Method of selection for treatment of subjects at risk of invasive breast cancer

Abstract

To provide a method for treatment of subjects at risk of invasive breast cancer.SOLUTION: A method of treating a subject comprises: identifying a subject with DCIS that has an elevated level of activity in a k-ras pathway; and administering an aggressive breast cancer therapy to the subject, wherein the aggressive breast cancer therapy is not radiation.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] [Related Applications] This application claims priority to U.S. Provisional Application No. 62 / 731,316, filed Sep. 14, 2018, which is incorporated by reference in its entirety. [Technical field] The present technology generally relates to whether subjects at risk for invasive breast cancer will respond to various forms of cancer treatment. [Background technology]

[0002] There are various markers that can be used to identify the tumor of a subject.In addition, there are various markers that can be used to predict tumor progression.For example, US Patent Publication Nos. 2010 / 0003189, 2012 / 0003639, 2012 / 0003639, and 20170350895 disclose various markers that, when tested in various combinations, can predict the possibility that a subject has DCIS and / or invasive breast cancer. Summary of the Invention

[0003] In some embodiments, a method of treating a subject is provided, the method comprising: identifying a subject with DCIS that has high activity levels in the k-ras pathway; and administering to the subject an aggressive breast cancer therapy, wherein the aggressive breast cancer therapy is not radiation. In some embodiments, the k-ras pathway is high when there is an elevated level of at least one of K-ras, RAF, MAPK, MEK, ETS, or SIAH.

[0004] In some embodiments, a method of treating a subject is provided, the method comprising identifying a subject with DCIS that is HER2-positive and SIAH2-positive; and administering to the subject an aggressive breast cancer treatment.

[0005] In some embodiments, a method for identifying a subject not responding to radiation therapy is provided, the method comprising: identifying a subject with DCIS at high risk of invasive breast cancer; and determining whether the subject is HER2 (or EGFR) and SIAH2 positive; wherein if the subject is HER2 and SIAH2 positive, administering an aggressive therapy to the subject, wherein the aggressive therapy is not radiation therapy, and the aggressive therapy is selected from the group consisting of an antibody against HER2 or trastuzumab.

[0006] In some embodiments, a method is provided for identifying a subject for aggressive cancer treatment, the method comprising: identifying a subject with DCIS at high risk for invasive breast cancer; and determining whether the subject is HER2 and SIAH2 positive, where if the subject is HER2 and SIAH2 positive, administering an aggressive therapy to the subject, where the aggressive therapy is not radiation therapy, and the aggressive therapy is selected from the group consisting of an antibody against HER2 or trastuzumab.

[0007] In some embodiments, a method is provided for determining a recommended treatment regimen for a subject, the method comprising: identifying a subject with DCIS at high risk for invasive breast cancer; and determining whether the subject is HER2 positive and SIAH2 positive; where if the subject is HER2 and SIAH2 positive, recommending aggressive therapy for the subject, where the aggressive therapy is not radiation therapy, and where the aggressive therapy is selected from the group consisting of an antibody against HER2 or trastuzumab.

[0008] In some embodiments, a method for treating a subject is provided. The method includes providing a DCIS sample from the subject; analyzing the DCIS sample for levels of at least PR and at least either a) at least HER2 and SIAH2 or b) at least FOXA1; providing a prognosis based on at least PR, HER2 and SIAH2, or based on at least PR and FOXA1 (wherein if the sample is PR positive, further analyzing the sample for levels of COX2, where COX2 positivity accompanied by at least FOXA1 positivity indicates an increased risk of invasive breast cancer); determining whether the subject is HER2 positive; and if the subject is HER2 positive, administering an aggressive therapy to the subject; wherein the aggressive therapy is not radiation therapy, and the aggressive therapy is selected from the group consisting of an antibody against HER2 or trastuzumab.

[0009] In some embodiments, a method is provided for reducing the risk of invasive breast cancer events in a subject. The method includes: providing a DCIS sample from a subject; analyzing the DCIS sample for at least the level of PR, and at least one of analyzing the sample for HER2 and SIAH2 or analyzing the sample for FOXA1; providing a prognosis based on at least PR, HER2 and SIAH2, or at least PR and FOXA1; if the sample is PR positive and FOXA1 negative, further analyzing the sample for Ki67, size, or Ki67 and size levels, where the sample is Ki67 positive, DCIS size is greater than 5mm, or both indicate high risk of invasive breast cancer; and if the subject is both HER2 positive and Ki67 positive, DCIS is greater than 5mm, or combinations thereof and FOXA1 negative, administering an aggressive therapy to the subject, where the aggressive therapy is not radiation therapy, and the aggressive therapy is selected from the group consisting of an antibody against HER2 or trastuzumab.

[0010] In some embodiments, a method of providing a benefit of radiation therapy is provided, the method comprising identifying a subject with DCIS who is at high risk of invasive breast cancer, and administering radiation therapy if the subject is HER2 negative, or not administering radiation therapy to the subject if the subject is HER2 positive.

[0011] In some embodiments, a method is provided for reducing the risk of a stage 1A invasive breast cancer event in a subject, the method comprising: providing a DCIS sample from the subject; analyzing the DCIS sample for at least a level of PR and at least one of a) at least HER2 and SIAH2 or b) at least FOXA1; providing a prognosis based on at least PR, HER2 and SIAH2 or at least PR and FOXA1, and if the sample is PR positive, further analyzing the sample for a level of COX2; where FOXA1 is at least positive and COX2 is positive indicates an increased risk of invasive breast cancer, and if the increased risk of invasive breast cancer is present, providing the subject with a more aggressive therapy than standard therapy.

[0012] In some embodiments, a method for determining whether insurance will cover the cost of radiation therapy is provided, the method comprising: identifying a subject with DCIS at high risk for invasive breast cancer; determining whether the subject is HER2 positive; not covering the cost of radiation therapy for the subject if the subject is HER2 positive, and covering the cost of radiation therapy for the subject if the subject is HER2 negative.

[0013] In some embodiments, a method of providing reimbursement for radiation therapy is provided, the method including: identifying a subject who has DCIS and is at high risk for invasive breast cancer; determining whether the subject is HER2 positive and SIAH2 positive; and, if the subject is HER2 negative or SIAH2 negative, providing reimbursement for the costs of radiation therapy to the subject.

[0014] In some embodiments, a method is provided for providing treatment to a subject not treated by current standard therapies, comprising identifying a subject with DCIS, where the subject is at high risk for developing invasive breast cancer; and, if the subject is HER2+ and SIAH+, administering to the subject chemotherapy, an antibody against HER2, and / or trastuzumab.

[0015] In some embodiments, a method for selecting a therapy for a subject is provided, the method comprising identifying a subject at high risk for invasive breast cancer and having DCIS;

[0016] identifying whether the subject is HER2 positive or HER2 negative, wherein if the subject is HER2 positive, administering an aggressive therapy to the subject, wherein the aggressive therapy is not radiation therapy, and the aggressive therapy is selected from the group consisting of an antibody against HER2 or trastuzumab; and if the subject is HER2 negative, not administering the aggressive therapy to the subject, thereby reducing the subject's risk of a cardiovascular event.

[0017] In some embodiments, a method of treating a subject refractory to radiation therapy is provided, the method comprising identifying a subject with HER2-positive and SIAH2-positive DCIS; and administering to the subject a therapy other than radiation therapy. The present invention may also include the following aspects: [Section 1] 1. A method of treating a subject, comprising: Identifying subjects with DCIS who have high levels of activity in the k-ras pathway; and administering to the subject an aggressive breast cancer treatment; the active breast cancer treatment is not radiation; [Section 2] The method according to item 1, wherein the k-ras pathway is high when the level of at least one of K-ras, RAF, MAPK, MEK, ETS or SIAH is high; [Section 3] 1. A method of treating a subject, comprising: To identify subjects with HER2-positive and SIAH2-positive DCIS; and administering to said subject an aggressive breast cancer treatment. A method comprising: [Section 4] 1. A method for identifying a subject who is not responsive to radiation therapy, comprising: Identifying subjects with DCIS who are at high risk for invasive breast cancer; and determining whether the subject is HER2 (or EGFR) positive and SIAH2 positive; Including, if the subject is HER2 positive and SIAH2 positive, administering aggressive therapy to the subject; the active therapy is not radiation therapy; wherein said active therapy is selected from the group consisting of an antibody against HER2 or trastuzumab; [Section 5] 1. A method for identifying a subject for aggressive cancer treatment, comprising: Identifying subjects with DCIS who are at high risk for invasive breast cancer; and determining whether the subject is HER2 positive and SIAH2 positive; Including, if the subject is HER2 positive and SIAH2 positive, administering aggressive therapy to the subject; the active therapy is not radiation therapy; wherein said active therapy is selected from the group consisting of antibodies against HER2 or trastuzumab; [Section 6] 1. A method for determining a recommended treatment regimen for a subject, comprising: To identify subjects with DCIS who are at high risk for invasive breast cancer; and determining whether the subject is HER2 positive and SIAH2 positive; Including, if the subject is HER2 and SIAH2 positive, recommending aggressive therapy to the subject; wherein said active therapy is not radiation therapy and said active therapy is selected from the group consisting of: antibodies against HER2 or trastuzumab; [Section 7] 1. A method for treating a subject, comprising: Providing a DCIS sample from the subject; At least PR level, and a) at least HER2 and SIAH2, or b) at least FOXA1 analyzing the DCIS sample for at least one of: providing a prognosis based on at least PR, HER2 and SIAH2, or based on PR and FOXA1, further comprising analyzing the sample for levels of COX2 if said sample is PR positive, wherein COX2 positivity accompanied by at least FOXA1 positivity indicates an increased risk of invasive breast cancer; determining whether the subject is HER2 positive; and if the subject is HER2 positive, administering aggressive therapy to the subject; a method, wherein said active therapy is not radiation therapy and said active therapy is selected from the group consisting of an antibody against HER2 or trastuzumab; [Section 8] 1. A method for reducing a risk of an invasive breast cancer event in a subject, comprising: Providing a DCIS sample from the subject; analyzing the DCIS sample for at least the level of PR; and a) analyzing said sample for at least HER2 and SIAH2; or b) analyzing said sample for at least FOXA1. At least one of the following; providing a prognosis based on at least PR, HER2 and SIAH2, or based on at least PR and FOXA1; If the sample is PR positive and FOXA1 negative, further analyzing the sample for Ki67 level, size, or Ki67 level and size, where Ki67 positivity of the sample, DCIS size greater than 5 mm, or both, indicate an increased risk of invasive breast cancer; and The subject a) If HER2 positive, b) Ki67 positive, DCIS size >5mm, or a combination of both, and FOXA1 negative and if both, administering active therapy to the subject; a method wherein said active therapy is not radiation therapy, and said active therapy is selected from the group consisting of an antibody against HER2, or trastuzumab; [Section 9] Any one of the methods provided above, wherein said active therapy comprises an anti-HER2 antibody; [Section 10] Item 10. The method according to item 9, wherein the antibody is trastuzumab; [Section 11] 1. A method of providing a radiation therapy benefit comprising: To identify subjects with DCIS who are at high risk for invasive breast cancer; and administering radiation therapy to the subject if the subject is HER2 negative, and not administering radiation therapy to the subject if the subject is HER2 positive; [Section 12] 1. A method for reducing a risk of a stage 1A invasive breast cancer event in a subject, comprising: Providing a DCIS sample from the subject; analyzing the DCIS sample for at least the level of PR; and a) analyzing said sample for at least HER2 and SIAH2; or b) analyzing said sample for at least FOXA1. At least one of the following; and providing a prognosis based on at least PR, HER2 and SIAH2, or based on at least PR and FOXA1; if the sample is PR positive, further analyzing the sample for the level of COX2; Positive FOXA1 and positive COX2 are indicative of an increased risk of invasive breast cancer. providing said subject with a therapy that is more aggressive than standard therapy if said subject is at high risk for invasive breast cancer; [Section 13] 13. A method of determining whether insurance will cover the cost of radiation therapy, comprising: To identify subjects with DCIS who are at high risk for invasive breast cancer; determining whether the subject is HER2 positive; and not covering the cost of radiation therapy for said subject if said subject is HER2 positive, and covering the cost of radiation therapy for said subject if said subject is HER2 negative; [Section 14] 1. A method of providing reimbursement for radiation therapy comprising: To identify subjects with DCIS who are also at high risk for invasive breast cancer; determining whether the subject is HER2 positive and SIAH2 positive; and providing reimbursement for the costs of radiation therapy to the subject if the subject is HER2 negative or SIAH2 negative; [Section 15] 1. A method of providing treatment to a subject not treated under current standard therapy, comprising: Identifying a subject with DCIS, wherein the subject is at high risk for developing invasive breast cancer; and if the subject is HER2+ and SIAH+, administering to the subject chemotherapy, an antibody to HER2, and / or trastuzumab; [Section 16] 1. A method for selecting a therapy for a subject, comprising: To identify subjects with DCIS who are at high risk for invasive breast cancer; determining whether the subject is HER2 positive or HER2 negative, wherein administering an aggressive therapy to the subject if the subject is HER2 positive; the active therapy is not radiation therapy, and the active therapy is selected from the group consisting of an antibody against HER2 or trastuzumab; if said subject is HER2 negative, then refraining from administering active therapy to said subject, thereby reducing said subject's risk of a cardiovascular event; [Section 17] 1. A method of treating a subject refractory to radiation therapy comprising: To identify subjects with HER2-positive and SIAH2-positive DCIS; and administering to said subject a treatment other than radiation therapy; [Section 18] Item 18. The method according to any one of items 1 to 17, wherein the subject is at high risk if the subject is PR positive and has a very high level of FOXA1; [Section 19] 18. The method according to any one of items 1 to 17, further comprising, if the sample is PR positive, analyzing the sample for Ki67 level, size, or both Ki67 level and size; [Section 20] 18. The method according to any one of clauses 1 to 17, wherein if the sample is PR positive and FOXA1 negative, the sample is further analyzed for Ki67 level, size, or Ki67 level and size, and Ki67 positivity, DCIS with a size greater than 5 mm, or both, indicate high risk; [Section 21] The method according to any one of items 1 to 17, further comprising analyzing p16, COX2, and Ki67; [Section 22] Item 18. The method according to any one of items 1 to 17, wherein the analysis of each marker is carried out in parallel with each other; [Section 23] Item 18. The method according to any one of items 1 to 17, wherein the analysis of each marker is performed at overlapping times; [Section 24] 18. The method according to any one of clauses 1 to 17, wherein a PR analysis is performed first and any further analysis is performed depending on the results of the PR analysis; [Section 25] Item 18. The method according to any one of items 1 to 17, wherein no additional markers are examined to determine treatment. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a set of graphs showing that almost all high-risk patients receive significant benefit from radiation therapy.

[0019] [Diagram 2] FIG. 2 is a set of graphs showing that there is a subset of HER2+ and SIAH2+ people who do not respond to radiation therapy.

[0020] [Diagram 3] Figure 3 shows SIAH2 IHC assay (top, negative, on UUH TMA; bottom, positive, Biomax BR8011 TMA). Figure 3 shows IHC assay images showing several embodiments of negative (top) and positive (bottom) staining results for SIAH2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Although there have been many advances in determining the risk of a subject developing ductal carcinoma in situ (DCIS) and / or invasive breast cancer, analysis often focuses on stratifying various subjects according to risk alone, rather than how a particular subject will be receptive to a particular treatment. The present disclosure further provides a state-of-the-art methodology, allowing a particular subject to be appropriately matched to a particular treatment. In some embodiments, by providing a subject with DCIS at high risk of invasive breast cancer, the subject can then be tested for its receptivity to radiation therapy via one or more markers provided herein. Thus, subjects at high risk of invasive breast cancer can be appropriately matched with a treatment that will work for them. This can be particularly useful in determining which subjects should be administered radiation therapy, since there is a significant number of refractory subjects in the high risk category of invasive breast cancer. Ideally, such subjects should not receive radiation therapy, but instead receive alternative forms of therapy to prevent and / or reduce the risk of invasive breast cancer events, such as anti-HER2 antibody therapy, ERBB therapy, and e.g., ERBB1234. The disclosure provides a concise set of definitions and embodiments, followed by a detailed description of the processes and various embodiments surrounding the processes, and then concludes with some examples.

[0022] [Definitions and optional embodiments] The term "and / or" is to be regarded as providing explicit support for either an both or either meaning.

[0023] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of other elements, integers or steps, or group of elements, integers or steps.

[0024] The following explanation of terms and methods is provided to better explain the present disclosure and guide those skilled in the art in the practice of the present disclosure. The singular forms "A", "an" and "The" refer to one or more, unless the context clearly dictates otherwise. For example, the term "comprising a nucleic acid molecule" includes single or multiple 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 or a combination of two or more elements of the described alternative elements, unless the context clearly dictates otherwise. As used herein, "means" includes "means". Thus, "comprising A or B", "comprising A, B, or A and B", does not exclude additional elements. Unless otherwise stated, the definitions provided herein control when this definition may differ from other possible definitions.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.All HUGO Gene Nomenclature Committee (HGNC) Identifiers (IDs) mentioned herein are incorporated by reference in their entirety.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, but suitable methods and materials are described below.The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0026] "Antibody" refers to a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. In some embodiments, an antibody is composed of a heavy chain and a light chain, each of which is a variable heavy chain (V H ) region and the variable light chain (V L ) region. H Area and V L Together, these regions are responsible for binding to the antigen that is recognized by the antibody. Antibodies include intact immunoglobulins, Fab' fragments, and F(ab)' fragments. 2It includes variants and portions thereof, such as fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region are joined by a linker, while dsFv has been mutated to introduce a disulfide bond into the chains to stabilize their association. The term also includes engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3.sup.rd Ed., WH Freeman & Co., New York, 1997. Various antibodies can be used to detect the markers, including, for example, those in the following table, Table 0.1. [Table A] [Table B]

[0027] In some embodiments, any of the methods, kits, etc. provided herein that test for the presence or absence of any of the target proteins listed in Table 0.1 may use any one or more of the corresponding antibodies to that target protein.

[0028] In some embodiments, each heavy and light chain comprises a constant region and a variable region (regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind to an antigen. The light and heavy chain variable regions comprise a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs".

[0029] "V HThe terms "V" and "VH" refer to the variable region of an immunoglobulin heavy chain (including the variable region of an Fv, scFv, dsFv or Fab). L The terms "VL" and "VL" refer to the variable region of an immunoglobulin light chain (including the variable region of an Fv, scFv, dsFv or Fab).

[0030] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those of skill in the art, for example, by creating hybrid antibody-forming cells from the fusion of a myeloma cell with an immune spleen cell. Monoclonal antibodies include humanized monoclonal antibodies.

[0031] A "polyclonal antibody" is an antibody derived from different B cell lines. A polyclonal antibody is a mixture of immunoglobulin molecules secreted against a particular antigen, each recognizing a different epitope. These antibodies are produced by methods known to those skilled in the art, for example, by inducing B lymphocytes to produce IgG immunoglobulins specific for the antigen by injection of the antigen into a suitable mammal (e.g., mouse, rabbit or goat), followed by purification from the mammal's serum.

[0032] "Chimeric antibodies" have framework residues derived from one species (eg, human) and CDRs (which generally confer antigen binding) derived from another species (eg, murine antibodies).

[0033] A "humanized" immunoglobulin is an immunoglobulin that includes a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". As an example, in a humanized immunoglobulin, all CDRs are derived from a donor immunoglobulin. Constant regions need not be present, but if present, they are substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized immunoglobulin (except possibly the CDRs) are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. Humanized immunoglobulins can be constructed by means of genetic engineering (see, e.g., U.S. Pat. No. 5,585,089).

[0034] The term "array" refers to an arrangement of molecules, such as biopolymers (such as peptides or nucleic acid molecules) or biological samples (such as tissue sections), at addressable locations on or within a substrate. A "microarray" is an array that is miniaturized to require or be aided by microscopic examination for evaluation or analysis. Arrays are sometimes called chips or biochips.

[0035] Arrays of molecules allow for a large number of analyses to be performed on a sample at one time. In some embodiments, an array of one or more molecules (e.g., oligonucleotide probes) occurs multiple times (e.g., twice) on the array, for example, to provide an internal control. The number of addressable locations on the array can be, for example, at least 1, at least 2, at least 5, 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, at least 550, at least 600, at least 800, at least 1000, at least 10,000, or more. In certain examples, the array comprises nucleic acid molecules, such as oligonucleotide sequences, at least 15 nucleotides in length, for example, about 15-40 nucleotides in length. In certain examples, the array comprises oligonucleotide probes or primers that can be used to detect at least one of the markers described herein, for example, the markers in Tables 1-9, 11, and 13-15 provided herein.

[0036] In some embodiments, each sample arranged in an array can be addressable in that its location can be reliably and consistently determined within at least two dimensions of the array. An addressable array can be computer readable in that a computer can be programmed to correlate a specific address on the array with information about the sample at that location (e.g., hybridization or binding data, including signal intensity). In some examples of computer readable formats, the individual features in the array are arranged in a regular manner, for example, in a Cartesian grid pattern, and the Cartesian grid pattern can be correlated to address information by a computer.

[0037] Protein-based arrays include arrays in which the probe molecule is or comprises a protein, or the target molecule is or comprises a protein, and the array comprises a nucleic acid to which the protein binds, or vice versa. In some examples, the array comprises an antibody against a marker provided herein, such as at least one of those in Tables 1-9, 11, and 13-15.

[0038] As used herein, the term "gene" refers to a nucleic acid in a subject's genome that can be expressed to produce mRNA and / or protein, in addition to any intervening intron sequences, and in addition to regulatory regions that control expression of the gene, e.g., a promoter or fragments thereof.

[0039] As used herein, the term "diagnosis" and its variants, such as "diagnose" or "diagnosing", are intended to include, but are not limited to, a primary diagnosis of a clinical condition or any primary diagnosis of a clinical condition. The diagnostic assays described herein are also useful for assessing the remission of a subject, or for monitoring disease recurrence, or tumor recurrence (e.g., after surgery, radiation therapy, adjuvant therapy, or chemotherapy), or for determining the appearance of metastases of a primary tumor.

[0040] In some embodiments, the prognostic assays described herein are useful for assessing the likelihood of treatment benefit, disease recurrence, tumor recurrence, or metastasis of a primary tumor, such as after 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 tests can be used to predict whether a patient will develop

[0041] The term "breast tumor" refers to a neoplastic condition of breast tissue that may be benign or malignant, and the term "tumor" is synonymous with "neoplasm" and "lesion." Exemplary breast tumors include invasive breast cancer, DCIS, lobular carcinoma in situ (LCIS), and atypical ductal hyperplasia (ADH).

[0042] The term "cancer" refers to a malignant neoplasm that undergoes characteristic anaplasia with loss of differentiation, increased growth rate, invasion of surrounding tissues, and is capable of metastasis. The term "cancer" is intended to include diseases characterized by uncontrolled growth of cells within a subject, such as, but not limited to, invasive breast cancer.

[0043] The term "intraductal lesion" refers to a tumor that is confined to the inside of a milk duct and is therefore not an invasive breast cancer. Exemplary intraductal lesions include ADH and DCIS.

[0044] ADH is a neoplastic intraductal (non-invasive) lesion characterized by the proliferation of uniformly distributed monomorphic mammary epithelial cells.

[0045] DCIS is an intraductal (non-invasive) neoplastic lesion characterized by increased proliferation of the breast epithelium and minimal cellular atypia. DCIS is classified as low, intermediate, or high grade based on factors such as nuclear atypia, intraluminal necrosis, and mitotic activity. Low-grade DCIS and ADH are morphologically identical, and ADH is differentiated from DCIS based on the extent of the disease, which is determined by its size and / or the number of ducts involved. DCIS is typically initially diagnosed from tissue biopsy triggered by suspicious findings on mammography and / or ultrasound imaging (e.g., microcalcifications, abnormal masses, tissue distortion or asymmetry, etc.). It may be by routine screening imaging or, rarely, by imaging triggered by a positive physical exam (e.g., palpable mass, nipple discharge, skin changes, etc.) or significant changes in a previously identified mass.

[0046] Cell proliferation in DCIS is confined to the breast ducts. If the proliferative cells invade through the basement membrane of the myoepithelial cell (MEC) layer lining the ducts and are thus present in the surrounding stroma, the lesion is considered invasive breast cancer even though DCIS is also present. Invasion is sometimes very minimal (microinvasion) or the only evidence of invasion is disruption of the MEC layer (e.g., by observing discontinuities in MEC-specific protein marker staining such as SMMHC and / or p63). Typically, these microinvasive cases are treated as invasive breast cancer, although there is some controversy in the treatment of these cases.

[0047] It is difficult to estimate the recurrence rate in DCIS with current treatments. However, approximately 20% of patients who undergo lumpectomy without any further treatment will experience a recurrent event within 10 years, split almost evenly between DCIS and invasive events, while patients who undergo mastectomy have less than a 2% chance of experiencing a recurrence. The standard treatment following lumpectomy is to receive adjuvant radiation therapy (RT). There is evidence from several randomized clinical trials that adjuvant radiation therapy after lumpectomy approximately halves the risk of recurrence for both DCIS and invasive event types, and current clinical and pathological assessment techniques do not identify low-risk subgroups that would not benefit from radiation therapy. Radiation is not usually administered after mastectomy. Importantly, although radiation reduces the risk of recurrent events, a survival benefit has not been established with radiation, as it does for invasive breast cancer.

[0048] LCIS ​​is a noninvasive lesion originating from the terminal ductal-lobular unit of the breast and is typically composed of small, often loosely aggregated cells. When it spreads within the ducts, it can be distinguished from DCIS on the basis of morphology and / or marker staining.

[0049] The term "invasive breast cancer" refers to a non-overlapping malignancy distinct from ADH and DCIS, in which tumor cells have invaded adjacent tissue outside the ductal structures. It can be divided into stages (I, IIA, IIB, IIIA, IIIB, and IV).

[0050] Surgery is a method of treatment for breast tumors and is often involved in diagnosis. The type of surgery depends on how widespread the tumor is at the time of diagnosis (tumor stage), the type and grade of the tumor. The term "treatment" as provided herein does not require a complete or 100% cure of the subject. Instead, it encompasses a broader concept such as delaying the onset of one or more symptoms, prolonging the life and / or quality of life of the subject, reducing the severity of one or more symptoms, etc.

[0051] "Risk" herein refers to the likelihood that a subject diagnosed with DCIS will have an ipsilateral breast event after having an initial DCIS event. Primary treatment for DCIS can include surgery, radiation therapy, or adjuvant chemotherapy. In some embodiments, the initial DCIS can be removed. The event can be a DCIS event or an invasive breast cancer event. "Risk of invasive breast cancer" refers to the risk of developing (or being diagnosed with) a subsequent invasive breast cancer in the same (ipsilateral) breast. This also applies to "risk of DCIS" or total risk. In some embodiments, early DCIS can be excluded.

[0052] In some embodiments, surgery to treat DCIS breast tumors and / or prevent or reduce the risk of subsequent ipsilateral invasive breast cancer may include lumpectomy, mastectomy, and / or bilateral mastectomy.

[0053] Adjuvant chemotherapy is often used after surgery to treat any residual disease. Systemic chemotherapy often includes platinum derivatives in combination with taxanes. Adjuvant chemotherapy is also used to treat subjects with recurrent or metastatic disease.

[0054] "Adjuvant DCIS treatment" refers to appropriate treatment for subjects who are likely to subsequently develop a DCIS event, and includes less aggressive to more aggressive treatment options, ranging from frequent monitoring with subsequent lumpectomy planned upon early detection of a breast event, to lumpectomy without radiation, additional lumpectomy, wide excision, depending on the risk profile and patient's perception of benefit. In some embodiments, subjects who do not have invasive breast cancer but are at risk for DCIS recurrence can receive adjuvant DCIS treatment (optionally in combination with any of the embodiments provided herein).

[0055] "Adjuvant invasive breast cancer treatment" means any treatment appropriate for a subject likely to develop invasive breast cancer, including lumpectomy with radiation, lumpectomy with receptor-targeted chemotherapy, lumpectomy with radiation with receptor-targeted chemotherapy, mastectomy, mastectomy with receptor-targeted chemotherapy, mastectomy with radiation, mastectomy with radiation and receptor-targeted chemotherapy, surgery with chemotherapy, etc. In some embodiments, a subject who does not have invasive breast cancer but is at risk for DCIS recurrence can receive adjuvant DCIS treatment (optionally in combination with any of the embodiments provided herein).

[0056] "Marker" refers to a measured biological component, such as a protein, mRNA transcript, or DNA amplification level. The risk of a subsequent ipsilateral breast event can be predicted through various sets or combinations of markers that allow prediction of whether a subject with DCIS is likely to experience ipsilateral DCIS recurrence, subsequent ipsilateral invasive breast cancer, or both, or whether they are likely to experience no treatment after treatment for DCIS.

[0057] The term "control" refers to a sample or standard used for comparison with a sample being tested, treated, characterized, analyzed, etc. In some embodiments, a control is a sample obtained from a healthy patient, or a non-tumor tissue sample obtained from a patient diagnosed with a breast tumor. In some embodiments, a control is a historical control or standard reference value or range of values ​​(such as previously tested control samples, such as a group of breast tumor patients with poor prognosis, or a group of samples representing baseline or normal values, such as the level of a cancer-associated gene in non-tumor tissue).

[0058] 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 before an event occurs to one or more covariates that may be associated with that amount 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. The "Cox hazard ratio" is the ratio of hazard rates corresponding to a condition described by a two-level explanatory variable (covariate), calculated using the Cox proportional hazards model. The Cox hazard ratio is the ratio of survival hazard to one unit change in the covariate. For example, the Cox hazard ratio may be the ratio of survival hazard to one unit change in log gene expression level. Thus, the larger the number, the more impact it has on the hazard rate of the event being evaluated, such as survival or disease recurrence. In some embodiments, a hazard ratio (HR) greater than 1 indicates that an increase in the level of the covariate is associated with a worse patient outcome, where the covariate level is a marker expression level. In some embodiments, an HR less than 1 indicates that a decrease in the covariate level is associated with an improved patient outcome, where the covariate level is a marker expression level.

[0059] As used herein, the term "non-tumor tissue sample" includes a sample from a normal or healthy cell or tissue, or a dataset generated using information from a normal or healthy cell or tissue. For example, the non-tumor sample can be selected from the group including: (i) a sample containing non-tumor cells; (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 dataset comprising measurements of modified chromatin and / or gene expression for a healthy individual or a population of healthy individuals; (vi) a dataset comprising measurements of modified chromatin and / or gene expression for a normal individual or a population of normal individuals; and (vii) a dataset comprising measurements of modified chromatin and / or gene expression from a subject to be tested, where the measurements are determined in a matched sample with normal cells. Preferably, the non-tumor sample is (i) or (ii) or (v) or (vii).

[0060] As used herein, the term "subject" includes any animal, preferably a mammal, including humans. 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 animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). Preferably, the mammal is a human or a primate. More preferably, the mammal is a human.

[0061] Detecting the expression of a gene product refers to determining the expression level in either a qualitative or quantitative manner that can detect a nucleic acid molecule or protein. Exemplary methods include, but are not limited to, microarray analysis, RT-PCR, Northern blot, Western blot, next generation sequencing, and mass spectrometry.

[0062] "Diagnosis" refers to the process of identifying a disease from the signs, symptoms, and results of various tests. The conclusion reached during this process is also called "diagnosis". Commonly performed forms of testing include biopsy for collection of DCIS. In some embodiments, diagnosis includes determining whether a subject with DCIS has a good or bad prognosis. In some embodiments, the prognosis can be a high or low likelihood of a subsequent DCIS event (within the next 10, 15, or 20 years). In some embodiments, the prognosis can be a high or low likelihood of an invasive breast cancer event (within the next 10, 15, or 20 years). In some embodiments, the prognosis can be a high or low likelihood of a subsequent DCIS event (within the next 10 years) and a high or low likelihood of an invasive breast cancer event (within the next 10 years).

[0063] "Differential or altered expression" refers to a difference or change, such as an increase or decrease in the amount of RNA, conversion of mRNA to protein, the level of protein in a system, or any combination thereof. In some instances, the difference is compared to a control or reference value or range of values, such as the amount of gene expression expected in a subject without DCIS and / or 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 or a change in protein levels.

[0064] The term "expression" refers to the process by which the coded information of a gene is converted into operational, non-operational, or structural parts of a cell, such as the synthesis of RNA and / or proteins. Gene expression can be influenced by external signals. For example, exposure of a cell to a hormone can stimulate the expression of hormone-induced genes. Different types of cells can respond differently to the same signal. Expression of a gene can be regulated anywhere along the pathway from DNA to RNA to protein. Regulation can include control by transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or by their activation, inactivation, compartmentalization, or degradation after specific protein molecules are produced. In some embodiments, gene expression can be monitored to determine the diagnosis and / or prognosis of a subject with DCIS, for example, to determine or predict the likelihood that the subject will subsequently develop DCIS or invasive breast cancer. In some embodiments, mRNA expression can be monitored to determine the diagnosis and / or prognosis of a subject with DCIS, for example, to determine or predict the likelihood that the subject will subsequently develop DCIS or invasive breast cancer. In some embodiments, protein expression can be monitored to determine the diagnosis and / or prognosis of a subject with DCIS, e.g., to determine or predict the likelihood that a subject will subsequently develop DCIS or invasive breast cancer.

[0065] The expression of a nucleic acid molecule in a sample can be altered compared 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. Alterations in the expression of a nucleic acid molecule can be associated with, and can actually cause, alterations in the expression of the corresponding protein.

[0066] In some embodiments, protein expression may also be altered in some manner such that it differs from the expression of the protein in a normal (e.g., non-DCIS) context, including, but not necessarily limited to: (1) expression of an increased amount of protein compared to a control or standard amount; (2) expression of a decreased amount of protein compared to a control or standard amount; (3) change in subcellular localization or targeting of the protein; (4) change in temporally regulated expression of the protein (expression where the protein is not normally expressed, or alternatively, not expressed where it is normally expressed); (5) change in stability of the protein due to an increase in the time the protein remains localized within the cell; and (6) change in localization (such as organ or tissue specific or subcellular localization) of the protein (such as not being expressed where the protein is normally expressed, or being expressed where it would not normally be expressed), respectively, as compared to a control or standard.

[0067] Controls or standards for comparison with samples for determining differential expression include samples that are considered normal (in the sense that they have not been altered to meet the desired characteristics, such as samples from subjects who do not have DCIS or who have had DCIS but have not experienced DCIS and / or invasive breast cancer 10 years after the DCIS event). It should also be noted that experimental values ​​(e.g., ranges of values) may also vary from laboratory to laboratory, although they may be set arbitrarily. Laboratory standards and values ​​may be set based on known or determined population values ​​and may be provided in the form of graphs or tables to allow comparison of measured values ​​with experimentally determined values.

[0068] As will be appreciated by those skilled in the art, any of the above controls or standards may be provided for any of the methods (e.g., treatment, analysis, or prognosis) and any of the compositions or methods provided herein. These may be positive or negative controls or standards (e.g., indicating what is a high or normal level of expression or presence of a molecule). The controls may also be adapted to the relevant molecule type (e.g., RNA vs. protein). In some embodiments, the controls and / or standards may be for COX-2, Ki-67, p16, PR, SIAH2, FOXA1, and / or HER2. In some embodiments, the controls and / or standards may be for COX-2, Ki-67, p16, ER, SIAH2, FOXA1, and / or HER2. In some embodiments, any of the PR embodiments provided herein may be replaced with ER as a marker.

[0069] The term "gene expression profile" (or signature) refers to differential or altered gene expression detectable by changes in detectable amounts of gene expression (such as cDNA, mRNA, or protein) or by changes in detectable amounts of proteins expressed by those genes. A distinct or identifiable pattern of gene expression, such as a pattern of high and low expression of a defined set of genes or gene indicator nucleic acids such as ESTs. In some instances, as few as two genes provide a profile, but more genes can be used in the profile, for example, at least 3, 4, 5, 6, or 7 markers (e.g., genes) can be used to provide a prognosis for the risk of subsequent DCIS and / or subsequent invasive breast cancer. Gene expression profiles can include absolute as well as relative expression levels of particular genes and can be viewed in the context of a test sample compared to a baseline or control sample profile (e.g., a sample from the same tissue from a subject without a tumor). In some embodiments, the gene expression profile in a subject is read on an array (such as a nucleic acid or protein array). For example, the gene expression profile can be read on a genomic array such as an Affymetrix array. TM (Santa Clara, California) Human Genome GeneChip TMThis can be done using commercially available arrays such as arrays. In some embodiments, any two or more of the markers shown in any one of Tables 1-9, 11, and 13-15 can be used as a profile. "Hybridization" means, for example, the formation of base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex. Hybridization conditions that result in a particular degree of stringency will vary depending on the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. In general, the temperature of hybridization and the ionic strength (e.g., sodium concentration) of the hybridization buffer determine the stringency of hybridization. Calculations regarding hybridization conditions to achieve a particular degree of stringency are described in Sambrook et al. (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY (chapters 9 and 11).

[0070] The term "isolated" as used in reference to an "isolated" biological component (e.g., a nucleic acid molecule, a protein, or a cell) is one that has been substantially separated or purified from other biological components, such as other chromosomal and extrachromosomal DNA and RNA, proteins, cells, etc., contained within the cell of the organism in which it naturally occurs, or within the organism itself. "Isolated" nucleic acid molecules and proteins include nucleic acid molecules and proteins that have been purified by standard purification methods. The term also encompasses 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, the isolated cells are DCIS cells that have been substantially separated from other breast cell types, such as non-tumor breast cells.

[0071] The term "label" or "probe" refers to a substance that can be detected, for example, by ELISA, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to a nucleic acid molecule or protein (e.g., one that can hybridize or bind to any of the markers in any one or more of Tables 1-9, 11, and 13-15), thereby allowing detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the selection of appropriate labels for various purposes are described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1989) and Ausubel et al. (in Current Protocols In Molecular Biology, John Wiley & Sons, New York, 1998). In some embodiments, the label is conjugated to a binding agent that specifically binds to one or more of the markers disclosed in any one or more of Tables 1-9, 11, and 13-15, allowing for detection of the presence of the marker in a subject or a DCIS sample derived from a subject.

[0072] 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.

[0073] A nucleic acid array is an arrangement of nucleic acids (such as DNA or RNA) at assigned locations on a matrix, such as found in a cDNA array or an oligonucleotide array.

[0074] A "nucleic acid molecule representing a gene" is any nucleic acid, e.g., DNA (introns or exons or both), cDNA, or RNA (such as mRNA), of any length suitable for use as a probe or other indicator molecule, which provides information about the corresponding gene, such as those listed in Tables 1-9, 11, and 13-15.

[0075] "Polymerase chain reaction" (PCR) is an in vitro amplification technique that increases the copy number of a nucleic acid molecule (e.g., a nucleic acid molecule in a sample or specimen), such as the amplification of a nucleic acid molecule listed in Tables 1-9, 11, and 13-15. The product of 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, e.g., DNA oligonucleotides 10-100 nucleotides in length, e.g., about 15, 20, 25, 30, or 50 nucleotides in length or more (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, e.g., those listed in Tables 1-9, 11, and 13-15). Primers can be selected that include at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides of the markers 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).

[0076] The term "prognosis" refers to a prediction of the course of a disease. In some embodiments provided herein, when the phrase is used in the context of a person who already has DCIS, it refers to the likelihood (within 10, 15, or 20 years) that a subject with DCIS will have a) ipsilateral DCIS event following surgical resection of the primary DCIS, b) ipsilateral invasive breast cancer, c) both events, or d) neither a) nor b). This prediction can include determining 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 treatment (e.g., radiation) will prevent an ipsilateral breast event, d) the optimal treatment method to help prevent an ipsilateral breast event that matches the severity of the most likely event, or e) a combination thereof.

[0077] The phrase "specific binding agent" refers to an agent that substantially or preferentially binds only to a defined target, such as a protein, enzyme, polysaccharide, oligonucleotide, DNA, RNA, recombinant vector, or small molecule. In one example, a "specific binding agent" can bind to at least one of the disclosed markers (such as those listed in Tables 1-9, 11, and 13-15). In some embodiments, a specific binding agent can bind to a downstream factor regulated by at least one of the disclosed markers (such as those listed in Tables 1-9, 11, and 13-15). Thus, a nucleic acid specific binding agent substantially binds only to a defined nucleic acid, such as an RNA, or to a specific region within a nucleic acid. For example, a "specific binding agent" includes an antisense compound (e.g., an antisense oligonucleotide, siRNA, miRNA, shRNA, or ribozyme) that substantially binds to a specific RNA.

[0078] A "protein specific binding agent" binds substantially only to a defined protein or to a specific region within a protein. For example, "specific binding agents" include antibodies and other substances that bind substantially to a specific polypeptide. The antibodies may be monoclonal or polyclonal antibodies specific for the polypeptide and its immunologically effective portions ("fragments"). The determination that a particular agent binds substantially only to a particular polypeptide can be readily made by using or adapting routine procedures. One suitable in vitro assay utilizes Western blotting procedures (described in many standard texts, such as Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

[0079] Cyclooxygenase-2 ("prostaglandin-endoperoxide synthase 2", "PTGS2", and "COX-2"; HGNC:9605), referred to herein as COX-2, is an enzyme encoded by the PTGS2 gene. Unless otherwise indicated, the term may encompass DNA, RNA, and / or protein versions. Thus, the level of a marker indicated may refer to, for example, RNA levels or protein levels. Use of the general terminology herein (such as "level of COX-2") refers to all of the above options together and individually (e.g., COX-2 protein levels and COX-2 RNA levels, or COX-2 protein levels, or COX-2 RNA levels).

[0080] Proliferation marker Ki-67 ("MKI67" and "MIB-1"; HGNC:7107), referred to herein as "Ki-67", is a protein encoded by the MKI67 gene. Unless otherwise indicated, the term can encompass DNA, RNA, and / or protein versions. Thus, the level of a marker indicated can refer to, for example, RNA levels or protein levels. Use of general terms herein (such as "levels of p16") refers to all of the above options together and individually (e.g., Ki-67 protein levels and Ki-67 RNA levels, or Ki-67 protein levels, or Ki-67 RNA levels).

[0081] Cyclin-dependent kinase inhibitor 2A p16 isoform ("cyclin-dependent kinase inhibitor 2A", "p16 / INK4A", "CDKN2A", and "MTS1"; HGNC:1787), referred to herein as "p16", is a tumor suppressor protein encoded by the CDKN2A gene. Unless otherwise specified, the term can encompass DNA, RNA, and / or protein versions. Thus, the level of a given marker can refer to, for example, RNA levels or protein levels. Use of the general terminology herein (such as "p16 levels") refers to all of the above options together and individually (e.g., p16 protein levels and p16 RNA levels, or p16 protein levels, or p16 RNA levels).

[0082] Progesterone receptor ("NR3C3", "PR", "PGR", "HGNC:8910"), referred to herein as "PR", is a protein encoded by the PGR gene. Unless otherwise indicated, the term can encompass DNA, RNA, and / or protein versions. Thus, the level of a marker indicated can indicate, for example, RNA levels or protein levels. Use of a general term (such as "PR level") herein indicates all of the above options together and individually (e.g., PR protein level and PR RNA level, or PR protein level, or PR RNA level).

[0083] Estrogen receptor 1 ("ESR1", "ER", "ESR", "Era", "ESRA", "ESTRR", and "NR3A1"; "HGNC:3467"), referred to herein as "ER", is a protein encoded by the ESR1 gene. Unless otherwise indicated, DNA, RNA, and / or protein versions can be encompassed. Thus, the level of a marker indicated can indicate, for example, RNA levels or protein levels. In this specification, the use of a general term (such as "ER level") indicates all of the above options together and individually (e.g., ER protein level and ER RNA level, or ER protein level, or ER RNA level).

[0084] SIAH2 E3 ubiquitin protein ligase 2 ("SIAH2" and "seven in absentia [Drosophila] homolog 2"; HGNC:10858), referred to herein as "SIAH2", is an enzyme encoded by the SIAH2 gene. Unless otherwise indicated, the term may encompass DNA, RNA, and / or protein versions. Thus, the level of a marker indicated may refer to, for example, RNA levels or protein levels. Use of the general term (e.g., SIAH2 "level") herein refers to all of the above options together and individually (e.g., SIAH2 protein level and SIAH2 RNA level, or SIAH2 protein level, or SIAH2 RNA level).

[0085] Forkhead Box A1 ("FOXA1"; HGNC:5021), referred to herein as "FOXA1", is a protein encoded by the FOXA1 gene. Unless otherwise indicated, the term can encompass DNA, RNA, and / or protein versions. Thus, the level of a marker indicated can refer to, for example, RNA levels or protein levels. Use of the general terminology herein (such as "level of FOXA1") refers to all of the above options together and individually (e.g., FOXA1 protein level and FOXA1 RNA level, or FOXA1 protein level, or FOXA1 RNA level).

[0086] v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 ("ERBB2", "HER2" [human epidermal growth factor receptor 2], "NEU", and "CD340"; HGNC:3430), referred to herein as "HER2", is a protein encoded by the ERBB2 gene. Unless otherwise indicated, the term can encompass DNA, RNA, and / or protein versions. Thus, the level of a given marker can refer to, for example, RNA levels or protein levels. Use of the general term herein (such as "level of HER2") refers to all of the above options together and individually (e.g., HER2 protein level and HER2 RNA level, or HER2 protein level, or HER2 RNA level).

[0087] Subjects with "post-menopausal" status may be identified by cessation of menstruation (if known) or by age, eg, greater than 50, eg, greater than 55 (if menstrual status is unknown).

[0088] The term "radiotherapy" refers to therapy relating to or including any form of radiation in an amount that is therapeutic to a subject.

[0089] The term "non-radiotherapy" refers to a therapy suitable for treating or reducing the risk of invasive breast cancer in a subject, and whose therapeutic effect is not due to radiation. Examples of such therapies include chemotherapy, targeted and non-targeted, immune and non-immunomodulatory, monoclonal, other targeted and non-targeted genomic therapies, antibody therapeutics, including HER2 antibodies, including trastuzumab. Often, in this application, "non-radiotherapy" is referred to as "other therapies."

[0090] The term "aggressive" as used herein means that the treatment is appropriate for subjects who are at high risk of developing the indicated event. Thus, aggressive breast cancer therapy is a therapy for subjects who are understood to be most likely to develop breast cancer. Such therapies are generally more extensive in nature than other therapies. Examples of such therapies include: aggressive radiation therapy, and aggressive non-radiation therapy.

[0091] [General description of various embodiments] Provided herein are methods for identifying and treating various subjects with appropriate forms of therapy, both in terms of the risk to the subject and the likelihood that the subject will respond to the treatment. It is recognized that not all subjects, even those at high risk of invasive breast cancer, will respond to various forms of therapy, especially radiation therapy. Thus, various embodiments provided herein allow subjects at increased risk of invasive breast cancer to determine whether they should receive radiation therapy or some other therapy instead.

[0092] In some embodiments, a method of treating a subject is provided. The method includes identifying a subject with DCIS. The subject has an elevated level of activity of the k-ras pathway. The subject then undergoes (or is treated with) aggressive breast cancer treatment. In some embodiments, the k-ras pathway is elevated when the level of at least one of K-ras, RAF, MAPK, MEK, ETS, or SIAH2 is elevated. In some embodiments, elevated refers to at least a 10% or greater increase in protein or RNA levels of one or more of K-ras, RAF, MAPK, MEK, ETS, or SIAH2. In some embodiments, the risk of developing invasive breast cancer is at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500% or greater than the level in a non-elevated subject. In some embodiments, if the subject has elevated levels of at least one of K-ras, RAF, MAPK, MEK, ETS, or SIAH, the subject is further treated with an active therapy without radiation. In some embodiments, the subject also has DCIS. In some embodiments, the subject has DCIS, is HER2 positive, and has elevated levels of 2, 3, 4, 5, or all 6 of K-ras, RAF, MAPK, MEK, ETS, and SIAH. In some embodiments, the subject has DCIS, is HER2 positive, and has elevated levels of 1, 2, 3, 4, 5, or all 6 of K-ras, RAF, MAPK, MEK, ETS, and SIAH2, and is then treated with a non-radiotherapy, e.g., a HER2 antibody such as trastuzumab.

[0093] In some embodiments, a method of treating a subject is provided. The method includes identifying a subject with DCIS, i.e., HER2 positive and SIAH2 positive, and administering to the subject an aggressive breast cancer treatment. In some embodiments, the therapy is not radiation therapy. In some embodiments, the aggressive breast cancer treatment is chemotherapy, such as a Her2 Ab, such as trastuzumab.

[0094] In some embodiments, a method for identifying a subject who does not respond to radiation therapy is provided. The method includes identifying a subject with DCIS who is at high risk of invasive breast cancer, and determining whether the subject is HER2 (or EGFR) positive and SIAH2 positive. If the subject is HER2 positive and SIAH2 positive, then the subject is administered an aggressive therapy (or receives an aggressive therapy if the person is a patient). The aggressive therapy is not radiation therapy, and may be selected from one or more of the group consisting of an antibody against HER2 or trastuzumab.

[0095] In some embodiments, a method for identifying a subject for aggressive cancer treatment is provided. The method includes identifying a subject with DCIS at high risk of invasive breast cancer, and determining whether the subject is HER2 positive and SIAH2 positive. In some embodiments, if the subject is HER2 positive and SIAH2 positive, the subject is administered (or prescribed) aggressive therapy. The aggressive therapy is not radiation therapy. In some embodiments, the aggressive therapy is selected from one or more of the group consisting of an antibody against HER2, trastuzumab, a cytotoxic drug, and an ERBB2-directed compound (such as an antibody against ERBB2).

[0096] In some embodiments, a method for treating a subject is provided. The method includes at least: providing a DCIS sample from a subject; analyzing the DCIS sample for at least the level of PR, and at least one of a) analyzing the sample for at least HER2 and SIAH2, or b) analyzing the sample for at least FOXA1; providing a prognosis based on at least PR, HER2 and SIAH2, or at least PR and FOXA1; and if the sample is PR positive, further analyzing the sample for the level of COX2, and COX2 positivity that is at least FOXA1 positive indicates a high risk of invasive breast cancer. The method further includes determining whether the subject is HER2 positive, and administering an aggressive therapy to the subject if the subject is HER2 positive. The aggressive therapy is not radiation therapy. In some embodiments, the aggressive therapy is selected from one or more of the group consisting of an antibody against HER2 and trastuzumab.

[0097] In some embodiments, a method is provided for reducing the risk of invasive breast cancer events in a subject. The method includes providing a DCIS sample from a subject; analyzing the DCIS sample for at least the level of PR, and at least one of a) analyzing the sample for at least HER2 and SIAH2, or b) analyzing the sample for at least FOXA1; and then providing a prognosis based on at least PR, HER2 and SIAH2, or at least PR and FOXA1. Then, if the sample is PR positive and FOXA1 negative, the sample is analyzed for Ki67 level, size, or Ki67 level and size. If the sample is Ki67 positive, DCIS size is greater than 5mm, or both, it indicates an increased risk of invasive breast cancer. The method further includes administering an aggressive therapy to the subject if the subject is a) HER2 positive, and b) FOXA1 negative, and Ki67 positive, DCIS size is greater than 5mm, or a combination thereof. The aggressive therapy is not radiation therapy. In some embodiments, the active therapy is selected from one or more of the group consisting of an antibody against HER2 and trastuzumab.

[0098] In some embodiments, a method is provided for providing the benefit of radiation therapy to a subject.The method includes: identifying the subject with DCIS that is at high risk of invasive breast cancer; if the subject is HER2 negative, administering radiation therapy to the subject; in some embodiments, if the subject is HER2 positive, not administering radiation therapy to the subject.

[0099] In some embodiments, a method is provided for determining which treatment method to recommend to a subject. The method includes identifying a subject with DCIS who is at high risk of invasive breast cancer; determining whether the subject is HER2 and SIAH2 positive; If the subject is HER2 and SIAH2 positive, an aggressive therapy is recommended for the subject, and the aggressive therapy is not radiation therapy. In some embodiments, the aggressive therapy for breast cancer is selected from the group consisting of an antibody against HER2 or trastuzumab, or any other option described herein. If the subject is HER2 negative or SIAH2 negative and still at high risk of invasive breast cancer, radiation therapy is recommended.

[0100] In some embodiments, the recommendation is made to the subject by a physician. In some embodiments, this is made separately, by a healthcare provider, or via an insurance company, following analysis of the markers. In some embodiments, the recommendation process is provided via the selection and / or administration of a particular treatment to the subject. In some embodiments, the recommendation process is provided via the approval of reimbursement and / or payment for a non-radiation therapy for the subject.

[0101] In some embodiments, a method for selecting a treatment for a subject is provided. The method includes identifying a subject with DCIS at high risk of invasive breast cancer and determining whether the subject is HER2 positive or HER2 negative. If the subject is HER2 positive, then the subject can be administered an aggressive therapy. In some embodiments, the aggressive therapy is not radiation therapy. In some embodiments, the aggressive therapy is selected from the group consisting of an antibody against HER2 or trastuzumab or at least one of the other options disclosed herein. If the subject is HER2 negative, one does not administer the aggressive therapy to the subject. This combination allows the subject's risk of a cardiovascular event to be reduced, while maintaining the therapeutic effect for those who need or would benefit from the particular treatment.

[0102] In some embodiments, a method is provided for providing treatment to a subject who would not otherwise be treated under the current (2018 US) standard of care. The method includes identifying a subject who has DCIS. The subject is also at high risk for developing invasive breast cancer. The method further includes administering chemotherapy to the subject. The chemotherapy can include, for example, an antibody against HER2 and / or trastuzumab. This is done when the subject is HER2+ and SIAH+.

[0103] In some embodiments, a method for treating a subject refractory to radiation therapy is provided. The method includes identifying a subject with DCIS, i.e., HER2 positive and SIAH2 positive, and administering a therapy other than radiation therapy to the subject. In some embodiments, the therapy other than radiation therapy is an antibody against HER2 and / or trastuzumab and / or a cytotoxic drug, and an ERBB2-directed compound (such as an antibody against ERBB2).

[0104] In some embodiments, a method is provided for reducing the risk of stage 1A invasive breast cancer events in a subject. The method includes providing a DCIS sample from the subject; analyzing the DCIS sample for at least the level of PR, and at least one of a) analyzing the sample for at least HER2 and SIAH2, or b) analyzing the sample for at least FOXA1. The method further includes providing a prognosis based on at least PR, HER2, and SIAH2, or at least PR and FOXA1. If the sample is PR positive, it further includes analyzing the sample for the level of COX2. If the sample is COX2 positive and FOXA1 positive, it indicates an increased risk of invasive breast cancer. If the risk of invasive breast cancer is increased, the subject is provided with a more aggressive therapy than the standard therapy to treat DCIS in the United States as of 2018 (e.g., only 1A DCIS).

[0105] In some embodiments, a method for determining whether insurance will cover the cost of radiation therapy is provided, the method includes identifying a subject with DCIS who is at high risk for invasive breast cancer; determining whether the subject is HER2 positive; and not covering the cost of radiation therapy for the subject if the subject is HER2 positive, and covering the cost of radiation therapy for the subject if the subject is HER2 negative.

[0106] In some embodiments, a method of determining whether insurance will cover the cost of radiation therapy is provided, the method including identifying a subject with DCIS who is at high risk for invasive breast cancer; determining whether the subject is HER2 positive and SIAH2 positive; not covering the cost of radiation therapy for the subject if the subject is HER2 positive and SIAH2 positive; and covering the cost of radiation therapy for the subject if the subject is HER2 negative and / or SIAH2 negative.

[0107] In some embodiments, a method of paying for radiation therapy is provided, the method including identifying a subject at high risk of invasive breast cancer and having DCIS; determining whether the subject is HER2 positive; not paying for radiation therapy for the subject if the subject is HER2 positive; and paying (at least in part) for radiation therapy for the subject if the subject is HER2 negative.

[0108] In some embodiments, a method of paying for radiation therapy is provided, the method including identifying a subject at high risk of invasive breast cancer and having DCIS; determining whether the subject is HER2 positive and SIAH2 positive; not paying for radiation therapy for the subject if the subject is HER2 positive and SIAH2 positive; and paying (at least in part) for radiation therapy for the subject if the subject is HER2 negative and / or SIAH2 negative.

[0109] In some embodiments, a method of providing reimbursement for radiation therapy is provided, the method including: identifying a subject who has DCIS and is at higher risk for invasive breast cancer; determining whether the subject is HER2 positive and SIAH2 positive; and providing the subject with reimbursement for the cost of radiation therapy if the subject is HER2 negative or SIAH2 negative.

[0110] In some embodiments, a method of providing reimbursement for non-radiotherapy is provided, the method comprising: identifying a subject who has DCIS and is at higher risk for invasive breast cancer; determining whether the subject is HER2 positive and SIAH2 positive; and, if the subject is HER2 positive and SIAH2 positive, providing the subject with reimbursement for the cost of non-radiotherapy.

[0111] In some embodiments, aggressive therapy as used herein is a therapy suitable for subjects at high risk of developing invasive breast cancer, which therapy is sufficient to address and significantly reduce the risk of invasive breast cancer. In some embodiments, the therapy is chemotherapy. In some embodiments, the chemotherapy is selected from the group consisting of HER2 and / or trastuzumab and / or cytotoxic drugs, and ERBB2-directed compounds (such as antibodies against ERBB2). In some embodiments, the therapy is a therapy that includes an anti-HER2 antibody, such as trastuzumab. In some embodiments, the therapy includes trastuzumab.

[0112] In some embodiments, the analysis of each marker is performed in parallel with each other. In some embodiments, the analysis of each marker is performed at overlapping times. In some embodiments, the PR analysis is performed first, and any further analysis is performed depending on the results of the PR analysis. In some embodiments, no additional markers are examined to determine the specific therapy to be administered. In some embodiments, the HER2 and / or SIAH2 analysis is performed first. In some embodiments, the sample and / or subject is first determined to have DCIS, and only then is it determined whether there is an elevated risk of invasive cancer, and only then is it determined whether there is refractory to radiation therapy (i.e., HER2+ and SIAH+).

[0113] In some embodiments, a further factor that can be considered to determine appropriate therapy is one that indicates elevation of the K-ras pathway. Subjects with high levels can be determined to be refractory to radiation therapy (if they also have DCIS).

[0114] Exemplary embodiments of "high risk" subjects and their identification Exemplary embodiments for identifying subjects at high risk of developing invasive breast cancer are provided below. This is not exhaustive, but is representative of how one of skill in the art may identify such individuals. In some embodiments, any of the methods provided herein for identifying subjects who should receive "elevated risk", or "high risk", or "aggressive" treatment indicate that the subject is at elevated risk of invasive breast cancer and would benefit from the classification of subjects with DCIS provided herein and determining whether they are refractory to radiation therapy. Thus, any of the embodiments provided herein (regarding treating, identifying, etc., subjects at risk for invasive breast cancer) can be used in embodiments to determine which treatment should be administered to the subject. In some embodiments, any subject identified as having a high risk can also be screened for whether they will respond to radiation therapy or should instead receive non-radiation therapy (such as HER2 antibodies). Only those that indicate "invasive risk" for the prognoses listed in the table below are relevant to the process provided herein, some of whom already have DCIS. In some embodiments, other categories of subjects indicate subjects that would not benefit from the radiation refractory analysis provided herein. Thus, the present disclosure provides guidance as to who will benefit from this analysis and who will not: once elevated risk is established, the sample is then further tested to determine appropriate treatment for the subject, by options described herein (e.g., testing for HER2 and SIAH2). [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10]

[0115] Table 9 outlines marker combinations that indicate that a subject diagnosed with DCIS is at increased risk of experiencing an ipsilateral DCIS event following surgical resection of the primary DCIS (for use in this table only).

[0116] In some embodiments, Table 11 provides a summary of combinations that indicate that a subject diagnosed with DCIS is at increased risk of experiencing ipsilateral DCIS and / or invasive breast cancer following surgical resection of the primary DCIS. [Table 11-1] [Table 11-2]

[0117] Table 11 summarizes relevant markers for identifying the level of risk that a subject with DCIS will experience invasive breast cancer. Table 11 provides a summary of combinations that indicate an increased risk of experiencing invasive breast cancer and / or recurrence of DCIS for subjects who already have DCIS. The results above (Tables 1-9, 11, 13-15) are expressly contemplated for all embodiments of the various methods, kits, compositions, etc. provided herein. In some embodiments, only those meeting an increased risk of invasive breast cancer are analyzed for HER2 and SIAH2 levels.

[0118] In some embodiments, any of the described combinations of the above markers or variables can be used for compositions or methods related to risk of DCIS recurrence and / or invasive breast cancer (as indicated). As described herein, various combinations indicate that the subject has a relatively high (or low) risk of experiencing DCIS recurrence and / or invasive breast cancer. Thus, in some embodiments, this can be practically employed in terms of, for example, appropriate prognosis of the subject, advanced treatment methods for the subject (e.g., taking an approach that not only resolves the DCIS that the subject currently has, but also appropriately addresses the risk level of DCIS recurrence and / or invasive breast cancer), methods for analyzing samples (e.g., DCIS samples for various markers), compositions and kits that enable the above methods, etc. In some embodiments, in a PR- background, low FOXA1 correlates with invasive events, and in a PR+ background, high FOXA1 correlates with invasive recurrence. Both correlations are reversed for DCIS events. In some embodiments, separate biomarker-based risk models (algorithms) can be used to predict invasive and DCIS events, with at least some biomarkers being evaluated differently in the context of other markers, rather than being assigned a single weight in a linear algorithm. In some embodiments, the PR / FOXA1 combination identifies a subset of patients who will experience a significant benefit from RT compared to the rest of the patients. As shown, these studies demonstrate that the present approach to risk stratification modeling can accurately identify patients at risk for DCIS or invasive events after a primary DCIS diagnosis. In some embodiments, the models presented herein (e.g., in the Examples below) are the basis for a comprehensive multimarker panel. Note that in certain embodiments, the risk models can be run on a computing device, while in other embodiments, the risk models can be run manually.

[0119] In some embodiments, a method of analyzing a sample is provided, which includes analyzing a human DCIS tissue sample for PR, and either or both of analyzing the sample for at least HER2 and SIAH2, and / or analyzing the sample for at least FOXA1. In some embodiments, depending on the nature of the result, this indicates that the subject who provided the sample is at high or elevated risk of invasive breast cancer (see, e.g., Tables 1-9, 11, 13, and 15).

[0120] In some embodiments, a method of analyzing a sample is provided, comprising analyzing a human DCIS tissue sample for at least the levels of SIAH2 and FOXA1. In some embodiments, depending on the nature of the result, this indicates that the subject providing the sample is at high risk or elevated risk of invasive breast cancer (see, e.g., Tables 1-9, 11, 13, and 15).

[0121] In some embodiments, a method of analyzing a sample is provided. The method includes providing a DCIS sample from a subject with DCIS; 1) analyzing the DCIS sample for SIAH2, and analyzing the DCIS sample for at least one of HER2, PR, FOXA1, or any combination thereof, or 2) analyzing the DCIS sample for FOXA1 and PR. In some embodiments, depending on the nature of the result, this indicates that the subject who provided the sample is at high risk or elevated risk of invasive breast cancer (see, e.g., Tables 1-9, 11, 13, and 15).

[0122] In some embodiments, a method is provided for prognosing the risk of an invasive breast cancer event in a subject. The method includes providing a DCIS sample from the subject; analyzing the DCIS sample for at least the level of PR, and at least one of analyzing the sample for at least HER2 and SIAH2 or analyzing the sample for at least FOXA1. The method further includes providing a prognosis based on at least PR, HER2 and SIAH2, or based on at least PR and FOXA1. In some embodiments, depending on the nature of the result, this indicates that the subject who provided the sample is at high risk or elevated risk of invasive breast cancer (see, e.g., Tables 1-9, 11).

[0123] In some embodiments, a method is provided for prognosing the risk of an invasive breast cancer event in a subject. The method includes providing a DCIS sample from the subject; analyzing the sample for at least the levels of SIAH2 and FOXA1; and prognosing the subject as having an elevated risk of invasive breast cancer based on the levels of at least SIAH2 and FOXA1. In some embodiments, depending on the nature of the result, this indicates that the subject who provided the sample is at high risk or elevated risk of invasive breast cancer (see, e.g., Tables 1-9, 11).

[0124] In some embodiments, a method is provided for prognosing the risk of invasive breast cancer events in a subject.The method includes: providing a DCIS sample from a subject; analyzing the sample for a) PR, HER2, and SIAH2, or b) PR and FOXA1; and prognosing the subject as being at high risk of invasive breast cancer events if the sample has at least one of a) PR-, HER2-, and SIAH2-, b) PR+, FOXA1+, or c) PR+, FOXA1-, and Ki67+.

[0125] In some embodiments, a method is provided for treating a subject at risk of having invasive breast cancer, the method comprising providing a subject with DCIS; the subject has DCIS that is at least one of a) PR-, HER2-, and SIAH2-, b) PR+, FOXA1+, or c) PR+, FOXA1-, and Ki67+; and administering to the subject a therapy that is more aggressive than a standard therapy for DCIS.

[0126] In some embodiments, a method is provided for prognosing the risk of an invasive breast cancer event in a subject. The method may include, as an additional conversion process, applying an algorithm to the protein and / or mRNA expression levels, thereby providing a signature for the marker. The method includes providing a DCIS sample from the subject; analyzing the DCIS sample for at least the level of PR, and at least one of analyzing the sample for at least HER2 and SIAH2 or analyzing the sample for at least FOXA1. The method further includes providing a prognosis based on at least the PR, HER2 and SIAH2 signature or based on at least the PR and FOXA1 signature. In some embodiments, depending on the nature of the result, this indicates that the subject who provided the sample is at high risk or elevated risk of invasive breast cancer (see, for example, Tables 1-9, 11, 13, and 15).

[0127] In some embodiments, a method is provided for prognosing the risk of an invasive breast cancer event in a subject. The method includes providing a DCIS sample from the subject, analyzing the sample for at least SIAH2 and FOXA1 levels, and prognosing the subject as having an elevated risk of invasive breast cancer based on the signature of at least SIAH2 and FOXA1. In some embodiments, depending on the nature of the result, this indicates that the subject who provided the sample is at high or elevated risk of invasive breast cancer (see, e.g., Tables 1-9, 11, 13, and 15).

[0128] In some embodiments, a method is provided for prognosing the risk of invasive breast cancer events in a subject. The method includes: providing a DCIS sample from a subject; analyzing the sample for a) PR, HER2, and SIAH2, or b) PR and FOXA1; and prognosing the subject as having an elevated risk of invasive breast cancer events using a signature comprising at least one of a) PR-, HER2-, and SIAH2-, b) PR+, FOXA1+, or c) PR+, FOXA1-, and Ki67+. In some embodiments, depending on the nature of the result, this indicates that the subject who provided the sample is at high risk or high risk of invasive breast cancer (see, for example, Tables 1-9, 11, 13, and 15).

[0129] In some embodiments, any of the methods, compositions, kits, systems, etc. described herein may be employed with one or more and / or combinations of the following markers (and / or other described variations), as outlined in Tables 1-9 11, 14, and 15 for markers associated with DCIS (Table 9 for the combinations listed), and Tables 11, 13, and 15 for markers associated with invasive breast cancer. The HER2 and SIAH2 analysis process described above to determine the appropriate therapy to administer may be part or a combination of any of these techniques, thereby identifying subjects at elevated risk for invasive breast cancer (through which techniques, for example, may determine whether to receive radiation therapy or any non-radiotherapy treatment).

[0130] In some embodiments, any of the above methods can be combined with any one or more of the following additional aspects relating to the method.

[0131] In some embodiments, the subject is at high risk if PR positive and there is a very high level of FOXA1 (e.g., "elevated risk"). In some embodiments, if the sample is PR positive, the sample is further analyzed for Ki67, size, or both Ki67 and size. In some embodiments, if the sample is PR positive and FOXA1 negative, the sample is further analyzed for Ki67, size, or Ki67 and size levels, where Ki67 positivity, DCIS size greater than 5mm, or both indicate high risk (e.g., "elevated risk"). In some embodiments, the method further comprises analyzing for p16, COX2, and Ki67 to determine an elevated risk level.

[0132] In some embodiments, any of the methods and / or compositions for determining elevated risk of invasive breast cancer may be used to identify subjects at high risk of invasive breast cancer as described in U.S. Patent Publication Nos. 20100003189, 20120003639, 20120003639, and 20170350895, each of which is incorporated by reference in its entirety herein.

[0133] In some embodiments, one or more of the indicated markers may be analyzed and / or assayed. In some embodiments, the markers HER2 and SIAH2 are assayed separately from other markers. In some embodiments, HER2 and SIAH2 are assayed as part of a process to determine elevated risk in a subject and / or sample. In some embodiments, HER2 and SIAH2 are assayed with one or more of ER, PR, COX-2, FOXA1, Ki67, and / or p16. In some embodiments, for subjects with DCIS, any of the following assays for markers may be performed in combination with HER2 and / or SIAH2.

[0134] In some embodiments, if the sample is PR positive, the sample may be further analyzed for the level of COX-2. In some embodiments, if the sample is PR positive, the sample may be further analyzed for Ki67 or size, or both Ki67 and size. In some embodiments, the sample may be analyzed for p16 with Ki-67 or p16 with COX-2. In some embodiments, the method further comprises analyzing at least the following combinations: a) PR, HER2, and SIAH2, b) PR, FOXA1, and COX-2, and c) PR, FOXA1, and Ki67. In some embodiments, it is determined whether a subject is HER2 positive and SIAH2 positive and has DCIS, and then it is determined whether the subject will receive radiation therapy or whether a non-radiation therapy such as an antibody against HER2 should be used.

[0135] In some embodiments, markers other than those disclosed and described herein may be analyzed and / or assayed. These additional markers are disclosed and described in U.S. Patent Application No. 12 / 373,047, filed May 13, 2009, and U.S. Patent Application No. 13 / 094,729, filed April 26, 2011, the contents of both applications being incorporated herein by reference in their entireties.

[0136] In some embodiments, the method further comprises analyzing at least COX-2, Ki67, p16, PR and HER2. In some embodiments, if the sample is PR positive, the sample may be further analyzed for COX-2 levels, where COX-2+ with at least FOXA1+ indicates an increased risk of invasive breast cancer. In some embodiments, if the sample is PR positive and there are very high levels of FOXA1, the risk of invasive breast cancer is increased. In some embodiments, if the sample is PR positive, the sample may be further analyzed for Ki67, size, or both Ki67 and size. In some embodiments, if the sample is PR positive and FOXA1-, the sample may be further analyzed for Ki67 levels, size, or both Ki67 and size levels. Ki67+, DCIS size greater than 5mm, or both indicate an increased risk of invasive breast cancer. In some embodiments, the method further comprises analyzing the sample for p16, COX-2, and Ki67. In some embodiments, it is determined whether a subject is HER2 positive and SIAH2 positive and has DCIS, and then it is determined whether the subject will receive radiation therapy or whether a non-radiation therapy, such as an antibody against HER2, should be used.

[0137] In some embodiments, the analysis of each marker is performed in parallel with one another, hi some embodiments, the analysis of each marker is performed at overlapping times.

[0138] In some embodiments, the PR analysis is performed first, and any further analysis is performed depending on the results of the PR analysis. In some embodiments, there is no order constraint on any of the testing and / or analysis for any of the markers provided herein.

[0139] In some embodiments, the method further comprises determining a prognosis for the subject with DCIS, where levels of at least SIAH2 and FOXA1 compared to non-DCIS controls indicate a poor prognosis for the subject, or no significant difference in expression of SIAH2 and FOXA1 compared to non-tumor controls indicates a good prognosis for the subject.

[0140] In some embodiments, the DCIS sample is further analyzed for COX-2. In some embodiments, the DCIS sample is further analyzed for p16. In some embodiments, the DCIS sample is further analyzed for at least SIAH2, FOXA1, and PR, and in some embodiments, the DCIS sample is further analyzed for HER2. In some embodiments, the DCIS sample is further analyzed for COX-2. In some embodiments, the DCIS sample is further analyzed for Ki67. In some embodiments, the DCIS sample is further analyzed for p16. In some embodiments, it is determined whether a subject is HER2 positive and SIAH2 positive and has DCIS, and then it is determined whether the subject will receive radiation therapy or whether a non-radiation therapy, such as an antibody against HER2, should be used.

[0141] In some embodiments, the analysis (staining and / or scoring) includes SIAH2 and FOXA1. In some embodiments, the analysis further includes at least one of the following additional combinations: 1) p16, 2) Ki67, 3) PR, 4) COX-2, 5) HER2, 6) p16 and Ki67, 7) p16 and PR, 8) p16 and COX-2, 9) p16 and HER2, 10) HER2 and Ki67, 11) HER2 and PR, 12) HER2 and COX-2, 13) p16, COX-2 and HER2, 14) HER2, COX-2, and Ki67, 15) HER2, COX-2, and PR, 16) HER2 and COX-2, 17) p16, Ki-67, COX-2, and HER2, 18) Ki-67, HER2, COX-2, and PR, and / or 19) Ki-67, HER2, COX-2, PR, and p16. In some embodiments, a subject is HER2 positive and SIAH2 positive, and it is determined whether the subject has DCIS, and then it is determined whether the subject can undergo radiation therapy or whether a non-radiation therapy, such as an antibody against HER2, should be used.

[0142] In some embodiments, the DCIS lesions are further analyzed for grade, necrosis, size, and / or margin status.

[0143] In some embodiments, the methods further include a prognosis of risk by including subject age, menopausal status, mammographic density, tumor palpability.

[0144] In some embodiments, any of the "PR" steps, methods, compositions, etc. provided herein can be interchangeable with an ER step (where ER staining and / or scoring is performed).

[0145] [Report / Recommendation] In some embodiments, any of the methods can further include preparing a report regarding the risk associated with the human DCIS tissue sample. In some embodiments, the report is a written report providing the risk of invasive breast cancer. In some embodiments, the report is generated from and / or includes one or more of the marker combinations provided in Tables 1-9, 11, and 13-15. In some embodiments, the report also details whether the subject should undergo radiation therapy or employ non-radiation therapy, such as an antibody against HER2.

[0146] In some embodiments, any of the methods further include providing a report regarding the level of risk of a subsequent DCIS event. In some embodiments, the report is a written report providing the risk of a subsequent DCIS event. In some embodiments, the report is generated from and / or includes one or more of the marker combinations provided in Tables 1-9, 11, and 13-15.

[0147] In some embodiments, any of the methods further include recommending a given treatment in light of the results obtained from analyzing the DCIS sample for SIAH2 and at least one of HER2, PR, FOXA1, or any combination thereof. In some embodiments, the treatment is less aggressive than another treatment that would have been recommended in the absence of a method for predicting a low likelihood of invasive breast cancer. In some embodiments, the treatment is more aggressive than another treatment that would have been recommended in the absence of a method for predicting a high likelihood of invasive breast cancer. In some embodiments, the treatment is less aggressive than another treatment that would have been recommended in the absence of a method for predicting a low likelihood of DCIS recurrence. In some embodiments, the treatment is more aggressive than another treatment that would have been recommended in the absence of a method for predicting a high likelihood of DCIS recurrence. In some embodiments, the report also details whether the subject should receive radiation therapy or use a non-radiation therapy, such as an antibody against HER2 (e.g., depending on the results of HER2 and SIAH2).

[0148] In some embodiments, the method further includes determining the risk of DCIS, invasive breast cancer, or both. In some embodiments, the method further includes providing a written report regarding the risk of DCIS, invasive breast cancer, or both (e.g., in accordance with Tables 1-9, 11, and 13-15) and whether the subject should receive non-radiotherapy (such as an antibody against HER2) or radiation therapy.

[0149] [Treatment] In some embodiments, any of the above methods can include and / or be followed by appropriate treatment for the subject, taking into account the reclassified risk of subsequent DCIS and / or invasive breast cancer, and / or responsiveness to radiation therapy (for those with high risk of invasive breast cancer category). In some embodiments, such treatment can be appropriate to reduce the risk of invasive breast cancer if there is a risk of invasive breast cancer. In some embodiments, for those with high risk of invasive breast cancer, appropriate treatment of non-radiation (for those who are HER2+ and SIAH+) or radiation therapy (for those who are not both HER2+ and SIAH+) can be provided to or received by the subject. In some embodiments, the non-radiation therapy is an antibody against HER2, such as trastuzumab.

[0150] In some embodiments, the treatment suitable for reducing the risk of DCIS recurrence includes at least one of surgical resection, radiation therapy, and anti-hormonal therapy. In some embodiments, the treatment method may be suitable when the subject knows that the probability of an invasive event is low, but would not be suitable when the subject knows that the probability of an invasive breast cancer event is high and the subject knows how likely it is that the subject will be refractory to radiation therapy.

[0151] In some embodiments, the treatment suitable for reducing the risk of invasive breast cancer comprises at least one of mastectomy, targeted HER therapy, and receptor-targeted chemotherapy. In some embodiments, such a treatment method may be suitable when the subject knows that the subject has a high probability of invasive events, but is not suitable when the subject knows that the subject has a low probability of invasive breast cancer events. In some embodiments, the treatment is suitable when the subject does not respond to the treatment. In some embodiments, the subject predicted to be refractory to radiation therapy does not receive or is not administered radiation therapy.

[0152] In some embodiments, any of the above methods can be followed by "watchful waiting" or other relatively minimally / intrusive treatments. For example, if none of the high-risk categories for invasive breast cancer are met and the subject is not at risk for DCIS (or is not concerned about being at risk for DCIS), the approach to treating DCIS is to not take immediate action and may include more frequent breast imaging to identify ipsilateral breast events early.

[0153] Additional aspects and approaches regarding possible therapeutic actions specific to this DCIS subject are provided below.

[0154] [kit] In some embodiments, a kit is provided. The kit can include a FOXA1 probe and a SIAH2 probe. In some embodiments, the kit further includes a COX-2 probe, a Ki67 probe, a p16 probe, a PR probe, and a HER2 probe. In some embodiments, the probe is an isolated antibody. In some embodiments, the probe is a nucleic acid that suitably selectively hybridizes to FOXA1, SIAH2, COX-2, Ki67, p16, PR, or HER2. In some embodiments, the kit includes a sufficient amount of probe and / or the probe has sufficient sensitivity and / or selectivity such that the "+" and "-" states of one or more of the markers in Tables 1-9, 11, and 13-15 can be suitably distinguished from one another. In some embodiments, any of the kits provided herein includes at least sufficient probes for HER2 and SIAH. In some embodiments, the HER2 and SIAH2 probes can be part of the kit themselves or can be implemented separately.

[0155] In some embodiments, an antibody composition is provided that includes an isolated FOXA1 antibody and an isolated SIAH2 antibody. In some embodiments, the antibody composition further includes an isolated COX-2 antibody, an isolated Ki67 antibody, an isolated p16 antibody, an isolated PR antibody, and an isolated HER2 antibody. In some embodiments, the HER2 antibody and the SIAH2 antibody are provided in combination with one or more other antibodies.

[0156] In some embodiments, a solid support is provided that comprises a probe or antibody specific for at least SIAH2 and FOXA1. In some embodiments, the probe or antibody consists essentially of a probe or antibody specific for predicting DCIS or invasive breast cancer in subjects with DCIS, comprising at least HER2 and SIAH. In some embodiments, a solid support is provided that comprises a probe or antibody specific for at least SIAH2 and HER2.

[0157] In some embodiments, the subject and / or sample being analyzed may be (or may be derived from) a patient. In some embodiments, the subject has or has had DCIS. In some embodiments, the sample is derived from DCIS in the subject in question. There are various ways in which such subjects may be identified.

[0158] [sample] In some embodiments, the DCIS sample itself may be processed in any number of ways to prepare it for screening for markers. In some embodiments, the DCIS sample is surgically removed from the patient and stored. In some embodiments, the DCIS sample is obtained by surgical removal. In some embodiments, the DCIS sample is cut into one or more blocks, such as 2, 3, 4, 5 or more blocks.

[0159] In some embodiments, the levels of SIAH2 and HER2, and / or PR, FOXA1, or any combination thereof, are at least one of an RNA level, a DNA level, or a protein level. In some embodiments, the levels of SIAH2 and HER2, and / or PR, FOXA1, or any combination thereof, are at least one of an RNA level, a DNA level, or a protein level. In some embodiments, the levels of SIAH2 and HER2 and FOXA1 are at least one of an RNA level, a DNA level, or a protein level.

[0160] In some embodiments, the signature comprising the levels of SIAH2 and HER2, PR, FOXA1, or a combination thereof is at least one of the following: RNA level, DNA level, protein level. In some embodiments, the signature comprising the levels of SIAH2 and HER2, and at least one of PR or FOXA1 is at least one of the following: RNA level, DNA level, protein level. In some embodiments, the signature comprising the levels of SIAH2 and HER2, and at least one of FOXA1 is at least one of the following: RNA level, DNA level, protein level.

[0161] In some embodiments, a method of preparing a sample is provided, comprising obtaining a DCIS sample from a subject and preparing the sample so that its DNA, RNA, and / or protein can be analyzed for at least SIAH2 and HER2 and / or FOXA1.

[0162] In some embodiments, the sample is preserved. In some embodiments, the sample is preserved by freezing. In some embodiments, the sample is embedded in a chemical such as optimal cutting temperature (OCT) compound or undergoes fixation (or not) with a chemical (including but not limited to formalin, formaldehyde, quaternary ammonium salts, alcohol, acetone, or other chemicals that preserve or extract DNA, RNA, and / or protein). In some embodiments, the technique used is one that allows the DNA, RNA, and / or protein of SIAH2 and HER2 and / or FOXA1 to be preserved in the appropriate amount and conditions so that SIAH2 and HER2 and / or FOXA1 can be analyzed as provided herein.

[0163] In some embodiments, DCIS samples are processed to allow for immunohistochemistry for at least SIAH2 and HER2 and / or FOXA1. In some embodiments, at least three such samples (such as in the form of slices) can be prepared.

[0164] In some embodiments, analyzing the sample includes determining the amount of a particular RNA in the sample. The amount of RNA for each marker can be determined by any number of techniques, some of which are discussed elsewhere in this application. In some embodiments, the RNA level is determined by at least one of the following assays: nucleic acid microarray, reverse transcriptase-polymerase chain reaction, in situ nucleic acid detection, or next-generation sequencing. In some embodiments, the expression of at least one of SIAH2, HER2, and FOXA1 is measured by real-time quantitative polymerase chain reaction or microarray analysis.

[0165] In some embodiments, RNA levels are determined by assays including nucleic acid microarrays, reverse transcriptase-polymerase chain reaction, in situ nucleic acid detection, or next generation sequencing.

[0166] In some embodiments, analyzing the DCIS sample includes determining the amount of a particular protein in the sample. The amount of protein for each marker may be determined by any number of techniques, some of which are discussed elsewhere in this application. In some embodiments, the protein levels are determined by immunohistochemistry, immunofluorescence, or mass spectrometry.

[0167] In some embodiments, patient specimens used for biomarker detection can be surgically removed breast tissue cut into small blocks and immersed in fixative. In some embodiments, after fixation, the blocks can be dehydrated and then embedded in paraffin wax. In some embodiments, the small blocks are no more than 20 mm in length and no more than 5 mm in thickness to allow complete penetration of the fixative. In some embodiments, the specimens are fixed in 10% neutral buffered formalin at room temperature for 24-48 hours to preserve tissue architecture and compartmentalization of the various markers. However, other fixatives and fixation times (e.g., 6-72 hours) may also be compatible with the marker assay. In some embodiments, the assay is optimized to use specimens that are flash frozen (e.g., in liquid nitrogen) rather than fixed and embedded.

[0168] In some embodiments, the process of sample processing may include dehydration and embedding, which may be done manually or automated using tissue processing equipment. In either case, the aqueous portion of the tissue and fixative solution may be replaced by passing the block through a series of increasingly more concentrated alcohol solutions. After reaching 100% alcohol, the alcohol is replaced with a chemical such as xylene (or a xylene-free equivalent), followed by the introduction of molten low-melting point (e.g., about 45° C.) paraffin wax for embedding. FFPE blocks may be stored for many years before analysis. In some embodiments, DCIS tissue "cores" may be excised from these blocks using a hollow needle and then inserted in an array format into separate paraffin blocks. Such "tissue microarrays" (TMAs) allow the evaluation of multiple tissues on a single section / microscope slide.

[0169] In some embodiments, a microtome can be used to cut ultrathin sections, approximately 3-5 micrometers thick, from formalin-fixed, paraffin-embedded (FFPE) tumor blocks. Sections can be mounted on glass microscope slides to ensure that the tissue does not fold or fragment and interfere with the assay. The glass slides can have a positively charged surface to bind the negatively charged tissue sections, although other methods of tissue attachment, including adhesives, can also be adapted.

[0170] In some embodiments, dewaxing and rehydration of the tissue sections can then be performed. These processes can be performed manually or automated using specific staining equipment. Wax can be removed from the tissue sections on the slides by heating and / or immersion in a solution of xylene (or an equivalent xylene-free solution such as, for example, Novocastra Bond Dewaxing Solution). Rehydration can be achieved by passing the slides through a series of low-concentration alcohol solutions until the concentration reaches 0% (pure water). Following dewaxing and rehydration, the tissue sections can be stained with hematoxylin and eosin (H&E) and evaluated by a pathologist or histologist as described below after staining for various molecular markers using immunohistochemistry (IHC) and / or in situ hybridization (ISH) assays. The above processing steps can be performed for any of the methods provided herein for various markers (HER2 and SIAH2, and at least one of COX-2, Ki-67, PR, p16, and FOXA1).

[0171] [DCIS diagnosis and evaluation of pathological factors - hematoxylin and eosin staining] In some embodiments, the subject and / or sample is identified as a DCIS sample or a subject with DCIS by any of a variety of methods known to those of skill in the art. This can (in some embodiments) be done prior to any of the other method steps provided herein. Provided herein is a non-exhaustive set of options for identifying a person with DCIS.

[0172] In some embodiments, hematoxylin and eosin (H&E) may be used to stain at least one tissue section from each patient (or set of sequenced patients) to confirm the DCIS diagnosis, to evaluate certain pathological features (nuclear grade, architectural pattern, and presence or absence of necrosis), and as a reference for the interpretation of molecular marker assays. This histological staining allows for the differentiation of the nuclei and cytoplasm of individual cells, as well as the various cellular and interstitial tissue components, based on the color of the stain. Staining may be performed manually or automated using dedicated staining instruments. In either case, the sections may be immersed in a hematoxylin solution for about 4 minutes to stain the nuclei blue, and then rinsed with tap water (alkaline). In some embodiments, the sections may then be exposed briefly (typically just a few seconds) to an acidic alcohol solution to remove the background staining of the hematoxylin, and then rinsed with tap water (alkaline). A "blueing" solution (e.g., lithium carbonate for 30-60 seconds) may then be applied to enhance the blue color of the hematoxylin in the nuclei, followed by a water rinse. Eosin solution is then applied for about 2 minutes to stain other (eosinophilic) cellular components, followed by rinsing with water. The tissue is dehydrated in an alcohol series, cleared in xylene (or equivalent), and a cover slip is attached using a mounting medium. Other options are known to those skilled in the art. In some embodiments, any method for confirming the current presence of DCIS can be used on the sample. In some embodiments, no confirmation process is required. The above variations can be modified as appropriate by those skilled in the art.

[0173] DCIS itself can be identified in a sample or subject in a variety of ways. Intraductal proliferative lesions include a group of cytologically and architecturally diverse epithelial proliferations originating from terminal duct lobular units (TDLUs) and can be associated with an increased risk (of varying magnitude) of subsequent invasive breast cancer development. DCIS can be considered as a true precursor lesion that may lead to invasive breast cancer. However, as has been demonstrated, not all DCIS events progress to form invasive breast cancer. There are various grades of DCIS (all of which are encompassed within the term "DCIS" unless otherwise indicated).

[0174] "Low-grade DCIS" is composed of small monomorphic cells that grow in a variety of patterns, including arcaded, micropapillary, cribriform, and solid. Nuclei are generally uniform in size, display regular chromatin patterns, nucleoli are inconspicuous, and mitotic figures are rare. Low-grade DCIS requires either two spaces or involvement of one or more duct cross sections greater than 2 mm in diameter. Although exfoliated cells within the duct lumen may be present, overt necrotic / comedo-type histological features are not typical of low-grade DCIS. In some embodiments, the definition is the CAP definition.

[0175] The cytologic features of DCIS include: a uniform, monotonous population of round cells; a slightly increased nuclear-cytoplasmic ratio; equidistant or highly organized nuclear distribution; round nuclei; and hyperchromasia, with or without. Architectural features may include arcadian, cribriform, solid, and / or micropapillary.

[0176] "Intermediate-grade DCIS" is often composed of cells similar to low-grade DCIS cytologically, forming a solid, cribriform, or micropapillary pattern, but with some ducts containing intraluminal necrosis. Some may show intermediate-grade nuclei, sometimes with nucleoli and coarse chromatin; necrosis may or may not be present.

[0177] "High-grade DCIS" is usually larger than 5 mm, but even a single <1 mm duct with typical morphological features is sufficient for diagnosis. It is composed of cells proliferating as a layer and forming a highly amorphous, micropapillary, cribriform or solid pattern. The nuclei are high grade, markedly pleomorphic and poorly polarized, with irregular contours and distribution, coarse, clumped chromatin and prominent nucleoli. Mitotic figures are usually common, but their presence is not required. Comedoncrosis is often associated with high-grade DCIS, but is not required for diagnosis. A single layer of highly undifferentiated cells lining the duct in a flat manner is sufficient.

[0178] "Atypical ductal hyperplasia" is different from DCIS. The morphological characteristics of atypical ductal hyperplasia are identical to those of low-grade DCIS, but the size of ADH is limited. There are two quantitative criteria that distinguish ADH from low-grade DCIS: the presence of homogeneous involvement of two or less membrane-bound spaces; or a size of 2 mm or less. The use of either or both criteria is considered appropriate by the authors of the WHO classification. In some embodiments, the definition is the CAP definition.

[0179] [Histological confirmation of DCIS] In some embodiments, the subject has at least one form of DCIS. In some embodiments, the presence of DCIS can be confirmed by any of a variety of techniques, including, for example, using slide-mounted tissue sections stained with hematoxylin and eosin (H&E) or equivalent histological stains (see above). In some embodiments, the evaluation can be performed according to the WHO classification of breast tumors (Lakhani SR. WHO classification of tumours of the breast. Lyon: International Agency for Research on Cancer, 2012, and Tavassoli FA, Devilee P. Pathology and genetics of tumours of the breast and female genital organs. Lyon: IARC Press, 2003) - see the section on the definition of DCIS. These references include example images and review the features of DCIS and its differential diagnosis with other breast entities such as invasive breast cancer (including microinvasion defined as invasion ≦1 mm), lobular carcinoma in situ (LCIS), Paget's disease in situ, atypical ductal hyperplasia (ADH), sclerosing adenosis, etc. These references are incorporated herein by reference in their entireties.

[0180] In some embodiments, if the histologic features are not sufficient for a diagnosis of DCIS, the diagnosis may be confirmed by a second pathologist. Additional tissue blocks may be used for morphologic review.

[0181] [Cases suspected to be invasive (or microinvasive) carcinoma based on morphological characteristics:] Normal breast ducts and lobules, as well as intraductal epithelial proliferations, are composed of two epithelial layers. Loss of the outer myoepithelial layer is characteristic of invasive carcinoma of the breast. The outer myoepithelial layer is preserved in ductal carcinoma in situ and all benign proliferative processes alike. Thus, identifying the presence or absence of myoepithelium using antibodies against myoepithelial-specific proteins may be useful in distinguishing in situ from invasive carcinoma in situations where morphology is equivocal (Kalof AN et al., Kalof AN, Tam D, Beatty B, Cooper K. Immunostaining patterns of myoepithelial cells in breast lesions: a comparison of CD10 and smooth muscle myosin heavy chain. J Clin Pathol 2004; 57, 625-629; Barbareschi M et al., Barbareschi M, Pecciarini L, Cangi MG et al. p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol; 25, 1054-1060, 2001).

[0182] In some embodiments, if there is clear morphological evidence of invasion, including microinvasion, the patient may be considered unsuitable for prognostic DCIS testing (and may not be tested or excluded from the assay). In some embodiments, if there is a question of microinvasion upon morphological examination of the tumor lesion, additional myoepithelial marker immunostudies (p63 and / or smooth muscle myosin heavy chain (SMMHC) immunostaining) may be performed to examine the continuity of the myoepithelial cell layer and to confirm and / or exclude the presence of (micro)invasive carcinoma. In some embodiments, other follow-up procedures may be performed for confirmation, if necessary.

[0183] [Cases suspected to be of lobular origin based on morphological features:] It has been demonstrated that even in the setting of nonclassical morphologic findings, loss of expression of E-cadherin by immunohistochemistry can confirm the diagnosis of lobular carcinoma in histologic situations where ductal and lobular neoplasms may be confused, especially in the setting of carcinoma in situ (Acs G et al., Acs G, Lawton TJ, Rebbeck TR, LiVolsi VA, Zhang PJ. Differential expression of E-cadherin in lobular and ductal neoplasms of the breast and its biologic and diagnostic implications. Am J Clin Pathol 2001; 115, 85-98, 2001). In lobular neoplasms, mutations in the E-cadherin gene result in loss of expression of E-cadherin, a cell surface adhesion molecule present in normal breast epithelium and ductal carcinomas. The role of E-cadherin in homotypic cell-cell junctions, and loss of expression of this cell surface protein are responsible for the characteristic noncohesive growth pattern of lobular carcinoma.

[0184] In some embodiments, when examining intraductal epithelial proliferation, if it is unclear whether the intraductal tumor is ductal or lobular in nature, E-cadherin immunostaining can be performed to confirm ductal or lobular differentiation. In some embodiments, if lobular carcinoma in situ (LCIS) is confirmed histologically or by loss of E-cadherin by immunohistochemistry, the patient may not be eligible for further prognostic testing (e.g., may not be tested or may be excluded from the assay). In some embodiments, if the subject currently has LCIS and does not have DCIS, the subject is not treated with the method. In some embodiments, if there is no evidence of DCIS or invasive carcinoma, additional tissue blocks may be requested for morphological review.

[0185] In some embodiments, a sample or subject is excluded from the method if one or more of the following apply: a) no DCIS identification, b) identification of invasive or microinvasive carcinoma, c) identification of LCIS rather than DCIS, c) quantitative criteria for low / intermediate DCIS are not met: 1) presence of homogeneous involvement of more than 2 membrane-bound spaces and / or size ≦2 mm (quantitative criteria not required for high-grade DCIS), or 2) there is folded tissue in the region of interest - not possible to score adequately.

[0186] [Determination of nuclear grade] In some embodiments, DCIS nuclear grade may be determined by using slide-mounted tissue sections stained with Hematoxylin and Eosin (H&E) or equivalent histological stain. In some embodiments, the evaluation is performed according to the College of American Pathologists "Protocol for Examination from Patients with Ductal Carcinoma in Situ (DCIS) of Breast" (Lester SC, Bose S, Chen YY et alArch Pathol Lab Med 2009; 133, 15-25). Nuclear grades of I (low), II (intermediate), and / or III (high) are described according to Table 12: [Table 12]

[0187] It is not uncommon to find a mixture of DCIS of various grades in the same biopsy. In some embodiments, if more than one grade of DCIS is present, the percentage of each grade (percentage in decimals) is noted. In some embodiments, a subject with extensive disease and high-grade DCIS would not be considered to be at low risk for a subsequent ipsilateral breast event. In some embodiments, any of the methods provided herein can begin with first determining whether the subject has DCIS and / or DCIS nuclear grade. In some embodiments, the method does not include determining the nuclear grade.

[0188] [Necrosis determination] In some embodiments, the presence and extent of necrosis in DCIS can be examined using slide-mounted tissue sections stained with hematoxylin and eosin (H&E) or equivalent histological stain. This evaluation is performed based on the references in the College of American Pathologists "Protocol for the Examination of Specimens from Patients with Ductal Carcinoma in Situ (DCIS) of the Breast" (June 2012). In some embodiments, necrosis can be classified as follows: A) unspecified: no evidence of necrosis, B) focal (punctate): small foci, unclear at low magnification, or single-cell necrosis, or C) intermediate (comedo-like / extensive): the central portion of the involved lumen is replaced by an area of ​​widespread necrosis that is easily detected at low magnification. Ghost cells and karyorrhectic debris are generally present. Central necrosis is commonly associated with high-grade nuclear (comedo DCIS), but can also occur in low or intermediate nuclear grade DCIS. In some embodiments, any of the methods provided herein can include determining necrosis.

[0189] [IHC marker staining / scoring] When formalin-based fixation methods are used, they create molecular bridges in the protein, thereby masking epitopes from recognition by antibodies, and other fixation / preservation methods may also mask epitopes. In such embodiments, epitope retrieval may be a pretreatment step that allows for unmasking of epitopes by at least partially reversing the changes introduced by fixation / preservation. Thus, in some embodiments, any of the methods and / or kits provided herein may include a step or component for epitope retrieval.

[0190] Epitope retrieval can be performed in different ways by varying the chemicals in the solution (e.g., buffers, proteases, chelating agents, etc.), the pH of the solution, the temperature of the solution (e.g., as applied by a water bath, pressure oven, autoclave, or microwave), and / or the time in the solution, etc. In addition to the specific methods described in the examples below, many of these other methods can be used to achieve substantially equivalent results, depending on the tissue source, primary antibody, and other factors.

[0191] For each protein marker described herein (COX-2, Ki67, HER2, p16, PR, SIAH2, and FOXA1, or their products in Table 0.1), multiple antibodies are commercially available and / or reported in the literature, but new antibodies can also be generated. In some embodiments, the antibodies are raised against and / or recognize different epitopes on the protein markers (COX-2, Ki67, HER2, p16, PR, SIAH2, and FOXA1), and in other cases, the antibodies are raised against and / or recognize the same (or similar) epitope. The usefulness of an individual antibody in an assay depends on its affinity and specificity for the epitope, as well as the accessibility of the epitope in the assay (e.g., after epitope retrieval in an IHC assay). Some antibodies recognize more than one protein marker and are therefore typically not suitable for specific marker assays. Alternatively, an antibody may have low affinity or recognize an epitope that remains inaccessible in a particular sample, making it unsuitable for a particular assay type, e.g., an antibody that can be used for immunoblotting of fresh protein lysates may not be useful for an IHC assay on FFPE tissues because it cannot unmask its epitope through epitope retrieval.

[0192] The concentrations of primary antibody concentrates available from manufacturers vary depending on the method of production (e.g., tissue culture supernatant, ascites, or total antiserum) and whether any purification has been performed (e.g., affinity purification), but generally range from about 0.1 to 10 mg / ml. The optimal final primary antibody concentration for incubation on sections depends on factors such as the binding properties of the specific antibody, the incubation time and temperature, and other factors specific to individual laboratories, but generally ranges from 0.1 to 10 μg / ml, with dilutions ranging from about 1:10 to 1:1,000 being commonly used. In some embodiments, staining results can be obtained with antibodies over a range of incubation times, temperatures, and final primary antibody concentrations.

[0193] In some embodiments, the Novocastra Bond Refine Polymer system can be used for detection of the primary antibody. This system includes a polymer backbone to which multiple secondary antibodies (against rabbit IgG) and enzymes are attached, as well as a rabbit anti-mouse IgG linker (when used with mouse primary antibodies). The enzyme catalyzes a chemical reaction with DAB to form a brown precipitate, which is visualized during marker scoring. In some embodiments, one of several other detection methods can provide suitable results, including systems that utilize avidin-biotin complex (ABC), labeled streptavidin-biotin (LSAB), catalytic signal amplification, and / or other technologies available in various formats from many different manufacturers. In some embodiments, ABC and other polymer-based technologies (e.g., Dako EnVision+) can be used (for detection) with similar results.

[0194] In some embodiments, the chromogen DAB can be used for final visualization of the marker through an enzymatic reaction of horseradish peroxidase (HRP) which produces a brown precipitate at the antibody binding site. In some embodiments, HRP can be used in combination with the chromogen 3-amino-9-ethylcarbazole (AEC) to produce a red coloration with substantially equivalent results. Alternatively, the enzyme alkaline phosphatase in combination with the chromogens nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) or the enzyme glucose oxidase in combination with NBT also produce a blue-purple coloration. There are a variety of enzyme / chromogen combinations that can produce results.

[0195] The following sections outline various representative embodiments for staining and scoring seven markers (PR, HER2, COX-2, Ki-67, SIAH2, FOXA1, and p16). In some embodiments, other markers that perform the same function can be used in place of any one or more of these markers. In some embodiments, any one or more of these markers can be used in combinations suggested in the attached tables (Tables 1-9, 11, and 13-15). In some embodiments, one or more of the listed markers can be used (e.g., for staining and / or scoring), but a corresponding staining technique and / or a corresponding score is used instead (as outlined herein). In some embodiments, a method of performing HER2 and SIAH2 analysis to determine whether a person is at high risk for DCIS can then use the same HER2 and SIAH2 data to determine whether the subject will respond to radiation therapy or should instead receive a non-radiation therapy, such as a HER2 antibody, such as trastuzumab.

[0196] [PR staining] In some embodiments, any technique for PR staining may be used as long as it is sufficient to observe the degree of PR variation provided and described herein. In some embodiments, protein levels may be checked. In some embodiments, mRNA levels may be checked. In some embodiments, DNA levels may be checked. In some embodiments, both protein and mRNA levels may be checked. A high level may be a level above a control or standardized level, e.g., a level in a non-DCIS sample. Similarly, a reduced level may be a level below a control or standardized level, e.g., a level in a non-DCIS sample. In some embodiments, a "positive" or "high" result is more than a "negative" or "low" result (in the context of scoring).

[0197] In some embodiments, to assess progesterone receptor (PR; PGR; HGNC:8910) by IHC, the following steps can be performed using a Leica BOND-MAX automated staining instrument, with washing between each step: Dewaxed and rehydrated tissue sections can be treated with Novocastra Peroxide Block (3-4% hydrogen peroxide) (peroxidase blocking step), followed by treatment with Novocastra Bond Epitope Retrieval Solution 1 (10 mM sodium citrate buffer + 0.05% Tween 20, pH 6.0 solution base) at 95°C-100°C for 30 minutes (epitope retrieval step). The tissue sections are then incubated with mouse monoclonal antibody PgR 636 (Dako M3569) diluted 1:50 in Novocastra Primary Antibody Diluent for 30 minutes at room temperature (primary antibody step), followed by Novocastra Post Primary solution for 15 minutes (rabbit anti-mouse antibody to introduce rabbit anti-IgG linker), followed by Novocastra Bond Refine Polymer for 15 minutes (anti-rabbit poly-HRP-IgG) (secondary detection step), followed by 3,3'-diaminobenzidine (DAB) for 5 minutes (chromogen visualization step), followed by <0.1% hematoxylin for 7 minutes (nuclear counterstain step). Finally, a coverslip is mounted using mounting medium. In other embodiments, other options can be used.

[0198] In some embodiments, PR can be detected by a number of primary antibodies from a number of different manufacturers, and most will produce suitable results. In some embodiments, mouse monoclonal 1A6 and rabbit monoclonal SP2 from Novocastra and Lab Vision can be used. Other options include mouse monoclonals PgR1294, 16, 1A6, 1E2, Ab-8, Ab-9, hPRa2, hPRa3, and PR88; rabbit monoclonals SP2, SP42, Y85, and EP2; and rabbit polyclonal A0097 (Dako), as well as others from manufacturers such as Dako (Agilent), Novocastra (Leica), Ventana Medical Systems (Roche), Cell Marque (Sigma-Aldrich), Lab Vision (Thermo Scientific), BioGenex, Biocare, and Epitomics. In some embodiments, other options can be used.

[0199] In some embodiments, high pH epitope retrieval (pH 9) can be performed using microwave heating in Tris-EDTA buffer. The titer of each lot of Dako PgR 636 antibody can be adjusted by the manufacturer to a reference lot to ensure consistent staining performance at the same dilution factor, and the 1:50 dilution used in the above example is suggested by the manufacturer. However, a range of dilutions (e.g., 1:10 to 1:500) with similar performance can be used. Other dilutions may be optimal for other antibody preparations, and in some cases, some preparations are provided pre-diluted (ready to use). In some embodiments, other options can be used.

[0200] [PR Scoring] In some embodiments, any technique for PR scoring can be used, so long as it is sufficient to observe the degree of PR variation provided and described herein.

[0201] In some embodiments, PR status is determined from IHC stained slides based on the percentage of DCIS tumor cells with nuclear signal. In some embodiments, all areas of the tissue section containing DCIS can be evaluated to arrive at the percentage. In some embodiments, at least three DCIS-containing ducts or 1 mm of DCIS tissue can be used to score the markers.

[0202] In some embodiments, the intensity of the nuclear signal may be reported as weak (1+), moderate (2+), or strong (3+). Intensity is the average intensity of DCIS tumor cell nuclei with signal across the entire tissue section relative to the intensity of a positive control run in the same staining batch. In some embodiments, the selection and / or scoring of the DCIS areas to be scored is performed manually by a pathologist and / or automatically using a computer on scanned images of the slides.

[0203] In some embodiments, DCIS with less than 10% of tumor cells having a nuclear signal can be considered negative, while DCIS with more than 10% of tumor cells having a nuclear signal can be considered positive when assayed by the system or examples described herein. In some embodiments, a sample is considered negative only in the presence of appropriately stained external and internal controls. In some embodiments, any specimen lacking a negative internal control element (normal ductal epithelium) should be reported as uninterpretable (rather than negative) and repeated using another tumor specimen from the same or an alternative tumor block.

[0204] In some embodiments, alternative thresholds can be applied based on the technique used for staining and / or analysis (e.g., 0% vs. >0%, <1% vs. ≥1%, ≤1% vs. >1%, <5% vs. ≥5%, ≤5% vs. >5%, <10% vs. ≥10%, <15% vs. ≥15%, ≤15% vs. >15%, <20% vs. ≥20%, ≤20% vs. >20%, <25% vs. ≥25%, ≤25% vs. >25%, <30% vs. ≥30% or other thresholds). In some embodiments, alternative techniques and / or scoring methods can be used for detection, which can result in corresponding but different cut-off ranges for high and / or low risk. In such situations, the ranges provided herein for the present technique can be correlated with other techniques (for "corresponding values") by analyzing the same sample (or two samples from the same DCIS sample) by two different techniques and identifying them as equivalent to each other. Alternative scoring methods, such as the Allred scoring system, immunoscore, and other methods that combine rate and intensity scoring components, also show validation.

[0205] In some embodiments, PR scoring can be as follows: negative is less than 5 percent positive in the percentage scoring of IHC; positive is 10 percent or more positive in the percentage scoring of IHC. In some embodiments, the percentage difference between positive and negative can be compressed so that any sample is either positive or negative. In some embodiments, the difference between positive and negative can be dropped or ignored if the sample is within the range. In some embodiments, any value between positive and negative can be selected as the absolute discrimination line between positive and negative (e.g., 5, 6, 7, 8, 9, or 10).

[0206] In some embodiments, FOXA1 scoring can be as follows: negative is less than 100 immunoscore (intensity x percentage) in IHC scoring; positive is greater than 100 immunoscore (intensity x percentage) in IHC scoring. In some embodiments, the percentage difference (100) between positive and negative can be compressed so that any sample is either positive or negative. In some embodiments, if a sample is within a range, the difference between positive and negative can be dropped or ignored. In some embodiments, any value between positive and negative can be selected as the absolute discrimination line between positive and negative (e.g., 100 or less is negative while 100 or more is positive).

[0207] In some embodiments, SIAH2 scoring can be as follows: negative is less than 10 percent positive in the IHC percentage scoring; positive is more than 20 percent positive in the IHC percentage scoring. In some embodiments, the percentage difference between positive and negative can be compressed so that any sample is either positive or negative. In some embodiments, if a sample is within the range, the difference between positive and negative can be dropped or ignored. In some embodiments, any value between positive and negative can be selected as the absolute discrimination line between positive and negative (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0208] In some embodiments, HER2 scoring can be as follows: negative is less than a 3+ score by the HercepTest scoring criteria for IHC; positive is a 3+ score by the HercepTest scoring criteria for IHC.

[0209] In some embodiments, Ki67 scoring can be as follows: negative is less than 10 percent positive for IHC percentage scoring; positive is more than 15 percent positive for IHC percentage scoring. In some embodiments, the percentage difference between positive and negative can be compressed so that any sample is either positive or negative. In some embodiments, if a sample is within the range, the difference between positive and negative can be dropped or ignored. In some embodiments, any value between positive and negative can be selected as the absolute discrimination line between positive and negative (e.g., 10, 11, 12, 13, 14, or 15).

[0210] In some embodiments, p16 scoring can be as follows: negative is less than 20 percent positive in the percentage scoring of IHC; positive is more than 25 percent positive in the percentage scoring of IHC. In some embodiments, the percentage difference between positive and negative can be compressed so that any sample is either positive or negative. In some embodiments, if a sample is within the range, the difference between positive and negative can be dropped or ignored. In some embodiments, any value between positive and negative can be selected as the absolute discrimination line between positive and negative (e.g., 20, 21, 22, 23, 24, or 25).

[0211] In some embodiments, COX-2 scoring can be as follows: negative is less than 6 by the Allred scoring criteria for COX-2 by IHC; positive is greater than 6 by the Allred scoring criteria for COX-2 by IHC.

[0212] In some embodiments, the above ranges are for an IHC assay. In some embodiments, the above ranges are for a FISH assay.

[0213] In some embodiments, the positivity threshold can be higher, depending on the combination of markers used in concert as determined by one of skill in the art to incorporate into a particular test.

[0214] In some embodiments, SIAH2, FOXA1, and COX-2 can be further divided into high (very high, high) and low (very low, low) subcategories. This allows finer lines to be drawn regarding risk combinations. In some embodiments, this is simplified for analysis by placing very high and high in the "high" group and very low and low in the "low" group.

[0215] [HER2 staining] [HER2 IHC] In some embodiments, any technique for HER2 staining can be used as long as it is sufficient to observe the degree of HER2 variation provided and described herein. In some embodiments, protein levels can be checked. In some embodiments, mRNA levels can be checked. In some embodiments, DNA levels can be checked. In some embodiments, both protein and mRNA levels can be checked. A high level can be a level higher than a control or standardized level, e.g., a level in a non-DCIS sample. Similarly, a reduced level can be a level lower than a control or standardized level, e.g., a level in a non-DCIS sample. In some embodiments, a "positive" or "high" result is one that exceeds a "negative" or "low" result (in the context of scoring).

[0216] In some embodiments, to assess v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2; HGNC:3430; HER2 [human epidermal growth factor receptor 2]; NEU) by IHC, the following steps can be performed (through the chromogen visualization step) with rinsing between each step on a Dako Autostainer: Dewaxed and rehydrated tissue sections can be treated with Dako Peroxidase Blocking Reagent (peroxidase blocking step) followed by treatment with Dako HercepTest Epitope Retrieval Solution (10 mM citrate buffer + detergent, pH 6) in a 95°C-99°C water bath for 40 minutes (epitope retrieval step). The tissue sections are then incubated with pre-diluted rabbit polyclonal anti-HER2 antibody (Dako K5207) for 30 min at room temperature (primary antibody step), followed by incubation with Dako HercepTest Visualization Reagent for 30 min (secondary detection step), followed by incubation with 3,3'-diaminobenzidine (DAB) for 10 min (chromogen visualization step), followed by incubation with hematoxylin (nuclear counterstain step). Finally, the sections are cover slipped using mounting medium.

[0217] In some embodiments, HER2 can be detected by various antibodies against HER2. In some embodiments, mouse monoclonal TAB250 and rabbit monoclonal SP3 can be used from Invitrogen and Lab Vision. Other options include mouse monoclonals CB-11, TA9145, Ab-17, 3B5, and PN2A, and rabbit monoclonals SP3, 4B5, EP1045Y, and EP3, as well as many others from manufacturers such as Dako (Agilent), Novocastra (Leica), Ventana Medical Systems (Roche), Cell Marque (Sigma-Aldrich), Lab Vision (Thermo Scientific), BioGenex, etc.

[0218] In some embodiments, heat-induced epitope retrieval can be performed by microwave or water bath. In the above example, the HER2 primary antibody is provided in a pre-diluted (ready-to-use) format, so no dilution is performed. However, other preparations of HER2 primary antibodies are available in concentrated formats that require dilution. For example, a 200 μg / ml Ig concentrate can be diluted 1:400-1:800 to a final Ig concentration of 0.25-0.50 μg / ml, or alternative concentrates can be diluted 1:50-1:500 with similar results achieved at a dilution range of 1:100, with the optimal dilution being 1:100.

[0219] [HER2-ISH] In some embodiments, an alternative method for evaluating HER2 may be ISH. Fluorescent ISH (FISH) assay using Vysis PathVysion HER-2 DNA Probe Kit (Abbott Molecular) may include the following steps with washing between each step. Dewaxed tissue sections may be treated with 0.2N HCl for 20 minutes, then Vysis Pretreatment Solution (1N sodium isothiocyanate) for 30-60 minutes at 80°C (pretreatment step), followed by Vysis Protease Solution for 10-60 minutes at 37°C (protease step), followed by 10% neutral buffered formalin (fixation step), followed by treatment with increasing concentrations of alcohol solutions (dehydration step).

[0220] In some embodiments, the DNA probe mixture is applied to the tissue section under a sealed coverslip. The mixture contains a Locus Specific Identifier for HER2 (190-Kb Spectrum Orange directly-labeled, fluorescent DNA probe specific for the HER-2 gene locus at 17q11.2-q12) and a Chromosome Enumeration Probe for chromosome 17 (CEP17; a 5.4 Kb Spectrum Green directly-labeled, fluorescent DNA probe specific for the alpha satellite DNA sequence at the centromeric region of chromosome 17 at 17p11.1-q11.1), and also contains unlabeled blocking DNA to suppress sequences contained in the target locus common to other chromosomes. The slide is then placed in a Thermobrite device and exposed to 73°C for 5 minutes (DNA denaturation step), followed by incubation at 37°C for 14-24 hours (DNA hybridization step).

[0221] In some embodiments, the tissue is treated with two washes of 2x saline sodium citrate (SSC) + 0.3% NP-40, the first for 2-5 min at room temperature and the second for 2 min at 71-73°C (post-hybridization wash step). Finally, nuclei are stained blue with 4,6 diamidino-2-phenylindole (DAPI) solution (counterstain step).

[0222] In some embodiments, an alternative ISH method based on silver deposition (SISH; Ventana Medical Systems Inform HER2 kit) can be used according to the manufacturer's instructions to obtain substantially equivalent results. In some embodiments, other HER2 ISH methods, such as chromogenic ISH (CISH), can also be used.

[0223] [HER2 scoring--HER2 IHC scoring] In some embodiments, any technique for HER2 scoring provided and described herein can be used, so long as it is sufficient to observe the degree of HER2 variation.

[0224] In some embodiments, HER2 status is determined from IHC stained slides according to College of American Pathologists-American Society of Clinical Oncology (CAP-ASCO) guidelines (Wolff et al. J Clin Oncol 31:3997, 2013) with modifications for scoring for intraductal tumor cells. HER2 scores of 0 to 3+ are defined as follows: 0 is defined as no staining observed or incomplete membrane staining, faintly / barely discernible, in the range of ≦10% of tumor cells; 1+ is defined by incomplete membrane staining, faintly / barely discernible, in the range of >10% of tumor cells; 2+ is defined by incomplete and / or weak / moderate (observed in homogeneous and continuous population) and peri-membrane staining that is within >10% of tumor cells, or strong and complete and peri-membrane staining that is within ≦10% of tumor cells. 3+ is defined by complete and intense pericellular membrane staining (observed in a homogenous and contiguous population, observed within >10% of tumor cells, and easily recognized with a low magnification objective).

[0225] In some embodiments, the selection and / or scoring of the DCIS area to be scored is performed manually by a pathologist and / or automatically using a computer on the scanned image of the slide. In some embodiments, a result of 0, 1+, or 2+ is considered negative, and 3+ is considered positive. In some embodiments, a result of 2+ is considered equivocal and can trigger a HER2 ISH assay that determines the status through quantification of HER2 gene amplification (amplified patients are positive). In some embodiments, HER2 IHC can be completely replaced by a HER2 ISH assay. Both of these alternative approaches have shown utility in relevant studies.

[0226] [HER2 ISH scoring] In some embodiments, HER2 status is determined by counting orange HER2 signals and green CEP17 signals in at least 20 DCIS cell nuclei from FISH stained slides and then calculating the ratio. DCIS is considered HER2 non-amplified (negative) if there are equal numbers of orange and green signals or if the ratio of orange to green is less than 2.0 and the average HER2 copy number per cell is less than 4.0. DCIS is considered HER2 amplified (positive) if the ratio of orange to green signals is greater than 2.0. Cells are also considered amplified (positive) if the ratio of orange to green signals is less than 2.0 and the average HER2 copy number per cell is 6.0 or greater. If the ratio of orange to green signals is less than 2.0 and the average HER2 copy number per cell is 4.0 or greater but less than 6.0, count another 20 cells and recalculate the ratio for all 40 cells, applying the threshold of <2.0 (negative) vs. ≧2.0 (positive).

[0227] [Ki-67 staining] In some embodiments, any technique for Ki-67 staining can be used as long as it is sufficient to observe the degree of Ki-67 variation provided and described herein. In some embodiments, protein levels can be checked. In some embodiments, mRNA levels can be checked. In some embodiments, DNA levels can be checked. In some embodiments, both protein and mRNA levels can be checked. A high level can be a level higher than a control or standardized level, e.g., a level in a non-DCIS sample. Similarly, a reduced level can be a level lower than a control or standardized level, e.g., a level in a non-DCIS sample. In some embodiments, a "positive" or "high" result is one that exceeds a "negative" or "low" result (in the context of scoring).

[0228] In some embodiments, to assess Ki-67 (MKI67; MIB-1; HGNC:7107) levels by IHC, the following steps can be performed using a Leica BOND-MAX automated staining instrument, with washing between each step: In some embodiments, dewaxed and rehydrated tissue sections can be treated with Novocastra Peroxide Block (3-4% hydrogen peroxide) (peroxidase blocking step), followed by treatment with Novocastra Bond Epitope Retrieval Solution 2 (10 mM Tris base and 1 mM ethylenediaminetetraacetic acid [EDTA] buffer + 0.05% Tween 20, pH 9.0 solution base) at 95°C-100°C for 30 minutes (epitope retrieval step). In some embodiments, the tissue sections are then incubated with mouse monoclonal antibody MIB-1 (Dako M7240) diluted 1:50 in Novocastra Primary Antibody Diluent for 30 minutes at room temperature (primary antibody step), followed by Novocastra Post Primary solution for 15 minutes (rabbit anti-mouse antibody to introduce rabbit anti-IgG linker), followed by Novocastra Bond Refine Polymer for 15 minutes (anti-rabbit poly-HRP-IgG) (secondary detection step), followed by 3,3'-diaminobenzidine (DAB) for 5 minutes (chromogen visualization step), followed by <0.1% hematoxylin for 7 minutes (nuclear counterstain step). Finally, a cover slip is mounted using mounting medium.

[0229] In some embodiments, Ki-67 can be detected by any Ki-67 specific antibody. In some embodiments, the antibody can be mouse monoclonal MM1, K-2; 7B11, BGX-297, Ki88, Ki-S5, and DVB-2; rabbit monoclonal SP6, 30-9, EPR3611; and many other monoclonal NCL-Ki67p from manufacturers such as Dako (Agilent), Novocastra (Leica), Ventana Medical Systems (Roche), Cell Marque (Sigma-Aldrich), Lab Vision (Thermo Scientific), BioGenex, and Biocare.

[0230] In some embodiments, a dilution of 1:50 is used for the Dako MIB-1 antibody, although other dilutions can be used, including dilutions ranging from 1:40 to 1:600, e.g., 1:75 to 1:150. Similarly, other antibody manufacturers report recommended dilutions ranging from 1:100 to 1:200 for their antibody preparations, and some preparations are provided pre-diluted (ready to use).

[0231] [Ki-67 scoring] In some embodiments, any technique for Ki-67 scoring may be used, so long as it is sufficient to observe the degree of Ki-67 variation provided and described herein.

[0232] In some embodiments, Ki-67 status is determined from IHC stained slides based on the percentage of DCIS tumor cells with nuclear signal. In some embodiments, all areas of the tissue section containing DCIS are evaluated to calculate the percentage. In some embodiments, at least three DCIS-containing vessels or 1 mm of DCIS tissue are used to score the marker. In some embodiments, the intensity of the signal is also reported as weak (1+), moderate (2+), or strong (3+). In some embodiments, the intensity is the average intensity of DCIS tumor cell nuclei with signal across the entire tissue section. In some embodiments, the selection and / or scoring of the DCIS areas to be scored is performed manually by a pathologist and / or automatically using a computer on scanned images of the slide.

[0233] In some embodiments, DCIS with less than 10% of tumor cells having a nuclear signal is considered negative, while DCIS with more than 10% of tumor cells having a nuclear signal is considered positive. A sample is considered negative only in the presence of an appropriately stained positive control.

[0234] In some embodiments, alternative thresholds (e.g., 0% vs. >0%, <1% vs. ≥1%, ≤1% vs. >1%, <5% vs. ≥5%, ≤5% vs. >5%, <10% vs. ≥10%, <15% vs. ≥15%, ≤15% vs. >15%, <20% vs. ≥20%, ≤20% vs. >20%, <25% vs. ≥25%, ≤25% vs. >25%, <30% vs. ≥30%, etc.) can be used based on the technique used for staining and / or analysis. In some embodiments, alternative techniques and / or scoring methods can be used for detection, resulting in corresponding but different cutoff ranges for high and / or low risk. In such situations, the ranges provided herein for the present techniques can be correlated (for "corresponding values") with other techniques by analyzing the same sample (or two samples from the same DCIS sample) by two different techniques and identifying them as equivalent to each other. The Allred scoring system, immune scores, and other scoring methods that combine percentage and intensity scoring elements also show validation.

[0235] [p16 / INK4A staining] In some embodiments, any technique for p16 staining may be used as long as it is sufficient to observe the degree of p16 variation provided and described herein. In some embodiments, protein levels may be checked. In some embodiments, mRNA levels may be checked. In some embodiments, DNA levels may be checked. In some embodiments, both protein and mRNA levels may be checked. A high level may be a level higher than a control or standardized level, e.g., a level in a non-DCIS sample. Similarly, a reduced level may be a level lower than a control or standardized level, e.g., a level in a non-DCIS sample. In some embodiments, a "positive" or "high" result is one that exceeds a "negative" or "low" result (in the context of scoring).

[0236] In some embodiments, to assess the p16 isoform of cyclin-dependent kinase inhibitor 2A (p16 / INK4A; CDKN2A; MTS1; HGNC:1787) by IHC, the following steps can be performed using a Leica BOND-MAX automated staining instrument, with washing between each step: In some embodiments, dewaxed and rehydrated tissue sections can be treated with Novocastra Peroxide Block (3-4% hydrogen peroxide) (peroxidase blocking step), followed by treatment with Novocastra Bond Epitope Retrieval Solution 2 (10 mM Tris base and 1 mM EDTA buffer + 0.05% Tween 20, pH 9.0 solution base) at 95°C-100°C for 30 minutes (epitope retrieval step). The tissue sections are then incubated with pre-diluted mouse monoclonal antibody E6H4 (Ventana Medical Systems CINtec p16 Histology Kit) for 30 min at room temperature (primary antibody step), followed by 15 min incubation with Novocastra Post Primary solution (rabbit anti-mouse antibody to introduce rabbit anti-IgG linker), followed by 15 min incubation with Novocastra Bond Refine Polymer (anti-rabbit poly-HRP-IgG) (secondary detection step), followed by 5 min incubation with 3,3'-diaminobenzidine (DAB) (chromogen visualization step), followed by 7 min incubation with <0.1% hematoxylin (nuclear counterstain step). Finally, the coverslips are mounted using mounting medium.

[0237] In some embodiments, p16 / INK4A can be detected by a primary antibody. In some embodiments, mouse monoclonal DCS-50.1 / A7 (Neomarkers) can be used. In some embodiments, mouse monoclonals 6H12, JC8, 16PO4;16PO7, and G175-405, as well as rabbit monoclonal EPR1473, and others can be used. In some embodiments, p16 / INK4A primary antibodies are provided in a pre-diluted (ready-to-use) format, and thus no dilution is performed. However, other preparations of p16 / INK4A primary antibodies are available in concentrated formats, and dilutions are recommended by the manufacturers in the range of 1:75 to 1:500, or even 1:25 to 1:800, depending on the particular protocol.

[0238] [p16 / INK4A scoring] In some embodiments, any technique for p16 scoring may be used, so long as it is sufficient to observe the degree of p16 variation provided and described herein.

[0239] In some embodiments, p16 / INK4A status is determined from an IHC stained slide based on the percentage of DCIS tumor cells with nuclear signal, conditioned by the intensity of the signal. In some embodiments, the intensity of the signal is reported as weak (1+), medium (2+), or strong (3+). In some embodiments, the intensity is the average intensity of DCIS tumor cell nuclei with signal across the entire tissue section relative to the intensity of a positive control performed with the same staining batch. In some embodiments, cells with nuclear signal of at least medium (2+) intensity are considered positive. In some embodiments, cells with no or weak staining (1+) are considered negative. In some embodiments, all areas of the tissue section containing DCIS are evaluated to calculate the percentage. In some embodiments, at least three DCIS-containing tubes or 1 mm of DCIS tissue can be used to score the marker. In some embodiments, the selection and / or scoring of the DCIS areas to be scored is performed manually by a pathologist and / or automatically using a computer on scanned images of the slide.

[0240] In some embodiments, DCIS with 25% or less tumor cells with at least moderate intensity nuclear signal is considered negative, while DCIS with more than 25% tumor cells with at least moderate intensity nuclear signal is considered positive. A sample is considered negative only in the presence of an appropriately stained positive control. Alternative thresholds (e.g., <20% vs. ≧20%, ≦20% vs. >20%, <25% vs. ≧25%, <30% vs. ≧30%, ≦30% vs. >30%, <40% vs. ≧40%, etc.) and other thresholds can be used based on the technique used for staining and / or analysis. In some embodiments, alternative techniques and / or scoring methods can be used for detection, which can result in corresponding but different cut-off ranges for high and / or low risk. In such situations, the ranges provided herein for the present technology can be correlated with other techniques (for "corresponding values") by analyzing the same sample (or two samples from the same DCIS sample) by two different techniques and identifying them as equivalent to each other. The Allred scoring system, immune scores, and other scoring methods that combine percentage and intensity scoring elements also show validation.

[0241] The staining pattern of p16 / INK4A can be nuclear and cytoplasmic and is often heterogeneous in nature. Furthermore, p16 / INK4A staining can be present in both DCIS tumor cells and surrounding stromal cells.

[0242] [COX-2 staining] In some embodiments, any technique for COX-2 staining may be used, as long as it is sufficient to observe the degree of COX-2 variation described herein. In some embodiments, protein levels may be checked. In some embodiments, mRNA levels may be checked. In some embodiments, DNA levels may be checked. In some embodiments, both protein and mRNA levels may be checked. A high level may be a level higher than a control or standardized level, e.g., a level in a non-DCIS sample. Similarly, a reduced level may be a level lower than a control or standardized level, e.g., a level in a non-DCIS sample. In some embodiments, a "positive" or "high" result is greater than a "negative" or "low" result (in the context of scoring).

[0243] In some embodiments, prostaglandin-endoperoxide synthase 2 (PTGS2; cyclooxygenase-2 [COX-2]; HGNC:9605) may be assessed by IHC; the following steps may be performed using a Leica BOND-MAX automated staining instrument, with washing between each step. In some embodiments, dewaxed and rehydrated tissue sections may be treated with Novocastra Peroxide Block (3-4% hydrogen peroxide) (peroxidase blocking step), followed by treatment with Novocastra Bond Epitope Retrieval Solution 1 (10 mM sodium citrate buffer + 0.05% Tween 20, pH 6.0 solution base) at 95°C-100°C for 30 minutes (epitope retrieval step). In some embodiments, the tissue sections are then incubated with rabbit monoclonal antibody SP21 (Cell Marque 24OR-16) diluted 1:50 in Novocastra Primary Antibody Diluent for 30 minutes at room temperature (primary antibody step), followed by Novocastra Bond Refine Polymer (anti-rabbit poly-HRP-IgG) for 15 minutes (secondary detection step), followed by 3,3'-diaminobenzidine (DAB) for 5 minutes (chromogen visualization step), followed by <0.1% hematoxylin for 7 minutes (nuclear counterstain step). Finally, a coverslip is mounted using mounting medium.

[0244] In some embodiments, COX-2 can be detected by a primary antibody. In some embodiments, mouse monoclonal CX-294 (Dako) can be used. In some embodiments, mouse monoclonal 4H12 (Novocastra) and rabbit monoclonal SP21 from manufacturers such as Ventana Medical Systems (Roche), Cell Marque (Sigma-Aldrich), Lab Vision (Thermo Scientific), and Biocare can be used.

[0245] In some embodiments, a dilution of 1:50 can be used for Cell Marque SP21 antibody. In some embodiments, dilutions ranging from 1:100 to 1:500, or 1:50 to 1:500, 1:50 to 1:200 can be used. Additionally, some preparations are provided in a pre-diluted (ready to use) form.

[0246] [COX-2 scoring] In some embodiments, any technique for COX-2 scoring may be used, so long as it is sufficient to observe the degree of COX-2 variation provided and described herein.

[0247] In some embodiments, COX-2 status may be determined from IHC stained slides based on the percentage of DCIS tumor cells with cytoplasmic signal and the intensity of the signal, in the form of an Allred score. In some embodiments, intensity is reported as absent (0), weak (1), intermediate (2), or strong (3) and represents the average signal intensity across the entire tissue section relative to the intensity of a positive control run in the same staining batch. In some embodiments, the percentages are converted to a percentage score as follows: 0, 0% positive; 1, ≦1% positive; 2, >1-10% positive; 3, 11-33% positive; 4, 34-66% positive; 5, 67-100% positive. The Allred score is the sum of the intensity score and the percentage score, ranging from 0 to 8.

[0248] In some embodiments, all areas of the tissue section containing DCIS are evaluated. In some embodiments, at least three DCIS-containing vessels or 1 mm of DCIS tissue are used to score the markers. The selection and / or scoring of the DCIS areas to be scored can be done manually by a pathologist and / or automatically using a computer on a scanned image of the slide.

[0249] In some embodiments, DCIS with an Allred score of 0-6 is considered negative, and DCIS with an Allred score of 7 or 8 is considered positive. In some embodiments, samples are considered negative in the presence of an appropriately stained positive control. In some embodiments, alternative techniques and / or scoring methods can be used for detection, which can result in corresponding but different cut-off ranges for high and / or low risk. In such situations, the ranges provided herein for the present technique can be correlated with other techniques (for "corresponding values") by analyzing the same sample (or two samples from the same DCIS sample) by two different techniques and identifying them as equivalent to each other. In some embodiments, alternative scoring methods such as the Allred scoring system, immune scores, and others that combine percentage and intensity scoring elements also show efficacy.

[0250] [FOXA1 staining] In some embodiments, any technique for FOXA1 staining may be used as long as it is sufficient to observe the degree of FOXA1 variation provided and described herein. In some embodiments, protein levels may be checked. In some embodiments, mRNA levels may be checked. In some embodiments, DNA levels may be checked. In some embodiments, both protein and mRNA levels may be checked. A high level may be a level higher than a control or standardized level, e.g., a level in a non-DCIS sample. Similarly, a reduced level may be a level lower than a control or standardized level, e.g., a level in a non-DCIS sample. In some embodiments, a "positive" or "high" result is greater than a "negative" or "low" result (in the context of scoring).

[0251] In some embodiments, to assess Forkhead box A1 (FOXA1; HGNC:5021) by IHC, the following steps can be performed using a Leica BOND-MAX automated staining instrument, with washing between each step: In some embodiments, dewaxed and rehydrated tissue sections can be treated with Novocastra Peroxide Block (3-4% hydrogen peroxide) (peroxidase blocking step), followed by treatment with Novocastra Bond Epitope Retrieval Solution 2 (10 mM Tris base and 1 mM ethylenediaminetetraacetic acid [EDTA] buffer + 0.05% Tween 20, pH 9.0 solution base) at 95°C-100°C for 30 minutes (epitope retrieval step). The tissue sections are then incubated with mouse monoclonal antibody 2F83 (Cell Marque 405M-16) diluted 1:25 in Novocastra Primary Antibody Diluent for 30 min at room temperature (primary antibody step), followed by Novocastra Post Primary solution (anti-mouse IgG to introduce rabbit anti-mouse IgG), followed by Novocastra Refine Polymer (anti-rabbit poly-HRP-IgG) for 15 min (secondary detection step), followed by 3,3'-diaminobenzidine (DAB) for 5 min (chromogen visualization step), followed by <0.1% hematoxylin for 7 min (nuclear counterstain step). Finally, coverslips are mounted using mounting medium.

[0252] In some embodiments, FOXA1 mouse monoclonal antibody 2F83 from Abcam can be used at a dilution of 1:450 (or, for example, 1:2,000) which gives useful results, or at a dilution of 1:25 (based on manufacturer recommended starting dilution range of 1:25-1:100, initial estimated Ig concentration of 2.5-25.0 μg / ml, final estimated optimal Ig concentration range of 0.1-1.0 μg / ml). In some embodiments, mouse monoclonal antibodies can include 2F83, 3A8, 1B1, 2D7, 3C1, and 4F6, as well as rabbit monoclonals SP88 and EPR10881 from Thermo Scientific, Spring Bioscience, Epitomics, Millipore, and various other manufacturers. With other antibody preparations, various dilutions can be used together (e.g., Millipore recommends a 1:500 dilution of their 1 mg / ml version of 2F83, and Thermo Scientific recommends a 1:20 to 1:200 dilution of their 1 mg / ml preparation of clone 3A8), and some FOXA1 antibody preparations are provided pre-diluted (ready to use).

[0253] [FOXA1 Scoring] In some embodiments, any technique for scoring FOXA1 can be used, so long as it is sufficient to observe the degree of FOXA1 variation provided and described herein.

[0254] In some embodiments, FOXA1 status can be determined from IHC stained slides in the form of an immunoscore based on the percentage of DCIS tumor cells with nuclear signal and the intensity of the signal. Intensity can be reported as absent (0), weak (1), intermediate (2), or strong (3) and can represent the average signal intensity across the tissue section relative to the intensity of a positive control run in the same staining batch. In some embodiments, the immunoscore can be the product of an intensity score and a percentage score on a scale of 0 to 300.

[0255] In some embodiments, all areas of the tissue section containing DCIS are evaluated. In some embodiments, three DCIS-containing vessels or 1 mm of DCIS tissue are used to score the markers. The selection and / or scoring of the DCIS areas to be scored can be done manually by a pathologist and / or automatically using a computer on a scanned image of the slide.

[0256] In some embodiments, DCIS with an immunoscore below 100 is considered FOXA1 low, DCIS with an immunoscore between 100 and 250 is considered FOXA1 intermediate, and DCIS with an immunoscore above 250 is considered FOXA1 high. Alternative lower (e.g., 40 or 150) and / or higher (e.g., 150 or 250) thresholds, as well as alternative scoring methods, also show utility. Furthermore, utility may vary based on predicted outcome (i.e., DCIS or invasive event in the ipsilateral breast). In some embodiments, FOXA1 is simply treated as either "negative" (below 100) or positive (above 100).

[0257] In some embodiments, alternative techniques and / or scoring methods can be used for detection, which can result in corresponding but different cut-off ranges for high and / or low risk. In such situations, the ranges provided herein for this technique can be correlated with other techniques (for "corresponding values") by analyzing the same sample (or two samples from the same DCIS sample) by two different techniques and identifying them as equivalent to each other. In some embodiments, alternative scoring methods such as the Allred scoring system, immune scores, and others that combine percentage and intensity scoring elements also show validity.

[0258] [SIAH2 staining] In some embodiments, any technique for SIAH2 staining may be used as long as it is sufficient to observe the degree of SIAH2 variation provided and described herein. In some embodiments, protein levels may be checked. In some embodiments, mRNA levels may be checked. In some embodiments, DNA levels may be checked. In some embodiments, both protein and mRNA levels may be checked. A high level may be a level higher than a control or standardized level, e.g., a level in a non-DCIS sample. Similarly, a reduced level may be a level lower than a control or standardized level, e.g., a level in a non-DCIS sample. In some embodiments, a "positive" or "high" result is greater than a "negative" or "low" result (in the context of scoring).

[0259] In some embodiments, to assess SIAH2 E3 ubiquitin protein ligase 2 (SIAH2; resting 7 [Drosophila] homolog 2; HGNC:10858) by IHC, the following steps can be performed using a Leica BOND-MAX automated staining instrument, with washing between each step: In some embodiments, dewaxed and rehydrated tissue sections can be treated with Novocastra Peroxide Block (3-4% hydrogen peroxide) (peroxidase blocking step), followed by treatment with Novocastra Bond Epitope Retrieval Solution 1 (10 mM sodium citrate buffer + 0.05% Tween 20, pH 6.0 solution base) at 95°C-100°C for 30 minutes (epitope retrieval step). In some embodiments, the tissue sections are then incubated with mouse monoclonal antibody 24E6H3 (Santa Cruz Biotechnology sc-81787) diluted 1:200 in Novocastra Primary Antibody Diluent for 30 minutes at room temperature (primary antibody step), followed by Novocastra Post Primary solution for 15 minutes (anti-mouse IgG to introduce rabbit anti-mouse IgG), followed by Novocastra Refine Polymer for 15 minutes (anti-rabbit poly-HRP-IgG) (secondary detection step), followed by 3,3'-diaminobenzidine (DAB) for 5 minutes (chromogen visualization step), followed by <0.1% hematoxylin for 7 minutes (nuclear counterstain step). Finally, a cover slip is mounted using mounting medium.

[0260] In some embodiments, epitope retrieval may be performed in EDTA-containing buffers at higher pH (8.0) in either a 98°C water bath or steam chamber using both mouse monoclonal antibody 24E6H3 and its parent clone 24E6 in related studies. In some embodiments, alternative antibodies may be used, including those from Novus Biologicals. With different preparations of these antibodies, different dilutions may be used, including dilutions in the range of 1:40 to 1:200. In some embodiments, other primary antibodies may be used, such as mouse monoclonal 35F7I4 (Creative Diagnostics), which is effective for IHC (recommended dilution of 1:40 to 1:50), as well as mouse monoclonals 1F5, 2G6, and SIAH2-369 (available from Abnova, Epigentek, US Biologicals, Sigma-Aldrich, and Creative Diagnostics).

[0261] [SIAH2 Scoring] In some embodiments, any technique for SIAH2 scoring may be used, so long as it is sufficient to observe the degree of SIAH2 variation provided and described herein.

[0262] In some embodiments, SIAH2 status is determined from an IHC stained slide based on the percentage of DCIS tumor cells with nuclear signal. In some embodiments, all areas of the tissue section containing DCIS are evaluated to calculate the percentage. In some embodiments, at least three DCIS-containing ducts or 1 mm of DCIS tissue are used to score the marker. In some embodiments, the intensity of the signal may be reported as weak (1+), moderate (2+), or strong (3+). In some embodiments, the intensity is the average intensity of DCIS tumor cell nuclei with signal across the entire tissue section. In some embodiments, the selection and / or scoring of the DCIS areas to be scored is performed manually by a pathologist and / or automatically using a computer on scanned images of the slide.

[0263] In some embodiments, DCIS in which less than 20% of the tumor cells have a nuclear signal is considered negative, while DCIS in which 20% or more of the tumor cells have a nuclear signal is considered positive.

[0264] In some embodiments, a sample is considered negative only in the presence of an appropriately stained positive control.

[0265] In some embodiments, alternative techniques and / or scoring methods can be used for detection, which can result in corresponding but different cut-off ranges for high and / or low risk. In such situations, the ranges provided herein for the above-mentioned techniques can be correlated with other techniques ("corresponding values") by analyzing the same sample (or two samples from the same DCIS sample) by two different techniques and identifying them as equivalent to each other. In some embodiments, alternative scoring methods such as the Allred scoring system, immune scores, and others that combine percentage and intensity scoring elements also show efficacy. In some embodiments, alternative thresholds (e.g., ≦20% vs. >20%, <25% vs. ≧25%, ≦25% vs. >25%, <30% vs. ≧30%, ≦30% vs. >30%, <40% vs. ≧40%, ≦40% vs. >40%, etc.) and other thresholds can be used. In some embodiments, alternative scoring methods such as the Allred scoring system, immune scores, and others that combine percentage and intensity scoring elements can also be used.

[0266] An example of SIAH2 staining and scoring is shown in FIG. 3: FIG. 3 shows a SIAH2 IHC assay (top, negative on UUH TMA; bottom, positive on Biomax BR8011 TMA).

[0267] [Statistical analysis] Kaplan-Meier survival analysis can be used to estimate the proportion of patients who experience a first event (DCIS or invasive recurrence) after initial DCIS diagnosis / surgery. Hazard ratios (HRs) can be determined using Cox proportional hazards analysis. At the time of the first event (DCIS or invasive), patients can be censored for other event types. Patients can also be censored if an event is detected within 6 months after surgery or on the first mammogram after surgery, as this can be considered persistent disease rather than recurrence. In some embodiments, subjects with persistent disease are excluded from the various methods provided herein.

[0268] [How to evaluate markers] Although the above assay and scoring systems are presented, in some embodiments, alternative techniques and / or scoring methods can be used for detection, which can result in corresponding but different cut-off ranges for high and / or low risk. In such situations, the ranges provided herein for the present techniques can be correlated with other techniques (for one or more "corresponding values" of COX-2, Ki67, p16, SIAH2, FOXA1, PR, and / or HER2) by analyzing the same sample (or two samples from the same DCIS sample) by two different techniques and identifying them as being consistent or equal to each other. In some embodiments, alternative scoring methods such as the Allred scoring system, immune scores, and others that combine percentage and intensity scoring elements also show efficacy. In some embodiments, there may be corresponding techniques and / or scores for one or more of COX-2, Ki67, p16, SIAH2, FOXA1, PR, and / or HER2. In some embodiments, there may be corresponding techniques for one or more of COX-2, Ki67, p16, SIAH2, FOXA1, PR, and / or HER2. In some embodiments, there may be corresponding scores for one or more of COX-2, Ki67, p16, SIAH2, FOXA1, PR, and / or HER2. As will be appreciated by those of skill in the art, these corresponding scores and / or techniques may be used in any of the embodiments provided herein and are expressly contemplated as alternatives to each and every disclosure regarding the markers described.

[0269] In some embodiments, protein detection assays may be used, including, for example, immunohistochemistry, immunofluorescence, mass spectrometry, or other assays discussed in more detail below. In some embodiments, nucleic acid hybridization-based methods such as Northern blots, gene expression arrays, quantitative real-time polymerase chain reaction (qPCR), nCounter, in situ nucleic acid detection, and mRNA detection assays including next-generation RNA sequencing (RNA-Seq) may be used. In some embodiments, techniques for detecting changes at the DNA level may be used, including, for example, polymerase chain reaction (PCR), in situ hybridization (ISH), next-generation sequencing (NGS), and others as would be readily understood by one of skill in the art.

[0270] In some embodiments, corresponding values ​​(including alternative scoring or alternative assays for analyzing samples) are used, while the relative results (e.g., high vs. low or very high vs. medium vs. low) are maintained between the various techniques for analysis or scoring systems. Thus, a score "high" in one system correlates with a score "high" in another system without great complexity or difficulty. Thus, various results can be ported from one system to another, as desired, as long as the levels in the sense of relatively high vs. low (for example) are maintained. Similarly, in some embodiments, protein levels can be used for one marker, while a second marker may be analyzed via DNA, and a third marker may be analyzed via mRNA, for example. Thus, the nature of the molecules being tested may change the properties of the molecules being tested within the test, as needed.

[0271] In some embodiments, the expression level is determined by detecting the level of mRNA transcribed from the gene.

[0272] In some embodiments, mRNA in a sample is first transcribed into cDNA using reverse transcriptase.

[0273] In some embodiments, the sample is subjected to an amplification reaction (e.g., using methods based on polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand displacement amplification (SDA), etc.), probe amplification (e.g., using methods based on ligase chain reaction (LCR), cleavage enzyme invader, etc.), signal amplification (e.g., using methods based on branched DNA probes [bDNA], hybrid capture, etc.), and others as would be readily appreciated by one of skill in the art.

[0274] In some embodiments, mRNA in a sample is detected by hybridizing a nucleic acid probe or primer that can selectively hybridize to the mRNA transcript of interest, or cDNA derived therefrom, and then detecting hybridization using a detection device or system.

[0275] In this context, the term "selective hybridization" means that hybridization of a probe or primer occurs with a higher frequency or rate than hybridization of the same probe or primer to any other nucleic acid, or has a higher maximum reaction rate. Preferably, the probe or primer does not hybridize to another nucleic acid at a detectable level under the reaction conditions used.

[0276] Since transcripts of the genes described herein are detected using mRNA or cDNA derived therefrom, assays that detect changes in mRNA can be used (e.g., Northern hybridization, RT-PCR, NASBA, TMA or ligase chain reaction).

[0277] In some embodiments, mRNA quantification can be performed on a gene expression array platform, including Agilent's Bioanalyzer™ system, Affymetrix's GeneChip™, GeneTitan™ or GeneAtlas™ systems, and others that would be readily apparent to one of skill in the art.

[0278] In some embodiments, mRNA quantification can be performed by real-time qPCR. Such reactions can be performed with a variety of reporters, including non-specific DNA-binding fluorescent dyes (e.g., SYBR™ Green) or fluorescent reporter probes that selectively hybridize to the sequence of interest (e.g., TaqMan™ probes). In some cases, reactions are performed in parallel on multiple partitions of the same sample (digital PCR). Real-time qPCR platforms include ThermoFisher Scientific / Applied Biosystems' FAST, QuantStudio and related systems, Hologic / Gen-Probe's DTS system, Roche / Idaho Technology's LightCycler™ system, Qiagen's Rotor-Gene™ system, Bio-Rad's CFX and related systems, and others as would be readily understood by one of skill in the art.

[0279] In some embodiments, quantification of mRNA can be performed without reverse transcription or amplification steps, such as NanoString's in vitro nCounter™ platform or various in situ hybridization (ISH) approaches including paired-probe ISH (e.g., RNAscope™ and Quanti-Gene RNAview), single-tag multi-probe ISH (e.g., Stellaris™), or locked nucleic acid (LNA) probes.

[0280] In some embodiments, mRNA quantification may be performed using RNA-Seq next generation sequencing technologies, including sequencing-by-synthesis (e.g., Illumina's HiSeq™ and NextSeq™ systems), single molecule real-time sequencing (e.g., Pacific Biosciences' RS system), ion semiconductor sequencing (e.g., ThermoFisher Scientific's Ion Torrent™ system, etc.), sequencing-by-ligation (e.g., ThermoFisher Scientific's SOLiD™ system), pyrosequencing (e.g., Roche / 454 Life Sciences), and others, as would be readily understood by one of skill in the art.

[0281] Methods of RT-PCR include, for example, those described in Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995). Essentially, this method involves performing a PCR reaction using cDNA produced by reverse transcribing mRNA from a cell using a reverse transcriptase enzyme. The above PCR methods are intended to be applied mutatis mutandis to this embodiment of the invention.

[0282] Similarly, PCR can be performed using cDNA. One or more probes or primers used in the reaction hybridize specifically to the transcript of interest.

[0283] The method of TMA or self-sustaining sequence replication (3SR) uses two or more oligonucleotides that flank a target sequence, an RNA polymerase, RNase H, and reverse transcriptase. One oligonucleotide (which also contains an RNA polymerase binding site) hybridizes to an RNA molecule containing the target sequence, and reverse transcriptase produces a cDNA copy of this region. RNase H is used to digest the RNA in the RNA-DNA complex, and a second oligonucleotide is used to generate a copy of the cDNA. RNA polymerase is then used to generate an RNA copy of the cDNA, and the process is repeated.

[0284] The NASBA system selectively amplifies a target mRNA sequence by the simultaneous action of three enzymes: reverse transcriptase, RNase H, and RNA polymerase. The mRNA template is transcribed into cDNA by reverse transcription using an oligonucleotide that hybridizes to the target sequence and contains an RNA polymerase binding site at its 5' end. The template RNA is digested with RNase H, synthesizing double-stranded DNA. RNA polymerase then makes multiple RNA copies of the cDNA, and the process is repeated.

[0285] In some embodiments, microarrays can be used to determine the expression levels of one or more of the nucleic acids described herein. Such methods allow for the detection of a large number of different nucleic acids, thereby providing multi-pane testing and improving the sensitivity and / or accuracy of the diagnostic assays of the invention.

[0286] In some embodiments, the expression level is determined by detecting the level of a protein encoded by a nucleic acid in a gene described herein.

[0287] In this regard, the present embodiments are not necessarily limited to the detection of proteins comprising the specific amino acid sequences recited herein. Rather, the invention encompasses the detection of variant sequences (e.g., having at least about 80% or 90% or 95% or 98% amino acid sequence identity), or the detection of immunogenic fragments or epitopes of said proteins.

[0288] The amount, level and / or presence of a polypeptide may be determined using any of a variety of techniques known to those skilled in the art, such as, for example, a technique selected from the group of immunohistochemistry, immunofluorescence, immunoblot, Western blot, dot blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption / ionization time of flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry such as, for example, LC MS / MS), biosensor technology, evanescent fiber optic technology or protein chip technology.

[0289] In some embodiments, the assay used to determine the amount or level of a protein is a semi-quantitative assay. In some embodiments, the assay used to determine the amount or level of a protein is a quantitative assay. As is clear from the above description, such an assay may include the use of appropriate controls, for example from normal individuals or matched normal controls.

[0290] In some embodiments, standard solid-phase ELISA or FLISA formats can be useful in determining the concentration of proteins from various samples.

[0291] In one form, such an assay involves immobilizing a biological sample on a solid matrix (e.g., polystyrene or polycarbonate microwells or dipsticks), a membrane, or a glass support (e.g., a glass slide). An antibody that specifically binds to a protein described herein is directly contacted with the immobilized biological sample and forms a direct bond with any of its target proteins present in the sample. The antibody is generally labeled with a detectable reporter molecule, such as a fluorescent label (e.g., FITC or Texas Red) or a fluorescent semiconductor nanocrystal (described in U.S. Pat. No. 6,306,610) in the case of FLISA, or an enzyme (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP) or β-galactosidase) in the case of ELISA, or a second labeled antibody that binds to the first antibody may be used. After washing to remove unbound antibody, the label is detected either directly in the case of fluorescent labels, or by addition of a substrate in the case of enzyme labels (e.g., hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indole-β-D-galaotopyranoside (x-gal)).

[0292] In some embodiments, ELISA or FLISA involves immobilizing an antibody or ligand that specifically binds to the protein on a solid matrix, such as a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick, or a glass support. The sample is then physically associated with the antibody, which binds or "captures" the polypeptide. The bound protein is then detected using a labeled antibody. For example, a labeled antibody that binds to a different epitope than the first (capture) antibody is used to detect the captured protein. Alternatively, a third labeled antibody that binds to the second (detection) antibody can be used.

[0293] In some embodiments, for example, a biosensor instrument (e.g., BIAcore™, Pharmacia Biosensor, Piscataway, NJ) is used to detect the presence or concentration of a protein in a bodily fluid. In such an assay, an antibody or ligand that specifically binds to a protein is immobilized on the surface of a receptor chip. For example, the antibody or ligand is covalently attached to a dextran fiber attached to a gold film in a flow cell of the biosensor device. The test sample is passed through the cell. Any antigens present in the bodily fluid sample bind to the immobilized antibody or ligand, causing a change in the refractive index of the medium above the gold film, which is detected as a change in the surface plasmon resonance of the gold film.

[0294] In some embodiments, the presence or level of a protein or its fragment or epitope is detected using a protein and / or antibody chip. To prepare such a chip, an antibody or ligand that binds to an antigen of interest is attached to a solid support (e.g., glass, polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, gold or silicon nitride). This immobilization can be direct (e.g., covalent (e.g., Schiff base formation, disulfide bond, or amide or urea bond formation, etc.)) or indirect.

[0295] To bind proteins to solid supports, it is often useful to treat the solid support to create chemically reactive groups on the surface, for example with aldehyde-containing silane reagents or calixcrown derivatives as described in Lee et al., Proteomics, 3: 2289-2304, 2003. Streptavidin chips are also useful for capturing biotin-bound proteins and / or peptides and / or nucleic acids and / or cells (e.g., as described in Pavlickova et al., Biotechniques, 34: 124-130, 2003). Alternatively, peptides can be captured on microfabricated polyacrylamide gel pads and accelerated within the gel using microelectrophoresis, as described in Arenkov et al. (Anal. Biochem. 278:123-131, 2000).

[0296] Other assay formats are also contemplated, such as flow-through immunoassays (PCT / AU2002 / 01684), lateral flow immunoassays (US20040228761, US20040248322 or US20040265926), fluorescence polarization immunoassays (FPIA) (U.S. Pat. Nos. 4,593,089, 4,492,762, 4,668,640 and 4,751,190), homogeneous microparticle immunoassays ("HMI") (e.g., U.S. Pat. Nos. 5,571,728, 4,847,209, 6,514,770 and 6,248,597), or chemiluminescent microparticle immunoassays ("CMIA"). The contents of all patent applications and patents disclosed in this paragraph are incorporated herein by reference in their entirety. In some embodiments, mRNA may be used to assay for HER2. In some embodiments, mRNA is not used to assay for SIAH2.

[0297] In some embodiments, "high risk" for DCIS is defined as detailed in Gorringe and Fox, Ductal Carcinoma in Situ Biology, Biomarkers, and Diagnosis, Frontiers in Oncology, Vol. 71-74 (October 2017), which is incorporated herein by reference in its entirety. In some embodiments, any one or more of the conditions, markers, etc. described in Gorringe and Fox are used to define the high risk aspects employed and to initially identify the subject as being in the high-risk group. From among them, other tests, analyses, and / or treatment options described herein can be advanced. High-risk aspects can be defined. In some embodiments, "high risk" for DCIS is defined as high risk under DCISionRT analysis. In some embodiments, the DCISionRT analysis is described in Bremer et al., "A Biologic Signature for Breast Ductal Carcinoma in situ to Predict Radiation Therapy benefit and Assess Recurrence Risk," American Cancer Society, Clinical Cancer Research, doi: 10.1158 / 1078-0432.CCR-18-0842, July 27, 2018. In some embodiments, the high risk (for DCIS recurrence or invasive breast cancer in subjects with DCIS) is determined by any one or more of the compositions or techniques in U.S. Patent Publication No. 2017 / 0350895, the entire contents of which are incorporated herein by reference.

[0298] [Treatment based on test results] For subjects at risk of a subsequent invasive ipsilateral breast event, they may be treated with at least BCS+radiotherapy (RT) and may receive additional adjuvant therapy of at least hormone therapy (such as tamoxifen or aromatase inhibitors) and / or HER2 therapy (such as trastuzumab). Which option is chosen depends on whether the subject responds to radiation therapy (e.g., elevated k-ras members and / or HER2+ and SIAH+) based on the present disclosure. In some embodiments, if the subject is likely to have both an invasive ipsilateral breast event and a DCIS ipsilateral breast event, the subject may be treated with mastectomy, and if the subject is likely not to respond to radiation therapy (e.g., elevated k-ras pathway and / or HER2+ and SIAH+), the subject may receive additional adjuvant therapy of at least hormone therapy and / or HER2 therapy.

[0299] In some embodiments, rather than relying on standard clinical and pathological factors to determine treatment or interpret risk profile, biomarker signatures can be used to direct the administration of treatment. This feature allows for a superior treatment approach without the need to interpret risk profile and then determine treatment. That is, compared to the current DCIS standard treatment (breast-conserving surgery (BCS) with adjuvant radiation therapy), more appropriate treatment can be administered to the patient according to the signature that recommends the patient's treatment based on the biological profile detailed and described in Tables 13-15 and included herein as examples. In some embodiments, the subject is treated with BCS or frequent breast imaging (watchful waiting) for early detection of ipsilateral breast events to reduce the likelihood of subsequent ipsilateral breast events. In some embodiments, the induction treatment is at least BCS + radiation therapy and may include additional adjuvant therapy of at least hormone therapy (e.g., tamoxifen or aromatase inhibitor) and / or HER2 therapy (e.g., trastuzumab) to reduce the likelihood of invasive ipsilateral breast events. In some embodiments, if the subject is SIAH+ and HER2+ and / or has an elevated k-ras pathway, radiation therapy is not used and HER2 therapy can be used instead. In some embodiments, the subject can be treated with mastectomy and can receive additional adjuvant therapy of at least hormone therapy and / or HER2 therapy to reduce the likelihood that the subject / patient will have an invasive ipsilateral breast event and / or a DCIS ipsilateral breast event (especially if the subject is refractory to radiation therapy). [Table 13-1] [Table 13-2] [Table 14-1] [Table 14-2]

Table 14-3

Table 14-4

[0300] In some embodiments, the marker signatures disclosed and described herein can be used to comprehensively determine the overall risk of recurrence or progression, and / or the likelihood that a subject will respond or not respond to radiation therapy, thereby determining the appropriate treatment. (a) The markers listed in Table 13 are used to assess the risk of invasive breast cancer, and (b) the markers listed in Table 14 are used to assess the risk of DCIS events. In combination, the overall risk, including the risk of invasive events and the risk of DCIS events, can be determined and used to recommend more appropriate treatment. For example, if a patient is found to be at low risk for invasive events listed in Table 13, but at high risk for DCIS events listed in Table 14, the patient will have a high overall risk of recurrence. As disclosed and described herein, various methods and compositions can determine the likelihood of (a) recurrent breast events, (b) progression to invasive or metastatic cancer, or (c) whether a subject / patient currently with DCIS will not experience further DCIS / invasive breast cancer, will experience DCIS, and / or will experience invasive breast cancer, so that patients diagnosed with DCIS can be treated in a more appropriate manner.Similar applications can be made for Table 15 as combination options.Then, for example, based on the k-ras pathway level and / or HER2 and SIAH2 status of the sample, it can be further determined whether the subject will respond to radiation therapy, thereby determining whether the subject should receive radiation therapy or non-radiation therapy, such as HER2 antibodies such as trastuzumab. Thus, in some embodiments, the optional option for determining risk level can be combined with the further option (if appropriate) of determining the k-ras pathway level and / or HER2 and SIAH2 status of the sample to determine whether the subject will respond to radiation therapy, and thereby determine whether the subject should receive radiation therapy or a non-radiation therapy (as described in more detail herein), such as a HER2 antibody such as trastuzumab.

[0301] In some embodiments, the marker signatures disclosed and described herein can be used to comprehensively treat patients in a manner that appropriately reduces the risk of total recurrence or progression, including invasive events and / or DCIS events. For example, (a) the marker signatures in Table 13 can be used to determine the treatment that reduces the risk of invasive events, and (b) the marker signatures in Table 14 can be used to determine the treatment that reduces the risk of DCIS events, and / or the treatment that reduces the total risk according to Tables 13 and 14. The same can be applied in Table 15 as a combination option. Subjects with an elevated risk of invasive events can then be treated with either radiation therapy or non-radiation therapy, as outlined herein.

[0302] Below is a non-exhaustive list of examples of patient treatment options based on the patient's individual risk profile, as described in Tables 13-15. EXAMPLES

[0303] Example 1 DCIS samples from subjects with DCIS are analyzed for at least one of SIAH2 and HER2, and PR and FOXA1, or (alternatively) FOXA1 and PR. The results are compared to the matrix in Tables 9 and / or 11 to determine whether the subject is at increased risk of invasive breast cancer, DCIS recurrence, or neither. If the subject is at increased risk of invasive breast cancer, SIAH2 and HER2 levels are considered. Subjects who are HER2+ and SIAH2+ do not receive radiation therapy, but instead receive trastuzumab.

[0304] Example 2 DCIS samples from subjects are analyzed for at least PR, HER2 and SIAH2 levels, or (alternatively) samples are analyzed for at least FOXA1. Prognosis is provided based on at least PR, HER2 and SIAH2, or at least PR and FOXA1. Depending on the nature of the result, this indicates that the subject who provided the sample is at high or elevated risk of invasive breast cancer (see, for example, Tables 9 and / or 11). If the subject is at high risk of invasive breast cancer, SIAH2 and HER2 levels are considered. Subjects who are HER2+ and SIAH2+ will not receive radiation therapy, but will instead receive anti-HER2 antibodies.

[0305] Example 3 DCIS samples from subjects are analyzed for at least SIAH2 and FOXA1 levels. Subjects at high risk of invasive breast cancer are provided with a prognosis based on at least SIAH2 and FOXA1 levels. If a subject is at high risk of invasive breast cancer, SIAH2 and HER2 levels are considered. Subjects who are HER2+ and SIAH2+ will not receive radiation therapy, but will instead receive an anti-HER2 antibody.

[0306] Example 4 DCIS samples from subjects are analyzed for A) PR, HER2, and SIAH2, or (alternatively) b) PR and FOXA1. If the subject has at least one of a) PR-, HER2- and SIAH2-, b) PR+, FOXA1+, or c) PR+, FOXA1- and Ki67+, a prognosis is provided for the subject as being at high risk for an invasive breast cancer event. If the subject is at high risk for invasive breast cancer, SIAH2 and HER2 levels are considered. Subjects who are HER2+ and SIAH2+ will not receive radiation therapy, but will instead receive an anti-HER2 antibody.

[0307] Example 5 DCIS samples are analyzed for at least one of a) SIAH2 and FOXA1, b) SIAH2 and at least one of i) PR and ii) HER2, c) SIAH2 and postmenopausal status; or (alternatively) d) PR and FOXA1. If at least one of a) SIAH2+ and FOXA1+, b) SIAH2+ and HER2+ or PR-; SIAH2+ and postmenopausal; or PR+ and FOXA1-; is present in the DCIS sample, a prognosis is provided for the subject as being at increased risk of a DCIS event. If the subject is at high risk of invasive breast cancer, SIAH2 and HER2 levels are considered. Subjects who are HER2+ and SIAH2+ will not receive radiation therapy, but will instead receive an anti-HER2 antibody.

[0308] Example 6 DCIS samples are analyzed for at least one of the following: a) PR-, HER2-, and SIAH2-; b) PR+, FOXA1+; or c) PR+, FOXA1-, and Ki67+. If the analysis indicates an increased risk of invasive breast cancer (see Tables 9 and / or 11), the subject is given more aggressive therapy than standard therapy for DCIS. If the subject is at increased risk of invasive breast cancer, SIAH2 and HER2 levels are considered. Subjects who are HER2+ and SIAH2+ will not receive radiation therapy, but will instead receive anti-HER2 antibodies.

[0309] Example 7 DCIS samples are analyzed for at least one of a) SIAH2+ and FOXA1+, b) SIAH2+ and HER2+ or PR-, c) SIAH2+ and postmenopausal status, or (alternatively) d) PR+ and FOXA1-. If the analysis indicates a high likelihood of invasive breast cancer, the subject is given more aggressive therapy than standard therapy for a single DCIS event. If the subject is at high risk for invasive breast cancer, SIAH2 and HER2 levels are considered. Subjects who are HER2+ and SIAH2+ will not receive radiation therapy, but will instead receive an anti-HER2 antibody.

[0310] Example 8 The subject provides a document and is analyzed for at least one of the combinations outlined in any one of Tables 13-15 to provide a risk level for the subject. The corresponding therapy is then administered based on the risk level indicated for the subject, as outlined in the appropriate row of Tables 13-15. If the subject is at high risk for invasive breast cancer, SIAH2 and HER2 levels are considered. Subjects who are HER2+ and SIAH2+ will not receive radiation therapy, but will instead receive an anti-HER2 antibody.

[0311] Example 9 As shown in Figure 1, the majority of subjects with DCIS who are at high risk for invasive breast cancer will benefit significantly from radiation therapy. However, there is a non-responsive subgroup of these people who do not respond to radiation therapy, as shown in Figure 2. These individuals are HER2+ and SIAH+.

Claims

1. 1. A method for identifying targets for breast cancer treatment, comprising: Providing a DCIS sample from a subject with primary DCIS, wherein the subject is at elevated risk of a subsequent ipsilateral breast event; and analyzing said DCIS sample from the subject by measuring expression levels of HER2 and SIAH2; Identifying and selecting subjects for breast cancer treatment based on positive expression levels of both HER2 and SIAH2 to reduce an elevated risk of subsequent ipsilateral breast events; Wherein the breast cancer treatment is: (i) surgical removal of the primary DCIS; and (ii) one or more of active radiation therapy or non-radiation therapy; The method of claim 1,

2. 2. The method of claim 1, wherein the non-radiation therapy comprises chemotherapy or HER2 therapy.

3. 3. The method of claim 2, wherein the HER2 therapy is a HER2 antibody therapy, optionally wherein the HER2 antibody therapy is trastuzumab.

4. The method of any one of claims 1 to 3, comprising analysing the sample for expression of Ki67, optionally determining that the sample is Ki67 positive.

5. The method of any one of claims 1 to 4, wherein the subsequent ipsilateral breast event comprises subsequent ipsilateral invasive breast cancer.

6. The method of any one of claims 1 to 4, wherein the subsequent ipsilateral breast event comprises an ipsilateral DCIS recurrence.

7. The method according to any one of claims 1 to 6, wherein HER2 positivity corresponds to a score of 3+.

8. The method of any one of claims 1 to 7, wherein the DCIS sample is PR positive and FOXA1 positive.

9. 8. The method of any one of claims 1 to 7, comprising analyzing the DCIS sample for expression levels of one or more of PR, FOXA1, COX-2, and p16, and optionally determining that the subject with primary DCIS is at elevated risk of a subsequent ipsilateral breast event based on the analysis of one or more of PR, FOXA1, COX-2, and p16.

10. The method of any one of claims 1 to 9, comprising analysing the DCIS sample for grade, necrosis, size and / or margin status.

11. An anti-HER-2 antibody for use in the preparation of a medicament for treating a subject, wherein the subject has DCIS and is at elevated risk of a subsequent ipsilateral breast event, and it has been determined that the subject is appropriate for aggressive breast cancer treatment to reduce the risk of the subsequent ipsilateral breast event based on the subject being HER2 positive and SIAH2 positive.

12. The anti-HER-2 antibody of claim 11, which is trastuzumab.

13. The anti-HER-2 antibody of claim 11 or 12, wherein the subsequent ipsilateral breast event comprises subsequent ipsilateral invasive breast cancer or ipsilateral DCIS recurrence.

14. A kit for analyzing DCIS samples from subjects who have DCIS and are at high risk of a subsequent ipsilateral breast event, the kit comprising: a HER2 probe; and a SIAH2 probe.

15. The kit of claim 14, comprising one or more probes for FOXA1, COX-2, Ki67, p16, and PR.

16. The kit of claim 15, comprising probes for FOXA1, COX-2, Ki67, p16 and PR.

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