EP2205977A1 - Use of tenascin-w as a biomarker for colon cancer - Google Patents

Abstract

The present invention 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. Kits and antibodies therefore are also provided.

Description

Use of Tenascin-W as a Biomarker for colon cancer

Field of the invention

The present invention provides a novel blood, plasma or serum biomarker for colon cancer.

Background of the invention

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| 4}. In recent years it has been recog nized that cancer cells are able to alter and activate their adjacent stroma by secreting soluble factors promoting the formation of a growth-permissive microenvironment required for full neoplastic manifestation ^5"7.

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. Moreover, tenascin-C can trigger oncogenic signalling pathways such as EGFR ^{14 15}, ERK/MAPK and Wnt ¹⁶. Importantly, 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 teπascin-C expression in most cancers may be reflected in elevated tenascin-C levels in body fluids. Indeed, elevated levels of tenascin-C have 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. However, 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. Moreover, high serum tenascin-C levels were clearly correlated with non-cancerous liver diseases including hepatitis and liver cirrhosis ^26"29, and with elevated levels of C- reactive protein (CRP) ²¹, an acute phase protein expressed as a consequence of infection, tissue damage or other inflammatory conditions ^{30 31}. Therefore, a need remains for reliable blood or serum biomarkers for cancer.

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. Using oncogene-induced mammary tumour models, it was shown that 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. Recently, the inventors confirmed the presence of tenascin-W in the stroma of human breast cancer tissues ³⁶.

Summary of the invention

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 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.

Hence, 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.

Description of the figures

Figure 1 - Overview of the antibod ies raised against tenascin-W

(a) Schematic representation of the domain structure of human TNW. The recombinant full- length TNW and the fragments that served as antigens to raise polyclonal and monoclonal antibodies are indicated by lines. The following symbols have been used to identify the structural domains: heptad repeats (wavy line), EGF-like repeats (diamonds), FNHI domains (boxes), FNIiI domains generated by duplication (dark boxes), fibrinogen giobe (circle), (b) Coomassie-stained gel (lanes 1-5) and western blot (lanes 6-10). (1 , 6) TNW conditioned medium 10 μl, (2, 7) fibronectin 2.5 μg, (3, 8) fibrinogen 2,5 μg, (4, 9) purified TNC 2.5 μg, (5) purified TNW 2.5 μg and (10) purified hT NW 500 ng. lmmunoblot analysis shows that the detection antibody pAb (FL) specifically recognizes TNW and that it does not cross-react with the other tested proteins, (c) The reactivity of pAb (FL) with purified tenascin-W (W) can be blocked by incubation with 10 μg/ml of purified teπascin-W (FLhTNW)₁ but not with 10 μg/ml of the bacteriaily expressed FNIlI 3F/4 fragment, while the reactivity of pAb (3F/4) can be blocked by both tenascin-W preparations (d) Log-log presentation of a typical standard curve using mAb 29A as capture antibody, pAb (FL) as detection antibody, and a serial dilution of the purified TNW is shown. Detection limit was reached at TNW concentrations of about 0.005 mg/l. Figure 2 - Tenascin-W serum levels

(a) TNW serum levels of heaithy volunteers (n= 25, mean: 0.389 +/- 0.145 mg/l) and of non- metastatic colorectal cancer patients (n=17, mean: 0.794 +/- 0.38 mg/l) are shown. There is a statisticaily significant increase in mean tenascin-W level (bar) in colorectal cancer patients compared to that in volunteers, (b) Serum TNC levels of healthy individuals and of colorectal cancer patients is displayed. They show an increase of the mean serum TNC level (n=17, mean: 0.98 +/- 0.24 mg/l) compared to that in healthy individuals {n=13, mean: 0.798 +/- 0.24 mg/l). (c) Serum TNW levels in non-metastatic breast cancer patients (n=16, mean: 0.682 +/- 0.44 mg/l) and of healthy volunteers (n=25, mean: 0.389 +/- 0.145 mg/l) are shown. The bars indicate the mean tenascin values.

Figure 3 - Tenascin-W expression in colorectal cancer

(a) Eleven colorectal cancer extracts (T) and three normal colon tissues (N) were tested on immunoblots for the presence of TNW and TNC. Analysis revealed a basal expression of low molecular-weight isoform of TNC in the normal mucosa and up-regulation of mostly large TNC variants in the tumor tissue. The position of the recombinant high molecular weight TNC isoform loaded for comparison is indicated (arrowhead). TNW is detectable in a large fraction of tumoral tissues but not in normal colon mucosae, (b) lmmunohistochemical analysis of frozen sections of patient L confirmed immunoblot results and showed strong staining in the tumor stroma for both tenascins. Whereas no staining was detectable in the normal tissue with respect to TNW, strong staining was observed for TNC in the muscularis mucosa. Calibration bar: 250 μm. N: normal; T: tumor.

Figure 4 - Frozen colon tissue microarray lmmunohistochem istry with a mAb against TNC₁ 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) is negative for TNW, but positive for TNC. 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. In some patients, 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.

Detailed description of the invention

Recently, 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)). In the broader aspects of the invention, 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. For example, to control for the quality of each sample, another enzymatic activity, such as alkaline phosphatase, can serve as an internal control. In addition, an internal standard can be measured concurrently with tenasciπ-W in the sample as a control for assay conditions. Thus, 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. In this invention, there are no limitations on the type of assay used to measure tenascin-W or tenascin-W activity. For example, 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. Preferably, the blood, plasma or serum concentration of tenascin-W is measured by ELISA in a manner well- known in the art.

Methods for sample concentration and protein purification are described in the literature (see Scopes, 1987). For example, if desired, the teπascin-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 . In such cases, the tenascin-W and protein yield after each purification step needs to be considered in determining the amount of teπascin-W in a sample.

in the methods of the invention, 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 teπascin-W protein in the blood, plasma or serum of healthy individuals is indicative for a higher likelihood of recurrence of a colon cancer. The person skilled in the art will understand that 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. For the purpose of the present invention, "about" means +/- 10%.

In some embodiments of the methods of the invention, 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. The concentration of tenascin-C can be measured similarly than that of tenascin-W. Alternatively, different methods can be used to measure both proteins in a blood, plasma or serum sample.

In the methods of the invention, 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 teπascin-C. For instance, 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.

Also provided by the present invention are 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.

It is well known that if 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. Methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art and described in the scientific and patent literature, see, e.g., Coligan, Current Protocols in Immunology, Wiley /Green, NY (1991); Stites <eds.) Basic and Clinical Immunology (7th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein (Stites); Goding, Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press, New York, NY (1986); and Kohler (1975) Nature 256: 495. Such techniques include selection of antibodies from libraries of recombinant antibodies displayed in phage or similar on ceils. See, Huse (1989) Science 246: 1275 and Ward (1989) Nature 341: 544. Recombinant antibodies can be expressed by transient or stable expression vectors in mammalian cells, as in Norderhaug (1997) J. Immunol. Methods 204: 77-87.

For the purposes of the present invention, 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. For example, F(ab')2 results if the antibody of this invention is digested with pepsin, and Fab results if digested with papain. Fab¹ 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. In particular, humanized antibodies are also envisioned. For the purpose of this invention, 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 tβnascin-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. In an alternative embodiment, 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. For example, useful labels can include ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵L ¹³¹I₁ 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.

immunological binding assays are known in the art. For a review, see Methods in Cell Biology Vol. 37; Antibodies in Cell Biology, Asai, (Ed.) Academic Press, Inc. New York (1993).

Throughout the assays of the invention, 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). However, 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.

In an embodiment, 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 tenasciπ-W. The kit of the Invention may further comprise an antibody, or a fragment thereof, specific for tenascin-C. Such 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. Alternatively, the primary aπti-tenascin-W antibodies can be labelled for direct detection.

Unless otherwise defined, ali technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention beiongs. Although methods and materials simϋar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Examples

Materials and Methods

Recombinant proteins

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).

Anti-tenascin-W antibody production

Two polyclonal antisera recognizing human tenascin-W were generated. The first antiserum , called pAb (FL), was raised in rabbits against the purified full-length human tenascin-W ³⁶ (Fig. 1a, indicated by the long line). To raise the second polyclonal antisera in rabbits, a recombinant fragment of human tenascin-W was cloned, bacteriaily expressed, and purified. To clone the recombinant fragment, specific primers were designed to amplify the sequence encoding the last two fibronectin type 111 domains with the Expand High Fidelity PCR System (Roche, Rotkreuz, Switzerland). The cDNA of the full-length human tenascin-W (described above) was used as template and the PCR was performed with the primer set 5'- GAGGATCCGAAATTGACGGCCCCAAAAACC-3' / 5'-

AT AAGCTTATGTGGAGAGGGTGGTGGA-S'. 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. Purification was performed under native conditions and elution by 250 mM imidazole (pH 6.9), The bacterially expressed fragment of teπascin-W was then used to raise polyclonal antisera (pAb (3F/4)) in rabbits using standard immunization procedures. To raise monoclonal antibodies (mAbs) in mice, a recombinant fragment of human tenasciπ- W containing the last three fibronectin type III domains (Fig. 1a, short line above the molecule) was cloned, bacterially expressed, and purified as described above. To clone the recombinant fragment, cDNA was synthesized from total RNA that was extracted from Saos- 2 cells (ATCC # HTB-85) by Trizol™ reagent (Invitrogen, Carlsbad, CA₁ USA). Primers amplifying the last three FN type III domains, -were used for nested PCR with the Expand High Fidelity System (Roche, Rotkreuz, 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'-

CTAAGCTTTCATGTGGAGAGGGTGGTGGATAC-S'. 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 μg of the purified recombinant tenascin-W fragment emulsified with STIMUNE (ID-Lelystad, Institute for Animal Sciences and Health, Lelystad, NL). For boosting , mice were injected twice with a 4-week interval with 25 μg 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 μl of mAb 29A (capture antibody, 10 μg/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 μt of serum samples or standard {purified human tenascin-W), both diluted in blocking solution, were incubated for 1 hour at 37⁰C. Each serum was assayed in a 2-fold dilution series ranging from 1 :2 to 1 :8. Samples were discarded and the plates were washed extensively four times with washing buffer. After incubation with 50 μi of pAb (FL) (detection antibody, 1 :500 in blocking solution, 1 hour at 37⁰C)₁ the plates were washed again four times and incubated with peroxidase-labeled goat anti-rabbit IgG (1 :2000 in blocking solution, 1^~hour at 37°C). After washing the plates four times with washing buffer and twice with PBS, the enzymatic color reaction was carried out by adding 100 μl of reaction buffer (0.1 M citric acid, 0.2 M Na₂HPO₄, 4 mM 1 , 2- phenylendiamine, 0.003 % H₂O₂). The reaction was stopped with 50 μl of 4 M H₂SO₄ and the plates were read with a VERSAMAX Microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 490 nm against 620 nm.

To monitor serum tenascin~C, 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.

Patient population and serum preparation

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.

Healthy volunteers were gender mixed and between 30 and 76 years old, without history of cancer, chronic disease or medication other than hormonal contraception. Although the average age of 43 was lower than that of the cancer patients, there was no indication of an increase of tenascin-W levels with age. Ethical and scientific approvals were given by the local Ethics Committee of the Lausanne University Hospital. Written informed consent was obtained from all the patients and healthy volunteers. For serum preparation, blood was collected in tubes without anticoagulant. After a 10 minute centrifugation at 1 ,00Og, the serum was extracted and frozen at -80° C until use. Histological diagnosis and grade of all tumors was reviewed by one pathologist (SW) according to the WHO Classification of Tumors ³⁹. Tumor stage was determined using the International Union Against Cancer (TNM classification of malignant tumors) ⁴⁰.

Human tissue extracts and western blot analysis

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, Rotkreuz, Switzerland)). Insoluble material was pelleted and reducing SDS-PAGE sample buffer was added to the supernatant and boiled for 5 minutes at 95°C. After electrophoresis on 6 % polyacrylamide gels, 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. After blocking for 1 hour at room temperature in TBS containing 0.05 % Tween and 5 % skim milk powder, 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.

lmmunohistochemistry and Frozen Tissue Microarray

A frozen tissue microarray (TMA) was constructed from frozen tissue samples of 32 colon carcinoma and 6 normal colon mucosae. Pathological features of these samples are summarized in 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 driϋ for recipient hole making instead of the conventional hoilow needle. AII immunostainings were performed using the Discovery XT automated stainer (Ventana Medical Systems, Tucson, AZ, USA), with DAB Map detection kit {Ventana). Frozen tissue slides were dried for 1 hour at room temperature, fixed for 10 minutes at 4⁰C 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).

Results

Precision and sensitivity of the sandwich-ELISA

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). To further rule out that 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. Furthermore, 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).

Testing a serial dilution of their purified tenascin-W, the present inventors concluded that with their newly established sandwich-ELISA, serum tenascin-W concentrations as low as 0.005 mg/l could be detected. A typical standard curve for tenascin-W is shown as log-log presentation in Figure 1 d. When the present inventors tested a different capture antibody (mAb 56O) they obtained the same results as with mAb 29A (data not shown) and decided to use mAb 29A for all further sandwich -ELISAs. The precision of the assay was estimated by testing 4 serum samples three times in one assay ("within-run", Table Ib) or in duplicates in two consecutive assays ("between -run", Table ic). 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.

Table I

TABLE Ia - COMPARISON OF TWO DIFFERENT DETECTION ANTIBODIES Sample Tenascin-W (mg/l) pAb(FL) pAb(3F/4) serum 1 0 339 +/- 0.04 0.396 +/- 0 08 serum 2 08BO +/- 005 0824 +/- 0.08 serum 3 0 184 +/- 0.05 0,176 +/- 0.01 serum 4 0.496 +/- 0.06 0 522 +/- 0.07

Serum TNW ϊevelε 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 Ihθ two detection antibodies as revealed by student's L-test (p>0 05}

TABLE Ib - PRECISION OF THE ASSAY: WITHIN-RUN

Sample Tenascin-W (mg/l) mean +/- SD CV (%) serum 1 0.268 +/- 0.012 4.0 serum 2 0 288 +/- 0.023 6.9 serum 3 0.310 +/- 0014 4.5 serum 4 0 452 +/- 0.028 6.2

Precision was estimated by assaying 4 serum samples 3 times in one assay (within run). SD = standard deviation, CV (%) = coefficient of variation

TABLE Ic - PRECISION OF THE ASSAY: BETWEEN-RUN

Sample Tenascin-W (mg/l) mean +/- SD CV (%) serum 1 0.268 +/- 0.001 0 4 serum 2 0 180 +/- 0.027 15.2 serum 3 0.194 +/- 0 014 7.4 serum 4 0.460 +/- 0.024 5.2

Precision was estimated by assaying 4 serum samples in duplicate in 2 consecutive assays (between run) SD - standard deviation, CV (%) = coefficient of variation Tenascin-W is up-regulated in the serum of colorectal and breast cancer patients

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 tenasciπ-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. Although both tenascins showed the same tendency of increased levels in colorectal cancer patients, the difference in the mean value in cancer sera compared to volunteers was much more pronounced for tenascin-W. However, not every patient with a high tenascin-C serum level also contained high levels of tenascin-W (Table II), indicating that there is no strict correlation between serum levels of the two tenascins in colorectal cancer patients .Although, there was no overt correlation between elevated serum tenascin-W levels and staging of the colorectal tumors, it is worth noting that 4 out of 5 patients who developed tumor recurrence after surgery had high levels of tenascin-W. Mean serum tenascin-W level of recurrent patients (n=5) was 1.02 +/- 0.34 mg/l which corresponds to a 1.5-fold increase compared to non-recurrent colorectal cancer patients (n=12, mean=0.7G0 +/- 0.37 mg/I) and a 2.6-fold increase compared to healthy volunteers (Table II). Table il

Table I! - CLINiCOPATHOLOGICAL FEATURES AND TENASCSN LEVELS OF THE COLORECTAL CANCER PATIENTS

Patient Age Sex Localization Blood collection Grade TNM TNW TNC Recurrence

(dd/mm/yy) (ma/I) (mg/l) (May 2007)

1³ 49 F Rectum n.a. n.a. T3N1 M0 0.380 0.820 no

2 89 M Left π.a. 2 T4N1M0 0.330 0.810 no

3 48 F Left n.a. 2 T3N2W0 0.382 1.080 no

4 60 M Left 13/02/03 1 T1 N0M0 0.560 0.674 yes

5 83 M Right 27/02/03 3 T3N0M0 1.100 0.920 yes

6 81 F Rectum 17/04/03 1 T2N0M0 0.800 0.848 yes

7 62 F Left 12/05/03 2 T3N2M0 1.230 1.230 yes

8 54 M Left 03/07/03 _ 2 T2N1M0 0.500 0.674 no

9 62 M Left 10/07/03 2 T4N1M0 0.473 0.571 no

10 60 M Right 11/08/03 2 T4N2M0 1.400 1.150 yes

11^b 73 M Left 15/08/03 2 T4N0M0 1.130 0.733 no

12 68 M Right 15/08/03 2 T3N0M0 1.380 1.240 no

13 81 M Left 08/10/03 2 T2N2M0 0.920 1.190 no

14 75 F Left n.a. 2 T3NOM0 1.270 0.908 no

15 67 F Left 09/12/03 2 T3NOM0 0.560 0.915 no

16 89 F Transverse 09/01/04 2 T2N0M0 0.563 1.620 no

17 94 M Right 14/01/04 2 T4N1 M0 0.520 1.280 no n a not available. Grade = tumor grade according to WHO Classification of Tumors, 2000, TNM ( rumor, Nαde,Metastasιs) = tumor stage according to UICC Classification, 2002, TNW, TNC' protein levels inserum deduced by sandwich ELISA.

* Neoadjuvant radiotherapy (25/11/02 - 12/12/02)

* Adjuvant radiochamotherapy with 5-Fluorouracιl (5-FU) (23/09/03 ■ 31/10/03)

To test whether 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 expression in normal colon and cancer tissue

To determine whether increased expression of tenascin-W in the tumor tissue could explain the elevated serum levels in colorectal cancer patients, the present inventors performed immunoblots on 11 colorectal tumor tissue extracts. As shown in Figure 3a, a large fraction of the tumor tissue samples contained detectable levels of tenascin-C (9/11 ; 82%). Furthermore, tenascin-W was also highly expressed in these tumor extracts (9/11 ; 82%). In two cases (patients E and F) tenascin-W and teπascin-C were absent or barely detectable while all other samples tested showed co-expression of the two tenascins (Fig. 3a). Besides tumor tissue, corresponding normal mucosa was available for patients K and L, and for patient J the present inventors obtained normal tissue only. In all normai colon mucosae tested, 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. For tenascin-C, the present inventors detected different isoforms, whereas for tenascin-W they mostly observed a single band in the extracts tested. However, there was a faint second lower tenascin-W band detectable in tumor extracts of patients B and I. This smaller band is likely to represent degradation products of tenascin-W, but the present inventors cannot exclude that it might be due to a low level of alternative splicing. Tenascin-C isoform expression differed between tumor and normal tissues. In normal colon mucosae, only the low molecular-weight isoform could be observed, whereas in tumor extracts low and high molecular-weight isoforms were detectable. In summary, the majority of human colorectal tumors express both tenascins, but their relative amount varies greatly between patients.

In order to confirm expression of tenascin-W and tenascin-C in tumor tissues and to localize their presence within the tissues, the present inventors 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. Taken together, 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. To extend this analysis to a larger number of samples the present inventors stained a frozen colon tissue micro array (TMA) that contained 32 cancer spots and 6 spots of normal colon. Without exception all cancer tissue sections were stained for tenascin-W as well as tenascin- C, while all normal tissues were negative for tenascin-W, but positive for tenascin-C (Table III). 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 (patient 15) 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). In 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.

Table IS! - CLIN)COPATHOLOGiC FEATURES AND

TENASCIN STAINING OF THE FROZEN TMA

Patient Ag^e Localization Grade TNM TNW TNC staining staining

1 59 rectum 2 T3N0Mx ++ +++

2 71 left 3 T3N2Mx ++ +++

3 77 right 2 T2N0M1 ++ ++

4 66 rectum 2 T3N1 Mx ++ ++

5 31 right 2 T2N0M0 +++ +++

6 78 rectum 3 T4N1 MX + +

7 68 left 2 T2N1 MX ++ ++

8 45 left 2 T3N2M1 ++ ++

9 79 left 2 T2N0MX + +

10 80 left 2 T4N0Mx +

11 83 left 2 T3N0MX +++

12 82 right 3 T4N2M1 +++

13 78 right 2 T4N2M1 + ++

14 67 right 3 T3N1 Mx + ++

15 82 right 3 T4N2Mx +++ +++

16 89 left 2 T4N0MX ++ +

17 64 left 2 TSNOMx ++ ++

18 88 right 2 T3N1 Mx +++ +++

19 81 right 2 T3N2Mx ++ ++

20 80 rectum 2 T2N1 MX +++ +++

21 68 right 2 T2N0M0 ++ ++

22 66 right 2 T3N0Mx + +

23 54 left 3 T3N1 MX + +++

24 62 right 3 T3N2MX + +

25 83 right 2 T3N0Mx ++

26 81 rectum 3 T2N1 Mx + +

27 45 left 2 T4IM1 M1 ++ ₊₊

28 74 left 2 T4N2M1 ++ +÷+

29 85 rectum 2 T3N1 Mx + ++

30 65 left 2 T3N0Mx + ++

31 87 rectum 2 T3N0Mx +++ +++

32 46 left 2 T3N3M1 +++ +++

33 69 normal +

34 93 normal - +

35 83 normal +

36 70 normal - +++

37 93 normal - +

38 82 normal - +

Grade = tumor grade according to WHO Classification of Tumors 2000, TNM {Tumor, Node, Metastasis) = tumor stage according to UICC Classification, 2002, Classification in categories +-*^•+ >B0% of tumor area is stained. ++ >20% of tumor area is stained, + <20% of tumor area is stained See examples of each category in Figure 4. Discussion

In 2006, 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. Because of the low accuracy and sensitivity of the classical fecal occult blood test, several biochemical markers have been developed and explored over the years for colorectal cancer screening, including carcinoembryonic antigen (CEA) serum levels ^4S, galactose-N acetylgalactosamine fecal levels ⁴⁵, and K-Ras mutations in human stool ^{47| 4β} , but none is enoug h sensitive and specific for early cancer detection. Recently, 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. Nevertheless, the most effective available method to prevent and detect colorectal cancer today is colonoscopy. However, this investigation is invasive, time- consuming, uncomfortable, and relatively expensive ⁵⁴. Moreover, 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. Thus surrogate markers of colon cancer detectable in the blood may be a convenient method for screening and early cancer detection.

In search for new diagnostic or prognostic tumor markers, 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, In comparison, serum levels of tenascin-C in controls were 2~fold higher and scattered over a broad range. Interestingly, the present inventors observed a clear increase in mean tenascin-W levels in colorectal cancer sera (0.794 +/- 0.38 mg/l). The histogram in Figure 2a (right) shows that within the cohort of the colorectal cancer patients, there are two subpopulations: one with elevated tenascin-W levels (approximately 2.5 times the mean of contra! values), the other with levels comparable to the controls. These groups may represent different kinetics of tenascin-W elevation in serum, whereby 'normal' levels would become elevated later during disease progression. Another important issue is whether the level of tenascin-W is associated with more unfavorable disease progression. In this regard, it is noteworthy that the follow-up studies of the colorectal cancer patients revealed that 4^"out of 5 patients who developed tumor recurrence after treatment had high tenascin-W levels (i.e. above mean levels) in their sera (Table (I). This additionally suggests a potential value of tenascin-W levels in sera in predicting recurrence or progression.

It is also important to consider that the presence of tenascin-W, or of any other biomarker in serum, could be influenced by any co-morbidity (e.g. cardiovascular diseases, inflammatory disease) or through indirect effects of the tumor on other organs (e.g. bone marrow, liver). For instance 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. To test for expression of tenascin-W in the tumor, 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, however, was absent from normai colon mucosa, in contrast to tenascin-C, which is expressed (at reduced levels) in the norma! tissue as well. Interestingly, in the normal tissue, only the low molecular-weight isoform of tenascin-C is present, whereas in the tumor tissue, low and/or high molecular- weight isoforms are over-expressed. This result implies differential tenascin-C isoform expression in normal and neoplastic human colon tissue. This is in agreement with previous reports showing expression of the small isoform in normal stable tissues ^5y, while larger variants are detectable in diseased tissues including neoplasia, wound healing or inflammation ^58'61. lmmunohistochemical analysis of frozen sections of the same patients revealed prominent stromal staining for both tenascins. The 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.

As is apparent to one of ordinary skill in the art, variations in the above-described methods can be introduced with ease to attain the same objective. Various incubating conditions, labels, apparatus and materials can be chosen according to individual preference. All publications referred to herein are incorporated by reference in their entirety as if each were referred to individually.

References

1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70.

2. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med 2004 ;10:789- 99.

3. Bissell MJ, Radisky D. Putting tumours in context. Nat Rev Cancer 2001 ;1 :46-54.

4. Mueller MIW, Fusenig NE. Friends or foes - bipolar effects of the tumour stroma in cancer. Nat Rev Cancer 2004;4:839-49. 5 Beacham DA₁ Cukiβrman E. Stromageπesis: the changing face of fibroblastic microenvironments during tumor progression. Semin Cancer Biol 2005:15:329-41.

6. Bhowmick NA, Moses HL. Turnor-stroma interactions. Curr Opin Genet Dev 2Q05;15:97-101.

7 Kallurϊ R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer 2006;6:392-401.

8. Chiquet-Ehrismaπn R, Mackie EJ, Pearson CA, Sakakura T. Tenascin: an extracellular matrix protein involved in tissue interactions during fetal development and oncogenesis. Cell 1986;47:131-9.

9. Orend G. Potential oncogenic action of tenascin-C in tumorigenesis. int J Biochem CeIi Biol 2005;37:1066-83. Epub 2005 Jan 18.

10. Huang W, Chiquet-Ehrismann R, Moyano JV₁ Garcia-Pardo A₁ Orend G. Interference of tenascin-C with syndecan-4 binding to fibronectin blocks cell adhesion and stimulates tumor cell proliferation. Cancer Res 2001 ,61 :8586-94.

11. Midwood KS, Valenick LV, Hsia HC, Schwarzbauer JE. Coregulation of fibronectin signaling and matrix contraction by tenascin-C and syndecan-4. MoI Biol CeIf 2004;15:5670-7.

12. Zagzag D, Shift B, JaIIo Gl, Greco MA, Blanco C, Cohen H, Hukin J, Allen JC, Friedlander DR. Tenascin-C promotes microvascular cell migration and phosphorylation of focal adhesion kinase. Cancer Res 2002;62 2660-8.

13. Sarkar S, Nuttall RK, Liu S₁ Edwards DR, Yong VW. Tenascin-C stimulates glioma cell invasion through matrix metalloproteinase-12. Cancer Res 2006;66:11771-80.

14. Jones PL, Crack J, Rabinovitch M. Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the alpha v beta 3 integriπ to promote epidermal growth factor receptor phosphorylation and growth. J Cell Biol 1997; 139:279-93.

15. Swindle CS, Tran KT, Johnson TD, Banerjee P₁ Mayes AM, Griffith L₁ Wells A. Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor. J Cell Biol 2001 ;154:459-68.

16. Ruiz C₁ Huang W, Hegi ME, Lange K, Hamou MF, Fluri E₁ Oakeley EJ, Chiquet-Ehrismann R, Orend G. Growth promoting signaling by tenascin-C [corrected]. Cancer Res 2004:64:7377-85. Minn AJ, Gupta GP, Siege! PM, Bos PD, Shu W, Gin DD, Viale A, Olshen AB, Gerald WL, Massague J Genes that mediate breast cancer metastasis to lung Nature 2005,436 518-24

Chiquet-Ehπsmann R, Chiquet M Tenascins. regulation and putative functions during pathological stress J Patho! 2003,200 488-99

Herlyn M, Graeven U, Speicher D, SeIa BA, Bennicelli JL, Kath R, Guerry D Characterization of tenascin secreted by human melanoma cells Cancer Res 1991 ,51 4853-8

Riedl S Bodenmuller H, Hinz U₁ HoIIe R, Moller P, Schlag P, Herfarth C, Faissner A Significance of tenascin serum level as tumor marker in primary colorectal carcinoma lnt J Cancer 1995,64 65-9

Scheπk S, Muser J₁ Vollmer G, Chiquet-Ehπsmaπn R Tenascin-C in serum a questionable tumor marker lnt J Cancer 1995,61 443-9

Pauli C, Stieber P₁ Schmitt UM, Andratschke M₁ Hoffmann K, Wollenberg B The significance of Tenascin-C serum level as tumor marker m squamous cell carcinoma of the head and neck Anticancer Res 2002,22 3093-7

Ishiwata T, Takahashi K, Shimanuki Y, Ohashi R, Cui R, Takahashi F, Shimizu K, Miura K, Fukuchi Y Serum tenascin-C as a potential predictive marker of angiogenesis in non-small cell lung cancer Anticancer Res 2005,25 489-95

Takeda A, Otani Y, lseki H₁ Takeuchi H, Aikawa K₁ Tabuchi S₁ Shinozuka N, Saeki T, Okazaki Y, Koyama I Clinical Significance of large tenascin-C spliced variant as a potential biomarker for colorectal cancer World J Surg 2007,31 388-94

Takeda A, Otani Y₁ Hirooka E, Okada K, Torn T, Shinozuka N, Koyarna I Plasma large Tenascin-C spliced variant as a possible biomarker for the prediction of hepatic recurrence in colorectal cancer Surgery 2007,141 124-5

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 N₁ Fujita N, Hoπike S, Wataπabe 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

Lebensztejn DM, Sobaniec-Lotowska ME, Kaczmarski M₁ Voelker M, Schuppan D Matrix- derived serum markers in monitoring liver fibrosis in children with chronic hepatitis B treated with interferon alpha World J Gastroenterol 2006,12 3338-43

El-Karef A, Kaito M, Tanaka H, lkeda K, Nishioka T, Fujita N, lnada H, Adachi Y₁ Kawada N, Nakajima Y Imanaka-Yoshida K, Yoshida T Expression of large tenascin-C splice variants by hepatic stellate cells/myofibroblasts in chrϋnic hepatitis C J Hepatol 2006,46 664-73

Szalai AJ, Agrawal A, Greenhough TJ, Volanakis JE C-reactive protein structural biology, gene expression, and host defense function Immunol Res 1997,16 127-36

Vermeire S, Van Assche G, Rutgeerts P The role of C-reactive protein as an inflammatory marker in gastrointestinal diseases Nat CIm Pract Gastroenterol Hepatol 2005,2 580-6

Weber P, Montag D, Schachner M, Bernhardt RR Zebrafish tenascin-W, a new member of the tenascin family J Neurobiol 1998,35 1 -16

Scherberich A, Tucker RP, Samandari E, Brown-Luedi M₁ Martin D, Chiquet-Ehπsmann R Murine tenascin-W a novel mammalian tenascin expressed in kidney and at sites of bone and smooth muscle development J Cell Sci 2004,117 571 -81

Meloty-Kapella CV, Degen M, Chiquet-Ehrismann R, Tucker RP Avian tenascin-W expression in smooth muscle and bone, and effects on calvaπal cell spreading and adhesion in vitro Dev Dyn 2006,235 1532-42

Scherberich A, Tucker RP, Degen M, Brown-Luedi M, Andres AC, Chiquet-Ehπsmann R ^Tenascιn-W is found in malignant mammary tumors, promotes alphaδ integrin-dependent motility and requires p38MAPK activity for BMP-2 and TNF-alpha induced expression in vitro Oncogene 2005,24 1525-32

Degen M, Brellier F, Kain R, Ruiz C, Terracciano L, Orend G, Chiquet-Ehπsmann R Tenascin- W is a Novel Marker for Activated Tumor Stroma in Low-Grade Human Breast Cancer and Influences Cell Behavior Cancer Res 2007,67 9169-79 Lange K, Kammerer, M , Hegi, M , Grotegut, S , Dittmann, A , Huang, W , Fluri, E Yip, G W , Goette, M , Ruiz, C , and Orend, G Endothelin Receptor Type B Counteracts Tenascm-C- Inducβd Endothelin Receptor Type A-Depeπdent Focal Adhesion and Actin Stress Fiber Disorganization Cancer Res 2007,67

Pearson CA, Pearson D, Shibahara S, Hofsteenge J₁ Chiquet-Ehrismann R Tenascin cDNA cloning and induction by TGF-beta Embo J 1988,7 2977-82

Hamilton SR, Aaltonen LA World Health Organization Classification of Tumours Pathology and Genetics of Tumours of the Digestive-Systemed Lyon, France lARCPress, 2000.

Sobin LH, Wittekind C₁ eds TNM classification of malignant tumors, 6^th ed New York Wiley- Liss, 2002

Schoenberg Fejzo M, Slamon DJ Frozen tumor tissue microarray technology for analysis of tumor RNA, DNA, and proteins Am J Pathol 2001 ,159 1645-50

Washizu K, Kimura S, Hiraiwa H, Matsunaga K₁ Kuwabara M₁ Aπyoshi Y₁ Kato K, Takeuchi K Development and application of an enzyme immunoassay for tenascin CIm Chim Acta 1993,219 15-22

Ferlay J, Autier P, Boniol M, Heanue M, Colombet M, Boyle P Estimates of the cancer incidence and mortality in Europe in 2006 Ann Oncol 2007

Fearon ER, Vogelstein B A genetic model for colorectal tumoπgenesis Cell 1990,61.759-67

Rosandic M, Skegro M, Paar V, Paar D₁ Scukanec-Spoljar M, Juricic M Vucelic B, Pulanic R, Rustemovic N, Ostojic R, Ljubojevic N Quantitative tissue carcinoembryonic antigen (T CEA) assay as a screening test for severe dysplasia in colorectal adenomas Acta Med Austπaca 1999,26 89-92

Shamsuddin AM A simple mucus test for cancer screening Anticancer Res 1996,18 2193-9

Doolittle BR, Emanuel J, Tuttle C, Costa J Detection of the mutated K-Ras biomarker in colorectal carcinoma Exp MoI Pathol 2001 ,70 289-301 48. Rennert G, Kislitsiπ D, Brenner DE, Rennert HS, Lev Z. Detecting K-ras mutations in stool from fecal occult blood test cards in multiphasic screening for colorectal cancer. Cancer Lett 2007.

49. Hardt PD₁ Ngoumou BK, Rupp J, Schπell-Kretschmer H, Kloer HU. Tumor M2-pyruvate kinase: a promising tumor marker in the diagnosis of gastro-intestinal cancer. Anticancer Res 2000;20:4965-8.

50. Schulze G. The tumor marker tumor M2-PK: an application in the diagnosis of gastrointestinal cancer. Anticancer Res 2000;20:4961 -4.

51. Schneider J, Schulze G. Comparison of tumor M2-ρyruvate kinase (tumor M2-PK), carcinoembryonic antigen (CEA)₁ carbohydrate antigens CA 19-9 and CA 72-4 in the diagnosis of gastrointestinal cancer. Anticancer Res 2003;23:5089-93.

52. Hardt PD₁ Toepler M, Ngoumou B, Rupp J, Kloer HU. Measurement of fecal pyruvate kinase type M2 (tumor M2-PK) concentrations in patients with gastric cancer, colorectal cancer, colorectal adenomas and controls. Anticancer Res 2003;23:851-3.

53. Ewald N₁ Toepier M, Akinci A, Kloer HU, Bretzel RG, Hardt PD. [Pyruvate kinase M2 (tumor M2-PK) as a screening tool for colorectal cancer (CRC). A review of current published data], Z Gastroenterol 2005;43:1313-7.

54. Rennert G. Prevention and early detection of colorectal cancer-new horizons. Rέcent Results Cancer Res 2007;174:179-87.

55. Schenk S, Muser J, Vollmer G, Chiquet-Ehrismann R. Tenascin-C in serum: an acute-phase protein or a carcinoma marker? [letter], lnt J Cancer 1995;60: 145.

56. Gazzaniga P, Nofroni I₁ 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.

57. Mackie EJ₁ Tucker RP. Tenascin in bone morphogenesis: expression by osteoblasts and cell type- specific expression of splice variants. J Cell Sci 1992; 103:765-71. BOΓSI L, Csrnemol'a B, Nicolo G, Spina B, Tanara G₁ Zardi L Expression of different tenasαn isoforms in normal, hyperplastic and neoplastic human breast tissues int J Cancer 1992,52 683-92

Hindermann W, Berndt A, Borsi L, Luo X, Hyckel P, Katenkamp D, Kosmeh! H Synthesis and protein distribution of the unspliced large tenascin-C isoform in oral squamous cell carcinoma J Pathol 1999,189475-80

Matsumoto K, Hiraiwa N, Yoshiki A, Ohnishi M, Kusakabe M Tenascin-C expression and splice variant in habu snake venom-induced glomerulonephritis Exp MoI Pathol 2002,72 186- 95

Tsunoda T, lnada H, Kalembeyi I, Imanaka-Yoshida K, Sakakibara M, Okada R, Katsuta K, Sakakura T, Majima Y, Yoshida T Involvement of large tenascin-C splice variants in breast cancer progression Am J Pathol 2003,162 1857-67

Claims

CJairns

1. A method of detecting, diagnosing or prog nosing a colon cancer comprising:

(a) 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.

2. The method of claim 1 wherein an at least two-fold increase of the concentration of tenascin-W measured in step (a) as compared to the standard value for the concentration of tenascin-W protein in the blood, plasma or serum of heajthy individuals is indicative for a higher likelihood of recurrence of a colon cancer.

3. The method of claim 1 or 2 further comprising the steps of:

(c) measuring the concentration of tenascin-C protein in the blood, plasma or serum sample obtained from the individual, and

(d) comparing the concentration measured in step (c) with a standard vaiue for the concentration of tenascin-C protein in the blood, plasma or serum of healthy individuals.

4. The method of claim 1 , 2 or 3 wherein said concentration of tenascin-W and/or tenascin-C protein is measured using an antibody, or a fragment thereof, specific for tenascin-W and/or tenascin-C.

5. The method of claim 4 wherein said concentration of tenascin-W and/or teπascin-C protein is measured by ELISA.

6. A kit for detecting, diagnosing or prog nosing a colon cancer in a blood, plasma or serum sample comprising an antibody, or a fragment thereof, specific for tenascin-W.

7. The kit of claim 6 further comprising an antibody, or a fragment thereof, specific for tenascin-C.

8. Use of the blood, plasma or serum concentration of tenascin-W protein as a biomarker for colon cancer.

9. An antibody, or a fragment thereof, specific for tenascin-W for use in diagnostic for colon cancer by measuring blood, piasma or serum concentration of tenasciπ-W protein.

10. Tenascin-W as a blood, plasma or serum biomarker for colon cancer.