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WO2005095978A1 - Utilisation de la pyrroline-5-carboxylate reductase en tant que marqueur pour le cancer colorectal - Google Patents

Utilisation de la pyrroline-5-carboxylate reductase en tant que marqueur pour le cancer colorectal Download PDF

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Publication number
WO2005095978A1
WO2005095978A1 PCT/EP2005/003263 EP2005003263W WO2005095978A1 WO 2005095978 A1 WO2005095978 A1 WO 2005095978A1 EP 2005003263 W EP2005003263 W EP 2005003263W WO 2005095978 A1 WO2005095978 A1 WO 2005095978A1
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proc
colorectal cancer
diagnosis
protein
sample
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PCT/EP2005/003263
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Michael Tacke
Johannes Auer
Ursula Brehm
Johann Karl
Stefan Palme
Wolfgang Rollinger
Werner Zolg
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Roche Diagnostics Gmbh
F. Hoffmann-La Roche Ag
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Publication of WO2005095978A1 publication Critical patent/WO2005095978A1/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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)

Definitions

  • the present invention relates to the diagnosis of colorectal cancer. It discloses the use of the protein PROC (pyrroline-5-carboxylate reductase) in the diagnosis of colorectal cancer. Furthermore, it especially relates to a method for diagnosis of colorectal cancer from a stool sample, derived from an individual by measuring PROC in said sample. Measurement of PROC can, e.g., be used in the early detection or diagnosis of colorectal cancer.
  • PROC pyrroline-5-carboxylate reductase
  • 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, D.A., 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 PROC under conditions appropriate for formation of a complex between said binding agent and PROC, 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 PROC.
  • 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 PROC under conditions appropriate for formation of a complex between said binding agent and PROC, and d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • the method comprises using a stool sample obtained from an individual in step (a).
  • 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 PROC under conditions appropriate for formation of a complex between said binding agent and PROC, 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 PROC.
  • 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 coloncytes, c) contacting said processed sample with a specific binding agent for PROC under conditions appropriate for formation of a complex between said binding agent and PROC, and d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • the protein PROC (Pyrroline-5-carboxylate reductase, also known as PYCR1; Swiss-PROT: P32322) is characterized by the sequence given SEQ ID NO:l.
  • PROC catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate to proline.
  • Merrill, M. J., et al., T. Biol. Chem. 264 (1989) 9352-9358 studied the properties of human erythrocyte pyrroline-5-carboxylate reductase. They concluded that in addition to the traditional role of catalyzing the obligatory and final unidirectional step in pyrroline biosynthesis, the enzyme may play a physiologic role in the generation of NADP(+) in some cell types including human erythrocytes.
  • the present invention shall not be construed to be limited to the full-length protein PROC of SEQ ID NO :1.
  • PROC 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 PROC 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 PROC are directly correlated to tumor burden. After chemotherapy a short term (few hours to 14 days) increase in PROC may serve as an indicator of tumor cell death. In the follow-up of patients (from 3 months to 10 years) an increase of PROC 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 PROC 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 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).
  • 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.
  • early diagnosis of CRC refers to a diagnosis at a pre-malignant state (adenoma) or at a tumor stage where no metastases at all
  • Tj S denotes carcinoma in situ.
  • the detection of PROC 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
  • PROC 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 PROC, a lectin binding to PROC or an antibody to PROC.
  • a receptor for PROC e.g., a receptor for PROC
  • a lectin binding to PROC e.g., an antibody to PROC.
  • specific is used to indicate that other biomolecules present in the sample do not significantly bind to with the binding agent specific for PROC. A level of less than 5% cross-reactivity is considered not significant.
  • a specific binding agent preferably is an antibody reactive with PROC.
  • 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.
  • clearly also polyclonal antibodies from different species e.g.
  • 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 PROC in a method according to the present invention is yet another preferred embodiment.
  • PROC 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 PROC 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.
  • 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 the marker PROC.
  • PROC is detected in a sandwich type assay format.
  • a first specific binding agent is used to capture PROC 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.
  • PROC can be measured from a stool sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker PROC in the diagnosis of CRC.
  • Antibodies to PROC with great advantage can also be used in established procedures, e.g., to detect colorectal cancer cells in situ, in biopsies, or in immunohistological procedures.
  • an antibody to PROC is used in a qualitative (PROC present or absent) or quantitative (PROC amount is determined) immunoassay.
  • the present invention relates to use of protein PROC as a marker molecule in the diagnosis of colorectal cancer from a stool sample obtained from an individual.
  • the term marker molecule is used to indicate that an increased level of the analyte PROC as measured from a bodily fluid or especially from a processed stool sample obtained from an individual marks the presence of CRC.
  • novel marker PROC in the early diagnosis of colorectal cancer.
  • the use of protein PROC itself represents a significant progress to the challenging field of CRC diagnosis from stool. Combining measurements of PROC 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 PROC as a marker molecule for colorectal cancer in combination with at least one other marker molecule 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 PROC may be combined are hemoglobin and/or 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 PROC and auxiliary reagents for measurement of PROC.
  • 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 (number of true-positive test results) / (number of true-positive + number of false- negative test results)]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • the false-positive fraction On the x- axis is the false-positive fraction, or 1 - specificity [defined as (number of false- positive results) / (number of true-negative + number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup. Because the true- and false-positive fractions are calculated entirely separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of disease in the sample. Each point on the ROC plot represents a sensitivity/ 1 -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. (If the ROC plot falls completely below the 45° diagonal, this is easily remedied by reversing the criterion for "positivity" from "greater than” to "less than” or vice versa.) Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test.
  • One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number.
  • the diagnostic method based on measurement of PROC alone in comparison to the established marker hemoglobin alone has been found to have an at least as good a diagnostic accuracy (sensitivity/specificity profile) as demonstrated by the area under the curve.
  • Figure 1 shows a typical example of a 2D-gel, loaded with a tumor sample (left side, A), and a gel, loaded with a matched control sample (right side, B) obtained from adjacent healthy mucosa.
  • the apparent molecular weight and the isoelectric point of PROC correspond to the theoretical values of 33.4 kDa and 7.2, respectively.
  • the circle in the enlarged section of these gels indicates the position for the protein PROC. This protein is not detectable by the same method in healthy mucosa.
  • Figure 2 shows a typical example of a Western-Blot.
  • the gel is loaded with tissue lysates from colorectal tumor tissue and adjacent healthy control tissue from 3 patients (subject 5: colon ca (carcinoma), Dukes B; subject 7: colon ca, Dukes C; and subject 13: colon ca, Dukes B). Presence of PROC in the samples is tested using a polyclonal rabbit anti-PROC serum. Lanes containing tumor lysates are indicated with "T", lanes containing normal control tissue with "N”. The marker lane containing a molecular weight protein standard is indicated by "M”. The arrow indicates the position in the gel of the PROC band. All tumor samples give a strong signal at the position of PROC, whereas almost no signal can be detected in the lysates from adjacent normal control tissue. This strong overexpression of PROC in tumor tissue from colorectal cancer patients is shown in 10 out of 10 subjects tested.
  • 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 [Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 1 873 580]
  • 3 ml of the homogenate are subjected to a sucrose-density centrifiigation (10-60% sucrose) for 1 h at 4,500 x g. After this centrifiigation step three fractions are obtained. The fraction on top of the gradient contains the soluble proteins and is used for further analysis.
  • IPG strips pH 6-9 (Amersham Biosciences, Freiburg, Germany) overnight.
  • the IEF is performed using the following gradient protocol: 1.) 1 minute to 500 N; 2.) 2 h to 3,500 N; 3.) 22 h at constant 3,500 N giving rise to 82 kVh. After IEF, strips are stored at -80°C or directly used for SDS-PAGE.
  • the strips Prior to SDS-PAGE the strips are incubated in equilibration buffer (6 M urea, 50 mM Tris/HCl, pH 8.8, 30% glycerol, 2% SDS), for reduction DTT (15 min, + 50 mg DTT/10 ml), and for alkylation IAA (15 min, + 235 mg iodacetamide/10 ml) is added.
  • equilibration buffer 6 M urea, 50 mM Tris/HCl, pH 8.8, 30% glycerol, 2% SDS
  • DTT 15 min, + 50 mg DTT/10 ml
  • alkylation IAA 15 min, + 235 mg iodacetamide/10 ml
  • the strips are put on 12.5% polyacrylamide gels and subjected to electrophoresis at 1 W/gel for 1 h and thereafter at 17 W/gel. Subsequently, the gels are fixed (50% methanol, 10% acetate) and stained overnight with ⁇ ovex Colloidal Blue St
  • protein PROC is found to be specifically expressed or strongly overexpressed in tumor tissue and not detectable or less strongly expressed in healthy control tissue.
  • Polyclonal antibody to the colorectal cancer marker protein PROC is generated for further use of the antibody in the measurement of serum, and plasma, and blood and stool levels of PROC 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 (www.proteoexpert.com).
  • the "RTS 100 E. coli Linear Template Generation Set, His-tag” (Roche Diagnostics GmbH, Mannheim, Germany, Cat.No.
  • His-PROC fusion protein Purification of His-PROC fusion protein is done using a Ni-chelate column following standard procedures. Briefly, 1 1 of bacteria culture containing the expression vector for the His-PROC fusion protein is pelleted by centrifiigation. 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 centrifiigation and the supernatant is applied to a Ni-chelate chromatographic column.
  • mice 12 week old A/J mice are initially immunized intraperitoneally with 100 ⁇ g PROC. This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 ⁇ .g PROC adsorbed to aluminum hydroxide and 10 germs of Bordetella pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 ⁇ g PROC 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 2x10 7 myeloma cells (P3_X63-Ag8-653, ATCC CRL1580) and centriftiged ( 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 centriftiged again at 400 x g in a 50 ml conical tube.
  • FCS fetal calf serum
  • the sedimented cells are taken up in RPMI 1640 medium containing 10% FCS and sown in hypoxanthine-azaserine selection medium (lOO mmol/1 hypoxanthine, 1 ⁇ g/ml azaserine in RPMI 1640+10% FCS).
  • Interleukin 6 at 100 U/ml is added to the medium as a growth factor.
  • PROC- 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 1O0 U/ml is added to the medium as a growth additive. c) Immunoglobi lin isolation from the cell culture supernatants
  • the hybridoma cells obtained are sown at a density of lxlO 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 Bruck, C, et al., Methods in Enzymology 121 (1986) 587-695).
  • a fresh emulsion of the protein solution (100 ⁇ g/ml PROC protein) 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-PROC 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 centrifiigation (8,000 x g, 15 min, 4°C). The supernatant is discarded.
  • the pellet is dissolved in 10 mM NaH PO 4 /NaOH, pH 7.5, 30 mM INfaCl 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,
  • 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, Germany, Cat. No. 1 333 054) (3.8 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex® 200 (10 mM NaH 2 P0 /NaOH, pH 7.5, 30 mM NaCl). The fractions containing digoxigenylated IgG are collected. Monoclonal antibodies are labeled with digoxigenin according to the same procedure.
  • Tissue lysates from tumor samples and healthy control samples are prepared as described in Example 1, "Tissue preparation”.
  • tissue lysate For each tissue sample tested, 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-PROC 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 an POD-conjugated polyclonal sheep anti- rabbit IgG antibody, diluted to 10 mU/ml in 0.5x Roti ® -Block blocking buffer. After incubation for 1 h, the membranes are washed 6 times in PBS/0.05% Tween-
  • the membrane is incubated with the Lumi-Light Western Blotting Substrate (Order-No. 2015196, Roche Diagnostics GmbH, Mannheim, Germany) and exposed to an autoradiographic film.
  • Lumi-Light Western Blotting Substrate Order-No. 2015196, Roche Diagnostics GmbH, Mannheim, Germany
  • a sandwich ELISA For detection of PROC in a processed human stool sample, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-PR.OC polyclonal antibody (see Example 2) are conjugated with biotin and digoxygenin, respectively. Streptavidin-coated 96-well microwell plates are incubated with 100 ⁇ l biotinylated anti-PROC 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 PROC, plates are washed three times with 0.9% NaCl, 0.1% Tween-20. For specific detection of bound PROC, wells are incubated with 100 ⁇ l of digoxygenylated anti-PROC polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 M 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 M phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. Plates are subsequently washed three times with the same buffer. For detection of antigen-antibody complexes, wells are incubated with 20 mU/ml anti- digoxigenin-POD conjugates (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1633716) for 60 min in 10 M phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. Plates are subsequently washed three times with the same buffer. For detection of antigen-antibody complexes, wells are incubated with 20 mU/ml anti- digoxigenin-POD conjugates (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1633716) for 60 min in 10 M
  • 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 T ls -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.
  • PROC 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.

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Abstract

L'invention concerne le diagnostic du cancer colorectal et, plus particulièrement, l'utilisation de la protéine pyrroline-5-carboxylate réductase (PROC) dans le diagnostic du cancer colorectal. L'invention concerne également une méthode de diagnostic du cancer colorectal à partir d'un échantillon de selles tiré d'un sujet, cette méthode consistant à mesurer la protéine PROC dans ledit échantillon. La mesure de la protéine PROC peut, par exemple, être utilisée dans la détection ou le diagnostic précoce du cancer colorectal.
PCT/EP2005/003263 2004-03-30 2005-03-29 Utilisation de la pyrroline-5-carboxylate reductase en tant que marqueur pour le cancer colorectal WO2005095978A1 (fr)

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EP04007621 2004-03-30
EP04007621.8 2004-03-30

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WO2005095978A1 true WO2005095978A1 (fr) 2005-10-13

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007071366A1 (fr) * 2005-12-21 2007-06-28 Roche Diagnostics Gmbh Méthode d'évaluation du cancer colorectal par mesure de l'hémoglobine et de la pyruvate-kinase m2 dans un echantillon de selles
WO2008138522A1 (fr) * 2007-05-10 2008-11-20 Roche Diagnostics Gmbh Utilisation de timp-1 comme marqueur du cancer colorectal
EP2059607A4 (fr) * 2006-08-23 2010-03-24 Ca Nat Research Council Procédé moléculaire destiné au diagnostic du cancer du colon

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002099043A2 (fr) * 2001-06-05 2002-12-12 Exelixis, Inc. P5crs identifies comme des modificateurs de la voie p53 et procedes d'utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002099043A2 (fr) * 2001-06-05 2002-12-12 Exelixis, Inc. P5crs identifies comme des modificateurs de la voie p53 et procedes d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEVIN BERNARD ET AL: "Emerging technologies in screening for colorectal cancer: CT colonography, immunochemical fecal occult blood tests, and stool screening using molecular markers.", CA: A CANCER JOURNAL FOR CLINICIANS. 2003 JAN-FEB, vol. 53, no. 1, January 2003 (2003-01-01), pages 44 - 55, XP002333004, ISSN: 0007-9235 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007071366A1 (fr) * 2005-12-21 2007-06-28 Roche Diagnostics Gmbh Méthode d'évaluation du cancer colorectal par mesure de l'hémoglobine et de la pyruvate-kinase m2 dans un echantillon de selles
EP2059607A4 (fr) * 2006-08-23 2010-03-24 Ca Nat Research Council Procédé moléculaire destiné au diagnostic du cancer du colon
WO2008138522A1 (fr) * 2007-05-10 2008-11-20 Roche Diagnostics Gmbh Utilisation de timp-1 comme marqueur du cancer colorectal

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