WO2006114659A1

WO2006114659A1 - Glycosylation markers for cancer diagnosing and monitoring

Info

Publication number: WO2006114659A1
Application number: PCT/IB2005/002531
Authority: WO
Inventors: Raymond A. Dwek; Rafael De Llorens; Rosa Peracaula; Catherine M. Radcliffe; Louise Royle; Pauline M. Rudd; Nicole Zitzmann
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-04-26
Filing date: 2005-06-24
Publication date: 2006-11-02
Anticipated expiration: 2007-10-26
Also published as: JP2008539412A; EP1886144A1

Abstract

A. method of determining one or more glycosylation markers of cancer comprises obtaining a diseased sample and a control sample, wherein the diseased sample is a sample from a subject diagnosed with cancer and the control sample is a sample from healthy control; releasing a diseased glycan pool of total glycoproteins from the diseased sample and a control glycan pool of total glycoproteins from the control sample without purifying the glycoproteins and without exposing the diseased sample and the control sample to hydrazinolysis; measuring a diseased glycoprofile of the diseased glycan pool and a control glycoprofile of the control glycan pool using chromatography, mass spectrometry or a combination thereof; comparing the diseased glycoprof:ïle and the control glycoprofiles to determine said one or more glycosylation markers of cancer. A method for diagnosing and monitoring cancer in a subject comprising obtaining a sample of body fluid or a body tissue of the subject; releasing a glycan pool of total glycoproteins from the sample without purifying the glycoproteins; measuring a glycoprofile of the glycan pool.

Description

GLYCOSYLATION MARKERS FOR CANCER DIAGNOSING

AND MONITORING

This application claims priority to US provisional patent application No. 60/674,723 "Glycosylation markers for cancer diagnosing and monitoring" to Dwek et. al. filed April 26, 2005, incorporated hereby by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to methods of diagnosing and monitoring cancer and, in particular, to methods of diagnosing and monitoring cancer based on detailed glycosylation analysis.

Presymptomatic screening to detect early-stage cancer reduces cancer-related mortality and treatment-related morbidity. Although many cancers can be treated and cured if they are diagnosed while tumors are still localized, most cancers are not detected until after they have invaded the surrounding tissue or metastasized to distant sites. For example, only 50% of breast cancers, 56% of prostate cancers and 35% of colorectal cancers are localized at the time of diagnosis, see Watkins, B., Szaro, R., Ball, S., Knubovets, T., Briggman, J., Hlavaty, J. J., Kusinitz, F., Stieg, A., and Wu, Y. (2001) Detection of early-stage cancer by serum protein analysis. American Laboratory. June, 32-36. incorporated herein by reference in its entirety. The situation is much worse for other, less treatable types of cancer. For example, about 80% of pancreatic cancers are already metastatic at the time of diagnosis which results in 1-year survival rate after diagnosis of about 19% and 5-year survival rate of about 4%. Similar 5-year survival rates (<5%) were reported for hepatocellular carcinoma. As therapeutic options for cancer treatment increase, early detection of cancer becomes important for improving prognosis.

In recent years, several serum protein markers have been developed for certain types of cancer. For example, prostate specific antigen (PSA), a glycoprotein secreted by prostate cells that is found in serum in prostate pathologies, is currently used as a tumor marker for prostate cancer. Other protein markers for cancer diagnostics and monitoring are alpha-fetoprotein for hepatocellular carcinoma and testicular cancer, NMP22 for bladder cancer, catecholamines for neuroblastoma, immunoglobulins for multiple myeloma, carcinoembryonic antigen (CEA) for colorectal cancer, HER-2, CA 15-3 and CA 27-29 for breast cancer, CA 125 for ovarian cancer, CAl 9-9 for pancreatic cancer, see Keesee et. al. Crit. Rev. Eukaryotic Gene Expr, 1996, 6(2&3): 189-214; Diamandis, Clin. Lab. News 1996, 22: 235-239, Stein et. al. J. Urol 1998, 160(3, pt l):645-659. Although the development of the serum markers facilitated the clinical management of certain types of cancer, the assays of these biomarkers are neither sensitive nor specific enough for use as the sole screening method for cancer diagnostics. Thus, it is highly desirable to develop new cancer-related biomarkers that will be more sensitive and more specific to detect recurrence and metastases at the earliest stages for both diagnosing and monitoring cancer progression.

Methods of developing new cancer-related biomarkers were suggested based on the difference in glycosylation in glycoproteins from cancer patients and healthy controls. For example, Block et. al. was comparing glycosylation profiles in immunoglobulin G (IgG) depleted sera from hepatitis B virus infected subjects (humans and woodchucks) with hepatocellular carcinoma and from respective healthy controls to identify particular glycoproteins with glycosylation changes as cancer- related biomarkers, see Block, T. M., Comunale, M. A., Lowman, M., Steel, L. F., Romano, P. R., Fimmel, C, Tennant, B. C, London, W. T., Evans, A. A., Blumberg, B. S., Dwek, R. A., Mattu, T. S. and Mehta, A. S. (2005). "Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans." Proc Natl Acad Sd USA 102: 779-84, incorporated herein by reference.

Particular differences in glycosylation profiles of purified glycoproteins between diseased patients and healthy controls can serve themselves as markers of the disease. For example, a clear correlation between rheumatoid arthritis and the percentage of the galactosylation on N-glycans released from purified immunoglobulin G (IgG) has been established in Parekh et ah, see "Association of Rheumatoid Arthritis and Primary Osteoarthritis with Changes in the Glycosylation Pattern of Total Serum IgG, "Nature, 316, pp. 452-457, 1985, incorporated herein by reference in its entirety. Alterations in glycosylation profiles of purified glycoproteins were also reported for certain types of cancer. For example, glycosylation was found to be different for glycans released from purified PSA from seminal plasma and from purified PSA secreted by the tumor prostate cell line LNCaP, see Peracaula R, Tabares G, Royle L, Harvey DJ₃ Dwek RA, Rudd, PM, de Llorens R. (2003). Altered glycosylation pattern allows the distinction between Prostate Specific Antigen (PSA) from normal and tumor origins, Glycobiology, 13, 457-470, incorporated herein by reference in its entirety. Completely different glycosylation profiles were found for pancreatic ribonuclease (RNase 1) isolated from healthy pancreas and from pancreatic adenocarcinoma tumor cells (Capan-1 and MDAPanc-3), see Peracaula R, Royle L, Tabares G, Mallorqui-Fernandez G, Barrabes S, Harvey D, Dwek RA, Rudd, PM, de Llorens R. (2003) "Glycosylation of human pancreatic ribonuclease: differences between normal and tumour states", Glycobiology, 13, 227-244, incorporated herein by reference in its entirety. Thus, glycosylation analysis can be a powerful tool for identifying cancer-related biomarkers, however, currently used methods involve purifying glycoproteins, a step which can be time consuming and which can require a large amount of sample material from patients. Accordingly, it is highly desirable to develop methods for identifying cancer-related glycosylation markers and related methods for diagnosing and monitoring cancer that would not comprise purifying glycoproteins. Performing glycosylation analysis on whole, i.e. not depleted and not purified, samples can be particularly beneficial for cancer diagnostics and monitoring. Although differences in the glycosylation profile can be associated with the presence in samples of cancer patients of glycoproteins specifically associated with cancer, such as alpha-fetoprotein (see e.g. Johnson, P. J., T. C. Poon, et al. (2000). "Structures of disease-specific serum alpha-fetoprotein isoforms." Br J Cancer 83(10): 1330-7; and Chan, M. H., M. M. Shing, et al. (2000). "Alpha-fetoprotein variants in a case of pancreatoblastoma." Ann Clin Biochem 37 ( Pt 5): 681-5), many other tumor glycoproteins, i.e. glycoproteins that are not specific inflammatory markers of cancer, can be expected to carry altered glycosylation because glycosylation pathways are usually disturbed in tumor cells, see e.g. "Effects of N-Glycosylation on in vitro Activity of Bowes Melanoma and Human Colon Fibroblast Derived Tissue Plasminogen Activator" Art Wittwer, Susan Howard, Linda S. Carr, Nikos K. Harakas, Joseph Feder Raj B. Parekh, Pauline M. Rudd, Raymond A. Dwek and Thomas W. Rademacher Biochemistry, 1989, 28, 7660-7669; "N-Glycosylation and in vitro Enzymatic Activity of Human Recombinant Tissue Plasminogen Activator Expressed in Chinese Hamster Ovary Cells and a Murine Cell line" Raj B. Parekh, Raymond A. Dwek, Pauline M. Rudd, Jerry R. Thomas, T. W. Rademacher, T. Warren, T.C. Wun, B. Herbert, B. Reitz, M. Palmier, T. Ramabhadran and D.C. Teimeir Biochemistry 1989, 28, 7670-7679, both incorporated herein in their entirety. Based on the above, performing detailed glycosylation analysis on samples of whole body fluid or body tissue, without isolating or purifying specific glycoproteins, can be expected to identify glycosylation markers of cancer amplified compared with glycosylation analysis of purified glycoproteins.

SUMMARY OF THE INVENTION

One embodiment of the invention is a method of determining one or more glycosylation markers of cancer comprising obtaining a diseased sample and the control sample, wherein the diseased sample is a sample from a subject diagnosed with cancer and the control sample is a sample from healthy control, releasing a diseased glycan pool of total glycoproteins from the diseased sample and a control glycan pool of total glycoproteins from the control sample without purifying the glycoproteins and without exposing the diseased sample and the control sample to hydrazinolysis; measuring a diseased glycoprofile of the diseased glycan pool and a control glycoprofile of the control glycan pool by chromatography, mass spectrometry or a combination thereof; comparing the diseased glycoprofiles and the control glycoprofiles to determine the one or more glycosylation markers of cancer.

Another embodiment of the invention is a method for diagnosing and monitoring cancer in a subject comprising obtaining a sample of the subject; releasing a glycan pool of total glycoproteins from the sample without purifying the glycoproteins; measuring a glycoprofile of the glycans.

Yet another embodiment of the invention method for optimizing a dosage of an existing therapeutic agent against cancer comprising obtaining a first sample of a body fluid or a body tissue from a cancer patient before administering the therapeutic agent to the patient; obtaining a second sample of a body fluid or a body tissue from the cancer patient after administering the therapeutic agent to the patient; releasing glycans of glycoproteins from the first and the second samples without purifying the glycoproteins; measuring a first glycoprofile of the glycans from the first sample and a second glycoprofile of the glycans from the second sample; comparing a level of a glycosylation marker of the cancer in the first glycoprofile and the second glycoprofile.

And yet another embodiment of the invention is a method of testing a new therapy or a new therapeutic agent for treating cancer comprising obtaining a first sample of a body fluid or a body tissue from a cancer patient before exposing the patient to the new therapy or the new therapeutic agent; obtaining a second sample of a body fluid or a body tissue from the cancer patient after exposing the patient to the new therapy or the new therapeutic agent; releasing glycans of glycoproteins from the first and the second samples without purifying the glycoproteins; measuring a first glycoprofile of the glycans from the first sample and a second glycoprofile of the glycans from the second sample; comparing a level of a glycosylation marker of the cancer in the first glycoprofile and the second glycoprofile.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates determination of glycosylation marker for breast cancer.

FIG. 2 illustrates determination of glycosylation marker for pancreatic cancer.

FIG. 3 illustrates determination of glycosylation marker for prostate cancer.

FIG. 4 illustrates determination of glycosylation marker for hepatocellular carcinoma in hepatitis C virus (HCV) infected patients.

FIG. 5 illustrates determination of glycosylation marker for ovarian cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is related to methods of diagnosing and monitoring cancer and, in particular, to methods of diagnosing and monitoring cancer based on detailed glycosylation analysis.

This application incorporates by reference in their entirety US provisional patent application No. 60/674,724 "An automated glycofmgerprinting strategy" to Dwek et. al. filed 04/26/2005 and US provisional patent application No. 60/674,722 "High Throughput Glycan Analysis for Diagnosing and Monitoring Rheumatoid Arthritis and Other Autoimmune Diseases" to Dwek et. al. filed 04/26/2005.

Unless otherwise specified, "a" or "an" means "one or more".

"Glycoprofile" or "glycosylation profile" means a presentation of glycan structures (oligosaccharides) present in a pool of glycans . A glycoprofile can be presented, for example, as a plurality of peaks corresponding to glycan structures present in a pool of glycans.

"Glycosylation marker" means a particular difference in glycosylation between a sample of a subject diagnosed with cancer or cancer condition and a sample from healthy control.

The inventors recognized that, in cancer tumor cells, glycosylation can be altered not for one or a few, but for many glycoproteins and, therefore, performing detailed glycosylation analysis on samples of whole body fluid or body tissue, without isolating or purifying specific glycoproteins; will identify glycosylation markers of cancer amplified compared with glycosylation analysis of isolated glycoproteins. The inventors also realized that treating glycans of total glycoproteins with one or more exoglycosidase enzymes could allow the glycosylation markers of cancer to be segregated by shifting glycan structures that do not carry the glycosylation markers from the measured region of the glycoprofile. Furthermore, the inventors recognized that the glycosylation markers could be present on more than one glycan structure in the total glycan pool. Therefore, treating glycans with one or more exoglycosidase enzymes can also amplify the glycosylation markers by digesting away one or more monosaccharides that are attached to some of the marker oligosaccharides but are not an essential feature of the marker. Accordingly, methods for determining one or more glycosylation markers of cancer and related methods for diagnosing and monitoring cancer are provided.

One embodiment of the invention is a method of determining one or more glycosylation markers of cancer comprising obtaining a diseased sample and a control sample, wherein the diseased sample is a sample of a body fluid or a body tissue from patients diagnosed with cancer and the control sample is a sample of the body fluid or the body tissue from healthy control; releasing a diseased glycan pool of total glycoproteins from the diseased sample and a control glycan pool of total glycoproteins from the control sample without purifying glycoproteins in the diseased and the control samples; measuring a diseased glycoprofile of the diseased glycan pool and a control glycoprofile of the control glycan pool by chromatography, mass spectrometry or a combination thereof, and comparing the diseased glycoprofile and the control glycoprofile to determine said one or more glycosylation markers of cancer. In some embodiments, comparing the diseased glycoprofile and the control glycoprofile can comprise comparing peak ratios in the diseased glycoprofile and in the control glycoprofile. In some embodiments, the method of determining one or more glycosylation markers of cancer can further comprise selecting a best glycosylation marker of cancer out of the one or more glycosylation marker of cancer, wherein the best glycosylation marker has the highest correlation with one or parameters of the subject diagnosed with cancer. The parameters of the subject diagnosed with cancer can be, for example, diagnosis, age, sex, cancer stage, response to therapy, medical history or any combination thereof. In some embodiments, comparing the diseased glycoprofile and the control glycoprofile can be carried out following digestion of the diseased glycan pool and the control glycan pool with one or more exoglycosidase enzymes in any combination. For example, digestion of the diseased glycan pool and the control glycan pool can be a sequential digestion , or with an array comprising one or more exoglycosidase enzymes. Digestion of the diseased glycan pool and the control glycan pool with one or more glycosidase in any combination can be used to amplify and/or segregate the glycosylation markers of cancer. The determined glycosylation markers of cancer can be used for diagnosing, monitoring and/or prognosticating cancer in a subject based on a detailed glycosylation analysis using chromatography, mass spectrometry or a combination thereof. The determined glycosylation markers can be also used for isolating in a body fluid or a body tissue one or more glycoproteins that are specific biomarkers of cancer. The determined glycosylation markers can be also used for diagnosing, monitoring and/or prognosticating cancer using analytical techniques other than the techniques used to determine the glycosylation marker of cancer. These other analytical techniques can be, for example, capillary electrophoresis or lectin chromatography.

Another embodiment of the invention is a method for diagnosing and monitoring cancer in a subject comprising obtaining a sample of body fluid or a body tissue of the subject such as human being; releasing glycans of glycoproteins from the sample without purifying the glycoproteins; measuring a glycoprofile of the glycans. The method for diagnosing and monitoring can further comprise determining a clinical status of the subject from a level of a glycosylation marker of cancer in the glycoprofile. The glycosylation marker can be, for example, a marker determined by the above method. When the glycosylation marker is determined, measuring the glycoprofile can be carried out by any suitable technique, i.e. not necessarily by the technique used to determine the glycosylation marker. For example, measuring the glycoprofile can be carried out by capillary electrophoresis or lectin chromatography. The clinical status of the subject can be, for example, selected from the group consisting of cancer, precancerous condition, a benign condition or no condition. A clinical status can be a particular stage of cancer, such as tumor, lymph node or metastases. A clinical status can be also a particular substage of tumor, lymph node or metastases stage.

The methods of the present invention can be applied to cancers, such as prostate cancer, pancreatic cancer, breast cancer, bladder cancer, renal cancer, colon cancer, ovarian cancer, hepatocellular carcinoma, stomach cancer, lung cancer.

A sample of a body fluid or a body tissue can be, for example, a sample of whole serum, blood plasma, urine, seminal fluid, seminal plasma, feces, or saliva. Particular type of the body fluid or body tissue used depends on the type of cancer. In some embodiments, samples of body fluid or body tissue can be obtained from tumor cells.

Releasing glycans.

Glycans can be released from a sample of a body fluid or a body tissue, such as a sample of whole serum, blood plasma, urine, seminal fluid, seminal plasma, feces or saliva. The released glycans can be N-glycans or O-glycans. In some embodiments, releasing a glycan pool of glycoproteins from a sample of a body fluid or a body tissue can be carried out without purifying the glycoproteins. In other words, the released glycans are glycans of all or substantially all of the glycoproteins present in the sample of a body fluid or a body tissue rather than of one or more purified and isolated glycoproteins. In some embodiments, substantially all of the glycoproteins can mean all the glycoproteins that are recovered, yet in some embodiments substantially all of the glycoproteins can mean all the glycoproteins except those that are specifically removed. Releasing glycans can be carried out without exposing a sample of a body fluid or a body tissue to hydrazinolysis. In some embodiments, releasing glycans can be carried out from a very small sample of a body fluid. In some embodiments, samples of a body fluid can be less than 100 microliters, yet preferably less than 50 microliters, yet more preferably less than 20 microliters, yet more preferably less than 10 microliters, yet most preferably less than 5 microliters. The present methods of releasing can be optimized to work with body fluid samples of less than 1 microliters.

In some embodiments, releasing glycans can comprise releasing glycans from total glycoproteins of a body fluid or a body tissue in solution. Yet in some embodiments, releasing glycans can comprise immobilizing total glycoproteins of a body fluid or a body tissue, for example, on protein binding membrane or in a gel. The protein binding membrane can be any protein binding membrane, for example, polyvinyldene fluoride (PVDF) membrane, nylon membrane or Polytetrafluoroethylene (PTFE) membrane. In some embodiments, releasing glycans can further comprise releasing glycans from the total glycoproteins immobilized on the protein binding membrane or in the gel. When released glycans are iV-lmked glycans, releasing glycans from the immobilized glycoproteins can be carried out using enzymatic release with, for example, peptide N glycosidase F. When the glycoproteins are immobilized in the gel, releasing glycans can comprise separating the gel into a plurality of bands and selecting one or more bands from the plurality of bands from which the glycans are subsequently released (in gel band method). In some embodiments, releasing glycans from the gel can be carried out from the total gel, i.e. without separating gel into the bands. In some embodiments, releasing glycans is carried out by chemical release methods, such as /^-elimination or ammonia-based /^-elimination, which can be used for releasing iV-linked or O-linked glycans from glycoproteins in solution or from glycoproteins immobilized on protein binding membrane. For using the methods of this invention in a high throughput format, it may be preferred to release a glycan pool from total glycoproteins immobilized in a gel or on a protein binding membrane as it can allow to use smaller samples of body fluid or body tissue.

The details of some of the release methods and their applicability to both N- glycans and O-glycans are discussed below, however, it should be understood that the present invention is not limited to the discussed below release methods.

In-gel-band: This method can be used for N-glycan release from single glycopeptides in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) gel bands and is based on the method described in Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A. and Harvey, D. J. (1997) "Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high- performance liquid chromatography." Anal-Biochem 250: 82-101, incorporated herein by reference in its entirety. Samples can be reduced and alkylated by adding 4μl of 5X sample buffer (5X -sample buffer: 0.04g Bromophenol blue, 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water), 2μl of 0.5M dithiothreitol (DTT) and water to make up to 20μl in total, incubated at 7O⁰C for lOmin, then alkylated by addition of 2μl of 10OmM iodoacetamide and incubated for 30 min in the dark at room temperature. Samples can be then separated on SDS-PAGE gels after which the proteins are stained with Coomassie brilliant blue, the band of interest is excised and destained. Subsequently, the gel band can be cut into lmm³ pieces and frozen for 2 hours or more (this can help break down the gel matrix). This gel band can be then washed alternatively with ImI of acetonitrile then ImI of digestion buffer (2OmM NaHCO₃ pH 7), which can be repeated twice before the gel plug can be then dried. PNGase F buffer solution (30μl of 100 U/ml) is added (this is enough for 10-15mm³ gel), more enzyme solution is added if larger gel bands can be used. The PNGaseF and gel pieces can be incubated overnight at 37⁰C. The supernatant can be recovered along with 3 x 200 μl water washes (with sonication with gel pieces for 30 mins each) followed by an acetonitrile wash (to squeeze out the gel), another water wash and a final acetonitrile wash. Samples may or may not be desalted using, for example, 50 μl of activated AG- 50(H⁺), filtered through a 0.45 μm LH Millipore filter and dried down for fluorescent labeling.

In-gel-block: To avoid the problems with clean up of samples following solution phase enzymatic glycan release an in-gel-block release from protein mixtures can be used. Briefly, the whole protein mixture (e.g. serum or plasma) can be reduced and alkylated as in the In-gel oligosaccharide release described above, then set into 15% SDS-gel mixture but without bromophenol blue. A total volume of gel of 185 μl can be used (initially set into a 48 well plate, then removed for cutting up) with 300 μl of 100 U/ml of PNGaseF. The washing procedures can be similar to those used for in-gel-band release. This procedure can be more suitable for automated glycan release than in-solution PNGaseF release, and can be the preferred method for high throughput glycan analysis. This system can be easily further modified to work with smaller amounts of gel set into a 96 well plate. Enzymatic release ofN-glycansfrom PVDF membranes

The glycoproteins in reduced and denatured serum samples can be attached to a hydrophobic PVDF membrane in a 96 well plate by simple filtration. The samples can be then washed to remove contaminates, incubated with PNGaseF to release the glycans based on the methods described in Papac, D. L, et. al. Glycobiology 8: 445- 54, 1998, and in Callewaert, N., et. al Electrophoresis 25: 3128-31, 2004, both incorporated herein by reference in their entirety. The iV-glycans can be then washed from the bound protein, collected and dried down ready for fluorescent labeling. N- glycans can be released in situ from the glycoproteins by incubation with PNGaseF and by chemical means. Chemical release of N- and 0-glycans

In contrast to the advantages that enzymatic release of N-glycans affords to N- glycan analysis, no enzymatic methodology currently exists for the release of structurally intact O-glycans. Chemical release by reductive β-elimination can require the concomitant reduction of the released oligosaccharides to their alditol derivatives (Amano, J. et. al. Methods Enzymol 179: 261-70, 1989) to prevent degradation (peeling). This reduction precludes the use of any post-release labeling so that detection is limited to mass spectrometry, pulsed amperometric detection and/or radioactivity.

Ammonia-based β-elimination can be used to release both N- and O-glycans by a modification of the classical β-elimination (Huang, Y. et. al. Analytical Chemistry 73: 6063-6069, 2001) which can be applied to glycoproteins in solution or on PVDF membranes. Ammonia-based β-elimination can be done from PVDF membranes. This strategy, can be optimized for high throughput, and can provide a powerful approach for releasing both N- and O-glycans in their correct molar proportions and in an open ring form suitable for post-release labeling.

Release of N- and O-glycans from protein binding PVDF membranes by ammonia based beta-elimination.

Samples of glycoprotein, mixtures of glycoproteins, whole serum or other body fluids are reduced and alkylated by adding 4μl of 5X sample buffer (5X sample buffer: 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water), 2μl of 0.5M dithiothreitol (DTT) and water to make up to 20 μl in total, incubated at 7O⁰C for lOmin, then alkylated by addition of 2μl of 10OmM iodoacetamide and incubated for 30 min in the dark at room temperature. Protein binding PVDF membranes (Durapore 13mm x 0.45 μm HVHP, Millipore) in Swinnex filter holders (Millipore) are pre-washed with 2 x 2.5 ml water using an all-polypropylene 2.5 ml syringe (Sigma), followed by a syringe full of air to remove most of the liquid from the membrane. The reduced and alkylated sample is then applied directly to the membrane and left to bind for 5 min before washing by pushing through 2 x 2.5 ml water slowly with a syringe, followed by a syringe full of air to remove most of the liquid from the membrane. The filter with the bound glycoprotein samples is then carefully removed from the filter holder and placed in a 1.5 ml screw capped polypropylene tube with a molded PTFE cap. 1 ml of ammonium carbonate saturated 29.2% aqueous ammonium hydroxide, plus lOOmg ammonium carbonate is added to the tube. This is incubated for 40 hours at 6O⁰C, then cooled in the fridge. The liquid is then transferred to a clean tube and evaporated to dryness. The released glycans are re-dissolved in water and re-dried until most of the salts are removed. 100 μl of 0.5M boric acid is added to the glycans and incubated at 37⁰C for 30 min. The glycans are then dried under vacuum, ImI methanol added, re-dried, a further 1 ml methanol added and re-dried to remove the boric acid.

Quantitatively analyzing the glycans.

Labeling of glycans.

In some embodiments, upon releasing, the glycans can be labeled with, for example, a fluorescent label or a radioactive label. The fluorescent label can be, for example, 2-aminopyridine (2-AP), 2-aminobenzamide (2-AB), 2-aminoanthranilic acid (2-AA), 2-aminoacridone (AMAC) or 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS). Labeling of glycans with fluorescent labels is described, for example, by Bigge, J. C, et. άl. "Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid." Anal Biochem 230: 229-38, 1995, incorporated herein reference in its entirety, and Anumula, K. R. (2000). High- sensitivity and high-resolution methods for glycoprotein analysis. Analytical Biochemistry 283: 17-26, incorporated by reference in its entirety. Fluorescent labels can label all glycans efficiently and non-selectively and can enable detection and quantification of glycans in the sub picomole range. The choice of fluorescent label depends on the separation technique used. For example, a charged label is specifically required for capillary electrophoresis. In particular, 2-AB label can be preferred for chromatographic, enzymatic and mass spectroscopic processes and analyses, while 2- AA label can be preferred for electrophoretic analyses. Unlabelled glycans can be also detected by, for example, mass spectrometry, however, fluorescent labelling may aid glycan ionisation, see e.g. Harvey, D. J. (1999). "Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates." Mass Spectrom Rev 18: 349-450.; Harvey, D. J. (2000). Electrospray mass spectrometry and fragmentation of N-lmked carbohydrates derivatized at the reducing terminus. J Am Soc Mass Spectrom 11 : 900-915.

Measuring glycoproflle of the released glycans.

Glycoprofile of the glycans means a presentation of particular glycan structures in the glycans. Measuring glycoprofile of the glycans can be carried out by quantitative analytical technique, such as chromatography, mass spectrometry, electrophoresis or a combination thereof. In particular, the chromatographic technique can be high performance anion exchange chromatography (HPAEC), weak ion exchange chromatography (WAX), gel permeation chromatography (GPC), high performance liquid chromatography (HPLC), normal phase high performance liquid chromatography (NP-HPLC), reverse phase HPLC (RP-HPLC), or porous graphite carbon HPLC (PGC-HPLC). The mass spectrometry technique can be, for example, matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI- TOF-MS), electrospray ionization time of flight mass spectrometry (ESI-TOF-MS), positive or negative ion mass spectrometry or liquid chromatography mass spectrometry (LC-MS). The electrophoretic technique can be, for example, gel electrophoresis or capillary electrophoresis. The use of these quantitative analytical techniques for analyzing glycans is described, for example, in the following publications:

1) Guile, G. R., Wong, S. Y. and Dwek, R. A. (1994). "Analytical and preparative separation of anionic oligosaccharides by weak anion-exchange high-performance liquid chromatography on an inert polymer column." Analytical Biochemistry 222: 231-5 for HPLC, incorporated herein by reference in its entirety;

2) Butler, M., Quelhas, D., Critchley, A. J., Carchon, H., Hebestreit, H. F., Hibbert, R. G., Vilarinho, L., Teles, E., Matthijs, G., Schollen, E., Argibay, P., Harvey, D. J., Dwek, R. A., Jaeken, J. and Rudd, P. M. (2003). "Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis." Glycobiology 13: 601-22, for MALDI-MS, NP-HPLC and ESI-liquid chromatography/MS, incorporated herein by reference in its entirety;

3) Jackson, P., Pluskal, M. G. and Skea, W. (1994). "The use of polyacrylamide gel electrophoresis for the analysis of acidic glycans labeled with the fluorophore 2- aminoacridone." Electrophoresis 15: 896-902, for polyacrylamide gel electrophoresis (PAGE), incorporated herein by reference in its entirety;

4) Hardy, M. R. and Townsend, R. R. (1994). "High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates." Methods Enzymol 230: 208- 25., for HPAEC using pulsed amperometric detection (PAD), incorporated herein by reference in its entirety;

5) Callewaert, N., Contreras, R., Mitnik-Gankin, L., Carey, L., Matsudaira, P. and Ehrlich, D. (2004). "Total serum protein N-glycome profiling on a capillary electrophoresis-microfluidics platform." Electrophoresis 25: 3128-31 for capillary electrophoresis, incorporated herein by reference in its entirety;

6) Guile, G. R., Rudd, P. M., Wing, D. R., Prime, S. B. and Dwek, R. A. (1996). "A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles." Anal Biochem 240: 210-26, for HPLC, incorporated herein by reference in its entirety;

7) Caesar, J. P., Jr., Sheeley, D. M. and Reinhold, V. N. (1990). "Femtomole oligosaccharide detection using a reducing-end derivative and chemical ionization mass spectrometry." Anal Biochem 191: 247-52, for LC-MS, incorporated herein by reference in its entirety;

8) Mattu, T. S., Royle, L., Langridge, J., Wormald, M. R., Van den Steen, P. E., Van Damme, J., Opdenakker, G., Harvey, D. J., Dwek, R. A. and Rudd, P. M. (2000). "O- glycan analysis of natural human neutrophil gelatinase B using a combination of normal phase-HPLC and online tandem mass spectrometry: implications for the domain organization of the enzyme." Biochemistry 39: 15695-704, for NP-HPLC and MS, incorporated herein by reference in its entirety; 9) Royle, L., Mattu, T. S., Hart, E., Langridge, J. I., Merry, A. H., Murphy, N., Harvey, D. J., Dwek, R. A. and Rudd, P. M. (2002). "An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins." Anal Biochem 304: 70-90, for NP-HPLC and MS₅ incorporated herein by reference in its entirety;

10) Anumula, K. R. and Du, P. (1999). "Characterization of carbohydrates using highly fluorescent 2- aminobenzoic acid tag following gel electrophoresis of glycoproteins." Anal Biochem 275: 236-42, for gel electrophoresis, incorporated herein by reference in its entirety;

11) Huang, Y. and Mechref, Y. (2001). "Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis." Analytical Chemistry 73: 6063-6069, for a combination of MALDI- MS and capillary electrophoresis, incorporated herein by reference in its entirety;

12) Burlingame, A. L. (1996). "Characterization of protein glycosylation by mass spectrometry." Curr Opin Biotechnol 7: 4-10, for mass spectrometry, incorporated herein by reference in its entirety;

13) Costello, C. E. (1999). "Bioanalytic applications of mass spectrometry." Curr Opin Biotechnol 10: 22-8, for mass spectrometry, incorporated herein by reference in its entirety;

14) Davies, M. J. and Hounsell, E. F. (1996). "Comparison of separation modes of high-performance liquid chromatography for the analysis of glycoprotein- and proteoglycan-derived oligosaccharides." J Chromatogr A 720: 227-33, for HPLC, incorporated herein by reference in its entirety;

15) El Rassi, Z. (1999). "Recent developments in capillary electrophoresis and capillary electrochromatography of carbohydrate species." Electrophoresis 20: 3134- 44, for capillary electrophoresis and capillary electrochromatography, incorporated herein by reference in its entirety;

16) Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A. and Harvey, D. J. (1997). "Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography." Anal- Biochem 250: 82-101, for NP-HPLC and MALDI-MS, incorporated herein by reference in its entirety;

17) Reinhold, V. N., Reinhold, B. B. and Chan, S. (1996). "Carbohydrate sequence analysis by electrospray ionization-mass spectrometry." Methods Enzymol 271: 377- 402, for ESI-MS, incorporated herein by reference in its entirety;

18) Mattu, T. S., Pleass, R. J., Willis, A. C, Kilian, M., Wormald, M. R., Lellouch, A. C, Rudd, P. M., Woof, J. M. and Dwek, R. A. (1998). "The glycosylate and structure of human serum IgAl, Fab, and Fc regions and the role of N-glycosylation on Fc alpha receptor interactions." Journal of Biological Chemistry 273: 2260-72, for WAX and NP-HPLC, incorporated herein by reference in its entirety.

19) Callewaert, N., Schollen, E., Vanhecke, A., Jaeken, J., Matthijs, G., and Contreras, R. (2003). Increased fucosylation and reduced branching of serum glycoprotein N-glycans in all known subtypes of congenital disorder of glycosylation I. Glycobiology 13: 367-375, incorporated herein by reference in its entirety;

20) Block, T.M. Comunale, M.A., Lowman, M., Steel, L.F., Romano, P.R.,, Fimmel, C, Teimant, B.C. London, A.A. Evans, B.S. Blumberg, R.A. Dwek, T.S. Mattu and A.S. Mehta , "Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans". PNAS USA (2005) 102, 779- 784, incorporated herein by reference in its entirety;

21) D. J. Harvey, Fragmentation of negative ions from carbohydrates: Part 1; Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from iV-linked carbohydrates, J. Am. Soc. Mass Spectrom., 2005, 16, 622-630, incorporated herein by reference in its entirety;

22) D. J. Harvey, Fragmentation of negative ions from carbohydrates: Part 2, Fragmentation of high-mannose iV-linked glycans, J. Am. Soc. Mass Spectrom., 2005, 16, 631-646, incorporated herein by reference in its entirety;

23) D. J. Harvey, Fragmentation of negative ions from carbohydrates: Part 3, Fragmentation of hybrid and complex iV-linked glycans, J. Am. Soc. Mass Spectrom., 2005, 16, 647-659, incorporated, herein by reference in its entirety.

Although many techniques can be used for measuring glycoprofϊle, in some embodiments it can be preferred to measure a glycoprofϊle by high performance liquid chromatography (HPLC) (e.g. normal phase) alone or in combination with mass spectrometry. HPLC measured glycoprofile can trace all glycan structures present in a glycan pool in correct molar proportions. Polar functional groups of stationary phase of HPLC can interact with the hydroxyl groups of the glycans in a manner that is reproducible for a particular monosaccharide linked in a specific matter. For example, the contribution of the outer arm fucose addition is much greater than the addition of a core fucose residue; a core fucose residue always contributes 0.5 glucose units (gu) to the overall elution position. The characteristic incremental values associated with different monosaccharide additions can allow to assign a predicted structure for a particular peak present in the glycoprofile. This structure can be then confirmed by digestion with exoglycosidase arrays and/or mass spectrometry. Other techniques, such as capillary electrophoresis are not predictable as HPLC. Although, CE migration times can be calibrated with standards, the migration times of unknown structures can not be predicted. Measuring glycoprofiles by HPLC can be also preferred for the following reason. Digestion a glycan pool with one or more exoglycosidases removes monosaccharide residues and, thus, decreases the retention times or associated gu values in the glycoprofile measured by HPLC. In some embodiments, this can enable to segregate the peaks that are associated with one or glycosylation markers by shifting away peaks that are not related to the glycosylation changes away from the measured region of the glycoprofile.

In some embodiments, glycoprofile can be presented as a plurality of peaks corresponding to glycan structures in the glycans. In the method of determining a glycosylation marker, a peak ratio means a ratio between any one or more peaks and any other one or more peaks within the same glycosylation profile. In the method of determining a glycosylation marker, comparing peak ratios can mean comparing peaks intensities or comparing integrated areas under the peaks. In some embodiments of the method of determining glycosylation marker, comparing peak ratios can be carried for glycans of the diseased and control samples which were not digested with exoglycosidase. In some embodiments, comparing peak ratios can be carried out on the glycans which were digested with exoglycosidase. In some embodiments, comparing peak ratios can be carried out for the glycans which were not digested with exoglycosidase and for the glycans digested with exoglycosidase.

Measuring glycoprofile of the glycans with the above described methods can allow for detection of a particular glycan structure present in the glycans in subpicomole levels. Accordingly, in some of the embodiments, measuring glycoprofiles of the glycans is carried out using a technique able to detect a glycan structure present in the glycans in amount of 1 picomole, preferably 0.1 picomole, yet more preferably 0.01 picomole.

Measuring glycoprofile of the glycans can comprise constructing a database of glycan structures of the glycans. The parameters of this database can be, for example, glycan structure along with: elution times (from HPLC data); mass and composition (from MS data); experimentally determined and/or predicted glycan structures, elution times, mass and composition, following treatment with exoglycosidase enzymes; experimentally determined and/or predicted glycan structures, mass and composition following MS fragmentation. The database can, for example, make preliminary and final assignments of the glycan structures as well as recommend the appropriate exoglycosidase arrays to confirm preliminary assignments. The use of databases in measuring glycoprofiles is described, for example, in the following references:

1) Mattu, T. S., Royle, L., Langridge, J., Wormald, M. R., Van den Steen, P. E., Van Damme, J., Opdenakker, G., Harvey, D. H., Dwek, R. A. and Rudd, P. M. (2000). "The O-glycan analysis of natural human neutrophil gelatinase B using a novel strategy combining normal phase- HPLC and on-line tandem mass spectrometry: implications for the domain organization of the enzyme." Biochemistry 39: 15695-704.", incorporated herein by reference in its entirety;

2) Royle, L., Mattu, T. S., Hart, E., Langridge, J. L, Merry, A. H., Murphy, N., Harvey, D. J., Dwek, R. A. and Rudd, P. M. (2002). "An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins." Anal Biochem 304: 70-90, incorporated herein by reference in its entirety;

3) Butler, M., Quelhas, D., Critchley, A. J., Carchon, H., Hebestreit, H. F., Hibbert, R. G., Vilarinho, L., Teles, E., Matthijs, G., Schollen, E., Argibay, P., Harvey, D. J., Dwek, R. A., Jaeken, J. and Rudd, P. M. (2003). "Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis." Glycobiology 13: 601-22, incorporated herein by reference in its entirety;

4) Peracaula, R., Royle, L., Tabares, G., Mallorqui-Fernandez, G., Barrabes, S., Harvey, D. J., Dwek, R. A., Rudd, P. M. and de Llorens, R. (2003). "Glycosylation of human pancreatic ribonuclease: differences between normal and tumor states." Glycobiology 13: 227-44, incorporated herein by reference in its entirety; 5) Peracaula, R., Tabares, G., Royle, L., Harvey, D. J., Dwek, R. A., Rudd, P. M. and de Llorens, R. (2003). "Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins." Glycobiology 13: 457-70.

Exoglycosidase digestion to amplify/segregate glycosylation markers

In some embodiments, the released glycans can be subjected to further enzymatic digestion with one or more enzymes. The enzymatic digestion can be done using any suitable enzymes, such as glycosidases. Examples of suitable glycosidases include, but are not limited to, N-glycosidase F (PNGase F), sialidase, β- galactosidase, fucosidase αl-6,2»3,4, αl-3,4, αl-2 fucosidase, alpha-amylase, beta- amylase, glucan 1,4-alpha-glucosidase, cellulase, endo-l,3(4)-beta-glucanase, inulinase, endo-l,4-beta-xylanase, oligosaccharide alpha- 1,6-glucosidase, dextranase, chitinase, polygalacturonase, lysozyme, exo-alpha-sialidase, alpha-glucosidase, beta- glucosidase, alpha-galactosidase, beta-galactosidase, alpha-mannosidase, beta- mannosidase, beta-fructofuranosidase, alpha,alpha-trehalase, beta-glucuronidase, xylan endo-l,3-beta-xylosidase, amylo-alpha- 1,6-glucosidase, hyaluronoglucosaminidase, hyaluronoglucuronidase, xylan 1,4-beta-xylosidase, beta- D-fucosidase, glucan endo-l,3-beta-D-glucosidase, alpha-L-rhamnosidase, pullulanase, GDP-glucosidase, beta-L-rhamnosidase, fucoidanase, glucosylceramidase, galactosylceramidase, galactosylgalactosylglucosylceramidase, sucrose alpha-glucosidase, alpha-N-acetylgalactosaminidase, alpha-N- acetylglucosaminidase, alpha-L-fucosidase, beta-N-acetylhexosaminidase, beta-N- acetylgalactosaminidase, cyclomaltodextrinase, alpha-L-arabinofuranosidase, glucuronosyl-disulfoglucosamine glucuronidase, isopullulanase, glucan 1,3-beta- glucosidase, glucan endo-l,3-alpha-glucosidase, glucan 1,4-alpha- maltotetrahydrolase, mycodextranase, glycosylceraniidase, 1,2-alpha-L-fucosidase, 2,6-beta-fructan 6-levanbiohydrolase, levanase, quercitrinase, galacturan 1,4-alpha- galacturonidase, isoamylase, glucan 1 ,6-alpha-glucosidase, glucan endo-l,2-beta- glucosidase, xylan 1,3-beta-xylosidase, licheninase, glucan 1,4-beta-glucosidase, glucan endo-l,6-beta-glucosidase, L-iduronidase, mannan l,2-(l,3)-alpha- mannosidase, mannan endo-l,4-beta-mannosidase, fructan beta-fructosidase, agarase, exo-poly-alpha-galacturonosidase, kappa-carrageenase, glucan 1,3-alpha-glucosidase, 6-phospho-beta-galactosidase, 6-phospho-beta-glucosidase, capsular-polysaccharide endo-l,3-alpha-galactosidase, beta-L-arabinosidase, arabinogalactan endo-l,4-beta- galactosidase, cellulose 1,4-beta-cellobiosidase, peptidoglycan beta-N- acetylniuramidase, alpha,alpha-phosphotrehalase, glucan 1,6-alpha-isomaltosidase, dextran 1,6-alpha-isomaltotriosidase, mannosyl-glycoprotein endo-beta-N- acetylglucosamidase, glycopeptide alpha-N-acetylgalactosaminidase, glucan 1,4- alpha-maltohexaosidase, arabinan endo-l,5-alpha-L-arabinosidase, mannan 1,4-beta- mannobiosidase, mannan endo-l,6-beta-mannosidase, blood-group-substance endo- 1,4-beta-galactosidase, keratan-sulfate endo-l,4-beta-galactosidase, steryl-beta- glucosidase, strictosidine beta-glucosidase, mannosyl-oligosaccharide glucosidase, protein-glucosylgalactosylhydroxylysine glucosidase, lactase, endogalactosaminidase, mucinaminylserine mucinaminidase, 1,3-alpha-L-fucosidase, 2-deoxyglucosidase, mannosyl-oligosaccharide 1,2-alpha-mannosidase, mannosyl-oligosaccharide 1,3-1,6- alpha-mannosidase, branched-dextran exo-l^-alpha-glucosidase, glucan 1,4-alpha- maltotriohydrolase, amygdalin beta-glucosidase, prunasin beta-glucosidase, vicianin beta-glucosidase, oligoxyloglucan beta-glycosidase, polymannuronate hydrolase, maltose-6'-phosphate glucosidase, endoglycosylceramidase, 3-deoxy-2- octulosonidase, raucaffricine beta-glucosidase, coniferin beta-glucosidase, 1,6-alpha- L-rucosidase, glycyrrhizinate beta-glucuronidase, endo-alpha-sialidase, glycoprotein endo-alpha-l,2-mannosidase, xylan alpha- 1,2-glucuronosidase, chitosanase, glucan 1,4-alpha-maltohydrolase, difructose-anhydride synthase, neopullulanase, glucuronoarabinoxylan endo-l,4-beta-xylanase, mannan exo- 1,2-1, 6-alpha- mannosidase, anhydrosialidase, alpha-glucosiduronase, lacto-N-biosidase, 4-alpha-D- {(l->4)-alpha-D-glucano}trehalose trehalohydrolase, limit dextrinase, poly(ADP- ribose) glycohydrolase, 3-deoxyoctulosonase, galactan 1,3-beta-galactosidase, beta- galactofuranosidase, thioglucosidase, ribosylhomocysteinase, beta-primeverosidase.

Most preferably, enzymatic digestion is carried out with one or more exoglycosidases listed in table 1.

In some embodiments, the enzymatic digestion can be sequential, so not all monosaccharides are removed at once. The digested glycans can be analyzed after each digestion step to obtain a glycosylation profile. In some embodiments, the enzymatic digestion can be digestion with an array comprising one or more exoglycosidases. Digestion with an array means using a panel of exoglycosidases together in a single digestion on a pool of glycans. Each exoglycosidase enzyme removes specific terminal monosaccharides attached in defined linkages.

Digestion of a glycan pool with one or more exoglycosidases which can be used in any combination is important for two reasons. First, digestion with one or more exoglycosidase can segregate the glycosylation marker by shifting glycans that do not contain the marker from the measured region of the glycoprofile. Second, digestion with one or more exoglycosidase can be used to amplify the markers by digesting away monosaccharides that are attached to some of the markers oligosaccharides but are not essential feature of the markers. For example, a glycosylation marker of which the essential part consists of a LeX epitope may be present on more than one glycan structure, e.g., it can be present on both oligosaccharide A that has a core fucose and on oligosaccharide B that does not have a core fucose. By digesting away the core fucose, structures A and B merge, thus, amplifying the signal associated with the glycosylation marker.

Using the glycosylation markers of cancer to identify and isolate glycoproteins In some embodiments, the determined glycosylation marker of cancer can be used for identifying and isolating one or more glycoprotein biomarkers, i.e. glycoproteins that are specific for particular type of cancer. The glycoprotein biomarker of the disease carries the glycosylation marker of cancer. The isolation of the glycoprotein biomarkers of the cancer can be carried out using lectins or monoclonal antibodies. For example, lectins were used to isolate gp73, a glycoprotein marker of hepatitis B associated with liver cancer in "Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans". T.M. Block, M.A. Comunale, M. Lowman, L.F. Steel, P.R. Romano, C. Fimmel, B.C. Tennant, W.T. London, A.A. Evans, B.S. Blumberg, R.A. Dwek, T.S. Mattu and A.S. Mehta (2005) Proc. Natl. Acad. Sci. USA₅ 102, 779-784.

The methodology for diagnosing and monitoring cancer can be illustrated in more details by the following examples, however, it should be understood that the present invention is not limited thereto.

Example 1. Breast Cancer.

Breast cancer is the leading cause of cancer deaths in females with a million new cases diagnosed annually, worldwide, see e.g. Mazor, Y., Keydar, L, and Benhar, I. MoI Immunol. 2005 Jan;42(l):55-69, incorporated herein by reference in its entirety. Humanization and epitope mapping of the H23 anti-MUCl monoclonal antibody reveals a dual epitope specificity. The panel of serum markers which is currently in practice mainly comprises of CAl 5-3 and/or C All.29 in combination with CEA (Carcinoembryogenic Antigen), see e.g. Duffy, MJ. (1999) CAl 5.3 and related mucins as circulating markers in breast cancer. Ann. Clin. Biochem., 36, 579- 586; Perkins, G.L., Slater, E.D., Sanders, G.K., and Prichard, J.G. (2003) Serum tumor markers. American Family Physician, 68, 1075-1082, both incorporated herein by reference. Both CAl 5-3 and CA27.29 are directed against MUCl, see Klee, G.G. and Schreiber, W.E. (2004) MUCl gene-derived glycoprotein assays for monitoring breast cancer (CA 15-3), CA 27.29, BR): Are They Measuring the Same Antigen? Arch. Pathol. Lab. Med., 128, 1131-1135, a mucin which is overly expressed and aberrantly glycosylated in breast cancer, see Taylor-Papadimitriou, J., Burchell, J., Miles, D. W., and Dalziel, M. (1999) MUCl and cancer. Biochem. Biophys. Acta., 1455, 301-313.

Analysis of Glycosylation Profiles of GIy cans Released from whole sera of a breast cancer patient and healthy controls.

Glycosylation profiles of glycans released from whole serum of controls and breast cancer patients were compared to detect a potential glycosylation marker differentiating the two groups. In addition to that, total serum glycans from a single breast cancer patient, but at two different stages of malignancy, were analyzed to correlate the detected marker with breast cancer progression.

Samples of serum from breast cancer patient were obtained from a single donor (LD) with her consent before and after mastectomy. The healthy control serum was obtained from pooled blood bank serum.

Glycoproteins in reduced and denatured serum samples were attached to a hydrophobic PVDF membrane in a 96 well plate (Multiscreen_IP, 0.45μm hydrophobic, high polyvinyldene fluoride (PVDF) membranes ,Millipore, Bedford, MA, USA) by simple filtration. The samples were then washed to remove contaminates, incubated with PNGaseF to release the glycans based on the methods described in Papac, D. L, et. al. Glycobiology 8: 445-54, 1998, and in Callewaert, N., et. al. Electrophoresis 25: 3128-31, 2004, both incorporated herein by reference in their entirety. The iV-glycans were then washed from the bound protein, collected and dried down ready for fluorescent labeling. Released glycans were labeled with 2- aminobenzamide (2-AB) fluorescent label with or without a commercial kit (e.g. Ludger Ltd, Oxford, UK) as described in Bigge, J. C, Patel, T. P., Bruce, J. A., Goulding, P. N., Charles, S. M., and Parekh, R. B. (1995). Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Analytical Biochemistry 230: 229-238, incorporated herein by reference in its entirety, and run by normal phase high performance liquid chromatography (NP-HPLC) on a 4.6 x 250 mm TSK Amide-80 column (Anachem, Luton, UK) using a Waters 2695 separations module equipped with a Waters 2475 fluorescence detector (Waters, Milford, MA, USA) as described in Guile, G. R., Rudd, P. M., Wing, D. R., Prime, S. B., and Dwek, R. A. (1996). A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Analytical Biochemistry 240: 210-226, incorporated herein by reference in its entirety. Prior to NP-HPLC analysis, glycans were subsequently digested with a series of exoglycosidases.

Figure IA shows glycosylation profiles of undigested glycans released from serum of a healthy control and a breast cancer patient. Sample 1 of the breast cancer patient is taken before surgery and Sample 2 is taken after surgery with liver metastases. Both glycosylation profiles from breast cancer samples demonstrate an increase in the peak at 10.5 glucose units (GU) compared to the glycosylation profile from the control sample (Figure IA). The 10.5 GU peak shifts down to 7.5 GU following digestion with sialidase, β 1-3,4,6 galactosidase and αl-2 link specific fucosidase, and has a higher percentage in the patient sample compared to the control (Figure IB). The peak at GU 7.5 is then completely digested by the combination of sialidase, βl-4 galactosidase (in place of β 1-3,4,6 galactosidase) and αl-3/4 link specific fucosidase in the control and patient samples indicating the presence of outer arm αl-3/4 fucosylation. This demonstrates an increased amount of LewisX epitope in the cancer (Figure 1C). After surgery the marker decreased from 3.9% to 3.3% suggesting that the prognosis may be poor.

Conclusion: a glycosylation marker of breast cancer was identified by comparing glycosylation profiles of glycans released from whole serum of breast cancer patient and of glycans released from whole serum of a healthy control. Digestion with exoglycosidases amplifies/segregates the glycosylation marker of breast cancer. The glycosylation marker is elevated in disease.

Example 2. Pancreatic cancer.

Pancreatic cancer is the fifth leading cause of cancer death in the United States killing about 30,000 people each year. About an equal number of new cases of pancreatic cancer are diagnosed each year, which corresponds to about 9 new cases per 100,000 people. While treatment options have advanced, pancreatic cancer remains very difficult to treat as evidenced by the high mortality rate. Specifically, the 1-year survival rate of pancreatic cancer patients is 19% and the 5-year survival rate is 4%. This high mortality rate is due, in part, to the fact that about 80% of pancreatic cancers are already metastatic at the time of diagnosis. See Yeo et ah, CURRENT PROBLEMS IN CANCER 26(4): (2002).

A variety of serum tumor markers, especially CAl 9-9, a Lewis blood group- related mucin, have been proposed for diagnosing and monitoring of this type of neoplasia, but their application remains experimental, see Lillemoe, K.D., Yeo, C.J., and Cameron, JX. (2000) Pancreatic cancer: state-of-the art care. Cancer J. Clin., 50, 241-268, incorporated herein by reference in its entirety.

In pancreatic cancer, both cell surface glycoproteins and secreted pancreatic ribonuclease (RNase 1) are aberrantly glycosylated, see e.g. Peracaula R, Royle L, Tabares G, Mallorqui-Fernandez G, Barrabes S, Harvey D, Dwek RA, Rudd, PM, de Llorens R. (2003) "Glycosylation of human pancreatic ribonuclease: differences between normal and tumour states", Glycobiology, 13, 227-244, incorporated herein by reference in its entirety. In particular, one of the most distinctive features was that RNase 1 glycans from established adenocarcinoma pancreatic cell lines, Capan-1 and MDAPanc-3, contained sialylated structures, which were completely absent in the RNase 1 from healthy pancreas. These differences provide distinct epitopes that were clearly detected using monoclonal antibodies against carbohydrate antigens. Monoclonal antibodies to Lewis^y reacted only with normal pancreatic RNase 1, whereas, in contrast, monoclonal antibodies to sialyl-Lewis^x and sialyl-Lewis^a reacted only with RNase 1 secreted from the tumor cells.

Analysis of Glycans Released from whole sera of Pancreatic cancer patients and corresponding healthy controls. In this study, glycosylation profiles of glycans released from whole serum of controls and pancreatic cancer patients were compared to detect a potential glycosylation marker differentiating the two groups.

Sample of serum from pancreatic cancer patient was obtained from a patient with neoplastic cancer, high creatinine levels and ,vascular affectations. Samples of healthy control serum were obtained as discarded clinical material from individuals undergoing routine employee health screening.

Glycoproteins in reduced and denatured serum samples were attached to a hydrophobic PVDF membrane in a 96 well plate (Multiscreen_IP, 0.45 μm hydrophobic, high polyvinyldene fluoride (PVDF) membranes, Millipore, Bedford, MA, USA) by simple filtration. The samples were then washed to remove contaminates, incubated with PNGaseF to release the glycans based on the methods described in Papac, D. L, et. al. Glycobiology 8: 445-54, 1998, and in Callewaert, N., et. al. Electrophoresis 25: 3128-31, 2004, both incorporated herein by reference in their entirety. The iV-glycans were then washed from the bound protein, collected and dried down ready for fluorescent labeling.

Released glycans were labeled with 2-aminobenzamide (2-AB) fluorescent label with or without a commercial kit (from e.g. Ludger Ltd, Oxford, UK) as described in Bigge, J. C, Patel, T. P., Bruce, J. A., Goulding, P. K, Charles, S. M,, and Parekh, R. B. (1995). Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Analytical Biochemistry 230: 229- 238, incorporated herein by reference in its entirety, and run by normal phase high performance liquid chromatography (NP-HPLC) on a 4.6 x 250 mm TSK Amide-80 column (Anachem, Luton, UK) using a Waters 2695 separations module equipped with a Waters 2475 fluorescence detector (Waters, Milford, MA, USA) as described in Guile, G. R., Rudd, P. M., Wing, D. R., Prime, S. B., and Dwek, R. A. (1996). A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Analytical Biochemistry 240: 210-226, incorporated herein by reference in its entirety. Prior to NP-HPLC analysis, glycans were subsequently digested with a series of exoglycosidases. Resulting glycosylation profiles are presented on Figure 2. In particular, left panel of Figure 2 demonstrates glycosylation profiles of glycans digested with sialidase, β-galactosidase and fucosidase with the arm specificity αl- 6,2»3,4. The glycosylation profile from the pancreatic cancer sample has additional peaks not present in the control sample. The glycans were further subjected to a digestion with αl-3,4 fucosidase. The results of this digestion are presented on the right panel of Figure 2. The digestion with αl-3,4 fucosidase removes the additional peaks previously present in the pancreatic cancer sample but not in the control sample, thus, demonstrating that there is an increase in the outer arm αl-3,4 fucosylation for the glycans released from the whole serum of the pancreatic cancer patient.

Conclusion: a glycosylation marker of pancreatic cancer was identified by comparing glycosylation profiles of glycans released from whole serum of pancreatic cancer patients and of glycans released from whole serum of a healthy control. The glycosylation marker of pancreatic cancer can be amplified/segregated by digesting with an array of exoglycosidases.

Example 3. Prostate Cancer.

Prostate cancer is the most common cancer in men in Western countries and is the second leading cause of cancer death. In fact, prostate cancer is the sixth most common cause of death overall for men in the U.S. It is estimated that 232,090 new cases of prostate cancer will be diagnosed in 2005 with 30,350 deaths attributed to prostate cancer. One in six men will be diagnosed with prostate cancer, and 1 in 33 men will die of the disease.

As other types of cancer, prostate cancer is classified based on how far the cancer has progressed. A number of scales exist, but the TNM (tumor, lymph node, and metastases) scale is a standard scale commonly referred to in the medical literature. In the Tl stage, the tumor cannot be seen on scans or felt during examination. These tumors are typically detected using a needle biopsy after an abnormal PSA test, which is discussed in more detail below. At the T2 stage, the tumor can be seen or felt but remains inside the prostate gland. T3 stage tumors have broken through the capsule of the prostate gland, and T4 stage tumors have spread into other body organs, such as the rectum or bladder. The N stages follow similar classifications of tumor spread as follows: (a) NO - no cancer cells found in any lymph nodes; (b) Nl - one positive lymph node smaller than 2cm across; (c) N2 - more than one positive lymph node or a tumor that is between 2 and 5cm across; and (d) N3 - any positive lymph node that is bigger than 5 cm across. Finally, a cancer is MO if no cancer has spread outside the pelvis and Ml if cancer has spread outside the pelvis.

Prostate-specific antigen (PSA), a glycoprotein secreted by prostate cells that is found in serum in prostate pathologies, is the currently used tumour marker for prostate cancer diagnostics, see e.g. Diamandis E. (1998) Prostate-Specific antigen: Its Uselfulness in Clinical Medicine. TEM, 9, 310-316, incorporated herein by reference. However, PSA is still not specific enough for diagnosing prostate cancer, as other prostatic pathologies, like benign prostate hyperplasia (BPH), can show serum PSA elevations. Different approaches have been tried to improve this situation but so far only with limited success, see e.g. Brawer MK. (1999) Prostate- specific antigen: current status. CA Cancer J Clin, 49, 264-281, incorporated herein by reference in its entirety.

Glycosylation has been found to be different between purified PSA from seminal plasma and when secreted by the tumour prostate cell line LNCaP, see e.g. Peracaula R, Tabares G, Royle L, Harvey DJ, Dwek RA, Rudd, PM, de Llorens R. (2003) Altered glycosylation pattern allows the distinction between Prostate Specific Antigen (PSA) from normal and tumor origins, Glycobiology, 13, 457-470.

Analysis of Glycans Released from Whole sera of Prostate cancer patients and corresponding healthy controls.

In this study, glycosylation profiles of glycans released from whole serum of healthy control and prostate cancer patient were compared to detect a potential glycosylation marker differentiating the two groups. Samples of tumor serum were obtained from a patient with prostate cancer with elevated levels of serum PSA (1.8 micrograms/ml). The healthy control serum was obtained from pooled blood bank serum.

Glycoproteins in reduced and denatured serum samples were attached to a hydrophobic PVDF membrane in a 96 well plate (Multiscreen_IP, 0.45μm hydrophobic, high polyvinyldene fluoride (PVDF) membranes, Millipore, Bedford, MA, USA) by simple filtration. The samples were then washed to remove contaminates, incubated with PNGaseF to release the glycans based on the methods described in Papac, D. L, et. al. Glycobiology 8: 445-54, 1998, and in Callewaert, N., et. al. Electrophoresis 25: 3128-31, 2004, both incorporated herein by reference in their entirety. The iV-glycans were then washed from the bound protein, collected and dried down ready for fluorescent labeling.

Released glycans were labeled with 2-aminobenzamide (2-AB) fluorescent label (Ludger Ltd, Oxford, UK) as described in Bigge, J. C, Patel, T. P., Bruce, J. A., Goulding, P. N., Charles, S. M., and Parekh, R. B. (1995). Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Analytical Biochemistry 230: 229-238, incorporated herein by reference in its entirety, and run by normal phase high performance liquid chromatography (NP-HPLC) on a 4.6 x 250 mm TSK Amide-80 column (Anachem, Luton, UK) using a Waters 2695 separations module equipped with a Waters 2475 fluorescence detector (Waters, Milford, MA, USA) as described in Guile, G. R., Rudd, P. M., Wing, D. R., Prime, S. B., and Dwek, R. A. (1996). A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Analytical Biochemistry 240: 210-226, incorporated herein by reference in its entirety. Prior to NP-HPLC analysis, glycans were subsequently digested with a series of exoglycosidases.

Resulting glycosylation profiles are presented on Figure 3. hi particular, the left panel of figure 3 demonstrates the glycosylation profiles of undigested glycans, the upper right panel of figure 3 demonstrates the glycosylation profiles of glycans digested with array of sialidase, β-galactosidase, and fucosidase with the arm specificity αl-6,2»3,4, while the lower left panel of figure 3 demonstrates the glycosylation profiles of glycans further digested with αl-3,4 fucosidase. For undigested glycans, a difference between glycosylation profiles from prostate cancer samples and from healthy control sample was observed in the region of ~ 9.5 to ~ 11 GU (Figure 3, left panel). Following the digestion with sialidase, β-galactosidase, and fucosidase αl-6,2»3,4, the glycosylation profile from prostate cancer sample demonstrates a stronger peak (in intensity and peak area) at ~ 6.5 GU compared to the control glycosylation profile. The stronger peak at -6.5 GU indicates a higher content of tetra antennary glycans in the prostate cancer sample. Further digestion with αl- 3,4 fucosidase removes/reduces the peaks at 7-7.5 GU in the glycosylation profiles of prostate cancer samples, while the peaks of the control sample are almost not affected. This indicates that there is an increase in the outer arm αl-3,4 fucosylation in the glycans released from a whole serum of prostate cancer patient.

Conclusion: a glycosylation marker of prostate cancer was identified by comparing glycosylation profiles of glycans released from whole serum of prostate cancer patient and of glycans released from whole serum of a healthy control. Digestion of glycans with exoglycosidases amplifies/segregates the glycosylation marker of prostate cancer.

Example 4. Hepatocellular Carcinoma in hepatitis C virus infected patients.

Hepatitis C is a serious worldwide health problem. Globally, an estimated 170 million people have been infected with the hepatitis C virus (HCV). Chronic HCV infection can result in fibrosis, cirrhosis, hepatocellular carcinoma (HCC) and hepatic decompensation. HCV related end-stage liver disease is the leading indication for liver transplants in the United States. There is no vaccine available against HCV. Liver biopsy is considered the gold standard for assessment of liver damage and determining the need of treatment, although it is expensive, invasive, and subject to interpretive variation. Treatment for HCV, a 6 to 12 month course of pegylated interferon and ribavirin, can lead to potentially severe side effects and only eradicates HCV in about half of patients. Current surveillance techniques are suboptimal for early diagnosis of HCC, which occurs in 1-4% of cirrhosis annually. There is an urgent need for non-invasive testing methods, as well as the identification of prognostic markers and more effective screening methods for early diagnosis of HCC.

Glycosylation profiles of glycans released from whole serum of controls and hepatitis C virus (HCV) infected patients with hepatocellular carcinoma were compared to detect a potential glycosylation marker differentiating the two groups. Two samples from healthy controls, including both pooled and individual sera, were analyzed. A specific database containing NP-HPLC serum glycan profiles for both sialylated and neutral glycans contains more than 38 glycans. The same procedure was applied to patient sera and the glycosylation marker of hepatocellular carcinoma in HCV patients was identified by comparison the database of glycans released from whole serum of HCV infected patients with the database of glycans released from whole serum of healthy controls

Samples of serum from HCV infected patients with hepatocellular carcinoma were obtained from HCV infected patients with moderate or severe fibrosis/cirrhosis. Samples of healthy control serum were obtained as discarded clinical material from individuals undergoing routine health screening

Glycoproteins in reduced and denatured serum samples were attached to a hydrophobic PVDF membrane in a 96 well plate (MultiscreenJGP, 0.45μm hydrophobic, high polyvinyldene fluoride (PVDF) membranes, Millipore, Bedford, MA, USA) by simple filtration. The samples were then washed to remove contaminates, incubated with PNGaseF to release the glycans based on the methods described in Papac, D. L, et. al. Glycobiology 8: 445-54, 1998, and in Callewaert, N., et. al. Electrophoresis 25: 3128-31, 2004, both incorporated herein by reference in their entirety. The iV-glycans were then washed from the bound protein, collected and dried down ready for fluorescent labeling.

Released glycans were labeled with 2-aminobenzamide (2-AB) fluorescent label with or without a commercial kit (from e.g. Ludger Ltd, Oxford, UK) as described in Bigge, J. C, Patel, T. P., Bruce, J. A., Goulding, P. N., Charles, S. M., andParekh, R. B. (1995). Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Analytical Biochemistry 230: 229- 238, incorporated herein by reference in its entirety, and run by normal phase high performance liquid chromatography (NP-HPLC) on a 4.6 x 250 mm TSK Amide-80 column (Anachem, Luton, UK) using a Waters 2695 separations module equipped with a Waters 2475 fluorescence detector (Waters, Milford, MA, USA) as described in Guile, G. R., Rudd, P. M., Wing, D. R., Prime, S. B., and Dwek, R. A. (1996). A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Analytical Biochemistry 240: 210-226, incorporated herein by reference in its entirety. Prior to NP-HPLC analysis, glycans were subsequently digested with a series of exoglycosidases .

Figure 4 presents NP-HPLC profiles of glycans released from control sample Con_9 and from sample of HCV infected patient with hepatocellular carcinoma HCV_42. On figure 4, panel (A) corresponds to glycoprofiles of whole serum glycans not exposed to any exoglycosidase digestion, panel (B) to glycoprofiles following digestion with an array of α2-3,6,8-sialidase, βl-4 galactosidase and β-iV- acetylgmcosaminidase. Panel (C) of figure 4 demonstrates that the marker correlated with the diagnosis of hepatocellular carcinoma in HCV patients is the percentage of core fucosylated glycans measured after digestion with α2-3,6,8-sialidase, βl-4 galactosidase and β-N-acetylglucosaminidase. Healthy control sera contains between 15 and 17% of these glycans, while sera of HCV infected patients with hepatocellular carcinoma contained more than 19% of these glycans. The correlation was also observed between the stage of the disease and the percentage of core fucosylated glycans measured after digestion with α2-3,6,8-sialidase, βl-4 galactosidase and β-iV- acetylglucosaminidase. HCV infected patients in moderate stage of hepatocellular carcinoma had the percentage of core fucosylated glycans measured after digestion with α2-3,6,8-sialidase, βl-4 galactosidase and β-JV-acetylglucosaminidase of 20- 22%, while patients in severe stages of disease, such as severe fibrosis/ cirrhosis, had this glycan marker above 25 % on average.

Conclusion: a glycosylation marker of hepatocellular carcinoma in HCV patients was identified by comparing glycosylation profiles of glycans released from whole serum of HCV patients with hepatocellular carcinoma and of glycans released from whole serum of healthy controls. The glycosylation marker of hepatocellular carcinoma in HCV patients is the percentage of core fucosylated glycans measured after digestion with α2-3,6,8-sialidase, βl-4 galactosidase and β-iV- acetylglucosaminidase. The marker correlates with the disease diagnosis and the disease severity. Digestion of glycans with exoglycosidases amplifies/segregates the glycosylation marker of hepatocellular carcinoma in HCV patients. Example 5. Ovarian Cancer

Ovarian cancer is the fourth leading cause of cancer related deaths in women in USA and the leading cause of gynecologic cancer death. Ovarian cancer is characterized by few early symptoms, presentation at an advanced stage, and poor survival. Despite being one tenth as common as breast cancer, ovarian cancer is three times more lethal. It is estimated that in 2005 22,220 women will be newly diagnosed with ovarian cancer, and 16,210 will die from the disease, see Jemal, A., Murray, T., Ward, E., Samuels, A., Tiwari, R., Ghafoor, A., Feuer EX. & Thun, MJ. (2005) CA Cancer J. Clin. 55, 10-30, incorporated hereby by reference in its entirety. The high mortality rate is due to the difficulties with the early detection of ovarian cancer. Indeed, ~ 80% of patients are diagnosed currently with advanced staged disease.

As other types of cancer, ovarian cancer is classified based on how far the cancer has progressed. A number of scales exist, but the TNM (tumor, lymph node, and metastases) scale is a standard scale commonly referred to in the medical literature. At Tl stage, cancer is confined to the ovaries - one or both. At T2 stage, cancer is in one or both ovaries and is extending into pelvic tissues and/or has also spread to the surface of the pelvic lining. At T3 stage, cancer is in one or both ovaries and has spread to the abdominal lining outside the pelvis. N categories indicate whether or not the cancer has spread to regional (nearby) lymph nodes and, if so, how many lymph nodes are involved. For ovarian cancer, NO stage means no lymph node involvement and Nl stage means that cancer cells found in regional lymph nodes close to tumor. M categories indicate whether or not the cancer has spread to distant organs, such as the liver, lungs, or non-regional lymph nodes. For ovarian cancer, MO stage means no distant spread is observed and Ml means that distant spread is present. Ovarian cancer is also often stages of disease from stage I (the least advanced) to stage IV (the most advanced stage). More details of grading ovarian and other cancers can be found, for example, at the American Cancer Society website (www.cancer.org^*).

Serum biomarkers that are elevated in women with ovarian cancer include carcinoembrionic antigen, ovarian cystadenocarcinoma antigen, lipid-associated sialic acid, NB/70, TAG 72.3, CA-15.3 and CA-125. see e.g. G. Mor et al, PNAS, v. 102, pp. 7677-7682, 2005. CA 125 is elevated in 82% of women with advanced ovarian cancer, however, it has a low predictive value for early stages of cancer, see Kozak et. al. PNAS, v.100, pp. 12343-12348, 2003.

In this study, glycosylation profiles of glycans released from whole serum of healthy control and ovarian cancer patient were compared to detect a potential glycosylation marker differentiating the two groups.

Samples of tumor serum were obtained from a patient with advanced malignant tumor. The healthy control serum was obtained from pooled blood bank serum.

Released glycans were labeled with 2-aminobenzamide (2-AB) fluorescent label (Ludger Ltd, Oxford, UK) as described in Bigge, J. C, Patel, T. P., Bruce, J. A., Goulding, P. N., Charles, S. M., and Parekh, R. B. (1995). Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Analytical Biochemistry 230: 229-238, incorporated herein by reference in its entirety, and run by normal phase high performance liquid chromatography (NP-HPLC) on a 4.6 x 250 mm TSK Amide-80 column (Anachem, Luton, UK) using a Waters 2695 separations module equipped with a Waters 2475 fluorescence detector (Waters, Milford, MA, USA) as described in Guile, G R., Rudd, P. M., Wing, D. R., Prime, S. B., and Dwek, R. A. (1996). A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Analytical Biochemistry 240: 210-226, incorporated herein by reference in its entirety. Prior to NP-HPLC analysis, glycans were subsequently digested with a series of exoglycosidases.

Resulting glycosylation profiles are presented on Figure 5. In particular, Figure 5A demonstrates the glycosylation profiles of undigested glycans, Figure 5B demonstrates the glycosylation profiles of glycans digested with sialidase, β 1-3,4,6 galactosidase and αl-2 link specific fucosidase, while Figure 5C demonstrates the glycosylation profiles of glycans further digested with sialidase, βl-4 galactosidase (in place of βl-3,4,6 galactosidase) and αl-3/4 link specific fucosidase. For undigested glycans, a difference between glycosylation profiles from ovarian cancer sample and from healthy control sample was observed in the region of ~ 9.5 to ~ 11 GU (Figure 5A). In particular, a strong peak was observed ~ 10.5 GU in the ovarian cancer patient which was weaker in the control sample. Upon digestion with sialidase, βl-3,4,6 galactosidase and αl-2 link specific fucosidase, the ~1Q.5 GU peak shifted to ~ 7.5 GU (Figure 5B). The 7.5 GU peak has a higher percentage (—11.8%) in the ovarian cancer sample than in the control sample. The peak at GU 7.5 is then completely digested by the combination of sialidase, βl-4 galactosidase (in place of βl-3,4,6 galactosidase) and αl-3/4 link specific fucosidase in the patient samples indicating the presence of outer arm αl-3 fucose (i.e. Lewis x epitope) (Figure 5C).

Conclusion: a glycosylation marker of ovarian cancer was identified by comparing glycosylation profiles of glycans released from whole serum of ovarian cancer patient and of glycans released from whole serum of a healthy control. Digestion with exoglycosidases amplifies/segregates the glycosylation marker of ovarian cancer. Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention.

All of the publications, patent applications and patents cited in this specification are incorporated herein by reference in their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of determining one or more glycosylation markers of cancer comprising obtaining a diseased sample and a control sample, wherein the diseased sample is a sample from a subject diagnosed with cancer and the control sample is a sample from healthy control; releasing a diseased glycan pool of total glycoproteins from the diseased sample and a control glycan pool of total glycoproteins from the control sample without purifying the glycoproteins and without exposing the diseased sample and the control sample to hydrazinolysis; measuring a diseased glycoprofile of the diseased glycan pool and a control glycoprofile of the control glycan pool using chromatography, mass spectrometry or a combination thereof; comparing the diseased glycoprofile and the control glycoprofiles to determine said one or more glycosylation markers of cancer.

2. The method of claim 1, wherein comparing the diseased glycoprofile and the control glycoprofile comprises comparing peak ratios in the diseased glycoprofile and in the control glycoprofile.

3. The method of claim 1 , further comprising selecting a best glycosylation marker out of said one or more glycosylation markers of cancer, wherein the best glycosylation marker has a highest correlation with one or more parameters of the subject diagnosed with cancer.

4. The method of claim 3, wherein the parameters of the subject diagnosed with cancer are diagnosis, disease stage, disease severity, age, sex, medical history, response to therapy or a combination thereof.

5. The method of claim 3, wherein the parameter is diagnosis.

6. The method of claim 1, wherein the cancer is pancreatic cancer, prostate cancer, breast cancer, hepatocellular carcinoma, ovarian cancer, bladder cancer, renal cancer, colon cancer, stomach cancer or lung cancer.

7. The method of claim 1, wherein said cancer is pancreatic cancer.

8. The method of claim 1, wherein said cancer is prostate cancer.

9. The method of claim 1, wherein said cancer is breast cancer.

10. The method of claim 1, wherein said cancer is hepatocellular carcinoma.

11. The method of claim 1, wherein said cancer is ovarian cancer.

12. The method of claim I₅ further comprising amplifying and segregating the glycosylation marker by digesting the diseased glycan pool and the control glycan pool with one or more exoglycosidases.

13. The method of claim 1 , further comprising amplifying and segregating the glycosylation marker by sequential digesting the diseased glycan pool and the control glycan pool with one or more exoglycosidases.

14. The method of claim 1, further comprising amplifying and segregating the glycosylation marker by digesting the diseased glycan pool and the control glycan pool with an array comprising one or more exoglycosidases.

15. The method of claim 1 , wherein the diseased glycan pool and the control glycan pool are pools of JV-linked glycans.

16. The method of claim 15, wherein said releasing is releasing of JV- glycans from a gel. i

17. The method of claim 1 , wherein the diseased glycan pool and the control glycan pool are pools of O-linked glycans.

18. The method of claim 1, wherein said releasing comprises attaching glycoproteins to polyvinyldene fluoride membranes.

19. The method of claim 18, wherein said releasing is releasing by ammonia-based β-eleimination from the polyvinyldene fluoride membranes.

20. The method of claim 1, further comprising labeling glycans in the diseased glycan pool and the control glycan pool with a radioactive or fluorescent label.

21. The method of claim 20, wherein the fluorescent label is 2- aminopyridine, 2-ammobenzamide, 2-aminoanthranilic acid, 2-aminoacridone or 8- aminonaphthalene-l,3,6-trisulfonic acid.

22. The method of claim 20, wherein the fluorescent label is 2- aminobenzamide.

23. The method of claim 1, wherein the diseased sample and the control sample are samples of a body fluid.

24. The method of claim 23, wherein the body fluid is whole serum, blood plasma, urine, seminal fluid or saliva.

25. The method of claim 23, wherein the body fluid is whole serum.

26. A method for diagnosing and monitoring cancer in a subject comprising obtaining a sample of body fluid or a body tissue of the subject; releasing a glycan pool of total glycoproteins from the sample without purifying the glycoproteins; measuring a glycoprofile of the glycan pool.

27. The method of claim 26, further comprising determining a clinical status of the subject from a level of a glycosylation marker of cancer in the glycoprofile.

77 28. The method of claim 27, wherein the clinical status is a stage of

78 cancer.

79 29. The method of claim 27, wherein the clinical status is selected from the so group consisting of cancer, precancerous condition, a benign condition or no

81 condition.

82 30. The method of claim 26, wherein cancer is pancreatic cancer, prostate

83 cancer, breast cancer, hepatocellular carcinoma, ovary cancer, bladder cancer, renal

84 cancer, colon cancer, stomach cancer or lung cancer.

85 31. The method of claim 26, wherein cancer is hepatocellular carcinoma.

86 32. The method of claim 31 , wherein the glycosylation marker is a

87 percentage of core fucosylated glycans measured after digestion with α2-3,6,8-

88 sialidase, βl-4 galactosidase and β-JV-acetylglucoaminidase in the glycans.

89 33. The method of claim 26, wherein cancer is prostate cancer.

90 34. The method of claim 26, wherein cancer is breast cancer.

91 35. The method of claim 26, wherein cancer is ovarian cancer.

92 36. The method of claim 26, wherein cancer is pancreatic cancer.

93 37. The method of claim 26, wherein the body fluid is whole serum, blood

94 plasma, urine, seminal fluid or saliva.

95 38. The method of claim 26, wherein the body fluid is serum.

96 39. The method of claim 26, wherein releasing a glycan pool comprises

97 preparing a gel from the sample.

98 40. The method of claim 39, wherein the glycan pool is a pool of N-

99 glycans and releasing a glycan pool further comprises releasing the pool of iV-glycans oo from the gel using PNGase F enzyme.

101 41. The method of claim 26, wherein releasing the glycan pool comprises

102 attaching the total glycoproteins to polyvinyldene fluoride membranes.

103 42. The method of claim 41 , wherein the glycan pool is a pool of N-

104 glycans and releasing the glycan pool further comprises incubating the polyvinyldene

105 fluoride membranes with PNGaseF enzyme.

106 43. The method of claim 41 , wherein releasing the glycan pool further

107 comprises chemically releasing the glycan pool by /^-elimination

108 44. The method of claim 41 , wherein releasing the glycan pool further

109 comprises releasing the glycan pool by ammonia-based /^-elimination.

no 45. The method of claim 26, further comprising digesting the glycans with

111 one or more exoglycosidase.

112 46. The method of claim 26, further comprising sequential digesting the

113 glycans with one or more exoglycosidase.

114 47. The method of claim 26, further comprising digesting the glycans with

115 an array comprising more than one exoglycosidase.

116 48. The method of claim 26, wherein measuring the glycoprofile is carried

117 out by chromatography, mass spectrometry or a combination thereof.

118 49. A method for optimizing a dosage of a existing therapeutic agent

119 against cancer comprising

120 obtaining a first sample of a body fluid or a body tissue from a cancer patient

121 before administering the therapeutic agent to the patient;

122 obtaining a second sample of a body fluid or a body tissue from the cancer

123 patient after administering the therapeutic agent to the patient;

124 releasing glycans of glycoproteins from the first and the second samples

125 without purifying the glycoproteins and without exposing the first and the second

126 sample to hydrazinolysis;

127 measuring a first glycoprofile of the glycans from the first sample and a 128 second glycoprofile of the glycans from the second sample;

129 comparing a level of a glycosylation marker of the cancer in the first

130 glycoprofile and the second glycoprofile.

131 50. A method oftesting a new therapy or a new therapeutic agent for

132 treating cancer comprising

133 obtaining a first sample of a body fluid or a body tissue from a cancer patient

134 before exposing the patient to the new therapy or the new therapeutic agent;

135 obtaining a second sample of a body fluid or a body tissue from the cancer

136 patient after exposing the patient to the new therapy or the new therapeutic agent;

137 releasing glycans of glycoproteins from the first and the second samples

138 without purifying the glycoproteins and without exposing the first and the second

139 samples to hydrazinolysis;

140 measuring a first glycoprofile of the glycans from the first sample and a

141 second glycoprofile of the glycans from the second sample;

142 comparing a level of a glycosylation marker of the cancer in the first

143 glycoprofile and the second glycoprofile.

144 51. A database comprising

145 glycan structures of glycans of glycoproteins, wherein the glycans are released

146 from a sample of a body fluid or a body tissue of a subject diagnosed with cancer and

147 wherein releasing the glycans is carried out without purifying the glycoproteins.

148 52. The database of claim 49, wherein the glycans are N-glycans.

149 53. The database of claim 49, wherein the glycans are O-glycans.

150