PATENT ABSTRACT
The present invention discloses twenty two 22 protein biomarkers of breast cancer. More specifically, the present invention discloses the identities, specificities, and uses of up to twenty two (22) protein biomarkers in blood serum for distinguishing between patients with earlier and later stages of breast cancer, patients with benign breast diseases or abnormalities, and normal individuals lacking breast abnormalities. More specifically, the present invention relates to specificities of isoforms of up to 22 protein biomarkers in blood serum for distinguishing between patients with earlier and later stages of breast cancer, patients with benign breast diseases or abnormalities, and normal individuals lacking breast abnormalities.

PATENT DESCRIPTION
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority under 35 U.S.C. §120 to pending non-provisional U.S. Ser. No. 11/635,281, filed on Dec. 7, 2006, which claims benefit of priority under 35 U.S.C. §119(e) of provisional U.S. Ser. No. 60/834,649, filed Aug. 1, 2006, now abandoned, and of provisional U.S. Ser. No. 60/754,441, filed Dec. 27, 2005, now abandoned. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    This invention relates to twenty two (22) protein biomarkers of breast cancer. More specifically, the invention relates to the differential expression of up to 22 protein biomarkers in blood serum that can be used in diagnosis, determination of disease severity, and monitoring of therapeutic response of patients with breast cancer. The method is based on the use of two-dimensional (2D) gel electrophoresis to separate the complex mixture of proteins found in blood serum, the quantitation of up to 22 identified protein spots, and statistical analysis, to distinguish between patients with early and later stages of breast cancer, patients with benign breast disease or abnormalities, and normal women, for the purpose of screening, diagnosis, for determination of disease severity, and for treatment response monitoring. 
         [0004]    2. Description of the Related Art 
         [0005]    There is an urgent need for objective diagnostic tests to detect breast cancer in its earliest stages. By the time a patient is diagnosed with breast cancer by mammography and subsequent biopsy, the patient has had the disease for an average 6-10 years (Spratt, J. S. et al. 1986,  Cancer Research  4.6, 970-974, A. Hollingsworth, personal communication Dec. 2, 2004 re Spratt et al). In addition, when mammography is the only screening tool utilized, it has to be remembered that sensitivity here is only 70% overall even with digital technology, and mammography was recently found in a major trial to have a mere 41% sensitivity when a 15-month follow-up period was used to define false-negatives. (Pisano et al. 2005,  N Engl J Med  353, 1773-1783). MRI detects breast cancer earlier, and with much greater sensitivity, than mammograms (Hollingsworth, A. B. et al. 2003,  J. OK. St. Med. Assoc.  96, Hollingsworth A. B. et al. 2004  Amer. J. Surgery  187 349-362). Genetic mutational tests (BRCA 1 and 2 genes) detect genetic disposition of breast cancer risk, but aggressive screening, usually with breast MRI, is chosen more often than preventive mastectomy by patients who tests BRCA-positive (Hollingsworth A. B. et al. 2004; Robson, M. E. et al. 2004,  JAMA  292, 1368-1370). Whereas the need for imaging of breast tumors will always be required for localization and treatment, a sensitive early detection screening test with cost comparable to mammograms is needed to justify the high cost and insurance reimbursement for auxiliary imaging with ultrasound and/or MRI. 
         [0006]    There has been a tremendous interest in the potential ability of proteomic technology to fulfill the unmet needs of effective strategies for early diagnosis of cancer (Alaiya, A. et al. 2005,  J. Proteome Res.  4: 1213-1222) with a special emphasis on cancer detection in biological fluids from patients, including ovarian cancer (Emmanuel F. Petricoin, A. M. Ardekani, B. A. Hitt et al. 2002,  Lancet  359: 572-577) and breast cancer (Paweletz C. P. et al 2001,  Dis. Markers  17: 301-307; Henry M. Kuerer, H. M. et al. 2002,  Cancer  95: 2276-2282). Proteomics is a new field of medical research wherein proteins are identified and linked to biological functions, including roles in a variety of disease states. With the completion of the mapping of the human genome, the identification of unique gene products, or proteins, has increased exponentially. In addition, molecular diagnostic testing for the presence of proteins already known to be involved in certain biological functions has progressed from research applications alone to use in disease screening and diagnosis for clinicians. However, proteomic testing for diagnostic purposes remains in its infancy. 
         [0007]    Detection of abnormalities in the genome of an individual can reveal the risk or potential risk for individuals to develop a disease. The transition from gene based risk to emergence of disease can be characterized as an expression of genomic abnormalities in the proteome. In fact, whether arising from genetic, environmental, or other factors, the appearance of abnormalities in the proteome signals the beginning of the process of cascading effects that can result in the deterioration of the health of the patient. Therefore, detection of proteomic abnormalities at an early stage is desired in order to allow for detection of disease processes either before the disease is established or in its earliest stages where treatment may be more effective. 
         [0008]    Recent progress using a novel form of mass spectrometry called surface enhanced laser desorption and ionization time of flight (SELDI-TOF) for the testing of ovarian cancer and Alzheimer&#39;s disease has led to an increased interest in proteomics as a diagnostic tool (Petrocoin, E. F. et al. 2002 . Lancet  359:572-577, Lewczuk, P. et al. 2004 . Biol. Psychiatry  55:524-530). Furthermore, proteomics has been applied to the study of breast cancer through use of 2D gel electrophoresis and image analysis to study the development and progression of breast carcinoma in patients&#39; breast ductal fluid specimens (Kuerer, H. M. et al. 2002 . Cancer  95:2276-2282) and in plasma (Goufman, et al. 2006 . Biochemistry  2006, 71(4):354-60). In the case of breast cancer, breast ductal fluid specimens were used to identify distinct protein expression patterns in bilateral matched pair ductal fluid samples of women with unilateral invasive breast carcinoma (Kuerer, H. M. et al. 2002). 
         [0009]    Detection of biomarkers is an active field of research. For example, U.S. Pat. No. 5,958,785 discloses a biomarker for detecting long-term or chronic alcohol consumption. The biomarker disclosed is a single biomarker and is identified as an alcohol-specific ethanol glycoconjugate. U.S. Pat. No. 6,124,108 discloses a biomarker for mustard chemical injury. The biomarker is a specific protein band detected through gel electrophoresis and the patent describes use of the biomarker to raise protective antibodies or in a kit to identify the presence or absence of the biomarker in individuals who may have been exposed to mustard poisoning. U.S. Pat. No. 6,326,209 B1 discloses measurement of total urinary 17 ketosteroid-sulfates as biomarkers of biological age. U.S. Pat. No. 6,693,177 B1 discloses a process for preparation of a single biomarker specific for O-acetylated sialic acid and useful for diagnosis and outcome monitoring in patients with lymphoblastic leukemia. 
         [0010]    Two-dimensional (2D) gel electrophoresis has been used in research laboratories for biomarker discovery since the 1970&#39;s (Margolis J. et al. 1969,  Nature.  1969 221: 1056-1057; Orrick, L. R. et al. 1973 ; Proc Nat&#39;l Acad. Sci. USA.  70: 1316-1320; Goldknopf, I. L. et al. 1975,  J Biol Chem.  250: 7182-7187; Goldknopf, I. L. et al. 1977 , Proc Nat&#39;l Acad Sci USA.  74: 5492-5495; O′Farrell, P. H. 1975,  J. Biol. Chem.  250: 4007-4021; Anderson, L. 1977 , Proc Nat&#39;l Acad Sci USA.  74: 864-868; Klose, J. 1975,  Human Genetic.  26: 231-243). The advent of much faster identification of proteins spots by in-gel digestion and mass spectroscopy ushered in the accelerated development of proteomic science through large-scale application of these techniques (Aebersold R. 2003,  Nature,  422: 198-207; Kuruma, H. et al. 2004 , Prostate Cancer and Prostatic Disease  1: 1-8; Kuncewicz, T. et al. 2003,  Molecular  &amp;  Cellular Proteomics  2: 156-163). With the advent of bioinformatics, progression of proteomics towards diagnostics and personalized medicine has become feasible (White, C. N. et al. 2004  Clinical Biochemistry,  37: 636-641; Anderson N. L. et al. 2002,  Molecular  &amp;  Cellular Proteomics  1:845-867). Clinical proteomics is maturing fast into a powerful approach for comprehensive analyses of disease mechanisms and disease markers (Kuruma, H. et al. 2004; Sheta, E. A. et al. 2006 , Expert Rev. Proteomics  3: 45-62). We have recently applied 2D gel proteomics of human serum combined with discriminant biostatistics to the differential diagnosis of neurodegenerative diseases (Goldknopf, I. L. et al. 2006,  Biochem. Biophys. Res. Commun.  342: 1034-1039; Sheta, E. A. et al. 2006). In the present invention, we use the same approach to monitor the concentrations of 22 protein biomarkers, resolved and quantitated by 2D gel electrophoresis of blood serum, to distinguish between patients who have been diagnosed with earlier and later stages of breast cancer, with benign breast disease, and with no breast abnormalities as normal controls. 
       SUMMARY OF THE INVENTION 
       [0011]    The present invention relates to 22 protein biomarkers in blood serum for screening, diagnosis, determination of disease severity, and monitoring response to treatment, of breast cancer. More specifically, the present invention consists of up to 22 protein biomarkers in blood and their use in diagnostic assays for differentiating between patients with earlier and later stages of breast cancer, patients having benign breast disease or abnormalities, and normal individuals. The method comprises collecting a biological sample from patients having biopsy confirmed and histological staged breast cancer, patients having benign breast disease or abnormalities, and patients having no evidence of breast disease or breast abnormality, then determining the concentrations of up to 22 protein biomarkers identified as related to breast cancer. Patients are then sorted into these respective groupings based on a statistical analysis of the concentration in blood serum of up to 22 protein biomarkers. 
         [0012]    One aspect of the present invention is the use of up to 22 biomarkers for screening a patient for breast cancer. The method includes: collecting a biological sample from a patient, determining the concentrations of up to 22 protein biomarkers identified as related to breast cancer, and determining whether or not the patient has breast cancer, based on a statistical analysis of the concentration in blood serum of one or more of the selected 22 protein biomarkers. This aspect of the invention can be used as an early blood screen in patients to complement mammography, such that a negative mammogram but a positive blood test would signal the need for more sensitive imaging such as breast MRI. In the case of an equivocal mammogram, the predictive power of a blood test would help the radiologist to decide whether or not to proceed with biopsy. 
         [0013]    Another aspect of the present invention is the use of up to 22 protein biomarkers for determining the severity of breast cancer and/or monitoring the response to treatment of a patient. The method includes: collecting a biological sample from a patient, determining the concentrations of up to 22 protein biomarkers identified as related to breast cancer, and determining the severity of breast cancer and/or response of the patient to treatment based on the concentrations in blood serum of up to 22 protein biomarkers. For example, this aspect of the invention can be used to help the oncologist make decisions about specific chemotherapeutic and/or anti-hormonal regimens, and/or therapeutic antibodies and/or other therapeutic agents and regimens, and to monitor the response of the patient to treatment. 
         [0014]    Another aspect of the present invention is the use of up to 22 biomarkers for determining the biological mechanism of disease of a patient and/or the drug target of the patient for treatment of breast cancer. The method includes: collecting a biological sample from a patient, determining the concentrations of up to 22 protein biomarkers identified as related to breast cancer, and determining the mechanism of disease active in the patient and/or identifying the drug target appropriate for treatment of the patient, based on the concentration in blood serum of up to 22 protein biomarkers. 
         [0015]    The foregoing has outlined rather broadly several aspects of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and the specific embodiments disclosed might be readily utilized as a basis for modifying or redesigning the methods for carrying out the same purposes as the invention. It should be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]    For a more complete understanding of the present invention, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which: 
           [0017]      FIG. 1 : Representative 2D gel electrophoretic image of human serum proteins with the positions of 4 of the 22 protein biomarker spots, the electrophoretic isoforms of the Inter-alpha-trypsin inhibitor heavy chain (H4) related 35 KD (ITI (H4) RP 35 KD) protein spots B2422, B2505, B3410, and B4404, indicated by arrows, circles and numbers. 
           [0018]      FIGS. 2A-2D : Statistical box and whiskers plots (constructed using Analyze-it software for Microsoft XL) of blood serum concentrations (2D gel spot density, PPM) of the four electrophoretic isoforms of the Inter-alpha-trypsin inhibitor heavy chain (H4) related 35 KD (ITI (H4) RP 35 KD) protein spots:  FIG. 2A : B2422;  FIG. 2B : B2505;  FIG. 2C : B3410; and  FIG. 2D : B4404, as depicted in  FIG. 1 , from patients with breast cancer (BC), benign breast abnormalities or disease (B9), and normal controls subjects (N). B2505 is up-regulated in breast cancer and B2422, B3410 and B4404 are down-regulated in breast cancer. Summary statistics are illustrated in Table XXXIII a-d. 
           [0019]      FIGS. 3A-3D : Statistical box and whiskers plots and Receiver Operator Characteristics (ROC) plot (constructed using Analyze-it software for Microsoft XL) of blood serum concentrations of the sum of the four electrophoretic isoforms of the biomarker Inter-α-Trypsin Heavy Chain Related (H4) Protein, 35 KD, processing product (ITI (H4) RP 35 KD), corresponding to the sum of biomarker spots (B2422+B2505+B3410+B4404) in normal control subjects (N), patients with benign breast abnormalities or disease (B9), and breast cancer patients (BC), expressed both as:  FIG. 3A ,  FIG. 3B : concentration=2D gel spot density (PPM); and as  FIG. 3C ,  FIG. 3D ]: differential expression from normal=fold of average normal 2D gel spot density (PPM) (i.e. Normalized to the average of the normal concentrations). Values for retrospective and prospective samples determined separately and then combined for statistical analysis. Summary statistics are depicted: for  FIGS. 3A ,  3 B in Table XXXIII e and Table XXXIV a; and for  FIGS. 3C ,  3 D in Table XXXIV b. 
           [0020]      FIGS. 4A-4D : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations (as fold of average normal blood serum concentration (2D gel spot density PPM) of the four electrophoretic isoforms of Inter-α-Trypsin Heavy Chain (H4) Related 35 KD protein spots:  FIG. 4A : B2422;  FIG. 4B : B2505;  FIG. 4C : B3410; and  FIG. 4D : B4404, in normal control subjects (N), patients with benign breast abnormalities or disease (B9), combined non-breast cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC). Summary statistics are depicted in Table XXXV. 
           [0021]      FIGS. 5A-5D : Receiver Operator Characteristics (ROC) of the patients in  FIGS. 4A-4D , including sensitivities and specificities of diagnosis based on the individual performances of the four electrophoretic isoforms of Inter-α-Trypsin Heavy Chain (H4) Related 35 KD protein spots:  FIG. 5A : B2422;  FIG. 5B : B2505;  FIG. 5C : B3410; and  FIG. 5D : B4404. 
           [0022]      FIG. 6 : A representative 2D gel electrophoretic image of human serum proteins with the positions of the 22 protein biomarker spots: B1322; B1418; B2317; B2422; B2505; B3406; B3410; B4404; B5539; B6519; B6605; B7408; B1512; B2412; B4008; B4206; B3506; B4414; B5713; B6014; B6218; and B7108, indicated by arrows, circles and numbers. 
           [0023]      FIGS. 7A-7B : illustrates:  FIG. 7A : the estimation of the molecular weights (MW) of protein biomarker spots: B1322; B1418; B2317; B2422; B2505; B3406; B3410; B4404; B5539; B6519; B6605; B7408; B1512; B2412; B4008; B4206; B3506; B4414; B5713; B6014; B6218; and B7108, by 2D gel electrophoresis (relative migration in the SDS second dimension) employing protein standards of known molecular weights; and  FIG. 7B : the estimation of isoelectric points of protein biomarker spots: B1322; B1418; B2317; B2422; B2505; B3406; B3410; B4404; B5539; B6519; B6605; B7408; B1512; B2412; B4008; B4206; B3506; B4414; B5713; B6014; B6218; and B7108, by 2D gel electrophoresis (relative focusing position in the isoelectric focusing first dimension between the extremes of the pH gradient, pH 5-8). Summary data are depicted in Tables III-V. 
           [0024]      FIGS. 8A-8B  (Sheet 11/38):  FIG. 8A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations (as fold of average normal blood serum concentration (2D gel spot density, PPM) of immunoglobulin lambda (λ) light chain spot B1322, in normal control subjects, patients with benign breast abnormalities, combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 8B : Receiver Operator Characteristics (ROC) of immunoglobulin lambda (λ) light chain spot B1322 for the patients in  FIGS. 8A-8B , including sensitivities and specificities of diagnosis for differentiation between N vs. B9; N vs. Non-DCIS BC; and N vs. DCIS BC. Summary statistics are depicted in Table XXXVI. 
           [0025]      FIGS. 9A-9B :  FIG. 9A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations (as fold of average normal blood serum concentration (2D gel spot density, PPM) of alpha-1-microglobulin protein spot B1418 in normal control subjects, patients with benign breast abnormalities, combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 9B : Receiver Operator Characteristics (ROC) of alpha-1-microglobulin protein spot B1418 with sensitivities and specificities of diagnosis for differentiation of N vs. DCIS BC; N vs. Non-DCIS BC; and N vs. combined BC. Summary statistics are depicted in Table XXXVII. 
           [0026]      FIGS. 10A-10B :  FIG. 10A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations (as fold of average normal blood serum concentration (fold of 2D gel spot density, PPM) of Apolipoprotein A-I protein spot B2317, in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 10B : Receiver Operator Characteristics (ROC) of Apolipoprotein A-I protein spot B2317 with sensitivities and specificities of diagnosis for distinguishing DCIS BC vs. N; DCIS BC vs. B9; B9 vs. N; Non-DCIA BC vs. B9; Non-DCIS BC vs. N; and Combined BC vs. N+B9. Summary statistics are depicted in Table XXXVIII. 
           [0027]      FIGS. 11A-11B :  FIG. 11A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations (as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Apolipoprotein E3 protein spot B3406 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 11B : Receiver Operator Characteristics (ROC) of Apolipoprotein E3 protein spot B3406 with sensitivities and specificities of diagnosis for distinguishing DCIS BC vs. N+B9. Summary statistics are depicted in Table XXXIX. 
           [0028]      FIGS. 12A-12B :  FIG. 12A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations (as fold of average normal blood serum concentration (concentration=normal 2D gel spot density, PPM) of Serum Albumin protein spot B5539 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 12B : Receiver Operator Characteristics (ROC) of Serum Albumin protein spot B5539, with sensitivities and specificities of diagnosis for distinguishing Non-DCIS BC vs. N+B9. Summary statistics are depicted in Table XL. 
           [0029]      FIGS. 13A-13B :  FIG. 13A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations (as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Lectin P35 protein spot B6519 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 13B : Receiver Operator Characteristics (ROC) of Lectin P35 protein spot B6519, with sensitivities and specificities of diagnosis for distinguishing Combined BC vs. N. Summary statistics are depicted in Table XLIX. 
           [0030]      FIGS. 14A-14B :  FIG. 14A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations (as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Transferrin protein spot B6605 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 14B : Receiver Operator Characteristics (ROC) of Transferrin protein spot B6605, with sensitivities and specificities of diagnosis for distinguishing B9 vs. N and DCIS BC vs. N. Summary statistics are depicted in Table XLI. 
           [0031]      FIGS. 15A-15B :  FIG. 15A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Complement C4A protein spot B7408 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 15B : Receiver Operator Characteristics (ROC) of Complement C4A protein spot B7408, with sensitivities and specificities of diagnosis for distinguishing DCIS BC vs. N, B9 vs. N, and for not distinguishing Non-DCIS BC vs. N. Summary statistics are depicted in Table L. 
           [0032]      FIGS. 16A-16D :  FIG. 16A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Haptoglobin protein spot B1512 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 16B-16D : Receiver Operator Characteristics (ROC) of Haptoglobin protein spot B1512, with sensitivities and specificities of diagnosis for distinguishing Non-DCIS BC vs. N, DCIS BC vs. B9 vs. N, vs. N+B9 vs. Combined BC. Summary statistics are depicted in Table XLIII. 
           [0033]      FIGS. 17A-17B :  FIG. 17A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations (as fold of average normal blood serum concentration (2D gel spot density, PPM) of Apoptosis Inhibitor CD5L protein spot B2412 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 17B : Receiver Operator Characteristics (ROC) of Apoptosis Inhibitor CD5L protein spot B2412, with sensitivities and specificities of diagnosis for distinguishing Combined BC vs. N+B9. Summary statistics are depicted in Table LI. 
           [0034]      FIGS. 18A-18B :  FIG. 18A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations (as fold of average normal blood serum concentration (2D gel spot density, PPM) of Haptoglobin protein spot B4008 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 18B : Receiver Operator Characteristics (ROC) of Haptoglobin protein spot B4008, with sensitivities and specificities of diagnosis for distinguishing Combined BC vs. N. Summary statistics are depicted in Table XLV. 
           [0035]      FIGS. 19A-19B :  FIG. 19A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Haptoglobin protein spot B4206 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 19B : Receiver Operator Characteristics (ROC) of Haptoglobin protein spot B4206, with sensitivities and specificities of diagnosis for distinguishing DCIS BC vs. N. Summary statistics are depicted in Table XLVI. 
           [0036]      FIGS. 20A-20B :  FIG. 20A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Haptoglobin Related Protein spot B4424 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 20B : Receiver Operator Characteristics (ROC) of Haptoglobin Related Protein spot B4424, with lack of sensitivity and specificity of diagnosis for distinguishing N+B9 vs. BC. 
           [0037]    Summary statistics are depicted in Table XLVIII. 
           [0038]      FIGS. 21A-21B :  FIG. 21A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Haptoglobin Related Protein spot B3506 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 21B : Receiver Operator Characteristics (ROC) of Haptoglobin Related Protein spot B3506, with lack of sensitivity and specificity of diagnosis for distinguishing N+B9 vs. Combined BC. Summary statistics are depicted in Table XLVII. 
           [0039]      FIGS. 22A-22B :  FIG. 22A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Serotransferrin protein spot B5713 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 22B : Receiver Operator Characteristics (ROC) of Serotransferrin protein spot B5713, with sensitivities and specificities of diagnosis for distinguishing Combined BC vs. Normal. Summary statistics are depicted in Table XLII. 
           [0040]      FIGS. 23A-23C :  FIG. 23A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Haptoglobin protein spot B6014 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC) wherein  FIG. 23B : 62.2% of the Non-DCIS BC patients have detectable levels of Haptoglobin protein spot B6014 as compared to 32.3% of N+B9 patients and 33.3% of DCIS BC patients, and  FIG. 23C : Receiver Operator Characteristics (ROC) with sensitivities and specificities of diagnosis for distinguishing Non-DCIS BC vs. N+B9 and Non-DCIS BC vs. DCIS BC based on detection of Haptoglobin protein spot B6014. Summary statistics are depicted in Table XLIV. 
           [0041]      FIGS. 24A-24B :  FIG. 24A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of Ribosomal and Nucleolar protein L27a protein spot B6218 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIG. 24B : Receiver Operator Characteristics (ROC) of Ribosomal and Nucleolar protein L27a spot B6218, with sensitivities and specificities of diagnosis for distinguishing DCIS BC vs. N+B9 and Non-DCIS BC vs. N+B9. Summary statistics are depicted in Table LII. 
           [0042]      FIGS. 25A-25D :  FIG. 25A : Statistical box and whiskers plots of the differential expression from normal of the blood serum concentrations as fold of average normal blood serum concentration (concentration=2D gel spot density, PPM) of NSB protein spot B7108 in normal control subjects (N), patients with benign breast abnormalities (B9), combined non-cancer controls (N+B9), combined breast cancer patients (DCIS BC+Non-DCIS BC); “purely invasive” breast cancer without in-situ breast cancer (Non-DCIS BC) and breast cancer patients with in-situ breast cancer (DCIS BC); and  FIGS. 25B-25D : Receiver Operator Characteristics (ROC) of NSB protein spot B7108, with sensitivities and specificities of diagnosis for distinguishing N vs. B9 vs. N+B9 vs. Combined BC vs. DCIS BC vs. Non-DCIS BC. Summary statistics are depicted in Table LIII. 
           [0043]      FIGS. 26A-26B : Median differential expression profiles of blood serum concentrations of:  FIG. 26A : the 22 breast cancer biomarkers; and  FIG. 26B : 4 isoforms of the ITI (H4) RP 35 KD protein (protein spots B2505, B2422, B4404, B3410); 4 isoforms of a Haptoglobin protein (protein spots B6014, B1512; B4008, B4206); and 2 isoforms of a Haptoglobin related protein (B3506, B4424), as median fold of average mean spot concentration (concentration=2D gel spot density, PPM) for N, B9, DCIS-BC and Non-DCIS BC. Summary statistics are depicted in Table LIV. 
           [0044]      FIGS. 27A-27B : Illustration of Gold Standard Immunoassay Procedure showing the derivation of antigens for immunization ( FIG. 27A ) and immunization and antibody specificity using an ELISA employing monoclonal antibodies developed to recognize either: Both the ITI (H4) RP 35 KD protein and the PK120 precursor, using recombinant protein as immunogenic agent and protein standard (Antigen 1), or only the ITI (H4) RP 35 KD and not recognizing the PK120 precursor, using a synthetic peptide as immunogenic agent (Antigen 3), and using a recombinant protein with the his-tag removed from the amino-terminus, as protein standard (Antigen 2). 
           [0045]      FIGS. 28A-28B   FIG. 28A : Box and whisker plot of μg per ml of serum of breast cancer biomarkers as determined by ELISA employing monoclonal antibodies recognizing a) specifically the Inter-alpha-trypsin inhibitor heavy chain (H4) related 35 KD protein (Spots B2422, B2505, B3410, and B4404 depicted in  FIG. 1 ); and  FIG. 28B : The same protein as in A as well as the precursor Inter-alpha-trypsin inhibitor heavy chain (H4) related 110 KD protein. The upper and lower edges of the box are the 1 st  and 3 rd  quartile. The median, denoted as a line within the box, falls between these boundaries. The dashed line connects the nearest observation within 1.5 IQRs (inter-quartile ranges IRQ=Q3−Q1). The red crosses and circles indicate possible outliers more than 1.5 and 3.0 IQRs. The graphs were created in Microsoft Excel using Analyse-it software for Excel. Summary statistics for the graphs are illustrated in Table LVI, LVII d, e, and the Receiver Operator Characteristics (ROC) are found in Table LVIII. 
           [0046]      FIGS. 29A-29C : Quantitative 2D Gel analysis of Total ITI (H4) RP 35 KD proteins (sum of the densities of spots B2422+B2505+B3410+B4404, in 2D gels, PPM; 35 KD specific Elisa immunoassay ( FIG. 29A ), yg protein per ml of blood serum ( FIG. 29B ); and Fold of average normal of the sum of the densities ( FIG. 29C ) of spots B2422+B2505+B3410+B4404 in 2D gels) in retrospective blood serum samples. The graphs were created in Microsoft Excel using Analyse-it software for Excel. Summary statistics are illustrated in Table LVI, LVII a-c. 
           [0047]      FIGS. 30A-30E : Cross-validation of concentration of total ITI (H4) RP 35 KD proteins (Fold of average normal of the sum of the densities of spots B2422+B2505+B3410+B4404 in 2D gels) of retrospective samples ( FIG. 30A ); prospective samples ( FIG. 30B ), combined retrospective and prospective samples with non-DCIS and DCIS breast cancer in separate groups ( FIG. 30C ), and Elisa immunoassay, Fold of μg protein per ml of blood serum using the 35 KD specific ( FIG. 30D ), and the 35 KD+PK120 precursor ( FIG. 30E ) specific antibodies (Table LVII). 
           [0048]      FIGS. 31A-31F : Cross validation of quantitative 2D gel electrophoresis: Total Inter-alpha trypsin inhibitor heavy chain (H4) related 35 KD protein (Fold of average normal of the sum of the densities of spots B2422+B2505+B3410+B4404 in 2D gels): Serum samples collected in Red/Gray cap tubes with clot accelerator and polymer gel ( FIGS. 31A ,  31 D); Serum samples collected in Red cap tubes without accelerator or gel (B, C, E, F); gels stained with SyproRuby™ ( FIGS. 31A ,  31 B,  31 D,  31 E, Bio-Rad); gels stained with Lava Purple™ ( FIGS. 31C ,  31 F, Fluorotechnics) fluorescent protein stains; protein staining patterns digitized using an FX-Imager ( FIGS. 31A ,  31 D, Bio-Rad); an FLA 7000 Imager ( 31 B,  31 C,  31 E,  31 F, Fuji); PPM stain intensity ( FIGS. 31A ,  31 B,  31 C) and fold of average normal stain intensity (PPM,  FIGS. 31D ,  31 E,  31 F) Summary statistics for the graphs are found in Table LIX. 
       
    
    
       [0049]    Table I: Staging of Breast Cancer 
         [0050]    Table II: Isoelectric points (pI) and molecular weights (Da) of standard protein mixture with isoforms separated as spots on 2D gels. 
         [0051]    Table III: Molecular weights (MW) of the 22 breast cancer biomarker protein spots, based upon migration relative to the 10 KD protein standard in the SDS 2″ dimension of the 2D gel electrophoresis as depicted in  FIG. 7A . 
         [0052]    Table IV: Isoelectric points (pI) of the 22 breast cancer biomarker protein spots, based upon their relative mobility, i.e. their position between the pH 5.0 and pH 8.0 range attained by isoelectric focusing in the 1 st  dimension of the 2D gel electrophoresis as depicted in  FIG. 7B . 
         [0053]    Table V: Protein biomarker spot molecular weights (MW) and isoelectric points (pI) as determined from 2D gels ( FIGS. 7A-7B ) as compared to the values calculated from the amino acid sequences as identified by LC MS/MS of the in-gel tryptic digests of the spots (Tables VI-XXXII, SEQ ID NOS: 1-22). 
         [0054]    Table VI: The 22 Breast Cancer Biomarkers—Protein Identification by LC MSMS of 2D gel spot in-gel trypsin digests ( FIGS. 1 ,  6 , Tables VI-XXXII, SEQ ID NOS: 1-22). 
         [0055]    Table VII: Single letter amino acid sequence (SEQ ID NO:1) of Immunoglobulin Lambda Chain protein spot B1322. 
         [0056]    Table VIII: Single letter amino acid sequence (SEQ ID NO: 2) of Alpha-1-microglobulin protein spot B1418. Also shown is its placement in the single letter amino acid sequence of the precursor, which also contains the protein bikunin (SEQ ID NO: 24). 
         [0057]    Table IX: Single letter amino acid sequence (SEQ ID NO: 3) of Apolipoprotein A-I protein spot B2317. 
         [0058]    Table X: Amino acid sequence of Inter-alpha-Trypsin inhibitor heavy chain (H4) related protein (ITIHRP, PK120), the precursor to the 35 KD biomarker protein spots B2422, B2505, B3410, and B4404 (SEQ ID NO: 25). The placement of the Inter-alpha-trypsin Inhibitor Heavy Chain (H4) Related 35 KD Protein, and the corresponding 75 KD protein, are indicated within the sequence of the PK120 precursor. 
         [0059]    Table XI: Single letter amino acid sequences of isoforms 1 (SEQ ID NO: 4) and 2 (SEQ ID NO: 5) of the Inter-alpha-trypsin Inhibitor Heavy Chain (H4) Related 35 KD protein spots B2422, B2505, B3410, and B4404. 
         [0060]    Table XII: Single letter amino acid sequence alignment of the Inter-alpha-trypsin Inhibitor Heavy Chain (H4) Related 35 KD Protein Isoform 1 (SEQ ID NO: 26) and Isoform 2 (SEQ ID NO: 26). Identical sequences are marked with stars while unmatched sequences are marked by dashes. 
         [0061]    Table XIII: Single letter amino acid sequence (SEQ ID NO: 6) of Apolipoprotein E3 protein spot B3406. 
         [0062]    Table XIV: Single letter amino acid sequence (SEQ ID NO: 7) of human albumin protein spot B5539. 
         [0063]    Table XV: Single letter amino acid sequence (SEQ ID NO: 8) of human Lectin P35 3 protein spot B6519. 
         [0064]    Table XVI: Single letter amino acid sequence (SEQ ID NO: 9) of Transferrin protein spot B6605. 
         [0065]    Table XVII: Single letter amino acid sequence (SEQ ID NO: 10) of Complement C4A gamma protein spot B7408. 
         [0066]    Table XVIII: Single letter amino acid sequence of parental protein Complement C4A (SEQ ID NO: 28). 
         [0067]    Table XIX: Single letter amino acid sequence (SEQ ID NO: 11) of Haptoglobin protein spots B1512; B4008; B4206; and B6014. 
         [0068]    Table XX: Single letter amino acid sequence (SEQ ID NO: 12) of Haptoglobin-related protein spots B3506 and B4424. 
         [0069]    Table XXI: Single letter amino acid sequences of peptides identified by LC MS/MS of in-gel tryptic digests of protein spot B2412. 
         [0070]    Table XXII: Single letter amino acid sequence (SEQ ID NO: 13) of AIM protein spot B2412. 
         [0071]    Table XXIII: Single letter amino acid sequence (SEQ ID NO: 14) of CD5L protein alternate sequence of protein spot B2412. 
         [0072]    Table XXIV: Single letter amino acid sequence (SEQ ID NO: 23) of Serotransferrin protein B5713. 
         [0073]    Table XXV: Single letter amino acid sequence (SEQ ID NO: 15) of nucleolar/ribosomal protein L27a protein spot B6218. 
         [0074]    Table XXVI: Alternate single letter amino acid sequence (SEQ ID NO: 16) of nucleolar/ribosomal protein L27a protein spot B6218. 
         [0075]    Table XXVII: Alternate single letter amino acid sequence (SEQ ID NO: 17) of nucleolar/ribosomal protein L27a protein spot B6218. 
         [0076]    Table XXVIII: Single letter amino acid sequence (SEQ ID NO: 18) of Reticulon-4 precursor to protein spot B7108. 
         [0077]    Table XXIX: Single letter amino acid sequence (SEQ ID NO: 19) of Reticulon-4 protein spot B7108. 
         [0078]    Table XXX: Alternate single letter amino acid sequence (SEQ ID NO: 20) of Reticulon-4 protein spot B7108. 
         [0079]    Table XXXI: Alternate single letter amino acid sequence (SEQ ID NO: 21) of Reticulon-4 protein spot B7108. 
         [0080]    Table XXXII: Alternate single letter amino acid sequence (SEQ ID NO: 22) of Reticulon-4 protein spot B7108. 
         [0081]    Table XXXIII: Summary statistics for ITI (H4) RP 35 KD isoform electrophoretic variants ( FIG. 1 ) as depicted in graphs in  FIG. 2 , and for the sum of the isoforms ( FIG. 3A , graph, retrospective samples, N, B9, BC). 
         [0082]    Table XXXIV: Summary statistics for the Total ITI (H4) RP 35 KD proteins equal to the sum of the blood serum concentrations of protein spots B2422+B2505+B3410+B4404: a. measured as 2D gel spot density (PPM) as depicted in  FIG. 3A ; b measured as differential expression from normal as depicted in  FIG. 3B , wherein differential expression from normal=fold of average normal concentration, and wherein concentration=2D gel spot density, PPM. 
         [0083]    Table XXXV: Summary statistics of the differential expression of the Individual ITI (H4) RP 35 KD Protein Spots B2422, B2505, B3410, and B4404, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graphs in  FIGS. 4A-4D . 
         [0084]    Table XXXVI: Summary statistics of the differential expression of Immunoglobulin lambda chain protein spot B1322, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 8A . 
         [0085]    Table XXXVII: Summary statistics of the differential expression of Alpha-1-microglobulin protein spot B1418, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 9A . 
         [0086]    Table XXXVIII: Summary statistics of the differential expression of Apolipoprotein A1 protein spot B2317, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 10A . 
         [0087]    Table XXXIX: Summary statistics of the differential expression of Apolipoprotein E3 protein spot B3406, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 11A . 
         [0088]    Table XL: Summary statistics of the differential expression of Serum albumin protein spot B5539, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 12A . 
         [0089]    Table XLI: Summary statistics of the differential expression of protein Transferrin protein spot B6605, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 14A . 
         [0090]    Table XLII: Summary statistics of the differential expression of Serotransferrin protein spot B5713, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 22A . 
         [0091]    Table XLIII: Summary statistics of the differential expression of Haptoglobin protein spot B1512, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 16A . 
         [0092]    Table XLIV: Summary statistics of the differential expression of Haptoglobin protein spot B6014, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 23A . 
         [0093]    Table XLV: Summary statistics of the differential expression of Haptoglobin protein spot B4008, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 18A . 
         [0094]    Table XLVI: Summary statistics of the differential expression of Haptoglobin protein spot B4206, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 19A . 
         [0095]    Table XLVII: Summary statistics of the differential expression of Haptoglobin related protein spot B3506, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 21A . 
         [0096]    Table XLVIII: Summary statistics of the differential expression of Haptoglobin related protein spot B4424, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 20A . 
         [0097]    Table XLIX: Summary statistics of the differential expression of Lectin P35 3 protein spot B6519, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 13A . 
         [0098]    Table L: Summary statistics of the differential expression of Complement C4A gamma protein spot B7408, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 15A . 
         [0099]    Table LI: Summary statistics of the differential expression of Apoptosis Inhibitor (CD5L) protein spot B2412, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 17A . 
         [0100]    Table LII: Summary statistics of the differential expression of Nucleolar/ribosomal protein spot B6218, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 24A . 
         [0101]    Table LIII: Summary statistics of the differential expression of Reticulon-4 protein spot 
         [0102]    B7108, equal to the fold of average normal blood serum concentration (concentration measured as protein spot density, PPM) as depicted in the graph in  FIG. 25A . 
         [0103]    Table LIV: Linear discriminant biostatistics of the differential expression in blood serum: a. the 9 Step Disk biomarkers and b. the total 22 breast cancer protein biomarkers. 
         [0104]    Table LV: The 22 breast cancer protein biomarker disease median profiles as depicted in the graphs in  FIGS. 26A-26B . 
         [0105]    Table LVI: Calibration of blood serum concentrations of the total of inter-alpha-trypsin inhibitor heavy chain (H4) related 35 KD protein, equal to the sum of the concentrations of corresponding biomarker protein spots B2422+B2505+B3410+B4404 from 2D gels ( FIG. 1 ) and from Elisa employing the monoclonal antibody specific for the inter-alpha-trypsin inhibitor heavy chain (H4) related 35 KD proteins only ( FIG. 3A ). Retrospective samples were employed. The conversion from 2D gel digital image PPM to μg protein/ml of blood serum is based on the Elisa μg protein/ml, which was calibrated with the purified recombinant protein reference standard ( FIGS. 27A-27B ). These results are illustrated graphically in  FIGS. 28A-28B  (Elisa) and  29 A (2D gels). 
         [0106]    Table LVII: Summary statistics for the graphs in  FIGS. 30A-30E . 
         [0107]    Table LVIII: Receiver Operator Characteristics (ROC, AUC±S.E.) of Elisa Immunoassay vs. Quantitative 2D Electrophoresis of Total ITI (H4) RP 35 KD 
         [0108]    Table LIX: Summary statistics for the graphs in  FIGS. 31A-31F . 
       DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0109]    The present invention is a diagnostic assay for differentiating between patients having earlier and/or later stages of breast cancer, patients with benign breast disease and/or abnormalities, and normal control individuals. The method is based on the use of two-dimensional (2D) gel electrophoresis to separate the complex mixture of proteins found in blood serum and the quantitation of a group of identified biomarkers to differentiate between patients having earlier or later stages of breast cancer, patients with benign breast disease or abnormalities, and normal control individuals. 
         [0110]    In the context of the present invention breast cancer consists of biopsy confirmed and histological staged disease. The breast cancer may be from a plurality of stages, wherein staging is the process physicians use to assess the size and location of a patient&#39;s cancer. Identifying the cancer stage is one of the most important factors in selecting treatment options. It should be noted that a patient may have more than stage of breast cancer at any one time, further complicating treatment and outcomes for the patient. 
         [0111]    In the present invention, the stages of breast cancer are defined as shown in Table I. 
         [0112]    In the context of the present invention, the “protein expression profile” corresponds to the steady state level of the various proteins in biological samples that can be expressed quantitatively. These steady state levels are the result of the combination of all the factors that control protein concentration in a biological sample. These factors include but are not limited to: the rates of transcription of the genes encoding the hnRNAs; processing of the hnRNAs into mRNAs; The rates of splicing and the splicing variations during the processing of the hnRNAs into mRNAs which govern the relative amounts of the protein sequence isoforms; the rates of processing of the various mRNAs by 3′-polyadenylation and 5′-capping; the rates of transport of the mRNAs to the sites of protein synthesis; the rate of translation of the mRNA&#39;s into the corresponding proteins; the rates of protein post-translational modifications, including but not limited to phosphorylation, nitrosylation, methylation, acetylation, glycosylation, poly-ADP-ribosylation, ubiquitinylation, and conjugation with ubiquitin Like proteins; the rates of protein turnover via the ubiquitin-proteosome system and via proteolytic processing of the parent protein into various active and inactive subcomponents; the rates of intracellular transport of the proteins among compartments, such as but not limited to the nucleus, the lysosomes, golgi, the membrane, and the mitochondrion; the rates of secretion of the proteins into the interstitial space; the rates of secretion related protein processing; and the stability and rates of proteolytic processing and degradation of the proteins in the biological sample before and after the sample is taken from the patient. 
         [0113]    In the context of the present invention, a “biomarker” corresponds to a protein or protein fragment present in a biological sample from a patient, wherein the quantity of the biomarker in the biological sample provides information about whether the patient exhibits an altered biological state such as earlier breast cancer such as ductal carcinoma in situ (DCIS, Stage 0), later breast cancer (Invasive, Stages I, II, III, IV), or combinations thereof, such as breast cancer that includes ductal carcinoma in situ (DCIS DCIS-BC), or breast cancer that does not include ductal carcinoma in-situ (Non-DCIS-BC), or benign breast disease or abnormalities (B9). 
         [0114]    A “normal” sample is a sample, preferably a normal serum sample, is taken from an individual with no known breast disease and/or no known breast abnormalities. 
         [0115]    The present invention is based on the quantification of specified proteins. Preferably the proteins are separated and identified by 2D gel electrophoresis. In the past, this method has been considered highly specialized, labor intensive and non-reproducible. 
         [0116]    Only recently with the advent of integrated supplies, robotics, and software combined with bioinformatics has progression of this proteomics technique in the direction of diagnostics become feasible. The promise and utility of 2D gel electrophoresis is based on its ability to detect changes in protein expression and to discriminate protein isoforms that arise due to variations in amino acid sequence and/or post-synthetic protein modifications such as phosphorylation, nitrosylation, ubiquitination, conjugation with ubiquitin-Like proteins, acetylation, and glycosylation. These are important variables in cell regulatory processes involved in disease states. 
         [0117]    There are few comparable alternatives to 2D gels for tracking changes in protein expression patterns related to disease progression. The introduction of high sensitivity fluorescent staining, digital image processing and computerized image analysis has greatly amplified and simplified the detection of unique species and the quantification of proteins. By using known protein standards as landmarks within each gel run, computerized analysis can detect unique differences in protein expression and modifications between two samples from the same individual or between several individuals. 
       Materials and Methods: 
     Sample Collection and Preparation 
       [0118]    Serum samples were prepared from blood acquired by venipuncture. The blood was allowed to clot at room temperature for 30-60 minutes, centrifuged at 1200×g for 15 minutes, and the separated serum was divided into aliquots, and frozen at −40° C. or below until shipment. Samples were shipped on dry ice and were delivered within 24 hours of shipping. 
         [0119]    Once the serum samples were received, logged in, and assigned a sample number; they were further processed in preparation for 2D gel electrophoresis. All samples were stored at −80° C. or below. When the serum samples were removed from storage, they were placed on ice for thawing and kept on ice for further processing. 
       Separation of Proteins in Patient Samples 
       [0120]    The serum protein from patients and normal control subjects analyzed in the present invention were separated using 2D gel electrophoresis. Other various techniques known in the art for separating proteins can also be used. These other techniques include but are not limited to gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography, or any of the various centrifugation techniques well known in the art. In some cases, a combination of one or more chromatography or centrifugation steps may be combined via electrospray or nanospray with mass spectroscopy or tandem mass spectroscopy, or any protein separation technique that determines the pattern of proteins in a mixture either as a one-dimensional, two-dimensional, three-dimensional or multi-dimensional pattern or list of proteins present. 
       Two Dimensional Gel Electrophoresis of Samples 
       [0121]    Preferably the protein profiles of the present invention are obtained by subjecting biological samples to two-dimensional (2D) gel electrophoresis to separate the proteins in the biological sample into a two-dimensional array of protein spots. 
         [0122]    Two-dimensional gel electrophoresis is a useful technique for separating complex mixtures of proteins and can be performed using a variety of methods known in the art (see, e.g., U.S. Pat. Nos. 5,534,121; 6,398,933; and 6,855,554). 
         [0123]    Preferably, the first dimensional gel is an isoelectric focusing gel and the second dimension gel is a denaturing polyacrylamide gradient gel. 
         [0124]    Proteins are amphoteric, containing both positive and negative charges and like all ampholytes exhibit the property that their charge depends on pH. At low pH (acidic conditions), proteins are positively charged while at high pH (basic conditions) they are negatively charged. For every protein there is a pH at which the protein is uncharged, the protein&#39;s isoelectric point. When a charged molecule is placed in an electric field it will migrate towards the opposite charge. 
         [0125]    In a pH gradient such as those used in the present invention, containing a reducing agent such as dithiothreitol (DTT), a protein will migrate to the point at which it reaches its isoelectric point and becomes uncharged. The uncharged protein will not migrate further and stops. Each protein will stop at its isoelectric point and the proteins can thus be separated according to their isoelectric points. In order to achieve optimal separation of proteins, various pH gradients may be used. For example, a very broad range of pH, from about 3 to 11 or 3 to 10 can be used, or a more narrow range, such as from pH 4 to 7 or 5 to 8 or 7 to 10 or 6 to 11 can be used. The choice of pH range is determined empirically and such determinations are within the skill of the ordinary practitioner and can be accomplished without undue experimentation. 
         [0126]    In the second dimension, proteins are separated according to molecular weight by measuring mobility through a uniform or gradient polyacrylamide gel in the detergent sodium dodecyl sulfate (SDS). In the presence of SDS and a reducing agent such as dithiothreitol (DTT), the proteins act as though they are of uniform shape with the same charge to mass ratio. When the proteins are placed in an electric field, they migrate into and through the gel from one edge to the other. As the proteins migrate though the gel, individual proteins move at different speeds with the smaller ones moving faster than the larger ones. This process is stopped when the fastest moving components reach the other side of the gel. At this point, the proteins are distributed across the gel with the higher molecular weight proteins near the origin and the low molecular weight proteins near the other side of the gel. 
         [0127]    It is well known in the art that various concentration gradients of acrylamide may be used for such protein separations. For example, a gradient of about 5% to 20% may be used in certain embodiments or any other gradient that achieves a satisfactory separation of proteins in the sample may be used. Other gradients would include but not be limited to about 5 to 18%, 6 to 20%, 8 to 20%, 8 to 18%, 8 to 16%, 10 to 16%, or any range as determined by one of skill. 
         [0128]    The end result of the 2D gel procedure is the separation of a complex mixture of proteins into a two dimensional array, a pattern of protein spots, based on the differences in their individual characteristics of isoelectric point and molecular weight. 
       Reagents 
       [0129]    Protease inhibitor cocktail were from Roche Diagnostics Corporation (Indianapolis, Ind.), Protein assay and purification reagents were from Bio-Rad Laboratories (Hercules, Calif.). Immobilon-P membranes and ECL reagents were from Pierce (Rockford, Ill.). All other chemicals were from Sigma Chemical (St. Louis, Mo.). 
       2D Gel Standards 
       [0130]    Purified proteins having known characteristics are used as internal and external standards and as a calibrator for 2D gel electrophoresis. The standards consist of seven reduced, denatured proteins that can be run either as spiked internal standards or as external standards to test the ampholyte mixture and the reproducibility of the gels. A set mixture of proteins (the “standard mixture”) is used to determine pH gradients and molecular weights for the two dimensions of the electrophoresis operation. Table II lists the isoelectric point (pI) values and molecular weights for the proteins included in a standard mixture. 
         [0131]    In addition, standard mixtures such as Precision Plus Protein Standards (Bio-Rad Laboratories), a mixture of 10 recombinant proteins ranging from 10-250 kD, are typically added as external molecular weight standards for the second dimension, or the SDS-PAGE portion of the system. The Precision Plus Protein Standards have an r 2  value of the R f  vs. log molecular weight plot of &gt;0.99. 
       Separation of Proteins in Serum Samples 
       [0132]    An appropriate amount of isoelectric focusing (IEF) loading buffer (LB-2), was added to the diluted serum sample, incubated at room temperature and vortexed periodically until the pellet was dissolved to visual clarity. The samples were centrifuged briefly before a protein assay was performed on the sample. 
         [0133]    Approximately 100 μg of the serum proteins were suspended in a total volume of 184 μl of IEF loading buffer containing 5 M urea, 2 M Thiourea, 1% CHAPS, 2% ASB-14, 0.25% Tween 20, 100 mM DTT, 1% ampholytes pH 3-10, 5% glycerol, 1×EDTA-free protease inhibitor cocktail and 1 — 1 Bromophenol Blue as a color marker to monitor the process of gel electrophoresis. Each sample was loaded onto an 11 cm IEF strip (Bio-Rad Laboratories), pH 5-8, and overlaid with 1.5-3.0 ml of mineral oil to minimize the sample buffer evaporation. Using the PROTEAN® IEF Cell, an active rehydration was performed at 50V and 20° C. for 12-18 hours. 
         [0134]    IEF strips were then transferred to a new tray and focused for 20 min at 250V followed by a linear voltage increase to 8000V over 2.5 hours. A final rapid focusing was performed at 8000V until 20,000 volt-hours were achieved. Running the IEF strip at 500V until the strips were removed finished the isoelectric focusing process. 
         [0135]    Isoelectric focused strips were incubated on an orbital shaker for 15 min with equilibration buffer (2.5 ml buffer/strip). The equilibration buffer contained 6M urea, 2% SDS, 0.375M HCl, and 20% glycerol, as well as freshly added DTT to a final concentration of 30 mg/ml. An additional 15 min incubation of the IEF strips in the equilibration buffer was performed as before, except freshly added iodoacetamide (C 2 H 4 INO) was added to a final concentration of 40 mg/ml. The IPG strips were then removed from the tray using clean forceps and washed five times in a graduated cylinder containing the Bio Rad Laboratories running buffer 1× Tris-Glycine-SDS. 
         [0136]    The washed IEF strips were then laid on the surface of Bio Rad pre-cast CRITERION SDS-gels 8-16%. The IEF strips were fixed in place on the gels by applying a low melting agarose. A second dimensional separation was applied at 200V for about one hour. After running, the gels were carefully removed and placed in a clean tray and washed twice for 20 minutes in 100 ml of pre-staining solution containing 10% methanol and 7% acetic acid. 
       Staining and Analysis of the 2D Gels 
       [0137]    Once the 2D gel patterns of the serum samples are obtained, the protein spots resolved in the gels are visualized with either a fluorescent or colored stain. In the preferred embodiment, the fluorescent dye Lava Purple (Fluorotechnics) is the fluorescent stain. In another embodiment, another fluorescent stain, such as SyproRuby™ (Bio-Rad Laboratories) is employed. Once the protein spots are stained, the gel is scanned by a digital fluorescent scanner. In a preferred embodiment the FLA-7000 (Fujifilm) is the fluorescent scanner. In another embodiment, another fluorescent scanner, such as an FX-Imager (Bio-Rad Laboratories) is employed, or when visible dyes, such as silver or Coomassie Blue, are employed, a digital visible light scanner, such as a GS-800 densitometer (Bio-Rad Laboratories) is employed. The fluorescent or visible digital image of the protein spot pattern of the 2D gel, i.e. a protein expression profile of the sample, is thus obtained. 
         [0138]    The digital image of the scanned gel is processed using PDQuest™ (Bio-Rad Laboratories) image analysis software to first detect the proteins, locate the selected biomarkers, and then to quantitate the protein in each of the selected spots. The scanned image is cropped and filtered to eliminate artifacts, using the image editing control. Individual cropped and filtered images are then placed in a matched set for comparison to other images and controls. 
         [0139]    This process allowed quantitative and qualitative spot comparisons across gels and the determination of protein biomarker molecular weight and isoelectric point values. Multiple gel images were normalized to allow an accurate and reproducible comparison of spot quantities across two or more gels. The gels were normalized using the “total of all valid (detected and confirmed by the operator) spots method” in that a small percentage of the 1200 protein spots detected and verified change between serum samples, and that all spots detected and verified is a good estimate to correct for any differences in total protein amount applied to each gel. The quantitative amounts of the selected biomarkers present in each sample were then exported for further analysis using statistical programs. 
       Tryptic Digestion, MALDI/MS, and LC-MS/MS 
       [0140]    Following software analysis, unique spots were excised from the gel using the ProteomeWorks™ robotic spot cutter (Bio-Rad). In-gel spots were subjected to proteolytic digestion on a ProGest™ (Genomic Solutions, Ann Arbor, Mich.). A portion of the resulting digest supernatant was used for MALDI/MS analysis. Peptide solutions were concentrated and desalted using μ-C18 Ziplips™ (Millipore). Peptides were eluted with MALDI matrix alpha-cyano 4-hydroxycinnamic acid prepared in 60% acetonitrile, 0.2% TFA. Samples were robotically spotted onto MALDI chip, using ProMS™ (Genomic Solutions, Ann Arbor, Mich.). 
         [0141]    MALDI/MS data was acquired on an Applied Biosystems Voyger DE-STR instrument and the observed m/z values were submitted to ProFound (Proteometrics software package) for peptide mass fingerprint searching using NCBInr database. 
         [0142]    For LC/MS/MS, samples were analyzed by nano-LC/MS/MS on a Micromass Q-TOF 2. Aliquots of 15 μl of hydrolysate were processed on a 75 mm C18 column at a flow rate of 200 mL/min. MS/MS data were searched using a local copy of MASCOT, using peptide mass tolerance of ±100 ppm and fragment mass tolerance of ±0.1 Da, fixed modification of carbamidomethyl (C) and variables, including oxidation (M), acetyl (N-term), Pyro-glu (N-term Q), Pyro-glu (N-term E) and max missed cleavages of trypsin of 1. 
       Biostatistical Analysis 
       [0143]    Statistical significance of differences in biomarker blood serum concentrations between different patient and control groups is performed using methods well known in the art, such as Box and Whiskers plots, Receiver Operator Characteristics (ROC), and analysis of variance, employing a standard off the shelf software package, such as “Analyze-it” in Microsoft XL. 
         [0144]    Discriminant analysis is a well-validated multivariate analysis procedure. Discriminant analysis identifies sets of linearly independent functions that will successfully classify individuals into a well-defined collection of groups. The statistical model assumes a multivarate normal distribution for the set of biomarkers identified from each disease group. Let  x   ij  be the p-tuple vector of biomarkers from the i th  patient in the j th  group, j=1, 2 Let x j  be the p-tuple centroid of the j th  group, made up of the mean biomarker values from the j th  disease group. S is the estimate of the within group variance-covariance matrix. The discriminant function is then that set of linear functions determined by the vector a that maximizes the quantity: 
         [0000]    
       
         
           
             
               
                 
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         [0145]    The outcome of the discriminant analysis is a collection of m−1 linear functions of the biomarkers (m) that maximize the ability to separate individuals into disease groups. The vector  a  is the p-tuple vector which contains the coefficients that, when multiplied by an individual&#39;s biomarkers, produces the linear discriminant function, or index that is used to classify that individual. 
         [0146]    In general, if there are m biomarkers, there will be a maximum of (m−1, g−1) discriminant functions where g is the number of groups. Let  a   j (k) be the k th  p-tuple discriminant function. Then the value of that discriminator for the i th  patient is  a   j (k)′ x   i . Thus for each patient there are k such values computed, which are used in a classification analysis. The discriminant functions themselves are linearly independent, i.e., for each pair of the m discriminant functions,  a   j (k) and  a   j (l), then,  a   j (k)′ a   j (l)=0. Thus, the m−1 discriminant functions provide incremental and non-redundant discriminant ability. 
         [0147]    Identifying the discriminant function involves identifying the coefficients λ from the linear algebraic system of equations |H−λ i (H+E)|=0 where H and E are the one way analysis of variance hypotheses and error matrices respectively. It is this computation that is provided by SAS. SAS identifies the collection of best discriminators using a forward entry procedure where the p-value to enter and the p value to stay in the model are each 0.15. 
         [0148]    While the discrimination procedure is fairly robust in the presence of mild departures from the normality assumption, it is very sensitive to the assumption of homogeneity of variance. This means that the variance-covariance matrices of the groups between which discrimination is sought must be equal. In this circumstance, these variance-covariance matrices can be pooled. However, in the situation where the variance-covariance matrices are not equal (multivariate heteroscedasticity), this pooling procedure is sub-optimal. In this circumstance, the individual variance-covariance matrices are used. 
         [0149]    The use of the two within-group variance-covariance matrices is an important complication in the computation of discriminant functions. When the homoscedasticity assumption is appropriate, the within group variance-covariance matrices can be pooled, producing a linear discriminant function. The use of the within-group variance-covariance matrices produces a quadratic discriminant function, (i.e., where the discriminant function is a function of the squares of the proteomic measures). Both linear and quadratic statistical functions are illustrated in the embodiments of this invention. 
       Classification Analysis 
       [0150]    Discriminant analysis was applied to the training set, from which the contribution of each individual biomarker was determined. The SAS® statistical software program was then used to determine the linear combinations of biomarkers that provided an optimum classification of individuals into disease groups. Alternatively, the programmer manually selected different combinations of biomarkers to be incorporated into a linear or quadratic discriminant function to optimize the classification of individuals into disease groups. 
         [0151]    The output of discriminant analysis (DA) is a classification table that permits the calculation of clinical sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV):
       Clinical Sensitivity is how often the test is positive in diseased patients.   Clinical Specificity is how often the test is negative in non-diseased individuals.   Negative Predictive Value (NPV) is the probability that the patient will not have the disease when restricted to all individuals who test negative.   Positive Predictive Value (PPV) is the probability that the patient has the disease when restricted to those individuals who test positive.       
 
         [0156]    NPV and PPV were not assessed in the case of the present study as these values are dependent upon patient mix and the present study used different numbers of patients in each category, due to sample availability. 
       2D Gel Electrophoretic Controls 
       [0157]    Representative samples from individuals with known cases of breast cancer, benign breast disease, or normal controls, were run as positive and negative reference controls. Serum containing all of the selected biomarkers was also provided as a reference standard. A reference control was periodically run as an external standard and for tracking overall performance and reproducibility. In addition, 2D gel images from samples classified as breast cancer, benign breast disease, or normal controls, were used for reference. The spot locations for the selected biomarkers were illustrated in  FIG. 1 . 
       Samples Analyzed 
       [0158]    The present invention is a two-dimensional gel electrophoresis assay of patient blood serum samples, employing the 22 biomarker spots, combined with multi variate biostatistics, is used to distinguish between subjects with normal breasts, patients with benign breast disease, and patients with breast cancer. 
         [0159]    The 2D gel electrophoresis of the human blood serum samples of this study separated &gt;1200 spots in the pH 5-8 range, 22 of which (FIGS.  1  and  8 A- 8 B, numbered spots: B1322, B1418, B2317, B2422, B2525, B3406, B3410, B4404, B5539, B6505, B6519, B7408, B1512, 2412, B4008, B4206, B3506, B4424, B5713, B6014, B6218, and B7108) displayed differences in serum concentrations between samples from normal subjects, patients with benign breast disease or abnormalities, and patients with breast cancer. 
         [0160]    When the 22 biomarker spots were robotically excised, subjected to in-gel trypsin digestion and the peptides analyzed by LC-MS/MS fingerprint identification, (Tables III), comparison of the 2D gel measured and the protein sequence calculated masses and isoelectric points of the biomarker spots, with the peptides identified by LC-MS/MS, indicated that some of the biomarker protein spots appear on 2D gels as smaller components of parent molecules, i.e. smaller than the original translation products of the mRNA, whereas others are the full length translated products, including those with additional molecular weight contribution from post-synthetic modifications, such as glycosylation, etc ( FIGS. 1 ,  6 ,  7 A- 7 B, Tables VII-XXXII, SEQ ID NOS: 1-22). 
         [0161]    Spot identification by LC MS/MS of in-gel trypsin digests, and pI and Molecular Weight estimations from 2D gels and amino acid sequences ( FIGS. 1 ,  6 ,  7 A- 7 B, Tables III-VI) indicated that biomarker protein spots B2422, B2505, B3410, and B4404 ( FIGS. 1 ,  6 ) correspond to electrophoretic variants of the 35 KD processing product of Inter-alpha-trypsin inhibitor heavy chain (H4) related protein, isoforms 1 and 2 (Tables VI, X-XII, SEQ ID NOS: 4-5). 
         [0000]    Normal Controls vs. Benign Breast Abnormalities vs. Breast Cancer 
         [0162]    These four spots corresponding to the 35 KD isoforms of the Inter-alpha-trypsin inhibitor Heavy Chain (H4) related protein, individually  FIGS. 2A-2D ) and collectively (=B25422+B2505+B3410+B4404,  FIG. 3A ), demonstrated differences in blood serum concentrations between normal controls (N), patients with benign breast disease or abnormalities (B9), and patients with breast cancer (BC) (Table XXXIII and XXXIV). 
         [0163]      FIGS. 2A-2D  illustrate that when these four spots corresponding to the 35 KD isoforms of the Inter-alpha-trypsin inhibitor Heavy Chain (H4) related protein were analyzed for individual performance by 2D gel electrophoresis ( FIGS. 2A : B2422;  2 B: B2505;  2 C: B3410;  2 D: B4404), three of the four,  2 A: B2422;  2 B: B3410; and  2 D: B4404, demonstrated down-shifts in blood serum concentration in breast cancer patients (BC) vs. normal controls (N) and patients with benign breast disease or abnormalities (B9) (Table XXXIII a, c, d). Conversely, the other isoform spot (B2505,  FIG. 2B ) actually displayed an increase in concentration in breast cancer patients (Table XXXIII b). 
         [0164]      FIG. 3A  and Table XXXIII e illustrates that when all four isoforms are analyzed as the total sum, the combined effect is a more modest down-shift (Table XXXIII e), masking the differences in performance between the isoforms seen in  FIGS. 2A-2B . Furthermore, as also illustrated in  FIG. 3A  and Table XXXIV, there is a difference between the concentrations in the retrospective samples vs. the concentrations in the prospective samples, such that the normal (N) and breast cancer (BC) prospective samples both have higher concentrations of the combined 35 KD isoforms of the Inter-alpha-trypsin inhibitor Heavy Chain (H4) related protein biomarkers (sum of the concentrations of B2422+B2505+B3410+B4404), than that of the retrospective samples (Table XXXIV). This renders the retrospective samples no longer capable of performing as a model to diagnose the prospective samples ( FIG. 3A  arrow). This in part explains why so many protein biomarkers, originally discovered in retrospective biological samples, such as blood serum stored in freezers, fail to validate clinically upon fresh prospective samples. 
         [0165]    While the use of absolute values of concentrations of the protein biomarkers (for example 2D gel spot density, PPM) do not provide for consistency between retrospective and prospective databases, another embodiment of the invention consists of determining the differential expression on the basis of the fold value of the normal concentrations, wherein:
       Differential Expression: The deviation in biomarker concentration from the normal state as a function of disease, and wherein:       
 
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         [0167]    In this embodiment of the invention, comparison of prospective and retrospective samples on a fold differential expression basis provides for consistent results, as illustrated in  FIGS. 3B ,  3 C. 
         [0168]      FIGS. 3B ,  3 C illustrates a comparison for the retrospective samples, wherein the pattern of differential expression is essentially unaltered when converted from protein concentration as 2D gel protein spot density (PPM,  FIG. 3A ) to differential expression as fold of average 2D gel protein spot density ( FIGS. 3B ,  3 C). 
         [0169]    As also illustrated in  FIGS. 3B and 3C , when retrospective and prospective samples are separately placed on a differential expression (fold of average normal) basis, the normal means coincide at 1.0 fold, and the differential expression of the prospective samples is now consistent with and readable on the retrospective samples ( FIG. 3A , compared to  FIG. 3B , Table XXXIV). 
         [0000]    Ductal Carcinoma In Situ Breast Cancer (DCIS BC) vs. Non-Ductal Carcinoma In-Situ Breast Cancer (Non-DCIS BC) 
         [0170]    Illustrated in  FIGS. 4A-4D  and  5 A- 5 D and Table XXXV a-d are the differential expression (in fold of average normal concentrations) of the individual biomarkers, the isoform spots of the 35 KD isoforms of the Inter-alpha-trypsin inhibitor heavy chain (H4) related protein biomarkers ( FIGS. 4A ,  5 A: B2422,  FIGS. 4A ,  5 B: B2505,  FIGS. 4A ,  5 C: B3410,  FIGS. 4A ,  5 D: B4404), wherein retrospective and prospective samples are combined after fold conversion. When these biomarkers are considered individually and earlier (DCIS BC) and later (Non-DCIS BC) stages of breast cancer are considered separately, isoform specific and stage specific differences in the differential expression from the normal controls are revealed. The non-DCIS breast cancer (Non-DCIS BC) concentrations are down-regulated, and the DCIS breast cancer (DCIS BC) concentrations are up-regulated in the blood serum of patients relative to the normal samples ( FIGS. 4A-4D ,  5 A- 5 D, Table XXXV). Furthermore, the individual biomarker performance is not identical for each of the four isoforms, in that different degrees of up and/or down-regulation are found with statistically significant single variable biostatistics ( FIGS. 4A-4D ,  5 A- 5 D, Table XXXV). This is illustrated by the less significant down-regulation of protein biomarker spot B2505 ( FIG. 4B ,  FIG. 5B , Table XXXV b*) in non-DCIS breast cancer, relative to the other isoforms B2422, B3410, and B4404 ( FIGS. 4A ,  4 C, and  4 D, Table XXXV a, c, d). 
         [0171]    Thus, in a preferred embodiment of the invention, the blood serum concentrations of the different electrophoretic isoforms with the same protein amino acid sequence are nonetheless determined separately for greater diagnostic performance. Also in a preferred embodiment of the invention, DCIS, DCIS breast cancer, and non-DCIS breast cancer may be considered as separate groups for the purposes of the invention. 
       Additional Protein Biomarkers 
       [0172]    Additional spot identifications by LC MS/MS of in-gel trypsin digests, and pI and Molecular Weight estimations from 2D gels and amino acid sequences ( FIGS. 6 ,  7 A- 7 B and Tables III-VI) indicated that:
       Biomarker protein spot B1322 ( FIG. 6 ) corresponds to an Immunoglobulin Lambda protein (Tables VI-VII, SEQ ID NO: 1); and   Biomarker protein spot B1418 ( FIG. 6 ) corresponds to an Alpha-1-microglobulin protein (Tables VI and VIII, SEQ ID NO: 2); and   Biomarker protein spot B2317 ( FIG. 6 ) corresponds to an Apolipoprotein A-1 protein (Tables VI, IX, SEQ ID NO: 3); and   Biomarker protein spot B3406 ( FIG. 6 ) corresponds to an Apolipoprotein E3 protein (Tables VI, XIII, SEQ ID NO: 6); and   Biomarker protein spot B5539 ( FIG. 6 ) corresponds to a human Albumin protein (Tables VI, XIV, SEQ ID NO: 7); and   Biomarker protein spot B6519 ( FIG. 6 ) corresponds to a human Albumin protein (Tables VI, XV, SEQ ID NO: 8); and   Biomarker protein spot B6605 ( FIG. 6 ) corresponds to a Transferrin protein (Tables VI, XVI, SEQ ID NO: 9); and   Biomarker protein spot B7408 ( FIG. 6 ) corresponds to a Complement C4A gamma protein (Tables VI, XVII-XVIII, SEQ ID NO: 10); and   Biomarker protein spots B1512, B4008, B4206, and B6014 ( FIG. 6 ) correspond to electrophoretic isoforms of a Haptoglobin alpha chain and/or a Haptoglobin beta chain protein (Tables VI, XIX, SEQ ID NO: 11); and   Biomarker protein spots B3507, and B4424 ( FIG. 6 ) correspond to electrophoretic isoforms of a Haptoglobin related protein (Tables VI, XX, SEQ ID NO: 12); and   Biomarker protein spots B2412 ( FIG. 6 ) correspond to an Apoptosis Inhibitor protein (AIM) and/or a CD5L protein (Tables VI, XXI-XXII, SEQ ID NOS: 13-14); and   Biomarker protein spot B5713 ( FIG. 6 ) corresponds to a Serotransferrin protein (Tables VI, XXIV, SEQ ID NO: 23); and   Biomarker protein spot B6218 ( FIG. 6 ) corresponds to a Nucleolar and Ribosomal protein L27a protein (Tables VI, XXV-XXVII, SEQ ID NOS: 15-17); and   Biomarker protein spot B2412 ( FIG. 6 ) corresponds to a Reticulon-4 protein (Tables VI, XXVIII-XXXII, SEQ ID NOS: 18-22).       
 
         [0187]    As shown in  FIGS. 8A-8B , the blood serum concentrations of Immunoglobulin lambda (λ) light chain biomarker protein spot B1322 ( FIG. 8A ,  8 B, Table XXXVI) demonstrates a modest down shift in blood serum concentration, between that of normal controls (N) and that of both patients with benign breast disease or abnormalities (B9), and patients with Breast Cancer (BC). 
         [0188]    As shown in  FIGS. 9A-9B , the blood serum concentrations of Alpha-1-microglobulin biomarker protein spot B1418 ( FIGS. 9A ,  9 B, Table XXXVII) demonstrates a modest and progressive up shift in blood serum concentration, from normal controls (N) to those of patients with benign breast disease or abnormalities (B9), and patients with Breast Cancer (Combined BC). The concentration appears to be maximal in DCIS BC (Table XXXVI). 
         [0189]    As shown in  FIGS. 10A-10B , Apolipoprotein A-I biomarker protein spot B2317, ( FIGS. 10A ,  10 B, Table XXXVIII), demonstrates a down shift in blood serum concentration between normal controls (N) and patients with benign breast disease or abnormalities (B9), and conversely demonstrated an up-shift between normal controls (N) and patients with DCIS breast cancer (DCIS BC). 
         [0190]    As shown in  FIGS. 11A-11B , Apolipoprotein E3 biomarker protein spot B3406 ( FIGS. 11A ,  11 B, Table XXXIX), demonstrates an down shift in blood serum concentration between normal controls (N) and patients with benign breast disease or abnormalities (B9), and conversely demonstrated an up-shift between patients with benign breast disease or abnormalities (B9) and patients with DCIS breast cancer (DCIS BC), and a corresponding return to normal levels in patients with Non-DCIS breast cancer (Non-DCIS BC). 
         [0191]    As shown in  FIGS. 12A-12B , Serum albumin biomarker protein spot B5539 ( FIGS. 14  A, B, Table XL) demonstrated an up-shift in blood serum concentration between normal controls (N) and patients with benign breast disease or abnormalities (B9), and conversely demonstrated a progressive down-shift between patients with benign breast disease or abnormalities (B9), patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC) to levels below normal (N). 
         [0192]    As shown in  FIGS. 13A-13B , Lectin P35 biomarker protein spot B6519 ( FIGS. 13A ,  13 B, Table XLIX) demonstrated a progressive up shift in blood serum concentration from that of normal controls (N) and patients with benign breast disease or abnormalities (B9), to that of patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC). 
         [0193]    As shown in  FIGS. 14A-14B , Transferrin biomarker protein spot B6605 ( FIGS. 14A ,  14 B, Table XLI) demonstrated an up-shift in blood serum concentration between that of normal controls (N) and that of patients with benign breast disease or abnormalities (B9), patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC). The effect appeared to be maximal in patients with benign breast disease or abnormalities (B9) and to be progressively lower in patients with DCIS breast cancer (DCIS BC) and Non-DCIS breast cancer (Non-DCIS BC). 
         [0194]    As shown in  FIGS. 15A-15B , Complement C4A biomarker protein spot B7408 ( FIGS. 15A ,  15 B, Table L) demonstrated an up-shift in blood serum concentration between that of normal controls (N) and that of patients with benign breast disease or abnormalities (B9), patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC). The effect appeared to be maximal in patients with DCIS breast cancer (DCIS BC). 
         [0195]    As shown in  FIGS. 16A-16D , Haptoglobin biomarker protein spot B1512 ( FIGS. 161A-16D , Table XLIII) a progressive up shift in blood serum concentration from that of normal controls (N) to that of patients with benign breast disease or abnormalities (B9), to that of patients with DCIS breast cancer (DCIS BC), and maximally to that of patients with Non-DCIS breast cancer (Non-DCIS BC). 
         [0196]    As shown in  FIGS. 17A-17B , Apoptosis Inhibitor (AIM and/or CD5L) biomarker protein spot B2412 ( FIGS. 17A ,  17 B, Table LI) demonstrated a progressive up-shift in blood serum concentration between that of normal controls (N) and that of patients with benign breast disease or abnormalities (B9), patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC). The effect appeared to be maximal in patients with DCIS breast cancer (DCIS BC). 
         [0197]    As shown in  FIGS. 18A-18B , Haptoglobin biomarker protein spot B4008 ( FIGS. 18A ,  18 B, Table XLV) demonstrated an up-shift in blood serum concentration from that of normal controls (N) to that of patients with benign breast disease or abnormalities (B9), patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC). 
         [0198]    As shown in  FIGS. 19A-19B , Haptoglobin biomarker protein spot B4206 ( FIGS. 19A ,  19 B, Table XLVI) demonstrated an up-shift in blood serum concentration from that of normal controls (N) to that of patients with benign breast disease or abnormalities (B9), patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC). The effect appeared to be maximal in patients with DCIS breast cancer (DCIS BC). 
         [0199]    As shown in  FIGS. 20A-20B , Haptoglobin related biomarker protein spot B4424 ( FIGS. 20A ,  20 B, Table XLVIII) demonstrated a slight down-shift in blood serum concentration from that of normal controls (N) and patients with benign breast disease or abnormalities (B9), to that of patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC). The effect appeared to be slightly more pronounced in that of patients with DCIS breast cancer (DCIS BC) than that of patients with Non-DCIS breast cancer (Non-DCIS BC). 
         [0200]    As shown in  FIGS. 21A-21B , Haptoglobin related biomarker protein spot B3506 ( FIGS. 21A ,  21 B, Table XLVII) demonstrated a slight down-shift in blood serum concentration from that of normal controls (N) to that of patients with benign breast disease or abnormalities (B9), patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC). 
         [0201]    As shown in  FIGS. 22A-22B , Serotransferrin biomarker protein spot B5713 ( FIGS. 22A ,  22 B, Table XLII) demonstrated a down-shift in blood serum concentration from that of normal controls (N) to that of patients with benign breast disease or abnormalities (B9), patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC). 
         [0202]    As shown in  FIGS. 23A-23C , Haptoglobin biomarker protein spot B6014 ( FIGS. 23A ,  23 B,  23 C, Table XLIV) demonstrated differential expression wherein a greater number (62.2%) of samples contained detectable blood serum levels of this biomarker in Non-DCIS breast cancer (Non-DCIS BC), than in normal controls and patients with benign breast disease or abnormalities (N+B9, 32.3%) and in patients with DCIS breast Cancer (DCIS BC,). 
         [0203]    As shown in  FIGS. 24A-24B , Nucleolar and/or Ribosomal protein L27a biomarker protein spot B6218 ( FIGS. 24A ,  24 B, Table LII) demonstrated an up-shift in blood serum concentration from that of normal controls (N) and patients with benign breast disease or abnormalities (B9), to that of patients with DCIS breast cancer (DCIS BC), and patients with Non-DCIS breast cancer (Non-DCIS BC). The effect appeared to be maximal in patients with DCIS breast cancer (DCIS BC). 
         [0204]    As shown in  FIGS. 25A-25D , Nucleolar and/or Reticulon-4 biomarker protein spot B7108 ( FIGS. 25A-25D , Table LIII) demonstrated a progressive down-shift in blood serum concentration from that of normal controls (N) to that of patients with benign breast disease or abnormalities (B9), to that of patients with DCIS breast cancer (DCIS BC), and most pronounced in that of patients with Non-DCIS breast cancer (Non-DCIS BC). 
         [0205]    While individual single variable non-parametric statistics of each of the 22 protein biomarkers in blood serum indicated significant disease specific differential expression, no single biomarker was capable of fully distinguishing between all the normal samples, benign samples, and breast cancer samples. However, the individual biomarkers performed differently from one another and when used together, employing multivariate linear discriminant analysis (Table X), the 22 biomarkers employed as a group were capable of synergistic discrimination of the three groups from each other (3-way,  25 A &amp;  25 B) and between cancer and not cancer (2 way,  25 C &amp;  25 D) with higher sensitivities and specificities (Table LIV). Furthermore, a group of 9 biomarkers selected by the Step Disc function of the linear discriminant analysis was essentially as good as the entire group of 22 biomarkers (Table LIV, compare a and b). 
         [0206]    As shown in  FIGS. 26A-26B , ( FIGS. 26A ,  26 B, Table LIV), the median differential expression profiles (median fold of mean normal blood serum concentration, where concentration=median 2D gel spot density, PPM) showed distinct differences between normal controls (Normal Median), patients with benign breast disease or abnormalities (B9 Median), patients with DCIS breast cancer (DCIS BC Median), and patients with Non-DCIS breast cancer (Non-DCIS BC Median). Furthermore, when these profiles are displayed in order of selection by the Step Disk function ( FIG. 26A ), a pattern is revealed wherein: 
         [0207]    Apolipoprotein A-1 biomarker protein spot B2317 preferentially separates DCIS-BC from N+B9+Non-DCIS BC; followed by
       ITI (H4) RP 35 KD protein isoform biomarker protein spot B2505, which preferentially separates DCIS-BC and to a lesser extent Non-DCIS BC from N+B9; followed by   Nucleolar and/or Ribosomal protein L27a biomarker protein spot B6218, which preferentially separates DCIS-BC and Non-DCIS BC from N+B9; followed by   Haptoglobin biomarker protein spot B6014, which preferentially separates Non-DCIS BC from N+B9+DCIS BC; followed by   Haptoglobin biomarker protein spot B1512, which preferentially separates Non-DCIS BC from N+B9+DCIS BC; followed by   Reticulon-4 biomarker protein spot B7108, which preferentially separates Non-DCIS BC from N+B9+DCIS BC; followed by   Serum Albumin protein spot B5539, which preferentially separates Non-DCIS BC from N+B9+DCIS BC; followed by   ITI (H4) RP 35 KD protein isoform biomarker protein spot B2422, which preferentially separates DCIS-BC and Non-DCIS BC from N+B9; followed by   ITI (H4) RP 35 KD protein isoform biomarker protein spot B2422, which preferentially separates DCIS-BC from N+B9+Non-DCIS BC.       
 
         [0216]    The aforementioned Step Disc series of biomarkers (below the arrow,  FIG. 26A ) outlines how each new biomarker is synergistic with the previously selected biomarkers, arriving at the utility of specificity and sensitivity of the multivariate biostatistical analysis of the invention. 
         [0217]    The additional 13 of the 22 biomarkers not selected by the Step Disc function are also displayed (below the dotted line,  FIG. 26A ) which also show distinct differences in separation between the groups of patients and controls. However, Based upon the slight increases in sensitivities and specificities obtained when they are also employed in the multivariate analysis (Table LIV b), these differences are largely redundant with the other nine biomarkers. 
         [0218]      FIG. 26B  further illustrates this redundancy when the individual isoforms are displayed in the order that they were selected into the Step Disk function, wherein:
       Step Disk selected ITI (H4) RP 35 KD isoform spots B2505, B2422, and B4404, but not isoform spot B3410; and wherein   Step Disk selected Haptoglobin isoform spots B6014 and B1512, but not isoform spots B4008 nor B4206; and wherein   Step Disk selected neither Haptoglobin related protein isoform spots B3506 nor B4424.         
         [0222]    On the other hand, when additional patient samples are added to the database, these additional “redundant” biomarkers provide further synergy to the invention. 
         [0223]    The serum samples may also be subjected to various other techniques known in the art for separating and quantitating proteins. Such techniques include, but are not limited to gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography (typically in an HPLC or FPLC apparatus), or any of the various centrifugation techniques well known in the art. Certain embodiments would also include a combination of one or more chromatography or centrifugation steps combined via electrospray or nanospray with mass spectrometry or tandem mass spectrometry of the proteins themselves, or of a total digest of the protein mixtures. Certain embodiments may also include surface enhanced laser desorption mass spectrometry or tandem mass spectrometry, or any protein separation technique that determines the pattern of proteins in the mixture either as a one-dimensional, two-dimensional, three-dimensional or multi-dimensional protein pattern, and or the pattern of protein post synthetic modification isoforms. 
         [0224]    Quantitation of a protein by antibodies directed against that protein is well known in the field. The techniques and methodologies for the production of one or more antibodies to the proteins are routine in the field and are not described in detail herein. 
         [0225]    As used herein, the term antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or 1 gM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. 
         [0226]    Monoclonal antibodies (MAbs) are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred. The invention thus provides monoclonal antibodies of human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies are generally preferred. However, “humanized” antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. 
         [0227]    The term “antibody” thus also refers to any antibody-like molecule that has 20 an antigen binding region, and includes antibody fragments such as Fab′, Fab, F(ab′)2, single domain antibodies (DABS), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g.,  Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). 
         [0228]    Antibodies to the one or more of the 22 protein biomarkers may be used in a variety of assays in order to quantitate the protein in serum samples, or other fluid or tissue samples. Well known methods include immunoprecipitation, antibody sandwich assays, ELISA and affinity chromatography methods that include antibodies bound to a solid support. Such methods also include microarrays of antibodies or proteins contained on a glass slide or a silicon chip, for example. 
         [0229]    It is contemplated that arrays of antibodies to up to 22 protein biomarkers, or peptides derived, may be produced in an array and contacted with the serum samples or protein fractions of serum samples in order to quantitate the proteins. The use of such microarrays is well known in the art and is described, for example in U.S. Pat. No. 5,143,854, incorporated herein by reference. 
         [0230]    The present invention includes a screening assay for breast cancer based on the up-regulation and/or down-regulation of the 22 protein biomarkers. One embodiment of the assay will be constructed with antibodies recognizing up to 22 protein biomarkers. One or more antibodies targeted to antigenic determinants of up to 22 protein biomarkers will be spotted onto a surface, such as a polyvinyl membrane or glass slide. As the antibodies used will each recognize an antigenic determinant of up to 22 protein biomarkers, incubation of the spots with patient samples will permit attachment of up to 22 protein biomarkers to the antibody. 
         [0231]    The binding of up to 22 protein biomarkers can be reported using any of the known reporter techniques including radioimunoassays (RIA), stains, enzyme linked immunosorbant assays (ELISA), sandwich ELISAs with a horseradish peroxidase (HRP)-conjugated second antibody also recognizing up to 22 protein biomarkers, the pre-binding of fluorescent dyes to the proteins in the sample, or biotinylating the proteins in the sample and using an HRP-bound streptavidin reporter. The HRP can be developed with a chemiluminescent, fluorescent, or colorimetric reporter. Other enzymes, such as luciferase or glucose oxidase, or any enzyme that can be used to develop light or color can be utilized at this step. 
         [0232]    As shown in Table X, the N-terminal of the of ITI (H4) RP PK-120 precursor is different from the ITI (H4) RP 35 KD isoforms, wherein the sequence containing the 35 KD (PK-120), corresponds to biomarkers B2422, B2505, B3410, and B44.04 of the present invention is located in the C-terminal sequence. The lack of homology is maintained throughout the 35 KD product. For high throughput immunoassays, biomarker specific antibodies can be developed using truncated cDNA sequences to produce recombinant antigens in bacterial or mammalian systems, containing only the epitopes of the 35 KD biomarkers without the epitopes of the upstream region of the parent molecules. These antigens in turn can be used to immunize rabbits, sheep, chickens, or goats, for polyclonal antibodies, or mice to produce monoclonal antibodies either with classic hybridoma technologies or phage display methods. The recombinant antigens can also be employed as affinity agents to purify antibodies and as reagent controls in assays. 
         [0233]    Alternatively, antibodies could be raised to the upstream portions of the parent molecule that would not cross react with the ITI (H4) RP 35 KD isoforms (Table X). Such antibodies could be used as affinity capture agents to isolate from serum or other sources the intact PK 120. Subsequent treatment of this group with plasma Kallikrein, which selectively cleaves out the ITI (H4) RP 35 KD isoforms would release the 35 KD isoforms, which would not bind the antibodies and thus the biomarkers, in native purified form, can be obtained from a biological sample. 
         [0234]    Alternatively, the approach illustrated in  FIGS. 27A-27B  can be employed. In this embodiment of the invention a recombinant protein, for example containing the sequence of the ITI (H4) RP 35 KD protein isoforms (SEQ ID NOS: 4 or 5), is made via an expression vector, such that the protein contains a his tag at its N terminus ( FIG. 27A : Antigen 1). Another antigen is constructed as a synthetic peptide derived from the amino terminal sequence as it exists without the his tag at the N terminus ( FIG. 27A , Antigen 3). Monoclonal antibodies are then obtained from mice immunized with either Antigen 1 or Antigen 3. The monoclonal antibodies are screened by Elisa using Antigen 1 and Antigen 2, wherein Antigen 2 is obtained by removing the his tag from Antigen 1 ( FIGS. 27A ,  27 B). From the anti-Antigen 1 monoclonal antibodies, a monoclonal antibody is selected that reacts with both the ITI (H4) RP 35 KD Isoforms and the PK 120 precursor ( FIG. 27B , Anti-Antigen 1 Antibody). From the anti-Antigen 3 monoclonal antibodies, a monoclonal antibody is selected that reacts with only the ITI (H4) RP 35 KD Isoforms and not the PK 120 precursor, which does not contain the free N terminals amino acid sequence of Antigens 2 and 3 ( FIG. 27B , Anti-Antigen 3 Antibody). 
         [0235]    As illustrated in  FIGS. 28A-28B , an Elisa immunoassay using the ITI (H4) RP 35 KD protein specific anti-Antigen 3 monoclonal antibody detects the decreased blood serum concentration (μg protein/ml of serum) of the ITI (H4) RP 35 KD protein isoforms ( FIG. 28A ). On the other hand, the Elisa performed with the anti-Antigen 1 monoclonal antibody, that also detects the PK 120 precursor (Table X), does not show a decrease in blood serum concentration ( FIG. 28B ). Thus in an embodiment of the invention, isoform specific antibodies selectively detect and measure differences in concentration of antibodies for diagnosis of disease, such as breast cancer, that. 
         [0236]    As illustrated in  FIGS. 29A-29C  and  30 A- 30 E, the differential expression from normal (in fold of average normal μg protein/ml of serum,  FIG. 30D ) using the Elisa with anti-Antigen 3 specific for the ITI (H4) RP 35 KD proteins is comparable to that determined by quantitative 2D gel electrophoresis (fold of average normal 2D gel spot density, PPM,  FIGS. 29A ,  30 A- 30 C). 
         [0237]    There is also a plurality method variations in the preferred embodiment using 2D gel electrophoresis. As shown in  FIGS. 31A-31F , variations in blood serum preparation methods ( FIGS. 31A ,  31 D, vs.  FIGS. 31B ,  31 C,  31 E, and  31 F), in digital imaging equipment ( FIGS. 31A ,  31 D, vs.  FIGS. 31B ,  31 C,  31 E, and  31 F), in protein stains ( FIGS. 31A ,  31 B,  31 D, and  31 E vs.  FIGS. 31C ,  31 F), can all be employed to perform embodiments of the invention. 
         [0238]    A comparison of  FIG. 3B  and  FIGS. 30A ,  30 B,  30 D,  30 E to  FIGS. 2A-2D  and  4 A- 4 D, illustrates how the preferred embodiment of the invention, employing quantitative 2D gel electrophoresis, distinguishes specificities related to differences in the differential expression of the individual isoforms of the ITI (H4) RP 35 KD protein spots B2422, B2505, B3410, and B4.404, not seen using the anti-Antigen 3 monoclonal antibody, which was directed to the 35 KD amino acid sequence common to all four, not the source of the electrophoretic separation of these isoforms. Thus in a preferred embodiment of the invention, employment of protein separation techniques, and/or more specific antibodies to distinguish between proteins with the same amino acid sequence enhance the diagnostic specificity and sensitivity of the invention. 
         [0239]    Similar approaches are available for the other of up to 22 biomarkers whose amino acid sequences are defined in some of the accompanying tables. 
         [0240]    All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. 
         [0241]    More specifically, it is well recognized in the art that the statistical data, including but not limited to the mean, standard error, standard deviation, median, interquartile range, 95% confidence limits, results of analysis of variance, non-parametric median tests, discriminant analysis, etc., will vary as data from additional patients are added to the database or antibodies are utilized to determine concentrations of one or more of the 22 biomarkers of the present invention, or any biomarker. Therefore changes in the statistical values of one or more of the 22 protein biomarkers do not depart from the concept, spirit and scope of the invention. 
         [0242]    Also more specifically, it is disclosed (in cross referenced U.S. Utility patent applications by Goldknopf, I. L., et al. Ser. Nos. 11/507,337 and 11/503,881, US Provisional Patent Applications by Goldknopf et al. Ser. No. 60/708,992 and 60/738,710, and referenced in Goldknopf, I. L et al. 2006 and Sheta et al. 2006, hereby incorporated as reference) that blood serum concentrations of protein biomarkers, including an inter alpha trypsin inhibitor family heavy chain (H4) related protein 35 KD and Apolipoprotein E3, can be used in combination with other biomarkers for diagnosis, differential diagnosis, and screening. Consequently, the use of one or more of the 22 protein biomarkers in conjunction with one or more additional biomarkers not disclosed in the present invention does not depart from the concept, spirit and scope of the invention. 
         [0243]    It is also well recognized in the art that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. 
       Tables I-LV 
       [0244]      
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 Table I: Staging Breast Cancer 
               
             
          
           
               
                   
                   
                 Lymph Node 
                 Metastasis* 
                   
               
               
                 Stage 
                 Tumor Size 
                 Involvement 
                 (Spread) 
                 AKA 
               
               
                   
               
               
                 0 
                 In situ 
                 No 
                 No 
                 Carcinoma 
               
               
                   
                 (DCIS, LCIS) 
                   
                   
                 in situ 
               
               
                 I 
                 Less than 2 cm 
                 No 
                 No 
                 Invasive 
               
               
                   
                   
                   
                   
                 carcinoma 
               
               
                 II 
                 Between 2-5 cm 
                 No or in same 
                 No 
               
               
                   
                   
                 side of breast 
               
               
                 III 
                 More than 5 cm 
                 Yes, on same 
                 Yes 
               
               
                   
                   
                 side of breast 
               
               
                 IV 
                 Not applicable 
                 Not applicable 
               
               
                   
               
               
                 *No = not detected 
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE II 
               
               
                   
                   
               
               
                   
                   
                   
                 Molecular 
               
               
                   
                 Protein 
                 pI 
                 Weight (Da) 
               
               
                   
                   
               
             
             
               
                   
                 Hen egg white conalbumin 
                 6.0, 6.3, 6.6 
                 76,000 
               
               
                   
                 Bovine serum albumin 
                 5.4, 5.5, 5.6 
                 66,200 
               
               
                   
                 Bovine muscle actin 
                 5.0, 5.1 
                 43,000 
               
               
                   
                 Rabbit muscle GAPDH 
                 8.3, 8.5 
                 36,000 
               
               
                   
                 Bovine carbonic anhydrase 
                 5.9, 6.0 
                 31,000 
               
               
                   
                 Soybean trypsin inhibitor 
                 4.5 
                 21,500 
               
               
                   
                 Equine myoglobin conalbumin 
                 7.0 
                 17,500 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE III 
               
               
                   
                   
               
               
                   
                   
                 Relative Mobility (Rf) 
                   
               
               
                   
                 Biomarker 
                 (Fold of 10,000 MW 
                 y = 13043x − 
               
               
                   
                 Spot 
                 distance from origin) 
                 1.0128 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 B1322 
                 0.604 
                 21,738 
               
               
                   
                 B1418 
                 0.474 
                 27,780 
               
               
                   
                 B2713 
                 0.630 
                 20,830 
               
               
                   
                 B2422 
                 0.448 
                 29,411 
               
               
                   
                 B2505 
                 0.429 
                 30,766 
               
               
                   
                 B3410 
                 0.468 
                 28,170 
               
               
                   
                 B4404 
                 0.487 
                 27,029 
               
               
                   
                 B3406 
                 0.442 
                 29,849 
               
               
                   
                 B5539 
                 0.325 
                 40,755 
               
               
                   
                 B6519 
                 0.422 
                 31,245 
               
               
                   
                 B6605 
                 0.253 
                 52,417 
               
               
                   
                 B7408 
                 0.461 
                 28,572 
               
               
                   
                 B1512 
                 0.325 
                 40,755 
               
               
                   
                 B2412 
                 0.506 
                 25,977 
               
               
                   
                 B4008 
                 1.091 
                 11,943 
               
               
                   
                 B4206 
                 0.740 
                 17,687 
               
               
                   
                 B3507 
                 0.396 
                 33,321 
               
               
                   
                 B4424 
                 0.403 
                 32,777 
               
               
                   
                 B5713 
                 0.169 
                 79,034 
               
               
                   
                 B6014 
                 0.896 
                 14,576 
               
               
                   
                 B6218 
                 0.792 
                 16,513 
               
               
                   
                 B7108 
                 0.948 
                 13,767 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE IV 
               
             
             
               
                   
               
               
                 Calculation of pI From 2D Gel 
               
               
                 Electrophoresis 
               
             
          
           
               
                   
                   
                 Relative 
                   
               
               
                   
                 Spot 
                 Focusing 
                 pI 
               
               
                   
                   
               
             
          
           
               
                   
                 Acidic end 
                 0.0000 
                 5.00 
               
               
                   
                 Basic end 
                 1.0000 
                 8.00 
               
               
                   
                 B1322 
                 0.0875 
                 5.26 
               
               
                   
                 B1418 
                 0.0798 
                 5.24 
               
               
                   
                 B2317 
                 0.1787 
                 5.54 
               
               
                   
                 B2422 
                 0.2015 
                 5.60 
               
               
                   
                 B2505 
                 0.1445 
                 5.43 
               
               
                   
                 B3410 
                 0.2700 
                 5.81 
               
               
                   
                 B4404 
                 0.3536 
                 6.06 
               
               
                   
                 B3406 
                 0.2510 
                 5.75 
               
               
                   
                 B5539 
                 0.4715 
                 6.41 
               
               
                   
                 B6519 
                 0.6464 
                 6.94 
               
               
                   
                 B6605 
                 0.6008 
                 6.80 
               
               
                   
                 B7408 
                 0.7338 
                 7.20 
               
               
                   
                 B1512 
                 0.0760 
                 5.23 
               
               
                   
                 B2412 
                 0.1293 
                 5.39 
               
               
                   
                 B4008 
                 0.3574 
                 6.07 
               
               
                   
                 B4206 
                 0.3422 
                 6.03 
               
               
                   
                 B3506 
                 0.2852 
                 5.86 
               
               
                   
                 B4424 
                 0.4639 
                 6.39 
               
               
                   
                 B5713 
                 0.5513 
                 6.65 
               
               
                   
                 B6014 
                 0.6768 
                 7.03 
               
               
                   
                 B6218 
                 0.6768 
                 7.03 
               
               
                   
                 B7108 
                 0.7452 
                 7.24 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE V 
               
             
             
               
                   
                   
               
               
                   
                   
                   
                 Amino Acid 
                   
               
               
                   
                 2D Gel 
                   
                 Sequence 
               
             
          
           
               
                   
                 MW 
                 pI 
                 MW 
                 pI 
               
               
                   
                   
               
             
          
           
               
                   
                 B1322 
                 21,738 
                 5.26 
                 24,489 
                 5.8 
               
               
                   
                 B1418 
                 27,780 
                 5.24 
                 20,433 
                 5.8 
               
               
                   
                 B2713 
                 20,830 
                 5.54 
                 28,962 
                 5.4 
               
               
                   
                 B2422 
                 29,411 
                 5.60 
                 26,970 
                 6.3 
               
               
                   
                   
                   
                   
                 28,253 
                 7.1 
               
               
                   
                 B2505 
                 30,766 
                 5.43 
                 26,970 
                 6.3 
               
               
                   
                   
                   
                   
                 28,253 
                 7.1 
               
               
                   
                 B3410 
                 28,170 
                 5.81 
                 26,970 
                 6.3 
               
               
                   
                   
                   
                   
                 28,253 
                 7.1 
               
               
                   
                 B4404 
                 27,029 
                 6.06 
                 26,970 
                 6.3 
               
               
                   
                   
                   
                   
                 28,253 
                 7.1 
               
               
                   
                 B3406 
                 29,849 
                 5.75 
                 34,364 
                 5.5 
               
               
                   
                 B5539 
                 40,755 
                 6.41 
                 44,994 
                 5.9 
               
               
                   
                 B6519 
                 31,245 
                 6.94 
                 34,019 
                 6.1 
               
               
                   
                 B6605 
                 52,417 
                 6.80 
                 77,051 
                 6.8 
               
               
                   
                 B7408 
                 28,572 
                 7.20 
                 33,074 
                 6.4 
               
               
                   
                 B1512 
                 40,755 
                 5.39 
                 45,206 
                 6.1 
               
               
                   
                 B2412 
                 25,977 
                 5.42 
                 38,088 
                 5.3 
               
               
                   
                   
                   
                   
                 38,130 
                 5.3 
               
               
                   
                 B4008 
                 11,943 
                 6.07 
                 45,206 
                 6.1 
               
               
                   
                 B4206 
                 17,687 
                 6.03 
                 45,206 
                 6.1 
               
               
                   
                 B3507 
                 33,321 
                 5.86 
                 39,008 
                 6.4 
               
               
                   
                 B4424 
                 32,777 
                 6.39 
                 39,008 
                 6.4 
               
               
                   
                 B5713 
                 79,034 
                 6.65 
                 77,051 
                 6.8 
               
               
                   
                 B6014 
                 14,576 
                 7.03 
                 45,206 
                 6.1 
               
               
                   
                 B6218 
                 16,513 
                 7.03 
                 16,430 
                 11.0 
               
               
                   
                   
                   
                   
                 16,044 
                 10.7 
               
               
                   
                 B7108 
                 13,767 
                 7.24 
                 12,932 
                 4.8 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE VI 
               
               
                   
               
               
                 Spot # 
                 Protein ID 
                 Accession # 
                 # of peptides 
                 Sequence # 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 B1332 
                 Immunoglobulin lambda chain 
                 106653 
                 2 
                  1 
               
               
                 B1418 
                 Alpha-1-microglobulin 
                 223373 
                 3 
                  2 
               
               
                 B2317 
                 Apolipoprotein A1 
                 178775 
                 9 
                  3 
               
               
                 B2422 
                 Inter-α-trypsin inhibitor family heavy chain 
                 1483187 
                 5 
                 4, 5 
               
               
                   
                 related protein (ITIHRP) 
               
               
                 B2505 
                 Inter-α-trypsin inhibitor family heavy chain 
                 1483187 
                 3 
                 4, 5 
               
               
                   
                 related protein (ITIHRP) 
               
               
                 B3406 
                 Apolipoprotein E3 
                 178849 
                 3 
                  6 
               
               
                   
                   
                 1942471 
                 4 
               
               
                 B3410 
                 Inter-α-trypsin inhibitor family heavy chain 
                 1483187 
                 4 
                 4, 5 
               
               
                   
                 related protein (ITIHRP) 
               
               
                 B4404 
                 Inter-α-trypsin inhibitor family heavy chain 
                 1402590 
                 3 
                 4, 5 
               
               
                   
                 related protein (ITIHRP) 
               
               
                 B5539 
                 Serum Albumin Protein 
                 28590 
                 5 
                  7 
               
               
                 B6519 
                 Lectin P35 3 
                 1669349 
                 3 
                  8 
               
               
                 B6605 
                 Transferrin 
                 4557871 
                 9 
                  9 
               
               
                 B7408 
                 Complement component C4A 
                 179674 
                 2 
                 10 
               
               
                 B1512 
                 Haptoglobin precursor [Contains: Haptoglobin 
                 P00738 
                 24 
                 11 
               
               
                   
                 alpha chain; Haptoglobin beta chain] 
               
               
                 B2412 
                 Apoptosis inhibitor expressed by Macrophages 
                 4102235 
                 9 
                 13, 14 
               
               
                   
                 Human secreted protein CD5L [ Homo sapiens ] 
                 37182111 
               
               
                 B4008 
                 Haptoglobin precursor [Contains: Haptoglobin 
                 P00738 
                 9 
                 11 
               
               
                   
                 alpha chain; Haptoglobin beta chain] 
               
               
                 B4206 
                 Haptoglobin precursor [Contains: Haptoglobin 
                 P00738 
                 11 
                 11 
               
               
                   
                 alpha chain; Haptoglobin beta chain] 
               
               
                 B3506 
                 Haptoglobin-related protein precursor 
                 P00739 
                 8 
                 12 
               
               
                 B4424 
                 Haptoglobin-related protein precursor 
                 P00739 
                 5 
                 12 
               
               
                 B5713 
                 Serotransferrin precursor (Transferrin) 
                 P02787 
                 6 
                 23 
               
               
                   
                 (Siderophilin) (Beta-1-metal-binding globulin) 
               
               
                 B6014 
                 Haptoglobin precursor [Contains: Haptoglobin 
                 P00738 
                 10 
                 11 
               
               
                   
                 alpha chain; Haptoglobin beta chain] 
               
               
                 B6218 
                 Unknown (protein for IMAGE: 3543815) [60S 
                 18042923 
                 1 
                 15-17 
               
               
                   
                 ribosomal protein L27a] 
               
               
                 B7108 
                 Reticulon-4 (Neurite outgrowth inhibitor) 
                 Q9NQC3 
                 1 
                 18-22 
               
               
                   
                 (Nogo protein) (Foocen) (Neuroendocrine- 
               
               
                   
                 specific protein) (NSP) (Neuroendocrine- 
               
               
                   
                 specific protein C homolog) (RTN-x) 
               
               
                   
                 (Reticulon-5) -  Homo sapiens  (Human) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE VII 
               
               
                   
               
             
             
               
                 span of LC/MS/MS identified peptides underlined 
               
               
                 MAWTVLLLGL LSHCTGSVTS YVLTQPPSVS VAPGKTASIT 
               
               
                   
               
               
                 CGGNNIGSKS VHWYQQKPGQ APVLVVYDDS DRPSGIPER F   
               
               
                   
               
               
                 
                   SGSNSGNTAT LTISRVEAGD EADYYCQVWD SSSDVVFGGG 
                 
               
               
                   
               
               
                 
                   TKLTVLGQPK AAPSVTLFPP SSEELQANKA TLVCLISDFY 
                 
               
               
                   
               
               
                   PGAVTVAWKA DSSPVKAGVE TTTPSK QSNN KYAASSYLSL 
               
               
                   
               
               
                 TPEQWKSHRS YSCQVTHEGS TVEKTVAPTE CS (SEQ ID NO: 1) 
               
               
                   
               
               
                 pI of Protein: 5.8 
               
               
                 Protein MW: 24489 
               
               
                 Accession #106653 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                 TABLE VIII 
               
               
                   
               
             
             
               
                 GPVPTPPDNI QVQENFNISR IYGKWYNLAI GSTCPLKIMD  RMTVSTLVLG EGATEAEISM TSTRWRKGVC   
                   
               
               
                   
               
               
                 
                   EETSGAYEKT DTDGKFLYHK SKWNITMESY VVHTNYDEYA IFLTKKFSRH HGPTITAKLY 
                 
               
               
                   
               
               
                   GRAPQLRETL LQDFRVVAQG VGIPEDSIFT MADR GECVPG EQEPEPILIP R (SEQ ID NO: 2) 
               
               
                   
               
               
                 MS-Digest Search Results: span of LC/MS/MS identified peptides underlinedpI of Protein: 5.8 
               
               
                 Protein MW: 20433 
               
               
                 Accession #223373 
               
               
                 Protein alternative names: 
               
               
                 HCP; IATIL; ITIL; OTTHUMP00000063975; UTI 
               
               
                 ALPHA-1 MICROGLOBULIN/BIKUNIN PRECURSOR 
               
               
                 Alpha-1-microglobulin/bikunin precursor (inter-alpha-trypsin inhibitor, light chain; protein HC) 
               
               
                 Alpha--microglobulin/bikunin precursor; inter-alpha-trypsin 
               
               
                 COMPLEX-FORMING GLYCOPROTEIN HETEROGENEOUS IN CHARGE INTER-ALPHA-TRYPSIN INHIBITOR 
               
               
                 The alpha-1-microglobulin (Protein HC) is a 31-kD, single chain plasma glycoprotein, which appears to 
               
               
                 be involved in regulation of the inflammatory process (Mendez et al., 1986). The alpha-1-microglobulin/ 
               
               
                 bikunin precursor gene (AMBP) codes for a precursor that splits into alpha-1-microglobulin, which 
               
               
                 belongs to the lipocalin superfamily, and bikunin (formerly HI-30, urinary trypsin inhibitor, inhibitor 
               
               
                 subunit of inter-alpha-trypsin inhibitor). The amino acid sequence of the parental protein is provided 
               
               
                 below: 
               
               
                 Parental precursor protein alternative names: 
               
               
                 Alpha-1-microglobulin (Protein HC) (Complex-forming glycoprotein heterogeneous in charge)/Inter-alpha- 
               
               
                 trypsin inhibitor light chain (ITI-LC) (Bikunin) (HI-30)] complex 
               
             
          
           
               
                 Parental protein sequence: span of LC/MS/MS identified peptides underlined: 
                   
                   
               
               
                 Signal peptide (italics): 
               
               
                 
                   MRSLGALLL LSACLAVSA 
                   G PVPTPPDNIQ VQENFNISRI YGKWYNLAIG STCPWLKKIM 
                 
                 60 
                 Alpha-1-microglobulin 
               
               
                   
                   
                 (bold letters) 
               
               
                 
                   D 
                   
                     RMTVSTLVL GEGATEAEIS MTSTRWRKGV CEETSGAYEK TDTDGKFLYH KSKWNITMES 
                   
                 
                 120 
               
               
                 
                   
                     YVVHTNYDEY AIFLTKKFSR HHGPTITAKL YGRAPQLRET LLQDFRVVAQ GVGIPEDSIF 
                   
                 
                 180 
               
               
                     TMADRGECVP GEQEPEPILI PR   VRR AVLPQ EEEGSGGGQL VTEVTKKEDS CQLGYSAGPC   
                 240 
                 Inter-α-Trypsin Inhibitor- 
               
               
                 
                   MGMTSRYFYN GTSMACETFQ YGGCMGNGNN FVTEKECLQT CRTVAACNLP IVRGPCRAFI 
                 
                 300 
                 light chain (Bikunin) 
               
               
                   QLWAFDAVKG KCVLFPYGGC QGNGNKFYSE KECREYCGVP GDGDEELLRF SN  (SEQ ID NO: 24) 
                 352 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
             
           
               
                 TABLE IX 
               
               
                   
               
             
             
               
                 Protein alternative names: 
               
               
                 Amyloidosis 
               
               
                 APOLIPOPROTEIN OF HIGH DENSITY LIPOPROTEIN APOA1/APOC3 FUSION GENE 
               
               
                 Apolipoprotein A-I 
               
               
                 Apolipoprotein A-I precursor 
               
               
                 Proapolipoprotein 
               
               
                 Parental Protein Full Sequence: NCBI accession # 178775: 
               
               
                 Span of LC/MS/MS identified peptides underlined: 
               
             
          
           
               
                 1 
                 RHFWQQDEPP QSPWDRVK   DL ATVYVDVLKD SGRDYVSQFE GSALGKQLNL KLLDNWDSVT     
                 Sequence identical to 
                   
               
               
                 61 
                 
                   
                     STFSKLREQL GPVTQEFWDN LEKETEGLRQ EMSKDLEEVK AKVQPYLDDF QKKWQEEMEL 
                   
                 
                 apolipoprotein A1 lacking 
               
               
                 121 
                 
                   
                     YRQKVEPLRA ELQEGARQKL HELQEKLSPL GEEMRDRARA HVDALRTHLA PYSDELRQRL 
                   
                 
                 the n-terminal signal 
               
               
                 181 
                 
                   
                     AARLEALKEN GGARLAEYHA KATEHLSTLS EKAKPALEDL RQGLLPVLES FKVSFLSALE 
                   
                 
                 peptide [MKAAVLTLAVLFLTGSQA] 
               
               
                 241 
                     EYTK   KLNTQ (SEQ ID NO: 3) 
               
               
                   
               
             
          
           
               
                 MS-Digest Search Results 
                   
               
               
                 pI of Protein: 5.4 
               
               
                 Protein MW: 28962 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE X 
               
               
                   
               
             
             
               
                 Protein Alternative Names: 
               
               
                 IHRP; ITIHL1,2; PK120 
               
               
                 INTER-ALPHA-TRYPSIN INHIBITOR, HEAVY CHAIN 4 
               
               
                 INTER-ALPHA-TRYPSIN INHIBITOR, HEAVY CHAIN-LIKE, 1,2 
               
               
                 INTER-ALPHA-TRYPSIN INHIBITOR, HEAVY CHAIN-RELATED PROTEIN 
               
               
                 PLASMA KALLIKREIN-SENSITIVE GLYCOPROTEIN 120 
               
               
                 Inter-alpha (globulin) inhibitor H4 (plasma Kallikrein-sensitive glycoprotein) 
               
               
                 Parental Protein Full Sequence: NCBI accession # 1483187: The tryptic peptide 35 kD processing product of ITIHRP is underlined 
               
               
                 SIGNAL PEPTIDE 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 The amino acid sequence of the inter-alpha-trypsin inhibitor heavy chain (H4) related protein composed of 930 amino acids (Mwt 103.4 kDa). The N-terminal 28 residues corresponded to a signal peptide for secretion. The N-terminal 600 residues of the mature form exhibited considerable homology to those of Inter-alpha trypsin inhibitor (ITI) heavy chains, while the C-terminal 300 residues showed no homology with the heavy chains and low homology with ATP-dependent proteases. Inter-alpha-trypsin inhibitor heavy chain (H4) related protein is readily cleaved into 75- and 35-kDa fragments when plasma is incubated at 37 degrees C. The cleaved site, Arg-Arg-Leu (RRL), is within a proline-rich region (Saguchi et al, J Biochem (1995)117:14-18). The 35-kDa cleavage fragment (underlined), expands the amino acid sequence starting at Arginine (R)-689 to Leucine (L)-930, is the fragment detected on 2D gel electrophoresis, marked as spots # 2422, 2505, 3410, and 4404 (Mwt 35 KD), it is most likely that the 4 protein spots corresponds to the 35 KD processing product in depicted in FIG. 1. [00510050] The sequence of peptides also exists in proteins with NCBI accession numbers: 1483187; 4096840; 7770149; 13432192; 55620443; 55732844, which belong to “Inter-alpha-trypsin inhibitor family heavy chain (H4) related protein family (ITIHRP; ITIH4). 
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XI 
               
               
                   
               
             
             
               
                 LC/MS/MS identified peptides span (underlined): 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XII 
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XIII 
               
               
                   
               
             
             
               
                 Protein alternative names: 
               
               
                 AD2; BROAD-BETALIPOPROTEINEMIA; FLOATING-BETALIPOPROTEINEMIA; 
               
               
                 MGC1571; apoprotein 
               
               
                 APOE APOLIPOPROTEIN E, DEFICIENCY OR DEFECT OF 
               
               
                 Alzheimer disease 2 (APOE*E4-associated, late onset) 
               
               
                 CORONARY ARTERY DISEASE, SEVERE, SUSCEPTIBILITY TO 
               
               
                 DYSBETALIPOPROTEINEMIA DUE TO DEFECT IN APOLIPOPROTEIN E-d 
               
               
                 FAMILIAL HYPERBETA- AND PREBETALIPOPROTEINEMIA 
               
               
                 FAMILIAL HYPERCHOLESTEROLEMIA WITH HYPERLIPEMIA 
               
               
                 HYPERLIPEMIA WITH FAMILIAL HYPERCHOLESTEROLEMIC XANTHOMATOSIS 
               
               
                 HYPERLIPOPROTEINEMIA, TYPE III 
               
               
                 Apolipoprotein E 
               
               
                 Apolipoprotein E precursor 
               
               
                 Apolipoprotein E3 
               
               
                 Parental Protein Full Sequence: NCBI accession # 1669349 and accession #178849: 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 pI of Protein: 5.5 
               
               
                 Protein MW: 34364 
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XIV 
               
               
                   
               
             
             
               
                 Protein alternative names: 
               
               
                 DKFZp779N1935; PRO1341 
               
               
                 ALB DYSALBUMINEMIC HYPERTHYROXINEMIA 
               
               
                 HYPERTHYROXINEMIA, DYSALBUMINEMIC 
               
               
                 PRO0883 protein 
               
               
                 albumin precursor 
               
               
                 serum albumin 
               
               
                 Cell growth inhibiting protein 42 
               
               
                 Protein sequence of Human Albumin precursor, NCBI accession # 28590: 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 * Protein sequence that corresponds to spot B5539 has an estimated molecular weight of ~45 kD and pI of ~6.2, which is calculated to correspond to albumin fragment sequence that starts at Aspartic acid (D) residue number 211* extends to the C-terminal Leucine (L) residue # 609 and expands the LC-MS/MS identified peptides (underlined). 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE XV 
               
               
                   
               
             
             
               
                 Protein alternative names: 
               
               
                 Ficolin-2 precursor (Collagen/fibrinogen domain-containing protein 2) 
               
               
                 (Ficolin-B) (Ficolin B) (Serum Lectin p35) (EBP-37) (Heckling) (L-Ficolin). 
               
               
                 Parental Protein Full Sequence: NCBI accession # 1669349: 
               
               
                 Span op LC/MS/MS identified peptides underlined: 
               
             
          
           
               
                 1 
                 MELDRAVGVL GAATLLLSFL GMAWALQAAD TCPEVK   MVGL EGSDKLTILR GCPGLPGAPG     
                   
               
               
                   
               
               
                 61 
                 
                     DKGEAGTNGK RGERGPPGPP GKAGPPGPNG  APGEPQPCLT GPRTCKDLLD RGHFLSGWHT 
                 
               
               
                   
               
               
                 121 
                 
                   
                     IYLPDCRPLT VLCDMDTDGG GWTVFQRRVD GSVDFYRDWA TYKQGFGSRL GEFWLGNDNI 
                   
                 
               
               
                   
               
               
                 181 
                     HALTAQGTSE LRVDLVDFED NYQFAK   YRSF KVADEAEKYN LVLGAFVEGS AGDSLTFHNN 
               
               
                   
               
               
                 241 
                 QSFSTKDQDN DLNTGNCAVM FQGAWWYKNC HVSNLNGRYL RGTHGSFANG INWKSGKGYN 
               
               
                   
               
               
                 301 
                 YSYKVSEMKV RPA (SEQ ID NO: 8) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
               
               
               
             
               
               
             
           
               
                 TABLE XVI 
               
               
                   
               
             
             
               
                 Span of LC/MS/MS identified peptides underlined 
                   
               
               
                 Protein Sequence: NCBI Accession# 4557871 
               
             
          
           
               
                 1 
                 MRLAVGALLV CAVLGLCLAV PDKTVRWCAV SEHEATKCQS FRDHMKSVIP SDGPSVACVK 
                   
               
               
                 61 
                 KASYLDCIRA IAANEADAVT LDAGLVYDAY LAPNNLKPVV    AEFYGSKEDP QTFYYAVAVV     
               
               
                 121 
                 
                   
                     KKDSGFQMNQ LRGKKSCHTG LGRSAGWNIP IGLLYCDLPE PRKPLEKAVA NFFSGSCAPC 
                   
                 
               
               
                 181 
                 
                   
                     ADGTDFPQLC QLCPGCGCST LNQYFGYSGA FKCLKDGAGD VAFVKHSTIF ENLANKADRD 
                   
                 
               
               
                 241 
                 
                   
                     QYELLCLDNT RKPVDEYKDC HLAQVPSHTV VARSMGGKED LIWELLNQAQ EHFGKDKSKE 
                   
                 
               
               
                 301 
                 
                   
                     FQLFSSPHGK DLLFKDSAHG FLKVPPRMDA KMYLGYEYVT AIRNLREGTC PEAPTDECKP 
                   
                 
               
               
                 361 
                 
                   
                     VKWCALSHHE RLKCDEWSVN SVGKIECVSA ETTEDCIAKI MNGEADAMSL DGGFVYIAGK 
                   
                 
               
               
                 421 
                 
                   
                     CGLVPVLAEN YNKSDNCEDT PEAGYFAVAV VKKSASDLTW DNLKGKKSCH TAVGRTAGWN 
                   
                 
               
               
                 481 
                 
                   
                     IPMGLLYNKI NHCRFDEFFS EGCAPGSKKD SSLCKLCMGS GLNLCEPNNK EGYYGYTGAF 
                   
                 
               
               
                 541 
                 
                   
                     RCLVEKGDVA FVKHQTVPQN TGGKNPDPWA KNLNEKDYEL LCLDGTRKPV EEYANCHLAR 
                   
                 
               
               
                 601 
                 
                   
                     APNHAVVTRK DKEACVHKIL RQQQHLFGSN VTDCSGNFCL FRSETKDLLF RDDTVCLAKL 
                   
                 
               
               
                 661 
                     HDRNTYEKYL GEEYVK   AVGN LRKCSTSSLL EACTFRRP (SEQ ID NO: 9) 
               
               
                   
               
             
          
           
               
                 pI of the Protein: 6.8 
                   
               
               
                 Molecular Weight: 77050 Da 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XVII 
               
               
                   
               
             
             
               
                 Protein alternative names: 
               
               
                 C4A2; C4A3; C4A4; C4A6; C4S; CO4 
               
               
                 C4A anaphylatoxin 
               
               
                 COMPLEMENT COMPONENT 4S 
               
               
                 RODGERS FORM OF C4 COMPLEMENT COMPONENT 4A DEFICIENCY 
               
               
                 acidic C4 
               
               
                 c4 propeptide 
               
               
                 complement component 4A preproprotein 
               
               
                 complement component C4B 
               
               
                 Span of LC/MS/MS Tryptic peptides underlined 
               
               
                 1          11         21         31         41         51         61         71 
               
               
                 EAPK   VVEEQE SR VHYTVCIW RNGKVGLSGM AIADVTLLSG FHALRADLEK LTSLSDRYVS HFETEGPHVL LYFDSVPTSR   
               
               
                   
               
               
                 81         91         101        111        121        131        141        151 
               
               
                 
                   ECVGFEAVQE VPVGLVQPAS ATLYDYYNPE RRCSVFYGAP SKSRLLATLC SAEVCQCAEG KCPRQRRALE R GLQDEDGYR   
                 
               
               
                   
               
               
                 161        171        181        191        201        211        221        231 
               
               
                 MKFACYYPRV EYGFQVKVLR EDSRAAFRLF ETKITQVLHF TKDVKAAANQ MRNFLVRASC RLRLEPGKEY LIMGLDGATY 
               
               
                   
               
               
                 241        251        261        271        281        291 
               
               
                 DLEGHPQYLL DSNSWIEEMP SE R LC R ST R Q  R AACAQLNDF LQEYGTQGCQ  V  (SEQ ID NO: 10) 
               
               
                   
               
               
                 pI of Protein: 6.4 
               
               
                 Protein MW: 33074 Da 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XVIII 
               
               
                   
               
             
             
               
                 NCBI Accession # 179674 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
           
               
                 TABLE XIX 
               
               
                   
               
             
             
               
                 Accession # P00738. Haptoglobin precu...[gi: 123508] 
                   
               
               
                 Precursor Contains: Haptoglobin alpha chain; Haptoglobin beta chain 
               
               
                 MSALGAVIALLLWGQLFAVDSGNDVTDIADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGD 
               
               
                   
               
               
                 GVYTLNDKKQWINKAVGDKLPECEADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVY 
               
               
                   
               
               
                 TLNNEKQWINKAVGDKLPECEAVCGKPKNPANPVQRILGGHLDAKGSFPWQAKMVSHHNLTT 
               
               
                   
               
               
                 GATLINEQWLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLHPNYSQVDIGLIK 
               
               
                   
               
               
                 LKQKVSVNERVMPICLPSKDYAEVGRVGYVSGWGRNANFKFTDHLKYVMLPVADQDQCIRHY 
               
               
                   
               
               
                 EGSTVPEKKTPKSPVGVQPILNEHTFCAGMSKYQEDTCYGDAGSAFAVHDLEEDTWYATGILSF 
               
               
                   
               
               
                 DKSCAVAEYGVYVKVTSIQDWVQKTIAEN (SEQ ID NO: 11) 
               
               
                   
               
               
                 pI of Protein: 6.1 
               
               
                 Protein MW: 45206 
               
               
                 2D gel Results 
               
               
                 B1512: MW 38648; 
               
               
                 B4008: MW 12257 
               
               
                 B4206: MW 17699 
               
               
                 B6014: MW 14768 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XX 
               
               
                   
               
             
             
               
                 P00739. Haptoglobin-relat...[gi: 123510] 
               
               
                 MSDLGAVISLLLWGRQLFALYSGNDVTDISDDRFPKPPEIANGYVEHLFRYQCKNYYRLRTEGD 
               
               
                   
               
               
                 GVYTLNDKKQWINKAVGDKLPECEAVCGKPKNPANPVQRILGGHLDAKGSFPWQAKMVSHHN 
               
               
                   
               
               
                 LTTGATLINEQWLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLHPNYHQVDIGL 
               
               
                   
               
               
                 IKLKQKVLVNERVMPICLPSKNYAEVGRVGYVSGWGQSDNFKLTDHLKYVMLPVADQYDCITH 
               
               
                   
               
               
                 YEGSTCPKWKAPKSPVGVQPILNEHTFCVGMSKYQEDTCYGDAGSAFAVHDLEEDTWYAAGIL 
               
               
                   
               
               
                 SFDKSCAVAEYGVYVKVTSIQDWVQKTIAEN (SEQ ID NO: 12) 
               
               
                   
               
               
                 pI of Protein: 6.4 
               
               
                 Protein MW: 39008 
               
               
                 ZD gel Results 
               
               
                 B3606: MW 32011; 
               
               
                 B4424: MW 31521 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE XXI 
               
             
             
               
                   
               
               
                 Peptides identified by LC MS/MS of 
               
               
                 in-gel tryptic digests: 
               
             
          
           
               
                 Accession 
                 Sequence 
                 Name 
               
               
                   
               
               
                 gi|4102235; 
                 CSGEEQSLEQCQHR 
                 AIM [ Homo sapiens ]; CD5L 
               
               
                 gi|37182111 
                 LVGGDNLCSGR 
                 [ Homo sapiens ] 
               
               
                   
                 IWLDNVR 
               
               
                   
                 CYGPGVGR 
               
               
                   
                 EATLQDCPSGPWGK 
               
               
                   
                 CSGEEQSLEQCQHR 
               
               
                   
                 HQNQWY 
               
               
                   
                 IWLDNVR 
               
               
                   
                 IWLDNVR 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
           
               
                 TABLE XXII 
               
               
                   
               
             
             
               
                 Accession #AAD01446 [gi: 4102235] Sequence #13: 
                   
               
               
                 Span of LC/MS/MS identified peptides underlined 
               
               
                 MALLFSLILAICTRPGFLASPSGVRLVGGLHRCEGRVEVEQKGQWGTVCDDGWDIKDVAVLCRE 
               
               
                   
               
               
                 LGCGAASGTPSGILYEPPAEKEQKVLIQSVSCTGTEDTLAQCEQEEVYDCSHDEDAGASCENPES 
               
               
                   
               
               
                 SFSPVPEGVRLADGPGHCKGRVEVK   HQNQWYTVCQTGWSLRAAKVVCRQLGCGRAVLTQK     
               
               
                   
               
               
                 
                   
                     RCNKHAYGRKPIWLSQMSCSGREATLQDCPSGPWGKNTCNHDEDTWVECEDPFDLRLVG 
                   
                 
               
               
                   
               
               
                 
                   
                     GDNLCSGRLEVLHKGVWGSVCDDNWGEKEDQVVCKQLGCGKSLSPSFRDRKCYGPGVG 
                   
                 
               
               
                   
               
               
                     RIWLDNVRCSGEEQSLEQCQHR   FWGFHDCTHQEDVAVICSG (SEQ ID NO: 13) 
               
               
                   
               
               
                 pI of Protein: 5.3 
               
               
                 Protein MW: 38088 
               
               
                 2D gel Results: 
               
               
                 B2412: MW 25359 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
           
               
                 TABLE XXIII 
               
               
                   
               
             
             
               
                 Accession #AAQ88858. [gi: 37182111]; Sequence #14: 
                   
               
               
                 Span of LC/MS/MS identified peptides underlined 
               
               
                 MALLFSLILAICTRPGFLASPSGVRLVGGLHRCEGRVEVEQKGQWGTVCDDGWDIKDVAVLCRE 
               
               
                   
               
               
                 LGCGAASGTPSGILYEPPAEKEQKVLIQSVSCTGTEDTLAQCEQEEVYDCSHDEDAGASCENPES 
               
               
                   
               
               
                 SFSPVPEGVRLADGPGHCKGRVEVK   HQNQWYTVCQTGWSLRAAKVVCRQLGCGRAVLTQK     
               
               
                   
               
               
                 
                   
                     RCNKHAYGRKPIWLSQMSCSGREATLQDCPSGPWGKNTCNHDEDTWVECEDPFDLRLVG 
                   
                 
               
               
                   
               
               
                 
                   
                     GDNLCSGRLEVLHKGVWGSVCDDNWGEKEDQVVCKQLGCGKSLSPSFRDRKCYGPGVG 
                   
                 
               
               
                   
               
               
                     RIWLDNVRCSGEEQSLEQCQHR   FWGFHDCTHQEDVAVICSV (SEQ ID NO: 14) 
               
               
                   
               
               
                 pI of Protein: 5.3 
               
               
                 Protein MW: 38130 
               
               
                 2D gel Results: 
               
               
                 B2412: MW 25359 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
           
               
                 TABLE XXIV 
               
               
                   
               
             
             
               
                 MRLAVGALLV CAVLGLCLAV PDKTVRWCAV SEHEATKCQS FRDHMKSVIP SDGPSVACVK 
                   
               
               
                   
               
               
                 KASYLDCIRA IAANEADAVT LDAGLVYDAY LAPNNLKPVV AEFYGSKEDP QTFYYAVAVV 
               
               
                   
               
               
                 KKDSGFQMNQ LRGKKSCHTG LGRSAGWNIP IGLLYCDLPE PRKPLEKAVA NFFSGSCAPC 
               
               
                   
               
               
                 ADGTDFPQLC QLCPGCGCST LNQYFGYSGA FKCLKDGAGD VAFVKHSTIF ENLANKADRD 
               
               
                   
               
               
                 QYELLCLDNT RKPVDEYKDC HLAQVPSHTV VARSMGGKED LIWELLNQAQ EHFGKDKSKE 
               
               
                   
               
               
                 FQLFSSPHGK DLLFKDSAHG FLKVPPRMDA KMYLGYEYVT AIRNLREGTC PEAPTDECKP 
               
               
                   
               
               
                 VKWCALSHHE RLKCDEWSVN SVGKIECVSA ETTEDCIAKI MNGEADAMSL DGGFVYIAGK 
               
               
                   
               
               
                 CGLVPVLAEN YNKSDNCEDT PEAGYFAVAV VKKSASDLTW DNLKGKKSCH TAVGRTAGWN 
               
               
                   
               
               
                 IPMGLLYNKI NHCRFDEFFS EGCAPGSKKD SSLCKLCMGS GLNLCEPNNK EGYYGYTGAF 
               
               
                   
               
               
                 RCLVEKGDVA FVKHQTVPQN TGGKNPDPWA KNLNEKDYEL LCLDGTRKPV EEYANCHLAR 
               
               
                   
               
               
                 APNHAVVTRK DKEACVHKIL RQQQHLFGSN VTDCSGNFCL FRSETKDLLF RDDTVCLAKL 
               
               
                   
               
               
                 HDRNTYEKYL GEEYVKAVGN LRKCSTSSLL EACTFRRP (SEQ ID NO: 23) 
               
               
                   
               
               
                 pI of Protein: 6.8 
               
               
                 Protein MW: 77051 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
           
               
                 TABLE XXV 
               
               
                   
               
             
             
               
                 Accession AAH20169. [gi: 18042923] 
                   
               
               
                 Span of LC/MS/MS identified peptides underlined: 
               
               
                 PSRLRKTRKLRGHVSHGHGRIGKHRKHPGGRGNAGGLHHHRINFDKYHPGYFGKVGMKHYHLKRNQSFCP 
               
               
                   
               
               
                 TVNLDKLWTLVSEQTRVNAAKNKTGAAPIIDVVRSGYYKVLGKGK   LPKQPVIVK   AKFFSRRAEEKIKSVG 
               
               
                   
               
               
                 GACVLVA (SEQ ID NO: 15) 
               
               
                   
               
               
                 gb|AAH20169.1|AAH20169 Unknown (protein for IMAGE: 3543815) [ Homo sapiens ] 
               
               
                 Length = 147 
               
               
                 pI of Protein: 11.0 
               
               
                 Protein MW: 16430 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
           
               
                 TABLE XXVI 
               
               
                   
               
             
             
               
                 Accession NP_000981 [gi: 4506625] 
                   
               
               
                 Span of LC/MS/MS identified peptides underlined: 
               
               
                 MPSRLRKTQKLRGHVSHGHGRIGKLQKHPRGHSNAGGMHHHRINFNKYYPGYFGKVGMRYYLKRNQTVSL 
               
               
                   
               
               
                 DKLWTLVSEQTQVNAAKNKPGAAPLIDVVQSGYYKVLGKEK   LPKQPVIVK   AKFFSRRAEKIKGVKGTCVL 
               
               
                   
               
               
                 VA (SEQ ID NO: 16) 
               
               
                   
               
               
                 ref|NP_000981.1|ribosomal protein L27a [ Homo sapiens ] 
               
               
                 sp|P46776|RL27A HUMAN 60S ribosomal protein L27a 
               
               
                 gb|AAA85656.1|ribosomal protein L27a 
               
               
                 dbj|BAA77361.1|ribosomal protein L27A [ Homo sapiens ] 
               
               
                 gb|AAH05326.1|Ribosomal protein L27a [ Homo sapiens ] 
               
               
                 gb|EAW68619.1|ribosomal protein L27a [ Homo sapiens ] 
               
               
                 prf||2113200C ribosomal protein L27a 
               
               
                 Length = 148 
               
               
                 pI of Protein: 10.7 
               
               
                 Protein MW: 16044 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
           
               
                 TABLE XXVII 
               
               
                   
               
             
             
               
                 Accession EAW75952 hCG38472 [gi: 119596358][[;]] 
                   
               
               
                 Span of LC/MS/MS identified peptides underlined: 
               
               
                 MPSRLRKTQKLRGHVSHGHGRIGKLQKHPRGHSNAGGMHHHRINFNKYYPGYFGKVGMRYYL 
               
               
                   
               
               
                 KRNQTVSLDKLWTLVSEQTQVNAAKNKPGAAPLIDVVQSGYYKVLGKEK   LPKQPVIVK   AKFFS 
               
               
                   
               
               
                 RRAEKIKGVKGTCVLVA (SEQ ID NO: 16) 
               
               
                   
               
               
                 gb|EAW75952.1|hCG38472 [ Homo sapiens ] 
               
               
                 Length = 142 
               
               
                 pI of Protein: 10.7 
               
               
                 Protein MW: 16044 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
           
               
                 TABLE XXVIII 
               
               
                   
               
             
             
               
                 Accession Q9NQC3. [gi: 17369290]; 
                   
               
               
                 Span of LC/MS/MS identified peptides underlined: 
               
               
                 MEDLDQSPLV SSSDSPPRPQ PAFKYQFVRE PEDEEEEEEE EEEDEDEDLE ELEVLERKPA 
               
               
                   
               
               
                 AGLSAAPVPT APAAGAPLMD FGNDFVPPAP RGPLPAAPPV APERQPSWDP SPVSSTVPAP 
               
               
                   
               
               
                 SPLSAAAVSP SKLPEDDEPP ARPPPPPPAS VSPQAEPVWT PPAPAPAAPP STPAAPKRRG 
               
               
                   
               
               
                 SSGSVDETLF ALPAASEPVI RSSAENMDLK EQPGNTISAG QEDFPSVLLE TAASLPSLSP 
               
               
                   
               
               
                 LSAASFKEHE YLGNLSTVLP TEGTLQENVS EASKEVSEKA KTLLIDRDLT EFSELEYSEM 
               
               
                   
               
               
                 GSSFSVSPK   A ESAVIVANPR    EEIIVKNKDE EEKLVSNNIL HNQQELPTAL TKLVKEDEVV 
               
               
                   
               
               
                 SSEKAKDSFN EKRVAVEAPM REEYADFKPF ERVWEVKDSK EDSDMLAAGG KIESNLESKV 
               
               
                   
               
               
                 DKKCFADSLE QTNHEKDSES SNDDTSFPST PEGIKDRSGA YITCAPFNPA ATESIATNIF 
               
               
                   
               
               
                 PLLGDPTSEN KTDEKKIEEK KAQIVTEKNT STKTSNPFLV AAQDSETDYV TTDNLTKVTE 
               
               
                   
               
               
                 EVVANMPEGL TPDLVQEACE SELNEVTGTK IAYETKMDLV QTSEVMQESL YPAAQLCPSF 
               
               
                   
               
               
                 EESEATPSPV LPDIVMEAPL NSAVPSAGAS VIQPSSSPLE ASSVNYESIK HEPENPPPYE 
               
               
                   
               
               
                 EAMSVSLKKV SGIKEEIKEP ENI NAAL QET EAPYISIACD LIKETKLSAE PAPDFSDYSE 
               
               
                   
               
               
                 MAKVEQPVPD HSELVEDSSP DSEPVDLFSD DSIPDVPQKQ DETVMLVKES LTETSFESMI 
               
               
                   
               
               
                 EYENKEKLSA LPPEGGKPYL ESFKLSLDNT KDTLLPDEVS TLSKKEKIPL QMEELSTAVY 
               
               
                   
               
               
                 SNDDLFISKE AQIRETETFS DSSPIEIIDE FPTLISSKTD SFSKLAREYT DLEVSHKSEI 
               
               
                   
               
               
                 ANAPDGAGSL PCTELPHDLS LKNIQPKVEE KISFSDDFSK NGSATSKVLL LPPDVSALAT 
               
               
                   
               
               
                 QAEIESIVKP KVLVKEAEKK LPSDTEKEDR SPSAIFSAEL SKTSVVDLLY WRDIKKTGVV 
               
               
                   
               
               
                 FGASLFLLLS LTVFSIVSVT AYIALALLSV TISFRIYKGV IQAIQKSDEG HPFRAYLESE 
               
               
                   
               
               
                 VAISEELVQK YSNSALGHVN CTIKELRRLF LVDDLVDSLK FAVLMWVFTY VGALFNGLTL 
               
               
                   
               
               
                 LILALISLFS VPVIYERHQA QIDHYLGLAN KNVKDAMAKI QAKIPGLKRK AE 
               
               
                 (SEQ ID NO: 18) 
               
               
                   
               
               
                 pI of Protein: 4.4 
               
               
                 Protein MW: 129932 
               
               
                 Alternative names for B7108: (Neurite outgrowth inhibitor) (Nogo protein) (Foocen) 
               
               
                 (Neuroendocrine-specific protein) (NSP) (Neuroendocrine-specific protein C homolog) (RTN-x) 
               
               
                 (Reticulon-5) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XXIX 
               
               
                   
               
             
             
               
                 Span of LC/MS/MS identified peptides underlined: 
               
               
                 DETLFALPAA SEPVIRSSAE NMDLKEQPGN TISAGQEDFP SVLLETAASL PSLSPLSAAS 
               
               
                   
               
               
                 FKEHEYLGNL STVLPTEGTL QENVSEASKE VSEKAKTLLI DRDLTEFSEL EYSEMGSSFS 
               
               
                   
               
               
                 VSPK   AESAVI VANPR    (SEQ ID NO: 19) 
               
               
                   
               
               
                 pI of Protein: 4.3 
               
               
                 Protein MW: 14420 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XXX 
               
               
                   
               
             
             
               
                 Span of LC/MS/MS identified peptides underlined: 
               
               
                     AESAVI VANPR   EEIIV KNKDEEEKLV SNNILHNQQE LPTALTKLVK EDEVVSSEKA 
               
               
                   
               
               
                 KDSFNEKRVA VEAPMREEYA DFKPFERVWE VKDSKEDSDM LAAGGKIESN LESKVDKK 
               
               
                   
               
               
                 (SEQ ID NO: 20) 
               
               
                   
               
               
                 pI of Protein: 4.8 
               
               
                 Protein MW: 12932 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XXXI 
               
               
                   
               
             
             
               
                 Span of LC/MS/MS identified peptides underlined: 
               
               
                     AESAVI VANPR   EEIIV KNKDEEEKLV SNNILHNQQE LPTALTKLVK EDEVVSSEKA 
               
               
                   
               
               
                 KDSFNEKRVA VEAPMREEYA DFKPFERVWE VKDSKEDSDM LAAGGKIESN LESKVDKK 
               
               
                   
               
               
                 CF ADSLEQTNHE  K DSESSNDDT SFPSTPEGI K  D R  (SEQ ID NO: 21) 
               
               
                   
               
               
                 pI of Protein: 4.6 
               
               
                 Protein MW: 16701 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
           
               
                 TABLE XXXII 
               
               
                   
               
             
             
               
                 Span of LC/MS/MS identified peptides underlined: 
               
               
                     AESAVI VANPR   EEIIV KNKDEEEKLV SNNILHNQQE LPTALTKLVK EDEVVSSEKA 
               
               
                   
               
               
                 KDSFNEKRVA VEAPMREEYA DFKPFERVWE VKDSKEDSDM LAAGGKIESN LESKVDKK 
               
               
                   
               
               
                 CF ADSLEQTNHE  K  (SEQ ID NO: 22) 
               
               
                   
               
               
                 pI of Protein: 4.8 
               
               
                 Protein MW: 14435 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XXXIII 
               
               
                   
               
             
             
               
                 a): B2422, down-regulated in breast cancer 
               
             
          
           
               
                 ITI(H4) RP 35 KD 
                   
                   
                   
                   
                   
                   
               
               
                 Isoform Protein 
               
               
                 Spot B2422 
               
               
                 Retrospective Samples 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 N 
                 192 
                 64 
                 45.8 
                 3.63 
                 32.0 
                 39.3 
               
               
                 B9 
                 344 
                 115 
                 45.8 
                 2.61 
                 32.0 
                 64.1 
               
               
                 BC 
                 294 
                 98 
                 34.6 
                 3.09 
                 8.8 
                 56.1 
               
               
                   
               
             
          
           
               
                 b): B2505, up-regulated in breast cancer* 
               
             
          
           
               
                 ITI(H4) RP 35 KD 
                   
                   
                   
                   
                   
                   
               
               
                 Isoform Protein 
               
               
                 Spot B2505 
               
               
                 Retrospective Samples 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 N 
                 192 
                 64 
                 104.3 
                 4.39 
                 95.5 
                 54.0 
               
               
                 B9 
                 344 
                 115 
                 101.7 
                 3.10 
                 88.2 
                 74.9 
               
               
                 BC 
                 294 
                 98 
                 114.6 
                 5.02 
                 89.8 
                 92.6 
               
               
                   
               
             
          
           
               
                 c): B3410, down-regulated in breast cancer 
               
             
          
           
               
                 ITI(H4) RP 35 KD 
                   
                   
                   
                   
                   
                   
               
               
                 Isoform Protein 
               
               
                 Spot B3410 
               
               
                 Retrospective Samples 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 N 
                 192 
                 64 
                 19.3 
                 1.69 
                 13.3 
                 18.5 
               
               
                 B9 
                 344 
                 115 
                 17.6 
                 1.17 
                 10.1 
                 29.2 
               
               
                 BC 
                 294 
                 98 
                 14.6 
                 1.41 
                 0.0 
                 26.6 
               
               
                   
               
             
          
           
               
                 d): B4404, down-regulated in breast cancer 
               
             
          
           
               
                 ITI(H4) RP 35 KD 
                   
                   
                   
                   
                   
                   
               
               
                 Isoform Protein 
               
               
                 Spot B4404 
               
               
                 Retrospective Samples 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 N 
                 192 
                 64 
                 21.2 
                 1.43 
                 17.0 
                 19.4 
               
               
                 B9 
                 344 
                 115 
                 23.1 
                 1.41 
                 16.9 
                 16.5 
               
               
                 BC 
                 294 
                 98 
                 16.0 
                 1.35 
                 10.0 
                 21.9 
               
               
                   
               
             
          
           
               
                 e) Sum of B2422 + B2505 + B3410 + B4404: 
               
               
                 “down-regulated” in breast cancer* 
               
             
          
           
               
                 ITI(H4) RP 35 KD 
                   
                   
                   
                   
                   
                   
               
               
                 PPM Sum of Isoforms 
               
               
                 B2422 + B2505 + 
               
               
                 B3410 + B4404 
               
               
                 Retrospective Samples 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 N 
                 192 
                 64 
                 190.6 
                 9.35 
                 168.5 
                 108.5 
               
               
                 B9 
                 344 
                 115 
                 188.2 
                 6.10 
                 161.8 
                 144.3 
               
               
                 BC 
                 294 
                 98 
                 179.9 
                 9.46 
                 117.4 
                 137.4 
               
               
                   
               
               
                 *One of the isoforms that make up the sum, B2505 (b), is actually up-regulated. This is due to the lack of a significant down-regulation of B2505 in non-DCIS breast cancer patients (FIG. 4b; Table XXXVb). Thus, the up-regulation observed comes from the contribution from the more pronounced up regulation in the DCIS breast cancer patients within the breast cancer group. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE XXXIV 
               
               
                   
               
               
                 Total ITI (H4) RP 35 KD Proteins = Sum of Protein Spots 
               
               
                 B2422 + B2505 + B3410 + B4404 
               
               
                 Blood Serum Concentration Measured as 2D Gel Spot Density (PPM) 
               
               
                 Retrospective vs. Prospective Samples 
               
               
                   
               
             
             
               
                 a) Concentration in 2D gel spot density: 
               
               
                 Total ITI (H4) RP 35 KD Proteins = Sum of 2D gel spot density (PPM) of protein spots 
               
               
                 B2422 + B2505 + B3410 + B4404 
               
               
                   
               
             
          
           
               
                   
                 ITI(H4) RP 35 KD 
                   
                   
                   
                   
                   
                   
               
               
                   
                 PPM Sum of Isoforms 
               
               
                   
                 B2422 + B2505 + B3410 + B4404 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 Retrospective 
                 N 
                 192 
                 64 
                 190.6 
                 9.35 
                 168.5 
                 108.5 
               
               
                 Samples 
                 B9 
                 344 
                 115 
                 188.2 
                 6.10 
                 161.8 
                 144.3 
               
               
                   
                 N + B9 
                 536 
                 179 
                 189.1 
                 5.15 
                 165.5 
                 127.7 
               
               
                   
                 BC 
                 294 
                 98 
                 179.9 
                 9.46 
                 117.4 
                 137.4 
               
               
                 Prospective 
                 N 
                 48 
                 16 
                 282.2 
                 21.96 
                 273.0 
                 163.4 
               
               
                 Samples 
                 BC 
                 36 
                 12 
                 212.6 
                 12.16 
                 223.8 
                 118.6 
               
               
                   
               
               
                   
                 Total ITI (H4) RP 35 KD 
               
               
                   
                 Isoform Spots = B2422 + 
               
               
                   
                 B2505 + B2410 + B4404 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 Retrospective 
                 N 
                 240 
                 80 
                 209.74 
                 15.11 
                 177.29 
                 108.23 
               
               
                 and Prospective 
                 B9 
                 327 
                 109 
                 188.00 
                 10.46 
                 165.76 
                 148.48 
               
               
                 Combined 
                 N + B9 
                 567 
                 189 
                 197.20 
                 8.80 
                 171.88 
                 136.39 
               
               
                 With and 
                 Combined BC 
                 312 
                 104 
                 188.70 
                 15.33 
                 127.71 
                 161.10 
               
               
                 Without DCIS 
                 Non-DCIS BC 
                 222 
                 74 
                 148.96 
                 13.51 
                 106.10 
                 117.82 
               
               
                   
                 DCIS BC 
                 90 
                 30 
                 286.72 
                 36.00 
                 218.14 
                 291.90 
               
               
                   
               
             
          
           
               
                 b) Differential Expression in Fold of Average Normal Concentration; 
               
               
                 Concentration = Fold of Average 2D Gel Spot Density (PPM)* 
               
               
                 Total ITI(H4) RP 35 KD = Protein Spots B2422 + B2505 + B3410 + B4404 
               
               
                   
               
             
          
           
               
                   
                 Retrospective 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 Min 
                 Max 
                 IQR 
               
               
                   
               
               
                 b1 
                 N 
                 192 
                 64 
                 1.000 
                 0.049 
                 0.884 
                 0.199 
                 5.714 
                 0.569 
               
               
                   
                 B9 
                 344 
                 115 
                 0.988 
                 0.032 
                 0.849 
                 0.148 
                 3.292 
                 0.757 
               
               
                   
                 BC 
                 294 
                 98 
                 0.944 
                 0.050 
                 0.616 
                 0.020 
                 4.933 
                 0.721 
               
               
                   
               
               
                   
                 Prospective 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 Min 
                 Max 
                 IQR 
               
               
                   
               
               
                 b2 
                 N 
                 51 
                 17 
                 1.000 
                 0.075 
                 0.931 
                 0.214 
                 2.628 
                 0.534 
               
               
                   
                 BC 
                 39 
                 13 
                 0.775 
                 0.041 
                 0.848 
                 0.277 
                 1.203 
                 0.392 
               
               
                   
               
               
                   
                 Combined +/− DCIS 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 Min 
                 Max 
                 IQR 
               
               
                   
               
               
                 b3 
                 N + B9 
                 567 
                 189 
                 1.003 
                 0.044 
                 0.876 
                 0.211 
                 4.965 
                 0.664 
               
               
                   
                 Non-DCIS BC 
                 234 
                 78 
                 0.747 
                 0.085 
                 0.538 
                 0.048 
                 3.624 
                 0.601 
               
               
                   
                 DCIS BC 
                 90 
                 30 
                 1.482 
                 0.191 
                 1.130 
                 0.231 
                 4.548 
                 1.566 
               
               
                   
               
               
                 *Determined separately for prospective and retrospective samples, then combined in b3 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XXXV 
               
               
                   
               
               
                 Fold of Average Normal PPM or μg protein/ml of blood serum 
               
               
                   
               
             
             
               
                 a. 
               
             
          
           
               
                 ITI (H4) RP 35 KD 
                   
                   
                   
                   
                   
                   
               
               
                 Isoform Spot B2422 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 N 
                 240 
                 80 
                 1.008 
                 0.110 
                 0.812 
                 0.828 
               
               
                 B9 
                 327 
                 109 
                 1.011 
                 0.099 
                 0.758 
                 1.452 
               
               
                 N + B9 
                 567 
                 189 
                 1.009 
                 0.073 
                 0.803 
                 1.144 
               
               
                 Combined BC 
                 312 
                 104 
                 0.799 
                 0.108 
                 0.312 
                 1.241 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.567 
                 0.105 
                 0.181 
                 0.800 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.372 
                 0.242 
                 1.131 
                 2.016 
               
               
                   
               
             
          
           
               
                 b. 
               
             
          
           
               
                 ITI (H4) RP 35 KD 
                   
                   
                   
                   
                   
                   
               
               
                 Isoform Spot B2505 
                 Gels 
                 Patients 
                 *Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 N 
                 240 
                 80 
                 1.001 
                 0.059 
                 0.944 
                 0.481 
               
               
                 B9 
                 327 
                 109 
                 0.968 
                 0.050 
                 0.850 
                 0.687 
               
               
                 N + B9 
                 567 
                 189 
                 0.982 
                 0.038 
                 0.888 
                 0.590 
               
               
                 Combined BC 
                 312 
                 104 
                 1.102 
                 0.077 
                 0.841 
                 0.820 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.915 
                 0.063 
                 0.758 
                 0.684 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.564 
                 0.196 
                 1.161 
                 1.166 
               
               
                   
               
             
          
           
               
                 c. 
               
             
          
           
               
                 ITI (H4) RP 35 KD 
                   
                   
                   
                   
                   
                   
               
               
                 Isoform Spot B3410 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 N 
                 240 
                 80 
                 1.006 
                 0.117 
                 0.803 
                 0.919 
               
               
                 B9 
                 327 
                 109 
                 0.920 
                 0.103 
                 0.588 
                 1.591 
               
               
                 N + B9 
                 567 
                 189 
                 0.957 
                 0.077 
                 0.752 
                 1.487 
               
               
                 Combined BC 
                 312 
                 104 
                 0.806 
                 0.116 
                 0.229 
                 1.353 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.535 
                 0.102 
                 0.000 
                 0.731 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.474 
                 0.281 
                 1.226 
                 2.383 
               
               
                   
               
             
          
           
               
                 d. 
               
             
          
           
               
                 ITI (H4) RP 35 KD 
                   
                   
                   
                   
                   
                   
               
               
                 Isoform Spot B4404 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 N 
                 240 
                 80 
                 1.004 
                 0.130 
                 0.809 
                 0.734 
               
               
                 B9 
                 327 
                 109 
                 1.084 
                 0.108 
                 0.847 
                 0.675 
               
               
                 N + B9 
                 567 
                 189 
                 1.050 
                 0.083 
                 0.824 
                 0.719 
               
               
                 Combined BC 
                 312 
                 104 
                 0.727 
                 0.091 
                 0.482 
                 0.901 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.548 
                 0.096 
                 0.350 
                 0.735 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.170 
                 0.189 
                 0.860 
                 1.306 
               
               
                   
               
               
                 *Insignificant down-regulation of b. B2505 in non-DCIS breast cancer patients, as compared to a. B2422, c. B3410, and d. B4404. 
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XXXVI 
               
               
                   
               
               
                 Immunoglobulin Lambda 
                   
                   
                   
                   
                   
                   
               
               
                 Chain Protein 
               
               
                 Spot B1322 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 1.004 
                 0.054 
                 0.911 
                 0.572 
               
               
                 B9 
                 327 
                 109 
                 0.915 
                 0.039 
                 0.816 
                 0.447 
               
               
                 N + B9 
                 567 
                 189 
                 0.953 
                 0.032 
                 0.852 
                 0.477 
               
               
                 Combined BC 
                 312 
                 104 
                 0.931 
                 0.043 
                 0.841 
                 0.483 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.916 
                 0.049 
                 0.818 
                 0.506 
               
               
                 DCIS BC 
                 90 
                 30 
                 0.966 
                 0.086 
                 0.898 
                 0.344 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XXXVII 
               
               
                   
               
               
                 Alpha-1-microglobulin 
                   
                   
                   
                   
                   
                   
               
               
                 Protein Spot B1418 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 1.000 
                 0.035 
                 0.934 
                 0.427 
               
               
                 B9 
                 327 
                 109 
                 1.092 
                 0.046 
                 0.956 
                 0.619 
               
               
                 N + B9 
                 567 
                 189 
                 1.053 
                 0.031 
                 0.944 
                 0.557 
               
               
                 Combined BC 
                 312 
                 104 
                 1.212 
                 0.069 
                 1.053 
                 0.522 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 1.192 
                 0.088 
                 1.009 
                 0.568 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.259 
                 0.102 
                 1.168 
                 0.361 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XXXVIII 
               
               
                   
               
               
                 Apolipoprotein A1 
                   
                   
                   
                   
                   
                   
               
               
                 Protein B2317 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 0.996 
                 0.037 
                 0.984 
                 0.378 
               
               
                 B9 
                 327 
                 109 
                 0.842 
                 0.033 
                 0.794 
                 0.516 
               
               
                 N + B9 
                 567 
                 189 
                 0.907 
                 0.025 
                 0.904 
                 0.445 
               
               
                 Combined BC 
                 312 
                 104 
                 1.095 
                 0.071 
                 0.943 
                 0.550 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.950 
                 0.051 
                 0.874 
                 0.478 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.453 
                 0.198 
                 1.242 
                 0.497 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XXXIX 
               
               
                   
               
               
                 Apolipoprotein E3 
                   
                   
                   
                   
                   
                   
               
               
                 Protein Spot B3406 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 0.988 
                 0.066 
                 0.827 
                 0.725 
               
               
                 B9 
                 327 
                 109 
                 0.970 
                 0.069 
                 0.871 
                 0.918 
               
               
                 N + B9 
                 567 
                 189 
                 0.977 
                 0.049 
                 0.860 
                 0.835 
               
               
                 Combined BC 
                 312 
                 104 
                 1.023 
                 0.070 
                 0.856 
                 0.753 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.947 
                 0.071 
                 0.825 
                 0.695 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.211 
                 0.164 
                 0.948 
                 0.878 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XL 
               
               
                   
               
               
                 Serum Albumin 
                   
                   
                   
                   
                   
                   
               
               
                 Protein Spot B5539 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 1.001 
                 0.093 
                 0.948 
                 0.342 
               
               
                 B9 
                 327 
                 109 
                 1.170 
                 0.032 
                 1.139 
                 0.404 
               
               
                 N + B9 
                 567 
                 189 
                 1.098 
                 0.044 
                 1.017 
                 0.355 
               
               
                 Combined BC 
                 312 
                 104 
                 0.896 
                 0.034 
                 0.892 
                 0.465 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.854 
                 0.034 
                 0.856 
                 0.379 
               
               
                 DCIS BC 
                 90 
                 30 
                 0.999 
                 0.081 
                 1.081 
                 0.599 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XLI 
               
               
                   
               
               
                 Transferrin Protein 
                   
                   
                   
                   
                   
                   
               
               
                 Spot B6605 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 1.003 
                 0.039 
                 0.926 
                 0.406 
               
               
                 B9 
                 327 
                 109 
                 1.186 
                 0.046 
                 1.151 
                 0.537 
               
               
                 N + B9 
                 567 
                 189 
                 1.109 
                 0.032 
                 1.045 
                 0.506 
               
               
                 Combined BC 
                 312 
                 104 
                 1.107 
                 0.055 
                 1.034 
                 0.615 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 1.086 
                 0.062 
                 0.993 
                 0.597 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.157 
                 0.116 
                 1.167 
                 0.681 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XLII 
               
               
                   
               
               
                 Serotransferin Protein 
                   
                   
                   
                   
                   
                   
               
               
                 Spot B5713 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 0.992 
                 0.081 
                 0.866 
                 0.723 
               
               
                 B9 
                 327 
                 109 
                 0.856 
                 0.059 
                 0.682 
                 0.771 
               
               
                 N + B9 
                 567 
                 189 
                 0.914 
                 0.048 
                 0.747 
                 0.737 
               
               
                 Combined BC 
                 312 
                 104 
                 0.833 
                 0.066 
                 0.612 
                 0.790 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.841 
                 0.084 
                 0.587 
                 0.841 
               
               
                 DCIS BC 
                 90 
                 30 
                 0.816 
                 0.099 
                 0.652 
                 0.578 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XLIII 
               
               
                   
               
               
                 Haptoglobin Protein 
                   
                   
                   
                   
                   
                   
               
               
                 Spot B1512 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 0.995 
                 0.058 
                 0.957 
                 0.814 
               
               
                 B9 
                 327 
                 109 
                 1.206 
                 0.060 
                 1.128 
                 0.836 
               
               
                 N + B9 
                 567 
                 189 
                 1.116 
                 0.043 
                 1.063 
                 0.817 
               
               
                 Combined BC 
                 312 
                 104 
                 1.418 
                 0.068 
                 1.354 
                 0.865 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 1.483 
                 0.083 
                 1.426 
                 0.897 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.259 
                 0.115 
                 1.125 
                 0.954 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XLIV 
               
               
                   
               
               
                 Haptoglobin Protein 
                   
                   
                   
                   
                   
                   
               
               
                 Spot B6014 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 1.013 
                 0.198 
                 0.000 
                 1.345 
               
               
                 B9 
                 327 
                 109 
                 0.749 
                 0.155 
                 0.000 
                 0.131 
               
               
                 N + B9 
                 567 
                 189 
                 0.860 
                 0.122 
                 0.000 
                 1.061 
               
               
                 Combined BC 
                 312 
                 104 
                 1.821 
                 0.319 
                 0.085 
                 2.784 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 2.091 
                 0.405 
                 0.322 
                 3.066 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.154 
                 0.455 
                 0.000 
                 2.086 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XLV 
               
               
                   
               
               
                 Haptoglobin Protein 
                   
                   
                   
                   
                   
                   
               
               
                 Spot B4008 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 0.977 
                 0.086 
                 0.855 
                 1.054 
               
               
                 B9 
                 327 
                 109 
                 1.277 
                 0.130 
                 1.019 
                 1.329 
               
               
                 N + B9 
                 567 
                 189 
                 1.150 
                 0.084 
                 0.933 
                 1.231 
               
               
                 Combined BC 
                 312 
                 104 
                 1.311 
                 0.139 
                 0.976 
                 0.947 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 1.210 
                 0.100 
                 0.994 
                 0.942 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.561 
                 0.417 
                 0.859 
                 0.962 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XLVI 
               
               
                   
               
               
                 Haptoglobin Protein 
                   
                   
                   
                   
                   
                   
               
               
                 Spot B4206 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 0.975 
                 0.110 
                 0.864 
                 1.196 
               
               
                 B9 
                 327 
                 109 
                 1.394 
                 0.134 
                 1.133 
                 1.308 
               
               
                 N + B9 
                 567 
                 189 
                 1.217 
                 0.091 
                 0.982 
                 1.352 
               
               
                 Combined BC 
                 312 
                 104 
                 1.579 
                 0.167 
                 1.274 
                 1.880 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 1.390 
                 0.167 
                 1.094 
                 2.290 
               
               
                 DCIS BC 
                 90 
                 30 
                 2.045 
                 0.396 
                 1.654 
                 1.230 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XLVII 
               
               
                   
               
               
                 Haptoglobin Related 
                   
                   
                   
                   
                   
                   
               
               
                 Protein Spot B3506 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 1.013 
                 0.099 
                 0.762 
                 1.383 
               
               
                 B9 
                 327 
                 109 
                 1.002 
                 0.187 
                 0.706 
                 1.214 
               
               
                 N + B9 
                 567 
                 189 
                 1.006 
                 0.115 
                 0.710 
                 1.294 
               
               
                 Combined BC 
                 312 
                 104 
                 0.940 
                 0.094 
                 0.701 
                 1.443 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.960 
                 0.115 
                 0.847 
                 1.514 
               
               
                 DCIS BC 
                 90 
                 30 
                 0.892 
                 0.162 
                 0.638 
                 1.122 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XLVIII 
               
               
                   
               
               
                 Haptoglobin Related 
                   
                   
                   
                   
                   
                   
               
               
                 Protein Spot B4424 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 0.999 
                 0.104 
                 0.800 
                 1.166 
               
               
                 B9 
                 327 
                 109 
                 1.045 
                 0.080 
                 0.955 
                 0.945 
               
               
                 N + B9 
                 567 
                 189 
                 1.025 
                 0.063 
                 0.918 
                 1.077 
               
               
                 Combined BC 
                 312 
                 104 
                 0.930 
                 0.069 
                 0.893 
                 0.816 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.953 
                 0.087 
                 0.887 
                 0.813 
               
               
                 DCIS BC 
                 90 
                 30 
                 0.875 
                 0.109 
                 0.895 
                 0.829 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE XLIX 
               
               
                   
               
               
                 Lectin P35 3 Protein 
                   
                   
                   
                   
                   
                   
               
               
                 Spot B6519 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 0.995 
                 0.041 
                 0.986 
                 0.478 
               
               
                 B9 
                 327 
                 109 
                 1.269 
                 0.167 
                 0.992 
                 0.572 
               
               
                 N + B9 
                 567 
                 189 
                 1.153 
                 0.098 
                 0.992 
                 0.522 
               
               
                 Combined BC 
                 309 
                 103 
                 1.214 
                 0.135 
                 1.030 
                 0.558 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 1.143 
                 0.111 
                 1.038 
                 0.531 
               
               
                 DCIS BC 
                 87 
                 29 
                 1.393 
                 0.391 
                 0.977 
                 0.605 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE L 
               
               
                   
               
               
                 Complement 
                   
                   
                   
                   
                   
                   
               
               
                 C4A gamma 
               
               
                 Protein Spot B7408 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 1.008 
                 0.069 
                 0.862 
                 0.863 
               
               
                 B9 
                 327 
                 109 
                 1.273 
                 0.077 
                 1.058 
                 0.918 
               
               
                 N + B9 
                 567 
                 189 
                 1.161 
                 0.054 
                 0.992 
                 0.903 
               
               
                 Combined BC 
                 312 
                 104 
                 1.320 
                 0.104 
                 1.062 
                 0.864 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 1.180 
                 0.106 
                 0.992 
                 0.830 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.664 
                 0.238 
                 1.177 
                 1.440 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE LI 
               
               
                   
               
               
                 Apoptosis 
                   
                   
                   
                   
                   
                   
               
               
                 Inhibitor (CD5L) 
               
               
                 Protein Spot B2412 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 1.002 
                 0.031 
                 0.989 
                 0.329 
               
               
                 B9 
                 327 
                 109 
                 1.052 
                 0.036 
                 0.938 
                 0.431 
               
               
                 N + B9 
                 567 
                 189 
                 1.031 
                 0.025 
                 0.967 
                 0.361 
               
               
                 Combined BC 
                 312 
                 104 
                 1.181 
                 0.058 
                 1.056 
                 0.521 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 1.154 
                 0.070 
                 1.046 
                 0.556 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.250 
                 0.101 
                 1.093 
                 0.389 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE LII 
               
               
                   
               
               
                 Nucleolar 
                   
                   
                   
                   
                   
                   
               
               
                 Ribosomal Protein 
               
               
                 L27a Spot B6218 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 0.909 
                 0.114 
                 0.672 
                 1.217 
               
               
                 B9 
                 327 
                 109 
                 0.835 
                 0.089 
                 0.539 
                 1.106 
               
               
                 N + B9 
                 567 
                 189 
                 0.866 
                 0.070 
                 0.604 
                 1.147 
               
               
                 Combined BC 
                 312 
                 104 
                 1.514 
                 0.170 
                 1.010 
                 1.873 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 1.383 
                 0.193 
                 0.989 
                 1.905 
               
               
                 DCIS BC 
                 90 
                 30 
                 1.838 
                 0.346 
                 1.143 
                 2.017 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE LIII 
               
               
                   
               
               
                 Neuroendocrie 
                   
                   
                   
                   
                   
                   
               
               
                 Specific (NSP) 
               
               
                 Protein Spot B7108 
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 N 
                 240 
                 80 
                 1.003 
                 0.051 
                 0.908 
                 0.571 
               
               
                 B9 
                 327 
                 109 
                 0.844 
                 0.050 
                 0.768 
                 0.474 
               
               
                 N + B9 
                 567 
                 189 
                 0.911 
                 0.036 
                 0.816 
                 0.516 
               
               
                 Combined BC 
                 312 
                 104 
                 0.748 
                 0.047 
                 0.717 
                 0.640 
               
               
                 Non-DCIS BC 
                 222 
                 74 
                 0.722 
                 0.061 
                 0.630 
                 0.722 
               
               
                 DCIS BC 
                 90 
                 30 
                 0.812 
                 0.066 
                 0.746 
                 0.467 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE LIV 
               
             
             
               
                   
               
               
                 Number of Observations and Percent Classified into 
                 Number of Observations and Percent Classified 
               
               
                 Diagnosis Step Disk 9 Biomarkers 
                 All 22 Biomarkers 
               
             
          
           
               
                 From Diagnosis 
                 Control (N + B9) 
                 Combined BC 
                 Total 
                 From Diagnosis 
                 Control (N + B9) 
                 Combined BC 
               
               
                   
               
             
          
           
               
                 N + B9 
                 141 
                 48 
                 189 
                 N + B9 
                 143 
                 46 
               
               
                   
                 74.60% 
                 25.40% 
                   
                   
                 75.66% 
                 24.34% 
               
               
                 DCIS BC 
                 6 
                 24 
                 30 
                 DCIS BC 
                 5 
                 25 
               
               
                   
                 20.00% 
                 80.00% 
                   
                   
                 16.67% 
                 83.33% 
               
               
                 Non-DCIS BC 
                 19 
                 55 
                 74 
                 Non-DCIS BC 
                 19 
                 55 
               
               
                   
                 25.68% 
                 74.32% 
                   
                   
                 25.68% 
                 74.32% 
               
               
                 Combined BC 
                 24 
                 80 
                 104 
                 Combined BC 
                 24 
                 80 
               
               
                   
                 23.08% 
                 76.92% 
                   
                   
                 23.08% 
                 76.92% 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE LV 
               
               
                   
                   
               
               
                   
                   
                 Normal 
                 B9 
                 DCIS BC 
                 Non-DCIS BC 
               
               
                   
                   
                 Median 
                 Median 
                 Median 
                 Median 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 B2317 
                 0.984 
                 0.794 
                 1.242 
                 0.874 
               
               
                   
                 B2505 
                 0.944 
                 0.850 
                 1.161 
                 0.758 
               
               
                   
                 B6218 
                 0.672 
                 0.539 
                 1.143 
                 0.989 
               
               
                   
                 B6014 
                 0.000 
                 0.000 
                 0.000 
                 0.322 
               
               
                   
                 B1512 
                 0.957 
                 1.128 
                 1.125 
                 1.426 
               
               
                   
                 B7108 
                 0.908 
                 0.768 
                 0.746 
                 0.630 
               
               
                   
                 B5539 
                 0.948 
                 1.139 
                 1.081 
                 0.856 
               
               
                   
                 B2422 
                 0.812 
                 0.758 
                 1.131 
                 0.181 
               
               
                   
                 B4404 
                 0.809 
                 0.847 
                 0.860 
                 0.350 
               
               
                   
                 B3410 
                 0.803 
                 0.588 
                 1.226 
                 0.000 
               
               
                   
                 B7408 
                 0.862 
                 1.058 
                 1.177 
                 0.992 
               
               
                   
                 B4008 
                 0.855 
                 1.019 
                 0.859 
                 0.994 
               
               
                   
                 B4206 
                 0.864 
                 1.133 
                 1.654 
                 1.094 
               
               
                   
                 B2412 
                 0.989 
                 0.938 
                 1.093 
                 1.046 
               
               
                   
                 B1322 
                 0.911 
                 0.816 
                 0.898 
                 0.818 
               
               
                   
                 B1418 
                 0.934 
                 0.956 
                 1.168 
                 1.009 
               
               
                   
                 B3406 
                 0.827 
                 0.871 
                 0.948 
                 0.825 
               
               
                   
                 B6519 
                 0.986 
                 0.992 
                 1.055 
                 1.038 
               
               
                   
                 B6605 
                 0.926 
                 1.151 
                 1.167 
                 0.993 
               
               
                   
                 B3506 
                 0.762 
                 0.706 
                 0.638 
                 0.847 
               
               
                   
                 B4424 
                 0.800 
                 0.955 
                 0.895 
                 0.887 
               
               
                   
                 B5713 
                 0.866 
                 0.682 
                 0.652 
                 0.587 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE LVI 
               
             
             
               
                   
               
               
                 Blood Serum Concentration: 
               
               
                 ? g 35 KD Protein 
               
               
                 (Isoforms N2422 + B2505 + B3410 + B4404)/ml of 
               
               
                 Blood Serum 
               
             
          
           
               
                   
                 Quantitative 2D Gels 
                 Anti-ITI(H4)RP 
               
               
                   
                 Electrophoresis 
                 35 KD 
               
             
          
           
               
                   
                 PPM Spot 
                   
                 Protein ELISA 
               
             
          
           
               
                 ITI (H4) RP 35 KD Sum = 
                 Density 
                 ug/ml of Serum* 
                 ug/ml of Serum 
               
             
          
           
               
                 B2422 + B2505 + B3410 + B4404 
                 Mean 
                 ± 
                 S.E. 
                 Mean* 
                 ± 
                 S.E. 
                 Mean 
                 ± 
                 S.E. 
               
               
                   
               
               
                 N 
                 190.6 
                   
                 9.35 
                 17.50 
                   
                 0.86 
                 17.50 
                   
                 0.85 
               
               
                 B9 
                 188.2 
                   
                 6.10 
                 17.28 
                   
                 0.56 
                 19.19 
                   
                 0.61 
               
               
                 BC 
                 179.9 
                   
                 9.46 
                 16.51 
                   
                 0.87 
                 14.15 
                   
                 0.48 
               
               
                   
               
               
                 Conversion Factor* = (190.6 PPM/17.50 ug) = 10.89 PPM/ug protein per ml of serum 
               
               
                 *Based on Normal Controls and Lava Purple Stain 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE LVII 
               
               
                   
               
             
             
               
                 Total ITI (H4) RP 35 KD = Protein Spots B2422 + B2505 + B3410 + B4404 
               
               
                 Fold of Average Normal PPM or ug/ml of Serum 
               
             
          
           
               
                   
                   
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 Min 
                 Max 
                 IQR 
               
               
                   
               
               
                   
                 Retrospective 
               
               
                 a: 
                 N 
                 192 
                 64 
                 1.000 
                 0.049 
                 0.884 
                 0.199 
                 5.714 
                 0.569 
               
               
                   
                 B9 
                 344 
                 115 
                 0.988 
                 0.032 
                 0.849 
                 0.146 
                 3.292 
                 0.757 
               
               
                   
                 BC 
                 294 
                 98 
                 0.944 
                 0.050 
                 0.616 
                 0.020 
                 4.933 
                 0.721 
               
               
                   
                 Prospective 
               
               
                 b: 
                 N 
                 51 
                 17 
                 1.000 
                 0.075 
                 0.931 
                 0.214 
                 2.628 
                 0.534 
               
               
                   
                 BC 
                 39 
                 13 
                 0.775 
                 0.041 
                 0.848 
                 0.277 
                 1.203 
                 0.392 
               
               
                   
                 Combined +/− DCIS 
               
               
                 c: 
                 N + B9 
                 567 
                 189 
                 1.008 
                 0.044 
                 0.876 
                 0.211 
                 4.965 
                 0.664 
               
               
                   
                 Non-DCIS BC 
                 234 
                 78 
                 0.747 
                 0.065 
                 0.538 
                 0.048 
                 3.624 
                 0.601 
               
               
                   
                 DCIS BC 
                 90 
                 30 
                 1.482 
                 0.191 
                 1.130 
                 0.231 
                 4.548 
                 1.566 
               
               
                   
               
             
          
           
               
                 Total ITI (H4) RP 35 KD = Protein Spots B2422 + B2505 + B3410 + B4404 
               
               
                 Fold of Average Normal μg/ml of Serum 
               
               
                 by Elisa Employing Monoclonal 
               
               
                 Antibodies to: 
               
             
          
           
               
                   
                 Total ITI (H4) RP Elisa 
                   
                   
                   
                   
                   
                   
               
               
                   
                 B2422 + B2505 + B3410 + B4404 
                 Diagnosis 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
               
               
                 d: 
                 ITI (H4) RP 35 KD 
                 N 
                 177 
                 1.000 
                 0.048 
                 0.797 
                 0.709 
               
               
                   
                   
                 B9 
                 244 
                 1.097 
                 0.035 
                 0.990 
                 0.639 
               
               
                   
                   
                 BC 
                 277 
                 0.809 
                 0.028 
                 0.724 
                 0.330 
               
               
                 e: 
                 ITI (H4) RP 35 KD + 
                 N 
                 177 
                 1.000 
                 0.028 
                 0.959 
                 0.346 
               
               
                   
                 PK 120 Precursor 
                 B9 
                 244 
                 0.939 
                 0.020 
                 0.881 
                 0.383 
               
               
                   
                   
                 BC 
                 277 
                 1.024 
                 0.022 
                 0.951 
                 0.395 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE LVIII 
               
               
                   
               
               
                   
                   
                 Anti-ITI (H4) 
                 Anti-ITI (H4) 
               
               
                   
                 Retrospective 
                 RP 35 KD 
                 RP 35 KD + 
               
               
                 Analytical Method 
                 Samples 
                 Only 
                 PK120 Precursor 
               
               
                   
               
             
             
               
                 Elisa 
                 BC &lt; B9 
                 0.69 ± 0.024 
                 0.57 ± 0.025 
               
               
                   
                 BC &lt; N 
                 0.58 ± 0.029 
                 0.52 ± 0.029 
               
               
                 2D Gel 
                 BC &lt; N + B9 
                 0.59 ± 0.022 
               
               
                 Electrophoresis 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE LIX 
               
               
                   
                   
               
               
                   
                 Gels 
                 Patients 
                 Mean 
                 SE 
                 Median 
                 IQR 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 ITI (H4) RP 35 KD Sum 
                   
                   
                   
                   
                   
                   
               
               
                 SyproRuby FX 
               
               
                 PPM 
               
               
                 B2422 + B2505 + B3410 + B4404 
               
               
                 N 
                 27 
                 27 
                 1968.3 
                 158.44 
                 1907.2 
                 881.2 
               
               
                 B9 
                 144 
                 144 
                 1492.9 
                 89.65 
                 1208.8 
                 1050.8 
               
               
                 BC 
                 16 
                 16 
                 1299.0 
                 181.80 
                 1069.4 
                 1089.2 
               
               
                 ITI (H4) RP 35 KD Sum 
               
               
                 SyproRuby FLA 
               
               
                 PPM 
               
               
                 B2422 + B2505 + B3410 + B4404 
               
               
                 N 
                 84 
                 28 
                 296.661 
                 19.578 
                 242.730 
                 223.928 
               
               
                 B9 
                 90 
                 30 
                 166.086 
                 9.001 
                 149.802 
                 141.296 
               
               
                 BC 
                 60 
                 20 
                 166.506 
                 16.754 
                 130.312 
                 121.587 
               
               
                 ITI (H4) RP 35 KD Sum 
               
               
                 Lava Purple FLA 
               
               
                 PPM 
               
               
                 B2422 + B2505 + B3410 + B4404 
               
               
                 N 
                 84 
                 28 
                 177.783 
                 10.435 
                 155.611 
                 104.783 
               
               
                 B9 
                 90 
                 30 
                 108.789 
                 4.217 
                 106.598 
                 55.072 
               
               
                 BC 
                 60 
                 20 
                 121.783 
                 6.593 
                 110.603 
                 65.583 
               
               
                 ITI (H4) RP 35 KD Sum 
               
               
                 SyproRuby FX 
               
               
                 Fold of Average N 
               
               
                 B2422 + B2505 + B3410 + B4404 
               
               
                 N 
                 27 
                 27 
                 1.000 
                 0.080 
                 0.969 
                 0.448 
               
               
                 B9 
                 144 
                 144 
                 0.758 
                 0.046 
                 0.614 
                 0.534 
               
               
                 BC 
                 16 
                 16 
                 0.660 
                 0.092 
                 0.543 
                 0.553 
               
               
                 ITI (H4) RP 35 KD Sum 
               
               
                 SyproRuby FLA 
               
               
                 Fold of Average N 
               
               
                 B2422 + B2505 + B3410 + B4404 
               
               
                 N 
                 84 
                 28 
                 1.000 
                 0.0660 
                 0.818 
                 0.755 
               
               
                 B9 
                 90 
                 30 
                 0.560 
                 0.0303 
                 0.505 
                 0.476 
               
               
                 BC 
                 60 
                 20 
                 0.561 
                 0.0565 
                 0.439 
                 0.410 
               
               
                 ITI (H4) RP 35 KD Sum 
               
               
                 Lava Purple FLA 
               
               
                 Fold of Average N 
               
               
                 B2422 + B2505 + B3410 + B4404 
               
               
                 N 
                 84 
                 28 
                 1.000 
                 0.0587 
                 0.875 
                 0.589 
               
               
                 B9 
                 90 
                 30 
                 0.612 
                 0.0237 
                 0.600 
                 0.310 
               
               
                 BC 
                 60 
                 20 
                 0.685 
                 0.0371 
                 0.622 
                 0.369 
               
               
                   
               
             
          
         
       
     
       REFERENCES 
       [0000]    
       
         1. Tan, P. K. et al. Evaluation of gene expression measurements from commercial microarray platforms.  Nucleic Acids Res  31, 5676-5684 (2003). 
         2. Marshall, E. Getting the noise out of gene arrays. Science 306, 630-631 (2004). 
         3. Shi, L. et al. The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.  Nat Biotechnol  24, 1151-1161 (2006). 
         4. Guo, L. et al. Rat toxicogenomic study reveals analytical consistency across microarray platforms.  Nat Biotechnol  24, 1162-1169 (2006). 
         5. Canales, R. D. et al. Evaluation of DNA microarray results with quantitative gene expression platforms.  Nat Biotechnol  24, in press (2006). 
         6. Shippy, R. et al. Using RNA sample titrations to assess microarray platform performance and normalization techniques.  Nat Biotechnol  24, 1123-1131 (2006). 
         7. Patterson, T. A. et al. Performance comparison of one-color and two-color platforms within the Microarray Quality Control (MAQC) project.  Nat Biotechnol  24, 1140-1150 (2006). 
         8. Tong, W. et al. Evaluation of external RNA controls for the assessment of microarray performance.  Nat Biotechnol  24, 1132-1139 (2006). 
         9. Casciano, D. A. &amp; Woodcock, J. Empowering microarrays in the regulatory setting.  Nat Biotechnol  24, 1103 (2006). 
         10. Making the most of microarrays.  Nat Biotechnol  24, 1039 (2006). 
         11. Frueh, F. W. Impact of microarray data quality on genomic data submissions to the FDA.  Nat Biotechnol  24, 1105-1107 (2006). 
         12. Dix, D. J. et al. A framework for the use of genomics data at the EPA.  Nat Biotechnol  24, 1108-1111 (2006). 
         13. Ji, H. &amp; Davis, R. W. Data quality in genomics and microarrays.  Nat Biotechnol  24, 1112-1113 (2006). 
         14. Reid, L. H. &amp; Warrington, J. A. A note on nomenclature.  Nat Biotechnol  24, ii (2006). 
         15. Strauss, E. Arrays of hope.  Cell  127, 657-659 (2006). 
         16. Eisenstein, M. Microarrays: quality control.  Nature  442, 1067-1070 (2006). 
         17. Couzin, J. Genomics. Microarray data reproduced, but some concerns remain.  Science  313, 1559 (2006). 
         18. Kiermer, V. Microarray quality in the spotlight again.  Nat Methods  3, 772 (2006). 
         19. Sage, L. Do microarrays measure up?  Anal Chem  78, 7358-7360 (2006). 
         20. Michiels, S., Koscielny, S. &amp; Hill, C. Prediction of cancer outcome with microarrays: a multiple random validation strategy.  Lancet  365, 488-492 (2005). 
         21. Ioannidis, J. P. Microarrays and molecular research: noise discovery?  Lancet  365, 454-455 (2005). 
         22. Ein-Dor, L., Zuk, 0. &amp; Domany, E. Thousands of samples are needed to generate a robust gene list for predicting outcome in cancer.  Proc Natl Acad Sci USA  103, 5923-5928 (2006). 
         23. Simon, R. Development and evaluation of therapeutically relevant predictive classifiers using gene expression profiling.  J Natl Cancer Inst  98, 1169-1171 (2006).