WO2017181163A2

WO2017181163A2 - Methods and compositions for detection and diagnosis of breast cancer

Info

Publication number: WO2017181163A2
Application number: PCT/US2017/027834
Authority: WO
Inventors: Karen Chapman; Maria PRENDES; Karen COPELAND; Lyndal HESTERBERG
Original assignee: Oncocyte Corporation
Priority date: 2016-04-16
Filing date: 2017-04-17
Publication date: 2017-10-19
Also published as: WO2017181163A3

Abstract

The present disclosure provides methods, compositions and kits for the detection and diagnosis of breast cancer.

Description

METHODS AND COMPOSITIONS FOR DETECTION AND DIAGNOSIS

OF BREAST CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. provisional patent application serial number 62/323,695 filed on April 16, 2017, incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure relates to cancer and the diagnosis and treatment of cancer.

BACKGROUND

[0003] In the United States, breast cancer is the second leading cause of cancer-related death in women. During diagnosis, in order to determine if a breast suspicious area might be cancerous, the common diagnostic route consists of a diagnostic mammogram, followed by a MRI (Magnetic Resonance Imaging) or an ultrasound, and if required, a biopsy of the breast. The American College of Radiology's standard practice of describing mammogram findings, the Breast Imaging Reporting and Data System (BIRAD), places breast anomalies into 6 categories. A BIRAD >4 indicates a suspicious finding that might require a biopsy.

[0004] Approximately 10% of women are recalled from screening mammography for further testing; of those 10% approximately 95% are false positives. Over the course of 10 years of screening, one out of every two women will experience a false positive diagnosis (i.e., detection of a cancer that would not cause a woman any harm in her lifetime and that would not have progressed or otherwise been detected in the absence of the mammogram or ultrasound) with 7% to 17% of these women undergoing unnecessary biopsies, resulting in additional testing and anxiety. These false positive diagnoses come at a high cost, as the cost of a breast biopsy typically ranges from $l,000-$4,000.

[0005] In addition, 5% of breast biopsies provide false negative results, and 6% to 46% of women with invasive cancer have negative mammograms. These false negative mammograms are more prevalent in young women and in women with dense breast tissue. The consequence of a false negative diagnostic delays treatment and impacts mortality.

[0006] With respect to mammograms, questions have been raised about under-diagnosis of cancers and/or over-diagnosis of benign conditions, as well as the risks associated with exposure to radiation, even though the dose required for a mammogram is very small. Clinicians have long recognized that mammography's precision as a diagnostic tool can be hindered by its technical limitations, including variability in the radiologists' diagnostic interpretations, clinical accuracies, and management recommendations.

[0007] One of the reasons for the debate over the efficiency of mammograms is related to breast density. Mammographic detection of breast cancer is impaired in areas of dense breast tissue; thus, there is a risk that certain cancers will go undetected by a mammogram, in women with dense breast tissue. In addition, high breast density per se is one of the strongest known risk factors for developing breast cancer, second to age and carrying a BRCA1 or BRCA2 mutation. Women with dense tissue in 75% or more of the breast have a risk of breast cancer four to six times greater than the risk among women with little or no dense tissue. Estimates of attributable risk show that densities in more than 50% of the breast might account for about a third of all breast cancers. Although breast density is influenced by genetics, it is also affected by other factors, such as weight, drugs and alcohol consumption. In most women breast density will change overtime, decreasing with age.

[0008] Extensive mammographic density is also associated with the risk of benign breast disease. In women with mammographic density in more than 75% of the breast, the risks for hyperplasia without atypia, with atypia, and in situ ductal carcinoma is increased by 95%. Breast lesions with atypia or ductal carcinoma in situ (DCIS) are associated with significant higher risks of subsequent invasive carcinoma, and women with these findings may require additional surveillance to reduce their risks.

[0009] The use of ultrasound for the detection of breast cancer is a common tool to evaluate abnormal findings from a screening or diagnostic mammogram. This method is more useful than mammography in women with dense breasts, but it also increases the likelihood of false-positives, resulting in overdiagnosis and unnecessary treatment.

[0010] In addition to the aforementioned handicaps of mammograms and ultrasounds, a MRI of the breast requires special equipment, just as mammography uses x-ray machines designed specially to image the breasts. However, many hospitals and imaging centers do not have dedicated breast MRI equipment available, requiring the scan to be repeated at another facility if a biopsy is necessary, increasing the already high cost of a MRI. Additionally, guidelines recommend MRI screening for approximately 6 million women who either have a family history, a BRCA gene mutation, or dense breast history, since mammograms have been shown to miss cases of cancer in patients that meet these profiles.

[0011] For the aforementioned reasons, the current clinical practice for the diagnosis of breast cancer is in need of a confirmatory breast cancer test that will allow the elimination of unnecessary biopsies, offer a degree of certainty about the nature of the pathology, and distinguish benign from malignant neoplasms with accuracy (i.e., a test having high sensitivity and high specificity). Such an improved confirmatory test should be cost-effective; should allow an accurate diagnosis that is unaffected by breast density (which, as stated above, is a confounding factor in diagnosis), and will offer a diagnostic opportunity for women who cannot have a mammogram.

SUMMARY

[0012] Disclosed herein are, inter alia, simple liquid biopsy-based tests for breast cancer, requiring small sample volumes. The procedures are non-invasive, consisting of acquisition of a bodily fluid (e.g., a blood draw) and do not require expensive instrumentation or equipment. Analysis and interpretation of complex results is not required; thereby offering a rapid, objective, and accurate answer that will spare patients the stress associated with a false positive diagnosis and reduce costs to the health system.

[0013] The methods and compositions for detection and diagnosis of breast cancer disclosed herein will reduce the dependence on imaging technologies (which are affected by the density of breast tissue) and invasive surgical procedures (e.g., biopsies).

[0014] The methods and compositions disclosed herein are based on the identification of biomarkers that are specific to breast cancer. Accordingly, several panels of biomarkers, whose presence and levels can be measured in patient samples, are provided. The biomarkers provided in the panels disclosed herein represent genes whose products are detected at different (higher or lower) levels in samples (e.g. serum samples) obtained from subjects with breast cancer vs. subjects with benign breast pathologies/no cancer.

[0015] Embodiments of the disclosure provide methods of diagnosis, prognosis and/or detection of cancer. Other embodiments provide compositions relating to the diagnosis, prognosis and/or detection of cancer. The methods and compositions may be used for diagnosing and/or detecting cancers, for example, breast cancer, including ductal atypia, ductal hyperplasia, ductal carcinoma in situ (DCIS), invasive (infiltrating) ductal carcinoma, lobular carcinoma, inflammatory breast cancer and Paget disease.

[0016] In certain embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject {i.e., a "subject sample"); b) contacting the sample from the subject with one or more agents that detect one or more markers differentially expressed in breast cancer; c) contacting a control sample with one or more agents from b); and d) comparing the expression levels of the one or more markers between the sample obtained from the subject and the control sample, wherein a differential expression (i.e., overexpression or underexpression) of the one or more markers in the subject sample relative to the control sample indicates that the subject has breast cancer. Suitable markers include gene products {i.e., mRNAs and/or polypeptides) encoded by any one or more of the genes listed in Tables 1-6.

[0017] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject {i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more of the genes listed in any of Tables 1-6; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0018] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject {i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more of the genes listed in Table 1; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer. [0019] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more of the genes listed in Table 2; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0020] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more of the genes listed in Table 3; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0021] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more of the genes listed in Table 4; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0022] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more of the genes listed in Table 5; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0023] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more of the genes listed in Table 6; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0024] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more, any two or more, any three or more, any four or more, any five or more, any six or more, any seven or more, any eight or more, any nine or more, any ten or more, any eleven or more, any twelve or more, any thirteen or more, any fourteen or more, or any fifteen or more of the genes listed in Table 3; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0025] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of the CRP, ApoE2, C5, cathepsin D, uPAR, CTGF, PTN, MIG, Factor I, IL-1R4/ST2, IL-18Rbeta, MK13, Carbonic anhydrase 6, CYTN, PPIE and SLAF6 genes in the subject sample; c) measuring the expression level of the genes of step b) in a control sample; and d) comparing the expression level of the genes in the subject sample relative to the expression level of the same genes in the control sample, wherein a differential expression (overexpression or underexpression) of the genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0026] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more, any two or more, any three or more, any four or more, any five or more, any six or more, any seven or more, any eight or more, any nine or more, any ten or more, any eleven or more, any twelve or more, any thirteen or more, or any fourteen or more of the genes listed in Table 4; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0027] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of the cathepsin D, IL-18Rbeta, SLAF6, CTGF, C5, CYTN, ApoE2, PTN, MK13, PPIE, Factor I, IL-1R4/ST2, Carbonic anhydrase 6, CRP, and uPAR genes in the subject sample; c) measuring the expression level of the genes of step b) in a control sample; and d) comparing the expression level of the genes in the subject sample relative to the expression level of the same genes in the control sample, wherein a differential expression (overexpression or underexpression) of the genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0028] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more, any two or more, any three or more, any four or more, or any five or more of the genes listed in Table 5; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0029] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of the C5, cathepsin D, CTGF, IL-18Rbeta, MK13 and SLAF6 genes in the subject sample; c) measuring the expression level of the genes of step b) in a control sample; and d) comparing the expression level of the genes in the subject sample relative to the expression level of the same genes in the control sample, wherein a differential expression (overexpression or underexpression) of the genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0030] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of one or more genes in the subject sample, wherein the one or more genes are selected from any one or more, any two or more, any three or more, any four or more, or any five or more of the genes listed in Table 6; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0031] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of the C5, cathepsin D, MIG, IL-18Rbeta, MK13 and ApoE2 genes in the subject sample; c) measuring the expression level of the genes of step b) in a control sample; and d) comparing the expression level of the genes in the subject sample relative to the expression level of the same genes in the control sample, wherein a differential expression (e.g., overexpression) of one or more of the genes in the subject sample relative to the control sample indicates the subject has breast cancer.. [0032] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level in the subject sample of one or more genes chosen from C5, cathepsin D, IL-18Rbeta, and MK13; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0033] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of the C5, cathepsin D, IL-18Rbeta, and MK13 genes in the subject sample; c) measuring the expression level of the genes of step b) in a control sample; and d) comparing the expression level of the genes in the subject sample relative to the expression level of the same genes in the control sample, wherein a differential expression (overexpression or underexpression) of one or more of the genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0034] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level in the subject sample of one or more genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, CTGF and SLAF6; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0035] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of the C5, cathepsin D, IL-18Rbeta, MK13, CTGF and SLAF6 genes in the subject sample; c) measuring the expression level of the genes of step b) in a control sample; and d) comparing the expression level of the genes in the subject sample relative to the expression level of the same genes in the control sample, wherein a differential expression (overexpression or underexpression) of one or more of the genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0036] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level in the subject sample of one or more genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, MIG and ApoE2; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression ) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0037] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level of the C5, cathepsin D, IL-18Rbeta, MK13, MIG and ApoE2 genes in the subject sample; c) measuring the expression level of the genes of step b) in a control sample; and d) comparing the expression level of the genes in the subject sample relative to the expression level of the same genes in the control sample, wherein a differential expression (overexpression or underexpression) of one or more of the genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0038] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) measuring the expression level in the subject sample of one or more genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR; c) measuring the expression level of the one or more genes of step b) in a control sample; and d) comparing the expression level of the one or more genes in the subject sample relative to the expression level of the same one or more genes in the control sample, wherein a differential expression (overexpression or underexpression) of the one or more genes in the subject sample relative to the control sample indicates the subject has breast cancer. [0039] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e. , a "subject sample"); b) measuring the expression levels of the C5, cathepsin D, IL- 18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL- 1R4/ST2, carbonic anhydrase 6, CRP and uPAR genes in the subject sample; c) measuring the expression level of the genes of step b) in a control sample; and d) comparing the expression level of the genes in the subject sample relative to the expression level of the same genes in the control sample, wherein a differential expression (overexpression or underexpression) of one or more of the genes in the subject sample relative to the control sample indicates the subject has breast cancer.

[0040] In certain embodiments, measuring the expression level of a gene is accomplished by measuring levels of the protein product of the gene. Such can be achieved using agents that specifically bind to the protein of interest, for example, antibodies (e.g. , monoclonal antibodies, humanized antibodies) or aptamers (e.g. , nucleic acid aptamers, peptide aptamers) and testing for binding of the agent to a polypeptide in the sample.

[0041] In certain embodiments, measuring the expression level of a gene is accomplished by measuring levels of the nucleic acid product of the gene (e.g. , mRNA, cDNA). Such can be achieved using agents that specifically bind to the nucleic acid of interest (for example, labeled nucleic acid probes), and testing for binding of the agent to a nucleic acid in the sample. Nucleic acid probes can be labeled using any label known in the art, including but not limited to radioactive, colorimetric, enzymatic, fluorometric and magnetic labels.

[0042] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) contacting the subject sample with one or more reagents that specifically bind to a polypeptide encoded by one or more of the genes listed in Tables 1-6, and testing for binding to determine the level of the one or more polypeptides in the subject sample; c) contacting a control sample with one or more of the reagents from b) and testing for binding to determine the level of the one or more polypeptides in the control sample; and d) comparing the level of the one or more polypeptides in the subject sample with the level of the one or more polypeptides in the control sample, wherein presence of the one or more polypeptides in the subject sample at a higher or lower level compared to the control sample indicates that the subject has breast cancer. [0043] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) contacting the subject sample with one or more reagents that specifically bind to a polypeptide encoded by one or more of the genes listed in Tables 1-6, wherein at least one of the reagents specifically binds to a polypeptide encoded by one or more of the C5, cathepsin D, IL- 18Rbeta, MK13, SLAF6, CTGF, CYTN, MIG, ApoE2, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR genes and testing for binding to determine the level of the one or more polypeptides in the subject sample; c) contacting a control sample with one or more of the reagents from b) and testing for binding to determine the level of the one or more polypeptides in the control sample; and d) comparing the level of the one or more polypeptides in the subject sample with the level of the one or more polypeptides in the control sample, wherein presence of the one or more polypeptides in the subject sample at a higher or lower level compared to the control sample indicates that the subject has breast cancer.

[0044] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) contacting the subject sample with one or more reagents that specifically bind to a polypeptide encoded by one or more of the C5, cathepsin D, IL-18Rbeta, MK13, SLAF6 and CTGF genes and testing for binding to determine the level of the one or more polypeptides in the subject sample; c) contacting a control sample with one or more of the reagents from b) and testing for binding to determine the level of the one or more polypeptides in the control sample; and d) comparing the level of the one or more polypeptides in the subject sample with the level of the one or more polypeptides in the control sample, wherein presence of the one or more polypeptides in the subject sample at a higher or lower level compared to the control sample indicates that the subject has breast cancer.

[0045] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) contacting the subject sample with one or more reagents that specifically bind to a polypeptide encoded by one or more of the C5, cathepsin D, IL-18Rbeta, MK13, MIG and ApoE2 genes and testing for binding to determine the expression level of the one or more polypeptides in the subject sample; c) contacting a control sample with one or more of the reagents from b) and testing for binding to determine the expression level of the one or more polypeptides in the control sample; and d) comparing the level of the one or more polypeptides in the subject sample with the level of the one or more polypeptides in the control sample, wherein presence of the one or more polypeptides in the subject sample at a higher or lower level compared to the control sample indicates that the subject has breast cancer.

[0046] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) contacting the subject sample with one or more reagents that specifically bind to the polypeptides encoded by the C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR genes and testing for binding to determine the level of the polypeptides in the subject sample; c) contacting a control sample with one or more of the reagents from b) and testing for binding to determine the level of the polypeptides in the control sample; and d) comparing the levels of the C5, cathepsin D, IL- 18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and suPAR polypeptides in the subject sample with the levels of the C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL- 1R4/ST2, carbonic anhydrase 6, CRP and suPAR polypeptides in the control sample, wherein differential levels of one or more of the C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and suPAR polypeptides in the subject sample compared to the control sample indicates that the subject has breast cancer.

[0047] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) contacting the subject sample with one or more reagents that specifically bind to the polypeptides encoded by the C5, cathepsin D, IL-18Rbeta, MK13, SLAF6 and CTGF genes and testing for binding to determine the levels of the polypeptides in the subject sample; c) contacting a control sample with one or more of the reagents from b) and testing for binding to determine the levels of the polypeptides in the control sample; and d) comparing the level of the C5, cathepsin D, IL-18Rbeta, MK13, SLAF6 and CTGF polypeptides in the subject sample with the levels of the C5, cathepsin D, IL-18Rbeta, MK13, SLAF6 and CTGF polypeptides in the control sample, wherein differential levels of one or more of the C5, cathepsin D, IL-18Rbeta, MK13, SLAF6 and CTGF genes in the subject sample compared to the control sample indicates that the subject has breast cancer.

[0048] In some embodiments, the present disclosure provides a method for detecting breast cancer in a subject comprising: a) obtaining a sample from a subject (i.e., a "subject sample"); b) contacting the subject sample with one or more reagents that specifically bind to the polypeptides encoded by the C5, cathepsin D, IL-18Rbeta, MK13, MIG and ApoE2 genes and testing for binding to determine the levels of the polypeptides in the subject sample; c) contacting a control sample with one or more of the reagents from b) and testing for binding to determine the levels of the polypeptides in the control sample; and d) comparing the levels of the C5, cathepsin D, IL-18Rbeta, MK13, MIG and ApoE2 polypeptides in the subject sample with the levels of the C5, cathepsin D, IL-18Rbeta, MK13, MIG and ApoE2 polypeptides in the control sample, wherein differential levels of one or more of the C5, cathepsin D, IL-18Rbeta, MK13, MIG and ApoE2 polypeptides in the subject sample compared to the control sample indicates that the subject has breast cancer.

[0049] In other embodiments, the present disclosure provides a method of detecting breast cancer in a subject comprising: a) obtaining a sample from a subject and b) detecting the presence of a breast cancer associated marker or markers in the sample, wherein the breast cancer associated marker or markers is a gene product, a nucleic acid, a fragment thereof or a complement thereof, chosen from markers encoded by C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, MIG, ApoE2, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR genes and/or any one or more genes listed in any of Tables 1-6.

[0050] In other embodiments, the present disclosure provides a method of detecting breast cancer in a subject comprising: a) obtaining a sample from a subject and b) detecting the presence of a breast cancer associated marker or markers in the sample, wherein the breast cancer associated marker or markers is a polypeptide or a fragment thereof, chosen from markers encoded by C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, MIG, ApoE2, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR genes and/or any one or more genes listed in any of Tables 1-6.

[0051] With regard to the embodiments described in the preceding paragraphs, the sample may be any sample as described infra, for example, a bodily fluid, such as blood, plasma, serum or urine. The sample may be a cellular sample or the extract of a cellular sample. The sample may be a tissue sample. Nucleic acids and/or proteins may be isolated from the sample. Nucleic acids such as RNA (e.g. , mRNA) may be transcribed into cDNA. The agent may be one or more molecules that bind specifically to one or more proteins expressed by the cancer cell, or the agent may be one or more molecules that bind specifically to one or more nucleic acids expressed by the cancer cell. For example, the agent may be a protein (e.g. , an antibody), or an aptamer, that binds specifically to the protein expressed by one of the marker genes identified infra. The agent may be one or more nucleic acids that hybridize to a nucleic acid expressed by the cancer cell. The nucleic acid expressed by the cancer cell may be an RNA molecule, e.g., an mRNA.

[0052] In addition to a comparison between a subject and control sample, differential expression (i.e., overexpression or underexpression) of the breast cancer markers identified infra can be determined based upon a cutoff value, wherein a value higher or lower than the cutoff value indicates the subject has breast cancer. Examples of non-limiting cutoff values are described herein. In some embodiments, whether the subject has breast cancer can be determined by measuring the expression of the upregulated genes vs. downregulated genes within a single sample, followed by obtaining a simple ratio of: i)the sum total of the expression values of the upregulated genes within a single sample, to ii) the sum total of the expression values of the downregulated genes within the single sample, or an inverse ratio thereof, wherein a ratio larger or smaller than a selected cutoff value indicates the subject has breast cancer.

[0053] In certain embodiments the present disclosure provides compositions of matter useful in distinguishing a breast cancer cell from a non-cancerous cell comprising one or more molecules that specifically bind to a molecule expressed at higher levels by a breast cancer cell compared to a non-cancer cell. In certain embodiments, the composition comprises a protein or an aptamer that binds to one or more molecules expressed by the breast cancer cell at higher levels compared to the non-cancer cell. In certain embodiments, the protein or an aptamer binds to one or more molecules present at higher levels in the cancerous sample as compared to the benign sample. In other embodiments, the protein or an aptamer binds to one or more molecules present at higher levels in the benign sample as compared to the cancerous sample. In certain embodiments, the composition comprises a nucleic acid that binds to one or more molecules expressed by the breast cancer cell at higher levels compared to the non-cancer cell. In certain embodiments, the nucleic acid binds to one or more molecules present at higher levels in the cancerous sample as compared to the benign sample. In other embodiments, the nucleic acid binds to one or more molecules present at higher levels in the benign sample as compared to the cancerous sample.

[0054] In some embodiments the present disclosure provides a composition of matter comprising one or more proteins, such as an antibody, or one or more aptamers, that specifically binds to a breast cancer marker chosen from any one or more of the markers listed in any of Tables 1-6.

[0055] In some embodiments the present disclosure provides a composition of matter comprising one or more proteins, such as an antibody, or one or more aptamers, that specifically binds to a breast cancer marker chosen from C5, cathepsin D, IL-18Rbeta and MK13. In some embodiments, the marker may be expressed by the breast cancer cell, either at a level that is higher or lower than the level of the same marker expressed by a non-cancerous cell. In other embodiments, the marker may be present at elevated (or alternatively, reduced) levels in biological fluids obtained from a subject, such as blood, plasma, serum or urine.

[0056] In some embodiments the present disclosure provides a composition of matter comprising one or more proteins, such as an antibody, or one or more aptamers, that specifically binds to a breast cancer marker chosen from C5, cathepsin D, IL-18Rbeta, MK13, CTGF and SLAF6. In some embodiments, the marker may be expressed by the breast cancer cell, either at a level that is higher or lower than the level of the same marker expressed by a non-cancerous cell. In other embodiments, the marker may be present at elevated (or alternatively, reduced) levels in biological fluids obtained from a subject, such as blood, plasma, serum or urine.

[0057] In some embodiments the present disclosure provides a composition of matter comprising one or more proteins, such as an antibody, or one or more aptamers, that specifically bind to a breast cancer marker chosen from C5, cathepsin D, IL-18Rbeta, MK13, MIG and ApoE2. In some embodiments, the marker may be expressed by the breast cancer cell, either at a level that is higher or lower than the level of the same marker expressed by a non-cancerous cell. In other embodiments, the marker may be present at elevated (or alternatively, reduced) levels in biological fluids obtained from a subject, such as blood, plasma, serum or urine.

[0058] In some embodiments the present disclosure provides a composition of matter comprising one or more proteins, such as an antibody, or one or more aptamers, that specifically bind to a breast cancer marker chosen from one or more of C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and suPAR. In some embodiments, the marker may be expressed by the breast cancer cell, either at a level that is higher or lower than the level of the same marker expressed by a noncancerous cell. In other embodiments, the marker may be present at elevated (or alternatively, reduced) levels in biological fluids obtained from a subject, such as blood, plasma, serum or urine.

[0059] In further embodiments the present disclosure provides a composition of matter comprising a plurality of proteins, such as a plurality of antibodies, or a plurality of aptamers, that specifically bind to a panel of markers wherein the panel of markers comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more molecules (e.g., proteins) encoded by the genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR. Any of the individual markers within a panel of markers may be present in the subject sample at a level that is higher or lower than the level of the same marker in a control sample. In other embodiments, the panel of markers or any individual markers within a panel of markers may be present at elevated (or alternatively, reduced) levels in biological fluids obtained from a subject, such as blood, serum or urine.

[0060] In other embodiments the present disclosure provides a composition of matter comprising a nucleic acid that specifically binds to a marker molecule, such as a mRNA molecule or its complement, encoded by one or more genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR. The marker molecule may be expressed by the breast cancer cell at level that is higher or level than the level expressed by a non-cancerous cell. In other embodiments, the marker molecule may be present at elevated (or alternatively, reduced) levels in biological fluids obtained from a subject, such as blood, serum or urine.

[0061] In further embodiments the present disclosure provides a composition of matter comprising a plurality of nucleic acids that specifically bind to a panel of markers wherein the panel of markers comprises nucleic acids encoded by two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more of genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL- 1R4/ST2, carbonic anhydrase 6, CRP and uPAR. The panel of markers may be expressed at a level that is higher or lower than the level of the panel of markers in a non-cancerous cell. In other embodiments, the panel of markers may be present at elevated (or alternatively, reduced) levels in biological fluids obtained from a subject, such as blood, serum or urine.

[0062] In still further embodiments the present disclosure provides a method of determining if a breast cancer in a subject is advancing comprising a) measuring the expression level of one or more markers associated with breast cancer at a first time point; b) measuring the expression level of the one or more markers measured in a) at a second time point, wherein the second time point is subsequent to the first time point; and c) comparing the expression level measured in a) and b), wherein an increase or decrease in the expression level of the one or more markers in b) compared to a) indicates that the subject's breast cancer is advancing. Suitable markers include those markers encoded by the genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR; and/or any one or more genes listed in any of Tables 1-6.

[0063] In some embodiments the present disclosure provides antigens (i.e., cancer- associated polypeptides) associated with breast cancer as targets for diagnostic and/or therapeutic antibodies. In some embodiments, the antigen may be chosen from a protein encoded by a gene chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR and/or any one or more genes listed in any of Tables 1-6, a fragment of said protein, or a combination of proteins or protein fragments encoded by any of the genes listed infra.

[0064] In some embodiments the present disclosure provides antigens (i.e., cancer- associated polypeptides) associated with breast cancer as targets for diagnostic and/or therapeutic antibodies. In some embodiments, the antigen(s) may include a panel of proteins encoded by one or more of the genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR and/or any one or more genes listed in any of Tables 1-6, or one or more fragments of the one or more proteins listed above.

[0065] In yet other embodiments the present disclosure provides a method of eliciting an immune response to a breast cancer cell comprising contacting a subject with a protein or protein fragment that is expressed by a breast cancer cell thereby eliciting an immune response to the breast cancer cell. As an example the subject may be contacted intravenously or intramuscularly with protein or protein fragment.

[0066] In further embodiments the present disclosure provides a method of eliciting an immune response to a breast cancer cell comprising contacting a subject with one or more proteins or protein fragments that is encoded by a gene chosen from C5, cathepsin D, IL- 18 Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL- 1R4/ST2, carbonic anhydrase 6, CRP and uPAR and/or any one or more genes listed in any of Tables 1-6, thereby eliciting an immune response to a breast cancer cell. As an example the subject may be contacted with the protein or the protein fragment intravenously or intramuscularly.

[0067] In yet other embodiments present disclosure provides a kit for detecting breast cancer cells in a sample. The kit may comprise one or more agents that detect expression of any of the cancer associated markers (e.g. , polypeptides, nucleic acids) disclosed infra. The agents may bind to one or more of the cancer associated markers disclosed infra. The kit may include agents that are proteins and/or nucleic acids for example. In one embodiment the kit provides a plurality of agents. The agents may be able to detect the panel of markers encoded by the genes comprising C5, cathepsin D, IL- 18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL- 1R4/ST2, carbonic anhydrase 6, CRP and uPAR and/or any one or more genes listed in any of Tables 1-6.

[0068] In still other embodiments the present disclosure provides a kit for detecting breast cancer in a sample comprising a plurality of agents that specifically bind to a plurality of molecules encoded for by a plurality of genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR; and/or any one or more genes listed in any of Tables 1-6.

[0069] In other embodiments the present disclosure provides a kit for detection of breast cancer in a sample obtained from a subject. The kit may comprise one or more agents that bind specifically to one or more of the markers encoded for by one or more of the genes listed in any of Tables 1-6. The kit may comprise one or more containers and instructions for determining if the sample is positive for cancer. The kit may optionally contain one or more multiwell plates, a detectable substance such as a dye, a radioactively labeled molecule, a chemiluminescently labeled molecule and the like. The detectible substance may be linked the agent that specifically binds to a molecule expressed by a breast cancer cell. The kit may further contain a positive control (e.g. , one or more breast cancer cells; or specific known quantities of the molecule expressed by the breast cancer cell) and/or a negative control (e.g., a tissue or cell sample that is non-cancerous).

[0070] In some embodiments the present disclosure provides a kit for the detection of breast cancer comprising one or more agents that specifically bind one or more markers encoded by genes chosen from C5, cathepsin D, IL- 18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL- 1R4/ST2, carbonic anhydrase 6, CRP and uPAR and/or any one or more genes listed in any of Tables 1-6. The agent may be a protein, such as an antibody. Alternatively, the agent may be an aptamer that binds to the one or more protein(s) of choice. Alternatively, the agent may be a nucleic such as a DNA molecule or an RNA molecule. The kit may comprise one or more containers and instructions for determining if the sample is positive for cancer. The kit may optionally contain one or more multiwell plates, a detectable substance such as a dye, a radioactively labeled molecule, a chemiluminescently labeled molecule and the like. The detectable substance may be linked to the agent that specifically binds the one or more markers disclosed infra. The kit may further contain a positive control (e.g. , one or more breast cancer cells; or specific known quantities of the molecule expressed by the breast cancer cell) and/or a negative control (e.g. , a tissue or cell sample that is non-cancerous). As an example the kit may take the form of an ELISA or a DNA microarray. In some embodiments the kit may include one or more antibodies suitable for use in a fluorescent activated cell sorter, e.g. use in flow cytometry.

[0071] Some embodiments are directed to a method of treating breast cancer in a subject, the method comprising administering to a subject in need thereof a therapeutic agent modulating the activity of a breast cancer associated protein, wherein the cancer associated protein is encoded by a gene selected from C5, cathepsin D, IL- 18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR; and/or any one or more genes listed in any of Tables 1-6, homologs thereof, combinations thereof, or a fragment thereof. In some embodiments, the therapeutic agent binds to the cancer associated protein. In some embodiments, the therapeutic agent is an aptamer. In some embodiments, the therapeutic agent is an antibody. In some embodiments, the antibody may be a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is a humanized or human antibody. In some embodiments the antibody may be conjugated with a drug or a toxin.

[0072] In some embodiments, a method of treating breast cancer in a subject may comprise administering to a subject in need thereof a therapeutic agent that modulates the expression of one or more genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR and/or any one or more genes listed in any of Tables 1-6, fragments thereof, homologs thereof, and/or complements thereof.

[0073] In further embodiments, the present disclosure provides a method of treating breast cancer comprising a gene knockdown of one or more genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR and/or any one or more genes listed in any of Tables 1-6, fragments thereof, homologs thereof, and or complements thereof.

[0074] In still other embodiments, the present disclosure provides methods of screening a drug candidate for activity against breast cancer, the method comprising: (a) contacting a cell that expresses one or more breast cancer associated genes chosen from C5, cathepsin D, IL- 18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR and/or any one or more genes listed in any of Tables 1-6, with a drug candidate; (b) detecting an effect of the drug candidate on expression of the one or more breast cancer associated genes in the cell from a); and (c) comparing the level of expression of one or more of the genes recited in a) in the absence of the drug candidate to the level of expression of the one or more genes recited in a) in the presence of the drug candidate; wherein a decrease in the expression of the breast cancer associated gene that is upregulated in breast cancer or an increase in the expression of a breast cancer associated gene that is downregulated in breast cancer in the presence of the drug candidate indicates that the drug candidate has activity against breast cancer.

[0075] In some embodiments, the present disclosure provides methods of visualizing a breast cancer tumor comprising a) targeting one or more breast cancer associated proteins with a labeled molecule that binds specifically to the breast cancer tumor, wherein the breast cancer associated protein is selected from a protein encoded for by one or more genes chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL- 1R4/ST2, carbonic anhydrase 6, CRP and uPAR and/or any one or more genes listed in any of Tables 1-6; and b) detecting the labeled molecule, wherein the labeled molecule visualizes the tumor. Visualization may be done in vivo, or in vitro. [0076] In yet other embodiments, the present disclosure provides methods of visualizing a breast cancer tumor comprising a) targeting one or more breast cancer associated genes or gene products, (e.g. , mRNA) with a labeled molecule (e.g. , a labeled nucleic acid) that binds specifically to the breast cancer associated gene or gene product, wherein the breast cancer associated gene or gene product is chosen from C5, cathepsin D, IL-18Rbeta, MK13, SLAF6, CTGF, CYTN, ApoE2, MIG, PTN, PPIE, Factor I, IL-1R4/ST2, carbonic anhydrase 6, CRP and uPAR; and/or any one or more genes listed in any of Tables 1-6, and b) detecting the labeled molecule, wherein the labeled molecule visualizes the tumor. Visualization may be done in vivo, or in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] For a fuller understanding of the nature and advantages of the present invention, reference should be had to the following detailed description taken in connection with the accompanying drawings, in which:

[0078] FIG. 1 shows analysis of robust FDR (false discovery rate) LogWorth by effect size for analysis of 1,310 markers for suitability as breast cancer diagnostics.

[0079] FIG. 2A shows CDF (cumulative distribution function) of benign vs. cancerous samples for the C5 marker.

[0080] FIG. 2B shows a density plot (derived from a scatter plot of the marker on a loglO scale) to illustrate the ability of the C5 marker to distinguish between benign vs. cancerous samples.

[0081] FIG. 3 shows comparisons of ROC (receiver operating characteristic) curves for three preliminary algorithms for distinguishing cancerous samples from benign samples. The curves represent the 15-marker panel shown in Table 4 (top black curve), the 6-marker panel A as shown in Table 5 (lower black curve), and the 6-marker panel B as shown in Table 6 (gray curve).

[0082] FIG. 4A shows data and violin plots of model scores (i.e., probability of being a cancer sample) for the 15-marker panel.

[0083] FIG. 4B shows data and violin plots of model scores (i.e., probability of being a cancer sample) for the 6-marker panel A.

[0084] FIG. 4C shows data and violin plots of model scores (i.e., probability of being a cancer sample) for the 6-marker panel B. DETAILED DESCRIPTION

[0085] Before the compositions and methods of the present disclosure are described, it is to be understood that the invention or inventions disclosed herein are not limited to the particular processes, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure, the preferred methods, devices, and materials are now described. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0086] As used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a "therapeutic" is a reference to one or more therapeutics and equivalents thereof known to those skilled in the art, and so forth.

[0087] As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45% to 55%.

[0088] "Administering," when used in conjunction with a therapeutic, means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient whereby the therapeutic treats the tissue to which it is targeted. Thus, as used herein, the term "administering," when used in conjunction with a therapeutic, can include, but is not limited to, providing the therapeutic into or onto the target tissue; providing the therapeutic systemically to a patient by, e.g., intravenous injection whereby the therapeutic reaches the target tissue; providing the therapeutic in the form of the encoding sequence thereof to the target tissue (e.g., by so-called gene-therapy techniques). "Administering" a composition may be accomplished by oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, transdermal diffusion or electrophoresis, local injection, extended release delivery devices including locally implanted extended release devices such as bioerodible or reservoir-based implants, as protein therapeutics or as nucleic acid therapeutic via gene therapy vectors, topical administration, or by any of these methods in combination with other known techniques. Such combination techniques include, without limitation, heating, radiation and ultrasound.

[0089] "Agent" as used herein refers to a molecule that specifically binds to a cancer associated sequence or a molecule encoded by a cancer associated sequence or a receptor that binds to a molecule encoded by a cancer associated sequence. Examples of agents include nucleic acid molecules (such as DNA), proteins (such as antibodies) and aptamers. The agent may be linked with a label or detectible substance as described infra. The agent may be linked with a therapeutic agent or a toxin.

[0090] The term "amplify" as used herein means creating an amplification product which may include, for example, additional target molecules, or target-like molecules or molecules complementary to the target molecule, which molecules are created by virtue of the presence of the target molecule in the sample. In the situation where the target is a nucleic acid, an amplification product can be made enzymatically with DNA or RNA polymerases or reverse transcriptases, or any combination thereof.

[0091] The terms "animal," "patient" or "subject" as used herein include, but are not limited to, humans, non-human primates and non-human vertebrates such as wild, domestic and farm animals including any mammal, such as cats, dogs, cows, sheep, pigs, horses, rabbits, rodents such as mice and rats. In some embodiments, the term "subject," "patient" or "animal" refers to a male. In some embodiments, the term "subject," "patient" or "animal" refers to a female.

[0092] The term "antibody", as used herein, means an immunoglobulin or a part thereof, and encompasses any polypeptide comprising an antigen binding site regardless of the source, method of production, or other characteristics. The term includes for example, polyclonal, monoclonal, monospecific, polyspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutated, and CDR grafted antibodies. A part of an antibody can include any fragment which can bind antigen, for example, an Fab, F (ab')2, Fv, scFv.

[0093] The term "biological sources" as used herein refers to the sources from which the target polynucleotides or proteins or peptide fragments may be derived. The source can be of any form of "sample" as described infra, including but not limited to, cell, tissue or fluid. "Different biological sources" can refer to different cells/tissues/organs of the same individual, or cells/tissues/organs from different individuals of the same species, or cells/tissues/organs from different species.

[0094] The term "capture reagent" refers to a reagent, for example an antibody or antigen binding protein, capable of binding a target molecule or analyte to be detected in a sample.

[0095] The term "gene expression result" refers to a qualitative and/or quantitative result regarding the expression of a gene or gene product. Any method known in the art may be used to quantitate a gene expression result. The gene expression result can be an amount or copy number of the gene, the RNA encoded by the gene, the mRNA encoded by the gene, the protein product encoded by the gene, or any combination thereof. The gene expression result can also be normalized or compared to a standard. The gene expression result can be used, for example, to determine if a gene is expressed, overexpressed, or differentially expressed in two or more samples by comparing the gene expression results from 2 or more samples or one or more samples with a standard or a control.

[0096] The term "homology," as used herein, refers to a degree of complementarity. There may be partial homology or complete homology. The word "identity" may substitute for the word "homology." A partially complementary nucleic acid sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as "substantially homologous." The inhibition of hybridization of the completely complementary nucleic acid sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency. A substantially homologous sequence or hybridization probe will compete for and inhibit the binding of a completely homologous sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that nonspecific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% homology or identity). In the absence of non-specific binding, the substantially homologous sequence or probe will not hybridize to the second non- complementary target sequence.

[0097] As used herein, the term "hybridization" or "hybridizing" refers to hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementary," as used herein in reference to nucleic acid molecules, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that a nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. A nucleic acid compound is specifically hybridizable when there is binding of the molecule to the target, and there is a sufficient degree of complementarity to avoid non-specific binding of the molecule to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[0098] The term "inhibiting" includes the administration of a compound of the present disclosure to prevent the onset of the symptoms, alleviating the symptoms, or eliminating the disease, condition or disorder. The term "inhibiting" may also refer to lowering the expression level of gene, such as a gene encoding a cancer associated sequence. Expression level of RNA and/or protein may be lowered.

[0099] The terms "label" and/or "detectable substance" refer to a composition capable of producing a detectable signal indicative of the presence of the target polynucleotide or a polypeptide or protein in an assay sample. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by a device or method, such as, but not limited to, a spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical detection device or any other appropriate device. In some embodiments, the label may be detectable visually without the aid of a device. The term "label" is used to refer to any chemical group or moiety having a detectable physical property or any compound capable of causing a chemical group or moiety to exhibit a detectable physical property, such as an enzyme that catalyzes conversion of a substrate into a detectable product. The term "label" also encompasses compounds that inhibit the expression of a particular physical property. The label may also be a compound that is a member of a binding pair, the other member of which bears a detectable physical property.

[00100] A "microarray" is a linear or two-dimensional array of, for example, discrete regions, each having a defined area, each optionally containing a polynucleotide of defined sequence, formed on the surface of a solid support. The density of the discrete regions on a microarray is determined by the total numbers of target polynucleotides to be detected on the surface of a single solid phase support, preferably at least about 50/cm² more preferably at least about 100/cm², even more preferably at least about 500/cm², and still more preferably at least about 1,000/cm². As used herein, a DNA microarray is an array of oligonucleotide primers placed on a chip or other surfaces used to identify, amplify, detect, or clone target polynucleotides. Since the position of each particular group of oligonucleotides in the array is known, the identities of the target polynucleotides can be determined based on their binding to a particular position in the microarray.

[00101] As used herein, the term "naturally occurring" refers to sequences or structures that may be in a form normally found in nature, or to phenomena that inevitably occur in nature in all circumstances. "Naturally occurring" may include sequences in a form normally found in any animal.

[00102] The use of "nucleic acid," "polynucleotide" or "oligonucleotide" or equivalents herein means at least two nucleotides covalently linked together. In some embodiments, an oligonucleotide is an oligomer of 6, 8, 10, 12, 20, 30 or up to 100 nucleotides. In some embodiments, an oligonucleotide is an oligomer of at least 6, 8, 10, 12, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides. A "polynucleotide" or "oligonucleotide" may comprise DNA, RNA, PNA (peptide nucleic acid) or a polymer of nucleotides linked by phosphodiester and/or any alternate bonds.

[00103] As used herein, the term "optional" or "optionally" refers to embodiments where the subsequently described structure, event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not. [00104] The phrases "percent homology," "% homology," "percent identity," or "% identity" refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (LASERGENE software package, DNASTAR). The MEGALIGN program can create alignments between two or more sequences according to different methods, e.g., the Clustal Method. (Higgins, D. G. and P. M. Sharp (1988) Gene 73:237- 244.) The Clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be calculated by the Clustal Method, or by other methods known in the art, such as the Jotun Hein Method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.

[00105] By "pharmaceutically acceptable", it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[00106] "Recombinant protein," as used herein, means a protein made using recombinant techniques, for example, but not limited to, through the expression of a recombinant nucleic acid as described infra. A recombinant protein may be distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure. For example, an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample. A substantially pure protein comprises about 50-75%, about 80%, or about 90%. In some embodiments, a substantially pure protein comprises about 80-99%, 85-99%, 90- 99%, 95-99%, or 97-99% by weight of the total protein. A recombinant protein can also include the production of a cancer associated protein from one organism (e.g. human) in a different organism (e.g. yeast, E. coli, or the like) or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed herein. Recombinant proteins may also differ from naturally-occurring proteins with respect to one or more post- translational modifications such as, for example, phosphorylation, glycosylation or ubiquitination.

[00107] The abbreviation "ROC" as used herein refers to "receiver operating characteristic". A receiver operating characteristic graph is used in the statistical analysis of binary classifiers. The ROC curve is created by plotting the sensitivity (true positive rate) versus 1- specificity (false positive rate). The area under the ROC curve ("AUC") is the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. In simple terms, the AUC of a ROC curve provides a means of reducing classifier ROC performance to a single value. Random classification yields an AUC of 0.5 whereas perfect classification (no classification errors) yields an AUC of 1.

[00108] As used herein, the term "sample" refers to composition that is being tested or treated with a reagent, agent, capture reagent, binding partner and the like. Samples may be obtained from subjects. In some embodiments, the sample may be blood, plasma, serum, urine or any combination thereof. A sample may be derived from blood, plasma, serum, urine or any combination thereof. Other typical samples include, but are not limited to, any bodily fluid obtained from a mammalian subject, tissue biopsy, sputum, lymphatic fluid, blood cells (e.g., peripheral blood mononuclear cells), tissue or fine needle biopsy samples, peritoneal fluid, colostrum, breast milk, fetal fluid, fecal material, tears, pleural fluid, or cells therefrom. The sample may be processed in some manner before being used in a method described herein, for example a particular component to be analyzed or tested according to any of the methods described infra. One or more molecules (e.g. , nucleic acids, proteins) may be isolated from a sample.

[00109] The terms "specific binding," "specifically binds," and the like, refer to instances where two or more molecules form a complex that is measurable under physiologic or assay conditions and is selective. An antibody or antigen binding protein or other molecule is said to "specifically bind" to a protein, antigen, or epitope if, under appropriately selected conditions, such binding is not substantially inhibited, while at the same time non-specific binding is inhibited. Specific binding is characterized by a high affinity and is selective for the compound, protein, epitope, or antigen. Nonspecific binding usually has a low affinity. Examples of specific binding include the binding of enzyme and substrate, an antibody and its antigenic epitope, a cellular signaling molecule and its respective cell receptor.

[00110] As used herein, a polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which is comprised of a sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding to a region of the designated nucleotide sequence. "Corresponding" means homologous to or complementary to the designated sequence. Preferably, the sequence of the region from which the polynucleotide is derived is homologous to or complementary to a sequence that is unique to a cancer associated gene.

[00111] As used herein, the term "tag," "sequence tag" or "primer tag sequence" refers to an oligonucleotide with a specific nucleic acid sequence that serves to identify a batch of polynucleotides bearing such tags therein. Polynucleotides from the same biological source are covalently tagged with a specific sequence tag so that in subsequent analysis the polynucleotide can be identified according to its source of origin. The sequence tags also serve as primers for nucleic acid amplification reactions.

[00112] The term "support" refers to conventional supports such as beads, particles, dipsticks, fibers, filters, membranes, and silane or silicate supports such as glass slides.

[00113] As used herein, the term "therapeutic" or "therapeutic agent" means an agent that can be used to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient. In part, embodiments of the present disclosure are directed to the treatment of cancer or the decrease in proliferation of cells. In some embodiments, the term "therapeutic" or "therapeutic agent" may refer to any molecule that associates with or affects the target marker or cancer associated sequence disclosed infra, its expression or its function. In various embodiments, such therapeutics may include molecules such as, for example, a therapeutic cell, a therapeutic peptide, a therapeutic gene, a therapeutic compound, or the like, that associates with or affects the target marker or cancer associated sequence disclosed infra, its expression or its function. [00114] A "therapeutically effective amount" or "effective amount" of a composition is a predetermined amount calculated to achieve the desired effect, i.e., to inhibit, block, or reverse the activation, migration, metastasis, or proliferation of cells. In some embodiments, the effective amount is a prophylactic amount. In some embodiments, the effective amount is an amount used to medically treat the disease or condition. The specific dose of a composition administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the composition administered, the route of administration, and the condition being treated. It will be understood that the effective amount administered will be determined by the physician in the light of the relevant circumstances including the condition to be treated, the choice of composition to be administered, and the chosen route of administration. A therapeutically effective amount of composition of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the targeted tissue.

[00115] The terms "treat," "treated," or "treating" as used herein can refer to both therapeutic treatment or prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, symptom, disorder or disease, or to obtain beneficial or desired clinical results. In some embodiments, the term may refer to both treating and preventing. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[00116] The term "tissue" refers to any aggregation of similarly specialized cells that are united in the performance of a particular function.

Cancer Associated Sequences [00117] In some embodiments, the present disclosure provides for nucleic acid and protein sequences that are associated with cancer, herein termed "cancer associated" or "CA" sequences. In some embodiments, the present disclosure provides nucleic acid and protein sequences that are associated with breast cancers such as, without limitation, ductal atypia, ductal hyperplasia, ductal carcinoma in situ (DCIS), invasive (infiltrating) ductal carcinoma, lobular carcinoma, inflammatory breast cancer and Paget disease. However, as will be appreciated by those skilled in the art, sequences that are identified in one type of cancer may have a strong likelihood of being involved in other types of cancers as well. Thus, while the sequences outlined herein may be initially identified as correlated with one or more types of cancers, they may also be found in other types of cancers as well. The method of diagnosing may comprise measuring the level of expression of a cancer associated marker disclosed herein. The method may further comprise comparing the expression level of the cancer associated sequence with a standard and/or a control. The standard may be from a sample known to contain breast cancer cells. The control may include known breast cancer cells and/or non-cancerous cells, such as non-cancer cells derived from breast tissue.

[00118] Cancer associated sequences may include those that are up-regulated (i.e. , expressed at a higher level), as well as those that are down-regulated (i.e. , expressed at a lower level), in cancers. Cancer associated sequences can also include sequences that have been altered (i.e. , translocations, truncated sequences or sequences with substitutions, deletions or insertions, including, but not limited to, point mutations) and show either the same expression profile or an altered profile. In some embodiments, the cancer associated sequences are from humans; however, as will be appreciated by those in the art, cancer associated sequences from other organisms may be useful in animal models of disease and drug evaluation; thus, other cancer associated sequences may be useful, including those obtained from any subject, such as, without limitation, sequences from vertebrates, including mammals, such as rodents (rats, mice, hamsters, guinea pigs, etc.), primates, and farm animals (including sheep, goats, pigs, cows, horses, etc.). Cancer associated sequences from other organisms may be obtained using the techniques outlined herein.

[00119] Examples of cancer associated sequences include the nucleic acid and amino acid sequences encoded by the genes listed in Tables 1-6.

[00120] In some embodiments, the cancer associated sequences are nucleic acids. As will be appreciated by those skilled in the art and as described herein, cancer associated sequences of embodiments herein may be useful in a variety of applications including diagnostic applications to detect nucleic acids or their expression levels in a subject, therapeutic applications or a combination thereof. Further, the cancer associated sequences of embodiments herein may be used in screening applications; for example, generation of biochips (e.g., microarrays) comprising nucleic acid probes that specifically bind to the cancer associated sequences.

[00121] A nucleic acid of the present disclosure may include phosphodiester bonds, although in some cases, as outlined below (for example, in antisense applications or when a nucleic acid is a candidate drug agent), nucleic acid analogues may have alternate backbones, comprising, for example, phosphoramidate (Beaucage et al., Tetrahedron 49(10): 1925 (1993) and references therein); Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81 :579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26: 141 91986)); phosphorothioate (Mag et al., Nucleic Acids Res. 19: 1437 (1991) and U.S. Pat. No. 5,644,048); phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111 :2321 (1989); O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and/or peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114: 1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31 : 1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996),). Other nucleic acid analogues include those with positively-charged backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non- ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13: 1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34: 17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp. 169-176). Several nucleic acid analogues are described in Rawls, C & E News Jun. 2, 1997 page 35. These modifications of the ribose-phosphate backbone may be made for a variety of reasons, for example to increase the stability and half-life of such molecules in physiological environments for use in anti-sense applications or as probes on a biochip.

[00122] As will be appreciated by those skilled in the art, such nucleic acid analogues may be used in some embodiments of the present disclosure. In addition, mixtures of naturally occurring nucleic acids and nucleic acid analogues can be made; alternatively, mixtures of different nucleic acid analogues, and mixtures of naturally occurring nucleic acids and analogues may be made.

[00123] In some embodiments, the nucleic acids may be single stranded or double stranded or may contain portions of both double stranded or single stranded sequence. As will be appreciated by those skilled in the art, the depiction of a single strand also defines the sequence of the other (complementary) strand; thus the sequences described herein also includes the complement of the sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, etc. As used herein, the term "nucleoside" includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino- modified nucleosides. In addition, "nucleoside" includes non-naturally occurring analogue structures. Thus, for example, the subject units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.

[00124] In some embodiments, cancer associated sequences may include both nucleic acid and amino acid sequences. In some embodiments, the cancer associated sequences may include sequences having at least about 60% homology with the disclosed sequences. In some embodiments, the cancer associated sequences may have at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, about 99.8% homology with the disclosed sequences. In some embodiments, the cancer associated sequences may be "mutant nucleic acids". As used herein, "mutant nucleic acids" refers to, for example, deletion mutants, insertions, point mutations, substitutions, and translocations.

[00125] In some embodiments, the cancer associated sequences may be recombinant nucleic acids. The term "recombinant nucleic acid," as used herein, refers to nucleic acid molecules, originally formed in vitro, in general, by the manipulation of nucleic acid by polymerases, ligases, kinases and/or endonucleases, in a form not normally found in nature. Thus a recombinant nucleic acid may be an isolated nucleic acid, in a linear form, or cloned in a vector formed in vitro by ligating DNA molecules that are not normally joined, both of which are considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it can replicate using the in vivo cellular machinery of the host cell rather than by in vitro manipulation; however, such nucleic acids, once produced recombinantly, although subsequently replicated in vivo, are still considered recombinant or isolated for the purposes of the invention. As used herein, a "polynucleotide" or "nucleic acid" is a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides or a mixture thereof. This term includes double- and single- stranded DNA and RNA. It also includes known types of modifications, for example, labels which are known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with a nucleotide analogue, internucleotide modifications-such as, for example, those with uncharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including e.g., nucleases, toxins, antibodies, signal peptides, poly- L- lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide.

[00126] The use of microarray analysis of gene expression allows the identification of host sequences associated with breast cancer. These sequences may then be used in a number of different ways, including diagnosis, prognosis, screening for modulators (including both agonists and antagonists), antibody generation (for immunotherapy and imaging), etc. However, as will be appreciated by those skilled in the art, sequences that are identified in one type of cancer may have a strong likelihood of being involved in other types of cancers as well. Thus, while the sequences outlined herein are initially identified as correlated with breast cancers, they may also be found in other types of cancers as well.

[00127] Some embodiments described herein are directed to the use of cancer associated sequences for diagnosis and treatment of breast cancer. In some embodiments, the cancer associated sequence is selected from any one or more genes (or the complement thereof) listed in Tables 1-6. In some embodiments, these cancer associated sequences may be associated with breast cancers including, without limitation, ductal atypia, ductal hyperplasia, ductal carcinoma in situ (DCIS), invasive (infiltrating) ductal carcinoma, lobular carcinoma, inflammatory breast cancer and Paget disease, recurrent and metastatic breast cancer, or combinations thereof.

[00128] In some embodiments, the cancer associated sequences are DNA sequences encoding mRNA encoded by any one or more of the genes listed in Tables 1-6. Alternatively, a cancer associated sequence can be a cancer-associated associated protein or cancer associated polypeptide expressed by the aforementioned mRNAs or homologues thereof. In some embodiments, the cancer associated sequence may be a nucleic acid that is a mutant version of the above disclosed sequences. In some embodiments, the homologue may have at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% identity with the disclosed polypeptide sequence.

[00129] In some embodiments, an isolated nucleic acid comprises at least 10, 12, 15, 20 or 30 contiguous nucleotides of a sequence (or complement thereof) selected from the group consisting of the cancer associated polynucleotide sequences corresponding to any one or more genes listed in Tables 1-6.

[00130] In some embodiments, the polynucleotide, or its complement or a fragment thereof, further comprises a detectable label, is attached to a solid support, is prepared at least in part by chemical synthesis, is an antisense fragment, is single stranded, is double stranded or is comprised in a microarray.

[00131] In some embodiments, the present disclosure provides an isolated polypeptide, encoded within an open reading frame of a cancer associated sequence selected from the polynucleotide sequences (or complements thereof) corresponding to any one or more of the genes listed in Tables 1-6. In some embodiments, the present disclosure provides an isolated polypeptide, wherein said polypeptide comprises the amino acid sequence encoded by a polynucleotide selected from the group consisting of sequences (or complements thereof) corresponding to any one or more gene(s) listed in Tables 1-6. In some embodiments, the invention provides an isolated polypeptide, wherein said polypeptide comprises the amino acid sequence encoded by a cancer associated polypeptide as described herein.

[00132] In some embodiments, the present disclosure further provides an isolated polypeptide, comprising the amino acid sequence of an epitope of the amino acid sequence of a cancer associated polypeptide disclosed herein. The polypeptide or fragment thereof may be attached to a solid support. In some embodiments the present disclosure provides an isolated antibody (monoclonal or polyclonal) or antigen binding fragment thereof, that binds to such a polypeptide. The isolated antibody or antigen binding fragment thereof may be attached to a solid support. The isolated antibody or antigen binding fragment thereof may further comprise a detectable substance.

[00133] Some embodiments also provide for antigens (e.g., cancer-associated polypeptides) associated with a variety of cancers as targets for diagnostic and/or therapeutic antibodies, e.g. breast cancer antigens. These antigens may also be useful for drug discovery (e.g. , small molecules) and for further characterization of cellular regulation, growth, and differentiation.

Methods of Detecting and Diagnosing Breast Cancer

[00134] In some embodiments, a method of detecting or diagnosing breast cancer may comprise assaying gene expression in a subject in need of said diagnosis. Any method known in the art may be used to assay gene expression of one or more markers disclosed herein. In some embodiments, detecting a level of a cancer associated sequence may comprise techniques such as, but not limited to, polymerase chain reaction (PCR), mass spectroscopy, microarray, gel electrophoresis, and/or hybridization using one more probes that specifically bind a nucleic acid encoding a cancer associated sequence disclosed herein. Information relating to expression of a receptor can also be useful in determining therapies aimed at up- or down-regulating the cancer associated sequence's signaling using agonists or antagonists.

[00135] In some embodiments, a method of diagnosing breast cancer may comprise detecting a level of the cancer associated protein in a subject. In some embodiments, a method of screening for cancer may comprise detecting a level of the cancer associated protein. In some embodiments, the cancer associated protein is encoded by a nucleotide sequence (or fragment thereof, or complement thereof) selected from a sequence corresponding to any one or more of the genes listed in Tables 1-6. In some embodiments, a method of detecting cancer in a sample may comprise contacting the sample obtained from a subject with an antibody that specifically binds a cancer-associated protein as disclosed herein. In some embodiments, the antibody may be a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody may be a humanized or a recombinant antibody. In some embodiments, an antibody specifically binds to one or more of a molecule, such as protein or peptide, encoded by one or more cancer associated sequences disclosed herein.

[00136] In some embodiments, the antibody binds to an epitope from a protein encoded by any one or more of the genes listed in Tables 1-6. In some embodiments, the epitope is a fragment of a protein sequence encoded by the nucleotide sequence of any of the cancer associated sequences disclosed herein. In some embodiments, the epitope comprises about 1- 10, 1-20, 1-30, 3-10, or 3-15 residues of the cancer associated sequence. In some embodiments, the epitope is not linear. In some embodiments, the epitope is discontinuous.

[00137] In some embodiments, the antibody binds to the regions described herein or a peptide with at least 90, 95, or 99% homology or identity to the region. In some embodiments, the fragment of the regions described herein is 5- 10 residues in length. In some embodiments, the fragment of the regions (e.g. , epitope) described herein are 3-5 residues in length. The fragments are described based upon the length provided. In some embodiments, the epitope is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 residues in length.

[00138] In some embodiments, the sequence to which the antibody binds may include both nucleic acid and amino acid sequences. In some embodiments, the sequence to which the antibody binds may include sequences having at least about 60% homology with the disclosed sequences. In some embodiments, the sequence to which the antibody binds may have at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 99.8% homology with the disclosed sequences. In some embodiments, the sequences may be referred to as "mutant nucleic acids" or "mutant peptide sequences."

[00139] In some embodiments, a subject can be diagnosed with breast cancer by detecting the presence, in a sample obtained from the subject, of a cancer associated sequence, or a fragment or complement thereof, e.g. , a sequence corresponding to any one or more of the genes listed in Tables 1-6. As discussed, cancer associated sequences may be detected in any type of sample, including, but not limited to, serum, blood, tumor and the like. The sample may be any type of sample as described herein.

[00140] Any assay known in the art may be used to screen for the presence, absence or expression level of one or more proteins encoded for by a cancer associated sequence described infra. In some embodiments the assay may be, for example, an ELISA, a radio-immuno assay, a western blot, a flow cytometry assay and the like. [00141] In certain embodiments, a cancer-associated protein is detected by using an aptamer that specifically binds to the protein of interest. Aptamers are unique short nucleic acid (e.g., DNA, RNA) or peptide sequences that can be obtained by randomized synthesis followed by multiple rounds of selection for binding to a target. Certain aptamers, known as slow off-rate modified aptamers, or SOMAmers^®, comprise unique short DNA sequences that incorporate several bases that have been modified to include "protein-like" side chains, and a 5'-linker. Aptamers are high-affinity binding reagents which are very specific for their targets (e.g. , polypeptides, nucleic acids, small organic molecules) and allow for extremely high multiplexing of protein measurements in a high throughput and reproducible manner with very small sample volume requirements.

[00142] In some embodiments, the present disclosure provides a method of diagnosing breast cancer or a neoplastic condition in a subject, the method comprising obtaining, from a sample derived from the subject, a gene expression result for one or more cancer associated sequences selected from sequences corresponding to any one or more of the genes listed in Tables 1-6; and diagnosing breast cancer or a neoplastic condition in the subject based on the cancer associated sequence gene expression result, wherein the subject is diagnosed as having breast cancer or a neoplastic condition if the cancer associated sequence is expressed at a level that is 1) higher than its expression level in a negative control such a non-cancerous breast tissue or cell sample and/or 2) higher than or equivalent to its expression level in a standard or positive control wherein the standard or positive control is known to contain breast cancer cells.

[00143] Some embodiments are directed to a biochip (e.g. , a microarray) comprising one or more nucleic acid sequences which encode one or more cancer associated proteins. In some embodiments, a biochip comprises a nucleic acid molecule which encodes at least a portion of a cancer associated protein. In some embodiments, the cancer associated protein is encoded by a sequence selected from sequence corresponding to any one or more of the genes listed in Tables 1-6; or a fragment thereof, or a complement thereof, or a homologues thereof, or combinations thereof. In some embodiments, the nucleic acid molecule specifically hybridizes with a nucleic acid sequence selected from a sequence corresponding to any one or more of the genes listed in Tables 1-6, a fragment thereof, or a complement thereof. In some embodiments, the biochip comprises first and second nucleic molecules wherein the first nucleic acid molecule specifically hybridizes with a first sequence selected from a cancer associated sequence disclosed herein and the second nucleic acid molecule specifically hybridizes with a second sequence selected from a cancer associated sequences disclosed herein, wherein the first and second sequences are not the same sequence. In some embodiments, the present disclosure provides methods of detecting or diagnosing cancer, such as breast cancer, comprising detecting the expression of a nucleic acid sequence selected from sequences corresponding to any one or more of the genes listed in Tables 1-6, or a fragment or a complement thereof, wherein a sample is contacted with a biochip comprising a sequence selected from sequences corresponding to any one or more of the genes listed in Tables 1-6 or a fragment or a complement thereof.

[00144] Also provided herein is a method for diagnosing or determining the propensity to cancers, for example breast cancer, by measuring the expression level of one or more of the sequences upregulated in breast cancer, disclosed herein, in a sample and comparing the expression level of the one or more cancer associated sequences in the sample with expression level of the same cancer associated sequences in a non-cancerous cell. A higher level of expression of one or more of the cancer associated sequences disclosed herein in the sample compared to the noncancerous cell indicates a propensity for the development of cancer, e.g., breast cancer.

[00145] In some embodiments, the present disclosure provides a method for detecting a cancer associated sequence by expression of a polypeptide in a test sample, comprising detecting a level of expression of at least one polypeptide such as, without limitation, a cancer associated protein encoded by a sequence disclosed herein, or a fragment thereof. In some embodiments, the method comprises comparing the level of expression of the polypeptide in the test sample with a level of expression of polypeptide in a normal sample, i.e., a non-cancerous sample, wherein a higher level of expression of the polypeptide in the test sample relative to the level of polypeptide expression in the normal sample is indicative of the presence of cancer in the test sample. In some embodiments, the polypeptide expression is compared to a cancer sample, wherein a level of expression in the test sample that is at least as high as the level of expression in the cancer sample is indicative of the presence of cancer in the test sample. In some embodiments, the sample is a cell sample. In some embodiments the sample is a tissue sample. In some embodiments the sample is a bodily fluid. Examples of suitable bodily fluids, include, but are not limited to, blood, serum, plasma, saliva and urine. In some embodiments the sample is a blood sample. In some embodiments the sample is a serum sample. In some embodiments the sample is a urine sample. [00146] In some embodiments, the present disclosure provides a method for detecting cancer by detecting the presence of an antibody in a test serum sample. In some embodiments, the antibody recognizes a polypeptide or an epitope of a cancer associated sequence disclosed herein. In some embodiments, the method comprises detecting a level of an antibody against an antigenic polypeptide such as, without limitation, a cancer associated protein such as a protein encoded by a cancer associated sequence disclosed herein, or an antigenic fragment thereof. In some embodiments, the method comprises comparing the level of the antibody in the test sample with a level of the antibody in the control sample, wherein an altered level of antibody in said test sample relative to the level of antibody in the control sample is indicative of the presence of cancer in the test sample. In some embodiments, the control sample is a sample derived from a non-cancerous sample, e.g. , blood or serum obtained from a subject that is cancer free. In these cases, a higher level of antibody in the test sample, compared to the non-cancerous control sample, indicates the presence of cancer in the test sample. In some embodiments, the control is derived from a cancer sample, and, in these cases, levels or amount of antibody that are the same or greater in the test sample compared to the cancer control sample are indicative of the presence of cancer in the test sample.

[00147] In some embodiments, a method for diagnosing cancer or a neoplastic condition comprises a) determining the expression of one or more genes comprising a nucleic acid sequence (or a fragment thereof or a complement thereof) selected from the group consisting of the human genomic and mRNA sequences corresponding to any one or more of the genes listed in Tables 1- 6, in a first sample type (e.g. tissue, bodily fluid, etc.) of a first individual; and b) comparing said expression of said gene(s) with their expression in a second normal sample type from said first individual or a sample from a second unaffected individual; wherein an increase in said expression in the first sample, compared to either the (i) second normal sample from the first individual or (ii) the sample from the second unaffected individual indicates that the first individual has cancer.

[00148] In some embodiments, the present disclosure also provides a method for detecting presence or absence of cancer cells in a subject. In some embodiments, the method comprises contacting one or more cells from the subject with an antibody as described herein. The antibody may be conjugated to a detectible substance. In some embodiments the antibody that binds to a protein encoded by a cancer associated sequence disclosed herein may bind to a second antibody wherein the second antibody is conjugated to a detectable substance. In some embodiments the antibody that binds to a protein encoded for by a cancer associated sequence disclosed herein is bound to a solid support. In some embodiments, the method comprises detecting a complex of a cancer associated protein and the antibody, wherein detection of the complex indicates with the presence of cancer cells in the subject. The complex may include a detectable substance as described herein. The complex may include a solid support, such as bead, a chip, a magnet, a multiwell plate and the like.

[00149] In some embodiments, the present disclosure provides methods of detecting cancer in a test sample, comprising: (i) detecting a level of activity of at least one polypeptide that is a gene product; and (ii) comparing the level of activity of the polypeptide in the test sample with a level of activity of polypeptide in a normal sample, wherein an increased level of activity of the polypeptide in the test sample relative to the level of polypeptide activity in the normal sample is indicative of the presence of cancer in the test sample, wherein said gene product is a product of a gene selected from one or more of the cancer associated sequences provided herein.

Capture Reagents and Specific Binding Partners

[00150] The present disclosure provides for specific binding partners and capture reagents that bind specifically to cancer associated sequences disclosed herein and the polypeptides or proteins encoded by those sequences. The capture reagents and specific binding partners may be used in diagnostic assays as disclosed herein and/or in therapeutic methods described herein, as well as in drug screening assays disclosed infra. Capture reagents include for example nucleic acids and proteins. Suitable proteins include antibodies. Capture reagents and binding partners can also include aptamers.

[00151] As used herein, the term "specifically binds" or "specifically binding" means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding is indicated if the molecule has measurably higher affinity for cells expressing a protein encoded by a cancer associated sequence disclosed herein than for cells that do not express the same protein encoded by the cancer associated sequences disclosed herein. Specificity of binding can be determined, for example, by competitive inhibition of a known binding molecule. [00152] The term "specifically binding," as used herein, includes both low and high affinity specific binding. Specific binding can be exhibited, for example, by a low affinity homing molecule having a Kd of at least about 10^"4M. Specific binding also can be exhibited by a high affinity homing molecule, for example, a homing molecule having a Kd of at least about 10^"5 M. Such a molecule can have, for example, a Kd of at least about 10^"6 M, at least about 10^"7 M, at least about 10^"8 M, at least about 10^"9 M, at least about 10^"10 M, or can have a Kd of at least about 10^"11 M or 10^"12 M or greater. Both low and high affinity homing molecules are useful and are encompassed by the present disclosure. Low affinity homing molecules are useful in targeting, for example, multivalent conjugates. High affinity homing molecules are useful in targeting, for example, multivalent and univalent conjugates.

[00153] In some embodiments the specific binding partner or capture reagent is an antibody. Binding in IgG antibodies, for example, is generally characterized by an affinity of at least about 10^"7 M or higher, such as at least about 10 ^s M or higher, or at least about 10^"9 M or higher, or at least about 10^"10M or higher, or at least about 10^"11 M or higher, or at least about 10^" ¹² M or higher. The term is also applicable where, e.g., an antigen-binding domain is specific for a particular epitope that is not carried by numerous antigens, in which case the antibody or antigen binding protein carrying the antigen-binding domain will generally not bind other antigens. In some embodiments, the capture reagent has a Kd equal or less than 10^"9 M, 10^"10 M, or 10^"11 M for its binding partner (e.g. antigen). In some embodiments, the capture reagent has a Ka greater than or equal to 10⁹ M^"1 for its binding partner. Capture reagent can also refer to, for example, antibodies. Intact antibodies, also known as immunoglobulins, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each, and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and kappa, exist in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins are assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. Each light chain is composed of an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain is composed of an N-terminal V domain (VH), three or four C domains (CHs), and a hinge region. The CH domain most proximal to VH is designated CHI. The VH and VL domains consist of four regions of relatively conserved sequences named framework regions (FRl, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequences (complementarity determining regions, CDRs). The CDRs contain most of the residues responsible for specific interactions of the antibody or antigen binding protein with the antigen. CDRs are referred to as CDR1, CDR2, and CDR3. Accordingly, CDR constituents on the heavy chain are referred to as HI, H2, and H3, while CDR constituents on the light chain are referred to as LI, L2, and L3. CDR3 is the greatest source of molecular diversity within the antibody or antigen binding protein-binding site. H3, for example, can be as short as two amino acid residues or greater than 26 amino acids. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Eds. Harlow et al., 1988. One of skill in the art will recognize that each subunit structure, e.g., a CH, VH, CL, VL, CDR, and/or FR structure, comprises active fragments. For example, active fragments may consist of the portion of the VH, VL, or CDR subunit that binds the antigen, i.e., the antigen-binding fragment, or the portion of the CH subunit that binds to and/or activates an Fc receptor and/or complement.

[00154] Non-limiting examples of binding fragments encompassed within the term "antigen- specific antibody" used herein include: (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated CDR. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be recombinantly joined by a synthetic linker, creating a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv)). The most commonly used linker is a 15-residue (Gly₄Ser)3 peptide, but other linkers are also known in the art. Single chain antibodies are also intended to be encompassed within the terms "antibody or antigen binding protein," or "antigen-binding fragment" of an antibody. The antibody can also be a polyclonal antibody, monoclonal antibody, chimeric antibody, antigen-binding fragment, Fc fragment, single chain antibody, or any derivatives thereof.

[00155] Antibodies can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody diversity is created by multiple germline genes encoding variable domains and a variety of somatic events. The somatic events include recombination of variable gene segments with diversity (D) and joining (J) gene segments to make a complete VH domain, and the recombination of variable and joining gene segments to make a complete VL domain. The recombination process itself is imprecise, resulting in the loss or addition of amino acids at the V (D) J junctions. These mechanisms of diversity occur in the developing B cell prior to antigen exposure. After antigenic stimulation, the expressed antibody genes in B cells undergo somatic mutation. Based on the estimated number of germline gene segments, the random recombination of these segments, and random VH-VL pairing, up to 1.6X10⁷ different antibodies may be produced (Fundamental Immunology, 3rd ed. (1993), ed. Paul, Raven Press, New York, N.Y.). When other processes that contribute to antibody diversity (such as somatic mutation) are taken into account, it is thought that upwards of 1X10¹⁰ different antibodies may be generated (Immunoglobulin Genes, 2nd ed. (1995), eds. Jonio et al., Academic Press, San Diego, Calif.). Because of the many processes involved in generating antibody diversity, it is unlikely that independently derived monoclonal antibodies with the same antigen specificity will have identical amino acid sequences.

[00156] Antibodies, or antigen binding protein molecules, capable of specifically interacting with the antigens, epitopes, or other molecules described herein may be produced by methods well known to those skilled in the art. For example, monoclonal antibodies can be produced by generation of hybridomas in accordance with known methods. Hybridomas formed in this manner can then be screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and Biacore analysis, to identify one or more hybridomas that produce an antibody that specifically interacts with a molecule or compound of interest. As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody to a polypeptide of the present disclosure may be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a polypeptide of the present disclosure to thereby isolate immunoglobulin library members that bind to the polypeptide. Techniques and commercially available kits for generating and screening phage display libraries are well known to those skilled in the art. Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody or antigen binding protein display libraries can be found in the literature.

[00157] Examples of chimeric antibodies include, but are not limited to, humanized antibodies. The antibodies described herein can also be human antibodies. In some embodiments, the capture reagent comprises a detection reagent. The detection reagent can be any reagent that can be used to detect the presence of the capture reagent binding to its specific binding partner. The capture reagent can comprise a detection reagent directly or the capture reagent can comprise a particle that comprises the detection reagent. In some embodiments, the capture reagent and/or particle comprises a color, colloidal gold, radioactive tag, fluorescent tag, or a chemiluminescent substrate. The particle can be, for example, a viral particle, a latex particle, a lipid particle, or a fluorescent particle.

[00158] The capture reagents (e.g. antibody) of the present disclosure can also include an anti-antibody, i.e. an antibody that recognizes another antibody but is not specific to an antigen, such as, but not limited to, anti-IgG, anti-IgM, or ant-IgE antibody. This non-specific antibody can be used as a positive control to detect whether the antigen specific antibody is present in a sample.

[00159] Nucleic acid capture reagents include DNA, RNA and PNA molecules for example. The nucleic acid may be about 5 nucleotides long, about 10 nucleotides long, about 15 nucleotides long, about 20 nucleotides long, about 25 nucleotides long, about 30 nucleotides long, about 35 nucleotides long about 40 nucleotides long. The nucleic acid may be greater than 30 nucleotides long. The nucleic acid may be less than 30 nucleotides long.

Treatment of Breast Cancer

[00160] In some embodiments, breast cancers expressing one or more of the cancer associated sequences disclosed herein may be treated by antagonizing the cancer associated sequence's activity. In some embodiments, a method of treating breast cancer may comprise administering a therapeutic such as, without limitation, antibodies that antagonize the ligand binding to the cancer associated sequence, small molecules that inhibit the cancer associated sequence's expression or activity, siRNAs directed towards the cancer associated sequence, or the like.

[00161] In some embodiments, a method of treating cancer (e.g. breast or other types of cancer) comprises detecting the presence of a cancer associated sequence's receptor and administering a cancer treatment. The treatment may specifically bind to the cancer associated sequence's receptor. The cancer treatment may be any cancer treatment or one that specifically inhibits the action of a cancer associated sequence. For example, various cancers are tested to determine if a specific molecule is present before giving a cancer treatment. In some embodiments, therefore, a sample is obtained from the patient and tested for the presence of a cancer associated sequence or the overexpression of a cancer associated sequence as described herein. In some embodiments, if a cancer associated sequence is found to be overexpressed then a breast cancer treatment or therapeutic is administered to the subject. The breast cancer treatment may be a conventional non-specific treatment, such as chemotherapy, or the treatment may comprise a specific treatment that only targets the activity of the cancer associated sequence or the receptor to which the cancer associated sequence binds. These treatments can be, for example, an antibody that specifically binds to the cancer associated sequence and inhibits its activity. The treatment may be a nucleic acid that downregulates or silences the expression of the cancer associated sequence.

[00162] Some embodiments herein describe methods of treating cancer or a neoplastic condition comprising administering, to a subject, an antibody that binds to the cancer associated sequence. In some embodiments, the antibody may be monoclonal or polyclonal. In some embodiments, the antibody may be humanized or recombinant. In some embodiments, the antibody may neutralize biological activity of the cancer associated sequence by binding to and/or interfering with the cancer associated sequence's receptor. In some embodiments the antibody may bind to site on the protein encoded by the cancer associated DNA sequence that is not the receptor. In some embodiments, administering the antibody may be to a biological fluid or tissue, such as, without limitation, blood, urine, serum, plasma, tumor tissue, or the like.

[00163] In some embodiments, a method of treating cancer may comprise administering an agent that interferes with the synthesis, secretion, receptor binding or receptor signaling of cancer associated proteins or its receptors. In some embodiments, the cancer may be selected from, including, without limitation, ductal atypia, ductal hyperplasia, ductal carcinoma in situ (DCIS), invasive (infiltrating) ductal carcinoma, lobular carcinoma, inflammatory breast cancer, Paget disease, recurrent and metastatic breast cancer, or a combination thereof.

[00164] In some embodiments, the cancer cell may be targeted specifically with a therapeutic based upon the differentially expressed gene or gene product. For example, in some embodiments, the differentially expressed gene product may be an enzyme, which can convert an anti-cancer prodrug into its active form. Therefore, in normal cells, where the differentially expressed gene product is not expressed or expressed at significantly lower levels, the prodrug may be either not activated or activated in a lesser amount, and may be, therefore less toxic to normal cells. Therefore, the cancer prodrug may, in some embodiments, be given in a higher dosage so that the cancer cells can metabolize the prodrug, which will, for example, kill the cancer cell, and the normal cells will not metabolize the prodrug or not as well, and, therefore, the prodrug will be less toxic to the patient. An example of the use of this type of treatment is for tumor cells that overexpress a metalloprotease, which is described in Atkinson et al., British Journal of Pharmacology (2008) 153, 1344-1352. Using proteases to target cancer cells is also described in Carl et al., PNAS, Vol. 77, No. 4, pp. 2224-2228, April 1980. For example, doxorubicin or other types of chemotherapeutic can be linked to a peptide sequence that is specifically cleaved or recognized by the differentially expressed gene product. The doxorubicin or other type of chemotherapeutic is then cleaved from the peptide sequence and is activated such that it can kill or inhibit the growth of the cancer cell whereas in the normal cell the chemotherapeutic is never internalized into the cell or is not metabolized as efficiently, and is, therefore, less toxic.

[00165] In some embodiments, a method of treating breast cancer may comprise gene knockdown of one or more cancer associated sequences described herein. Gene knockdown refers to techniques by which the expression of one or more of an organism's genes is reduced, either through genetic modification (a change in the DNA of one of the organism's chromosomes such as, without limitation, chromosomes encoding cancer associated sequences) or by treatment with a reagent such as a short DNA or RNA oligonucleotide with a sequence complementary to either an mPvNA transcript or a gene. In some embodiments, the oligonucleotide used may be selected from RNase-H competent antisense, such as, without limitation, ssDNA oligonucleotides, ssRNA oligonucleotides, phosphorothioate oligonucleotides, or chimeric oligonucleotides; RNase- independent antisense, such as morpholino oligonucleotides, 2'-0-methyl phosphorothioate oligonucleotides, locked nucleic acid oligonucleotides, or peptide nucleic acid oligonucleotides; RNAi oligonucleotides, such as, without limitation, siRNA duplex oligonucleotides, or shRNA oligonucleotides; or any combination thereof. In some embodiments, a plasmid may be introduced into a cell, wherein the plasmid expresses either an antisense RNA transcript or an shRNA transcript. The oligonucleotide introduced or transcript expressed may interact with the target mRNA by complementary base pairing (a sense-antisense interaction).

[00166] The specific mechanism of silencing may vary with the oligonucleotide chemistry. In some embodiments, the binding of a oligonucleotide described herein to the active gene or its transcripts may cause decreased expression through blocking of transcription, degradation of the mRNA transcript (e.g. by small interfering RNA (siRNA) or RNase-H dependent antisense) or blocking either mRNA translation, pre-mRNA splicing sites or nuclease cleavage sites used for maturation of other functional RNAs such as miRNA (e.g. by morpholino oligonucleotides or other RNase-H independent antisense). For example, RNase-H competent antisense oligonucleotides (and antisense RNA transcripts) may form duplexes with RNA that are recognized by the enzyme RNase-H, which cleaves the RNA strand. As another example, RNase- independent oligonucleotides may bind to the mRNA and block the translation process. In some embodiments, the oligonucleotides may bind in the 5'-UTR and halt the initiation complex as it travels from the 5'-cap to the start codon, preventing ribosome assembly. A single strand of RNAi oligonucleotides may be loaded into the RISC complex, which catalytically cleaves complementary sequences and inhibits translation of some mRNAs bearing partially- complementary sequences. The oligonucleotides may be introduced into a cell by any technique including, without limitation, electroporation, microinjection, salt-shock methods such as, for example, CaCl₂ shock; transfection of anionic oligonucleotides by cationic lipids such as, for example, Lipofectamine^®; transfection of uncharged oligonucleotides by endosomal release agents such as, for example, Endo-Porter; or any combination thereof. In some embodiments, the oligonucleotides may be delivered from the blood to the cytosol using techniques selected from nanoparticle complexes, virally-mediated transfection, oligonucleotides linked to octaguanidinium dendrimers (morpholino oligonucleotides), or any combination thereof.

[00167] In additional embodiments, all or a portion of the sequence of any of the cancer- associated genes listed in any of Tables 1-6 can be deleted, so as to prevent expression of the cancer-associated sequence. Methods for targeted deletion of cellular sequences are known in the art and include zinc finger nucleases, TALENs and the CRISPR-Cas9 system. Delivery of the aforementioned reagents to tumor cells can be accomplished, e.g., with viral vectors (e.g., adenovirus, AAV).

[00168] In some embodiments, a method of treating breast cancer comprises treating a subject with a suitable reagent to knockdown or inhibit expression of a gene encoding the mRNA disclosed in sequences corresponding to any one or more of the genes listed in Tables 1-6, a fragment thereof, a complement thereof, or a combination thereof. In other embodiments the present disclosure provide for the in vitro knockdown of the expression of one or more of the genes disclosed in sequences corresponding to any one or more of the genes listed in Tables 1-6, or a fragment thereof or a complement thereof.

[00169] In some embodiments, breast cancers are treated by modulating the activity or expression of sequences corresponding to any one or more of the genes listed in Tables 1-6 or a fragment thereof or a complement thereof, or the gene product thereof.

[00170] In some embodiments, a method of treating breast cancer comprises administering an antibody (e.g. monoclonal antibody, human antibody, humanized antibody, recombinant antibody, chimeric antibody, and the like) that specifically binds to a cancer associated protein that is expressed on a cell surface. In some embodiments, the antibody binds to an extracellular domain of the cancer associated protein. In some embodiments, the antibody binds to a cancer associated protein differentially expressed on a cancer cell surface relative to a normal cell surface, or, in some embodiments, to at least one human cancer cell line. In some embodiments, the antibody is linked to a therapeutic agent or a toxin.

[00171] In some embodiments, implementation of an immunotherapy strategy for treating, reducing the symptoms of, or preventing cancer or neoplasms, (e.g., a vaccine) may be achieved using many different techniques available to the skilled artisan.

[00172] Immunotherapy or the use of antibodies for therapeutic purposes has been used in recent years to treat cancer. Passive immunotherapy involves the use of monoclonal antibodies in cancer treatments. See, for example, Cancer: Principles and Practice of Oncology, 6th Edition (2001) Chapter 20 pp. 495-508. Inherent therapeutic biological activity of these antibodies include direct inhibition of tumor cell growth or survival, and the ability to recruit the natural cell killing activity of the body's immune system. These agents may be administered alone or in conjunction with radiation or chemotherapeutic agents. Alternatively, antibodies may be used to make antibody conjugates in which the antibody is linked to a toxic agent and directs that agent to the tumor by specifically binding to the tumor.

Screening for Cancer Therapeutics

[00173] The present disclosure provides for screening assays to determine if a candidate molecule has an inhibitory effect on the growth and or metastasis of breast cancer cells. Suitable candidates include proteins, peptides, nucleic acids such as DNA, RNA shRNA smRNA and the like, small molecules including small organic molecules and small inorganic molecules. A small molecule may include molecules less than 50 kd, less than 25 kD, less than 10 kD, less than 5 kD, less than 2.5 kD, or less than 1 kD.

[00174] In some embodiments, a method of identifying an anti-cancer agent is provided, wherein the method comprises contacting a candidate agent with a sample; and determining the cancer associated sequence's activity in the sample. In some embodiments, the candidate agent is identified as an anti-cancer agent if the cancer associated sequence's activity is reduced in the sample after the contacting. In other embodiments the candidate agent reduces the expression level of one or more cancer associated sequences disclosed infra.

[00175] In some embodiments, the candidate agent is an antibody. In some embodiments, the method comprises contacting a candidate antibody that binds to the cancer associated sequence with a sample, and assaying for the cancer associated sequence's activity, wherein the candidate antibody is identified as an anti-cancer agent if the activity of the cancer associated sequence is reduced in the sample after the contacting. A cancer associated sequence's activity can be any activity of the cancer associated sequence. An example of an activity may include enzymatic activity either of the cancer associated sequence itself or of an enzyme that interacts with or is modulated by the cancer associated sequence either at the nucleic acid level or the protein level.

[00176] In some embodiments, the present disclosure provides methods of identifying an anti-cancer (e.g. breast cancer) agent comprising contacting a candidate agent to a cell sample; and determining activity of a cancer associated sequence, wherein the candidate agent is identified as an anti-cancer agent if the cancer associated sequence's activity is reduced in the cell sample after the contacting. In some embodiments, the present disclosure provides methods of identifying an anti-cancer agent, the method comprising contacting a cell sample with a candidate agent that binds to a cancer associated sequence (or a fragment thereof, a complement thereof, or combination thereof) selected from any one or more of the genes listed in Tables 1-6, and assaying for the cancer associated sequence's activity or expression level, wherein the candidate agent is identified as an anti-cancer agent if the cancer associated sequence's activity is modulated in the cell sample after the contacting.

[00177] In some embodiments, a method of screening drug candidates includes comparing the level of expression of the cancer-associated sequence in the absence of the drug candidate to the level of expression in the presence of the drug candidate. [00178] Some embodiments are directed to a method of screening for a therapeutic agent capable of binding to a cancer-associated sequence (nucleic acid or protein), the method comprising combining the cancer-associated sequence and a candidate therapeutic agent, and determining the binding of the candidate agent to the cancer-associated sequence.

[00179] Further provided herein is a method for screening for a therapeutic agent capable of modulating the activity of a cancer-associated sequence. In some embodiments, the method comprises combining the cancer-associated sequence and a candidate therapeutic agent, and determining the effect of the candidate agent on the bioactivity of the cancer-associated sequence. An agent that modulates the bioactivity of a cancer associated sequence may be used as a therapeutic agent capable of modulating the activity of a cancer-associated sequence.

[00180] In certain embodiments the present disclosure provides a method of screening for anticancer activity comprising: (a) contacting a cell that expresses a cancer associated gene selected from one or more cancer associated sequences disclosed in any of Tables 1-6, homologues thereof, combinations thereof, or fragments thereof with an anticancer drug candidate; (b) detecting an effect of the anticancer drug candidate on an expression of the cancer associated sequence in the cell (either at the nucleic acid or protein level); and (c) comparing the level of expression in the absence of the drug candidate to the level of expression in the presence of the drug candidate; wherein an effect on the expression of the cancer associate gene indicates that the candidate has anticancer activity. For example the drug candidate may lower the expression level of the cancer associated sequence in the cell.

[00181] In some embodiments, a method of evaluating the effect of a candidate cancer drug may comprise administering the drug to a patient and removing a cell sample from the patient. The expression profile of the cell is then determined. In some embodiments, the method may further comprise comparing the expression profile of the patient to an expression profile of a healthy individual. In some embodiments, the expression profile comprises measuring the expression of one or more or any combination thereof of the genes disclosed in any of Tables 1-6. In some embodiments, if the expression profile of one or more or any combination thereof of the sequences disclosed in any of Tables 1-6 is modified (increased or decreased) the candidate cancer drug is said to be effective.

[00182] In some embodiments, the present disclosure provides a method of screening for anticancer activity comprising: (a) providing a cell that expresses a cancer associated gene (or a fragment thereof or a complement thereof) that encodes a nucleic acid sequence selected from the group consisting of the cancer associated sequences chosen from sequences corresponding to any one or more of the genes listed in Tables 1-6, (b) contacting the cell, which can be derived from a cancer cell, with an anticancer drug candidate; (c) monitoring an effect of the anticancer drug candidate on expression of the cancer associated sequence in the cell sample, and optionally (d) comparing the level of expression in the absence of said drug candidate to the level of expression in the presence of the drug candidate; wherein, if expression in the presence of said anticancer drug candidate is less than expression in the absence of said anticancer drug candidate, the anticancer drug candidate has anti-cancer activity.

[00183] Suitable drug candidates include, but are not limited to an inhibitor of transcription, a G-protein coupled receptor antagonist, a growth factor antagonist, a serine- threonine kinase antagonist, and/or a tyrosine kinase antagonist. In some embodiments, in which the candidate modulates (e.g. , inhibits) the expression of the cancer associated sequence, the candidate is said to have anticancer activity. In some embodiments, the anticancer activity is determined by measuring cell growth. In some embodiments, the candidate inhibits or retards cell growth and is said to have anticancer activity. In some embodiments, the candidate causes the cell to die, and thus, the candidate is said to have anticancer activity.

[00184] In some embodiments, the present disclosure provides a method of screening for activity against breast cancer. In some embodiments, the method comprises contacting a cell that overexpresses a cancer associated gene which is complementary to a cancer associated sequence selected from cancer associated sequences disclosed in any of Tables 1-6, homologues thereof, combinations thereof, or fragments thereof with a breast cancer drug candidate. In some embodiments, the method comprises detecting an effect of the breast cancer drug candidate on an expression of the cancer associated polynucleotide in the cell or an effect on the cell's growth or viability. In some embodiments, the method comprises comparing the level of expression, cell growth, or viability in the absence of the drug candidate to the level of expression, cell growth, or viability in the presence of the drug candidate; wherein an effect on the expression of the cancer associated polynucleotide, cell growth, or viability indicates that the candidate has activity against a breast cancer cell that differentially expresses (e.g., overexpresses) a cancer associated gene, wherein said gene is selected from any one or more of the genes listed in Tables 1-6, complements thereof, homologues thereof, combinations thereof, or fragments thereof. In some embodiments, the drug candidate may include, for example, a transcription inhibitor, a G-protein coupled receptor antagonist, a growth factor antagonist, a serine-threonine kinase antagonist, or a tyrosine kinase antagonist.

Methods of Identifying Breast Cancer Markers

[00185] The pattern of gene expression in a particular living cell may be characteristic of its current state. Nearly all differences in the state or type of a cell are reflected in the differences in RNA levels of one or more genes. Comparing expression patterns of uncharacterized genes may provide clues to their function. High throughput analysis of expression of hundreds or thousands of genes can help in (a) identification of complex genetic diseases, (b) analysis of differential gene expression over time, between tissues and disease states, and (c) drug discovery and toxicology studies. Increase or decrease in the levels of expression of certain genes correlate with cancer biology. For example, oncogenes are positive regulators of tumorigenesis, while tumor suppressor genes are negative regulators of tumorigenesis. (Marshall, Cell, 64: 313-406 (1991); Weinberg, Science, 254: 1138-1146 (1991)). Accordingly, some embodiments herein provide for polynucleotide and polypeptide sequences involved in cancer and, in particular, in oncogenesis.

[00186] Oncogenes are genes that can cause cancer. Carcinogenesis can occur by a wide variety of mechanisms, including infection of cells by viruses containing oncogenes, activation of protooncogenes in the host genome, and mutations of protooncogenes and tumor suppressor genes. Carcinogenesis is fundamentally driven by somatic cell evolution (i.e. mutation and natural selection of variants with progressive loss of growth control). The genes that serve as targets for these somatic mutations are classified as either protooncogenes or tumor suppressor genes, depending on whether their mutant phenotypes are dominant or recessive, respectively.

[00187] Some embodiments of the present disclosure are directed to cancer associated sequences ("target markers"). Some embodiments are directed to methods of identifying novel target markers useful in the diagnosis and treatment of cancer wherein expression levels of mRNAs, miRNAs, proteins, or protein post translational modifications including but not limited to phosphorylation and sumoylation are compared between five categories of cell types: (1) immortal pluripotent stem cells (such as embryonic stem ("ES") cells, induced pluripotent stem ("iPS") cells, and germ-line cells such as embryonal carcinoma ("EC") cells) or gonadal tissues; (2) ES, iPS, or EC-derived clonal embryonic progenitor ("EP") cell lines, (3) nucleated blood cells including but not limited to CD34+ cells and CD133+ cells; (4) normal mortal somatic adult- derived tissues and cultured cells including: skin fibroblasts, vascular endothelial cells, normal non-lymphoid and non-cancerous tissues, and the like, and (5) malignant cancer cells including cultured cancer cell lines or human tumor tissue. mRNAs, miRNAs, or proteins that are generally expressed (or not expressed) in categories 1, 3, and 5, or categories 1 and 5 but not expressed (or expressed) in categories 2 and 4 are candidate targets for cancer diagnosis and therapy. Some embodiments herein are directed to human applications, non-human veterinary applications, or a combination thereof.

[00188] Another method of identifying cancer-associated sequences (i.e., cancer- associated markers, cancer-associated genes) is to compare gene expression in cancerous cells to gene expression in non-cancerous cells and identify genes whose expression is greater in cancerous cells. Gene expression can be measured as either mRNA or protein. The Examples provided herein describe application of such a method to identify the cancer-associated sequences listed in Tables 1-6.

[00189] Once expression is determined, the gene sequence results may be further filtered by considering fold-change in cancer cell lines vs. normal tissue; general specificity; secreted or not, level of expression in cancer cell lines; and signal to noise ratio.

[00190] It will be appreciated that there are various methods of obtaining expression data and uses of the expression data. For example, the expression data that can be used to detect or diagnose a subject with cancer can be obtained experimentally. In some embodiments, obtaining the expression data comprises obtaining the sample and processing the sample to experimentally determine the expression data. The expression data can comprise expression data for one or more of the cancer associated sequences described herein. The expression data can be experimentally determined by, for example, using a microarray or quantitative amplification method such as, but not limited to, those described herein. In some embodiments, obtaining expression data associated with a sample comprises receiving the expression data from a third party that has processed the sample to experimentally determine the expression data.

[00191] Detecting a level of expression or similar steps that are described herein may be done experimentally or provided by a third-party as is described herein. Therefore, for example, "detecting a level of expression" may refer to experimentally measuring the data and/or having the data provided by another party who has processed a sample to determine and detect a level of expression data.

Techniques for Analyzing Samples

[00192] Any technique known in the art may be used to analyze a sample according to the methods disclosed infra such as methods of detecting or diagnosing cancer in a sample or identifying a new cancer associated sequence. Exemplary techniques are provided below.

[00193] Gene Expression Assays: Measurement of the gene expression levels may be performed by any known methods in the art, including but not limited to quantitative PCR, or microarray gene expression analysis, bead array gene expression analysis and RNA blot (Northern) analysis. The gene expression levels may be represented as relative expression normalized to the ADPRT gene (Accession number NM_001618.2), GAPD gene (Accession number NM_002046.2), or other housekeeping genes known in the art. In the case of microarrayed probes of mRNA expression, the gene expression data may also be normalized by a median of medians method. In this method, each array gives a different total intensity. Using the median value is a robust way of comparing cell lines (arrays) in an experiment. As an example, the median was found for each cell line and then the median of those medians became the value for normalization. The signal from the each cell line was made relative to each of the other cell lines.

[00194] RNA extraction: Cells of the present disclosure may be incubated with 0.05% trypsin and 0.5 mM EDTA, followed by collecting in DMEM (Gibco, Gaithersburg, MD) with 0.5% BSA. Total RNA may be purified from cells using the RNeasy Mini kit (Qiagen, Hilden, Germany).

[00195] Isolation of total RNA and miRNA from cells: Total RNA or samples enriched for small RNA species may be isolated from cell cultures that undergo serum starvation prior to harvesting RNA to approximate cellular growth arrest observed in many mature tissues. Cellular growth arrest may be performed by changing to medium containing 0.5% serum for 5 days, with one medium change 2-3 days after the first addition of low serum medium. RNA may be harvested according to the vendor's instructions for Qiagen RNEasy kits to isolate total RNA or Ambion mirVana kits to isolate RNA enriched for small RNA species. The RNA concentrations may be determined by spectrophotometry and RNA quality may be determined by denaturing agarose gel electrophoresis to visualize 28S and 18S RNA. Samples with clearly visible 28S and 18S bands without signs of degradation and at a ratio of approximately 2: 1, 28S: 18S may be used for subsequent miRNA analysis.

[00196] Assay for miRNA in samples isolated from human cells: The miRNAs may be quantitated using a Human Panel TaqMan^® MicroRNA Assay from Applied Biosystems, Inc. This is a two-step assay that uses stem-loop primers for reverse transcription (RT) followed by realtime TaqMan^®. The assay includes two steps, reverse transcription (RT) and quantitative PCR. Real-time PCR may be performed on an Applied Biosystems 7500 Real-Time PCR System. The copy number per cell may be estimated based on the standard curve of synthetic mir-16 miRNA and assuming a total RNA mass of approximately 15pg/cell.

[00197] The reverse transcription reaction may be performed using lx cDNA archiving buffer, 3.35 units MMLV reverse transcriptase, 5mM each dNTP, 1.3 units AB RNase inhibitor, 2.5 nM 330-plex reverse primer (RP), and 3 ng of cellular RNA in a final volume of 5 ul. The reverse transcription reaction may be performed on a BioRad or MJ thermocycler with a cycling profile of 20 °C for 30 sec; 42 °C for 30 sec; 50 °C for 1 sec, for 60 cycles followed by one cycle of 85 °C for 5 min.

[00198] Real-time PCR. Two microliters of 1:400 diluted Pre-PCR product may be used for a 20 ul reaction. All reactions may be duplicated. Because the method is very robust, duplicate samples may be sufficient and accurate enough to obtain values for miRNA expression levels. TaqMan^® universal PCR master mix of ABI may be used according to manufacturer's suggestion. Briefly, lx TaqMan^® Universal Master Mix (ABI), 1 uM Forward Primer, 1 uM Universal Reverse Primer and 0.2 uM TaqMan^® Probe may be used for each real-time PCR. The conditions used may be as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and 60°C for 1 min. All the reactions may be run on ABI Prism 7000 Sequence Detection System.

[00199] Microarray hybridization and data processing. cDNA samples and cellular total RNA (5 μg in each of eight individual tubes) may be subjected to the One-Cycle Target Labeling procedure for biotin labeling by in vitro transcription (IVT) (Affymetrix, Santa Clara, CA) or using the niumina Total Prep RNA Labelling kit. For analysis on Affymetrix gene chips, the cRNA may be subsequently fragmented and hybridized to the Human Genome U133 Plus 2.0 Array (Affymetrix) according to the manufacturer's instructions. The microarray image data may be processed with the GeneChip Scanner 3000 (Affymetrix) to generate CEL data. The CEL data may be then subjected to analysis with dChip software, which has the advantage of normalizing and processing multiple datasets simultaneously. Data obtained from the eight nonamplified controls from cells, from the eight independently amplified samples from the diluted cellular RNA, and from the amplified cDNA samples from 20 single cells may be normalized separately within the respective groups, according to the program's default setting. The model based expression indices (MBEI) may be calculated using the PM/MM difference mode with log-2 transformation of signal intensity and truncation of low values to zero. The absolute calls (Present, Marginal and Absent) may be calculated by the Affymetrix Microarray Software 5.0 (MAS 5.0) algorithm using the dChip default setting. The expression levels of only the Present probes may be considered for all quantitative analyses described below. The GEO accession number for the microarray data is GSE4309. For analysis on Illumina Human HT-12 v4 Expression Bead Chips, labeled cRNA may be hybridized according to the manufacturer's instructions.

[00200] Calculation of coverage and accuracy. A true positive is defined as probes called Present in at least six of the eight nonamplified controls, and the true expression levels are defined as the log-averaged expression levels of the Present probes. The definition of coverage is (the number of truly positive probes detected in amplified samples)/(the number of truly positive probes). The definition of accuracy is (the number of truly positive probes detected in amplified samples)/(the number of probes detected in amplified samples). The expression levels of the amplified and nonamplified samples may be divided by the class interval of 20.5 (20, 20.5, 21, 21.5...), where accuracy and coverage are calculated. These expression level bins may be also used to analyze the frequency distribution of the detected probes.

[00201] Analysis of gene expression profiles of cells: The unsupervised clustering and class neighbor analyses of the microarray data from cells may be performed using GenePattern software (http://www.broad.mit.edu/cancer/software/genepattern/), which performs the signal-to- noise ratio analysis/T-test in conjunction with the permutation test to preclude the contribution of any sample variability, including those from methodology and/or biopsy, at high confidence. The analyses may be conducted on the 14, 128 probes for which at least 6 out of 20 single cells provided Present calls and at least 1 out of 20 samples provided expression levels >20 copies per cell. The expression levels calculated for probes with Absent/Marginal calls may be truncated to zero. To calculate relative gene expression levels, the Ct values obtained with Q-PCR analyses may be corrected using the efficiencies of the individual primer pairs quantified either with whole human genome (BD Biosciences) or plasmids that contain gene fragments. The relative expression levels may be further transformed into copy numbers with a calibration line calculated using the spike RNAs included in the reaction mixture (loglO[expression level] = 1.05 x logl0[copy number] + 4.65). The Chi-square test for independence may be performed to evaluate the association of gene expressions with Gata4, which represents the difference between cluster 1 and cluster 2 determined by the unsupervised clustering and which is restricted to PE at later stages. The expression levels of individual genes measured with Q-PCR may be classified into three categories: high (>100 copies per cell), middle (10-100 copies per cell), and low (<10 copies per cell). The Chi-square and P-values for independence from Gata4 expression may be calculated based on this classification. Chi squared is defined as follows: χ2 =∑∑ (n fij - fi fj)2/n fi fj, where i and j represent expression level categories (high, middle or low) of the reference (Gata4) and the target gene, respectively; fi, fj, and fij represent the observed frequency of categories i, j and ij, respectively; and n represents the sample number (n = 24). The degrees of freedom may be defined as (r - 1) x (c - 1), where r and c represent available numbers of expression level categories of Gata4 and of the target gene, respectively.

Generating an Immune Response Against Breast Cancer

[00202] In some embodiments, antigen presenting cells (APCs) may be used to activate T lymphocytes in vivo or ex vivo, to elicit an immune response against cells expressing a cancer associated sequence. APCs are highly specialized cells and may include, without limitation, macrophages, monocytes, and dendritic cells (DCs). APCs may process antigens and display their peptide fragments on the cell surface together with molecules required for lymphocyte activation. In some embodiments, the APCs may be dendritic cells. DCs may be classified into subgroups, including, e.g., follicular dendritic cells, Langerhans dendritic cells, and epidermal dendritic cells. In other embodiments the present disclosure provides a method of eliciting an antibody response to one or more of the cancer associated sequences disclosed infra. The method may comprise administering a protein or a peptide fragment encoded by one or more of the cancer associated sequences disclosed infra to a subject.

[00203] Some embodiments are directed to the use of cancer associated polypeptides and polynucleotides encoding a cancer associated sequence, a fragment thereof, or a mutant thereof, and antigen presenting cells (such as, without limitation, dendritic cells), to elicit an immune response against cells expressing a cancer-associated polypeptide sequence, such as, without limitation, cancer cells, in a subject. In some embodiments, the method of eliciting an immune response against cells expressing a cancer associated sequence comprises (1) isolating a hematopoietic stem cell, (2) genetically modifying the cell to express a cancer associated sequence, (3) differentiating the cell into DCs; and (4) administering the DCs to the subject (e.g., human patient). In some embodiments, the method of eliciting an immune response includes (1) isolating DCs (or isolation and differentiation of DC precursor cells), (2) pulsing the cells with a cancer associated sequence, and; (3) administering the DCs to the subject. These approaches are discussed in greater detail, infra. In some embodiments, the pulsed or expressing DCs may be used to activate T lymphocytes ex vivo. These general techniques and variations thereof are within the skill of those in the art (see, e.g., W097/29182; WO 97/04802; WO 97/22349; WO 96/23060; WO 98/01538; Hsu et al., 1996, Nature Med. 2:52-58), and still other variations may be discovered in the future. In some embodiments, the cancer associated sequence is contacted with a subject to stimulate an immune response. In some embodiments, the immune response is a therapeutic immune response so as to treat a subject as described infra. In some embodiments, the immune response is a prophylactic immune response. For example, the cancer associated sequence can be contacted with a subject under conditions effective to stimulate an immune response. The cancer associated sequence can be administered as, for example, a DNA molecule {e.g. DNA vaccine), RNA molecule, or polypeptide, or any combination thereof. The identity of particular sequences useful in stimulating an immune response against breast cancer cells {e.g., sequences of the markers disclosed in Tables 1-6) was not known prior to the present disclosure. Any sequence or combination of sequences disclosed herein or a homologue thereof can be administered to a subject to stimulate an immune response.

[00204] In some embodiments, dendritic cell precursor cells are isolated for transduction with a cancer associated sequence, and induced to differentiate into dendritic cells. The genetically modified DCs express the cancer associated sequence, and may display peptide fragments on the cell surface.

[00205] In some embodiments, the cancer associated sequence expressed comprises a sequence of a naturally occurring protein. In some embodiments, the cancer associate sequence does not comprise a naturally occurring sequence. As already noted, fragments of naturally occurring proteins may be used; in addition, the expressed polypeptide may comprise mutations such as deletions, insertions, or amino acid substitutions when compared to a naturally occurring polypeptide, so long as at least one peptide epitope can be processed by the DC and presented on a MHC class I or II surface molecule. In some embodiments, it may be desirable to use sequences other than "wild type," in order to, for example, increase antigenicity of the peptide or to increase peptide expression levels. In some embodiments, the introduced cancer associated sequences may encode variants such as polymorphic variants (e.g., a variant expressed by a particular human patient) or variants characteristic of a particular cancer (e.g., a cancer in a particular subject).

[00206] In some embodiments, a cancer associated sequence may be introduced (transduced) into DCs or stem cells in any of a variety of standard methods, including transfection, recombinant vaccinia viruses, adeno-associated viruses (AAVs), retroviruses, etc.

[00207] In some embodiments, the transformed DCs of the present disclosure may be introduced into the subject (e.g., without limitation, a human patient) where the DCs may induce an immune response. Typically, the immune response includes a cytotoxic T-lymphocyte (CTL) response against target cells bearing antigenic peptides (e.g., in a MHC class I/peptide complex). These target cells are typically cancer cells.

[00208] In some embodiments, when the DCs are to be administered to a subject, they may preferably isolated from, or derived from precursor cells from, that subject (i.e., the DCs may administered to an autologous subject). However, the cells may be infused into HLA-matched allogeneic or HLA-mismatched allogeneic subject. In the latter case, immunosuppressive drugs may be administered to the subject.

[00209] In some embodiments, the cells may be administered in any suitable manner. In some embodiments, the cell may be administered with a pharmaceutically acceptable carrier (e.g., saline). In some embodiments, the cells may be administered through intravenous, intra- articular, intramuscular, intradermal, intraperitoneal, or subcutaneous routes. Administration (i.e., immunization) may be repeated at time intervals. Infusions of DC may be combined with administration of cytokines that act to maintain DC number and activity (e.g., GM-CSF, IL-12).

[00210] In some embodiments, the dose administered to a subject may be a dose sufficient to induce an immune response as detected by assays which measure T cell proliferation, T lymphocyte cytotoxicity, and/or effect a beneficial therapeutic response in the patient over time, e.g., to inhibit growth of cancer cells or result in reduction in the number of cancer cells or the size of a tumor. [00211] In some embodiments, DCs are obtained (either from a patient or by in vitro differentiation of precursor cells) and pulsed with antigenic peptides having a cancer associated sequence. The pulsing results in the presentation of peptides onto the surface MHC molecules of the cells. The peptide/MHC complexes displayed on the cell surface may be capable of inducing a MHC-restricted cytotoxic T-lymphocyte response against target cells expressing cancer associated polypeptides (e.g., without limitations, cancer cells).

[00212] In some embodiments, cancer associated sequences used for pulsing may have a length of at least about 6 or 8 amino acids and fewer than about 30 amino acids or fewer than about 50 amino acid residues. In some embodiments, an immunogenic peptide sequence may have from about 8 to about 12 amino acids. In some embodiments, a mixture of human protein fragments may be used; alternatively a particular peptide of defined sequence may be used. The peptide antigens may be produced by de novo peptide synthesis, enzymatic digestion of purified or recombinant human peptides, by purification of the peptide sequence from a natural source (e.g., a subject or tumor cells from a subject), or expression of a recombinant polynucleotide encoding a human peptide fragment.

[00213] In some embodiments, the amount of peptide used for pulsing DC may depend on the nature, size and purity of the peptide or polypeptide. In some embodiments, an amount of from about 0.05 ug/ml to about 1 mg/ml, from about 0.05 ug/ml to about 500 ug/ml, from about 0.05 ug/ml to about 250 ug/ml, from about 0.5 ug/ml to about 1 mg/ml, from about 0.5 ug/ml to about 500 ug/ml, from about 0.5 ug/ml to about 250 ug/ml, or from about 1 ug/ml to about 100 ug/ml of peptide may be used. After adding the peptide antigen(s) to the cultured DC, the cells may then be allowed sufficient time to take up and process the antigen and express antigen peptides on the cell surface in association with either class I or class II MHC. In some embodiments, the time to take up and process the antigen may be about 18 to about 30 hours, about 20 to about 30 hours, or about 24 hours.

[00214] Numerous examples of systems and methods for predicting peptide binding motifs for different MHC Class I and II molecules have been described. Such prediction could be used for predicting peptide motifs that will bind to the desired MHC Class I or II molecules. Examples of such methods, systems, and databases that those of ordinary skill in the art might consult for such purpose include: [00215] 1. Peptide Binding Motifs for MHC Class I and II Molecules; William E. Biddison, Roland Martin, Current Protocols in Immunology, Unit II (DOI: 10.1002/0471142735.ima01is36; Online Posting Date: May, 2001).

[00216] Reference 1 above provides an overview of the use of peptide-binding motifs to predict interaction with a specific MHC class I or II allele, and gives examples for the use of MHC binding motifs to predict T-cell recognition.

[00217] One skilled in the art of peptide-based vaccination may determine which peptides would work best in individuals based on their HLA alleles (e.g., due to "MHC restriction"). Different HLA alleles will bind particular peptide motifs (usually 2 or 3 highly conserved positions out of 8-10) with different affinities which can be predicted theoretically or measured as dissociation rates. Thus, a skilled artisan may be able to tailor the peptides to a subject's HLA profile.

[00218] In some embodiments, the present disclosure provides methods of eliciting an immune response against cells expressing a cancer associated sequence comprising contacting a subject with a cancer associated sequence under conditions effective to elicit an immune response in the subject, wherein said cancer associated sequence comprises a sequence or fragment thereof of a gene selected from one or more of the cancer associated sequences disclosed herein.

Transfecting Cells With Cancer Associated Sequences

[00219] Cells may be transfected with one or more of the cancer associated sequences disclosed herein. Transfected cells may be useful in screening assays, diagnosis and detection assays. Transfected cells expressing one or more cancer associated sequences as disclosed herein may be used to obtain isolated nucleic acids encoding cancer associated sequences and/or isolated proteins or peptide fragments encoded by one or more cancer associated sequences.

[00220] Electroporation may be used to introduce the cancer associated nucleic acids described herein into mammalian cells (Neumann, E. et al. (1982) EMBO J. 1, 841-845), plant and bacterial cells, and may also be used to introduce proteins (Marrero, M.B. et al. (1995) J. Biol. Chem. 270, 15734-15738; Nolkrantz, K. et al. (2002) Anal. Chem. 74, 4300-4305; Rui, M. et al. (2002) Life Sci. 71, 1771-1778). Cells suspended in a buffered solution of the purified protein of interest are placed in a pulsed electrical field. Briefly, high-voltage electric pulses result in the formation of small (nanometer- sized) pores in the cell membrane. Proteins enter the cell via these small pores or during the process of membrane reorganization as the pores close and the cell returns to its normal state. The efficiency of delivery may be dependent upon the strength of the applied electrical field, the length of the pulses, the temperature and the composition of the buffered medium. Electroporation is successful with a variety of cell types, even some cell lines that are resistant to other delivery methods, although the overall efficiency is often quite low. Some cell lines may remain refractory even to electroporation unless partially activated.

[00221] Microinjection may be used to introduce femtoliter volumes of DNA directly into the nucleus of a cell (Capecchi, M.R. (1980) Cell 22, 470-488) where it can be integrated directly into the host cell genome, thus creating an established cell line bearing the sequence of interest. Proteins such as antibodies (Abarzua, P. et al. (1995) Cancer Res. 55, 3490-3494; Theiss, C. and Meller, K. (2002) Exp. Cell Res. 281, 197-204) and mutant proteins (Naryanan, A. et al. (2003) J. Cell Sci. 116, 177-186) can also be directly delivered into cells via microinjection to determine their effects on cellular processes firsthand. Microinjection has the advantage of introducing macromolecules directly into the cell, thereby bypassing exposure to potentially undesirable cellular compartments such as low-pH endosomes.

[00222] Several proteins and small peptides have the ability to transduce or travel through biological membranes independent of classical receptor-mediated or endocytosis-mediated pathways. Examples of these proteins include the HIV-1 TAT protein, the herpes simplex virus 1 (HSV-1) DNA-binding protein VP22, and the Drosophila Antennapedia (Antp) homeotic transcription factor. In some embodiments, protein transduction domains (PTDs) from these proteins may be fused to other macromolecules, peptides or proteins such as, without limitation, a cancer associated polypeptide or fragment thereof, to successfully transport the polypeptide into a cell (Schwarze, S.R. et al. (2000) Trends Cell Biol. 10, 290-295). Exemplary advantages of using fusions of these transduction domains is that protein entry is rapid, concentration-dependent and appears to work with cell types that are difficult to transduce using other methods (Fenton, M. et al. (1998) J. Immunol. Methods 212, 41-48).

[00223] In some embodiments, liposomes may be used as vehicles to deliver oligonucleotides, DNA (gene) constructs and small drug molecules into cells (Zabner, J. et al. (1995) J. Biol. Chem. 270, 18997-19007; Feigner, P.L. et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7413-7417). Certain lipids, when placed in an aqueous solution and sonicated, form closed vesicles consisting of a circularized lipid bilayer surrounding an aqueous compartment. The vesicles or liposomes of embodiments herein may be formed in a solution containing the molecule to be delivered. In addition to encapsulating DNA in an aqueous solution, cationic liposomes may spontaneously and efficiently form complexes with DNA, with the positively charged head groups on the lipids interacting with the negatively charged backbone of the DNA. The exact composition and/or mixture of cationic lipids used can be altered, depending upon the macromolecule of interest and the cell type used (Feigner, J.H. et al. (1994) J. Biol. Chem. 269, 2550-2561). The cationic liposome strategy has also been applied successfully to protein delivery (Zelphati, O. et al. (2001) J. Biol. Chem. 276, 35103-35110). Because proteins are more heterogeneous than DNA, the physical characteristics of the protein, such as its charge and hydrophobicity, may influence the extent of its interaction with the cationic lipids.

Kits

[00224] Also provided by the invention are kits and systems for practicing the subject methods, as described above, such components configured to diagnose cancer in a subject, treat cancer in a subject, detect cancer in a sample, or perform basic research experiments on cancer cells (e.g., either derived directly from a subject, grown in vitro or ex vivo, or from an animal model of cancer. The various components of the kits may be present in separate containers or certain compatible components may be pre-combined into a single container, as desired.

[00225] In some embodiments, the present disclosure provides a kit for diagnosing the presence of cancer in a test sample, said kit comprising at least one polynucleotide that selectively hybridizes to a cancer associated polynucleotide sequence chosen from any one or more of the genes listed in Tables 1-6, or a fragment or a complement thereof. In another embodiment the invention provides an electronic library comprising a cancer associated polynucleotide, a cancer associated polypeptide, or fragment thereof, disclosed infra. In some embodiments the kit may include one or more capture reagents or specific binding partners of one or more cancer associated sequences disclosed infra.

[00226] The subject systems and kits may also include one or more other reagents for performing any of the subject methods. The reagents may include one or more matrices, solvents, sample preparation reagents, buffers, desalting reagents, enzymatic reagents, denaturing reagents, probes, polynucleotides, vectors (e.g., plasmid or viral vectors), etc., where calibration standards such as positive and negative controls may be provided as well. As such, the kits may include one or more containers such as vials or bottles, with each container containing a separate component for carrying out a sample processing or preparing step and/or for carrying out one or more steps for producing a normalized sample according to the present disclosure.

[00227] In addition to above-mentioned components, the subject kits typically further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e. , associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[00228] In addition to the subject database, programming and instructions, the kits may also include one or more control samples and reagents, e.g. , two or more control samples for use in testing the kit.

[00229]

EMR2 ADGRE2 NM_013447.3 Q9UHX3 30817

BASI BSG NM_001728.2 P35613 682

NLGNX NLGN4X NM_020742.2 Q8N0W4 57502

C5a C5 NM_001735.2 P01031 727

C5b, 6 C5 C6 NM_001735.2, P01031,P13671 727,729 Complex NM_000065.1

Fibronectin FN1 NM_002026.2 P02751 2335

HSP90b HSP90AB 1 NM_007355.2 P08238 3326

MMP-3 MMP3 NM_002422.3 P08254 4314

Renin REN NM_000537.2 P00797 5972

SAA SAA1 NM_000331.3 P0DJI8 6288

C2 C2 NM_000063.3 P06681 717

4-lBB TNFSF9 NM_003811.2 P41273 8744 ligand

Ephrin-A3 EFNA3 NM_004952.3 P52797 1944

FGF-10 FGF10 NM_004465.1 015520 2255

Macrophage MRC1 NM_002438.1 P22897 4360 mannose

receptor

Galectin-4 LGALS4 NM_006149.2 P56470 3960

IL-22BP IL22RA2 NM_052962.2 Q969J5 116379

MAPK14 MAPK14 NM_139012.1 Q16539 1432

RS3 RPS3 NM_001005.3 P23396 6188

URB CCDC80 NM_199511.1 Q76M96 151887

AIP AIP NM_003977.1 000170 9049

FUT5 FUT5 NM_002034.2 Q11128 2527

HSP90a/b HSP90AA1 NM_001017963.2, P07900 P08238 3320, 3326

HSP90AB 1 NM_007355.2

Cathepsin D CTSD NM_001909.3 P07339 1509

HSP90b HSP90AB 1 NM_007355.2 P08238 3326

SAA SAA1 NM .000331.3 P0DJI8 6288

Vitronectin VTN NM_000638.3 P04004 7448 suPAR PLAUR NM_002659.2 Q03405 5329

CTGF CTGF NM .001901.2 P29279 1490

PTN PTN NM_002825.5 P21246 5764

CD38 CD38 NM_001775.2 P28907 952

IP- 10 CXCL10 NM_001565.2 P02778 3627

MIG CXCL9 NM_002416.1 Q07325 4283 sTie-1 TIE1 NM_005424.2 P35590 7075

Factor I CFI NM_000204.2 P05156 3426

IL-1R4/ST2 IL1RL1 NM_003856.2 Q01638 9173

FGF10 FGF10 NM_004465.1 015520 2255

Macrophage MRC1 NM_002438.1 P22897 4360 mannose

receptor

IL-18Rbeta IL18RAP NM_003853.2 095256 8807

Galectin-4 LGALS4 NM_006149.2 P56470 3960

MK13 MAPK13 NM_002754.3 015264 5603

KPC1 RNF123 NM_022064.4 P41743 5584

GDF2 GDF2 NM_016204.1 Q9UK05 2658

EMR2 ADGRE2 NM_013447.3 Q9UHX3 30817

Carbonic CA6 NM_001215.2 P23280 765 anhydrase 6

URB CCDC80 NM_199511.1 Q76M96 151887

CYTN CST1 NM_001898.2 P01037 1469

PPIE PPIE NM_203457.1 Q9UNP9 10450

NLGNX NLGN4X NM_020742.2 Q8N0W4 57502

SLAF6 SLAMF6 NM_052931.3 Q96DU3 114836

HSP90a/b HSP90AA1 NM_001017963.2, P07900 P08238 3320, 3326

HSP90AB 1 NM_007355.2

CYTN CST1 NM_001898.2 P01037 1469

C5 C5 NM_001735.2 P01031 727

Cathepsin CTSD NM_001909.3 P07339 1509 D

suPAR PLAUR NM_002659.2 Q03405 5329

CTGF CTGF NM_001901.2 P29279 1490

PTN PTN NM_002825.5 P21246 5764

MIG CXCL9 NM_002416.1 Q07325 4283

Factor I CFI NM_000204.2 P05156 3426

IL- IL1RL1 NM_003856.2 Q01638 9173

1R4/ST2

IL-18Rbeta IL18RAP NM_003853.2 095256 8807

MK13 MAPK13 NM_002754.3 015264 5603

Carbonic CA6 NM_001215.2 P23280 765 anhydrase

6

CYTN CST1 NM_001898.2 P01037 1469

PPIE PPIE NM_203457.1 Q9UNP9 10450

SLAF6 SLAMF6 NM_052931.3 Q96DU3 114836

suPAR PLAUR NM_002659.2 Q03405 5329

EXAMPLES

[00230] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.

Example 1: Cancer gene expression data set

[00231] Progress in biomarker research shows that in many complex diseases, changes in expression of multiple biomarkers, rather than a single marker, are required to provide a better understanding of disease patterns. Furthermore, different diseases (including different cancers and different occurrences of the same cancer type) will exhibit changes in expression of different biomarkers. Therefore, one of the most challenging and important tasks in biomarker research consists on identifying the best panel of biomarkers giving the highest sensitivity and specificity to confirm a diagnosis. The specific number of biomarkers, as well as the particular biomarkers to be used in combination, are determined by a statistical algorithm employed to select the minimal number of biomarkers needed showing the highest differences among the populations tested, while maintaining the highest sensitivity and specificity.

[00232] Many of the genes associated with the normal growth of embryonic stem cells are abnormally reactivated by cancer cells. Based on this premise, a gene expression dataset was established, using messenger RNA and micro RNA microarray technology. The dataset contains expression levels of over 47,000 genes from over 600 unique samples, representing both normal and cancerous tissues and cell lines, including multiple human embryonic stem cell lines. This broad, bioinformatics-based approach has permitted the identification of numerous genes abnormally activated in cancer or tumor cells. Many of these genes have not been previously associated with cancer. Moreover, expression of a large subset of these genes is found across numerous cancer types, such as cancers of the lung, breast, bladder, colon, and ovaries, suggesting that these genes may control fundamental processes during cancer growth and progression.

[00233] This gene expression data presents numerous diagnostic test opportunities. The inventors have determined that the proteins produced from a subset of these genes are detected at different (higher or lower) levels in the blood or urine of subjects with cancer, as compared to levels of the same gene products in the blood or urine of healthy subjects. This technology is known as "liquid biopsy" in which, instead of examining a sample from tumor tissue, blood and/or urine are examined for the presence of DNA and proteins shed by tumors or cancer cells into the blood or urine.

Example 2: Identification of breast cancer biomarkers

[00234] Two populations were selected for the assessment of distinguishable differences in breast cancer biomarker expression in serum: 1) subjects with pathology-confirmed malignant breast cancer and 2) subjects with pathology-confirmed benign neoplasms. For this initial analysis, the malignant breast cancer samples were infiltrating ductal carcinomas, as they represent the most common breast cancer (80% of cases). Thus, a set of 100 samples, 50 pathology-confirmed benign (of mixed types) and 50 pathology-confirmed infiltrating ductal carcinomas, were tested. The differences were measured in terms of expression of protein biomarkers by using the SOMAscan^® platform (SomaLogic, Inc., Boulder, CO), testing 1,310 biomarkers simultaneously.

[00235] Following analysis of the aforementioned 1,310 biomarkers in the cancerous and benign samples, a number of statistical techniques were used to develop a list of markers with potential for distinguishing between cancerous and benign samples. No single marker was identified that, by itself, could sufficiently discriminate between the cancerous and benign samples. However, combined in an algorithm, subsets or panels of the identified markers were able to distinguish cancerous vs. benign samples, as discussed infra.

[00236] To identify markers that are useful in a breast cancer diagnostic panel, multiple methods were employed. First an initial "response screening" process was used. This process ran each of the 1,310 markers one at a time testing each marker against the entire group of markers. An adjusted p-value (i.e., false discovery rate (FDR) corrected) was obtained and a plot of the FDR LogWorth (i.e., -logio[/?-value]) by effect size identified markers with the largest effects (labeled with the marker name in the plot). The results, shown in Figure 1, provided a number of breast cancer markers which were subjected to further analysis.

[00237] The highest- scoring marker in this analysis was the C5 gene. However, testing by cumulative distribution function (CDF) (Figure 2A) and density (Figure 2B) showed that this single marker was insufficient to distinguish cancer and benign samples with sufficient precision to be of clinical use. This confirms the need for multiple markers in combination to build a diagnostic test.

[00238] Therefore, a second method was used to screen for breast cancer markers. This was a predictor screening method, which utilizes random forests (i.e. , partitioning); and evaluates multiple markers in combination, rather than one at a time, as in the response screening process described above. When both the response screening and predictor screening methods were used, lists of 33 markers (Table 1) and 36 markers (Table 2) were identified to be further tested in the modeling (algorithm development) stage.

Example 3: Modeling and algorithm development

[00239] Focusing on the 36 markers shown in Table 2 that were identified in the screening process, generalized linear modeling was used to develop three potential diagnostic models. K- fold validation (using either 3 or 5 folds) was used to guard against over fitting of the models with this initial data set. A 15-marker model (shown in Table 4) resulted in an AUC (area under curve) of 0.92 with specificity of approximately 76% when sensitivity is 90%. Two 6-marker models (shown in Tables 5 and 6) both provided an AUC of 0.85 with specificity of 56% and 64%, respectively, for a sensitivity of 90%. The two 6-marker models had four markers in common (cathepsin D, IL-18Rbeta, C5, MK13), similar AUC but slightly different ROC curve shape resulting in a shifted specificity when sensitivity was set at 90%. The 15-marker model also contained the same four common markers (cathepsin D, IL-18Rbeta, C5, MK13). The ROC curves are shown in Figure 3 and the model results are illustrated in Figures 4A, 4B and 4C.

[00240] A similar analysis provided a 16-marker panel, as shown in Table 3.

[00241] The models (i.e., marker panels) described above demonstrate the feasibility of developing multi-marker diagnostics for cancer, based on 6 to 15 markers. The two 6-marker models demonstrate the flexibility in the exact markers used for such a model. Such marker flexibility is a benefit when moving from a research platform to a clinical platform, as all markers may not transfer with comparable performances.

[00242] These models described above also demonstrate that increasing the number of markers in the model often will improve the specificity of the model for a fixed sensitivity, and that the shape of the ROC curve (not just the AUC) is important to review.

Example 4: Clinical Protocols

[00243] Data from clinical studies are used to support the use of the breast cancer diagnostic test described above by clinicians to aid in determining the malignancy potential of suspicious mammography findings. In a prospective, single-blind, multicenter study; serum samples are collected to evaluate the panels of serum biomarkers, disclosed above, used in the diagnosis of breast cancer. Subjects who have provided informed consent have a single blood sample collected. In addition to providing a complete health history, they provide the report from their current mammogram and any other laboratory and histology reports from any resulting biopsy of the breast.

Claims

1. A method of detecting breast cancer in a subject comprising

a) contacting a sample from a subject with one or more reagents that specifically bind to a protein encoded by one or more of the genes listed in Tables 3-6, wherein at least one of the reagents specifically binds to a protein encoded by one or more of the C5, cathepsin D, IL-18Rbeta and MK13 genes, and testing for binding to determine the level of the one or more proteins in the subject sample;

b) contacting a control sample with one or more of the reagents from b) and testing for binding to determine the level of the one or more proteins in the control sample; and

c) comparing the level of the one or more genes in the subject sample with the expression level of the one or more proteins in the control sample;

wherein a presence of the one or more proteins at a higher or lower level in the subject sample compared to the control sample indicates that the subject has breast cancer.

2. The method of claim 1 , wherein the subject sample and control sample are a bodily fluid selected from the group consisting of blood, plasma, serum and urine.

3. The method of claim 1 , wherein the subject sample and control sample are a cell or tissue sample.

4. The method of claim 1 , wherein the subject is a human.

5. The method of claim 1 , wherein the reagent is selected from the group consisting of an antibody and an aptamer.

6. The method of claim 5, wherein the reagent is an antibody and said testing for binding is by ELISA.

7. The method of claim 5, wherein the aptamer comprises an

oligonucleotide, optionally further comprising one or more modified bases.

8. The method of claim 5, wherein the aptamer comprises a peptide.

9. The method of claim 1 , wherein the control sample comprises noncancerous breast cells.

10. The method of claim 1 , wherein the control sample is obtained from a non- breast tissue of the subject.

11. The method of claim 1 , wherein at least one of the reagents specifically binds to a protein encoded by one or more of the CTGF and SLAF6 genes.

12. The method of claim 1 , wherein at least one of the reagents specifically binds to a protein encoded by one or more of the MIG and ApoE2 genes.

13. The method of claim 1 , wherein at least one of the reagents specifically binds to a protein encoded by one or more of the SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL-1 R4/ST2, carbonic anhydrase 6, CRP and uPAR genes.

14. A kit for detecting breast cancer in a sample, the kit comprising one or more aptamers that specifically bind to a protein encoded by one or more of the C5, cathepsin D, IL-18Rbeta and MK13 genes.

15. The kit of claim 14, further comprising one or more aptamers that specifically bind to a protein encoded by one or more of the CTGF and SLAF6 genes.

16. The kit of claim 14, further comprising one or more aptamers that specifically bind to a protein encoded by one or more of the MIG and ApoE2 genes.

17. The kit of claim 14, further comprising one or more aptamers that specifically bind to a protein encoded by one or more of the SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL-1 R4/ST2, carbonic anhydrase 6, CRP and uPAR genes.

18. The kit of any of claims 14-17, wherein the aptamer is a nucleic acid.

19. The kit of any of claims 14-17, wherein the aptamer is a peptide.

20. A kit for detecting breast cancer in a sample, the kit comprising one or more antibodies that specifically bind to a protein encoded by one or more of the C5, cathepsin D, IL-18Rbeta and MK13 genes.

21. The kit of claim 20, further comprising one or more antibodies that specifically bind to a protein encoded by one or more of the CTGF and SLAF6 genes.

22. The kit of claim 20, further comprising one or more antibodies that specifically bind to a protein encoded by one or more of the MIG and ApoE2 genes.

23. The kit of claim 20, further comprising one or more antibodies that specifically bind to a protein encoded by one or more of the SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL-1 R4/ST2, carbonic anhydrase 6, CRP and uPAR genes.

24. A method of detecting breast cancer in a subject comprising

a) contacting a sample from a subject with one or more reagents that specifically bind to a nucleic acid encoded by one or more of the genes listed in Tables 3-6, wherein at least one of the reagents specifically binds to a nucleic acid encoded by one or more of the C5, cathepsin D, IL-18Rbeta and MK13 genes, and testing for binding to determine the expression level of the one or more genes in the subject sample;

b) contacting a control sample with one or more of the reagents from b) and testing for binding to determine the expression level of the one or more genes in the control sample; and

c) comparing the expression level of the one or more genes in the subject sample with the expression level of the one or more genes in the control sample;

wherein a higher level of expression of the one or more genes in the subject sample compared to the control sample indicates that the subject has breast cancer.

25. The method of claim 23, wherein the subject sample and the control sample is a bodily fluid selected from the group consisting of blood, plasma, serum and urine.

26. The method of claim 23, wherein the subject sample and the control sample is a cell or tissue sample.

27. The method of claim 23, wherein the sample comprises a mRNA or cDNA preparation from the subject.

28. The method of claim 23, wherein the subject is a human.

29. The method of claim 23, wherein the reagent is a labeled nucleic acid probe.

30. The method of claim 28, wherein the label is selected from the group consisting of radioactive, colorimetric, enzymatic, fluorometric, chemiluminescent and magnetic.

31. The method of claim 23, wherein the control sample comprises noncancerous breast cells.

32. The method of claim 23, wherein the control sample is obtained from a non-breast tissue of the subject.

33. The method of claim 23, wherein at least one of the reagents specifically binds to a nucleic acid encoded by one or more of the CTGF and SLAF6 genes.

34. The method of claim 23, wherein at least one of the reagents specifically binds to a nucleic acid encoded by one or more of the MIG and ApoE2 genes.

35. The method of claim 23, wherein at least one of the reagents specifically binds to a nucleic acid encoded by one or more of the SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL-1 R4/ST2, carbonic anhydrase 6, CRP and uPAR genes.

36. A kit for detecting breast cancer in a sample, the kit comprising one or more nucleic acids that specifically bind to a nucleic acid encoded by one or more of the C5, cathepsin D, IL-18Rbeta and MK13 genes.

37. The kit of claim 35, further comprising one or more nucleic acids that specifically bind to a nucleic acid encoded by one or more of the CTGF and SLAF6 genes.

38. The kit of claim 35, further comprising one or more nucleic acids that specifically bind to a nucleic acid encoded by one or more of the MIG and ApoE2 genes.

39. The kit of claim 35, further comprising one or more nucleic acids that specifically bind to a nucleic acid encoded by one or more of the SLAF6, CTGF, CYTN, ApoE2, PTN, PPIE, Factor I, IL-1 R4/ST2, carbonic anhydrase 6, CRP and uPAR genes.