USE OF C3A AND DERIVATIVES THEREOF AS BIOMARKER FOR COLORECTAL ADENOMA AND/OR CARCINOMA ; DIAGNOSIS METHODS AND ASSAYS USING THE SAME

Description

[0001] The present invention relates to the field of detection of colorectal adenoma and/or colorectal carcinoma.

[0002] Colorectal carcinoma is the third most frequently diagnosed carcinoma (9.4 %) worldwide. In 2003 nearly 945 000 new cases of colorectal carcinoma were diagnosed worldwide and approximately 492 000 people died of this disease. The incidence of colorectal carcinoma is increasing, while the mortality rate of colorectal carcinoma is decreasing. Incidence of colorectal carcinoma increases with age, beginning at around 40 years of age, and it is higher for men than for women (40.6 for men versus 30.6 for women, per 100 000 per year) (World cancer report, 2003, Ed. BW. Stewart and P. Kleihues. IARC Press, Lyon).

[0003] In most patients, development of colorectal carcinoma follows a multistep progression from premalignant adenoma to invasive malignancies that have the propensity for metastasis. There is evidence that reduction in colorectal carcinoma morbidity and mortality can be achieved through detection and treatment of early-stage colorectal carcinomas and identification and removal of colorectal adenomatous polyps, which are precursors of colorectal carcinoma.

[0004] So far only invasive colorectal screening tests such as colonoscopy have been shown to achieve detection of early stage colorectal carcinoma and its precursors, i. e. adenomatous polyps and/or flat neoplastic areas. Several tests are available as options for colorectal carcinoma screening. The screening tests encompass faecal occult blood testing (FOBT), flexible sigmoidoscopy, FOBT combined with flexible sigmoidoscopy and colonoscopy. The various screening tests differ from each other regarding performance, effectiveness, possible screening frequency, test complications, costs and acceptance by patients.

[0005] Screening by the faecal occult blood test is currently considered to be the optimal screening strategy in terms of cost-effectiveness. Occult blood in stool can be detected by chemical agents such as guaiac, through hemeporphyrin or immunological methods. The guaiac slide test Hemoccult (II) available from SmithKline Diagnostics is most widely used.

[0006] Various technical factors affect its clinical performance. Hemoccult has about 50 % sensitivity for colorectal carcinomas and about 98 % specificity, however, the sensitivity is low for polyps, at around 10 % (Simon JB. (1998) Gastroenterologist 6:66-78. Review). Another important drawback of occult blood screening is the poor accuracy of prediction, only 10 % of positive reactions prove to be due to colorectal carcinoma (Simon JB. (1998) Gastroenterologist 6:66-78. Review; Mandel JS et al. (1999) J. Natl. Cancer Inst. 91:434-437; Hardcastle JD et al. (1996) Lancet 348:1472-1477; Kronborg O et al. (1996) Lancet 348:1467-71; Winawer SJ et al. (1997) Gastroenterology 112:594-642; Fletcher RH (1998) N. Engl. J. Med. 338:1153-1154).

[0007] Moreover, a faecal occult blood test does only provide results after progression of the disease to a certain stage. It would be desirable to have a test system allowing the detection of colorectal adenoma and/or colorectal carcinoma at an earlier point in time.

[0008] More recently developed immunological tests generally have high sensitivity, however, a poor specificity which remains an important problem. Other methods, such as genetic testings of stool samples for KRAS oncogenes and for p53 protein, are not yet cost-effective and have a low sensitivity (Calistri D et al. (2003) Clin. Gastroenterol. Hepatol. 1:377-383; Schoen RE (2002) Nat. Rev. Cancer 2:65-70).

[0009] Endoscopy (Kavanagh AM (1998) Cancer Causes Control 9:455-462), using either the flexible sigmoidoscope or the colonoscope (Lieberman DA (1997) Gastroenterol. Clin. N. Am. 26: 71-83), is the most definitive means of detection, but has limitations. The false negative rate for flat neoplastic lesions has been recognized and remains to be high (Kudo S (1997) Gastrointest. Endosc. Clin. N. Am. 7:87-98.). Colonoscopy permits examination of the colon with a low false negative rate for polypoid lesions of at least 10 mm in diameter. For this reason, the intervals allowed before reexamination are relatively long after a negative assessment (up to ten years) or up to five years after polypectomy.

[0010] However, patient compliance with such recommendation for reexamination after colonoscopy is poor. Moreover, a colonoscopy is costly and cumbersome. In view of the high costs of a generalized examination and the limited acceptance of a colonoscopy by the population this examination method has a limited application.

[0011] Isolated tissue samples, which are collected, can be tested for colorectal carcinoma and its precursor, colorectal adenoma, by various methods. DE 197 11 111A discloses a method using an in vitro determination of intraepithelial colon bacteria, components and reaction products thereof. Another method using HERG gene expression in tissue samples is disclosed in DE 102 24 534.

[0012] CEA-(Carcinoembryonic antigen)-levels in blood samples have been used to detect colon carcinoma. However, CEA levels are not specifically elevated in colon carcinoma and have been shown to be elevated also in patients with other malignant diseases (e.g., cancers of the stomach, pancreas, breast, and lung) and with various nonmalignant conditions (e.g., alcoholic liver disease, inflammatory bowel disease, heavy cigarette smoking, chronic bronchitis, and pancreatitis). (Posner MR, Mayer RJ: The use of serologic tumor markers in gastro intestinal malignancies. Hematol Oncol Clin North Am 8:533, 1994). Further, the CEA-levels are not elevated in colon adenomas.

[0013] An object of the invention is to provide means allowing an early detection of colon adenoma and/or colon carcinoma.

[0014] It is a further object to provide means of allowing an selective and specific detection of colon adenoma and/or colon carcinoma by a non-invasive method.

[0015] It is a further object to provide a biomarker which can be used in the detection of colorectal adenoma and/or carcinoma.

[0016] Another object of the present invention is to provide a test system for detecting colorectal adenoma or carcinoma which is cost effective and can be widely used.

[0017] Moreover, the test system should be easy to handle and more convenient for the individual to be examined for colorectal adenoma and/or carcinoma.

[0018] The objects underlying the present invention are solved by the use of C3a or a derivative thereof as a biomarker for the detection of colorectal adenoma and/or colorectal carcinoma in an individual.

[0019] The objects are further solved by a method for detecting colorectal adenoma and/or colorectal carcinoma comprising the steps:

a) providing an isolated sample material which has been taken from an individual,

b) determining the level of C3a or a derivative thereof in said isolated sample material,

c) comparing the determined level of C3a or a derivative thereof with one or more reference values.

[0020] The objects are further solved by a method for discriminating between colorectal adenoma and colorectal carcinoma comprising the steps:

a) providing an isolated sample material which has been taken from an individual,

b) determining the level of C3a or a derivative thereof in said isolated sample material,

c) comparing the determined level of C3a or a derivative thereof with one or more reference values.

[0021] The objects are also solved by a method for monitoring the development and/or the course and/or the treatment of colorectal adenoma and/or colorectal carcinoma comprising the steps:

a) providing an isolated sample material which has been taken from an individual,

b) determining the level of C3a or a derivative thereof in said isolated sample material,

c) comparing the determined level of C3a or derivative thereof with one ore more reference values.

[0022] In a preferred embodiment the effectiveness of a surgical or therapeutical procedure is controlled in order to decide as to whether the colorectal adenoma and/or colorectal carcinoma is completely removed. In another embodiment the therapy of an colorectal adenoma and/or colorectal cancer patient with one or more chemical substances, antibodies, antisense-RNA, radiation, e.g. X-rays or combinations thereof is controlled in order to control the effectiveness of the treatment.

[0023] The objects are solved as well by providing a test system for detecting colorectal adenoma and/or colorectal cancer in a sample of an individual comprising:

a) an antibody or a receptor which binds to an epitope of C3a or a derivative thereof,

b) a solid support which supports said antibody or receptor,

c) a reagent for detecting the binding of said epitope of C3a or a derivative thereof to said antibody or receptor.

[0024] The objects are furthermore solved by the provision of an array comprising detection molecules for detecting of colorectal adenoma and/or colorectal carcinoma in an individual comprising as detection molecule:

a) a nucleic acid probe immobilized to a solid support for binding to and detecting mRNA encoding C3a or a derivative thereof and/or for binding to and detecting C3a proteins or derivatives thereof, or

b) an antibody immobilized to a solid support for binding to and detecting of an epitope of C3a or a derivative thereof, or

c) a receptor immobilized to a solid support for binding to and detecting of an epitope of C3a or a derivative thereof,

wherein preferably each different amounts of detection molecules are immobilized to the solid support to increase the accuracy of the quantification.

[0025] The nucleic acid probe is for example selected from the group consisting of single-stranded or double-stranded DNA or RNA, aptamers and combinations thereof. Aptamers are single-stranded oligonucleotides that assume a specific, sequence-dependent shape and bind to protein targets with high specificity and affinity. Aptamers are identified using the SELEX process (Tuerk C. and Gold L. (1990) Science 249: 505-510; Ellington AD ans Szostak JW. (1990) Nature 346: 818-822).

[0026] Preferred embodiments are specified in dependent claims.

[0027] According to the present invention the term "sample material" is also designated as "sample".

[0028] Pursuant to the present invention the term "biomarker" is meant to designate a protein or protein fragment or a nucleic acid which is indicative for the incidence of the colorectal adenoma and/or colorectal carcinoma. That means the "biomarker" is used as a mean for detecting colorectal adenoma and/or colorectal carcinoma.

[0029] The term "individual" or "individuals" is meant to designate a mammal. Preferably, the mammal is a human being such as a patient.

[0030] The term "healthy individual" or "healthy individuals" is meant to designate individual(s) not diseased of colorectal adenoma and/or colorectal carcinoma. That is to say, the term "healthy individual(s)" is used only in respect of the pathological condition of colorectal adenoma and/or colorectal carcinoma and does not exclude the individual to suffer from diseases other than colorectal adenoma and/or colorectal carcinoma.

[0031] The term "derivative thereof" is meant to describe any modification on DNA, mRNA or protein level comprising e.g. the truncated gene, fragments of said gene, a mutated gene, or modified gene. The term "gene" includes nucleic acid sequences, such as DNA, RNA, mRNA or protein sequences or oligopeptide sequences or peptide sequences. The derivative can be a modification which is an result of a deletion, substitution or insertion of the gene. The gene modification can be a result of the naturally occurring gene variability. The term "naturally occurring gene variability" means modifications which are not a result of genetic engineering. The gene modification can be a result of the processing of the gene or gene product within the body and/ or a degradation product. The modification on protein level can be due to enzymatic or chemical modification within the body. For example the modification can be a a glycosylation or phosphorylation or famesylation. Preferably, the derivative codes for or comprises at least 5 amino acids, more preferably 10 amino acids, most preferably 20 amino acids of the unmodified protein. In one embodiment the derivative codes for at least one epitope of the respective protein.

[0032] The term "C3a or a derivative thereof" as used in the present invention also comprises truncated C3a, fragments of C3a, mutated C3a, modified C3a or the precursor C3 (Fig. 1, SEQ ID No.1) or fragments of C3. In one embodiment the derivative has a protein sequence identity of 80%, preferably 90%, more preferably 98% with the sequence SEQ-ID-No. 2 (Fig 2A, SEQ ID No. 2). The modification of "C3a" can be due to enzymatic or chemical modification. In particular, the term C3a or a derivative thereof especially comprises a truncated C3a-protein preferably having a molecular weight in the range of 8,950 ± 25 Da; more preferably in the range of 8,950 ± 20 Da. In a preferred embodiment the truncated C3a-protein has a molecular weight of 8,939 Da. Preferably, the C3a-protein has no C-terminal Arginin and optionally a molecular weight in the range of 8,950 ± 20 Da. In one embodiment the C3a derivative is C3a-desArg (Fig 2B, SEQ ID No. 3). In one embodiment the C3a derivative is obtained by cleavage of C3a by mastcell-chymase. In another embodiment the C3a is obtained by cleavage of C3 by C3-convertase.

[0033] C3a belongs to the group of anaphylatoxins. C3a, C4a and C5a are proteolytic products of serine proteases of the complement system. C3a (SEQ-ID-No.2) is derived from the third component (C3) (SEQ-ID-NO.1) of the blood complement system during complement activation. C3a is a hormon with local effectiveness. Approximately 40% of the amino acid residues in C3a are involved in a helical conformation. Serum anaphylatoxins are involved in a variety of cellular immune responses, as well as being potent proinflammartory agents. C3a produces powerful effects on blood vessel walls, contraction of smooth muscle and an increase in vascular permeability. The C-terminal arginine in C3a is of fundamental importance for its biological activity. Anaphylatoxins are regulated by carboxypeptidase N (anaphylatoxin inactivator), which removes within seconds the carboxyterminal arginine. This mechanism converts the intact anaphylatoxin into a less active C3a-desArg form (SEQ ID No.3).

[0034] The term "epitope" is meant to designate any structural element of a protein or peptide or any proteinaceous structure allowing the specific binding of an antibody, an antibody fragment, a protein or peptide structure or a receptor.

[0035] The methods of the present invention are carried out with sample material such as a body fluid or tissue sample which already has been isolated 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.

[0036] It has been surprisingly discovered by the present inventors that the protein C3a or a derivative thereof can be used as a biomarker for the detection of colorectal adenoma and/or carcinoma. The inventors have now surprisingly found that the level protein C3a or a derivative thereof in a body fluid is elevated in individuals having colorectal adenoma and/or carcinoma. Furthermore, the protein C3a level or a derivative thereof in a body fluid can be used to distinguish healthy people from people having colorectal adenoma and/or carcinoma as well as people having colorectal adenoma from people having colorectal carcinoma.

[0037] Pursuant to the present invention, sample material can be tissue, cells or a body fluid. Preferably the sample material is a body fluid such as blood, blood plasma, blood serum, bone marrow, stool, synovial fluid, lymphatic fluid, cerebrospinal fluid, sputum, urine, mother milk, sperm, exudate and mixtures thereof. In a preferred embodiment the body fluids are fractionated with anion exchange chromatography. The C3a protein is for example eluted at pH 9.0. The transthyretin protein (p13,776) is for example eluted at pH 4.0.

[0038] Preferably, the body fluid has been isolated before carrying out the methods of the present invention. The methods of the invention are preferably carried out in vitro by a technician in a laboratory.

[0039] According to a preferred embodiment of the invention, C3a is measured in blood plasma or blood serum. Blood serum can be easily obtained by taking blood from an individual to be medically examined and separating the supernatant from the clotted blood.

[0040] The level of C3a or a derivative thereof in the body fluid, preferably blood serum, is higher with progressive formation of colorectal adenoma. The colorectal adenoma is a benign neoplasma which may become malign. When developing colorectal cancer from benign colorectal adenoma, the level of C3a or a derivative thereof in body fluids, preferably blood serum, further is elevated.

[0041] After transformation of colorectal adenoma into colorectal cancer, the pathological condition of the afflicted individual can be further exacerbated by formation of metastasis.

[0042] The present invention provides an early stage biomarker which allows to detect the neoplastic disease at an early and still benign stage, neoplastic disease at an early stage or benign stage and/or early tumor stages. The early detection enables the physician to timely remove the colorectal adenoma and to dramatically increase the chance of the individual to survive.

[0043] Moreover, the present invention allows to monitor the level of C3a or a derivative thereof in a body fluid such as blood serum over an extended period of time, such as years.

[0044] The long term monitoring allows to differentiate between healthy individuals and colorectal adenoma and/or colorectal carcinoma. The level of C3a or a derivative thereof can be routinely checked, for example, once or twice a year. If an increase of the level of C3a or a derivative thereof is detected this can be indicative for colorectal adenoma and/or early colorectal carcinoma. A further increase of the level of C3a or a derivative thereof can then be indicative for the transformation into malign colorectal carcinoma.

[0045] Moreover, the course of the disease and/or the treatment can be monitored. If the level of C3a or a derivative thereof further increases, for example after removal of the colorectal adenoma, this can be indicative for exacerbation of the pathological condition.

[0046] That means, the level of C3a or a derivative thereof is a valuable clinical parameter for detecting and/or monitoring of colorectal adenoma and/or colorectal carcinoma. The level of C3a or a derivative thereof in body fluids is higher after incidence of colorectal adenoma. Therefore, the level of C3a or a derivative thereof is an important clinical parameter to allow an early diagnosis and, consequently, an early treatment of the disease. In a preferred embodiment patients with elevated C3a levels or derivatives thereof are subsequently examinated by colonoscopy.

[0047] The method of the invention for detection of colorectal adenoma and/or colorectal carcinoma comprises the step of providing an isolated sample material which has been taken from an individual, then determining the level of C3a or a derivative thereof in the isolated sample material, and finally comparing the determined level of C3a or a derivative thereof with one or more reference values. In one embodiment, one or more further biomarker(s) is/are additionally detected in an isolated sample material which has been taken from an individual, the level of the biomarker(s) is/are determined and compared with one or more respective reference values.

[0048] The reference value can be calculated as the average level of C3a or a derivative thereof determined in a plurality of isolated samples of healthy individuals or individuals suffering from colorectal adenoma and/or colorectal carcinoma. This reference value can be established as a range to be considered as normal meaning that the person is healthy or suffers from colorectal adenoma and/or colorectal carcinoma. A specific value within a range can then be indicative for healthy condition or the pathological condition of colorectal adenoma and/or colorectal carcinoma. This range of reference value can be established by taking a statistically relevant number of body fluid samples, such as serum samples, of healthy individuals as it is done for any other medical parameter range such as, e.g., blood sugar. Preferably, two reference values are calculated which are designated as negative control and positive control 1. The reference value of the negative control is calculated from healthy individuals and the positive control is calculated from individuals suffering from colorectal adenoma or colorectal carcinoma. More preferably, three reference values are calculated which are designated as negative control and positive control 1 and positive control 2. Positive control 1 can be calculated from individuals suffering from colorectal carcinoma and positive control 2 can be calculated from individuals suffering from colorectal adenoma.

[0049] In an another embodiment of the present invention, the reference values can be individual reference values calculated as the average level of C3a or a derivative thereof determined in a plurality of isolated samples taken from the individual over a period of time.

[0050] When monitoring the level of C3a or a derivative thereof over an extended period of time, such as months or years, it is possible to establish an individual average level. The C3a or a derivative thereof level can be measured, for example, from the same blood serum sample when measuring blood sugar and can be used to establish an individual calibration curve allowing to specifically detect any individual increase of the level of C3a or a derivative thereof.

[0051] The reference value for further biomarkers can also be calculated in the same way as described for C3a. The average levels of C3a or further biomarkers may be the mean or median level.

[0052] In another aspect the present invention further provides a test system for detecting colorectal adenoma and/or colorectal carcinoma in an isolated sample material of an individual. The test system is based either on the specificity of an antibody or a receptor to specifically bind to an epitope or a suitable structural element of C3a or a derivative thereof or a fragment of thereof. A receptor can be any structure able to bind specifically to C3a or a derivative thereof. The receptor can be, for example, an antibody fragment such as an Fab or an F(ab')₂ fragment or any other protein or peptide structure being able to specifically bind to C3a or a derivative thereof.

[0053] The antibody, antibody fragment or receptor is bound to a solid support such as, e.g., a plastic surface or beads to allow binding and detection of C3a or a derivative thereof. For example, a conventional microtiter plate can be used as a plastic surface. The detection of the binding of C3a or a derivative thereof can be effected, for example, by using a secondary antibody labelled with a detectable group. The detectable group can be, for example, a radioactive isotop or an enzyme like horseradish peroxidase or alkaline phosphatase detectable by adding a suitable substrate to produce, for example, a colour or a fluorescence signal.

[0054] The test system can be an immunoassay such as an enzyme-linked immunosorbentassay (ELISA) or an radio immunoassay (RIA) or luminescence immunossay (LIA). However, any other immunological test system using the specificity of antibodies or fragments of antibodies can be used such as Western blotting or immuno precipitation.

[0055] The present invention also provides an array comprising detection molecules for detecting colorectal adenoma and/or colorectal carcinoma in an individual, wherein the detection molecule can be a nucleic acid probe immobilized on a solid support for binding to and detecting of mRNA encoding C3a, fragments, mutations, variants or derivatives thereof, or an antibody immobilized on a solid support for binding to and detecting of an epitope of C3a or a derivative thereof, or a receptor immobilized on a solid support for binding to and detecting of an epitope of C3a or a derivative thereof. Preferably, the array comprises further detection molecules which are biomarkers for detecting colorectal adenoma and colorectal carcinoma.

[0056] The nucleic acid probe can be any natural occurring or synthetic oligonucleotide or chemically modified oligonucleotides, as well as cDNA, cRNA, aptamer and the like. Alternatively, the present invention also comprises an inverse array comprising patient samples immobilized on a solid support which can be detected by the above defined detection molecules.

[0057] Preferably the array comprises detection molecules which are immobilized to a solid surface at identifiable positions.

[0058] The term "array" as used in the present invention refers to a grouping or an arrangement, without being necessarily a regular arrangement. An array comprises preferably at least 2, more preferably 5 different sets of detection molecules or patient samples. Preferably, the array of the present invention comprises at least 50 sets of detection molecules or patient samples, further preferred at least 100 sets of detection molecules or patient samples. Pursuant to another embodiment of the invention the array of the present invention comprises at least 500 sets of detection molecules or patient samples. The detection molecule can be for example a nucleic acid probe or an antibody or a receptor.

[0059] The described array can be used in a test system according to the invention. The array can be either a micro array or a macro array.

[0060] The detection molecules are immobilised to a solid surface or support or solid support surface. This array or microarray is then screened by hybridising nucleic acid probes prepared from patient samples or by contacting the array with proteinaceous probes prepared from patient samples.

[0061] The support can be a polymeric material such as nylon or plastic or an inorganic material such as silicon, for example a silicon wafer, or ceramic. Pursuant to a preferred embodiment, glass (SiO₂) is used as solid support material. The glass can be a glass slide or glass chip. Pursuant to another embodiment of the invention the glass substrate has an atomically flat surface.

[0062] For example, the array can be comprised of immobilized nucleic acid probes able to specifically bind to mRNA of C3a or a derivative thereof or antibodies specifically bind to C3a protein or derivatives thereof being present in a body fluid such as serum. Another preferred embodiment is to produce cDNA by reverse transcription of C3a encoding mRNA or of mRNA encoding a derivative of C3a and to specifically detect the amount of respective cDNA with said array. The array technology is known to the skilled person. A quantification of the measured mRNA or cDNA or proteins, respectively, can be effected by comparison of the measured values with a standard or calibration curve of known amounts of C3a or a derivative thereof mRNA or cDNA or proteins.

[0063] Preferably, different amounts of detection molecules are immobilized each on the solid support to allow an accurate quantification of the level of C3a or a derivative thereof.

[0064] Pursuant to another embodiment of the invention, the level of C3a or a derivative thereof is determined by mass spectroscopy.

[0065] Mass spectroscopy allows to specifically detect C3a or a derivative thereof via its molecular weight and to quantify the amount of C3a or a derivative thereof very easily.

[0066] Any suitable ionization method in the field of mass spectroscopy known in the art can be employed to ionize the C3a or a derivative thereof molecule, fragments, mutations, variants or derivatives thereof. The ionization methods comprise electron impact (EI), chemical ionization (CI), field ionization (FDI), electrospray ionization (ESI), laser desorption ionization (LDI), matrix assisted laser desorption ionization (MALDI) and surface enhanced laser desorption ionization (SELDI).

[0067] Any suitable detection method in the field of mass spectroscopy known in the art can be employed to determine the molecular mass of C3a or a derivative thereof. The detection methods comprise quadrupol mass spectroscopy (QMS), fourier transform mass spectroscopy (FT-MS) and time-of-flight mass spectroscopy (TOF-MS).

[0068] Preferably, the mass spectroscopy is a surface enhanced laser desorption ionization-time of flight-mass spectroscopy (SELDI-TOF-MS). Before carrying out a SELDI-TOF-MS, the C3a or a derivative thereof in the isolated sample is preferably immobilized on a chip or solid support with an activated surface. The activated surface comprises preferably immobilized antibodies against anti- C3a or a derivative thereof such as, for example, rabbit polyclonal-antibodies. After binding of the C3a or a derivative thereof to the antibodies, a time-of-flight analysis in a SELDI-TOF mass spectrometer is carried out, which delivers intensity signals for determination of the C3a or a derivative thereof level.

[0069] Moreover, mass spectroscopy allows to simultaneously detect other proteins which can have a relevance with respect to the detection of colorectal adenoma and/or colorectal cancer.

[0070] In an embodiment of the present invention the sensitivity and/or specificity of the detection of colorectal adenoma and/or colorectal carcinoma is enhanced by additionally detection of a further biomarker. In particular, in one embodiment the sensitivity and/or specificity of the detection of colorectal adenoma and/or colorectal carcinoma is enhanced by detection of another protein or nucleic acid in combination with C3a or a derivative thereof.

[0071] Preferably, the sensitivity and specifity of the methods, arrays, test systems and uses according to the present invention are increased by the combination of detecting C3a and derivatives thereof with transthyretin and derivatives thereof.

[0072] The term "transthyretin or a derivative thereof" as used in the present invention also comprises truncated transthyretin, fragments of transthyretin, mutated transthyretin, or modified transthyretin. The modification of "transthyretin" can be due to enzymatic or chemical modification. Moreover, the term "transthyretin" is also used to designate monomeric or multimeric forms of transthyretin. For example, the term "transthyretin" especially covers the monomeric protein chain usually being part of the homotetrameric protein transthyretin.

[0073] Transthyretin is also designated as prealbumin. Transthyretin is a tetrameric protein having a molecular weight of about 54,000 Da that is synthesized mainly in the liver Transthyretin is normally a homotetramer comprising four protein chains having each a molecular weight of about 14,000 Da. Using mass spectroscopy the inventors have detected several variants of the transthyretin protein chains having a molecular weight of inter alia 13,776 Da, 13,884 Da or 14,103 Da. The inventors have found out that especially the level of molecular variants of transthyretin having a molecular weight of 13,776 Da and 13,884 Da is decreased in a body fluid such as serum in case of incidence of colorectal adenoma and/or colorectal carcinoma.

[0074] In a further embodiment of the present invention the sensitivity and/or specificity of the detection of colorectal adenoma and/or colorectal carcinoma is enhanced by additionally detection of p53, CEA (carcinoembryonic antigen) and/or CA 19-9, CA 15-3, Kras, mutated E-cadherin, β-Catenin or derivatives thereof in combination with C3a or a derivative thereof.

[0075] In a further embodiment of the present invention the sensitivity and/or specificity of the detection of colorectal adenoma and/or colorectal carcinoma can be enhanced by additionally detection of mutations in DNA mismatch genes, e.g. MSH2, MSH3, MLH1, PMS1, PMS2, MSH6, microsatellite instability of e.g. MHL1 or MSH2, SNPs (single nucleotide polymorphysm) or C-reactive protein plasma concentrations.

[0076] In a further embodiment of the present invention the sensitivity and/or specificity of the detection of colorectal adenoma and/or colorectal carcinoma is optionally enhanced by detection of CA15-3, CA-125 and/or Her-2/neu in combination with C3a or a derivative thereof. CA15-3 is an oncofetal antigen, which is expressed by several carcinomas, and is often measured with other tumor markers. Both CA15-3 and CA-125 are prognostic indicators, mainly for breast cancer, but also in addition to visceral metastases. The amplification of Her-2/neu in breast carcinoma is associated with poor prognosis, short disease-free interval and short survival time. Little is known up to now about the starting point of amplification and the progress of Her-2/neu up to now.

[0077] In a preferred embodiment colorectal adenoma and/or colorectal carcinoma are detected by the combination of the biomarkers C3a and transthyretin or derivatives thereof. This allows the detection of colorectal adenoma and/or colorectal carcinoma with an increased sensitivity and/or specificity. Further, the detection method is well accepted by the patients, since the detection method is non-invasive.

[0078] The sensitivity and specificity are defined as follow:

The sensitivity is the number of true positive patients (%) with regard to the number of all patients (100 %). The patients are individuals having colorectal adenoma and/or colorectal carcinoma.

[0079] The specificity is the number of true negative individuals (%) with regard to the number of all healthy individuals (100%).

[0080] The sensitivity and specificity can be alternatively defined by the following formulas:

		diagnosis
		+	-
test	+	TP	FP
	-	FN	TN

TP: True positive (test positiv, diagnosis correct); FP: False positive (test positiv, diagnosis incorrect); TN: True negative (test negative, diagnosis correct); FN: False negative (test negative, diagnosis incorrect);

[0081] The sensitivity is calculated by the following formula:


and the specificity is calculated by the following formula:

[0082] The result of each analysis group, which is selected from TP, FP, TN, FN, is calculated for a plurality of isolated samples selected from the group consisting of healthy individuals, colorectal adenoma patients and/or colorectal carcinoma patients. TP, FP, TN, FN relates to number of individuals that are correlated with the status true positive, false positive, true negative, false negative, respectively.

[0083] The methods of the present invention can be carried out in combination with other diagnostic methods for detection of colorectal adenoma and/or colorectal carcinoma to increase the overall sensitivity and/or specificity. The detection of C3a allows an very early detection of colorectal adenoma and can therefore be used as an very early marker.

[0084] Preferably, the methods of the present invention are carried out as an early detection and/or monitoring method. If the results of the methods of the present invention should indicate the incidence of colorectal adenoma and/or colorectal adenoma, further examinations such as colonoscopy should be carried out.

[0085] The following polyclonal anti-transthyretin antibodies and C3a antibodies can be used when practicing the invention:

• Anti-transthyretin: PC 066 available from The Binding Site Ltd., Birmingham, England and A 0002, available from DAKO, Hamburg, Germany.
• Anti-C3a-desArg: available with the Quidel immunoassay (Quidel Corporation, 10165 McKellar Court, San Diego, CA 92121, USA).

Figures

[0086] 

Fig. 1 shows the C3 protein sequence.

Fig 2. shows (A) the C3a protein sequence and (B) the C3a-desArg protein sequence.

Fig. 3 shows a schematic diagram for fractionating and profiling of serum samples.

Fig. 4 shows the quantification of C3a-desArg using an ELISA. Serum samples from non-cancer (n=28), adenoma (n=28) and colorectal cancer patients (n=28) were assayed in dublicate in the Quidel C3a Enzyme Immunoassay. (A = adenoma group, N = healthy control group, T = cancer group)

Fig. 5 shows the analysis of C3a-desArg by A) ELISA and B) SELDI-TOF MS. The mean C3a-desArg concentrations are significantly higher in the adenoma and cancer group compared to the non-cancerous control group. (A = adenoma group, N = healthy control group, T = cancer group)

Fig. 6 shows the quantification of transthyretin by A) radial immunodiffusion and B) SELDI-TOF MS analysis. The mean transthyretin concentrations are significantly lower in the adenoma and cancer group compared to the non-cancerous control group. Further, the mean transthyretin concentrations are significantly lower in the cancer group compared with the adenoma group as measured by radial immunodiffusion. (A =adenoma group, N =healthy control group, T = cancer group)

Figure 7 shows the correlation between SELDI-TOF MS and immunoassay data. C3a-desArg (A) was analyzed by ELISA, transthyretin (B) by radial immunodiffusion.

Examples

[0087] Unless otherwise stated all methods were carried out following the protocol of the manufacturer of the analytical systems.

Serum collection and serum fractionation

[0088] Serum from three groups of human patients were collected and investigated.

[0089] Group 1 consisted of 28 patients which were surgical patients treated for non-cancerous diseases such as inguinal hernia, gall bladder stones or diverticulitis. These individuals of group 1 were taken as the group of healthy individuals, i. e. those, who did not suffer from colorectal adenoma and/or colorectal carcinoma.

[0090] Group 2 consisted of 28 patients, who were all surgical patients treated for undefined tumors, which turned out to be benign colorectal adenoma.

[0091] Group 3 consisted of 28 patients, who were patients having colorectal carcinoma. All these 28 patients suffered from TNM stage III (Tumor, Node, Metastasis stage III) colorectal carcinoma.

[0092] Ethical guidelines and patient confidentiality have been strictly assured and all patients gave written consent to participate in this study. All patients had comparable preoperative preparations such as fasting time and medication at time of surgery.

[0093] Serum from each patient was fractionated by anion exchange chromatography (Serum Fractionation Kit / Q HyperD resin, Ciphergen Biosystems, Inc.) using a 96 well format automation approach (Biomek2000, Ciphergen), according to the protocol of the manufacturer, to reduce some of the interference by the most abundant proteins. As shown in Fig. 3, the fractionation produced 6 fractions containing proteins separated roughly on the basis of the protein pl-value.

[0094] The C3A-desArg protein (p8,960 Da) was eluted with fraction 1 at pH 9.0 wash solution (50 mM Tris-HCl with 0,1 % OGP (Octyl-β-D-glucopyranoside, pH 9.0) (according to the conditions defined by Ciphergen Biosystems Inc. expression difference mapping kit-serum fractionation cat. no K100-0007). The transthyretin protein (p 13,776 Da was eluted with fraction 4 at pH 4.0 (100 mM Sodium acetate with 0.1 % OGP, pH 4.0 conditions (according to the conditions defined by Ciphergen Biosystems Inc. expression difference mapping kit-serum fractionation cat. no K100-0007).

SELDI-TOF-MS analysis

[0095] CM10 protein arrays were processed in a bioprocessor (Ciphergen Biosystems, Inc.) according to the protocol of the manufacturer. Chips were equilibrated with CM10 binding buffer (Ciphergen Biosystems, Inc.) for 2x5 minutes and were subsequently incubated with the serum fractions (which had been diluted 1:10 in CM10 binding buffer). After 45 minutes the unbound material was removed and the chips were washed 3 times with CM10 binding buffer and 2 times with water. After drying at room temperature for 10 minutes, 2 applications of 0.05 M sinapinic acid (1.0 µl) were added and the chips were analyzed with the Ciphergen Protein ChipReader (model PBSII).

[0096] The Protein ChipReader is a time-of-flight mass spectrometer. The mass values and signal intensities for the detected proteins are transferred to a software, which is supplied by Ciphergen for further in-depth analysis by the ProteinChip Data Analysis Program and Biomarker Wizard Program.

[0097] To minimize data variability, measurement was performed within two days using samples from all patient groups randomly distributed on the chips. As a standard control for normalization, pooled normal serum was used parallel to all measurements.

[0098] The mass spectra of proteins were generated by using an average of 195 laser shots at a laser intensity of 185. The detector was run at a sensitivity of 7. For data acquisition, the detection size range was set between 2,000 and 40,000 Da. The laser was focused at 10,000 Da. The data were analyzed with the ProteinChip Data Analysis Program (version 3.1, Ciphergen Biosystems) and with the Biomarker Wizard Program (version 3.1, Ciphergen Biosystems).The peak intensities were normalized to the total ion current. It should be noted that the measured molecular weights may vary from measurement to measurement and may be dependent on the specifics of the used mass spectroscope. The measured molecular weight of C3desArg may be within the range of 8,950 ± 25 Da.

C3a ELISA analysis

[0099] For the quantitation of the C3a-desArg fragment in serum an enzyme immunoassay from Quidel was used (Quidel Corporation, 10165 McKellar Court, San Diego, CA 92121, USA). The ELISA was performed according to the manufacturer's instructions.

[0100] The microtiter strips included in the kit are coated with a monoclonal antibody (included in the immunoassay from Quidel) specific for human C3a-desArg. Samples were diluted 1:500 and incubated for one hour at 18-25°C. During this incubation, C3a-desArg in the specimen will bind to the monoclonal antibody. After rinsing off the unbound native C3, peroxidase-conjugated rabbit anti-C3a was used for the detection of bound C3a-desArg. Excess conjugate is removed through a washing step, and the amount of C3a-desArg in the serum sample was quantified using the peroxidase reaction and a standard curve.

Radial immunodiffusion for the quantification of Transthyretin

[0101] The radial immunodiffusion assay (Tina-Quant Prealbumin assay, Immunoturbidometric assay for the determination of prealbumin, Roche diagnostics GmbH, Mannheim, Germany, Cat.-No. 11660519) was performed in an clinical service laboratory. Serum was applied to a cylindrical well cut in a gel matrix containing a uniform concentration of monospecific antibodies. Antigen placed in the well diffuses radially, producing a precipitin ring. Precipitin rings can be read any time after overnight incubation, or endpoint. Results were quantitated by comparing the diameter of the precipitin ring produced by the sample to the precipitin rings produced by standards of known concentrations.

Statistical evaluation of the data

[0102] For the three patient groups cut-off values are calculated by the C&RT(CART)algorithmus on the basis of decision-tree analysis (Breiman, L., Friedman, J. H., Olshen, R. A., & Stone, C. J. (1984). Classification and regression trees. Monterey, CA: Wadsworth & Brooks/Cole Advanced Books & Software). The Cutoff-values have been calculated in order to select and specify the limiting values between the different analysis groups. The evaluation has been performed with STATISTICA Software Vs 7.1 from STATSOFT INC, the decision-tree analysis is performed with Data-Miner Modul subprogramm Standard Classification Trees (CAndRT) (StatSoft, Inc. (2005). STATISTICA (data analysis software system), version 7.1. www.statsoft.com.)

[0103] The statistical data are evaluated on the basis on the mean value and standard deviation. Further, the Figures show a confidence interval of mean ± 95, indicating to find the true mean values of prospect patient groups with 95% probability within this interval. The statistical evaluation is performed by the T-Test. The tests were considered as significant at p values p< 0,05. The whiskers of the box plots show the standard deviation.

Example 1

[0104] In this experiment, the expression of C3a-desArg between serum from non-carcinoma patients (Group 1, n=28 patients) and serum from patients with colorectal adenoma (Group 2, n=28 patients) and colorectal carcinoma (Group 3, n=28 patients) were identified in duplicate by ELISA (Quidel C3a Enzyme immuno assay) and by SELDI-TOF MS analysis as described above.

Example 2

[0105] The expression of transthyretin was quantified by Radial immunodiffusion and by SELDI-TOF MS analysis as described above with the same patient collective.

Results and statistical evaluation

[0106] As shown in Figure 4, the intensity of the concentration of C3a-desArg [ng/ml] differs significantly between the three groups. The C3a-desArg level increases from healthy individuals over colorectal adenoma patients to colorectal carcinoma patients.

[0107] Table 1 shows the distribution of serum levels of C3a-desArg [ng/ml], and transthyretin [g/l] among 84 serum samples using SELDI-TOF MS and immunoassays (C3a-desArg-ELISA, transthyretin immunodiffusion) for validation. 28 serum samples in each group were measured.

Table 1

Variable	Method	Mean ± Std.Dev. N	Mean ± Std.Dev. A	Mean ± Std.Dev. T	Mean ± Std.Dev. A+T	p-Value N vs. A	p-Value N vs. T	p-Value N vs. A+T	p-Value A vs. T
Transthyretin	Immunodi ffusion	0.250 ± 0.043	0.191 ± 0.035	0.154 ± 0.056	0.172 ± 0.05	0.000001	0.000001	0.000001	0.0046
Transthyretin MG 13,776 Da	SELDI	1.728 ± 0.462	1.123 ± 0.287	0.996 ± 0.497	1.060 ± 0.407	0.000001	0.000001	0.000001	0.247
C3A-desArg	ELISA	863.51 ± 678.93	1871.12 ± 1090.62 ±	2513.333 ± 2060.46	2192.23 ± 1665.25	0.00011	0.000179	0.000117	0.150
C3a-desArg MG 8,960 Da	SELDI	0.5724 ± 0.387	1.363 ± 1.012	1.552 ± 1.271	1.458 ± 1.143	0.00030	0.000268	0.00015	0.541

[0108] Serum concentrations of C3a-desArg measured by ELISA were significantly higher in adenoma and carcinoma patients compared to the non-cancerous control group (Figure 5A). According to these data, a discrimination of healthy individuals from the cancer patients was possible. Furthermore, a discrimination between the healthy individuals and the adenoma patients was also possible. The results were very similar to the data achieved by SELDI-TOF MS analysis (Figure 5B). In Figure 7 the scatter plot of both methods (SELDI-TOF MS and ELISA) is shown, demonstrating a good correlation (Fig. 7A; r = 0,7) between the intensity of p8,960 measured by SELDI-TOF MS and the concentration of C3a-desArg measured by ELISA of each patient as well as a good correlation (Fig. 7B, r=0.81) between the intensity of p13,776 measured by SELDI-TOF MS and the concentration of transthyretin measured by ELISA of each patient. This indicates that the results are independent from the analysis method.

[0109] Transthyretin-data were generated by SELDI-TOF MS analysis (Figure 6B) and were confirmed by radial immunodiffusion (Figure 6A). Among patients with colorectal cancer transthyretin was significantly lower compared to non-cancer patients. Moreover, the transthyretin concentrations in serum of adenoma patients is still significantly lower than in normal serum.

[0110] Table 2 shows the comparison of sensitivity and specificity of C3a-desArg, measured by SELDI-TOF MS and ELISA, respectively, for the discrimination between healthy controls and adenoma/tumor patients.

Table 2

C3a-desArg (healthy controls versus Adenoma+Tumor patients)
	ELISA
Sensitivity	75 %
Specificity	78 %

[0111] Table 3 shows the comparison of sensitivity and specificity of transthyretin, measured by SELDI-TOF MS and radial immunodiffusion, respectively, for the discrimination between healthy controls and adenoma/tumor patients.

Table 3

Transthyretin (healthy controls versus Adenoma+Tumor patients)
	SELDI-TOF MS	Radial Imunodiffusion
Sensitivity	75%	88 %
Specificity	90%	70%

[0112] Table 4 shows the comparison of sensitivity and specificity of both biomarkers (C3a-desArg/transthyretin) in combination. C3a-desArg and transthyretin were measured by immunoasay for the discriminiation between healthy controls and Adenoma/Tumor patients. Cutoff values for transthyretin (TTR) and C3a-desArg are shown in brackets.

Table 4

Combination of C3a-desArg and transthyretin (healthy controls versus Adenoma+Tumor patients)
	SELDI-TOF MS	Radial Immunodiffusion/ELISA (TTR <0.22 and C3a-desArg >1000)
Sensitivity	70%	67 %
Specificity	100%	89 %

[0113] Table 5 shows the sensitivity and specificity of C3a-desArg as single biomarker. C3a-desArg levels were measured by ELISA for the discrimination between healthy controls and adenoma and/or tumor patients. The cutoff values are shown in brackets.

Table 5

C3a-desArg
	Normal vs. Adenoma	Normal vs. Tumor	Normal vs. Adenoma+Tumor
Sensitivity	79 %	61%	75 %
	(</> 990)	(</> 1786)	(</> 990)
Specificity	78 %	93 %	78 %

[0114] Table 6 shows the sensitivity and specificity of transthyretin as single biomarker. Transthyretin levels were measured by radial immunodiffusion for the discrimination between healthy controls and adenoma and/or tumor patients.The cutoff values are shown in brackets.

Table 6

Transthyretin
	Normal vs. Adenoma	Normal vs. Tumor	Normal vs. Adenoma+Tumor
Sensitivity	86%	61 %	88 %
	(</> 0.225)	(</> 0.165)	(</> 0.22)
Specificity	68 %	100%	70%

[0115] Table 7 shows the sensitivity and specificity of C3a-desArg and transthyretin (TTR) in combination. Transthyretin and C3a-desArg levels were measured by ELISA and radial immunodiffusion, respectively. The cutoff values are shown in brackets.

Table 7

Combination of Transthyretin (TTR)and C3a-desArg
	Normal vs. Adenoma
Sensitivity	97 %
	(</> 0.225 TTR)
	and (</> 1974 C3a-
	desArg)
Specificity	70 %

[0116] These data show that C3a, optionally in combination with transthyretin, is(are) an excellent biomarker(s) for the detection of colorectal adenoma and/or colorectal carcinoma. In contrast to already known biomarkers CEA and CA 19-9 it is possible to discriminate between healthy individuals and adenoma patients. The sensitivity and specifity of the C3a test is high and allows an early specific detection of adenomas without a colonoscopy. In particular, the combination of the biomarkers C3a and transthyretin allows the detection of adenomas with an excellent sensitivity and high specifity.

Claims

1. A method for detecting colorectal adenoma and/or colorectal carcinoma comprising the steps:

a) providing an isolated sample material which has been taken from an individual,

b) determining the level of C3a or a derivative thereof having the same function in said isolated sample material,

c) comparing the determined level of C3a or a derivative thereof having the same function with one or more reference values.

2. A method for discriminating between colorectal adenoma and colorectal carcinoma comprising the steps:

a) providing an isolated sample material which has been taken from an individual,

b) determining the level of C3a or a derivative thereof having the same function in said isolated sample material,

c) comparing the determined level of C3a or a derivative thereof having the same function with one or more reference values.

3. A method for monitoring the development and/or the course and/or the treatment of colorectal adenoma and/or colorectal carcinoma comprising the steps:

a) providing an isolated sample material which has been taken from an individual,

b) determining the level of C3a or a derivative thereof having the same function in said isolated sample material,

c) comparing the determined level of C3a or a derivative thereof having the same function with one or more reference values.

4. The method according to any one of claims 1 to 3, wherein the level of C3a or a derivative thereof in said sample material taken from a colorectal adenoma or colorectal carcinoma patient is higher compared to a sample material of a healthy individual.

5. The method according to any one of claims 1 to 4, wherein a first increase of the level of C3a or a derivative thereof in a first sample material is indicative for colorectal adenoma and wherein a second increase of the level of C3a or a derivative thereof in a second sample material, isolated from said individual at a later point in time than said first sample material, is indicative for colorectal carcinoma, with the proviso that said second increase is stronger than said first increase.

6. The method according to any one of claims 1 to 5, wherein in step (b) one or more further biomarker(s) for detecting colorectal adenoma and/or colorectal carcinoma is/are determined in said isolated sample material and wherein in step (c) the determined level of said biomarker(s) is/are compared with one or more respective reference values.

7. The method according to any one of claims 1 to 6, wherein at least one further biomarker(s) for detecting colorectal adenoma and/or colorectal carcinoma is optionally selected from the group consisting of transthyretin, p53, CEA, CA 19-9, CA 15-3, CA-125, Kras, β-Catenin, Her-2/neu, C-reactive protein plasma and derivatives thereof and
mutations in E-cadherin, MSH2, MSH3, MLH1, PMS1, PMS2, MSH6 genes
and
microsatellite instability of MHL1 or MSH2 and
SNPs and combinations thereof.

8. The method according to any one of claims 1 to 7, wherein the reference value(s) of C3a and/or derivative(s) thereof and optionally the reference value(s) of the further biomarker(s) and/or derivative(s) thereof is/are calculated as the average level of C3a and/or a derivative thereof and optionally further biomarker(s) and/or derivative(s) thereof in a plurality of isolated samples of a respective group of individuals, wherein the group of individuals are healthy individuals, colorectal adenoma patients and/or colorectal carcinoma patients.

9. The method according to any one of claims 1 to 7, wherein the reference value is/are individual reference value(s) calculated as the average level of C3a and/or a derivative thereof and optionally of further biomarker(s) and/or derivative(s) thereof determined in a plurality of isolated sample material taken from said individual over a period of time.

10. The method according to any one of the preceding claims, wherein the isolated sample material is a body fluid and is optionally selected from the group consisting of blood, blood plasma, serum, bone marrow, stool, synovial fluid, lymphatic fluid, cerebro spinal fluid, sputum, urine, mother milk, sperm, exudate and mixtures thereof.

11. The method according to any one of the preceding claims, wherein the level of C3a and/or a derivative thereof and optionally of further biomarker(s) in said sample material is determined on DNA, mRNA and/or protein level.

12. The method according to any one of the preceding claims, wherein the level of C3a and/or a derivative thereof and optionally of further biomarker(s) in said sample material is(are) determined by a nucleic acid hybridisation technique, immunological methods or proteomics technique, and/or mass spectroscopy.

13. The method according to any one of the preceding claims, wherein the method is carried out in combination with other diagnostic methods for colorectal adenoma and/or colorectal carcinoma to increase sensitivity and/or specificity.

14. A use of C3a or a derivative thereof having the same function as a biomarker for detection of colorectal adenoma and/or colorectal carcinoma in an isolated sample of an individual.

15. The use according to claim 14 for an early detection of colorectal adenoma and/or colorectal carcinom in an isolated sample of an individual.

16. The use according to any one of claims 14 or 15, in combination with one or more further biomarkers for colorectal adenoma and/or colorectal carcinoma to increase sensitivity and/or specificity.

17. The use according to claim 16, wherein the at least one further biomarker is optionally selected from the group consisting of transthyretin, p53, CEA, CA 19-9, CA 15-3, CA-125, Kras, β-Catenin, Her-2/neu, C-reactive protein plasma and derivatives thereof and
mutations in E-cadherin, MSH2, MSH3, MLH1, PMS1, PMS2, MSH6 genes
and
microsatellite instability of MHL1 or MSH2 and
SNPs and combinations thereof.

18. An immunoassay for detecting colorectal adenoma and/or colorectal carcinoma in an isolated sample material of an individual comprising:

a) an antibody or a receptor which binds to an epitope of C3a or a derivative thereof having the same function,

b) a solid support which supports said antibody or receptor,

c) a reagent for detecting the binding of said epitope of C3a or a derivative thereof having the same function to said antibody or receptor,

wherein said immunoassay comprises one or more antibodies or receptors for detection of one or more further biomarkers for colorectal adenoma and/or colorectal carcinoma.

19. The immunoassay according to claim 18, wherein at least one further biomarker is optionally selected from the group consisting of transthyretin, p53, CEA, CA 19-9, CA 15-3, CA-125, Kras, β-Catenin, Her-2/neu, C-reactive protein plasma and derivatives thereof and
mutations in E-cadherin, MSH2, MSH3, MLH1, PMS1, PMS2, MSH6 genes
and
microsatellite instability of MHL1 or MSH2 and
SNPs and combinations thereof.

20. An array comprising detection molecules for detecting of colorectal adenoma and/or colorectal carcinoma in an individual comprising as detection molecule:

a) A nucleic acid probe immobilized to a solid support for binding to and detecting of mRNA encoding C3a or a derivative thereof having the same function, or

b) an antibody immobilized to a solid support for binding to and detecting of an epitope of C3a or a derivative thereof having the same function, or

c) a receptor immobilized to a solid support for binding to and detecting of an epitope of C3a or a derivative thereof having the same function,

wherein preferably each different amounts of detection molecules are immobilized to the solid support to increase the accuracy of the quantification,
wherein said array comprises one or more antibodies or receptors for detection of one or more further biomarkers for colorectal adenoma and/or colorectal carcinoma.

21. The array according to claim 20, wherein the at least one further biomarker is optionally selected from the group consisting of transthyretin, p53, CEA, CA 19-9, CA 15-3, CA-125, Kras, β-Catenin, Her-2/neu, C-reactive protein plasma and derivatives thereof and
mutations in E-cadherin, MSH2, MSH3, MLH1, PMS1, PMS2, MSH6 genes and microsatellite instability of MHL1 or MSH2 and SNPs and combinations thereof.

Ansprüche

1. Verfahren zum Nachweisen von kolorektalem Adenom und/oder kolorektalem Karzinom, umfassend die folgenden Stufen:

a) das Bereitstellen eines isolierten Probenmaterials, das einem Individuum entnommen worden ist,

b) das Bestimmen der Konzentration von C3a oder einem Derivat davon mit der gleichen Funktion im isolierten Probenmaterial und

c) das Vergleichen der bestimmten Konzentration von C3a oder einem Derivat davon mit der gleichen Funktion mit einem oder mehreren Referenzwerten.

2. Verfahren zum Unterscheiden zwischen kolorektalem Adenom und kolorektalem Karzinom, umfassend die folgenden Stufen:

a) das Bereitstellen eines isolierten Probenmaterials, das einem Individuum entnommen worden ist,

b) das Bestimmen der Konzentration von C3a oder einem Derivat davon mit der gleichen Funktion im isolierten Probenmaterial und

c) das Vergleichen der bestimmten Konzentration von C3a oder einem Derivat davon mit der gleichen Funktion mit einem oder mehreren Referenzwerten.

3. Verfahren zum Überwachen der Entwicklung und/oder des Verlaufs und/oder der Behandlung von kolorektalem Adenom und/oder kolorektalem Karzinom, umfassend die folgenden Stufen:

a) das Bereitstellen eines isolierten Probenmaterials, das einem Individuum entnommen worden ist,

b) das Bestimmen der Konzentration von C3a oder einem Derivat davon mit der gleichen Funktion im isolierten Probenmaterial und

c) das Vergleichen der bestimmten Konzentration von C3a oder einem Derivat davon mit der gleichen Funktion mit einem oder mehreren Referenzwerten.

4. Verfahren nach einem der Ansprüche 1 bis 3, wobei die Konzentration von C3a oder einem Derivat davon im Probenmaterial, das einem Patienten mit kolorektalem Adenom oder kolorektalem Karzinom entnommen worden ist, höher ist als in einem Probenmaterial eines gesunden Individuums.

5. Verfahren nach einem der Ansprüche 1 bis 4, wobei ein erster Anstieg der Konzentration von C3a oder einem Derivat davon in einem ersten Probenmaterial ein Indiz für kolorektales Adenom darstellt und wobei ein zweiter Anstieg der Konzentration von C3a oder einem Derivat davon in einem zweiten Probenmaterial, das dem Individuum zu einem späteren Zeitpunkt als das erste Probenmaterial entnommen worden ist, ein Indiz für kolorektales Karzinom darstellt, mit der Maßgabe, dass der zweite Anstieg stärker als der erste Anstieg ist.

6. Verfahren nach einem der Ansprüche 1 bis 5, wobei in Stufe b) ein oder mehr weitere Biomarker zum Nachweis von kolorektalem Adenom und/oder kolorektalem Karzinom im isolierten Probenmaterial bestimmt werden und wobei in Stufe c) die bestimmte Konzentration des oder der Biomarker mit einem oder mehreren entsprechenden Referenzwerten verglichen wird.

7. Verfahren nach einem der Ansprüche 1 bis 6, wobei mindestens ein weiterer Biomarker zum Nachweisen von kolorektalem Adenom und/oder kolorektalem Karzinom gegebenenfalls aus der Gruppe ausgewählt wird, die besteht aus Transthyretin, p53, CEA, CA 19-9, CA 15-3, CA-125, Kras, β-Catenin, Her-2/neu, C-reaktivem Proteinplasma und Derivaten davon und
Mutationen in den E-Cadherin-, MSH2-, MSH3-, MLH1-, PMS1-, PMS2- und MSH6-Genen und
Mikrosatelliten-Instabilität von MHL1 oder MSH2 und
SNPs und Kombinationen davon.

8. Verfahren nach einem der Ansprüche 1 bis 7, wobei der oder die Referenzwerte von C3a und/oder Derivaten davon und gegebenenfalls der oder die Referenzwerte der weiteren Biomarker und/oder Derivate davon als Mittelwert von C3a und/oder einem Derivat davon und gegebenenfalls von einem oder mehreren weiteren Biomarkern und/oder Derivaten davon in einer Mehrzahl von isolierten Proben einer entsprechenden Gruppe von Individuen berechnet werden, wobei es sich bei der Gruppe von Individuen um gesunde Individuen, Patienten mit kolorektalem Adenom und/oder Patienten mit kolorektalem Karzinom handelt.

9. Verfahren nach einem der Ansprüche 1 bis 7, wobei es sich bei den Referenzwerten um individuelle Referenzwerte handelt, die als durchschnittliche Konzentration von C3a und/oder einem Derivat davon und gegebenenfalls von einem oder mehreren weiteren Biomarkern und/oder Derivaten davon berechnet werden, die in einer Mehrzahl von isolierten Probenmaterialien, die dem Individuum im Verlauf einer Zeitspanne entnommen werden, bestimmt werden.

10. Verfahren nach einem der vorstehenden Ansprüche, wobei es sich beim isolierten Probenmaterial um eine Körperflüssigkeit handelt, die optional aus der Gruppe ausgewählt ist, die aus Blut, Blutplasma, Serum, Knochenmark, Stuhl, Synovialflüssigkeit, lymphatischer Flüssigkeit, zerebrospinaler Flüssigkeit, Sputum, Urin, Muttermilch, Sperma, Exsudat und Gemischen davon besteht.

11. Verfahren nach einem der vorstehenden Ansprüche, wobei die Konzentration von C3a und/oder einem Derivat davon und optional von einem oder mehreren weiteren Biomarkern im Probenmaterial durch die DNA-, mRNA-und/oder Proteinkonzentration bestimmt wird.

12. Verfahren nach einem der vorstehenden Ansprüche, wobei die Konzentration von C3a und/oder einem Derivat davon und gegebenenfalls von einem oder mehreren weiteren Biomarkern im Probenmaterial durch eine Nucleinsäure-Hybridisierungstechnik, immunologische Verfahren oder Proteomtechnik und/oder Massenspektroskopie bestimmt wird.

13. Verfahren nach einem der vorstehenden Ansprüche, wobei das Verfahren in Kombination mit anderen diagnostischen Verfahren für kolorektales Adenom und/oder kolorektales Karzinom durchgeführt wird, um die Empfindlichkeit und/oder Spezifität zu erhöhen.

14. Verwendung von C3a oder einem Derivat davon mit der gleichen Funktion als Biomarker zum Nachweisen von kolorektalem Adenom und/oder kolorektalem Karzinom in einer isolierten Probe eines Individuums.

15. Verfahren nach Anspruch 14 zum frühen Nachweisen von kolorektalem Adenom und/oder kolorektalem Karzinom in einer isolierten Probe eines Individuums.

16. Verwendung nach einem der Ansprüche 14 oder 15 in Kombination mit einem oder mehreren weiteren Biomarkern für kolorektales Adenom und/oder kolorektales Karzinom zur Erhöhung der Empfindlichkeit und/oder Spezifität.

17. Verwendung nach Anspruch 16, wobei der mindestens eine weitere Biomarker optional aus der Gruppe ausgewählt wird, die besteht aus Transthyretin, p53, CEA, CA 19-9, CA 15-3, CA-125, Kras, β-Catenin, Her-2/neu, C-reaktivem Proteinplasma und Derivaten davon und
Mutationen in den E-Cadherin-, MSH2-, MSH3-, MLH1-, PMS1-, PMS2- und MSH6-Genen und
Mikrosatelliten-Instabilität von MHL1 oder MSH2 und
SNPs und Kombinationen davon.

18. Immunoassay zum Nachweisen von kolorektalem Adenoma und/oder kolorektalem Karzinom in einem isolierten Probenmaterial eines Individuums, umfassend die folgenden Stufen:

a) ein Antikörper oder ein Rezeptor, der an ein Epitop von C3a oder ein Derivat davon mit der gleichen Funktion bindet,

b) ein fester Träger, der den Antikörper oder Rezeptor trägt und

c) ein Reagenz zum Nachweisen der Bindung des Epitops von C3a oder eines Derivats davon mit der gleichen Funktion an den Antikörper oder Rezeptor,

wobei der Immunoassay einen oder mehrere Antikörper oder Rezeptoren zum Nachweisen von einem oder mehreren weiteren Biomarkern für kolorektales Adenom und/oder kolorektales Karzinom umfasst.

19. Immunoassay nach Anspruch 18, wobei der mindestens eine weitere Biomarker optional aus der Gruppe ausgewählt wird, die besteht aus Transthyretin, p53, CEA, CA 19-9, CA 15-3, CA-125, Kras, β-Catenin, Her-2/neu, C-reaktivem Proteinplasma und Derivaten davon und
Mutationen in den E-Cadherin-, MSH2-, MSH3-, MLH1-, PMS1-, PMS2- und MSH6-Genen und
Mikrosatelliten-Instabilität von MHL1 oder MSH2 und
SNPs und Kombinationen davon.

20. Array, umfassend Nachweismoleküle zum Nachweisen von kolorektalem Adenom und/oder kolorektalem Karzinom in einem Individuum, umfassend als Nachweismolekül:

a) eine Nucleinsäuresonde, die an einem festen Träger immobilisiert ist, um eine für C3a oder einem Derivat mit der gleichen Funktion kodierende mRNA zu binden und nachzuweisen, oder

b) einen Antikörper, der an einem festen Träger immobilisiert ist, um ein Epitop von C3a oder einem Derivat davon mit der gleichen Funktion zu binden und/oder nachzuweisen, oder

c) einen Rezeptor, der an einem festen Träger immobilisiert ist, um ein Epitop von C3a oder einem Derivat davon mit der gleichen Funktion zu binden und nachzuweisen,

wobei vorzugsweise jeweils verschiedene Mengen an Nachweismolekülen am festen Träger immobilisiert werden, um die Genauigkeit der quantitativen Bestimmung zu erhöhen,
wobei das Array einen oder mehrere Antikörper oder Rezeptoren zum Nachweisen von einem oder mehreren weiteren Biomarkern für kolorektales Adenom und/oder kolorektales Karzinom umfasst.

21. Array nach Anspruch 20, wobei der mindestens eine weitere Biomarker optional aus der Gruppe ausgewählt ist, die besteht aus Transthyretin, p53, CEA, CA 19-9, CA 15-3, CA-125, Kras, β-Catenin, Her-2/neu, C-reaktivem Proteinplasma und Derivaten davon und
Mutationen in den E-Cadherin-, MSH2-, MSH3-, MLH1-, PMS1-, PMS2- und MSH6-Genen und
Mikrosatelliten-Instabilität von MHL1 oder MSH2 und
SNPs und Kombinationen davon.

Revendications

1. Procédé permettant de détecter l'adénome colorectal et/ou le carcinome colorectal comprenant les étapes consistant à :

a) fournir un échantillon isolé qui a été prélevé d'un individu,

b) déterminer le taux de C3a ou d'un dérivé de celui-ci ayant la même fonction dans ledit échantillon isolé,

c) comparer le taux déterminé de C3a ou d'un dérivé de celui-ci ayant la même fonction avec une ou plusieurs valeurs de référence.

2. Procédé permettant de faire la distinction entre l'adénome colorectal et le carcinome colorectal comprenant les étapes consistant à :

a) fournir un échantillon isolé qui a été prélevé d'un individu,

b) déterminer le taux de C3a ou d'un dérivé de celui-ci ayant la même fonction dans ledit échantillon isolé,

c) comparer le taux déterminé de C3a ou d'un dérivé de celui-ci ayant la même fonction avec une ou plusieurs valeurs de référence.

3. Procédé permettant de suivre le développement et/ou l'évolution et/ou le traitement de l'adénome colorectal et/ou du carcinome colorectal comprenant les étapes consistant à :

a) fournir un échantillon isolé qui a été prélevé d'un individu,

b) déterminer le taux de C3a ou d'un dérivé de celui-ci ayant la même fonction dans ledit échantillon isolé,

c) comparer le taux déterminé de C3a ou d'un dérivé de celui-ci ayant la même fonction avec une ou plusieurs valeurs de référence.

4. Procédé selon l'une quelconque des revendications 1 à 3, dans lequel le taux de C3a ou d'un dérivé de celui-ci dans ledit échantillon ayant été prélevé d'un malade atteint d'adénome colorectal ou de carcinome colorectal est supérieur par rapport à un échantillon d'un individu sain.

5. Procédé selon l'une quelconque des revendications 1 à 4, dans lequel une première augmentation du taux de C3a ou d'un dérivé de celui-ci dans un premier échantillon est indicative d'adénome colorectal et dans lequel une seconde augmentation du taux de C3a ou d'un dérivé de celui-ci dans un second échantillon, isolé dudit individu à un stade ultérieur que ledit premier échantillon, est indicative de carcinome colorectal, à condition que ladite seconde augmentation soit plus intense que ladite première augmentation.

6. Procédé selon l'une quelconque des revendications 1 à 5, dans lequel à l'étape (b) un ou plusieurs autre(s) biomarqueur(s) servant à détecter l'adénome colorectal et/ou le carcinome colorectal est/sont déterminé(s) dans ledit échantillon isolé et dans lequel à l'étape (c) le taux déterminé dudit (desdits) biomarqueur(s) est/sont comparé(s) avec une ou plusieurs valeurs de référence correspondantes.

7. Procédé selon l'une quelconque des revendications 1 à 6, dans lequel au moins un autre biomarqueur servant à détecter l'adénome colorectal et/ou le carcinome colorectal est éventuellement choisi dans le groupe formé par la transthyrétine, la p53, le CEA, le CA 19-9, le CA 15-3, le CA-125, Kras, la caténine β, Her-2/neu, du plasma contenant la protéine C réactive et des dérivés de ceux-ci et
des mutations dans les gènes de la cadhérine E, de MSH2, MSH3, MLH1, PMS1, PMS2, MSH6
et
l'instabilité des microsatellites de MHL1 ou MSH2 et
les SNP et des combinaisons de ceux-ci.

8. Procédé selon l'une quelconque des revendications 1 à 7, dans lequel la (les) valeur(s) de référence de C3a et/ou du (des) dérivé(s) de celui-ci et éventuellement la (les) valeur(s) de référence du (des) autre(s) biomarqueur(s) et/ou du (des) dérivé(s) de celui-ci (ceux-ci) est/sont calculé(s) sous la forme du taux moyen de C3a et/ou d'un dérivé de celui-ci et éventuellement d'un autre (d'autres) biomarqueur(s) et/ou dérivé(s) de celui-ci (ceux-ci) dans une pluralité d'échantillons isolés d'un groupe correspondant d'individus, dans lequel le groupe d'individus consiste en des individus sains, des malades atteints d'adénome colorectal et/ou de malades atteints de carcinome colorectal.

9. Procédé selon l'une quelconque des revendications 1 à 7, dans lequel la (les) valeur(s) de référence est/sont une (des) valeur(s) de référence individuelle(s) calculée(s) sous la forme du taux moyen de C3a et/ou d'un dérivé de celui-ci et éventuellement d'un autre (d'autres) biomarqueur(s) et/ou dérivé(s) de celui-ci (ceux-ci) déterminés dans une pluralité d'échantillons isolés prélevés dudit individu sur un intervalle de temps.

10. Procédé selon l'une quelconque des revendications précédentes, dans lequel l'échantillon isolé est un liquide biologique et est éventuellement choisi dans le groupe formé par le sang, le plasma sanguin, le sérum, la moelle osseuse, les selles, le liquide synovial, le liquide lymphatique, le liquide céphalorachidien, les expectorations, l'urine, le lait maternel, le sperme, les exsudats et des mélanges de ceux-ci.

11. Procédé selon l'une quelconque des revendications précédentes, dans lequel le taux de C3a et/ou d'un dérivé de celui-ci et éventuellement d'un autre (d'autres) biomarqueur(s) dans ledit échantillon est déterminé au niveau de l'ADN, de l'ARNm et/ou protéique.

12. Procédé selon l'une quelconque des revendications précédentes, dans lequel le(s) taux de C3a et/ou d'un dérivé de celui-ci et éventuellement d'un autre (d'autres) biomarqueur(s) dans ledit échantillon est (sont) déterminé(s) au moyen d'une technique d'hybridation d'acides nucléiques, de procédés immunologiques ou d'une technique propre à la protéomique, et/ou par spectroscopie de masse.

13. Procédé selon l'une quelconque des revendications précédentes, dans lequel le procédé est exécuté en combinaison avec d'autres procédés diagnostiques pour l'adénome colorectal et/ou le carcinome colorectal afin d'accroître la sensibilité et/ou la spécificité.

14. Utilisation de C3a ou d'un dérivé de celui-ci ayant la même fonction en tant que biomarqueur pour la détection de l'adénome colorectal et/ou du carcinome colorectal dans un échantillon isolé chez un individu.

15. Utilisation selon la revendication 14 pour une détection précoce de l'adénome colorectal et/ou du carcinome colorectal dans un échantillon isolé d'un individu.

16. Utilisation selon l'une quelconque des revendications 14 ou 15, en combinaison avec un ou plusieurs autres biomarqueurs de l'adénome colorectal et/ou du carcinome colorectal afin d'accroître la sensibilité et/ou la spécificité.

17. Utilisation selon la revendication 16, dans laquelle au moins un autre biomarqueur est éventuellement choisi dans le groupe formé par la transthyrétine, la p53, le CEA, le CA 19-9, le CA 15-3, le CA-125, Kras, la caténine β, Her-2/neu, du plasma contenant la protéine C réactive et des dérivés de ceux-ci et
des mutations dans les gènes de la cadhérine E, de MSH2, MSH3, MLH1, PMS1, PMS2, MSH6
et
l'instabilité des microsatellites de MHL1 ou MSH2 et
les SNP et des combinaisons de ceux-ci.

18. Dosage immunologique servant à détecter l'adénome colorectal et/ou le carcinome colorectal dans un échantillon isolé d'un individu comprenant :

a) un anticorps ou un récepteur se liant à un épitope de C3a ou d'un dérivé de celui-ci ayant la même fonction,

b) un support solide qui supporte ledit anticorps ou récepteur,

c) un réactif permettant de détecter la liaison dudit épitope de C3a ou d'un dérivé de celui-ci ayant la même fonction audit anticorps ou récepteur,

ledit dosage immunologique comprenant un ou plusieurs anticorps ou récepteurs permettant de détecter un ou plusieurs autres biomarqueurs de l'adénome colorectal et/ou du carcinome colorectal.

19. Dosage immunologique selon la revendication 18, dans lequel au moins un autre biomarqueur est éventuellement choisi dans le groupe formé par la transthyrétine, la p53, le CEA, le CA 19-9, le CA 15-3, le CA-125, Kras, la caténine β, Her-2/neu, du plasma contenant la protéine C réactive et des dérivés de ceux-ci et
des mutations dans les gènes de la cadhérine E, de MSH2, MSH3, MLH1, PMS1, PMS2, MSH6
et
l'instabilité des microsatellites de MHL1 ou MSH2 et
les SNP et des combinaisons de ceux-ci.

20. Puce comprenant des molécules de détection servant à détecter l'adénome colorectal et/ou le carcinome colorectal chez un individu comportant en guise de molécule de détection :

a) une sonde d'acide nucléique immobilisée sur un support solide pour la liaison à, et la détection de, ARNm codant pour C3a ou un dérivé de celui-ci ayant la même fonction, ou

b) un anticorps immobilisé sur un support solide pour la liaison à, et la détection de, un épitope de C3a ou d'un dérivé de celui-ci ayant la même fonction, ou

c) un récepteur immobilisé sur un support solide pour la liaison à, et la détection de, un épitope de C3a ou d'un dérivé de celui-ci ayant la même fonction,

dans laquelle, de préférence, des quantités différentes de chaque molécule de détection sont immobilisées sur le support solide afin d'accroître la précision de la quantification,
ladite puce comprenant un ou plusieurs anticorps ou récepteurs permettant de détecter un ou plusieurs autres biomarqueurs de l'adénome colorectal et/ou du carcinome colorectal.

21. Puce selon la revendication 20, dans laquelle au moins un autre biomarqueur est éventuellement choisi dans le groupe formé par la transthyrétine, la p53, le CEA, le CA 19-9, le CA 15-3, le CA-125, Kras, la caténine β, Her-2/neu, du plasma contenant la protéine C réactive et des dérivés de ceux-ci et
des mutations dans les gènes de la cadhérine E, de MSH2, MSH3, MLH1, PMS1, PMS2, MSH6
et
l'instabilité des microsatellites de MHL1 ou MSH2 et
les SNP et des combinaisons de ceux-ci.

Drawing