Biosignatures for breast cancer treatment and methods of use therof

Abstract

Methods of predicting treatment benefit for reducing risk of recurrence for breast cancer is provided. In some embodiments, a method can include providing a breast tissue sample from a subject with invasive, hormone receptor (HR)-positive, HER2-negative breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1, where the analyzed biosignature predicts the benefit from a breast cancer treatment with adjuvant therapy (e.g., radiotherapy followed by endocrine therapy) to reduce the risk of breast cancer recurrence in the subject. In some embodiments, the method includes administering the breast cancer treatment with adjuvant therapy to the subject.

Description

PRLUD.024WO PATENT BIOSIGNATURES FOR BREAST CANCER TREATMENT AND METHODS OF USE THEROF INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57. This Application claims priority to U.S. Provisional Application No. 63 / 564411, filed March 12, 2024. The contents of this related application is incorporated by reference herein. BACKGROUND

[0002] The landscape of breast cancer management is evolving with emphasis on the need for personalized treatment regimens to tailor treatment in early-stage, hormone positive (HR+) invasive breast cancer (BC) patients. SUMMARY

[0003] The methods disclosed herein each have several aspects, no single one of which is solely responsible for their desirable attributes. Without limiting the scope of the claims, some prominent features will now be discussed briefly. Numerous other embodiments are also contemplated, including embodiments that have fewer, additional, and / or different components, steps, features, objects, benefits, and advantages. The components, aspects, and steps may also be arranged and ordered differently. After considering this discussion, and particularly after reading the section entitled “Detailed Description”, one will understand how the features of the devices and methods disclosed herein provide advantages over other known devices and methods.

[0004] Provided herein is a method of treating invasive breast cancer, the method including providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a first biosignature comprising at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1; selecting an adjuvant therapy for a breast cancer treatment basedon the analysis of the first biosignature, wherein the adjuvant therapy comprises radiotherapy (RT); and administering the breast cancer treatment to the subject, thereby reducing a risk of breast cancer recurrence.

[0005] Also provided is a method of treating invasive breast cancer, the method including: providing a breast tissue sample of an invasive, hormone receptor-positive, HER2- negative (HR(+) / HER2(-)) breast cancer; having the breast tissue sample analyzed for a first biosignature comprising at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1; and receiving a breast cancer treatment based on the analysis of the biosignature, wherein the breast cancer treatment comprises an adjuvant therapy comprising radiotherapy (RT), thereby reducing a risk of breast cancer recurrence.

[0006] Further provided is a method of treating invasive breast cancer, the method including: providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising the following biomarkers: (a) PD-1 and IRF9; and (b) at least: (i) GLUT1 and Ki67; or (ii) PR and FOXA1; selecting a breast cancer treatment comprising an adjuvant therapy based on the analysis of the biosignature, wherein radiotherapy (RT) is selected for the adjuvant therapy based on the analysis of at least PD-1 and IRF9; and administering the breast cancer treatment to the subject, thereby reducing a risk of breast cancer recurrence.

[0007] Provided herein is a method of identifying a subject for breast cancer treatment, the method including providing a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67; and identifying the subject for a breast cancer treatment to reduce a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis, based on the analyzed biosignature.

[0008] Also provided is a method of diagnosis, the method including providing a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67; based on the analyzed biosignature being GLUT1(-) and Ki67(-), identifying the subject as benefiting from a breast cancer treatment comprising an adjuvant therapy comprising radiotherapy (RT) and endocrine therapy (ET) toreduce a risk of LRR, or a risk of LRR and distal metastasis, optionally wherein the adjuvant therapy comprises RT followed by ET.

[0009] Provided herein is a method of diagnosis, the method including, providing a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67, or at least the biomarkers PR and FOXA1; based on the analyzed biosignature being: (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+), identifying the subject as benefiting from a breast cancer treatment comprising an adjuvant therapy comprising radiotherapy (RT) and endocrine therapy (ET) to reduce a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis.

[0010] Further provided is a combination of detection reagents for detecting expression of biomarkers comprising at least GLUT1 and Ki67 in a breast tissue sample, for use in any one of the methods provided herein. Also provided is a kit including reagents for detecting expression of biomarkers comprising at least GLUT1 and Ki67 in a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer.

[0011] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals, if any, may correspond to similar, though perhaps not identical, components. For the sake of brevity, any reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a plot showing some embodiments of locoregional recurrence (LRR) rate for breast cancer based on clinicopathological (CP) risk factors and biosignature scores.

[0013] FIG.2 is a plot showing some embodiments of LRR for breast cancer based on CP factors and biosignatures.

[0014] FIG. 3 is a plot showing some embodiments of combined LRR and distant recurrence risk for breast cancer based on CP risk factors and biosignature scores.

[0015] FIG.4 is a plot showing some embodiments of LRR for breast cancer based on CP risk factors and biosignature scores.

[0016] FIG.5 is a plot showing some embodiments of LRR for breast cancer based on CP risk factors and biosignature scores.

[0017] FIG. 6 is a plot showing some embodiments of combined LRR and distant recurrence risk for breast cancer based on CP risk factors and biosignature scores.

[0018] FIG. 7 is a plot showing some embodiments of combined LRR and distant recurrence risk for breast cancer based on CP risk factors and biosignature scores.

[0019] FIG. 8 is a plot showing some embodiments of combined LRR and distant recurrence risk for breast cancer based on CP risk factors and biosignature scores.

[0020] FIG.9 is a plot showing some embodiments of LRR for breast cancer based on CP risk factors and biosignature scores.

[0021] FIG.10 is a plot showing some embodiments of combined LRR and distant recurrence risk for breast cancer based on CP risk factors and biosignature scores.

[0022] FIG. 11 depicts a flow diagram of a method of treating invasive breast cancer according to a non-limiting embodiment.

[0023] FIG. 12 depicts a flow diagram of a method of treating invasive breast cancer according to a non-limiting embodiment.

[0024] FIG. 13 depicts a flow diagram of a method of treating invasive breast cancer according to a non-limiting embodiment.

[0025] FIG. 14 depicts a flow diagram of a method of identifying a subject for breast cancer treatment according to a non-limiting embodiment.

[0026] FIG. 15 depicts a flow diagram of a method of diagnosing according to a non-limiting embodiment.

[0027] FIG. 16 depicts a flow diagram of a method of diagnosing according to a non-limiting embodiment. DETAILED DESCRIPTION

[0028] The landscape of breast cancer management is evolving with emphasis on the need for personalized treatment regimens to tailor treatment in early-stage, hormone positive (HR+) invasive breast cancer (BC) patients. Given that locoregional recurrence (LRR)rates are lower in HR+ BC patients, multiple studies have evaluated the role of adjuvant radiation therapy (RT) and / or endocrine therapy (ET) in these patients.

[0029] Ongoing studies are exploring the integration of clinical and biological factors to better assess patient-specific recurrence risk profiles to individualize treatment in the adjuvant setting.

[0030] The present disclosure provides that interaction of cellular pathways can be used as part of a biosignature to predict recurrence risk and the RT and ET benefit for early- stage HR+ HER2-negative BC. For some embodiments, this can be used as a diagnostic or identifier of subjects to receive ET. In some embodiments, the method can further comprise administering the ET to the subject upon their identification.

[0031] Methods of predicting treatment benefit for reducing risk of recurrence for breast cancer is provided. A method of the present disclosure can include providing a breast tissue sample from a subject with invasive, hormone receptor (HR)-positive, HER2-negative breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1, where the analyzed biosignature predicts the benefit from a breast cancer treatment with adjuvant therapy (e.g., radiotherapy followed by endocrine therapy) to reduce the risk of breast cancer recurrence in the subject. In some embodiments, the method includes administering the breast cancer treatment with adjuvant therapy to the subject. Terms

[0032] As used herein, the term “diagnosis”, and variants thereof, such as, but not limited to “diagnose” or “diagnosing” shall include, but not be limited to, a primary diagnosis of a clinical state or any primary diagnosis of a clinical state. Furthermore, this term denotes the process of identifying a disease by its signs, symptoms and results of various tests. The conclusion reached through that process is also called “a diagnosis.” Forms of testing commonly performed include biopsy for the collection of the tumor. In some embodiments, the prognosis can be a high or low likelihood of a subsequent (within the next 10 years, 15, or 20 years) invasive breast cancer event.

[0033] As used herein, the term “prognosis” denotes an outcome or course of a disease. In some embodiments provided herein, the phrase, when used in the context of a person already having invasive breast cancer, denotes the likelihood that a subject having theinvasive breast cancer will go on (within a following ten, fifteen, or twenty year period) to have a subsequent ipsilateral invasive breast cancer event after surgical removal of the primary tumor. The outcome can include a) the likelihood of an ipsilateral breast event, b) the likelihood of an ipsilateral breast event in a particular amount of time (e.g., 1, 2, 3 or 5 years), c) the likelihood that a particular therapy (e.g., radiation) will prevent an ipsilateral breast event, d) an optimal treatment to help prevent an ipsilateral event that matches the severity of the most likely event, or e) combinations thereof.

[0034] As used herein. the term “breast tumor” denotes a neoplastic condition of breast tissue that can be benign or malignant. The term “tumor” is synonymous with “neoplasm” and “lesion”. Exemplary breast tumors include invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and atypical ductal hyperplasia (ADH).

[0035] As used herein, the term “cancer” denotes a malignant neoplasm that has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and is capable of metastasis. The term “cancer” shall be taken to include a disease that is characterized by uncontrolled growth of cells within a subject, such as, but not limited to, invasive breast cancer. In some embodiments, invasion of the surrounding tissue is the invasion of the basement membrane.

[0036] As used herein, the term “invasive breast cancer” denotes that the neoplastic (tumor) cells have invaded through the epithelial basement membrane. This distinguishes invasive breast cancer from other hyperplastic (ductal hyperplasia) or dysplastic (atypical ductal hyperplasia, ADH) or non-invasive neoplastic (DCIS, LCIS) breast lesions which are characterized by an intact (non-invaded) basement membrane. It can be divided into stages (I, IIA, IIB, IIIA, IIIB, and IV). It can be divided into different molecular subtypes based on the presence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2) in / on the tumor cells. In some embodiments, any of the methods provided herein can be applied to invasive breast cancer to determine the success of radiotherapy for preventing an invasive breast cancer recurrence. Rates of recurrence in invasive breast cancer varies between 3-10% post breast conserving surgery (BCS) + radiotherapy (RT). Meta analysis of the landmark randomized clinical trials provide evidence that adjuvant radiation therapy following lumpectomy reduces recurrence risk by approximately half. It furtherdemonstrated that the absolute benefits of radiotherapy varied between subgroups defined by age, hormone receptor status, and other risk factors suggesting that the current clinical and pathologic assessment techniques cannot identify a low-risk sub-group in which there is no benefit from radiation therapy.

[0037] When cancer is diagnosed, health care professionals attempt to ascertain the cancer’s spread by a process called staging. A typical staging system used for breast cancer is the American Joint Committee of Cancer (AJCC) TNM system, effective January 2018. Understanding a Breast Cancer Diagnosis, ACS, November 8, 2011, pp. 24-30. For staging under the TNM system, 7 pieces of information are used: the extent (size) of the tumor (T); the spread to nearby lymph nodes (N); the spread (metastasis) to distant sites (M); Estrogen Receptor (ER) status; Progesterone Receptor (PR) status; HER2 status; and grade of the cancer. When examining the primary tumor, T categories for breast cancer are used to denote the tumor’s size, if it has spread to the skin or to the chest wall under the breast: TX (primary tumor cannot be assessed); T0 (no evidence of a primary tumor); Tis (carcinoma in situ, e.g., DCIS or Paget disease of the breast with no associated tumor mass); T1 (includes T1a, T1b, and T1c), wherein the tumor is 2 cm (3 / 4 of an inch) or less across; T2 (tumor is more than 2 cm but not more than 5 cm (2 inches) across); T3 (tumor is more than 5 cm across); T4 (includes T4a, T4b, T4c, and T4d), wherein the tumor is of any size growing into the chest wall or skin.

[0038] Part of the staging process also involves examining nearby lymph nodes (often through surgical removal and histopathological examination) to check for the presence of cancer cells. If cancer cells are not found in these lymph nodes, the patient is considered "node-negative" (often denoted as N0 in staging terminology). This status generally indicates a lower stage of cancer, suggesting that the disease has not spread beyond the primary tumor site or to distant parts of the body. Consequently, node-negative patients may have a better prognosis and could be candidates for less aggressive treatment options compared to those with node-positive cancer, where cancer cells have been found in the lymph nodes, indicating a higher stage of disease.

[0039] As used herein, the terms “HER2 +ve” and / or “HER2 enhanced” breast cancer refers to breast cancers wherein a least a portion of the cancer cells overexpress human epidermal growth factor 2 (HER2) on the surface of the cancer cells. HER2 is a receptortyrosine kinase involved in the regulation of cell growth and differentiation. HER2 +ve breast cancers are characterized by the enhanced HER2 expression assayed using IHC or FISH than the invasive breast cancers that are not HER2+ve. As used herein, “HER2-negative” or “HER2(-)” denotes a subject that is not HER2+ve.

[0040] As used herein, the term “HR +ve,” “HR-positive,” or “HR(+)” breast cancer refers to breast cancers wherein ER and / or PR are present on the surface of cancer cells. In some embodiments, ER is present on the surface of cancer cells in a HR(+) cancer. In some embodiments, PR is present on the surface of cancer cells in a HR(+) cancer. In some embodiments, ER or PR is present on the surface of cancer cells in a HR(+) cancer. In some embodiments, ER and PR is present on the surface of cancer cells in a HR(+) cancer. In some embodiments, at least 1% of cells in the tumor expresses ER and / or PR in a HR(+) cancer. In some embodiments, more than 1% of cells in the tumor expresses ER in a HR(+) cancer. In some embodiments, more than 1% of cells in the tumor expresses PR in a HR(+) cancer. In some embodiments, more than 1% of cells in the tumor expresses ER or PR in a HR(+) cancer. In some embodiments, more than 1% of cells in the tumor expresses ER and PR in a HR(+) cancer.

[0041] As used herein, the term “radiation therapy” (RT) denotes a therapy that involves or includes some form of radiation in an amount that is therapeutic to the subject.

[0042] As used herein, the terms “RT boost” or “boost,” the latter when used within the context of RT, includes an additional / extra dose of radiation applied to a site. The site may be a tumor bed. The site may be an initial tumor site. The site may be any tissue or location requiring the additional or extra dose of radiation. The additional / extra dose may be simultaneously integrated / concomitant (i.e., given together with the standard radiotherapy) or sequential (i.e., given after standard radiotherapy). The additional / extra dose of radiation may be at least 10 Gy if given sequentially or at least 5 Gy if simultaneously integrated / concomitant. The additional / extra dose of radiation may be no more than 16 Gy. The additional / extra dose may be provided using external beam radiotherapy, internal beam radiotherapy / brachytherapy, stereotactic radiotherapy, or intraoperative radiotherapy.

[0043] Surgery is a treatment for a breast tumor and is frequently involved in diagnosis. The type of surgery depends upon how widespread the tumor is when diagnosed (the tumor stage), as well as the type and grade of tumor.

[0044] The terms “treatment” and “therapy” as provided herein are used interchangeably and does not require the complete or 100% curing of the subject. Instead, it encompasses the broader concept or delaying the onset of one or more symptoms, extending the life and / or quality of life of the subject, reducing the severity of one or more symptoms, etc.

[0045] “Risk of invasive breast cancer,” denotes a risk of developing (or being diagnosed with) a subsequent invasive breast cancer in the same (a.k.a. ipsilateral) breast.

[0046] Adjuvant chemotherapy is often used after surgery to treat any residual disease. Systemic chemotherapy often includes a platinum derivative with a taxane. Adjuvant chemotherapy is also used to treat subjects who have a recurrence or metastasis.

[0047] “Adjuvant invasive breast cancer treatment” denotes any treatment that is appropriate for a subject that is likely to have an invasive breast cancer occurrence, which can include, lumpectomy with radiation, to lumpectomy with a receptor targeted chemotherapy, to lumpectomy with radiation with a receptor targeted chemotherapy, to mastectomy, to mastectomy with a receptor targeted chemotherapy, to mastectomy with radiation, to mastectomy with radiation and a receptor targeted chemotherapy, to surgery with a chemotherapy. In some embodiments, the targeted therapy can include: endocrine therapy (blocking hormone receptors); anti-HER2 therapy (blocking the HER2 receptor which is overexpressed in some breast cancers); immunotherapy (blocking molecules which inhibit the immune response); conjugated monoclonal antibodies (an antibody conjugated to chemotherapy (the antibody will function as a homing device to bring the chemotherapy specifically to the cancer cells); tyrosine kinase inhibitors (receptors relying on tyrosine kinase signaling); anti-PDGFRb antibodies (blocks PDGFRb); PARP inhibitor therapy (blocking poly ADP ribose polymerase (PARP) activity), etc.

[0048] As used herein, “chemotherapy” (CT) refers to one kind of treatment for treating cancers, wherein a therapeutic amount of one or more chemotherapeutic agents is administered to target and destroy cancerous cells, particularly those that have proliferated beyond the primary tumor site and metastasized at distant locations within the subject’s body.

[0049] As used herein, “endocrine therapy” (ET) refers to a kind of treatment for treating cancers that involves hormonal modulation. A purpose of ET is to reduce the level of the body’s natural hormones (particularly estrogen) or to inhibit their supportive effects ontumors. ET includes, but is not limited by, Selective Estrogen Receptor Modulators (SERMs), Aromatase Inhibitors (AIs), Estrogen Receptor Downregulators (ERDs), Complete Estrogen Receptor Antagonists (CERANs), and Ovarian Suppression or Ablation. ET can be combined with other therapies. In some non-limiting examples, ET can be used as adjuvant therapy to reduce risk of recurrence or as palliative therapy.

[0050] “Standard of care” or “standard” as used herein, with reference to a therapy or treatment, have the ordinary and customary meaning to one of ordinary skill in the art in view of the present disclosure. In some embodiments, standard of care denotes a therapy or treatment option recommended for a patient under a guideline such as that provided by NCCN, ESMO, ESTRO, ASTRO, Clinical Practice Recommendations Australia, or NICE guideline, and optionally, any one or more of the respective guidelines as of March 2024. In some embodiments, a therapy recommended under standard of care for a patient does not take into account guidance provided by analysis of markers as disclosed herein.

[0051] The terms “standard radiation therapy” and “standard radiotherapy” are used interchangeably herein and denote a therapy that involves or includes some form of radiation in an amount that is therapeutic to the subject under the current standard of care for breast cancer. In some embodiments, the standard of care is any one that is provided in NCCN, ESMO, ESTRO, ASTRO, Clinical Practice Recommendations Australia, or NICE guideline, and optionally, any one or more of the respective guidelines as of March 2024. In some embodiments, the standard of care is any one of those provided in Tables 1 and 2 below. Table 1. Radiotherapy GuidelinesTable 2. ESTRO Radiotherapy Guidelines (2022)

[0052] As used herein, the term “radiotherapy de-intensification” includes radiotherapy omission (i.e., not giving any radiotherapy at all) or simply reducing the radiation dose or the number of fractions of a given dose fractionation scheme.

[0053] The term “non-radiation therapy” denotes a therapy that is adequate for addressing or reducing the risk of invasive breast cancer in a subject, and that does not derive its therapeutic effect by radiation. Examples of such therapy include, chemo therapeutics, targeted and non-targeted, immune and non-immune modulated, monoclonal, other targetedand non-targeted, genomic therapies, antibody therapeutics, including, HER2 antibodies, including Trastuzumab. Often, in the present application, “non-radiation therapy” is denoted as “other therapy”.

[0054] As used herein, the term “local recurrence” denotes that a recurrence is in the operated breast. As used herein, local recurrence is interchangeable with ipsilateral breast tumor recurrence (IBTR).

[0055] As used herein, the term “regional recurrence” denotes that a recurrence is in regional lymph nodes (axillary, supraclavicular, infraclavicular, intrapectoral or internal mammary lymph nodes).

[0056] As used herein, the term “distant metastasis” refers to all other recurrences outside the above types of recurrences (local or regional). In other words, distant metastasis refers to recurrences in all other tissues of the body.

[0057] As used herein, “marker” and “biomarker” are used interchangeably and refer to a measured biological component such as a protein, an mRNA transcript, or a level of DNA amplification.

[0058] “Programmed death 1”, “PD-1”, “PD1”, “PDCD1” are used interchangeably herein and refers to the gene or gene product of PDCD1. In some embodiments, PD-1 is human PD-1. PD-1 is a cell surface receptor approximately 50 to 55 kDa. Structurally, PD-1 is composed of an extracellular domain that interacts with its ligands, PD-L1 and PD-L2, a transmembrane domain, and an intracellular domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine- based switch motif (ITSM). In breast cancer, PD-1 and its ligands are overexpressed, which leads to suppression of the immune response against tumor cells, which enables tumor cells to proliferate and spread.

[0059] Interferon Regulatory Factor 9 (IRF9) is a member of the interferon regulatory factor family and is approximately 48 kDa. IRF9 contains a DNA-binding domain and acts as a transcription factor as part of the ISGF3 complex that mediates type I interferon antiviral response by regulating the downstream expression of interferon-stimulated genes. In the context of breast cancer, IRF9 may be involved in pathways that contribute to tumor progression and resistance to therapy.

[0060] Aldehyde dehydrogenase A1 (ALDHA1) is a member of the aldehyde dehydrogenase family of enzymes that play a crucial role in the oxidative metabolism of aldehydes into their corresponding carboxylic acids. ALDHA1 is a cytosolic enzyme that is pivotal in detoxifying aldehydes derived from alcohol metabolism and lipid peroxidation, thus contributing significantly to cellular homeostasis and protection against oxidative stress. Structurally, ALDHA1 exhibits a highly conserved active site that facilitates the catalytic conversion of aldehydes using NAD(P)+ as a cofactor, leading to the production of NADH (or NADPH) and the corresponding acid. High concentrations of this enzyme are found in the liver, where it is involved in the metabolism of ethanol, as well as in other organs contributing to the detoxification of endogenously produced and exogenously derived aldehydic compounds. Without being bound by theory, the upregulation of ALDHA1 is associated with breast cancer aggressiveness and poor prognosis due to its high expression in a tumor, which may provide a route for tumors to resist chemotherapy.

[0061] Glucose transporter 1 (GLUT1) is an integral membrane protein that facilitates the diffusion of glucose across the cellular membranes of mammalian cells. GLUT1 is characterized by its 12-transmembrane spanning domains with both of its N- and C-termini predicted to lie intracellularly. GLUT1 is encoded by the SLC2A1 gene is one of a family of 14 genes encoding GLUT proteins. GLUT1 is ubiquitously expressed in various tissues, with particularly high levels in erythrocytes and in the blood-brain barrier, where it ensures the efficient and continuous transport of glucose from the bloodstream into the brain and other cells. This protein is crucial for maintaining glucose homeostasis, enabling cells to uptake glucose in a concentration-dependent manner without the need for ATP. Without being bound by theory, elevated glucose levels in cancer cells are thought to be attributed to the upregulation of GLUT1, which may regulate signaling cascades involved in tumorigenesis of breast cancer.

[0062] Ki67 is a nuclear protein that is present in the late gap 1 (G1), synthesis (S), gap 2 (G2), and mitosis (M) phases of the cell cycle and is associated with cellular proliferation and is absent in quiescent cells (G0 phase). In humans, Ki67 is encoded by the MK167 gene. Initially, two isoforms of Ki67 with MW of 345 and 396 kDa were identified. The presence of Ki67 during the cell cycle is regulated by a precise balance between synthesis and degradation.

[0063] Progesterone receptor (PR) is member of a nuclear receptor superfamily. When activated by progesterone, the PR dimerizes and binds to specific DNA sequencesknown as progesterone response elements (PREs), which are involved in cell growth, differentiation, and reproductive function.

[0064] Forkhead box A1 protein (FOXA1) is a member of the forkhead-family of transcription factors. FOXA1 binds to highly compact heterochromatin, hence its role as a pioneer factor, which causes certain genomic regions to be exposed to other transcription factors, which effectively modulates the transcriptional activity of a wide array of genes. FOXA1 mediates the attachment of estrogen and androgen receptors attachment to chromatin. In breast tissues, FOXA1 co-localizes with estrogen receptors or androgen receptors in the nuclei and has a key role in the development of the breast tissue.

[0065] The term “control” refers to a sample or standard used for comparison with a sample which is being examined, processed, characterized, analyzed, etc. In some embodiments, the control is a sample obtained from a healthy patient or a non-tumor tissue sample obtained from a patient diagnosed with a breast tumor. In some embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of breast tumor patients with poor prognosis, or group of samples that represent baseline or normal values, such as the level of cancer- associated genes or proteins in non-tumor tissue).

[0066] The “Cox hazard ratio” is derived from the Cox proportional hazards model. Proportional hazards models are a class of survival models in statistics. Survival models relate the time that passes before some event occurs to one or more covariates that may be associated with that quantity of time. In the Cox proportional hazards model, the unique effect of a unit increase in a covariate is multiplicative with respect to the hazard rate. A “Cox hazard ratio” is the ratio of the hazard rates corresponding to the conditions described by two levels of an explanatory variable -- a covariate, that is calculated using the cox proportional hazards model. The cox hazard ratio is the ratio of survival hazards for a one-unit change in the covariate. For example, the Cox hazard ratio may be the ratio of survival hazards for a 1 unit change in the logarithmic gene expression level. Thus, a larger value has a greater effect on survival or the hazard rate of the event being assessed, such as disease recurrence. In some embodiments, a hazard ratio (HR) greater than 1 indicates that an increased covariate level is associated with a worse patient outcome, where the covariate level is a marker expression level.In some embodiments, a HR less than 1 indicates that a decreased covariate level is associated with a better patient outcome, where the covariate level is a marker expression level.

[0067] As used herein, the term “non-tumor tissue sample” shall be taken to include any sample from or including a normal or healthy cell or tissue, or a data set produced using information from a normal or healthy cell or tissue. For example, the non-tumor sample may be selected from the group comprising or consisting of: (i) a sample comprising a non-tumor cell; (ii) a sample from a normal tissue; (iii) a sample from a healthy tissue; (iv) an extract of any one of (i) to (iii); (v) a data set comprising measurements of modified chromatin and / or gene expression for a healthy individual or a population of healthy individuals; (vi) a data set comprising measurements of modified chromatin and / or gene expression for a normal individual or a population of normal individuals; and (vii) a data set comprising measurements of the modified chromatin and / or gene expression from the subject being tested wherein the measurements are determined in a matched sample having normal cells. Preferably, the non- tumor sample is (i) or (ii) or (v) or (vii).

[0068] Detecting expression of a gene product denotes determining of a level of expression in either a qualitative or quantitative manner. Exemplary methods include, but are not limited to: microarray analysis, RT-PCR, Northern blot, in situ hybridization, Western blot, immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), multiplex immunofluorescence (MIF), enzyme-linked immunosorbent assay (ELISA), next generation sequencing (NGS), NGS-RNA seq, and mass spectrometry. For immunohistochemistry, immunofluorescence, and related technologies, the H-Score provides a scoring system that quantifies the abundance of a marker by capturing both the intensity and the proportion of the marker of interest from a captured image of the labeled sample.

[0069] As used herein, the term “multiplex immunofluorescence” (MIF), refers to a technique that simultaneously interrogates proteins within a single sample or tissue by using fluorescently labeled antibodies designed to be specific to the target of interest. In MIF, each target (specifically, the target’s antigen) is recognized by a unique antibody having a unique fluorescent label. By using antibodies labeled with different fluorescent dyes, MIF allows for the observation and analysis (including, but not limited to, spatial and volumetric analysis) of multiple antigens within the same biological sample at the same time.

[0070] The term “expression” denotes the process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of an RNA or protein. Gene expression can be influenced by external signals. For instance, exposure of a cell to a hormone may stimulate expression of a hormone-induced gene. Different types of cells can respond differently to an identical signal. Expression of a gene also can be regulated anywhere in the pathway from DNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, protein transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization.

[0071] The expression of a nucleic acid molecule or protein in a sample can be altered relative to a control sample, such as a normal or non-tumor sample. Alterations in gene expression, such as differential expression, include but are not limited to: (1) overexpression; (2) underexpression; or (3) suppression of expression.

[0072] As used herein, the term “intraductal lesion” refers to tumors that are confined to the interior of the mammary ducts and are, therefore, not invasive breast cancers. Exemplary intraductal lesions include ADH and DCIS.

[0073] As used herein, ADH is a neoplastic intraductal (non-invasive) lesion characterized by proliferation of evenly distributed, monomorphic mammary epithelial cells.

[0074] As used herein, DCIS is a neoplastic intraductal (non-invasive) lesion characterized by increased mammary epithelial proliferation with subtle to marked cellular atypia. DCIS has been divided into grades (low, intermediate, and high) based on factors such as nuclear atypia, intraluminal necrosis, mitotic activity etc. Low-grade DCIS and ADH are morphologically identical, and ADH is distinguished from DCIS based on the extent of the lesion, as determined by its size and / or the number of involved ducts. DCIS is initially typically diagnosed from a tissue biopsy triggered by a suspicious finding (e.g., microcalcifications, unusual mass, tissue distortion or asymmetry, etc.) on a mammogram and / or ultrasound imaging test. It may be from routine screening imaging or, more rarely, from diagnostic imaging triggered by a positive physical examination (e.g., a palpable mass, nipple discharge, skin change, etc.) or by a significant change in a previously identified mass.

[0075] Cellular proliferation in DCIS is confined to the milk ducts. If the proliferating cells have invaded through the basement membrane of the myoepithelial cell(MEC) layer lining the duct, thus appearing in the surrounding stroma, then the lesion is considered an invasive breast cancer, even if DCIS is also present. In some cases, the invasion is very minimal (microinvasion) or the only evidence of invasion is disruption of the MEC layer (e.g., by observing discontinuities in MEC-specific protein marker stains such as SMMHC and / or p63). Typically, these microinvasive cases are treated as invasive breast cancers, although there is some controversy in the treatment of these cases.

[0076] Recurrence rates in DCIS with current treatments are difficult to estimate. However, it is likely that about 20% of patients who receive lumpectomies without any further treatment would experience recurrence events within 10 years, approximately evenly split between DCIS and invasive events, while about 5% of patients who receive nipple sparing skin sparing mastectomies would experience recurrence. Standard of care with lumpectomy is to receive adjuvant radiation therapy (RT). Several randomized clinical trials provide evidence that adjuvant radiation therapy following lumpectomy reduces recurrence risk by approximately half for both DCIS and invasive event types, and that current clinical and pathologic assessment techniques cannot identify a low-risk sub-group in which there is no benefit from radiation therapy.

[0077] The term “label” or “probe” denotes an agent or reagents capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to a nucleic acid molecule or protein (such as one that can hybridize or bind to any of the markers provided herein, for example, any one of GLUT1, Ki67, PR, FOXA1, PD-1, IRF9, ALDHA1), thereby permitting detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). In some embodiments, a label is conjugated to a binding agent that specifically binds to a biomarker (for example, any one of GLUT1, Ki67, PR, FOXA1, PD-1, IRF9, ALDHA1) to allow for detecting the presence of the marker in a subject or a sample from the subject. In some embodiments, a label is conjugated to a binding agent that specifically binds to a biomarker(for example, any one of GLUT1, Ki67, PR, FOXA1, PD-1, IRF9, ALDHA1) to allow for detecting the presence of the marker in a subject or a sample from the subject.

[0078] As used herein, the term “subject” encompasses any animal including humans, preferably a mammal. Exemplary subjects include but are not limited to humans, primates, livestock (e.g. sheep, cows, horses, donkeys, pigs), companion animals (e.g. dogs, cats), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs, hamsters), captive wild animals (e.g. fox, deer). Preferably the mammal is a human or primate. More preferably the mammal is a human. “Subject” and “patient” are used interchangeably herein. In some embodiments, the subject is a female subject.

[0079] The term “and / or” shall be taken to provide explicit support for both meanings or for either meaning.

[0080] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0081] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms “a,” “an,” and “the” refer to one or more than one, unless the context clearly dictates otherwise. For example, the term “comprising a nucleic acid molecule” includes single or plural nucleic acid molecules and is considered equivalent to the phrase “comprising at least one nucleic acid molecule.” The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements. Unless otherwise specified, the definitions provided herein control when the present definitions may be different from other possible definitions. METHODS

[0082] Methods of treating invasive breast cancer are provided. With reference to FIG.11, a method 1100 of treating invasive breast cancer can include providing 1110 a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative(HR(+) / HER2(-)) breast cancer. The method can include analyzing 1120 the breast tissue sample for a first biosignature including at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1. The method can include selecting 1130 an adjuvant therapy for a breast cancer treatment based on the analysis of the first biosignature, wherein the adjuvant therapy comprises radiotherapy (RT) (e.g., standard radiotherapy). Then, the method can include administering 1140 the breast cancer treatment (or the adjuvant therapy) to the subject. The method can reduce a risk of breast cancer recurrence (e.g., risk of local regional recurrence (LRR), or a combined risk for LRR and distal metastasis). In some embodiments, the risk of breast cancer recurrence is a 10-year risk of recurrence. In some embodiments, the adjuvant therapy also includes endocrine therapy (ET). In some embodiments, the adjuvant therapy includes RT followed by ET. In some embodiments, the adjuvant therapy includes ET after RT.

[0083] With reference to FIG.12, a method 1200 of treating invasive breast cancer is provided. The method can include providing 1210 a breast tissue sample of an invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer (e.g., a subject or patient can have a biopsy taken from a HR(+) / HER2(-) tumor in breast tissue). The method can include having 1220 the breast tissue sample analyzed for a first biosignature comprising at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1. The method can include receiving 1230 a breast cancer treatment based on the analysis of the biosignature, wherein the breast cancer treatment comprises an adjuvant therapy comprising radiotherapy (RT) (e.g., standard radiotherapy). The method can reduce a risk of breast cancer recurrence (e.g., risk of local regional recurrence (LRR), or a combined risk for LRR and distal metastasis). In some embodiments, the risk of breast cancer recurrence is a 10-year risk of recurrence.

[0084] In some embodiments, the biosignature (e.g., the first biosignature) includes GLUT1(-) and Ki67(-). In some embodiments, the risk of breast cancer recurrence is a risk of LRR, or a risk of LRR and distal metastasis (e.g., a combined risk for LRR or distal metastasis), when the biosignature (e.g., the first biosignature) includes GLUT1(-) and Ki67(-). In some embodiments, the biosignature (e.g., the first biosignature) includes (a) GLUT1(-) and Ki67(- ); or (b) PR(+) and FOXA1(+). In some embodiments, the risk of breast cancer recurrence is a risk of LRR, or a risk of LRR and distal metastasis (e.g., a combined risk for LRR or distalmetastasis) when the biosignature (e.g., the first biosignature) includes (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+). In some embodiments, the risk of breast cancer recurrence is a 10-year risk of LRR. In some embodiments, the risk of breast cancer recurrence is a 10-year risk of LRR and distal metastasis.

[0085] In some embodiments, the method includes analyzing the breast tissue sample for a second biosignature that includes one or more of the following biomarkers: PD- 1, IRF9, ALDHA1. In some embodiments, the second biosignature includes PD-1 and IRF9. In some embodiments, the second biosignature includes the biomarker GLUT1. In some embodiments, the method includes assessing a risk of breast cancer recurrence based on the analysis of the second biosignature. In some embodiments, the method includes selecting RT for the adjuvant therapy based on the analysis of the second biosignature. Suitable, non- limiting options for assessing the risk of breast cancer recurrence based on the analysis of the second biosignature and / or selecting RT for the adjuvant therapy based on the analysis of the second biosignature includes those set forth in PCT Publication No. WO 2024 / 173438, which is hereby incorporated by reference in its entirety. In some embodiments, the method including the second biosignature as described herein can include conducting any one or more of the steps in a single row shown in Tables 3 and 4. In the methods related to diagnostics, the methods can include using, in Table 3 for example, for every row, the columns designated as Biomarker or Biomarker Algorithm, Method of Quantitation, Population (group), Finding, Prognostic for recurrence (in the context of a medical recommendation), and RT Treatment Benefit (in the context of a medical recommendation). In the methods related to treatments, the methods can include using, in Table 3 for example, for every row, the columns designated as Class, Biomarker or Biomarker Algorithm, Method of Quantitation, Population, Finding, Prognostic for recurrence, and RT Treatment Benefit. Table 3† National Institute for Health and Care Excellence. Early and locally advanced breast cancer: diagnosis and management. July 18, 2018 (www.nice.org.uk / guidance / ng101) Gradishar WJ, Anderson BO, Balassanian R, et al. NCCN guidelines insights breast cancer, version 1.2017. J Natl Compr Canc Netw 2017;15:433-451.

[0086] With reference to FIG.13, a method 1300 of treating invasive breast cancer is provided. The method can include providing 1310 a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer. The method can include analyzing 1320 the breast tissue sample for a biosignature comprising the following biomarkers: (a) PD-1 and IRF9; and (b) at least: (i) GLUT1 and Ki67; or (ii) PR and FOXA1. The method can further include selecting 1330 a breast cancer treatment comprising an adjuvant therapy based on the analysis of the biosignature, wherein radiotherapy (RT) is selected for the adjuvant therapy based on the analysis of at least PD-1 and IRF9 (e.g., as disclosed herein, including Tables 3 and 4). The method can also include administering 1330 the breast cancer treatment to the subject. The method can reduce a risk of breast cancer recurrence (e.g., risk of local regional recurrence (LRR), or a combined risk for LRR and distal metastasis). In some embodiments, the risk of breast cancer recurrence is a 10-year risk of recurrence. In some embodiments, the adjuvant therapy comprises RT and endocrine therapy(ET). In some embodiments, the adjuvant therapy includes ET after RT. In some embodiments, RT and endocrine therapy (ET) are selected for the adjuvant therapy based on the analysis of at least: (i) GLUT1 and Ki67; or (ii) PR and FOXA1. In some embodiments, RT is followed by ET (or ET is after RT). In some embodiments, the biosignature includes ALDHA1 in (a).

[0087] In some embodiments, the biosignature includes GLUT1(-) and Ki67(-), wherein the risk of breast cancer recurrence is a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis (e.g., a combined risk for LRR or distal metastasis). In some embodiments, the biosignature comprises: (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+), wherein the risk of breast cancer recurrence comprises a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis (e.g., a combined risk for LRR or distal metastasis). In some embodiments, the risk of breast cancer recurrence is a 10-year risk of LRR. In some embodiments, the risk of breast cancer recurrence is a 10-year risk of LRR and distal metastasis.

[0088] In some embodiments, a biosignature of the present disclosure can be used to identify a subject for breast cancer treatment. With reference to FIG. 14, a method 1400 of identifying a subject for breast cancer treatment is provided. The method can include providing 1410 a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2- negative (HR(+) / HER2(-)) breast cancer. The method can include analyzing 1420 the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67. The method can further include identifying 1430 the subject for a breast cancer treatment to reduce a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis, based on the analyzed biosignature. In some embodiments, the analyzed biosignature being GLUT1(-) and Ki67(-) indicates that the subject will benefit from the breast cancer treatment that comprises an adjuvant therapy comprising radiotherapy (RT) and endocrine therapy (ET) to reduce a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis. In some embodiments, RT is followed by ET. In some embodiments, the risk of breast cancer recurrence is a 10-year risk of LRR. In some embodiments, the risk of breast cancer recurrence is a 10-year risk of LRR and distal metastasis.

[0089] Methods of diagnosis are also provided. The method of diagnosis can include providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue samplefor a biosignature comprising at least the biomarkers GLUT1 and Ki67; and determining a benefit to the subject of administering a breast cancer treatment comprising an adjuvant therapy based on the analyzed biosignature.

[0090] With reference to FIG. 15, a method 1500 of diagnosis can include providing 1510 a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer. The method can include analyzing 1520 the breast tissue sample for a biosignature that includes at least the biomarkers GLUT1 and Ki67. The method can also include identifying the subject as benefiting from a breast cancer treatment comprising an adjuvant therapy comprising radiotherapy (RT) and endocrine therapy (ET) to reduce a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis, based on the analyzed biosignature being GLUT1(-) and Ki67(-). In some embodiments, the adjuvant therapy comprises RT followed by ET. In some embodiments, the risk of breast cancer recurrence is a 10-year risk of LRR. In some embodiments, the risk of breast cancer recurrence is a 10-year risk of LRR and distal metastasis.

[0091] With reference to FIG. 16, a method 1600 of diagnosis can include providing 1610 a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer. The method can include analyzing 1620 the breast tissue sample for a biosignature that includes at least the biomarkers GLUT1 and Ki67, or at least the biomarkers PR and FOXA1. In some embodiments, the method further includes identifying 1630 the subject as benefiting from a breast cancer treatment comprising an adjuvant therapy comprising radiotherapy (RT) and endocrine therapy (ET) to reduce a risk of LRR, or a risk of LRR and distal metastasis (e.g., a combined risk for LRR and distal metastasis), based on the analyzed biosignature being: (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+). In some embodiments, the adjuvant therapy comprises RT followed by ET. In some embodiments, the risk of breast cancer recurrence is a 10-year risk of LRR. In some embodiments, the risk of breast cancer recurrence is a 10-year risk of LRR and distal metastasis.

[0092] In any method provided herein, in some embodiments, the breast cancer treatment includes lumpectomy (or breast conserving surgery (BCS)) or mastectomy. In some embodiments, the breast cancer treatment includes surgical removal of the tumor (e.g., lumpectomy or mastectomy). As used herein, “lumpectomy” and “breast conserving surgery”(or “BCS”) are used interchangeably. In some embodiments, the breast cancer treatment includes lumpectomy and the adjuvant therapy (e.g., RT and / or ET), as described herein. In some embodiments, the breast cancer treatment includes lumpectomy or mastectomy and the adjuvant therapy (e.g., RT and / or ET), as described herein. In some embodiments, the breast cancer treatment includes lumpectomy and the adjuvant therapy that includes RT followed by ET. In any method of treatment herein, in some embodiments, where the subject has been administered or has received lumpectomy or mastectomy, administering the breast cancer treatment involves administering the adjuvant therapy (e.g., if benefit is predicted based on the analysis of the biosignature as described herein).

[0093] In some embodiments, the subject has (or has been diagnosed with) T1 / T2N0M0 breast cancer. In some embodiments, the subject is a female subject. In some embodiments, the subject is, is about, or is at least 20, 30, 40, 45, 50, 55, 60, 65, 70, or 75 years old.

[0094] Analyzing the breast tissue sample for a biosignature can be done using any suitable option. In some embodiments, analyzing the breast tissue sample for a biosignature includes an expression level of a gene product (e.g., mRNA, protein, etc.) of a biomarker in the biosignature in the breast tissue sample or a portion thereof. Detecting an expression level of a gene product denotes determining of a level of expression in either a qualitative or quantitative manner. Exemplary methods include, but are not limited to: microarray analysis, RT-PCR, Northern blot, in situ hybridization, Western blot, immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), multiplex immunofluorescence (MIF), ELISA, next generation sequencing, NGS-RNA seq, and mass spectrometry. In some embodiments, analyzing the breast tissue sample for a biosignature includes contacting the breast tissue sample or a portion thereof with a detection reagent for each of the biomarkers of the biosignature; and detecting binding of the detection agent, to determine the biosignature. Any suitable detection reagent can be used. In some embodiments, the detection reagent includes an antibody or nucleic acid probe to one or more of the biomarkers. In some embodiments, the detection reagent includes an antibody to one or more of the biomarkers. Any suitable antibody for detecting the biomarker in the breast tissue sample can be used.

[0095] In any method provided herein, in some embodiments, GLUT1(-) denotes an expression level of GLUT1 in cells of the breast tissue sample from the subject at or below a threshold level. Any suitable threshold level of GLUT1 expression can be used. In some embodiments, the expression level of GLUT1 is an H score. In some embodiments, the threshold level is or is about a 25 percentile of GLUT1 expression levels in patients in a cohort (e.g., a cohort of patients with invasive HR(+) / HER2(-) breast cancer). In some embodiments, the threshold level of GLUT1 is, is about, or is at most a 75, 60, 50, 40, 30, 25, 20, 15, 10, 5, or 1 percentile of GLUT1 expression levels in cells of the breast tissue sample from patients in a cohort (e.g., a cohort of patients with invasive HR(+) / HER2(-) breast cancer), or optionally is a percentile in a range defined by any two of the listed levels (e.g., 1-75 percentile, 5-50 percentile, 10-40 percentile, 15-30 percentile, 20-30 percentile, etc.). In some embodiments, GLUT1 expression level is a GLUT1 protein expression level. In some embodiments, GLUT1 expression level is a GLUT1 nucleic acid expression level (e.g., a GLUT1 mRNA expression level).

[0096] In any method provided herein, in some embodiments, Ki67(-) denotes a percentage of Ki67-expressing cells in a tumor of the breast tissue sample at or below a threshold percentage. Any suitable threshold percentage of Ki67-expressing cells can be used. In some embodiments, the threshold percentage is or is about a 25 percentile of Ki67- expressing cells in patients in a cohort (e.g., a cohort of patients with invasive HR(+) / HER2(- ) breast cancer). In some embodiments, the threshold level of Ki67-expressing cells in patients in a cohort (e.g., a cohort of patients with invasive HR(+) / HER2(-) breast cancer) is, is about, or is at most a 75, 60, 50, 40, 30, 25, 20, 15, 10, 5, or 1st percentile of the percentages of Ki67- expressing cells in a tumor of the breast tissue sample from patients in the cohort, or optionally is a percentile in a range defined by any two of the listed levels (e.g., 1-75 percentile, 5-50 percentile, 10-40 percentile, 15-30 percentile, 20-30 percentile, etc.). In some embodiments, the Ki67 expression is a Ki67 protein expression. In some embodiments, Ki67 expression is a Ki67 nucleic acid expression (e.g., Ki67 mRNA expression).

[0097] In any method provided herein, in some embodiments, PR(+) denotes a percentage of PR-expressing cells in a tumor of the breast tissue sample at or above a threshold percentage. Any suitable threshold percentage of PR-expressing cells can be used. In some embodiments, the threshold percentage is or is about a 50 percentile of percentages of PR-expressing cells in patients in a cohort (e.g., a cohort of patients with invasive HR(+) / HER2(- ) breast cancer). In some embodiments, the threshold level is, is about, or is at least the 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, or 90thpercentile of the percentages of PR-expressing cells in patients in the cohort, or optionally is a percentile in range defined by any of the listed levels (e.g., 10-90 percentile, 20-80 percentile, 30-70 percentile, 40-60 percentile, etc.). In some embodiments, PR expression level is a PR protein expression level. In some embodiments, PR expression level is a PR nucleic acid expression level (e.g., a PR mRNA expression level).

[0098] In some embodiments, FOXA1(+) denotes an expression level of FOXA1 at or above a threshold level. In some embodiments, the expression level of FOXA1 is an H score. Any suitable threshold level of FOXA1 expression can be used. In some embodiments, the threshold level is or is about a 75 percentile of FOXA1 expression levels in patients in a cohort (e.g., a cohort of patients with invasive HR(+) / HER2(-) breast cancer). In some embodiments, the threshold level is, is about, or is at least a 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, or 90th percentile of FOXA1 expression levels in patients in the cohort, or optionally, the threshold is in a range defined by any two of the listed levels (e.g., 10-90 percentile, 20-80 percentile, 30-70 percentile, 40-60 percentile, etc.). In some embodiments, FOXA1 expression level is a FOXA1 protein expression level. In some embodiments, FOXA1 expression level is a FOXA1 nucleic acid expression level (e.g., a FOXA1 mRNA expression level).

[0099] In some embodiments, the biosignature includes one or more of PD-1, IRF9, and ALDHA1. In some embodiments, one or more biomarkers are assessed (e.g., to analyze the breast tissue sample for a biosignature). In some embodiments, the one or more biomarkers include Programmed death 1 (PD-1). In some embodiments, the one or more biomarkers include Interferon Regulatory Factor 9 (IRF9). In some embodiments, the one or more biomarkers include Aldehyde dehydrogenase A1 (ALDHA1). In some embodiments, the one or more biomarkers include Glucose transporter 1 (GLUT1). In some embodiments, the one or more biomarkers include Ki67. In some embodiments, the one or more biomarkers include PR. In some embodiments, the one or more biomarkers include FOXA1.

[0100] In some embodiments, two or more biomarkers are assessed. In some embodiments, the two or more biomarkers include any two of the following biomarkers: PD-1, IRF9, ALDHA1, GLUT1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1 and IRF9. In some embodiments, the two or more biomarkers include PD-1 and ALDHA1. In some embodiments, the two or more biomarkers include PD-1 and GLUT1. In some embodiments, the two or more biomarkers include PD-1 and Ki67. In some embodiments, the two or more biomarkers include PD-1 and PR. In some embodiments, the two or more biomarkers include PD-1 and FOXA1. In some embodiments, the two or more biomarkers include IRF9 and ALDHA1. In some embodiments, the two or more biomarkers include IRF9 and GLUT1. In some embodiments, the two or more biomarkers include IRF9 and Ki67. In some embodiments, the two or more biomarkers include IRF9 and PR. In some embodiments, the two or more biomarkers include IRF9 and FOXA1. In some embodiments, the two or more biomarkers include ALDHA1 and GLUT1. In some embodiments, the two or more biomarkers include ALDHA1 and Ki67. In some embodiments, the two or more biomarkers include ALDHA1 and PR. In some embodiments, the two or more biomarkers include ALDHA1 and FOXA1. In some embodiments, the two or more biomarkers include GLUT1 and Ki67. In some embodiments, the two or more biomarkers include GLUT1 and PR. In some embodiments, the two or more biomarkers include GLUT1 and FOXA1. In some embodiments, the two or more biomarkers include Ki67 and PR. In some embodiments, the two or more biomarkers include Ki67 and FOXA1. In some embodiments, the two or more biomarkers include PR and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, and ALDHA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, and GLUT1. In some embodiments, the two or more biomarkers include PD-1, IRF9, and Ki67. In some embodiments, the two or more biomarkers include PD-1, IRF9, and PR. In some embodiments, the two or more biomarkers include PD-1, IRF9, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, and GLUT1. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, and Ki67. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, and PR. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, GLUT1, and Ki67. In some embodiments, the two or more biomarkers include PD-1, GLUT1, and PR. In some embodiments, the two or more biomarkers include PD-1, GLUT1, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, Ki67, and PR. In some embodiments,the two or more biomarkers include PD-1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, PR, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, and GLUT1. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, and Ki67. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, and PR. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, GLUT1, and Ki67. In some embodiments, the two or more biomarkers include IRF9, GLUT1, and PR. In some embodiments, the two or more biomarkers include IRF9, GLUT1, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, Ki67, and PR. In some embodiments, the two or more biomarkers include IRF9, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, PR, and FOXA1. In some embodiments, the two or more biomarkers include ALDHA1, GLUT1, and Ki67. In some embodiments, the two or more biomarkers include ALDHA1, GLUT1, and PR. In some embodiments, the two or more biomarkers include ALDHA1, GLUT1, and FOXA1. In some embodiments, the two or more biomarkers include ALDHA1, Ki67, and PR. In some embodiments, the two or more biomarkers include ALDHA1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include ALDHA1, PR, and FOXA1. In some embodiments, the two or more biomarkers include GLUT1, Ki67, and PR. In some embodiments, the two or more biomarkers include GLUT1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include GLUT1, PR, and FOXA1. In some embodiments, the two or more biomarkers include Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, and GLUT1. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, and Ki67. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, and PR. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, GLUT1, and Ki67. In some embodiments, the two or more biomarkers include PD-1, IRF9, GLUT1, and PR. In some embodiments, the two or more biomarkers include PD-1, IRF9, GLUT1, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, Ki67, and PR. In some embodiments, the two or more biomarkers include PD-1, IRF9, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, PR, and FOXA1. In someembodiments, the two or more biomarkers include PD-1, ALDHA1, GLUT1, and Ki67. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, GLUT1, and PR. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, GLUT1, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, Ki67, and PR. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, GLUT1, Ki67, and PR. In some embodiments, the two or more biomarkers include PD-1, GLUT1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, GLUT1, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, GLUT1, and Ki67. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, GLUT1, and PR. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, GLUT1, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, Ki67, and PR. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, PR, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, GLUT1, Ki67, and PR. In some embodiments, the two or more biomarkers include IRF9, GLUT1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, GLUT1, PR, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include ALDHA1, GLUT1, Ki67, and PR. In some embodiments, the two or more biomarkers include ALDHA1, GLUT1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include ALDHA1, GLUT1, PR, and FOXA1. In some embodiments, the two or more biomarkers include ALDHA1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include GLUT1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, GLUT1, and Ki67. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, GLUT1, and PR. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, GLUT1, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, Ki67, and PR. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, Ki67, andFOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, GLUT1, Ki67, and PR. In some embodiments, the two or more biomarkers include PD-1, IRF9, GLUT1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, GLUT1, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, GLUT1, Ki67, and PR. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, GLUT1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, GLUT1, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, GLUT1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, GLUT1, Ki67, and PR. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, GLUT1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, GLUT1, PR, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, GLUT1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include ALDHA1, GLUT1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, GLUT1, Ki67, and PR. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, GLUT1, Ki67, and FOXA1. In some embodiments, the two or more biomarkers include PD- 1, IRF9, ALDHA1, GLUT1, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, GLUT1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, ALDHA1, GLUT1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include IRF9, ALDHA1, GLUT1, Ki67, PR, and FOXA1. In some embodiments, the two or more biomarkers include PD-1, IRF9, ALDHA1, GLUT1, Ki67, PR, and FOXA1.

[0101] In some embodiments, an interaction between RT and one or more biomarkers is assessed. In some embodiments, an interaction between ET and one or morebiomarkers is assessed. In some embodiments, a biosignature includes one or more biomarkers. In some embodiments, the biosignature includes two or more biomarkers.

[0102] With reference to Figure 4, in some embodiments, the biosignature includes Ki67 and GLUT1, wherein a <25th percentile of Ki67 expression for patients in a cohort, and a <25th percentile of GLUT1 expression for patients in a cohort is predictive of benefit of ET after RT, wherein the local regional recurrence (LRR) risk is reduced.

[0103] With reference to Figure 7, in some embodiments, the biosignature includes Ki67 and GLUT1, wherein a <25th percentile of Ki67 expression for patients in a cohort, and a <25th percentile of GLUT1 expression for patients in a cohort is predictive of benefit of ET after RT, wherein the combined LRR and distant risk is reduced.

[0104] With reference to Figure 8, in some embodiments, the biosignature includes Ki67 and GLUT1, wherein a <25th percentile of Ki67 expression for patients in a cohort, and a <25th percentile of GLUT1 expression for patients in a cohort is predictive for benefit of ET after RT, wherein the combined LRR and distant risk is reduced. In some embodiments, the biosignature is predictive for ET benefit after RT for LRR risk. In some embodiments, the biosignature includes PR and FOXA1, wherein a >50th percentile of PR expression for patients in a cohort, and a >75th percentile of FOXA1 expression for patients in a cohort is predictive for benefit of ET after RT, wherein the combined LRR and distant risk is reduced. In some embodiments, the biosignature is predictive for ET benefit after RT for LRR risk.

[0105] In some embodiments, an additional biosignature is predictive for the benefit of ET after RT to reduce Local Regional recurrence risk (ET:RT:biosignature_ET.1), see Figure 4 LRR risk. In some embodiments, (the biosignature ET.1) patients with: low percentage of KI67 positive cells in the tumor, less than 25th percentile of the ki67 expression for patients in the cohort, AND low expression of Glut1 (H-score) in tumor cells, less than 25th percentile of the GLUT1 expression for patients in the cohort the biosignature_ET.1 was not prognostic for LRR risk.

[0106] The biosignature_ET.1 was also not predictive of RT benefit for local / regional risk reduction (FIGURE 5) or combined LRR / distant risk reduction (Figure 6).

[0107] In some embodiments, an additional biosignature is predictive for the benefit of ET after RT to reduce combined Local Regional recurrence / distant risk (ET:RT:biosignature_ET.1), see Figure 7 LRR / distant risk. In some embodiments, (thebiosignature ET.1) patients with: low percentage of KI67 positive cells in the tumor, less than 25th percentile of the ki67 expression for patients in the cohort, AND low expression of Glut1 (H-score) in tumor cells, less than 25th percentile of the GLUT1 expression for patients in the cohort the biosignature_ET.1 was not prognostic for LRR / distant risk.

[0108] In some embodiments, an additional biosignature is predictive for the benefit of ET after RT to reduce combined Local Regional recurrence / distant risk (ET:RT:biosignature_ET.2), see Figure 8 LRR / distant risk. In some embodiments, the biosignature (ET.2) patients with: low percentage of KI67 positive cells in the tumor, less than 25th percentile of the ki67 expression for patients in the cohort, AND low expression of Glut1 (H-score) in tumor cells, less than 25th percentile of the GLUT1 expression for patients in the cohort OR high percentage of PR positive cells in the tumor, more than 50th percentile of the pr expression for patients in the cohort, AND high expression of FOXA1 (H-score) in tumor cells, more than 75th percentile of the FOXA1 expression for patients in the cohort.

[0109] The biosignature_ET.2 was predictive for ET benefit after RT for LRR / distant risk, but was not prognostic for recurrence risk after breast conserving surgery (BCS). Further, biosignature_ET.2 was predictive for ET benefit after RT for LRR risk, but was not prognostic for recurrence risk after BCS. See figure 9. Thus, in some embodiments, an assay or method checking these markers can be used to identify the subject to be accordingly treated and / or further include the appropriate ET.

[0110] Biosignature_ET.2 was predictive for ET benefit after RT for LRR / distant risk, biosignature (previous application) was predictive for RT benefit for LRR / distant risk. Biosignature_ET.2 was but was not prognostic for recurrence risk after BCS. See figure 10.

[0111] In any of the methods (e.g., for treating or diagnosing a patient or subject for breast cancer) as provided herein, a breast tissue sample is a core biopsy, fine-needle aspiration (FNA) sample, excisional biopsy or a sample from surgery. In some embodiments, a breast tissue sample obtained from a subject is provided for performing a method of the present disclosure, e.g., for determining expression of one or more markers in the sample. In some embodiments, the method (e.g., at least the part of a method involving analysis of one or more biomarkers as disclosed herein) is performed using breast tissue sample from a pre- operative tumor, e.g., a biopsy obtained from the subject before breast-conserving surgery (BCS). In some embodiments, the breast tissue sample is a biopsy obtained from the subjectbefore operating on a tumor, e.g., before BCS. In some embodiments, the method (e.g., at least the part of a method involving analysis of one or more biomarkers as disclosed herein) is performed on a post-operative tumor, e.g., a tumor obtained from the subject after breast- conserving surgery (BCS). In some embodiments, the breast tissue sample is a preserved sample. In some embodiments, the breast tissue sample is a fresh frozen or formalin-fixed, paraffin-embedded tissue. In some embodiments, the breast tissue sample is paraffin embedded. mRNA-BINDING NUCLEIC ACIDS, ANTIBODIES, AND COMPOSITIONS THEREOF

[0112] Provided herein are detection reagents for detecting the presence and / or expression level of a biomarker of the present disclosure in a sample (e.g., breast tissue sample).

[0113] Provided herein are mRNA binding nucleic acids and antibodies for use in the diagnosis and / or treatment of invasive breast cancer. A mRNA-binding nucleotide (or nucleic acid) or an antibody for use in the diagnosis and / or treatment of breast cancer is provided, where the nucleotide (or nucleic acid) or the antibody is used for quantifying the level of a biomarker (including, without limitation, one or more of GLUT1, Ki67, PR, FOXA1, PD-1, IRF9, ALDHA1) that is expressed in a breast cancer sample, where whether the subject will benefit from adjuvant therapy that includes RT and ET to reduce a risk of breast cancer recurrence is determined by taking in to account the level of (a) GLUT1 and Ki67, or (b) PR and FOXA1. Also provided is a mRNA-binding nucleotide (or nucleic acid) or an antibody for use in the diagnosis of breast cancer is provided, where the nucleotide (or nucleic acid) or the antibody is used for quantifying the level of a biomarker (including, without limitation, one or more of GLUT1, Ki67, PR, FOXA1, PD-1, IRF9, ALDHA1) that is expressed in a breast cancer sample, and where, in some embodiments, GLUT1(-) and Ki67(-), or PR(+) and FOXA1(+) indicates that the patients belong to a patient subgroup that will benefit from adjuvant therapy that includes RT and ET (e.g., RT followed by ET) to reduce a risk of breast cancer recurrence. The mRNA-binding nucleotide (or nucleic acid) or antibody provided herein also finds use in selecting a treatment option for a subject with breast cancer, based on the diagnosis. Also provided is an antibody for use in the diagnosis of breast cancer, wherein the antibody is used for quantifying the level of, e.g., GLUT1, Ki67, PR, or FOXA1, that isexpressed in a breast cancer sample from a patient. Also provided is a mRNA binding nucleotide (or nucleic acid) or antibody for use in the diagnosis of breast cancer, wherein the antibody is used for quantifying the level of, e.g., GLUT1, Ki67, PR, or FOXA1, that is expressed in a breast cancer sample from a patient, and where whether the subject will benefit from adjuvant therapy that includes RT and ET (e.g., RT followed by ET) to reduce a risk of breast cancer recurrence is determined by taking in to account the level of (a) GLUT1 and Ki67, or (b) PR and FOXA1. Also provided is an antibody for use in the diagnosis of breast cancer, wherein the antibody is used for quantifying the level of, e.g., GLUT1, Ki67, PR, or FOXA1, that is expressed in a breast cancer sample from a patient, and where, in some embodiments, GLUT1(-) and Ki67(-), or PR(+) and FOXA1(+) indicates that the patients belong to a patient subgroup that will benefit from adjuvant therapy that includes RT and ET (e.g., RT followed by ET) to reduce a risk of breast cancer recurrence. Also provided is a mRNA binding nucleotide (or nucleic acid) for use in the diagnosis of breast cancer, wherein the nucleotide (or nucleic acid) is used for quantifying the level of, e.g., GLUT1, Ki67, PR, or FOXA1, that is expressed in a breast cancer sample from a patient.

[0114] Also provided are compositions that include mRNA binding nucleic acids, nucleotides, and antibodies of the present disclosure. In some embodiments, a composition of the present disclosure includes detection reagents for detecting expression of biomarkers comprising at least GLUT1 and Ki67 in a breast tissue sample, for use in any one of the methods herein. Also provided is a combination of detection reagents for detecting expression of biomarkers comprising at least GLUT1 and Ki67 in a breast tissue sample, for use in any one of the methods herein. In some embodiments, the biomarkers include PD-1 and IRF9. In some embodiments, the biomarkers include ALDHA1. KITS

[0115] Also provided are kits that find use in diagnosing a subject or assaying a tumor sample, according to some embodiments of the present disclosure. The kit can include a mRNA-binding nucleotide (or nucleic acid) and / or an antibody that binds to a biomarker disclosed herein (including, without limitation, one or more of GLUT1, Ki67, PR, FOXA1, PD-1, IRF9, ALDHA1) in a breast tissue sample. Also provided is a kit that includes reagents for detecting expression of biomarkers comprising at least GLUT1 and Ki67 in a breast tissuesample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer. In some embodiments, the biomarkers include PD-1 and IRF9. In some embodiments, the biomarkers include ALDHA1. Additional embodiments

[0116] Non-limiting embodiments of the present disclosure are provided by the following numbered embodiments. 1. A method of treating invasive breast cancer, the method comprising: providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a first biosignature comprising at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1; selecting an adjuvant therapy for a breast cancer treatment based on the analysis of the first biosignature, wherein the adjuvant therapy comprises radiotherapy (RT); and administering the breast cancer treatment to the subject, thereby reducing a risk of breast cancer recurrence. 2. The method of embodiment 1, wherein the adjuvant therapy further comprises endocrine therapy (ET). 3. The method of embodiment 1 or 2, wherein the adjuvant therapy comprises RT followed by ET. 4. The method of any one of embodiments 1-3, wherein the first biosignature comprises GLUT1(-) and Ki67(-), wherein the risk of breast cancer recurrence comprises a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis. 5. The method of any one of embodiments 1-3, wherein the first biosignature comprises the following biomarkers: (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+), wherein the risk of breast cancer recurrence comprises a risk of LRR, or a risk of LRR and distal metastasis. 6. The method of embodiment 5, wherein PR(+) denotes a percentage of PR- expressing cells in a tumor of the breast tissue sample at or above a threshold percentage. 7. The method of embodiment 6, wherein the threshold percentage is a 50 percentile of percentages of PR-expressing cells in patients in a cohort.8. The method of any one of embodiments 5-7, wherein FOXA1(+) denotes an expression level of FOXA1 at or above a threshold level, optionally wherein the expression level is an H score. 9. The method of embodiment 8, wherein the threshold level is a 75 percentile of FOXA1 expression levels in patients in a cohort. 10. The method of any one of embodiments 4-9, wherein GLUT1(-) denotes an expression level of GLUT1 at or below a threshold level, optionally wherein the expression level is an H score. 11. The method of embodiment 10, wherein the threshold level is a 25 percentile of GLUT1 expression levels in patients in a cohort. 12. The method of any one of embodiments 4-11, wherein Ki67(-) denotes a percentage of Ki67-expressing cells in a tumor of the breast tissue sample at or below a threshold percentage. 13. The method of embodiment 12, wherein the threshold percentage is a 25 percentile of percentages of Ki67-expressing cells in patients in a cohort. 14. The method of any one of the preceding embodiments, comprising: analyzing the breast tissue sample for a second biosignature that comprises one or more of the following biomarkers: PD-1, IRF9, ALDHA1, optionally wherein the second biosignature comprises PD-1 and IRF9; and at least: assessing a risk of breast cancer recurrence based on the analysis of the second biosignature; and / or selecting RT for the adjuvant therapy based on the analysis of the second biosignature. 15. The method of embodiment 14, wherein the second biosignature comprises the biomarker GLUT1. 16. A method of treating invasive breast cancer, the method comprising: providing a breast tissue sample of an invasive, hormone receptor-positive, HER2- negative (HR(+) / HER2(-)) breast cancer; having the breast tissue sample analyzed for a first biosignature comprising at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1; andreceiving a breast cancer treatment based on the analysis of the biosignature, wherein the breast cancer treatment comprises an adjuvant therapy comprising radiotherapy (RT), thereby reducing a risk of breast cancer recurrence. 17. The method of embodiment 16, wherein the adjuvant therapy further comprises endocrine therapy (ET). 18. The method of embodiment 16 or 17, wherein the adjuvant therapy comprises RT followed by ET. 19. The method of any one of embodiments 16-18, wherein the first biosignature comprises GLUT1(-) and Ki67(-), wherein the risk of breast cancer recurrence is a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis. 20. The method of any one of embodiments 16-18, wherein the first biosignature comprises: (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+), wherein the risk of breast cancer recurrence comprises a risk of LRR, or a risk of LRR and distal metastasis. 21. The method of embodiment 20, wherein PR(+) denotes a percentage of PR- expressing cells in a tumor of the breast tissue sample at or above a threshold percentage. 22. The method of embodiment 21, wherein the threshold percentage is a 50 percentile of percentages of PR-expressing cells in patients in a cohort. 23. The method of any one of embodiments 20-22, wherein FOXA1(+) denotes an expression level of FOXA1 at or above a threshold level, optionally wherein the expression level is an H score. 24. The method of embodiment 23, wherein the threshold level is a 75 percentile of FOXA1 expression levels in patients in a cohort. 25. The method of any one of embodiments 19-24, wherein GLUT1(-) denotes an expression level of GLUT1 at or below a threshold level, optionally wherein the expression level is an H score. 26. The method of embodiment 25, wherein the threshold level is a 25 percentile of GLUT1 expression levels in patients in a cohort. 27. The method of any one of embodiments 19-26, wherein Ki67(-) denotes a percentage of Ki67-expressing cells in a tumor of the breast tissue sample at or below a threshold percentage.28. The method of embodiment 27, wherein the threshold percentage is a 25 percentile of percentages of Ki67-expressing cells in patients in a cohort. 29. The method of any one of embodiments 16-28, comprising: having the breast tissue sample analyzed for a second biosignature comprising one or more of the following biomarkers: PD-1, IRF9, ALDHA1, optionally wherein the second biosignature comprises PD-1 and IRF9, wherein RT is selected for the adjuvant therapy based on the analysis of the second biosignature. 30. A method of treating invasive breast cancer, the method comprising: providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising the following biomarkers: (a) PD-1 and IRF9; and (b) at least: (i) GLUT1 and Ki67; or (ii) PR and FOXA1; selecting a breast cancer treatment comprising an adjuvant therapy based on the analysis of the biosignature, wherein radiotherapy (RT) is selected for the adjuvant therapy based on the analysis of at least PD-1 and IRF9; and administering the breast cancer treatment to the subject, thereby reducing a risk of breast cancer recurrence. 31. The method of embodiment 30, wherein RT and endocrine therapy (ET) are selected for the adjuvant therapy based on the analysis of at least: (i) GLUT1 and Ki67; or (ii) PR and FOXA1, optionally wherein RT is followed by ET. 32. The method of embodiment 30 or 31, wherein the biosignature comprises GLUT1(-) and Ki67(-), wherein the risk of breast cancer recurrence is a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis. 33. The method of embodiment 30 or 31, wherein the biosignature comprises: (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+), wherein the risk of breast cancer recurrence comprises a risk of LRR, or a risk of LRR and distal metastasis.34. The method of embodiment 33, wherein PR(+) denotes a percentage of PR- expressing cells in a tumor of the breast tissue sample at or above a 50 percentile of percentages of Ki67-expressing cells in patients in a cohort. 35. The method of embodiment 33 or 34, wherein FOXA1(+) denotes an expression level of FOXA1 at or above a 75 percentile of FOXA1 expression levels in patients in a cohort, optionally wherein the expression level is an H score. 36. The method of any one of embodiments 32-35, wherein GLUT1(-) denotes an expression level of GLUT1 at or below a 25 percentile of GLUT1 expression levels in patients in a cohort, optionally wherein the expression level is an H score. 37. The method of any one of embodiments 32-36, wherein Ki67(-) denotes a percentage of Ki67-expressing cells in a tumor of the breast tissue sample at or below a 25 percentile of percentages of Ki67-expressing cells in patients in a cohort. 38. A method of identifying a subject for breast cancer treatment, comprising: providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67; and identifying the subject for a breast cancer treatment to reduce a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis, based on the analyzed biosignature. 39. The method of embodiment 38, wherein the analyzed biosignature being GLUT1(-) and Ki67(-) indicates that the subject will benefit from the breast cancer treatment that comprises an adjuvant therapy comprising radiotherapy (RT) and endocrine therapy (ET) to reduce a risk of LRR, or a risk of LRR and distal metastasis, optionally wherein RT is followed by ET. 40. A method of diagnosis, comprising: providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67; based on the analyzed biosignature being GLUT1(-) and Ki67(-), identifying the subject as benefiting from a breast cancer treatment comprising an adjuvant therapycomprising radiotherapy (RT) and endocrine therapy (ET) to reduce a risk of LRR, or a risk of LRR and distal metastasis, optionally wherein the adjuvant therapy comprises RT followed by ET. 41. A method of diagnosis, comprising: providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67, or at least the biomarkers PR and FOXA1; based on the analyzed biosignature being: (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+), identifying the subject as benefiting from a breast cancer treatment comprising an adjuvant therapy comprising radiotherapy (RT) and endocrine therapy (ET) to reduce a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis. 42. The method of embodiment 41, wherein PR(+) denotes a percentage of PR- expressing cells in a tumor of the breast tissue sample at or above a threshold percentage. 43. The method of embodiment 42, wherein the threshold percentage is a 50 percentile of percentages of PR-expressing cells in patients in a cohort. 44. The method of any one of embodiments 41-43, wherein FOXA1(+) denotes an expression level of FOXA1 at or above a threshold level, optionally wherein the expression level is an H score. 45. The method of embodiment 44, wherein the threshold level is a 75 percentile of FOXA1 expression levels in patients in a cohort. 46. The method of any one of embodiments 40-45, wherein GLUT1(-) denotes an expression level of GLUT1 at or below a threshold level, optionally wherein the expression level is an H score. 47. The method of embodiment 46, wherein the threshold level is a 25 percentile of GLUT1 expression levels in patients in a cohort. 48. The method of any one of embodiments 40-47, wherein Ki67(-) denotes a percentage of Ki67-expressing cells in a tumor of the breast tissue sample at or below a threshold percentage. 49. The method of embodiment 48, wherein the threshold percentage is a 25 percentile of percentages of Ki67-expressing cells in patients in a cohort.50. The method of any one of embodiments 40-49, comprising administering the breast cancer treatment to the subject. 51. The method of any one of the preceding embodiments, wherein the subject has T1 / T2N0M0 breast cancer. 52. The method of any one of the preceding embodiments, wherein the breast cancer treatment comprises lumpectomy or mastectomy. 53. The method of any one of the preceding embodiments, wherein the breast tissue sample is analyzed by: contacting the breast tissue sample with a detection reagent for each of the biomarkers of the biosignature, optionally wherein the detection reagent comprises an antibody or nucleic acid probe to each of the biomarkers; and detecting binding of the detection agent, to determine the biosignature. 54. A combination of detection reagents for detecting expression of biomarkers comprising at least GLUT1 and Ki67 in a breast tissue sample, for use in the method of any one of the preceding embodiments. 55. A kit comprising reagents for detecting expression of biomarkers comprising at least GLUT1 and Ki67 in a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer. 56. The combination of embodiment 54, or the kit of embodiment 55, wherein the biomarkers comprise at least PD-1 and IRF9, optionally wherein the biomarkers comprise ALDHA1. EXAMPLES Example 1

[0117] This non-limiting example summarizes the findings and conclusions of the studies set forth in Examples 2-4.

[0118] Purpose / Objective(s): The landscape of breast cancer management is evolving with emphasis on the need for personalized treatment regimens to tailor treatment in early-stage, hormone positive (HR+) invasive breast cancer (BC) patients. Given that locoregional recurrence (LRR) rates are lower in HR+ BC patients, multiple studies have evaluated the role of adjuvant radiation therapy (RT) and / or endocrine therapy (ET) in these patients. Ongoing studies are exploring the integration of clinical and biological factors tobetter assess patient-specific recurrence risk profiles to individualize treatment in the adjuvant setting. The present study hypothesizes that the interaction of cellular pathways influencing tumor biology can be used as part of a biosignature to predict recurrence risk and the RT and ET benefit for early-stage HR+ HER2-negative BC.

[0119] Methods: A subset of 704 HR+ T1 / T2N0M0 patients with BC who underwent BCS with or without RT between 1987-2002 were identified from a multi- institutional cohort of women. A biosignature that calculates an individualized risk profile was developed and cross-validated. The association between the biosignature and the 10-yr LRR rate was assessed. RT and ET benefit were assessed as a function of the biosignature score, using survival analyses and multivariable Cox proportional hazards adjusted for treatment, clinicopathologic (CP) risk factors: age, grade, and tumor size. (FIGs.1-10)

[0120] Results: Median follow-up was 126 months and median age was 63 yrs old for the cohort with 93% (n=661) having T1 disease.36% of patients (n=253) received ET, 82% (n=584) received RT, with 29% (n=205) receiving both. On multivariable analysis, higher grade (p=.04), younger age (p=.05), and receipt of RT (p=.007) were associated with LRR. However, in a multivariable analysis that incorporated treatment and CP factors, the biosignature was prognostic (p<.001), while the CP factors were not associated with the LRR rate. The biosignature also predicted the benefit from RT (interaction with RT, p<.001) and incremental ET benefit after RT (interaction with ET, p=.0018) over 10 years. Collectively, the biosignature identified a) a Low-Risk Group with a 1% 10-yr LRR rate + / - RT (HR~1, p=0.99, n=146), b) an Elevated-Risk Group with a 17% LRR rate without RT and 10% with RT (HR=0.45, p=0.018, n=462) and c) a Residual-Risk Group with a 31% LRR rate without RT and 23% with RT (HR=0.62, p=.4, n=96).

[0121] Conclusions: The novel biosignature was prognostic for LRR after BCS and identified clinically meaningful risk groups, predicting differential RT and incremental ET benefit associated with 10-yr LRR. This initial validation indicates that the biosignature may be useful tool to aid in the assessment of the benefit of adjuvant therapy in early-stage HR+ HER2-negative IBC. Example 2

[0122] This non-limiting example shows an assessment of a biosignature for radiotherapy (RT) benefit for predicting benefit of endocrine therapy (ET) after RT to reduce recurrence risk.

[0123] Biosignatures for RT benefit was assessed for predicting benefit of endocrine therapy (ET) to reduce local regional recurrence (LRR) risk in hormone positive (HR+) HER2-negative T1 / T2N0M0 patients with BC. As shown in Fig. 1, a previously identified biosignature for radiation therapy (RT) was not predictive of the benefit of endocrine therapy (ET) to reduce the LRR risk.

[0124] FIG. 1: Biosignature for RT was not predictive for benefit of ET to reduce Local Regional recurrence risk (ET:biosignature).

[0125] The biosignature for RT benefit was assessed for predicting benefit of ET after RT to reduce local regional recurrence (LRR) risk in hormone positive (HR+) HER2- negative T1 / T2N0M0 patients with BC. As shown in Fig. 2, the biosignature for RT was not predictive for the benefit of ET after RT to reduce LRR risk in HR+, HER2-negative T1 / T2N0M0 patients.

[0126] FIG. 2: Biosignature for RT was not predictive for benefit of ET after RT to reduce Local Regional recurrence risk (ET:RT:biosignature).

[0127] The biosignature for RT benefit was assessed for predicting benefit of ET after RT to reduce combined local regional and distant recurrence risk in HR+, HER2-negative T1 / T2N0M0 patients with BC. As shown in Fig.3, the biosignature for RT was predictive for the benefit of ET after RT to reduce local regional recurrence risk (LRR) and distant recurrence risk (DRR) in HR+, HER2-negative T1 / T2N0M0 patients with BC.

[0128] FIG. 3: Biosignature for RT was predictive for benefit of ET after RT to reduce combined Local Regional and distant recurrence risk (ET:RT:biosignature) Example 3

[0129] This non-limiting example shows a novel biosignature for predicting the benefit of ET after RT to reduce the local regional recurrence risk.

[0130] Biosignatures were assessed for predicting the benefit of ET LRR risk. As shown in Fig. 4, the biosignature of ET:RT:biosignature_ET.1 was predictive for the benefit of ET after RT to reduce LRR risk in HR+, HER2-negative T1 / T2N0M0 patients with BC.The biosignature ET.1 were patients with: 1) a low percentage of KI67 positive cells in the tumor (less than 25th percentile of the KI67 expression for patients in the cohort), AND 2) low expression of Glut1 (H-score) in tumor cells (less than 25th percentile of the GLUT1 expression for patients in the cohort). The biosignature_ET.1 was not prognostic for LRR risk.

[0131] FIG.4: An additional biosignature was predictive for the benefit of ET after RT to reduce Local Regional recurrence risk (ET:RT:biosignature_ET.1).

[0132] The novel biosignature was assessed for predicting the RT benefit for local / regional risk reduction. As shown in Fig. 5, the biosignature_ET.1 was not predictive of the benefit of radiation therapy (RT) for local regional recurrence risk (LRR) reduction in hormone positive (HR+) HER2-negative T1 / T2N0M0 patients with BC.

[0133] FIG. 5: The biosignature_ET.1 was also not predictive of RT benefit for local / regional risk reduction.

[0134] The novel biosignature was assessed for predicting the benefit of radiation therapy (RT) for LRRdistal risk reduction. As shown in Fig.6, the biosignature_ET.1 was not predictive for the benefit of RT for local / regional risk reduction or combined (LRR) / distant risk reduction in HR+, HER2-negative T1 / T2N0M0 patients with BC.

[0135] FIG. 6: The biosignature_ET.1 was also not predictive of RT benefit for combined LRR / distant risk reduction.

[0136] The novel biosignature was assessed for predicting the benefit of endocrine therapy (ET) after radiation therapy (RT) to reduce combined local regional recurrence / distant risk in HR+, HER2-negative T1 / T2N0M0 patients with BC. As shown in Fig. 7, the biosignature ET:RT:biosignature_ET.1 was predictive for the benefit of ET after RT to reduce combined local regional recurrence / distant risk. The biosignature ET.1 were patients with low percentage of Ki67 positive cells in the tumor (less than 25th percentile of the Ki67 expression for patients in the cohort), AND low expression of Glut1 (H-score) in tumor cells (less than 25th percentile of the GLUT1 expression for patients in the cohort). The biosignature_ET.1 was not prognostic for LRR / distant risk.

[0137] FIG.7: An additional biosignature was predictive for the benefit of ET after RT to reduce combined Local Regional recurrence / distant risk (ET:RT:biosignature_ET.1). Example 4

[0138] This non-limiting example shows a novel biosignature for predicting the benefit of ET after RT to reduce the local regional recurrence risk.

[0139] Biosignatures were assessed for predicting the benefit of ET after RT to reduce combined local regional recurrence / distant risk. As shown in Figure 8, the ET:RT:biosignature_ET.2 was predictive for the benefit of ET after RT to reduce combined local regional recurrence / distant risk in HR+, HER2-negative T1 / T2N0M0 patients with BC. The biosignature ET.2 were patients with low percentage of KI67 positive cells in the tumor (less than 25th percentile of the KI67 expression for patients in the cohort), AND low expression of Glut1 (H-score) in tumor cells (less than 25th percentile of the GLUT1 expression for patients in the cohort) OR high percentage of PR positive cells in the tumor (more than 50th percentile of the PR expression for patients in the cohort), AND high expression of FOXA1 (H-score) in tumor cells (more than 75th percentile of the FOXA1 expression for patients in the cohort). The biosignature_ET.2 was predictive for ET benefit after RT for LRR / distant risk, but was not prognostic for recurrence risk after BCS.

[0140] FIG.8: An additional biosignature was predictive for the benefit of ET after RT to reduce combined Local Regional recurrence / distant risk (ET:RT:biosignature_ET.2).

[0141] The novel biosignature was assessed for predicting ET benefit after RT for LRR / distant risk in HR+ HER2-negative T1 / T2N0M0 patients with BC. As shown in Fig. 9, the biosignature_ET.2 was predictive for ET benefit after RT for LRR risk, but was not prognostic for recurrence risk after BCS.

[0142] FIG. 9: biosignature_ET.2 was predictive for ET benefit after RT for LRR risk, but was not prognostic for recurrence risk after BCS.

[0143] The novel biosignature was assessed for predicting ET benefit after RT for LRR / distant risk in HR+, HER2-negative T1 / T2N0M0 patients with BC. As shown in Fig.10, biosignature_ET.2 was predictive for ET benefit after RT for LRR / distant risk, the previously identified biosignature (Example 2) was predictive for RT benefit for LRR / distant risk. Biosignature_ET.2 was but was not prognostic for recurrence risk after BCS.

[0144] FIG. 10: Biosignature_ET.2 was predictive for ET benefit after RT for LRR / distant risk, biosignature (previous application) was predictive for RT benefit for LRR / distant risk. Biosignature_ET.2 was but was not prognostic for recurrence risk after BCSExample 5

[0145] This non-limiting example provides a method of treating invasive breast cancer.

[0146] A breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer is provided. The breast tissue sample is analyzed for a first biosignature that includes at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1. An adjuvant therapy for a breast cancer treatment is selected based on the analysis of the first biosignature, wherein the adjuvant therapy includes radiotherapy (RT). The breast cancer treatment is administered to the subject. The administered breast cancer treatment can reduce the subject’s risk of breast cancer recurrence. Example 6

[0147] This non-limiting example provides a method of treating invasive breast cancer.

[0148] A breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer is provided. The subject has the breast tissue sample analyzed for a first biosignature that includes at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1. The subject receives a breast cancer treatment based on the analysis of the biosignature. The breast cancer treatment includes an adjuvant therapy that can be radiotherapy (RT). The breast cancer treatment can reduce the subject’s risk of breast cancer recurrence. Example 7

[0149] This non-limiting example provides a method of treating invasive breast cancer.

[0150] A breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer is provided. The breast tissue sample is analyzed for a biosignature that includes the following biomarkers: (a) PD-1 and IRF9; and (b) at least: (i) GLUT1 and Ki67; or (ii) PR and FOXA1. A breast cancer treatment comprising an adjuvant therapy is selected based on the analysis of the biosignature, where radiotherapy (RT) is selected for the adjuvant therapy based on the analysis of at least PD-1 and IRF9. Thebreast cancer treatment is administered to the subject. The administered breast cancer treatment can reduce the subject’s risk of breast cancer recurrence. Example 8

[0151] This non-limiting example provides a method of identifying a subject for breast cancer treatment.

[0152] A breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer is provided. The breast tissue sample is analyzed for a biosignature that includes at least the biomarkers GLUT1 and Ki67. The subject is identified for a breast cancer treatment to reduce a risk of local regional recurrence (LRR) based on the analyzed biosignature. Example 9

[0153] This non-limiting example provides a method of diagnosis.

[0143] A breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer is provided. The breast tissue sample is analyzed for a biosignature that includes at least the biomarkers GLUT1 and Ki67. Based on the analyzed biosignature being GLUT1(-) and Ki67(-), the subject is identified as benefiting from a breast cancer treatment that includes an adjuvant therapy that includes radiotherapy (RT) and endocrine therapy (ET). The adjuvant therapy can reduce a risk of local regional recurrence (LRR). The adjuvant therapy can include RT followed by ET. Example 10

[0154] This non-limiting example provides a method of diagnosis.

[0155] A breast tissue sample from a subject with invasive, hormone receptor (HR)-positive, HER2-negative (HR(+) / HER2(-)) breast cancer is provided. The breast tissue sample is analyzed for a biosignature that includes at least the biomarkers GLUT1 and Ki67, or at least the biomarkers PR and FOXA1. Based on the analyzed biosignature being (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+), the subject is identified as benefiting from a breast cancer treatment that includes an adjuvant therapy that includes a RT and ET. The adjuvant therapy can reduce a risk of local regional recurrence (LRR) and distal metastasis.

[0156] The described embodiments and examples of the present disclosure are intended to be illustrative rather than restrictive, and are not intended to represent every embodiment or example of the present disclosure, and thus, are not to be limited in scope by the specific embodiments and examples described herein. While the fundamental novel features of the disclosure as applied to various specific embodiments thereof have been shown, described, and pointed out, it will also be understood that various omissions, substitutions, and changes in the details of the methods that are disclosed, may become apparent and may be made by those skilled in the art without departing from the spirit of the disclosure. For example, it is expressly intended that all combinations of those method steps that perform substantially the same function in substantially the same way to achieve the same results are within the scope of the disclosure. Additionally, one or more of the disclosed method steps may be repeated any number of times. Moreover, it should be recognized that method steps shown and / or described in connection with any disclosed form or embodiment of the disclosure may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice. Further, in at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. Further, various modifications and variations can be made without departing from the spirit or scope of the disclosure.

Claims

WHAT IS CLAIMED IS:

1. A method of treating invasive breast cancer, the method comprising: providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a first biosignature comprising at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1; selecting an adjuvant therapy for a breast cancer treatment based on the analysis of the first biosignature, wherein the adjuvant therapy comprises radiotherapy (RT); and administering the breast cancer treatment to the subject, thereby reducing a risk of breast cancer recurrence.

2. The method of claim 1, wherein the adjuvant therapy further comprises endocrine therapy (ET).

3. The method of claim 2, wherein the adjuvant therapy comprises RT followed by ET.

4. The method of claim 3, wherein the first biosignature comprises GLUT1(-) and Ki67(-), wherein the risk of breast cancer recurrence comprises a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis.

5. The method of claim 3, wherein the first biosignature comprises the following biomarkers: (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+), wherein the risk of breast cancer recurrence comprises a risk of LRR, or a risk of LRR and distal metastasis.

6. The method of claim 5, wherein PR(+) denotes a percentage of PR-expressing cells in a tumor of the breast tissue sample at or above a threshold percentage.

7. The method of claim 6, wherein the threshold percentage is a 50 percentile of percentages of PR-expressing cells in patients in a cohort.

8. The method of claim 5, wherein FOXA1(+) denotes an expression level of FOXA1 at or above a threshold level, optionally wherein the expression level is an H score.

9. The method of claim 8, wherein the threshold level is a 75 percentile of FOXA1 expression levels in patients in a cohort.

10. The method of claim 4, wherein GLUT1(-) denotes an expression level of GLUT1 at or below a threshold level, optionally wherein the expression level is an H score.

11. The method of claim 10, wherein the threshold level is a 25 percentile of GLUT1 expression levels in patients in a cohort.

12. The method of claim 4, wherein Ki67(-) denotes a percentage of Ki67- expressing cells in a tumor of the breast tissue sample at or below a threshold percentage.

13. The method of claim 12, wherein the threshold percentage is a 25 percentile of percentages of Ki67-expressing cells in patients in a cohort.

14. The method of any one of the preceding claims, comprising: analyzing the breast tissue sample for a second biosignature that comprises one or more of the following biomarkers: PD-1, IRF9, ALDHA1, optionally wherein the second biosignature comprises PD-1 and IRF9; and at least: assessing a risk of breast cancer recurrence based on the analysis of the second biosignature; and / or selecting RT for the adjuvant therapy based on the analysis of the second biosignature.

15. The method of claim 14, wherein the second biosignature comprises the biomarker GLUT1.

16. A method of treating invasive breast cancer, the method comprising: providing a breast tissue sample of an invasive, hormone receptor-positive, HER2- negative (HR(+) / HER2(-)) breast cancer; having the breast tissue sample analyzed for a first biosignature comprising at least the biomarkers GLUT1 and Ki67 or at least the biomarkers PR and FOXA1; and receiving a breast cancer treatment based on the analysis of the biosignature, wherein the breast cancer treatment comprises an adjuvant therapy comprising radiotherapy (RT), thereby reducing a risk of breast cancer recurrence.

17. The method of claim 16, wherein the adjuvant therapy further comprises endocrine therapy (ET).

18. The method of claim 17, wherein the adjuvant therapy comprises RT followed by ET.

19. The method of claim 18, wherein the first biosignature comprises GLUT1(-) and Ki67(-), wherein the risk of breast cancer recurrence is a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis.

20. The method of claim 18, wherein the first biosignature comprises: (a) GLUT1(- ) and Ki67(-); or (b) PR(+) and FOXA1(+), wherein the risk of breast cancer recurrence comprises a risk of LRR, or a risk of LRR and distal metastasis.

21. The method of claim 20, wherein PR(+) denotes a percentage of PR-expressing cells in a tumor of the breast tissue sample at or above a threshold percentage.

22. The method of claim 21, wherein the threshold percentage is a 50 percentile of percentages of PR-expressing cells in patients in a cohort.

23. The method of claim 20, wherein FOXA1(+) denotes an expression level of FOXA1 at or above a threshold level, optionally wherein the expression level is an H score.

24. The method of claim 23, wherein the threshold level is a 75 percentile of FOXA1 expression levels in patients in a cohort.

25. The method of claim 19, wherein GLUT1(-) denotes an expression level of GLUT1 at or below a threshold level, optionally wherein the expression level is an H score.

26. The method of claim 25, wherein the threshold level is a 25 percentile of GLUT1 expression levels in patients in a cohort.

27. The method of claim 19, wherein Ki67(-) denotes a percentage of Ki67- expressing cells in a tumor of the breast tissue sample at or below a threshold percentage.

28. The method of claim 27, wherein the threshold percentage is a 25 percentile of percentages of Ki67-expressing cells in patients in a cohort.

29. The method of any one of claims 16-28, comprising: having the breast tissue sample analyzed for a second biosignature comprising one or more of the following biomarkers: PD-1, IRF9, ALDHA1, optionally wherein the second biosignature comprises PD-1 and IRF9, wherein RT is selected for the adjuvant therapy based on the analysis of the second biosignature.

30. A method of treating invasive breast cancer, the method comprising: providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer;analyzing the breast tissue sample for a biosignature comprising the following biomarkers: (a) PD-1 and IRF9; and (b) at least: (i) GLUT1 and Ki67; or (ii) PR and FOXA1; selecting a breast cancer treatment comprising an adjuvant therapy based on the analysis of the biosignature, wherein radiotherapy (RT) is selected for the adjuvant therapy based on the analysis of at least PD-1 and IRF9; and administering the breast cancer treatment to the subject, thereby reducing a risk of breast cancer recurrence.

31. The method of claim 30, wherein RT and endocrine therapy (ET) are selected for the adjuvant therapy based on the analysis of at least: (i) GLUT1 and Ki67; or (ii) PR and FOXA1, optionally wherein RT is followed by ET.

32. The method of claim 31, wherein the biosignature comprises GLUT1(-) and Ki67(-), wherein the risk of breast cancer recurrence is a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis.

33. The method of claim 31, wherein the biosignature comprises: (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+), wherein the risk of breast cancer recurrence comprises a risk of LRR, or a risk of LRR and distal metastasis.

34. The method of claim 33, wherein PR(+) denotes a percentage of PR-expressing cells in a tumor of the breast tissue sample at or above a 50 percentile of percentages of Ki67- expressing cells in patients in a cohort.

35. The method of claim 33, wherein FOXA1(+) denotes an expression level of FOXA1 at or above a 75 percentile of FOXA1 expression levels in patients in a cohort, optionally wherein the expression level is an H score.

36. The method of claim 32, wherein GLUT1(-) denotes an expression level of GLUT1 at or below a 25 percentile of GLUT1 expression levels in patients in a cohort, optionally wherein the expression level is an H score.

37. The method of claim 32, wherein Ki67(-) denotes a percentage of Ki67- expressing cells in a tumor of the breast tissue sample at or below a 25 percentile of percentages of Ki67-expressing cells in patients in a cohort.

38. A method of identifying a subject for breast cancer treatment, comprising:providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67; and identifying the subject for a breast cancer treatment to reduce a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis, based on the analyzed biosignature.

39. The method of claim 38, wherein the analyzed biosignature being GLUT1(-) and Ki67(-) indicates that the subject will benefit from the breast cancer treatment that comprises an adjuvant therapy comprising radiotherapy (RT) and endocrine therapy (ET) to reduce a risk of LRR, or a risk of LRR and distal metastasis, optionally wherein RT is followed by ET.

40. A method of diagnosis, comprising: providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67; based on the analyzed biosignature being GLUT1(-) and Ki67(-), identifying the subject as benefiting from a breast cancer treatment comprising an adjuvant therapy comprising radiotherapy (RT) and endocrine therapy (ET) to reduce a risk of LRR, or a risk of LRR and distal metastasis, optionally wherein the adjuvant therapy comprises RT followed by ET.

41. A method of diagnosis, comprising: providing a breast tissue sample from a subject with invasive, hormone receptor- positive, HER2-negative (HR(+) / HER2(-)) breast cancer; analyzing the breast tissue sample for a biosignature comprising at least the biomarkers GLUT1 and Ki67, or at least the biomarkers PR and FOXA1; based on the analyzed biosignature being: (a) GLUT1(-) and Ki67(-); or (b) PR(+) and FOXA1(+), identifying the subject as benefiting from a breast cancer treatment comprising an adjuvant therapy comprising radiotherapy (RT) and endocrine therapy (ET) to reduce a risk of local regional recurrence (LRR), or a risk of LRR and distal metastasis.

42. The method of claim 41, wherein PR(+) denotes a percentage of PR-expressing cells in a tumor of the breast tissue sample at or above a threshold percentage.

43. The method of claim 42, wherein the threshold percentage is a 50 percentile of percentages of PR-expressing cells in patients in a cohort.

44. The method of claim 41, wherein FOXA1(+) denotes an expression level of FOXA1 at or above a threshold level, optionally wherein the expression level is an H score.

45. The method of claim 44, wherein the threshold level is a 75 percentile of FOXA1 expression levels in patients in a cohort.

46. The method of claim 40, wherein GLUT1(-) denotes an expression level of GLUT1 at or below a threshold level, optionally wherein the expression level is an H score.

47. The method of claim 46, wherein the threshold level is a 25 percentile of GLUT1 expression levels in patients in a cohort.

48. The method of claim 40, wherein Ki67(-) denotes a percentage of Ki67- expressing cells in a tumor of the breast tissue sample at or below a threshold percentage.

49. The method of claim 48, wherein the threshold percentage is a 25 percentile of percentages of Ki67-expressing cells in patients in a cohort.

50. The method of claim 40, comprising administering the breast cancer treatment to the subject.

51. The method of any one of the preceding claims, wherein the subject has T1 / T2N0M0 breast cancer.

52. The method of any one of the preceding claims, wherein the breast cancer treatment comprises lumpectomy or mastectomy.

53. The method of any one of the preceding claims, wherein the breast tissue sample is analyzed by: contacting the breast tissue sample with a detection reagent for each of the biomarkers of the biosignature, optionally wherein the detection reagent comprises an antibody or nucleic acid probe to each of the biomarkers; and detecting binding of the detection agent, to determine the biosignature.

54. A combination of detection reagents for detecting expression of biomarkers comprising at least GLUT1 and Ki67 in a breast tissue sample, for use in the method of any one of the preceding claims.

55. A kit comprising reagents for detecting expression of biomarkers comprising at least GLUT1 and Ki67 in a breast tissue sample from a subject with invasive, hormone receptor-positive, HER2-negative (HR(+) / HER2(-)) breast cancer.

56. The combination of claim 54, or the kit of claim 55, wherein the biomarkers comprise at least PD-1 and IRF9, optionally wherein the biomarkers comprise ALDHA1.

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