WO2019074920A1 - Méthodes et compositions pour la détection et le diagnostic du cancer du sein - Google Patents

Méthodes et compositions pour la détection et le diagnostic du cancer du sein Download PDF

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Publication number
WO2019074920A1
WO2019074920A1 PCT/US2018/055007 US2018055007W WO2019074920A1 WO 2019074920 A1 WO2019074920 A1 WO 2019074920A1 US 2018055007 W US2018055007 W US 2018055007W WO 2019074920 A1 WO2019074920 A1 WO 2019074920A1
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WIPO (PCT)
Prior art keywords
protein
sample
breast cancer
tables
proteins
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PCT/US2018/055007
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English (en)
Inventor
Lyndal HESTERBERG
Philip MCQUARY
Karen COPELAND
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Oncocyte Corporation
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Publication of WO2019074920A1 publication Critical patent/WO2019074920A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • breast cancer is the second leading cause of cancer-related death in women.
  • 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.
  • MRI Magnetic Resonance Imaging
  • ultrasound ultrasound
  • BIRAD Breast Imaging Reporting and Data System
  • Such an improved confirmatory detection 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 breast cancer detection and diagnostic opportunity for women who cannot have a mammogram.
  • 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).
  • the methods and compositions disclosed herein are based on the identification of proteins used in the disclosed detection methods as protein biomarkers that can be differentially expressed proteins (DEPs), but may not be either necessarily unique cancer specific proteins nor proteins unique to non-cancerous breast tissue or cancerous breast tissue. 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 protein products are detected at different (higher or lower) levels in samples (e.g. serum samples and other bodily fluid samples) obtained from subjects with breast cancer vs. subjects with benign breast pathologies/no cancer.
  • the samples can also include DEPs within cells, tissues, cancerous cells, tumors and tumorous tissues.
  • Embodiments of the disclosure provide methods of detection and/or diagnosis and prognosis of cancer.
  • Other embodiments provide compositions relating to the detection and/or diagnosis, prognosis of cancer and methods to assess treatment efficacy of cancer treatments.
  • the methods and compositions may be used for detection and/or diagnosis and prognosis of 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.
  • the present disclosure provides methods and compositions for detecting and diagnosing breast cancer in a subject comprising: a) obtaining a sample from an individual; b) obtaining a non-breast cancer control sample; c) detecting protein levels in the individual' s sample, the proteins comprising at least two or more protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12-14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14; and d) detecting protein levels in the non-breast cancer control sample, the proteins comprising at least two or more protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14.
  • the method further provides diagnosing the individual as having breast cancer when the protein levels from the individual's sample differ significantly from corresponding protein levels in the non-breast cancer control sample; wherein: the detection of aberrant protein levels compared to a non-breast cancer control sample is a confirmatory breast cancer diagnosis; the diagnosis is achieved absent a surgical breast biopsy procedure; and there is an essential absence of cutaneous scaring of the individual's breast(s).
  • the present disclosure provides methods and compositions for detection of breast cancer utilizing two or more protein biomarkers selected from the group of protein biomarkers listed in UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, and P48061.
  • the methods and compositions for detection further utilizes one or more protein biomarkers listed in UniProt Nos. Q9C035, Q04637, P10747, P22301, P52823, 060880, and P78410.
  • the individual sample and the non-breast cancer control sample can be a bodily fluid selected from the group consisting of whole blood, plasma, serum, interstitial fluid, cerebrospinal fluid, lymph fluid, saliva, sputum, and urine.
  • the non-breast cancer control sample can also be non-cancerous breast cells or non-breast tissue of the individual and the samples can be of human origin.
  • the present disclosure provides methods and compositions for detecting breast cancer in an individual by determining protein levels of selected protein biomarkers.
  • the methods and compositions comprises contacting a fraction of the bodily fluid sample to a set of reagents comprising antibodies that specifically bind to at least two or more protein biomarkers selected from the group consisting of proteins listed in UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061.
  • the methods and compositions further comprise at least one reagent that specifically binds to a protein biomarker of one of the proteins listed in UniProt Nos. Q9C035, Q04637, P10747, P22301, P52823, 060880, P78410.
  • the present disclosure provides methods and compositions for detecting breast cancer in an individual by binding an antibody or an aptamer reagent specific for a protein biomarker listed in Tables 7, 8, and 12-14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14.
  • the method and compositions can comprise an antibody and testing for binding of the antibody to its specific protein biomarker utilizing a method selected from the group consisting of ELISA, immunofluorescent microscopy, and radio-immuno assay.
  • the method and compositions employ an aptamer(s) having an oligonucleotide composition and optionally further having one or more modified nucleobases.
  • the method and compositions employ an aptamer(s) having a peptide composition.
  • the present disclosure provides a kit for detecting breast cancer in a sample, the kit having one or more aptamers that specifically bind to a protein encoded by one or more of the genes encoding protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14.
  • the kit can further have one or more aptamers that specifically bind to one or more proteins encoded by genes encoding proteins listed in UniProt Nos. Q9UMR7, Q8NHI6, 094992, Q6DN72, P48061.
  • the kit can have one or more aptamers that specifically bind to a protein encoded by genes encoding proteins listed in UniProt Nos. Q9C035, Q04637, P10747, P22301, P52823, 060880, P78410.
  • the aptamer can have an oligonucleotide composition.
  • the aptamer can have a peptide composition.
  • 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 to the aptamer agent that specifically binds to a protein differentially expressed in a bodily fluid or by a breast cancer cell or can specifically bind to a nucleic acid encoding a protein differentially expressed in a bodily fluid or by a breast cancer cell.
  • the kit may further contain a positive control (e.g. , a bodily fluid, one or more breast cancer cells; or specific known quantities of the differentially expressed protein or known quantities of mRNA or cDNA encoding a differentially expressed protein present in a bodily fluid from a breast cancer individual' s sample) and/or a negative control (e.g. , a bodily fluid, tissue or cell sample or specific known quantities of the differentially expressed protein or known quantities of mRNA or cDNA encoding a differentially expressed protein present in a bodily fluid from a non-cancerous individual).
  • a positive control e.g. , a bodily fluid, one or more breast cancer cells; or specific known quantities
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. In other embodiments, the composition comprises a molecule that binds to one or more proteins circulating in a bodily fluid, expressed by a cancerous cell or tissue or a non-cancerous cell or tissue.
  • the protein can be a protein biomarker listed in Tables 7, 8 and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14.
  • the present disclosure provides a kit for detecting breast cancer in a sample, the kit having one or more one or more antibodies that specifically bind to one or more proteins encoded by genes encoding protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1 , 4, and 7- 14.
  • the kit can further have one or more antibodies that specifically bind to one or more proteins encoded by genes encoding proteins listed in UniProt Nos.
  • the kit can have one or more antibodies that specifically bind to a protein encoded by genes encoding proteins listed in UniProt Nos. Q9C035, Q04637, P10747, P22301 , P52823, 060880, P78410.
  • the kit can have one or more containers and instructions for determining if the individual' s 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 to the antibody agent that specifically binds to a protein differentially expressed in a bodily fluid or by a breast cancer cell.
  • the kit may further contain a positive control (e.g. , a bodily fluid, one or more breast cancer cells; or specific known quantities of the differentially expressed protein in a bodily fluid from a breast cancer individual' s sample) and/or a negative control (e.g. , a bodily fluid, tissue or cell sample or specific known quantities of the differentially expressed protein in a bodily fluid from a non-cancerous individual).
  • a positive control e.g. , a bodily fluid, one or more breast cancer cells; or specific known quantities of the differentially expressed protein in a bodily fluid from a breast cancer individual' s sample
  • a negative control e.g. , a bodily fluid, tissue or cell sample or specific known quantities of the differentially expressed protein in a bodily fluid from a non-cancerous individual.
  • the present disclosure provides a method of detecting breast cancer in an individual by a) obtaining a first circulating blood sample from a first individual, b) obtaining a second circulating blood sample from a second individual, wherein the second sample is from a breast cancer individual, and c) detecting protein levels for each member of a list of proteins in the first and second samples, the list comprising protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14.
  • the method further comprises diagnosing said first individual as having breast cancer when the protein levels from the first individual' s sample do not differ significantly from the second individual' s breast cancer control sample; wherein the detection of comparable protein levels in the first sample compared to the second individual' s breast cancer control sample is a confirmatory breast cancer diagnosis; the diagnosis is achieved absent a surgical breast biopsy procedure; and there is an essential absence of cutaneous scaring of the individual' s breast(s).
  • the list of proteins comprises no more than 20 proteins; the method can further have the transmission of a report of detection to a health practitioner, and the results can either provide a diagnosis of breast cancer or a diagnosis of no evidence of breast cancer.
  • the present disclosure provides a method of detecting breast cancer in an individual by a) obtaining a sample from an individual; b) obtaining a non-breast cancer control sample; c) detecting protein levels in the individual's sample, the proteins comprising at least two or more protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14; and d) detecting protein levels in the non-breast cancer control sample, the proteins comprising at least two or more protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12-14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14.
  • the method further provides diagnosing the individual as having breast cancer when the protein levels from the individual's sample differ significantly from corresponding protein levels in the non-breast cancer control sample; wherein: the detection of aberrant protein levels compared to a non-breast cancer control sample is a confirmatory breast cancer diagnosis; the diagnosis is achieved absent a surgical breast biopsy procedure; and there is an essential absence of cutaneous scaring of the individual's breast(s).
  • the disclosed method of detecting breast cancer in an individual uses samples from the individual and the non-breast cancer control obtained from a bodily fluid selected from the group consisting of blood, plasma, serum, interstitial fluid, cerebrospinal fluid, lymph fluid, saliva, sputum, and urine.
  • the samples can be a cell or tissue sample.
  • the sample further has a mRNA or cDNA preparation from the individual and non-breast cancer control samples.
  • the mRNA or cDNA preparation encodes for at least two or more protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14.
  • the samples are human and the non-breast cancer control sample can be obtained from a non-breast tissue of the individual.
  • the detection of protein levels comprises contacting a fraction of the bodily fluid sample to a set of reagents comprising antibodies that specifically bind to at least two or more protein biomarkers selected from the group consisting of protein biomarkers listed in UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061.
  • at least one of the reagents specifically binds to a protein biomarker of one of the proteins listed in UniProt Nos.
  • the reagent can be a labeled nucleic acid probe and the label can be selected from the group consisting of colorimetric, enzymatic, fluorometric, chemiluminescent, radioactive, and magnetic labels.
  • the reagent antibodies specifically bind to: protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14; a nucleic acid encoding a protein selected from UniProt protein Nos.
  • Q9UMR7, Q8NHJ6, 094992, Q6DN72, and P48061 a nucleic acid encoding a protein selected from UniProt protein Nos. Q9C035, Q04637, P10747, P22301, P52823, 060880, and P78410.
  • 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.
  • measuring the expression level of a gene is accomplished by measuring levels of the protein product (the differentially expressed proteins disclosed infra) of the gene.
  • levels of the protein product the differentially expressed proteins disclosed infra
  • 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.
  • the differentially expressed protein(s) level(s) can measure protein levels for protein(s) found to be circulating extracellular, within a bodily fluid, intracellular, within cancerous cells, within tumors and within tumorous tissues.
  • the present disclosure provides a kit for detection of breast cancer in a sample obtained from an individual.
  • the kit can comprise one or more nucleic acids that specifically bind to one or more nucleic acids encoding one or more protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12-14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14.
  • the kit can further have one or more nucleic acids that specifically bind to a nucleic acid encoding a protein selected from UniProt protein Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061.
  • the kit can further have one or more nucleic acids that specifically bind to a nucleic acid encoding a protein selected from UniProt protein Nos. Q9C035, Q04637, P10747, P22301, P52823, 060880, P78410.
  • the kit can 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 to the antibody agent that specifically binds to a protein differentially expressed in a bodily fluid or by a breast cancer cell.
  • the kit may further contain a positive control (e.g. , a bodily fluid, one or more breast cancer cells; or specific known quantities of the differentially expressed protein in a bodily fluid from a breast cancer individual' s sample) and/or a negative control (e.g. , a bodily fluid, tissue or cell sample or specific known quantities of the differentially expressed protein in a bodily fluid from a noncancerous individual).
  • a positive control e.g. , a bodily fluid, one or more breast cancer cells; or specific known quantities of the differentially expressed protein in a bodily fluid from a breast cancer individual' s sample
  • a negative control e.g. , a bodily fluid, tissue or cell sample or specific known quantities of the differentially expressed protein in a bodily fluid from a noncancerous individual.
  • the present disclosure provides a method for monitoring efficacy of a breast cancer treatment methodology by a) obtaining a first sample comprising circulating blood from the individual at a first time point; b) administering the treatment methodology to the individual: c) obtaining a second sample comprising circulating blood from the individual at a second time point; d) determining protein levels for each member of a list of proteins comprising at least two or more protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14 in said first sample; and e) determining protein levels for each member of the list of proteins comprising at least two or more protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1 , 4, and 7- 14 in said second sample.
  • the first sample can be obtained by drawing blood from a vein or artery of the individual.
  • the treatment methodology can be selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, and genetic editing and combinations thereof.
  • Gene editing can alter a gene' s expression by a method consisting of gene: silencing, knockdown, inhibition, mutation, deletion, and combinations thereof, of one or more nucleic acid sequences in the gene encoding at least one protein biomarker.
  • the listed protein biomarkers can further comprise proteins listed in UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, and P48061. Additional protein biomarkers can comprise proteins listed as UniProt Nos.
  • the method for monitoring efficacy of a breast cancer treatment methodology can have a change in the treatment methodology when protein levels for the first sample are the same as the protein levels for the second sample.
  • the treatment methodology can also be repeating of the treatment methodology when protein levels for the first sample are the same as the protein levels for the second sample.
  • the treatment methodology can also be discontinuing the treatment methodology when protein levels of the second sample return to levels corresponding to a healthy individual.
  • 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.
  • 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.
  • 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 disclosed protein biomarkers at a first time point; b) measuring the expression level of the one or more disclosed protein biomarkers 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 disclosed protein biomarkers in b) compared to a) indicates that the subject's breast cancer is advancing.
  • Suitable protein biomarkers include those protein biomarkers from UniProt Nos.
  • the present disclosure provides antigens (i.e. , cancer- associated polypeptides) associated with breast cancer as targets for diagnostic and/or therapeutic antibodies.
  • the antigen may be chosen from a protein biomarker chosen from UniProt Nos.
  • the present disclosure provides antigens (i.e. , cancer- associated polypeptides) associated with breast cancer as targets for diagnostic and/or therapeutic antibodies.
  • the antigen(s) may include a panel of proteins encoded by one or more of the protein biomarkers chosen from UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061, Q9C035, Q04637, P10747, P22301, P52823, 060880, and P78410 and/or any one or more protein biomarkers listed in any of Tables 1, 4, and 7-14, or one or more fragments of the one or more proteins listed above.
  • 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.
  • a subject may be contacted intravenously or intramuscularly with protein or protein fragment.
  • 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 chosen from UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061, Q9C035, Q04637, P10747, P22301, P52823, 060880, and P78410 and/or any one or more protein biomarkers listed in any of Tables 1, 4, and 7-14, thereby eliciting an immune response to a breast cancer cell.
  • the subject may be contacted with the protein or the protein fragment intravenously or intramuscularly.
  • the 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 differentially expressed protein biomarkers (e.g., polypeptides, and the nucleic acids encoding the polypeptides) disclosed infra.
  • the agents may bind to one or more of the differentially expressed protein biomarkers disclosed infra.
  • the kit may include agents that are proteins and/or nucleic acids for example.
  • the kit provides a plurality of agents. The agents may be able to detect the panel of protein biomarkers comprising one or more proteins or protein fragments chosen from UniProt Nos.
  • 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 encoding protein biomarkers one or more proteins chosen from UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061, Q9C035, Q04637, P10747, P22301 , P52823, 060880, and P78410 and/or any one or more protein biomarkers listed in any of Tables 1 , 4, and 7- 14.
  • 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 protein biomarkers chosen from UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061 , Q9C035, Q04637, P10747, P22301 , P52823, 060880, and P78410 and/or any one or more protein biomarkers listed in any of Tables 1, 4, and 7- 14.
  • 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).
  • the present disclosure provides a kit for the detection of breast cancer comprising one or more agents that specifically bind one or more protein biomarkers of one or more proteins chosen from UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061 , Q9C035, Q04637, P10747, P22301 , P52823, 060880, and P78410 and/or any one or more protein biomarkers listed in any of Tables 1 , 4, and 7- 14.
  • the agent may be a protein, such as an antibody.
  • the agent may be an aptamer that binds to the one or more protein(s) of choice.
  • 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 protein biomarkers 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).
  • the kit may take the form of an ELISA or a DNA microarray.
  • the kit may include one or more antibodies suitable for use in a fluorescent activated cell sorter, e.g. use in flow cytometry.
  • 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 and/or a differentially expressed protein, wherein the cancer associated protein and/or the differentially expressed protein is encoded by a gene selected from chosen from UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061, Q9C035, Q04637, P10747, P22301, P52823, 060880, and P78410 and/or any one or more protein biomarkers listed in any of Tables 1, 4, and 7- 14, homo logs thereof, combinations thereof, or a fragment thereof.
  • the therapeutic agent binds to the cancer associated protein and/or the differentially expressed protein.
  • the therapeutic agent is an aptamer.
  • the therapeutic agent is an antibody.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the antibody is a humanized or human antibody.
  • the antibody may be conjugated with a drug or a toxin.
  • 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 encoding one or more proteins chosen from UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061, Q9C035, Q04637, P10747, P22301, P52823, 060880, and P78410 and/or any one or more protein listed in any of Tables 1, 4, and 7- 14, fragments thereof, homologs thereof, and/or complements thereof.
  • a therapeutic agent that modulates the expression of one or more genes encoding one or more proteins chosen from UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061, Q9C035, Q04637, P10747, P22301, P52823, 060880, and P78410 and/or any one or more protein listed in any of Tables 1, 4, and 7- 14, fragments thereof, homologs thereof
  • the present disclosure provides a method of treating breast cancer comprising a gene knockdown of one or more genes encoding one or more proteins chosen from UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061, Q9C035, Q04637, P10747, P22301, P52823, 060880, and P78410 and/or any one or more protein listed in any of Tables 1, 4, and 7-14, fragments thereof, homo logs thereof, and or complements thereof.
  • 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 differentially expressed genes encoding the proteins chosen from UniProt Nos.
  • the present disclosure provides methods of visualizing a breast cancer tumor comprising a) targeting one or more differentially expressed proteins with a labeled molecule that binds specifically to the breast cancer tumor, wherein the differentially expressed protein is selected from a protein encoded for by one or more genes chosen from UniProt Nos. Q9UMR7, Q8NHJ6, 094992, Q6DN72, P48061, Q9C035, Q04637, P10747, P22301, P52823, 060880, and P78410 and/or any one or more gene(s) encoding the proteins listed in any of Tables 1, 4, and 7-14; and b) detecting the labeled molecule, wherein the labeled molecule visualizes the tumor. Visualization may be done in vivo, or in vitro.
  • the present disclosure provides methods of visualizing a breast cancer tumor comprising a) targeting one or more differentially expressed genes encoding the disclosed differentially expressed proteins infra or gene products, ⁇ e.g., mRNA) with a labeled molecule ⁇ e.g. , a labeled nucleic acid) that binds specifically to the differentially expressed genes encoding the disclosed differentially expressed proteins or gene products, wherein the differentially expressed genes encoding the disclosed differentially expressed proteins infra or gene products is chosen from UniProt Nos.
  • the present disclosure provides methods of detecting expressed gene product levels comprising a) obtaining a biological sample from a subject, and performing an assay on the biological sample ⁇ e.g., a bodily fluid) to detect expressed gene product levels of at least two or more protein biomarkers selected from the group consisting of the panels of protein biomarkers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14, wherein the expressed gene product levels are detected through the presence of nucleic acids, antibodies, or proteins in the subject's biological sample.
  • FIG. 1 shows analysis of robust FDR (false discovery rate) LogWorth by effect size for analysis of 1,012 markers for suitability as breast cancer diagnostics.
  • FIG. 2A shows data and violin plots of model scores ⁇ i.e., probability of being a cancer sample) for a 5-marker panel (AllPnl).
  • FIG. 2C through FIG. 2M illustrate individual data plots for the five AllPnl model markers.
  • FIGS. 2C, 2D, 2E, 21 and 2J illustrate oneway analysis of each of the five markers to distinguish between benign vs. malignant samples.
  • FIGS. 2F, 2G, 2H, 2L, and 2M illustrate the CDF (cumulative distribution function) plots of benign vs. malignant samples for each of the five markers.
  • FIGS. 2K and 2N illustrate the combination of the five markers in the AllPnl model in a oneway analysis and in a CDF plot showing how the combination of markers is much stronger at separation (benign vs. malignant) than each individual marker.
  • FIG. 3 shows comparisons of ROC (receiver operating characteristic) curves for the 11 OLink panels where each panel' s 92 markers were used as a candidate marker set and a generalized regression model using a "fast fitting" routine that zeros out terms to build "quick" models. Of interest is the most upper-left curve that is well above all other curves. This was from the model built from the immune response (IR) panel. The initial immune response panel model had 27 markers evaluated for distinguishing benign vs. malignant samples.
  • IR immune response
  • FIG. 4 shows analysis of one factor at a time with a FDR (false discovery rate) LogWorth by effect size for analysis of 23/27 IR markers for suitability as breast cancer diagnostics, identifying five markers of interest.
  • FDR false discovery rate
  • FIG. 5A through FIG.5R illustrate individual data plots for six IR model markers.
  • FIGS. 5A, 5B, 5C, 5J, 5K and 5L illustrate oneway analysis of each of the six markers to distinguish between benign vs. malignant samples.
  • FIGS. 5D, 5E, 5F, 5M, 5N, and 50 illustrate the CDF (cumulative distribution function) plots of benign vs. malignant samples for each of the six markers.
  • FIGS. 5G, 5H, 51, 5P, 5Q, and 5R illustrate density plots (derived from a scatter plot of each of the six markers on a log 10 scale) to illustrate the ability of the six markers to distinguish between benign vs. malignant samples.
  • FIG. 6B shows data and violin plots of model scores (i.e., probability of being a cancer sample) for the 11 -marker model panel (ImRspl).
  • FIG. 7B shows data and violin plots of model scores (i.e., probability of being a cancer sample) for the 7-marker model panel (ImRsp2).
  • FIG. 8A through FIG. 8L illustrate individual data plots for four additional IR model markers listed in Table 8 but not represented in FIGS. 2C-2R.
  • FIGS. 8 A, 8B, 8C, and 8D illustrate oneway analysis of each of the four markers to distinguish between benign vs. malignant samples.
  • FIGS. 8E, 8F, 8G, and 8H illustrate the CDF (cumulative distribution function) plots of benign vs. malignant samples for each of the four markers.
  • FIGS. 81, 8 J, 8K, and 8L illustrate density plots (derived from a scatter plot of each of the four markers on a log 10 scale) to illustrate the ability of the four markers to distinguish between benign vs. malignant samples. Little separation as stand alone markers is observed.
  • FIG. 9 illustrates markers order by ordered of significance (16 markers not zeroed out) from left to right as a result of fitting a candidate set of 68 markers to evaluate 24 markers from the immune response panel with low variation (Table 10) in a generalized regression model using the lasso fitting routine to zero out non- significant terms and then fitted with 500 bootstrap samples from the data set.
  • the 16 markers are in Table 11.
  • FIG. 10B shows data and violin plots of model scores (i.e., probability of being a cancer sample) for the 16-marker model panel (ImRsp2).
  • FIG. 12A through FIG. 12D show data and violin plots of model scores (i.e., probability of being a cancer sample) for the potential robustness for each of the models constructed.
  • FIGS. 13A through FIG. 13D show data plots of the four models constructed with controls with model scores (i.e., probability of being a cancer sample) for the potential robustness for each of the four models constructed.
  • 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%.
  • 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.
  • 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.
  • Agent 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.
  • amplify 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.
  • an amplification product can be made enzymatically with DNA or RNA polymerases or reverse transcriptases, or any combination thereof.
  • animal refers to, 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.
  • the term "subject,” “patient” or “animal” refers to a male.
  • the term “subject,” “patient” or “animal” refers to a female.
  • antibody 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, bi-specific, 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.
  • biological sources 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.
  • capture reagent refers to a reagent, for example an antibody or antigen binding protein, capable of binding a target molecule, including but not limited to a protein marker, or analyte to be detected in a sample.
  • the phrase "differentially expressed protein” can refer to one or more proteins whose expression in a test sample, including and without limitation, the measured level of protein expressed, can be either higher or lower compared to the same one or more measured levels of proteins expressed in a non-disease sample, for example but not limited to, bodily fluid and/or cell sample from a non-cancerous breast and/or ovarian individual, as compared to a bodily fluid, cell, tumor cell, cancerous tissue or tumorous tissue sample from an individual who will develop, be diagnosed or has been diagnosed with breast and/or ovarian cancer.
  • a non-disease sample for example but not limited to, bodily fluid and/or cell sample from a non-cancerous breast and/or ovarian individual, as compared to a bodily fluid, cell, tumor cell, cancerous tissue or tumorous tissue sample from an individual who will develop, be diagnosed or has been diagnosed with breast and/or ovarian cancer.
  • DEPs Due to a seemingly progressive decline in biological processes including functioning tumor suppressor genes and apoptotic genes as well as up-regulation of normal genes promoting cell growth, i.e., oncogenes, earlier detection, using DEPs, of the decline or up-regulation in one or more of these gene's biological processes can have the potential to elucidate the development of neoplasms and oncogenisis.
  • the identification of DEPs can also serve as early indicators of metastatic processes and neoplastic growths by measuring their levels of DEPs in an individual' s bodily fluid and/or within a cell sample. DEP levels can measure protein levels for DEPs found circulating extracellularly in bodily fluid(s), intracellular, within cancerous cells, within tumors and within tumorous tissues.
  • 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 corresponding to a known positive breast cancer control sample.
  • 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.
  • hybridization or “hybridizing” refers to hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • 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.
  • “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.
  • 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.
  • inhibitors 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.
  • label and/or “detectable substance” can 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.
  • 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.
  • 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.
  • 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.
  • a "microarray” as used herein can be 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/cm 2 more preferably at least about 100/cm 2 , even more preferably at least about 500/cm 2 , and still more preferably at least about 1,000/cm 2 .
  • 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 micro array.
  • 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.
  • nucleic acid means at least two nucleotides covalently linked together.
  • an oligonucleotide is an oligomer of 6, 8, 10, 12, 20, 30 or up to 100 nucleotides.
  • 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.
  • 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.
  • Percent homology refers 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 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.
  • 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.
  • Recombinant protein 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.
  • 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.
  • 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.
  • 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.
  • ROC 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.
  • AUC area under the ROC curve
  • 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.
  • sample refers to a 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 a bodily fluid, including but not limited to blood, plasma, serum, urine, saliva, cerebral spinal fluid (CSF), or any combination thereof. A sample may be derived from blood, plasma, serum, urine or any combination thereof.
  • CSF cerebral spinal fluid
  • samples include, but are not limited to, any bodily fluid obtained from a mammalian subject, tissue biopsy, tumor tissue, 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
  • 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.
  • 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.
  • 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.
  • sequence tag 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.
  • support refers to conventional supports such as beads, particles, dipsticks, fibers, filters, membranes, and silane or silicate supports such as glass slides.
  • 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.
  • embodiments of the present disclosure are directed to the treatment of cancer or the decrease in proliferation of cells.
  • 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.
  • 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.
  • a "therapeutically effective amount” or “effective amount” of a composition can be a predetermined amount calculated to achieve the desired effect, i.e., to inhibit, block, or reverse the activation, migration, metastasis, or proliferation of cells.
  • the effective amount is a prophylactic amount.
  • 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.
  • 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.
  • treat 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.
  • the term may refer to both treating and preventing.
  • 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.
  • tissue refers to any aggregation of similarly specialized cells that are united in the performance of a particular function.
  • the present disclosure provides for nucleic acids and extracellular proteins that can be differentially expressed but are not necessarily either associated with cancer or unique cancer specific proteins, herein termed “differentially expressed protein” and “differentially expressed protein biomarkers” referred to as “DE” proteins or “DE biomarkers”, respectively.
  • the present disclosure provides nucleic acid and proteins that have been found to be 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.
  • proteins that are identified in one type of cancer may have a strong likelihood of being involved in other types of cancers as well.
  • the proteins 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 detecting, diagnosing and/or prognosticating may comprise measuring the level of expression of a differentially expressed marker disclosed herein.
  • the method may further comprise comparing the expression level of the differentially expressed protein 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.
  • the control may also comprise a benchmark level of marker expression, wherein one or more measured marker expression levels in a biological sample taken from a subject that surpasses the benchmark may indicate a particular condition.
  • Differentially expressed sequences and proteins 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.
  • Differentially expressed 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 protein expression profile or an altered protein expression profile.
  • differentially expressed sequences and protein markers are from humans; however, as will be appreciated by those in the art, differentially expressed sequences and protein markers from other organisms may be useful in animal models of disease and drug evaluation; thus, other differentially expressed sequences and protein markers 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.). Differentially expressed sequences and protein markers from other organisms may be obtained using the techniques outlined herein.
  • differentially expressed proteins include the nucleic acid sequences encoding the proteins listed in Tables 7, 8, and 12-14 and combinations thereof.
  • the differentially expressed sequences are nucleic acids.
  • differentially expressed sequences of embodiments herein may be useful in a variety of applications including diagnostic applications to detect nucleic acids or their expression levels and to detect the proteins they encode in a subject, therapeutic applications or a combination thereof. Further, the differentially expressed 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 differentially expressed sequences.
  • 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; phosphorothioate; phosphorodithioate; O- methylphosphoroamidite linkages, and/or peptide nucleic acid backbones and linkages.
  • Other nucleic acid analogues include those with positively-charged backbones; non-ionic backbones and non-ribose backbones.
  • Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids.
  • nucleic acid analogues may be used in some embodiments of the present disclosure.
  • 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.
  • the nucleic acids may be single stranded or double stranded or may contain portions of both double stranded or single stranded sequence.
  • 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.
  • nucleoside includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino-modified nucleosides.
  • nucleoside includes non- naturally occurring analogue structures.
  • differentially expressed sequences may include both nucleic acid and amino acid sequences and the encoded proteins.
  • the differentially expressed sequences may include sequences having at least about 60% homology with the disclosed sequences.
  • the differentially expressed sequences may have at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 99.8% homology with the disclosed sequences.
  • the differentially expressed sequences may be "mutant nucleic acids".
  • mutant nucleic acids refers to, for example, deletion mutants, insertions, point mutations, substitutions, and translocations.
  • the differentially expressed proteins may include proteins having at least about 60% homology with the disclosed differentially expressed proteins. In some embodiments, the proteins may have at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98.5%, about 99%, and about 99.8% homology with the disclosed differentially expressed proteins. In some embodiments, the differentially expressed proteins may be "variant proteins". As used herein, "variant proteins" can refer to, for example, a differentially expressed protein having one or more amino acid deletion(s), insertion(s), substitution(s), and incorporation of non-naturally occurring amino acid(s).
  • the differentially expressed 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.
  • 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.
  • nucleic acid 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.
  • 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.
  • 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.
  • proteins including e.g., nucleases
  • sequences associated with breast cancer 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.
  • sequences that are identified in one type of cancer may have a strong likelihood of being involved in other types of cancers as well.
  • sequences outlined herein are initially identified as correlated with breast cancers, they may also be found in other types of cancers as well.
  • differentially expressed sequences are directed to the use of differentially expressed sequences for detection, diagnosis and treatment of breast cancer.
  • the differentially expressed sequence is selected from any one or more genes (or the complement thereof) encoding the protein markers listed in Tables 7, 8, and 12-14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14.
  • these differentially expressed 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.
  • the differentially expressed sequences are DNA sequences encoding mRNA encoding any one or more of the protein markers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14.
  • a differentially expressed sequence can be a differentially expressed protein or cancer associated polypeptide expressed by the aforementioned mRNAs or homologues thereof.
  • the differentially expressed sequence may be a nucleic acid that is a mutant version of the above disclosed sequences.
  • 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 96%, at least about 97%, at least about 98%, at least about 99%, and at least about 99.5% identity with the disclosed protein markers.
  • 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 differentially expressed polynucleotide sequences corresponding to and encoding a portion of any one or more protein markers listed in Tables 7, 8 and 12-14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14.
  • 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.
  • the present disclosure provides an isolated protein, encoded within an open reading frame of a differentially expressed nucleic acid sequence selected from the polynucleotide sequences (or complements thereof) corresponding to and encoding any one or more protein markers listed in Tables 7, 8 and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14.
  • the present disclosure provides an isolated protein, wherein said protein comprises the amino acid sequence encoded by a polynucleotide selected from the group consisting of sequences (or complements thereof) corresponding to and encoding any one or more protein marker(s) listed in Tables 7, 8 and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14.
  • the invention provides an isolated protein, wherein said protein comprises the amino acid sequence encoded by a differentially expressed protein as described herein.
  • the present disclosure further provides an isolated protein, comprising the amino acid sequence of an epitope of the amino acid sequence of a differentially expressed protein disclosed herein.
  • the protein or fragment thereof may be attached to a solid support.
  • the present disclosure provides an isolated antibody (monoclonal or polyclonal) or antigen binding fragment thereof, that binds to such a protein.
  • 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.
  • Some embodiments also provide for antigens (e.g. , differentially expressed polypeptides and proteins) 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.
  • antigens e.g. , differentially expressed polypeptides and proteins
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • mass spectroscopy mass spectroscopy
  • microarray microarray
  • gel electrophoresis and/or hybridization using one more probes that specifically bind a nucleic acid encoding a cancer associated sequence disclosed herein.
  • detecting a level of a differentially expressed protein marker may comprise techniques such as, but not limited to, ELISA, immunofluorescent microscopy, a radio-immuno assay, a western blot, and a flow cytometry assay, and the like. Information relating to expression of a marker can also be useful in determining therapies aimed at up- or down-regulating the differentially expressed protein's signaling using agonists or antagonists.
  • a method of diagnosing breast cancer may comprise detecting a level of the differentially expressed protein in a subject.
  • a method of screening for cancer may comprise detecting a level of the differentially expressed protein.
  • the differentially expressed protein is selected from a protein marker corresponding to any one or more of the protein markers listed in Tables 7, 8, and 12- 14 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14.
  • 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.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the antibody may be a humanized or a recombinant antibody.
  • an antibody specifically binds to one or more of a molecule, such as protein or peptide, of one or more protein markers disclosed herein.
  • the antibody binds to an epitope from a protein marker listed in Tables 7, 8, and 12- 14 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14.
  • the epitope is a fragment of a protein sequence encoded by the nucleotide sequence of any of the differentially expressed proteins disclosed herein.
  • the epitope comprises about 1- 10, 1-20, 1-30, 3-10, or 3- 15 residues of the differentially expressed sequence.
  • the epitope is not linear. In some embodiments, the epitope is discontinuous.
  • the antibody binds to the regions described herein or a peptide with at least 90%, 95%, or 99% homology or identity to the region.
  • the fragment of the regions described herein is 5- 10 residues in length.
  • 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.
  • the epitope is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 residues in length.
  • 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", “mutant peptide sequences" or "variant peptide sequences”.
  • a subject can be diagnosed with breast cancer by detecting the presence, in a sample obtained from the subject, of a differentially expressed protein marker, or a fragment thereof, e.g. , a protein marker corresponding to any one or more of the protein markers listed in Tables 7, 8, and 12-14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14.
  • differentially expressed markers may be detected in any type of sample, including, but not limited to, serum, blood, plasma, urine, tumor and the like.
  • the sample may be any type of sample as described herein.
  • 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 differentially expressed nucleic acid sequence described infra.
  • the assay may be, for example, an ELISA, immunofluorescent microscopy, a radio-immuno assay, a western blot, a flow cytometry assay and the like.
  • a differentially expressed 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.
  • 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 differentially expressed sequences encoding a protein corresponding to any one or more of the protein markers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14; and diagnosing breast cancer or a neoplastic condition in the subject based on the differentially expressed protein marker expression result, wherein the subject is diagnosed as having breast cancer or a neoplastic condition if the differentially expressed protein marker is expressed at a level that is 1) higher than its expression level in a negative control such a noncancerous breast tissue, bodily fluid 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.
  • a biochip ⁇ e.g., a microarray
  • a biochip comprises a nucleic acid molecule which encodes at least a portion of a cancer associated protein.
  • the cancer associated protein is encoded by a nucleic acid sequence, or a fragment thereof, or a complement thereof, or a homologue thereof, or combinations thereof which encodes a protein or a fragment thereof corresponding to any one or more of the protein markers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14..
  • the nucleic acid molecule specifically hybridizes with a nucleic acid sequence which encodes a protein or a fragment thereof corresponding to any one or more of the protein markers listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14.
  • 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.
  • the present disclosure provides methods of detecting or diagnosing cancer, such as breast cancer, comprising detecting the expression of a protein encoded by a nucleic acid sequence, or a fragment thereof, or a complement thereof, or a homologue thereof, or combinations thereof which encodes a protein or a fragment thereof corresponding to any one or more of the protein markers listed in Tables 7, 8, and 12-14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14, wherein a sample is contacted with a biochip comprising a sequence, or a fragment thereof, or a complement thereof, or a homologue thereof, or combinations thereof which encodes a protein or a fragment thereof corresponding to any one or more of the protein markers or fragments thereof listed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7-14.
  • 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 proteins upregulated in breast cancer, disclosed herein, in a sample and comparing the expression level of the one or more cancer associated proteins in the sample with expression level of the same cancer associated proteins in a non-cancerous cell.
  • a higher level of expression of one or more of the cancer associated proteins disclosed herein in the sample compared to the non-cancerous cell indicates a propensity for the development of cancer, e.g. , breast cancer.
  • the present disclosure provides a method for detecting a differentially expressed nucleic acid 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 differentially expressed protein disclosed herein, or a fragment thereof wherein the protein or a fragment thereof is encoded by a nucleic acid sequence as is known to one of skill in the art.
  • 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 noncancerous sample is indicative of the presence of cancer in the test sample.
  • 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.
  • the sample is a cell sample.
  • the sample is a tissue sample.
  • the sample is a bodily fluid.
  • suitable bodily fluids include, but are not limited to, blood, serum, plasma, saliva and urine.
  • the sample is a blood sample.
  • the sample is a serum sample.
  • the sample is a plasma sample.
  • the sample is a urine sample.
  • the present disclosure provides a method for detecting cancer by detecting the presence of an antibody in a test serum sample.
  • the antibody recognizes a polypeptide or an epitope of a differentially expressed protein disclosed herein.
  • the method comprises detecting a level of an antibody against an antigenic polypeptide such as, without limitation, a differentially expressed protein such as a protein disclosed herein, or an antigenic fragment thereof encoded by a nucleic acid sequence as is known to one of skill in the art.
  • 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.
  • 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.
  • a method for diagnosing cancer or a neoplastic condition comprises a) determining the expression of one or more protein markers selected from the group consisting of any one or more of the protein markers listed in Tables 7, 8, and 12- 14 and combinations of protein bio markers listed in Tables 1, 4, and 7-14, in a first sample type (e.g.
  • tissue, bodily fluid, etc. of a first individual; and b) comparing said expression of said protein(s) with their expression in a second normal sample type from said first individual or a sample from a second unaffected (non-cancerous) 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.
  • the present disclosure also provides a method for detecting presence or absence of cancer cells in a subject.
  • 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.
  • the antibody that binds to a protein encoded by a differentially expressed sequence disclosed herein may bind to a second antibody wherein the second antibody is conjugated to a detectable substance.
  • the antibody that binds to a differentially expressed protein marker or fragment thereof listed in Tables 7, 8, and 12- 14, can be bound to a solid support.
  • the method comprises detecting a complex of a differentially expressed 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.
  • the method comprises detecting extracellular, circulating differentially expressed proteins and the measurement of the differentially expressed protein levels can indicate protein expression from cDNA and mRNA. The measured differentially expressed protein levels can be detected independent from the presence of cancer cells, either circulating or not, in the subject.
  • detection of the circulating, extracellular differentially expressed proteins can be proteins that are differentially expressed but may not be either associated with cancer or cancer specific proteins.
  • 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 encoding its corresponding differentially expressed protein marker disclosed in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14, as provided herein.
  • the present disclosure provides for specific binding partners and capture reagents that bind specifically to differentially expressed polypeptides or proteins disclosed herein and the nucleic acid sequences encoding these polypeptides or proteins 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.
  • 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.
  • 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 "4 M. 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 " M, at least about 10 " M, at least about 10 M, at least about 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.
  • 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 "8 M or higher, or at least about 10 "9 M or higher, or at least about 10 "10 M 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.
  • 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 10 9 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.
  • 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.
  • 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.
  • each subunit structure e.g., a CH, VH, CL, VL, CDR, and/or FR structure
  • 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.
  • 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; (hi) 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.
  • 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 (Gly 4 Ser) 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.
  • 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.
  • 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.
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • Biacore analysis to identify one or more hybridomas that produce an antibody that specifically interacts with a molecule or compound of interest.
  • 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.
  • a recombinant combinatorial immunoglobulin library e.g., an antibody phage display library
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • breast cancers expressing one or more of the differentially expressed proteins disclosed herein, or fragments thereof may be treated by antagonizing the differentially expressed protein' s or fragment's activity.
  • a method of treating breast cancer may comprise administering a therapeutic such as, without limitation, antibodies that antagonize the ligand binding to the differentially expressed protein or fragment thereof, small molecules that inhibit the differentially expressed protein's or fragment's expression or activity, siRNAs directed towards the nucleic acid sequence encoding the differentially expressed protein, or the like.
  • a method of treating cancer comprises detecting the presence of a differentially expressed protein' s receptor and administering a cancer treatment.
  • the treatment may specifically bind to the differentially expressed protein's receptor.
  • the cancer treatment may be any cancer treatment or one that specifically inhibits the action of a differentially expressed protein.
  • 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 differentially expressed protein or the overexpression of a differentially expressed protein as described herein.
  • 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 differentially expressed protein or the receptor to which the differentially expressed protein binds.
  • These treatments can be, for example, an antibody that specifically binds to the differentially expressed protein and inhibits its activity.
  • the treatment may be a nucleic acid that downregulates or silences the expression of the differentially expressed protein.
  • Some embodiments herein describe methods of treating cancer or a neoplastic condition comprising administering, to a subject, an antibody that binds to the differentially expressed protein.
  • the antibody may be monoclonal or polyclonal.
  • the antibody may be humanized or recombinant.
  • the antibody may neutralize biological activity of the differentially expressed protein by binding to and/or interfering with the differentially expressed protein's receptor.
  • the antibody may bind to site on the differentially expressed protein encoded by the DNA sequence that is not the receptor.
  • administering the antibody may be to a biological fluid or tissue, such as, without limitation, blood, urine, serum, plasma, tumor tissue, or the like.
  • a method of treating cancer may comprise administering an agent that interferes with the synthesis, secretion, receptor binding or receptor signaling of differentially expressed proteins or its receptors.
  • 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.
  • the cancer cell may be targeted specifically with a therapeutic based upon the differentially expressed gene or gene product.
  • 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.
  • 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.
  • 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.
  • a method of treating breast cancer may comprise gene knockdown of one or more nucleic acid sequences encoding differentially expressed proteins 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 mRNA transcript or a gene.
  • 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 -O-methyl phosphorothioate oligonucleotides, locked nucleic acid oligonucleotides, or peptide nucleic acid oligonucleo tides; RNAi oligonucleotides, such as, without limitation, siRNA duplex oligonucleotides, or shRNA oligonucleotides; or any combination thereof.
  • RNase-H competent antisense such as, without limitation, ssDNA oligonucleotides, ssRNA oligonucleotides, phosphoroth
  • 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).
  • the specific mechanism of silencing may vary with the oligonucleotide chemistry.
  • 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).
  • siRNA small interfering RNA
  • RNase-H dependent antisense e.g. by RNase-H dependent antisense
  • RNase- H competent antisense oligonucleotides may form duplexes with RNA that are recognized by the enzyme RNase-H, which cleaves the RNA strand.
  • 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.
  • 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, CaCi 2 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.
  • 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 oligonucleo tides), or any combination thereof.
  • all or a portion of the nucleic acid sequence encoding its corresponding differentially expressed protein of any one of the protein markers listed in any of Tables 7, 8, and 12-14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14 can be deleted, so as to prevent expression of the differentially expressed protein.
  • Methods for targeted deletion of cellular sequences 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).
  • a method of treating breast cancer comprises treating a subject with a suitable reagent to knockdown or inhibit expression of a gene, a fragment thereof, a complement thereof, or a combination thereof encoding the mRNA disclosed in sequences encoding any one or more of the corresponding differentially expressed proteins listed in Tables 7, 8, and 12-14, or a fragment thereof and combinations of protein biomarkers listed in Tables 1, 4, and 7-14 or a fragment thereof.
  • the present disclosure provides for the in vitro knockdown of the expression of one or more of the genes, a fragment thereof, a complement thereof, or a combination thereof encoding any one or more of the corresponding differentially expressed proteins listed in Tables 7,8, and 12- 14 and combinations of proteins thereof listed in Tables 1, 4, and 7- 14.
  • breast cancers are treated by modulating the activity or expression of sequences encoding the corresponding differentially expressed gene products or fragments thereof to any one or more of the protein biomarkers in Tables 7, 8, and 12- 14 and combinations of proteins thereof listed in Tables 1, 4, and 7- 14.
  • 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 differentially expressed protein that is expressed on a cell surface.
  • the antibody binds to an extracellular domain of the differentially expressed protein.
  • the antibody binds to a differentially expressed 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.
  • the antibody is linked to a therapeutic agent or a toxin.
  • implementation of an immunotherapy strategy for treating, reducing the symptoms of, or preventing cancer or neoplasms, may be achieved using many different techniques available to the skilled artisan.
  • 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.
  • 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
  • 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.
  • a method of identifying an anti-cancer agent comprises contacting a candidate agent with a sample; and determining the cancer associated sequence's activity in the sample.
  • 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.
  • the candidate agent reduces the expression level of one or more differentially expressed proteins disclosed infra.
  • the candidate agent is an antibody.
  • the method comprises contacting a candidate antibody that binds to the differentially expressed protein within a sample, and assaying for the differentially expressed protein's activity and/or level, wherein the candidate antibody is identified as an anti-cancer agent if the activity and/or level of the differentially expressed protein is reduced in the sample after the contacting.
  • a differentially expressed protein's activity and/or level can be any activity and/or level of the differentially expressed protein.
  • An example of an activity may include enzymatic activity either of the differentially expressed protein itself or of an enzyme that interacts with or is modulated by the differentially expressed protein either at the nucleic acid level or the protein level.
  • 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 differentially expressed protein, wherein the candidate agent is identified as an anti-cancer agent if the differentially expressed protein's activity and/or level is reduced in the cell sample after the contacting.
  • an anti-cancer e.g. breast cancer
  • 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 differentially expressed nucleic acid sequence (or a fragment thereof, a complement thereof, or combination thereof) encoding the corresponding differentially expressed protein or fragment thereof selected from any one of the panels of protein biomarkers in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14, and assaying for the differentially expressed protein's activity or expression level, wherein the candidate agent is identified as an anti-cancer agent if the differentially expressed protein's activity or expressed level is modulated in the cell sample after the contacting.
  • a candidate agent that binds to a differentially expressed nucleic acid sequence (or a fragment thereof, a complement thereof, or combination thereof) encoding the corresponding differentially expressed protein or fragment thereof selected from any one of the panels of protein biomarkers in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14,
  • a method of screening drug candidates includes comparing the level of expression of the differentially expressed protein in the absence of the drug candidate to the level of expression in the presence of the drug candidate.
  • Some embodiments are directed to a method of screening for a therapeutic agent capable of binding to a differentially expressed sequence (nucleic acid or protein), the method comprising combining the differentially expressed sequence and a candidate therapeutic agent, and determining the binding of the candidate agent to the differentially expressed sequence.
  • the method comprises combining the differentially expressed sequence and a candidate therapeutic agent, and determining the effect of the candidate agent on the bioactivity of the differentially expressed sequence.
  • An agent that modulates the bioactivity of a differentially expressed sequence may be used as a therapeutic agent capable of modulating the activity of a differentially expressed nucleic acid sequence.
  • the present disclosure provides a method of screening for anticancer activity comprising: (a) contacting a cell that expresses a differentially expressed gene, homologues thereof, combinations thereof, or fragments thereof encoding the corresponding differentially expressed gene product or fragment thereof selected from any one of the protein biomarkers in Tables 7, 8, and 12-14 and combinations of protein biomarkers listed in Tables 1, 4, and 7- 14,with an anticancer drug candidate; (b) detecting an effect of the anticancer drug candidate on an expression of the differentially expressed 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 differentially expressed gene indicates that the candidate has anticancer activity.
  • the drug candidate may lower the expression level of the differentially expressed sequence in the cell encoding for a differentially expressed protein whose level can be detected either intracellular and/or extracellular.
  • 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 differentially expressed gene products or fragments thereof of the protein biomarkers in Tables 7, 8, and 12- 14 and combinations of protein biomarkers listed in Tables 1 , 4, and 7- 14.
  • the candidate cancer drug is said to be effective.
  • the present disclosure provides a method of screening for anticancer activity comprising: (a) providing a cell that expresses a differentially expressed gene (or a fragment thereof or a complement thereof) having a nucleic acid sequence encoding the corresponding differentially expressed gene product from the group consisting of the differentially expressed proteins chosen from proteins corresponding to any one or more of the differentially expressed proteins in Tables 7, 8, and 12- 14 and combinations of differentially expressed proteins from within Tables 1, 4 and 7- 14, (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 differentially expressed protein 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.
  • 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.
  • the candidate modulates (e.g. , inhibits) the expression of the differentially expressed sequence (nucleic acid or protein)
  • the candidate in which the candidate modulates (e.g. , inhibits) the expression of the differentially expressed sequence (nucleic acid or protein), the candidate is said to have anticancer activity.
  • the anticancer activity is determined by measuring cell growth.
  • the candidate inhibits or retards cell growth and is said to have anticancer activity.
  • the candidate causes the cell to die, and thus, the candidate is said to have anticancer activity.
  • the present disclosure provides a method of screening for activity against breast cancer.
  • the method comprises contacting a cell that overexpresses a differentially expressed gene, homologues thereof, combinations thereof, or fragments thereof, which can be complementary to a differentially expressed nucleic acid sequence encoding the corresponding differentially expressed gene product or fragment thereof selected from any one of the protein biomarkers in Tables 7, 8, and 12- 14 and combinations of differentially expressed proteins listed in Tables 1 , 4 and 7- 14, with a breast cancer drug candidate.
  • the method comprises detecting an effect of the breast cancer drug candidate on an expression of the polynucleotide encoding the differentially expressed protein in the cell, extracellularly 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 polynucleotide encoding the differentially expressed protein, on cell growth, or viability indicates that the candidate has activity against a breast cancer cell that differentially expresses (e.g., overexpresses) a gene encoding a differentially expressed protein, wherein the protein can be a cancer associated protein, a protein that is not unique to a cancer or a protein that can be a unique cancer specific protein, wherein said gene is selected from any one or more of the genes,, complements thereof, homologues thereof, combinations thereof, or fragments thereof encoding the
  • 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, and the like.
  • the present disclosure can be used in methods to determine if a breast cancer treatment method has been successful. Success can be seen as an inhibitory effect on the growth and/or metastasis of breast cancer cells.
  • the disclosed panels can be used to assess efficacy.
  • Efficacy can be substantiated when a treatment methodology results in arresting an increase and/or achieves a decline in the number of breast cancer cells.
  • the treatment methodology's efficacy can assist an oncologist, the breast cancer patient and other health care practitioners to evaluate if the treatment methodology should be continued, alter the dosage level of the therapeutic (increase or decrease), substituted with an alternative therapeutic or treatment methodology, or stopped due to success in treating a breast cancer 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.
  • Efficacy can be assessed by comparing the differentially expressed biomarker proteins' levels using one or more of the protein biomarker panels listed in Tables 7, 8 and 12- 14 as well as combinations of the biomarker proteins listed in Tables 1, 4, and 7- 14.
  • the comparison can be based on the differentially expressed biomarker proteins' levels optionally determined prior to initiation of a treatment methodology, a first sample time point, and at a second sample time point (e.g., about 1 week, about 2 weeks, about 3 weeks, and about 4 weeks and so on) after initiation of the treatment methodology.
  • a second sample time point e.g., about 1 week, about 2 weeks, about 3 weeks, and about 4 weeks and so on
  • comparison of biomarker protein panel(s) of differentially expressed proteins' levels can be evaluated between patient samples collected prior to (optionally) and during the treatment relative to a control sample from the patient undergoing treatment using a noncancerous tissue sample or to results for a non-cancerous reference sample.
  • a method of monitoring efficacy of a cancer treatment methodology comprises determining protein levels of differentially expressed biomarker proteins from one or more biomarker protein panels listed in Tables 7, 8 and 12-14 as well as combinations of the biomarker proteins listed in Tables 1, 4, and 7-14.
  • the differentially expressed biomarker proteins' levels can exhibit either an increase or decrease relative to a non-cancerous control sample (patient or reference) when compared to patient's levels prior to initiation and levels during cancer treatment.
  • the cancer treatment may be any cancer treatment or one that specifically inhibits the action of a differentially expressed biomarker protein as discussed supra.
  • the treatment methodology can comprises a treatment methodology selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, genetic editing by silencing, knockdown, inhibition, mutation or deletion of one or more nucleic acid sequences encoding differentially expressed biomarker proteins described herein, antibody interference methods, and combinations thereof.
  • the treatment can further include surgical removal, resection and/or amputation of a cancerous cyst, tissue, tumor, or neoplasm. In some embodiments, therefore, a sample is obtained from the patient and tested for the level of differentially expressed biomarker proteins as described herein.
  • the cancer treatment methodology shows a lack of efficacy and the treatment methodology can be changed.
  • the cancer treatment methodology can be repeated to verify if there is a lack or gradual evidence of treatment efficacy or if the treatment dose should be increased in order to achieve efficacy.
  • the cancer treatment methodology demonstrates efficacy.
  • the protein biomarkers' protein expression levels that have been measured in the panels of Tables 7, 8 and 12-14 and combinations of protein biomarkers from any of Tables 1, 4 and 4- 14 can have either all elevated or all decreased levels of expression when comparisons of protein expression levels were done between non-cancerous samples and individuals' samples with an unknown cancer detection determination.
  • the protein biomarkers' protein expression levels that have been measured within the panels of Tables 7, 8 and 12- 14 and combinations of protein biomarkers from any of Tables 1, 4 and 4- 14 have indicated that three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 11 or more, 12 or more, 13 or more, and 14 or more measured protein levels were either elevated or decreased in level of expression when comparisons of protein expression levels were done between non-cancerous samples and individuals' samples with an unknown cancer detection determination.
  • 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.
  • some embodiments herein provide for polynucleotide and polypeptide sequences involved in cancer and, in particular, in oncogenesis.
  • 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.
  • Some embodiments of the present disclosure are directed to cancer associated sequences ("target markers", either nucleic acid or protein). 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 three categories of cell types: (1) nucleated blood cells including but not limited to CD34+ cells and CD133+ cells; (2) normal mortal somatic adult-derived tissues and cultured cells including: skin fibroblasts, vascular endothelial cells, normal non-lymphoid and noncancerous tissues, and the like, and (3) malignant cancer cells including cultured cancer cell lines or human tumor tissue.
  • target markers either nucleic acid or protein.
  • mRNAs, miRNAs, or proteins that are generally expressed (or not expressed) in categories 1, and 3 but not expressed (or expressed) in category 2 are candidate targets for cancer diagnosis and therapy.
  • Each of the cell types can be found in a bodily fluid.
  • cancer-associated sequences i.e. , cancer- associated markers, cancer-associated genes and the corresponding encoded cancer-associate polypeptides
  • Another method of identifying cancer-associated sequences is to compare gene expression in cancerous cells to gene expression in noncancerous 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 and differentially expressed protein markers listed in Tables 7, 8, and 12- 14 as well as additional protein biomarkers listed in Tables 1, 4, and 9- 11.
  • the gene expression 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.
  • the expression data that can be used to detect or diagnose a subject with cancer can be obtained experimentally.
  • 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 protein markers listed in Tables 1, 4, and 7-14, 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, and as is known to one of skill in the art.
  • 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.
  • 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.
  • 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 (nucleic acid or protein). Exemplary techniques are provided below.
  • 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.
  • 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.
  • 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).
  • 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' s RNEasy kits to isolate total RNA or Ambion's mirVana kits to isolate RNA enriched for small RNA species.
  • 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.
  • 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 real-time TaqMan ® assays. 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 15 pg/cell.
  • the reverse transcription reaction may be performed using lx cDNA archiving buffer, 3.35 units MMLV reverse transcriptase, 5 mM 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 ⁇ .
  • 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.
  • cDNA samples and cellular total RNA may be subjected to the One-Cycle Target Labeling procedure for bio tin labeling by in vitro transcription (IVT) (Affymetrix, Santa Clara, CA) or using the Illumina Total Prep RNA Labelling kit (Illumina, San Diego, CA).
  • IVT in vitro transcription
  • 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.
  • labeled cRNA may be hybridized according to the manufacturer's instructions.
  • 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 samp les)/( 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.
  • the unsupervised clustering and class neighbor analyses of the microarray data from cells may be performed using GenePattern software (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.
  • 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 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 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.
  • antigen presenting cells 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.
  • 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.
  • the present disclosure provides a method of eliciting an antibody response to one or more of the differentially expressed proteins disclosed infra.
  • the method may comprise administering the differentially expressed protein or a peptide fragment or an inhibitor of the protein(s) identified from the UniProt number for each protein listed in Tables 1, 2, and 4-14 for the designated protein bio markers listed in Tables 1, 4, and 7, -14, disclosed infra to a subject.
  • Some embodiments are directed to the use of cancer associated and/or differentially expressed polypeptides and polynucleotides encoding a cancer associated and/or differentially expressed 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 and/or differentially expressed polypeptide sequence, such as, without limitation, cancer cells, in a subject.
  • antigen presenting cells such as, without limitation, dendritic cells
  • 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).
  • 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.
  • the pulsed or expressing DCs may be used to activate T lymphocytes ex vivo.
  • the cancer associated sequence is contacted with a subject to stimulate an immune response.
  • the immune response is a therapeutic immune response so as to treat a subject as described infra.
  • the immune response is a prophylactic immune response.
  • 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.
  • a DNA molecule e.g. DNA vaccine
  • RNA molecule e.g. DNA vaccine
  • 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 the encoded peptide fragments on the cell surface.
  • the cancer associated sequence and/or the differentially expressed protein comprises a sequence of a naturally occurring protein. In some embodiments, the cancer associate sequence and/or the differentially expressed protein does not comprise a naturally occurring sequence.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • CTL cytotoxic T- lymphocyte
  • the DCs 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.
  • the cells may be administered in any suitable manner.
  • the cell may be administered with a pharmaceutically acceptable carrier (e.g., saline).
  • the cells may be administered through intravenous, intraarticular, 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).
  • 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.
  • 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).
  • 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.
  • an immunogenic peptide sequence may have from about 8 to about 12 amino acids.
  • 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.
  • 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.
  • the cells 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.
  • 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.
  • 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.
  • the present disclosure provides methods of eliciting an immune response against cells expressing a differentially expressed protein disclosed infra comprising contacting a subject with a differentially expressed protein under conditions effective to elicit an immune response in the subject, wherein said differentially expressed protein comprises a sequence or fragment thereof of a protein selected from one or more of the protein listed in Tables 1, 4, and 7-14 as disclosed herein.
  • Cells may be transfected with one or more of the nucleic acid sequences encoding the differentially expressed proteins disclosed herein. Transfected cells may be useful in screening assays, diagnosis and detection assays. Transfected cells expressing one or more nucleic acid sequences encoding the differentially expressed proteins as disclosed herein may be used to obtain isolated proteins or peptide fragments encoded by one or more of the nucleic acid sequences encoding the differentially expressed proteins.
  • Electroporation may be used to introduce the nucleic acid sequences encoding the differentially expressed proteins 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 the differentially expressed 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.
  • 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.
  • 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.
  • proteins and small peptides have the ability to transduce or travel through biological membranes independent of classical receptor-mediated or endocytosis-mediated pathways.
  • 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.
  • protein transduction domains (PTDs) from these proteins may be fused to other macromolecules, peptides or proteins such as, without limitation, a differentially expressed 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.
  • liposomes may be used as vehicles to deliver oligonucleotides, DNA (gene) constructs and small drug molecules into cells.
  • 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.
  • 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.
  • composition and/or mixture of cationic lipids used can be altered, depending upon the macromolecule of interest and the cell type used.
  • the cationic liposome strategy has also been applied successfully to protein delivery. 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 and systems for practicing the subject methods are provided by the disclosed invention, such components configured to diagnose cancer in a subject, detect cancer in a subject, treat cancer in a subject, detect cancer in a sample, evaluate therapeutics in e.g., in vitro or in vivo cancer cells or tumors, monitor therapeutic efficacy 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.
  • 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, fragment or a complement thereof, encoding a protein chosen from any one or more of the protein listed in Tables 1, 4, and 7-14.
  • the invention provides an electronic library comprising a differentially expressed protein, a nucleic acid encoding a differentially expressed protein, or fragment(s) thereof, disclosed infra.
  • the kit may include one or more capture reagents or specific binding partners of one or more differentially expressed proteins disclosed infra.
  • the present disclosure provides a kit for diagnosing the presence of cancer in a test sample, said kit comprising at least one antibody, polypeptide or protein that selectively binds to a differentially expressed protein sequence chosen from any one or more of the protein listed in Tables 1, 4, and 7-14, or a fragment thereof.
  • the invention provides an electronic library comprising a differentially expressed polynucleotide or fragment thereof, encoding a differentially expressed polypeptide, disclosed infra.
  • the kit may include one or more capture reagents or specific binding partners of one or more differentially expressed proteins disclosed infra.
  • 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.
  • 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.
  • 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.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (/ ' . e. , associated with the packaging or sub-packaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc.
  • 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.
  • kits may also include one or more control samples and reagents, e.g. , two or more control samples for use in testing the kit.
  • 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 of 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.
  • the gene expression data has been used in selecting the disclosed DEP proteins.
  • the identified DEPs present numerous diagnostic test opportunities including determination and findings of breast cancer earlier than traditional clinical testing methods such as mammography, ultrasonography, surgical biopsy and magnetic resonance imaging (MRI).
  • 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. It can also be deduced that the gene expression of DEPs would also be comparable within in circulating cancer cells, tumor cells and tumorous tissues.
  • 136 patient samples were selected for identifying breast cancer markers. There were 39 malignant samples and 87 benign samples for the assessment of distinguishable differences in breast cancer biomarker expression in serum: 1) 20/39 patient samples with pathology-confirmed malignant breast cancer had been previously tested on the SOMAscan ® platform (SomaLogic, Inc., Boulder, CO) and 2) 87 patient samples 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).
  • Each of the biomarkers were assessed for their value as predictors of distinguishing between the cancerous and benign samples from each of the eleven panels.
  • 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.
  • OLink results were reported as the LOD when they were too low to measure.
  • subsets or panels of the identified markers were able to distinguish cancerous vs. benign samples, as discussed infra.
  • the differentially expressed sequences (polypeptide and nucleic acid) as disclosed in Tables 7, 8, and 12-14 can be identified by searching the UniProt website by the disclosed UniProt ID number. Links to the polypeptide, nucleic acid genomic, mRNA and CDS sequences found in UniProtKB database and other listed sequence databases as well as databases providing information regarding structure, function, names and taxonomy, variant(s), gene expression, mutation(s), and the like, can be found within its UniProt ID entry.
  • Marker screening with bootstrap partition models is a second screening method that was used. Bootstraping adds a multivariate dimension to the screening process. 25 top biomarkers were identified from three rounds of screening (each marker had an average rank ⁇ 30) are listed in Table 4. These 25 markers were then used as a candidate set for modeling. 13/25 were retained in the model shown in Table 5 (ImRspO). The AUC was 0.87 for this model. The model was then fit with bootstrap samples 250 times and the marker p-values were recorded. 12/13 had a median p-value ⁇ 1.0 (i.e., the marker was non-zero in at least 50% of the 250 models). The results are presented in Table 6. The 12 markers were then used to build a model (ImRspl) with PRDX5 dropping out leaving a model based on 11 markers (Table 7).
  • Figure 13A illustrates a visual comparison of the four ImRsp models
  • Figure 13B illustrates a visual comparison of the four ImRsp models with controls. It is noted that the controls were not used in the fitting of the models.

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Abstract

L'invention concerne des méthodes, des compositions et des trousses pour la détection et le traitement du cancer du sein.
PCT/US2018/055007 2017-10-10 2018-10-09 Méthodes et compositions pour la détection et le diagnostic du cancer du sein WO2019074920A1 (fr)

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