WO2014183777A1 - Procédés de détection de polypes ou de carcinomes colorectaux et procédés de traitement des polypes ou des carcinomes colorectaux - Google Patents

Procédés de détection de polypes ou de carcinomes colorectaux et procédés de traitement des polypes ou des carcinomes colorectaux Download PDF

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WO2014183777A1
WO2014183777A1 PCT/EP2013/059845 EP2013059845W WO2014183777A1 WO 2014183777 A1 WO2014183777 A1 WO 2014183777A1 EP 2013059845 W EP2013059845 W EP 2013059845W WO 2014183777 A1 WO2014183777 A1 WO 2014183777A1
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colorectal
level
patient
sample
polyps
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PCT/EP2013/059845
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English (en)
Inventor
Markus Neurath
Benno WEIGMANN
Hansjörg Schild
Alexandra WLODARSKI
Stefan Tenzer
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Biontech Ag
Universitaetsklinikum Erlangen
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Priority to PCT/EP2013/059845 priority Critical patent/WO2014183777A1/fr
Publication of WO2014183777A1 publication Critical patent/WO2014183777A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine

Definitions

  • the present invention relates to a method of detecting colorectal polyps or colorectal carcinoma, a method of differentiating between non-cancerous or pre-cancerous colorectal polyps and cancerous colorectal polyps, a method of monitoring the course of a colorectal cell proliferative disorder, such as colorectal polyps or colorectal carcinoma, a method of treating colorectal polyps or colorectal carcinoma, and a method of identifying a compound suitable for the treatment of colorectal polyps or colorectal carcinoma.
  • the present invention further relates to a kit useful for conducting the above methods.
  • Colorectal cancer is a leading cause of cancer death in westernized societies, and for a long time screening programs have been shown to reduce mortality from this disease.
  • Strategies for reducing the toll from colorectal cancer focus on early detection and removal of potential precancerous lesions. Therefore, colonoscopy is currently regarded as the gold standard for the detection of polyps and cancers in the colon as well as in the rectum and is the preferred method of screening for colorectal cancer in Europe and the USA.
  • non-invasive stool tests for colorectal cancer (CRC) screening exist: Fecal occult blood testing (FOBT), such as guaiac-based FOBT (gFOBT) or immunochemical FOBT, genetic stool tests, and M2-PK.
  • FOBT Fecal occult blood testing
  • gFOBT guaiac-based FOBT
  • immunochemical FOBT genetic stool tests
  • M2-PK M2-PK
  • Filtertests which detect blood in the stool rely on the fact that colorectal neoplasms tend to bleed more often than normal mucosa.
  • FOBT blood in the stool
  • the most widely used tests are guaiac-based FOBT's. If blood is present in the stool, the hemoglobin with its pseudoperoxidase activity will result in a color change of the test field in the presence of hydrogen peroxide. Because colorectal neoplasms tend to bleed intermittingly, testing several stool samples increases the yield. It has become standard to test three consecutive stools, i.e. to use three test slides for screening purposes. A test is positive, if one or more of the six test fields turn the color. A positive test has to be followed up by complete colonoscopy.
  • Stool DNA testing was added to the colorectal cancer screening recommendations in 2008. However, the test is recommended primarily for the detection of cancer, not for polyps or precancerous lesions. Cells that line the lower intestinal tract are constantly being shed into the colon, picked up by feces, and excreted in bowel movements. When normal cells change into precancerous or cancerous ones, their DNA changes or mutates. By looking at markers for genes that are commonly changed in cancers and precancerous polyps, DNA stool tests can identify patients who need further colonoscopy testing.
  • the M2-PK Stool Test based on the ELISA method, established in 2010, is a more specific test, which is much more sensitive than previous laboratory bowel cancer screening tests.
  • Tumor M2-PK pyruvate kinase isoenzyme type M2
  • the present inventors were successful in developing such a method. They found that the determination of the level of Alpha-1-Microglobulin/Bikunin Precursor (AMBP) and/or C- Reactive Protein (CRP) in a sample isolated from a patient allows for the detection of colorectal polyps or carcinoma. In particular, they figured out that the determination of the level of AMBP or CRP in a sample isolated from a patient allows for the detection of colonic polyps or carcinoma with high sensitivity and specificity. In addition, they found that with the determination of the level of both AMBP and CRP, the sensitivity of the method can actually be further increased.
  • AMBP Alpha-1-Microglobulin/Bikunin Precursor
  • CRP C- Reactive Protein
  • AMBP as well as CRP are highly expressed in colorectal polyps and even more highly expressed in colorectal carcinoma. In the absence of colorectal polyps or carcinoma, AMBP as well as CRP are only weakly expressed. Thus, the level of AMBP and/or CRP in a sample isolated from a patient functions as a precise and specific indicator for the presence of colorectal polyps or carcinoma.
  • patients with a high risk for colorectal polyps or carcinoma, or having colorectal polyps or carcinoma can be identified with a fast, cost-efficient, and non-invasive screening method which is based on the determination of the level of AMBP and/or CRP in a sample isolated from said patients. Said method further reduces unnecessary colonoscopic examinations leading to cost savings and improved safety.
  • the specificity and sensitivity of the AMBP and/or CRP-based assay system permits the straight forward use for a preventive screening ahead of endoscopy as well as in the post-operative relapse phase.
  • the AMBP- and/or CRP-based test will serve as a marker for postoperative recurrence or tumor persistence.
  • These findings may be used for the indication of endoscopic surveillance colonoscopy. Alternatively, these findings may be used to decide whether adjuvant chemotherapy in colorectal cancer patients is necessary.
  • the present invention relates to a method of detecting colorectal polyps comprising the step of determining the level of:
  • CRP C-Reactive Protein
  • a level of AMBP and/or CRP which is above a threshold value indicative for healthiness indicates that the patient is at risk of developing or has colorectal polyps.
  • a level of AMBP and/or CRP which is above a threshold value indicative for healthiness and below a threshold value indicative for colorectal carcinoma indicates that the patient is at risk of developing or has colorectal polyps.
  • the level of AMBP and/or CRP is compared to a reference level of AMBP and/or CRP.
  • the reference level is the level determined by measuring one or more reference samples from one or more subjects. Said subjects may be
  • a level which is higher compared to the reference level in healthy subjects indicates that the patient is at risk of developing or has colorectal polyps.
  • a level which is higher compared to the reference level in healthy subjects and lower compared to the reference level in subjects having colorectal carcinoma indicates that the patient is at risk of developing or has colorectal polyps.
  • the colorectal polyps are adenomas or adenomatous polyps.
  • the present invention relates to a method of detecting colorectal carcinoma comprising the step of determining the level of Alpha-1 -Microglobulin/Bikunin Precursor (AMBP) and C-Reactive Protein (CRP) in a sample isolated from a patient.
  • AMBP Alpha-1 -Microglobulin/Bikunin Precursor
  • CRP C-Reactive Protein
  • a level of AMBP and CRP which is above a threshold value indicative for healthiness and/or above a threshold value indicative for colorectal polyps indicates that the patient is at risk of developing or has colorectal carcinoma.
  • the level of AMBP and CRP is compared to a reference level of AMBP and CRP.
  • the reference level is the level determined by measuring one or more reference samples from one or more subjects. The subjects may be
  • the present invention relates to a method of differentiating between noncancerous or pre-cancerous colorectal polyps and cancerous colorectal polyps comprising the step of determining the level of:
  • AMBP Alpha-1-Microglobulin/Bikunin Precursor
  • CRP C-Reactive Protein
  • a level of AMBP and/or CRP which is
  • the level of AMBP and/or CRP is compared to a reference level of AMBP and/or CRP.
  • the reference level is the level determined by measuring one or more reference samples from one or more subjects. The subjects may be
  • the present invention relates to a method of monitoring the course of a colorectal cell proliferative disorder in a patient comprising the step of determining the level of:
  • CRP C-Reactive Protein
  • the colorectal cell proliferative disorder is selected from the group consisting of colorectal polyps and colorectal carcinoma.
  • said monitoring comprises determining the level of AMBP and/or CRP in a sample at a first point in time and in at least one further sample at a later point in time and comparing said levels determined at the different time points.
  • a level which increases over time indicates that
  • the patient was healthy at the first point in time and a level which increases over time indicates that the patient has acquired a risk of developing colorectal polyps or carcinoma or that the patient has developed colorectal polyps or carcinoma,
  • the patient has received a therapeutic treatment for colorectal polyps or carcinoma and a level which does not change or increases over time indicates that the patient does not respond to said treatment.
  • the therapeutic treatment for colorectal polyps or carcinoma is selected from the group consisting of surgery, radiofrequency ablation, cryotherapy, chemotherapy, radiation therapy, and targeted therapy.
  • the colorectal polyps are adenomas or adenomatous polyps.
  • the present invention relates to a method of identifying a compound suitable for the treatment of colorectal polyps or colorectal carcinoma in a patient comprising the steps of:
  • test system comprising a level of Alpha-1 -Microglobulin/Bikunin Precursor (AMBP), C-Reactive Protein (CRP) or a combination thereof,
  • AMBP Alpha-1 -Microglobulin/Bikunin Precursor
  • CRP C-Reactive Protein
  • test compound is identified as a compound suitable for the treatment of colorectal polyps or colorectal carcinoma in the patient, when the effect of the test compound on the test system comprises a decrease in the level of AMBP and/or CRP compared to
  • test or control system comprises or consists of
  • the colorectal polyps are adenomas or adenomatous polyps.
  • the present invention relates to a method of treating colorectal polyps in a patient comprising the steps of:
  • the treatment of colorectal polyps is selected from the group consisting of surgery, cryotherapy, and targeted therapy.
  • the colorectal polyps are adenomas or adenomatous polyps.
  • the present invention relates to a method of treating colorectal carcinoma in a patient comprising the steps of:
  • the treatment of colorectal carcinoma is selected from the group consisting of surgery, radiofrequency ablation, cryotherapy, chemotherapy, radiation therapy, and targeted therapy.
  • the patient is a mammal, preferably a human or a rodent.
  • the sample is a body fluid sample or a tissue sample. In one preferred embodiment,
  • the body fluid sample is selected from the group consisting of a blood sample, cerebrospinal fluid (CSF) sample, urine sample, sputum sample, breast milk sample, cerumen (earwax) sample, endolymph sample, perilymph sample, gastric juice sample, mucus sample, peritoneal fluid sample, pleural fluid sample, saliva sample, semen sample, sweat sample, and tears sample, or
  • the tissue sample is selected from the group consisting of a tissue section sample and tissue explant sample.
  • the blood sample is a whole blood sample or a blood fraction sample.
  • the blood fraction sample is a blood cell fraction, blood serum, or blood plasma sample.
  • the level of AMBP and/or CRP is the expression level.
  • the expression level is determined with an immunoassay, gel electrophoresis, spectrometry, chromatography, in situ hybridization, or a combination thereof. In one more preferred embodiment,
  • the immunoassay is an enzyme immunoassay, preferably an enzyme-linked immunosorbent assay (ELISA), a Western Blot (immunoblot), a radio immunoassay (RIA), or a luminescence immunoassay (LIA),
  • ELISA enzyme-linked immunosorbent assay
  • Western Blot immunoblot
  • RIA radio immunoassay
  • LIA luminescence immunoassay
  • the gel electrophoresis is 1 D or 2D gel electrophoresis
  • the spectrometry is mass spectrometry (MS), preferably tandem mass spectrometry (MS/MS),
  • the chromatography is liquid chromatography (LC) or affinity chromatography,
  • the in situ hybridization is a silver in situ hybridization (SISH), chromogenic in situ hybridization (CISH), or fluorescence in situ hybridization (FISH),
  • the chromatography is combined with spectrometry, preferably mass spectrometry (MS), and is more preferably liquid chromatography-mass spectrometry (LC-MS) and most preferably liquid chromatography-tandem mass spectrometry (LC-MS/MS), or
  • the gel electrophoresis is combined with an immunoassay and is more preferably a 2D immunoblot.
  • the mass spectrometry is an electrospray ionization mass spectrometry (ESI-MS), a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), or an electron capture dissociation mass spectrometry (ECD- MS).
  • ESI-MS electrospray ionization mass spectrometry
  • MALDI-MS matrix-assisted laser desorption/ionization mass spectrometry
  • ECD- MS electron capture dissociation mass spectrometry
  • the present invention relates to a kit comprising means for determining the level of
  • CRP C-Reactive Protein
  • said kit is useful for conducting the methods of all aspects of the present invention.
  • said kit further comprises
  • said data carrier is an electronical or a non-electronical data carrier.
  • said data carrier e.g. electronical or non- electronical data carrier, comprises instructions on how to carry out the methods of all aspects of the present invention.
  • the patient is a mammal, preferably a human or a rodent and/or the subject is a mammal, preferably a human or a rodent.
  • Said subject may be a subject having colorectal polyps or colorectal carcinoma.
  • the sample is a body fluid sample or a tissue sample. In one preferred embodiment,
  • the body fluid sample is selected from the group consisting of a blood sample, cerebrospinal fluid (CSF) sample, urine sample, sputum sample, breast milk sample, cerumen (earwax) sample, endolymph sample, perilymph sample, gastric juice sample, mucus sample, peritoneal fluid sample, pleural fluid sample, saliva sample, semen sample, sweat sample, and tears sample, or
  • the tissue sample is selected from the group consisting of a tissue section sample and tissue explant sample.
  • the blood sample is a whole blood sample or a blood fraction sample.
  • the blood fraction sample is a blood serum or blood plasma sample.
  • the level of AMBP and/or CRP is the expression level.
  • Figure 1 Shows the patients characteristics. A total of 30 patients were used for the proteome study (discovery set), 75 for the ELISA experiments (validation set).
  • FIG. 2 Shows fully automated HPLC based depletion of high and moderate abundant proteins (HAP and MAP).
  • HAP high and moderate abundant proteins
  • CRC colorectal adenoma and 10 patients with colorectal carcinoma
  • MAP moderate abundant proteins
  • the collected flow-through is highly enriched with low abundant proteins, which represents the majority of biomarkers.
  • the low abundant protein fraction (LAP) is then digested with trypsin before labelfree identification and quantification by LC-MS/MS. Potential biomarker candidates were further confirmed by Western blot and ELISA and statistical analysis to check their clinical potential.
  • B+C shows the names of the six strongest up-regulated proteins (C-reactive protein (CRP), Complement component 9 (C9), Alpha-1-Microglobulin/Bikunin Precursor (AMBP), Leucine rich alpha 2 glycoprotein (A2GL), Plasma protease C1 inhibitor (IC1 ), Haptoglobin (HPT), Alpha-1-antichymotrypsin (AACT), Monocyte differentiation antigen CD14 (CD14)) and four strongest down-regulated proteins (Tenscin X (TENXA), Pregnancy zone protein (PZP), Transthyretin (TTHY), Profilin 1 (PROF1 ), Kallistatin (KAIN), Corticosteroid binding globulin (CBG), Insulin like growth factor binding protein complex acid labile subunit (ALS)).
  • CRP C-reactive protein
  • ABP Alpha-1-Microglobulin/Bikunin Precursor
  • A2GL Plasma protease C1 inhibitor
  • HPT Hap
  • FIG. 4 Shows the differentially detected proteins Alpha-1-Microglobulin/Bikunin Precursor (AMBP) and CRP (C-reactive protein) with their average log2 ratios (adenoma vs. controls, carcinoma vs. controls, carcinoma vs. adenoma). Both proteins belong to the strongest up- regulated proteins detected by mass spectrometry (MS) analysis as shown in Figure 3.
  • AMBP Alpha-1-Microglobulin/Bikunin Precursor
  • CRP C-reactive protein
  • Figure 7 Shows the correlation of CRP and AMBP between carcinoma & adenomas and control group in ROC-curves (AMBP: gray ROC-curve; CRP: black ROC-curve).
  • CRP and AMBP were analyzed by ELISA as shown in Figure 6.
  • Data-points are mean ⁇ standard deviation.
  • FIG. 8 Shows the correlation of AMBP and CRP in combination between carcinoma & adenoma and control group in ROC-curves (B,C,D).
  • AMBP and CRP were each analyzed by ELISA and the combined values were calculated in arbitrary units (A).
  • Data-points are mean ⁇ standard deviation.
  • Figure 9 Shows an overview of the calculated AUC values from ROC analysis (as shown in Figure 7+8).
  • Figure 10 Shows sensitivity and specificity pairs for AMBP, CRP, and both AMBP and CRP.
  • FIG. 11 Shows Immunofluorescence staining of AMBP in human tumor/normal colon tissue. Representative Immunohistochemistry stainings for AMBP are shown. Paraffinsections of the colonic specimens were incubated with a specific AMBP antibody and a secondary biotinylated antibody followed by incubation with Streptavidin-Dylight 648. Cells were counterstained with Hoechst dye upon analysis by fluorescence microscopy. A higher number of positive cells can be observed in colon carcinoma tissue. Detailed description of the invention
  • the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (lUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.
  • the present inventors were successful in developing a non-invasive and laboratory-based method for the detection of colorectal polyps or carcinoma. They found that the determination of the level of Alpha-1 -Microglobulin/Bikunin Precursor (AMBP) and/or C-Reactive Protein (CRP) in a sample isolated from a patient allows for the detection of colorectal polyps or carcinoma. In particular, they figured out that the determination of the level of AMBP or CRP in a sample isolated from a patient allows for the detection of colonic polyps or carcinoma with high sensitivity and specificity. In addition, they found that with the determination of the level of both AMBP and CRP, the sensitivity of the method can actually be further increased.
  • AMBP Alpha-1 -Microglobulin/Bikunin Precursor
  • CRP C-Reactive Protein
  • AMBP as well as CRP are highly expressed in colorectal polyps and even more highly expressed in colorectal carcinoma. In the absence of colorectal polyps or carcinoma, AMBP as well as CRP are only weakly expressed. Thus, the level of AMBP and/or CRP in a sample isolated from a patient functions as a precise and specific indicator for the presence of colorectal polyps or carcinoma.
  • the present inventors provide with AMBP and/or CRP early stage biomarkers which allow the detection of colorectal polyps or carcinoma at an early stage.
  • the early detection enables the physician to further analyze the patient by colonoscopy and/or to remove the colorectal polyps or carcinoma and, thus, to dramatically increase the chance of the patient to survive.
  • colonal cell proliferative disorder refers to a condition in which the unregulated and/or abnormal growth of colorectal cells can lead to the development of an unwanted colorectal condition or disease, which can be cancerous, e.g. colorectal carcinoma, or non-cancerous, e.g. benign colorectal polyps.
  • the colorectal cell proliferative disorder may be selected from the group consisting of "colorectal polyps" or "colorectal carcinoma”.
  • colon encompasses the colon, rectum (parts of the large intestine) as well as the appendix.
  • colonal polyps refers to polyps (fleshy growths) occurring on the lining of the colon or rectum (parts of the large intestine). Colorectal polyps can be classified in hyperplastic polyps, neoplastic polyps such as adenomas, hamartomatous polyps and inflammatory polyps. Said term, as used herein, encompasses benign/non- malignant polyps (also designated as non-cancerous polyps), e.g. hyperplastic polyps, hamartomatous polyps or inflammatory polyps, pre-malignant polyps (also designated as pre-cancerous polyps), e.g.
  • neoplastic polyps such as adenomas like tubular adenomas, and/or malignant polyps (also designated as cancerous polyps), e.g. colorectal carcinoma.
  • adenomas like tubular adenomas
  • malignant polyps also designated as cancerous polyps
  • colorectal carcinoma e.g. colorectal carcinoma
  • the colorectal polyps are colorectal adenomas.
  • Colorectal adenomas may also be designated as adenomatous polyps herein.
  • Neoplastic polyps may be found in the distal colon and rectum. They have no malignant potential, which means that they are no more likely than normal tissue to eventually become cancer.
  • Neoplastic polyps consist of a neoplasm tissue.
  • a neoplasm is a tissue whose cells have lost its normal differentiation. They can be either benign growths or malignant growths. The malignant growths can either have primary or secondary causes.
  • Neoplastic polyps include adenomas.
  • An adenoma is tumor glandular tissue that has not (yet) gained the properties of cancer. An adenoma has, however, the potential to become cancer.
  • the adenoma may be a tubular, tubulovillous, villous and sessile serrated (SSA) adenoma.
  • the villous adenoma is associated with the highest malignant potential because it generally has the largest surface area. This is because the "villi" are projections into the lumen and hence have a bigger surface area. However, a villous adenoma is no more likely than a tubular or tubulovillous adenoma to become cancerous if the sizes are all the same.
  • Hamartomatous polyps as well as inflammatory polyps have also no malignant potential.
  • Hamartomatous polyps are growths like tumors found in organs as a result of faulty development. They are normally made up of a mixture of tissues. They grow at the normal rate of the host tissue and rarely cause problems such as compression.
  • a common example of a hamartomatous lesion is a strawberry naevus.
  • Hamartomatous polyps are often found by chance; occurring in syndromes such as Koz-Jegher Syndrome or Juvenile Polyposis Syndrome.
  • inflammatory polyps are polyps which are associated with inflammatory conditions such as Ulcerative Colitis and Crohns disease.
  • colonal polyps also refers to polyps which may be classified according to the following levels:
  • Level 1 high degree of dysplasia and penetration of malignant glands through the muscularis mucosa into the submucosa, within the polyp head,
  • Level 2 high degree of dysplasia and the same submucosal invasion, but present at the junction of the head to the stalk, or
  • Level 3 high degree of dysplasia and invasion of the stalk.
  • Colorectal polyps of level 4 are characterized by a high degree of dysplasia and an invasion of the stalk's base at the connection to the colonic wall. This level corresponds to Dukes stage "A" of the classification system for colorectal cancer of the British pathologist Cuthbert Dukes. Dukes stage “A” means that the cancer is only in the innermost lining of the colon or rectum, or slightly growing into the muscle layer. Colorectal polyps of level 4 are preferably not encompassed by the term “colorectal polyps”, as used herein.
  • the term “hyperplasia”, as used herein, means increase in number of cells/proliferation of cells. It may result in the gross enlargement of an organ.
  • displasia means in pathology an abnormality of development. It involves the change of cell phenotype (size, shape, and organization of tissue).
  • colon/bowel carcinoma also known as colon/bowel carcinoma or colon/bowel cancer
  • colon/bowel carcinoma relates to carcinoma in the colon, rectum (parts of the large intestine), or appendix.
  • colonal carcinoma also relates to a disease in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). These three malignant properties differentiate the colorectal carcinoma from a benign/non-malignant tumor, which is self- limited and does not invade and/or metastasize.
  • colonal carcinoma covers malignant polyps (also designated as cancerous polyps) or malignant polyp accumulations. It also covers colorectal polyps of level 4 which are characterized by a high degree of dysplasia and an invasion of the stalk's base at the connection to the colonic wall. This level corresponds to Dukes stage "A” (see above).
  • colonal carcinoma as used herein, preferably does not comprise cancer metastasis originating therefrom.
  • the colorectal carcinoma may be classified according to the above mentioned Dukes classification system which classifies the cancer into Dukes stages "A”, “B”, “C”, and “D” (see Dukes CE, "The classification of cancer of the rectum”, Journal of Pathological Bacteriology, 1932, 35:323).
  • the colorectal carcinoma may also be staged according to the more detailed and most common Tumor, Node, and Metastasis (TNM) staging system from the American Joint Committee on Cancer (AJCC) (see AJCC Cancer Staging Manual, sixth edition, Springer-Verlag New York, Inc. 2002).
  • This staging system describes the size of a primary tumor (T), whether any lymph nodes contain cancer cells (N), and whether the cancer has spread to another part of the body (M).
  • stage 0 relates to carcinoma in situ (e.g. intramucosal carcinoma, wherein malignant cells invade the mucosal lamina basement).
  • carcinoma in situ e.g. intramucosal carcinoma, wherein malignant cells invade the mucosal lamina basement.
  • the criteria for stages I, II, and III are larger tumor size and/or spread of the cancer beyond the organ in which it first developed to nearby lymph nodes and/or organs adjacent to the location of the primary tumor.
  • the criteria for stage IV are that the cancer has spread to another organ(s).
  • the colorectal carcinoma is a carcinoma of at least stage 0, at least stage I, at least stage II, or at least stage III. Accordingly, the colorectal carcinoma may be a colorectal carcinoma of stage I, II, III, or IV.
  • non-cancerous colorectal polyps refers to benign/non-malignant polyps occurring on the lining of the colon or rectum. Said polyps show a high degree of hyperplasia. They have no malignant potential, e.g. hyperplastic polyps.
  • pre-cancerous colorectal polyps refers to pre-malignant polyps occurring on the lining of the colon or rectum. Said polyps have not (yet) gained the properties of cancer, e.g. neoplastic polyps like tubular adenoma. Said polyps have, however, the potential to become cancer.
  • pre-cancerous colorectal polyps also refers to polyps which can be classified according to the following levels:
  • Level 1 high degree of dysplasia and penetration of malignant glands through the muscularis mucosa into the submucosa, within the polyp head,
  • Level 2 high degree of dysplasia and the same submucosal invasion, but present at the junction of the head to the stalk, or
  • Level 3 high degree of dysplasia and invasion of the stalk.
  • cancer refers to malignant polyps occurring on the lining of the colon or rectum. Said polyps have gained the properties of cancer.
  • Dukes stage “A” means that the cancer is only in the innermost lining of the colon or rectum, or slightly growing into the muscle layer (see also above).
  • the Duke Dukes classification system (see Dukes CE, "The classification of cancer of the rectum", Journal of Pathological Bacteriology, 1932, 35:323) or the Tumor, Node, and Metastasis (TNM) staging system from the American Joint Committee on Cancer (AJCC) (see AJCC Cancer Staging Manual, sixth edition, Springer-Verlag New York, Inc. 2002) may be used (see also above).
  • AJCC American Joint Committee on Cancer
  • the kit of the present invention comprises means for determining the level of AMBP and/or CRP.
  • the AMBP gene encodes a complex glycoprotein secreted in plasma.
  • AMBP is a precursor which is proteolytically processed into distinct functioning proteins: (A1 M) Alpha-1 - Microglobulin, which belongs to the superfamily of lipocalin transport proteins and may play a role in the regulation of inflammatory processes, and Bikunin, which is a urinary trypsin inhibitor belonging to the superfamily of Kunitz-type protease inhibitors.
  • A1 M is thought to play a role as a carrier for small hydrophobic molecules and may have a central role in the network of immune regulation.
  • Bikunin is a serine protease inhibitor of the Kunitz superfamily and is thought to participate in the control of such events as endothelial cell growth or extracellular matrix stabilization.
  • both A1 M and Bikunin originate from a shared precursor polypeptide named the Alpha-1- Microglobulin/Bikunin Precursor (AMBP) which is cleaved by a furin-like protease to release the two mature molecules. After AMBP cleavage late in the trans-Golgi network, A1 M and Bikunin are secreted into the blood as separate proteins.
  • the AMBP polypeptide is encoded by a single copy gene, which can be expressed in the liver.
  • the human AMBP gene is located on chromosome 9 in a cluster of lipocalin genes and consists of 10 exons which span 1 .3 kb and nine introns with an aggregate length of about 16.5 kb.
  • the AMBP gene is under the major control of a potent and liver specific enhancer made of nine clustered boxes that are proven targets for several hepatocyte-enriched nuclear factors (HNF) designated HNF-1 , -3, and -4. This enhancer is in a remote location, 2.7 kb away from the gene promoter which displays a weak and ubiquitous activity.
  • HNF hepatocyte-enriched nuclear factors
  • the determination of the level of AMBP encompasses the determination of the level of the Alpha-1 -Microglobulin/Bikunin precursor protein, the level of Alpha-1-Microglobulin and/or the level of Bikunin.
  • the Alpha-1 - Microglobulin/Bikunin precursor protein is proteolytically processed into Alpha-1- Microglobulin as well as Bikunin in the trans-Golgi network.
  • AMBP is human AMBP. It preferably has an amino acid sequence according to SEQ ID NO: 1 or is a variant thereof. Said variant may has an amino acid sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence according to SEQ ID NO: 1 (human AMBP, accession number P02760, version P02760.1 and Gl:122801 ).
  • the variant may have an amino acid sequence which is at least 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the amino acid sequence according to SEQ ID NO: 1 .
  • sequence identity is over a continuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 220, 250, 280, 300, 320, 350, or more amino acids, preferably over the whole length of the amino acid sequence according to SEQ ID NO: 1.
  • Alignment tools are well known to the person skilled in the art and can be, for example, obtained on the World Wide Web, e.g., ClustalW (www.ebi.ac.uk/clustalw) or Align (http://www.ebi.ac.uk/emboss/align/index.html) using standard settings, preferably for Align EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • CRP is an acute-phase protein and a marker for inflammation.
  • the synthesis of CRP in the cells may be regulated by pro-inflammatory cytokines like interleukin-1 , interleukin-6, and tumor-necrosis factor, which are also reported for different malignancies. Therefore, these pro-inflammatory cytokines are currently the subject of intense studies as influencing factors in various types of tumors. It is increasingly recognized that in addition to tumor stage, the disease progression depends on a complex interaction between the tumor and the host's inflammatory response.
  • CRP is human CRP. It preferably has an amino acid sequence according to SEQ ID NO: 2 or is a variant thereof. Said variant may has an amino acid sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence according to SEQ ID NO: 2 (human CPR, accession number P02741 , version: P02741.1 and Gl:1 17486).
  • the variant may have an amino acid sequence which is at least 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the amino acid sequence according to SEQ ID NO: 2.
  • sequence identity is over a continuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 220, or more amino acids, preferably over the whole length of the amino acid sequence according to SEQ ID NO: 2.
  • sample material such as blood plasma or serum.
  • Said sample material may be isolated from a patient, e.g. from the human body. Subsequently, the sample material can be fractionated and/or purified. It is, for example, possible to store the sample material to be tested in a freezer and to carry out the methods of the present invention at an appropriate point in time after thawing the respective sample material. It has been surprisingly found by the present inventors that AMBP and/or CRP can be used as a biomarker for the detection of colorectal polyps and/or colorectal carcinoma.
  • the level of AMBP and/or CRP in a sample e.g. blood plasma or serum
  • a sample e.g. blood plasma or serum
  • the determination of the level of AMBP and/or CRP in a sample is preferably carried out in vitro by a technician in a laboratory. Blood serum can be easily obtained by taking blood from a patient to be medically examined and separating the supernatant from the clotted blood.
  • the level of AMBP and/or CRP in a sample e.g.
  • blood plasma or serum can be assayed with commercial available AMBP and/or CRP ELISA Kits according to the protocol provided by the manufacturer.
  • the level of AMBP and/or CRP in a sample e.g. blood plasma or serum, is higher with polyps and carcinoma.
  • sample refers to any biological sample which may be isolated from a patient and used for analysis purposes, in particular for the determination of the level of AMBP and/or CRP.
  • Said sample may be a body fluid sample, a tissue sample, or a cell sample.
  • samples encompassed by the present invention are tissue (e.g. section or explant) samples, single cell samples, cell colony samples, cell culture samples, blood (e.g. whole blood or blood fraction such as blood cell fraction, serum or plasma) samples, urine samples, or samples from other peripheral sources.
  • tissue e.g. section or explant
  • single cell samples single cell samples
  • cell colony samples cell culture samples
  • blood e.g. whole blood or blood fraction such as blood cell fraction, serum or plasma
  • urine samples or samples from other peripheral sources.
  • Said samples may be mixed or pooled, e.g. a sample may be a mixture of a blood sample and an urine sample.
  • Said samples may be provided by removing a body fluid, cell(s), cell colonies, an explant, or a section from a patient, but may also be provided by using a previously isolated sample.
  • a tissue sample may be removed from a patient by conventional biopsy techniques or a blood sample may be taken from a patient by conventional blood collection techniques.
  • the sample e.g. tissue sample or blood sample, may be obtained from a patient prior to initiation of the therapeutic treatment, during the therapeutic treatment, and/or after the therapeutic treatment.
  • the sample is a tissue sample, preferably a tissue section sample or a tissue explant sample. It is preferred that the tissue sample has a weight of between 0.1 and 500 mg, more preferably of between 0.5 and 250 mg, and most preferably of between 1 and 50 mg.
  • the sample is a body fluid sample.
  • body fluid sample refers to any liquid sample derived from the body of a patient.
  • Said body fluid sample may be a blood sample, urine sample, sputum sample, breast milk sample, cerebrospinal fluid (CSF) sample, cerumen (earwax) sample, endolymph sample, perilymph sample, gastric juice sample, mucus sample, peritoneal fluid sample, pleural fluid sample, saliva sample, sebum (skin oil) sample, semen sample, sweat sample, tears sample, vaginal secretion sample, or vomit sample including components or fractions thereof.
  • CSF cerebrospinal fluid
  • cerumen earwax
  • endolymph sample perilymph sample
  • gastric juice sample mucus sample
  • mucus sample mucus sample
  • peritoneal fluid sample pleural fluid sample
  • saliva sample sebum (skin oil) sample
  • semen sample semen sample
  • sweat sample tears sample
  • a body fluid sample may be a mixture of a blood and an urine sample or a mixture of a blood and cerebrospinal fluid sample.
  • Said body fluid sample may be provided by removing a body liquid from a patient, but may also be provided by using previously isolated body fluid sample material.
  • Said "body fluid sample” allows for a noninvasive analysis of a patient. It is further preferred that the body fluid sample has a volume of between 0.01 and 20 ml, more preferably of between 0.1 and 10 ml, even more preferably of between 0.5 and 8 ml, and most preferably of between 1 and 5 ml.
  • the blood sample is a whole blood sample or a blood fraction sample such as a blood cell fraction, blood serum, or blood plasma sample.
  • Blood cells also known as hemopoietic cells may be used. Hemopoietic cells are mature cell types and their immature precursors are identifiable either by morphology or, mostly, by a distinct pattern of cell surface markers. Said term is used to distinguish these cells from other cell types found in the body and also includes T-cells and distinctive subsets, which are the only hematopoietic cells that are not generated in the bone marrow. Said blood cells may be erythrocytes, leukocytes, and/or thrombocytes, e.g.
  • PBMCs Peripheral blood mononuclear cells
  • the blood serum or plasma sample has a volume of between 0.01 and 20 ml, more preferably of between 0.1 and 10 ml, even more preferably of between 0.5 and 8 ml and most preferably of between 1 and 5 ml.
  • patient refers to any organism such as vertebrate, particularly any mammal, including both a human and another mammal, e.g. an animal such as a rodent, a rabbit, or a monkey.
  • the rodent may be a mouse, rat, hamster, guinea pig, or chinchilla.
  • the patient is a human or a rodent.
  • the present inventors figured out that the determination of the AMBP level allows for the detection of colonic polyps and carcinoma with a sensitivity of 85.7 % and a specificity of 84% and that the determination of the CRP level allows for the detection of colonic polyps and carcinoma with a sensitivity of 78% and a specificity of 68%.
  • the sensitivity of the method can actually be increased to 91 .8%.
  • sensitivity refers to the number of true positive patients (%) with regard to the number of all patients (100%).
  • the patients may be individuals having polyps, or carcinoma. More specifically, the patients may be individuals having non-cancerous or pre-cancerous polyps, or the patients may be individuals having cancerous polyps.
  • the sensitivity is calculated by the following formula:
  • the term "specificity”, as used herein, relates to the number of true negative individuals (%) with regard to the number of all healthy individuals (100%).
  • the result of each analysis group is calculated from a plurality of isolated samples, i.e. from at least 2 isolated samples, preferably from between 2 and 20, more preferably from between 10 and 60, and even more preferably from between 50 and 100 isolated samples, selected from the group consisting of healthy individuals (patients), patients having colorectal polyps and patients having colorectal carcinoma.
  • the methods of the present invention can be carried out in combination with other diagnostic methods for the detection of colorectal polyps or carcinoma to increase the overall sensitivity and/or specificity.
  • the detection of AMBP and/or CRP allows a very early detection of colorectal polyps or carcinoma and can, therefore, be used as a very early marker.
  • AUC relates to an abbreviation for the area under a curve. In particular, it refers to the area under a Receiver Operating Characteristic (ROC) curve.
  • ROC Receiver Operating Characteristic
  • ROC Receiver Operating Characteristic
  • the area under an ROC curve is a measure for the accuracy of a diagnostic test (the larger the area the better, optimum is 1 , a random test would have a ROC curve lying on the diagonal with an area of 0.5 (see, for reference, for example, JP. Egan. Signal Detection Theory and ROC Analysis).
  • the level of AMBP and/or CRP is above and/or below a threshold value.
  • Said threshold value may be indicative for healthiness, colorectal carcinoma, or colorectal polyps.
  • Said threshold value may also be indicative for non-cancerous or pre-cancerous colorectal polyps, or for cancerous colorectal polyps.
  • healthiness refers to a healthy condition, i.e. a condition not represented by colorectal polyps, colorectal carcinoma, non-cancerous or pre-cancerous colorectal polyps, and/or cancerous colorectal polyps. However, the presence of other diseases not tested or known is not excluded.
  • the threshold value indicative for healthiness is 2.06 ⁇ g/ml for AMBP, 2.20 ⁇ g/ml for CRP or 2.53 marker units for AMBP and CRP.
  • a level, e.g. serum or plasma level, of AMBP which is above a threshold value indicative for healthiness of 2.06 ⁇ g/ml indicates that the patient is at risk of developing or has colorectal polyps, e.g. colorectal adenomas, and/or is at risk of developing or has colorectal carcinoma.
  • a level e.g.
  • CRP serum or plasma level, of CRP which is above a threshold value indicative for healthiness of 2.20 ⁇ / ⁇ indicates that the patient is at risk of developing or has colorectal polyps, e.g. colorectal adenomas, and/or is at risk of developing or has colorectal carcinoma.
  • a combined level, e.g. serum or plasma level, of AMBP and CRP which is above a threshold value indicative for healthiness of 2.53 marker units indicates that the patient is at risk of developing or has colorectal polyps, e.g. colorectal adenomas, and/or is at risk of developing or has colorectal carcinoma.
  • Marker Units were defined as a linear combination of weighted concentrations of AMBP c(AMBP) and concentration of CRP (c(CRP) in plasma or serum. Weighting coefficients K C RP and KAMBP were determined based on the initial study dataset and are defined as followed:
  • threshold value may belong to the mean value calculated from a number of healthy subjects, subjects having colorectal polyps, subjects having colorectal carcinoma, subjects having non-cancerous or pre-cancerous colorectal polyps, or subjects having cancerous colorectal polyps.
  • Said threshold value may represent an amount or a concentration of AMBP and/or CRP or any combination of these values or any calculated value derived thereof.
  • subject relates to any organism such as vertebrate, particularly any mammal, including both a human and another mammal, e.g. an animal such as a rodent, a rabbit, or a monkey.
  • the rodent may be a mouse, rat, hamster, guinea pig, or chinchilla.
  • the subject is a human or a rodent.
  • the term "healthy subject”, as used herein, refers to a subject known to be healthy, i.e. not having colorectal polyps, colorectal carcinoma, non-cancerous or pre-cancerous colorectal polyps, and/or cancerous colorectal polyps. However, a subject being healthy according to the above definition may possibly suffer from another disease not tested or known.
  • the methods of detecting colorectal polyps of detecting colorectal carcinoma or of differentiating between non-cancerous or pre-cancerous colorectal polyps and cancerous colorectal polyps of the present invention (see first, second and third aspect), where the level of AMBP and/or CRP is above a threshold value indicative for
  • the threshold value is preferably the upper value of the 95% confidence interval and belonging to the mean value calculated from subjects being healthy,
  • the threshold value is preferably the upper value of the 95% confidence interval and belonging to the mean value calculated from subjects having colorectal polyps,
  • the threshold value is preferably the upper value of the 95% confidence interval and belonging to the mean value calculated from subjects having non-cancerous or pre-cancerous colorectal polyps, and/or
  • the threshold value is preferably the lower value of the 95% confidence interval and belonging to the mean value calculated from subjects having colorectal carcinoma, or
  • the threshold value is preferably the lower value of the 95% confidence interval and belonging to the mean value calculated from subjects having cancerous colorectal polyps.
  • the 95% confidence interval and the mean value may be determined by techniques known in the art.
  • At least two subjects preferably at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, or at least 2000 subjects, are tested.
  • the level of AMBP and/or CRP is compared to a reference level of AMBP and/or CRP, i.e. the level of AMBP is compared to a reference level of AMBP, the level of CRP is compared to a reference level of CRP, or the level of AMBP is compared to a reference level of AMBP and the level of CRP is compared to a reference level of CRP.
  • Said reference level is the level which may be determined by measuring one or more reference samples, such as at least 1 , at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, or at least 2000 reference samples, from one or more subjects, such as at least 1 , at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, or at least 2000 subjects.
  • reference samples such as at least 1 , at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50,
  • one reference sample per subject is measured. It is further preferred that at least two subjects are tested. Said subjects may be healthy subjects, subjects having colorectal polyps, subjects having colorectal carcinoma, subjects having non-cancerous or pre-cancerous colorectal polyps, or subjects having cancerous colorectal polyps.
  • Said reference level may be an average level (or mean level).
  • the average level may be determined by measuring one reference sample from each one of the at least two subjects, preferably at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, or at least 2000 subjects, and calculating the middle value of the reference levels determined therein.
  • reference sample refers to any biological sample which is used for comparison purposes, in particular for comparison with the "sample” to be tested. Statements made with regard to the preferred embodiments of the “sample” fully analogously apply to the preferred embodiments of the “reference sample”. It is preferred that the source and the amount of the "sample” as well as of the “reference sample” are the same.
  • a level which is higher compared to the reference level means that the level is preferably by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 1 10%, by at least 120%, by at least 130%, by at least 140%, by at least at least 150%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, or by at least 500% higher compared to the reference level.
  • a level which is lower compared to the reference level means that the level is preferably by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 1 10%, by at least 120%, by at least 130%, by at least 140%, by at least at least 150%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, or by at least 500% lower compared to the reference level.
  • a level of AMBP which is at least 2.2 fold higher (log2 ratio of 1.13) and/or a level of CRP which is at least 4.3 fold higher (log2 ratio of 2.12) compared to the reference level in healthy subjects indicates that the patient is at risk of developing or has colorectal polyps, e.g. colorectal adenomas.
  • a level of AMBP which is at least 2.2 fold higher (log2 ratio of 1.13) and/or a level of CRP which is at least 4.3 fold higher (log2 ratio of 2.12) compared to the reference level in healthy subjects indicates that the patient is at risk of developing or has colorectal polyps, e.g. colorectal adenomas.
  • colorectal adenomas a level of AMBP which is at least 2.2 fold higher (log2 ratio of 1.13) and/or a level of CRP which is at least 4.3 fold higher (log2 ratio of 2.12) compared to the reference level in healthy subjects and a level of AMBP which is at least 1 .5 fold lower (log2 ratio of 0.62) and/or a level of CRP which is at least 6.1 fold lower (log2 ratio of 2.61 ) compared to the reference level in subjects having colorectal carcinoma indicates that the patient is at risk of developing or has colorectal polyps, e.g. colorectal adenomas.
  • a level of AMBP which is at least 2.4 fold higher (log2 ratio of 1.24) and a level of CRP which is at least 9.4 fold higher (log2 ratio of 3.23) compared to the reference level in healthy subjects indicates that the patient is at risk of developing or has colorectal carcinoma.
  • a level of AMBP which is at least 1.5 fold higher (log2 ratio of 0.62) and a level of CRP which is at least 6.1 fold higher (log2 ratio of 2.61 ) compared to the reference level in subjects having colorectal polyps, e.g. colorectal adenomas indicates that the patient is at risk of developing or has colorectal carcinoma.
  • the present invention relates to a method of differentiating between non-cancerous or precancerous colorectal polyps and cancerous colorectal polyps.
  • the term "differentiating between non-cancerous or pre-cancerous colorectal polyps and cancerous colorectal polyps", as used herein, relates to the discrimination between both disease states based on observations made with respect to the level of AMBP and/or CRP.
  • patients having polyps can be classified as having non-cancerous or pre-cancerous colorectal polyps, or as having cancerous polyps.
  • the patient is classified as having cancerous colorectal polyps, or if the level of AMBP and/or CRP is below a threshold value indicative for cancerous colorectal polyps, the patient is classified as having non-cancerous or pre-cancerous colorectal polyps.
  • the method of differentiating between non-cancerous or pre-cancerous colorectal polyps and cancerous colorectal polyps is performed on a patient which is already diagnosed as having colorectal polyps.
  • the present invention relates to a method of differentiating between adenomas (adenomatous polyps) and cancerous colorectal polyps.
  • the method of detecting colorectal polyps or carcinoma may also be designated as a method of diagnosing colorectal polyps or carcinoma.
  • the method of differentiating between non-cancerous or pre-cancerous colorectal polyps and cancerous polyps may also be designated as a method of differentially diagnosing between non-cancerous or precancerous colorectal polyps and cancerous polyps.
  • the present invention further relates to a method of monitoring the course of a colorectal proliferative disorder, e.g. colorectal polyps or colorectal carcinoma. It allows monitoring of the level of AMBP and/or CRP in a sample isolated from a patient, e.g. body fluid such as blood serum, over an extended period of time, such as years.
  • the long term monitoring allows differentiating between healthy patients, patients having polyps and patients having colorectal carcinoma.
  • It further allows detecting whether a colorectal carcinoma is progressing in the patient. Said progression may be in such a way that cancer cells from its original site have spread within the colon or to other parts of the body.
  • metastasis The spreading of cancer cells from its original site to another part of the body is called "metastasis".
  • metastasis The formation of metastasis is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, a new tumor, i.e. a secondary tumor or metastatic tumor, is formed at the target site. This process depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor because tumor cells or components may remain and develop metastatic potential.
  • the level of AMBP and/or CRP which is still increased after surgery may be indicative for the incomplete removal of a colorectal carcinoma.
  • the level of AMBP and/or CRP can be routinely checked, for example twice a year, in a patient.
  • the detection of an increase of the level of AMBP and/or CRP over time can be indicative for the development of colorectal polyps, e.g. colorectal adenomas, or colorectal carcinoma in said patient.
  • an increase in the level of AMBP by at least 2.2 fold (log2 ratio of 1 .13) and/or an increase in the level of CRP by at least 4.3 fold (log2 ratio of 2.12) over time may be indicative for the development of colorectal polyps, e.g. colorectal adenomas, while an increase in the level of AMBP by at least 2.4 fold (log2 ratio of 1 .24) and/or an increase in the level of CRP by at least 9.4 fold (log2 ratio of 3.23) over time may be indicative for the development of colorectal carcinoma.
  • a further increase in the level of AMBP and/or CRP in a patient having colorectal polyps, e.g. colorectal adenomas can be indicative for the transformation of colorectal polyps, e.g. colorectal adenomas, into colorectal carcinoma.
  • an increase in the level of AMBP by at least 1.5 fold (log2 ratio of 0.62) and/or an increase in the level of CRP by at least 6.1 fold (log2 ratio of 2.61 ) over time may be indicative for the development of colorectal polyps, e.g. colorectal adenomas, into colorectal carcinoma.
  • the level of AMBP and/or CRP is a valuable clinical parameter for monitoring colorectal polyps as well as colorectal carcinoma. It is an important clinical parameter to allow an early diagnosis and consequently an early treatment of the disease. Patients with an increased level of AMBP and/or CRP should be examined by colonoscopy.
  • the therapeutic treatment of said disorder can be monitored.
  • a further increase of the level of AMBP and/or CRP after therapeutic treatment e.g. chemotherapy, surgery, or targeted therapy
  • a new increase in the level of AMBP and/or CRP after therapeutic treatment e.g. chemotherapy, surgery, or targeted therapy
  • a further colonoscopy may be recommended.
  • the monitoring comprises the determination of the level of AMBP and/or CRP in a sample at a first point in time and in at least one further sample at a later point in time.
  • the levels determined at the different points in time are then compared. Said comparison may show that the level of AMBP and/or CRP increases, decreases, or does not change over time.
  • the level of AMBP and/or CRP increases by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 1 10%, by at least 120%, by at least 130%, by at least 140%, by at least at least 150%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, or by at least 500% over time.
  • the level of AMBP and/or CRP decreases by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 1 10%, by at least 120%, by at least 130%, by at least 140%, by at least at least 150%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, or by at least 500% over time.
  • the level of AMBP and/or CRP does not change over time
  • the level of AMBP and/or CRP preferably means that the level of AMBP and/or CRP varies between 0 and ⁇ 20%, e.g. 0, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 19.9, 19.99, or 19.999%.
  • the level of AMBP and/or CRP is constant over time.
  • the time period between the different points in time preferably amounts to at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days (1 week), at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 1 1 months, at least 12 months (1 year), at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years.
  • the patient may be tested once or twice a year.
  • Said first point in time may be before the initiation of a therapeutic treatment and said later point in time may be during a therapeutic treatment, e.g. targeted treatment, surgery or chemotherapy, and/or after a therapeutic treatment, e.g. targeted treatment, surgery or chemotherapy.
  • the comparison of the levels of AMBP and/or CRP at the different points in time allows the evaluation of the effectiveness of a therapeutic treatment, e.g. targeted treatment, surgery or chemotherapy, the determination, whether a patient responds to a therapeutic treatment or not, e.g. targeted treatment, surgery or chemotherapy, and/or the detection of a relapse of the colorectal cell proliferative disorder such as colorectal polyps or colorectal carcinoma. Determining the level of AMBP and/or CRP after surgery may help, for example, to evaluate whether the colorectal polyps have been completely removed or whether the colorectal carcinoma has been completely removed.
  • (therapeutic) treatment of colorectal polyps or carcinoma relates to any treatment which improves the health status and/or prolongs (increases) the lifespan of a patient.
  • Said treatment may eliminate the colorectal polyps or carcinoma in a patient, reduce the size or the number of colorectal polyps or carcinoma in a patient, arrest or slow the development of colorectal polyps or carcinoma in a patient, inhibit or slow the development of new colorectal polyps or carcinoma in a patient, decrease the frequency or severity of symptoms in a patient, and/or decrease recurrences in a patient who currently has or who previously has had colorectal polyps or carcinoma.
  • the (therapeutic) treatment of colorectal polyps or carcinoma is selected from the group consisting of surgery, radiofrequency ablation, cryotherapy, chemotherapy, radiation therapy, and targeted therapy.
  • the term "surgery”, as used herein, refers to the removal of colorectal polyps or colorectal carcinoma in an operation. It is the most common treatment for all stages of colorectal carcinoma. A surgeon may remove the colorectal polyps or colorectal carcinoma using local excision. To treat a colorectal carcinoma, the resection of the colon with anastomosis or colostomy is also possible. The operation technique usually depends on the severity of the disorder. Colorectal polyps are commonly removed during colonoscopy or sigmoidoscopy, e.g. using a wire loop that cuts the stalk of the polyp and cauterises it to prevent bleeding.
  • the term "radiofrequency ablation” relates to the use of a special probe with tiny electrodes that kill cancer cells. Sometimes the probe is inserted directly through the skin and only local anaesthesia is needed. In other cases, the probe is inserted through an incision in the abdomen. This is done in the hospital with general anaesthesia.
  • ..cryotherapy refers to the use of an instrument that freezes and destroys abnormal tissue. This type of treatment is also called cryosurgery.
  • ..chemotherapy refers to the use of drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing.
  • chemotherapy is taken by mouth or injected into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy).
  • systemic chemotherapy When chemotherapy is placed directly into the cerebrospinal fluid, an organ, or a body cavity such as the abdomen, the drugs mainly affect cancer cells in those areas (regional chemotherapy).
  • radiation therapy refers to the use of high- energy x-rays or other types of radiation to kill cancer cells or keep them from growing.
  • radiation therapy uses a machine outside the body to send radiation toward the cancer.
  • Internal radiation therapy uses a radioactive substance sealed in needles, seeds, wires, or catheters that are placed directly into or near the cancer. The way the radiation therapy is given depends on the type and stage of the cancer being treated. This treatment is particularly used to treat colorectal carcinoma.
  • Targeted therapy relates to the use of drugs or other substances to identify and attack specific cancer cells without harming normal cells.
  • Types of targeted therapies used in the treatment of cancerous tissue include monoclonal antibodies and angiogenesis inhibitors.
  • Monoclonal antibodies are made in the laboratory from a single type of immune system cell. These antibodies can identify substances on cancer cells or normal substances that may help cancer cells grow. The antibodies attach to the substances and kill the cancer cells, block their growth, or keep them from spreading.
  • Monoclonal antibodies are usually given by infusion. They may be used alone or to carry drugs, toxins, or radioactive material directly to cancer cells.
  • Angiogenesis inhibitors stop the growth of new blood vessels that tumors need to grow.
  • targeted therapy also refers to the use of drugs or other substances which prevent the increase in number of cells/proliferative cells not having a malignant potential, e.g. in benign colorectal polyps.
  • the determination of the level of AMBP and/or CRP allows for the detection of colonic polyps or carcinoma and for differentiating between non-cancerous or pre-cancerous colorectal polyps and cancerous polyps with high sensitivity and specificity. Said sensitivity and/or specificity may be further enhanced by the additional detection of Complement component 9 (C9), Leucine rich alpha 2 glycoprotein 1 (A2GL), Plasma protease C1 inhibitor (IC1 ), Transthyretin (TTHY), Profilin 1 (PROF1 ), Haptoglobin (HPT) and/or Insulin like growth factor binding protein complex acid labile subunit (ALS) (as shown in Figure 3 C+D).
  • C9 Complement component 9
  • A2GL Leucine rich alpha 2 glycoprotein 1
  • IC1 Plasma protease C1 inhibitor
  • TTHY Transthyretin
  • PROF1 Profilin 1
  • HPT Haptoglobin
  • ALS Insulin like growth factor binding protein complex acid labile
  • the immune markers are selected from the group consisting of cytotoxic and memory T cells (CD8/CD45RO). It has been shown that a very important clinical translation is the establishment of an immune score based on the density of two lymphocyte populations, cytotoxic and memory T cells (CD8/CD45RO), both in the centre and the invasive margin of tumors, to establish prognosis of clinical outcome in patients, even when there is no cancer associated prognostic marker such as in early tumor stage (l/ll) patients.
  • CD8/CD45RO cytotoxic and memory T cells
  • T H 1 /cytotoxic memory T lymphocytes located both in the centre and the invasive margin of the primary tumor is associated with long disease free and overall survival and low risk of relapse and metastasis. This was particularly illustrated in colorectal cancer, and should be applicable to most human tumors.
  • this immune score classification may help identify the high-risk patients who would benefit the most from adjuvant therapy (see Galon J. et al. "The immune score as a new possible approach for the classification of cancer", Journal of translational medicine, 2012, 10:1 ).
  • the present invention further provides a method of identifying a compound suitable for the treatment of colorectal polyps or colorectal carcinoma in a patient.
  • Said method comprises the steps of (i) providing a test system comprising a level of Alpha-1 -Microglobulin/Bikunin Precursor (AMBP), C-Reactive Protein (CRP), or a combination thereof and (ii) contacting the test system with a test compound.
  • AMBP Alpha-1 -Microglobulin/Bikunin Precursor
  • CRP C-Reactive Protein
  • test compound means any compound suitable for pharmaceutical delivery.
  • the test compound is preferably selected from the group consisting of cells, viruses, bacteria, genetically modified cells, nucleic acids (e.g. vectors comprising a transgene), proteins, peptides, hormones, antibodies, RNA, preferably siRNA or dsRNA, small molecules such as small organic or inorganic molecules, preferably about 800 Daltons more preferably about 500 Daltons, drugs, pharmaceutically active substances, metabolites, natural compounds, or samples of soil, plants or marine origin.
  • Test compounds may be designed specifically or may be derived from libraries already available.
  • library refers to a collection of samples.
  • the test compound is provided in form of a chemical compound library.
  • Chemical compound libraries include a plurality of chemical compounds and have been assembled from any of multiple sources, including chemical synthesized molecules or natural products, or have been generated by combinatorial chemistry techniques. They are especially suitable for high-throughput screening and may be comprised of chemical compounds of a particular structure or compounds of a particular organism such as a plant.
  • synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), ChemBridge Corporation (San Diego, CA), or Aldrich (Milwaukee, Wl).
  • a natural compound library is, for example, available from TimTec LLC (Newark, DE).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts can be used.
  • the test system may be contacted with a test compound over a time period of at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes (1 hour), at least 2 hours , at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 1 1 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours (1 day), at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days (1 week), at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7
  • test compound means that the test compound is applied or administered to the test system.
  • the test system comprises or consists of a cell sample, such as a cell culture sample
  • the application may comprise cell medium supplementation, cell electroporation, or cell lipofection.
  • the administration may comprise oral, nasal, rectal, parenteral, or topical administration.
  • the parental administration may comprise subcutaneous, intracutaneous, intramuscular, intravenous or intraperitoneal administration.
  • an effect of the test compound on the test system is determined.
  • the determination of an effect of the test compound on the test system usually comprises the determination of the level of AMBP and/or CRP in the test system after contact with the test compound.
  • the test compound is identified as a compound suitable for the treatment of colorectal polyps or colorectal carcinoma in the patient, when the effect of the test compound on the test system comprises a decrease in the level of AMBP and/or CRP compared to (i) the level of AMBP and/or CRP comprised in the test system provided in (i), and/or (ii) the level of AMBP and/or CRP comprised in a control system not contacted with the test compound.
  • a decrease in the level of AMBP and/or CRP preferably means a decrease by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 1 10%, by at least 120%, by at least 130%, by at least 140%, by at least at least 150%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, or by at least 500%.
  • test system refers to any system which is used for testing purposes, e.g. for testing whether a compound is suitable for the treatment of colorectal polyps or colorectal carcinoma in a patient. Said system is contacted with a test compound.
  • said system comprises or consists of a sample isolated from a subject having colorectal polyps or colorectal carcinoma, or comprises or consists of a subject having colorectal polyps or colorectal carcinoma.
  • the level of AMBP and/or CRP which is comprised in the test system is above zero (> 0).
  • the test system may comprise or consist of a TruCulture Blood collection and Whole Blood Culture System (Myriad RBM). Said system allows for blood collection, provision of nutrients for up to 48 hours incubation time and investigation of interactions of the test compound with the sample within the same tube.
  • the test system may also comprise or consist of a model organism. Model organisms for colorectal carcinoma are well known in the art and include, for example, rodent, particularly mouse or rat, model systems. For example, mice with mutations in the adenomatous polyposis coli (APC) gene or 1 ,2-dimethylhydrazine (DMH)/azoxymethane (AOM) rats are known animal models for colorectal carcinoma.
  • APC adenomatous polyposis coli
  • DMH 1,2-dimethylhydrazine
  • AOM azoxymethane
  • control system refers to any system comprising a level of AMBP and/or CRP.
  • the control system is not contacted with a test compound.
  • the control system may be contracted with a placebo (compound).
  • Said placebo may be an aqueous solution or a salt solution such as a physiologic salt solution.
  • said system comprises or consists of a sample isolated from a subject having colorectal polyps or colorectal carcinoma, or comprises of consists of a subject having colorectal polyps or colorectal carcinoma. It is preferred that the "control system” is the same as the "test system” but not contacted with the test compound.
  • the subject mentioned above may be any organism such as vertebrate, particularly any mammal, including both a human and another mammal, e.g. an animal such as a rodent, a rabbit, or a monkey.
  • the rodent may be a mouse, rat, hamster, guinea pig, or chinchilla.
  • the test system contacted with a test compound comprises or consists of a subject having colorectal polyps or colorectal carcinoma
  • a sample is taken from said subject after contact with the test compound in order to determine the level of AMBP and/or CRP and, thus, the effect of the test compound on said subject, e.g. by removing body fluids, cell(s), cell colonies, an explant, or a section from said subject.
  • a tissue sample may be removed from said subject by conventional biopsy techniques or a blood sample may be taken from said subject by conventional blood collection techniques.
  • the level of AMBP and/or CRP is the expression level.
  • expression level refers to the expression level of the gene of AMBP and/or CRP.
  • the expression level of the gene of AMBP and/or CRP may be determined on the mRNA level (transcriptional level) or protein level (translational level), for example, by measuring the transcribed mRNA (e.g. via northern blot), by measuring the produced protein (e.g. via Western Blot), by directly or indirectly staining the protein (e.g. via immunohistochemistry) or by directly staining the mRNA (e.g. via in situ hybridization).
  • Said level of AMBP and/or CRP, particularly expression level of AMBP and/or CRP may be indicated as (relative) concentration of AMBP and/or CRP (mRNA or protein), (relative) amount of AMBP and/or CRP (mRNA or protein), or (relative) extinction units such as relative fluorescence units.
  • Said expression level may be determined with an immunoassay, gel electrophoresis, spectrometry, chromatography, in situ hybridization, or a combination thereof.
  • the immunoassay is an enzyme immunoassay, preferably an enzyme-linked immunosorbent assay (ELISA), a Western Blot (immunoblot), a radio immunoassay (RIA), or a luminescence immunoassay (LIA),
  • ELISA enzyme-linked immunosorbent assay
  • RIA radio immunoassay
  • LIA luminescence immunoassay
  • the gel electrophoresis is 1 D or 2D gel electrophoresis
  • the spectrometry is mass spectrometry (MS), preferably tandem mass spectrometry (MS/MS),
  • the chromatography is liquid chromatography (LC) or affinity chromatography,
  • the in situ hybridization is a silver in situ hybridization (SISH), chromogenic in situ hybridization (CISH), or fluorescence in situ hybridization (FISH),
  • the chromatography is combined with spectrometry, preferably mass spectrometry (MS), and is more preferably liquid chromatography-mass spectrometry (LC-MS) and most preferably liquid chromatography-tandem mass spectrometry (LC-MS/MS), or
  • the gel electrophoresis is combined with an immunoassay and is more preferably a 2D immunoblot.
  • 1 D (One-dimensional) gel electrophoresis includes protein separation techniques such as Sodium dodecyl sulfate Dolyacrylamide gel electrophoresis (SDS-PAGE), native gel electrophoresis and isoelectric focusing.
  • SDS-PAGE Sodium dodecyl sulfate Dolyacrylamide gel electrophoresis
  • native gel electrophoresis native gel electrophoresis
  • isoelectric focusing is a technique for separating proteins based on their ability to move within an electrical current, which is a function of the length of their polypeptide chains or of their molecular weight.
  • SDS detergent to these samples gives the proteins the same electrical charge.
  • SDS-PAGE allows for separation of proteins from a wide range of samples including cells, tissues and whole blood.
  • Native Gel Electrophoresis is a technique used mainly in protein electrophoresis where the proteins are not denatured and therefore separated based on their charge-to-mass ratio.
  • the main types of native gels used in protein electrophoresis are polyacrylamide gels and agarose gels. It should be noted that unlike SDS-PAGE type electrophoreses, native gel electrophoresis does not use a charged denaturing agent.
  • the proteins being separated therefore, differ in molecular mass and intrinsic charge and experience different electrophoretic forces dependent on the ratio of the two. Since the proteins remain in the native state they may be visualised not only by general protein staining reagents but also by specific enzyme-linked staining.
  • 2D gel electrophoresis refers to a form of gel electrophoresis commonly used to analyze proteins in two dimensions.
  • 2D gel electrophoresis begins with 1 D electrophoresis but then separates the molecules by a second property in a direction 90 degrees from the first.
  • 1 D electrophoresis proteins are separated in one dimension, so that all the proteins/molecules will lie along a lane but that the molecules are spread out across a 2D gel.
  • the two dimensions that proteins are separated into using this technique can be isoelectric point, protein complex mass in the native state, and protein mass.
  • the first dimension is isoelectric focusing (IEF) and the second dimension is SDS-PAGE.
  • isoelectric focusing (also known as electrofocusing)
  • electrofocusing relates to a technique in order to separate the proteins by their isoelectric point. Thereby, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pH-stages other than the isoelectric point, proteins will be charged. If the proteins are positively charged, they will be pulled towards the more negative end of the gel and if the proteins are negatively charged they will be pulled to the more positive end of the gel. The proteins applied in the IEF will move along the gel and will accumulate at their isoelectric point; that is, the point at which the overall charge on the protein is 0 (a neutral charge).
  • IEF is preferably carried out using immobilized pH gradient (iPG) gels, or iPG strips, more preferably dry and rehydratable iPG strips.
  • iPG immobilized pH gradient
  • Microfluidic chip based isoelectric focusing may also be used (Sommer and Hatch, Electrophoresis. 2009 Mar;30(5):742-5.).
  • the term "Western blotting”, as used herein, relates to a technique which allows the detection of specific proteins (native or denatured) from extracts made from cells or tissues or body liquid samples, before or after any purification steps. Proteins are generally separated by size using gel electrophoresis before being transferred to a synthetic membrane (typically nitrocellulose or PVDF) via dry, semi-dry, or wet blotting methods. The membrane can then be probed using antibodies using methods similar to immunohistochemistry, but without a need for fixation. Detection is typically performed using reporter enzyme linked antibodies, e.g. peroxidase linked antibodies to catalyze a chemiluminescent reaction or alkaline phosphatase linked antibodies to catalyze a colorimetric reaction.
  • reporter enzyme linked antibodies e.g. peroxidase linked antibodies to catalyze a chemiluminescent reaction or alkaline phosphatase linked antibodies to catalyze a colorimetric reaction.
  • Western blotting is a routine molecular biology method that can be used to semi-quantitatively or quantitatively compare protein levels between extracts. The size separation prior to blotting allows the protein molecular weight to be gauged as compared with known molecular weight markers.
  • Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The colorimetric detection method may depend on incubation of the Western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody.
  • the reporter enzyme such as peroxidase
  • enzyme-linked immunosorbent assay or ELISA relates to a method for quantitatively or semi-quantitatively determining protein concentrations from a sample, e.g. blood plasma, serum or cell/tissue extracts, in a multi-well plate format (usually 96-wells per plate). Broadly, proteins in solution are adsorbed to ELISA plates. Antibodies specific for the protein of interest are used to probe the plate. Background is minimized by optimizing blocking and washing methods (as for IHC), and specificity is ensured via the presence of positive and negative controls. Detection methods are usually colorimetric or chemiluminescence based.
  • mass spectrometry refers to the use of an ionization source to generate gas phase ions from a sample on a surface and detecting the gas phase ions with a mass spectrometer.
  • laser desorption mass spectrometry refers to the use of a laser as an ionization source to generate gas phase ions from a sample on a surface and detecting the gas phase ions with a mass spectrometer.
  • the mass spectrometry may be a matrix-assisted laser desorption/ionization mass spectrometry or MALDI.
  • the analyte is typically mixed with a matrix material that, upon drying, co-crystallizes with the analyte.
  • the matrix material absorbs energy from the energy source which otherwise would fragment the labile biomolecules or analytes.
  • the mass spectrometry may also be a surface- enhanced laser desorption/ionization mass spectrometry or SELDI.
  • SELDI the surface on which the analyte is applied plays an active role in the analyte capture and/or desorption.
  • tandem mass spectrometry refers to multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation.
  • one mass analyzer can isolate one peptide from many entering a mass spectrometer.
  • a second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID).
  • CID collision-induced dissociation
  • a third mass analyzer sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time, as in a quadrupole ion trap.
  • fragmenting molecules for tandem MS including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), blackbody infrared radiative dissociation (BIRD), electron- detachment dissociation (EDD) and surface-induced dissociation (SID).
  • CID collision-induced dissociation
  • ECD electron capture dissociation
  • ETD electron transfer dissociation
  • IRMPD infrared multiphoton dissociation
  • BIRD blackbody infrared radiative dissociation
  • ETD electron- detachment dissociation
  • SID surface-induced dissociation
  • ELISA Kits can be used when practicing the methods of the present invention: (i) huC-reactive protein (CRP), (#USC-E90821 HU) from USCN Life Science, Wuhan, PR China), and/or huAMBP ELISA Kit, (#CSB-E1 1316h) from Cusabio, Wuhan, PR China.
  • CCP huC-reactive protein
  • USC-E90821 HU from USCN Life Science, Wuhan, PR China
  • huAMBP ELISA Kit (#CSB-E1 1316h) from Cusabio, Wuhan, PR China.
  • the present invention further relates to a kit comprising means for determining the level of AMBP and/or CRP in a sample isolated from a patient. Said kit is useful for conducting the methods of the present invention.
  • kit of parts in short: kit
  • kit is understood to be any combination of at least some of the components identified herein, which are combined, coexisting spatially, to a functional unit, and which can contain further components.
  • Said means for determining the level of AMBP and/or CRP may be antibodies or fragments thereof, which are capable of specially binding to an epitope or a suitable structural element of AMBP or CRP.
  • Said antibody fragments may be Fab or F(ab)2 fragments.
  • Said means for determining the level of AMBP and/or CRP may also be receptors, which are capable of specifically binding any structure of AMBP or CRP.
  • the receptor can be, for example, any protein or peptide structure which is capable of specially binding to AMBP or CRP.
  • the AMBP and/or CRP antibody or antibody fragment may be bound to a solid support, e.g. a plastic surface, to allow binding and detection of AMBP and/or CRP.
  • a solid support e.g. a plastic surface
  • a microtiter plate can be used as a plastic surface.
  • the detection of the binding of AMBP and/or CRP can be effected by using a secondary antibody labeled with a detectable group.
  • the detectable group can be an enzyme like a Horseradish peroxidase (HRP) or alkaline phosphatase detectable by adding a suitable substrate to produce a colour or a fluorescence signal.
  • HRP Horseradish peroxidase
  • alkaline phosphatase detectable by adding a suitable substrate to produce a colour or a fluorescence signal.
  • Said kit may further comprise (i) a container, and/or (ii) a data carrier.
  • Said data carrier may be a non-electronical data carrier, e.g. a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronical data carrier.
  • the access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized database.
  • said kit may comprise materials desirable from a commercial and user standpoint including a buffer(s), a reagent(s) and/or a diluent(s) for determining the level of AMBP and/or CRP.
  • the above mentioned data carrier may comprise a threshold value or reference level of AMBP and/or CRP.
  • the data carrier comprises an access code which allows the access to a database
  • said threshold value or reference level is deposited in this database.
  • the data carrier may comprise information or instructions on how to carry out the methods of the present invention.
  • Plasma samples were obtained from consenting patients between October 2008 and October 2009 at the University Medical Center Mainz and between October 2009 and February 2013 at the University of Er Weg-Nuernberg. Samples were taken immediately prior to routine screening colonoscopy. The study was performed according to the requirements of the local ethics committee. Patients with known chronic diseases, other malignant disorders, autoimmune pathologies, diabetics or smokers were excluded from the study. The results from the screening endoscopy including histopathologic analysis of biopsies were taken to classify the patients into the three followings groups: colorectal carcinoma (CRC), colorectal adenoma and controls.
  • CRC colorectal carcinoma
  • CRC colorectal adenoma
  • plasma samples of 75 patients were used and analysed by ELISA and Western blotting (validation set, Figure 1 ).
  • 29 were diagnosed with colon cancer (stages ll-IV) and 21 with colonic adenomas.
  • the control group consisted of 25 patients without detection of colonic adenomas or CRC on screening endoscopy and medical personnel and lab members.
  • the plasma sample was then depleted of highly abundant proteins using the ProteomeLab lgY12 antibody system (Beckman Coulter, Fullerton, CA; now Sigma-Aldrich) consisting of microbead bound IgY polyclonal antibodies to abundant plasma proteins (albumin, IgG, alpha-1 -antitrypsin, IgA, IgM, Transferrin, Haptoglobin, alpha-1-acid glycoprotein (Orosomucoid), alpha-2-macroglobulin, HDL (apolipoprotein A1 &A2), and fibrinogen) and the Seppro Supermix (Genway Biotech) which depletes the next 77 moderately abundant proteins.
  • the lgY12- and SuperMix depletion columns were used in tandem using an AKTA FPLC system (Amersham Biosciences).
  • the flow-through fractions (20 ⁇ g) were precipitated using the ProteoExtractKit (Merck, Darmstadt, Germany) according to the manufacturer ' s instructions. Precipitated proteins were solubilised in 25mM ammonium bicarbonate containing 0.1 % RapiGest (Waters, Eschborn, Germany) for 15 min at 80°C. Proteins were reduced by adding 5mM DTT (45 min, 56°C) and free cysteins alkylated with iodoacetamid (Sigma, Taufkirchen, Germany) (25mM, 25°C, 1 h in the dark).
  • porcine sequencing grade trypsin Promega, Mannheim, Germany
  • RapiGest was hydrolyzed by adding 10mM HCI (37°C, 10 min), the resulting precipitate was removed by centrifugation (13000g, 15 min, 4°C), and the supernatant was transferred into an autosampler vial for peptide analysis via LC-MS.
  • LC Capillary liquid chromatography
  • Mass spectrometry (MS) analysis of tryptic peptides was performed using a Waters Q-TOF Premier API system, operated in V-mode with typical resolving power of at least 10,000. All analyses were performed by using positive mode ESI using a NanoLockSpray source. The lock mass channel was sampled every 30 s. Accurate mass LC-MS data were collected in an alternating, low-energy (MS) and elevated energy (MSE) mode of acquisition. The spectral acquisition time in each mode was 0.7 s with a 0.05 s interscan delay. In low-energy MS mode, data were collected at a constant collision energy of 3 eV. In MSE mode, collision energy was ramped from 16 to 36 eV during each 0.7 s data collection cycle. One cycle of MS and MSE data was acquired every 1 .5 s.
  • MS mass spectrometry
  • the continuum LC-MSE data were processed and searched using the IDENTITYE-Algorithm of ProteinLynx Global Server (PLGS) version 2.5.
  • the resulting peptide and protein identifications were evaluated by software using statistical models. Protein identifications were assigned by searching the human taxon of the UniProtKB/SwissProt database (release 2010_09) supplemented with known possible contaminants and standard proteins (porcine trypsin, yeast enolase) using the precursor and fragmentation data afforded by the LC-MS acquisition method.
  • the search parameter values for each precursor and associated fragment ions were set by the software using the measured mass error obtained from processing the raw continuum data.
  • Peptide identifications were restricted to tryptic peptides with no more than one missed cleavage.
  • Carbamidomethyl cysteine was set as fixed modification, and oxidized methionine, protein N-acetylation, and deamidation of asparagine and glutamine were searched as variable modifications.
  • a database search was performed allowing a maximal mass deviation of 15 ppm for precursor ions and 30 ppm for fragmentions.
  • For valid protein identification at least two peptides had to be detected with a total of at least seven fragments. All reported peptide identifications provided by the IDENTITYE-algorithm were correct with >95% probability.
  • the false positive rate for protein identification was set to 3% based on search of a 5x randomized database, which was generated automatically using PLGS 2.5 by randomizing the sequence of each entry.
  • Plasma CRP and AMBP levels were assayed by commercially available CRP and AMBP ELISA Kits (USCN Life Science, China; Cusabio Biotech) according to the protocol provided by the manufacturer.
  • Plasma samples for AMBP ELISA were examined at a dilution of 1 :10, plasma samples for CRP ELISA at a dilution of 1 :1000 to ensure signal in the linear range of the reference standard curve.
  • VDF polyvinylidene difluoride
  • Blots were washed with TBS/0.05 % Tween 20, incubated with a horseradish peroxidise-conjugated secondary antibody (Thermo-Fisher Scientific), washed with TBS/0.05 % Tween 20 and once with TBS, and developed by enhanced chemoluminescence (BioRad GelDoc System). Antibodies for Western blot analysis were specific for CRP and AMBP (Abnova, Taiwan, 1 :1000).
  • AMBP Staining for AMBP was done by using a mouse monoclonal antibody to AMBP (Abnova, clone 3F1 ) at a concentration of 1 :250 and incubation was performed for 10 hours at 4°C. Before incubation, cryosections were deparaffinised, fixed with acetone and unspecific binding of proteins was avoided by use of protein and peroxidase blocking reagents (Vector labs) for 20 minutes. Afterwards, slides were incubated with a biotinylated anti-mouse secondary antibody (BioLegend) and treated with streptavidin-Dylight 648 (Invitrogen) according to the manufacturer's guidelines. Nuclei were stained with Hoechst 33342 (Life Technologies). Analysis of the slides was performed using an Olympus Type IX 70 microscope.
  • ROC Receiver operating characteristics
  • SEQ ID NO: 1 human AMBP, 352 amino acids, P02760
  • SEQ ID NO: 2 (human CRP, 224 amino acids, P02741 )

Abstract

La présente invention concerne un procédé de détection de polypes colorectaux ou de carcinomes colorectaux, un procédé de différenciation entre les polypes colorectaux non cancéreux ou pré-cancéreux et les polypes colorectaux cancéreux, un procédé de surveillance de l'évolution d'un syndrome prolifératif des cellules colorectales, tel que les polypes colorectaux ou un carcinome colorectal, un procédé de traitement des polypes colorectaux ou d'un carcinome colorectal, et un procédé d'identification d'un composé approprié pour le traitement des polypes colorectaux ou d'un carcinome colorectal. La présente invention concerne en outre un kit utile pour réaliser les procédés ci-dessus.
PCT/EP2013/059845 2013-05-13 2013-05-13 Procédés de détection de polypes ou de carcinomes colorectaux et procédés de traitement des polypes ou des carcinomes colorectaux WO2014183777A1 (fr)

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US20170269089A1 (en) * 2014-12-11 2017-09-21 Wisconsin Alumni Research Foundation Methods for Detection and Treatment of Colorectal Cancer
JP2017538938A (ja) * 2014-12-11 2017-12-28 ウイスコンシン・アルムニ・リサーチ・ファンデーション 大腸癌の検出及び処置方法
JP2022058635A (ja) * 2014-12-11 2022-04-12 ウイスコンシン・アルムニ・リサーチ・ファンデーション 大腸癌の検出及び処置方法
KR102457151B1 (ko) * 2014-12-11 2022-10-19 위스콘신 얼럼나이 리서어치 화운데이션 대장암의 검출 및 치료를 위한 방법
US11513123B2 (en) 2014-12-11 2022-11-29 Wisconsin Alumni Research Foundation Methods for detection and treatment of colorectal cancer
US11933785B2 (en) * 2016-06-10 2024-03-19 Wisconsin Alumni Research Foundation Methods for detection, staging, and surveillance of colorectal adenomas and carcinomas
CN111684282A (zh) * 2017-12-05 2020-09-18 迪森德克斯公司 结直肠癌生物标志物的稳健小组
CN113358872A (zh) * 2021-06-03 2021-09-07 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） 用于评估肿瘤免疫治疗疗效的标志物组及系统
CN113358872B (zh) * 2021-06-03 2022-10-21 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） 用于评估肿瘤免疫治疗疗效的标志物组及系统

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