WO2010148145A1 - Procédés et kits pour détecter un cancer ovarien à partir de sang - Google Patents

Procédés et kits pour détecter un cancer ovarien à partir de sang Download PDF

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Publication number
WO2010148145A1
WO2010148145A1 PCT/US2010/038899 US2010038899W WO2010148145A1 WO 2010148145 A1 WO2010148145 A1 WO 2010148145A1 US 2010038899 W US2010038899 W US 2010038899W WO 2010148145 A1 WO2010148145 A1 WO 2010148145A1
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protein
subject
ovarian cancer
levels
sample
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PCT/US2010/038899
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English (en)
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Samir M. Hanash
Vitor M. Faca
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Fred Hutchinson Cancer Research Center
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Publication of WO2010148145A1 publication Critical patent/WO2010148145A1/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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the technical area of the description pertains to methods of screening for or detecting ovarian cancer in subjects, including screening for or detecting blood-based marker proteins.
  • Ovarian cancer is the fifth leading cause of cancer death among women in the United States and has the highest mortality rate of all gynecologic cancers. It is estimated that 21,650 new cases of ovarian cancer will be diagnosed in the United States in 2008, and 15,520 women will die of this disease. The median age at diagnosis is 63. The prognosis for survival from ovarian cancer largely depends on the extent of disease at diagnosis. The overall five-year survival rate for ovarian cancer is lower than fifty percent. Fewer than one fourth of women present with localized disease at diagnosis.
  • CA125 Proteins detectable in serum and plasma are commonly relied upon to diagnose, monitor cancer response to therapy, and disease recurrence, CA125, the current ovarian cancer marker proteins detected in circulation, represent a secreted protein that occurs in low abundance in plasma.
  • CA125 is a tumor-associated antigen that is used clinically to monitor patients with epithelial ovarian carcinomas.
  • elevated CA125 levels are not specific to ovarian cancer and have been observed in patients with nongynecological cancers and in the presence of other conditions.
  • the sensitivity of the CA125 test for the detection of ovarian cancer was estimated to be 35 U/mL ranged. Elevated levels were found in 20% to 57% of ovarian cancer patients within the first three years of follow-up with a specificity of 95%.
  • plasma profiling represents a substantial challenge.
  • plasma is one of the most accessible biological materials, it contains vast assemblies of proteins and complexes and exhibits considerable heterogeneity between and within subjects that hinder proteomic analysis of low abundance proteins.
  • the present invention provides a method of diagnosing, prognosing or screening for ovarian cancer in a subject.
  • the method can be carried out on a biological sample such as a blood or tissue sample obtained from the subject.
  • One aspect of the invention is a method comprising the steps of:
  • Another aspect of the invention is a method comprising: contacting blood of a subject with at least one antibody to measure plasma levels of at least one protein selected from the group consisting of GRN 1 IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and CD14.
  • Another aspect of the invention is a method comprising: administering to a subject a therapeutic regime for treatment of ovarian cancer, wherein said subject is identified by blood levels of at least one protein selected from the group consisting of GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and CD14.
  • Another aspect of the invention is a method comprising: (a) receiving a blood sample that was collected a subject having ovarian cancer; (b) contacting said blood sample with at least one antibody to measure levels of at least one protein selected from the group consisting of GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and CD14, in which the expression levels indicates a therapy suitable for the subject.
  • An embodiment of the invention is the method further comprising determining a difference in levels of GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, or CD 14 in a biological sample of a subject and a control sample, wherein said the difference in levels indicates that the subject is at risk of ovarian cancer.
  • An additional embodiment is the method in which the controlled sample is from said subject or from a different subject.
  • Another aspect of the invention is a method comprising; (a) receiving a blood sample that was collected from a subject having ovarian cancer; (b) contacting said blood with a multiplicity of antibodies to measure levels of at least one protein selected from the group consisting of GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and CD14; and (c) using a computer to transform said levels into a probability that the subject has an ovarian cancer.
  • Another aspect of the invention is a method comprising: (a) measuring blood levels of at least one protein selected from the group consisting of GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and CD14 from a subject having ovarian cancer; (b) using a computer to transform said levels into a probability that the subject has an ovarian cancer; (c) repeating steps (a) and (b) at one or more time points during treatment of said subject for ovarian cancer, wherein a low probability value indicates effective treatment.
  • Another aspect of the invention is a method of manufacturing a report comprising: (a) contacting a sample of blood, or material derived from the blood, with a multiplicity of antibodies; (b) measuring reactivity of said antibodies to at least one protein selected from the group consisting of GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and CD14; (c) transforming by a computing means said reactivity into a level of at least one said protein in blood; and (d) producing a report in a tangible medium describing said level of said protein.
  • kits comprising: one or more means of detecting at least one protein selected from the group consisting of GRN, IGFBP2, TGFB 1 , THBS 1 , RARRES2, LCN2, TIMPl , and CD14 in blood of a subject at risk for ovarian cancer.
  • An embodiment of the kit comprises at least one antibody for measuring levels of the protein.
  • CA125 alone, a combination marker of GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, and
  • TIMPl, and CA125 + combination marker are shown for A) all ovarian cancer cases vs. healthy controls and B) early stage ovarian cancer cases vs. healthy controls.
  • altered level or altered levels as used with respect to marker proteins herein refers to an increased level (e.g., a one or two fold increase, or more) or a decreased level (e.g., a one or two-fold decrease, or more) in the quantity of one or more marker proteins detectable in or via a biological sample from a subject, as compared to a level or levels of one ore more marker proteins in a corresponding subject not afflicted with a ovarian disease such as ovarian cancer.
  • Biological sample refers to any material taken from the body of a subject that may carry the target compound or compounds of the tests described herein, including both tissue samples and biological fluids such as blood samples, saliva samples, urine samples, etc.
  • Blood sample refers to whole blood or any fraction thereof that may contain detectable levels of marker proteins therein (if marker proteins are present in the whole blood sample from which said fraction is obtained), and in particular embodiments refers to a blood sera or blood plasma sample.
  • Diagnosing means providing an indication that a subject may be afflicted with or at risk of developing a disease, particularly a ovarian disease such as ovarian cancer, and includes other terms such as screening for a disease, providing a risk assessment for disease, etc. It will be appreciated that no such technique is perfect and that such diagnosis, prognosis or the like may be confirmed by other procedures including physical examination, imaging, and histological examination of tissue samples. Most generally a “diagnosis” is conducted by a medical doctor based on a variety of information generated by a variety of procedures.
  • prognosing includes providing an assessment or indication of disease in response to treatment (such as surgical, radiation therapy, chemotherapy, and combinations thereof) after initial diagnosis, as an indication of the efficacy of the treatment, risk of the disease returning, severity of disease following treatment, or the like.
  • Marker protein refers to any protein that can be detected, directly or indirectly (e.g., via an analog, metabolite, fragment or breakdown product) in a biological sample from a subject, an increase or decrease of the amount of which, compared to amounts found in similar subjects without disease, is indicative of the presence or risk of ovarian cancer in a subject.
  • Marker proteins described herein include any protein listed in Table 1 herein. The analog, metabolite, fragment or breakdown product of the marker protein may or may not possess die functional activity of the marker protein listed.
  • CA125 is a known marker protein for ovarian cancer, and can be detected in accordance with known techniques, including but not limited to those described in U.S. Patent Nos. 6,716,595; 6,030,341; and 7,205,142.
  • “Ovarian cancer” as described herein refers to any type of cancerous or precancerous tissues arising from normal tissues of the ovaries, and spreading to other regions within the abdomen, including die ovaries, fallopian tubes and uterus, or other organs, including, e.g., liver and intestine.
  • Protein test refers to a group of individual laboratory tests that are related in some way, including, but not limited to, the medical condition they are designed to detect (e.g., ovarian cancer), the specimen type (e.g., blood), and the methodology employed by the test (e.g., detection of altered level of a target protein or proteins).
  • biomarkers are defined as follows:
  • GRN Granulins are a family of secreted, glycosylated peptides mat are cleaved from a single precursor protein with 7.5 repeats of a highly conserved 12- cysteine granulin/epithelin motif.
  • the 88 kDa precursor protein, progranulin is also called proepithelin and PC cell-derived growth factor (PCDGF).
  • Cleavage of the signal peptide produces mature granulin which be further cleaved into a variety of active, 6 kDa peptides. These smaller cleavage products are named granulin A, B, C, D, E, F, and G..
  • Epithelins 1 and 2 are synonymous with granulins A and B, respectively.
  • IGFBP2 Insulin-like growth factor (IGF)-binding protein 2 is a protein that in humans is encoded by the IGFBP2 gene. IGF-binding proteins prolong the half-life of the IGFs and have been shown to either inhibit or stimulate the promoting effects of the IGFs on cell culture. They alter the interaction of IGFs with their cell surface receptors. UniProtKB Entry P18065.
  • TGF- ⁇ l Transforming growth factor beta 1
  • TGF- ⁇ l Transforming growth factor beta 1
  • cytokines a polypeptide member of the transforming growth factor beta superfamily of cytokines. It is a secreted protein that performs many cellular functions, including the control of cell growth, cell proliferation, cell differentiation and apoptosis.
  • TGF- ⁇ l is encoded by the TGFB I gene. UniProtKB Entry P0U37.
  • Thrombospondin 1 is a subunit of a disulfide-linked homotrimeric protein. This protein is an adhesive glycoprotein mat mediates cell-to-cell and cell-to-matrix interactions. This protein can bind to fibrinogen, fibronectin, laminin, type V collagen and integrins alpha- V/beta-1. This protein has been shown to play roles in platelet aggregation, angiogenesis, and tumorigenesis. UniProtKB Entry P07996
  • RARRES2 Retinoic acid receptor responder (tazarotene induced) 2 protein is a secreted chemotactic protein that initiates chemotaxis via the ChemR23 G protein-coupled seven-transmembrane domain ligand. Expression of this gene is upregulated by the synthetic retinoid tazarotene and occurs in a wide variety of tissues. The active protein has several roles, including that as an adipokine, and is truncated on both termini from the proprotein. UniProtKB Entry Q99969.
  • LCN2 Lipocalin-2 (LCN2), also known as oncogene 24p3 or neutrophil gelatinase-associated lipocalin, is a protein that in humans is encoded by the LCN2 gene.
  • LCN2 Lipocalin-2
  • the binding of lipocalin-2 to bacterial siderophores is important in the innate immune response to bacterial infection. Upon encountering invading bacteria the toll-like receptors on immune cells stimulate the synthesis and secretion of Iipocalin-2. Secreted lipocalin-2 then limits bacterial growth by sequestering iron-containing siderophores.
  • TIMPl TIMP metallopeptidase inhibitor 1, a tissue inhibitor of metalloproteinases, is a glycoprotein that is expressed from the several tissues of organisms. This protein a member of the TIMP family. The glycoprotein is a natural inhibitor of the matrix metalloprote ⁇ nases, a group of peptidases involved in degradation of the extracellular matrix. In addition to its inhibitory role against most of the known MMPs, the encoded protein is able to promote cell proliferation in a wide range of cell types, and may also have an anti- apoptotic function. UniProtKB Entry P01033.
  • CD14 The CD 14 protein is a component of the innate immune system, and exists in two forms, membrane-anchored by a glycosylphosphatidylinositol tail or soluble. Soluble CD14 either appears after shedding of membrane-anchored CD14 (48 KDa) or is directly secreted from intracellular vesicles (56 KDa). CD14 is included in the "cluster of differentiation" group of cell surface marker proteins. CD 14 acts as a co-receptor (along with the Toll-like receptor TLR 4 and MD- 2) for the detection of bacterial lipopolysaccharide. CD14 can bind LPS only in the presence of lipopolysaccharide-binding protein. Although LPS is considered its main ligand, CD14 also recognizes other pathogen- associated molecular patterns. UniProtKB Entry P08571.
  • Subjects as described herein are generally human subjects and includes “patients".
  • the subjects may be male or female and may be of any race or ethnicity, including but not limited to Caucasian, African- American, African, Asian, Hispanic, Indian, etc.
  • the subjects may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric.
  • Subjects may also include animal subjects, particularly mammalian subjects such as dog, cat, horse, mouse, rat, etc., screened for veterinary medicine or pharmaceutical drug development purposes.
  • Subjects include but are not limited to those who may have, possess, have been exposed to, or have been previously diagnosed as afflicted with one or more risk factors for ovarian cancer.
  • Risk factors include age, gender, race, smoking, diet, obesity, diabetes, chronic pancreatitis, work exposure, family history, and stomach problems. These risk factors may be considered in combination with the disclosed methods of detecting ovarian cancer for a diagnosis, prognosis or screening.
  • the disclosed methods of detecting ovarian cancer for a diagnosis, prognosis or screening may also be used in combination with other diagnostic methods, including, but not limited to, scanning of the pancreas by an ultrasound or CT scan of the abdomen, detection of bilirubin and other substances, physical signs of jaundice, performing a biopsy, and screening for indicators of the possibility of ovarian cancer such as CA125.
  • the subject's biological sample is provided, preferably a body fluid, most preferably a sample from blood.
  • the body fluid can be blood and fractions thereof, blood serum, blood plasma, urine, excreta, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), pleural effusion, tears, saliva, sputum, sweat, biopsy, ascites, cerebrospinal fluid, amniotic fluid, lymph, marrow, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions, breast secretions, ovarian cyst secretions, hair, and tissue extract samples, including paraffin-embedded (FFPE) tissue homogenates.
  • FFPE paraffin-embedded
  • a blood sample from a subject can be carried out by phlebotomy or any other suitable technique, with the blood sample processed further to provide a serum sample or other suitable blood fraction.
  • the step of determining the presence of an altered level of a marker protein in the sample, and/or depressed level of a marker protein in the sample can also be carried out either directly or indirectly in accordance with known techniques, including, but not limited to, mass spectrometry, chromatography, electrophoresis, sedimentation, isoelectric focusing, and antibody assay.
  • Methods for detection and quantitation of proteins include both "capture” and "non-capture” methods, and may or may not include specific antibodies.
  • non-antibody affinity agents may include aptamers, peptoids, and lectins.
  • Non-capture methods may include mass spectrometry.
  • Methods may include direct or indirect detection of specific marker proteins may be performed with
  • binding reaction may be detected through any of several tags, including fluorescent tags, enzyme tags, biotin/ubiquitin affinity methods, quantum dots;
  • non-array-based methods such as ELISA
  • array-based methods such as antibody arrays
  • alternative multiplex assay methods such as Luminex bead-based assays
  • biosensors such as aptamer biosensors.
  • Marker proteins may also be identified by two-dimensional electrophoresis (2-D electrophoresis).
  • 2D-electrophoresis is a technique comprising denaturing electrophoresis, followed by isoelectric focusing; this generates a two-dimensional gel (2D gel) containing a plurality of separated proteins. Altered levels of marker proteins in a first sample or sample set with respect to a second sample or sample set can be determined when 2D gel electrophoresis gives a different signal when applied to the first and second samples or sample sets.
  • Altered levels of marker proteins may be present in first sample or sample sets at increased, elevated, depressed or reduced levels as compared to the second sample or sample sets.
  • increased level it is meant (a) any level of a marker protein when that marker protein is not present in a normal subject without ovarian cancer, as well as (b) an elevated level (e.g., a two- or three-fold increase in detected quantity) of marker protein or a particular isoform of a marker protein when that protein or a particular isoform is present in a normal subject without ovarian cancer.
  • depression level it is meant (a) an absence of a particular marker protein or isoform of a particular marker protein when that marker protein is present in a normal subject without ovarian cancer, as well as (b) a reduced level (e.g., a two- or three-fold reduction in detected quantity) of a marker protein or isoform of a marker protein when that protein or isoform is present in a normal subject without ovarian cancer.
  • the steps of (a) assaying a sample for an elevated level of a marker protein and/or depressed level of a marker protein, and (b) correlating an elevated level of a marker protein and/or a depressed level of a marker protein in said sample with ovarian cancer can be carried out in accordance with known techniques or variations thereof that will be apparent to persons skilled in the art.
  • Signals obtained upon analyzing a biological sample or sample set from subjects having ovarian cancer relative to signals obtained upon analyzing a biological sample or sample set from normal subjects without ovarian cancer will depend upon the particular analytical protocol and detection technique that is used.
  • each laboratory will establish a reference range for each marker protein identifier (e.g., pi and/or MW) in normal subjects without ovarian cancer according to the analytical protocol and detection technique in use, as is conventional in the diagnostic art.
  • marker protein identifier e.g., pi and/or MW
  • Protein levels can be measure by the use of antibodies.
  • Antibody assays may, in general, be homogeneous assays or heterogeneous assays.
  • the immunological reaction usually involves the specific antibody, a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution.
  • Immunochemical labels that may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
  • the reagents are usually the specimen, the antibody to the marker protein and a system or means for producing a detectable signal. Similar specimens as described above may be used.
  • the antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase.
  • the support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal.
  • the signal is related to the presence of the analyte in the specimen.
  • Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, and so forth.
  • an antibody that binds to tfiat site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step.
  • the presence of the detectable group on the solid support indicates the presence of the antigen in the test sample.
  • suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like. Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof, which may be useful for carrying out the methods disclosed herein.
  • Antibodies for immunoassays can be polyclonal or monoclonal antibodies, Fab fragments, humanized antibodies and chimeric antibodies (including fragments thereof) and can be produced in accordance with known techniques, based on one or more marker protein.
  • monoclonal antibodies may be produced in a hybridoma cell line according.
  • Monoclonal Fab fragments may be produced in Escherichia coli from the known sequences by recombinant techniques known to those skilled in the art.
  • Polyclonal antibodies can be produced in animals such as goats, rabbits and horses by administration of one or more marker protein, optionally in combination with an adjuvant, as an immunogen, optionally administering booster doses thereof, and collecting the polyclonal antibodies from the animal.
  • Kits for diagnosis, prognosis or screening for ovarian cancer include at least one biochemical material and/or reagent, such as buffers and/or binding partners that are capable of specifically binding with one or more marker proteins embodied herein.
  • biochemical material and/or reagent such as buffers and/or binding partners that are capable of specifically binding with one or more marker proteins embodied herein.
  • these can provide a means for determining binding between the biochemical material and one or more marker proteins, whereby at least one analysis to determine a presence of one or more marker proteins, analyte thereof, or a biochemical material specific thereto, is carried out on a biological sample.
  • analysis or analyses may be carried out with the additional use of detection devices for immunoassay, chromatography, spectrometry, electrophoresis, sedimentation, isoelectric focusing, or any combination thereof.
  • the kit may optionally include instructions for performing the method or assay. Additionally the kit may optionally include depictions or photographs that represent the appearance of positive and negative results. In some embodiments, the components of the kit may be packaged together in a common container.
  • the marker proteins described herein can be detected individually or in panels with one another or other additional markers for ovarian cancer such as described above.
  • the levels of the various markers are optionally but preferably tested from the same biological sample obtained from the subject (e.g., by detecting the quantities or amounts of various proteins in the same blood sample obtained from a patient).
  • the panel test may include determining an altered level for each of 2, 3, 4, 5, or 6 different marker proteins, up to 38 or more different proteins (e.g., a panel of some or all proteins set forth in Table 1 below).
  • the combination of multiple marker proteins in a panel test serves to reduce the number of false positives and false negatives should an aberrant value for one particular member of the panel be found.
  • Kits for diagnosis, prognosis or screening for ovarian cancer include at least one biochemical material and/or reagent, such as buffers and/or binding partners, that is capable of specifically binding with one or more marker proteins from Table 1 included in a panel.
  • biochemical material and/or reagent such as buffers and/or binding partners
  • these can provide a means for determining binding between the biochemical material and one or more marker proteins of the panel, whereby at least one analysis to determine a presence of one or more marker proteins, analyte thereof, or a biochemical material specific thereto, is carried out on a biological sample.
  • analysis or analyses may be carried out with the additional use of detection devices for immunoassay, chromatography, spectrometry, electrophoresis, sedimentation, isoelectric focusing, or any combination thereof.
  • kits may optionally comprise instructions for performing the method or assay. Additionally the kit may optionally comprise depictions or photographs that represent the appearance of positive and negative results, In some embodiments, the components of the kit may be packaged together in a common container.
  • Proteomic profiling of ovarian cancer cell populations including cell lines and fresh tumor cells enriched from ascites fluid resulted in the identification of several thousand proteins and elucidated the repertoire of proteins expressed on the cell surface and proteins released into the extra-cellular milieu.
  • Proteome analysis has uncovered shedding of extra-cellular domains and highly dynamic processes of protein secretion.
  • Test agents and compounds include but are not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies, e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g., Fab and F(ab)2, and epitope-binding fragments thereof), nucleic acids (e.g., antisense, interference RNA) and small organic or inorganic molecules.
  • the agents or compounds may be endogenous physiological compounds or natural or synthetic compounds.
  • the invention also provides a method for assessing the potential efficacy of a test agent for inhibiting ovarian cancer in a patient.
  • This method comprises comparing levels of a one or more marker proteins, including GRN, IGFBP2, TGFB 1 , THBS 1 , RARRES2, LCN2, TIMP 1 , and (optionally) CA125, and/or polynucleotides encoding same, in the first sample, relative to the second sample, to determine if the test agent is potentially efficacious for inhibiting ovarian cancer in the patient.
  • a one or more marker proteins including GRN, IGFBP2, TGFB 1 , THBS 1 , RARRES2, LCN2, TIMP 1 , and (optionally) CA125, and/or polynucleotides encoding same, in the first sample, relative to the second sample, to determine if the test agent is potentially efficacious for inhibiting ovarian cancer in the patient.
  • the first and second samples may be portions of a single sample obtained from a patient or portions of pooled samples obtained from a patient.
  • the invention provides a method of selecting an agent for inhibiting ovarian cancer in a patient comprising:
  • test agents which alters die levels of die protein markers, optionally CA125, and/or polynucleotides encoding same, in the aliquot containing that test agent relative to odier test agents.
  • Still another aspect of the present invention provides a method of conducting a drug discovery business comprising:
  • step (b) conducting therapeutic profiling of agents identified in step (a), or further analogs diereof, for efficacy and toxicity in animals;
  • step (c) formulating a pharmaceutical preparation including one or more agents identified in step (b) as having an acceptable therapeutic profile.
  • die subject method can also include a step of establishing a distribution system for distributing die pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing die pharmaceutical preparation.
  • the invention also contemplates a method of assessing the ovarian carcinogenic potential of a test compound comprising:
  • markers comprise or are selected from the group consisting of GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and CD14.
  • Computer readable media comprising a plurality of protein markers, and optionally CA125, is also provided.
  • Computer readable media refers to any medium that can be read and accessed directly by a computer, including but not limited to magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD- ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • the invention contemplates computer readable medium having recorded thereon markers identified for patients and controls, "Recorded” refers to a process for storing information on computer readable medium. The skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising information on a plurality of protein markers, and optionally CA125.
  • a variety of data processor programs and formats can be used to store information on a plurality of protein markers, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl and optionally CA125, on computer readable medium.
  • the information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.
  • Any number of data processor structuring formats e.g., text file or database
  • marker information in computer readable form
  • one skilled in the art can use the information in computer readable form to compare marker information obtained during or following therapy with the information stored within the data storage means.
  • the invention provides a medium for holding instructions for performing a method for determining whether a patient has ovarian cancer or a pre-disposition to ovarian cancer, comprising determining the presence or absence of a plurality of protein markers, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, optionally CA125, and/or polynucleotides encoding same, and based on the presence or absence of the plurality of protein markers, optionally CA125, and/or polynucleotides encoding same, determining whether the patient has ovarian cancer or a pre-disposition to ovarian cancer, and optionally recommending treatment for the ovarian cancer or pre-ovarian cancer condition.
  • a plurality of protein markers including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, optionally CA125, and/or polynucleotides encoding same, and based on the presence or absence of the
  • the invention also provides in an electronic system and/or in a network, a method for determining whether a subject has ovarian cancer or a pre-disposition to ovarian cancer associated with a plurality of protein markers, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and optionally CA125, and/or polynucleotides encoding same, comprising determining the presence or absence of a plurality of protein markers, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and optionally CA125, and/or polynucleotides encoding same, and based on the presence or absence of the plurality of protein markers, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl 1 and optionally CA125, and/or polynucleotides encoding same, determining whether the subject has ovarian cancer or a pre-dis
  • the invention further provides in a network, a method for determining whether a subject has ovarian cancer or a pre-disposition to ovarian cancer associated with a plurality of protein markers, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, optionally CA125 and/or polynucleotides encoding same, comprising: (a) receiving phenotypic information on the subject and information on a plurality of protein markers, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TlMPl, optionally CA125 and/or polynucleotides encoding same associated with samples from the subject; (b) acquiring information from the network corresponding to the plurality of protein markers, including GRN, IGFBP2, TGFBl 1 THBSl, RARRES2, LCN2, TIMPl, optionally CA125, and/or polynucleotides encoding same; and (c) based on the
  • a system of the invention generally comprises a digital computer; a database server coupled to the computer; a database coupled to the database server having data stored therein, the data comprising records of data comprising a plurality of protein markers, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, optionally CA 125, and/or polynucleotides encoding same, and a code mechanism for applying queries based upon a desired selection criteria to the data file in the database to produce reports of records which match the desired selection criteria.
  • a code mechanism for applying queries based upon a desired selection criteria to the data file in the database to produce reports of records which match the desired selection criteria.
  • a method for detecting an ovarian cancer cell using a computer having a processor, memory, display, and input/output devices comprising the steps of: (a) creating records of a plurality of protein markers, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, optionally CA125, and/or polynucleotides encoding same, isolated from a sample suspected of containing an ovarian cancer cell;
  • step (c) using a code mechanism for applying queries based upon a desired selection criteria to the data file in the database to produce reports of records of step (a) which provide a match of the desired selection criteria of the database of step (b) the presence of a match being a positive indication that the markers of step (a) have been isolated from a cell that is an ovarian cancer cell.
  • the invention contemplates a method of manufacturing a report comprising (a) contacting a sample of blood, or material derived from the blood, with a multiplicity of antibodies; (b) measuring reactivity of said antibodies to at least one protein selected from the group consisting of GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and CD14; and (c) transforming by a computing means said reactivity into a level of at least one said protein in blood; and (d) producing a report describing said levels of said protein in a tangible medium.
  • a major benefit of the report is to provide a means of diagnosing or measuring die presence of a disease in a clinical setting, to thereby accelerate treatment of the disease to an earlier stage than afforded by conventional diagnostic means.
  • a report will provide improved health care for the patient, increased certainty for the health care provider's diagnosis, decreased cost of health care for both health care managers and insurance providers, and improved public health.
  • Any individual having an interest in measuring the presence of a disease in a subject may cause a report to be manufactured, including a subject or patient, a medical doctor, a physician, a health management organization (HMO), a clinic, a health care provider, a health insurer, a company involved in reimbursing an insurance claim or in negotiating the cost of a diagnostic service.
  • a pharmaceutical company may cause a report to be manufactured during a clinical trial to measure the extent of efficacy of an experimental therapy, hi a health care setting, the cost of the report may be paid by an insurance provider who causes the report to conform to certain specifications which are required for payment.
  • the doctor or odier health care provider may cause the report to contain information relevant to a diagnosis or prognosis.
  • the offer can be made through advertisement by computer or printed media, preferably to a group of doctors or physicians, or by specific contact with a group of medical service providers.
  • the offer can be made with a demand for payment.
  • the payment may be made by the person requesting the product report.
  • the requester can be a subject being tested, or the subject's health care provider.
  • the payment can be received from the requester or from a third party, e.g., a group health insurance provider.
  • the biological sample will be provided to the manufacturer of the report.
  • the sample may be provided directly or through agents who manage transportation within the quality needed to maintain the viability of the biological sample.
  • the sample may be provided to the manufacturer of the report at the doctor's office or through collection centers.
  • the report must contain information about the levels of at least one of the protein markers in the blood.
  • the levels of the protein marker in the blood can described with respect to an absolute concentration or amount, or may be relative to normal levels in the population or relative to prior measurements in the same subject.
  • the report may be a product manufactured from a network corresponding to (a) the plurality of protein markers, optionally CA125, and/or polynucleotides encoding same; and (b) from phenotypic information, information on a plurality of protein markers, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, optionally CA125, and/or polynucleotides encoding same, and acquired information, determining whether the subject has ovarian cancer or a pre-disposition to ovarian cancer; and (c) optionally recommending treatment or a treatment modality for the ovarian cancer or pre-ovarian cancer condition.
  • the report may instead or in addition contain a probability assessment that evaluates the relative risk of ovarian cancer, or the extent of an existing cancer.
  • the report may provide information to the requestor that enables a diagnosis or prognosis related to ovarian cancer.
  • the manufactured report can also contain a diagnosis or prognosis resulting from analysis performed by a diagnostic service provider utilizing information about the levels of at least one of the protein markers in die blood..
  • the report may be produced in a writing.
  • the writing can be in a printed form, or through an electronic means. If electronic, it may be through an email account, a secure web sight or an external FTP site Kits
  • the methods described herein may be performed by utilizing pre-packaged diagnostic kits comprising at least a plurality of protein, nucleic acids or binding agents (e.g. antibodies) or CA125 nucleic acids or binding agents described herein, which may be conveniently used, e.g., in clinical settings, to screen and diagnose patients, and to screen and identify those individuals afflicted with or exhibiting a predisposition to ovarian cancer.
  • nucleic acids or binding agents e.g. antibodies
  • CA125 nucleic acids or binding agents described herein which may be conveniently used, e.g., in clinical settings, to screen and diagnose patients, and to screen and identify those individuals afflicted with or exhibiting a predisposition to ovarian cancer.
  • kits for carrying out the methods of the invention typically comprise two or more components required for performing a diagnostic assay.
  • Components include but are not limited to compounds, reagents, containers, and/or equipment.
  • a container with a kit comprises binding agents as described herein.
  • the kit may contain antibodies specific for a one or more proteins, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and optionally CA125, antibodies against the antibodies labeled with enzymes; and substrates for the enzymes.
  • the kit may also contain microtiter plate wells, standards, assay diluent, wash buffer, adhesive plate covers, and/or instructions for carrying out a method of the invention using the kit.
  • the kit includes antibodies or antibody fragments which bind specifically to epitopes of each of one or more proteins, including GRN, IGFBP2, TGFB 1 , THBSl, RARRES2, LCN2, TIMPl, and optionally CA125, and means for detecting binding of the antibodies to epitopes associated with tumor cells, either as concentrates (including lyophilized compositions), which may be further diluted prior to use or at the concentration of use, where the vials may include one or more dosages.
  • single dosages may be provided in sterilized containers, having the desired amount and concentration of agents. Containers that provide a formulation for direct use, usually do not require other reagents, as for example, where the kit contains radiolabelled antibody preparations for in vivo imaging.
  • kits may be designed to detect the level of polynucleotides encoding proteins, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and optionally CA125 polynucleotides, in a sample.
  • kits generally comprise oligonucleotide probes or primers, as described herein, that hybridize to a plurality of polynucleotides encoding protein polypeptides, including GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl and optionally CA125.
  • Such oligonucleotides may be used, for example, within a PCR or hybridization procedure.
  • kits include second oligonucleotides and/or diagnostic reagents to facilitate detection of a plurality polynucleotides encoding protein polypeptides, and optionally CA125 polynucleotides.
  • reagents suitable for applying the screening methods of the invention to evaluate compounds may be packaged into convenient kits described herein providing the necessary materials packaged into suitable containers.
  • LSL-K-ras GI D/+ Pten loxP oxP mice were generated and ovarian tumors were induced by Adeno-Cre adenovirus as previously described (Dinulescu, e ⁇ ⁇ /.(2005) Nat Med 11:63-70).
  • Control mice were littermates that underwent the same surgical procedure but were injected instead with Adeno-empty virus.
  • Plasma samples from AdenoCre-infected K-ras/Pten mice and Adeno-empty injected controls were subjected to quantitative proteomic analysis to determine differences in plasma protein levels. Cancer cases were mice with ⁇ 0.5cm ovarian tumors, with pelvic and peritoneal metastasis.
  • the sample was fractionated identically in two dimensions: first by anion exchange and second by reverse phase as described previously. Digestion with trypsin was performed following reconstitution of lyophilized aliquots of a total 144 pools. The samples were then analyzed by shotgun LC-MS/MS analysis on a LTQ- FT (ThermoFisher Scientific) mass spectrometer equipped with a nano-LC system (Waters). The nano-LC was equipped with a 25 cm column packed with Magic C18 packing material (Michrom)). Spectra were acquired in data dependent mode with a MS 1 m/z range of 400 to 1800, followed by selection of the 5 most abundant doubly or triply protonated ions in each MS 1 spectrum for MS/MS analysis.
  • LTQ- FT ThermoFisher Scientific
  • Quantitative ratios were obtained for peptides containing cysteine residues labeled with heavy and light acrylamide isotopes to obtain the relative quantification from MSl spectra for each pair of peptides identified. Calculation of ratios between cancer and normal were fraction- centric ⁇ i.e., per LC-MS/MS run). All identified peptide measured ratios were processed such that multiple measurements for a given peptide in one individual fraction were log 2 averaged, resulting in a dataset containing one ratio per peptide per each individual fraction. A global normalization factor was then computed as the mode of the peptide ratio histogram. All peptide ratios for a specific protein present in a particular fraction were then normalized and log- averaged to obtain the local relative protein ratio. Statistical significance of protein quantitation was assigned by two methods as described herein.
  • IPAS Ingenuity Pathways Analysis
  • MetaCore from GeneGo Inc.
  • a feature of the IPAS platform is that extensive fractionation allows de-complexing of the samples into individual fractions to allow identification and quantification of proteins present in the plasma over 6-7 orders of magnitude.
  • a dataset containing accession numbers and the corresponding cancer-to-control ratios was uploaded into each application where all proteins identified in the IPAS experiment were used as a reference set.
  • Each accession number was mapped to its corresponding gene object in the IPAS knowledge base or MetaCore's manually curated data base.
  • IPAS IP-based advanced genome sequence
  • these genes were designated as focus genes and were overlaid onto a global molecular network developed from information contained in the IPAS knowledge base. Networks of these focus genes were then algorithmically generated based on their connectivity. A score is generated for each network based on the fit between the focus genes and each network. The score is the -log(p-value) calculated based on a hypergeometric distribution with the right-tailed Fisher's Exact Test.
  • MetaCore the gene list of proteins found to be up-regulated in the mouse plasma and secreted/shed in mouse and human cancer cell lines (total of 58 genes) was submitted to an enrichment and network workflow.
  • Enrichment analysis was conducted across three GeneGo curated ontologies along with Gene Ontology to provide a quantitative analysis of the most relevant biological functions represented by the data. Networks and the statistics for each, were generated using the analyze network algorithm, one of the nine network building algorithms in MetaCore.
  • PeptideProphet an empirical statistical modeling program, was used to estimate the accuracy of peptide identifications. Factors determined by the search algorithm were weighted to assign a single number for each peptide identification mat can be then compared to other peptide identifications.
  • ProteinProphet a program that applies a statistical model to infer protein groups from peptide identifications and validates these groups with a probability assignment, was also utilized. A protein group may contain one or more protein sequence, wim each sequence being indistinguishable based on the identified peptides. Proteins with a ProteinProphet score corresponding to 5% error rate (-3.5% false discovery rate as determined by ProteinProphet) were retained.
  • protein For each protein group (herein, "protein"), a representative gene symbol was chosen. Some 1,725,000 mass spectra were collected and analyzed. Statistical significance of protein quantification by mass spectrometry was determined by two methods. Proteins for which multiple paired MS events of heavy and light acrylamide were observed, a one-sample t-test was used to calculate a p- value for the mean ratio of the whole protein across all fractions. Secondly, the probability for the ratio for each MS event was calculated from the distribution of ratios in a control-control experiment in which the same sample was labeled with heavy and light acrylamide. If the p- value for each individual event was ⁇ 0.05, the overall protein ratio was considered statistically significant. 1,031 unique proteins were identified with high confidence. Of these, 106 proteins were upregulated 1.5-fold or greater (p-value ⁇ 0.05)
  • upregulated proteins contained a signal peptide for secretion, whereas 17% encompassed in their corresponding gene sequence a trans-membrane domain. 28% of the upregulated proteins were previously identified in proteome profiling of mouse liver tissue, a major source of plasma proteins. Nine percent had no human ortholog as determined based on the Mouse Genome Database.
  • a proteomic analysis of three human ovarian cancer cell lines (OVCAR3, CAOV3, and ES2) and of tumor cells from ascites fluid obtained from an ovarian cancer patient was conducted.
  • the ascites derived tumor cells were collected from a patient with serous ovarian cancer.
  • Cells were isotopically labeled in culture using SILAC to ascertain the cellular origin of proteins identified in media.
  • upregulated mouse plasma proteins By comparing the upregulated mouse plasma proteins with the list of proteins enriched in the surface or secreted cellular compartments from human ovarian cancer cells, 55% (58/106) of upregulated proteins in mouse plasma were found to be released from ovarian cancer cells through secretion or shedding or enriched in the ovarian cancer cell surface compartment.
  • Candidate markers for ovarian cancer that were identified in both mouse plasma and ovarian cancer cells included WFDC2 (HE4), IGFBP2, and LCN2. The balance of proteins not identified in ovarian cancer cell analyses consisted primarily of inflammatory and immune response related proteins.
  • upregulated proteins in the mouse plasma analysis 58 were found to be also enriched in the surface or secreted sub-cellular compartments of human ovarian cancer cells.
  • Pathway analysis of the 48 proteins that were found to be upregulated in mouse plasma with tumor development, but not identified as secreted or surface membrane proteins in ovarian cancer cells yielded three significant networks with Ingenuity, including inflammatory proteins such as haptoglobin, S100A8, and CCL8,
  • the categorization of upregulated proteins in the plasma based on enrichment in ovarian cancer cells sub- fractions provided a means for assessing which upregulated proteins in plasma were more likely derived from cancer cells, and which proteins were more likely related to host-response.
  • Proteins identified in ovarian cancer cells and found to be upregulated in plasma from tumor bearing mice were selected for immunoblot analysis. Expression pattern of key proteins from the IPAS analysis was analyzed in conditioned media of human ovarian cancer cell lines and human primary ovarian tumors freshly collected from patients undergoing surgery. In addition, western blot analysis was performed on ovarian tumors collected from two mouse models of ovarian cancer: K-ras/Pten and Pten/Apc.
  • Specimens consisted of tumor tissue collected from mouse models, conditioned media from human ovarian cancer cell lines, and primary ovarian tumors from human subjects.
  • tissue lysates were prepared from normal ovaries and tumors taken from the same animal (a-d) along with four-five tumors from separate animals.
  • tissue lysates were prepared from four tumor samples paired with their respective normal tissue from the same patient and three tumor samples, in which no normal samples were available.
  • Patient samples are as follows: one patient had independent bilateral serous borderline tumors in her ovaries, three were patients with papillary serous carcinomas, one patient had an epithelial borderline tumor of the M ⁇ llerian-type, with endocervical mucinous and serous differentiation, one patient had a clear cell carcinoma, and one patient had an endometrioid adenocarcinoma.
  • Protein concentration was determined for each specimen. Samples were resolved by electrophoresis and transferred to membranes, and incubated with primary antibodies. Horseradish peroxidase-conjugated secondary antibodies were applied and detected by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology Inc, Rockford, IL).
  • TIMPl TIMPl
  • LCN2 IGFBP2, PFNl
  • SPARC EEF1B2, CLU
  • FBLN2 FBLN2
  • Increased protein levels for Timpl, Lcn2, Igfbp2, PmI, and Sparc were observed in ovarian tumor tissues isolated from K-ras/Pten and Pten/Apc ovarian cancer mouse models compared to control tissue lysates.
  • TIMPl, LCN2, IGFBP2, PFNl, SPARC, EEF1B2, CLU, and FBLN2 was observed in CM collected from ovarian cancer cell lines derived from serous adenocarcinomas (OVCAR-3, SKOV3, CaOV3, OVCAR-5, OVCAR-8), endometrioid carcinoma (TOVl 12D), clear cell carcinoma (ES-2, IGROVl) compared to conditioned media derived from human ovarian surface epithelium (HOSE).
  • expression levels of these same proteins were elevated in ovarian tumor lysates compared to control tissue freshly collected from patients undergoing surgery.
  • Plasma Levels of Mouse TlMPl and LCN2 Significantly Increase During Ovarian Cancer GEM
  • the subset of proteins found to be upregulated in tumor bearing mice were tested for their levels in human and murine plasma and to compare performance of proteins found to be secreted by ovarian cancer cells with other proteins.
  • ELISA For validation studies using ELISA, we collected plasma from infected mice at various stages of tumor progression and controls. In addition, we also collected ascites or ovarian tumor fluid extracted from late stage tumors. Timpl concentrations in murine plasma were measured using a Quanitikine- Mouse Timpl ELISA Kit, while mouse Lcn2 levels were detected with a DuoSet ELISA Kit (R&D Systems, Minneapolis, MN USA).
  • mice were grouped according to disease stage: control, Stages I-II (early stage) and Stage IU-IV (late stage).
  • Human plasma samples were collected from women. 163 samples were obtained in total consisting of: 68 women newly diagnosed with ovarian cancer (55 collected at the time of surgery, 13 collected in advance of surgery), 56 healthy controls (collected under non-surgical conditions from apparently healthy women attending regular breast cancer screening exams), 11 surgical controls (patients undergoing gynecologic surgery for a variety of conditions but with normal ovarian pathology), and 28 samples from patients with benign ovarian disease (collected at the tune of surgery). The same specimen processing protocol was used for all samples.
  • Plasma levels of vWF (1:100) (American Diagnostics, Stamford, CT, USA), GRN (1:200) (Adipogen, Seoul, South Korea), sICAM2 (1:20) (Abeam, Cambridge, MA, USA), and LCN2 (1:500) (BioPorto Diagnostics, Gentofte, Denmark) were also measured. Plasma levels of CAI 25 were measured using a bead- based assay.
  • proteins levels were normalized to eliminate batch-to-batch variation in measurements. Marker levels were normalized to give healthy controls a mean of 0 and a standard deviation of 1. Different groups were compared using a two-tailed Student's t-test analysis.
  • Statistical calculations for the CAl 25 and combination marker included generation of a variable indicating the presence of cancer and fitting of a logistic regression model to the data according to the formula: ⁇ cl*CA125 + c2*proteinl + c3*protein2 + c4*protein3 + c5* ⁇ rotein4 + c6*protein5 + c7*protein6 + c8*protein7.
  • the fitted equation was then utilized to predict the probability of having cancer for each woman based on her marker levels and the fitted probabilities were used as the combination marker.
  • the regression model was fit using R 2.6.0 (R Development Core Team (2008)) and ROC curves were produced using the ROCR package (R package version 1.0-1, ed., 2005), Timpl levels in Stage I/II and Stage III/IV samples were increased 5.9-fold (p ⁇ 0.0001) and 9.5-fold ( ⁇ 0.0001) compared to controls, respectively. Similar findings were observed for Lcn2.
  • Timpl and Lcn2 were measured and found enriched in peritoneal ascites. Timpl levels were elevated more than 400-fold and 250 fold in ovarian tumor fluid and peritoneal ascites compared to murine plasma, respectively. Likewise, Lcn2 had a similar pattern with a 79- and 19-fold increased levels in ovarian tumor fluid and peritoneal ascites, respectively, consistent with their active secretion into surrounding fluids by tumor cells.
  • Plasma levels of human THBSl, VWF, GRN, CD14, TIMPl, PPBP, IGFBP2, RARRES2, LCN2, NRCAM, and TGFBl increase in cancer
  • Table 1 shows proteins assayed in human ovarian cancer patient and control samples. Results included mouse plasma cancer/control ratios and ratios of subcellular compartments in the cell line data. A "
  • ELISA p-values for the respective groups are summarized as follows: * ⁇ ⁇ 0.05, ** ⁇ ⁇ 0.01, and *** p ⁇ 0.0001. NS represents not significant.
  • results from assays in human plasma showed that levels of THBSl, VWF, GRN, CD14, TIMPl, PPBP, IGFBP2, RARRES2, LCN2, NRCAM 1 and TGFBl, were statistically significantly elevated in plasma samples from newly diagnosed subjects with ovarian cancer compared to controls.
  • THBSl, VWF, GRN, CD14, TIMPl, IGFBP2, and NRCAM also showed statistically significant increased levels in early stage cases.
  • GRN 1 IGFBP2, TGFBl, THBSl, LCN2, RARRES2, TIMPl, and CD14 were found to be significantly upregulated in the human ovarian cancer plasma assays.
  • AXL, DKK3, ICAM2, ALCAM, VCAMl, IGFlR, and LYVEl exhibited statistically significant decreased levels in cases compared to controls. Their reduced levels contrast with their occurrence as shed or on the surface of ovarian cancer cells.
  • Six of these seven proteins contained a trans-membrane domain.
  • NrCAM was found to be upregulated primarily in early ovarian cancer plasmas.
  • Plasma THBSl levels have been previously reported to be elevated in patients with gynecologic malignancies (Nathan, etal. (1994) Cancer 73:2853-58).
  • VWF is a known inflammatory protein. Interestingly it is also expressed in ovarian cancer cells and has been previously reported to be elevated in ovarian cancer patient serum (Gadducci, et al. (1994) Gynecol Oncol 53:352-356).
  • GRN has been described as a putative novel growth factor for ovarian cancer, and was found to be highly secreted by ovarian cancer cells (Jones, et al. (2003) Gynecol Oncol 88:S136-39).
  • CD14, IGFBP2, and LCN2 have been previously assayed in serum from ovarian cancer patients (Palmer, et al. (2008) PLoS ONE 3:e2633; Baron-Hay, et al. (2004) CHn Cancer Res 10:1796-1806; Flyvbjerg, et al (199I) J CHn Endocrinol Metab 82:2308-13; Lim, et al. (2007) Int J Cancer 120:2426-34; Gadducci, et al. (1995) Gynecol Oncol 58:184-88).
  • Figure 1 shows the receiver operator characteristic (ROC) curves for a combination panel of the seven secreted proteins with or without CA125 with area under the curves (AUCs) of 0.98 and 0.89 respectively, for all cases.
  • ROC receiver operator characteristic
  • AUCs area under the curves
  • curves for CA125 alone, a combination marker of GRN, IGFBP2, TGFBl, THBSl, RARRES2, LCN2, TIMPl, and CA125 combination marker are shown for A) all ovarian cancer cases vs. healdiy controls and B) early stage ovarian cancer cases vs. healthy controls.
  • the p- value achieved for differences between the two ROC curves with all cases and controls in the test set was 0.095 based on statistical comparison of two ROC-curve estimates obtained from partially-paired datasets.
  • ROC curves are also provided for early cases versus controls ( Figure IB).
  • the subset of proteins that were validated in human ovarian cancer sera represented proteins found to be upregulated in plasma from tumor bearing mice and found to be secreted in human ovarian cancer cells.
  • Elevated levels of the protein CD14 in plasma were found in from ovarian cancer patients vs from controls.

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Abstract

La complexité du protéome plasmatique humain représente un défi substantiel pour la mise au point de nouvelles méthodes diagnostiques. L'invention porte sur un procédé consistant à mettre en contact le sang d'un sujet avec au moins un anticorps pour mesurer les taux plasmatiques d'au moins une protéine choisie dans le groupe constitué par GRN, IGFBP2, TGFB1, THBS1, RARRES2, LCN2, TIMP1 et CD14. Un ordinateur est utile pour transformer la réactivité à l'anticorps en une valeur de taux sanguin. Les taux plasmatiques de ces protéines sont utiles pour sélectionner une thérapie appropriée pour des sujets atteints d'un cancer ovarien, ou pour surveiller l'efficacité de la thérapie. L'invention porte également sur un procédé d'élaboration d'un compte-rendu dans un milieu tangible décrivant ledit taux de ladite protéine.
PCT/US2010/038899 2009-06-16 2010-06-16 Procédés et kits pour détecter un cancer ovarien à partir de sang WO2010148145A1 (fr)

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JP7154523B2 (ja) 2017-07-31 2022-10-18 国立大学法人福井大学 子宮肉腫診断のための迅速測定法
WO2020193403A1 (fr) * 2019-03-22 2020-10-01 Katholieke Universiteit Leuven Méthodes de diagnostic du cancer de l'ovaire

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