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WO2006114659A1 - Marqueurs de glycosylation pour un diagnostic et une surveillance du cancer - Google Patents

Marqueurs de glycosylation pour un diagnostic et une surveillance du cancer Download PDF

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Publication number
WO2006114659A1
WO2006114659A1 PCT/IB2005/002531 IB2005002531W WO2006114659A1 WO 2006114659 A1 WO2006114659 A1 WO 2006114659A1 IB 2005002531 W IB2005002531 W IB 2005002531W WO 2006114659 A1 WO2006114659 A1 WO 2006114659A1
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WIPO (PCT)
Prior art keywords
cancer
glycans
sample
glycoprofile
diseased
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PCT/IB2005/002531
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English (en)
Inventor
Raymond A. Dwek
Rafael De Llorens
Rosa Peracaula
Catherine M. Radcliffe
Louise Royle
Pauline M. Rudd
Nicole Zitzmann
Original Assignee
Dwek Raymond A
Rafael De Llorens
Rosa Peracaula
Radcliffe Catherine M
Louise Royle
Rudd Pauline M
Nicole Zitzmann
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Application filed by Dwek Raymond A, Rafael De Llorens, Rosa Peracaula, Radcliffe Catherine M, Louise Royle, Rudd Pauline M, Nicole Zitzmann filed Critical Dwek Raymond A
Priority to EP05770420A priority Critical patent/EP1886144A1/fr
Priority to JP2008508313A priority patent/JP2008539412A/ja
Priority to US11/411,232 priority patent/US8039208B2/en
Priority to US11/411,246 priority patent/US7892752B2/en
Publication of WO2006114659A1 publication Critical patent/WO2006114659A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Definitions

  • 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.
  • prostate specific antigen 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.
  • 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., Consale, M. A., Lowman, M., Steel, L. F., Romano, P. R., Fimmel, C, Tennant, B. C, London, W. T., Evans, A. A., Blumberg, B.
  • IgG immunoglobulin G
  • glycosylation profiles of purified glycoproteins 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.
  • IgG immunoglobulin G
  • 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 DJ 3 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.
  • PSA Prostate Specific Antigen
  • pancreatic ribonuclease pancreatic ribonuclease 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • glycosylation 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.
  • 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.
  • comparing the diseased glycoprofile and the control glycoprofile can comprise comparing peak ratios in the diseased glycoprofile and in the control glycoprofile.
  • 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.
  • 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.
  • 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.
  • measuring the glycoprofile can be carried out by any suitable technique, i.e. not necessarily by the technique used to determine the glycosylation marker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PVDF polyvinyldene fluoride
  • PTFE Polytetrafluoroethylene
  • releasing glycans can further comprise releasing glycans from the total glycoproteins immobilized on the protein binding membrane or in the gel.
  • releasing glycans from the immobilized glycoproteins can be carried out using enzymatic release with, for example, peptide N glycosidase F.
  • 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).
  • releasing glycans from the gel can be carried out from the total gel, i.e. without separating gel into the bands.
  • 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.
  • 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.
  • 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 4 ⁇ l 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), 2 ⁇ l of 0.5M dithiothreitol (DTT) and water to make up to 20 ⁇ l in total, incubated at 7O 0 C for lOmin, then alkylated by addition of 2 ⁇ l of 10OmM iodoacetamide and incubated for 30 min in the dark at room temperature.
  • 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
  • DTT dithiothreitol
  • 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 lmm 3 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 NaHCO 3 pH 7), which can be repeated twice before the gel plug can be then dried. PNGase F buffer solution (30 ⁇ l of 100 U/ml) is added (this is enough for 10-15mm 3 gel), more enzyme solution is added if larger gel bands can be used. The PNGaseF and gel pieces can be incubated overnight at 37 0 C.
  • the supernatant can be recovered along with 3 x 200 ⁇ l 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 ⁇ l of activated AG- 50(H + ), filtered through a 0.45 ⁇ m LH Millipore filter and dried down for fluorescent labeling.
  • 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 ⁇ l can be used (initially set into a 48 well plate, then removed for cutting up) with 300 ⁇ l of 100 U/ml of PNGaseF. The washing procedures can be similar to those used for in-gel-band release.
  • the whole protein mixture e.g. serum or plasma
  • a total volume of gel of 185 ⁇ l can be used (initially set into a 48 well plate, then removed for cutting up) with 300 ⁇ l 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
  • Ammonia-based ⁇ -elimination can be used to release both N- and O-glycans by a modification of the classical ⁇ -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 ⁇ -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.
  • Samples of glycoprotein, mixtures of glycoproteins, whole serum or other body fluids are reduced and alkylated by adding 4 ⁇ l 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), 2 ⁇ l of 0.5M dithiothreitol (DTT) and water to make up to 20 ⁇ l in total, incubated at 7O 0 C for lOmin, then alkylated by addition of 2 ⁇ l of 10OmM iodoacetamide and incubated for 30 min in the dark at room temperature.
  • 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
  • DTT dithiothreitol
  • Protein binding PVDF membranes (Durapore 13mm x 0.45 ⁇ m 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 6O 0 C, 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 ⁇ l of 0.5M boric acid is added to the glycans and incubated at 37 0 C 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.
  • the glycans upon releasing, 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. ⁇ l.
  • 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.
  • 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.
  • 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.
  • 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.
  • HPLC high performance liquid chromatography
  • mass spectrometry 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.
  • 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.
  • glycoprofile can be presented as a plurality of peaks corresponding to glycan structures in the glycans.
  • a peak ratio means a ratio between any one or more peaks and any other one or more peaks within the same glycosylation profile.
  • comparing peak ratios can mean comparing peaks intensities or comparing integrated areas under the peaks.
  • comparing peak ratios can be carried for glycans of the diseased and control samples which were not digested with exoglycosidase.
  • 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:
  • 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.
  • suitable glycosidases include, but are not limited to, N-glycosidase F (PNGase F), sialidase, ⁇ - galactosidase, fucosidase ⁇ l-6,2»3,4, ⁇ l-3,4, ⁇ l-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, polygalactur
  • enzymatic digestion is carried out with one or more exoglycosidases listed in table 1.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 2-AB 2- aminobenzamide
  • 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).
  • NP-HPLC normal phase high performance liquid chromatography
  • 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, ⁇ 1-3,4,6 galactosidase and ⁇ l-2 link specific fucosidase, and has a higher percentage in the patient sample compared to the control ( Figure IB).
  • 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).
  • 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.
  • 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.
  • 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 ⁇ m 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.
  • 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).
  • 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).
  • 2-AB 2-aminobenzamide
  • 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.
  • 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.
  • TNM tumor, lymph node, and metastases
  • 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.
  • 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.
  • 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 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.
  • PSA is still not specific enough for diagnosing prostate cancer, as other prostatic pathologies, like benign prostate hyperplasia (BPH), can show serum PSA elevations.
  • BPH benign prostate hyperplasia
  • 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 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, 0.45 ⁇ m 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.
  • 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).
  • NP-HPLC normal phase high performance liquid chromatography
  • glycans were subsequently digested with a series of exoglycosidases.
  • 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, ⁇ -galactosidase, and fucosidase with the arm specificity ⁇ l-6,2»3,4
  • the lower left panel of figure 3 demonstrates the glycosylation profiles of glycans further digested with ⁇ l-3,4 fucosidase.
  • 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.
  • 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.
  • 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
  • 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).
  • 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).
  • NP-HPLC normal phase high performance liquid chromatography
  • 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.
  • 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 ⁇ 2-3,6,8-sialidase, ⁇ l-4 galactosidase and ⁇ -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 ⁇ 2-3,6,8-sialidase, ⁇ l-4 galactosidase and ⁇ -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.
  • HCV infected patients in moderate stage of hepatocellular carcinoma had the percentage of core fucosylated glycans measured after digestion with ⁇ 2-3,6,8-sialidase, ⁇ l-4 galactosidase and ⁇ -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.
  • 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 ⁇ 2-3,6,8-sialidase, ⁇ l-4 galactosidase and ⁇ -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.
  • 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.
  • ovarian cancer is classified based on how far the cancer has progressed.
  • TNM tumor, lymph node, and metastases
  • Tl stage cancer is confined to the ovaries - one or both.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Glycoproteins in reduced and denatured serum samples were attached to a hydrophobic PVDF membrane in a 96 well plate (Multiscreen_IP, 0.45 ⁇ m 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.
  • 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).
  • NP-HPLC normal phase high performance liquid chromatography
  • glycans were subsequently digested with a series of exoglycosidases.
  • Figure 5A demonstrates the glycosylation profiles of undigested glycans
  • Figure 5B demonstrates the glycosylation profiles of glycans digested with sialidase, ⁇ 1-3,4,6 galactosidase and ⁇ l-2 link specific fucosidase
  • Figure 5C demonstrates the glycosylation profiles of glycans further digested with sialidase, ⁇ l-4 galactosidase (in place of ⁇ l-3,4,6 galactosidase) and ⁇ l-3/4 link specific fucosidase.
  • 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.

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Abstract

L'invention concerne une méthode de détermination d'au moins un marqueur de glycosylation du cancer. Cette méthode consiste à obtenir un échantillon malade et un échantillon témoin. L'échantillon malade est un échantillon provenant d'un patient qui a été diagnostiqué avec le cancer, et l'échantillon témoin est un échantillon provenant d'un patient témoin sain. Cette méthode consiste à libérer un ensemble de glycans malades de glycoprotéines totales provenant de l'échantillon malade, et un ensemble de glycans témoins de glycoprotéines totales provenant de l'échantillon témoin, sans purifier les glycoprotéines et sans exposer l'échantillon malade et l'échantillon témoin à une hydrazinolyse; à mesurer un glycoprofil malade de l'ensemble de glycans malades et un glycoprofil témoin de l'ensemble de glycans témoins, à l'aide d'une chromatographie, d'une spectrométrie de masse ou d'une combinaison de deux; à comparer le glycoprofil malade et les glycoprofils témoins pour déterminer au moins un marqueur de glycosylation du cancer. L'invention concerne une méthode pour diagnostiquer et pour surveiller le cancer chez un patient. Cette méthode consiste à obtenir un échantillon de fluide corporel ou de tissu corporel du patient; à libérer un ensemble de glycans de glycoprotéines totales provenant de l'échantillon, sans purifier les glycoprotéines; et à mesurer un glycoprofil de l'ensemble de glycans.
PCT/IB2005/002531 2005-04-26 2005-06-24 Marqueurs de glycosylation pour un diagnostic et une surveillance du cancer WO2006114659A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009013538A2 (fr) * 2007-07-20 2009-01-29 National Institute For Bioprocessing Research And Training Marqueurs de glycosylation pour le cancer et l'inflammation chronique
WO2009044213A1 (fr) * 2007-10-05 2009-04-09 National Institute For Bioprocessing Research And Training Marqueurs de glycosylation associés à une pancréatite, à une sepsie et à un cancer du pancréas
US20090117106A1 (en) * 2005-07-20 2009-05-07 Glykos Finland Oy Cancer Specific Glycans and Use Thereof
JP2009168470A (ja) * 2008-01-10 2009-07-30 Wako Pure Chem Ind Ltd 膵臓癌マーカーおよび膵臓癌の検査方法
WO2009136506A1 (fr) * 2008-05-09 2009-11-12 住友ベークライト株式会社 Procédé de diagnostic du cancer du pancréas à l’aide de chaînes de sucre de type à liaison n
US9804163B2 (en) 2010-08-06 2017-10-31 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Biomarkers for prostate cancer and methods for their detection
EP3467503A4 (fr) * 2016-05-18 2020-02-05 Tosoh Corporation Procédé de détection du cancer du poumon et trousse de détection
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EP2909632B1 (fr) 2012-10-17 2021-11-24 Hexal AG Procédé amélioré de mise en correspondance de glycanes de glycoprotéines
EP3802624A4 (fr) * 2018-06-01 2022-03-23 Musc Foundation for Research Development Analyse de glycanes de protéines et de cellules

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US10948482B2 (en) * 2018-06-07 2021-03-16 National Synchrotron Radiation Research Center Method for cancer grading
JP2021032562A (ja) * 2019-08-13 2021-03-01 株式会社島津製作所 分析用試料の調製方法および分析方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008760A1 (fr) 2000-07-19 2002-01-31 Biotron Limited Procede permettant d'identifier des marqueurs du cancer et utilisations de ceux-ci pour diagnostiquer un cancer
WO2003016464A2 (fr) 2001-08-17 2003-02-27 Biotie Therapies Corp. Sequences d'oligosaccharides specifiques pour le cancer et leur utilisation
WO2004019040A1 (fr) 2002-08-20 2004-03-04 Proteome Systems Intellectual Property Pty Ltd Procede d'identification de marqueurs diagnostiques de maladies et leurs utilisations dans le diagnostic du cancer
WO2004066808A2 (fr) * 2002-12-20 2004-08-12 Momenta Pharmaceuticals, Inc. Marqueurs de glycane utilises dans le diagnostic et la surveillance d'une maladie

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008760A1 (fr) 2000-07-19 2002-01-31 Biotron Limited Procede permettant d'identifier des marqueurs du cancer et utilisations de ceux-ci pour diagnostiquer un cancer
WO2003016464A2 (fr) 2001-08-17 2003-02-27 Biotie Therapies Corp. Sequences d'oligosaccharides specifiques pour le cancer et leur utilisation
WO2004019040A1 (fr) 2002-08-20 2004-03-04 Proteome Systems Intellectual Property Pty Ltd Procede d'identification de marqueurs diagnostiques de maladies et leurs utilisations dans le diagnostic du cancer
WO2004066808A2 (fr) * 2002-12-20 2004-08-12 Momenta Pharmaceuticals, Inc. Marqueurs de glycane utilises dans le diagnostic et la surveillance d'une maladie

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
"Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates", MASS SPECTROM REV, vol. 18, pages 349 - 450
AMANO, J., METHODS ENZYMOL, vol. 179, 1989, pages 261 - 70
ART WITTWER ET AL.: "Effects of N-Glycosylation on in vitro Activity of Bowes Melanoma and Human Colon Fibroblast Derived Tissue Plasminogen Activator", BIOCHEMISTRY, vol. 28, 1989, pages 7662 - 7669
CIOLCZYK-WIERZBICKA DOROTA ET AL: "The structure of the oligosaccharides of N-cadherin from human melanoma cell lines", GLYCOCONJUGATE JOURNAL, vol. 20, no. 7-8, 2004, pages 483 - 492, XP002362375, ISSN: 0282-0080 *
DIAMANDIS, CLIN. LAB. NEWS, vol. 22, 1996, pages 235 - 239
HARVEY, D. J.: "Electrospray mass spectrometry and fragmentation of N-linked carbohydrates derivatized at the reducing terminus", JAM SOC MASS SPECTROM, vol. 11, 2000, pages 900 - 915
KEESEE, CRIT. REV. EUKARYOTIC GENE EXPR, vol. 6, no. 2, 3, 1996, pages 189 - 214
PERACAULA R ET AL.: "Altered glycosylation pattern allows the distinction between Prostate Specific Antigen (PSA) from normal and tumor origins", GLYCOBIOLOGY, vol. 13, 2003, pages 457 - 470
PERACAULA R ET AL.: "Glycosylation of human pancreatic ribonuclease: differences between normal and tumour states", GLYCOBIOLOGY, vol. 13, 2003, pages 227 - 244
PERACAULA ROSA ET AL: "Glycosylation of human pancreatic ribonuclease: Differences between normal and tumor states.", GLYCOBIOLOGY, vol. 13, no. 4, 1 April 2003 (2003-04-01), pages 227 - 244, XP002362434, ISSN: 0959-6658 *
RAJ B. PAREKH ET AL.: "N-Glycosylation and in vitro Enzymatic Activity of Human Recombinant Tissue Plasminogen Activator Expressed in Chinese Hamster Ovary Cells and a Murine Cell line", BIOCHEMISTRY, vol. 28, 1989, pages 7670 - 7679
STEIN ET AL., J. UROL, vol. 160, 1998, pages 645 - 659
TAYLOR-PAPADIMITRIOU, J. ET AL.: "MUC1 and cancer", BIOCHEM. BIOPHYS. ACTA., vol. 1455, 1999, pages 301 - 313
YEO ET AL., CURRENT PROBLEMS IN CANCER, vol. 26, no. 4, 2002, pages 176 - 275

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WO2009013538A2 (fr) * 2007-07-20 2009-01-29 National Institute For Bioprocessing Research And Training Marqueurs de glycosylation pour le cancer et l'inflammation chronique
WO2009013538A3 (fr) * 2007-07-20 2009-04-02 Nat Inst For Bioproc Res And T Marqueurs de glycosylation pour le cancer et l'inflammation chronique
WO2009044213A1 (fr) * 2007-10-05 2009-04-09 National Institute For Bioprocessing Research And Training Marqueurs de glycosylation associés à une pancréatite, à une sepsie et à un cancer du pancréas
JP2009168470A (ja) * 2008-01-10 2009-07-30 Wako Pure Chem Ind Ltd 膵臓癌マーカーおよび膵臓癌の検査方法
JP2009270996A (ja) * 2008-05-09 2009-11-19 Sumitomo Bakelite Co Ltd Ｎ結合型糖鎖を利用した膵臓癌の診断方法
WO2009136506A1 (fr) * 2008-05-09 2009-11-12 住友ベークライト株式会社 Procédé de diagnostic du cancer du pancréas à l’aide de chaînes de sucre de type à liaison n
US8372647B2 (en) 2008-05-09 2013-02-12 Sumitomo Bakelite Company Limited Method of diagnosing pancreatic cancer with the use of N-binding type sugar chains
US9804163B2 (en) 2010-08-06 2017-10-31 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Biomarkers for prostate cancer and methods for their detection
EP2909632B1 (fr) 2012-10-17 2021-11-24 Hexal AG Procédé amélioré de mise en correspondance de glycanes de glycoprotéines
EP3467503A4 (fr) * 2016-05-18 2020-02-05 Tosoh Corporation Procédé de détection du cancer du poumon et trousse de détection
US11137405B2 (en) 2016-05-18 2021-10-05 Tosoh Corporation Lung cancer detection method and detection kit
EP3802624A4 (fr) * 2018-06-01 2022-03-23 Musc Foundation for Research Development Analyse de glycanes de protéines et de cellules
CN113480629A (zh) * 2021-06-16 2021-10-08 复旦大学 区分肝癌和正常人的末端唾液酸化异构糖肽标志物及其筛查方法
CN113480629B (zh) * 2021-06-16 2022-08-19 复旦大学 区分肝癌和正常人的末端唾液酸化异构糖肽标志物及其筛查方法

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