Panel of ER regulatory genes for use in monitoring endocrine therapy in breast cancer

Abstract

The present disclosure relates to a panel of ER-regulated genes and a method for using the panel to monitor the response of an individual with cancer to treatment with amsenestrant, the method comprising: determining a first ER activity score from a sample from the individual at a first time point prior to treatment with amsenestrant using the panel; then determining a second ER activity score from a sample from the individual at a second time point after administration of amsenestrant using the panel; and comparing the first ER activity score to the second ER activity score, wherein a decrease in the second ER activity score relative to the first ER activity score is predictive of target engagement of amsenestrant.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 117,229, filed November 23, (此处原文本似乎有误,推测应该是2020年)2020; U.S. Provisional Application No. 63 / 173,107, filed April 9, 2021; and European Application No. 21315066.7, filed April 16, 2021, the contents of each of which are hereby incorporated by reference in their entirety for all purposes.

Background Art

[0002] The present disclosure describes a panel of ER - regulated genes useful for monitoring ER - target engagement, including the on - target activity of the endocrine therapy amcenestrant, and related kits. Shomali et al., SAR439859, a Novel Selective Estrogen Receptor Degrader (SERD), Demonstrates Effective and Broad Antitumor Activity in Wild - Type and Mutant ER - Positive Breast Cancer Models, Mol. Cancer Ther. (2021) is hereby incorporated by reference in its entirety for all purposes.

[0003] Breast cancer is the most frequently diagnosed cancer in women. Estrogen receptor - positive (ER+) cancer accounts for approximately 75% of all breast cancers.

[0004] Because it relies on ER signaling, the primary therapeutic approach to ER+ breast cancer has been to block estrogen function. Anti-hormone therapies that directly antagonize the function of estrogen receptor alpha (ERα; such as tamoxifen), or therapies that block the production of its ligand, estrogen (such as aromatase inhibitors), are the go-to therapies for ER-positive (ER+) breast cancer. While these treatments significantly reduce the risk of recurrence from early-stage disease and improve outcomes for patients with advanced disease, recurrence after long-term treatment is common. Recently, recurrent mutations in the ligand-binding domain of ERα have been identified in approximately 25–40% of patients who have relapsed after receiving one or more previous hormone therapies. These mutations confer estrogen-independent constitutive activity of ERα, induction of tumor growth, reduced efficacy to anti-ERα therapy, and complete resistance to aromatase inhibitors.

[0005] Some ligands targeting ERα can increase the steady-state level of the ERα protein through biological feedback mechanisms such as transcriptional compensation or increased thermodynamic stability upon ligand binding. For example, tamoxifen induces ERα protein stabilization and adopts a conformation that may trigger agonist signaling. Furthermore, some mutations in ERα, such as mutations affecting the Y537S or D538G amino acids, have been suggested to be involved in ERα stabilization. In addition, increased ERα stability may lead to leakage of ERα signaling if a sustained therapeutic range is not achieved. Thus, in addition to ERα antagonism, there is a rationale for the degradation of the ERα protein to affect the biological properties of ERα and the efficacy of ERα-targeted therapies.

[0006] To modulate ER activity, selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders (SERDs) have been developed.

[0007] Fulvestrant was developed to overcome the partial regulation of ER transcriptional activity by SERMs. Selective estrogen receptor degraders (SERDs) like fulvestrant not only bind to ERα and antagonize ERα function, but also induce structural changes that trigger proteasome-mediated degradation, more effectively inhibiting ERα signaling (Non-Patent Literature 1). Fulvestrant is an approved SERD indicated for the treatment of ER+ metastatic breast cancer in postmenopausal women whose disease has progressed after anti-estrogen therapy. Fulvestrant has been demonstrated to be preclinically and clinically useful after other hormonal therapies have failed. However, fulvestrant, a neutral steroid with lipophilic side chains, requires a non-conventional, long-acting intramuscular depot formulation, and its dose and exposure are limited to maximize receptor engagement. The clinical benefit of fulvestrant is limited by its pharmaceutical properties and the burden of intramuscular administration.

[0008] To address the aforementioned pharmacological drawbacks caused by fulvestrant, several SERDs, including GDC-0810 (NCT 01823835), AZD9496 (NCT 02248090), AZD9833 (NCT 03616586), GDC-0927 (NCT 02316509), and GDC-9545 (NCT 03916744, NCT 03332797), have been initiated into clinical trials. These novel SERDs, which are chemically distinct from fulvestrant, can be classified into two main groups based on the chemical structure of their key side chains that promote the degradation of ERα. GDC-0927 is characterized by a fluoroalkylamine side chain, while GDC-0810, AZD9496, and LSZ102 each have a cinnamic acid side chain. It is not well understood whether their different side chains and / or ability to induce ERα degradation lead to differences in their biological and antitumor activity. Furthermore, these SERDs present conflicting data regarding their relative ability to induce ERα agonist activity or to promote complete ERα degradation.

[0009] To better define the molecular properties necessary to achieve optimal clinical activity of SERDs, it is crucial to understand the relationship between the drug's molecular structure, the level of ERα degradation, and its subsequent impact on antitumor activity. This specification describes amsenestrant (laboratory code SAR439859), a novel nonsteroidal, orally bioavailable SERD with a fluoropropylpyrrolidinyl side chain, unlike SERDs with a cinnamic acid side chain. SAR439859 exhibits strong ERα antagonist activity, potently inducing its degradation, resulting in improved efficacy in both in vitro and in vivo ER+ breast cancer models.

[0010] Currently, clinical SERD activity is measured by degradation using IHC and by inhibition of the ER target gene progesterone receptor (PGR). Since estrogen, which acts as an agonist, also promotes the degradation of estrogen receptors, degradation of the ER alone is insufficient to determine SERD activity in the ER pathway. Although PGR is a well-established target gene of the ER, the ER is a transcription factor that regulates the transcription of many genes, and PGR may not completely capture the activity of these molecules on the ER pathway.

[0011] Therefore, there is a need for tools to monitor the response of cancer patients being treated with SERDs, including amsenestrant, by predicting targeted engagement, including on-target activity. Since ER is a transcription factor that regulates the expression of a set of many genes, it is thought that monitoring the expression levels of a subset of these genes (i.e., gene signatures) before treatment, and the regulation of these gene signatures after treatment with amsenestrant, would reveal targeted engagement, including on-target activity.

[0012] Genetic signatures provide information about the expression levels of specific gene groups in cells or tissues. In breast cancer, genetic signatures can complement classical prognostic factors (age, positive lymph nodes, tumor size, etc.) to provide predictive and prognostic values. For example, in breast cancer, 70-gene signatures (MammaPrint) and 21-gene signatures (OncoType) are used to identify patients for whom adjuvant therapy is most effective and are widely used in Europe and the United States. The SET index, used to predict the effectiveness of endocrine therapy in breast cancer, is used for metastatic breast cancer. ER / PR Additional gene signatures have been developed, such as the ER activity score published by Genentech / Hoffman-La Roche (Non-Patent Document 1), as described in Non-Patent Document 2 and Non-Patent Document 3.

[0013] Abbreviation DMSO (Dimethyl Sulfoxide) ER estrogen receptor ERα (Estrogen Receptor Alpha) IHC immunohistochemistry IL Interleukin LBD Ligand Binding Domain NA Not applicable QD once a day Q2W Every 2 weeks RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) SERD Selective ER Degrading Agent WT wild type [Prior art documents] [Patent Documents]

[0014] [Patent Document 1] International Publication No. 2020 / 037203 [Non-patent literature]

[0015] [Non-Patent Document 1] Heidari et al., Pharmacodynamic Imaging Guides Dosing of a Selective Estrogen Receptor Degrader. Clin. Cancer Res. (2015) [Non-Patent Document 2] W. Symmans et al., Journal of Clinical Oncology, Vol. 28, No. 27, September 20, 2010, pp. 4111-4119. [Non-Patent Document 3] B. Sinn et al., in Breast Cancer, (2019) 5:16 [Brief explanation of the drawing]

[0016] [Figure 1-1] A. Figure shows heatmaps of 1022 genes differentially expressed in the HCC1428 LTED breast cancer cell line in the absence of exogenous estrogen at two doses (30 nM and 300 nM). Data were normalized by log2, followed by standardization and hierarchical clustering; B. Figure shows comparison of ER scores and six SERDs / evaluation of the regulation of ER transcriptional activity by SERDs. ER transcriptional activity is expressed as an ER activity score from GSVA using ER signatures (Wilcoxon test is used to compare the mean of groups depicted with ** P < 0.01 and *** P < 0.001). C. D. Figure shows reverse transcription-quantitative polymerase chain reaction analysis of the effects of different selective ER degradation molecules on the expression of ERα target genes CXCL12 and Bcas1. *Significance (P < 0.01) is shown when compared using unpaired t-tests. [Figure 1-2] Continuation of Figure 1-1. [Figure 1-3] Continuation of Figure 1-2. [Figure 2]Figure showing the analysis of PGR gene expression in tumors collected 8 hours after the final administration of tamoxifen (30 mg / kg), fulvestrant (200 mg / kg), GDC0810 (100 mg / kg), and SAR439859 (100 mg / kg). For Figure 2, *P<0.05; indicates significance compared to the vehicle treatment group at the end of the study using an unpaired t-test. [Figure 3] Figure showing the correlation of gene expression profile similarities among SERDs in the HCI013 PDX tumor model. A panel of 1022 genes identified as differentially expressed in the comparison of at least one compound to DMSO was used for hierarchical clustering of compound treatment data normalized to the DMSO control at each dose and time point. The similarity of the expression profiles between compounds was evaluated by Pearson correlation of differentially expressed genes. [Figure 4] Figure showing the evaluation of target gene regulation of ERα ligands in an endocrine therapy-resistant tumor model. (A) Gene expression analysis of CXCL12 and (B) ER activity score 8 hours after the final administration of tamoxifen (30 mg / kg, QD), GDC0810 (100 mg / kg, QD), fulvestrant (200 mg / kg, Q2W), and SAR439859 (100 mg / kg, QD) in the HCI013 PDX tumor model. Data represent the mean and standard deviation of three replicates of CXCL12 gene expression. The ER signature of 87 genes was regulated by estradiol, then blocked by SERMs, and the ER activity score was calculated using SERD compounds and evaluated using gene set variation analysis (GSVA). Data represent the mean and deviation from the mean for each of the treatments described. [[ID=#7]] [Figure 5] Figure showing the ER activity signature. The heatmap shows the DMSO-normalized gene expression of 87 genes in the ER activity signature. 66 genes were induced by E2 and downregulated by SERD / SERM, and 21 genes were suppressed by E2 and upregulated by SERD / SERM. [Figure 6-1] A diagram showing on - target estrogen receptor degradation / pathway inhibition during amcenestrant therapy, indicated by changes from screening to cycle 2, day 28 (i.e., before and after treatment). In A, it shows the ER nuclear H - score by IHC (IHC ER expression); in B, it shows the PgR nuclear H - score by IHC; and in C, it shows the percentage of positive cells with Ki67 protein expression by IHC; in D, it shows the pre - and post - GSVA ER activation score (ER activity score based on RNA - seq data from paired tumor biopsies). *Each row in each of FIGS. 6A - D shows one patient with available data at baseline / screening and after baseline. 8 patients are shown in FIG. 6A. 8 patients are shown in FIG. 6B; some lines in FIG. 6B overlap. 8 patients are shown in FIG. 6C. 5 patients are shown in FIG. 6D. C1D1, cycle 1, day 1; C2D28, cycle 2, day 28; CB, clinical benefit (complete response + partial response + stable disease for 24 weeks or more); ER, estrogen receptor; ESR1, estrogen receptor 1; GSVA, gene set variation analysis; IHC, immunohistochemistry; PgR, progesterone receptor. [Figure 6-2] Continuation of FIG. 6 - 1. [[ID=?]] Summary of the Invention Problems to be Solved by the Invention

[0017] In this specification, a set of 87 genes having higher and lower expression levels respectively identified from transcriptional profiling of cell lines is provided to identify genes sensitive to estradiol and ER antagonists including SERD. This set of ER - regulated genes would be useful for monitoring the response of individuals with cancer to treatment with amcenestrant. The present disclosure includes, for example, any one or combination of the following embodiments: Means for Solving the Problems

[0018] Embodiment 1. A panel of ER regulatory genes having N genes, where N is at least 7 and equal to about 87 or less than 87 (87 or less), and the N genes include any 2 to 21 of the following upregulated genes: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO; and the N genes include any 2 to 21 of the following downregulated genes: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, F RK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, S LC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, Includes any 5 to 66 of the following: SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.

[0019] Embodiment 2. N is 87, and the 87 genes include the following 21 upregulated genes: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO; and the 87 genes also include the following 66 downregulated genes: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CA P2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1 H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, A panel of Embodiment 1, including TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.

[0020] Embodiment 3. A method for monitoring the response of an individual with cancer to treatment with amsenestrant, the method comprising: (a) determining a first ER activity score from a sample from the individual at a first time point prior to treatment with amsenestrant; (b) determining a second ER activity score from a sample from the individual at a second time point following administration of amsenestrant, following step (a); and (c) comparing the first ER activity score with a second ER activity score, wherein the decrease in the second ER activity score relative to the first ER activity score is a predictor of target engagement with amsenestrant. Here, determining the first and second ER activity scores involves analyzing a panel of ER regulatory genes having N genes, where N is at least 7 and less than or equal to 87, and the N genes include any 2 to 21 of the following upregulated genes: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO; and the N genes include any 2 to 21 of the following downregulated genes: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H 3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2 This includes any 5 to 66 of the following: SLC39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.

[0021] Embodiment 4. The ER regulatory gene panel has 87 genes, the 87 genes include the following 21 upregulated genes: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO; and the 87 genes include the following 66 downregulated genes: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, S FXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H The method according to Embodiment 3, comprising 3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.

[0022] Embodiment 5. The method according to Embodiment 3 or 4, wherein the decrease in the second ER activity score relative to the first ER activity score is a predictor of the on-target activity of amsenestrant.

[0023] Embodiment 6. The method according to Embodiment 5, wherein the on-target activity of amsenestrant includes ER degradation and ER inhibition.

[0024] Embodiment 7. The method according to any one of Embodiments 3 to 6, comprising bringing a sample from an organism into contact with the probe.

[0025] Embodiment 8. The method according to any one of Embodiments 3 to 7, comprising detecting the RNA expression level of each of N genes in a sample from an individual.

[0026] Embodiment 9. The method according to any one of Embodiments 3 to 8, comprising RNA sequencing.

[0027] Embodiment 10. The method according to any one of Embodiments 3 to 9, wherein the individual has ER+ breast cancer.

[0028] Embodiment 11. The method according to any one of Embodiments 3 to 10, wherein the individual has HER2-breast cancer.

[0029] Embodiment 12. The method according to any one of Embodiments 3 to 11, wherein the individual has progressive or metastatic breast cancer.

[0030] Embodiment 13. The method according to any one of Embodiments 3 to 12, wherein the sample is a tumor biopsy.

[0031] Embodiment 14. The method according to any one of Embodiments 3 to 12, wherein the sample is a blood sample.

[0032] Embodiment 15. The method according to any one of Embodiments 3 to 12, wherein the sample is a serum sample.

[0033] Embodiment 16. The method according to Embodiment 13, wherein the tumor biopsy is a formalin-fixed tumor biopsy, a paraffin-embedded tumor biopsy, a formalin-fixed paraffin-embedded (FFPE) tumor biopsy, a fresh-frozen (FF) tumor biopsy, a frozen tumor biopsy, or a fresh tumor biopsy.

[0034] Embodiment 17. A kit comprising a set of probes for detecting the expression of upregulated and downregulated genes; Here, the upregulated genes include any 2 to 21 of PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO; and The downregulated genes are H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SY Includes any 5 to 66 of BU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.

[0035] Embodiment 18. A set of probes for detecting the expression of upregulated and downregulated genes includes probes for detecting the upregulated genes PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO; and the downregulated genes H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, and SFX N2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1 H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, TMEM1 The kit according to Embodiment 17, comprising probes for detecting 64, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH. [Modes for carrying out the invention]

[0036] definition The term "ER" or "estrogen receptor" refers to estrogen receptor α (gene name ESR1), a protein that binds to estradiol (E2). ER activates the transcription of numerous genes and is a major determinant of overall gene transcription in breast cancer. When bound to estrogen, ER can promote cell proliferation. Estrogen receptor-positive (ER+) breast cancer typically indicates that the proliferation of breast cancer cells is dependent on estrogen.

[0037] As used herein, “panel” or “set” refers to a group. For example, as used herein, “panel (or set) of ER regulatory genes” means a group of genes regulated by the ER.

[0038] The term "upregulated gene" refers to a gene that shows increased expression after administration of a monotherapy agent such as amsenestrant, compared to treatment with a control such as DMSO, or compared to an earlier pre-treatment point in the treated individual.

[0039] The term "downregulated gene" refers to a gene that shows reduced expression after administration of a monotherapy agent such as amsenestrant, compared to treatment with a control such as DMSO, or compared to an earlier pre-treatment point in the treated individual.

[0040] As used herein, the term “ER activity score” refers to a score obtained using a calculation method such as GSVA on a gene expression dataset obtained from a biological sample (for a description of calculating the score using the GSVA method, see Hanzelmann et al., GSVA: gene set variation analysis for microarray and RNA-seq data, BMC Bioinformatics, 2013; 14:7, incorporated herein by reference in its entirety). The E2 induction score and E2 repression score are calculated separately. The E2 induction score is calculated using a calculation method such as GSVA on an upregulated set of genes from a gene expression dataset obtained from a biological sample. The E2 repression score is calculated using a calculation method such as GSVA on a downregulated set of genes from a gene expression dataset obtained from a biological sample. The ER activity score is calculated as follows: ER activity score = ER induction score - E2 repression score.

[0041] "On-target activity of amsenestrant" refers to the degradation and / or inhibition of the ER, and can be indicated, for example, by immunohistochemistry by reducing ER protein expression, by reducing the expression of proteins regulated by the ER signaling pathway (progesterone receptor, Ki67), by immunohistochemistry by GSVA, and / or by the ER activity score.

[0042] The term "advanced breast cancer" refers to cancer where the tumor is not in a localized area (i.e., outside the site of the primary tumor) or cannot be surgically removed.

[0043] The term "metastatic breast cancer" refers to cancer that has spread to other parts of the body, such as the liver, lungs, bones, brain, and / or other sites.

[0044] ER regulatory genes In some embodiments, a panel of ER regulatory genes is included, where the panel of ER regulatory genes has N genes, N is at least 7 and about 87 or less, and the N genes include any 2 to 21 of the following upregulated genes: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO; and the N genes include any 2 to 21 of the following downregulated genes: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3 , SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L , SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC Includes any 5 to 66 of the following: 39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.

[0045] In some embodiments, a panel of ER regulatory genes is included, where the panel of ER regulatory genes has N genes, where N is approximately 87 or less, and is at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, and fewer. At least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, At least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 8 It includes 2, at least 83, at least 84, at least 85, at least 86, or 87 genes, and N genes are any 2-21, 3-21, 4-21, 5-21, or 6-21 of the following upregulated genes: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO.The genes include 7-21, 8-21, 9-21, 10-21, 11-21, 12-21, 13-21, 14-21, 15-21, 16-21, 17-21, 18-21, 19-21, 20-21, or 21; and N genes include the following downregulated genes: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERP INA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, F HL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, H Any 5-66, 6-66, 7-66, 8-66, 9-66, 10-66, 11-66, 12-66, 13-66, 14-66, 15-66, 16-66, 17-66, 18-66, 19-66, 20-66, 21-66, 22-66, 23-66, 24-66, 25-66, 26-66, 27-66, 28- 66 pieces, 29~66 pieces, 30~66 pieces, 31~66 pieces, 32~66 pieces, 33~66 pieces, 34~66 pieces, 35~66 pieces, 36~66 pieces, 37 ~66 pieces, 38~66 pieces, 39~66 pieces, 40~66 pieces, 41~66 pieces, 42~66 pieces, 43~66 pieces, 44~66 pieces, 45~66 pieces, 4 6~66 pieces, 47~66 pieces, 48~66 pieces, 49~66 pieces, 50~66 pieces, 51~66 pieces, 52~66 pieces, 53~66 pieces, 54~66 pieces, 55~66 pieces, 56~66 pieces, 57~66 pieces, 58~66 pieces, 59~66 pieces, 60~66 pieces, 61~66 pieces, 62~66 pieces, 63~66 pieces,Includes 64-66, 65-66, or 66 items.

[0046] In some embodiments, the panel of ER regulatory genes includes PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, SYNPO, H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, and DSC. It has approximately 87 genes, including C1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.Of approximately 87 genes, 21 were upregulated: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO; and 66 were upregulated: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, and W DR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH are downregulated genes.

[0047] method In some embodiments, methods are provided herein for monitoring the response of individuals with cancer to treatment with amsenestrant, the methods comprising: (a) determining a first ER activity score from a sample from the individual at a first time point prior to treatment with amsenestrant; (b) determining a second ER activity score from a sample from the individual at a second time point following administration of amsenestrant, following step (a); and (c) comparing the first ER activity score with the second ER activity score, wherein a decrease in the second ER activity score relative to the first ER activity score indicates target engagement of amsenestrant. The process includes a step in which the predictors of ER activity score are determined, wherein determining a first ER activity score and a second ER activity score involves analyzing a panel of ER regulatory genes having N genes, where N is at least 7 and about 87 or less, and the N genes include any 2 to 21 of the following upregulated genes: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO;Furthermore, the following N genes were downregulated: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, R Includes any 5 to 66 of the following: GS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.

[0048] In some embodiments, methods are provided herein for monitoring the response of individuals with cancer to treatment with amsenestrant, the methods comprising: (a) determining a first ER activity score from a sample from the individual at a first time point prior to treatment with amsenestrant; (b) determining a second ER activity score from a sample from the individual at a second time point following administration of amsenestrant, following step (a); and (c) comparing the first ER activity score with the second ER activity score, wherein the second ER activity score is compared to the first ER activity score. The decrease in the ER activity score is a predictor of target engagement of amsenestrant, and the steps include determining the first and second ER activity scores, which include analyzing a panel of ER regulatory genes having N genes, where N is approximately 87 or less, and at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, and at least 2 0, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, fewer 48 each, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75,It contains at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, or 87 genes, and N genes are upregulated genes such as: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC2830 70, PSCA, BAK1, PLIN2, and any 2-21, 3-21, 4-21, 5-21, 6-21, 7-21, 8-21, 9-21, 10-21, 11-21, 12-21, 13-21, 14-21, 15-21, 16-21, 17-21, 18-21, 19-21, 20-21, or 21 of the following genes; and N genes including the following downregulated genes: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, R AD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, Any 5 to 66, 6 to 66, 7 to 66, 8 to 66, 9 to 66, 10 to 66 of HIST1H1D and HIST1H2BH, 11~66 pieces, 12~66 pieces, 13~66 pieces, 14~66 pieces, 15~66 pieces, 16~66 pieces, 17~66 pieces, 18~66 pieces, 19~66 pieces,20~66 pieces, 21~66 pieces, 22~66 pieces, 23~66 pieces, 24~66 pieces, 25~66 pieces, 26~66 pieces, 27~66 pieces, 28~66 pieces, 29~66 pieces, 30~66 pieces, 31~66 pieces , 32~66 pieces, 33~66 pieces, 34~66 pieces, 35~66 pieces, 36~66 pieces, 37~66 pieces, 38~66 pieces, 39~66 pieces, 40~66 pieces, 41~66 pieces, 42~66 pieces, 43~66 pieces Includes 1, 44-66, 45-66, 46-66, 47-66, 48-66, 49-66, 50-66, 51-66, 52-66, 53-66, 54-66, 55-66, 56-66, 57-66, 58-66, 59-66, 60-66, 61-66, 62-66, 63-66, 64-66, 65-66, or 66.

[0049] In some embodiments, methods are provided herein for monitoring the response of an individual with cancer to treatment with amsenestrant, the methods comprising: (a) determining a first ER activity score from a sample from the individual at a first time point prior to treatment with amsenestrant; (b) determining a second ER activity score from a sample from the individual at a second time point following administration of amsenestrant, following step (a); and (c) comparing the first ER activity score with the second ER activity score, wherein a decrease in the second ER activity score relative to the first ER activity score indicates amsenestrant The process includes predictors of nestrant target engagement, wherein determining a first ER activity score and a second ER activity score involves analyzing a panel of ER regulatory genes having approximately 87 genes, as well as 21 upregulated genes, including PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO;In addition, 66 genes were downregulated: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4 Includes C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.

[0050] In some embodiments, the decrease in the second ER activity score relative to the first ER activity score is a predictor of the on-target activity of amsenestrant. In some embodiments, the on-target activity of amsenestrant includes ER degradation and ER inhibition.

[0051] Assay / RNA sequencing In some embodiments, the gene expression level may be a nucleic acid expression level, such as an RNA expression level, an mRNA expression level, or a DNA expression level. Any suitable method for determining the nucleic acid expression level can be used. In some embodiments, the nucleic acid expression level is determined using RNA-seq. For example, the nucleic acid expression level may be determined using the RNA ACCESS protocol or the TRUSEQ RIBO-ZERO00 protocol (ILLUMINA), RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technology, or a combination thereof.

[0052] Methods for evaluating intracellular mRNA are well known and include, for example, RNA sequencing (RNA-seq), whole-genome sequencing (WGS), sequential gene expression analysis (SAGE), and various nucleic acid amplification assays such as RT-PCR using complementary primers specific to a given set of genes. In some embodiments, qRT-PCR is used. Furthermore, such methods may include one or more steps that allow for the determination of the level of target mRNA in a biological sample by simultaneously examining the levels of comparison control mRNA sequences of "housekeeping" genes, such as actin family members. In some embodiments, the sequence of the amplified target cDNA can be determined. Optional methods include protocols for examining or detecting mRNA, such as target mRNA, in tissue or cell samples using microarray technology. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured so that the sequence and position of each member of the array are known. For example, the selection of genes whose expression correlates with targeted engagement of a therapy including amsenestrant, including on-target activity, can be aligned on a solid support. For instance, any of the 87 genes in Table 3 can be aligned on a solid support. Hybridization of a labeled probe with a specific array member indicates that the sample from which the probe originates expresses that gene.

[0053] Biological samples Any biological sample containing one or more tumor cells can be used in the methods disclosed herein. In some embodiments, the sample is selected from tumor biopsies, blood samples, serum samples, or any combination thereof. In some embodiments, the sample is a tumor biopsy. In some embodiments, the sample obtained from the subject is a formalin-fixed tumor biopsy. In some embodiments, the sample obtained from the subject is a formalin-fixed paraffin-embedded tumor (FFPE) biopsy or a paraffin-embedded tumor biopsy. In some embodiments, the sample obtained from the subject is a fresh-frozen (FF) tumor biopsy. In some embodiments, the sample is a fresh tumor biopsy. In some embodiments, the sample is an archived tumor biopsy. In some embodiments, the sample is a frozen tumor biopsy.

[0054] In some examples of any method and assay, the sample is obtained from the individual prior to the administration of the amsenestrant described herein. In some embodiments, the sample is obtained minutes, hours, days, weeks, months, or years after the administration of the amsenestrant described herein. In other words, the sample may be a baseline sample. In some embodiments, the sample is obtained from the individual after the administration of the amsenestrant described herein. In some cases, the sample from the individual is obtained within 30 hours after the administration of the endocrine therapy. In some embodiments, the sample is obtained minutes, hours, or days after the administration of the amsenestrant described herein. In some embodiments, for example, multiple samples are obtained from the same individual at different time points before and after the administration of the amsenestrant described herein.

[0055] SERD compound In some embodiments, the SERD compound is SAR439859 (amsenestrant). The synthesis of SAR439859 is described in the International Publication of the Patent Application No. 2017140669 (Example 51). SAR439859 (amsenestrant) is 6-(2,4-dichlorophenyl)-5-[4-[(3S)-1-(3-fluoropropyl)pyrrolidine-3-yl]oxyphenyl]-8,9-dihydro-7H-benzo[7]annelen-2-carboxylic acid. The chemical formula for SAR439859 (amsenestrant) is as follows: [ka]

[0056] Additional anti-cancer therapy In some embodiments, the methods disclosed herein further include administering amsenestrant and additional anticancer therapy. Additional anticancer therapy may include any therapy known in the art for the treatment of tumors in a subject and / or any standard treatment therapy. In some embodiments, additional anticancer therapy includes surgery, radiotherapy, chemotherapy, immunotherapy, hormone therapy, or any combination thereof. In some embodiments, additional anticancer therapy includes chemotherapy. In some embodiments, additional anticancer therapy includes immunotherapy. In some embodiments, additional anticancer therapy includes hormone therapy.

[0057] In some embodiments, the anticancer therapy is palbociclib. In some embodiments, the anticancer therapy is alpelisib.

[0058] cancer In some embodiments, this disclosure relates to a method for monitoring the response of individuals with cancer to treatment with amsenestrant.

[0059] In some embodiments, the cancer is breast cancer, preferably ER+ / HER2- advanced or metastatic breast cancer.

[0060] Kits and manufactured products In some embodiments, kits or products comprising materials useful for monitoring the response of individuals with cancer to treatment with amsenestrant are provided herein. In some embodiments, the kit or product comprises a first set of probes for detecting the expression of upregulated and downregulated genes; wherein the upregulated genes are PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MAT Including any 2-21, 3-21, 4-21, 5-21, 6-21, 7-21, 8-21, 9-21, 10-21, 11-21, 12-21, 13-21, 14-21, 15-21, 16-21, 17-21, 18-21, 19-21, 20-21, or 21 of N2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO;And the downregulated genes include any 5 to 66, 6 to 66, 7 to 66, 8 to 66, 9 to 66, 10 to 66, 11 to 66, 12 to 66, 13 to 66, 14 to 66, 15 to 66, 16 to 66, 17 to 66, 18 to 66, 19 to 66, 20 to 66, 21 to 66, 22 to 66, 23 to 66, 24 to 66, 25 to 66, 26 to 66, 27 to 66, 28 to 66, 29 to 66, 30 to 66, 31 to 66, 32 to 66, 33 to 66, 34 to 66, 35 to 66, 36 to 66, 37 to 66, 38 to 66, 39 to 66, 40 to 66, 41 to 66, 42 to 66, 43 to 66, 44 to 66, 45 to 66, 46 to 66, 47 to 66, 48 to 66, 49 to 66, 50 to 66, 51 to 66, 52 to 66, 53 to 66, 54 to 66, 55 to 66, 56 to 66, 57 to 66, 58 to 66, 59 to 66, 60 to 66, 61 to 66, 62 to 66, 63 to 66, 64 to 66, 65 to 66, or 66 of H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.;

[0061] In some embodiments, the kit or product comprises a first set of probes for detecting the expression of upregulated and downregulated genes; the upregulated genes here include PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO; and the downregulated genes include H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, and SCNN1 B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SD K2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SL Includes C39A8, SKP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH.

[0062] In some embodiments, the kit or product may include one or more reagents for preparing a sample for RNA sequencing analysis. In some embodiments, the sample is selected from a tumor biopsy, a blood sample, a serum sample, or any combination thereof. In some embodiments, the sample is a tumor biopsy. In some embodiments, the sample obtained from the subject is a formalin-fixed tumor biopsy. In some embodiments, the sample obtained from the subject is a formalin-fixed paraffin-embedded tumor (FFPE) biopsy or a paraffin-embedded tumor biopsy. In some embodiments, the sample obtained from the subject is a fresh-frozen (FF) tumor biopsy. In some embodiments, the sample is a fresh tumor biopsy. In some embodiments, the sample is an archived tumor biopsy. In some embodiments, the sample is a frozen tumor biopsy.

[0063] In some embodiments, the kit or product further comprises one or more reagents for determining the ER activity score from a sample.

[0064] In some embodiments, the kit or manufactured product may include instructions for using the kit to monitor and / or evaluate the response of individuals with breast cancer to treatment with amsenestrant, as described herein.

[0065] In some embodiments, the kit or product may include a container, a label on the container, and a composition contained within the container, wherein the composition comprises one or more polynucleotides that hybridize to complements of genes listed herein (e.g., any of the genes in Table 3) under stringent conditions, the label on the container indicates that the composition can be used to assess the activity of the set of genes listed in Table 3 herein in a sample, and the kit includes instructions for using the polynucleotide(s) to assess the presence of RNA or DNA of the genes in a particular sample type.

[0066] In some embodiments, the kit or product is oligonucleotide-based and may include, for example, (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide that hybridizes to a nucleic acid sequence encoding a protein, or (2) a pair of primers useful for amplifying nucleic acid molecules. In some embodiments, the kit or product may also include buffers, preservatives, or protein stabilizers. In some embodiments, the kit or product may further include components necessary for detecting the detectable label, e.g., enzymes or substrates. In some embodiments, the kit or product may also include a control sample or a set of control samples that can be assayed and compared with the test sample. In some embodiments, each component of the kit or product may be sealed in an individual container, and all of the various containers may be sealed in a single package along with instructions for interpreting the results of assays performed using the kit or product. [Examples]

[0067] material and method The main details of the materials and methods used are as follows.

[0068] Cell culture and reagents MCF7, CAMA-1, ZR-75~1, MDAMB134VI, MDAMB361, BT474, BT473, MDAMB415, EFM19, HCC1428, HCC1500, HEK293T, MDAMB231, and SUM44PE cells were purchased from ATCC or Asterand and authenticated using short tandem repeat (STR) DNA profiling in Idexx. The HCC1428 LTED cell line was obtained from Carlos Arteaga (TW Miller et al., Clinical Cancer Research, 2011; 17: pp. 2024-2034). All cell lines were routinely screened for mycoplasma contamination using Lonza Mycoalert and Stratagene Mycosensor. Unless otherwise specified, tissue culture supplements and media were purchased from Hyclone, Corning, or Invitrogen. Cells were maintained according to ATCC recommendations. HCC1428-LTED was maintained in phenol red-free modified Eagle medium (IMEM, Corning) containing 10% dextran-charcoal treated (CSS) FBS (Hyclone). SUM44PE was maintained in IMEM containing 2% CSS as previously described. HCI-013 was established from the pleural effusion of a 53-year-old woman with metastatic ER+ / progesterone receptor-positive / human epidermal growth factor receptor 2-negative invasive lobular carcinoma (donated by Alana Welms). Fulvestrant, 4OH-tamoxifen, raloxifene, bazedoxifene, and 17β-estradiol were purchased from Sigma Aldrich.

[0069] compound SAR439859 was synthesized as described in International Publication No. 2017140669 (Example 51) of the patent application. GDC0810 and AZD9496 were synthesized as described in International Publication No. 2012037410 (Example 111) and International Publication No. 2014191726 (Example 1), respectively. Both AZD-SAR and GDC-SAR were prepared as described in International Publication No. 2018091153 (Examples 255 and 256, respectively).

[0070] RNA isolation and quantitative polymerase chain reaction (qPCR) RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's instructions, quantified using NanoDrop 8000 (ThermoScientific), and reverse transcribed using the cDNA archiving kit (Applied Biosystems). Taqman gene expression assays (Applied Biosystems) were used to quantify PGR (Hs00172183_m1), Bcas1 (Hs00952822_m1), CXCL12 (Hs03676656_mH), BLNK1 (Hs00929914_m1), IL20 (Hs00218888_m1), and the house-keeping genes PGK (Hs00391480_m1) and GAPDH (Hs99999905_m1). The relative amounts were determined using the ΔΔ threshold cycle (ΔΔCt) according to the manufacturer's instructions for use (Applied Biosystems).

[0071] RNA sequencing / development of ER regulatory gene panels To develop a panel of ER regulatory genes, the following experiments were conducted. First, MCF-7 cells were treated individually with DMSO, estradiol (E2), two SERMs, and five SERD compounds at three different concentrations. See Table 1 below for the treatment scheme for MCF-7 cells with monotherapy (DMSO, estradiol, SERM, or SERD). The unit of concentration is nM.

[0072] [Table 1]

[0073] Secondly, MCF-7 cells were treated with a combination of estradiol and one of the SERD compounds. See Table 2 below for a treatment scheme for MCF-7 cells using estradiol in addition to two SERMs and one of five SERDs. The unit of concentration is nM.

[0074] [Table 2]

[0075] RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's protocol. RNA sample concentrations were determined using NanoDrop 8000 (ThermoScientific), and RNA integrity was determined using 4200 TapeStation (Agilent Technologies).

[0076] RNA-seq libraries of 96 SERD RNA samples were prepared using a kit from the NuGen Ovation® Universal RNA-Seq System (Illumina® Platform) in accordance with the manufacturer's protocol. The libraries were sequenced on an Illumina HiSeq 2500 in rapid mode using a 50 bp rapid single-read flow cell. RNA-seq data were processed as follows: FASTQ files of RNA sequencing were mapped to the reference genome GRCh 38 using a Spliced ​​Transcripts Alignment to a Reference (STAR) aligner (Dobin et al., Bioinformatics, January 2013, Vol. 29, No. 1, pp. 15-21) and Cufflinks to generate gene-level estimates of expression in TPMs per million transcripts (TPMs). TPMs were calculated as computed values ​​as units of expression, then quantile-normalized and log2-transformed. RNA-seq data generated from this cell line analysis identified genes that were significantly upregulated and downregulated by E2 compared to DMSO. ER regulatory genes were identified using two comparisons. First, genes regulated by one or more SERM or SERD compounds and opposedly regulated by estradiol were identified ([log2 FC]>2 and FDR<0.05 vs. DMSO control) in Table 1. Two-factor (treatment and dose) analysis of variance (ANOVA) models were used at respective doses and time points to identify genes differentially expressed between the SERD compound treatment group and the dimethyl sulfoxide (DMSO; control) treatment group. The treatment factor was fixed at seven levels: six SERD compounds and DMSO control. The dose factor was also fixed at two levels: low and high. All samples were treated over 24 hours. Post-hoc contrast analyses were performed between each SERD compound treatment and the DMSO control at respective dose and time levels. Using cutoff levels of absolute chromosome change ≥ 1.5 and false detection rate adjusted p-value ≤ 0.05, differentially expressed genes (DEGs) were selected, and a panel of 1022 DEGs was obtained.This analysis was performed using Array Studio (Qiagen). A panel of 1022 genes identified as differentially expressed in comparisons of at least one compound against DMSO was used for hierarchical clustering of compound-therapeutic data normalized to DMSO controls at each dose and time point. Full linkage clustering was performed based on Pearson correlation coefficients. Similarity of expression profiles between compounds was assessed by Pearson correlations of differentially expressed genes.

[0077] Secondly, genes regulated by one or more SERM or SERD molecules in the presence of estradiol were identified ([log2 FC]>2 and FDR<0.05 vs. estradiol alone) (see Table 2). 87 genes were regulated in both comparisons. Of these 87 genes, 66 were induced by E2 and downregulated by SERD / SERM, and 21 were repressed by E2 and upregulated by SERD / SERM. This signature can be used to measure ER regulation by SERMs and SERDs. In general, the 87 genes were more strongly regulated by SERD compounds than by SERM compounds; see Figure 5.

[0078] result To evaluate ER regulation by amsenestrant and other SERDs, HCC1428-LTED (long-term estrogen deficiency) breast cancer cell lines were treated with 300 nM (high) or 30 nM (low) SERD compounds for 24 hours. To elucidate ER-specific activity, we then evaluated gene expression after compound treatment in HCC1428-LTED cells, the hormone-deficient cell line. Overall mRNA expression changes were assessed 24 hours post-treatment. Hierarchical clustering in a selected panel of 1022 transcripts identified two groups with distinct signatures: one group included GDC-0810 and AZD9496, and the other group included fulvestrant, SAR439859, GDC-SAR, and AZD-SAR (Figure 1A). Our ER activity score analysis showed that two SERDs, fulvestrant and SAR439859, potently reduced ER activity scores, similar to the two hybrid molecules, GDC-SAR and AZD-SAR. In contrast, GDC-0810 and AZD9496 only partially reduced ER activity scores. Statistical correlation revealed that the expression profile induced by SAR439859 was closely correlated with that of fulvestrant. On the other hand, the transcriptional profiles of GDC-0810 and AZD9496 showed only a weak correlation with the transcriptional profile of fulvestrant. Interestingly, the profiles of the hybrid molecules GDC-SAR and AZD-SAR also closely correlated with those of fulvestrant and SAR439859 (Figure 1A; Figure 3).

[0079] Generally, 87 genes were more strongly regulated by SERD compounds than by SERM compounds. Of these 87 genes, 66 were induced by E2 and downregulated by SERD / SERM, and 21 were repressed by E2 and upregulated by SERD / SERM.

[0080] To evaluate the relative ER regulatory activity of compounds, a panel of ER regulatory genes (87 genes, Table 3) was developed by transcriptional profiling of multiple cell lines, identifying genes regulated by estradiol and subsequently blocked by SERMs and SERDs. This panel of ER regulatory genes can be used to measure ER regulation by SERMs and SERDs. Subsequently, ER activity scores were evaluated using gene set variation analysis (GSVA) ​​of the ER signature (BMC Bioinformatics. 2013;14:7).

[0081] Fulvestrant and SAR439859 demonstrated deep inhibition of ER activity, while GDC-0810 and AZD9496 only partially inhibited ERα activity, further supporting the above observation. Interestingly, both hybrid molecules, GDC-SAR and AZD-SAR, also potently inhibited ERα transcriptional activity (Figure 1B). The findings were confirmed by measuring the expression of two well-validated ER target genes, CXCL12 and BCAS1, by RT-qPCR. Gene expression analysis provided further confirmation of the differential response of these molecules to well-validated ERα target genes (Elife. 2016;5, Endocr Relat Cancer. 2015;22(5):713-24). SAR439859, GDC-SAR, and AZD-SAR inhibited CXCL12 expression and induced Bcas1 expression, while GDC-0810 and AZD9496 did not induce any significant changes in the expression of these genes (Figure 1C, D). Surprisingly, SAR439859 and fulvestrant induced significant regulation of ERα-specific activity in the absence of E2, suggesting potent inverse agonist activity of these compounds.

[0082] The list provided below lists the genes that are downregulated or upregulated by SERD, and more specifically by treatment with amsenestrant.

[0083] [Table 3-1] [Table 3-2]

[0084] RT-qPCR analysis of individual ERα suppressor genes such as PGR and CXCL12 confirmed more potent inhibition by SAR439859 compared to fulvestrant or GDC-0810 in HCI013 tumors (Figures 2 and 4A, respectively). ERα transcriptional activity was analyzed in the HCI013 model using a panel of genes corresponding to ER gene signatures derived from in vitro cell line analysis, as previously described. Both SAR439859 and GDC-0810 treatments demonstrated repressive activity of ERα signaling, with SAR439859 achieving more pronounced ERα suppression compared to GDC-0810 (Figure 4B). [Examples]

[0085] This study was an open-label, phase 1 / 2 trial in postmenopausal women with ER+ / HER2- advanced / metastatic breast cancer, evaluating the safety, antitumor activity, pharmacokinetics, and pharmacodynamic properties of amsenestrant, administered as monotherapy in dose-escalation cohorts (Part A) and dose-expansion cohorts (Part B), and as combination therapy in other arms of the study. A screening period was included, from 28 days prior to baseline to baseline. The treatment period consisted of 28-day therapy cycles with SAR439859. A follow-up period was followed after the completion of treatment. In the Part A dose-escalation cohort, SAR439859 was administered as a PO (oral) QD (once daily) of 20–600 mg. In the Part B dose-expansion cohort, SAR439859 was administered as a PO QD of 400 mg.

[0086] Paired tumor biopsies were collected at screening of patients from Part B who consented to collection and at the end of Cycle 2 (Cycle 2, Day 28). For each biopsy, formalin-fixed paraffin-embedded (FFPE) tissue (block or slide) was collected, 5 μm each for immunohistochemical (IHC) analysis and 10 μm each for RNA extraction and subsequent RNA-seq analysis. IHC ER, progesterone receptor (PgR), and Ki67 protein expression slides were evaluated by two specialist pathologists (VP ​​and A-LB). IHC staining was performed on the Ventana Discovery XT IHC platform using anti-ER clone SP1 (CONFIRM anti-estrogen receptor, Roche, ref. 790-4325), anti-PgR clone 1E2 (CONFIRM anti-progesterone receptor, Roche, ref. 790-4296), and anti-Ki67 clone 30-9 (CONFIRM anti-Ki-67, Roche, Ref. 790-4286). Stained slides were evaluated using a standard light microscope with a manual scoring system. Staining was evaluated only in tumor cells. H scores were calculated for ER and PgR expression. For Ki67 protein expression, the percentage of positive tumor cells was calculated. Total RNA was extracted from FFPE tissue (10 μm slides). After applying a targeted enrichment RNA-seq approach using KAPA RNA HyperPrep and the whole exome SeqCap kit (Roche), sequencing reads were generated using NextSeq 500 (Illumina). RNA-seq FASTQ files were processed using STAR aligner and Cufflinks to generate gene-level fragments per kilobase of transcript per million mapped reads (FPKM). These FPKM values ​​were converted to gene-level estimates of expression in total product per million (TPM). The TPM data were then quantile-normalized and log2-transformed. The decrease in ER activation score, measured by gene set variation analysis (GSVA) ​​as previously described, was determined by RNA-seq analysis.

[0087] Plasma samples were collected at the end of cycle 1, day 1 and cycle 2, day 28 to evaluate ESR1 mutations.

[0088] The status of wild-type and mutant ESR1 in circulating free DNA (cfDNA) was determined. cfDNA was measured at baseline and day 56 of the treatment period. cfDNA was evaluated for pathogenic ESR1 mutations (single nucleotide variants) in the ligand-binding domain of ESR1 by a multiplex central droplet digital polymerase chain reaction (dd-PCR) BEAMing 12-gene assay using the OncoBEAM® platform from Sysmex Inostics (Baltimore, MD, USA). cfDNA was also measured by alternative methods at baseline and at the end of treatment using the Roche AVENIO expanded panel and a 77-gene NGS panel (Ambry Genetics). Germ cell mutations were evaluated at baseline. Salivary mutations are referred to for reference regarding the potential for germ cell mutations in cfDNA.

[0089] Amsenestrant induced ER degradation / signaling inhibition, as demonstrated in paired tumor biopsies (collected at baseline and after two 28-day cycles of amsenestrant administration). In one patient, a biopsy was collected on day 15 of the first administration cycle during treatment.

[0090] Amsenestrant demonstrated robust on-target activity, as indicated by an overall decrease in IHC ER expression (ER degradation) (Figure 6A; median relative change from baseline was -58% [range -93% to -44%] among patients with non-zero values ​​in available data at screening and cycle 2, day 28; n=6), a decrease in PgR expression (Figure 6B; median relative change from screening was -88% [range -100% to -38%]; n=3), a decrease in Ki67 protein expression (Figure 6C; median change from screening in percentage of Ki67-positive cells was -8% [range -30% to +30%]; n=7), and a decrease in GSVA ER activation score (Figure 6D; median change from screening was -0.4 [range -1.1 to +0.4]; n=5). The H score for the ER or PgR nucleus decreased at cycle 2, day 28 in all patients with a non-zero screening value. Of the patients whose H score for the ER or PgR nucleus was equal to zero at screening, one patient showed an increase in the PgR nucleus H score at cycle 2, day 28. The ER activation score decreased or remained stable only among patients who showed clinical benefit (median change from screening was -0.6 [range -1.1 to 0]; n=4), while the ER activation score increased in patients who did not show clinical benefit (change from screening: +0.4). The numbers in "(n=)" above indicate the number of patients for which descriptive statistics (median change or median relative change) were calculated. The number of patients for whom data is available at baseline / screening and after baseline (each line in Figures 6A-D represents one patient) may differ from the numbers in "(n=)" above, because, at least, the median relative change could not be calculated when baseline / screening was equal to zero.

[0091] Responses for clinical benefit assessment were evaluated according to RECIST v1.1.

[0092] ER activity scores (assessed using five pairs of biopsies) decreased or remained stable in patients who showed clinical benefit, but increased in patients who did not. Patients who showed clinical benefit but not partial response showed a decrease in ER activity scores. One patient who achieved both clinical benefit and partial response showed no change in activation score (and therefore a very low baseline score, indicating low ER activity).

[0093] ER activity scores correlated more closely with clinical benefit (CB) than ER protein IHC. Of all the methods used to assess amsenestrant on-target activity in tumor biopsies (immunohistochemical and transcriptome analysis), ER activity scores most clearly differentiated between patients who achieved clinical benefit. IHC ER protein levels did not necessarily correlate with ER pathway status.

[0094] The application of ER activity scores to preclinical and clinical studies demonstrates their potential value as biomarkers, such as targeted engagement pharmacodynamic (PD) biomarkers for SERD.

[0095] The strengths of this study include a combined evaluation of safety, antitumor activity, and pharmacokinetics / pharmacodynamics, demonstrating potent targeted engagement and ER degradation / pathway inhibition in ER+ / HER2- advanced breast cancer.

Claims

1. A panel of ER regulatory genes having 87 genes, wherein the 87 genes are upregulated into the following 21 genes: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA , BAK1, PLIN2, and SYNPO; and 87 genes, with the following 66 downregulated genes: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SE RPINA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, S Includes KP2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH, The panel refers to genes in which upregulated and downregulated genes show increased and decreased expression after administration of amsenestrant compared to treatment with a control.

2. A method for monitoring the response of an individual with cancer to treatment with amsenestrant, the method comprising: (a) determining a first ER activity score from a sample from the individual at a first time point prior to treatment with amsenestrant; (b) determining a second ER activity score from a sample from the individual at a second time point following administration of amsenestrant, following step (a); and (c) comparing the first ER activity score to a second ER activity score, wherein the decrease in the second ER activity score relative to the first ER activity score is a predictor of target engagement of amsenestrant. Here, determining the first ER activity score and the second ER activity score involves analyzing a panel of ER regulatory genes. The ER regulatory gene panel consists of 87 genes, and these 87 genes have been upregulated into the following 21 genes: PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BA It includes K1, PLIN2, and SYNPO; and the 87 genes are downregulated by the following 66 genes: H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERP INA5, SFXN2, CAP2, HIST1H2BM, CDC45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, H IST1H4D, HIST1H4C, RGS22, SYBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SK Includes P2, HIST1H3B, TMEM164, AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH, The method, wherein upregulated and downregulated genes refer to genes that show increased and decreased expression after administration of amsenestrant compared to treatment with a control.

3. The method according to claim 2, wherein the decrease in the second ER activity score relative to the first ER activity score is a predictor of the on-target activity of amsenestrant.

4. The method according to claim 3, wherein the on-target activity of amsenestrant comprises ER degradation and ER inhibition.

5. The method according to claim 2, comprising bringing a sample from an individual into contact with a probe.

6. The method according to any one of claims 2 to 5, comprising detecting the RNA expression level of each gene in a sample from an individual.

7. The method according to any one of claims 2 to 6, comprising RNA sequencing.

8. The method according to any one of claims 2 to 7, wherein the individual has ER + breast cancer.

9. The method according to any one of claims 2 to 8, wherein the individual has HER2-breast cancer.

10. The method according to any one of claims 2 to 9, wherein the individual has progressive or metastatic breast cancer.

11. The method according to any one of claims 2 to 10, wherein the sample is a tumor biopsy.

12. The method according to any one of claims 2 to 10, wherein the sample is a blood sample.

13. The method according to any one of claims 2 to 10, wherein the sample is a serum sample.

14. Tumor biopsies include formalin-fixed tumor biopsies, paraffin-embedded tumor biopsies, and formalin-fixed paraffin-embedded tumor biopsies. The method according to claim 11, wherein the biopsy is a fin-embedded (FFPE) tumor biopsy, a fresh-frozen (FF) tumor biopsy, a frozen tumor biopsy, or a fresh tumor biopsy.

15. A kit comprising a set of probes for detecting the expression of upregulated and downregulated genes; Includes probes for detecting upregulated genes PTPRT, CAMK1D, BCAS1, ABCG1, KRT81, TLE2, SALL4, ITGB6, BBC3, IL1R1, CORO2A, GRM4, ATP6V0A4, MATN2, PPFIBP2, BMF, LOC283070, PSCA, BAK1, PLIN2, and SYNPO. The downregulated genes H19, MYBL1, MGP, SLC5A8, PKIB, PGR, TMPRSS3, IGSF1, COL21A1, FSIP1, KLHL4, EGR3, NPY1R, HIST1H3C, GRIK3, SCNN1B, FRK, PRSS23, SERPINA5, SFXN2, CAP2, HIST1H2BM, CD C45, WDR62, DSCC1, GJA1, FAM196A, NTRK2, RAD54L, SDK2, SLC4A10, HIST1H4D, HIST1H4C, RGS22, S YBU, CA8, DIAPH3, FHL2, HIST1H1B, KCNH1, RAD51AP1, FANCD2, SLC39A8, SKP2, HIST1H3B, TMEM164, Includes probes for detecting AURKB, DEPTOR, XRCC2, C1QTNF6, ERCC6L, ORC1, HIST2H2AC, RRM2, EME1, HIST1H2BF, CLSPN, EXO1, SDC2, HIST1H3I, HIST2H2AB, HIST1H4B, ASCL1, ATAD5, HIST1H1D, and HIST1H2BH. The kit, wherein upregulated and downregulated genes refer to genes that show increased and decreased expression after administration of amsenestrant compared to treatment with a control.

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