Lasofoxifene combined treatment of ER + breast cancer progressing after treatment of CDK4 / 6 inhibitor

Abstract

The present invention provides a method of slowing the progression of ER + breast cancer in a patient comprising administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i) wherein breast cancer: (i) is estrogen receptor positive (ER +); (ii) having at least one function-acquired missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) have progressed and / or have oncogenic mutations in genes other than ESR1 during previous treatment with a CDK4 / 6 inhibitor.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 345,843 filed on May 25, 2022, U.S. Provisional Application No. 63 / 411,633 filed on September 30, 2022, U.S. Provisional Application No. 63 / 426,737 filed on November 19, 2022, U.S. Provisional Application No. 63 / 430,194 filed on December 5, 2022, and U.S. Provisional Application No. 63 / 446,760 filed on February 17, 2023, the entire disclosures of each of which are incorporated herein by reference. Background Art

[0003] Estrogen receptor positive (ER + )Breast cancer expresses estrogen receptor alpha (ERα), which is encoded by the ESR1 gene. Approximately 70% of breast cancers are ER + , therefore, treatment with drugs that deplete circulating estrogen levels or block estrogen signaling in cancer cells (collectively known as endocrine therapy). Endocrine therapy has led to + The prognosis for women with breast cancer has improved dramatically. However, the effectiveness of endocrine therapy is limited by intrinsic and, to a significant extent, acquired endocrine resistance. In response to the selective pressure exerted by endocrine therapy, particularly aromatase inhibitors (AIs), ER + Tumors develop various escape mechanisms. These include the acquisition of gain-of-function mutations in the ESR1 gene, which alter the ligand-binding domain of the ERα receptor, allowing the receptor to remain constitutively active in the presence of low levels or absence of estrogen. Despite the benefits of endocrine therapy, most ER + Tumors will eventually acquire drug resistance and progress.

[0004] Lasofoxifene is a third-generation selective estrogen receptor modulator (SERM) that has been shown to reduce the risk of aggressive ER in women with wild-type estrogen receptors. + Risk of breast cancer: postmenopausal women who are receiving osteoporosis treatment and have no history of breast cancer. LaCroix et al., J. Natl. Cancer Inst. 102: 1706-1715 (2010). Later, it was shown that lasofoxifene retains the ability to inhibit ERα receptors (ESR1 gene) that have a gain-of-function mutation in the ligand binding domain. +The ability of cancer to progress. U.S. Patent Nos. 10,258,605 and 10,905,659; WO 2019 / 199891; Lainé et al., Breast Cancer Res. 23(1):54 (2021). A Phase 2 clinical trial NCT03781063 (ELAINE trial) is currently underway to confirm the efficacy of lasofoxifene as a single agent in the treatment of locally advanced or metastatic ER cancer with ESR1 gain-of-function mutations. + Outcomes in premenopausal and postmenopausal women with breast cancer.

[0005] In the past decade, a new class of drugs, cyclin-dependent kinase 4 / 6 inhibitors (CDK4 / 6i), has been marketed for the treatment of patients with (ER + ) women with breast cancer. Three CDK4 / 6i have been approved in many countries around the world, including the United States, for use in combination with endocrine therapy to treat ER + Cancer: palbociclib (IBRANCE, Pfizer), ribociclib (KISQALI, Novartis), and abemaciclib (VERZENIO, Eli Lilly). However, ER + Tumors have been shown to become resistant to CDK4 / 6i and eventually progress.

[0006] There remains a need for effective treatments for ER-positive patients with ESR mutations who have become resistant to endocrine therapy and CDK4 / 6 inhibitors and who therefore progress + New treatments for tumors. Summary of the invention

[0007] The ongoing ELAINE 2 clinical trial is an open-label, multicenter study evaluating the combination of the third-generation SERM lasofoxifene and the CDK4 / 6 inhibitor abemaciclib in the treatment of locally advanced or metastatic ER + / HER2 -Efficacy, safety, and tolerability in premenopausal and postmenopausal women with breast cancer (with an ESR1 mutation) who had disease progression following first-line, second-line, or third-line hormonal therapy for metastatic disease. To be eligible, patients had to have disease progression following up to three of the following metastatic breast cancer therapies: an aromatase inhibitor (AI) and / or fulvestrant, as monotherapy or in combination with any marketed CDK4 / 6i; and / or the combination of fulvestrant and apellix; and / or tamoxifen; and / or the combination of exemestane / everolimus; and up to first-line chemotherapy in the metastatic setting (48%). The majority of patients enrolled in the trial (28 / 29) had progressed on prior CDK4 / 6 inhibitor combination therapy. We have now found that the combination of lasofoxifene with the CDK4 / 6 inhibitor (CDK4 / 6i) abemaciclib was well tolerated and demonstrated strong and meaningful efficacy in these patients to reduce ER cancer with locally advanced or metastatic ER cancer that harbors an ESR1 gain-of-function mutation. + Breast cancer progression in women with breast cancer who have progressed on prior CDK4 / 6i therapy.

[0008] Furthermore, we have now found that the combination of lasofoxifene and the CDK4 / 6 inhibitor (CDK4 / 6i) abemaciclib can reduce the incidence of ER in patients with locally advanced or metastatic disease harboring ESR gain-of-function mutations and oncogenic mutations in one or more genes other than the ESR1 gene. + Breast cancer progression in women with breast cancer.

[0009] Therefore, in a first aspect, the present invention provides a method for slowing down the progression of breast cancer in a patient, comprising:

[0010] administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i),

[0011] The breast cancer:

[0012] (i) Estrogen receptor positive (ER + );

[0013] (ii) having at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and

[0014] (iii) have progressed during previous CDK4 / 6 inhibitor therapy.

[0015] In one aspect, the present invention provides a method for slowing the progression of breast cancer in a patient, comprising:

[0016] administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i),

[0017] The breast cancer:

[0018] (i) Estrogen receptor positive (ER + );

[0019] (ii) having at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and

[0020] (iii) having an oncogenic mutation in one or more genes other than the ESR1 gene.

[0021] In one aspect, the present invention provides a method for slowing the progression of breast cancer in a patient, comprising:

[0022] administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i),

[0023] The breast cancer:

[0024] (i) Estrogen receptor positive (ER + );

[0025] (ii) having at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and

[0026] (iii) having increased expression of one or more genes other than the ESR1 gene.

[0027] In one aspect, provided herein is a method of monitoring a patient receiving treatment for breast cancer, comprising:

[0028] (a) determining a quantitative measure of the mutant allele frequency (MAF) of at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene (ESR1ctDNA) in circulating tumor DNA (ctDNA) of a biological sample of a patient, wherein the quantitative measure is performed over a period of time at defined time intervals; and

[0029] (b) determining a positive predictive value (PPV) for a clinical benefit of stable disease from a cancer treatment, wherein the PPV represents responsiveness to the cancer treatment, and

[0030] The cancer treatment comprises an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i).

[0031] The method of any of the preceding embodiments, wherein ER + Breast cancer is HER2 - .

[0032] The method of any of the preceding embodiments, wherein the ER + Breast cancer is either locally advanced or metastatic.

[0033] The method of any of the preceding embodiments, wherein lasofoxifene is administered as lasofoxifene tartrate.

[0034] The method of any of the preceding embodiments, wherein lasofoxifene is administered orally at 5 mg / day.

[0035] The method of any of the preceding embodiments, wherein the CDK4 / 6i administered to the patient is selected from palbociclib, ribociclib, and abemaciclib.

[0036] The method of any of the foregoing embodiments, 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.

[0037] The method of any of the preceding embodiments, 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.

[0038] The method of any of the preceding embodiments, wherein the cancer has been previously identified as having at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

[0039] The method of any one of the preceding embodiments, further comprising the following earlier steps:

[0040] The patient is determined to have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

[0041] The method of any of the preceding embodiments, wherein the at least one gain-of-function missense mutation is located in any one of amino acids D538, Y537, L469, L536, P535, V534, S463, V392, and E380. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description and accompanying drawings, in which:

[0043] Figure 1 is a swimmer graph presenting data on individual patient responses to lasofoxifene and abemaciclib treatment in the ELAINE2 clinical trial (NCT04432454) at the first interim date.

[0044] Figure 2 Shown with Figure 1 Maximum tumor response of patients at the same time point (PD=progressive disease; SD=stable disease; PR=partial response).

[0045] Figure 3 is the Swimmer Plot, which shows the Figure 1 and 2 Data from the ELAINE2 clinical trial (NCT04432454) at a later interim date on patients’ responses to treatment with lasofoxifene and abemaciclib provide additional information on each subject’s pre-enrollment treatment.

[0046] Figure 4 Depicts the duration of response to prior second- and third-line cancer therapy and the duration of response to prior second- and third-line cancer therapy for each individual participant in the ELAINE 2 trial prior to enrollment. Figure 3 The duration of the test for response to the combination of lasofoxifene and abemaciclib at the same time point in the study.

[0047] Figure 5 Presents the prevalence of oncogenic mutations in a panel of genes, including ESR1 and other genes known or suspected to cause tumorigenesis at baseline in subjects enrolled in ELAINE 2, as well as the clinical benefit (CB) and median progression-free survival (mPFS) achieved by subjects subsequently treated with lasofoxifene and abemaciclib in the ELAINE 2 trial, as of the end of the trial. Figure 3 mid-term date.

[0048] Figure 6 Individual MAF kinetics of the most common mutESR1 variants are shown. Shown are the Y537C, Y537N, Y537S, and D538G variants. Variants with low baseline MAF (solid lines with diamond ends) use the left vertical axis (y-axis), and variants with high baseline MAF (dashed lines with circle ends) use the right vertical axis (y-axis). X-axis: timeline from baseline to week 4. BL: baseline; MAF: mutant allele fraction.

[0049] Figure 7 is the Swimmer Plot, showing the Figure 1-4Data on the response of patients to treatment with lasofoxifene and abemaciclib in the ELAINE2 clinical trial (NCT04432454) are shown at the interim date of the study. Additional information about each subject's pre-enrollment treatment is also shown.

[0050] Fig. 8A and 8B Shows the Figure 5 Figure 2. Exemplary copy number variation (CNV) types of various oncogenes, including CCND1, detected in circulating tumor DNA (ctDNA) of subjects in the ELAINE 2 clinical trial (NCT04432454) as of date of publication. Fig. 8A The CNV events detected for each gene are shown. Figure 8B The copy number distribution of each gene for the CNV events shown on the left panel is shown. CNVs are annotated as "focal" or "aneuploidy", or "amplification" where the focal or aneuploidy status is uncertain.

[0051] Fig. 9 Shows that after Figure 5 The prevalence of oncogenic mutations in a panel of genes (including ESR1 and other genes known or suspected to cause tumors in subjects enrolled in ELAINE 2) at baseline as of the interim date of the trial, as well as the clinical benefit (CB) and median progression-free survival (mPFS) achieved by subjects subsequently treated with lasofoxifene and abemaciclib in the ELAINE 2 trial.

[0052] Figures 10A-10B is the Swimmer Plot, which shows the patient's Figure 1-4 Data on response to treatment with lasofoxifene and abemaciclib in the ELAINE 2 clinical trial (NCT04432454) will be available at an interim date after 7 and 12 months. Fig. 10B yes Fig. 10A A copy of the study is provided, including individual patient (although de-identified) assignment numbers. Additional information on each subject's pre-enrollment treatment is also shown. DETAILED DESCRIPTION

[0053] The ongoing ELAINE 2 clinical trial is an open-label, multicenter study evaluating the combination of the third-generation SERM lasofoxifene and the CDK4 / 6 inhibitor abemaciclib in the treatment of patients with locally advanced or metastatic ER + / HER2 -Efficacy, safety, and tolerability in premenopausal and postmenopausal women with breast cancer (with an ESR1 mutation) and disease progression following first-line, second-line, or third-line hormonal therapy for metastatic disease. To be eligible, patients had to have disease progression on no more than three of the following therapies for metastatic breast cancer: aromatase inhibitors (AIs) and / or fulvestrant, either as monotherapy or in combination with any marketed CDK4 / 6i; and / or fulvestrant in combination with apellis; and / or tamoxifen; and / or exemestane / everolimus combination.

[0054] All but one (28 / 29) of the patients enrolled in the trial had disease progression after prior CDK4 / 6 inhibitor combination therapy. We have found that the combination of lasofoxifene and the CDK4 / 6 inhibitor (CDK4 / 6i) abemaciclib was well tolerated and showed strong and meaningful efficacy in these patients, reducing the incidence of advanced ER + Progression of breast cancer in women with breast cancer harboring gain-of-function mutations in the ESR1 ligand-binding domain who have progressed after prior CDK4 / 6i therapy.

[0055] Furthermore, we have found that the combination of lasofoxifene and the CDK4 / 6 inhibitor (CDK4 / 6i) abemaciclib can reduce the incidence of ER + Progression of breast cancer in women with ESR1 gain-of-function mutations and oncogenic mutations in one or more genes other than ESR1.

[0056] 5.1. Treatment

[0057] Therefore, in a first aspect, the present invention discloses a method for slowing down (or reducing) the progression of breast cancer in a patient by administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), wherein the breast cancer: (i) is 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 during prior CDK4 / 6 inhibitor therapy.

[0058] 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.

[0059] Also disclosed herein is a method for treating breast cancer in a patient by administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), wherein the breast cancer: (i) is estrogen receptor positive (ER + ); (ii) at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) 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's ER + Breast cancer is metastatic.

[0060] 5.1.1. Patients with ER + Cancer patients

[0061] According to the treatment provided, patients with ER + Breast cancer. In various embodiments, the patient is diagnosed with ER by immunohistochemistry (IHC) performed on a sample of the patient's cancer. + Breast cancer.

[0062] In some embodiments, the patient is premenopausal, perimenopausal, or postmenopausal. In some embodiments, the patient is premenopausal and has locally advanced or metastatic ER + Breast cancer. In some embodiments, the patient is perimenopausal and has locally advanced or metastatic ER + Breast cancer. In some embodiments, the patient is postmenopausal and has locally advanced or metastatic ER + Breast cancer.

[0063] In some embodiments of the provided treatment methods, the patient's breast cancer is HER2- (ER + / HER2 - In certain embodiments, the patient has locally advanced or metastatic ER + / HER2 - Breast cancer.

[0064] ESR1 gene mutation

[0065] According to the treatment methods provided, cells of the patient's cancer have acquired at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

[0066] In some embodiments, the mutation results in ligand-independent activity of the estrogen receptor. In some embodiments, the mutation results in increased 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 increases the metastatic activity of cancer. In some embodiments, the mutation increases ER + Further metastatic activity of metastatic breast cancer.

[0067] In various embodiments, the mutation is derived from a rare and undetectable pre-existing clone. In some embodiments, the mutation is de novo acquired during endocrine therapy treatment. In some embodiments, the mutation is de novo acquired after multiple lines of endocrine therapy treatment. In some embodiments, the mutation is de novo acquired after multiple lines of endocrine therapy treatment of metastatic breast cancer. In various embodiments, the mutant clone expands to become a more dominant clone during continuous endocrine therapy treatment.

[0068] 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 truncation 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.

[0069] In some embodiments, the patient has ER + A breast cancer having at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the ESR1 gene. In various embodiments, at least one mutation is located in an amino acid selected from the group consisting of D538, Y537, L536, P535, V534, L469, S463, V392, and E380, wherein the amino acids are numbered according to the ESR1 protein with NCBI accession number NP_000116.2.

[0070] 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.

[0071] In certain embodiments, the mutation is in amino acid D538. In certain preferred embodiments, the mutation is D538G. In certain embodiments, ER +The breast cancer has at least one D538G mutation and at least one amino acid mutation selected from Y537, L536, P535, V534, L469, S463, V392 and / or E380. In certain embodiments, ER + The breast cancer has at least one D538 mutation and at least one Y537 mutation.

[0072] In certain embodiments, the mutation is in amino acid Y537. + Breast cancer has at least one amino acid Y537 mutation and at least one amino acid mutation selected from D538, L536, P535, V534, L469, S463, V392 and / or E380. In some of these embodiments, mutation is Y537S, Y537N, Y537C or Y537Q. In some preferred embodiments, mutation is Y537S. In some preferred embodiments, mutation is Y537C. In some preferred embodiments, mutation is Y537N. In some preferred embodiments, mutation is Y537Q.

[0073] In certain embodiments, the mutation is located in amino acid L469. + The breast cancer has at least one mutation in amino acid L469 and at least one mutation in amino acid selected from D538, L536, Y537, P535, V534, S463, V392 and / or E380. In certain preferred embodiments, the mutation is L469V.

[0074] In some embodiments, the mutation is amino acid L536. + The breast cancer has at least one mutation in amino acid L536 and at least one mutation in 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.

[0075] In some embodiments, the mutation is at amino acid P535. + The breast cancer has at least one mutation in amino acid P535 and at least one mutation in amino acids selected from D538, Y537, L536, V534, L469, S463, V392 and / or E380. In certain embodiments, the mutation is P535H.

[0076] In some embodiments, the mutation is at amino acid V534. + The breast cancer has at least one mutation in amino acid V534 and at least one mutation in amino acids selected from D538, Y537, L536, P535, L469, S463, V392 and / or E380. In certain embodiments, the mutation is V534E.

[0077] In some embodiments, the mutation is at amino acid S463. + The breast cancer has at least one mutation in amino acid S463 and at least one mutation in an amino acid selected from D538, Y537, L536, P535, V534, L469, V392 and / or E380. In certain embodiments, the mutation is S463P.

[0078] In some embodiments, the mutation is amino acid V392. + The breast cancer has at least one mutation in amino acid V392 and at least one mutation in amino acids selected from D538, Y537, L536, P535, V534, L469, S463 and / or E380. In certain embodiments, the mutation is V392I.

[0079] In some embodiments, the mutation is amino acid E380. + The breast cancer has at least one mutation of amino acid E380 and at least one mutation of an amino acid selected from D538, Y537, L536, P535, V534, L469, S463 and / or S463. In certain embodiments, the mutation is E380Q.

[0080] 5.1.2.1 Detection of ESR1 gene mutation

[0081] In various embodiments, the patient's ER + Breast cancer has previously been identified as having at least one mutation in the ESR1 gene.Some embodiments of the methods described herein further comprise an earlier step of detecting a mutation in the ESR1 gene.

[0082] In some embodiments, massively parallel next generation sequencing (NGS) is used to detect estrogen receptor mutations in patient cancer. In some embodiments, the entire genome is sequenced. In some embodiments, a selected genome of cancer-related genes is sequenced. In some embodiments, all coding exons within a given gene set are sequenced. In some embodiments, known "hotspot" regions within a given gene set are sequenced. However, the inherent error rate of current next generation sequencing technology is as high as 1%, limiting the sensitivity and specificity of detection. In some embodiments, targeted sequencing is used to detect the presence of ESR1 mutations. Although targeted sequencing allows deeper sequencing, it is currently also limited by a 1% error rate. In some embodiments, a method with a reduced sequencing error rate is used. In a specific embodiment, a safe sequencing system (Safe-SeqS) is used, which marks each template molecule to allow for confident identification of rare variants. See Kinde et al., Proceedings of the National Academy of Sciences 108 (23): 9530-9535 (2011). In a specific embodiment, ultrasensitive double-stranded sequencing is used, which independently marks and sequences each of the two strands of the DNA double strand. See Schmitt et al., Proceedings of the National Academy of Sciences 109(36):14508-14513(2012). In some embodiments, digital droplet PCR is used, which emulsifies DNA in thousands to millions of droplets to encapsulate single DNA molecules designed with mutation-specific primers. 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).

[0083] In some embodiments, the detection of an ESR1 mutation occurs concurrently with initial diagnosis. In some embodiments, the detection of a mutation occurs during assessment of disease progression, relapse, or recurrence. In some embodiments, the detection of a mutation occurs during disease progression. In some embodiments, the detection of a mutation occurs during stable disease.

[0084] In some embodiments, one or more biological samples are obtained from a patient to detect mutations. In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a tumor biopsy. In some embodiments, the tissue sample is a biopsy of a metastasis. In some other embodiments, the biological sample is a body fluid, such as a body fluid obtained from peripheral blood (liquid biopsy). In some embodiments, the liquid biopsy includes circulating tumor cells (CTC). In some embodiments, the liquid biopsy includes cell-free DNA.

[0085] In a specific embodiment of the method provided herein, ESR1 mutations are monitored by analyzing circulating tumor DNA (ctDNA). In some implementations, ctDNA analysis is performed, for example, intermittently or regularly, during the entire treatment period. In some of these embodiments, ctDNA is extracted from a patient blood sample. In certain embodiments, ctDNA is assessed by performing digital PCR analysis on ESR1 mutations.

[0086] In some embodiments, ctDNA analysis is performed by a liquid biopsy assay as a companion diagnostic device to identify breast cancer patients who lack or have an ESR1 mutation. Exemplary liquid biopsy assays include CDx (2021, FDA-approved panel or specialized service panel [guardant360cdx.com / gene-list / ]), Guardant360ResponseTM (2021, [ncbi.nlm.nih.gov / gtr / tests / 593444 / ]) and Liquid CDx (2021, [assets.ctfassets.net / w98cd481qyp0 / wVEm7VtICYR0sT5C1VbU7 / fd055e0476183a6acd4eae6b583e3a00 / F1LCDx_Technical_Specs_072021.pdf]), the entire contents of which are incorporated herein by reference.

[0087] 5.1.3. Patients with oncogenic mutations other than ESR1 mutations

[0088] In one aspect, disclosed herein is a method for slowing down (or reducing) the progression of breast cancer in a patient by administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), wherein the breast cancer: (i) is estrogen receptor positive (ER +); (ii) having 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. In some embodiments, the oncogenic mutation is 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 body fluid (e.g., 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.

[0089] In certain embodiments, the patient's ER + The breast cancer is locally advanced. In certain embodiments, the patient's ER + Breast cancer is metastatic.

[0090] 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.

[0091] In certain embodiments, in patients with at least one or more oncogenic mutations, the use of lasofoxifene and CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib) combined therapy can provide 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 longer median progression-free survival (mPFS). In some embodiments, the use of lasofoxifene and abemaciclib combined therapy can provide patients with at least 24 weeks of mPFS. In some embodiments, the individual patient has one or more oncogenic mutation genes selected from the following: 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 detected in the patient's ctDNA or cancer.

[0092] In certain embodiments, in patients with at least one or more oncogenic mutations, the use of lasofoxifene in combination with CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib) provides clinical benefit (CB; defined as stable disease for ≥24 weeks, or confirmed partial or complete remission) for 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, the use of lasofoxifene in combination with abemaciclib provides CB (defined as stable disease for ≥24 weeks, or confirmed partial or complete remission) for about 90%, about 95%, or about 100% of the patient population. In some embodiments, the individual patient has one or more oncogenic mutation genes 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.

[0093] In some embodiments, the individual patient has one or more oncogenic mutation genes 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 in each of the genes TP53, PIK3CA, CCND1, ARID1A, FGFR1, CCNE1 and ERBB2 are detected in the ctDNA or cancer of the individual patient. In some embodiments, the oncogenic mutations are selected from single nucleotide variations (SNVs), insertions and deletions (indels), copy number variations (CNVs) (focal, aneuploidy, amplification), fusions and combinations thereof.

[0094] In some embodiments, 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 has a positive response to the combined treatment with lasofoxifene and CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib) (e.g., reaching CB, defined as stable disease for ≥24 weeks, or confirming partial or complete remission). In some embodiments, in a patient population that has a positive response (e.g., reaching CB) to the combined treatment with lasofoxifene and CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib), the prevalence of one or more genes with oncogenic mutations is at least 17%. In some embodiments, the 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.

[0095] 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.

[0096] In one aspect, disclosed herein is a method for slowing down (or reducing) the progression of breast cancer in a patient by administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), wherein the breast cancer: (i) is estrogen receptor positive (ER + ); (ii) at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) increased expression of one or more genes other than the ESR1 gene as compared to expression in non-cancerous breast cells of the subject or to 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's ER +The breast cancer is metastatic. In some embodiments, at least one of the one or more genes whose expression levels are increased in the patient's ctDNA or cancer is selected from 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, FGF R2, 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, NF 2. 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 whose expression is increased is selected from 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, 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, NK X2-1, 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, one or more genes with increased expression are detected in the patient's ctDNA or cancer, and the one or more genes are selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1, or mTOR.

[0097] In one aspect, disclosed herein is a method for monitoring a patient receiving treatment for breast cancer, by (a) determining a quantitative measurement of the mutant allele frequency (MAF) of at least one gain-of-function missense mutation in the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene (ESR1 ctDNA) in the circulating tumor DNA (ctDNA) of the patient's biological sample, wherein the quantitative measurement is performed over a period of time at a determined time interval; and (b) determining a positive predictive value (PPV) for clinical benefit (CB, defined as stable disease ≥ 24 weeks, or confirmed partial or complete remission), wherein the PPV represents responsiveness to cancer treatment, wherein the cancer treatment comprises an effective amount of lasofoxifene or a pharmaceutically 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, 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 determines that MAF decreases 4 weeks after treatment. In some embodiments, the patient determines that MAF decreases 24 weeks after treatment.

[0098] In some embodiments, the patient is a postmenopausal woman. In some embodiments, the patient is a premenopausal woman. In some embodiments, the patient has osteoporosis or is at a higher risk of developing osteoporosis.

[0099] In various embodiments, in response to the combined treatment of lasofoxifene and CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib), the patient obtains clinical benefit (CB, defined as stable condition ≥ 24 weeks, or confirms partial or complete remission), stable condition 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 longer. In various embodiments, after the patient receives lasofoxifene and CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib) combined treatment, the duration of stable condition is at least about 20%, 15% or 10% longer than the second-line or third-line treatment previously received. In some embodiments, the patient has been treated with one or more CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib) before. In some embodiments, breast cancer progresses after receiving one or more previous endocrine therapies, and the endocrine therapies are selected from SERMs other than selective ER degraders (SERDs), selective ER regulators (SERMs), optionally lasofoxifene, aromatase inhibitors (AIs), mTOR inhibitors and / or PI3K inhibitors. In some embodiments, breast cancer progresses after receiving the previous treatment of Fulvestrant. In some embodiments, breast cancer progresses after previously using sirolimus, temsirolimus, everolimus or lidaformolimus for treatment. In some embodiments, breast cancer progresses after previously using everolimus for treatment. In some embodiments, breast cancer progresses after previously using mTOR inhibitors and CDK4 / 6i (e.g., palbociclib, ribociclib, abemaciclib) for treatment. In some embodiments, breast cancer progresses after previously using everolimus and palbociclib for treatment.

[0100] 5.1.4. Previous treatment

[0101] 5.1.4.1 Previous endocrine therapy

[0102] In various embodiments of the methods provided herein, the patient has previously received one or more endocrine therapies. In certain embodiments, the patient's cancer has recurred or progressed after prior treatment.

[0103] In some embodiments, previous endocrine therapy is to use the selective ER regulator (SERM) except lasofoxifene.In various embodiments, SERM is selected from tamoxifen, raloxifene, toremifene, ospemifene, bromidrol, batexifene and ormeloxifene.In certain embodiments, previous endocrine therapy is to use tamoxifen.

[0104] In some embodiments, the previous endocrine therapy is the administration of a selective ER degrader (SERD). In some embodiments, the selective ER degrader is selected from fulvestrant, elastrant (RAD1901), ARN-810 (GDC-0810), giressant (GDC-9545), amixistrant (SAR439859), lintostrant (G1T48), LSZ102, imarustrant (LY3484356), zN-c5, D-0502, SHR9549, camistran (AZD9833) and AZD9496. In certain embodiments, the previous endocrine therapy is the administration of fulvestrant.

[0105] In some embodiments, the prior endocrine therapy was administration of an aromatase inhibitor (AI). In some embodiments, the aromatase inhibitor is selected from exemestane ( ), letrozole ( ) and anastrozole ( ).

[0106] In some embodiments, the prior endocrine therapy is ovarian suppression. In various embodiments, ovarian suppression is achieved by oophorectomy or administration of a GnRH antagonist.

[0107] In some embodiments, the patient's cancer relapses or progresses after treatment with tamoxifen. In some embodiments, the patient's cancer relapses or progresses after treatment with fulvestrant. In some embodiments, the patient's cancer relapses or progresses after treatment with an aromatase inhibitor (AI). In some embodiments, the patient's cancer relapses or progresses after treatment with an AI in combination with a CDK4 / 6i. In certain embodiments, the patient's cancer relapses or progresses after treatment with an AI in combination with palbociclib or ribociclib. In some of these embodiments, the patient's cancer relapses or progresses after multiple lines of endocrine therapy.

[0108] 5.1.4.2 Prior endocrine therapy for metastatic disease

[0109] In certain embodiments, the patient's ER + The breast cancer is metastatic and has progressed on first-line (1L), second-line (2L), or third-line (3L) therapy for metastatic disease. In some embodiments, the metastatic disease is localized or metastatic to lymph nodes, or metastatic to an internal organ (e.g., lung, pleural effusion, liver, ascites, central nervous system). In some embodiments, the patient's cancer has recurred or progressed after one or more second-line or third-line (3L) cancer therapies, such as Figure 4As shown. In some embodiments, the patient's cancer relapses or progresses after treatment of metastatic disease with at least one CDK 4 / 6 inhibitor and at least one 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 histone deacetylase (HDAC) inhibitor. In some embodiments, the patient's metastatic breast cancer relapses or progresses after treatment with at least one tamoxifen, fulvestrant, capecitabine, everolimus, apellis, talazopanib, palbociclib, ribociclib or abemaciclib (alone or in combination).

[0110] In certain embodiments, progression has occurred in one or two of the following prior treatments for metastatic breast cancer: aromatase inhibitors (AI) and / or fulvestrant as monotherapy or in combination with any commercially approved CDK 4 / 6 inhibitor (CDKi); and / or a combination of fulvestrant and apellis; and / or tamoxifen; and / or a combination of exemestane / everolimus. In some embodiments, the previously administered CDK 4 / 6 inhibitor is abemaciclib, ribociclib, or palbociclib. In some embodiments, the progression of metastatic cancer occurred in at least one prior treatment of abemaciclib, palbociclib, or ribociclib. In some embodiments, the progression of metastatic cancer occurred in a prior abemaciclib treatment. In some embodiments, the progression of metastatic cancer occurred in a prior palbociclib treatment. In some embodiments, the progression of metastatic cancer occurred in a prior ribociclib treatment. In some embodiments, the cancer has metastasized to internal organs.

[0111] In certain embodiments, the metastatic cancer progressed while taking a nonsteroidal aromatase inhibitor (AI); a SERD (eg, fulvestrant); a combination of an AI and a CDK4 / 6 inhibitor; or a combination of a SERD (eg, fulvestrant) and a CDK4 / 6 inhibitor.

[0112] In certain embodiments, progression occurs on a CDK 4 / 6 inhibitor as monotherapy or in combination therapy.

[0113] 5.2. Pharmaceutical Compositions

[0114] Estrogen receptor positive (ER + ) A method for treating breast cancer comprises administering to a patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i).

[0115] In some embodiments, lasofoxifene is administered as lasofoxifene tartrate.

[0116] In some embodiments, the CDK4 / 6i is selected from palbociclib, abemaciclib and ribociclib. In certain embodiments, the CDK4 / 6i is abemaciclib.

[0117] The term "pharmaceutically acceptable salt" refers to nontoxic pharmaceutically acceptable salts. However, other salts well 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, saccharinic acid or trifluoroacetic acid. Representative organic or inorganic bases include, but are not limited to, alkaline or cationic salts, such as benzathine penicillin, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.

[0118] Embodiments also include prodrugs of the active compounds disclosed herein. In general, such prodrugs will be functional derivatives of the compound that can be easily converted into the desired compound in vivo. Therefore, in the therapeutic methods of the present invention, the term "administering" should include treating the various diseases described with a specifically disclosed compound or with a compound that may not be specifically disclosed but is converted into a specified compound in vivo after administration to a subject. Conventional procedures for selecting and preparing suitable prodrug derivatives are described in, for example, "Design of Prodrugs", H.Bundgaard, Elsevier, 1985.

[0119] Some crystalline forms of the compounds exist in the form of polymorphs and are therefore intended to be included in the present invention. In addition, some solid forms of the active compounds exist in the form of solvates, such as solvates with water (i.e., hydrates) or common organic solvents, and embodiments of the present invention encompass such solvates.

[0120] When the preparation process of the active compound used in the method provided by the invention produces a mixture of stereoisomers, in some embodiments, these isomers are separated by conventional techniques (e.g., preparative chromatography). In some embodiments, the compound is in racemic form or as a single enantiomer or diastereomer by stereospecific synthesis or by splitting preparation. In some embodiments, the compound is split into its component enantiomers or diastereomers by standard techniques, such as by forming a stereoisomer pair with an optically active base to form a salt, followed by fractional crystallization and regeneration of free acid. In some embodiments, the compound is split by forming stereoisomer esters or amides, followed by chromatographic separation and removal of chiral auxiliary agents. Alternatively, a chiral HPLC column is used to split the compound. It should be understood that the composition comprising all stereoisomers, racemic mixtures, diastereomers, cis-trans isomers and enantiomers thereof is included in the embodiments herein.

[0121] In certain embodiments, the active compound is formulated in a separate pharmaceutical composition. In addition to the active compound, the pharmaceutical preparation or composition also contains one or more 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 efficacy of the active ingredient. The exact nature of the carrier or other material depends on the route of administration, such as oral, intravenous, transdermal, vaginal topical or vaginal ring.

[0122] In certain embodiments, the pharmaceutical composition for oral administration is in the form of tablets, capsules, powders or liquids. In certain embodiments, the tablet comprises a solid carrier, such as gelatin or an adjuvant. In certain embodiments, the liquid pharmaceutical composition comprises a liquid carrier, such as water, petroleum, animal oil, vegetable oil, mineral oil or synthetic oil. It may also include physiological saline solution, glucose or other sugar solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.

[0123] For parenteral administration, the composition is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions well using, for example, isotonic vehicles (e.g., sodium chloride injection, Ringer's injection, lactated Ringer's injection). Preservatives, stabilizers, buffers, antioxidants and / or other additives may be added as needed.

[0124] Treatment options

[0125] In the methods described herein, the term "treating / treatment" and its grammatical variants are used in the broadest sense understood in the clinical field. Therefore, these terms do not require a cure or complete alleviation of the disease, and include obtaining any clinically desired pharmacological and / or physiological effects. In certain embodiments, the effect is a partial or complete response of breast cancer, slowing down or inhibiting the progression of cancer; or causing cancer regression.

[0126] As used herein with respect to combination therapy, the term "effective amount" refers to individual doses of lasofoxifene and CDK4 / 6i, each of which, when used in combination, produces the desired effect for which they are administered.

[0127] 5.3.1. Combined administration

[0128] In some embodiments of the provided method, lasofoxifene or its pharmaceutically acceptable salt and CDK4 / 6i (e.g., abemaciclib, palbociclib, ribociclib) are used as a separate dosage form. In some of these embodiments, lasofoxifene or its salt and CDK4 / 6i are used alone simultaneously (simultaneously). In some other embodiments, lasofoxifene or its salt and CDK4 / 6i are used at different times (e.g., sequentially or according to an unrelated schedule) as a separate dosage form.

[0129] In certain embodiments, lasofoxifene is administered in a single dosage form comprising lasofoxifene or a salt thereof and a CDK4 / 6i. In certain embodiments, the CDK4 / 6i is abemaciclib.

[0130] 5.3.1.1 Administration of lasofoxifene

[0131] In various embodiments of the above-described methods of treatment, lasofoxifene or a pharmaceutically acceptable salt thereof is administered orally.

[0132] In some embodiments, lasofoxifene or a pharmaceutically acceptable salt thereof is administered to a patient by oral administration (oral; po), and the lasofoxifene dosage is about 0.5 mg / day orally to about 10 mg / day orally, for example, about 0.5 mg / day orally to about 5 mg / day orally, about 1 mg / day orally to about 5 mg / day orally, about 2 mg / day orally to about 5 mg / day orally, about 3 mg / day orally to about 5 mg / day orally, about 4 mg / day orally to about 5 mg / day orally, about 0.5 mg / day orally to about 4 mg / day oral, about 1 mg / day oral to about 4 mg / day oral, about 2 mg / day oral to about 4 mg / day oral, about 3 mg / day oral to about 4 mg / day oral, about 0.5 mg / day oral to about 3 mg / day oral, about 1 mg / day oral to about 3 mg / day oral, about 2 mg / day oral to about 3 mg / day oral, about 0.5 mg / day oral to about 2 mg / day oral, about 1 mg / day oral to about 2 mg / day oral, or about 0.5 mg / day oral to about 1 mg / day oral.

[0133] In some embodiments, lasofoxifene or its pharmaceutically acceptable salt is orally administered with about 0.5mg lasofoxifene / day. In some embodiments, lasofoxifene or its pharmaceutically acceptable salt is orally administered with about 1mg lasofoxifene / day. In some embodiments, the oral dose of lasofoxifene or its pharmaceutically acceptable salt is about 1.5mg lasofoxifene / day, about 2mg lasofoxifene / day, about 2.5mg lasofoxifene / day, about 3mg lasofoxifene / day, about 3.5mg lasofoxifene / day, about 4mg lasofoxifene / day, about 4.5mg lasofoxifene / day, about 5mg lasofoxifene / day, about 6mg lasofoxifene / day, about 7mg lasofoxifene / day, about 8mg lasofoxifene / day, about 9mg lasofoxifene / day or about 10mg lasofoxifene / day. In some other embodiments, lasofoxifene or its pharmaceutically acceptable salt is orally administered with the dosage exceeding 10mg lasofoxifene / day.

[0134] In some embodiments, lasofoxifene or its pharmaceutically acceptable salt is applied with a dosage of 0.5mg / day to 10mg / day. In some embodiments, the dosage of lasofoxifene or its pharmaceutically acceptable salt is 0.5mg lasofoxifene / day, 1mg lasofoxifene / day, 1.5mg lasofoxifene / day, 2mg lasofoxifene / day, 2.5mg lasofoxifene / day, 3mg lasofoxifene / day, 3.5mg lasofoxifene / day, 4mg lasofoxifene / day, 5mg lasofoxifene / day, 5.5mg lasofoxifene / day, 6mg lasofoxifene / day, 6.5mg lasofoxifene / day, 7mg lasofoxifene / day, 7.5mg lasofoxifene / day, 8mg lasofoxifene / day, 8.5mg lasofoxifene / day, 9mg lasofoxifene / day, 9.5mg lasofoxifene / day or 10mg lasofoxifene / day. In a currently preferred embodiment, lasofoxifene or a pharmaceutically acceptable salt thereof is orally administered at 5 mg lasofoxifene / day. In a specific embodiment, lasofoxifene tartrate is orally administered at 5 mg lasofoxifene / day.

[0135] In certain embodiments, lasofoxifene is administered once a day. In certain embodiments, lasofoxifene is administered once every two days. In certain embodiments, lasofoxifene is administered once every three days. In certain embodiments, lasofoxifene is administered once every four days. In certain embodiments, lasofoxifene is administered once every five days. In certain embodiments, lasofoxifene is administered once every six days. In certain embodiments, lasofoxifene is administered once a week. In certain embodiments, lasofoxifene is administered once every two weeks. In certain embodiments, lasofoxifene is administered once every three weeks. In certain embodiments, lasofoxifene is administered once a month.

[0136] In some embodiments, lasofoxifene is administered to the patient via vaginal ring administration. In these embodiments, lasofoxifene is administered once every two weeks. In these embodiments, lasofoxifene is administered once every three weeks. In these embodiments, lasofoxifene is administered once a month. In these embodiments, lasofoxifene is administered once every two months. In these embodiments, lasofoxifene is administered once every three months. In these embodiments, lasofoxifene is administered once every four months.

[0137] In some embodiments, lasofoxifene is administered to the ER + Breast cancer patients until their cancer progresses on treatment, they are in complete remission, or side effects become unbearable.

[0138] 5.3.1.2 Administration of Abemaciclib

[0139] In each of the above-mentioned therapeutic methods, the patient receives the combined administration of lasofoxifene (e.g., lasofoxifene tartrate) and a CDK4 / 6 inhibitor. In a typical embodiment, the CDK4 / 6 inhibitor is selected from palbociclib, ribociclib and abemaciclib. In a currently preferred embodiment, the patient is orally administered lasofoxifene tartrate with 5mg lasofoxifene / day, and with 50mg to 200mg BID, 100mg to 200mg BID or 150mg BID oral abemaciclib.

[0140] In various embodiments, abemaciclib is administered orally.

[0141] In some embodiments, abemaciclib is administered at about 25 mg / day orally to about 600 mg / day orally, e.g., about 50 mg / day orally to about 200 mg / day orally, e.g., about 25 mg / day orally, about 50 mg / day orally, about 100 mg / day orally, about 150 mg / day orally, about 200 mg / day orally, about 250 mg / day orally, about 300 mg / day orally, about 350 mg / day orally, about 400 mg / day orally, about 450 mg / day orally, or about 600 mg / day orally.

[0142] In some embodiments, abemaciclib is administered once daily. In certain embodiments, abemaciclib is administered twice daily.

[0143] In certain embodiments, abemaciclib is administered twice daily (BID). In typical embodiments, the daily dose is administered in two equal doses. In some embodiments, abemaciclib 300 mg / day is administered orally in two separate 150 mg doses (e.g., one 150 mg tablet is taken in the morning and one 150 mg tablet is taken in the evening).

[0144] In some embodiments, abemaciclib is administered at a starting dose and then adjusted downward according to a first dose reduction plan. In certain embodiments, the abemaciclib dose is subsequently reduced according to a second dose reduction plan. In certain embodiments, the abemaciclib dose is subsequently reduced according to a third dose reduction plan. In certain embodiments of these embodiments, each dose reduction is 50 mg per dose. For example, according to the first dose reduction, the dose is reduced by 300 mg / day (150 mg orally twice a day), so that a 200 mg / day dose is administered twice a day (100 mg orally twice a day).

[0145] Clinical endpoints

[0146] In various embodiments, the method comprises administering a combination of lasofoxifene and a CDK4 / 6i, wherein the combination is effective to treat ER with at least one ESR1 mutation. +Breast cancer. In certain embodiments, the method is effective in reducing the incidence of ER cancer with at least one ESR1 mutation. + Progression of breast cancer.

[0147] In certain embodiments, the combination is effective to increase ER + In certain embodiments, the combination is effective in reducing ER + In certain embodiments, the combination is effective to increase ER + In certain embodiments, the combination is effective to reduce ER + In certain embodiments, the combination is effective in prolonging ER + Progression-free survival in breast cancer patients. In certain embodiments, efficacy is determined by comparison with standard of care.

[0148] In some embodiments, the methods provided herein can prolong the survival of an ER cell line having one or more of the ESR1 mutations discussed herein. + In some embodiments, the method can reduce the selection pressure and prevent ER during treatment. + Expansion of endocrine-resistant clones in locally advanced or metastatic breast cancer.

[0149] In some embodiments, lasofoxifene or a salt thereof and abemaciclib are administered to a patient until the patient's cancer is in complete remission, progresses on treatment, or until the side effects are unbearable.

[0150] In some embodiments, lasofoxifene or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor (e.g., abemaciclib, ribociclib, palbociclib) are administered at an effective dose and for a time to provide clinical benefit (CB, defined as stable disease for ≥24 weeks, or confirmed partial or complete remission) in patients whose cancer has relapsed or progressed after one or more 2L or 3L cancer therapies, such as Figure 1 and 3And shown in Examples 2 and 3. In some embodiments, lasofoxifene and CDK 4 / 6 inhibitors (e.g., abemaciclib, ribociclib, palbociclib) are administered with an effective dose and time providing a response duration longer than any previous 2L or 3L therapy. In some embodiments, lasofoxifene and CDK 4 / 6 inhibitors (e.g., lasofoxifene and abemaciclib) stabilize CB 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 longer. In some embodiments, lasofoxifene combined with abemaciclib treatment can stabilize the 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 longer. In some embodiments, lasofoxifene and CDK 4 / 6 inhibitor (e.g., lasofoxifene / abecib) treatment can stabilize the disease (defined as stable disease ≥ 24 weeks, or confirmed partial or complete remission) 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 longer. In some embodiments, lasofoxifene and CDK 4 / 6 inhibitor (e.g., lasofoxifene / abecib) treatment can achieve complete or partial remission of CB, wherein the patient's tumor is significantly reduced, such as Figure 2 shown.

[0151] Example

[0152] 6.1. Example 1: Phase 2 Clinical Study (ELAINE 2)

[0153] The phase 2 clinical study NCT04432454 (ELAINE 2) is currently underway to evaluate the combination of lasofoxifene and abemaciclib in patients with locally advanced or metastatic estrogen receptor-positive (ER-positive) ovarian cancer with ESR1 mutations. + ) / human epidermal growth factor 2 negative (HER2-)(ER + / HER2 - The study was designed to evaluate the efficacy, safety, and tolerability of levofloxacin in postmenopausal and selected premenopausal women with breast cancer. As described in Section 5.1.1 below, the study enrolled patients whose tumors had progressed on prior CDK4 / 6 inhibitor therapy.

[0154] 6.1.1. Summary of experimental observations

[0155] ELAINE 2 is an open-label, phase 2, multicenter trial designed to evaluate the safety and efficacy of LAS in combination with the CDK4 / 6i abemaciclib (Abema). + / HER2 - Premenopausal and postmenopausal women with mBC (metastatic breast cancer) and acquired ESR1 mutations (determined by ctDNA testing) whose disease progressed after first-line or two-line hormonal therapy for metastatic disease (with or without CDK4 / 6i, including Abema). Patients received oral lasofoxifene (LAS) 5mg / day and abemaciclib 150mg 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).

[0156] 29 patients were enrolled at 16 trial sites in the United States (October 2020 to June 2021). The mean age was 58.3 years (range, 35-79 years); 86% were Caucasian. Most patients had disease progression after at least 2 hormonal therapies (80%). All but 1 patient had received prior CDK4 / 6i therapy, 72% had received fulvestrant (FVT); 48% had received metastatic chemotherapy. Four patients discontinued the trial due to adverse events (AEs, n=2), consent withdrawal (n=1), or investigator withdrawal (n=1). No deaths occurred during the study, and few grade 3 / 4 AEs were observed. The most common AEs were diarrhea, nausea, and leukopenia. The abemaciclib dose was reduced from 150 mg BID to 100 mg BID in 5 patients. As of the first interim date, 11 patients had disease progression and 14 patients continued treatment. The median PFS after review was 13.9 months (95% CI, 8.0-NE), the ORR was 33.3% (95% CI, 16.3-56.3), with 6 patients confirmed with partial response, and the CBR was 62.1% (95% CI, 44.0-77.3).

[0157] like Figure 1-4 Results showed that patients derived clinical benefit (CB, defined as stable disease for ≥24 weeks or confirmed partial or complete response) from the combination of lasofoxifene and abemaciclib, even after disease progression following prior treatment with a cyclin-dependent kinase 4 / 6 inhibitor (CDK4 / 6i), including prior treatment with ribociclib, palbociclib, and / or abemaciclib.

[0158] Conclusion: Lasofoxifene plus abemaciclib combination therapy is well tolerated and effective in patients with ER + / HER2 -Strong and meaningful efficacy was demonstrated in women with metastatic breast cancer (mBC) and ESR1 mutations who had progressed on prior CDK4 / 6i therapy.

[0159] Study design

[0160] This is an open-label, multicenter study evaluating the combination of lasofoxifene and abemaciclib in the treatment of patients with locally advanced or metastatic ER + / HER2 - Efficacy, safety, and tolerability in premenopausal and postmenopausal women with breast cancer (with an ESR1 mutation) and disease progression following first-line, second-line, or third-line hormonal therapy for metastatic disease. To be eligible, patients had to have disease progression on no more than three of the following therapies for metastatic breast cancer: AIs and / or fulvestrant, either as monotherapy or in combination with any marketed CDK4 / 6i; and / or the combination of fulvestrant and apellis; and / or tamoxifen; and / or the combination of exemestane / everolimus.

[0161] On the day of enrollment (Day 1), subjects took lasofoxifene 5 mg orally once daily and abemaciclib 150 mg orally twice daily. Study medication continued until documented breast cancer progression or withdrawal from the study for any reason.

[0162] Treatment continued until disease progression, death, radiographic or clinical evidence of unacceptable toxicity, or withdrawal from the study for any reason. Enrolled subjects were observed every 2 weeks for the first two months of treatment and then monthly until disease progression. Efficacy assessments were performed every 8 weeks. Safety assessments were performed at weeks 2, 4, 6, and 8 after enrollment and then monthly until disease progression.

[0163] 6.1.3. Medication schedule

[0164] Subjects will take 5 mg lasofoxifene (one tablet) orally once daily and 150 mg abemaciclib (3 tablets) orally twice daily, either with or without food. Study medication will continue until breast cancer progression is documented or the patient is withdrawn from the study for any reason.

[0165] Lasofoxifene

[0166] The active drug 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.

[0167] Chemical Structure:

[0168]

[0169] Molecular formula: C 28 H 31 NO2·C4H6O6

[0170] Molecular weight: 563.64 Daltons

[0171] Physical appearance: white to off-white solid

[0172] Lasofoxifene is supplied as a white to off-white solid 5 mg tablet using the D-(-)-tartrate salt.

[0173] Abemaciclib

[0174] 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.

[0175] Molecular formula: C 27 H 32 F2N8

[0176] Molecular weight: 506.59 Daltons

[0177] Physical appearance: white to yellow powder

[0178] 6.1.3.1 Dose Adjustment for Treatment Toxicity

[0179] Lasofoxifene

[0180] The dose was not reduced for any toxicity associated with lasofoxifene. If a grade 3 or 4 adverse event related to lasofoxifene occurred, treatment was suspended until the toxicity resolved to < grade 2 or baseline, and lasofoxifene was resumed at the designated dose. If the patient required fasting, lasofoxifene and abemaciclib were suspended until the subject could take oral fluids. If the subject could not tolerate lasofoxifene or did not take lasofoxifene for 3 consecutive weeks, they should be withdrawn from the study.

[0181] Appearance: White to yellow powder

[0182] Abemaciclib

[0183] Two dose adjustments of abemaciclib were permitted for adverse responses:

[0184] Starting dose – 150 mg twice daily (3 tablets twice daily)

[0185] First dose reduction – 100 mg twice daily (2 tablets twice daily)

[0186] Second dose reduction – 50 mg twice daily (1 tablet twice daily)

[0187] · Third dose reduction – not allowed

[0188] If a subject is unable to tolerate abemaciclib 50 mg twice daily, that 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 needs to be withdrawn from the study. Table 1 below summarizes dose modifications for treatment toxicity.

[0189]

[0190]

[0191] 6.1.4. Primary and secondary endpoints

[0192] The primary endpoint was to evaluate the efficacy of lasofoxifene plus abemaciclib in the treatment of patients with locally advanced or metastatic ER cancer based on the incidence, severity, and mortality of adverse events. + / HER2 - Safety and tolerability in postmenopausal women with breast cancer and ESR1 mutations. The pharmacokinetic endpoint was to determine if there were any drug interactions between lasofoxifene and abemaciclib compared with steady-state drug concentrations achieved in previous clinical trials.

[0193] Secondary endpoints included 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 duration of response. For subjects with measurable disease at baseline, progression was defined according to RECIST criteria.

[0194] 6.1.5. Inclusion and Exclusion Criteria

[0195] Inclusion criteria included:

[0196] 1. Premenopause or postmenopause.

[0197] Postmenopausal women are defined as:

[0198] a. Aged ≥ 60 years and no vaginal bleeding in the past year, or

[0199] b. < 60 years of age and have "premature menopause" or "premature ovarian failure" manifested by secondary amenorrhea for at least 1 year with follicle stimulating hormone (FSH) and estradiol levels in the postmenopausal range according to institutional criteria, or

[0200] c. Surgical menopause, bilateral oophorectomy.

[0201] Note: Premenopausal women who meet all other inclusion criteria must maintain ovarian suppression (eg, Lupron) during the study and are advised to use appropriate contraception to prevent pregnancy.

[0202] 2. If possible, a biopsy of metastatic breast cancer tissue will be obtained for evaluation by a local laboratory based on the American Society of Clinical Oncology / College of American Pathologists guidelines using slides, paraffin blocks, or paraffin specimens to provide ER + and HER2 - Histologic or cytologic confirmation of disease. If a biopsy is not possible, confirmation that the subject is ER positive from tissue obtained at the time of initial diagnosis must be provided. + and HER2 - .

[0203] 3. Locally advanced or metastatic breast cancer with radiographic or clinical evidence of disease progression after first-line and / or second-line hormonal therapy for metastatic disease. No more than 2 of the following endocrine therapies for metastatic breast cancer may have caused disease progression: aromatase inhibitors (AI) and / or fulvestrant, as monotherapy or in combination with any commercially approved CDK4 / 6i; and / or fulvestrant in combination with apellis; and / or tamoxifen; and / or exemestane / everolimus in combination. (Note: Subjects should stop using any CDK4 / 6i for at least 21 days prior to starting study treatment)

[0204] 4. Subjects must have no evidence of disease progression for at least 6 months during their first hormonal therapy for advanced breast cancer.

[0205] 5. At least one or more of the following ESR1 point mutations assessed in cell-free circulating tumor DNA (ctDNA) obtained from blood or tissue specimens: Y537S, Y537C, D538G, E380Q, S463P, V534E, P535H, L536H, L536P, L536R, L536Q, or Y537N. Note: Sponsor's blood ctDNA assay must be used, but tissue sequencing (if done) can be performed with a validated commercial assay.

[0206] Note: This inclusion criterion is met if a positive ESR1 mutation is detected in tissue or ctDNA using a validated commercial assay prior to or at disease progression. However, blood must still be obtained for genomic analysis using the sponsor's ctDNA assay.

[0207] 6. Locally advanced or metastatic breast cancer, with measurable (according to RECIST 1.1 [Eisenhauer et al. New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1). European Journal of Cancer. 2009; 45: 228-47]) or non-measurable lesions.

[0208] 7. Subjects who have received one cytotoxic chemotherapy regimen for metastatic disease and subjects who have received one cytotoxic chemotherapy regimen in neoadjuvant or adjuvant therapy before participating in the trial can be enrolled, but must not have any acute toxicity of chemotherapy before participating in the study, excluding alopecia and grade 2 peripheral neuropathy. A washout period of at least 21 days is required between the last chemotherapy dose and entry into the study.

[0209] 8. Patients with stable brain metastases from breast cancer are allowed to enroll as long as they have received radiotherapy and there is no evidence of progression of brain metastases within at least 3 months after completing radiotherapy.

[0210] 9. ECOG physical fitness score is 0 or 1.

[0211] 10. Adequate organ function, as manifested by:

[0212] a. Absolute neutrophil count (ANC) ≥ 1,500 cells / mm 3

[0213] b. Platelet count ≥ 100,000 cells / mm 3

[0214] c. Hemoglobin ≥8.0g / dl

[0215] d. ALT and AST levels ≤3 upper limit of normal (ULN) or ≤5 in case of liver metastasis

[0216] e. Serum total bilirubin ≤ 0.5X ULN (≤ 3.0X ULN for subjects known to have Gilbert's syndrome)

[0217] f. Alkaline phosphatase level ≤ 3X ULN

[0218] g. Creatinine clearance is 40 ml / min or higher (calculated by Cockcroft-Gault formula)

[0219] h. International normalized ratio (INR) and activated partial thromboplastin time (aPTT) <2.0X ULN

[0220] 11. Ability to swallow pills.

[0221] 12. Be able to understand and voluntarily sign a written informed consent prior to any screening procedures.

[0222] Exclusion criteria. Subjects meeting any of the following criteria will be excluded from the trial.

[0223] 1. Lymphangitic carcinomatosis involving the lungs.

[0224] 2. Visceral crisis requiring cytotoxic chemotherapy assessed by the investigator.

[0225] 3. Received radiotherapy within 30 days before participating in the trial, except for local radiotherapy for analgesia or lytic lesions with risk of fracture, which can be completed within 7 days before participating in the trial. Subjects must have recovered from radiotherapy toxicity before participating in the trial.

[0226] 4. Subjects with known inactivating RB1 mutations or deletions (RB1 mutation screening is not required to participate in the trial).

[0227] 5. History of long QTC syndrome or QTC>480 milliseconds.

[0228] 6. History of pulmonary embolism (PE) or deep vein thrombosis (DVT) or any known thrombotic tendency in the past 6 months. Subjects who are using anticoagulants for maintenance therapy can be enrolled as long as DVT and / or PE occurred more than 6 months before enrollment and there is no evidence of active thrombosis. Low-dose ASA is allowed.

[0229] 7. Subjects who are concurrently taking strong CYP3A4 inhibitors, such as clarithromycin, telithromycin, nefazodone, itraconazole, ketoconazole, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, tipranavir.

[0230] 8. Subjects receiving treatment with 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.

[0231] 9. Any major comorbidity that may affect the study or subject safety. Since the occurrence of interstitial lung disease (ILD) has been reported with CDK4 / 6i, subjects with a history of ILD or severe dyspnea at rest or requiring oxygen therapy should not participate in the study.

[0232] 10. Subject has active systemic bacterial or fungal infection (requiring intravenous [IV] antibiotics at the time of initiation of treatment).

[0233] 11. History of positive human immunodeficiency virus (HIV) or hepatitis B virus (HBV) test [can be enrolled without screening].

[0234] 12. Subjects with hepatitis C virus (HCV) and still have viral load at screening. Subjects who have previously received treatment and achieved HCV cure (no viral load) can participate in the study.

[0235] 13. History of malignant tumor in the past 5 years (excluding breast cancer), except for basal cell carcinoma or squamous cell carcinoma of the skin or early cervical cancer cured by surgery.

[0236] 14. Positive pregnancy test (premenopausal only).

[0237] 15. History of non-compliance with medical regimen.

[0238] 16. Unwilling or unable to abide by agreements.

[0239] 17. Participation in any clinical research trial involving investigational drugs or devices within the past 30 days.

[0240] 6.1.6. Efficacy analysis

[0241] PFS was presented as Kaplan-Meier curves, and median PFS was estimated. Clinical benefit rate (CBR) was defined as the percentage of subjects with complete or partial response or stable disease for ≥24 weeks, expressed with 95% confidence intervals. ORR, defined as the percentage of subjects with complete or partial response, was summarized in a similar manner. Duration of response and duration of response (DoR) were provided for each individual responder.

[0242] 6.1.7. Security analysis

[0243] For the safety population, descriptive summaries of AEs, clinical laboratory data, vital signs, and ECGs are provided.

[0244] Verbatim descriptions of AEs reported during the study were mapped to the appropriate system organ class and preferred term using the Medical Dictionary for Regulatory Activities (MedDRA). All reported AEs were tabulated and graded according to CTCAE version 5.0. AEs were summarized by worst grade and grade for each subject. All treatment-emergent adverse events (TEAEs) (i.e., adverse events occurring during or after the first dose of study drug) were summarized in frequency tables. Treatment-emergent serious AEs and TEAEs that led to early discontinuation of the study were listed and summarized in frequency tables. The number and proportion of subjects who discontinued one or both study treatments prematurely due to AEs were reported with 95% confidence intervals. Subjects who experienced major adverse events were similarly summarized. For the 24 evaluable subjects, the upper half width of the 95% confidence interval for the Wilson score was estimated to be within 20%. The probability of an adverse event occurring at least once with an incidence of 7% was 0.82.

[0245] All adverse events resulting in death are listed by subject and are accompanied by narratives.

[0246] Clinical laboratory test results were summarized using descriptive statistics and summarized in cumulative shift tables as absolute values ​​and change from baseline values.

[0247] 6.1.8. Analysis of pharmacokinetic variables

[0248] Pharmacokinetic sampling of lasofoxifene and abemaciclib concentrations was performed before each visit, starting from Visit 0 (Day 1) until the last / ET visit. Pharmacokinetic concentrations of lasofoxifene, abemaciclib, and 3 abemaciclib metabolites (LSN2839567, LSN3106726, and LSN3106729) were summarized and presented as mean, median, SD, and range for each visit and compared with previously obtained PK results.

[0249] 6.2. Example 2: First interim results of the ELAINE 2 clinical trial

[0250] A total of twenty-nine (29) patients were enrolled. Patient disposition is summarized in Table 2. Patient demographics and baseline characteristics are summarized in Table 3, and patients' prior cancer treatments are summarized in Table 4.

[0251] 6.2.1 Patient Disposition

[0252] Table 2

[0253]

[0254] 6.2.2 Demographics and baseline characteristics n = 29

[0255]

[0256] 6.2.3 Previous breast cancer treatment

[0257]

[0258] *Data are presented as n (%) unless otherwise stated. CDK4 / 6i, cyclin-dependent kinase 4 / 6 inhibitor. 26 patients (89.7%) had received prior radiotherapy.

[0259] 6.2.4. ELAINE 2 Swimmer Diagram

[0260] Figure 1 Patient responses to lasofoxifene and CDK 4 / 6 inhibitor treatment as of the first interim date are summarized.

[0261] refer to Figure 1 As of the first interim date, approximately 68.9% (20 / 29) of subjects treated with laso / abema had clinical benefit (CB), stable disease, and a complete or partial response for at least 24 weeks after treatment (vertical dashed line). Of these patients, approximately 65% ​​(13 / 20) had an ongoing response (arrow) as of the first interim date. Patients 29, 21, 16, 6 ( Figure 1 Patients marked with an asterisk in the table had progressed on abemaciclib prior to enrollment. Of note, 75% (3 / 4) of these patients achieved CB and stable disease until weeks 40 (patient 16), 48 (patient 21), and 68 (patient 29). Patients 16 and 6 had disease progression and withdrew at weeks 40 and 8, respectively.

[0262] Approximately 48% (14 / 29) of patients had disease progression (squares) while receiving fulvestrant treatment prior to enrollment. These patients generally respond well to laso / abema treatment. As of the first interim date, approximately 64% (9 / 14) achieved CB and had complete or partial remission to laso / abema treatment, lasting up to 62 weeks. For example, patients 28, 27, 24, 19, and 14 had stable disease, with remission to laso / abema (arrows) lasting to 62 weeks, 58 weeks, 54 weeks, 50 weeks, 42 weeks, and 38 weeks, respectively. Patients 15 and 13 had stable disease with progression at 36 weeks and 32 weeks, respectively.

[0263] All patients had at least one ESR1 missense mutation at enrollment. Approximately 69% (20 / 29) of patients had the Y537S mutation, of which approximately 65% ​​(13 / 20) achieved CB, complete or partial remission, and stable disease until 62 weeks. Patients 28, 27, 25, 24, 23, 21, 20, 19, 18, 17, 11, and 10 had stable disease, with sustained remissions lasting until 62 weeks, 58 weeks, 54 weeks, 48 ​​weeks, 48 ​​weeks, 48 ​​weeks, 48 ​​weeks, 40 weeks, 40 weeks, 40 weeks, 32 weeks, and 32 weeks, respectively. Patients 15 and 13 had stable disease with progressive disease at 32 weeks.

[0264] Notably, 75% (12 / 16) of patients with visceral metastases benefited from laso / abema treatment and achieved CB, even though their cancer had metastasized to visceral organs before enrollment.

[0265] 6.2.5. Progression-free survival

[0266] Table 5 summarizes the progression-free survival (PFS) of the patients.

[0267]

[0268] 6.2.6. Tumor Response Waterfall Plot

[0269] Figure 2 Preliminary data of the maximum tumor response evaluated as of the first interim date are shown. The maximum percentage change of the sum of the target lesion sizes of patients with complete or partial remission to laso / abema treatment was up to 80%. 9 (9 / 18) patients with measurable lesions had partial remissions, and the objective response rate (ORR) was 50% (95% confidence, 29.0-71.0). Table 6 summarizes the ORR of the patients (ORR=50%), and Table 7 summarizes the DOR and TTR of the patients.

[0270] 6.2.7. ORR, DOR, and TTR

[0271] Table 6 summarizes the ORR of the patients (ORR=50%), and Table 7 summarizes the DOR of the patients.

[0272]

[0273]

[0274] 1 Number of subjects with measurable target lesions

[0275] 2 Event or inspection date – first PR date

[0276] 3 First PR date – randomization date + 1

[0277] 6.2.8. Clinical benefit rate (CBR) N = 29

[0278] Table 8 summarizes the clinical benefit rate (CBR) of the patients.

[0279]

[0280] * SD quantity does not include PR quantity

[0281] 6.2.9. Number of subjects experiencing the most common adverse events (AEs)

[0282] Table 9 summarizes the subjects with the highest grade counts.

[0283]

[0284]

[0285] 6.2.10. Number of subjects experiencing hematological adverse events (AEs)

[0286] Table 10 summarizes the number of subjects experiencing hematologic adverse events (AEs).

[0287]

[0288] 6.2.11. Number of subjects experiencing hepatic adverse events (AEs)

[0289] The number of subjects who experienced hepatic adverse events is summarized in Table 11.

[0290]

[0291]

[0292] 6.2.12. Grade 3 and 4 Toxicity (n=29)

[0293] Table 12 summarizes grade 4 and 5 toxicities.

[0294]

[0295] 6.2.13. Adverse events (AEs) of special interest

[0296] Table 13 summarizes adverse events (AEs) of special interest.

[0297]

[0298]

[0299] 6.2.14. Summary of Dose Reductions

[0300] Lasofoxifene dose not reduced as per protocol

[0301] · Abemaciclib

[0302] o No patient was reduced to 50 mg BID

[0303] o Reduce from 150 mg BID to 100 mg BID

[0304] 4 cases due to AE

[0305] oHyponatremia

[0306] o Dizziness, fatigue, vomiting, weight loss

[0307] o Increased creatinine

[0308] oAnorexia, fatigue, nausea, muscle weakness

[0309] 1 case due to investigator's discretion

[0310] 6.2.15. Comparison of ELAINE 2 interim PFS data with other trials

[0311] As shown in Table 14 below, compared to published and publicly presented data in patients who had received CDK4 / 6i treatment, the combination of lasofoxifene and abemaciclib provided a longer median PFS and comparable efficacy to CDK4 / 6i-naive patients; more specifically, compared to abemaciclib alone, both in CDK-naive patients (Monarch 1 trial) and in the post-CDK population (Abema trial); Piqray / fulvestrant in patients with PIK3CA mutations (Bylieve trial); camistran plus palbociclib (Serena-1 trial), amcenestrant / CDK4 / 6i (Ameera-1 trial); fulvestrant plus abemaciclib (Monarch 2 trial), and fulvestrant plus palbociclib (Paloma-3 trial)

[0312]

[0313] 6.2.16. Efficacy in patients whose tumors have progressed after prior CDK4 / 6i therapy

[0314] Many patients whose tumors had progressed after prior treatment with CDK4 / 6 inhibitors have been enrolled. The combination of lasofoxifene and abemaciclib reduced progression in patients whose breast cancer had progressed after prior treatment with abemaciclib.

[0315] Patient 29

[0316] ο40 years old, bone metastasis: letrozole for 3 years; letrozole / palbociclib for 3 years;

[0317] Fulvestrant / abecib 12 weeks; capecitabine 7 months

[0318] οD538G 6.855% mutant allele fraction (MAF)

[0319] Stable disease at 68 weeks

[0320] Patient 21

[0321] ο42 years old: chemotherapy / Herceptin; tamoxifen for 10 years; letrozole / palbociclib for 2 years and 8 months; abemaciclib for 16 weeks

[0322] ο24 mm liver mass

[0323] οY537S 0.248% MAF

[0324] ο Confirmed partial response at 48 weeks, 71% reduction in liver lesions at 40 weeks

[0325] Patient 16

[0326] ο78 years old, taking letrozole / palbociclib for 2 years and 2 months; fulvestrant / abecib for 1 year and 3 months; capecitabine for 1 month

[0327] ο 18 mm target liver lesions, pleura, lymph nodes and bone metastases

[0328] οD538G 0.3% MAF

[0329] o Progressive disease at 40 weeks, stable disease (6% reduction in target lesions)

[0330] Patient 6

[0331] ο59 years old, taking fulvestrant / abecib for 2 years; capecitabine for 1 month

[0332] ο35 mm liver metastasis

[0333] οD538G 1.28MAF

[0334] o Progression at 8 weeks (stable liver lesions but new lesions)

[0335] 6.3 Example 3: Second interim results of the ELAINE 2 clinical trial

[0336] As of the second, later interim date, two of the four patients (50%) who had progressed on prior abemaciclib therapy continued to benefit from the combination of lasofoxifene and abemaciclib (laso / abema). Figure 3 Patient 29 (2011-01) had a D538G mutation and had previously been treated with palbociclib and fulvestrant, and remained in remission with stable disease until week 88. Patient 21 (2005-02) had a Y537S missense mutation, visceral metastases, and had disease progression after prior treatment with abemaciclib and palbociclib, and had stable disease after laso / abema until week 56, when her cancer did not progress. Figure 3As shown, patients whose disease progressed after receiving other CDK 4 / 6 inhibitors (e.g., palbociclib, ribociclib) prior to enrollment also achieved clinical benefit (CB). The majority of patients received palbociclib, with 70% (17 / 24) achieving CB, while 100% (2 / 2) of patients previously treated with ribociclib achieved CB. The data suggest that patients with ESR1 mutations who progressed after receiving CDK4 / 6i therapy have a high likelihood of achieving clinical benefit from the combination of lasofoxifene and abemaciclib, regardless of prior CDK 4 / 6i treatment. Patient 2004-03 had visceral metastases and had previously received palbociclib, achieving CB and stable disease, but withdrew early at 56 weeks due to noncompliance (diamonds). Table 15 summarizes the second interim results for patients enrolled after prior progression on abemaciclib.

[0337]

[0338]

[0339] Patients in the study who had received other related treatments prior to laso / abema treatment also benefited from the combination therapy. Figure 3 , the patient 2001-01 had been treated with apellix, and achieved CB at 32 weeks with stable disease. The patient 2009-02 had been treated with apellix, and achieved CB and stable disease to 36 weeks after combined treatment with lasofoxifene and abemaciclib. The patient 2016-01 had been treated with a PARP inhibitor (talzenna), and achieved CB and confirmed partial remission at 32 weeks after combined treatment with lasofoxifene and abemaciclib. The patient 2018-01 had been treated with ribociclib, and achieved CB and stable disease at 60 weeks after combined treatment with lasofoxifene and abemaciclib. The patient 2004-06 had previously received ribociclib / fulvestrant for 9 months, and confirmed partial remission after treatment with lasofoxifene and abemaciclib, and the treatment lasted for 64 weeks.

[0340] Patients in this study generally achieved better responses on the combination of lasofoxifene and abemaciclib than on their previous 2L and 3L therapies, which is surprising because historically, later-line therapies often provide shorter-lasting benefits than patients’ previous therapies. Figure 4As shown, patients treated with laso / abema had longer duration of stable disease than those who received prior 2L and 3L therapy. As of the second interim date, subjects treated with laso / abema had an average duration of stable disease of approximately 8.7 months. All 29 patients had received prior 2L therapy, with an average duration of stable disease of approximately 4.2 months. Nine of these subjects also received 3L therapy prior to enrollment, with an average duration of stable disease of approximately 7.2 months. The duration of stable disease in patients treated with the combination of lasofoxifene and abemaciclib increased by an average of approximately 51.2% compared with prior 2L therapy and by approximately 16.9% compared with prior 3L therapy. For example, patient 2011-00001 (patient 29) had stable disease for 22 months after treatment with laso / abemicaiclib, compared with 7 months of stable disease on prior 2L therapy and 3 months of stable disease on prior 3L therapy. Patient 2005-00002 (patient 21) had stable disease for 14 months after treatment with lasofoxifene and abemaciclib, compared with 4 months of stable disease on prior 3L therapy. Patient 2015-00001 had stable disease for 19 months after treatment with laxodil / abecib, whereas previous 2L and 3L treatments lasted 10 and 2 months, respectively.

[0341] The results showed that the combination of lasofoxifene and abemaciclib had acceptable tolerability, safety, and efficacy for patients with metastatic breast cancer who had at least one ESR1 mutation and whose disease had progressed after treatment with one or more CDK4 / 6 inhibitors and endocrine therapy.

[0342] 6.4. Example 4: ESR1 mutations in circulating tumor DNA (ctDNA) of patients in the ELAINE 2 clinical trial

[0343] This example investigates the efficacy of lasofoxifene and abemaciclib in the ELAINE 2 clinical trial in ER patients. + / HER2 - ESR1 mutations in circulating tumor DNA (ctDNA) of 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).

[0344] ER + Long-term endocrine therapy (ET) for breast cancer often leads to acquired ESR1 mutations (mutESR1), which cause endocrine resistance, tumor progression, and poor prognosis. +ELAINE 2 is an open-label, phase 2, multicenter trial designed to evaluate the efficacy of lasofoxifene (LAS [selective estrogen receptor modulator]) plus abemaciclib (Abema [CDK4 / 6i], provided by Eli Lilly) for the treatment of ER. + / HER2 - The safety and efficacy of LAS in patients with mutESR1 mBC who had progressed after prior ET were evaluated. Preliminary data of LAS combined with 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 acceptable safety and tolerability.

[0345] Methods: ELAINE 2 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 study withdrawal. ctDNA was assessed by the Sysmex-Inostics SafeSeq assay, which detects low allele fraction of mutESR1 at BL, every 4 weeks, and at the end of treatment. MAF changes from BL to week 4 were described as decreased (ESR1 MAF decreased or not detected [ND]), increased (MAF increased), or equivocal (some MAF trends of both increase and decrease in patients with polyclonality [>1mutESR1]). The correlation of MAF changes at week 4 with CB at week 24 was explored.

[0346] A total of 29 patients (median of 2 prior metastatic therapies: 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 29 patients had evaluable BL and week 4 ctDNA results: 21 patients had a decrease in MAF (81%, 14 cleared [54% with ND]), 3 (12%) had an increase, and 2 (8%) had equivocal ESR1 MAF changes, as described in Table 16 below.

[0347]

[0348]

[0349] CI, confidence interval; MAF, mutant allele fraction; ND, not detected; NPV, negative predictive value; PPV, positive predictive value.

[0350] *Sensitivity and specificity analyses did not include allele results.

[0351] Of the four patients who had progressed on prior Abema-based therapy, three had mESR1 clearance observed at week 4, and all three achieved CB. MAF reduction / clearance was frequently observed for all commonly detected mESR1 variants, including Y537S, D538G, Y537N, and Y537C variants, after four weeks of LAS plus abema. Figure 6 ). CB at 24 weeks was observed in 17 patients with decreased ESR1 MAF, 2 patients with increased ESR1 MAF, and 1 patient with equivocal MAF change. The sensitivity of predicting CB based on the direction of ESR1 MAF change was 89.5%, specificity was 20%, and the positive likelihood rPBatio (LR+) was 1.1. The positive predictive value (PPV) of CB with decreased MAF was 81%, while the negative predictive value (NPV) of increased MAF was 33%. Of the 14 (54%) ND ESR1 MAF patients, 13 had CB, so increased ESR MAF had a sensitivity of approximately 87%, a specificity of approximately 50%, a PPV of approximately 93%, and an NPV of approximately 33%. Compared with decreased MAF, mutESR1 clearance at week 4 had similar sensitivity (approximately 87%) and higher PPV (approximately 93%) for CB prediction, with an LR+ of 1.7. All 9 patients with objective response (OR) showed complete clearance of mESR1 (n=5) or a decrease of ESR1 MAF of 50%-93% (n=4) at week 4.

[0352] In the ELAINE 2 clinical trial, 81% of patients achieved mutESR1 reduction / clearance (ND) after four (4) weeks of LAS plus Abema treatment, which was associated with clinical benefit. All mutESR1 detected appeared to be a target for this therapy. High sensitivity and favorable PPV were observed in patients with reduced MAF, and even more so in patients with ND MAF; however, increased MAF had lower specificity and did not predict treatment failure.

[0353] In conclusion, analysis of ctDNA data from ELAINE 2 showed that mutESR1 variants, including the difficult-to-treat Y537S, were reduced / cleared in the majority (~81%) of patients after 4 weeks of LAS plus Abema. ESR1 MAF reduction / clearance was associated with CB and OR, with high sensitivity (89%) and favorable PPV (81%) for predicting CB.

[0354] The PPV for mutESR1 clearance was higher (93%). Increased MAF was less specific and did not predict treatment failure. The results suggest strong targeting of mutESR1 by LAS plus Abema. Overall, the results suggest that ESR1 liquid biopsy assessment is a suitable non-invasive surrogate marker for monitoring patient response or resistance to this novel LAS-Abema combination.

[0355] 6.5. Example 5: Oncogenic mutations in circulating tumor DNA (ctDNA) from patients in the ELAINE 2 clinical trial

[0356] This example investigates the efficacy of lasofoxifene plus abemaciclib in the ELAINE 2 clinical trial in ER patients. + Oncogenic mutations in genes other than ESR1 in circulating tumor DNA (ctDNA) of patients with HER2- / metastatic breast cancer (mBC). Figure 5 The gene sets being tested are summarized, and these genes are present in the ELAINE 2 patient population. ESR1 gain-of-function mutations (mutESR1; top row) are included in the positive control. The data in this example show the correlation between the prevalence of oncogenic mutations in genes other than mutESR1 and clinical benefit (CB) and median progression-free survival (mPFS).

[0357] At baseline (BL), approximately 5 mL of whole blood samples were collected from individual ELAINE 2 clinical trial patients in Streck Cell-Free DNA Blood Collection Tubes (BCT). The individual patients had been previously diagnosed with ER by an oncologist. + mBC. In order to meet the eligibility requirements, patients must have a past medical history indicating the presence of mutESR1, or have been detected with mutESR1 (whether intrinsic or acquired) using the assay described in Example 4. Oral lasofoxifene 5mg / day and abemaciclib 150mg BID until disease progression, death, unacceptable toxicity or withdrawal from the study. The samples are processed for plasma separation and cell-free DNA (cfDNA) extraction, which may contain circulating tumor DNA. About 5-30ngcfDNA is used to prepare a sequencing library enriched by hybrid capture. The enriched library is then sequenced using next-generation sequencing, for example on an Illumina NextSeq 550 platform. Sequencing data are analyzed using a bioinformatics process designed to detect single nucleotide variations (SNVs), insertions and deletions (indels), copy number amplifications (CNAs) and fusions. Pathogenic (e.g., carcinogenic) germline variations and somatic variations are detected.

[0358] like Figure 5As shown, a total of 41 genes with one or more oncogenic mutations were detected in blood samples of the ELAINE 2 patient population. The prevalence of each gene mutation was at least about 3% of the patient population. The mutation was a germline mutation, a somatic mutation, or both. In addition to ESR1, the most common genes with one or more oncogenic mutations 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 one or more oncogenic mutations in these genes achieved clinical benefit (CB) after treatment with lasofoxifene and abemaciclib, i.e., stable disease and mPFS of at least 24 weeks. For example, approximately 100% of patients with CCND1 oncogenic mutations achieved CB and mPFS of at least 56 weeks, while 76% of patients with TERT oncogenic mutations achieved CB and mPFS of at least 44 weeks. Overall, patients with oncogenic mutations in at least one gene 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 can achieve mPFS of at least 24 weeks. Patients with one or more mutations in at least one gene 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. Notably, patients with oncogenic mutations in one or more genes selected from GNAS, RHEB, NTRK3, IDH2, and / or mTOR had no CB and mPFS of 8 weeks or less.

[0359] Interestingly, at these baseline measurements, ESR1 mutations were only detected in approximately 90% of enrolled subjects. In at least some of the subjects, enrollment into the clinical trial was based on a prior history of ESR1 mutations, rather than detection of ESR1mut at enrollment, which is likely attributable to patient response to prior therapy. This is consistent with our observations in the ELAINE 2 clinical trial itself, where approximately 68.9% (20 of 29) of subjects showed a reduction or disappearance (ND) of ESR1 mutations at week 4 of treatment with LAS+abema.

[0360] For example, patient 29 (2011-00001) had an ESR1 D538G mutation at baseline and had previously received abemaciclib and other first-line or second-line endocrine therapies ( Figure 1 and 3 Patient 29, who received the combination of lasofoxifene and abemaciclib, had ND results for ESR1 mutations in ctDNA. Patient 29 continued to respond to treatment and had clinical benefit, with stable disease for at least 88 weeks ( Figure 3 ).

[0361] Patient 16 (2003-00001) had a past medical history suggesting the presence of the ESR1 gain-of-function mutation D538G. Patient 16 had also received prior abemaciclib therapy and other first-line or second-line endocrine therapies ( Figure 1 and 3 ). At baseline, patient 16 had no detectable mutESR1 (ESR1 mutation detection result was ND) 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.

[0362] The data in this example show that lasofoxifene combined with abemaciclib is effective in treating metastatic ER cancer with ESR1 gene mutations and oncogenic mutations in one or more genes other than ESR1. + Breast cancer is effective; lasofoxifene combined with abemaciclib is used to treat metastatic ER cancer without detectable ESR1 mutations and with oncogenic mutations in one or more genes other than ESR1 + Breast cancer effective.

[0363] 6.6. Example 6: Third interim results of the ELAINE 2 clinical trial

[0364] As of the third, later interim date, 20 of 29 patients (approximately 69%) had developed CB on the combination of lasofoxifene and abemaciclib (laso / abema). Figure 7, the updated Swimmer Plot, and Figure 3 In contrast, 11 of the 20 patients with CB (55%) had their status updated. Of these patients, 8 of the 11 patients (approximately 72.7%) had sustained or partial remission and stable disease at weeks 68 to 100 since the last update. For example, patient 29 (2011-01) had sustained remission and stable disease to 100 weeks, while patient 2005-01 had sustained remission and stable disease to 84 weeks, patient 2002-01 had sustained remission and stable disease to 80 weeks, patients 2001-05 and 2016-03 had sustained remission and stable disease to 72 weeks, patient 2018-01 had sustained remission and stable disease to 68 weeks, and patient 2017-01 had partial remission and stable disease to 80 weeks. Patient 2015-01 had partial remission and stable disease to 88 weeks. Two of the 20 patients (10%) had disease progression 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 the 11 patients (approximately 63.6%) had the difficult-to-treat Y537S mutation at baseline. The data showed that all 7 patients had sustained or partial responses to laso / abema combination therapy and had stable disease for at least 72 weeks. Laso / abema efficacy was observed even in patients with visceral metastases at baseline (approximately 42.8%, 3 of 7 patients). At least 7 of the 11 patients (approximately 63.6%) had oncogenic mutations in one or more genes other than ESR1, and the prevalence of at least one oncogenic mutation in the patient population was greater than 20% (e.g., HNF1A, TERT, TP53, APC, PIK3CA, ATM, CCND1, see also Figure 5 ).

[0365]

[0366]

[0367] With reference to Table 17, it is noteworthy that patients with mutations previously associated with endocrine resistance or CDK4 / 6i resistance achieve clinical benefit (CB), stable condition, and show complete or partial remission of at least 72 weeks.For example, patients with at least one of FGFR1, ERBB2, CCND1, CCNE1, ARD1A, PIK3CA and TP53 have consistent and strong clinical remission to lasofoxifene / abecib combination in Elaine 2.Patient 2005-01 with TP53, CCND1 and CCNE1 mutations or CNV has complete remission and stable condition to 84 weeks.Patient 2014-01 with TP53, CCND1 and ARID1A mutations or CNV has partial remission and progress occurs at 72 weeks.Patient 2001-02 suffers from PIK3CA mutation or CNV, partially remitted and progress occurs at 72 weeks.Patient 2001-05 suffers from PIK3CA mutation or CNV, complete remission and stable condition to 72 weeks. Notably, these four patients also had the difficult-to-treat Y537S mutESR1 variant.

[0368] These results were unexpected and suggest that the combination of lasofoxifene and abemaciclib may be effective in reducing or preventing tumor progression in patients at high risk for or who have developed resistance to endocrine therapy or CDK4 / 6i therapy.

[0369] The results are consistent with our observations presented in Example 3 and further demonstrate that the combination of lasofoxifene and abemaciclib has acceptable tolerability, safety, and efficacy for patients with metastatic breast cancer who carry at least one ESR1 mutation and often at least one or more other gene mutations and whose disease has progressed after treatment with one or more CDK4 / 6 inhibitors and / or one or more endocrine therapies.

[0370] 6.7. Example 7: Copy number variation of ESR1 mutations and oncogenic mutations in circulating tumor DNA (ctDNA) from patients in the ELAINE 2 clinical trial

[0371] This example investigates the efficacy of lasofoxifene plus abemaciclib in the ELAINE 2 clinical trial in ER patients. + / HER2 - ESR1 mutations and copy number variations (CNVs) of oncogenic mutations in circulating tumor DNA (ctDNA) of patients with metastatic breast cancer (mBC), as described in Example 5. The data in this example demonstrate the correlation of the presence of ESR1 mutation variants and / or oncogenic mutation variants with clinical benefit (CB).

[0372] A total of 29 samples (batch 1: 25 samples / patient; batch 2: 4 samples / patient) from 29 patients were collected from the ELAINE 2 clinical trial. Patients took oral lasofoxifene 5mg / day and abemaciclib 150mg BID until disease progression, death, unacceptable toxicity or withdrawal from the study. As described in Example 5, plasma separation and cell-free DNA (ctDNA) extraction and sequencing were performed on the sample. The bioinformatics flow analysis sequencing data designed for detection of single nucleotide variants (SNV), insertions and deletions (indel), copy number variations (CNV) and fusions was used. In batch 1, copy number amplification (CNA) was detected in 19 genes, including CCND1. Amplification types were annotated as focal, aneuploidy or focal / aneuploidy state uncertain amplification. The results are listed in Table 18.

[0373]

[0374] Fig. 8 shows examples of copy number variation events detected for each gene. The left panel of Fig. 8 shows copy number variation (CNV) events detected for each gene, including CCND1, CCNE1, CDK4, EGFR, FGFR1, MYC. The right panel of Fig. 8 shows the copy number distribution of each gene of the CNV events shown in the left panel. 5 CNV events and more than 8 copy numbers were detected in CCND1, 3 CNV events and more than 7 copy numbers were detected in FGFR1, and 2 CNV events and about 5 copy numbers were detected in CDK4.

[0375] 6.8. Example 8: Interim Results of Oncogenic Mutations in Circulating Tumor DNA (ctDNA) of Patients in the ELAINE 2 Clinical Trial

[0376] This example provides data on ER treated with lasofoxifene plus abemaciclib in the ELAINE 2 clinical trial described in Example 5 as of an interim date subsequent to the date of Example 5. + / HER2 - Oncogenic mutations in genes other than ESR1 are present in circulating tumor DNA (ctDNA) in patients with metastatic breast cancer (mBC). The results are summarized in Fig. 9 For completeness, ESR1 gain-of-function mutations (mutESR1; top row) are included.

[0377] like Fig. 9As shown, a total of 41 genes with one or more oncogenic mutations were detected in blood samples of the ELAINE 2 patient population. At least 87.8% (36 of 41) of patients with oncogenic mutations in one or more of the above genes achieved clinical benefit (CB) after treatment with lasofoxifene and abemaciclib, that is, stable disease for ≥24 weeks, or confirmed partial or complete remission. 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, approximately 100% of patients with CCND1 oncogenic mutations continued to achieve CB and mPFS for at least 72 weeks, 100% of patients with FGFR1 oncogenic mutations continued to achieve CB and mPFS for at least 72 weeks, and 76% of patients with TERT oncogenic mutations 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) in CCND1 and FGFR1 and variants in PIK3CA and TP53 responded to lasofoxifene and abemaciclib.

[0378] The data in this example show that lasofoxifene combined with abemaciclib is effective in treating metastatic ER with ESR1 gene mutations and oncogenic mutations in one or more genes other than ESR1. + breast cancer, and maintain this subgroup of patients and those with metastatic ER + CB and mPFS in patients with breast cancer without detectable ESR1 mutation and with oncogenic mutations in one or more genes other than ESR1.

[0379] 6.9. Example 9: Fourth interim results of the ELAINE 2 clinical trial

[0380] As of the fourth, later date following the date of Example 6, 20 of the 29 patients (approximately 68.9%) continued to experience CB on the combination of lasofoxifene and abemaciclib (laso / abema). Figures 10A-10B , updated Swimmer Plot, with Figure 7In contrast, 8 of 20 CB patients (40%) had their status updated. Of these patients, 6 of 8 patients (75%) had ongoing or partial remission with stable disease at weeks 96 to 128, while 2 of 8 patients (25%) had ongoing or partial remission with progressive disease since the last update. For example, patient 29 (2011-01) had ongoing remission with stable disease to 128 weeks, while patient 2002-01 had ongoing remission with stable disease to 100 weeks, and patients 2001-05 and 2018-01 had ongoing remission with stable disease to 96 weeks. Patient 2015-01 had partial remission with stable disease to 120 weeks. Patient 2016-03 had partial remission with stable disease to 96 weeks. Patient 2005-01 had ongoing remission but progressive disease at week 104. Patient 2017-01 had partial remission with progressive disease at week 100. Table 19 summarizes the efficacy of the lasofoxifene / abecib combination in patients who achieved clinical benefit, including those described in Table 17. As shown, 13 of 20 patients (65%) who had the difficult-to-treat Y537S mutation at baseline had sustained disease or partial remission and stable disease up to 120 weeks (e.g., patient 2015-01). The efficacy of lasofoxifene / abecib was observed even in patients with visceral metastases at baseline (60%, 12 of 20 patients). All 20 patients (100%) who had clinical benefit also had oncogenic mutations in one or more genes other than ESR1, and at least one oncogenic mutation had a prevalence of more than 17% in the patient population (e.g., HNF1A, TERT, TP53, APC, PIK3CA, ATM, CCND1, FGFR1, see also Fig. 9 ).

[0381]

[0382]

[0383]

[0384]

[0385] With reference to Table 19, it is noteworthy that patients with mutations previously associated with endocrine resistance or CDK4 / 6i resistance (e.g., FGFR1, ERBB2, CCND1, CCNE1, ARD1A, PIK3CA, and TP53) continue to have consistent strong clinical remission for the lasofoxifene / abexiclib combination in Elaine 2. Subject 2011-01 with D538G mutESR1 variant and TP53 and CCND1 mutations or copy number variation (CNV) continues to have complete remission and stable condition to 128 weeks. Patient 2017-01 with E380Q and L469V mutESR1 variant and CCNE1 mutation or CNV continues to have partial remission and progress at 100 weeks. Patient 2016-03 suffers from D538G mutESR1 variant and ERBB2 mutation or CNV, continues to have partial remission and stable condition at 96 weeks. Patient 2002-02 had a partial response until progression at week 32 despite having multiple mutESR1 variants, including Y537N, L536H, D538G, and E380Q, and a CCNE mutation or CNV.

[0386] Notably, patients with the difficult-to-treat Y537S mutESR1 variant continued to have consistently strong clinical responses to the lasofoxifene / abecib combination. Patient 2015-01, who had the Y537S mutESR1 variant and FGFR1 and TP53 mutations or CNVs, continued to have a partial response and stable disease through week 120. Patient 2005-01, who had the Y537S, Y537N, and D538G mutESR1 variants and TP53, CCND1, and CCNE1 mutations or CNVs, continued to have a complete response and stable disease and had progression at week 104. Patient 2001-02, who had the Y537S, Y537N, Y537D, and D538G mutESR1 variants and PIK3CA, FGFR1, and CCND1 mutations or CNVs, achieved a partial response and had progression at week 72. Patient 2014-01 had Y537S, Y537N, and D538GmutESR1 variants and TP53, CCND1, and ARID1A mutations or CNVs, achieved a partial response and progressed at 72 weeks. Patient 2004-03 had Y537SmutESR1 variant and ERBB2 mutations or CNVs, achieved a complete response and stable disease at 56 weeks but withdrew early due to noncompliance. Patient 2005-02 had Y537SmutESR1 variant and PIK3CA and ARID1A mutations or CNVs, achieved a partial response and progressed at 56 weeks. Patient 2008-01 had Y537S and D538GmutESR1 variants and CCND1 and FGFR1 mutations or CNVs, achieved a partial response and progressed at 32 weeks. Patient 2016-01, who had Y537S, Y537C, L536P, and D538G mutESR1 variants and TP53, CCND1, and FGFR1 mutations or CNVs, achieved a partial response and progressed at week 32. Patient 2001-05, who had a Y537SmutESR1 variant and a PIK3CA mutation or CNV, maintained a complete response with stable disease through week 96. Patient 2001-01, who had Y537S and D538G mutESR1 variants and a PIK3CA mutation or CNV, achieved a partial response and progressed at week 32. Patient 2009-02, who had a Y537S mutESR1 variant and TP53 and PIK3CA mutations or CNVs, achieved a complete response and stable disease and progressed at week 36.

[0387] Patients with target lesions at baseline were monitored for changes in the study. Of note, 10 of the 20 patients (50%) had a confirmed partial response and achieved clinical benefit. Patient 2001-01 had target lesions in the lungs (11 mm) at baseline, and the lesions were reduced 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 of 72 mm), and the lesions were reduced by 74% at 32 weeks. Patient 2004-06 had target lesions in the liver, bones, and pleural cavity at baseline (total diameter of 79 mm), and the lesions were reduced by 47% at 64 weeks. Patient 2005-02 had target lesions in the liver (total diameter of 24 mm) at baseline, and the lesions were reduced by 33% at 56 weeks. Patient 2008-01 had target lesions in the liver (total diameter of 56 mm) at baseline, and the lesions were reduced by 50% at 32 weeks. Patient 2014-01 had target lesions in the liver and spleen (41 mm), which shrank by 22% at 64 weeks. Patient 2015-01 had target lesions in the liver (56 mm in total diameter), which shrank by 52% at 104 weeks. Patient 2016-01 had target lesions in the liver (47 mm), which shrank by 47% at 32 weeks. Patient 2016-03 had target lesions in the liver (67 mm in total diameter), which shrank by 67% at 88 weeks. Patient 2017-01 had target lesions in the left paracentesis (15 mm in total diameter), which shrank by 40% at 96 weeks. The data suggest that the lasofoxifene / abexiclib combination is effective in slowing tumor progression and / or inhibiting tumor growth.

[0388] The results are consistent with our observations in Examples 3 and 7, and further indicate that for patients with metastatic breast cancer who carry at least one ESR1 mutation and typically carry at least one or more other gene mutations and who progress after receiving one or more CDK4 / 6 inhibitors and / or one or more endocrine therapy treatments, lasofoxifene and abemaciclib combined therapy has acceptable tolerability, safety and effectiveness. In addition, the results further indicate that lasofoxifene and abemaciclib combined therapy is effective for patients carrying refractory mutESR1 variants. For patients with one or more oncogenic mutations or biomarker CNVs associated with endocrine therapy or CDK4 / 6i resistance at the same time, the results are unexpected.

[0389] 7. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0390] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

[0391] All references, issued patents, and patent applications cited throughout the text of this specification are incorporated herein by reference in their entirety for all purposes.

Claims

1. A method for slowing down the progression of breast cancer in a patient, comprising: administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), The breast cancer: (i) Estrogen receptor positive (ER + ); (ii) having 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 during previous CDK4 / 6 inhibitor therapy.

2. The method according to claim 1, wherein ER + Breast cancer is HER2 - .

3. The method according to claim 1 or claim 2, wherein the ER + The breast cancer is locally advanced.

4. The method according to claim 1 or claim 2, wherein the ER + Breast cancer is metastatic.

5. The method of any one of claims 1-4, wherein lasofoxifene is administered as lasofoxifene tartrate.

6. The method according to any one of claims 1-5, wherein lasofoxifene is administered orally at 5 mg / day.

7. The method according to any one of claims 1-6, wherein the CDK4 / 6i administered to the patient is selected from palbociclib, ribociclib and abemaciclib.

8. The method of claim 7, wherein the CDK4 / 6i administered to the patient is abemaciclib.

9. The method of claim 8, wherein abemaciclib is administered orally at 50 mg to 200 mg BID.

10. The method of claim 9, wherein abemaciclib is administered orally at 100 mg to 200 mg BID.

11. The method of claim 10, wherein abemaciclib is administered orally at 150 mg BID.

12. The method of claim 1, wherein the previously administered CDK4 / 6 inhibitor is selected from palbociclib, ribociclib, and abemaciclib.

13. The method of claim 12, wherein the previously administered CDK4 / 6 inhibitor is abemaciclib.

14. The method of any one of claims 1-13, wherein the cancer has been previously identified as having at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

15. The method according to any one of claims 1 to 14, further comprising the following earlier steps: The patient is determined to have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

16. The method of any one of claims 1-15, wherein the at least one gain-of-function missense mutation is located in any one of amino acids D538, Y537, L469, L536, P535, V534, S463, V392, and E380.

17. The method of claim 16, wherein the at least one gain-of-function missense mutation is located in amino acid D538.

18. The method of claim 17, wherein the mutation is D538G.

19. The method of claim 16, wherein the at least one gain-of-function missense mutation is located in amino acid Y537.

20. The method of claim 19, wherein the mutation is Y537S, Y537N, Y537C or Y537Q.

21. The method of claim 20, wherein the mutation is Y537C.

22. The method of claim 16, wherein the at least one gain-of-function missense mutation is located in amino acid L469.

23. The method of claim 22, wherein the mutation is L469V.

24. The method of claim 16, wherein the at least one gain-of-function missense mutation is located in amino acid L536.

25. The method of claim 24, wherein the mutation is L536R or L536Q.

26. The method of claim 16, wherein the at least one gain-of-function missense mutation is located in amino acid P535.

27. The method of claim 26, wherein the mutation is P535H.

28. The method of claim 16, wherein the at least one gain-of-function missense mutation is located in amino acid V534.

29. The method of claim 28, wherein the mutation is V534E.

30. The method of claim 16, wherein the at least one gain-of-function missense mutation is located in amino acid S463.

31. The method of claim 30, wherein the mutation is S463P.

32. The method of claim 16, wherein the at least one gain-of-function missense mutation is located in amino acid V392.

33. The method of claim 32, wherein the mutation is V392I.

34. The method of claim 16, wherein the at least one gain-of-function missense mutation is located in amino acid E380.

35. The method of claim 34, wherein the mutation is E380Q.

36. The method of claim 4, wherein the ER + Breast cancer is viscerally metastatic.

37. The method of any one of claims 1-36, wherein the breast cancer has progressed on one or more prior endocrine therapies.

38. The method of claim 37, wherein the prior endocrine therapy is 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.

39. The method of claim 38, wherein the SERD is fulvestrant.

40. The method of any one of claims 1-39, wherein the patient achieves 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 or longer after initiating treatment with lasofoxifene or a pharmaceutically acceptable salt thereof and CDK4 / 6i.

41. The method of claim 40, wherein the patient is in complete or partial remission to treatment with lasofoxifene or a pharmaceutically acceptable salt thereof and a CDK4 / 6i.

42. The method of any one of claims 1-41, wherein the patient achieves stable disease for a duration that is at least about 20%, 15%, or 10% longer than the patient's prior second-line or third-line therapy.

43. A method for slowing the progression of breast cancer in a patient, comprising: administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), The breast cancer: (i) Estrogen receptor positive (ER + ); (ii) having 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.

44. The method of claim 43, wherein the breast cancer has progressed during prior CDK4 / 6 inhibitor treatment.

45. The method of claim 43 or 44, wherein ER + Breast cancer is HER2 - .

46. ​​The method of claim 45, wherein the ER + The breast cancer is locally advanced.

47. The method of claim 45, wherein the ER + Breast cancer is metastatic.

48. The method of any one of claims 43-47, wherein lasofoxifene is administered as lasofoxifene tartrate.

49. The method of any one of claims 43-48, wherein lasofoxifene is administered orally at 5 mg / day.

50. The method of any one of claims 43-49, wherein the CDK4 / 6i administered to the patient is selected from palbociclib, ribociclib, and abemaciclib.

51. The method of claim 50, wherein the CDK4 / 6i administered to the patient is abemaciclib.

52. The method of claim 51, wherein abemaciclib is administered orally at 50 mg to 200 mg BID.

53. The method of claim 52, wherein abemaciclib is administered orally at 100 mg to 200 mg BID.

54. The method of claim 53, wherein abemaciclib is administered orally at 150 mg BID.

55. The method of claim 44, or any one of claims 45-54 as appended to claim 44, wherein the previously administered CDK4 / 6 inhibitor is selected from palbociclib, ribociclib and abemaciclib.

56. The method of claim 55, wherein the previously administered CDK4 / 6 inhibitor is abemaciclib.

57. The method of any one of claims 43-56, further comprising a prior step of detecting oncogenic mutations in one or more genes with oncogenic mutations in circulating tumor DNA (ctDNA).

58. The method of claim 57, wherein 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.

59. The method of claim 58, wherein 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.

60. The method of any one of claims 43-59, 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, or PDGFRA.

61. The method of any one of claims 43-59, wherein the one or more genes having oncogenic mutations are selected from CCND1, FGFR1, CCNE1, AR, ALK, MAPK3, KIT, SMAD4, NOTCH1, RB1, BRAF, RAF1, PTEN, AKT1, CDH1, BRCA1, MYC, CDKN2A, or PDGFRA.

62. The method of any one of claims 43-59, 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, BRCA2, STK11, FGFR1, ARID1A, CCNE1, AR, ALK, ERBB2, KIT, BRAF, or CDK4.

63. The method of claim 62, wherein the one or more genes having oncogenic mutations are selected from TP53, PIK3CA, CCND1, ARID1A, FGFR1, CCNE1, and ERBB2.

64. The method of claim 63, wherein the oncogenic mutation is PIK3CA.

65. The method of any one of claims 43-59, wherein the one or more genes having oncogenic mutations are selected from HNF1A, TERT, GATA3, CDK12, MAPK3, GNAS, RAF1, RHEB, or NTRK3.

66. The method of any one of claims 43-65, wherein the cancer has been previously determined to have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

67. The method according to any one of claims 43 to 66, further comprising the earlier step of: The patient is determined to have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

68. The method of any one of claims 43-67, wherein the at least one gain-of-function missense mutation is located in any one of amino acids D538, Y537, L469, L536, P535, V534, S463, V392, and E380.

69. The method of claim 68, wherein the at least one gain-of-function missense mutation is located in amino acid D538.

70. The method of claim 69, wherein the mutation is D538G.

71. The method of claim 68, wherein the at least one gain-of-function missense mutation is located in amino acid Y537.

72. The method of claim 71, wherein the mutation is Y537S, Y537N, Y537C or Y537Q.

73. The method of claim 72, wherein the mutation is Y537C.

74. The method of claim 72, wherein the mutation is Y537S.

75. The method of claim 68, wherein the at least one gain-of-function missense mutation is located in amino acid L469.

76. The method of claim 75, wherein the mutation is L469V.

77. The method of claim 68, wherein the at least one gain-of-function missense mutation is located in amino acid L536.

78. The method of claim 77, wherein the mutation is L536R or L536Q.

79. The method of claim 68, wherein the at least one gain-of-function missense mutation is located in amino acid P535.

80. The method of claim 79, wherein the mutation is P535H.

81. The method of claim 68, wherein the at least one gain-of-function missense mutation is located in amino acid V534.

82. The method of claim 81, wherein the mutation is V534E.

83. The method of claim 68, wherein the at least one gain-of-function missense mutation is located in amino acid S463.

84. The method of claim 83, wherein the mutation is S463P.

85. The method of claim 68, wherein the at least one gain-of-function missense mutation is located in amino acid V392.

86. The method of claim 85, wherein the mutation is V392I.

87. The method of claim 68, wherein the at least one gain-of-function missense mutation is located in amino acid E380.

88. The method of claim 87, wherein the mutation is E380Q.

89. The method of claim 47, wherein the ER + Breast cancer is viscerally metastatic.

90. The method of any one of claims 43-89, wherein the breast cancer has progressed following one or more prior endocrine therapies.

91. The method of claim 90, wherein the prior endocrine therapy is 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.

92. The method of claim 91, wherein the SERD is fulvestrant.

93. The method of any one of claims 43-92, wherein the patient achieves 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 or longer after initiating treatment with lasofoxifene or a pharmaceutically acceptable salt thereof and CDK4 / 6i.

94. The method of claim 93, wherein the patient is in complete or partial remission to treatment with lasofoxifene or a pharmaceutically acceptable salt thereof and a CDK4 / 6i.

95. The method of any one of claims 43-94, wherein the patient achieves stable disease for a duration of at least about 20%, 15%, or 10% longer with lasofoxifene or a pharmaceutically acceptable salt thereof and a CDK4 / 6i than the patient's previous second-line or third-line therapy.

96. A method for slowing the progression of breast cancer in a patient, comprising: administering to the patient an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i), The breast cancer: (i) Estrogen receptor positive (ER + ); (ii) having at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene; and (iii) having increased expression of one or more genes other than the ESR1 gene.

97. The method of claim 96, wherein the breast cancer has progressed during prior CDK4 / 6 inhibitor treatment.

98. The method of claim 96 or 97, wherein ER + Breast cancer is HER2 - .

99. The method of claim 98, wherein the ER + The breast cancer is locally advanced.

100. The method of claim 98, wherein the ER + Breast cancer is metastatic.

101. The method of any one of claims 96-100, wherein lasofoxifene is administered as lasofoxifene tartrate.

102. The method of any one of claims 96-101, wherein lasofoxifene is administered orally at 5 mg / day.

103. The method of any one of claims 96-102, wherein the CDK4 / 6i administered to the patient is selected from palbociclib, ribociclib, and abemaciclib.

104. The method of claim 103, wherein the CDK4 / 6i administered to the patient is abemaciclib.

105. The method of claim 104, wherein abemaciclib is administered orally at 50 mg to 200 mg BID.

106. The method of claim 105, wherein abemaciclib is administered orally at 100 mg to 200 mg BID.

107. The method of claim 106, wherein abemaciclib is administered orally at 150 mg BID.

108. The method of any one of claims 96-107, wherein the previously administered CDK4 / 6 inhibitor is selected from palbociclib, ribociclib, and abemaciclib.

109. The method of claim 108, wherein the previously administered CDK4 / 6 inhibitor is abemaciclib.

110. The method of any one of claims 96-109, further comprising an earlier step of detecting increased expression of one or more genes other than ESR1 in circulating tumor DNA (ctDNA).

111. The method of claim 110, wherein at least one 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, 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.

112. The method of claim 111, wherein each of the one or more genes whose expression is increased 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, 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.

113. The method of any one of claims 96-112, wherein the one or more genes with increased expression are selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1, or mTOR.

114. The method of any one of claims 96-113, wherein the cancer has been previously determined to have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

115. The method of any one of claims 96-114, further comprising the earlier step of: The patient is determined to have at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

116. The method of any one of claims 96-115, wherein the at least one gain-of-function missense mutation is located in any one of amino acids D538, Y537, L469, L536, P535, V534, S463, V392, and E380.

117. The method of claim 116, wherein the at least one gain-of-function missense mutation is located in amino acid D538.

118. The method of claim 117, wherein the mutation is D538G.

119. The method of claim 116, wherein the at least one gain-of-function missense mutation is located in amino acid Y537.

120. The method of claim 119, wherein the mutation is Y537S, Y537N, Y537C or Y537Q.

121. The method of claim 120, wherein the mutation is Y537C.

122. The method of claim 120, wherein the mutation is Y537S.

123. The method of claim 116, wherein the at least one gain-of-function missense mutation is located in amino acid L469.

124. The method of claim 123, wherein the mutation is L469V.

125. The method of claim 116, wherein the at least one gain-of-function missense mutation is located in amino acid L536.

126. The method of claim 125, wherein the mutation is L536R or L536Q.

127. The method of claim 116, wherein the at least one gain-of-function missense mutation is located in amino acid P535.

128. The method of claim 127, wherein the mutation is P535H.

129. The method of claim 116, wherein the at least one gain-of-function missense mutation is located in amino acid V534.

130. The method of claim 129, wherein the mutation is V534E.

131. The method of claim 116, wherein the at least one gain-of-function missense mutation is located in amino acid S463.

132. The method of claim 131, wherein the mutation is S463P.

133. The method of claim 116, wherein the at least one gain-of-function missense mutation is located in amino acid V392.

134. The method of claim 133, wherein the mutation is V392I.

135. The method of claim 116, wherein the at least one gain-of-function missense mutation is located in amino acid E380.

136. The method of claim 135, wherein the mutation is E380Q.

137. The method of claim 100, wherein the ER + Breast cancer is viscerally metastatic.

138. The method of any one of claims 96-137, wherein the breast cancer has progressed following one or more prior endocrine therapies.

139. The method of claim 138, wherein the prior endocrine therapy is 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.

140. The method of claim 139, wherein the SERD is fulvestrant.

141. The method of claim 139, wherein the mTOR inhibitor is selected from sirolimus, temsirolimus, everolimus, or riboside.

142. The method of claim 141, wherein the mTOR inhibitor is everolimus.

143. The method of any one of claims 96-142, wherein the patient achieves 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 or longer after initiating treatment with lasofoxifene or a pharmaceutically acceptable salt thereof and CDK4 / 6i.

144. The method of claim 143, wherein the patient is in complete or partial response to treatment with lasofoxifene or a pharmaceutically acceptable salt thereof and a CDK4 / 6i.

145. The method of any one of claims 96-144, wherein the patient's stable disease duration after treatment with lasofoxifene or a pharmaceutically acceptable salt thereof and CDK4 / 6i is at least about 20%, 15% or 10% longer than the patient's previous second-line or third-line treatment.

146. A method of monitoring a patient being treated for breast cancer, comprising: (a) determining a quantitative measure of the mutant allele frequency (MAF) of at least one gain-of-function missense mutation within the ligand binding domain (LBD) of the estrogen receptor 1 (ESR1) gene (ESR1ctDNA) in circulating tumor DNA (ctDNA) of a biological sample of the patient, wherein the quantitative measure is performed over a period of time at defined time intervals; and (b) determine the positive predictive value (PPV) of clinical benefit of cancer treatment in terms of disease stabilization, wherein the PPV represents responsiveness to a cancer treatment, and The cancer treatment comprises an effective amount of lasofoxifene or a pharmaceutically acceptable salt thereof and an effective amount of a CDK4 / 6 inhibitor (CDK4 / 6i).

147. The method of claim 146, wherein the patient has a decrease in MAF after treatment.

148. The method of any one of claims 146-147, wherein the quantitative measurement of MAF is determined at 0 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks and / or 24 weeks.

149. The method of claim 148, wherein the quantitative measurement of MAF is determined every 4 weeks.

150. The method of any one of claims 146-149, wherein the patient achieves a 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 or longer.

151. The method of any one of claims 146-150, wherein the biological sample is blood, plasma, or serum.

152. The method of any one of claims 146-151, 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.

153. The method of claim 152, wherein the at least one gain-of-function missense mutation is located in any one of amino acids D538, Y537, L469, L536, P535, V534, S463, V392, and E380.

154. The method of claim 153, wherein the at least one gain-of-function missense mutation is located in amino acid D538.

155. The method of claim 154, wherein the mutation is D538G.

156. The method of claim 152, wherein the at least one gain-of-function missense mutation is located in amino acid Y537.

157. The method of claim 152, wherein the mutation is Y537S, Y537N, Y537C or Y537Q.

158. The method of claim 157, wherein the mutation is Y537C.

159. The method of claim 157, wherein the mutation is Y537S.

160. The method of claim 152, wherein the at least one gain-of-function missense mutation is located in amino acid L469.

161. The method of claim 160, wherein the mutation is L469V.

162. The method of claim 152, wherein the at least one gain-of-function missense mutation is located in amino acid L536.

163. The method of claim 162, wherein the mutation is L536R or L536Q.

164. The method of claim 152, wherein the at least one gain-of-function missense mutation is located in amino acid P535.

165. The method of claim 164, wherein the mutation is P535H.

166. The method of claim 152, wherein the at least one gain-of-function missense mutation is located in amino acid V534.

167. The method of claim 166, wherein the mutation is V534E.

168. The method of claim 152, wherein the at least one gain-of-function missense mutation is located in amino acid S463.

169. The method of claim 168, wherein the mutation is S463P.

170. The method of claim 152, wherein the at least one gain-of-function missense mutation is located in amino acid V392.

171. The method of claim 170, wherein the mutation is V392I.

172. The method of claim 152, wherein the at least one gain-of-function missense mutation is located in amino acid E380.

173. The method of claim 172, wherein the mutation is E380Q.

174. The method of any one of claims 146-173, wherein the breast cancer is ER + Breast cancer.

175. The method of claim 174, wherein the ER + Breast cancer is HER2 - .

176. The method of claim 174 or 175, wherein the ER + The breast cancer is locally advanced.

177. The method of claim 174 or 175, wherein the ER + Breast cancer is metastatic.

178. The method of any one of claims 146-177, wherein lasofoxifene is administered as lasofoxifene tartrate.

179. The method of any one of claims 146-178, wherein lasofoxifene is administered orally at 5 mg / day.

180. The method of any one of claims 146-179, wherein the CDK4 / 6i administered to the patient is selected from palbociclib, ribociclib, and abemaciclib.

181. The method of claim 180, wherein the CDK4 / 6i administered to the patient is abemaciclib.

182. The method of claim 181, wherein abemaciclib is administered orally at 50 mg to 200 mg BID.

183. The method of claim 182, wherein abemaciclib is administered orally at 100 mg to 200 mg BID.

184. The method of claim 183, wherein abemaciclib is administered orally at 150 mg BID.

185. The method of any one of claims 146-184, wherein the subject has progressed during prior CDK4 / 6 inhibitor treatment.

186. The method of claim 185, wherein the CDK4 / 6i administered to the patient is selected from palbociclib, ribociclib, and abemaciclib.

187. The method of claim 186, wherein the CDK4 / 6i administered to the patient is abemaciclib.

188. The method of any one of claims 146-187, wherein the subject has an oncogenic mutation in one or more genes other than ESR1.

189. The method of claim 188, wherein 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.

190. The method of claim 189, wherein 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.

191. The method of any one of claims 146-190, 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, or PDGFRA.

192. The method of any one of claims 146-190, wherein the one or more genes having oncogenic mutations are selected from CCND1, FGFR1, CCNE1, AR, ALK, MAPK3, KIT, SMAD4, NOTCH1, RB1, BRAF, RAF1, PTEN, AKT1, CDH1, BRCA1, MYC, CDKN2A, or PDGFRA.

193. The method of any one of claims 146-190, 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, BRCA2, STK11, FGFR1, ARID1A, CCNE1, AR, ALK, ERBB2, KIT, BRAF, or CDK4.

194. The method of claim 193, wherein the one or more genes having oncogenic mutations are selected from TP53, PIK3CA, CCND1, ARID1A, FGFR1, CCNE1, and ERBB2.

195. The method of claim 194, wherein the oncogenic mutation is PIK3CA.

196. The method of any one of claims 146-190, wherein the one or more genes having oncogenic mutations are selected from HNF1A, TERT, GATA3, CDK12, MAPK3, GNAS, RAF1, RHEB, or NTRK3.

197. The method of any one of claims 146-196, wherein the subject has increased expression of one or more genes other than ESR1.

198. The method of claim 197, wherein at least one of the one or more genes whose expression is increased 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, 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.

199. The method of claim 198, wherein each of the one or more genes whose expression is increased 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, 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.

200. The method of any one of claims 197-199, wherein the one or more genes with increased expression are selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1, or mTOR.

201. The method of claim 177, wherein the ER + Breast cancer is viscerally metastatic.

202. The method of any one of claims 146-201, wherein the breast cancer has progressed on one or more prior endocrine therapies.

203. The method of claim 202, wherein the prior endocrine therapy is a selective ER degrader (SERD), a selective ER modulator (SERM), an aromatase inhibitor (AI), an mTOR inhibitor, and / or a PI3K inhibitor.

204. The method of claim 203, wherein the SERD is fulvestrant.

205. The method of claim 203, wherein the mTOR inhibitor is selected from sirolimus, temsirolimus, everolimus, and riboside.

206. The method of claim 205, wherein the mTOR inhibitor is everolimus.

Images