 treatment), H3B 6545 (H3 Biomedicine), SAR439859 (Sanofi), aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, MEGASE® medroxyprogesterone acetate, AROMASIN® (exemestane), formestanie, fadrozole, RIVISOR® (Vorozole), FEMARA® (letrozole), and ARIMIDEX® (anastrozole); antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxane nucleoside cytosine analogue);and antisense oligonucleotides, specifically those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, and combinations of two or more of the above.

[0065] Examples of chemotherapy agents (and as applicable non-endocrine therapies) include (but are not limited to) mammalian mTOR inhibitors of rapamycin, such as sirolimus (also known as rapamycin), temsirolimus (also known as CCI-779 or TORISEL®), everolimus (also known as RAD001 or AFINITOR®), ridaforolimus (also known as AP-23573, MK-8669 or deforolimus), OSI-027, AZD8055, and INK128; and phosphatidylinositol 3-kinase (PI3K) inhibitors, such as idelalisib (also known as GS-1101 or CAL-101), BKM120, and perifosine. (Also known as KRX-0401); phosphatidylinositol 3-kinase (PI3K) / mTOR inhibitors, such as XL765, GDC-0980, BEZ235 (also known as NVP-BEZ235), BGT226, GSK2126458, PF-04691502, and PF-05212384 (also known as PKI-587); and cyclin-dependent kinase (CDK) 4 / 6 inhibitors, such as abemaciclib (VERZENIO®), palbociclib (IBRANCE®), ribociclib (KISQALI®), and trilaciclib. (G1T28); anthracyclines, such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin; taxanes, including paclitaxel and docetaxel; podophyllotoxin; gemcitabine (GEMZAR®); 5-fluorouracil (5-FU); cyclophosphamide (CYTOXAN®); platinum analogs, such as cisplatin and carboplatin; vinorelbine (NAVELBINE®); capecitabine (XELODA®); ixabepilone (IXEMPRA®);And eribulin (HALAVEN®); ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase type 1 inhibitor; ABARELIX® rmRH; vinorelbine and esperamicin (see U.S. Patent No. 4,675,187), any of the compounds described in Section IV-A below, and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0066] Other exemplary chemotherapeutic agents (and non-endocrine therapies) include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; azacyclopropanes such as benzodopa, carboquone, meturedopa, and uredopa; ethylimines and methyl melamines, including altretamine, triethylmelamine, triethylphosphamide, triethylthiophosphamide, and trimethyltrimethylcyanamide; acetogenin (especially bullatacin and bullatacinone); and delnabinol (MARINOL®). ); β-lapachone; lapacol; colchicine; betulinic acid; camptothecin (including synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopoletin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptococcin (Specifically, cryptococcal 1 and cryptococcal 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards, such as chlorambucil, chlornaphazine, chlorphosphatamine, estramustine, ifosfamide, mechlorethamine, mechlorethamine dimethyl diethylamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωIl). (See, for example, Nicolaou et al., Angew. Chem Intl. Ed. Engl. , 33: 183-186 (1994)); CDP323, an oral α-4 integrin inhibitor; dynemicin, including dynemicin A; esperamycin;And neocarzinostatin chromophores and related chromoproteins, aldenyne antibiotic chromophores), aclacinomysin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin C, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, doxorubicin (including ADRIAMYCIN®, morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolidone doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposome doxorubicin TLC D-99) (MYOCET®), pegylated liposomal doxorubicin (CAELYX® and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid (Acid), nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate, gemcitabine (GEMZAR®), tegafur. UFTORAL®, capecitabine (XELODA®), epothilone, and 5-fluorouracil (5-FU); cobetastatin; folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine.Pyrimidine analogues, such as ancitabine, azacitidine, 6-azouridine, carmofur, cytarabine, doxifluridine, enocitabine, and fluxuridine; androgens, such as calusterone, dromostanolone, epitiostanol, mepitiostane, and testolactone; antiadrenergic agents, such as amylometazone, mitotane, and trilostane; and folic acid supplements, such as folinic acid. acid); aceglatone; aldoxyphosphatidylglycerol; aminoacetopropionic acid; eniluracil; amsacrine; bestrabucil, bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate); Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansine and Ansamitocin; Mitoguazone; Mitoantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; 2-Ethylacetazine; Procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Razoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic acid acid); triaziquone; 2,2',2'-trichlorotriethylamine;Trichothecene (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactalol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxanes, such as TAXOL® (Bristol-Myers Squibb Oncology, Princeton, NJ), TAXOL's albumin-engineered nanoparticle formulation (ABRAXANETM), and TAXOTERE® (Rhome-Poulene Rorer). Antony, France); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum preparations, such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vinca, which inhibits tubulin polymerization to prevent microtubule formation, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; donomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids, such as retinoic acid, including bexarotene (TARGRETIN®);Bisphosphonates, such as clophosphonates (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronic acid (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); trisatabine (a 1,3-dioxane-1,3-cytosine analog); antisense oligonucleotides, specifically those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R) (e.g., erlotinib (TARCEVA™)). )); and VEGF-A to reduce cell proliferation; vaccines, such as THERATOPE® vaccine and gene therapy vaccines (e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine); topoisomerase type 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib); Bayer; SU-11248 (sunitinib), SUTENT®, Pfizer); perifoxine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium. (GENASENSE®); pixantrone; EGFR inhibitors; tyrosine kinase inhibitors; serine-threonine kinase inhibitors, such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors, such as lonafarnib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisolone (abbreviations).FOLFOX is an abbreviation for a treatment regimen utilizing oxaliplatin (ELOXATINTM) in combination with 5-FU and folate, or a pharmaceutically acceptable salt, acid, or derivative thereof; and combinations of two or more of the above.

[0067] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include (but are not limited to) radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, donomycin, or other inserters); growth inhibitors; enzymes and their fragments, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including their fragments and / or variants; and various antitumor or anticancer agents disclosed below. Cytotoxic agents can be non-endocrine agents.

[0068] "Article" refers to any article (e.g., package or container) or kit containing at least one reagent, such as a drug for treating a disease or condition (e.g., cancer, e.g., breast cancer, e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), and / or a probe for specifically detecting the biomarkers described herein. In some embodiments, the article or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0069] As used herein, the term "biomarker" refers to an indicator, such as a predictive, forecasting, and / or pharmacodynamic indicator, that can be detected in a sample (e.g., a tissue sample, such as a tumor tissue sample, such as formalin-fixed and paraffin-embedded (FFPE), fresh-frozen (FF), archived, fresh, or frozen tumor tissue sample). Biomarkers can be used as indicators of specific subtypes of diseases or conditions characterized by certain molecular, pathological, histological, and / or clinical features (e.g., breast cancer, HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). In some embodiments, the biomarker is a gene or gene set. Biomarkers include (but are not limited to) polynucleotides (e.g., DNA and / or RNA), polynucleotide copy number alterations (e.g., DNA copy number), peptides, peptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrate and / or glycolipid molecular markers. Exemplary sets of biomarkers are shown in Tables 1 through 6.

[0070] In this document, the terms "biomarker signature," "signature," "biomarker performance signature," or "performance signature" are used interchangeably and refer to a combination of biomarkers whose performance is, for example, a predictive, forecasting, and / or pharmacodynamic indicator (e.g., a 41-gene signature (e.g., a combination of genes described in Tables 3 and 6), a 19-gene signature (e.g., a combination of genes described in Tables 2 and 5), or a 14-gene signature (e.g., a combination of genes described in Tables 1 and 4)). Biomarker signatures can be used as indicators of specific subtypes of diseases or conditions characterized by certain molecular, pathological, histological, and / or clinical features (e.g., cancer, such as breast cancer, such as HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or ductal B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). In some embodiments, the biomarker signature is a "genetic signature." The terms "genetic signature" and "genetic expression signature" are used interchangeably and refer to combinations of polynucleotides whose expression serves as, for example, predictive, diagnostic, and / or predictive indicators. In some embodiments, the biomarker signature is a "protein signature." The terms "protein signature" and "protein expression signature" are used interchangeably and refer to combinations of peptides whose expression serves as, for example, predictive, predictive, and / or pharmacological indicators.

[0071] Unless otherwise specified, the term "AGR3" refers to any initial pregradient 3 protein disulfide isomerase family member from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats)). The term encompasses "full-length" untreated AGR3 as well as any form of AGR3 produced by cellular treatment. The term also encompasses naturally occurring variants of AGR3, such as splice variants or paired gene variants. In this art, AGR3 is also referred to as protein disulfide isomerase family A member 18, breast cancer membrane protein 11, BCMP11, PDIA18, HAG-3, HAG3, AG-3, AG3, and pregradient protein 3 homologs. An exemplary human AGR3 nucleic acid sequence is shown under NCBI reference sequence: NM_176813.4 or in SEQ ID NO: 1. The amino acid sequence of an exemplary protein encoded by human AGR3 is shown under UniProt registration number Q8TD06 or in SEQ ID NO: 2.

[0072] Unless otherwise specified, the term "AMZ1" refers to any naïve Archaelysin family metallopeptidase 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated AMZ1 as well as any form of AMZ1 produced through cellular processing. The term also encompasses naturally occurring variants of AMZ1, such as splice variants or paired gene variants. AMZ1 is also referred to in this art as Archaemetzincin-1, Archaemetzincin-1, and KIAA1950. The nucleotide sequence of an exemplary human AMZ1 is shown under NCBI reference sequence: NM_133463.3 or in SEQ ID NO: 3. The amino acid sequence of an exemplary protein encoded by human AMZ1 is shown under UniProt registration number Q400G9 or in SEQ ID NO: 4.

[0073] Unless otherwise specified, the term "AREG" refers to any initial amphiregulin from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated AREG as well as any form of AREG produced through cellular processing. The term also encompasses naturally occurring variants of AREG, such as splice variants or paired gene variants. In this art, AREG is also referred to as colorectal cell-derived growth factor, schwannoma-derived growth factor, amphiregulin B, AREGB, CRDGF, and SDGF. The nucleic acid sequence of an exemplary human AREG is shown under NCBI reference sequence: NM_001657.3 or in SEQ ID NO: 5. The amino acid sequence of an exemplary protein encoded by human AREG is shown under UniProt registration number P15514 or in SEQ ID NO: 6.

[0074] Unless otherwise specified, the term "C5AR2" refers to any initial complement component 5a receptor 2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated C5AR2 and any form of C5AR2 produced by cellular processing. The term also encompasses naturally occurring variants of C5AR2, such as splice variants or paired gene variants. In this art, C5AR2 is also referred to as complement component 5a receptor 2, G protein-coupled receptor 77, GPR77, C5L2, C5a anaphylatoxin chemokine receptor C5L2, and GPF77. An exemplary human C5AR2 nucleic acid sequence is shown under NCBI reference sequence: NM_001271749.1 or in SEQ ID NO: 7. The amino acid sequence of an exemplary protein encoded by human C5AR2 is shown under UniProt registration number Q9P296 or in SEQ ID NO: 8.

[0075] Unless otherwise specified, the term "CELSR2" refers to any naïve cadherin EGF LAG seven-channel G receptor 2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated CELSR2 as well as any form of CELSR2 produced by cellular treatment. The term also encompasses naturally occurring variants of CELSR2, such as splice variants or paired gene variants. In this technique, CELSR2 is also referred to as multiple epidermal growth factor-like domain protein 3, adhesion G protein-coupled receptor C2, epidermal growth factor-like protein 2, multiple EGF domain-like protein 3, cadherin family member 10, Flamingo homolog 3, EGF-like protein 2, CDHF10, EGFL2, MEGF3, Flamingo1, KIAA0279, and ADGRC2. The nucleic acid sequence of an exemplary human CELSR2 is shown under NCBI reference sequence: NM_001408.2 or in SEQ ID NO: 9. The amino acid sequence of an exemplary protein encoded by human CELSR2 is shown under UniProt registration number Q9HCU4 or in SEQ ID NO: 10.

[0076] Unless otherwise specified, the term "CT62" refers to any initial cancer / testis antigen 62 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) . The term covers "full-length" untreated CT62 as well as any form of CT62 produced by cell treatment. The term also covers naturally occurring variants of CT62, such as splice variants or paired gene variants. The nucleic acid sequence of an exemplary human CT62 is shown under NCBI reference sequence: XM_006720429 or in SEQ ID NO: 11. The amino acid sequence of an exemplary protein encoded by human CT62 is shown under UniProt registration number P0C5K7 or in SEQ ID NO: 12.

[0077] Unless otherwise specified, the term "FKBP4" refers to any initial FK506-binding protein 4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated FKBP4 as well as any form of FKBP4 produced by cellular processing. The term also encompasses naturally occurring variants of FKBP4, such as splice variants or paired gene variants. In this technique, FKBP4 is also referred to as gyroisomerase, FKBP51, FKBP52, FKBP59, HBI, peptidyl-prolyl cis-trans isomerase FKBP4, T-cell FK506-binding protein (59kD), HSP-binding immunoaffinity, immunoaffinity FKBP52, PPIase FKBP4, PPIASE, Hsp56, P52, and P59. The nucleic acid sequence of an exemplary human FKBP4 is shown under NCBI reference sequence: NM_002014.3 or in SEQ ID NO: 13. The amino acid sequence of an exemplary protein encoded by human FKBP4 is shown under UniProt registration number Q02790 or in SEQ ID NO: 14.

[0078] Unless otherwise specified, the term "FMN1" refers to any initial Formin 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) . The term encompasses "full-length" untreated FMN1 as well as any form of FMN1 produced by cellular processing. The term also encompasses naturally occurring variants of FMN1, such as splice variants or paired gene variants. In this art, FMN1 is also referred to as limb deformity protein homolog, FMN, and LD. An exemplary human FMN1 nucleic acid sequence is shown under NCBI reference sequence: NM_001277313.1 or in SEQ ID NO: 15. An exemplary protein encoded by human FMN1 is shown under UniProt registration number Q68DA7 or in SEQ ID NO: 16.

[0079] Unless otherwise specified, the term "GREB1" refers to any initial estrogen regulation of breast cancer 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) . The term encompasses "full-length" untreated GREB1 and any form of GREB1 produced by cell treatment. The term also encompasses naturally occurring variants of GREB1, such as splice variants or paired gene variants. GREB1 is also referred to in this art as the gene regulatory protein of breast cancer 1 and KIAA0575. An exemplary human GREB1 nucleic acid sequence is shown under NCBI reference sequence: NM_014668.3 or in SEQ ID NO: 17. An exemplary protein encoded by human GREB1 is shown under UniProt registration number Q4ZG55 or in SEQ ID NO: 18.

[0080] Unless otherwise specified, the term "IGFBP4" refers to any naïve insulin-like growth factor binding protein 4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated IGFBP4 and any form of IGFBP4 produced by cellular treatment. The term also encompasses naturally occurring variants of IGFBP4, such as splice variants or paired gene variants. In this art, IGFBP4 is also referred to as IGF-binding protein 4, IBP-4, HT29-IGFBP, and BP-4. The nucleotide sequence of an exemplary human IGFBP4 is shown under NCBI reference sequence: NM_001552.2 or in SEQ ID NO: 19. The amino acid sequence of an exemplary protein encoded by human IGFBP4 is shown under UniProt registration number P22692 or in SEQ ID NO: 20.

[0081] Unless otherwise specified, the term "NOS1AP" refers to any initial nitric oxide synthase 1 adaptor protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated NOS1AP as well as any form of NOS1AP produced by cellular processing. The term also encompasses naturally occurring variants of NOS1AP, such as splice variants or paired gene variants. In this art, NOS1AP is also referred to as the C-terminal PDZ ligand of a neuronal nitric oxide synthase protein, nitric oxide synthase 1 (neuronal) adaptor protein, CAPON, a ligand of a neuronal nitric oxide synthase protein with a C-terminal PDZ domain, 6330408P19Rik, and KIAA0464. An exemplary human NOS1AP nucleic acid sequence is shown under NCBI reference sequence: NM_014697.2 or in SEQ ID NO: 21. The amino acid sequence of an exemplary protein encoded by human NOS1AP is shown under UniProt registration number O75052 or in SEQ ID NO: 22.

[0082]

[0005] "NXPH3" refers to any naïve neurotrophin 3 derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated NXPH3 as well as any form of NXPH3 produced through cellular processing. The term also encompasses naturally occurring variants of NXPH3, such as splice variants or paired gene variants. NXPH3 is also referred to as NPH3 and KIAA1159 in this technique. An exemplary human NXPH3 nucleic acid sequence is shown under NCBI reference sequence: NM_007225.2 or in SEQ ID NO: 23. An exemplary protein encoded by human NXPH3 is shown under UniProt registration number O95157 or in SEQ ID NO: 24.

[0083] "OLFM1" refers to any initial olfactomedin 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) . The term encompasses "full-length" untreated OLFM1 as well as any form of OLFM1 produced by cell treatment. The term also encompasses naturally occurring variants of OLFM1, such as splice variants or paired gene variants. In this technique, OLFM1 is also referred to as neuronal olfactomedin-associated ER localizer, Noelin, NOE1, olfactomedin-associated ER localizer, neuroblastoma protein, Pancortin 1, Pancortin, NOELIN1, NOEL1, OlfA, and AMY. An exemplary human OLFM1 nucleic acid sequence is shown under NCBI reference sequence: NM_014279.4 or in SEQ ID NO: 25. The amino acid sequence of an exemplary protein encoded by human OLFM1 is shown under UniProt registration number Q99784 or in SEQ ID NO: 26.

[0084] Unless otherwise specified, the term "PGR" refers to any naïve progesterone receptor from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated PGR as well as any form of PGR produced by cellular processing. The term also encompasses naturally occurring variants of PGR, such as splice variants or paired gene variants. In this art, PGR is also referred to as nuclear receptor subfamily 3C member 3, NR3C3, and PR. The nucleic acid sequence of an exemplary human PGR is shown under NCBI reference sequence: NM_000926.4 or in SEQ ID NO: 27. The amino acid sequence of an exemplary protein encoded by a human PGR is shown under UniProt registration number P06401 or in SEQ ID NO: 28.

[0085] Unless otherwise specified, the term "PPM1J" refers to any initial protein phosphatase Mg2+ / Mn2+ dependent 1J from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats)). This term encompasses "full-length" untreated PPM1J and any form of PPM1J produced by cell treatment. The term also encompasses naturally occurring variants of PPM1J, such as splice variants or paired gene variants. In this art, PPM1J is also referred to as protein phosphatase 1J (containing the PP2C domain), protein phosphatase 2Cζ, EC 3.1.3.16, PP2C-ζ, protein phosphatase 2a, catalytic subunit, ζ isoform, protein phosphatase 1J, PP2Cζ, and PP2CZ. An exemplary human PPM1J nucleic acid sequence is shown under NCBI reference sequence: NM_005167.5 or in SEQ ID NO: 29. The amino acid sequence of an exemplary protein encoded by human PPM1J is shown under UniProt register number Q5JR12 or in SEQ ID NO: 30.

[0086] Unless otherwise specified, the term "RAPGEFL1" refers to any initial Rap-like guanine nucleotide exchange factor 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated RAPGEFL1 and any form of RAPGEFL1 produced through cellular treatment. The term also encompasses naturally occurring variants of RAPGEFL1, such as splice variants or paired gene variants. In this art, RAPGEFL1 is also referred to as linked guanine nucleotide exchange factor II, Rap-like guanine nucleotide exchange factor (GEF) 1, and linked GEFII. An exemplary human RAPGEFL1 nucleic acid sequence is shown under NCBI reference sequence: NM_001303533.1 or in SEQ ID NO: 31. The amino acid sequence of an exemplary protein encoded by human RAPGEFL1 is shown under UniProt register number Q9UHV5 or in SEQ ID NO: 32.

[0087] Unless otherwise specified, the term "RBM24" refers to any initial RNA-binding motif protein 24 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated RBM24 as well as any form of RBM24 produced through cellular processing. The term also encompasses naturally occurring variants of RBM24, such as splice variants or paired gene variants. In this technique, RBM24 is also referred to as containing RNA-binding regions (RNP1, RRM) 6, RNPC6, RNA-binding protein 24, and DJ259A10.1. An exemplary human RBM24 nucleic acid sequence is shown under NCBI reference sequence: NM_001143942.1 or in SEQ ID NO: 33. The amino acid sequence of an exemplary protein encoded by human RBM24 is shown under UniProt register number Q9BX46 or in SEQ ID NO: 34.

[0088] Unless otherwise specified, the term "RERG" means any initial RAS-like estrogen-regulated growth inhibitor derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers "full-length" untreated RERG as well as any form of RERG produced by cellular treatment. The term also covers naturally occurring variants of RERG, such as splice variants or paired gene variants. The nucleic acid sequence of an exemplary human RERG is shown under NCBI reference sequence: NM_032918.2 or in SEQ ID NO: 35. The amino acid sequence of an exemplary protein encoded by human RERG is shown under UniProt registration number Q96A58 or in SEQ ID NO: 36.

[0089] Unless otherwise specified, the term "RET" refers to any naïve Ret proto-oncogene from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated RET as well as any form of RET produced by cell treatment. The term also encompasses naturally occurring variants of RET, such as splice variants or paired gene variants. In this technique, RET is also referred to as cadherin-associated family member 16, transfection rearrangement, RET receptor tyrosine kinase, cadherin family member 12, proto-oncogene C-Ret, EC 2.7.10.1, CDHF12, CDHR16, PTC, multiple endocrine neoplasia and medullary thyroid carcinoma 1, EC 2.7.10, RET-ELE1, HSCR1, MEN2A, MEN2B, RET51, and MTC1. The nucleic acid sequence of an exemplary human RET is shown under NCBI reference sequence: NM_020975.5 or in SEQ ID NO: 37. The amino acid sequence of an exemplary protein encoded by human PET is shown under UniProt registration number P07949 or in SEQ ID NO: 38.

[0090] Unless otherwise specified, the term "SGK3" refers to any initial serum / glucocorticoid-regulated kinase family member 3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated SGK3 as well as any form of SGK3 produced by cellular treatment. The term also encompasses naturally occurring variants of SGK3, such as splice variants or paired gene variants. SGK3 is also referred to in this art as cytokine-independent survival kinase, EC 2.7.11.1, SGKL, CISK, EC 2.7.11, serine / threonine protein kinase Sgk3, and SGK2. An exemplary human SGK3 nucleic acid sequence is shown under NCBI reference sequence: NM_001033578.2 or in SEQ ID NO: 39. The amino acid sequence of an exemplary protein encoded by human SGK3 is shown under UniProt registration number Q96BR1 or in SEQ ID NO: 40.

[0091] Unless otherwise specified, the term "SLC9A3R1" refers to any initial SLC9A3 regulator from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats)1. The term covers "full-length" untreated SLC9A3R1 as well as any form of SLC9A3R1 produced by cellular treatment. The term also covers naturally occurring variants of SLC9A3R1, such as splice variants or paired gene variants. In this technology, SLC9A3R1 is also referred to as solute carrier family 9, subfamily A (NHE3, antiporter 3), member 3 regulator 1, Na(+) / H(+) exchange regulatory cofactor, NHERF-1, EBP50, NHERF, Ezrin-Radixin-Moesin-binding phosphoprotein-50, sodium-hydrogen exchange regulator 1, and NPHLOP2. An exemplary human SLC9A3R1 nucleic acid sequence is shown under NCBI reference sequence: NM_004252.4 or in SEQ ID NO: 41. An exemplary protein encoded by human SLC9A3R1 is shown under UniProt accession number O14745 or in SEQ ID NO: 42.

[0092] Unless otherwise specified, the term "TFF1" refers to any initial trefoil factor 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated TFF1 as well as any form of TFF1 produced through cellular treatment. The term also encompasses naturally occurring variants of TFF1, such as splice variants or paired gene variants. TFF1 is also referred to in this art as breast cancer estrogen-inducible protein, polypeptide P1.A, protein PS2, HP1.A, PNR-2, BCEI, PS2, gastrointestinal trefoil protein PS2, D21S21, and HPS2. An exemplary human TFF1 nucleic acid sequence is shown under NCBI reference sequence: NM_003225.2 or in SEQ ID NO: 43. The amino acid sequence of an exemplary protein encoded by human TFF1 is shown under UniProt registration number P04155 or in SEQ ID NO: 44.

[0093] Unless otherwise specified, the term "ZNF703" refers to any initial zinc finger protein 703 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated ZNF703 and any form of ZNF703 produced through cellular processing. The term also encompasses naturally occurring variants of ZNF703, such as splice variants or paired gene variants. In this art, ZNF703 is also referred to as zinc finger elbow-associated proline domain protein 1, ZEPPO1, ZPO1, ZNF503L, and NLZ1. Exemplary human ZNF703 nucleic acid sequences are shown under NCBI reference sequence: NM_025069.2 or in SEQ ID NO: 45. The amino acid sequence of an exemplary protein encoded by human ZNF703 is shown under UniProt register number Q9H7S9 or in SEQ ID NO: 46.

[0094] Unless otherwise specified, the term "BAMBI" refers to any initial BMP and activin membrane-binding inhibitor from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated BAMBI and any form of BAMBI produced by cellular processing. The term also encompasses naturally occurring variants of BAMBI, such as splice variants or paired gene variants. In this art, BAMBI is also referred to as the putative transmembrane protein NMA, nontransferable gene A protein, and homologs of NMA and BMP and activin membrane-binding inhibitors. The nucleic acid sequence of an exemplary human BAMBI is shown under NCBI reference sequence: NM_012342.2 or in SEQ ID NO: 47. The amino acid sequence of an exemplary protein encoded by human BAMBI is shown under UniProt registration number Q13145 or in SEQ ID NO: 48.

[0095] Unless otherwise specified, the term "BCAS1" refers to any initial breast cancer amplification sequence1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated BCAS1 and any form of BCAS1 produced by cell processing. The term also encompasses naturally occurring variants of BCAS1, such as splice variants or paired gene variants. In this art, BCAS1 is also referred to as amplified and overexpressed in breast cancer, novel amplification in breast cancer1, AIBC1, and NABC1. An exemplary human BCAS1 nucleic acid sequence is shown under NCBI reference sequence: NM_003657.3 or in SEQ ID NO: 49. An exemplary protein encoded by human BCAS1 is shown under UniProt registration number O75363 or in SEQ ID NO: 50.

[0096] Unless otherwise specified, the term "CCNG2" refers to any naïve cyclin G2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers "full-length" untreated CCNG2 as well as any form of CCNG2 produced by cell treatment. The term also covers naturally occurring variants of CCNG2, such as splice variants or paired gene variants. An exemplary human CCNG2 nucleic acid sequence is shown under NCBI reference sequence: XM_011532399.2 or in SEQ ID NO: 51. An exemplary protein encoded by human CCNG2 is shown under UniProt registration number Q16589 or in SEQ ID NO: 52.

[0097] Unless otherwise specified, the term "DDIT4" refers to any initial DNA damage from any vertebrate source (including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats)) that induces transcript 4. The term encompasses "full-length" untreated DDIT4 as well as any form of DDIT4 produced by cellular treatment. The term also encompasses naturally occurring variants of DDIT4, such as splicing variants or paired gene variants. DDIT4 is also referred to in this art as developmental regulatory proteins and DNA damage response 1, HIF-1 response proteins RTP801, REDD1, RTP801, and Dig2. The nucleotide sequence of an exemplary human DDIT4 is shown under NCBI reference sequence: NM_019058.3 or in SEQ ID NO: 53. The amino acid sequence of an exemplary protein encoded by human DDIT4 is shown under UniProt registration number Q9NX09 or in SEQ ID NO: 54.

[0098] Unless otherwise specified, the term "EGLN3" refers to any naïve Egl-9 family hypoxia-inducible factor 3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats)). The term encompasses "full-length" untreated EGLN3 as well as any form of EGLN3 produced by cellular treatment. The term also encompasses naturally occurring variants of EGLN3, such as splice variants or paired gene variants. In this technique, EGLN3 is also referred to as protein 3 containing a prolyl hydroxylase domain, hypoxia-inducible factor prolyl hydroxylase 3, HIF-prolyl hydroxylase 3, HPH-1, HPH-3, PHD3, Egl 9-like protein 3 isoform, Egl 9 homolog 3, EC 1.14.11.29, EC 1.14.11, HIFP4H3, and HIFPH3. The nucleic acid sequence of an exemplary human EGLN3 is shown under NCBI reference sequence: NM_022073.3 or in SEQ ID NO: 55. The amino acid sequence of an exemplary protein encoded by human EGLN3 is shown under UniProt registration number Q9H6Z9 or in SEQ ID NO: 56.

[0099] Unless otherwise specified, the term "FAM171B" refers to any initially sequence-similar member of family 171B from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers "full-length" untreated FAM171B and any form of FAM171B produced by cellular processing. The term also covers naturally occurring variants of FAM171B, such as splice variants or paired gene variants. In this art, FAM171B is also referred to as KIAA1946 and the protein FAM171B. An exemplary human FAM171B nucleic acid sequence is shown under NCBI reference sequence: NM_177454.3 or in SEQ ID NO: 57. An exemplary protein encoded by human FAM171B is shown under UniProt registration number Q6P995 or in SEQ ID NO: 58.

[0100] Unless otherwise specified, the term "GRM4" refers to any initial glutamate metabolite receptor 4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated GRM4 as well as any form of GRM4 produced by cellular processing. The term also encompasses naturally occurring variants of GRM4, such as splice variants or paired gene variants. GRM4 is also referred to as GPRC1D, MGLUR4, and MGlu4 in this art. An exemplary human GRM4 nucleic acid sequence is shown under NCBI reference sequence: NM_000841.4 or in SEQ ID NO: 59. An exemplary protein encoded by human GRM4 is shown under UniProt registration number Q14833 or in SEQ ID NO: 60.

[0101] Unless otherwise specified, the term "IL1R1" refers to any naïve interleukin-1 receptor type 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated IL1R1 as well as any form of IL1R1 produced by cellular treatment. The term also encompasses naturally occurring variants of IL1R1, such as splice variants or paired gene variants. In this technique, IL1R1 is also referred to as CD121 antigen family member A, interleukin-1 receptor α, IL-1R-α, IL1RA, IL1R, P80, CD121a antigen, D2S1473, CD121A, and IL1RT1. An exemplary human IL1R1 nucleic acid sequence is shown under NCBI reference sequence: NM_001288706.1 or in SEQ ID NO: 61. The amino acid sequence of an exemplary protein encoded by human IL1R1 is shown under UniProt register number P14778 or in SEQ ID NO: 62.

[0102] Unless otherwise specified, the term "LIPH" refers to any initial lipase H from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated LIPH as well as any form of LIPH produced by cellular processing. The term also encompasses naturally occurring variants of LIPH, such as splice variants or paired gene variants. LIPH is also referred to in this art as membrane-associated phosphatidic acid selective phospholipase A1-α, PD lipase-associated protein, phospholipase A1 member B, MPA-PLA1 α, LPDLR, membrane-bound phosphatidic acid selective phospholipase A1, lipase member H, EC 3.1.1.3, C 3.1., ARWH2, HYPT7, LAH2, and AH. Exemplary human LIPH nucleic acid sequences are shown under NCBI reference sequence: XM_006713529.4 or in SEQ ID NO: 63. The amino acid sequence of an exemplary protein encoded by human LIPH is shown under UniProt registration number Q8WWY8 or in SEQ ID NO: 64.

[0103] Unless otherwise specified, the term "NBEA" refers to any initial protein kinase-anchored protein antibody (Neurobeachin) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated NBEA as well as any form of NBEA produced through cell processing. The term also encompasses naturally occurring variants of NBEA, such as splice variants or paired gene variants. NBEA is also referred to in this art as lysosomal transport regulator 2, BCL8B, LYST2, protein BCL8B, EC 1.14.14.5, EC 6.1.1.11, and KIAA1544. Exemplary human NBEA nucleic acid sequences are shown under NCBI reference sequence: NM_015678.4 or in SEQ ID NO: 65. The amino acid sequence of an exemplary protein encoded by human NBEA is shown under UniProt registration number Q8NFP9 or in SEQ ID NO: 66.

[0104] Unless otherwise specified, the term "PNPLA7" refers to any initial phospholipase domain 7 containing the potato tuber storage protein (Patatin) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated PNPLA7 as well as any form of PNPLA7 produced by cell treatment. The term also encompasses naturally occurring variants of PNPLA7, such as splice variants or paired gene variants. In this art, PNPLA7 is also referred to as C9orf111, protein 7 containing the potato tuber storage protein phospholipase domain, chromosome 9 open reading frame 111, EC 3.1.1.5, NTE-R1, and NTEL1. An exemplary human PNPLA7 nucleic acid sequence is shown under NCBI reference sequence: NM_001098537.2 or in SEQ ID NO: 67. The amino acid sequence of an exemplary protein encoded by human PNPLA7 is shown under UniProt register number Q6ZV29 or in SEQ ID NO: 68.

[0105] Unless otherwise specified, the term "PSCA" refers to any initial prostate stem cell antigen from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated PSCA as well as any form of PSCA produced through cell processing. The term also encompasses naturally occurring variants of PSCA, such as splice variants or paired gene variants. PSCA is also referred to as PRO232 in this technique. The nucleic acid sequence of an exemplary human PSCA is shown under NCBI reference sequence: NM_005672.4 or in SEQ ID NO: 69. The amino acid sequence of an exemplary protein encoded by human PSCA is shown under UniProt registration number O43653 or in SEQ ID NO: 70.

[0106] Unless otherwise specified, the term "SEMA3E" refers to any naïve semaphorin 3E from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated SEMA3E as well as any form of SEMA3E produced through cellular processing. The term also encompasses naturally occurring variants of SEMA3E, such as splice variants or paired gene variants. In this art, SEMA3E is also referred to as the Sema domain, immunoglobulin domain (Ig), short base domain, secreted (semaphorin) 3E, SEMAH, semaphorin-3E, M-Sema H, KIAA0331, M-SemaK, and Coll-5. An exemplary human SEMA3E nucleic acid sequence is shown under NCBI reference sequence: NM_012431.2 or in SEQ ID NO: 71. The amino acid sequence of an exemplary protein encoded by human SEMA3E is shown under UniProt registration number O15041 or in SEQ ID NO: 72.

[0107] Unless otherwise specified, the term "SSPO" refers to any initial SCO-spondin from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated SSPO as well as any form of SSPO produced through cellular processing. The term also encompasses naturally occurring variants of SSPO, such as splice variants or paired gene variants. SSPO is also referred to in this art as SCO protein, containing the Thrombospondin domain, sub-organ spondin, EC 3.4.24.82, EC 3.4.21.9, and KIAA2036. An exemplary human SSPO nucleic acid sequence is shown under NCBI reference sequence: BN000852.1 or in SEQ ID NO: 73. The amino acid sequence of an exemplary protein encoded by human SSPO is shown under UniProt registration number A2VEC9 or in SEQ ID NO: 74.

[0108] Unless otherwise specified, the term "STON1" refers to any initial stonein 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated STON1 and any form of STON1 produced by cell treatment. The term also encompasses naturally occurring variants of STON1, such as splice variants or paired gene variants. STON1 is also referred to in this art as Stoned B-like factor, SALF, SBLF, STN1, Stoned B homolog 1, and STNB1. The nucleotide sequence of an exemplary human STON1 is shown under NCBI reference sequence: NM_001198595.1 or in SEQ ID NO: 75. The amino acid sequence of an exemplary protein encoded by human STON1 is shown under UniProt registration number Q9Y6Q2 or in SEQ ID NO: 76.

[0109] Unless otherwise specified, the term "TGFB3" refers to any initial transgenic growth factor β3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated TGFB3 as well as any form of TGFB3 produced through cellular processing. The term also encompasses naturally occurring variants of TGFB3, such as splice variants or paired gene variants. In this art, TGFB3 is also referred to as pretransgenic growth factor β-3, arrhythmic right ventricular dysplasia 1, TGF-β-3, ARVD1, LDS5, RNHF, and ARVD. The nucleotide sequence of an exemplary human TGFB3 is shown under NCBI reference sequence: NM_003239.4 or in SEQ ID NO: 77. The amino acid sequence of an exemplary protein encoded by human TGFB3 is shown under UniProt register number P10600 or in SEQ ID NO: 78.

[0110] Unless otherwise specified, the term "TP53INP1" refers to any initial tumor protein P53-inducible nucleoprotein 1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) . The term encompasses "full-length" untreated TP53INP1 as well as any form of TP53INP1 produced by cellular treatment. The term also encompasses naturally occurring variants of TP53INP1, such as splice variants or paired gene variants. TP53INP1 is also referred to in this art as P53-dependent damage-inducible nucleoprotein 1, stress-induced protein, P53DINP1, SIP, P53-inducible P53DINP1, TP53DINP1, TP53INP1A, TP53INP1B, and Teap. An exemplary human TP53INP1 nucleic acid sequence is shown under NCBI reference sequence: NM_033285.3 or in SEQ ID NO: 79. The amino acid sequence of an exemplary protein encoded by human TP53INP1 is shown under UniProt registration number Q96A56 or in SEQ ID NO: 80.

[0111] Unless otherwise specified, the term "TP53INP2" refers to any initial tumor protein P53-inducible nucleoprotein 2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated TP53INP2 as well as any form of TP53INP2 produced by cellular processing. The term also encompasses naturally occurring variants of TP53INP2, such as splice variants or paired gene variants. TP53INP2 is also referred to in this art as P53-inducible protein U, C20orf110, PIG-U, PINH, DOR, chromosome 20 open reading frame 110, diabetes and obesity regulatory gene, and DJ1181N3.1. An exemplary human TP53INP2 nucleic acid sequence is shown under NCBI reference sequence: NM_021202.2 or in SEQ ID NO: 81. The amino acid sequence of an exemplary protein encoded by human TP53INP2 is shown under UniProt registration number Q8IXH6 or in SEQ ID NO: 82.

[0112] Unless otherwise specified, the term "GUSB" refers to any initial glucuronidase β from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated GUSB as well as any form of GUSB produced by cellular processing. The term also encompasses naturally occurring variants of GUSB, such as splice variants or paired gene variants. GUSB is also referred to in this art as EC 3.2.1.31, β-G1, β-D-glucuronidase, MPS7, and BG. The nucleic acid sequence of an exemplary human GUSB is shown under NCBI reference sequence: NM_000181.3 or in SEQ ID NO: 83. The amino acid sequence of an exemplary protein encoded by human GUSB is shown under UniProt registration number P08236 or in SEQ ID NO: 84.

[0113] Unless otherwise specified, the term "PPIA" refers to any initial peptidyl-prolyl isomerase A from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" untreated PPIA as well as any form of PPIA produced through cellular processing. The term also encompasses naturally occurring variants of PPIA, such as splice variants or paired gene variants. In this technique, PPIA is also referred to as Cyclosporin A binding protein, Cyclophilin A, gyrase A, EC 5.2.1.8, PPIase A, CYPA, epididymal secretory sperm-binding protein Li 69p, peptidyl-prolyl cis-trans isomerase A, T-cell cyclophilin, HEL-S-69p, and CYPH. The nucleic acid sequence of an exemplary human PPIA is shown under NCBI reference sequence: NM_021130.4 or in SEQ ID NO: 85. The amino acid sequence of an exemplary protein encoded by human PPIA is shown under UniProt registration number P62937 or in SEQ ID NO: 86.

[0114] Unless otherwise specified, the term "UBC" refers to any initial ubiquitin C from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" untreated UBC as well as any form of UBC produced through cellular processing. The term also encompasses naturally occurring variants of UBC, such as splice variants or paired gene variants. UBC is also referred to as polyubiquitin-C and HMG20 in this technique. The nucleic acid sequence of an exemplary human UBC is shown under NCBI reference sequence: NM_021009.6 or in SEQ ID NO: 87. The amino acid sequence of an exemplary protein encoded by human UBC is shown under UniProt registration number P0CG48 or in SEQ ID NO: 88.

[0115] Unless otherwise specified, the term "SDHA" refers to succinate dehydrogenase complex flavoprotein subunit A from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers "full-length" untreated SDHA as well as any form of SDHA produced through cellular processing. The term also covers naturally occurring variants of SDHA, such as splice variants or paired gene variants. The nucleic acid sequence of an exemplary human SDHA is shown under NCBI reference sequence: NM_001330758 or in SEQ ID NO: 89. The amino acid sequence of an exemplary protein encoded by human SDHA is shown under UniProt registration number P31040 or in SEQ ID NO: 90.

[0116] The terms "cancer" and "carcinoma" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include (but are not limited to) carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of these cancers include (but are not limited to) breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS and / or metastatic or locally advanced breast cancer); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; bladder cancer (e.g., urethral epithelial bladder cancer (UBC), muscle-invasive bladder cancer (MIBC), and BCG-refractory non-muscle-invasive bladder cancer (NMIBC)); kidney cancer (e.g., renal cell carcinoma (RCC)); urethral cancer; prostate cancer, such as castration-resistant prostate cancer (CRPC); peritoneal cancer; hepatocellular carcinoma; gastric cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; hepatocellular carcinoma; colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; prostate cancer; vulvar cancer; thyroid cancer; hepatic cancer. Carcinoma; anal cancer; penile cancer; melanoma, including superficial diffuse melanoma, lentiginesic malignant melanoma, peripheral lentiginesic melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL) and small lymphocytic (SL) lymphoma). NHL, moderately malignant / follicular NHL, moderately malignant diffuse NHL, highly malignant immunoblastic NHL, highly malignant lymphoblastic NHL, highly malignant small non-lytic cell NHL, mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic medulloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndrome (MDS), as well as abnormal angiogenesis, edema (such as those associated with brain tumors), Meigs' syndrome, brain cancer, head and neck cancer and related metastases associated with leukoblastic disease.

[0117] As used herein, the term "breast cancer" refers to a histologically or cytologically confirmed cancer of the breast. In some embodiments, the breast cancer is carcinoma. In some embodiments, the breast cancer is adenocarcinoma. In some embodiments, the breast cancer is sarcoma. In some embodiments, the breast cancer is HR+ breast cancer. In some embodiments, the HR+ breast cancer is ER+ breast cancer. In some embodiments, the ER+ breast cancer is ductal A type breast cancer. In some embodiments, the ER+ breast cancer is ductal B type breast cancer. In some embodiments, the breast cancer is metastatic or locally advanced breast cancer.

[0118] "Locally advanced breast cancer" refers to cancer that has spread from the site of origin of breast cancer to nearby tissues or lymph nodes, but not to other parts of the body.

[0119] The term "metastatic breast cancer" refers to cancer that has spread from the breast to other parts of the body, such as bones, liver, lungs, or brain. Metastatic breast cancer may also be referred to as stage IV breast cancer.

[0120] The term "ductal carcinoma in situ" or (DCIS cancer) refers to breast cancer known in this technique as being intraductal, non-avoidable, and pre-invasive, pre-existing primary tumor.

[0121] The terms "cell proliferation disorder" and "proliferative disorder" refer to a condition associated with a certain degree of abnormal cell proliferation. In one embodiment, the cell proliferation disorder is breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). In another embodiment, the cell proliferation disorder is a tumor.

[0122] As used herein, the term "combined administration" refers to the administration of two or more therapeutic agents, wherein the administration of these agents overlaps at least partially in time. Therefore, combined administration includes a dosing regimen in which one or more agents are continued after the administration of one or more other agents has been discontinued.

[0123] As used herein, “delayed progression” of a condition or disease means the postponement, inhibition, slowing, deceleration, stabilization, and / or delay of the development of a disease or condition (e.g., breast cancer, such as HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). This delay can be of varying lengths, depending on the patient’s medical history and / or the patient’s current treatment. It will be apparent to those skilled in this technique that a sufficient or significant delay can effectively cover prevention, as the patient does not develop the disease.

[0124] The terms “determination”, “detection”, and their grammatical variations include any method of determination or detection, including direct and indirect determination or detection.

[0125] "Symptom" or "disease" means any condition from which one would benefit from treatment, including (but not limited to) chronic and acute symptom or disease, including those pathological conditions that predispose mammals to the symptom in question (e.g., cancer, e.g., breast cancer, e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal type A or ductal type B breast cancer)), DCIS and / or metastatic or locally advanced breast cancer).

[0126] As used in this article, the term "diagnosis" refers to the identification or classification of a molecular or pathological state, disease, or symptom (e.g., cancer, e.g., breast cancer, e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal type A or ductal type B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). For example, "diagnosis" can refer to the identification of a specific type of breast cancer. "Diagnosis" can also refer to the classification of a specific subtype of breast cancer, e.g., by histopathological criteria, or by a subtype characterized by a combination of molecular features (e.g., by the expression of a combination of biomarkers (e.g., specific genes or proteins encoded by those genes)).

[0127] An "effective amount" of a compound (e.g., an endocrine therapy as described herein) or a combination thereof (e.g., a pharmaceutical composition) is the minimum amount required to achieve a measurable increase in overall survival (OS) or progression-free survival (PFS) for a desired therapeutic or preventative outcome, such as a specific disease or condition (e.g., breast cancer, e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). An effective amount, as used herein, may vary depending on factors such as an individual's disease state, age, sex, and weight, and the antibody's ability to elicit a desired response in a subject. An effective amount is also any toxic or adverse effect that is exceeded by a therapeutically effective effect. For preventative use, beneficial or desired outcomes include outcomes such as eliminating or reducing the risk of disease (including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during disease development), reducing disease severity, or delaying disease onset. An effective amount may be administered in one or more doses. For the purposes described herein, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventative or therapeutic treatment. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be combined with another drug, compound, or pharmaceutical composition. Therefore, an "effective amount" can be considered in the context of administration of one or more therapeutic agents, and it can be assumed that if combined with one or more other agents, the effective amount provides a single agent that achieves or achieves the desired result. For example, an effective amount of endocrine therapy for cancer treatment, as described herein, may reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow down to a certain extent and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow down to a certain extent and preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more of the symptoms associated with the disease to a certain extent. To the extent that a drug can prevent growth and / or kill existing cancer cells, it may be cytotoxic and / or cell-inhibiting. For cancer therapy, in vivo efficacy can be measured, for example, by assessing the duration of survival, time to disease progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0128] In this document, the terms "expression level," "quantity," or "level" of biomarkers used interchangeably refer to the detectable level of a biological sample. "Expression" generally refers to the process of converting information (e.g., genetically encoded and / or exogenous) into structures present and manipulated within the cell. Therefore, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modification of a polypeptide) should also be considered expressions, regardless of whether they originate from transcripts produced by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides, such as proteolysis. "Expressed genes" include those transcribed into polynucleotides such as mRNA and then translated into polypeptides, and those also transcribed into RNA but not translated into polypeptides (e.g., transport and ribosomal RNA). The degree of expression can be measured using methods known to those skilled in the art and also disclosed herein. The degree or level of expression of a biomarker can be used to identify / characterize subjects with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer) who respond to or benefit from a particular therapy (e.g., therapies including endocrine therapies (e.g., SERMs (e.g., SERDs), GnRH agonists, and / or AIs)). The degree or level of expression of the biomarkers provided herein in subjects with the breast cancers described herein can also be used to determine and / or track the efficacy of administered endocrine therapies over time.

[0129] Performance levels can be measured using detection and techniques suitable for measuring RNA levels. For example, performance levels can be measured using RNA-Seq kits, which are applicable to kits as described herein. Exemplary techniques that can be used to measure performance levels herein include (but are not limited to) the RNA ACCESS® protocol or the TRUSEQ® RIBO-ZERO® protocol (ILLUMINA®), RT-qPCR, qPCR, multiplex qPCR (e.g., fluidigm), nanowire technology, RT-qPCR, microarray analysis, SAGE, or MassARRAY.

[0130] As used herein, the term "E2-induction score" refers to a numerical value reflecting the degree of aggregation of a predetermined set of genes, the induction of which reflects estrogen receptor (ER) pathway activity. For example, an E2-induction score may reflect at least 5, 6, 7, or 8 of the genes listed in Table 1 (i.e., AMZ1, C5AR2, CELSR2, FKBP4, GREB1, OLFM1, SLC9A3R1, and TFF1), at least 5, 6, 7, 8, 9, 10, or 11 of the genes listed in Table 2 (i.e., AMZ1, AREG, C5AR2, CELSR2, FKBP4, FMN1, GREB1, OLFM1, RBM24, SLC9A3R1, and TFF1), or at least 5, 6, 7, 8, 9, 10, or 11 of the genes listed in Table 3. The degree of aggregation of genes 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 (i.e., AGR3, AMZ1, AREG, C5AR2, CELSR2, CT62, FKBP4, FMN1, GREB1, IGFBP4, NOS1AP, NXPH3, OLFM1, PGR, PPM1J, RAPGEFL1, RBM24, RERG, RET, SGK3, SLC9A3R1, TFF1, and ZNF703) reflects the activity of the estrogen receptor (ER) pathway. The degree of aggregation of a predetermined gene set can be determined (e.g.) by measuring the mean z-score of the predetermined gene set obtained from samples (e.g., tissue samples, such as tumor tissue samples, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples) obtained from individuals (e.g., individuals with cancer, such as breast cancer (e.g., breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or ductal B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer)). However, it should be understood that methods known in this art, such as, for example, the mean expression of genes described in a reference population space, or, for example, the degree of aggregation relative to the mean expression of genes in untreated or mediated tumors (expressed as fold change, as appropriate), can be used to determine the degree of aggregation.

[0131] As used herein, the "E2 repression score" refers to a numerical value reflecting the degree of aggregation of a predetermined set of genes, the inhibition of which reflects estrogen receptor (ER) pathway activity. For example, the E2 repression score may reflect at least 3, 4, 5, or 6 of the genes listed in Table 4 (i.e., BCAS1, CCNG2, IL1R1, PNPLA7, SEMA3E, and STON1), at least 4, 5, 6, 7, or 8 of the genes listed in Table 5 (i.e., BCAS1, CCNG2, IL1R1, NBEA, PNPLA7, SEMA3E, STON1, and TP53INP1), or at least 4, 5, 6, 7, 8, 9, or 1 of the genes listed in Table 6. The degree of aggregation of genes 0, 11, 12, 13, 14, 15, 16, 17, or 18 (i.e., BAMBI, BCAS1, CCNG2, DDIT4, EGLN3, FAM171B, GRM4, IL1R1, LIPH, NBEA, PNPLA7, PSCA, SEMA3E, SSPO, STON1, TGFB3, TP53INP1, and TP53INP2) is calculated, and the inhibition of these genes reflects estrogen receptor (ER) pathway activity. The degree of aggregation of a predetermined gene set can be calculated as described above for E2 induction scoring of HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or ductal B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer.

[0132] The "Estrogen Receptor Pathway Activity Score," "ER Pathway Activity Score," and "Comprehensive ER Pathway Activity Score" refer to numerical values ​​that reflect the mathematical difference between the E2 induction score and the E2 inhibition score. The ER pathway activity score can be used as a predictive, forecasting, and / or pharmacodynamic biomarker (e.g., to identify individuals with breast cancer who may benefit from treatments including endocrine therapy or to monitor the responsiveness of individuals with breast cancer to treatments including endocrine therapy).

[0133] "Reference Estrogen Receptor Pathway Activity Score" and "Reference ER Pathway Activity Score" refer to ER pathway activity scores compared with another ER pathway activity score, for example, to make predictions, forecasts, and / or treatment decisions. For example, the reference ER pathway activity score could be the ER pathway activity score of a reference sample, the ER pathway activity score of a reference population (e.g., a population of patients with HR+ breast cancer), and / or a predetermined value. In some instances, the reference ER pathway activity score is a cutoff value that significantly separates individuals with breast cancer who have ER pathway activity from those with breast cancer who have low or no ER pathway activity (e.g., a reference ER pathway activity score equal to or higher than -1.0 (e.g., -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, or higher)). In some instances, the reference ER pathway activity score is a cutoff value that significantly distinguishes individuals with breast cancer who respond to treatment including endocrine therapy as described herein from those who do not (e.g., a reference ER pathway activity score equal to or greater than -1.0 (e.g., -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, or higher)). Those skilled in the art will understand that the value of the reference ER pathway activity score can depend on variations in: the type of breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or ductal B breast cancer)), DCIS and / or metastatic or locally advanced breast cancer), the method used to measure the ER pathway activity score, the specific genetic characteristics examined (e.g., combinations of genes described in Tables 1 to 6), and / or the statistical methods used to generate the ER pathway activity score. For example, the activity score described in this paper can be calculated by calculating the z-score of the reference population and using the following formula to rescale the expression of each gene across samples to a mean of 0 and a standard deviation of 1. The performance data of a given patient can then be superimposed onto the z-score reference space as described in this paper.

[0134] The z-score can be described by the formula: z = (x - μ) / σ, where z is the rescaled score, x is the measured gene expression level, μ is the mean gene expression calculated from the self-reference population, and σ is the standard deviation of gene expression calculated from the self-reference population.

[0135] In some embodiments, a reference estrogen receptor pathway activity score is calculated with reference to a standard control as defined herein.

[0136] The term "reference E2-inducible activity score" refers to an E2-inducible activity score that is compared with another E2-inducible activity score, for example, to make predictions, forecasts, and / or treatment decisions. For example, the reference E2-inducible activity score could be the reference E2-inducible activity score of a reference sample, the reference E2-inducible activity score of a reference population (e.g., a population of patients with HR+ breast cancer), and / or a predetermined value. In some instances, the reference E2-inducible activity score is used as a cutoff value that significantly distinguishes individuals with breast cancer who have E2-inducible activity from those with breast cancer who have low or no E2-inducible activity (e.g., a reference E2-inducible activity score equal to or higher than -2.0 (e.g., -2.0, -1.9, -1.8, -1.7, -1.6, -1.5, -1.4, -1.3, -1.2, -1.1, -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2 or higher)). In some instances, the reference E2-induced activity score is used as a cutoff value that significantly distinguishes individuals with breast cancer who respond to treatment including endocrine therapy as described herein from those who do not (e.g., a reference E2-induced activity score equal to or greater than -2.0 (e.g., -2.0 (e.g., -2.0, -1.9, -1.8, -1.7, -1.6, -1.5, -1.4, -1.3, -1.2, -1.1, -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2 or higher)). Those skilled in this art will understand that the value of the reference E2-inducible activity score can depend on the following variations: the type of breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS and / or metastatic or locally advanced breast cancer), the method used to measure the E2-inducible activity score, the specific genetic characteristics examined (e.g., combinations of genes described herein and, for example, those described in Tables 1 to 6), and / or the statistical method used to generate the E2-inducible activity score. The E2-inducible activity score can be calculated as described herein for estrogen receptor pathway activity scoring. In some embodiments, the E2-inducible activity score is calculated with reference to a standard control as defined herein.

[0137] The ability to distinguish (e.g., to calculate an activity score as defined herein) is relative to the discrimination / characterization / quantification of the expression of the genes described herein and is not by means of a form of detection used to determine the degree of expression of such genes.

[0138] As used herein, "reference gene" refers to a gene or genome (e.g., 1, 2, 3 or more genes) used for comparison purposes, such as housekeeping genes. "Housekeeping gene" in this document refers to a gene or genome (e.g., 1, 2, 3 or more genes) that encodes proteins whose activation is essential for maintaining cellular function and is generally similar across all cell types. Exemplary housekeeping genes include SDHA, GUSB, PPIA, and UBC.

[0139] As used herein, the terms "individual," "patient," and "subject" are used interchangeably and refer to any single animal, preferably a mammal in need of treatment (including non-human animals such as dogs, cats, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates). In some embodiments, the individual, patient, or subject is a human.

[0140] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found in cells containing nucleic acid molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0141] When used herein, the word "tag" refers to a detectable compound or composition. The tag is typically conjugated or fused directly or indirectly with a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the conjugated or fused reagent. The tag itself may be detectable (e.g., a radioisotope tag or a fluorescent tag) or, in the case of an enzyme tag, may catalyze a chemical change in the recipient compound or composition that results in a detectable product.

[0142] As used herein, the term "modifier" refers to an agent that interacts directly or indirectly with a target. Such interactions include, but are not limited to, interactions between agonists, partial agonists, inverse agonists, antagonists, degraders, or combinations thereof. In some embodiments, the modifier is an antagonist. In some embodiments, the modifier is a degrader.

[0143] As used herein, the term "degrading agent" refers to an agent that binds to a nuclear hormone receptor and subsequently reduces the steady-state protein level of that receptor. In some embodiments, the degrading agent as described herein reduces the steady-state estrogen receptor level by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the degrading agent as described herein reduces the steady-state estrogen receptor level by at least 65%. In some embodiments, the degrading agent as described herein reduces the steady-state estrogen receptor level by at least 85%.

[0144] The term "nucleic acid" refers to deoxynucleotides or ribonucleotides in single- or double-stranded form, their polymers, and their complement. The term "polynucleotide" refers to a straight-chain sequence of a nucleotide. The term "nucleotide" generally refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxynucleotides, or modified forms thereof. Examples of polynucleotides covered herein include single- and double-stranded DNA, single- and double-stranded RNA (including siRNA), and hybrid molecules having mixtures of single- and double-stranded DNA and RNA. As used herein, nucleic acid also refers to nucleic acids having the same basic chemical structure as naturally occurring nucleic acids. These analogues have modified sugars and / or modified ring substituents, but retain the same basic chemical structure as naturally occurring nucleic acids. Nucleic acid analogues refer to chemical compounds having a structure different from the general chemical structure of nucleic acids, but functioning in a manner similar to naturally occurring nucleic acids. Examples of such analogues include (but are not limited to) thiophosphates, phosphatidylamine, methyl phosphate, palmitic methyl phosphate, 2-o-methylribonucleotides, and peptide nucleic acids (PNAs). The term "oligonucleotide" refers to relatively short polynucleotides (e.g., less than about 250 nucleotides in length), including (but not limited to) single-stranded deoxynucleotides, single-stranded or double-stranded ribonucleotides, RNA:DNA hybrids, and double-stranded DNA. Oligonucleotides (such as single-stranded DNA probe oligonucleotides) are often synthesized by chemical methods, for example using commercially available automated oligonucleotide synthesizers. However, oligonucleotides can also be prepared by various other methods, including in vitro recombinant DNA-mediated techniques, and by the expression of DNA in cells and organisms.

[0145] The term "packaging insert" refers to the instruction leaflet commonly included in the commercial packaging of therapeutic products, which contains information about indications, uses, dosage, administration, combination therapy, contraindications and / or warnings about the use of such therapeutic products.

[0146] The term "medical formulation" refers to a preparation in which the biological activity of the active ingredient contained therein is permitted and which does not contain any additional components that would be unacceptably toxic to the subject to which the formulation will be administered.

[0147] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical formulation (excluding the active ingredient) that are non-toxic to the subjects. Pharmaceutically acceptable carriers include (but are not limited to) buffers, excipients, stabilizers, or preservatives.

[0148] As used herein, unless otherwise specified, the term "protein" means any raw protein of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) . The term covers "full-length" unprocessed proteins as well as any form of proteins produced by cellular processing. The term also covers naturally occurring variants of proteins, such as splice variants or paired gene variants.

[0149] The "sequence identity percentage (%)" for a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence. If necessary, the maximum sequence identity percentage is achieved after sequence alignment and gap introduction, without considering any conserved substitutions as part of sequence identity. Alignments for the purpose of determining the nucleic acid or amino acid sequence identity percentage can be achieved in various ways familiar with this technique, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Those skilled in this technique can determine suitable parameters for sequence alignment, including any algorithms required to achieve maximum alignment of the full length of the sequences being compared. However, for the purposes of this document, the sequence alignment computer program ALIGN-2 is used to generate the sequence identity % value. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc. and its source code was submitted to the US Copyright Office, Washington DC, 20559, and registered under US Copyright registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems including Digital UNIX V4.0D. All sequence comparison parameters are established by the ALIGN-2 program and remain unchanged.

[0150] When using ALIGN-2 for nucleic acid or amino acid sequence comparison, the sequence identity percentage of a given nucleic acid or amino acid sequence A with or against a given nucleic acid or amino acid sequence B is calculated as follows (or it can be phrased as a given nucleic acid or amino acid sequence A having or containing a certain sequence identity percentage with or against a given nucleic acid or amino acid sequence B): 100 multiplied by the fraction X / Y, Where X represents the number of nucleic acid or amino acid residues that received the same matching score in the A and B alignments using the sequence alignment program ALIGN-2, and Y represents the total number of nucleic acid or amino acid residues in B. It should be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the sequence identity % between A and B will not be equal to the sequence identity % between B and A. Unless otherwise explicitly specified, all sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described immediately preceding the paragraph.

[0151] "Correlate / correlating" means comparing the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol in any way. For example, we can use the results of a first analysis or protocol to conduct a second protocol and / or we can use the results of a first analysis or protocol to determine whether a second analysis or protocol should be conducted. Regarding examples of peptide analysis or protocols, we can use the results of peptide performance analysis or protocols to determine whether a specific treatment protocol should be administered. Regarding examples of polynucleotide analysis or protocols, we can use the results of polynucleotide performance analysis or protocols to determine whether a specific treatment protocol should be administered.

[0152] As used interchangeably in this document, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. Such nucleotides may be deoxynucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogues, or any substrate that may be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction.

[0153] Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogues. If present, these modifications can be applied to the nucleotide structure before or after polymer assembly. The sequence of the nucleotide can be broken down by non-nucleotide components. Polynucleotides can be further modified post-synthesis, such as by conjugation with tags. Other types of modifications include, for example, the "cap" substitution of one or more naturally occurring nucleotides with analogues; internal nucleotide modifications, such as those having non-electrolyzed bonds (e.g., methyl phosphate, triphosphate, phosphatamine, carbamate, etc.) and those having charged bonds (e.g., thiophosphate, dithiophosphate, etc.); those containing attached moieties (e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.)); those containing intercalators (e.g., acridine, psoralen, etc.); those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.); those containing alkylating agents; those having modified bonds (e.g., α-mutant isomers of nucleic acids, etc.); and unmodified forms of polynucleotides. Furthermore, any of the hydroxyl groups typically present in sugars can be replaced, for example, by phosphonate or phosphate groups, protected by a standard protecting group, or activated to prepare additional bonds for additional nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' OH groups can be phosphorylated or partially replaced by an amine or an organic end-capping group of 1 to 20 carbon atoms. Other hydroxyl groups can also be derived into standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose known in this art, including, for example, 2'-o-methyl-, 2'-o-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-mutantoses, epimeric sugars (such as arabinose, xylose, or lythose), piperanose, furanose, sedoheptulose, acyclic analogs, and non-basic nucleoside analogs (such as methylnucleosides). One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include (but are not limited to) examples of phosphate esters replaced by P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR2 ("acetylamine"), P(O)R, P(O)OR', CO, or CH2 ("formaldehyde"), wherein each R or R' is independently H or, where applicable, contains an ether (-O-)-linked substituted or unsubstituted alkyl (1 to 20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl group. Not all links in a polynucleotide must be identical. The above applies to all polynucleotides mentioned herein, including RNA and DNA.

[0154] As used herein, the technique of "polymerase chain reaction" or "PCR" generally refers to a procedure in which amplifies small amounts of specific fragments of nucleic acid, RNA, and / or DNA, as described in U.S. Patent No. 4,683,195, issued July 28, 1987. Generally, sequence information from the ends or beyond the region of interest must be available to allow the design of oligonucleotide primers; these primers will be identical or similar in sequence to the opposite strand of the template to be amplified. The 5' nucleotides of both primers may coincide with the ends of the amplified material. PCR can also be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences. See also Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich (ed.), PCR Technology, (Stockton Press, NY, 1989). As used in this article, PCR is considered one example of a nucleic acid polymerase reaction method for amplifying nucleic acid test samples, but not the only example. This method includes using known nucleic acids (DNA or RNA) as primers and using nucleic acid polymerases to amplify or generate specific fragments of nucleic acids or amplify or generate specific fragments of nucleic acids complementary to specific nucleic acids.

[0155] As used herein, the terms "reverse transcriptase polymerase chain reaction" or "RT-PCR" refer to the replication and amplification of RNA sequences. In this method, reverse transcription is coupled to PCR, for example, as described in U.S. Patent No. 5,322,770, the entire contents of which are incorporated herein by reference. In RT-PCR, an RNA template is converted into cDNA by the reverse transcriptase activity of an enzyme, and then amplified using the polymerase activity of the same or different enzymes. Both thermostable and thermolabile reverse transcriptases and polymerases can be used. "Reverse transcriptase" (RT) can include reverse transcriptases derived from retroviruses, other viruses, and DNA polymerases exhibiting reverse transcriptase activity.

[0156] As used in this article, the terms "reverse transcriptase quantitative polymerase chain reaction" or "RT-qPCR" refer to the form of PCR, in which the nucleic acid to be amplified is RNA that has been reverse transcribed into cDNA for the first time, and the amount of PCR products is measured at each step of the PCR reaction.

[0157] "Quantitative real-time polymerase chain reaction" or "qRT-PCR" refers to a form of PCR in which the amount of PCR product is measured at each step of the PCR reaction. This technique has been described in various publications, including Cronin et al., Am. J. Pathol. 164(1):35-42 (2004); and Ma et al., Cancer Cell 5:607-616 (2004).

[0158] The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acids obtained from a single source (e.g., an individual) that uses more than one set of primers for the purpose of amplifying two or more DNA sequences in a single reaction.

[0159] The term "RNA-seq" (also known as "Whole Transcriptome Shotgun Sequencing (WTSS)") refers to the use of high-throughput sequencing technologies to sequence and / or quantify cDNA to obtain information about the RNA content of a sample. Publications describing RNA-seq include: Wang et al., "RNA-Seq: a revolutionary tool for transcriptomics," Nature Reviews Genetics 10 (1): 57-63 (January 2009); Ryan et al., BioTechniques 45 (1): 81-94 (2008); and Maher et al., "Transcriptome sequencing to detect gene fusions in cancer," Nature 458 (7234): 97-101 (January 2009). Exemplary RNA-seq protocols include the use of the RNA ACCESS® protocol or the TRUSEQ® RIBO-ZERO® protocol (ILLUMINA®).

[0160] "Response to treatment," "responsiveness to treatment," or "benefit from treatment" can be assessed using any endpoint that indicates benefit to the individual, including (but not limited to) (1) some degree of inhibition of disease progression (e.g., breast cancer progression), including slowing and complete cessation; (2) reduction in tumor size; (3) inhibition (i.e., reduction, slowing, or complete cessation) of cancer cell infiltration into adjacent peripheral organs and / or tissues; (4) inhibition (i.e., reduction, slowing, or complete cessation) of metastasis; (5) some degree of relief of one or more symptoms associated with the disease or condition (e.g., cancer); (6) increased or prolonged survival, including recurrence-free survival (RFS), disease-free survival (DFS), overall survival (OS HR < 1), and progression-free survival (PFS HR < 1); and / or (7) a reduction in mortality at a given time point following treatment (e.g., treatment with endocrine therapy (SERMs, e.g., SERDs, GnRH agonists, and / or AIs)). The response to treatment may also be referred to as a pharmacodynamic response or pathway (e.g., ER pathway) response and may be assessed using methods known in this technique.

[0161] As used herein, "progression-free survival" or "PFS" refers to the length of time during and after treatment during which the disease being treated (e.g., breast cancer, e.g., HR+ breast cancer, e.g., ER+ breast cancer, e.g., ductal A or B breast cancer, e.g., advanced or metastatic breast cancer) does not progress or worsen. PFS may include the amount of time an individual experiences a complete or partial response, and the amount of time an individual experiences disease stabilization.

[0162] As used in this article, “overall survival” or “OS” refers to an individual who is alive after a specific time period (e.g., 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years or more since diagnosis or treatment).

[0163] As used in this article, "recurrence-free survival" or "RFS" refers to the length of time a patient survives without any signs, symptoms, or recurrence of tumor in the same location or region after the initial treatment.

[0164] As used in this article, "disease-free survival" or "DFS" refers to the length of time a patient survives without any signs, symptoms, or recurrence of tumors in any region, including the progression of distant metastases, after initial treatment.

[0165] As used in this article, "complete response" or "CR" refers to the disappearance of all signs of cancer in response to treatment. This does not necessarily mean that the cancer has been cured.

[0166] As used in this article, "partial response" or "PR" refers to a reduction in the size of one or more tumors or lesions or the degree of cancer in the body in response to treatment.

[0167] As used herein, “hazard ratio” or “HR” is a statistical definition of event rate. For the purposes provided herein, the hazard ratio is defined as the probability of an event (e.g., PFS or OS) in the experimental (e.g., treatment) group / arm at any given time point divided by the probability of the event in the control group / arm. An HR of 1 indicates that the relative risk of the endpoint (e.g., death) is equal in both the treatment and control groups; a value greater than 1 indicates that the risk is greater in the treatment group than in the control group; and a value less than 1 indicates that the risk is greater in the control group than in the treatment group. The hazard ratio (i.e., PFS HR) in progression-free survival analysis is an overview of the difference between two progression-free survival curves, representing the reduction in the risk of death in the treatment group compared to the control group during the follow-up period. The hazard ratio (i.e., OS HR) in overall survival analysis is an overview of the difference between two overall survival curves, representing the reduction in the risk of death in the treatment group compared to the control group during the follow-up period.

[0168] "Prolonged survival" means an increase in overall survival or progression-free survival in treated individuals compared to untreated individuals (i.e., compared to individuals who have not received any medication), or compared to individuals who do not show a biomarker at a specified level, and / or compared to individuals treated with an approved anticancer therapy. Objective response refers to a measurable response, including complete response (CR) or partial response (PR).

[0169] "Reduction or inhibition" means the ability to cause a total reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduction or inhibition can refer to symptoms of a disease being treated (e.g., breast cancer, e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), the presence or size of metastases, or the size of the primary tumor. When referring to a tumor pathway, reduction or inhibition means a reduction in the expression or activity of any component of the pathway (e.g., a reduction in the expression of ER, a reduction in the activity of ER, or a degradation of ER).

[0170] As used herein, “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue” refers to a sample, cell, tissue, standard, or level used for comparative purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual. In another embodiment, the reference sample is obtained from one or more individuals who are not the subject or individual. In any of the above embodiments, one or more individuals from whom the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained have breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or ductal B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). In some embodiments, one or more individuals who have obtained a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue have breast cancer and have previously been treated with anticancer therapies (e.g., endocrine therapies (e.g., SERM (e.g., SERD), GnRH agonists, and / or AIs) at one or more doses). In other embodiments, one or more individuals who have obtained a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue have breast cancer and are undergoing initial treatment. In any of the above embodiments, the subject / individual and one or more individuals who are not the subject or individual have the same breast cancer.

[0171] As used herein, a "standard control" referring to the level of expression of one or more genes means the level of expression measured in a control subject (e.g., in a sample from a control subject) or a control subject population. In an embodiment, the control subject is a healthy control subject relative to the subject being tested, wherein the healthy control subject does not have cancer. In an embodiment, the control subject is a test subject prior to treatment of the test subject, wherein both the test subject and the control subject have breast cancer. For example, in an embodiment, the test subject has been treated with an anticancer agent for breast cancer and the control subject is a test subject prior to treatment. In an embodiment, the control subject population is a variety of collections of healthy and diseased subjects, wherein the level of expression of the test subject is compared to the level of expression of the control subject population (e.g., the average level of expression of the control subject population). In an embodiment, the control subject population is a collection of healthy subjects without breast cancer, wherein the level of expression of the test subject is compared to the level of expression of the control subject population (e.g., the average level of expression of the control subject population). In one embodiment, the control group is a collection of subjects who have been treated for breast cancer, wherein the performance level of the test subjects is compared with the performance level of the control group (e.g., the average performance level of the control group).

[0172] As used herein, the term "sample" refers to a composition obtained from or derived from a subject and / or individual of interest who contains cellular and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase "disease sample" and its various variations refer to any sample obtained from a subject of interest who is expected or known to contain cellular and / or molecular entities to be characterized. Samples include (but are not limited to) tissue extracts (such as homogenized tissue), tumor tissue, cell extracts, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media and combinations thereof.

[0173] "Tissue sample" or "cell sample" means a collection of similar cells obtained from the tissue of a subject or individual. The source of tissue or cell samples may be solid tissue, tissue samples, biopsies, and / or aspiration from FFPE, FF, fresh, frozen, and / or preserved organs; blood or any blood component, such as plasma; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of the subject's pregnancy or development. Tissue samples may also be primary or cultured cells or cell lines. Where appropriate, tissue or cell samples may be obtained from disease (e.g., breast cancer, such as HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer) tissues / organs. Tissue samples may contain compounds that do not substantially mix with the tissue naturally, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

[0174] For the purposes of this article, a “slice” of a tissue sample refers to a single portion or section of a tissue sample, such as a thin slice of tissue or cells cut from a tissue sample. It should be understood that multiple slices of a tissue sample can be used and analyzed, provided that the same slice of the tissue sample can be analyzed at both the morphological and molecular levels, or at both the peptide and polynucleotide levels.

[0175] As used herein, "treatment" (and its grammatical variations, such as "treat / treating") refers to a clinical intervention that attempts to alter the natural course of a subject being treated, and may be directed at prevention or during the course of clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease (e.g., breast cancer, such as HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), alleviating symptoms, reducing any direct or indirect pathological outcome of the disease, preventing metastasis, reducing the rate of disease progression, improving or mitigating the disease state, and reducing or improving prognosis. In some embodiments, the treatments described herein are used to delay disease progression or slow the progression of disease (e.g., breast cancer, such as HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). In some instances, this treatment can increase overall survival (OS) (e.g., by approximately 20% or greater, approximately 25% or greater, approximately 30% or greater, approximately 35% or greater, approximately 40% or greater, approximately 45% or greater, approximately 50% or greater, approximately 55% or greater, approximately 60% or greater, approximately 65% ​​or greater, approximately 70% or greater, approximately 75% or greater, approximately 80% or greater, approximately 85% or greater, approximately 90% or greater, approximately 95% or greater, approximately 96% or greater, approximately 97% or greater, approximately 98% or greater, or approximately 99% or greater). In some instances, the treatment can increase OS by, for example, about 5% to about 500%, about 10% to about 450%, about 20% to about 400%, about 25% to about 350%, about 30% to about 400%, about 35% to about 350%, about 40% to about 300%, about 45% to about 250%, about 50% to about 200%, about 55% to about 150%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, or about 98% to about 100%. In some instances, this treatment can increase progression-free survival (PFS) (e.g., by approximately 20% or greater, approximately 25% or greater, approximately 30% or greater, approximately 35% or greater, approximately 40% or greater, approximately 45% or greater, approximately 50% or greater, approximately 55% or greater, approximately 60% or greater, approximately 65% ​​or greater, approximately 70% or greater, approximately 75% or greater, approximately 80% or greater, approximately 85% or greater, approximately 90% or greater, approximately 95% or greater, approximately 96% or greater, approximately 97% or greater, approximately 98% or greater, or approximately 99% or greater).In some instances, the treatment may increase PFS by, for example, about 5% to about 500%, about 10% to about 450%, about 20% to about 400%, about 25% to about 350%, about 30% to about 400%, about 35% to about 350%, about 40% to about 300%, about 45% to about 250%, about 50% to about 200%, about 55% to about 150%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, or about 98% to about 100%.

[0176] As used in this article, "tumor" refers to all proliferative cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues. As mentioned in this article, the terms "cancer," "carcinoma," "proliferative disorder," "proliferative disease," and "tumor" are not mutually exclusive. [III.] [method] []

[0177] This document provides methods and assays for: identifying individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer) who may benefit from treatment including endocrine therapies as described herein; selecting a therapy for the individual with breast cancer; treating the individual with breast cancer based on the diagnostic methods provided herein; and monitoring the efficacy of the endocrine therapy. In one embodiment, the endocrine therapy is a compound as described herein. In another embodiment, the endocrine therapy is a SERM, SERD, AI, or a combination thereof.

[0178] The methods and assays described herein are based on findings that estradiol (E2) induction scores or estrogen receptor (ER) pathway activity scores measured from samples taken from an individual (e.g., tissue samples, such as tumor tissue samples, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples) can be used to predict the therapeutic efficacy of the endocrine therapies described herein. Any of these methods may further include administering endocrine therapy to the individual (e.g., as described in Section IV-A below).

[0179] Therefore, this document also provides methods and assays for determining E2 induction scores and / or ER pathway activity scores from individual samples. Any of the methods provided herein may include administering anticancer therapy to the individual in addition to endocrine therapy (e.g., as described in Section IV-A below). Any of these methods may further include administering an effective dose of an additional therapeutic agent as described herein. A. Diagnostic methods and testing

[0180] Predictive diagnostic methods and tests

[0181] In specific instances, the methods and assays provided herein can be used to identify individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer) who may benefit from treatment including endocrine therapy as described herein. The method includes measuring an ER pathway activity score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as formalin-fixed paraffin-embedded (FFPE), fresh-frozen (FF), archived, fresh, or frozen tumor tissue sample), wherein an ER pathway activity score equal to or higher than a reference ER pathway activity score identifies the individual as a recipient of treatment including endocrine therapy as described herein.

[0182] In specific instances, the methods and assays provided herein can be used to identify individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer), DCIS, and / or metastatic or locally advanced breast cancer) who may benefit from treatment including endocrine therapy as described herein. The method includes measuring an E2-inducible score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an E2-inducible score equal to or higher than a reference E2-inducible score identifies the individual as a potential beneficiary of treatment including endocrine therapy as described herein.

[0183] In specific instances, the methods and assays provided herein can be used to select a therapy for an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), the method comprising determining an ER pathway activity score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an ER pathway activity score equal to or higher than a reference ER pathway activity score identifies the individual as a potential beneficiary of treatment including endocrine therapy as described herein.

[0184] In specific instances, the methods and assays provided herein can be used to select a therapy for an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), the method comprising determining an E2 induction score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an E2 induction score equal to or higher than a reference E2 induction score identifies the individual as a potential beneficiary of a treatment including endocrine therapy as described herein.

[0185] In specific instances, the methods and assays provided herein can be used to identify individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer), DCIS, and / or metastatic or locally advanced breast cancer) who may benefit from treatment including endocrine therapies as described herein), the method comprising measuring an ER pathway activity score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an ER pathway activity score lower than a reference ER pathway activity score classifies the individual as less likely to benefit from treatment including endocrine therapies as described herein.

[0186] In specific instances, the methods and assays provided herein can be used to identify individuals with breast cancer who may benefit from treatment including endocrine therapies as described herein. The method includes determining an E2-inducible score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an E2-inducible score lower than a reference E2-inducible score identifies the individual as less likely to benefit from treatment including endocrine therapies as described herein.

[0187] In specific instances, the methods and assays provided herein can be used to select therapies for individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). The method includes determining an ER pathway activity score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an ER pathway activity score lower than a reference ER pathway activity score classifies the individual as less likely to benefit from treatments including endocrine therapies as described herein. For example, the method involves selecting anticancer therapies for the individual other than endocrine therapy.

[0188] In specific instances, the methods and assays provided herein can be used to select therapies for individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). The method includes determining an E2-inducible score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an E2-inducible score lower than a reference E2-inducible score classifies the individual as less likely to benefit from treatments including endocrine therapies as described herein. For example, the method involves selecting anticancer therapies for the individual other than endocrine therapy.

[0189] In specific instances, the methods and assays provided herein can be used to identify individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer), DCIS, and / or metastatic or locally advanced breast cancer) who may benefit from treatment including anticancer therapies other than endocrine therapy), wherein the method includes measuring an ER pathway activity score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an ER pathway activity score lower than a reference ER pathway activity score identifies the individual as a recipient of treatment including anticancer therapies other than endocrine therapy).

[0190] In specific instances, the methods and assays provided herein can be used to identify individuals with breast cancer who may benefit from treatment including anticancer therapies other than endocrine therapy. The method includes measuring an E2-inducible score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an E2-inducible score lower than a reference E2-inducible score identifies the individual as a beneficiary of treatment including anticancer therapies other than endocrine therapy.

[0191] In specific instances, the methods and assays provided herein can be used to select therapies for individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). The method includes determining an ER pathway activity score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an ER pathway activity score lower than a reference ER pathway activity score identifies the individual as a potential beneficiary of treatments including anticancer therapies other than endocrine therapy.

[0192] In specific instances, the methods and assays provided herein can be used to select therapies for individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). The method includes determining an E2 induction score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein an E2 induction score lower than a reference E2 induction score identifies the individual as a potential beneficiary of treatments including anticancer therapies other than endocrine therapy.

[0193] In any of the above examples, the reference ER pathway activity score may be the ER pathway activity score of a reference population of individuals with HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer. In some examples, the reference population is a group of individuals who have not yet received treatment including endocrine therapy as described herein. In some examples, the reference population is a group of individuals who have not yet received prior endocrine therapy as described herein. In some examples, the reference population is a group of individuals who are not currently receiving anticancer treatment (including endocrine therapy as described herein). In some examples, the reference ER pathway activity score may be a pre-specified reference ER pathway activity score. In some examples, the reference ER pathway activity score may be equal to or greater than -1.0 (e.g., -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, or higher). For example, in some instances, the reference ER pathway activity score may be equal to or greater than -0.9. In some instances, the reference ER pathway activity score may be equal to or greater than -0.8. In some instances, the reference ER pathway activity score may be equal to or greater than -0.7. In some instances, the reference ER pathway activity score may be equal to or greater than -0.6. In some instances, the reference ER pathway activity score may be equal to or greater than -0.5. In some instances, the reference ER pathway activity score may be equal to or greater than -0.4. In some instances, the reference ER pathway activity score may be equal to or greater than -0.3. In some instances, the reference ER pathway activity score may be equal to or greater than -0.2. In some instances, the reference ER pathway activity score may be between about -1.0 and about -0.2 (e.g., between about -0.9 and about -0.2, between about -0.8 and about -0.2, between about -0.7 and about -0.2, between about -0.6 and about -0.2, between about -0.5 and about -0.2, between about -0.4 and about -0.2, or between about -0.3 and about -0.2).

[0194] In any of the above examples, the ER pathway activity score of a sample from an individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample) may be equal to or greater than -1.0 (e.g., -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, or higher). For example, in some examples, the ER pathway activity score may be equal to or greater than -0.9. In some examples, the ER pathway activity score may be equal to or greater than -0.8. In some examples, the ER pathway activity score may be equal to or greater than -0.7. In some examples, the ER pathway activity score may be equal to or greater than -0.6. In some examples, the ER pathway activity score may be equal to or greater than -0.5. In some examples, the ER pathway activity score may be equal to or greater than -0.4. In some examples, the ER pathway activity score may be equal to or greater than -0.3. In some instances, the ER pathway activity score may be equal to or greater than -0.2. In some instances, the ER pathway activity score may be between approximately -1.0 and approximately -0.2 (e.g., between approximately -0.9 and approximately -0.2, between approximately -0.8 and approximately -0.2, between approximately -0.7 and approximately -0.2, between approximately -0.6 and approximately -0.2, between approximately -0.5 and approximately -0.2, between approximately -0.4 and approximately -0.2, or between approximately -0.3 and approximately -0.2). In some instances, the ER activity score of the sample may be less than -1.0.

[0195] In any of the above examples, the reference E2-inducing score may be the E2-inducing score of a reference population of individuals with hormone receptor (HR)+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B type breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer. In some examples, the reference population is a group of individuals who have not yet received treatment including endocrine therapy as described herein. In some examples, the reference E2-inducing score may be a pre-specified reference E2-inducing score. In some examples, the reference E2-inducing score may be equal to or greater than -2.0 (e.g., -2.0, -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, or higher). For example, in some examples, the reference E2-inducing score may be equal to or greater than -1.0. In some examples, the reference E2-inducing score may be equal to or greater than -0.9. In some instances, the reference E2 induced score may be equal to or higher than -0.8. In some instances, the reference E2 induced score may be equal to or higher than -0.7. In some instances, the reference E2 induced score may be equal to or higher than -0.6. In some instances, the reference E2 induced score may be equal to or higher than -0.5. In some instances, the reference E2 induced score may be equal to or higher than -0.4. In some instances, the reference E2 induced score may be equal to or higher than -0.3. In some instances, the reference E2 induced score may be equal to or higher than -0.2. In some instances, the reference E2 induced score may be equal to or higher than -0.1. In some instances, the reference E2 induced score may be between about -2.0 and about -0.1 (e.g., between about -1.0 and about -0.1, between about -0.7 and about -0.1, between about -0.6 and about -0.1, between about -0.5 and about -0.1, between about -0.4 and about -0.1, between about -0.3 and about -0.1, or between about -0.2 and about -0.1).

[0196] In any of the above examples, the E2-inducing score of a sample from an individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample) may be equal to or greater than -2.0 (e.g., -2.0, -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, -0.1, or higher). For example, in some instances, the E2-inducing score may be equal to or greater than -1.0. In some instances, the E2-inducing score may be equal to or greater than -0.9. In some instances, the E2-inducing score may be equal to or greater than -0.8. In some instances, the E2-inducing score may be equal to or greater than -0.7. In some instances, the E2-inducing score may be equal to or greater than -0.6. In some instances, the E2-inducing score may be equal to or greater than -0.5. In some instances, the E2-induced score may be equal to or greater than -0.4. In some instances, the E2-induced score may be equal to or greater than -0.3. In some instances, the E2-induced score may be equal to or greater than -0.2. In some instances, the E2-induced score may be equal to or greater than -0.1. In some instances, the E2-induced score may be between approximately -2.0 and approximately -0.1 (e.g., between approximately -1.0 and approximately -0.1, between approximately -0.7 and approximately -0.1, between approximately -0.6 and approximately -0.1, between approximately -0.5 and approximately -0.1, between approximately -0.4 and approximately -0.1, between approximately -0.3 and approximately -0.1, or between approximately -0.2 and approximately -0.1). In some instances, the E2-induced score of the sample may be less than -2.0.

[0197] In any of the above-described prediction methods and detections, such methods and detections may further include administering endocrine therapy to the individual (e.g., as described in Section IV-A below). In a specific instance, when the ER pathway activity score of a sample from the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples) is equal to or higher than a reference ER pathway activity score, the method further includes administering endocrine therapy to the individual. In a specific instance, when the E2 induction score of a sample from the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples) is equal to or higher than a reference E2 induction score, the method further includes administering endocrine therapy to the individual.

[0198] In any of the above-described prediction methods and detections, these methods and detections may further include administering anticancer therapy to the individual other than endocrine therapy (e.g., as described in Section IV-A below). In a specific instance, when the ER pathway activity score of a sample from the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples) is lower than a reference ER pathway activity score, the method further includes administering anticancer therapy to the individual other than endocrine therapy. In a specific instance, when the E2 induction score of a sample from the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples) is lower than a reference E2 induction score, the method further includes administering anticancer therapy to the individual other than endocrine therapy.

[0199] The methods provided herein may include determining ER pathway activity scores from samples taken from an individual (e.g., tissue samples, such as tumor tissue samples, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples). In some instances, the sample may be an FFPE tumor tissue sample. In some instances, the sample may be an FF tumor tissue sample.

[0200] In any of the above examples, the individual may have HR+ breast cancer. In some examples, the HR+ breast cancer may be ER+ breast cancer. In some examples, the individual may have ER+ breast cancer selected from, for example, ductal carcinoma type A or ductal carcinoma type B. In some examples, the breast cancer may be advanced or metastatic breast cancer.

[0201] In some instances of the methods or tests described above that involve determining the ER pathway activity score and / or E2 induction score from a sample of an individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples), the individual has previously received endocrine therapy as described herein. In other instances, the individual has not previously received endocrine therapy. In another embodiment, the individual has received one or more prior therapies prior to performing the methods and tests described herein, wherein such therapy may be endocrine therapy or non-endocrine therapy as described herein.

[0202] In some instances, these methods further include generating reports, such as electronic reports, web-based reports, or paper reports, for individuals or entities, caregivers, physicians, oncologists, hospitals, clinics, third-party payers, insurance companies, pharmaceutical or biotechnology companies, or government agencies. In some embodiments, the report includes the output of the method, which includes evaluating ER pathway activity scores and / or E2 induction scores.

[0203] Pharmacodynamic diagnostic methods

[0204] Pharmacodynamic methods are also provided in this article. In some instances, these methods may involve monitoring an individual's response to treatment with endocrine therapies as described herein.

[0205] In some instances, the method includes: (a) determining an ER pathway activity score from a sample (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample) from the individual at a first time point; (b) after step (a), determining a second ER pathway activity score from a sample from the individual at a second time point following administration of endocrine therapy as described herein; and (c) comparing the first ER pathway activity score with the second ER pathway activity score, wherein a reduction in the second ER pathway activity score relative to the first ER pathway activity score (e.g., a reduction in ER pathway activity score of about 0.1, 0.2, 0.3, or greater) predicts an individual's response to treatment with endocrine therapy (e.g., SERM (e.g., SERD), GnRH agonists, and / or AI). In some instances, a reduction in the ER pathway activity score refers to an overall reduction of at least 0.1 in the ER pathway activity score. In some instances, a reduction in the ER pathway activity score refers to an overall reduction of at least 0.2 in the ER pathway activity score. In some instances, a reduction in ER pathway activity score refers to an overall reduction of at least 0.3 in ER pathway activity score.

[0206] In some instances, the method further includes administering one or more additional doses of endocrine therapy if the second ER pathway activity score of a sample from the individual (e.g., a tissue sample, such as a tumor tissue sample, such as a formalin-fixed paraffin-embedded (FFPE), fresh-frozen (FF), archived, fresh, or frozen tumor tissue sample) is reduced relative to the first ER pathway activity score. In some instances, a reduced ER pathway activity score refers to an overall reduction of at least 0.1 in the ER pathway activity score (e.g., a reduction of 0.1, 0.2, 0.3, or greater). In some instances, a reduced ER pathway activity score refers to an overall reduction of at least 0.2 in the ER pathway activity score. In some instances, a reduced ER pathway activity score refers to an overall reduction of at least 0.3 in the ER pathway activity score.

[0207] In some instances of any of the methods described above, the first ER pathway activity score is an ER pathway activity score measured from an individual's sample (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample) obtained prior to administration of the first dose of endocrine therapy as described herein. In other words, the sample may be a baseline sample. In other instances, the first ER pathway activity score is an ER pathway activity score measured from an individual's sample obtained at a previous time point, wherein the previous time point is after administration of the first dose of endocrine therapy as described herein. In other instances, the first ER pathway activity score is an ER pathway activity score measured from an individual's sample obtained at a previous time point, wherein the previous time point is after administration of the first dose of non-endocrine therapy as described herein. In other instances, the first ER pathway activity score is a predetermined ER pathway activity score.

[0208] In some instances, the method includes: (a) determining an E2-induced score at a first time point on a sample from the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample); (b) after step (a), determining a second E2-induced score at a second time point on a sample from the individual following administration of endocrine therapy as described herein; and (c) comparing the first E2-induced score with the second E2-induced score, wherein a reduction in the second E2-induced score relative to the first E2-induced score (e.g., a reduction of at least 0.1, 0.2, 0.3, or greater) predicts a response to treatment with the endocrine therapy as described herein. In some instances, a reduction in the E2-induced score refers to an overall reduction of at least 0.1. In some instances, a reduction in the E2-induced score refers to an overall reduction of at least 0.2. In some instances, a reduction in the E2-induced score refers to an overall reduction of at least 0.3.

[0209] In some instances, the method further includes administering one or more additional doses of one or more endocrine therapies as described herein if the second E2-inducing score of the sample (e.g., a tissue sample, such as a tumor tissue sample, such as an FFPE, FF, archived, fresh, or frozen tumor tissue sample) is reduced relative to the first E2-inducing score. In some instances, a reduced E2-inducing score refers to an overall reduction of at least 0.1 in the E2-inducing score (e.g., a reduction of 0.1, 0.2, 0.3, or greater). In some instances, a reduced E2-inducing score refers to an overall reduction of at least 0.2 in the E2-inducing score. In some instances, a reduced E2-inducing score refers to an overall reduction of at least 0.3 in the E2-inducing score.

[0210] In some instances of any of the methods described above, the first E2-induced score is an E2-induced score measured from a sample (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample) obtained before the administration of a first dose of anticancer therapy (e.g., endocrine therapy as described herein). In other words, the sample may be a baseline sample. In other instances, the first E2-induced score is an E2-induced score measured from a sample obtained at a previous time point, wherein the previous time point is after the administration of the first dose of endocrine therapy as described herein. In still other instances, the first E2-induced score is a predetermined E2-induced score.

[0211] In some instances, the second ER pathway activity score of a sample from an individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples) is reduced relative to the first ER pathway activity score, and the method or detection further involves administering an additional dose of endocrine therapy as described herein to the individual. In some instances, the second E2 induction score of a sample from an individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples) is reduced relative to the first E2 induction score, and the method or detection further involves administering an additional dose of endocrine therapy as described herein to the individual.

[0212] The methods provided herein may include determining ER pathway activity scores from samples taken from an individual (e.g., tissue samples, such as tumor tissue samples, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples). In some instances, the sample may be an FFPE tumor tissue sample. In some instances, the sample may be an FF tumor tissue sample.

[0213] In any of the above examples, the individual may have HR+ breast cancer. In some examples, the HR+ cancer may be ER+ breast cancer. In some examples, the individual may have ER+ breast cancer, selected from, for example, ductal A or ductal B breast cancer. In some examples, the breast cancer may be advanced or metastatic breast cancer.

[0214] In some instances of any of the methods or assays described above involving the determination of ER pathway activity scores and / or E2 induction scores from samples taken from an individual (e.g., tissue samples, such as tumor tissue samples, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples), the individual has previously received endocrine therapy as described herein. In other instances, the individual has not previously received endocrine therapy. In some instances of any of these methods and assays, the sample (e.g., tissue samples, such as tumor tissue samples, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples) was obtained from an individual prior to administration of endocrine therapy as described herein (e.g., minutes, hours, days, weeks, months, or years ago). In other words, the sample may be a baseline sample. In some instances of any of the methods described above, the sample was obtained from an individual after administration of endocrine therapy (e.g., minutes, hours, or days later). In some instances, the sample from the individual was obtained within 30 hours of administration of endocrine therapy. In some instances, multiple samples were obtained from the same individuals at different time points (e.g., before and after administration of endocrine therapy).

[0215] In some instances, these methods further include generating reports, such as electronic reports, web-based reports, or paper reports, for individuals or entities, caregivers, physicians, oncologists, hospitals, clinics, third-party payers, insurance companies, pharmaceutical or biotechnology companies, or government agencies. In some instances, the report includes the output of the method, which includes evaluating ER pathway activity scores and / or E2 induction scores.

[0216] In one sample, a method is provided for detecting estrogen receptor (ER) pathway activity in a subject with breast cancer. The method includes detecting the expression levels of at least five genes described in Table 1 and at least five genes described in Table 4; at least five genes described in Table 2 and at least five genes described in Table 5; or at least five genes described in Table 3 and at least five genes described in Table 6.

[0217] The controls used in the methods presented herein are valuable for determining the significance of data. For example, if the value of a given parameter varies widely in controls, the variation in the test sample will not be considered significant. In some instances of the disclosed methods, when assessing the expression level of any of the genes provided in Tables 1 through 6, that expression level is compared to the control expression level of that gene. The control expression level refers to the expression level of a gene from samples or subjects lacking breast cancer, samples or subjects in a selection phase of breast cancer or a cancer state, or in the absence of a specific variable (such as a treatment agent). Alternatively, the control level includes a known amount of the gene. This known amount is associated with the average level of subjects lacking breast cancer, in a selection phase of breast cancer or a cancer state, or in the absence of a specific variable (such as a treatment agent). The control level also includes the expression level of genes from one or more selected samples or subjects as described herein. For example, the control level includes an assessment of the expression level of genes from samples from subjects who do not have breast cancer, are in a selection phase of breast cancer or a cancer state, or have breast cancer but have not yet received breast cancer treatment. Another exemplary control level includes an assessment of gene expression levels in samples taken from multiple subjects who do not have breast cancer, are in the cancer selection phase, or have breast cancer but have not yet received breast cancer treatment. In an embodiment, the threshold for elevated gene expression levels is above the median expression level in a control sample group, wherein the control sample is, in this case, a group of subjects with breast cancer.

[0218] In one embodiment, the method includes detecting the expression levels of at least five genes (e.g., 5, 6, 7, 8, etc.) described in Table 1 and at least five genes described in Table 4. In another embodiment, the method includes detecting the expression levels of at least five genes described in Table 2 and at least five genes described in Table 5. In yet another embodiment, the method includes detecting the expression levels of at least five genes described in Table 3 and at least five genes described in Table 6.

[0219] In the embodiments, the expression levels of at least five genes described in Table 1, at least five genes described in Table 2, or at least five genes described in Table 3 are greater than those of the standard control group. In the embodiments, the expression levels of all genes described in Table 1, all genes described in Table 2, or all genes described in Table 3 are greater than those of the standard control group. In the embodiments, the expression levels of at least five genes described in Table 1 are greater than those of the standard control group. In the embodiments, the expression levels of all genes described in Table 1 are greater than those of the standard control group. In the embodiments, the expression levels of at least five genes described in Table 2 are greater than those of the standard control group. In the embodiments, the expression levels of all genes described in Table 2 are greater than those of the standard control group. In the embodiments, the expression levels of at least five genes described in Table 3 are greater than those of the standard control group. In the embodiments, the expression levels of all genes described in Table 3 are greater than those of the standard control group.

[0220] In this embodiment, the threshold value for elevated gene expression (e.g., expression of any of the genes described in Tables 1 to 6) is above the median expression level in a control group, wherein the control group is, in this case, a group of subjects with breast cancer. In this embodiment, it is above the first quartile of gene expression in the control group, wherein the control group is, in this case, a group of subjects with breast cancer. In this embodiment, it is above the third quartile of gene expression in the control group, wherein the control group is, in this case, a group of subjects with breast cancer. In this embodiment, it is above the fifth percentile of gene expression in the control group, wherein the control group is, in this case, a group of subjects with breast cancer. In this embodiment, it is above the tenth percentile of gene expression in the control group, wherein the control group is, in this case, a group of subjects with breast cancer. In this embodiment, it is above the twentieth percentile of gene expression in the control group, wherein the control group is, in this case, a group of subjects with breast cancer. In one embodiment, the gene expression of the control sample group is at or above the 30th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is at or above the 40th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is at or above the 45th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is at or above the 50th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is at or above the 60th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is at or above the 70th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is at or above the 80th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In one embodiment, the gene expression is above the 90th percentile in a control group, wherein the control group is, in some cases, a group of subjects with breast cancer.

[0221] In the embodiments, the expression levels of at least five genes described in Table 4, at least five genes described in Table 5, or at least five genes described in Table 6 are lower than those of the standard control group. In the embodiments, the expression levels of all genes described in Table 4, all genes described in Table 5, or all genes described in Table 6 are lower than those of the standard control group. In the embodiments, the expression levels of at least five genes described in Table 4 are lower than those of the standard control group. In the embodiments, the expression levels of all genes described in Table 4 are lower than those of the standard control group. In the embodiments, the expression levels of at least five genes described in Table 5 are lower than those of the standard control group. In the embodiments, the expression levels of all genes described in Table 5 are lower than those of the standard control group. In the embodiments, the expression levels of at least five genes described in Table 6 are lower than those of the standard control group. In the embodiments, the expression levels of all genes described in Table 6 are lower than those of the standard control group.

[0222] In an embodiment, the threshold value for reduced gene expression (e.g., expression of any of the genes described in Tables 1 to 6) is below the median expression level in a control group, wherein the control group is, in this case, a group of subjects with breast cancer. In an embodiment, it is below the first quartile of gene expression in the control group, wherein the control group is, in this case, a group of subjects with breast cancer. In an embodiment, it is below the third quartile of gene expression in the control group, wherein the control group is, in this case, a group of subjects with breast cancer. In an embodiment, it is below the fifth percentile of gene expression in the control group, wherein the control group is, in this case, a group of subjects with breast cancer. In an embodiment, it is below the tenth percentile of gene expression in the control group, wherein the control group is, in this case, a group of subjects with breast cancer. In an embodiment, it is below the twentieth percentile of gene expression in the control group, wherein the control group is, in this case, a group of subjects with breast cancer. In one embodiment, the gene expression of the control sample group is below the 30th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is below the 40th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is below the 45th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is below the 50th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is below the 60th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is below the 70th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In another embodiment, the gene expression of the control sample group is below the 80th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer. In one embodiment, the gene expression of the control sample group is below the 90th percentile, wherein the control sample is, in this case, a group of subjects with breast cancer.

[0223] In one embodiment, the subject had already undergone endocrine therapy prior to the test. In another embodiment, the subject underwent endocrine therapy after the test.

[0224] In one embodiment, the method includes detecting the expression levels of all genes described in Table 1 and all genes described in Table 4. In another embodiment, the method includes detecting the expression levels of all genes described in Table 2 and all genes described in Table 5. In another embodiment, the method includes detecting the expression levels of all genes described in Table 3 and all genes described in Table 6. In another embodiment, the method includes detecting the expression levels of all genes described in Table 1 and all genes described in Table 4 without detecting the expression levels of any other genes in the subject. In another embodiment, the method includes detecting the expression levels of all genes described in Table 2 and all genes described in Table 5 without detecting the expression levels of any other genes in the subject. In another embodiment, the method includes detecting the expression levels of all genes described in Table 3 and all genes described in Table 6 without detecting the expression levels of any other genes in the subject.

[0225] In one embodiment, the subject was treated with an endocrine therapy, wherein the endocrine therapy was a selective estrogen receptor degrader.

[0226] In one embodiment, the method includes determining an estrogen receptor (ER) pathway activity score from a sample of a subject. In another embodiment, an ER pathway activity score equal to or higher than a reference ER pathway activity score identifies an individual as a potential beneficiary of treatment including endocrine therapy. In yet another embodiment, the method includes comparing ER pathway activity scores of samples; an ER pathway activity score equal to or higher than a reference ER pathway activity score identifies an individual as a potential beneficiary of treatment including endocrine therapy.

[0227] In one embodiment, a method is provided, comprising detecting, by means of one or more processors, a first expression level of at least five genes described in Table 1, at least five genes described in Table 2, or at least five genes described in Table 3; detecting, by means of one or more processors, a second expression level of at least five genes described in Table 4, at least five genes described in Table 5, or at least five genes described in Table 6; and detecting estrogen receptor (ER) pathway activity in a subject with cancer, at least based on the first expression level and / or the second expression level. In one embodiment, the first expression level is increased relative to the second expression level. In another embodiment, the first expression level is decreased relative to the second expression level.

[0228] In an embodiment, the threshold value for an elevated level of expression of a first gene (e.g., the first level of expression of any gene described in Tables 1 to 6) is above the second level of expression. In an embodiment, it is above the first quartile of the second gene expression level. In an embodiment, it is above the third quartile of the second gene expression level. In an embodiment, it is above the 5th percentile of the second gene expression level. In an embodiment, it is above the 10th percentile of the second gene expression level. In an embodiment, it is above the 20th percentile of the second gene expression level. In an embodiment, it is above the 30th percentile of the second gene expression level. In an embodiment, it is above the 40th percentile of the second gene expression level. In an embodiment, it is above the 45th percentile of the second gene expression level. In an embodiment, it is above the 50th percentile of the second gene expression level. In an embodiment, it is above the 60th percentile of the second gene expression level. In an embodiment, it is above the 70th percentile of the second gene expression level. In one embodiment, it is above the 80th percentile of the expression level of the second gene. In another embodiment, it is above the 90th percentile of the expression level of the second gene.

[0229] In an embodiment, the threshold value for reduced expression of the first gene (e.g., the first expression level of any gene described in Tables 1 to 6) is below the second expression level. In an embodiment, it is below the first quartile of the second gene expression level. In an embodiment, it is below the third quartile of the second gene expression level. In an embodiment, it is below the 5th percentile of the second gene expression level. In an embodiment, it is below the 10th percentile of the second gene expression level. In an embodiment, it is below the 20th percentile of the second gene expression level. In an embodiment, it is below the 30th percentile of the second gene expression level. In an embodiment, it is below the 40th percentile of the second gene expression level. In an embodiment, it is below the 45th percentile of the second gene expression level. In an embodiment, it is below the 50th percentile of the second gene expression level. In an embodiment, it is below the 60th percentile of the second gene expression level. In an embodiment, it is below the 70th percentile of the second gene expression level. In one embodiment, it is below the 80th percentile of the second gene expression level. In another embodiment, it is below the 90th percentile of the second gene expression level.

[0230] In the embodiments, the expression levels of at least five genes described in Table 1, at least five genes described in Table 2, or at least five genes described in Table 3 are greater than those of the standard control group. In the embodiments, the expression levels of at least five genes described in Table 4, at least five genes described in Table 5, or at least five genes described in Table 6 are less than those of the standard control group.

[0231] In one embodiment, the method includes treating the subject with endocrine therapy prior to the test. In another embodiment, the method includes treating the subject with endocrine therapy based at least on the estrogen receptor (ER) pathway activity detected in the subject. B. Treatment methods

[0232] This document also provides methods for treating individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer). Therefore, in some instances, the methods provided herein include administering endocrine therapy as described herein to the individual. In other instances, the methods provided herein include administering an anticancer agent to the individual in addition to endocrine therapy. Any of the anticancer agents described herein (e.g., in Section IV below) or known in this art may be used in combination with these methods.

[0233] This article provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal type A or ductal type B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising (i) determining an ER pathway activity score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein the ER pathway activity score is determined to be at or above a reference ER pathway activity score (e.g., a reference ER pathway activity score of a reference population, such as a reference ER pathway activity score equal to or greater than -1.0); and (ii) administering an effective dose of endocrine therapy as described herein to the individual.

[0234] This article provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising administering endocrine therapy as described herein to the individual, wherein the individual has been identified by one or more of the predictive diagnostic methods described in Section III-A above as being more likely to benefit from treatment including endocrine therapy.

[0235] This article provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising administering endocrine therapy as described herein to the individual, wherein the individual has been determined by any of the predictive diagnostic methods described in Section III-A above to have an ER pathway activity score equal to or higher than a reference ER pathway activity score.

[0236] This article provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal type A or ductal type B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising (i) determining an E2-inducible score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein the E2-inducible score is determined to be at or above a reference E2-inducible score (e.g., a reference E2-inducible score of a reference population, such as a reference E2-inducible score of -2.0 or above); and (ii) administering an effective dose of endocrine therapy as described herein to the individual.

[0237] This article provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising administering endocrine therapy as described herein to the individual, wherein the individual has been identified by any of the predictive diagnostic methods described in Section III-A above as having an E2 induction score equal to or higher than a reference E2 induction score.

[0238] This article also provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal type A or ductal type B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising (i) measuring an ER pathway activity score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein the measured ER pathway activity score is below a reference ER pathway activity score (e.g., a reference ER pathway activity score of a reference population, such as a reference ER pathway activity score below -1.0); and (ii) administering an effective dose of anticancer therapy other than endocrine therapy to the individual.

[0239] This article provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising administering to the individual an effective dose of anticancer therapy other than endocrine therapy, wherein the individual has been determined by one or more of the predictive diagnostic methods described in Section III-A above to be less likely to benefit from treatment including endocrine therapy.

[0240] This article provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising administering anticancer therapy to the individual in addition to endocrine therapy, wherein the individual has been identified by one or more of the predictive diagnostic methods described in Section III-A above as being more likely to benefit from treatment including anticancer therapy in addition to endocrine therapy.

[0241] This article provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising administering to the individual an effective dose of anticancer therapy other than endocrine therapy, wherein the individual has been identified by any of the predictive diagnostic methods described in Section III-A above as having an ER pathway activity score lower than a reference ER pathway activity score.

[0242] This article provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal type A or ductal type B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising (i) determining an E2-inducible score from a sample of the individual (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample), wherein the E2-inducible score is determined to be below a reference E2-inducible score (e.g., a reference E2-inducible score of a reference population, such as a reference E2-inducible score below -2.0); and (ii) administering an effective dose of anticancer therapy other than endocrine therapy to the individual.

[0243] This article provides a method for treating an individual with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer), comprising administering to the individual an effective dose of anticancer therapy other than endocrine therapy, wherein the individual has been identified by any of the predictive diagnostic methods described in Section III-A above as having an E2 induction score lower than a reference E2 induction score.

[0244] In any of the above examples, the reference ER pathway activity score may be the ER pathway activity score of a reference population of individuals with HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer. In some examples, the reference population is a group of individuals who have not yet received treatment including endocrine therapy (including those described herein). In some examples, the reference ER pathway activity score may be a pre-specified reference ER pathway activity score. In some examples, the reference ER pathway activity score may be equal to or greater than -1.0 (e.g., -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, and -0.2 or higher). For example, in some examples, the reference ER pathway activity score may be equal to or greater than -0.9. In some examples, the reference ER pathway activity score may be equal to or greater than -0.8. In some instances, the reference ER pathway activity score may be equal to or greater than -0.7. In some instances, the reference ER pathway activity score may be equal to or greater than -0.6. In some instances, the reference ER pathway activity score may be equal to or greater than -0.5. In some instances, the reference ER pathway activity score may be equal to or greater than -0.4. In some instances, the reference ER pathway activity score may be equal to or greater than -0.3. In some instances, the reference ER pathway activity score may be equal to or greater than -0.2. In some instances, the reference ER pathway activity score may be between about -1.0 and about -0.2 (e.g., between about -0.9 and about -0.2, between about -0.8 and about -0.2, between about -0.7 and about -0.2, between about -0.6 and about -0.2, between about -0.5 and about -0.2, between about -0.4 and about -0.2, or between about -0.3 and about -0.2).

[0245] In any of the above examples, the ER pathway activity score of a sample from an individual (e.g., a tissue sample, such as a tumor tissue sample, such as a formalin-fixed paraffin-embedded (FFPE), fresh-frozen (FF), archived, fresh, or frozen tumor tissue sample) may be equal to or greater than -1.0 (e.g., -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, or higher). For example, in some examples, the ER pathway activity score may be equal to or greater than -0.9. In some examples, the ER pathway activity score may be equal to or greater than -0.8. In some examples, the ER pathway activity score may be equal to or greater than -0.7. In some examples, the ER pathway activity score may be equal to or greater than -0.6. In some examples, the ER pathway activity score may be equal to or greater than -0.5. In some examples, the ER pathway activity score may be equal to or greater than -0.4. In some instances, the ER pathway activity score may be equal to or greater than -0.3. In some instances, the ER pathway activity score may be equal to or greater than -0.2. In some instances, the ER pathway activity score may be between approximately -1.0 and approximately -0.2 (e.g., between approximately -0.9 and approximately -0.2, between approximately -0.8 and approximately -0.2, between approximately -0.7 and approximately -0.2, between approximately -0.6 and approximately -0.2, between approximately -0.5 and approximately -0.2, between approximately -0.4 and approximately -0.2, or between approximately -0.3 and approximately -0.2). In some instances, the ER activity score of the sample may be less than -1.0.

[0246] In any of the above examples, the reference E2-inducing score may be the E2-inducing score of a reference population of individuals with HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer. In some examples, the reference population is a group of individuals who have not yet received treatment including endocrine therapy as described herein. In some examples, the reference E2-inducing score may be a pre-specified reference E2-inducing score. In some examples, the reference E2-inducing score may be equal to or greater than -2.0 (e.g., -2.0, -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, or higher). For example, in some examples, the reference E2-inducing score may be equal to or greater than -1.0. In some examples, the reference E2-inducing score may be equal to or greater than -0.9. In some instances, the reference E2 induced score may be equal to or higher than -0.8. In some instances, the reference E2 induced score may be equal to or higher than -0.7. In some instances, the reference E2 induced score may be equal to or higher than -0.6. In some instances, the reference E2 induced score may be equal to or higher than -0.5. In some instances, the reference E2 induced score may be equal to or higher than -0.4. In some instances, the reference E2 induced score may be equal to or higher than -0.3. In some instances, the reference E2 induced score may be equal to or higher than -0.2. In some instances, the reference E2 induced score may be equal to or higher than -0.1. In some instances, the reference E2 induced score may be between about -2.0 and about -0.1 (e.g., between about -1.0 and about -0.1, between about -0.7 and about -0.1, between about -0.6 and about -0.1, between about -0.5 and about -0.1, between about -0.4 and about -0.1, between about -0.3 and about -0.1, or between about -0.2 and about -0.1).

[0247] In any of the above examples, the E2 induction score of a sample from an individual (e.g., a tissue sample, such as a tumor tissue sample, such as a formalin-fixed paraffin-embedded (FFPE), fresh-frozen (FF), archived, fresh, or frozen tumor tissue sample) may be equal to or greater than -2.0 (e.g., -2.0, -1.0, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, -0.1, or higher). In some examples, the E2 induction score may be equal to or greater than -1.0. In some examples, the E2 induction score may be equal to or greater than -0.9. In some examples, the E2 induction score may be equal to or greater than -0.8. For example, in some examples, the E2 induction score may be equal to or greater than -0.7. In some examples, the E2 induction score may be equal to or greater than -0.6. In some examples, the E2 induction score may be equal to or greater than -0.5. In some instances, the E2-induced score may be equal to or greater than -0.4. In some instances, the E2-induced score may be equal to or greater than -0.3. In some instances, the E2-induced score may be equal to or greater than -0.2. In some instances, the E2-induced score may be equal to or greater than -0.1. In some instances, the E2-induced score may be between approximately -2.0 and approximately -0.1 (e.g., between approximately -1.0 and approximately -0.1, between approximately -0.7 and approximately -0.1, between approximately -0.6 and approximately -0.1, between approximately -0.5 and approximately -0.1, between approximately -0.4 and approximately -0.1, between approximately -0.3 and approximately -0.1, or between approximately -0.2 and approximately -0.1). In some instances, the E2-induced score of the sample may be less than -2.0.

[0248] The methods provided herein may include determining ER pathway activity scores from samples taken from an individual (e.g., tissue samples, such as tumor tissue samples, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples). In some instances, the sample may be an FFPE tumor tissue sample. In some instances, the sample may be an FF tumor tissue sample.

[0249] In any of the above examples, the individual may have HR+ breast cancer. In some examples, the HR+ cancer may be ER+ breast cancer. In some examples, the individual may have ER+ breast cancer, selected from, for example, ductal A or ductal B breast cancer. In some examples, the breast cancer may be advanced or metastatic breast cancer.

[0250] In some instances involving the determination of ER pathway activity scores and / or E2 induction scores from samples of an individual (e.g., tissue samples, such as tumor tissue samples, such as formalin-fixed paraffin-embedded (FFPE), fresh-frozen (FF), archived, fresh, or frozen tumor tissue samples), the individual has previously received endocrine therapy as described herein. In other instances, the individual has not previously received endocrine therapy.

[0251] In some instances, these methods further include generating reports, such as electronic reports, web-based reports, or paper reports, for individuals or entities, caregivers, physicians, oncologists, hospitals, clinics, third-party payers, insurance companies, pharmaceutical or biotechnology companies, or government agencies. In some instances, the report includes the output of the method, which includes evaluating ER pathway activity scores and / or E2 induction scores. C. Exemplary methods for determining E2 induction score, E2 inhibition score, and ER pathway activity score

[0252] The methods and assays provided herein may include determining E2 induction scores, E2 inhibition scores, and / or ER pathway activity scores based on the expression levels of a predetermined set of genes (e.g., biomarkers) from individuals (e.g., individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal A or ductal B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer)). These genes may be tissue samples (e.g., tumor tissue samples, e.g., FFPE, FF, archived, fresh, or frozen tumor tissue samples). In some instances, the predetermined set of genes is the set listed in any of Tables 1 to 3. In some instances, the predetermined set of genes is the set listed in Tables 1 and 4 (e.g., 14 - gene characteristics). In some instances, the predetermined set of genes is the set listed in Tables 2 and 5 (e.g., 19 - gene characteristics). In some instances, the predetermined set of genes is the set listed in Tables 3 and 6 (e.g., 41 - gene characteristics). In some instances, the predetermined gene set is the gene set listed in Tables 1 and 4 (e.g., 14 - gene trait) and has no other genes. In some instances, the predetermined gene set is the gene set listed in Tables 2 and 5 (e.g., 19 - gene trait) and has no other genes. In some instances, the predetermined gene set is the gene set listed in Tables 3 and 6 (e.g., 41 - gene trait) and has no other genes.

[0253] In some embodiments, the ER pathway activity score is calculated using an 8-gene signature by subtracting the E2 repression score, which is determined by the average z-score of the five E2-inducible genes described in Table 3, from the average z-score of the three E2-repressive genes described in Table 6.

[0254] In some embodiments, the ER pathway activity score is calculated using a 9-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the five E2-inducing genes described in Table 3, from the E2 repression score determined by the average z-score performance of the four E2-repressing genes described in Table 6. In some embodiments, the ER pathway activity score is calculated using a 9-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the six E2-inducing genes described in Table 3, from the E2 repression score determined by the average z-score performance of the three E2-repressing genes described in Table 6.

[0255] In some embodiments, the ER pathway activity score is calculated using a 10-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the seven E2-inducing genes described in Table 3, from the E2 repression score, determined by the average z-score performance of the three E2-repressing genes described in Table 6. In some embodiments, the ER pathway activity score is calculated using a 10-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the four E2-repressing genes described in Table 6, from the E2-inducing score, determined by the average z-score performance of the six E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 10-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the five E2-inducing genes described in Table 6, from the E2 repression score, determined by the average z-score performance of the five E2-repressing genes described in Table 6.

[0256] In some embodiments, the ER pathway activity score is calculated using an 11-gene feature by subtracting the E2 repression score, determined by the average z-score performance of three of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of eight of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using an 11-gene feature by subtracting the E2 repression score, determined by the average z-score performance of four of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of seven of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 9-gene feature by subtracting the E2 repression score, determined by the average z-score performance of four of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of five of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 11-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the six E2-inducible genes described in Table 3, from the average z-score performance of the five E2-repressive genes described in Table 6.

[0257] In some embodiments, the ER pathway activity score is calculated using 12-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of three of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of nine of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 12-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of four of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of eight of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 12-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of five of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of seven of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 12-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the six E2-inducible genes described in Table 3, from the average z-score performance of the six E2-repressive genes described in Table 6.

[0258] In some embodiments, the ER pathway activity score is calculated using 13-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of three of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of ten of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 13-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of four of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of nine of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 13-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of five of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of eight of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 13-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the seven E2-inducible genes described in Table 3, from the average z-score performance of the six E2-repressive genes described in Table 6.

[0259] In some embodiments, the ER pathway activity score is calculated using a 14-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the 11 E2-inducing genes described in Table 3, from the E2 induction score measured by the average z-score performance of the 3 E2 repressor genes described in Table 6. In some embodiments, the ER pathway activity score is calculated using a 14-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the 10 E2-inducing genes described in Table 3, from the E2 repression score, determined by the average z-score performance of the 4 E2 repressor genes described in Table 6. In some embodiments, the ER pathway activity score is calculated using a 14-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the 9 E2-inducing genes described in Table 3, from the E2 repression score, determined by the average z-score performance of the 5 E2 repressor genes described in Table 6. In some embodiments, the ER pathway activity score is calculated using a 14-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the eight E2-inducing genes described in Table 3, from the E2 repression score, determined by the average z-score performance of the six E2-repressing genes described in Table 6. In some embodiments, the ER pathway activity score is calculated using a 14-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the eight E2-inducing genes described in Table 1, from the E2 repression score, determined by the average z-score performance of the six E2-repressing genes described in Table 4. In some embodiments, the ER pathway activity score is calculated using a 14-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the seven E2-inducing genes described in Table 3, from the E2 repression score, determined by the average z-score performance of the seven E2-repressing genes described in Table 6.

[0260] In some embodiments, the ER pathway activity score is calculated using a 15-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 12 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 15-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 4 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 11 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 15-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 5 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 10 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 15-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the 6 E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of the 9 E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 15-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the 7 E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of the 8 E2 inducible genes described in Table 3.

[0261] In some embodiments, the ER pathway activity score is calculated using a 16-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 13 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 16-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 4 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 12 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 16-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 5 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 11 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 16-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 10 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 16-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 7 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 9 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 16-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 8 of the E2 inducible genes described in Table 3, from the E2 repression score, from the E2 repressor genes described in Table 6.

[0262] In some embodiments, the ER pathway activity score is calculated using 17-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 14 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 17-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 4 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 13 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 17-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 5 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 12 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 17-gene characteristic by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes, from the average z-score performance of 11 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 17-gene characteristic by subtracting the average z-score performance of 7 of the E2 repressor genes, from the average z-score performance of 10 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 17-gene characteristic by subtracting the average z-score performance of 8 of the E2 repressor genes, from the average z-score performance of 9 of the E2-inducing genes described in Table 3.

[0263] In some embodiments, the ER pathway activity score is calculated using 18-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes, from the average z-score performance of 15 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 18-gene characteristics by subtracting the average z-score performance of 4 of the E2 repressor genes, from the average z-score performance of 14 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 18-gene characteristics by subtracting the average z-score performance of 5 of the E2 repressor genes, from the average z-score performance of 13 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 18-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes, from the average z-score performance of 12 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 18-gene characteristics by subtracting the average z-score performance of 7 of the E2 repressor genes, from the average z-score performance of 11 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 18-gene characteristics by subtracting the average z-score performance of 8 of the E2 repressor genes, from the average z-score performance of 10 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 18-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the nine E2-inducible genes described in Table 3, from the average z-score performance of the nine E2-repressive genes described in Table 6.

[0264] In some embodiments, the ER pathway activity score is calculated using the 19-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 16 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 19-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 4 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 15 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 19-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 5 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 14 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 19-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes, from the average z-score performance of 13 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 19-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 7 of the E2 repressor genes, from the average z-score performance of 12 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 19-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 8 of the E2 repressor genes, from the average z-score performance of 11 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 19-gene characteristic by subtracting the E2 repression score, determined by the average z-score performance of the 11 E2-inducing genes described in Table 5, from the E2-inducing score determined by the average z-score performance of the 11 E2-inducing genes described in Table 2. In some embodiments, the ER pathway activity score is calculated using the 19-gene characteristic by subtracting the average z-score performance of the 9 E2-repression genes described in Table 6 from the E2-inducing score determined by the average z-score performance of the 10 E2-inducing genes described in Table 3.

[0265] In some embodiments, the ER pathway activity score is calculated using a 20-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 17 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 20-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 4 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 16 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 20-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 5 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 15 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 20-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 14 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 20-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 7 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 13 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 20-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 8 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 12 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 20-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the 9 E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of the 11 E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 20-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the 10 E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of the 10 E2 repressor genes described in Table 3.

[0266] In some embodiments, the ER pathway activity score is calculated using 21-gene features by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 18 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 21-gene features by subtracting the E2 repression score, determined by the average z-score performance of 4 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 17 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 21-gene features by subtracting the E2 repression score, determined by the average z-score performance of 5 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 16 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 21-gene features by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes, from the average z-score performance of 15 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 21-gene features by subtracting the average z-score performance of 7 of the E2 repressor genes, from the average z-score performance of 14 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 21-gene features by subtracting the average z-score performance of 8 of the E2 repressor genes, from the average z-score performance of 13 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 21-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of the 9 E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of the 12 E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 21-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of the 10 E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of the 11 E2 inducible genes described in Table 3.

[0267] In some embodiments, the ER pathway activity score is calculated using 22-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes, from the average z-score performance of 19 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 22-gene characteristics by subtracting the average z-score performance of 4 of the E2 repressor genes, from the average z-score performance of 18 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 22-gene characteristics by subtracting the average z-score performance of 5 of the E2 repressor genes, from the average z-score performance of 17 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 22-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 16 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 22-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 7 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 15 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 22-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 8 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 14 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 22-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 9 of the E2 repressor genes, from the average z-score performance of 13 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 22-gene characteristics by subtracting the average z-score performance of 10 of the E2 repressor genes, from the average z-score performance of 12 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 22-gene characteristics by subtracting the average z-score performance of 11 of the E2-inducing genes, from the average z-score performance of 11 of the E2-repressor ...inducing genes, from the average z-score performance of

[0268] In some embodiments, the ER pathway activity score is calculated using the 23-gene characteristic by subtracting the E2 repression score, which is determined by the average z-score performance of 3 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 20 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 23-gene characteristic by subtracting the E2 repression score, which is determined by the average z-score performance of 4 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 19 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 23-gene characteristic by subtracting the E2 repression score, which is determined by the average z-score performance of 5 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 18 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 23-gene characteristic by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes, from the average z-score performance of 17 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 23-gene characteristic by subtracting the average z-score performance of 7 of the E2 repressor genes, from the average z-score performance of 16 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 23-gene characteristic by subtracting the average z-score performance of 8 of the E2 repressor genes, from the average z-score performance of 15 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 23-gene characteristic by subtracting the E2 repression score, determined by the average z-score performance of the 9 E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of the 14 E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 23-gene characteristic by subtracting the E2 repression score, determined by the average z-score performance of the 10 E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of the 13 E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 23-gene characteristic by subtracting the E2 repression score, determined by the average z-score performance of the 11 E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of the 12 E2 inducible genes described in Table 3.

[0269] In some embodiments, the ER pathway activity score is calculated using a 24-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 21 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 24-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 4 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 20 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 24-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 5 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 19 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 24-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes, from the average z-score performance of 18 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 24-gene feature by subtracting the average z-score performance of 7 of the E2 repressor genes, from the average z-score performance of 17 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 24-gene feature by subtracting the average z-score performance of 8 of the E2 repressor genes, from the average z-score performance of 16 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 24-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 9 of the E2 repressor genes described in Table 6, from the average z-score performance of 15 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 24-gene feature by subtracting the average z-score performance of 10 of the E2 repressor genes described in Table 6 from the average z-score performance of 14 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 24-gene feature by subtracting the average z-score performance of 11 of the E2 repressor genes described in Table 6 from the average z-score performance of 13 of the E2-inducing genes described in Table 3.In some embodiments, the ER pathway activity score is calculated using 24-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of the 12 E2-inducible genes described in Table 6, from the E2-inducible score determined by the average z-score performance of the 12 E2-repressive genes described in Table 3.

[0270] In some embodiments, the ER pathway activity score is calculated using the 25-gene characteristic by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes, from the average z-score performance of 22 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 25-gene characteristic by subtracting the average z-score performance of 4 of the E2 repressor genes, from the average z-score performance of 21 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 25-gene characteristic by subtracting the average z-score performance of 5 of the E2 repressor genes, from the average z-score performance of 20 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 25-gene characteristic by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes, from the average z-score performance of 19 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 25-gene characteristic by subtracting the average z-score performance of 7 of the E2 repressor genes, from the average z-score performance of 18 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 25-gene characteristic by subtracting the average z-score performance of 8 of the E2 repressor genes, from the average z-score performance of 17 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 25-gene characteristic by subtracting the E2 repression score, determined by the average z-score performance of 9 of the E2 repressor genes, from the average z-score performance of 16 of the E2-inducing genes described in Table 3, from the average z-score performance of 9 of the E2-repressor genes ...In some embodiments, the ER pathway activity score is calculated using 25-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of the 13 E2-inducible genes described in Table 3, from the average z-score performance of the 12 E2-repressive genes described in Table 6.

[0271] In some embodiments, the ER pathway activity score is calculated using the 26-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 3 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 23 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 26-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 4 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 22 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 26-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 5 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 21 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 26-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 6 of the E2 repressor genes, from the average z-score performance of 20 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 26-gene feature by subtracting the average z-score performance of 7 of the E2 repressor genes, from the average z-score performance of 19 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using the 26-gene feature by subtracting the average z-score performance of 8 of the E2 repressor genes, from the average z-score performance of 18 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 26-gene feature by subtracting the E2 repression score, determined by the average z-score performance of 9 of the E2 repressor genes described in Table 6, from the average z-score performance of 17 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 26-gene feature by subtracting the average z-score performance of 10 of the E2 repressor genes described in Table 6 from the average z-score performance of 16 of the E2-inducing genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using a 26-gene feature by subtracting the average z-score performance of 11 of the E2 repressor genes described in Table 6 from the average z-score performance of 15 of the E2-inducing genes described in Table 3.In some embodiments, the ER pathway activity score is calculated using 26-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 12 of the E2 repressor genes described in Table 6, from the E2 induction score determined by the average z-score performance of 14 of the E2 inducible genes described in Table 3. In some embodiments, the ER pathway activity score is calculated using 26-gene characteristics by subtracting the E2 repression score, determined by the average z-score performance of 13 of the E2 inducible genes described in Table 3, from the E2 repression score determined by the average z-score performance of 13 of the E2 repressor genes described in Table 6.

[0272] In some embodiments, the ER pathway activity score is calculated using 27-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of 4 of the E2 repression genes described in Table 6, from the E2 induction score determined by the average z-score performance of 23 of the E2 inducible genes described in Table 3.

[0273] In some embodiments, the ER pathway activity score is calculated using 28-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of 5 of the E2 repression genes described in Table 6, from the E2 induction score determined by the average z-score performance of 23 of the E2 inducible genes described in Table 3.

[0274] In some embodiments, the ER pathway activity score is calculated using 29-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of 6 of the E2 repression genes described in Table 6, from the E2 induction score determined by the average z-score performance of 23 of the E2 inducible genes described in Table 3.

[0275] In some embodiments, the ER pathway activity score is calculated using 30-gene features by subtracting the E2 repression score, which is determined by the average z-score performance of 7 of the E2 repression genes described in Table 6, from the E2 induction score determined by the average z-score performance of 23 of the E2 inducible genes described in Table 3.

[0276] In some embodiments, the ER pathway activity score is calculated using 31-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the 23 E2-inducible genes described in Table 3, from the average z-score performance of the 8 E2-repressive genes described in Table 6.

[0277] In some embodiments, the ER pathway activity score is calculated using 32-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the 23 E2-inducible genes described in Table 3, from the average z-score performance of the 9 E2-repressive genes described in Table 6.

[0278] In some embodiments, the ER pathway activity score is calculated using 33-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the 23 E2-inducible genes described in Table 3, from the average z-score performance of the 10 E2-repressive genes described in Table 6.

[0279] In some embodiments, the ER pathway activity score is calculated using 34-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the 23 E2-inducible genes described in Table 3, from the average z-score performance of the 11 E2-repressive genes described in Table 6.

[0280] In some embodiments, the ER pathway activity score is calculated using 35-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the 23 E2-inducible genes described in Table 3, from the average z-score performance of the 12 E2-repressive genes described in Table 6.

[0281] In some embodiments, the ER pathway activity score is calculated using 36-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the 23 E2-inducible genes described in Table 3, from the average z-score performance of the 13 E2-repressive genes described in Table 6.

[0282] In some embodiments, the ER pathway activity score is calculated using 37-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the 23 E2-inducible genes described in Table 3, from the average z-score performance of the 14 E2-repressive genes described in Table 6.

[0283] In some embodiments, the ER pathway activity score is calculated using 38-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of 23 of the E2-inducible genes described in Table 3, from the average z-score performance of 15 of the E2-repressive genes described in Table 6.

[0284] In some embodiments, the ER pathway activity score is calculated using 39-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the 23 E2-inducible genes described in Table 3, from the average z-score performance of the 16 E2-repressive genes described in Table 6.

[0285] In some embodiments, the ER pathway activity score is calculated using 40-gene features by subtracting the E2 repression score, which is determined by the average z-score performance of the 23 E2-inducible genes described in Table 3, from the average z-score performance of the 17 E2-repressive genes described in Table 6.

[0286] In some embodiments, the ER pathway activity score is calculated using 41-gene characteristics by subtracting the E2 repression score, which is determined by the average z-score performance of the 23 E2-inducible genes described in Table 3, from the average z-score performance of the 18 E2-repressive genes described in Table 6.

[0287] In some embodiments, the ER pathway activity score is calculated using a 14-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the eight E2-inducing genes described in Table 4 from the average z-score performance of the eight E2-inducing genes described in Table 1. In some embodiments, the ER pathway activity score is calculated using a 19-gene feature by subtracting the E2 repression score, determined by the average z-score performance of the eleven E2-inducing genes described in Table 5 from the average z-score performance of the eleven E2-inducing genes described in Table 2. In some embodiments, the ER pathway activity score is calculated using a 41-gene feature by subtracting the average z-score performance of the eleven E2-inducing genes described in Table 6 from the average z-score performance of the twenty-three E2-inducing genes described in Table 3 from the average z-score performance of the eighteen E2-repression genes described in Table 6. The ER pathway activity score can be used as a surrogate biomarker for the ER pathway activity of an individual's tumor.

[0288] In any of the methods or assays provided herein, in which the expression level of a predetermined gene set is determined in a sample from an individual (e.g., a tissue sample, such as a tumor tissue sample, such as formalin-fixed paraffin-embedded (FFPE), fresh-frozen (FF), archived, fresh, or frozen tumor tissue sample), it should be understood that the expression level of the predetermined gene set can be normalized, for example, to a reference gene, such as a housekeeping gene. In some instances, the reference gene is SDHA, GUSB, PPIA, and / or UBC. In some instances, the expression level of more than one gene of interest (e.g., the predetermined gene set listed in any of Tables 1 to 6) can be determined by aggregation methods known to those skilled in the art and disclosed herein (including, for example, by calculating the median or average of all expression levels of the gene of interest). Prior to aggregation, the expression levels of each gene of interest can be standardized using statistical methods known to those skilled in this technique and disclosed herein, including, for example, standardization to the expression levels of one or more housekeeping genes, to the median or mean expression levels of genes measured across the reference population, standardization to the total library size, or standardization to the median or mean expression levels of all genes measured. In some instances, prior to aggregation across multiple genes of interest, the standardized expression levels of each gene of interest can be standardized using statistical methods known to those skilled in this technique and disclosed herein, including, for example, by calculating a Z-score for the standardized expression levels of each gene of interest, or by, for example, scaling the standardized expression levels of each gene to their respective scales of expression levels in the reference population.

[0289] Samples from individuals may be FFPE samples, FF samples, fresh samples, frozen samples, or archived samples. In some instances, the sample is an FFPE sample. In some instances, the sample is an FF sample. The degree of expression of the predetermined gene set can be quantified based on any suitable criteria known in this technique, including (but not limited to) measures of the number of mRNA, DNA, cDNA, and / or gene copies in an individual.

[0290] Table 1: 8 E2-induced genes with 14-gene characteristics

[0291] Table 2: 11 E2-induced genes with 19-gene characteristics

[0292] Table 3: 41 - 23 E2-induced genes with gene characteristics

[0293] Table 4: Six E2 repressor genes with 14-gene characteristics

[0294] Table 5: 19-Genetic Characteristics of 8 E2 Repressor Genes

[0295] Table 6: 41 - Genetic Characteristics of 18 E2 Repressor Genes

[0296] In some instances of the methods and assays described above, the degree of gene expression may be the degree of nucleic acid expression (e.g., RNA expression (e.g., mRNA expression) or DNA expression). Any suitable method for determining the degree of nucleic acid expression may be used, for example, as described in Section VII below. In some instances, the degree of nucleic acid expression is determined using RNA-seq (e.g., using the RNA ACCESS® protocol or the TRUSEQ® RIBO-ZERO® protocol (ILLUMINA®)), RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, or combinations thereof.

[0297] Methods for evaluating mRNA in cells are well-known and include, for example, RNA sequencing (RNA-seq), whole-genome sequencing (WGS), serial analysis of gene expression (SAGE), and various nucleic acid amplification assays (such as RT-PCR (e.g., qRT-PCR)) using complementary primers specifically targeting a predetermined set of genes. Furthermore, these methods may include one or more steps that allow us to determine the level of target mRNA in a biological sample (e.g., by simultaneously examining the level of a comparative control mRNA sequence of a "housekeeping" gene (such as a member of the actin family). Where appropriate, the sequence of the amplified target cDNA may be determined. Options may include protocols for examining or detecting mRNA (such as target mRNA) in tissue or cell samples using microarray technology. Using a nucleic acid microarray, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. These probes are then hybridized to a nucleic acid array immobilized on a solid support. The array is configured such that the sequence and location of each member of the array are known. For example, the selection of genes whose expression is associated with increased or decreased clinical benefit from treatments including immunotherapy and inhibitory matrix antagonists can be arranged on a solid support. Hybridization of a labeled probe with a specific array member indicates that the sample from which the probe is derived expresses the gene.

[0298] In some instances of the methods and assays, a sample (e.g., a tissue sample, such as a tumor tissue sample, such as FFPE, FF, archived, fresh, or frozen tumor tissue sample) is obtained from the individual prior to (e.g., minutes, hours, days, weeks, months, or years prior to) the administration of endocrine therapy as described herein. In other words, this sample may serve as a baseline sample. In some instances of the methods described above, a sample is obtained from the individual after the administration of endocrine therapy (e.g., minutes, hours, or days later). In some instances, a sample is obtained from the individual within 30 hours of the administration of endocrine therapy. In some instances, multiple samples are obtained from the same individual at different time points (e.g., before and after the administration of endocrine therapy).

[0299] In any of the above examples, the individual may have HR+ breast cancer. In some examples, the HR+ cancer may be ER+ breast cancer. In some examples, the individual may have ER+ breast cancer, selected from, for example, ductal A or ductal B breast cancer. In some examples, the breast cancer may be advanced or metastatic breast cancer.

[0300] In some instances involving the determination of ER pathway activity scores and / or E2 induction scores from samples of an individual (e.g., tissue samples, such as tumor tissue samples, such as formalin-fixed paraffin-embedded (FFPE), fresh-frozen (FF), archived, fresh, or frozen tumor tissue samples), the individual has previously received endocrine therapy as described herein. In other instances, the individual has not previously received endocrine therapy.

[0301] In some instances, these methods further include generating reports, such as electronic reports, web-based reports, or paper reports, for individuals or entities, caregivers, physicians, oncologists, hospitals, clinics, third-party payers, insurance companies, pharmaceutical or biotechnology companies, or government agencies. In some instances, the report includes the output of the method, which includes evaluating ER pathway activity scores and / or E2 induction scores. [IV.] [Anticancer agents] []

[0302] This article provides methods for treating individuals with breast cancer (e.g., HR+ breast cancer (e.g., ER+ breast cancer (e.g., ductal type A or ductal type B breast cancer)) and / or metastatic or locally advanced breast cancer). Any of the above methods may be based on the determination of E2 induction scores and / or ER pathway activity scores from samples taken from the individual (e.g., tissue samples, such as tumor tissue samples, such as FFPE, FF, archived, fresh, or frozen tumor tissue samples).

[0303] In some instances of any of the methods described above, the anticancer therapeutic agent used in the methods described herein may be administered via, for example, orally, intramuscularly, subcutaneously, intravenously, intradermally, percutaneously, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intrasheathically, intranasally, intravaginally, intrarectally, intratumorally, intratumorally, intraperitoneally, subconjunctivally, intracysticly, intramucosally, intraperitoneally, intraumbilically, intraocularly, intraorbitally (e.g., by intravitreal injection), by eye drops, locally, transdermally, non-enterically, by inhalation, by injection, by inhibition of transplantation, by infusion, by continuous infusion, by direct local perfusion of target cells, by catheter, by irrigation, as a cream or lipid composition. In some instances of any of the methods described above, the anticancer therapeutic agents used in the methods described herein may be administered orally. For example, in some instances, endocrine therapy as described herein may be administered orally or intramuscularly. In some instances, SERM may be administered orally or intramuscularly. In some instances, SERD may be administered orally or intramuscularly. In some instances of any of the methods described above, the anticancer therapeutic agents used in the methods described herein may be administered intramuscularly. For example, in some instances, endocrine therapy as described herein may be administered orally. In some instances, SERM may be administered orally. In some instances, SERD may be administered orally. The anticancer therapeutic agents used in the methods described herein may also be administered systemically or locally. The administration method may vary depending on various factors (e.g., the anticancer therapeutic agent being administered and the severity of the condition, disease, or symptom being treated (e.g., breast cancer)).

[0304] Anticancer agents (including endocrine agents (and any additional therapeutic agents) as described herein) may be formulated, dosed, and administered in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of drug delivery, the method of administration, the timing of administration, and other factors known to the medical practitioner. Anticancer agents need not, but may be formulated and / or administered concurrently with one or more agents currently used for the prevention or treatment of the disease in question. The effective amount of these other agents depends on the amount of anticancer agent present in the formulation, the type of disease or treatment, and the other factors discussed above. These are generally administered at the same dose and via the route of administration as described herein, or approximately 1 to 99% of the dose described herein, or at any dose and via any route deemed appropriate by empirical / clinical determination.

[0305] For the prevention or treatment of breast cancer (e.g., HR+ breast cancer, e.g., ER+ breast cancer, e.g., ductal A or B breast cancer)), DCIS, and / or metastatic or locally advanced breast cancer, the appropriate dose of the anticancer agent (e.g., endocrine therapy) described herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type, severity, and course of the disease to be treated (whether the anticancer agent is administered for preventative or therapeutic purposes), prior therapy, the individual's clinical history and response to the anticancer agent, and the attending physician's discretion. The anticancer agent may be administered to the individual in a single dose or over a series of treatments. For repeated administrations over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms is achieved. An initial, faster-acting dose may be administered, followed by one or more lower doses. However, other dosing regimens may be available. Progression of this therapy is easily monitored using known techniques and assays. In some instances, the endocrine therapies provided herein are administered at doses of about 1 mg / kg to about 100 mg / kg. In another instance, the endocrine therapies provided herein are administered at doses of about 100 mg / kg to about 1000 mg / kg. In yet another instance, the endocrine therapies described herein are administered at doses of about 1000 mg / kg to about 2000 mg / kg. Some endocrine therapies described herein may be administered via a cyclical route as understood in the art, for example, for 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive days, followed by rest periods of 1, 2, 3, 4, 5, 6, 7, or more days. In yet another instance, the endocrine therapies described herein are administered according to a package insert. A. Endocrine therapy

[0306] In some instances of any of the methods described above, endocrine therapy may be administered to an individual.

[0307] Exemplary endocrine therapies used in the methods described herein include compounds that modulate the activity of estrogen receptors. In some instances, the compounds described herein include selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), aromatase inhibitors (AIs), or combinations thereof.

[0308] In some instances, the endocrine therapy includes an aromatase inhibitor. The aromatase inhibitor may be a pharmaceutical agent known in this art. For example, in one instance, the aromatase inhibitor is letrozole, anastrozole, exemestane, or testosterone, or a pharmaceutically acceptable salt thereof, or a combination thereof.

[0309] In another instance, the endocrine therapy includes SERM.

[0310] This endocrine therapy may include compounds that are tetrasubstituted olefins known to have antagonistic activity against ER. For example, the endocrine therapy may be tamoxifen, including its derivatives, such as hydroxytamoxifen. In one example, the endocrine therapy includes nafoxidine. In another example, the endocrine therapy includes clomiphene, toremifene, raloxifene, diacetylcholine, bardoxifene, bromoestradiol, cyclofennig, lasoxifene, olmexifen, acobifene, elacestrant, brilanestrant, oflomiphene, droloxifene, etacstil, or oselmifene, or pharmaceutically acceptable salts thereof, or combinations thereof. In yet another example, the endocrine therapy includes G1T48 or pharmaceutically acceptable salts thereof.

[0311] In another instance, the endocrine therapy includes SERD.

[0312] In one instance, the endocrine therapy included fulvestrant. , Or a medicinally acceptable salt.

[0313] In one state, the endocrine therapy includes a compound having formula (1): , (1) in: Z is either -OH or -OR10; R2 is a C1-4 alkyl, C1-4 fluoroalkyl, C1-4 deuterated alkyl, C3-6 cycloalkyl, or C1-4 alkyl-W; W represents hydroxyl, halogen, CN, C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy, C1-4 haloalkoxy, or C3-6 cycloalkyl; Each R3 is independently a halogen, a C1-4 alkyl, or a C1-4 fluoroalkyl; Each R4 is independently a halogen, -CN, -OR9, -S(O)2R10, C1-4 alkyl, C1-4 fluoroalkyl, or C1-4 heteroalkyl; Each R5 is independently a halogen, -CN, -OR9, -S(O)2R10, C1-4 alkyl, C1-4 fluoroalkyl, or C1-4 heteroalkyl; R6 is H, C1-4 alkyl, or halogen; R7 is H, C1-4 alkyl, or halogen; R9 is H, C1-6 alkyl, C1-6 fluoroalkyl, or C3-6 cycloalkyl; R10 is a C1-6 alkyl group; m is 0, 1, or 2; n is 0, 1, 2, 3, or 4; and p is 0, 1, or 2.

[0314] In another example of a compound of formula (1), Z is -OH or -OR10; R2 is a C1-4 alkyl, C1-4 fluoroalkyl, C1-4 deuterated alkyl, C3-6 cycloalkyl, or C1-4 alkyl-W, wherein W is hydroxyl, halogen, CN, or C1-4 alkyl; each R3 is independently a halogen, C1-4 alkyl, or C1-4 fluoroalkyl; each R4 is independently a halogen, -CN, -OR9, -S(O)2R10, C1-4 alkyl, C1-4 fluoroalkyl, or C1-4 heteroalkyl; each R5 is independently a halogen, -CN, -OR9, -S(O)2R10, C1-4 alkyl, C1-4 fluoroalkyl, or C1-4 heteroalkyl; R6 is H, C1-4 alkyl, or halogen; R7 is H, C1-4 Alkyl or halogen; R9 is H, C1-6 alkyl, C1-6 fluoroalkyl or C3-6 cycloalkyl; R10 is C1-6 alkyl; m is 0, 1 or 2; n is 0, 1, 2, 3 or 4; and p is 0, 1 or 2.

[0315] In another example of a compound of formula (1), Z is -OH. In another example of a compound of formula (1), Z is -OR10. In another example of a compound of formula (1), Z is -OH, -OCH3, or -OCH2CH3.

[0316] In another example of a compound of formula (1), R6 is H, -CH3, F, or Cl. In another example of a compound of formula (1), R6 is H. In another example of a compound of formula (1), R7 is H, -CH3, F, or Cl. In another example of a compound of formula (1), R7 is H.

[0317] In another example of a compound of formula (1), R3 is independently a halogen, a C1-4 alkyl, or a C1-4 fluoroalkyl. In another example of a compound of formula (1), each R3 is independently F, Cl, or -CH3. In another example of a compound of formula (1), each R4 is independently a halogen, -CN, -OH, -OR9, -S(O)2R10, a C1-4 alkyl, a C1-4 fluoroalkyl, or a C1-4 heteroalkyl. In another example of a compound of formula (1), each R4 is independently a halogen, -CN, -OH, -S(O)2CH3, -S(O)2CH2CH3, -CH3, -CH2CH3, -CF3, -CH2OH, -OCF3, -OCH3, or -OCH2CH3. In another example of a compound of formula (1), each R4 is independently F, Cl, -CN, -OH, -CH3, -CH2CH3, -CF3, -CH2OH, -OCF3, -OCH3, or -OCH2CH3. In another example of a compound of formula (1), each R4 is independently F or Cl. In another example of a compound of formula (1), each R5 is independently a halogen, a C1-4 alkyl, or a C1-4 fluoroalkyl. In another example of a compound of formula (1), each R5 is independently F, Cl, or -CH3.

[0318] In another example of a compound of formula (1), m is 0 or 1. In another example of a compound of formula (1), m is 0. In another example of a compound of formula (1), m is 1. In another example of a compound of formula (1), n ​​is 0, 1, or 2. In another example of a compound of formula (1), n ​​is 0. In another example of a compound of formula (1), n ​​is 1. In another example of a compound of formula (1), n ​​is 2. In another example of a compound of formula (1), p is 0 or 1. In another example of a compound of formula (1), p is 0. In another example of a compound of formula (1), p is 1.

[0319] In another example of a compound of formula (1), Z is -OH; R6 is H, -CH3, F or Cl; R7 is H, -CH3, F or Cl; each R3 is independently a halogen, C1-4 alkyl or C1-4 fluoroalkyl; each R4 is independently a halogen, -CN, -OR9, -S(O)2R10, C1-4 alkyl, C1-4 fluoroalkyl or C1-4 heteroalkyl; each R5 is independently a halogen, C1-4 alkyl or C1-4 fluoroalkyl; m is 0 or 1; n is 0, 1 or 2; and p is 0 or 1.

[0320] In another example of a compound of formula (1), R2 is a C1-4 alkyl, C1-4 fluoroalkyl, C1-4 deuterated alkyl, C3-6 cycloalkyl, or C1-4 alkyl-W; W is a hydroxyl, halogen, CN, C1-4 alkoxy, or C3-6 cycloalkyl. In another example of a compound of formula (1), R2 is a C1-4 alkyl, C1-4 fluoroalkyl, or C1-4 deuterated alkyl. In another example of a compound of formula (1), R2 is a C1-4 alkyl. In another example of a compound of formula (1), R2 is -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH2CH2CH3, -CH2CH(CH3)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CD3, -CH2CD3, -CD2CD3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, -CH2-W, or -CH2CH2-W; W is hydroxyl, F, Cl, -CN, -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH3)2, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In another example of a compound of formula (1), W is hydroxyl, F, Cl, -CN, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In another example of a compound of formula (1), R2 is -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH2CH2CH3, -CH2CH(CH3)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CD3, -CH2CD3, -CD2CD3, -CH2-W, or -CH2CH2-W. In another example of a compound of formula (1), R2 is -CH3, -CH2CH3, -CH2F, -CHF2, -CF3, -CH2CF3, -CD3, -CD2CD3, -CH2CD3, or cyclopropyl.

[0321] In another example of the compound of formula (1), Z is -OH; R6 is H; R7 is H; m is 0; n is 0, 1 or 2; and p is 0.

[0322] In another example of a compound of formula (1), the compound of formula (1) has the structure of formula (1a) or a pharmaceutically acceptable salt or N-oxide thereof: . Equation (1a)

[0323] In another embodiment, the endocrine therapy includes the compounds described in U.S. Patent No. 8,299,112 (e.g., in Table 1), the entire contents of which are incorporated herein by reference for all purposes.

[0324] In another embodiment, the endocrine therapy comprises compounds having the following formula: (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)ethyl acrylate; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-benzo[d][1,2,3]triazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-chlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-chlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(3-methoxyphenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(3-(hydroxymethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-(hydroxymethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-(hydroxymethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-(hydroxymethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl) (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(p-tolyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(p-tolyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(p-tolyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(2-methoxyphenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(2-methoxyphenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl) (E)-2-(4-methoxyphenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(3-chlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(3-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(2-ethylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(2-(trifluoromethyl)phenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-4-chloro-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-fluorophenyl)-1-(1H-indazol-4-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-) (E)-3-(4-(E)-2-(2,4-difluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-(E)-2-(2-chloro-3-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-(E)-2-(2-chloro-3-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-(E)-2-cyclopropyl-1-(1H-indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid; (E)-3-(4-(E)-2-(4-fluoro-2-methylphenyl)-1-(1H-indazol-5-yl) (E)-3-(4-(E)-2-(2,6-difluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-(E)-2-(2,6-dichlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-(E)-4,4,4-trideuterated-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-(E)-2-(4-fluoro-3-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(5-fluoro-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2,3-difluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2,5-difluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2,5-difluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(2-chloro-6-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(7-chloro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(4-methyl-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(7-methyl-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(7-methyl-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(6-methyl) -1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(3-methyl-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(3-chloro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-chloro-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-phenylprop-1-en-1-yl) (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-phenylpent-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(3-cyanophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-cyanophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-4-hydroxy-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((Z)-1-(1H-indola-5-yl)-3-methoxy-2-phenylprop-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(4-fluoro-1H-indola-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indola-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indola-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(6-chloro-1H-indola-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-1-(1H-indazol-5-yl)-4-methyl-2-phenylpent-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-benzo[d][1,2,3]triazol-5-yl)-2-(2-chloro-4-fluorophenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(4-chloro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-cyclopentyl-1-(1H-indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid; (E)-3-(4-((E)-2-cyclohexyl-1-( 1H-Indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-3-methyl-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-3-cyclopropyl-1-(1H-Indazol-5-yl)-2-phenylprop-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chlorophenyl)-2-cyclopropyl-1-(1H-Indazol-5-yl)vinyl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(6-fluoro-1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4 -((E)-1-(1H-benzo[d]imidazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-phenylhex-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-3-cyclopentyl-1-(1H-indazol-5-yl)-2-phenylprop-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(7-fluoro-1H-indazol-5-yl)-2-phenylbut-1-yl)acrylic acid; (E)-3-(4-((E)-1-(7-fluoro-1H-indole-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)-4-methylpent-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((Z)-3,3-difluoro-1-(1H-indazol-5-yl)-2-phenylprop-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(7-fluoro-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-4-fluoro-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-4-chloro-2-(2-chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((Z)-3,3,3-trifluoro-1-(1H-indazol-5-yl)-2-phenylprop-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(4-fluoro-1H-indazol-5-yl)-2-(4-fluoro-2-methylphenyl)but-1-en-1-yl)phenyl)acrylic acid;( (E)-3-(4-((E)-2-(4-chloro-2-methylphenyl)-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-cyclopropyl-1-(4-fluoro-1H-indazol-5-yl)-2-(4-fluoro-2-methylphenyl)vinyl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-chloro-2-methylphenyl)-2-cyclopropyl-1-(4-fluoro-1H-indazol-5-yl)vinyl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(4-chloro-1H-indazol-5-yl)-2-(2-chloro-4-fluorophenyl)but-1-en-)phenyl)acrylic acid; (E)-3-(4-((E)-1-(4-chloro-1H-indazol-5-yl)-2-(2-chloro-4-fluorophenyl)but-1-en-)phenyl)acrylic acid; (E)-3-(4-((E)-1-(4-chloro-1H-indazol-5-yl)-2-(2-chloro-4-fluorophenyl)but-1-en-)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-3,3-difluoro-1-(1H-indazol-5-yl)prop-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-cyclopropyl-1-(4-fluoro-1H-indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid; (E)-3-(4-((E)-4-chloro-1-(4-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-4-chloro-2-(2-chloro-4-fluorophenyl)-1-(4-fluoro-1H-indazol-5-yl)but- 1-En-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-4-fluoro-2-(4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-4-fluoro-1-(4-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-5-methoxy-2-phenylpent-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-6-methoxy-2-phenylhex-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)-3-methylbut-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(3-(trifluoromethoxy)phenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-cyclobutyl-1-(1H-indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-2-cyclobutyl-1-(1H-indazol-5-yl)vinyl)phenyl)acrylic acid; (E)-3-(4 -((E)-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-2-cyclobutyl-1-(3-fluoro-1H-indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(3-fluoro-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-2-cyclobutyl-1-(3-fluoro-1H-indazol-5-yl)vinyl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-2-cyclobutyl-1-(3-fluoro-1H-indazol-5-yl)vinyl)phenyl)acrylic acid;(E)-3-(4-((E)-2- (4-Fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)ethyl acrylate; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(2-methoxyphenyl)but-1-en-1-yl)phenyl)ethyl acrylate; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(4-methoxyphenyl)but-1-en-1-yl)phenyl)ethyl acrylate; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-2-methacrylic acid; (E)-3-(4-((Z)-1-(1H-indazol-5-yl)-2-... (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)-2-methylphenyl)acrylic acid; (E)-3-(4-((Z)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)-2-chlorophenyl)acrylic acid; (Z)-3-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-2-fluoroacrylic acid; (Z)-3-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-2-chloroacrylic acid;(E)-3-(4-((Z)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)-3-fluorophenyl)acrylic acid; (E)-3-(4-((Z)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)-2-fluorophenyl)acrylic acid; (E)-3-(4-((Z)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)-2-(trifluoromethyl)phenyl)acrylic acid; (E)-3-(4-((Z)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)-3-methoxyphenyl)acrylic acid; (E)-3-(4-((Z)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)-3-methoxyphenyl)acrylic acid; (E)-3-(4-((Z)-1-(1H-indazol-5-yl)-2-phenyl) (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylate ethyl hydrochloride; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylate; (E)-3-(4-((E)-2-(2,4-dichlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylate ethyl hydrochloride; (E)-3-(4-((E)-2-(2,4-dichlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylate ethyl hydrochloride; (E)-3-(4-((E)-2-(2,4-dichlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylate ethyl hydrochloride; (E)-3-(4-((E)-2-(2,4-dichlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl) (E)-3-(4-((E)-2-(4-chloro-2-(trifluoromethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-2-cyclopropyl-1-(1H-indazol-5-yl)vinyl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-fluoro-2-(trifluoromethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-(1-(4-fluoro-1H-indazol-5-yl)-2-(4-fluoro-2-(trifluoromethyl)phenyl)butyl)phenyl)acrylic acid ;(E)-3-(4-((E)-2-(2,4-dichlorophenyl)-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(4-chloro-2-(trifluoromethyl)phenyl)-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-4-fluoro-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(2-chloro-4-methoxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(2,4-dichlorophenyl)-4-fluoro-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-cyclopropyl-2-(2,4-dichlorophenyl)-1-(4-fluoro-1H-indazol-5-yl)vinyl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-2-cyclopropyl-1-(4-fluoro-1H-indazol-5-yl)vinyl)phenyl)acrylic acid; (E)-3-(4-((E)-2-cyclopropyl-2-(2,4-dichlorophenyl)-1-(1H-indazol-5-yl)vinyl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-chloro-2-methylphenyl)-2-cyclopropyl-1-(1H-indazol-5-yl)vinyl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(2-methyl-5-(methanesulfonyl)phenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(4-methoxy-2-methylphenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-fluoro-4-methoxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)- 3-(4-((E)-2-(2-chloro-5-methoxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-fluoro-4-(methanesulfonyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2,4-dichlorophenyl)-3,3,4,4,4-pentadeuter-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(3-(methanesulfonyl)phenyl)but-1-en-1-yl)phenyl)propionic acid Acrylic acid; (E)-3-(4-((E)-2-(2,4-dichlorophenyl)-1-(7-fluoro-1H-indole-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-3-methoxyphenyl)-1-(1H-indole-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2,4-dichlorophenyl)-1-(7-fluoro-1H-indole-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(7-fluoro-1H-indole-5-yl)but-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-3,3,4,4,4-pentadeuterated-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-chloro-2-cyanophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-cyano-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-cyano-4-(trifluoromethyl)phenyl)-1-(1H-indazol-5-yl))phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-cyano-4-(trifluoromethyl)phenyl)-1-(1H-indazol-5-yl))phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-cyanophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(3-cyano-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-cyano-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-cyano-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(5-cyano-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-cyano-4-methoxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(3-hydroxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-hydroxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-hydroxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)) (E)-3-(4-(1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)propionic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(4-(2-methoxyethoxy)phenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(4-(3-methoxypropoxy)phenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(3-(2-methoxyethoxy)phenyl)but-1-en-1-yl)phenyl)acrylic acid;(E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(3-(3-methoxypropoxy)phenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(3-(cyclohexyloxy)phenyl)-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(3-butoxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(3-) (E)-3-(4-((E)-2-(3-(hexyloxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-butoxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(4-(pentoxy)phenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-butoxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2- (4-(hexyloxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(4-(2-hydroxyethoxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-1-(1H-indazol-5-yl)-2-(2-(methanesulfonyl)phenyl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chlorophenyl)-1-(1-methyl-1H-indazol-5-yl)but-1-en-1-yl) (E)-3-(4-((E)-2-cyclobutyl-1-(1-methyl-1H-indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(1-methyl-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; (E)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(1-(difluoromethyl)-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid; or a pharmaceutically acceptable salt or N-oxide thereof, or a combination thereof.

[0325] In one instance, the endocrine therapy includes brilanestrant (GDC-0810) having the following structure: , Or a medicinally acceptable salt.

[0326] In another embodiment, the endocrine therapy includes compounds described in USPN 9,499,538 or 9,586,952, the full text of which is incorporated herein by reference for all purposes.

[0327] In another embodiment, the endocrine therapy comprises compounds described in USPN 7,612,114 or 8,399,520, the full text of which is incorporated herein by reference for all purposes. In one example, the endocrine therapy comprises elacestrant (RAD1901): , Or a medicinally acceptable salt.

[0328] In another embodiment, the endocrine therapy comprises the compounds described in International Patent Application No. WO2018077260 (e.g., in Table 2), the entire contents of which are incorporated herein by reference for all purposes. In one example, the endocrine therapy comprises LX-039. In another example, the endocrine therapy comprises a compound having the following structure: , Or a medicinally acceptable salt.

[0329] In another embodiment, the endocrine therapy includes compounds described in International Patent Application Nos. WO2017136688, WO2017162206, WO2017140669, WO2017216280, WO2017216279, WO2018091153, WO2018019793, and WO2018077630, the full text of which is incorporated herein by reference for all purposes.

[0330] In another example, the endocrine therapy includes AZ9496 having the following structure: , Or a medicinally acceptable salt.

[0331] In another embodiment, the endocrine therapy comprises a compound as described in U.S. Patent Application No. 20150284357, the entire contents of which are incorporated herein by reference for all purposes. In another embodiment, the endocrine therapy comprises a compound as described in USPN 9,475,791, the entire contents of which are incorporated herein by reference for all purposes. In yet another embodiment, the endocrine therapy comprises a compound as described in International Patent Application Nos. WO2018081168 or WO2018129387, the entire contents of which are incorporated herein by reference for all purposes. In one embodiment, the endocrine therapy comprises a compound having the following formula: Or its stereoisomers or pharmaceutically acceptable salts.

[0332] In another embodiment, the endocrine therapy includes compounds described in USPN 8,703,810 (e.g., in the table of compounds provided therein), the full text of which is incorporated herein by reference for all purposes.

[0333] In one state, the endocrine therapy includes a compound having formula (2): , Equation (2) Or a medicinally acceptable salt, wherein: R1 is H, F, C1-C4 alkyl, or C1-C4 fluoroalkyl; R3 is H, halogen, C1-C4 alkyl, C3-C6 cycloalkyl, or C1-C4 fluoroalkyl; Each R4 group is independently selected from H, halogens, -CN, -OH, -OR9, -SR9, -S(O)R10, -S(O)2R10, -C(O)R10, -C(O)OH, -C(O)OR10, -C(O)NHR10, -C(O)N(R10)2, substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C1-C6 fluoroalkyl groups, substituted or unsubstituted C1-C6 fluoroalkoxy groups, substituted or unsubstituted C1-C6 alkoxy groups, and substituted or unsubstituted C1-C6 heteroalkyl groups; Each R5 group is independently selected from H, halogens, -CN, -OH, -OR9, -SR9, -S(O)R10, -S(O)2R10, substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C1-C6 fluoroalkyl groups, substituted or unsubstituted C1-C6 fluoroalkoxy groups, substituted or unsubstituted C1-C6 alkoxy groups, and substituted or unsubstituted C1-C6 heteroalkyl groups; Each R6 group is independently selected from H, halogens, -CN, -OH, -OR9, -SR9, -S(O)R10, -S(O)2R10, substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C1-C6 fluoroalkyl groups, substituted or unsubstituted C1-C6 fluoroalkoxy groups, substituted or unsubstituted C1-C6 alkoxy groups, and substituted or unsubstituted C1-C6 heteroalkyl groups; It is pyrrolidinyl or azahexacyclic butyl; R7 is H or a C1-C4 alkyl group; Each R8 group is independently selected from F, Cl, -CN, -OH, -OR9, -SR9, -S(O)R10, -S(O)2R10, substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C1-C6 fluoroalkyl groups, substituted or unsubstituted C1-C6 fluoroalkoxy groups, substituted or unsubstituted C1-C6 alkoxy groups, and substituted or unsubstituted C1-C6 heteroalkyl groups; Alternatively, one R8 atom, together with R1 and the intervening atom connecting R8 to R1, can form a 5-, 6-, or 7-membered ring; Each R9 group is independently selected from H, -C(O)R10, -C(O)OR10, -C(O)NHR10, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C1-C6 fluoroalkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C2-C10 heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -C1-C2 pentenyl- (substituted or unsubstituted C3-C10 cycloalkyl), -C1-C2 pentenyl- (substituted or unsubstituted C2-C10 heterocycloalkyl), -C1-C2 pentenyl- (substituted or unsubstituted aryl), and -C1-C2 pentenyl- (substituted or unsubstituted heteroaryl); or Each R10 is independently selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C1-C6 fluoroalkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C2-C10 heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -C1-C2 pentenyl- (substituted or unsubstituted C3-C10 cycloalkyl), -C1-C2 pentenyl- (substituted or unsubstituted C2-C10 heterocycloalkyl), -C1-C2 pentenyl- (substituted or unsubstituted aryl), and -C1-C2 pentenyl- (substituted or unsubstituted heteroaryl); Y is -O-, -S-, or -NR11-; R11 is H, -C(O)R10, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl. X is -O-, -S-, -CH2-, -NH-, or -N(C1-C6 alkyl)-; m can be 0, 1, 2, 3, or 4; n is 0, 1, 2, or 3; p is 0, 1, 2, 3, or 4; and t can be 1, 2, 3 or 4.

[0334] In one example, the compound of formula (2) is a compound wherein R1 is H or C1-C4 alkyl; R3 is C1-C4 alkyl or C1-C4 fluoroalkyl; each R4 is independently selected from H, halogen, -CN, -OH, -OR9, -SR9, -S(=O)R10, -S(=O)2R10, C1-C4 alkyl, C1-C4 fluoroalkyl, C1-C4 fluoroalkoxy, C1-C4 alkoxy and C1-C4 heteroalkyl; each R5 is independently selected from H, F, Cl, -OH, -CH3, -CF3, -OCF3 and -OCH3; each R6 is independently selected from H, F, Cl, -OH, -CH3, -CF3, -OCF3 and -OCH3; R7 is H; each R8 The components are independently selected from H, F, Cl, -OH, C1-C4 alkyl, C1-C4 fluoroalkyl, C1-C4 fluoroalkoxy, C1-C4 alkoxy, and C1-C4 heteroalkyl; Y is -O- or -S-; X is -O-, -S-, -CH2-, -NH-, or -N(CH3)-; and p is 0, 1, or 2.

[0335] In another embodiment, the endocrine therapy comprises compounds having the following formula: 3-(3-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; (S)-3-(3-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; (R)-3-(3-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol 3-(3-hydroxyphenyl)-4-methyl-2-(4-((R)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-hydroxyphenyl)-4-methyl-2-(4-(2-((S)-3-methylpyrrolidin-1-yl)ethoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((S)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-hydroxyphenyl)-4-methyl-2-(4-((R)-2-)ethoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-hydroxyphenyl)-4-methyl-2-(4-((R)-2-)ethoxy)phenyl)-2H-benzopiperan-6-ol; ((S)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 2-(4-((S)-2-(3,3-dimethylpyrrolidin-1-yl)propoxy)phenyl)-3-(3-hydroxyphenyl)-4-methyl-2H-benzopiperan-6-ol; 2-(4-(2-(3,3-dimethylpyrrolidin-1-yl)ethoxy)phenyl)-3-(3-hydroxyphenyl)-4-methyl-2H-benzopiperan-6-ol; 3-(3-hydroxyphenyl)-4-methyl-2-(4-(2-((R)-2-methylpyrrolidin-1-yl)ethoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-hydroxyphenyl)-4-methyl-2-( 4-((S)-2-((R)-2-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((S)-2-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-7-ol; 3-(4-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-7-ol;4-Methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-3-phenyl-2H-benzopiperan-6-ol; 3-(4-fluorophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; (S)-3-(4-fluorophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; (R)-3-(4-fluorophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(4-Hydrophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-fluorophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-fluoro-4-Hydrophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 2-(2-fluoro-4-((S)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-Hydrophenyl)-4-methyl-2H-benzopiperan-6-ol; 3-(3-Hydrophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-Hydrophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(4-Hydrophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(4-Hydrophenyl)-4-methyl-2-(4-((S)-2-((R)- 3-Methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(4-hydroxy-3-methylphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3-fluoro-5-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(4-chlorophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol;3-(2-fluoro-4-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3,4-difluorophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3,5-difluoro-4-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(2,4-difluoro-3-hydroxyphenyl)-4-methyl-2-(4-((S))-4-methylpropoxy)phenyl)-2H-benzopiperan-6-ol -2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(3,4-difluoro-5-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(2-chloro-4-fluorophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(2,4-difluorophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H- Benzo[a]piperan-6-ol; 3-(4-bromophenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzo[a]piperan-6-ol; 4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-3-(o-tolyl)-2H-benzo[a]piperan-6-ol; 3-(4-fluoro-3-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzo[a]piperan-6-ol; 3-(4-ethynylphenyl)-4-methyl-2-(4-((S)-2-((R)) -3-Methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 4-Methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-3-(4-(methanesulfonyl)phenyl)-2H-benzopiperan-6-ol; 3-(2-fluoro-3-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 5-fluoro-3-(3-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol;3-(2-fluoro-5-hydroxyphenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 2-(4-((S)-2-((R)-3-fluoropyrrolidin-1-yl)propoxy)phenyl)-3-(3-hydroxyphenyl)-4-methyl-2H-benzopiperan-6-ol; 3-(4-hydroxyphenyl)-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-4-(trifluoro) 3-(3-hydroxyphenyl)-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-4-(trifluoromethyl)-2H-benzopiperan-6-ol; 3-(3-hydroxy-4-(trifluoromethyl)phenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; 3-(4-hydroxy-3-(trifluoromethyl)phenyl)-4-methyl-2-(4-((S)-2-((R)-3-methylpyrrolidin-1-yl)propoxy)phenyl)-2H-benzopiperan-6-ol; or a pharmaceutically acceptable salt thereof, or a combination thereof.

[0336] In one example, the endocrine therapy includes GDC-0927 (SRN-0927) having the following structure: , Or a medically acceptable salt.

[0337] In another embodiment, the endocrine therapy includes compounds described in USPN 9,980,947 (e.g., in Table 1 therein), the full text of which is incorporated herein by reference for all purposes.

[0338] In one state, the endocrine therapy includes compounds of formula (3): , (3) Or its stereoisomers, tautomers, or pharmaceutically acceptable salts, wherein: Y1 is either CRb or N; Y2 can be -(CH2)-, -(CH2CH2)-, or NRa; Y3 is either NRa or C(Rb)2; One of Y1, Y2, and Y3 is N or NRa; Ra is H, C1-C6 alkyl, C2-C8 alkenyl, propargyl, C3-C6 cycloalkyl, or C3-C6 heterocyclic group, which may be substituted, as appropriate, by one or more groups independently selected from the group consisting of F, Cl, Br, I, CN, OH, OCH3, and SO2CH3; Rb is H, -O (C1-C3 alkyl), C1-C6 alkyl, C2-C8 alkenyl, propargyl, -(C1-C6 alkyldiyl)-(C3-C6 cycloalkyl), C3-C6 cycloalkyl, or C3-C6 heterocyclic, which may be substituted, as appropriate, by one or more groups independently selected from the group consisting of F, Cl, Br, I, CN, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH2CH2F, OH, OCH3, and SO2CH3; Rc is H, C1-C6 alkyl, allyl, or propargyl, which may be substituted, as appropriate, by one or more groups independently selected from the group consisting of F, Cl, Br, I, CN, OH, OCH3, and SO2CH3; Z1 is a CRaRb, C(O) or bond; Cy can be C6-C20 aryldiyl, C3-C12 carbocyclic diyl, C2-C20 heterocyclic diyl, or C1-C20 heteroaryldiyl; Z2 can be O, S, NRa, C1-C6 alkyldiyl, C1-C6 fluoroalkyldiyl, O-(C1-C6 alkyldiyl), O-(C1-C6 fluoroalkyldiyl), C(O) or a bond; R1, R2, R3 and R4 are independently H, F, Cl, Br, I, -CN, -CH3, -CH2 CH3, -CH(CH3 )2, -CH2 CH(CH3 )2, -CH2 OH, -CH2 OCH3, -CH2 CH2 OH, -C(CH3 )2 OH, -CH(OH)CH(CH3 )2 , -C(CH3 )2 CH2 OH, -CH2 CH2 SO2 CH3, -CH2 OP(O)(OH)2, -CH2 F, -CHF2, -CH2 NH2, -CH2 NHSO2 CH3, -CH2 NHCH3, -CH2 N(CH3 )2, -CF3, -CH2 CF3, -CH2 CHF2, -CH(CH3)CN, -C(CH3 )2 CN, -CH2 CN, -CO2 H, -COCH3 , -CO2 CH3 , -CO2 C(CH3 )3 , -COCH(OH)CH3 , -CONH2 , -CONHCH3 , -CONHCH2 CH3 , -CONHCH(CH3 )2 , -CON(CH3 )2 , -C(CH3 )2 CONH2 , -NH2 , -NHCH3 , -N(CH3 )2 , -NHCOCH3 , -N(CH3 )COCH3 , -NHS(O)2 CH3 , -N(CH3 )C(CH3 )2 CONH2 , -N(CH3 )CH2 CH2 S(O)2 CH3 , -NO2 , =O, -OH, -OCH3 , -OCH2 CH3 , -OCH2 CH2 OCH3 , -OCH2 CH2 OH, -OCH2 CH2 N(CH3 )2、-OP(O)(OH)2、-S(O)2N(CH3)2、-SCH3、-S(O)2CH3、-S(O)3H、cyclopropyl、cyclopropylamide、cyclobutyl、oxo-1-yl、azacyclobutyl、1-methylazacyclobutyl-3-yl)oxy、N-methyl-N-oxo-1-ylamino、azacyclobutyl-1-ylmethyl、benzyloxyphenyl、pyrrolidin-1-yl、pyrrolidin-1-yl-methyl ketone、piperazin-1-yl、morpholinylmethyl、morpholinyl ketone or morpholinyl; R5 is H, C1-C9 alkyl, C3-C9 cycloalkyl, C3-C9 heterocyclic, C6-C9 aryl, C6-C9 heteroaryl, -(C1-C6 alkyldiyl)-(C3-C9 cycloalkyl), -(C1-C6 alkyldiyl)-(C3-C9 heterocyclic), C(O)Rb, C(O)NRa, SO2 Ra or SO2 NRa, which may be substituted, as appropriate, with one or more of a halogen, CN, ORa, N(Ra)2, C1-C9 alkyl, C3-C9 cycloalkyl, C3-C9 heterocyclic, C6-C9 aryl, C6-C9 heteroaryl, C(O)Rb, C(O)NRa, SO2 Ra or SO2 NRa; R6 is F, Cl, Br, I, -CN, -CH3 , -CH2 CH3 , -CH(CH3 )2 , -CH2 CH(CH3 )2 , -CH2 OH, -CH2 OCH3 , -CH2 CH2 OH, -C(CH3 )2 OH, -CH(OH)CH(CH3 )2 , -C(CH3 )2 CH2 OH, -CH2 CH2 SO2 CH3 , -CH2 OP(O)(OH)2 , -CH2 F , -CHF2 , -CH2 NH2 , -CH2 NHSO2 CH3 , -CH2 NHCH3 , -CH2 N(CH3 )2 , -CF3 , -CH2 CF3 , -CH2 CHF2 , -CH(CH3 )CN , -C(CH3 )2 CN , -CH2 CN , -CO2 H , -COCH3 , -CO2 CH3 , -CO2 C(CH3 )3 , -COCH(OH)CH3 , -CONH2 , -CONHCH3 , -CONHCH2 CH3 , -CONHCH(CH3 )2 , -CON(CH3 )2 , -C(CH3 )2 CONH2 , -NH2 , -NHCH3 , -N(CH3 )2 , -NHCOCH3 , -N(CH3 )COCH3 , -NHS(O)2 CH3 , -N(CH3 )C(CH3 )2 CONH2 , -N(CH3 )CH2 CH2 S(O)2 CH3 , -NO2 , =O, -OH, -OCH3 , -OCH2 CH3 , -OCH2 CH2 OCH3 , -OCH2 CH2 OH, -OCH2 CH2 N(CH3 )2 , -OP(O)(OH)2 -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, -S(O)3H, cyclopropyl, cyclopropylamide, cyclobutyl, oxo-1-yl, aziridine-1-yl, 1-methylaziridine-3-yl)oxy, N-methyl-N-oxo-3-ylamino, aziridine-1-ylmethyl, benzyloxyphenyl, pyrrolidin-1-yl, pyrrolidin-1-yl-methyl ketone, piperazine-1-yl, morpholinylmethyl, morpholinyl ketone or morpholinyl; and m can be 0, 1, 2, 3, or 4; The alkyl dienes, fluoroalkyl dienes, aryl dienes, carbocyclic dienes, heterocyclic dienes, and heteroaryl dienes are, as appropriate, independently substituted by one or more of the following groups: F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH2CH2F, -CH(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2 , -CH2 NHSO2 CH3 , -CH2 NHCH3 , -CH2 N(CH3 )2 , -CO2 H , -COCH3 , -CO2 CH3 , -CO2 C(CH3 )3 , -COCH(OH)CH3 , -CONH2 , -CONHCH3 , -CON(CH3 )2 , -C(CH3 )2 CONH2 , -NH2 , -NHCH3 , -N(CH3 )2 , -NHCOCH3 , -N(CH3 )COCH3 , -NHS(O)2 CH3 , -N(CH3 )C(CH3 )2 CONH2 , -N(CH3 )CH2 CH2 S(O)2 CH3 , -NO2 , =O, -OH, -OCH3 , -OCH2 CH3 , -OCH2 CH2 OCH3 , -OCH2 CH2 OH, -OCH2 CH2 N(CH3)2, -OP(O)(OH)2, -S(O)2 N(CH3)2, -SCH3, -S(O)2 CH3, -S(O)3 H, cyclopropyl, cyclopropylamide, cyclobutyl, oxo-1-yl, aziridine-1-yl, 1-methylaziridine-3-yl)oxy, N-methyl-N-oxo-3-ylamino, aziridine-1-ylmethyl, benzyloxyphenyl, pyrrolidin-1-yl, pyrrolidin-1-yl-methyl ketone, piperazine-1-yl, morpholinylmethyl, morpholinyl ketone and morpholinyl.

[0339] In one example of a compound of formula (3), Y1 is CRb and Y3 is NRa. In another example of a compound of formula (3), Y1 is N and Y3 is C(Rb)2. In another example of a compound of formula (3), Y2 is -(CH2)-. In another example of a compound of formula (3), Y2 is -(CH2CH2)-.

[0340] In another example ...

Claims

1. Use of an endocrine therapy drug for preparing a drug for treating an individual with breast cancer, wherein the individual has been identified as a likely beneficiary of treatment containing the endocrine therapy drug, wherein the endocrine therapy drug is a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor covalent antagonist (SERCA), a selective human estrogen receptor agonist (ShERPA), or an aromatase inhibitor (AI), or a combination of two or more thereof, the method comprising: determining an estrogen receptor (ER) pathway activity score from a sample of the individual, wherein if the ER pathway activity score from the sample is equal to or higher than a reference ER pathway activity score, the individual is identified as a beneficiary of treatment containing the endocrine therapy drug, wherein the ER pathway activity score is determined by subtracting an E2 repressed score from an E2-induced score. The E2 repression score is calculated by the mean z-score of at least six E2 repressor genes selected from the following groups: BAMBI, BCAS1, CCNG2, DDIT4, EGLN3, FAM171B, GRM4, IL1R1, LIPH, NBEA, PNPLA7, PSCA, SEMA3E, SSPO, STON1, TGFB3, TP53INP1, and TP53INP2; the E2 induction score is calculated by the mean z-score of at least six E2 repressor genes selected from the following groups: BAMBI, BCAS1, CCNG2, DDIT4, EGLN3, FAM171B, GRM4, IL1R1, LIPH, NBEA, PNPLA7, PSCA, SEMA3E, SSPO, STON1, TGFB3, TP53INP1, and TP53INP2. The score was determined by the average z-score of at least eight E2-inducible genes selected from the following groups: AGR3, AMZ1, AREG, C5AR2, CELSR2, CT62, FKBP4, FMN1, GREB1, IGFBP4, NOS1AP, NXPH3, OLFM1, PGR, PPM1J, RAPGEFL1, RBM24, RERG, RET, SGK3, SLC9A3R1, TFF1, and ZNF703.

2. Use of an endocrine therapy drug for the preparation of a drug for treating an individual with breast cancer, the individual being determined to have an ER pathway activity score equal to or higher than a reference ER pathway activity score, wherein the ER pathway activity score is calculated by subtracting an E2 repression score from an E2 induction score; the E2 repression score is determined by the mean z-score of at least six E2 repression genes selected from the following groups: BAMBI, BCAS1, CCNG2, DDIT4, EGLN3, FAM171B, GRM4, IL1R1, LIPH, NBEA, PNPLA7, PSCA, SEMA3E, SSPO, STON1, TGFB3, TP53INP1, and TP53INP2; the E2 induction score is determined by the mean z-score of at least eight E2 induction genes selected from the following groups: AGR3, AMZ1, AREG, C5AR2, CELSR2, CT62, FKBP4, FM N1, GREB1, IGFBP4, NOS1AP, NXPH3, OLFM1, PGR, PPM1J, RAPGEFL1, RBM24, RERG, RET, SGK3, SLC9A3R1, TFF1, and ZNF703, wherein the reference ER pathway activity score is: (a) an ER pathway activity score measured from a sample obtained from the individual prior to administration of the first dose of the endocrine therapy drug; (b) an ER pathway activity score measured from a sample obtained from the individual at a previous time point, wherein the previous time point is after administration of the first dose of the endocrine therapy drug; or (c) a pre-specified ER pathway activity score, wherein the endocrine therapy drug is a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor covalent antagonist (SERCA), a selective human estrogen receptor agonist (ShERPA), or an aromatase inhibitor (AI), or a combination of two or more thereof.

3. As requested in item 2, wherein the pre-specified ER pathway activity score is obtained from a reference population of individuals with hormone receptor-positive (HR+) breast cancer.

4. Use of an endocrine therapy drug for preparing a drug for treating an individual with breast cancer, wherein the endocrine therapy drug is a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor covalent antagonist (SERCA), a selective human estrogen receptor agonist (ShERPA), or an aromatase inhibitor (AI), or a combination of two or more thereof, wherein an E2-induced score from a sample of the individual is determined by E2 induction from a group selected from the following groups. The mean z-score of 23 genes was used to determine the following genes: AGR3, AMZ1, AREG, C5AR2, CELSR2, CT62, FKBP4, FMN1, GREB1, IGFBP4, NOS1AP, NXPH3, OLFM1, PGR, PPM1J, RAPGEFL1, RBM24, RERG, RET, SGK3, SLC9A3R1, TFF1, and ZNF703, among which the E2 induction score from this sample was equal to or higher than the reference E2 induction score.

5. As requested in item 4, wherein the reference E2 induced score is the E2 induced score of the reference group.

6. As requested in item 5, wherein the reference group is a group of individuals with HR+ breast cancer tumors.

7. Use of an endocrine therapy drug for the preparation of a drug for treating an individual with breast cancer, wherein the endocrine therapy drug is a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), a selective estrogen receptor covalent antagonist (SERCA), a selective human estrogen receptor agonist (ShERPA), an aromatase inhibitor (AI), or a combination of two or more thereof, wherein: (a) At the first time point, the first E2 induction score of the sample from the individual was determined by the mean z-score of 23 E2 induction genes selected from the following groups: AGR3, AMZ1, AREG, C5AR2, CELSR2, CT62, FKBP4, FMN1, GREB1, IGFBP4, NOS1AP, NXPH3, OLFM1, PGR, PPM1J, RAPGEFL1, RBM24, RERG, RET, SGK3, SLC9A3R1, TFF1 and ZNF703; (b) At a second time point following the administration of endocrine therapy drugs after step (a), a second E2 induction score from a sample of the individual is determined by the mean z-score of 23 E2 induction genes selected from the following groups: AGR3, AMZ1, AREG, C5AR2, CELSR2, CT62, FKBP4, FMN1, GREB1, IGFBP4, NOS1AP, NXPH3, OLFM1, PGR, PPM1J, RAPGEFL1, RBM24, RERG, RET, SGK3, SLC9A3R1, TFF1, and ZNF703; and (c) the first E2 induction score is compared with the second E2 induction score, wherein a decrease in the second E2 induction score relative to the first E2 induction score is predicted to indicate that the system may respond to treatment with endocrine therapy drugs.

8. As claimed in claim 7, wherein the first E2-induced score is: (a) an E2-induced score measured from a sample of the individual prior to administration of the first dose of the endocrine therapy drug; (b) an E2-induced score measured from a sample of the individual at a previous time point, wherein the previous time point is after administration of the first dose of the endocrine therapy drug; or (c) a pre-specified E2-induced score.

9. For the purposes of any of claims 1 to 8, wherein the breast cancer is ER+ breast cancer.

10. As requested in claim 9, wherein the ER+ breast cancer is ductal type A or ductal type B breast cancer.

11. For the purposes of any of claims 1 to 8, wherein the breast cancer is advanced or metastatic breast cancer.

12. The use as claimed in any of claims 1 to 8, wherein the endocrine therapy drug is a selective estrogen receptor degrader (SERD).

13. The use of any of the claims 1 to 8, wherein the individual is a human being.

14. The use of any of claims 1 to 8, wherein the endocrine therapy drug is a compound having the following formula: , or a pharmaceutically acceptable salt thereof.

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