NL2008707C2 - Biomarkers. - Google Patents

Description

BIOMARKERS Field of the Invention

The present invention relates to the identification of biomarkers associated with cancer, in particular colorectal cancer and precursors thereof, i.e. high risk adenomas, and their 5 use in screening methods, arrays for use in colorectal cancer screening methods, methods of treating colorectal cancer, compounds for use in treating colorectal cancer, and kits for use in colorectal cancer screening methods.

Background of the Invention

Colorectal cancer (CRC) is a significant health problem. It is the 3rd most common 10 cancer worldwide, with over 1.200.000 new cases each year, with a fatal outcome for almost half the patients (Karsa LV, et al. Best Pract Res Clin Gastroenterol 2010; 24:381-96.). Because CRC develops over many years, there is an excellent window of opportunity to detect the disease in an early, curable, or even premalignant stage. This can be achieved by screening of asymptomatic individuals. However, the currently 15 available screening tests are either cumbersome or carry risk of complications and over treatment like colonoscopy.

The immunochemical fecal occult blood test (iFOBT), further referred to as fecal immunochemical test (FIT), is a widely used test that detects small traces of blood in stool, derived from lesions such as tumors that bleed in the colon. The FIT is a non-20 invasive method that makes use of an antibody directed against hemoglobin. However, not all colon tumors bleed and therefore the FIT has a sensitivity that leaves room for improvement (Duffy MJ, et al. Int J Cancer 2011; 128:3-11; van Veen W, Mali WP. [Colorectal cancer screening: advice from the Health Council of the Netherlands]. Ned Tijdschr Geneeskd 2009; 153:A1441.). Additional markers that detect other tumor 25 characteristics besides bleeding in the stool could increase this sensitivity and the chance of identifying a colon tumor. Molecular changes resulting from the neoplastic process include changes in protein expression. The proteins for which expression is increased have the potential to serve as informative biomarkers with high diagnostic performance.

30 Several studies have been performed to identify proteins in stool and blood that can be used for the early detection of CRC and high risk colorectal adenomas, but most of these proteins have not been validated in a screening setting or failed to improve current tests for early detection, such as Carcinoembryonic antigen (CEA) or Calprotectin (Bosch LJ, et al. Molecular tests for colorectal cancer screening. Clin 35 Colorectal Cancer 2011; 10:8-23.). There therefore remains a need for further tumor- 2 specific protein markers to improve the current available non-invasive screening possibilities for CRC and high risk adenomas.

Recent technological advances in mass spectrometry can boost the discovery of novel protein markers (de Wit M, etal. Gut 2011; Jimenez CR, etal. J Proteomics 2010; 73: 5 1873-95). Tandem mass spectrometry-based approaches now have the power to analyze complex protein samples and to detect proteins in low concentrations (Cox J, Mann M. Annu Rev Biochem 2011; 80: 273-99). Although most biomarker discovery studies have been performed using tissue and/or cell line material followed by validation in stool or blood, the chemical composition of the biological sample used for 10 screening may significantly affect the nature of biomarkers that can be identified. This holds especially for stool samples, in which the low pH, the protease- and glycosidase activities of bacteria, enzymes and other substances can disturb the specific detection of these markers (Young GP, Bosch LJW. Curr Colorectal Cancer Rep 2011; 7: 62-70). Measuring molecules directly in the biological sample that will be used for screening, 15 i.e. stool, could therefore be a valuable approach to provide us with reliable biomarkers that are stable in the fecal environment. A recent study by Ang et al. has shown the feasibility of protein biomarker discovery in human stool samples using mass spectrometry (Ang CS, Nice EC. J Proteome Res 2010; 9:4346-55).

The present invention aims to overcome some or all of the problems associated with 20 the prior art.

Summary of the Invention

According to a first aspect of the invention there is provided a method for screening for colorectal cancer, the method comprising: screening a biological sample obtained from an individual for one or more 25 biomarkers selected from the group defined in Table 1, wherein the presence of or increased expression of the one or more biomarkers relative to a control sample is indicative that the individual is at risk of suffering from or is suffering from colorectal cancer.

In one embodiment, the one or more biomarkers is a protein.

30 Thus, the present invention provides protein signatures for diagnosing or predicting colorectal cancer in a subject. The present invention thus allows for detection of advanced and high-risk colonic adenomas and adenocarcinomas.

In one embodiment, the presence of the one or more biomarkers is indicative that the individual is at risk of suffering from or is suffering from colorectal cancer.

3

In an alternative embodiment, the increased expression of the one or more biomarkers relative to a control sample is indicative that the individual is at risk of suffering from or is suffering from colorectal cancer.

5 The one or more biomarkers may be selected from the group defined in Table 2. The biomarkers in Table 2 represent a preferred subset of the gene products of Table 1, for which the levels of differential expression in CRC or high risk colorectal adenoma samples relative to control samples are considered statistically significant.

The one or more biomarkers may be selected from the group defined in Table 3. The 10 biomarkers of Table 3 are differentially expressed in FIT-negative CRC samples relative to a control sample.

This particular subset of the markers of Table 1 are of particular importance as these markers have the potential to be used for the detection of CRC or susceptibility to CRC (i.e. detection of high risk colorectal adenomas) in those patients for whom the fecal 15 immunochemical test, or any other test that aims to detect hemoglobin (such as the guaiac-based Fecal Occult Blood Test), comes back negative.

The one or more biomarkers may have a higher discriminative power than hemoglobin. By “higher discriminative power” it is meant that a biomarker has a higher sensitivity and specificity than hemoglobin.

20 Thus, in one embodiment, the one or more biomarkers may be selected from the group consisting of: S100 calcium binding protein A8 (S100A8), complement component C4B (Chido blood group) 2 (C4A/C4B), transferrin (TF), alpha-2-macroglobulin (A2M), S100 calcium binding protein A9 (S100A9), proteinase 3 (PRTN3), Azurocidin (AZU1), lactotransferrin (LTF), hemopexin (HPX)and defensin, alpha 1 (DEFA1).

25 The one or more biomarkers may be selected from the group defined in Table 4. The group of biomarkers in Table 4 represent a subset of Table 1, and are markers which have been found to be expressed only in CRC samples and not in control samples.

In one embodiment, the biological sample is a stool sample.

The stool sample may be screened for the one or more biomarkers using (targeted) 30 mass spectrometry.

The stool sample may be screened for the one or more biomarkers using a binding agent capable of binding to the one or more biomarkers.

The binding agent may be an antibody or fragment thereof. The antibody or fragment thereof may be a recombinant antibody or fragment thereof. The antibody or fragment 35 thereof may be selected from the group consisting of: scFv; Fab; a binding domain of an immunoglobulin molecule.

4

In one embodiment, the binding agent may be an aptamer.

The screening may be performed using an array. The array may be a bead-based array. The array may be a surface-based array.

The control sample may comprise a stool sample from an individual known to be free 5 from colorectal cancer or high risk colorectal adenomas.

The stool sample may also be analysed by the fecal immunochemical test.

According to a second aspect of the present invention there is provided the use of one or more biomarkers selected from the group defined in Table 1 for diagnosing or predicting colorectal cancer in an individual.

10 The one or more biomarkers of the second aspect may be selected from the group defined in Table 2.

The one or more biomarkers of the second aspect may be selected from the group defined in Table 3.

The one or more biomarkers of the second aspect may have a higher discriminative 15 power than hemoglobin.

Thus, the one or more biomarkers of the second aspect may be selected from the group consisting of: S100 calcium binding protein A8 (S100A8), complement component C4B (Chido blood group) 2 (C4A/C4B), transferrin (TF), alpha-2-macroglobulin (A2M), S100 calcium binding protein A9 (S100A9), proteinase 3 20 (PRTN3), Azurocidin (AZU1), lactotransferrin (LTF), hemopexin (HPX) and defensin, alpha 1 (DEFA1).

The one or more biomarkers of the second aspect may be selected from the group defined in Table 4.

According to a third aspect of the invention there is provided an array for determining 25 whether an individual is at risk of suffering from or is suffering from colorectal cancer, the array comprising one or more binding agent as defined according to certain embodiments of the first aspect of the invention.

The array may be for use in a method according to the first aspect of the invention. According to a fourth aspect of the invention there is provided a method for treating 30 colorectal cancer, the method comprising: screening a stool sample obtained from an individual for one or more biomarkers selected from the group defined in Table 1, wherein the presence of or increased expression of the one or more biomarkers relative is indicative that the individual is suffering from colorectal cancer; and 35 administering a therapeutically active amount of a cancer therapeutic agent.

5

The one or more biomarkers of the fourth aspect may be selected from the group defined in Table 2.

The one or more biomarkers of the fourth aspect may be selected from the group defined in Table 3.

5 The one or more biomarkers of the fourth aspect may have a higher discriminative power than hemoglobin.

Thus, the one or more biomarkers of the fourth aspect may be selected from the group consisting of: S100 calcium binding protein A8 (S100A8), complement component C4B (Chido blood group) 2 (C4A/C4B), transferrin (TF), alpha-2-macroglobulin (A2M), S100 10 calcium binding protein A9 (S100A9), proteinase 3 (PRTN3), Azurocidin (AZU1), lactotransferrin (LTF), hemopexin (HPX) and defensin, alpha 1 (DEFA1).

The one or more biomarkers of the fourth aspect may be selected from the group defined in Table 4.

According to a fifth aspect of the invention there is provided a cancer therapeutic agent 15 for use in a method for treating colorectal cancer, the method comprising: screening a stool sample obtained from an individual for one or more biomarkers selected from the group defined in Table 1, wherein the presence of or increased expression of the one or more biomarkers relative is indicative that the individual is suffering from colorectal cancer; and 20 administering a therapeutically active amount of the cancer therapeutic agent.

The cancer therapeutic agent of the fourth or fifth aspect may comprise one or more therapeutic monoclonal antibody, one or more small molecule inhibitor or one or more chemotherapeutic agent or any combination thereof.

The one or more therapeutic monoclonal antibody may comprise one or more of 25 bevacizumab, cetuximab or panitumumab or any combination thereof.

The one or more small molecule inhibitor may comprise one or more of erlotinib, sorafenib or alisertib or any combination thereof.

The one or more chemotherapeutic agent may comprise one or more of 5-FU, capecitabine, irinotecan oxaliplatin, or leucovorin or any combination thereof.

30 The one or more biomarkers of the fifth aspect may be selected from the group defined in Table 2.

The one or more biomarkers of the fifth aspect may be selected from the group defined in Table 3.

The one or more biomarkers of the fifth aspect may have a higher discriminative power 35 than hemoglobin.

6

Thus, the one or more biomarkers of the fifth aspect may be selected from the group consisting of: S100 calcium binding protein A8 (S100A8), complement component C4B (Chido blood group) 2 (C4A/C4B), transferrin (TF), alpha-2-macroglobulin (A2M), S100 calcium binding protein A9 (S100A9), proteinase 3 (PRTN3), Azurocidin (AZU1), 5 lactotransferrin (LTF), hemopexin (HPX) and defensin, alpha 1 (DEFA1).

The one or more biomarkers of the fifth aspect may be selected from the group defined in Table 4.

According to a sixth aspect of the invention there is provided a kit for screening for colorectal cancer in an individual, the kit comprising: 10 (a) One or more binding agent which selectively bind to one or more biomarkers as defined in Table 1, or an array according to the third aspect; (b) Instructions for performing the method as defined in the first aspect.

The kit may, for example, be an ELISA kit. The one or more binding agent may, for 15 example, comprise an antibody. The one or more binding agent may comprise an aptamer.

Any one or more features described for any aspect of the present invention or preferred embodiments or examples thereof, described herein, may be used in conjunction with any one or more other features described for any other aspect of the present invention 20 or preferred embodiments or examples thereof described herein. The fact that a feature may only be described in relation to one aspect or embodiment or example does not limit its relevance to only that aspect or embodiment or example if it is technically relevant to one or more other aspect or embodiment or example.

7

Detailed Description of the Invention

Colorectal cancer

The most common colon cancer cell type is adenocarcinoma which accounts for 95% of cases. Other, rarer types include lymphoma and squamous cell carcinoma. 5 Colorectal adenocarcinoma arises from precursor lesions called adenomas, of which only a minority progress to cancer. Adenomas that progress to cancer are referred to as high risk adenomas.

Protein markers have great potential to be applied for stool-based CRC screening, because they can be measured in small sample volumes with simple and relatively 10 cheap assays, of which the widely used FIT is an excellent example (Bosch LJ, et at. Molecular tests for colorectal cancer screening. Clin Colorectal Cancer 2011; 10; 8-23; Young GP, Bosch LJW. Curr Colorectal Cancer Rep 2011; 7: 62-70; Oort FA, et al. Aliment Pharmacol Ther 2010; 31: 432-9).

The present study has identified novel protein biomarkers by applying in-depth 15 proteomics to stool samples. From a total of 830 detected human proteins, 134 were significantly enriched in stool samples from CRC patients compared to control stool samples, of which several showed higher discriminative power than hemoglobin and/or complementarity to hemoglobin.

The approach of measuring molecules directly in stool is of significance to reveal 20 biomarkers that are stable in the fecal environment and detectable in the background of bacterial- and food-related molecules.

The present invention is advantageously used for screening for colorectal cancer, that is adenocarcinoma found in the colon. However, the methods of the invention should not be considered as being limited solely to the detection of colonic adenocarcinomas. 25 Rather, the methods of the invention are also useful in the detection of advanced or high-risk colonic adenomas, thus enabling the identification of an individual at risk of developing colorectal cancer due to the presence of an advanced or a high-risk adenoma.

References herein to screening for colorectal cancer thus may include screening for 30 advanced colonic adenomas and high-risk adenomas as well as colonic adenocarcinoma.

It is also expected that the biomarkers identified by the present invention may also find application for the diagnosis of adenocarcinomas present higher up the gastrointestinal tract.

35 Thus, the present invention may also provide a method for screening for gastrointestinal disease or gastrointestinal cancer, the method comprising: screening a 8 biological sample, for example a stool sample, from an individual for one or more biomarkers selected from the group defined in Table 1, wherein the presence of or increased expression of the one or more biomarkers relative to a control sample is indicative that the patient is at risk of suffering from or is suffering from gastrointestinal 5 disease or gastrointestinal cancer.

Sample for screening

The sample for screening may include cell lines, biopsies, whole blood, blood serum, sputum, stool, urine, synovial fluid, wound fluid, cerebral-spinal fluid, tissue from eyes, intestine, kidney, brain, skin, heart, prostate, lung, breast, liver, muscle or connective 10 tissue, the said tissue being optionally embedded in paraffin, histologic object slides, and all possible combinations thereof.

The preferred biological sample is stool.

The sample may be prepared by any conventional method for extracting proteins from a biological sample. One exemplary method can be found in Ang CS, Nice EC. J 15 Proteome Res. 2010; 9:4346-55, the contents of which are incorporated herein by reference.

Biomarkers

The present invention provides a set of biomarkers which may be detected directly from a stool sample and which have been shown to be reliable indicators for the 20 presence of advanced colonic adenomas or adenocarcinomas in an individual.

The biomarkers identified by this study are listed in Table 1. In one embodiment, the methods of the invention screen for more than one biomarker from the group defined in Table 1, for example two, three, four, five, six, seven, eight, nine, ten of the biomarkers of the group defined in Table 1. In an alternative embodiment, the methods of the 25 invention screen for more than ten of the biomarkers of the group defined in Table 1, for example, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred of the biomarkers of the group defined in Table 1.

Thus, the methods of the invention screen for the presence of or increased expression 30 of one or more of: complement component C4B (Chido blood group) 2 (C4A/C4B); glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2) (GOT2); glucose-6-phosphate isomerase (GPI); transketolase (TKT); N-acylaminoacyl-peptide hydrolase (APEH); histone cluster 1, H4c (HIST4H4 (includes others)); Fatty acid-binding protein 5 (psoriasis-associated) (FABP5); hexosaminidase B (beta 35 polypeptide) (HEXB); epithelial cell adhesion molecule (EPCAM); NME1-NME2 walkthrough (NME1-NME2); Superoxide dismutase 2, mitochondrial (SOD2); Tu 9 translation elongation factor, mitochondrial (TUFM); Glutathione synthetase (GSS); annexin A2 (ANXA2); ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B); 10 kDa heat shock protein (chaperonin 10) (HSPE1); glyoxalase I (GL01); histone cluster 2, H2be (HIST2H2BE (includes others)); S100 calcium 5 binding protein A4 (S100A4); S100 calcium binding protein A11 (S100A11); latexin (LXN); dehydrogenase/reductase (SDR family) member 11 (DHRS11); N- acetylglucosaminidase, alpha (NAGLU); Translin (TSN); Proteasome (prosome, macropain) subunit alpha type-4 (PSMA4); Proteasome (prosome, macropain) subunit alpha type-6 (PSMA6); ras-related C3 botulinum toxin substrate 1 (rho family, small 10 GTP binding protein Rad) (RAC1); Adenosylhomocysteinase (AHCY); fucosidase, alpha-L- 1, tissue (FUCA1); S100 calcium binding protein P (S100P); Proteasome (prosome, macropain) subunit beta type-2 (PSMB2); X-prolyl aminopeptidase (aminopeptidase P) 1 (XPNPEP1); Keratin 18 (KRT18); Nuclear cap-binding protein subunit 1 80 kDa (NCBP1); mannosidase, alpha, class 2B, member 1 (MAN2B1); S100 15 calcium binding protein A6 (S100A6); valosin containing protein (VCP); quinolinate phosphoribosyltransferase (QPRT); major histocompatibility complex, class I, B (HLA-B); phosphoglycerate mutase 1 (brain) (PGAM1); ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3); serpin peptidase inhibitor, clade B (ovalbumin), member 10 (SERPINB10); myeloperoxidase (MPO); creatine kinase, 20 mitochondrial 1B (CKMT1A/CKMT1B); proteinase 3 (PRTN3); elastase, neutrophil expressed (ELANE); MORC family CW-type zinc finger 1 (MORC1); ubiquitin B (UBB); phospholipase A2, group IIA (platelets, synovial fluid) (PLA2G2A); carbonic anhydrase IV (CA4); G elongation factor, mitochondrial 2 (GFM2); S100 calcium binding protein A7 (S100A7); Bactericidal permeability-increasing protein (BPI); collagen, type VI, 25 alpha 5 (COL6A5); LIM homeobox 8 (LHX8); cysteine-rich secretory protein 3 (CRISP3); Azurocidin (AZU1); hemicentin 1 (HMCN1); Transglutaminase 3 (E polypeptide, protein-glutamine gamma-glutamyltransferase) (TGM3); CDC42 binding protein kinase alpha (DMPK-like) (CDC42BPA); Cathepsin G (CTSG); Resistin (RETN); methylmalonyl CoA mutase (MUT); armadillo repeat containing, X-linked 4 30 (ARMCX4); Integrin alpha-M (complement component 3 receptor 3 subunit) (ITGAM); Calcium channel, voltage dependent, R-type alpha-1 E subunit (CACNA1E); T-cell lymphoma invasion and metastasis 2 (TIAM2); HIR histone cell cycle regulation defective homolog A (S. cerevisiae) (HIRA); dopey family member 2 (DOPEY2); integrin beta 1 binding protein 3 (ITGB1BP3); Sodium channel, voltage-gated, type VII, 35 alpha (SCN7A); Rab3C, member RAS oncogene family (RAB3C); chromosome 9 open reading frame 79 (C9orf79); nuclear factor of activated T-cells, cytoplasmic, 10 calcineurin-dependent 4 (NFATC4); UDP-glucose glycoprotein glucosyltransferase 2 (UGGT2); Cornulin (CRNN); kielin/chordin-like protein (KCP); CD1E molecule (CD1E); coiled-coil domain-containing 18 (CCDC18); leukotriene A-4 hydrolase (LTA4H); albumin (ALB); alpha-2-macroglobulin (A2M); complement component 3 (C3); 5 hemoglobin, beta (HBB); transferrin (TF); hemoglobin, alpha 1 (HBA1/HBA2); lactotransferrin (LTF); ceruloplasmin (ferroxidase) (CP); catalase (CAT); group-specific component (vitamin D-binding protein) (GC); serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1); fibrinogen gamma chain (FGG); S100 calcium binding protein A8 (S100A8); ferritin, light polypeptide (FTL); actin, beta (ACTB); 10 fibronectin 1 (FN1); defensin, alpha 1 (DEFA1 (includes others)); serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 (SERPING1); retinol binding protein 4, plasma (RBP4); peroxiredoxin 2 (PRDX2); fibrinogen alpha chain (FGA); serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2 (SERPINF2); carbonic anhydrase II (CA2); orosomucoid 1 (ORM1/ORM2); 15 lactate dehydrogenase A (LDHA); vitronectin (VTN); kininogen-1 (KNG1); actin, alpha, cardiac muscle 1 (ACTC1); leucine-rich alpha-2-glycoprotein 1 (LRG1); gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) (GGH); enolase 1, (alpha) (EN01); profilin 1 (PFN1); serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 7 (SERPINA7); alpha-1-microglobulin/bikunin 20 precursor (AMBP); lamin A/C (LMNA); apolipoprotein D (APOD); thyroid hormone receptor interactor 11 (TRIP11); complement component 4 binding protein, alpha (C4BPA); tropomyosin 4 (TPM4); filamin A, alpha (FLNA); haptoglobin (HP); hemopexin (HPX); hemoglobin, delta (HBD); fibrinogen beta chain (FGB); S100 calcium binding protein A9 (S100A9); complement component 5 (C5); solute carrier 25 family 26, member 3 (SLC26A3); complement component 9 (C9); amyloid P component, serum (APCS); alpha-1-B glycoprotein (A1BG); complement C3-like (LOC100133511); inter-alpha (globulin) inhibitor H4 (plasma Kallikrein-sensitive glycoprotein) (ITIH4); complement component C8, alpha polypeptide (C8A); inter-alpha (globulin) inhibitor H1 (ITIH1); acyl-CoA dehydrogenase, very long chain (ACADVL); 30 cDNA FLJ60317, highly similar to Aminoacylase-1 (ACY1); Ankyrin repeat domain-containing protein 35 (ANKRD35); baculoviral IAP repeat-containing 6 (BIRC6); Bleomycin hydrolase (BLMH); bone marrow stromal cell antigen 12 (BST1); hypothetical protein LOC643677 (C13orf40); Cytidine deaminase (CDA); chitinase 1 (chitotriosidase) (CHIT1); cathepsin C (CTSC); Cathepsin S (CTSS); Isoform 2 of 35 Dedicator of cytokinesis protein 4 (DOCK4); Glutathione reductase (GSR); hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHCI)-like 11 domain (RLD) 1 (HERC1); heet domain and RLD 2 (HERC2); major histocompatibility complex, class II, DR beta 5 (HLA-DRB5); isocitrate dehydrogenase 1 (NADP+), soluble (IDH1); inter-alpha (globulin) inhibitor H2 (ITIH2); Uncharacterized protein KIAA1797 (KIAA1797); Lysozyme C (LYZ); Nebulin (NEB); NIMA (never in mitosis 5 gene a)- related kinase 10 (NEK10); peptidase D (PEPD); quiescin Q6 sulfhydryl oxidase 1 (QSOX1); ribonuclease T2 (RNASET2); serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1); serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (SERPINA3); serpin peptidase inhibitor, clade B (ovalbumin), member 3 (SERPINB3); SET domain containing 2 10 (SETD2); Shugoshin-like 2 (SGOL2); sialic acid acetylesterase (SIAE); spectrin repeat containing, nuclear envelope 1 (SYNE1); Transaldolase 1 (TALD01); Taste receptor type 2 member 42 (TAS2R42); triosephosphate isomerase 1 (TPM); Vinculin (VCL); Zymogen granule membrane protein 16 (ZG16); hypothetical protein LOC79887 (PLBD1); Isoform 1 of Serine/threonine-protein phosphatase 6 regulatory ankyrin 15 repeat subunit A (ANKRD28); Cystatin-C (CST3); D-dopachrome decarboxylase (DDT); Synapse-associated protein 1 (SYAP1); Proteasome subunit alpha type-2 (PSMA2); SUB1 homolog (S. cerevisiae) (SUB1); Microfibril-associated glycoprotein 3 (MFAP3); Cathepsin D (CTSD); proteasome (prosome, macropain) subunit, beta type, 1 (PSMB1); proteasome (prosome, macropain) subunit, beta type, 5 (PSMB5); cDNA 20 FLJ61112, highly similar to BTB/POZ domain-containing protein KCTD15 (KCTD15); prolyl 4-hydroxylase, beta polypeptide (P4HB); glutathione peroxidase 1 (GPX1); serpin peptidase inhibitor, clade B (ovalbumin), member 5 (SERPINB5); Isoform 1 of collagen, type IV, alpha 3 (Goodpasture antigen) binding protein (COL4A3BP); proteasome (prosome, macropain) subunit, beta type, 6 (PSMB6); Keratin 20 (KRT20); 25 Calpain small subunit 1 (CAPNS1); peroxiredoxin 3 (PRDX3); NACC family member 2, BEN and BTB (POZ) domain containing (NACC2); Rho GDP-dissociation inhibitor 2 (ARHGDIB); Macrophage migration inhibitory factor (MIF); Ran-binding protein 6 (RANBP6); spinster homolog 3 (Drosophila) (SPNS3); minichromosome maintenance complex component 2 (MCM2); Fumarylacetoacetase (FAH); heat shock 70kDa protein 30 8 (HSPA8); brain abundant, membrane attached signal protein 1 (BASP1); Branched- chain-amino-acid aminotransferase (BCAT2); Moesin (MSN); serpin peptidase inhibitor, clade B (ovalbumin), member 8 (SERPINB8); glucose-6-phosphate dehydrogenase (G6PD); Isoform 1 of UPF0557 protein C10orf119 (C10orf119); Prosaposin (PSAP); eukaryotic translation elongation factor 1 gamma (EEF1G); four 35 and a half LIM domains 1 (FHL1); carboxypeptidase, vitellogenic-like (CPVL); tubulin tyrosine ligase-like family, member 3 (TTLL3); 12 IPI:IPI00942608.1|ENSEMBL:ENSP0000 (unmapped; 26kDa protein); proline-rich protein BstNI subfamily 2 (PRB1/PRB2); Protocadherin-8 (PCDH8); Alpha-2-macroglobulin-like protein 1 (A2ML1); Guanine deaminase (GDA); Lipocalin-1 (LCN1); Histone H1.4 (HIST1H1E); IPI:IPI00937064.1|REFSEQ:XP_002342720 (ZAN); 5 heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1); ); Endoplasmic reticulum aminopeptidase 2 (ERAP2); 14-3-3 protein zeta/delta (YWHAZ); G-protein coupled receptor 39 (GPR39); similar to KIAA1783 protein (KIAA1783 protein); apolipoprotein A-l binding protein (APOA1BP); pleckstrin and Sec7 domain containing 2 (PSD2); prolylcarboxypeptidase (angiotensinase C) (PROP); Tubulin alpha-1 C chain 10 (TUBA1C); Calmodulin-like protein 5 (CALML5); ARP3 actin-related protein 3 homolog (yeast) (ACTR3); myosin, light chain 6, alkali, smooth muscle and non-muscle (MYL6); Vasodilator-stimulated phosphoprotein (VASP); ARP2 actin-related protein 2 homolog (yeast) (ACTR2); Rheumatoid factor (RF-IP18); Phosphoglycerate kinase 1 (PGK1); Solute carrier family 35 member F1 (SLC35F1); Solute carrier family 35 member F1 15 (SLC35F1); alkaline phosphatase, liver/bone/kidney (ALPL); I tropomyosin 3 (TPM3);

Hexokinase-3 (HK3); Vimentin (VIM); Annexin A1 (ANXA1); IPUPI00930073.1 |TREMBL:B2R853 (KRT6C); Keratin, type II cytoskeletal 6C (KRT6C); myosin, heavy chain 13, skeletal muscle (MYH13); cell cycle progression 1 (CCPG1); Hypothetical protein (H-INV); calcium channel, voltage-dependent, L type, 20 alpha 1D subunit (CACNA1D); LY6/PLAUR domain containing 5 (LYPD5); aarF domain containing kinase 2 (ADCK2); Myosin-lc (MY01C); amyloid beta precursor protein (cytoplasmic tail) binding protein 2 (APPBP2); integrin, alpha 2b (platelet glycoprotein lib of llb/llla complex, antigen CD41) (ITGA2B); tubulin, beta 6 (TUBB6); synaptotagmin-like 4 (SYTL4); aquaporin 4 (AQP4); cell division cycle 42 (GTP binding 25 protein, 25kDa) (CDC42); myosin, light chain 12B, regulatory (MYL12B); protein L-Myc- 2-like (LOC100293553); RAP1B, member of RAS oncogene family (RAP1B); glycoprotein IX (platelet) (GP9); Destrin (DSTN); complement component 1, q subcomponent, C chain (C1QC); epidermal growth factor receptor pathway substrate 8 (EPS8); dual specificity phosphatase 3 (DUSP3); ras homolog gene family, member A 30 (RHOA); myosin, light chain 9, regulatory (MYL9); peptidylprolyl isomerase A (cyclophilin A) (PPIA); Cofilin-1 (CFL1).

In one embodiment, the methods of the invention screen for one or more biomarkers, the presence of which in a sample is indicative that the individual is at risk of suffering from or is suffering from colorectal cancer.

35 In an alternative embodiment, the methods of the invention screen for one or more biomarkers, the increased expression of which in a sample relative to a control sample 13 is indicative that the individual is at risk of suffering from or is suffering from colorectal cancer.

In a preferred embodiment, the methods of the invention screen for one or more biomarkers from the group defined in Table 2. The biomarkers provided in Table 2 5 represent a preferred subset of those defined in Table 1, the differential expression for which relative to control samples is considered statistically significant. Therefore, this group of biomarkers represents a panel of biomarkers from which any number of biomarkers may be selected for screening.

Thus, in one embodiment, the methods of the invention may screen for more than one 10 biomarker from the group defined in Table 2, for example two, three, four, five, six, seven, eight, nine, ten of the biomarkers of the group defined in Table 2. In an alternative embodiment, the methods of the invention screen for more than ten of the biomarkers of the group defined in Table 2, for example, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, thirty, forty, fifty, sixty, 15 seventy, eighty, ninety, one hundred of the biomarkers of the group defined in Table 2. Thus, the methods of the invention screen for the presence of or increased expression of one or more of: complement component C4B (Chido blood group) 2 (C4A/C4B); glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2) (GOT2); glucose-6-phosphate isomerase (GPI); transketolase (TKT); N-acylaminoacyl-20 peptide hydrolase (APEH); histone cluster 1, H4c (HIST4H4 (includes others)); Fatty acid-binding protein 5 (psoriasis-associated) (FABP5); hexosaminidase B (beta polypeptide) (HEXB); epithelial cell adhesion molecule (EPCAM); Nucleoside diphosphate kinase (NME1-NME2); Superoxide dismutase 2, mitochondrial (SOD2); Tu translation elongation factor, mitochondrial (TUFM); Glutathione synthetase (GSS); 25 annexin A2 (ANXA2); ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B); 10 kDa heat shock protein (chaperonin 10) (HSPE1); glyoxalase I (GL01); histone cluster 2, H2be (HIST2H2BE (includes others)); S100 calcium binding protein A4 (S100A4); S100 calcium binding protein A11 (S100A11); latexin (LXN); dehydrogenase/reductase (SDR family) member 11 (DHRS11); N- 30 acetylglucosaminidase, alpha (NAGLU); Translin (TSN); Proteasome (prosome, macropain) subunit alpha type-4 (PSMA4); Proteasome (prosome, macropain) subunit alpha type-6 (PSMA6); ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rad) (RAC1); Adenosylhomocysteinase (AHCY); fucosidase, alpha-L- 1, tissue (FUCA1); S100 calcium binding protein P (S100P); Proteasome 35 (prosome, macropain) subunit beta type-2 (PSMB2); X-prolyl aminopeptidase (aminopeptidase P) 1 (XPNPEP1); Keratin 18 (KRT18); Nuclear cap-binding protein 14 subunit 1 80 kDa (NCBP1); mannosidase, alpha, class 2B, member 1 (MAN2B1); S100 calcium binding protein A6 (S100A6); valosin containing protein (VCP); quinolinate phosphoribosyltransferase (QPRT); major histocompatibility complex, class I, B (HLA-B); phosphoglycerate mutase 1 (brain) (PGAM1); ectonucleotide 5 pyrophosphatase/phosphodiesterase 3 (ENPP3); serpin peptidase inhibitor, clade B (ovalbumin), member 10 (SERPINB10); myeloperoxidase (MPO); creatine kinase, mitochondrial 1B (CKMT1A/CKMT1B); proteinase 3 (PRTN3); elastase, neutrophil expressed (ELANE); MORC family CW-type zinc finger 1 (MORC1); ubiquitin B (UBB); phospholipase A2, group IIA (platelets, synovial fluid) (PLA2G2A); carbonic anhydrase 10 IV (CA4); G elongation factor, mitochondrial 2 (GFM2); S100 calcium binding protein A7 (S100A7); Bactericidal permeability-increasing protein (BPI); collagen, type VI, alpha 5 (COL6A5); LIM homeobox 8 (LHX8); cysteine-rich secretory protein 3 (CRISP3); Azurocidin (AZU1); hemicentin 1 (HMCN1); Transglutaminase 3 (E polypeptide, protein-glutamine gamma-glutamyltransferase) (TGM3); CDC42 binding 15 protein kinase alpha (DMPK-like) (CDC42BPA); Cathepsin G (CTSG); Resistin (RETN); methylmalonyl CoA mutase (MUT); armadillo repeat containing, X-linked 4 (ARMCX4); Integrin alpha-M (ITGAM); Calcium channel, voltage dependent, R-type alpha-1 E subunit (CACNA1E); T-cell lymphoma invasion and metastasis 2 (TIAM2); HIR histone cell cycle regulation defective homolog A (S. cerevisiae) (HIRA); dopey 20 family member 2 (DOPEY2); integrin beta 1 binding protein 3 (ITGB1BP3); Sodium channel, voltage-gated, type VII, alpha (SCN7A); Rab3C, member RAS oncogene family (RAB3C); chromosome 9 open reading frame 79 (C9orf79); nuclear factor of activated T-cells, calcineurin-dependent 4 (NFATC4); UDP-glucose glycoprotein glucosyltransferase 2 (UGGT2); Cornulin (CRNN); kielin/chordin-like protein (KCP); 25 CD1E molecule (CD1E); coiled-coil domain-containing 18 (CCDC18); leukotriene A-4 hydrolase (LTA4H); albumin (ALB); alpha-2-macroglobulin (A2M); complement component 3 (C3); hemoglobin, beta (HBB); transferrin (TF); hemoglobin, alpha 1 (HBA1/HBA2); lactotransferrin (LTF); ceruloplasmin (ferroxidase) (CP); catalase (CAT); group-specific component (vitamin D-binding protein) (GC); serpin peptidase inhibitor, 30 clade C (antithrombin), member 1 (SERPINC1); fibrinogen gamma chain (FGG); S100 calcium binding protein A8 (S100A8); ferritin, light polypeptide (FTL); actin, beta (ACTB); fibronectin 1 (FN1); defensin, alpha 1 (DEFA1 (includes others)); serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 (SERPING1); retinol binding protein 4, plasma (RBP4); peroxiredoxin 2 (PRDX2); fibrinogen alpha chain (FGA); 35 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2 (SERPINF2); carbonic anhydrase II (CA2); orosomucoid 1 15 (0RM1/0RM2); lactate dehydrogenase A (LDHA); vitronectin (VTN); kininogen-1 (KNG1); actin, alpha, cardiac muscle 1 (ACTC1); leucine-rich alpha-2-glycoprotein 1 (LRG1); gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) (GGH); enolase 1, (alpha) (EN01); profilin 1 (PFN1); serpin peptidase inhibitor, clade A 5 (alpha-1 antiproteinase, antitrypsin), member 7 (SERPINA7); alpha-1-microglobulin/bikunin precursor (AMBP); lamin A/C (LMNA); apolipoprotein D (APOD); thyroid hormone receptor interactor 11 (TRIP11); complement component 4 binding protein, alpha (C4BPA); tropomyosin 4 (TPM4); filamin A, alpha (FLNA); haptoglobin (HP); hemopexin (HPX); hemoglobin, delta (HBD); fibrinogen beta chain (FGB); S100 10 calcium binding protein A9 (S100A9); complement component 5 (C5); solute carrier family 26, member 3 (SLC26A3); complement component 9 (C9); amyloid P component, serum (APCS); alpha-1 -B glycoprotein (A1BG); complement C3-like (LOC100133511); inter-alpha (globulin) inhibitor H4 (plasma Kallikrein-sensitive glycoprotein) (ITIH4); complement component C8, alpha polypeptide (C8A); inter-alpha 15 (globulin) inhibitor H1 (ITIH1).

In one embodiment, the method of the invention screens for one or more biomarkers capable of diagnosing or predicting CRC in an individual who tested negative in the fecal immunochemical test.

Thus, the methods of the invention may screen for one or more biomarkers selected 20 from the group defined in Table 3. The biomarkers provided in Table 3 represent a preferred subset of those defined in Table 1, which have been found to be present in significantly higher levels in CRC samples which came back negative from the fecal immunochemical test. Thus, the group of Table 3 represents a panel of biomarkers from which any number of biomarkers may be selected for screening.

25 Thus, in one embodiment, the methods of the invention may screen for more than one biomarker from the group defined in Table 3, for example two, three, four, five, six, seven, eight, nine, ten of the biomarkers of the group defined in Table 3. In an alternative embodiment, the methods of the invention screen for more than ten of the biomarkers of the group defined in Table 3, for example, eleven, twelve, thirteen, 30 fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred of the biomarkers of the group defined in Table 3. Thus, the methods of the invention may screen for the presence of or increased expression of one or more of: complement component C4B (Chido blood group) 2 (C4A/C4B); glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate 35 aminotransferase 2) (GOT2); glucose-6-phosphate isomerase (GPI); transketolase (TKT); N-acylaminoacyl-peptide hydrolase (APEH); hexosaminidase B (beta 16 polypeptide) (HEXB); epithelial cell adhesion molecule (EPCAM); NME1-NME2 readthrough (NME1-NME2); Tu translation elongation factor, mitochondrial (TUFM); Glutathione synthetase (GSS); glyoxalase I (GL01); latexin (LXN); Proteasome (prosome, macropain) subunit alpha type-4 (PSMA4); fucosidase, alpha-L- 1, tissue 5 (FUCA1); Keratin 18 (KRT18); Nuclear cap-binding protein subunit 1 80 kDa (NCBP1); mannosidase, alpha, class 2B, member 1 (MAN2B1); S100 calcium binding protein A6 (S100A6); major histocompatibility complex, class I, B (HLA-B); ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3); serpin peptidase inhibitor, clade B (ovalbumin), member 10 (SERPINB10); myeloperoxidase (MPO); proteinase 3 10 (PRTN3); phospholipase A2, group IIA (platelets, synovial fluid) (PLA2G2A); carbonic anhydrase IV (CA4); G elongation factor, mitochondrial 2 (GFM2); S100 calcium binding protein A7 (S100A7); Bactericidal permeability-increasing protein (BPI); collagen, type VI, alpha 5 (COL6A5); LIM homeobox 8 (LHX8); Azurocidin (AZU1); hemicentin 1 (HMCN1); methylmalonyl CoA mutase (MUT); armadillo repeat 15 containing, X-linked 4 (ARMCX4); Integrin alpha-M (ITGAM); Calcium channel, voltage dependent, R-type alpha-1 E subunit (CACNA1E); T-cell lymphoma invasion and metastasis 2 (TIAM2); HIR histone cell cycle regulation defective homolog A (S. cerevisiae) (HIRA); dopey family member 2 (DOPEY2); Sodium channel, voltagegated, type VII, alpha (SCN7A); chromosome 9 open reading frame 79 (C9orf79); 20 UDP-glucose glycoprotein glucosyltransferase 2 (UGGT2); Cornulin (CRNN); coiled-coil domain-containing 18 (CCDC18); alpha-2-macroglobulin (A2M); complement component 3 (C3); hemoglobin, beta (HBB); transferrin (TF); hemoglobin, alpha 1 (HBA1/HBA2); lactotransferrin (LTF); ceruloplasmin (ferroxidase) (CP); catalase (CAT); fibrinogen gamma chain (FGG); S100 calcium binding protein A8 (S100A8); ferritin, 25 light polypeptide (FTL); fibronectin 1 (FN1); defensin, alpha 1 (DEFA1 (includes others)); serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 (SERPING1); retinol binding protein 4, plasma (RBP4); peroxiredoxin 2 (PRDX2); serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2 (SERPINF2); lactate dehydrogenase (LDHA); vitronectin (VTN); kininogen-1 (KNG1); 30 leucine-rich alpha-2-glycoprotein 1 (LRG1); gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) (GGH); serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 7 (SERPINA7); alpha-1-microglobulin/bikunin precursor (AMBP); thyroid hormone receptor interactor 11 (TRIP11); complement component 4 binding protein, alpha (C4BPA); filamin A, alpha (FLNA); hemopexin 35 (HPX); S100 calcium binding protein A9 (S100A9); complement component 5 (C5); solute carrier family 26, member 3 (SLC26A3); complement component 9 (C9); amyloid 17 P component, serum (APCS); complement C3-like (LOC100133511); serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (SERPINA3); Cathepsin S (CTSS); Cytidine deaminase (CDA); Shugoshin-like 2 (SGOL2); serpin peptidase inhibitor, clade B (ovalbumin), member 3 (SERPINB3); hect domain and 5 RLD 2 (HERC2); triosephosphate isomerase 1 (TPI1); Isoform 1 of collagen, type IV, alpha 3 (Goodpasture antigen) binding protein (COL4A3BP); ribonuclease T2 (RNASET2); Glutathione reductase (GSR); spinster homolog 3 (Drosophila) (SPNS3); cDNA FLJ60317, highly similar to Aminoacylase-1 (ACY1); serpin peptidase inhibitor, clade B (ovalbumin), member 8 (SERPINB8); Branched-chain-amino-acid 10 aminotransferase (BCAT2); sialic acid acetylesterase (SIAE); peptidase D (PEPD); major histocompatibility complex, class II, DR beta 5 (HLA-DRB5); SET domain containing 2 (SETD2); hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHCI)-like domain (RLD) 1 (HERC1); isocitrate dehydrogenase 1 (NADP+), soluble (IDH1); cathepsin C (CTSC); serpin peptidase inhibitor, clade A 15 (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1); spectrin repeat containing, nuclear envelope 1 (SYNE1); Ankyrin repeat domain-containing protein 35 (ANKRD35); NIMA (never in mitosis gene a)- related kinase 10 (NEK10); inter-alpha (globulin) inhibitor H2 (ITIH2); acyl-CoA dehydrogenase, very long chain (ACADVL); Nebulin (NEB); Zymogen granule membrane protein 16 (ZG16); Vinculin (VCL); 20 Isoform 2 of Dedicator of cytokinesis protein 4 (DOCK4); hypothetical protein LOC643677 (C13orf40); Uncharacterized protein KIAA1797 (KIAA1797); baculoviral IAP repeat-containing 6 (BIRC6); Transaldolase 1 (TALD01); Taste receptor type 2 member 42 (TAS2R42); chitinase 1 (chitotriosidase) (CHIT1); quiescin Q6 sulfhydryl oxidase 1 (QSOX1); bone marrow stromal cell antigen 12 (BST1); Bleomycin hydrolase 25 (BLMH); Lysozyme C (LYZ).

In one embodiment, the method of the invention screens for one or more biomarkers having a higher discriminatory power than haemoglobin. In other words, the one or more biomarkers have a higher sensitivity and higher specificity than haemoglobin in detecting the presence of advanced adenoma or adenocarcinoma in a sample.

30 Thus, in one embodiment, the method of the invention screens for one or more biomarker, for example two, three, four, five, six, seven, eight, nine, ten of the biomarkers selected from the group consisting of: S100 calcium binding protein A8 (S100A8), complement component C4B (Chido blood group) 2 (C4A/C4B), transferrin (TF), alpha-2-macroglobulin (A2M), S100 calcium binding protein A9 (S100A9), 35 proteinase 3 (PRTN3), Azurocidin (AZU1), lactotransferrin (LTF), hemopexin (HPX) and defensin, alpha 1 (DEFA1).

18

In one embodiment, the method of the invention screens for one or more biomarkers, the presence of which in a sample is indicative that the individual is at risk of suffering from or is suffering from colorectal cancer.

Thus, in one embodiment, the method of the invention screens for one or more 5 biomarker, for example two, three, four, five, six, seven, eight, nine, ten of the biomarkers selected from the group defined in Table 4. The biomarkers provided in Table 4 represent a preferred subset of those defined in Table 1, which have been found to be expressed only in CRC samples, not in control samples. Thus, the group of Table 4 represents a panel of biomarkers from which any number of biomarkers may 10 be selected for screening.

Preferably, the method of the invention has an accuracy of at least 75%, for example 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% accuracy. Preferably, the method of the invention has a sensitivity of at least 75%, for example 15 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sensitivity. Preferably, the method of the invention has a specificity of at least 75%, for example 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% specificity.

20 By "accuracy" it is meant the proportion of correct outcomes of a diagnosis, by "sensitivity" it is meant the proportion of all positive diagnoses that are correctly classified as positives, and by "specificity" it is meant the proportion of all negative diagnoses that are correctly classified as negatives. The methods of the invention may be verified by subsequent colonoscopy.

25

Screening methods

In one embodiment, the biological sample, for example a stool sample, may be screened for the one or more biomarkers using (targeted) mass spectrometry.

Protein marker discovery and screening by LC-MS/MS and hypothesis-based protein 30 marker detection by SRM-MS has been applied before on murine and human stool samples, respectively (Ang CS, Nice EC. J Proteome Res 2010; 9: 4346-55; Ang CS, et at. Electrophoresis 2011; 32: 1926-38; Ang CS, et al. J Chromatogr A 2010; 1217: 3330-40).

Despite the complexity of stool material, the present invention has successfully 35 detected several of these previously reported human stool proteins. In addition, the number of included samples and corresponding identified proteins in the current study 19 exceeds that of previous studies. Therefore the data presented here is the largest overview of the human stool proteome to date.

While sample screening by the mass spectrometric techniques described herein has been proven to be effective, a preferred screening method comprises an antibody 5 based screening assay. The fecal immunochemical test (FIT) comprises an antibody based screening assay and so an antibody based screening assay for one or more of the biomarkers identified in the present application provides a complementary screen which can be readily incorporated into the existing FIT assay.

Thus, in one embodiment of the methods of the invention, the biological sample is 10 screened for the one or more biomarkers using a binding agent capable of binding to the one or more biomarkers.

Binding agents (also referred to as binding molecules) can be selected from a library, based on their ability to bind a given motif, as discussed below.

In one embodiment, the first binding agent is an antibody or a fragment thereof. Thus, a 15 fragment may contain one or more of the variable heavy (VH) or variable light (VL) domains. For example, the term antibody fragment includes Fab-like molecules (Better et al Science 1988; 240, 1041); Fv molecules (Skerra et al Science 1988; 240, 1038); single-chain Fv (ScFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al Science 1988; 242,423; Huston et al Proc. Natl. Acad. 20 Sci. USA 1988; 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al Nature 1989; 341,544).

The term "antibody variant" includes any synthetic antibodies, recombinant antibodies or antibody hybrids, such as but not limited to, a single-chain antibody molecule produced by phage-display of immunoglobulin light and/or heavy chain variable and/or 25 constant regions, or other immunointeractive molecule capable of binding to an antigen in an immunoassay format that is known to those skilled in the art.

A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein Nature 1991; 349, 293-299.

30 In one embodiment, the antibody or fragment thereof is a recombinant antibody or fragment thereof (such as an scFv). By "ScFv molecules" it is meant molecules wherein the VH and VL partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than whole antibodies, are several-fold. Effector functions of whole antibodies, such as complement binding, are 35 removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and 20 secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.

Whole antibodies, and F(ab')2 fragments are "bivalent". By "bivalent" it is meant that the said antibodies and F(ab')2 fragments have two antigen combining sites. In contrast, 5 Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining site.

The antibodies may be monoclonal or polyclonal. Suitable antibodies may be prepared by known techniques and need no further discussion.

Additionally or alternatively the binding agent may be an aptamer. Suitable aptamers 10 may be prepared by known techniques and need no further discussion.

In one embodiment, the one or more biomarker(s) in the test sample is labelled with a detectable moiety. In one embodiment, the one or more biomarker(s) in the control sample is labelled with a detectable moiety (which may be the same or different from the detectable moiety used to label the test sample).

15 A "detectable moiety" is one which may be detected and the relative amount and/or location of the moiety (for example, the location on an array) determined.

Detectable moieties are well known in the art. A detectable moiety may be a fluorescent and/or luminescent and/or chemiluminescent moiety which, when exposed to specific conditions, may be detected. For example, a fluorescent moiety may need to 20 be exposed to radiation (i.e. light) at a specific wavelength and intensity to cause excitation of the fluorescent moiety, thereby enabling it to emit detectable fluorescence at a specific wavelength that may be detected.

Alternatively, the detectable moiety may be an enzyme which is capable of converting a (preferably undetectable) substrate into a detectable product that can be visualised 25 and/or detected. Examples of suitable enzymes are discussed in more detail below in relation to, for example, ELISA assays.

Alternatively, the detectable moiety may be a radioactive label, which may be incorporated by methods well known in the art.

Arrays 30 In one embodiment, the methods of the present invention may be carried out on an array.

Arrays per se are well known in the art. Typically they are formed of a linear or two-dimensional structure having spaced apart (i.e. discrete) regions ("spots"), each having a finite area, formed on the surface of a solid support. An array can also be a bead 35 structure where each bead can be identified by a molecular code or colour code or identified in a continuous flow. Analysis can also be performed sequentially where the 21 sample is passed over a series of spots each adsorbing the class of molecules from the solution.

The solid support is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. 5 The solid supports may be in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane (e.g. plastic, polymer, perspex, silicon, amongst others), a plurality of polymeric pins, or a plurality of microtitre wells, or any other surface suitable for immobilising proteins, antibodies and 10 other suitable molecules and/or conducting an immunoassay.

The binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing a protein molecule, antibody or the like to the solid support. By using well-known techniques, such as contact or non-contact printing, masking or photolithography, the location of each spot can be defined.

15 Once suitable binding molecules (discussed above) have been identified and isolated, the skilled person can manufacture an array using methods well known in the art of molecular biology.

In one embodiment, the screening may comprise using an assay comprising a second binding agent capable of binding to the one or more biomarkers, the second binding 20 agent having a detectable moiety.

In one embodiment, the second binding agent is an antibody or a fragment thereof (for example, as described above in relation to the first binding agent).

Typically, the assay is an ELISA (Enzyme Linked Immunosorbent Assay) which usually involves the use of enzymes which give a coloured reaction product, usually in solid 25 phase assays. Enzymes such as horseradish peroxidase and phosphatase have been widely employed. A way of amplifying the phosphatase reaction is to use NADP as a substrate to generate NAD which now acts as a coenzyme for a second enzyme system. Pyrophosphatase from Escherichia coli provides a good conjugate because the enzyme is not present in tissues, is stable and gives a good reaction colour. Chemi-30 luminescent systems based on enzymes such as luciferase can also be used.

Conjugation with the vitamin biotin is also employed used since this can readily be detected by its reaction with enzyme-linked avidin or streptavidin to which it binds with great specificity and affinity.

It will be appreciated by persons skilled in the art that there is a degree of fluidity in the 35 biomarker composition of the signatures of the invention. Thus, different combinations of the biomarkers may be equally useful in the diagnosis, prognosis and/or 22

characterisation of colorectal cancer. In this way, each biomarker (either alone or in combination with one or more other biomarkers) makes a contribution to the signature. Compounds and methods for treating CRC

The identification of the biomarkers as defined in Table 1 allows not only the detection 5 of advanced colonic adenomas and colonic adenocarcinomas (colorectal cancer), but enables also methods of treating colorectal cancers as defined in the fourth aspect of the invention, and also provides for compounds for use in methods of treating colorectal cancers as defined in the fifth aspect of the invention.

While early diagnosis of colorectal cancer often allows for curative surgical removal of 10 the tumour, later diagnosis may result in a (chemo)therapeutic treatment instead. Therapeutic agents used to treat colorectal cancer include monoclonal antibodies, small molecule inhibitors and chemotherapeutic agents.

Typical therapeutic monoclonal antibodies include but are not limited to bevacizumab, cetuximab or panitumumab. Typical small molecule inhibitors include but are not 15 limited to erlotinib, sorafenib or alisertib. Typical chemotherapeutic agents include but are not limited to 5-FU, capecitabine, irinotecan oxaliplatin, or leucovorin or any combination thereof. Combination therapies of, for example, a therapeutic monoclonal antibody and a small molecule inhibitor may be used. Thus, any combination of two or more of a monoclonal antibody, a small molecule inhibitor and a chemotherapeutic 20 agent is envisaged.

Kit for performing the method

The kit for performing the method according to the invention may be selected from any suitable assay and data processing apparatus and equipments.

The suitable selection will be well within the ability of those skilled in the art, and further 25 description is not necessary here.

“Comprising”

The term “comprising” and related terms herein is to be interpreted as embracing “consisting essentially of’ and “consisting of, these two expressions being interchangeable with “comprising” in all definitions and discussion in this patent in order 30 to specify alternative extents of exclusion of unspecified elements additional to the recited elements.

The term “comprising” and related expressions means “including” and therefore leaves open the option of including unspecified elements, whether essential or not. The term “consisting essentially of and related expressions permits the presence of elements 35 that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. The term “consisting of and related expressions means 23 “consisting only of and therefore excludes any element not recited in that description of the embodiment.

Brief Description of the Figures

The invention shall now be further described by the following example with reference to 5 the attached figures. The example is provided by way of example only, without any intended limitation of the scope of the invention. All cited references are incorporated herein by reference in their entireties.

Figure 1A shows the number of human proteins detected in stool samples from 12 CRC patients and from 10 control patients. The Venn diagram shows the number of 10 proteins detected in CRC or control stool samples and their overlap;

Figure 1B shows the subcellular location of the identified human proteins. The pie chart shows the percentage of different subcellular locations of the 830 human proteins that were identified in 22 stool samples. The ratios were similar for CRC patients and control patients; 15 Figure 2A shows a scatterplot of protein quantifications of hemoglobin alpha as measured by LC-MS/MS compared to hemoglobin quantification by FIT of individual stool samples;

Figure 2B shows a scatterplot of protein quantifications of hemoglobin beta as measured by LC-MS/MS compared to hemoglobin quantification by FIT of individual 20 stool samples;

Figure 2C shows a scatterplot of protein quantifications of hemoglobin alpha and beta as measured by LC-MS/MS of individual stool samples;

Figure 2D shows a scatterplot of protein quantifications of S100A8 and S100A9 as measured by LC-MS/MS of individual stool samples. Filled circles represent stool 25 samples from subjects with CRC (n=12), crosses represent stool samples from subjects without colon neoplasia (n=10); and

Figure 3 shows a scatterplot of protein quantifications of Complement component 4B measured by LC-MS/MS in relation to FIT values of individual stool samples. The dotted line shows the cut-off of 75 ng/ml for FIT. A FIT value less than 75 ng/ml is 30 regarded as a negative test result (Van Veen, NTG, 2009). Filled circles represent stool samples from subjects with CRC (n=12), crosses represent stool samples from subjects without colon neoplasia (n=10).

TABLES

Accession Gene Symbol Entrez Gene Name Number 24 IPI00892604 C4A/C4B complement component 4B (Chido blood group) IPI00018206 GOT2 glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2) IPI00027497 GPI glucose-6-phosphate isomerase IPI00643920 TKT transketolase IPI00337741 APEH N-acylaminoacyl-peptide hydrolase IPI00453473 HIST4H4 histone cluster 1, H4c (includes others) IPI00007797 FABP5 fatty acid binding protein 5 (psoriasis- associated) IPI00012585 HEXB hexosaminidase B (beta polypeptide) IPI00296215 EPCAM epithelial cell adhesion molecule IPI00604590 NME1-NME2 NME1-NME2 readthrough IPI00022314 SOD2 superoxide dismutase 2, mitochondrial IPI00027107 TUFM Tu translation elongation factor, mitochondrial IPI00010706 GSS glutathione synthetase IPI00418169 ANXA2 annexin A2 IPI00303476 ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide IPI00220362 HSPE1 heat shock 10kDa protein 1 (chaperonin 10)

IPI00220766 GL01 glyoxalase I

IPI00003935 HIST2H2BE histone cluster 2, H2be (includes others) IPI00032313 S100A4 S100 calcium binding protein A4 IPI00013895 S100A11 S100 calcium binding protein A11 IPI00106687 LXN latex in IPI00034280 DHRS11 dehydrogenase/reductase (SDR family) member 11 IPI00008787 NAGLU N-acetylglucosaminidase, alpha IPI00018768 TSN translin 25 IPI00299155 PSMA4 proteasome (prosome, macropain) subunit, alpha type, 4 IPI00029623 PSMA6 proteasome (prosome, macropain) subunit, alpha type, 6 IPI00010271 RAC1 ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rad) IPI00012007 AHCY adenosylhomocysteinase IPI00385751 FUCA1 fucosidase, alpha-L-1, tissue

IPI00017526 S100P S100 calcium binding protein P

IPI00028006 PSMB2 proteasome (prosome, macropain) subunit, beta type, 2 IPI00793375 XPNPEP1 X-prolyl aminopeptidase (aminopeptidase P) 1, soluble IPI00554788 KRT18 keratin 18 IPI00019380 NCBP1 nuclear cap binding protein subunit 1, 80kDa IPI00012989 MAN2B1 mannosidase, alpha, class 2B, member 1 IPI00027463 S100A6 S100 calcium binding protein A6 IPI00022774 VCP valosin-containing protein IPI00300086 QPRT quinolinate phosphoribosyltransferase

IPI00472073 HLA-B major histocompatibility complex, class I, B

IPI00453476 PGAM1 phosphoglycerate mutase 1 (brain) IPI00020999 ENPP3 ectonucleotide

pyrophosphatase/phosphodiesterase 3 IPI00010304 SERPINB10 serpin peptidase inhibitor, clade B

(ovalbumin), member 10 IPI00007244 MPO myeloperoxidase

IPI00877726 CKMT1A/CKMT1B creatine kinase, mitochondrial 1B

IPI00027409 PRTN3 proteinase 3 IPI00027769 ELANE elastase, neutrophil expressed IPI00004362 MORC1 MORC family CW-type zinc finger 1

IPI00719280 ÜBB ubiquitin B

IPI00026962 PLA2G2A phospholipase A2, group IIA (platelets, synovial fluid) 26

IPI00027466 CA4 carbonic anhydrase IV

IPI00071703 GFM2 G elongation factor, mitochondrial 2 IPI00219806 S100A7 S100 calcium binding protein A7 IPI00827847 BPI bactericidal/permeability-increasing protein IPI00176125 COL6A5 collagen, type VI, alpha 5 IPI00470355 LHX8 LIM homeobox 8 IPI00942117 CRISP3 cysteine-rich secretory protein 3 IPI00022246 AZU1 azurocidin 1 IPI00045512 HMCN1 hemicentin 1 IPI00300376 TGM3 transglutaminase 3 (E polypeptide, protein- glutamine-gamma-glutamyltransferase) IPI00640468 CDC42BPA CDC42 binding protein kinase alpha (DMPK-like)

IPI00028064 CTSG cathepsin G

IPI00006988 RETN resistin IPI00024934 MUT methylmalonyl CoA mutase IPI00552983 ARMCX4 armadillo repeat containing, X-linked 4 IPI00217987 ITGAM integrin, alpha M (complement component 3 receptor 3 subunit)

IPI00165045 CACNA1E calcium channel, voltage-dependent, R

type, alpha 1E subunit IPI00018363 TIAM2 T-cell lymphoma invasion and metastasis 2 IPI00217560 HIRA HIR histone cell cycle regulation defective homolog A (S. cerevisiae) IPI00294653 DOPEY2 dopey family member 2 IPI00004401 ITGB1BP3 integrin beta 1 binding protein 3 IPI00300117 SCN7A sodium channel, voltage-gated, type VII, alpha IPI00061114 RAB3C RAB3C, member RAS oncogene family IPI00168442 C9orf79 chromosome 9 open reading frame 79 IPI00385215 NFATC4 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 IPI00024467 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 IPI00297056 CRNN cornulin 27 IPI00914663 KCP kielin/chordin-like protein IPI00418592 CD1E CD1e molecule IPI00642206 CCDC18 coiled-coil domain containing 18 IPI00514090 LTA4H leukotriene A4 hydrolase IPI00745872 ALB albumin IPI00478003 A2M alpha-2-macroglobulin IPI00783987 C3 complement component 3 IPI00654755 HBB hemoglobin, beta IPI00022463 TF transferrin IPI00410714 HBA1/HBA2 hemoglobin, alpha 1 IPI00298860 LTF lactotransferrin IPI00017601 CP ceruloplasmin (ferroxidase) IPI00465436 CAT catalase

IPI00555812 GC group-specific component (vitamin D

binding protein)

IPI00032179 SERPINC1 serpin peptidase inhibitor, clade C

(antithrombin), member 1 IPI00219713 FGG fibrinogen gamma chain IPI00007047 S100A8 S100 calcium binding protein A8 IPI00375676 FTL ferritin, light polypeptide IPI00021439 ACTB actin, beta IPI00022418 FN1 fibronectin 1 IPI00005721 DEFA1 (includes defensin, alpha 1 others) IPI00291866 SERPING1 serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 IPI00022420 RBP4 retinol binding protein 4, plasma IPI00027350 PRDX2 peroxiredoxin 2 IPI00021885 FGA fibrinogen alpha chain IPI00029863 SERPINF2 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2

IPI00218414 CA2 carbonic anhydrase II

IPI00020091 ORM1/ORM2 orosomucoid 1

IPI00217966 LDHA lactate dehydrogenase A

28 IPI00298971 VTN vitronectin IPI00215894 KNG1 kininogen 1 IPI00023006 ACTC1 actin, alpha, cardiac muscle 1 IPI00022417 LRG1 leucine-rich alpha-2-glycoprotein 1 IPI00023728 GGH gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) IPI00465248 EN01 enolase 1, (alpha) IPI00216691 PFN1 profilin 1 IPI00292946 SERPINA7 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 7 IPI00022426 AMBP alpha-1 -microglobulin/bikunin precursor

IPI00021405 LMNA lamin A/C

IPI00006662 APOD apolipoprotein D

IPI00003515 TRIP11 thyroid hormone receptor interactor 11 IPI00021727 C4BPA complement component 4 binding protein, alpha IPI00010779 TPM4 tropomyosin 4 IPI00302592 FLNA filamin A, alpha IPI00641737 HP haptoglobin IPI00022488 HPX hemopexin IPI00473011 HBD hemoglobin, delta IPI00298497 FGB fibrinogen beta chain IPI00027462 S100A9 S100 calcium binding protein A9 IPI00032291 C5 complement component 5 IPI00031036 SLC26A3 solute carrier family 26, member 3 IPI00022395 C9 complement component 9 IPI00022391 APCS amyloid P component, serum IPI00022895 A1BG alpha-1 -B glycoprotein IPI00887739 LOC100133511 complement C3-like IPI00218192 ITIH4 inter-alpha (globulin) inhibitor H4 (plasma

Kallikrein-sensitive glycoprotein) IPI00011252 C8A complement component 8, alpha polypeptide IPI00292530 mRÏ inter-alpha (globulin) inhibitor H1 IPI00937735 ACADVL acyl-CoA dehydrogenase, very long chain 29 IPI00009268 ACY1 cDNA FLJ60317, highly similar to

Aminoacylase-1 IPI00166331 ANKRD35 Ankyrin repeat domain-containing protein 35 IPI00299635 BIRC6 baculoviral IAP repeat-containing 6 IPI00219575 BLMH Bleomycin hydrolase IPI00026240 BST1 bone marrow stromal cell antigen 1 IPI00929313 C13orf40 hypothetical protein LOC643677 IPI00027983 CDA Cytidine deaminase IPI00852865 CHIT1 chitinase 1 (chitotriosidase) IPI00022810 CTSC Dipeptidyl-peptidase 1

IPI00299150 CTSS Cathepsin S

IPI00006024 DOCK4 Isoform 2 of Dedicator of cytokinesis protein 4 IPI00016862 GSR Glutathione reductase IPI00941901 HERC1 hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHCI)-like domain (RLD) 1 IPI00005826 HERC2 hect domain and RLD 2 IPI00746667 HLA-DRB5 HLA class II histocompatibility antigen, DRB1-11 beta chain IPI00027223 IDH1 Isocitrate dehydrogenase [NADP] cytoplasmic IPI00305461 ITIH2 inter-alpha (globulin) inhibitor H2 IPI00748360 KIAA1797 Uncharacterized protein KIAA1797

IPI00019038 LYZ Lysozyme C

IPI00303335 NËB Nebulin IPI00065454 NEK10 NIMA (never in mitosis gene a)- related kinase 10(NEK10);

IPI00257882 PEPD peptidase D

IPI00003590 QSOX1 quiescin Q6 sulfhydryl oxidase IPI00939604 RNASET2 Ribonuclease T2 IPI00553177 SERPINA1 serpin peptidase inhibitor, clade A (alpha-1 30 antiproteinase, antitrypsin), member 1 IPI00550991 SERPINA3 serpin peptidase inhibitor, clade A (alpha-1

antiproteinase, antitrypsin), member 3 IPI00022204 SERPINB3 serpin peptidase inhibitor, clade B

(ovalbumin), member 3 IPI00307733 SETD2 SET domain containing 2 IPI00218013 SGOL2 Shugoshin-like 2 IPI00010949 SIAE sialic acid acetylesterase IPI00386444 SYNE1 spectrin repeat containing, nuclear envelope 1 IPI00744692 TALD01 Transaldolase 1 IPI00375371 TAS2R42 Taste receptor type 2 member 42 IPI00465028 TPI1 triosephosphate isomerase 1 IPI00291175 VCL Vinculin IPI00029647 ZG16 Zymogen granule membrane protein 16 IPI00016255 PLBD1 hypothetical protein LOC79887 IPI00477505 ANKRD28 Isoform 1 of Serine/threonine-protein

phosphatase 6 regulatory ankyrin repeat subunit A

IPI00032293 CST3 Cystatin-C

IPI00293867 DDT D-dopachrome decarboxylase IPI00059242 SYAP1 Synapse-associated protein 1 IPI00219622 PSMA2 Proteasome subunit alpha type-2 IPI00221222 SUB1 SUB1 homolog (S. cerevisiae) IPI00022791 MFAP3 Microfibril-associated glycoprotein 3

IPI00011229 CTSD Cathepsin D

IPI00025019 PSMB1 proteasome (prosome, macropain) subunit, beta type, 1 IPI00479306 PSMB5 proteasome (prosome, macropain) subunit, beta type, 5

IPI00234793 KCTD15 cDNA FLJ61112, highly similar to BTB/POZ

domain-containing protein KCTD15 IPI00010796 P4HB prolyl 4-hydroxylase, beta polypeptide IPI00927606 GPX1 glutathione peroxidase 1

IPI00783625 SERPINB5 serpin peptidase inhibitor, clade B

31 (ovalbumin), member 5 IPI00024701 COL4A3BP collagen, type IV, alpha 3 (Goodpasture antigen) binding protein IPI00000811 PSMB6 proteasome (prosome, macropain) subunit, beta type, 6 IPI00021298 KRT20 Keratin 20 IPI00025084 CAPNS1 Calpain small subunit 1 IPI00024919 PRDX3 peroxiredoxin 3

IPI00059930 NACC2 NACC family member 2, BEN and BTB

(POZ) domain containing IPI00003817 ARHGDIB Rho GDP-dissociation inhibitor 2 IPI00293276 MIF Macrophage migration inhibitory factor IPI00514622 RANBP6 Ran-binding protein 6 IPI00333141 SPNS3 spinster homolog 3 (Drosophila) IPI00922181 MCM2 minichromosome maintenance complex component 2 IPI00031708 FAH Fumarylacetoacetase IPI00003865 HSPA8 heat shock 70kDa protein 8 IPI00299024 BASP1 brain abundant, membrane attached signal protein 1 IPI00181135 BCAT2 Branched-chain-amino-acid aminotransferase IPI00219365 MSN Moesin

IPI00032134 SERPINB8 serpin peptidase inhibitor, clade B

(ovalbumin), member 8 IPI00853547 G6PD glucose-6-phosphate dehydrogenase isoform a IPI00478758 C10orf119 Isoform 1 of UPF0557 protein C10orf119 IPI00873020 PSAP Prosaposin IPI00000875 EEF1G eukaryotic translation elongation factor 1 gamma IPI00014398 FHL1 four and a half LIM domains 1 IPI00301395 CPVL carboxypeptidase, vitellogenic-like IPI00790262 TTLL3 tubulin tyrosine ligase-like family, member 3 IPI00942608 26kDaprotein IPI:IPI00942608.1|ENSEMBL:ENSP0000 32 IPI00023038 PRB1/PRB2 proline-rich protein BstNI subfamily 2 IPI00001895 PCDH8 Protocadherin-8 IPI00419215 A2ML1 Alpha-2-macroglobulin-like protein 1 IPI00644409 GDA Guanine deaminase IPI00009650 LCN1 Lipocalin-1 IPI00217467 HIST1H1E Histone H1.4 IPI00937064 ZAN IPI:IPI00937064.1|REFSEQ:XP_002342720 IPI00396378 HNRNPA2B1 heterogeneous nuclear ribonucleoprotein A2/B1 IPI00465261 ERAP2 endoplasmic reticulum aminopeptidase 2 IPI00021263 YWHAZ 14-3-3 protein zeta/delta IPI00011241 GPR39 G-protein coupled receptor 39 IPI00786880 KIAA1783 similar to KIAA1783 protein IPI00168479 APOA1BP apolipoprotein A-l binding protein IPI00031084 PSD2 pleckstrin and Sec7 domain containing 2 IPI00001593 PRCP prolylcarboxypeptidase (angiotensinase C) IPI00218343 TUBA1C Tubulin alpha-1 C chain IPI00021536 CALML5 Calmodulin-like protein 5 IPI00028091 ACTR3 ARP3 actin-related protein 3 homolog (yeast) IPI00796366 MYL6 myosin, light chain 6, alkali, smooth muscle and non-muscle IPI00301058 VASP Vasodilator-stimulated phosphoprotein IPI00470573 ACTR2 ARP2 actin-related protein 2 homolog (yeast) IPI00884078 RF-IP18 Rheumatoid factor RF-IP18 IPI00169383 PGK1 Phosphoglycerate kinase 1 IPI00299619 SLC35F1 Solute carrier family 35 member F1 IPI00419916 ALPL alkaline phosphatase, liver/bone/kidney IPI00218319 TPM3 tropomyosin 3 IPI00005118 HK3 Hexokinase-3 IPI00418471 VIM Vimentin IPI00218918 ANXA1 Annexin A1 IPI00930073 KRT6C IPMPI00930073.1 |TREMBL:B2R853

IPI00299145 KRT6C Keratin, type II cytoskeletal 6C

33 IPI00007858 MYH13 myosin, heavy chain 13, skeletal muscle IPI00297235 CCPG1 cell cycle progression 1 IPI00927726 H-INV Hypothetical protein

IPI00009008 CACNA1D calcium channel, voltage-dependent, L

type, alpha 1D subunit IPI00304554 LYPD5 LY6/PLAUR domain containing 5 IPI00815807 ADCK2 aarF domain containing kinase 2 IPI00743335 MY01C Myosin-lc IPI00007393 APPBP2 amyloid beta precursor protein (cytoplasmic tail) binding protein 2 IPI00295976 ITGA2B integrin, alpha 2b (platelet glycoprotein Mb of llb/llla complex, antigen CD41) IPI00641706 TUBB6 tubulin, beta 6 IPI00060201 SYTL4 synaptotagmin-like 4 IPI00908634 AQP4 aquaporin 4 IPI00016786 CDC42 cell division cycle 42 (GTP binding protein, 25kDa) IPI00033494 MYL12B myosin, light chain 12B, regulatory IPI00409756 LOC100293553 protein L-Myc-2-like IPI00015148 RAP1B RAP1B, member of RAS oncogene family IPI00027502 GP9 glycoprotein IX (platelet) IPI00473014 DSTN Destrin IPI00022394 C1QC complement component 1, q subcomponent, C chain IPI00290337 EPS8 epidermal growth factor receptor pathway substrate 8 IPI00018671 DUSP3 dual specificity phosphatase 3

IPI00478231 RHOA ras homolog gene family, member A

IPI00220278 MYL9 myosin, light chain 9, regulatory IPI00419585 PPIA peptidylprolyl isomerase A (cyclophilin A) IPI00012011 CFL1 Cofilin-1

Table 1: Biomarkers indicative of colorectal cancer.

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Accession Gene Symbol Entrez Gene Name

Number IPI00022426 AMBP alpha-1 -microglobulin/bikunin precursor IPI00022246 AZU1 azurocidin 1 IPI00827847 BPI bactericidal/permeability-increasing protein IPI00892604 C4A/C4B complement component 4B (Chido blood group) IPI00021727 C4BPA complement component 4 binding protein, alpha IPI00168442 C9orf79 chromosome 9 open reading frame 79

IPI00165045 CACNA1E calcium channel, voltage-dependent, R

type, alpha 1E subunit IPI00642206 CCDC18 coiled-coil domain containing 18 IPI00297056 CRNN comulin IPI00296215 EPCAM epithelial cell adhesion molecule IPI00219713 FGG fibrinogen gamma chain IPI00302592 FLNA filamin A, alpha

IPI00220766 GL01 glyoxalase I

IPI00018206 GOT2 glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2) IPI00010706 GSS glutathione synthetase IPI00217560 HIRA HIR histone cell cycle regulation defective homolog A (S. cerevisiae) IPI00472073 HLA-B major histocompatibility complex, class

I, B

IPI00022488 RPX hemopexin IPI00217987 ITGAM integrin, alpha M (complement component 3 receptor 3 subunit) IPI00215894 KNG1 kininogen 1 IPI00554788 KRT18 keratin 18

IPI00217966 LDHA lactate dehydrogenase A

IPI00470355 LHX8 LIM homeobox 8 IPI00887739 LOC100133511 complement C3-like 47 IPI00019380 NCBP1 nuclear cap binding protein subunit 1, 80kDa IPI00604590 NME1-NME2 NME1-NME2 readthrough IPI00299155 PSMA4 proteasome (prosome, macropain) subunit, alpha type, 4 IPI00300117 SCN7A sodium channel, voltage-gated, type VII, alpha

IPI00292946 SERPINA7 serpin peptidase inhibitor, clade A

(alpha-1 antiproteinase, antitrypsin), member 7 IPI00291866 SERPING1 serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 IPI00024467 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 IPI00298971 VTN vitronectin IPI00478003 A2M alpha-2-macroglobulin IPI00022391 APCS amyloid P component, serum IPI00337741 APEH N-acylaminoacyl-peptide hydrolase IPI00552983 ARMCX4 armadillo repeat containing, X-linked 4 IPI00783987 C3 complement component 3 IPI00032291 C5 complement component 5 IPI00022395 C9 complement component 9

IPI00027466 CA4 carbonic anhydrase IV

IPI00465436 CAT catalase IPI00176125 COL6A5 collagen, type VI, alpha 5 IPI00017601 CP ceruloplasmin (ferroxidase) IPI00005721 DEFAI(includesothers) defensin, alpha 1 IPI00294653 DOPEY2 dopey family member 2 IPI00020999 ENPP3 ectonucleotide pyrophosphatase/phosphodiesterase 3 IPI00022418 FN1 fibronectin 1 IPI00375676 FTL ferritin, light polypeptide IPI00385751 FUCA1 fucosidase, alpha-L-1, tissue IPI00071703 GFM2 G elongation factor, mitochondrial 2 IPI00023728 GGH gamma-glutamyl hydrolase (conjugase, 48 folylpolygammaglutamyl hydrolase) IPI00027497 GPI glucose-6-phosphate isomerase IPI00410714 HBA1/HBA2 hemoglobin, alpha 1 IPI00654755 HBB hemoglobin, beta IPI00012585 HEXB hexosaminidase B (beta polypeptide) IPI00045512 HMCN1 hemicentin 1 IPI00022417 LRG1 leucine-rich alpha-2-glycoprotein 1 IPI00298860 LTF lactotransferrin IPI00106687 LXN latex in IPI00012989 MAN2B1 mannosidase, alpha, class 2B, member IPI00007244 MPO myeloperoxidase IPI00024934 MUT methylmalonyl CoA mutase IPI00026962 PLA2G2A phospholipase A2, group IIA (platelets, synovial fluid) IPI00027350 PRDX2 peroxiredoxin 2 IPI00027409 PRTN3 proteinase 3 IPI00022420 RBP4 retinol binding protein 4, plasma IPI00027463 S100A6 S100 calcium binding protein A6 IPI00219806 S100A7 S100 calcium binding protein A7 IPI00007047 S100A8 S100 calcium binding protein A8 IPI00027462 S100A9 S100 calcium binding protein A9

IPI00010304 SERPINB10 serpin peptidase inhibitor, clade B

(ovalbumin), member 10

IPI00029863 SERPINF2 serpin peptidase inhibitor, clade F

(alpha-2 antiplasmin, pigment epithelium derived factor), member 2 IPI00031036 SLC26A3 solute carrier family 26, member 3 IPI00022463 TF transferrin IPI00018363 TIAM2 T-cell lymphoma invasion and metastasis 2 IPI00643920 TKT transketolase IPI00003515 TRIP11 thyroid hormone receptor interactor 11 IPI00027107 TUFM Tu translation elongation factor, mitochondrial 49 IPI00181135 BCAT2 branched chain amino-acid transaminase 2, mitochondrial IPI00024701 COL4A3BP collagen, type IV, alpha 3 (Goodpasture antigen) binding protein

IPI00032134 SERPINB8 serpin peptidase inhibitor, clade B

(ovalbumin), member 8 IPI00333141 SPNS3 spinster homolog 3 (Drosophila) IPI00937735 ACADVL acyl-CoA dehydrogenase, very long chain IPI00009268 ACY1 Unmapped by Ingenuity IPI00166331 ANKRD35 ankyrin repeat domain 35 IPI00299635 BIRC6 baculoviral IAP repeat-containing 6 IPI00219575 BLMH bleomycin hydrolase IPI00026240 BST1 bone marrow stromal cell antigen 1 IPI00929313 C13orf40 chromosome 13 open reading frame 40 IPI00027983 CDA cytidine deaminase IPI00852865 CHIT1 chitinase 1 (chitotriosidase)

IPI00022810 CTSC cathepsin C

IPI00299150 CTSS cathepsin S

IPI00006024 DOCK4 dedicator of cytokinesis 4 IPI00016862 GSR glutathione reductase

IPI00941901 HERC1 hect (homologous to the E6-AP

(UBE3A) carboxyl terminus) domain and RCC1 (CHCI)-like domain (RLD) 1 IPI00005826 HERC2 hect domain and RLD 2 IPI00746667 HLA-DRB5 major histocompatibility complex, class II, DR beta 5 IPI00027223 IDH1 isocitrate dehydrogenase 1 (NADP+), soluble IPI00305461 ITIH2 inter-alpha (globulin) inhibitor H2 IPI00748360 KIAA1797 KIAA1797 IPI00019038 LYZ lysozyme IPI00303335 NËB nebulin IPI00065454 NEK10 NIMA (never in mitosis gene a)- related kinase 10 50

IPI00257882 PEPD peptidase D

IPI00003590 QSOX1 quiescin Q6 sulfhydryl oxidase 1 IPI00939604 RNASET2 ribonuclease T2

IPI00553177 SERPINA1 serpin peptidase inhibitor, clade A

(alpha-1 antiproteinase, antitrypsin), member 1

IPI00550991 SERPINA3 serpin peptidase inhibitor, clade A

(alpha-1 antiproteinase, antitrypsin), member 3

IPI00022204 SERPINB3 serpin peptidase inhibitor, clade B

(ovalbumin), member 3 IPI00307733 SETD2 SET domain containing 2 IPI00218013 SGOL2 shugoshin-like 2 IPI00010949 SIAE sialic acid acetylesterase IPI00386444 SYNE1 spectrin repeat containing, nuclear envelope 1 IPI00744692 TALD01 transaldolase 1 IPI00375371 TAS2R42 taste receptor, type 2, member 42 IPI00465028 TPI1 triosephosphate isomerase 1 IPI00291175 VCL vinculin IPI00029647 ZG16 zymogen granule protein 16

Table 3 - A subset of biomarkers from Table 1 which are differentially expressed in FIT-negative CRC samples relative to a control sample

Accession Gene Symbol Entrez Gene Name Number IPI00892604 C4A/C4B complement component 4B (Chido blood group) IPI00018206 GOT2 glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2) IPI00007797 FABP5 fatty acid binding protein 5 (psoriasis-associated) IPI00296215 EPCAM epithelial cell adhesion molecule IPI00604590 NME1-NME2 NME1-NME2 readthrough IPI00022314 SOD2 superoxide dismutase 2, mitochondrial 51 IPI00010706 GSS glutathione synthetase IPI00418169 ANXA2 annexin A2 IPI00220362 HSPE1 heat shock 10kDa protein 1 (chaperonin 10)

IPI00220766 GL01 glyoxalase I

IPI00032313 S100A4 S100 calcium binding protein A4 IPI00013895 S100A11 S100 calcium binding protein A11 IPI00018768 TSN translin IPI00299155 PSMA4 proteasome (prosome, macropain) subunit, alpha type, 4 IPI00029623 PSMA6 proteasome (prosome, macropain) subunit, alpha type, 6 IPI00010271 RAC1 ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rad) IPI00012007 AHCY adenosylhomocysteinase

IPI00017526 S100P S100 calcium binding protein P

IPI00028006 PSMB2 proteasome (prosome, macropain) subunit, beta type, 2 IPI00793375 XPNPEP1 X-prolyl aminopeptidase (aminopeptidase P) 1, soluble IPI00554788 KRT18 keratin 18 IPI00019380 NCBP1 nuclear cap binding protein subunit 1,80kDa IPI00022774 VCP valosin-containing protein IPI00300086 QPRT quinolinate phosphoribosyltransferase

IPI00472073 HLA-B major histocompatibility complex, class I, B

IPI00453476 PGAM1 phosphoglycerate mutase 1 (brain) IPI00827847 BPI bactericidal/permeability-increasing protein IPI00470355 LHX8 LIM homeobox 8 IPI00942117 CRISP3 cysteine-rich secretory protein 3 IPI00022246 AZU1 azurocidin 1 IPI00300376 TGM3 transglutaminase 3 (E polypeptide, protein-

glutamine-gamma-glutamyltransferase) IPI00028064 CTSG cathepsin G

IPI00006988 RETN resistin IPI00217987 ITGAM integrin, alpha M (complement component 3 receptor 3 subunit) 52 IPI00165045 CACNA1E calcium channel, voltage-dependent, R type, alpha 1E subunit IPI00217560 HIRA HIR histone cell cycle regulation defective homolog A (S. cerevisiae) IPI00004401 ITGB1BP3 integrin beta 1 binding protein 3 IPI00300117 SCN7A sodium channel, voltage-gated, type VII, alpha IPI00061114 RAB3C RAB3C, member RAS oncogene family IPI00168442 C9orf79 chromosome 9 open reading frame 79 IPI00385215 NFATC4 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 IPI00024467 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 IPI00297056 CRNN cornulin IPI00914663 KCP kielin/chordin-like protein IPI00418592 CD1E CD1e molecule IPI00642206 CCDC18 coiled-coil domain containing 18 IPI00514090 LTA4H leukotriene A4 hydrolase IPI00555812 GC group-specific component (vitamin D binding protein) IPI00219713 FGG fibrinogen gamma chain IPI00291866 SERPING1 serpin peptidase inhibitor, clade G (C1 inhibitor), member 1

IPI00217966 LDHA lactate dehydrogenase A

IPI00298971 VTN vitronectin IPI00215894 KNG1 kininogen 1 IPI00216691 PFN1 profilin 1 IPI00292946 SERPINA7 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 7 IPI00022426 AMBP alpha-1 -microglobulin/bikunin precursor

IPI00021405 LMNA lamin A/C

IPI00021727 C4BPA complement component 4 binding protein, alpha IPI00010779 TPM4 tropomyosin 4 IPI00302592 FLNA filamin A, alpha IPI00022488 HPX hemopexin IPI00473011 HBD hemoglobin, delta IPI00298497 FGB fibrinogen beta chain 53 IPI00022895 A1BG alpha-1-B glycoprotein IPI00887739 complement C3-like LOC100133511 IPI00218192 ITIH4 inter-alpha (globulin) inhibitor H4 (plasma

Kallikrein-sensitive glycoprotein) IPI00011252 C8A complement component 8, alpha polypeptide IPI00292530 iTÏHÏ inter-alpha (globulin) inhibitor H1 IPI00016255 PLBD1 hypothetical protein LOC79887 IPI00477505 ANKRD28 Isoform 1 of Serine/threonine-protein phosphatase

6 regulatory ankyrin repeat subunit A IPI00032293 CST3 Cystatin-C

IPI00293867 DDT D-dopachrome decarboxylase IPI00059242 SYAP1 Synapse-associated protein 1 IPI00219622 PSMA2 Proteasome subunit alpha type-2 IPI00221222 SUB1 SUB1 homolog (S. cerevisiae) IPI00022791 MFAP3 Microfibril-associated glycoprotein 3

IPI00011229 CTSD Cathepsin D

IPI00025019 PSMB1 proteasome (prosome, macropain) subunit, beta type, 1 IPI00479306 PSMB5 proteasome (prosome, macropain) subunit, beta type, 5

IPI00234793 KCTD15 cDNA FLJ61112, highly similar to BTB/POZ

domain-containing protein KCTD15 IPI00010796 P4HB prolyl 4-hydroxylase, beta polypeptide IPI00927606 GPX1 glutathione peroxidase 1 IPI00783625 SERPINB5 serpin peptidase inhibitor, clade B (ovalbumin), member 5 IPI00024701 COL4A3BP collagen, type IV, alpha 3 (Goodpasture antigen) binding protein IPI00000811 PSMB6 proteasome (prosome, macropain) subunit, beta type, 6 IPI00021298 KRT20 Keratin 20 IPI00025084 CAPNS1 Calpain small subunit 1 IPI00024919 PRDX3 peroxiredoxin 3 IPI00059930 NACC2 NACC family member 2, BEN and BTB (POZ) 54 domain containing IPI00003817 ARHGDIB Rho GDP-dissociation inhibitor 2 IPI00293276 MIF Macrophage migration inhibitory factor IPI00514622 RANBP6 Ran-binding protein 6 IPI00333141 SPNS3 Isoform 1 of Protein spinster homolog 3 IPI00922181 MCM2 minichromosome maintenance complex component 2 IPI00031708 FAH Fumarylacetoacetase IPI00003865 HSPA8 heat shock 70kDa protein 8 IPI00299024 BASP1 brain abundant, membrane attached signal protein IPI00181135 BCAT2 Branched-chain-amino-acid aminotransferase IPI00219365 MSN Moesin IPI00032134 SERPINB8 Serpin B8 IPI00853547 G6PD glucose-6-phosphate dehydrogenase IPI00478758 C10orf119 Isoform 1 of UPF0557 protein C10orf119 IPI00873020 PSAP Prosaposin IPI00000875 EEF1G eukaryotic translation elongation factor 1 gamma IPI00014398 FHL1 four and a half LIM domains 1 IPI00301395 CPVL carboxypeptidase, vitellogenic-like IPI00790262 TTLL3 tubulin tyrosine ligase-like family, member 3 IPI00942608 26kDaprotein IPI:IPI00942608.1|ENSEMBL:ENSP0000 IPI00023038 PRB1/PRB2 proline-rich protein BstNI subfamily 2 IPI00001895 PCDH8 Protocadherin-8 IPI00419215 A2ML1 Alpha-2-macroglobulin-like protein 1 IPI00644409 GDA Guanine deaminase IPI00009650 LCN1 Lipocalin-1 IPI00217467 HIST1H1E Histone H1.4 IPI00937064 ZAN IPI:IPI00937064.1|REFSEQ:XP_002342720 IPI00396378 HNRNPA2B1 heterogeneous nuclear ribonucleoprotein A2/B1 IPI00465261 ERAP2 endoplasmic reticulum aminopeptidase 2 IPI00021263 YWHAZ 14-3-3 protein zeta/delta IPI00011241 GPR39 G-protein coupled receptor 39 IPI00786880 KIAA1783 similar to KIAA1783 protein IPI00168479 APOA1BP apolipoprotein A-l binding protein 55 IPI00031084 PSD2 pleckstrin and Sec7 domain containing 2 IPI00001593 PRCP prolylcarboxypeptidase (angiotensinase C) IPI00218343 TUBA1C Tubulin alpha-1 C chain IPI00021536 CALML5 Calmodulin-like protein 5 IPI00028091 ACTR3 ARP3 actin-related protein 3 homolog (yeast) IPI00796366 MYL6 myosin, light chain 6, alkali, smooth muscle and non-muscle IPI00301058 VASP Vasodilator-stimulated phosphoprotein IPI00470573 ACTR2 ARP2 actin-related protein 2 homolog (yeast) IPI00884078 RF-IP18 Rheumatoid factor RF-IP18 IPI00169383 PGK1 Phosphoglycerate kinase 1 IPI00299619 SLC35F1 Solute carrier family 35 member F1 IPI00419916 ALPL alkaline phosphatase, liver/bone/kidney IPI00218319 TPM3 tropomyosin 3 IPI00005118 HK3 Hexokinase-3 IPI00418471 VIM Vimentin IPI00218918 ANXA1 Annexin A1 IPI00930073 KRT6C IPUPI00930073.1 |TREMBL:B2R853

IPI00299145 KRT6C Keratin, type II cytoskeletal 6C

IPI00007858 MYH13 myosin, heavy chain 13, skeletal muscle IPI00297235 CCPG1 cell cycle progression 1 IPI00927726 H-INV Hypothetical protein IPI00009008 CACNA1D calcium channel, voltage-dependent, L type, alpha 1D subunit IPI00304554 LYPD5 LY6/PLAUR domain containing 5 IPI00815807 ADCK2 aarF domain containing kinase 2 IPI00743335 MY01C Myosin-lc IPI00007393 APPBP2 amyloid beta precursor protein (cytoplasmic tail) binding protein 2 IPI00295976 ITGA2B integrin, alpha 2b (platelet glycoprotein lib of llb/llla complex, antigen CD41) IPI00641706 TUBB6 tubulin, beta 6 IPI00060201 SYTL4 synaptotagmin-like 4 IPI00908634 AQP4 aquaporin 4 IPI00016786 CDC42 cell division cycle 42 (GTP binding protein, 25kDa) 56 IPI00033494 MYL12B myosin, light chain 12B, regulatory IPI00409756 LOC100293553 protein L-Myc-2-like IPI00015148 RAP1B RAP1B, member of RAS oncogene family IPI00027502 GP9 glycoprotein IX (platelet) IPI00473014 DSTN Destrin

IPI00022394 C1QC complement component 1, q subcomponent, C

chain IPI00290337 EPS8 epidermal growth factor receptor pathway substrate 8 IPI00018671 DUSP3 dual specificity phosphatase 3

IPI00478231 RHOA ras homolog gene family, member A

IPI00220278 MYL9 myosin, light chain 9, regulatory IPI00419585 PPIA peptidylprolyl isomerase A (cyclophilin A) IPI00012011 CFL1 Cofilin-1

Table 4 - A subset of biomarkers from Table 1 which were found to be expressed on y in CRC samples and not in control samples 57

Example

Materials & Methods

Stool samples and protein extraction

Written informed consent was obtained from all subjects who provided stool samples 5 and this study was approved by the Medical Ethical Committee of the VU University Medical Center, The Netherlands. Partial stool samples were collected from referral subjects who underwent colonoscopy between November 2003 and June 2006 at the VU University Medical Center in Amsterdam, The Netherlands. Partial stool samples were collected from before colonoscopy or following diagnosis at colonoscopy and prior 10 to surgical resection of their tumors (see Table 5 for patient characteristics).

Patient number8 Age Gender Category Tumor Size (mm) Stageb Tumor location Positive FITC

_1__74__f__CRC__40___coecum__+_ _2__85__m__CRC__35___right sided__;_ _3__86__m__CRC__30___descendens/sigmoid__:_ 4__82__f CRC__59___right sided__- _5__60__m__CRC__15___rectosigmoid__+_ _6__85__m__CRC__60___right sided__+_ _7__83__m__CRC__70___flexura hepatica__:_ _8__71__f__CRC__35___sigmoid__+_ _9__63__f__CRC__50___rectosigmoid__+_ _10__59__m__CRC__52___coecum__+_ _11__84__f__CRC__50___ileocoecaal__+_ _12__86__m__CRC__68___ascendens__+_ _13__69__m control__na__na__na__;_ _14__75__m__control__na__na__na__:_ _15__68__f__control__na__na__na__-_ _16__73__f control__na__na__na__:_ _17__75__f__control__na__na__na__:_ _18__67__f__control__na__na__na__:_ _19__62__m__control__na__na__na__-_ _20__60__f__control__na__na__na__:_ _21__60__m__control__na__na__na__:_ 22 58 m control na na na I -

Table 5 Clinicopathological characteristics of patients a

This column represents an anonymization number code b

As per the Modified Astler Coller classification (Compton and Greene, CA Cancer J Clin 2004) 15 c cut-off > 75 ng/ul (van Veen, Ned tijdschriftGeneeskd, 2009)

At collection, stool samples were immediately stored at 4°C and transferred to -20°C within 36 hours. Stool samples were thawed and after performing FIT (fecal immunochemical test; FIT), ~1 g stool was sampled from each stool sample for protein extraction. For this study stool samples from 10 subjects without colon neoplasia and 4 20 stool samples from CRC patients with a negative FIT score were selected next to 8 stool samples from CRC patients with a positive FIT score.

Stool proteins were extracted as described before (Ang CS, Nice EC. Targeted in-gel MRM: a hypothesis driven approach for colorectal cancer biomarker discovery in human feces. J Proteome Res 2010; 9: 4346-55) with few adaptations. In short, 58 samples were homogenized in a two-fold excess volume of PBS by vortexing and centrifuged at 4°C for 15 minutes at 16.000 G. The supernatants were centrifuged once more at 4°C for 10 minutes at full speed. Following the last spin cycle the supernatants were cleaned from remaining particles by filtering through a 0.22 pM PVDF filter 5 (Millipore, Billerica, MA, USA). Finally, the samples were concentrated to approximately 200 pi using a 3 kDa cut-off filter (Amicon Ultra, Millipore Corporation, Billerica, MA, USA).

1D-SDS gel electrophoresis and sample processing for proteomics analysis

Equal protein amounts (~30 pg) were loaded and separated on precast 4-12% gradient 10 gels (Invitrogen, Carlsbad, USA), fixed in 50% ethanol containing 3% phosphoric acid washed and stained overnight with Coomassie R-250. After staining the gels were washed in MilliQ water and stored at 4°C until processing for in-gel digestion. Each lane was cut in 10 equal individual bands and each band was further processed into tryptic peptides as described before (Albrethsen J, etal. Mol Cell Proteomics 2010; 9: 15 988-1005; Piersma SR, et al. J Proteome Res 2010; 9: 1913-22).The peptides were extracted and the volume of the desalted peptide fractions was reduced to 50 pi in a vacuum centrifuge.

nanoLC-MS/MS proteomics analysis

Peptides were separated by an Ultimate 3000 nanoLC system (Dionex LC-Packings, 20 Amsterdam, The Netherlands) equipped with a 20 cm x 75 pm ID fused silica column custom packed with 3 pm 120 A ReproSil Pur C18 aqua (Dr Maisch GMBH, Ammerbuch-Entringen, Germany) as described previously (Piersma SR, et al. J Proteome Res 2010; 9: 1913-22). Peptides were trapped on a 5 mm x 300 pm ID Pepmap C18 cartridge (Dionex LC-Packings, Amsterdam, The Netherlands) and 25 separated at 300 nl/min in a 60 min gradient. Intact peptide mass spectra and fragmentation spectra (top 5) were acquired on a LTQ-FT hybrid mass spectrometer (Thermo Fisher, Bremen, Germany). Dynamic exclusion was applied with a repeat count of 1 and an exclusion time of 30 seconds.

Database searching and Statistical analysis 30 MS/MS spectra were searched against the human IPI database 3.62 (83.947 entries) using Sequest (version 27, rev 12). Scaffold version 3.00 (Proteomesoftware, Portland, OR) was used to organize the data and to validate peptide identifications using the PeptideProphet (probability >95%) and ProteinProphet (probability of >99% with 2 peptides or more in one sample) (Keller A, et al. Anal Chem 2002; 74: 5383-92; 35 Nesvizhskii Al, et al. Anal Chem 2003; 75: 4646-58). The software programs SecretomeP and SignalP were used for prediction of non-classical secretion and 59 presence of a signal peptide (Bendtsen JD, et al. Protein Eng Des Sel 2004; 17: 349-56; Bendtsen JD, et al. J Mol Biol 2004; 340: 783-95).

For each protein the number of assigned MS/MS spectra was summed across the 10 fractions and exported to Excel. Normalized counts were calculated by dividing the 5 counts per protein by the sum of all counts per sample and multiciplication by the average sum across all samples. Differential analysis of proteins present in stool samples from CRC patients versus stool samples from healthy controls was performed using the beta-binomial test. The beta-binomial test takes into account the within-sample variation and the between-sample variation in a single statistical model (Pham 10 TV, et al. Bioinformatics 2010; 26: 363-9).

Fecal Immunochemical Test analysis FIT samples (OC-sensor®, Eiken Chemical Co., Tokyo, Japan) were processed with the OC sensor MICRO desktop analyser (Eiken Chemical) and analyzed according to the manufacturer’s instructions. A cut-off of 75 ng/ml was used to determine a positive 15 test result (van Veen W, Mali WP. [Colorectal cancer screening: advice from the Health Council of the Netherlands]. Ned Tijdschr Geneeskd 2009; 153:A1441).

60

Results

Human protein identification in stool samples

In total 830 human proteins were identified in at least one of the 22 stool samples. Of these, 624 proteins were identified both in CRC and control stool samples, 164 proteins 5 were detected only in CRC stool samples, and 42 proteins were detected only in control stool samples (see Figure 1A). On average the number of identified proteins was consistent across all samples and did not differ significantly between CRC and control samples (326 and 296 proteins, respectively).

The primary annotation of subcellular localization of the proteins was mainly the 10 cytoplasm (35%) and the extracellular space (16%) and did not differ between CRC and control samples (see Figure 1B). When the protein sequences were examined for secretion signals with the software tools signalP indicating signal peptides and secretomeP indicating non-classical secretion, 38% of proteins were predicted to have a signal peptide and the majority of proteins 64% were predicted to be a secreted 15 protein. These data indicate that a high percentage of human proteins in stool samples consists of secreted proteins, e.g. in comparison to only 13% of proteins with a signal peptide in a cell line lysate (Piersma SR, et al. J Proteome Res 2010; 9:1913-22). Verification ofLC-MS/MS protein quantification

The fecal immunochemical test (FIT) is used in many countries as a non-invasive test 20 for the early detection of CRC, and is based on the detection of hemoglobin. To verify the results obtained by LC-MS/MS, hemoglobin spectral counts were compared with FIT values (ng/ml) determined in the same stool samples. The adult hemoglobin protein is a heterodimer consisting of two a and two |3 chains (Schechter AN. Blood 2008; 112: 3927-38). As can be seen in Figures 2A and 2B, LC-MS/MS data on both 25 the a and p chain of hemoglobin significantly correlated to FIT values (both p = 0.04). In addition, a very strong correlation was observed between the spectral counts of the a and p chain (see Figure 2C; Pearson correlation of 0.98, p = 1.1*10'14).

Another protein which has been frequently detected in stool and associated to CRC is 30 Calprotectin (Bosch LJ, et al. Molecular tests for colorectal cancer screening. Clin Colorectal Cancer 2011; 10:8-23). Calprotectin is a heterodimer as well, consisting of S100A8 and S100A9 subunits (Yui S, etal. Biol Pharm Bull 2003; 26: 753-60). A strong correlation was observed between spectral counts of S100A8 and S100A9 (see Figure 2D; Pearson correlation of 0.92, p =1.2*10"9), further validating the LC-MS/MS data 35 obtained from stool.

61

These results on hemoglobin and calprotectin show that LC-MS/MS on stool samples provides us with robust quantification of proteins, and is a valid approach for protein marker discovery.

Origin of human stool proteins 5 The mechanisms of how tumor-derived biomarkers end up in stool can be broadly divided in leaked markers, secreted markers and exfoliated markers (Osborn NK, Ahlquist DA. Gastroenterology 2005; 128: 192-206). Hemoglobin is a typical example of a leaked marker from disturbed blood vessels in a neoplastic lesion. Secreted and exfoliated markers can be derived from the epithelial cells lining the colorectal lumen, 10 but can also originate from cells in the surrounding stroma, such as immune cells. An overlap analysis with a previously obtained dataset of plasma proteins (unpublished data) together with the public available Human Proteome Organisation (HUPO), Human Plasma Proteome Project (HPPP) database fwww.hupo.org/research/hppp: Omenn GS, etal. Proteomics 2005; 5: 3226-45; States DJ, et al. Nat Biotechnol 2006; 15 24: 333-8) (high confidence list) revealed that 21.6 % of the 830 identified proteins possibly originate from blood. To estimate how many of the 830 identified human proteins originate from epithelial cells, an overlap analysis was performed with proteins detected in CRC cell lines (unpublished data). Almost half of these proteins were also identified in the CRC cell lines suggesting an epithelial origin.

20 Stool proteins that discriminate CRC patients from control subjects

Unsupervised hierarchical cluster analysis of human proteins identified in all CRC and control stool samples revealed two clusters. Cluster one grouped nine CRC stool samples together, and cluster two grouped all ten control stool samples and three CRC stool samples together (data not shown). Stool samples from CRC patients thus show 25 a specific protein expression pattern as compared to stool from control subjects. This protein expression pattern can therefore be applied to discriminate most of the CRC stool samples from control stool samples without further selection of specific proteins. The complete stool protein dataset was analyzed with beta binomial statistics to identify potential cancer-associated proteins (Pham TV, et al. Bioinformatics 2010; 26: 363-9). 30 From the 830 human stool proteins, 221 proteins were differentially detected, of which the levels of 134 proteins were significantly higher in CRC compared to control stool samples (p< 0.05; Table 2), while 87 proteins were significantly lower in controls compared to CRC stool samples.

Cluster analysis based on the 221 differentially detected proteins in CRC and control 35 stool samples revealed two clusters as well (data not shown). Again, the nine CRC stool samples clustered together in one cluster, and the same three CRC stool samples 62 mentioned above clustered together with the control stool samples in the second cluster.

Within the second cluster, the three CRC stool samples grouped together with one control sample. The protein expression pattern of these three CRC stool samples 5 contained aspects of both the control stool samples and the other nine CRC stool samples, indicating that a selection of these proteins would be able to discriminate these CRC samples from controls.

In addition, three out of four FIT negative CRC patients clustered together with FIT positive CRC patients. Therefore this group of proteins contains potential candidate 10 biomarkers that complement FIT.

Proteins that complement FIT for detecting CRC

Novel biomarkers for early detection of CRC should perform significantly better than FIT, or should complement FIT, in order to increase diagnostic accuracy. As expected from their correlation with FIT, hemoglobin a and p were both significantly more 15 abundantly present in CRC compared to control stool samples. The data revealed several proteins of which levels were significantly higher in CRC stool samples compared to control stool samples with a higher discriminative power than hemoglobin. It is of interest to see if these proteins detect the same CRC samples as hemoglobin does, or if they detect other CRC samples. Figure 3 shows an example of one of these 20 proteins (C4A/C4B) that detects all FIT-negative CRC patients, without detecting control subject, thereby reaching a 100% sensitivity and specificity in this setting. However, not only proteins with a higher discriminative power than hemoglobin have potential to complement FIT.

For this reason, proteins that showed significantly higher levels in the FIT negative 25 CRC stool samples compared to all controls were investigated. Indeed, out of the 134 proteins with significantly higher levels in CRC versus control stool samples, around 90% also showed significantly higher levels in the FIT negative CRC stool samples. This indicates that these candidates have high potential to be of added value to the current detection of hemoglobin.

30 The present study has delivered in-depth proteomic analysis on human stool samples, providing a list of 134 human proteins that were significantly enriched in stool samples from CRC patients, of which several showed highly significant discriminative power. Thus, discriminative markers for improving current FIT tests have been identified.

Industrial Applicability 63

The present invention thus provides a set of biomarkers for screening for colorectal cancer, or susceptibility to colorectal cancer. The biomarkers enable early diagnosis such that early stage surgical removal of a tumor before metastasis is possible.

The foregoing broadly describes the present invention without limitation to particular 5 embodiments. Variations and modifications as will be readily apparent to those skilled in the art are intended to be within the scope of the invention as defined by the following claims.

Claims (52)

A method for screening for colorectal cancer, the method comprising: screening a biological sample taken from an individual for one or more biomarkers selected from the group defined in Table 1, the presence of which of, whether the increased expression of the one or more biomarkers relative to a reference sample is indicative of the individual's risk of developing colorectal cancer or is already suffering from it. 10 The method of claim 1, wherein the presence of the one or more biomarkers is indicative of the fact that the individual is at risk of, or already suffers from, colorectal cancer. The method of claim 1, wherein the increased expression of the one or more biomarkers relative to a reference sample is indicative of the fact that the individual is at risk of, or already suffers from, colorectal cancer. 4. A method according to any one of the preceding claims, wherein the one or more biomarkers is or are selected from the group defined in Table 2. The method of any one of claims 1-3, wherein the one or more biomarkers is or are selected from the group defined in Table 3. A method according to any one of the preceding claims, wherein the one or more biomarkers is or are selected from the group consisting of: S100 calcium binding protein A8 (S100A8), complement component C4B (Chido blood group) 2 (C4A / C4B), transferrin ( TF), alpha-2 macroglobulin (A2M), S100 calcium binding protein A9 (S100A9), proteinase 3 (PRTN3), Azurocidine (AZU1), lactotransferrin (LTF), hemopexine (HPX), and defensin, alpha 1 (DEFA1) . The method of claim 1 or claim 2, wherein the one or more biomarkers is or are selected from the group defined in Table 4. 8. A method according to any one of the preceding claims, wherein the biological sample comprises a stool sample. The method of any one of the preceding claims, wherein the stool sample is screened for the one or more biomarkers by using targeted mass spectrometry. 10 The method of any one of claims 1-8, wherein the stool sample is screened on the one or more biomarkers by using a binding agent capable of binding to the one or more biomarkers. The method of claim 10, wherein the binding agent is an antibody or a fragment thereof. The method of claim 11, wherein the antibody or fragment thereof is a recombinant antibody or fragment thereof. 20 The method of claim 12, wherein the antibody or fragment thereof is selected from the group consisting of: scFv; Fab; a binding domain of an immunoglobulin molecule. The method of any one of claims 10-13, wherein the screening is performed using an array. The method of claim 14, wherein the array is an array based on beads. The method of claim 14, wherein the array is a surface-based array. The method of any one of the preceding claims, wherein the reference sample comprises a biological sample from an individual known to be free from colorectal cancer and / or from advanced adenomas. The method of any one of the preceding claims, wherein the stool sample is also analyzed using the fecal immunochemical test. 19. Use of one or more biomarkers selected from the group defined in Table 1 as a biomarker for diagnosing or predicting colorectal cancer in an individual. 20. Use according to claim 19, wherein the presence of the one or more biomarkers is indicative of the fact that the individual is at risk of, or already suffers from, colorectal cancer. Use according to claim 19, wherein the increased expression of the one or more biomarkers relative to a reference sample is indicative of the fact that the individual is at risk of, or already suffers from, colorectal cancer. 20 Use according to any of claims 19-21, wherein the one or more biomarkers is or are selected from the group defined in Table 2. 23. Use according to any one of claims 19-21, wherein the one or more biomarkers is or are selected from the group defined in Table 3. Use according to any of claims 19-23, wherein the one or more biomarkers is or are selected from the group consisting of: S100 calcium binding protein A8 (S100A8), complement component C4B (Chido blood group) 2 (C4A / C4B), Transferline (TF), alpha-2 macroglobulin (A2M), SI00 calcium binding protein A9 (S100A9), proteinase 3 (PRTN3), Azurocidin (AZU1), lactotransferrin (LTF), hemopexin (HPX), and defensin, alpha 1 ( DEFA1). SI00 calcium binding protein A9 (S100A9), proteinase 3 (PRTN3), Azurocidine (AZU1), lactotransferrin (LTF), hemopexine (HPX), and defensin, alpha 1 (DEFA1). The use of claim 19 or claim 20, wherein the one or more biomarkers is or are selected from the group defined in Table 4. An array for determining whether an individual is at risk of, or is already suffering from, colorectal cancer, the array comprising one or more binding agents as defined in any one of claims 10-13. An array according to claim 26, wherein the array is for use in a method according to any one of claims 1-18. 28. A method for treating colorectal cancer, the method comprising: screening a biological sample taken from an individual for one or more biomarkers selected or selected from the group defined in Table 1, wherein the presence of, or the increased expression of, one or more biomarkers relative to a reference sample is indicative of the individual's suffering from colorectal cancer. 29. The method of claim 28, wherein the biological sample comprises a stool sample. 30. A method according to claim 28 or claim 29, wherein the anti-cancer therapeutic agent comprises one or more monoclonal antibodies, one or more small molecule inhibitors, or one or more chemotherapeutic agents, or a combination of the foregoing. The method of claim 30, wherein the one or more therapeutic monoclonal antibodies comprise one or more of the following: bevacizumab, cetuximab, or panitumumab, or a combination thereof. 30 The method of claim 30, wherein the one or more small molecule inhibitors comprise one or more of the following: erlotinib, sorafenib, or alisertib, or a combination thereof. The method of claim 30, wherein the one or more chemotherapeutic agents comprise one or more of the following: 5-FU, capecitabine, irinotecan, oxaliplatin, or leucovorin, or a combination thereof. The method of any one of claims 28-33, wherein the presence of the one or more biomarkers is indicative of the fact that the individual is at risk of, or already suffers from, colorectal cancer. 35. A method according to any one of claims 28-33, wherein the increased expression of the one or more biomarkers relative to a reference sample is indicative of the fact that the individual is at risk of, or already suffers from, colorectal cancer. 36. A method according to any one of claims 28-33, wherein the one or more biomarkers is or are selected from the group defined in Table 2. 20 The method of any one of claims 28 to 35, wherein the one or more biomarkers is or are selected from the group defined in Table 3. 38. A method according to any one of claims 28-37, wherein the one or more biomarkers is or are selected from the group consisting of: S100 calcium binding protein A8 (S100A8), complement component C4B (Chido blood group) 2 (C4A / C4B) , transferline (TF), alpha-2 macroglobulin (A2M), SI00 calcium binding protein A9 (S100A9), proteinase 3 (PRTN3), Azurocidin (AZU1), lactotransferrin (LTF), hemopexin (HPX), and defensin, alpha 1 ( DEFA1). 30 The method of any one of claims 28 to 34, wherein the one or more biomarkers is or are selected from the group defined in Table 4. 40. Anti-cancer therapeutic agent, for use in a colorectal cancer treatment method, the method comprising: screening a stool sample taken from an individual, on one or more biomarkers selected or selected from the group which is defined in Table 1, wherein the presence of, or the elevated expression of, one or more biomarkers relative to a reference sample is indicative of the individual's suffering from colorectal cancer; and administering a therapeutically effective amount of the anti-cancer therapeutic agent. The anti-cancer therapeutic agent, for use according to claim 40, wherein the biological sample comprises a stool sample. The anti-cancer therapeutic agent, for use according to claim 40 or claim 41, wherein the anti-cancer therapeutic agent comprises one or more monoclonal antibodies, one or more small molecule inhibitors, or one or more chemotherapeutic agents, or a combination thereof. The anti-cancer therapeutic agent, for use according to claim 42, wherein the one or more therapeutic monoclonal antibodies comprise one or more of the following: bevacizumab, cetuximab, or panitumumab, or a combination thereof. The anti-cancer therapeutic agent, for use according to claim 42, wherein the one or more small molecule inhibitors comprise one or more of the following: erlotinib, sorafenib, or alisertib, or a combination thereof. The anti-cancer therapeutic agent, for use according to claim 42, wherein the one or more chemotherapeutic agents comprise one or more of the following: 5-FU, capecitabine, irinotecan, oxaliplatin, or leucovorin, or a combination thereof. An anti-cancer therapeutic, for use according to any one of claims 40 to 45, wherein the presence of the one or more biomarkers is indicative of the fact that the individual is at risk of, or already suffers from, colorectal cancer. 47. Anti-cancer therapeutic agent, for use according to any one of claims 40-45, wherein the elevated expression of the one or more biomarkers relative to a reference sample is indicative of the risk of the individual getting colorectal cancer, or already suffers from it. 48. Anti-cancer therapeutic agent, for use according to any of claims 40-47, wherein the one or more biomarkers is or are selected from the group defined in Table 2. Anti-cancer therapeutic agent, for use according to any of claims 40-47, wherein the one or more biomarkers is or are selected from the group defined in Table 3. 20 An anti-cancer therapeutic agent, for use according to any of claims 40-47, wherein the one or more biomarkers is or are selected from the group consisting of: S100 calcium-binding protein A8 (S100A8), complement component C4B (Chido blood group) 2 (C4A / C4B), transferrin (TF), alpha-2 macroglobulin (A2M), 51. Anti-cancer therapeutic agent, for use according to any of claims 40-46, wherein the one or more biomarkers is or are selected from the group defined in Table 4. A kit for screening an individual for colorectal cancer, the kit comprising: a) one or more binding agents as defined in any one of claims 9-12, or an array according to claim 24 or claim 25; b) instructions for carrying out the method according to any one of claims 1-17. 5