Lasofoxifene combination treatment for ER+ breast cancer progressing with CDK4 / 6 inhibitors

Abstract

1. A method for reducing the progression of ER+ breast cancer in a patient, comprising administering to the patient an effective amount of lasofoxifene, or a pharma- ceutical acceptable salt thereof, and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), wherein the breast cancer is: (i) estrogen receptor positive (ER+) + (ii) have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) have progressed on prior CDK4 / 6 inhibitor treatment and / or have an oncogenic mutation in a gene other than ESR1.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 345,843, filed May 25, 2022; U.S. Provisional Patent Application No. 63 / 411,633, filed September 30, 2022; U.S. Provisional Patent Application No. 63 / 426,737, filed November 19, 2022; U.S. Provisional Patent Application No. 63 / 430,194, filed December 5, 2022; and U.S. Provisional Patent Application No. 63 / 446,760, filed February 17, 2023, the entire disclosures of each of which are incorporated by reference herein. [Background technology]

[0002] 2. Background of the invention Estrogen receptor positive (ER + Breast cancers express estrogen receptor alpha (ERα), which is encoded by the ESR1 gene. Approximately 70% of breast cancers + and therefore are treated with agents that deplete circulating estrogen levels or block estrogen signaling in cancer cells (collectively, endocrine therapy). Endocrine therapy reduces the ER + The outcomes of women with breast cancer have been significantly improved. However, the effectiveness of endocrine therapy is limited by intrinsic and, importantly, acquired endocrine resistance. In response to the selective pressure imposed by endocrine therapy, particularly aromatase inhibitors (AIs), ER + Tumors evolve various evasion mechanisms. Among these is the acquisition of gain-of-function mutations in the ESR1 gene that alter the ligand-binding domain of the ERα receptor, rendering the receptor constitutively active when estrogen levels are low or absent. Despite the benefits of endocrine treatment, ER + The majority of tumors eventually acquire resistance and progress.

[0003] Lasofoxifene, a third-generation selective estrogen receptor modulator (SERM), inhibits invasive ER tumors in women with wild-type estrogen receptors, i.e., postmenopausal women with no history of breast cancer who are being treated for osteoporosis. + Lasofoxifene has been shown to reduce the risk of breast cancer. LaCroix et al., J. Natl. Cancer Inst. 102:1706-1715 (2010). Lasofoxifene has subsequently been shown to inhibit ERα receptors that have a gain-of-function mutation in the ligand-binding domain of the ERα receptor (ESR1 gene). + It has been shown that ESR1 retains the ability to suppress cancer progression. U.S. Patent Nos. 10,258,605 and 10,905,659; WO2019 / 199891; Laine et al., Breast Cancer Res. 23(1):54(2021). + The efficacy of lasofoxifene as a single agent in treating pre- and postmenopausal women with breast cancer is currently being confirmed in a Phase 2 clinical trial, NCT03781063 (ELAINE trial). Over the past decade, a new class of drugs, cyclin-dependent kinase 4 / 6 inhibitors (CDK4 / 6i), has emerged to treat (ER) + ) have become commercially available for the treatment of women with breast cancer. Three CDK4 / 6i drugs for the treatment of cancer in combination with endocrine therapy have been approved in several countries around the world, including the United States: palbociclib (IBRANCE, Pfizer), ribociclib (KISQALI, Novartis), and abemaciclib (VERZENIO, Eli Lilly). However, ER + Tumors have been shown to develop resistance to CDK4 / 6i and ultimately progress. Carrying mutations in ESR1 and developing resistance to endocrine therapy and CDK4 / 6 inhibitors, and therefore advanced ER + There remains a need for new effective therapies for the treatment of tumors. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 10,258,605 [Patent Document 2] U.S. Patent No. 10,905,659 [Patent Document 3] International Publication No. 2019 / 199891 [Non-patent literature]

[0005] [Non-Patent Document 1] LaCroix et al., J. Natl. Cancer Inst. 102:1706-1715 (2010) [Non-Patent Document 2] Laine et al.,Breast Cancer Res.23(1):54(2021) Summary of the Invention [Means for solving the problem]

[0006] 3. Overview of the Invention The ongoing ELAINE2 clinical trial is evaluating patients with locally advanced or metastatic ER who harbor an ESR1 mutation and have progressed on first-, second-, or third-line hormonal treatment for metastatic disease. + / HER2 -This is an open-label, multicenter study evaluating the efficacy, safety, and tolerability of the combination of the third-generation SERM lasofoxifene and the CDK4 / 6 inhibitor abemaciclib for the treatment of pre- and postmenopausal women with breast cancer. To be eligible for enrollment, progression occurred after up to three of the following treatments for metastatic breast cancer: aromatase inhibitors (AIs) and / or fulvestrant (either as monotherapy or in combination with any commercially available CDK4 / 6i); and / or a combination of fulvestrant and alpelisib; and / or tamoxifen; and / or an exemestane / everolimus combination; and up to one line of chemotherapy in the metastatic setting (48%). The majority of patients enrolled in the study (28 / 29) had progressed on prior CDK4 / 6 inhibitor combination treatment. The inventors herein demonstrate that the combination of lasofoxifene and the CDK4 / 6 inhibitor (CDK4 / 6i) abemaciclib is well tolerated and demonstrates robust and meaningful efficacy in patients with locally advanced or metastatic ER disease harboring ESR1 gain-of-function mutations who have progressed on prior CDK4 / 6i therapy. + It has been found to reduce the progression of breast cancer in women with breast cancer.

[0007] Furthermore, the inventors have now discovered that a combination of lasofoxifene and the CDK4 / 6 inhibitor (CDK4 / 6i) abemaciclib reduces breast cancer progression in women with locally advanced or metastatic ER+ breast cancer harboring an ESR1 gain-of-function mutation and an oncogenic mutation in one or more genes other than the ESR1 gene.

[0008] Thus, in a first aspect, there is provided a method of reducing progression of breast cancer in a patient, comprising: administering to said patient an effective amount of lasofoxifene, or a pharma- ceutically acceptable salt thereof, and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i); The breast cancer is (i) Estrogen receptor positive (ER + ) and; (ii) have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) have progressed on prior CDK4 / 6 inhibitor therapy, methods are provided herein.

[0009] In one embodiment, there is provided a method of reducing the progression of breast cancer in a patient, comprising: administering to said patient an effective amount of lasofoxifene, or a pharma- ceutically acceptable salt thereof, and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i); The breast cancer is (i) Estrogen receptor positive (ER + ) and; (ii) have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) having an oncogenic mutation in one or more genes other than the ESR1 gene, a method is provided herein.

[0010] In one embodiment, there is provided a method of reducing the progression of breast cancer in a patient, comprising: administering to said patient an effective amount of lasofoxifene, or a pharma- ceutically acceptable salt thereof, and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i); The breast cancer is (i) Estrogen receptor positive (ER + ) and; (ii) have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) A method is provided herein, wherein the expression of one or more genes other than the ESR1 gene is increased.

[0011] In one embodiment, a method of monitoring a patient during breast cancer treatment comprises: (a) determining a quantitative measure of the mutant allele frequency (MAF) in circulating tumor DNA (ctDNA) (ESR1 ctDNA) of at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene in the patient's biological sample, the quantitative measure being performed at determined intervals over a period of time; and (b) determining a positive predictive value (PPV) for clinical benefit with stable disease of said cancer treatment, the PPV indicates responsiveness to the cancer treatment; determining that the cancer treatment comprises an effective amount of lasofoxifene or a pharma- ceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i); A method is provided herein, comprising:

[0012] ER + Breast cancer is HER2 - 3. The method of any preceding embodiment, wherein

[0013] ER + The method of any preceding embodiment, wherein the breast cancer is locally advanced or metastatic.

[0014] The method of any preceding embodiment, wherein the lasofoxifene is administered as lasofoxifene tartrate.

[0015] The method of any preceding embodiment, wherein lasofoxifene is administered at 5 mg / day per os.

[0016] The method of any preceding embodiment, wherein the CDK4 / 6i administered to the patient is selected from palbociclib, ribociclib, and abemaciclib.

[0017] The method of any preceding embodiment, wherein the CDK4 / 6i administered to the patient is abemaciclib. In certain embodiments, abemaciclib is administered orally at 50 mg to 200 mg BID. In certain embodiments, abemaciclib is administered orally at 100 mg to 200 mg BID. In certain embodiments, abemaciclib is administered orally at 150 mg BID.

[0018] The method of any preceding embodiment, wherein the previously administered CDK4 / 6 inhibitor is selected from palbociclib, ribociclib, and abemaciclib. In certain of these embodiments, the previously administered CDK4 / 6 inhibitor is abemaciclib.

[0019] The method of any preceding embodiment, wherein the cancer has previously been determined to have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

[0020] The method of any preceding embodiment, further comprising the earlier step of determining that the patient has at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

[0021] The method of any preceding embodiment, wherein said at least one gain-of-function missense mutation is present at any one of amino acids D538, Y537, L536, P535, V534, L469, S463, V392, and E380.

[0022] 4. A brief description of some figures in the drawing These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and accompanying drawings. [Brief description of the drawings]

[0023] [Figure 1]FIG. 1 is a swimmer plot showing data on individual patient response to treatment with lasofoxifene and abemaciclib at the first interim date in the ELAINE2 clinical trial (NCT04432454).

[0024] [Diagram 2] FIG. 2 shows the maximum tumor response of the patients at the same time points as FIG. 1 (PD=progressive disease; SD=stable disease; PR=partial response).

[0025] [Diagram 3] FIG. 3 is a swimmer plot showing data regarding patient response to treatment with lasofoxifene and abemaciclib at a later interim date than FIGS. 1 and 2 in the ELAINE 2 clinical trial (NCT04432454), with additional information regarding each subject's pre-enrollment treatment.

[0026] [Figure 4-1] Figure 4 shows, for each individual subject enrolled in the ELAINE2 trial, a comparison of the subject's pre-enrollment duration of response on prior second-line and third-line cancer therapies with the on-study duration of response to the combination of lasofoxifene and abemaciclib at the same time points as in Figure 3. [Figure 4-2] Figure 4 shows, for each individual subject enrolled in the ELAINE2 trial, a comparison of the subject's pre-enrollment duration of response on prior second-line and third-line cancer therapies with the on-study duration of response to the combination of lasofoxifene and abemaciclib at the same time points as in Figure 3.

[0027] [Diagram 5]FIG. 5 shows the prevalence of oncogenic mutations in a gene panel including the ESR1 gene and other genes known or suspected to contribute to neoplasia at baseline among enrolled ELAINE2 subjects as of the midpoint date in FIG. 3, and the subjects' subsequent achievement of clinical benefit (CB) and median progression-free survival (mPFS) upon treatment with lasofoxifene and abemaciclib in the ELAINE2 trial.

[0028] [Figure 6-1] Figure 6 shows individual MAF kinetics for the most commonly observed mutESR1 variants. Y537C, Y537N, Y537S, and D538G variants are shown. Variants with low baseline MAF (solid line with double ended diamonds) use the left vertical axis (y-axis) and variants with high baseline MAF (dashed line with double ended circle) use the right vertical axis (y-axis). X-axis: timeline from baseline to week 4. BL: baseline; MAF: mutant allele fraction. [Figure 6-2] Figure 6 shows individual MAF kinetics for the most commonly observed mutESR1 variants. Y537C, Y537N, Y537S, and D538G variants are shown. Variants with low baseline MAF (solid line with double ended diamonds) use the left vertical axis (y-axis) and variants with high baseline MAF (dashed line with double ended circle) use the right vertical axis (y-axis). X-axis: timeline from baseline to week 4. BL: baseline; MAF: mutant allele fraction.

[0029] [Figure 7] Figure 7 is a swimmer plot showing data regarding patient response to treatment with lasofoxifene and abemaciclib in the ELAINE 2 clinical trial (NCT04432454) at a later interim date than in Figures 1-4. Additional information regarding each subject's pre-enrollment treatment is also shown.

[0030] [Figure 8]Figures 8A and 8B show exemplary types of copy number variations (CNVs) of various cancer genes, including CCND1, detected in circulating tumor DNA (ctDNA) of subjects in the ELAINE2 clinical trial (NCT04432454) as of the date of Figure 5. Figure 8A shows the CNV events detected per gene. Figure 8B shows the copy number distribution per gene for the CNV events shown in the left panel. CNVs are annotated as "focal" or "aneuploidy", or "amplification" when the focal or aneuploidy status is uncertain.

[0031] [Figure 9] FIG. 9 shows the prevalence of oncogenic mutations in a gene panel including the ESR1 gene and other genes known or suspected to contribute to neoplasia at baseline in enrolled ELAINE2 subjects at the median date later than FIG. 5, and the subjects' subsequent achievement of clinical benefit (CB) and median progression-free survival (mPFS) upon treatment with lasofoxifene and abemaciclib in the ELAINE2 trial.

[0032] [Figure 10-1] Figures 10A-10B are swimmer plots showing data regarding patient response to treatment with lasofoxifene and abemaciclib in the ELAINE2 clinical trial (NCT04432454) at a later interim date than Figures 1-4 and 7. Figure 10B is a reproduction of Figure 10A including the designation numbers of individual patients (but anonymized). Additional information regarding each subject's pre-enrollment treatment is also shown. [Figure 10-2] Figures 10A-10B are swimmer plots showing data regarding patient response to treatment with lasofoxifene and abemaciclib in the ELAINE2 clinical trial (NCT04432454) at a later interim date than Figures 1-4 and 7. Figure 10B is a reproduction of Figure 10A including the designation numbers of individual patients (but anonymized). Additional information regarding each subject's pre-enrollment treatment is also shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] 5. Detailed Description of the Invention The ongoing ELAINE2 clinical trial is evaluating patients with locally advanced or metastatic ER who harbor an ESR1 mutation and whose disease has progressed on first-, second-, or third-line hormonal treatment for metastatic disease. + / HER2 - This is an open-label, multicenter study evaluating the efficacy, safety, and tolerability of the combination of the third-generation SERM lasofoxifene and the CDK4 / 6 inhibitor abemaciclib for the treatment of pre- and postmenopausal women with breast cancer.To be eligible for enrollment, progression occurred on ≤3 of the following treatments for metastatic breast cancer: an aromatase inhibitor (AI) and / or fulvestrant (either as monotherapy or in combination with any commercially available CDK4 / 6i); and / or a combination of fulvestrant and alpelisib; and / or tamoxifen; and / or a combination of exemestane / everolimus.

[0034] All but one (28 / 29) of the patients enrolled in the study had progressed on prior CDK4 / 6 inhibitor combination therapy. We herein demonstrate that the combination of lasofoxifene with the CDK4 / 6 inhibitor (CDK4 / 6i) abemaciclib was well tolerated and demonstrated robust and meaningful efficacy in such patients harboring gain-of-function mutations in the ligand-binding domain of ESR1 and with advanced ER disease who had progressed on prior CDK4 / 6i therapy. + It has been found to reduce the progression of breast cancer in women with breast cancer.

[0035] Furthermore, the inventors have now discovered that a combination of lasofoxifene and the CDK4 / 6 inhibitor (CDK4 / 6i) abemaciclib reduces breast cancer progression in women with locally advanced or metastatic ER+ breast cancer harboring an ESR1 gain-of-function mutation and an oncogenic mutation in one or more genes other than the ESR1 gene. 5.1. Treatment method

[0036] Thus, in a first aspect, there is provided a method of reducing (or delaying) the progression of breast cancer in a patient by administering to the patient an effective amount of lasofoxifene, or a pharma- ceutical acceptable salt thereof, and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), wherein the breast cancer is: (i) estrogen receptor positive (ER) + (ii) have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) have progressed on previous CDK4 / 6 inhibitor therapy.

[0037] In certain embodiments, the patient's breast cancer is human epidermal growth factor receptor 2 negative (HER2 - In certain embodiments, the patient's breast cancer is locally advanced. In certain embodiments, the patient's breast cancer is metastatic.

[0038] 1. A method of treating breast cancer in a patient by administering to the patient an effective amount of lasofoxifene, or a pharma- ceutical acceptable salt thereof, and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), wherein the breast cancer is: (i) estrogen receptor positive (ER) + (ii) have at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) have an oncogenic mutation in one or more genes other than the ESR1 gene. In certain embodiments, the patient's cancer is human epidermal growth factor receptor 2 negative (HER2 - In certain embodiments, the patient's ER + The breast cancer is locally advanced. In certain embodiments, the patient has + The breast cancer is metastatic. 5.1.1.ER + Patients with cancer

[0039] According to the treatment method provided, the patient was admitted to the ER. +In various embodiments, the patient has breast cancer. + Has been diagnosed with breast cancer.

[0040] In some embodiments, the patient is premenopausal, perimenopausal, or postmenopausal. In some embodiments, the patient is premenopausal and has locally advanced or metastatic ER. + In some embodiments, the patient is peri-menopausal and has locally advanced or metastatic ER. + In some embodiments, the patient is postmenopausal and has locally advanced or metastatic ER cancer. + Having breast cancer.

[0041] In some embodiments of the provided methods of treatment, the patient's breast cancer is HER2 - (ER + / HER2 - In certain embodiments, the patient has locally advanced or metastatic ER. + / HER2 - Having breast cancer. Mutations in the ESR1 gene

[0042] According to the provided method of treatment, cells of the patient's cancer have acquired at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

[0043] In some embodiments, the mutation results in ligand-independent activity of the estrogen receptor. In some embodiments, the mutation results in enhanced ligand-stimulated activity of the estrogen receptor. In some embodiments, the mutation results in resistance to endocrine therapy. In some embodiments, the mutation promotes tumor growth. In some embodiments, the mutation enhances metastatic activity of a cancer. In some embodiments, the mutation enhances ER activation. + Enhances the further metastatic activity of metastatic breast cancer.

[0044] In various embodiments, the mutation arises from a rare, undetectable pre-existing clone. In some embodiments, the mutation is acquired de novo during the course of endocrine therapy treatment. In some embodiments, the mutation is acquired de novo after multiple rounds of endocrine therapy treatment. In some embodiments, the mutation is acquired de novo after multiple rounds of endocrine therapy treatment for metastatic breast cancer. In various embodiments, the mutated clone expands to become a more dominant clone during successive rounds of endocrine therapy.

[0045] In some embodiments, the mutation in the ESR1 gene is a missense point mutation. In some embodiments, the mutation in the ESR1 gene is a truncating mutation. In some embodiments, the mutation in the ESR1 gene is a gene amplification. In some embodiments, the mutation in the ESR1 gene is a genomic rearrangement.

[0046] In some embodiments, the patient has at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the ESR1 gene. + In various embodiments, at least one of the mutations is present at an amino acid selected from D538, Y537, L536, P535, V534, L469, S463, V392 and E380, said amino acids being numbered according to ESR1 protein having NCBI accession number NP_000116.2.

[0047] In certain embodiments, the mutation increases the stability of the agonist conformation of helix 12 of the ERα protein. In some of these embodiments, the mutation increases the binding of the estrogen receptor to its coactivator. In some of these embodiments, the mutation results in hormone-independent activity of the estrogen receptor. In some of these embodiments, the mutation results in resistance to tamoxifen, fulvestrant, and / or aromatase inhibitors.

[0048] In certain embodiments, the mutation is at amino acid D538. In certain preferred embodiments, the mutation is D538G. + The breast cancer has at least one D538G mutation and at least one mutation at an amino acid selected from Y537, L536, P535, V534, L469, S463, V392, and / or E380. + The breast cancer harbors at least one D538 mutation and at least one Y537 mutation.

[0049] In certain embodiments, the mutation is present at amino acid Y537. + The breast cancer has at least one mutation at amino acid Y537 and at least one mutation at amino acid selected from D538, L536, P535, V534, L469, S463, V392, and / or E380.In some of these embodiments, the mutation is Y537S, Y537N, Y537C, or Y537Q.In certain preferred embodiments, the mutation is Y537S.In certain preferred embodiments, the mutation is Y537C.In certain preferred embodiments, the mutation is Y537N.In certain preferred embodiments, the mutation is Y537Q.

[0050] In some embodiments, the mutation is present at amino acid L469. + The breast cancer has at least one mutation at amino acid L469 and at least one mutation at an amino acid selected from D538, L536, Y537, P535, V534, S463, V392, and / or E380. In certain preferred embodiments, the mutation is L469V.

[0051] In some embodiments, the mutation is at amino acid L536. +The breast cancer has at least one mutation at amino acid L536 and at least one mutation at amino acid selected from D538, Y537, P535, V534, L469, S463, V392, and / or E380.In certain embodiments, the mutation is L536R or L536Q.In certain embodiments, the mutation is L536R.In certain embodiments, the mutation is L536Q.In certain embodiments, the mutation is L536P.In certain embodiments, the mutation is L536H.

[0052] In some embodiments, the mutation is at amino acid P535. + The breast cancer has at least one mutation at amino acid P535 and at least one mutation at an amino acid selected from D538, Y537, L536, V534, L469, S463, V392, and / or E380. In certain embodiments, the mutation is P535H.

[0053] In some embodiments, the mutation is at amino acid V534. + The breast cancer has at least one mutation at amino acid V534 and at least one mutation at an amino acid selected from D538, Y537, L536, P535, L469, S463, V392, and / or E380. In certain embodiments, the mutation is V534E.

[0054] In some embodiments, the mutation is at amino acid S463. + The breast cancer has at least one mutation at amino acid S463 and at least one mutation at an amino acid selected from D538, Y537, L536, P535, V534, L469, V392, and / or E380. In certain embodiments, the mutation is S463P.

[0055] In some embodiments, the mutation is present at amino acid V392. +The breast cancer has at least one mutation at amino acid V392 and at least one mutation at an amino acid selected from D538, Y537, L536, P535, V534, L469, S463, and / or E380. In certain embodiments, the mutation is V392I.

[0056] In some embodiments, the mutation is at amino acid E380. + The breast cancer has at least one mutation at amino acid E380 and at least one mutation at an amino acid selected from D538, Y537, L536, P535, V534, L469, S463, and / or S463. In certain embodiments, the mutation is E380Q. 5.1.2.1 Detection of ESR1 gene mutations

[0057] In various embodiments, the patient's ER + Breast cancer has previously been determined to have at least one mutation in the ESR1 gene. Some embodiments of the methods described herein further include an earlier step of detecting a mutation in the ESR1 gene.

[0058] In some embodiments, massively parallel next generation sequencing (NGS) is used to detect estrogen receptor mutations in the patient's cancer. In certain embodiments, the entire genome is sequenced. In certain embodiments, a selected gene panel of cancer-related genes is sequenced. In certain embodiments, all coding exons in a given gene set are sequenced. In certain embodiments, known "hotspot" regions in a given gene set are sequenced. However, the inherent error rate of current next generation sequencing technology is up to 1%, limiting the sensitivity and specificity of detection. In some embodiments, targeted sequencing is used to detect the presence of ESR1 mutations. Targeted sequencing allows for deeper sequencing, but is also currently limited by a 1% error rate. In some embodiments, methods with reduced sequencing error rates are used. In certain embodiments, the Safe-Sequencing System (Safe-SeqS) is used, which tags each template molecule to ensure rare variants are identified. See Kinde et al., Proceedings of the National Academy of Sciences 108(23):9530-9535(2011). Certain embodiments use ultrasensitive duplex sequencing, which independently tags and sequences each of the two strands of a DNA duplex. See Schmitt et al., Proceedings of the National Academy of Sciences 109(36):14508-14513(2012). Some embodiments use digital droplet PCR, designed with mutant-specific primers, which emulsifies DNA in thousands to millions of droplets to encapsulate a single DNA molecule. See Vogelstein and Kinzler, Proceedings of the National Academy of Sciences 96(16):2322-2326(1999) and Huggett et al., Clinical Chemistry 61(1):79-88(2014).

[0059] In some embodiments, detection of ESR1 mutations is performed with initial diagnosis. In some embodiments, detection of mutations is performed at the time of evaluating disease progression, relapse, or recurrence. In some embodiments, detection of mutations is performed at the time of disease progression. In some embodiments, detection of mutations is performed at the time of disease stability.

[0060] In some embodiments, one or more biological specimens are obtained from a patient for detection of mutations. In certain embodiments, the biological specimen is a tissue specimen. In certain embodiments, the tissue specimen is a tumor biopsy. In certain embodiments, the tissue specimen is a biopsy of a metastasis. In some other embodiments, the biological specimen is a body fluid (liquid biopsy), for example, from peripheral blood. In certain embodiments, the liquid biopsy comprises circulating tumor cells (CTCs). In certain embodiments, the liquid biopsy comprises cell-free DNA.

[0061] In certain embodiments of the method provided herein, ESR1 mutation is monitored by analyzing circulating tumor DNA (ctDNA).In some embodiments, ctDNA analysis is performed, for example, intermittently or periodically during treatment.In some of these embodiments, ctDNA is extracted from patient's blood sample.In certain embodiments, ctDNA is evaluated by digital PCR analysis of ESR1 mutation.

[0062] In some embodiments, ctDNA analysis is performed with a liquid biopsy assay as a companion diagnostic device to identify patients with breast cancer that lack or have ESR1 mutations. Exemplary liquid biopsy assays are Guardant360® CDx (2021, FDA approved panel or professional services panel [guardant360cdx.com / gene-list / ]), Guardant360 Response™ (2021, [ncbi.nlm.nih.gov / gtr / tests / 593444 / ]), and FoundationOne® Liquid CDx (2021, [assets.ctfassets.net / w98cd481qyp0 / wVEm7VtICYR0sT5C1VbU7 / fd055e0476183a6acd4eae6b583e3a00 / F1LCDx_Technical_Specs_072021.pdf]), each of which is incorporated by reference in its entirety herein. 5.1.3.Patients with oncogenic mutations other than ESR1 mutations

[0063] In one embodiment, a method of reducing (or delaying) the progression of breast cancer in a patient by administering to the patient an effective amount of lasofoxifene or a pharma- ceutical acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), wherein the breast cancer is: (i) estrogen receptor positive (ER)-positive + (ii) have at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) have oncogenic mutations in one or more genes other than the ESR1 gene. In some embodiments, the oncogenic mutations are detected in circulating tumor DNA (ctDNA) in a biological sample obtained from the patient. In some embodiments, the biological sample is blood, plasma, serum, or a bodily fluid (such as saliva, tears, semen, cervical fluid, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, amniotic fluid, or extracellular fluid). In some embodiments, the biological sample is plasma.

[0064] In certain embodiments, the patient's ER + The breast cancer is locally advanced. In certain embodiments, the patient has + The breast cancer is metastatic.

[0065] In some embodiments, at least one of the one or more genes having an oncogenic mutation is selected from HNF1A, TERT, GATA3, CDK12, MAPK3, GNAS, RAF1, RHEB, NTRK3, TP53, PIK3CA, APC, ATM, MET, CCND1, EGFR, ROS1, BRCA2, STK11, FGFR1, ARID1A, CCNE1, AR, ALK, ERBB2, KIT, CDK4, SMAD4, NOTCH1, RB1, BRAF, PTEN, AKT1, CDH1, BRCA1, MYC, CDKN2A, IDH2, MTOR, or PDGFRA. In some embodiments, each of the one or more genes having an oncogenic mutation is selected from HNF1A, TERT, GATA3, CDK12, MAPK3, GNAS, RAF1, RHEB, NTRK3, TP53, PIK3CA, APC, ATM, MET, CCND1, EGFR, ROS1, BRCA2, STK11, FGFR1, ARID1A, CCNE1, AR, ALK, ERBB2, KIT, CDK4, SMAD4, NOTCH1, RB1, BRAF, PTEN, AKT1, CDH1, BRCA1, MYC, CDKN2A, IDH2, MTOR, or PDGFRA.

[0066] In certain embodiments, treatment with lasofoxifene in combination with CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib) in patients with at least one or more oncogenic mutations provides a median progression-free survival (mPFS) of at least 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 ​​weeks, 52 weeks, 56 weeks, 60 weeks, 64 weeks, 68 weeks, 72 weeks, 76 weeks, 80 weeks, 84 weeks, 88 weeks, 92 weeks, 96 weeks, 100 weeks, or more. In some embodiments, treatment with lasofoxifene in combination with abemaciclib provides patients with an mPFS of at least 24 weeks. In some embodiments, an individual patient has one or more genes with oncogenic mutations detected in the patient's ctDNA or cancer, and the one or more genes are selected from HNF1A, TERT, GATA3, CDK12, MAPK3, TP53, PIK3CA, APC, ATM, MET, CCND1, EGFR, ROS1, STK11, FGFR1, ARID1A, CCNE1, AR, ALK, ERBB2, KIT, CDK4, SMAD4, NOTCH1, RB1, BRAF, RAF1, PTEN, AKT1, CDH1, BRCA1, MYC, CDKN2A, or PDGFRA.

[0067] In certain embodiments, treatment with lasofoxifene in combination with CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib) in patients with at least one or more oncogenic mutations results in clinical benefit (CB; defined as stable disease for 24 weeks or more, or a confirmed partial or complete response) in at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the patient population. In some embodiments, treatment with lasofoxifene in combination with abemaciclib results in CB (defined as stable disease for 24 weeks or more, or a confirmed partial or complete response) in about 90%, about 95%, or about 100% of the patient population. In some embodiments, an individual patient has one or more genes with oncogenic mutations detected in the patient's ctDNA or cancer, and the one or more genes are selected from CCND1, FGFR1, CCNE1, AR, ALK, MAPK3, KIT, SMAD4, NOTCH1, RB1, BRAF, RAF1, PTEN, AKT1, CDH1, BRCA1, MYC, CDKN2A, or PDGFRA.

[0068] In some embodiments, an individual patient has one or more genes with oncogenic mutations detected in the patient's ctDNA or cancer, and the one or more genes are selected from TP53, PIK3CA, CCND1, ARID1A, FGFR1, CCNE1, and ERBB2, and combinations thereof. In some embodiments, one or more oncogenic mutations of each of the genes TP53, PIK3CA, CCND1, ARID1A, FGFR1, CCNE1, and ERBB2 are detected in the individual patient's ctDNA or cancer. In some embodiments, the oncogenic mutations are selected from single nucleotide variants (SNVs), insertions and deletions (indels), copy number variations (CNVs) (focal, aneuploidy, amplification), fusions, and combinations thereof.

[0069] In some embodiments, the one or more genes with oncogenic mutations have a prevalence of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a patient population that responds positively (e.g., achieves stable disease for 24 weeks or more, or CB defined as a confirmed partial or complete response) to treatment with lasofoxifene in combination with a CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib). In some embodiments, the one or more genes with oncogenic mutations have a prevalence of at least 17% in a patient population that responds positively (e.g., achieves CB) to treatment with lasofoxifene in combination with CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib). In some embodiments, an individual patient has one or more genes with oncogenic mutations detected in the patient's ctDNA or cancer, and the one or more genes are selected from HNF1A, TERT, GATA3, CDK12, MAPK3, TP53, PIK3CA, APC, ATM, MET, CCND1, EGFR, ROS1, BRCA2, STK11, FGFR1, ARID1A, CCNE1, AR, ALK, ERBB2, KIT, BRAF, or CDK4.

[0070] In some embodiments, one or more genes with oncogenic mutations are detected in the patient's ctDNA or cancer, and the one or more genes are selected from HNF1A, TERT, GATA3, CDK12, MAPK3, GNAS, RAF1, RHEB, or NTRK3. In some embodiments, the ctDNA or cancer does not have oncogenic mutations in one or more genes selected from GNAS, RHEB, NTRK3, IDH2, or mTOR. In some embodiments, the ctDNA or cancer does not have oncogenic mutations in IDH2 or mTOR.

[0071] In one embodiment, a method of reducing (or delaying) the progression of breast cancer in a patient by administering to the patient an effective amount of lasofoxifene or a pharma- ceutical acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), wherein the breast cancer is: (i) estrogen receptor positive (ER)-positive + (ii) have at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) have increased expression of one or more genes other than the ESR1 gene compared to expression in non-cancerous breast cells in the subject or expression levels in subjects without breast cancer. In certain embodiments, the patient's ER + The breast cancer is locally advanced. In certain embodiments, the patient has +In some embodiments, the breast cancer is metastatic. At least one of the one or more genes with increased expression levels detected in the patient's ctDNA or cancer is selected from the group consisting of ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4, FG FR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, HRAS, IDH1, IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4, MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2 , NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2, or VHL.In some embodiments, each of the one or more genes with increased expression is selected from the group consisting of ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FGF4, FGFR5, FGFR6, FGFR7, FGFR8, FGFR9, FGFR10, FGFR11, FGFR12, FGFR13, FGFR14, FGFR15, FGFR16, FGFR17, FGFR18, FGFR19, FGFR20, FGFR21, FGFR22, FGFR23, FGFR24, FGFR25, FGFR26, FGFR27, FGFR28, FGFR29 ... LT3, FRS2, HIF1A, HRAS, IDH1, IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4, MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2, NKX2-1, selected from NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2, or VHL. In some embodiments, the one or more genes with increased expression are detected in the patient's ctDNA or the cancer, and the one or more genes are selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1, or mTOR.

[0072] In one embodiment, disclosed herein is a method of monitoring a patient during breast cancer treatment by: (a) determining a quantitative measure of mutant allele frequency (MAF) in circulating tumor DNA (ctDNA) (ESR1 ctDNA) of at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene in a biological sample of the patient, said quantitative measure being performed at determined intervals over a period of time; and (b) determining a positive predictive value (PPV) for clinical benefit (CB defined as stable disease for 24 weeks or more, or confirmed partial or complete response), wherein said PPV indicates responsiveness to a cancer treatment, said cancer treatment comprising an effective amount of lasofoxifene or a pharmacologic acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i). In some embodiments, the method further comprises determining that the patient has at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene. In certain embodiments, the at least one gain-of-function missense mutation is selected from D538, Y537, L536, P535, V534, L469, S463, V392, and E380. In some embodiments, the gain-of-function mutation is D538G, Y537N, or Y537S. In some embodiments, the patient has a reduced MAF after treatment when determined at 4 weeks. In some embodiments, the patient has a reduced MAF after treatment when determined at 24 weeks.

[0073] In some embodiments, the patient is a postmenopausal female. In some embodiments, the patient is a premenopausal female. In some embodiments, the patient has osteoporosis or is at higher risk for osteoporosis.

[0074] In various embodiments, patients respond to treatment with lasofoxifene in combination with a CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib) and obtain clinical benefit with stable disease for at least 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 ​​weeks, 52 weeks, 56 weeks, 60 weeks, 64 weeks, 68 weeks, 72 weeks, 74 weeks, 78 weeks, 82 weeks, 86 weeks, 88 weeks, or more (CB defined as stable disease for 24 weeks or more, or confirmed partial or complete response). In various embodiments, patients respond to treatment with lasofoxifene in combination with a CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib) and obtain a mean stable disease duration that is at least about 20%, 15%, or 10% longer than that during the preceding second-line or third-line treatment. In some embodiments, the patient has undergone one or more prior treatments with CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib). In some embodiments, the breast cancer has progressed on one or more prior endocrine therapies selected from a selective ER degrader (SERD), a selective ER modulator (SERM), optionally a SERM other than lasofoxifene, an aromatase inhibitor (AI), an mTOR inhibitor, and / or a PI3K inhibitor. In some embodiments, the breast cancer has progressed on prior treatment with fulvestrant. In some embodiments, the breast cancer has progressed on prior treatment with sirolimus, temsirolimus, everolimus, or ridaforolimus. In some embodiments, the breast cancer has progressed on prior treatment with everolimus. In some embodiments, the breast cancer has progressed on prior treatment with an mTOR inhibitor and a CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib). In some embodiments, the breast cancer has progressed upon prior treatment with everolimus and palbociclib. 5.1.4. Previous treatment 5.1.4.1 Previous endocrine treatment

[0075] In various embodiments of the methods provided herein, the patient has been previously treated with one or more series of endocrine therapies. In certain embodiments, the patient's cancer has recurred or progressed after the previous treatment.

[0076] In some embodiments, the previous endocrine treatment is administration of a selective ER modulator (SERM) other than lasofoxifene.In various embodiments, the SERM is selected from tamoxifen, raloxifene, toremifene, ospemifene, broparestrol, bazedoxifene, and ormeloxifene.In certain embodiments, the previous endocrine treatment is administration of tamoxifen.

[0077] In some embodiments, the previous endocrine treatment is the administration of a selective ER degrader (SERD). In some embodiments, the selective ER degrader is selected from fulvestrant, elacestrant (RAD1901), ARN-810 (GDC-0810), gildedestrant (GDC-9545), amsenestrant (SAR439859), lindestrant (G1T48), LSZ102, imrunestrant (LY3484356), zN-c5, D-0502, SHR9549, camizestrant (AZD9833), and AZD9496. In certain embodiments, the previous endocrine treatment is the administration of fulvestrant.

[0078] In some embodiments, the prior endocrine treatment is administration of an aromatase inhibitor (AI). In some embodiments, the aromatase inhibitor is selected from exemestane (Aromasin®), letrozole (Femara®), and anastrozole (Arimidex®).

[0079] In some embodiments, the prior endocrine treatment is ovarian suppression. In various embodiments, ovarian suppression is achieved by oophorectomy or administration of a GnRH antagonist.

[0080] In some embodiments, the patient's cancer has recurred or progressed after tamoxifen treatment. In some embodiments, the patient's cancer has recurred or progressed after fulvestrant treatment. In some embodiments, the patient's cancer has recurred or progressed after aromatase inhibitor (AI) treatment. In some embodiments, the patient's cancer has recurred or progressed after AI treatment in combination with CDK4 / 6i. In certain embodiments, the patient's cancer has recurred or progressed after AI treatment in combination with palbociclib or ribociclib. In some of these embodiments, the patient's cancer has recurred or progressed after multiple courses of endocrine therapy. 5.1.4.2 Previous endocrine treatment for metastatic disease

[0081] In certain embodiments, the patient's ER + The breast cancer is metastatic and has progressed during treatment for first (1L), second (2L), or third (3L) line metastatic disease. In some embodiments, the metastatic disease is regional or lymph node metastasis, or visceral (e.g., lung, pleural effusion, liver, ascites, CNS) metastasis. In some embodiments, the patient's cancer has recurred or progressed after one or more second or third line (3L) cancer therapies, as shown in FIG. 4. In some embodiments, the patient's cancer has recurred or progressed after treatment of the metastatic disease with at least one CDK4 / 6 inhibitor, and at least one of endocrine therapy (mammalian target of rapamycin (mTOR) inhibitor, phosphatidylinositol-3-kinase (PI3K) inhibitor, heat shock protein 90 (HSP90) inhibitor, poly(ADP-ribose)polymerase (PARP) inhibitor, AKT inhibitor, or mammalian target of histone deacetylase (HDAC) inhibitor). In some embodiments, the patient's metastatic breast cancer has recurred or progressed after treatment with at least one of the following, alone or in combination: tamoxifen, fulvestrant, capecitabine, everolimus, alpelisib, talazoparib, palbociclib, ribociclib, or abemaciclib.

[0082] In certain embodiments, the metastatic cancer has progressed on one or two previous treatments for metastatic breast cancer: aromatase inhibitor (AI) and / or fulvestrant, either as monotherapy or in combination with any commercially approved CDK4 / 6 inhibitor (CDKi); and / or a combination of fulvestrant and alpelisib; and / or tamoxifen; and / or a combination of exemestane / everolimus. In some embodiments, the previously administered CDK4 / 6 inhibitor is abemaciclib, ribociclib, or palbociclib. In some embodiments, the metastatic cancer has progressed on at least one previous treatment with abemaciclib, palbociclib, or ribociclib. In some embodiments, the metastatic cancer has progressed on a previous abemaciclib treatment. In some embodiments, the metastatic cancer has progressed on a previous palbociclib treatment. In some embodiments, the metastatic cancer has progressed on a previous ribociclib treatment. In some embodiments, the cancer has metastasized to the viscera.

[0083] In certain embodiments, the metastatic cancer has progressed on a non-steroidal aromatase inhibitor (AI); a SERD (e.g., fulvestrant); an AI in combination with a CDK4 / 6 inhibitor; or a SERD in combination with a CDK4 / 6 inhibitor (e.g., fulvestrant).

[0084] In certain embodiments, progression occurred on a CDK4 / 6 inhibitor, either as monotherapy or in combination with therapy. 5.2. Pharmaceutical Compositions

[0085] The estrogen receptor positive (ER) + ) A method for treating breast cancer comprises administering to a patient an effective amount of lasofoxifene or a pharma- ceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i).

[0086] In some embodiments, lasofoxifene is administered as lasofoxifene tartrate.

[0087] In some embodiments, the CDK4 / 6i is selected from palbociclib, abemaciclib, and ribociclib. In certain embodiments, the CDK4 / 6i is abemaciclib.

[0088] The term "pharmaceutically acceptable salt" refers to a non-toxic pharmaceutically acceptable salt. However, other salts known to those skilled in the art may be used. Representative organic or inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, hydroxyethanesulfonic acid, benzenesulfonic acid, oxalic acid, pamoic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, salicylic acid, saccharic acid, or trifluoroacetic acid. Representative organic or inorganic bases include, but are not limited to, basic or cationic salts such as benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0089] The examples also include prodrugs of the active compounds disclosed herein. In general, such prodrugs are functional derivatives of the compounds that are readily convertible in vivo to the required compound. Thus, in the treatment methods of the present invention, the term "administering" is intended to encompass treatment of the various disorders described with a specifically disclosed compound or with a compound that may not be specifically disclosed, but that is converted to the specified compound in vivo after administration to a subject. Conventional procedures for the selection and preparation of stable prodrug derivatives are described, for example, in "Design of Prodrugs", H. Bundgaard, Elsevier, 1985.

[0090] Some of the crystalline forms of the compounds may exist as polymorphs and, as such, are intended to be included in the present invention. Additionally, some of the solid forms of the active compounds may exist as solvates, for example with water (i.e., hydrates) or common organic solvents, and such solvates are included in embodiments of the present invention.

[0091] When the preparation process of the active compound administered in the methods provided herein produces a mixture of stereoisomers, in some embodiments, these isomers are separated by conventional techniques such as preparative chromatography. In some embodiments, the compound is prepared in racemic form, or as individual enantiomers or diastereomers, either by stereospecific synthesis or resolution. In some embodiments, the compound is resolved into its component enantiomers or diastereomers by standard techniques, such as forming a stereoisomeric pair by salt formation with an optically active base, followed by fractional crystallization and regeneration of the free acid. In some embodiments, the compound is resolved by forming an ester or amide of the stereoisomer, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compound is resolved using a chiral HPLC column. It should be understood that compositions containing all of its stereoisomers, racemic mixtures, diastereomers, cis-trans isomers, and enantiomers are encompassed by the embodiments herein.

[0092] In certain embodiments, active compounds are formulated into separate pharmaceutical compositions.In addition to active compounds, pharmaceutical preparations or compositions further comprise one or more of pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with the effectiveness of active ingredients.The exact nature of carriers or other materials depends on the route of administration (e.g., oral, intravenous, transdermal, vaginal topical, or vaginal ring).

[0093] In certain embodiments, pharmaceutical compositions for oral administration are in the form of tablets, capsules, powders, or liquids.In certain embodiments, tablets contain solid carriers such as gelatin or adjuvants.In certain embodiments, liquid pharmaceutical compositions contain liquid carriers such as water, petroleum, animal oils, vegetable oils, mineral oils, or synthetic oils.Saline, dextrose, or other saccharide solutions or glycols (such as ethylene glycol, propylene glycol, or polyethylene glycol) can also be included.

[0094] For parenteral administration, the composition is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability.Those skilled in the art can fully prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as necessary. 5.3. Treatment regimen

[0095] In the methods described herein, the terms "treating," "treatment," and grammatical variations thereof are used in the broadest sense understood in the clinical art. Thus, these terms do not require a cure or complete remission of the disease, but encompass obtaining any clinically desirable pharmacological and / or physiological effect. In certain embodiments, the effect is a partial or complete response of breast cancer, a delay or inhibition of the progression of the cancer; or a regression of the cancer.

[0096] The term "effective amount" as used herein with respect to combination therapy means individual dosages of each of the lasofoxifene and CDK4 / 6i that are administered in combination to produce the desired effect. 5.3.1. Combination Administration

[0097] In some embodiments of the provided methods, lasofoxifene or a pharma- ceutically acceptable salt thereof and CDK4 / 6i (e.g., abemaciclib, palbociclib, ribociclib) are administered as separate dosage forms. In certain of these embodiments, lasofoxifene or a salt thereof and CDK4 / 6i are administered separately at the same time (simultaneously). In some other embodiments, lasofoxifene or a salt thereof and CDK4 / 6i are administered as separate dosage forms at separate times (e.g., sequentially or on an unrelated schedule).

[0098] In certain embodiments, lasofoxifene is administered in a single dosage form that includes both lasofoxifene or a salt thereof and a CDK4 / 6i. In certain embodiments, the CDK4 / 6i is abemaciclib. 5.3.1.1 Lasofoxifene Administration

[0099] In various embodiments of the above methods of treatment, lasofoxifene or a pharma- ceutically acceptable salt thereof is administered by oral administration.

[0100] In some embodiments, lasofoxifene or a pharma- ceutically acceptable salt thereof is administered orally (per os; po) at a dose of about 0.5 mg / day per os to about 10 mg / day per os, e.g., about 0.5 mg / day per os to about 5 mg / day per os, about 1 mg / day per os to about 5 mg / day per os, about 2 mg / day per os to about 5 mg / day per os, about 3 mg / day per os to about 5 mg / day per os, about 4 mg / day per os to about 5 mg / day per os, about 0.5 mg / day per os to about 4 mg / day per os, about 1 mg / day per os to about 4 mg / day per os, about 2 mg / day per os to about 4 mg / day per os, about 3 mg / day per os to about 4 mg / day per os, about 0.5 mg / day per os to about 3 mg / day per os, about 1 mg / day per os The patient is administered lasofoxifene dosages of about 0.5 mg / day per os to about 2 mg / day per os, about 1 mg / day per os to about 2 mg / day per os, or about 0.5 mg / day per os to about 1 mg / day per os.

[0101] In some embodiments, lasofoxifene or a pharma- ceutically acceptable salt thereof is administered at about 0.5 mg lasofoxifene / day per os. In some embodiments, lasofoxifene or a pharma- ceutically acceptable salt thereof is administered at about 1 mg lasofoxifene / day per os. In some embodiments, about 1.5 mg lasofoxifene / day per os, about 2 mg lasofoxifene / day per os, 2.5 mg lasofoxifene / day per os, about 3 mg lasofoxifene / day per os, about 3.5 mg lasofoxifene / day per os, about 4 mg lasofoxifene / day per os, about 4.5 mg lasofoxifene / day per os, about 5 mg lasofoxifene / day per os, about 6 mg lasofoxifene / day per os, about 7 mg lasofoxifene / day per os, about 8 mg lasofoxifene / day per os, about 9 mg lasofoxifene / day per os, or about 10 mg lasofoxifene / day per os of lasofoxifene or a pharma- ceutically acceptable salt thereof. In some other embodiments, the lasofoxifene or a pharma- ceutically acceptable salt thereof is administered at a dose of greater than 10 mg lasofoxifene / day per os.

[0102] In some embodiments, lasofoxifene or a pharma- ceutically acceptable salt thereof is administered at 0.5 mg / day lasofoxifene to 10 mg / day. In some embodiments, lasofoxifene or a pharma- ceutically acceptable salt thereof is administered at 0.5 mg lasofoxifene / day, 1 mg lasofoxifene / day, 1.5 mg lasofoxifene / day, 2 mg lasofoxifene / day, 2.5 mg lasofoxifene / day, 3 mg lasofoxifene / day, 3.5 mg lasofoxifene / day, 4 mg lasofoxifene / day, 5 mg lasofoxifene / day, 5.5 mg lasofoxifene / day, 6 mg lasofoxifene / day, 6.5 mg lasofoxifene / day, 7 mg lasofoxifene / day, 7.5 mg lasofoxifene / day, 8 mg lasofoxifene / day, 8.5 mg lasofoxifene / day, 9 mg lasofoxifene / day, 9.5 mg lasofoxifene / day, or 10 mg lasofoxifene / day. In a currently preferred embodiment, lasofoxifene or a pharma- ceutically acceptable salt thereof is administered orally at 5 mg lasofoxifene / day.In a particular embodiment, lasofoxifene tartrate is administered orally at 5 mg lasofoxifene / day.

[0103] In certain embodiments, lasofoxifene is administered once every day. In certain embodiments, lasofoxifene is administered once every 2 days. In certain embodiments, lasofoxifene is administered once every 3 days. In certain embodiments, lasofoxifene is administered once every 4 days. In certain embodiments, lasofoxifene is administered once every 5 days. In certain embodiments, lasofoxifene is administered once every 6 days. In certain embodiments, lasofoxifene is administered once every week. In certain embodiments, lasofoxifene is administered once every 2 weeks. In certain embodiments, lasofoxifene is administered once every 3 weeks. In certain embodiments, lasofoxifene is administered once every month.

[0104] In some embodiments, lasofoxifene is administered to the patient by intravaginal ring administration. In some of these embodiments, lasofoxifene is administered once every two weeks. In some of these embodiments, lasofoxifene is administered once every three weeks. In some of these embodiments, lasofoxifene is administered once every month. In some of these embodiments, lasofoxifene is administered once every two months. In some of these embodiments, lasofoxifene is administered once every three months. In some of these embodiments, lasofoxifene is administered once every four months.

[0105] In some embodiments, lasofoxifene is administered to the patient in an ER-positive state until the patient's cancer progresses during treatment, goes into complete remission, or becomes intolerant to the side effects. + It is administered to breast cancer patients. 5.3.1.2 Abemaciclib Administration

[0106] In each of the above treatment methods, the patient is administered lasofoxifene (e.g., lasofoxifene tartrate) in combination with a CDK4 / 6 inhibitor. In an exemplary embodiment, the CDK4 / 6 inhibitor is selected from palbociclib, ribociclib, and abemaciclib. In a currently preferred embodiment, the patient is orally administered lasofoxifene tartrate at 5 mg lasofoxifene / day and abemaciclib at 50 mg-200 mg BID, 100 mg-200 mg BID, or 150 mg BID.

[0107] In various embodiments, abemaciclib is administered orally.

[0108] In some embodiments, abemaciclib is administered at about 25 mg / day per os to about 600 mg / day per os, e.g., about 50 mg / day per os to about 200 mg / day per os, e.g., about 25 mg / day per os, about 50 mg / day per os, about 100 mg / day per os, about 150 mg / day per os, about 200 mg / day per os, about 250 mg / day per os, about 300 mg / day per os, about 350 mg / day per os, about 400 mg / day per os, about 450 mg / day per os, or about 600 mg / day per os.

[0109] In some embodiments, abemaciclib is administered once daily. In certain embodiments, abemaciclib is administered twice daily.

[0110] In certain embodiments, abemaciclib is administered twice daily (BID).In typical embodiments, the daily dose is administered in two equally divided doses.In some embodiments, abemaciclib 300mg / day per os is orally administered in two separate 150mg doses (e.g., one 150mg tablet in the morning and one 150mg tablet in the evening).

[0111] In some embodiments, abemaciclib is administered at a starting dose, and then adjusted downward according to a first dose reduction schedule. In certain embodiments, the dose of abemaciclib is then reduced according to a second dose reduction schedule. In certain embodiments, the dose of abemaciclib is then reduced according to a third dose reduction schedule. In some of these embodiments, each dose reduction is 50 mg per dose. The dose of 300 mg / day (150 mg per os administered twice daily) is reduced according to a first dose reduction schedule, so that the dose of 200 mg / day is administered twice daily (100 mg per os administered twice daily). 5.4. Clinical Endpoints

[0112] In various embodiments, the method comprises administering a combination of lasofoxifene and a CDK4 / 6i, wherein the combination is administered to an ER patient having at least one ESR1 mutation. + In certain embodiments, the method is effective in treating breast cancer. + It is effective in reducing the progression of breast cancer.

[0113] In certain embodiments, the combination is + In certain embodiments, the combination is effective in increasing disease-free survival in breast cancer patients. + In certain embodiments, the combination is effective in reducing the regression of breast cancer. + In certain embodiments, the combination is effective in increasing the time to regression of breast cancer. + In certain embodiments, the combination is effective in reducing metastasis of breast cancer. + It is effective in increasing the duration of progression-free survival in breast cancer patients. In certain embodiments, the comparison to determine efficacy is made against a standard of care.

[0114] In some embodiments, the methods provided herein involve treating an ER patient with one or more of the ESR1 mutations discussed herein. + In some embodiments, the method includes increasing disease-free survival, decreasing relapse, increasing time to relapse, decreasing metastasis, and / or increasing duration of progression-free survival in patients with locally advanced or metastatic breast cancer. + Reduces selective pressure in locally advanced or metastatic breast cancer and prevents the expansion of endocrine-resistant clones.

[0115] In some embodiments, the patient is administered lasofoxifene or a salt thereof and abemaciclib until the patient's cancer goes into complete remission, progresses during treatment, or becomes intolerant to side effects.

[0116] In some embodiments, as shown in Figures 1 and 3 and Examples 2 and 3, lasofoxifene or a pharma- ceutically acceptable salt thereof and a CDK4 / 6 inhibitor (e.g., abemaciclib, ribociclib, palbociclib) are administered at a dose and for a duration effective to obtain clinical benefit (CB defined as stable disease for 24 weeks or more, or confirmed partial or complete response) in patients whose cancer has recurred or progressed after one or more 2L or 3L cancer therapies. In some embodiments, lasofoxifene and a CDK4 / 6 inhibitor (e.g., abemaciclib, ribociclib, palbociclib) are administered at a dose and for a duration effective to provide a longer duration of response than any prior 2L or 3L therapy. In some embodiments, lasofoxifene and a CDK4 / 6 inhibitors (e.g., lasofoxifene and abemaciclib) confer CB with stable disease for at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 52 weeks, at least 56 weeks, at least 60 weeks, at least 64 weeks, at least 68 weeks, at least 72 weeks, at least 76 weeks, at least 80 weeks, at least 84 weeks, at least 88 weeks, or more. In some embodiments, lasofoxifene + abemaciclib treatment confer stable disease for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more. In some embodiments, lasofoxifene and CDK4 / 6 inhibitor (e.g., lasofoxifene / abemaciclib) treatment confers CB (defined as stable disease for 24 weeks or more, or a confirmed partial or complete response) for at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 11 years, at least 12 years, or more.In some embodiments, lasofoxifene and CDK4 / 6 inhibitor (e.g., lasofoxifene / abemaciclib) treatment confers CB with complete or partial responses in patients with significant tumor reduction as shown in FIG. 2. EXAMPLES

[0117] 6. Working Example 6.1. Example 1: Phase 2 Clinical Study (ELAINE2) Locally advanced or metastatic estrogen receptor-positive (ER) tumors with ESR1 mutations + ) / Human epidermal growth factor 2 negative (HER2 - )(ER + / HER2 - ) A phase 2 clinical study, NCT04432454 (ELAINE2), is currently underway to evaluate the efficacy, safety, and tolerability of the combination of lasofoxifene and abemaciclib for the treatment of postmenopausal and certain premenopausal women with breast cancer. As discussed in Section 5.1.1 below, the study is enrolling patients whose tumors had progressed on prior CDK4 / 6 inhibitor treatment. 6.1.1. Summary of Experimental Observations

[0118] ELAINE2 is an open-label, phase 2, multicenter study evaluating the safety and efficacy of LAS in combination with abemaciclib (Abema), a CDK4 / 6i. Study participants had ER disease with acquired ESR1 mutations (identified by ctDNA testing). + / HER2 - Pre- and postmenopausal women with mBC (metastatic breast cancer) whose disease had progressed on 1 or 2 lines of hormonal therapy for metastatic disease with or without CDK4 / 6i (including Abema). Patients received oral lasofoxifene (LAS) 5 mg / day and abemaciclib 150 mg BID. Treatment continued until evidence of disease progression, death, unacceptable toxicity, or withdrawal from the study. The primary endpoint was safety, and secondary endpoints were progression-free survival (PFS), objective response rate (ORR), and clinical benefit rate (CBR).

[0119] Twenty-nine (29) patients were enrolled at 16 sites in the United States (October 2020-June 2021). Mean age was 58.3 years (range 35-79); 86% were white. Most had progressed on at least two prior hormonal treatments (80%). All but one patient had received a prior CDK4 / 6i, 72% had received prior fulvestrant (FVT); 48% had received chemotherapy in the metastatic setting. Four patients discontinued the study due to adverse events (AEs, n=2), consent withdrawal (n=1), or investigator-initiated withdrawal (n=1). There were no deaths during the study, and grade 3 / 4 AEs were rare. The most common AEs were diarrhea, nausea, and leukopenia. The abemaciclib dose was reduced from 150 mg to 100 mg BID in five patients. As of the first interim date, 11 patients had progressed and 14 remained on treatment.At the time of censoring, median PFS was 13.9 months (95% CI, 8.0-NE), ORR was 33.3% (95% CI, 16.3-56.3) (six confirmed partial responses), and CBR was 62.1% (95% CI, 44.0-77.3).

[0120] As shown in Figures 1 - 4 , patients obtained clinical benefit (CB defined as stable disease for ≥24 weeks or confirmed partial or complete response) from lasofoxifene in combination with abemaciclib, even after progression on prior cyclin-dependent kinase 4 / 6 inhibitor (CDK4 / 6i) treatment (including prior treatment with ribociclib, palbociclib, and / or abemaciclib).

[0121] Conclusions: Lasofoxifene in combination with abemaciclib was well tolerated and improved ER progression on prior CDK4 / 6i therapy. + / HER2 - Robust and meaningful efficacy was demonstrated in women with metastatic breast cancer (mBC) and ESR1 mutations. Study design

[0122] This is in patients with locally advanced or metastatic ER who harbor ESR1 mutations and have disease progression on first-, second-, or third-line hormonal treatment for metastatic disease. + / HER2 - This is an open-label, multicenter study evaluating the efficacy, safety, and tolerability of the combination of lasofoxifene and abemaciclib for the treatment of premenopausal and postmenopausal women with breast cancer.To be eligible for enrollment, progression occurred on ≤3 of the following treatments for metastatic breast cancer: AI and / or fulvestrant (either as monotherapy or in combination with any commercially available CDK4 / 6i); and / or the combination of fulvestrant and alpelisib; and / or tamoxifen; and / or the combination of exemestane / everolimus.

[0123] On the day of enrollment (Day 1), subjects received 5 mg oral lasofoxifene once daily and 150 mg abemaciclib twice daily. Study medication will continue until breast cancer progression is documented or subjects withdraw from the study for any reason.

[0124] Treatment will continue until disease progression, death, radiological or clinical evidence of unacceptable toxicity, or withdrawal from the study for any reason. Enrolled subjects will be seen every 2 weeks for the first 2 months of treatment, then monthly until progression. Efficacy assessments will be performed every 8 weeks. Safety assessments will be performed at weeks 2, 4, 6, and 8 after enrollment, then monthly until disease progression. 6.1.3. Drug Schedules

[0125] Subjects will receive oral lasofoxifene 5 mg once daily (1 tablet) and oral abemaciclib 150 mg twice daily (3 tablets) with or without food. Study medication will continue until breast cancer progression is documented or subjects withdraw from the study for any reason.

[0126] Lasofoxifene

[0127] The active pharmaceutical ingredient is lasofoxifene. Its chemical name is 6S-phenyl-5R-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalen-ol, 2S,3S-dihydroxy-succinic acid. Chemical structure: [ka] Molecular formula:C 28 H 31 NO 2 C 4 H 6 O 6 Molecular weight: 563.64 Daltons Appearance: White to off-white solid Lasofoxifene is supplied as a white to off-white solid 5 mg tablet using the D-(-)-tartrate salt.

[0128] Abemaciclib

[0129] Abemaciclib is a kinase inhibitor with the chemical name 2-pyrimidinamine, N-[5-[(4-ethyl-1-piperazinyl)methyl]-2-pyridinyl]-5-fluoro-4-[4-fluoro-2-methyl-1-(1-methylethyl)-1H-benzimidazol-6-yl. Molecular formula: C27H32F2N8 Molecular weight: 506.59 Daltons Appearance: White to yellow powder 6.1.3.1 Dose Modifications for Treatment Toxicity

[0130] Lasofoxifene

[0131] There will be no dose reductions due to any lasofoxifene-related toxicity. If a grade 3 or 4 AE event occurs related to lasofoxifene, treatment will be withheld until toxicity is less than grade 2 or has returned to baseline, and lasofoxifene will be resumed at the dose specified. Should a patient require NPO, lasofoxifene and abemaciclib will be withheld until the subject is able to take fluids by mouth. If a subject is unable to tolerate lasofoxifene or has not taken lasofoxifene for 3 consecutive weeks, the subject should be withdrawn from the study.

[0132] Appearance: White to yellow powder

[0133] Abemaciclib

[0134] Two abemaciclib dose modifications for adverse reactions are permitted: Starting dose: 150 mg twice daily (3 tablets twice daily) First dose reduction - 100mg twice daily (2 tablets twice daily) Second dose reduction - 50 mg twice daily (1 tablet twice daily) Third dose reduction – not possible If a subject is unable to tolerate abemaciclib 50 mg twice daily, this treatment should be discontinued; however, lasofoxifene may be continued until disease progression is documented. However, if lasofoxifene is discontinued, abemaciclib cannot be continued as monotherapy and the subject will need to be withdrawn from the study. Dose modifications for treatment toxicities are summarized in Table 1 below. [Table 1-1] [Table 1-2] 6.1.4. Primary and secondary endpoints

[0135] The primary endpoint was locally advanced or metastatic ER, based on the number of adverse events, severity of adverse events, and mortality due to adverse events. + / HER2 - Evaluation of the safety and tolerability of the combination of lasofoxifene and abemaciclib for the treatment of postmenopausal women with breast cancer and ESR1 mutations. The pharmacokinetic endpoint is to determine whether there are any drug-drug interactions between lasofoxifene and abemaciclib when compared to steady-state drug concentrations obtained in previous clinical trials.

[0136] Secondary endpoints include: progression-free survival (PFS); clinical benefit (CB defined as stable disease for ≥24 weeks or confirmed partial or complete response) rate (CBR), duration of response, objective response rate (ORR), quality of life (QoL), and time to response. For subjects with measurable disease at baseline, progression will be determined according to RECIST criteria. 6.1.5. Inclusion and Exclusion Criteria

[0137] Inclusion criteria included: 1. Pre- or post-menopausal. Post-menopausal women are defined as: a. 60 years of age or older and have had no vaginal bleeding in the past year, or b. Under age 60, with "premature menopause" or "premature ovarian failure" with secondary amenorrhea evident for at least 1 year and follicle-stimulating hormone (FSH) and estradiol levels in the postmenopausal range according to institutional standards, or c. Surgical menopause by bilateral oophorectomy. NOTE: Premenopausal women who meet all other inclusion criteria must maintain ovarian suppression (e.g., Lupron) for the duration of the study and subjects must be instructed to use adequate contraception to prevent pregnancy. 2. If possible, biopsy metastatic breast cancer tissue with histologic or cytologic confirmation of ER+ and HER2- disease assessed by a local laboratory using slides, paraffin blocks, or paraffin samples according to American Society of Clinical Oncology / College of American Pathologists guidelines. If biopsy is not possible, ER and HER2 status from tissue obtained at the time of initial diagnosis must confirm that the subject is ER+ and HER2-. 3. Locally advanced or metastatic breast cancer with evidence of radiological or clinical progression on first-line and / or second-line hormonal therapy for metastatic disease. May have progressed on no more than two of the following endocrine treatments for metastatic breast cancer: aromatase inhibitors (AI) and / or fulvestrant, either as monotherapy or in combination with any commercially approved CDK4 / 6i; and / or fulvestrant in combination with alpelisib; and / or tamoxifen; and / or exemestane / everolimus combination. (Note: Subjects should have stopped any CDK4 / 6i for at least 21 days prior to initiation of study treatment.) 4. Subjects must have no evidence of progression for at least 6 months during their first hormonal treatment for advanced breast cancer. 5. At least one or more of the following ESR1 point mutations as assessed in cell-free circulating tumor DNA (ctDNA) from blood or tissue samples: Y537S, Y537C, D538G, E380Q, S463P, V534E, P535H, L536H, L536P, L536R, L536Q, or Y537N. Note: Sponsor's blood ctDNA assay must be used, however tissue sequencing (if performed) may be performed with any validated commercially available assay. Note: A positive ESR1 mutation in tissue or ctDNA using a validated commercially available assay may be accepted as fulfilling this entry criterion if performed prior to or at the time of disease progression; however, blood must still be collected for genomic analysis using the sponsor's ctDNA assay. 6.Locally advanced or metastatic breast cancer with either measurable (per RECIST 1.1 [Eisenhauer et al. New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). European Journal of Cancer. 2009; 45: 228-47]) or non-measurable disease. 7. Subjects who may have received one cytotoxic chemotherapy regimen for metastatic disease and who have received one cytotoxic chemotherapy regimen in neoadjuvant or adjuvant therapy prior to study entry may be enrolled, but must be free of all acute toxicities of chemotherapy, except for alopecia and grade 2 peripheral nerve neuropathy, prior to study entry. A washout period of at least 21 days is required between the last chemotherapy dose and study entry. 8. Stable metastases of breast cancer to the brain are acceptable as long as the subject has undergone radiation therapy and has not demonstrated any evidence of progression of brain metastases for at least 3 months after completion of radiation therapy. 9.ECOG performance score of 0 or 1. 10. Adequate organ function as demonstrated by: Absolute neutrophil count (ANC) of 1,500 cells / mm 3 End B. Platelet count 100,000 cells / mm 3 End C. Hemoglobin is 8.0 g / dl or more d. ALT and AST levels below 3 upper limit of normal (ULN) or below 5 if liver metastases are present e. Total serum bilirubin ≦0.5×ULN (≦3.0×ULN for subjects known to have Gilbert's syndrome) f. Alkaline phosphatase level below 3 × ULN Creatinine clearance calculated by the Cockcroft-Gault formula is 40 ml / min or greater h. International normalized ratio (INR) and activated partial thromboplastin time (aPTT) less than 2.0 × ULN 11. I can swallow pills. 12. Able to understand and willingly sign a written informed consent prior to any screening procedures.

[0138] Exclusion Criteria. Subjects meeting any of the following criteria will be excluded from participation in the study. 1. Lymphatic carcinomatosis involving the lungs. 2. Visceral crisis requiring cytotoxic chemotherapy as assessed by the investigator. 3. Subjects have received radiation therapy within 30 days prior to study entry, except for local radiation therapy for pain relief or to lytic lesions at risk for fracture, in which case subsequent local radiation therapy may be completed within 7 days prior to study entry. Subjects must have recovered from the toxicity of radiation therapy prior to entry. 4. Subjects with known inactivating RB1 mutations or deletions (screening for RB1 mutations is not required for participation). 5. History of long QTC syndrome or QTC >480 ms. 6. History of pulmonary embolism (PE) or deep vein thrombosis (DVT) within the past 6 months, or any known thrombophilia. Subjects stable on maintenance anticoagulation are eligible, as long as the onset of DVT and / or PE occurred more than 6 months prior to enrollment and there is no evidence of active thrombosis. Use of low-dose ASA is permitted. 7. Subjects taking strong CYP3A4 inhibitors, such as clarithromycin, telithromycin, nefazodone, itraconazole, ketoconazole, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, or tipranavir. 8. Subjects receiving strong and moderate CYP3A4 inducers such as amprenavir, barbituates, carbamazepine, clotrimazole, dexamethasone, efavirenz, ethosuximide, griseofulvin, modafinil, nevirapine, oxcarbazepine, phenobarbital, phenytoin, chronic prednisone treatment, primidone, rifabutin, rifampin, rifapentine, ritonavir, and topiramate. 9. Any significant comorbidity that may affect the study or subject safety. Because CDK4 / 6i has been reported to cause interstitial lung disease (ILD), subjects with a history of ILD, severe dyspnea at rest, or who require oxygen therapy should not participate in the study. 10. Subject has an active systemic bacterial or fungal infection (requiring intravenous [IV] antibiotics at the start of treatment). 11. History of positive human immunodeficiency virus (HIV) or hepatitis B virus (HBV) test (screening is not required for enrollment). 12. Subjects who have Hepatitis C Virus (HCV) at screening and still have a viral load. Subjects who have been previously treated and have achieved cure for HCV (no viral load) may participate in the study. 13. History of malignant disease (except breast cancer) within the past 5 years, except for basal or squamous cell carcinoma of the skin cured by surgery, or early stage cervical cancer. 14. Positive pregnancy test (premenopausal only). 15. History of non-adherence to medical regimens. 16. Unwilling or unable to comply with the protocol. 17. Currently participating in any clinical research trial involving an investigational drug or investigational device within the past 30 days. Efficacy Analysis

[0139] Kaplan-Meier curves for PFS are shown with estimated median PFS. Clinical benefit rates (CBR), defined as the percentage of subjects with complete or partial response or stable disease for ≥24 weeks, are shown with 95% confidence intervals. ORR, defined as the percentage of subjects with complete or partial response, is similarly summarized. Time to response and duration of response (DoR) are shown for each responder. 6.1.7. Safety analysis

[0140] For the safety population, a narrative summary of AEs, clinical laboratory data, vital signs, and ECGs will be presented.

[0141] Verbatim descriptions of AEs reported during the study are mapped to the appropriate system organ class and preferred term using the Medical Dictionary for Clinical Trials (MedDRA). All reported AEs are tabulated and graded according to CTCAE version 5.0. AEs are summarized by worst grade and grade per subject. All treatment-emergent adverse events (TEAEs) (i.e., those occurring during or after the first dose of study drug) are summarized in frequency tables. Treatment-emergent serious AEs and TEAEs that led to early study discontinuation are listed and summarized in frequency tables. The number and proportion of subjects who prematurely discontinued either or both study treatments due to AEs are reported with 95% confidence intervals. Subjects who experienced significant adverse events of interest are similarly summarized. With 24 evaluable subjects, the width of the upper half of the 95% confidence interval for the Wilson score is estimated to be within 20%. The probability of experiencing at least one adverse event with a 7% incidence is 0.82.

[0142] All AEs leading to death are listed by subject with a narrative.

[0143] Laboratory test results are summarized using descriptive statistics for absolute values ​​and in cumulative shift tables for changes from baseline. 6.1.8. Analysis of Pharmacokinetic Variables

[0144] Pharmacokinetic sampling for lasofoxifene and abemaciclib concentrations will be performed pre-dose at every visit starting at Visit 0 (Day 1) through the Final / ET visit. Pharmacokinetic concentrations of lasofoxifene, abemaciclib, and three abemaciclib metabolites (LSN2839567, LSN3106726, and LSN3106729) will be summarized and the mean, median, SD, and range at each time point will be presented and compared to previous PK results. 6.2. Example 2: First Interim Results of the ELAINE2 Clinical Trial

[0145] A total of twenty-nine (29) patients were enrolled. The patient disposition is summarized in Table 2. The patient demographics and baseline characteristics are summarized in Table 3, and the patients' previous cancer treatments are summarized in Table 4. 6.2.1. Patient breakdown [Table 2] 6.2.2. Demographics and baseline characteristics n=29 [Table 3] 6.2.3. Previous breast cancer treatment [Table 4] * Data are expressed as n (%) unless otherwise stated. CDK4 / 6i, cyclin-dependent kinase 4 / 6 inhibitors. Twenty-six (89.7%) patients had received prior radiotherapy. 6.2.4.ELAINE2 Swimmer Plot

[0146] Patient responses to lasofoxifene and CDK4 / 6 inhibitor treatment at the first median date are summarized in Figure 1 .

[0147] Referring to Figure 1, at the first median date, approximately 68.9% (20 / 29) of patients treated with lasofoxifene and abemaciclib (laso / abema) had clinical benefit (CB) with stable disease and complete or partial response for at least 24 weeks after treatment (vertical dotted line). Of these patients, approximately 65% ​​(13 / 20) had a sustained response (arrow) at the first median date. Patients 29, 21, 16, and 6 (patients marked with an asterisk in Figure 1) had progressed on abemaciclib prior to enrollment. Of note, 75% (3 / 4) of these patients had CB and stable disease by 40 weeks (patient 16), 48 weeks (patient 21), and 68 weeks (patient 29). Patients 16 and 6 progressed and were withdrawn at 40 and 8 weeks, respectively.

[0148] Approximately 48% (14 / 29) of patients had progressed on previous, pre-enrollment fulvestrant treatment (squares). These patients generally responded well to laso / abema treatment. Approximately 64% (9 / 14) achieved CB and had a complete or partial response to laso / abema treatment for up to 62 weeks at the first median date. For example, patients 28, 27, 24, 19, and 14 had stable disease and sustained response (arrows) to laso / abema for up to 62 weeks, 58 weeks, 54 weeks, 50 weeks, 42 weeks, and 38 weeks, respectively. Patients 15 and 13 had stable disease and progressed at 36 and 32 weeks, respectively.

[0149] All patients had at least one missense mutation in ESR1 at enrollment. Approximately 69% (20 / 29) of patients had a Y537S mutation, and approximately 65% ​​(13 / 20) of these patients achieved CB and had complete or partial responses and stable disease through 62 weeks. Patients 28, 27, 25, 24, 23, 21, 20, 19, 18, 17, 11, and 10 had stable disease and durable responses through 62 weeks, 58 weeks, 54 weeks, 48 ​​weeks, 48 ​​weeks, 48 ​​weeks, 40 weeks, 40 weeks, 32 weeks, and 32 weeks, respectively. Patients 15 and 13 had stable disease and progressed at 32 weeks.

[0150] Notably, 75% (12 / 16) of patients with visceral metastases benefited from laso / abema treatment and achieved CB, even if their cancer had spread to the viscera prior to enrollment. 6.2.5.Progression-free survival

[0151] Abemaciclib / Laso combination. Table 5 summarizes the progression-free survival (PFS) of patients. [Table 5] 6.2.6. Tumor Response Waterfall Plots

[0152] Preliminary data of maximum tumor response assessed at the first interim date are shown in Figure 2. Patients with a complete or partial response to laso / abema treatment had a maximum percent change in the sum of target lesion dimensions of up to 80%. Nine (9 / 18) patients with measurable lesions had a partial response, resulting in an objective response rate (ORR) of 50% (95% confidence interval, 29.0-71.0). Table 6 summarizes the ORR of patients (ORR=50%), and Table 7 summarizes the DOR and TTR of patients. 6.2.7.ORR, DOR, and TTR

[0153] Table 6 summarizes the ORR of the patients (ORR = 50%), and Table 7 summarizes the DOR of the patients. [Table 6] [Table 7] 1 Number of subjects with measurable target lesions 2 Event date or censoring date - first PR date 3 Date of first PR - Date of randomization + 1 6.2.8.Clinical profit rate (CBR)N=29

[0154] Table 8 summarizes the clinical benefit rates (CBRs) for patients. [Table 8] * Number of patients with SD excluding those with PR 6.2.9. Number of Subjects with Most Common Adverse Events (AEs)

[0155] Subjects with the highest grade counts are summarized in Table 9. [Table 9] 6.2.10. Number of Subjects with Hematological Adverse Events (AEs)

[0156] The number of subjects with hematological adverse events (AEs) is summarized in Table 10. [Table 10] 6.2.11. Number of subjects with hepatic adverse events (AEs)

[0157] Subjects with hepatic AEs are summarized in Table 11. [Table 11] Grade 3 and 4 Toxicity (n=29)

[0158] Table 12 summarizes grade 4 and 4 toxicities. [Table 12] 6.2.13. Adverse events of special interest (AEs)

[0159] Table 13 summarizes adverse events (AEs) of special interest. [Table 13] 6.2.14. Overview of dose reductions The dose of lasofoxifene was not reduced according to protocol Abemaciclib Patients were not reduced to 50 mg BID Reduce from 150mg BID to 100mg BID 4 cases due to AE Hyponatremia Dizziness, fatigue, vomiting, weight loss Increased creatinine Loss of appetite, fatigue, nausea, generalized muscle weakness One at the investigator's discretion 6.2.15. Comparison of ELAINE2 Interim PFS Data with Other Studies

[0160] As shown in Table 14 below, lasofoxifene in combination with abemaciclib shows longer median PFS and comparable efficacy to CDK4 / 6i naive treated patients compared to published and publicly available data in patients previously treated with CDK4 / 6i, more specifically, abema alone in either CDK naive patients (Monarch 1 study) or post-CDK population (Abema study); Piqray / fulvestrant in patients with PIK3CA mutations (Bylieve study); camizestrant + palbociclib (Serena-1 study), amizestrant / CDK4 / 6i (Ameera-1 study); fulvestrant + abemaciclib (Monarch 2 study), and fulvestrant + palbociclib (Paloma-3 study). [Table 14] 1 Damodaran et al ASCO 2022; 2 Dickler at al Clin Cancer Res 2017 Sep 1:23(17):5219-5224; 3 Wander et al, JNCCN, 10:6004, March 24, 2021; 4 Rugo et al Lancet Onc 2021; 5 Oliveria ASCO 2022; 6 Chandarlapaty ASCO 2021 7 Sledge et al. JCO 2017 8 Cristofanilli et al Lancet Onc 2016 * Calculated from reported swimmer plots 6.2.16. Efficacy in Patients with Tumor Progression on Prior CDK4 / 6i Therapy

[0161] A large number of patients whose tumors had progressed on previous CDK4 / 6 inhibitor treatment were enrolled. The combination of lasofoxifene and abemaciclib reduced breast cancer progression in patients who had previously progressed on abemaciclib treatment. ·Patient 29 40 years old with bone metastases: Letrozole for 3 years; Letrozole / palbociclib for 3 years; Fulvestrant / abemaciclib for 12 weeks; Capecitabine for 7 months The mutant allele fraction (MAF) of D538G is 6.855% Stable disease at 68 weeks ·Patient 21 Age 42: chemotherapy / Herceptin; tamoxifen for 10 years; letrozole / palbociclib for 2 years and 8 months; abemaciclib for 16 weeks 〇24mm liver mass 〇Y537S has a MAF of 0.248% Partial response was confirmed at 48 weeks, and liver lesions were reduced by 71% at 40 weeks. ·Patient 16 Age 78: Letrozole / palbo for 2 years and 2 months; Fulvestrant / abemaciclib for 1 year and 3 months; Capecitabine for 1 month 18mm target liver lesion, metastases to pleura, LN, and bone D538G has a MAF of 0.3% Progressed at 40 weeks with stable disease (target lesions decreased by 6%) ·Patient 6 Age 59: fulvestant / abemaciclib for 2 years; capecitabine for 1 month 〇35mm liver metastasis 〇D538G has a MAF of 1.28 Progression in 8 weeks (liver lesions were stable, but new lesions were found) 6.3. Example 3: Second Interim Results of the ELAINE2 Clinical Trial

[0162] At the second late median date, two of four patients (50%) who had progressed on previous abemaciclib treatment continued to benefit from treatment with the combination of lasofoxifene and abemaciclib (laso / abema). With reference to Figure 3, patient 29 (2011-01), who had a D538G mutation and had previously been treated with palbociclib and fulvestrant, had a sustained response and stable disease until 88 weeks. Patient 21 (2005-02), who had a Y537S missense mutation, visceral metastases, and had progressed on previous abemaciclib and palbociclib treatment, had stable disease in response to laso / abema until 56 weeks, at which point his cancer progressed. As shown in Figure 3, patients who had progressed on other CDK4 / 6 inhibitors (e.g., palbociclib, ribociclib) prior to enrollment also achieved clinical benefit (CB). Most patients received palbociclib, and 70% (17 / 24) of these patients achieved CB, while 100% (2 / 2) of those previously treated with ribociclib achieved CB. The data demonstrate that patients with ESR1 mutations who progressed on CDK4 / 6i have a high probability of clinical benefit with combination treatment with lasofoxifene and abemaciclib, independent of prior CDK4 / 6i. Patient 2004-03 with visceral metastases and prior palbociclib treatment achieved CB and stable disease, but withdrew early at 56 weeks due to non-compliance (diamond). Table 15 summarizes the second interim results of patients enrolled after progression on prior abemaciclib. [Table 15]

[0163] Patients who had received other relevant treatments prior to laso / abema treatment on the study also benefited from the combination treatment. With reference to Figure 3, patient 2001-01, who had received previous alpelisib treatment, achieved CB with stable disease for up to 32 weeks. Patient 2009-02, who had received previous alpelisib, achieved CB with stable disease for up to 36 weeks upon combination of lasofoxifene and abemaciclib. Patient 2016-01, who had received previous PARP inhibitor (Talzenna) treatment, achieved CB for up to 32 weeks upon combination of lasofoxifene and abemaciclib with a confirmed partial response. Patient 2018-01, who had received previous ribociclib treatment, achieved CB with stable disease for 60 weeks upon combination treatment of lasofoxifene and abemaciclib. Patient 2004-06, who had previously been treated with ribociclib / fulvestrant for 9 months, had a partial response to lasofoxifene and abemaciclib that lasted for 64 weeks.

[0164] Patients in this study routinely achieved better responses with the combination of lasofoxifene and abemaciclib than with prior 2L and 3L therapy, which is surprising because, historically, subsequent treatment lines often have a shorter duration of benefit than those obtained with the patient's earlier treatment lines. As shown in Figure 4, patients with laso / abema had a longer duration of stable disease than with their respective prior 2L and 3L treatments. At the second median date, subjects treated with laso / abema had a mean duration of stable disease of about 8.7 months. All 29 patients who had prior 2L therapy had a mean duration of stable disease of about 4.2 months. Nine of these subjects had also received 3L therapy prior to enrollment and had a mean duration of stable disease of about 7.2 months. Patients treated with the combination of lasofoxifene and abemaciclib had, on average, an increase in the duration of stable disease of about 51.2% compared to the preceding 2L therapy and an increase in the duration of stable disease of about 16.9% compared to the preceding 3L therapy. For example, patient 2011-00001 (patient 29) had stable disease for up to 22 months with laso / abema compared to 7 months with the preceding 2L therapy and 3 months with the preceding 3L therapy. Patient 2005-00002 (patient 21) had stable disease for up to 14 months with lasofoxifene and abemaciclib compared to 4 months with the preceding 3L therapy. Patient 2015-00001 had stable disease for up to 19 months with laso / abema compared to 10 months and 2 months with the preceding 2L and 3L therapy, respectively.

[0165] The results demonstrate that combination treatment with lasofoxifene and abemaciclib is tolerable, safe, and effective in patients with metastatic breast cancer harboring at least one ESR1 mutation who had progressed on one or more CDK4 / 6 inhibitors and endocrine therapy. 6.4. Example 4: ESR1 Mutations in Circulating Tumor DNA (ctDNA) from Patients in the ELAINE2 Clinical Trial

[0166] This example illustrates the efficacy and safety of lasofoxifene plus abemaciclib in patients with ER in the ELAINE2 clinical trial. + / HER2 - We investigate ESR1 mutations in circulating tumor DNA (ctDNA) in patients with metastatic breast cancer (mBC). The data in this example demonstrate a correlation between changes in ESR1 mutant allele frequency (MAF) and clinical benefit (CB).

[0167] The use of long-term endocrine therapy (ET) for ER+ breast cancer often results in the acquisition of ESR1 mutations (mutESR1) that cause endocrine resistance, tumor progression, and poor prognosis. There is an unmet clinical need for treating ER+ mBC patients with mutESR1, especially after progression on CDK4 / 6 inhibitors (CDK4 / 6i). ELAINE2 is an open-label, phase 2, multicenter study evaluating the safety and efficacy of lasofoxifene (LAS [selective estrogen receptor modulator]) + abemaciclib (Abema [CDK4 / 6i], provided by Eli Lilly) in patients with ER+ / HER2- and mutESR1 mBC that progressed after previous ET. Preliminary data with LAS + Abema showed a median progression-free survival of 55.7 weeks, an objective response rate of 50%, and a 24-week clinical benefit (CB) rate of 69%, with an acceptable safety and tolerability profile.

[0168] ELAINE2 clinical trial patients with detectable ctDNA mutESR1 at baseline (BL) were analyzed. Oral LAS 5 mg / day and Abema 150 mg BID were administered until disease progression, death, unacceptable toxicity, or withdrawal from study. ctDNA was assessed by the Sysmex-Inostics SafeSeq assay, which detects mutESR1 at low allele fractions, at BL, every 4 weeks, and end of treatment. Changes in MAF from BL to week 4 were characterized as decreasing (decreased or undetectable ESR1 MAF [ND]), increasing (increased MAF), or unstable (some MAFs show increasing and decreasing trends in polyclonal patients [mutESR1 >1]). Correlation of MAF change at 4 weeks with CB at 24 weeks was investigated.

[0169] A total of 29 patients (median of 2 prior metastatic treatments: 97% CDK4 / 6i, 79% fulvestrant, 48% chemotherapy) had BL mutESR1 with Y537S (66%), D538G (45%), Y537N (28%), Y537C (10%), and other less frequently detected mutations; 14 (48.3%) patients were polyclonal. Twenty-six (26) of the 29 patients had evaluable BL and ctDNA results at week 4: As summarized in Table 16 below, 21 patients had decreased MAF (81%, 14 lost [54% ND]), 3 (12%) had increased, and 2 (8%) had an undetermined change in ESR1 MAF. [Table 16] CI, confidence interval; MAF, variant allele fraction; ND, not detected; NPV, negative predictive value; PPV, positive predictive value. * Sensitivity and specificity analyses did not include indeterminate results.

[0170] mESR1 loss at week 4 was observed in 3 of 4 patients who had progressed during previous Abema-based therapy, and all 3 achieved CB. Reduced / lost MAF was frequently observed after 4 weeks of LAS+abema for all commonly detected mESR1 variants, including Y537S, D538G, Y537N, and Y537C variants (Figure 6). CB at 24 weeks was observed in 17 patients with decreased ESR1 MAF, 2 with increased, and 1 with an indeterminate change in MAF. To predict CB based on the direction of ESR1 MAF change, we calculated a sensitivity of 89.5%, specificity of 20%, and positive likelihood rPBatio(LR+) of 1.1. The positive predictive value (PPV) of CB with decreased MAF was 81%, and the negative predictive value (NPV) of increased MAF was 33%. Of 14 (54%) patients with ND ESR1 MAF, 13 had CB, resulting in approximately 87% sensitivity, 50% specificity, 93% PPV, and 33% NPV for increased ESR MAF. MutESR1 loss at week 4 had similar sensitivity (approximately 87%) and higher PPV (approximately 93%) for CB prediction compared with decreased MAF, with an LR+ of 1.7. All nine patients with objective response (OR) showed complete loss of mESR1 (n=5) or a 50%–93% decrease in ESR1 MAF (n=4) at week 4.

[0171] In the ELAINE2 clinical trial, 81% of patients had a reduction / loss (ND) of mutESR1 after four (4) weeks of LAS+Abema, which correlated with clinical benefit. Any detected mutESR1 appears to be targeted by this treatment. High sensitivity and good PPV were observed in patients with reduced MAF, and even more so in patients with ND MAF; however, increased MAF was less specific and did not predict treatment failure.

[0172] In summary, analysis of ctDNA data in ELAINE2 demonstrated that mutated ESR1 variants, including the difficult-to-treat Y537S, were lost / lost in most (~81%) patients 4 weeks after LAS+Abema. Lost / lost ESR1 MAF was associated with CB and OR, with high sensitivity (89%) and good PPV (81%) for predicting CB.

[0173] In the case of mutESR1 loss, the PPV was higher (93%). Increased MAF was less specific and not predictive of treatment failure. The results show robust target engagement of LAS+Abema with mutESR1. Overall, the results demonstrate that ESR1 liquid biopsy assessment is a suitable non-invasive surrogate marker for monitoring patient treatment response or resistance to this novel LAS-Abema combination. 6.5. Example 5: Oncogenic Mutations in Circulating Tumor DNA (ctDNA) from Patients in the ELAINE2 Clinical Trial

[0174] This example investigates oncogenic mutations of genes other than ESR1 in circulating tumor DNA (ctDNA) in patients with ER+ / HER2- metastatic breast cancer (mBC) treated with lasofoxifene + abemaciclib in the ELAINE2 clinical trial. Figure 5 summarizes the panel of genes tested and present in the ELAINE2 patient population. Gain-of-function mutations of ESR1 (mutESR1; top row) are included as a positive control. The data in this example demonstrate the correlation of the prevalence of oncogenic mutations of genes other than mutESR1 with clinical benefit (CB) and median progression-free survival (mPFS).

[0175] Approximately 5 mL whole blood samples were collected in Streck cell-free DNA blood collection tubes (BCT) from individual ELAINE2 clinical trial patients at baseline (BL). Individual patients had previously been diagnosed by an oncologist as having ER+ mBC. To be eligible for enrollment, patients must have had a prior medical history indicating the presence of mutESR1 or be detected to have endogenous or acquired mutESR1 using the assay described in Example 4. Oral lasofoxifene 5 mg / day and abemaciclib 150 mg BID were administered until disease progression, death, unacceptable toxicity, or withdrawal from the study. Samples were processed for plasma separation and extraction of cell-free DNA (cfDNA), which may include circulating tumor DNA. Approximately 5-30 ng of cfDNA was used to prepare enriched sequencing libraries by hybridization capture. The enriched libraries were then sequenced using next-generation sequencing, for example, on an Illumina NextSeq 550 platform. Sequencing data were analyzed using a bioinformatics pipeline designed to detect single nucleotide variants (SNVs), insertions and deletions (indels), copy number amplifications (CNAs), and fusions. Both pathogenic (e.g., oncogenic) germline and somatic alterations were detected.

[0176] As shown in Figure 5, a total of 41 genes with one or more oncogenic mutations were detected in blood samples from the ELAINE2 patient population. Each gene mutation had a prevalence of at least about 3% of the patient population. Mutations were either germline, somatic, or both. The most common genes with one or more oncogenic mutations other than ESR1 were HNF1A (62%), TERT (59%), TP53 (41%), APC (28%), PIK3CA (28%), ATM (24%), CCND1 (21%), MET (17%), EFGR (17%), FGFR1 (17%), GATA3 (17%), and BRCA1 (17%). At least 43% of patients with oncogenic mutations in one or more of these genes achieved clinical benefit (CB)-stable disease and mPFS of at least 24 weeks in response to lasofoxifene and abemaciclib treatment. For example, approximately 100% of patients with oncogenic mutations in CCND1 achieved CB and an mPFS of at least 56 weeks, while 76% of patients with oncogenic mutations in TERT achieved CB and an mPFS of at least 44 weeks. In general, patients with oncogenic mutations in at least one of the genes selected from HNF1A, TERT, GATA3, CDK12, MAPK3, TP53, PIK3CA, APC, ATM, MET, CCND1, EGFR, ROS1, STK11, FGFR1, ARID1A, CCNE1, AR, ALK, ERBB2, KIT, CDK4, SMAD4, NOTCH1, RB1, BRAF, RAF1, PTEN, AKT1, CDH1, BRCA1, MYC, CDKN2A, or PDGFRA achieved an mPFS of at least 24 weeks. Patients with one or more mutations in at least one of the genes selected from CCND1, FGFR1, CCNE1, AR, ALK, MAPK3, KIT, SMAD4, NOTCH1, RB1, BRAF, RAF1, PTEN, AKT1, CDH1, BRCA1, MYC, CDKN2A, or PDGFRA achieved 100% clinical benefit and mPFS of at least 32 weeks.Of note, patients with oncogenic mutations in one or more of the genes selected from GNAS, RHEB, NTRK3, IDH2, and / or mTOR had no CB and mPFS of 8 weeks or less.

[0177] Interestingly, these baseline measurements detected ESR1 mutations in only about 90% of the enrolled subjects. At least in some of the subjects whose enrollment in the clinical trial was based on a history of ESR1 mutations rather than on the detection of ESR1mut at enrollment, this may be due to the patient's response to previous treatment. This would be consistent with our observations in the ELAINE2 clinical trial itself, where about 68.9% of subjects (20 out of 29) showed a reduction or disappearance (ND) of ESR1 mutations at week 4 of LAS+abema treatment.

[0178] For example, patient 29 (2011-00001) had an ESR1 D538G mutation at baseline after previous (pre-enrollment) abemaciclib treatment and other first- or second-line endocrine therapies (Figures 1 and 3). Patient 29 achieved ND for the ESR1 mutation in ctDNA at week 4 upon combination treatment with lasofoxifene and abemaciclib. Patient 29 continues to respond to treatment and has clinical benefit with stable disease for at least 88 weeks (Figure 3).

[0179] Patient 16 (2003-00001) had a medical history indicating the presence of the ESR1 gain-of-function mutation D538G. Patient 16 had also received previous abemaciclib treatment and other first- or second-line endocrine therapy (Figures 1 and 3). At baseline, patient 16 had no detectable mutESR1 (ND for ESR1 mutations) using the assay described in Example 4. At baseline, patient 16 was detected to have oncogenic mutations in TERT, ATM, and MAPK3 (data not shown). Patient 16 maintained ND for ESR1 mutations in ctDNA until the end of treatment at week 40.

[0180] The data in this example demonstrate that lasofoxifene in combination with abemaciclib is effective in treating metastatic ER+ breast cancer with an ESR1 gene mutation and one or more oncogenic mutations in genes other than ESR1; and that lasofoxifene in combination with abemaciclib is effective in treating metastatic ER+ breast cancer with no detectable ESR1 mutation and one or more oncogenic mutations in genes other than ESR1. 6.6. Example 6: Third Interim Results of the ELAINE2 Clinical Trial

[0181] At the third subsequent interim date, 20 of the 29 patients (approximately 69%) had CB resulting from treatment with the combination of lasofoxifene and abemaciclib (laso / abema). Referring to Figure 7, the updated swimmer plot, 11 of the 20 patients (55%) who had CB had an updated status compared to Figure 3. Of these patients, 8 of the 11 patients (approximately 72.7%) had a sustained response or partial response and stable disease at 68-100 weeks since the last update. For example, patient 29 (2011-01) continued to have a sustained response and stable disease through 100 weeks, while patient 2005-01 had a sustained response and stable disease through 84 weeks, patient 2002-01 had a sustained response and stable disease through 80 weeks, patients 2001-05 and 2016-03 had a sustained response and stable disease through 72 weeks, patient 2018-01 had a sustained response and stable disease through 68 weeks, and patient 2017-01 had a partial response and stable disease through 80 weeks. Patient 2015-01 had a partial response and stable disease through 88 weeks. Two (2) of 20 patients (10%) progressed at week 72 (patient 2001-02 and patient 2014-01), and one patient (5%) withdrew early (patient 2004-06). As shown in Table 17 below, 7 of 11 patients (about 63.6%) had the difficult-to-treat Y537S mutation at baseline. The data show that all 7 patients had a continuous or partial response to laso / abema combination treatment and had stable disease for up to at least 72 weeks. Laso / abema efficacy was observed even in patients with visceral metastases at baseline (about 42.8%, 3 of 7). At least 7 of 11 patients (about 63.6%) had oncogenic mutations in one or more genes other than ESR1, and at least one of the oncogenic mutations had a prevalence of more than 20% in the patient population (e.g., HNF1A, TERT, TP53, APC, PIK3CA, ATM, CCND1, see also Figure 5). [Table 17-1] [Table 17-2] * TBD: Oncogenic mutations to be determined

[0182] With reference to Table 17, note that patients with mutations previously associated with endocrine resistance or CDK4 / 6i resistance achieved clinical benefit (CB) with stable disease and showed complete or partial response for at least 72 weeks. For example, patients with at least one of FGFR1, ERBB2, CCND1, CCNE1, ARD1A, PIK3CA, and TP53 had consistent and robust clinical responses to the lasofoxifene / abemaciclib combination in Elaine2. Patient 2005-01, who had TP53, CCND1, and CCNE1 mutations and CNV, had a complete response and stable disease until 84 weeks. Patient 2014-01, who had TP53, CCND1, and ARID1A mutations or CNV, had a partial response and progressed at 72 weeks. Patient 2001-02, who had PIK3CA mutations or CNV, had a partial response and progressed at 72 weeks. Patient 2001-05, who had a PIK3CA mutation or CNV, had a complete response and stable disease through 72 weeks. Note that these four patients also had the difficult-to-treat Y537S mutESR1 variant.

[0183] These results are unexpected and demonstrate that combination treatment with lasofoxifene and abemaciclib is effective in reducing or preventing tumor progression in patients who are at higher risk of developing resistance to endocrine therapy or CDK4 / 6i treatment or who have developed resistance to endocrine therapy or CDK4 / 6i treatment.

[0184] The results are consistent with our findings shown in Example 3 and further demonstrate acceptable tolerability, safety, and efficacy of combination treatment with lasofoxifene and abemaciclib in patients with metastatic breast cancer harboring at least one ESR1 mutation, often harboring at least one or more mutations in other genes, and who have progressed on one or more CDK4 / 6 inhibitors, and / or one or more endocrine therapies. 6.7. Example 7: Copy number variations of ESR1 mutations and oncogenic mutations in circulating tumor DNA (ctDNA) from patients in the ELAINE2 clinical trial

[0185] This example investigates copy number variations (CNVs) of ESR1 mutations and oncogenic mutations in circulating tumor DNA (ctDNA) in patients with ER+ / HER2- metastatic breast cancer (mBC) treated with lasofoxifene + abemaciclib in the ELAINE2 clinical trial described in Example 5. The data in this example demonstrate a correlation between the presence of ESR1 mutation variants and / or oncogenic mutation variants and clinical benefit (CB).

[0186] A total of 29 samples from 29 patients (Batch 1: 25 samples / patient; Batch 2: 4 samples / patient) were collected from the ELAINE2 clinical trial. Patients were administered oral lasofoxifene 5 mg / day and abemaciclib 150 mg BID until disease progression, death, unacceptable toxicity, or withdrawal from the study. Samples were processed for plasma separation and cell-free DNA (ctDNA) extraction and sequencing as described in Example 5. Sequencing data were analyzed using a bioinformatics pipeline designed to detect single nucleotide variants (SNVs), insertions and deletions (indels), copy number variations (CNVs), and fusions. In Batch 1, copy number amplifications (CNAs) were detected in 19, including CCND1. Amplification types were annotated as focal, aneuploid, or amplified (if focal / aneuploidy status was uncertain). Results are provided in Table 18. [Table 18]

[0187] Figure 8 shows examples of copy number variation events detected per gene. The left panel of Figure 8 shows copy number variation (CNV) events detected per gene, including CCND1, CCNE1, CDK4, EGFR, FGFR1, MYC. The right panel of Figure 8 shows the copy number distribution per gene for the CNV events shown on the left panel. Five CNV events and more than 8 copy numbers were detected in CCND1, three CNV events and more than 7 copy numbers were detected in FGFR1, while two CNV events and about 5 copy numbers were detected in CDK4. 6.8. Example 8: Interim Results of Oncogenic Mutations in Circulating Tumor DNA (ctDNA) from Patients in the ELAINE2 Clinical Trial

[0188] This example provides oncogenic mutations in genes other than ESR1 present in circulating tumor DNA (ctDNA) in patients with ER+ / HER2- metastatic breast cancer (mBC) treated with lasofoxifene + abemaciclib in the ELAINE2 clinical trial described in Example 5, as of a median date after the date of Example 5. The results are summarized in Figure 9. ESR1 gain-of-function mutations (mutESR1; top row) are included for completeness.

[0189] As shown in Figure 9, a total of 41 genes with one or more oncogenic mutations were detected in blood samples from the ELAINE2 patient population. At least 87.8% (36 of 41) of patients with oncogenic mutations in one or more of these genes achieved clinical benefit (CB) (defined as stable disease for ≥24 weeks or confirmed partial or complete response) in response to lasofoxifene and abemaciclib treatment. Patients with one or more mutations in TERT, APC, ATM, CCND1, MET, EGFR, FGFR1, GATA3, STK11, ROS1, ERBB2, CCNE1, AR, SMAD4, ALK BRAF, KIT, CDK4, AKT1, CDH1, BRCA1, MYC, and PDGFRA continued to achieve CB and mPFS. For example, about 100% of patients with oncogenic mutations in CCND1 continued to achieve CB and mPFS for at least 72 weeks, 100% of patients with oncogenic mutations in FGFR1 continued to achieve CB and mPFS for at least 72 weeks, while 76% of patients with oncogenic mutations in TERT continued to achieve CB and mPFS for at least 56 weeks. Patients with one or more mutations in TP53 or PIK3CA achieved CB and mPFS for at least 36 and 34 weeks, respectively. Notably, patients with coexisting copy number variants (CNVs) of CCND1 and FGFR1 and alterations of PIK3CA and TP53 responded to lasofoxifene and abemaciclib.

[0190] The data in this example demonstrate that lasofoxifene in combination with abemaciclib is effective in treating metastatic ER+ breast cancer with ESR1 gene mutations and one or more oncogenic mutations in genes other than ESR1, and in maintaining CB and mPFS in this subset of patients, and in patients with metastatic ER+ breast cancer without detectable ESR1 mutations and with one or more oncogenic mutations in genes other than ESR1. 6.9. Example 9: Fourth Interim Results of the ELAINE2 Clinical Trial

[0191] As of date 4, which is later than the date of Example 6, 20 of the 29 patients (approximately 68.9%) continued to have CB from treatment with the combination of lasofoxifene and abemaciclib (laso / abema). Referring to the updated swimmer plots of Figures 10A-10B, 8 of the 20 patients (40%) who had CB had an updated status compared to Figure 7. Of these patients, 6 of the 8 patients (75%) had ongoing or partial response and stable disease 96-128 weeks after the last update, while 2 of the 8 patients (25%) had ongoing or partial response and progressive disease. For example, patient 29 (2011-01) had a sustained response and stable disease until 128 weeks, while patient 2002-01 had a sustained response and stable disease until 100 weeks, and patients 2001-05 and 2018-01 had a sustained response and stable disease until 96 weeks. Patient 2015-01 had a partial response and stable disease until 120 weeks. Patient 2016-03 had a partial response and stable disease until 96 weeks. Patient 2005-01 had a sustained response but progressed at 104 weeks. Patient 2017-01 had a partial response and progressed at 100 weeks. Table 19 summarizes the efficacy of the lasofoxifene / abemaciclib combination in patients who achieved clinical benefit (including those listed in Table 17). As shown, 13 of 20 patients (65%) had the difficult-to-treat Y537S mutation at baseline, but had ongoing or partial responses and stable disease for up to 120 weeks (e.g., patient 2015-01). Efficacy of lasofoxifene / abemaciclib was observed even in patients with visceral metastases at baseline (60%, 12 of 20). All 20 patients (100%) who had clinical benefit also had oncogenic mutations in one or more genes other than ESR1, with at least one of the oncogenic mutations having a prevalence of >17% in the patient population (e.g., HNF1A, TERT, TP53, APC, PIK3CA, ATM, CCND1, FGFR1, see also Figure 9). [Table 19-1] [Table 19-2] [Table 19-3]

[0192] With reference to Table 19, note that patients with mutations previously associated with endocrine resistance or CDK4 / 6i resistance (such as FGFR1, ERBB2, CCND1, CCNE1, ARD1A, PIK3CA, and TP53) continued to have consistent and robust clinical responses to the lasofoxifene / abemaciclib combination in Elaine2. Subject 2011-01 with D538G mutESR1 variant and TP53 and CCND1 mutations or copy number variants (CNVs) continued to have a complete response and stable disease through 128 weeks. Patient 2017-01 with E380Q and L469V mutESR1 variants and CCNE1 mutations or CNVs continued to have a partial response and progressed at 100 weeks. Patient 2016-03, who had a D538G mutESR1 variant and an ERBB2 mutation or CNV, continued to have a partial response and stable disease at 96 weeks. Patient 2002-02, who had multiple mutESR1 variants (including Y537N, L536H, D538G, and E380Q) but also had a CCNE mutation or CNV, had a partial response until progression at 32 weeks.

[0193] Of note, patients with the difficult-to-treat Y537S mutESR1 variant continued to have consistent and robust clinical efficacy to the lasofoxifene / abemaciclib combination. Patient 2015-01, with the Y537S mutESR1 variant and FGFR1 and TP53 mutations or CNVs, continued to have partial responses and stable disease through 120 weeks. Patient 2005-01, with the Y537S, Y537N, and D538G mutESR1 variants and TP53, CCND1, and CCNE1 mutations or CNVs, continued to have complete responses and stable disease and progressed at 104 weeks. Patient 2001-02, with the Y537S, Y537N, Y537D, and D538G mutESR1 variants and PIK3CA, FGFR1, and CCND1 mutations or CNVs, had a partial response and progressed at 72 weeks. Patient 2014-01, with Y537S, Y537N, and D538G mutESR1 variants and TP53, CCND1, and ARID1A mutations or CNVs, had a partial response and progressed at 72 weeks. Patient 2004-03, with Y537S mutESR1 variants and ERBB2 mutations or CNVs, had a complete response and stable disease at 56 weeks but withdrew early due to non-compliance. Patient 2005-02, with Y537S mutESR1 variants and PIK3CA and ARID1A mutations or CNVs, had a partial response and progressed at 56 weeks. Patient 2008-01, with Y537S and D538G mutESR1 variants and CCND1 and FGFR1 mutations or CNVs, had a partial response and progressed at 32 weeks. Patient 2016-01, with Y537S, Y537C, L536P, and D538G mutESR1 variants and TP53, CCND1, and FGFR1 mutations or CNVs, had a partial response and progressed at 32 weeks. Patient 2001-05, with Y537S mutESR1 variants and PIK3CA mutations or CNVs, continued to have a complete response and stable disease through 96 weeks. Patient 2001-01, with Y537S and D538G mutESR1 variants and PIK3CA mutations or CNVs, had a partial response and progressed at 32 weeks.Patient 2009-02, who harbored the Y537S mutESR1 variant and TP53 and PIK3CA mutations or CNVs, had a complete response and stable disease with progression at 36 weeks.

[0194] Patients with target lesions at baseline were monitored for changes during the study. Note that 10 of 20 (50%) had confirmed partial responses and achieved clinical benefit. Patient 2001-01 had target lesions in the lungs at baseline (11 mm), which decreased by 55% at 64 weeks. Patient 2002-02 had target lesions in the liver (20 mm), lungs (23 mm, 29 mm) at baseline (total diameter 72 mm), which decreased by 74% at 32 weeks. Patient 2004-06 had target lesions in the liver, bone, and pleural cavity at baseline (total diameter 79 mm), which decreased by 47% at 64 weeks. Patient 2005-02 had lesions in the liver (total diameter 24 mm) at baseline, which decreased by 33% at 56 weeks. Patient 2008-01 had target lesions in the liver (total diameter 56 mm) at baseline, which decreased by 50% at 32 weeks. Patient 2014-01 had target lesions in the liver and spleen (41 mm), which decreased by 22% at 64 weeks. Patient 2015-01 had target lesions in the liver (total diameter 56 mm), which decreased by 52% at 104 weeks. Patient 2016-01 had target lesions in the liver (total diameter 47 mm), which decreased by 47% at 32 weeks. Patient 2016-03 had target lesions in the liver (total diameter 67 mm), which decreased by 67% at 88 weeks. Patient 2017-01 had target lesions in the left paraceliac (total diameter 15 mm), which decreased by 40% at 96 weeks. The data demonstrate that combination treatment with lasofoxifene / abemaciclib is effective in reducing tumor progression and / or inhibiting tumor growth.

[0195] The results are consistent with our findings in Examples 3 and 7, and further demonstrate acceptable tolerability, safety, and efficacy of combination treatment with lasofoxifene and abemaciclib in metastatic breast cancer patients who carry at least one ESR1 mutation, often carry at least one or more mutations in other genes, and have progressed on one or more CDK4 / 6 inhibitors, and / or one or more endocrine therapies. Moreover, the results further demonstrate that combination treatment with lasofoxifene and abemaciclib is effective in patients who carry difficult-to-treat mutESR1 variants. The results are unexpected in patients who also carry one or more oncogenic mutations or CNVs of biomarkers associated with endocrine therapy or CDK4 / 6i resistance. 7. Equivalents and Incorporation by Reference

[0196] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0197] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A combination for reducing the progression of breast cancer in a patient, comprising rasofoxifen or a pharmaceutically acceptable salt thereof and a CDK4 / 6 inhibitor (CDK4 / 6i), The aforementioned breast cancer, (i) Estrogen receptor positive (ER ＋ ) and; (ii) Having at least one gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) A combination having oncogenic mutations in one or more genes other than the ESR1 gene.

2. The combination according to claim 1, wherein the breast cancer has progressed during previous treatment with a CDK4 / 6 inhibitor.

3. The aforementioned ER ＋ Breast cancer is HER2 － The combination described in claim 1.

4. The aforementioned ER ＋ The combination according to claim 3, wherein the breast cancer is locally advanced.

5. The aforementioned ER ＋ The combination according to claim 3, wherein the breast cancer is metastatic or visceral.

6. The combination according to claim 1, characterized in that rasofoxifen is administered as rasofoxifen tartrate.

7. The combination according to claim 1, characterized in that rasofoxifen is administered orally at a dose of 5 mg / day.

8. The combination according to claim 1, wherein the CDK4 / 6i administered to the patient is selected from palbociclib, ribociclib, and abemaciclib.

9. The combination according to claim 8, wherein the CDK4 / 6i administered to the patient is abemaciclib.

10. The combination according to claim 9, characterized in that abemaciclib is administered orally in a BID of 50 mg to 200 mg.

11. The combination according to claim 2, wherein the previously administered CDK4 / 6 inhibitor is selected from palbociclib, ribociclib, and abemaciclib.

12. The combination according to claim 1, wherein oncogenic mutations in one or more genes having oncogenic mutations in circulating tumor DNA (ctDNA) are detected prior to administration of the combination.

13. The combination according to claim 12, wherein at least one of the one or more genes having oncogenic mutations is selected from HNF1A, TERT, GATA3, CDK12, MAPK3, GNAS, RAF1, RHEB, NTRK3, TP53, PIK3CA, APC, ATM, MET, CCND1, EGFR, ROS1, BRCA2, STK11, FGFR1, ARID1A, CCNE1, AR, ALK, ERBB2, KIT, CDK4, SMAD4, NOTCH1, RB1, BRAF, PTEN, AKT1, CDH1, BRCA1, MYC, CDKN2A, IDH2, MTOR, and PDGFRA.

14. The combination according to claim 1, wherein the one or more genes having oncogenic mutations are selected from HNF1A, TERT, GATA3, CDK12, MAPK3, TP53, PIK3CA, APC, ATM, MET, CCND1, EGFR, ROS1, STK11, FGFR1, ARID1A, CCNE1, AR, ALK, ERBB2, KIT, CDK4, SMAD4, NOTCH1, RB1, BRAF, RAF1, PTEN, AKT1, CDH1, BRCA1, MYC, CDKN2A, and PDGFRA.

15. The combination according to claim 14, wherein the one or more genes having oncogenic mutations are selected from TP53, PIK3CA, CCND1, ARID1A, FGFR1, CCNE1, and ERBB2.

16. The combination according to claim 15, wherein the oncogenic mutation is PIK3CA.

17. The combination according to claim 1, wherein the at least one gain-of-function missense mutation is present in any one of the amino acids selected from D538, Y537, L469, L536, P535, V534, S463, V392, and E380.

18. The combination according to claim 17, wherein the mutation is Y537S, Y537N, Y537C, Y537Q, L469V, L536R, L536Q, P535H, V534E, S463P, V392I, or E380Q.

19. The combination according to claim 1, wherein the breast cancer progressed during one or more prior endocrine treatments selected from a selective ER degrading agent (SERD), a selective ER modifier (SERM), a SERM other than rasofoxifene as needed, an aromatase inhibitor (AI), an mTOR inhibitor, and / or a PI3K inhibitor.

20. The combination according to claim 19, wherein the SERD is full bestrant.

21. The combination according to any one of claims 1 to 20, wherein the patient obtains a clinical benefit with a stable disease for at least 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 ​​weeks, 52 weeks, 56 weeks, 60 weeks, 64 weeks, 68 weeks, 72 weeks, 74 weeks, 78 weeks, 82 weeks, 86 weeks, or longer, after initiation of treatment with rasofoxifen or a pharmaceutically acceptable salt thereof and CDK4 / 6i.

22. The combination according to claim 21, wherein the patient has a complete or partial response to the treatment with rasofoxifen or a pharmaceutically acceptable salt thereof and CDK4 / 6i.

23. The combination according to any one of claims 1 to 20, wherein the patient, when using rasofoxifen or a pharmaceutically acceptable salt thereof and CDK4 / 6i, obtains a duration of stable disease at least about 20%, 15%, or 10% longer than the patient received prior second-line or third-line treatment.

24. A composition for reducing the progression of breast cancer in a patient, comprising rasofoxifen or a pharmaceutically acceptable salt thereof, characterized in that the composition is administered in combination with a CDK4 / 6 inhibitor (CDK4 / 6i), The aforementioned breast cancer, (i) Estrogen receptor positive (ER+); (ii) Having at least one gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) A composition having oncogenic mutations in one or more genes other than the ESR1 gene.

25. A composition for reducing the progression of breast cancer in a patient, comprising a CDK4 / 6 inhibitor (CDK4 / 6i), wherein the composition is administered in combination with rasofoxifen or a pharmaceutically acceptable salt thereof. The aforementioned breast cancer, (i) Estrogen receptor positive (ER+); (ii) Having at least one gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) A composition having oncogenic mutations in one or more genes other than the ESR1 gene.

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