Abstract:
This invention provides methods, reagents, and diagnostic and prognostic markers useful for minimally invasive identification, diagnosis, and therapeutic intervention in individuals with colorectal cancers, or individuals who may be susceptible to developing colorectal cancers.

Description:
[0001]    The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: 14-1895-WO_SeqList_ST25.txt, date recorded: Dec. 10, 2015, file size 21 KB). 
       BACKGROUND OF THE INVENTION 
       [0002]    Field of the Invention 
         [0003]    This invention provides methods, reagents, and diagnostic and prognostic markers useful for minimally invasive identification, diagnosis, and therapeutic intervention in individuals with colorectal cancers, or individuals who may be susceptible to developing colorectal cancers. Particular embodiments of the invention employ serum biomarkers present in proteomic and transcriptome screens that identify individuals likely to develop colorectal cancer and provide a basis for making decisions regarding more invasive diagnostic methods (such as colonoscopies), particularly in individuals without other indicators for such procedures. Additional particular embodiments provide a panel of serum biomarkers for use in the methods provided herein. 
         [0004]    Description of Related Art 
         [0005]    Colorectal cancer is a major cause of cancer-related morbidity and mortality in modernized nations, and is increasing in frequency in the developing world (Jemal et al.,  CA Cancer J Clin  2011, 61, (2), 69-90.). While early detection of localized colorectal cancer often leads to complete cure by polypectomy or surgery, the modalities for early detection are currently limited in sensitivity and specificity, have low patient adherence to screening recommendations, and place a strain on the capacity of clinical gastroenterologists (Hopchik,  Gastroenterol Nurs  2013, 36, (4), 289-90, 331; Kriza et al.,  Eur J Radiol  2013, 82, (11), e629-36). The current recommended screening procedures (colonoscopy, CT scan, or Fecal Occult Blood Test) can be non-specific, insensitive for the earliest operable lesions, or highly invasive (Leng et al.,  J Gerontol A Biol Sci Med Sci  2008, 63, (8), 879-84; Burt et al.,  J Natl Compr Canc Netw  2013, 11, (12), 1538-75). By contrast, a detection modality based upon blood or serum samples can achieve much broader patient compliance and clinical coverage. 
       SUMMARY OF THE INVENTION 
       [0006]    This invention provides methods, reagents, and diagnostic and prognostic markers useful for minimally invasive identification, diagnosis, and therapeutic intervention in individuals with colorectal cancers, or individuals who may be susceptible to developing colorectal cancers. In certain embodiments the invention provides serum biomarkers, and methods of using those serum biomarkers, including methods of screening, detection, monitoring, treatment, and prognostic evaluation of colorectal cancers. Other embodiments provide synthetic peptides useful for minimally invasive identification, diagnosis, and therapeutic intervention in patients with colorectal cancers, or those patients who may be susceptible to developing colorectal cancers. 
         [0007]    In one aspect, provided herein are methods for identifying a subject with cancerous or pre-cancerous lesions in the colon, the method comprising: (a) assaying a biosample from the subject for one or a plurality of protein biomarkers, wherein the protein biomarkers are epidermal growth factor receptor, leucine-rich alpha-2 glycoprotein, inter-alpha trypsin inhibitor heavy chain 3, inter-alpha trypsin inhibitor heavy chain 4, dipeptidyl peptidase 4, peptidase inhibitor 16, coagulation factor V, C-reactive protein, Rho-GDP dissociation inhibitor 1 isoform A, hemopexin, extracellular superoxide dismutase[Cu—Zn], thrombospondin-4, collagen alpha-1(l) chain, cadherin-2, or vitronectin; (b) determining the level of one or a plurality of the protein biomarkers in the biosample; and (c) identifying the subject as having a lesion of the colon when the level of one or a plurality of the protein biomarkers is different than a level detected in a subject without polyp formation in the colon. 
         [0008]    In particular embodiments, the lesion identified and treated by the presently disclosed methods comprises a pre-cancerous condition in the colon. For example, embodiments of the present disclosure provide methods of identifying dysplasia, an aberrant crypt, or a benign polyp in the colon. 
         [0009]    In some embodiments, the lesion identified by the presently disclosed methods comprises polyp formation. In particular embodiments, the polyp is an adenoma or a carcinoma. In other embodiments, the carcinoma is classified as stage 1, stage 2, stage 3, or stage 4. In still other embodiments, methods of the present disclosure are useful for identifying a carcinoma as stage 1, stage 2, stage 3, or stage 4 carcinoma. 
         [0010]    In particular embodiments, the methods are performed on biosamples such as, without limitation, blood, plasma, or serum. 
         [0011]    In other embodiments, the biosample is assayed by a method comprising: (a) selecting one or more synthetic peptides with homology to one or a plurality of the protein biomarkers; (b) combining the synthetic peptides with the biosample; and (c) subjecting the combination to a physical separation method. In particular embodiments the physical separation method is liquid chromatography. In other particular embodiments, the synthetic peptides are isotopically labeled. In yet other particular embodiments the assaying step comprises an immunologic assay such as enzyme-linked immunosorbent assay. 
         [0012]    In other embodiments the determining step comprises mass spectrometry. 
         [0013]    In other embodiments, the method further comprises the step of administering treatment to a subject identified as having polyp formation in the colon. In yet other embodiments, the method further comprises the step of performing a colonoscopy to a subject identified as having a having polyp formation in the colon. In other embodiments, the method is non-invasive. 
         [0014]    In another aspect, provided herein is a method for screening individuals to determine a need for a colonoscopy comprising (a) assaying a biosample from the subject for one or a plurality of protein biomarkers, wherein the protein biomarkers are epidermal growth factor receptor, leucine-rich alpha-2 glycoprotein, inter-alpha trypsin inhibitor heavy chain 3, inter-alpha trypsin inhibitor heavy chain 4, dipeptidyl peptidase 4, peptidase inhibitor 16, coagulation factor V, C-reactive protein, Rho-GDP dissociation inhibitor 1 isoform A, hemopexin, extracellular superoxide dismutase[Cu—Zn], thrombospondin-4, collagen alpha-1(l) chain, cadherin-2, and vitronectin; (b) determining the level of one or a plurality of the protein biomarkers in the biosample; and (c) identifying the individual as needing a colonoscopy when the level of one or a plurality of the protein biomarkers is different than the level detected in a subject without polyp formation in the colon. 
         [0015]    In still other embodiments, methods are provided for identifying individuals who would benefit from further clinical assessment or treatment, including but not limited to, further assessment or treatment by colonoscopy or polypectomy procedures. In other embodiments, post-surgical or post-polypectomy patient monitoring is provided. In still other embodiments, the disclosed methods are useful for monitoring responsiveness of a patient to chemopreventative or chemotherapeutic agents. 
         [0016]    In addition, methods are provided that are capable of enhancing utility of currently existing colorectal screening, diagnosis, prognosis and treatment methodologies Accordingly, certain embodiments disclosed here are useful in combination with other techniques known to the art, including, without limitation, colonoscopy, sigmoidoscopy, CT scan, or Fecal Occult Blood Test, Fecal Immunochemical Test, and other Fecal-based screening or diagnostic techniques. 
         [0017]    Also provided here are protein biomarkers with clinical application, including without limitation application to screening, diagnosis, prognosis, and treatment of colorectal cancers and precancerous conditions. In certain embodiments, protein biomarkers comprise epidermal growth factor receptor, leucine-rich alpha-2 glycoprotein, inter-alpha trypsin inhibitor heavy chain 3, inter-alpha trypsin inhibitor heavy chain 4, dipeptidyl peptidase 4, peptidase inhibitor 16, coagulation factor V, C-reactive protein, Rho-GDP dissociation inhibitor 1 isoform A, hemopexin, extracellular superoxide dismutase[Cu—Zn], thrombospondin-4, collagen alpha-1(l) chain, cadherin-2, or vitronectin or any combination thereof useful for prognosis, diagnosis or treatment. 
         [0018]    In certain embodiments, a panel of protein biomarkers is provided comprised of a subset of the protein biomarkers disclosed herein. In particular embodiments, methods are provided comprising (a) assaying a biosample from the subject for the levels of a panel of protein biomarkers; (b) determining the levels of the panel of protein biomarkers in the biosample; and (c) identifying the individual as needing a colonoscopy when the level of one or a plurality of the protein biomarkers in the panel is different than the levels detected in a subject without polyp formation in the colon. In some embodiments, a panel of protein biomarkers is provided comprising at least the protein biomarkers: LRG1, F5, VTN, MMP7, MMP10, CD44, ITIH3, ITIH4, HPX, CFI, SOD3, and COL1A1. In other embodiments, a panel of protein biomarkers is provided comprising at least the protein biomarkers: EGFR, LRG1, ITIH4, and F5. In still other embodiments, a panel of protein biomarkers is provided comprising at least the protein biomarkers: DPP4, LRG1, ITIH4, VTN, HPX, EGFR and F5. In yet further embodiments, a panel of protein biomarkers is provided comprising at least the protein biomarkers: EGFR, LRG1, ITIH3, ITIH4, DPP4, PI16, F5, CRP, and ARHGDIA. 
         [0019]    These and other features and advantages of the present invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1  illustrates the workflow of a targeted quantitative proteomics experiment. Stable isotope labeled reference standards are spiked into a protein extract prior to enzymatic digestion. Peptides are chromatographically separated by reversed-phase chromatography followed by analysis in-line with a triple quadrupole mass spectrometer (QQQ-MS) where targeted precursor and fragment ion masses (transitions) are selected. Quantification occurs by comparing the extracted ion chromatogram areas of the endogenous and reference standard fragment ions. Such a targeted quantitative proteomics procedure is also known as a selected reaction monitoring (SRM), or multiple reaction monitoring (MRM), procedure. 
           [0021]      FIG. 2  presents the criteria for the selection of peptides for targeted analyses. “Tier 1” criteria must be met in order to use a peptide for a targeted analysis. “Tier 2” criteria maximize chances that the endogenous peptide is visible in the targeted mass spectrometry assay. “Tier 3” criteria should be considered for targeted analysis in multiple species. 
           [0022]      FIG. 3  illustrates selected results of gene expression profiling in normal colonic tissue and tumor tissue in an Apc Pirc/+  rat model (“Pirc rats”). Gene transcripts upregulated in tumor compared to normal tissue were identified by Agilent Whole Genome Microarray. These candidates represent genes which: 1) show a 5-fold or greater upregulation in mRNA expression levels in tumors, 2) code for known or predicted secreted proteins, and 3) have some known biological significance to human colon cancer. 
           [0023]      FIG. 4  presents a summary of candidates that were selected for selected reaction monitoring mass spectrometry experiment (SRM-MS) validation screening. Using SRM-MS, the endogenous forms of peptides from each of these proteins were successfully identified at quantifiable levels in the serum of F 1 -Pirc rats. 
           [0024]      FIG. 5A  presents protein biomarker expression in serum displayed over a time course. 
           [0025]      FIG. 5B  presents protein biomarker expression in serum displayed as a function of large intestinal tumor burden. 
           [0026]      FIG. 6A  present Receiver Operator Characteristic (ROC) analyses of biomarker EGFR indicating diagnostic utility as a protein biomarker to detect tumors in F 1 -Pirc rat serum. 
           [0027]      FIG. 6B  present Receiver Operator Characteristic (ROC) analyses of biomarker LRG1 indicating diagnostic utility as a protein biomarker to detect tumors in F 1 -Pirc rat serum. 
           [0028]      FIG. 6C  present Receiver Operator Characteristic (ROC) analyses of biomarker ITIH4 indicating diagnostic utility as a protein biomarker to detect tumors in F 1 -Pirc rat serum. 
           [0029]      FIG. 6D  present Receiver Operator Characteristic (ROC) analyses of biomarker ITIH3 indicating diagnostic utility as a protein biomarker to detect tumors in F 1 -Pirc rat serum. 
           [0030]      FIG. 7  presents an ROC analysis of a panel comprised of epidermal growth factor receptor (EGFR), Leucine-rich alpha-2-glycoprotein (LRG1), inter-alpha trypsin inhibitor, heavy chain H4 (ITIH4), and coagulation factor V (F5) for detecting tumors in F 1 -Pirc rats from serum. 
           [0031]      FIG. 8  presents the design of a human clinical study to determine the ability of biomarkers identified in animal models to predict, diagnose, and determine prognosis for humans with colorectal cancer, or those with precancerous stages of colon cancer. 
           [0032]      FIGS. 9A-9J  present the results for the indicated biomarkers studied in a human population comprised of a healthy (“control”) group, and individuals with precancerous (stage 0) or cancerous (stage 1-3) colon lesions. Top panels show bar plots of the observed biomarker levels in patient groups assessed relative to synthetic reference standards. Data are expressed as a ratio of endogenous to the synthetic peptide reference standards. Patient groups comprise healthy subjects (control), or those with stage 0, stage 1, stage 2, or stage 3 lesions. Average (“Avg.”) Cancers comprise stages 0-3 combined. The “n-” value denotes the number of patients in each group. Error bars represent average biological standard error. Bottom panels show bar plots of the ratio of observed biomarker expression levels in subjects with colon lesions (stages 0-3, and the combined average) relative to the same biomarker expression levels in healthy control subjects. Each bar shows the fold change of the indicated biomarkers between control subjects and those with colon lesions. Quantitated ratios are shown for each bar plot, along with P-values from 2-tailed t-tests for each ratio. 
           [0033]      FIG. 10  shows relative Col1a1 biomaker expression levels in 12 patients with known adenomas. Top panel shows expression prior to polyp removal (“pre-poly”) and 3-4 weeks after polyp removal. Bottom panel presents the same data as the fold-change in Col1a1 biomarker data after polyp removal. 
           [0034]      FIG. 11  presents post-polypectomy reversion toward normal expression levels for LRG1 and ITIH3. Patients 1 and 2 in these graphs each had three growing adenomas. The bar graphs display the area ratio between the internal reference standard and the endogenous serum peptide. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0035]    All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes. 
         [0036]    Provided herein are non-invasive methods for identifying and treating subjects with cancerous or pre-cancerous lesions of the colon. A “lesion” as used herein refers to an abnormal region of the colon, and includes dysplasia, aberrant crypts, as well as benign or cancerous polyps. 
         [0037]    A “polyp” as used herein refers to a polyp present in any of the four stages of colorectal cancer, or to a polyp of a precancerous condition. As used herein, “colorectal cancer” refers to a malignant condition comprised of any of the 4 stages, ranging from stage 1 to stage 4, classified by the American Joint Committee on Cancer (AJCC) according to the TNM system (which evaluates histological properties (T), tumor presence in nearby lymph nodes (N), and metastatic spread (M)) (Gunderson, L. L. et al.,  J Clin Oncol  2010, 28, (2), 264-71.; Greene, F. L.,  Bull Am Coll Surg  2002, 87, (7), 13-5). (Table 1.) 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 TNM staging system by the American Joint Committee on Cancer (AJCC), 6th Edition 
               
             
          
           
               
                 AJCC Stage 
                 TNM Stage 
                 TNM staging criteria 
               
               
                   
               
               
                 Stage 0 
                 Tis N0 M0 
                 Tis: Tumor is confined to mucosal layer. Cancer-in-situ 
               
               
                 Stage I 
                 T1 N0 M0 
                 T1: Tumor invades submucosa 
               
               
                 Stage I 
                 T2 N0 M0 
                 T2: Tumor invades muscle layer 
               
               
                 Stage II-A 
                 T3 N0 M0 
                 T3: Tumor invades serosa or beyond without metastasis to other organs 
               
               
                 Stage II-B 
                 T4 N0 M0 
                 T4: Tumor invades adjacent organs or perforates the visceral peritoneum 
               
               
                 Stage III-A 
                 T1-2 N1 M0 
                 N1: Metastasis to 1-3 lymphnodes. T1 or T2 
               
               
                 Stage III-B 
                 T3-4 N1 M0 
                 N1, and T3 or T4 
               
               
                 Stage III-C 
                 any T, N2 M0 
                 N2: Metastasis to 4 or more regional lymphnodes. Any T 
               
               
                 Stage IV 
                 any T, any N, M1 
                 M1: Distant metastases, Any T, any N 
               
               
                   
               
             
          
         
       
     
         [0038]    A “pre-cancerous condition” as used herein refers to a patient with a pre-invasive, pre-metastatic lesion that disposes a person to colon cancer. Examples include dysplasia, the presence of aberrant crypts, and the presence of adenomas. The AJCC formally characterizes adenomas as pre-cancerous polyps (“Stage 0”) by a T-stage of “Tis”, where the “is” stands for carcinoma in situ. Tis adenomas are characterized by a polyp sitting in the large intestinal mucosa, with no invasion of the intestinal wall. (Table 1.) 
         [0039]    In some embodiments, methods are provided for analyzing the clinical stage of lesions in the colon of a subject. As used herein, “stage” or “staging” refers to one or more clinical classification systems used to describe the progression and severity of cancerous or pre-cancerous lesions in the colon. Colorectal tumor stage describes the location and level of tumor invasion into the intestinal wall, regional lymph nodes, and adjacent tissues. 
         [0040]    A nonexclusive example of a tumor staging system used in connection with embodiments of the present disclosure is the TNM system (Gunderson, L. L. et al.,  J Clin Oncol  2010, 28, (2), 264-71.; Greene, F. L.,  Bull Am Coll Surg  2002, 87, (7), 13-5) (Table 1). In the TNM system, stages 1 and 2 have T-stages of T1 or T2 and T3 or T4, respectively, with no invasion of lymph nodes or metastasis (N0 M0). Stage 1 lesions have passed into the submucosa and possibly the muscle layer. Stage 2 lesions have invaded the serosa and may have grown through the intestinal wall but has not invaded any nearby lymph nodes. Stage 3 lesions are very complex with three sub classifications, but can broadly be characterized by having any T-stage and the invasion of some or many nearby lymph nodes. Stage 4, constituting malignant, metastatic colon cancer, can have any T or any N classification but has metastasized other organs, most commonly the liver. (Table 1.) 
         [0041]    Those skilled in the art will recognize alternative staging systems useful in connection with the presently disclosed methods. Examples of other staging systems include the Duke&#39;s classification system (Dukes, C. E.,  Journal of Pathological Bacteriology  1932, 35:323), and the Astler-Coller classification system (Astler V. B. and Coller F. A.,  Ann Surg  1954, 139:846). 
         [0042]    In some embodiments, methods are provided for analyzing the clinical grade of lesions in the colon of a subject. As used herein, “tumor grade” refers to a histological assessment that describes the degree to which the tumor cells have differentiated into normal colon tissue cells. Current tumor grade classifications are part of the TNM guidelines of colon cancer classification and range from G1 to G4. Cells rated G1 histologically look the most like healthy colon tissue cells. G2 rated cells are moderately differentiated, G3 rated cells are poorly differentiated, and G4 cells are undifferentiated. Higher-grade cells tend to grow more rapidly and can influence the method of cancer treatment. In some embodiments, the protein biomarkers and methods provided herein can be used to assess the level of cellular differentiation (tumor grade) and influence patient treatment strategies. 
         [0043]    In one particular aspect, provided herein are methods for identifying a subject with polyp formation in the colon, the method comprising (a) assaying a biosample from the subject for one or a plurality of protein biomarkers; (b) determining the level of one or a plurality of the protein biomarkers in the biosample; and (c) identifying the subject as having polyp formation in the colon when the level of one or a plurality of the protein biomarkers is different than a level detected in a subject without polyp formation in the colon. Suitable protein biomarkers include epidermal growth factor receptor, leucine-rich alpha-2 glycoprotein, inter-alpha trypsin inhibitor heavy chain 3, inter-alpha trypsin inhibitor heavy chain 4, dipeptidyl peptidase 4, peptidase inhibitor 16, coagulation factor V, C-reactive protein, Rho-GDP dissociation inhibitor 1 isoform A, hemopexin, extracellular superoxide dismutase[Cu—Zn], thrombospondin-4, collagen alpha-1(l) chain, cadherin-2, vitronectin, maltase glucoamylase, isocitrate dehydrogenase, pyruvate kinase m2, vitamin D binding protein, CD44 antigen, CEACAM5, cathespin B, serum amyloid P, fetuin B, matrilysin, complement factor 1, heparin cofactor 2, sulfhydryl oxidase 1, thrombospondin 4, and receptor-type tyrosine-protein phosphastase mu. 
       Non-Exclusive NCBI Accession Data for Certain Exemplary Biomarkers Presented Herein 
       [0044]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 Protein Name 
                 Protein Symbol 
                 NCBI RefSeq Number (Mouse/Rat/Human) 
               
               
                   
               
             
             
               
                 epidermal growth factor receptor 
                 EGFR 
                 NP_997538.1/NP_113695/NP_958439 
               
               
                 leucine-rich alpha-2-glycoprotein 
                 LRG1 
                 NP_084072/NP_001009717/NP_443204 
               
               
                 inter-alpha-trypsin inhibitor heavy 
                 ITIH3 
                 NP_032433/NP_059047/NP_002208 
               
               
                 inter alpha-trypsin inhibitor, heavy  
                 ITIH4 
                 NP_061216/NP_062242/NP_001159921 
               
               
                 Dipeptidyl peptidase-4 
                 DPP4 
                 NP_034204/NP_036921/NP_001926 
               
               
                 Peptidase inhibitor 16 
                 PI16 
                 NP_076223/NP_001163952/NP_699201 
               
               
                 coagulation factor V 
                 F5 
                 NP_032002.1/NP_001041343/NP_000121 
               
               
                 C-reactive protein 
                 CRP 
                 NP_031794.3/NP_058792/NP_000558 
               
               
                 rho GDP-dissociation inhibitor 1 
                 ARHGDIA 
                 NP_598557.3/NP_001007006/NP_004300 
               
               
                 hemopexin 
                 HPX 
                 NP_059067.2/NP_445770/NP_000604 
               
               
                 Extracellular superoxide dismutase 
                 SOD3 
                 NP_035565/NP_037012/NP 003093 
               
               
                 [Cu—Zn] 
                   
                   
               
               
                 Thrombospondin-4 
                 THBS4 
                 NP_035712/NP_058829/NP_003239 
               
               
                 collagen alpha-1(I) chain 
                 COL1A1 
                 NP_031768.2/NP_445756/NP_000079 
               
               
                 Cadherin-2 
                 CDH2 
                 NP_031690/NP_112623/NP_001783 
               
               
                 vitronectin 
                 VTN 
                 NP_035837/NP_062029/NP_000629 
               
               
                   
               
             
          
         
       
     
         [0045]    In another aspect, Inter-alpha-trypsin inhibitors, heavy chain H3 (ITIH3) and heavy chain 4, isoform 1 (ITIH4) are provided as biomarkers useful to practice the present methods. The inter-alpha trypsin inhibitors are involved in the covalent binding and stabilization of hyaluronic acid on the extracellular matrix (Chen, L. et al.,  J Biol Chem  1994, 269, (45), 28282-7). Hyaluronan is a large epithelial glycosaminoglycan complex known to increase in size with the growth of colonic polyps and tumors (Misra, S. et al.,  Connect Tissue Res  2008, 49, (3), 219-24). In addition, ITIH3 has previously been identified as upregulated in the plasma of human gastric cancer patients and has a predicted role in the prevention of metastasis and tumor invasion activities in colon cancer (Misra, S. et al.,  Connect Tissue Res  2008, 49, (3), 219-24; Chong, P. K. et al.,  J Proteome Res  2010, 9, (7), 3671-9). 
         [0046]    In addition, epidermal growth factor receptor (EGFR) is provided as a biomarker useful to practice the present methods. EGFR is implicated in poor tumor prognosis (Lieto, E. et al.,  Ann Surg Oncol  2008, 15, (1), 69-79). 
         [0047]    Certain enzymes implicated in the inflammation response are provided as biomarkers useful to practice the present methods. Alpha-1-antitrypsin 1-5 is a protease inhibitor that helps protect tissues from the release of inflammatory enzymes and often rises in concentration during acute inflammation reactions (Foell, D. et al.,  Gut  2009, 58, (6), 859-68). This protein has been identified at increased levels in stool samples and in serum from human colon cancer patients (Foell, D. et al.,  Gut  2009, 58, (6), 859-68; Ward, D. G. et al.,  Br J Cancer  2006, 94, (12), 1898-905). Leucine-rich alpha-2-glycoprotein (LRG1) and fetuin-B are thought to play a role in acute phase response and inflammation (Hsu, S. J. et al.,  Genome  2004, 47, (5), 931-46.; Shirai, R. et al.  Biochem Biophys Res Commun  2009, 382, (4), 776-9). LRG1 has shown upregulation in multiple mouse studies and has been shown to be upregulated in the plasma of human colon cancer patients (Hung, K. E. et al.,  Cancer Prev Res  ( Phila ) 2009, 2, (3), 224-33.; Chong, P. K. et al.,  J Proteome Res  2010, 9, (7), 3671-9; Shirai, R. et al.  Biochem Biophys Res Commun  2009, 382, (4), 776-9); Ladd, J. J. et al.,  Cancer Prev Res  ( Phila ) 2012, 5, (4), 655-64). 
         [0048]    LRG1 is an acute phase response protein that is upregulated in the blood of humans and murine models of colon cancer (Ivancic, M. M. et al.,  J Proteome Res  2013, 12, (9), 4152-66; Chong, P. K. et al.,  J Proteome Res  2010, 9, (7), 3671-9; Ladd, J. J. et al.,  Cancer Prev Res  ( Phila ) 2012, 5, (4), 655-64; Ivancic, M. M. et al.,  Cancer Prev Res  2014, 55, 7(11); 1160-9). Studies have shown that this protein is also upregulated in the serum of patients with ulcerative colitis, suggesting that LRG1 may also be a systemic indicator of intestinal disease (Serada, S. et al.,  Inflamm Bowel Dis  2012, 18, (11), 2169-79). One study showed that LRG1 promotes endothelial cell formation via signaling by the TGF-β pathway through interactions with ALK1-SMAD 1, 5, and 8, thus inducing an angiogenic state (Wang, X. et al.,  Nature  2013, 499, (7458), 306-11). Angiogenesis, one of the fundamental attributes of tumor invasion and metastasis, can be triggered very early in tumor formation (Hanahan, D. et al.,  Cell  2011, 144, (5), 646-74). Other studies have shown that circulating levels of LRG1 in blood plasma may be useful to diagnose colorectal cancer and identify regional tumor localization within the colon, rectosigmoid junction, and the rectum (Surinova, S. et al.,  EMBO Mol Med  2015, 7, 1153-1165; Surinova, S. et al.,  EMBO Mol Med  2015, 7, 1166-1178). 
         [0049]    Maltase-glucoamylase (MGAM) is also provided as one of the biomarkers useful to practice the present methods. MGAM is an intestinal protein necessary for catalyzing the final steps in starch catabolism (Real, F. X. et al.,  Int J Cancer  1992, 51, (2), 173-81; Young, G. P. et al.,  J Gastroenterol Hepatol  1992, 7, (4), 347-54). An immunohistochemical study that compared MGAM expression on human colonic epithelium to normal epithelium showed reduced expression of this enzyme (Real, F. X. et al.,  Int J Cancer  1992, 51, (2), 173-81; Young, G. P. et al.,  J Gastroenterol Hepatol  1992, 7, (4), 347-54). MGAM, while quite specific to the intestine, is a very large protein (1827 amino acids) with a single transmembrane pass and a very small domain inside the cell (Sim, L. et al.,  J Mol Biol  2008, 375, (3), 782-92). The brush border activity of the enzyme is localized completely external to the cell. Thus, this protein may be released into the blood. 
         [0050]    In addition, Collagen-1 type 1(I) alpha 1 (COL1A1) is provided as one of the biomarkers useful to practice the present methods. Downregulated COL1A1 has been previously reported in cancer studies, and it is thought to play a role as part of oncogenic transformation (Sengupta, P. et al.  J Biol Chem  2005, 280, (22), 21004-14). Among its many functions, COL1A1 is a positive regulator of the canonical WNT signaling pathway, the pathway that is constitutively active in early stages of colon cancer (Medici, D. et al.,  Matrix Biol  2010, 29, (3), 161-5). COL1A1 and LRP5 expression are commonly linked in bone matrix formation and are misregulated in bone disease. LRP5 is a co-receptor with the frizzled receptor in the WNT signaling pathway. 
         [0051]    Coagulation factor V (F5) is also provided as one of the biomarkers useful to practice the present methods. F5 is a cofactor for activated coagulation factor X (Xa) which assists in cleaving prothrombin to form an active thrombin protein which is vital for blood clotting (Davie, E. W. et al.,  Biochemistry  1991, 30, (43), 10363-70). Perturbation in hemostasis is a commonly observed side effect of cancer, with venous thromboembolism as a documented complication in colon cancer patients (Falanga, A. et al.,  J Thromb Haemost  2013, 11, (2), 223-33; Alcalay, A. et al.,  J Clin Oncol  2006, 24, (7), 1112-8). Coagulants such as fibrinogen, F5, and other coagulation factors have increased levels in colon cancer patients (Paspatis, G. A. et al.,  Pathophysiol Haemost Thromb  2002, 32, (1), 2-7; Vossen, C. Y. et al.,  J Clin Oncol  2011, 29, (13), 1722-7). In addition, F5 is most known for its association with the Factor V Leiden coagulation disease. Factor V Leiden is caused by a single nucleotide polymorphism (SNP) involving an R506Q mutation. This mutation reduces the ability of the activated protein C anticoagulant protein from binding F5. Normal interactions between activated protein C and F5 lead to the degradation of F5. However, in the absence of this interaction, F5 levels increase and cause excessive coagulation. Patients homozygous for the factor V Leiden mutation show a nearly 6-fold increased risk for colorectal cancer (Vossen, C. Y. et al.,  J Clin Oncol  2011, 29, (13), 1722-7). A recent biomarker study has indicated that F5 may be a blood plasma marker to distinguish localized versus metastatic colorectal cancers (Surinova, S. et al.,  EMBO Mol Med  2015, 7, 1153-1165). 
         [0052]    In another aspect, Vitronectin (VTN) is provided as one of the biomarkers useful to practice the present methods. Vitronectin has been shown to promote cell adhesion and spreading, and is indicated in tumor malignancy (Felding-Habermann, B. et al.,  Curr Opin Cell Biol  1993, 5, (5), 864-8). This protein also inhibits the membrane-damaging effect of some proteins involved in the terminal cytolytic complement pathway through binding to several serpin serine protease inhibitors (Milis, L.; Morris, C. A. et al.,  Clin Exp Immunol  1993, 92, (1), 114-9). Large and consistent upregulation of several complement factors in murine models, including complement factor B, complement C5, and complement C4-B, suggests that vitronectin could partially mitigate the damaging effects of these upregulated proteins. Vitronectin may also be a marker for regional tumor localization within the colon, rectosigmoid junction and the rectum (Surinova, S. et al.,  EMBO Mol Med  2015, 7, 1153-1165). 
         [0053]    Additionally, Cathepsins B and E are provided as biomarkers useful to practice the present methods. Cathepsin E is a gastric aspartyl protease that is found at highest levels on the mucosal producing epithelial cells of the stomach (Caruso, M. et al.,  Virchows Arch  2009, 454, (3), 291-302). It is commonly upregulated in gastric cancers. Cathepsin B has been identified in elevated amounts on the surface of colon tumor cells, in plasma, and has previously been linked to negative colon cancer prognosis (Hung, K. E. et al.,  Cancer Prev Res  ( Phila ) 2009, 2, (3), 224-33; Cavallo-Medved, D. et al.,  Neoplasia  2003, 5, (6), 507-19). Alpha-2-macroglobulin, a protease inhibitor (and a biomarker useful to practice the present methods), has been shown to interact with these two cathepsins (Shibata, M. et al.,  Eur J Biochem  2003, 270, (6), 1189-98.; Mason, R. W.,  Arch Biochem Biophys  1989, 273, (2), 367-74). 
         [0054]    Certain aspects of the invention provide assaying the biosample for protein biomarkers wherein the assaying step comprises extracting a desired peptide from a biosample and separating the extracted peptide mixture. In particular embodiments, the protein is extracted from a biological material of interest and the isolated proteins are enzymatically digested with a protease to generate peptide fragments. The complex peptide mixture is chromatographically separated using reversed-phase chromatography. In a particular embodiment the reversed phase chromatography is high pH reversed phase chromatography. Alternatively, the complex peptide mixture is chromatographically separated using offline ion exchange chromatography or high pH reversed-phase chromatography. Furthermore, those of skill in the art will recognize that other extraction and separation techniques are suitable for practicing embodiments of the present methods. 
         [0055]    In particular embodiments, a stable isotope labeled standard is spiked into the protein extract prior to an enzymatic digest. The reference standard can be used for relative or absolute quantification (Yocum, A. K. and Chinnaiyan, A. M.,  Brief Funct Genomic Proteomic  2009, 8, (2), 145-57). A common absolute quantification method is known as AQUA (standing for Absolute QUAntification). AQUA peptides are identical in sequence to the endogenous peptide with the exception of a heavy stable isotope amino acid spiked into a sample at a known concentration. Thus, the exact concentration of the endogenous peptide, when compared to the AQUA peptide, can be determined (Gerber, S. A. et al.,  Proc Natl Acad Sci USA  2003, 100, (12), 6940-5). When the exact concentration of stable isotope-labeled peptide is unknown, the peptide can be spiked into the sample at a known ratio and used for relative quantification. Reference standards can also be made as whole proteins or synthetic concatenated tryptic peptides in vivo using stable isotope labeled proteins (PSAQ) or concatemers (QconCAT), respectively (Kaiser, S. E. et al.,  Nat Methods  2011, 8, (8), 691-6, 130). 
         [0056]    Particular embodiments disclosed herein employ reversed-phase chromatography that is optimized to resolve low-level endogenous peptides and optimize peak shapes for quantitative peak integration. For example, in certain embodiments the HPLC system is an Eksigent Nano 2D LC equipped with a Nanoflex cHiPLC system. The Nanoflex system is optionally equipped with C18 microfluidic chips that are used for trapping and chromatographically eluting peptides in a reversed-phase gradient. In addition, the Nanoflex system is optionally equipped with a column heater to optimize the effect of temperature on peak resolution. 
         [0057]    In particular embodiments, the methods provided herein use optimized chromatography gradient lengths to identify low abundance endogenous peptides by shifting the number of co-eluting species and reducing localized sample complexity. Accordingly, in a particular embodiment the methods herein provide an effective gradient length of 90-minutes for chromatographic separations. 
         [0058]    To achieve high specificity, the peptide amino acid sequence of the reference standard is unique to the protein biomarker (Lange, V. et al.,  Mol Syst Biol  2008, 4, 222). Peptide length is kept between approximately 6-20 amino acids to achieve good chromatographic peak shape, proper ionization, and optimal fragmentation (Picotti, P. et al.,  Nature  2013, 494, (7436), 266-70; Elias, J. E. et al.,  Nat Methods  2005, 2, (9), 667-75; Kirkpatrick, D. S. et al.,  Methods  2005, 35, (3), 265-73). In certain embodiments, peptide collision energies are optimized to provide the most intense fragment ions, and a scheduling method is implemented so that only a limited number of transitions are analyzed over a given cycle time. Those skilled in the art will recognize that scheduling has the capacity to increase dwell times (length of time a transition is analyzed) in order to maximize signal for a particular ion. In one embodiment, a scheduling window of 5-7 minutes is chosen resulting in dwell times of at least 20 ms or more within a 1.5-second cycle time for peptides used in the present methods. Alternatively, scheduling windows of different lengths are also contemplated. 
         [0059]    The use of a stable isotope as reference standard provides the ability to directly compare two or more samples within the same analysis, thus eliminating problems associated with the run-to-run variability observed in label-free methods. These standards, unique to the target protein biomarker, contain a heavy stable isotope labeled amino acid to differentiate it from the target endogenous peptide biomarker. Further, these reference standards also have the ability to assist in identifying the correct peptide isomer of interest when multiple similar peptide sequences exist in a complex protein digest, thus contributing to the specificity of the assay (Banack, S. A. et al.,  Toxicon  2010, 56, (6), 868-79). 
         [0060]    In certain embodiments, the levels of the one or plurality of the protein biomarkers in the biosample are determined using mass spectrometry. In particular embodiments, the levels of protein biomarkers are determined using selected reaction monitoring mass spectrometry (SRM-MS). In other embodiments, the levels of the one or plurality of the protein biomarkers in the biosample are determined using other quantitative mass spectrometry techniques, including, without limitation, spectral counting, isobaric mass tagging, or ion mobility mass spectrometry. 
         [0061]    In further embodiments, the absolute concentration of the one or a plurality of protein biomarkers is determined. In some embodiments, absolute concentration of the one or a plurality of protein biomarkers is determined using SRM-MS in combination with the AQUA method. 
         [0062]    In other embodiments, the determining step of the claimed methods employs alternatives to mass spectrometry. For example, in certain embodiments, a level of protein biomarker is determined using routine immunoassay techniques known to the art. Such immunoassay techniques include, without limitation, Enzyme-Linked immunosorbent assay (ELISA), protein arrays, Western blotting, flow cytometry cell sorting, immunohistochemstry, immunocytochemistry, or immunocytometry. In some embodiments of the presently disclosed methods, the determining step comprises variations on routine immunoassay techniques, including, without limitation, microfluidic chip-based ELISAs or Westerns. 
         [0063]    In still other embodiments, the determining step of the presently disclosed methods employ quantification by electrophoresis. For example, in some embodiments, the determining step comprises, without limitation, one- or two-dimensional electrophoresis, or capillary electrophoresis. Those skilled in the art will recognize still further quantitative electrophoresis methods suitable for practicing the present disclosure. 
         [0064]    In still further embodiments, the levels of the one or plurality of the protein biomarkers in the biosample are determined by traditional protein quantification techniques. For example, in certain embodiments the levels of one or a plurality of biomarkers are determined using, without limitation, UV-VIS spectroscopy, Bradford, BCA, or Lowry Assays. In some embodiments, determining the levels of one or a plurality of biomarkers is accomplished after the biomarker is purified from the biosample. 
         [0065]    In other embodiments, the determining step of the present disclosure comprises subjected the biosample to one or more chromatographic quantitation techniques. Examples of liquid chromatography methods include cation exchange, anion exchange, reversed-phase, and size exclusion chromatography. Those skilled in the art recognize that the area under a chromatographic peak is representative of the relative amount of a biomarker present in a biosample. 
         [0066]    A subject is identified as having polyp formation in the colon when the expression level of one or a plurality of the protein biomarkers is different than an expression level detected in a subject without polyp formation in the colon. In certain embodiments, levels of a subject without a polyp formation in the colon are derived from a database of protein markers from previously tested subjects who did not have polyp formation or colon cancer. Alternatively, the levels of differentially expressed biomarkers in the biosample of a subject are measured relative to a biosample from a subject without cancer or precancer. 
         [0067]    “Differentially expressed” as used herein refers to a comparison between a biomarker determined in two or more biosamples, or between a biomarker determined in a biosample and a biomarker reference standard, wherein expression levels of a measured biomarker are different between the compared biosamples, or between the biosample and the reference standard. In some embodiments, differential expression comprises an increase in a compared biomarker level. In other embodiments, differential expression comprises a decrease in a compared biomarker level. In still other embodiments, differential expression comprises a change in a compared biomarker over time. In yet other embodiments, differential expression comprises a change in a compared biomarker between different stages of polyps or tumors present in the colon of a subject. In still other embodiments, differential expression comprises a change in a compared biomarker during treatment of a lesion present in the colon of a subject. 
         [0068]    In embodiments, differential expression of one or a plurality of biomarkers of the present disclosure is used to determine the presence, or stage, of lesions in the colon of a subject. In particular embodiments, differential expression comprises a deviation in the level of one or a plurality of biomarkers in a biosample from a reference biosample, or from a biomarker reference standard. In some embodiments, a deviation in one or a plurality of biomarkers of about 10%, about 20%, about 30%, about 40% about 50%, about 60%, about 70%, about 80%, or about 90%, from the corresponding reference amount, is indicative of the presence or stage of a lesion in the colon of a subject. In alternative embodiments, a deviation in one or a plurality of biomarkers of about 2-fold, about 4-fold, about 8-fold, about 10-fold, about 20-fold, about 40-fold, about 80-fold, or about 100-fold, from the corresponding reference amount, is indicative of the presence or stage of a lesion in the colon of a subject. 
         [0069]    Methods are provided to determine the level of one or a plurality of protein biomarkers in a biosample collected from a human, non-human primate, mouse, rat, dog, cat, horse, or cow. As used herein, a “biosample” is comprised of biologic material isolated from a subject and includes, without limitation, blood, serum, tissue, plasma or blood cells. 
         [0070]    Notably, the biomarkers useful for the presently disclosed methods comprise a bodily response at times occurring distant from the tumor or adenoma or polyp cells. Major examples include hepatically produced acute-phase and inflammatory response proteins. Acute phase, inflammatory and immune responses have been identified as a common response to tumor presence (Mantovani, A. et al.,  Nature  2008, 454, (7203), 436-44; 215), and cell adhesion represents an important function related to cancer metastasis. Hyaluronan-binding proteins such as the inter-alpha-trypsin inhibitors provide vital transport of this glycosaminoglycan to growing tumors. These are just a few examples presented here relating to the systemic response to cancer. Accordingly, one of skill in the art will recognize that the biosamples of the present invention are derived from both tumor and non-tumor cells. Furthermore, one of skill in the art will recognize that the biosamples of the present invention are optionally isolated from a broad range of materials, including without limitation blood, serum, plasma, tissue, ascites fluid, urine, and fecal matter. 
         [0071]    Embodiments of the present disclosure provide a biosample-based test for colorectal cancer that has the sensitivity and specificity to provide an alternative to routine screening using colonoscopy as a primary diagnostic mechanism. Embodiments of the method of screening use a high-throughput targeted mass spectrometry procedure, which multiplexes many protein markers into a single quantitative screening assay. 
         [0072]    In particular embodiments, the biosample-based test for colorectal cancer using SRM-MS advantageously provides a reduced cost per biomarker, potential for increased throughput in a biomarker panel analysis, and increased sensitivity and specificity. 
         [0073]    Certain embodiments provide methods for routine screening of populations for the presence of pre-cancerous or cancerous conditions. These methods include routine collection of blood and other materials useful for diagnostic purposes. In addition, in certain embodiments the biosample of the present invention is obtained during, or coincident to, a colonoscopy or polypectomy procedure. In still further embodiments, the biosample of embodiments of the present invention is obtained periodically following colonoscopy or polypectomy. In still further embodiments, the biosample is obtained prior to colonoscopy and levels of protein biomarkers determined to identify patients requiring colonoscopy. 
         [0074]    Embodiments of the present methods are useful for routine screening of patient populations. The present methods are particularly advantageous in cases where compliance of eligible subjects with existing screening recommendations is low, primarily because existing screening methods can be invasive, expensive, and unavailable in rural areas. In addition, embodiments of the present methods are useful for screening of patient populations that do not present elevated risk factors for colorectal cancer (e.g. family history), or would not otherwise be indicated for currently existing screening or diagnostic methods. 
         [0075]    In still other embodiments, methods are provided for identifying individuals who would benefit from further clinical assessment or treatment, including but not limited to, further assessment or treatment by colonoscopy or polypectomy procedures. In other embodiments, post-surgical or post-polypectomy patient monitoring is provided. In still other embodiments, the present methods are useful for monitoring responsiveness of a patient to chemopreventative or chemotherapuetic agents. 
         [0076]    In addition, methods are provided that are capable of enhancing the diagnostic and prognostic utility of currently existing colorectal screening, diagnostic, prognostic and treatment techniques. Accordingly, certain embodiments disclosed here are useful in combination with other techniques known to the art, including colonoscopy, CT scan, or Fecal Occult Blood Test. 
         [0077]    In a further aspect, the disclosure is directed to a kit for the determination of colorectal lesions in a subject. The kit includes one or more detecting reagents for detecting the one or a plurality of biomarkers of the present disclosure, and optionally includes a set of standard values for one or a plurality of protein biomarkers associated with the presence or absence of cancerous or pre-cancerous colorectal lesions. 
         [0078]    Moreover, the present disclosure relates to a kit adapted for carrying out methods of the present disclosure referred to above comprising; a) means for determining the amounts of the one or a plurality of biomarkers in a biosample of the present disclosure, b) means for comparing the amounts determined in step a) with reference amounts, whereby the presence or stage of lesions present in the colon of a subject are to be determined. In some embodiments, the kit comprises instructions for carrying out methods of the present disclosure. 
         [0079]    The term “kit” as used herein refers to a collection of the aforementioned means, suitably, provided separately or within a single container. The container, also suitably, comprises instructions for carrying out methods of the present disclosure. 
         [0080]    The present disclosure also relates to the use of a kit or kits as cited beforehand, for: identifying a subject with cancerous or pre-cancerous lesions in the colon; or for identifying a colorectal carcinoma as stage 1, stage 2, stage 3, or stage 4 carcinoma; or for determining and administering a course of treatment to a subject identified as having polyp formation in the colon; or for screening individuals to determine a need for a colonoscopy. 
         [0081]    The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. 
         [0082]    The examples that follow are illustrative of specific embodiments of the invention and various uses thereof. They are set forth for explanatory purposes only and are not to be taken as limiting the invention. 
       Examples 
     Example 1: Apc Pirc/+  Rat Microarray and Longtitudinal Analysis 
       [0083]    Animal Breeding and Maintenance. 
         [0084]    Only male rats were utilized for the microarray and proteomics studies to eliminate potential confounding by estrus cycling in female rats. A 12:12 hour light:dark cycle was maintained throughout the experiments, and rats were all dissected within a four-hour window to control for any variation due to circadian cycles. F 1  generation (ACIxF344)-Apc Pirc/+  rats were generated by breeding female ACI Apc +/+  rats (Harlan) to male F344N/Tac coisogenic Apc Pirc/+  (Pirc) rats (developed in the laboratory of WFD and now commercially available through Taconic) (Amos-Landgraf, J. M. et al.,  Proc Natl Acad Sci USA  2007, 104, (10), 4036-41). These “F 1 -Pirc” rats show an increased tumor multiplicity and decreased time to tumor emergence compared to the standard coisogenic F344N/Tac-Pirc rat. One group of 97-day old F 1 -Pirc rats was used for the microarray study. An additional two groups, an F 1 -Pirc and a (ACI X F344) F 1  Apc +/+  “F 1 -wildtype” cohorts, were followed longitudinally from 60 to 135 days of age for the proteomics study. 
         [0085]    The Microarray Rat Cohort. 
         [0086]    The microarray experiments follow the nomenclature, descriptions, and data sharing recommended by the MIAME Guidelines (Brazma, A. et al.,  Nat Genet  2001, 29, (4), 365-71). Data have been deposited in NCBI&#39;s Gene Expression Omnibus (Edgar, R. et al.,  Nucleic Acids Res  2002, 30, (1), 207-10) and are accessible through GEO Series accession number GSE54035. To measure the levels of transcripts that were differentially expressed in tumors, RNA was isolated from 10 colonic tumor samples and 4 matched normal tissue samples from four F 1 -Pirc rats. Tumor samples were obtained by harvesting one-quarter of the tumor. For the collection of normal intestinal tissue, a scalpel blade was used to gently scrape the luminal surface of the distal colon. A minimum 2 mm barrier surrounding any tumor was required for normal tissue collection. 
         [0087]    Total RNA (100 ng) was labeled with a Low Input Quick Amp kit with Cy3 dye (Agilent Technologies) according to the manufacturer&#39;s instructions. RNA collected from normal tissue from a cohort of rats was labeled with Cy5 dye. Samples were evenly distributed and hybridized to Agilent 4x44k Whole Genome microarrays. Following incubation, arrays were scanned on an Agilent High-Resolution Microarray Scanner at 3 μm resolution with a 20 bit data format. Files were extracted using Agilent Feature Extraction version 10.7. Data were then imported into Genome Suite software for analysis (Partek). A list of genes differentially expressed between normal colonic tissue and tumor tissue was generated using the criterion of differential expression equal to or greater than 5-fold with a false discovery rate (FDR) equal to or less than 5%. 
         [0088]    The Longitudinal Rat Cohorts. 
         [0089]    14 F 1 -Pirc and 10 F 1 -wildtype rats underwent endoscopy at 60 and 90 days of age to determine the multiplicity and growth pattern of colonic tumors in vivo. Rats were then sacrificed at 135 days to determine total intestinal tumor multiplicity. Blood was collected from each animal at 60, 90 and 135 days of age. For endoscopy, the animal was anesthetized with 3% isoflurane and placed on a sterile surgical field, ventral side down. The colon was flushed with warm saline to remove any fecal material and to provide lubrication. Tumors were examined at each of the two endoscopy visits and any tumor that was seen at both visits was given one of three scores: growing, static, or regressing. A consensus score was generated for each tumor based on agreement between at least two of three blinded observers. After sacrifice at 135 days of age, formalin-fixed tumors in the small intestine and colon were counted at 10× magnification on an Olympus dissecting microscope. 
       Example 2: Liquid Chromatography Selected Reaction Monitoring Mass Spectrometry 
       [0090]    Protein Candidate Selection. 
         [0091]    Serum proteins for targeted mass spectrometry analysis were chosen using two strategies. First, protein candidates were chosen corresponding to transcripts up-regulated in colon tumors in the microarray study. These candidates were nominated using three criteria: those with RNA levels up-regulated at least 5-fold in colonic neoplasms compared to normal tissue after filtering to a 0.05 false discovery rate; proteins predicted or known to be secreted (Edgar, R. et al.,  Nucleic Acids Res  2002, 30, (1), 207-10); and proteins with potential biological significance to colon cancer (Vogelstein, B. et al.,  Science  2013, 339, (6127), 1546-58). The second strategy of candidate selection used quantitative proteomic data from the serum of the Apc Min/+  mouse compared to wildtype, as previously described (Ivancic, M. M. et al.,  J Proteome Res  2013, 12, (9), 4152-66). Two protein candidates that arose in both detection strategies were chosen, to increase the chance of identifying differentially expressed blood proteins. 
         [0092]    Synthetic Peptides for Targeted Analysis. 
         [0093]    Peptides were designed and synthesized using a three tiered selection process. ( FIG. 2 .) Tier 1 selection criteria included sequence uniqueness (Altschul, S. F. et al.,  J Mol Biol  1990, 215, (3), 403-10), length, relative hydrophobicity (Yang, F. et al.,  Expert Rev Proteomics  2012, 9, (2), 129-34), and absence of known post-translational modification (unless that modification is targeted for analysis). 
         [0094]    Tier 2 selection criteria included empirical mass spectrometry data indicating suitability of proteotypic peptide sequence, including untargeted data collected from in-house shotgun proteomics studies, and data found in open-source mass spectrometry repositories such as PeptideAtlas, which stores proteomic data from yeast, mice, and humans (Deutsch, E. W. et al.,  EMBO Rep  2008, 9, (5), 429-34). Peptides routinely identified within these databases have a greater chance of being identified in a targeted analysis. These mass spectrometry data resources can also identify proteins with peptides that are prone to missed cleavage during enzymatic digestion. Tier 2 criteria also comprised predictive algorithms to identify peptides that are good for targeted SRM analyses on triple quadrupole instruments. For example, SRMAtlas uses the predictive algorithm PABST to assist in selecting proteotypic peptides for SRM analyses. PABST uses both theoretical and empirical data to score peptides for their usability in an SRM-MS experiment for yeast, mice, or humans. 
         [0095]    Tier 3 criteria are relevant to designing peptides that have cross-species significance. The peptides targeted must be unique within the proteome of the experimental model species, but must also be identical to the orthologous protein in humans, thus facilitating extrapolation of animal data to methods and reagents useful in humans. 
         [0096]    In a first iteration, 61 peptides were selected from rat transcriptome candidates (Example 3, infra). Of the 61 tested, 9 peptides satisfied additional screening criteria. In a second round of screening, 30 peptides were selected and tested and 27 satisfied additional screening criteria. A complete list of peptides is provided in Table 2. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Peptide Sequence 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 (*indicates  13 C 15 N  
                   
                   
                   
                   
                   
                   
                   
                   
                 Top 
                   
               
               
                   
                 aminoacid in  
                   
                   
                   
                   
                 Discovery 
                 Endo- 
                   
                   
                 Peptide 
                   
               
               
                   
                 reference 
                   
                   
                   
                   
                 study  
                 genous 
                   
                   
                 ions 
                   
               
               
                   
                 standard 
                   
                   
                 Percent 
                   
                 (rat 
                 Ovserved 
                 Dominant 
                   
                 monitored 
                 Peptide 
               
               
                   
                 unless notes 
                   
                 Gene 
                 Purity of 
                 Sequence 
                 and/or 
                 in SRM 
                 Charge 
                 Collision 
                 (Qtrap 
                 Ion 
               
               
                 Number 
                 otherwise 
                 Name 
                 symbol 
                 standard 
                 Homology 
                 mouse) 
                 assay 
                 State 
                 Energy 
                 5500) 
                 quantified 
               
               
                   
               
             
             
               
                  1 
                 TSWGLENEALV*R 
                 Interluken 1 
                 IL1RL1 
                  95.6 
                 Rat 
                 Trans- 
                 No 
                 +2 
                 38.2 
                 y8, y9,  
                 N/A 
               
               
                   
                   
                 receptor- 
                   
                   
                   
                 criptome 
                   
                   
                   
                 y10, b8 
                   
               
               
                   
                   
                 like 1 
                   
                   
                   
                 (Rat) 
                   
                   
                   
                   
                   
               
               
                  2 
                 FTHTENGTNYIV*TATR 
                   
                   
                 100 
                 Rat 
                   
                 No 
                 +3 
                 26.0 
                 y6, b8, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 b9, b10 
                   
               
               
                  3 
                 SFTV*EEK 
                   
                   
                  97.6 
                 Mouse 
                   
                 No 
                 +2 
                 19.7 
                 y4, y5, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                 b2, b3 
                   
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                  4 
                 AHMSYLFICK* 
                   
                   
                 96.1 
                 Rat 
                   
                 No 
                 +2 
                 30.7 
                 y7, y8, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 b6, b7 
                   
               
               
                   
               
               
                  5 
                 FLVDQIV*K 
                 Matrix 
                 MMP7 
                 100 
                 Rat 
                 Trans- 
                 Yes 
                 +2 
                 23.2 
                 y6, y7, b7 
                 y6 
               
               
                  6 
                 IVSYTTDLP*R 
                 Metallo- 
                   
                  96.1 
                 Rat 
                 criptome 
                 No 
                 +2 
                 29.0 
                 y6, y7, 
                 N/A 
               
               
                   
                   
                 proteinase- 
                   
                   
                   
                 (Rat) 
                   
                   
                   
                 y8, y9 
                   
               
               
                 80 
                 DLPHTVD*R 
                 7 (Matrily 
                   
                  83 
                 Human 
                   
                 No 
                 +3 
                 20.9 
                 y4, y5, b4 
                 Monitoring 
               
               
                   
                   
                 sin) 
                   
                   
                   
                   
                   
                   
                   
                   
                 y5 
               
               
                   
               
               
                  7 
                 TYFFVGDK* 
                 Matrix 
                 MMP10 
                 99.5 
                 Mouse 
                 Trans- 
                 No 
                 +2 
                 23.6 
                 y5, y6, y7 
                 N/A 
               
               
                   
                   
                 Metallo- 
                   
                   
                 and 
                 criptome 
                   
                   
                   
                   
                   
               
               
                   
                   
                 proteinase- 
                   
                   
                 Rat 
                 (Rat) 
                   
                   
                   
                   
                   
               
               
                  8 
                 TVTHTLK* 
                 10 
                   
                  91.3 
                 Rat 
                   
                 No 
                 +2 
                 18.5 
                 y6, y7, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 y8, b8 
                   
               
               
                  9 
                 QDHSTMCKAQQYL*EK 
                   
                   
                  99.1 
                 Rat 
                   
                 No 
                 +3 
                 25.8 
                 y3, y4, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 y5, b5 
                   
               
               
                 10 
                 LDSNTEMMHKP*R 
                   
                   
                  99 
                 Rat 
                   
                 No 
                 +4 
                 19.2 
                 y5, y6, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 y7, y8 
                   
               
               
                 11 
                 FLGLE&lt;TGK* 
                   
                   
                  97.7 
                 Mouse 
                   
                 No 
                 +2 
                 24.1 
                 y5, y7, y8 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 MMP3/10 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 Rat 
                   
                   
                   
                   
                   
                   
               
               
                 12 
                 IDAAV*FEK 
                   
                   
                  96.9 
                 Mouse 
                   
                 Yes 
                 +2 
                 21.2 
                 y4, y5, 
                 Too low to 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                 y6, y7 
                 quantify 
               
               
                   
                   
                   
                   
                   
                 Rat 
                   
                   
                   
                   
                   
                   
               
               
                 13 
                 GSQFWAV*R 
                   
                   
                  99.9 
                 Mouse 
                   
                 Yes 
                 +2 
                 22.9 
                 y4, y5, y6 
                 Too low to 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                 quantify 
               
               
                   
                   
                   
                   
                   
                 Rat 
                   
                   
                   
                   
                   
                   
               
               
                 14 
                 SNSWLL*C 
                   
                   
                  98.8 
                 Mouse 
                   
                 Yes 
                 +2 
                 20.8 
                 b4, b5, b6 
                 b5 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 Rat 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 15 
                 DDAFFIGSTLATIASTV*YSK 
                 CD44 
                 CD44 
                 100 
                 Rat 
                 Trans- 
                 Yes 
                 +2 
                 28.9 
                 y7, y9, b5 
                 Too low to 
               
               
                   
                   
                 antigen 
                   
                   
                   
                 criptome 
                   
                   
                   
                   
                 quantify 
               
               
                   
                   
                   
                   
                   
                   
                 (Rat) 
                   
                   
                   
                   
                   
               
               
                 16 
                 EPTETPDQFMTADET*R 
                   
                   
                 100 
                 Rat 
                   
                 No 
                 +2 
                 49.0 
                 y8, y9, b5 
                 N/A 
               
               
                 17 
                 TQWMPIHSNPEVLLQTTT*R 
                   
                   
                 100 
                 Rat 
                   
                 No 
                 +3 
                 30.2 
                 y7, y8, b9 
                 N/A 
               
               
                 18 
                 STPEGYILHTDLPTSQP*TGDR 
                   
                   
                 100 
                 Rat 
                   
                 Yes 
                 +3 
                 30.7 
                 y5, y8, y9 
                 Too low to 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 quantify 
               
               
                 19 
                 KPSELNGEASK* 
                   
                   
                  90.7 
                 Mouse 
                   
                 No 
                 +3 
                 19.1 
                 y4, y5, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                 b4, b5 
                   
               
               
                   
                   
                   
                   
                   
                 Rat 
                   
                   
                   
                   
                   
                   
               
               
                 20 
                 NLQSVDMK* 
                   
                   
                  98.2 
                 Mouse 
                   
                 Yes 
                 +2 
                 22.4 
                 y8, y9, 
                 Too low to 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                 y10, b5 
                 quantify 
               
               
                   
                   
                   
                   
                   
                 Rat 
                   
                   
                   
                   
                   
                   
               
               
                 21 
                 LVINSGNGTV*EDR 
                   
                   
                  86.1 
                 Rat 
                   
                 No 
                 +2 
                 34.9 
                 y8, y9, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 y10, y11 
                   
               
               
                 73 
                 YGFIGHVVIP*R 
                   
                   
                  82 
                 Rat 
                   
                 Yes 
                 +3 
                 22.8 
                 y4, y5, y8 
                 y4 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 22 
                 AFPAFVL*R 
                 Wnt 
                 Wit1 
                  95 
                 Rat 
                 Trans- 
                 Yes 
                 +2 
                 22.0 
                 y4, y5, y6 
                 y6 
               
               
                 23 
                 LGTVPHK* 
                 Inhibitory 
                   
                 100 
                 Mouse 
                 criptome 
                 No 
                 +2 
                 17.2 
                 y4, b4, b5 
                 N/A 
               
               
                   
                   
                 Factor 1 
                   
                   
                 rat 
                 (Rat) 
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 24 
                 ASVVQVGFPCL*GK 
                   
                   
                  99.2 
                 Mouse 
                   
                 No 
                 +2 
                 34.6 
                 y7, y8, y9 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 25 
                 YGASLMHAPRPAGAGL*ER 
                   
                   
                 100 
                 Mouse 
                   
                 No 
                 +3 
                 26.3 
                 y10, b9, b10 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                 26 
                 TPQNAI*FFK 
                   
                   
                 100 
                 Mouse 
                   
                 No 
                 +2 
                 26.1 
                 y5, y6, y7 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 27 
                 TCQQAECP*GGCR 
                   
                   
                  95.2 
                 Mouse 
                   
                 No 
                 +2 
                 35.3 
                 y5, y7, y8 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 28 
                 ADAGQPPEESLYLWI*DAHQAR 
                   
                   
                 100 poor 
                 Mouse 
                   
                 No 
                 +3 
                 31.6 
                 y7, y8, y9 
                 N/A 
               
               
                   
                   
                   
                   
                 peak 
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                 shape 
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                 29 
                 LWSILPCLLLL*R 
                   
                   
                  97.6 
                 Mouse 
                   
                 Yes 
                 +2 
                 38.4 
                 y7, y8, y10 
                 Too low to 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                 quantify 
               
               
                   
               
               
                 30 
                 VVGGKPAEMGDYPWQVAI*K 
                 Complement 
                 CFI 
                  99.3 
                 Rat 
                 Trans- 
                 Yes 
                 +3 
                 28.2 
                 y7, y8, 
                 y7 
               
               
                   
                   
                 Factor I 
                   
                   
                   
                 criptome 
                   
                   
                   
                 b9, b11 
                   
               
               
                 31 
                 LPYQCP*K 
                   
                   
                  96.3 
                 Rat 
                 and 
                 Yes 
                 +2 
                 21.6 
                 y4, y5, y6 
                 y8 
               
               
                   
                   
                   
                   
                   
                 and 
                 Proteome 
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 32 
                 VFCQP*WQK 
                   
                   
                  99.9 
                 Rat 
                   
                 Yes 
                 +2 
                 26.9 
                 y5, y6, y7 
                 y6 
               
               
                 33 
                 GYPTYCHLK* 
                   
                   
                  98.8 
                 Rat 
                   
                 Yes 
                 +2 
                 28.2 
                 y5, y6, y7 
                 y7 
               
               
                 34 
                 SFECLHPEIK* 
                   
                   
                  99.6 
                 Mouse 
                   
                 Yes 
                 +2 
                 31.7 
                 y7, 78, b6 
                 b6 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                 35 
                 FNIPVNHK* 
                   
                   
                 100 
                 Rat 
                   
                 Yes 
                 +3 
                 17.1 
                 y3, y4 
                 y5 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 y6, b3 
                   
               
               
                 36 
                 INSTECLHVR* 
                   
                   
                  99.8 
                 Rat 
                   
                 Yes 
                 +3 
                 30.8 
                 y6, y7, y8 
                 N/A 
               
               
                 37 
                 FNVSLIYGSTDTEGIVQV*K 
                   
                   
                  95.4 
                 Rat 
                   
                 Yes 
                 +2 
                 28.5 
                 y8, y9 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 y10, y12 
                   
               
               
                 81 
                 VFSLQWGEV*K 
                   
                   
                  75 
                 Human 
                   
                 Yes 
                 +2 
                 28.3 
                 y5, y6, 
                 y6 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 y7, y8 
                   
               
               
                   
               
               
                 38 
                 ISHELESSSSEV*N 
                 Secreted 
                 SPP1 
                  94.9 
                 Mouse 
                 Trans- 
                 No 
                 +2 
                 36.2 
                 b6, b7, 
                 N/A 
               
               
                   
                   
                 Phospho- 
                   
                   
                 and 
                 criptome 
                   
                   
                   
                 b9, b11 
                   
               
               
                   
                   
                 protein-1 
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 39 
                 SISTINVPHQY*SR 
                 S100 calcium 
                 S100A9 
                 100 
                 Rat 
                 Trans- 
                 Yes 
                 +3 
                 24.3 
                 y6, y7 
                 Too low 
               
               
                   
                   
                   
                   
                   
                   
                 criptome 
                   
                   
                   
                 y8, b7 
                 to quantify 
               
               
                 40 
                 YGHPDTLNK* 
                 binding 
                   
                  94.5 
                 Rat 
                   
                 No 
                 +3 
                 17.9 
                 y4, y5, 
                 N/A 
               
               
                   
                   
                 protein A9 
                   
                   
                   
                   
                   
                   
                   
                 y6, b8 
                   
               
               
                   
               
               
                 41 
                 LSTSWTEEDNVDNTL*FK 
                 Follistatin 
                 FST 
                  96.8 
                 Mouse 
                 Trans- 
                 Yes 
                 +3 
                 27.8 
                 y7, y8, 
                   
               
               
                   
                   
                   
                   
                   
                 rat 
                 criptome 
                   
                   
                   
                 y9, y10 
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 42 
                 ATCLL*GR 
                   
                   
                 100 
                 Mouse 
                   
                 No 
                 +2 
                 18.3 
                 y4, y5, b4 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 43 
                 EECCST*GR 
                   
                   
                 100 
                 Mouse 
                   
                 No 
                 +2 
                 24.2 
                 y5, y6, y7 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 44 
                 WMIFNGGAPNCIP*CK 
                   
                   
                 100 
                 Mouse 
                   
                 No 
                 +2 
                 46.1 
                 y3, y9, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                 y10, y11 
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 45 
                 SIGLAYEGK* 
                   
                   
                 100 
                 Mouse 
                   
                 No 
                 +2 
                 22.5 
                 y4, y5, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                 y6, y7 
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 46 
                 EAACSSGVLLEV*K 
                   
                   
                  95.1 
                 Mouse 
                   
                 No 
                 +2 
                 34.6 
                 y7, y8, y9 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 47 
                 CSLCDELCPDSK* 
                   
                   
                 100 
                 Mouse 
                   
                 No 
                 +2 
                 38.0 
                 y7, y8, y9 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 48 
                 SCEDIQCGGGK* 
                   
                   
                 100 
                 Mouse 
                   
                 No 
                 +2 
                 30.3 
                 y7, y8, y9 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 49 
                 EACLDPEAPMV*QK 
                 Chemokine 
                 Cxcl1 
                  99.2 
                 Rat 
                 Trans- 
                 No 
                 +2 
                 34.6 
                 y6, y9, y10 
                 N/A 
               
               
                   
                   
                 (C-X-C 
                   
                   
                   
                 criptome 
                   
                   
                   
                   
                   
               
               
                   
                   
                 motif)  
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 ligand 1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 50 
                 LDQNQV*R 
                 Chemokine 
                 Cd2 
                 100 
                 Rat 
                 Trans- 
                 No 
                 +2 
                 20.6 
                 y4, y5, 
                 N/A 
               
               
                   
                   
                 (C-C 
                   
                   
                   
                 criptome 
                   
                   
                   
                 y6, b5 
                   
               
               
                 51 
                 MI*PMSR 
                 motif)  
                   
                 100 
                 Mouse 
                   
                 Yes 
                 +2 
                 16.7 
                 y3, y4, y5 
                 Too low 
               
               
                   
                   
                 ligand 2 
                   
                   
                 and 
                   
                   
                   
                   
                   
                 to quantify 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 52 
                 TLFLLALLGGVSGL*R 
                 Leucine- 
                 Lng1 
                  95.6 
                 Rat 
                 Trans- 
                 Yes 
                 +2 
                 39.4 
                 y10, y11 
                 Too low 
               
               
                   
                   
                 Rich alpha- 
                   
                   
                   
                 criptome 
                   
                   
                   
                 y12, b9 
                 to quantify 
               
               
                 53 
                 SSAALNTLVL*R 
                 2- 
                   
                  97.7 
                 Rat 
                 and 
                 Yes 
                 +2 
                 28.4 
                 y6, y7 
                 y6 
               
               
                   
                   
                 glycoprotein 
                   
                   
                   
                 Proteome 
                   
                   
                   
                 y8, y9 
                   
               
               
                 54 
                 LLDVAELGT*L 
                   
                   
                  98.2 
                 Rat 
                   
                 No 
                 +1 
                 55.2 
                 b6, b7, b8 
                 N/A 
               
               
                 55 
                 SLPPGL*FR 
                   
                   
                  99.4 
                 Rat 
                   
                 Yes 
                 +2 
                 21.0 
                 y4, y5, y6 
                 Too low 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 to quantify 
               
               
                 56 
                 DLVDL*CR 
                   
                   
                 100 
                 Rat 
                   
                 No 
                 +2 
                 21.1 
                 y4, y5, b4 
                 N/A 
               
               
                 57 
                 LHL*EGNR 
                   
                   
                 96.1 
                 Mouse 
                   
                 No 
                 +2 
                 19.7 
                 y4, y5, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                 y6, b4 
                   
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                 58 
                 ENQL*QEASAR 
                   
                   
                 95.8 
                 Rat 
                   
                 No 
                 +2 
                 26.4 
                 y6, y7, 
                 N/A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 y8, b5 
                   
               
               
                 76 
                 VAAGAFQGL* 
                   
                   
                 83 
                 Human 
                   
                 Yes 
                 +2 
                 26.7 
                 y5, y7 
                 y8 
               
               
                   
                 (13C labeled 
                   
                   
                   
                   
                   
                   
                   
                   
                 y8, y9 
                   
               
               
                   
                 only) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 59 
                 NLYLSCV*MK 
                 Interleukin- 
                 Il1b 
                  99 
                 Mouse 
                 Trans- 
                 No 
                 +2 
                 27.9 
                 b8, b9, b10 
                 N/A 
               
               
                   
                   
                 1 beta 
                   
                   
                 and 
                 criptome 
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                 60 
                 CLVLSDPCEL*K 
                   
                   
                  99 
                 Rat 
                   
                 No 
                 +2 
                 33.8 
                 y5, y6, y7 
                 N/A 
               
               
                 61 
                 DGTPTLQESV*DPK 
                   
                   
                 100 
                 Mouse 
                   
                 Yes 
                 +2 
                 38.5 
                 y8, y9, 
                 Too low 
               
               
                   
                   
                   
                   
                   
                 and 
                   
                   
                   
                   
                 y11, b3 
                 to quantify 
               
               
                   
                   
                   
                   
                   
                 rat 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 62 
                 SLSQQIENI*R 
                 Collagen 
                 Col1a1 
                  86 
                 Mouse 
                 Proteome 
                 Yes 
                 +2 
                 34.3 
                 y6, y7, 
                 y6 
               
               
                   
                   
                 alpha-1(I) 
                   
                   
                 rat and 
                   
                   
                   
                   
                 y8, y9 
                   
               
               
                   
                   
                 chain 
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 63 
                 IPLENLQII*R 
                 Epidermal 
                 EGFR 
                  96 
                 Mouse 
                 Proteome 
                 Yes 
                 +2 
                 34.6 
                 y5, y6, 
                 y7 
               
               
                   
                 (13C labeled 
                 Growth 
                   
                   
                 rat and 
                   
                   
                   
                   
                 y7, y8 
                   
               
               
                   
                 only) 
                 Factor 
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                 92 
                 NYVVTDHGSCV*R 
                 Recptor 
                   
                  70 
                 Mouse 
                   
                 Yes 
                 +3 
                 23.1 
                 y6, y7, y8 
                 y6 
               
               
                   
                   
                   
                   
                   
                 rat and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 64 
                 EVSFDVEL*PK 
                 Inter-alpha- 
                 ITIH3 
                  92 
                 Mouse 
                 Proteome 
                 Yes 
                 +2 
                 29.8 
                 y5, y6, 
                 y5 
               
               
                   
                 (13C Labeled 
                 trypsin 
                   
                   
                 rat and 
                   
                   
                   
                   
                 y7, y8 
                   
               
               
                   
                 only) 
                 inhibitor 
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 heavy 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 chain H3 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 65 
                 AYVAFPD*FFR 
                 Maltase 
                 Mgam 
                  88 
                 Mouse 
                 Proteome 
                 Yes, 
                 +2 
                 34.1 
                 y5, y6, 
                 y5 
               
               
                   
                   
                 Glucomylase 
                   
                   
                 and 
                   
                 mouse, no 
                   
                   
                 y7, y8 
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                 human 
                   
                   
                   
                   
               
               
                 83 
                 SSVYANAFPSTPVNPL*R 
                   
                   
                  82 
                 Human 
                 Proteome 
                 No 
                 +2 
                 38.6 
                 y3, y6, 
                 N/A 
               
               
                   
                 (13C Labeled 
                   
                   
                   
                   
                   
                   
                   
                   
                 y9, b8 
                   
               
               
                   
                 only) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 66 
                 NFFNPPII*SR 
                 Coagulation 
                 F5 
                  93 
                 Mouse 
                 Proteome 
                 Yes 
                 +2 
                 30.6 
                 y6, y7, 
                 y6 
               
               
                   
                   
                 factor V 
                   
                   
                 rat and 
                   
                   
                   
                   
                 y8, b8 
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 67 
                 LWWLDL*K 
                 Hemopexin 
                 Hps 
                 not 
                 Mouse 
                 Proteome 
                 Yes 
                 +2 
                 26.4 
                 y4, y5, 
                 y5 
               
               
                   
                 (13C labeled 
                   
                   
                 deter- 
                 rat and 
                   
                   
                   
                   
                 y6, b8 
                   
               
               
                   
                 only) 
                   
                   
                 mined 
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 68 
                 TIEAEAAHGTV*TR 
                 Isocitrate 
                 Idh2 
                  98 
                 Mouse 
                 Proteome 
                 No 
                 +3 
                 22.2 
                 y5, y6, 
                 N/A 
               
               
                   
                   
                 dehyro- 
                   
                   
                 rat and 
                   
                   
                   
                   
                 y7, y8 
                   
               
               
                   
                   
                 genase  
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 [NADP], 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 mitochon- 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 drial 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 69 
                 EAEAAIYHLQLFEEL*R 
                 Pyruvate 
                 Pkm2 
                  85 
                 Mouse 
                 Proteome 
                 No 
                 +3 
                 37.7 
                 y5, y6, 
                 N/A 
               
               
                   
                 (13C labeled 
                 Kinase, M2 
                   
                   
                 rat and 
                   
                   
                   
                   
                 y7, y8 
                   
               
               
                   
                 only) 
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 70 
                 VLEPTL*K 
                 Vitamin 
                 Gc 
                  94 
                 Mouse 
                 Proteome 
                 Yes 
                 +2 
                 23.3 
                 y4, y5, y6 
                 y4 
               
               
                   
                   
                 D-binding 
                   
                   
                 and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 protein 
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 71 
                 FEDGVLDPDYP*R 
                 Vitronectin 
                 VTN 
                  99 
                 Rat and 
                 Proteome 
                 Yes 
                 +2 
                 34.5 
                 y5, y6, 
                 y5 
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                 y7, y8 
                   
               
               
                   
               
               
                 72 
                 FAHTVVT*SR 
                 Inter-alpha- 
                 ITIH4 
                  88 
                 Mouse 
                 Proteome 
                 Yes 
                 +3 
                 16.1 
                 y3, y4, 
                 y3 
               
               
                   
                   
                 trypsin 
                   
                   
                 rat and 
                   
                   
                   
                   
                 b3, b4 
                   
               
               
                   
                   
                 inhibitor, 
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 Heavy chain 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 4 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 74 
                 TLTLLSV*TR 
                 CEACAM5 
                 CEA 
                  77 
                 Human, 
                 N/A Human 
                 No 
                 +2 
                 27.9 
                 y5, y6, 
                 Monitoring 
               
               
                   
                   
                   
                   
                   
                 No 
                 Prognostic 
                   
                   
                   
                 y8, b5 
                 y5 
               
               
                   
                   
                   
                   
                   
                 Murine 
                 marker 
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 homo- 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 logs 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 75 
                 LCGTFLGGPKPP*QR 
                 Cathepsin 
                 Ctsb 
                  98 
                 Human 
                 Proteome 
                 Yes 
                 +3 
                 26.3 
                 y5, y6, 
                 y8 
               
               
                   
                   
                 B 
                   
                   
                   
                   
                   
                   
                   
                 y7, y8 
                   
               
               
                   
               
               
                 77 
                 GYVIKPL*VWV 
                 Serum 
                 APCS 
                  87 
                 Human 
                 Proteome 
                 Yes 
                 +2 
                 27.0 
                 y6, y7, 
                 b9 
               
               
                   
                   
                 Amyloid P 
                   
                   
                   
                   
                   
                   
                   
                 y8, b9 
                   
               
               
                   
               
               
                 78 
                 IFFESVYGQC*K 
                 Fetuin B 
                 FetuB 
                  77 
                 Human 
                 Proteome 
                 Yes 
                 +2 
                 31.7 
                 y6, y7, 
                 y9 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 y8, y9 
                   
               
               
                   
               
               
                 79 
                 ESDYSVVSL*K 
                 C-reactiv 
                 Crp 
                  99 
                 Human 
                 Proteome 
                 Yes 
                 +2 
                 28.2 
                 y5, y6, y8 
                 y6 
               
               
                   
                 (13C labeled 
                 protein 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 only) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 82 
                 FTVDRPFLFLIY*EHR 
                 heparin 
                 SerpinD1 
                  87 
                 Human 
                 Proteome 
                 Yes 
                 +3/+4 
                 30.0/24.2 
                 y5, y6, 
                 y5 
               
               
                   
                   
                 cofactor 2 
                   
                   
                   
                   
                   
                   
                   
                 y7, b8 
                   
               
               
                   
               
               
                 84 
                 LAGAPSEDPQFP*K 
                 Sulflhydryl 
                 QSOX1 
                  92 
                 Human 
                 Proteome 
                 Yes 
                 +2 
                 31.3 
                 y5, y7, 
                 y9 
               
               
                   
                   
                 Osucase 1 
                   
                   
                   
                   
                   
                   
                   
                 y9, b4 
                   
               
               
                   
               
               
                 85 
                 AEEYEFLTPVEEAP*K 
                 Rho-GDP 
                 Arhgdia 
                  88 
                 Human 
                 Proteome 
                 Yes 
                 +2 
                 38.4 
                 y7, y9, 
                 y7 
               
               
                   
                   
                 Dissociation 
                   
                   
                   
                   
                   
                   
                   
                 y10 
                   
               
               
                   
                   
                 Inhibitor 1, 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 Isoform a 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 (RHOGDI) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 86 
                 WDEELAAFA*K 
                 Peptidase 
                 PI16 
                 Peak 1 is 
                 Human 
                 Proteome 
                 Yes 
                 +2 
                 29.1 
                 y6, y8, y9 
                 y8 
               
               
                   
                   
                 inhibitor 16 
                   
                 21% and 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                 Peak 2 is 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                 66% 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 87 
                 GPFPQEL*VR 
                 Cadherin-2- 
                 Cdh2 
                  90 
                 Mouse 
                 Proteome 
                 Yes 
                 +2 
                 25.6 
                 y6, y7, b3 
                 y6 
               
               
                   
                 (13C Labeled 
                 (N-Cadherin) 
                   
                   
                 rat and 
                   
                   
                   
                   
                   
                   
               
               
                   
                 only) 
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 88 
                 WEYYDSVY*TER 
                 Dipeptidyl 
                 DPP4 
                 75 
                 Mouse 
                 Proteome 
                 Yes 
                 +2 
                 37.1 
                 y7, y8, 
                 y9 
               
               
                   
                   
                 peptidase 4 
                   
                   
                 rat and 
                   
                   
                   
                   
                 y9, b5 
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 89 
                 VTGVVL*FR 
                 extra- 
                 Sod3 
                  93 
                 Human 
                 Proteome 
                 Yes 
                 +2 
                 23.9 
                 y4, y5, 
                 y6 
               
               
                   
                 (13C Labeled 
                 cellular 
                   
                   
                   
                   
                   
                   
                   
                 y6, y7 
                   
               
               
                   
                 only) 
                 superoxide 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 dismutase 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 [Cu-Zn] 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 90 
                 DVDIDSYPDEELPCSA*R 
                 Thrombo- 
                 Thbs4 
                  92 
                 Mouse 
                 Proteome 
                 Yes 
                 +2 
                 41.5 
                 y5, y9, 
                 y10 
               
               
                   
                   
                 spondin 
                   
                   
                 rat and 
                   
                   
                   
                   
                 y10, b7 
                   
               
               
                   
                   
                   
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 91 
                 GFGPPATN*QFTTK 
                 receptor- 
                 Ptprm 
                  98 
                 Mouse 
                 Proteome 
                 Yes 
                 +2 
                 37.5 
                 y7, y9, b4 
                 y9 
               
               
                   
                   
                 type 
                   
                   
                 rat and 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 tyrosin- 
                   
                   
                 human 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 protein 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 phosphatase 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 mu 
               
               
                   
               
             
          
         
       
     
         [0097]    After peptide selection, a synthetic form of each targeting peptide of interest, containing one heavy labeled stable isotope amino acid, was synthesized. This peptide can either be used for absolute quantitative analysis, or it can be used for relative quantitative analysis. The embodiments in the examples infra use relative quantification with unpurified synthetic reference standards spiked into the samples at known dilution factors. In complex mixtures such as serum, multiple isobaric peptides from different proteins will produce peaks at similar elution times. A heavy reference standard can assist in identifying the correct retention time and transition order of the endogenous peptide, thus preventing quantification of the incorrect peptide. An isotopically labeled peptide reference standard unique to each selected biomarker candidate was synthesized by the UW-Madison Biotechnology Center&#39;s peptide synthesis core facility, with the incorporation of at least one of  13 C and  15 N labeled amino acids in each reference peptide. (Table 2.) 
         [0098]    Sample Collection. 
         [0099]    Blood samples were collected, processed and stored following the standard operating procedure published by the Early Detection Research Network (Tuck, M. K. et al.,  J Proteome Res  2009, 8, (1), 113-7). Approximately 1.5 ml of blood was collected from the retro-orbital sinus into Protein LoBind tubes (Eppendorf). The time of day for blood collection was controlled across the study, with all blood samples collected within a 2-hour window. Blood was left to clot at room temperature for 30-60 minutes before centrifugation at room temperature for 20 minutes at 1,200 g (Eppendorf 5415c). The serum was then transferred to new Protein LoBind tubes using sterile LoRetention Dualfilter pipet tips (Eppendorf) and frozen at −80° C. until use. 
         [0100]    Sample Preparation. 
         [0101]    Serum was washed five times with 10 kDa MWCO Amicon Centriprep units with 5 mL of 20% acetonitrile/80% Milli-Q H 2 O at 1500 g for 1 h at 4° C. followed by lyophilization. For murine studies, the major blood proteins (albumin, transferrin, and IgG) were removed from a 2 mg aliquot of resolublized serum, using a 4.6 mm×100 mm mouse MARS column (Agilent Technologies) according to the manufacturer&#39;s protocol. (Human sample preparation described in Example 4, infra.) Proteins not retained by the column were collected, concentrated, and precipitated with trichloroacetic acid as previously described (Ivancic, M. M. et al.,  J Proteome Res  2013, 12, (9), 4152-66). A Pierce™ BCA protein concentration assay was performed on resolublized samples according to the manufacturer&#39;s instructions (Thermo Fisher Scientific). 
         [0102]    A 100 μg aliquot of serum protein from each sample underwent reduction and alkylation of cysteine residues, followed by digestion using sequencing grade porcine trypsin (Promega) at a 1:50 trypsin-protein ratio. Prior to reduction and alkylation, the stable isotope labeled peptide reference standard of each target endogenous peptide was added to the serum protein sample. Trypsin digestions were performed at 37° C. overnight. The resultant peptides were desalted on SPEC C18 Pipette Tips (Agilent Technologies) according to manufacturer&#39;s instructions. Eluted peptides were dried using a vacuum centrifuge. 
         [0103]    LC-SRM-MS Method. 
         [0104]    Synthetic peptides were resolubilized in 0.1% formic acid, 5% acetonitrile, and water to a concentration of 1 μg/μl endogenous peptides. Liquid chromatography separation was achieved using a NanoLC ultra 2D (Eksigent) equipped with a nanoflex cHiPLC. The microfluidic chip was a 75 μm diameter 15 cm length column with C18 3 μm resin at a 120 Å pore size and the temperature of the cHiPLC system was set to 37° C. A 90 minute gradient at a flow rate of 300 nl/min was applied as follows: starting conditions were set at 97% 0.1% formic acid in water (buffer A) and 3% 0.1% formic acid in acetonitrile (buffer B) and increased linearly to 15% B by 30 minutes. Buffer B was increased linearly to 35% by 60 minutes and then a steeper gradient to 50% B was applied to 85 minutes. The gradient was switched back to starting conditions at 90 minutes. Peptides were eluted directly into a 5500 QTrap (AbSciex). Peptide precursors were selected in Q1 followed by fragmentation in q2 and subsequent monitoring of the top 3-4 transitions for each peptide in Q3. All Q1 and Q3 masses were measured at unit resolution. To maximize dwell times, a 5-minute scheduling window was applied with a 1.5 second cycle time. Method development and peak analysis was done using Skyline software. 
         [0105]    Data Processing. 
         [0106]    Mass spectrometry results were imported into Skyline and peaks integrated. Each peptide was evaluated using the average peak area of the most intense transition over three technical replicates. For each protein, an average ratio of F 1 -Pirc/F 1 -wildtype was calculated for each of the peptides. P-values were obtained using a two-tailed Student&#39;s t-test assuming a normal distribution. 
         [0107]    The diagnostic capability of serum protein markers on an individual level and as a panel was determined by Receiver Operator Characteristic (ROC) analysis using the JROCFIT web-based calculator (see, Eng J., ROC analysis: web-based calculator for ROC curves, provided on the Johns Hopkins University School of Medicine website), using the same test set of 14 F 1 -Pirc and 10 F 1 -wildtype animals. Data format 2 (binary response with confidence rating) was used with a total of three rating categories: 1=low confidence; 2=intermediate confidence; and 3=high confidence. First, each protein was rated for its diagnostic capacity as an individual protein. Next, a group of four specific proteins, chosen on the basis of their individual ROC analyses, was evaluated for its diagnostic potential as a panel. More details of the ROC analysis of single proteins and a panel are described below. 
         [0108]    In proteomics, relative quantification often relies on the use of fold changes derived from a comparison of one biological condition (e.g. cancer) to another biological condition (e.g. no cancer). Most researchers assign an arbitrarily defined threshold expression change for the data being analyzed. Recently, reasonable threshold assignments for protein upregulation were defined by Serang and colleagues (Serang, O. et al.,  J Proteome Res  2013, 12, (10), 4556-65). They determined that a 1.2-fold change is a reasonable cut-off to consider a quantified protein upregulated. Because ROC analysis relies on quantitative cutoffs to determine a positive or negative test, the guideline set by Serang et. al. as a framework for assigning positive and negative results in the ROC analysis was used. 
         [0109]    Proteins were partitioned into those expected to be upregulated or downregulated based on the two discovery studies (transcriptome/proteome). Tables 3 and 4 show the confidence ratings assigned (1-3) for each protein expression ratio (required by analysis format 2, binary response with confidence rating, on www.jrocfit.org). It was expected that LRG1, F5, VTN, MMP7, MMP10, CD44, ITIH3, ITIH4, HPX, and CFI would be upregulated based on discovery data. Accordingly, their protein ratings were based off of the 1.2-fold threshold (Serang, O. et al.,  J Proteome Res  2013, 12, (10), 4556-65). While below the 1.2 expression threshold, proteins upregulated in the 1.16-1.19 category with low confidence were considered to reduce the possibility of false negatives in the ROC analysis. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Confidence ratings for expected upregulated colon cancer biomarkers 
               
             
          
           
               
                 Fold change/ 
                   
               
               
                 Confidence rating 
                 Description of rating 
               
               
                   
               
               
                 1.1 or less = 3 
                 Indicates high positivity that there are no tumors 
               
               
                 1.11-1.15 = 2 
                 Indicates fairly high positivity that there are  
               
               
                   
                 no tumors 
               
               
                 1.16-1.19 = 1 
                 Indicates can&#39;t definitively tell whether or not  
               
               
                   
                 there are tumors, marked as a pos. test 
               
               
                 1.20-1.29 = 2 
                 Indicates fairly high positivity that there are tumors  
               
               
                   
                 present 
               
               
                 1.30 or greater = 3 
                 Indicates very positive that the are tumors present 
               
               
                   
               
             
          
         
       
     
         [0110]    The work by Serang and colleagues did not set a fold-change value considered reasonably significant for downregulation. Therefore, an expression ratio of 0.86 or lower was set as the cutoff for reasonable downregulation. While above the 0.86 threshold, proteins with a 0.87-0.89 expression ratio were considered downregulated with a low level of confidence to avoid introducing false negatives into the analysis. The downregulation cutoffs presented below for proteins were used: 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Confidence ratings for expected downregulated colon cancer biomarkers 
               
             
          
           
               
                 Fold change/ 
                   
               
               
                 Confidence rating 
                 Description of rating 
               
               
                   
               
               
                 0.95 or greater = 3 
                 Indicates high positivity that there are no tumors 
               
               
                 0.90-0.94 = 2 
                 Indicates fairly high positivity that there are no  
               
               
                   
                 tumors 
               
               
                 0.87-0.89 = 1 
                 Indicates can&#39;t definitively tell whether or not  
               
               
                   
                 there are tumors, marked as a pos. test 
               
               
                 0.80-0.86 = 2 
                 Indicates fairly high positivity that there are tumors  
               
               
                   
                 present 
               
               
                 0.79 or less = 3 
                 Indicates very positive that the are tumors present 
               
               
                   
               
             
          
         
       
     
         [0111]    ROC Analysis of Candidates as a Panel. 
         [0112]    The murine models indicated that EGFR, LRG1, ITIH4, and F5 had the greatest diagnostic potential as determined by their individual ROC analyses and the low variance in their wildtype concentrations (see Example 3, infra.). Therefore, these 4 proteins were selected for ROC analysis as a panel. Three different analyses for EGFR, LRG1, ITIH4, and F5 as a panel were done based on the number of individual positives for these proteins using the rating system above. The first analysis was the least stringent, requiring that only 1 protein show differential expression of the four in the panel. The second and third analyses required at least 2 and 3 positive values, respectively. As with the analysis using single proteins, format 2 (binary response with confidence rating) on JROCFIT was used. Confidence ratings were assigned based on the number of positive markers in the panel. Tables 5-7 are the ratings used for each of the three stringency levels tested. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Confidence ratings for a panel in which 1 of 4 proteins must be positive 
               
             
          
           
               
                 Confidence rating 
                 Description of rating 
               
               
                   
               
               
                 3 
                 At least 3 markers are positive (Pos diagnosis) or  
               
               
                   
                 all are negative (neg. diagaosis) 
               
               
                 2 
                 2 markers are positive (pos diagnosis) 
               
               
                 1 
                 1 marker is positive (pos diagnosis) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Confidence ratings for a panel in which 2 of 4 proteins must be positive 
               
             
          
           
               
                 Confidence rating 
                 Description of rating 
               
               
                   
               
               
                 3 
                 At least 3 are positive (pos diagnosis), or all are 
               
               
                   
                 negative (neg diagnosis) 
               
               
                 2  
                 2 markers are positive (Pos diagnosis) 
               
               
                 1 
                 1 is positive (neg diagnosis) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Confidence ratings for a panel in which 3 of 4 proteins must be positive 
               
             
          
           
               
                 Confidence rating 
                 Description of rating 
               
               
                   
               
               
                 3 
                 All are positive (pos diagnosis), or all are negative 
               
               
                   
                 (neg. diagnosis) 
               
               
                 2 
                 3 are positive, 1 is negative (Pos diagnosis) or 1 is 
               
               
                   
                 positive (negative diagnosis) 
               
               
                 1 
                 2 are positive (neg diagnosis) 
               
               
                   
               
             
          
         
       
     
       Example 3: Serum Biomarkers Predict Tumor Formation in a Murine Model of Colon Cancer 
       [0113]    Transcriptomic and Proteomic Discovery Studies Identified Protein Biomarker Candidates for Validation in F 1 -Pirc Rats. 
         [0114]    A total of 928 microarray probes were differentially expressed by at least 5-fold between normal colonic tissue and tumors from F 1 -Pirc rats. In total, 543 probes were more highly expressed in tumor tissue, while the remaining 415 probes were more highly expressed in normal tissue. For the purposes of this study, only those probes upregulated in tumor were considered. The list of probes was narrowed to 12 transcriptome candidates (15 total probes) by selecting those whose gene products are secreted and suggested to have potential biological significance to colon cancer based on published literature ( FIG. 3 ). During SRM-MS method development, the endogenous proteins for 5 of the 12 transcriptome candidates and 9 of the 11 proteomic candidates were visible and quantifiable by mass spectrometry (see also Table 1). The final list of 12 proteins selected for validation included 3 candidates from the F 1 -Pirc rat tumor transcriptome analysis, 7 from the Apc Min/+  mouse serum proteomic study, with CFI and LRG1 shared between the two discovery strategies ( FIG. 4 ). 
         [0115]    Protein Expression Over Time Reveals Differential Expression Concordant with Increases in Tumor Multiplicity. 
         [0116]    Quantitative proteomics revealed that MMP7, LRG1, ITIH4, VTN, HPX and F5 proteins show increased levels in blood serum over time. ( FIG. 5 , Table 8.) Average EGFR expression in F 1 -Pirc rats was significantly downregulated at 135 days, as observed in the prior proteomics discovery study (Ivancic, M. M. et al.,  J Proteome Res  2013, 12, (9), 4152-66). In all, seven proteins showed significant changes in levels of serum in tumor-bearing Pirc rats. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Summary of protein expression and statistical analysis for individual biomarker candidates 
               
             
          
           
               
                   
                   
                   
                   
                 Average 
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                 expression 
                   
                   
                   
                   
                 F 1 -Wildtype 
               
               
                   
                 Protein 
                   
                 Time point 
                 ratio 
                   
                   
                   
                   
                 variance 
               
               
                 Protein name 
                 symbol 
                 NCBI Number 
                 (days of age) 
                 (Pirc/WT) 
                 p-value 
                 Sensitivity 
                 Specificity 
                 AUC 
                 over time 
               
               
                   
               
             
          
           
               
                 Matrix 
                 MMP7 
                 NP_036996 
                 60 
                 1.12 
                 0.46 
                 ND a   
                 ND a   
                 ND a   
                 25.7% 
               
               
                 metalloproteinase-7 
                   
                   
                 90 
                 1.38 
                 0.04 
                 57.1% 
                 80.0% 
                 0.664 
                   
               
               
                   
                   
                   
                 135 
                 1.74 
                 0.004 
                 85.7% 
                 80.0% 
                 0.843 
                   
               
               
                 Leucine-rich alpha-2 
                 LRG1 
                 NP_001009717 
                 60 
                 1.07 
                 0.06 
                 16.7% 
                 100.0% 
                 0.674 
                 12.9% 
               
               
                 glycoprotein 
                   
                   
                 90 
                 1.21 
                 0.03 
                 64.3% 
                 90.0% 
                 0.857 
                   
               
               
                   
                   
                   
                 135 
                 1.43 
                 &lt;0.001 
                 92.9% 
                 90.0% 
                 0.907 
                   
               
               
                 Inter-alpha trypsin 
                 ITIH4 
                 NP_062242 
                 60 
                 1.11 
                 0.06 
                 50.0% 
                 83.3% 
                 0.701 
                 15.0% 
               
               
                 inhibitor, heavy chain 4 
                   
                   
                 90 
                 1.14 
                 0.03 
                 28.6% 
                 100.0% 
                 0.649 
                   
               
               
                   
                   
                   
                 135 
                 1.37 
                 0.001 
                 78.6% 
                 90.0% 
                 0.871 
                   
               
               
                 Vitronectin 
                 VTN 
                 NP_062029 
                 60 
                 1.03 
                 0.61 
                 8.3% 
                 91.7% 
                 0.504 
                 16.2% 
               
               
                   
                   
                   
                 90 
                 1.12 
                 0.001 
                 35.7% 
                 100.0% 
                 0.821 
                   
               
               
                   
                   
                   
                 135 
                 1.20 
                 0.02 
                 71.4% 
                 90.0% 
                 0.854 
                   
               
               
                 Hemopexin 
                 HPX 
                 NP_445770 
                 60 
                 1.15 
                 0.006 
                 50.0% 
                 100.0% 
                 0.708 
                 23.3% 
               
               
                   
                   
                   
                 90 
                 1.26 
                 &lt;0.001 
                 78.6% 
                 100.0% 
                 0.882 
                   
               
               
                   
                   
                   
                 135 
                 1.43 
                 0.0027 
                 85.7% 
                 80.0% 
                 0.792 
                   
               
               
                 Epidermal growth 
                 EGFR 
                 NP_113695 
                 60 
                 0.97 
                 0.33 
                 8.3% 
                 100.0% 
                 0.632 
                 11.8% 
               
               
                 factor receptor 
                   
                   
                 90 
                 0.87 
                 0.002 
                 50.0% 
                 100.0% 
                 0.832 
                   
               
               
                   
                   
                   
                 135 
                 0.65 
                 &lt;0.001 
                 100.0% 
                 80.0% 
                 0.939 
                   
               
               
                 Coagulation factor V 
                 F5 
                 NP_001041343 
                 60 
                 1.00 
                 0.94 
                 8.3% 
                 100.0% 
                 0.545 
                 11.5% 
               
               
                   
                   
                   
                 90 
                 1.08 
                 0.08 
                 21.4% 
                 100.0% 
                 0.679 
                   
               
               
                   
                   
                   
                 135 
                 1.24 
                 0.007 
                 64.3% 
                 90.0% 
                 0.757 
                   
               
               
                 Inter-alpha trypsin 
                 ITIH3 
                 NP_059047 
                 60 
                 1.03 
                 0.57 
                 25.0% 
                 91.7% 
                 0.615 
                 16.3% 
               
               
                 inhibitor, heavy chain 
                   
                   
                 90 
                 1.07 
                 0.02 
                 14.3% 
                 100.0% 
                 0.679 
                   
               
               
                 H3 
                   
                   
                 135 
                 1.05 
                 0.34 
                 14.3% 
                 90.0% 
                 0.428 
                   
               
               
                 Complement Factor I 
                 CFI 
                 NP_077071 
                 60 
                 1.04 
                 0.59 
                 16.7% 
                 91.7% 
                 0.576 
                 23.9% 
               
               
                   
                   
                   
                 90 
                 1.08 
                 0.26 
                 21.4% 
                 90.0% 
                 0.867 
                   
               
               
                   
                   
                   
                 135 
                 1.13 
                 0.24 
                 50.0% 
                 80.0% 
                 0.820 
                   
               
               
                 Collagen, Type, I 
                 COL1A1 
                 NP_445756 
                 60 
                 1.11 
                 0.09 
                 8.3% 
                 91.7% 
                 0.309 
                 57.6% 
               
               
                 Alpha 1 
                   
                   
                 90 
                 1.1 
                 0.11 
                 7.1% 
                 80.0% 
                 0.371 
                   
               
               
                   
                   
                   
                 135 
                 0.91 
                 0.18 
                 42.9% 
                 70.0% 
                 0.592 
                   
               
               
                 Matrix 
                 MMP10 
                 NP_598198 
                 60 
                 1.02 
                 0.81 
                 8.3% 
                 83.3% 
                 0.462 
                 12.0% 
               
               
                 Metalloproteinase 10 
                   
                   
                 90 
                 1.03 
                 0.30 
                 7.1% 
                 100.0% 
                 0.561 
                   
               
               
                   
                   
                   
                 135 
                 0.97 
                 0.48 
                 0.0% 
                 90.0% 
                 0.482 
                   
               
               
                 CD44 Antigen 
                 CD44 
                 NP_037056 
                 60 
                 1.05 
                 0.48 
                 16.7% 
                 75.0% 
                 0.286 
                 17.8% 
               
               
                   
                   
                   
                 90 
                 1.07 
                 0.25 
                 21.4% 
                 90.0% 
                 0.755 
                   
               
               
                   
                   
                   
                 135 
                 0.91 
                 0.33 
                 7.1% 
                 80.0% 
                 0.672 
               
               
                   
               
             
          
         
       
     
         [0117]    At the 60, 90, and 135-day time points, F 1 -Pirc rats averaged 2±2, 7±4, and 19±5 colonic tumors, respectively. (Table 9.) Tumor counts for the small intestine could be obtained only upon dissection at the terminal time point of 135 days, and averaged 13±6 tumors. Of the 26 colonic tumors monitored by colonoscopy, 21 (81%) grew, 4 (15%) were static, and 1 regressed. The magnitude of expression change compared to wildtype rats was generally proportional to tumor burden. ( FIG. 5B .) 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Tumor counts at the 60, 90, and 135-day time points 
               
             
          
           
               
                   
                 Large 
                 Large 
                 Large 
                 Small 
                 Total 
               
               
                   
                 Intestine 
                 intestine 
                 intestine 
                 intestine 
                 intestine 
               
               
                 Pirc Rat* 
                 60 days 
                 90 days 
                 135 days 
                 135 days 
                 135 days 
               
               
                   
               
             
          
           
               
                  1 
                 0 
                 5 
                 16 
                 4 
                 20 
               
               
                  2 
                 2 
                 5 
                 15 
                 5 
                 20 
               
               
                  3 
                 2 
                 6 
                 15 
                 7 
                 22 
               
               
                  4 
                 2 
                 8 
                 17 
                 6 
                 22 
               
               
                  5 
                 2 
                 6 
                 17 
                 6 
                 23 
               
               
                  6 
                 3 
                 6 
                 18 
                 5 
                 23 
               
               
                  7 
                 5 
                 15 
                 20 
                 3 
                 23 
               
               
                  8 
                 1 
                 2 
                 17 
                 12 
                 29 
               
               
                  9 
                 2 
                 7 
                 19 
                 13 
                 32 
               
               
                 10 
                 1 
                 2 
                 24 
                 8 
                 32 
               
               
                 11 
                 9 
                 15 
                 33 
                 9 
                 42 
               
               
                 12 
                 2 
                 6 
                 13 
                 7 
                 20 
               
               
                 13 
                 0 
                 9 
                 24 
                 2 
                 26 
               
               
                 14 
                 3 
                 7 
                 21 
                 7 
                 28 
               
               
                   
               
             
          
         
       
     
         [0118]    Protein Candidates have Diagnostic and Prognostic Utility for Detection of Colorectal Cancers and Precancerous Conditions. 
         [0119]    The diagnostic ability of each biomarker to identify the presence of intestinal tumors was evaluated in two ways. First, the statistical significance of the ratio of average protein expression in F 1 -Pirc rats compared to F 1 -wildtype rats was determined. (Table 8.) The average area ratios of MMP7, LRG1, ITIH4, VTN, HPX, EGFR and F5 each changed significantly (p&lt;0.05) by 135 days. Except for F5, each of these proteins also shows a significant change by 90 days. A published histological review of colon polyps from F 1 -Pirc rats shows that the vast majority of tumors are noninvasive adenomas within the time range studied (Amos-Landgraf, J. M. et al.,  Proc Natl Acad Sci USA  2007, 104, (10), 4036-41), thus suggesting that the differentially expressed proteins can hold potential to identify polyps at the early adenoma stage. Further, the lack of protein expression changes at 60 days gives increased confidence that changes detected at the 90 and 135-day time points are directly or indirectly owing to the presence of the polyps and not due to an extra-tumoral effect of the Apc mutation. 
         [0120]    ROC analysis was then used to evaluate the potential of each protein to diagnose early colonic neoplasia among the group of 14 F 1 -Pirc and 10 F 1 -wildtype rats. Table 8 summarizes the sensitivity, specificity, and area under the curve (AUC) of each protein biomarker at 60, 90 and 135 days. (See also  FIG. 6 .) As with the analysis by p-values, AUCs showed greater diagnostic potential at 90 and 135 days than at 60 days, with the sensitivity increasing as tumor burden increased. The most predictive proteins were LRG1 and EGFR, which had 1 and 0 false negatives, respectively, at 135 days. These proteins also had very few false positives (1 and 2, respectively), again indicating that their changes in expression in serum are tumor-specific. Among other proteins that show sensitivity and specificity at the 135-day time point are MMP7, ITIH4 and HPX, MMP10, and CD44. 
         [0121]    A Protein Panel has High Sensitivity and Specificity for Identifying Early-Stage Colon Adenomas. 
         [0122]    To improve the overall sensitivity for detecting the earliest adenomas, several of the proteins were analyzed for their predictive ability as a panel. LRG1, ITIH4, EGFR and F5 were chosen because they showed significant differential expression in F 1 -Pirc rats and showed the least variance in F 1 -wildtype protein concentration over time (15% or less).  FIG. 7  and Table 10 highlight the sensitivity and specificity of this panel to identify rats with colonic polyps. Sensitivity was highest when the threshold for positive diagnosis was set to require only a single protein in the panel to show a positive result. Importantly, at 60 and 90 days the sensitivity increased using the four-protein panel. The panel reduced the number of false negatives from 6 (ITIH4 alone) to 4 at 60 days, and reduced it even further at 90 days from 5 (LRG1 alone) to 2. Maximally, 2/10 samples (20%) showed false positives at 60, 90, and 135 days. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Summary of ROC analysis for a panel of four biomarkers 
               
               
                 (F5, EGFR, LRG1, and ITIH4) 
               
             
          
           
               
                   
                 Minimum number 
                   
                   
                   
                   
               
               
                   
                 of positive 
                   
                   
                   
                   
               
               
                   
                 markers to make 
                 Time  
                   
                   
                   
               
               
                   
                 positive diagnosis 
                 point 
                 Sensitivity 
                 Specificity 
                 AUC 
               
               
                   
                   
               
             
          
           
               
                   
                 1 Positive 
                  60 
                 66.7% 
                 83.3% 
                 0.764 
               
               
                   
                   
                  90 
                 85.7% 
                 90.0% 
                 0.900 
               
               
                   
                   
                 135 
                  100% 
                 80.0% 
                 0.932 
               
               
                   
                 2 Positives 
                  60 
                 16.7% 
                  100% 
                 0.764 
               
               
                   
                   
                  90 
                 42.9% 
                  100% 
                 0.843 
               
               
                   
                   
                 135 
                 85.7% 
                 80.0% 
                 0.914 
               
               
                   
                 3 or more 
                  60 
                   0% 
                  100% 
                 0.764 
               
               
                   
                 Positives 
                  90 
                 21.4% 
                  100% 
                 0.911 
               
               
                   
                   
                 135 
                 78.6% 
                 90.0% 
                 0.904 
               
               
                   
                   
               
             
          
         
       
     
         [0123]    A more stringent criterion for a positive diagnosis is that two or more proteins must show a positive result. With this criterion, the number of false positives decreased, as expected, and the number of false negatives increased significantly. Since the major goal is to detect the presence of colonic tumors with high sensitivity and no false negatives, it is counterproductive to require simultaneous changes in multiple positive markers. Therefore, the ROC analysis method was used to understand the sensitivity and specificity of each protein individually or in a panel, aiming to minimize the number of false negatives. The AUC value assumes that the sensitivity and specificity measurements are equally important (Grund, B. and Sabin, C.,  Curr Opin HIV AIDS  2010, 5, (6), 473-9). Accordingly, both sensitivity and specificity values (Table 10) are contemplated in the present methods to better assess the markers under consideration. 
       Example 4: Serum Biomarkers Predict Clinical Outcome in Human Colon Cancer Patients 
       [0124]    Biomarker Candidate and Selection. 
         [0125]    The peptides identified and validated in animal models were used to conduct targeted proteomic analysis in humans. A list of more than 40 candidate proteins was identified by longitudinal study of blood proteins in tumor-bearing mice and rats (Example 3, supra) (Ivancic, M. M. et al.,  J Proteome Res  2013, 12, (9), 4152-66; Ivancic, M. M. et al.,  Cancer Prev Res  2014, 55, 7(11); 1160-9). Some of these markers overlap with other colon cancer biomarker discovery studies done in animals and humans (Hung, K. E. et al.,  Cancer Prev Res  ( Phila ) 2009, 2, (3), 224-33; Chong, P. K. et al.,  J Proteome Res  2010, 9, (7), 3671-9; Ladd, J. J. et al.,  Cancer Prev Res  ( Phila ) 2012, 5, (4), 655-64; Surinova, S. et al.,  EMBO Mol Med  2015, 7, 1153-1165). The candidate list of proteins from all of these studies was pared down to 30 proteins for biomarker screening in a human population. An emphasis was placed on selecting proteins with overlap across multiple biomarker studies and those with potential biological significance to colon cancer based on published literature. (Table 11.) 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 List of candidate biomarkers selected for SRM-MS analysis 
               
             
          
           
               
                   
                   
                   
                 Endogenous 
               
               
                   
                   
                   
                 indentified in 
               
               
                   
                   
                   
                 human serum 
               
               
                   
                   
                 Studies in which protein was 
                 during SRM 
               
               
                   
                 Protein 
                 indentified as a colon cancer 
                 method 
               
               
                 Protein 
                 Symbol 
                 biomarker in blood 
                 development? 
               
               
                   
               
               
                 Collagen alpha-1(I) chain 
                 COL1A1 
                 Mouse, Rat 
                 yes 
               
               
                 Epidermal Growth Factor 
                 EGFR 
                 Mouse, Rat, Chong et. al. 
                 yes 
               
               
                 Receptor 
                   
                   
                   
               
               
                 Inter-alpha-trypsin 
                 ITIH3 
                 Mouse, Rat, Chong et. al. 
                 yes 
               
               
                 inhibitor heavy chain H3 
                   
                   
                   
               
               
                 Maltase Glucoamylase 
                 MGAM 
                 Mouse 
                 no 
               
               
                 Coagulation factor V 
                 F5 
                 Mouse, Rat, Hung et. al.,  
                 yes 
               
               
                   
                   
                 Surinova et. al 
                   
               
               
                 Hemopexin 
                 HPX 
                 Mouse, Rat 
                 yes 
               
               
                 Isocitrate 
                 IGH2 
                 Mouse 
                 no 
               
               
                 dehydrogenase [NADP], 
                   
                   
                   
               
               
                 mitochondrial 
                   
                   
                   
               
               
                 Pyruvate Kinase, M2 
                 PKM2 
                 Mouse, Hung et. al. 
                 no 
               
               
                 Vitamin D-binding 
                 GC 
                 Mouse 
                 yes 
               
               
                 protein 
                   
                   
                   
               
               
                 Inter-alpha-trypsin 
                 ITIH4 
                 Mouse, Rat, Surinova et. al. 
                 yes 
               
               
                 inhibitor heavy chain H4 
                   
                   
                   
               
               
                 CD44 
                 CD44 
                 Rat, Surinova et. al 
                 yes 
               
               
                 CEACAM5 
                 CEA 
                 Neither-Prognostic marker 
                 no 
               
               
                 Cathapsin B 
                 CTSB 
                 Mouse, Hung et. al. 
                 yes 
               
               
                 Leucine-rich alpha-2- 
                 LRG1 
                 Mouse, Rat, Hung et. al. Ladd, et. 
                 yes 
               
               
                 glycoprotein 
                   
                 al. and Chong et. al, Surinova et. al. 
                   
               
               
                 Serum Amyloid P 
                 APCS 
                 Mouse, Chong et. al. 
                 yes 
               
               
                 Fetuin B 
                 FETUB 
                 Mouse, Surinova et. al. 
                 yes 
               
               
                 C-reactive protein 
                 CRP 
                 Mouse 
                 yes 
               
               
                 Matrilysin 
                 MMP7 
                 Rat 
                 no 
               
               
                 Complement factor I 
                 CFI 
                 Mouse, Rat, Hung et. al. 
                 yes 
               
               
                 heparin cofactor 2 
                 SERPIND1 
                 Mouse 
                 yes 
               
               
                 Sulfhydryl Oxidase 1 
                 QSOX1 
                 Mouse 
                 yes 
               
               
                 Rho-GDP Dissociation 
                 ARHGDIA 
                 Mouse 
                 yes 
               
               
                 Inhibitor 1, Isoform a 
                   
                   
                   
               
               
                 (RhoGDI) 
                   
                   
                   
               
               
                 Peptidase inhibitor 16 
                 PI16 
                 Mouse 
                 yes 
               
               
                 Cadherin-2 (N-Cadherin) 
                 CDH2 
                 Mouse 
                 yes 
               
               
                 Dipeptidyl peptidase 4 
                 DPP4 
                 Mouse 
                 yes 
               
               
                 extracellular superoxide 
                 SOD3 
                 Mouse 
                 yes 
               
               
                 dismulase [Cu—Zn] 
                   
                   
                   
               
               
                 Thrombospondin-4 
                 THBS4 
                 Mouse 
                 yes 
               
               
                 receptor-type tyrosine- 
                 PTPRM 
                 Mouse 
                 yes 
               
               
                 protein phospatase mu 
               
               
                   
               
             
          
         
       
     
         [0126]    Patient Population. 
         [0127]    The design of the clinical study is presented in  FIG. 8 . Serum samples collected from human subjects were divided into four different categories: (1) polyp-free control (n=23), (2) pre- and (3) post-polypectomy from growing adenoma (n=14), and (4) non-metastatic colon cancer (stages 1-3) (n=20). Subjects with a history of inflammatory bowel disease and metastatic colon cancer were excluded from this study. Those within the polyp-free control group were verified as such by colonoscopy. Tumor staging within the non-metastatic cancer group was achieved from pathology results from tumors removed at the time of surgical resection. The subjects with known growing adenomas were identified by longitudinal analysis of their polyps using computed tomography (CT) colonography. At the first patient visit, polyps were identified and 3-dimensional size was measured. If the polyp volume was identified as growing at a 5-year follow-up visit, the patient was enrolled in the study, blood collected, and the polyp was removed. Subjects returned for a second blood-draw post-polypectomy approximately one month later. All patients undergoing blood draws prior to polypectomy or surgery were fasted following guidelines for a standard colonoscopy or colectomy preparation (Wexner, S. D. et al.,  Gastrointest Endosc  2006, 63, (7), 894-909). In the post-polypectomy group, approximately half of the patients fasted overnight before the procedure. In a study done by Hsieh et. al. evaluating different collection procedures for samples undergoing proteomic profiling, the differences between fasting and non-fasting serum were minimal (Hsieh, S. Y. et al.,  Proteomics  2006, 6, (10), 3189-98). Thus, unless non-fasted patient samples clearly behave differently from fasted samples, all will be included in the study. Patient accruals are presented in Table 12. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Accruals of control, adenoma, and colon cancer subjects 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                 # of samples 
               
               
                   
                 Current # of 
                   
                   
                   
                   
                   
                 analyzed by mass 
               
               
                   
                 samples 
                   
                   
                 Number of 
                 Cancer 
                 Average Pre- 
                 spectrometry 
               
               
                   
                 (Updated 
                   
                 Median Age, 
                 Polyps 
                 Stage 
                 Operative CEA 
                 (as of November 
               
               
                 Group: 
                 Dec. 11, 2014) 
                 Sex (M/F) 
                 (range) 
                 (1/2/3) 
                 (1/2/3) 
                 Level, (range) 
                 2014) 
               
               
                   
               
               
                 Polyp-free Control 
                 59 
                 34/25 
                 59.5, (50-80) 
                 N/A 
                 N/A 
                 N/A 
                 23 
               
               
                 Colon cancer 
                 20 
                 10/10 
                 72.5, (49-86) 
                 N/A 
                 9/7/4 
                 2.9, (&lt;0.5-6.3) 
                 20 
               
               
                 (Stages 0-3) 
               
               
                 Adenoma (Pre- 
                 29 (paired) 
                 17/7  
                   60, (42-76) 
                 10/11/2 
                 N/A 
                 N/A 
                 14 Pre/12 Post 
               
               
                 and Post-polypectomy) 
               
               
                   
               
             
          
         
       
     
         [0128]    Sample Preparation. 
         [0129]    Serum samples were thawed at room temperature, allowed to sit for a minimum of 30 min at room temperature, and all samples processed within a 4-hour window. Immunodepletion of the top 6 most abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin and antitrypsin) was achieved using a 4.6×100 mm Agilent Multi-Affinity Removal Column according to manufacturer&#39;s instructions. Briefly, a 60 μl aliquot of serum was solubilized in 400 μl of Agilent Buffer A, was filtered and injected onto a Waters 1740 HPLC equipped with a photodiode array detector. Both the 215 nm and 280 nm wavelengths were monitored. The flow-through fraction containing low-abundance proteins and bound high abundant protein fraction were both collected. The low abundance proteins were concentrated and precipitated using a trichloroacetic acid protein precipitation as described in Example 2, supra. A Pierce™ BCA protein concentration assay was performed on resolubilized samples according to the manufacturer&#39;s instructions (Thermo Fisher Scientific). A 100 μg aliquot of serum proteins was digested with trypsin in the presence of stable isotope labeled reference standards as described in Example 2, supra, and desalted using SPEC C18 solid-phase extraction tips (Agilent) according to manufacturer instructions (Ivancic, M. M. et al.,  Cancer Prev Res  2014, 55, 7(11); 1160-9). 
         [0130]    Mass Spectrometry Assay. 
         [0131]    Chromatographic separation of a 2 μg peptide sample was achieved by reversed phase chromatography using a NanoLC Ultra 2D HPLC (Eksigent) equipped with a Nanoflex cHiPLC set to 37° C. A 90-minute gradient was used for peptide separation, as described in detail in Example 2, supra, followed by elution directly into a 5500 QTrap mass spectrometer (AbSciex). Peptide precursors were selected in quadrupole 1 (Q1), fragmented in q2, and the top 3-4 transitions were selected for monitoring in Q3. All Q1 and Q3 masses were measured at unit resolution. A 7-minute scheduling window was applied with a 2-second cycle time. Method development and peak analysis were done using Skyline software (MacLean, B. et al.,  Bioinformatics  2010, 26, (7), 966-8). 
         [0132]    Data Processing and Analysis. 
         [0133]    Mass spectrometry results were imported into Skyline and peaks integrated. All peak areas from reference standards and endogenous transitions were evaluated using the AuDIT algorithm to identify the transition with the lowest coefficient of variance (Abbatiello, S. E. et al.,  Clin Chem  2010, 56, (2), 291-305). Peptide quantities were assessed using the average peak area of the transition with the lowest variance over three technical replicates. Relative changes in protein expression were determined by taking a ratio of (cancer/control) for each protein. The growing adenoma samples were compared to both the polyp-free control group and their paired post-polypectomy sample. A two-tailed student&#39;s t-test assuming a normal distribution was used to assess the significance in protein expression changes. A change was considered significant if the p-value was less than or equal to 0.05. 
         [0134]    Serum Biomarkers have Diagnostic and Prognostic Utility in Human Colon Cancer. 
         [0135]    The serum levels of several biomarkers correlated with cancer incidence and outcomes in the patient study. ( FIG. 9A-J , Table 13). EGFR, DPP4, and PI16 were down-regulated in subjects with adenomas and in stages 1-3 colon cancer, and ARHGDIA (RhoDG1) was down-regulated in patients with adenomas and early stage cancers. LRG1, ITIH3, ITIH4, F5, and CRP were up-regulated in patients with colon cancer. (Table 10, statistically significant differences indicated in bold-face.) 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 Relative protein expression in subjects with adenomas and  
               
               
                 non-metastatic carcinomas compared to polyp-free controls 
               
               
                 (bolded data denote statistical significance) 
               
             
          
           
               
                   
                 Avg.  
                   
                   
                   
                   
               
               
                 Protein 
                 Cancers 
                 Adenomas 
                   
                   
                   
               
               
                 Symbol 
                 (stages 1-3) 
                 (Stage 0) 
                 Stage 1 
                 Stage 2 
                 Stage 3 
               
               
                   
               
             
          
           
               
                 EGFR 
                 
                   0.85 
                 
                 0.97 
                 
                   0.87 
                 
                 
                   0.82 
                 
                 0.88 
               
               
                 LRG1 
                 
                   1.67 
                 
                 1.08 
                 
                   1.41 
                 
                 
                   1.79 
                 
                 
                   2.03 
                 
               
               
                 ITIH3 
                 
                   1.32 
                 
                 0.88 
                 1.26 
                 1.29 
                 
                   1.49 
                 
               
               
                 ITIH4 
                 
                   1.28 
                 
                 1.14 
                 
                   1.24 
                 
                 
                   1.29 
                 
                 
                   1.37 
                 
               
               
                 DPP4 
                 
                   0.78 
                 
                 0.95 
                 0.82 
                 
                   0.76 
                 
                 
                   0.7  
                 
               
               
                 PI16 
                 
                   0.79 
                 
                 0.85 
                 0.84 
                 0.82 
                 
                   0.64 
                 
               
               
                 F5 
                 
                   1.19 
                 
                 
                   1.27 
                 
                 1.22 
                 1.21 
                 1.1  
               
               
                 CRP 
                 
                   2.55 
                 
                 1.47 
                 1.71 
                 2.41 
                 
                   4.67 
                 
               
               
                 ARHGDIA 
                 0.77 
                 
                   0.49 
                 
                 0.65 
                 0.65 
                 1.24 
               
               
                 HPX 
                 1.05 
                 0.92 
                 1.13 
                 1.02 
                 0.95 
               
               
                 SOD3 
                 0.89 
                 0.91 
                 0.82 
                 1.06 
                 0.74 
               
               
                 THBS4 
                 0.92 
                 1.01 
                 0.98 
                 0.99 
                 0.68 
               
               
                 COL1A1 
                 0.97 
                 0.92 
                 0.88 
                 1.11 
                 0.92 
               
               
                 CDH2 
                 1.13 
                 0.93 
                 1.21 
                 1.15 
                 0.94 
               
               
                 VTN 
                 1.05 
                 1.05 
                 1.08 
                 0.98 
                 1.08 
               
               
                   
               
             
          
         
       
     
         [0136]    Serum Biomarkers with Pre- and Post-Polypectomy Prognostic Relevance. 
         [0137]    Paired pre- and post-polypectomy serum samples from patients with growing adenomas were compared to assess changes in protein expression. Two patients with three growing adenomas were used to assess the ability of these biomarkers to differentiate between pre- and post-polypectomy samples. The post-polypectomy blood draws occurred at 21 days (patient 1) and 30 days (patient 2) after polyp removal. 
         [0138]    LRG1, ITIH3, APCS, SERPIND1, THBS4, and F5 all showed differences in expression between pre- and post-polypectomy samples. Of these six proteins, LRG1 and ITIH3 proteins are upregulated in humans with cancer (Table 13). LRG1 and ITIH3 showed reduced expression levels post-polypectomy in both patient samples ( FIG. 11 ). In addition, Col1a1 shows consistent, statistically significant increase in this protein&#39;s expression after polyp removal ( FIG. 10 ). 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Exemplary Peptide Sequences Useful in Disclosed Embodiments 
               
             
          
           
               
                 SEQ ID 
                   
                   
               
               
                 NO 
                 Peptide Sequence 
                 Protein Biomarker Name 
               
               
                   
               
               
                  1 
                 TSWGLENEALIVR 
                 Interleukin 1 receptor-like 1 
               
               
                  2 
                 FTHTENGTNYIVTATR 
                   
               
               
                  3 
                 SFTVEEK 
                   
               
               
                  4 
                 AHMSYLFIDK 
                   
               
               
                   
               
               
                  5 
                 FLVDQIVK 
                 Matrix Metalloproteinase-7 
               
               
                  6 
                 IVSYTTDLPR 
                 (Matrilysin) 
               
               
                 80 
                 DLPHITVDR 
                   
               
               
                   
               
               
                  7 
                 TYFFVGDK 
                 Matrix Metalloproteinase-10 
               
               
                  8 
                 TVTHTLK 
                   
               
               
                  9 
                 QDHSTMDLAQQYLEK 
                   
               
               
                 10 
                 LDSNTVEMMHKPR 
                   
               
               
                 11 
                 FLGLEMTGK 
                   
               
               
                 12 
                 IDAAVFEK 
                   
               
               
                 13 
                 GSQFWAVR 
                   
               
               
                 14 
                 SNSWLLC 
                   
               
               
                   
               
               
                 15 
                 DDAFFIGSTLATIASTVYSK 
                 CD44 antigen 
               
               
                 16 
                 EPTETPDQFMTADETR 
                   
               
               
                 17 
                 TQWNPIHSNPEVLLQTTTR 
                   
               
               
                 18 
                 STPEGYILHTDLPTSQPTGDR 
                   
               
               
                 19 
                 KPSELNGEASK 
                   
               
               
                 20 
                 NLQSVDMK 
                   
               
               
                 21 
                 LVINSGNGTVEDR 
                   
               
               
                 73 
                 YGFIEGHVVIPR 
                   
               
               
                   
               
               
                 22 
                 AFPAFVLR 
                 Wnt Inhibitory Factor 1 
               
               
                 23 
                 LGTVPHK 
                   
               
               
                 24 
                 ASVVQVGFPCLGK 
                   
               
               
                 25 
                 YGASLMHAPRPAGAGLER 
                   
               
               
                 26 
                 TPQNAIFFK 
                   
               
               
                 27 
                 TCQQAECPGGCR 
                   
               
               
                 28 
                 ADAGQPPEESLYLWIDAHQAR 
                   
               
               
                 29 
                 LWSILPCLLLLR 
                   
               
               
                   
               
               
                 30 
                 VVGGKPAEMGDYPWQVAIK 
                 Complement Factor I 
               
               
                 31 
                 LPYQCPK 
                   
               
               
                 32 
                 VFCQPWQK 
                   
               
               
                 33 
                 GYPTYCHLK 
                   
               
               
                 34 
                 SFECLHPEIK 
                   
               
               
                 35 
                 FNIPVNHK 
                   
               
               
                 36 
                 INSTECLHVR 
                   
               
               
                 37 
                 FNVSLIYGSTDTEGIVQVK 
                   
               
               
                 81 
                 VFSLQWGEVK 
                   
               
               
                   
               
               
                 38 
                 ISHELESSSSEVN 
                 Secreted Phosphoprotein-1 
               
               
                   
               
               
                 39 
                 SISTIINVFHQYSR 
                 S100 calcium binding protein 
               
               
                 40 
                 YGHPDTLNK 
                 A9 
               
               
                   
               
               
                 41 
                 LSTSWTEEDVNDNTLFK 
                 Follistatin 
               
               
                 42 
                 ATCLLGR 
                   
               
               
                 43 
                 EECCSTGR 
                   
               
               
                 44 
                 WMIFNGGAPNCIPCK 
                   
               
               
                 45 
                 SIGLAYEGK 
                   
               
               
                 46 
                 EAACSSGVLLEVK 
                   
               
               
                 47 
                 CSLCDELCPDSK 
                   
               
               
                 48 
                 SCEDIQCGGGK 
                   
               
               
                   
               
               
                 49 
                 EACLDPEAPMVQK 
                 Chemokine (C-X-C motif) 
               
               
                   
                   
                 ligand 1 
               
               
                   
               
               
                 50 
                 LDQNQVR 
                 Chemokine (C-C motif) ligand 
               
               
                 51 
                 MIPMSR 
                 2 
               
               
                   
               
               
                 52 
                 TLFLLALLGGVSGLR 
                 Leucine-Rich alpha-2-  
               
               
                 53 
                 SSAALNTLVLR 
                 glycoprotein 
               
               
                 54 
                 LLDVAELGTL 
                   
               
               
                 55 
                 SLPPGLFR 
                   
               
               
                 56 
                 DLVDLCR 
                   
               
               
                 57 
                 LHLEGNR 
                   
               
               
                 58 
                 ENQLQEASAR 
                   
               
               
                 76 
                 VAAGAFQGLR 
                   
               
               
                   
               
               
                 59 
                 NLYLSCVMK 
                 Interleukin-1 beta 
               
               
                 60 
                 CLVLSDPCELK 
                   
               
               
                 61 
                 DGTPTLQLESVDPK 
                   
               
               
                   
               
               
                 62 
                 SLSQQIENIR 
                 Collagen alpha-1(I) chain 
               
               
                   
               
               
                 63 
                 IPLENLQIIR 
                 Epidermal Growth Factor 
               
               
                 92 
                 NYVVTDHGSCVR 
                 Receptor 
               
               
                   
               
               
                 64 
                 EVSFDVELPK 
                 Inter-alpha-trypsin inhibitor 
               
               
                   
                   
                 heavy chain H3 
               
               
                   
               
               
                 65 
                 AYVAFPDFFR 
                 Maltase Glucoamylase 
               
               
                 83 
                 SSVYANAFPSTPVNPLR 
                   
               
               
                   
               
               
                 66 
                 NFFNPPIISR 
                 Coagulation factor V 
               
               
                   
               
               
                 67 
                 LWWLDLK 
                 Hemopexin 
               
               
                   
               
               
                 68 
                 TIEAEAAHGTVTR 
                 Isocitrate dehydrogenase 
               
               
                   
                   
                 [NADP], mitochondrial 
               
               
                   
               
               
                 69 
                 EAEAAIYHLQLFEELR 
                 Pyruvate Kinase, M2 
               
               
                   
               
               
                 70 
                 VLEPTLK 
                 Vitamin D-binding protein 
               
               
                   
               
               
                 71 
                 FEDGVLDPDYPR 
                 Vitronectin 
               
               
                   
               
               
                 72 
                 FAHTVVTSR 
                 Inter-alpha-trypsin inhibitor, 
               
               
                   
                   
                 Heavy chain 4 
               
               
                   
               
               
                 74 
                 TLTLLSVTR 
                 CEACAM5 
               
               
                   
               
               
                 75 
                 LCGTFLGGPKPPQR 
                 Cathepsin B 
               
               
                   
               
               
                 77 
                 GYVIIKPLVWV 
                 Serum Amyloid P 
               
               
                   
               
               
                 78 
                 IFFESVYGQCK 
                 Fetuin B 
               
               
                   
               
               
                 79 
                 ESDTSYVSLK 
                 C-reactive protein 
               
               
                   
               
               
                 82 
                 FTVDRPFLFLIYEHR 
                 heparin cofactor 2 
               
               
                   
               
               
                 84 
                 LAGAPSEDPQFPK 
                 Sulfhydryl Oxidase 1 
               
               
                   
               
               
                 85 
                 AEEYEFLTPVEEAPK 
                 Rho-GDP Dissociation 
               
               
                   
                   
                 Inhibitor 1, Isoform a 
               
               
                   
                   
                 (ARHGDIA) 
               
               
                   
               
               
                 86 
                 WDEELAAFAK 
                 Peptidase inhibitor 16 
               
               
                   
               
               
                 87 
                 GPFPQELVR 
                 Cadherin-2 (N-Cadherin) 
               
               
                   
               
               
                 88 
                 WEYYDSVYTER 
                 Dipeptidyl peptidase 4 
               
               
                   
               
               
                 89 
                 VTGVVLFR 
                 extracellular superoxide 
               
               
                   
                   
                 dismutase [Cu-Zn] 
               
               
                   
               
               
                 90 
                 DVDIDSYPDEELPCSAR 
                 Thrombospondin-4 
               
               
                   
               
               
                 91 
                 GFGPPATNQFTTK 
                 receptor-type tyrosine-protein 
               
               
                   
                   
                 phosphatase mu 
               
               
                   
               
             
          
         
       
     
         [0139]    Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.