FR3001805A1 - METHOD FOR DETECTING COLORECTAL CANCER - Google Patents

Abstract

The invention relates to a method for the in vitro diagnosis of colorectal cancer in a patient by determining the presence of the prolidase in a biological sample of the patient.

Description

The present invention relates to the diagnosis of colorectal cancer in a human patient. Colorectal cancer (CRC) is a major public health problem. Its worldwide incidence was estimated at 1,234,216 new cases in 2008, compared with 875,000 in 1996. In France, the number of new cases per year was estimated in 2011 at 40,500. The standardized incidence rate at the global level was in 2011, 36.3 per 100,000 men and 24.7 per 100,000 women. Colorectal cancer is the third most common cancer category (second-highest cancer in women and third-highest in men). The 5-year survival rate, at all stages, is close to 56%. According to estimates, the number of colorectal cancers is expected to increase in the coming years, reaching 45 000 new annual cases in France in 2020. In 2008, colorectal cancer was the most common cancer in a dozen European countries, including Czech Republic, Hungary, Slovakia, Denmark, Norway, Italy, the Netherlands, Germany, Belgium and Slovenia, with a standardized incidence rate to the world population of 35.5 and 43 per 100 000. France is one of the countries in which the risk of colorectal cancer is high, as are the other countries of Western Europe, the USA, Australia and, more recently, Japan. Only an early diagnosis offers the hope of a cure. However, at present, there is no serological screening test or specific diagnosis that is early. In France, as in a number of European countries, the mass screening policy put in place for colorectal cancer is based on the carrying out of the blood test in the stool, by a biennial paraclinical test in the stool (Faecal Occult Blood test, FOBT, marketed for example under the name of Hemoccult®). This technique has demonstrated its clinical utility. When used every 30 years for 30 people aged 50 to 74, it can reduce colorectal cancer mortality by 15 to 20%. For this, it is necessary that more than half of the population concerned regularly participate in screening and that a colonoscopy is done in case of a positive test, possibly followed by appropriate treatment. Nevertheless, this screening technique suffers from a certain number of handicaps: the major disadvantage of this test is its poor sensitivity, especially for adenomas (pre-cancerous dysplastic lesion) which, if they are large, or in severe dysplasia, will lead in 1 out of 10 cases to the development of a cancer. - The appearance of blood in the stool can be linked to a non-tumoral condition: recto-colic hemorrhages, hemorrhoids, fistulas, ... - It is difficult to obtain an important acceptability, because it is a test in the stool. As a result, effective mass screening relies on a highly structured organization with systematic reminders. Finally, the tests for blood detection in stools based on a guaiac test, of the Hemoccult® type, are not specific for human hemoglobin. They are difficult to interpret, and must therefore be read in specialized centers by qualified and competent staff. Immunological tests specific for human hemoglobin (OCSensor, FOB Gold, Magstream, etc.) have also been described. They represent an improvement over the Hemoccult® test, allowing in particular an increase in sensitivity for both cancers and adenomas, but with a decrease in specificity, and consequently an increase in the number of unnecessary colonoscopies. . However, it is still a blood test in the stool with the problems inherent in such tests: detection of non-specific bleeding, variation in bleeding time, and especially risk of poor adhesion of some reluctant patients to perform a test in the stool. In the United States, there is no systematic mass screening policy for colorectal cancer. There are, however, 25 recommendations for screening, by one or more existing methods. In particular, in addition to the blood test in the stool (by an immunological test or guaiac), one can achieve a sigmoidoscopy associated or not with a FOBT test, a colonoscopy, a virtual colonoscopy or a double contrast barium enema. New methods, such as the search for mutations or methylation of DNA in the stool, are still under study. Systematic colonoscopy after 50 years theoretically reduces colorectal cancer mortality. This is the most sensitive and specific examination for the detection of cancers, which also allows the possible excision of precancerous lesions. But the acceptability of this test in healthy subjects is too low for a screening policy using endoscopy to reduce mortality (the acceptability for colonoscopy in European countries having implemented this strategy of screening is around 2%). There is a significant risk (1% 0) of perforation and hemorrhage of the colon and death (1/10000), as well as a high cost for public health. In addition, colonoscopy requires a very demanding pre-colonic preparation, which largely explains the low adhesion. Immunoassay-measurable tumor markers have been described for a long time in colorectal cancer. These include carcinoembryonic antigen (CEA) and CA19-9. ACE is used for monitoring. It can not be used for screening or early diagnosis of colorectal cancer because its sensitivity and specificity are insufficient. Indeed, this marker is expressed by other types of cancers and in benign pathologies, and especially it detects only a small number of colorectal cancers. The Applicant has now surprisingly demonstrated a new colorectal cancer marker which overcomes the aforementioned drawbacks, in particular the lack of specificity and sensitivity, and which is therefore a good candidate both for diagnosis and for cancer screening. colorectal even at very early stages and to a stage yet non-cancerous adenoma. Thus, the present invention relates to a method of diagnosis or screening for colorectal cancer by determining the presence of prolidase in a patient. The method of the invention can also be used for the early diagnosis of colorectal cancer in a patient, or for the detection of carcinomas at the stage in situ (Tis TO) or for the detection of colorectal adenoma. According to a first variant of any of the methods of the invention above, the method is applied to the diagnosis, or early diagnosis, of colorectal cancer, in vitro, or to the detection of colorectal adenoma or d a carcinoma in situ, in vitro, by determining the presence of the prolidase in at least one biological sample of the patient. The above methods can be practiced, but not limited to, patients suspected of cancer, for example, colorectal cancer.

The invention also relates to a method for screening, in vitro, a colorectal cancer in a population by determining the presence of the prolidase in a biological sample of an individual. By determining the presence of the prolidase, it is understood that the prolidase and / or at least one tracer representative of the prolidase is detected. This tracer is characteristic of the presence of the prolidase. Depending on the level of prolidase in the patient or in the biological sample and according to the technique used, the detection of a tracer above may be more relevant than that of the prolidase per se. Thus, a tracer can be chosen from the precursors of the prolidase, the metabolites of the prolidase, or to an immune response due to the prolidase. Thus, a tracer of the prolidase is preferably selected from peptides, preferably mixtures of peptides of the prolidase; nucleic acids, preferably messenger RNAs or a modification of the gene encoding prolidase, such as a methylation of its promoter, or a modification of the nucleic acid leading to a modification of the expression of the prolidase; antibodies specifically recognizing prolidase and produced in the patient in response to a presence of the prolidase, by immunogenic reaction. More particularly, according to a method of diagnosis or early diagnosis, colorectal cancer, or detection of a colorectal adenoma, or detection of carcinoma in situ, of the invention, the prolidase and / or less a representative tracer of the prolidase in said biological sample. A method of the invention may further include demonstrating a variation of prolidase expression. Advantageously, a decrease in the expression of the prolidase is demonstrated. This variation can be observed by comparison with a so-called control reference population, of patients not suspected of CRC, for example patients who have had a colonoscopy which has proved normal, or compared to a predetermined reference value. Preferably, the variation is compared to a reference population, with samples of the same origin, as defined below. The invention also relates to the use of any of the above methods, both in the early diagnosis, screening, therapeutic monitoring, prognosis, as well as in the diagnosis of relapses in the context of colorectal cancer.

By biological sample is meant any sample likely to contain the prolidase and / or at least one tracer representative of the prolidase. This sample may come from a sample of any biological fluid, such as whole blood, serum, plasma, urine, cerebrospinal fluid, organic secretions, saliva, stool for example, or a tissue sample or isolated cells. Preferably, the sample is remote from the potential tumor. The determination of the presence of the prolidase in a biological sample, remote or otherwise from the tumor, then makes it possible to conclude that the desired pathology is present. One of the advantages of the method of the invention therefore lies in the possibility of using a sample remote from the potential tumor as a diagnostic sample, which allows a simple and non-invasive diagnosis whereas a tissue diagnosis requires a biopsy taken invasively. Indeed, the study of tissue markers, for example on a tissue section (immunohistochemistry), may be of interest for prognosis but has no interest for screening or early diagnosis of colorectal cancer. A method of the invention may also be associated with the determination of the presence, in addition to the prolidase, of at least one other marker. Advantageously, this other marker is chosen from the following zo markers: ACE; Aminoacylase 1; Apolipoprotein A1; Apolipoprotein A2; Beta 2 Microglobulin; CA242; CA50; CA72-2; CA19-9; Calgranulin C; calreticulin; CCSA-3 (colon cancer specific antigen); CCSA-4; CD24; CD26; Cripto-1; Cytokeratin 20; Epithelial-Cadherin; ezrin; Galectin-3; GST Pi (GSTP1); HSP60; Intelectin-1; Intestinal Fatty Acid-Binding Protein; Keratin type 1 Cytoskeletal 18; Keratin type 1 Cytoskeletal 19; Keratin type II Cytoskeletal 8; Leukocyte Elastase Inhibitor, associated or not with Proteinase 3 (PRN3); Liver Fatty AcidBinding Protein; L-Lactate Dehydrogenase Chain B (LDHB); maspin; MIF 30; MUC5AC; osteopontin; I-plastin; 20S proteasome; (Pro) defensin-A5; (Pro) defensin-A6; Protein Disulfide Isomerase (PDI); Pyruvate kinase M2-PK; Regenerating Islet-Derived Protein 3 Alpha; If 00A8; If 00A9; testosterone; TIMP-1; Translationally Controlled Tumor Protein; Tumor-Associated Calcium Signal 35 Transducer 1; Tumor-Associated Calcium Signal Transducer 1; villin; vimentin; Heat Shock Cognate 71 kDa (HSP71), BIP (or GRP-78), Peroxiredoxin-5, Glyceraldehyde-3-phosphate dehydrogenase (G3PDH); Alpha-Enolase; Methyl-DNA in the blood, preferably the methylated DNA of the AXL4 gene (Aristaless-like Homeobox-4 Gene Methylation) or the methylated DNA of the Septin-9 gene; specific alterations of DNA fragments in the stool such as specific DNA mutations in the stool or specific alterations of the DNA methylation pattern in the stool; - haptoglobin in the stool; human hemoglobin in the stool. More preferably, the presence, in addition to the prolidase, of the aforementioned marker associations is determined. As will be illustrated in the examples, some associations are advantageous. Thus, the invention also relates to a method according to which the presence of the prolidase and that of at least the following associations of markers: ACE and CA19-9, LFABP and PDI, LFABP, CA19-9 are determined. and ACE, LFABP, ACE and Timp1, LFABP, ACE and M2PK, L-FABP, CA19-9, ACE and PDI LFABP, ACE, M2PK and Timp1, LFABP, ACE, CA19-9 and PDI, LFABP, ACE, M2PK, Timp1, CA19-9 and PDI, LFABP, ACE, M2PK, Timp1, CA19-9, Villine and PDI, LFABP, ACE, M2PK, Timp1, CA19-9, G3PDH and PDI, LFABP, ACE, M2PK, Timp1, CA19-9, Villine and G3PDH, LFABP, ACE, M2PK, Timp1, CA19-9, Villine, G3PDH and PDI. The invention is hereinafter described in more detail. Prolidase, also called Proline dipeptidase, Xaa-pro dipeptidase, Imidodipeptidase, Peptidase D, PEPD (Gene ID: 5184, Uniprot Accession number P12955, EC = 3.4.13.9) is a member of the peptidase family, more particularly the metalloids. -proteinases (matrix metallo protein, MMP). It is a manganese dependent exopeptidase, which forms a homodimer which hydrolyzes imidodipeptides or imidotripeptides which contain in C-terminal a proline or hydroxy-proline residue (X-Pro or X-Hyp, where X represents any amino acid ). Found in plasma, it allows the release of proline as the final stage of protein catabolism, and particularly collagen, the most important source of these imidodipeptides. This enzyme plays an important role in the recycling of proline, and its absence is a limiting factor for collagen production. It plays an important role in remodeling and cell growth. The prolidase gene (PEPD) is located on chromosome 19 (chromosomal location 19q13.11) and encodes a polypeptide of 493 amino acids with a molecular weight of 54 kDa. The mature form of the enzyme is a dimer composed of 2 identical subunits. The gene has many allelic polymorphisms, without any impact on the enzymatic activity. Rare mutations in the gene found on exons 7, 8, 12 and 14 are responsible for prolidase deficiency, which is a characteristic pathology by urinary excretion of a large amount of proline-containing peptides (imidopeptiduria) , cutaneous involvement, recurrent infections, mental retardation and the presence at a high concentration of proline dipeptides in plasma.

In the articles of the literature, the levels of prolidase are obtained by measuring the enzymatic activity of the protein, by measuring the amount of proline released per minute and per milligram of protein [http: //www.cancer. gov / cancertopics / factsheet / Detection / colorectal-screening; Screening for Colorectal Cancer: A Targeted, Updated Systematic Review for the U.S. Preventive Services Task Force. Ann Intem Med. 2008; 149 (9): 638 658]. Thus, the enzymatic activity of prolidase has been evaluated in the serum of patients with endometrial cancer, ovarian cancer or breast cancer, and in all cases, Enzyme activity was greater in these patients than in control subjects. The enzymatic activity of prolidase has been evaluated in these various cancers in the serum (comparison with other pathologies) or in the tissue (comparison of the cancerous tissue with respect to the "healthy" tissue). The expression of prolidase has been evaluated in lung cancer tissues [Kama E, Surazynski A, Palka J., Int J Exp Pathol. 2000 Oct; 81 (5): 341-7. Collagen gen metabolism is accompanied by an increase in prolidase activity in lung carcinoma planoepithelial] and stomach [Guszczyn T, Sobolewski K., Deregulation of collagen gen metabolism in human. stomach cancer. Pathobiology 2004; 71: 308-13] semi-quantitatively, by Western Blot, and was increased. On the other hand, the expression of prolidase has never been measured at a distance from the tumor, as for example in blood or serum, whatever the pathology associated with its expression. In addition, in such a fluid, there is not necessarily a parallel between the enzymatic activity of a protein and its expression. Nor is there necessarily any correlation between the tissue expression of a protein and the measurement of the expression levels of this same protein at a distance from the tumor. Markers whose presence can be determined, in addition to the prolidase, according to variants of the methods of the invention are hereinafter described in more detail. It should be noted that the following markers, namely Alpha-Enolase, G3PDH, BIP (GRP78), HSP71 (HSC70), Peroxy-5 and PDI, constitute as such markers of colorectal cancer and that may, alone or in combination with each other and / or with the prolidase and / or any other marker, be used in a method of diagnosing colorectal cancer in a patient. For this purpose, the presence of at least one of the above markers is determined in said patient. According to a variant of this method, in vitro a colorectal cancer is diagnosed in a patient by determining the presence of at least one of these markers in a biological sample of the patient. Like prolidase, the determination of the presence of these markers, alone or in combination with each other, with the prolidase and / or any other marker, makes it possible to diagnose an invasive colorectal cancer early or to detect a colorectal adenoma, or to determine the presence of carcinoma in situ, TO, in a patient or in a biological sample of a patient, in vitro. The individual scores of each of the aforementioned markers for the detection of CRC are presented in Example 6. The definition of biological sample given previously with reference to prolidase applies to any of the aforementioned markers. The determination of the presence of these markers, alone or in combination, can be carried out by any method well known to those skilled in the art, and in particular those described in the present description with reference to the prolidase.

Thus, as for prolidase, the determination of their presence may result from a direct detection of these markers and / or from a detection of at least one of the tracers representative of these markers, such as those chosen from the precursors of these markers. the metabolites of these markers, the products of degradation or cleavage of these markers, and any molecule associated with at least one activity of these markers, for example an immune response. Thus, a tracer of such a marker is preferably selected from peptides, preferably peptide mixtures such as those described below for each of the markers; nucleic acids, preferably messenger RNAs or a modification of the gene coding for said marker, such as a methylation of its promoter, or a modification of the nucleic acid leading to a modification of the expression of the marker; antibodies specifically recognizing this marker and produced in the patient in response to a presence of this marker, by immunogenic reaction.

According to a variant of the method comprising the determination of the presence of several markers, as described above, the markers, and / or their tracers, can be detected in the same biological sample, in different biological samples but coming from a fluid or of a tissue of the same nature, in different biological samples and of different nature. Among these markers, those described in more detail below are preferred markers: Alpha-Enolase Alpha-Enolase, also known as 2-phospho-D-glycerate hydro-lyase, C-myc promoter-binding protein, Enolase 1 or Non-neural enolase (ENOA HUMAN, Gene ID: EN01, Uniprot accession number P06733, EC = 4.2.1.11) belongs to the family of Enolases. It is a multifunctional enzyme, which plays a role in glycolysis, and is involved in many processes such as growth control, tolerance to hypoxia and allergic responses. It is in particular a metabolic enzyme involved in the synthesis of pyruvate. It stimulates the production of immunoglobulins. It can also serve as a receptor and activator of plasminogen on the surface of several cell types, such as leucocytes and neurons. As a plasminogen receptor, it allows the action of plasmin and the degradation of the extra-cellular matrix. in tumor processes. It promotes tumor invasion and may have diagnostic and prognostic value. The Alpha-Enolase gene encodes a polypeptide of 434 amino acids with a molecular weight of 47 kDa. It is a protein of 493 amino acids. Enolase is in mammals composed of 3 subunits that can form homo or heterodimers. Their association is specific for cell subtypes and stage of development. It is localized in the cytoplasm, with a possible translocation to the plasma membrane. Alpha / alpha homodimers are expressed in most adult and embryonic tissues. Alpha-Enolase has been shown to be inducible for lymphoma, and is up-regulated in response to enterovirus 71 infection. The alpha / gamma heterodimer is a marker of cerebral hypoxia after cardiac arrest. A splice variant of the gene gives a shorter isoform that can bind the c-myc promoter and function as a tumor suppressor. Antibodies to Alpha-Enolase have been found in the sera of cancer patients accompanied by "cancer-associated retinopathy" (CAR, paraneoplastic pathology mainly associated with small cell lung cancer). The EN01 gene has been shown to have a central role as a "knot" of connection between different genes during cancerous processes. Alpha-Enolase appears to have a role in cell transformation and invasion via the induction of CCL20 expression, and its overexpression is related to prognosis in head and neck cancers. Overexpression of Alpha-Enolase has been reported to correlate with tumor progression, particularly of the lung and neuroblastoma, and has been reported in hepatocarcinoma and analyzed in colonic lineages. Tissue expression of Alpha-Enolase has been studied in The Human Protein Atlas (http://www.proteinatlas.org/ENSG00000074800) using antibody CAB018614 (confidence in markings is uncertain according to quality control criteria of the site). The expression of Alpha-Enolase is cytoplasmic, and occasionally nuclear. It is moderate in normal digestive tissues in more than 75% of cases, as well as in tumor colorectal tissues. In contrast, the serum Alpha-Enolase assay has never been described for diagnosis, colorectal cancer monitoring, screening, or prognosis assessment.

The Applicant has now demonstrated that the levels of Alpha-Enolase in the sera of patients, assayed for example by a multiple Reaction Monitoring mass spectrometry technology, are increased compared to those of control subjects. And if we associate the scores obtained for Alpha-Enolase with those obtained for prolidase, we improve the diagnosis of colorectal cancer. G3PDH No. Swiss Prot Protein Name Names of Protein Gene Names P04406 Glyceraldehyde-3-phosphate dehydrogenase Peptidyl-cysteine Snitrosylase GAPDH GAPDH GAPDH GAPD This is a protein of 335 amino acids, of 36 KDalton. GAPDH is a homotetramer that belongs to the glyceraldehyde-3-phosphate dehydrogenase family, and has both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, and plays a role in glycolysis and nuclear functions, respectively. It modulates the cytoskeleton assembly, stimulates the binding of CHP1 to microtubules, and binds itself to microtubules. Furthermore, GAPDH is a key enzyme for glycolysis, which catalyzes the first step by converting D-glyceraldehyde 3-phosphate zo (G3P) to phospho-D-glyceroyl phosphate, with NAD as the substrate. At the cellular level, it is expressed in both the cytoplasm, the nucleus and the membrane, and is related to the cytoskeleton. The GAPDH gene is known to be a normalizing gene in semi-quantitative analyzes in Northern blot or PCR experiments. It is therefore used as an internal standard in many publications concerning the quantification of mRNA type markers, as it is known to have a stable expression level. Unexpectedly, GAPDH expression has been shown to be increased in colorectal cancer tissues relative to adjacent healthy tissues, and similarly in cancerous lines. This high expression may play a role in cancer development and metastasis, and GAPDH may be a therapeutic target. But other authors have studied hypoxia regulation of GAPDH, as GAPDH is used as a standardization gene. It appears that GAPDH is not regulated by hypoxia in hepatocarcinoma, lung cancer or colon cancer lines, and therefore does not seem to be a good therapeutic target. GAPDH has never been shown as a diagnostic marker for colorectal cancer. The Applicant has now shown that the levels of GAPDH in the sera of patients, assayed for example by a multiple Reaction Monitoring mass spectrometry technology, are increased compared to those of control subjects. And if we associate the scores obtained for GAPDH with those obtained for prolidase, we improve the diagnosis of colorectal cancer.

BEEP N ° Swiss Protein name Names synonymous with protein Prot gene names P11021 BiP 78 kDa glucose-regulated protein HSPA5 GRP-78 Heat shock 70 kDa protein 5 GRP78 lmmunoglobulin heavy chain- Endoplasmic lumenal reticulum binding protein Ca (2+) -binding protein It is a protein of 654 amino acids, of 72.3 KDalton; it belongs to the Heat Shock protein 70 family. It has a probable role in facilitating the assembly of multimeric protein complexes in the endoplasmic reticulum, and plays a role as an autoantigen in rheumatoid arthritis. GRP78 is part of a large chaperone protein complex comprising JB11, HSP90B1, HYOU, PDIA2, PDIA4, PDIA6, PPIB, SDF2L1, UGT1A1. As a chaperone protein, it has an anti-apoptotic role, and can bind to calcium. GRP78 promotes cell proliferation, survival, metastasis and resistance to chemotherapy. It could have a role as a therapeutic target. It has recently been suggested that the level of tissue expression of GRP78, as well as the presence of autoantibodies in patient serum, can be used to stratify patients and predict response to treatment [Mintz PJ, Kim J, Do KA, et al. Fingerprinting the circulating directory of antibodies from cancer patients. Nat Biotechnol 2003; 21: 57-63; Gonzalez-Gronow M, Cuchacovich M, Llanos C, Urzua C, Gawdi G, Pizzo SV. Prostate cancer cell proliferation in vitro is modulated by antibodies against glucose-regulated protein 78 isolated from patient serum. Cancer Res 2006; 66: 11424-31]. Anti-GRP78 auto-Ab was found in 3 of 15 colon cancers, 17/60 of gastric cancers, and absent in healthy subjects (0/20). GRP78 was identified in 1997 in a two-dimensional electrophoresis analysis of the LIM 1215 colonic cancer cell line [Ji H, Reid GE, Moritz RL, JS Eds, Burgess AVV, Simpson RJ, Electrophoresis. 1997 Mar-Apr; 18 (3-4): 605-13. A two-dimensional gel database of human colon carcinoma proteins.], Without it being possible to conclude diagnostic properties. It is relatively ubiquitously expressed on the surface of cells. It has also been shown that carcinogens such as nicotine impact the promoter of GRP78, a reflection of the stress induced on the endoplasmic reticulum [Nicotine increases oxidative stress, activates NF-kappaB and GRP78, apoptosis and sensitizes cells to genotoxic / xenobiotic stresses by a multiple stress inducer, deoxycholate: relevance to colon carcinogenesis. Crowley-Weber CL, Dvorakova K, Crowley C, Bernstein H, Bernstein C, Wal H Station, Payne CM. Chem Biol Interact. 2003 Mar 6; 145 (1): 53-66]. GRP78 and HSP90 have been shown to be constitutively expressed in gastrointestinal cancer lines. Ehrenfried JA, BE Herron, Townsend CM Jr, BM Evers. Surg Oncol. 1995 Aug; 4 (4): 197-203], with differential expression in gastric, pancreatic and colic cancers, and may be as having a protective role. Xioming Xing et al. studied the expression of GRP78 at the protein level and the expression of mRNA in colonic tissues. While there does not appear to be a difference in the expression of GRP78 mRNA, the protein is overexpressed during colonic carcinogenesis, but without correlation with differentiation in cancerous tissues [Xing X, Lai M, Wang Y, Xu E, Huang Q. Clin Chim Acta. 2006 Feb; 364 (1-2): 308-15. Epub 2005 Sep 21]; according to the same authors, this overexpression seems associated with the proliferation of cancer cells and their resistance to apoptosis [Xing X, Li Y, Liu H, Wang L, Sun L. Acta Histochem. 2011 Dec; 113 (8): 777-82. doi: 10.1016 / j.acthis.2010.11.006. Epub 2010 Dec 14].

In patent application WO2004 / 005928A2, expression changes of various proteins, including GRP78 for the detection of cancer cells not dependent on steroids, are described. The Applicant has now unexpectedly demonstrated that the levels of GRP78 in the sera of the patients, assayed for example by a multiple Reaction Monitoring mass spectrometry technology, are reduced compared to those of control subjects. And if we associate the scores obtained for GRP78 with those obtained for prolidase, we improve the diagnosis of colorectal cancer.

HSP71 (HSC70) No. Swiss Prot Protein name Protein synonyms Gene name P11142 Heat shock cognate 71 kDa protein Heat shock 70 kDa protein 8 HSPA8 HSC70, H5P73, HSPA10 Heat shock protein cognate 71 kDa (HSC70) belongs to the HSP70 family which comprises a dozen proteins with a molecular weight between 66 and 78kDa. HSP70 are chaperone proteins, ie molecules that, by temporarily binding to a newly formed protein within a living cell, allow it to acquire its characteristic form.

Each HSP70 protein has a common structure that contains two domains: a highly conserved amino terminal end with ATPase activity and a less conserved carboxy terminus that binds peptides. In cooperation with other chaperones, HSP7O5 stabilizes pre-existing proteins against aggregation and alters the refolding of newly translated polypeptides into the cytosol as well as within the organelles. The length of the HSC70 sequence is 646 AA, and it is 70.9 KDalton. HSC70 is rapidly translocated from the cytoplasm to the nucleus, and particularly to the nucleolus, under the effect of the temperature rise.

Recently, overexpression in colorectal cancer of the HSC70 protein has been demonstrated in a study using two-dimensional electrophoresis of micro-dissected tissues, with no indication of a possible diagnostic role. Another study found similar results, with increased expression of HSC70 in colonic tumor tissues, associated with increased expression of HSP90, with no correlation with cancer stage. In contrast, the serum HSP71 (HSC70) assay has never been described for the diagnosis or monitoring of colorectal cancer, or for screening or prognosis assessment. The Applicant has now unexpectedly demonstrated that HSP71 levels in patient sera, assayed, for example, by multiple Reaction Monitoring mass spectrometry technology, are increased relative to those of control subjects. And if we associate the scores obtained for HSP71 with those obtained for prolidase, we improve the diagnosis of colorectal cancer. Peroxy-5 Peroxiredoxin-5 protein name, Imitochondrial Alu corepressor 1 P30044 Antioxidant enzyme B166 Prx-V PRDX5 Peroxiredoxin V Peroxy-5 ACR1 Thioredoxin peroxidase PMP20 Thioredoxin reductase Peroxyredoxin-5 is a 214 amino acid protein of 22 KDaltons. It belongs to the family of peroxiredoxins 2. It is of cytoplasmic or mitochondrial localization. It is involved in intracellular oxidation / reduction (redox) pathways by reducing hydrogen peroxides. It has been described in the mouse as a peroxidase capable of inhibiting p53-induced apoptosis. It has never been described as a diagnostic tool for colorectal cancer. The Applicant has now demonstrated that the levels of Peroxy-5 in the sera of patients, assayed for example by a Multiple Reaction Monitoring mass spectrometry technology, are increased compared to those of control subjects. And that if one associates No. Swiss Prot Names synonymous with the protein Names of the gene the scores obtained for Peroxy-5 with those obtained for prolidase, one improves the diagnosis of colorectal cancer. PDI No. Swiss Prot Protein Name Gene Name P07237 Protein disulfide-EC 5.3.4.1 P4HB isomerase PDI PDI, PDIA1, Prolyl 4-hydroxylase subunit beta Cellular thyroid hormone-binding protein PO4DB p55 PDI is a multi-functional protein which catalyzes the formation, disruption and rearrangement of intramolecular disulfide bridges. At the cell surface it acts as a reductase and cleaves the disulfide bridges of the proteins attached to the cells. Inside the cells, it is a soluble molecule located in the endoplasmic reticulum lumen, where it forms and rearranges the disulfide bonds of neosynthesized proteins. It is a multi-functional protein with 2 catalytic domains of the Thioredoxin type with a characteristic CXXC motif. At high concentration the PDI functions as a chaperone protein that inhibits the aggregation of misfolded proteins. At low concentration, it has an antagonistic role and facilitates aggregation. PDI also forms the structural subunit of various enzymes, such as Prolyl Hydroxylase, which catalyzes the hydroxylation of proline residues of pro-alpha pro-collagen chains.

PDI has been found to be particularly abundant on the surface of cancer cells. Post translational modifications have been observed in the case of colon cancer by T. Tom onaga et al. [T. Tomonaga et al. Clin. Canc. Reas. 2004, 2007-2014]. R. Stierum et al. [R. Stierum et al. Biochimica and Biophysica Acta, 2003, 73-91] showed that the differentiation of the Caco-2 colonic cancer cell line was accompanied by an increase in PDI cell concentration. The Applicant has now shown that the PDI levels in the patient's sera, assayed, for example, by a Multiple Reaction Monitoring mass spectrometry technology, are reduced compared with those of control subjects. And if we associate the scores obtained for PDI with those obtained for prolidase, we improve the diagnosis of colorectal cancer. The various methods that are the subject of the invention and defined above can be implemented by any appropriate technique that is well known to those skilled in the art. Thus, the prolidase can be assayed by a quantitative technique of mass spectrometry, or by any immunological technique, or a combination of both techniques. For example, the LC-MRM-MS technique which combines liquid chromatography and mass spectrometry in MRM (Mutiple Reaction Monitoring) mode can be used. By determination of the presence of the prolidase is meant the direct detection of the prolidase, or the culture of cells or tissue sensitive to the activity of the prolidase, or any other method of determining the presence of a prolidase. protein in a sample, known to those skilled in the art. As stated above, the method for determining the presence of the prolidase which is the subject of the present invention also applies to methods for determining the presence of the markers chosen from Alpha-Enolase, G3PDH, BIP, HSP71, Peroxy-5, PDI. . The determination of the presence of the prolidase by direct detection of the prolidase constitutes a particular embodiment of the invention. By direct detection of the prolidase is meant the detection of the prolidase itself in the biological sample. The direct detection of the prolidase in the biological sample can be carried out by any means known to those skilled in the art, for example by immunoassay or by mass spectrometry, which constitutes a particular embodiment of the invention. 'invention. The immunoassay may be any test widely known to those skilled in the art involving immunological reactions, i.e., reactions between the prolidase and a specific binding partner of the prolidase. The specific binding partners of the prolidase are any partner likely to bind to the prolidase. By way of example, mention may be made of antibodies, antibody fractions, receptors, mimotopes, synthetic antibodies, recombinant antibodies, aptamers and any other molecule capable of binding to the prolidase. The binding partner antibodies are, for example, either polyclonal antibodies or monoclonal antibodies, or antibody analogues and in particular the affinity proteins with competitive properties known under the name Nanofitins. The polyclonal antibodies can be obtained by immunizing an animal with prolidase, followed by the recovery of the desired antibodies in purified form, by taking the serum of said animal, and separating said antibodies from the other constituents of the serum, in particular by chromatography. affinity on a column to which is fixed an antigen specifically recognized by the antibodies, in particular the prolidase. The monoclonal antibodies can be obtained by the hybridoma technique, the general principle of which is recalled below. Firstly, an animal, usually a mouse (or cells cultured in the context of in vitro immunizations), is immunized with prolidase, whose B lymphocytes are then capable of producing antibodies against said antigen. These antibody-producing lymphocytes are then fused with "immortal" myeloma cells (murine in the example) to give rise to hybridomas. From the heterogeneous mixture of cells thus obtained, the cells capable of producing a particular antibody and of multiplying indefinitely are then selected. Each hybridoma is multiplied in the clone form, each leading to the production of a monoclonal antibody whose prolidase recognition properties can be tested, for example by ELISA, by immunoblotting in one or two dimensions, in vitro. immunofluorescence, or using a biosensor. The monoclonal antibodies thus selected are subsequently purified, in particular according to the affinity chromatography technique described above. The monoclonal antibodies can also be recombinant antibodies obtained by genetic engineering, by techniques well known to those skilled in the art.

Examples of anti-prolidase antibodies are known and are available in particular in the catalog of Abnova, and USCN Life Science Inc. The specific binding partners of the prolidase may be labeled for the disclosure of the prolidase / binding partner binding when the binding partner is used as a detection reagent, and therefore for the direct detection of the prolidase in the biological sample. By labeling the binding partners is meant the fixing of a marker capable of directly or indirectly generating a detectable signal. A nonlimiting list of these markers consists of: the enzymes which produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, α-galactosidase, glucose-6-phosphate dehydrogenase, chromophores such as fluorescent compounds, luminescent compounds, dyes, radioactive molecules such as 32P, 35S or 1251, and fluorescent molecules such as alexa or phycocyanins; nucleic acids which can then be amplified for get an increase in the signal. Indirect systems can also be used, such as ligands capable of reacting with an anti-ligand. The ligand / anti-ligand pairs are well known to those skilled in the art, which is the case, for example, of the following pairs: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin, polynucleotide / complementary polynucleotide. In this case, it is the ligand that carries the binding partner. The anti-ligand can be detectable directly by the markers described in the preceding paragraph or be itself detectable by a ligand / anti-ligand. These indirect detection systems can lead, under certain conditions, to amplification of the signal. This signal amplification technique is well known to those skilled in the art, and reference may be made to prior patent applications FR2781802A1 or WO95 / 08000A2 of one of the Applicants or to the article by J. Chevalier et al. al. [J. Knight, J. Yi, O. Michel, and XM Tang, J. Histochem. Cytochem. 45: 481-491, 1997]. Depending on the type of labeling used, those skilled in the art will add reagents allowing the visualization of the marking.

By way of example of immunological tests as defined above, mention may be made of "sandwich" methods such as ELISA, IRMA and RIA, so-called competitive methods and direct immunodetection methods such as immunohistochemistry, immunocytochemistry, Western-blot and Dot-blot. It is also possible to use so-called "digital" methods to increase the detection sensitivity, by making it possible to detect single molecules.

In the case of so-called competition methods, the prolidase is labeled as described above for the binding partner. Examples of immunoassays in the form of commercial kits can be found: for example the prolidase could also be assayed using microplate format assays such as kits marketed by USCN Life Science Inc. (ELISA Kit for Peptidase D, ref. 81011). Or using the antibody pairs marketed by Abnova (PEPD Matched Antibody Pair, Catalog #: H00005184-AP11). Mass spectrometry can also be used for the direct detection of prolidase in the biological sample. The principle of spectrometry is widely known to those skilled in the art and is described for example in Patterson, S., 2000, Mass spectrometry and proteomics. Physiological Genomics 2, 59-65. To do this, the biological sample previously treated or not is passed through a mass spectrometer and the spectrum obtained is compared with that of the prolidase. An example of preliminary treatment of the sample consists in passing it on an immunocapture support, comprising one of the binding partners of the prolidase, for example an antibody directed against the prolidase. Another example of pre-treatment of the sample may be the zo pre-fractionation of the biological sample in order to separate the proteins from the sample. In techniques well known to those skilled in the art, one can for example first of all deplete the majority proteins of the sample. The mass spectrometry to be used in the process of the invention is widely known to those skilled in the art as a powerful tool for the analysis and detection of different types of molecules. In general, any type of molecule that can be ionized can be detected as a function of its molecular mass using a mass spectrometer. Depending on the nature of the molecule to be detected, of protein or metabolic origin, some mass spectrometry technologies may be more suitable. Nevertheless, whatever the mass spectrometry method used for the detection, the latter comprises a step of ionization of the target molecule into so-called molecular ions, in this case a step of ionization of the characterization markers, and a step separation of the molecular ions obtained as a function of their mass. All mass spectrometers therefore comprise: i) an ionization source for ionizing the markers present in the sample to be analyzed, that is to say, to give a positive or negative charge to these markers; ii) a mass analyzer for separating ionized markers, or molecular ions, as a function of their mass-to-charge ratio (m / z); iii) a detector for measuring the signal produced either directly by the molecular ions, or by ions produced from the molecular ions, as detailed below. The ionization step necessary for the implementation of mass spectrometry can be carried out by any method known to those skilled in the art. The ionization source makes it possible to bring the molecules to be assayed under a gaseous and ionized state. An ionization source can be used either in positive mode to study positive ions, or in negative mode to study negative ions. Several types of sources exist and will be used depending on the desired result and the molecules analyzed. These include: electronic ionization (EI), chemical ionization (CI) and chemical desorption-ionization (DCI) fast atom bombardment (FAB), metastable atoms (MAB) or ions (SIMS, LSIMS inductively coupled plasma (ICP) atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) electrospray or electrospray (ESI) matrix-assisted laser desorption-ionization (MALDI), activated by a surface (SELDI) or on silicon (DIOS) ionization-desorption by interaction with metastable species (DART) In particular, the ionization can be implemented as follows: The sample containing the target molecules is introduced into a source of ionization, where the molecules are ionized in the gaseous state and thus transformed into molecular ions that correspond to the initial molecules. An electrospray ionization source (ESI for ElectroSpray Ionization) makes it possible to ionize a molecule while passing it from a liquid state to a gaseous state. The molecular ions obtained then correspond to the molecules present in the liquid state, with positive mode one, two or even three or more additional protons and therefore carry one, two or even three or more charges. For example, when the target molecule is a protein, ionization of the proteotypic peptides obtained after fractionation of the target protein, by means of an electrospray-type source operating in a positive mode, leads to gaseous polypeptide ions, with one, two or even three or more additional protons and which therefore carry one, two or even three or more charges, and allows a transition from a liquid state to a gaseous state. This type of source is particularly well suited when the target molecules or proteotypic peptides obtained are previously separated by reverse phase liquid chromatography. Nevertheless, the ionization efficiency of the molecules present in the sample may vary according to the concentration and nature of the different species present. This phenomenon results in a matrix effect well known to those skilled in the art.

A MALDI ionization source will ionize molecules from a solid state sample. The mass analyzer in which is implemented the step of separating the ionized markers according to their mass / charge ratio (m / z) is any mass analyzer known to those skilled in the art. We can mention low-resolution analyzers, quadrupole or quadrupole (Q), 3D (IT) or linear (LIT), also called ion trap, and high-resolution analyzers, which measure the exact mass of the analytes. which use in particular the magnetic sector coupled to an electrical sector, the flight time (TOF).

The separation of the molecular ions as a function of their m / z ratio can be carried out only once (simple mass spectrometry or MS), or several successive MS separations can be carried out. When two successive MS separations are performed, the analysis is called MS / MS or MS2. When three successive MS separations are performed, the analysis is called MS / MS / MS or MS3 and more generally, when n successive MS separations are made, the analysis is called MSn. Among the techniques implementing several successive separations, the modes SRM (Selected Reaction Monitoring) in case of detection or determination of a single target molecule, or MRM (Multiple Reaction Monitoring) in case of detection or determination of several target molecules, are particular uses of MS2 separation. Similarly MRM3 mode is a particular use of separation in MS / MS / MS. This is called targeted mass spectrometry. In the case of detection in simple MS mode, it is the mass / charge ratio of the molecular ions obtained which is correlated with the target molecule to be detected. In the case of detection in MS / MS mode, essentially two steps are added, relative to an MS assay, which are: i) fragmentation of the molecular ions, then called precursor ions, to give ions called fragment ions of 1st generation, and ii) a separation of the so-called fragment ion ions of the first generation as a function of their mass (m / z) 2, the ratio (m / z) 1 corresponding to the ratio (m / z) of the precursor ions. It is then the mass / charge ratio of the first generation fragment ions thus obtained which correlates with the target molecule to be detected. The term "first generation fragment ion" means an ion derived from the precursor ion, following a fragmentation step and whose mass-to-charge ratio m / z is different from the precursor ion. Couples (m / z) 1 and (m / z) 2 are called transitions and are representative of the characteristic ions to be detected.

The choice of characteristic ions that are detected to be correlated to the target molecule is made by those skilled in the art according to the standard methods. Their selection will lead advantageously to the most sensitive dosages, the most specific and the most robust possible, in terms of reproducibility and reliability. In the methods developed for the selection of proteotypic peptides (m / z) 1, and first generation fragment (m / z) 2, the choice is essentially based on the intensity of the response. For more details, see V. Fusaro et al. [V. Fusaro et al. Nature Biotech. 27; 2009; 190-198]. Commercial software, such as MIDAS and MRM Pilote software from Applied Biosytems or MRMaid [J. Mead et al., MCP, Nov. 15, 2008, E-pub] may be used by one skilled in the art to enable him to predict all the possible couples of transitions. It will also be possible to use a database called PeptideAtlas, built by F. Desiere et al. [F. Desiere et al. Nucleic Acids Res. 2006, Jan 1; 34 (database issue): D655-8] to compile the set of MRM transitions of peptides described by the scientific community. This PeptideAtlas database is available for free access on the internet. For non-protein molecules, it is also possible to use databases, such as for example that accessible through Cliquid software Applied Biosytems (USA). An alternative approach to select the proteotypic peptides, (m / z) 1 and (m / z) 2, is to use the MS / MS fragmentation spectra obtained from other work. This work can be, for example, the discovery and identification phases of biomarkers by proteomic analysis. This approach was proposed by Thermo Scientific at a user meeting. It makes it possible to generate a list of candidate transitions from the peptides identified experimentally by the SIEVE software (Thermo Scientific). Some criteria have been detailed in the literature for the choice of ions (m / z) 1 and (m / z) 2 and are detailed below: peptides with internal cleavage sites, that is to say with Lysine or internal Arginine, should be avoided, unless Lysine or Arginine is followed by Proline, - Peptides with Asparagine or Glutamine should be avoided as they may occur. dimerize, - Peptides with Glutamine or Glutamic Acid in N-terminal should be avoided because they can cyclize spontaneously, 20 - Peptides with Methionine should be avoided as they can be oxidized, - Peptides with Cysteine should be avoided because they can be modified in a non-reproducible way during a possible step of denaturation, reduction and blocking of the thiol functions. Proline peptides can be considered favorable because they produce intense fragments of MS / MS with only one very large peak. However, a single very large fragment does not validate the identity of the transition in a complex mixture. Indeed, only the simultaneous presence of several characteristic fragments makes it possible to verify that the desired precursor ion is well detected. The peptides have a Proline adjacent to the C-terminal (position n-1) or in second position with respect to C-terminal. terminal (position n-2) should be avoided because, in this case, the size of the first generation fragment peptide is generally considered too small to be sufficiently specific. The selection of fragments having a mass greater than the precursor is to be preferred for promote specificity. For this, it is necessary to select a dicharged precursor ion and select the most intense first generation fragment ion having a mass greater than the precursor, ie a single-generation fragment ion unloaded. Fragmentation of the selected precursor ions is carried out in a fragmentation cell such as the triple quadrupole type, or ion trap type, or type of flight time (TOF), which also allow the separation of ions. The fragmentation or fragmentation will be conventionally carried out by collision with an inert gas such as argon or nitrogen, within an electric field, by photoexcitation or photodissociation using an intense light source, collision with electrons or radical species, by applying a potential difference, for example in a flight time tube, or by any other mode of activation.

The characteristics of the electric field condition the intensity and the nature of the fragmentation. Thus, the electric field applied in the presence of an inert gas, for example in a quadrupole, conditions the collision energy supplied to the ions. This collision energy will be optimized, by the skilled person, to increase the sensitivity of the transition to be assayed. For example, it is possible to vary the collision energy between 5 and 180 e-V in q2 in an AB SCIEX QTRAP® 5500 mass spectrometer from Applied Biosystems (Foster City, USA). Similarly, the duration of the collision step and the excitation energy within, for example, an ion trap will be optimized, by the skilled person, to lead to the most sensitive assay. For example, it is possible to vary this duration, called excitation time, between 0.010 and 50 ms and the excitation energy between 0 and 1 (Arbitrary unit) in Q3 in an AB SCIEX mass spectrometer. QTRAP® 5500 from App lied Biosystems. Finally, the detection of the selected characteristic ions is done in a conventional manner, in particular thanks to a detector and a treatment system. The detector collects ions and produces an electrical signal whose intensity depends on the amount of ions collected. The signal obtained is then amplified so that it can be processed by computer. A computer set of data processing makes it possible to transform the information received by the detector into a mass spectrum.

The principle of the SRM mode, or even the MRM mode, is to specifically select a precursor ion, to fragment it, and then to specifically select one of its fragment ions. For such applications, devices of the triple quadrupole type or triple quadrupole hybrids with ion trap are generally used. In the case of a triple quadrupole device (Q1q2Q3) used in MS2 mode, for the purpose of assaying or detecting a target protein, the first quadrupole (Q1) makes it possible to filter the molecular ions corresponding to the proteotypic peptides characteristic of the protein to be assayed and obtained during a previous step of digestion, according to their mass-to-charge ratio (m / z). Only the peptides having the mass / charge ratio of the desired proteotypic peptide, called ratio (m / z) 1, are transmitted in the second quadrupole (q2) and act as precursor ions for the subsequent fragmentation. The q2 analyzer makes it possible to fragment the peptides of mass / charge ratio (m / z) 1 into first generation fragment ions. Fragmentation is generally achieved by collision of the precursor peptides with an inert gas, such as nitrogen or argon in q2. The first generation fragment ions are transmitted in a third quadrupole (Q3) which filters the first generation fragment ions according to a mass-to-specific charge ratio called ratio (m / z) 2. Only the first generation fragment ions having the mass / charge ratio of a characteristic fragment of the desired proteotypic peptide (m / z) 2 are transmitted in the detector to be detected or even quantified. This mode of operation has a double selectivity, in relation to the selection of the precursor ion on the one hand and the selection of the first generation fragment ion on the other hand. Mass spectrometry in SRM or MRM mode is therefore advantageous for quantification When the mass spectrometry used in the method of the invention is a tandem mass spectrometry (M52, M53, M54 or M55), several analyzers of mass can be coupled together. For example, a first analyzer separates the ions, a collision cell makes it possible to fragment the ions, and a second analyzer separates the fragment ions. Some analyzers, such as ion traps or FT-ICR, are several analyzers in one and can fragment the ions and analyze the fragments directly.

According to preferred embodiments of the invention, the process of the invention comprises one or more of the following characteristics: mass spectrometry, used for the typing properties, potential resistance to at least one antimicrobial and factor virulence, is an MS / MS type spectrometry, which has the advantage of generating a specific fragment of the molecule to be detected or quantified, and thus to bring a high specificity to the assay method; MS / MS spectrometry is MRM, which has the advantage of using an analysis cycle time in the mass spectrometer of a few tens of milliseconds, which makes it possible to detect or quantify with great sensitivity, and in a multiplexed manner, a large number of different molecules; the determination of the typing properties, antimicrobial resistance and virulence factor is carried out in the same mass spectrometry apparatus, preferably simultaneously, which has the advantage of reducing the analysis time and the cost of the instrument, it also facilitates the processing and rendering of results. When the markers to be assayed in a biological fluid are of protein origin, upstream of the detection by mass spectrometry, the sample to be analyzed is preferentially treated beforehand to generate peptides from all the proteins present in the sample. sample to fragment these proteins into peptides, for example by digestion with a proteolytic enzyme (protease), or by action of a chemical reagent. Indeed, the cleavage of the proteins can be done by a physico-chemical treatment, a biological treatment or a combination of the two treatments. Among the treatments that can be used, mention may be made of treatment with hydroxyl radicals, in particular with H 2 O 2. Treatment with hydroxyl radicals causes cleavage of the peptide bonds which occurs randomly on any peptide bond of the protein. The concentration of hydroxyl radicals determines the number of cleavages operated and therefore the length of the peptide fragments obtained. Other chemical treatments may also be used such as, for example, cyanogen bromide (CNBr) treatment which specifically cleaves the peptide bonds at the carboxylic group of the methionyl residues. It is also possible to carry out partial acid cleavage at the aspartyl residues by heating at 100 ° C a solution of proteins in trifluoroacetic acid.

Protein treatment by enzymatic digestion is nevertheless preferred over physicochemical treatment because it preserves the structure of proteins more, and is easier to control. By "enzymatic digestion" is meant the single or combined action of one or more enzymes under appropriate reaction conditions. Proteolysis enzymes, called proteases, cut proteins at specific locations. Each protease generally recognizes a sequence of amino acids in which it always performs the same cut. Some proteases recognize a single amino acid or a sequence of two amino acids between which they cleave; other proteases only recognize longer sequences. These proteases may be endoproteases or exoproteases. Among the proteases known include, such as those described in patent application WO2005 / 098017A1: specific enzymes such as trypsin which cleaves the peptide bond at the carboxylic group of the Arg and Lys residues, the endolysin which cleaves the linkage peptide of the -CO group of lysines, the chymotrypsin which hydrolyzes the peptide bond at the carboxylic group of the aromatic residues (Phe, Tyr and Trp), the pepsin which cuts at the level of the NH 2 group of the aromatic residues (Phe, Tyr and Trp) the V8 protease of Staphylococcus aureus strain V8 which cleaves the peptide bond at the carboxylic group of the Glu residue; non-specific enzymes such as thermolysin from the bacteria Bacillus thermoproteolyticus which hydrolyzes the peptide bond of the NH 2 group of hydrophobic amino acids (Xaa-Leu, Xaa-Ie, Xaa-Phe), subtilisin and pronase which are proteases which hydrolyze substantially all the bonds and can transform the proteins into oligopeptides under controlled reaction conditions (enzyme concentration and reaction time). Several proteases can be used simultaneously, if their modes of action are compatible, or they can be used successively. In the context of the invention, the digestion of the sample is preferably carried out by the action of a protease enzyme, for example trypsin. The generation of peptides using a chemical reagent or a protease can be obtained by simple reaction in solution. It can also be carried out with a microwave oven, or under pressure, or even with an ultrasonic device. In these last three cases, the protocol will be much faster. Among the peptides thus obtained, the peptides specific for the protein are called proteotypic peptides. They will be dosed by mass spectrometry. According to one embodiment of the invention, the characterization markers are dosable proteins in a biological fluid. In particular, said proteins are digested into peptides, preferably by an enzyme, more preferably by trypsin. Similarly, the sample containing markers of protein origin may also be pretreated for purification purposes. When the markers are of protein origin, this pretreatment purification may be carried out before or after the peptide generation step as described above. The prior purification of the sample is widely known to those skilled in the art and may in particular implement centrifugation, filtration, electrophoresis or chromatography techniques. These separative techniques can be used alone or combined with one another to achieve multidimensional separation. For example, multidimensional chromatography can be used by combining ion exchange chromatography separation with reverse phase chromatography, as described by T. Fortin et al. [T. Fortin et al., 2009, Mol. This!! Proteomics, 8 (5): 1006-1015], or H. Keshishian et al [H. Keshishian et al., 2007, Mol. This!! Proteomics, 2212-2229]. In these publications, the chromatographic medium may be in column or cartridge (solid phase extraction). The electrophoretic or chromatographic fraction (or the retention time in mono or multidimensional chromatography) of the proteotypic peptides is characteristic of each peptide and the implementation of these techniques therefore makes it possible to select the proteotypic peptide (s) to be assayed. Such fractionation of the generated peptides makes it possible to increase the specificity of the subsequent assay by mass spectrometry. For all mass spectrometry methods described above, calibration is required to correlate the measured peak area, corresponding to the intensity of current induced by the detected ions, with the amount of target molecule to be assayed. For this purpose, the calibrations conventionally used in mass spectrometry can be implemented in the context of the invention. MRM assays are typically calibrated using external standards or, preferably, using internal standards as described by T. Fortin et al. supra. In the case where the target molecule is a proteotypic peptide, which makes it possible to assay a protein of interest, the correlation between the quantitative measurement and the quantity of target proteotypic peptide, and subsequently of the protein of interest, is obtained by calibrating the signal measured with respect to a standard signal for which the quantity to be determined is known. Calibration can be performed by means of a calibration curve, for example obtained by successive injections of standard prototypic peptide at different concentrations (external calibration), or preferentially by internal calibration using a heavy peptide, as standard internal, for example according to the AQUA, QconCAT or PSAQ methods detailed below. By "heavy peptide" is meant a peptide corresponding to the proteotypic peptide, but in which one or more carbon atoms 12 (12C) is (are) replaced by carbon 13 (13C), and / or one or more Nitrogen atoms 14 (14N) are (are) replaced by nitrogen (15N). The use of heavy peptides as internal standards (AQUA) has also been proposed in US2004 / 0229283A1. The principle is to artificially synthesize proteotypic peptides with amino acids with heavier isotopes than the usual natural isotopes. Such amino acids are obtained, for example, by replacing some of the carbon atoms 12 (12C) with carbon 13 (13C), or by replacing some of the nitrogen atoms 14 (14N) with nitrogen (15N). ). The artificial peptide (AQUA) thus synthesized has the same physicochemical properties as the natural peptide (with the exception of a higher mass). It is generally added, at a given concentration, to the sample, upstream of the mass spectroscopy assay, for example between the treatment resulting in the cleavage of the proteins of the sample of interest and the fractionation of the peptides obtained after the analysis. treatment stage. As a result, the AQUA peptide is co-purified with the natural peptide to be assayed, during the fractionation of the peptides. The two peptides are therefore injected simultaneously into the mass spectrometer for the assay. They then undergo the same ionization yields in the source. The comparison of the peak areas of the natural peptides and AQUA, the concentration of which is known, makes it possible to calculate the concentration of the natural peptide and thus to go back to the concentration of the protein to be assayed. A variant of the AQUA technique has been proposed by J.-M. Pratt et al. [J.-M. Pratt et al., 2006, Nat. Protoc., 1: 10291043] as QconCat. This variant is also described in the patent application WO2006 / 128492A1. It consists of concatenating different AQUA peptides and producing the artificial polypeptide as a heavy recombinant protein. The recombinant protein is synthesized with amino acids having heavy isotopes. In this way, it is possible to obtain a standard for calibrating the simultaneous determination of several proteins at a lower cost. The QconCAT standard is added from the beginning, upstream of the treatment leading to the cleavage of the proteins and before the steps of protein fractionation, denaturation, reduction and then blocking of the thiol functions of the proteins, if these are present. The QconCAT standard thus undergoes the same treatment cycle leading to cleavage of proteins as the natural protein, which allows to take into account the yield of the treatment step leading to cleavage of proteins. Indeed, the treatment, in particular by digestion, of the natural protein may not be complete. In this case the use of an AQUA standard would underestimate the amount of natural protein. For an absolute dosage, it may therefore be important to consider processing efficiencies leading to protein cleavage. However, V. Brun et al. [V. Brun et al., 2007, Mol. This!! Proteomics, 2139-2149] have shown that sometimes the QconQAT standards do not exactly reproduce the treatment yield, particularly by digestion of the natural protein, probably because of a different three-dimensional conformation of the QconCAT protein.

V. Brun et al. then proposed to use a method called PSAQ and described in the patent application WO2008 / 145763A1. In this case, the internal standard is a recombinant protein, having the same sequence as the natural protein but synthesized with heavy amino acids. The synthesis is carried out ex-vivo with heavy amino acids. This standard has the exact same physicochemical properties as the natural protein (except for a higher mass). It is added from the beginning, before the protein fractionation step, when the latter is present. It is therefore co-purified with the native protein during the protein fractionation step. It has the same processing yield, especially by digestion, as the native protein. The heavy peptide obtained after cleavage is also co-purified with the natural peptide, if a peptide fractionation step is performed. The two peptides are therefore injected simultaneously into the mass spectrometer, for quantitative determination. They then undergo the same ionization yields in the source. The comparison of the peak areas of the natural peptides and the reference peptides in the PSAQ method makes it possible to calculate the concentration of the protein to be assayed taking into account all the steps of the assay procedure. All of these techniques, namely AQUA, QconCAT or PSAQ or any other calibration technique, used in mass spectrometry assays and in particular in the MRM or MS assays, can be implemented to carry out the calibration, in the scope of the invention. Preferably, the mass spectrometry used in the detection method according to the invention is of the MS / MS type. More preferably, mass spectrometry is MRM. In a preferred embodiment of the invention, the peak areas of the natural peptides (sample to be assayed) and of the internal standard added to the sample, the concentration of which is known, are measured. The result is expressed as the ratio of natural peptide area / internal standard area. To obtain an absolute quantification during the same experiment, a fixed quantity of standard is added to all the analyzed samples. There is a standard to measure for each transition followed. Dosage is understood to mean any analytical method that makes it possible to quantify a given protein, whether it be an MRM assay, an ELISA assay, or any other method known to those skilled in the art. Quantification can be absolute. The protein dose is then expressed in one unit of the international system, such as ng / ml. Or relative, the dose is then expressed in Arbitrary Units, when one is calibrated with respect to a reference molecule. This is the case, for example, for MRM quantification, where the ratio of the areas of the chromatographic peaks of the natural transition divided by the area of the heavy transition: light area / heavy area is performed. For the CA19-9, the quantity is estimated relative to an international standard, as described in the instructions for the Vidas® CA19-9 kit of the Applicant. When using mass spectrometry, and in particular the LC-MRM-MS technique described above, the doses for several transitions of the same protein are summerized to obtain a single assay of the protein. By summerization and summeriser, it is meant to combine several independent assays of the same protein, which correlate with each other, to obtain a single dose which is representative of the dose of the protein in the biological sample studied.

The summerization can be done for example by the method of median polish as with the package r which uses the procedure of Tukey Package stats version 2.15.0 I nd ex; http://stat.ethz.ch/R-manual/R- patched / library / stats / html / medpolish.html. The sample on which the process of the invention may be used is any sample capable of containing the prolidase and the other markers of interest, or any of their tracers. It may then correspond to a biological fluid sample (whole blood, serum, plasma, urine, saliva, cerebrospinal fluid, organic secretion, stool for example), in liquid form or deposited for example on a paper-like collection paper. Watman (dried blood spot), a tissue sample or isolated cells. This sample may be used as it is insofar as the prolidase, one of its tracers, or one of the other markers or their tracers, are available in the sample tested, or it may be subjected to analysis beforehand. enrichment type preparation, extraction, concentration, purification, culture, according to methods known to those skilled in the art. The biological sample used for the direct detection of the prolidase, which may contain prolidase as such, may consist of biological fluid or tissue derived from the biopsy of the tumor, lymph nodes or metastases of the patient considered .

As a biological fluid, mention may be made of whole blood or its derivatives such as serum or plasma, bone marrow, milk, saliva, stool, cerebrospinal fluid, urine and effusions. Blood or its derivatives, saliva, urine and stool are preferred. For the detection of the prolidase, the biological fluid, which constitutes a particular embodiment of the invention, may require a particular treatment. Indeed, the biological fluid may contain the prolidase itself, or it may contain circulating tumor cells that contain prolidase, and / or circulating tumor cells that are capable of secreting the prolidase.

Thus, according to one embodiment of the invention, the biological fluid is pretreated to isolate the circulating tumor cells contained in said fluid. By isolating the circulating tumor cells, it is intended to obtain a cell fraction enriched in circulating tumor cells. The treatment of the fluid to isolate the circulating tumor cells can be carried out by cell sorting in a flow cytometer, by enrichment on Ficoll, by enrichment with magnetic beads coated with specific antibodies, by filtration according to the size of the cells or their physico-chemical properties, or by any other specific enrichment method known to those skilled in the art. In the case of blood or bone marrow as a biological fluid, circulating tumor cells can be isolated by a Ficoll cell separation technique associated with depletion of blood cells using anti-CD45 antibodies coupled to magnetic beads ( Dynal Biotech ASA, Norway). The direct detection of the prolidase and the other markers of interest can then be carried out directly from circulating tumor cells isolated from the biological fluid, for example by immunocytochemical labeling of these cells with an anti-prolidase antibody, after having deposited the tumor cells. circulating on a slide by cytospin. The direct detection of the prolidase can also be carried out directly in the circulating tumor cells using the flow cytometry method as described in Métézeau P, Ronot X, Noan-Merdrignac G, Ratinaud MH, flow cytometry for the study of the normal or pathological cell (Volume I), Eds Medsi-MacGrawhill. Under these conditions, said circulating cells can be treated under conditions permitting the blocking of the prolidase within said cells. Such treatment is described for example in Intracellular Flow Cytometry, Applied Reagents and Techniques, pp 1-21, BD Pharmingen. The detection of the prolidase is then made after making the cell membrane permeable to return the specific binding partners of the prolidase. The direct detection of the prolidase from the circulating cells can also be carried out using the method described in the patent application WO 03/076942 A2 filed by one of the Applicants.

The direct detection of the prolidase in the tumor cells can also be carried out in the culture medium of said cells after having grown under conditions such that they secrete prolidase. These culture conditions are conventional conditions such as 37 ° C under humid atmosphere and 5% CO2. When the biological sample to be tested is tissue derived from the tumor biopsy, lymph nodes or metastases of the patient, which constitutes a particular embodiment of the invention, the direct detection of the prolidase is carried out directly on the cuts obtained, without prior treatment of said fabric. Another mode of detecting the presence of the prolidase consists in the culture, in the presence of the biological sample, of cells sensitive to prolidase, which constitutes a particular embodiment of the invention. In this case, the detection of the presence of the prolidase in a biological sample is evidenced by the reaction of the prolidase sensitive cells. By prolidase-sensitive cells is meant any cell sensitive to the biological activity of the prolidase. The biological sample that can be used for the detection of the presence of the prolidase by culture of prolidase sensitive cells may be any sample as described above. Thus, the biological sample may consist of biological fluid, if necessary pretreated to isolate the circulating tumor cells, which can then be cultured under conditions such that they secrete prolidase, as previously described. Another mode of detecting the presence of prolidase in the biological samples consists in the detection of the mRNA of the prolidase in said sample, which constitutes another embodiment of the invention. By nucleic acid are meant oligonucleotides, deoxyribonucleic acids and ribonucleic acids, as well as their derivatives. The term "oligonucleotide" refers to a sequence of at least 2 nucleotides (deoxyribonucleotides or ribonucleotides, or both), natural or modified, capable of hybridizing, under appropriate hybridization conditions, with an at least partially complementary oligonucleotide. . By modified nucleotide is meant, for example, a nucleotide comprising a modified base and / or comprising a modification at the level of the internucleotide link and / or at the level of the backbone. As an example of a modified base, mention may be made of inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, diamino-2,6-purine and bromo-5-deoxyuridine. To illustrate a modified internucleotide linkage, mention may be made of phosphorothioate, N-alkylphosphoramidate, alkylphosphonate and alkylphosphodiester linkages. Alpha-oligonucleotides such as those described in FRA-2,607,507, LNAs such as phosphorothioate-LNA and 2'-thio-LNA described in Bioorganic & Medicinal Chemistry Letters, Volume 8, Issue 16, August 18, 1998, pages 2219 -2222, and the NAPs that are the subject of the article by Mr. Egholm et al., [Mr. Egholm et al., J. Am. Chem. Soc. (1992), 114, 1895-1897], are examples of oligonucleotides consisting of nucleotides whose backbone is modified. Another modification may be, for example, a variation in the splicing of the mRNAs resulting in a change in the structure of the protein. For example, the prolidase can be expressed in truncated form. Detection of mRNA in a liquid sample is widely known to those skilled in the art. It can for example be implemented by hybridization reactions between the target mRNA and a nucleic acid capable of binding with the target mRNA. The revelation of the hybridization reactions can be carried out by labeling the binding nucleic acids, as previously illustrated. Before hybridization with the binding nucleic acid, the target mRNA can be extracted by methods known to those skilled in the art, and then optionally amplified, for example by RT-PCR or by NASBA [Maleck, L., and al., 1994, Methods in Molecular Biology, 28, ch 36, Isaac Ed PG, Humana Press, Inc., Totowa, NJ]. The biological sample that can be used for detecting the presence of prolidase by detection of prolidase mRNA can be any sample as described above. Thus, the biological sample may consist of tissue derived from tumor biopsy, ganglion or metastasis, or biological fluid, if necessary previously treated to isolate circulating tumor cells, as described above. Another way of detecting the presence of prolidase in the biological samples is the detection of all or part of the prolidase gene in said sample, having undergone modifications leading to a modification in the expression of the prolidase, which is another embodiment of the invention. Such a modification may be, for example, a methylation of the promoter of the prolidase gene. Other modifications may be the presence of mutations in the nucleotide chain or mRNA splicing variations, resulting in a change in expression of the prolidase or the expression of a modified form of the prolidase. In a preferred embodiment of the invention, it is possible to detect a tracer of the prolidase following the presence of a modified form of the prolidase. For example, the presence of autoantibodies to the prolidase can be detected in a sample remote from the tumor. The present invention is now illustrated by the following examples which identify the interests and advantages.

EXAMPLE 1 Detection of Prolidase by the LC-MRM-MS Technique 1) Methodology of the Multiple Reaction Monitoring (MRM) Technique The principle of this assay is based on the association between liquid chromatography and a triple hybrid mass spectrometer. quadrupole / ion trap (5500QT from AB Sciex Toronto, Canada) operating in MRM mode (Q1q2Q3). In principle, for each protein of interest, the tryptic peptides of interest are chosen. Transitions are predicted for example using Skyline software (MIT Cambridge USA) or MRM pilotTM from AB Sciex. The prolidase is quantified by the quantification of one or more of its peptides. These peptides are chosen according to their physicochemical characteristics, and their ability to be detected in the mass spectrometer. The selected peptides are isolated in the first quadrupole Q1, to be fragmented in the collision cell of the mass spectrometer (q2). From the generated son ions, the most representative and specific were chosen, and the intensity of these son ions was monitored after selection in the third quadrupole Q3. The parent ion / ion ion mass pair is referred to as the transition, and it is the association of the transition with the corresponding chromatographic retention time that defines the specificity of the assay relative to the target protein. To obtain a quantitative assay, an internal standard must be added (for example an identical "heavy" peptide which will serve as a standard, in known quantity, which is added to the sample and is eluted at the same chromatographic time and which allows the The peptides were selected according to their sensitivity (better response in the mass spectrometer) and their specificity in the matrix Peptides of identical sequences to the selected target peptides were synthesized with lysine or labeled arginine (13C and 15N): +8 Dalton for lysine, +10 Da for arginine, in order to be able to perform quantitative assays.In fact, these heavy peptides have the same physicochemical properties as the target peptides, and are eluted at the same chromatographic retention times In order to be able to lower the detection limit to a few ng / ml, an improved method of MRM-MS has been implemented, it comprises the following successive stages: tryptic digestion; SPE (Solid-Phase Extraction) fractionation of peptides; and liquid chromatography (LC) coupled to the MRMMS and described hereinafter. The development was carried out on overloaded samples by adding the synthetic peptides. Calibration To carry out a relative quantitative study of the prolidase, a fixed amount (10 μl) of a pool of heavy peptides of the protein to be assayed synthesized with 13C-containing Arginine or Lysine was added to each sample. 15N. Quantification is relative and not absolute because the amount of peptides synthesized and added is fixed for all samples, but it is only estimated and not known precisely.

Enzymatic digestion Serum samples are denatured in a 6M urea solution buffered with 10 mM Tris pH 8 and containing 30 mM dithiothreitol, for 40 minutes at 40 ° C, and then alkylated with 50 mM iodoacetamide at room temperature. ambient, for 40 minutes, in the dark. They are diluted 6 times in water, then the tryptic digestion is carried out at 37 ° C, overnight, using a substrate enzyme ratio of 1:30 (Promega). The digestion is stopped by adding formic acid to a final concentration of 0.5%. The digested samples are desalted by Solid Phase Extraction (SPE) using Oasis HLB 3cc reverse phase cartridges (60 mg) (Waters, Manchester UK). After application of the sample, the cartridges are washed with 0.1 ml of 0.1% formic acid, and then the elution is carried out with a methanol / water mixture (80/20 v / v) containing 0.1% d formic acid. The eluates are dried under vacuum. SPE fractionation The dry samples are taken up in 1 ml of acetate buffer and loaded onto the mixed (hydrophobic and cation exchange) Oasis MCX (mixed cation exchange) 60 mg cartridges (Waters, Manchester UK) pre-equilibrated with acetate and methanol buffer. The cartridges are washed with 1 ml of acetate buffer and 1 ml of methanol. The peptides of interest (Table 1) are eluted with 1 ml of a methanol / acetate buffer mixture (50/50 v / v). The pH of the acetate buffer is chosen according to the isoelectric point of the peptide of interest. The eluates are dried under vacuum, dissolved in 200 μl of a solution of acetonitrile / water (3/97 v / v) containing 0.1% formic acid. A 50 μl aliquot was injected into the LC coupled to an MS-MS system.

Liquid Chromatography and Mass Spectrometry LC-MS analysis was performed on an HP 1100 series high pressure chromatography (HPLC) system with binary pump and injector (Agilent Technologies, Santa Clara California USA) coupled to a mass spectrometer, AB Sciex 5500 QTrap (MS Triple Quadrupole Hybrid - Ionic Trap) (AB Sciex, Toronto Canada) for improved sensitivity. LC separation was performed on a C18 Symmetry column (Waters, Manchester UK), at an elution rate of 300 pl / min. (Eluent A = 0.1% formic acid in water, eluent B = 0.1% formic acid in acetonitrile, linear gradient from 5% B to 50% B in 25 min, then from 50% B to 100 % B in 3 min). MS analysis is performed in positive ionization mode at a voltage of 5500 V applied to the needle allowing ionization in the source. Instrument control and data acquisition are performed with Analyst 1.4.1 software. The flow rates of nebulizer (air) and curtain gas (nitrogen) are 30 and 20 psi, respectively. The Turbo VTM ion source is set at 400 ° C, the auxiliary nitrogen flow at 40 psi. The collision energy (CE), the DP (declustering potential) and the collision cell exit potential (CXP) are optimized for each of the selected MRM transitions. All parent ions di- or tri-charged theoretical tryptic peptides in a mass range of 800 to 3000 Da and all possible fragment ions of type y or b. For each protein, each possible transition was tested to determine the most sensitive and specific transitions. The result of this selection is summarized in Table 1. The MCX chromatographic separation was carried out at different pHs, the pH chosen for the rest of the experiments being the pH which makes it possible to obtain the area of the highest peak. 2) Example of the peptides and transitions monitored for the determination of prolidase Table 1 prolidase (Xaapro) Sequences pl TR Q1 Q3 Q1 Q3 DP EP CE CXP (peptide (peptide (peptide (peptide light heavy peptide standard internal standard) heavy standard internal) natural ) natural) IDEPGLR 4'4 9.8 400.2 442.3 405.2 452.3 101 10 17 12 (SEQ ID NO: 1) 571.3 581.3 106 10 15 14 686.3 696.3 101 10 15 16 AVYEAVLR 6'38 11 460.76 375.7 465.75 380.7 85 4 19 20 (SEQ ID NO: 2) 458.3 468.3 85 4 25 25 587.4 597.4 85 4 25 48 750.4 760.4 85 4 19 42 GVNTDSGSVCR (SEQ ID NO: 3) 5.8 11 576.3 665.3 581.3 675.3 126 10 25 32 780.3 790.3 126 10 23 16 881.4 891.4 126 10 21 20 It is possible to perform the determination of the prolidase by following and quantifying one or more transitions as described above. In a preferred embodiment of the invention, the transitions monitored are averaged by any means known to those skilled in the art. In a preferred embodiment of the invention, it is possible, for example, to average the transitions used for the assay by the median polish method. In a preferred embodiment of the invention, it is possible to use a package r according to Tukey's procedure for producing the median polish.

In another preferred embodiment of the invention, it is possible to assay the prolidase in samples following a single transition, for example the 400.2 / 686.3 transition of the peptide of SEQ ID NO: 1, and the transition of the corresponding heavy peptide. . For each transition, the ratio of the area under the curve of the natural transition chromatographic peak divided by the area under the curve of the heavy transition chromatographic peak will be established to obtain a dose per transition for each sample, and then summerized. between them or not the doses obtained for the different transitions. The doses that will be used in Examples 3 and 4 for obtaining doses and prolidase score were obtained by following and quantifying the 400.2 / 686.3 transition of the peptide of SEQ ID NO: 1. Example 2: Marker detection The principle of the assays using the Multiple Reaction Monitoring MRM assay method described in Example 1 for the determination of the prolidase can be applied in the same way to the determination of any protein label which has been confirmed. sequence. These assays can be used to assay all types of biological samples, such as for example blood, serum, plasma, stool, urine, etc. As described in Example 1, for a given protein it is possible to quantify a specific marker by following one or more transitions. Each transition can be independently monitored, or an average can be made between the transitions, using any summerization method known to those skilled in the art. In the same way for the calibration, a relative quantitative study of each marker is carried out, a fixed amount (10 μl) of a pool of heavy peptides of the protein to be assayed synthesized with Arginine or Lysine having 13C and 15N. Quantification is relative and not absolute because the amount of peptides synthesized and added is fixed for all samples, but it is only estimated and not known precisely. To obtain the doses in Arbitrary Units used in combination in Example 4, the ratio of the areas of the chromatographic peaks of the natural transition divided by the area of the heavy transition was performed. The Alpha-Enolase protein can be assayed, for example, by following the transitions listed in Table 2 below: Table 2 Alpha -Enolase Sequences pl TR Q1 Q3 Q1 (internal heavy peptide) Q3 DP EP CE CXP (peptide (peptide ( light peptide light heavy standard internal) natural) natural) YISPDQLADLYK 4'2 18'1 713.37 531.8 717.35 535.8 181 4 31 14 (SEQ ID NO: 4) 575.3 579.3 181 4 29 14 609.3 617.2 181 4 39 14 IEEELGSK 4'3 8'3 452.73 533.3 456.75 541.1 95 5 26 28 (SEQ ID NO: 5) 662.3 670.2 95 5 22 In a preferred embodiment of the invention, the 452.7 / 662.2 transition of the peptide IEEELGSK (SEQ ID NO: 5) will be followed, and the transition of the corresponding heavy peptide will be established for each transition the ratio of the areas of the chromatographic peaks of the natural transition divided by the area of the heavy transition to obtain a dose per transition for each sample, then summerisera between them or not the doses obtained for different transitions. The doses which will be used in Example 4 for the performance of a score in combination with the prolidase, and in Example 6 for obtaining a dose and an individual score, were obtained by following the transition 452.7 / 662.2 of the peptide of SEQ ID NO: 5. The protein 78 kDa glucose-regulated protein (BIP, GRP78) can be assayed by following, for example, the transitions listed in Table 3 below: Table 3 BIP Sequences pl TR Q1 Q3 Q1 Q3 DP EP CE CXP (peptide (peptide (peptide (heavy light peptide standard internal standard) heavy standard internal) natural) natural) ELEEIVQPIISK 4.3 17.3 699.4 557.4 703.4 565.4 144 3 36.7 7 (SEQ ID NO: 6) 685.4 693.4 144 3 27 8 784.5 792.5 144 3 31 9 ITPSYVAFTPEGER (SEQ ID NO: 7) 4.5 15.2 783.9 587.3 788.9 597.3 152.3 4 35.8 7 835.4 845.4 152.3 4 37.1 9 906.4 916.4 152.3 4 37.3 10 LTPEEIER 4.3 10 , 6,493.8,386.7 498.75 391.7 86.8 4 19.2 9 (SEQ ID NO: 8) 675.4 685.4 86.8 4 27.2 8 772.4 782.4 86 , 8 4 19.4 9 In a preferred mode of the invention, the transitions of the peptide of SEQ ID NO: 6 and their transitions of the corresponding heavy peptide will be followed, the ratio of the areas of the chromatographic peaks of the natural transition divided by the area of the heavy transition will be established for each transition. to obtain a dose per transition for each sample, then summerisera between them or not the doses obtained for the different transitions. The doses which will be used in Example 4 for carrying out a score in combination with the prolidase, and in Example 6 for obtaining a dose and an individual score, were obtained by summerizing between them the doses of the 3 transitions of the peptide of SEQ ID NO: 6, using the median polish method.

The G3PDH protein (GAPDH) can be determined by following, for example, the transitions listed in Table 4 below: Table 4 G3PDH Sequences I p I I Q I I Q3 I Q3 I DP IEICI CX (peptid (peptide (peptide (peptide PE light pe e light heavy heavy natural) natural standard standard internal) internal) GALQNIIPASTGAAK 8.7 13.1 706.40 597.3 710.4 613.4 120 6 27 29 (SEQ ID NO: 9) 815.4 823, 4 120 6 32 37 VIPELNGK 6 10.6 435.3 560.2 439.25 568.2 116 4 23 14 (SEQ ID NO: 10) 657.2 665.2 116 4 13 16 VPTANVSVVDLTCR 5.8 13.9 765.9 483.3 770 9770 '483.1 85 4 40 12 (SEQ ID NO: 11) 9 549.3 559.3 85 4 53 14 949.5 959.5 85 4 44 24 VGVNGFGR 9.7 10 , 3 403.22 436.2 408.2 446.2 101 10 19 12 (SEQ ID NO: 12) 550.3 560.3 101 10 19 14 706.3 716.3 101 10 17 16 In a preferred embodiment of the In the invention, the transitions of the peptide of SEQ ID NO: 9, or the 403,2 / 550,3 and 403,2 / 706,3 transitions of the peptide of SEQ ID NO: 12, or the 435,3 / 657 transition will be followed. , 2 of the peptide of SEQ ID NO: 10, or the transit 765.9 / 949.5 ion of the peptide of SEQ ID NO: 11, and their transitions of the corresponding heavy peptides, will be established for each transition the ratio of the areas of the chromatographic peaks of the natural transition divided by the area of the heavy transition to obtain a dose per transition for each sample, then summerisera between them or not the doses obtained for the different transitions. The doses which will be used in Example 4 for carrying out a score in combination with the prolidase, and in Example 6 for obtaining a dose and an individual score, were obtained by summerizing between them the doses of the transitions 403,2 / 550,3 and 403,2 / 706,3 of the peptide of SEQ ID NO: 12, the doses of the 765,9 / 949,5 transition of the peptide of SEQ ID NO: 11, and the 435.3 / 657.2 transition doses of the peptide of SEQ ID NO: 10, using the median polish method. The HSP71 protein (HSC70) can be assayed, for example, by following the zo transitions listed in Table 5 below: Table 5 HSP71 Sequences pl TR Q1 Q3 Q1 Q3 DP CE CXP (peptide (peptide heavy light heavyweight peptide internal standard) standard natural) natural) (internal peptide) FEELNADLFR 4.1 18 627.3 735.3 632.3 745.3 156 4 25 16 (SEQ ID NO: 13) 848.4 858.4 156 4 25 20 977.5 987.4 156 4 21 24 DAGTIAGLNVLR 5.8 17,600.3 671.4 605.35 681.4 69 2 32 17 (SEQ ID NO: 14) 742.5 752.5 69 2 30 18 855, 6,865.6 69 2 30 22 SQIHDIVLVGGSTR (SEQ ID NO: 15) 6.5 12.8 494.6 477.3 497.9 487.3 75 3 19 13 576.3 586.3 75 3 18 16 793, In a preferred embodiment of the invention, the 600.3 / 671.4 and 600.3 / 742.5 transitions of the peptide of SEQ ID NO: 14, or the transition 494.6 /, will be followed. 793.4 of the peptide of SEQ ID NO: 15, and their transitions of the corresponding heavy peptides, the ratio of the areas of the chromatographic peaks of the nature transition will be established for each transition. It is divided by the area of the heavy transition to obtain a dose per transition for each sample, then summerisera between them or not the doses obtained for the different transitions.

The doses which will be used in Example 4 for carrying out a score in combination with the prolidase, and in Example 6 for obtaining a dose and an individual score, were obtained by summerizing between them the doses of the transitions 600.3 / 671.4 and 600.3 / 742.5 of the peptide of SEQ ID NO: 14, and the dose of the transition 494,6 / 793,4 of the peptide of SEQ ID NO : 15, using the median polish method. The PDI protein can be determined by following, for example, the transitions listed in Table 6 below: Table 6 PDI Sequences I p I Q1 I Q3 I Q1 (peptide I Q3 (peptide I DP I EP I CE I CXP ( peptide (light heavy heavy peptide standard standard natural standard) natural) internal) internal) IFGGEIK 6 9.45 382.2 260.2 386.2 268.2 75 6 25 13 (SEQ ID NO: 16) 503.3 511, 3 75 6 17 25 650.4 658.4 75 6 17 13 ENLLDFIK 4.4 19.5 496.3 407.3 500.3 415.3 89 3 22 12 (SEQ ID NO: 17) 522.3 530, In a preferred embodiment of the invention, the transitions of the peptide of SEQ ID NO: 17 will be followed, and their transitions of the corresponding heavy peptide will be established for each transition. the ratio of the areas of the chromatographic peaks of the natural transition divided by the area of the heavy transition to obtain a dose per transition for each sample, then summerisera between them or not the doses obtained for the different transitions. The doses which will be used in Example 4 for carrying out a score in combination with the prolidase, and in Example 6 for obtaining a dose and an individual score, were obtained by summerizing between them the doses of the different transitions of the peptide of SEQ ID NO: 17, using the median polish method. The peroxy-5 protein can be assayed, for example, by following the transitions listed in Table 7 below: Table 7 Peroxi-5 Sequences pl TR Q1 Q3 Q1 Q3 DP EP CE CXP (peptide. (Peptide (peptide (light-light peptide heavy heavy natural) natural) standard standard internal) internal) THLPGFVEQAEALK (SEQ ID NO: 18) 16,513.9 659.4 516.6 667.4 79.9 3 16.2 17 653.4 653.4 79.9 3 18.7 13 788.4 796.4 79.9 3 16.2 19 LLADPTGAFGK 5.8 14.8 545.3 677.4 549.3 685.4 103 3 25.1 15 (SEQ ID NO: 19) ) 792.4 800.4 103 3 20 17 863.4 871.4 103 3 17.1 20 VNLAELFK 18.9 467.3 607.4 471.3 615.4 104.2 3 16.5 15 (SEQ ID NO: 20) 720.4 728.4 104.2 3 15.4 15.3 834.5 842.5 104.2 3 14.4 In a preferred embodiment of the invention, the 545.3 transition will be followed. / 677.4 of the peptide of SEQ ID NO: 19, or the transitions 513,9 / 653,4 and 513,9 / 788,4 of the peptide of SEQ ID NO: 18, and their transitions of the corresponding heavy peptides, will be established for each transition the ratio of the areas of the chromatographic peaks of the a natural transition divided by the area of the heavy transition to obtain a dose per transition for each sample, then summerisera between them or not the doses obtained for the different transitions.

The doses which will be used in Example 4 for carrying out a score in combination with the prolidase, and in Example 6 for obtaining a dose and an individual score, were obtained by summerizing between them the doses of the 545.3 / 677.4 transition of the peptide of SEQ ID NO: 19, and the doses of the transitions 513.9 / 653.4 and 513.9 / 788.4 of the peptide of SEQ ID NO : 18, using the median polish method. Example 3 Use of Serum Assays in Immunoassays 1) Selection of Proteins and Dosages The Applicant has shown in Example 3 that abnormally decreased doses of protein prolidase could be observed in the bloodstream of certain patients with colorectal cancer. . On the other hand, the Applicant has shown in patent applications WO2009024691, WO2009019365, WO2009019368, WO2009019369, WO2009019366, WO2009019370, WO2009019367, WO2010004214, WO2010112777 that abnormally high or abnormally decreased doses of other tumor markers such as Ezrin, L- FABP, Apo A1, Apo A2, Beta2 Microglobulin, ACE, CA19-9, Galectin-3, 20S Proteasome (Prosomes), Protein Disulfide Isomerase (PDI) and ProDefensin A6 could be observed in the bloodstream of some patients with colorectal cancer. The methods of assaying these tumor markers have been described in the patent applications raised.

In practice, the doses of the different labels assayed in ELISA used for the combination with the prolidase in Example 4 were obtained in the following way: The method of assaying Timp1 was implemented with the ELISA Timp1 kit of IBL International (No. RE54081) according to the manufacturer's instructions, for the determination of Timp1 protein in EDTA plasma of patients and controls. The process for assaying M2PK was carried out with the Schebo M2PK ELISA kit (ScheBo® Tumor M2-PKTM EDTA Plasma Test Cat No. 08) according to the manufacturer's instructions, for the assay of the M2PK protein in the EDTA plasma of patients and controls. The assay procedure for the HSP60 marker is described in the article by Hamelin et al. [Céline Hamelin, Emilie Comut, Florence Poirier, Sylvie Pons, Corinne Beaulieu, Jean-Philippe Charrier, Hader Haidous, Eddy Cotte, Claude Lambert, Francoise Piard, Yasemin Ataman-Onal, Genevieve Choguet-Kastylevsky. Of a potential serum marker for colorectal cancer. FEBS J. 2011 Dec; 278 (24): 4845-4859]. The ApoA1 and ApoA2 proteins were assayed in the sera with the kits marketed by Abnova (AP0A1 ELISA Kit, Catalog #: 20 KA0460, AP0A2 ELISA Kit, Catalog #: KA0461), according to the manufacturer's instructions. The tumor markers ACE, 132 Microglobulin and CA19-9 were assayed using the assay kits of the Applicant, Vidas® CEA (s), Vidas®B2 Microglobulin, Vidas® CA19-9, respectively, according to US Pat. operating protocol specific to each kit. The proteins Galectine 3 and Proteasome 20S (Prosomes) were assayed as described in the applicant's patent, WO2010004214, with sandwich ELISA tests developed on the Vidas® automaton. The Ezrin protein was assayed as described in the patent WO 2009019365, with a sandwich ELISA test developed on the Vidas® automaton. The LFABP protein was assayed as described below: Assaying LFABP Obtaining Monoclonal Antibodies Directed Against LFABP Obtaining the Recombinant Protein To carry out immunizations and obtain monoclonal antibodies against the L-FABP protein we have everything first obtained recombinant protein L-FABP. The complementary DNA, the expression vectors, the cloning and the obtaining of the recombinant LFABP protein (expression and purification) were carried out in the same way as described in the patent WO2010 / 004214. Immunizations Animal model The immunization experiments were performed in female BALB / c (H-2d) mice aged 6 to 8 weeks at the time of the first immunization. Immunogens and immunizations In order to increase the immune responses obtained in the mice and to be able to generate monoclonal antibodies, the L-FABP protein was produced in the form of recombinant proteins produced according to the procedures described in the patent application WO2010 / 004214. The protein was mixed volume for volume with Freund's adjuvant (Sigma), prepared as a water-in-oil emulsion and which is known to have good immunogenicity. Three mice were immunized. The mice received 3 successive doses of 10 μg of the immunogens at 0, 2 and 4 weeks. All injections were done subcutaneously. The first injection is made in admixture with the complete Freund's adjuvant, the next two are mixed with the incomplete Freund's adjuvant. Between day 50 and day 70 after the first injection, the humoral responses were restimulated with an intravenous injection of 100 μg of the recombinant protein. Monitoring of the appearance of the humoral response In order to follow the appearance of the antibodies, blood samples are regularly taken from the mice. The presence of anti-tumor marker antibodies is tested using an ELISA. The protein of interest is used in uptake (1 μg / well), after saturation the antigen is reacted with different dilutions of the sera to be tested (incubation at 37 ° C. for 1 hour). Specific antibodies present in the serum are revealed by alkaline phosphatase-conjugated AffiniPure mouse anti-IgG goat antibody (H + L, Jackson Immunoresearch, Cat No. 115-055-146), which binds to the desired antibodies. (0.1 μg / well). Obtaining Monoclonal Antibodies Three days after the last injection, for each tumor marker, one of the immunized mice was sacrificed; blood and spleen were removed. Splenocytes obtained from the spleen were cultured with Sp2 / 0-Ag14 myeloma cells to fuse and immortalize, according to the protocol described by Kohler and Milstein [G. Kohler and C. Milstein, 1975, Nature, 256, 495-497; G. Lier and C. Milstein, 1976, Eur J Immunol, 6, 511519). After a 12-14 day incubation period, the resulting hybridoma supernatants were screened for the presence of anti-tumor marker antibodies using the ELISA test described in point 3 of this example. Positive hybridoma colonies were subcloned twice according to the limiting dilution technique, well known to those skilled in the art. Characterization of the monoclonal antibodies The monoclonal antibodies obtained against L-FABP are the following: 2E5G9, 3A7B4, 5A8H2, 2C9G6, 3D6G1, 8B10B8, 7D8A11 They were analyzed by the Western blot technique. Complementarity of Antibodies In order to be able to construct the ELISA test for the determination of patient samples, the complementarity of the antibodies was first tested. These are the same antibodies that are used in capture or revelation (after biotinylation). Each biotinylated revelation antibody is tested with each capture antibody (VIDAS cone antibody). The pairs are tested with the recombinant L-FABP protein described above. Table 8 summarizes the results obtained for the different pairs of antibodies.

Table 8 Cross-tabulation showing the results obtained for each pair of antibodies with the recombinant L-FABP protein (average of the signal - average of the RFV background noise). (RFV or Relative Fluorescence Value is the signal read by Vidas®).

Detection Capture 2E5G9 3A7B4 5A8H2 2C9G6 3D6G1 8B10B8 7D8A11 2E5G9 10.5 16.5 25 10537 10377.5 5066 0.5 3A7B4 42 198 707.5 10071 9923 10009 24.5 5A8H2 36 109 513 10404 10283.5 9930 18.5 2C9G6 10753.5 10815 10708.5 1417.5 213 10774.5 1562 3D6G1 10136.5 10707.5 10684.5 606.5 138 10462 1038.5 8B10B8 I 8846 9940.5 9677 10385.5 10637.5 87 363.5 7D8A11 21.5 14 8053.5 8094.5 128 4.5 At the end of these tests, we retain 6 pairs of complementary antibodies: 3A7B4 / 3D6G1 5A8H2 / 3D6G1 2C9G6 / 5A8H2 3D6G1 / 5A8H2 8B10B8 / 5A8H2 2C9G6 / 2E5G9 Characterization epitopes The epitopes recognized by the monoclonal antibodies have been characterized using Spotscan techniques, peptide library screening carried by phages, and the construction of chimeric proteins (fragment library). - Methodology The Spotscan technique, adapted from Frank and Dörring, makes it possible to simultaneously synthesize a large number of peptides fixed on a cellulose membrane. These peptides reproduce the sequence of the target antigen as peptides of 8 to 12 amino acids, overlapping by 1 to 4 residues. These peptides are then brought into contact with the antibody to be studied in a blot-type colorimetric test, and the identification of the immunoreactive peptides makes it possible to deduce the minimal sequence of the epitope of the antibody and to locate it precisely on the antigen. The synthesis is carried out on a cellulose membrane uniformly carrying polyethylene glycol (PEG) arms with a length of 8 to 10 units, having a free NH 2 function at the end of the chain. It proceeds from the C-terminus to the N-terminus of the peptides. The amino acids have their amino function protected by a Fmoc (9-fluoremethyloxycarbonyl) group, and their side chains, which are capable of reacting during synthesis, are also protected by trityl, t-butyl or t-butyl ether groups. The stock solutions of amino acids are prepared at a concentration of 0.33 M in NMP (N-methyl-pyrrolidone) containing 0.5 M HOBt (hydroxybenzotriazole). The amino acid deposition is carried out using the ASP 222 robot (Abimed, Langenfeld, Germany), controlled via the AutoSpot XL software. The use of this robot makes it possible to simultaneously make up to 4 membranes of 96 spots, ie 384 peptides. For a coupling cycle of an amino acid, the robot deposits 0.7 μl of the activated amino acid solution extemporaneously (a volume of 1.1 M diisopropyl carbodiimide solution diluted in NMP for 3 volumes of stock solution amino acid) on the membranes. This deposit is repeated a second time, then the membranes are rinsed in DMF (N, N-dimethylformamide). The unreacted NH 2 groups are then acetylated by 4 to 6 incubations of 10 minutes in a solution of 10% DMF acetic anhydride, in order to avoid the appearance of abortive or truncated peptides. After 3 washes for 2 minutes in DMF, the Fmoc groups protecting the amino function of the amino acids are cleaved by incubation for 5 minutes in a solution of piperidine 20% DMF. After 4 washes in DMF, the spots are stained with 1% DMF bromophenol blue solution, then the membrane is rinsed 3 times with methanol and dried in the open air before the next coupling cycle. . This protocol is repeated for the addition of each new amino acid. After coupling the last amino acid, the peptides are acetylated to allow blocking of all free NH 2 groups, thereby preventing the addition of another amino acid. Then the side chains of all the peptides are deprotected by incubating the membranes in a trifluoroacetic acid / dichloromethane / triisobutylsilane bath (5: 5: 0.3) for 1 hour. The membranes are then rinsed 4 times with dichloromethane, 3 times with DMF and 3 times with methanol before being dried in the open air and stored at 25 ° -20 ° C. until immunorevelation. To immunorelease the spots with a monoclonal antibody, the membranes are first rinsed in methanol, then washed in TBS (50 mM Tris-HCl pH 8.0, 140 mM NaCl, 3 mM KCl) before being incubated overnight. at room temperature in the saturation solution (10X 30 casein concentrate solution (Western Blocking reagent, Roche) diluted in TBS-Tween 20 0.05% (TBS-T) and containing 5% sucrose). After washing for 10 minutes in TBS-T, the membranes are incubated for 1 h 30 at 37 ° C. with the monoclonal antibody diluted to 20 μg / ml in saturation solution. The membranes are then washed 3 times with TBS-T and are then incubated with the anti-mouse conjugate conjugated to alkaline phosphatase (Jackson Immunoresearch), diluted to 1 / 2000th in saturation solution. After 2 washes of 10 minutes in TBS-T, then 2 washes in CBS (10 mM citric acid pH 7, 140 mM NaCl, 3 mM KCl), the developer, prepared extemporaneously (5-bromo, 4-chloro, 3-indoyl). 600 μM phosphate, thiazolyl blue tetrazolium bromide 720 μM, and 5 mM MgCl2 in CBS) is contacted with the membrane for 30-45 minutes in the dark. The immunoreactive peptides appear in blue-violet. After 3 rinses in distilled water, the membranes are scanned and then preserved in water until regeneration. The regeneration makes it possible to eliminate the antibodies and the conjugates fixed on the peptides, which thus makes it possible to carry out a new immunoreactivity test vis-à-vis another antibody. The membranes undergo a series of washings of 10 minutes each: 1 washing in distilled water, 6 washes in DMF, 3 washes in buffer of regeneration A (8 M urea, 35 mM SDS (sodium dodecyl sulfate), p-mercaptoethanol 0.1 %), 3 washes in regeneration buffer B (distilled water / ethanol / acetic acid 4: 5: 1), then 2 washes in methanol. The membranes are then dried in the open air before being stored at -20 ° C. Characterization of epitopes by screening phage-borne peptide libraries was performed using the New England Biolabs PhD12 Phage Display Peptide Library Kit (Cat No. E # 8110S), following the instructions provided with the kit. , version 2.7 of the protocol dating from November 2007. A third epitope localization technology was used: the construction of a library of fragments of recombinant L-FABP protein against which the affinity of the antibodies of interest was tested. Results Table 9 summarizes the epitopes recognized by L-FABP antibodies whose epitope has been analyzed by the three techniques mentioned. Table 9 Antibody Epitope Sequence Epitope Type Epitope Technique 2C9G6 1 N-term 1-56 SEQ ID NO: 21 Contextual fragment library 3D6G1 1 N-term 1-56 SEQ ID NO: 21 Contextual fragment library 881088 1 N-term 1-56 SEQ ID NO: 21 Contextual fragment library 5A8H2 2 N-term 1-65 SEQ ID NO: 22 Sequence Fragment library required for (AA 57 to 65 required) binding 3 HLSEYHPVVHPRA SEQ ID NO : 23 Mimotope Epitope Screening phage library conformation nel 7D8A11 4 TELNGDIITNTMTLGDIVF KRISKRI Spotscan linear epitope and fragment library SEQ ID NO: 24 (102-127) aAbstract amino acid sequence of L-FABP binding region to antibody tested . The numbers in parentheses correspond to the position of the epitope on the amino acid sequence of LFABP, the numbering starting at the initial methionine.

The epitopes recognized by the antibodies 3A7B4 and 5A8H2 could not be determined by the Spotscan technique, indicating that they are not linear. Screening of phage-borne peptide libraries allowed for the selection of a mimotope (linear sequence mimicking an epitope) that reacts with the 5A8H2 antibody. The consensus sequence of this mimotope was aligned to determine a consensus sequence which is shown in Table 9, which represents the minimal sequence recognized by the antibody. It was not possible to find this consensus sequence in the primary structure (or peptide sequence) of L-FABP. Thus, this consensus sequence corresponds to residues located at different locations on the primary structure of the protein but which share a proximity in its three-dimensional structure to form a conformational epitope. In addition, the use of the library of recombinant protein fragments made it possible to locate the epitope for the antibodies 2C9G6, 3D6G1 and 8B10B8 in the first 56 amino acids of the protein. The 5A8H2 antibody requires 9 additional amino acids (AA 1-65) to bind to the binding site opposite the binding site of the 7D8A11 antibody. The 7D8A11 antibody is the only one that has a linear epitope that can be characterized as a spotcan. Development of sandwich ELISA assays against the LFABP protein on the Vidas® automaton The LFABP protein was assayed using the antibodies described in Example 1 and a sandwich immunoassay using, for example, Vidas @ ELISA automaton (bioMérieux). This type of test can also be performed microplate, automated or manual. In a first step, the various available antibodies were tested to select a functional antibody pair, that is, at least one antibody capable of capturing the biomarker protein (capture antibody) and at least one antibody capable of reveal the biomarker molecule (revealing antibody). To do this, the ELISA test was constructed using the reagents of the Vidas @ HBs Ag Ultra kit (bioMérieux, Cat No. 30315). The reagents were used as described in the relevant leaflet (Cat.Nr. 11728 D - EN - 2005/05), as amended: The reagents were used as described in the corresponding leaflet (cat.no 11728D-EN-2005/05 ), with the following modifications: - The cones were sensitized with the selected 5 capture Acids used at a concentration of 10 μg / ml. - The contents of the second well of the HBS Ag Ultra cartridge were replaced with 300 μl of biotin-conjugated antibody, diluted to 1 μg / ml in the second well buffer of the Vidas® HBs Ag Ultra kit (buffer). with goat serum and 1 g / l sodium azide). - The serum sample (50p1) was diluted directly into the second well of the HBS Ag Ultra cartridge. The ELISA reaction was carried out using the Vidas® automaton and the HBS protocol whose incubation step of the sample with the capture and revelation antibodies had been increased to 100 cycles. The results were obtained in the form of raw values after subtraction of the background noise (reading of the substrate before reaction). A standard curve was established by assaying a concentration range of the recombinant biomarker protein. The standard curve was plotted by plotting on the abscissa the concentration of the biomarker protein and on the ordinate the signal read by Vidas® (RFV or Relative Fluorescence Value). The concentration of the biomarker protein present in the body fluid to be assayed (blood, serum, plasma, saddle) was calculated by plotting the concentration corresponding to the Vidas® read RFV signal. Equivalence of the various ELISA tests developed The aim was to evaluate the correlation between the various sandwich immunoassays developed. The Vidas® tests were compared with each other and also compared to the test marketed by Hycult biotechnology for assaying human L-FABP protein (Cat No. HK404), as described in WO 2009019368. equivalence were achieved through serum samples from patients with colorectal cancer (CRC) or from healthy subjects blood donor controls (EFS), respectively obtained in hospital departments under 2 protocols Huriet law, and in a French institution blood bank (healthy subjects). TABLE 10 Doses of LFABP in ng / ml obtained with the commercial Hycult biotechnology test and with the various Vidas® tests developed Patient Hycult 2C9G6 / 5A8H2 5A8H2 / 3D6G1 2C9G6 / 2E5G9 3A7B4 / 3D6G1 3D6G1 / 5A8H2 8B10B8 / 5A8H2 CBSE004 7.78 2, 87 1.74 2.57 CBSE011 7.73 1.92 1.33 1.72 CBSE012 0.11 2.31 1.83 2.07 CBSE018 1.35 0.75 1.19 CBSE019 6.65 4.48 3.41 3.98 CBSE023 8.21 4.60 4.92 4.07 CBSE025 16.38 7.88 7.45 6.87 CLSP043 2.89 1.94 2.49 1.70 2.47 2, 29 0.22 CLSP047 1.94 1.39 0.00 1.22 CLSP078 2.56 13.01 8.27 11.33 CLSP090 3.77 1.18 0.98 1.04 CLSP164 6.54 4.18 4.77 4.60 4.49 4.14 1.54 CLSP165 7.86 1.27 1.68 1.80 2.32 1.82 1.64 CLSP167 8.17 0.00 0.43 0.00 2.01 0.00 0.52 CLSP170 6.02 4.36 4.26 4.61 4.16 3.60 2.12 CLSP174 23.64 5.57 6.73 6.26 7.80 4.67 7.39 CLSP182 7.25 4.47 4.61 5.50 4.46 4.21 4.06 CLSP183 16.90 11.07 10.12 9.79 9.32 7.08 15.94 CLSP185 19, 63 5.35 7.01 6.36 8.41 4.44 5.32 CLSP186 7.06 4.59 4.42 5.35 4.44 3.39 2.72 CLSP189 5.34 0.00 0, 25 0.00 1.16 0.00 0.69 CLSP194 13 , 09 5.38 5.65 6.71 5.40 5.17 5.52 CLSP198 8.16 2.20 3.10 3.14 4.28 2.88 1.94 CLSP207 6.02 4.60 4 , 71 5.58 4.67 4.01 1.91 GHBD020 2.24 5.46 3.08 4.81 GHBD021 1.84 1.58 1.41 GHBD029 2.75 3.42 2.74 3.05 N000533 3.46 0.59 1.18 0.96 1.51 1.21 1.85 N002301 7.78 5.54 5.19 4.88 N005702 2.74 1.04 0.79 0.91 N009944 3 , 49 1.41 2.06 2.66 1.90 1.98 1.69 N011147 2.86 0.92 1.52 1.82 1.41 1.50 1.16 N011968 2.06 1.26 1 , 56 1.64 1.62 1.65 0.97 N011984 5.29 0.87 1.32 1.21 1.80 1.45 1.19 N014106 2.66 0.45 0.91 0.66 1 , 0.89 0.74 N015402 4.41 1.34 0.90 1.18 N017234 4.04 1.90 2.49 3.33 2.41 2.61 2.48 N017269 2.89 1.46 2.13 2.38 2.00 1.97 2.01 N018544 2.42 0.99 1.47 2.17 1.49 1.67 1.11 N018552 2.28 0.00 0.15 0.00 0.52 0.00 0.47 N020501 11.37 2.61 2.90 2.34 N045730 2.29 0.19 0.69 0.63 0.89 0.61 0.74 N054582 1.84 1, 45 1.65 1.28 N057046 5.59 0.82 0.00 0.70 N057054 21.05 2.69 3.25 2.40 N074598 2.53 1.53 1.13 1.36 N286613 4.11 2.38 2.54 3.30 2.56 3.00 3.83 N286656 7.95 1.77 2.26 2.73 3.24 2.60 4.12 N286680 4.58 2.7 5 2.86 3.30 2.85 3.39 1.45 N318050 3.93 1.56 2.16 2.18 2.05 N318341 4.46 1.72 2.38 2.86 2.12 2, 40 1.86 N318384 1.63 0.60 1.19 1.19 1.12 1.18 0.71 N318421 3.86 0.69 1.15 1.45 1.47 1.14 1.56 N329630 6 , 90 1.24 1.69 2.34 2.28 1.82 4.04 N376488 4.73 0.64 1.20 1.30 1.57 1.14 1.57 N376912 2.55 0.27 0 , 84 0.94 1.15 0.80 1.07 N40776- 3.46 0.62 1.00 0.98 1.35 1.15 1.21 N418599 2.60 1.11 1.46 1.94 1.49 1.63 2.01 N46043- 4.18 0.67 1.34 1.18 1.72 1.03 0.90 N461993 2.92 0.00 0.16 0.01 0.69 0, 00 0.73 N462187 3.96 0.79 1.32 1.18 1.79 1.43 1.23 N483535 6.42 0.00 0.30 0.00 1.28 0.00 0.83 N491678 7 , 73 3.86 3.38 3.43 N494801 4.38 2.96 2.64 2.65 N511498 2.24 0.46 0.81 0.71 1.03 0.78 0.51 N520547 7.74 3.03 3.74 2.70 N527039 2.36 0.31 0.79 0.41 1.03 0.70 0.60 N530485 6.80 0.00 0.32 0.00 1.40 0.00 0.66 N593116 2.38 0.99 1.41 1.95 1.42 1.49 1.46 N734641 3.04 8.30 7.31 7.24 N748022 2.14 0 0.17 0.00 0 , 46 0.00 0.47 N828353 8.21 4.52 4.11 4.01 N831245 7.74 2.77 2.32 2.48 N862300 3.59 1.79 4.75 3.53 2.33 The correlates of these different tests compared to the 2C9G6 / 5A8H2 test are reported in Table 11 below: Table 11 Correlation data between different ELISA tests Parameter 5A8H2 / 3D6G1 2C9G6 / 2E5G9 3A7B4 / 3D6G1 3D6G1 / 5A8H2 8B10B8 / 5A8H2 Hycult Number of pairs XY 47 45 47 45 47 47 Correlation of Spearman 0.9864 0.9783 0.9244 0.9909 0.8091 0.5298 Confidence Interval 0.9752 to 0.9926 0.9598 to 0.9883 0.8653 to 0.9581 0.9831 to 0.9951 0.6752 to 0.8914 0.2781 to 0.7135 95% Value of P value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0001 (bilateral) Is the correlation Yes Yes Yes Yes Yes significant? (alpha = 0.05) The various tests developed in Vidas® have a good correlation between them and can all be used to determine the LFABP protein. EXAMPLE 4 Detection of Patients With Colorectal Cancers and Patients Controlled by the Prolidase MRM Assay 1) Patients and Samples The collection of blood samples was carried out through a network of 8 clinical centers distributed throughout France, in France. the framework of 2 Huriet law protocols for patients with colorectal cancer. For control patients, negative colonoscopies, a multicentric study involving about 30 gastroenterologists was set up to collect the blood of HemoccultTM positive patients, aged 50 to 75, before carrying out the colonoscopy and any colonic preparation. Healthy blood donors were collected at the French Blood Establishment (EFS) in Lyon, and at EFS Grenoble. Patients labeled "MedGe" are patients aged 51 to 74, who were collected in Poland as part of a general medicine consultation.

All samples were taken in compliance with current legislation and Good Clinical Practice, following a process of storage and monitoring in accordance with good laboratory practices, colorectal cancer patients and negative colonoscopy checks being taken exactly under the same conditions . In order to obtain serum, the blood sample is taken on a dry tube. After coagulation, the tube is centrifuged for 10 min at 1000 g, the serum is removed, aliquoted and stored at -80 ° C. The samples are well documented for the clinical history of patients.

To obtain plasma, the blood sample is taken on an EDTA tube. After centrifugation for 10 min at 1000 g, the plasma is removed, aliquoted and stored at -80 ° C. The samples are well documented for the clinical history of patients. A total of 331 patients were tested: 102 patients with invasive colorectal cancer, stages I-IV, 11 patients with Tis-TO, 3 patients with colorectal adenomas and 215 control patients: 70 patients with total colonoscopy normal (ColoNeg), 38 healthy donors from EFS Grenoble (EFS Gre) 37 healthy donors from EFS Lyon (EFS Ly) 70 controls collected in general medical consultation (MedGe) 2) Prolidase Clinical Samples Prolidase protein was assayed with MRM technology, as described in Example 1. In practice only the 400.2 / 686.3 transition of the peptide of SEQ ID NO: 1 was followed and quantified, as well as the corresponding heavy transition. The results are expressed in Arbitrary unit, realizing the ratio of the area under the curve of the peak of the natural transition divided by the area under the curve of the chromatographic peak of the heavy transition: light area / heavy area. Table 12 Serum Levels of Prolidase in Colorectal Cancer Patients (CC R) and Controls (ColoNeq, EFS Ly, EFS Gre, MedGe) Patient Age Gender Class 60 57 Male Tis 57 Male Tis 50, Tis Female 60 Male f Tis 59 Man Tis 57 Man Tis 58 Man Tis 59 Man Tis 56 Man Tis 58 Woman Tis 57 Woman Tis 56 Woman CCR 74 Man CCR 76 Woman CCR 63 Woman CCR 55 Man CCR 84 Woman CCR 71 Woman CCR 51 Man CCR 64 Woman CCR 59 Woman CCR 58 Female CCR 58 Male CCR 54 Male CCR 65 Male CCR 59 Male CCR 56 Male CCR 56 Female CCR 51 Female CCR 52 Female CCR 55 Female CCR 61 Male CCR 60 Male CCR 61 Male CCR 55 Female CCR 50 Female CCR Prolidase Stadium Dose Ratio Unit Arbitrary TO 0,113 TO 0,105 TO 0,109 TO 0,123 TO 0,096 TO 0,109 TO 0,122 TO 0,115 TO 0,132 TO 0,135 TO 0,148 I 0,098 I 0,091 I 0,10759 I 0,163 I 0,103 I 0,080 I 0,079 I 0,123 I 0,143 I 0,105 I 0,093, .. I 0,141 I 0,111 I 0,144 Il 0,168 Il 0,101 Il 0,133 Il 0,110 Il 0,113 Il 0,097 Il 0,109 Il 0,077 Il 0,065 Il 0,084 Il 0,089 CBSE011 CL5P234 CLSP286 CLSP300 CLSP315 CL5P367 CL5P724 CNSE003 CNSE004 GHBD015 GHBD036 CBSE001 CBSE016 CLSP047 CLSP162 CLSP196 CL5P224 CLSP235 CL5P263 CLSP340 CL5P344 CL5P364 CL5P376 GHBD003 GHBD094 CLSP075 CLSP076 CLSP080 CLSP096 CLSP110 CLSP117 CLSP130 CLSP133 CLSP143 CLSP147 CLSP154 CLSP155 63 Man JRC H 0,065 CLSP157 58 Female JRC H 0.097 CLSP165 58 Female CCR H 0.114 CLSP170 60 Female CCR H 0.070 CLSP173 61 Male CCR H 0.093 CLSP177 60 Male CCR H 0.057 CLSP178 60, i CCR H 0.109 Male CLSP179 61 Male CCR He 0.095 CLSP186 63 Female CCR H 0.131 CLSP194 60 Male CCR H 0.093 CLSP201 55 Male CCR H 0.086 CLSP207 55 Male CCR H 0.077 CLSP209 64 Male CCR H 0.118 CLSP220 53 Male CCR H 0.090 CLSP222 58 Male CCR H 0.083 CLSP253 61 Female CCR H 0.10650 CLSP256 86 Female CCR H 0.088 CLSP280 62 Male CCR H 0.103 .. ., CLSP296 56 Male CCR H 0.169. ,, CLSP310 81 Male CCR H 0.122 CLSP327 64 Male CCR H 0.063 CLSP333 60 Male CCR H 0.116 CLSP341 53 Male CCR H 0.079 CLSP346 66 Male CCR H 0.105 CLSP347 49 Female CCR H 0.132 CLSP380 60 Male CCR H 0.083 CNSE002 62 Female CCR H 0.087 GHBD021 71 Female CCR H 0.117 GHBD043 71 Male CCR H 0.084 GHBD064 83 Female CCR H 0.099 GHBD066 82 Female CCR H 0.138 GHBD075 58 Male CCR H 0.104 GHBD087 62 Female CCR H 0.123 GHBD090 59 Male CCR H 0.191 GHBD096 62 Female CCR H 0.110 GHBD100 75 Male CCR H 0.067 GHBD103 68 Male CCR H 0.192 CBSE023 76 Male CCR III 0.141 CLSP044 80 Male CCR III 0.113 CLSP074 79 Female CCR III 0.058 CLSP078 72 Female CCR III 0.078 CLSP081 78 Female CCR III 0.059 CLSP098 61 Male CCR III 0.057 CLSP174 77 Male CCR III 0.086 CLSP227 52 Male CCR III 0.093 CLSP233 59, i CCR III 0.078 Male CLSP255 42 Female CCR III 0.078 CLSP289 52 Male CCR III 0.129 CLSP320 70 Male CCR III 0.104 CLSP324 53 Male CCR III 0.061 CLSP383 78 Male CCR III 0.131 CLSP384 37 Male CCR III 0.144 CNSE001 78 Male CCR III 0.131 CNSE016 65 Male CCR III 0.089 CNSE017 77 Female CCR III 0.093 CSEM015 65 Female CCR III 0.147 CSEM029 63 Female CCR III 0 09 8 _._ GHBD004 58 Male CCR III 0.092 GHBD017 70 Male CCR III 0.078 GHBD034 48 Female CCR III 0.139 GHBD042 77 Female CCR III 0.140 GHBD045 73 Male CCR III 0.032 GHBD089 54 Female CCR III 0.093 GHBD105 79 Male CCR III 0.118 CBSE012 37 Female CCR IV 0.095 CBSE026 72 Male CCR IV 0.073 CLSP156 59 Male CCR IV 0.066 CLSP159 69 Female CCR IV 0.170 CLSP167 81 Male CCR IV 0.089 CLSP260 57 Female CCR IV 0.120 CLSP334 54 Female CCR IV 0.189 CLSP357 78 Male CCR IV 0.125 CLSP365 64 Male CCR IV 0.176 CLSP366 45 Male CCR IV 0.157 GHBD022 56 Male CCR IV 0.145 GHBD071 61 Male CCR IV 0.149 CLSP197 68 Female CCR NA 0.128 HGED005 68 Male Control ColoNeg 0.111 PROMIS 21-01-005 68 Male Control ColoNeg 0.124 PROMIS 21-01-006 62 Male Witness ColoNeg 0,177 t PROMIS 21-01-009 51 Female Witness ColoNeg 0,149 PROMIS 21-01-011 60 Female Witness ColoNeg 0,209 PROMIS 21-01-012 55 Female Witness ColoNeg 0,129 PROMIS 21-02-001 73 Female Witness ColoNeg 0,159 PROMIS 21- 02-003 55, Witness ColoNeg 0.188 Woman PROMIS 2 1-02-004 57 Male Witness ColoNeg 0.294 PROMIS 21-02-006 51 Male Witness ColoNeg 0.130 PROMIS 21-02-008 55 Male Witness ColoNeg 0.197 PROMIS 21-02-011 55 Female Witness ColoNeg 0.120 PROMIS 21-02-014 58 Male Witness ColoNeg 0,123 PROMIS 21-02-016 62 Male Witness ColoNeg 0,191 PROMIS 21-03-004 61 Male Witness ColoNeg 0,136 PROMIS 21-03-013 52 Female Witness ColoNeg 0,279 PROMIS 21-03-015 74 Female Witness ColoNeg 0,194 PROMIS 21 -04-002 67 Male Witness ColoNeg 0.117 PROMIS 21-04-006 72 Male Witness ColoNeg 0.111 ..., "PROMIS 21-04-007 66 Female Witness ColoNeg 0.171 PROMIS 21-04-008 62 Female Witness ColoNeg 0.237 PROMIS 21- 04-009 58 Male Witness ColoNeg 0,203 PROMIS 21-04-013 63 Female Witness ColoNeg 0,121 PROMIS 21-07-004 66 Male Witness ColoNeg 0,149 PROMIS 21-12-001 59 Male Witness ColoNeg 0,159 PROMIS 21-12-007 53 Female Witness ColoNeg 0,10716 PROMIS 21-12-011 52 Male Witness ColoNeg 0,169 PROMIS 21-22-001 61 Female Witness ColoNeg 0,195 PROMIS 21-22-006 65 Female Witness ColoN eg 0,155 PROMIS 21-22-008 57 Woman Witness ColoNeg 0,129 PROMIS 37-03-004 53 Woman Witness ColoNeg 0,110 PROMIS 37-04-001 65 Man Witness ColoNeg 0,252 PROMIS 37-04-005 69 Woman Witness ColoNeg 0,112 PROMIS 37-05 -008 55 Male Witness ColoNeg 0.160 PROMIS 37-06-001 65 Female Witness ColoNeg 0.144 PROMIS 37-09-002 50 Female Witness ColoNeg 0.167 PROMIS 37-11-001 56 Male Witness ColoNeg 0.10969 PROMIS 37-11-002 59 Male Witness ColoNeg 0,170 PROMIS 37-11-011 71 Male Witness ColoNeg 0,121 PROMIS 37-11-016 50 Female Witness ColoNeg 0,153 PROMIS 37-12-002 63 Male Witness ColoNeg 0,222 PROMIS 37-13-003 53 Female ', Witness ColoNeg 0,174 PROMIS 37-13-006 53 Male Witness ColoNeg 0.160 PROMIS 37-13-008 61 Male Witness ColoNeg 0.203 PROMIS 37-14-009 63 Male Witness ColoNeg 0.132 PROMIS 37-14-018 60 Male Witness ColoNeg 0.087 PROMIS 37-14-021 61 , Witness ColoNeg 0,129 Woman PROMIS 37-15-002 68 Woman Witness ColoNeg 0,165 PROMIS 37-15-006 67 Woman Witness ColoNeg 0,239 PROMIS 37-16-001 57 Man e Witness ColoNeg 0,256 PROMIS 37-17-001 52 Male Witness ColoNeg 0,184 PROMIS 37-17-003 55 Male Witness ColoNeg 0,140 PROMIS 37-17-004 58 Woman Witness ColoNeg 0,139 PROMIS 37-18-001 53 Male Witness ColoNeg 0,134 PROMIS 37 -18-003 50 Female Witness ColoNeg 0.158 PROMIS 37-18-004 57 Male Witness ColoNeg 0.188 PROMIS 37-18-005 55 Female Witness ColoNeg 0.111 PROMIS 37-18-024 Male Witness ColoNeg 0.128 ..., "PROMIS 37-19 -006 50 Man Witness ColoNeg 0.122 PROMIS 37-19-007 51 Woman Witness ColoNeg 0.136 PROMIS 37-20-004 53 Woman Witness ColoNeg 0.179 PROMIS 71-02-003 60 Woman Witness ColoNeg 0.159 PROMIS 71-04-003 75 Woman Witness ColoNeg 0,161 PROMIS 71-09-007 52 Male Witness ColoNeg 0,175 PROMIS 71-10-001 56 Female Witness ColoNeg 0,171 PROMIS 71-10-007 71 Female Witness ColoNeg 0,162 PROMIS 71-11-001 63 Male Witness ColoNeg 0,191 PROMIS 71-11- 006 59 Male Witness ColoNeg 0.155 PROMIS 71-11-008 54 Male Witness ColoNeg 0.181 PROMIS 71-11-009 60 Female Witness ColoNeg 0.140 801725-SER 4 3 Male Witness EFS Gre 0.148 801726-SER 45 Male Witness EFS Gre 0.163 801727-SER 34 Male Witness EFS Gre 0.131 801729-SER 51 Female Witness EFS Gre 0.132 801730-SER 44 Female Witness EFS Gre 0.148 801731-SER 65 Female Witness EFS Gre 0,182 801732-SER 24 Female Witness EFS Gre 0.150 801733-SER 37 Female Witness EFS Gre 0.127 801734-SER 36 Male Witness EFS Gre 0.193 801735-SER 61 Female Witness EFS Gre 0.171 801736-SER 56 Male Witness EFS Gre 0.178 t 801737-SER 51 Man Witness EFS Gre 0.125 801739-SER 45 Woman Witness EFS Gre 0.150 801740-SER 52 Man Witness EFS Gre 0.112 801741-SER 46 Man Witness EFS Gre 0.182 801742-SER 36, Witness EFS Gre 0.146 Man 801743-SER 57 Woman Witness EFS Gre 0.178 801744-SER 52 Male Witness EFS Gre 0.191 801746-SER 60 Female Witness EFS Gre 0.171 801747-SER 60 Female Witness EFS Gre 0.247 801748-SER 58 Male Witness EFS Gre 0.366 801749-SER 18 Female Witness EFS Gre 0.124 801750-SER 61 Male Witness EFS Gre 0.175 801751-SER 30 Female Witness EFS Gre 0.165 801752-SER 22 Female Witness EFS Gre 0.141 801753-SER 40 Female Witness EFS Gre 0.124 801754-SER 57 Male Witness EFS Gre 0.154 ..., 801757-SER 26 Male Witness EFS Gre 0.184 .. ', 801758-SER 38 Female Witness EFS Gre 0.157 801759-SER 52 Female Witness EFS Gre 0.115 801760-SER 42 Male Witness EFS Gre 0.173 801761-SER 18 Female Witness EFS Gre 0.190 801762-SER 22 Female Witness EFS Gre 0.140 801763-SER 23 Female Witness EFS Gre 0.109 801764 -SER 36 Female Witness EFS Gre 0.195 801765-SER 47 Male Witness EFS Gre 0.163 801766-SER 39 Female Witness EFS Gre 0.187 801767-SER 28 Female Witness EFS Gre 0.178 P040 02 011 DL 64 Male Witness MedGe 0.114 P040 02 012 CR 69 Male Control MedGe 0.219 P040 02 014 MG 62 Male Control MedGe 0.137 P040 02 015 HT 61 Male Control MedGe 0.143 P040 02 017 JS 60 Male Control MedGe 0.150 P040 02 018 PB 59 Male Control MedGe 0.158 P040 02 019 PK 52 Male Control MedGe 0.144 P040 02 020 KA 54 Male Control MedGe 0.100 P040 02 021 JL 63 Male Control MedGe 0.232 P040 02 022 MK 69 Male Witness MedGe 0,158 P040 02,023 CM 67 Male Witness MedGe 0,117 P040 02,024 TM 58 Male Witness MedGe 0,242 P040 02,025 PA 53 Male Witness MedGe 0,214 P040 02,026 MR 59 Male Witness MedGe 0,121 P040 02,027 HF 57 Male MedGe control 0.100 P040 02 030 Z, J 60 Male MedGe control 0.149 P040 02 031 P \ N 56 Male MedGe control 0.115 P040 02 032 \ NR 51 Male MedGe control 0.141 P040 02 033 \ NZ 68 Male MedGe control 0.171 P040 02 034 SJ 59 Male Control MedGe 0,132 P040 02,035 KF 70 Male Control MedGe 0,115 P040 02,036 PZ 52 Male Control MedGe 0,203 P040 02,027 GB 62 Male Control MedGe 0,126 P040 02,038 VVS 74 Male Control MedGe 0,160 P040 02,039 KK 54 Male Control MedGe 0,184 P040 02 040 RT 59 Male MedGe Witness 0.171 P040 02 041 DD 62 Male MedGe Witness 0.118 P040 02 042 \ NZ 63 Male MedGe Witness 0.128 P040 03 011 BH 61 Male MedGe Witness 0.146 P040 03 012 BJ 59 Male MedGe Witness 0.152 P040 03 013 JE 52 Male Witness MedGe 0.246 P040 03 014 GT 51 Male Witness n MedGe 0.146 P040 03 015 SM 64 Male Control MedGe 0.103 P040 03 016 CB 64 Male Control MedGe 0.144 P040 03 018 SS 62 Male Control MedGe 0.162 P040 03 019 RH 57 Male Control MedGe 0.152 P040 03 020 TJ 56 Male Control MedGe 0.140 P040 03 021 R \ N 51 Male Control MedGe 0.124 P040 03 022 ZZ 60 Male Control MedGe 0.171 P040 03 023 PA 63 Male Control MedGe 0.152 P040 03 024 LS 52 Male Control MedGe 0.157 P040 03 026 PS 69 Male Control MedGe 0.165 P040 03 027 FZ 62 Male Control MedGe 0.200 P040 03 028 VVT 52 Male Control MedGe 0.124 P040 03 029 BZ 64 Male Control MedGe 0.149 P040 03 030 MR 53 Male Control MedGe 0.169 P040 03 031 TM 52 Male Control MedGe 0.101 P040 03 032 B \ N 55 Male Control MedGe 0.117 P040 03 033 JR 52 Male Control MedGe 0.158 P040 03 034 RJ 66 Male Control MedGe 0.136 P040 03 036 CZ 61 Male Control MedGe 0.147 P040 03 037 SZ 52 Male Control MedGe 0.153 P040 03 038 BT 60 Male Control MedGe 0.128 P040 03 039 CJ 62 Male Witness MedGe 0,168 P040 03,040 DF 59 Male Control MedGe 0.213 P040 03 041 MN 56 Male Control MedGe 0.130 P040 03 042 SA 67 Male Control MedGe 0.119 P040 03 043 KJ 61 Male Control MedGe 0.124 P040 03 044 LA 53 Male Control MedGe 0.136 P040 03 045 BM 52 Male Control MedGe 0.144 P040 03 046 TS 55 Male Control MedGe 0.146 P040 03 047 NJ 59 Male Control MedGe 0.170 P040 03 048 KZ 58 Male Control MedGe 0.203 P040 03 049 CK 63 Male Control MedGe 0.157 P040 03 051 DJ ND Male Control MedGe 0.162 P040 03 052 DJ ND Male Witness MedGe 0.246 P040 03 053 SZ ND Male Witness MedGe 0.156 P040 06 005 SH 61 Male Witness MedGe 0.153 P040 06 006 SM 71 Male Witness MedGe 0.141 P040 06 007 KJ 70 Male Witness MedGe 0.136 N0037612 54 Male Witness EFS Ly 0.126 N0037639 36 Female Witness EFS Ly 0.093 N0037751 54 Male Witness EFS Ly 0.184 N011968 50 Male Witness EFS Ly 0.180 N0247243 42 Male Witness EFS Ly 0.155 N0247366 32 Female Witness EFS Ly 0.152 N027871X 59 Female Witness EFS Ly 0.131 N0352209 65 Female Witness EFS Ly 0.136 N0352217 54 Female Witness EFS Ly 0,127 N0352305 59 Male Witness EFS Ly 0,176 N0352524 62 Female Witness EFS Ly 0,173 N0352604 61 Female Witness EFS Ly 0,142 N0356509 44 Female Witness EFS Ly 0,136 N0389490 49 Male Witness EFS Ly 0,124 N041234 57 Male Witness EFS Ly 0,180 N042229 54 Male Witness EFS Ly 0,121 N057046 51 Female Witness EFS Ly 0,230 N1200020 49 Male Witness EFS Ly 0,208 N1200039 64 Female Witness EFS Ly 0,215 N144032 59 Female Witness EFS Ly 0,169 N146687 51 Female Witness EFS Ly 0,10751 N353910 52 Male Witness EFS Ly 0 , 10717 N461790 57 Male Control EFS Ly 0,158 N462187 56 Male Control EFS Ly 0,10822 N483770 58, Control EFS Ly 0,163 Male N491627 51 Control Male EFS Ly 0,184 N50798X 59 Control EFS Ly 0,200 N53016X 59 Control EFS Ly 0,130 N530442 56 Male Witness EFS Ly 0,149 N530485 53 Female Witness EFS Ly 0,177 N543710 57 Female Witness EFS Ly 0,148 N545062 56 Male Witness EFS Ly 0,171 N555658 53 Female Witness EFS Ly 0,145 N59426X 52 Female Witness EFS Ly 0 , 146 N626516 53 Male Control EFS Ly 0.138 N828361 63 Female Control EFS Ly 0.090 N830656 64 Male Control EFS Ly 0.162 Prolidase was decreased in patients with invasive CRC and patients with carcinoma in situ Tis TO compared to control patients with the MRM technique used. In this example with the MRM test used, we set the threshold for the detection of invasive colorectal cancers and Tis at 0.10696 (dose strictly lower than that of the second ColoNeg control of the cohort for the sample / standard ratio of the quantification of peak of the 400.2 / 686.3 transition of the peptide of SEQ ID NO: 1. 59 of the 113 CCR or Tis patients are below the threshold and are detected as colorectal cancer, whereas only 1 ColoNeg control, 3 MedGe controls and 2 EFS Ly controls are below the threshold, ie 52.2% sensitivity (42.61% to 61.70%) for 97% specificity (94.03% to 98.97%) Table 13 15 Serum assays Prolidase in Patients Colorectal Adenomas N ° Patient Age Gender Class Histology Prolidase Dose Ratio Arbitrary Unit CLSP337 63 Female Adenoma High Risk High Grade Dysplasia 0.079 CLSP378 75 Male Adenoma High Risk High Grade Dysplasia 0.1736 GHBD079 76 Male Adenoma Low Risk Low grade dysplasia 0.115 Keeping the threshold set above at 0.10696, the prolidase is decreased in one patient with colorectal adenoma out of 3.

Example 5: Use of serum assays of tumor markers in combination 1) Selection of proteins and assays The Applicant has shown in Example 4 that abnormally decreased doses of tracer peptides of prolidase could be observed in the bloodstream of some patients. patients with colorectal cancer. On the other hand, the Applicant has shown in patent applications WO 2009024691, WO2009019365, WO2009019368, WO2009019369, WO2009019366, WO2009019370, WO2009019367, WO2010004214, WO2010112777 that abnormally high or abnormally decreased doses of other tumor markers such as Ezrin. , LFABP, Apo A1, Apo A2, Beta2 Microglobulin, ACE, CA19-9, Galectin-3, 20S Proteasome, Protein Disulfide Isomerase (PDI) and ProDefensin A6 could be seen in the bloodstream of some cancer patients. colorectal. The methods of assaying these tumor markers have been described in the patent applications raised. The L-FABP, Apo A1, Apo A2, Beta 2 Microglobulin, HSP60, ACE, CA19-9, Galectin-3, Ezrin, Proteasome 20S (prosomes), Timp1 and M2PK proteins were assayed as described in Example 3 The assays can also be carried out using the MRM mass spectrometry assay technology, as described in Examples 1, 2 and 4. In particular, the prolidase (PEPD) was assayed as described in Examples 1 and 4. , and the protein Alpha-Enolase, the protein 78 kDa glucose-regulated protein (BIP, GRP78), the protein Glyceraldehyde-3-phosphate dehydrogenase (G3PDH, GAPDH), the heat shock protein cognate 71 kDa protein (HSC70), the Disulfite isomerase protein (PDI), Peroxiredoxin-5 protein, mitochondrial (Peroxy-5) were assayed in patient sera and controls as described in Example 2. 2) Patients and specimens Patients tested for marker combination are the 113 color cancer patients ectal invasive (CCR) or Tis and the 70 negative colonoscopy controls (ColoNeg) that were described in Example 4. The samples tested are sera, except for the Timp1 and M2PK proteins for which they are the plasma of the same patients , collected at the same time following the procedure described in example 4. 3) Combination methodology and scores Surprisingly, the increase or decrease in the blood dose of two given markers is not systematically observed in the same patients . As a result, the combination of several tumor markers makes it possible to increase the number of patients identified as having colorectal cancer. Thus a patient A may have an increase or decrease in one or more tumor markers (group X), said group X markers may be normal in a patient B; in the same patient B one or more other tumor markers (group Y) can be raised or lowered, said group Y markers may be normal in patient A. The various tumor markers assayed by the Applicant can thus be combined by means of various mathematical algorithms well known to those skilled in the art. By way of illustration and without this example being exhaustive, the following method has been implemented: A threshold value has been set for each tumor marker, according to a desired minimum specificity. - When the blood level of the tumor marker was increased in case of colorectal cancer, the blood dose obtained for a given patient was divided by its threshold value. When the blood dose of the tumor marker was decreased in case of colorectal cancer, the blood dose obtained for a given patient was reversed and then multiplied by its threshold value. - When the ratio, blood dose divided by threshold value, was greater than 1, the ratio was multiplied by a coefficient, for example 10. The value thus obtained was named "score" for the patient studied tumor marker considered. The score is therefore systematically positive, whether the tumor marker is increased or decreased in the blood circulation of patients with colorectal cancer. The scores obtained for different tumor markers were added by weighting them by a factor specific to each marker. In the case of the example below all the weighting factors were set to 1. - The sum of the scores was divided by the total number of scores summed and the value thus obtained was called the "total score". - The patient is diagnosed as having colorectal cancer when his total score is increased compared to a threshold score, again fixed according to the desired specificity.

With this method, it is therefore sufficient for a given patient that a single marker has a positive score for the total score in combination is positive. The total scores for selection of different 2-marker combinations including prolidase are given in Table 16. The total scores for selection of different 3, 4, 5 and 8 marker combinations comprising prolidase are given in Table 15. The total scores for examples of 3- and 4-label combinations not including prolidase are given in Table 16. The combination of markers was evaluated on the same group of 113 patients with invasive colorectal cancer or Tis, and 70 controls 25 controls with a colonoscopy totally negative. Table 14 gives the results for prolidase in dose (reduced marker dose in certain invasive CCR colorectal cancers and some Tis) and in score (increased score,> 1, in some invasive CCR colorectal cancers and some Tis). Table 14 Patient Class Stage Prolidase Dose Ratio Prolidase Score Arbitrary Unit Threshold 0.10696 1 CBSE011 Tis TO 0.111 0.95 CL5P234 Tis TO 0.105 16.2 CL5P286 Tis TO 0.109 0.98 CLSP300 Tis TO 0.123 0.87 CLSP315 Tis TO 0.096 17.8 CLSP367 Tis TO 0.109 0.98 CLSP724 Tis TO 0.122 0.88 CNSE003 Tis TO 0.115 0.93 CNSE004 Tis TO 0.132 0.81 GHBD015 Tis TO 0.135 0.79 GHBD036 Tis TO 0.148 0.72 CBSE001 CCR I 0.098 17 , CBSE016 CCR I 0.091 18.8 CLSP047 CCR I 0.108 0.99 CLSP162 CCR I 0.163 0.65 CLSP196 CCR I 0.103 16.7 CLSP224 CCR I 0.080 21.5 CLSP235 CCR I 0.079 21.6 CLSP263 CCR I 0.123 0, 87 CLSP340 CCR I 0.143 0.75 CLSP344 CCR I 0.105 16.4 CLSP364 CCR I 0.093 18.4 CLSP376 CCR I 0.141 0.76 GHBD003 CCR I 0.11 0.97 GHBD094 CCR I 0.144 0.7 CLSP075 CCR It 0.168 0.64 CLSP076 CCR Il 0,101 16,9 CLSP080 CCR Il 0,133 0,80 CLSP096 CCR Il 0,110 0,97 CLSP110 CCR Il 0,113 0,94 CLSP117 CCR It 0,097 17,6 CLSP130 CCR Il 0,109 0,98 CLSP133 CCR It 0,077 22,2 CLSP143 CCR It 0.065 26.2 CLSP147 CCR It 0.084 20, 5 CLSP154 CCR It 0.089 19.2 CLSP155 CCR It 0.065 26.2 CLSP157 CCR It 0.097 17.7 CLSP165 CCR It 0.114 0.93 CLSP170 CCR It 0.070 24.4 CLSP173 CCR It 0.093 18.4 CLSP177 CCR It 0.057 30.3 CLSP178 CCR It 0.109 0.98 CLSP179 CCR It 0.095 18.0 CLSP186 CCR It 0.131 0.81 CLSP194 CCR It 0.093 18.3 CLSP201 CCR It 0.086 20.0 CLSP207 CCR It 0.077 22.3 CLSP209 CCR It 0.118 0.90 CLSP220 CCR It 0.090 19.0 CLSP222 CCR It 0.083 20.7 CLSP253 CCR It 0.106 16.1 CLSP256 CCR It 0.088 19.4 CLSP280 CCR It 0.103 16.5 CLSP296 CCR It 0.169 0.63 CLSP310 CCR It 0.122 0.88 CLSP327 CCR It 0.063 27.0 CLSP333 CCR It 0.116 0.92 CLSP341 CCR It 0.079 21.6 CLSP346 CCR It 0.105 16.3 CLSP347 CCR It 0.132 0.81 CLSP380 CCR It 0.083 20.7 CNSE002 CCR It 0.087 19.7 GHBD021 CCR It 0.117 0.92 GHBD043 CCR It 0.084 20.4 GHBD064 CCR It 0.099 17.2 GHBD066 CCR It 0.138 0.78 GHBD075 CCR It 0.104 16.4 GHBD087 CCR It 0.123 0.87 GHBD090 CCR It 0.191 0.56 GHBD096 CCR It 0.110 0.98 GHBD100 CCR It 0.067 25.4 GHBD103 CCR It 0.192 0.56 CBSE023 CC R III 0.141 0.76 CLSP044 CCR III 0.113 0.95 CLSP074 CCR III 0.058 29.4 CLSP078 CCR III 0.078 21.8 CLSP081 CCR III 0.059 28.8 CLSP098 CCR III 0.057 30.0 CLSP174 CCR III 0.086 19.8 CLSP227 CCR III 0.093 18.5 CLSP233 CCR III 0.078 21.9 CLSP255 CCR III 0.078 22.0 CLSP289 CCR III 0.129 0.83 CLSP320 CCR III 0.104 16.4 CLSP324 CCR III 0.061 28.2 CLSP383 CCR III 0.131 0.82 CLSP384 CCR III 0.144 0.74 CNSE001 CCR III 0.131 0.82 CNSE016 CCR III 0.089 19.2 CNSE017 CCR III 0.093 18.4 CSEM015 CCR III 0.147 0.73 CSEM029 CCR III 0.098 17.5 GHBD004 CCR III 0.092 18.6 GHBD017 CCR III 0.078 22.0 GHBD034 CCR III 0.139 0.77 GHBD042 CCR III 0.140 0.77 GHBD045 CCR III 0.032 54.3 GHBD089 CCR III 0.093 18.4 GHBD105 CCR III 0.118 0.90 CBSE012 CCR IV 0.095 18.0 CBSE026 CCR IV 0.073 23 , 3 CLSP156 CCR IV 0.066 26.1 CLSP159 CCR IV 0.170 0.63 CLSP167 CCR IV 0.089 19.3 CLSP260 CCR IV 0.120 0.89 CLSP334 CCR IV 0.189 0.57 CLSP357 CCR IV 0.125 0.85 CLSP365 CCR IV 0.176 0, 61 CLSP366 CCR IV 0.157 0.68 GHBD022 CCR IV 0.145 0.74 GHBD071 CCR IV 0.149 0.72 CLSP197 CCR NA 0.128 0.83 HGED005 ColoNeg indicator 0.112 0.96 PROMIS 21-01-005 ColoNeg indicator 0.124 0.86 PROMIS 21-01-006 ColoNeg indicator 0.177 0.60 PROMIS 21 -01-009 ColoNeg indicator 0,149 0,72 PROMIS 21-01-011 ColoNeg indicator 0,209 0,51 PROMIS 21-01-012 ColoNeg indicator 0,129 0,83 PROMIS 21-02-001 ColoNeg indicator 0,159 0,67 PROMIS 21-02 -003 ColoNeg indicator 0,188 0,57 PROMIS 21-02-004 ColoNeg indicator 0,294 0,36 PROMIS 21-02-006 ColoNeg indicator 0,130 0,82 PROMIS 21-02-008 ColoNeg indicator 0,197 0,54 PROMIS 21-02-011 Witness ColoNeg 0.120 0.89 PROMIS 21-02-014 Witness ColoNeg 0.123 0.87 PROMIS 21-02-016 Witness ColoNeg 0.191 0.56 PROMIS 21-03-004 Witness ColoNeg 0.136 0.79 PROMIS 21-03-013 Witness ColoNeg 0,279 0,38 PROMIS 21-03-015 Witness ColoNeg 0,194 0,55 PROMIS 21-04-002 Witness ColoNeg 0,117 0,92 PROMIS 21-04-006 Witness ColoNeg 0,111 0,96 PROMIS 21-04-007 Witness ColoNeg 0,171 0 , 62 PROMIS 21-04-008 Witness ColoNeg 0.237 0.45 PROMIS 21-04-009 Witness Col oNeg 0,203 0,53 PROMIS 21-04-013 Witness ColoNeg 0,121 0,88 PROMIS 21-07-004 Witness ColoNeg 0,149 0,72 PROMIS 21-12-001 Witness ColoNeg 0,159 0,67 PROMIS 21-12-007 Witness ColoNeg 0 , 10716 0,998 PROMIS 21-12-011 Witness ColoNeg 0,169 0,63 PROMIS 21-22-001 Witness ColoNeg 0,195 0,55 PROMIS 21-22-006 Witness ColoNeg 0,155 0,69 PROMIS 21-22-008 Witness ColoNeg 0,129 0, 83 PROMIS 37-03-004 ColoNeg indicator 0,110 0,98 PROMIS 37-04-001 ColoNeg indicator 0,252 0,42 PROMIS 37-04-005 ColoNeg indicator 0,112 0,96 PROMIS 37-05-008 ColoNeg indicator 0,160 0,67 PROMIS 37-06-001 ColoNeg indicator 0,144 0,74 PROMIS 37-09-002 ColoNeg indicator 0,167 0,64 PROMIS 37-11-001 ColoNeg indicator 0,10696 1,000 PROMIS 37-11-002 ColoNeg indicator 0,170 0,63 PROMIS 37- 11-011 ColoNeg indicator 0,121 0,88 PROMIS 37-11-016 ColoNeg indicator 0,153 0,70 PROMIS 37-12-002 ColoNeg indicator 0,222 0,48 PROMIS 37-13-003 ColoNeg indicator 0,174 0,62 PROMIS 37-13- 006 ColoNeg indicator 0.160 0,67 PROMIS 37-13-008 ColoNeg indicator 0,203 0,53 PROMIS 37- 14-009 ColoNeg indicator 0,132 0,81 PROMIS 37-14-018 ColoNeg indicator 0,087 19,7 PROMIS 37-14-021 ColoNeg indicator 0,129 0,83 PROMIS 37-15-002 ColoNeg indicator 0,165 0,65 PROMIS 37-15- 006 ColoNeg indicator 0.239 0.45 PROMIS 37-16-001 ColoNeg indicator 0.256 0.42 PROMIS 37-17-001 ColoNeg indicator 0.184 0.58 PROMIS 37-17-003 ColoNeg indicator 0.140 0.76 PROMIS 37-17-004 Indicator ColoNeg 0,139 0,77 PROMIS 37-18-001 Witness ColoNeg 0,134 0,80 PROMIS 37-18-003 Witness ColoNeg 0,158 0,68 PROMIS 37-18-004 Witness ColoNeg 0,188 0,57 PROMIS 37-18-005 Witness ColoNeg 0,111 0,97 PROMIS 37-18-024 Witness ColoNeg 0,128 0,84 PROMIS 37-19-006 Witness ColoNeg 0,122 0,88 PROMIS 37-19-007 Witness ColoNeg 0,136 0,79 PROMIS 37-20-004 Witness ColoNeg 0,179 0, 60 PROMIS 71-02-003 Witness ColoNeg 0.159 0.67 PROMIS 71-04-003 Witness ColoNeg 0.161 0.67 PROMIS 71-09-007 Witness ColoNeg 0.175 0.61 PROMIS 71-10-001 Witness ColoNeg 0.171 0.63 PROMIS 71-10-007 Witness ColoNeg 0.162 0.66 PROMIS 71-11-001 Witness ColoNeg 0.191 0.5 6 PROMIS 71-11-006 ColoNeg indicator 0.155 0.69 PROMIS 71-11-008 ColoNeg indicator 0.181 0.59 PROMIS 71-11-009 ColoNeg indicator 0.140 0.76 4) Results of combinations of prolidase with another marker in Patients and controls Following the same score principle, the results obtained for prolidase can be combined with the results obtained with other markers, 2 to 2. For a patient, if a marker is retained as being above of a threshold set in advance according to the desired specificity, the final score will be considered as significantly positive.

The association of tumor markers prolidase and Galectine 3 thus makes it possible to obtain for the same group of 183 patients increased total scores "2a" in 65 patients with invasive colorectal adenocarcinoma or Tis for 3 patients with Negative Colonoscopy. above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the single Ga13 assay was increased in 13 patients with invasive colorectal adenocarcinoma or a Tis for 2 Negative Colonoscopy patients above the threshold. The association of tumor markers prolidase and LFABP thus makes it possible to obtain for the same group of 183 patients increased total scores "2b" in 77 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative Colonoscopy patients. above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the single LFABP assay was increased in 42 patients with invasive colorectal adenocarcinoma or a Tis for 2 Negative Colonoscopy patients above the threshold. The combination of tumor markers prolidase and ACE thus makes it possible to obtain, for the same group of 183 patients, total scores "" increased in 71 patients with invasive colorectal adenocarcinoma or Tis for 3 patients Negative Coloscopy above threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the single ACE assay was increased in 29 patients with invasive colorectal adenocarcinoma or a Tis for 2 Negative Colonoscopy patients above the threshold. The combination of tumor markers prolidase and HSP60 thus makes it possible to obtain for the same group of 183 patients 'total' scores increased in 68 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative Colonoscopy patients above threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the single HSP60 assay was increased in 25 patients with invasive colorectal adenocarcinoma or a Tis for 2 Negative Colonoscopy patients above the threshold. The combination of tumor markers prolidase and Alpha-Enolase thus makes it possible to obtain for the same group of 183 patients "total" total scores increased in 71 patients with invasive colorectal adenocarcinoma or Tis for 3 patients Negative colonoscopy above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the only Alpha-Enolase assay was increased in 17 patients with adenocarcinoma invasive colorectal or Tis for 2 Negative Coloscopy patients above the threshold. The combination of prolidase and BIP tumor markers makes it possible to obtain, for the same group of 183 patients, total scores "" increased in 67 patients with invasive colorectal adenocarcinoma or Tis for 3 patients Negative Coloscopy above threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the only score of the BIP marker was increased in 24 patients with adenocarcinoma invasive colorectal or Tis for 2 Negative Coloscopy patients above the threshold.

The combination of the prolidase and G3PDH tumor markers makes it possible to obtain, for the same group of 183 patients, increased total "2g" scores in 76 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative Colonoscopy patients. above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the only G3PDH assay was increased in 31 patients with invasive colorectal adenocarcinoma or a Tis for 2 Negative Colonoscopy patients above the threshold. The combination of tumor markers Prolidase and HSP71 thus makes it possible to obtain for the same group of 183 patients a total score "2h" increased in 67 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative Colonoscopy patients. above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the single HSP71 assay was increased in 13 patients with invasive colorectal adenocarcinoma or a Tis for 2 Negative Colonoscopy patients above the threshold. The combination of tumor markers Prolidase and Peroxi-5 allows to obtain for the same group of 183 patients total scores "2i" increased in 71 patients with invasive colorectal adenocarcinoma or Tis for 3 patients Negative colonoscopy above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the single Peroxi-5 assay was increased in 24 patients with adenocarcinoma invasive colorectal or Tis for 2 Negative Coloscopy patients above the threshold. The association of tumor markers prolidase and CA19-9 thus makes it possible to obtain for the same group of 183 patients a total score "2i" increased in 71 patients with invasive colorectal adenocarcinoma or Tis for 2 patients Negative colonoscopy above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the only CA19-9 assay was increased in 23 patients with adenocarcinoma invasive colorectal or Tis for 1 Negative Colonoscopy patient above the threshold.

The combination of prolidase and ApoA1 tumor markers makes it possible to obtain, for the same group of 183 patients, increased "2k" total scores in 64 patients with invasive colorectal adenocarcinoma or Tis for 2 Negative Colonoscopy patients. above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the only score for the ApoA1 marker was increased in 18 patients with adenocarcinoma invasive colorectal or Tis for 1 Negative Colonoscopy patient above the threshold. The combination of prolidase and ApoA2 tumor markers makes it possible to obtain, for the same group of 183 patients, increased total scores "21" in 65 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative Colonoscopy patients. above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the only score for the ApoA2 marker was increased in 10 patients with adenocarcinoma invasive colorectal or Tis for 2 Negative Coloscopy patients above the threshold. The combination of tumor markers Prolidase and Prosomes makes it possible to obtain for the same group of 183 patients a total score "2" '"increased in 68 patients with invasive colorectal adenocarcinoma or Tis for 3 patients Negative colonoscopy above the threshold, whereas the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Colonoscopy patient with a score above the threshold, and the only Prosomes assay was increased in 14 patients with of invasive colorectal adenocarcinoma or Tis for 2 Negative Coloscopy patients above the threshold The combination of the prolidase and M2PK tumor markers thus makes it possible to obtain for the same group of 183 patients total scores "" increased at 72 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative Coloscopy patients above the threshold, whereas the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the only M2PK assay was increased in 24 patients with invasive colorectal adenocarcinoma or Tis for 2 Negative Colonoscopy patients above the threshold.

The combination of tumor markers prolidase and Timp1 thus makes it possible to obtain, for the same group of 183 patients, total scores "2 °" increased in 67 patients with invasive colorectal adenocarcinoma or Tis for 3 patients with Negative Colonoscopy. above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the single Timp1 assay was increased in 24 patients with invasive colorectal adenocarcinoma or a Tis for 2 Negative Colonoscopy patients above the threshold. The combination of the prolidase and Ezrin tumor markers makes it possible to obtain, for the same group of 183 patients, increased "2P" total scores in 65 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative Colonoscopy patients. above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the single Ezrin dosage was increased in 17 patients with invasive colorectal adenocarcinoma or a Tis for 2 Negative Colonoscopy patients above the threshold. The combination of prolidase and PDI tumor markers makes it possible to obtain, for the same group of 183 patients, an overall "2" score increased in 72 patients with invasive colorectal adenocarcinoma or Tis for 2 patients with Negative Colonoscopy. above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the only PDI score was increased in 29 patients with colorectal adenocarcinoma Invasive or Tis for 1 Negative Colonoscopy patient with a score above the threshold. The combination of tumor markers prolidase and Beta2-microglobulin thus makes it possible to obtain for the same group of 183 patients a total score "21-" increased in 69 patients with invasive colorectal adenocarcinoma or Tis for 2 patients. Negative above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, and the single Beta2-microglobulin assay was increased in 29 patients with adenocarcinoma invasive colorectal or Tis for 1 Negative Colonoscopy patient above the threshold. Table 15: Scores of associations of each marker taken alone in combination with prolidase Patient Pathology Stage Score 20 Score 2 ° Score 20 Score 20 Score 20 Score 2 "Score 20 Score 2" Score 2 Score 2i Score 2 "Score 2 'Score r Score 20 Score 2 ° Score 20 Score 2 ° Score 2 "CBSE011 ris TO 0.66 0.65 0.72 0.47 0.88 0.88 9.86 0.68 0.88 0.55 0.72 0.94 0.75 19.9 10.7 9.5 0.87 9.60 CLSP234 Tus TO 8.49 17.6 8.29 8.12 8.34 8.57 8.47 8.44 8.33 8.18 8,31 8,51 8,55 8,28 8,54 8,28 8,57 16,3 CLSP286 ris TO 0,85 0.54 0.49 0.49 9.26 0.98 11.0 8.54 9 , 29 0.60 0.90 0.83 0.67 0.76 0.76 0.72 8.98 0.79 CLSP300 Tis TO 0.47 0.86 0.62 0.43 0.64 9.36 0, 71 0.60 0.64 0.49 0.62 0.75 0.76 0.76 0.71 0.63 11.6 0.75 CLSP315 fis TO 9.19 18.2 8.99 8.91 9 , 06 17.8 9.10 9.03 9.11 9.98 9.01 9.26 9.03 9.18 9.22 9.08 9.38 17.1 CLSP367 Tm ro 0.83 0.87 0, 60 0.92 0.49 0.89 14.8 9.85 10.0 0.60 11.5 9.08 0.73 11.6 8.89 0.95 0.83 11.5 CLSP724 Tis TO 0.82 10, 4 0.55 0.44 9.47 0.85 14.0 10.5 0.82 11.9 0.63 0.91 0 , 88 0.92 0.80 0.79 0.82 0.72 CNSE003 Tm TO 0.95 15.4 0.48 141 0.74 0.94 0.79 0.67 9.30 0.60 0, 76 0.78 0.64 16.0 0.79 0.77 8.83 8.49 CNSE004 -fis TO 0.77 0.63 0.40 0.62 0.71 8.80 0.75 0.58 0.73 0.44 0.78 0.82 0.81 0.87 0.66 0.83 8.70 0.70 GHBD015 loses TO 0.88 11.1 0.49 8.98 10.2 0.82 14.8 0.67 12.9 0.54 0.61 0.77 0.88 0.82 0.81 19.37 0.81 0.77 GHBD036 Tus TO 0.56 0.69 0.68 0.67 11.3 9.06 12.1 0.79 10.2 0.46 0.53 0.72 0.49 0.55 8:64 0.55 8.73 9.48 CBSE001 CCR I 9.12 21.2 9.22 29.1 9.22 9.23 9.23 9.10 9.20 8.82 17.5 17.5 17.5 9.17 19.9 16.9 9.19 9.11 CBSE016 CCR I 9.81 23.9 22.2 18.3 9.70 9.79 9.75 9.60 9.85 9 , 42 9.61 9.83 9.64 9.71 20.3 17.9 9.80 9.85 CLSP047 CCR 0.77 0.51 0.62 0.50 0.63 0.98 0.79 0.88 0.90 0.52 0.99 0.99 0.99 0.99 0.99 0.90 0.79 9.90 0.88 CLSP162 CCR 0.67 0.65 0.33 0.33 0.58 0.75 8, 73 0.63 0.44 0.34 22.8 0.85 0.71 0.59 0.59 0.72 0.75 0.53 CLSP195 CCR 8.62 17.0 8.77 8.55 8.53 8.75 8, 62 8.55 8.73 8.58 8.81 8.74 8.73 8.67 8.67 8.54 8.77 8.73 CLSP224 CCR 11.1 46.2 19.5 50.6 11.0 11.2 11.1 11.0 11.2 10.9 24.1 11.2 23.1 19.5 11.2 11.2 11.2 20.5 CLSP235 CCR 11.0 10.9 10.8 10.8 10 , 9 22.5 10.8 10.8 11.0 10.9 11.2 11.2 10.9 10.9 10.9 10.8 25.7 10.9 CLSP263 CCR 9.61 0.85 0.56 0.77 0.79 0.79 12.5 9.32 0.78 12.5 0.70 0.80 13.0 0.64 0.77 0.73 0.78 10.9 CLSP340 CCR 0.72 0.58 0.53 0.55 9.82 0.65 918 10.9 0.85 0.45 0.61 0.72 0.67 0.55 0.64 0.78 0.67 0.73 CLSP344 CCR 8.44 8.38 8.35 8.18 8.43 8.59 8.80 8.41 8.46 8.23 8.50 8.60 8.57 8.33 8.47 25.29 8.64 8.54 CLSP384 CCR 9.48 9, 25 9.33 9.18 9.51 9.48 9.63 9.49 17.6 9.30 9.30 9.50 9.46 9.41 9.54 9.48 9.48 9.85 CLSP376 CCR 0.68 0.51 0 , 52 0.38 0.74 0.71 0.84 0.69 0.73 0.70 0.76 0.76 0.76 0.77 0.64 0.71 0.66 0.75 GHBD003 CCR 0.83 0.53 0.60 0.48 0.97 0.86 0 , 75 0.70 0.78 0.55 0.69 0.90 0.64 0.61 0.75 0.64 0.86 0.74 GHBD094 CCR 0.99 0.77 0.50 0.37 0.54 0, 00 0.00 0.00 0.00 0.46 0.58 0.73 0.68 0.78 0.88 0.35 0.74 0.79 CLSP075 CCR Il 0.62 0.59 0.41 0.32 0.81 0.74 0.63 0.54 0.72 0.39 0.64 0.64 0.64 0.53 0.69 0.79 0.79 0.88 82 CLSP076 CCR H 8.77 17.1 8.58 20.8 8.89 8.95 8.77 8.67 8.76 8.48 16.9 18.9 16.9 8.71 8.88 8.69 8 , 93 8,81 CLSP080 CCR Il 0.64 0.45 0.47 0.40 0.58 0.77 0.66 0.82 0.72 0.51 0.54 9.15 0.64 0.62 0.67 0.55 0.77 0.80 CLSP098 CCR It 0.73 0.95 24.4 0.49 0.78 0.95 0.66 0.85 0.75 0.67 0.82 0.71 0.62 13.4 0.96 0.75 0.93 11.7 CLSP110 CCR It 0, 68 9.18 0.47 0.47 0.73 9.05 0.74 0.63 0.80 0.53 0.77 0.81 0.74 0.77 0.87 0.66 9.41 0.90 CLSP117 CCR H 8.98 25.7 9.04 9.11 17.0 9.13 21.8 21.3 9.28 8.92 40.8 9.04 8.76 17.6 18.5 9.0 9.07 20.3 CLSP130 CCR H 10.6 9.82 0, 83 0.49 0.76 0.96 0.76 0.64 0.70 0.58 0.70 0.84 0.83 0.73 0.98 0.73 0.96 9.74 CLSP133 CCR H 11 , 4 11.3 11.2 11.1 11.2 19.9 11.3 11.2 11.2 11.2 20.2 11.5 22.2 11.4 11.3 11.2 20.2 11.4 CLSP143 CCR H 13.4 13.4 23.4 13.1 13.3 13.6 12.9 13.2 13.3 13.2 13.4 13.4 13.3 13.5 13.4 13.3 23.7 13.5 CLSP147 CCR H 19.5 10.4 10.4 10.6 10.5 10.7 10.5 10.5 10.5 10.3 21.1 10.7 33.0 32.4 19.2 10.6 19.5 18.8 CLSP154 CCR H 9.76 9.91 30.4 9.58 9.84 18.1 9.89 9.83 9.90 9.79 9.90 10.1 9.86 9.88 9.90 9.86 10.1 9.98 CLSP155 CCR Il 13.5 13.3 34.9 13.1 13.3 13.5 13.3 13.3 13.5 28.3 13.4 13.4 13.2 13.5 13.4 132 13.8 13.5 CLSP157 CCR H 9.10 9.30 8.94 8.88 9.06 9.18 18.7 9.00 18.8 8.89 9.21 9.24 8.98 9 , 13 9.11 9.24 9.33 9.03 CLSP165 CCR II 0.62 0.76 0.47 0.47 0.76 9.10 0.74 0.61 0.74 0.53 0.93 8, 76 0.68 0.66 0.80 0.63 9.32 0.70 CLSP170 CCR It 12.3 23.1 21.8 122 12.4 12.6 12.7 12.4 12.4 12.4 12.4 12.7 12, 8 12.5 12.6 12.3 12.6 12.4 CLSP173 CCR Il 9.70 9.24 9.29 9.42 9.56 9.70 17.3 9.42 18.4 9.43 9.55 17.3 9.45 9.56 9.64 9.71 9.64 9.61 CLSP177 CCR H 15.5 15.6 15.3 27.4 26.3 15.5 30.5 15.8 34.2 27.0 29.3 15.6 15.8 30.3 25.6 27.5 15.3 15.5 CLSP178 CCR H 0.80 112.0 39.5 25.8 0.88 0.88 0.97 0.84 0.83 0.71 0.84 0.94 0.70 0.98 0 , 86 0.74 0.84 0.83 CLSP179 CCR H 19.2 17.3 20.0 8.99 9.15 9.44 919 914 9.28 9.20 9.27 9.45 9.16 9.37 9.23 9 , 9.39 9.33 CLSP186 CCR H 0.73 11.7 0.58 0.41 0.60 0.81 0.54 0.82 0.68 0.48 0.61 0.71 0.67 0.58 0.85 0, 74 0.86 12.7 CLSP194 CCR H 9.39 25.1 9.27 9.3 5 9.40 9.55 9.48 9.32 9.53 9.20 9.26 9.56 18.3 9.26 9.45 9.41 17.6 18.1 CLSP201 CCR H 10.2 23 , 6 10.3 79.4 10.2 10.4 10.2 102 10.4 10.0 10.4 10.2 10.2 10.1 26.1 10.2 10.3 22.7 CLSP207 CCR It 11.5 23, 8 11.3 11.1 11.3 20.3 11.3 11.3 11.4 21.6 22.3 22.3 22.3 11.3 11.5 11.3 11.6 11.4 CLSP209 CCR H 0.83 15.4 0, 57 0.69 0.88 8.74 0.78 0.72 0.73 0.52 0.69 0.76 9.42 0.59 12.5 0.6 8.71 0.73 CLSP220 CCR H 18.2 18.4 23.7 9.51 9.72 18.9 9.96 9.71 9.78 9.83 9.69 19.8 9.68 9.82 9.95 9.76 9.98 9.92 CLSP222 CCR Il 10.6 28 , 0 20.7 24.9 10.7 18.9 10.7 10.6 18.6 10.4 10.7 10.6 10.7 24.9 19.5 10.7 10.8 19.7 CL5P253 CCR H 8.33 8.38 8.22 8, 03 8.25 17.2 8.29 8.22 8.41 8.10 16.1 16.1 16.1 8.33 8.36 8.21 16.3 8.41 CLSP256 CCR H 10.2 10.1 10.1 74.3 10.0 10.1 102 10.0 10.1 9.97 10.0 10.0 10.2 37.9 17.9 10.2 10.1 19.1 CLSP280 CCR It 8 , 72 8.57 8.53 8.27 8.58 8.66 24.6 21.2 18.5 8.49 8.66 8.65 8.65 8.55 17.7 8.6 8.65 8.67 CLSP296 CCR H 0.48 8.63 0.54 0.71 0, 51 8.80 0.56 0.60 0.52 29.7 32.8 0.71 0.61 0.42 16.5 0.5 0.80 0.59 83 CLSP310 CCR II 0.68 0.59 0.66 0.44 0.70 0.84 0.77 0.68 0.75 0.62 0.72 0.88 0.79 0.65 0.87 0, 66 0.93 0.83 CLSP327 CCR H 13.8 13.9 13.6 13.8 13.8 13.9 21.7 13.8 13.9 13.5 13.7 13.9 13.7 14.0 13.9 13.9 14.0 13.8 CLSP333 CCR H 0.79 8.84 0.65 0.46 0.68 0.82 0.91 0.75 0.85 0.95 0.84 0.82 0.93 0.64 0.84 0.78 0.78 8.58 CLSP341 CCR H 11.1 11.0 10.9 22.0 11.2 11.2 20.7 11.2 11.1 10.9 11.1 11.2 11.0 11.0 11.2 11.0 11.3 11.2 CLSP346 CCR Il 8.52 8.44 21.7 8.15 17.5 8.42 18.1 8.55 8.62 24.2 8.38 8.43 8.53 8.63 8.56 8.34 8.42 16.8 CLSP347 CCR H 8.60 0.74 14.4 0.41 10.5 0.69 13.0 0.73 0.90 0.44 0.50 0.76 8.43 0.79 0.86 0.84 0.69 0.79 CLSP380 CCR H 10.7 10.8 10.4 10.8 10.7 10.7 23.1 10.8 10.8 10.4 10.7 10.7 10.7 19.2 10.6 19, 4 10.7 10.8 CNSE002 CCR H 10.1 9.92 10.3 9.84 10.1 10.2 10.1 10.0 10.2 0.95 33.2 19.6 10.3 19.7 10.3 10.1 102 10.2 GHBD021 CCR H 0.72 0.51 0.52 0.62 0.70 0.93 0.73 0.66 0.83 0.51 0.83 0.85 0.92 0.61 0.77 0.66 0.87 0.80 GHBD043 CCR Il 10.5 10.5 10.5 10.5 10.4 18.3 10.5 10.4 10.4 10.4 10.5 10.5 10.4 10.6 30.7 10.5 10.7 21.5 GHBD064 CCR H 17.3 27.1 48.4 9.03 8.84 9.00 9.04 8.82 9.02 8.76 8.75 9.10 9.03 21.9 9.07 19.36 9.07 18.6 GHBD066 CCR It 0.76 0.75 0.46 0.66 0.65 0.85 0.77 0.59 0.56 0.44 0.64 8.91 0.86 0.70 0.84 0.80 0.84 0.76 GHBD075 CCR H 8.55 8.54 8.51 20.1 8.48 8.70 8.49 8,38 8.54 8,26 8,52 8,64 8,50 8,53 8,45 8,58 8,82 8,43 GHBD087 CCR H 0.78 0.00 0.69 0.80 0.84 0.90 0, 61 0.57 0.64 0.58 0.84 0.69 0.64 0.61 0.75 0.58 8.47 0.71 GHBD090 CCR It 0.44 0.53 0.37 028 0.45 0.63 0.43 0.50 0.54 0.37 0.37 0.59 0.55 0.65 0.61 0.45 0.60 0.55 GHBD096 CCR H 0.64 0.84 0.85 0.49 0.59 0.98 0.59 0.56 0.76 0.53 0, 90 8.82 0.70 12.0 0.82 0.59 9.99 0.83 GHBD100 CCR H 16.4 21.9 12.9 22.1 13.0 25.4 25.4 25.4 25.4 12, 8 20.7 13.1 12.9 26.8 25.8 13.2 25.4 26.2 GHBD103 CCR H 0.58 8.99 0.40 0.28 0.56 0.72 0.77 0.56 0.63 0.36 0.60 0.70 0.70 0.49 0.60 0.57 0.65 8.90 CB3E023 CCR HI 8.88 9.67 0.65 0.63 10.1 0.88 14 , 5 0.80 9.81 0.61 0) 37 0.76 9.79 0.78 0.76 0.83 0.82 0.73 CLSP044 CC R HI 0.78 0.72 140.3 0.47 0.79 0.82 0.90 0.73 11.3 0.58 0.68 0.75 0.95 0.67 0.77 0.73 0.86 0.79 CLSP074 CCR Hl 15.0 14.9 14.8 31.3 14.9 23.4 15.0 14.9 28.2 14.9 29.4 29.4 29.4 15.1 24.7 15.0 15 , 2 23.1 CLSP078 CCR HI 11.3 29.1 11.0 31.1 21.8 11.4 11.1 11.1 19.1 11.0 11.2 11.1 11.2 11.3 11, 3 11.1 18.9 11.3 CLSP081 CCR HI 14.7 14.6 14.8 52.2 14.8 14.8 22.9 14.7 23.0 14.5 28.8 28.8 28 , 8 28.8 28.8 14.8 14.8 23.5 CLSP098 CCR HI 15.2 15.2 15.1 15.5 15.0 24.9 24.2 15.2 15.4 15.1 26, 2 15.5 151 15.2 152 24.7 25.9 15.3 CLSP174 CCR HI 20.0 27.0 69.0 102 19.5 10.2 19.1 19.2 26.5 116.2 10.3 10.3 182 19, 8 30.7 21.8 10.2 10.2 CL6P227 CCR HI 9.73 48.8 36.6 9.24 9.49 18.3 9.53 9.45 19.5 9.42 19.0 9.61 9.51 17.5 9.55 9.59 9.82 18.8 CLSP233 CCR III 11.3 11.1 11.1 11.0 11.2 22.4 11.2 11.1 11.2 11.1 21 , 9 21.9 21.9 11.2 11.2 11.1 19.3 11.2 CLSP255 CCR HI 11.3 27.8 11.1 11.2 11.2 11.5 11.3 11.2 11.4 37.4 52.0 11.4 11.1 11.2 11.4 11.2 11.5 11.4 CLSP289 CCR HI 0.70 0.85 0.52 0.42 0.73 0.89 0.79 0.67 0.71 0.47 0 , 76 0.87 0.85 0.63 0.68 0.61 0.86 0.85 84 CLSP320 CCR III 8.42 8.40 8.41 8.21 8.32 8.65 8.51 8, 43 8.40 8.28 8.30 8.52 8.58 8.57 8.47 8.50 16.8 8.45 CLSP324 CCR III 14.3 14.2 22.7 14.4 23.9 14.6 24 5 23.8 22.9 14.5 37.8 14.5 22.9 14.5 14.5 14.4 14.5 14.4 CLSP383 CCR III 0.69 0.47 0.63 0.64 0.68 0.79 0.89 0.74 0.71 0.45 0.82 0.82 0.82 10.5 10.5 0.7 0.82 0.80 CLSP384 CCR III 0.58 13.5 129.7 0.37 0.66 0.75 8.65 0.76 0.68 11.1 10.9 0.73 12.9 24.0 0.71 0.81 0.68 0.78 CNSE001 CCR III 0.66 0.72 12.8 8.55 0.79 0.89 0 , 73 0.63 0.89 16.6 27.2 0.83 0.63 0.69 0.80 0.82 9.36 0.71 CNSE016 CCR III 9.93 73.3 48.4 101.5 10.0 9.96 9.94 9.85 9.99 46.2 9.89 10.0 9.96 20.5 9.91 18.71 9.80 9.93 CNSE017 CCR III 9.54 22.5 9.30 9, 53 9,43 18,5 9,34 9,33 9,52 9,28 20,9 9,52 9,45 9,43 9,52 18,13 20,9 9,57 CSEM015 CCR III 0.66 10.0 0.53 0 , 36 0.57 0.85 0.52 0.48 0.51 0.39 0.85 0.75 0.56 0.73 0.73 0.52 9.54 0.62 CSEM029 CCR III 9.10 9.07 9.02 9, 01 8.96 9.22 8.97 8.98 9.15 8.88 9.02 9.16 9.18 9.16 17.6 9.0 18.8 9.12 GH B0004 CCR III 9,43 9,42 40.0 18.2 18.6 9.77 9.39 9.41 9.51 9.48 9.51 9.71 9.53 9.53 9.82 9.47 9 , 72 9.67 GHBD017 CCR III 11.2 11.0 11.1 11.4 11.2 11.4 11.3 11.2 11.2 11.1 11.2 11.3 11.3 11.3 19.6 11.3 11.4 11.4 GHBD034 CCR III 0 , 79 0.41 0.45 0.69 8.74 0.86 16.7 10.3 0.87 0.48 0.69 0.60 0.63 0.58 0.75 9.57 0.85 0.63 GHBD042 CCR III 12.2 0.64 0.44 0.38 0.67 0.81 0.70 0.53 10.7 0.73 0.65 0.86 0.54 0.63 0.73 0.61 0.78 0.76 GHBD045 CCR III 27.5 27.2 27.2 27.2 27.3 27.6 27.3 27.3 27.3 27.2 27.5 27.5 27.4 27.8 27.6 27.3 27.5 36.7 GHBD089 CCR III 9,46 9,58 55.4 9.22 9,47 9.71 9,46 9,33 9,45 9,47 9,47 17.6 9,33 9,45 9,47 9,39 17.9 9.59 GHBD105 CCR HI 0 , 71 0.76 0.61 0.45 0.69 0.95 0.78 0.59 0.66 15.5 0.87 0.79 13.2 15.2 8.58 0.67 0.78 0.92 CBSE012 CCR IV 9.26 9.25 9, 50 32.1 9.22 17.2 9.12 9.17 9.17 27.0 19.1 9.48 18.0 17.3 9.42 9.32 17.7 9.25 CBSE026 CCR IV 12.0 35.4 640.7 109.0 12.1 12.0 12.0 11.9 12.1 9596 12.1 12.0 27.9 23.3 23.3 32.7 12.0 23.4 CLSP156 CCR IV 25.2 13.5 39.5 13.0 13.4 13.5 27.2 21.6 13.5 30.4 13.3 13.5 13.5 24.1 13.4 13.3 21.4 13.5 CLSP159 CCR IV 0.89 10.3 84.1 43.8 0.69 0.77 0, 69 0.61 34.0 68.5 0.82 0.71 0.68 21.7 0.78 0.72 0.74 0.80 CLSP167 CCR IV 9.94 9.74 9.86 9.64 9.85 18, 8 9.94 9.83 9.94 23.8 10.1 9.97 9.90 9.91 18.0 9.9 18.8 18.5 CLSP260 CCR IV 0.57 59.0 57.0 14.7 9, 18 0.86 0.94 0.76 8.67 0.89 0.81 0.70 11.4 0.84 0.75 11.74 0.90 0.92 CLSP334 CCR IV 0.53 0.46 0.48 0.28 10 , 3 0.60 12.1 8.85 10.2 0.38 0.84 0.76 8.29 8.84 0.53 0.72 0.65 0.46 CLSP357 CCR IV 0.73 0.66 18, 1 0.43 0.78 0.70 9.85 0.87 0.83 31.1 0.64 0.81 0.87 0.90 0.71 0.84 0.73 0.91 CLSP365 CCR IV 0.81 0.64 56.0 16, 6 0.70 0.68 0.62 0.54 0.60 456.0 0.57 0.73 11.4 11.5 0.79 8.77 0.64 0.68 CLSP388 CCR IV 0.50 0.58 0.65 0.78 0.72 0.71 8.79 0.72 0, 79 21.4 0.68 0.73 0.54 9.36 0.65 0.60 0.74 0.63 GHBD022 CCR iv 0.67 11.7 2584 41.4 9.12 0.78 0.74 0, 82 0.64 9.03 0.48 0.79 0.81 0.81 9.88 0.86 0.70 0.87 GHBD071 CCR IV 9.77 32.4 72.1 65.1 18.8 8.63 11.0 8.61 12, 2 16.9 0.70 11.3 0.85 12.7 0.66 43.03 0.81 0. 61 CLSP197 CCR NA 0.73 0.79 0.49 0.42 0.71 0.83 0.83 0.83 0.83 0.67 0.71 0.79 0.82 0.87 0.49 0.89 0, 83 0.70 85 HGED005 CobNeg control 0.88 0.87 0.72 0.88 8.85 0.88 0.87 0.84 0.98 0.59 0.98 0.98 0.98 0.94 0.97 11 , 09 0.82 0.89 PROMIS 21-01-005 CobNeg Control 0.64 0.74 0.49 0.43 0.68 0.91 0.89 0.58 0.63 0.86 0.69 0.86 0.70 0.67 0.75 0.67 0.85 0.78 PROMIS 21-01-008 ColoNeg Indicator 0.47 0.44 0.30 0.30 0.34 0.74 0.46 0.55 0.48 0.35 0.39 0.69 0.59 0.48 0.62 0.57 0.76 0.54 PROMIS 21-01-009 CobNeg Control 0.58 0.81 0.44 0.38 0.58 0.87 0.48 0.81 0.75 0.62 0.58 0.63 0.44 0.44 0.58 0.73 0.74 0.47 PROMIS 21-01-011 CobNeg Indicator 0.44 0.28 0.26 0.28 0.35 0.61 0.43 0.39 0.46 0.36 0.49 0.63 17.0 0.38 0.56 0.59 0.60 0.47 PROMIS 21-01-012 ColoNeg indicator 0.71 0.52 0.66 0.42 0.76 0.75 0.74 0.68 0.76 0.64 0.54 0.72 0.58 0.63 0.66 0.67 0.81 0.67 PROMIS 21-02-001 ColoNeg Indicator 11.8 9.59 0.47 0.34 0.65 0.87 0.84 0 , 54 0.69 0.48 0.87 0.67 0.87 0.79 13.9 0.7 0.59 12.1 PROMIS 21-02-003 CobNeg Indicator 0.56 0.46 0.38 0.28 0.38 0.67 0.44 0.40 0.50 0.78 0.63 0.73 0.51 0.48 0.59 0.51 0.69 0.56 PROMIS 21-02-004 ColoNeg indicator 0.39 0 , 56 0.18 0.18 0.36 0.45 0.88 0.68 0.62 0.24 0.38 0.58 0.58 0.41 0.48 0.46 0.38 0.44 PROMIS 21-02 -008 ColoNeg indicator 0.57 0.82 0.41 0.41 0.57 9.29 0.91 8.68 0.91 0.50 0.64 0.85 0.64 0.65 0.85 0 , 46 9,90 0,66 PROMIS 21-02-008 ColoNeg indicator 0,58 8,82 0.40 0.27 0.48 0.69 0.50 27.6 0.44 0.46 0.36 8.67 8.53 0.54 0.54 0.58 0.67 0.54 PROMIS 21-02-011 ColoNeg indicator 0.67 0.55 0.63 0.45 0.60 0.82 0.66 0.60 0.61 0.79 0.61 0.92 0.60 0.89 0.89 0.77 0.89 0.71 PROMIS 21-02-014 Witness ColoNeg 0.60 0.78 0.53 0.43 0.60 0.93 0.60 0.63 0.63 0.48 0.87 0.87 0.87 0.87 0.87 0.64 0.88 0.70 PROMIS 21-02-016 ColoNeg Indicator 0.63 0.30 0.68 0.53 0.60 0 , 87 0.59 0.44 0.58 0.37 0.37 0.58 0.60 0.70 0.57 0.54 0.61 0.55 PROMIS 21-03-004 ColoNeg indicator 0.56 0.84 0.80 0.63 0.62 0.75 0, 60 0.57 0.70 0.45 0.79 0.79 0.79 0.84 0.76 0.73 0.79 0.63 PROMIS 21-03-013 ColoNeg indicator 0.36 0.35 0.19 0.19 0.49 0.66 0.44 0.43 0.46 0.22 0.44 0.58 0.40 0.38 0.59 0.40 0.69 0.47 PROMIS 21-03-015 ColoNeg indicator 0.47 0.81 0.38 0.28 0.45 0.88 0.42 0.46 0.45 0.34 0.55 0.78 0.36 0.47 0.65 0.46 0.87 0.89 PROMIS 21-04-002 ColoNeg indicator 0.80 0.77 0.58 0.46 0.70 0.98 0.88 0.73 0.65 0.70 0.84 0.87 0.73 0.94 0.92 0.64 0.84 0.71 PROMIS 21-04-008 CobNeq indicator 0.77 0.50 0.56 0.48 0.88 0,95 0,84 0.70 0.84 0.54 0.85 0.92 0.84 0.63 0.70 0.84 0.98 0.76 PROMIS 21-04-007 ColoNeg indicator 0.87 0.65 0.41 0, 31 0.44 0.65 0.50 0.50 0.57 0.39 0.40 0.70 0.68 0.85 0.76 0.57 0.70 0.68 PROMIS 21-04-008 Witness ColoNeg 0.41 0.50 0.35 0.23 0.72 0.55 0.50 0.46 0.48 0.25 0.56 0.63 0.63 0.49 0.53 0.42 0.58 0.52 PROMIS 21-04-009 CobNeg indicator 0.47 0.46 0.39 0, 26 0.45 0.60 0.44 0.48 0.51 0.56 0.50 0.64 0.66 0.76 0.84 0.53 0.58 0.67 PROMIS 21-04-013 Witness ColoNeg 0.80 0.70 0.80 0.44 0.56 0.79 0.52 0.57 0.55 0.78 0.88 0.88 0.88 0.71 0.71 0.59 0.82 0, 77 PROMIS 21-07-004 ColoNeg indicator 0.53 0.88 0.52 0.86 0.57 0.75 0.53 0.54 0.6 7 0.46 0.54 0.77 0.58 0.57 0.71 0.60 0.68 0.83 PROMIS 21-12-001 CobNeg Indicator 0.52 0.49 0.58 0.34 0.78 0.78 0.85 0 , 48 0.81 0.59 0.45 0.64 0.51 12.2 0.58 0.59 0.71 0.61 PROMIS 21-12-007 ColoNeg indicator 0.75 0.80 0.50 0.50 0.70 9.51 0.59 0.61 0.68 0.57 0.78 0.93 0.65 1.00 0.75 0.65 1.00 0.75 PROMIS 21-12-011 ColoNeg indicator 0.81 0.41 0.82 0.32 0, 53 0.87 0.50 0.48 0.56 0.53 0.48 0.81 0.48 0.67 0.59 0.52 0.87 0.57 PROMIS 21-22-001 ColoNeg indicator 0.57 0.38 0.61 0.27 0, 47 0.58 0.48 0.44 0.67 0.71 0.60 0.58 0.59 0.45 8.49 0.50 0.62 0.56 PROMIS 21-22-006 ColoNeg indicator 0.71 0.89 0.47 0.34 0, 49 0.81 0.50 0.47 0.48 0.40 0.69 0.69 0.69 0.64 0.61 0.51 0.77 0.73 86 PROMIS 21-22-008 Witness ColoNeg 0.77 0.69 0.41 0.41 0.68 0.78 0.71 0.62 0.70 0.48 0.91 0.84 0.68 0.59 0.68 0.59 0.75 0, 75 PROMIS 37-03-004 CobNeg indicator 0.74 0.66 0.58 0.49 0.74 0.87 0.82 0.77 0.85 0.54 0.58 0.78 0.77 0.72 0.77 0.78 0.90 0.73 PROMIS 37- 04-001 ColoNeg indicator 0.54 0.68 9.95 0.21 0.50 0.58 0.53 0.44 0.48 0.33 0.56 9.4 4 0.43 0.58 0.62 0.47 0.48 0.71 PROMIS 37-04-005 ColoNeg indicator 0.85 0.82 0.59 0.48 0.83 0.87 0.59 0.62 0 , 87 0.57 0.96 0.96 0.98 0.98 0.96 0.75 0.82 0.94 PROMIS 37-05-008 CobNeg Indicator 0.60 0.70 0.33 0.69 0.69 0.77 0.76 0.63 0.66 0.41 0.49 0.54 0.66 0.59 0.61 0.83 0.77 0.89 PROMIS 37-06-001 ColoNeg indicator 0.62 0.72 0.55 0.37 0.66 0.71 0 , 52 0.58 0.61 0.51 0.79 0.79 0.68 0.82 0.62 0.59 0.72 0.68 PROMIS 37-09-002 ColoNeg indicator 0.53 0.40 0.41 0.32 0 , 52 0.72 0.59 0.49 0.54 10.8 0.53 0.77 0.54 0.55 0.77 0.56 0.72 0.59 PROMIS 37-11-001 Witness CobNeg 0.78 0.66 0.50 9.95 0.70 0.93 0.74 0.63 0.67 0.54 0.66 0.80 0.77 0.70 0.77 18.16 0.95 0.77 PROMIS 37-11-002 ColoNeg Indicator 0 , 55 0.44 0.31 0.31 0.49 0.76 0.64 0.50 0.47 0.36 0.61 0.74 0.71 0.78 0.65 0.56 0.78 0, 69 PROMIS 37-11-011 ColoNeg indicator 0.57 0.69 0.59 0.44 0.59 0.92 0.81 0.57 0.56 0.45 0.72 0.89 0.57 0.65 0 , 74 0.65 0.78 0.76 PROMIS 37-11-016 CobNeg Indicator 0.68 0.45 0.35 0.35 0.55 0.72 0.54 0.49 0.64 0.48 0.70 0.52 0.58 9.41 0.84 0 , 56 0.73 0.67 PROMIS 37-12-002 ColoNeg indicator 0.55 0.40 0.33 0.24 0.41 0.59 0.57 0.39 0.50 0.31 0.64 0.67 0, 59 0.45 0.51 0.45 0.60 0.57 PROMIS 37-13-003 CobNeg Control 0.61 0.62 0.42 0.48 0.46 0.69 0.48 0.47 0.47 0 , 49 0.60 0.74 0.54 0.57 0.71 0.50 0.67 0.66 PROMIS 37-13-008 ColoNeg indicator 0.40 0.78 0.44 0.45 0.57 0.78 0.52 0.48 0.57 0.37 0.57 0.80 0.83 0.62 0.83 0.58 0.78 0.59 PROMIS 37-13-008 ColoNeg indicator 11.4 0.48 0.26 026 0.55 0.58 0.73 0.60 0.48 0.29 0, 55 0.60 0.53 0.42 0.51 0.49 0.64 0.57 PROMIS 37-14-009 CobNeg indicator 0.59 0.78 0.52 0.58 0.56 0.85 0, 50 0.64 0.55 0.42 0.60 0.89 0.62 0.56 0.72 0.58 0.80 0.65 PROMIS 37-14-018 ColoNeg indicator 10.1 10.2 9.90 9 , 83 9.93 10.3 9.92 9.91 10.0 9.90 10.1 10.2 9.94 19.7 19.7 9.9 10.3 10.1 PROMIS 37-14-021 Control CobNeg 0.65 0.45 0.52 0.42 0.53 0.87 0.50 0.49 0.51 0.51 0.75 0.85 0.70 0.57 0.69 0.58 0.79 0.69 PROMIS 37-15 -002 CobNeg Control 0.51 0.49 0.40 0.43 26.8 0.65 0.50 0.48 0.51 0.42 0.63 0.69 0.62 0.46 0.82 0.52 0.65 0.60 PROMIS 3 7-15-006 ColoNeg Indicator 0.44 0.53 0.35 0.22 0.37 0.61 0.40 0.34 0.33 0.28 0.54 0.87 0.36 0.47 0 , 67 0.44 0.64 0.80 PROMIS 37-16-001 ColoNeg indicator 0.48 0.42 0.30 0.21 0.40 0.53 8.96 0.57 0.64 0.30 0.64 0.59 0.81 0.66 0.47 0.48 0.54 0.49 PROMIS 37-17-001 ColoNeg indicator 0.48 0.51 0.51 029 0.49 0.69 0.60 0.52 0.64 0.41 0.42 0, 66 0.46 0.48 0.55 0.52 0.62 0.55 PROMIS 37-17-003 ColoNeg indicator 0.58 0.52 11.3 0.38 0.50 0.75 0.60 0.48 0 , 61 0.42 0.63 0.70 0.52 0.73 0.63 0.54 0.67 0.64 PROMIS 37-17-004 CobNeg indicator 0.60 0.66 0.50 0.38 0.60 0.70 0.56 0.60 0.52 0.46 0.69 0.81 0.64 0.76 0.65 0.63 0.77 0.69 PROMIS 37-18-001 ColoNeg indicator 0.58 0.83 0.46 0.40 0.57 0.83 0.66 0.63 0.85 0.49 0.62 0.83 0 , 55 0.55 0.64 0.57 0.80 0.62 PROMIS 37-18-003 ColoNeg indicator 0.64 0.70 0.41 0.34 0.48 0.77 0.57 0.52 0.55 0, 40 0.53 0.73 0.75 0.48 0.62 0.47 0.80 0.53 PROMIS 37-18-004 CobNeg indicator 0.42 0.48 0.28 0.28 0.43 0, 67 0.82 0.43 0.50 0.34 0.58 0.82 0.70 0.48 0.67 0.49 0.71 0.56 PROMIS 37-18-005 Witness Co loNeg 0.63 0.87 0.58 0.48 0.61 0.92 0.67 0.64 0.67 0.56 0.77 0.87 0.65 0.65 0.76 0.63 0.87 0.81 PROMIS 37-18-024 CobNeg indicator 0.52 0.55 0.55 0, 42 0.52 0.84 0.53 0.59 0.86 0.53 0.84 0.77 0.62 0.72 0.75 0.55 0.88 0.71 87 PROMIS 37-19-006 Witness CobNeg 0.69 0.89 0.44 0.44 0.54 0.78 0.59 0.57 0.58 0.48 0.67 0.88 0.63 0.88 0.70 0.59 0, 73 0.73 PROMIS 37-19-007 ColoNeg indicator 0.56 0.57 0.47 0.39 0.55 0.78 0.54 0.51 0.67 0.41 0.82 0.85 0.58 0.53 0.69 0.81 0, 83 0.68 PROMIS 37-20-004 ColoNeg indicator 0.56 0.45 0.44 0.30 0.58 0.65 0.77 0.55 11.1 0.32 0.80 0.63 0.52 0.77 0.64 0.63 0.64 0.56 PROMIS 71-02-003 ColoNeg Indicator 0.47 0.42 0.40 0.34 0.45 0.68 0.55 0.48 0.54 0.37 0.80 0.82 0.51 0.71 0.59 0.59 0.75 0.82 PROMIS 71-04-003 ColoNeg indicator 0.58 0.70 0.40 0.53 0.62 0.74 0.53 0.78 15, 5 0.47 0.52 0.71 0.51 0.58 0.61 0.56 0.75 0.67 PROMIS 71-09-007 ColoNeg indicator 0.51 0.54 0.41 0.31 0.51 0.72 0.63 0 , 56 0.72 0.43 0.51 0.59 0.47 0.44 0.66 0.44 0.69 0.82 PROMIS 71-10-001 CobNeg Indicator 0.51 0.42 0.45 52.7 0.63 0.58 0.56 0.59 0.57 0.69 0.45 0.68 0.49 0.57 0.55 0.55 0.65 0.61 PROMIS 71-10 -007 ColoNeg indicator 0.83 0.37 0.68 0.33 0.83 0.67 9.28 0.59 0.54 0.41 0.66 0.66 0.68 0.70 0.69 0.62 0.66 0.81 PROMIS 71-11-001 ColoNeg control 0.47 0.47 0.45 0.52 0.59 0.60 0.62 0.50 0.57 0.36 0.61 0.63 0.89 0.51 0.59 0.57 0.58 0 , 66 PROMIS 71-11-008 ColoNeg indicator 0.61 0.56 0.42 0.34 0.59 0.64 0.58 0.51 0.52 0.40 0.62 0.75 0.87 0.50 0.60 0.55 0.72 0.84 PROMIS 71-11-008 ColoNeg indicator 0.58 0.43 0.40 0.30 0.60 0.39 0.48 0.57 0.40 0.59 0.55 0.44 0.56 0.47 0.63 0.55 PROMIS 71-11-009 CobNeg Indicator 0.52 0.70 0.44 0.38 0.53 0.79 0.54 0.53 0.58 0.58 11.1 0.78 0.50 0 , 57 0.63 0.55 0.76 0.72 Performance Score e Score e Score e Score 2 ° Score 2 ° Score e Score 2 ° Score e Score 21 Score 2 'Score e Score 21 Score r Score e Score 2 ° Score 2 ° Score e Score 2` Threshold 0.99 0,95 0,85 0.92 0,97 0,98 0,97 0.87 0,96 0.95 0,99 0.99 0.99 1,00 0,98 0,95 1 0.94 Sensitivity% 57.5 68.1 62.8 60.2 62.8 58 , 4 66.4 58.4 61.9 61.9 58.6 57.5 60.2 63.7 59.3 56.6 63.7 61.1 Specificity% 95.7 95.7 95.7 95.7 95.7 95.7 95.7 95.7 95.7 97.2 97.2 95.7 95.7 95.7 95.7 95.7 97.2 97.2 CCR invasive and Tis above threshold 65 77 71 68 71 67 76 67 71 71 64 65 68 72 67 65 72 69 ColoNeg above threshold 3 3 3 3 3 3 3 3 3 2 2 3 3 3 3 3 2 2 Tis: Patients with tumor in situ ( intra-mucosal carcinoma TO), CCR = patients with invasive colorectal cancer of stage TNM I to IV (Adenocarcinoma), ColoNeg- = control subjects, with a totally normal colonoscopy, and a positive FOBT test Score 2: Association GaI3, prolidase Score 2b: association LFABP, prolidase Score 2C: association ACE, prolidase Score 2d: association HSP60, -prolidase Score 2e: association Alpha-Enolase, prolidase Score 2: association BIP, prolidase oo 1-1 00 o U1 Score 2: association G3PDH, prolidase Score 2h: association HSP71, prolidase Score 2: association Peroxy-5, prolidase Score 2: association CA19-9, prolidase Score 2k: Association ApoA1, Prolidase Score 21: Association ApoA2, Prolidase Score 2m. Association Prosomes, Prolidase Score 2 °: Association M2PK, Prolidase Score 2 °: Association Timp1, Prolidase Score 2P: Ezrin combination, Prolidase Score 2g: PDI combination, Prolidase Score 2r: Beta2-microglobulin combination, Prolidase 88 5) Results of the combinations of prolidase with several markers in patients and controls The Applicant has also associated the performance of prolidase with several markers. Prolidase is particularly useful for the detection of colorectal cancer because it complements the detection of other markers of colorectal cancer. See Tables 15 and 16. By way of example, mention may be made of the following combinations: The association of the prolidase, LFABP and ACE tumor markers thus makes it possible to obtain for the same group of 183 patients total scores "" increased in 83 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative Coloscopy patients above the threshold. While the only prolidase score was increased in 59 patients with colorectal adenocarcinoma, for a Negative Coloscopy patient with a score above the threshold, the single LFABP assay was increased in 42 patients with invasive colorectal adenocarcinoma or of one Tis for 2 Negative Colonoscopic patients above the threshold, and the single ACE assay was increased in 25 patients with invasive colorectal adenocarcinoma or Tis without Negative Colonoscopy patient above the threshold.

The combination of the prolidase, LFABP, CA19-9 and ACE tumor markers makes it possible to obtain, for the same group of 183 patients, increased total scores "" in 85 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative colonoscopy patients above the threshold. While the only prolidase score was increased in 59 patients with invasive colorectal adenocarcinoma or Tis for a Negative Coloscopy patient with a score above the threshold, the single LFABP assay was increased in 39 patients with Invasive colorectal adenocarcinoma or Tis for 1 Negative Coloscopy patient above the threshold, the only CA19-9 assay was increased in 23 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Colonoscopy patient. above the threshold and the single ACE assay was increased in 25 patients with invasive colorectal adenocarcinoma or Tis without Negative Colonoscopy patient above the threshold. The combination of the prolidase, LFABP, M2PK and ACE tumor markers makes it possible to obtain, for the same group of 183 patients, total scores "" increased in 86 patients with invasive colorectal adenocarcinoma or Tis for 3 patients. Negative above the threshold. While the only prolidase score was increased in 59 patients with invasive colorectal adenocarcinoma or Tis for a Negative Coloscopy patient with a score above the threshold, the single LFABP assay was increased in 39 patients with Invasive colorectal adenocarcinoma or Tis for 1 Negative Colonoscopy patient above the threshold, the only M2PK assay was increased in 18 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Colonoscopy patient over the threshold. threshold and the single ACE assay was increased in 25 patients with invasive colorectal adenocarcinoma or Tis without Negative Colonoscopy patient above the threshold. The association of tumor markers prolidase, LFABP, Timp1 and ACE thus makes it possible to obtain for the same group of 183 patients total scores "" increased in 87 patients with invasive colorectal adenocarcinoma or Tis for 3 patients. Negative above the threshold. While the only prolidase score was increased in 59 patients with invasive colorectal adenocarcinoma or Tis for a Negative Coloscopy patient with a score above the threshold, the single LFABP assay was increased in 39 patients with Invasive colorectal adenocarcinoma or Tis for 1 Negative Colonoscopy patient above the threshold, the single Timp1 assay was increased in 24 patients with invasive colorectal adenocarcinoma or Tis for 2 Negative Colonoscopy patients over the threshold. threshold and the single ACE assay was increased in 25 patients with invasive colorectal adenocarcinoma or Tis without Negative Colonoscopy patient above the threshold. The combination of the prolidase, LFABP, M2PK, Timp1 and ACE tumor markers makes it possible to obtain, for the same group of 183 patients, a total of "5" increased scores in 88 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative Colonoscopy patients above the threshold. While the only prolidase score was increased in 59 patients with invasive colorectal adenocarcinoma or Tis for a Negative Coloscopy patient with a score above the threshold, the single LFABP assay was increased in 39 patients with Invasive colorectal adenocarcinoma or Tis for 1 Negative Colonoscopy patient above the threshold, the only M2PK assay was increased in 18 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Colonoscopy patient over the threshold. threshold, the single Timp1 assay was increased in 22 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Coloscopy patient above the threshold and the single ACE assay was increased in 25 patients with invasive colorectal adenocarcinoma or a Tis without Negative Colonoscopy patient above the threshold. The association of the prolidase, LFABP, ACE, PDI and CA19-9 tumor markers makes it possible to obtain, for the same group of 183 patients, increased total "5wb" scores in 93 patients with invasive colorectal adenocarcinoma or Tis for 3 Negative Colonoscopy patients above the threshold. While the only prolidase score was increased in 59 patients with invasive colorectal adenocarcinoma or Tis for a Negative Coloscopy patient with a score above the threshold, the single LFABP assay was increased in 39 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Colonoscopy patient above threshold, the ACE single assay was increased in 25 patients with invasive colorectal adenocarcinoma or Tis without Negative Colonoscopy patient above the threshold , the only PDI score was increased in 29 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Coloscopy patient above the threshold, and the only CA19-9 assay was increased in 23 patients with invasive colorectal adenocarcinoma or Tis for 1 patient Negative colonoscopy above the threshold. The association of tumor markers prolidase, LFABP, M2PK, Timp1, Villine, CA19-9, G3PDH and ACE thus makes it possible to obtain, for the same group of 183 patients, total scores "" increased in 94 patients with colorectal adenocarcinoma. Invasive or Tis for 3 Negative Colonoscopy patients above the threshold. While the only prolidase score was increased in 59 patients with invasive colorectal adenocarcinoma or Tis for a Negative Coloscopy patient with a score above the threshold, the single LFABP assay was increased in 39 patients with Invasive colorectal adenocarcinoma or Tis for 1 Negative Colonoscopy patient above the threshold, the only M2PK assay was increased in 18 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Colonoscopy patient over the threshold. threshold, the only Timp1 assay was increased in 4 patients with invasive colorectal adenocarcinoma or Tis without Negative Coloscopy patient above the threshold, the Villin single dose was increased in 5 patients with invasive colorectal adenocarcinoma or of a Tis without Negative Colonoscopy Patient Above Threshold, the Single G3PDH Assay was Increased in 24 Patients With Invasive Colorectal Adenocarcinoma or T1 s Without Patients Negative Colonoscopy Above Threshold, the Single CA19-9 Assay was Increased in 21 Patients With Invasive Colorectal Adenocarcinoma or Tis Without Patient Negative Colonoscopy Above Threshold and the Single ACE Assay Was Increased in 25 patients with invasive colorectal adenocarcinoma or Tis without Negative Colonoscopy patient above the threshold. 6) Prolidase alone can replace the combination of several markers The Applicant has also shown that by its performance prolidase advantageously replaces alone a combination of several markers. For example, the combination of LFABP, ACE, and the M2PK marker results in a total of 183 patients with a total score of "3 without Prolidase Y" increased in 58 patients with invasive colorectal adenocarcinoma. of a Tis for 2 Negative Colonoscopic Patients Above the Threshold, with the Single ACE Assay Increased in 25 Colorectal Adenocarcinoma Patients Without Patients Negative Colonoscopy Above the Threshold, the Single M2PK Assay Increased in 18 Patients With Colorectal Adenocarcinoma invasive or Tis for 1 Negative Colonoscopic patient above the threshold, and only increased LFABP assay in 39 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Coloscopy patient above the threshold. In this same group of patients, prolidase alone can detect 59 patients for a Negative Colonoscopy below the threshold, as described in Example 1. With another example, the association of ACE, CA19 9, of the Timp1 marker and of the M2PK marker thus makes it possible to obtain, for the same group of 183 patients, increased total "4 without prolidase" scores in 54 patients with invasive colorectal adenocarcinoma or Tis for 3 patients. Negative colonoscopy above the threshold, with the single ACE assay increased in 25 patients with colorectal adenocarcinoma without patient Negative colonoscopy above the threshold, the only M2PK assay increased in 18 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Coloscopy patient above threshold, the only Timp1 assay increased in 22 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Coloscopy patient above the threshold, and the only dose increased CA19-9 in 23 patients with invasive colorectal adenocarcinoma or Tis for 1 Negative Coloscopy patient above the threshold. In this same group of patients, prolidase alone can detect 59 patients for a Negative Colonoscopy below the threshold, as described in Example 1. This example can be repeated with various known markers that allow in combination of detect about 50% of colorectal cancers. Table 16 Patient Class Stage Score 3s Score 4t Score 4u Score 4v Score 5wa Score 5wb Score 8x Score 3 without prolidase y Score 3 without prolidase z CBSE011 Tis TO 0.55 0.44 8.99 5.52 11.2 0.51 6.78 11.7 13.7 CL5P234 Tis TO 11.8 8.56 8.61 8.75 7.05 7.03 4.51 6.07 0.36 CL5P286 Tis TO 0.36 0.31 0, 39 0.40 0.42 3.65 4.73 0.19 0.30 CLSP300 Tis TO 0.67 0.51 0.63 0.62 0.61 4.86 0.42 0.55 0.37 CLSP315 Tis TO 12,2 8,86 8,96 8,99 7,29 7,27 4,57 6,00 0,33 CL5P367 Tis TO 0,63 0,50 5,37 4,66 7,57 0,54 6,01 6,83 9,09 CL5P724 Tis TO 6,99 9,30 5,13 5,10 4,25 7,59 5,81 6,55 6,14 CNSE003 Tis TO 10,3 7,26 14, 1 7.37 11.4 9.16 7.21 18.5 7.10 CNSE004 Tis TO 0.42 0.32 0.51 0.44 0.51 3.57 0.41 0.42 0.35 GHBD015 Tis TO 7,47 5,31 5,44 5,46 4,52 4,41 6,12 6,99 0,50 GHBD036 Tis TO 0.62 0.49 0.53 4.59 3.65 3.74 3.03 0.47 4.28 CBSE001 CCR I 14.4 10.4 10.6 16.0 12.8 8.51 5.51 8.27 5.88 CBSE016 CCR I 22.2 16.2 16, 3 21.6 17.3 13.1 8.44 15.5 10.1 CLSP047 CCR I 0.40 0.31 0.40 0.50 0.50 4.01 0.42 0.10 0.34 CLSP162 CCR I 0, 43 0.32 0.43 0.45 0.45 0.43 0.41 0.36 0.25 CLSP196 CCR I 11.5 8.44 8.51 8.53 6.94 6.93 4.45 5 , 79 0.59 CL5P224 CCR I 31.1 22.2 22.4 22.4 18.1 18.0 11.4 22.7 0.74 CL5P235 CCR I 7.28 5.49 5.49 5.53 4.45 10.4 2.81 0.13 0.15 CL5P263 CCR I 0.63 5.08 0.55 0.63 0.57 4.20 3.17 0.45 6.33 CLSP340 CCR I 0, 46 0.37 0.42 0.47 0.43 0.41 0.43 0.30 0.30 CL5P344 CCR I 5.67 4.27 4.31 4.39 3.56 3.60 2.33 0 , 0.30 CL5P364 CCR I 6.24 4.72 4.78 4.86 3.96 3.90 2.56 0.25 0.39 CL5P376 CCR I 0.41 0.42 0.48 0.43 0.48 0.45 0.45 0.38 0.51 GHBD003 CCR I 0.41 0.33 0.36 0.44 0.39 0.41 0.31 0.16 0.26 GHBD094 CCR I 0, 50 0.43 0.54 0.54 0.57 0.51 0.37 0.54 0.50 CLSP075 CCR It 0.44 0.34 0.41 0.50 0.47 0.46 0.40 0 , 34 0.34 CLSP076 CCR It 11.4 8.30 8.40 8.50 6.88 6.83 4.40 5.56 0.36 CLSP080 CCR It 0.33 0.29 0.34 0.38 0.38 0.38 0.36 0.19 0.31 CLSP096 CCR Il 12.3 9.27 14.9 9.44 12.1 7.60 7.61 19.5 14.8 CLSP110 CCR Il 6, 12 4.33 4.43 4.51 3.70 7.04 2.38 5.59 0.34 CLSP117 CCR II 17.2 12.4 16.5 17.2 17.1 10.1 10.6 16 , 0 6.49 CLSP130 CCR It 6.61 4.69 4.77 4.90 4.00 3.94 2.55 6.03 0.44 CLSP133 CCR It 7.55 5.69 5.80 5.79 4.75 8.18 3.02 0.35 0.35 CLSP143 CCR Il 9.26 6.96 7.11 7.06 5.78 9.79 3.58 0.74 0.56 CLSP147 CCR It 7.04 5.29 15, 1 9.75 15.5 7.95 7.71 13.2 14.2 CLSP154 CCR It 16.7 12.6 12.7 12.7 10.3 10.3 6.54 10.5 8.00 CLSP155 CCR It 19.4 20.4 14.7 14.7 11.9 16.5 7.59 10.9 15.9 CLSP157 CCR It 6.24 4.68 4.79 4.79 3.93 3.93 4.67 0.48 0.29 CLSP165 CCR It 0.51 0.39 0.46 0.54 0.50 3.86 0.43 0.30 0.28 CLSP170 CCR It 15.7 11.5 11, 6 11.6 9.4 9.38 5.99 7.29 0.63 CLSP173 CCR It 6.20 4.73 4.80 4.86 4.01 3.96 5.60 0.26 0.50 CLSP177 CCR It 10.5 12.4 10.5 13.1 13.0 9.99 8.74 0.55 14.8 CLSP178 CCR It 93.7 66.7 66.9 66.8 53.6 53.6 33.7 88.8 14.7 CLSP179 CCR Il 11.8 5.07 5.15 5.10 4.21 4.21 2.73 0.88 0.63 CLSP186 CCR It 7.87 5.57 5, 61 5.76 4.66 4.63 2.96 7.21 0.39 CLSP194 CCR II 16.8 12.1 12.1 12.2 9.8 13.1 6.24 10.1 0.25 CLSP201 CCR It 15.9 11.5 11.5 19.5 15.5 9.28 8.19 8.67 7.98 CLSP207 CCR It 16.0 11.8 11.6 11.8 9.5 9.63 6.01 8.07 5.53 CLSP209 CCR Il 10.4 7.31 7.34 13.3 10.6 9.15 3.92 9.49 6.02 CLSP220 CCR Il 19.2 14.2 14.2 14 3 11.6 11.6 7.36 12.6 5.69 CLSP222 CCR Il 19.0 13.7 20.1 18.2 19.6 11.1 10.3 19.9 11.2 CLSP253 CCR It 5 , 68 4.27 4.37 4.41 3.63 6.74 2.30 0.48 0.38 CLSP256 CCR Il 6.96 5.31 17.6 9.29 14.3 4.40 9.04 17.1 13.0 CLSP280 CCR It 5.84 4.45 4.49 4.89 9.08 7.26 3.71 6.07 0.47 4.91 CLSP296 CCR It 5.86 11.7 0.53 8, 59 6.73 9.57 6.31 0.49 22.7 CLSP310 CCR II 0.50 0.44 0.47 0.59 0.54 0.55 0.42 0.33 0.47 CLSP327 CCR It 9 , 6.97 7.17 7.15 5.88 5.77 3.80 0.57 0.44 CLSP333 CCR It 5.98 0.73 0.62 0.73 0.65 0.71 0.56 0.53 0.58 CLSP341 CCR It 7.37 5.55 5.61 5.70 4.63 4.62 5.11 0.27 0.34 CLSP346 CCR It 12.2 15.3 9.38 9, 37 7.67 12.4 7.13 7.08 13.4 CLSP347 CCR Il 7.30 5.47 5.63 5.69 4.68 4.49 5.74 7.24 5.51 CLSP380 CCR It 7 , 21 5.42 5.64 5.52 4.61 4.47 5.72 0.62 0.44 CNSE002 CCR It 6.83 5.17 6.83 5.36 5.35 4.29 3.14 0.41 0.60 GHBD021 CCR It 0.37 0.30 0.34 0.43 0.39 0.40 0.36 0.15 0.26 GHBD043 CCR It 7.14 5.43 5.51 15.6 12.4 4.53 5.90 0.54 10.4 GHBD064 CCR It 37.4 27.5 33.3 27.7 26.8 22.2 16.9 38.7 20.7 GHBD066 CCR H 0.53 0.40 0.52 0.61 0.60 0.51 0.45 0.44 0.41 GHBD075 CCR H 5.84 4.39 4.51 4.49 3.70 3.67 2 , 39 0.54 0.39 GHBD087 CCR H 0.72 0.58 0.60 0.68 0.61 3.67 0.44 0.52 0.39 GHBD090 CCR H 0.40 0.33 0.45 0.46 0.49 0.39 0.35 0.42 0.40 GHBD096 CCR H 0.74 0.56 5.64 0.71 4.64 4.25 2.95 7.20 5.41 GHBD100 CCR H 9.40 5.93 13.8 14.5 16.6 10.8 7.14 13.8 12.4 GHBD103 CCR H 6.05 4.29 4.35 4.42 3.61 3.58 2 , 34 5.62 0.33 CBSE023 CCR III 6.58 4.72 6.18 6.18 6.18 3.95 7.38 8.88 0.43 CLSP044 CCR III 68.5 51.4 51.5 51.5 41.3 41.3 26.0 68.3 51.3 CLSP074 CCR III 10.0 7.56 7.66 12.5 10.0 6.24 4.07 0.40 5.16 CLSP078 CCR III 19.4 14.0 14.1 14.2 11.5 14.4 7.28 11.6 0.43 CLSP081 CCR III 9.85 7.41 9.84 9.84 9.84 6.09 8 , 03 0.37 0.21 CLSP098 CCR III 10.2 7.67 7.75 7.77 6.30 10.5 6.01 0.34 0.29 CLSP174 CCR III 46.8 75.3 46.0 44.9 44.6 60.3 40.5 59.2 113.1 CLSP227 CCR III 44.5 32.2 32.4 32.3 26.0 25.9 16.3 37.0 10.5 CLSP233 CCR III 7.46 5.63 5.71 5.69 4.64 7.85 2.95 0.30 0.31 CLSP255 CCR III 18.6 23.5 13.5 13.6 10.9 19.0 10.1 10.6 13.6 CLSP289 CCR III 0.62 0.47 0.54 0.58 0.54 0.56 0.49 0.45 0.29 CLSP320 CCR III 5.70 4.29 4 43 4.40 3.64 6.87 2.36 0.43 0.40 CLSP324 CCR III 9.72 7.44 7.49 7.52 6.17 6.11 6.25 0.59 0.81 CLSP383 CCR III 0.42 0.33 4.76 5.34 7.72 0.43 2.62 6.07 9.44 CLSP384 CCR III 71.9 57.6 63.9 53.7 51.3 46.2 32.3 85.0 63.1 CNSE001 CCR III 6.40 11.0 4.91 4.99 4.08 12.4 2.77 6.28 12.8 CNSE016 CCR III 67.8 62.8 53 6 48.9 43.0 50.3 31.4 65.0 37.4 CNSE017 CCR III 15.0 10.9 10.9 11.0 8.9 13.3 5.58 8.44 0.29 CSEM015 CCR III 6.74 4.76 6.33 6.33 6.33 7.47 3.26 9.13 0.14 CSEM029 CCR III 6.17 4.65 4.79 9.03 7.26 7.74 2.61 0.54 4.60 GHBD004 CCR III 21.2 16.0 16.0 16.1 12.9 12.9 8.10 15.1 11.5 GHBD017 CCR III 7.40 5.59 5, 70 9.85 7.91 4.63 3.02 0.26 4.41 GHBD034 CCR III 0.31 0.26 0.32 0.41 0.39 0.39 3.94 0.16 0.32 GHBD042 CCR III 0.45 0.47 0.44 0.51 0.49 0.53 0.38 0.33 0.47 GHBD045 CCR III 18.2 13.6 13.8 13 , 9 11.2 11.0 7.02 0.33 0.46 GHBD089 CCR III 28.8 21.7 21.7 21.8 17.5 20.9 11.0 22.8 17.2 GHBD105 CCR III 0.58 6.18 6.95 4.49 5.75 5.08 3.78 8.96 14.3 CBSE012 CCR IV 6.40 11.7 5.02 4.99 4.17 12.8 4 87 0.68 9.58 CBSE026 CCR IV 329.6 3920 328.6 328.6 328.6 3136 1723 481.3 10043 CLSP156 CCR IV 21.9 23.0 21.2 16.5 17.1 21.8 16.0 19.6 23.3 CLSP159 CCR IV 47.6 60.7 44.8 35.6 36.0 48.8 30.7 59.6 73.3 CLSP167 CCR IV 6.60 10.4 5, 06 9.15 7.32 12.0 2.85 0.32 11.4 CLSP260 CCR IV 66.9 48.4 48.4 48.4 38.9 38.9 24.5 64.3 21.2 CLSP334 CCR IV 0.40 0.33 0.53 0.42 0.52 0.41 2.95 0.52 0.48 CLSP357 CCR IV 9.04 18.5 6.98 6.92 5.70 14.9 9.40 9.03 22.1 CLSP365 CCR IV 27.5 195.3 25.6 20.9 20.6 156.3 68.5 33.9 253.3 CLSP366 CCR IV 0.54 8.46 0, 64 0.55 0.64 6.93 3.09 0.63 11.0 GHBD022 CCR IV 1265.0 948.6 948.5 953.0 762.5 759.0 474.6 1264.5 951.9 GHBD071 RCC IV 56.5 47.7 46.8 41.5 37.5 38.3 27.9 62.1 40.0 CLSP197 CCR NA 0.43 0.40 0.53 0.43 0.52 0.51 4.20 0.53 0.47 HGED005 ColoNeg indicator 0.57 0.46 0.62 0.67 0.69 0 , 51 0.54 0.51 0.59 PROMIS 21-01-005 ColoNeg indicator 0.53 0.55 0.49 0.54 0.51 0.61 0.43 0.36 0.49 PROMIS 21-01 -006 ColoNeg indicator 0.29 0.24 0.29 0.37 0.36 0.37 0.23 0.19 0.26 PROMIS 21-01-009 ColoNeg indicator 0.58 0.52 0.45 0, 53 0.45 0.57 0.43 0.37 0.30 PROMIS 21-01-011 ColoNeg indicator 0.19 0.18 0.19 0.29 0.27 0.28 0.22 0.09 0, 26 PROMIS 21-01-012 Witness ColoNeg 0.47 0.43 0.44 0.47 0.45 0.50 0.36 0.31 0.42 PROMIS 21-02-001 Witness ColoNeg 6.46 4.59 4.72 11.3 9.05 3.78 2.61 6.08 6.87 PROMIS 21-02-003 ColoNeg Indicator 0.35 0.45 0.34 0.41 0.39 0.52 0.31 0.26 0.51 PROMIS 21-02-004 ColoNeg indicator 0.37 0.29 0.37 0.42 0.41 0.31 0.39 0.37 0.28 PROMIS 21-02-006 ColoNeg indicator 0 , 41 0.34 0.41 0.42 0.42 4.07 0.36 0.27 0.27 PROMIS 21-02-008 ColoNeg indicator 5.95 0.51 0.58 0.58 0.58 0 , 56 0.43 0.59 0.29 PROMIS 21-02-011 ColoNeg indicator 0.46 0.47 0.45 0.45 0.45 0.55 0.38 0.23 0.48 PROMIS 21-02 -014 ColoNeg indicator 0.57 0.43 0.55 0.55 0.55 0.53 0.33 0.40 0.12 PROMIS 21-02-016 ColoNeg indicator 0.39 0.33 0, 48 0.44 0.50 0.39 0.40 0.46 0.52 PROMIS 21-03-004 ColoNeg indicator 0.76 0.58 0.75 0.74 0.75 0.62 0.49 0, 74 0.56 PROMIS 21-03-013 ColoNeg indicator 0.23 0.18 0.25 0.37 0.36 0.34 0.25 0.21 0.29 PROMIS 21-03-015 ColoNeg indicator 0.46 0.35 0.42 0.52 0.48 0.44 0.34 0.37 0.34 PROMIS 21-04-002 ColoNeg indicator 0.56 0.51 0.63 0.55 0.63 0.56 0.58 0.53 0.49 PROMIS 21-04-006 ColoNeg indicator 0.44 0.35 0.39 0.44 0.40 0.48 0.37 0.20 0.24 PROMIS 21-04-007 ColoNeg indicator 0.48 0.38 0.50 0.57 0.57 0.46 0.37 0.46 0.44 PROMIS 21-04-008 ColoNeg indicator 0.39 0.30 0.40 0.44 0 , 44 0.38 0.38 0.38 0.32 PROMIS 21-04-009 ColoNeg indicator 0.37 0.38 0.49 0.46 0.54 0.42 0.41 0.48 0.60 PROMIS 21-04-013 ColoNeg indicator 0.65 0.59 0.59 0.61 0.58 0.63 0.39 0.50 0.53 PROMIS 21-07-004 ColoNeg indicator 0.65 0.51 0, 57 0.64 0.59 0.53 0.38 0.51 0.37 PROMIS 21-12-001 ColoNeg indicator 0.44 0.43 5.58 0.45 4.56 0.49 2.93 7, 21 5.58 PROMIS 21-12-007 ColoNeg indicator 0.53 0.42 0.52 0.51 0.50 0.53 0.33 0.28 0.20 PROMIS 21-12-011 ColoNe indicator g 0.52 0.47 0.54 0.52 0.54 0.52 0.38 0.51 0.58 PROMIS 21-22-001 ColoNeg indicator 0.42 0.48 0.39 4.42 0, 51 0.52 0.37 0.34 0.67 PROMIS 21-22-006 ColoNeg indicator 0.52 0.40 0.51 0.51 0.51 0.49 0.36 0.45 0.34 PROMIS 21 -22-008 ColoNeg indicator 0.46 0.36 0.41 0.47 0.43 0.42 0.36 0.28 0.24 PROMIS 37-03-004 ColoNeg indicator 0.48 0.38 0.46 0.50 0.48 0.47 0.48 0.29 0.30 PROMIS 37-04-001 ColoNeg indicator 0.74 0.58 0.70 0.74 0.72 0.57 0.53 0.79 0.64 PROMIS 37-04-005 ColoNeg indicator 0.60 0.47 0.58 0.58 0.58 0.51 0.34 0.40 0.17 PROMIS 37-05-008 ColoNeg indicator 0.46 0 , 36 0.45 0.48 0.47 0.46 0.42 0.38 0.28 PROMIS 37-06-001 ColoNeg indicator 0.57 0.47 0.61 0.54 0.59 0.51 0 , 39 0.57 0.45 PROMIS 37-09-002 ColoNeg indicator 0.31 0.48 0.33 0.46 0.44 0.55 0.41 0.23 5.58 PROMIS 37-11-001 Witness ColoNeg 0.44 0.34 0.41 0.46 0.44 0.45 0.33 0.22 0.24 PROMIS 37-11-002 ColoNeg indicator 0.29 0.23 0.42 0.38 0, 47 0.37 0.38 0.35 0.39 PROMIS 37-11-011 ColoNeg indicator 0.53 0.40 0.48 0.54 0.50 0.45 0.40 0.35 0.30 PROMIS 37 -11 to 016 ColoNeg indicator 0.30 0.27 0.47 0.47 0.57 0.37 0.37 0.39 0.55 PROMIS 37-12-002 ColoNeg indicator 0.31 0.25 0.32 0.36 0 , 36 0.35 0.32 0.27 0.29 PROMIS 37-13-003 ColoNeg indicator 0.46 0.41 0.45 0.54 0.52 0.47 0.35 0.40 0.44 PROMIS 37-13-006 ColoNeg indicator 0.57 0.43 0.54 0.66 0.63 0.51 0.39 0.50 0.43 PROMIS 37-13-008 ColoNeg indicator 0.32 0.24 0, 0.36 0.34 0.35 0.33 0.23 0.20 PROMIS 37-14-009 ColoNeg indicator 0.57 0.42 0.49 0.58 0.51 0.50 0.31 0, 38 0,27 PROMIS 37-14-018 ColoNeg indicator 6,80 5,12 6,79 6,79 6,79 4,26 3,43 0,35 0,12 PROMIS 37-14-021 ColoNeg indicator 0,35 0.30 0.33 0.40 0.37 0.39 0.26 0.16 0.29 PROMIS 37-15-002 ColoNeg indicator 0.36 0.30 0.33 0.52 0.46 0.38 0.32 0.22 0.38 PROMIS 37-15-006 ColoNeg indicator 0.41 0.32 0.41 0.52 0.50 0.42 0.32 0.40 0.40 PROMIS 37-16-001 ColoNeg indicator 0.32 0.27 0.43 0.36 0.45 0.35 0.43 0.44 0.40 PROMIS 37-17-001 ColoNeg indicator 0.45 0.37 0.41 0.46 0 , 43 0.43 0.36 0.35 0.35 PROMIS 37-17-003 ColoNeg indicator 0.68 0.52 0.66 0.63 0.63 0.53 0.42 0.62 0.55 PRO MIS 37-17-004 ColoNeg indicator 0,50 0,40 0,53 0,50 0,53 0,47 0,35 0,45 0,38 PROMIS 37-18-001 ColoNeg indicator 0,45 0,36 0 , 40 0.45 0.41 0.45 0.33 0.26 0.25 PROMIS 37-18-003 ColoNeg indicator 0.50 0.39 0.42 0.51 0.45 0.49 0.31 0 , 34 0.25 PROMIS 37-18-004 ColoNeg indicator 0.32 0.26 0.32 0.43 0.41 0.37 0.30 0.24 0.30 PROMIS 37-18-005 ColoNeg indicator 0, 49 0.39 0.44 0.50 0.46 0.47 0.31 0.26 0.29 PROMIS 37-18-024 ColoNeg indicator 0.43 0.36 0.45 0.48 0.49 0, 47 0.31 0.32 0.39 PROMIS 37-19-006 ColoNeg indicator 0.59 0.44 0.54 0.56 0.53 0.47 0.37 0.42 0.25 PROMIS 37-19- 007 ColoNeg indicator 0.42 0.31 0.37 0.45 0.41 0.42 0.29 0.23 0.24 PROMIS 37-20-004 ColoNeg indicator 0.37 0.28 0.48 0.44 0.52 0.36 0.52 0.44 0.44 PROMIS 71-02-003 ColoNeg indicator 0.31 0.24 0.39 0.36 0.42 0.36 0.31 0.30 0.33 PROMIS 71-04-003 ColoNeg indicator 0,50 0,41 0,46 0,50 0,48 0,50 0,33 0,39 0,33 PROMIS 71-09-007 ColoNeg indicator 0,41 0,35 0 , 36 0.47 0.42 0.43 0.35 0.28 0.33 PROMIS 71-10-001 ColoNeg indicator 0.34 0.40 0.37 0.37 0.39 0.45 0.35 0.28 0.47 PROMIS 71-10-007 ColoNeg Indicator 0.41 0.34 0.47 0.48 0.51 0.40 0.45 0.41 0.50 PROMIS 71-11-001 ColoNeg Indicator 0 , 40 0.32 0.39 0.44 0.43 0.38 0.33 0.34 0.35 PROMIS 71-11-006 ColoNeg indicator 0.41 0.33 0.37 0.43 0.40 0 , 41 0.31 0.27 0.25 PROMIS 71-11-008 ColoNeg indicator 0.34 0.33 0.31 0.38 0.35 0.40 0.24 0.22 0.33 PROMIS 71-11 -009 ColoNeg indicator 0.50 0.43 0.44 0.49 0.45 0.50 0.35 0.34 0.32 Performance Score 3s Score 4t Score 4u Score 4v Score Score Score Score 3 without prolidaseY Score 3 without prolidasez 5wa 5wb 8) <Threshold 0.76 0.73 0.75 0.75 0.75 0.63 0.58 0.88 0.81 Sensitivity% 73.5 75.2 76.1 77 77.9 82 , 3 83.2 51.3 47.8 Specificity% 95.7 97.2 95.7 95.7 95.7 95.7 95.7 97.2 95.7 CCR invasive and Tis above threshold 83 85 86 87 88 93 94 58 54 ColoNeg above threshold 3 2 3 3 3 3 3 2 3 Tis: Patients with tumor in situ (intra mucosal carcinoma TO), CCR = patients with colorectal cancer of stage TNM I to IV (Adenocarcinoma), ColoNeg- = healthy subjects, with u n a positive FOBT test and a completely normal colonoscopy.

3S Score: Prolidase Association, LFABP, ACE Score 4t: Prolidase Association, LFABP, ACE, CA19-9 4U Score: Prolidase Association, LFABP, ACE, M2PK Score 4 ": Prolidase Association, LFABP, ACE, Timp1 Score 5wa: Prolidase Association , LFABP, ACE, M2PK, Timp1 5wb Score: Prolidase Association, LFABP, ACE, PDI, CA19-9 8x Score: Prolidase Association, LFABP, ACE, M2PK, Timp1, CA19-9, Villin, G3PDH Score 3 without Prolidase: Association LFABP, ACE, M2PK Score 3 without prolidasez: association ACE, M2PK, C19-9, Timp1.

Example 6: Detection of colorectal cancer patients and control patients by the MRM assay of the enolase, G3PDH, BIP, HSP71, Peroxv-5 and PDI markers For each of these markers, the scores were obtained in the same conditions than those used to obtain the prolidase scores and described in Example 5 (Table 14).

Table 17 Patient Class Stage Alpha-Alpha-G3PDH Dose Ratio Arbitrary Unborn G3PDH BIP BIP HSP71 Dose Ratio Unke "Arbitrary HSP71 Peroxy-5 Dose Ratio Arbitrary Unit Peroxy- 5 Score PDI Dose Ratio Arbitrary Unit PDI In olase Enolase Score Dose Score Score Score Dose Ratio Score Ratio Unit Arbitrary Arbitrary Unit CBSE011 Tis TO 0.184 0.816 0.454 18.773 0.059 0.802 0.010 0.418 0.023 0.880 0.070 0.799 CLSP234 Tis TO 0.100 0.443 0.275 0.711 0.052 0.909 0.015 0.635 0.012 0.426 0.062 0.893 CLSP286 Tis TO 0.247 17.540 0.507 20.986 0.049 0.971 0.024 16.098 0.032 17.608 0.052 16.988 CLSP300 Tis TO 0.092 0.467 0.215 0.555 0.043 17.840 0.008 0.328 0.012 0.406 0.040 22.285 CLSP315 Tis TO 0.071 0.316 0.147 0.381 0.043 17.852 0.006 0.244 0.012 0.400 0.059 0.941 CLSP367 Tis TO 0.000 0.000 0.693 28.701 0.060 0.796 0.028 18.715 0.035 19.042 0.083 0.668 CLSP724 Tis TO 0.254 18.070 0.654 27.083 0.058 0.816 0.030 20.193 0.022 0.751 0.074 0.753 CNSE003 Tis TO 0.123 0.546 0.256 0.661 0.05 0 0.960 0.010 0.407 0.032 17.676 0.053 16.737 CNSE004 Tis TO 0.140 0.620 0.264 0.683 0.045 16.792 0.007 0.309 0.019 0.648 0.054 16.584 GHBD015 lilies TO 0.277 19.689 0.698 28.877 0.056 0.854 0.013 0.541 0.045 24.993 0.068 0.817 GHBD036 Tis TO 0.308 21.894 0.569 23.570 0.044 17.400 0.020 0.847 0.036 19.646 0.053 16.734 CBSE001 CCR I 0.218 0.968 0.377 0.976 0.049 0.979 0.017 0.716 0.027 0.913 0.062 0.891 CBSE016 CCR I 0.139 0.618 0.274 0.710 0.059 0.800 0.010 0.418 0.027 0.912 0.068 0.821 CLSP047 CCR I 0.061 0.269 0.226 0.586 0.049 0.969 0.008 0.333 0.024 0.815 0.047 18.797 CLSP162 CCR I 0.116 0.513 0.406 16.810 0.056 0.847 0.014 0.602 0.007 0.227 0.065 0.851 CLSP196 CCR I 0.090 0.401 0.223 0.576 0.057 0.842 0.010 0.438 0.023 0.789 0.063 0.886 CLSP224 CCR I 0.122 0.543 0.281 0.726 0.054 0.884 0.012 0.498 0.023 0.791 0.058 0.953 CLSP235 CCR I 0.058 0.256 0.028 0.071 0.032 23.467 0.003 0.128 0.015 0.509 0.030 29.846 CLSP263 CCR I 0.159 0.706 0.587 24.283 0.066 0.716 0.026 17.775 0.020 0.690 0 , 080 0.695 CLSP340 CCR I 0.266 18.897 0.426 17.617 0.087 0.549 0.031 21.062 0.028 0.955 0.093 0.598 CLSP344 CCR I 0.113 0.503 0.327 0.847 0.057 0.832 0.011 0.467 0.016 0.557 0.060 0.930 CLSP364 CCR I 0.147 0.652 0.347 0.897 0.084 0.569 0.015 0.625 0.031 16.834 0.093 0.596 CLSP376 CCR I 0.165 0.731 0.359 0.928 0.071 0.688 0.015 0.624 0.020 0.702 0.098 0.567 100 GHBD003 CCR I 0.000 0.204 0.529 0.062 0.761 0.010 0.425 0.017 0.584 0.074 0.748 GHBD094 CCR I 0.0 0.0 0.0 0.0 0.0 0.0 CLSP075 CCR H 0.132 0.585 0.243 0.628 0.057 0.840 0.011 0.441 0.024 0.810 0.059 0.935 CLSP076 CCR H 0.101 0.448 0.238 0.616 0.049 0.966 0.010 0.411 0.017 0.597 0.059 0.936 CLSP080 CCR H 0.081 0.381 0.199 0.516 0.065 0.734 0.010 0.432 0.019 0.639 0.075 0.739 CLSP096 CCR H 0.133 0.589 0.133 0.343 0.052 0.917 0.008 0.335 0.016 0.535 0.062 0.896 CLSP110 CCR II 0.116 0.514 0.206 0.533 0.044 17.162 0.007 0.308 0.019 0.651 0.050 17.881 CLSP117 CCR H 0.231 16.410 0.628 25.978 0.070 0.678 0.037 24.949 0.028 0.970 0.101 0.5 CLSP130 CCR H 0.120 0.533 0.208 0.537 0.050 0.945 0.007 0.286 0.012 0.420 0.060 0.931 CLSP133 CCR II 0.058 0.256 0.152 0.394 0.043 17.692 0.006 0.273 0.008 0.267 0.049 18.147 CLSP143 CCR It 0.083 0.370 -0.167 -0.433 0.051 0.937 0.006 0.251 0.013 0.445 0.042 21.119 CLSP147 CCR H 0.116 0.517 0.227 0.587 0.054 0.886 0.014 0.569 0.015 0.517 0.048 18.569 CLSP154 CCR II 0.116 0.516 0.241 0.622 0.045 17.058 0.012 0.497 0.019 0.642 0.057 0.981 CLSP155 CCR H 0.077 0.344 0.194 0.502 0.054 0.882 0.008 0.321 0.022 0.742 0.061 0.910 CLSP157 CCR H 0.093 0.413 0.476 19.720 0.074 0.639 0.007 0.292 0.036 19.859 0.059 0.949 CLSP165 CCR II 0.130 0.579 0.210 0.542 0.044 17.268 0.007 0.294 0.016 0.552 0.050 17.712 CLSP170 CCR It 0.086 0.380 0.365 0.944 0.055 0.864 0.010 0.420 0.014 0.493 0.068 0.818 CLSP173 CCR H 0.157 0.697 0.391 16.186 0.049 0.971 0.010 0.414 0.033 18.412 0.065 0.861 CLSP177 CCR Il 0.314 22.303 0.742 30.716 0.072 0.658 0.023 0.960 0.070 38.215 0.131 0.424 CLSP178 CCR It 0.178 0.788 0, 369 0.954 0.061 0.776 0.017 0.693 0.020 0.685 0.062 0.896 CLSP179 CCR H 0.069 0.307 0.150 0.388 0.053 0.899 0.007 0.285 0.017 0.576 0.070 0.794 CLSP186 CCR H 0.085 0.378 0.100 0.270 0.080 0.798 0.010 0.430 0.016 0.556 0.062 0.896 CLSP194 CCR H 0.109 0.484 0.253 0.655 0.060 0.787 0.008 0.338 0.022 0.758 0.053 16.847 CLSP201 CCR H 0.115 0.511 0.127 0.328 0.053 0.897 0.012 0.498 0.023 0.778 0.100 0.556 CLSP207 CCR H 0.075 0.331 0.096 0.249 0.041 18.380 0.006 0.242 0.015 0.509 0.061 0.919 CLSP209 CCR H 0.101 0.449 0.237 0.613 0.046 16.569 0.013 0.530 0.016 0.551 0.054 16.512 CLSP220 CCR H 0.095 0.420 0.352 0.911 0.041 18.783 0.009 0.398 0.016 0.544 0.059 0.945 CLSP222 CCR H 0.144 0.639 0.318 0.822 0.044 17.177 0.014 0.598 0.030 16.469 0.063 0.887 CLSP253 CCR H 0.098 0.437 0.200 0.517 0.042 18.236 0.009 0.366 0.022 0.743 0.054 16.627 CLSP256 CCR H 0.142 0.629 0.375 0.969 0.053 0.906 0.016 0.660 0.022 0.744 0.070 0.797 CLSP280 CCR It 0.138 0.613 0.790 32.707 0.082 0.771 0.039 25.938 0.037 20 , 417 0.074 0.746 101 CLSP296 CCR H 0.085 0.379 0.187 0.483 0.045 16.965 0.013 0.566 0.012 0.467 0.057 0.967 CLSP310 CCR H 0.119 0.529 0.254 0.657 0.059 0.800 0.010 0.430 0.018 0.627 0.056 0.985 CLSP327 CCR II 0.133 0.590 0.395 16.366 0.063 0.755 0.014 0.589 0.021 0.724 0.059 0.947 CLSP333 CCR H 0.098 0.434 0.351 0.908 0.065 0.727 0.014 0.575 0.023 0.775 0.086 0.649 CLSP341 CCR H 0.189 0.841 0.476 19.687 0.064 0.746 0.017 0.696 0.016 0.558 0.062 0.891 CLSP346 CCR It 0.263 18.663 0.482 19.932 0.089 0.537 0.019 0.803 0.028 0.951 0.101 0.549 CLSP347 CCR II 0.284 20.164 0.609 25.215 0.083 0.574 0.015 0.648 0.029 0.997 0.100 0.558 CLSP380 CCR H 0.177 0.786 0.618 25.586 0.060 0.797 0.021 0.884 0.028 0.977 0.086 0.647 CNSE002 CCR D 0.099 0.439 0.216 0.558 0.064 0.747 0.009 0.377 0.021 0.736 0.069 0.805 GHBD021 CCR H 0.111 0.492 0.212 0.548 0.051 0.937 0.010 0.405 0.021 0.735 0.067 0.831 GHBD043 CCR I] 0.087 0.388 0.188 0.487 0.047 16.085 0.007 0.292 0.014 0.471 0.059 0.92 GHBD064 CCR H 0.101 0.447 0.331 0.856 0.061 0.776 0.010 0.418 0.024 0.818 0.061 0.913 GHBD066 CCR H 0.118 0.522 0.293 0.757 0.052 0.920 0.010 0.400 0.010 0.342 0.061 0.909 GHBD075 CCR H 0.122 0.540 0.211 0.545 0.049 0.973 0.008 0.327 0.019 0.659 0.069 0.805 GHBD087 CCR It 0.095 0.420 0.134 0.347 0.051 0.930 0.006 0.267 0.012 0.416 0.055 16.074 GHBD090 CCR H 0.079 0.351 0.118 0.305 0.068 0.702 0.011 0.441 0.015 0.516 0.087 0.642 GHBD096 CCR It 0.046 0.206 0.083 0.214 0.048 0.990 0.003 0.142 0.016 0.544 0.047 18.998 GHBD100 CCR II 0.0 0.0 0.0 0, 0 0.0 0.0 GHBD103 CCR H 0.128 0.567 0.377 0.975 0.054 0.874 0.013 0.556 0.021 0.706 0.075 0.737 CBSE023 CCR III 0.272 19.346 0.682 28.243 0.049 0.969 0.020 0.840 0.034 18.866 0.063 0.889 CLSP044 CCR III 0.143 0.634 0.328 0.849 0.068 0.696 0.012 0.510 0.039 21.613 0.071 0.779 CLSP074 CCR III 0.099 0.439 0.218 0.564 0.044 17.414 0.009 0.382 0.049 27.042 0.058 0.952 CLSP078 CCR III 0.0 0.163 0.421 0.048 0.993 0.011 0.448 0.030 16.472 0.055 16.083 CLSP081 CCR III 0.183 0 , 814 0.412 17.065 0.059 0.804 0.016 0.677 0.031 17.187 0.067 0.835 CLSP098 CCR III 0.000 0.000 0.445 18.408 0.038 19.881 0.009 0.385 0.023 0.806 0.041 21.746 CLSP174 CCR III 0.269 19.13 133 0.446 18.447 0.083 0.570 0.028 18.599 0.060 33.217 0.107 0.520 CLSP227 CCR III 0.116 0.517 0.226 0.584 0.042 18.049 0.010 0.436 0.037 20.495 0.073 0.762 0.033 0.091 0.391 0.193 0.499 0.033 22.944 0.007 0.295 0.014 0.480 0.053 16.726 CLSP255 CCR III 0.077 0.341 0.204 0.528 0.051 0.929 0.008 0.326 0.024 0.811 0.059 0.941 CLSP289 CCR III 0.143 0.634 0.290 0.749 0.050 0.946 0.012 0.498 0.017 0.590 0.062 0.895 CLSP320 CCR III 0.049 0.218 0.233 0.603 0.053 0.894 0.011 0.442 0.011 0.376 0.052 17.187 102 CLSP324 CCR III 0.278 19.717 0.504 20.843 0.051 0.939 0.029 19.513 0.032 17.559 0.070 0.799 CLSP383 CCR III 0.120 0.533 0.369 0.955 0.062 0.761 0.016 0.662 0.017 0.596 0.068 0.822 CLSP384 CCR III 0.129 0.573 0.400 16.556 0.063 0.758 0.018 0.768 0.018 0.617 0.097 0.571 CNSE001 CCR III 0.174 0.772 0.24 0.634 0.049 0.961 0.011 0.447 0.028 0.955 0.050 17.903 CNSE016 CCR III 0.182 0.889 0.267 0.691 0.066 0.722 0.012 0.509 0.023 0.789 0.137 0.405 CNSE017 CCR III 0.094 0.418 0.088 0.228 0.041 18.482 0.005 0.209 0.017 0.600 0.038 23.266 CSEM015 CCR III 0.092 0.408 0.121 0.314 0.049 0.965 0.006 0.235 0.009 0.300 0.048 18.344 CSEM029 CCR III 0.084 0.373 0.158 0.409 0.052 0.906 0.009 0.377 0.022 0.752 0.044 20.098 GHBD004 CCR III 0.0 0.055 0.141 0.053 0.905 0.005 0.192 0.011 0.388 0.069 0.804 GHBD017 CCR III 0.103 0.459 0.270 0.700 0.058 0.815 0.011 0.479 0.015 0.501 0.069 0.809 GHBD034 CCR III 0.235 16.711 0.787 32.590 0.050 0.947 0.029 19.769 0.028 0.979 0.060 0.925 GHBD042 CCR III 0.128 0.568 0.244 0.630 0.055 0.861 0.007 0.285 0.038 20.725 0.070 0.794 GHBD045 CCR III 0.051 0.228 0.108 0.278 0.050 0.942 0.008 0.325 0.011 0.363 0.087 0.642 GHBD089 CCR III 0.113 0.502 0.189 0.490 0.048 0.990 0.005 0.220 0.014 0.474 0.051 17.437 GHBD105 CCR III 0.110 0.487 0.252 0.652 0.048 0.996 0.007 0.27 4 0.012 0.423 0.084 0.664 CBSE012 CCR IV 0.090 0.400 0.078 0.202 0.047 16.281 0.007 0.297 0.009 0.312 0.051 17.288 CBSE026 CCR IV 0.182 0.809 0.290 0.751 0.068 0.700 0.012 0.520 0.022 0.773 0.077 0.718 CLSP156 CCR IV 0.181 0.802 0.682 28.221 0.055 0.869 0.025 17.075 0.025 0.845 0.053 16.789 CLSP159 CCR IV 0.171 0.759 0.289 0.749 0.052 0.907 0.014 0.585 0.123 67.466 0.065 0.858 CLSP167 CCR IV 0.095 0.422 0.233 0.603 0.042 18.288 0.009 0.377 0.017 0.587 0.049 18.280 CLSP260 CCR IV 0.246 17.461 0.384 0.994 0.057 0.830 0.015 0.635 0.030 16.454 0.061 0.906 CLSP334 CCR IV 0.283 20.085 0.571 23.624 0.076 0.624 0.026 17.141 0.036 19.787 0.078 0.734 CLSP357 CCR IV 0.161 0.713 0.455 18.843 0.086 0.553 0.021 0.896 0.024 0.815 0.091 0.644 CLSP365 CCR IV 0.179 0.796 0.248 0.642 0.063 0.750 0.011 0.473 0.017 0.594 0.082 0.680 CLSP366 CCR IV 0.169 0.751 0.408 16.904 0.064 0.743 0.018 0.770 0.026 0.901 0.070 0.797 GHBD022 CCR IV 0.246 17.501 0.291 0.753 0.058 0.825 0.012 0.514 0.016 0.539 0.084 0.665 GHBD071 CCR IV 0.518 36.822 0.512 21.194 0.046 16.552 0.025 16.504 0.043 23.621 0.061 0.905 CLSP197 CCR NA 0.0 0.0 0.0 0.0 0.0 0.0 HGED005 ColoNeg control 0.236 16.744 0.304 0.786 0.062 0.767 0.017 0.726 0.028 0.967 0.082 0,678 PROMIS 21-01-005 ColoNeg indicator 0,111 0,491 0,199 0,515 0,050 0,960 0.007 0.300 0.011 0.389 0.066 0.846 PROMIS 21-01-006 ColoNeg indicator 0.109 0.482 0.123 0.317 0.054 0.879 0.012 0.503 0.009 0.313 0.061 0.913 103 PROMIS 21-01-009 ColoNeg indicator 0.091 0.403 0.094 0.242 0.077 0.615 0.012 0.501 0.023 0.791 0.073 0.760 PROMIS 21-01-011 Control ColoNeg 0.042 0.187 0.135 0.350 0.067 0.708 0.006 0.263 0.012 0.416 0.080 0.698 PROMIS 21-01-012 Control ColoNeg 0.155 0.687 0.252 0.653 0.070 0.676 0.012 0.499 0.020 0.693 0.071 0.786 PROMIS 21-02-001 ColoNeg indicator 0.140 0.620 0.387 1.000 0.072 0.664 0.010 0.415 0.020 0.698 0.158 0.516 PROMIS 21-02-003 ColoNeg indicator 0.045 0.198 0.119 0.307 0.062 0.766 0.005 0.227 0.013 0.439 0.068 0.813 PROMIS 21-02-004 ColoNeg indicator0,0 0,386 0,999 0,088 0,543 0,024 1,000 0,025 0,871 0,137 0,406 PROMIS 21-02-006 ColoNeg indicator 0,072 0,320 0,382 0,988 0,043 17,748 0,025 16,545 0.029 1,000 0.047 18,985 PROMIS 21-02-008 ColoNeg indicator 0,085 0,379 0,178 0,460 0,057 0,833 0,081 54,590 0,010 0.338 0.070 0.794 PROMIS 21-02-011 ColoNeg Indicator 0.072 0.319 0.164 0.425 0.064 0.747 0.008 0.316 0.010 0.339 0.063 0.887 PROMIS 21-02-014 ColoNeg Indicator 0.075 0.331 0.128 0.330 0.048 0.993 0.009 0.397 0.011 0.388 0.063 0.888 PROMIS 21-02-016 Indicator ColoNeg 0.144 0.631 0.239 0.618 0.062 0.773 0.008 0.327 0.016 0.551 0.085 0.654 PROMIS 21-03-004 ColoNeg Control 0.101 0.448 0.159 0.412 0.066 0.717 0.008 0.346 0.018 0.613 0.069 0.801 PROMIS 21-03-013 ColoNeg Control 0.136 0.604 0.194 0.501 0.051 0.938 0.011 0.476 0.016 0.540 0.056 0.994 PROMIS 21-03-015 ColoNeg indicator 0.079 0.349 0.108 0.279 0.059 0.804 0.009 0.359 0.010 0.339 0.070 0.791 PROMIS 21-04-002 ColoNeg indicator 0.110 0.489 0.327 0.845 0.048 1.000 0.013 0.543 0.011 0.381 0.073 0.765 PROMIS 21-04-006 ColoNeg Indicator 0.172 0.762 0.278 0.720 0.051 0.932 0.011 0.450 0.021 0.729 0.056 0.996 PROMIS 21-04-007 ColoNeg Indicator 0.057 0.253 0.147 0.381 0.071 0.672 0.009 0.378 0.015 0.514 0.071 0.780 PROMIS 21-04-008 ColoNeg Indicator 0.223 0.988 0.211 0.545 0.073 0.641 0.011 0.471 0.015 0.511 0.078 0.709 PROMIS 21-04-009 ColoNeg Control 0.086 0.380 0.137 0.354 0.070 0.677 0.010 0.402 0.014 0.494 0.095 0.587 PROMIS 21-04-013 ColoNeg Control 0.055 0.245 0.058 0.151 0.068 0.701 0.006 0.264 0.007 0.227 0.073 0.762 PROMIS 21-07-004 ColoNeg Indicator 0.094 0.416 0.131 0.340 0.061 0.777 0.008 0.353 0.018 0.614 0.087 0.642 PROMIS 21-12-001 ColoNeg Indicator 0.199 0.881 0.242 0.626 0.056 0.845 0.007 0.292 0.016 0.553 0.073 0.757 PROMIS 21-12-007 ColoNeg Indicator 0.090 0.400 0.073 0.189 0.042 18.020 0.005 0.214 0.011 0.364 0.058 1.000 PROMIS 21-12-011 ColoNeg Control 0.097 0.430 0.141 0.365 0.067 0.713 0.008 0.328 0.014 0.492 0.077 0.718 PROMIS 21-22-001 ColoNeg Control 0.089 0, 394 0.155 0.402 0.079 0.602 0.008 0.327 0.023 0.793 0.080 0.694 PROMIS 21-22-006 ColoNeg Control 0.065 0.290 0.118 0.306 0.052 0.923 0.006 0.246 0.008 0.267 0.065 0.856 PROMIS 21-22-008 ColoNeg Control 0.113 0.500 0.226 0.584 0.065 0.737 0.010 0.409 0.017 0.579 0.083 0.674 PROMIS 37-03-004 ColoNeg Indicator 0.116 0.514 0.257 0.664 0.061 0.774 0.014 0.568 0.021 0.725 0.068 0.822 PROMIS 37-04-001 ColoNeg Indicator 0.128 0.568 0.248 0.641 0.068 0.700 0.011 0.455 0.015 0.530 0.105 0.532 PROMIS 37-04-005 ColoNeg Indicator 0.067 0.296 0.083 0.215 0.060 0.794 0.007 0.285 0.011 0.375 0.082 0.680 PROMIS 37-05-008 ColoNeg Control 0.162 0.720 0.327 0.845 0.055 0.867 0.014 0.588 0.019 0.657 0.084 0.870 104 PROMIS 37-06-001 ColoNeg Control 0.131 0.582 0.11 0.288 0.071 0.673 0.010 0.419 0.014 0.478 0.079 0.703 PROMIS 37-09-002 ColoNeg control 0.089 0.394 0.210 0.544 0.059 0.801 0.008 0.335 0.013 0.431 0.069 0.880 PROMIS 37-11-001 ColoNeg control 0.088 0.391 0.188 0.486 0.055 0.860 0.006 0.255 0.010 0.350 0.062 0.890 PROMIS 37-11-002 ColoNeg Indicator 0.081 0.361 0.256 0.661 0.053 0.898 0.009 0.375 0.009 0.317 0.060 0.932 PROMIS 37-11-011 ColoNeg Indicator 0.066 0.293 0.287 0.742 0.049 0.964 0.006 0.250 0.007 0.245 0.082 0.677 PROMIS 37-11-016 ColoNeg Indicator 0.092 0.409 0.146 0.378 0.064 0.742 0.007 0.283 0.017 0.588 0.073 0.766 PROMIS 37-12-002 ColoNeg Control 0.077 0.342 0.255 0.660 0.069 0.690 0.007 0.297 0.015 0.511 0.078 0.715 PROMIS 37-13-003 ColoNeg Control 0.070 0.311 0.135 0.348 0.062 0.769 0.008 0.318 0.009 0.317 0.077 0.719 PROMIS 37-13-006 ColoNeg Indicator 0.106 0.469 0.141 0.365 0.053 0.890 0.007 0.296 0.014 0.469 0.065 0.853 PROMIS 37-13-008 ColoNeg Indicator 0.127 0.566 0.360 0.931 0.075 0.632 0.016 0.670 0.013 0.436 0.074 0.754 PROMIS 37-14-009 ColoNeg Indicator 0.071 0.314 0.071 0.183 0.053 0.899 0.011 0.478 0.009 0.297 0.070 0.793 PROMIS 37-14-018 ColoNeg Control 0.044 0.193 0.068 0.169 0.051 0.924 0.004 0.149 0.012 0.415 0.065 0.850 PROMIS 37-14-021 ColoNeg Control 0.053 0.2 0.067 0.174 0.052 0.912 0.004 0.158 0.006 0.199 0.074 0.749 PROMIS 37-15-002 ColoNeg Control 0.746 52.949 0.137 0.356 0.072 0.658 0.008 0.319 0.011 0.368 0.084 0.661 PROMIS 37-15-006 ColoNeg Control 0.065 0.290 0.136 0.353 0.081 0.778 0.005 0.229 0.006 0.218 0.067 0.831 PROMIS 37-16-001 ColoNeg Control 0.084 0.372 0.423 17.500 0.075 0.635 0.017 0.729 0.025 0.869 0.085 0.655 PROMIS 37-17-001 ColoNeg Control 0.090 0.400 0.242 0.626 0.059 0.804 0.011 0.453 0.020 0.699 0.085 0.655 PROMIS 37-17-003 ColoNeg Indicator 0.052 0.230 0.167 0.431 0.064 0.740 0.004 0.155 0.013 0.463 0.098 0.569 PROMIS 37-17-004 ColoNeg Control 0.098 0.435 0.134 0.347 0.076 0.625 0.010 0.431 0.008 0.273 0.071 0.780 PROMIS 37-18-001 ColoNeg Control 0.076 0.335 0.197 0.511 0.056 0.857 0.011 0.462 0.026 0.899 0.069 0,801 PROMIS 37-18-003 ColoNeg control light 0.082 0.275 0.179 0.463 0.055 0.864 0.009 0.359 0.012 0.429 0.061 0.915 PROMIS 37-18-004 ColoNeg control light 0.084 0.285 0.257 0.664 0.061 0.774 0.007 0.283 0.012 0.421 0, 066 0.843 PROMIS 37-18-005 ColoNeg indicator 0.057 0.251 0.143 0.369 0.054 0.887 0.007 0.312 0.011 0.383 0.071 0.782 PROMIS 37-18-024 ColoNeg indicator 0.047 0.207 0.090 0.233 0.056 0.843 0.008 0.351 0.026 0.887 0.063 0.888 PROMIS 37-19-006 ColoNeg indicator 0.047 0.209 0.117 0.304 0.070 0.682 0.006 0.261 0.008 0.284 0.095 0.585 PROMIS 37-19-007 ColoNeg Indicator 0.071 0.313 0.117 0.302 0.062 0.772 0.006 0.237 0.016 0.547 0.064 0.870 PROMIS 37-20-004 ColoNeg Indicator 0.125 0.554 0.365 0.943 0.069 0.693 0.012 0.492 0.039 21.553 0.082 0.680 PROMIS 71-02-003 ColoNeg indicator 0.051 0.226 0.167 0.432 0.070 0.684 0.007 0.289 0.012 0.402 0.067 0.833 PROMIS 71-04-003 ColoNeg indicator 0.128 0.569 0.152 0.392 0.058 0.817 0.022 0.903 0.055 30.325 0.066 0.839 PROMIS 71-09-007 ColoNeg indicator 0.090 0.401 0.251 0.648 0.057 0.837 0.012 0.509 0.024 0.820 0.072 0.769 PROMIS 71-10-001 ColoNeg control 0.0 0.192 0.497 0.091 0.525 0.013 0.560 0.015 0.505 0.083 0.671 105 PROMIS 71-10-007 ColoNeg indicator 0225 1.000 0.4 33 17.902 0.070 0.681 0.012 0.523 0.012 0.424 0.084 0.683 PROMIS 71-11-001 ColoNeg Control 0.141 0.628 0.264 0.684 0.074 0.641 0.010 0.432 0.017 0.577 0.093 0.599 PROMIS 71-11-006 ColoNeg Control 0.109 0.485 0.179 0.462 0.080 0.592 0.008 0.339 0.011 0.361 0.075 0.742 PROMIS 71-11-008 ColoNeg Control 0.046 0.206 0.077 0.199 0.075 0.637 0.009 0.362 0.016 0.551 0.084 0.663 PROMIS 71-11-009 ColoNeg Control 0.067 0.299 0.123 0.318 0.058 0.820 0.007 0.299 0.012 0.398 0.074 0.754 Performances Alpha-Enolase Score G3PDH Score BIP Score HSP71 Score Peroxy-5 Score PDI Score Threshold 0.988 0.999 1 1 1 0.996 Sensitivity% 15.0 27.4 21.2 11.5 21.2 25.7 Specificity% 97.1 97.1 97.1 97.1 97, 1 98.6 CCR + above the threshold 17 31 24 13 24 29 ColoNeg above the threshold 2 2 2 2 2 1 .r 5 woo ii 00 o U1

Claims (27)

REVENDICATIONS1. A method of diagnosing, in vitro, colorectal cancer in a patient by determining the presence of the prolidase in a biological sample of the patient. 2. A method for early diagnosis, in vitro, of colorectal cancer in a patient, by determining the presence of the prolidase, in a biological sample of the patient. 3. A method for detecting a colorectal Tis tumor in a biological sample of a patient by determining the presence of the prolidase in a biological sample of the patient. 4. A method for detecting a colorectal adenoma in a biological sample of a patient by determining the presence of the prolidase in a biological sample of the patient. 5. A method for in vitro screening of colorectal cancer in a population by determining the presence of prolidase in a biological sample of an individual. 6. Method according to any one of claims 1 to 4, characterized in that the patient is suspected of cancer, for example colorectal cancer. 7. Process according to any one of Claims 1 to 6, characterized in that the prolidase and / or at least one tracer representative of the prolidase and selected from peptides, nucleic acids and antibodies are detected in said biological sample. 8. Process according to any one of the preceding claims, characterized in that the presence of the prolidase is determined and a variation in its expression is shown. 9. Process according to claim 8, characterized in that a decrease in the expression of the prolidase is demonstrated. 10. Process according to any one of the preceding claims, characterized in that the presence of the prolidase and that of at least one marker chosen from the following markers is determined: ACE; Aminoacylase 1; Apolipoprotein A1; Apolipoprotein A2; Beta 2 Microglobulin; CA242; CA50; CA72-2; CA19-9; Calgranulin C; calreticulin; CCSA-3 (colon cancer specific antigen); CCSA-4; CD24; CD26; Cripto-1; Cytokeratin 20; Epithelial-Cadherin; ezrin; Galectin-3; GST Pi 35 (GSTP1); HSP60; Intelectin-1; Intestinal Fatty Acid-Binding Protein; Keratin type I Cytoskeletal 18; Keratin type I Cytoskeletal 19; Keratin type II7 Cytoskeletal 8; Leukocyte Elastase Inhibitor, associated or not with Proteinase 3 (PRN3); Liver Fatty Acid-Binding Protein; L-Lactate Dehydrogenase Chain B (LDHB); maspin; MIF; MUC5AC; osteopontin; Plastin-1; 20S proteasome; (Pro) defensin-A5; (Pro) defensin-A6; Protein Disulfide Isomerase (PDI); Pyruvate kinase M2-PK; Regenerating Islet-Derived Protein 3 Alpha; S100A8; SI 00A9; testosterone; TIMP-1; Translationally-Controlled Tumor Protein; Tumor-Associated Calcium Signal Transducer 1; Tumor-Associated Calcium Signal Transducer 1; villin; vimentin; Heat Shock 71 kDa cognate (HSP71), BIP (GRP-78), Peroxiredoxin-5, Glyceraldehyde-3-phosphate dehydrogenase (G3PDH); Alpha-Enolase; Methylated DNA in the blood, preferably the methylated DNA of the AXL4 gene (Aristaless-like Homeobox-4 Gene Methylation) or the methylated DNA of the Septin-9 gene; specific alterations of stool DNA fragments, such as specific mutations in the stool DNA or specific alterations in the DNA methylation pattern in the stool; - haptoglobin in the stool; human hemoglobin in the stool. 11. Process according to claim 10, characterized in that the presence of the prolidase and that of at least one marker chosen from L-FABP, CA19-9, ACE, Beta-2 Microglobulin, Timp1, M2PK, is determined. HSP60, (Pro) defensin A6, BIP (GRP78), G3PDH, GaI3, PDI, ApoA1 and ApoA2, 12. Process according to claim 11, characterized in that the presence of the prolidase and that of at least the following associations of markers: ACE and CA19-9, LFABP and PDI, L-FABP, CA19-9 are determined. and ACE, LFABP, ACE and Timp1, LFABP, ACE and M2PK, LFABP, CA19-9, ACE and PDI LFABP, ACE, M2PK and Timp1, LFABP, ACE, CA19-9 and PDI. LFABP, ACE, M2PK, Timp1, CA19-9 and PDI. LFABP, ACE, M2PK, Timp1, CA19-9, Villine and PDI. LFABP, ACE, M2PK, Timp1, CA19-9, G3PDH and PDI.8 LFABP, ACE, M2PK, Timp1, CA19-9, Villine and G3PDH. LFABP, ACE, M2PK, Timp1, CA19-9, Villine, G3PDH and PDI. 13. Method according to any one of the preceding claims, characterized in that the biological sample of the patient is selected from blood, serum, plasma, urine, saliva, tissue and stool. 14. The method of claim 13, characterized in that the biological sample is remote from the tumor and is preferably selected from blood, serum and plasma. 15. Process according to any one of Claims 1 to 14, characterized in that the presence of the prolidase is determined by a mass spectrometry technique. 16. Process according to claim 15, characterized in that the presence of the prolidase is determined by the LC-MRM-MS technique which combines liquid chromatography and mass spectrometry in MRM (Mutiple Reaction Monitoring) mode. 17. Method according to any one of claims 1 to 14, characterized in that the presence of the prolidase is determined by an immunological technique. 18. Process according to any one of Claims 1 to 14, characterized in that the presence of the prolidase is determined by combining an immunological technique and a mass spectrometry technique. 19. Method according to any one of claims 15 to 18, characterized in that a peptide or a mixture of peptides selected from the peptides of sequence SEQ ID NO: 1 to 3 is detected. 20. Process according to claim 19, characterized in that the peptides of the prolidase are detected by following the transitions listed in Table 1. 21. The method of claim 20, wherein the 400.2 / 686.3 transition of the peptide of SEQ ID NO: 1, and the corresponding heavy peptide transition is followed to determine the presence of the prolidase. 22. Method according to any one of claims 10 to 21, characterized in that one follows the transition 452.7 / 662.2 of the peptide of SEQ ID NO: 5, and the transition of the corresponding heavy peptide, to determine the presence of the Alpha-enolase. 35 23. Method according to any one of claims 10 to 22, characterized in that one follows the transitions 699.4 / 557.4; 699.4 / 685.4; 9,699.4 / 784.5 of the peptide of SEQ ID NO: 6, and the corresponding heavy peptide transition, to determine the presence of the BIP protein (GRP78). The method according to any one of claims 10 to 23, wherein the transitions 403.2 / 550.3 and 403.2 / 706.3 of the peptide of SEQ ID NO: 12 or the transition 435.3 / are followed. 657.2 of the peptide of SEQ ID NO: 10, and the corresponding heavy peptide transitions, to determine the presence of G3PDH. 25. A method according to any one of claims 10 to 24, wherein the 494,6 / 793,4 transition of the peptide of SEQ ID NO: 15 is followed, and the corresponding heavy peptide transition, to determine the presence of HSP71 (HSC70). 26. The method of any one of claims 10 to 25 wherein the 496.3 / 407.3 transitions are followed; 496.3 / 522.3; 496.3 / 635.4 peptide peptide of SEQ ID NO: 17, and corresponding heavy peptide transitions, to determine the presence of PDI. 27. Use of a method according to any one of claims 1 to 26, in screening, early diagnosis, therapeutic monitoring, prognosis and diagnosis of relapses in the context of colorectal cancer.