WO2005015225A1 - Utilisation de la proteine de liaison a element amont eloigne (fuse) (fubp) comme marqueur du cancer colorectal - Google Patents

Utilisation de la proteine de liaison a element amont eloigne (fuse) (fubp) comme marqueur du cancer colorectal Download PDF

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WO2005015225A1
WO2005015225A1 PCT/EP2004/008765 EP2004008765W WO2005015225A1 WO 2005015225 A1 WO2005015225 A1 WO 2005015225A1 EP 2004008765 W EP2004008765 W EP 2004008765W WO 2005015225 A1 WO2005015225 A1 WO 2005015225A1
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fubp
colorectal cancer
diagnosis
protein
marker
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PCT/EP2004/008765
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English (en)
Inventor
Michael Tacke
Peter Berndt
Marie-Luise Hagmann
Johann Karl
Hanno Langen
Stefan Palme
Markus Roessler
Wolfgang Rollinger
Werner Zolg
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Roche Diagnostics Gmbh
F. Hoffmann-La Roche Ag
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Publication of WO2005015225A1 publication Critical patent/WO2005015225A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity

Definitions

  • CRC colorectal cancer
  • the prognosis in advanced stages of tumor is poor. More than one third of the patients will die from progressive disease within five years after diagnosis, corresponding to a survival rate of about 40% for five years.
  • Current treatment is only curing a fraction of the patients and clearly has the best effect on those patients diagnosed in an early stage of disease.
  • CRC colorectal cancer
  • a protein encoded by a rare mRNA may be found in very high amounts and a protein encoded by an abundant mRNA may nonetheless be hard to detect and find at all. This lack of correlation between mRNA-level and protein level is due to reasons like mRNA stability, efficiency of translation, stability of the protein, etc.
  • WO 02/078636 reports about nine colorectal cancer-associated spots as found by surface- enhanced laser desorption and ionization (SELDI). These spots are seen more frequently in sera obtained from patients with CRC as compared to sera obtained from healthy controls. However, the identity of the molecule(s) comprised in such spot, e.g., its (their sequence), is not known.
  • a new diagnostic marker as a single marker should be at least as good as the best single marker known in the art. Or, a new marker should lead to a progress in diagnostic sensitivity and/or specificity either if used alone or in combination with one or more other markers, respectively.
  • the diagnostic sensitivity and/or specificity of a test is best assessed by its receiver-operating characteristics, which will be described in detail below.
  • CEA carcinoembryonic antigen
  • a tumor-associated glycoprotein a tumor-associated glycoprotein
  • Samples taken from stool have the advantage that such sampling is easily possible by non-invasive means.
  • the guaiac test is currently most widely used as a screening assay for CRC from stool.
  • the guaiac test however, has both poor sensitivity as well as poor specificity.
  • the sensitivity of the guaiac-based fecal occult blood tests is ⁇ 26%, which means 74% of patients with malignant lesions will remain undetected (Ahlquist, DA.., Gastroenterol. Clin. North Am. 26 (1997) 41-55).
  • the hemoglobin assay has an unsatisfactory sensitivity for the detection of colorectal neoplasms. Whereas cancer in its progressed carcinoma stage is detected with a sensitivity of about 87% the earlier tumor stages are not detected with a sufficient sensitivity.
  • the hemoglobin-haptoglobin complex assay was more sensitive in the detection of earlier stages of CRC. This more sensitive detection was accompanied by a poor specificity. Since poor specificity, however, translates to a high number of unnecessary secondary investigations, like colonoscopy, an assay with a poor accuracy also does not meet the requirements of a generally accepted screening assay.
  • the present invention therefore relates to a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual, b) contacting said sample with a specific binding agent for FUBP under conditions appropriate for formation of a complex between said binding agent and FUBP, and c) correlating the amount of complex formed in (b) to the diagnosis of colorectal cancer.
  • the stool sample is processed to obtain a processed sample liquid which is more convenient to handle than a stool specimen. Such processed sample is then incubated with the specific binding agent for FUBP.
  • the present invention therefore also relates to a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual, b) processing said sample to obtain a processed liquid sample, c) contacting said processed liquid sample with a specific binding agent for FUBP under conditions appropriate for formation of a complex between said binding agent and FUBP, and d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • a preferred method uses a stool sample obtained from an individual.
  • Another preferred embodiment of the invention is a method for the diagnosis of colorectal cancer comprising the steps of a) processing a stool sample obtained from an individual to obtain a processed liquid sample b) contacting said processed liquid sample with a specific binding agent for FUBP under conditions appropriate for formation of a complex between said binding agent and FUBP, and c) correlating the amount of complex formed in (b) to the diagnosis of colorectal cancer.
  • the stool sample is processed to retrieve colonycytes which are then smeared on a microscopic slide. Such processed sample is then incubated with the specific binding agent for FUBP.
  • the present invention therefore also relates to a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual, b) processing said sample to retrieve colonycytes, c) contacting said processed sample with a specific binding agent for FUBP under conditions appropriate for formation of a complex between said binding agent and FUBP, and d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • the protein FUBP "Fuse-binding protein", i.e. far upstream element-binding protein; far upstream element binding protein; FBP) (TREMBL: Q12828) is characterized by the sequence given SEQ ID NO:l.
  • FUBP is expressed in proliferating cells from a variety of lineages, but not in quiescent cells.
  • T cells and fibroblasts were stimulated to transit from GO into the cell cycle, there was a dramatic rise of both FUBP mRNA and DNA sequence specific nuclear FUBP binding activity, which correlated with the appearance of c- myc mRNA.
  • both FUBP and c-myc expression were sustained for more than 24 h.
  • the coordinate expression of FUBP and c-myc throughout all phases of the cell cycle is consistent with the role of FUBP as a growth- dependent regulator of c-myc expression.
  • the present invention shall not be construed to be limited to the full-length protein FUBP of SEQ ID NO:l.
  • Physiological or artificial fragments of FUBP, secondary modifications of FUBP, as well as allelic variants of FUBP are also encompassed by the present invention.
  • Artificial fragments preferably encompass a peptide produced synthetically or by recombinant techniques, which at least comprises one epitope of diagnostic interest consisting of at least 6 contiguous amino acids as derived from the sequence disclosed in SEQ ID NO:l. Such fragment may advantageously be used for generation of antibodies or as a standard in an immunoassay. More preferred the artificial fragment comprises at least two epitopes of interest appropriate for setting up a sandwich immunoassay.
  • the novel marker FUBP may be used for monitoring as well as for screening purposes. Its use for screening purposes is most preferred.
  • the diagnostic method according to the present invention may help to assess tumor load, efficacy of treatment and tumor recurrence in the follow-up of patients.
  • Increased levels of FUBP are directly correlated to tumor burden. After chemotherapy a short term (few hours to 14 days) increase in FUBP may serve as an indicator of tumor cell death. In the follow- up of patients (from 3 months to 10 years) an increase of FUBP can be used as an indicator for tumor recurrence in the colorectum.
  • the diagnostic method according to the present invention is used for screening purposes. I.e., it is used to assess subjects without a prior diagnosis of CRC by measuring the level of FUBP in a stool sample and correlating the level measured to the presence or absence of CRC.
  • the staging of cancer is the classification of the disease in terms of extent, progression, and severity. It groups cancer patients so that generalizations can be made about prognosis and the choice of therapy.
  • TNM system is the most widely used classification of the anatomical extent of cancer. It represents an internationally accepted, uniform staging system. There are three basic variables: T (the extent of the primary tumor), N (the status of regional lymph nodes) and M (the presence or absence of distant metastases).
  • T the extent of the primary tumor
  • N the status of regional lymph nodes
  • M the presence or absence of distant metastases.
  • TNM criteria are published by the UICC (International Union against Cancer), Sobin, L.H., Wittekind, Ch. (eds): TNM Classification of Malignant Tumours, fifth edition, 1997. What is especially important is, that early diagnosis of CRC translates to a much better prognosis. Malignant tumors of the colorectum arise from benign tumors, i.e. from adenoma. Therefore, best prognosis have those patients diagnosed at the adenoma stage.
  • early diagnosis of CRC refers to a diagnosis at a pre-malignant state (adenoma) or at a tumor stage where no metastases at all (neither proximal nor distal), i.e., adenoma, Tj s , NO, M0 or Tl-4; NO; M0 are present.
  • T ls denotes carcinoma in situ.
  • the detection of FUBP is used to diagnose CRC as early as in the adenoma stage.
  • the diagnostic method according to the present invention is based on a stool sample which is derived from an individual.
  • the stool sample is extracted and FUBP is specifically measured from this processed stool sample by use of a specific binding agent.
  • a specific binding agent is, e.g., a receptor for FUBP, a lectin binding to FUBP or an antibody to FUBP.
  • a specific binding agent has at least an affinity of 10 7 1/mol for its corresponding target molecule.
  • the specific binding agent preferably has an affinity of 10 8 1/mol or even more preferred of 10 9 1/mol for its target molecule.
  • specific is used to indicate that other biomolecules present in the sample do not significantly bind to with the binding agent specific for FUBP.
  • the level of binding to a biomolecule other than the target molecule results in a binding affinity which is only 10%, more preferably only 5% of the affinity of the target molecule or less.
  • a most preferred specific binding agent will fulfill both the above minimum criteria for affinity as well as for specificity.
  • a specific binding agent preferably is an antibody reactive with FUBP.
  • the term antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of such antibodies, as well as to genetic constructs comprising the binding domain of an antibody.
  • Antibodies are generated by state of the art procedures, e.g., as described in Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam).
  • Tijssen Tejssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam.
  • the skilled artisan is well aware of methods based on immunosorbents that can be used for the specific isolation of antibodies. By these means the quality of polyclonal antibodies and hence their performance in immunoassays can be enhanced. (Tijssen, P., supra, pages 108-115).
  • polyclonal antibodies raised in rabbits have been used.
  • polyclonal antibodies from different species e.g. rats or guinea pigs
  • monoclonal antibodies can also be used. Since monoclonal antibodies can be produced in any amount required with constant properties, they represent ideal tools in development of an assay for clinical routine.
  • the generation and use of monoclonal antibodies to FUBP in a method according to the present invention is yet another preferred embodiment.
  • FUBP has been identified as a marker which is useful in the diagnosis of CRC
  • alternative ways may be used to reach a result comparable to the achievements of the present invention.
  • alternative strategies to generate antibodies may be used.
  • Such strategies comprise amongst others the use of synthetic peptides, representing an epitope of FUBP for immunization.
  • DNA Immunization also known as DNA vaccination may be used.
  • the stool sample is obtained from an individual. An aliquot of the stool sample may be used directly. Preferably an aliquot of the stool sample is processed to yield a liquid sample.
  • the stool sample is preferably used or processed directly after sampling or stored cooled or more conveniently stored frozen. Frozen stool samples can be processed by thawing, followed by dilution in an appropriate buffer, mixing and centrifugation. Supernatants are used as liquid sample for subsequent measurement of marker FUBP.
  • An aliquot of the processed stool sample is incubated with the specific binding agent for FUBP under conditions appropriate for formation of a binding agent FUBP-complex. Such conditions need not be specified, since the skilled artisan without any inventive effort can easily identify such appropriate incubation conditions.
  • the amount of complex is measured and correlated to the diagnosis of CRC.
  • CRC diagnosis of CRC.
  • the skilled artisan will appreciate there are numerous methods to measure the amount of specific binding agent FUBP-complex all described in detail in relevant textbooks (cf., e.g., Tijssen P., supra, or Diamandis, E.P., et al., eds. (1996) Immunoassay,
  • FUBP is detected in a sandwich type assay format.
  • a first specific binding agent is used to capture FUBP on the one side and a second specific binding agent, which is labeled to be directly or indirectly detectable is used on the other side.
  • FUBP can be measured from a stool sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker FUBP in the diagnosis of CRC.
  • an antibody to FUBP is used in a qualitative (FUBP present or absent) or quantitative (FUBP amount is determined) immunoassay.
  • the present invention relates to use of protein FUBP as a marker molecule in the diagnosis of colorectal cancer from a stool sample obtained from an individual.
  • marker molecule is used to indicate that an increased level of the analyte
  • FUBP as measured from a bodily fluid or especially a processed stool sample obtained from an individual marks the presence of CRC.
  • the use of protein FUBP itself represents a significant progress to the challenging field of CRC diagnosis from stool. Combining measurements of FUBP with other known markers, like hemoglobin or the hemoglobin-haptoglobin complex, or with other markers of CRC yet to be discovered, leads to further improvements. Therefore in a further preferred embodiment the present invention relates to the use of FUBP as a marker molecule for colorectal cancer in combination with one or more other marker molecules for colorectal cancer in the diagnosis of colorectal cancer from a stool sample obtained from an individual.
  • Preferred selected other CRC markers with which the measurement of FUBP may be combined are hemoglobin and/or the hemoglobin-haptoglobin complex. Very preferred the marker FUBP is used in combination with hemoglobin. Also very preferred the marker FUBP is used in combination with the hemoglobin-haptoglobin complex.
  • Diagnostic reagents in the field of specific binding assays like immunoassays, usually are best provided in the form of a kit, which comprises the specific binding agent and the auxiliary reagents required to perform the assay.
  • the present invention therefore also relates to an immunological kit comprising at least one specific binding agent for FUBP and auxiliary reagents for measurement of FUBP.
  • ROC receiver-operating characteristics
  • the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as
  • Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
  • One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number.
  • Clinical utility of the novel marker FUBP has been assessed in comparison to and in combination with the established marker hemoglobin using a receiver operator curve analysis (ROC; Zweig, M. H., and Campbell, G., Clin. Chem. 39 (1993) 561- 577).
  • ROC receiver operator curve analysis
  • This analysis has been based on well-defined patient cohorts consisting of 30 samples each from patients in Tl-3; NO; M0, more progressed tumor, i.e., T4 and/or various severity of metastasis (N+ and/or M+), and healthy controls, respectively.
  • Figure 1 shows a typical example of a 2D-gel, loaded with a tumor sample (left side), and a gel, loaded with a matched control sample (right side) obtained from adjacent healthy mucosa.
  • the molecular weight inferred from the position of the protein spot in the gel is about 68 kDa (less), the isoelectric point is at about pH 7.
  • the circle in the enlarged section of these gels indicates the position for the protein FUBP. This protein was not detectable by the same method in healthy mucosa.
  • Figure 2 Typical example of a Western-Blot.
  • a polyacrylamide gel was loaded with tissue lysates from colorectal tumor tissue and adjacent healthy control tissue from 4 patients (subject 4: colon ca (carcinoma), Dukes C; subject 10: colon ca, Dukes C; subject 13: colon ca, Dukes B; and subject 14: colon ca, Dukes B) and after electrophoresis the proteins were blotted onto a nitrocellulose membrane. Presence of FUBP in the samples was tested using a monoclonal mouse anti-FUBP antibody. Lanes containing tumor lysates are indicated with "T”, lanes containing normal control tissue with "N". The arrow indicates the position in the gel of the FUBP band. All tumor samples give a strong signal at the position of FUBP, whereas only a weak signal can be detected in the lysates from adjacent normal control tissue.
  • tissue specimen from 10 patients suffering from colorectal cancer are analyzed. From each patient three different tissue types are collected from therapeutic resections: tumor tissue (> 80% tumor) (T), adjacent healthy tissue (N) and stripped mucosa from adjacent healthy mucosa (M). The latter two tissue types serve as matched healthy control samples. Tissues are immediately snap frozen after resection and stored at - 80°C before processing. Tumors are diagnosed by histopathological criteria.
  • 0.8-1.2 g of frozen tissue are put into a mortar and completely frozen by liquid nitrogen.
  • the tissue is pulverized in the mortar, dissolved in the 10-fold volume (w/v) of lysis buffer (40 mM Na-citrate, 5 mM MgCl 2 , 1% Genapol X-080, 0.02% Na-azide, Complete ® EDTA-free [Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 1 873 580] ) and subsequently homogenized in a Wheaton® glass homogenizer (20 x loose fitting, 20 x tight fitting).
  • lysis buffer 40 mM Na-citrate, 5 mM MgCl 2 , 1% Genapol X-080, 0.02% Na-azide, Complete ® EDTA-free
  • Each patient is analyzed separately by image analysis with the ProteomeWeaver® software (Definiens AG, Germany, M ⁇ nchen).
  • ProteomeWeaver® software Detiens AG, Germany, M ⁇ nchen.
  • all spots of the gel are excised by a picking robot and the proteins present in the spots are identified by
  • MALDI-TOF mass spectrometry (Ultraflex Tof/Tof, Bruker Daltonik GmbH, Bremen, Germany).
  • 4 gels from the tumor sample are compared with 4 gels each from adjacent normal and stripped mucosa tissue and analyzed for distinctive spots corresponding to differentially expressed proteins.
  • protein FUBP is found to be specifically expressed or strongly overexpressed in tumor tissue and not detectable or less strongly expressed in healthy control tissue. It therefore - amongst many other proteins - qualifies as a candidate marker for use in the diagnosis of colorectal cancer.
  • Polyclonal antibody to the colorectal cancer marker protein FUBP is generated for further use of the antibody in the measurement of serum and plasma and blood and stool levels of FUBP by immunodetection assays, e.g. Western Blotting and ELISA.
  • recombinant expression of the protein is performed for obtaining immunogens.
  • the expression is done applying a combination of the RTS 100 expression system and E.coli.
  • the DNA sequence is analyzed and recommendations for high yield cDNA silent mutational variants and respective PCR-primer sequences are obtained using the "ProteoExpert RTS E.coli HY" system. This is a commercial web based service
  • the "RTS 100 E. coli Linear Template Generation Set, His-tag” (Roche Diagnostics GmbH, Mannheim, Germany, Cat.No. 3186237) system to generate linear PCR templates from the cDNA and for in-vitro transcription and expression of the nucleotide sequence coding for the FUBP protein is used.
  • the expressed protein contains a His-tag. The best expressing variant is identified. All steps from PCR to expression and detection are carried out according to the instructions of the manufacturer.
  • the respective PCR product, containing all necessary T7 regulatory regions is cloned into the pBAD TOPO ® vector (Invitrogen, Düsseldorf,
  • the construct is transformed into E. coli BL 21 (DE 3) (Studier, F.W., et al., Methods Enzymol. 185 (1990) 60-89) and the transformed bacteria are cultivated in a 1 1 batch for protein expression.
  • His-FUBP fusion protein Purification of His-FUBP fusion protein is done following standard procedures on a Ni-chelate column. Briefly, 1 1 of bacteria culture containing the expression vector for the His-FUBP fusion protein is pelleted by centrifugation. The cell pellet is resuspended in lysis buffer, containing phosphate, pH 8.0, 7 M guanidium chloride, imidazole and thioglycerole, followed by homogenization using a Ultra-Turrax ® . Insoluble material is pelleted by high speed centrifugation and the supernatant is applied to a Ni-chelate chromatographic column. The column is washed with several bed volumes of lysis buffer followed by washes with buffer, containing phosphate, pH 8.0 and Urea. Finally, bound antigen is eluted using a phosphate buffer containing SDS under acid conditions.
  • mice 12 week old A/J mice are initially immunized intraperitoneally with 100 ⁇ g FUBP. This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 ⁇ g FUBP adsorbed to aluminum hydroxide and 10 9 germs of Bordetella pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 ⁇ g FUBP in PBS buffer for each.
  • Spleen cells of the mice immunized according to a) are fused with myeloma cells according to Galfre, G., and Milstein, C, Methods in Enzymology 73 (1981) 3-46.
  • ca. 1*10 8 spleen cells of the immunized mouse are mixed with 2xl0 7 myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10 min at 300 x g and 4°C).
  • the cells are then washed once with RPMI 1640 medium without fetal calf serum (FCS) and centrifuged again at 400 x g in a 50 ml conical tube.
  • FCS fetal calf serum
  • FUBP- positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter. In this process again interleukin 6 at 100 U/ml is added to the medium as a growth additive.
  • the hybridoma cells obtained are sown at a density of lxl 0 5 cells per ml in RPMI 1640 medium containing 10% FCS and proliferated for 7 days in a fermenter (Thermodux Co., Wertheim/Main, Model MCS-104XL, Order No. 144-050). On average concentrations of 100 ⁇ g monoclonal antibody per ml are obtained in the culture supernatant. Purification of this antibody from the culture supernatant is carried out by conventional methods in protein chemistry (e.g. according to Brack, C, et al., Methods in Enzymology 121 (1986) 587-695).
  • a fresh emulsion of the protein solution (100 ⁇ g/ml protein FUBP) and complete Freund's adjuvant at the ratio of 1:1 is prepared.
  • Each rabbit is immunized with 1 ml of the emulsion at days 1, 7, 14 and 30, 60 and 90. Blood is drawn and resulting anti-FUBP serum used for further experiments as described in examples 3 and 4.
  • IgG immunoglobulin G
  • rabbit serum is diluted with 4 volumes of acetate buffer (60 mM, pH 4.0). The pH is adjusted to 4.5 with 2 M Tris-base. Caprylic acid (25 ⁇ l/ml of diluted sample) is added drop-wise under vigorous stirring. After 30 min the sample is centrifuged (13,000 x g, 30 min, 4°C), the pellet discarded and the supernatant collected. The pH of the supernatant is adjusted to 7.5 by the addition of 2 M Tris-base and filtered (0.2 ⁇ m).
  • the immunoglobulin in the supernatant is precipitated under vigorous stirring by the drop-wise addition of a 4 M ammonium sulfate solution to a final concentration of 2 M.
  • the precipitated immunoglobulins are collected by centrifugation (8,000 x g, 15 min, 4°C).
  • the supernatant is discarded.
  • the pellet is dissolved in 10 mM NaH 2 PO /NaOH, pH 7.5, 30 mM NaCl and exhaustively dialyzed.
  • the dialysate is centrifuged (13,000 x g, 15 min, 4°C) and filtered (0.2 ⁇ m).
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl. Per ml IgG solution 50 ⁇ l Biotin -N-hydroxysuccinimide (3.6 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex 200 (10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl). The fraction containing biotinylated IgG are collected. Monoclonal antibodies are biotinylated according to the same procedure.
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, 30 mM NaCl, pH 7.5.
  • Per ml IgG solution 50 ⁇ l digoxigenin-3-O-methylcarbonyl- ⁇ - aminocaproic acid-N-hydroxysuccinimide ester (Roche Diagnostics, Mannheim,
  • Tissue lysates from tumor samples and healthy control samples are prepared as described in Example 1, "Tissue preparation”.
  • SDS-PAGE and Western-Blotting are carried out using reagents and equipment of Invitrogen, Düsseldorf, Germany.
  • 10 ⁇ g of tissue lysate are diluted in reducing NuPAGE ® (Invitrogen) SDS sample buffer and heated for 10 min at 95°C.
  • Samples are run on 4-12% NuPAGE® gels (Tris-Glycine) in the MES running buffer system.
  • the gel-separated protein mixture is blotted onto nitrocellulose membranes using the Invitrogen XCell II Blot Module (Invitrogen) and the NuPAGE ® transfer buffer system.
  • the membranes are washed 3 times in PBS/0.05% Tween-20 and blocked with Roti®-Block blocking buffer (A151.1; Carl Roth GmbH, Düsseldorf, Germany) for 2 h.
  • the primary antibody, polyclonal rabbit anti-FUBP serum (generation described in Example 2), is diluted 1:10,000 in Roti®-
  • Block blocking buffer and incubated with the membrane for 1 h The membranes are washed 6 times in PBS/0.05% Tween-20.
  • the specifically bound primary rabbit antibody is labeled with a POD-conjugated polyclonal sheep anti-rabbit IgG antibody, diluted to 10 mU/ml in 0.5 x Roti ® -Block blocking buffer. After incubation for 1 h, the membranes are washed 6 times in PBS/0.05% Tween-20.
  • the membrane is incubated with the Lumi-Light us Western Blotting Substrate (Order-No. 2015196, Roche Diagnostics GmbH, Mannheim, Germany) and exposed to an autoradiographic film.
  • the stool samples are thawed, tenfold diluted with a phosphate buffer, pH 7.4 and thoroughly mixed in order to yield a suspension of the stool sample in the extraction buffer. After centrifugation for 15 min at 12,000 x g, the upper portion of the supernatant is saved for further analysis. An aliquot of this processed stool sample is used for quantification of FUBP by ELISA.
  • a sandwich ELISA For detection of FUBP in human a processed stool sample, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-FUBP polyclonal antibody (see example 2) are conjugated with biotin and digoxygenin, respectively.
  • Streptavidin-coated 96-well microtiter plates are incubated with 100 ⁇ l biotinylated anti-FUBP polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. After incubation, plates are washed three times with 0.9% NaCl , 0.1% Tween-20. Wells are then incubated for 2 h with either a serial dilution of the recombinant protein (see Example 2) as standard antigen or with diluted stool samples from patients. After binding of FUBP, plates are washed three times with 0.9% NaCl, 0.1% Tween-20.
  • wells are incubated with 100 ⁇ l of digoxygenylated anti- FUBP polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. Thereafter, plates are washed three times to remove unbound antibody.
  • wells are incubated with 20 mU/ml anti- digoxigenin-POD conjugates (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1633716) for 60 min in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. Plates are subsequently washed three times with the same buffer.
  • antigen-antibody complexes wells are incubated with
  • Accuracy is assessed by analyzing individual stool samples obtained from well- characterized patient cohorts, i.e., 30 patients having undergone colonoscopy and found to be free of adenoma or CRC, 30 patients diagnosed and staged as Tl-3, NO, M0 of CRC, and 30 patients diagnosed with progressed CRC, having at least tumor infiltration in at least one proximal lymph node or more severe forms of metastasis, respectively.
  • FUBP is measured as described above in a stool sample obtained from each of these individuals.
  • ROC-analysis is performed according to Zweig, M. H., and Campbell, supra.
  • Discriminatory power for differentiating patients in the group Tj s -3, NO, M0 from healthy individuals for the combination of FUBP with the established marker hemoglobin is calculated by regularized discriminant analysis (Friedman, J. H., Regularized Discriminant Analysis, Journal of the American Statistical Association 84 (1989) 165-175).

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Abstract

La présente invention concerne le diagnostic du cancer colorectal. L'invention se rapporte à l'utilisation de la protéine FUBP (« protéine de liaison Fuse ») dans le diagnostic du cancer colorectal. L'invention porte sur un procédé qui permet de diagnostiquer le cancer colorectal à partir d'un prélèvement de selles issu d'un individu en mesurant la FUBP dans ledit prélèvement. La mesure de la FUBP peut, par exemple, être utilisée pour la détection ou le diagnostic précoce du cancer colorectal.
PCT/EP2004/008765 2003-08-06 2004-08-05 Utilisation de la proteine de liaison a element amont eloigne (fuse) (fubp) comme marqueur du cancer colorectal WO2005015225A1 (fr)

Applications Claiming Priority (2)

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EP03017526 2003-08-06
EP03017526.9 2003-08-06

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WO2005015225A1 true WO2005015225A1 (fr) 2005-02-17

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580760A (en) * 1993-02-22 1996-12-03 The United States Of America As Represented By The Department Of Health And Human Services FUSE binding protein and cDNA therefor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580760A (en) * 1993-02-22 1996-12-03 The United States Of America As Represented By The Department Of Health And Human Services FUSE binding protein and cDNA therefor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HE L ET AL: "Loss of FBP function arrests cellular proliferation and extinguishes c-myc expression", EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 19, no. 5, 2000, pages 1034 - 1044, XP002975839, ISSN: 0261-4189 *
HUTH JEFFREY R ET AL: "NMR-driven discovery of benzoylanthranilic acid inhibitors of far upstream element binding protein binding to the human oncogene c-myc promoter.", JOURNAL OF MEDICINAL CHEMISTRY. 23 SEP 2004, vol. 47, no. 20, 23 September 2004 (2004-09-23), pages 4851 - 4857, XP002302467, ISSN: 0022-2623 *
ROCHLITZ CHRISTOPH F ET AL: "Overexpression and amplification of c-myc during progression of human colorectal cancer", ONCOLOGY (BASEL), vol. 53, no. 6, 1996, pages 448 - 454, XP008037560, ISSN: 0030-2414 *

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