An in vitro method for detecting cancer

Abstract

The present invention relates to an in vitro method for detecting cancer in a subject, for predicting the clinical outcome of a subject suffering from cancer, or for monitoring the treatment of a subject suffering from cancer, based on the tumoral carbohydrate Ca10 (Ca10) levels measured in a biofluid sample isolated from the subject.

Description

[0001] AN IN VITRO METHOD FOR DETECTING CANCER

[0002] The invention refers to an in vitro method for cancer detection based on the tumor- associated carbohydrate Ca10 (Ca10) levels in a biological fluid sample. Thus, the present invention relates to the technical field of cancer, specifically, to tumor serum markers thereof.

[0003] BACKGROUND ART

[0004] Malignant transformation is accompanied by changes in tumor cell surface carbohydrates, including aberrant or incomplete glycosylation of heavily glycosylated structures. These structures can be released from tumor cells and detected in the biological fluids of cancer patients, making them useful as tumor markers. In addition, their presence may be associated with cancer prognosis by influencing metastasis and tumor progression.

[0005] The carbohydrate Ca10 is a highly glycosylated structure present on the cell surface of murine Ehrlich tumor (ET) cells. Ca10 was defined by its reactivity with the monoclonal antibody A10, obtained from hybridomas derived from mice immunized with ET cells. Ca10 is spontaneously released from ET cells and can be detected in the serum of solid ET-bearing mice.

[0006] Therefore, there is the necessity in the state to the art to increase the insights on tumor escape mechanisms in order to develop new tools in the detection of cancer.

[0007] DESCRIPTION OF THE INVENTION

[0008] By studying the serum levels of murine Ca10 (mCa10) in mice bearing solid murine Ehrlich carcinoma, a strong correlation between mCa10 levels and tumor mass was established. mCa10 is a proteoglycan with a heparan sulfate glycosaminoglycan structure, sensitive to certain heparinases (heparinase 3). The heparan sulfate structure found in mCa10 is peculiar for its extremely low sulfatation degree. It would be related to the non-sulfated precursor of this glycosaminoglycan, heparosan, normally absent on the cell surface of mammalian cells. The analysis of the main carbohydrate component in mCa10, either by nuclear magnetic resonance (NMR) or by chromatography of the released fragments after complete hydrolysis with heparinases only shows a significant proportion (20-40%) of non-acetylated glucosamine residues. It does not show sulfation of either glucosamine or glucuronic acid residues, contrary to what could be expected for normally processed heparan sulfate glycosaminoglycan present in healthy tissues. This discrepancy suggests a typical processing or modification in the mCa10 heparan sulfate glycosaminoglycan.

[0009] Surprisingly, circulating levels of a human homologue (Ca10H) of the murine tumor glycosaminoglycan mCa10 are detected in the serum of cancer patients with tumors originating from different tissues. Apart from their common reactivity with A10, the structural relationship between mCa10 and Ca10H is based on their high sensitivity to heparinase 3, which is highly specific for glycosaminoglycans of heparan sulfate. Circulating Ca10H levels are found to be elevated in serum of cancer patients, on a cut-off established with sera from control subjects. Of note, in patients with prostate cancer, it has been possible to establish a correlation between serum Ca10H levels and bone metastases.

[0010] Since Ca10H levels in serum vary among cancer patients with different or the same histological tumor type, Ca10H could represent a new “tumor marker” with additional clinical usefulness as a prognostic factor.

[0011] Uses of Ca10 as a tumor marker.

[0012] In view of the foregoing, the present invention discloses Ca10H as a “tumor marker”, this is, a substance that is found in high levels in biofluid sample of some people with cancer in comparison with non-suffering cancer people. Tumor markers are produced either by the cancer cells themselves or by the body in response to the presence of cancer or certain benign (noncancerous) conditions. Based on the above, a first aspect the present invention relates to an in vitro method for detecting cancer in a subject, hereinafter “detection method of the invention”, comprising:

[0013] (a) quantifying the tumoral carbohydrate Ca10 levels in a biofluid sample isolated from the subject; and

[0014] (b) comparing the Ca10 levels obtained in step (a) with a control level,

[0015] Wherein if the Ca10 levels in the subject are increased with respect to the control levels, then the subject suffers from cancer.

[0016] As used herein, the term “cancer detection” refers to the identification of a tumoral tissue by examination or parameters or biomarkers in a subject. In the context of the present invention, the parameter required for detecting cancer is the Ca10H levels in a biofluid sample of the subject.

[0017] As use herein, the term “cancer” refers to the disease resulting from the uncontrolled proliferation of cells giving rise to a malignant tumour or to a malignant tumoral tissue. A tumour is considered malignant when the tumour cells can grow rapidly, show anaplasia and / or are able to infiltrate the tissue, invade adjacent tissues or even spread to other parts of the body, a process known as metastasis. Thus, in one particular embodiment, the cancer is a metastatic cancer.

[0018] On the other hand, the tumour (or cancer) can be located or originate in any tissue or organ of the body. Thus, any cancer that releases Ca1 OH to reach a biological sample is susceptible to detection by the method of the invention, regardless of its stage of development, its origin or its location. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood- forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord. The cancer may be a solid tumor or a non-solid tumor, including metastasis. In a preferred embodiment, patients diagnosed with selected cancers, such as carcinomas and adenocarcinomas, had on average above five times higher levels of Ca10H than healthy controls. Specifically, the mean serum levels of Ca10H in these patients was 4.0 Arbitrary Units (AU) / mL with a standard error of the mean (SEM) of 0.6, compared to a mean of 0.7 AU / mL with a SEM of 0.0 in the control group (see Figure 9b). Thus, in a particular embodiment, the cancer is a carcinoma or an adenocarcinoma.

[0019] Examples of cancer include, but are not limited to, a lung tumor or cancer, such as a small cell or non-small cell lung cancer, or an adenocarcinoma, squamous cell carcinoma or a large cell carcinoma. Further particular non-limiting examples of a tumor or cancer include a carcinoma, sarcoma, lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma, mesothelioma, reticuloendothelial, lymphatic or haematopoietic neoplasia, tumor, cancer or malignancy. Additional particular non-limiting examples of tumor or cancer is a lung, thyroid, head or neck, nasopharynx, throat, nose or sinuses, brain, spine, breast, adrenal gland, pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), genito-urinary tract (uterus, ovary, cervix, endometrial, bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood, muscle, or skin neoplasia, tumor, or cancer. Still further particular non-limiting examples of a tumor or cancer include a breast cancer, prostate cancer, pancreas cancer, gastric cancer, pleural mesothelioma, colon cancer, rectal cancer, large bowel cancer, small intestinal cancer, esophageal cancer, duodenal cancer, lingual cancer, pharyngeal cancer, salivary gland cancer, cerebral tumor, schwanoma, liver cancer, kidney cancer, bile duct cancer, endometrial cancer, cervical cancer, uterine body cancer, ovarian cancer, bladder cancer, urethral cancer, skin cancer, angioma, malignant lymphoma, malignant melanoma, thyroid cancer, parathyroid cancer, nasal cancer, paranasal cancer, auditory organ cancer, carcinoma of oral floor, laryngeal cancer, parotid cancer, submandibular cancer, bone tumor, angiofibroma, retinal sarcoma, penile cancer, testicular tumor, pediatric solid cancer, Kaposi's sarcoma, tumor of maxillary sinus, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, lymphoma, multiple myeloma or leukemia. Nevertheless, in a particular embodiment of the detection method of the invention, alone or in combination with all or each one of the previous particular embodiments, the cancer is prostate cancer, cervix cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, endometrial cancer, skin cancer, thyroid cancer, bladder cancer, ovarian cancer, liver cancer, esophageal cancer, or colorectal cancer.

[0020] The first step of the detection method of the invention comprises quantifying the Ca10 levels in a biological sample isolated from the subject.

[0021] The term “tumoral carbohydrate Ca10” or “Ca10” encompasses both the murine tumoral carbohydrate derived from Ehrlich’s tumor cells (mCa10), as well as any other homologue tumoral carbohydrate equivalent to mCa10 in other mammal, such as its human counterpart, i.e. the human tumoral carbohydrate Ca10 or Ca10H.

[0022] The “tumoral carbohydrate Ca10” or “Ca10” refers to a proteoglycan with a heparan sulfate glycosaminoglycan structure, sensitive to certain heparinases (heparinase 3), that reacts with the monoclonal antibody A10 (Gil, J. etal. 1990. Cancer Res 50, 7301- 7306). In particular, the mCa10 shows the following features:

[0023] The principal polysaccharide component of mCa10 is an incompletely processed heparosan, precursor of heparan sulfate, and hydrolysable with heparinases, with an average molecular size over 100 kDa, as measured by NMR diffusion ordered spectroscopy (DOSY) where N-acetylglucosamine residues are deacetylated between 20 to 40% and the total sulfation observed below 0,2% as measured by NMR and chromatographic methods coupled to mass spectrometry. The generic polymeric structure of mCa10 can be described as a polysaccharide comprising a combination of tetramers of formula: wherein each tetramer is

[0024] (a) x= 2 and y= 0; or

[0025] (b) x=1 and y= 1 ; or

[0026] (c) x=0 and y=2; and wherein represents the point of attachment to another tetramer; and wherein

[0027] The ratio £x / y in the polysaccharide is from 4 / 1 to 3 / 2; and wherein

[0028] The total number of tetramers in the polysaccharide is from 50 to 150.

[0029] The polysaccharide may comprise attach to one of ends a group -OH, a residue beta- Glucuronyl or a glycosaminoglycan protein attachment sequence.

[0030] Thus, the main polysaccharide component of mCa10 is constituted by the disaccharide repeating units of heparosan, [3-D-glucuronic acid (GlcA) and a-D-N- acetylglucosamine (GIcNAc), where a proportion between 20% to 40% of N- acetylglucosamine residues are deacetylated (GlcN), according to the simplified formula: [—>4) [3-D-GlcA (1 — 4)-a-D-GlcNAc / GlcN (1 — >] . The mCa10 comprising the polysaccharide of the above-mentioned formula is capable of inducing the production of Tregs. Without wishing to be bound by any theory, and based on the results shown in the present invention, the inventors consider that the human counterpart of mCa10, i.e. Ca10H, shows a similar structure to one of mCa10.

[0031] Methods for quantifying the levels or amount of carbohydrates in a sample are widely known in the state of the art, and any of them can be used to carry out step a) of the cancer detection method of the invention. Non-limiting examples of these methods include: Lectin based methods, NMR, chromatography, mass spectrometry and colorimetric methods depending of the nature of the carbohydrate components. For example, heparan-sulfate quantification and analysis in plasma samples can be performed by liquid chromatography coupled to mass spectrometry. Nevertheless, in a particular embodiment of the cancer detection method of the invention, alone or in combination with all or each one of the previous particular embodiments, the method used for quantifying the level of Ca10H is by ELISA (Enzyme-Linked ImmunoSorbent Assay), more particularly, by ELISA using the monoclonal antibody A10 (Gil, J. et al. 1990. Cited ad supra).

[0032] The cancer detection method of the present invention can be performed on any suitable biological sample. As used herein, a "biofluid sample" refers to any fluid sample of biological material derived from an animal such as, but not limited to blood, serum, plasma, pleural, bronchioalveolar, peritoneal liquids, urine, and sputum The biofluid sample may be tested directly or may require some form of treatment prior to testing or may be partially purified or otherwise enriched prior to analysis. Thus, in a particular embodiment, alone or in combination with all or each one of the previous particular embodiments, the biofluid sample is blood, serum, plasma, pleural liquid, bronchioalveolar liquid, peritoneal liquid, urine, or sputum.

[0033] Likewise, the cancer detection method of the present invention can be carried out on any subject. As used herein, the term “subject” refers to all animals classified as mammals and includes but is not limited to domestic and farm animals, primates and humans, for example, human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. In a particular embodiment of the cancer detection method of the invention, alone or in combination with all or each one of the previous particular embodiments, the subject is a mammal, more preferably, a human. Next, in step b) of the detection method of the invention, the levels of Ca10 quantified in step a) are compared with the ones in a control level. In the context of the present invention, the “control level” refers to the levels of Ca10 in sample isolated from a subject not suffering from cancer or not comprising tumoral tissue. As the skilled person in the art understands, the control level may also refer to the median value of the Ca10 levels in a collection of biofluid samples from subjects not suffering from cancer or not comprising tumoral tissue. In this case, said control level is typically obtained by combining equal amounts of samples from a population of subjects who are clinically well documented. In the context of the present invention, the terms “control level” and “reference value” are equivalents and refer to the same concept.

[0034] In the context of the present invention, it is understood that the Ca10 levels are increased with respect to the control level when a value is greater or higher than the other. In particular, it is understood that the Ca10 levels are “increased” when the levels of the Ca10 in the subject sample are, at least, 1.1 -fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold higher, or even more, than the Ca10 levels in the control sample. Finally, depending on the result of said comparison, it can be concluded whether the subject suffers or not from cancer. In this way, if the Ca10 levels in the subject are increased with respect to the control level, then the subject suffers from cancer.

[0035] In another aspect, the present invention relates to an in vitro method for predicting the clinical outcome of a subject suffering from cancer, or for monitoring the treatment of a subject suffering from cancer, hereinafter “second method of the invention”, comprising

[0036] (a) quantifying the Ca10 levels in a biofluid sample isolated from the subject; and

[0037] (b) comparing the Ca10 levels obtained in step (a) with a control level, wherein if the Ca10 levels in the subject are increased with respect to the control level, then the clinical outcome of the subject is negative and either the subject is going to suffer from metastasis or the tumoral mass is going to increase; or the treatment is not being effective.

[0038] The terms “cancer” and “subject” have been defined un previous paragraphs, and said definitions together with their particular embodiments are applicable to the second method of the invention.

[0039] The term "prediction" is used herein to refer to the likelihood that a patient will have a particular clinical outcome. As will be explained later, the clinical outcome may be positive or negative. The predictive method of the present invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient. The predictive method of the present invention is a valuable tool in predicting if a patient is likely to respond favorably to a treatment regimen. As used herein, the expression “clinical outcome” is understood as the expected course of a disease. It denotes the doctor's prediction of how a subject's disease will progress, and whether there is chance of recovery o recurrence.

[0040] As used herein, the expression “monitoring the treatment” refers to determine the response of a subject to a therapy, i.e. it refers to the assessment of the results of a therapy in a patient with cancer in response to a therapy. The response of the subject to the therapy may be negative (the subject does not respond to the therapy) or positive (the subject responds to the therapy).

[0041] A first step of the second method of the invention comprises quantifying the Ca10 levels in a biofluid sample isolated from the subject. The terms “Ca10”, “biofluid sample”, “subject” has been defined previously in the present description together with their particular embodiments. Likewise, methods for quantifying the level of Ca10 have been also disclosed in previous paragraphs.

[0042] Next, the second method of the invention comprises comparing the Ca10 levels obtained in the first step with a control level. When the second method of the invention refers to predict the clinical outcome of the subject, the term “control level” refers to the amount of Ca10 in a subject suffering from cancer but no suffering from metastasis. As the skilled person understands, the control level may also refer to the median value of the Ca10 levels in a collection of biological samples from subjects suffering from cancer (or comprising tumoral tissue) but no suffering from metastasis. In this case, said control level is typically obtained by combining equal amounts of samples from a population of subjects who are clinically well documented.

[0043] Alternatively, when the second method of the invention refers to monitor the treatment administered to a subject, the term “control level” refers to the Ca10 levels in the same subject previously to be treated with a therapy.

[0044] Once the Ca10 levels between the subject and the control level are compared, the skilled person in the art may conclude that if the Ca10 levels in the subject are increased with respect to the control level, then the clinical outcome of the subject is negative and either the subject is going to suffer from metastasis or the tumoral mass is going to increase; or the treatment is not effective.

[0045] Thus, in the context of the second method of the invention, the subject has “negative clinical outcome” (or “the clinical outcome is negative” or “has poor prognosis”) when the subject is going to suffer from metastasis, i.e. the cancer is going to evolve to metastasis (the tumor of the subject is a malignant tumor which is going to undergo metastasis), or the tumoral mass in going to increase in size, i.e. the tumor keep growing unaffected by the therapy, likely undergoing metastasis and reaching other organs.

[0046] On the other hand, as used in the second method of the invention, the expression “the treatment is not effective” means that the subject is not responding to the therapy, i.e. despite of the treatment administered to the subject, the cancer has evolved to metastasis, or the tumoral mass has increased in size. In the context of the second method of the invention, the expression “the tumoral mass has increased in size” means that the tumoral mass after the therapy is stabilized or the tumour keep growing.

[0047] In another aspect, the present invention relates to the use of a kit for detecting cancer in vitro, or for predicting in vitro the clinical outcome of a subject suffering from cancer, or for monitoring in vitro the treatment of a subject suffering from cancer, wherein the kit comprises elements for quantifying the Ca10 levels in a biological sample of a subject.

[0048] The terms “cancer”, “biofluid sample”, “subject”, “prediction”, “clinical outcome”, “monitoring”, etc. have been defined previously, and both their definition as well as their particular embodiments are applicable to the use of a kit for detecting cancer in vitro.

[0049] As used in the present invention, the term “Kit” refers to a product comprising the different reagents or elements necessary to carry out the detection method of the invention, or to quantify the Ca10 levels in a biofluid sample of a subject which are packaged to enable them to be transported and stored.

[0050] Therefore, in another particular embodiment of the use of a kit for the in vitro detection of cancer, or for predicting in vitro the clinical outcome of a subject suffering from cancer, or for monitoring in vitro the treatment of a subject suffering from cancer, the cancer is a carcinoma or an adenocarcinoma.

[0051] In another particular embodiment of the use of a kit for the in vitro detection of cancer, or for predicting in vitro the clinical outcome of a subject suffering from cancer, or for monitoring in vitro the treatment of a subject suffering from cancer, the cancer is prostate cancer, cervix cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, endometrial cancer, skin cancer, thyroid cancer, bladder cancer, ovarian cancer, lever cancer, oesophageal cancer, or colorectal cancer.

[0052] In another particular embodiment of the use of a kit for the in vitro detection of cancer, or for predicting in vitro the clinical outcome of a subject suffering from cancer, or for monitoring in vitro the treatment of a subject suffering from cancer, the biofluid sample is blood, serum or plasma pleural liquid, bronchioalveolar liquid, peritoneal liquid, urine, or sputum

[0053] Examples of reagents or elements for detecting Ca10H or quantifying the Ca10H level in a biological sample of a subject include, without being limited to, an antibody, in particular, anti-Ca10H antibodies; Lectins or structures designed to bind to characteristic Ca10H sites ([^4) p-D-GIcA (1 — > 4)-a-D-GlcNAc / GlcN (1 —>].), Fv fragments, nanobodies, peptides and aptamers, all of them with affinity for Ca10H.

[0054] In another particular embodiment of the use of a kit for the in vitro detection of cancer, or for predicting in vitro the clinical outcome of a subject suffering from cancer, or for monitoring in vitro the treatment of a subject suffering from cancer, the elements are antibodies, or a fragment thereof; peptides; or aptamers; all of them with affinity for Ca10H.

[0055] The kit may further include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc. Suitable materials for packaging the components of the kit include glass, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, vials, paper, sachets and the like. Additionally, the kits of the invention may contain instructions for simultaneous, sequential or separate use of the different components in the kit. Said instructions may be in the form of printed material or in the form of an electronic medium capable of storing instructions so that they can be read by a subject, such as electronic storage media, optical media and the like.

[0056] DESCRIPTION OF THE DRAWINGS

[0057] Figure 1. a, Subcutaneous inoculation protocol with ET cells in mice, b, Correlation of serum Ca10 levels with tumor size (mm3) (

[0058] Figure 2. a, Two dimensional (2D) Diffusion Ordered Spectroscopy DOSY and one dimensional, 1 D-1 H spectra of Ca10 (Control, grey) and Ca10 subjected to oxidation with NalO4 (Ca10 OX, black). 2D-DOSY spectrum of pullulan 100 kDa is included as size standard; all of them acquired a 298 K; vertical axis corresponds to logarithm of diffusion coefficient. Inset, A10 mAb activity before (Ca10) and after oxidation (Ca10 OX) determined by ELISA, b, 2D-DOSY and 1 D-1 H spectra of Ca10 (control, grey), Ca10 subjected to degradation treatment with heparinase (HPSE treat., black). 2D- DOSY spectra of Pullulan 100 kDa (black) and AUA-GIcNAc, 0,38 kDa are included as size standards; all of them acquired a 303 K. Inset, A10 activity before (Ca10) and after treatment with HPSE (HPSE treat.) determined by ELISA, c, Heteronuclear single quantum coherence spectra (HSQC) spectra of heparinase-digested Ca10 disaccharides with corresponding peak assignments (unlabelled signals correspond to buffer components tris and glycerol), d, Mean fluorescence intensity (MFI) for A10+ET cells and Ca10 concentration in supernatants after 24 h (All / 106ET cells) in the presence of glycosaminoglycan biosynthesis inhibitors O-acetyl-4-fluoro-4-deoxy- GIcNAc, 4-fluoro-4-deoxy-GlcNAc, Sodium chlorate and Xyloside (n = 3-7). Values are mean ± SEM. Statistical significance was determined using Unpaired Student t test (d) and Paired Student t test (e-g). *P < 0.05, **P < 0.01 , and ***P < 0.001.

[0059] Figure 3. a-b, HSQC spectra of heparan sulfate disaccharide standards AUA-GIcN (a) and AUA-GIcNAc (b) with the corresponding peak assignments.

[0060] Figure 4. A10 mAb activity before (Ca10) and after pronase treatment (Ca10+pronase) determined by ELISA (n = 4). Values are mean ± SEM. Statistical significance was determined using Paired Student t test (a). **P < 0.01.

[0061] Figure 5. Regression line used to calculate serum circulating Ca10 levels (Ca10H) in human sera.

[0062] Figure 6. a, Scheme of the protocol to determine human Ca10 homologue (Ca10H) concentrations in serum from healthy donors and cancer patients, b, Concentrations of Ca10H in serum from healthy controls (n=131), and patients diagnosed with prostate cancer (n=248), colorectal cancer (n=66) or other types of cancer (n=71). c, Concentrations of Ca10H in serum from prostate cancer patients with (n=42) or without (n=30) metastases, d, Correlations between Ca10H and alkaline phosphatase serum levels in prostate cancer patients with or without metastases, e, Concentrations of Ca10H in untreated, HPSE or Sialidase treated serum from different prostate cancer patients (n=3). Values are mean ± SEM. Statistical significance was determined using Mann-Whitney test (b and c), Spearman test (d) and Unpaired Student f testl). **P < 0.01 , and ***P < 0.001.

[0063] Figure 7. Serum levels of Ca10H in healthy control subjects and in patients with various types of cancer at any clinical stage. Values are mean ± SEM. Figure 8. Serum levels of Ca10H in healthy control subjects and in patients with prostate cancer (PCa) or benign prostatic hyperplasia (BPH) at any clinical stage. Values are mean ± SEM. Statistical significance was determined using Mann-Whitney test.

[0064] Figure 9. Correlation between the serum levels of Ca10H and PSA (prostate-specific antigen) in patients with prostate cancer at any clinical stage. Correlation was determined using Spearman rank correlation coefficient.

[0065] Figure 10. Serum levels of Ca10H in healthy control subjects and in patients with fibroadenoma (FA) or breast cancer at any clinical stage. Values are mean ± SEM. Statistical significance was determined using Mann-Whitney test.

[0066] Figure 11. Correlation between the serum levels of Ca10H and CA 15.3 (a) or CEA (Carcinoembryonic antigen) (b) in patients with breast cancer at any clinical stage. Correlation was determined using Spearman rank correlation coefficient.

[0067] Figure 12. Serum levels of Ca10H in healthy control subjects and in patients with colon polyps or colorectal cancer (Colon Ca) at any clinical stage. Values are mean ± SEM. Statistical significance was determined using Mann-Whitney test.

[0068] Figure 13. Correlation between the serum levels of Ca10H and CA 19.9 (a) or CEA (Carcinoembryonic antigen) (b) in patients with colorectal cancer at any clinical stage. Correlation was determined using Spearman rank correlation coefficient.

[0069] Examples

[0070] Example 1. Serum Ca10 levels correlate with the tumor mass in Ehrlich tumor-bearing mice.

[0071] Material and Methods

[0072] Ehrlich tumor (ET) cells, initially originated from hyperdiploid Ehrlich-Lettre mouse ascite tumor cells, are derived from a cloned cell variant selected for its high reactivity with the monoclonal antibody A10. The monoclonal antibody A10 (Gil, J. et al.1990. Cancer Res 50, 7301-7306) was purified from the hybridoma culture supernatant as follows:

[0073] BALB / c mice were immunized with mitomycin-treated ET cells (Subiza, J. L., Coll, J., Alvarez, R., Valdivieso, M., and De la Concha, E. G. Cancer Immunol. Immunother., 25: 87-92. 1987). Cell fusion was carried out with spleen cells from an immunized mouse (3 days after the last dose), following the method of Kohler and Milstein (Kohler, G., and Milstein, C. Nature (Lond.). 256:495-497, 1975.) with minor modifications. Briefly, mouse spleen cells (5 x 107) were fused with FO (nonsecreting myeloma) cells (2 x 107) in polyethylene glycol (PEG 4000; Merck, Darmstadt, Federal Republic of Germany). The fused cells were distributed into 96-well microtiter plates and cultured in hypoxanthine-azaserine-RPMI at 37°C in 5% CO2 incubator. Hybridoma culture supernatants were assayed for anti-ET cell surface carbohydrates by ELISA as described below. Cultures showing IgM reactivity towards these carbohydrates were cloned twice by limiting dilution using thymocytes as feeder layers, picking up only from those wells where a single clone could be assured microscopically. Stable cultures of antibody-producing hybridomas were expanded and monoclonal antibodies produced in pristane- trated BALB / c mice. Cell-free ascites fluid from each hybridoma was recovered in sterile conditions, filtered (0.45 pm; Millipore, Molsheim, France), decomplemented (40 minutes at 56°C) and stored at -40°C in aliquots until used

[0074] Ca10 was obtained from 24 h supernatants of ET cells growing in vitro in serum-free medium. Briefly, the pooled supernatants collected by centrifugation were subjected to tangential ultrafiltration on 300 kDa membranes under high ionic strength (1 M NaCI). The Ca10-enriched fraction recovered in the retentate, was subsequently diafiltrated against distilled water on 300 kDa membranes and lyophilized, having a relative composition of 85±5% carbohydrate and 10±5% protein with no significant amounts of nucleic acids or lipids.

[0075] C57BL / 6J mice were inoculated with 105ET cells by subcutaneous injection into the left groin. Tumor size was monitored weekly by calculating the tumor volume measured with a Vernier caliper. Mice were sacrificed at different time points (days) of tumor development to measure Ca10 in sera (ELISA)

[0076] Results

[0077] Ehrlich tumor (ET)-bearing mice show increasing amounts of Ca10 in their sera as the tumor develops. A strong positive correlation was observed between serum Ca10 levels and tumor size was confirmed (Figure 1 b).

[0078] Example 3. Murine Ehrlich tumor-derived Ca10 is a heparan sulfate-related glycosaminoglycan.

[0079] Material and Methods

[0080] NMR spectra were acquired using a Bruker AVANCE 600 MHz spectrometer equipped with a triple resonance TXI cryogenic probe and processed with TOPSIN 3.0 software (Bruker SA). NMR samples were prepared in deuterium oxide (D2O). One dimensional proton (1 D-1H) spectra and two-dimensional (2D) Diffusion-Ordered Spectroscopy (DOSY), 2D Heteronuclear1H-13C Single Quantum Coherence (1H-13C HSQC) and 2D homonuclear1H-1H Total Correlation SpectroscopY(TOCSY) spectra were acquired using standard pulse sequences included in TOPSPIN software to characterize the structure (TOCSY and HSQC) and hydrodynamic behaviour (DOSY) of Ca10 samples.

[0081] To monitor periodate oxidation, Ca10 was dissolved in 50 mM sodium periodate (NalO4) in D2O at a final concentration of 5 mg / mL in an NMR tube and was introduced in the spectrometer probe adjusted at 298 K. One dimensional 1 D-1H and 2D DOSY spectra were acquired at sequential times to follow the oxidation reaction during 8 h. DOSY spectra were performed using the standard Bruker pulse sequence (Iedbpgp2s), acquiring 16 gradient points, with 128 scans each, between 2 and 95% gradient intensity using a diffusion time delay of 0.25 s and 2500 ps wide pulse gradient.

[0082] To monitor heparinase digestion, 5 pL of Bacteroides heparinases II and III (400 UA / mL and 700 UA / mL, respectively) were added to the NMR tube containing 5 mg / mL Ca10 sample in heparinase buffer and the tube was introduced in the spectrometer probe adjusted at 303 K. To follow the enzymatic reaction, 1 D-1H and 2D DOSY spectra were acquired at sequential times during 24 h. The 1 D-1H spectra of 128 scans of 32 K size were recorded applying the TOPSIN zgesgp pulse program that includes a gradient sculpting water suppression procedure. The 2D DOSY experiments were performed using the pulse sequence Iedbpgp2s acquiring 32 gradient points, with 128 scans each between 2 and 95% gradient intensity. The diffusion time delay and gradient duration were 600 ms and 2500 ps, respectively, before the starting of the reaction and 170 ms and 1700 ps at end point of the reaction. Enzyme digestions. A collection of glycosidases was obtained; N-glycosidase (PNGase F), O-glycanase (endo-galactosaminidase), endo-p-acetylglucosaminidase, exoglycosidases (a- and p- galactosidases), a-mannosidase, glucosidases, glucosaminidase, a-fucosidase, sialidase (NZYTECH), chitosanase 8B, heparinases (New England Biolabs) and Chondroitinase ABC. Working conditions with each enzyme have been set up using model glycoproteins such as a1 -acid glycoprotein, fetuin, asialofetuin, ribonuclease B and commercial oligosaccharides. Enzymatic digestions were analysed by NMR, Polyacrylamide gel electrophoresis (SDS-PAGE) and Ca10-epitope sandwich ELISA evaluation.

[0083] The sample of heparinases-digested Ca10 was filtered using Vivaspin™ 50K Centrifugal Concentrators (Sartorius), and the filtrate was lyophilized and resuspended in D2O for NMR analysis. 1 D-1H, 2D TOCSY,1H-13C HSQC and DOSY spectra were acquired. For comparison a collection of heparan sulfate disaccharide standards were also NMR analyzed: AUA,2S - GlcNAc,6S (l-A) AUA - GlcNS,6S (II- S), AUA - GIcNS (IV-S), AUA - GIcNAc (IV-A), AUA,2S - GIcNAc (II l-A) (Iduron, UK) and AUA - GlcN (IV-H) (Santa Cruz Biotechnology). Samples of each of them at 1 mM concentration in D2O were prepared and 1 D-1H, 2D1H-13C HSQC and DOSY spectra acquired for comparison.

[0084] To analyse the contribution of the carbohydrate structure, hmoDCs were stimulated with 20 pg / mL of Ca10 (without treatment), oxidized Ca10 (Ca10 OX), Ca10 treated with Heparinase (HPSE treat.) or Ca10 treated with pronase (Ca10+pronase) for 18 h. Results

[0085] Ca10 derived from Ehrlich tumor cells is a heavily glycosylated high molecular weight structure, being the carbohydrate epitope recognized by mAb A10 sensitive to NalC>4 oxidation (Figure 2a), which specifically cleaves bonds between vicinal carbons bearing unsubstituted hydroxyl or amino groups. Treatment of Ca10 with NalC>4 completely abolished the mAb A10 reactivity and resulted in significant degradation and thus molecular size reduction of the carbohydrate component as observed by NMR diffusion ordered spectroscopy (DOSY) (Figure 2a). To gain insight into the carbohydrate structure contained in Ca10 and considering that the NMR profiles of Ca10 saccharides are consistent with glycosaminoglycans (GAGs), different enzymes cleaving glycans at different positions were used (Table 2). Exoglycosidases had no effect on Ca10, nor had PNGase-F or O-Glycanase. Among all the tested enzymes, Ca10 was only degraded by the GAG-lyases of heparinase type, pointing to the glycan structure of Ca10 being a heparan sulfate-related GAG (Table 2). This was confirmed by the fact that treatment of Ca10 with heparinase ll / lll (HPSE treat.) completely abolished mAb A10 reactivity (Figure 2b) and also hydrolyzed the carbohydrate component into low molecular weight fragments as observed by diffusion NMR (Figure 2b). Likewise, the NMR profile of the major fraction of the resulting disaccharide fragments upon digestion matches the profiles of AUA-GlcNH2 and AUA-GIcHNHAc related to heparan sulfate-forming disaccharides (Figure 2c and Figures 3a, 3b), confirming that the main carbohydrate moiety of Ca10 is a GAG of heparan type.

[0086] Table 2. Enzymatic digestion of Ca10 preparations.

[0087] Supporting the above data, ET cells growth in the presence of specific inhibitors of heparan sulfate biosynthesis showed significantly lower Ca10 surface expression and lower levels of soluble Ca10 than untreated cells (Figure 3d).). The treatment of Ca10 with pronase only slightly reduced mAb A10 reactivity without significant differences observed (Figure 4).

[0088] Example 4. Patients with different carcinomas show higher serum levels of a human Ca10 homologue (Ca10H) than healthy controls.

[0089] Material and Methods

[0090] Sera from patients diagnosed with prostate adenocarcinoma (n = 248), colorectal adenocarcinoma (n = 66) and other types of cancer (n = 71) at any stage of the disease, were obtained from the Central Laboratory of Hospital Clinico San Carlos (Madrid, Spain) during routine follow-up. Prostate cancer patients with (n=42) or without (n = 30) bone metastases from the Urology Department were selected to measure their serum levels of Ca10H, as described above for Ca10, and alkaline phosphatase by spectrophotometry. The serum samples from the healthy donors (n = 131) used as controls were from the Blood Donor Unit of the Hospital Clinico San Carlos.

[0091] Ca10H was measured in human sera by a A10-based sandwich ELISA. In this assay, purified mAb A10 (IgMK) from culture supernatants of the mAb A10-producing hybridoma was used as capture and detection antibody (HRP-labeled). Plates were coated overnight at 4°C with 5 pg / mL of mAb A10 in 0.05 M carbonate-bicarbonate buffer. Then, they were washed with PBS 0.25% Tween-20 (PBS-Tw) and incubated 2 h at room temperature (RT) with the samples diluted in PBS-Tw. After a wash step, a dilution of HRP-mAb A10 (1 :1000 dilution) in PBS-Tw, 5% FBS was added and incubated for 2 h at RT. After a final washing, the peroxidase substrate (OPD, 0.63 mg / mL) was added in 0.1 M citrate buffer with 0.03% H-O2 - pH 5.5. The enzymatic reaction was allowed to develop for 30 min and stopped by adding a 10% HCI solution. Ca10H concentration was expressed as arbitrary units (AU) per mL by extrapolating the OD (495 nm) to a reference curve established with Ca10. The detection and quantitation limits were established in 0.03 and 0.05 AU / mL respectively, with a linear range between 0.03 to 1.56 AU / mL (R2>0,98). Sensivity and specifity (% of recovery=99.85) assays were performed to validate its use for Ca10 measurement (Figure 5)

[0092] Results

[0093] To assess the presence of circulating human Ca10 homologue (Ca10H) and to quantify its serum levels in cancer patients and healthy controls, the same ELISA as for murine Ca10 was used as described in material and methods (Figure 6a). Sera from patients with different types of cancer under follow-up at any stage of the disease were tested. As shown, the Ca10H levels in patients with prostate (n=248), colorectal (n=66) or other cancer types (n=71) were significantly increased compared to those detected in healthy donors (n=131) (Figure 6b). To gain preliminary insight into the potential clinical relevance of Ca10H levels in cancer patients, different serum samples from prostate cancer patients with (n=42) or without (n=30) bone metastases were tested. As shown in Figure 6c, prostate cancer patients with bone metastases showed higher levels of Ca10H than those without metastases. Supporting these data, serum Ca10H levels correlated with serum alkaline phosphatase in patients with bone metastases, a well-recognized bone turnover biomarker (Figure 6d). Notably, treatment of different sera from prostate cancer patients with heparinase, but not with sialidase used as control, completely abolished Ca10H detection in those sera (Figure 6e), verifying the relationship of human Ca10H to murine Ca10 and to heparan sulfate, at least in serum from prostate cancer patients. Figure 7 shows the serum Ca10H levels in a second cohort of cancer patients, distinct from those presented in Figure 6a. This confirms that the mean values of Ca10H levels are elevated in almost all patients with epithelial cancers, compared to those in healthy controls (Figure 7).

[0094] In the case of prostate cancer, serum Ca10H values were significantly higher compared to both healthy controls and patients with benign prostatic hyperplasia (Figure 8). The correlation between Ca10H and serum PSA (prostate-specific antigen) in this type of cancer was weak (r<0.5), yet still statistically significant (Figure 9).

[0095] In breast cancer, mean serum Ca10H levels were also elevated relative to healthy controls (Figure 10). In the case of fibroadenomas, a benign breast tumor, these levels were also significantly higher than in healthy controls, although to a lesser extent (Figure 10). The correlation between the two commonly used tumor markers in breast cancer (CA 15.3 and CEA) and Ca10H was weak (r<0.5) in both cases, although statistically significant (Figure 11).

[0096] In patients with colorectal cancer, serum Ca10H levels were elevated compared to healthy controls (Figure 12). These levels were also increased in patients with colon polyposis compared to controls (Figure 12). No correlation was observed between the two commonly used tumor markers in colorectal cancer (CA 19.9 and CEA) and Ca10H (Figure 13).

Claims

CLAIMS1. An in vitro method for detecting cancer in a subject, comprising:(a) quantifying the tumoral carbohydrate Ca10 (Ca10) levels in a biofluid sample isolated from the subject; and(b) comparing the Ca10 levels obtained in step (a) with a control level,Wherein if the Ca10 levels in the subject are increased with respect to the control levels, then the subject suffers from cancer.

2. An in vitro method for predicting the clinical outcome of a subject suffering from cancer, or for monitoring the treatment of a subject suffering from cancer, comprising(a) quantifying the tumoral carbohydrate Ca10 (Ca10) levels in a biological sample isolated from the subject; and(b) comparing the Ca10 levels obtained in step (a) with a control level,Wherein if the Ca10 levels in the subject are increased with respect to the control level, then the clinical outcome of the subject is negative and either the subject is going to suffer from metastasis or the tumoral mass is going to increase; or the treatment is not effective.

3. The method according to claim 1 or 2, wherein the cancer is a carcinoma or an adenocarcinoma.

4. The method according to any one of claims 1 to 3, wherein the cancer is prostate cancer, cervix cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, endometrial cancer, skin cancer, thyroid cancer, bladder cancer, ovarian cancer, lever cancer, esophageal cancer, or colorectal cancer.

5. The method according to any one of claims 1 to 4, wherein the biofluid sample is blood, serum, plasma, pleural liquid, bronchioalveolar liquid, peritoneal liquid, urine, or sputum.

6. Use of a kit for detecting cancer in vitro, or for predicting in vitro the clinical outcome of a subject suffering from cancer, or for monitoring in vitro the treatment of a subject suffering from cancer, wherein the kit comprises elements for quantifying the Ca10 levels in a biological sample of a subject.

7. Use of a kit for detecting cancer in vitro, or for predicting in vitro the clinical outcome of a subject suffering from cancer, or for monitoring in vitro the treatment of a subject suffering from cancer, according to claim 6, wherein the cancer is a carcinoma or an adenocarcinoma.

8. Use of a kit for detecting cancer in vitro, or for predicting in vitro the clinical outcome of a subject suffering from cancer, or for monitoring in vitro the treatment of a subject suffering from cancer, according to claim 6 or 7, wherein the cancer is prostate cancer, cervix cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, endometrial cancer, skin cancer, thyroid cancer, bladder cancer, ovarian cancer, lever cancer, esophageal cancer, or colorectal cancer.

9. Use of a kit for detecting cancer in vitro, or for predicting in vitro the clinical outcome of a subject suffering from cancer, or for monitoring in vitro the treatment of a subject suffering from cancer, according to any one of claims 6 to 8, wherein the biological sample is blood, serum or plasma pleural liquid, bronchioalveolar liquid, peritoneal liquid, urine, or sputum.

10. Use of a kit for detecting cancer in vitro, or for predicting in vitro the clinical outcome of a subject suffering from cancer, or for monitoring in vitro the treatment of a subject suffering from cancer, according to any one of claims 6 to 9, wherein the elements are antibodies, or a fragment thereof, peptides or aptamers, all of them with affinity for Ca10.

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