Cancer Biomarkers

Abstract

The present invention relates to a method for screening for cancer in a subject, comprising determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, said sample being obtained from said subject.

Description

[Technical field]

[0001] The present invention relates to cancer biomarkers and methods of screening for cancer, which involve determining the levels and / or composition of certain biomarkers indicative of cancer in a subject.

[0002] The number of cancer cases is predicted to increase substantially in the near future. The increase in the cancer population dictates an urgent need to improve the current diagnostic status of cancer. In particular, affordable and practical tools for cancer diagnosis are needed to assist medical professionals in the early detection of high-risk cancers that typically correlate with more favorable clinical outcomes, or to guide the treatment of current cancer patients.

[0003] Circulating biomarkers are molecules that can be measured in an individual's available body fluids, such as blood or urine, and whose levels are useful for aiding in diagnosis and / or prognosis and / or prediction of response to treatment. Examples of widely used biomarkers are prostate-specific antigen (PSA) for prostate cancer, carcinoembryonic antigen (CEA) for colorectal cancer, and carbohydrate antigen 125 for ovarian cancer. However, the clinical value of these biomarkers for diagnosing cancer is actively debated. For example, the standard PSA test for detecting prostate adenocarcinoma in men over 50 years of age with average risk has typical values ​​of sensitivity and specificity equal to 21% (51% for high-grade lesions with Gleason score 8 or higher) and 91%, respectively, assuming a cutoff value equal to 4ng / ml (Wolf,AM,et al.,2010 CA Cancer J Clin 60,70-98).

[0004] What is needed in the art is a new method for screening (e.g., diagnosing) cancer. Identification of novel biomarkers for cancer could potentially have clinical implications for a large number of patients and is an important clinical advance. Here, we provide evidence for blood and / or urine markers of cancer that can be used in highly specific (e.g., 98% specificity has been demonstrated) and highly sensitive assays for detecting cancer. Thus, advantageously, such methods are non-invasive, performed on readily available samples, and highly accurate.

[0005] The availability of such tests would also be valuable for several medical decisions, such as: determining the risk of progression in newly diagnosed cancers, guiding treatment options in cancer patients with uncertain clinical risk, monitoring cancer before and after surgery or drug treatment, preventing recurrence of disease for longer periods after a patient has typically been declared cured, assessing the occurrence of cancer in at-risk populations, such as individuals with a genetic predisposition or who exhibit risk factors or who exhibit symptoms, confirming whether metastasis is attributable to a particular cancer, predicting recurrence or relapse in patients with early-stage cancer, distinguishing lesions suspicious for cancer from non-malignant disease, detecting a particular stage (e.g., early stage) or grade (e.g., low grade) of cancer, determining the tissue of origin of the cancer, or screening for cancer in the general population.

[0006] The inventors have found that the levels of certain glycosaminoglycans (GAGs), particularly the levels of the protein-free fractions of certain GAGs such as chondroitin sulfate (CS) and heparan sulfate (HS), and / or the chemical composition of said GAGs, particularly the chemical composition of the protein-free fractions of said GAGs, are found at differential levels in body fluid samples from cancer patients compared to control subjects. These differential levels of GAGs CS or HS, or the differential chemical composition of GAGs CS or HS (GAG profile) can serve as a biomarker for cancer and are therefore useful for screening for cancer in subjects. Thus, the inventors have found that GAG profiles from accessible fluids are suitable for use as biomarkers / diagnostic markers for cancer.

[0007] Surprisingly and advantageously, the inventors have found that changes in the levels of GAGs CS and / or HS are observed in accessible body fluids of cancer patients, especially when the protein-free fraction of GAGs is analyzed, and that these GAG ​​profiles are suitable for use as biomarkers of cancer. The inventors have also shown that in addition to the overall (total) levels or concentrations of CS and / or HS, other changes in chemical composition, such as specific disaccharide sulfation patterns of CS and / or HS (especially when the protein-free fraction of GAGs is analyzed), are also observed between cancer and normal samples, and can be used very effectively to diagnose cancer.

[0008] Surprisingly, it has not been previously recognized that, for example, the analysis of the protein-free fraction of GAGs as described herein, as opposed to the total GAG population (including both protein-free and protein-bound GAGs), can result in an effective diagnosis of cancer. This discovery by the inventors advantageously results in improvements and efficiencies in the GAG ​​processing steps required for GAG-based cancer screening methods. For example, in the preferred method of the present invention, it is not necessary to contact the sample with a proteolytic agent (or other suitable agent) to "release" or "liberate" or "convert" the protein-bound fraction of GAGs into a protein-free or less protein-free fraction so that it can be analyzed. Thus, in the preferred method of the present invention, it is specifically the protein-free fraction (or the naturally protein-free fraction) of GAGs in the body fluid sample that is analyzed.

[0009] The inventors have also found that other improvements and efficiencies in the processing steps of GAGs can be made. For example, it has been surprisingly found that a method in which the purification step is omitted, in which the GAGs in a sample are purified on the basis of their negative charge, for example using a method such as anion exchange chromatography or using an anion exchange resin, results in improved processing and measurement of GAGs, regardless of whether the protein-free or total (protein-free + protein-bound) fraction of GAGs is analyzed.

[0010] Clearly, the discovery that cancer diagnosis can be performed in an available body fluid sample from a subject, such as blood or urine, would be highly advantageous, where we have observed systemic changes in GAG composition associated with cancer.

[0011] Advantageously, the inventors have also shown that the identified markers, specific for cancer development and calculated based on measurements in accessible body fluids, are accurate and robust predictors of the disease.

[0012] Thus, in one aspect, the present invention provides a method of screening for cancer in a subject, comprising determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, said sample being obtained from said subject.

[0013] A preferred method of the present invention provides a method for screening for cancer selected from the group consisting of genitourinary cancer (e.g., kidney, prostate or bladder cancer), respiratory tract cancer (e.g., lung cancer), brain tumors, hematological cancers (e.g., lymphoma), colorectal cancer, uterine cancer, gastrointestinal neuroendocrine tumors, breast cancer, ovarian cancer and head and neck cancer.

[0014] In some embodiments, the genitourinary cancer is kidney cancer (eg, renal cell carcinoma), prostate cancer, or bladder cancer.

[0015] In some embodiments, the respiratory tract cancer is lung cancer or respiratory tract head and neck cancer.

[0016] In some embodiments, the brain tumor is a glioma (eg, diffuse glioma).

[0017] In some embodiments, the hematological cancer is lymphoma.

[0018] In some embodiments, the breast cancer is invasive ductal carcinoma.

[0019] In some embodiments, the uterine cancer is cervical squamous cell carcinoma or endometrial cancer.

[0020] In some embodiments, the head and neck cancer is head and neck squamous cell carcinoma.

[0021] In some embodiments, the prostate cancer may be prostate adenocarcinoma.

[0022] In some embodiments, the colorectal cancer may be colorectal carcinoma.

[0023] In some embodiments, the gastrointestinal neuroendocrine tumor can be a small intestinal neuroendocrine tumor.

[0024] In some embodiments, the endometrial cancer can be an endometrial adenocarcinoma.

[0025] In some embodiments, the ovarian cancer can be ovarian epithelial cancer.

[0026] Another preferred method of the present invention provides a method for screening for cancer selected from the group consisting of bladder cancer, invasive ductal carcinoma, cervical squamous cell carcinoma, chronic lymphocytic leukemia, colorectal cancer, endometrial cancer, diffuse glioma, gastrointestinal neuroendocrine tumor, head and neck squamous cell carcinoma, diffuse large B-cell lymphoma, non-small cell lung cancer, ovarian cancer, prostate cancer and renal cell carcinoma. Diffuse large B-cell lymphoma may also be referred to herein as non-follicular lymphoma.

[0027] References herein to "cancer" can refer to any type of cancer, or to one or more of the types of cancer described in the paragraph above.

[0028] In some embodiments, genitourinary cancers are not screened for. In some embodiments, respiratory tract cancers are not screened for. In some embodiments, brain tumors are not screened for. In some embodiments, blood cancers are not screened for. In some embodiments, genitourinary cancers are not screened for. In some embodiments, colorectal cancer is not screened for. In some embodiments, colorectal cancer is not screened for. In some embodiments, uterine cancer is not screened for. In some embodiments, gastrointestinal endocrine tumors are not screened for. In some embodiments, small intestinal endocrine tumors are not screened for. In some embodiments, breast cancer is not screened for. In some embodiments, head and neck cancer is not screened for. In some embodiments, ovarian cancer is not screened for. In some embodiments, ovarian epithelial cancer is not screened for. In some embodiments, renal cell carcinoma is not screened for. In some embodiments, prostate cancer is not screened for. In some embodiments, prostate adenocarcinoma is not screened for. In some embodiments, bladder cancer is not screened for. In some embodiments, lung cancer is not screened for. In some embodiments, glioma is not screened for. In some embodiments, lymphoma is not screened for. In some embodiments, invasive ductal carcinoma is not screened. In some embodiments, cervical squamous cell carcinoma is not screened. In some embodiments, endometrial cancer is not screened. In some embodiments, endometrial adenocarcinoma is not screened. In some embodiments, chronic lymphocytic leukemia is not screened. In some embodiments, diffuse glioma is not screened. In some embodiments, head and neck squamous cell carcinoma is not screened. In some embodiments, diffuse large B-cell lymphoma is not screened. In some embodiments, non-small cell lung cancer is not screened. In some embodiments, renal cell carcinoma (or renal cell carcinoma) is not screened. In some embodiments, thyroid cancer is not screened. In some embodiments, pancreatic cancer is not screened. In some embodiments, liver cancer is not screened. In some embodiments, cholangiocarcinoma is not screened. In some embodiments, gastric cancer is not screened. In some embodiments, esophageal cancer is not screened. In some embodiments, skin cancer is not screened. In some embodiments, melanoma is not screened.In some embodiments, neuroendocrine tumors are not screened for.

[0029] In some embodiments, one or more (or all) of kidney cancer (eg, renal cell carcinoma), skin cancer (eg, melanoma), and prostate cancer are not screened for.

[0030] In a preferred method of the invention, an alteration in the level and / or chemical composition of chondroitin sulfate (CS) and / or heparan sulfate (HS) in the protein-free fraction compared to a control level and / or chemical composition is indicative of cancer in the subject.

[0031] In some embodiments of certain methods of the invention, alterations in the levels and / or chemical composition of chondroitin sulfate (CS) and / or heparan sulfate (HS) compared to control levels and / or chemical composition are indicative of cancer in the subject.

[0032] In preferred methods of the invention, both the level and the chemical composition are determined, in other preferred methods of the invention, only the chemical composition is determined, or in other preferred methods, the level (total level or concentration) of only CS and / or HS is determined.

[0033] The methods of the invention include determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample. In some embodiments, the level and / or chemical composition of one of the GAGs is determined. In some embodiments, the level and / or chemical composition of chondroitin sulfate (CS) is determined. In some embodiments, the level and / or chemical composition of heparan sulfate (HS) is determined. In some embodiments, the level and / or chemical composition of chondroitin sulfate (CS) and heparan sulfate (HS) are determined.

[0034] In some preferred methods of the invention, the method comprises determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample. Thus, in some embodiments, the level and / or chemical composition of the protein-free fraction of chondroitin sulfate (CS) is determined. In some embodiments, the level and / or chemical composition of the protein-free fraction of heparan sulfate (HS) is determined. In some embodiments, the level and / or chemical composition of the protein-free fraction of chondroitin sulfate (CS) and heparan sulfate (HS) is determined.

[0035] In some embodiments, hyaluronic acid (HA) levels and / or chemical composition are further determined.

[0036] Glycosaminoglycans (GAGs) are sugar-containing molecules that can be attached to proteins on serine residues, i.e. form part of proteoglycans. They are formed from linear or unbranched chains of monosaccharides that can be sulfated (i.e. are polysaccharides). Heparan sulfate (HS), chondroitin sulfate (CS), keratan sulfate (KS), hyaluronic acid (HA) and heparin are common types of GAGs, of which HS and CS are examples of sulfated GAGs. Different types of GAGs are distinguished by different repeating disaccharide units.

[0037] When linked or attached to proteins, CS and HS are GAGs that share a common biosynthetic pathway in linking to a core protein, but then they differ in their polymerization in that the CS repeating disaccharide is composed of repeating N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) residues, whereas the repeating disaccharide in HS is typically composed of repeating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) residues. Each monosaccharide is attached by a specific enzyme allowing multiple levels of regulation over GAG synthesis.

[0038] Although GAGs can be bound to proteins, i.e., in the protein-bound or proteoglycan form, GAGs can also exist in a "free" form, i.e., non-protein-bound or non-proteoglycan form. Such "free" forms of GAGs are referred to herein as "protein-free GAGs."

[0039] Thus, in a body fluid (or body sample), there is typically a protein-free fraction of GAGs (or a protein-free pool of GAGs) and a protein-bound fraction of GAGs (or a protein-bound pool of GAGs). The two fractions together (i.e., the protein-free fraction + the protein-bound fraction) may be referred to as the total GAG fraction or total GAG pool.

[0040] As discussed elsewhere herein, in a preferred method of the invention, the level or composition of the protein-free fraction of one or both of the GAGs chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample is determined.

[0041] Protein-free fraction GAG (or disaccharide units derived therefrom as discussed elsewhere herein) for analysis can be obtained by any suitable means.

[0042] As discussed elsewhere herein, bodily fluid samples are typically processed prior to analysis. Such processing typically involves subjecting the GAGs to a processing step to obtain disaccharide units for analysis. Such processing typically involves contacting the sample (or the GAGs in the sample) with an enzyme (e.g., a GAG lyase such as chondroitinase or heparinase) that digests (or fragments) the GAGs into disaccharide units. Without wishing to be bound by theory, such enzymes are unable to access protein-bound GAGs, but rather act only (or essentially only) on (or use as a substrate for) protein-free GAGs. When a proteoglycan (which is a protein with GAGs attached or bound to it) is contacted with a proteolytic agent (e.g., a protease such as proteinase K), the protein component is digested by the proteolytic agent and the protein-bound GAGs are liberated or released, meaning that the protein-bound GAGs are in (or converted to) a "free" or "released" form, which is then available for digestion (or fragmentation) by an enzyme such as a GAG lyase. In certain preferred methods of the invention, what is determined is specifically the level and / or composition of the protein-free fraction (or native protein-free fraction) of one or both of the GAGs CS and HS, and thus the preferred methods of the invention do not involve contacting the sample with a proteolytic agent (e.g., a protease such as proteinase K). Thus, omitting the proteolytic agent during processing of the sample for analysis is a way to obtain (or obtain only, or obtain essentially only) the protein-free fraction of GAGs (or disaccharides subsequently derived therefrom) for analysis. Protein-bound GAGs (proteoglycan GAGs) that have been (or are) "liberated" or "released" by the action of a proteolytic agent (eg, a protease) are not protein-free GAGs in accordance with the present invention.

[0043] Thus, protein-free GAGs (or protein-free fractions of GAGs) are GAGs (or fractions of GAGs) that are already (or naturally) free (i.e., not bound to proteins) in the absence (or absence) of a sample that has been treated with a proteolytic agent (e.g., a protease). In other words, protein-free GAGs (or protein-free fractions of GAGs) are GAGs (or fractions of GAGs) that are free (i.e., not bound to proteins) in the original, initial, or untreated sample. For example, protein-free GAGs (or protein-free fractions of GAGs) can be GAGs (or fractions of GAGs) that are present in a sample, e.g., an original or untreated sample, and that are susceptible to or accessible to or available for (e.g., are substrates for) digestion (or fragmentation) into disaccharide units as described elsewhere herein, e.g., using an enzyme, such as a lyase enzyme.

[0044] As indicated above, protein-free GAGs (or protein-free fractions of GAGs) may be considered to be non-protein-bound (or non-protein-bound fractions) or non-proteoglycan (or non-proteoglycan fractions) forms of GAGs. In other words, protein-free GAGs (or protein-free fractions of GAGs) are GAGs that do not modify proteoglycans in the original, initial, or untreated sample.

[0045] Thus, in some preferred embodiments, the methods include determining the level and / or chemical composition of protein-free fractions of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, wherein the sample is subjected to a treatment prior to determining said level and / or composition, and wherein said treatment does not include contacting the sample with a proteolytic agent (e.g., a protease such as proteinase K) or other agent capable of releasing protein-free GAGs from protein-bound GAGs.

[0046] From another perspective, in some preferred embodiments, the methods include determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, wherein the sample is subjected to a treatment prior to determining said level and / or composition, and wherein said treatment does not include contacting the sample with a proteolytic agent (e.g., a protease such as proteinase K) or other agent capable of releasing GAGs (or GAG chains) from proteoglycans.

[0047] Thus, in some preferred embodiments, the method comprises determining the level and / or chemical composition of the protein free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, said sample being obtained from said subject and subjected to a treatment prior to determining said level and / or chemical composition, said treatment comprising: (a) fragmenting said one or both GAGs into disaccharide units, (b) without contacting the sample with a proteolytic agent prior to (a).

[0048] As noted above, protein-bound GAGs (or protein-bound fractions of GAGs) may be considered to be proteoglycan GAGs (or proteoglycan fractions of GAGs). From another perspective, protein-bound GAGs (or protein-bound fractions of GAGs) may be considered to be GAGs that typically require the protein to which they are bound so that they can be liberated or released (or become liberated or released) upon contact with a proteolytic agent (e.g., a protease, such as a non-specific protease).

[0049] In some other methods of the invention, the level and / or chemical composition of the total fraction (or total pool) of either or both of GAGs, CSs and HSs in a bodily fluid sample may be determined (i.e., protein-free GAGs + protein-bound GAGs). In such embodiments, the sample is typically contacted with a proteolytic agent during sample processing.

[0050] The "level" of HS or CS referred to herein generally refers to the total level or amount (e.g., concentration) of HS or CS present in a sample. The level of CS and / or HS in a sample can be measured or determined by any suitable method known and described in the art. Some methods involve electrophoresis, particularly capillary electrophoresis, such as capillary electrophoresis with fluorescence detection, such as laser-induced fluorescence detection. Other suitable methods are gel electrophoresis, such as agarose gel electrophoresis (e.g., FACE, fluorophore-assisted carbohydrate electrophoresis) or mass spectrometry or liquid chromatography, such as HPLC, optionally combined with mass spectrometry (HPLC-MS). A preferred method involves high performance liquid chromatography (HPLC), preferably ultra HPLC (UHPLC), in combination with mass spectrometry, such as MS / MS or triple quadrupole mass spectrometry. A preferred method includes ultra high performance liquid chromatography (UHPLC) in combination with an electrospray ionization triple quadrupole mass spectrometry system.

[0051] Conveniently, these levels may be measured as concentrations (e.g. actual or absolute levels or concentrations), for example as micrograms per mL (μg / ml), however, again, any suitable measure of levels may be used.

[0052] In a preferred method of the invention, the levels of HS and / or CS, preferably the levels of the protein-free fraction of HS and / or CS, are determined separately or individually. In other words, the method does not involve measuring the total GAG level in the sample, or the total level of all GAGs present, for example, in combination in the protein-free GAG ​​fraction, but rather the level of one or more of the individual GAGs, HS or CS.

[0053] In certain embodiments, the level (e.g., total level or concentration) of CS and / or HS, e.g., the level of the protein-free fraction of CS and / or HS, can be determined, e.g., in a blood or urine sample.

[0054] In some embodiments of the invention, an increase in the level (or concentration) of CS in a bodily fluid sample and / or an increase in the level (or concentration) of HS in a bodily fluid sample is indicative of cancer in said subject and can be used to screen, diagnose, etc., the subject as described elsewhere herein.

[0055] In some embodiments of the invention, an increase in the level (or concentration) of CS in a blood (e.g., plasma) sample is indicative of a cancer selected from the group consisting of uterine cancer (e.g., cervical squamous cell carcinoma or endometrial cancer), blood cancer (e.g., lymphoma such as chronic lymphocytic leukemia), brain cancer (e.g., diffuse glioma), gastrointestinal endocrine tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer), ovarian cancer, bladder cancer, prostate cancer, and kidney cancer (e.g., renal cell carcinoma) in said subject, and can be used to screen and diagnose the subject, as described elsewhere herein.

[0056] In some embodiments, an increased level (or concentration) of CS in a urine sample is indicative of a cancer selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), head and neck cancer (e.g., head and neck squamous cell carcinoma), kidney cancer (e.g., renal cell carcinoma) and bladder cancer in the subject, and can be used to screen, diagnose, etc., the subject as described elsewhere herein.

[0057] In some embodiments, an increased level (or concentration) of HS in a urine sample is indicative of a cancer selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), head and neck cancer (e.g., head and neck squamous cell carcinoma), and kidney cancer (e.g., renal cell carcinoma) in said subject, and may be used to screen, diagnose, etc., the subject, as described elsewhere herein.

[0058] The individual monosaccharide units that make up CS and HS may have different sulfation patterns in terms of the position and amount / number of sulfate molecules. In the case of CS, sulfation may occur most commonly at one or more of the following positions: 2 of GlcA and 4 and 6 of GalNAc. In the case of HS, sulfation may occur at one or more of the following positions: 2 of GlcA after epimerization to IdoA (iduronic acid), 3 and 6 of GlcNAc, and N-sulfation of GlcNAc. Thus, each individual disaccharide in the GAG ​​chain may have 0 (i.e., unsulfated), 1, 2, 3 or 4 (only in HS) sulfated forms, which in turn results in different overall chemical compositions of the GAG ​​chain in terms of sulfation levels and specific disaccharide sulfation patterns.

[0059] As described elsewhere herein, preferred embodiments of the present invention involve the determination of the chemical composition of one or both of CS and HS, in particular the chemical composition of the protein-free fraction of one or both of CS and HS. The term "chemical composition" as used herein can refer to both the level of GAGs as well as the disaccharide sulfation composition of GAGs. In particular, the term includes the determination of one or more specific forms, such as sulfated forms, of the disaccharides that constitute the CS or HS GAGs. In other words, the term "chemical composition" refers to the amount or level of one or more of the various sulfated and / or non-sulfated forms of CS or HS disaccharides as described elsewhere herein, as well as some other characteristic of the individual GAGs present (e.g., total HS or CS GAG levels), or other characteristics related to GAG sulfation (e.g., HS charge or CS charge). Such chemical composition analyzed or determined in the present invention can also be referred to herein as GAG profile, GAG form, GAG signature, GAG characteristics, GAG-ome, GAG-ome characteristics.

[0060] Thus, for example, the term "chemical composition" as used herein can refer to the determination or analysis of the sulfation pattern (e.g., one or more of the sulfated forms) of the disaccharides that make up the CS and / or HS.

[0061] For example, there are eight major sulfated and non-sulfated forms (sulfation patterns, disaccharide sulfate forms) of CS, which are 0s CS (also called non-sulfated CS or CS 0 unit), 2s CS (also called chondroitin-2-sulfate), 4s CS (also called chondroitin-4-sulfate or CS A unit), 6s CS (also called chondroitin-6-sulfate or CS C unit), 2s4s CS (also called chondroitin-2-4-sulfate), 2s6s CS (also called chondroitin-2-6-sulfate or CS D unit), 4s6s CS (also called chondroitin-4-6-sulfate or CS E unit) and Tris CS (also called chondroitin-2-4-6-sulfate or trisulfated CS).

[0062] Each of the above are forms of CS GAG (CS GAG forms or properties) that may be measured in the methods of the present invention. One or more of these forms may be measured, for example, up to eight of these sulfated forms may be measured, for example, 1, 2, 3, 4, 5, 6, 7 or all eight of these sulfated forms. In some embodiments, measurement of all eight of these sulfated forms is preferred.

[0063] Another GAG profile for CS that may be measured in the methods of the invention is the total concentration of CS (also referred to herein as CS tot or Tot CS or total CS) or the total level of CS. This is typically measured as a concentration, e.g., absolute concentration in μg / ml, as described elsewhere herein. In some embodiments, total CS is measured by summing the levels of all measured CS disaccharide forms listed above. In embodiments of the invention in which the total concentration of CS is measured as one of the GAG ​​profiles, it is preferred that at least one other GAG or CS profile is measured, e.g., a profile that is not based on the total level of other individual GAGs present (e.g., not total HS or total HA). In some embodiments, the total concentration of CS is not measured. In some embodiments, the measurement of one or more CS GAG profiles is preferred.

[0064] "Charged CS" is another GAG form or property that can be measured in the present invention, for example, as part of a GAG profile. "Charged CS" refers to the total fraction of sulfated disaccharides of CS, i.e., the fraction of sulfated disaccharides of CS present or measured in a sample out of the total CS disaccharides present or measured in the sample (i.e., sulfated CS disaccharides / sulfated + non-sulfated CS disaccharides). In some embodiments, "charged CS" refers to the weighted sum of the concentrations of all CS disaccharides divided by total CS, where the weight is the number of sulfo groups in the disaccharide, i.e., 0 for 0s CS, 1 for 4s CS, 6s CS, and 2s CS, 2 for 2s6s CS, 4s6s CS, and 2s4s CS, and 3 for Tris CS (this is the definition of "charged CS" used in connection with the term "charged CS" in the Examples section of this specification).

[0065] Because the measurement of "charged CS" depends on the measurement of other properties, namely the levels of sulfated and non-sulfated CS disaccharides, this property is not referred to herein as an independent GAG or CS property. Thus, up to nine independent CS properties may be measured in the methods of the invention, which are the eight sulfated and non-sulfated forms listed above, along with total CS. In some embodiments, all nine of these independent CS properties are measured. In some embodiments, only the eight sulfated and non-sulfated forms of the CS disaccharides listed above are measured.

[0066] In some embodiments, it is preferred to measure up to eight (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) or all eight of the sulfated forms of CS (i.e., sulfated and non-sulfated forms) along with total CS and charged CS. In preferred embodiments, at least one (or at least 2, 3, 4, 5, 6, 7, or 8) sulfated forms of CS are measured.

[0067] In some embodiments, one or more (or all) of the following GAG properties may be measured or determined: the relative level of 4s CS to 6s CS (e.g., 4s CS / 6s CS ratio or 6s CS / 4s CS inverse ratio), the relative level of 6s CS to 0s CS (e.g., 6s CS / 0s CS ratio or 0s CS / 6s CS inverse ratio), or the relative level of 4s CS to 0s CS (e.g., 4s CS / 0s CS ratio or 0s CS / 4s CS inverse ratio). In some embodiments, one or more of the following GAG properties may be measured or determined: the relative level of 6s CS to 0s CS (e.g., 6s CS / 0s CS ratio or 0s CS / 6s CS inverse ratio), or the relative level of 4s CS to 0s CS (e.g., 4s CS / 0s CS ratio or 0s CS / 4s CS inverse ratio). In some embodiments, the relative levels of 4s CS to 6s CS (eg, the 4s CS / 6s CS ratio or the inverse 6s CS / 4s CS ratio) are not measured or determined.

[0068] For example, there are eight major sulfated and non-sulfated forms (sulfation patterns, disaccharide sulfate forms) of HS, which are 0s HS (also called non-sulfated HS), 2s HS (sulfated at position 2 of GlcA), Ns HS (sulfated at position N of GlcNAc), 6s HS (sulfated at position 6 of GlcNAc), 2s6s HS (sulfated at positions 2 of GlcA and 6 of GlcNAc), Ns6s HS (sulfated at positions 6 and N of GlcNAc), Ns2s HS (sulfated at positions 2 of GlcA and 6 and N of GlcNAc), and Tris HS (sulfated at positions 2 of GlcA and 6 and N of GlcNAc, also called trisulfated HS). Note that sulfate at position 3 of GlcNAc is also possible but is rarely observed.

[0069] Each of the above are forms of HS GAG (HS GAG forms or properties) that may be measured or determined in the methods of the invention. However, due to its rarity, in preferred embodiments of the invention, the sulfated form having sulfate at position 3 of GlcNAc is not measured. Thus, in the methods of the invention, one or more (or all) of these nine (or preferably eight) forms may be measured, for example, up to nine (or preferably up to eight) of these sulfated forms, for example, 1, 2, 3, 4, 5, 6, 7, 8 or all nine. In some embodiments, measurement of all eight of these sulfated forms (excluding the sulfated form having sulfate at position 3 of GlcNAc) is preferred.

[0070] Another GAG profile for HS that may be measured in the methods of the invention is the total concentration of HS (also referred to herein as HS tot or Tot HS or total HS) or the total level of HS. This is typically measured as a concentration, e.g., absolute concentration in μg / ml, as described elsewhere herein. In some embodiments, the total HS is measured by summing the levels of all measured HS disaccharide forms listed above. In embodiments of the invention in which the total concentration of HS is measured as one of the GAG ​​profiles, it is preferred that at least one other GAG profile or HS profile is measured, e.g., a profile that is not based on the total level of other individual GAGs present (e.g., not total CS or total HA). In some embodiments, the total concentration of HS is not measured. Measurement of one or more HS GAG profiles is preferred in the methods of the invention, e.g., when the sample is blood or urine, particularly when the sample is a urine sample.

[0071] "Charged HS" is another GAG form or property that can be measured in the present invention, for example, as part of a GAG profile. "Charged HS" refers to the total fraction of sulfated disaccharides of HS, i.e., the fraction of sulfated disaccharides of HS present or measured in a sample out of the total HS disaccharides present or measured in the sample (i.e., sulfated HS disaccharides / sulfated + non-sulfated HS disaccharides). In some embodiments, "charged HS" refers to the weighted sum of the concentrations of all HS disaccharides divided by total HS, where the weight is the number of sulfo groups in that disaccharide, i.e., 0 for 0s HS, 1 for 4s CS, 6s HS, and 2s HS, 2 for 2s6s CS, 4s6s HS, and 2s4s HS, and 3 for Tris HS (this is the definition of "charged HS" used in connection with the term "charged HS" in the Examples section of this specification).

[0072] Because the measurement of "charged HS" depends on the measurement of other properties, namely sulfated and non-sulfated HS disaccharides, this property is not referred to herein as an independent GAG or HS property. Thus, up to 10 independent HS properties can be measured in the methods of the invention, which are the nine sulfated and non-sulfated forms listed above (preferably excluding the sulfated form having sulfate at position 3 of GlcNAc) along with total HS. Thus, in some embodiments, nine independent HS properties are measured (the eight major sulfated and non-sulfated HS forms + total HS). In some embodiments, only the eight sulfated and non-sulfated forms of the HS disaccharides listed above are measured.

[0073] In some embodiments, it is preferred to measure up to eight (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) or all eight of the major sulfated forms of HS (i.e., the sulfated and non-sulfated forms listed above, excluding the sulfated form having sulfate at position 3 of GlcNAc), along with total HS and charged HS. In preferred embodiments, at least one (or at least 2, 3, 4, 5, 6, 7, or 8) sulfated forms of HS are measured.

[0074] In some embodiments, 9 independent HS profiles are measured (8 major sulfated and non-sulfated HS forms + total HS) and 9 independent CS profiles are measured, i.e., 18 independent GAG profiles.

[0075] As described elsewhere herein, in some embodiments, the level and / or chemical composition of hyaluronic acid (HA) may further be determined in the body fluid sample. Hyaluronic acid (HA) is typically not sulfated. Thus, when HA is measured according to the present invention, it is typically and preferably the level (total level or concentration) of HA that is measured (also referred to herein as HA tot or Tot HA or total HA). This is typically measured as a concentration, e.g., in μg / ml, as described elsewhere herein. In some embodiments of the present invention where the total concentration of HA is measured as one of the GAG ​​characteristics, at least one other GAG characteristic (e.g., CS and / or HS characteristic) is measured, e.g., a characteristic that is not based on the total level of other individual GAGs present (e.g., not total CS or total HS). In some embodiments, HA is not measured. In some embodiments, HA is not measured in urine. In some embodiments, HA is not measured in blood.

[0076] In some embodiments, 9 independent HS profiles are measured (8 major sulfated and non-sulfated HS forms + total HS), 9 independent CS profiles are measured (8 major sulfated and non-sulfated CS forms + total CS), and total HA is measured, i.e., 19 independent GAG profiles are measured.

[0077] In some embodiments, 8 independent HS profiles are measured (8 major sulfated and non-sulfated HS forms), 8 independent CS profiles are measured (8 major sulfated and non-sulfated CS forms), and total HA is measured, i.e., 17 independent GAG profiles are measured.

[0078] In some embodiments, eight independent HS profiles are measured (eight major sulfated and non-sulfated HS forms) and eight independent CS profiles are measured (eight major sulfated and non-sulfated CS forms), i.e., 16 independent GAG profiles are measured.

[0079] CS (total CS) and HS (total HS) are typically measured in absolute concentrations, e.g., μg / ml, as described elsewhere herein.

[0080] The various CS and HS sulfated forms can also be measured in terms of absolute concentrations, e.g., in μg / ml. Thus, in some embodiments, the level (or concentration) of a given CS sulfated form or a given HS sulfated form is the absolute level or concentration of a given CS sulfated form or a given HS sulfated form.

[0081] However, the various CS and HS sulfated forms may alternatively or additionally be measured in terms of relative concentrations (or levels). Thus, in some embodiments, the level (or concentration) of a given CS sulfated form or a given HS sulfated form is a relative level or concentration.

[0082] Thus, the "level" or "concentration" of a sulfated GAG form can be its absolute concentration or its relative concentration. In some embodiments, both the absolute and relative concentrations of one or more sulfated GAG forms are measured (or determined).

[0083] "Relative concentration" can be considered as the mass fraction (e.g., %) of a given CS sulfated form or a given HS sulfated form obtained (or calculated or determined) by normalizing its absolute concentration to the total concentration of the relevant GAG class, i.e., by normalizing its absolute concentration to the total CS concentration or total HS concentration (as appropriate).

[0084] Therefore, the relative concentration of a given sulfated form of CS can be considered as the mass fraction (e.g., %) of said given sulfated form of CS obtained (or calculated or determined) by normalizing its absolute concentration with the total CS concentration.

[0085] The relative concentration of a given sulfated form of HS can be considered as the mass fraction (e.g., in %) of said given sulfated form of HS obtained (or calculated or determined) by normalizing its absolute concentration by the total HS concentration.

[0086] The relative concentration can be expressed as a percentage (%).

[0087] Thus, in some embodiments, the level (or concentration) of a given CS sulfated form or a given HS sulfated form can be an absolute and / or relative concentration of a given CS sulfated form or a given HS sulfated form. In some embodiments, the level (or concentration) of a given CS sulfated form or a given HS sulfated form is an absolute concentration of a given CS sulfated form or a given HS sulfated form. In some embodiments, the level (or concentration) of a given CS sulfated form or a given HS sulfated form is a relative concentration of a given CS sulfated form or a given HS sulfated form.

[0088] Alternatively, the relative concentration can be considered as a "fraction" or "mass fraction" or "proportion" or "relative measure," as discussed below.

[0089] As mentioned above, the GAG ​​profile or GAG form, e.g., disaccharide sulfated forms (excluding total CS or total HS), may be measured not as absolute levels or concentrations, but as fraction sizes or fractions or mass fractions (e.g., μg / μg) or percentages or relative measurements, e.g., given a value less than 1 or normalized to 1 (or expressed as a %) depending on the level of all sulfated forms of the relevant GAG class (or all major sulfated forms of the relevant GAG class) measured in the sample. In other words, the level of each of the desired sulfated forms is measured independently and then normalized to 1. In other words, the level of each of the desired sulfated forms is measured independently and then its mass fraction or volume fraction or molar fraction is calculated. These fractions may be expressed as percentages. In other words, these fractions may be normalized to 100. For example, in some embodiments, the fraction size of a given sulfated or non-sulfated CS form can be determined by measuring the level of the given sulfated or non-sulfated CS form and dividing it by the sum of the levels of all CS sulfated forms (or all of the major sulfated forms) and non-sulfated CS forms measured (or present) in the sample. In some embodiments, the fraction size of a given sulfated or non-sulfated HS form can be determined by measuring the level of the given sulfated or non-sulfated HS form and dividing it by the sum of the levels of all HS sulfated forms (or major sulfated forms) and non-sulfated HS forms measured (or present) in the sample. When calculating such fractions, it is preferred that at least the major sulfated forms of CS or HS are measured so that the fractions of a particular individual sulfated form can be normalized to 1.

[0090] In some embodiments, preferably, at least the unsulfated form of HS or CS is measured as the predominant sulfated form.

[0091] Relative measurements may be easier to interpret, for example an 0s CS measurement of 0.6 indicates that 60% of the measured CS disaccharides are unsulfated. However, absolute levels may also be measured. Indeed, in some embodiments, it is preferred to measure the absolute concentration of one or more sulfated forms.

[0092] In some preferred embodiments of the invention, the disaccharide composition (e.g., a particular sulfation pattern (e.g., sulfated form)) of one or more (or all) of the disaccharides constituting the CS and / or HS is measured or determined. In more preferred embodiments, the sulfation properties or forms of one or more (or all) of the CS and / or HS, such as those outlined above (e.g., 0s CS, 2s CS, etc.), are measured or determined. Suitable methods for doing this are well known to the skilled artisan and any of these can be used. However, a convenient method for achieving such quantification of the disaccharide composition or suitable properties or forms of the CS or HS (and separation of the disaccharide forms) is to use electrophoresis, in particular capillary electrophoresis, for example capillary electrophoresis with fluorescence detection, for example capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). An alternative method is liquid chromatography, preferably HPLC (high performance liquid chromatography), for example SAX HPLC. Preferably, mass spectrometry (e.g., HPLC-MS), for example electrospray ionization mass spectrometry (ESI-MS), is also used. Alternatively, mass spectrometry can be used without chromatography (e.g., liquid chromatography). One example is capillary electrophoresis with laser-induced fluorescence detection. Another example is HPLC ESI-MS. A preferred method involves high performance liquid chromatography (HPLC), preferably ultra HPLC (UHPLC), in combination with mass spectrometry, e.g., MS / MS or triple quadrupole mass spectrometry. A preferred method includes ultra performance liquid chromatography (UHPLC) coupled with electrospray ionization triple quadrupole mass spectrometry. Particularly preferred methods are outlined in the examples.

[0093] In some methods of the invention where the level of one or more individual disaccharide forms is measured, the GAG ​​is subjected to a processing step (e.g. a step of fragmentation or cleavage or digestion (e.g. by chemical digestion or enzymatic treatment)) to obtain the disaccharide units, which are then analyzed. The enzyme can be a GAG lyase, such as a chondroitinase or a heparinase, or a combination of chondroitinases, or a combination of heparinases, or a combination of one or more chondroitinases and one or more heparinases. Preferably, the chondroitinase is chondroitinase ABC or chondroitinase B. Preferably, the heparinase is heparinase I-II-III. In a preferred embodiment, one or more chondroitinases and one or more heparinases, preferably chondroitinase ABC and heparinase I-II-III, are used.

[0094] In some methods of the invention, GAGs in a sample are subjected to a step of extraction (e.g., using a proteolytic agent, such as a protease, such as a non-specific protease, such as proteinase K) and / or purification, such as using an anion exchange resin (or other means for purifying GAGs based on the negative charge of the GAGs).

[0095] However, in preferred methods of the invention, one or both of these steps are not performed (i.e., there is no such extraction and / or no such purification). For example, in preferred embodiments of the invention, such protein digestion (extraction) steps are not performed, e.g., when the level and / or composition of the protein-free fraction of one or more GAGs is determined. In other words, the method does not involve a processing step in which the sample is contacted with a proteolytic agent, e.g., a protease, e.g., proteinase K. As discussed elsewhere herein, omitting a processing step in which the sample is contacted with a proteolytic agent means that the protein-free fraction of GAGs can be specifically analyzed.

[0096] In another preferred embodiment of the invention, the method does not involve a processing step in which GAGs (e.g., GAGs, CS and / or HS) are purified from the sample based on the negative charge of the GAGs, for example, using an anion exchange resin. Without wishing to be bound by theory, omitting a processing step in which GAGs (e.g., GAGs, CS and / or HS) are purified from the sample based on the negative charge of the GAGs (e.g., using an anion exchange resin) can simplify the method and provide efficiencies in terms of the yield of GAGs obtained during processing of a body fluid sample.

[0097] In a preferred method of the invention, the method does not involve a processing step of contacting the sample with a proteolytic agent, such as a protease, such as proteinase K, and does not involve a processing step of purifying GAGs (e.g., GAG, CS and / or HS) from the sample based on the negative charge of the GAGs, for example using an anion exchange resin (or other means of purifying GAGs based on the negative charge of the GAGs).

[0098] In some methods of the invention, the GAGs in the sample (e.g., the various different GAG forms in the sample) are subjected to a separation and / or quantification step, as described elsewhere herein. For example, HPLC in combination with mass spectrometry may be used in preferred embodiments, as discussed elsewhere herein. A particularly preferred method includes ultra-high performance liquid chromatography (UHPLC) coupled with electrospray ionization triple quadrupole mass spectrometry.

[0099] Other methods that may be used are known in the art, however, examples include analytical techniques that involve the use of antibodies against various GAG forms, such as techniques such as Western blot, ELISA or FACS, or methods involving agarose gel electrophoresis (e.g., fluorophore-assisted carbohydrate electrophoresis (FACE)) or polyacrylamide gel electrophoresis (PAGE).

[0100] A preferred method of the present invention provides a method for screening for cancer in a subject, said method comprising determining or measuring the amount or level and / or chemical composition of GAGs in a body fluid sample, preferably in a protein-free fraction, said determining comprising determining the level, preferably in said protein-free fraction, of one or more GAG ​​characteristics selected from the group consisting of one or more (or all) of specific sulfated or non-sulfated forms of CS or HS disaccharides, charged HS, charged CS, total concentration of CS or total concentration of HS.

[0101] A preferred method of the present invention provides a method for screening for cancer in a subject, said method comprising determining or measuring the amount or level and / or chemical composition in a body fluid sample, preferably in the protein free fraction, of GAGs, said determining comprising determining or measuring the amount or level and / or chemical composition of 0s CS, 2s CS, 6s CS, 4s CS, 2s6s CS, 2s4s CS, 4s6s CS, Tris CS, 0s HS, 2s HS, 6s HS, 2s6s HS, Ns HS, Ns2s HS, Ns6s HS, Tris HS, ratio of 4s CS to 6s CS, ratio of 6s CS to 0s CS and ratio of 4s CS to 0s CS, charged HS, charged CS, total concentration of CS (CStot) and total concentration of HS (HS The method includes determining the level, preferably in the protein-free fraction, of one or more (or all) of the GAG ​​characteristics selected from the group consisting of: ...

[0102] As noted above, the levels (or concentrations) of particular sulfated or non-sulfated forms of CS and HS can be absolute or relative concentrations.

[0103] Thus, in some embodiments, a GAG characteristic selected from the group consisting of 0s CS, 2s CS, 4s CS, 6s CS, 2s4s CS, 2s6s CS, 4s6s CS, Tris CS, charged CS, total CS, 0s HS, 2s HS, Ns HS, 6s HS, 2s6s HS, Ns6s HS, Ns2s HS, Tris HS, HS sulfated at position 3 of GlcNAc, total HS, charged HS may be measured or determined in the methods of the invention. As described elsewhere herein, preferably, HS sulfated at position 3 of GlcNAc is not measured or determined. Thus, in some embodiments, a GAG profile selected from the group consisting of 0s CS, 2s CS, 4s CS, 6s CS, 2s4s CS, 2s6s CS, 4s6s CS, Tris CS, charged CS, total CS, 0s HS, 2s HS, Ns HS, 6s HS, 2s6s HS, Ns6s HS, Ns2s HS, Tris HS, total HS, charged HS may be measured or determined in the methods of the invention. In some embodiments, the level and / or chemical composition of hyaluronic acid (HA) may further be determined in the bodily fluid sample.

[0104] In some embodiments, for example when the levels of various GAGs are determined in the protein free fraction, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 6s CS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of:

[0105] In some embodiments, for example when the levels of various GAGs are determined in the protein free fraction, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 6s CS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of:

[0106] In some embodiments, for example when the levels of various GAGs are determined in the protein free fraction, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, absolute concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s CS, relative concentration of Ns HS, absolute concentration of 6Ns HS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s HS, absolute concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s CS, relative concentration of Ns HS, absolute concentration of 6Ns HS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS.

[0107] In some embodiments, for example when the levels of various GAGs are determined in the protein free fraction, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, absolute concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s CS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of:

[0108] In some embodiments, for example when the levels of various GAGs are determined in the protein free fraction and said body fluid sample is urine, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 6s CS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of:

[0109] In some embodiments, for example when the levels of various GAGs are determined in the protein free fraction and said body fluid sample is urine, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 6s CS, absolute concentration of 2s6s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of:

[0110] In some embodiments, for example when the levels of various GAGs are determined in the protein free fraction and said body fluid sample is urine, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, CS tot, HS tot., specific sulfated or non-sulfated forms of CS or HS disaccharides.

[0111] In some embodiments, for example when the levels of various GAGs are determined in the protein free fraction and said body fluid sample is urine, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 0s HS, relative concentration of 6s CS, relative concentration of Ns HS, relative concentration of 0s HS, relative concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 0s CS, absolute concentration of 4s CS, absolute concentration of 6s CS, absolute concentration of 0s CS, ratio of 6s CS to 0s CS, CS tot, HS tot., specific sulfated or non-sulfated forms of CS or HS disaccharides.

[0112] In some embodiments, for example, when the levels of various GAGs are determined in a protein-free fraction and the body fluid sample is blood, e.g., plasma, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 4s CS, relative concentration of 4s CS, ratio of 4s CS to 0s CS, CS Tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of:

[0113] In some embodiments, for example, when the levels of various GAGs are determined in a protein-free fraction and the body fluid sample is blood, e.g., plasma, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 4s CS, relative concentration of 4s CS, CS Tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of:

[0114] In some embodiments, for example, when the levels of various GAGs are determined in a protein-free fraction and the body fluid sample is blood, e.g., plasma, the method of the invention comprises measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 0s CS, relative concentration of 4s CS, ratio of 4s CS to 0s CS, specific sulfated or non-sulfated forms of CS or HS disaccharides.

[0115] In some embodiments, for example, when the levels of various GAGs are determined in a protein-free fraction, the GAG ​​profile can be determined from both blood (e.g., plasma) and urine. Preferred exemplary GAG profiles are as outlined in the paragraph above, and any combination of these blood and urine profiles can be used. Such a determination involving both blood (e.g., plasma) and urine samples can be referred to as a combined determination, and can result in, for example, a combined score.

[0116] In some embodiments, for example, when the levels of various GAGs are determined in a protein-free fraction and both blood (e.g., plasma) and urine fluid samples are used, the method of the invention comprises: Measuring or determining in said urine sample one or more (or all) of the GAG ​​characteristics selected from the group consisting of the relative concentration of 0s CS, the absolute concentration of 0s HS, the absolute concentration of Ns HS, the relative concentration of 2s6s CS, the relative concentration of 0s HS, the relative concentration of Ns HS (HS tot), and charged CS; -measuring or determining one or more (or all) of the GAG ​​characteristics selected from the group consisting of absolute concentration of 0s CS, relative concentration of 4s CS, and CS tot.

[0117] In some embodiments, for example, when the levels of various GAGs are determined in a protein-free fraction and both blood (e.g., plasma) and urine fluid samples are used, the method of the invention comprises: detecting or determining in said urine sample one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 0s HS, relative concentration of Ns HS, relative concentration of 0s CS, relative concentration of 0s HS, absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 4s CS, relative concentration of 6s CS, absolute concentration of 6s CS, ratio of 6s CS to 0s CS, CS tot, and charged CS; Measuring or determining one or both of the GAG ​​characteristics selected from the group consisting of the ratio of 4s CS to 0s CS, and CS tot in said blood, e.g., plasma sample.

[0118] In some embodiments, for example when the levels of various GAGs are determined in a protein-free fraction, the method of the invention comprises measuring or determining in a urine sample one or more (or all) of the GAG ​​characteristics selected from the group consisting of absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 4s CS, absolute concentration of 6s CS. In some such embodiments, an increase in the absolute concentration of 0s HS or absolute concentration of 4s CS in a urine sample, for example compared to a control level, is indicative of cancer in the subject. In some such embodiments, a decrease in the absolute concentration of Ns HS or absolute concentration of 6s CS in a urine sample, for example compared to a control level, is indicative of cancer in the subject.

[0119] In some embodiments, for example when the levels of various GAGs are determined in a protein-free fraction, the methods of the invention include measuring or determining the absolute concentration of 0s CS in a blood (e.g., plasma) sample. In some embodiments, in the method of screening for cancer, an increase in the absolute concentration of 0s CS in a blood (e.g., plasma) sample, for example compared to a control level, is indicative of cancer in said subject.

[0120] In some embodiments, for example, when the levels of various GAGs are determined in a protein-free fraction and both blood (e.g., plasma) and urine body fluid samples are used, the method of the present invention comprises measuring or determining in the urine sample one or more (or all) of the GAG ​​characteristics selected from the group consisting of absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 4s CS, absolute concentration of 6s CS, and measuring or determining in the blood (e.g., plasma) sample the absolute concentration of 0s CS. In some such embodiments, an increase in the absolute concentration of 0s HS or absolute concentration of 4s CS in the urine sample, for example, compared to a control level, is indicative of cancer in the subject. In some such embodiments, a decrease in the absolute concentration of Ns HS or absolute concentration of 6s CS in the urine sample, for example, compared to a control level, is indicative of cancer in the subject. In some embodiments, in a method of screening for cancer, an increase in the absolute concentration of 0s CS in the blood (e.g., plasma) sample, for example, compared to a control level, is indicative of cancer in the subject.

[0121] In some embodiments, for example when the levels of various GAGs are determined in a protein-free fraction, a preferred method of the invention comprises measuring or determining the level of one or both of the GAG ​​characteristics selected from the group consisting of the absolute concentration of 0s CS and CS Tot.

[0122] In some embodiments, for example, when the levels of various GAGs are determined in the protein-free fraction, an increase (e.g., compared to a control level) in the level of one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 6s CS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, CS tot, HS tot, a particular sulfated or non-sulfated form of a CS or HS disaccharide selected from the group consisting of is indicative of cancer (preferred cancers are as described elsewhere herein, e.g., in Table A).

[0123] In some embodiments, for example, when the levels of various GAGs are determined in the protein-free fraction, a decrease in the level of one or more (or all) of the GAG ​​characteristics selected from the group consisting of the relative concentration of charged CS and 6s CS, e.g., compared to the control level, is indicative of cancer (preferred cancers are as described elsewhere herein, e.g., in Table A).

[0124] In some embodiments, for example, when the levels of various GAGs are determined in a protein-free fraction and the body fluid sample is blood, e.g., plasma, an increase (e.g., compared to a control level) in the level of one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 4s CS, relative concentration of 4s CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of CS Tot, is indicative of cancer (preferred cancers are as described elsewhere herein, e.g., in Table A).

[0125] In some embodiments, for example, levels of various GAGs are determined in a protein-free fraction and where the body fluid sample is blood, e.g., plasma, a reduction in charged CS, e.g., compared to control levels, is indicative of cancer (preferred cancers are as described elsewhere herein, e.g., in Table A).

[0126] In some embodiments, for example, when the levels of various GAGs are determined in the protein-free fraction and the body fluid sample is urine, an increase (e.g., compared to a control level) in the level of one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s Cs, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 6s CS, absolute concentration of 2s6s CS, relative concentration of 2s6s CS, CS tot, HS tot, a particular sulfated or non-sulfated form of a CS or HS disaccharide selected from the group consisting of is indicative of cancer (preferred cancers are as described elsewhere herein, e.g., in Table A).

[0127] In some embodiments, for example, when the levels of various GAGs are determined in the protein-free fraction and the body fluid sample is urine, a decrease in the level of one or both of the relative concentrations of GAG-specific charged CS or 6s CS, e.g., compared to control levels, is indicative of cancer (preferred cancers are as described elsewhere herein, e.g., in Table A).

[0128] Although the methods of the invention may involve determining the level of only one of the listed GAG characteristics, preferably the methods include determining the level of more than one of the GAG ​​characteristics, and more preferably the methods include determining the levels of two or more, three or more, four or more, or all of the GAG ​​characteristics. In some embodiments, the methods include determining the levels of up to eight or all of the sulfated and non-sulfated CS forms: 0s CS, 2s CS, 6s CS, 4s CS, 2s6s CS, 2s4s CS, 4s6s CS, and Tris CS, optionally together with total CS and / or charged CS, and / or determining the levels of up to eight or all of the sulfated and non-sulfated HS forms: 0s HS, 2s HS, 6s HS, 2s6s HS, Ns HS, Ns2s HS, Ns6s HS, and Tris HS, optionally together with total HS and / or charged HS.

[0129] In some embodiments, particularly preferred GAG forms measured or determined in the methods of the invention are one or more (or all) of the absolute concentration of 0s CS, CS Tot, absolute concentration of 4s CS, and absolute concentration of 0s HS. In some such embodiments, an alteration, preferably an increase, in the level of one or more (or all) of the above GAG ​​forms, e.g., compared to a control level, is indicative of cancer.

[0130] In some embodiments, the particularly preferred GAG forms measured or determined in the method of the present invention are the absolute concentration of 0s CS and / or CS Tot. In some such embodiments, a change, preferably an increase, in the level of one or both of the GAG ​​forms, e.g., compared to a control level, is indicative of cancer. Typically and preferably, the level of one or both of the GAG ​​forms is in blood (e.g., plasma) or urine. Thus, in some embodiments, the level of one or both of the GAG ​​forms is in a blood (e.g., plasma) sample. Thus, in some embodiments, the level of one or both of the GAG ​​forms is in a urine sample.

[0131] In some embodiments in which one or both of the absolute concentration of 0s CS and CS Tot are determined, preferably the cancer is selected from the group consisting of uterine cancer (e.g., cervical squamous cell carcinoma or endometrial cancer), blood cancer (e.g., lymphoma such as chronic lymphocytic leukemia), brain tumor (e.g., diffuse glioma), gastrointestinal endocrine tumor, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, and kidney cancer (e.g., renal cell carcinoma).

[0132] In some embodiments where one or both of the absolute concentration of 0s CS and CS Tot are determined and the body fluid sample is blood (e.g., plasma), preferably the cancer is selected from the group consisting of uterine cancer (e.g., cervical squamous cell carcinoma or endometrial cancer), plasma cancer (e.g., lymphoma such as chronic lymphocytic leukemia), brain cancer (e.g., diffuse glioma), gastrointestinal endocrine tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer), ovarian cancer, and renal cancer (e.g., renal cell carcinoma).

[0133] In some embodiments where one or both of the absolute concentration of 0s CS and CS Tot are determined and the body fluid sample is urine, preferably the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), bladder cancer, and kidney cancer (e.g., renal cell carcinoma).

[0134] In a preferred embodiment, an increase in the level of one or both of the absolute concentration of 0s CS and CS Tot, e.g., compared to a control level, is indicative of cancer (e.g., one or more of the above cancer types mentioned in the paragraph above in connection with the embodiment in which the absolute concentration of 0s CS and one or both of CS Tot are determined).

[0135] In some embodiments, a particularly preferred GAG form measured or determined in the method of the present invention is the absolute concentration of 0s CS. In some such embodiments, a change, preferably an increase, in the level of said GAG form, e.g., compared to a control level, is indicative of cancer. Typically and preferably, the level of said GAG form is a level in blood (e.g., plasma) or urine. Thus, in some embodiments, the level of said GAG form is a level in a blood (e.g., plasma) sample. Thus, in some embodiments, the level of said GAG form is a level in a urine sample.

[0136] In some embodiments in which the absolute concentration of 0s CS is determined, preferably the cancer is selected from the group consisting of breast cancer, colorectal cancer, uterine cancer (e.g., cervical squamous cell carcinoma or endometrial cancer), blood cancer (e.g., lymphoma such as chronic lymphocytic leukemia or diffuse large B-cell lymphoma), brain cancer (e.g., diffuse glioma), gastrointestinal endocrine tumors, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, and kidney cancer (e.g., renal cell carcinoma).

[0137] In some embodiments where the absolute concentration of 0s CS is determined and the body fluid sample is blood (e.g., plasma), preferably the cancer is selected from the group consisting of breast cancer, colorectal cancer, uterine cancer (e.g., cervical squamous cell carcinoma or endometrial cancer), blood cancer (e.g., lymphatic such as chronic lymphocytic leukemia or diffuse large B cell lymphoma), brain cancer (e.g., diffuse glioma), gastrointestinal endocrine tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer), ovarian cancer, and renal cancer (e.g., renal cell carcinoma).

[0138] In some embodiments in which the absolute concentration of 0s CS is determined and the body fluid sample is urine, preferably the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), bladder cancer, and kidney cancer (e.g., renal cell carcinoma).

[0139] In a preferred embodiment, an increase in the absolute concentration of 0s CS, e.g., compared to a control level, is indicative of cancer (e.g., one or more of the cancer types mentioned in the paragraph above in connection with the embodiment in which the absolute concentration of 0s CS is determined).

[0140] In some embodiments, the particularly preferred GAG form measured or determined in the method of the present invention is CS Tot. In some such embodiments, a change, preferably an increase, in the level of said GAG form, e.g., compared to a control level, is indicative of cancer. Typically and preferably, the level of said GAG form is a level in blood (e.g., plasma) or urine. Thus, in some embodiments, the level of said GAG form is a level in a blood (e.g., plasma) sample. Thus, in some embodiments, the level of said GAG form is a level in a urine sample.

[0141] In some embodiments in which CS Tot is determined, preferably the cancer is selected from the group consisting of uterine cancer (e.g., cervical squamous cell carcinoma or endometrial cancer), blood cancer (e.g., lymphoma such as chronic lymphocytic leukemia), brain cancer (e.g., diffuse glioma), gastrointestinal endocrine tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer), ovarian cancer, bladder cancer, and kidney cancer (e.g., renal cell carcinoma).

[0142] In some embodiments where CS Tot is determined and the body fluid sample is blood (e.g., plasma), preferably the cancer is selected from the group consisting of uterine cancer (e.g., cervical squamous cell carcinoma or endometrial cancer), plasma cancer (e.g., lymphoma such as chronic lymphocytic leukemia), brain cancer (e.g., diffuse glioma), gastrointestinal endocrine tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer), ovarian cancer, bladder cancer, prostate cancer, and kidney cancer (e.g., renal cell carcinoma).

[0143] In some embodiments where CS Tot is determined and the body fluid sample is urine, preferably the cancer is selected from the group consisting of head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer), bladder cancer, and kidney cancer (e.g., renal cell carcinoma).

[0144] In preferred embodiments, an increase in CS Tot, eg, compared to a control level, is indicative of cancer (eg, one or more of the above cancer types mentioned in the paragraph above in connection with the embodiment in which CS Tot is determined).

[0145] In some embodiments, a particularly preferred GAG form measured or determined in the method of the present invention is the absolute concentration of 4s CS. In some such embodiments, a change, preferably an increase, in the level of said GAG form, e.g., compared to a control level, is indicative of cancer. Typically and preferably, the level of said GAG form is a level in blood (e.g., plasma) or urine. Thus, in some embodiments, the level of said GAG form is a level in a blood (e.g., plasma) sample. Thus, in some embodiments, the level of said GAG form is a level in a urine sample.

[0146] In some embodiments in which the absolute concentration of 4s CS is determined, preferably the cancer is a genitourinary cancer or a respiratory tract cancer.

[0147] In some embodiments in which the absolute concentration of 4s CS is determined, preferably the cancer is selected from the group consisting of head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer) and bladder cancer.

[0148] In some embodiments where the absolute concentration of 4s CS is determined and the body fluid sample is blood (eg, plasma), preferably the cancer is a genitourinary cancer or a respiratory tract cancer.

[0149] In some embodiments where the absolute concentration of 4s CS is determined and the body fluid sample is blood (e.g., plasma), preferably the cancer is selected from the group consisting of head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer), bladder cancer, prostate cancer, and kidney cancer (e.g., renal cell carcinoma).

[0150] In some embodiments in which the absolute concentration of 4s CS is determined and the bodily fluid sample is urine, preferably the cancer is selected from the group consisting of head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer) and bladder cancer.

[0151] In a preferred embodiment, an increase in the absolute concentration of 0s CS, e.g., compared to a control level, is indicative of cancer (e.g., one or more of the cancer types mentioned in the paragraph above in connection with the embodiment in which the absolute concentration of 0s CS is determined).

[0152] In some embodiments, a particularly preferred GAG form that is measured or determined in the methods of the invention is the absolute concentration of 0s HS. In some such embodiments, an alteration, preferably an increase, in the level of said GAG form, e.g., compared to a control level, is indicative of cancer.

[0153] In some embodiments in which the absolute concentration of 0s HS is determined, where the bodily fluid sample is urine, preferably the cancer is selected from the group consisting of head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer), and kidney cancer (e.g., renal cell carcinoma).

[0154] In a preferred embodiment, an increase in the absolute concentration of 0s HS, e.g., compared to a control level, is indicative of cancer (e.g., one or more of the above cancer types mentioned in the paragraph above in connection with the embodiment in which the absolute concentration of 0s HS is determined).

[0155] In some preferred embodiments, the particularly preferred GAG forms measured or determined in the method of the present invention are non-sulfated GAG forms, 0s CS and / or 0s HS, typically the absolute concentration of said GAG forms. Preferably, an increase in the level of one or both of said GAG forms, e.g. compared to a control level, is indicative of cancer (e.g., a cancer described elsewhere herein). Typically and preferably, said non-sulfated GAG form level is in blood (e.g., plasma) or urine. Thus, in some embodiments, said non-sulfated GAG form level is in a blood (e.g., plasma) sample. Thus, in some embodiments, said non-sulfated GAG form level is in a urine sample.

[0156] In some embodiments, particularly preferred GAG forms measured or determined in the methods of the invention are one or more (or all) of the absolute concentration of 4s CS, the absolute concentration of 0s CS, and CS Tot (e.g., in blood, e.g., plasma, sample). In some embodiments, particularly preferred GAG forms measured or determined in the methods of the invention are one or more (or all) of the absolute concentration of 4s CS and the absolute concentration of 0s CS (e.g., in blood, e.g., plasma, sample). In some such embodiments, a change, preferably an increase, in the level of one or more (or all) of the above GAG ​​forms, e.g., compared to a control level, is indicative of cancer (e.g., one or more of the above cancer types mentioned in the paragraph above in connection with the embodiment in which the absolute concentration of 4s CS or the absolute concentration of 0s CS or CS Tot is determined).

[0157] In some embodiments, the GAG ​​forms measured or determined in the methods of the invention are one or more of the GAG ​​forms shown in Table A herein. In some embodiments, an increase in the level of one or more of the GAG ​​forms reported in Table A herein as being increased in cancer samples compared to healthy samples (preferably compared to control levels) is indicative of cancer. In some embodiments, a decrease in the level of one or more of the GAG ​​forms reported in Table A herein as being decreased in cancer samples compared to healthy samples (preferably compared to control levels) is indicative of cancer.

[0158] In some embodiments, a particularly preferred GAG form that is measured or determined in the methods of the invention is the absolute concentration of 4s CS. In some such embodiments, an alteration, preferably an increase, in the level of said GAG form, e.g., compared to a control level, is indicative of cancer.

[0159] In some embodiments in which the level of 0s CS is determined (or the screening, e.g., diagnosis, is based on an altered level of 0s CS), it may be preferable that the cancer is not a kidney cancer (e.g., renal cell carcinoma) and / or is not a prostate cancer.

[0160] In some embodiments in which the level of 0s CS is determined (or the screening, e.g., diagnosis, is based on an altered level of 0s CS) and the body fluid is urine, it may be preferred that the cancer is not renal cancer (e.g., renal cell carcinoma) and / or is not prostate cancer.

[0161] In some embodiments in which the level of 0s CS is determined (or the screening, eg diagnosis, is based on an altered level of 0s CS) and the body fluid is plasma, it may be preferred that the cancer is not prostate cancer.

[0162] In some embodiments in which the level of 4s CS is determined (or a screening, e.g., diagnosis, is based on a change in the level of 4s CS) and the body fluid is plasma, it may be preferable that the cancer is not a kidney cancer (e.g., renal cell carcinoma) and / or is not a skin cancer (e.g., melanoma).

[0163] In any embodiment referring to determining the level of one or more from a particular list of GAG forms (or GAG properties), in some such embodiments the levels of all listed GAG forms (or GAG properties) may be determined.

[0164] In some embodiments, the level of a single GAG ​​form (GAG profile) is determined.

[0165] In one embodiment of the cancer screening method, the method comprises determining the level in the sample of one or more GAG ​​features (GAG characteristics) identified in Table A herein as significantly altered between cancer and healthy samples, i.e. features having a "% in ROPE" (area of ​​practical equivalence) of less than 5.00 (corresponding to a value of less than 0.05 in the "ROPE percentage" column of Table A, preferably the value in the "ROPE percentage" column of Table A can be less than 0.04, 0.03, 0.02, 0.01, or even a value of 0.00).

[0166] In other embodiments of the invention, the level of more than one of the GAG ​​forms (GAG signature) is determined (e.g., the levels of two or more GAG ​​forms, or three or more GAG ​​forms, or four or more GAG ​​forms, or five or more GAG ​​forms are determined). "More than one" means 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, etc. In any list of markers or GAG signatures provided herein, it is a preferred embodiment that all are measured. Also, a determination of the level of each and every possible combination of GAG forms may be made.

[0167] Thus, in some embodiments, a multi-marker approach is implemented. Determining the levels of multiple GAG ​​forms (biomarker multiplexing) can improve screening (e.g., diagnostic) accuracy.

[0168] Thus, although the markers (GAG forms) can be used individually in the methods of the invention, they can also be used in combination (eg, in the form of a multi-marker assay).

[0169] In some embodiments involving determining the level in the sample of more than one GAG ​​feature (GAG characteristic), each of the more than one GAG ​​features includes determining the level in the sample of a feature that can be identified in Table A herein as significantly altered between cancer and healthy samples, i.e., a feature having a "% in ROPE" (area of ​​practical equivalence) of less than 5.00 (corresponding to a value of less than 0.05 in the "ROPE percentage" column of Table A), preferably, the value in the "ROPE percentage" column of Table A can be less than 0.04, 0.03, 0.02, 0.01, or even 0.00.

[0170] In some embodiments, the level of a single GAG ​​form (GAG signature) is used as the basis for screening for cancer, e.g., diagnosis, prognosis, in some embodiments, may be made based on the level of a single GAG ​​form. In other embodiments, more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, etc.) GAG forms are used as the basis for screening for cancer, e.g., diagnosis, prognosis, in some embodiments, may be made based on the level of more than one GAG ​​form, e.g., any one of the groups (or subgroups) of GAG forms described herein. In some embodiments, when one GAG ​​form or a group (or subgroup or subset) of GAG forms is used as the basis for screening for cancer (e.g., diagnosis or prognosis), the levels of one or more (or all) of the other GAG forms (or GAG signatures) described herein may be further determined or measured.

[0171] Based on the observed changes in the levels of various GAG forms in cancer patients versus healthy patients, a scoring method, scoring system, marker or formula can be designed that uses such levels of various GAG forms to arrive at an index, e.g., in the form of a value or score, as needed, which can then be used for screening (e.g., diagnosis, etc.). Appropriate scoring systems and parameters (e.g., GAG forms) to be measured can be easily designed, e.g., based on the data described herein, e.g., the data in Table A, e.g., based on one or more of the characteristics or properties of individual GAGs that show significant differences in a particular sample (blood (e.g., plasma) or urine) shown in Table A. In particular, the GAG ​​properties of Table A that are most different between cancer samples and healthy samples (e.g., one or more or all properties with a % ROPE value of 0.00 or close to 0.00, e.g., one or more or all properties with a % ROPE value of 5.00 or 4.00 or 3.00 or 2.00 or less than 1.00) can be selected. The % for ROPE values ​​of 5.00 or 4.00 or 3.00 or 2.00 or 1.00 or less corresponds to the values ​​of 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, and 0.01 or less in the "ROPE_Percentage" column of Table A.

[0172] In some embodiments according to the present invention, the chemical composition may be expressed in terms of a score (or GAG score), said score being based on the measured levels of one or more (preferably two or more) or all of the GAG ​​characteristics (or groups of GAG characteristics) described herein.

[0173] In some embodiments, the score (or GAG score) may be based on (or may be derived or calculated or computed using) one or more (or all) measured (or determined) GAG properties selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, absolute concentration of 6s CS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS, and specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, absolute concentration of 6s CS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS.

[0174] In some embodiments, the score (or GAG score) may be based on (or may be derived or calculated or computed using) one or more (or all) measured (or determined) GAG properties selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS, and specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS.

[0175] In some embodiments, where the bodily fluid sample is a blood (e.g., plasma) sample, the score (or GAG score or blood GAG score or plasma GAG score) may be based on (or may be derived or calculated or computed using) one or more (or all) measured (or determined) GAG characteristics selected from the group consisting of: absolute concentration of 0s CS, relative concentration of 4s CS, ratio of 4s CS to 0s CS.

[0176] In some embodiments, where the bodily fluid sample is a urine sample, the score (or GAG score or urine GAG ​​score) may be based on (or may be derived or calculated or computed using) one or more (or all) measured (or determined) GAG characteristics selected from the group consisting of: absolute concentration of 4s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, CS tot, HS tot.

[0177] In some embodiments, where the bodily fluid sample is a urine sample, the score (or GAG score or urine GAG ​​score) may be based on (or may be derived or calculated or computed using) one or more (or all) measured (or determined) GAG characteristics selected from the group consisting of: absolute concentration of 0s HS, relative concentration of 6s CS, relative concentration of Ns HS, relative concentration of 0s HS, relative concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 0s CS, absolute concentration of 4s CS, absolute concentration of 6s CS, absolute concentration of 0s CS, ratio of 6s CS to 0s CS, CS tot, HS tot.

[0178] In some embodiments, the score may be based on (or derived or calculated or computed using) measured levels of one or more GAG ​​profiles in two or more types of bodily fluid samples (e.g., blood (e.g., plasma) and urine). In some such embodiments, the GAG ​​profiles measured in each type of bodily fluid may be the same or different.

[0179] In some embodiments, the score (or GAG score or combined GAG score) may be based on (or may be derived or calculated or computed using) (i) one or more (or all) measured (or determined) GAG characteristics in a blood (e.g., plasma) sample selected from the group consisting of absolute concentration of 0s CS, relative concentration of 4s CS, and CS tot; (ii) one or more (or all) measured (or determined) GAG characteristics in a urine sample selected from the group consisting of relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, relative concentration of 2s6s CS, relative concentration of 0s HS, relative concentration of Ns HS, HS tot, and charged CS.

[0180] In some embodiments, the score (or GAG score or combined GAG score) may be based on (or may be derived or calculated or computed using) one or both of the GAG ​​characteristics measured (or determined) in a blood (e.g., plasma) sample selected from the group consisting of: the ratio of 4s CS to 0s CS, and CS tot, and (ii) one or more (or all) of the GAG ​​characteristics measured (or determined) in a urine sample selected from the group consisting of: absolute concentration of 0s HS, relative concentration of Ns HS, relative concentration of 0s CS, relative concentration of 0s HS, absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 4s CS, relative concentration of 6s CS, absolute concentration of 6s CS, ratio of 6s CS to 0s CS, CS tot, and charged CS.

[0181] In some embodiments, the score (or GAG score or combined GAG score) may be based on (or may be derived or calculated or computed using) (i) the measured (or determined) absolute concentration of 0s CS in a blood (e.g., plasma) sample, and (ii) one or more (or all) measured (or determined) GAG characteristics in a urine sample selected from the group consisting of the absolute concentration of 0s HS, the absolute concentration of Ns HS, the absolute concentration of 4s CS, and the absolute concentration of 6s CS.

[0182] A score based on one or more GAG ​​characteristics measured in a urine sample may be referred to as a urine score (or a urine GAG ​​score). A score based on one or more GAG ​​characteristics measured in a blood (e.g., plasma) sample may be referred to as a blood score or a plasma score (or a blood GAG score or a plasma GAG score), as appropriate. A score based on one or more GAG ​​characteristics measured in more than one type of body fluid sample (e.g., blood (e.g., plasma) and urine) may be referred to as a combined score (or a combined GAG score).

[0183] In some embodiments, a preferred blood (e.g., plasma) score (or blood GAG score or plasma GAG score) may be based on (or may be derived or calculated or computed using) the measured (or determined) GAG characteristics described in connection with the plasma scores described in the Examples section herein. In some embodiments, a preferred urine score (or urine GAG ​​score) may be based on (or may be derived or calculated or computed using) the measured (or determined) GAG characteristics described in connection with the urine scores described in the Examples section herein. In some embodiments, a preferred combined score (or combined GAG score) may be based on (or may be derived or calculated or computed using) the measured (or determined) GAG characteristics described in connection with the combined scores described in the Examples section herein.

[0184] In some embodiments, suitable threshold or cut-off values ​​for use with the score (e.g., used to declare a sample positive or negative) can be designed by one skilled in the art. As an example, the cut-off (or threshold) value can be calculated based on an ROC curve. In an embodiment where the goal is to classify subjects as having high-risk cancer (e.g., having poor or poorer overall survival prospects) or low-risk cancer (e.g., having good or better overall survival prospects), an example of a cut-off (or threshold) value can be the median score across a population of cancer subjects or multiple cancer samples. Alternatively, a maximally selected rank statistic can be used to identify the cut-off value (or cut-off score).

[0185] In some embodiments, screening (e.g., diagnosis, etc.) may be performed by comparing a given score for the sample (or subject) to be diagnosed to a threshold or cutoff value and assigning a result (e.g., a positive or negative diagnosis, etc.) based on whether the determined score is above or below (e.g., significantly above or significantly below) the cutoff value.

[0186] In some cases, a high score results in a positive screening, diagnosis, etc. for the presence of cancer. Where the objective is to provide a scoring system in which a high score indicates the presence of cancer, conveniently the scoring system can be designed as a ratio or fraction in which the numerator is the sum of values ​​associated with one or more GAG ​​features (GAG forms) associated with cancer and the denominator is the sum of values ​​associated with one or more GAG ​​features (GAG forms) associated with a healthy state.

[0187] Of course, alternative scoring systems (or formulas) can be similarly designed, for example, where the numerator is the sum of values ​​associated with one or more GAG ​​characteristics (GAG forms) associated with a healthy state and the denominator is the sum of values ​​associated with one or more GAG ​​characteristics (GAG forms) associated with cancer, with a lower score indicating the presence of cancer.

[0188] Any scoring method, scoring system, marker or formula that includes any suitable combination of GAG characteristics, for example in the form of a value or score, can be used to arrive at an index, which can then be used to screen (e.g., diagnose) cancer. For example, the above method or the like can be an algorithm that includes any suitable combination of GAG characteristics as input, for example, for performing pattern recognition of samples, for example, to arrive at an index, for example in the form of a value or score, which can then be used to screen (e.g., diagnose) cancer. Non-limiting examples of such algorithms include machine learning or deep learning algorithms that implement classification (algorithmic classifiers), such as linear classifiers (e.g., Fisher's linear discriminant, logistic regression, naive Bayes classifier, perceptron), support vector machines (e.g., least squares support vector machines), quadratic classifiers, kernel estimation (e.g., k-nearest neighbors), boosting, decision trees (e.g., random forests), neural networks, learning vector quantization.

[0189] The use of such classifiers, such as machine learning classifiers, such as random forest classifiers, will be within the skill of those skilled in the art.For example, such classifiers can be conveniently trained on GAG signatures from a training set of samples, and then tested for accuracy on a test set of samples.Classifiers can generate black-box models trained on the most important GAG signatures, and can therefore be used to identify the most important GAG signatures that can be used to arrive at an accurate screening (e.g., diagnosis) of cancer.

[0190] In some embodiments, a multivariate logistic regression model is used to calculate the GAG ​​score. In such embodiments, the GAG ​​score is equal to the log odds for cancer versus control (response or dependent variable) considering one or more GAG ​​features used as predictors (i.e. explanatory or independent or regressor variables) in the logistic regression model. In some embodiments, one GAG ​​feature can be used as an explanatory variable. In some embodiments, two or more GAG ​​features can be used as explanatory variables. In some embodiments, only plasma GAG features are used as explanatory variables. In such cases, plasma GAG scores are derived. In some embodiments, only urinary GAG features are used as explanatory variables. In such cases, urinary GAG scores are derived. In some embodiments, both plasma and urinary GAG features are used as explanatory variables. In such cases, combined GAG scores are derived. The GAG ​​features used as predictors can be selected from the totality of GAG features measured in the sample using a suitable feature selection method. Examples of feature selection methods include filter methods, wrapper methods, and embedding methods, and are known to those skilled in the art. In some embodiments, a multivariable logistic regression model with embedded feature selection is developed using predictive projection modeling (see, e.g., Piironen, J., Paasiniemi, M. and Vehtari, A. "Projective inference in high-dimensional problems: Prediction and feature selection", Electronic Journal of Statistics, 14, 2155-2197 (2020), available at https: / / arxiv.org / abs / 1810.02406). In some embodiments, the response calculated from the linear predictor developed using predictive projection modeling is a GAG score. In some embodiments, for a given GAG score, a particular GAG feature selected as an explanatory variable in the underlying logistic regression model is positively associated with cancer if the corresponding coefficient is positive conditional on the values ​​of all other GAG features selected as explanatory variables.Conversely, in some embodiments, a particular GAG signature selected as an explanatory variable in the underlying logistic regression model is negatively associated with cancer if the corresponding coefficient is negatively conditional on the values ​​of all other GAG signatures selected as explanatory variables.

[0191] As described elsewhere herein, screening for cancer according to the present invention may involve using a score (or GAG score, e.g., a blood (e.g., plasma) GAG score, a urinary GAG score, or a combined GAG score), or using a score (or GAG score) to represent the level and / or chemical composition. As described elsewhere herein, in some such embodiments, a change in the score (e.g., which may be an increase or decrease, in some embodiments preferably an increase) compared to a control score (or cutoff or threshold level) is indicative of cancer in the subject.

[0192] As indicated above, a preferred cancer according to the present invention is prostate cancer (PCa).

[0193] In some embodiments, in the method of screening for prostate cancer, the particularly preferred GAG forms measured or determined in the method of the present invention are one or more (or all) of the following: total CS, the relative concentration of 4s CS, the absolute concentration of 4s CS, and the relative concentration of 2s6s CS. In some such embodiments, a change in the level of one or more (or all) of the above GAG ​​forms, for example compared to a control level, is indicative of prostate cancer.

[0194] In some embodiments, in the method of screening for prostate cancer, an increase in one or more (or all) of total CS, relative concentration of 4s CS, and absolute concentration of 4s CS in a blood sample, e.g., compared to a control level, is indicative of prostate cancer in the subject.

[0195] In some embodiments, in methods of screening for prostate cancer, for example, an increase in the relative concentration of 2s6s CS in a urine sample compared to a control level is indicative of prostate cancer in said subject.

[0196] As indicated above, a preferred cancer according to the present invention is colorectal cancer (CRC).

[0197] In some embodiments, in the screening method for colorectal cancer, the particularly preferred GAG forms measured or determined in the method of the present invention are one or more (or all) of the absolute concentration of 0s CS, the relative concentration of charged CS and 0s CS. In some such embodiments, a change in the level of one or more (or all) of the above GAG ​​forms, e.g., compared to a control level, is indicative of prostate cancer.

[0198] In some embodiments, in the method of screening for colorectal cancer, an increase in either or both of the absolute concentration of 0s CS and the relative concentration of 0s CS in a blood sample, e.g., compared to a control level, is indicative of colorectal cancer in the subject.

[0199] In some embodiments, in the methods of screening for colorectal cancer, a reduction in the blood sample in charged CS, eg, compared to a control level, is indicative of colorectal cancer in said subject.

[0200] As indicated above, preferred cancers according to the present invention are neuroendocrine cancers (eg, gastroenterological endocrine tumors, GNETs).

[0201] In some embodiments, in methods of screening for neuroendocrine cancer (e.g., gastrointestinal endocrine tumors), particularly preferred GAG forms measured or determined in the methods of the invention are the absolute concentration of 0s CS and / or total CS. In some such embodiments, an alteration in the level of one or both of the above GAG ​​forms, e.g., compared to a control level, is indicative of neuroendocrine cancer (e.g., gastrointestinal endocrine tumors).

[0202] In some embodiments, in a method for screening for neuroendocrine cancer (e.g., gastrointestinal endocrine tumors), an increase in the absolute concentration of either or both of 0s CS and total CS in a blood sample, e.g., compared to a control level, is indicative of neuroendocrine cancer (e.g., gastrointestinal endocrine tumors) in the subject.

[0203] As indicated above, preferred cancers according to the present invention are hematological cancers (eg, chronic lymphocytic leukemia LL, or diffuse large B-cell lymphoma NHL).

[0204] In some embodiments, in methods of screening for hematological cancer (e.g., chronic lymphocytic leukemia or diffuse large B-cell lymphoma), a particularly preferred GAG form that is measured or determined in the methods of the invention is the absolute concentration of 0s CS. In some such embodiments, a change in the level of the GAG ​​form, e.g., compared to a control level, is indicative of hematological cancer (e.g., chronic lymphocytic leukemia or diffuse large B-cell lymphoma).

[0205] In some embodiments, in a method of screening for hematological cancer (e.g., chronic lymphocytic leukemia or diffuse large B-cell lymphoma), an increase in the absolute concentration of 0s CS in a blood sample, e.g., compared to a control level, is indicative of hematological cancer (e.g., chronic lymphocytic leukemia or diffuse large B-cell lymphoma) in the subject.

[0206] In some embodiments, in methods of screening for chronic lymphocytic leukemia, particularly preferred GAG forms measured or determined by the methods of the invention are the absolute concentration of 0s CS and / or total CS. In some such embodiments, a change in the level of one or both of the above GAG ​​forms, e.g., compared to a control level, is indicative of chronic lymphocytic leukemia.

[0207] In some embodiments, in a method for screening for chronic lymphocytic leukemia, an increase in the absolute concentration of either or both of 0s CS and total CS in a blood sample, e.g., compared to a control level, is indicative of chronic lymphocytic leukemia in the subject.

[0208] In some embodiments, in the method of screening for diffuse large B-cell lymphoma, the particularly preferred GAG forms measured or determined by the method of the present invention are one or both of the absolute concentrations of 0s CS and charged CS. In some such embodiments, an alteration in the level of one or both of the above GAG ​​forms, e.g., compared to a control level, is indicative of diffuse large B-cell lymphoma.

[0209] In some embodiments, in the method of screening for diffuse large B-cell lymphoma, an increase in the absolute concentration of 0s CS in a blood sample, e.g., compared to a control level, is indicative of diffuse large B-cell lymphoma in the subject.

[0210] In some embodiments, in the method of screening for diffuse large B-cell lymphoma, a reduction in charged CS in a blood sample, e.g., compared to a control level, is indicative of diffuse large B-cell lymphoma in the subject.

[0211] As noted above, a preferred cancer according to the present invention is bladder cancer (BCa).

[0212] In some embodiments, in the method of screening for bladder cancer, the particularly preferred GAG forms measured or determined by the method of the present invention are one or more (or all) of total CS, the relative concentration of 4s CS, the absolute concentration of 4s CS, the absolute concentration of 0s CS, the absolute concentration of 4s CS, the relative concentration of 6s CS and the relative concentration of 0s CS. In some such embodiments, a change in the level of one or more (or all) of the above GAG ​​forms, e.g., compared to a control level, is indicative of prostate cancer.

[0213] In some embodiments, in a method for screening for bladder cancer, an increase in one or more (or all) of total CS, relative concentration of 4s CS, and absolute concentration of 4s CS in a blood sample, e.g., compared to a control level, is indicative of bladder cancer in the subject.

[0214] In some embodiments, in the method of screening for bladder cancer, an increase in one or more (or all) of total CS, absolute concentration of 0s CS, absolute concentration of 4s CS, and relative concentration of 0s CS in a urine sample, e.g., compared to a control level, is indicative of bladder cancer in the subject.

[0215] In some embodiments, in methods of screening for bladder cancer, a decrease in the relative concentration of 6s CS in a urine sample, eg, compared to a control level, is indicative of bladder cancer in said subject.

[0216] As indicated above, a preferred cancer according to the present invention is breast cancer (eg, invasive ductal carcinoma, BC).

[0217] In some embodiments, in methods of screening for breast cancer (e.g., invasive ductal carcinoma, BC), a particularly preferred GAG form measured or determined in the methods of the invention is the absolute concentration of 0s CS. In some such aspects, a change in the level of said GAG form, e.g., compared to a control level, is indicative of breast cancer.

[0218] In some embodiments, in methods of screening for breast cancer (eg, invasive ductal carcinoma, BC), an increase in the absolute concentration of 0s CS in a blood sample, for example compared to a control level, is indicative of breast cancer in said subject.

[0219] As indicated above, a preferred cancer according to the present invention is ovarian cancer (OV).

[0220] In some embodiments, in the methods of screening for ovarian cancer, the particularly preferred GAG forms measured or determined in the methods of the invention are one or both of the absolute concentrations of total CS and 0s CS. In some such embodiments, an alteration in the level of one or both of the above GAG ​​forms, e.g., compared to a control level, is indicative of ovarian cancer.

[0221] In some embodiments, in the method of screening for ovarian cancer, an increase in the absolute concentration of either or both of total CS and 0s CS in a blood sample, eg, compared to a control level, is indicative of ovarian cancer in said subject.

[0222] As indicated above, a preferred cancer according to the present invention is uterine cancer (eg endometrial cancer EC, or cervical cancer, preferably cervical squamous cell carcinoma CST).

[0223] In some embodiments, in methods of screening for uterine cancer (e.g., endometrial cancer EC, or cervical cancer, preferably cervical squamous cell carcinoma CST), particularly preferred GAG forms measured or determined in the methods of the invention are one or both of the absolute concentrations of total CS and 0s CS. In some such embodiments, a change in the level of one or both of the above GAG ​​forms, e.g., compared to a control level, is indicative of uterine cancer (e.g., endometrial cancer EC, or cervical cancer, preferably cervical squamous cell carcinoma CST).

[0224] In some embodiments, in a method of screening for uterine cancer (e.g., endometrial cancer EC, or cervical cancer, preferably cervical squamous cell carcinoma, CST), an increase in the absolute concentration of either or both of total CS and 0s CS in a blood sample, e.g., compared to a control level, is indicative of uterine cancer (e.g., endometrial cancer EC, or cervical cancer, preferably cervical squamous cell carcinoma, CST) in the subject.

[0225] As indicated above, a preferred cancer according to the present invention is a brain tumor (eg, diffuse glioma DG).

[0226] In some embodiments, in methods of screening for brain tumors (e.g., diffuse glioma, DG), particularly preferred GAG forms measured or determined in the methods of the invention are one or both of the absolute concentrations of total CS and 0s CS. In some such embodiments, an alteration in the level of one or both of the above GAG ​​forms, e.g., compared to a control level, is indicative of a brain tumor (e.g., diffuse glioma, DG).

[0227] In some embodiments, in a method of screening for brain tumors (e.g., diffuse glioma, DG), an increase in the absolute concentration of one or both of total CS and 0s CS in a blood sample, e.g., compared to a control level, is indicative of a brain tumor (e.g., diffuse glioma, DG) in the subject.

[0228] As indicated above, a preferred cancer according to the present invention is lung cancer (eg, non-small cell lung cancer, NSCLC).

[0229] In some embodiments, in the method of screening for lung cancer (e.g., non-small cell lung cancer), the particularly preferred GAG forms measured or determined in the method of the present invention are one or more (or all) of total CS, absolute concentration of 0s CS, absolute concentration of 4s CS, absolute concentration of Ns HS, total HS, absolute concentration of 0s HS, absolute concentration of 2s6s CS, absolute concentration of 4s CS, relative concentration of 6s CS, and absolute concentration of 6s CS. In some such embodiments, a change in the level of one or more (or all) of the above GAG ​​forms, e.g., compared to a control level, is indicative of lung cancer (e.g., non-small cell lung cancer).

[0230] In some embodiments, in a method of screening for lung cancer (e.g., non-small cell lung cancer), an increase in one or more (or all) of total CS, absolute concentration of 0s CS, and absolute concentration of 4s CS in a blood sample, e.g., compared to a control level, is indicative of lung cancer (e.g., non-small cell lung cancer) in the subject.

[0231] In some embodiments, in a method of screening for lung cancer (e.g., non-small cell lung cancer), an increase in a urine sample of one or more (or all) of the absolute concentration of Ns HS, total CS, total HS, absolute concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of 2s6s CS, absolute concentration of 4s CS, and absolute concentration of 6s CS, for example, compared to a control level, is indicative of lung cancer (e.g., non-small cell lung cancer) in the subject.

[0232] In some embodiments, in a method of screening for lung cancer (e.g., non-small cell lung cancer), a decrease in the relative concentration of 6s CS in a urine sample, e.g., compared to a control level, is indicative of lung cancer (e.g., non-small cell lung cancer) in the subject.

[0233] As noted above, a preferred cancer according to the present invention is renal cancer (eg, renal cell carcinoma, RCC).

[0234] In some embodiments, in methods of screening for kidney cancer (e.g., renal cell carcinoma), particularly preferred GAG forms measured or determined in the methods of the invention are one or more (or all) of total CS, absolute concentration of 0s CS, relative concentration of 4s CS, absolute concentration of 4s CS, charged CS, total HS, relative concentration of 0s CS, absolute concentration of 0s HS, and relative concentration of 6s CS. In some such embodiments, a change in the level of one or more (or all) of the above GAG ​​forms, e.g., compared to a control level, is indicative of kidney cancer (e.g., renal cell carcinoma).

[0235] In some embodiments, in a method of screening for kidney cancer (e.g., renal cell carcinoma), for example, an increase in one or more (or all) of total CS, absolute concentration of 0s CS, relative concentration of 4s CS, and absolute concentration of 4s CS compared to a control level is indicative of kidney cancer (e.g., renal cell carcinoma) in the subject.

[0236] In some embodiments, in a method of screening for renal cancer (e.g., renal cell carcinoma), an increase in one or more (or all) of total HS, the relative concentration of 0s CS, the absolute concentration of 0s CS, the absolute concentration of 0s HS, and total CS in a urine sample compared to a control level is indicative of renal cancer (e.g., renal cell carcinoma) in the subject.

[0237] In some embodiments, in a method of screening for renal cancer (e.g., renal cell carcinoma), a decrease in the relative concentration of one or both of charged CS and 6s CS in a urine sample, e.g., compared to a control level, is indicative of renal cancer (e.g., renal cell carcinoma) in the subject.

[0238] As noted above, a preferred cancer according to the present invention is head and neck cancer (eg, head and neck squamous cell carcinoma, HN).

[0239] In some embodiments, in methods of screening for head and neck cancer (e.g., head and neck squamous cell carcinoma), particularly preferred GAG forms measured or determined by the methods of the invention are one or more (or all) of total CS, absolute concentration of 4s CS, absolute concentration of 0s CS, total HS, absolute concentration of Ns HS, and absolute concentration of 0s HS. In some such embodiments, an alteration in the level of one or more (or all) of the above GAG ​​forms, e.g., compared to a control level, is indicative of head and neck cancer (e.g., head and neck squamous cell carcinoma).

[0240] In some embodiments, in a method of screening for head and neck cancer (e.g., head and neck squamous cell carcinoma), an increase in one or more (or all) of total CS, absolute concentration of 4s CS, and absolute concentration of 0s CS in a blood sample, e.g., compared to a control level, is indicative of head and neck cancer (e.g., head and neck squamous cell carcinoma) in the subject.

[0241] In some embodiments, in methods of screening for head and neck cancer (e.g., head and neck squamous cell carcinoma), an increase in a urine sample of one or more (or all) of total HS, absolute concentration of 4s CS, absolute concentration of Ns HS, absolute concentration of total CS and absolute concentration of 0s HS, e.g., compared to a control level, is indicative of head and neck cancer (e.g., head and neck squamous cell carcinoma) in the subject.

[0242] As discussed herein, the method of the present invention may include determining or measuring "one or more specific GAG forms (or a group of GAG forms)" selected from the group consisting of "certain specific GAG forms (or a group of GAG forms)" as set forth herein. For the avoidance of doubt, in some embodiments in which one or more of the specific GAG forms (or a group of GAG forms) or one or more of the specific genes (or a group of genes) discussed herein are measured or determined, one or more GAG ​​forms or one or more other (or distinct) genes and / or one or more other biomarkers may be further measured or determined. Thus, "selected from the group consisting of" may be an "open" term. In some embodiments, only one or more of the specific GAG forms (or a group of GAG forms) discussed herein are measured or determined (e.g., other GAG forms or other biomarkers are not measured or determined).

[0243] As described above, the present invention provides a method for screening for cancer in a subject. In another aspect, the present invention provides a method for diagnosing cancer in a subject. In another aspect, the present invention provides a method for prognosticating (prognosticating the future severity, course and / or outcome of cancer) cancer in a subject. In another aspect, the present invention provides a method for predicting the occurrence of cancer in a subject over a certain period of time. In another aspect, the present invention provides a method for estimating the risk of occurrence of cancer in a subject over a certain period of time. In another aspect, the present invention provides a method for monitoring the occurrence of cancer in a subject at risk. In another aspect, the present invention provides a method for monitoring the progression of cancer in a subject. In another aspect, the present invention provides a method for determining the clinical severity of cancer in a subject. In another aspect, the present invention provides a method for determining or estimating the stage of cancer (e.g., stage I, stage II, stage III or stage IV cancer). In another aspect, the present invention provides a method for determining or estimating the grade of cancer (e.g., low grade or high grade, grade 1 or grade 2 or grade 3 or grade 4). In another aspect, the present invention provides a method for determining the risk of progression of cancer in a subject. In another aspect, the present invention provides a method for guiding treatment based on a risk assessment of cancer in a subject. In another aspect, the present invention provides a method for predicting a subject's response to a cancer treatment. In another aspect, the present invention provides a method for determining the efficacy of a treatment or surgical plan for cancer in a subject. In another aspect, the present invention provides a method for detecting recurrence or relapse of cancer (e.g., in a patient with early stage cancer). In another aspect, the present invention provides a method for patient or treatment selection, for example, a means for distinguishing patients with small cancerous masses that are not cancerous, e.g., non-malignant, benign or painless masses (but may show some problematic symptoms and be suspected to be cancerous), from patients with cancer. Thus, in another aspect, the present invention provides a method for distinguishing cancer from non-malignant diseases. In some embodiments, the present invention provides a method for determining whether metastasis is due to a particular cancer. In another aspect, the present invention provides a method for predicting whether metastasis is expected from a given cancer.In another aspect, the present invention provides a method for screening for cancer in a general population. In another aspect, the present invention provides a method for screening for cancer in a population at risk of having or developing cancer (e.g., individuals with a genetic predisposition or individuals exhibiting risk factors or individuals exhibiting symptoms). In another aspect, the present invention provides a method for predicting or determining the tissue of origin of a cancer. In another aspect, the present invention provides a method for predicting the prognosis of a cancer patient. In another aspect, the present invention provides a method for predicting the prognosis of a patient with a given cancer. In another aspect, the present invention provides a method for predicting or determining whether a cancer is metastatic.

[0244] Thus, the cancer screening methods of the present invention can be used, for example, to diagnose cancer, prognose cancer, predict the occurrence of cancer, estimate the risk of occurrence of cancer, monitor the occurrence of cancer in a subject at risk, monitor the progression of cancer, determine the clinical severity of cancer, determine or estimate the stage of cancer (e.g., stage I, stage II, stage III or stage IV cancer), determine or estimate the grade of cancer (e.g., low grade or high grade, grade 1 or grade 2 or grade 3 or grade 4), predict the response of a subject to treatment for cancer, determine the therapeutic regimen or therapeutic modality used to treat cancer, It can be used to determine the effectiveness of a surgical regimen, to detect the recurrence or relapse of cancer, for patient or treatment selection, to distinguish small cancer masses suspicious of cancer from other non-malignant diseases, to determine if metastasis is due to a given cancer, to screen for cancer in the general population, to screen for cancer in populations at risk of having or developing cancer (e.g., individuals who are genetically predisposed or who present risk factors or who present symptoms), to predict or determine if a cancer will be metastatic, or to predict or determine the tissue of origin of a cancer.

[0245] Thus, in one aspect, the present invention provides a method for diagnosing cancer in a subject. In some embodiments, a positive diagnosis (i.e., the presence of cancer) is made when the level of one or more of the GAG ​​forms in the sample is altered (possibly increased or decreased) compared to a control level. GAG forms whose increased levels are indicative of (e.g., diagnostic of) cancer are described elsewhere herein. GAG forms whose reduced levels are indicative of (e.g., diagnostic of) cancer (e.g., prostate cancer) are described elsewhere herein. Alternatively, multiple different GAG forms or characteristics are analyzed as described elsewhere herein, for example, to arrive at a diagnosis using a scoring system or method (e.g., GAG score). When a scoring system (e.g., GAG score) is used, a positive diagnosis (i.e., the presence of cancer) can be made when the score is altered (or significantly altered) compared to a control score or a cutoff (or threshold) level.

[0246] In another aspect, the present invention provides a method for the prognosis of cancer in a subject. In such a method, the level (or score) of one or more of the above-mentioned GAG forms in a sample indicates the future severity, course and / or outcome of the cancer. For example, a change (possibly an increase or a decrease) in the level (or score) of one or more GAG ​​forms in a sample compared to a control level (or control score or cut-off level) may indicate a poor prognosis. For example, a high increase in the level (or score, if a score is used) compared to a control level (or score or cut-off level) may indicate a particularly poor prognosis. Thus, in some embodiments, an increase in the level of one or more GAG ​​forms, where an increase in the level is indicative of cancer, suggests a poor prognosis. In some embodiments, a decrease in the level of one or more GAG ​​forms, where a decrease in the level is indicative of cancer, suggests (i.e. indicates) a poor prognosis. Conversely, if one or more GAG ​​forms have unchanged levels (or essentially unchanged levels), this may indicate a good prognosis. In some embodiments, if a scoring system is used, an increase in the score, where an increase in the score is indicative of cancer, suggests a poor prognosis. In some embodiments, when a scoring system is used, a decrease in the score is indicative of cancer and is suggestive (i.e., indicative of) a poor prognosis. Conversely, if one or more GAG ​​scores remain unchanged (or essentially unchanged), this may indicate a good prognosis.

[0247] Continuous (periodic) measurement of the level of one or more GAG ​​forms (biomarkers) according to the present invention can also be used for prognostic purposes, looking for either an increase or decrease in the level (or score) over time. In some embodiments, a change (optionally an increase or decrease) in the level or score of one or more of the GAG ​​forms or scores over time (compared to a control level or score or cut-off level, e.g., a level or score that moves away from the control level or score or cut-off level) can indicate a worsening prognosis. In some embodiments, a change (optionally an increase or decrease) in the level of one or more of the GAG ​​forms or scores over time (compared to a control level or score or cut-off level, e.g., a level that moves closer to the control level or score or cut-off level) can indicate an improving prognosis.

[0248] In some embodiments, a change (possibly an increase or decrease) in the level and / or chemical composition (or score) of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a bodily fluid sample (e.g., compared to a control level or composition or score) may be indicative of prognosis.

[0249] Thus, the prognostic methods of the present invention can be used to predict (or estimate) a prognosis, e.g., whether a subject has a good (or better) prognosis, or whether a subject has a poor (or worse) prognosis. In some embodiments, a change (possibly an increase or a decrease) in the level and / or chemical composition (or score) of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample can indicate a good (or better) prognosis relative to the prognosis for the control subject or control population (or the average (e.g. median) control subject or level in a control or reference population) from which the control level (or score) was obtained (or derived). In some embodiments, a change (possibly an increase or decrease) in the level and / or chemical composition (or score) of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a bodily fluid sample may indicate a poor (or worse) prognosis relative to the prognosis for the control subject or control population (or the average (e.g., median) control subject or level in a control or reference population) from which the control level (or score) was obtained (or derived).

[0250] Prognosis can be considered as an assessment of a subject's chances of survival (e.g., over a given period of time, e.g., 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 or 33 months, or e.g., 5-year survival). Thus, from another perspective, the present invention in one aspect provides a method for predicting (or determining) the chances of survival of a subject with cancer. In some embodiments, "survival" can be calculated as the time (e.g., number of days) between the date of sampling (the date a sample is obtained from the subject) and the time of the "event". In some embodiments, "survival" can be calculated from the start of cancer treatment, e.g., the start of medical therapy or the date of surgery. From another perspective, prognosis can be considered as an assessment of the prospects of mortality for a subject or the risk of progression to a worse condition. Thus, the present invention in one aspect provides a method for predicting (or determining) the risk of death or progression of a subject with cancer.

[0251] In one embodiment, "survival" is "overall survival" (OS). In such an embodiment, the "event" is the date of death. Thus, in some embodiments, the prognostic methods of the invention can be used to predict (or estimate) the probability of death occurring in a particular future period (e.g., 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, 30 months, or 33 months, or, for example, 5-year survival). In some embodiments, the prognostic methods of the invention can be used to predict or estimate the amount of time before death. In some embodiments, "survival" is "progression-free survival" or "disease-free survival" or "recurrence-free survival" or "treatment-free survival".

[0252] In one aspect, the present invention provides a method for monitoring the occurrence of cancer in subjects at risk of developing cancer.Such a method and the GAG ​​form (or score) measured are similar to the diagnostic method as described herein, but are carried out on subjects at a certain risk of developing cancer, and therefore can benefit from closer monitoring.Such "at risk" subjects are easily identified by those skilled in the art, and include, for example, subjects with a family history of cancer or a genetic predisposition to cancer, or subjects who have remission from cancer, or subjects with recognized risk factors for cancer. For example, for some cancers, a recognized risk factor is age, e.g., over 40 years old, or over 50 years old, or over 55 years old, or preferably over 65 years old, e.g., 40-65, or 50-65, or 55-65, or 60-65, or 70-75, or 40-75, or 50-75, or 55-75, or 60-75, or 65-75 (e.g., 66-71), or 70-75, or 40-85, or 50-85, or 55-85, or 60-85, or 65-85, or 70-85 years. For example, with prostate cancer, men in such age ranges may be particularly at risk.

[0253] Thus, in some embodiments of the invention, it is understood that the method can be performed on "healthy" patients (subjects) or at least patients (subjects) who have not yet developed any clinical symptoms of cancer, such as patients with very early or preclinical stages of cancer, such as patients whose primary tumors are so small that they cannot be assessed or detected, or whose cells have undergone precancerous changes associated with cancer but have not yet become malignant. Thus, in another aspect, the invention provides a method of predicting the development of cancer in a subject (e.g., over a given period of time, e.g., 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, or 60 months). In another aspect, the invention provides a method of estimating the risk of developing cancer in a subject (e.g., over a given period of time, e.g., 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, or 60 months).

[0254] Thus, the method of the present invention can also be used to monitor disease progression. Such monitoring can be performed before, during or after treatment of cancer by surgery or therapy (e.g., pharmaceutical therapy). Thus, in another aspect, the present invention provides a method for monitoring the progression of cancer in a subject. In some embodiments of the method for monitoring the progression of cancer (e.g., renal cancer or other cancers described herein) in a subject, the level (or score or composition) of one or more of the GAG ​​forms indicates the progression of the cancer. In some preferred embodiments, the level of the absolute concentration of 0s CS (or the determined chemical composition including the absolute concentration of 0s CS, or the score used using (or including) the absolute concentration of 0s CS) indicates the progression of the cancer, and preferably, a high (or higher) or increased (or increasing) level (e.g., measured over time, e.g., by performing serial measurements) indicates the progression of the cancer (worsening of the cancer).

[0255] The methods of the present invention may also be used to predict (or determine) whether a particular cancer (e.g., renal cancer or other cancers discussed herein) is metastatic. In some preferred embodiments, the level of absolute concentration of 0s CS (or the determined chemical composition including the absolute concentration of 0s CS, or the score used using (or including) the absolute concentration of 0s CS) is indicative of metastasis, and preferably high (or higher) or increased (or increasing) levels (e.g., measured over time, e.g., by making serial measurements) are indicative of metastasis. Thus, in some embodiments, the absolute concentration of 0s CS (e.g., in blood, e.g., plasma samples, or in urine samples) may be indicative of metastatic cancer.

[0256] The methods of the invention can be used in the active monitoring of patients not undergoing surgery or treatment, for example to monitor the progression of cancer in untreated patients. Again, serial measurements allow assessment of whether or to what extent the cancer is worsening, and thus allow more sound decisions to be made, for example, regarding whether therapeutic or surgical intervention is necessary or advisable.

[0257] As noted above, monitoring can also be carried out to obtain early, ideally pre-clinical, signs of cancer, for example in individuals thought to be at risk of developing cancer, such as healthy individuals.

[0258] In another aspect, the present invention provides a method for determining the clinical severity of cancer in a subject. In such a method, the level (or score derived therefrom) of one or more GAG ​​forms in a sample shows an association with the severity of cancer. Thus, the level (or score) of one or more GAG ​​forms shows the severity of cancer. In some embodiments, the more the level (or score derived therefrom) of one or more GAG ​​forms changes (possibly increases or decreases) compared to the control level (or score), the more likely the cancer is of a severe form. Thus, in some embodiments, the method of the present invention can be used in the selection of patients for treatment.

[0259] Serial (periodic) measurement of the levels (or scores derived therefrom) of one or more GAG ​​forms (biomarkers) can also be used to monitor the severity of cancer, looking for either an increase or decrease in levels over time. Observation of altered levels (possibly an increase or decrease) can also be used to guide and monitor treatment in the context of asymptomatic disease, i.e., before treatment or surgery, e.g., before the initiation of pharmaceutical treatment or surgery, or in the context of "follow-up" during or after treatment to assess the effectiveness of treatment and look for signs of treatment failure.

[0260] Therefore, the present invention also provides a method for predicting the success of a subject in treatment or surgery.For example, subjects with less severe forms or early stages of cancer (e.g., prostate cancer), as determined by the level (or score derived therefrom) of one or more GAG ​​forms in sample according to the present invention, are generally more likely to be successful in treatment or surgery, particularly surgery.In such a method, the selection of treatment or surgery can be guided by knowledge of the level (or score derived therefrom) of one or more GAG ​​forms in sample.In some embodiments of the method for predicting the success of a subject in treatment or surgery, the level of HA is not measured or determined.

[0261] The present invention also provides a method for patient or treatment selection, since it provides a means for distinguishing high-risk cancer patients from low-risk cancer patients. Thus, from another perspective, the method of the present invention provides a method for distinguishing high-risk cancer from low-risk cancer, and can guide appropriate treatment.

[0262] In some embodiments, the present invention provides a method for distinguishing between high (or higher) risk cancer (e.g., prostate cancer, e.g., with a Gleason score of 8 or higher) and low (or lower) risk cancer (e.g., prostate cancer, e.g., with a Gleason score of 6 or lower) in subjects diagnosed with cancer (e.g., prostate cancer) (e.g., recently diagnosed, e.g., less than 1 month or less than 6 months or less than 1 year since diagnosis). High (or higher) risk can mean that the subject has a poor (or worse) prognosis, and low (or lower) risk can mean that the subject has a good (or better) prognosis. Intermediate risk subjects (e.g., prostate cancer subjects with a Gleason score of 7) can also be identified. The method of the present invention can be used to assess the severity, aggressiveness, metastatic potential or risk level in subjects diagnosed with cancer (e.g., prostate cancer) (e.g., recently diagnosed).

[0263] In some embodiments, the invention provides methods of monitoring (e.g., continuously monitoring or performing active surveillance) a subject having cancer (e.g., a subject being treated for cancer). Such monitoring can guide which treatment to use, or whether no treatment should be given.

[0264] In some embodiments, the more altered (and possibly increased or decreased) the level (or score) of one or more of the GAG ​​forms compared to a control level (or score or cutoff level), the more likely a high-risk cancer is (or the less likely a low-risk cancer is). Conversely, in some embodiments, the less altered (and possibly increased or decreased) the level (or score) of one or more of the GAG ​​forms compared to a control level (or score cutoff level), the less likely a high-risk cancer is (or the more likely a low-risk cancer is).

[0265] In some embodiments, low (or lower) risk patients may be placed under observation or active surveillance and may not be given treatment (e.g., drug therapy or surgery). In some embodiments, high (or higher) risk patients may receive treatment, such as resection (e.g., prostatectomy in the case of prostate cancer), radiation therapy, hormone therapy, or other treatment (e.g., as detailed elsewhere herein).

[0266] The present invention also provides a method for determining (or monitoring) the effectiveness of a therapeutic regimen used to treat cancer, in other words, a method for tracking or monitoring the response to treatment. In such a method, a change (possibly an increase or a decrease) in the level (or score) of one or more GAG ​​forms according to the present invention indicates the effectiveness of the therapeutic regimen used. For example, if the level (or score derived therefrom) of one or more GAG ​​forms, where an increased level (or score) indicates cancer, decreases during (or after) treatment, this indicates an effective therapeutic regimen. Conversely, if the level (or score derived therefrom) of one or more GAG ​​forms, where a decreased level (or score) indicates cancer, increases during (or after) treatment, this indicates an effective therapeutic regimen. In such a method, continuous (periodic) measurement of the level of one or more GAG ​​forms (biomarkers) over time can also be used to determine the effectiveness of the therapeutic regimen being used. A similar method can be used to provide a method for determining (or monitoring) the effectiveness of a surgical regimen being used to treat cancer.

[0267] The present invention also provides methods for detecting cancer recurrence (relapse), e.g., in subjects who previously had cancer but were successfully treated, e.g., by surgery or therapy (e.g., drug therapy) such that the cancer is deemed to be in remission or cured, or for predicting metastatic relapse, e.g., in patients under follow-up. Such subjects form an "at risk" category and can well benefit from regular monitoring for cancer. Such methods for detecting cancer recurrence (or relapse) use the diagnostic methods described herein to detect the presence or absence of cancer.

[0268] The present invention also provides a method for patient or treatment selection, to provide a means for distinguishing cancer patients from non-malignant disease (e.g., non-malignant prostate disease) patients. Thus, in another aspect, the method of the present invention provides a method for distinguishing cancer from non-malignant disease.

[0269] Such methods of patient or treatment selection, or methods for distinguishing cancer from non-malignant disease, use diagnostic methods as described herein to detect the presence or absence of cancer.

[0270] The present invention also provides a method for predicting or determining the tissue of origin of a cancer. In some such methods, the level and / or composition (or score) of one or more of the GAG ​​forms described herein may indicate the tissue of origin of a cancer. Predicting or determining the tissue of origin of a cancer may guide further diagnosis and / or treatment and / or surgical follow-up (e.g., by recommending a particular type of scan (e.g., CT scan or MRI scan) for a particular part of the body (e.g., chest CT scan vs. abdominal CT scan vs. brain MRI scan). Predicting or determining the tissue of origin may be performed as described in the Examples section herein. Predicting or determining the tissue of origin may use any suitable modeling tool or method that uses the measured levels of GAG forms as input, for example, a multi-class classification machine learning method may be used with the measured levels of GAG forms as input. Non-limiting examples of machine learning methods are learning linear classifiers (e.g., Fisher's Linear Discriminant, Logistic Regression, Naive Bayes Classifier, Perceptron), support vector machines (e.g., Least Squares Support Vector Machines), quadratic classifiers, kernel estimation (e.g., k-nearest neighbors), boosting, decision trees (e.g., Random Forests), neural networks, deep neural networks, vector quantization.

[0271] The features and discussion herein relating to cancer screening methods (eg, with respect to preferred GAG forms or combinations thereof or scores for measurement) apply to other related methods of the invention (eg, methods of diagnosing cancer, etc.).

[0272] In some embodiments, the invention provides for the use of the methods of the invention (such as, for example, screening, diagnostic or prognostic methods as described herein) in combination with other known screening, diagnostic or prognostic methods for cancer, such as radiological imaging (e.g., computed tomography, CT, or positron emission tomography, PET scan) or magnetic resonance imaging (MRI scan), or histological evaluation, for example, using a tumor biopsy. As an example, in the case of prostate cancer, a PSA (prostate specific antigen) test or a DRE (digital) test or a Prostate Core Mitomic Test (Prostate Core Mitomic Test) may be used. TM and other methods that may be used in conjunction with the methods of the present invention. Thus, for example, the methods of the present invention may be used to confirm a diagnosis of cancer in a subject. In some embodiments, the methods of the present invention are used alone.

[0273] The level of the GAG ​​form in question can be determined or measured by analyzing a sample obtained or removed from the subject by appropriate means. Measurements are typically performed in vitro.

[0274] The level of one or more GAG ​​forms in a sample can be measured (determined) by any suitable assay or technique or method, several of which are well known in the art, including: It has been documented. Electrophoresis, such as agarose gel electrophoresis or capillary electrophoresis (especially capillary electrophoresis with fluorescence detection such as CE-LIF), is a technique that can be used to measure (determine) the level of one or more GAG ​​forms according to the invention. Liquid chromatography, in particular HPLC (high performance liquid chromatography) combined with mass spectrometry (MS), is a preferred technique for measuring (determining) the level of one or more GAG ​​forms according to the invention.

[0275] Suitable electrophoretic (e.g., capillary electrophoresis) and liquid chromatography (e.g., HPLC) techniques for GAG conformation analysis, as well as suitable mass spectrometry methods (and associated data processing techniques), are well known and documented in the art.

[0276] One method that can be used in the present invention is capillary electrophoresis with laser-induced fluorescence detection, CE-LIF (e.g., as described in Galeotti et al., 2014, Electrophoresis 35:811-818; and Kottler et al., 2013, Electrophoresis 34:2323-2336). HPLC can also be used in combination with post-column derivatization and fluorimetric detection, as described, for example, in Volpi 2006, Curr Pharm Des 12:639-658, HPLC in combination with ESI-MS (electrospray ionization-mass spectrometry) can also be used, as described, for example, in Volpi and Linhardt, 2010, Nature protocols 5:993-1004, or HPLC in combination with fluorimetric detection, as described, for example, in Galeotti and Volpi, 2011, Anal Chem 83:6770-6777 or Volpi et al., 2014, Nature Protocols 9:541-558. Agarose gel electrophoresis can also be used, e.g., FACE (fluorophore-assisted carbohydrate electrophoresis) as described in Volpi and Maccari, 2006, Analyt Technol Biomed Life Sci, 834:1-13, and Volpi and Maccari, 2002, Electrophoresis 23:4060-4066. Particularly preferred methods for determining the level of one or more GAG ​​forms in a sample are described in the Examples herein. Thus, preferred methods may involve high performance liquid chromatography (HPLC), preferably ultra HPLC (UHPLC), in combination with mass spectrometry, such as MS / MS or triple quadrupole mass spectrometry. Particularly preferred methods include ultra high performance liquid chromatography (UHPLC) coupled with electrospray ionization triple quadrupole mass spectrometry system. Examples of such methods are described in Tamburro et al., 2012, bioRxiv, doi:10.1101 / 2021.02.04.429694.

[0277] Certain methods of sample preparation (or processing), such as GAG extraction and purification, are also known and described in the art, for example in Volpi and Maccari, 2005, Biomacromolecules 6:3174-3180 and Clin Chim Acta 356:125-133, Coppa et al., 2011 Glycobiology 21:295-303. However, such reported technique-based sample preparation (or processing) methods involve protease treatment (protease extraction) and a purification step based on the use of anion exchange resin. In some methods of the invention, such a protease treatment step and / or a purification step using anion exchange resin may be performed. However, as discussed elsewhere herein, in preferred methods, a protease treatment step and / or a purification step using anion exchange resin is not performed. In particular, in preferred methods where the protein-free fraction of GAGs is analyzed, a protease treatment step is not performed.

[0278] In some embodiments, HPLC and mass spectrometry (and associated data processing techniques) are used to obtain the fraction of the level of one or more specific GAG forms (e.g., sulfated or non-sulfated disaccharide forms) in a sample compared to the total amount. For example, after sample preparation, GAGs can be digested using enzymes, separated on an HPLC column, and identified using MS. As described elsewhere herein, the amount of one or more individual GAG forms (e.g., specific sulfated or non-sulfated disaccharide forms) can be conveniently normalized (i.e., divided) by the sum of all the amounts of the individual GAG forms measured to obtain a fraction (or proportion or relative concentration). However, the absolute concentration (or absolute level) of an individual GAG form (e.g., GAG sulfated form) can alternatively or additionally be measured.

[0279] According to the present invention, a quantitative, semi-quantitative or qualitative assessment (determination) of the levels of one or more GAG ​​forms can be performed.

[0280] Suitable methods for doing this are well known to those skilled in the art and any of these can be used. However, a convenient way to achieve such quantification of the disaccharide composition or the relevant properties or forms of CS or HS (and separation of the disaccharide forms) is to use electrophoresis, in particular capillary electrophoresis, for example capillary electrophoresis with fluorescence detection, for example capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) (e.g., Galeotti 2014, supra, or Kottler 2013, supra). An alternative method that is preferred in some embodiments is to use liquid chromatography, preferably HPLC (High Performance Liquid Chromatography), for example SAX HPLC, or for example as described in Volpi 2006 (supra), Galeotti and Volpi 2011 (supra), Volpi et al., 2014 (supra), or Volpi and Linhardt, 2010 (supra). Preferably, mass spectrometry (HPLC-MS), such as electrospray ionization mass spectrometry (ESI-MS), such as HPLC ESI-MS, is also used. Particularly preferred methods are outlined in the Examples. One particular method is capillary electrophoresis (e.g. capillary electrophoresis with laser-induced fluorescence detection). Another particularly preferred example is HPLC followed by MS (HPLC-MS), such as HPLC ESI-MS. Preferred HPLC-MS methods are discussed elsewhere herein.

[0281] Thus, in a preferred method of the invention, said level or chemical composition of said GAG or GAG profile is determined by HPLC and mass spectrometry. Preferably, said HPLC is ultra HPLC and / or said mass spectrometry is triple quadrupole mass spectrometry. In a particular preferred method, said level or chemical composition of said GAG or GAG profile is determined by a combination of high performance liquid chromatography (HPLC), preferably ultra HPLC (UHPLC), and mass spectrometry, such as MS / MS or triple quadrupole mass spectrometry. A preferred method comprises ultra high performance liquid chromatography (UHPLC) coupled (or combined) with electrospray ionization triple quadrupole mass spectrometry.

[0282] Generally, the determination of GAG properties or forms according to the present invention does not involve measuring the GAG ​​molecules in the exact same form as found in the subject's bodily fluid (e.g., does not involve measuring the GAG ​​in its naturally occurring form). For example, such native or naturally occurring GAG molecules are often found in biological samples (e.g., bodily fluid samples) in the form of long glycans that may either be bound to proteins (also referred to herein as protein-bound or proteoglycan GAGs) or may not be bound to proteins (also referred to herein as free or non-protein-bound GAGs).

[0283] In some embodiments, the methods of the invention may include a step of processing the sample. Thus, in some embodiments, the methods of the invention may be performed on such processed samples or material derived from such processed samples. Thus, in general, the methods of the invention are performed on samples that have been processed in some way (e.g., artificial samples rather than natural samples).

[0284] Processing steps include, but are not limited to, extraction or purification of GAGs from the sample, fragmentation or cleavage or digestion of proteins present in the sample, e.g., via the use of proteases such as proteinase K, as a means of separating or extracting or removing GAGs from proteins to which they are bound, purification of GAGs, e.g., using anion exchange resins, isolation of cells from the sample, isolation of cellular components from the sample, extraction (e.g., isolation or purification) of proteins / peptides from the sample. Thus, the above processing steps may also include steps performed on the body fluid sample to prepare it for analysis, e.g., in the case of a blood sample, such steps may include preparing appropriate blood components for analysis, e.g., plasma or serum, or in the case of a urine sample, removal of cells or other impurities. Processing steps may involve one or more of digestion, extraction, purification, boiling, filtration, lyophilization, fractionation, centrifugation, concentration, dilution, inactivation of interfering components, addition of reagents, derivatization, complexation, etc. Exemplary processing steps are described in the Examples.

[0285] While certain methods of the invention may involve a step of fragmentation or cleavage or digestion of proteins present in the sample (e.g. as a means of separating or extracting or removing GAGs from the proteins to which they are bound, via the use of a protease such as proteinase K) and / or purification of the GAGs (e.g. using an anion exchange resin), as will be apparent from the discussion elsewhere herein, certain preferred methods do not involve a step of fragmentation / cleavage / digestion of proteins and / or a step of purification (e.g. using an anion exchange resin). In particular, preferred methods in which the level and / or chemical composition of the protein-free fraction of GAGs is determined do not involve a step of fragmentation / cleavage / digestion of proteins.

[0286] Generally, a GAG-containing body fluid sample obtained from a subject is subjected to at least one processing step before determining the level and / or chemical composition according to the methods of the invention. In particular, in methods in which the level of one or more of the specific sulfated or non-sulfated forms of CS or HS disaccharides is determined, the GAG ​​is preferably subjected to a processing step to obtain the disaccharide units for analysis.

[0287] In some such methods of the invention in which the levels of specific individual disaccharide forms are measured, the GAG ​​(e.g., a full length GAG molecule, or a polymeric polysaccharide chain of GAG, or a chain of repeating disaccharide units of GAG) is subjected to a processing step, such as a step of fragmentation or cleavage or digestion (e.g., by chemical digestion or enzymatic treatment). Suitable methods of digestion or enzymatic treatment, such as the use of one or more GAG ​​lyase enzymes, e.g., one or more chondroitinase enzymes, such as chondroitinase ABC or chondroitinase B, and / or the use of one or more heparinase enzymes, such as heparinase I-II-III, to obtain the disaccharide units that are subsequently analyzed, are known to those of skill in the art.

[0288] Other methods for determining the level or composition of GAGs are known in the art that can be used, however, examples include analytical techniques involving the use of antibodies against various GAG forms, such as techniques such as Western blot, ELISA or FACS, or methods involving agarose gel electrophoresis (e.g., fluorophore-assisted carbohydrate electrophoresis (FACE)) or polyacrylamide gel electrophoresis (PAGE).

[0289] In some embodiments, the level of one or more GAG ​​forms (e.g., specific sulfated or non-sulfated forms of CS or HS disaccharides, e.g., derived by fragmentation, cleavage or digestion from a full-length GAG molecule or a chain of repeating disaccharide units of a GAG molecule) associated with (e.g., physically associated with, complexed with, derivatized with, or labeled with) a reagent (e.g., 2-aminoacridone) being used to detect the GAG ​​form is determined. Thus, in some embodiments, the level of a complex of the GAG ​​form with the reagent used to detect the GAG ​​form is determined. Suitable reagents for detecting specific GAG forms are discussed elsewhere herein, but include, for example, antibodies or certain fluorophores (or other detectable labels or dyes) that are attached to (or used to derivatize) the GAG ​​form in question to render it detectable by a fluorometer (or other detection device). Thus, purely by way of example, in some embodiments, the level of a GAG form associated with (e.g., complexed with, or derivatized by) an antibody or fluorophore, etc., can be determined. In some embodiments, the level of a GAG form associated (eg, complexed or derivatized) with 2-aminoacridone can be determined.

[0290] A preferred method of the invention comprises a step of determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, and therefore advantageously does not require separation or extraction of GAG molecules from the proteins to which they are bound. Instead, the protein-free fraction (and only the protein-free fraction) of GAGs in a body fluid sample can be analyzed from the sample without any such treatment to separate GAGs from proteins, for example by digesting the proteins. Thus, a preferred method does not comprise a treatment step of contacting said sample with a proteolytic agent, such as a protease.

[0291] Other preferred methods do not include a step in which GAGs are purified from a sample based on the negative charge of said GAGs (eg, using an anion exchange resin).

[0292] Thus, in a preferred method of the invention, said sample has been obtained from said subject and has been subjected to a treatment prior to determining said level and / or chemical composition, The above process is (a) fragmenting said one or both GAGs into disaccharide units, (b) Before (a), (i) contacting the sample with a proteolytic agent; and (ii) purifying said one or both GAGs in said sample based on the negative charge of said GAG.

[0293] A still further aspect of the present invention provides a method of screening for cancer in a subject, comprising determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, the sample has been obtained from the subject and has been subjected to processing prior to determining the level and / or chemical composition; The above process is (a) fragmenting said one or both GAGs into disaccharide units, (b) Before (a), (i) contacting the sample with a proteolytic agent; and (ii) purifying said one or both GAGs in said sample based on the negative charge of said GAG.

[0294] Embodiments of other aspects of the invention described elsewhere in this specification may be applied to this aspect of the invention mutatis mutandis (e.g. preferred GAG form (or group of GAG forms), preferred cancer (or group of cancers), preferred treatment steps, preferred body fluids, etc.).

[0295] In a preferred method, the fragmentation in (a) is conveniently carried out by contacting the one or both GAGs with one or more GAG ​​lyase enzymes (e.g., as discussed elsewhere herein). For example, the fragmentation in (a) can be carried out by contacting the one or both GAGs with one or more chondroitinase enzymes and / or one or more heparinase enzymes.

[0296] In methods based on the art, the contacting step of (b)(i) is conveniently performed by contacting the sample with one or more protease enzymes, e.g., proteinase K. Thus, in preferred methods of the invention, such a step is not performed. The proteolytic agent of (b)(i) may be a protease (e.g., a non-specific protease such as proteinase K). Thus, preferably, the method of the invention does not include a step of contacting the sample with a protease (e.g., a non-specific protease, e.g., proteinase K) prior to (a).

[0297] In the art based methods, the purifying step of (b)(ii) is conveniently carried out by using an anion exchange resin. Thus, in the preferred method of the invention, such a step is not carried out. Thus, preferably, the method of the invention does not include a step prior to (a) of purifying the one or both GAGs in the sample using an anion exchange resin.

[0298] In a preferred method of the invention, the method does not include the contacting of (b)(i).

[0299] In a preferred method of the invention, the method does not include purifying (b)(ii).

[0300] In other preferred methods of the invention, neither step (b)(i) nor (b)(ii) is performed. Thus, in particularly preferred embodiments, the method does not include the contacting of (b)(i) or the purifying of (b)(ii).

[0301] A still further aspect of the invention provides a method of screening for cancer in a subject, comprising determining the level and / or chemical composition of non-proteoglycan fractions of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a sample of a body fluid, said sample being obtained from said subject. Embodiments of other aspects of the invention described elsewhere herein may be applied to this aspect of the invention mutatis mutandis (e.g. preferred GAG form (or group of GAG forms), preferred cancer (or group of cancers), preferred processing steps, preferred body fluids, etc.).

[0302] A still further aspect of the invention provides a method of screening for cancer in a subject, comprising determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, wherein determining said level and / or chemical composition comprises analyzing disaccharide units derived from said GAGs that are not bound to proteins in said body fluid sample, and does not include analyzing disaccharide units derived from GAGs that are bound to proteins in said body fluid sample. Embodiments of other aspects of the invention described elsewhere herein may be applied to this aspect of the invention mutatis mutandis (e.g. preferred GAG form (or group of GAG forms), preferred cancer (or group of cancers), preferred processing steps, preferred body fluids, etc.).

[0303] A still further aspect of the invention provides a method of screening for cancer in a subject comprising determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, wherein determining said level and / or chemical composition comprises analyzing a population of disaccharide units consisting essentially of disaccharide units derived from a non-proteoglycan fraction (or protein-free fraction) of said GAG in said body fluid sample. Embodiments of other aspects of the invention described elsewhere herein may be applied to this aspect of the invention mutatis mutandis (e.g. preferred GAG form (or group of GAG forms), preferred cancer (or group of cancers), preferred processing steps, preferred body fluids, etc.).

[0304] In another aspect, the present invention provides a method for screening for cancer in a subject, comprising determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, said sample being obtained from said subject. In a preferred embodiment, a change in the level and / or chemical composition of chondroitin sulfate (CS) and / or heparan sulfate (HS) in said sample compared to a control level and / or chemical composition is indicative of cancer in said subject. Embodiments of other aspects of the invention described elsewhere herein may be applied to this aspect of the invention (e.g. GAG form (or group of GAG forms), cancer (or group of cancers), processing steps, body fluids, fractions of GAGs analyzed, type of method, etc.) mutatis mutandis. Any features (e.g. preferred features) described elsewhere herein in relation to any other aspect of the invention may be applied to this aspect of the invention mutatis mutandis.

[0305] Thus, in some embodiments of certain preferred methods of the invention, the methods comprise determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS), although it is not essential in all aspects of the invention that the level and / or chemical composition of the "protein-free fraction" in particular be determined.

[0306] In some embodiments, the present invention provides a method for screening for cancer in a subject, comprising determining the absolute concentration of 0s CS in a body fluid sample (e.g., blood, e.g., plasma sample or urine sample), said sample being obtained from said subject. In a preferred embodiment, a change in the absolute concentration of 0s CS in said sample, preferably an increase in the absolute concentration of 0s CS, compared to a control level is indicative of cancer in said subject. Preferred cancers are described elsewhere herein.

[0307] In some embodiments, the only GAG form used on the basis of screening is 0s CS (i.e., in some embodiments, the level of a single GAG ​​form (GAG profile) is used on the basis of screening for cancer, e.g., diagnosis, prognosis may be based on the level of a single GAG ​​form, where that single GAG ​​form is the absolute concentration of 0s CS). In other embodiments, more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, etc.) GAG forms are used on the basis of screening for cancer, e.g., diagnosis, prognosis may be based on the levels of two or more GAG ​​forms, where in some embodiments the one GAG ​​form used is the absolute concentration of 0s CS, e.g., in some embodiments screening may be based on any one of the groups (or subgroups) of GAG forms described herein that include the absolute concentration of 0s CS. In some embodiments, when one GAG ​​form or group (or subgroup or subset) of GAG forms is used as the basis for screening (e.g., diagnosis or prognosis) for cancer, the levels of one or more (or all) of the other GAG forms (or GAG characteristics) described herein may be further determined or measured.

[0308] A change in the level (or composition or score) of one or more GAG ​​forms (GAG features) as described herein includes any measurable change or alteration of the GAG ​​form (biomarker) (or score) in question when the GAG ​​form in question is compared to a control level. A level (or score) change includes an increase or decrease in the level (or score). Preferably, the level (or score) is significantly altered compared to the level (or score or cut-off level) found in an appropriate control (e.g., a control sample or subject or population). More preferably, a significant change in level or composition or score is statistically significant, preferably with a p-value of <0.05 or a % ROPE value of ≦5.00.

[0309] In some embodiments, a change in level (or score) of > 2%, > 3%, > 5%, > 10%, > 25%, > 50%, > 75%, > 100%, > 200%, > 300%, > 400%, > 500%, > 600%, > 700%, > 800%, > 900%, > 1000%, > 2000%, > 5000%, or > 10,000% compared to the level (or score) found in a suitable control sample or subject or population (i.e., when compared to a control level) may indicate the presence of cancer.

[0310] An "increase" or "increased" level of one or more of the GAG ​​forms (GAG signatures) or scores described herein includes any measurable increase or elevation of the GAG ​​form (biomarker) (or score) in question when the GAG ​​form (or score) in question is compared to a control level (or control score or cut-off level). Preferably, the level (or score) is significantly increased compared to the level (or score or cut-off level) found in an appropriate control (e.g., a control sample or subject or population). More preferably, a significant increase in the level (or score) is statistically significant, preferably with a p-value < 0.05 or a % ROPE value ≦ 5.00.

[0311] In some embodiments, an increase in the level (or score) compared to a level (or score) found in a suitable control sample or subject or population (i.e., when compared to a control level or control score or cut-off level) of ≧2%, ≧3%, ≧5%, ≧10%, ≧25%, ≧50%, ≧75%, ≧100%, ≧200%, ≧300%, ≧400%, ≧500%, ≧600%, ≧700%, ≧800%, ≧900%, ≧1000%, ≧2000%, ≧5000%, or ≧10,000% may indicate the presence of cancer.

[0312] A "reduction" or "reduced" level of one or more of the GAG ​​forms (GAG signatures) or scores described herein includes any measurable decrease or reduction in the GAG ​​form (biomarker) (or score) in question when the GAG ​​form (or score) in question is compared to a control level (or control score or cut-off level). Preferably, the level (or score) is significantly reduced compared to the level (or score or cut-off level) found in an appropriate control (e.g., a control sample or subject or population). More preferably, a significant reduction in the level (or score) is statistically significant, preferably with a p-value < 0.05 or a % ROPE value ≦ 5.00.

[0313] In some embodiments, a decrease in the level (or score) compared to the level (or score) found in a suitable control sample or subject or population (i.e., compared to a control level or control score or cut-off level) of ≧2%, ≧3%, ≧5%, ≧10%, ≧25%, ≧50%, ≧75%, ≧100%, ≧200%, ≧300%, ≧400%, ≧500%, ≧600%, ≧700%, ≧800%, ≧900%, ≧1000%, ≧2000%, ≧5000%, or ≧10,000% may indicate the presence of cancer.

[0314] A "control level" is a level of a GAG form (GAG profile) in a control subject or population (e.g., in a sample obtained from a control subject or population). Suitable control subjects or samples for use in the methods of the present invention are easily identified by the skilled artisan, e.g., suitable control groups are as described in the Examples. Such subjects may also be referred to as "normal" subjects or reference populations. Examples of suitable populations of control subjects include healthy subjects (e.g., individuals who have no history of any form of cancer and no other co-morbid diseases). Other preferred control subjects include individuals who do not suffer from cancer, and preferably do not have a history of cancer (e.g., do not suffer from any of the types of cancer mentioned herein, and preferably do not have a history of cancer). When a specific cancer is involved, suitable control subjects include individuals who do not have a history of any form of disease (e.g., cancer) in organs or tissues related to that specific cancer, and preferably do not have other co-morbid diseases. Preferably, such control subjects also do not suffer from inflammatory conditions. Preferably, control subjects are not regular users of any drug. In a preferred embodiment, the control subject is a healthy subject.

[0315] The control level may correspond to the level of an equivalent GAG form in a suitable control subject or sample, for example, a cut-off level or threshold level or range found in a control or reference population. Alternatively, the control level may correspond to the level of the marker in question (GAG form) in the same individual subject or a sample from said subject, measured at an earlier time point (e.g., compared to a "baseline" level in that subject). This type of control level (i.e., a control level from an individual subject) is particularly useful for embodiments of the invention in which continuous or periodic measurements of GAG forms in either healthy or diseased individuals are made to explore changes in the level of GAG forms. In this regard, a suitable control level is the individual's own baseline, stable value, null value, previous value or dry value (as appropriate), as opposed to a control or cut-off level found in the overall control population. A control level may also be referred to as a "normal" level or a "reference" level. A control level may be a discrete number or a range.

[0316] A control level for comparison may be derived by testing a suitable set of control subjects, although the methods of the invention do not necessarily involve performing activity tests on control subjects as part of the methods of the invention, but will generally involve comparison with a control level previously determined from the control subjects and known to the person performing the methods of the invention.

[0317] A "control chemical composition" is a chemical composition in a control subject or population (e.g., in a sample obtained from a control subject or population). The discussion above regarding "control levels" (e.g., appropriate control subjects, control samples, control populations, etc.) can be applied mutatis mutandis to a "control chemical composition."

[0318] As described elsewhere herein, cancer screening according to the present invention may involve using a score (or GAG score) or expressing a level and / or chemical composition using a score (or GAG score). In some such embodiments, a change in the score (e.g., an increase or decrease, as the case may be) compared to a control score (or cut-off or threshold level) is indicative of cancer in said subject. The above discussion regarding "control levels" (e.g., suitable control subjects, control samples, control populations, etc.) may be applied mutatis mutandis to "control scores."

[0319] As described elsewhere herein, in some preferred methods of the invention, the method comprises determining the level and / or chemical composition (or score based thereon) of the protein-free fraction of one or both of the GAGs chondroitin sulfate (CS) and heparin sulfate (HS). In some such embodiments, a change in the level and / or chemical composition of the protein-free fraction (or score based thereon) compared to the level and / or chemical composition of the protein-free fraction of one or both of the GAGs in a control (e.g., a control sample or a control score or cut-off level) is indicative of cancer. The above discussion regarding "control level" or "control chemical composition" or "control score" (e.g., a suitable control subject, control sample, control population, etc.) may be applied mutatis mutandis to embodiments of the invention comprising determining the level and / or chemical composition of the protein-free fraction of one or both of the GAGs (or a score based thereon).

[0320] The screening, diagnosis, etc. methods of the present invention are directed to cancer. Some of the screening, etc. methods of the present invention are generally applicable to a wide variety of cancers, rather than any particular type of cancer, and can be used, if desired, to screen, etc., for the presence or possibility of cancer in a subject. After the method of the present invention indicates the possible presence of cancer, follow-up tests can be performed, if necessary, to identify the specific cancer present in an individual subject.

[0321] Also described herein are methods for screening for certain cancers.

[0322] The method of the present invention can be performed at any stage of cancer, for example, it can be used for early or early stage cancer, or advanced or late stage cancer disease. In another aspect, the method of the present invention can be performed for any grade of cancer, for example, it can be used for low grade cancer, intermediate grade cancer, or high grade cancer.

[0323] Thus, the method of the present invention may be performed on a cancer selected from the group consisting of stage I cancer, stage II cancer, stage III cancer, stage IV cancer and cancer of unspecified stage.

[0324] In some embodiments, the methods of the present invention can be advantageously used to screen for early or stage I cancer or low-grade cancer. In some embodiments, the methods of the present invention can be used to screen for stage II cancer. In some embodiments, the methods of the present invention can be used to screen for stage III cancer. In some embodiments, the methods of the present invention can be used to screen for stage IV cancer or high-grade cancer.

[0325] The classification of cancer at a given stage may follow any definition recognized and accepted in the art.

[0326] For example, depending on the type of cancer, stage I or low-grade cancer may be classified as FIGO stage I, Ann Arbor stage 1, Binet stage A, breast cancer grade 1-2, low-grade (non-glioblastoma) glioma, Gleason grade sum <7, TNM stage I, or ENETS grade 1.

[0327] Depending on the type of cancer, stage II cancer may be classified as FIGO stage II, Ann Arbor stage 2, Binet stage B, or TNM stage II or IIA or IIB.

[0328] Depending on the type of cancer, stage III cancer may be classified as FIGO stage III, Ann Arbor stage 3, or TNM stage III or IIIA or IIIB or IIIC.

[0329] Depending on the type of cancer, stage IV or high-grade cancer may be classified as FIGO stage IV, Ann Arbor stage 4, Binet stage C, breast cancer grade 3, high-grade (glioblastoma) glioma, Gleason grade sum > 7, TNM stage IV or IVA or IVB or IVC, or ENETS grade 2.

[0330] Those of skill in the art are familiar with which staging / grading systems or conventions are appropriate for which types of cancer, and can readily use an appropriate staging / grading system based on the type of cancer.

[0331] Classification of cancer at a given risk can be performed by any definition recognized and accepted in the art. For example, risk can be evaluated by determining the stage of cancer in clinical diagnosis (e.g., using the staging system described above), or by determining the stage of cancer in pathological diagnosis (e.g., using the staging system described above), or by evaluating biopsy results (e.g., by evaluating tumor size, and / or tumor grade and / or tumor volume), or by evaluating the results of other tests (X-ray, CT and / or MRI scan, or bone scan), or by any combination of the above. Those skilled in the art can easily determine risk based on one or more of the above evaluations. As an example, prostate cancer can be considered as low risk if Gleason score is 6 or less, intermediate risk if Gleason score is 7, and high risk if Gleason score is 8 or more.

[0332] In some embodiments, the cancer may be a non-metastatic form of cancer. In some embodiments, the cancer may be a metastatic form of cancer (as opposed to localized or regional cancer).

[0333] The method of the present invention can be performed on any suitable bodily fluid sample. In this regard, the present invention is exemplified by blood (e.g., plasma) and urine, but suitable GAG ​​forms to be measured in other types of bodily fluid samples can be determined by one of skill in the art following the teachings provided herein. Typically, the sample is obtained (removed) from a subject (e.g., as described elsewhere herein), preferably a human subject. In other aspects, the method further comprises the step of obtaining a sample from the subject.

[0334] Reference herein to a "body fluid" includes reference to any fluid derived from the body of a subject. Exemplary fluids include blood (including all blood-derived components, e.g., plasma, serum, etc.), urine, saliva, tears, bronchial secretions, or mucus. Preferably, the body fluid is a circulatory fluid (particularly blood or blood components) or urine. Particularly preferred body fluids are blood (e.g., plasma) or urine. Particularly preferred body fluids are plasma or urine. In some preferred embodiments, the sample is a blood sample (e.g., a plasma or serum sample). In some preferred embodiments, the sample is a plasma sample. In some embodiments, the plasma sample is a platelet poor plasma sample. In some preferred embodiments, the sample is a serum sample. In some preferred embodiments, the sample is a urine sample. In some embodiments, the sample is not a urine sample. In some embodiments, the sample is not a blood sample (e.g., not a plasma sample). The body fluid or sample may be in the form of a liquid biopsy.

[0335] The term "sample" also encompasses any material obtained by processing a body fluid sample, e.g., obtained by processing a blood (e.g., plasma) or urine sample. Processing of a biological sample to obtain a test sample may involve one or more of digestion, boiling, filtration, distillation, centrifugation, lyophilization, fractionation, extraction, concentration, dilution, purification, inactivation of interfering components, addition of reagents, derivatization, complexation, etc., as described elsewhere herein. Appropriate processing steps may be selected depending on the characteristics of the method to be performed.

[0336] Any suitable method for isolating a bodily fluid sample (eg, a urine or blood (eg, serum or plasma) sample) may be used.

[0337] Any sample directly or indirectly affected by a suspected cancer (e.g., tumor) may be used. The sample (e.g., original or unprocessed sample) typically includes a protein-free fraction of GAGs and a protein-bound fraction of GAGs (as discussed elsewhere herein). In a preferred method of the present invention, the level and / or chemical composition of the protein-free fraction of one or both of the GAGs chondroitin sulfate (CS) and heparan sulfate (HS) are determined. Thus, in a preferred embodiment, the body fluid sample has been (or is to be) processed such that only (or essentially only) the protein-free fraction of the GAGs (typically disaccharide units derived therefrom) is subsequently analyzed (i.e., the level and / or chemical composition of the protein-free fraction of one or both of the GAGs (typically disaccharide units derived therefrom) is subsequently determined). In other methods of the invention, the sample is (or has been) processed such that the total (protein-free + protein-bound) fraction or pool of one or both of the GAGs is then analyzed (i.e., the level and / or chemical composition of the total (protein-free + protein-bound) fraction or pool of one or both of the GAGs (typically the disaccharide units derived therefrom) is then determined).

[0338] In some embodiments, the sample may contain circulating tumor cells (eg, metastatic tumor cells).

[0339] The term "sample" also encompasses any material obtained by processing a biological sample (e.g., as described above), the obtained material comprising a disaccharide unit (or a population of disaccharide units) obtained by processing a GAG (e.g., as described elsewhere herein).

[0340] In some embodiments, the method of the invention may include a step of processing the sample. Thus, in some embodiments, the method of the invention may be performed on such a processed sample or on material derived from such a processed sample. Thus, in some embodiments, the method of the invention may be performed on a processed sample. Processing steps include, but are not limited to, isolating cells from the sample, isolating cellular components from the sample, and extracting (e.g., isolating or purifying) proteins / peptides (although preferred methods of the invention involve the determination of a protein-free GAG ​​fraction, and extraction of proteins or removal of protein components from proteoglycans (protein-bound GAGs) present in the sample (e.g., by digesting or otherwise removing protein components) is preferably not performed). Processing steps may involve one or more of filtration, distillation, centrifugation, extraction, concentration, dilution, purification, inactivation of interfering components, addition of reagents, derivatization, amplification, adapter ligation, etc.

[0341] The sample can be used immediately or can be stored (eg, at -80°C) for later use.

[0342] The method of the present invention described herein can be carried out on any type of subject that may suffer from cancer.The method is generally carried out on mammals, such as humans, primates (e.g. monkeys), laboratory mammals (e.g. mice, rats, rabbits, guinea pigs), domestic mammals (e.g. horses, cows, sheep, pigs) or domestic pets (e.g. cats, dogs).Preferably, the subject is a human.For prostate cancer screening, typically, the subject is a male mammal, such as a male human.

[0343] In one embodiment, the subject (e.g., a human) is a subject at risk of developing cancer or at risk for developing cancer, such as a healthy subject or a subject not exhibiting any symptoms of disease, or any other suitable "at risk" subject as described elsewhere herein. In another embodiment, the subject is a subject who has cancer, is suspected of having (or will develop) cancer, or may have (or develop) cancer.

[0344] In some aspects, the methods of the invention may further comprise the initial step of selecting a subject (e.g., a human subject) at risk of developing cancer, or at risk of developing cancer, or suspected of having or having (or developing), or likely to have (or develop), cancer. Subsequent method steps may be performed on a sample from such a selected subject.

[0345] In some aspects, the method of the present invention is provided further comprising a step of treating the cancer by therapy (e.g., drug therapy) or surgery. For example, if the result of the method of the present invention indicates cancer in the subject (e.g., a positive diagnosis of cancer is made), a further step of treating the cancer by therapy or surgery can be performed. For example, if the result of the method of the present invention indicates high-risk (or high-grade) cancer in the subject (e.g., a positive diagnosis of high-risk cancer is made), an additional step of treating the cancer by therapy or surgery can be performed. For example, if the result of the method of the present invention indicates low-risk (or low-grade) cancer in the subject (e.g., a positive diagnosis of low-risk cancer is made), an additional step of follow-up or active surveillance can be performed. Methods of treating cancer by therapy or surgery are known in the art. For example, one surgical option for prostate cancer is prostatectomy, which aims to eradicate the tumor and can be either curative (complete removal) or partial. Pharmaceutical treatments can include standard chemotherapy and immunotherapy and hormonal therapy, in addition to treatments that are undergoing ongoing clinical trials.Pharmaceutical treatments may include standard chemotherapy (e.g. gemcitabine, vinblastine, floxuridine, 5-fluorouracil, or capecitabine), targeted therapy (tyrosine kinase inhibitors, mTOR pathway inhibitors, VEGF pathway inhibitors, Janus kinase inhibitors, ALK inhibitors, Bcl-2 inhibitors, PARP inhibitors, PI3K inhibitors, Braf inhibitors, MEK inhibitors, CDK inhibitors, Hsp90 inhibitors, or more specific examples such as imatinib, gefitinib, erlotinib, sorafenib, sunitinib, dasatinib, lapatinib, nilotinib, bortezomib, tamoxifen, tofacitinib, crizotinib, navitoclax, gossypol, iniparib, olapatinib, perifosine, apatinib, zoptarelin, doxorubicin (AN-152), [D -Lys(6)]-LHRH conjugated to doxorubicin, vemurafenib, dabrafenib, LGX818, trametinib, pazopanib, cabozantinib, axitinib, temsirolimus, everolimus, vemurafenib, dabrafenib, lenvatinib), immunotherapy (e.g., interferon-gamma, interleukin-2, interferon-alpha, or PD-1 or PD-L1 blockade) Treatment options may include drug withdrawal (e.g., pembrolizumab, ipilimumab, nivolumab), other monoclonal antibody therapies (e.g., rituximab, trastuzumab, alemtuzumab, cetuximab, panitumumab, bevacizumab), and hormonal treatments (e.g., orchiectomy, luteinizing hormone releasing hormone agonists or antagonists, antiandrogens, estrogens, or ketoconazole). Other forms of treatment include radiation therapy and cryotherapy, and vaccine treatments (such as chimeric antigen receptor modified (CAR) T cells).

[0346] Thus, in some embodiments, the methods of the invention (e.g., screening or diagnostic methods) further comprising a step of treating cancer comprise administering to the patient a therapeutically effective amount of a chemotherapeutic agent selected from the group consisting of, for example, gemcitabine, vinblastine, floxuridine, 5-fluorouracil, or capecitabine, such as a tyrosine kinase inhibitor, an mTOR pathway inhibitor, a VEGF pathway inhibitor, a Janus kinase inhibitor, an ALK inhibitor, a Bcl-2 inhibitor, a PARP inhibitor, a PI3K inhibitor, or a combination thereof. agents, Braf inhibitors, MEK inhibitors, CDK inhibitors, Hsp90 inhibitors, or more specific examples thereof include imatinib, gefitinib, erlotinib, sorafenib, sunitinib, dasatinib, lapatinib, nilotinib, bortezomib, tamoxifen, tofacitinib, crizotinib, navitoclax, gossypol, iniparib, olaparib, perifosine, apatinib, zoptarelin, doxorubicin (AN-152), [D-Lys (6)]-A drug for targeted therapy selected from doxorubicin, vemurafenib, dabrafenib, LGX818, trametinib, pazopanib, cabozantinib, axitinib, temsirolimus, everolimus, vemurafenib, dabrafenib, lenvatinib, or, for example, interferon-gamma, interleukin-2, interferon-alpha, or PD-1 or PD-L1 blockers, for example, pembrolizumab, ipilimumab, nibortezomi ... This may include administering to the subject one or more agents selected from a mab, or an agent for hormonal therapy, such as a luteinizing hormone releasing hormone agonist (e.g., leuprolide, goserelin, triptorelin, histrelin) or antagonist (e.g., degarelix or a CYP17 inhibitor), an antiandrogen (e.g., flutamide, bicalutamide, nilutamide) or an estrogen or an agent for immunotherapy selected from ketoconazole.

[0347] Alternatively, there is provided a method of the invention further comprising the step of performing a further diagnostic procedure, such as a CT scan (or a PSA test in the case of prostate cancer).

[0348] Thus, in some embodiments, the methods of the invention (e.g., screening or diagnostic methods) that further include a step of treating cancer may include administering to the subject a therapeutically effective amount of one or more agents selected from the group consisting of a chemotherapeutic agent, an agent for targeted therapy, or an agent for immunotherapy, or an agent for hormonal therapy, or a dose of radiation therapy, or a dose of cryotherapy.

[0349] In some embodiments, if the level of one or more GAG ​​signatures in the sample, or a score based on these levels, is altered to a particular degree compared to a control level or score (or cut-off level), a further step of administering a therapeutically effective amount of a pharmaceutical agent (such as a chemotherapeutic agent as described above) to the patient is performed and / or surgery is performed. Preferred degrees of alteration are discussed elsewhere herein.

[0350] In some embodiments, if the subject has already undergone drug therapy (e.g., chemotherapy or other therapy as described above), and the level of one or more GAG ​​signatures in the sample or the score based on these levels changes to a certain degree (or does not actually change) compared to the control level (e.g., compared to the level or score previously recorded for the same subject), this may indicate that the current therapeutic agent is not effective and that a therapeutic agent other than the previous therapeutic agent should be used. Thus, a step may be performed in which the subject is administered a therapeutically effective amount of a therapeutic agent other than the previously administered therapeutic agent (e.g., a chemotherapeutic agent as described above, etc.).

[0351] In some embodiments, if the methods of the present invention reveal that a current treatment regimen is ineffective, e.g., if continuous or periodic measurements of one or more GAG ​​signatures in a sample, or a score based on their levels, reveal that the treatment is ineffective, a step may be taken to alter (e.g., increase) the dosage of the therapeutic agent.

[0352] More specifically, for example, in some such aspects of the present invention, methods are provided that include determining the level of one or more GAG ​​signatures in a sample, and if one or more levels or a score based on these levels are determined to be greater than an appropriate cut-off level, e.g., a cut-off level pre-specified to maximize the accuracy of a positive diagnosis of cancer, the method may include the further step of performing surgery or an additional diagnostic procedure (e.g., a CT scan) or administering a therapeutically effective amount of a recommended drug for the treatment of cancer. The agent may be, for example, a standard chemotherapy (e.g., gemcitabine, vinblastine, floxuridine, 5-fluorouracil, or capecitabine), a targeted therapy (tyrosine kinase inhibitors, mTOR pathway inhibitors, VEGF pathway inhibitors, Janus kinase inhibitors, ALK inhibitors, Bcl-2 inhibitors, PARP inhibitors, PI3K inhibitors, Braf inhibitors, MEK inhibitors, CDK inhibitors, Hsp90 inhibitors, or more specific examples such as imatinib, vemurafenib, LGX818, trametinib, pazopanib, olapatinib, perifosine, apatinib, zoptarelin, doxorubicin (AN-152), doxorubicin linked to [D-Lys(6)]-LHRH, These may include immunotherapy (e.g., interferon-gamma, interleukin-2, interferon-alpha, or PD-1 or PD-L1 blockers, e.g., pembrolizumab, ipilimumab, nivolumab), other monoclonal antibody therapies (e.g., rituximab, trastuzumab, alemtuzumab, cetuximab, panitumumab, bevacizumab), or hormonal therapy, such as luteinizing hormone releasing hormone agonists (e.g., leuprolide, goserelin, triptorelin, histrelin) or antagonists (e.g., degarelix or CYP17 inhibitors), antiandrogens (e.g., flutamide, bicalutamide, nilutamide) or estrogens or ketoconazole.

[0353] Conversely, methods are provided that include determining the level of one or more GAG ​​signatures in a sample, and if one or more levels or a score based on these levels are determined to be lower than an appropriate cut-off level (e.g., a cut-off level pre-specified to maximize predictive value for a negative diagnosis of cancer), the method may include the further step of not performing surgery, or performing a further diagnostic procedure (e.g., a CT scan), or modifying a current dosage of a drug, or administering a therapeutically effective amount of a separate recommended drug for cancer (e.g., prostate cancer) to one already in use. The agent may be, for example, a standard chemotherapy (e.g., gemcitabine, vinblastine, floxuridine, 5-fluorouracil, or capecitabine), a targeted therapy (tyrosine kinase inhibitors, mTOR pathway inhibitors, VEGF pathway inhibitors, Janus kinase inhibitors, ALK inhibitors, Bcl-2 inhibitors, PARP inhibitors, PI3K inhibitors, Braf inhibitors, MEK inhibitors, CDK inhibitors, Hsp90 inhibitors, or more specific examples such as imatinib, vemurafenib, LGX818, trametinib, pazopanib, olapatinib, perifosine, apatinib, zoptarelin, doxorubicin (AN-152), doxorubicin linked to [D-Lys(6)]-LHRH, These may include immunotherapy (e.g., interferon-gamma, interleukin-2, interferon-alpha, or PD-1 or PD-L1 blockers, e.g., pembrolizumab, ipilimumab, nivolumab), other monoclonal antibody therapies (e.g., rituximab, trastuzumab, alemtuzumab, cetuximab, panitumumab, bevacizumab), or hormonal therapy, such as luteinizing hormone releasing hormone agonists (e.g., leuprolide, goserelin, triptorelin, histrelin) or antagonists (e.g., degarelix or CYP17 inhibitors), antiandrogens (e.g., flutamide, bicalutamide, nilutamide) or estrogens or ketoconazole.

[0354] A still further aspect provides a kit for screening for cancer (e.g., for diagnosing or determining the severity or prognosis of cancer) comprising one or more agents suitable for determining the level of one or more of the GAG ​​characteristics (GAG forms) described herein in a sample. A still further aspect provides a kit for screening for cancer (e.g., for diagnosing or determining the severity or prognosis of cancer) comprising one or more reagents (or components) for processing a body fluid sample containing GAG whose level and / or chemical composition is to be determined according to the present invention. In a preferred aspect, the kit is for use in the method of the present invention described herein. Preferably, the kit comprises instructions for use of the kit components, e.g., in screening (e.g., diagnosis).

[0355] In one aspect, the present invention provides a method for detecting (or determining) the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, wherein the sample is obtained from a subject.

[0356] In one aspect, the invention provides a method for detecting (or determining) the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS), the method comprising: (a) obtaining a body fluid sample from a human patient; (b) detecting (or determining) the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in the sample.

[0357] A still further aspect of the invention provides a method for detecting (or determining) the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, comprising: the sample has been obtained from the subject and has been subjected to processing prior to determining the level and / or chemical composition; The above process is (a) fragmenting said one or both GAGs into disaccharide units, (b) Before (a), (i) contacting the sample with a proteolytic agent; and (ii) purifying said one or both GAGs in said sample based on the negative charge of said GAG.

[0358] The features and discussion herein relating to cancer screening methods (e.g., diagnostic methods, prognostic methods, etc.), e.g., preferred GAG forms or combinations thereof for measurement, can be applied mutatis mutandis to the detection methods of the present invention.

[0359] Where the terms "comprise," "comprises," "comprising," "has," or "having," or other equivalent terms are used herein, in some more specific embodiments these terms include the terms "consists of" or "consists essentially of," or other equivalent terms. [Brief description of the drawings]

[0360] The invention will now be further described with reference to the following non-limiting examples and with reference to the following figures.

[0361] [Figure 1A]A) ROC curves in discovery (60% samples) and validation (40% samples) sets for plasma pan-cancer GAG score across different stage / grade groups and B) plasma pan-cancer GAG score. C)-D) Shown as A) and B) for urine pan-cancer GAG score. E)-F) Shown as A) and B) for combined pan-cancer GAG score. G) Sensitivity at 98% specificity for plasma, urine, and combined pan-cancer GAG score across different stage / grade groups in discovery and validation sets. H) Tissue of origin (TOO) prediction in validation set (N=74, 5 cancer types) using a Bayesian additive regression tree model trained on combined GAGomes in discovery set (N=110). Legend: See Table 1. [Figure 1B] A) ROC curves in discovery (60% samples) and validation (40% samples) sets for plasma pan-cancer GAG score across different stage / grade groups and B) plasma pan-cancer GAG score. C)-D) Shown as A) and B) for urine pan-cancer GAG score. E)-F) Shown as A) and B) for combined pan-cancer GAG score. G) Sensitivity at 98% specificity for plasma, urine, and combined pan-cancer GAG score across different stage / grade groups in discovery and validation sets. H) Tissue of origin (TOO) prediction in validation set (N=74, 5 cancer types) using a Bayesian additive regression tree model trained on combined GAGomes in discovery set (N=110). Legend: See Table 1. [Figure 1C]A) ROC curves in discovery (60% samples) and validation (40% samples) sets for plasma pan-cancer GAG score across different stage / grade groups and B) plasma pan-cancer GAG score. C)-D) Shown as A) and B) for urine pan-cancer GAG score. E)-F) Shown as A) and B) for combined pan-cancer GAG score. G) Sensitivity at 98% specificity for plasma, urine, and combined pan-cancer GAG score across different stage / grade groups in discovery and validation sets. H) Tissue of origin (TOO) prediction in validation set (N=74, 5 cancer types) using a Bayesian additive regression tree model trained on combined GAGomes in discovery set (N=110). Legend: See Table 1. [Figure 1D] A) ROC curves in discovery (60% samples) and validation (40% samples) sets for plasma pan-cancer GAG score across different stage / grade groups and B) plasma pan-cancer GAG score. C)-D) Shown as A) and B) for urine pan-cancer GAG score. E)-F) Shown as A) and B) for combined pan-cancer GAG score. G) Sensitivity at 98% specificity for plasma, urine, and combined pan-cancer GAG score across different stage / grade groups in discovery and validation sets. H) Tissue of origin (TOO) prediction in validation set (N=74, 5 cancer types) using a Bayesian additive regression tree model trained on combined GAGomes in discovery set (N=110). Legend: See Table 1. [Figure 1E]A) ROC curves in discovery (60% samples) and validation (40% samples) sets for plasma pan-cancer GAG score across different stage / grade groups and B) plasma pan-cancer GAG score. C)-D) Shown as A) and B) for urine pan-cancer GAG score. E)-F) Shown as A) and B) for combined pan-cancer GAG score. G) Sensitivity at 98% specificity for plasma, urine, and combined pan-cancer GAG score across different stage / grade groups in discovery and validation sets. H) Tissue of origin (TOO) prediction in validation set (N=74, 5 cancer types) using a Bayesian additive regression tree model trained on combined GAGomes in discovery set (N=110). Legend: See Table 1. [Figure 1F] A) ROC curves in discovery (60% samples) and validation (40% samples) sets for plasma pan-cancer GAG score across different stage / grade groups and B) plasma pan-cancer GAG score. C)-D) Shown as A) and B) for urine pan-cancer GAG score. E)-F) Shown as A) and B) for combined pan-cancer GAG score. G) Sensitivity at 98% specificity for plasma, urine, and combined pan-cancer GAG score across different stage / grade groups in discovery and validation sets. H) Tissue of origin (TOO) prediction in validation set (N=74, 5 cancer types) using a Bayesian additive regression tree model trained on combined GAGomes in discovery set (N=110). Legend: See Table 1. [Figure 1G]A) ROC curves in discovery (60% samples) and validation (40% samples) sets for plasma pan-cancer GAG score across different stage / grade groups and B) plasma pan-cancer GAG score. C)-D) Shown as A) and B) for urine pan-cancer GAG score. E)-F) Shown as A) and B) for combined pan-cancer GAG score. G) Sensitivity at 98% specificity for plasma, urine, and combined pan-cancer GAG score across different stage / grade groups in discovery and validation sets. H) Tissue of origin (TOO) prediction in validation set (N=74, 5 cancer types) using a Bayesian additive regression tree model trained on combined GAGomes in discovery set (N=110). Legend: See Table 1. [Figure 1H] A) ROC curves in discovery (60% samples) and validation (40% samples) sets for plasma pan-cancer GAG score across different stage / grade groups and B) plasma pan-cancer GAG score. C)-D) Shown as A) and B) for urine pan-cancer GAG score. E)-F) Shown as A) and B) for combined pan-cancer GAG score. G) Sensitivity at 98% specificity for plasma, urine, and combined pan-cancer GAG score across different stage / grade groups in discovery and validation sets. H) Tissue of origin (TOO) prediction in validation set (N=74, 5 cancer types) using a Bayesian additive regression tree model trained on combined GAGomes in discovery set (N=110). Legend: See Table 1. [Diagram 2] Performance of the tissue of origin (TOO) classifier in the discovery cohort. Numbers in boxes represent the number of samples classified as belonging to the predicted TOO. Overall and balanced accuracy in predicting cancer tissue of origin was 77.3% and 81.3%, respectively. N=110. Legend: See Table 1 [Figure 3A] Kaplan-Meier curves for overall survival in cancer patients stratified into "high" vs. "low" risk by GAG score values. A) Plasma score. B) Urine score. C) Combined score. [Figure 3B]Kaplan-Meier curves for overall survival in cancer patients stratified into "high" vs. "low" risk by GAG score values. A) Plasma score. B) Urine score. C) Combined score. [Figure 3C] Kaplan-Meier curves for overall survival in cancer patients stratified into "high" vs. "low" risk by GAG score values. A) Plasma score. B) Urine score. C) Combined score. [Figure 4A] Kaplan-Meier survival curves for overall survival for different cancer types where patients are stratified into "high" vs. "low" risk based on A) plasma, B) urine, and C) combined GAG scores above / below optimal cutoff. Legend: BC-breast cancer; BCa-bladder cancer; CRC-colorectal cancer; CST-cervical cancer; DG-diffuse glioma; EC-endometrial cancer; GNET-small intestinal neuroendocrine tumors; HN-head and neck cancer; LL-B cell leukemia; NHL-non-follicular lymphoma; NSCLC-non-small cell lung cancer; OC-ovarian cancer; PCa-prostate cancer; RCC-renal cell carcinoma. [Figure 4B] Kaplan-Meier survival curves for overall survival for different cancer types where patients are stratified into "high" vs. "low" risk based on A) plasma, B) urine, and C) combined GAG scores above / below optimal cutoff. Legend: BC-breast cancer; BCa-bladder cancer; CRC-colorectal cancer; CST-cervical cancer; DG-diffuse glioma; EC-endometrial cancer; GNET-small intestinal neuroendocrine tumors; HN-head and neck cancer; LL-B cell leukemia; NHL-non-follicular lymphoma; NSCLC-non-small cell lung cancer; OC-ovarian cancer; PCa-prostate cancer; RCC-renal cell carcinoma. [Figure 4C]Kaplan-Meier survival curves for overall survival for different cancer types where patients are stratified into "high" vs. "low" risk based on A) plasma, B) urine, and C) combined GAG scores above / below optimal cutoff. Legend: BC-breast cancer; BCa-bladder cancer; CRC-colorectal cancer; CST-cervical cancer; DG-diffuse glioma; EC-endometrial cancer; GNET-small intestinal neuroendocrine tumors; HN-head and neck cancer; LL-B cell leukemia; NHL-non-follicular lymphoma; NSCLC-non-small cell lung cancer; OC-ovarian cancer; PCa-prostate cancer; RCC-renal cell carcinoma. [Figure 5A] Changes in biofluid glycosaminoglycans during cancer progression in mice. A) Experimental design. B) Principal component analysis of plasma (left panel) and urinary (right panel) GAGomes at different time points of cancer progression. C) Longitudinal levels of plasma and urinary 0s CS, respectively, per individual mouse at different time points of cancer progression. [Figure 5B] Changes in biofluid glycosaminoglycans during cancer progression in mice. A) Experimental design. B) Principal component analysis of plasma (left panel) and urinary (right panel) GAGomes at different time points of cancer progression. C) Longitudinal levels of plasma and urinary 0s CS, respectively, per individual mouse at different time points of cancer progression. [Figure 5C] Changes in biofluid glycosaminoglycans during cancer progression in mice. A) Experimental design. B) Principal component analysis of plasma (left panel) and urinary (right panel) GAGomes at different time points of cancer progression. C) Longitudinal levels of plasma and urinary 0s CS, respectively, per individual mouse at different time points of cancer progression. [Figure 6A]Plasma, urine, and combined pan-cancer GAG scores from Example 2. A), B), C) Plasma, urine, and combined pan-cancer GAG scores across different stage / grade groups, respectively. Crossbars indicate median and 25 / 75 quantiles. D) ROC curves for plasma, urine, and combined GAG scores. E), F), G) Kaplan-Meier curves for overall survival across all cancer patients stratified into "low" (undetected) vs. "high" (detected) pan-cancer plasma (E), urine (F), and combined (G) GAG score value groups, respectively. For each score, patients with a score higher than the cutoff at 95% specificity were assigned to "high" vs. "low" groups. Panels show the numbers at risk in each group. Plasma pan-cancer GAG score (cut-off score = 1.259, HR = 0.61 [95% CI = 0.44 to 0.85], p = 0.0031. N = 370, 13 cancer types), urine (cut-off score = 0.255, HR = 0.50 [95% CI = 0.25 to 0.98], p = 0.0441. N = 162, 4 cancer types). Combined (cut-off score = 0.662, HR = 0.86 [95% CI = 0.50 to 1.49], p = 0.595). N = 152, 4 cancer types). Legend: S1 = stage I / low grade, S2 = stage II, S3 = stage III, S4 = stage IV / high grade, H = healthy. [Figure 6B]Plasma, urine, and combined pan-cancer GAG scores from Example 2. A), B), C) Plasma, urine, and combined pan-cancer GAG scores across different stage / grade groups, respectively. Crossbars indicate median and 25 / 75 quantiles. D) ROC curves for plasma, urine, and combined GAG scores. E), F), G) Kaplan-Meier curves for overall survival across all cancer patients stratified into "low" (undetected) vs. "high" (detected) pan-cancer plasma (E), urine (F), and combined (G) GAG score value groups, respectively. For each score, patients with a score higher than the cutoff at 95% specificity were assigned to "high" vs. "low" groups. Panels show the numbers at risk in each group. Plasma pan-cancer GAG score (cut-off score = 1.259, HR = 0.61 [95% CI = 0.44 to 0.85], p = 0.0031. N = 370, 13 cancer types), urine (cut-off score = 0.255, HR = 0.50 [95% CI = 0.25 to 0.98], p = 0.0441. N = 162, 4 cancer types). Combined (cut-off score = 0.662, HR = 0.86 [95% CI = 0.50 to 1.49], p = 0.595). N = 152, 4 cancer types). Legend: S1 = stage I / low grade, S2 = stage II, S3 = stage III, S4 = stage IV / high grade, H = healthy. [Figure 6C]Plasma, urine, and combined pan-cancer GAG scores from Example 2. A), B), C) Plasma, urine, and combined pan-cancer GAG scores across different stage / grade groups, respectively. Crossbars indicate median and 25 / 75 quantiles. D) ROC curves for plasma, urine, and combined GAG scores. E), F), G) Kaplan-Meier curves for overall survival across all cancer patients stratified into "low" (undetected) vs. "high" (detected) pan-cancer plasma (E), urine (F), and combined (G) GAG score value groups, respectively. For each score, patients with a score higher than the cutoff at 95% specificity were assigned to "high" vs. "low" groups. Panels show the numbers at risk in each group. Plasma pan-cancer GAG score (cut-off score = 1.259, HR = 0.61 [95% CI = 0.44 to 0.85], p = 0.0031. N = 370, 13 cancer types), urine (cut-off score = 0.255, HR = 0.50 [95% CI = 0.25 to 0.98], p = 0.0441. N = 162, 4 cancer types). Combined (cut-off score = 0.662, HR = 0.86 [95% CI = 0.50 to 1.49], p = 0.595). N = 152, 4 cancer types). Legend: S1 = stage I / low grade, S2 = stage II, S3 = stage III, S4 = stage IV / high grade, H = healthy. [Figure 6D]Plasma, urine, and combined pan-cancer GAG scores from Example 2. A), B), C) Plasma, urine, and combined pan-cancer GAG scores across different stage / grade groups, respectively. Crossbars indicate median and 25 / 75 quantiles. D) ROC curves for plasma, urine, and combined GAG scores. E), F), G) Kaplan-Meier curves for overall survival across all cancer patients stratified into "low" (undetected) vs. "high" (detected) pan-cancer plasma (E), urine (F), and combined (G) GAG score value groups, respectively. For each score, patients with a score higher than the cutoff at 95% specificity were assigned to "high" vs. "low" groups. Panels show the numbers at risk in each group. Plasma pan-cancer GAG score (cut-off score = 1.259, HR = 0.61 [95% CI = 0.44 to 0.85], p = 0.0031. N = 370, 13 cancer types), urine (cut-off score = 0.255, HR = 0.50 [95% CI = 0.25 to 0.98], p = 0.0441. N = 162, 4 cancer types). Combined (cut-off score = 0.662, HR = 0.86 [95% CI = 0.50 to 1.49], p = 0.595). N = 152, 4 cancer types). Legend: S1 = stage I / low grade, S2 = stage II, S3 = stage III, S4 = stage IV / high grade, H = healthy. [Figure 6E]Plasma, urine, and combined pan-cancer GAG scores from Example 2. A), B), C) Plasma, urine, and combined pan-cancer GAG scores across different stage / grade groups, respectively. Crossbars indicate median and 25 / 75 quantiles. D) ROC curves for plasma, urine, and combined GAG scores. E), F), G) Kaplan-Meier curves for overall survival across all cancer patients stratified into "low" (undetected) vs. "high" (detected) pan-cancer plasma (E), urine (F), and combined (G) GAG score value groups, respectively. For each score, patients with a score higher than the cutoff at 95% specificity were assigned to "high" vs. "low" groups. Panels show the numbers at risk in each group. Plasma pan-cancer GAG score (cut-off score = 1.259, HR = 0.61 [95% CI = 0.44 to 0.85], p = 0.0031. N = 370, 13 cancer types), urine (cut-off score = 0.255, HR = 0.50 [95% CI = 0.25 to 0.98], p = 0.0441. N = 162, 4 cancer types). Combined (cut-off score = 0.662, HR = 0.86 [95% CI = 0.50 to 1.49], p = 0.595). N = 152, 4 cancer types). Legend: S1 = stage I / low grade, S2 = stage II, S3 = stage III, S4 = stage IV / high grade, H = healthy. [Figure 6F]Plasma, urine, and combined pan-cancer GAG scores from Example 2. A), B), C) Plasma, urine, and combined pan-cancer GAG scores across different stage / grade groups, respectively. Crossbars indicate median and 25 / 75 quantiles. D) ROC curves for plasma, urine, and combined GAG scores. E), F), G) Kaplan-Meier curves for overall survival across all cancer patients stratified into "low" (undetected) vs. "high" (detected) pan-cancer plasma (E), urine (F), and combined (G) GAG score value groups, respectively. For each score, patients with a score higher than the cutoff at 95% specificity were assigned to "high" vs. "low" groups. Panels show the numbers at risk in each group. Plasma pan-cancer GAG score (cut-off score = 1.259, HR = 0.61 [95% CI = 0.44 to 0.85], p = 0.0031. N = 370, 13 cancer types), urine (cut-off score = 0.255, HR = 0.50 [95% CI = 0.25 to 0.98], p = 0.0441. N = 162, 4 cancer types). Combined (cut-off score = 0.662, HR = 0.86 [95% CI = 0.50 to 1.49], p = 0.595). N = 152, 4 cancer types). Legend: S1 = stage I / low grade, S2 = stage II, S3 = stage III, S4 = stage IV / high grade, H = healthy. [Figure 6G]Plasma, urine, and combined pan-cancer GAG scores from Example 2. A), B), C) Plasma, urine, and combined pan-cancer GAG scores across different stage / grade groups, respectively. Crossbars indicate median and 25 / 75 quantiles. D) ROC curves for plasma, urine, and combined GAG scores. E), F), G) Kaplan-Meier curves for overall survival across all cancer patients stratified into "low" (undetected) vs. "high" (detected) pan-cancer plasma (E), urine (F), and combined (G) GAG score value groups, respectively. For each score, patients with a score higher than the cutoff at 95% specificity were assigned to "high" vs. "low" groups. Panels show the numbers at risk in each group. Plasma pan-cancer GAG score (cut-off score = 1.259, HR = 0.61 [95% CI = 0.44 to 0.85], p = 0.0031. N = 370, 13 cancer types), urine (cut-off score = 0.255, HR = 0.50 [95% CI = 0.25 to 0.98], p = 0.0441. N = 162, 4 cancer types). Combined (cut-off score = 0.662, HR = 0.86 [95% CI = 0.50 to 1.49], p = 0.595). N = 152, 4 cancer types). Legend: S1 = stage I / low grade, S2 = stage II, S3 = stage III, S4 = stage IV / high grade, H = healthy.

[0362] Example 1 Introduction We investigated urinary and / or plasma glycosaminoglycan (GAG) profiles or GAGomes, including the group of chondroitin sulfate (CS), heparan sulfate (HS), and hyaluronic acid (HA) disaccharides, as biomarkers of cancer metabolism for the early detection of 14 cancers in a total of 979 urinary and / or plasma cancer vs. healthy subjects. In this study, GAGomes were tested in protein-free fractions of plasma and urine samples. We observed extensive cancer-specific changes in biofluid GAGomes that were recapitulated in an in vivo model of cancer progression. We developed a machine learning model that detected any stage, any type of cancer with 98% specificity, up to 40.5% sensitivity, predicted tissue of origin with 74% accuracy, and correlated independently with overall survival. Overall, GAGomes were powerful and versatile metabolic biomarkers for cancer, representing a new avenue for liquid biopsy.

[0363] Study design and GAGome measurements Study design and patient recruitment The design was a case-control study with mixed retrospective and prospective cohorts. The study population included cases defined as patients with a confirmed cancer diagnosis (no history of cancer across 14 cancer types, active disease [treatment naive or metastatic disease]), self-assessed healthy subjects (adjusted to be very well, with no medical or known family history of cancer) and controls defined as cancer-free patients with initial suspicion of colorectal cancer.

[0364] A retrospective cohort with plasma samples was obtained from patients with ductal invasive carcinoma (BC), colorectal cancer (CRC), cervical squamous cell carcinoma (CST), diffuse glioma (DG), small intestinal neuroendocrine tumors (GNET), endometrial adenocarcinoma (EC), chronic lymphocytic leukemia (LL), diffuse large B-cell lymphoma (NHL), non-small cell lung cancer (NSCLC), ovarian epithelial carcinoma (OV) and prostate adenocarcinoma (PCa), as well as cancer-free patients initially suspected of CRC as an additional control group. Inclusion criteria were as follows: cases - diagnosed with any of the cancer types mentioned above, active disease at the time of sampling [treatment-naïve or metastatic disease], age >18 years at sampling, controls - patients initially suspected of CRC and cancer-free at final diagnosis, age >18 years at sampling, exclusion criteria were as follows: history of non-basal cell carcinoma, samples collected before 2015 for all diagnostic groups except CRC (2012) and LL (2014). Patients were identified retrospectively by random selection based on the eligibility criteria, resulting in the inclusion of 14–40 patients per cancer type, of which 50% were early / low grade and 50% were late / high grade (defined below).

[0365] A prospective cohort with plasma and urine samples was obtained from patients with bladder cancer (BCa), NSCLC, head and neck squamous cell carcinoma (HN), renal cell carcinoma (RCC), PCa and healthy controls. Eligibility criteria were as follows: For NSCLC and HN: Inclusions: ECOG performance status 0-2, diagnosis of non-small cell lung cancer or head and neck squamous cell carcinoma, metastatic disease, expected life expectancy over 2 months, informed consent, and exclusion: lack of adequate compliance to accept sampling, in case of PCa: Inclusion: Referral for robotic-assisted laparoscopic prostatectomy for prostate cancer, serum PSA at clinical diagnosis available, transrectal ultrasound-guided biopsy report available, suitable for all protocol procedures and informed consent, Exclusion Criteria: None, For RCC: Inclusions: referral for partial or radical nephrectomy for suspected renal cell carcinoma, predicted life expectancy over 2 months, standard imaging evaluation 12 weeks prior to inclusion, planned for standard imaging within 16 weeks after initiation of treatment, and informed consent, Exclusions: lack of adequate compliance to accept serial sampling, for BCa: Inclusions: Referral for transurethral resection of the bladder (TURB) for BCa, informed consent, urine cytology available at time of diagnosis, suitability to undergo all protocol procedures, Exclusions: age under 18 years, history of bladder or prostate radiation, previous diagnosis of cancer, for healthy controls: Inclusion: self-rated health of at least "fair", age 21-78 years, informed consent, suitable to undergo all protocol procedures, Exclusion: medical history of cancer (excluding non-melanoma skin cancer), family history of cancer among first degree relatives, and for men, total serum PSA ≥ 0.5 ng / mL within the past 5 years or at the time of enrollment.

[0366] For all cases, clinical data relating to age, sex, eligibility criteria, and date of death or last known alive, diagnosis, and tumor grade or stage were retrieved from patient diaries in the case group and through a questionnaire in the healthy control group. Cases were grouped by cancer type and stage / grade. Specifically, we classified cases as early / low grade vs. stage IV / high grade as follows: TNM I-III vs. TNM IV in BC, CRC, NSCLC, RCC, BCa, HN; G1 (mitotic count (10 HPF)<2 and Ki67<2) vs. G2 (mitotic count (10 HPF)2-20 and Ki67 3-20) in GNET; low-grade glioma vs. glioblastoma multiforme in DG; stage I vs. stage II-IV in CST; and stage I-II vs. stage III-IV in EC / OV; Binet stage A-B vs. C in LL; Anna Arbor stage I-II vs. III-IV in NHL; pathological Greenson grade <7 vs. >=7 in PCa. Further subsets were: stage I / low grade, including all early stage / low grade except TNM II-III, FIGO stage II, Binet stage B, and Ann Arbor stage II; stage II, including TNM II stage, FIGO stage II, Binet stage B, and Ann Arbor stage II; and stage III, including TNM III, FIGO stage III, and Ann Arbor stage III.

[0367] All participants provided informed consent at the recruitment site under an ethical committee-approved protocol.

[0368] We conducted a case-control study including a total of 553 cancer patients across 14 types (N = 14-104, median per type = 28) and 426 healthy subjects with similar demographic characteristics across a multi-center international cohort. Thirty-four percent of all cancer patients were classified as stage I / low grade (6%-66% across all types, median per type = 41%). Patient characteristics are summarized in Table 1.

[0369] [Table 1]

[0370] We measured GAGomes in plasma and urine in a single blinded laboratory using a standardized UHPLC-MS / MS method (D. Tamburro, S. Bratulic, SA Shameh, NK Soni, A. Bacconi, F. Maccari, F. Galeotti, K. Mattsson, N. Volpi, J. Nielsen, F. Gatto, bioRxiv, in press, doi:10.1101 / 2021.02.04.429694). For a given fluid, the GAGome included concentrations of all disaccharide forms of chondroitin sulfate (CS), heparan sulfate (HS) and hyaluronic acid (HA), plus dependent properties such as CS and HS charge and total CS and HS concentration, for a total of 39 features. Details of sample collection, processing and GAG measurement are given below.

[0371] Sample collection Across all cohorts, we successfully analyzed a total of 942 plasma samples and 560 urine samples, split into 517 plasma samples in 14 cancer types and 220 urine samples in 5 cancer types for the case group, and 425 plasma samples and 340 urine samples for the control group. A subset of 184 cases (5 cancer types) and 339 healthy controls had matched plasma and urine samples. All samples were de-identified and registered according to applicable national legislation for biobanking.

[0372] Whole blood samples were collected in K2 EDTA coated tubes at room temperature and processed within 15 minutes. Generally, tubes were centrifuged (2500 RCF, 15 minutes at 4°C) and plasma was extracted and collected in a separate cryovial for storage at -80°C until transport in dry ice. In the healthy control cohort, plasma centrifugation was at 1100-1300 RCF, 10-20 minutes at room temperature. In the retrospective cohort, plasma centrifugation was at 2400 RCF, 7 minutes at room temperature. In the BCa cohort, plasma centrifugation was at 2000 RCF, 10 minutes at room temperature. Urine was any void spot collection in a polypropylene cup and 100-220 μL was aliquoted into a cryovial for storage at -20°C until transport in dry ice. In the BCa cohort, urine was also centrifuged at 2,000 RCF, 5 minutes at room temperature. Variations in sample collection are due to different protocols in the robotic collection of samples across sites and are not expected to have a significant effect on GAG measurements (data not shown).

[0373] Sample processing and measurement and analysis of GAGs Urine and plasma samples were processed to obtain GAG disaccharides for analysis.

[0374] Processing of GAGs was performed prior to ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry according to the instructions for the Elypta MIRAM™ Glycosaminoglycan Kit. All reagents and consumables used were included in the kit. The method was based on a previously established protocol for glycosaminoglycan (GAG) extraction and detection by Volpi et al. (Nature Protocols, 9, 541-558 (2014)). Briefly, the method consists of an enzymatic digestion assay using chondroitinase ABC and heparinase I-II-III to depolymerize GAGs in the sample into disaccharides. It should be noted that compared to Volpi et al., where the use of non-specific proteases is recommended for biological fluid analysis, the method used in this study omitted the addition of proteases and therefore the analysis was limited to the protein-free fraction of GAGs. It should be noted that compared to Volpi et al., the method used in this study omitted the step of purifying GAGs using an anion exchange resin.

[0375] The inventors verified that the absence of an anion exchange resin increased the extraction yield of GAGs from the samples compared to the absence of an anion exchange resin. The inventors prepared nine RCC and nine healthy urine samples using a step of purifying GAGs using an anion exchange resin (N 樹脂 = 18), 6 RCC and 6 healthy urine samples were prepared omitting that step (N 樹脂なし = 12) experiments were performed. In a multivariate linear regression, we estimated that omission of the anion exchange resin was associated with a 2.4-fold increase in total CS concentration (p = 0.0004), a 1.9-fold increase in the concentration of 0s CS (p = 0.04), a 2.6-fold increase in the concentration of 4s CS (p = 0.00003), and a 3.0-fold increase in the concentration of 6s CS (p = 0.000003), independent of RCC vs. healthy status. This suggested that purification of GAGs using anion exchange resin was less effective in terms of GAG extraction yield than direct enzymatic digestion of samples omitting the use of anion exchange resin, especially with respect to extraction of sulfated GAG disaccharides.

[0376] The GAG ​​disaccharides were then labeled with 2-aminoacridone.

[0377] The processed samples were then injected into an ultra-high performance liquid chromatography (UHPLC) coupled with an electrospray ionization triple quadrupole mass spectrometry system (ESI-MS / MS, Waters® 6 Acquity I-class Plus Xevo TQ-S micro) for disaccharide separation and detection. Multiple reaction monitoring (MRM) analysis performed in mass spectrometry software (Waters® 9 TargetLynx) was used to acquire peaks of 17 GAG disaccharides (listed below) at pre-specified retention times across six transitions. We used mass spectrometry software (Waters® TargetLynx) for peak integration, calibration curve construction, and quantification. We exported the processed data to Results in Excel format, which was imported into R (4.0.2) for secondary analysis.

[0378] The measured GAG profile (GAGomes) consisted of absolute concentrations of 17 GAG disaccharides corresponding to eight different sulfation patterns of chondroitin sulfate (CS) and heparan sulfate (HS), as well as hyaluronic acid (HA) disaccharides. Specifically, we quantified eight CS disaccharides (0s CS, 2s CS, 6s CS, 4s CS, 2s6s CS, 2s4s CS, 4s6s CS, Tris CS) and eight HS disaccharides (0s HS, 2s HS, 6s HS, Ns HS, Ns6s HS, Ns2s HS, 2s6s HS, Tris HS). The GAGome was characterized by a further 22 dependent features: The GAGome was expanded to include total CS and total HS concentrations as the sum of the corresponding disaccharide concentrations, charged CS and HS, two ratios (4s CS / 0s CS and 6s CS / 0s CS), and the relative concentration (or mass fraction, %) of each of the 16 CS and HS disaccharides by normalizing their absolute concentration by the total CS and HS concentration, respectively. Thus, for a plasma or urine sample collected at a given visit, the GAGome consisted of 39 GAG features.

[0379] GAGomes vs. physiological levels in any stage of cancer We next investigated whether differences existed in GAGome features across each cancer type relative to their physiological levels measured in healthy subjects. To this end, we used Bayesian mixed-effects linear regression with asymmetric response models to correlate each GAGome feature with each group as a predictor. We considered that GAGome features were indeed different from physiological levels in a given cancer type by defining a region of practical equivalence (ROPE) centered on the estimated level across healthy subjects. This analysis highlighted several GAGome features that were altered across multiple cancers. Most notably, we observed a near-universal increase in urinary and plasma concentrations of non-sulfated CS (0s CS). We further uncovered several cancer type-specific GAGome features, such as reduced plasma CS charge in colorectal cancer (CRC) and non-follicular lymphoma (NHL) and elevated urinary 2s6s CS in prostate cancer (PCa).

[0380] In plasma, increases in non-sulfated (0s) CS were observed in 12 of 14 cancer types (85%). In plasma, increases in total CS were observed in 11 of 14 cancer types (80%). In plasma, 4-sulfated (4s) CS was elevated in genitourinary and respiratory tract cancers (Gen: RCC, BCa, PCa, Resp: HN, NSCLC). In urine, increases in non-sulfated (0s) CS and HS were observed in 3 of 5 cancer types (60%). In urine, increases in total CS were observed in 4 of 5 cancer types (80%). In urine, increases in 6-sulfated CS, either monosulfated or disulfated (2s6s), were found in non-small cell lung cancer samples.

[0381] Table A below shows certain data demonstrating that the GAGome features analyzed in this study are significantly (ROPE<5%) altered in cancer versus healthy samples (plasma or urine samples). A number of <0.05 in the "ROPE_percentage" column means a ROPE value of <5%. A positive number in the "Difference column" means that the described GAGome feature is increased in cancer versus healthy samples. A negative number in the "Difference column" means that the described GAGome feature is decreased in cancer versus healthy samples. "μg.mL" = total GAG class concentration in micrograms / mL. Suffix "_conc" = GAGome feature in absolute concentration (micrograms / mL). No suffix = GAGome feature in relative concentration (micrograms / micrograms total for related GAG groups, e.g. CS) (also considered as "mass fraction" - relative concentration is also explained elsewhere herein). Cancer types were: BCa-bladder cancer, BC-breast invasive ductal carcinoma, CST-cervical squamous cell carcinoma, LL-chronic lymphocytic leukemia, CRC-colorectal cancer, EC-endometrial cancer, DG-diffuse glioma, GNET-gastroenterological endocrine tumors, HN-head and neck squamous cell carcinoma, NHL-diffuse large B-cell lymphoma, NSCLC-non-small cell lung cancer, OV-ovarian cancer, PCa-prostate cancer, RCC-renal cell carcinoma. "95%CI" = 95% confidence interval for the difference in values ​​between cancer types vs. healthy.

[0382] [Table 2-1]

[0383] [Table 2-2]

[0384] Further details of statistical analysis and Bayesian inference (ROPE estimation) are described here: We performed estimation of group differences in GAG features by Bayesian estimation and equivalence testing, BEST (Kruschke, J. Exp Psychol Gen, 142, 573-603 (2013)). Briefly, we modeled each individual standardized GAGome feature as a response with an asymmetric normal distribution in a mixed-effects model, where diagnosis was a fixed effect and experimental batch was treated as a random factor. We modeled group-specific variance as a multiplicative interaction between cancer type and experimental batch. We used uninformative prior distributions (Kruschke, J. Exp Psychol Gen, 142, 573-603 (2013)) for all predictors. We considered the convergence of Bayesian estimation acceptable if the effective sample size (ESS) was >5000 and the potential scale reduction factor R<1.001. We used the posterior samples to calculate the 95% confidence interval (95%CI) of the group median for each GAGome feature conditional on diagnosis. We then calculated the 95% confidence interval (95%CI) for the difference in median for each cancer diagnosis versus healthy. We considered a GAGome feature to be correlated with diagnosis versus healthy if the 95%CI of the mean difference did not cross 0 and 5% or less fell within the region of a pre-specified range of practical equivalence (ROPE) interval around 0. We defined the ROPE boundary as 0.2 of the overall standardized mean. Bayesian inference was performed using the brms(2.14.4) package (Burkner, Journal of Statistical Software, 80, 1-28(2017), Burkner, The R journal, 10, 395-411(2018)) and the tidybayes(Matthew Kay, Zenodo, 2020, https: / / zenodo.org / record / 4284191) packages (version) in R(4.0.3).

[0385] Development and validation of the GAG ​​score as a pan-cancer biomarker Having identified the presence of several shared and at least one altered GAGome feature in all cancer types, we sought to identify a minimal set of GAGome features to reliably distinguish any type of cancer from healthy controls.

[0386] We first split the dataset into a discovery (60%) and validation (40%) set and ensured that there were at least five samples for each cancer type in the validation set (Table 2).

[0387] Table 2 Number of samples with urine only GAGome features, plasma only GAGome features, or both urine and plasma ("combined") GAGome features were assigned by group to discovery versus validation set. We randomly assigned each sample to either discovery or validation set in a 60%:40% ratio, with the constraint that there were at least 5 samples of each cancer type in the validation set. H-healthy, BC-breast cancer, BCa-bladder cancer, CRC-colorectal cancer, CST-cervical cancer, DG-diffuse glioma, EC-endometrial cancer, GNET-small intestinal neuroendocrine tumor, HN-head and neck cancer, LL-B cell leukemia, NHL-non-follicular lymphoma, NSCLC-non-small cell lung cancer, OC-ovarian cancer, PCa-prostate cancer, RCC-renal cell carcinoma.

[0388] [Table 3]

[0389] Using the discovery set, we independently selected relevant features in urine, plasma, and combined GAGomes using projected predictor selection (Piironen, J., Paasiniemi, M., and Vehtari, A. Prediction and feature selection, Electronic Journal of Statistics, 14, 2155-2197 (2020)). We then fitted three reference (plasma only, urine only, and combined) Bayesian multivariate logistic regressions to cancer (aggregating all cancer types) vs. health, standardizing detectable GAGome features as predictors. Note that the plasma-only and urine-only models were used as input for GAGome features measured in only plasma or urine samples, respectively, while the combined model was used as input for both plasma and urine GAGome features. We used a heavy-tailed standard t-distribution (df=3) as a prior distribution for the intercept and coefficients for all predictors. We fitted the models using the rstanrm package (2.21.1) with four chains for a total of 4000 iterations (2000 warm-ups). R for urine, plasma and combined reference models 2 were 0.31, 0.45, and 0.66, respectively. Variable selection was then performed using leave-one-out cross-validation forward selection using the cv_varsel function from the projpred package version 2.0.2 (projpred: Projection Predictive Feature Selection (https: / / mc-stan.org / projpred / )). We selected the submodels of minimum size such that the estimated difference in the sum of log-predictive densities (ELPD) between the reference model and the submodels was at most one standard error away from 0 (default). Where necessary, we further constrained the size of the models such that the number of predictors never exceeded 1 / 10 of the number of cancer samples in the discovery set. Constraints on the maximum number of parameters were 31, 13, and 11 for plasma, urine, and combination, respectively. We then selected and projected the final set of submodels using either the default proposed model size (plasma, 3 features) or based on a maximum pre-specified number of features, which was 13 for urine and 11 for the combination model. Finally, we projected 400 plots of the submodel of the selected size and predicted the response using the linear predictor plot ("proj_linpred" function, averaging over all parameters). The response corresponding to the log odds of any cancer vs. healthy control is defined as the pan-cancer GAG score. When only three plasma GAGome features are used as input, the GAG ​​score is the plasma pan-cancer GAG score. Conversely, when 13 urine GAGome features are used as input, the GAG ​​score is the urine pan-cancer GAG score. Finally, when 11 combined GAGome features are used as input, the GAG ​​score is the combined pan-cancer GAG score.

[0390] The three GAGome features used for the plasma score were 0s CS (μg / ml), 4s / 0s CS and 4s CS (%), all measured in plasma samples.

[0391] The 13 GAGome features used for the urine score were 0s HS (μg / ml), 6s CS (%), Ns HS (μg / ml), 2s6s CS (%), Ns HS (%), 0s HS (%), total HS (μg / ml), 4s CS (%), 0s CS (%), 6s / 0s CS, 2s6s CS (μg / ml), 4s CS (μg / ml) and total CS (μg / ml), all measured in urine samples.

[0392] The 11 GAGome features used for the combined score were plasma total CS (μg / ml), urine 0s HS (μg / ml), urine Ns HS (μg / ml), urine 0s CS (%), plasma 4s CS (%), urine 2s6s CS (%), urine Ns HS (%), urine 0s HS (%), urine total HS (μg / ml), urine charged CS and plasma 0s CS (μg / ml).

[0393] For a given GAG score, a particular GAGome feature is positively (negatively) associated with any type of cancer and healthy state if the corresponding coefficient is positively (negatively) conditional on the values ​​of all other GAGome features in that GAG score.

[0394] For each pan-cancer GAG score, we estimated the metric of discrimination (in terms of the area under the receiver operating characteristic curve (ROC), AUC) and clinical utility (sensitivity at 98% specificity). Confidence intervals for sensitivity at 98% specificity were calculated by binomial approximation.

[0395] The pan-cancer GAG score distinguished any type of cancer from healthy subjects with AUC=0.83 (95% CI=0.80-0.86) for discovery and AUC=0.84 (95% CI=0.80-0.88) for validation in plasma (Figure 1A and 1B), AUC=0.88 (95% CI=0.85-0.93) for discovery and 0.82 (95% CI=0.76-0.87) for validation in urine (Figure 1C and 1D), and AUC=0.92 (95% CI=0.90-0.96) for discovery and 0.86 (95% CI=0.81-0.92) for validation in plasma and urine combined (Figure 1E and 1F). In the validation set, the sensitivity to any stage of cancer for plasma, urine, and combined pan-cancer GAG scores was 38.6% (95% CI = 18.0% to 47.8%), 34.6% (95% CI = 22.7% to 51.1%), and 40.5% (95% CI = 25.7% to 54.1%), respectively (Figure 1G). In the subset of stage I / low-grade tumors, the sensitivity was 30.9% (95% CI = 8.8% to 47.1%), 33.3% (95% CI = 12.5% ​​to 54.2%), and 33.3% (95% CI = 9.5% to 61.9%) for plasma, urine, and combined, respectively. All three scores showed similar sensitivity to stages I-II and I-III in the validation subset, revealing a weak dependency between GAGome alterations and tumor stage or grade (Figure 1G). Sensitivity was comparable across individual cancer types, ranging from 21% in breast cancer to 71% in renal cell carcinoma. In addition, all ROC curves were nearly symmetric, so that the opposite scenario, where sensitivity was fixed at 98%, yielded specificities ranging from 20% to 40%.

[0396] Glycosaminoglycans can predict tissue of origin Given the presence of characteristic GAGome patterns across cancer types, we investigated whether combined plasma and urine GAGomes could be used to identify the tissue of origin (TOO) of cancer. We performed a tissue of origin analysis (TOO) on a subset of cancers in the cohort, where we defined TOO as the cancer type. We preselected specific GAG forms (median measured concentrations were higher than 0.1 μg / mL). We trained a multinomial Bayesian additive regression tree (BART) (R. Sparapani et al., Journal of Statistical Software. 97, 1-66 (2021)) on cancer samples in the discovery set (60%, N=110 across five cancer types), where cancer type was the response and preselected GAGs were the predictors. We assigned the category with the highest average posterior probability as the predictive category (Figure 2). We verified the prediction accuracy on the validation set (40% remaining, N=74). Confidence intervals of accuracy and balanced accuracy were calculated by 5000 bootstrap iterations using normal approximation. In the validation set, the balanced accuracy of classification was 74.3% (95% CI=68.1%-80.3%) (Figure 1H). When tumors were grouped into airway (NSCLC and HN) vs. genitourinary (RCC, PCa and BCa), the accuracy of predicting TOO was 89.2% (95% CI: 82.2%-96.4%).

[0397] Pan-cancer GAG score was an independent predictor of overall survival To assess whether alterations in GAGome signatures in cancer suggest aggressive tumor biology, we correlated each pan-cancer GAG score with overall survival (OS). From the date of sample collection, the median follow-up time was 17 months in the plasma cohort (N=370 across 13 cancer types, range: 14–47 per type, N 死亡N deaths = 82, range: 1-18 per type) and 15 months in the urine cohort (N = 162 across 4 cancer types, range: 17-50 per type; N deaths = 33, range: 4-13 per type). In multivariate Cox regression, both plasma and urinary pan-cancer GAG scores were independent predictors of OS after adjusting for cancer type, age, sex, and late stage / high grade vs. early stage / low grade disease (HR = 1.30, 95% CI: 1.08-1.57, p = 0.006 and HR = 1.53, 95% CI: 1.07-2.18, p = 0.019, respectively).

[0398] We then determined optimized cutoffs for plasma, urine, and combined pan-cancer GAG scores using maximally selected rank statistics (p<0.01), which dichotomized patients into "high" versus "low" risk groups depending on whether their individual scores were above or below the cutoff. For all three scores, risk groups correlated with OS across and within cancer types (in plasma, HR=1.87 (95%CI=1.36-2.57), p<0.00013, Figures 3A and 4A); HR=2.50 (95%CI=1.50-4.16), urinary p<0.005, Figures 3B and 4B; HR=2.66 (95%CI=1.60-4.44), combined p<0.000168, Figures 3C and 4C). These findings link GAGome alterations to an aggressive cancer phenotype.

[0399] GAGomes dynamics in an in vivo model of cancer progression We sought to verify whether alterations in GAGomes, previously attributed to any type of cancer, are in fact mechanistically linked in vivo to cancer onset and progression. We performed longitudinal measurements of urinary and plasma GAGomes in BALB / c (BALB / CaNNCrl) mice in which Renca renal adenocarcinoma was orthotopically induced on day 0 (D0) (N=20 in 10 metabolic cages, FIG. 5A). Tumor-bearing kidneys were excised on day 7 and mice were sacrificed on day 20. This model was chosen because it recapitulates cancer progression from localized to metastatic recurrence after surgery (P. Sobczuk, et al., Transl Oncol. 13, 100745 (2020)). In principal component analysis (PCA), we observed changes in plasma GAGomes and to a lesser extent in urine GAGomes that coincided with progression from baseline (day 0) to local growth (day 6), post-operative resection (day 8), and metastasis (day 20) (Figure 5B). Consistent with the patterns observed above in human cancer samples, we reported a reliable linear increase in non-sulfated CS (0s CS) across time points in both plasma and urine (% change in metastasis vs. baseline = 148% [95% confidence interval: +91%, +231%] in plasma and 116% [95% confidence interval: +39%, +211%] in urine, Figure 5C). This suggests that the GAGome changes captured in the pan-cancer GAG score are causally related to cancer initiation and progression.

[0400] Further details regarding the mouse studies are as follows: Briefly, 28 female BALB / c (BALB / CaNNCrl) mice (5-6 weeks old at reception) were used as controls in experiment-3. RenCa tumors were orthotopically induced in 25 female BALB / c mice under anesthesia on day 0 (D0). Briefly, the abdomen of the animals was opened by a midline incision under sterile conditions. 5 × 106 5Mouse renal adenocarcinoma (RenCa) tumor cells (American Type Culture Collection, USA) were slowly injected into the subcapsular space of the left kidney. On day 7 (D7), the abdomen of the mice was opened and the kidney containing the injected RenCa cells was excised.

[0401] Blood was collected from 20 mice at each time point. Withdrawals by merciful termination were replaced. Blood (50 μL / sample) was collected into K2 EDTA tubes by jugular vein puncture. As a terminal procedure, intracardiac blood collection was performed under deep isoflurane gas anesthesia. Blood was collected from animals at the following time points: D-1 (24 h before engraftment), D6 (24 h before nephrectomy), D8 (24 h after nephrectomy) and D20 (day of mouse termination). Thus, each mouse generated up to four plasma samples. Blood was collected into collection tubes containing anticoagulant (K2 EDTA). The tubes were centrifuged (2000 g, 10 min, room temperature) to obtain plasma. Plasma samples were stored in propylene tubes at -20 °C until transportation.

[0402] Urine was collected from 20 mice divided into 10 metabolic cages, replacing withdrawals due to catastrophic termination. For pooled urine collection of 2 mice per cage, animals were confined in metabolic cages for 24 h at +4 °C. All urine was collected from animals at the following time points: D-2 to D-1 (24 h before OT engraftment), D5 to D6 (24 h before nephrectomy), D7 to D8 (24 h after nephrectomy) and D19 to D20 (24 h before mouse termination), collected in propylene tubes and stored at -20 °C until shipment. Each group of 2 mice produced 4 urine samples. All surviving mice were terminated on day 20 as described above.

[0403] Consideration Compared to physiological levels, we observed extensive changes in the GAGomes of cancer patients. Such changes were already observed at stage I. The rise in non-sulfated CS was particularly notable. These trends were reproduced in an in vivo mouse model, causally linking them to cancer immediately at its onset. Few small studies have investigated biofluid GAGomes in disease. Among these, respiratory failure and septic shock did not significantly alter plasma or urinary non-sulfated CS, despite the fact that these conditions directly affect the GAG-rich endothelial glycocalyx of the lung and kidney, respectively. Taken together, this suggests a tumor-associated origin for the increase in non-sulfated CS.

[0404] Here, we designed a liquid biopsy test to detect multiple types of cancer-induced GAGome alterations. Unlike genomics- and proteomics-based detection assays that investigate the full picture of potential alterations, a patient's GAGomes constitute a relatively small set of features. GAGome features could extract meaningful information about the spatial and temporal status of cancer from early stages. From a practical perspective, this results in a relatively low assay complexity and therefore low cost, a more feasible implementation in high-volume settings such as screening, and robust predictive performance. Using the pan-cancer GAG score, the sensitivity for stage I / low-grade tumors ranged from 31% to 33% with a specificity of 98%, comparable to or superior to recently reported genomics-based assays. Notably, although these genomics-based assays do not work well in cancers that shed little cell-free cDNA, such as genitourinary and brain tumors, we observed that GAGomes were altered in all cancer types tested, including low- and high-grade gliomas and renal cell carcinoma.

[0405] The metabolic nature of GAGomes and their ability to detect several cancer types at early stages makes them highly useful and makes them suitable for first-pass standalone tests or as combination tests in a multi-cancer screening setting.

[0406] Example 2 The study design, patient recruitment, study population characteristics, sample collection and processing, and GAGome measurements (GAG measurements) were essentially the same as those presented in Example 1. The main differences are outlined below.

[0407] sample Across all cohorts, we successfully analyzed a total of 969 plasma samples and 560 urine samples, split into 517 plasma samples in 14 cancer types and 220 urine samples in 5 cancer types for the case group, and 452 plasma samples and 340 urine samples for the control group. A subset of 184 cases (5 cancer types) and 339 healthy controls had plasma and urine samples available.

[0408] Development of pan-cancer GAG score We aimed to identify a minimal subset of GAGome features (also referred to herein as GAG features or GAG signatures) that inform the discrimination between cancer and healthy subjects. To this end, we used projected predictor selection to independently select relevant features in urine, plasma, and combined GAGomes. We then fitted three reference (plasma only, urine only, and combined) Bayesian multivariate logistic regressions on cancer (aggregating all cancer types) versus healthy, standardizing detectable GAGome features as predictors. We used a heavy-tailed standard t-distribution (df=3) as prior distribution for intercepts and coefficients for all predictors. We fitted the model using the rstanrm package (2.21.1) with 4 chains for a total of 4000 iterations (2000 warm-ups).

[0409] We then performed variable selection using leave-one-out cross-validation forward selection using the cv_varsel function from the projpred package version 2.0.223,24,39 (Kruschke, J. Exp Psychol Gen, 142,573-603 (2013)). We selected submodels of minimal size such that the estimated difference in the sum of log-predicted densities (ELPD) between the reference model and the submodels was at most one standard error away from 0 (default). Where necessary, we further constrained the size of the models such that the number of predictors never exceeded 1 / 10 of the number of cancer samples in the training cohort. We then selected and projected the final set of submodels with the default proposed model sizes (3 features for plasma, 13 features for urine, and -14 features for the combination).

[0410] Finally, for each model, 400 plots of the submodel of the selected size were projected and the response was predicted using a plot of the linear predictor (proj_linpred function averaged over all parameters). The effect size of the response, called the pan-cancer GAG score, was predicted as the log odds of any type of cancer. Confidence intervals for sensitivity at 95% specificity were calculated by binomial approximation.

[0411] When three plasma GAGome features are used as input, the GAG ​​score is the plasma pan-cancer GAG score. Conversely, when 13 urine GAGome features are used as input, the GAG ​​score is the urine pan-cancer GAG score. Finally, when 14 combined GAGome features are used as input, the GAG ​​score is the combined pan-cancer GAG score.

[0412] The three GAGome features used for the plasma score were 0s CS [ug / mL], 4s CS [%] and 4s / 0s CS, all measured in plasma samples.

[0413] The 13 GAGome features used for the urine score were 0s HS [μg / mL], 6s CS [%], Ns HS [%], 0s HS [%], 2s6s CS [%], total HS [μg / mL], 4s CS [%], 0s CS [%], 6s / 0s CS, 4s CS [μg / mL], 6s CS [μg / mL], total CS [μg / mL] and 0s CS [μg / mL], all measured in urine samples.

[0414] The 14 GAGome features used for the combined score were: Total CS plasma [ug / mL], 0s HS urine [ug / mL], Ns HS urine [%], 0s CS urine [%], 4s / 0s CS plasma, Charge CS urine [-], 0s HS urine [%], 4s CS urine [ug / mL], Total CS urine [ug / mL], 6s / 0s CS urine, 0s CS urine [ug / mL], 4s CS urine [%], 6s CS urine [%] and 6s CS urine [ug / mL].

[0415] Results for pan-cancer GAG score For each pan-cancer GAG score, we estimated the metrics of discrimination (in terms of area under the receiver operating characteristic curve (ROC), AUC) and clinical utility (sensitivity at 95% specificity). The pan-cancer GAG score distinguished any type of cancer from healthy subjects with AUC=0.83 (95% CI=0.8-0.86) for plasma (Figure 6A and D), AUC=0.88 (95% CI=0.85-0.91) for urine (Figure 6B and D), and AUC=0.93 (95% CI=0.9-0.95) for plasma and urine combined (Figure 6C and D). Sensitivity for any stage of cancer for plasma, urine, and the combined pan-cancer GAG score was 46.2% (95% CI = 41.9% to 50.6%), 66.8% (95% CI = 60.2% to 73.0%), and 65.8% (95% CI = 58.4% to 72.6%) at 95% specificity, respectively. In the subset of stage I / low-grade disease, sensitivities at 95% specificity were 41.6% (95% CI = 34.2% to 49.2%), 62.3% (95% CI = 47.9% to 75.2%), and 61.4% (95% CI = 45.5% to 75.6%) for plasma, urine, and the combined score, respectively. At 99% specificity, sensitivities were 25.7%, 25.0%, and 35.3% for plasma, urine, and the combined score, respectively. All three scores showed a weak dependency between free GAGome alterations and tumor stage or grade, with a slight increase in sensitivity in stages I-II and even stages I-III. Overall, we observed similar sensitivity of each score across individual cancer types. The top types detected were NHL, CRC, and chronic lymphocytic leukemia (LL) for the plasma pan-cancer GAG score (range: 23.4% in bladder cancer to 66.7% in NHL, LL, and CRC), and RCC and non-small cell lung cancer (NSCLC) for the urine or combined score (range: 47.1% in head and neck squamous cell carcinoma to 82.4%-84.6% in RCC, urine and combined, respectively).The following individual types of cancer were also analyzed individually using plasma scores, and for each individual cancer type, the median plasma score was found to be significantly altered (increased) compared to healthy samples (data not shown). The following individual types of cancer were also analyzed individually using urine scores, and for each individual cancer type, the median urine score was found to be significantly altered (increased) compared to healthy samples (data not shown). The following individual types of cancer were also analyzed individually using combined scores, and for each individual cancer type, the median combined score was found to be significantly altered (increased) compared to healthy samples (data not shown). Taken together, these findings indicate that free GAGomes are significantly altered from physiological levels across early and late stage cancers and may be useful for the early detection of multiple cancers.

[0416] Internal validation of the pan-cancer GAG score We validated the selection procedure by bootstrap. For this purpose, we analyzed 500 bootstraps for plasma and 1000 bootstraps for urine and combined datasets. In each bootstrap, we first fit a reference logistic Bayes regression model. We used the same priors and fitting parameters as above (t-distribution priors with df=3, 4000 iterations with 2000 warm-up samples). Secondly, we performed projected predictor selection using leave-one-out cross-validation. The projection used 400 samples, and the projected model size was either automatically determined by ELPD or set to 1 / 10 of the number of cases in the original dataset. Finally, we predicted the response (log odds of any type of cancer) using plots of linear predictors on three datasets: bootstrap, evaluation dataset (samples excluded from bootstrap) and original dataset. We recorded the selected model size, AUC, sensitivity at 95% specificity, and the stretched Brier metric for the full and projected models on the bootstrapped original and validation datasets.

[0417] Correlation between pan-cancer GAG score and overall survival To assess whether changes in cancer-associated GAGome signatures suggest aggressive tumor biology, we correlated each pan-cancer GAG score with overall survival (OS). From the date of sample collection, the median follow-up was 17 months in the plasma cohort (N=370 across 13 cancer types, range: 14-47 per type, Ndeath=82, range: 1-18 per type), 15 months in the urine cohort (N=162 across 4 cancer types, range: 17-50 per type, Ndeath=33, range: 4-13 per type), and 15 months in the combined cohort (N=152 across 4 cancer types, range: 17-50 per type, Ndeath=33, range: 4-13 per type). In multivariate Cox regression, plasma, as well as urine and combined pan-cancer GAG scores, were independent predictors of OS after adjusting for cancer type, age, sex, and stage IV / high-grade disease (for plasma, hazard ratio [HR] = 1.29, 95% CI: 1.06-1.56, p = 0.0009; for urine, HR = 1.79, 95% CI: 1.27-2.53, p = 0.0009; for combined, HR = 1.91, 95% CI: 1.33-1.73, p = 0.0004). These findings associate GAGome alterations with aggressive cancer phenotypes. Moreover, they suggested that patients with scores below the 95% specificity cutoff, in other words, undetected when using the pan-cancer GAG score, may have a better prognosis. To validate this for each score, we dichotomized patients into "high" versus "low" groups according to whether their individual scores were above or below the score-specific 95% specificity cutoff. For plasma and urinary GAG scores, Kaplan-Meier survival analysis suggested that "low" risk (undetected) patients had a 39%-50% lower risk of death than "high" risk (detected) patients (unadjusted HR=0.61 (95% CI=0.44-0.85), p=0.0031 in plasma; HR=0.50 (95% CI=0.25-0.98), p=0.0441 in urine (Fig. 6F). Results for survival differences between groups are shown in Fig. 6G for the combined GAG score (unadjusted HR=0.86 (95% CI=0.50-1.49), p=0.595, Fig. 6G).Importantly, we observed a lower risk of death in both the stage I-III / low-grade and stage IV / high-grade subsets, suggesting an independent correlation between free GAGomes and prognosis. Alternative dichotomization with a "high" vs. "low" risk cutoff optimized using maximally selected rank statistics resulted in statistically significant correlations with OS for all three scores across and within cancer types. Cumulatively, these survival analyses suggest that patients who go undetected using the pan-cancer GAG score have a better prognosis and are characterized by a less aggressive cancer phenotype, independent of tumor stage and grade.

Claims

1. 1. A method for screening for cancer in a subject, comprising determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, said sample being obtained from said subject; A method wherein an alteration in the level and / or chemical composition of chondroitin sulfate (CS) and / or heparan sulfate (HS) in the protein-free fraction compared to a control level and / or chemical composition is indicative of cancer in the subject.

2. 2. The method of claim 1, wherein the determining of the chemical composition comprises determining the level in the protein-free fraction of one or more GAG ​​characteristics selected from the group consisting of one or more (or all) of specific sulfated or non-sulfated forms of CS or HS disaccharides, charged HS, charged CS, total concentration of CS or total concentration of HS.

3. 2. The method of claim 1, wherein the determining of the chemical composition comprises determining the level in the protein free fraction of one or more (or all) of GAG characteristics selected from the group consisting of specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of 0s CS, 2s CS, 6s CS, 4s CS, 2s6s CS, 2s4s CS, 4s6s CS, Tris CS, 0s HS, 2s HS, 6s HS, 2s6s HS, Ns HS, Ns2s HS, Ns6s HS, Tris HS, the ratio of 4s CS to 6s CS, the ratio of 6s CS to 0s CS, and the ratio of 4s CS to 0s CS, charged HS, charged CS, the total concentration of CS (CS tot), and the total concentration of HS (HS tot).

4. The method of claim 1 , wherein the body fluid sample is blood (e.g. plasma) and / or urine.

5. The determination of the chemical composition comprises: (i) one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 6s CS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of; (ii) one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 6s CS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, CS tot, HS tot, charged CS, or a specific sulfated or non-sulfated form of a CS or HS disaccharide selected from the group consisting of:

3. The method of claim 1, comprising: (iii) determining in the protein free fraction the level of one or more (or all) of GAG characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS.

6. 2. The method of claim 1, wherein the determining of the chemical composition comprises determining the level in the protein free fraction of one or more (or all) of GAG characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, absolute concentration of 6s CS, ratio of 6s CS to 0s CS, ratio of 4s CS to 0s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides.

7. The body fluid sample is blood (e.g., plasma), and the determination of the chemical composition comprises: (i) one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 4s CS, relative concentration of 4s CS, ratio of 4s CS to 0s CS, CS Tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides; or (ii) Absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 4s CS, 4s one or more (or all) of the GAG ​​characteristics selected from the group consisting of the relative concentration of CS, CS Tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides, or 2. The method of claim 1, comprising: (iii) determining in the protein-free fraction the level of one or more (or all) of GAG characteristics selected from the group consisting of: absolute concentration of 0s CS; relative concentration of 4s CS; ratio of 4s CS to 0s CS; specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of:

8. the body fluid sample is urine, and the determination of the chemical composition comprises: (i) one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 6s CS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides; or (ii) one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 6s CS, absolute concentration of 2s6s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, CS tot, HS tot, charged CS, specific sulfated or non-sulfated forms of CS or HS disaccharides; or 3. The method of claim 1, comprising: (iii) determining in the protein free fraction the level of one or more (or all) of a GAG characteristic selected from the group consisting of specific sulfated or non-sulfated forms of CS or HS disaccharides selected from the group consisting of: absolute concentration of 4s CS, relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 2s6s CS, relative concentration of 6s CS, relative concentration of 0s HS, relative concentration of Ns HS, ratio of 6s CS to 0s CS, CS tot, HS tot.

9. 2. The method of claim 1, wherein the body fluid sample is urine and the determination of the chemical composition comprises determining the level of one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 0s HS, relative concentration of 6s CS, relative concentration of Ns HS, relative concentration of 0s HS, relative concentration of 2s6s CS, relative concentration of 4s CS, relative concentration of 0s CS, absolute concentration of 4s CS, absolute concentration of 6s CS, absolute concentration of 0s CS, ratio of 6s CS to 0s CS, CS tot, HS tot, specific sulfated or non-sulfated forms of CS or HS disaccharides.

10. The chemical composition of said one or both of said GAGs in said protein-free fraction of a blood (e.g. plasma) sample is determined, and the chemical composition of said one or both of said GAGs in said protein-free fraction of a urine sample functions, preferably Determining the chemical composition of the protein-free fraction of the urine sample comprises determining the level in the protein-free fraction of one or more (or all) of GAG characteristics selected from the group consisting of: relative concentration of 0s CS, absolute concentration of 0s HS, absolute concentration of Ns HS, relative concentration of 2s6s CS, relative concentration of 0s HS, relative concentration of Ns HS, HS tot, and charged CS; 2. The method of claim 1, wherein determining the chemical composition of the protein-free fraction of the blood (e.g. plasma) sample comprises determining the level in the protein-free fraction of one or more (or all) of GAG characteristics selected from the group consisting of: absolute concentration of 0s CS, relative concentration of 4s CS, and CStot.

11. The chemical composition of the one or both of the GAGs in the protein-free fraction of a blood (e.g., plasma) sample is determined, and the chemical composition of the one or both of the GAGs in the protein-free fraction of a urine sample is determined, Determining the chemical composition of the protein-free fraction of the urine sample comprises determining the level in the protein-free fraction of one or more (or all) of GAG characteristics selected from the group consisting of: absolute concentration of 0s HS, relative concentration of Ns HS, relative concentration of 0s CS, relative concentration of 0s HS, absolute concentration of 4s CS, absolute concentration of 0s CS, relative concentration of 4s CS, relative concentration of 6s CS, absolute concentration of 6s CS, ratio of 6s CS to 0s CS, CS tot, and charged CS; 2. The method of claim 1, wherein determining the chemical composition of the protein-free fraction of the blood (e.g., plasma) sample comprises determining the level in the protein-free fraction of one or both of a GAG profile selected from the group consisting of: the ratio of 4s CS to 0s CS, and CS tot.

12. The chemical composition of the one or both of the GAGs in the protein-free fraction of a blood (e.g., plasma) sample is determined, and the chemical composition of the one or both of the GAGs in the protein-free fraction of a urine sample is determined, Determining the chemical composition of the protein-free fraction of the urine sample comprises determining the level in the protein-free fraction of one or more (or all) of the GAG ​​characteristics selected from the group consisting of: absolute concentration of 0s HS, absolute concentration of Ns HS, absolute concentration of 4s CS, absolute concentration of 6s CS; 2. The method of claim 1, wherein determining the chemical composition of the protein-free fraction of the blood (e.g. plasma) sample comprises determining the level in the protein-free fraction of the absolute concentration of 0s CS.

13. 2. The method of claim 1, wherein determining the level and / or chemical composition of the protein-free fraction of the body fluid sample comprises determining the level in the protein-free fraction of one or both of a GAG profile selected from the group consisting of absolute concentration of 0s CS and CS Tot.

14. 2. The method of claim 1, comprising determining a level in the protein-free fraction of an absolute concentration of Os CS, wherein an increase in the absolute concentration of Os CS compared to a control level is indicative of cancer.

15. 3. The method of claim 2, wherein the chemical composition can be expressed in terms of a score, the score being based on the measured levels of one or more (preferably more than one) or all of the GAG ​​characteristics.

16. 2. The method of claim 1, wherein the method comprises determining more than one level of the GAG ​​signature, preferably the method comprises determining two or more, three or more, four or more, or all levels of the GAG ​​signature.

17. The method of claim 1 , wherein the sample has been obtained from the subject and subjected to at least one processing step prior to determining the level and / or chemical composition.

18. 3. The method of claim 2, wherein the levels of one or more of the specific sulfated or non-sulfated forms of CS or HS disaccharides are determined and the GAGs are or have been subjected to a processing step to obtain the disaccharide units for analysis.

19. 20. The method of claim 17, wherein the at least one processing step does not include contacting the sample with a proteolytic agent.

20. the sample has been obtained from the subject and has been subjected to processing prior to determining the level and / or chemical composition; The process, (a) fragmenting said one or both GAGs into disaccharide units, (b) before (a), (i) contacting the sample with a proteolytic agent; and The method of claim 1, which does not include at least one of: (ii) purifying the one or both GAGs in the sample based on the negative charge of the GAG.

21. 21. The method of claim 20, which does not include the contacting of (b)(i) or the purifying of (b)(ii).

22. 21. The method of claim 20, wherein the fragmentation in (a) is carried out by contacting the one or both GAGs with one or more GAG ​​lyase enzymes.

23. 23. The method of claim 22, wherein the one or more GAG ​​lyase enzymes are one or more chondroitinase enzymes and / or one or more heparinase enzymes.

24. 21. The method of claim 20, wherein the purification of (b)(ii) is carried out using an anion exchange resin.

25. The method of claim 1 , wherein the level or chemical composition of the GAGs or GAG profile is determined by HPLC and mass spectrometry.

26. 26. The method of claim 25, wherein the HPLC is an ultra HPLC.

27. 26. The method of claim 25, wherein the mass spectrometry is triple quadrupole mass spectrometry.

28. 10. The method of claim 1, wherein the subject is at risk of developing or developing cancer, or has or is suspected of having cancer.

29. 2. The method of claim 1, wherein the cancer is: (i) stage I cancer, stage II cancer, stage III cancer, stage IV cancer, and stage unspecified cancer; or (ii) is selected from the group consisting of genitourinary cancer (e.g., renal, prostate or bladder cancer), respiratory tract cancer (e.g., lung cancer), brain cancer, blood cancer (e.g., lymphoma), colorectal cancer, uterine cancer, gastrointestinal neuroendocrine tumors, breast cancer, ovarian cancer and head and neck cancer; or (iii) selected from the group consisting of bladder cancer, invasive ductal carcinoma, cervical squamous cell carcinoma, chronic lymphocytic leukemia, colorectal cancer, endometrial carcinoma, diffuse glioma, gastrointestinal endocrine tumors, head and neck squamous cell carcinoma, diffuse large B-cell lymphoma, non-small cell lung cancer, ovarian cancer, prostate cancer, and renal cell carcinoma.

30. 2. The method of claim 1, wherein the method is used to diagnose cancer, for the prognosis of cancer, to predict the occurrence of cancer, to estimate the risk of developing cancer, to monitor a subject at risk of developing cancer, to monitor the progression of cancer in a subject, to determine the clinical severity of cancer, to predict a subject's response to cancer therapy or surgery, to determine the effectiveness of a therapeutic or surgical regimen used to treat cancer, to detect the recurrence of cancer, to predict the tissue of origin of cancer, or to distinguish small masses suspected to be cancer from other non-malignant diseases.

31. 1. A method for screening for cancer in a subject, comprising determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample; the sample has been obtained from the subject and has been subjected to processing prior to determining the level and / or chemical composition; The process, (a) fragmenting said one or both GAGs into disaccharide units, (b) before (a), (i) contacting the sample with a proteolytic agent; and (ii) purifying said one or both GAGs in said sample based on the negative charge of said GAG.

32. 32. The method of claim 31 , which does not include the contacting of (b)(i) or the purifying of (b)(ii).

33. The method according to claim 31, wherein the method has one or more features according to any one of claims 1 to 30.

34. 1. A method for determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS), comprising: (a) obtaining a body fluid sample from a human patient; (b) detecting the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in the sample.

35. 1. A method for determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, comprising: the sample has been obtained from the subject and has been subjected to processing prior to determining the level and / or chemical composition; The process, (a) fragmenting said one or both GAGs into disaccharide units, (b) before (a), (i) contacting the sample with a proteolytic agent; and (ii) purifying said one or both GAGs in said sample based on the negative charge of said GAG.

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