An in vitro method for detecting cancer

Abstract

The present invention relates to an in vitro method for detecting cancer in a subject, predicting clinical outcome of a subject with cancer, or monitoring treatment of a subject with cancer in vitro based on the level of tumor carbohydrate Ca10 (Ca10) measured in a biological fluid sample isolated from the subject.

Description

[0001] This invention relates to an in vitro method for detecting cancer based on the level of tumor-associated carbohydrate Ca10 (Ca10) in a biological fluid sample. Therefore, this invention relates to the field of cancer technology, and more particularly, to tumor serum markers. Background Technology

[0002] Malignant transformation is accompanied by changes in carbohydrates on the surface of tumor cells, including aberrant or incomplete glycosylation of highly glycosylated structures. These structures can be released from tumor cells and detected in the bodily fluids of cancer patients, making them useful tumor markers. Furthermore, their presence may be associated with cancer prognosis by influencing metastasis and tumor progression.

[0003] The carbohydrate Ca10 is a highly glycosylated structure present on the surface of mouse Ehrlich ascites tumor (ET) cells. Ca10 is defined by its reactivity with the monoclonal antibody A10, which is derived from hybridomas of mice immunized with ET cells. Ca10 is spontaneously released from ET cells and can be detected in the serum of mice carrying solid ET cells.

[0004] Therefore, it is necessary to improve our understanding of tumor escape mechanisms in order to develop new tools for cancer detection.

[0005] Invention Description

[0006] By studying the serum Ca10 (mCa10) levels in mice with solid Ehrlich ascites tumors, a strong correlation between mCa10 levels and tumor quality was established.

[0007] mCa10 is a proteoglycan with a heparan sulfate glycosaminoglycan structure that is sensitive to certain heparinases (heparinase 3). The heparan sulfate structure found in mCa10 is unique due to its extremely low degree of sulfation. This may be related to the non-sulfated precursor heparin of this glycosaminoglycan, which is not typically present on the cell surface of mammalian cells. Analysis of the major carbohydrate components in mCa10 by nuclear magnetic resonance (NMR) or by chromatographic analysis of the fragments released after complete hydrolysis with heparinase revealed only a significant proportion (20% to 40%) of non-acetylated glucosamine residues. It did not show sulfation of glucosamine or glucuronic acid residues, contrary to the expectation of normally processed heparan sulfate glycosaminoglycans present in healthy tissues. This difference suggests typical processing or modification of the mCa10 heparan sulfate glycosaminoglycan.

[0008] Surprisingly, circulating levels of the human homologue of mouse tumor glycosaminoglycan mCa10 (Ca10H) were detected in the serum of cancer patients with tumors originating from different tissues. In addition to their co-reactivity with A10, the structural relationship between mCa10 and Ca10H is based on their high sensitivity to heparinase 3, which exhibits high specificity for the glycosaminoglycans of heparan sulfate. Elevated circulating Ca10H levels were found in the serum of cancer patients, with a cutoff in the serum of control subjects. Notably, in prostate cancer patients, it is possible to establish a correlation between serum Ca10H levels and bone metastasis.

[0009] Because serum Ca10H levels vary among cancer patients with different or the same histological tumor types, Ca10H may represent a novel “tumor marker” with additional clinical use as a prognostic factor.

[0010] Use of Ca10 as a tumor marker .

[0011] In view of the above, this invention discloses Ca10H as a "tumor marker," a substance found at high levels in biological fluid samples from some people with cancer compared to those without cancer. Tumor markers are produced by cancer cells themselves or by the body in response to the presence of cancer or certain benign (non-cancerous) conditions.

[0012] Based on the above, a first aspect of the present invention relates to an in vitro method for detecting cancer in a subject, hereinafter referred to as "the detection method of the present invention", comprising: (a) Quantifying tumor carbohydrate Ca10 levels in biofluid samples isolated from subjects; and (b) Compare the Ca10 levels obtained in step (a) with the control level. If a subject's Ca10 level is increased relative to the control level, the subject is considered to have cancer.

[0013] As used herein, the term "cancer detection" refers to the identification of tumor tissue through examination or parameters or biomarkers in a subject. In the context of this invention, the parameter required for cancer detection is the level of Ca10H in a subject's biofluid sample.

[0014] As used in this article, the term “cancer” refers to a disease caused by the uncontrolled proliferation of cells that produce malignant tumors or malignant tumor tissue.

[0015] A tumor is considered malignant when tumor cells are able to grow rapidly, exhibit anaplasia and / or are able to penetrate tissues, invade adjacent tissues, or even spread to other parts of the body; this process is called metastasis. Therefore, in a particular implementation, the cancer is referred to as metastatic cancer.

[0016] On the other hand, tumors (or cancers) can be located in or originate from any tissue or organ in the body. Therefore, any cancer that releases Ca10H to a biological sample is readily detectable by the methods of this invention, regardless of its stage of development, origin, or location. There are several major types of cancer. Carcinoma is a cancer that begins in the skin, or in tissues lining or covering internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemia is a cancer that begins in hematopoietic tissues (such as bone marrow), resulting in the production and entry of a large number of abnormal blood cells into the bloodstream. Lymphoma and multiple myeloma are cancers that begin in cells of the immune system. Central nervous system cancers are cancers that begin in the brain and spinal cord tissues. Cancers can be solid tumors or non-solid tumors, including metastatic tumors. In a preferred embodiment, patients diagnosed with selected cancers (e.g., carcinoma and adenocarcinoma) have Ca10H levels that are on average more than 5 times higher than those of healthy controls. Specifically, the mean serum Ca10H level in these patients was 4.0 AU / mL, with a standard error of 0.6 (SEM), while the mean in the control group was 0.7 AU / mL, with a SEM of 0.0 (see [link to relevant documentation]). Figure 9 b). Therefore, in a particular implementation, the cancer is carcinoma or adenocarcinoma.

[0017] Examples of cancer include, but are not limited to, lung tumors or lung cancer, such as small cell or non-small cell lung cancer, or adenocarcinoma, squamous cell carcinoma, or large cell carcinoma. Further specific, non-limiting examples of tumors or cancers include carcinoma, sarcoma, lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma, oligodendroglioma, mesothelioma, reticuloendothelioma, lymphoma or hematopoietic tumor, neoplasm, cancer, or malignant tumor. Other specific, non-limiting examples of tumors or cancers are: lungs, thyroid gland, head or neck, nasopharynx, larynx, nose or sinuses, brain, spine, breast, adrenal glands, pituitary gland, thyroid gland, lymph nodes, gastrointestinal tract (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), genitourinary tract (uterus, ovaries, cervix, endometrium, bladder, testes, penis, prostate), kidneys, pancreas, liver, bones, bone marrow, lymph nodes, blood, muscles, or skin tumors, tumors, or cancers. Further specific, non-limiting examples of tumors or cancers include breast cancer, prostate cancer, pancreatic cancer, stomach cancer, pleural mesothelioma, colon cancer, rectal cancer, colorectal cancer, small bowel cancer, esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer, salivary gland cancer, brain tumor, schwannoma, liver cancer, kidney cancer, bile duct cancer, endometrial cancer, cervical cancer, uterine cancer, ovarian cancer, bladder cancer, urethral cancer, skin cancer, hemangioma, malignant lymphoma, malignant melanoma, thyroid cancer, parathyroid cancer, nasal cancer, paranasal cancer, cancer of the auditory organs, floor of mouth cancer, laryngeal cancer, parotid gland cancer, submandibular cancer, bone tumors, angiofibroma, retinal sarcoma, penile cancer, testicular tumors, childhood solid tumors, Kaposi's sarcoma, maxillary sinus tumors, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, lymphoma, multiple myeloma, or leukemia.

[0018] However, in one specific embodiment of the detection method of the present invention, the cancer may be, alone or in combination with all or each of the foregoing specific embodiments, prostate cancer, cervical 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.

[0019] The first step of the detection method of the present invention includes quantifying the Ca10 level in a biological sample isolated from a subject.

[0020] The term “tumor carbohydrate Ca10” or “Ca10” includes mouse tumor carbohydrates derived from Ehrlich’s tumor cells (mCa10), as well as any other homologous tumor carbohydrates in other mammals that are equivalent to mCa10, such as their human counterparts, namely human tumor carbohydrate Ca10 or Ca10H.

[0021] "Tumor carbohydrate Ca10" or "Ca10" refers to a proteoglycan with a heparan sulfate glycosaminoglycan structure, sensitive to certain heparinases (heparinase 3), and reacting with the monoclonal antibody a10 (Gil, J. et al., 1990. Cancer Res 50, 7301-7306). In particular, mCa10 has the following characteristics: The main polysaccharide component of mCa10 is an incompletely processed heparin precursor, a precursor of heparan sulfate, which can be hydrolyzed by heparinase. Its average molecular size exceeds 100 kDa. NMR diffusion-ordered spectroscopy (DOSY) measurements show that 20% to 40% of the N-acetylglucosamine residues are deacetylated. Measurements using NMR and chromatography coupled with mass spectrometry indicate that total sulfation is less than 0.2%. The general polymeric structure of mCa10 can be described as a polysaccharide comprising a tetrameric combination of the following:

[0022] Each tetramer is

[0023] (a) x = 2, y = 0; or

[0024] (b) x = 1, y = 1; or

[0025] (c) x = 0, y = 2; and

[0026] in Represents the connection point with another tetramer; and in The ratio of ∑x / ∑y in polysaccharides ranges from 4 / 1 to 3 / 2; and in The total number of tetramers in polysaccharides ranges from 50 to 150.

[0027] Polysaccharides may include a -OH group, a β-glucuronic acid residue, or a glycosaminoglycan protein linker sequence attached to one end.

[0028] Therefore, the major polysaccharide component of mCa10 consists of the disaccharide repeating unit of heparin, β-D-glucuronic acid (GlcA) and α-DN-acetylglucosamine (GlcNAc), wherein 20% to 40% of the N-acetylglucosamine residues are deacetylated (GlcN) according to a simplified formula: [→4)β-D-GlcA (1 → 4)-α-D-GlcNAc / GlcN (1→). mCa10 containing the polysaccharide of the above formula can induce the generation of regulatory T cells (Tregs). Not wishing to be bound by any theory, based on the results shown in this invention, the inventors believe that the human counterpart of mCa10, namely Ca10H, exhibits a structure similar to that of mCa10.

[0029] Methods for quantifying the level or amount of carbohydrates in a sample are well known in the art, and any of them can be used to implement step a) of the cancer detection method of the present invention. Non-limiting examples of these methods include: lectin-based methods, NMR, chromatography, mass spectrometry, and colorimetry, depending on the nature of the carbohydrate components. For example, the quantification and analysis of heparan sulfate in plasma samples can be performed by liquid chromatography coupled with mass spectrometry. However, in one specific embodiment of the cancer detection method of the present invention, the method for quantifying Ca10H levels, alone or in combination with all or each of the foregoing specific embodiments, is by ELISA (enzyme-linked immunosorbent assay), more specifically, by ELISA using the monoclonal antibody A10 (Gil, J. et al., 1990, Cited). ad supra ).

[0030] The cancer detection method of the present invention can be performed on any suitable biological sample. As used herein, "biological fluid sample" means any fluid sample of biological material from an animal, such as, but not limited to, blood, serum, plasma, pleura, bronchoalveolar fluid, peritoneal fluid, urine, and sputum. Biological fluid samples may be tested directly, or require some form of treatment prior to testing, or may be partially purified or enriched prior to analysis. Thus, in one specific embodiment, alone or in combination with all or each of the foregoing specific embodiments, the biological fluid sample is blood, serum, plasma, pleural fluid, bronchoalveolar fluid, peritoneal fluid, urine, or sputum.

[0031] Similarly, the cancer detection method of the present invention can be performed on any subject. As used herein, the term "subject" refers to all animals classified as mammals, including but not limited to livestock and farm animals, primates, and humans, such as humans, non-human primates, cattle, horses, pigs, sheep, goats, dogs, cats, or rodents. In one specific embodiment of the cancer detection method of the present invention, the subject is a mammal, more preferably a human, either alone or in combination with all or each of the foregoing specific embodiments.

[0032] Next, in step b) of the detection method of the present invention, the Ca10 level quantified in step a) is compared with a control level. In the context of the present invention, "control level" refers to the level of Ca10 in a sample isolated from a subject who has never had cancer or does not contain tumor tissue. As will be understood by those skilled in the art, the control level may also refer to the median Ca10 level in a biofluid sample collection from a subject who has not had cancer or does not contain tumor tissue. In this case, the control level is typically obtained by pooling equal amounts of samples from a clinically well-documented population of subjects. In the context of the present invention, the terms "control level" and "reference value" are equivalent and refer to the same concept.

[0033] In the context of this invention, it should be understood that an increase in Ca10 level relative to a control level occurs when one value is greater than or higher than another. Specifically, it should be understood that an "increase" in Ca10 level occurs when the Ca10 level in the subject's sample is at least 1.1, 1.5, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or even more times higher than the Ca10 level in the control sample. Finally, based on the results of this comparison, a conclusion can be drawn regarding whether the subject has cancer. Thus, if the subject's Ca10 level is increased relative to the control level, the subject has cancer.

[0034] In another aspect, the present invention relates to an in vitro method for predicting clinical outcomes in subjects with cancer or for monitoring treatment in subjects with cancer, hereinafter referred to as "the second method of the invention," comprising:

[0035] (a) Quantifying Ca10 levels in biofluid samples isolated from subjects; and

[0036] (b) Compare the Ca10 levels obtained in step (a) with the control level. If the subject's Ca10 level increases relative to the control level, then - The subject's clinical outcome is negative, and the subject will suffer from metastasis or an increase in tumor mass; or - The treatment was ineffective.

[0037] The terms “cancer” and “subject” have been defined in the preceding paragraphs, and these definitions, along with their specific implementations, apply to the second method of the present invention.

[0038] The term "prediction" is used herein to refer to the likelihood that a patient will have a specific clinical outcome. As will be explained later, clinical outcomes can be positive or negative. The predictive method of this invention can be used clinically to make treatment decisions by selecting the most appropriate treatment for any given patient. The predictive method of this invention is a valuable tool for predicting whether a patient is likely to respond favorably to a treatment regimen. As used herein, the term "clinical outcome" is understood to refer to the expected course of a disease. It represents a physician's prediction of how a subject's disease will progress and whether there is a chance of recovery or relapse.

[0039] As used in this article, the term "monitoring treatment" refers to determining a subject's response to treatment; that is, it refers to assessing the treatment outcome in which a cancer patient responds to treatment. A subject's response to treatment can be negative (the subject does not respond to treatment) or positive (the subject responds to treatment).

[0040] The first step of the second method of the present invention includes quantifying the Ca10 level in a biofluid sample isolated from a subject. The terms “Ca10,” “biofluid sample,” and “subject” have been defined herein along with their specific embodiments. Similarly, the method for quantifying the Ca10 level has been disclosed in the preceding paragraphs.

[0041] Next, the second method of the invention involves comparing the Ca10 level obtained in the first step with a control level. When the second method of the invention relates to predicting clinical outcomes for subjects, the term "control level" refers to the amount of Ca10 in subjects with cancer that has not metastasized. As understood by those skilled in the art, the control level can also refer to the median Ca10 level in a collection of biological samples from subjects with cancer (or containing tumor tissue) that has not metastasized. In this case, the control level is typically obtained by pooling equal amounts of samples from a clinically well-documented population of subjects.

[0042] Alternatively, when the second method of the present invention relates to monitoring treatment given to subjects, the term "control level" refers to the Ca10 level of the same subject before receiving treatment.

[0043] Once the Ca10 levels of the subjects and controls have been compared, those skilled in the art can conclude that if the Ca10 levels of the subjects increased relative to the control levels, then...

[0044] - The subject's clinical outcome is negative, and the subject will suffer from metastasis or an increase in tumor mass; or

[0045] - The treatment was ineffective.

[0046] Therefore, in the case of the second method of the present invention, when the subject is about to suffer metastasis, that is, when the cancer will develop into metastasis (the subject's tumor is a malignant tumor that will undergo metastasis), or when the tumor mass will enlarge, that is, when the tumor continues to grow unaffected by treatment and may undergo metastasis and reach other organs, the subject has a "negative clinical outcome" (or "negative clinical outcome" or "poor prognosis").

[0047] On the other hand, as used in the second method of the present invention, the expression "poor treatment effect" means that the subject does not respond to the treatment, i.e., although the subject has been treated, the cancer has metastasized, or the size of the tumor mass has increased. In the context of the second method of the present invention, the expression "the size of the tumor mass has increased" means that the tumor mass has stabilized after treatment or the tumor continues to grow.

[0048] In another aspect, the present invention relates to the use of a kit in the in vitro detection of cancer, or in the in vitro prediction of clinical outcomes in subjects with cancer, or in the in vitro monitoring of treatment in subjects with cancer, wherein the kit includes components for quantifying Ca10 levels in a subject's biological sample.

[0049] The terms “cancer,” “biofluid sample,” “subject,” “prediction,” “clinical outcome,” and “monitoring” have been defined above, and their definitions and specific implementation schemes apply to the use of kits for in vitro cancer detection.

[0050] As used herein, the term "kit" refers to a product containing various reagents or components necessary for implementing the detection methods of the present invention or for quantifying the Ca10 level in a subject's biological fluid sample, and which are packaged to enable them to be transported and stored.

[0051] Therefore, in another specific implementation of the kit's use for in vitro detection of cancer, or for in vitro prediction of clinical outcomes in subjects with cancer, or for in vitro monitoring of treatment in subjects with cancer, the cancer is carcinoma or adenocarcinoma.

[0052] In another specific implementation of the kit for the in vitro detection of cancer, or for the in vitro prediction of clinical outcomes in subjects with cancer, or for the in vitro monitoring of treatment in subjects with cancer, the cancer is prostate cancer, cervical 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.

[0053] In another specific embodiment of the kit’s use for in vitro detection of cancer, or for in vitro prediction of clinical outcomes in subjects with cancer, or for in vitro monitoring of treatment in subjects with cancer, the biofluid sample is blood, serum or plasma, pleural fluid, bronchoalveolar fluid, peritoneal fluid, urine or sputum.

[0054] Examples of reagents or elements used to detect or quantify Ca10H levels in biological samples of subjects include, but are not limited to, antibodies, particularly anti-Ca10H antibodies; lectins or structures ([→4)β-D-GlcA(1 → 4)-α-D-GlcNAc / GlcN(1→]), Fv fragments, nanobodies, peptides, and aptamers designed to bind to characteristic Ca10H sites, all of which have an affinity for Ca10H.

[0055] In another specific embodiment of the kit for use in in vitro detection of cancer, or for in vitro prediction of clinical outcomes in subjects with cancer, or for in vitro monitoring of treatment in subjects with cancer, the components are antibodies or fragments thereof; peptides; or aptamers; all of which have an affinity for Ca10H.

[0056] The kit may further include, but is not limited to, buffer solutions, contamination prevention reagents, and protein degradation inhibitors. Suitable materials for packaging the kit components include glass, plastics (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, and pouches. Furthermore, the kit of the present invention may include instructions for the simultaneous, sequential, or separate use of the different components. These instructions may be in the form of printed material or an electronic medium capable of storing the instructions so that they can be read by the subject, such as electronic storage media, optical media, etc. Attached Figure Description

[0057] Figure 1 a. Protocol for subcutaneous inoculation of ET cells in mice. b. Serum Ca10 levels and tumor size (mm). 3 The correlation of ).

[0058] Figure 2 a. Two-dimensional (2D) diffusion-ordered spectra (DOSY) and one-dimensional (1D-1H) spectra of Ca10 (control, gray) and Ca10 oxidized with NaIO4 (Ca10 OX, black). The 2D-DOSY spectrum of amylopectin 100 kDa is included as a size standard; both yielded a ordinate of 298 K, corresponding to the logarithm of the diffusion coefficient. Inset: A10 mAb activity before (Ca10) and after (Ca10 OX) oxidation, as determined by ELISA. b. 2D-DOSY and 1D-1H spectra of Ca10 (control, gray) treated with heparinase degradation (HPSE-treated, black). The 2D-DOSY spectra of amylopectin 100 kDa (black) and ΔUA-GlcNAc, 0.38 kDa, are included as size standards; both yielded a ordinate of 303 K. Inset: A10 activity before (Ca10) and after (HPSE-treated) HPSE treatment, as determined by ELISA. c. Heteronuclear single quantum coherence (HSQC) spectra of Ca10 disaccharide digested by heparinase, with corresponding peak assignments (unlabeled signals correspond to the buffer components tris(hydroxymethylaminomethane) and glycerol). d. A10 after 24 hours in the presence of glycosaminoglycan biosynthesis inhibitors O-acetyl-4-fluoro-4-deoxy-GlcNAc, 4-fluoro-4-deoxy-GlcNAc, sodium chlorate, and xylosyl glycosides (n = 3-7). +Mean fluorescence intensity (MFI) of ET cells and Ca10 concentration in supernatant (AU / 10) 6 ET cells). Values ​​are mean ± SEM. Using unmatched students. t Test (d) and paired students t Test (e.g.) to determine statistical significance. P < 0.05, P < 0.01, and P <0.001.

[0059] Figure 3 HSQC spectra of heparan sulfate disaccharide standards ΔUA-GlcN (a) and ΔUA-GlcNAc (b), and their corresponding peak assignments.

[0060] Figure 4 A10 mAb activity (n = 4) was determined by ELISA before (Ca10) and after (Ca10+ streptomycin) treatment. Values ​​are mean ± SEM. Paired students were used. t Test (a) to determine statistical significance. P <0.01.

[0061] Figure 5 A regression line used to calculate the circulating Ca10 level (Ca10H) in human serum.

[0062] Figure 6 a. Protocol for determining the concentration of human Ca10 homologue (Ca10H) in the serum of healthy donors and cancer patients. b. Concentration of Ca10H in the serum of healthy controls (n=131) and patients diagnosed with prostate cancer (n=248), colorectal cancer (n=66), or other types of cancer (n=71). c. Concentration of Ca10H in the serum of prostate cancer patients with (n=42) or without (n=30) metastases. d. Correlation between serum Ca10H and alkaline phosphatase levels in prostate cancer patients with or without metastases. e. Concentration of Ca10H in untreated, HPSE-treated, or sialidase-treated serum from different prostate cancer patients (n=3). Values ​​are mean ± SEM. Mann-Whitney test (b and c), Spearman test (d), and unpaired student test were used. t The test is used to determine statistical significance. P < 0.01, P < 0.001.

[0063] Figure 7 Serum Ca10H levels in healthy controls and patients with various types of cancer at any clinical stage. Values ​​are mean ± SEM.

[0064] Figure 8 Serum Ca10H levels in healthy controls and patients with prostate cancer (PCa) or benign prostatic hyperplasia (BPH) at any clinical stage. Values ​​are presented as mean ± SEM. Statistical significance was determined using the Mann-Whitney test.

[0065] Figure 9 The correlation between serum Ca10H levels and PSA (prostate-specific antigen) in prostate cancer patients at different clinical stages was investigated. The correlation was determined using Spearman's rank correlation coefficient.

[0066] Figure 10 Serum Ca10H levels in healthy controls and patients with fibroadenoma (FA) or breast cancer at any clinical stage. Values ​​are presented as mean ± SEM. Statistical significance was determined using the Mann-Whitney test.

[0067] Figure 11 The correlation between serum Ca10H levels and CA15.3 (a) or CEA (carcinoembryonic antigen) (b) in breast cancer patients at different clinical stages was determined using Spearman's rank correlation coefficient.

[0068] Figure 12 Serum Ca10H levels in healthy controls and patients with colonic polyps or colonic cancer (colonic Ca) at any clinical stage. Values ​​are presented as mean ± SEM. Statistical significance was determined using the Mann-Whitney test.

[0069] Figure 13 The correlation between serum Ca10H levels and CA19.9 (a) or CEA (carcinoembryonic antigen) (b) in colorectal cancer patients at different clinical stages was determined using Spearman's rank correlation coefficient.

[0070] Example

[0071] Example 1. Serum Ca10 levels are associated with tumor quality in Ehrlich tumor-bearing mice.

[0072] Materials and methods

[0073] Ehrlich ascites tumor (ET) cells were originally derived from superdiploid Ehrlich-Lightley mouse ascites tumor cells, which were derived from a clonal cell variant selected for its high reactivity with the monoclonal antibody A10.

[0074] Monoclonal antibody A10 (Gil, J. et al., 1990. Cancer Res 50, 7301-7306) was purified from hybridoma culture supernatant as follows: BALB / c mice were immunized with mitomycin C-treated ET cells (Subiza, JL, Coll, J., Alvarez, R., Valdivieso, M., and De la Concha, EG Cancer Immunol. Immunother., 25: 87-92. 1987). Cell fusion was performed using spleen cells from immunized mice, following a slightly modified method by Köhler and Milstein (Köhler, G., and Milstein, C. Nature (Lond.). 256:495-497, 1975.). In short, mouse spleen cells (5 x 10⁻⁶) were used for fusion. 7 ) and FO (non-secretory myeloma) cells (2 x 10 7 In polyethylene glycol (PEG 4000; Merck, Darmstadt, Federal Republic of Germany). Fusion cells were aliquoted into 96-well microtiter plates and cultured in hypoxanthine-diazoserine-RPMI at 37°C, 5% CO2. Anti-ET cell surface carbohydrates in the hybridoma culture supernatant were determined by ELISA as described below. Using thymocytes as a feeder layer, clones of the culture that would show IgM responsiveness to these carbohydrates were double-diluted, and only those wells from which a single clone could be ensured under a microscope were selected. Stable cultures of antibody-producing hybridomas were amplified, and monoclonal antibodies were generated in phytane-treated BALB / c mice. Cell-free ascites fluid from each hybridoma was aseptically recovered, filtered (0.45m; Merck Millipore, Molsheim, France), reverse-engineered (at 56°C for 40 min), and aliquoted at -40°C until use.

[0075] Ca10 was obtained from the supernatant of ET cells grown in vitro in serum-free medium over 24 h. Briefly, the combined supernatant collected by centrifugation was subjected to tangential ultrafiltration on a 300 kDa membrane at high ionic strength (1 M NaCl). The Ca10-rich fraction recovered from the osmotic residue was subsequently percolated with distilled water on a 300 kDa membrane and lyophilized, having a relative composition of 85 ± 5% carbohydrates and 10 ± 5% protein, with no significant amounts of nucleic acids or lipids.

[0076] By 10 5C57BL / 6J mice were inoculated with ET cells subcutaneously injected into the left groin. Tumor size was monitored weekly by calculating tumor volume using calipers. Mice were sacrificed at different time points (days) of tumor development to measure serum Ca10 (ELISA).

[0077] result

[0078] As the tumor progresses, mice carrying Ehrlich ascites (ET) tumors show an increase in serum Ca10 levels. A strong positive correlation was observed between serum Ca10 levels and tumor size. Figure 1 b).

[0079] Example 3: Ca10 derived from Ehrlich ascites tumors in mice is a heparan sulfate-associated glycosaminoglycan.

[0080] Materials and methods

[0081] NMR spectra were obtained using a Bruker AVANCE 600 MHz spectrometer equipped with a triple resonance TXI cryogenic probe and processed using TOPSIN 3.0 software (Bruker SA). NMR samples were prepared in deuterium oxide (D₂O). One-dimensional protons (1D- 1 H) spectroscopy and two-dimensional (2D) diffusion-ordered spectroscopy (DOSY), 2D heteronuclear spectroscopy 1 H- 13 C single quantum coherence ( 1 H- 13 C HSQC) and 2D isonuclear 1 H- 1 H total correlation spectrum Y (TOCSY) spectra were obtained using standard pulse sequences included in the TOPSPIN software to characterize the structure (TOCSY and HSQC) and hydrodynamic behavior (DOSY) of the Ca10 samples.

[0082] To monitor periodate oxidation, Ca10 was dissolved in 50 mM sodium periodate (NaIO4) with D2O, resulting in a final concentration of 5 mg / mL in the NMR tube, and introduced into the spectrometer probe conditioned at 298 K. One-dimensional 1D- 1 H and 2D-DOSY spectra were acquired continuously to track the oxidation reaction over 8 hours. DOSY spectroscopy was performed using a standard Bruker pulse sequence (ledbpgp2s), acquiring 16 gradient points, 128 scans per interval, with gradient intensities between 2% and 95%, using a diffusion time delay of 0.25 s and a wide pulse gradient of 2500 μs.

[0083] To monitor heparinase digestion, 5 μL of Bacteroidetes heparinase II and III (400 UA / mL and 700 UA / mL, respectively) were added to an NMR tube containing 5 mg / mL Ca10 sample in heparinase buffer, and the tube was then introduced into a spectrometer probe adjusted to 303 K. To track the enzymatic reaction, 1D- PCR samples were continuously collected over 24 hours. 1 H and 2D-DOSY spectra. 128 scans of 32 K size 1D-DOSY spectra were recorded using the TOPSIN zgesgp pulse program. 1 H-spectroscopy was used, and the procedure included a gradient-based water suppression process. 2D DOSY experiments were performed using the pulse sequence ledbpgp2s, acquiring 32 gradient points and performing 128 scans, each between 2% and 95% gradient intensity. The diffusion time delay and gradient duration were 600 ms and 2500 μs before the reaction began, and 170 ms and 1700 μs at the reaction endpoint, respectively.

[0084] Enzymatic digestion. A set of glycosidases was obtained: n-glycosidase (PNGase F), o-glycanase (endogalactosaminease), endo-β-acetylglucosidase, exoglycosidase (α- and β-galactosidase), α-mannosidase, glucosidase, glucosidase, α-fucosidase, sialidase (NZYTECH), chitosanase 8B, heparinase (New England Biolabs), and chondroitinase ABC. Working conditions for each enzyme were established using model glycoproteins such as α1-acid glycoprotein, fetoglobulin, desialylated fetoglobulin, ribonuclease B, and commercial oligosaccharides. Enzymatic digestion was evaluated and analyzed by NMR, SDS-PAGE, and Ca10-epitope sandwich ELISA.

[0085] The heparinase-digested Ca10 sample was filtered using a Vivaspin™ 50K centrifuge (Sartorius), the filtrate was lyophilized and resuspended in D2O for NMR analysis. 1D- 1 H, 2D TOCSY, 1 H- 13C HSQC and DOSY spectra. For comparison, a group of heparan sulfate disaccharide standards were also analyzed by NMR: ΔUA, 2S-GlcNAc, 6S (IA), ΔUA-GlcNS, 6S (II-S), ΔUA-GlcNS (IV-S), ΔUA-GlcNAc (IV-A), ΔUA, 2S-GlcNAc (III-A) (Iduron, UK), and ΔUA-GlcN (IV-H) (Santa Cruz Biotechnology). Samples were prepared at a concentration of 1 mM in D2O, and 1D- 1 H, 2D 1 H- 13 C HSQC and DOSY spectra were used for comparison.

[0086] To analyze the contribution of carbohydrate structure, hmoDCs were stimulated for 18 hours with 20 μg / mL Ca10 (untreated), oxidized Ca10 (Ca10OX), Ca10 treated with heparinase (HPSE-treated), or Ca10 treated with streptomycin (Ca10+streptomycin).

[0087] result

[0088] Ca10, derived from Ehrlich ascites cells, is a highly glycosylated, high-molecular-weight structure and a carbohydrate epitope recognized by mAb A10, which is sensitive to NaIO4 oxidation. Figure 2 a) specifically cleaves the bonds between adjacent carbons containing unsubstituted hydroxyl or amino groups. Treatment of Ca10 with NaIO4 completely eliminated the reactivity of mAb A10 and led to significant degradation, resulting in a reduction in the molecular size of the carbohydrate component observed by NMR diffusion-ordered spectroscopy (DOSY). Figure 2 a). To gain a deeper understanding of the carbohydrate structure contained in Ca10, and considering the consistency of the NMR spectrum of Ca10 sugar with glycosaminoglycans (GAGs), different enzymes were used to cleave the polysaccharide at different locations (Table 2). Exoglycosidases had no effect on Ca10, nor did PNGase-F or O-glycanases. Of all the enzymes tested, Ca10 was degraded only by heparinase-type GAG-lyases, indicating that the glycan structure of Ca10 is a heparan sulfate-associated GAG (Table 2). Treatment of Ca10 with heparinase II / III (HPSE treatment) completely eliminated mAbA10 reactivity ( Figure 2 b), and also hydrolyzes carbohydrate components into low molecular weight fragments (as observed by diffusion NMR). Figure 2The fact in b) confirms this. Similarly, the NMR spectrum of the major portion of the disaccharide fragment produced after digestion matches the spectra of ΔUA-GlcNH2 and ΔUA-GlcHNHAc associated with the disaccharide formation of heparan sulfate. Figure 2 c and Figure 3 (a, 3b) confirms that the main carbohydrate component of Ca10 is heparin-type GAG.

[0089]

[0090] Table 2. Enzymatic digestion of Ca10 preparations

[0091] Supporting the above data is the fact that, compared with untreated cells, ET cells grown in the presence of a specific inhibitor of heparan sulfate biosynthesis showed significantly lower Ca10 surface expression and lower levels of soluble Ca10 (Ca10). Figure 3 d). Treatment of Ca10 with streptomycin only slightly reduced mAb A10 reactivity; no significant difference was observed. Figure 4 ).

[0092] Example 4: Patients with different cancers showed higher serum levels of human Ca10 homolog (Ca10H) than healthy controls.

[0093] Materials and methods

[0094] During routine follow-up, serum samples were obtained from patients diagnosed with any stage of prostate adenocarcinoma (n = 248), colorectal adenocarcinoma (n = 66), and other types of cancer (n = 71) from the central laboratory of the San Carlos Clinical Hospital (Clínico San Carlos) (Madrid, Spain). Patients with prostate cancer from the urology department with (n = 42) or without (n = 30) bone metastases had their serum Ca10H levels (as described above for Ca10) and alkaline phosphatase levels measured spectrophotometrically. Serum samples from healthy donors (n = 131) served as controls from the blood donation unit of the San Carlos Clinical Hospital.

[0095] Ca10H in human serum was determined by an A10-based sandwich ELISA. In this assay, purified mAbA10 (IgMκ) from the culture supernatant of hybridomas producing mAbA10 was used as the capture and detection antibody (HRP-labeled). Plates were coated with 5 μg / mL mAbA10 overnight in 0.05 M carbonate-bicarbonate buffer at 4°C. The plates were then washed with 0.25% Tween-20 PBS (PBS-Tw) and incubated at room temperature (RT) with the sample diluted in PBS-Tw for 2 hours. After the washing step, HRP-mAbA10 dilution (1:1000) in PBS-Tw, 5% FBS was added, and the plates were incubated at room temperature for 2 hours. After the final wash, peroxidase substrate (OPD, 0.63 mg / mL) was added to a solution containing 0.03% HRP. – 02 - In 0.1 M citrate buffer at pH 5.5. Allow the enzymatic reaction to proceed for 30 minutes and stop by adding 10% HCl solution. The Ca10H concentration was expressed as arbitrary units (AU) per milliliter by extrapolating the OD (495 nm) to a reference curve established with Ca10. The detection and quantitation limits were 0.03 AU / mL and 0.05 AU / mL, respectively, and the linear range was 0.03 AU / mL to 1.56 AU / mL (R0). 2 > 0.98). Sensitivity and specificity (recovery % = 99.85%) analyses were performed to validate its use in Ca10 measurement. Figure 5 ).

[0096] result

[0097] To assess the presence of the circulating human Ca10 homolog (Ca10H) and quantify its serum levels in cancer patients and healthy controls, the same ELISA used for mouse Ca10 was employed as described in Materials and Methods. Figure 6 a). Serum levels in patients with different types of cancer followed up at any stage of the disease were tested. As shown in the figure, Ca10H levels were significantly increased in patients with prostate cancer (n=248), colorectal cancer (n=66), or other cancer types (n=71) compared to those detected in healthy donors (n=131). Figure 6 b). To preliminarily understand 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. Figure 6 As shown in Figure c, prostate cancer patients with bone metastases exhibited higher Ca10H levels than those without metastases. Supporting these data is the correlation between serum Ca10H levels in patients with bone metastases and serum alkaline phosphatase, a well-established biomarker for bone turnover. Figure 6 d). Notably, treatment of different sera from prostate cancer patients with heparinase, but without using sialidase as a control, completely eliminated the detection of Ca10H in these sera. Figure 6 (e) confirmed the relationship between human Ca10H and mouse Ca10 and heparan sulfate, at least in the serum of prostate cancer patients. Figure 7 The serum Ca10H levels of the second group of cancer patients were shown, compared with... Figure 6 The difference in a. This confirms that, compared with the healthy control group, the mean Ca10H level was elevated in almost all patients with epithelial cancer ( Figure 7 ).

[0098] In the case of prostate cancer, serum Ca10H levels were significantly higher than those in healthy controls and patients with benign prostatic hyperplasia. Figure 8 In this type of cancer, the correlation between Ca10H and serum PSA (prostate-specific antigen) was weak (r < 0.5), but still statistically significant. Figure 9 ).

[0099] In breast cancer, the mean serum Ca10H level was also elevated compared to the healthy control group. Figure 10 In the case of fibroadenoma (a benign breast tumor), these levels, although lower, were significantly higher than in healthy controls. Figure 10 In both cases, the correlation between the two commonly used tumor markers (CA 15.3 and CEA) and Ca10H in breast cancer was weak (r < 0.5), although statistically significant. Figure 11 ).

[0100] Compared to healthy controls, patients with colorectal cancer had elevated serum Ca10H levels ( Figure 12 These levels were also increased in patients with colonic polyposis compared to the control group. Figure 12 No correlation was observed between two commonly used tumor markers (CA 19.9 and CEA) and Ca10H in colorectal cancer. Figure 13 ).

Claims

1. An in vitro method for detecting cancer in a subject, comprising: (a) Quantifying the level of tumor carbohydrate Ca10 (Ca10) in biofluid samples isolated from subjects; and (b) Compare the Ca10 levels obtained in step (a) with the control level. If a subject's Ca10 level is increased relative to the control level, the subject is considered to have cancer.

2. An in vitro method for predicting clinical outcomes in subjects with cancer, or for monitoring treatment in subjects with cancer, comprising: (a) Quantifying the levels of tumor carbohydrate Ca10 (Ca10) in biological samples isolated from subjects; and (b) Compare the Ca10 levels obtained in step (a) with the control level. If the subject's Ca10 level increases relative to the control level, then - The subject's clinical outcome is negative, and the subject will suffer from metastasis or an increase in tumor mass; or - The treatment was ineffective.

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

4. The method according to any one of claims 1 to 3, wherein the cancer is prostate cancer, cervical 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.

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

6. The kit is used in the in vitro detection of cancer, or in the in vitro prediction of clinical outcomes in subjects with cancer, or in the in vitro monitoring of treatment in subjects with cancer, wherein the kit includes components for quantifying Ca10 levels in a subject's biological sample.

7. Use of the kit according to claim 6 in in vitro detection of cancer, or in in vitro prediction of clinical outcomes in subjects with cancer, or in in vitro monitoring of treatment in subjects with cancer, wherein said cancer is carcinoma or adenocarcinoma.

8. Use of the kit according to claim 6 or 7 in the in vitro detection of cancer, or in the in vitro prediction of clinical outcomes in subjects with cancer, or in the in vitro monitoring of treatment in subjects with cancer, wherein said cancer is prostate cancer, cervical 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.

9. The kit according to any one of claims 6 to 8 for use in in vitro detection of cancer, or in in vitro prediction of clinical outcomes in subjects with cancer, or in in vitro monitoring of treatment in subjects with cancer, wherein the biological sample is blood, serum or plasma, pleural fluid, bronchoalveolar fluid, peritoneal fluid, urine or sputum.

10. The kit according to any one of claims 6 to 9 for use in in vitro detection of cancer, or in in vitro prediction of clinical outcomes in subjects with cancer, or in in vitro monitoring of treatment in subjects with cancer, wherein the components are antibodies or fragments thereof, peptides or aptamers, all of which have an affinity for Ca10.

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