WO2009053354A1 - Utilisation de la ténascine w en tant que biomarqueur pour le cancer du côlon - Google Patents

Utilisation de la ténascine w en tant que biomarqueur pour le cancer du côlon Download PDF

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WO2009053354A1
WO2009053354A1 PCT/EP2008/064199 EP2008064199W WO2009053354A1 WO 2009053354 A1 WO2009053354 A1 WO 2009053354A1 EP 2008064199 W EP2008064199 W EP 2008064199W WO 2009053354 A1 WO2009053354 A1 WO 2009053354A1
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Prior art keywords
tenascin
serum
concentration
protein
blood
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PCT/EP2008/064199
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English (en)
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Ruth Chiquet-Ehrismann
Martin Degen
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Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research
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Priority to CN200880112766A priority Critical patent/CN101836115A/zh
Priority to EP08843203A priority patent/EP2205977A1/fr
Priority to JP2010529415A priority patent/JP2011501147A/ja
Priority to CA2702623A priority patent/CA2702623A1/fr
Publication of WO2009053354A1 publication Critical patent/WO2009053354A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]

Definitions

  • the present invention provides a novel blood, plasma or serum biomarker for colon cancer.
  • Tumour development and progression is not only dependent on the acquisition and accumulation of mutations leading to genetic alterations in cancer cells (for reviews see ll 2 ), but in addition depends on the cross-talk between the transformed epithelium and the tumour stroma, consisting of various cell types (e.g. activated fibroblasts, angiogenic endothelial cells, infiltrating inflammatory cells) and modified extracellular matrix (ECM) 3
  • ECM extracellular matrix
  • the adhesion-modulating ECM molecule tenascin-C is expressed cfe novo by activated fibroblasts in most solid tumours 7"9 .
  • Tumour promoting activities of tenascin-C include abolishment of cancer cell spreading on fibronectin through blocking of syndecan-4 1Ol t1 promotion of cancer cell proliferation 10 , induction of angiogenesis 12 , and enhanced cell invasiveness by up-regulating MMP-12 13 .
  • tenascin-C can trigger oncogenic signalling pathways such as EGFR 14 15 , ERK/MAPK and Wnt 16 .
  • tenascin-C is part of the gene expression sig nature that identifies metastatic breast cancer cells preferentially metastasizing to the lung 1? . Since tenascin-C is absent or expressed at greatly reduced levels in the adult organism, but gets re-expressed in tumours 18 , it was reasonable to assume that increased te ⁇ ascin-C expression in most cancers may be reflected in elevated tenascin-C levels in body fluids.
  • tenascin-C has been reported in serum of patients with different cancer types, including glioma, prostate or colorectal carcinomas, metastatic melanoma, squamous cell carcinoma of head and neck, and non-small cell lung cancer 19 ⁇ 25 .
  • the values for tenascin-C in serum of cancer patients were scattered over a wide range and a significant fraction of these patients had normal concentrations, resulting in a low sensitivity of tenascin-C measurement for detecting cancers.
  • Tenascin-W was originally identified in zebrafish where it was shown to be co-expressed with tenascin-C by neural crest cells and in somites 32 . More recently, murine 33 and chicken 34 tenascin-W have been described and in both of these animals tenascin-W was expressed in developing and adult smooth muscle cells and bone. Chicken tenascin-W function includes modulation of calvarial cell adtiesiorrand spreading in vitro 34 . The first study linking tenascin-W expression to the presence of a tumour was performed in mice.
  • tenascin-W is highly expressed in the tumour stroma sharing its prominent expression with tenascin-C 35 (see also WO-A-03/080663) .
  • Functional studies identified mouse tenascin-W as a molecule promoting migration of mammary cancer cells 35 .
  • the inventors confirmed the presence of tenascin-W in the stroma of human breast cancer tissues 36 .
  • tenascin-W is not limited to the cancer stroma and that tenascin-W could be used as a blood or serum biomarker for colon cancers.
  • the blood or serum levels of tenascin-W are higher in patients having a higher risk of recurrence of colon cancer.
  • the present invention thus provides a method of detecting, diagnosing or prognosing a colon cancer comprising measuring the concentration of tenascin-W protein in a blood, plasma or serum sample obtained from an individual, and (b) comparing the concentration measured in step (a) with a standard value for the concentration of tenascin-W protein in the blood, plasma or serum of healthy individuals, wherein an increased concentration of tenascin-W as compared to the standard value for the concentration of tenascin-W protein in the blood, plasma or serum of healthy individuals is indicative for the presence of a colon cancer.
  • the present invention also encompasses a kit for detecting, diagnosing or prognosing a colon cancer in a blood, plasma or serum sample comprising an antibody, or a fragment thereof, specific for tenascin-W.
  • an embodiment of the present invention is the use of the blood, plasma or serum concentration of tenascin-W protein as a biomarker for colon cancer.
  • Another embodiment of the present invention encompasses an antibody, or a fragment thereof, specific for tenascin-W for use in diagnostic for colon cancer by measuring blood, plasma or serum concentration of tenascin-Wprotein,
  • a further embodiment of the present invention is tenascin-W as a blood, plasma or serum biomarker for colon cancer.
  • FIG. 1 Overview of the antibod ies raised against tenascin-W
  • FIG. 4 Frozen colon tissue microarray lmmunohistochem istry with a mAb against TNC 1 pAb (3F/4) against TNW and Hematoxylin and Eosin stained sections (H&E) of a frozen colon TMA of the patients indicated are shown (cf. Table 111).
  • Norma! colon mucosa patient 37
  • TNC is also strongly expressed in colon smooth muscle (patient 36).
  • Patient 26 shows an example of a border of a tumor with the adjacent normal tissue. In the tumor patients 1-32 (Table III) analyzed it was found that TNW is exclusively localized in the tumor stroma.
  • TNW-positive areas seemed to be a subset of the TNC-positive region (patient 12).
  • Patients 15 and 5 show large (+++) areas of tenascin positivity.
  • Patients 4 and 13 display intermediate (++) to low ⁇ +) areas, respectively, of tenascin-W staining. In general, the staining correlates with the amount of tumor stroma present.
  • the inventors assessed the presence of tenascin-W in the stroma of human breast cancer tissues . While pursuing their investigations, the present inventors have realised that, surprisingly, the presence of tenascin-W is not limited to the cancer stroma and that tenascin-W could be used as a blood or serum biomarker for colon cancers. Moreover, the present inventors realised that blood or serum levels of tenascin-W are higher in patients having a higher risk of recurrence of colon cancer.
  • the present invention thus provides a method of detecting, diagnosing or prog nosing a colon cancer comprising measuring the concentration of tenascin-W protein in a blood, plasma or serum sample obtained from an individual, and (b) comparing the concentration measured in step (a) with a standard value for the concentration of tenascin-W protein in the blood, plasma or serum of healthy individuals, wherein an increased concentration of tenascin-W as compared to the standard value for the concentration of tenascin-W protein in the blood, plasma or serum of healthy individuals is indicative for the possible presence of a colon cancer.
  • a “biood, plasma or serum sample” is the material being analyzed, or measured, which can be, but not necessarily, subjected to pretreatment to provide the tenascin-W in assayable form.
  • This entails methods which are known in the art (for example, see Scopes, Protein Purification: Principles and Practice, Second Edition ⁇ Springer-Verlag, N.Y., 1987)).
  • Scopes, Protein Purification: Principles and Practice, Second Edition ⁇ Springer-Verlag, N.Y., 1987) there is no limitation on the collection and handling of blood, plasma or serum samples as long as consistency is maintained.
  • the sample is obtained by methods known in the art.
  • Consistency of measurement of tenascin-W or tenascin-W activity in blood, piasma or serum samples can be ensured by using a variety of techniques.
  • another enzymatic activity such as alkaline phosphatase
  • an internal standard can be measured concurrently with tenasci ⁇ -W in the sample as a control for assay conditions.
  • the analyzing, or measuring , step can comprise detecting a control protein in the sample, optionally normalizing the value obtained for tenascin-W with a signal obtained with the control protein.
  • the presence of tenascin-W in the blood, plasma or serum sample can be determined by detecting the tenascin-W protein using methods known in the art.
  • tenascin-W can be detected by immunoassays using antibodies specific for tenascsn-W, The antibody can be used, for example, in Western blots of two dimensional gels where the protein is identified by enzymelinked immunoassay or in dot blot (Antibody Sandwich) assays of total cellular protein, or partially purified protein.
  • the blood, plasma or serum concentration of tenascin-W is measured by ELISA in a manner well- known in the art.
  • the te ⁇ ascin-W present in the blood, plasma or serum sample can be concentrated, by precipitating with ammonium sulfate or by passing the blood, plasma or serum sample through a commercially available protein concentration filter, e.g., an Amicon or Millipore, ultrafiltration unit.
  • the blood, plasma or serum sample can be applied to a suitable purification matrix, such as an anion or a cation exchange resin, or a gel filtration matrix, or subjected to preparative gel electrophoresis .
  • a suitable purification matrix such as an anion or a cation exchange resin, or a gel filtration matrix, or subjected to preparative gel electrophoresis .
  • the tenascin-W and protein yield after each purification step needs to be considered in determining the amount of te ⁇ ascin-W in a sample.
  • an at least about two-fold increase of the concentration of tenascin-W measured in step (a) as compared to the standard value for the concentration of te ⁇ ascin-W protein in the blood, plasma or serum of healthy individuals is indicative for a higher likelihood of recurrence of a colon cancer.
  • this threshold could be increased depending on the circumstances. The increase could hence for instance be at least about 2.5-, 3-, 3.5-, 4-, 4.5- or 5-fold.
  • "about” means +/- 10%.
  • the concentration of tenascin-C protein in the blood, plasma or serum sample obtained from the individual can also be measured, and compared to a standard value for the concentration of tenascin-C protein in the blood, plasma or serum of healthy individuals.
  • the concentration of tenascin-C can be measured as described herein for tenascin-W.
  • concentration of tenascin-C can be measured similarly than that of tenascin-W.
  • different methods can be used to measure both proteins in a blood, plasma or serum sample.
  • the concentration of tenascin-W and/or tenascin-C protein can be measured using an antibody, or a fragment thereof, specific for tenascin-W and/or te ⁇ ascin-C.
  • tenascin-W may be detected using an antibody specific for tenascin-W, and a control assay can be carried out using an antibody specific for another tenascin molecule.
  • antibodies, or a fragment thereof, specific for tenascin-W for use in diagnostic for colon cancer by measuring blood, plasma or serum concentration of tenascin-W protein.
  • Methods for producing antibodies are well known in the art.
  • An antibody specific for tenascin-W can be easily obtained by immunizing an animal with an immunogenic amount of the polypeptide. Therefore, an antibody of the invention embraces polyclonal antibodies and antiserum which are obtained by immunizing an animal, and which can be confirmed to specifically recognize tenascin-W by Western blotting, ELISA, immunostaining or other routine procedure known in the art.
  • a polyclonal antibody can be obtained by sensitization
  • a monoclonal antibody secreted by a hybridoma may be obtained from the lymphocytes of the sensitized animal (Chapter 6, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Therefore, monoclonal antibodies, or a fragment thereof, specific for tenascin- W for use in diagnostic for colon cancer by measuring blood, plasma or serum concentration of tenascin-W protein are also provided by the invention.
  • Recombinant antibodies can be expressed by transient or stable expression vectors in mammalian cells, as in Norderhaug (1997) J. Immunol. Methods 204: 77-87.
  • an antibody specific for tenascin-W for use in diagnostic for colon cancer by measuring blood, plasma or serum concentration of tenascin- W protein also embraces an active fragment thereof.
  • An active fragment means a fragment of an antibody having activity of antigen-antibody reaction. Specifically named, these are active fragments, such as F(ab')2, Fab', Fab, and Fv.
  • F(ab')2 results if the antibody of this invention is digested with pepsin
  • Fab results if digested with papain.
  • Fab 1 results if F(ab')2 is reduced with a reagent such as 2-mercaptoethanol and alkylated with monoiodoacetic acid.
  • Fv is a mono active fragment where the variable reg ion of heavy chain and the variable reg ion of light chain are connected with a linker.
  • a chimeric antibody is obtained by conserving these active fragments and substituting the fragments of another animal for the fragments other than these active fragments.
  • humanized antibodies are also envisioned.
  • the term "antibody” also encompasses scFv and antibody-like molecules able to specifically bind to tenascin-W, e.g. aptamers of CDRs grafted onto alternative scaffold, which are well-known to the skilled person.
  • Methods for detecting t ⁇ nascin-W embrace, for example, the use of an antibody as referred to above, optionally with the use of an enzyme reaction.
  • the antibody recognizing tenascin- W can be detected using secondary antibodies specific for the tenascin-W antibody, which are optionally labeled with a radiolabel, an enzyme, avidin or biotin, or fluorescent materials (FITC or rhodamine), for example.
  • the antibody, or fragment thereof, recognizing tenascin-W is itself labeled.
  • a label may be any detectable composition whereby the detection can be spectroscopic, photochemical, biochemical, immunochemical, physical or chemical.
  • useful labels can include 32 P, 35 S, 3 H, 14 C, 125 L 131 I 1 fluorescent dyes (e.g. FITC, rhodamine and lanthanide phosphors), electron-dense reagents, enzymes, e.g. as commonly used in ELiSA (e.g. horseradish peroxidase, beta- galactosidase, luciferase and alkaline phosphatase), biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available.
  • the label may be directly incorporated into a target compound to be detected, or it may be attached to a probe or antibody which binds to the target.
  • incubation and/or washing steps may be required after each application of reagent or incubation of combinations of reagents. Incubation steps may vary from about 5 minutes to several hours, perhaps from about 30 minutes to about 6 hours. However, the incubation time usually depends upon the assay format, analyte, volume of solution, concentrations, and so forth. Usually, the assays should be carried out at ambient temperature, although they may be conducted at temperatures; in the range 4°C to 40'C, for example.
  • a particularly preferred assay format is an enzyme-linked immunosorbent assay (ELlSA).
  • ELlSA enzyme-linked immunosorbent assay
  • the methods and uses of the present invention can be performed in vitro, e.g. ELISA, as well as ex vivo or in vivo, e.g. online monitoring using antibodies fixed on a support.
  • the present invention provides a kit for detecting, diagnosing or prognosing a colon cancer in a blood, plasma or serum sample comprising an antibody, or a fragment thereof, specific for tenasci ⁇ -W.
  • the kit of the Invention may further comprise an antibody, or a fragment thereof, specific for tenascin-C.
  • kits comprise reagents useful for carrying out the methods of the invention, for example, antibodies from one or more species specific for tenascin-W and tenascin-C.
  • Secondary antibodies that recognise either or both such primary anti-tenascin antibodies can also be included for the purpose of recognition and detection of primary antibody binding to a sample.
  • Such secondary antibodies can be labelled for detection e.g. with fluorophores, enzymes, radioactive labels or otherwise. Other detection labels will occur to those skilled in the art.
  • the primary a ⁇ ti-tenascin-W antibodies can be labelled for direct detection.
  • Human tenascin-W and tenascin-C were cloned, expressed and purified as described previously 3S 37 .
  • Fibronectin was purified from filter-sterilized horse serum using a gelatin- agarose column. After washing of the column with PBS, fibronectin was eluted with 4 M Urea and finally dialyzed against PBS.
  • Human fibrinogen was purchased from Sigma (Sigma, Buchs, Switzerland).
  • the forward primer included a BamHI restriction site and the reverse primer a stop codon immediately followed by a Hindi!! restriction site to enable the directional cloning into the bacterial expression vector pQE30 (Qiagen, Hilden, Germany), supplying a C-terminal His tag for the purification of the recombinant fragment.
  • the recombinant fragment corresponding to fibronectin type III domains 3F/4 (Fig. 1a, short line below the molecule) was expressed and purified by affinity chromatography to a Ni-NTA matrix (Qiagen, Hilden, Germany) following the supplier's instructions.
  • cDNA was synthesized from total RNA that was extracted from Saos- 2 cells (ATCC # HTB-85) by TrizolTM reagent (Invitrogen, Carlsbad, CA 1 USA). Primers amplifying the last three FN type III domains, -were used for nested PCR with the Expand High Fidelity System (Roche, Rotnch, Switzerland) using the Saos-2 cDNA as template.
  • the first reaction was performed with the primer set S'-GGGAAGGAGCAGAGTAGCACTG- 3' / 5'-CCGCCTCTGGAAGACAATCC-S', the second reaction with the primers 5'- AGGGATCCGACATTGACAGCCCCCAAAACC-3' / 5'-
  • the forward primer for the second PCR included a BamHI restriction site and the reverse primer a stop codon immediately followed by a Hindlli restriction site to enable the directional cloning into the bacteria! expression vector pQE30 (Qiagen, Hilden, Germany), supplying a C-terminal His tag for the purification of the recombinant fragment.
  • MAbs were obtained by immunizing female BALB/c mice with 34.6 ⁇ g of the purified recombinant tenascin-W fragment emulsified with STIMUNE (ID-Lelystad, Institute for Animal Sciences and Health, Lelystad, NL).
  • mice were injected twice with a 4-week interval with 25 ⁇ g tenascin-W fragment.
  • Splenic lymphocytes were fused with the myeloma cell line P3X63Ag8.653 (ATTC # CRL- 1580) and cultured according to standard protocols.
  • the hybridoma supernatants were analyzed by ELISA and western blot analysis of a tenascin-W fragment expressed in HEK293 cells using a construct containing the sequence of the last three FN III domains of tenascin-W fused to an N-terminal fragment of chicken tenascin-C containing its secretion signal and the epitope for the well characterized anti-chicken tenascin-C antibody mAb 60 38 IgGs from two mAb hybridoma clones (mAb 29A and mAb 560) were purified from conditioned medium by protein G sepharose (Amersham, Oteifingen, CH) and used in this study.
  • Sandwich-ELISA lmmunolon 1 flat-bottomed 96-weli plates (Dynatech Laboratories, Chantilly, VA, USA) were coated with 50 ⁇ l of mAb 29A (capture antibody, 10 ⁇ g/ml in PBS) for 1 hour at 37°C and blocked with 4 % mi!k in PBS (blocking and washing solution) for another hour at room temperature. After washing the plate twice, 50 ⁇ t of serum samples or standard ⁇ purified human tenascin-W), both diluted in blocking solution, were incubated for 1 hour at 37 0 C. Each serum was assayed in a 2-fold dilution series ranging from 1 :2 to 1 :8.
  • sandwich-ELlSAs were performed as described previously 21 using the anti-human tenascin-C mAb B28.13 as capture antibody, a polyclonal chicken anti-human tenascin-C as detection antibody (1 :500) and a peroxidase-labeled goat anti- chicken IgG (1 :15,000), and with purified human tenascin-C as standard.
  • Patients were between the age of 48 and 94 years (average age 70.3 years), presenting non-metastatic colorectal cancer (CRC) and scheduled for tumor resection.
  • Exclusion criteria included patients with less than 10 g/dl hemoglobin, leucopenia, thrombocytopenia, deep venous thrombosis during the last three months, long term vascular catheter, chronic vascular or inflammatory disease, surgery and chemotherapy during the last 4 and 6 months respectively.
  • Fresh human tissue was frozen on dry ice immediately after surgery. For the processing of the tissue, it was thawed on ice, minced and homogenized in lysis buffer (100 mM phosphate buffer pH 8.0, 300 mM NaCI, 8 M " Urea, 1 % Triton-X-100, 10 mM beta- mercaptoethanol, 50 mM Guanidium Hydrochloride and complete protease inhibitor cocktail (Roche, Rot Regen, Switzerland)). Insoluble material was pelleted and reducing SDS-PAGE sample buffer was added to the supernatant and boiled for 5 minutes at 95°C.
  • lysis buffer 100 mM phosphate buffer pH 8.0, 300 mM NaCI, 8 M " Urea, 1 % Triton-X-100, 10 mM beta- mercaptoethanol, 50 mM Guanidium Hydrochloride and complete protease inhibitor cocktail (Roche, Rotsville, Switzerland)
  • proteins were electro-transferred onto polyvinyldifluoride membrane (Millipore, Bedford, MA, USA) using a semi-dry blotting apparatus (Millipore, Bedford, MA, USA). After the transfer, membranes were stained with amido black to control equal protein loading.
  • membranes were incubated overnight with either the polyclonal tenascin-W antiserum (pAb (3F/4); 1 :750), the mAb 560 raised against tenascin-W (1 :1000), the mAb B28-13 raised against tenascin-C (1 :100) 2 ⁇ or the mAb against vinculin (1 :2000; Sigma, Buchs, Switzerland) followed by an incubation for 1 hour with anti-rabbit IgG or anti-mouse IgG coupled to horseradish peroxidase (1 :10,000), respectively. Blots ware developed using Super Signal (Pierce, Rockfcrd, SL, USA) and exposed to Kodak BioMax MR Films.
  • TMA frozen tissue microarray
  • Table 111 patients 1-38.
  • the TMA was constructed in frozen Tissue-Tek OCT compound (Miles Laboratories, Naperville, IL, USA) as described previously * ⁇
  • the present inventors optimized a commercial microarray device (Beecher Instruments, Sun Prairie, Wl, USA) by using a 0.6 mm dri ⁇ for recipient hole making instead of the conventional hoilow needle.
  • AII immunostainings were performed using the Discovery XT automated stainer (Ventana Medical Systems, Arlington, AZ, USA), with DAB Map detection kit ⁇ Ventana).
  • Frozen tissue slides were dried for 1 hour at room temperature, fixed for 10 minutes at 4 0 C in acetone and then introduced into the automate. No pretreatment was required for any staining. Slides were first blocked twice for 12 minutes with the AB Block reagent ⁇ Ventana). Then they were incubated for 1 hour at 37°C with mAb B28-13 against tenascin-C (1 :2500), and mAb 560 against tenascin-W (1 :800). Afterwards, slides were treated for 32 minutes at 37°C with a biotinylated universal secondary antibody ⁇ Ventana) and counterstained with hematoxylin and bluing reagent (Ventana).
  • Serum tenascin-W levels were analyzed by a sandwich-ELISA using mAb 29A as capture antibody and pAb (FL) as detection antibody ⁇ Fig. 1 a). Specificity of the detection antibody was assessed by western blot analysis of tenascin-W conditioned medium, purified fibronectin, fibrinogen, tenascin-C, and tenascin-W (Fig. 1 b).
  • the anti-tenascin-W antiserum pAb (FL) specifically reacts with the human tenascin-W conditioned medium and with the purified tenascin-W, but did not show any cross-reactivity with tenascin-C or any of the other related proteins tested, which are known to be present in serum (Fig. 1 b).
  • their detection antibody recognizes serum contaminants potentially binding to tenascin-W
  • the present inventors used two distinct polyclonal anti-human tenascin-W antibodies pAb (FL) and pAb (3F/4) for tenascin-W detection.
  • Table Ia shows that both polyclonal detection antibodies measure similar tenascin-W concentrations in the same serum samples making it highly unlikely that they cross-react with contaminating serum proteins possibly bound to tenascin-W.
  • pAb (FL) recognizes different epitopes of tenascin-W than pAb (3F/4) since, in contrast to pAb (3F/4) , its reactivity with tenascin-W can not be blocked by the addition of the bacterial expressed fragment used to raise pAb (3F/4) (Fig. 1c).
  • the coefficient of variation (CV) was 5.6 +/- 1.3 % in the within-run and 6.52 +/- 4.9 % in the between-run. Therefore, the present inventors have established a sensitive, specific and precise sandwich-ELISA to detect tenascin-W in human serum samples.
  • Serum TNW ⁇ evel ⁇ analyzed by sandwich-ELISA using two different detection antibodies pAb(FL) and pAb(3F/4 ⁇ Serum TNW loveis in all samples tested did not differ significantly between Ih ⁇ two detection antibodies as revealed by student's L-test (p>0 05 ⁇
  • Tenascin-C was shown to be elevated in serum samples of colorectal cancer patients 20 ' 25 . There was however no indication that the same could be true for tenascin-W. Nevertheless, the present inventors measured tenascin-W levels in serum of 17 non-metastatic (at time of diagnosis) colorectal cancer patients and compared it to the levels monitored in the serum of 25 healthy volunteers. The mean value of tenasci ⁇ -W in the control sera was 0.389 +/- 0.14 mg/l (Fig. 2a). This is about half of the published mean serum tenascin-C level in healthy individuals 21 ' 42 .
  • Serum tenascin-W concentrations in colorectal cancer patients were significantly elevated compared to those in healthy individuals (p ⁇ 0.01) with a mean level of 0.794 +/- 0.38 mg/l (Fig. 2a). This corresponds to a 2-fold increase in comparison to healthy volunteers, lmmunoprecipitation (IP) followed by immunoblot analysis of serum samples resulted in the detection of a single tenascin-W-specific band, corresponding to the size of the purified full-length tenascin-W (data not shown). This indicated that the present inventors were measuring intact mature tenascin-W protein in the serum.
  • the present inventors also assayed the same sera for tenascin-C and obtained a mean tenascin-C level in healthy volunteers of 0.775 +/- 0.25 mg/l (Fig. 2b), which nicely matches the range described in the literature 21 42 .
  • the mean tenascin-C value in colorectal cancer sera was 0.98 +/- 0.24 mg/l and corresponds to a 1.2 fold increase compared to control sera (Fig. 2b).
  • Clinicopathologic features of the colorectal cancer patients available for their study as well as their individually measured tenascin-W and tenascin-C levels are given in Table II.
  • tenascin-W might be a specific serum marker for colorectal cancers or whether tenascin-W might have a broader spectrum of application
  • the present inventors analyzed tenascin-W in serum of 16 non-metastatic (at time of diagnosis) breast cancer patients and compared it to the levels monitored in healthy volunteers.
  • the mean serum value of tenascin-W in the breast cancer cohort was 0.682 +/- 0.43 mg/l. This corresponds to a statistically significant (p ⁇ 0.02) 1.75-fold increase compared to healthy volunteers ⁇ Fig. 2c). From these data the present inventors conclude that serum levels of tenascin-W are elevated in non-metastatic breast and colon cancer patients at time of first curative-intent surgery compared to healthy individuals.
  • tenascin-W was not detectable even though the tumor tissue extracts of the same patients revealed strong tenascin-W expression ⁇ patients K and L). This is in contrast to tenascin-C which was also detectable in normal colon mucosae, but at a much reduced level compared to the corresponding tumor sample.
  • tenascin-C the present inventors detected different isoforms, whereas for tenascin-W they mostly observed a single band in the extracts tested.
  • tenascin-W and tenascin-C stained frozen sections of normal colon mucosa and tumor tissue of patients K and L by immunohistochemistry. The staining pattern was the same for both patients. As shown for patient L (Fig. 3b) tenascin-C as well as tenascin-W staining revealed a very strong expression in the tumor stroma, the specialized connective tissue surrounding cancer cells. The normal colon mucosa was positive for tenascin-C but not tenascin-W consistent with the immunoblot findings.
  • the tenascin-C expression was strong in the muscularis mucosa and was also present surrounding the glands.
  • immunohistochemical staining of frozen sections confirmed the result obtained by immunoblotting of the same colorectal tumor extracts and indicated that tenascin-W is a novel marker for activated tumor stroma in colorectal cancer.
  • TMA frozen colon tissue micro array
  • Fig. 4 Examples of the different staining patterns observed are s hown in Fig. 4. Most of the tenascin-C positivity in normal colon tissue is seen in smooth muscle (Fig. 4, patient 36). In some of the tumors tenascin-W and tenascin-C staining s seemed to overlap entirely (Fig. 4, patients 4, 5), whereas in others the tenascin-W positive areas were a subset of the tenascin-C positive region (Fig. 4, patient 12). As can be observed in Fig. 4.
  • the tenascin-C positive and tenascin-W negative area in tumors may also be due to the presence of smooth muscle, as can be seen at the boarder of a tumor and normal tissue (Fig. 4, patient 26).
  • the extent of staining always overlapped with the stromal compartment of the tumor as judged from the H&E stained sections (Fig.4).
  • Table III patient data are summarized and the staining patterns are classified according to the area of the staining observed. This shows that tenascin-C has a slightly broader distribution than tenascin-W. Since tenascin-C is strongly expressed in smooth muscle of the colon, this may reflect the presence of smooth muscle cells within the cancer stroma.
  • colorectal cancer was the second major cause of cancer deaths in Europe, accountable for 207,400 deaths (12.2 % of total cancer deaths) 43 .
  • Colorectal carcinomas develop in defined stages over several years M . Deaths from colorectal cancer may be prevented inmost cases if the tumor is detected at an early stage (polyp or non-invasive carcinoma) and removed by endoscopic polypectomy or surgery. In this context, effective screening tools are of utmost importance.
  • CCA carcinoembryonic antigen
  • the tumor M2-Pyruvate kinase a dimeric form of the enzyme that catalyzes the last reaction within the glycolytic pathway was found to be up-regulated in both plasma and fecal samples of patients suffering from gastrointestinal cancers, including colorectal cancers 49"53 .
  • the most effective available method to prevent and detect colorectal cancer today is colonoscopy.
  • this investigation is invasive, time- consuming, uncomfortable, and relatively expensive 54 .
  • the frequency of the colonoscopic exams every 3 to 5 years is limited by these factors and it is not rare to observe the development of cancer between two colonoscopies.
  • surrogate markers of colon cancer detectable in the blood may be a convenient method for screening and early cancer detection.
  • tenascin-C levels have often been analyzed in sera of csncer patients and its potential vslue as a biomarker has been evaluated 21"23 55 ' 58 . Although elevated tenascin-C serum levels have been found in certain cancers, it still remains a questionable tumor marker 21 . Its levels are scattered over a wide range with many cancer patients having normal tenascin-C concentrations and its expression strongly correlates with sites of inflammation or infection 18 . In this example the present inventors evaluate tenascin-W for its potential to serve as biomarker in colorectal cancer.
  • a sensitive, precise and tenascin-W-specific sandwich-ELISA was established using different anti-tenascin-W antibodies and the purified protein as a standard.
  • the present inventors were able to detect and measure tenascin-W in the serum of healthy volunteers and colorectal cancer patients.
  • the mean serum tenascin-W level in healthy individuals was 0.389 +/- 0.145 mg/l.
  • Homogeneously low levels in a control population is a strict requirement for a cancer biomarker,
  • serum levels of tenascin-C in controls were 2 ⁇ fold higher and scattered over a broad range.
  • tenascin-W or of any other biomarker in serum
  • any co-morbidity e.g. cardiovascular diseases, inflammatory disease
  • tenascin-C serum levels were shown to correlate with inflammation rather than tumorigenesis 21 , but no correlation could be found linking tenascin-W to the presence of infection or inflammation (own unpublished observations).
  • the present inventors confirmed the tendency of tenascin-C to be present at higher levels in the serum of colorectal cancer patients (0.98 +/- 0.24 mg/l), however, the increase was much more moderate than for tenascin-W.
  • the present inventors neither could observe a correlation between the levels of the two tenascins, nor with the classification and staging of the primary tumor.
  • the present inventors also monitored a clear increase (1.75-fold) in the mean serum tenascin-W level in non -metastatic breast cancer patients compared to that in healthy individuals. This indicates that tenascin-W is not a biomarker specific for adenocarcinomas of the colon, but might have a broader spectrum for possible applications.
  • the present inventors investigated tumor extracts of 11 colorectal cancer patients and for some of them, corresponding normal tissue by immunoblotting .
  • the present inventors could show that tenascin-W as well as tenascin-C was detectable in the tumor tissue in 82 % of the patients. Since the immunoblotting analysis of tumor tissue was performed on different colorectal cancer patients the present inventors cannot make direct correlations between local tenascin-W expression in the tumor and corresponding circulating levels in the serum.
  • Tenascin-W was absent from normai colon mucosa, in contrast to tenascin-C, which is expressed (at reduced levels) in the norma!
  • stromal staining of tenascin-C and tenascin-W was confirmed on a frozen TWIA containing 32 different colon cancer sections. This study revealed that all of the tumors were stained for both tenascins and that the staining correlated with the amount of tumor stroma present. Similar observations have been made in human breast tumors. Tenascin-W and tenascin-C were shown to be highly expressed in a large fraction of human breast tumors. In contrast to normal colon mucosae, which express tenascin-C, normal mammary parenchyma was negative for both tenascins 36 .
  • Yamauchi M Mizuhara Y, Maezawa Y, Toda G Serum tenascin levels in chronic liver disease Liver 1994,14 148-53 Tanaka H, El-Karef A, Katto M, Kinoshita N 1 Fujita N, Ho ⁇ ike S, Wata ⁇ abe S, Yoshida T, Adachi Y Circulating level of large splice variants of tenascin-C is a marker of piecemeal necrosis activity in patients with chronic hepatitis C Liver lnt 2006,26 31 1 8
  • the tumor marker tumor M2-PK an application in the diagnosis of gastrointestinal cancer. Anticancer Res 2000;20:4961 -4.
  • Gazzaniga P Nofroni I 1 Gandini O, Silvestri I, Frati L, Agliano AM, Gradilone A. Tenascin C and epidermal growth factor receptor as markers of circulating tumoral cells in bladder and colon cancer. Oncol Rep 2Q05;14:1199-202.

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Abstract

La présente invention propose un procédé pour détecter, diagnostiquer ou pronostiquer un cancer du côlon, qui consiste à mesurer la concentration de la protéine ténascine W dans un échantillon de sang, de plasma ou de sérum obtenu auprès d'un individu, et (b)à comparer la concentration mesurée à l'étape (a) avec une valeur standard pour la concentration de la protéine ténascine W dans le sang, le plasma ou le sérum d'individus sains, une concentration accrue de la ténascine W par comparaison avec la valeur standard pour la concentration de la protéine ténascine W dans le sang, le plasma ou le sérum d'individus sains étant indicative de la présence d'un cancer du côlon. L'invention concerne également des trousses et des anticorps destinés à cet effet.
PCT/EP2008/064199 2007-10-22 2008-10-21 Utilisation de la ténascine w en tant que biomarqueur pour le cancer du côlon WO2009053354A1 (fr)

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CN200880112766A CN101836115A (zh) 2007-10-22 2008-10-21 生腱蛋白-w用作结肠癌生物标记的用途
EP08843203A EP2205977A1 (fr) 2007-10-22 2008-10-21 Utilisation de la ténascine w en tant que biomarqueur pour le cancer du côlon
JP2010529415A JP2011501147A (ja) 2007-10-22 2008-10-21 結腸癌のバイオマーカーとしてのテネイシン−ｗの使用
CA2702623A CA2702623A1 (fr) 2007-10-22 2008-10-21 Utilisation de la tenascine w en tant que biomarqueur pour le cancer du colon

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WO2003080663A2 (fr) * 2002-03-27 2003-10-02 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Compositions a base de tenascine-w et leurs utilisations

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WO2003080663A2 (fr) * 2002-03-27 2003-10-02 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Compositions a base de tenascine-w et leurs utilisations

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AKIHIKO TAKEDA ET AL: "Clinical Significance of Large Tenascin-C Spliced Variant as a Potential Biomarker for Colorectal Cancer", WORLD JOURNAL OF SURGERY ; OFFICIAL JOURNAL OF THE INTERNATIONAL SOCIETY OF SURGERY/SOCIÉTÉ INTERNATIONALE DE CHIRURGIE, SPRINGER-VERLAG, NE, vol. 31, no. 2, 5 January 2007 (2007-01-05), pages 388 - 394, XP019472760, ISSN: 1432-2323 *
DEGEN MARTIN ET AL: "Tenascin-W is a novel marker for activated tumor stroma in low-grade human breast cancer and influences cell behavior", CANCER RESEARCH, vol. 67, no. 19, 1 October 2007 (2007-10-01), pages 9169 - 9179, XP002464177, ISSN: 0008-5472 *
DEGEN MARTIN ET AL: "Tenascin-W, a new marker of cancer stroma, is elevated in sera of colon and breast cancer patients", INTERNATIONAL JOURNAL OF CANCER, vol. 122, no. 11, June 2008 (2008-06-01), pages 2454 - 2461, XP002508351, ISSN: 0020-7136 *
SCHERBERICH ARNAUD ET AL: "Tenascin-W is found in malignant mammary tumors, promotes alpha8 integrin-dependent motility and requires p38MAPK activity for BMP-2 and TNF-alpha induced expression in vitro", ONCOGENE, vol. 24, no. 9, 24 February 2005 (2005-02-24), pages 1525 - 1532, XP002464178, ISSN: 0950-9232 *

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