P53 protein stabieizers inceuding MDM2 inhibitors as a "chemicae mastectomy" strategy in breast cancer BRCA1 / 2 mutation carriers

Abstract

A method may include a method for aiding in preventing breast cancer in a subject having a heterozygous germline mutation in BRCA1 or BRCA2, comprising: a. identifying a subject having a heterozygous germline mutation in BRCA1 or BRCA2; b. administering to the subject a therapeutically effective amount of a P53 protein stabilizer, wherein the P53 protein stabilizer comprises an MDM2 inhibitor.

Description

APPLICATION OF P53 PROTEIN STABILIZERS INCLUDING MDM2 INHIBITORS AS A “CHEMICAL MASTECTOMY” STRATEGY IN PREVENTING BREAST CANCER IN BRCA1 / 2 MUTATION CARRIERS Cross-Reference To Related Application

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No.63 / 643,152 entitled “APPLICATION OF P53 PROTEIN STABILIZERS INCLUDING MDM2 INHIBITORS AS A “CHEMICAL MASTECTOMY” STRATEGY IN PREVENTING BREAST CANCER IN BRCA1 / 2 MUTATION CARRIERS,” filed May 6, 2024, the disclosure of which is incorporated herein in its entirety by reference. Government Support

[0002] This invention was made with government support under Grant No. R01CA273696 awarded by the National Institute of Health. The government has certain rights in the invention. Field of the Disclosure

[0003] The present disclosure pertains to medical treatments and diagnostics, specifically focusing on methods and compositions for aiding in preventing breast cancer in individuals with BRCA1 or BRCA2 mutations and for identifying pathogenic variants of unknown significance (VUS) in these genes. Background of the Disclosure

[0004] Individuals carrying heterozygous germline mutations in the BRCA1 or BRCA2 genes are at a significantly elevated risk for developing breast and ovarian cancers. The standard preventive strategy often involves prophylactic surgeries, such as bilateral mastectomy, which can be psychologically and physically demanding. There exists a substantial need for alternative, less invasive preventive options. Moreover, the clinical management of BRCA mutation carriers is complicated by the difficulty in identifying pathogenic VUS, which hampers decision-making regarding preventive treatments.Summary of the Disclosure

[0005] This disclosure helps to address these challenges by introducing methods and compositions for a chemoprevention strategy using Nutlin 3a (4-[[(4S,5R)-4,5-Bis(4- chlorophenyl)-4,5-dihydro-2-[4-methoxy-2-(1-methylethoxy)phenyl]-1H-imidazol-1-yl]carbonyl]- 2-piperazinone) or a derivative thereof. Nutlin 3a is an MDM2 antagonist (IC50 = 90 nM); inhibits the MDM2-p53 interaction. The active enantiomer of Nutlin 3a (Cat. No.3984) induces expression for P53 regulated genes. Mouse double minute 2 homolog (MDM2) also known as E3 ubiquitin-protein ligase MDM2 is a protein that in humans is encoded by the MDM2 gene. MDM2 is an important negative regulator of the p53 tumor suppressor. MDM2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain (TAD) of the p53 tumor suppressor and as an inhibitor of p53 transcriptional activation. p53, also known as Tumor protein P53, cellular tumor antigen p53 or transformation-related protein 53 (TRP53) is a regulatory transcription factor protein that is often mutated in human cancers. The p53 proteins (originally thought to be, and often spoken of as, a single protein) are crucial in vertebrates, where they prevent cancer formation.

[0006] The TP53 gene is the most frequently mutated gene (>50%) in human cancer, indicating that the TP53 gene plays a crucial role in preventing cancer formation. TP53 gene encodes proteins that bind to DNA and regulate gene expression to prevent mutations of the genome. In addition to the full-length protein, the human TP53 gene encodes at least 12 protein isoforms.

[0007] This strategy exploits the specific vulnerability of BRCA1 / 2 heterozygous cells to fluctuations in P53 protein levels, offering a potential non-surgical (e.g., non-mastectomy) option for cancer prevention. Additionally, the disclosure encompasses methods for employing Nutlin 3a or a derivative thereof to ascertain the pathogenicity of VUS in BRCA genes, thereby facilitating personalized treatment decisions.

[0008] In some aspects, the techniques described herein relate to a method for aiding in preventing breast cancer in a subject having a heterozygous germline mutation in BRCA1 or BRCA2, including: a. identifying a subject having a heterozygous germline mutation in BRCA1 or BRCA2; b. determining the pathogenicity of the BRCA1 or BRCA2 mutation; c. administering to the subject a therapeutically effective amount of a P53 protein stabilizer, wherein the P53 protein stabilizer includes an MDM2 inhibitor.

[0009] In some aspects, the techniques described herein relate to a method for identifying a pathogenic variant of unknown significance (VUS) in a BRCA1 or BRCA2 gene in a subject, including: a. obtaining a sample of mammary epithelial cells from the subject; b. culturing themammary epithelial cells; c. treating the cultured mammary epithelial cells with a P53 protein stabilizer; d. assessing the sensitivity of the treated cells to the P53 protein stabilizer; and e. determining the pathogenicity of the VUS based on the sensitivity of the cells to the P53 protein stabilizer.

[0010] In some aspects, the techniques described herein relate to a pharmaceutical composition including a P53 protein stabilizer for use in treating a subject having a heterozygous germline mutation in BRCA1 or BRCA2 to prevent breast cancer, wherein the P53 protein stabilizer includes an MDM2 inhibitor.

[0011] In some aspects, the techniques described herein relate to a use of a P53 protein stabilizer in the manufacture of a medicament for preventing breast cancer in a subject having a heterozygous germline mutation in BRCA1 or BRCA2, wherein the P53 protein stabilizer includes an MDM2 inhibitor.

[0012] In some aspects, the techniques described herein relate to a diagnostic kit for identifying a pathogenic variant of unknown significance (VUS) in a BRCA1 or BRCA2 gene in a subject, including: a. reagents for culturing mammary epithelial cells; b. a P53 protein stabilizer; and c. instructions for treating the cultured mammary epithelial cells with the P53 protein stabilizer and assessing their sensitivity to determine the pathogenicity of the VUS. Brief Description of the Drawings

[0013] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present invention.

[0014] FIG.1A provides a table listing lines and the mutation information of various samples.

[0015] FIG.1B shows expression in BRCA2 heterozygous cells compared to the wildtype counterparts.

[0016] FIG.2A shows results of incubation of cells with HU and a decrease in the length of IdU.

[0017] FIG.2B is a graph showing that BRCA2mut / + cell lines exhibited increased sensitivity compared to BRCA2 wildtype lines when treated with HU

[0018] FIGS.3A and 3B show results of immunofluorescence (IF) analysis of p53 mean intensity

[0019] FIGS 3C and 3D show western blot analysis of p53 protein level in whole-cell lysates.

[0020] FIG.4A shows western blot analysis of blocking proteasome-mediated p53 degradation leading to increased p53 levels in four BRCA2 heterozygous lines.

[0021] FIG.4B is a western blot analysis showing that incubation with Nutlin-3a in BRCA2mut / + cells restored P53 protein level.

[0022] FIG.4C is a western blot analysis showing that restoration of P53 protein level in BRCA1mut / + cells is possible.

[0023] FIGS, 5A and 5B are graphs showing sensitivity of BRCA1 and BRCA2 heterozygous cells to Nutlin 3a treatment, relative to wildtype cells. Detailed Description

[0024] This disclosure helps to address the aforementioned challenges by introducing methods and compositions for a chemoprevention strategy using Nutlin 3a or a derivative thereof, which is an MDM2 inhibitor as a P53 protein stabilizer. This strategy exploits the specific vulnerability of BRCA1 / 2 heterozygous cells to fluctuations in P53 protein levels, offering a potential non-surgical (e.g., non-mastectomy) option for cancer prevention. Additionally, the disclosure encompasses methods for employing Nutlin 3a or a derivative thereof to ascertain the pathogenicity of VUS in BRCA genes, thereby facilitating personalized treatment decisions.

[0025] The chemoprevention method involves identifying subjects who have heterozygous germline mutations in BRCA1 or BRCA2 through standard genetic testing. As used herein a “subject” can include any mammalian subject such as a human, mouse, rat, monkey, Guinea pig, rabbit, or other mammal. Genetic testing for BRCA1 and BRCA2 involves several methods to identify mutations that increase the risk of breast and ovarian cancers. The process typically may with a consultation with a healthcare professional or genetic counselor, who may review personal and family history of cancer to determine if testing is appropriate. A sample of blood or saliva may be collected and sent to a laboratory for analysis. The lab uses techniques such as DNA sequencing to examine the BRCA1 and BRCA2 genes for harmful changes, also known as mutations. There are different types of genetic tests available, including single-gene tests that focus solely on BRCA1 and BRCA2, and multigene panel tests that look for mutations in several genes simultaneously. The results can be positive, indicating a mutation that increases cancer risk; negative, meaning no harmful mutation was found; or uncertain, where the significance of the detected variant is unclear

[0026] BRCA1 and BRCA2 are tumor suppressor genes, which means they help repair damaged DNA and prevent cells from growing uncontrollably. Mutations in these genes impair their ability to repair DNA damages, leading to an increased risk of cancer. Women with BRCA1or BRCA2 mutations have a significantly higher risk of developing breast and ovarian cancers compared to the general population. These mutations can also increase the risk of other cancers, such as prostate and pancreatic cancers.

[0027] If a pathogenic BRCA1 or BRCA2 mutation is identified, Nutlin 3a or a derivative thereof is then administered in a therapeutically effective amount, typically ranging from 100 mg to 500 mg daily, tailored to the patient's condition and response. Administration can be oral via tablets, capsules, or liquid suspensions. In some embodiments, Nutlin 3a can be administered subcutaneously. In some embodiments, Nutlin 3a administration can be intravenous. In some embodiments, Nutlin 3a administration can be through orthotopic injection, such as fat pad injection and intraductal injection. Subjects are monitored for any side effects associated with Nutlin 3a or a derivative thereof, with dosage adjustments made accordingly to optimize safety and efficacy.

[0028] The mechanism of action of Nutlin 3a or a derivative thereof involves the inhibition of MDM2, a protein that negatively regulates the tumor suppressor protein P53. By blocking MDM2, Nutlin 3a or a derivative thereof prevents the degradation of P53, thereby stabilizing and increasing the levels of P53 in cells. Elevated P53 levels facilitate the activation of DNA repair pathways and induce apoptosis in cells with damaged DNA, such as those found in BRCA1 / 2 heterozygous individuals. This mechanism is crucial for preventing the proliferation of potentially cancerous cells.

[0029] The diagnostic method for VUS includes obtaining mammary epithelial cells from subjects, typically via a minimally invasive biopsy, and culturing them in appropriate media. The cultured cells are then treated with Nutlin 3a or a derivative thereof at concentrations ranging from 1 µM to 10 µM. The sensitivity of the cells to Nutlin 3a or a derivative thereof is assessed, correlating with the pathogenicity of the VUS. Cells that show increased sensitivity are indicative of pathogenic mutations.

[0030] Related biomarkers include P53 levels, which serve as a primary biomarker for assessing the efficacy of the treatment. Monitoring MDM2 levels can provide insights into the effectiveness of Nutlin 3a or a derivative thereof in inhibiting its target and stabilizing P53. Additionally, biomarkers such as γ-H2AX, which indicate DNA damage, can be useful in evaluating the cellular response to Nutlin 3a or a derivative thereof treatment.

[0031] Pharmaceutical compositions containing Nutlin 3a or a derivative thereof are formulated for effective delivery, considering factors like stability and patient compliance. Diagnostic kits include all necessary reagents and instructions for performing the VUS sensitivityassay, potentially complemented by software for data analysis and interpretation, enhancing the utility and accuracy of the diagnostic process.

[0032] In some examples, the pharmaceutical composition can include a derivative of Nutlin 3a or a derivative thereof. For example, the pharmaceutical composition can include the structure according to Formula I. , wherein at each occurrence -H, substituted orunsubstituted (C1-C20) or -O(C1-C20)hydrocarbyl, substituted or unsubstituted (C1-C20)aryl. Additionally, at each occurrence, -X is independently a halogen. In some examples -X is independently chloride or bromide.

[0033] In some examples, the pharmaceutical composition includes Nutlin 3a or a derivative thereof represented according to Formula II.

[0034] The3a or a derivative thereof are specifically designed to ensure stability and patient compliance, with considerations made for the method of administration and the physical form of the medication. Additionally, diagnostic kits have been developed to support the implementation of the VUS sensitivity assay. These kits include all necessary reagents and instructions for the assay, and may also feature software tools designed to assist in the analysis and interpretation of results, thereby enhancing the utility and accuracy of the diagnostic process.

[0035] This disclosure offers significant advantages over existing preventive and diagnostic approaches for BRCA1 / 2 mutation carriers by providing a non-surgical option forcancer prevention and facilitating personalized treatment planning based on the pathogenicity of VUS. It utilizes Nutlin 3a or a derivative thereof, leveraging its known safety profile and mechanism of action for quicker clinical application.

[0036] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0037] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. A comma can be used as a delimiter or digit group separator to the left or right of a decimal mark; for example, “0.000,1” is equivalent to “0.0001.” All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0038] In the methods described herein, the acts can be carried out in any order without departing from the principles of the disclosure, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conductedsimultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0039] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0040] The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of” as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than or equal to about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0041] Pharmaceutical compositions contain an effective amount of a compound as described herein and optionally one or more other therapeutic agents included in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also can be commingled with the compounds, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

[0042] The phrase “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ or portion of the body to another organ or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the composition, not injurious to the patient, and substantially non-pyrogenic. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, castor oil, medium chain triglyceride oil, safflower oil, sesame oil, olive oil, cornoil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical compositions. Pharmaceutical compositions of the present disclosure are non- pyrogenic, i.e., do not induce significant temperature elevations when administered to a patient.

[0043] The drug can be present in the pharmaceutical composition in a therapeutically effective amount. A “therapeutically effective amount” (or “effective amount”) of a compound with respect to use in treatment, refers to an amount of the compound in a preparation which, when administered as part of a desired dosage regimen (to a mammal, such as a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0044] The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the patient of one or more compound of the disclosure. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, (e.g., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (e.g., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0045] As used herein, the term “kit” refers to a product (e.g. medicament, kit-of- parts) comprising one package or one or more separate packages of: (i). A pharmaceutical composition containing an active pharmaceutical ingredient and at least one further active pharmaceutical ingredient and optionally a medical device. The at least one further active pharmaceutical ingredient may be present in said pharmaceutical composition, i.e. the kit may comprise one or more packages, wherein each package comprises one pharmaceutical composition which comprises two or more active pharmaceutical ingredients. The further active pharmaceutical ingredient may also be present in a further pharmaceutical composition, i.e. the kit may comprise separate packages of two or more pharmaceutical compositions, wherein each pharmaceutical composition contain one active pharmaceutical ingredient. Or(ii). A pharmaceutical composition containing an active pharmaceutical ingredient and medical device.

[0046] A kit may comprise one package only or may comprise one or more separate packages. For example, the kit may be a product (e.g. medicament) containing two or more vials each containing a defined pharmaceutical composition, wherein each pharmaceutical composition contains at least one active pharmaceutical ingredient. For example, the kit may comprise (i.) a vial containing a defined pharmaceutical composition and (ii). further a tablet, capsule, powder or any other oral dosage form which contains at least one further active pharmaceutical ingredient. The kit may further comprise a package leaflet with instructions for how to administer the pharmaceutical composition and the at least one further active pharmaceutical ingredient.

[0047] As used herein, the term “medical device” means any instrument, apparatus, implant, in vitro reagent or similar or related article that is used to diagnose, prevent, or treat a disease of other condition, and does not achieve its purpose through pharmacological action within or on the body.

[0048] The term “organic group” as used herein refers to any carbon-containing functional group. Examples can include an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0-2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (C1-C100)hydrocarbyl, wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.

[0049] The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include,but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0-2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C1- C100)hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.

[0050] The term “alkyl” as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n- propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompasses n- alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0051] The term “alkenyl” as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2,-C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

[0052] The term “alkynyl” as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to –C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and -CH2C≡C(CH2CH3) among others.

[0053] The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is bonded to a hydrogen forming a “formyl” group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning herein. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group. An example is a trifluoroacetyl group.

[0054] The term “cycloalkyl” as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example,amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term “cycloalkenyl” alone or in combination denotes a cyclic alkenyl group.

[0055] The term “aryl” as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.

[0056] The term “aralkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

[0057] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec- butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

[0058] The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independentlyselected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein.

[0059] The term “amino group” as used herein refers to a substituent of the form - NH2, -NHR, -NR2, -NR3+, wherein each R is independently selected, and protonated forms of each, except for -NR3+, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group.

[0060] The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0061] The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like.

[0062] The term “hydrocarbon” or “hydrocarbyl” as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups. The term “hydrocarbyl” refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. Hydrocarbyl groups can be shown as (Ca-Cb)hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms. For example, (C1- C4)hydrocarbyl means the hydrocarbyl group can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and (C0-Cb)hydrocarbyl means in certain embodiments there is no hydrocarbyl group. A hydrocarbylene group is a diradical hydrocarbon, e.g., a hydrocarbon that is bonded at two locations.

[0063] As used herein, a medical device may be a syringe, an insulin injection system, an insulin infusion system, an insulin pump or an insulin pen injection device. As used herein, a medical device may be mechanically or electromechanically driven.

[0064] The ingredients in the pharmaceutical composition can be defined as being Generally Recognized as Safe (“GRAS”). A full list of GRAS ingredients can be found in the GRAS Substances (SCOGS) Database maintained by the United States Food and Drug Administration. About 50% to about 100% of the ingredients in the pharmaceutical composition can be classified as being GRAS ingredients, about 75% to about 100%, about 90% to about 100%, less than, equal to, or greater than about 50%, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100% of the ingredients in the pharmaceutical composition can be classified as being GRAS ingredients. Example 1: Derivation of BRCA1mut / +and BRCA2mut / +mammary epithelial cells

[0065] To obtain BRCA1mut / +and BRCA2mut / +mammary epithelial cells, five primary human MECs strains from prophylactic mastectomies were performed on BRCA2 mutation carrying (BRCA2mut / +) women. Two primary human MECs strains from prophylactic mastectomies were performed on BRCA1 mutation carrying (BRCA1mut / +) women. Five wildtype control human MECs were derived from reduction mammoplasty tissue. The list of lines and the mutation information are provided in Figure 1A.

[0066] To isolate human MECs, tissue samples harvested from prophylactic mastectomies or reduction mammoplasty were minced into small pieces in PBS. The minced tissue was then subjected to overnight digestion at 37 °C in MEGM media (Lonza) supplemented with 1 mg / ml of collagenase type III (Roche). Following digestion, the tissue was pelleted, and the isolated cells were cultured in MEGM media supplemented with 1% Pen / Strep. The BRCA2 protein expression in BRCA2mut / +and BRCA2 wildtype lines were compared. As shown in Figure 1B, BRCA2 heterozygous cells do express less BRCA2 protein compared to the wildtype counterparts. Example 2: BRCA2mut / +MECs Mammary Epithelial Cells Response to Replication Stress

[0067] Given the critical role of BRCA2 in suppressing replication stress, the effects of BRCA2 heterozygosity on stalled replication fork repair were studied. DNA fiber analysis was carried out to investigate whether BRCA2mut / +cells show evidence of increased fork degradation when treated with stalled fork inducing agents. The MEC lines were pulse labeled with 5-iodo-2′-deoxyuridine (IdU) for 20min, followed by HU treatment for 3 hour to induce stalled forks. Upon removal of HU, cells were treated with 5-chloro-2′-deoxyuridine (CldU) for 40min. Fork degradation was analyzed by measuring the length of IdU labeledDNA strand. As shown in Figure 2A, incubation with HU led to a notable decrease in the length of IdU, indicating increased fork degradation in human BRCA2mut / +mammary epithelial cells. To further evaluate the effects of BRCA2 heterozygosity on suppressing replication stress, the sensitivity of BRCA2mut / +and BRCA2 wildtype MECs to stalled fork inducing agent, HU, was tested using CellTiter-Glo based cell survival assays. Cells were treated with HU at doses ranging from 0.1mM to 0.4mM. As shown in Figure 2B, BRCA2mut / +cell lines exhibited increased sensitivity compared to BRCA2 wildtype lines when treated with HU.

[0068] Taken together, these results indicate that BRCA2 heterozygosity leads to haploinsufficiency for replication stress suppression . Example 3: Downregulation of endogenous p53 protein levels in BRCA1mut / +and BRCA2mut / +cells

[0069] The loss of p53 is strongly associated with BRCA1 and BRCA2 mutant cancer. Additionally, studies have demonstrated that p53 loss enables the survival of Brca2 null mouse embryos (Brca2- / -) for an additional one to two days until E9-E10 (Ludwig, Chapman et al.1997). These suggest two things: firstly, that loss of p53 is a strong pre-requisite for the development of BRCA breast cancer, and secondly, loss of p53 allows BRCA-deficient cells to survive in face of DNA damage, albeit at the expense of presumably increased genomic instability.

[0070] This example aimed to investigate whether BRCA heterozygous cells, and not BRCA wildtype cells, would downregulate the levels of p53 protein to ensure their survival under ongoing low level of endogenous replication stress. P53 protein level was tested using immunofluorescence (IF) analysis of p53 mean intensity (Figures 3A and 3B) and western blot analysis of p53 protein level in whole-cell lysates (Figures 3C and 3D). A significant downregulation of endogenous p53 protein expression in BRCA1mut / +and BRCA2mut / +cells was observed when compared to wildtype cells. Example 4: BRCA1 and BRCA2 heterozygosity induces p53 degradation through MDM2

[0071] This example aimed to investigate whether the reduction in p53 protein levels in BRCA1mut / +and BRCA2mut / +cells was a result of increased p53 protein degradation. BRCA2mut / +and wildtype cells were treated with MG132 for 3hr and 7hr, and protein extracts were analyzed for p53 protein level. As shown in Figure 4A, blocking proteasome-mediated p53 degradation led to increased p53 levels in all four BRCA2 heterozygous lines.

[0072] To explore whether the increased degradation of p53 observed in BRCA2 heterozygous lines was driven by MDM2, the main E3 ligase that mediates p53 degradation (Grossman, Perez et al.1998, Marine and Lozano 2010). Cells were treated with Nutlin-3a, a widely recognized MDM2 inhibitor known for its high specificity in blocking the interaction between p53 and MDM2 (Shen and Maki 2011), to block p53 degradation. As shown in Figure 4B, incubation with Nutlin-3a in BRCA2mut / +cells restored P53 protein level, almost back to the same level as observed in wildtype cells. Restoration of P53 protein level in BRCA1mut / +cells was also observed (Figure 4C).

[0073] Taken together, these studies indicate that BRCA1 and BRCA2 heterozygous cells, experiencing endogenous level of replication stress, are sensitive to the presence of p53, and demonstrate a tendency to downregulate p53 protein levels. Example 5: Evaluate sensitivity of BRCA1mut / +and BRCA2mut / +cells to p53 stabilization

[0074] This example aimed to evaluate whether stabilizing p53 by using Nutlin 3a would result in cell death in BRCA1 or BRCA2 heterozygous cells. Cells were treated with Nutlin 3a at doses ranging from 2 µM to 8 µM. As shown in Figures 5A and 5B, BRCA1 and BRCA2 heterozygous cells displayed extreme sensitivity to Nutlin 3a treatment, whereas wildtype cells exhibit minimal response.

[0075] This example indicates that due to their haploinsufficiency in suppressing replication stress, BRCA1mut / +and BRCA2mut / +cells experience ongoing challenges from endogenous replication stress, triggering p53 degradation for their survival. Stabilizing p53, by using p53 stabilizer such as Nutlin 3a, would cause the death of BRCA1 or BRCA2 heterozygous cells. Exemplary Clauses.

[0076] The following exemplary clauses are provided, the numbering of which is not to be construed as designating levels of importance:

[0077] Clause 1. A method for aiding in preventing breast cancer in a subject having a heterozygous germline mutation in BRCA1 or BRCA2, comprising: a. identifying a subject having a heterozygous germline mutation in BRCA1 or BRCA2; b. optionally determining a pathogenicity of the BRAC1 or BRAC2 mutation of a.; c. administering to the subject a therapeutically effective amount of a P53 protein stabilizer, wherein the P53 protein stabilizer comprises an MDM2 inhibitor.

[0078] Clause 2. The method of clause 1, wherein the P53 protein stabilizer is Nutlin 3a or a derivative thereof.

[0079] Clause 3. A method for identifying a pathogenic variant of unknown significance (VUS) in a BRCA1 or BRCA2 gene in a subject, comprising: a. obtaining a sample of mammary epithelial cells from the subject; b. culturing the mammary epithelial cells; c. treating the cultured mammary epithelial cells with a P53 protein stabilizer; d. assessing the sensitivity of the treated cells to the P53 protein stabilizer; and e. determining the pathogenicity of the VUS based on the sensitivity of the cells to the P53 protein stabilizer.

[0080] Clause 4. The method of clause 3, wherein the P53 protein stabilizer is Nutlin 3a or a derivative thereof.

[0081] Clause 5. A pharmaceutical composition comprising a P53 protein stabilizer for use in treating a subject having a heterozygous germline mutation in BRCA1 or BRCA2 to aide in or prevent breast cancer, wherein the P53 protein stabilizer comprises an MDM2 inhibitor.

[0082] Clause 6. The pharmaceutical composition of clause 5, wherein the P53 protein stabilizer is Nutlin 3a or a derivative thereof.

[0083] Clause 7. A use of a P53 protein stabilizer in the manufacture of a medicament for aiding in preventing breast cancer in a subject having a heterozygous germline mutation in BRCA1 or BRCA2, wherein the P53 protein stabilizer comprises an MDM2 inhibitor.

[0084] Clause 8. The use of clause 7, wherein the P53 protein stabilizer is Nutlin 3a or a derivative thereof.

[0085] Clause 9. A diagnostic kit for identifying a pathogenic variant of unknown significance (VUS) in a BRCA1 or BRCA2 gene in a subject, comprising: a. reagents for culturing mammary epithelial cells; b. a P53 protein stabilizer; and c. instructions for treating the cultured mammary epithelial cells with the P53 protein stabilizer and assessing their sensitivity to determine the pathogenicity of the VUS.

[0086] Clause 10. The diagnostic kit of clause 9, wherein the P53 protein stabilizer is Nutlin 3a or a derivative thereof.

[0087] Clause 11. The method of clause 1, wherein the subject is identified as having a heterozygous germline mutation in BRCA1 or BRCA2 through genetic testing.

[0088] Clause 12. The method of clause 1, further comprising: a. determining the expression level of P53 protein in the subject's cells prior to administering the P53 protein stabilizer; b. wherein the administration of the P53 protein stabilizer is based on the expression level of P53 protein.

[0089] Clause 13. The method of clause 1, wherein the dosage of the P53 protein stabilizer is in a range of from 100 mg and 500 mg administered daily.

[0090] Clause 14. The method of clause 3, wherein the P53 protein stabilizer is administered orally, by orthotopic injection, or both.

[0091] Clause 15. The method of clause 3, wherein the subject is a female aged between 18 and 55 years.

[0092] Clause 16. The method of clause 3, further comprising monitoring the subject for side effects associated with the P53 protein stabilizer, and adjusting the dosage based on the observed side effects.

[0093] Clause 17. The method of clause 3, wherein the treatment with the P53 protein stabilizer is continued for a period ranging from 6 months to 5 years.

[0094] Clause 18. The method of clause 3, further comprising treating cells with an additional therapeutic agent selected from the group consisting of PARP inhibitors, chemotherapy agents, and radiation therapy.

[0095] Clause 19. The diagnostic kit of clause 9, further comprising reagents for detecting additional biomarkers indicative of cancer risk, including but not limited to CA- 125, HER2, and estrogen receptor (ER) status.

[0096] Clause 20. The diagnostic kit of clause 9, wherein the instructions include guidelines for interpreting the sensitivity of the cells to the P53 protein stabilizer in conjunction with historical data on BRCA1 or BRCA2 mutations.

[0097] Clause 21. The diagnostic kit of clause 9, wherein the kit includes software for analyzing and reporting the results of the sensitivity assessment.

[0098] Clause 22. The use of clause 7, wherein the medicament is formulated as a tablet, capsule, or liquid suspension.

[0099] Clause 23. The use of clause 7, wherein the medicament is part of a combination therapy including at least one other agent effective in aiding the prevention of breast cancer.

Claims

CLAIMS What is claimed is:

1. A method for aiding in preventing breast cancer in a subject having a heterozygous germline mutation in BRCA1 or BRCA2, comprising: a. identifying a subject having a heterozygous germline mutation in BRCA1 or BRCA2; b. optionally determining a pathogenicity of the BRAC1 or BRAC2 mutation of a.; c. administering to the subject a therapeutically effective amount of a P53 protein stabilizer, wherein the P53 protein stabilizer comprises an MDM2 inhibitor.

2. The method of claim 1, wherein the P53 protein stabilizer is Nutlin 3a or a derivative thereof.

3. A method for identifying a pathogenic variant of unknown significance (VUS) in a BRCA1 or BRCA2 gene in a subject, comprising: a. obtaining a sample of mammary epithelial cells from the subject; b. culturing the mammary epithelial cells; c. treating the cultured mammary epithelial cells with a P53 protein stabilizer; d. assessing the sensitivity of the treated cells to the P53 protein stabilizer; and e. determining the pathogenicity of the VUS based on the sensitivity of the cells to the P53 protein stabilizer.

4. The method of claim 3, wherein the P53 protein stabilizer is Nutlin 3a or a derivative thereof.

5. A pharmaceutical composition comprising a P53 protein stabilizer for use in treating a subject having a heterozygous germline mutation in BRCA1 or BRCA2 to aide in or prevent breast cancer, wherein the P53 protein stabilizer comprises an MDM2 inhibitor.

6. The pharmaceutical composition of claim 5, wherein the P53 protein stabilizer is Nutlin 3a or a derivative thereof.

7. A use of a P53 protein stabilizer in the manufacture of a medicament for aiding in preventing breast cancer in a subject having a heterozygous germline mutation in BRCA1 or BRCA2, wherein the P53 protein stabilizer comprises an MDM2 inhibitor.

8. The use of claim 7, wherein the P53 protein stabilizer is Nutlin 3a or a derivative thereof.

9. A diagnostic kit for identifying a pathogenic variant of unknown significance (VUS) in a BRCA1 or BRCA2 gene in a subject, comprising: a. reagents for culturing mammary epithelial cells; b. a P53 protein stabilizer; andc. instructions for treating the cultured mammary epithelial cells with the P53 protein stabilizer and assessing their sensitivity to determine the pathogenicity of the VUS.

10. The diagnostic kit of claim 9, wherein the P53 protein stabilizer is Nutlin 3a or a derivative thereof.

11. The method of claim 1, wherein the subject is identified as having a heterozygous germline mutation in BRCA1 or BRCA2 through genetic testing.

12. The method of claim 1, further comprising: a. determining the expression level of P53 protein in the subject's cells prior to administering the P53 protein stabilizer; b. wherein the administration of the P53 protein stabilizer is based on the expression level of P53 protein.

13. The method of claim 1, wherein the dosage of the P53 protein stabilizer is in a range of from 100 mg and 500 mg administered daily.

14. The method of claim 3, wherein the P53 protein stabilizer is administered orally, by orthotopic injection, or both.

15. The method of claim 3, wherein the subject is a female aged between 18 and 55 years.

16. The method of claim 3, further comprising monitoring the subject for side effects associated with the P53 protein stabilizer, and adjusting the dosage based on the observed side effects.

17. The method of claim 3, wherein the treatment with the P53 protein stabilizer is continued for a period ranging from 6 months to 5 years.

18. The method of claim 3, further comprising treating cells with an additional therapeutic agent selected from the group consisting of PARP inhibitors, chemotherapy agents, and radiation therapy.

19. The diagnostic kit of claim 9, further comprising reagents for detecting additional biomarkers indicative of cancer risk, including but not limited to CA-125, HER2, and estrogen receptor (ER) status.

20. The diagnostic kit of claim 9, wherein the instructions include guidelines for interpreting the sensitivity of the cells to the P53 protein stabilizer in conjunction with historical data on BRCA1 or BRCA2 mutations.

21. The diagnostic kit of claim 9, wherein the kit includes software for analyzing and reporting the results of the sensitivity assessment.

22. The use of claim 7, wherein the medicament is formulated as a tablet, capsule, or liquid suspension.

23. The use of claim 7, wherein the medicament is part of a combination therapy including at least one other agent effective in aiding in the prevention of breast cancer.

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