KR20070051327A - Use of transthyretin as a biomarker for colorectal adenoma and/or carcinoma; method for detection and test system - Google Patents

Abstract

The present invention provides a method for preparing a test sample comprising: a) providing an isolated sample material, obtained from an individual, b) measuring the level of transcyretin in the separated sample material, c) adjusting the reference value and the measured level of transcyretin. Instructions for detecting colorectal adenomas and / or colorectal carcinoma comprising the step of comparing. The present invention also directs methods for the treatment of colorectal adenomas and / or colorectal carcinoma and / or methods of monitoring the progression of colorectal adenomas and / or colorectal carcinoma, as well as methods for identifying between colorectal adenomas and colorectal carcinoma. In addition, the present invention directs inspection methods and arrays for use in the method. The present invention also directs the use of transcyretin as a biomarker for the detection of colorectal adenomas and / or colorectal carcinoma in a subject.

Description

Use of transcyretin as a biomarker for colorectal adenomas and / or carcinomas; methods for detection and screening systems

The present invention relates to the field of detection of colorectal adenomas and / or colorectal carcinoma.

Colorectal carcinoma is the third most frequently diagnosed carcinoma and the second most common cause of death from carcinoma in Europe and the United States. In 1996, an estimated 133500 new cases of colorectal carcinoma were diagnosed in the United States, with approximately 54900 people dying from the disease. The incidence of colorectal carcinoma is increasing, while the mortality rate of colorectal carcinoma is decreasing. The incidence of colon carcinoma increases with age, starting at about 40 years and higher in men than in women (60.4 vs female 40.9 for 100,000 people per year).

In most patients, progression of colorectal carcinoma involves multi-step progression from premalignant adenoma to invasive malignancies with metastasis propensity. Reduction of colorectal carcinoma morbidity and mortality can be achieved through the detection and treatment of early colorectal carcinoma and the identification and removal of colorectal adenomatous polyps, which are precursors of colorectal carcinoma.

Colorectal screening tests have been shown to make accurate detection of precursors of early colorectal carcinoma, namely adenomatous polypes and / or flat neoplastic areas. Several screening tests are available to screen for colorectal carcinoma. The screening tests include faecal occult blood testing (FOBT), flexible sigmoidoscopy, FOBT in combination with flexible colonoscopy and colonoscopy. The various screening tests differ from each other in terms of performance, validity, possible screening frequency, test complications, costs and patient acceptance.

Screening by fecal occult blood test is generally considered to be an optimal screening method in terms of cost effectiveness. Fecal occult blood is a chemical such as guiac and can be detected through hemeporphyrin or immunological methods.

The guaiac slide test Hemoccult (II), available from SmithKline Diagnostics, is the most widely used.

Several technical factors affect his clinical performance. Hemoccult has about 50% sensitivity and about 98% specificity for colorectal carcinoma, but the sensitivity is about 10% lower for polypes (Simon JB. (1998) Gastroenterologist 6: 66-78.Review). Another significant drawback of occult blood screening is poor predictive accuracy, with only about 10% of the positive responses demonstrated by 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).

In addition, fecal occult blood test provides results only after the disease has progressed to a certain stage. It will be desirable to have an inspection system that allows for the detection of colorectal adenomas and / or colorectal carcinoma at the earliest point in time.

Although relatively recently developed immunological tests generally have high sensitivity, low specificity remains an important problem. Other methods, such as genetic testing of fecal samples for the KRAS oncogene and p53 protein, are not yet cost effective (Calistri D et al. (2003) Clin. Gastroenterol. Hepatol. 1: 377-383; Schoen RE (2002). Nat. Rev. Cancer 2: 65-70).

Endoscopy (Kavanagh AM (1998) Cancer Causes Control 9: 455-462), using either flexible sinus or colonoscopy (Lieberman DA (1997) Gastroenterol. Clin. N. Am. 26: 71-83) It is the clearest means of detection, but it has limitations. False negative rates for flat neoplastic lesions have been recognized, which is still high (Kudo S (1997) Gastrointest. Endosc. Clin. N. Am. 7: 87-98.) . Colonoscopy has a low false negative rate for polyp lesions of at least 10 mm diameter when examined. For this reason, the allowable interval from negative judgment to retest (up to 10 years), or from polypectomy to retest, is relatively long, up to 5 years.

However, the patient does not follow the recommendation of reexamination after colonoscopy. In addition, colonoscopy is expensive and cumbersome. Because of the high cost of general medical examination and people's poor acceptance of colonoscopy, the medical examination method is limited.

Collected, isolated tissue samples can be examined for colorectal carcinoma and rectal colon adenomas, which are precursors thereof, in a number of ways. German Patent Publication DE 197 11 111A discloses a method using in vitro measurement of intulapithelial colon bacteria, components and their reaction products. Another method of using HERG gene expression in tissue samples is disclosed in DE 102 24 534.

It is an object of the present invention to provide a means to allow for the initial detection of colon adenomas and / or colon carcinomas.

Another object is to provide biomarkers that can be used for the detection of colorectal adenomas and / or carcinomas.

It is yet another object of the present invention to provide an inspection system for the detection of colorectal adenomas or carcinomas that can be widely used at an efficient cost.

In addition, the test system should be easy to handle and more convenient for the subject to be tested for colorectal adenomas and / or carcinomas.

The object underlying the present invention is solved by the use of transcyretin as a biomarker for the detection of colorectal adenomas and / or colorectal carcinoma in a subject. The detection can be performed in vivo and in vitro. According to a preferred embodiment, the detection is carried out in vitro.

This object is also solved by a method of detecting colorectal adenomas and / or colorectal carcinoma comprising the steps of:

a) providing an isolated sample material obtained from the subject,

b) measuring transcyretin levels in the separated sample material,

c) comparing the reference value with the measured transcyretin level.

This object is also solved by a method of identifying between colorectal adenomas and colorectal carcinomas comprising the following steps:

a) providing an isolated sample material obtained from the subject,

b) measuring transcyretin levels in the separated sample material,

c) comparing the reference value with the measured transcyretin level.

The object is also solved by treatment of colorectal adenomas and / or colorectal carcinomas, and course monitoring methods of colorectal adenomas and / or colorectal carcinomas comprising the following steps:

a) providing an isolated sample material obtained from the subject,

b) measuring transcyretin levels in the separated sample material,

c) comparing the reference value with the measured transcyretin level.

In addition, this object is solved by the provision of an inspection system for the detection of colorectal adenomas and / or colorectal carcinoma in a subject comprising:

a) an antibody or receptor that binds to an epitope of transcyretin,

b) a solid support for supporting said antibody or receptor,

c) a reagent for detecting binding of an epitope of said transcyretin to said antibody or receptor.

Furthermore, this object is solved by an array facility comprising detection molecules for the detection of colorectal adenomas and / or colorectal carcinoma in a subject, including as detection molecules,

a) nucleic acid probes immobilized on a solid support for binding to and encoding mRNA encoding transcyretin,

b) an antibody immobilized on a solid support to bind to and detect an epitope of transcyretin, or

c) receptors immobilized on a solid support to bind to and detect epitopes of transcyretin

And, preferably, different amounts of detection molecules are immobilized on the solid support for increased quantitative accuracy.

Preferred embodiments are specified in the independent claims.

According to the present invention, the term "biomarker" refers to a protein or protein fragment which indicates the development of colorectal adenomas and / or colorectal carcinoma. This means that "biomarkers" are used as detection agents or detection molecules.

The term "individual" or "individuals" refers to a mammal. Preferably, the mammal is a human, such as a patient.

The term "healthy individual" or "healthy individuals" refers to designating the individual (s) who are not suffering from colorectal adenomas and / or colorectal carcinoma diseases. That is, the term "health individual (s)" is used only with respect to the pathological condition of colorectal adenomas and / or colorectal carcinoma and does not exclude individuals suffering from diseases other than colorectal adenomas and / or colorectal carcinoma.

In addition, the term "transthyretin" as used herein refers to truncated transcyretin, transcyretin fragments, mutated transcyretin or modified transcytes. Contains retin. The modification of "transcyretin" may be due to enzymatic or chemical modification. In addition, the term "transcyretin" is also used to denote the monomeric or multimeric form of transcyretin. For example, the term “transcyretin” in particular encompasses monomeric protein chains that are part of transcyretin, which is usually a homotrameric protein.

The term “epitope” refers to an antibody, antibody fragment, protein or peptide structure, or any structural element of transcyretin that allows specific binding to a receptor.

The sample material separation process is not part of the present invention. That is, the method of the present invention is carried out with already separated sample material such as body fluids. For example, the sample material to be tested can be stored in a freezer and the method of the invention can be carried out at an appropriate time after thawing of each sample material.

Surprisingly, it has been found by the inventors that the transcyretin protein can be used as a biomarker for the detection of colorectal adenomas and / or carcinomas.

Transcyretin is also called prealbumin. Transcyretin, a tetrameric protein, has a molecular weight of about 54 kDa and is mainly synthesized in the liver. Transcyretin consists of four protein chains, which may be identical, of about 14 kDa. That is, transcyretin can be considered a homo tetrameric protein. It basically functions as a binding protein for thyroxin and as a carrier protein for retinal binding protein, which is in turn a transporter of vitamin A (Schussler, GC Thyroid. (2000), 10: 372 Hamilton, JA and Benson, MD Cell of Life Sci. (2001) 58: 1491-1521).

In addition, transcyretin is reported to form a complex with amyloid β-protein. Aggregation of amyloid beta-proteins is associated with amyloid deposition in the brain, a pathological finding of Alzheimer's disease.

Transcyretin is also called an indicator of nutritional status because serum transcyretin levels decline with inadequate protein intake and during acute or chronic inflammation. In addition, transcyretin is suitable for monitoring nutritional status because of its short half-life of only 2-3 days (Pleban WE, Conn. Med. (1989) 53: 405-407; Bernstein LH and lngenbleek Y, Clin. Chem. Lab. Med. (2002) 40: 1344-1348).

WO 02/059621 discloses reagents that regulate transcyretin and reagents that bind to transcyretin gene products, thereby preventing, ameliorating, or correcting obesity and related dysfunctions. To start.

The monomeric transcyretin protein chain in human plasma or serum appeared as two variants with molecular weights of at least 13.736 Da and 13.858 Da, respectively, indicated by SELDI (surface enhanced laser desorption / ionisation) analysis.

These different molecular weights are believed to be due to several post-translational modifications such as cleavage, cysteinylation and / or glutationylation. Cysteinylated transcyretin has a molecular weight of about 13.871 ± 11 Da, and glutathionylated transcyretin has a molecular weight of about 14.069 ± 5 Da.

WO 03/001182 describes the detection and isolation of homocysteinylated transcyretin from biological body fluids and its use as a marker for the diagnosis of homocysteinemia, hyperhomocysteinemia and related diseases. Discuss the use.

In some studies, transcyretin has been discussed as a diagnostic marker for carcinoma. Suresh et al. (J. Clin. Pathol. (1991) 44: 573-575) are bronchopulmonary carcinoid because 75% of carcinoid tumors are positive for transcyretin. Transcyretin is a useful marker in tumors. In another study, transcyretin has been discussed as a prognostic marker in uterine epidermal carcinoma (Mahlck et al., Gynecol. Obstet. Invest. (1994) 37: 135-140).

Surprisingly, the inventors have now found that the level of transcyretin in the body fluid correlates with the development of colorectal adenomas and / or carcinomas in the individual.

According to the present invention, the transcyretin is composed of blood, blood plasma, blood serum, bone marrow, stool, synovial fluid, lymphatic fluid, cerebrospinal fluid (cerebrospinal fluid), sputum, urine, mother milk, sperm, exudates and mixtures thereof.

Preferably, the body fluid is separated before carrying out the method of the present invention. Preferably, the method of the present invention is performed in vitro by a technician in a laboratory.

According to a preferred embodiment of the invention, transcyretin is measured in plasma or serum. Serum can be readily obtained by taking blood from an individual to undergo medical examination and separating the supernatant from the coagulated blood.

Transcyretin levels in body fluids, preferably serum, decrease with progressive formation of colorectal adenomas. Rectal colon adenomas are benign neoplasms and can turn malignant. When developing from colorectal adenomas to colorectal cancer, transcyretin levels in body fluids, preferably serum, are further reduced.

After transformation from colorectal adenomas to colorectal cancer, the pathological state of the suffering individual can be further exacerbated by metastatic formation.

The present invention provides early biomarkers that enable the detection of neoplastic disease in early and still benign phases. Early detection allows the surgeon to remove colonic adenomas in a timely manner and dramatically increases the survival of the individual.

In addition, the present invention makes it possible to monitor transcyretin levels in body fluids such as serum over long periods of time, such as years.

Long-term monitoring helps to distinguish between colorectal adenomas and colorectal carcinoma. Transcyretin levels can be checked regularly, for example once or twice a year. If a decrease in transcyretin levels is found, it may indicate a colorectal adenomas. Thereafter, further decreases in transcyretin levels may indicate a transition to malignant colorectal carcinoma.

In addition, the course of the disease and / or treatment can be monitored. If the transcyretin levels are further reduced, for example after removal of colorectal adenomas, this may indicate a worsening of the pathology.

This means that transcyretin levels are useful clinical parameters for the detection and / or monitoring of colorectal adenomas and / or colorectal carcinomas. Transcyretin levels in body fluids decrease very early after the development of colorectal adenomas. Thus, transcyretin or transcyretin levels are important clinical indicators that enable early diagnosis and, as a result, early treatment of the disease.

The method of the present invention for the detection of colorectal adenomas and / or colorectal carcinoma comprises the steps of providing an isolated sample material obtained from an individual, then measuring transcyretin levels of the isolated sample material, and finally Comparing the measured transcyretin level with a reference value.

The reference value can be estimated as the mean value of transcyretin measured in separate samples of multiple healthy individuals. This reference value is established as a range that is considered normal, meaning that a person is healthy. The drop below this range may then indicate a pathological condition of colorectal adenomas and / or colorectal carcinoma. The range of reference values can be established by taking a statistically relevant number of bodily fluid samples, such as serum samples, of a healthy individual, for example, as established in other medical indicator ranges such as blood sugar. .

In another embodiment of the invention, the reference value may be an individual reference value calculated as the mean value of transcyretin measured in a plurality of separated samples obtained from the individual over a period of time.

Individual standard values can be established when monitoring transcyretin levels over long periods of time, such as months or years. For example, transcyretin levels can be measured from the same serum sample as in blood glucose measurements, in particular to establish a calibration curve that allows for detection of any individual decrease in transcyretin levels. Can be used.

In another aspect, the present invention also provides a test system for detecting colorectal adenomas and / or colorectal carcinoma in a subject. The test system is based on either the specificity of the receptor or antibody to bind to the transcyretin fragment or epitope of the transcyretin or a suitable structural element. The receptor can be any structure that can specifically bind to transcyretin. For example, the receptor may be an antibody fragment, such as a Fab or F (ab ′) ₂ fragment, any other protein or peptide structure capable of specifically binding to transcyretin.

The antibody, antibody fragment or receptor binds to a solid support, such as a plastic surface or bead, which allows for the binding and detection of transcyretin. For example, conventional microtiter plates can be used as the plastic surface. Binding detection of transcyretin can be accomplished, for example, by the use of secondary antibodies labeled with a detectable group. Detectable groups are, for example, radioactive isotope, enzymatic horseradish peroxidase, which can be detected by adding a suitable substrate to generate a color or fluorescent signal, for example. enzyme like horseradish peroxidase) or alkaline phosphatase.

The test system may be an immunoassay, such as an enzyme-linked immunosorbentassay (ELISA) or a radio immunoassay (RIA). However, antibody fragments such as Western blotting or immuno precipitation or any other immunological assay system that uses the specificity of the antibody can be used.

The present invention also provides an array comprising detection molecules for detecting colorectal adenomas and / or colorectal carcinoma in a subject, wherein said detection molecules are transcyretin, fragments, mutations. A nucleic acid probe immobilized on a solid support for binding to and detecting a variant or derivative thereof, or an antibody immobilized on a solid support for binding to and detecting an epitope of transcyretin It may be a receptor immobilized on a solid support to bind to and detect an epitope of retin.

The nucleic acid probe can be any naturally occurring oligonucleotide or synthetic oligonucleotide, as well as cDNA, cRNA, and the like.

The described array can be used in the inspection system of the present invention. The array may be either a micro array or a macro array. The support may be ceramic or silicon, for example an inorganic material such as a silicon wafer, a polymeric material such as plastic or nylon. According to a preferred embodiment, glass (SiO ₂ ) is used as the solid support material. The glass may be a glass plate or a glass chip. According to another embodiment of the invention, the glass substrate has an atomically flat surface.

For example, the array may comprise an immobilized nucleic acid probe capable of specifically binding to mRNA of transcyretin present in body fluids such as serum. Another preferred embodiment produces cDNA by reverse transcription of transcyretin mRNA, and specifically detects each cDNA amount in the array. The array technology is known to those skilled in the art. Quantification of the measured mRNA or cDNA can be achieved by comparing the measured values with standard or calibration curves of known amounts of transcyretin mRNA or cDNA, respectively.

Preferably, different amounts of detection molecules are each immobilized on a solid support, allowing for accurate quantification of transcyretin levels.

According to another embodiment of the present invention, the transcyretin level is measured by mass spectroscopy.

Mass spectrometry allows the detection of transcyretin or its monomeric protein chains specifically through their molecular weight and makes it very easy to quantify the amount of transcyretin.

Transcyretin is a homologous tetramer, usually containing four protein chains, each having a molecular weight of about 14 kDa.

We used mass spectroscopy to detect several variants of the transcyretin protein chain, particularly with molecular weights of 13.776 Da, 13.884 Da or 14.103 Da.

The inventors have found that levels of molecular modifications of transcyretin with molecular weights of 13.776 Da and 13.884 Da are reduced, especially in body fluids such as in the case of colorectal adenomas and / or colorectal carcinomas.

Any suitable ionization method in the art of mass spectroscopy known in the art can be used to ionize transcyretin molecules, fragments, mutations, modifications or derivatives thereof. The ionization methods include electron impact (El), chemical ionization (Cl), field ionization (FDI), electrospray ionization (ESI), laser desorption ionization (laser) desorption ionization (LDI), matrix assisted laser desorption ionization (MALDI), and surface enhanced laser desorption ionization (SELDI).

Any suitable detection method in the field of mass spectroscopy known in the art can be used to determine the molecular mass of a transcyretin, fragment, mutation, modification or derivative thereof. The detection methods include quadrupole mass spectroscopy (QMS), Fourier transform mass spectroscopy (FT-MS) and time-of-flight mass spectroscopy (TOF-MS). ).

Preferably, the mass spectroscopy is surface enhanced laser desorption ionization- time of flight-mass spectroscopy (SELDI-TOF-MS). Preferably, before performing SELDI-TOF-MS, the transcyretin in the separated sample is immobilized on a chip or solid support having an activated surface. The activated surface preferably comprises an immobilized, for example anti-transcyretin antibody, such as a rabbit polyclonal anti-transcyretin antibody. After binding of transcyretin to the antibody, in SELDI-TOF mass spectroscopy, a time-of-flight analysis is carried that carries signal strength for the measurement of transcyretin levels.

In addition, mass spectrometry can simultaneously detect other proteins that may be relevant for the detection of colorectal adenomas and / or colorectal cancer.

In another embodiment of the invention, the detection sensitivity and / or specificity of colorectal adenomas and / or colorectal carcinoma is enhanced by detecting other proteins in conjunction with transcyretin.

In addition to transcyretin levels, one can measure the level of another biomarker of colorectal adenomas and / or colorectal carcinoma, the other biomarkers comprising polypeptides or proteins having a molecular weight of 8.960 Da. The level of said 8.960 Da protein or polypeptide in the sample material has been found to be increased in colorectal adenomas and / or colorectal carcinoma compared to the levels of said protein or polypeptide in samples isolated from healthy individuals.

Therefore, in mass spectrometry, in addition to the level of transcyretin in body fluids such as serum, it is preferable to also measure the value of the protein having a molecular weight of 8960 ± 9 Da. Mass spectrometry is an optional method for detecting and quantifying the 8960 ± 9 Da protein levels.

In another embodiment of the present invention, detecting CEA (carcinoembryonic antigen) and / or CA 19-9 in combination with transcyretin, the sensitivity of detecting colorectal adenomas and / or colorectal carcinoma and / or Or specificity is improved. CEA and CA 19-9 are analyzed both as response indicators and prognostic factors in advanced colorectal carcinoma.

In another embodiment of the invention, detecting CA15-3, CA-125 and / or Her-2 / neu in conjunction with transcyretin, the sensitivity and / or sensitivity of detecting colorectal adenomas and / or colorectal carcinoma Specificity is improved. 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, primarily for breast cancer and also for visceral metastasis. Amplification of Her-2 / neu in breast carcinoma is associated with poor prognosis, short disease-free interval and short survival time. To date, little is known about the origin of Her-2 / neu amplification and progression.

The methods of the present invention can be performed in conjunction with other diagnostic methods for the detection of colorectal adenomas and / or colorectal carcinoma to improve overall sensitivity and / or specificity.

Preferably, the method of the invention is carried out as an initial detection and / or monitoring method. If the results of the method of the invention should direct the development of colorectal adenomas and / or colorectal carcinoma, further examinations such as colonoscopy and fecal occult blood examination (FOBT) should be performed.

The following polyclonal anti-transcyretin antibodies can be used in practicing the present invention: from PC 066 and DAKO (Hamburg, Germany), available from The Binding Site Ltd., Birmingham, England. A 0002 available.

The following figures and examples are given solely for illustrative purposes. The invention is not intended to be limited to the following examples.

1 shows a schematic for the fractionating and profiling of serum samples.

2 shows a SELDI-TOF profile.

3 shows the difference between adenomas / carcinoma individuals and healthy individuals.

4 shows the SELDI-TOF protein profile of serum fractions from three different individuals.

5 shows SELDI immunoaffinity capture of transcyretin and its potential derivatives.

6 shows immunoprecipitation with anti-transcyretin antibodies.

If not stated otherwise, all methods were performed according to the protocol of the analytical system manufacturer.

Serum collection and serum splitting ( Serum collection and serum fractionation )

Serum was collected and studied from three groups of human patients.

Group 1 consisted of 30 patients who were surgically treated for non-cancerous diseases such as inuinal hernia, gallbladder stones or diverticulitis. The individuals in group 1 were considered healthy populations, ie, those who did not have colorectal adenomas and / or colorectal carcinoma.

Both groups consisted of 29 patients who were surgically treated for undefined tumors that eventually became benign rectal adenomas.

Group 3 consisted of 28 patients with colorectal carcinoma. These 28 patients were all suffering from colorectal carcinoma in TNM stage Ⅲ (Tumor (T umor), Section (N ode), the transition phase (M etastasis stage) Ⅲ).

Ethical guidelines and patient confidentiality were strictly guaranteed, and all patients made informed consent to participate in this study. All patients received similar preoperative procedures such as fasting time and premedication.

Serum from each patient was subjected to anion exchange chromatography using a 96 well format automation approach (Biomek2000, Ciphergen) according to the manufacturer's protocol to partially reduce interference by most very large proteins. Serum split kit / Q HyperD resin, Ciphergen Biosystems, Inc.). As shown in FIG. 1, the cleavage produces six fractions containing roughly separated protein based on protein pl-value.

From eight patients, all fractions were examined using chromatographic surface ProteinChip ^® arrays from Ciphergen Biosystems, Inc. The array provides for the combination of a wide variety of proteins and peptides for relatively high doses, a wide variety of protein profiling and peptide mapping applications. Six serum fractions from eight patients were applied to three different protein arrays: SAX-2, a strong anion array with four quarternary amine functionality, IMAC, an immobilized metal with nitriloacetic acid surface Weak cation exchange array with immobilized metal capture array and CM10, carboxylate functionality. Two fractions (fraction 1, fraction 4) representing the largest number of evaluable peaks were selected for the on-chip profiling approach, which showed the lowest data variability (CM 10 array). The two fractions in all three groups were measured using Cyphergen ProteinChip ^® Array CM 10.

Cypherzen Protein Chip (Registered trademark) array preparation and SELDI - TOF - MS  analysis

The CM 10 protein array is processed in a bioprocessor (bioprocessor, Ciphergen Biosystems, Inc.) according to the manufacturer's protocol. The chip is equilibrated with CM10 binding buffer solution (Ciphergen Biosystems, Inc.) for 2x5 minutes and then incubated with serum fraction diluted 1:10 in CM10 binding buffer solution. After 45 minutes, unbound material is removed and the chip is washed three times with CM 10 binding buffer solution and twice with water. After drying at room temperature for 10 minutes, 0.05 M sinapinic acid (1.0 μl) was added twice (2 applications) and the chip was analyzed with a Ciphergen Protein ChipReader (model PBSII). .

Protein chip readers are time-of-flight mass spectroscopy. For further detailed analysis by the ProteinChip Data Analysis Program and the Biomarker Wizard Program, the mass values and signal intensities of the detected proteins are transferred to software supplied by Cyphergen.

In order to minimize data variability, measurements are performed within two days with samples from all patient groups randomly distributed on the chip. As a standard control for normalization, pooled normal serum was used in parallel for all measurements.

Mass spectra of the protein were generated using an average of 195 laser shots at a laser intensity of 185. The detector was run at sensitivity 7. For data acquisition, the detection size range was set between 2.000 and 40.000 Da. The laser focused at 10.000 Da. The data were analyzed by protein chip data analysis program (version 3.1, Ciphergen Biosystems) and biomarker wizard program (version 3.1, Ciphergen Biosystems). The peak intensity was normalized to the total ion current.

result

In this experiment, differently expressed proteins were identified between the sera of non-carcinoma patients (group 1) and the sera of patients with colorectal adenomas (group 2) and the patients with colorectal carcinoma (group 3). As shown in Table 1, six peaks in fraction 1 were identified, which represented a mean value (p <0.0000001) of very markedly different intensities between healthy patients (non-carcinoma patients), colorectal adenocarcinoma patients and colorectal carcinoma patients. Indicated. As shown in Table 2, Fraction 4 provides an additional set of potential biomarkers, significantly different between the three groups (p <0.0000001).

TABLE 1 Biomarkers selected after treatment of Fraction 1 of each serum sample on Ciphergen chip. p-values were generated as nonparametric tests from the Biomarker Wizzard application. N represents transcyretin levels in 'health' patients, A in colorectal adenomas, and Ca in colorectal carcinoma. StDev is the standard deviation of the measurement.

TABLE 2 Biomarkers selected after treatment of Fraction 4 of each serum sample on Cypergen chip. p-values were generated by parametric testing from the Biomarker Wizard application. N represents transcyretin levels in 'health' patients, A in colorectal adenomas, and Ca in colorectal carcinoma. StDev is the standard deviation of the measurement.

Several proteins were tested as a single biomarker and also in collaboration with other markers using the Biomarker Pattern Software. One protein with a molecular weight of 13.770 Da (p13.770) was identified with 75% sensitivity and 90% specificity between adenocarcinoma patients and health controls.

2 shows the SELDI profile demonstrating biomarkers. 3 shows the distinction of healthy patients from adenomas / carcinoma patients on a scatter plot. The intensity of biomarkers with a molecular weight of 13770 Da (p13.370) is significantly lower in the serum of adenomas / carcinoma patients than in healthy patients.

4 shows the SELDI-TOF protein profile of serum fraction 4 from healthy patients, adenomas patients and cancer patients. As can be seen, the intensity of the p13.370 peak is significantly reduced from healthy patients to rectal colon adenomas and to colorectal cancer patients.

In conjunction with the second marker protein (MW 8.960 Da, Fraction 1), the isolation of healthy patients from adenocarcinoma patients was markedly increased. Twenty patients were correctly isolated from twenty thirty non-adenomatous / carcinoma (health) patients, twenty nine patients with colorectal adenocarcinoma, as well as twenty eight patients with colorectal carcinoma. With this combination, 70% sensitivity and 100% specificity were achieved.

Transcyretin  discrimination

Using immunoaffinity capture and immunoprecipitation test, 13.770 Da protein was identified as transcyretin.

Transcyretin  Protein Chip Immunoaffinity Capture

Immunity capture was performed using Protein G Array (Ciphergen Biosystems, Inc.) following the manufacturer's protocol. Rabbit polyclonal anti-transcyretin antibody A0002 (Dako, Hamburg, Germany) was diluted to a concentration of 0.2 μg / μl in PBS. 2 μl per spot was pipetted onto the array and then incubated for 24 hours in a 4 ° C. wet room. Unbound antibody was removed by washing with wash buffer solution (0.5% (v / v) Triton X-100 in PBS). After drying, the array was inserted into a bioprocessor (Ciphergen Biosystems, Inc.) for sample incubation. 2 [mu] l of serum fraction (large colorectal adenocarcinoma patient versus healthy patient) containing large and small amounts of 13.770 Da protein was added to the chip and incubated in a horizontal shaker for 1 hour at room temperature. After washing with PBS and HEPES buffer solution, the array was air dried for 10 minutes and 0.6 μl of saturated cinafinic acid matrix was added to each spot. The array was measured using the following parameters: laser intensity set to 185 and sensitivity set to 7. The detection size range was set between 200 and 200.000 Da and the laser focused at 10000 Da.

After binding to the antibody-coated array, four protein peaks of different molecular weights (about 13.761 Da, 13.914 Da, 14.102 Da and 14.292 Da) were identified, suggesting that transcyretin causes several molecular variations. . 5 shows a comparison of signal intensity between healthy patients and carcinoma patients.

Finally, immunoprecipitation was performed. Serum fraction 4 from 50 μl of other patients was incubated at 4 ° C. for 2 hours with polyclonal antibody to 15 μl (6,75 μg) of transcyretin. Negative controls were performed in the same manner without antibody. 50 μl of Protein A / G agarose (Pierce, Rockford, IL) was added and incubated at 4 ° C. overnight. After centrifugation at 3300 μg, the supernatants were transferred and stored respectively. To detach the protein from protein A / G agarose, the pellet was resuspended in 20 μl buffer (9M urea, 2% Chaps, 50 mM Tris-HCl, pH 9). Precipitates as well as supernatants are diluted in a CM10 binding buffer solution at a 1:10 ratio. Three proteins with various molecular weights (13.776 Da, 13.884 Da, 14.103 Da) were immunoprecipitated with anti-transcyretin antibodies. As shown in FIG. 6, the amount of both strains (13.776 Da and 13.884 Da) was significantly reduced in the supernatant, while the amount of strain of the largest molecular weight (14.103 Da) was unchanged.

This indicates that transcyretin protein chains having molecular weights of about 13.776 Da and 13.884 Da are important biomarkers for the detection of colorectal adenomas and / or rectal colon carcinomas.

Claims (28)

a) providing an isolated sample material obtained from the subject, b) measuring transcyretin levels in the separated sample material, c) comparing the measured transthyretin levels with a reference value, Colorectal adenoma and / or colorectal carcinoma detection method comprising a. a) providing an isolated sample material obtained from the subject, b) measuring transcyretin levels in the separated sample material, c) comparing the measured transcyretin level with a reference value, Identification method between rectal adenomas and colorectal carcinoma comprising a. a) providing an isolated sample material obtained from the subject, b) measuring transcyretin levels in the separated sample material, c) comparing the measured transcyretin level with a reference value, A method of monitoring the progression of colorectal adenomas and / or colorectal carcinoma comprising, and / or a method of treating colorectal adenomas and / or colorectal carcinoma. The method of claim 1, wherein the transcyretin level of the sample material is reduced compared to the sample material of a healthy individual. The second sample of claim 2, wherein the first decrease in transcyretin levels in the first sample material is indicative of colorectal adenomas and is separated from the subject at a later point in time than the first sample material. Wherein the second decrease in transcyretin levels in the substance is indicative of colorectal carcinoma, wherein the second decrease is conditionally stronger than the first decrease. The method of claim 1, wherein the reference value is calculated as an average value of transcyretin measured in separate samples of a plurality of healthy individuals. The method of claim 1, wherein the reference value is the reference value of the individual, calculated as the mean value of transcyretin measured in a plurality of separate samples obtained from the individual over a period of time. 8. The method of claim 1, wherein the transcyretin consists of one or four protein chains, which may be the same or different from each other. 9. The method of claim 1, wherein the transcyretin is truncated, modified and / or mutated. The method of claim 1, wherein the separated sample material is a body fluid. The method of claim 10, wherein the body fluid is selected from the group consisting of blood, plasma, serum, bone marrow, feces, lubricating fluid, lymph, cerebrospinal fluid, sputum, urine, breast milk, semen, exudate and mixtures thereof. The method of claim 1, wherein the transcyretin level in the sample material is measured by mass spectrometry. The method of claim 1, wherein the cancer embryo antigen (CEA) level in the sample material is further measured and the cancer embryo antigen level in the sample material is increased by colorectal adenomas and / or colorectal carcinoma. How to be. The method of claim 1, wherein the CA 19-9 value in the sample material is further measured and the CA 19-9 level in the sample material is increased by colorectal adenomas and / or colorectal carcinoma. How. The method according to any one of claims 1 to 14, wherein the value of the protein or polypeptide having a molecular weight of 8960 ± 9 Da is additionally determined and the level of the 8960 ± 9 Da protein or polypeptide in the sample material is determined by colorectal adenomas and / or Or increased by colorectal carcinoma. The method according to any one of claims 1 to 11, wherein transcyretin levels in the sample material are measured by immunoassay, preferably ELISA or RIA. The method of claim 1, wherein the transcyretin level in the sample material is measured by numerical measurement of mRNA encoding transcyretin in the sample material. The method of claim 1, wherein the method is performed in concert with other diagnostic methods of colorectal adenomas and / or colorectal carcinoma to increase sensitivity and / or specificity. Use of transcyretin as a biomarker for the detection of colorectal adenomas and / or colorectal carcinoma in a subject. The use of claim 19 for early detection of colorectal adenomas and / or colorectal carcinoma. Use according to claim 19 or 20, wherein the transcyretin is truncated, modified and / or mutated. Use according to claim 19-21, in combination with one or more additional biomarkers for colorectal adenomas and / or colorectal carcinoma to increase sensitivity and / or specificity. The use of claim 22, wherein the at least one biomarker comprises a cancer embryo antigen (CEA) and the level in the sample material is increased by colorectal adenomas and / or rectal colon carcinoma. The use of claim 22, wherein the at least one biomarker comprises CA 19-9 and the level in the sample material is increased by colorectal adenomas and / or colorectal carcinoma. The use of claim 22, wherein the at least one biomarker has a molecular weight of 8960 ± 9 Da and the level in the sample material is increased by colorectal adenomas and / or colorectal carcinoma. a) an antibody or receptor that binds to an epitope of transcyretin, b) a solid support for supporting said antibody or receptor, c) a reagent for detecting binding of the epitope of said transcyretin to said antibody or receptor, An inspection system for the detection of colorectal adenomas and / or colorectal carcinoma in a subject. The test system of claim 26, wherein the test system comprises one or more antibodies or receptors for detection of one or more biomarkers for colorectal adenomas and / or colorectal carcinoma. An array comprising detection molecules for the detection of colorectal adenomas and / or colorectal carcinoma in a subject, a) a nucleic acid probe immobilized on a solid support to bind to and encode an mRNA encoding transcyretin, or b) an antibody that binds to an epitope of transcyretin and is immobilized on a solid support to detect it, or c) a receptor immobilized on a solid support for binding to and detecting an epitope of transcyretin, Wherein the array is preferably one in which different amounts of detection molecules are immobilized on a solid support for increased quantitative accuracy.

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