Methods of reducing tumor size

Abstract

The present invention relates to methods of reducing tumor size by administering a combination therapy comprising testosterone and anastrozole. In some aspects, the present invention relates to treating ductal carcinoma in situ (DCIS) by administering a combination therapy comprising testosterone and anastrozole, wherein the individual does not receive a mastectomy.

Description

Attorney Docket No.: 356042000140METHODS OF REDUCING TUMOR SIZE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Application No.63 / 755,823, filed February 7, 2025, the contents of which are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] The present disclosure relates to methods of treating ductal carcinoma in situ (DCIS) by administering a combination therapy comprising testosterone and anastrozole. In some aspects, the present disclosure relates to treating DCIS by administering a combination therapy comprising testosterone and anastrozole, wherein the individual does not receive a mastectomy.BACKGROUND OF THE INVENTION

[0003] Prior to the introduction of population-based screening mammography, DCIS constituted less than 5% of breast cancer diagnoses. Today, DCIS represents 25-30% of screen-detected breast cancer. Although it is typically referred to as a “breast cancer,” DCIS is, in fact, a precancerous disease that, like invasive cancers, is heterogeneous. DCIS cases span a spectrum of biology, from scattered small, low-grade HR+ lesions to large palpable wall-to-wall lesions that are hormone receptor negative. Although the degree of risk for progression that this pose can vary widely, standard DCIS treatment consists of aggressive local therapy identical to that of invasive cancer, e.g., mastectomy or lumpectomy and radiation. Importantly, women with DCIS are at elevated risk for developing invasive breast cancer, even after breast conservation. But despite good outcomes (95% DRFS with treatment), DCIS presents a major clinical dilemma. Our inability to predict which lesions are destined to progress to invasive cancer and which will remain subclinical means that many women with DCIS are overtreated.

[0004] Thus, there is a need for new breast-conserving methods of treating DCIS that reduce the necessity for mastectomy.SUMMARY OF THE INVENTION

[0005] In some embodiments, provided herein is a method of reducing tumor size in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to1MF-366504339Attorney Docket No.: 356042000140about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the tumor size of the DCIS is reduced following at least one administration.

[0006] In some embodiments, provided herein is a method of treating DCIS in an individual comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, and (a) performing active surveillance of the DCIS in the individual, or (b) performing a lumpectomy on the individual, wherein the individual does not receive a mastectomy.

[0007] In some embodiments, provided herein is a method of conserving breast tissue in an individual having DCIS comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, and (a) performing active surveillance of the DCIS in the individual or (b) performing a lumpectomy on the individual, wherein the individual does not receive a mastectomy.

[0008] In some embodiments, provided herein is a method of increasing the probability of breast conserving surgery in an individual having DCIS comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the probability of breast conserving surgery in the individual is increased.

[0009] In some embodiments, provided herein is a method of avoiding a mastectomy in an individual having DCIS comprising administering about 100 mg testosterone and about 4 mg anastrozole to the individual about every three months, wherein the individual does not receive a mastectomy.

[0010] In some embodiments, provided herein is a method of treating ductal carcinoma in situ (DCIS) in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months; and performing active surveillance on the individual, wherein the individual is on active surveillance for at least 7 months.

[0011] In some of any of the above embodiments, the individual does not receive surgical intervention while on active surveillance.2MF-366504339Attorney Docket No.: 356042000140

[0012] In some of any of the above embodiments, the tumor size is reduced after 1, 2, or 3 administrations of the testosterone and the anastrozole.

[0013] In some of any of the above embodiments, the testosterone and the anastrozole are administered to the individual about every three months for up to about 36 months.

[0014] In some of any of the above embodiments, non-mass enhancement in ductal distribution is reduced following at least one administration of the testosterone and the anastrozole.

[0015] In some of any of the above embodiments, non-mass enhancement in ductal distribution is reduced after 1, 2, or 3 administrations of the testosterone and the anastrozole.

[0016] In some of any of the above embodiments, the testosterone and the anastrozole are administered subcutaneously.

[0017] In some of any of the above embodiments, the testosterone and the anastrozole are formulated as pellet. In some embodiments, the pellet is cylindrical. In some embodiments, the pellet is inserted subcutaneously in the upper outer gluteal region or iliac fossa of the individual.

[0018] In some of any of the above embodiments, the DCIS is hormone receptor positive DCIS.

[0019] In some of any of the above embodiments, the DCIS is ER+ / PR+ DCIS.

[0020] In some of any of the above embodiments, the individual is post-menopausal. In some of any of the above embodiments, the individual is in perimenopause.

[0021] In some of any of the above embodiments, the DCIS is intermediate grade DCIS.

[0022] In some of any of the above embodiments, the individual is a woman.

[0023] In some of any of the above embodiments, the individual is categorized as low risk by MRI after 3 or 6 months of treatment.

[0024] In some of any of the above embodiments, the individual does not progress to invasive ductal carcinoma.

[0025] In some of any of the above embodiments, the individual receives MRI and or mammogram about every six months.3MF-366504339Attorney Docket No.: 356042000140

[0026] In some of any of the above embodiments, the individual receives MRI and / or mammogram about every six months for about five years.

[0027] In some of any of the above embodiments, about 100 mg testosterone and about 4 mg anastrozole is administered to the individual.

[0028] In some of any of the above embodiments, the method further comprises determining tumor size. In some embodiments, the tumor size is determined by MRI. In some embodiments, the tumor size is determined by mammography.

[0029] In some of any of the above embodiments, background parenchymal enhancement is reduced following at least one administration of the testosterone and the anastrozole.

[0030] In some of any of the above embodiments, one or more focal lesions is reduced following at least one administration of the testosterone and the anastrozole.

[0031] In some of any of the above embodiments, the number or extent of calcifications is reduced following at least one administration of the testosterone and the anastrozole.

[0032] In some of any of the above embodiments, wherein breast density is reduced following at least one administration of the testosterone and the anastrozole.

[0033] In some of any of the above embodiments, the individual is administered testosterone, or an equivalent amount of a pharmaceutically acceptable salt or ester thereof.

[0034] In some of any of the above embodiments, the DCIS exhibits aromatase expression. In some embodiments, the aromatase expression of the DCIS is detectable in epithelial and / or stromal breast tissue of the individual.

[0035] In some of any of the above embodiments, the DCIS exhibits expression of at least one 5a-reductase isoenzyme.

[0036] In some of any of the above embodiments, the administration of the testosterone and the anastrozole to the individual results in a reduction in estrogenic signaling relative to androgenic signaling within the DCIS.

[0037] In some of any of the above embodiments, the administration of the testosterone and the anastrozole to the individual promotes androgen receptor-mediated transcriptional activity within DCIS tissue.4MF-366504339Attorney Docket No.: 356042000140

[0038] In some of any of the above embodiments, the administration of the testosterone and the anastrozole to the individual reduces aromatase-mediated conversion of testosterone to estradiol within DCIS tissue.

[0039] In some of any of the above embodiments, after administration of the testosterone and the anastrozole to the individual, testosterone is converted within the breast tissue of the individual to one or more 5a-reduced androgens. In some embodiments, the one or more Sa-reduced androgens comprise dihydrotestosterone (DHT).

[0040] In some of any of the above embodiments, the administration of the testosterone and the anastrozole to the individual results in alteration of intraductal steroid metabolism. In some embodiments, alteration of intraductal steroid metabolism corresponds to categorization of the individual as low risk for progression of disease and continuation of active surveillance.

[0041] In some of any of the above embodiments, the DCIS harbors early PI3K pathway activation.

[0042] In some of any of the above embodiments, the DCIS comprises a PIK3CA mutation.

[0043] In some of any of the above embodiments, the DCIS comprises altered PI3K pathway signaling.

[0044] In some of any of the above embodiments, the administration of the testosterone and the anastrozole reprograms PI3K-associated survival signaling.

[0045] In some of any of the above embodiments, the administration of the testosterone and the anastrozole provides sustained androgen receptor agonism.

[0046] In some of any of the above embodiments, the administration of the testosterone and the anastrozole suppresses estrogen formation.

[0047] In some of any of the above embodiments, the administration of the testosterone and the anastrozole restores endocrine responsiveness.

[0048] In some of any of the above embodiments, the administration of the testosterone and the anastrozole enables regression or stability of disease that is compatible with active surveillance.5MF-366504339Attorney Docket No.: 356042000140

[0049] In some of any of the above embodiments, the regression or stability of disease is evaluated and / or defined by imaging.

[0050] In some of any of the above embodiments, the individual is not administered a PI3K inhibitor.

[0051] In some of any of the above embodiments, the testosterone administered to the individual is testosterone or a pharmaceutically acceptable salt or ester thereof.BRIEF DESCRIPTION OF DRAWINGS

[0052] FIG. 1A shows a top down MRI image of a patient’s breasts prior to treatment.FIG. IB shows a top down MRI image of the patient’s breasts following six months of treatment with testosterone and anastrozole.

[0053] FIG. 2A shows a profile MRI image of a patient’ s right breast prior to treatment.FIG. 2B shows a profile MRI image of the patient’ s right breast following six months of treatment with testosterone and anastrozole.DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention is based, at least in part, on the surprising finding that treatment with a combination of testosterone and anastrozole effectively reduces tumor size in individuals with DCIS. In some aspects, the present invention relates to a method that effectively treats patients with DCIS using a combination of testosterone and anastrozole such that the likelihood of the patient needing to undergo a mastectomy is reduced. The combination therapy may reduce the tumor size so that less breast tissue needs to be removed and a lumpectomy rather than a mastectomy can be performed. Accordingly, one benefit of the present therapy is that it is a breast-conserving therapy. In further aspects, the patient may undergo active surveillance following treatment as an alternative to surgery.

[0055] The following description is provided in relation to several embodiments that may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combined with one or more features of other embodiments. In addition, a single feature or combination of features in certain embodiments may constitute additional embodiments. Specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the disclosed embodiments and variations of those embodiments.6MF-366504339Attorney Docket No.: 356042000140

[0056] The subject headings used in the detailed description are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.Definitions

[0057] ‘Tumor” as used herein refers to a mass of abnormal cells that can be detected by imaging. Tumors include solid cancer masses as well as pre-cancerous masses, such as those present in DCIS.

[0058] ‘Tumor size” as used herein refers to the size of a tumor as determined by the imagining technique used. For example, tumor size may be calculated as a tumor volume, based upon MRI imaging or as a linear measurement when mammography is used.

[0059] ‘Individual” or “patient” as used herein is a human. In some embodiments, an individual is a woman that has DCIS.

[0060] “Active surveillance” as used herein incorporates physical examination and imaging at intervals appropriate to the clinical presentation of the disease and the protocols recommended within the treating institution.

[0061] The term “pellet” means a solid formulation implant comprising an effective amount of testosterone and anastrozole. The pellet or implant may have different suitable shapes and sizes, for example, spherical, cylindrical, rectangular, square or combinations thereof. It may have angular edges or round edges. In certain embodiments, the pellet or implant may he compressed. In certain embodiments, the pellet is subcutaneously administered. In certain embodiments, the pellet is inserted subcutaneously in the upper outer gluteal region or iliac fossa of the individual.

[0062] The term “breast cancer” is understood to mean a malignant proliferation of epithelial cells lining the ducts or lobules of the breast.

[0063] The term “breast tissue” is understood to mean the collection of epithelial cells, stromal cells, extracellular matrix, and / or migratory cells, located within and in the vicinity of the breast.

[0064] The term “effective amount” or “pharmaceutically effective amount” of an agent or compound as provided herein is understood to mean a sufficient amount of the agent or7MF-366504339Attorney Docket No.: 356042000140compound to provide the desired therapeutic effect and is nontoxic, has an acceptable nontoxic profile and / or an acceptable side effects profile. The amount required may vary from patient to patient, depending, for example, on age, general condition of the patient, the severity of the condition being treated, the particular agent or compound administered one or more combinations of these factors and the like. An appropriate "effective amount” typically in an individual case may be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and / or using routine experimentation.

[0065] It is understood that aspect and embodiments of the invention described herein include “consisting” and / or “consisting essentially of’ aspects and embodiments. As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise.

[0066] In this application, the use of “or” means “and / or” unless expressly stated or understood by one skilled in the art. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Any example(s) following the term “e.g.,” or “for example” is not meant to be exhaustive or limiting.

[0067] It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0068] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one8MF-366504339Attorney Docket No.: 356042000140or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival. Also encompassed by “treatment” is a reduction of a pathological consequence of a cancer. The methods of the invention contemplate any one or more of these aspects of treatment.

[0069] The following patents and patent application publications are incorporated herein by reference in their entireties: WO2024 / 234056, WO2020 / 243777, WO2016 / 61615, WO2017 / 66827, W02007 / 45027, U.S. Patent Nos. 9,168,302; 9,351,977; 9,616,072;10,064,874; 10,155,005; 10,471,073; 10,525,063; 10,588,912; 10,765,684; 11,040,044; 11,883,414; 12,383,563; 11,524,014; 12,128,055; and U.S. Patent Application Publication US20250228872A1.Methods of Treatment

[0070] In some embodiments, provided herein is a method of treating ductal carcinoma in situ (DCIS), the method comprising administering a combination therapy comprising testosterone and anastrozole.

[0071] DCIS is a non-invasive form of breast cancer where abnormal cells are found in the lining of a breast duct but have not spread beyond the duct, and is considered the earliest form of breast cancer. DCIS can include, for example, a heterogenous group of neoplastic lesions. The lesions may be confined to the breast ducts and lobules. DCIS can be detected using, for example, screening mammography, and is highly treatable but can progress to invasive breast cancer if not managed appropriately. By non-invasive cancer is meant a cancer or carcinoma that while involving unregulated cell proliferation, has not spread or metastasized from its site of origin to surrounding breast structures or from ducts, lobules or glands of the breast. In contrast, a malignant cancer is one that has metastasized to other area(s) of the breast or body of the subject, or has spread from a duct, lobule or gland of the breast to surrounding breast tissues.

[0072] To prevent the progression of DCIS to invasive breast cancer, treatment options for DCIS can include, but are not limited to, surgery, e.g., mastectomy, lumpectomy, breastconserving surgery, endocrine therapy, and radiotherapy. However, these standard of care treatment options can be excessive in view of the early stage of disease, as not all DCIS lesions are guaranteed to progress to invasive breast cancer. Excessive treatment of individuals having DCIS can have a severely negative impact on patient experience, quality9MF-366504339Attorney Docket No.: 356042000140of life, and wellbeing, with significant physical and emotional consequences. Thus, there is an ongoing need to develop therapeutic approaches for DCIS that improve disease management such that overtreatment, unnecessary surgical intervention, and excessive side effects are avoided. In particular, there is an ongoing need to identify candidates who are suitable for long-term active surveillance, during which unnecessary surgical intervention may be deferred or entirely avoided.

[0073] An individual treated according to the methods described herein can have an early stage of breast cancer, e.g., ductal carcinoma in situ (DCIS). In some embodiments, the individual has hormone receptor positive DCIS. In some embodiments, the DCIS is ER+ / PR+ DCIS. In some embodiments, the individual has DCIS with or without microinvasion. In some embodiments, the individual is in perimenopause. In some embodiments, the individual is post-menopausal. In some embodiments, the DCIS is intermediate grade DCIS. In some embodiments, the individual is a woman. In some embodiments, the individual is categorized as low risk by MRI after 3 or 6 months of treatment according to the methods described herein. In some embodiments, the individual treated according to the methods described herein does not have invasive carcinoma or identification of a mass on MRI that is subsequently biopsied and found to be invasive cancer.

[0074] Provided herein are methods of treatment including a combination therapy comprising an androgenic agent and an aromatase inhibitor. In some embodiments, an androgenic agent as described herein is a chemical that increases androgenic activity or synthesis. In some embodiments, an androgenic agent is a compound that binds to and activates the androgen receptor, either directly or indirectly, thereby regulating androgen-responsive gene expression. For example, androgenic agents can include testosterone and other steroidal or non-steroidal compounds with androgen receptor activity, including synthetic and non-synthetic hormones. In some embodiments, an androgenic agent is a steroid hormone or non- steroid hormone that binds with high affinity (in the pM or nM range) and specificity to its intracellular mediator, the androgen receptor, to stimulate transactivation activity and thus regulate the expression of target genes. Non-limiting examples of androgenic agents include testosterone, isomers, metabolites, derivatives, precursors of testosterone or combinations thereof; synthetic hormones and / or non-synthetic hormones; and / or selective androgen receptor modulators (SARM). In some embodiments, an androgenic agent includes testosterone and other forms of steroid and non-steroid10MF-366504339Attorney Docket No.: 356042000140hormones that bind to the androgen receptor (e.g., in at least the nM range) to stimulate transactivation activity and thus regulate the expression of target genes, including synthetic and non-synthetic hormones, and physiologically active forms of testosterone, non-limiting forms of which include precursors of testosterone, and derivatives, isomers and esters of testosterone. The androgenic agent may, for example, be selected from the group consisting of: testosterone, methyl testosterone, testosterone undecanoate, testosterone propionatedihydrotestosterone, 5a-dihydrotestosterone, or alternatively androstenediol androstenediol-3-acetate, androstenediol- 17-acetate, androstenediol-3, 17-diacetate, androstenediol- 17-benzoate, androstenediol-3-acetate- 17-benzoate, androstenedione, adrenosterone, androsterone acetate, androsterone propionate, androsterone benzoate, dehydroepiandrosterone, sodium dehydroepiandrosterone sulfate, oxymetholone, fluoxymesterone, methandrostenolone, testolactone, pregnenolone, 17a-methylnortestosterone, norethandrolone, dromostanolone, dromostanolone propionate, nandrolone, nandrolone phenpropionate, nandrolone decanoate, nandrolone furylpropionate, nandrolone cyclohexanepropionate, nandrolone benzoate, nandrolone cyclohexanecarboxylate, danazol, oxymetholone, androsterone, stanozolol, ethylestrenol, oxandrolone, bolasterone, mesterolone, testosterone cypionate, testosterone phenylacetate, testosterone enanthate, testosterone acetate, testosterone buciclate, testosterone heptanoate, testosterone decanoate, testosterone caprate, testosterone isocaprate, and isomers, metabolites, derivatives, precursors of the aforementioned compounds, or combinations thereof. In addition to the pharmaceutically acceptable esters of testosterone, esters of dihydrotestosterone, include, but are not limited to, the enanthate, propionate, cypionate, phenylacetate, acetate, isobutyrate, buciclate, heptanoate, decanoate, undecanoate, caprate and / or isocaprate esters. The androgenic agent may be selected from the group consisting of testosterone, methyltestosterone, testosterone undecanoate, testosterone propionate, a selective androgen receptor modulator or combinations thereof. The androgenic agent may be, for example, testosterone, methyltestosterone, testosterone undecanoate, testosterone propionate, dehydroepiandrosterone, or sodium dehydroepiandrosterone sulfate, or a metabolic precursor, metabolite, or derivative thereof. In some embodiments, the androgenic agent comprises testosterone.

[0075] In some embodiments, an androgenic agent that releases testosterone into the body of the individual, e.g., into the serum or plasma, is administered. Testosterone-releasing11MF-366504339Attorney Docket No.: 356042000140androgenic agents include, for example, androgenic agents that function as prodrugs or precursors of testosterone and release free testosterone in the body of an individual as described herein following administration. Non-limiting examples of testosterone-releasing compounds include testosterone itself, pharmaceutically acceptable salts of testosterone, esters of testosterone, and metabolic precursors of testosterone that are enzymatically converted to testosterone following administration.

[0076] In some embodiments, the androgenic agent that releases testosterone into the body is testosterone. In other embodiments, the androgenic agent that releases testosterone into the body is a testosterone-releasing compound, for example, a compound that, upon administration, is enzymatically or metabolically converted in the body of an individual as described herein to free testosterone. For example, in some embodiments, the androgenic agent that releases testosterone into the body is testosterone or a pharmaceutically acceptable salt or ester thereof. Esters of testosterone may include, but are not limited to, enanthate, propionate, cypionate, phenylacetate, acetate, isobutyrate, buciclate, heptanoate, decanoate, undecanoate, caprate, and isocaprate esters. In some embodiments, an ester of testosterone is subject to enzymatic hydrolysis following administration. In some embodiments, a metabolic precursor of testosterone is enzymatically converted to testosterone following administration. Non-limiting examples of testosterone-releasing androgenic agents include testosterone, testosterone enanthate, testosterone cypionate, testosterone propionate, testosterone undecanoate, testosterone phenylacetate, testosterone acetate, testosterone buciclate, testosterone heptanoate, testosterone decanoate, testosterone caprate, testosterone isocaprate, androstenedione, and androstenediol, or combinations thereof.

[0077] In some embodiments, the androgenic agent is an androgen receptor agonist that does not release testosterone but exerts androgenic activity directly, including dihydrotestosterone and its esters, synthetic anabolic steroids, selective androgen receptor modulators, and related compounds.

[0078] In some embodiments, an aromatase inhibitor as described herein is a chemical compound, hormone or polypeptide that blocks and / or inhibits the activity of aromatase which is an enzyme that converts androgens to estrogens. The aromatase inhibitor may, for example, be selected from the group consisting of anastrozole, exemestane, and letrozole. In some embodiments, an aromatase inhibitor is an inhibitor of aromatase enzyme for blocking aromatization of the androgenic agent into an estrogen, such as estradiol. An aromatase12MF-366504339Attorney Docket No.: 356042000140inhibitor for use in a method of the present disclosure may be selected from non-steroidal aromatase inhibitors, steroidal aromatase inhibitors and mechanism-based steroidal inhibitors. Non-steroidal aromatase inhibitors can be divided into three classes: amino glutethimide-like molecules, imidazole / triazole derivatives, and flavonoid analogs. Examples of non-steroidal aromatase inhibitors include, but are not limited to, anastrozole (Arimidex; 2,2'-[5-(lH-l,2,4-triazol-l-ylmethyl)-l,3-phenylene]bis(2-methylpropanenitrile)); letrozole (Femara; (4,4'-((lH-l,2,4-triazol-l yl)methylene)dibenzonitrile); vorozole (Rivizor) andfadrozole (Afema).In some embodiments, the androgenic agent comprises testosterone. Exemestane (Aromasin; (6-methylenandrosta-l,4-diene-3, 17-dione)) and formestane (Lentaron; 4-hydroxyandrost-4-ene-3, 17-dione)) are examples of steroidal aromatase inhibitors that may be employed. In some embodiments, the aromatase inhibitor used in a method of the present disclosure is selected from the group consisting of anastrozole and letrozole. In some embodiments, the aromatase inhibitor comprises anastrozole.

[0079] Typically, a combination therapy as described herein is administered by subcutaneous implant (e.g., subcutaneous pellet(s)) by subcutaneous pellet inserted into the lower abdomen, upper gluteal region, or other location as deemed suitable. In some embodiments, the pellet is inserted subcutaneously in the upper outer gluteal region or iliac fossa of the individual. In some embodiments, the pellet is a cylindrical pellet. In some embodiments, the cylindrical pellet has a diameter of about 4 mm, about 4.25 mm, about 4.5 mm, about 4.75 mm, about 5 mm, about 5.25 mm, about 5.5 mm, about 5.75 mm, about 6 mm, about 6.25 mm, about 6.35 mm, about 6.5 mm, about 6.75 mm, or about 7 mm. In some embodiments, the cylindrical pellet has a diameter of between about 4.55 mm to about 6.35 mm. Subcutaneous pellets useful for administration of the androgenic agent and aromatase inhibitor of the present combination therapy are, for instance, described further in U.S. Patent Nos. 10,525,063; 11,040,044; 11,883,414; 10,064,874; 12,383,563; 11,524,014; 12,128,055; U.S. Patent Application Publication US20250228872A1; and International Patent Application Publications W02016 / 061615 and WO2020 / 243777. U.S. Patent Nos. 10,525,063;11,040,044; 11,883,414; 10,064,874; 12,383,563; 11,524,014; 12,128,055; U.S. Patent Application Publication US20250228872A1; and International Patent Application Publications WO 2016 / 061615 and WO 2020 / 243777 are specifically incorporated herein in their entireties. In particular, the androgenic agent and aromatase inhibitor are preferably administered in the form of a multi-phasic composition as described in U.S. Patent No.13MF-366504339Attorney Docket No.: 35604200014011,524,014 and the use of all such compositions, dosage forms, implants and pellets as described therein are expressly encompassed.

[0080] In certain embodiments, the pharmaceutical formulation can be an implant or pellet formulated by direct compression. For example, the pellet formulations may incorporate diluents, binders, lubricants and, disintegrators as well as the active ingredients. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Natural and synthetic gums are also convenient, including acacia, alginates, methylcellulose, and / or polyvinylpyrrolidine.Polyethylene glycol, ethyl cellulose and waxes may also serve as binders. A lubricant may be necessary for a pellet formulation and may be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and / or hydrogenated vegetable oils. Typically, the implant or pellet will have an implant or pellet hardness in a range of from about 6 Kg / N to about lOKg / N, preferably from about 7Kg / N to about 9Kg / N, most preferable about 8Kg / N and all such dosage forms within this range are expressly provided for herein.

[0081] By compressing the formulation into a solid dosage form such as an implant in the form of pellet for subcutaneous administration, a sustained release multi-phasic concentration pattern as described herein may be obtained. Knowing that such a release pattern of an androgenic agent and aromatase inhibitor may be obtained, a person of ordinary skill in the art will be able to utilize different ingredients in the formulation in view of the disclosures provided herein and provide such pharmaceutical formulations in accordance with the present disclosure.

[0082] In certain embodiments, the implant or pellet may be inserted into the subcutaneous fat of the subject’s pelvis, the subcutaneous fat of the subject’s breast, the subcutaneous fat of the subject’s buttocks, the subcutaneous fat of the subject’s abdomen or combinations thereof. In certain embodiments, the implant or pellet may be inserted into the subcutaneous fat of the subject’s lower abdominal wall. In certain embodiments, the implant or pellet may be inserted into the subcutaneous fat of the subject’s upper gluteal region.

[0083] As will be understood, the androgenic agent and aromatase inhibitor may be provided in pharmaceutical formulations other than for subcutaneous administration either together in the same formulation or in separate formulations for use in combination14MF-366504339Attorney Docket No.: 356042000140treatments as described herein, such formulations comprising one or more of suitable filler(s), lubricants, binders, disintegrants and / or pharmaceutically acceptable carriers or excipients as described above or used in the provision of pharmaceutical formulations for the desired route of administration and / or for obtaining the desired release pattern (e.g., a sustained release multi-phasic release pattern) of the active(s).

[0084] In particularly preferred embodiments, the combination therapy is administered to the subject by subcutaneous pellet comprising testosterone in an amount of from about 40 mg to about 120 mg and anastrozole in an amount of from 2 mg to about 6 mg. In some embodiments, the amount of testosterone in the pellet comprises an amount between about 40 mg to about 50 mg, about 50 mg to about 60 mg, about 60 mg to about 70 mg, about 70 mg to about 80 mg, about 80 mg to about 90 mg, about 90 mg to about 100 mg, about 100 mg to about 110 mg, or about 110 mg to about 120 mg. In some embodiments, the amount of anastrozole in the pellet comprises an amount between about 2 mg to about 2.5 mg, about 2.5 mg to about 3 mg, about 3 mg to about 3.5 mg, about 3.5 mg to about 4 mg, about 4 mg to about 4.5 mg, about 4.5 mg to about 5 mg, about 5 mg to about 5.5 mg, or about 5.5 mg to about 6 mg. Typically, the pellet comprises testosterone in an amount from about 60 mg to about 120 mg, and most preferably about 80 mg of testosterone or about 100 mg of testosterone, and anastrozole in an amount of from about 3 mg to about 5 mg and most preferably, about 4 mg.

[0085] In some embodiments, the methods provided herein comprise treating an individual with DCIS by administering about 100 mg testosterone and about 4 mg anastrozole about every three months. In some embodiments, about 100 mg testosterone and about 4 mg anastrozole are administered twice, three times, four times, five times, six times, seven times, eight times, nine times, 10 times, 11 times, or 12 times. In some embodiments, about 100 mg testosterone and about 4 mg anastrozole are administered every three months for about one year or about 12 months, for about two years or about 24 months, or for about three years or 36 months. In some embodiments, about 100 mg testosterone and about 4 mg anastrozole are administered every three months for up to about three years, or about 36 months.

[0086] In some embodiments, one or more MRI features in an individual as described herein are measured by a radiologist. In some embodiments, lesion conspicuity, BPE, change in lesion between MRIs, change in BPE between MRIs, and likelihood of invasive cancer are15MF-366504339Attorney Docket No.: 356042000140measured. In some embodiments, BPE is assessed per the standard BI-RADS categories of minimal, mild, moderate, and marked.Treatment Outcomes

[0087] Certain aspects of the present disclosure relate to active surveillance of an individual having DCIS. In some embodiments, active surveillance comprises physical examination and imaging, e.g., MRI, of an individual. In some embodiments, active surveillance is conducted at regular intervals to monitor an individual having DCIS. In some embodiments, active surveillance is performed at intervals appropriate to the clinical presentation of disease. In some embodiments, active surveillance comprises the absence of certain treatment measures for DCIS. Treatment measures for DCIS may include, but are not limited to, surgery, e.g., mastectomy, lumpectomy, hormone therapy, and radiotherapy. In some embodiments, active surveillance comprises an absence of mastectomy for the individual. In some embodiments, active surveillance comprises an absence of surgical intervention for the individual.

[0088] In some embodiments, the methods described herein impact the length of time that an individual having DCIS is on active surveillance. For example, provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months; and performing active surveillance on an individual, wherein the individual is on active surveillance for at least 7 months. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, continues on active surveillance for a longer period of time, i.e., is on long-term active surveillance, as compared to an individual having DCIS who is untreated or who is treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, continues on active surveillance for a longer period of time, i.e., is on longterm active surveillance, as compared to an individual having DCIS who is administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.16MF-366504339Attorney Docket No.: 356042000140

[0089] The period of time that an individual continues on active surveillance may be measured in days, weeks, months, or years. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, continues on active surveillance for at least 7 months. In some embodiments, the number of patients in a plurality of patients having DCIS who are suitable for long-term active surveillance is increased following treatment with a combination of testosterone and anastrozole, as described herein, as compared to the number of patients in a plurality of patients having DCIS who are suitable for long-term active surveillance who are untreated or who are treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, the number of patients in a plurality of patients having DCIS who are suitable for long-term active surveillance is increased following treatment with a combination of testosterone and anastrozole, as described herein, as compared to the number of patients in a plurality of patients having DCIS who are suitable for long-term active surveillance who are administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor as described herein. See, e.g., Example 1.

[0090] Certain aspects of the present disclosure relate to tumor size of an individual having DCIS. In some embodiments, tumor size is determined using MRI. In some embodiments, tumor size is determined using mammography. In some embodiments, MRI is performed on either 1.5 T or 3.0 T. In some embodiments, the MRI scan includes standard breast MRI sequences, including fat-suppressedT2-, non-fat suppressed T1-, fat-suppressed pre-contrast T1-, and fat suppressed T1 -weighted post-contrast images with at least two postcontrast time points. In some embodiments, an individual treated according to the methods described herein receives MRI and or mammogram about every six months for assessment of tumor size. In some embodiments, the individual receives MRI and / or mammogram about every six months for about five years.

[0091] In some embodiments, the methods described herein impact the tumor size of an individual having DCIS. For example, provided herein is a method of reducing tumor size in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the tumor size of the DCIS is reduced following at least one administration. In some embodiments, tumor size is reduced following a single treatment17MF-366504339Attorney Docket No.: 356042000140of testosterone and anastrozole. In some embodiments, tumor size is reduced following two, three, four or more treatments of testosterone and anastrozole. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, experiences a reduction in tumor size as compared to an individual having DCIS who is untreated or who is treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, experiences a reduction in tumor size as compared to an individual having DCIS who is administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor as described herein. See, e.g., Example 1. In some embodiments, the individual having DCIS who experiences a reduction in tumor size is treated with about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole every three months. In some embodiments, the individual having DCIS who experiences a reduction in tumor size is treated with about 100 mg testosterone and about 4 mg anastrozole. In some embodiments, the testosterone and the anastrozole are administered to the individual subcutaneously and are formulated as a pellet.

[0092] Certain aspects of the present disclosure relate to surgical intervention in an individual having DCIS. Surgical intervention can include, for example, mastectomy or lumpectomy. In some embodiments, the methods described herein impact the likelihood of surgical intervention for an individual having DCIS. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, has a reduced likelihood of surgical intervention as compared to an individual having DCIS who is untreated or who is treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, has a reduced likelihood of surgical intervention as compared to an individual having DCIS who is administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0093] Provided herein is a method of treating ductal carcinoma in situ (DCIS) in an individual comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, and (a) performing18MF-366504339Attorney Docket No.: 356042000140active surveillance of the DCIS in the individual, or (b) performing a lumpectomy on the individual, wherein the individual does not receive a mastectomy. In some embodiments, the individual having DCIS who undergoes a lumpectomy but not a mastectomy is treated with about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole every three months. In some embodiments, the individual having DCIS who undergoes a lumpectomy but not a mastectomy is treated with about 100 mg testosterone and about 4 mg anastrozole. In some embodiments, the testosterone and the anastrozole are administered to the individual subcutaneously and are formulated as a pellet. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole has an increased likelihood of receiving a lumpectomy and not a mastectomy, as compared to an individual having DCIS who is administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0094] In some embodiments, the methods described herein impact the conservation of breast tissue in an individual having DCIS. For example, provided herein is a method of conserving breast tissue in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, and (a) performing active surveillance of the DCIS in the individual or (b) performing a lumpectomy on the individual, wherein the individual does not receive a mastectomy. In some embodiments, the individual having DCIS who experiences conservation of breast tissue is treated with about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole every three months. In some embodiments, the individual having DCIS who experiences conservation of breast tissue is treated with about 100 mg testosterone and about 4 mg anastrozole. In some embodiments, the testosterone and the anastrozole are administered to the individual subcutaneously and are formulated as a pellet. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole has an increased potential for conservation of breast tissue, as compared to an individual having DCIS who is administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0095] In some embodiments, the methods described herein impact the probability of breast conserving surgery in an individual having DCIS. For example, provided herein is a19MF-366504339Attorney Docket No.: 356042000140method of increasing the probability of breast conserving surgery in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the probability of breast conserving surgery in the individual is increased. In some embodiments, the individual having DCIS who has an increased probability of breast conserving surgery is treated with about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole every three months. In some embodiments, the individual having DCIS who has an increased probability of breast conserving surgery is treated with about 100 mg testosterone and about 4 mg anastrozole. In some embodiments, the testosterone and the anastrozole are administered to the individual subcutaneously and are formulated as a pellet. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole has an increased probability of breast conserving surgery, as compared to an individual having DCIS who is administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0096] In some embodiments, the methods described herein impact the likelihood of mastectomy in an individual having DCIS. For example, provided herein is a method of avoiding a mastectomy in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the individual does not receive a mastectomy. In some embodiments, the individual having DCIS who does not receive a mastectomy is treated with about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole every three months. In some embodiments, the individual having DCIS who does not receive a mastectomy is treated with about 100 mg testosterone and about 4 mg anastrozole. In some embodiments, the testosterone and the anastrozole are administered to the individual subcutaneously and are formulated as a pellet. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole has a decreased likelihood of mastectomy, as compared to an individual having DCIS who is administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.20MF-366504339Attorney Docket No.: 356042000140

[0097] In some embodiments, the methods described herein impact the stability of disease progression. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, has a reduced progression of disease as compared to an individual having DCIS who is untreated or who is treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, has a reduced progression of disease as compared to an individual having DCIS who is administered a control treatment. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, does not progress to invasive ductal carcinoma. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, has a reduced likelihood of progressing to invasive ductal carcinoma as compared to an individual having DCIS who is untreated or who is treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, does not progress to invasive ductal carcinoma. In some embodiments, an individual having DCIS who is treated with a combination of testosterone and anastrozole, as described herein, has a reduced likelihood of progressing to invasive ductal carcinoma as compared to an individual having DCIS who is administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0098] In some embodiments, an individual having DCIS treated according to the methods described herein has reduced non-mass enhancement in ductal distribution following at least one administration of the testosterone and the anastrozole. In some embodiments, an individual having DCIS treated according to the methods described herein has reduced nonmass enhancement in ductal distribution following 1, 2, or 3 administrations of the testosterone and the anastrozole. In some embodiments, an individual having DCIS treated according to the methods described herein has reduced BPE following at least one administration of testosterone and anastrozole. In some embodiments, an individual having DCIS treated according to the methods described herein has a reduction in one or more focal lesions following at least one administration of testosterone and anastrozole. In some embodiments, an individual having DCIS treated according to the methods described herein21MF-366504339Attorney Docket No.: 356042000140has a reduction in the number or extent of calcifications following at least one administration of testosterone and anastrozole. In some embodiments, an individual having DCIS treated according to the methods described herein has a reduction in breast density following at least one administration of testosterone and anastrozole.

[0099] In some embodiments, the methods described herein impact the risk categorization of an individual by MRI. For example, provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the individual is categorized as low risk by MRI after 6 months of administration of the testosterone and the anastrozole. In some embodiments, the number of patients in a plurality of patients having DCIS who are treated with a combination of testosterone and anastrozole, as described herein, and who are categorized as low risk by MRI after 6 months of treatment, is increased as compared to the number of patients in a plurality of patients having DCIS who are untreated or who are treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, the number of patients in a plurality of patients having DCIS who are treated with a combination of testosterone and anastrozole, as described herein, and who are categorized as low risk by MRI after 6 months of treatment, is increased as compared to the number of patients in a plurality of patients having DCIS who are administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0100] Also provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the individual is categorized as low risk by MRI after 3 months of administration of the testosterone and the anastrozole. In some embodiments, the number of patients in a plurality of patients having DCIS who are treated with a combination of testosterone and anastrozole, as described herein, and who are categorized as low risk by MRI after 3 months of treatment, is increased as compared to the number of patients in a plurality of patients having DCIS who are untreated or who are treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, the number of patients in a plurality of patients having DCIS who are treated with a combination of testosterone and anastrozole, as described22MF-366504339Attorney Docket No.: 356042000140herein, and who are categorized as low risk by MRI after 3 months of treatment, is increased as compared to the number of patients in a plurality of patients having DCIS who are administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0101] In some embodiments, the low-risk categorization is measured by cases demonstrating endocrine responsiveness. In some embodiments, the low-risk categorization is determined based on lesion and background or lesion alone or lack of lesion and minimal background. In some embodiments, an individual is categorized as low risk at 6 months and has a subsequent low rate of invasive ductal cell carcinoma progression at 3 years.

[0102] In some embodiments, the methods described herein impact the Minimum Important Difference in overall quality of life (QoL) measured by PROP-R statistics (PROMIS) and FACT-ES (functional assessment of cancer therapy- endocrine symptoms, a QoL assessment). For example, provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the individual has a positive increase in overall quality of life as measured by PROMIS and FACT-ES. In some embodiments, the number of patients in a plurality of patients having DCIS who are treated with a combination of testosterone and anastrozole, as described herein, and who experience a minimum important difference in overall quality of life as measured by PROMIS and FACT-ES, is increased as compared to the number of patients in a plurality of patients having DCIS who are untreated or who are treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, the number of patients in a plurality of patients having DCIS who are treated with a combination of testosterone and anastrozole, as described herein, and who experience a minimum important difference in overall quality of life as measured by PROMIS and FACT-ES, is increased as compared to the number of patients in a plurality of patients having DCIS who are administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0103] In some embodiments, the methods described herein impact focal lesions (mass and non-mass enhancement (NME)). For example, provided herein is a method of treating23MF-366504339Attorney Docket No.: 356042000140DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the individual has a reduction in focal lesions. In some embodiments, the reduction in focal lesions is observed after 6 months of treatment with the testosterone and the anastrozole. In some embodiments, the reduction in focal lesions comprises a reduction in mass or non-mass enhancement (NME). In some embodiments, the reduction in focal lesions is measured using annotated Functional Tumor Volume.

[0104] In some embodiments, the methods described herein impact contralateral breast density. For example, provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the individual has a reduction in contralateral breast density. In some embodiments, the number of patients in a plurality of patients having DCIS who are treated with a combination of testosterone and anastrozole, as described herein, and who experience a reduction in contralateral breast density, is increased as compared to the number of patients in a plurality of patients having DCIS who are untreated or who are treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, the number of patients in a plurality of patients having DCIS who are treated with a combination of testosterone and anastrozole, as described herein, and who experience a reduction in contralateral breast density, is increased as compared to the number of patients in a plurality of patients having DCIS who are administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0105] In some embodiments, the methods described herein impact qualitative and automated BPE. For example, provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the individual has a contralateral reduction in qualitative and automated BPE. In some embodiments, the number of patients in a plurality of patients having DCIS who are treated with a combination of testosterone and anastrozole, as described herein, and who experience a contralateral reduction in qualitative and automated BPE, is increased as compared to the number of patients in a plurality of patients having DCIS who are untreated or who are24MF-366504339Attorney Docket No.: 356042000140treated with a therapy that does not comprise testosterone and anastrozole. In some embodiments, the number of patients in a plurality of patients having DCIS who are treated with a combination of testosterone and anastrozole, as described herein, and who experience a contralateral reduction in qualitative and automated BPE, is increased as compared to the number of patients in a plurality of patients having DCIS who are administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0106] In some embodiments, the methods described herein impact tolerability of therapy, e.g., time to discontinuation of therapy. For example, provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the individual has an increased time to discontinuation of therapy as compared to an individual who is administered a therapy that does not comprise testosterone and anastrozole. Also provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the individual has an increased time to discontinuation of therapy as compared to an individual who is administered a control treatment. In some embodiments, the control treatment comprises elacestrant, z-endoxifen, tamoxifen, and / or an aromatase inhibitor, as described herein. See, e.g., Example 1.

[0107] Certain aspects of the present disclosure relate to the presence and level, e.g., expression, of the enzymes 5a-reductase and aromatase in an individual having DCIS, and in particular, the presence and level of these enzymes in response to the methods of treatment described herein. For example, aromatase expression level may be increased in DCIS. In some embodiments, the individual having DCIS shows aromatase overexpression with intratumoral estrogen. In some embodiments, 5a-reductases, e.g., steroid 5a-reductase 1 / 2 (SRD5A1 / 2) are present in breast carcinoma and participate in local androgen production / action. Provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the DCIS exhibits aromatase expression detectable in epithelial and / or stromal breast tissue of the individual. Also provided herein is a method of treating DCIS in an individual, the method comprising25MF-366504339Attorney Docket No.: 356042000140administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the DCIS exhibits expression of at least one 5a-reductase isoenzyme. Also provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the administration of the testosterone and the anastrozole to the individual results in a reduction in estrogenic signaling relative to androgenic signaling within the DCIS. Also provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the administration of the testosterone and the anastrozole to the individual promotes androgen receptor-mediated transcriptional activity within DCIS tissue. Also provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the administration of the testosterone and the anastrozole to the individual reduces aromatase-mediated conversion of testosterone to estradiol within DCIS tissue. Also provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein after administration of the testosterone and the anastrozole to the individual, testosterone is converted within the breast tissue of the individual to one or more 5a-reduced androgens. In some embodiments, the one or more 5a-reduced androgens comprise dihydrotestosterone (DHT). Also provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the administration of the testosterone and the anastrozole to the individual results in alteration of intraductal steroid metabolism. In some embodiments, alteration of intraductal steroid metabolism corresponds to categorization of the individual as low risk for progression of disease and continuation of active surveillance.

[0108] Certain aspects of the present disclosure relate to the PI3K signaling pathway. In some embodiments, an individual as described herein has DCIS that harbors early PI3K pathway activation. Early PI3K pathway activation may occur in DCIS, for example, by PIK3CA mutation. In some embodiments, DCIS is associated with a high prevalence of26MF-366504339Attorney Docket No.: 356042000140PIK3CA mutations that alter PI3K pathway signaling. Early PI3K pathway activation can promote endocrine resistance, survival signaling, and persistence under standard estrogen receptor-directed therapies. In some embodiments, PIK3CA mutations are associated with low proliferation, persistence, and endocrine resistance in DCIS. In some embodiments, altered PI3K signaling in DCIS is associated with androgen receptor activation. DCIS with early PI3K pathway activation can limit suitability in individuals for active surveillance and breast conservation.

[0109] In some embodiments, the methods described herein impact PI3K signaling in DCIS. In some embodiments, the DCIS of the individual as described herein exhibits PI3K signaling pathways. Without wishing to be bound by theory, administration of the testosterone and the anastrozole as described herein may be able to target the PI3K pathway and DCIS harboring PI3K-related mutations, without the use of a PI3K inhibitor. For example, provided herein is a method of treating DCIS in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the DCIS has altered PI3K signaling, and wherein the administration of the testosterone and the anastrozole reprograms PI3K- associated survival signaling. In some embodiments, the DCIS exhibits altered PI3K signaling consistent with early PI3K pathway activation. In some embodiments, the DCIS comprises a PIK3CA mutation. In some embodiments, the administration of the testosterone and the anastrozole provides sustained androgen receptor agonism. In some embodiments, the administration of the testosterone and the anastrozole provides sustained androgen receptor agonism with concurrent suppression of estrogen formation. In some embodiments, the administration of the testosterone and the anastrozole reprograms PI3K-associated survival signaling and restores endocrine responsiveness. In some embodiments, the administration of the testosterone and the anastrozole enables regression or stability of disease that is compatible with active surveillance. In some embodiments, regression or stability of disease is evaluated and / or defined by imaging, e.g., MRI. In some embodiments, the individual having DCIS is administered the testosterone and the anastrozole, and is not administered a PI3K inhibitor.EXEMPLARY EMBODIMENTS

[0110] Exemplary embodiments of the methods described herein include:27MF-366504339Attorney Docket No.: 356042000140

[0111] Embodiment 1. A method of reducing tumor size in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the tumor size of the DCIS is reduced following at least one administration.

[0112] Embodiment 2. A method of treating ductal carcinoma in situ (DCIS) in an individual comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, and (a) performing active surveillance of the DCIS in the individual, or (b) performing a lumpectomy on the individual, wherein the individual does not receive a mastectomy.

[0113] Embodiment 3. A method of conserving breast tissue in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, and (a) performing active surveillance of the DCIS in the individual or (b) performing a lumpectomy on the individual, wherein the individual does not receive a mastectomy.

[0114] Embodiment 4. A method of increasing the probability of breast conserving surgery in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the probability of breast conserving surgery in the individual is increased.

[0115] Embodiment 5. A method of avoiding a mastectomy in an individual having ductal carcinoma in situ (DCIS) comprising administering about 100 mg testosterone and about 4 mg anastrozole to the individual about every three months, wherein the individual does not receive a mastectomy.

[0116] Embodiment 6. A method of treating ductal carcinoma in situ (DCIS) in an individual, the method comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months; and performing active surveillance on the individual, wherein the individual is on active surveillance for at least 7 months.

[0117] Embodiment 7. The method of embodiment 6, wherein the individual does not receive surgical intervention while on active surveillance.28MF-366504339Attorney Docket No.: 356042000140

[0118] Embodiment 8. The method of embodiment 1, wherein the tumor size is reduced after 1, 2, or 3 administrations of the testosterone and the anastrozole.

[0119] Embodiment 9. The method of any one of embodiments 1-8, wherein the testosterone and the anastrozole are administered to the individual about every three months for up to about 36 months.

[0120] Embodiment 10. The method of any one of embodiments 1-9, wherein non-mass enhancement in ductal distribution is reduced following at least one administration of the testosterone and the anastrozole.

[0121] Embodiment 11. The method of embodiment 10, wherein non-mass enhancement in ductal distribution is reduced after 1, 2, or 3 administrations of the testosterone and the anastrozole.

[0122] Embodiment 12. The method of any one of embodiments 1-11, wherein the testosterone and the anastrozole are administered subcutaneously.

[0123] Embodiment 13. The method of embodiment 12, wherein the testosterone and the anastrozole are formulated as pellet.

[0124] Embodiment 14. The method of embodiment 13, wherein the pellet is cylindrical.

[0125] Embodiment 15. The method of embodiment 13 or claim 14, wherein the pellet is inserted subcutaneously in the upper outer gluteal region or iliac fossa of the individual.

[0126] Embodiment 16. The method of any one of embodiments 1-15, wherein the DCIS is hormone receptor positive DCIS.

[0127] Embodiment 17. The method of any one of embodiments 1-16, wherein the DCIS is ER+ / PR+ DCIS.

[0128] Embodiment 18. The method of any one of embodiments 1-17, wherein the individual is in perimenopause.

[0129] Embodiment 19. The method of any one of embodiments 1-17, wherein the individual is post-menopausal.

[0130] Embodiment 20. The method of any one of embodiments 1-19, wherein the DCIS is intermediate grade DCIS.29MF-366504339Attorney Docket No.: 356042000140

[0131] Embodiment 21. The method of any one of embodiments 1-20, wherein the individual is a woman.

[0132] Embodiment 22. The method of any one of embodiments 1-21, wherein the individual is categorized as low risk by MRI after 3 or 6 months of treatment.

[0133] Embodiment 23. The method of any one of embodiments 1-22, wherein the individual does not progress to invasive ductal carcinoma.

[0134] Embodiment 24. The method of any one of embodiments 1-23, wherein the individual receives MRI and or mammogram about every six months.

[0135] Embodiment 25. The method of embodiment 24, wherein the individual receives MRI and / or mammogram about every six months for about five years.

[0136] Embodiment 26. The method of any one of embodiments 1-25, wherein about 100 mg testosterone and about 4 mg anastrozole is administered to the individual.

[0137] Embodiment 27. The method of any one of embodiments 1-26, further comprising determining tumor size.

[0138] Embodiment 28. The method of embodiment 27, wherein the tumor size is determined by MRI.

[0139] Embodiment 29. The method of embodiment 28, wherein the tumor size is determined by mammography.

[0140] Embodiment 30. The method of any one of embodiments 1-29, wherein background parenchymal enhancement is reduced following at least one administration of the testosterone and the anastrozole.

[0141] Embodiment 31. The method of any one of embodiments 1-30, wherein one or more focal lesions is reduced following at least one administration of the testosterone and the anastrozole.

[0142] Embodiment 32. The method of any one of embodiments 1-31, wherein the number or extent of calcifications is reduced following at least one administration of the testosterone and the anastrozole.30MF-366504339Attorney Docket No.: 356042000140

[0143] Embodiment 33. The method of any one of embodiments 1-32, wherein breast density is reduced following at least one administration of the testosterone and the anastrozole.

[0144] Embodiment 34. The method of any one of embodiments 1-33, wherein the testosterone administered to the individual is testosterone or a pharmaceutically acceptable salt or ester thereof.

[0145] Further exemplary embodiments of the methods described herein include:

[0146] Embodiment 1A. A method of reducing tumor size in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to about 120 mg testosterone, or an equivalent amount of a pharmaceutically acceptable salt or ester thereof, and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the tumor size of the DCIS is reduced following at least one administration.

[0147] Embodiment 2A. A method of treating ductal carcinoma in situ (DCIS) in an individual comprising administering about 80 mg to about 120 mg testosterone, or an equivalent amount of a pharmaceutically acceptable salt or ester thereof, and about 2 mg to about 4 mg anastrozole to the individual about every three months, and (a) performing active surveillance of the DCIS in the individual, or (b) performing a lumpectomy on the individual, wherein the individual does not receive a mastectomy.

[0148] Embodiment 3A. A method of conserving breast tissue in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to about 120 mg testosterone, or an equivalent amount of a pharmaceutically acceptable salt or ester thereof, and about 2 mg to about 4 mg anastrozole to the individual about every three months, and (a) performing active surveillance of the DCIS in the individual or (b) performing a lumpectomy on the individual, wherein the individual does not receive a mastectomy.

[0149] Embodiment 4A. A method of increasing the probability of breast conserving surgery in an individual having ductal carcinoma in situ (DCIS) comprising administering about 80 mg to about 120 mg testosterone, or an equivalent amount of a pharmaceutically acceptable salt or ester thereof, and about 2 mg to about 4 mg anastrozole to the individual about every three months, wherein the probability of breast conserving surgery in the individual is increased.31MF-366504339Attorney Docket No.: 356042000140

[0150] Embodiment 5A. A method of avoiding a mastectomy in an individual having ductal carcinoma in situ (DCIS) comprising administering about 100 mg testosterone, or an equivalent amount of a pharmaceutically acceptable salt or ester thereof, and about 4 mg anastrozole to the individual about every three months, wherein the individual does not receive a mastectomy.

[0151] Embodiment 6A. A method of treating ductal carcinoma in situ (DCIS) in an individual, the method comprising administering about 80 mg to about 120 mg testosterone, or an equivalent amount of a pharmaceutically acceptable salt or ester thereof, and about 2 mg to about 4 mg anastrozole to the individual about every three months; and performing active surveillance on the individual, wherein the individual is on active surveillance for at least 7 months.

[0152] Embodiment 7A. The method of embodiment 6A, wherein the individual does not receive surgical intervention while on active surveillance.

[0153] Embodiment 8 A. The method of embodiment 1A, wherein the tumor size is reduced after 1, 2, or 3 administrations of the testosterone and the anastrozole.

[0154] Embodiment 9 A. The method of any one of embodiments 1A-8A, wherein the testosterone and the anastrozole are administered to the individual about every three months for up to about 36 months.

[0155] Embodiment 10A. The method of any one of embodiments 1A-9A, wherein nonmass enhancement in ductal distribution is reduced following at least one administration of the testosterone and the anastrozole.

[0156] Embodiment 11A. The method of embodiment 10A, wherein non-mass enhancement in ductal distribution is reduced after 1, 2, or 3 administrations of the testosterone and the anastrozole.

[0157] Embodiment 12 A. The method of any one of embodiments 1A-11A, wherein the testosterone and the anastrozole are administered subcutaneously.

[0158] Embodiment 13 A. The method of embodiment 12A, wherein the testosterone and the anastrozole are formulated as pellet.

[0159] Embodiment 14A. The method of embodiment 13 A, wherein the pellet is cylindrical.32MF-366504339Attorney Docket No.: 356042000140

[0160] Embodiment 15A. The method of embodiment 13A or claim 14A, wherein the pellet is inserted subcutaneously in the upper outer gluteal region or iliac fossa of the individual.

[0161] Embodiment 16A. The method of any one of embodiments 1A-15A, wherein the DCIS is hormone receptor positive DCIS.

[0162] Embodiment 17A. The method of any one of embodiments 1A-16A, wherein the DCIS is ER+ / PR+ DCIS.

[0163] Embodiment 18A. The method of any one of embodiments 1A-17A, wherein the individual is in perimenopause.

[0164] Embodiment 19A. The method of any one of embodiments 1A-17A, wherein the individual is post-menopausal.

[0165] Embodiment 20A. The method of any one of embodiments 1A-19A, wherein the DCIS is intermediate grade DCIS.

[0166] Embodiment 21 A. The method of any one of embodiments 1A-20A, wherein the individual is a woman.

[0167] Embodiment 22A. The method of any one of embodiments 1A-21A, wherein the individual is categorized as low risk by MRI after 3 or 6 months of treatment.

[0168] Embodiment 23 A. The method of any one of embodiments 1A-22A, wherein the individual does not progress to invasive ductal carcinoma.

[0169] Embodiment 24A. The method of any one of embodiments 1A-23A, wherein the individual receives MRI and or mammogram about every six months.

[0170] Embodiment 25A. The method of embodiment 24A, wherein the individual receives MRI and / or mammogram about every six months for about five years.

[0171] Embodiment 26A. The method of any one of embodiments 1A-25A, wherein about 100 mg testosterone and about 4 mg anastrozole is administered to the individual.

[0172] Embodiment 27A. The method of any one of embodiments 1A-26A, further comprising determining tumor size.

[0173] Embodiment 28A. The method of embodiment 27A, wherein the tumor size is determined by MRI.33MF-366504339Attorney Docket No.: 356042000140

[0174] Embodiment 29A. The method of embodiment 28A, wherein the tumor size is determined by mammography.

[0175] Embodiment 30A. The method of any one of embodiments 1A-29A, wherein background parenchymal enhancement is reduced following at least one administration of the testosterone and the anastrozole.

[0176] Embodiment 31 A. The method of any one of embodiments 1A-30A, wherein one or more focal lesions is reduced following at least one administration of the testosterone and the anastrozole.

[0177] Embodiment 32A. The method of any one of embodiments 1A-31A, wherein the number or extent of calcifications is reduced following at least one administration of the testosterone and the anastrozole.

[0178] Embodiment 33A. The method of any one of embodiments 1A-32A, wherein breast density is reduced following at least one administration of the testosterone and the anastrozole.

[0179] Embodiment 34A. The method of any one of embodiments 1A-33A, wherein the DCIS exhibits aromatase expression.

[0180] Embodiment 35A. The method of embodiment 34A, wherein the aromatase expression of the DCIS is detectable in epithelial and / or stromal breast tissue of the individual.

[0181] Embodiment 36A. The method of any one of embodiments 1A-35A, wherein the DCIS exhibits expression of at least one 5a-reductase isoenzyme.

[0182] Embodiment 37A. The method of any one of embodiments 1A-36A, wherein the administration of the testosterone and the anastrozole to the individual results in a reduction in estrogenic signaling relative to androgenic signaling within the DCIS.

[0183] Embodiment 38A. The method of any one of embodiments 1A-37A, wherein the administration of the testosterone and the anastrozole to the individual promotes androgen receptor-mediated transcriptional activity within DCIS tissue.

[0184] Embodiment 39A. The method of any one of embodiments 1A-38A, wherein the administration of the testosterone and the anastrozole to the individual reduces aromatase-mediated conversion of testosterone to estradiol within DCIS tissue.34MF-366504339Attorney Docket No.: 356042000140

[0185] Embodiment 40A. The method of any one of embodiments 1A-39A, wherein after administration of the testosterone and the anastrozole to the individual, testosterone is converted within the breast tissue of the individual to one or more 5a-reduced androgens.

[0186] Embodiment 41A. The method of embodiment 40A, wherein the one or more Sa-reduced androgens comprise dihydrotestosterone (DHT).

[0187] Embodiment 42A. The method of any one of embodiments 1A-41A, wherein the administration of the testosterone and the anastrozole to the individual results in alteration of intraductal steroid metabolism.

[0188] Embodiment 43A. The method of embodiment 42A, wherein the alteration of intraductal steroid metabolism corresponds to categorization of the individual as low risk for progression of disease and continuation of active surveillance.

[0189] Embodiment 44A. The method of any one of embodiments 1A-43A, wherein the DCIS harbors early PI3K pathway activation.

[0190] Embodiment 45 A. The method of any one of embodiments 1-44A, wherein the DCIS comprises a PIK3CA mutation.

[0191] Embodiment 46A. The method of any one of embodiments 1A-45A, wherein the DCIS comprises altered PI3K pathway signaling.

[0192] Embodiment 47A. The method of any one of embodiments 1A-46A, wherein the administration of the testosterone and the anastrozole reprograms PI3K- associated survival signaling.

[0193] Embodiment 48A. The method of any one of embodiments 1A-47A, wherein the administration of the testosterone and the anastrozole provides sustained androgen receptor agonism.

[0194] Embodiment 49 A. The method of any one of embodiments 1A-48A, wherein the administration of the testosterone and the anastrozole suppresses estrogen formation.

[0195] Embodiment 50 A. The method of any one of embodiments 1A-49A, wherein the administration of the testosterone and the anastrozole restores endocrine responsiveness.35MF-366504339Attorney Docket No.: 356042000140

[0196] Embodiment 51 A. The method of any one of embodiments 1A-50A, wherein the administration of the testosterone and the anastrozole enables regression or stability of disease that is compatible with active surveillance.

[0197] Embodiment 52 A. The method of any one of embodiments 1A-51A, wherein the regression or stability of disease is evaluated and / or defined by imaging.

[0198] Embodiment 53 A. The method of any one of embodiments 1A-52A, wherein the individual is not administered a PI3K inhibitor.EXAMPLESExample 1 - DCIS: RECAST Trial Ductal Carcinoma In Situ: Re-Evaluating Conditions for Active Surveillance Suitability as TreatmentSummary

[0199] DCIS RECAST (Re-Evaluating Conditions for Active-Surveillance as Treatment) is a multicenter, randomized platform trial with a well-defined DCIS management strategy using active surveillance and response to endocrine risk-reduction treatment measured by non-invasive imaging (MRI and mammographic) endpoints. There are three objectives to the trial: (1) determine if next- generation endocrine treatments are more effective and / or more tolerable for risk reduction than standard agents (tamoxifen and letrozole), especially for luminal B disease; (2) validate the use of early imaging endpoints used to distinguish between patients at low risk for progression to invasive cancer (candidates for active surveillance) and those at high risk (candidates for surgical therapy); (3) better understand the underlying biology of responders and non-responders. The trial was sponsored by Quantum Leap Healthcare Collaborative.

[0200] This study incorporates both a neoadjuvant therapy phase (3-6 months), with patients at high risk for progression to invasive disease proceeding to surgery, followed by an extended surveillance phase for low-risk patients. The trial will test experimental agents to determine if they can increase the proportion of women who can remain on active surveillance after 7 months of endocrine therapy using the reduction of background parenchymal enhancement (BPE) and non-mass enhancement. Patients will receive experimental or standard endocrine risk-reduction (for premenopausal women: tamoxifen at 20 mg standard dose or 5 mg; for postmenopausal women: exemestane 25 mg daily or 336MF-366504339Attorney Docket No.: 356042000140times per week, letrozole 2.5 mg, anastrozole 1 mg orally, or 5 mg or 20 mg tamoxifen if an aromatase inhibitor is not tolerated). Measures of response will include change in BPE and focal lesions (mass or non-mass enhancement). Artificial intelligence imaging tools will be secondary endpoints. Women deemed to be low or intermediate risk after 6 months (absence of a distinct lesion, reduction of BPE, and lack of emergence of a focal lesion) will continue active surveillance and assigned endocrine therapy for 6 additional months. The concordance of “low-risk” categorization with a 3- and 5-year invasive breast cancer risk of under 15% will be assessed.

[0201] Patients will receive a baseline MRI, be stratified based on whether a distinct lesion is detected or not, randomly assigned to one of the endocrine therapy arms, and reevaluated by MRI at 3 and 6 months. Serial MRIs and imaging response will determine patient suitability for continuing on active surveillance or proceeding to surgery. Imaging is used to evaluate DCIS lesion response and BPE. If a lesion is distinct and stays distinct, the patient will be categorized as “high-risk” and definitive surgery will be recommended. If there is initially no distinct lesion, the background is responsive but the newly apparent lesion is not, and the lesion appears distinct at the second MRI, surgery will also be recommended. Those whose background and / or lesions decrease are in the “low-risk” group and can continue on active surveillance. Those in the intermediate group (stable lesion or stable background) may also continue surveillance (provider / patient decision) if they choose. After 6 months, patients continuing active surveillance will receive MRIs at 12, 24, 36, 48, and 60 months. All patients will also receive mammograms at 0 and 6 months; those continuing on active surveillance will also receive mammograms at 18, 30, 42, and 54 months. QOL will be surveyed every other week for the first 2 months and monthly thereafter.Treatment Regimens

[0202] The goal of this study is to see if active surveillance monitoring and hormonal therapy in patients diagnosed with Ductal cell Carcinoma In Situ (DCIS), an early stage of breast cancer, can be an effective management of the disease. The current management of most patients with DCIS involves surgical intervention with or without radiation, similar to more aggressive breast cancers. These treatments can come with some significant health effects. The main question this study aims to answer is: to determine whether novel endocrine therapy increases the fraction of patients who will be suitable for long-term active surveillance.37MF-366504339Attorney Docket No.: 356042000140

[0203] Participants will be asked to take one of three investigational study medications (Elacestrant, Testosterone + Anastrazole, or Z-Endoxifen) or receive control hormonal therapy (Tamoxifen or an aromatase inhibitor), depending on the treatment to which they have been randomized. Participants will be asked to return for evaluation with MRI at three months and six months. Depending on the evaluation, participants will have the option to continue on the treatment, with follow up evaluations of Mammogram and MRI at 6 month intervals. If the evaluation suggests surgery is recommended, the participant will discontinue the study treatment and will undergo surgery. In addition to the treatment and MRI evaluation, participants will be asked to: provide blood sample to understand their immune status, provide saliva sample for genetic testing, and provide the study with a portion of the tissue or slides generated from tissue removed during surgery performed as part of their standard of care. Participants will be followed annually for 10 years.

[0204] Control Arm:• For premenopausal women: 20 mg, 10 mg, or 5 mg tamoxifen orally• For postmenopausal women: standard oral doses of aromatase inhibitor (Al) of choice: exemestane 25 mg daily, letrozole 2.5 mg daily, or anastrozole 1 mg daily; or reduced exemestane dosing: 25 mg 3 times per week orally.• For postmenopausal women who are not tolerating an Al, investigators can change them to the low dose (5 mg or 10 mg) or standard dose (20 mg) of tamoxifen.• There is active follow up with MRI at baseline, 3 months, 6 months after treatment initiation, and every 6 months alternating MRI and mammogram for up to 5 years. Participants may continue treatment for up to 5 years.

[0205] Experimental Arms:• Testosterone and Anastrozole: For both pre- and post- menopausal subjects, 100 mg testosterone in combination with 4 mg anastrozole administered subcutaneously every 3 months for up to 3 years. Testosterone and anastrozole are administered as a white solid pellet for subcutaneous insertion consisting of 100 mg Testosterone and 4 mg Anastrozole, an aromatase inhibitor. A cylindrical pellet (4.5 mm diameter, 6.35 mm diameter) is inserted subcutaneously in the upper outer gluteal region or iliac fossa every 3 months, with treatment up to 3638MF-366504339Attorney Docket No.: 356042000140months. There is active follow up with MRI at baseline, 3 months, 6 months after treatment initiation, and every 6 months alternating MRI and mammogram for up to 5 years. Participants are followed for an additional 5 years.• Elacestrant: For both pre- and post-menopausal subjects, 400 mg PO elacestrant with food once daily up to 36 months.* (Z)-endoxifen: Both pre- and post- menopausal subjects, (z)-endoxifen lOmg delayed release capsule 1 hour before a meal or 2 hours after a meal once daily for up to 36 months.Inclusion Criteria• Female, at least 18 years old• Previous diagnosis of hormone receptor (HR)+ DCIS (at least 50% ER or PR and 2+;biopsy will have been performed previously at diagnosis) with or without microinvasion• Patients who have previously received endocrine therapy should have a washout period of 4-6 weeks prior to the screening MRI on the RECAST-DCIS trial• Bilateral mammogram performed within up to 4 months (120 days) of the start of trial treatment may be used for screening evaluation• MRI performed within up to 2 months (60 days) of the start of trial treatment for lesion evaluation• CBC w / diff, CMP, and Lipid Panel within normal limits within a year of the start of trial treatment. Abnormal labs to be repeated within 60 days prior to the start of trial treatment. Patients will be considered eligible for screening labs that are abnormal or out-of-range if the investigator has deemed the lab results not-clinically significant • Negative urine or serum pregnancy test within 1 month of the start of trial treatment • Controlled HIV positive patients are allowed as long as their current medication does not contraindicate the study's investigational agent• Informed consent provided by the patient• Willingness and ability to provide tumor samples for researchExclusion Criteria• Pregnant or actively breastfeeding women (must be documented by a pregnancy test during screening)39MF-366504339Attorney Docket No.: 356042000140• History of allergic reactions attributed to compounds of similar chemical or biologic composition to study agent based on review of the medical record and patient history • Invasive carcinoma or identification of a mass on MRI that is subsequently biopsied and found to be invasive cancer• Co-enrollment in clinical trials of pharmacologic agents requiring an IND• Ongoing treatment for DCIS other than what is specified in this protocol• Uncontrolled intercurrent illness, including psychiatric conditions, that would limit compliance with study requirements• Medical history or ongoing gastrointestinal disorders potentially affecting the absorption of investigational agent and / or tamoxifen. Active inflammatory bowel disease or chronic diarrhea, known active hepatitis A / B / C*, hepatic cirrhosis, short bowel syndrome, or any upper gastrointestinal surgery including gastric resection or banding procedureso *Active hepatitis, defined as: A (positive HA antigen or positive IgM); B (either positive HBs antigen or positive hepatitis B viral DNA test above the lower limit of detection of the assay); C (positive hepatitis C antibody result, and quantitative hepatitis C (HCV) ribonucleic acid (RNA) results greater than the lower limits of detection of the assay)• Participants unable to swallow normally or unable to take tablets and capsules.Predictable poor compliance to oral treatment.

[0206] Patients will be considered low risk if:• Lesion is not distinct or is non-mass enhancement (NME) and• NME or BPE respond to ET and,• There is moderate or marked BPE or NME is not conspicuous nor distinct• Patients will be considered intermediate risk if:• Lesion is not distinct or is NME and• Neither mass nor background respond to ET and• NME is not distinct from background

[0207] Patients will be considered high risk if:• There is a distinct lesion that is a mass or• Neither mass nor background respond to ET and NME is distinct from background or 40MF-366504339Attorney Docket No.: 356042000140• NME or BPE respond to ET and there is minimal BPE and NME is conspicuous or distinct from BPEPrimary Endpoint

[0208] Patients remaining on active surveillance at 7 months compared to control. Secondary Endpoints• Fraction of patients categorized as low risk by MRI after 6 months of treatment- Measured by cases demonstrating endocrine responsiveness (Determined based on lesion and background or lesion alone or lack of lesion and minimal background) • Fraction of patients categorized as low risk by MRI after 3 months of treatment- Measured by cases demonstrating endocrine responsiveness (Determined based on lesion and background or lesion alone or lack of lesion and minimal background) • Correlation of low-risk categorization at 6 months with subsequent rate of Invasive Ductal Cell Carcinoma progression at 3 years• Fraction of patients experiencing Minimum Important Difference in overall QOL measured by PROP-R statistics (PROMIS) and FACT-ES (functional assessment of cancer therapy- endocrine symptoms, a quality of life (QoL) assessment)• Rate of reduction in focal lesions (mass and non-mass enhancement (NME)) at 6 months and Rate of reduction in focal lesions using automated Functional Tumor Volume• % with contralateral reduction in qualitative and automated BPE and % reduction in contralateral breast density• Time to discontinuation of therapy (Tolerability of therapy) will be measured, and Adherence rates on each regimen, as well as PROP-R and FACT-ES score on each regimen will be measured. These assessments will be combined to evaluate efficacy and toxicity using a novel clinical benefit index.• Correlation of BPE change with agent and compare to quantitative imaging density Other Outcome Measures• Evaluate influence of polygenic risk on endocrine response and suitability for Active Surveillance41MF-366504339Attorney Docket No.: 356042000140• Response stratified by multiplex immune staining for Human Epidermal growth factor receptor-2 (HER2) isoforms and Response Predictive Subtypes (RPS)Imaging

[0209] MRIs will be obtained at 0, 3, 6, 12, 24, 36, 48, and 60 months. For premenopausal patients, it is preferred that MRIs will be performed 3-10 days after the start of menstruation in a given menstrual cycle.

[0210] Standardized BPE and lesion assessment: Evaluation of lesion stability vs. progression will be based on standardized qualitative interpretation using the algorithm developed in the analysis of our active surveillance data and proposed program of assessment. Imaging features will be assessed by two radiologists. If there is a discrepancy between the two assessments, a third radiologist will review and resolve the discrepancy. Table El: Imaging features to be assessed, asked for each MRITable E2: Imaging features to be assessed at each subsequent MRI (change assessment)Table E3: Mammogram imaging42MF-366504339Attorney Docket No.: 356042000140Table E4: Imaging for subsequent Mammograms - For each subsequent mammogram, there will be the following additional assessments:Example 2 - Preliminary Results

[0211] This Example provides interim results for a patient with DCIS who received 100 mg testosterone in combination with 4 mg anastrozole administered subcutaneously every 3 months in the RECAST study described in Example 1.

[0212] MRI was performed six months after the start of treatment with testosterone and anastrozole. At the time of imaging the patient had received two doses.

[0213] As shown in FIGS. 1A-1B, after two doses of testosterone and anastrozole, a significant reduction in non-mass enhancement (NME) was observed in the left breast. FIG.1A shows NME in a ductal distribution in the left breast (labeled with an arrow), at the pretreatment baseline. FIG. IB shows much less visible NME in the left breast (labeled with an arrow), after six months of treatment with testosterone and anastrozole (two doses).Similarly, as shown in FIGS. 2A-2B, prior to treatment, the patient had extensive DCIS with a low probability of breast conserving surgery due to involved margin (FIG. 2A). The patient had a likely poor cosmetic outcome due to the location of the DCIS in the breast. Following two doses of testosterone and anastrozole (six months of treatment), almost complete resolution of DCIS was achieved (FIG. 2B). Based upon the response, the patient appears to be eligible for breast conservation, with potentially only a minor surgical resection required.

[0214] These data demonstrate that treatment of DCIS with 100 mg testosterone in combination with 4 mg anastrozole administered subcutaneously every 3 months resulted in 43MF-366504339Attorney Docket No.: 356042000140reduced tumor size, a reduction in NME, and almost complete resolution of DCIS. Based upon this data combination therapy comprising testosterone and anastrozole may be appropriate for treating DCIS with minimal surgery by reducing tumor size. Patients who receive the combination therapy may be eligible for breast conserving surgery (i.e., a lumpectomy) and / or to undergo active surveillance rather than undergoing a mastectomy. Example 3 - Further Preliminary Results

[0215] This Example provides additional interim results for 50 patients enrolled in the RECAST study across the randomized arms as described in Example 1.

[0216] At the 3-month follow-up point of the study, 47 of 50 patients were undergoing or continuing with active surveillance / treatment. The remaining 3 of 50 patients stopped active surveillance / treatment. Two of these patients stopped for personal reasons. For one patient, imaging showed progression to invasive ductal carcinoma.

[0217] At the 6-month follow-up point of the study, 44 patients were undergoing or continuing with active surveillance / treatment. 19 of these 44 patients were prospectively continuing to 6 months. 25 of these 44 patients had reached or surpassed 6 months. 3 patients stopped active surveillance / treatment. For two of these patients, imaging showed progression to invasive ductal carcinoma. For one patient, surgery was elected at the 1 year time point and invasive disease (pTlbNO) was discovered.

[0218] At the long-term follow-up point of the study (median follow-up of 351 days), 22 / 25 patients remained on active surveillance.

[0219] 17 patients experienced any adverse event. All adverse events reported were Grade 1 or 2. No serious adverse events were reported and no safety signals were identified.

[0220] These results indicate that treatment with testosterone and anastrozole as described herein shows a clean early safety and feasibility profile within the RECAST study, with no observed invasive progression or active surveillance discontinuation in its initial cohort.44MF-366504339

Claims

Attorney Docket No.: 356042000140CLAIMS1. A method of reducing tumor size in an individual having ductal carcinoma in situ (DCIS) comprisingadministering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months,wherein the tumor size of the DCIS is reduced following at least one administration.

2. A method of treating ductal carcinoma in situ (DCIS) in an individual comprising administering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, and(a) performing active surveillance of the DCIS in the individual, or(b) performing a lumpectomy on the individual,wherein the individual does not receive a mastectomy.

3. A method of conserving breast tissue in an individual having ductal carcinoma in situ (DCIS) comprisingadministering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months, and(a) performing active surveillance of the DCIS in the individual or(b) performing a lumpectomy on the individual,wherein the individual does not receive a mastectomy.

4. A method of increasing the probability of breast conserving surgery in an individual having ductal carcinoma in situ (DCIS) comprisingadministering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months,wherein the probability of breast conserving surgery in the individual is increased.

5. A method of avoiding a mastectomy in an individual having ductal carcinoma in situ (DCIS) comprising45MF-366504339Attorney Docket No.: 356042000140administering about 100 mg testosterone and about 4 mg anastrozole to the individual about every three months,wherein the individual does not receive a mastectomy.

6. A method of treating ductal carcinoma in situ (DCIS) in an individual, the method comprisingadministering about 80 mg to about 120 mg testosterone and about 2 mg to about 4 mg anastrozole to the individual about every three months; andperforming active surveillance on the individual,wherein the individual is on active surveillance for at least 7 months.

7. The method of claim 6, wherein the individual does not receive surgical intervention while on active surveillance.

8. The method of claim 1, wherein the tumor size is reduced after 1, 2, or 3 administrations of the testosterone and the anastrozole.

9. The method of any one of claims 1-8, wherein the testosterone and the anastrozole are administered to the individual about every three months for up to about 36 months.

10. The method of any one of claims 1-9, wherein non-mass enhancement in ductal distribution is reduced following at least one administration of the testosterone and the anastrozole.

11. The method of claim 10, wherein non-mass enhancement in ductal distribution is reduced after 1, 2, or 3 administrations of the testosterone and the anastrozole.

12. The method of any one of claims 1-11, wherein the testosterone and the anastrozole are administered subcutaneously.

13. The method of claim 12, wherein the testosterone and the anastrozole are formulated as pellet.46MF-366504339Attorney Docket No.: 35604200014014. The method of claim 13, wherein the pellet is cylindrical.

15. The method of claim 13 or claim 14, wherein the pellet is inserted subcutaneously in the upper outer gluteal region or iliac fossa of the individual.

16. The method of any one of claims 1-15, wherein the DCIS is hormone receptor positive DCIS.

17. The method of any one of claims 1-16, wherein the DCIS is ER+ / PR+ DCIS.

18. The method of any one of claims 1-17, wherein the individual is in perimenopause.

19. The method of any one of claims 1-17, wherein the individual is post-menopausal.

20. The method of any one of claims 1-19, wherein the DCIS is intermediate grade DCIS.

21. The method of any one of claims 1-20, wherein the individual is a woman.

22. The method of any one of claims 1-21, wherein the individual is categorized as low risk by MRI after 3 or 6 months of treatment.

23. The method of any one of claims 1-22, wherein the individual does not progress to invasive ductal carcinoma.

24. The method of any one of claims 1-23, wherein the individual receives MRI and or mammogram about every six months.

25. The method of claim 24, wherein the individual receives MRI and / or mammogram about every six months for about five years.

26. The method of any one of claims 1-25, wherein about 100 mg testosterone and about 4 mg anastrozole is administered to the individual.47MF-366504339Attorney Docket No.: 35604200014027. The method of any one of claims 1-26, further comprising determining tumor size.

28. The method of claim 27, wherein the tumor size is determined by MRI.

29. The method of claim 28, wherein the tumor size is determined by mammography.

30. The method of any one of claims 1-29, wherein background parenchymal enhancement is reduced following at least one administration of the testosterone and the anastrozole.

31. The method of any one of claims 1-30, wherein one or more focal lesions is reduced following at least one administration of the testosterone and the anastrozole.

32. The method of any one of claims 1-31, wherein the number or extent of calcifications is reduced following at least one administration of the testosterone and the anastrozole.

33. The method of any one of claims 1-32, wherein breast density is reduced following at least one administration of the testosterone and the anastrozole.

34. The method of any one of claims 1-33, wherein the testosterone administered to the individual is testosterone or a pharmaceutically acceptable salt or ester thereof.48MF-366504339

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