Method of treating breast cancer using a breast cancer panel involving CD8, FOXP3, and / or PD-1 markers

Abstract

Aspects of the present disclosure generally relate to methods of diagnosing and treating subjects having cancer, for example breast cancer. Some aspects of the present disclosure relate to methods of predicting whether a tumor will be responsive to standard treatments based on the expression of one or more biomarkers, for example CD8, PD-1 and / or FOXP3.

Description

PRLUD.023WO PATENT METHOD OF TREATING BREAST CANCER USING A BREAST CANCER PANEL INVOLVING CD8, FOXP3, AND / OR PD-1 MARKERS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 596937, filed on November 7, 2023, the content of which is herein expressly incorporated by reference in its entirety. FIELD

[0002] Aspects of the present disclosure generally relate to methods of diagnosing and treating subjects having cancer. More specifically, some aspects of the present disclosure relate to methods of predicting whether a tumor will be responsive to standard of care treatments. BACKGROUND

[0003] Standard of care treatment recommendations for some early-stage cancers rely on clinicopathologic (CP) factors, which, due to the intricate underlying biology of cancer, can have limited utility in assessing the potential benefit of the treatment. Accordingly, there is a need for improved methods of predicting a subject’s response to cancer treatment, and methods of treatment utilizing such predictions. SUMMARY

[0004] Some embodiments disclosed herein relate to methods of treating invasive breast cancer, the methods comprising: providing a breast tissue sample from a subject with invasive hormone receptor-positive breast cancer, wherein the subject has T1 / T2N0M0 breast cancer; analyzing the breast tissue sample for an immune phenotype comprising expression levels of CD8 and PD-1; selecting a breast cancer treatment based on the subject’s immune phenotype; and administering the selected breast cancer treatment to the subject, thereby reducing a risk of ipsilateral breast recurrence (IBR). In some embodiments, the method further comprises determining that the subject’s immune phenotype comprises CD8High and PD- 1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype. In some embodiments, the immune phenotype comprises an expression level of FOXP3. In some embodiments, the method comprises determining that the subject’simmune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype. In some embodiments, the standard of care treatment is in line with a current guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline. In some embodiments the standard of care treatment comprises standard radiotherapy. In some embodiments, the method is performed on a pre-operative tumor. In some embodiments, the method is performed on a post-operative tumor. In some embodiments, one uses a representative control population as an external reference, wherein the expression levels of CD8, PD-1 and / or FOXP3 is compared to a population of stage I / IA breast cancer subjects, and wherein a control population is in line with the study population used in the present examples. In some embodiments, one uses housekeeping gene data as an internal reference. In some embodiments, if the method indicates that a subject will not respond or not respond adequately to standard radiotherapy, then one administers at least one of: a) a more intense level of therapy than that outlined in the NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines, b) the same NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines modalities given concurrently, e.g. RT + chemotherapy, RT+ targeted therapies, c) a radiation boost with higher dose levels or with broader indications than in current NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines d) a mastectomy, or e) concurrent radiochemotherapy.

[0005] Some embodiments disclosed herein relate to methods of identifying a subject for breast cancer treatment, comprising: providing a breast tissue sample from a subject with invasive hormone receptor-positive breast cancer, wherein the subject has T1 / T2N0M0 breast cancer; analyzing the breast tissue sample for an immune phenotype comprising expression levels of CD8, FOXP3, and PD-1; and selecting a breast cancer treatment for the subject based on a residual risk, for ipsilateral breast recurrence (IBR) predicted by the subject’s immune phenotype, were the subject to receive breast conserving surgery (BCS) and standard radiotherapy (RT) or chemotherapy. In some embodiments, the method further comprises predicting the residual risk for IBR based on the subject’s immune phenotype. In some embodiments, determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype. In some embodiments, the method comprisesadministering the selected breast cancer treatment to the subject, thereby reducing a risk of IBR. In some embodiments, the standard of care treatment is in line with a current guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline. In some embodiments the standard of care treatment comprises standard radiotherapy. In some embodiments, the method is performed on a pre-operative tumor. In some embodiments, the method is performed on a post-operative tumor. In some embodiments, one uses a representative control population as an external reference, wherein the expression levels of CD8, PD-1 and / or FOXP3 is compared to a population of stage I / IA breast cancer subjects, and wherein a control population is in line with the study population used in the present examples. In some embodiments, one uses housekeeping gene data as an internal reference. In some embodiments, if the method indicates that a subject will not respond or not respond adequately to standard radiotherapy, then one administers at least one of: a) a more intense level of therapy than that outlined in the NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines, b) the same NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines modalities given concurrently, e.g. RT + chemotherapy, RT+ targeted therapies, c) a radiation boost with higher dose levels or with broader indications than in current NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines d) a mastectomy, or e) concurrent radiochemotherapy.

[0006] Some embodiments disclosed herein relate to methods of identifying a subject for breast cancer treatment, comprising: identifying a subject with invasive hormone receptor-positive breast cancer; determining the immune phenotype of a breast tissue sample from the subject, wherein the immune phenotype comprises an expression level of two or more biomarkers comprising: CD8; and one or both of PD-1 and FOXP3; and identifying the subject for breast cancer treatment that is commensurate with a risk of ipsilateral breast recurrence (IBR) prognosed by the subject’s immune phenotype, and / or with responsiveness to standard radiotherapy (RT) predicted based on the subject’s immune phenotype. In some embodiments, the method further comprises determining that the subject’s immune phenotype comprises CD8High and PD-1Neg; and identifying the subject as benefiting from breast cancer treatment that comprises an alternative to standard radiotherapy, based on the determined immune phenotype. In some embodiments, determining that the subject’s immune phenotype comprises CD8High and FOXP3High; and identifying the subject as being at high risk of IBR, based onthe determined immune phenotype. In some embodiments, the method further comprises determining that the subject’s immune phenotype is CD8High, FOXP3High, and PD-1Neg; and identifying the subject as being at high risk of IBR and predicted to benefit from breast cancer treatment that comprises an alternative to standard radiotherapy, based on the determined immune phenotype. In some embodiments, the standard of care treatment is in line with a current guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline. In some embodiments the standard of care treatment comprises standard radiotherapy. In some embodiments, the method is performed on a pre-operative tumor. In some embodiments, the method is performed on a post- operative tumor. In some embodiments, one uses a representative control population as an external reference, wherein the expression levels of CD8, PD-1 and / or FOXP3 is compared to a population of stage I / IA breast cancer subjects, and wherein a control population is in line with the study population used in the present examples. In some embodiments, one uses housekeeping gene data as an internal reference. In some embodiments, if the method indicates that a subject will not respond or not respond adequately to standard radiotherapy, then one administers at least one of: a) a more intense level of therapy than that outlined in the NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines, b) the same NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines modalities given concurrently, e.g. RT + chemotherapy, RT+ targeted therapies, c) a radiation boost with higher dose levels or with broader indications than in current NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines d) a mastectomy, or e) concurrent radiochemotherapy.

[0007] Some embodiments herein are directed to methods of treating invasive breast cancer, the methods comprising: identifying a subject with invasive hormone receptor- positive breast cancer; determining an immune phenotype of a breast tissue sample from the subject, wherein the immune phenotype comprises the expression level of one or more biomarkers comprising CD8, PD-1, FOXP3, or a combination thereof; selecting a standard of care or intensified treatment based on the subject’s immune phenotype; and administering the selected standard of care or intensified treatment to the subject, thereby treating the invasive hormone receptor-positive breast cancer. In some embodiments, the method further comprises determining that the subject’s immune phenotype comprises CD8High and FOXP3High; andadministering the intensified treatment to the subject based on the determination of the subject’s immune phenotype. In some embodiments, the method further comprises determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD- 1Neg; and administering the intensified treatment to the subject based on the determination of the subject’s immune phenotype. In some embodiments, the standard of care treatment is in line with a current guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline. In some embodiments the standard of care treatment comprises standard radiotherapy. In some embodiments, the method is performed on a pre-operative tumor. In some embodiments, the method is performed on a post- operative tumor. In some embodiments, one uses a representative control population as an external reference, wherein the expression levels of CD8, PD-1 and / or FOXP3 is compared to a population of stage I / IA breast cancer subjects, and wherein a control population is in line with the study population used in the present examples. In some embodiments, one uses housekeeping gene data as an internal reference. In some embodiments, if the method indicates that a subject will not respond or not respond adequately to standard radiotherapy, then one administers at least one of: a) a more intense level of therapy than that outlined in the NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines, b) the same NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines modalities given concurrently, e.g. RT + chemotherapy, RT+ targeted therapies, c) a radiation boost with higher dose levels or with broader indications than in current NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines d) a mastectomy, or e) concurrent radiochemotherapy.

[0008] Some embodiments disclosed herein are directed to methods of identifying a subject with a high risk of ipsilateral breast recurrence (IBR), comprising: identifying a subject with invasive hormone receptor-positive breast cancer; determining an expression level of at least CD8 and FOXP3 in a breast tissue sample from the subject, and identifying the subject as being at high risk of IBR if the breast tissue sample from the subject is CD8High and FOXP3High.

[0009] Some embodiments disclosed herein are directed to methods of treating breast cancer, comprising: a) obtaining a breast tissue sample from a hormone receptor-positive breast cancer subject; b) determining an expression level of at least CD8 and PD-1 in the breasttissue sample; c) determining that the expression level of CD8 is above a first threshold and that the expression level of PD-1 is below a second threshold; and d) providing intensified treatment as intensified radiotherapy, intensified systemic therapy or mastectomy to the subject. In some embodiments, the first threshold is CD8 levels that are in the highest quartile of CD8 levels. In some embodiments, the second threshold is FOXP3 levels that are in the highest quartile of FOXP3 levels. In some embodiments, the third threshold is PD-1 levels that are in the lowest quartile of PD-1 levels.

[0010] Some embodiments disclosed herein are directed to methods of diagnosis, comprising: a) obtaining a breast tissue sample from a hormone receptor-positive breast cancer subject; b) analyzing the breast tissue sample for expression levels of at least CD8, and one or both of PD-1 and FOXP3; and c) determining that the expression level of CD8 is above a first threshold, that the expression level of FOXP3 is above a second threshold, and / or that the expression level of PD-1 is below a third threshold, thereby determining that the subject belongs to a group that would benefit from intensified radiotherapy. In some embodiments, the first threshold is CD8 levels that are in the highest quartile of CD8 levels. In some embodiments, the second threshold is FOXP3 levels that are in the highest quartile of FOXP3 levels. In some embodiments, the third threshold is PD-1 levels that are in the lowest quartile of PD-1 levels.

[0011] Some embodiments disclosed herein are directed to methods of treating a subject for breast cancer, comprising: identifying an incremental risk to a subject of ipsilateral breast recurrence (IBR) based on a level of CD8, PD-1 and FOXP3 in a breast tissue sample of an invasive hormone receptor-positive breast cancer in the subject; and administering an intensified breast cancer treatment to the subject based upon the incremental risk, wherein a higher incremental risk will increase a likelihood of an intensified breast cancer treatment that is at least more than what would be recommended by a guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline.

[0012] In some embodiments, the standard of care treatment includes breast conserving surgery followed by adjuvant therapy, such as standard radiation therapy. In some embodiments, the intensified treatment includes intensified, elevated, or aggressive radiation therapy. In some embodiments, the intensified systemic therapy comprises intensified systemic chemotherapy. In some embodiments, the intensified systemic chemotherapycomprises a platinum derivative with a taxane. In some embodiments, the intensified treatment comprises intensified radiotherapy. In some embodiments, the intensified radiotherapy is whole breast external radiotherapy, partial breast radiotherapy or brachytherapy or a combination thereof. In some embodiments, the intensified radiotherapy comprises a biologically effective dose (BED) of 73 Gy or more with a tumor alpha / beta ratio of 5, or a BED of 78 Gy or more with a tumor alpha / beta ratio of 4, or a BED of 87 Gy or more with a tumor alpha / beta ratio of 3, or a BED of 104 Gy or more with a tumor alpha / beta ratio of 2. In some embodiments, BED is defined as a measure of a true biological dose delivered by a combination of dose per fraction (d) and number of fractions (n) to a tissue characterized by a specific radiosensitivity (alpha / beta ratio): BED=n×d (1+d / (α / β)). BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In addition to the features described below, additional features and variations will be readily apparent from the following descriptions of the drawings and exemplary embodiments. It is to be understood that these drawings depict typical embodiments and are not intended to be limiting in scope.

[0014] FIG. 1 is a forest plot depicting some embodiments of a hazard ratio of subjects treated with breast conserving surgery with or without radiotherapy.

[0015] FIG. 2 is an image showing some embodiments of a calculation of ipsilateral breast cancer recurrence (IBR) risk in subjects with T1 / T2N0M0 breast cancer who underwent BCS with negative margins.

[0016] FIG.3 is an image showing a calculation of IBR risk in subjects with high levels of CD8 according to some embodiments disclosed herein.

[0017] FIG.4 is an image showing a calculation of IBR risk in subjects with high levels of CD8 according to some embodiments disclosed herein.

[0018] FIG.5 is an image showing a calculation of IBR risk in subjects with high levels of CD8 according to some embodiments disclosed herein.

[0019] FIG.6 is an image showing a calculation of IBR risk in subjects with high levels of CD8 according to some embodiments disclosed herein.

[0020] FIG. 7 is a flow chart depicting some non-limiting embodiments of a method of treating invasive breast cancer.

[0021] FIG. 8 is a flow chart depicting some non-limiting embodiments of a method of identifying a subject for breast cancer treatment .

[0022] FIG. 9 is a flow chart depicting some non-limiting embodiments of a method of treating invasive breast cancer.

[0023] FIG. 10 is a flow chart depicting some non-limiting embodiments of a method of identifying a subject for breast cancer treatment.

[0024] FIG. 11 is a flow chart depicting some non-limiting embodiments of a method of identifying a subject with a high risk of ipsilateral breast cancer recurrence (IBR).

[0025] FIG. 12 is a flow chart depicting some non-limiting embodiments of a method of treating breast cancer.

[0026] FIG. 13 is a flow chart depicting some non-limiting embodiments of a method of diagnosis.

[0027] FIG. 14 is a flow chart depicting some non-limiting embodiments of a method of treating a subject. DETAILED DESCRIPTION

[0028] Radiation therapy (RT) is a standard treatment for early-stage hormone receptor-positive invasive breast cancer (BC) following breast-conserving surgery (BCS). Current RT recommendations rely on clinicopathologic (CP) factors, including tumor size, margin status nodal status, and histological grade. While CP factors may provide valuable prognostic insights, their utility to assess RT benefit is limited. Ongoing studies are exploring the potential of integrating clinical and biological factors to effectively identify a subject’s recurrence risk profile with and without RT. However, the underlying biology of invasive breast cancer is intricate, as the tumor microenvironment plays a pivotal role in shaping the disease's biology and ultimately impacting treatment response. Accordingly, there is a need for improved methods of predicting a subject’s response to and treating cancer and methods of treatment utilizing such predictions.

[0029] Some embodiments disclosed herein are directed to methods of characterizing, diagnosing, and / or treating breast cancer. By identifying subjects who are more or less responsive to various types of therapy, including RT, various embodiments provided herein allow one to identify subjects who will be more responsive to a particular therapeutic approach to cancer (e.g., RT), thereby pairing a subject with the appropriate type of breastcancer therapy (e.g., low, standard, or intensified radiotherapy options) for treating various forms of breast cancer. Also provided are compositions or combinations and kits of detection reagents configured to detect markers of the present disclosure, that may find use in identifying subjects who will be more responsive to a particular therapeutic approach to cancer. Methods, compositions and kits of the present disclosure can be used to tailor treatment options for a subject with cancer, e.g., breast cancer, relative to the standard of care treatment, and can thereby reduce overtreatment or undertreatment from administration of the standard of care treatment. The appropriate treatment for the subject can be one that is expected to provide the desired reduction in risk of recurrence while reducing the risk of overtreatment or undertreatment. Terms

[0030] The term “and / or” shall be taken to provide explicit support for both meanings or for either meaning. “About” as used herein with reference to a value or an amount includes values or amounts plus or minus 10% of the listed value or amount, unless indicated otherwise.

[0031] 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.

[0032] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitablemethods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0033] There are a variety of markers for the identification of tumors in subjects. In addition, there are various markers that can be used for the prediction of neoplastic progression. For example, U.S. Pat. Pub. Nos. 2010 / 0003189, 2012 / 0003639, and 20170350895 disclose a variety of markers that when examined in various combinations can predict the likelihood that a subject will have ductal carcinoma in situ (DCIS) and / or invasive breast cancer.

[0034] Programmed death-1 (PD-1) is a so called “immune checkpoint” of the immune system. PD-1 inhibitory antibodies, such as KEYTRUDA (Pembrolizumab), are a treatment for many cancer forms. “Programmed death 1”, “PD-1”, “PD-1”, “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.

[0035] Programmed death ligand-1 (PD-L1) is a ligand of PD-1. When PD-L1 interacts with PD-1, T-lymphocyte proliferation and killing activity are negatively regulated. In the context of cancer, PD-1 / PD-L1 interaction leads to the inhibition of tumor cell killing by the immune system, thus leading to increased tumor cell resistance to pro-apoptotic signals and immune escape of tumor cells.

[0036] CD8 (cluster of differentiation 8) is a transmembrane glycoprotein that is present on cytotoxic / suppressor T-lymphocytes, a subset of medullary thymocytes, and a subpopulation of NK-cells. CD8 also serves as a co-receptor for the T-cell receptor (TCR). Along with the TCR, the CD8 co-receptor plays a role in T cell signaling and aids with cytotoxic T cell-antigen interactions. CD8+ T cells (often called cytotoxic T lymphocytes, or CTLs) are important for immune defense against intracellular pathogens, including viruses and bacteria, and for tumor surveillance. For example, when a CD8+ T cell recognizes its antigenand becomes activated, it secrets of cytokines, primarily TNF-α and IFN-γ, which can have anti-tumor and anti-viral / anti-microbial effects.

[0037] FOXP3 belongs to the forkhead / winged-helix family of transcriptional regulators and shares general structural features with other FoxP family members. FOXP3 functions as a master of transcription for the development of regulatory T-cells (Treg cells) both in humans and in mice.

[0038] As used herein, the term “array” denotes an arrangement of molecules, such as biological macromolecules (such as peptides or nucleic acid molecules) or biological samples (such as tissue sections), in addressable locations on or in a substrate. A “microarray” is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis. Arrays are sometimes called chips or biochips.

[0039] Utilizing an array of molecules makes it possible to carry out a very large number of analyses on a sample at one time. In some embodiments disclosed herein, arrays of one or more molecules (such as an oligonucleotide probe) will occur on the array a plurality of times (such as twice), for instance to provide internal controls. The number of addressable locations on the array can vary, for example from at least 1, to at least 2, to at least 5, to at least 10, at least 20, at least 30, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 500, least 550, at least 600, at least 800, at least 1000, at least 10,000, or more. In particular examples, an array includes nucleic acid molecules, such as oligonucleotide sequences that are at least 15 nucleotides in length, such as 15-40 nucleotides in length. In particular examples, an array includes oligonucleotide probes or primers which can be used to detect the markers noted herein, such as PD-1, CD8 and FOXP3.

[0040] In some embodiments, within an array, each arrayed sample can be addressable, in that its location can be reliably and consistently determined within at least two dimensions of the array. Addressable arrays can be computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (such as hybridization or binding data, including for instance signal intensity). In some examples of computer readable formats, the individual features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer.

[0041] As used herein, the term “gene” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and means nucleic acid in the genome of a subject capable of being expressed to produce a mRNA in addition to intervening intronic sequences and in addition to regulatory regions that control the expression of the gene, e.g., a promoter or fragment thereof.

[0042] 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. A diagnostic assay described herein is also useful for assessing the remission of a subject, or monitoring disease recurrence, or tumor recurrence, such as following surgery, radiation therapy, adjuvant therapy or chemotherapy, or determining the appearance of metastases of a primary tumor.

[0043] In some embodiments, a prognostic assay described herein is useful for assessing likelihood of treatment benefit, disease recurrence, tumor recurrence, or metastasis of a primary tumor, such as following surgery, radiation therapy, adjuvant therapy or chemotherapy. All such uses of the assays described herein are encompassed by the present disclosure. In some embodiments, the test can be used to predict if the subject will have an occurrence.

[0044] The term “cancer” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and 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.

[0045] The term “breast tumor” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and 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).

[0046] As used herein, “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. 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.

[0047] Breast cancers range from stage 0 (carcinoma in situ) to stage I (1) through IV (4). Generally, a lower number indicates the cancer is less spread whereas a higher number, such as stage IV generally indicates that a cancer has spread more. Within a given stage, a letter earlier in the alphabet generally indicates a lower stage. Although each subject’s cancer experience is unique, cancers with similar stages tend to have a similar outlook and are often treated in much the same way. In some embodiments disclosed herein, the breast cancer is staged using the American Joint Committee on Cancer (AJCC) TNM system, which is hereby incorporated by reference in its entirety. The AJCC system comprises both clinical and pathologic staging systems for breast cancer.

[0048] The pathologic stage (also called the surgical stage) is determined by examining tissue removed during an operation. Where surgery is not possible right away or at all, the cancer may be given a clinical stage based on the results of a physical exam, biopsy, and imaging tests. The clinical stage may be used to help plan treatment. Both the pathological and clinical staging systems analyze the extent (size) of the tumor (T); the extent to which the cancer has spread to nearby lymph nodes (N); the extent to which the cancer has spread or metastasized to distant sites (M); estrogen receptor (ER) status; progesterone receptor (PR) status; and HER2 status; the grade of the cancer (G), i.e., a determination of how much the cancer cells look like normal cells. Numbers or letters after T, N, and M provide more details about each of these factors. Higher numbers generally indicate that the cancer is more advanced.

[0049] In breast cancer staging, a “T” followed by a number from 0 to 4 describes the main (primary) tumor's size and indicates whether the tumor has spread to the skin or to the chest wall under the breast. Higher T numbers mean a larger tumor and / or wider spread to tissues near the breast. “TX” indicates that the primary tumor cannot be assessed. “T0”indicates that there is no evidence of a primary tumor. “Tis” indicates a carcinoma in situ (DCIS, or Paget disease of the breast with no associated tumor mass). “T1,” which includes T1a, T1b, and T1c indicates that the tumor is 2 cm (3 / 4 of an inch) or less across. “T2” indicates that the tumor is more than 2 cm but not more than 5 cm (2 inches) across. “T3” indicates that the tumor is more than 5 cm across. “T4,” which includes T4a, T4b, T4c, and T4d, indicates that a tumor of any size is growing into the chest wall or skin. This includes inflammatory breast cancer.

[0050] In breast cancer staging, a “N” followed by a number from 0 to 3 indicates whether the cancer has spread to lymph nodes near the breast and, if so, how many lymph nodes are involved. Generally, a deposit of cancer cells must contain at least 200 cells or be at least 0.2 mm across (less than 1 / 100 of an inch) for it to change the N stage. Where a cancer has spread into an area that is smaller than 0.2 mm (or fewer than 200 cells), the N stage will not change. Where the area of cancer spread is between 0.2 mm (or 200 cells) and 2 mm, it is called a micrometastasis. Micrometastases are counted only when there aren't any larger areas of cancer spread. Areas of cancer spread larger than 2 mm alter the N stage. These larger areas are may be referred to as macrometastases or metastases. “NX” indicates nearby lymph nodes cannot be assessed. “N0” indicates that the cancer has not spread to nearby lymph nodes. “N0(i+)” indicates that the area of cancer spread contains fewer than 200 cells and is smaller than 0.2 mm. The abbreviation "i+" indicates that a small number of cancer cells (called isolated tumor cells) were detected using immunohistochemistry. “N0(mol+)” indicates that the cancer cells cannot be seen in underarm lymph nodes, but traces of cancer cells were detected using RT-PCR. “N1” indicates that the cancer has spread to 1 to 3 axillary (underarm) lymph node(s), and / or that the cancer was detected in internal mammary lymph nodes on sentinel lymph node biopsy. “N1mi” indicates micrometastases (tiny areas of cancer spread) in the lymph nodes under the arm. The areas of cancer spread in the lymph nodes are at least 0.2mm across, but not larger than 2mm. “N1a” indicates that the cancer has spread to 1 to 3 lymph nodes under the arm with at least one area of cancer spread greater than 2 mm across. “N1b” indicates that the cancer has spread to internal mammary lymph nodes on the same side as the cancer, but this spread was only detected on sentinel lymph node biopsy, meaning that the cancer spread did not cause the lymph nodes to become enlarged. “N1c” indicates that both N1a and N1b apply. “N2” indicates that the cancer has spread to 4 to 9 lymph nodes under thearm, or cancer has enlarged the internal mammary lymph nodes. “N2a” indicates that the cancer has spread to 4 to 9 lymph nodes under the arm, with at least one area of cancer spread being larger than 2 mm. “N2b” indicates that the cancer has spread to one or more internal mammary lymph nodes, causing the lymph node(s) to become enlarged. “N3a” indicates that either the cancer has spread to 10 or more axillary lymph nodes, with at least one area of cancer spread greater than 2 mm, or that the cancer has spread to the lymph nodes under the collarbone (infraclavicular nodes), with at least one area of cancer spread greater than 2 mm. “N3b” indicates that either the cancer was detected in at least one axillary lymph node (with at least one area of cancer spread greater than 2 mm) and has enlarged the internal mammary lymph nodes, or that the cancer has spread to 4 or more axillary lymph nodes (with at least one area of cancer spread greater than 2 mm), and to the internal mammary lymph nodes on sentinel lymph node biopsy. “N3c” indicates that the cancer has spread to the lymph nodes above the collarbone (supraclavicular nodes) on the same side of the cancer with at least one area of cancer spread greater than 2 mm.

[0051] A “M” followed by a 0 or 1 indicates whether the cancer has spread to distant organs, for example, to the lungs, liver, or bones. “M0” indicates that no distant spread was detected on x-rays (or other imaging tests) or by physical exam. “cM0(i+)” indicates that small numbers of cancer cells were detected in the blood or bone marrow, or that areas of cancer spread no larger than 0.2 mm were detected in lymph nodes away from the underarm, collarbone, or internal mammary areas. “M1” indicates that the cancer has spread to distant organs, most often to the bones, lungs, brain, or liver, as seen on imaging tests or by physical exam, and / or a biopsy of one of these areas proves cancer has spread and is larger than 0.2mm.

[0052] 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.

[0053] The terms “treatment” and “therapy” as provided herein are used interchangeably and do not require the complete or 100% curing of the subject. Instead, the terms encompass the broader concept of 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.

[0054] “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.

[0055] 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.

[0056] “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.

[0057] “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] 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 subject or a non-tumor tissue sample obtained from a subject 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 subjects 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).

[0059] 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 unitchange 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 subject 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 subject outcome, where the covariate level is a marker expression level.

[0060] As used herein, the term “Alpha / beta (α / β) ratio” refers to a measure of intrinsic radiosensitivity of a specific tissue, measured in Gy.

[0061] 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).

[0062] 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.

[0063] 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 immunosorbentassay (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. 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.

[0064] 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.

[0065] “Differential or alteration in expression” denotes a difference or change, such as an increase or decrease, in the amount of RNA or protein. In some examples, the difference is relative to a control or reference value or range of values, such as an amount of gene expression that is expected in a subject who does not have an invasive breast cancer or in non-tumor tissue from a subject with a breast tumor. Detecting differential expression can include measuring a change in gene expression.

[0066] 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 caninclude controls on transcription, translation, RNA transport and processing, protein transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization.

[0067] 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.

[0068] Controls or standards for comparison to a sample, for the determination of differential expression, include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a subject who does not have invasive breast cancer in the 10 years following the event, as well as laboratory values (e.g., range of values), even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory. Laboratory standards and values can be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values. In some embodiments, the controls can be standardized levels set by one or more housekeeping genes.

[0069] As will be appreciated by one of skill in the art, any of the above controls or standards can be provided for any of the methods (such as treatment, analysis, or prognosis) provided herein, and for any of the compositions or methods. These can be positive or negative controls or standards (showing, for example, what a high level or normal level of expression or presence of the molecule is). The controls can be matched for the relevant molecule type as well (e.g., RNA, protein). In some embodiments, the control and / or standard can be for PD-1, CD8 and / or FOXP3.

[0070] The phrase “gene expression profile” (or signature) denotes a differential or altered gene expression that can be detected by changes in the detectable amount of gene expression (such as cDNA, mRNA, protein). A distinct or identifiable pattern of gene expression, for instance a pattern of high and low expression of a defined set of genes or gene- indicative nucleic acids such as ESTs. In some examples, as few as one gene provides a profile, but more genes can be used in a profile, for example, at least 2, 3, 4, 5, 6, or 7 markers (e.g., genes) can be employed to provide a prediction as to the effectiveness of a particular therapy. Gene expression profiles can include relative as well as absolute expression levels of specificgenes, and can be viewed in the context of a test sample compared to a baseline or control sample profile (such as a sample from the same tissue type from a subject who does not have a tumor). In some embodiments, a gene expression profile in a subject is read on an array (such as a nucleic acid). For example, a gene expression profile can be performed using a commercially available array such as Human Genome GeneChipTMarrays from AffymetrixTM(Santa Clara, Calif.). In some embodiments, any two or more of the markers indicated herein (including PD-1, CD8, FOXP3, and other markers or controls) can be employed as a profile or part of a profile analysis.

[0071] The term “hybridization” means to form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule, for example. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the sodium concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11).

[0072] The term “isolated” as used in an “isolated” biological component (such as a nucleic acid molecule, protein, or cell) is one that has been substantially separated or purified away from other biological components in the cell of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid molecules and proteins that have been “isolated” include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins. In some embodiments, an isolated cell is an invasive breast cancer cell that is substantially separated from other breast cell types, such as non-tumor breast cells.

[0073] The term “label” or “probe” denotes an agent 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 (including PD-1, CD8 and / or FOXP3)), thereby permittingdetection 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 PD-1, CD8 and / or FOXP3 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 PD-1, CD8 and / or FOXP3 to allow for detecting the presence of the marker in a subject or a sample from the subject.

[0074] The term “mammal” includes both human and non-human mammals. Examples of mammals include, but are not limited to: humans, pigs, cows, goats, cats, dogs, rabbits, rats, and mice.

[0075] A nucleic acid array is an arrangement of nucleic acids (such as DNA or RNA) in assigned locations on a matrix, such as that found in cDNA arrays, or oligonucleotide arrays.

[0076] “Nucleic acid molecules representing genes” refers to any nucleic acid, for example DNA (intron or exon or both), cDNA, or RNA (such as mRNA), of any length suitable for use as a probe or other indicator molecule, and that is informative about the corresponding gene.

[0077] “Polymerase chain reaction” (PCR) is an in vitro amplification technique that increases the number of copies of a nucleic acid molecule (for example, a nucleic acid molecule in a sample or specimen), such as amplification of a nucleic acid molecule encoding at least a portion of PD-1, CD8 or FOXP3. The product of a PCR can be characterized by standard techniques known in the art, such as electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and / or nucleic acid sequencing. In some examples, PCR utilizes primers, for example, DNA oligonucleotides 10-100 nucleotides in length, such as about 15, 20, 25, 30 or 50 nucleotides or more in length (such as primers that can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, such as a geneencoding PD-1, CD8 or FOXP3). Primers can be selected that include, include at least, or include about 15, 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of a marker provided herein. Methods for preparing and using nucleic acid primers are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990).

[0078] 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 the invasive 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.

[0079] The phrase “specific binding agent” denotes an agent that binds substantially or preferentially only to a defined target such as a protein, enzyme, polysaccharide, oligonucleotide, DNA, RNA, recombinant vector or a small molecule. In an example, a “specific binding agent” is capable of binding to at least one of the disclosed markers (such as PD-1, CD8 and / or FOXP3). In some embodiments, the specific binding agent is capable of binding to a downstream factor regulated by at least one of the disclosed markers (such as PD-1, CD8 and / or FOXP3). Thus, a nucleic acid-specific binding agent binds substantially only to the defined nucleic acid, such as RNA, or to a specific region within the nucleic acid. For example, a “specific binding agent” includes an antisense compound (such as an antisense oligonucleotide, siRNA, miRNA, shRNA or ribozyme) that binds substantially to a specified RNA.

[0080] The term “radiation therapy” denotes a therapy that involves or includes some form of radiation in an amount that is therapeutic to the subject, e.g., the subject having cancer.

[0081] “Standard of care” or “standard” as used herein, with reference to a therapy or treatment, have their 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 subject under a guideline such as that provided by NCCN, ESMO, ASTRO, Clinical Practice Recommendations Australia, or NICE guideline, and optionally, any one or more of the respective guidelines as of November of 2023. In some embodiments, a therapy recommended under standard of care for a subject does not take into account guidance provided by analysis of markers as disclosed herein.

[0082] 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, ASTRO, Clinical Practice Recommendations Australia, or NICE guideline, and optionally, any one or more of the respective guidelines as of November of 2023. In some embodiments, the standard of care is any one of those provided in Tables 1 and 2 below. Table 1. Radiotherapy Guidelines Guideline N- Surgery Volume Fractionation Boost (tumor bed) status on on on on if if^ N+= lymph node involvement ^ * APBI if low risk nd , e g ,tumor. However, late toxicity effects beyond 10 years are not currently defined. The optimal fractionation for the delivery of a boost is not known for this regimen. es ar is 50 n- sk I) BI ed al nd or th ly be le st on BI nd II, ad or e)• have a low absolute risk of local recurrence (defined as women aged 50 and over with tumours that are 3 cm or less, N0, ER-positive, HER2-negative and grade 1 to 2) and • have been advised to have adjuvant endocrine therapy for a minimum of 5 years

[2018] he th th .7, nd ve or er al he ve st ve st st le, orUltra-hypofractionation (26 Gy in five fractions) for chest wall irradiation without breast reconstruction can be offered as standard of care or within a randomised controlled trial or prospective registration cohort of or er, er st ry er ed or e. nd n. lyTable 2. ESTRO Radiotherapy Guidelines (2022) Panel: Final consensus statements st s, se, 1) astb  Moderate hypofractionation can be offered for chest wall irradiation regardless of time and type of breast reconstruction c Ultra-hypofractionation (26 Gy in five fractions) for chest wall irradiation without breast ial st on til ur ur ), ), nd in ed

[0083] 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 targeted and non-targeted, genomic therapies, antibody therapeutics, including, HER2 antibodies, including Trastuzumab. In some embodiments, in the present application, “non-radiation therapy” is denoted as “other therapy”, unless indicated otherwise. 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.

[0084] “Undertreatment” denotes an effect of a treatment for a disease that is not sufficiently or not significantly different from the effect of a relevant control treatment, wherethe subject’s prognosis with the control treatment is unfavorable (e.g., there is no significant relief from the disease or symptoms thereof). “Overtreatment” denotes an effect of a treatment for a disease that is not sufficiently or not significantly different from the effect of a relevant control treatment, where the subject’s prognosis with the control treatment is favorable (e.g., there is significant relief from the disease or symptoms thereof).

[0085] The term “Local recurrence” denotes that a recurrence is in the operated breast.

[0086] The term “Regional recurrence” denotes that a recurrence is in regional lymph nodes (axillary, supraclavicular, infraclavicular, intrapectoral or internal mammary lymph nodes).

[0087] 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.

[0088] “Benefit” as used herein includes a recognizable improvement in the course of disease over time. In some embodiments, a benefit includes a clinically significant improvement in one or more aspects of the disease. In some embodiments, a benefit includes a reduction in the risk of recurrence, e.g., risk of ipsilateral breast tumor recurrence after surgical removal of the primary tumor. In some embodiments, reduced risk of recurrence includes a delay in recurrence that is longer than that which would have been expected had the subject not received the benefit. In some embodiments, a benefit includes a statistically significant reduction in the risk of recurrence, e.g., risk of ipsilateral breast tumor recurrence after surgical removal of the primary tumor. In some embodiments, a benefit includes, without limitation, a delay in the onset of one or more symptoms, extension of the life and / or quality of life of the subject, or reduction in the severity of one or more symptoms. In some embodiments, a benefit includes a reduction in the level of risk of recurrence that is high enough to warrant the potential side effects from a treatment aimed at reducing the risk of recurrence.

[0089] In some embodiments, the methods provided herein are not applied to distant metastasis. In some embodiments, the methods provided herein are applied to local, regional, and / or local and regional recurrences.

[0090] Predictive vs prognostic effects. In some embodiments herein, the results are to provide a prognostic use. In some embodiments provided herein, the results provide a predictive use (e.g., which therapy will be most useful). In some embodiments, it is useful to separate a predictive from a prognostic effect. This distinction is known in the art of statistics. A prognostic biomarker provides information of a given subject’s outcome regardless of therapy and will only provide very crude estimates of absolute benefits from therapy based on the baseline risk of a disease event. A predictive biomarker on the other hand provides information of a given subject’s benefit from therapy which can be translated to a relative benefit. When combined with an assessment of a subject’s prognosis, the predictive biomarker can provide accurate information regarding a subject’s absolute benefit. Predictive effects can be tested through interaction tests as known in the art of statistics and determined significant if a p value below a threshold level is obtained. Prognostic effects are not tested through interaction tests. Some embodiments disclosed herein relate to biomarkers which are predictive for radiotherapy benefit. In some embodiments, predictive effects are analyzed by comparing the prognosis of a subject group characterized by a combination of one or more biomarkers (e.g., CD8High / FOXP3High / PD-1Neg) that is not treated with a given therapy (e.g., radiotherapy), or that is treated with a non-standard or intensified therapy, with a subject group with the same combination of the one or more biomarkers (e.g., CD8High / FOXP3High / PD- 1Neg) that is treated by the therapy (e.g., radiotherapy). In some embodiments, the radiotherapy predictive effect of the combined tested biomarkers disclosed herein provides information on how to use these biomarkers to optimize the individualization of radiotherapy in breast cancer (e.g., recommend or provide an appropriate therapy to achieve a desired level of risk of recurrence while avoiding over- and / or undertreatment).

[0091] The clinical utility of such predictive biomarkers for therapy decisions (in some embodiments for radiotherapy) is better than for prognostic biomarkers. A subject with a good prognosis who is determined (or predicted based on the predictive biomarkers) to derive a small relative benefit from radiotherapy, e.g., standard radiotherapy, can be omitted radiotherapy, or can be recommended or provided de-intensified radiotherapy relative to standard of care. On the other hand, a subject with a poor prognosis who is determined (or predicted based on the predictive biomarkers) to derive a small benefit from radiotherapy, e.g., standard radiotherapy, can be recommended or can be provided intensified therapy, e.g.,intensified relative to standard of care. Further, a subject with a poor prognosis who is determined to benefit from radiotherapy can be recommended standard radiotherapy. The biomarkers presented in this application provide both prognostic and predictive information which indicates clinical utility for individualizing radiotherapy. Non-limiting Embodiments

[0092] With reference to FIG. 7, some embodiments disclosed herein relate to a method 700 of treating breast cancer. In some embodiments, the breast cancer is invasive breast cancer. In some embodiments, the breast cancer is early-stage breast cancer (e.g., early-stage invasive breast cancer). In some embodiments, the breast cancer is T1 to T2, node negative, nonmetastatic (T1 / T2N0M0) breast cancer. The method can include providing 710 a breast tissue sample from a subject. In some embodiments, the tissue is from a subject with an invasive hormone receptor-positive breast cancer. In some embodiments, the tissue is from a T1 / T2N0M0 breast cancer. The tissue can be analyzed for an immune phenotype. In some embodiments, the immune phenotype is determined by determining the expression levels of one or more biomarkers. In some embodiments, the one or more biomarkers comprise any one, two or all three of PD-1, CD8 and FOXP3. The method can include analyzing 720 the breast tissue sample for an immune phenotype comprising expression levels of, for example, CD8 and PD-1. In some embodiments, the one or more biomarkers comprise one or more of PD-1, CD8 and FOXP3. In some embodiments, the one or more biomarkers includes PD-1, CD8 and FOXP3. In some embodiments, expression of CD8 (e.g., in the immune phenotype) comprises expression of CD8 in T cells, and / or expression of FOXP3 (e.g., in the immune phenotype) comprises expression of FOXP3 in regulatory T cells (Tregs), and / or expression of PD-1 in lymphocytes. A breast cancer treatment is then selected 730, based on the subject’s immune phenotype, and optionally administered 740 to the subject.

[0093] The method can include selecting 730 a breast cancer treatment based on the subject’s immune phenotype. In some embodiments, selecting a breast cancer treatment includes selecting the intensity of breast cancer treatment that takes into account the subject’s risk of ipsilateral breast recurrence (IBR) prognosed by the subject’s immune phenotype (e.g., based on CD8 and PD-1, or based on CD8 and FOXP3), as described herein. In some embodiments, the subject’s risk of IBR is independent of whether or not the subject receives adjuvant radiation therapy after BCS. In some embodiments, the subject’s risk of IBR isindependent of whether or not the subject receives adjuvant chemotherapy after BCS. In some embodiments, selecting a breast cancer treatment includes selecting the intensity and / or type of breast cancer treatment that takes into account the subject’s risk of ipsilateral breast recurrence (IBR) after receiving standard of care treatment (e.g., BCS with adjuvant therapy, such as adjuvant radiation therapy or adjuvant chemotherapy) that is predicted by the subject’s immune phenotype (e.g., based on CD8, PD-1, and FOXP3), as described herein. In some embodiments, selecting a breast cancer treatment includes selecting the intensity and / or type of breast cancer treatment that takes into account the subject’s responsiveness to standard of care treatment (e.g., BCS with adjuvant therapy, such as adjuvant radiation therapy or adjuvant chemotherapy) predicted by the subject’s immune phenotype (e.g., based on CD8, PD-1, and FOXP3), as described herein.

[0094] The method can further include administering 740 the selected breast cancer treatment to the subject, thereby reducing a risk of ipsilateral breast recurrence (IBR). In some embodiments, the selected breast cancer treatment has an intensity relative to the intensity of a standard of care treatment (without the present methods) that matches the desired reduction in risk of IBR given the prognosis for the subject. In some embodiments, the selected breast cancer treatment is an intensified breast cancer treatment (e.g., intensified relative to a standard of care treatment) when the reduction in risk of IBR with the standard of care treatment predicted for the subject based on the subject’s immune phenotype is less than the desired reduction (e.g., the predicted reduction in risk is less than the reduction that is predicted for a patient not having the subject’s immune phenotype).

[0095] In some embodiments, the method comprises determining that the subject’s immune phenotype comprises CD8Highand PD-1Neg; and selecting an intensified treatment for the breast cancer (e.g., intensified relative to a standard of care treatment the subject would have received without the present methods) based on the subject’s immune phenotype. In some embodiments, the method comprises determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype. In some embodiments, the treatment is or includes a radiotherapy. In some embodiments, the treatment is or includes an intensified radiotherapy.

[0096] As used herein, “hormone receptor-positive” has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and denotes the breast cancer is characterized by expression of estrogen receptor (ER) and / or progesterone receptor (PR). In any method herein, in some embodiments, the subject has invasive hormone receptor-positive breast cancer. In some embodiments, the subject has early- stage breast cancer (e.g., early-stage invasive breast cancer). In some embodiments, the subject has T1 to T2, node negative, nonmetastatic (T1 / T2N0M0) breast cancer. In some embodiments, the subject has hormone receptor-positive T1 / T2N0M0 breast cancer. In some embodiments, the subject has not received treatment for the breast cancer. In some embodiments, the subject has received treatment for the breast cancer. In some embodiments, the subject has received breast conserving surgery (BCS). In some embodiments, the subject has received chemotherapy for the breast cancer. In some embodiments, the subject has been treated with chemotherapy for the breast cancer. In some embodiments, the subject has received BCS with adjuvant therapy (e.g., adjuvant radiation therapy or adjuvant chemotherapy).

[0097] With reference to FIG.8, some embodiments herein relate to a method 800 of identifying a subject for breast cancer treatment. The method can include providing 810 a breast tissue sample from a subject with invasive hormone receptor-positive breast cancer. In some embodiments, the subject has T1 / T2N0M0 breast cancer. The method can comprise analyzing 820 the breast tissue sample for an immune phenotype comprising expression levels of CD8, FOXP3, and PD-1; and selecting 830 a breast cancer treatment for the subject based on a residual risk, or lack thereof, for ipsilateral breast recurrence (IBR) predicted by the subject’s immune phenotype, were the subject to receive breast conserving surgery (BCS) and standard radiotherapy (RT) or chemotherapy. In some embodiments, expression of CD8 (e.g., in the immune phenotype) comprises expression of CD8 in T cells, and / or expression of FOXP3 (e.g., in the immune phenotype) comprises expression of FOXP3 in regulatory T cells (Tregs), and / or expression of PD-1 in lymphocytes.

[0098] In some embodiments, the method comprises determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype. In someembodiments, administering the selected breast cancer treatment to the subject reduces the risk of IBR.

[0099] With reference to FIG. 10, some embodiments herein relate to a method 1000 of identifying a subject for breast cancer treatment. The method can include identifying 1010 a subject with invasive hormone receptor-positive breast cancer and determining 1020 the immune phenotype of a breast tissue sample from the subject. In some embodiments the cancer is T1 / T2N0M0 invasive breast cancer. In some embodiments, the immune phenotype comprises an expression level of two or more biomarkers. In some embodiments the biomarkers comprise CD8, PD-1, and / or FOXP3. In some embodiments, the biomarkers comprise CD8 and either one or both of PD-1 and FOXP3. In some embodiments, expression of CD8 (e.g., in the immune phenotype) comprises expression of CD8 in T cells, and / or expression of FOXP3 (e.g., in the immune phenotype) comprises expression of FOXP3 in regulatory T cells (Tregs), and / or expression of PD-1 in lymphocytes.

[0100] In some embodiments, the methods disclosed herein can also include identifying 1030 the subject for a breast cancer treatment that is commensurate with a risk of ipsilateral breast recurrence (IBR). In some embodiments, a breast cancer treatment that is commensurate with the risk of IBR for the subject includes intensified treatment (e.g., relative to standard of care treatment without the present methods) if the subject has an elevated risk of IBR (or higher than a desired risk of IBR) based on the subject’s immune phenotype.

[0101] In some embodiments, the method comprises identifying 1030 the subject for a breast cancer treatment that is commensurate with responsiveness to standard radiotherapy (RT). In some embodiments, the risk of ipsilateral breast recurrence (IBR) or responsiveness to standard radiotherapy (RT) is predicted based on the subject’s immune phenotype. In some embodiments, a breast cancer treatment that is commensurate with the risk of IBR for the subject includes intensified treatment (e.g., relative to standard of care treatment without the present methods) if the subject is predicted to have an elevated risk of IBR (or higher than a desired risk of IBR, or higher a risk of IBR for patients that do not have the subject’s immune phenotype and that have received standard of care treatment), e.g., were the standard of care treatment to be administered to the subject, based on the subject’s immune phenotype. In some embodiments, a breast cancer treatment that is commensurate with the risk of IBR for the subject includes intensified treatment (e.g., relative to standard of caretreatment without the present methods) if the subject is predicted to be inadequately responsive to standard of care treatment (e.g., BCS with adjuvant therapy, such as adjuvant radiation therapy or adjuvant chemotherapy) based on the subject’s immune phenotype. In some embodiments, a breast cancer treatment that is commensurate with the risk of IBR predicted for the subject includes standard of care treatment (e.g., treatment that would have been administered without the present methods) if the subject is predicted to have risk of IBR when given the standard of care treatment. In some embodiments, a breast cancer treatment that is commensurate with responsiveness to standard RT includes intensified treatment (e.g., relative to standard of care treatment without the present methods) if the subject is predicted to not be adequately responsive to standard RT (e.g., predicted not to sufficiently reduce the risk of IBR after BCS with standard RT or with chemotherapy).

[0102] In some embodiments, the method comprises determining that the subject’s immune phenotype comprises CD8Highand PD-1Neg. In some embodiments, identifying the subject as benefiting from breast cancer treatment that comprises an alternative to standard radiotherapy is based on the determined immune phenotype. In some embodiments, identifying the subject as benefiting from breast cancer treatment that comprises an alternative to standard radiotherapy is based on determining that the subject’s immune phenotype comprises CD8Highand FOXP3High. In some embodiments, identifying the subject as being at high risk of IBR and predicted to benefit from breast cancer treatment that comprises an alternative to standard radiotherapy comprises determining that the subject’s immune phenotype is CD8High, FOXP3High, and PD-1Neg.

[0103] With reference to FIG. 9, some embodiments herein are directed to a method 900 of treating invasive breast cancer. The method can include identifying 910 a subject with invasive hormone receptor-positive breast cancer and determining 920 the immune phenotype of a breast tissue sample from the subject. The immune phenotype can include the expression level of one or more biomarkers. In some embodiments, the one or more biomarkers comprise CD8, PD-1, FOXP3, or a combination thereof. In some embodiments, expression of CD8 (e.g., in the immune phenotype) comprises expression of CD8 in T cells, and / or expression of FOXP3 (e.g., in the immune phenotype) comprises expression of FOXP3 in regulatory T cells (Tregs), and / or expression of PD-1 in lymphocytes. A standard of care or intensified treatment is then selected 930 and administered 940 to the subject, thereby treatingthe invasive hormone receptor-positive breast cancer. In some embodiments, the standard of care or intensified treatment is selected based on the subject’s immune phenotype. In some embodiments, the standard of care treatment is in line with a current guideline, such as, for example, but not limited to, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guidelines.

[0104] With reference to FIG. 11, some embodiments herein are directed to a method 1100 of identifying a subject with a high risk of ipsilateral breast recurrence (IBR). The method can include identifying 1110 a subject with invasive hormone receptor-positive breast cancer; determining 1120 the expression of at least CD8 and FOXP3 in a breast tissue sample from the subject, and identifying 1130 the subject as being at high risk of IBR if the breast tissue sample from the subject is CD8Highand FOXP3High. In some embodiments, the method includes identifying the subject as being at high risk of IBR upon determining that the breast tissue sample from the subject is CD8Highand FOXP3High. In some embodiments, the method further comprises selecting a standard of care or intensified treatment based on the subject’s risk of IBR, e.g., the subject’s risk of IBR identified based on the determined expression of at least CD8 and FOXP3. In some embodiments, intensified treatment is selected based on a determination that the subject’s immune phenotype is CD8Highand FOXP3High. In some embodiments, the method includes further determining whether the breast tissue sample is PD-1Negor PD-1Pos(e.g., by analyzing the sample for PD-1 expression levels as described herein). In some embodiments, the method includes identifying the subject as being high risk of IBR and further having residual risk of IBR were the subject to receive standard of care treatment for the breast cancer when the breast tissue sample from the subject is CD8High, FOXP3Highand PD-1Neg. In some embodiments, the method includes identifying the subject as benefitting from intensified treatment, when the breast tissue sample from the subject is CD8High, FOXP3Highand PD-1Neg. In some embodiments, the method includes selecting the intensified treatment for the subject, when the breast tissue sample from the subject is CD8High, FOXP3Highand PD-1Neg.

[0105] With reference to FIG. 12, some embodiments herein are directed to a method 1200 of treating breast cancer. The method can comprise a) obtaining 1210 a breast tissue sample from a hormone receptor-positive breast cancer subject; b) determining 1220 a level of at least CD8 and PD-1 in the breast tissue sample; c) determining 1230 that the levelof CD8 is above a first threshold level and that the level of PD-1 is below a second threshold level; and d) providing 1240 intensified treatment as intensified radiotherapy, intensified systemic therapy or mastectomy to the subject. In some embodiments, the threshold levels are In some embodiments, the first threshold level for CD8 is the highest quartile of CD8 levels in a population of subjects having invasive breast cancer (e.g., where the CD8 levels are standardized levels set by one or more housekeeping genes). In some embodiments, the second threshold level for PD-1 is the lowest quartile of PD-1 levels in a population of subjects having invasive breast cancer (e.g., where the PD-1 levels are standardized levels set by one or more housekeeping genes).

[0106] With reference to FIG. 13, some embodiments herein are directed to a method 1300 of diagnosis. In some embodiments, the method comprises a) obtaining 1310 a breast tissue sample from a hormone receptor-positive breast cancer subject; b) analyzing 1320 the breast tissue sample for expression levels of at least CD8, and one or both of PD-1 and FOXP3; and c) determining 1330 that the expression level of CD8 is above a first threshold expression level, that the level of FOXP3 is above a second threshold expression level, and / or that the level of PD-1 is below a third threshold expression level, thereby determining that the subject belongs to a group that would benefit from intensified radiotherapy. In some embodiments, the first threshold level for CD8 is the highest quartile of CD8 levels in a population of subjects having invasive breast cancer (e.g., where the CD8 levels are standardized levels set by one or more housekeeping genes). In some embodiments, the second threshold level for FOXP3 is the highest quartile of FOXP3 levels in a population of subjects having invasive breast cancer (e.g., where the FOXP3 levels are standardized levels set by one or more housekeeping genes). In some embodiments, the third threshold level for PD-1 is the lowest quartile of PD-1 levels in a population of subjects having invasive breast cancer (e.g., where the PD-1 levels are standardized levels set by one or more housekeeping genes).

[0107] With reference to FIG. 14, some embodiments herein are directed to a method 1400 of treating a subject. The method can comprise identifying 1410 an incremental risk to a subject of ipsilateral breast recurrence (IBR) based on a level of CD8, PD-1 and FOXP3 in a breast tissue sample of an invasive hormone receptor-positive breast cancer in the subject. In some embodiments level of CD8, PD-1 and FOXP3 comprises expression of CD8 in T cells, and / or expression of FOXP3 in regulatory T cells (Tregs), and / or expression of PD-1 in lymphocytes. An intensified breast cancer therapy is then administered 1420 to the subject based upon the incremental risk, wherein a higher incremental risk will increase a likelihood of an intensive breast cancer treatment that is at least more intense than what would be recommended by a guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline.

[0108] Also provided are methods of recommending that insurance cover a therapy or treatment for breast cancer, or determining if insurance will cover a therapy for breast cancer. Provided herein is method comprising: providing a breast tissue sample from a subject with invasive hormone receptor-positive breast cancer, wherein the subject has T1 / T2N0M0 breast cancer; analyzing the breast tissue sample for an immune phenotype comprising expression levels of CD8 and PD-1; selecting a breast cancer treatment based on the subject’s immune phenotype; and recommending that insurance cover the cost of the selected breast cancer treatment. In some embodiments, the method includes determining that the subject’s immune phenotype comprises CD8Highand PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype. In some embodiments, the immune phenotype comprises an expression level of FOXP3. In some embodiments, the method includes determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype.

[0109] Provided herein is a method comprising: providing a breast tissue sample from a subject with invasive hormone receptor-positive breast cancer, wherein the subject has T1 / T2N0M0 breast cancer; analyzing the breast tissue sample for an immune phenotype comprising expression levels of CD8, FOXP3, and PD-1; selecting a breast cancer treatment for the subject based on a residual risk, or lack thereof, for ipsilateral breast recurrence (IBR) predicted by the subject’s immune phenotype, were the subject to receive breast conserving surgery (BCS) and standard radiotherapy (RT) or chemotherapy; and recommending that insurance cover the cost of the selected breast cancer treatment. In some embodiments, the method includes predicting the residual risk for IBR based on the subject’s immune phenotype. In some embodiments, the method includes determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype.

[0110] Also provided is a method that includes: identifying a subject with invasive hormone receptor-positive breast cancer; determining the immune phenotype of a breast tissue sample from the subject, wherein the immune phenotype comprises an expression level of two or more biomarkers comprising: CD8; and one or both of PD-1 and FOXP3; identifying the subject for breast cancer treatment that is commensurate with a risk of ipsilateral breast recurrence (IBR) prognosed by the subject’s immune phenotype, and / or with responsiveness to standard radiotherapy (RT) predicted based on the subject’s immune phenotype; and recommending that insurance cover the cost of the breast cancer treatment that is commensurate with the subject’s risk of IBR. In some embodiments, the method includes determining that the subject’s immune phenotype comprises CD8Highand PD-1Neg; and identifying the subject as benefiting from breast cancer treatment that comprises an alternative to standard radiotherapy, based on the determined immune phenotype. In some embodiments, the method includes determining that the subject’s immune phenotype comprises CD8Highand FOXP3High; and identifying the subject as being at high risk of IBR, based on the determined immune phenotype. In some embodiments, the method includes determining that the subject’s immune phenotype is CD8High, FOXP3High, and PD-1Neg; and identifying the subject as being at high risk of IBR and predicted to benefit from breast cancer treatment that comprises an alternative to standard radiotherapy, based on the determined immune phenotype.

[0111] Also provided is a method that includes: identifying a subject with invasive hormone receptor-positive breast cancer; determining an expression level of at least CD8 and FOXP3 in a breast tissue sample from the subject, identifying the subject as being at high risk of IBR if the breast tissue sample from the subject is CD8Highand FOXP3High; and recommending that insurance cover the cost of intensified treatment as intensified radiotherapy treatment, intensified systemic therapy or mastectomy to the patient.

[0112] Provided herein is a method that includes: a) obtaining a breast tissue sample from a hormone receptor-positive breast cancer subject; b) analyzing the breast tissue sample for expression levels of at least CD8, and one or both of PD-1 and FOXP3; c) determining that the expression level of CD8 is above a first threshold expression level, that the expression level of FOXP3 is above a second threshold expression level, and / or that the expression level of PD-1 is below a third threshold expression level; and d) recommending that insurance cover the cost of intensified treatment as intensified radiotherapy, intensifiedsystemic therapy or mastectomy to the subject. In some embodiments, the first threshold is the highest quartile of CD8 levels in a population of subjects having invasive breast cancer (e.g., where the CD8 levels are standardized levels set by one or more housekeeping genes). In some embodiments, the second threshold is the highest quartile of FOXP3 levels in a population of subjects having invasive breast cancer (e.g., where the FOXP3 levels are standardized levels set by one or more housekeeping genes). In some embodiments, the third threshold is the lowest quartile of PD-1 levels in a population of subjects having invasive breast cancer (e.g., where the PD-1 levels are standardized levels set by one or more housekeeping genes). In some embodiments, the population of subjects having invasive breast cancer includes subjects diagnosed with T1 / T2N0M0 breast cancer who had underwent BCS with negative margins).

[0113] In any method herein, in some embodiments, a standard of care treatment is selected, and optionally administered, based on the subject’s immune phenotype. In some embodiments, a standard of care treatment is selected and administered based on the subject’s immune phenotype. In some embodiments, the standard of care treatment is in line with a current guideline, such as, for example, but not limited to, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guidelines. In any method herein, in some embodiments, the method includes selecting a breast cancer treatment that is a standard of care treatment if the immune phenotype indicates that the subject does not have an elevated risk of IBR, and / or is adequately responsive to the standard of care treatment (e.g., BCS with adjuvant therapy, such as adjuvant radiation therapy or adjuvant chemotherapy). In some embodiments, a standard of care treatment is selected if the subject’s immune phenotype is not CD8Highand PD-1Neg; CD8Highand FOXP3High; or CD8High, FOXP3High, and PD-1Neg. In some embodiments, a standard of care treatment is selected if the subject’s immune phenotype is not CD8High, FOXP3High, and PD-1Neg. In some embodiments, a standard of care treatment is selected if the subject’s immune phenotype includes PD-1Pos. In some embodiments, a standard of care treatment is selected if the subject’s immune phenotype includes FOXP3Low. In some embodiments, a standard of care treatment is selected if the subject’s immune phenotype includes CD8Highand FOXP3Low. In some embodiments, a standard of care treatment is selected if the subject’s immune phenotype includes CD8High, and either one orboth of PD-1Posand FOXP3Low. In some embodiments, a standard of care treatment is selected if the subject’s immune phenotype is CD8High, FOXP3High, and PD-1Pos.

[0114] In some embodiments, the standard of care treatment comprises radiotherapy. In some embodiments, the standard of care treatment includes breast conserving surgery followed by adjuvant therapy, such as standard radiation therapy.

[0115] In some embodiments, a standard radiotherapy is administered. In some embodiments, the standard radiotherapy comprises a dose of 40-60 Gy. For example, in some embodiments, the standard radiotherapy comprises a dose of, or of about 40 Gy, 41 Gy, 42 Gy, 43 Gy, 44 Gy, 45 Gy, 46 Gy, 47 Gy, 48 Gy, 49 Gy, 50 Gy, 51 Gy, 52 Gy, 53 Gy, 54 Gy, 55 Gy, 56 Gy, 57 Gy, 58 Gy, 59 Gy, or 60 Gy, or a dosage that is in a range that is defined by any two of the preceding values. For example, in some embodiments, the standard radiotherapy comprises a dose of 40-60 Gy, 40-55 Gy, 40-50 Gy, 40-45 Gy, 45-60 Gy, 45-55 Gy, 45-50 Gy, 50-60 Gy, 50-55 Gy or 55-60 Gy. In some embodiments, the standard radiotherapy comprises a dose of, or of about 50 Gy. In some embodiments, the standard radiotherapy is administered over one or more fractions. For example, in some embodiments, the standard of care treatment is administered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fractions, or over a number of fractions that is in a range defined by any two of the preceding values. For example, in some embodiments, the standard radiotherapy is administered over 1-20, 1-15, 1- 10, 1-7, 1-5, 1-3, 3-20, 3-15, 3-10, 3-7, 7-20, 7-15, 7-10, 10-20, 10-15 or 15-20 fractions.

[0116] In any method herein, in some embodiments, treating the subject with intensified radiotherapy denotes a therapy that is more intense than the guidelines in at least one guideline, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or the NICE guideline, ignoring the CD8, PD-1 and / or FOXP3 marker state. In some embodiments, if the method indicates that a subject will not respond to standard radiotherapy, then one administers at least one of: a) a more intense level of therapy than that outlined in the NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines, b) the same NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines modalities given concurrently, e.g. RT + chemotherapy, RT+ targeted therapies, c) a radiation boost with higher dose levels or with broader indications than in current NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines d) a mastectomy, or e) concurrent radiochemotherapy.

[0117] In some embodiments, an intensified treatment (e.g., intensified relative to the standard of care treatment) is selected and / or administered based on the subject’s immune phenotype, e.g., including an expression level of one, two, or all three of CD8, FOXP3, and PD-1, as described herein. In some embodiments, an intensified treatment is selected and administered based on the subject’s immune phenotype. In some embodiments, an intensified treatment is selected (and optionally administered) when the subject’s immune phenotype includes CD8High, FOXP3High, and PD-1Neg. In some embodiments, an intensified treatment is selected (and optionally administered) when the subject’s immune phenotype includes CD8Highand PD-1Neg. In some embodiments, the intensified treatment includes intensified, elevated, or aggressive radiation therapy. In some embodiments, the intensified radiotherapy is whole breast external radiotherapy, partial breast radiotherapy or brachytherapy or a combination thereof. In some embodiments, the intensified treatment includes intensified systemic therapy, mastectomy, the additional use of a sensitizer to another therapy; a therapy above a level set by at least one of: the NCCN, ASTRO, ESMO, Clinical Practice Recommendations Australia, and / or NICE guidelines for the subject’s remaining indicators; or any combination thereof. In some embodiments, an intensified treatment comprises adding a boosting dose to a standard of care treatment for the subject when the standard of care treatment does not include a boosting dose, increasing a boosting dose beyond the standard of care amount for the subject, increasing the fraction dose on a per fraction basis above the standard for the subject, and increasing the number of fractions of a recommended dose above the standard for the subject.

[0118] In some embodiments, a systemic therapy is administered based on the subject’s immune phenotype, e.g., including an expression level of one, two, or all three of CD8, FOXP3, and PD-1, as described herein. In some embodiments, an intensified systemic therapy is selected (and optionally administered) when the subject’s immune phenotype includes CD8High, FOXP3High, and PD-1Neg. In some embodiments, an intensified systemic therapy is selected (and optionally administered) when the subject’s immune phenotype includes CD8Highand PD-1Neg. In some embodiments, the systemic therapy comprises systemic chemotherapy. In some embodiments, the systemic chemotherapy comprises a platinum derivative with a taxane. In some embodiments, an intensified treatment includes administering an intensified systemic therapy, e.g., systemic chemotherapy, such as a platinum derivative with a taxane.

[0119] In some embodiments, an intensified radiotherapy is administered. In some embodiments, the intensified radiotherapy comprises a dose of at least 60 Gy or more. For example, in some embodiments, the intensified radiotherapy comprises a dose of, of about, or of at least 60 Gy, 61 Gy, 62 Gy, 63 Gy, 64 Gy, 65 Gy, 66 Gy, 67 Gy, 68 Gy, 69 Gy, 70 Gy, 71 Gy, 72 Gy, 73 Gy, 74 Gy, 75 Gy, 76 Gy, 77 Gy, 78 Gy, 79 Gy, 80 Gy, 81 Gy, 82 Gy, 83 Gy, 84 Gy, 85 Gy, 86 Gy, 87 Gy, 88 Gy, 89 Gy, 90 Gy, 91 Gy, 92 Gy, 93 Gy, 94 Gy, 95 Gy, 96 Gy, 97 Gy, 98 Gy, 99 Gy, 100 Gy or a dosage that is in a range that is defined by any two of the preceding values. For example, in some embodiments, the intensified radiotherapy comprises a dose of 60-100 Gy, 60-95 Gy, 60-90 Gy , 60-85 Gy, 60-80 Gy, 60-75 Gy, 60-70 Gy, 60-65 Gy, 65-100 Gy, 65-95 Gy, 65-90, 65-85 Gy, 65-80 Gy, 65-75 Gy, 65-70 Gy, 70- 100 Gy, 70-95 Gy, 70-90 Gy, 70-85 Gy, 70-80 Gy, 70-75 Gy, 75-100 Gy, 75-95 Gy, 75-90 Gy, 75-85 Gy, 75-80 Gy, 80-100 Gy, 80-95 Gy, 80-90 Gy, 80-85 Gy, 85-100 Gy, 85-95 Gy, 85-90 Gy, 90-100 Gy, 90-95 Gy or 95-100 Gy. In some embodiments, the intensified radiotherapy is administered over one or more fractions. For example, in some embodiments, the intensified treatment is administered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fractions, or over a number of fractions that is in a range defined by any two of the preceding values. For example, in some embodiments, the intensified radiotherapy is administered over 1-20, 1-15, 1-10, 1-7, 1-5, 1-3, 3-20, 3-15, 3-10, 3-7, 7-20, 7-15, 7-10, 10- 20, 10-15 or 15-20 fractions.

[0120] In some embodiments, a biologically effective dose (BED) of an intensified radiotherapy is administered. In some embodiments, the BED is defined as a measure of a true biological dose delivered by a combination of dose per fraction (d) and number of fractions (n) to a tissue characterized by a specific radiosensitivity (alpha / beta ratio): BED=n×d (1+d / (α / β)). In some embodiments, a biologically effective dose (BED) includes 73 Gy or more with a tumor alpha / beta ratio of 5, or a BED of 78 Gy or more with a tumor alpha / beta ratio of 4, or a BED of 87 Gy or more with a tumor alpha / beta ratio of 3, or a BED of 104 Gy or more with a tumor alpha / beta ratio of 2.

[0121] In some embodiments, the expression level of CD8, PD-1 and / or FOXP3 is determined by detecting the amount of CD8, PD-1 and / or FOXP3 mRNA or protein in the breast tissue sample. In some embodiments, CD8, PD-1 and / or FOXP3 expression denotes a level of CD8, PD-1 and / or FOXP3 protein expression. In some embodiments, CD8, PD-1and / or FOXP3 expression denotes a level of CD8, PD-1 and / or FOXP3 mRNA present in the breast tissue sample. In some embodiments, high or low level of CD8, PD-1 and / or FOXP3 are defined by a comparison of CD8, PD-1 and / or FOXP3 protein levels from the breast tissue sample to a control sample. In some embodiments, the control sample does not include invasive cancer. In some embodiments, low, high, or both are determined by comparison to an internal control in the breast tissue sample. In some embodiments, the control includes a cell population that does not express CD8, PD-1 and / or FOXP3. In some embodiments, high or low level of CD8, PD-1 and / or FOXP3 are defined by a comparison to a standardized level set by a level of expression of a set of one or more housekeeping genes. In some embodiments, high CD8 and / or FOXP3 (or CD8Highand / or FOXP3High) denotes the subject has CD8 and / or FOXP3 levels in the highest quartile of CD8 and / or FOXP3 levels in a population of subjects having invasive breast cancer (e.g., where the CD8 and / or FOXP3 levels are each standardized levels set by one or more housekeeping genes). In some embodiments, two or more housekeeping genes are used from the breast tissue sample as a control comparison for CD8, PD-1 and / or FOXP3 level to determine high or low CD8, PD-1 and / or FOXP3 levels. In some embodiments, the population of subjects having invasive breast cancer includes subjects diagnosed with T1 / T2N0M0 breast cancer who had underwent BCS with negative margins).

[0122] In some embodiments, a high CD8 and / or FOXP3 level (or CD8Highand / or FOXP3High) is determined by the expression level of CD8 and / or FOXp3 being in the highest 25% of a population of subjects having invasive breast cancer. In some embodiments, high CD8 and / or FOXP3 level (or CD8Highand / or FOXP3High) denotes the subject has CD8 and / or FOXP3 levels in a highest quartile (or highest 50%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or a percentage in a range defined by any two of the preceding values) of CD8 and / or FOXP3 in a population of subjects having invasive breast cancer (e.g., where the CD8 and / or FOXP3 levels are standardized levels set by one or more housekeeping genes). In some embodiments, the population of subjects having invasive breast cancer includes subjects diagnosed with T1 / T2N0M0 breast cancer who had underwent BCS with negative margins).

[0123] In some embodiments, a low PD-1 level is determined by the expression level of PD-1 being in the lowest 25% of a population of subjects having invasive breast cancer. In some embodiments, PD-1Negis determined by the expression level of PD-1 being in the lowest 25% of a population of subjects having invasive breast cancer (where the PD-1 levelsare standardized levels set by one or more housekeeping genes). In some embodiments, low PD-1 denotes the subject has PD-1 levels in a lowest 85% or lowest three quartiles (or lowest 50%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or a percentage in a range defined by any two of the preceding values) of PD-1 levels in a population of subjects having invasive breast cancer (e.g., where the PD-1 levels are standardized levels set by one or more housekeeping genes). In some embodiments, low PD-1 denotes the subject has PD-1 levels in the lowest quartile of PD-1 levels in a population of subjects having invasive breast cancer (e.g., where the PD-1 levels are standardized levels set by one or more housekeeping genes). In some embodiments, PD-1Negdenotes the subject has PD-1 levels in the lowest quartile of PD-1 levels in a population of subjects having invasive breast cancer (e.g., where the PD-1 levels are standardized levels set by one or more housekeeping genes). In some embodiments, high level PD-1 denotes the subject has PD-1 levels in a highest quartile (or highest 50%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or a percentage in a range defined by any two of the preceding values) of PD-1 levels in a population of subjects having invasive breast cancer (e.g., where the PD-1 levels are standardized levels set by one or more housekeeping genes). In some embodiments, high level PD-1 denotes the subject has PD-1 levels in a highest three quartiles of PD-1 levels in a population of subjects having invasive breast cancer (e.g., where the PD-1 levels are standardized levels set by one or more housekeeping genes). In some embodiments, PD-1Posdenotes the subject has PD-1 levels in a highest three quartiles of PD-1 levels in a population of subjects having invasive breast cancer (e.g., where the PD-1 levels are standardized levels set by one or more housekeeping genes). In some embodiments, two or more housekeeping genes are used from the sample as a control comparison for PD-1 level to determine high, low, or high and low PD-1 levels. In some embodiments, a high or low PD-1 level is determined by low being in a lowest 50%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or a percentage in a range defined by any two of the preceding values, of a population. In some embodiments, a low PD-1 expression is defined as an amount less than the highest 50%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or a percentage in a range defined by any two of the preceding values, of a population of subjects having invasive breast cancer. In some embodiments, the threshold level of expression of PD-1 (e.g., for determining high or low PD- 1 levels) is the lowest quartile of PD-1 levels in a population of subjects having invasive breast cancer (e.g., where the PD-1 levels are standardized levels set by one or more housekeepinggenes). In some embodiments, the population of subjects having invasive breast cancer includes subjects diagnosed with T1 / T2N0M0 breast cancer who had underwent BCS with negative margins).

[0124] In some embodiments, a level of CD8, PD-1 and / or FOXP3 is analyzed as a continuous metric so that a continuous risk assessment is further provided to the subject. In some embodiments, a level of CD8, PD-1 and / or FOXP3 is determined by at least one of: IHC / immunofluorescence / western blot / laser capture, microdissection, RT-PCR, qPCR, PCR, deep sequencing, RNA-seq, a microarray assay, normalized and non-normalized probes, and NanoString. In some embodiments, a predictive value is continuous and therefore a more extreme result in terms of the level of CD8, PD-1 and / or FOXP3 correlates with a larger benefit (or lack thereof) of a corresponding therapy, wherein the corresponding therapy is the use of standard radiotherapy or an alternative to standard radiotherapy. In some embodiments, a representative control population is used as an external reference. In some embodiments, the expression levels of CD8, PD-1 and / or FOXP3 are compared to a population of stage I / IA breast cancer subjects.

[0125] The expression of biomarker (e.g., in the immune phenotype) in the tissue sample can include expression in or associated with any suitable cells in the tissue sample. In some embodiments, expression of CD8 (e.g., in the immune phenotype) comprises expression of CD8 in T cells (e.g., CD8-expressing T cells). In some embodiments, expression of FOXP3 (e.g., in the immune phenotype) comprises expression of FOXP3 in regulatory T cells (Tregs). In some embodiments, expression of PD-1 (e.g., in the immune phenotype) expression of PD- 1 in lymphocytes, e.g., tumor infiltrating lymphocytes (TILs).

[0126] In some embodiments, the methods disclosed herein include administering a treatment for the breast cancer, where the treatment is selected based on the subject’s risk of recurrence prognosed by the subject’s immune phenotype. In some embodiments, the methods disclosed herein include administering a treatment for breast cancer that is commensurate with the risk of recurrence, as determined based on the subject’s immune phenotype. In some embodiments, a subject having a higher risk of recurrence based on the subject’s immune phenotype is administered (or receives) a treatment for breast cancer that is more aggressive (or intense) than the standard of care treatment that otherwise would have been recommended or administered to the subject without the present method.

[0127] In some embodiments, the methods disclosed herein include administering a treatment for the breast cancer, where the treatment is selected based on the subject’s responsiveness to treatment as predicted by the subject’s immune phenotype. In some embodiments, a subject predicted to be less or inadequately responsive to standard of care treatment for breast cancer (e.g., a treatment that would be recommended to the subject without the present method) is administered (or receives) an alternative to standard of care treatment for breast cancer.

[0128] In some embodiments, the immune phenotype includes expression level of biomarkers, including one or more of CD8, FOXP3 and PD-1, in a tissue sample (e.g., breast tissue sample) from the subject.

[0129] In some embodiments, a method of treating a subject for cancer includes identifying a subject with breast cancer; and administering an intensified treatment for the breast cancer if the subject is determined to be CD8High / FOXP3High / PD-1Neg. In some embodiments, a method of treating a subject for cancer includes identifying a subject with breast cancer; and administering an alternative to standard of care treatment for the breast cancer if the subject is determined to be CD8High / FOXP3High / PD-1Neg.

[0130] In some embodiments, a method of treating a subject for cancer includes identifying a subject with breast cancer; and administering an intensified treatment for the breast cancer if the subject is determined to be CD8High / PD-1Neg. In some embodiments, a method of treating a subject for cancer includes identifying a subject with breast cancer; and administering an alternative to standard of care treatment for the breast cancer if the subject is determined to be CD8High / PD-1Neg.

[0131] In some embodiments, a method of treating a subject for cancer includes identifying a subject with breast cancer; and administering an intensified treatment for the breast cancer if the subject is determined to be CD8High / FOXP3High. In some embodiments, a method of treating a subject for cancer includes identifying a subject with breast cancer; and administering an alternative to standard of care treatment for the breast cancer if the subject is determined to be CD8High / FOXP3High.

[0132] In some embodiments, the alternative to standard of care treatment includes intensified treatment for the breast cancer, including, without limitation, intensified radiation therapy. In some embodiments, the alternative to standard of care treatment includes a therapyfor the breast cancer that is more aggressive (e.g., for radiotherapy: involving elevated radiation levels, adding a boosting dose, increasing a boosting dose, increasing the fraction dose on a per fraction basis above the standard for the subject, increasing the number of fractions of a recommended dose above the standard for the subject, etc.) than a therapy that otherwise would be selected for the subject, without analyzing CD8, FOXP3 and / or PD-1 as provided herein.

[0133] In some embodiments, the standard of care treatment includes breast conserving surgery followed by adjuvant therapy, such as standard radiation therapy. The term “standard radiation therapy” denotes 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, Clinical Practice Recommendations Australia, or NICE guideline, and optionally, any one or more of the respective guidelines as of November of 2023.

[0134] In some embodiments, the intensified treatment includes intensified, elevated, or aggressive radiation therapy. In some embodiments, intensified treatment includes intensified systemic therapy (or systemic chemotherapy) or mastectomy. In some embodiments, systemic chemotherapy often includes a platinum derivative with a taxane.

[0135] In some embodiments, the method further comprises determining the level of one or more additional biomarkers and / or clinicopathological characteristics. In some embodiments, the one or more additional biomarkers and / or clinicopathological characteristics comprises histological grade, age of the subject, endocrine treatment, and combinations thereof. In some embodiments, the method includes determining one or more additional biomarkers and / or clinicopathological characteristics, including, without limitation, histological grade, age of the subject, and endocrine treatment.

[0136] In some embodiments, the method comprises the diagnosis process described herein without the treatment step.

[0137] The breast tissue sample can be obtained using any suitable option. In some embodiments, a breast tissue sample is collected from a core biopsy, fine-needle aspiration (FNA), excisional biopsy or from surgery. In some embodiments, the tissue sample comprises or includes essentially of a core biopsy. In some embodiments, the sample consists of a core biopsy. In some embodiments, the sample is collected prior to a treatment of the subject forthe cancer. In some embodiments, the sample is collected prior to a therapeutic surgery of the subject for the cancer. In some embodiments, the method is performed on a pre-operative tumor. In some embodiments, the method is performed on a post-operative tumor. In some embodiments, the breast tissue sample comprises a formalin fixed paraffin embedded (FFPE) tissue sample.

[0138] In some embodiments, the subject has been subjected to breast conserving surgery. In some embodiments, the subject has not received breast conserving surgery.

[0139] Some embodiments herein relate to a composition of matter. In some embodiments, the composition comprises a combination of detection reagents for detecting expression of one or more biomarkers for use in the methods disclosed herein. In some embodiments, the biomarkers comprise CD8, PD-1, and / or FOXP3. In some embodiments, the composition comprises a combination of detection reagents for detecting expression of at least two of CD8, PD-1, and / or FOXP3. In some embodiments, the composition comprises a combination of detection reagents for detecting expression of CD8, PD-1, and FOXP3.

[0140] Some embodiments disclosed herein relate to a kit comprising a reagent for detecting expression of one or more biomarkers in a breast cancer tissue sample from a subject with invasive hormone receptor-positive breast cancer. In some embodiments, the kit comprises a reagent for detecting expression of at least two of CD8, PD-1, and / or FOXP3 in a breast cancer tissue sample from a subject with invasive hormone receptor-positive breast cancer. In some embodiments, the reagent is an isolated antibody. In some embodiments, the reagent is a nucleic acid that selectively hybridizes to any one of CD8, PD-1, and FOXP3 as appropriate.

[0141] In some embodiments, any one of the markers (e.g., biomarkers) can be combined with any other marker in a method of the present disclosure. In some embodiments, any one of the markers (e.g., immunological biomarkers) and additional factors can be combined with any other marker and additional factors in a method of the present disclosure. In some embodiments, any 1, 2, or all 3 of PD-1, CD8, and FOXP3 can be analyzed in the context of any one or more of tumor size, histological grade, genomic instability, proliferation rate of the tumor (e.g., Ki67 expression), estrogen receptor expression, progesterone receptor expression, biological subtype, age of the subject, treatment with endocrine therapy, level of lymphovascular invasion, lymph node status, radicality of surgery, HER2 status, genomic riskscore, to select intensified or de-intensified treatment (compared to standard of care), or standard of care treatment, as provided in any one of the tables or examples herein. Controls for Scoring

[0142] A sample, e.g., a breast cancer sample, can be scored for any of the biomarkers disclosed herein using any suitable option. Determining whether there is positive or negative staining for a biomarker in a sample can be done using any suitable option. In some embodiments, positive or negative staining for a biomarker in a sample can be determined by comparison to a suitable control. In some embodiments, a control includes a tissue or cell population having a known level of expression and / or staining for the biomarker. In some embodiments, a positive control includes a tissue or cell population known to express and / or to stain for the biomarker. In some embodiments, a negative control includes a tissue or cell population known not to express and / or to stain for the biomarker. In some embodiments, the control is an internal control. In some embodiments, the control is an external control or reference. Suitable external controls include, without limitation, tonsil, appendix, and liver tissues. In some embodiments, the control is normal tissue (e.g., non- pathological tissue). In some embodiments, the control does not include invasive cancer, e.g., invasive breast cancer. In some embodiment, the control is obtained from a population or cohort having similar clinical features as the study population used in the present examples (e.g., subjects diagnosed with T1 / T2N0M0 breast cancer who had underwent BCS with negative margins).

[0143] In some embodiments, the staining of the sample is done concurrently with staining of one or more controls. In some embodiments, a TMA section (e.g., sample to be tested) is stained for one or more markers with one, two, three, four or more tissues (e.g., tonsil, appendix, and liver tissue) as controls. In some embodiments, the control(s) are used to calibrate one or more parameters for the IHC staining process (e.g., pretreatment, concentration for the antibody, incubation times, and temperatures) to ensure optimal staining. In some embodiments, optimal staining is achieved when the protocol is sufficiently specific (e.g., target cells are stained), and / or sufficiently sensitive (e.g., captures low expression), and / or sufficiently high signal-to-noise ratio (e.g., clear contrast color). In some embodiments, staining is specific when a positive control is stained and a negative control is not stained under the same protocol. In some embodiments, staining is sensitive when a positive control havinglow expression (e.g., having expression level among the lower 25%, 20%, 15%, 10%, or 5% or less of a range of expression levels) is stained. In some cases, optimal staining may not be achieved. For example, if there is an incorrect calibration, it can generate false positive or negative results. In some embodiments, a final fine-tuning of the study material is performed before the staining process.

[0144] In some embodiments, an external control or reference can be used to determine the reproducibility of the staining. In some embodiments, normal tissue (e.g., non- pathologic tissue) is used as an external control or reference, as the tumor, the tissue of interest, can vary from patient to patient. In some embodiments, different normal tissues are combined to ensure the robustness of the external control or reference.

[0145] In some embodiments, the external (positive) control for PD-1 staining is human tonsil tissue. In some embodiments, staining reaction is seen in activated T cells, B cells, and myeloid cells in germinal centers. In some embodiments, no staining is observed in the squamous epithelium and stroma. In some embodiments, the negative control for PD-1 staining is normal tissue, for example normal lung tissue or normal placental tissue or superficial squamous epithelium of the tonsil.

[0146] In some embodiments, the external (positive) control for CD8 staining is human tonsil tissue (e.g., intense membrane staining) in suppressor / cytotoxic T cells. In some embodiments, no staining is seen in B cells, squamous cells, or the remaining stroma. In some embodiments, the negative control for CD8 staining is normal tissue, for example normal appendix tissue (e.g., the muscle layer).

[0147] In some embodiments, the external (positive) control for FOXP3 staining is human tonsil tissue. In some embodiments, staining in T cells is seen in the tonsils. . In some embodiments, B cells, squamous cell carcinoma, and stroma are expected to be negative. In some embodiments, the negative control for FOXP3 staining is normal tissue, for example normal appendix tissue (e.g., the muscle layer). PD-1-Expressing Lymphocyte Scoring

[0148] The level of PD-1-expressing lymphocytes in a sample can be analyzed using any suitable option. In some embodiments, expression of PD-1 by cells is analyzed based on protein expression. Any suitable option for detecting PD-1 protein expression by cells in a tissue can be used. In some embodiments, cell-surface expression of PD-1 by cells is analyzed.In some embodiments, one or more antibodies specific to PD-1 is used to detect expression of PD-1 by cells in a tissue. In some embodiments, positive or negative staining is determined by comparing with a suitable control. In some embodiments, positive or negative staining is determined by comparison to an internal control. In some embodiments, positive staining is determined by staining intensity that is comparable to staining intensity in a positive control known to have PD-1-expressing lymphocytes. In some embodiments, positive staining is determined by staining intensity that is stronger than the staining intensity in a negative control known to not have PD-1-expressing lymphocytes. In some embodiments, negative staining is determined by staining intensity that is comparable to staining intensity in a negative control known to not have PD-1-expressing lymphocytes. In some embodiments, negative staining is determined by staining intensity that is weaker than the staining intensity in a positive control known to have PD-1-expressing lymphocytes.

[0149] In some embodiments, expression of PD-1 in cells is analyzed based on mRNA expression. Any suitable option for detecting PD-1 mRNA expression by T cells in a tissue can be used. In some embodiments, PD-1 mRNA expression by cells in a sample is detected by in situ hybridization, e.g., using one of more probes specific to PD-1 mRNA.

[0150] In some embodiments, PD-1Highin a sample is evaluated as the proportion (or percentage) of lymphocytes expressing PD-1 protein or mRNA. In some embodiments, the proportion (or percentage) is recorded using semicontinuous values with the following categories: less than 0.1%, 0.1% to 0.5%, 0.5% to 1%, 1% to 9%, 10% to 49%, 50% to 74%, and 75% or greater. In some embodiments, PD-1Highdenotes that the proportion of the PD-1- expressing lymphocytes is 1% or greater. In some embodiments, PD-1Lowdenotes that the proportion of the PD-1-expressing lymphocytes is less than 1%.

[0151] In some embodiments, PD-1 expressing lymphocytes in a sample is evaluated as the number of lymphocytes expressing PD-1. In some embodiments, one counts a number of cells (e.g., lymphocytes) staining positively for PD-1 within a certain area of the tumor or stromal compartment.

[0152] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more tissue samples are analyzed per subject. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TMAs (e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different cores) per patient are analyzed. In someembodiments, the highest value for PD-1-expressing cells is used to infer the infiltration of PD-1-expressing lymphocytes. CD8+ T Cell Scoring

[0153] The level of CD8+ T cells in a sample can be analyzed using any suitable option. In some embodiments, expression of CD8 in cells is analyzed based on protein or mRNA expression. In some embodiments, expression of CD8 by cells is analyzed based on protein expression. Any suitable option for detecting CD8 protein expression by cells in a tissue can be used. In some embodiments, cell-surface expression of CD8 by cells is analyzed. In some embodiments, one or more antibodies specific to CD8 is used to detect expression of CD8 by cells in a tissue. In some embodiments, positive or negative staining is determined by comparing with a suitable control. In some embodiments, positive or negative staining is determined by comparison to an internal control. In some embodiments, positive staining is determined by staining intensity that is comparable to staining intensity in a positive control known to have CD8+ T cells. In some embodiments, positive staining is determined by staining intensity that is stronger than the staining intensity in a negative control known to not have CD8+ T cells. In some embodiments, negative staining is determined by staining intensity that is comparable to staining intensity in a negative control known to not have CD8+ T cells. In some embodiments, negative staining is determined by staining intensity that is weaker than the staining intensity in a positive control known to have CD8+ T cells.

[0154] In some embodiments, expression of CD8 in cells is analyzed based on mRNA expression. Any suitable option for detecting CD8 mRNA expression by T cells in a tissue can be used. In some embodiments, CD8 mRNA expression by cells in a sample is detected by in situ hybridization, e.g., using one of more probes specific to CD8 mRNA.

[0155] In some embodiments, CD8+ T cells in a sample is evaluated as the proportion (or percentage) of TILs occupied by the CD8-expressing cells. In some embodiments, the proportion (or percentage) is recorded using semicontinuous values with the following categories: less than 1%, 1% to 9%, 10% to 49%, 50% to 74%, and 75% or greater. In some embodiments, the proportion of the stromal area occupied by CD8+ cells is classified according to any suitable set of categories for percentage ranges. In some embodiments, any one of the categories has a percentage cutoff at 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or greater, or at a percentage in a range defined by any two of the preceding values. In some embodiments, the median value for the proportion is multiplied with the median stromal area occupied by the TILs to obtain a score of the calculated absolute stromal area occupied by CD8+ cells.

[0156] In some embodiments, CD8+ T cells in a sample is evaluated as the number of TILs expressing CD8. In some embodiments, one counts a number of cells (e.g., TILs) staining positively for CD8 within a certain area of the tumor or stromal compartment. In some embodiments, one estimates a stromal area or an intratumoral area occupied by the CD8+ cells (e.g., CD8+ TILs).

[0157] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more tissue samples are analyzed per subject. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TMAs (e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different cores) per patient are analyzed. In some embodiments, the highest value for CD8+ cells is used to infer the infiltration of CD8+ T cells.

[0158] In some embodiments, high or low CD8+ T cells in a sample can be determined based on a suitable percentage threshold, e.g., threshold for the percentage of the stromal area occupied by CD8+ cells as determined by a suitable option, such as immunohistochemistry. In some embodiments, high CD8+ T cells denotes at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or more, or a percentage in a range defined by any two of the preceding values, of the stromal area is occupied by CD8+ cells. In some embodiments, low CD8+ T cells denotes at most (or less than) 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, or a percentage in a range defined by any two of the preceding values, of the stromal area is occupied by CD8+ cells. In some embodiments, high CD8+ T cells denotes at least 10%, and low CD8+ T cells denotes less than 10%. In some embodiments, “proportion of lymphocytes expressing CD8” denotes the absolute stromal area occupied by CD8+ T cells, as disclosed herein. FOXP3+ Treg Cell Scoring

[0159] The level of FOXP3+ Treg cells in a sample can be analyzed using any suitable option. In some embodiments, expression of FOXP3 in cells is analyzed based on protein or mRNA expression. In some embodiments, expression of FOXP3 by cells is analyzed based on protein expression. Any suitable option for detecting FOXP3 protein expression by cells in a tissue can be used. In some embodiments, one or more antibodiesspecific to FOXP3 is used to detect expression of FOXP3 by cells in a tissue. In some embodiments, positive or negative staining is determined by comparing with a suitable control. In some embodiments, positive or negative staining is determined by comparison to an internal control. In some embodiments, positive staining is determined by staining intensity that is comparable to staining intensity in a positive control known to have FOXP3+ Treg cells. In some embodiments, positive staining is determined by staining intensity that is stronger than the staining intensity in a negative control known to not have FOXP3+ Treg cells. In some embodiments, negative staining is determined by staining intensity that is comparable to staining intensity in a negative control known to not have FOXP3+ Treg cells. In some embodiments, negative staining is determined by staining intensity that is weaker than the staining intensity in a positive control known to have FOXP3+ Treg cells.

[0160] In some embodiments, expression of FOXP3 in cells is analyzed based on mRNA expression. Any suitable option for detecting FOXP3 mRNA expression by Treg cells in a tissue can be used. In some embodiments, FOXP3 mRNA expression by cells in a sample is detected by in situ hybridization, e.g., using one of more probes specific to FOXP3 mRNA.

[0161] In some embodiments, FOXP3+ Treg cells in a sample is evaluated as the proportion (or percentage) of TILs occupied by the FOXP3-expressing cells. In some embodiments, the proportion (or percentage) is recorded using semicontinuous values with the following categories: less than 1%, 1% to 9%, 10% to 49%, 50% to 74%, and 75% or greater. In some embodiments, the proportion of the stromal area occupied by FOXP3+ cells is classified according to any suitable set of categories for percentage ranges. In some embodiments, any one of the categories has a percentage cutoff at 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 90%, 95% or greater, or at a percentage in a range defined by any two of the preceding values. In some embodiments, the median value for the proportion is multiplied with the median stromal area occupied by the TILs to obtain a score of the calculated absolute stromal area occupied by FOXP3+ cells.

[0162] In some embodiments, FOXP3+ Treg cells in a sample is evaluated as the number of TILs expressing FOXP3. In some embodiments, one counts a number of cells (e.g., Tregs) staining positively for FOXP3 within a certain area of the tumor or stromalcompartment. In some embodiments, one estimates a stromal area or an intratumoral area occupied by the FOXP3+ cells (e.g., FOXP3+ Tregs).

[0163] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more tissue samples are analyzed per subject. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TMAs (e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different cores) per patient are analyzed. In some embodiments, the highest value for FOXP3+ cells is used to infer the infiltration of FOXP3+ Treg cells.

[0164] In some embodiments, high or low FOXP3+ Treg cells in a sample can be determined based on a suitable percentage threshold, e.g., threshold for the percentage of the stromal area occupied by FOXP3+ cells as determined by a suitable option, such as immunohistochemistry. In some embodiments, high FOXP3+ Treg cells denotes at least 1%, 2%, 2.5 %, 3%, 4%, 5%, 7.5 %, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or more, or a percentage in a range defined by any two of the preceding values, of the stromal area is occupied by FOXP3+ cells. In some embodiments, low FOXP3+ Treg cells denotes at most (or less than) 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or a percentage in a range defined by any two of the preceding values, of the stromal area is occupied by FOXP3+ cells. In some embodiments, high FOXP3+ Treg cells denotes at least 5%, and low FOXP3+ Treg cells denotes less than 5%. In some embodiments, “proportion of lymphocytes expressing FOXP3” denotes the absolute stromal area occupied by FOXP3+ Treg cells, as disclosed herein. ER Scoring

[0165] Estrogen receptor (ER) expression in a sample can be analyzed using any suitable option. In some embodiments, ER expression denotes ER protein or mRNA expression. In some embodiments, ER positive denotes expression of ER detected in or above a predetermined threshold percentage of tumor cells in a sample. In some embodiments, ER negative denotes expression of ER detected below a predetermined threshold percentage of tumor cells in the sample. In some embodiments, the threshold is 1%, 2%, 5%, 10%, 15%, 20% or more, or a percentage in a range defined by any two of the preceding values. In some embodiments, ER is evaluated on a TMA or a whole-tissue section. Positive and negative staining can be determined based on a suitable control. In some embodiments, a positive control includes breast cancer tissue known to express ER, or a cell line known to express ER.PR Scoring

[0166] Progesterone receptor (PR) expression in a sample can be analyzed using any suitable option. In some embodiments, PR expression denotes PR protein or mRNA expression. In some embodiments, PR positive denotes expression of PR detected in or above a predetermined threshold percentage of tumor cells in a sample. In some embodiments, PR negative denotes expression of PR detected below a predetermined threshold percentage of tumor cells in the sample. In some embodiments, the threshold is 1%, 2%, 5%, 10%, 15%, 20% or more, or a percentage in a range defined by any two of the preceding values. In some embodiments, PR is evaluated on a TMA or a whole-tissue section. Positive and negative staining can be determined based on a suitable control. In some embodiments, a positive control includes breast cancer tissue known to express PR, or a cell line known to express PR. Numbered Arrangements

[0167] Some embodiments provided herein are described by way of the following provided numbered arrangements and also provided as possible combinations or overlapping 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 breast cancer, wherein the subject has T1 / T2N0M0 breast cancer; analyzing the breast tissue sample for an immune phenotype comprising expression levels of CD8 and PD-1; selecting a breast cancer treatment based on the subject’s immune phenotype; and administering the selected breast cancer treatment therapy to the subject, thereby reducing a risk of ipsilateral breast recurrence (IBR). 2. The method of arrangement 1, comprising: determining that the subject’s immune phenotype comprises CD8Highand PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype. 3. The method of arrangement 1, wherein the immune phenotype comprises an expression level of FOXP3. 4. The method of arrangement 3, comprising:determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype. 5. A method of identifying a subject for breast cancer treatment, comprising: providing a breast tissue sample from a subject with invasive hormone receptor- positive breast cancer, wherein the subject has T1 / T2N0M0 breast cancer; analyzing the breast tissue sample for an immune phenotype comprising expression levels of CD8, FOXP3, and PD-1; and selecting a breast cancer treatment for the subject based on a residual risk, or lack thereof, for ipsilateral breast recurrence (IBR) predicted by the subject’s immune phenotype, were the subject to receive breast conserving surgery (BCS) and standard radiotherapy (RT) or chemotherapy. 6. The method of arrangement 5, comprising predicting the residual risk for IBR based on the subject’s immune phenotype. 7. The method of arrangement 5 or 6, comprising: determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype. 8. The method of any one of arrangements 5-7, comprising administering the selected breast cancer treatment to the subject, thereby reducing a risk of IBR. 9. A method of identifying a subject for breast cancer treatment, comprising: identifying a subject with invasive hormone receptor-positive breast cancer; determining the immune phenotype of a breast tissue sample from the subject, wherein the immune phenotype comprises an expression level of two or more biomarkers comprising: CD8; and one or both of PD-1 and FOXP3; and identifying the subject for breast cancer treatment that is commensurate with a risk of ipsilateral breast recurrence (IBR) prognosed by the subject’s immune phenotype, and / or with responsiveness to standard radiotherapy (RT) predicted based on the subject’s immune phenotype.10. The method of arrangement 9, comprising: determining that the subject’s immune phenotype comprises CD8Highand PD-1Neg; and identifying the subject as benefiting from breast cancer treatment that comprises an alternative to standard radiotherapy, based on the determined immune phenotype. 11. The method of arrangement 9, comprising: determining that the subject’s immune phenotype comprises CD8Highand FOXP3High; and identifying the subject as being at high risk of IBR, based on the determined immune phenotype. 12. The method of any one of arrangements 9-11, comprising: determining that the subject’s immune phenotype is CD8High, FOXP3High, and PD-1Neg; and identifying the subject as being at high risk of IBR and predicted to benefit from breast cancer treatment that comprises an alternative to standard radiotherapy, based on the determined immune phenotype. 13. A method of treating invasive breast cancer, the method comprising: identifying a subject with invasive hormone receptor-positive breast cancer; determining an immune phenotype of a breast tissue sample from the subject, wherein the immune phenotype comprises the expression level of one or more biomarkers comprising CD8, PD-1, FOXP3, or a combination thereof; selecting a standard of care or intensified treatment based on the subject’s immune phenotype; and administering the selected standard of care or intensified treatment to the subject, thereby treating the invasive hormone receptor-positive breast cancer. 14. The method of arrangement 13, comprising: determining that the subject’s immune phenotype comprises CD8Highand FOXP3High; and administering the intensified treatment to the subject based on the determination of the subject’s immune phenotype. 15. The method of arrangement 13 or 14, comprising: determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and administering the intensified treatment to the subject based on the determination of the subject’s immune phenotype. 16. The method of any one of the preceding arrangements, wherein the standard of care treatment is in line with a current guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline, optionally wherein the standard of care treatment comprises standard radiotherapy.17. A method of identifying a subject with a high risk of ipsilateral breast recurrence (IBR), comprising: identifying a subject with invasive hormone receptor-positive breast cancer; determining an expression level of at least CD8 and FOXP3 in a breast tissue sample from the subject, and identifying the subject as being at high risk of IBR if the breast tissue sample from the subject is CD8Highand FOXP3High. 18. A method of treating breast cancer, comprising: a) obtaining a breast tissue sample from a hormone receptor-positive breast cancer subject; b) determining an expression level of at least CD8 and PD-1 in the breast tissue sample; c) determining that the expression level of CD8 is above a first threshold level and that the expression level of PD-1 is below a second threshold level; and d) providing intensified treatment as intensified radiotherapy, intensified systemic therapy or mastectomy to the subject. 19. A method of diagnosis, comprising: a) obtaining a breast tissue sample from a hormone receptor-positive breast cancer subject; b) analyzing the breast tissue sample for expression levels of at least CD8, and one or both of PD-1 and FOXP3; and c) determining that the expression level of CD8 is above a first threshold expression level, that the expression level of FOXP3 is above a second threshold expression level, and / or that the expression level of PD-1 is below a third threshold expression level, thereby determining that the subject belongs to a group that would benefit from intensified radiotherapy treatment. 20. The method of arrangement 19, wherein the third threshold is the lowest quartile of PD-1 levels in a population of subjects having invasive breast cancer. 21. The method of any one of arrangements 18-20, wherein the first threshold is the highest quartile of CD8 levels in a population of subjects having invasive breast cancer. 22. The method of any one of arrangements 18-21, wherein the second threshold is the highest quartile of FOXP3 levels in a population of subjects having invasive breast cancer.23. A method of treating a subject for breast cancer, comprising: identifying an incremental risk to a subject of ipsilateral breast recurrence (IBR) based on a level of CD8, PD-1 and FOXP3 in a breast tissue sample of an invasive hormone receptor- positive breast cancer in the subject; and administering an intensified breast cancer treatment to the subject based upon the incremental risk, wherein a higher incremental risk will increase a likelihood of an intensified breast cancer treatment that is at least more than what would be recommended by a guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline. 24. The method of any one of the preceding arrangements, wherein the standard of care treatment includes breast conserving surgery followed by adjuvant therapy, such as standard radiation therapy. 25. The method of any one of the preceding arrangements, wherein the intensified treatment includes intensified, elevated, or aggressive radiation therapy. 26. The method of any one of the preceding arrangements, wherein intensified treatment includes intensified systemic therapy, mastectomy, the additional use of a sensitizer to another therapy; a therapy above a level set by at least one of: the NCCN, ASTRO, ESMO, Clinical Practice Recommendations Australia, and / or NICE guidelines for the subject’s remaining indicators, or any combination thereof. 27. The method of arrangement 26, wherein the intensified systemic therapy comprises intensified systemic chemotherapy. 28. The method of arrangement 27, wherein the intensified systemic chemotherapy comprises a platinum derivative with a taxane. 29. The method of any one of the preceding arrangements, further comprising determining the level of one or more additional biomarkers and / or clinicopathological characteristics. 30. The method of arrangement 29, wherein the one or more additional biomarkers and / or clinicopathological characteristics comprises histological grade, age of the subject, endocrine treatment, and combinations thereof. 31. The method of any one of the preceding arrangements, wherein the intensified treatment comprises intensified radiotherapy.32. The method of arrangement 31, wherein the intensified radiotherapy is whole breast external radiotherapy, partial breast radiotherapy or brachytherapy or a combination thereof. 33. The method of arrangement 32, wherein the intensified radiotherapy comprises a biologically effective dose (BED) of 73 Gy or more with a tumor alpha / beta ratio of 5, or a BED of 78 Gy or more with a tumor alpha / beta ratio of 4, or a BED of 87 Gy or more with a tumor alpha / beta ratio of 3, or a BED of 104 Gy or more with a tumor alpha / beta ratio of 2. 34. The method of arrangement 33, wherein BED is defined as a measure of a true biological dose delivered by a combination of dose per fraction (d) and number of fractions (n)to a tissue characterized by a specific radiosensitivity (alpha / beta ratio): ^^^^^^ ൌ^^ ൈ ^^ ൫1 ^ ^^ / ^^^ / ^^^൯.of any one of the preceding arrangements, wherein the subject hasto conserving surgery. 36. The method of any one of the preceding arrangements, wherein the breast cancer is an early stage invasive breast cancer, optionally wherein the subject has T1 / T2N0M0 breast cancer. 37. The method of any one of the preceding arrangements, wherein the expression level of CD8, PD-1 and / or FOXP3 is determined by detecting the amount of CD8, PD-1 and / or FOXP3 mRNA or protein in the breast tissue sample. 38. The method of any one of the preceding arrangements, wherein CD8, PD-1 and / or FOXP3 expression denotes a level of CD8, PD-1 and / or FOXP3 protein expression. 39. The method of any one of the preceding arrangements, wherein CD8, PD-1 and / or FOXP3 expression denotes a level of CD8, PD-1 and / or FOXP3 mRNA present in the breast tissue sample, 40. The method of any one of the preceding arrangements, wherein high or low level of CD8, PD-1 and / or FOXP3 are defined by a comparison of CD8, PD-1 and / or FOXP3 protein levels from the breast tissue sample to a control sample. 41. The method of arrangement 40, wherein the control sample does not include invasive cancer. 42. The method of any one of the preceding arrangements, wherein low, high, or both are determined by comparison to an internal control in the breast tissue sample.43. The method of arrangement 42, wherein the control includes a cell population that does not express CD8, PD-1 and / or FOXP3. 44. The method of any one of the preceding arrangements, wherein high or low level of CD8, PD-1 and / or FOXP3 are defined by a comparison to a standardized level set by a level of expression of a set of one or more housekeeping genes. 45. The method of any one of the preceding arrangements, wherein treating the subject with intensified radiotherapy denotes a therapy above the guidelines in at least one guideline, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or the NICE guideline, ignoring the CD8, PD-1 and / or FOXP3 marker state. 46. The method of any one of the preceding arrangements, wherein treating the subject with intensified radiotherapy denotes a dose of at least one of: 67 Gy or more, adding a boosting dose to a standard of care treatment for the subject when the standard of care treatment does not include a boosting dose, increasing a boosting dose beyond the standard amount for the subject, increasing the fraction dose on a per fraction basis above the standard for the subject, and increasing the number of fractions of a recommended dose above the standard for the subject. 47. The method of any one of the preceding arrangements, wherein high CD8 and / or FOXP3 denotes the subject has CD8 and / or FOXP3 levels in the highest quartile of CD8 and / or FOXP3 levels in a population of subjects having invasive breast cancer. 48. The method of any one of the preceding arrangements, wherein two or more housekeeping genes are used from the breast tissue sample as a control comparison for CD8, PD-1 and / or FOXP3 level to determine high or low CD8, PD-1 and / or FOXP3 levels. 49. The method of any one of the preceding arrangements, wherein a high CD8 and / or high FOXP3 level is determined by the expression level of CD8 and / or FOXP3 being in the highest 25% of a population of subjects having invasive breast cancer . 50. The method of any one of the preceding arrangements, wherein a low or negative PD-1 level is determined by the expression level of PD-1 that is the lowest quartile of PD-1 expression levels in a population of subjects having invasive breast cancer. 51. The method of any one of the preceding arrangements, wherein a level of CD8, PD-1 and / or FOXP3 is analyzed as a continuous metric so that a continuous risk assessment is further provided to the subject.52. The method of any one of the preceding arrangements, wherein a level of CD8, PD-1 and / or FOXP3 is determined by at least one of: IHC / immunofluorescence / western blot / laser capture, microdissection, RT-PCR, QPCR, PCR, deep sequencing, RNA-seq, a microarray assay, normalized and non-normalized probes, and NanoString. 53. The method of any one of the preceding arrangements, wherein the cancer is T1 to T2, node negative, nonmetastatic (T1 / T2N0M0) invasive breast cancer. 54. The method of any one of the preceding arrangements, wherein a predictive value is continuous and therefore a more extreme result in terms of the level of CD8, PD-1 and / or FOXP3 correlates with a larger benefit (or lack thereof) of a corresponding therapy, wherein the corresponding therapy is the use of standard radiotherapy or an alternative to standard radiotherapy. 55. The method of any one of the preceding arrangements, wherein a breast tissue sample is collected from a core biopsy, fine-needle aspiration (FNA), excisional biopsy or from surgery. 56. The method of any one of the preceding arrangements, wherein the method is performed on a pre-operative tumor. 57. The method of any one of arrangements 1-55, wherein the method is performed on a post-operative tumor. 58. The method of any one of the preceding arrangements, wherein one uses a representative control population as an external reference, wherein the expression levels of CD8, PD-1 and / or FOXP3 is compared to a population of stage I / IA breast cancer subjects, and wherein a control population is in line with the study population used in the present examples. 59. The method of any one of the preceding arrangements, wherein one uses housekeeping gene data as an internal reference. 60. The method of any one of the preceding arrangements, wherein if the method indicates that a subject will not respond or not respond adequately to standard radiotherapy, then one administers at least one of: a) a more intense level of therapy than that outlined in the NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines, b) the same NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines modalities given concurrently, e.g. RT + chemotherapy, RT+ targeted therapies, c) a radiation boost withhigher dose levels or with broader indications than in current NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines d) a mastectomy, or e) concurrent radiochemotherapy. 61. The method of any one of the preceding arrangements, wherein expression of CD8 (e.g., in the immune phenotype) comprises expression of CD8 in T cells, and / or expression of FOXP3 (e.g., in the immune phenotype) comprises expression of FOXP3 in regulatory T cells (Tregs), and / or expression of PD-1 in lymphocytes. 62. The method of any one of the preceding arrangements, wherein analyzing the breast tissue sample for an immune phenotype comprises: contacting the breast tissue sample with a detection reagent for one or more of: CD8, PD-1, and FOXP3; and detecting binding of the detection reagent to the breast tissue sample, to determine the immune phenotype. 63. The method of any one of the preceding arrangements, wherein determining the immune phenotype of a breast tissue sample from the subject comprises: contacting the breast tissue sample with a detection reagent for one or more of: CD8, PD-1, and FOXP3; and detecting binding of the detection reagent to the breast tissue sample, to determine the immune phenotype. 64. The method of any one of the preceding arrangements, wherein determining the expression level of at least CD8 and FOXP3 in the breast tissue sample from the subject comprises: contacting the breast tissue sample with a detection reagent for one or more of: CD8, PD-1 and FOXP3; and detecting binding of the detection reagent to the breast tissue sample, to determine the expression of at least CD8 and FOXP3 in the breast tissue sample. 65. The method of any one of the preceding arrangements, wherein analyzing the breast tissue sample for expression levels of at least CD8, and one or both of PD-1 and FOXP3 comprises: contacting the breast tissue sample with a detection reagent for one or more of: CD8, PD-1 and FOXP3; anddetecting binding of the detection reagent to the breast tissue sample, to determine the expression levels. 66. A combination of detection reagents for detecting expression of at least two of CD8, PD-1, and / or FOXP3, for use in the method of any one of the preceding arrangements. 67. A kit comprising a reagent for detecting expression of at least two of CD8, PD- 1, and / or FOXP3 in a breast tissue sample from a subject with invasive hormone receptor- positive breast cancer. Examples Example 1 – Evaluation of immune phenotypes as predictive markers for radiotherapy

[0168] This non-limiting example evaluates the role of immune phenotypes as predictive markers for RT response as part of a biosignature for a well-defined cohort of hormone receptor-positive invasive breast cancer patients.

[0169] Methods: 428 patients (Sweden) diagnosed with T1 / T2N0M0 breast cancer who had underwent BCS with negative margins were included in the study. Patients were treated with BCS plus / minus RT (median total dose 50 Gy) based upon physician preference. Biomarkers were assayed in formalin fixed paraffin embedded tissue micro arrays or whole sections using multiplex immunofluorescence and multi-spectral imaging in a CLIA lab (Laguna Hills, CA). Immune phenotypes were defined using biomarkers CD8, FOXP3 and PD-1. Varied immune phenotypes were categorized into different risk groups and were analyzed for the ipsilateral breast recurrence (IBR) rate using Kaplan Meier analyses and Cox proportional hazards. Risk group prognosis and RT prediction was assessed for RT-risk group interaction in multivariable analysis, adjusting for grade, age and endocrine treatment. High CD8 expression (CD8High) denotes CD8 levels that are in the highest quartile of CD8 levels. High FOXP3 expression (FOXP3High) denotes FOXP3 levels that are in the highest quartile of FOXP3 levels. Negative PD-1 expression (PD-1Neg) denotes PD-1 levels that are in the lowest quartile of PD-1 levels.

[0170] FIG. 1 is a forest plot depicting some embodiments of a hazard ratio of patients treated with breast conserving surgery with or without radiotherapy.

[0171] FIG.2 is an image showing some embodiments of a calculation of IBR risk in patients with T1 / T2N0M0 breast cancer who underwent BCS with negative margins.

[0172] FIG. 3-6 are images showing some embodiments of a calculation of IBR risk in patients with high levels of CD8.

[0173] Results: Patients with high levels of CD8 (n=313) tended to show risk reduction after RT. Importantly among those with high CD8 expression, patients with high FOXP3 expression (CD8High / FOXP3High) had increased rates of 10-yr IBR (HR=1.95, 95%CI(1,3.8)) with and without RT (FIG.2). In multivariable analysis in patients with CD8Highexpression, including grade (2 and 3), age (>50 years), endocrine treatment, PD-1 expression and RT, those patients with high FOXP3 expression had an increased risk of recurrence with and without RT (HR=2.295%CI (1,4.3)) (FIG.3). In addition, patients with PD-1 expression trended (p=0.055) towards a higher risk of 10-yr IBR (HR=5.4, 95%CI(.96,30)) and preferentially benefited from RT (RT:PD-1 interaction HR=0.13, 95%CI(0.02,0.84)) (FIGs.1, 3; see also FIG.4). Of patients with higher CD8Highwho were treated with BCS plus RT, those with (CD8High / FOXP3High / PD-1Neg) had significantly higher 10-yr IBR rates (HR=2.5, 95%CI(1.0,5.8)) (FIG.6).

[0174] Conclusion: The data confirm that, in the subset of the patients with high CD8 expression, the immune phenotype (CD8High / FOXP3High) had increased 10-yr IBR risks with and without RT. However, the study further stratified the (CD8High) population, identifying a Residual Risk subtype CD8High / FOXP3High / PD-1Neg(those with higher risk of IBR remaining after BCS plus RT). Collectively, immune phenotypes improved identification of patients’ recurrence risk after BCS and can potentially aid in personalizing treatment of patients with early-stage breast cancer. Example 2

[0175] This non-limiting example relates to a method of treating invasive breast cancer. A breast tissue sample from a subject with invasive hormone receptor-positive breast cancer, for example T1 / T2N0M0 breast cancer, is provided and analyzed for an immune phenotype comprising expression levels of CD8 and PD-1. Where the immune phenotype comprises (a) CD8Highand PD-1Neg, or (b) CD8High, FOXP3High, and PD-1Neg, an intensified treatment for the breast cancer is selected and administered, thereby reducing a risk of ipsilateral breast recurrence (IBR).Example 3

[0176] This non-limiting example relates to a method of identifying a subject for breast cancer treatment. A breast tissue sample from a subject with invasive hormone receptor- positive breast cancer, for example T1 / T2N0M0 breast cancer, is provided and analyzed for an immune phenotype comprising expression levels of CD8, FOXP3, and PD-1. A residual risk, or lack thereof, for ipsilateral breast recurrence (IBR) (e.g., were the subject to receive breast conserving surgery (BCS) and standard radiotherapy (RT) or chemotherapy) is predicted by the subject’s immune phenotype. Where the immune phenotype indicates that there is a low risk of IBR, BCS is administered with, or without, RT or chemotherapy. Where the immune phenotype indicates that there is residual risk of IBR, for example, where the immune phenotype is CD8High, FOXP3High, and PD-1Neg, the subject is administered an intensified therapy. Example 4

[0177] This non-limiting example relates to a method of identifying a subject for breast cancer treatment. A subject with invasive hormone receptor-positive breast cancer is identified and a sample of their breast tissue is obtained. The breast tissue sample is analyzed to determine the immune phenotype, the immune phenotype comprising an expression level of two or more biomarkers comprising: CD8; and one or both of PD-1 and FOXP3. A risk of ipsilateral breast recurrence (IBR) (e.g., were the subject to receive breast conserving surgery (BCS) and standard radiotherapy (RT) or chemotherapy) and / or responsiveness to standard radiotherapy (RT) is estimated based on the determined immune phenotype. If the immune phenotype indicates an increased risk of IBR, and / or that the subject is unlikely to respond to RT, then an intensified treatment, for example an intensified radiotherapy, is administered. In some embodiments, the immune phenotype analysis indicates that the subject’s immune phenotype comprises CD8Highand PD-1Neg; CD8Highand FOXP3High; or CD8High, FOXP3High, and PD-1Negand the subject is identified as likely to benefit from a breast cancer treatment that comprises an alternative to standard radiotherapy, e.g., an intensified radiotherapy. Example 5

[0178] This non-limiting example relates to a method of treating invasive breast cancer. A subject is identified as having invasive hormone receptor-positive breast cancer. A breast tissue sample is obtained from the subject and analyzed for an immune phenotypecomprising the expression levels of one or more biomarkers comprising CD8, PD-1, FOXP3, or a combination thereof. The subject’s immune phenotype is determined to include CD8High, FOXP3High, and PD-1Neg. An intensified treatment (e.g., intensified radiotherapy, intensified systemic therapy, or mastectomy) is then selected and administered based upon the identified immune phenotype, thereby treating the invasive hormone receptor-positive breast cancer. Example 6

[0179] This non-limiting example relates to a method of identifying a subject for breast cancer treatment. A subject is identified as having invasive hormone receptor-positive breast cancer. A breast tissue sample is obtained and analyzed to determine an immune phenotype based upon the expression levels of CD8; and one or both of PD-1 and FOXP3. A breast cancer treatment that is commensurate with a risk of ipsilateral breast recurrence (IBR) prognosed by the subject’s immune phenotype, and / or with responsiveness to standard radiotherapy (RT) predicted based on the subject’s immune phenotype is then selected. Where the subject’s immune phenotype comprises CD8Highand PD-1Neg; the subject is identified as benefitting from a breast cancer treatment that comprises an alternative to standard radiotherapy (e.g., intensified radiotherapy, intensified systemic therapy, or mastectomy), based on the determined immune phenotype. Where the subject’s immune phenotype is CD8Highand FOXP3Highthe subject is identified as being at high risk of IBR, based on the determined immune phenotype. Where the subject’s immune phenotype is CD8High, FOXP3High, and PD-1Neg; the subject is identified as being at high risk of IBR and predicted to benefit from a breast cancer treatment that comprises an alternative to standard radiotherapy (e.g., intensified radiotherapy, intensified systemic therapy, or mastectomy), based on the determined immune phenotype. Example 7

[0180] This non-limiting example relates to a method of treating invasive breast cancer. A subject is identified as having invasive hormone receptor-positive breast cancer. A breast tissue sample is obtained from the subject and analyzed for an immune phenotype comprising the expression level of one or more biomarkers comprising CD8, PD-1, FOXP3, or a combination thereof. The subject’s immune phenotype is determined to include CD8High, FOXP3High, and PD-1Neg. An intensified treatment (e.g., intensified radiotherapy, intensifiedsystemic therapy, or mastectomy) is then selected and administered based on the subject’s immune phenotype. Example 8

[0181] This non-limiting example relates to a method of identifying a subject with a high risk of ipsilateral breast recurrence (IBR). A subject is identified as having an invasive hormone receptor-positive breast cancer. A breast tissue sample is obtained from the subject and analyzed for an expression level of at least CD8 and FOXP3. The subject is identified as being at high risk of IBR if the breast tissue sample from the subject is CD8Highand FOXP3High. Example 9

[0182] This non-limiting example relates to a method of treating breast cancer. A breast tissue sample from a hormone receptor-positive breast cancer subject is obtained and analyzed for an expression level of at least CD8 and PD-1. If it is determined that the expression level of CD8 is above a first threshold and that the level of PD-1 is below a second threshold, then an intensified treatment, for example an intensified radiotherapy, intensified systemic therapy or mastectomy, is then administered to the subject. Example 10

[0183] This non-limiting example relates to a method of diagnosis. A breast tissue sample is obtained from a hormone receptor-positive breast cancer subject and analyzed for the expression levels of at least CD8, and one or both of PD-1 and FOXP3. If it is determined that the expression level of CD8 is above a first threshold, that the expression level of FOXP3 is above a second threshold, and / or that the level of PD-1 is below a third threshold, then the subject is identified as belonging to a group that would benefit from intensified radiotherapy. Example 11

[0184] This non-limiting example relates to a method of treating a subject. A subject is identified as having invasive hormone receptor-positive breast cancer. A breast tissue sample is obtained and analyzed to determine the risk of ipsilateral breast recurrence (IBR) based on a level of CD8, PD-1 and FOXP3. A higher incremental risk of IBR indicates that the subject may benefit from an intensified breast cancer therapy that is at least more than what would be recommended by a guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline. The subject is then administered a therapy in line with the determined risk of IBR.

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 breast cancer, wherein the subject has T1 / T2N0M0 breast cancer; analyzing the breast tissue sample for an immune phenotype comprising expression levels of CD8 and PD-1; selecting a breast cancer treatment based on the subject’s immune phenotype; and administering the selected breast cancer treatment to the subject, thereby reducing a risk of ipsilateral breast recurrence (IBR).

2. The method of claim 1, comprising: determining that the subject’s immune phenotype comprises CD8Highand PD- 1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype.

3. The method of claim 1, wherein the immune phenotype comprises an expression level of FOXP3.

4. The method of claim 3, comprising: determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype.

5. A method of identifying a subject for breast cancer treatment, comprising: providing a breast tissue sample from a subject with invasive hormone receptor- positive breast cancer, wherein the subject has T1 / T2N0M0 breast cancer; analyzing the breast tissue sample for an immune phenotype comprising expression levels of CD8, FOXP3, and PD-1; and selecting a breast cancer treatment for the subject based on a residual risk, for ipsilateral breast recurrence (IBR) predicted by the subject’s immune phenotype, were the subject to receive breast conserving surgery (BCS) and standard radiotherapy (RT) or chemotherapy.

6. The method of claim 5, comprising predicting the residual risk for IBR based on the subject’s immune phenotype.

7. The method of claim 5, comprising: determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and selecting an intensified treatment for the breast cancer based on the subject’s immune phenotype.

8. The method of claim 5, comprising administering the selected breast cancer treatment to the subject, thereby reducing a risk of IBR.

9. A method of identifying a subject for breast cancer treatment, comprising: identifying a subject with invasive hormone receptor-positive breast cancer; determining the immune phenotype of a breast tissue sample from the subject, wherein the immune phenotype comprises an expression level of two or more biomarkers comprising: CD8; and one or both of PD-1 and FOXP3; and identifying the subject for breast cancer treatment that is commensurate with a risk of ipsilateral breast recurrence (IBR) prognosed by the subject’s immune phenotype, and / or with responsiveness to standard radiotherapy (RT) predicted based on the subject’s immune phenotype.

10. The method of claim 9, comprising: determining that the subject’s immune phenotype comprises CD8Highand PD-1Neg; and identifying the subject as benefiting from breast cancer treatment that comprises an alternative to standard radiotherapy, based on the determined immune phenotype.

11. The method of claim 9, comprising: determining that the subject’s immune phenotype comprises CD8Highand FOXP3High; and identifying the subject as being at high risk of IBR, based on the determined immune phenotype.

12. The method of claim 9, comprising: determining that the subject’s immune phenotype is CD8High, FOXP3High, and PD-1Neg; and identifying the subject as being at high risk of IBR and predicted to benefit from breast cancer treatment that comprises an alternative to standard radiotherapy, based on the determined immune phenotype.

13. A method of treating invasive breast cancer, the method comprising: identifying a subject with invasive hormone receptor-positive breast cancer;determining an immune phenotype of a breast tissue sample from the subject, wherein the immune phenotype comprises the expression level of one or more biomarkers comprising CD8, PD-1, FOXP3, or a combination thereof; selecting a standard of care or intensified treatment based on the subject’s immune phenotype; and administering the selected standard of care or intensified treatment to the subject, thereby treating the invasive hormone receptor-positive breast cancer.

14. The method of claim 13, comprising: determining that the subject’s immune phenotype comprises CD8Highand FOXP3High; and administering the intensified treatment to the subject based on the determination of the subject’s immune phenotype.

15. The method of claim 13, comprising: determining that the subject’s immune phenotype comprises CD8High, FOXP3High, and PD-1Neg; and administering the intensified treatment to the subject based on the determination of the subject’s immune phenotype.

16. The method of any one of the preceding claims, wherein the standard of care treatment is in line with a current guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline, optionally wherein the standard of care treatment comprises standard radiotherapy.

17. A method of identifying a subject with a high risk of ipsilateral breast recurrence (IBR), comprising: identifying a subject with invasive hormone receptor-positive breast cancer; determining an expression level of at least CD8 and FOXP3 in a breast tissue sample from the subject, and identifying the subject as being at high risk of IBR if the breast tissue sample from the subject is CD8Highand FOXP3High.

18. A method of treating breast cancer, comprising: a) obtaining a breast tissue sample from a hormone receptor-positive breast cancer subject; b) determining an expression level of at least CD8 and PD-1 in the breast tissue sample; c) determining that the expression level of CD8 is above a first threshold and that the expression level of PD-1 is below a second threshold; andd) providing intensified treatment as intensified radiotherapy, intensified systemic therapy or mastectomy to the subject.

19. A method of diagnosis, comprising: a) obtaining a breast tissue sample from a hormone receptor-positive breast cancer subject; b) analyzing the breast tissue sample for expression levels of at least CD8, and one or both of PD-1 and FOXP3; and c) determining that the expression level of CD8 is above a first threshold, that the expression level of FOXP3 is above a second threshold, and / or that the expression level of PD-1 is below a third threshold, thereby determining that the subject belongs to a group that would benefit from intensified radiotherapy.

20. The method of claim 19, wherein the third threshold is PD-1 levels that are in the lowest quartile of PD-1 levels.

21. The method of claim 19, wherein the first threshold is CD8 levels that are in the highest quartile of CD8 levels.

22. The method of claim 19, wherein the second threshold is FOXP3 levels that are in the highest quartile of FOXP3 levels.

23. A method of treating a subject for breast cancer, comprising: identifying an incremental risk to a subject of ipsilateral breast recurrence (IBR) based on a level of CD8, PD-1 and FOXP3 in a breast tissue sample of an invasive hormone receptor-positive breast cancer in the subject; and administering an intensified breast cancer treatment to the subject based upon the incremental risk, wherein a higher incremental risk will increase a likelihood of an intensified breast cancer treatment that is at least more than what would be recommended by a guideline, such as, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or NICE guideline.

24. The method of claim 13, wherein the standard of care treatment includes breast conserving surgery followed by adjuvant therapy, such as standard radiation therapy.

25. The method of claim 24, wherein the intensified treatment includes intensified, elevated, or aggressive radiation therapy.

26. The method of claim 2, wherein intensified treatment includes intensified systemic therapy, mastectomy, the additional use of a sensitizer to another therapy; a therapy above a level set by at least one of: the NCCN, ASTRO, ESMO, Clinical Practice Recommendations Australia, and / or NICE guidelines for the subject’s remaining indicators, or any combination thereof.

27. The method of claim 26, wherein the intensified systemic therapy comprises intensified systemic chemotherapy.

28. The method of claim 27, wherein the intensified systemic chemotherapy comprises a platinum derivative with a taxane.

29. The method of any one of the preceding claims, further comprising determining the level of one or more additional biomarkers and / or clinicopathological characteristics.

30. The method of claim 29, wherein the one or more additional biomarkers and / or clinicopathological characteristics comprises histological grade, age of the subject, endocrine treatment, and combinations thereof.

31. The method of claim 2, wherein the intensified treatment comprises intensified radiotherapy.

32. The method of claim 31, wherein the intensified radiotherapy is whole breast external radiotherapy, partial breast radiotherapy or brachytherapy or a combination thereof.

33. The method of claim 32, wherein the intensified radiotherapy comprises a biologically effective dose (BED) of 73 Gy or more with a tumor alpha / beta ratio of 5, or a BED of 78 Gy or more with a tumor alpha / beta ratio of 4, or a BED of 87 Gy or more with a tumor alpha / beta ratio of 3, or a BED of 104 Gy or more with a tumor alpha / beta ratio of 2.

34. The method of claim 33, wherein BED is defined as a measure of a true biological dose delivered by a combination of dose per fraction (d) and number of fractions (n)to a tissue characterized by a specific radiosensitivity (alpha / beta ratio): ^^^^^^ ൌ^^ ൈ ^^ ൫1 ^ ^^ / ^^^ / ^^^൯.of any one of the preceding claims, wherein the subject has been subjected to breast conserving surgery.

36. The method of claim 9, wherein the breast cancer is an early stage invasive breast cancer, optionally wherein the subject has T1 / T2N0M0 breast cancer.

37. The method of any one of the preceding claims, wherein the expression level of CD8, PD-1 and / or FOXP3 is determined by detecting the amount of CD8, PD-1 and / or FOXP3 mRNA or protein in the breast tissue sample.

38. The method of any one of the preceding claims, wherein CD8, PD-1 and / or FOXP3 expression denotes a level of CD8, PD-1 and / or FOXP3 protein expression.

39. The method of any one of the preceding claims, wherein CD8, PD-1 and / or FOXP3 expression denotes a level of CD8, PD-1 and / or FOXP3 mRNA present in the breast tissue sample, 40. The method of any one of the preceding claims, wherein high or low level of CD8, PD-1 and / or FOXP3 are defined by a comparison of CD8, PD-1 and / or FOXP3 protein levels from the breast tissue sample to a control sample.

41. The method of claim 40, wherein the control sample does not include invasive cancer.

42. The method of any one of the preceding claims, wherein low, high, or both are determined by comparison to an internal control in the breast tissue sample.

43. The method of claim 42, wherein the control includes a cell population that does not express CD8, PD-1 and / or FOXP3.

44. The method of any one of the preceding claims, wherein high or low level of CD8, PD-1 and / or FOXP3 are defined by a comparison to a standardized level set by a level of expression of a set of one or more housekeeping genes.

45. The method of any one of the preceding claims, wherein treating the subject with intensified radiotherapy denotes a therapy above the guidelines in at least one guideline, for example, NCCN, ASTRO, ESMO, ESTRO, Clinical Practice Recommendations Australia, or the NICE guideline, ignoring the CD8, PD-1 and / or FOXP3 marker state.

46. The method of any one of the preceding claims, wherein treating the subject with intensified radiotherapy denotes a dose of at least one of: 67 Gy or more, adding a boosting dose to a standard of care treatment for the subject when the standard of care treatment does not include a boosting dose, increasing a boosting dose beyond the standard amount for the subject, increasing the fraction dose on a per fraction basis above the standard for the subject, and increasing the number of fractions of a recommended dose above the standard for the subject.

47. The method of any one of the preceding claims, wherein high CD8 and / or FOXP3 denotes the subject has CD8 and / or FOXP3 levels in the highest quartile of CD8 and / or FOXP3 levels in a population of subjects having invasive breast cancer.

48. The method of any one of the preceding claims, wherein two or more housekeeping genes are used from the breast tissue sample as a control comparison for CD8, PD-1 and / or FOXP3 level to determine high or low CD8, PD-1 and / or FOXP3 levels.

49. The method of any one of the preceding claims, wherein a high CD8 and / or high FOXP3 level is determined by the expression level of CD8 and / or FOXP3 being in the highest 25% of a population of subjects having invasive breast cancer.

50. The method of any one of the preceding claims, wherein a low or negative PD- 1 level is determined by the expression level of PD-1 that is the lowest quartile of PD-1 expression levels in a population of subjects having invasive breast cancer.

51. The method of any one of the preceding claims, wherein a level of CD8, PD-1 and / or FOXP3 is analyzed as a continuous metric so that a continuous risk assessment is further provided to the subject.

52. The method of any one of the preceding claims, wherein a level of CD8, PD-1 and / or FOXP3 is determined by at least one of: IHC / immunofluorescence / western blot / laser capture, microdissection, RT-PCR, QPCR, PCR, deep sequencing, RNA-seq, a microarray assay, normalized and non-normalized probes, and NanoString.

53. The method of any one of the preceding claims, wherein the cancer is T1 to T2, node negative, nonmetastatic (T1 / T2N0M0) invasive breast cancer.

54. The method of any one of the preceding claims, wherein a predictive value is continuous and therefore a more extreme result in terms of the level of CD8, PD-1 and / or FOXP3 correlates with a larger benefit (or lack thereof) of a corresponding therapy, wherein the corresponding therapy is the use of standard radiotherapy or an alternative to standard radiotherapy.

55. The method of any one of the preceding claims, wherein a breast tissue sample is collected from a core biopsy, fine-needle aspiration (FNA), excisional biopsy or from surgery.

56. The method of any one of the preceding claims, wherein the method is performed on a pre-operative tumor.

57. The method of any one of claims 1-55, wherein the method is performed on a post-operative tumor.

58. The method of any one of the preceding claims, wherein one uses a representative control population as an external reference, wherein the expression levels of CD8, PD-1 and / or FOXP3 is compared to a population of stage I / IA breast cancer subjects, and wherein a control population is in line with the study population used in the present examples.

59. The method of any one of the preceding claims, wherein one uses housekeeping gene data as an internal reference.

60. The method of any one of the preceding claims, wherein if the method indicates that a subject will not respond or not respond adequately to standard radiotherapy, then one administers at least one of: a) a more intense level of therapy than that outlined in the NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines, b) the same NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines modalities given concurrently, e.g. RT + chemotherapy, RT+ targeted therapies, c) a radiation boost with higher dose levels or with broader indications than in current NCCN / ESMO / Clinical Practice Recommendations Australia / NICE guidelines d) a mastectomy, or e) concurrent radiochemotherapy.

61. The method of any one of the preceding claims, wherein expression of CD8 (e.g., in the immune phenotype) comprises expression of CD8 in T cells, and / or expression of FOXP3 (e.g., in the immune phenotype) comprises expression of FOXP3 in regulatory T cells (Tregs), and / or expression of PD-1 in lymphocytes.

62. The method of any one of the preceding claims, wherein analyzing the breast tissue sample for an immune phenotype comprises: contacting the breast tissue sample with a detection reagent for one or more of: CD8, PD-1, and FOXP3; and detecting binding of the detection reagent to the breast tissue sample, to determine the immune phenotype.

63. The method of any one of the preceding claims, wherein determining the immune phenotype of a breast tissue sample from the subject comprises:contacting the breast tissue sample with a detection reagent for one or more of: CD8, PD-1, and FOXP3; and detecting binding of the detection reagent to the breast tissue sample, to determine the immune phenotype.

64. The method of any one of the preceding claims, wherein determining the expression level of at least CD8 and FOXP3 in the breast tissue sample from the subject comprises: contacting the breast tissue sample with a detection reagent for one or more of: CD8, PD-1 and FOXP3; and detecting binding of the detection reagent to the breast tissue sample, to determine the expression of at least CD8 and FOXP3 in the breast tissue sample.

65. The method of any one of the preceding claims, wherein analyzing the breast tissue sample for expression levels of at least CD8, and one or both of PD-1 and FOXP3 comprises: contacting the breast tissue sample with a detection reagent for one or more of: CD8, PD-1 and FOXP3; and detecting binding of the detection reagent to the breast tissue sample, to determine the expression levels.

66. A combination of detection reagents for detecting expression of at least two of CD8, PD-1, and / or FOXP3, for use in the method of any one of the preceding claims.

67. A kit comprising a reagent for detecting expression of at least two of CD8, PD- 1, and / or FOXP3 in a breast tissue sample from a subject with invasive hormone receptor- positive breast cancer.

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